Patent Application
20100113591
Amyloidosis refers to a pathological condition characterized by the presence of amyloid fibrils. Amyloid is a generic term referring to a group of diverse but specific protein deposits (intracellular or extracellular) which are seen in a number of different diseases. Though diverse in their occurrence, all amyloid deposits have common morphologic properties, stain with specific dyes (e.g., Congo red), and have a characteristic red-green birefringent appearance in polarized light after staining. They also share common ultrastructural features and common X-ray diffraction and infrared spectra. Amyloid-related diseases can either be restricted to one organ or spread to several organs. The first instance is referred to as “localized amyloidosis” while the second is referred to as “systemic amyloidosis.”
Some amyloid diseases can be idiopathic, but most of these diseases appear as a complication of a previously existing disorder. For example, primary amyloidosis (AL amyloid) can appear without any other pathology or can follow plasma cell dyscrasia or multiple myeloma.
Secondary amyloidosis is usually seen associated with chronic infection (such as tuberculosis) or chronic inflammation (such as rheumatoid arthritis). A familial form of secondary amyloidosis is also seen in other types of familial amyloidosis, e.g., Familial Mediterranean Fever (FMF). This familial type of amyloidosis is genetically inherited and is found in specific population groups. In both primary and secondary amyloidosis, deposits are found in several organs and are thus considered systemic amyloid diseases.
“Localized amyloidoses” are those that tend to involve a single organ system. Different amyloids are also characterized by the type of protein present in the deposit. For example, neurodegenerative diseases such as scrapie, bovine spongiform encephalitis, Creutzfeldt-Jakob disease, and the like are characterized by the appearance and accumulation of a protease-resistant form of a prion protein (referred to as AScr or PrP-27) in the central nervous system. Similarly, Alzheimer's disease, another neurodegenerative disorder, is characterized by neuritic plaques and neurofibrillary tangles. In this case, the amyloid plaques found in the parenchyma and the blood vessel is formed by the deposition of fibrillar Aβ amyloid protein. Other diseases such as adult-onset diabetes (type II diabetes) are characterized by the localized accumulation of amyloid fibrils in the pancreas. Once these amyloids have formed, there is no known, widely accepted therapy or treatment which significantly dissolves amyloid deposits in situ, prevents further amyloid deposition or prevents the initiation of amyloid deposition.
Each amyloidogenic protein has the ability to undergo a conformational change and to organize into β-sheets and form insoluble fibrils which may be deposited extracellularly or intracellularly. Each amyloidogenic protein, although different in amino acid sequence, has the same property of forming fibrils and binding to other elements such as proteoglycan, amyloid P and complement component. Moreover, each amyloidogenic protein has amino acid sequences which, although different, show similarities such as regions with the ability to bind to the glycosaminoglycan (GAG) portion of proteoglycan (referred to as the GAG binding site) as well as other regions which promote β-sheet formation. Proteoglycans are macromolecules of various sizes and structures that are distributed almost everywhere in the body. They can be found in the intracellular compartment, on the surface of cells, and as part of the extracellular matrix. The basic structure of all proteoglycans is comprised of a core protein and at least one, but frequently more, polysaccharide chains (GAGs) attached to the core protein. Many different GAGs have been discovered including chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, and hyaluronan.
In specific cases, amyloid fibrils, once deposited, can become toxic to the surrounding cells. For example, the Aβ fibrils organized as senile plaques have been shown to be associated with dead neuronal cells, dystrophic neurites, astrocytosis, and microgliosis in patients with Alzheimer's disease. When tested in vitro, oligomeric (soluble) as well as fibrillar Aβ peptide was shown to be capable of triggering an activation process of microglia (brain macrophages), which would explain the presence of microgliosis and brain inflammation found in the brain of patients with Alzheimer's disease. Both oligomeric and fibrillar Aβ peptide can also induce neuronal cell death in vitro. See, e.g., M. P. Lambert, et al., Proc. Natl. Acad. Sci. USA 95, 6448-53 (1998).
In another type of amyloidosis seen in patients with type II diabetes, the amyloidogenic protein IAPP, when organized in oligomeric forms or in fibrils, has been shown to induce β-islet cell toxicity in vitro. Hence, appearance of IAPP fibrils in the pancreas of type II diabetic patients contributes to the loss of the β islet cells (Langerhans) and organ dysfunction which can lead to insulinemia.
Another type of amyloidosis is related to β2 microglobulin and is found in long-term hemodialysis patients. Patients undergoing long term hemodialysis will develop β2-microglobulin fibrils in the carpal tunnel and in the collagen rich tissues in several joints. This causes severe pains, joint stiffness and swelling.
Amyloidosis is also characteristic of Alzheimer's disease. Alzheimer's disease is a devastating disease of the brain that results in progressive memory loss leading to dementia, physical disability, and death over a relatively long period of time. With the aging populations in developed countries, the number of Alzheimer's patients is reaching epidemic proportions.
People suffering from Alzheimer's disease develop a progressive dementia in adulthood, accompanied by three main structural changes in the brain: diffuse loss of neurons in multiple parts of the brain; accumulation of intracellular protein deposits termed neurofibrillary tangles; and accumulation of extracellular protein deposits termed amyloid or senile plaques, surrounded by misshapen nerve terminals (dystrophic neurites) and activated microglia (microgliosis and astrocytosis). A main constituent of these amyloid plaques is the amyloid-β peptide (Aβ), a 39-43 amino-acid protein that is produced through cleavage of the β-amyloid precursor protein (APP). Extensive research has been conducted on the relevance of Aβ deposits in Alzheimer's disease, see, e.g., Selkoe, Trends in Cell Biology 8, 447-453 (1998). Aβ naturally arises from the metabolic processing of the amyloid precursor protein (“APP”) in the endoplasmic reticulum (“ER”), the Golgi apparatus, or the endosomal-lysosomal pathway, and most is normally secreted as a 40 (“Aβ1-40”) or 42 (“Aβ1-42”) amino acid peptide (Selkoe, Annu. Rev. Cell Biol. 10, 373-403 (1994)). A role for Aβ as a primary cause for Alzheimer's disease is supported by the presence of extracellular Aβ deposits in senile plaques of Alzheimer's disease, the increased production of Aβ in cells harboring mutant Alzheimer's disease associated genes, e.g., amyloid precursor protein, presenilin I and presenilin II; and the toxicity of extracellular soluble (oligomeric) or fibrillar Aβ to cells in culture. See, e.g., Gervais, Eur. Biopharm. Review, 40-42 (Autumn 2001); May, DDT 6, 459-62 (2001). Although symptomatic treatments exist for Alzheimer's disease, this disease cannot be prevented or cured at this time.
Alzheimer's disease is characterized by diffuse and neuritic plaques, cerebral angiopathy, and neurofibrillary tangles. Plaque and blood vessel amyloid is believed to be formed by the deposition of insoluble Aβ amyloid protein, which may be described as diffuse or fibrillary. Both soluble oligomeric Aβ and fibrillar Aβ are also believed to be neurotoxic and inflammatory.
Another type of amyloidosis is cerebral amyloid angiopathy (CAA). CAA is the specific deposition of amyloid-β fibrils in the walls of leptomingeal and cortical arteries, arterioles and veins. It is commonly associated with Alzheimer's disease, Down's syndrome and normal aging, as well as with a variety of familial conditions related to stroke or dementia (see Frangione et al., Amyloid: J. Protein Folding Disord. 8, Suppl. 1, 36-42 (2001)).
Presently available therapies for treatment of β-amyloid diseases are almost entirely symptomatic, providing only temporary or partial clinical benefit. Although some pharmaceutical agents have been described that offer partial symptomatic relief, no comprehensive pharmacological therapy is currently available for the prevention or treatment of, for example, Alzheimer's disease.
The present invention relates to the use of certain compounds in the treatment of amyloid-related diseases. In particular, the invention relates to a method of treating or preventing an amyloid-related disease in a subject comprising administering to the subject a therapeutic amount of a compound of the invention. The invention also pertains to each of the novel compounds of the invention as described herein. Among the compounds for use in the invention are those according to the following Formulae, such that, when administered, amyloid fibril formation, organ specific dysfunction (e.g., neurodegeneration), or cellular toxicity is reduced or inhibited.
In one embodiment, the invention pertains, at least in part to compounds of Formula I:
wherein:
R1 is a substituted or unsubstituted cycloalkyl, heterocyclic, aryl, arylcycloalkyl, bicyclic or tricyclic ring, a bicyclic or tricyclic fused ring group, or a substituted or unsubstituted C2-C10 alkyl group;
R2 is selected from a group consisting of hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, and benzoimidazolyl;
Y is SO3−X+, OSO3−X+, or SSO3−X+;
X+ is hydrogen, a cationic group, or an ester-forming group (i.e., as in a prodrug, which are described elsewhere herein); and
each of L1 and L2 is independently a substituted or unsubstituted C1-C5 alkyl group or absent, or a pharmaceutically acceptable salt thereof, provided that when R1 is alkyl, L1 is absent.
In another embodiment, the invention pertains, at least in part to compounds of Formula II:
wherein:
R1 is a substituted or unsubstituted cyclic, bicyclic, tricyclic, or benzoheterocyclic group or a substituted or unsubstituted C2-C10 alkyl group;
R2 is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, benzoimidazolyl, or linked to R1 to form a heterocycle;
Y is SO3−X+, OSO3−X+, or SSO3−X+;
X+ is hydrogen, a cationic group, or an ester forming moiety;
m is 0 or 1;
n is 1, 2, 3, or 4;
L is substituted or unsubstituted C1-C3 alkyl group or absent, or a pharmaceutically acceptable salt thereof, provided that when R1 is alkyl, L is absent.
In yet another embodiment, the invention pertains, at least in part to compounds of Formula III:
wherein:
A is nitrogen or oxygen;
R11 is hydrogen, salt-forming cation, ester forming group, —(CH2)x-Q, or when A is nitrogen, A and R11 taken together may be the residue of a natural or unnatural amino acid or a salt or ester thereof;
Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
x is 0, 1, 2, 3, or 4;
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
R3, R3a, R4, R4a, R5, R5a, R6, R6a, R7 and R7a are each independently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino, tetrazolyl, or two R groups on adjacent ring atoms taken together with the ring atoms form a double bond, provided that one of R3, R3a, R4, R4a, R5, R5a, R6, R6a, R7 and R7a is a moiety of Formula IIIa:
wherein:
m is 0, 1, 2, 3, or 4;
RA, RB, RC, RD, and RE are independently selected from a group of hydrogen, halogen, hydroxyl, alkyl, alkoxyl, halogenated alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano, thiazolyl, triazolyl, imidazolyl, tetrazolyl, benzothiazolyl, and benzoimidazolyl; and pharmaceutically acceptable salts and esters thereof, provided that said compound is not 3-(4-phenyl-1,2,3,6-tetrahydro-1-pyridyl)-1-propanesulfonic acid. In yet another embodiment, the invention pertains at least in part to compounds of Formula IV:
wherein:
A is nitrogen or oxygen;
R11 is hydrogen, salt-forming cation, ester forming group, —(CH2)x-Q, or when A is nitrogen, A and R11 taken together may be the residue of a natural or unnatural amino acid or a salt or ester thereof;
Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
x is 0, 1, 2, 3, or 4;
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
R4, R4a, R5, R5a, R6, R6a, R7, and R7a are each independently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino, tetrazolyl, R4 and R5 taken together, with the ring atoms they are attached to, form a double bond, or R6 and R7 taken together, with the ring atoms they are attached to, form a double bond;
m is 0, 1, 2, 3, or 4;
R8, R9, R10, R11, and R12 are independently selected from a group of hydrogen, halogen, hydroxyl, alkyl, alkoxyl, halogenated alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano, thiazolyl, triazolyl, imidazolyl, tetrazolyl, benzothiazolyl, and benzoimidazolyl, and pharmaceutically acceptable salts and esters thereof.
In another embodiment, the invention includes compounds of Formula V:
wherein:
A is nitrogen or oxygen;
R11 is hydrogen, salt-forming cation, ester forming group, —(CH2)x-Q, or when A is nitrogen, A and R11 taken together may be the residue of a natural or unnatural amino acid or a salt or ester thereof;
Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
x is 0, 1, 2, 3, or 4;
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
aa is a natural or unnatural amino acid residue;
m is 0, 1, 2, or 3;
R14 is hydrogen or protecting group;
R15 is hydrogen, alkyl or aryl, and pharmaceutically acceptable salts and prodrugs thereof.
In another embodiment, the invention includes compounds of the Formula VI:
wherein:
n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
A is oxygen or nitrogen;
R11 is hydrogen, salt-forming cation, ester forming group, —(CH2)x-Q, or when A is nitrogen, A and R11 taken together may be the residue of a natural or unnatural amino acid or a salt or ester thereof;
Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
x is 0, 1, 2, 3, or 4;
R19 is hydrogen, alkyl or aryl;
Y1 is oxygen, sulfur, or nitrogen;
Y2 is carbon, nitrogen, or oxygen;
R20 is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
R21 is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, benzoimidazolyl, or absent if Y2 is oxygen;
R22 is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, benzoimidazolyl; or R22 is hydrogen, hydroxyl, alkoxy or aryloxy if Y1 is nitrogen; or R22 is absent if Y1 is oxygen or sulfur; or R22 and R21 may be linked to form a cyclic moiety if Y1 is nitrogen;
R23 is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl, or absent if Y2 is nitrogen or oxygen;
or pharmaceutically acceptable salts thereof.
In another embodiment, the invention includes compounds of Formula VII:
wherein:
n is 2, 3, or 4;
A is oxygen or nitrogen;
R11 is hydrogen, salt-forming cation, ester forming group, —(CH2)x-Q, or when A is nitrogen, A and R11 taken together may be the residue of a natural or unnatural amino acid or a salt or ester thereof;
Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
x is 0, 1, 2, 3, or 4;
G is a direct bond or oxygen, nitrogen, or sulfur;
z is 0, 1, 2, 3, 4, or 5;
m is 0 or 1;
R24 is selected from a group consisting of hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, aroyl, alkylcarbonyl, aminoalkylcarbonyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, and benzoimidazolyl;
each R25 is independently selected from hydrogen, halogen, cyano, hydroxyl, alkoxy, thiol, amino, nitro, alkyl, aryl, carbocyclic, or heterocyclic, and pharmaceutically acceptable salts thereof.
In one embodiment, the compounds disclosed herein prevent or inhibit amyloid protein assembly into insoluble fibrils which, in vivo, are deposited in various organs, or it favors clearance of pre-formed deposits or slows deposition in patients already having deposits. In another embodiment, the compound may also prevent the amyloid protein, in its soluble, oligomeric form or in its fibrillar form, from binding or adhering to a cell surface and causing cell damage or toxicity. In yet another embodiment, the compound may block amyloid-induced cellular toxicity or macrophage activation. In another embodiment, the compound may block amyloid-induced neurotoxicity or microglial activation. In another embodiment, the compound protects cells from amyloid induced cytotoxicity of B-islet cells.
In another embodiment, the compound may enhance clearance from a specific organ, e.g., the brain or it decreases concentration of the amyloid protein in such a way that amyloid fibril formation is prevented in the targeted organ.
The compounds of the invention may be administered therapeutically or prophylactically to treat diseases associated with amyloid fibril formation, aggregation or deposition. The compounds of the invention may act to ameliorate the course of an amyloid related disease using any of the following mechanisms (this list is meant to be illustrative and not limiting): slowing the rate of amyloid fibril formation or deposition; lessening the degree of amyloid deposition; inhibiting, reducing, or preventing amyloid fibril formation; inhibiting neurodegeneration or cellular toxicity induced by amyloid; inhibiting amyloid induced inflammation; enhancing the clearance of amyloid; or favoring the degradation of amyloid protein prior to its organization in fibrils.
The compounds of the invention may be administered therapeutically or prophylactically to treat diseases associated with amyloid-β fibril formation, aggregation or deposition. The compounds of the invention may act to ameliorate the course of an amyloid-β related disease using any of the following mechanisms (this list is meant to be illustrative and not limiting): slowing the rate of amyloid-β fibril formation or deposition; lessening the degree of amyloid-β deposition; inhibiting, reducing, or preventing amyloid-β fibril formation; inhibiting neurodegeneration or cellular toxicity induced by amyloid-β; inhibiting amyloid-β induced inflammation; enhancing the clearance of amyloid-β from the brain; or favoring the degradation of amyloid-β protein prior to its organization in fibrils.
Therapeutic compounds of the invention may be effective in controlling amyloid-β deposition either following their entry into the brain (following penetration of the blood brain bather) or from the periphery. When acting from the periphery, a compound may alter the equilibrium of Aβ between the brain and the plasma so as to favor the exit of Aβ from the brain. It may also increase the catabolism of neuronal Aβ and change the rate of exit from the brain. An increase in the exit of Aβ from the brain would result in a decrease in Aβ brain and cerebral spinal fluid (CSF) concentration and therefore favor a decrease in Aβ deposition. Alternatively, compounds that penetrate the brain could control deposition by acting directly on brain Aβ e.g., by maintaining it in a non-fibrillar form, favoring its clearance from the brain, or by slowing down APP processing. These compounds could also prevent Aβ in the brain from interacting with the cell surface and therefore prevent neurotoxicity, neurodegeneration or inflammation. They may also decrease Aβ production by activated microglia. The compounds may also increase degradation by macrophages or neuronal cells.
In one embodiment, the method is used to treat Alzheimer's disease (e.g., sporadic, familial, or early AD). The method can also be used prophylactically or therapeutically to treat other clinical occurrences of amyloid-β deposition, such as in Down's syndrome individuals and in patients with cerebral amyloid angiopathy (“CAA”) or hereditary cerebral hemorrhage.
In another embodiment, the method is used to treat mild cognitive impairment. Mild Cognitive Impairment (“MCI”) is a condition characterized by a state of mild but measurable impairment in thinking skills, which is not necessarily associated with the presence of dementia. MCI frequently, but not necessarily, precedes Alzheimer's disease.
Additionally, abnormal accumulation of APP and of amyloid-β protein in muscle fibers has been implicated in the pathology of sporadic inclusion body myositis (IBM) (Askanas, et al., Proc. Natl. Acad. Sci. USA 93, 1314-1319 (1996); Askanas, et al., Current Opinion in Rheumatology 7, 486-496 (1995)). Accordingly, the compounds of the invention can be used prophylactically or therapeutically in the treatment of disorders in which amyloid-beta protein is abnormally deposited at non-neurological locations, such as treatment of IBM by delivery of the compounds to muscle fibers.
Additionally, it has been shown that Aβ is associated with abnormal extracellular deposits, known as drusen, that accumulate along the basal surface of the retinal pigmented epithelium in individuals with age-related macular degeneration (AMD). AMD is a cause of irreversible vision loss in older individuals. It is believed that Aβ deposition could be an important component of the local inflammatory events that contribute to atrophy of the retinal pigmented epithelium, drusen biogenesis, and the pathogenesis of AMD (Johnson, et al., Proc. Natl. Acad. Sci. USA 99(18), 11830-5 (2002)).
The present invention therefore relates to the use of compounds of Formulae I, II, III, IV, V, VI, VII, or otherwise described herein in the prevention or treatment of amyloid-related diseases, including, inter alia, Alzheimer's disease, cerebral amyloid angiopathy, mild cognitive impairment, inclusion body myositis, Down's syndrome, macular degeneration, as well as other types of amyloidosis like IAPP-related amyloidosis (e.g., diabetes), primary (AL) amyloidosis, secondary (AA) amyloidosis and β2 microglobulin-related (dialysis-related) amyloidosis.
In Type II diabetes related amyloidosis (IAPP), the amyloidogenic protein IAPP induces β-islet cell toxicity when organized in oligomeric forms or in fibrils. Hence, appearance of IAPP fibrils in the pancreas of type II diabetic patients contributes to the loss of the β islet cells (Langerhans) and organ dysfunction which leads to insulinemia. Primary amyloidosis (AL amyloid) is usually found associated with plasma cell dyscrasia and multiple myeloma. It can also be found as an idiopathic disease.
Secondary (AA) amyloidosis is usually seen associated with chronic infection (such as tuberculosis) or chronic inflammation (such as rheumatoid arthritis). A familial form of secondary amyloidosis is also seen in Familial Mediterranean Fever (FMF). β2 microglobulin-related (dialysis-related) amyloidosis is found in long-term hemodialysis patients. Patients undergoing long term hemodialysis will develop β2-microglobulin fibrils in the carpal tunnel and in the collagen rich tissues in several joints. This causes severe pains, joint stiffness and swelling. These deposits are due to the inability to maintain low levels of β2M in plasma of dialyzed patients. Increased plasma concentrations of β2M protein will induce structural changes and may lead to the deposition of modified β2M as insoluble fibrils in the joints.
The present invention relates to the use of compounds of Formulae I, II, III, IV, V, VI, VII, or compounds otherwise described herein in the treatment of amyloid-related diseases. For convenience, some definitions of terms referred to herein are set forth below.
Generally, AA amyloidosis is a manifestation of a number of diseases that provoke a sustained acute phase response. Such diseases include chronic inflammatory disorders, chronic local or systemic microbial infections, and malignant neoplasms. The most common form of reactive or secondary (AA) amyloidosis is seen as the result of long-standing inflammatory conditions. For example, patients with Rheumatoid Arthritis or Familial Mediterranean Fever (which is a genetic disease) can develop AA amyloidosis. The terms “AA amyloidosis” and “secondary (AA) amyloidosis” are used interchangeably. AA fibrils are generally composed of 8,000 Dalton fragments (AA peptide or protein) formed by proteolytic cleavage of serum amyloid A protein (ApoSAA), a circulating apolipoprotein which is mainly synthesized in hepatocytes in response to such cytokines as IL-1, IL-6 and TNF. Once secreted, ApoSAA is complexed with HDL. Deposition of AA fibrils can be widespread in the body, with a preference for parenchymal organs. The kidneys are usually a deposition site, and the liver and the spleen may also be affected. Deposition is also seen in the heart, gastrointestinal tract, and the skin.
Underlying diseases which can lead to the development of AA amyloidosis include, but are not limited to inflammatory diseases, such as rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome, Adult Still's disease, Behcet's syndrome, and Crohn's disease. AA deposits are also produced as a result of chronic microbial infections, such as leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronic pyelonephritis, osteomyelitis, and Whipple's disease. Certain malignant neoplasms can also result in AA fibril amyloid deposits. These include such conditions as Hodgkin's lymphoma, renal carcinoma, carcinomas of gut, lung and urogenital tract, basal cell carcinoma, and hairy cell leukemia. Other underlying conditions that may be associated with AA amyloidosis are Castleman's disease and Schnitzler's syndrome.
AL amyloid deposition is generally associated with almost any dyscrasia of the B lymphocyte lineage, ranging from malignancy of plasma cells (multiple myeloma) to benign monoclonal gammopathy. At times, the presence of amyloid deposits may be a primary indicator of the underlying dyscrasia. AL amyloidosis is also described in detail in Current Drug Targets, 2004, 5 159-171.
Fibrils of AL amyloid deposits are composed of monoclonal immunoglobulin light chains or fragments thereof. More specifically, the fragments are derived from the N-terminal region of the light chain (kappa or lambda) and contain all or part of the variable (VL) domain thereof. Deposits generally occur in the mesenchymal tissues, causing peripheral and autonomic neuropathy, carpal tunnel syndrome, macroglossia, restrictive cardiomyopathy, arthropathy of large joints, immune dyscrasias, myelomas, as well as occult dyscrasias. However, it should be noted that almost any tissue, particularly visceral organs such as the kidney, liver, spleen and heart, may be involved.
There are many forms of hereditary systemic amyloidoses. Although they are relatively rare conditions, adult onset of symptoms and their inheritance patterns (usually autosomal dominant) lead to persistence of such disorders in the general population. Generally, the syndromes are attributable to point mutations in the precursor protein leading to production of variant amyloidogenic peptides or proteins. Table 1 summarizes the fibril composition of exemplary forms of these disorders.
The data provided in Table 1 are exemplary and are not intended to limit the scope of the invention. For example, more than 40 separate point mutations in the transthyretin gene have been described, all of which give rise to clinically similar forms of familial amyloid polyneuropathy.
In general, any hereditary amyloid disorder can also occur sporadically, and both hereditary and sporadic forms of a disease present with the same characteristics with regard to amyloid. For example, the most prevalent form of secondary AA amyloidosis occurs sporadically, e.g. as a result of ongoing inflammation, and is not associated with Familial Mediterranean Fever. Thus general discussion relating to hereditary amyloid disorders below can also be applied to sporadic amyloidoses.
Transthyretin (TTR) is a 14 kiloDalton protein that is also sometimes referred to as prealbumin. It is produced by the liver and choroid plexus, and it functions in transporting thyroid hormones and vitamin A. At least 50 variant forms of the protein, each characterized by a single amino acid change, are responsible for various forms of familial amyloid polyneuropathy. For example, substitution of proline for leucine at position 55 results in a particularly progressive form of neuropathy; substitution of methionine for leucine at position 111 resulted in a severe cardiopathy in Danish patients.
Amyloid deposits isolated from heart tissue of patients with systemic amyloidosis have revealed that the deposits are composed of a heterogeneous mixture of TTR and fragments thereof, collectively referred to as ATTR, the full length sequences of which have been characterized. ATTR fibril components can be extracted from such plaques and their structure and sequence determined according to the methods known in the art (e.g., Gustaysson, A., et al., Laboratory Invest. 73: 703-708, 1995; Kametani, F., et al., Biochem. Biophys. Res. Commun. 125: 622-628, 1984; Pras, M., et al., PNAS 80: 539-42, 1983). Persons having point mutations in the molecule apolipoprotein A1 (e.g., Gly→Arg26; Trp→Arg50; Leu→Arg60) exhibit a form of amyloidosis (“Östertag type”) characterized by deposits of the protein apolipoprotein AI or fragments thereof (AApoAI). These patients have low levels of high density lipoprotein (HDL) and present with a peripheral neuropathy or renal failure.
A mutation in the alpha chain of the enzyme lysozyme (e.g., Ile→Thr56 or Asp→His57) is the basis of another form of Östertag-type non-neuropathic hereditary amyloid reported in English families. Here, fibrils of the mutant lysozyme protein (Alys) are deposited, and patients generally exhibit impaired renal function. This protein, unlike most of the fibril-forming proteins described herein, is usually present in whole (unfragmented) form (Benson, M. D., et al. CIBA Fdn. Symp. 199: 104-131, 1996).
Immunoglobulin light chains tend to form aggregates in various morphologies, including fibrillar (e.g., AL amyloidosis and AH amyloidosis), granular (e.g., light chain deposition disease (LCDD), heavy chain deposition disease (HCDD), and light-heavy chain deposition disease (LHCDD)), crystalline (e.g., Acquired Farconi's Syndrome), and microtubular (e.g., Cryoglobulinemia). AL and AH amyloidosis is indicated by the formation of insoluble fibrils of immunoglobulin light chains and heavy chain, respectively, and/or their fragments. In AL fibrils, lambda (λ) chains such as λ VI chains (λ6 chains), are found in greater concentrations than kappa (κ) chains. λIII chains are also slightly elevated. Merlini et al., Clin Chem Lab Med 39(11):1065-75 (2001). Heavy chain amyloidosis (AH) is generally characterized by aggregates of gamma chain amyloid proteins of the IgG1 subclass. Eulitz et al., Proc Natl Acad Sci USA 87:6542-46 (1990).
Comparison of amyloidogenic to non-amyloidogenic light chains has revealed that the former can include replacements or substitutions that appear to destabilize the folding of the protein and promote aggregation. AL and LCDD have been distinguished from other amyloid diseases due to their relatively small population monoclonal light chains, which are manufactured by neoplastic expansion of an antibody-producing B cell. AL aggregates typically are well-ordered fibrils of lambda chains. LCDD aggregates are relatively amorphous aggregations of both kappa and lambda chains, with a majority being kappa, in some cases κIV. Bellotti et al., Journal of Structural Biology 13:280-89 (2000). Comparison of amyloidogenic and non-amyloidogenic heavy chains in patients having AH amyloidosis has revealed missing and/or altered components. Eulitz et al., Proc Natl Acad Sci USA 87:6542-46 (1990) (pathogenic heavy chain characterized by significantly lower molecular mass than non-amyloidogenic heavy chains); and Solomon et al. Am J Hemat 45(2) 171-6 (1994) (amyloidogenic heavy chain characterized as consisting solely of the VH-D portion of the non-amyloidogenic heavy chain).
Accordingly, potential methods of detecting and monitoring treatment of subjects having or at risk of having AL, LCDD, AH, and the like, include but are not limited to immunoassaying plasma or urine for the presence or depressed deposition of amyloidogenic light or heavy chains, e.g., amyloid λ, amyloid κ, amyloid κIV, amyloid γ, or amyloid γ1.
The most frequent type of amyloid in the brain is composed primarily of Aβ peptide fibrils, resulting in dementia associated with sporadic (non-hereditary) Alzheimer's disease. In fact, the incidence of sporadic Alzheimer's disease greatly exceeds forms shown to be hereditary. Nevertheless, fibril peptides forming plaques are very similar in both types. Brain amyloidosis includes those diseases, conditions, pathologies, and other abnormalities of the structure or function of the brain, including components thereof, in which the causative agent is amyloid. The area of the brain affected in an amyloid-related disease may be the stroma including the vasculature or the parenchyma including functional or anatomical regions, or neurons themselves. A subject need not have received a definitive diagnosis of a specifically recognized amyloid-related disease. The term “amyloid related disease” includes brain amyloidosis.
Amyloid-β peptide (“Aβ”) is a 39-43 amino acid peptide derived by proteolysis from a large protein known as Beta Amyloid Precursor Protein (“PAPP”). Mutations in βAPP result in familial forms of Alzheimer's disease, Down's syndrome, cerebral amyloid angiopathy, and senile dementia, characterized by cerebral deposition of plaques composed of Aβ fibrils and other components, which are described in further detail below. Known mutations in APP associated with Alzheimer's disease occur proximate to the cleavage sites of β or γ-secretase, or within Aβ. For example, position 717 is proximate to the site of gamma-secretase cleavage of APP in its processing to Aβ, and positions 670/671 are proximate to the site of β-secretase cleavage. Mutations at any of these residues may result in Alzheimer's disease, presumably by causing an increase in the amount of the 42/43 amino acid form of Aβ generated from APP. The familial form of Alzheimer's disease represents only 10% of the subject population. Most occurrences of Alzheimer's disease are sporadic cases where APP and Aβ do not possess any mutation. The structure and sequence of Aβ peptides of various lengths are well known in the art. Such peptides can be made according to methods known in the art, or extracted from the brain according to known methods (e.g., Glenner and Wong, Biochem. Biophys. Res. Comm. 129, 885-90 (1984); Glenner and Wong, Biochem. Biophys. Res. Comm. 122, 1131-35 (1984)). In addition, various forms of the peptides are commercially available. APP is expressed and constitutively catabolized in most cells. The dominant catabolic pathway appears to be cleavage of APP within the Aβ sequence by an enzyme provisionally termed α-secretase, leading to release of a soluble ectodomain fragment known as APPsα. This cleavage precludes the formation of Aβ peptide. In contrast to this non-amyloidogenic pathway, APP can also be cleaved by enzymes known as β- and γ-secretase at the N- and C-termini of the Aβ, respectively, followed by release of Aβ into the extracellular space. To date, BACE has been identified as β-secretase (Vasser, et al., Science 286:735-741, 1999) and presenilins have been implicated in γ-secretase activity (De Strooper, et al., Nature 391, 387-90 (1998)). The 39-43 amino acid Aβ peptide is produced by sequential proteolytic cleavage of the amyloid precursor protein (APP) by the β and γ secretases enzyme. Although Aβ 40 is the predominant form produced, 5-7% of total Aβ exists as Aβ42 (Cappai et al., Int. J. Biochem. Cell Biol. 31. 885-89 (1999)).
The length of the Aβ peptide appears to dramatically alter its biochemical/biophysical properties. Specifically, the additional two amino acids at the C-terminus of Aβ 42 are very hydrophobic, presumably increasing the propensity of Aβ 42 to aggregate. For example, Jarrett, et al. demonstrated that Aβ42 aggregates very rapidly in vitro compared to Aβ40, suggesting that the longer forms of Aβ may be the important pathological proteins that are involved in the initial seeding of the neuritic plaques in Alzheimer's disease (Jarrett, et al., Biochemistry 32, 4693-97 (1993); Jarrett, et al., Ann. N.Y. Acad. Sci. 695, 144-48 (1993)).
This hypothesis has been further substantiated by the recent analysis of the contributions of specific forms of Aβ in cases of genetic familial forms of Alzheimer's disease (“FAD”). For example, the “London” mutant form of APP (APPV717I) linked to FAD selectively increases the production of Aβ 42/43 forms versus Aβ 40 (Suzuki, et al., Science 264, 1336-40 (1994)) while the “Swedish” mutant form of APP (APPK670N/M671L) increases levels of both Aβ40 and Aβ42/43 (Citron, et al., Nature 360, 672-674 (1992); Cai, et al., Science 259, 514-16, (1993)). Also, it has been observed that FAD-linked mutations in the Presenilin-1 (“PS1”) or Presenilin-2 (“PS2”) genes will lead to a selective increase in Aβ42/43 production but not Aβ40 (Borchelt, et al., Neuron 17, 1005-13 (1996)). This finding was corroborated in transgenic mouse models expressing PS mutants that demonstrate a selective increase in brain Aβ42 (Borchelt, op cit.; Duff, et al., Neurodegeneration 5(4), 293-98 (1996)). Thus the leading hypothesis regarding the etiology of Alzheimer's disease is that an increase in Aβ42 brain concentration due to an increased production and release of Aβ42 or a decrease in clearance (degradation or brain clearance) is a causative event in the disease pathology.
Multiple mutation sites in either Aβ or the APP gene have been identified and are clinically associated with either dementia or cerebral hemorrhage. Exemplary CAA disorders include, but are not limited to, hereditary cerebral hemorrhage with amyloidosis of Icelandic type (HCHWA-I); the Dutch variant of HCHWA (HCHWA-D; a mutation in Aβ); the Flemish mutation of Aβ; the Arctic mutation of Aβ; the Italian mutation of Aβ; the Iowa mutation of Aβ; familial British dementia; and familial Danish dementia. CAA may also be sporadic.
As used herein, the terms “β amyloid,” “amyloid-β,” and the like refer to amyloid β proteins or peptides, amyloid β precursor proteins or peptides, intermediates, and modifications and fragments thereof, unless otherwise specifically indicated. In particular, “Aβ” refers to any peptide produced by proteolytic processing of the APP gene product, especially peptides which are associated with amyloid pathologies, including Aβ1-39, Aβ1-40, Aβ1-41, Aβ1-42, and Aβ1-43. For convenience of nomenclature, “Aβ1-42” may be referred to herein as “Aβ(1-42)” or simply as “Aβ42” or “Aβ42” (and likewise for any other amyloid peptides discussed herein). As used herein, the terms “β amyloid,” “amyloid-β,” and “Aβ” are synonymous.
Unless otherwise specified, the term “amyloid” refers to amyloidogenic proteins, peptides, or fragments thereof which can be soluble (e.g., monomeric or oligomeric) or insoluble (e.g., having fibrillary structure or in amyloid plaque). See, e.g., M. P. Lambert, et al., Proc. Nat'l Acad. Sci. USA 95, 6448-53 (1998). “Amyloidosis” or “amyloid disease” or “amyloid-related disease” refers to a pathological condition characterized by the presence of amyloid fibers. “Amyloid” is a generic term referring to a group of diverse but specific protein deposits (intracellular or extracellular) which are seen in a number of different diseases. Though diverse in their occurrence, all amyloid deposits have common morphologic properties, stain with specific dyes (e.g., Congo red), and have a characteristic red-green birefringent appearance in polarized light after staining. They also share common ultrastructural features and common X-ray diffraction and infrared spectra.
Gelsolin is a calcium binding protein that binds to fragments and actin filaments. Mutations at position 187 (e.g., Asp→Asn; Asp→Tyr) of the protein result in a form of hereditary systemic amyloidosis, usually found in patients from Finland, as well as persons of Dutch or Japanese origin. In afflicted individuals, fibrils formed from gelsolin fragments (Agel), usually consist of amino acids 173-243 (68 kDa carboxyterminal fragment) and are deposited in blood vessels and basement membranes, resulting in corneal dystrophy and cranial neuropathy which progresses to peripheral neuropathy, dystrophic skin changes and deposition in other organs. (Kangas, H., et al. Human Mol. Genet. 5(9): 1237-1243, 1996). Other mutated proteins, such as mutant alpha chain of fibrinogen (AfibA) and mutant cystatin C (Acys) also form fibrils and produce characteristic hereditary disorders. AfibA fibrils form deposits characteristic of a nonneuropathic hereditary amyloid with renal disease; Acys deposits are characteristic of a hereditary cerebral amyloid angiopathy reported in Iceland (Isselbacher, Harrison's Principles of Internal Medicine, McGraw-Hill, San Francisco, 1995; Benson, et al.). In at least some cases, patients with cerebral amyloid angiopathy (CAA) have been shown to have amyloid fibrils containing a non-mutant form of cystatin C in conjunction with amyloid beta protein (Nagai, A., et al. Molec. Chem. Neuropathol. 33: 63-78, 1998).
Certain forms of prion disease are now considered to be heritable, accounting for up to 15% of cases, which were previously thought to be predominantly infectious in nature. (Baldwin, et al., in Research Advances in Alzheimer's Disease and Related Disorders, John Wiley and Sons, New York, 1995). In hereditary and sporadic prion disorders, patients develop plaques composed of abnormal isoforms of the normal prion protein (PrPSc). A predominant mutant isoform, PrPSc, also referred to as AScr, differs from the normal cellular protein in its resistance to protease degradation, insolubility after detergent extraction, deposition in secondary lysosomes, post-translational synthesis, and high β-pleated sheet content. Genetic linkage has been established for at least five mutations resulting in Creutzfeldt-Jacob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). (Baldwin, supra) Methods for extracting fibril peptides from scrapie fibrils, determining sequences and making such peptides are known in the art (e.g., Beekes, M., et al. J. Gen. Virol. 76: 2567-76, 1995).
For example, one form of GSS has been linked to a PrP mutation at codon 102, while telencephalic GSS segregates with a mutation at codon 117. Mutations at codons 198 and 217 result in a form of GSS in which neuritic plaques characteristic of Alzheimer's disease contain PrP instead of Aβ peptide. Certain forms of familial CJD have been associated with mutations at codons 200 and 210; mutations at codons 129 and 178 have been found in both familial CJD and FFI. (Baldwin, supra).
Local deposition of amyloid is common in the brain, particularly in elderly individuals. The most frequent type of amyloid in the brain is composed primarily of Aβ peptide fibrils, resulting in dementia or sporadic (non-hereditary) Alzheimer's disease. The most common occurrences of cerebral amyloidosis are sporadic and not familial For example, the incidence of sporadic Alzheimer's disease and sporadic CAA greatly exceeds the incidence of familial AD and CAA. Moreover, sporadic and familial forms of the disease cannot be distinguished from each other (they differ only in the presence or absence of an inherited genetic mutation); for example, the clinical symptoms and the amyloid plaques formed in both sporadic and familial AD are very similar, if not identical.
Cerebral amyloid angiopathy (CAA) refers to the specific deposition of amyloid fibrils in the walls of leptomingeal and cortical arteries, arterioles and veins. It is commonly associated with Alzheimer's disease, Down's syndrome and normal aging, as well as with a variety of familial conditions related to stroke or dementia (see Frangione et al., Amyloid: J. Protein Folding Disord. 8, Suppl. 1, 36-42 (2001)). CAA can occur sporadically or be hereditary.
Amyloid deposition, either systemic or focal, increases with age. For example, fibrils of wild type transthyretin (TTR) are commonly found in the heart tissue of elderly individuals. These may be asymptomatic, clinically silent, or may result in heart failure. Asymptomatic fibrillar focal deposits may also occur in the brain (Aβ), corpora amylacea of the prostate (β2 microglobulin), joints and seminal vesicles.
Plaques composed of β2 microglobulin (β2M) fibrils commonly develop in patients receiving long term hemodialysis or peritoneal dialysis. β2 microglobulin is a 11.8 kiloDalton polypeptide and is the light chain of Class I MHC antigens, which are present on all nucleated cells. Under normal circumstances, β2M is usually distributed in the extracellular space unless there is an impaired renal function, in which case β2M is transported into tissues where it polymerizes to form amyloid fibrils. Failure of clearance such as in the case of impaired renal function, leads to deposition in the carpal tunnel and other sites (primarily in collagen-rich tissues of the joints). Unlike other fibril proteins, β2M molecules are not produced by cleavage of a longer precursor protein and are generally present in unfragmented form in the fibrils. (Benson, supra). Retention and accumulation of this amyloid precursor has been shown to be the main pathogenic process underlying DRA. DRA is characterized by peripheral joint osteoarthropathy (e.g., joint stiffness, pain, swelling, etc.). Isoforms of β2M, glycated β2M, or polymers of β2M in tissue are the most amyloidogenic form (as opposed to native β2M). Unlike other types of amyloidosis, β2M is confined largely to osteoarticular sites. Visceral depositions are rare. Occasionally, these deposits may involve blood vessels and other important anatomic sites.
Despite improved dialysis methods for removal of β2M, the majority of patients have plasmatic β2M concentrations that remain dramatically higher than normal. These elevated β2M concentrations generally lead to Diabetes-Related Amyloidosis (DRA) and comorbidities that contribute to mortality.
Islet hyalinosis (amyloid deposition) was first described over a century ago as the presence of fibrous protein aggregates in the pancreas of patients with severe hyperglycemia (Opie, E L., J. Exp. Med. 5: 397-428, 1901). Today, islet amyloid, composed predominantly of islet amyloid polypeptide (IAPP), or amylin, is a characteristic histopathological marker in over 90% of all cases of Type II diabetes (also known as Non-Insulin Dependent Diabetes, or NIDDM). These fibrillar accumulations result from the aggregation of the islet amyloid polypeptide (IAPP) or amylin, which is a 37 amino acid peptide, derived from a larger precursor peptide, called pro-IAPP.
IAPP is co-secreted with insulin in response to β-cell secretagogues. This pathological feature is not associated with insulin-dependent (Type I) diabetes and is a unifying characteristic for the heterogeneous clinical phenotypes diagnosed as NIDDM (Type II diabetes).
Longitudinal studies in cats and immunocytochemical investigations in monkeys have shown that a progressive increase in islet amyloid is associated with a dramatic decrease in the population of insulin-secreting β-cells and increased severity of the disease. More recently, transgenic studies have strengthened the relationship between IAPP plaque formation and β-cell apoptosis and dysfunction, indicating that amyloid deposition is a principal factor in increasing severity of Type II diabetes.
IAPP has also been shown to induce β-islet cell toxicity in vitro, indicating that appearance of IAPP fibrils in the pancreas of Type II or Type I diabetic patients (post-islet transplantation) could contribute to the loss of the β-cell islets (Langerhans) and organ dysfunction. In patients with Type II diabetes, the accumulation of pancreatic IAPP leads to formation of oligomeric IAPP, leading to a buildup of IAPP-amyloid as insoluble fibrous deposits which eventually destroys the insulin-producing β cells of the islet, resulting in β cell depletion and failure (Westermark, P., Grimelius, L., Acta Path. Microbiol. Scand., sect. A. 81: 291-300, 1973; de Koning, E J P., et al., Diabetologia 36: 378-384, 1993; and Lorenzo, A., et al., Nature 368: 756-760, 1994). Accumulation of IAPP as fibrous deposits can also have an impact on the ratio of pro-IAPP to IAPP normally found in plasma by increasing this ratio due to the trapping of IAPP in deposits. Reduction of β cell mass can be manifested by hyperglycemia and insulinemia. This β-cell mass loss can lead to a need for insulin therapy.
Diseases caused by the death or malfunctioning of a particular type or types of cells can be treated by transplanting into the patient healthy cells of the relevant type of cell. This approach has been used for Type I diabetes patients. Often pancreatic islet cells from a donor are cultured in vitro prior to transplantation, to allow them to recover after the isolation procedure or to reduce their immunogenicity. However, in many instances islet cell transplantation is unsuccessful, due to death of the transplanted cells. One reason for this poor success rate is IAPP, which organizes into toxic oligomers. Toxic effects may result from intracellular and extracellular accumulation of fibril oligomers. The IAPP oligomers can form fibrils and become toxic to the cells in vitro. In addition, IAPP fibrils are likely to continue to grow after the cells are transplanted and cause death or dysfunction of the cells. This may occur even when the cells are from a healthy donor and the patient receiving the transplant does not have a disease that is characterized by the presence of fibrils. For example, compounds of the present invention may also be used in preparing tissues or cells for transplantation according to the methods described in International Patent Application (PCT) No. WO 01/003680.
The compounds of the invention may also stabilize the ratio of the concentrations of Pro-IAPP/IAPP, pro-Insulin/Insulin and C-peptide levels. In addition, as biological markers of efficacy, the results of the different tests, such as the arginine-insulin secretion test, the glucose tolerance test, insulin tolerance and sensitivity tests, could all be used as markers of reduced β-cell mass and/or accumulation of amyloid deposits. Such class of drugs could be used together with other drugs targeting insulin resistance, hepatic glucose production, and insulin secretion. Such compounds might prevent insulin therapy by preserving β-cell function and be applicable to preserving islet transplants.
Endocrine organs may harbor amyloid deposits, particularly in aged individuals. Hormone-secreting tumors may also contain hormone-derived amyloid plaques, the fibrils of which are made up of polypeptide hormones such as calcitonin (medullary carcinoma of the thyroid), and atrial natriuretic peptide (isolated atrial amyloidosis). Sequences and structures of these proteins are well known in the art.
There are a variety of other forms of amyloid disease that are normally manifest as localized deposits of amyloid. In general, these diseases are probably the result of the localized production or lack of catabolism of specific fibril precursors or a predisposition of a particular tissue (such as the joint) for fibril deposition. Examples of such idiopathic deposition include nodular AL amyloid, cutaneous amyloid, endocrine amyloid, and tumor-related amyloid. Other amyloid related diseases include those described in Table 1, such as familial amyloid polyneuropathy (FAP), senile systemic amyloidosis, Tenosynovium, familial amyloidosis, Ostertag-type, non-neuropathic amyloidosis, cranial neuropathy, hereditary cerebral hemorrhage, familial dementia, chronic dialysis, familial Creutzfeldt-Jakob disease; Gerstmann-Sträussler-Scheinker syndrome, hereditary spongiform encephalopathies, prion diseases, familial Mediterranean fever, Muckle-Well's syndrome, nephropathy, deafness, urticaria, limb pain, cardiomyopathy, cutaneous deposits, multiple myeloma, benign monoclonal gammopathy, maccoglobulinaemia, myeloma associated amyloidosis, medullary carcinomas of the thyroid, isolated atrial amyloid, and diabetes.
The compounds of the invention may be administered therapeutically or prophylactically to treat diseases associated with amyloid fibril formation, aggregation or deposition, regardless of the clinical setting. The compounds of the invention may act to ameliorate the course of an amyloid related disease using any of the following mechanisms, such as, for example but not limited to: slowing the rate of amyloid fibril formation or deposition; lessening the degree of amyloid deposition; inhibiting, reducing, or preventing amyloid fibril formation; inhibiting amyloid induced inflammation; enhancing the clearance of amyloid from, for example, the brain; or protecting cells from amyloid induced (oligomers or fibrillar) toxicity.
In an embodiment, the compounds of the invention may be administered therapeutically or prophylactically to treat diseases associated with amyloid-β fibril formation, aggregation or deposition. The compounds of the invention may act to ameliorate the course of an amyloid-β related disease using any of the following mechanisms (this list is meant to be illustrative and not limiting): slowing the rate of amyloid-β fibril formation or deposition; lessening the degree of amyloid-β deposition; inhibiting, reducing, or preventing amyloid-β fibril formation; inhibiting neurodegeneration or cellular toxicity induced by amyloid-β; inhibiting amyloid-β induced inflammation; enhancing the clearance of amyloid-β from the brain; or favoring greater catabolism of Aβ.
Compounds of the invention may be effective in controlling amyloid-β deposition either following their entry into the brain (following penetration of the blood brain barrier) or from the periphery. When acting from the periphery, a compound may alter the equilibrium of Aβ between the brain and the plasma so as to favor the exit of Aβ from the brain. An increase in the exit of Aβ from the brain would result in a decrease in Aβ brain concentration and therefore favor a decrease in Aβ deposition. In addition, compounds that penetrate the brain may control deposition by acting directly on brain Aβ, e.g., by maintaining it in a non-fibrillar form or favoring its clearance from the brain. The compounds may slow down APP processing; may increase degradation of Aβ fibrils by macrophages or by neuronal cells; or may decrease Aβ production by activated microglia. These compounds could also prevent Aβ in the brain from interacting with the cell surface and therefore prevent neurotoxicity, neurodegeneration, or inflammation.
In a preferred embodiment, the method is used to treat Alzheimer's disease (e.g., sporadic or familial AD). The method can also be used prophylactically or therapeutically to treat other clinical occurrences of amyloid-β deposition, such as in Down's syndrome individuals and in patients with cerebral amyloid angiopathy (“CAA”), hereditary cerebral hemorrhage, or early Alzheimer's disease.
In another embodiment, the method is used to treat mild cognitive impairment. Mild Cognitive Impairment (“MCI”) is a condition characterized by a state of mild but measurable impairment in thinking skills, which is not necessarily associated with the presence of dementia. MCI frequently, but not necessarily, precedes Alzheimer's disease. Additionally, abnormal accumulation of APP and of amyloid-β protein in muscle fibers has been implicated in the pathology of sporadic inclusion body myositis (IBM) (Askanas, V., et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1314-1319; Askanas, V. et al. (1995) Current Opinion in Rheumatology 7: 486-496). Accordingly, the compounds of the invention can be used prophylactically or therapeutically in the treatment of disorders in which amyloid-beta protein is abnormally deposited at non-neurological locations, such as treatment of IBM by delivery of the compounds to muscle fibers.
Additionally, it has been shown that Aβ is associated with abnormal extracellular deposits, known as drusen, that accumulate along the basal surface of the retinal pigmented epithelium in individuals with age-related macular degeneration (ARMD). ARMD is a cause of irreversible vision loss in older individuals. It is believed that Aβ deposition could be an important component of the local inflammatory events that contribute to atrophy of the retinal pigmented epithelium, drusen biogenesis, and the pathogenesis of ARMD (Johnson, et al., Proc. Natl. Acad. Sci. USA 99(18), 11830-5 (2002)).
In another embodiment, the invention also relates to a method of treating or preventing an amyloid-related disease in a subject (preferably a human) comprising administering to the subject a therapeutic amount of a compound according to the following Formulae or otherwise described herein, such that amyloid fibril formation or deposition, neurodegeneration, or cellular toxicity is reduced or inhibited. In another embodiment, the invention relates to a method of treating or preventing an amyloid-related disease in a subject (preferably a human) comprising administering to the subject a therapeutic amount of a compound according to the following Formulae or otherwise described herein, such that cognitive function is improved or stabilized or further deterioration in cognitive function is prevented, slowed, or stopped in patients with brain amyloidosis, e.g., Alzheimer's disease, Down's syndrome or cerebral amyloid angiopathy. These compounds can also improve quality of daily living in these subjects.
The therapeutic compounds of the invention may treat amyloidosis related to type II diabetes by, for example, stabilizing glycemia, preventing or reducing the loss of β cell mass, reducing or preventing hyperglycemia due to loss of β cell mass, and modulating (e.g., increasing or stabilizing) insulin production. The compounds of the invention may also stabilize the ratio of the concentrations of pro-IAPP/IAPP.
The therapeutic compounds of the invention may treat AA (secondary) amyloidosis and/or AL (primary) amyloidosis, by stabilizing renal function, decreasing proteinuria, increasing creatinine clearance (e.g., by at least 50% or greater or by at least 100% or greater), by leading to remission of chronic diarrhea or weight gain (e.g., 10% or greater), or by reducing serum creatinine. Visceral amyloid content as determined, e.g., by SAP scintigraphy may also be reduced.
The present invention relates, at least in part, to the use of certain chemical compounds (and pharmaceutical formulations thereof) in the prevention or treatment of amyloid-related diseases, including, inter alia, Alzheimer's disease, cerebral amyloid angiopathy, inclusion body myositis, Down's syndrome, diabetes related amyloidosis, hemodialysis-related amyloidosis (β2M), primary amyloidosis (e.g., λ or κ chain-related), familial amyloid polyneuropathy (FAP), senile systemic amyloidosis, familial amyloidosis, Ostertag-type non-neuropathic amyloidosis, cranial neuropathy, hereditary cerebral hemorrhage, familial dementia, chronic dialysis, familial Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, hereditary spongiform encephalopathies, prion diseases, familial Mediterranean fever, Muckle-Well's syndrome, nephropathy, deafness, urticaria, limb pain, cardiomyopathy, cutaneous deposits, multiple myeloma, benign monoclonal gammopathy, maccoglobulinaemia, myeloma associated amyloidosis, medullary carcinomas of the thyroid, and isolated atrial amyloid.
The chemical structures herein are drawn according to the conventional standards known in the art. Thus, where an atom, such as a carbon atom, as drawn appears to have an unsatisfied valency, then that valency is assumed to be satisfied by a hydrogen atom even though that hydrogen atom is not necessarily explicitly drawn. The structures of some of the compounds of this invention include stereogenic carbon atoms. It is to be understood that isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention unless indicated otherwise. That is, unless otherwise stipulated, any chiral carbon center may be of either (R)- or (S)-stereochemistry. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically-controlled synthesis. Furthermore, alkenes can include either the E- or Z-geometry, where appropriate. In addition, the compounds of the present invention may exist in unsolvated as well as solvated forms with acceptable solvents such as water, THF, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.
A “small molecule” refers to a compound that is not itself the product of gene transcription or translation (e.g., protein, RNA, or DNA) and preferably has a low molecular weight, e.g., less than about 2500 amu.
In general, the term “nucleophile” is art-recognized to mean a chemical group having a reactive pair of electrons that reacts with a compound by displacing a leaving group (commonly another nucleophile), such as commonly occur in aliphatic chemistry as unimolecular (known as “SN1”) or bimolecular (“SN2”) reactions. Examples of nucleophiles include uncharged compounds such as amines, mercaptans, and alcohols, and charged groups such as alkoxides, thiolates, carbanions, and a variety of organic and inorganic anions. Illustrative anionic nucleophiles include, inter alia, simple anions such as azide, cyanide, thiocyanate, acetate, formate, or chloroformate, and bisulfite. Organometallic reagents such as organocuprates, organozincs, organolithiums, Grignard reagents, enolates, and acetylides, will under appropriate reaction conditions, be suitable nucleophiles.
Similarly, an “electrophile” means an atom, molecule, or ion able to accept an electron pair, particularly a pair of electrons from a nucleophile, such as typically occurs during an electrophilic substitution reaction. In an electrophilic substitution reaction, an electrophile binds to a substrate with the expulsion of another electrophile, e.g., the substitution of a proton by another electrophile such as a nitronium ion on an aromatic substrate (e.g., benzene). Electrophiles include cyclic compounds such as epoxides, aziridines, episulfides, cyclic sulfates, carbonates, lactones, and lactams; and non-cyclic electrophiles include sulfates, sulfonates (e.g., tosylates), chlorides, bromides, and iodides. Generally, an electrophile may be a saturated carbon atom (e.g., a methylene group) bonded to a leaving group; however, an electrophile may also be an unsaturated group, such as an aldehyde, ketone, ester, or conjugated (α,β-unsaturated) analog thereof, which upon reaction with a nucleophile forms an adduct.
The term “leaving group” generally refers to a group that is readily displaced and substituted by a nucleophile (e.g., an amine, a thiol, an alcohol, or cyanide). Such leaving groups are well known and include carboxylates, N-hydroxysuccinimide (“NHS”), N-hydroxybenzotriazole, a halogen (fluorine, chlorine, bromine, or iodine), alkoxides, and thioalkoxides. A variety of sulfur-based leaving groups are routinely used in synthetic chemistry, including alkane sulfonyloxy groups (e.g., C1-C4 alkane such as methane sulfonyloxy, ethane sulfonyloxy, propane sulfonyloxy, and butane sulfonyloxy groups) and the halogenated analogs (e.g., halogeno(C1-C4 alkane) sulfonyloxy groups, such as trifluoromethane sulfonyloxy (i.e., triflate), 2,2,2-trichloroethane sulfonyloxy, 3,3,3-tribromopropane sulfonyloxy, and 4,4,4-trifluorobutane sulfonyloxy groups), as well as arylsulfonyloxy groups (e.g., C6-C10 aryl optionally substituted with 1 to 3 C1-C4 alkyl groups, such as benzene sulfonyloxy, α-naphthylsulfonyloxy, β-naphthylsulfonyloxy, p-toluenesulfonyloxy (i.e., tosylates), 4-tert-butylbenzene sulfonyloxy, mesitylene sulfonyloxy, and 6-ethyl-α-naphthylsulfonyloxy groups).
“Activated esters” may be represented by the formula -COL, where L is a leaving group, typical examples of which include N-hydroxysulfosuccinimidyl and N-hydroxysuccinimidyl groups; aryloxy groups substituted with electron-withdrawing groups (e.g., p-nitro, pentafluoro, pentachloro, p-cyano, or p-trifluoromethyl); and carboxylic acids activated by a carbodiimide to form an anhydride or mixed anhydride, e.g., —OCORa or —OCNRaNHRb, where Ra and Rb are independently C1-C6 alkyl, C5-C8 alkyl (e.g., cyclohexyl), C1-C6 perfluoroalkyl, or C1-C6 alkoxy groups. An activated ester may be formed in situ or may be an isolable reagent. Sulfosuccinimidyl esters, pentafluorothiophenol esters, and sulfotetrafluorophenol are preferred activated esters. However, the ester leaving group may be, for example, substituted or unsubstituted C1-C6 alkyl (such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl), or substituted or unsubstituted C6-C14 aryl or heterocyclic groups, such as 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl, 2,2-dibromoethyl, 2,2,2-trichloroethyl, 3-fluoropropyl, 4-chlorobutyl, methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, N-propoxymethyl, isopropoxymethyl, N-butoxymethyl, tert-butoxymethyl, 1-ethoxyethyl, 1-methyl-1-methoxyethyl, 1-(isopropoxy)ethyl, 3-methoxypropyl-4-methoxybutyl, fluoromethoxymethyl, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 3-fluoropropoxymethyl, 4-chlorobutoxyethyl, dibromomethoxyethyl, 2-chloroethoxypropyl, fluoromethoxybutyl, 2-methoxyethoxymethyl, ethoxymethoxyethyl, methoxyethoxypropyl, methoxyethoxybutyl, benzyl, phenethyl, 3-phenylpropyl, 4-phenylbutyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldipheylmethyl, 9-anthrylmethyl, 4-methylbenzyl, 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 2-nitrobenzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl, 4-cyanobenzyldiphenylmethyl, or bis(2-nitrophenyl)methyl groups.
The term “electron-withdrawing group” is art-recognized and describes the ability of a substituent to attract valence electrons (e.g., pi-electrons) from neighboring atoms, e.g., the substituent is more electronegative than neighboring atoms, or it draws electrons to itself more than a hydrogen atom would at the same position. The Hammett sigma value (σ) is an accepted measure of a group's electron-donating and withdrawing ability, especially the sigma para value (σp). See, e.g., “Advanced Organic Chemistry” by J. March, 5th Ed., John Wiley & Sons, Inc., New York, pp. 368-75 (2001). The Hammett constant values are generally negative for electron-donating groups (σp=−0.66 for NH2) and positive for electron-withdrawing groups (σp=0.78 for a nitro group), σp indicating para substitution. Exemplary electron-withdrawing groups include nitro, acyl (ketone), formyl (aldehyde), sulfonyl, trifluoromethyl, halogeno (e.g., chloro and fluoro), and cyano groups, among others. Conversely, an “electron-donating group” designates a substituent that contributes electrons more than hydrogen would if it occupied the same position in the molecule. Examples include amino (including alkylamino and dialkylamino), aryl, alkoxy (including aralkoxy), aryloxy, mercapto and alkylthio, and hydroxyl groups, among others.
As used herein, “alkyl” groups include saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), cyclic alkyl groups (or “cycloalkyl” or “alicyclic” or “carbocyclic” groups) (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl groups (e.g., alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups). The term “aliphatic group” includes organic moieties characterized by straight or branched-chains, typically having between 1 and 22 carbon atoms. In complex structures, the chains may be branched, bridged, or cross-linked. Aliphatic groups include alkyl groups, alkenyl groups, and alkynyl groups.
In certain embodiments, a straight-chain or branched-chain alkyl group may have 30 or fewer carbon atoms in its backbone, e.g., C1-C30 for straight-chain or C3-C30 for branched-chain. In certain embodiments, a straight-chain or branched-chain alkyl group may have 20 or fewer carbon atoms in its backbone, e.g., C1-C20 for straight-chain or C3-C20 for branched-chain, and more preferably 18 or fewer. Likewise, preferred cycloalkyl groups have from 4-10 carbon atoms in their ring structure, and more preferably have 4-7 carbon atoms in the ring structure. The term “lower alkyl” refers to alkyl groups having from 1 to 6 carbons in the chain, and to cycloalkyl groups having from 3 to 6 carbons in the ring structure. Unless the number of carbons is otherwise specified, “lower” as in “lower aliphatic,” “lower alkyl,” “lower alkenyl,” etc. as used herein means that the moiety has at least one and less than about 8 carbon atoms. In certain embodiments, a straight-chain or branched-chain lower alkyl group has 6 or fewer carbon atoms in its backbone (e.g., C1-C6 for straight-chain, C3-C6 for branched-chain), and more preferably 4 or fewer. Likewise, preferred cycloalkyl groups have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term “C1-C6” as in “C1-C6 alkyl” means alkyl groups containing 1 to 6 carbon atoms.
Moreover, unless otherwise specified the term alkyl includes both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl groups having substituents replacing one or more hydrogens on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, alkenyl, alkynyl, halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclic, alkylaryl, or aromatic (including heteroaromatic) groups.
An “arylalkyl” group is an alkyl group substituted with an aryl group (e.g., phenylmethyl (i.e., benzyl)). An “alkylaryl” moiety is an aryl group substituted with an alkyl group (e.g., p-methylphenyl (i.e., p-tolyl)). The term “n-alkyl” means a straight-chain (i.e., unbranched) unsubstituted alkyl group. An “alkylene” group is a divalent analog of the corresponding alkyl group. The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups analogous to alkyls, but which contain at least one double or triple carbon-carbon bond respectively. Suitable alkenyl and alkynyl groups include groups having 2 to about 12 carbon atoms, preferably from 2 to about 6 carbon atoms.
The term “aromatic group” or “aryl group” includes unsaturated and aromatic cyclic hydrocarbons as well as unsaturated and aromatic heterocycles containing one or more rings. Aryl groups may also be fused or bridged with alicyclic or heterocyclic rings that are not aromatic so as to form a polycycle (e.g., tetralin). An “arylene” group is a divalent analog of an aryl group. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).
The term “heterocyclic group” includes closed ring structures analogous to carbocyclic groups in which one or more of the carbon atoms in the ring is an element other than carbon, for example, nitrogen, sulfur, or oxygen. Heterocyclic groups may be saturated or unsaturated. Additionally, heterocyclic groups (such as pyrrolyl, pyridyl, isoquinolyl, quinolyl, purinyl, and furyl) may have aromatic character, in which case they may be referred to as “heteroaryl” or “heteroaromatic” groups.
Unless otherwise stipulated, aryl and heterocyclic (including heteroaryl) groups may also be substituted at one or more constituent atoms. Examples of heteroaromatic and heteroalicyclic groups may have 1 to 3 separate or fused rings with 3 to about 8 members per ring and one or more N, O, or S heteroatoms. In general, the term “heteroatom” includes atoms of any element other than carbon or hydrogen, preferred examples of which include nitrogen, oxygen, sulfur, and phosphorus. Heterocyclic groups may be saturated or unsaturated or aromatic.
Examples of heterocycles include, but are not limited to, acridinyl; azocinyl; benzimidazolyl; benzofuranyl; benzothiofuranyl; benzothiophenyl; benzoxazolyl; benzthiazolyl; benztriazolyl; benztetrazolyl; benzisoxazolyl; benzisothiazolyl; benzimidazolinyl; carbazolyl; 4aH-carbazolyl; carbolinyl; chromanyl; chromenyl; cinnolinyl; decahydroquinolinyl; 2H,6H-1,5,2-dithiazinyl; dihydrofuro[2,3-b]tetrahydrofuran; furanyl; furazanyl; imidazolidinyl; imidazolinyl; imidazolyl; 1H-indazolyl; indolenyl; indolinyl; indolizinyl; indolyl; 3H-indolyl; isobenzofuranyl; isochromanyl; isoindazolyl; isoindolinyl; isoindolyl; isoquinolinyl; isothiazolyl; isoxazolyl; methylenedioxyphenyl; morpholinyl; naphthyridinyl; octahydroisoquinolinyl; oxadiazolyl; 1,2,3-oxadiazolyl; 1,2,4-oxadiazolyl; 1,2,5-oxadiazolyl; 1,3,4-oxadiazolyl; oxazolidinyl; oxazolyl; oxazolidinyl; pyrimidinyl; phenanthridinyl; phenanthrolinyl; phenazinyl; phenothiazinyl; phenoxathiinyl; phenoxazinyl; phthalazinyl; piperazinyl; piperidinyl; piperidonyl; 4-piperidonyl; piperonyl; pteridinyl; purinyl; pyranyl; pyrazinyl; pyrazolidinyl; pyrazolinyl; pyrazolyl; pyridazinyl; pyridooxazole; pyridoimidazole; pyridothiazole; pyridinyl; pyridyl; pyrimidinyl; pyrrolidinyl; pyrrolinyl; 2H-pyrrolyl; pyrrolyl; quinazolinyl; quinolinyl; 4H-quinolizinyl; quinoxalinyl; quinuclidinyl; tetrahydrofuranyl; tetrahydroisoquinolinyl; tetrahydroquinolinyl; tetrazolyl; 6H-1,2,5-thiadiazinyl; 1,2,3-thiadiazolyl; 1,2,4-thiadiazolyl; 1,2,5-thiadiazolyl; 1,3,4-thiadiazolyl; thianthrenyl; thiazolyl; thienyl; thienothiazolyl; thienooxazolyl; thienoimidazolyl; thiophenyl; triazinyl; 1,2,3-triazolyl; 1,2,4-triazolyl; 1,2,5-triazolyl; 1,3,4-triazolyl; and xanthenyl. Preferred heterocycles include, but are not limited to, pyridinyl; furanyl; thienyl; pyrrolyl; pyrazolyl; pyrrolidinyl; imidazolyl; indolyl; benzimidazolyl; 1H-indazolyl; oxazolidinyl; benzotriazolyl; benzisoxazolyl; oxindolyl; benzoxazolinyl; and isatinoyl groups. Also included are fused ring and spiro compounds containing, for example, the above heterocycles.
A common hydrocarbon aryl group is a phenyl group having one ring. Two-ring hydrocarbon aryl groups include naphthyl, indenyl, benzocyclooctenyl, benzocycloheptenyl, pentalenyl, and azulenyl groups, as well as the partially hydrogenated analogs thereof such as indanyl and tetrahydronaphthyl. Exemplary three-ring hydrocarbon aryl groups include acephthylenyl, fluorenyl, phenalenyl, phenanthrenyl, and anthracenyl groups.
Aryl groups also include heteromonocyclic aryl groups, i.e., single-ring heteroaryl groups, such as thienyl, furyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, and pyridazinyl groups; and oxidized analogs thereof such as pyridonyl, oxazolonyl, pyrazolonyl, isoxazolonyl, and thiazolonyl groups. The corresponding hydrogenated (i.e., non-aromatic) heteromonocyclic groups include pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl and piperidino, piperazinyl, and morpholino and morpholinyl groups.
Aryl groups also include fused two-ring heteroaryls such as indolyl, isoindolyl, indolizinyl, indazolyl, quinolinyl, isoquinolinyl, phthalazinyl, quinoxalinyl, quinazolinyl, cinnolinyl, chromenyl, isochromenyl, benzothienyl, benzimidazolyl, benzothiazolyl, purinyl, quinolizinyl, isoquinolonyl, quinolonyl, naphthyridinyl, and pteridinyl groups, as well as the partially hydrogenated analogs such as chromanyl, isochromanyl, indolinyl, isoindolinyl, and tetrahydroindolyl groups. Aryl groups also include fused three-ring groups such as phenoxathiinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and dibenzofuranyl groups.
Some typical aryl groups include substituted or unsubstituted 5- and 6-membered single-ring groups. In another aspect, each Ar group may be selected from the group consisting of substituted or unsubstituted phenyl, pyrrolyl, furyl, thienyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl, pyridazinyl, and pyrimidinyl groups. Further examples include substituted or unsubstituted phenyl, 1-naphthyl, 2-naphthyl, biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl groups.
The term “amine” or “amino,” as used herein, refers to an unsubstituted or substituted moiety of the formula —NRaRb, in which Ra and Rb are each independently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb, taken together with the nitrogen atom to which they are attached, form a cyclic moiety having from 3 to 8 atoms in the ring. Thus, the term amino includes cyclic amino moieties such as piperidinyl or pyrrolidinyl groups, unless otherwise stated. Thus, the term “alkylamino” as used herein means an alkyl group having an amino group attached thereto. Suitable alkylamino groups include groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms. The term amino includes compounds or moieties in which a nitrogen atom is covalently bonded to at least one carbon or heteroatom. The term “dialkylamino” includes groups wherein the nitrogen atom is bound to at least two alkyl groups. The term “arylamino” and “diarylamino” include groups wherein the nitrogen is bound to at least one or two aryl groups, respectively. The term “alkylarylamino” refers to an amino group which is bound to at least one alkyl group and at least one aryl group. The term “alkaminoalkyl” refers to an alkyl, alkenyl, or alkynyl group substituted with an alkylamino group. The term “amide” or “aminocarbonyl” includes compounds or moieties which contain a nitrogen atom which is bound to the carbon of a carbonyl or a thiocarbonyl group.
The term “alkylthio” refers to an alkyl group, having a sulfhydryl group attached thereto. Suitable alkylthio groups include groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms.
The term “alkylcarboxyl” as used herein means an alkyl group having a carboxyl group attached thereto.
The term “alkoxy” as used herein means an alkyl group having an oxygen atom attached thereto. Representative alkoxy groups include groups having 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms, e.g., methoxy, ethoxy, propoxy, tert-butoxy and the like. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. The alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc., as well as perhalogenated alkyloxy groups.
The term “acylamino” includes moieties wherein an amino moiety is bonded to an acyl group. For example, the acylamino group includes alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.
The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” include alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone.
The term “carbonyl” or “carboxy” includes compounds and moieties which contain a carbon connected with a double bond to an oxygen atom. Examples of moieties which contain a carbonyl include aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.
The term “ether” or “ethereal” includes compounds or moieties which contain an oxygen bonded to two carbon atoms. For example, an ether or ethereal group includes “alkoxyalkyl” which refers to an alkyl, alkenyl, or alkynyl group substituted with an alkoxy group.
A “sulfonate” group is a —SO3H or —SO3−X+ group bonded to a carbon atom, where X+ is a cationic counter ion group. Similarly, a “sulfonic acid” compound has a —SO3H or —SO3−X+ group bonded to a carbon atom, where X+ is a cationic group. A “sulfate” as used herein is a —OSO3H or —OSO3−X+ group bonded to a carbon atom, and a “sulfuric acid” compound has a —SO3H or —OSO3−X+ group bonded to a carbon atom, where X+ is a cationic group. According to the invention, a suitable cationic group may be a hydrogen atom. In certain cases, the cationic group may actually be another group on the therapeutic compound that is positively charged at physiological pH, for example an amino group.
A “counter ion” is required to maintain electroneutrality. Examples of anionic counter ions include halide, triflate, sulfate, nitrate, hydroxide, carbonate, bicarbonate, acetate, phosphate, oxalate, cyanide, alkylcarboxylate, N-hydroxysuccinimide, N-hydroxybenzotriazole, alkoxide, thioalkoxide, alkane sulfonyloxy, halogenated alkane sulfonyloxy, arylsulfonyloxy, bisulfate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, or lactobionate. Compounds containing a cationic group covalently bonded to an anionic group may be referred to as an “internal salt.”
The term “nitro” means —NO2; the term “halogen” or “halogeno” or “halo” designates —F, —Cl, —Br or —I; the term “thiol,” “thio,” or “mercapto” means SH; and the term “hydroxyl” or “hydroxy” means —OH.
The term “acyl” refers to a carbonyl group that is attached through its carbon atom to a hydrogen (i.e., a formyl), an aliphatic group (e.g., acetyl), an aromatic group (e.g., benzoyl), and the like. The term “substituted acyl” includes acyl groups where one or more of the hydrogen atoms on one or more carbon atoms are replaced by, for example, an alkyl group, alkynyl group, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Unless otherwise specified, the chemical moieties of the compounds of the invention, including those groups discussed above, may be “substituted or unsubstituted.” In some embodiments, the term “substituted” means that the moiety has substituents placed on the moiety other than hydrogen (i.e., in most cases, replacing a hydrogen), which allow the molecule to perform its intended function. Examples of substituents include moieties selected from straight or branched alkyl (preferably C1-C5), cycloalkyl (preferably C3-C8), alkoxy (preferably C1-C6), thioalkyl (preferably C1-C6), alkenyl (preferably C2-C6), alkynyl (preferably C2-C6), heterocyclic, carbocyclic, aryl (e.g., phenyl), aryloxy (e.g., phenoxy), aralkyl (e.g., benzyl), aryloxyalkyl (e.g., phenyloxyalkyl), arylacetamidoyl, alkylaryl, heteroaralkyl, alkylcarbonyl and arylcarbonyl or other such acyl group, heteroarylcarbonyl, and heteroaryl groups, as well as (CR′R″)0-3NR′R″ (e.g., —NH2), (CR′R″)0-3CN (e.g., —CN), —NO2, halogen (e.g., —F, —Cl, —Br, or —I), (CR′R″)0-3C(halogen)3 (e.g., —CF3), (CR′R″)0-3CH(halogen)2, (CR′R″)0-3CH2(halogen), (CR′R″)0-3CONR′R″, (CR′R″)0-3(CNH)NR′R″, (CR′R″)0-3S(O)1-2NR′R″, (CR′R″)0-3CHO, (CR′R″)0-3O(CR′R″)0-3H, (CR′R″)0-3S(O)0-3R′ (e.g., —SO3H), (CR′R″)0-3O(CR′R″)0-3H (e.g., —CH2OCH3 and —OCH3), (CR′R″)0-3S(CR′R″)0-3H (e.g., —SH and —SCH3), (CR′R″)0-3OH (e.g., —OH), (CR′R″)0-3COR′, (CR′R″)0-3(substituted or unsubstituted phenyl), (CR′R″)0-3(C3-C8 cycloalkyl), (CR′R″)0-3CO2R′ (e.g., —CO2H), and (CR′R″)0-3OR′ groups, wherein R′ and R″ are each independently hydrogen, a C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, or aryl group; or the side chain of any naturally occurring amino acid.
In another embodiment, a substituent may be selected from straight or branched alkyl (preferably C1-C5), cycloalkyl (preferably C3-C8), alkoxy (preferably C1-C6), thioalkyl (preferably C1-C6), alkenyl (preferably C2-C6), alkynyl (preferably C2-C6), heterocyclic, carbocyclic, aryl (e.g., phenyl), aryloxy (e.g., phenoxy), aralkyl (e.g., benzyl), aryloxyalkyl (e.g., phenyloxyalkyl), arylacetamidoyl, alkylaryl, heteroaralkyl, alkylcarbonyl and arylcarbonyl or other such acyl group, heteroarylcarbonyl, or heteroaryl group, (CR′R″)0-10NR′R″ (e.g., —NH2), (CR′R″)0-10CN (e.g., —CN), NO2, halogen (e.g., F, Cl, Br, or I), (CR′R″)0-10C(halogen)3 (e.g., —CF3), (CR′R″)0-10CH(halogen)2, (CR′R″)0-10CH2(halogen), (CR′R″)0-10CONR′R″, (CR′R″)0-10(CNH)NR′R″, (CR′R″)0-10S(O)1-2NR′R″, (CR′R″)0-10CHO, (CR′R″)0-10O(CR′R″)0-10H, (CR′R″)0-10S(O)0-3R′ (e.g., —SO3H), (CR′R″)0-10O(CR′R″)0-10H (e.g., —CH2OCH3 and —OCH3), (CR′R″)0-10S(CR′R″)0-3H (e.g., —SH and —SCH3), (CR′R″)0-10H (e.g., —OH), (CR′R″)0-10COR′, (CR′R″)0-10(substituted or unsubstituted phenyl), (CR′R″)0-10(C3-C8 cycloalkyl), (CR′R″)0-10CO2R′ (e.g., —CO2H), or (CR′R″)0-10OR′ group, or the side chain of any naturally occurring amino acid; wherein R′and R″ are each independently hydrogen, a C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, or aryl group, or R′ and R″ taken together are a benzylidene group or a —(CH2)2—O—(CH2)2— group.
It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with the permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is meant to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more.
In some embodiments, a “substituent” may be selected from the group consisting of, for example, halogeno, trifluoromethyl, nitro, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkylcarbonyloxy, arylcarbonyloxy, C1-C6 alkoxycarbonyloxy, aryloxycarbonyloxy, C1-C6 alkylcarbonyl, C1-C6 alkoxycarbonyl, C1-C6 alkoxy, C1--C6 alkylthio, arylthio, heterocyclyl, aralkyl, and aryl (including heteroaryl) groups.
In one embodiment, the invention pertains to compounds of Formula I:
wherein:
R1 is a substituted or unsubstituted cycloalkyl, heterocyclic, aryl, arylcycloalkyl, bicyclic or tricyclic ring, a bicyclic or tricyclic fused ring group, or a substituted or unsubstituted C2-C10 alkyl group;
R2 is selected from a group consisting of hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, and benzoimidazolyl;
Y is SO3−X+, OSO3−X+, or SSO3−X+;
X+ is hydrogen, a cationic group, or ester-forming group; and
each of L1 and L2 is independently a substituted or unsubstituted C1-C5 alkyl group or absent, or a pharmaceutically acceptable salt thereof, provided that when R1 is alkyl, L1 is absent.
In a further embodiment, the invention pertains to compounds of Formula II:
wherein:
R1 is a substituted or unsubstituted cyclic, bicyclic, tricyclic, or benzoheterocyclic group or a substituted or unsubstituted C2-C10 alkyl group;
R2 is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, benzoimidazolyl, or linked to R1 to form a heterocycle;
Y is SO3−X+, OSO3−X+, or SSO3−X+;
X+ is hydrogen, a cationic group, or an ester forming moiety;
m is 0 or 1;
n is 1, 2, 3, or 4;
L is substituted or unsubstituted C1-C3 alkyl group or absent, or a pharmaceutically acceptable salt thereof, provided that when R1 is alkyl, L is absent.
In a further embodiment, R2 is hydrogen. In another further embodiment, R1 is straight chain alkyl, for example, ethyl, n-pentyl, n-heptyl, or n-octyl. In another embodiment, R1 is t-butyl. In yet another alternate embodiment, R1 is C7-C10 bicycloalkyl or tricycloalkyl, such as, for example, tricyclo[3.3.1.03,7]decyl (or adamantyl), bicyclo[2.1.2]heptyl, or indolyl. In another alternate embodiment, R1 is tetrahydronaphthyl.
In one embodiment, L2 is —(CH2)3—. In another further embodiment, L2 is —(CH2)4— or —(CH2)5—. In yet another further embodiment, L2 is —(CH2)2—. In yet another further embodiment, L2 is substituted alkyl, e.g., —CH2—(CHOH)—CH2—.
In another embodiment, L1 is CH2CH2 or absent.
In a further embodiment, R1 is branched alkyl, e.g., t-butyl. In another embodiment, R1 is adamanyl. In another embodiment, R1 is cyclic alkyl, e.g., cyclopropyl, cyclohexyl, cycloheptyl, cyclo-octyl, etc. The cycloalkyl moieties may be substituted further, e.g., with additional alkyl groups or other groups which allow the molecule to perform its intended function. In another embodiment, R1 is alkyl substituted with a propargyl moiety (e.g., HC≡C—). In another embodiment, R1 is cyclohexyl substituted with one or more methyl or propargyl groups.
In other embodiments, L1 is a C1-C2 alkyl linker group (e.g., —CH(CH3)— or —(CH2)2—. In a further embodiment, R1 is phenyl. In certain embodiments, R1 is substituted with a methoxy group. In other embodiments, L1 is C3, e.g., —(CH2)3— or C(CH3)2—. In certain embodiments, L1 is substituted, e.g., with an alkoxy, carboxylate (—COOH), benzyl, amido (—C═O—NH—), or ester (C═O—C—O) group. In certain embodiment, the ester group is a methyl, ethyl, propyl, butyl, cyclohexyl, or benzyl ester. In other embodiments, the ester group may be propenyl. In other embodiments, L1 is substituted with a carboxylate group. In a further embodiment, R1 is substituted with a substituted amido group, wherein the amido group is substituted with an alkyl, e.g., methyl, ethyl, propyl, butyl, pentyl, or hexyl group. In another embodiment, the alkyl R1 group is a substituted with a —C═O—NH—OH, C═O—NH2, or an amido group. In certain embodiments, the amido group is substituted with an alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclohexyl, etc.), a benzyl or an aryl group. In another embodiment, the amido group is substituted with a —CH(CH2)2 group. R1 itself may be substituted with a phenyl or may be branched or straight chain alkyl. In certain embodiments, R1 may also be substituted with a thioether moiety. Examples of thioethers include S-Me, S-Et, etc. In certain embodiments, the alkyl R1 moiety is substituted with both an aryl or a thioether moiety and an amido moiety. In other embodiments, the alkyl R1 moiety may be substituted with both a thioether and a carboxylate moiety. In other embodiments, alkyl R1 groups are substituted with hydroxyl. R1 groups, e.g., alkyl R1 groups, may also be substituted with both thioether and hydroxyl groups. In other embodiments, R1 groups, e.g., alkyl R1 groups are substituted with cyano groups. Examples of R1 groups including —CN moieties include —C(CH3)2CN, cyclohexyl substituted with one or more cyano groups, etc.
In other embodiments, alkyl R1 groups are substituted with aryl groups. The aryl groups may be substituted phenyl, for example. The substituted phenyl may be substituted with one or more substituents such as hydroxy, cyano and alkoxy. In other embodiments, alkyl R1 groups are substituted with tetrazolyl or substituted or unsubstituted benzyl. In a further embodiment, L1 is —C(CH3)2—(CH2)—. In another embodiment, L1 is —(C(CH3)2—CHOH—. In yet another embodiment, L1 is —(C(CH3)2CH(OMe)-. In another embodiment, R1 is substituted or unsubstituted phenyl. In a further embodiment, R1 is para-substituted phenyl. Examples of substitutents include but are not limited to fluorine, chlorine, bromine, iodine, methyl, t-butyl, alkoxy, methoxy, etc. In other embodiment, R1 is substituted at the meta position. Examples of substituents include methoxy, chloro, methyl, t-butyl, fluoro, alkyl, alkoxy, iodo, trifluoroalkyl, methoxy, etc. In another embodiment, R1 is phenyl substituted in the ortho position, with similar substituents. In another embodiment, L1 comprises a cycloalkyl moiety, e.g., cyclopentyl. In another embodiment, L1 comprises an alkyenyl group and, optionally, a substituted aryl group, with substitutents similar to those described about.
In certain embodiments, R1 is cyclopropyl or cyclohexyl. In certain embodiments, the cyclopropyl or cyclohexyl group is substituted with an ether group or an alkyl group. In certain further embodiments, the ether group is a benzyl ether group.
In another embodiment, wherein R1 is alkyl, it is substituted with groups such as phenyl, or hydroxy.
In other embodiments, the compound of the invention is selected from the group consisting of:
and pharmaceutically acceptable salts, esters, and prodrugs thereof.
In another embodiment, the invention pertains to compounds of Formula III:
wherein:
A is nitrogen or oxygen;
R11 is hydrogen, salt-forming cation, ester forming group, —(CH2)x-Q, or when A is nitrogen, A and R11 taken together may be the residue of a natural or unnatural amino acid or a salt or ester thereof;
Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
x is 0, 1, 2, 3, or 4;
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
R3, R3a, R4, R4a, R5, R5a, R6, R6a, R7 and R7a are each independently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino, tetrazolyl, or two R groups on adjacent ring atoms taken together with the ring atoms form a double bond, provided that one of R3, R3a, R4, R4a, R5, R5a, R6, R6a, R7 and R7a is a moiety of the Formula IIIa:
wherein:
m is 0, 1, 2, 3, or 4;
RA, RB, RC, RD, and RE are independently selected from a group of hydrogen, halogen, hydroxyl, alkyl, alkoxyl, halogenated alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano, thiazolyl, triazolyl, imidazolyl, tetrazolyl, benzothiazolyl, and benzoimidazolyl; and pharmaceutically acceptable salts and esters thereof, provided that said compound is not 3-(4-phenyl-1,2,3,6-tetrahydro-1-pyridyl)-1-propanesulfonic acid.
In a further embodiment, n is 2, 3 or 4.
In another embodiment, R11 is a salt-forming cation. Examples of salt forming cations include pharmaceutically acceptable salts described herein as well as lithium, sodium, potassium, magnesium, calcium, barium, zinc, iron, and ammonium. In another embodiment, R11 is an ester-forming group. An ester-forming group includes groups which when bound form an ester. Examples of such groups include substituted or unsubstituted alkyl, aryl, alkenyl, alkynyl, or cycloalkyl. In another embodiment, A is oxygen.
In another embodiment, R3 and R4 are taken together with the carbon atoms to which they are attached to form a double bond. In another embodiment, RA, RB, RC, RD, and RE are each hydrogen. RA, RB, RD, and RE are each hydrogen and RC is a halogen, such as fluorine, chlorine, iodine, or bromine
In another embodiment, R3 or R5a is a moiety of Formula IIIa.
In another embodiment, R4, R5, R6, and R7 are each hydrogen. In another further embodiment, R4a, R5a, R6a, and R7a are each hydrogen.
In another, R3a is hydroxyl, cyano, acyl, or hydroxyl.
In another further embodiment, R11 and A taken together are a natural or unnatural amino acid residue or a pharmaceutically acceptable salt or ester thereof. Examples of amino acid residues include esters and salts of phenylalanine and leucine.
In another embodiment, m is 0, 1, or 3.
Examples of compounds of Formula III include, but are not limited to:
and pharmaceutically acceptable salts, esters, and prodrugs thereof.
In another embodiment, the invention pertains to compounds of Formula IV:
wherein:
A is nitrogen or oxygen;
R11 is hydrogen, salt-forming cation, ester forming group, —(CH2)x-Q, or when A is nitrogen, A and R11 taken together may be the residue of a natural or unnatural amino acid or a salt or ester thereof;
Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
x is 0, 1, 2, 3, or 4;
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
R4, R4a, R5, R5a, R6, R6a, R7, and R7a are each independently hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, cyano, halogen, amino, tetrazolyl, R4 and R5 taken together, with the ring atoms they are attached to, form a double bond, or R6 and R7 taken together, with the ring atoms they are attached to, form a double bond;
m is 0, 1, 2, 3, or 4;
R8, R9, R10, R11, and R12 are independently selected from a group of hydrogen, halogen, hydroxyl, alkyl, alkoxyl, halogenated alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, cyano, thiazolyl, triazolyl, imidazolyl, tetrazolyl, benzothiazolyl, and benzoimidazolyl, and pharmaceutically acceptable salts and esters thereof.
In another embodiment, R11 is a salt-forming cation. Examples of salt forming cations include pharmaceutically acceptable salts described herein as well as lithium, sodium, potassium, magnesium, calcium, barium, zinc, iron, and ammonium. In another embodiment, R11 is an ester-forming group. An ester-forming group includes groups which when bound form an ester. Examples of such groups include substituted or unsubstituted alkyl, aryl, alkenyl, alkynyl, or cycloalkyl. In another embodiment, A is oxygen.
In another embodiment, m is 0 or 1. In another further embodiment, n is 2, 3, or 4. In another further embodiment, R4, R5, R6 and R7 are each hydrogen. R4a, R5a, R6a, and R7a also may be hydrogen. Examples of R8, R9, R10, R11, and R12 include hydrogen. In another embodiment R8, R9, R11, R12 are each hydrogen, and R10 is a halogen, (e.g., fluorine, chlorine, bromine, or iodine), nitro, or alkyl (e.g., methyl, ethyl, butyl).
In another embodiment, A-R11 may be the residue of an amino acid, e.g., a phenylalanine residue. In another embodiment, R9, R10, R11 and R12 are each hydrogen, and R8 is not hydrogen, e.g., halogen, e.g., fluorine, bromine, chlorine, or iodine.
In another embodiment, the compound is:
and pharmaceutically acceptable salts, esters, and prodrugs thereof.
In another embodiment, the invention pertains to compounds of Formula V:
wherein:
A is nitrogen or oxygen;
R11 is hydrogen, salt-forming cation, ester forming group, —(CH2)x-Q, or when A is nitrogen, A and R11 taken together may be the residue of a natural or unnatural amino acid or a salt or ester thereof;
Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
x is 0, 1, 2, 3, or 4;
n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
aa is a natural or unnatural amino acid residue;
m is 0, 1, 2, or 3;
R14 is hydrogen or protecting group;
R15 is hydrogen, alkyl or aryl, and pharmaceutically acceptable salts, esters and prodrugs thereof.
In another embodiment, R11 is a salt-forming cation. Examples of salt forming cations include pharmaceutically acceptable salts described herein as well as lithium, sodium, potassium, magnesium, calcium, barium, zinc, iron, and ammonium. In another embodiment, R11 is an ester-forming group. An ester-forming group includes groups which when bound form an ester. Examples of such groups include substituted or unsubstituted alkyl, aryl, alkenyl, alkynyl, or cycloalkyl. In another embodiment, A is oxygen.
In an embodiment, n is 2, 3 or 4. In certain embodiments, m is 0. In certain embodiments, A-R11 is a residue of a natural amino acid, or a salt or ester thereof. Examples of amino acid residues, include, but are not limited to, leucine or phenylalanine residues, and pharmaceutically acceptable salts and esters thereof. Examples of possible esters include methyl, ethyl, and t-butyl.
In another embodiment, m is 1. Examples of aa include natural and unnatural amino acid residues such as phenylalanine, glycine, and leucine.
In another embodiment, (aa)m is a residue of phe-phe, or an ester thereof.
In certain embodiments, R15 is hydrogen or substituted alkyl, e.g., arylalkyl. The term “unnatural amino acid” refers to any derivative of a natural amino acid including D forms, and α- and β-amino acid derivatives. It is noted that certain amino acids, e.g., hydroxyproline, that are classified as a non-natural amino acid herein, may be found in nature within a certain organism or a particular protein. Amino acids with many different protecting groups appropriate for immediate use in the solid phase synthesis of peptides are commercially available. In addition to the twenty most common naturally occurring amino acids, the following examples of non-natural amino acids and amino acid derivatives may be used according to the invention (common abbreviations in parentheses): β-alanine (β-ALA), γ-aminobutyric acid (GABA), 2-aminobutyric acid (2-Abu), α,β-dehydro-2-aminobutyric acid (8-AU), 1-aminocyclopropane-1-carboxylic acid (ACPC), aminoisobutyric acid (Aib), 2-amino-thiazoline-4-carboxylic acid, 5-aminovaleric acid (5-Ava), 6-aminohexanoic acid (6-Ahx), 8-aminooctanoic acid (8-Aoc), 11-aminoundecanoic acid (11-Aun), 12-aminododecanoic acid (12-Ado), 2-aminobenzoic acid (2-Abz), 3-aminobenzoic acid (3-Abz), 4-aminobenzoic acid(4-Abz), 4-amino-3-hydroxy-6-methylheptanoic acid (Statine, Sta), aminooxyacetic acid (Aoa), 2-aminotetraline-2-carboxylic acid (ATC), 4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA), para-aminophenylalanine (4—NH2-Phe), biphenylalanine (Bip), para-bromophenylalanine (4-Br-Phe), ortho-chlorophenylalanine](2-Cl-Phe), meta-chlorophenylalanine (3-Cl-Phe), para-chlorophenylalanine (4-Cl-Phe), meta-chlorotyrosine (3-Cl-Tyr), para-benzoylphenylalanine (Bpa), tert-butylglycine (TLG), cyclohexylalanine (Cha), cyclohexylglycine (Chg), 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutyric acid (Dbu), 3,4-dichlorophenylalanine (3,4-Cl2-Phe), 3,4-difluororphenylalanine (3,4-F2-Phe), 3,5-diiodotyrosine (3,5-I2-Tyr), ortho-fluorophenylalanine (2-F-Phe), meta-fluorophenylalanine (3-F-Phe), para-fluorophenylalanine (4-F-Phe), meta-fluorotyrosine (3-F-Tyr), homoserine (Hse), homophenylalanine (Hfe), homotyrosine (Htyr), 5-hydroxytryptophan (5-OH-Trp), hydroxyproline (Hyp), para-iodophenylalanine (4-I-Phe), 3-iodotyrosine (3-I-Tyr), indoline-2-carboxylic acid (Idc), isonipecotic acid (Inp), meta-methyltyrosine (3-Me-Tyr), 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal), para-nitrophenylalanine (4-NO2-Phe), 3-nitrotyrosine (3-NO2-Tyr), norleucine (Nle), norvaline (Nva), ornithine (Orn), ortho-phosphotyrosine (H2PO3-Tyr), octahydroindole-2-carboxylic acid (Oic), penicillamine (Pen), pentafluorophenylalanine (F5-Phe), phenylglycine (Phg), pipecolic acid (Pip), propargylglycine (Pra), pyroglutamic acid (PGLU), sarcosine (Sar), tetrahydroisoquinoline-3-carboxylic acid (Tic), thienylalanine, and thiazolidine-4-carboxylic acid (thioproline, Th). Additionally, N-alkylated amino acids may be used, as well as amino acids having amine-containing side chains (such as Lys and Orn) in which the amine has been acylated or or alkylated.
Examples of compounds of the invention include, but are not limited to:
and pharmaceutically acceptable salts, esters, and prodrugs thereof.
In another embodiment, the invention pertains, at least in part, to compounds of Formula VI:
wherein:
n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
A is oxygen or nitrogen;
R11 is hydrogen, salt-forming cation, ester forming group, —(CH2)x-Q, or when A is nitrogen, A and R11 taken together may be the residue of a natural or unnatural amino acid or a salt or ester thereof;
Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
x is 0, 1, 2, 3, or 4;
R19 is hydrogen, alkyl or aryl;
Y1 is oxygen, sulfur, or nitrogen;
Y2 is carbon, nitrogen, or oxygen;
R20 is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
R21 is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, benzoimidazolyl, or absent if Y2 is oxygen;
R22 is hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, benzoimidazolyl; or R22 is hydrogen, hydroxyl, alkoxy or aryloxy if Y1 is nitrogen; or R22 is absent if Y1 is oxygen or sulfur; or R22 and R21 may be linked to form a cyclic moiety if Y1 is nitrogen;
R23 is hydrogen, alkyl, amino, mercaptoalkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, tetrazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl, or absent if Y2 is nitrogen or oxygen;
or pharmaceutically acceptable salts thereof.
In another embodiment, R11 is a salt-forming cation. Examples of salt forming cations include pharmaceutically acceptable salts described herein as well as lithium, sodium, potassium, magnesium, calcium, barium, zinc, iron, and ammonium. In a further embodiment, the salt is a sodium salt. In a further, embodiment, A is oxygen.
In another embodiment, Y1 is oxygen or sulfur, and R22 is absent.
In another embodiment, Y2 is oxygen and R21 is absent. Examples of R20 include benzyl, aryl (e.g., phenyl), alkyl, cycloalkyl (e.g., adamantyl), etc. In other embodiment, Y2 is nitrogen and R21 is hydrogen. In other embodiment, R21 is benzyl. In another further embodiment, R20 and R21 are linked to form a pyridyl ring. In another embodiment, Y1 is sulfur.
Examples of compounds of the invention, include
and pharmaceutically acceptable salts, esters, and prodrugs thereof.
In another embodiment, the invention pertains to compounds of Formula VII:
wherein:
n is 2, 3, or 4;
A is oxygen or nitrogen;
R11 is hydrogen, salt-forming cation, ester forming group, —(CH2)x-Q, or when A is nitrogen, A and R11 taken together may be the residue of a natural or unnatural amino acid or a salt or ester thereof;
Q is hydrogen, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, or benzoimidazolyl;
x is 0, 1, 2, 3, or 4;
G is a direct bond or oxygen, nitrogen, or sulfur;
z is 0, 1, 2, 3, 4, or 5;
m is 0 or 1;
R24 is selected from a group consisting of hydrogen, alkyl, mercaptoalkyl, alkenyl, alkynyl, aroyl, alkylcarbonyl, aminoalkylcarbonyl, cycloalkyl, aryl, arylalkyl, thiazolyl, triazolyl, imidazolyl, benzothiazolyl, and benzoimidazolyl;
each R25 is independently selected from hydrogen, halogen, cyano, hydroxyl, alkoxy, thiol, amino, nitro, alkyl, aryl, carbocyclic, or heterocyclic, and pharmaceutically acceptable salts, esters, and prodrugs thereof.
In one embodiment, R11 is hydrogen. In another, A is oxygen. For example, n may be 3 and m may be 1. In other embodiments, R24 is hydrogen or benzyl.
In certain embodiments, z is 0, 2, or 3. In others, R25 is hydroxyl or alkoxy, e.g., methoxy, ethoxy, etc. In certain embodiments, two or more R25 substituents can be linked to form a fused ring (e.g., to form a methylendioxyphenyl moiety).
Examples of compounds of the invention include:
and pharmaceutically acceptable salts, esters, and prodrugs thereof.
Other compounds of the invention include
and pharmaceutically acceptable salts, esters, and prodrugs thereof.
The invention pertains to both salt forms and acid/base forms of the compounds of the invention. For example, the invention pertains not only to the particular salt forms of compounds shown herein as salts, but also the invention includes other pharmaceutically acceptable salts, and the acid and/or base form of the compound. The invention also pertains to salt forms of compounds shown herein.
Compounds of the invention are also shown in Table 2 below.
It should be noted that in the above table and throughout the application when an atom is shown without hydrogens, but hydrogens are required or chemically necessary to form a stable compound, hydrogens should be inferred to be part of the compound.
In one embodiment, the invention does not pertain to the compounds described in WO 00/64420 and WO 96/28187. In this embodiment, the invention does not pertain to methods of using the compounds described in WO 00/64420 and WO 96/28187 for the treatment of diseases or disorders described therein. In a further embodiment, the invention pertains to methods of using the compounds described in WO 00/64420 and WO 96/28187 for methods described in this application, which are not described in WO 00/64420 and WO 96/28187. Both of WO 00/64420 and WO 96/28187 are incorporated by reference herein in their entirety.
In another embodiment, the invention pertains to methods of the invention which use and pharmaceutical compositions comprising, the compounds of Table 2A. In another embodiment, the compounds of the invention do not include the compounds of Table 2A.
It should be understood that the use of any of the compounds described herein or in the applications identified in “The Related Applications” Section is within the scope of the present invention and is intended to be encompassed by the present invention and each of the applications are expressly incorporated herein at least for these purposes, and are furthermore expressly incorporated for all other purposes.
The term “subject” includes living organisms in which amyloidosis can occur, or which are susceptible to amyloid diseases, e.g., Alzheimer's disease, Down's syndrome, CAA, dialysis-related (β2M) amyloidosis, secondary (AA) amyloidosis, primary (AL) amyloidosis, hereditary amyloidosis, diabetes, etc. Examples of subjects include humans, chickens, ducks, peking ducks, geese, monkeys, deer, cows, rabbits, sheep, goats, dogs, cats, mice, rats, and transgenic species thereof. Administration of the compositions of the present invention to a subject to be treated can be carried out using known procedures, at dosages and for periods of time effective to modulate amyloid aggregation or amyloid-induced toxicity in the subject as further described herein. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the amount of amyloid already deposited at the clinical site in the subject, the age, sex, and weight of the subject, and the ability of the therapeutic compound to modulate amyloid aggregation in the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
In certain embodiments of the invention, the subject is in need of treatment by the methods of the invention, and is selected for treatment based on this need. A subject in need of treatment is art-recognized, and includes subjects that have been identified as having a disease or disorder related to amyloid-deposition or amyloidosis, has a symptom of such a disease or disorder, or is at risk of such a disease or disorder, and would be expected, based on diagnosis, e.g., medical diagnosis, to benefit from treatment (e.g., curing, healing, preventing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease or disorder, the symptom of the disease or disorder, or the risk of the disease or disorder).
In an exemplary aspect of the invention, the subject is a human. For example, the subject may be a human over 30 years old, human over 40 years old, a human over 50 years old, a human over 60 years old, a human over 70 years old, a human over 80 years old, a human over 85 years old, a human over 90 years old, or a human over 95 years old. The subject may be a female human, including a postmenopausal female human, who may be on hormone (estrogen) replacement therapy. The subject may also be a male human. In another embodiment, the subject is under 40 years old.
A subject may be a human at risk for Alzheimer's disease, e.g., being over the age of 40 or having a predisposition for Alzheimer's disease Alzheimer's disease predisposing factors identified or proposed in the scientific literature include, among others, a genotype predisposing a subject to Alzheimer's disease; environmental factors predisposing a subject to Alzheimer's disease; past history of infection by viral and bacterial agents predisposing a subject to Alzheimer's disease; and vascular factors predisposing a subject to Alzheimer's disease. A subject may also have one or more risk factors for cardiovascular disease (e.g., atherosclerosis of the coronary arteries, angina pectoris, and myocardial infarction) or cerebrovascular disease (e.g., atherosclerosis of the intracranial or extracranial arteries, stroke, syncope, and transient ischemic attacks), such as hypercholesterolemia, hypertension, diabetes, cigarette smoking, familial or previous history of coronary artery disease, cerebrovascular disease, and cardiovascular disease. Hypercholesterolemia typically is defined as a serum total cholesterol concentration of greater than about 5.2 mmol/L (about 200 mg/dL).
Several genotypes are believed to predispose a subject to Alzheimer's disease. These include the genotypes such as presenilin-1, presenilin-2, and amyloid precursor protein (APP) missense mutations associated with familial Alzheimer's disease, and α-2-macroglobulin and LRP-1 genotypes, which are thought to increase the risk of acquiring sporadic (late-onset) Alzheimer's disease. E.van Uden, et al., J. Neurosci. 22(21), 9298-304 (2002); J. J. Goto, et al., J. Mol. Neurosci. 19(1-2), 37-41 (2002). Another genetic risk factor for the development of Alzheimer's disease are variants of ApoE, the gene that encodes apolipoprotein E (particularly the apoE4 genotype), a constituent of the low-density lipoprotein particle. W J Strittmatter, et al., Annu. Rev. Neurosci. 19, 53-77 (1996). The molecular mechanisms by which the various ApoE alleles alter the likelihood of developing Alzheimer's disease are unknown, however the role of ApoE in cholesterol metabolism is consistent with the growing body of evidence linking cholesterol metabolism to Alzheimer's disease. For example, chronic use of cholesterol-lowering drugs such as statins has recently been associated with a lower incidence of Alzheimer's disease, and cholesterol-lowering drugs have been shown to reduce pathology in APP transgenic mice. These and other studies suggest that cholesterol may affect APP processing. ApoE4 has been suggested to alter Aβ trafficking (in and out of the brain), and favor retention of Aβ in the brain. ApoE4 has also been suggested to favor APP processing toward Aβ formation. Environmental factors have been proposed as predisposing a subject to Alzheimer's disease, including exposure to aluminum, although the epidemiological evidence is ambiguous. In addition, prior infection by certain viral or bacterial agents may predispose a subject to Alzheimer's disease, including the herpes simplex virus and chlamydia pneumoniae. Finally, other predisposing factors for Alzheimer's disease can include risk factors for cardiovascular or cerebrovascular disease, including cigarette smoking, hypertension and diabetes. “At risk for Alzheimer's disease” also encompasses any other predisposing factors not listed above or as yet identified and includes an increased risk for Alzheimer's disease caused by head injury, medications, diet, or lifestyle.
The methods of the present invention can be used for one or more of the following: to prevent Alzheimer's disease, to treat Alzheimer's disease, or ameliorate symptoms of Alzheimer's disease, or to regulate production of or levels of amyloid β (Aβ) peptides. In an embodiment, the human carries one or more mutations in the genes that encode β-amyloid precursor protein, presenilin-1 or presenilin-2. In another embodiment, the human carries the Apolipoprotein ε4 gene. In another embodiment, the human has a family history of Alzheimer's Disease or a dementia illness. In another embodiment, the human has trisomy 21 (Down's Syndrome). In another embodiment, the subject has a normal or low serum total blood cholesterol level. In another embodiment, the serum total blood cholesterol level is less than about 200 mg/dL, or less than about 180, and it can range from about 150 to about 200 mg/dL. In another embodiment, the total LDL cholesterol level is less than about 100 mg/dL, or less than about 90 mg/dL and can range from about 30 to about 100 mg/dL. Methods of measuring serum total blood cholesterol and total LDL cholesterol are well known to those skilled in the art and for example include those disclosed in WO 99/38498 at p. 11, incorporated by reference herein. Methods of determining levels of other sterols in serum are disclosed in H. Gylling, et al., “Serum Sterols During Stanol Ester Feeding in a Mildly Hypercholesterolemic Population”, J. Lipid Res. 40: 593-600 (1999).
In another embodiment, the subject has an elevated serum total blood cholesterol level. In another embodiment, the serum total cholesterol level is at least about 200 mg/dL, or at least about 220 mg/dL and can range from about 200 to about 1000 mg/dL. In another embodiment, the subject has an elevated total LDL cholesterol level. In another embodiment, the total LDL cholesterol level is greater than about 100 mg/dL, or even greater than about 110 mg/dL and can range from about 100 to about 1000 mg/dL.
In another embodiment, the human is at least about 40 years of age. In another embodiment, the human is at least about 60 years of age. In another embodiment, the human is at least about 70 years of age. In another embodiment, the human is at least about 80 years of age. In another embodiment, the human is at least about 85 years of age. In one embodiment, the human is between about 60 and about 100 years of age.
In still a further embodiment, the subject is shown to be at risk by a diagnostic brain imaging technique, for example, one that measures brain activity, plaque deposition, or brain atrophy.
In still a further embodiment, the subject is shown to be at risk by a cognitive test such as Clinical Dementia Rating (“CDR”), Alzheimer's Disease Assessment Scale-Cognition (“ADAS-Cog”), Disability Assessment for Dementia (“DAD”) or Mini-Mental State Examination (“MMSE”). The subject may exhibit a below average score on a cognitive test, as compared to a historical control of similar age and educational background. The subject may also exhibit a reduction in score as compared to previous scores of the subject on the same or similar cognition tests.
In determining the CDR, a subject is typically assessed and rated in each of six cognitive and behavioural categories: memory, orientation, judgement and problem solving, community affairs, home and hobbies, and personal care. The assessment may include historical information provided by the subject, or preferably, a corroborator who knows the subject well. The subject is assessed and rated in each of these areas and the overall rating, (0, 0.5, 1.0, 2.0 or 3.0) determined. A rating of 0 is considered normal. A rating of 1.0 is considered to correspond to mild dementia. A subject with a CDR of 0.5 is characterized by mild consistent forgetfulness, partial recollection of events and “benign” forgetfulness. In one embodiment the subject is assessed with a rating on the CDR of above 0, of above about 0.5, of above about 1.0, of above about 1.5, of above about 2.0, of above about 2.5, or at about 3.0.
Another test is the Mini-Mental State Examination (MMSE), as described by Folstein “Mini-mental state. A practical method for grading the cognitive state of patients for the clinician.” J. Psychiatr. Res. 12:189-198, 1975. The MMSE evaluates the presence of global intellectual deterioration. See also Folstein “Differential diagnosis of dementia. The clinical process.” Psychiatr Clin North Am. 20:45-57, 1997. The MMSE is a means to evaluate the onset of dementia and the presence of global intellectual deterioration, as seen in Alzheimer's disease and multi-infart dementia. The MMSE is scored from 1 to 30. The MMSE does not evaluate basic cognitive potential, as, for example, the so-called IQ test. Instead, it tests intellectual skills. A person of “normal” intellectual capabilities will score a “30” on the MMSE objective test (however, a person with a MMSE score of 30 could also score well below “normal” on an IQ test). See, e.g., Kaufer, J. Neuropsychiatry Clin. Neurosci. 10:55-63, 1998; Becke, Alzheimer Dis Assoc Disord. 12:54-57, 1998; Ellis, Arch. Neurol. 55:360-365, 1998; Magni, Int. Psychogeriatr. 8:127-134, 1996; Monsch, Acta Neurol. Scand. 92:145-150, 1995. In one embodiment, the subject scores below 30 at least once on the MMSE. In another embodiment, the subject scores below about 28, below about 26, below about 24, below about 22, below about 20, below about 18, below about 16, below about 14, below about 12, below about 10, below about 8, below about 6, below about 4, below about 2, or below about 1.
The Disability Assessment for Dementia (“DAD”) scale has been developed to measure a patient's ability to perform the activities of daily living (Gélinas I et al. Development of a Functional Measure for Persons with Alzheimer's Disease: The Disability Assessment for Dementia. Am. J. Occupational Therapy. 1999; 53: 471-481). Activities of daily living may be assessed according to self care (i.e., dressing and personal hygiene) and instrumental activities (e.g., housework, cooking, and using household devices). The objectives of the DAD scale include quantitatively measuring functional abilities in activities of daily living in individuals with cognitive impairments and to help delineate areas of cognitive deficits that may impair performance in activities of daily living. The DAD is administered through an interview with the caregiver. It measures actual performance in activities of daily living of the individual as observed over a 2 week period prior to the interview. The scale assesses the following domains of activities: hygiene, dressing, telephoning, continence, eating, meal preparation, outing activities, finance and correspondence, medication use, leisure and housework. A total score is obtained by adding the rating for each question and converting this total score out of 100. Higher scores represent less disability in ADL while lower scores indicate more dysfunction. In one embodiment, the subject scores below 100 at least once on the DAD. In another embodiment, the subject scores below about 95, below about 90, below about 85, below about 80, below about 75, below about 70, below about 65, below about 60, below about 55, below about 50, below about 45, below about 40, below about 30, below about 20, or below about 10.
Another means to evaluate cognition, particularly Alzheimer's disease, is the Alzheimer's Disease Assessment Scale (ADAS-Cog), or a variation termed the Standardized Alzheimer's Disease Assessment Scale (SADAS). It is commonly used as an efficacy measure in clinical drug trials of Alzheimer's disease and related disorders characterized by cognitive decline. SADAS and ADAS-Cog were not designed to diagnose Alzheimer's disease; they are useful in characterizing symptoms of dementia and are a relatively sensitive indicator of dementia progression. (See, e.g., Doraiswamy, Neurology 48:1511-1517, 1997; and Standish, J. Am. Geriatr. Soc. 44:712-716, 1996.) Annual deterioration in untreated Alzheimer's disease patients is approximately 8 points per year (See, e.g., Raskind, M. Prim. Care Companion J Clin Psychiatry 2000 August; 2(4):134-138).
The ADAS-cog is designed to measure, with the use of questionnaires, the progression and the severity of cognitive decline as seen in AD on a 70-point scale. The ADAS-cog scale quantifies the number of wrong answers. Consequently, a high score on the scale indicates a more severe case of cognitive decline. In one embodiment, a subject exhibits a score of greater than 0, greater than about 5, greater than about 10, greater than about 15, greater than about 20, greater than about 25, greater than about 30, greater than about 35, greater than about 40, greater than about 45, greater than about 50, greater than about 55, greater than about 60, greater than about 65, greater than about 68, or about 70. In another embodiment, the subject exhibits no symptoms of Alzheimer's Disease. In another embodiment, the subject is a human who is at least 40 years of age and exhibits no symptoms of Alzheimer's Disease. In another embodiment, the subject is a human who is at least 40 years of age and exhibits one or more symptoms of Alzheimer's Disease.
In another embodiment, the subject has Mild Cognitive Impairment. In a further embodiment, the subject has a CDR rating of about 0.5. In another embodiment, the subject has early Alzheimer's disease. In another embodiment, the subject has cerebral amyloid angiopathy.
By using the methods of the present invention, the levels of amyloid β peptides in a subject's plasma or cerebrospinal fluid (CSF) can be reduced from levels prior to treatment from about 10 to about 100 percent, or even about 50 to about 100 percent.
In an alternative embodiment, the subject can have an elevated level of amyloid Aβ40 and Aβ42 peptide in the blood and CSF prior to treatment, according to the present methods, of greater than about 10 pg/mL, or greater than about 20 pg/mL, or greater than about 35 pg/mL, or even greater than about 40 pg/mL. In another embodiment, the elevated level of amyloid Aβ42 peptide can range from about 30 pg/mL to about 200 pg/mL, or even to about 500 pg/mL. One skilled in the art would understand that as Alzheimer's disease progresses, the measurable levels of amyloid β peptide in the CSF may decrease from elevated levels present before onset of the disease. This effect is attributed to increased deposition, i.e., trapping of Aβ peptide in the brain instead of normal clearance from the brain into the CSF.
In an alternative embodiment, the subject can have an elevated level of amyloid Aβ40 peptide in the blood and CSF prior to treatment, according to the present methods, of greater than about 5 pg Aβ42/mL or greater than about 50 pg Aβ40/mL, or greater than about 400 pg/mL. In another embodiment, the elevated level of amyloid Aβ40 peptide can range from about 200 pg/mL to about 800 pg/mL, to even about 1000 pg/mL.
In another embodiment, the subject can have an elevated level of amyloid Aβ42 peptide in the CSF prior to treatment, according to the present methods, of greater than about 10 pg/mL, or greater than about 10 pg/mL, or greater than about 200 pg/mL, or greater than about 500 pg/mL. In another embodiment, the level of amyloid β peptide can range from about 10 pg/mL to about 1,000 pg/mL, or even about 100 pg/mL to about 1,000 pg/mL.
In another embodiment, the subject can have an elevated level of amyloid Aβ40 peptide in the CSF prior to treatment according to the present methods of greater than about pg/mL, or greater than about 50 pg/mL, or even greater than about 100 pg/mL. In another embodiment, the level of amyloid β peptide can range from about 10 pg/mL to about 1,000 pg/mL.
The amount of amyloid β peptide in the brain, CSF, blood, or plasma of a subject can be evaluated by enzyme-linked immunosorbent assay (“ELISA”) or quantitative immunoblotting test methods or by quantitative SELDI-TOF which are well known to those skilled in the art, such as is disclosed by Zhang, et al., J. Biol. Chem. 274, 8966-72 (1999) and Zhang, et al., Biochemistry 40, 5049-55 (2001). See also, A. K. Vehmas, et al., DNA Cell Biol. 20(11), 713-21 (2001), P. Lewczuk, et al., Rapid Commun. Mass Spectrom. 17(12), 1291-96 (2003); B. M. Austen, et al., J. Peptide Sci. 6, 459-69 (2000); and H. Davies, et al., BioTechniques 27, 1258-62 (1999). These tests are performed on samples of the brain or blood which have been prepared in a manner well known to one skilled in the art. Another example of a useful method for measuring levels of amyloid β peptides is by Europium immunoassay (EIA). See, e.g., WO 99/38498 at p. 11.
The methods of the invention may be applied as a therapy for a subject having Alzheimer's disease or a dementia, or the methods of the invention may be applied as a prophylaxis against Alzheimer's disease or dementia for subject with such a predisposition, as in a subject, e.g., with a genomic mutation in the APP gene, the ApoE gene, or a presenilin gene. The subject may have (or may be predisposed to developing or may be suspected of having) vascular dementia, or senile dementia, Mild Cognitive Impairment, or early Alzheimer's disease. In addition to Alzheimer's disease, the subject may have another amyloid-related disease such as cerebral amyloid angiopathy, or the subject may have amyloid deposits, especially amyloid-β amyloid deposits in the brain.
The present invention pertains to methods of using the compounds and pharmaceutical compositions thereof in the treatment and prevention of amyloid-related diseases. The pharmaceutical compositions of the invention may be administered therapeutically or prophylactically to treat diseases associated with amyloid (e.g., AL amyloid protein (λ or κ-chain related, e.g., amyloid λ, amyloid κ, amyloid κIV, amyloid λVI, amyloid γ, amyloid γ1), Aβ, IAPP, β2M, AA, or AH amyloid protein) fibril formation, aggregation or deposition.
The pharmaceutical compositions of the invention may act to ameliorate the course of an amyloid-related disease using any of the following mechanisms (this list is meant to be illustrative and not limiting): slowing the rate of amyloid fibril formation or deposition; lessening the degree of amyloid deposition; inhibiting, reducing, or preventing amyloid fibril formation; inhibiting neurodegeneration or cellular toxicity induced by amyloid; inhibiting amyloid induced inflammation; enhancing the clearance of amyloid from the brain; enhancing degradation of Aβ in the brain; or favoring clearance of amyloid protein prior to its organization in fibrils.
“Modulation” of amyloid deposition includes both inhibition, as defined above, and enhancement of amyloid deposition or fibril formation. The term “modulating” is intended, therefore, to encompass prevention or stopping of amyloid formation or accumulation, inhibition or slowing down of further amyloid formation or accumulation in a subject with ongoing amyloidosis, e.g., already having amyloid deposition, and reducing or reversing of amyloid formation or accumulation in a subject with ongoing amyloidosis; and enhancing amyloid deposition, e.g., increasing the rate or amount of amyloid deposition in vivo or in vitro. Amyloid-enhancing compounds may be useful in animal models of amyloidosis, for example, to make possible the development of amyloid deposits in animals in a shorter period of time or to increase amyloid deposits over a selected period of time. Amyloid-enhancing compounds may be useful in screening assays for compounds which inhibit amyloidosis in vivo, for example, in animal models, cellular assays and in vitro assays for amyloidosis. Such compounds may be used, for example, to provide faster or more sensitive assays for compounds. Modulation of amyloid deposition is determined relative to an untreated subject or relative to the treated subject prior to treatment.
“Inhibition” of amyloid deposition includes preventing or stopping of amyloid formation, e.g., fibrillogenesis, clearance of amyloid, e.g., soluble Aβ from brain, inhibiting or slowing down of further amyloid deposition in a subject with amyloidosis, e.g., already having amyloid deposits, and reducing or reversing amyloid fibrillogenesis or deposits in a subject with ongoing amyloidosis. Inhibition of amyloid deposition is determined relative to an untreated subject, or relative to the treated subject prior to treatment, or, e.g., determined by clinically measurable improvement, e.g., or in the case of a subject with brain amyloidosis, e.g., an Alzheimer's or cerebral amyloid angiopathy subject, stabilization of cognitive function or prevention of a further decrease in cognitive function (i.e., preventing, slowing, or stopping disease progression), or improvement of parameters such as the concentration of Aβ or tau in the CSF.
As used herein, “treatment” of a subject includes the application or administration of a composition of the invention to a subject, or application or administration of a composition of the invention to a cell or tissue from a subject, who has an amyloid-related disease or condition, has a symptom of such a disease or condition, or is at risk of (or susceptible to) such a disease or condition, with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term “treating” refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a subject's physical or mental well-being; or, in some situations, preventing the onset of dementia. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, a psychiatric evaluation, or a cognition test such as CDR, MMSE, DAD, ADAS-Cog, or another test known in the art. For example, the methods of the invention successfully treat a subject's dementia by slowing the rate of or lessening the extent of cognitive decline.
In one embodiment, the term “treating” includes maintaining a subject's CDR rating at its base line rating or at 0. In another embodiment, the term treating includes decreasing a subject's CDR rating by about 0.25 or more, about 0.5 or more, about 1.0 or more, about 1.5 or more, about 2.0 or more, about 2.5 or more, or about 3.0 or more. In another embodiment, the term “treating” also includes reducing the rate of the increase of a subject's CDR rating as compared to historical controls. In another embodiment, the term includes reducing the rate of increase of a subject's CDR rating by about 5% or more, about 10% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100%, of the increase of the historical or untreated controls.
In another embodiment, the term “treating” also includes maintaining a subject's score on the MMSE. The term “treating” includes increasing a subject's MMSE score by about 1, about 2, about 3, about 4, about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, or about 25 points. The term also includes reducing the rate of the decrease of a subject's MMSE score as compared to historical controls. In another embodiment, the term includes reducing the rate of decrease of a subject's MMSE score by about 5% or less, about 10% or less, about 20% or less, about 25% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less or about 100% or less, of the decrease of the historical or untreated controls.
In another embodiment, the term “treating” also includes maintaining a subject's score on the DAD. The term “treating” includes increasing a subject's DAD score by about 1, about 5, about 10, about 15, about 20, about 30, about 35, about 40, about 50, about 60, about 70, or about 80 points. The term also includes reducing the rate of the decrease of a subject's DAD score as compared to historical controls. In another embodiment, the term includes reducing the rate of decrease of a subject's DAD score by about 5% or less, about 10% or less, about 20% or less, about 25% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less or about 100% or less, of the decrease of the historical or untreated controls.
In yet another embodiment, the term “treating” includes maintaining a subject's score on the ADAS-Cog. The term “treating” includes decreasing a subject's ADAS-Cog score by about 1 point or greater, by about 2 points or greater, by about 3 points or greater, by about 4 points or greater, by about 5 points or greater, by about 7.5 points or greater, by about 10 points or greater, by about 12.5 points or greater, by about 15 points or greater, by about 17.5 points or greater, by about 20 points or greater, or by about 25 points or greater. The term also includes reducing the rate of the increase of a subject's ADAS-Cog score as compared to historical controls. In another embodiment, the term includes reducing the rate of increase of a subject's ADAS-Cog score by about 5% or more, about 10% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more or about 100% of the increase of the historical or untreated controls.
In another embodiment, the term “treating” e.g., for AA or AL amyloidosis, includes an increase in serum creatinine, e.g., an increase of creatinine clearance of 10% or greater, 20% or greater, 50% or greater, 80% or greater, 90% or greater, 100% or greater, 150% or greater, 200% or greater. The term “treating” also may induce remission of nephrotic syndrome (NS). It may also include remission of chronic diarrhea and/or a gain in body weight, e.g., by 10% or greater, 15% or greater, or 20% or greater.
Without wishing to be bound by theory, in some aspects the pharmaceutical compositions of the invention contain a compound that prevents or inhibits amyloid fibril formation, either in the brain or other organ of interest (acting locally) or throughout the entire body (acting systemically). Pharmaceutical compositions of the invention may be effective in controlling amyloid deposition either following their entry into the brain (following penetration of the blood brain bather) or from the periphery. When acting from the periphery, a compound of a pharmaceutical composition may alter the equilibrium of amyloidogenic peptide between the brain and the plasma so as to favor the exit of amyloidogenic peptide from the brain. It may also favor clearance (or catabolism) of the amyloid protein (soluble), and then prevent amyloid fibril formation and deposition due to a reduction of the amyloid protein pool in a specific organ, e.g., liver, spleen, pancreas, kidney, joints, brain, etc. An increase in the exit of amyloidogenic peptide from the brain would result in a decrease in amyloidogenic peptide brain concentration and therefore favor a decrease in amyloidogenic peptide deposition. In particular, an agent may lower the levels of amyloid β peptides, e.g., both Aβ40 and Aβ42 in the CSF and the plasma, or the agent may lower the levels of amyloid β peptides, e.g., Aβ 40 and Aβ42 in the CSF and increase it in the plasma. Alternatively, compounds that penetrate the brain could control deposition by acting directly on brain amyloidogenic peptide e.g., by maintaining it in a non-fibrillar form or favoring its clearance from the brain, by increasing its degradation in the brain, or protecting brain cells from the detrimental effect of amyloidogenic peptide. An agent can also cause a decrease of the concentration of the amyloid protein (i.e., in a specific organ so that the critical concentration needed to trigger amyloid fibril formation or deposition is not reached). Furthermore, the compounds described herein may inhibit or reduce an interaction between amyloid and a cell surface constituent, for example, a glycosaminoglycan or proteoglycan constituent of a basement membrane, whereby inhibiting or reducing this interaction produces the observed neuroprotective and cell-protective effects. For example, the compound may also prevent an amyloid peptide from binding or adhering to a cell surface, a process which is known to cause cell damage or toxicity. Similarly, the compound may block amyloid-induced cellular toxicity or microglial activation or amyloid-induced neurotoxicity, or inhibit amyloid induced inflammation. The compound may also reduce the rate or amount of amyloid aggregation, fibril formation, or deposition, or the compound lessens the degree of amyloid deposition. The foregoing mechanisms of action should not be construed as limiting the scope of the invention inasmuch as the invention may be practiced without such information.
The Aβ peptide has been shown by several groups to be highly toxic to neurons. Amyloid plaques are directly associated with reactive gliosis, dystrophic neurites and apoptotic cells, suggesting that plaques induce neurodegenerative changes. Neurotoxicity may eventually disrupt or even kill neurons. In vitro, Aβ has been shown to induce apoptosis in many different neuronal cell types, such as rat PC-12 cells, primary rat hippocampal and cortical cultures, and the predifferentiated human neurotype SH-SY5Y cell line (Dickson D W (2004) J Clin Invest 114:23-7; Canu et al. (2003) Cerebellum 2:270-278; Li et al. (1996) Brain Research 738:196-204). Numerous reports have shown that Aβ fibrils can induce neurodegeneration, and it has been shown that neuronal cells exposed in vitro to Aβ can become apoptotic (Morgan et al. (2004) Prog. Neurobiol. 74:323-349; Stefani et al. (2003) J. Mol. Med. 81:678-99; La Ferla et al. (1997) J. Clin. Invest. 100(2):310-320). In Alzheimer's disease, a progressive neuronal cell loss accompanies the deposition of Aβ amyloid fibrils in senile plaques.
In yet another aspect, the invention pertains to a method for inhibiting Aβ-induced neuronal cell death by administering an effective amount of a compound of the present invention.
Another aspect of the invention pertains to a method for providing neuroprotection to a subject having an Aβ-amyloid related disease, e.g., Alzheimer's disease, that includes administering an effective amount of a compound of the present invention to the subject, such that neuroprotection is provided.
In another aspect, methods for inhibiting Aβ-induced neuronal cell death are provided that include administration of an effective amount of a compound of the present invention to a subject such that neuronal cell death is inhibited.
In another aspect, methods for treating a disease state characterized by Aβ-induced neuronal cell death in a subject are provided, e.g., by administering an effective amount of a compound of the present invention. Non-limiting examples of such disease states include Alzheimer's disease and Aβ-amyloid related diseases.
The term “neuroprotection” includes protection of neuronal cells of a subject from Aβ-induced cell death, e.g., cell death induced directly or indirectly by an Aβ peptide. Aβ-induced cell death may result in initiation of processes such as, for example: the destabilization of the cytoskeleton; DNA fragmentation; the activation of hydrolytic enzymes, such as phospholipase A2; activation of caspases, calcium-activated proteases and/or calcium-activated endonucleases; inflammation mediated by macrophages; calcium influx into a cell; membrane potential changes in a cell; the disruption of cell junctions leading to decreased or absent cell-cell communication; and the activation of expression of genes involved in cell death, e.g., immediate-early genes.
The term “amyloid-β disease” (or “amyloidβ related disease,” which terms as used herein are synonymous) may be used for mild cognitive impairment; vascular dementia; early Alzheimer's disease; Alzheimer's disease, including sporadic (non-hereditary) Alzheimer's disease and familial (hereditary) Alzheimer's disease; age-related cognitive decline; cerebral amyloid angiopathy (“CAA”); hereditary cerebral hemorrhage; senile dementia; Down's syndrome; inclusion body myositis (“IBM”); or age-related macular degeneration (“ARMD”). According to certain aspects of the invention, amyloid-β is a peptide having 39-43 amino-acids, or amyloid-β is an amyloidogenic peptide produced from βAPP.
Mild cognitive impairment (“MCI”) is a condition characterized by a state of mild but measurable impairment in thinking skills, which is not necessarily associated with the presence of dementia. MCI frequently, but not necessarily, precedes Alzheimer's disease. It is a diagnosis that has most often been associated with mild memory problems, but it can also be characterized by mild impairments in other thinking skills, such as language or planning skills. However, in general, an individual with MCI will have more significant memory lapses than would be expected for someone of their age or educational background. As the condition progresses, a physician may change the diagnosis to “Mild-to-Moderate Cognitive Impairment,” as is well understood in this art.
Cerebral amyloid angiopathy (“CAA”) refers to the specific deposition of amyloid fibrils in the walls of leptomingeal and cortical arteries, arterioles and in capillaries and veins. It is commonly associated with Alzheimer's disease, Down's syndrome and normal aging, as well as with a variety of familial conditions related to stroke or dementia (see Frangione, et al., Amyloid: J. Protein Folding Disord. 8, Suppl. 1, 36-42 (2001)). CAA can occur sporadically or be hereditary. Multiple mutation sites in either Aβ or the APP gene have been identified and are clinically associated with either dementia or cerebral hemorrhage. Exemplary CAA disorders include, but are not limited to, hereditary cerebral hemorrhage with amyloidosis of Icelandic type (HCHWA-I); the Dutch variant of HCHWA (HCHWA-D; a mutation in Aβ); the Flemish mutation of Aβ; the Arctic mutation of Aβ; the Italian mutation of Aβ; the Iowa mutation of Aβ; familial British dementia; and familial Danish dementia. Cerebral amyloid angiopathy is known to be associated with cerebral hemorrhage (or hemorrhagic stroke).
Additionally, abnormal accumulation of APP and of amyloid-β protein in muscle fibers has been implicated in the pathology of sporadic inclusion body myositis (“IBM”) (Askanas, et al., Proc. Natl. Acad. Sci. USA 93, 1314-19 (1996); Askanas, et al., Current Opinion in Rheumatology 7, 486-96 (1995)). Accordingly, the compounds of the invention can be used prophylactically or therapeutically in the treatment of disorders in which amyloid-β protein is abnormally deposited at non-neurological locations, such as treatment of IBM by delivery of the compounds to muscle fibers.
Additionally, it has been shown that Aβ is associated with abnormal extracellular deposits, known as drusen, that accumulate along the basal surface of the retinal pigmented epithelium in individuals with age-related macular degeneration (ARMD). ARMD is a cause of irreversible vision loss in older individuals. It is believed that Aβ deposition could be an important component of the local inflammatory events that contribute to atrophy of the retinal pigmented epithelium, drusen biogenesis, and the pathogenesis of ARMD (Johnson, et al., Proc. Natl. Acad. Sci. USA 99(18), 11830-5 (2002)). Therefore, the invention also relates to the treatment or prevention of age-related macular degeneration.
Also, the invention relates to a method for preventing or inhibiting amyloid deposition in a subject. For example, such a method comprises administering to a subject a therapeutically effective amount of a compound capable of reducing the concentration of amyloid (e.g., AL amyloid protein (λ or κ-chain related, e.g., amyloid λ, amyloid κ, amyloid κIV, amyloid λVI, amyloid γ, amyloid γ1), Aβ, IAPP, β2m, AA, AH amyloid protein, or other amyloids), such that amyloid accumulation or deposition is prevented or inhibited.
In another aspect, the invention relates to a method for preventing, reducing, or inhibiting amyloid deposition in a subject. For example, such a method comprises administering to a subject a therapeutically effective amount of a compound capable of inhibiting amyloid (e.g., AL amyloid protein (λ or κ-chain related, e.g., amyloid λ, amyloid κ, amyloid κIV, amyloid λVI, amyloid γ, amyloid γ1), Aβ, IAPP, β2m, AA, AH amyloid protein, or other amyloids), such that amyloid deposition is prevented, reduced, or inhibited.
The invention also relates to a method for modulating, e.g., minimizing, amyloid-associated damage to cells, comprising the step of administering a compound capable of reducing the concentration of amyloid (e.g., AL amyloid protein (λ or κ-chain related, e.g., amyloid λ, amyloid κ, amyloid κIV, amyloid λVI, amyloid γ, amyloid γ1), Aβ, IAPP, β2m, AA, AH amyloid protein, or another amyloid), such that said amyloid-associated damage to cells is modulated. In certain aspects of the invention, the methods for modulating amyloid-associated damage to cells comprise a step of administering a compound capable of reducing the concentration of amyloid or reducing the interaction of an amyloid with a cell surface.
The invention also includes a method for directly or indirectly preventing cell death in a subject, the method comprising administering to a subject a therapeutically effective amount of a compound capable of preventing amyloid (e.g., AL amyloid protein (λ or κ-chain related, e.g., amyloid λ, amyloid κ, amyloid κIV, amyloid λVI, amyloid γ, amyloid γ1), Aβ, IAPP, β2m, AA, AH amyloid protein, or other amyloid) mediated events that lead, directly or indirectly, to cell death.
In an embodiment, the method is used to treat Alzheimer's disease (e.g. sporadic or familial AD). The method can also be used prophylactically or therapeutically to treat other clinical occurrences of amyloid-β deposition, such as in Down's syndrome individuals and in patients with cerebral amyloid angiopathy (“CAA”) or hereditary cerebral hemorrhage.
The compounds of the invention may be used prophylactically or therapeutically in the treatment of disorders in which amyloid-beta peptide is abnormally deposited at non-neurological locations, such as treatment of IBM by delivery of the compounds to muscle fibers, or treatment of macular degeneration by delivery of the compound(s) of the invention to the basal surface of the retinal pigmented epithelium.
The present invention also provides a method for modulating amyloid-associated damage to cells, comprising the step of administering a compound capable of reducing the concentration of Aβ, or capable of minimizing the interaction of Aβ (soluble oligomeric or fibrillary) with the cell surface, such that said amyloid-associated damage to cells is modulated. In certain aspects of the invention, the methods for modulating amyloid-associated damage to cells comprise a step of administering a compound capable of reducing the concentration of Aβ or reducing the interaction of Aβ with a cell surface.
In accordance with the present invention, there is further provided a method for preventing cell death in a subject, said method comprising administering to a subject a therapeutically effective amount of a compound capable of preventing Aβ-mediated events that lead, directly or indirectly, to cell death.
The present invention also provides a method for modulating amyloid-associated damage to cells, comprising the step of administering a compound capable of reducing the concentration of IAPP, or capable of minimizing the interaction of IAPP (soluble oligomeric or fibrillary) with the cell surface, such that said amyloid-associated damage to cells is modulated. In certain aspects of the invention, the methods for modulating amyloid-associated damage to cells comprise a step of administering a compound capable of reducing the concentration of IAPP or reducing the interaction of IAPP with a cell surface.
In accordance with the present invention, there is further provided a method for preventing cell death in a subject, said method comprising administering to a subject a therapeutically effective amount of a compound capable of preventing IAPP-mediated events that lead, directly or indirectly, to cell death.
This invention also provides methods and compositions which are useful in the treatment of amyloidosis. The methods of the invention involve administering to a subject a therapeutic compound which inhibits amyloid deposition. Accordingly, the compositions and methods of the invention are useful for inhibiting amyloidosis in disorders in which amyloid deposition occurs. The methods of the invention can be used therapeutically to treat amyloidosis or can be used prophylactically in a subject susceptible to (hereditary) amyloidosis or identified as being at risk to develop amyloidosis, e.g., hereditary, or identified as being at risk to develop amyloidosis. In certain embodiments, the invention includes a method of inhibiting an interaction between an amyloidogenic protein and a constituent of basement membrane to inhibit amyloid deposition. The constituent of basement membrane is a glycoprotein or proteoglycan, preferably heparan sulfate proteoglycan. A therapeutic compound used in this method may interfere with binding of a basement membrane constituent to a target binding site on an amyloidogenic protein, thereby inhibiting amyloid deposition.
In some aspects, the methods of the invention involve administering to a subject a therapeutic compound which inhibits amyloid deposition “Inhibition of amyloid deposition,” includes the prevention of amyloid formation, inhibition of further amyloid deposition in a subject with ongoing amyloidosis and reduction of amyloid deposits in a subject with ongoing amyloidosis Inhibition of amyloid deposition is determined relative to an untreated subject or relative to the treated subject prior to treatment. In an embodiment, amyloid deposition is inhibited by inhibiting an interaction between an amyloidogenic protein and a constituent of basement membrane. “Basement membrane” refers to an extracellular matrix comprising glycoproteins and proteoglycans, including laminin, collagen type IV, fibronectin, perlecan, agrin, dermatan sulfate, and heparan sulfate proteoglycan (HSPG). In one embodiment, amyloid deposition is inhibited by interfering with an interaction between an amyloidogenic protein and a sulfated glycosaminoglycan such as HSPG, dermatan sulfate, perlecan or agrin sulfate. Sulfated glycosaminoglycans are known to be present in all types of amyloids (see Snow, et al. Lab. Invest. 56, 120-23 (1987)) and amyloid deposition and HSPG deposition occur coincidentally in animal models of amyloidosis (see Snow, et al. Lab. Invest. 56, 665-75 (1987) and Gervais, F. et al. Curr. Med. Chem., 3, 361-370 (2003)). Consensus binding site motifs for HSPG in amyloidogenic proteins have been described (see, e.g., Cardin and Weintraub Arteriosclerosis 9, 21-32 (1989)).
The ability of a compound to prevent or block the formation or deposition of amyloid may reside in its ability to bind to non-fibrillar, soluble amyloid protein and to maintain its solubility.
The ability of a therapeutic compound of the invention to inhibit an interaction between an amyloidogenic protein and a glycoprotein or proteoglycan constituent of a basement membrane can be assessed by an in vitro binding assay, such as that described in U.S. Pat. No. 5,164,295, the contents of which are hereby incorporated by reference. Alternatively, the ability of a compound to bind to an amyloidogenic protein or to inhibit the binding of a basement membrane constituent (e.g., HSPG) to an amyloidogenic protein (e.g., Aβ) can be measured using a mass spectrometry assay where soluble protein, e.g., Aβ, IAPP, β2M is incubated with the compound. A compound which binds to, e.g., Aβ, will induce a change in the mass spectrum of the protein. Exemplary protocols for a mass spectrometry assay employing Aβ and IAPP can be found in the Examples, the results of which are provided in Table 3. The protocol can readily be modified to adjust the sensitivity of the data, e.g., by adjusting the amount of protein and/or compound employed. Thus, e.g., binding might be detected for test compounds noted as not having detectable binding employing less sensitive test protocols.
Alternative methods for screening compounds exist and can readily be employed by a skilled practitioner to provide an indication of the ability of test compounds to bind to, e.g., fibrillar Aβ. One such screening assay is an ultraviolet absorption assay. In an exemplary protocol, a test compound (20 μM) is incubated with 50 μM Aβ(1-40) fibers for 1 hour at 37° C. in Tris buffered saline (20 mM Tris, 150 mM NaCl, pH 7.4 containing 0.01 sodium azide). Following incubation, the solution is centrifuged for 20 minutes at 21,000 g to sediment the Aβ(1-40) fibers along with any bound test compound. The amount of test compound remaining in the supernatant can then be determined by reading the absorbance. The fraction of test compound bound can then be calculated by comparing the amount remaining in the supernatants of incubations with Aβ to the amount remaining in control incubations which do not contain Aβ fibers. Thioflavin T and Congo Red, both of which are known to bind to Aβ fibers, may be included in each assay run as positive controls. Before assaying, test compounds can be diluted to 40 μM, which would be twice the concentration in the final test, and then scanned using the Hewlett Packard 8453 UV/VIS spectrophotometer to determine if the absorbance is sufficient for detection.
In another embodiment, the invention pertains to a method for improving cognition in a subject suffering from an amyloid-related disease. The method includes administering an effective amount of a therapeutic compound of the invention, such that the subject's cognition is improved. The subject's cognition can be tested using methods known in the art such as the Clinical Dementia Rating (“CDR”), Mini-Mental State Examination (“MMSE”), Disability Assessment for Dementia (“DAD”), and/or the Alzheimer's Disease Assessment Scale-Cognition (“ADAS-Cog”).
In another embodiment, the invention pertains to a method for treating a subject for an amyloid-related disease. The method includes administering a cognitive test to a subject prior to administration of a compound of the invention, administering an effective amount of a compound of the invention to the subject, and administering a cognitive test to the subject subsequent to administration of the compound, such that the subject is treated for the amyloid-related disease, wherein the subject's score on said cognitive test is improved.
“Improvement,” “improved” or “improving” in cognition is present within the context of the present invention if there is a statistically significant difference in the direction of normality between the performance of subjects treated using the methods of the invention as compared to members of a placebo group, historical control, or between subsequent tests given to the same subject.
In one embodiment, a subject's CDR is maintained at 0. In another embodiment, a subject's CDR is decreased (e.g., improved) by about 0.25 or more, about 0.5 or more, about 1.0 or more, about 1.5 or more, about 2.0 or more, about 2.5 or more, or about 3.0 or more. In another embodiment, the rate of increase of a subject's CDR rating is reduced by about 5% or more, about 10% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more of the increase of the historical or untreated controls.
In one embodiment, a subject's score on the MMSE is maintained. Alternatively, the subject's score on the MMSE may be increased by about 1, about 2, about 3, about 4, about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, or about 25 points. In another alternative, the rate of the decrease of a subject's MMSE score as compared to historical controls is reduced. For example, the rate of the decrease of a subject's MMSE score may be reduced by about 5% or more, about 10% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more of the decrease of the historical or untreated controls.
In one embodiment, a subject's score on the DAD is maintained. Alternatively, the subject's score on the DAD may be increased by about 1, about 2, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 30, about 40, or about 50 or more points. In another alternative, the rate of the decrease of a subject's DAD score as compared to historical controls is reduced. For example, the rate of the decrease of a subject's DAD score may be reduced by about 5% or more, about 10% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more of the decrease of the historical or untreated controls.
In one embodiment, the invention pertains to a method for treating, slowing or stopping an amyloid-related disease associated with cognitive impairment, by administering to a subject an effective amount of a therapeutic compound of the invention, wherein the annual deterioration of the subject's cognition as measured by ADAS-Cog is less than 8 points per year, less the 6 points per year, less than 5 points per year, less than 4 points per year, or less than 3 points per year. In a further embodiment, the invention pertains to a method for treating, slowing or stopping an amyloid-related disease associated with cognition by administering an effective amount of a therapeutic compound of the invention such that the subject's cognition as measured by ADAS-Cog remains constant over a year. “Constant” includes fluctuations of no more than 2 points. Remaining constant includes fluctuations of two points or less in either direction. In a further embodiment, the subject's cognition improves by 2 points or greater per year, 3 points or greater per year, 4 point or greater per year, 5 points or greater per year, 6 points or greater per year, 7 points or greater per year, 8 points or greater per year, etc. as measured by the ADAS-Cog. In another alternative, the rate of the increase of a subject's ADAS-Cog score as compared to historical controls is reduced. For example, the rate of the increase of a subject's ADAS-Cog score may be reduced by about 5% or more, about 10% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more or about 100% of the increase of the historical or untreated controls.
In another embodiment, the ratio of Aβ42:Aβ40 in the CSF or plasma of a subject decreases by about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more. In another embodiment, the levels of Aβ in the subject's cerebrospinal fluid decrease by about 15% or more, about 25% or more, about 35% or more, about 45% or more, about 55% or more, about 75% or more, or about 90% or more.
It is to be understood that wherever values and ranges are provided herein, e.g., in ages of subject populations, dosages, and blood levels, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values in these values and ranges may also be the upper or lower limits of a range.
Furthermore, the invention pertains to any novel chemical compound described herein. That is, the invention relates to novel compounds, and novel methods of their use as described herein, which are within the scope of the Formulae disclosed herein, and which are not disclosed in the cited Patents and patent applications.
In general, the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here. Functional and structural equivalents of the compounds described herein and which have the same general properties, wherein one or more simple variations of substituents are made which do not adversely affect the essential nature or the utility of the compound are also included.
The compounds of the present invention may be readily prepared in accordance with the synthesis schemes and protocols described herein, as illustrated in the specific procedures provided. However, those skilled in the art will recognize that other synthetic pathways for forming the compounds of this invention may be used, and that the following is provided merely by way of example, and is not limiting to the present invention. See, e.g., “Comprehensive Organic Transformations” by R. Larock, VCH Publishers (1989). It will be further recognized that various protecting and deprotecting strategies will be employed that are standard in the art (See, e.g., “Protective Groups in Organic Synthesis” by Greene and Wuts). Those skilled in the relevant arts will recognize that the selection of any particular protecting group (e.g., amine and carboxyl protecting groups) will depend on the stability of the protected moiety with regards to the subsequent reaction conditions and will understand the appropriate selections.
Further illustrating the knowledge of those skilled in the art is the following sampling of the extensive chemical literature: “Chemistry of the Amino Acids” by J.P. Greenstein and M. Winitz, John Wiley & Sons, Inc., New York (1961); “Comprehensive Organic Transformations” by R. Larock, VCH Publishers (1989); T. D. Ocain, et al., J. Med. Chem. 31, 2193-99 (1988); E. M. Gordon, et al., J. Med. Chem. 31, 2199-10 (1988); “Practice of Peptide Synthesis” by M. Bodansky and A. Bodanszky, Springer-Verlag, New York (1984); “Protective Groups in Organic Synthesis” by T. Greene and P. Wuts (1991); “Asymmetric Synthesis: Construction of Chiral Molecules Using Amino Acids” by G. M. Coppola and H. F. Schuster, John Wiley & Sons, Inc., New York (1987); “The Chemical Synthesis of Peptides” by J. Jones, Oxford University Press, New York (1991); and “Introduction of Peptide Chemistry” by P. D. Bailey, John Wiley & Sons, Inc., New York (1992).
The synthesis of compounds of the invention is carried out in a solvent. Suitable solvents are liquids at ambient room temperature and pressure or remain in the liquid state under the temperature and pressure conditions used in the reaction. Useful solvents are not particularly restricted provided that they do not interfere with the reaction itself (that is, they preferably are inert solvents), and they dissolve a certain amount of the reactants. Depending on the circumstances, solvents may be distilled or degassed. Solvents may be, for example, aliphatic hydrocarbons (e.g., hexanes, heptanes, ligroin, petroleum ether, cyclohexane, or methylcyclohexane) and halogenated hydrocarbons (e.g., methylenechloride, chloroform, carbontetrachloride, dichloroethane, chlorobenzene, or dichlororbenzene); aromatic hydrocarbons (e.g., benzene, toluene, tetrahydronaphthalene, ethylbenzene, or xylene); ethers (e.g., diglyme, methyl-tert-butyl ether, methyl-tert-amyl ether, ethyl-tert-butyl ether, diethylether, diisopropylether, tetrahydrofuran or methyltetrahydrofurans, dioxane, dimethoxyethane, or diethyleneglycol dimethylether); nitriles (e.g., acetonitrile); ketones (e.g., acetone); esters (e.g., methyl acetate or ethyl acetate); and mixtures thereof.
In general, after completion of the reaction, the product is isolated from the reaction mixture according to standard techniques. For example, the solvent is removed by evaporation or filtration if the product is solid, optionally under reduced pressure. After the completion of the reaction, water may be added to the residue to make the aqueous layer acidic or basic and the precipitated compound filtered, although care should be exercised when handling water-sensitive compounds. Similarly, water may be added to the reaction mixture with a hydrophobic solvent to extract the target compound. The organic layer may be washed with water, dried over anhydrous magnesium sulphate or sodium sulphate, and the solvent is evaporated to obtain the target compound. The target compound thus obtained may be purified, if necessary, e.g., by recrystallization, reprecipitation, chromatography, or by converting it to a salt by addition of an acid or base.
The compounds of the invention may be supplied in a solution with an appropriate solvent or in a solvent-free form (e.g., lyophilized). In another aspect of the invention, the compounds and buffers necessary for carrying out the methods of the invention may be packaged as a kit, optionally including a container. The kit may be commercially used for treating or preventing amyloid-related disease according to the methods described herein and may include instructions for use in a method of the invention. Additional kit components may include acids, bases, buffering agents, inorganic salts, solvents, antioxidants, preservatives, or metal chelators. The additional kit components are present as pure compositions, or as aqueous or organic solutions that incorporate one or more additional kit components. Any or all of the kit components optionally further comprise buffers.
The term “container” includes any receptacle for holding the therapeutic compound. For example, in one embodiment, the container is the packaging that contains the compound. In other embodiments, the container is not the packaging that contains the compound, i.e., the container is a receptacle, such as a box or vial that contains the packaged compound or unpackaged compound and the instructions for use of the compound. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the therapeutic compound may be contained on the packaging containing the therapeutic compound, and as such the instructions form an increased functional relationship to the packaged product.
In another embodiment, the present invention relates to pharmaceutical compositions comprising agents according to any of the Formulae herein for the treatment of an amyloid-related disease, as well as methods of manufacturing such pharmaceutical compositions.
In general, the agents of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, in the patents and patent applications referred to herein, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here. Functional and structural equivalents of the agents described herein and which have the same general properties, wherein one or more simple variations of substituents are made which do not adversely affect the essential nature or the utility of the agent are also included.
The agents of the invention may be supplied in a solution with an appropriate solvent or in a solvent-free form (e.g., lyophilized). In another aspect of the invention, the agents and buffers necessary for carrying out the methods of the invention may be packaged as a kit. The kit may be commercially used according to the methods described herein and may include instructions for use in a method of the invention. Additional kit components may include acids, bases, buffering agents, inorganic salts, solvents, antioxidants, preservatives, or metal chelators. The additional kit components are present as pure compositions, or as aqueous or organic solutions that incorporate one or more additional kit components. Any or all of the kit components optionally further comprise buffers.
The therapeutic agent may also be administered parenterally, intraperitoneally, intraspinally, or intracerebrally. Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
To administer the therapeutic agent by other than parenteral administration, it may be necessary to coat the agent with, or co-administer the agent with, a material to prevent its inactivation. For example, the therapeutic agent may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., J. Neuroimmunol. 7, 27 (1984)).
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
Suitable pharmaceutically acceptable vehicles include, without limitation, any non-immunogenic pharmaceutical adjuvants suitable for oral, parenteral, nasal, mucosal, transdermal, intravascular (IV), intraarterial (IA), intramuscular (IM), and subcutaneous (SC) administration routes, such as phosphate buffer saline (PBS).
The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents are included, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
Sterile injectable solutions can be prepared by incorporating the therapeutic agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the therapeutic agent into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (i.e., the therapeutic agent) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The therapeutic agent can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The therapeutic agent and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the therapeutic agent may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the therapeutic agent in the compositions and preparations may, of course, be varied. The amount of the therapeutic agent in such therapeutically useful compositions is such that a suitable dosage will be obtained.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic agent for the treatment of amyloid deposition in subjects.
The present invention therefore includes pharmaceutical formulations comprising the agents of the Formulae described herein, including pharmaceutically acceptable salts thereof, in pharmaceutically acceptable vehicles for aerosol, oral and parenteral administration. Also, the present invention includes such agents, or salts thereof, which have been lyophilized and which may be reconstituted to form pharmaceutically acceptable formulations for administration, as by intravenous, intramuscular, or subcutaneous injection. Administration may also be intradermal or transdermal.
In accordance with the present invention, an agent of the Formulae described herein, and pharmaceutically acceptable salts thereof, may be administered orally or through inhalation as a solid, or may be administered intramuscularly or intravenously as a solution, suspension or emulsion. Alternatively, the agents or salts may also be administered by inhalation, intravenously or intramuscularly as a liposomal suspension.
Pharmaceutical formulations are also provided which are suitable for administration as an aerosol, by inhalation. These formulations comprise a solution or suspension of the desired agent of any Formula herein, or a salt thereof, or a plurality of solid particles of the agent or salt. The desired formulation may be placed in a small chamber and nebulized. Nebulization may be accomplished by compressed air or by ultrasonic energy to form a plurality of liquid droplets or solid particles comprising the agents or salts. The liquid droplets or solid particles should have a particle size in the range of about 0.5 to about 5 microns. The solid particles can be obtained by processing the solid agent of any Formula described herein, or a salt thereof, in any appropriate manner known in the art, such as by micronization. The size of the solid particles or droplets will be, for example, from about 1 to about 2 microns. In this respect, commercial nebulizers are available to achieve this purpose.
A pharmaceutical formulation suitable for administration as an aerosol may be in the form of a liquid, the formulation will comprise a water-soluble agent of any Formula described herein, or a salt thereof, in a carrier which comprises water. A surfactant may be present which lowers the surface tension of the formulation sufficiently to result in the formation of droplets within the desired size range when subjected to nebulization.
Peroral compositions also include liquid solutions, emulsions, suspensions, and the like. The pharmaceutically acceptable vehicles suitable for preparation of such compositions are well known in the art. Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, tragacanth, and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate. Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.
Pharmaceutical compositions may also be coated by conventional methods, typically with pH or time-dependent coatings, such that the subject agent is released in the gastrointestinal tract in the vicinity of the desired topical application, or at various times to extend the desired action. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, waxes, and shellac.
Other compositions useful for attaining systemic delivery of the subject agents include sublingual, buccal and nasal dosage forms. Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included.
The compositions of this invention can also be administered topically to a subject, e.g., by the direct laying on or spreading of the composition on the epidermal or epithelial tissue of the subject, or transdermally via a “patch”. Such compositions include, for example, lotions, creams, solutions, gels and solids. These topical compositions may comprise an effective amount, usually at least about 0.1%, or even from about 1% to about 5%, of an agent of the invention. Suitable carriers for topical administration typically remain in place on the skin as a continuous film, and resist being removed by perspiration or immersion in water. Generally, the carrier is organic in nature and capable of having dispersed or dissolved therein the therapeutic agent. The carrier may include pharmaceutically acceptable emollients, emulsifiers, thickening agents, solvents and the like.
In one embodiment, active agents are administered at a therapeutically effective dosage sufficient to inhibit amyloid deposition in a subject. A “therapeutically effective” dosage inhibits amyloid deposition by, for example, at least about 20%, or by at least about 40%, or even by at least about 60%, or by at least about 80% relative to untreated subjects. In the case of an Alzheimer's subject, a “therapeutically effective” dosage stabilizes cognitive function or prevents a further decrease in cognitive function (i.e., preventing, slowing, or stopping disease progression). The present invention accordingly provides therapeutic drugs. By “therapeutic” or “drug” is meant an agent having a beneficial ameliorative or prophylactic effect on a specific disease or condition in a living human or non-human animal.
In the case of AA or AL amyloidosis, the agent may improve or stabilize specific organ function. As an example, renal function may be stabilized or improved by 10% or greater, 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or by greater than 90%.
In the case of IAPP, the agent may maintain or increase β-islet cell function, as determined by insulin concentration or the Pro-IAPP/IAPP ratio. In a further embodiment, the Pro-IAPP/IAPP ratio is increased by about 10% or greater, about 20% or greater, about 30% or greater, about 40% or greater, or by about 50%. In a further embodiment, the ratio is increased up to 50%. In addition, a therapeutically effective amount of the agent may be effective to improve glycemia or insulin levels.
In another embodiment, the active agents are administered at a therapeutically effective dosage sufficient to treat AA (secondary) amyloidosis and/or AL (primary) amyloidosis, by stabilizing renal function, decreasing proteinuria, increasing creatinine clearance (e.g., by at least 50% or greater or by at least 100% or greater), remission of chronic diarrhea, or by weight gain (e.g., 10% or greater). In addition, the agents may be administered at a therapeutically effective dosage sufficient to improve nephrotic syndrome.
Furthermore, active agents may be administered at a therapeutically effective dosage sufficient to decrease deposition in a subject of amyloid protein, e.g., Aβ40 or Aβ42. A therapeutically effective dosage decreases amyloid deposition by, for example, at least about 15%, or by at least about 40%, or even by at least 60%, or at least by about 80% relative to untreated subjects.
In another embodiment, active agents are administered at a therapeutically effective dosage sufficient to increase or enhance amyloid protein, e.g., Aβ40 or Aβ 42, in the blood, CSF, or plasma of a subject. A therapeutically effective dosage increases the concentration by, for example, at least about 15%, or by at least about 40%, or even by at least 60%, or at least by about 80% relative to untreated subjects.
In yet another embodiment, active agents are administered at a therapeutically effective dosage sufficient to maintain a subject's CDR rating at its base line rating or at 0. In another embodiment, the active agents are administered at a therapeutically effective dosage sufficient to decrease a subject's CDR rating by about 0.25 or more, about 0.5 or more, about 1.0 or more, about 1.5 or more, about 2.0 or more, about 2.5 or more, or about 3.0 or more. In another embodiment, the active agents are administered at a therapeutically effective dosage sufficient to reduce the rate of the increase of a subject's CDR rating as compared to historical or untreated controls. In another embodiment, the therapeutically effective dosage is sufficient to reduce the rate of increase of a subject's CDR rating (relative to untreated subjects) by about 5% or greater, about 10% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 60% or greater, about 70% or greater, about 80% or greater, about 90% or greater or about 100% or greater.
In yet another embodiment, active agents are administered at a therapeutically effective dosage sufficient to maintain a subject's score on the MMSE. In another embodiment, the active agents are administered at a therapeutically effective dosage sufficient to increase a subject's MMSE score by about 1, about 2, about 3, about 4, about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, or about 25 points. In another embodiment, the active agents are administered at a therapeutically effective dosage sufficient to reduce the rate of the decrease of a subject's MMSE score as compared to historical controls. In another embodiment, the therapeutically effective dosage is sufficient to reduce the rate of decrease of a subject's MMSE score may be about 5% or less, about 10% or less, about 20% or less, about 25% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less or about 100% or less, of the decrease of the historical or untreated controls.
Another means to evaluate cognition, particularly Alzheimer's disease, is the Alzheimer's Disease Assessment Scale (ADAS-Cog), or a variation termed the Standardized Alzheimer's Disease Assessment Scale (SADAS). It is commonly used as an efficacy measure in clinical drug trials of Alzheimer's disease and related disorders characterized by cognitive decline. SADAS and ADAS-Cog were not designed to diagnose Alzheimer's disease; they are useful in characterizing symptoms of dementia and are a relatively sensitive indicator of dementia progression. (See, e.g., Doraiswamy, Neurology 48:1511-1517, 1997; and Standish, J. Am. Geriatr. Soc. 44:712-716, 1996.) Annual deterioration in untreated Alzheimer's disease patients is approximately 8 points per year (See, e.g., Raskind, M. Prim. Care Companion J Clin Psychiatry 2000 August; 2(4):134-138).
In yet another embodiment, active agents are administered at a therapeutically effective dosage sufficient to maintain a subject's score on the DAD. In another embodiment, the active agents are administered at a therapeutically effective dosage sufficient to increase a subject's DAD score by about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 40, or about 50 points or greater. In another embodiment, the active agents are administered at a therapeutically effective dosage sufficient to reduce the rate of the decrease of a subject's DAD score as compared to historical controls. In another embodiment, the therapeutically effective dosage is sufficient to reduce the rate of decrease of a subject's DAD score by about 5% or less, about 10% or less, about 20% or less, about 25% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less or about 100% or less, of the decrease of the historical or untreated controls.
In yet another embodiment, active agents are administered at a therapeutically effective dosage sufficient to maintain a subject's score on the ADAS-Cog. In another embodiment, the active agents are administered at a therapeutically effective dosage sufficient to decrease a subject's ADAS-Cog score by about 2 points or greater, by about 3 points or greater, by about 4 points or greater, by about 5 points or greater, by about 7.5 points or greater, by about 10 points or greater, by about 12.5 points or greater, by about 15 points or greater, by about 17.5 points or greater, by about 20 points or greater, or by about 25 points or greater. In another embodiment, the active agents are administered at a therapeutically effective dosage sufficient to reduce the rate of the increase of a subject's ADAS-Cog scores as compared to historical or untreated controls. In another embodiment, the therapeutically effective dosage is sufficient to reduce the rate of increase of a subject's ADAS-Cog scores (relative to untreated subjects) by about 5% or greater, about 10% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 60% or greater, about 70% or greater, about 80% or greater, about 90% or greater or about 100% or greater.
In another embodiment, active agents are administered at a therapeutically effective dosage sufficient to decrease the ratio of Aβ42:Aβ40 in the CSF or plasma of a subject by about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 50% or more.
In another embodiment, active agents are administered at a therapeutically effective dosage sufficient to lower levels of Aβ in the CSF or plasma of a subject by about 15% or more, about 25% or more, about 35% or more, about 45% or more, about 55% or more, about 75% or more, or about 95% or more.
Toxicity and therapeutic efficacy of such agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50, and usually a larger therapeutic index is more efficacious. While agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to unaffected cells and, thereby, reduce side effects.
It is understood that appropriate doses depend upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the subject. Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). It is furthermore understood that appropriate doses depend upon the potency. Such appropriate doses may be determined using the assays described herein. When one or more of these compounds is to be administered to an animal (e.g., a human), a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and any drug combination.
The ability of an agent to inhibit amyloid deposition can be evaluated in an animal model system that may be predictive of efficacy in inhibiting amyloid deposition in human diseases, such as a transgenic mouse expressing human APP or other relevant animal models where Aβ deposition is seen or for example in an animal model of AA amyloidosis. Likewise, the ability of an agent to prevent or reduce cognitive impairment in a model system may be indicative of efficacy in humans. Alternatively, the ability of an agent can be evaluated by examining the ability of the agent to inhibit amyloid fibril formation in vitro, e.g., using a fibrillogenesis assay such as that described herein, including a ThT, CD, or EM assay. Also the binding of an agent to amyloid fibrils may be measured using a MS assay as described herein. The ability of the agent to protect cells from amyloid induced toxicity is determined in vitro using biochemical assays to determine percent cell death induced by amyloid protein. The ability of an agent to modulate renal function may also be evaluated in an appropriate animal model system.
The therapeutic agent of the invention may be also be administered ex vivo to inhibit amyloid deposition or treat certain amyloid-related diseases, such as β2M amyloidosis and other amyloidoses related to dialysis. Ex vivo administration of the therapeutic agents of the invention can be accomplished by contacting a body fluid (e.g., blood, plasma, etc.) with a therapeutic compound of the invention such that the therapeutic compound is capable of performing its intended function and administering the body fluid to the subject. The therapeutic compound of the invention may perform its function ex vivo (e.g., dialysis filter), in vivo (e.g., administered with the body fluid), or both. For example, a therapeutic compound of the invention may be used to reduce plasma β2M levels and/or maintain β2M in its soluble form ex vivo, in vivo, or both.
The present invention is also related to prodrugs of the agents of the Formulae disclosed herein. Prodrugs are agents which are converted in vivo to active forms (see, e.g., R. B. Silverman, 1992, “The Organic Chemistry of Drug Design and Drug Action,” Academic Press, Chp. 8). Prodrugs can be used to alter the biodistribution (e.g., to allow agents which would not typically enter the reactive site of the protease) or the pharmacokinetics for a particular agent. For example, a carboxylic acid group, can be esterified, e.g., with a methyl group or an ethyl group to yield an ester. When the ester is administered to a subject, the ester is cleaved, enzymatically or non-enzymatically, reductively, oxidatively, or hydrolytically, to reveal the anionic group. An anionic group can be esterified with moieties (e.g., acyloxymethyl esters) which are cleaved to reveal an intermediate agent which subsequently decomposes to yield the active agent. The prodrug moieties may be metabolized in vivo by esterases or by other mechanisms to carboxylic acids.
Examples of prodrugs and their uses are well known in the art (see, e.g., Berge, et al., “Pharmaceutical Salts”, J. Pharm. Sci. 66, 1-19 (1977)). The prodrugs can be prepared in situ during the final isolation and purification of the agents, or by separately reacting the purified agent in its free acid form with a suitable derivatizing agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst.
Examples of cleavable carboxylic acid prodrug moieties include substituted and unsubstituted, branched or unbranched lower alkyl ester moieties, (e.g., ethyl esters, propyl esters, butyl esters, pentyl esters, cyclopentyl esters, hexyl esters, cyclohexyl esters), lower alkenyl esters, dilower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters, acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, dilower alkyl amides, and hydroxy amides.
Certain embodiments of the present agents can contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term “pharmaceutically acceptable salts” in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of agents of the present invention. These salts can be prepared in situ during the final isolation and purification of the agents of the invention, or by separately reacting a purified agent of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
Representative salts include the hydrohalide (including hydrobromide and hydrochloride), sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, 2-hydroxyethanesulfonate, and laurylsulphonate salts and the like. See, e.g., Berge et al., “Pharmaceutical Salts”, J. Pharm. Sci. 66, 1-19 (1977).
In other cases, the agents of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term “pharmaceutically acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of agents of the present invention.
These salts can likewise be prepared in situ during the final isolation and purification of the agents, or by separately reacting the purified agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
“Pharmaceutically acceptable salts” also includes, for example, derivatives of agents modified by making acid or base salts thereof, as described further below and elsewhere in the present application. Examples of pharmaceutically acceptable salts include mineral or organic acid salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent agent formed, for example, from non-toxic inorganic or organic acids. Such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acid; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic acid. Pharmaceutically acceptable salts may be synthesized from the parent agent which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts may be prepared by reacting the free acid or base forms of these agents with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
All acid, salt, base, and other ionic and non-ionic forms of the compounds described are included as compounds of the invention. For example, if a compound is shown as an acid herein, the salt forms of the compound are also included. Likewise, if a compound is shown as a salt, the acid and/or basic forms are also included.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention and covered by the claims appended hereto. The contents of all references, issued patents, and published patent applications cited throughout this application are hereby incorporated by reference. The invention is further illustrated by the following examples, which should not be construed as further limiting.
The test compounds were synthesized and screened by mass spectrometry (“MS”) assays, except for selected compounds which were purchased from a commercial source. The MS assay gives data on the ability of compounds to bind to proteins, in this example, to β-amyloid and IAPP.
In the MS assay for Aβ40, samples were prepared as aqueous solutions (adding 20% ethanol if necessary to solubilize in water), 200 μM of a test compound and 20 μM of solubilized Aβ40, or 400 μM of a test compound and 40 μM of solubilized Aβ40. The pH value of each sample was adjusted to 7.4 (±0.2) by addition of 0.1% aqueous sodium hydroxide. The solutions were then analyzed by electrospray ionization mass spectrometry using a Waters ZQ 4000 mass spectrometer. Samples were introduced by direct infusion at a flow-rate of 25 μL/min within 2 hr. after sample preparation. The source temperature was kept at 70° C. and the cone voltage was 20 V for all the analysis. Data were processed using Masslynx 3.5 software. The MS assay gives data on the ability of compounds to bind to soluble Aβ, whereas the ThT, EM and CD assays give data on inhibition of fibrillogenesis. The results from the assay for binding to Aβ are summarized in Table 3. In Table 3, a blank box means that a value was not determined for that compound in that assay.
The assay for IAPP was conducted under the same conditions except that 200 μM or 100 μM of test compound and 20 μM of solubilized IAPP were employed. The key below describes the codes used in Table 3A to quantify the binding based on the intensity of the absorption.
Compounds in Table 3B were tested under the following conditions: 30 μM Aβ peptide, 150 μM compound; 60-100% binding=(+++), 40-59% binding=(++), 20-39% binding=(+), <20%=(−), blank=not determined.
Compounds in Table 3C were tested under the following conditions 20 μM Aβ peptide, 100 μM compound; 75-100% binding=(+++), 40-74% binding=(++), 20-39% binding=(+), <20%=(−), blank=not determined.
Compounds in Table 3D were tested under the following conditions: 40 μM Aβ peptide, 400 μM compound; 75-100% binding=(+++), 40-74% binding=(++), 20-39% binding=(+), <20%=(−), blank=not determined.
Transgenic mice, TgCRND8, expressing the human amyloid precursor protein (hAPP) develop a pathology resembling Alzheimer's disease. In particular, high levels of Aβ40 and Aβ 42 have been documented in the plasma and the brain of these animals at 8-9 weeks of age, followed by early accumulation of amyloid plaques similar to the senile plaques observed in AD patients. These animals also display progressive cognitive deficits that parallel the appearance of degenerative changes. See, e.g., (Chishti, et al., J. Biol. Chem. 276, 21562-70 (2001).
The short term therapeutic effect of 19 compounds of the invention was studied. These compounds were administered over a 14 or 28 day period at the end of which the levels of Aβ peptides in the plasma and brain of TgCRND8 animals were determined.
Male and female transgenic mice from the 3rd and 4th B6C3F1 generations were used in this example and given daily subcutaneous or oral administrations of one of a series of compounds for 14 or 28 days. The following abbreviations are used to designate these animals from the 3rd and 4th generation backcross in the present protocol: TgCRND8-2.B6C3F1(N3); TgCRND8-2.B6C3F1(N4).
Baseline animals (Group 1) consisted of naive TgCRND8-2. B6C3F1(N3) at 11±1 weeks of age. These mice were used to determine the Aβ levels in the plasma and brain of naive transgenic animals at the initiation of treatment.
Starting at 11 weeks of age (±1 week) animals received daily administration of their respective treatment for a period of 14 or 28 days (groups 2-21), at a dose of 250 mg/kg at 10 ml/kg or of vehicle only (water; group 2) or 1% methyl cellulose only (group 21). The route of administration was subcutaneous for water-soluble compounds and oral for compounds solubilized in methylcellulose 1% (MC 1%). At the end of the treatment periods, plasma and perfused brains were collected for quantification of Aβ levels.
The mice used in this study were derived from a breeding colony at Institut Armand Frappier, and were well-acclimated to the animal facility environment prior to initiation of the study Animals were assigned, according to age and gender, into the following experimental groups:
All animals were examined daily for signs of ill health when handled in the morning for their daily treatment and twice a day for mortality checks (once daily during weekends and holidays). Detailed examinations were performed on the treatment initiation, weekly during the study, and once before terminal procedures. More frequent observations were undertaken when considered appropriate. Death and all individual clinical signs were individually recorded. Individual body weights were recorded at randomization, once weekly during the study, and once before terminal procedures.
At 11±1 weeks of age for the Baseline group, and at the end of the treatment period (14 or 28 days) for Groups 2 to 21, at 24 hours after the last compound administration animals were sacrificed and samples collected. An approximate blood volume of 500 μl was collected from the orbital sinus and kept on ice until centrifugation at 4° C. at a minimum speed of 3,000 rpm for 10 minutes. Plasma samples were immediately frozen and stored at −80° C. pending analysis. The brains were removed, frozen, and stored at −80° C. awaiting analysis.
Brains were weighted frozen and homogenized with 4 volumes of ice cold 50 mM Tris-Cl pH 8.0 buffer with protease inhibitor cocktail (4 mL of buffer for 1 g of wet brain). Samples were spun at 15000 g for 20 minutes and the supernatants were transferred to fresh tubes. One hundred fifty (150) μl from each supernatant were mixed with 250 μl of 8M guanidine-HCL/50 mM Tris-HCL pH 8.0 (ratio of 0.6 vol supernatant: 1 vol 8M guanidium/Tris-HCL 50 mM pH 8.0) and 400 μL 5 M guanidium/Tris-HCL 50 mM pH8.0 were added. The tubes were vortexed for 30 seconds and frozen at −80° C. In parallel, pellets were treated with 7 volumes of 5 M guanidine-HCL/50 mM Tris-HCL pH 8.0 (7 mL of guanidine for 1 g of wet brain), vortexed for 30 seconds and frozen at −80° C. Samples were thawed at room temperature, sonicated at 80° C. for 15 minutes and frozen again. This cycle was repeated 3 times to ensure homogeneity and samples were returned to −80° C. pending analysis.
Aβ levels were evaluated in plasma and brain samples by ELISA using Human Aβ40 and Aβ42 Fluorometric ELISA kits from Biosource (Cat. No. 89-344 and 89-348) according to manufacturer's recommended procedures. In short, samples were thawed at room temperature, sonicated for 5 minutes at 80° C. (sonication for brain homogenates; no sonication for plasma samples) and kept on ice. Aβ peptides were captured using 100 μl of the diluted samples to the plate and incubated without shaking at 4° C. overnight. The samples were aspirated and the wells were rinsed 4 times with wash buffer obtained from the Biosource ELISA kit. The anti-Aβ40 or anti-Aβ42 rabbit polyclonal antiserum (specific for the Aβ40 or Aβ42 peptide) was added (100 μl) and the plate was incubated at room temperature for 2 hours with shaking. The wells were aspirated and washed 4 times before adding 100 μl of the alkaline phosphatase labeled anti-rabbit antibody and incubating at room temperature for 2 hours with shaking. The plates were then rinsed 5 times and the fluorescent substrate (100 μl) was added to the plate. The plate was incubated for 35 minutes at room temperature and the plate was read using a titer plate reader at an excitation wavelength of 460 nm and emission at 560 nm
Compounds were scored based on their ability to modulate levels of Aβ peptides in the plasma and the cerebral soluble/insoluble levels in the brain. Levels of Aβ observed in the plasma and brain of treated animals were normalized using values from vehicle-treated (water) or methylcellulose-treated control groups and ranked according to the strength of the pharmacological effect. Results are shown in Tables 3 to 11. Increases in the levels of Aβ peptides are indicated using “+” symbols, while decreases in the levels of Aβ peptides are indicated using “−” symbols. The strongest effects are recorded as “+++” or “− − −” while the weakest are shown as “+” or “−”.
Specifically, increases in the levels of Aβ (relative to untreated control) of 20 to 39% are scored as “+”; increases of 40 to 69% are scored as “++”; and increases of 70% or higher are scored as “+ + +”. Decreases in the levels of Aβ of 5 to 19% are scored as “−”; decreases of 20 to 38% are scored as “− −”; and decreases of 39% or more are scored as “− − −”.
The data are presented in Tables 6-11. Treatment with these compounds after 14 and/or 28 days resulted in a significant change in the cerebral levels of Aβ40 and/or Aβ42 (Tables 8-11). Furthermore, treatment with these compounds, for instance, Compound BX (3-(t-butyl)amino-1-propanesulfonic acid), resulted after 14 and 28 days (Tables 6-7) in a significant increase in the levels of Aβ peptides in the plasma. This suggests that some of these compounds may act by a peripheral sink effect, sequestering Aβ peptides in the plasma and thereby facilitating their clearance from the CNS as previously suggested for treatment by passive immunization using anti-Aβ monoclonal antibody m266 (DeMattos et al., Science 295(5563):2264-7).
The tables show levels of Aβ peptides in the plasma and brain of TgCRND8 mice treated for 14 and 28 days with compounds of the invention.
Tables 6A and 6C show the data from Day 14 and Day 28 for the Plasma Vehicle group, respectively. Tables 6B and 7 show the data for the Plasma Methylcellulose group on Days 14 and 28, respectively. Tables 8 and 10 show the data on Days 14 and 28 for the Brain homogenate vehicle group, respectively. Tables 9 and 11 show the data for brain homogenate for the Methylcellulose group on Days 14 and 28, respectively.
Transgenic mice, TgCRND8, as those used in the short term treatment, overexpress a human APP gene with the Swedish and Indiana mutations leading to the production of high levels of the amyloid peptides and to the development of an early-onset, aggressive development of brain amyloidosis. The high levels of Aβ peptides and the relative overabundance of Aβ42 compared to Aβ40 are believed to be associated with the severe and early degenerative pathology observed. The pattern of amyloid deposition, presence of dystrophic neuritis, and cognitive deficit has been well documented in this transgenic mouse line. The levels of Aβ peptides in the brain of these mice increase dramatically as the animals age. While the total amyloid peptide levels increase from ˜1.6×105 pg/g of brain to ˜3.8×106 between 9 and 17 weeks of age.
While the early deposition of amyloid in this model allows the rapid testing of compounds in a relatively short time frame, the aggressivity of this model and the high levels of Aβ peptides renders therapeutic assessment in the longer term a more difficult task.
The long-term therapeutic effects of compounds of the present invention on cerebral amyloid deposition and β-amyloid (Aβ) levels in the plasma and in the brains of transgenic mice, TgCRND8, expressing the human amyloid precursor protein (hAPP) was studied. These compounds were administered over an 8 or 16 week period at the end of which the levels of Aβ peptides in the plasma and brain of TgCRND8 animals were determined. The goal of this study was to evaluate the efficacy of the compounds at modulating the progression of the amyloidogenic process in the brain and in the plasma of a transgenic mouse model of Alzheimer's disease (AD)
The mice used in the study consisted of animals bearing one copy of the hAPP gene (+/−) from the 2nd and 3rd generation progenies (N2 and N3) derived from backcrosses from TgCRND8.FVB(N2)AJ(N3) with B6AF1/J hybrid animals.
N1=TgCRND8.FVB(N2)AJ(N3)×B6AF1/J
N2=TgCRND8.FVB(N2)AJ(N3).B6AF1/J(N1)×B6AF1/J
N3=TgCRND8.FVB(N2)AJ(N3).B6AF1/J(N2)×B6AF1/a
The following abbreviations are used to designate these animals in the present study: TgCRND8.B6AF1/J(N2); TgCRND8.B6AF1/J(N3). Male and female transgenic mice were given daily subcutaneous (compound BX) or oral (compounds BW and BZ) administrations of the appropriate compounds for 8 or 16 weeks.
Baseline animals consisted of 9±1 week old naive TgCRND8.B6AF1/J animals from the 2nd and 3rd generations. These mice were used to determine the extent of cerebral amyloid deposits and Aβ levels in the plasma and brain of naive transgenic animals at the initiation of treatment.
Starting at 9 weeks of age (±1 week) animals received daily administration of their respective treatment for a period of 8 or 16 weeks, at a dose of 30 or 100 mg/kg at 10 ml/kg. The route of administration was subcutaneous for water-soluble compounds (Compound BX) and oral for compounds solubilized in methylcellulose 1% (MC 1%) (Compounds BW and BZ). At the end of the treatment periods, plasma and perfused brains were collected for quantification of Aβ levels.
Animal health was monitored, samples were collected and Aβ levels were measured as described above in the short term treatment study. Compounds were scored based on their ability to modulate levels of Aβ peptides in the plasma and the cerebral soluble/insoluble levels in the brain. Levels of Aβ observed in the plasma and brain of treated animals were compared to that of vehicle-treated (water) or methylcellulose-treated control groups and ranked according to the strength of the pharmacological effect. Results are shown in Table 12. Increases in the levels of Aβ peptides are indicated using “+” symbols, while decreases in the levels of Aβ peptides are indicated using “−” symbols. The strongest effects are recorded as “+++” or “−−−” while the weakest are shown as “+” or “−”.
Specifically, increases in the levels of Aβ (relative to vehicle treated control) of 5 to 14% are scored as “+”; increases of 15 to 29% are scored as “++”; and increases of 30% or higher are scored as “+++”. Decreases in the levels of Aβ of 5 to 14% are scored as “−”; decreases of 15 to 29% are scored as “−−”; and decreases of 30% or more are scored as “−−−”. Additionally, changes of 4% or less in either direction are scored as “0”.
Table 12 shows levels of Aβ peptides in the plasma and brain of TgCRND8 mice treated for 8 and 16 weeks with compounds of the invention. Treatment with these compounds after 8 and/or 16 weeks in many cases resulted in a change in the levels of Aβ and/or Aβ42 in the plasma and/or brain. For example, administration of compound BX generally resulted in a dramatic decrease in the amount of Aβ in the brain at both 8 and 16 weeks. Compound BW also resulted in a dramatic decrease in brain and plasma levels of Aβ at 8 weeks and plasma levels at 16 weeks.
For the ThioS studies, the plaques in the brains of the mice were quantified as follows. Mice were transcardially perfused with saline solution. Brains were dissected out and separated in 2 hemispheres. The left hemisphere was immersed in freshly-prepared 4% paraformaldehyde for 3 hrs at 4° C., then transferred into 30% sucrose at 4° C. When cryoprotection was achieved (24-48 hours), brains were frozen in isopentane at −45° C. and subsequently stored at −80° C. until sectioning. Coronal 40 μm-thick sections were performed, and stained with thioflavin S (1% solution in water) for 8 min After differentiation of the thioflavin S staining, sections were counterstained with hematoxylin for 5 minutes. Two sets of pictures were captured simultaneously. A first set of pictures was captured under brightfield illumination to obtain morphological details of the section; a second set of pictures was captured under a green, specific, fluorescent filter (fluorescein filter, Ex 495 nm, Em 525 nm). Image analysis to quantify the number of plaques and the area occupied by these plaques was performed using Image Pro Plus software (Media Cybernetics, MD, USA).
The data from the histological ThioS studies is summarized in Table 13. Increases in the areas and numbers of plaques are indicated using “+” symbols, while decreases in the areas and numbers of the plaques are indicated using “−” symbols. Preliminary histochemical experiments using ThioS staining of brain sections indicated that both the number of plaques and the area occupied by the plaques were decreased in mice treated with 30 mg/kg of compound BX.
Specifically, increases in the areas and numbers of plaques (relative to vehicle treated control) of 10 to 19.99% are scored as “+”. Decreases in the areas and numbers of plaques of 10 to 19.99% are scored as “−”. Additionally, changes of 9.99% or less in either direction are scored as “0”.
Recent findings have demonstrated that a high percentage of Alzheimer Disease (AD) patients also form Lewy bodies, most abundantly in the amygdala (Hamilton. 2000 Brain Pathol, 10:378; Mukaetova-Ladinska, et al. 2000. J Neuropathol Exp Neurol 59:408). Interestingly, the highly hydrophobic non-amyloid component (NAC) region of α-synuclein has also been described as the second most abundant component of amyloid plaques in the brain of AD patients, after. Alpha-synuclein has been shown to form fibrils in vitro. Furthermore it binds to Aβ and promotes its aggregation (Yoshimoto, et al. 1995. Proc Natl Acad Sci USA 92:9141). It was in fact originally identified as the precursor of the non-amyloid beta (Aβ) component (NAD) of AD plaques (Ueda, et al. 1993. Proc Natl Acad Sci USA 90:11282; Iwai. 2000. Biochem Biophys Acta 1502:95; Masliah, et al. 1996. Am J Pathol 148:201). NAC is a 35 amino acid long peptide with highly hydrophobic stretches which can self-aggregate and form fibrils in vitro. Moreover, these fibrils can efficiently seed the formation of Aβ fibrils in vitro (Han, et al. 1995. Chem. Biol. 2: 163-169; Iwai, et al. 1995. Biochemistry 34:10139). It is in fact through its NAC domain that alpha-synuclein retains its fibrillogenic properties. Modulating the properties of NAC or targeting NAC with the compounds of the invention could therefore be a valid therapeutic avenue to inhibit the formation of protein aggregates and inclusions associated with alpha-synucleopathies, as well as the formation of aggregates between the beta-amyloid peptide and NAC of alpha-synuclein.
The ability of the compounds of the present invention to bind to NAC peptide in aqueous solution was evaluated. The binding ability correlates to the intensities of the peptide-compound complex peaks observed by the Electrospray Mass Spectrum. Millipore distilled deionized water was used to prepare all aqueous solutions. For pH determination a Beckman Φ36 pH meter fitted with a Corning Semi-Micro Combination pH Electrode was employed.
NAC (MW 3260.6 Da) at 20 μM was first analyzed at pH 7.40 and the usual sodium clusters was observed at +2, +3 and +4 at m/z 1335.5, 1116.7 and 843.4 respectively. The optimal cone voltage was determined to be 20V.
Mass spectrometry—Mass spectrometric analysis was performed using a Waters ZQ 4000 mass spectrometer equipped with a Waters 2795 sample manager. MassLynx 4.0 (earlier by MassLynx 3.5) was used for data processing and analysis. Test compounds were mixed with disaggregated peptides in aqueous media (6.6% EtOH) at a 5:1 ratio (20 μM NAC: 100 μM of test compound or 40 μM NAC:200 μM of test compound). The pH of the mixture was adjusted to 7.4 (±0.2) using 0.1% NaOH (3-5 μL). Periodically, NAC peptide solution at 20 μM or 40 μM was also prepared in the same fashion and run as control. The spectra were obtained by introducing the solutions to the electrospray source by direct infusion using a syringe pump at a flow rate of 25 μl/min, and scanning from 100 to 2100 Da in the positive mode. The scan time was 0.9 sec per scan with an inter-scan delay of 0.1 sec and the run time was 5 min for each sample. All the mass spectra were sum of 300 scans. The desolvation and source temperature was 70° C. and the cone and capillary voltage were maintained at 20 V and 3.2 kV respectively.
The total area under the peaks for the bound NAC-compound complex divided by total area under the peaks for unbound NAC was determined for each compound tested. The results are summarized in Table 14 below.
The present invention also relates to novel compounds and the synthesis thereof. Accordingly, the following examples are presented to illustrate how some of those compounds may be prepared.
Isopropylamine (2.5 mL, 29 mmol) was added to a solution of 1,3-propane sultone (3.05 g, 25 mmol) in a mixture of dichloromethane/ether (40 mL, 1:1). The mixture was heated at reflux for 3 hours. The reaction mixture was cooled to room temperature and hexane (10 mL) was added. The solid material was collected by filtration, rinsed with ether (10 mL), and dried in vacuo. Compound CG was obtained as a fine white powder (2.98 g, 65.6% yield). m.p. 240-43° C. 1H NMR (500 MHz, D2O) δ 1.06 (d, J=6.3 Hz, 6H), 1.86 (qt, J=7.6 Hz, 2H), 2.76 (t, J=7.6 Hz, 2H), 3.14 (t, J=7.8 Hz, 2H), 3.13-3.21 (m, J=6.6 Hz, 1H). 13C NMR (125 MHz, D2O) δ 18.2, 21.5, 25.8, 43.4, 47.9, 50.8.
Cyclopropylamine (3.7 mL, 52 mmol) was added to a solution of 1,3-propane sultone (6.9 g, 55.3 mmol) in THF (60 mL). The mixture was heated with an oil-bath at 42° C. for 2 hours. Stirring was difficult and part of the solid formed a crust above the stirred mixture. The mixture was heated at reflux for 1 hour, cooled to room temperature. The solid material was collected by filtration, dried in a vacuum oven for 2 hours at 60° C. (4.95 g). The solid was recrystallized in methanol/water (35 mL/5 mL, v/v). The mixture was cooled in a fridge then the solid was collected by filtration, rinsed with methanol (15 mL), air-dried for 15 minutes, and further dried in a vacuum oven at 60° C. overnight. Compound CI was obtained as long, fine, white needles (3.74 g, 40% yield). m.p. 234-236° C. 1H NMR (500 MHz, D2O) δ 0.62-0.71 (m, 4H), 1.92 (qt, J=7.6 Hz, 2H), 2.51-2.55 (m, J=3.7 Hz, 1H), 2.78 (d, J=7.3 Hz, 2H), 3.09 (t, J=7.8 Hz, 2H). 13C NMR (125 MHz, D2O) δ 3.0, 21.2, 30.0, 46.8, 47.9. FT-IR (KBr) νmax 3051, 1570, 1465, 1039
Cyclopentylamine (3.95 mL, 40 mmol) was added to a solution of 1,3-propane sultone (5.5 g, 45 mmol) in THF (80 mL). The mixture was heated at reflux with an oil-bath for 4 hours. Stirring was difficult, some acetone and ethanol were added to restore stirring. The mixture was cooled to room temperature. The solid was collected by filtration, dried in a vacuum oven for 1 hour at 60° C. (5.47 g). The solid material was dissolved in methanol/water (35 mL/2.5 mL, v/v) at reflux. The mixture was cooled slowly to room temperature overnight, and further cooled in a fridge. The product was collected by filtration, rinsed with methanol (15 mL), air-dried for 15 minutes, and further dried in a vacuum oven at 60° C. overnight. The product, Compound CJ, was obtained as long fine white needles (4.79 g). A second crop was obtained from the combined crude and first recrystallization mother liquor (0.84 g). Both crops were pure and were combined, total 5.63 g, 68% yield. m.p. 280-82° C. 1H NMR (500 MHz, D2O) δ 1.34-1.43 (m, 4H), 1.46-1.54 (m, 2H), 1.82-1.90 (m, 4H), 2.76 (7, J=7.6 Hz, 2H), 2.95 (t, J=7.8 Hz, 2H), 3.35 (qt, J=7.2 Hz, 1H). 13C NMR (125 MHz, D2O) δ 21.5, 23.6, 29.3, 45.1, 47.9, 59.5. FT-IR (KBr) νmax 3558, 3501, 2972, 1647, 1587, 1466
Cycloheptylamine (3.9 mL, 30 mmol) was added to a solution of 1,3-propane sultone (4.1 g, 33 mmol) in THF (65 mL). The mixture was heated at reflux for 5 hours with a heating mantle. The mixture was cooled to room temperature and the solid was collected by filtration, and then dried in a vacuum oven for 1 hour at 60° C. (6.21 g). The solid material was dissolved in methanol/water (30 mL/3 mL, v/v). The solution was cooled slowly to room temperature, and further cooled with an ice-bath. The solid product was collected by filtration, rinsed with methanol, air-dried for 15 minutes, and further dried in a vacuum oven at 60° C. Compound CK was obtained as small white flakes (5.08 g, 72% yield). m.p. 341-43° C. 1H NMR (500 MHz, D2O) δ 1.21-1.42 (m, 8H), 1.45-1.51 (m, 2H), 1.79-1.89 (m, 4H), 2.76 (7, J=7.3 Hz, 2H), 2.96 (t, J=7.8 Hz, 2H), 3.35 (m, J=4.6 Hz, 1H). 13C NMR (125 MHz, D2O) δ 21.6, 23.3, 27.3, 30.5, 43.6, 48.0, 59.6. FT-IR (KBr) νmax 2924, 1615, 1464, 1243.
3-Amino-2-hydroxy-1-propanesulfonic acid (15.51 g, 100 mmol) was dissolved in water (150 mL) with the help of 1 equivalent of NaOH (4.08 g). A solution of CBZ-OSuc (27.4 g, 110 mmol) in MeCN (300 mL) was added. After stiffing for 4 hours at room temperature, the solvent was evaporated under reduced pressure. The wet cake (one equivalent in weight of water) was then suspended in acetone (400 mL) and heated under reflux for 20 minutes. The mixture was cooled to room temperature and the solid was collected by filtration, washed with acetone and dried overnight in then vacuum oven at 40° C. Compound AC was obtained as a white fine solid (28.85 g, 87.6 mmol, 88%). The 1H and 13C NMR were consistent with the structure.
4-Amino-1-butanesulfonic acid sodium salt (0.516 g, 2.95 mmol was dissolved in water for a final concentration of 0.5 M (slightly yellow solution). A solution of CBZ-OSuc in CH3CN (2 M, 1.55 mL, 3.1 mmol, 1.05 eq.) was added. The reagent precipitated. A mixture of 1,4-dioxane and ethanol was added until almost all of the solid was dissolved. After 3.75 h, the solvent was evaporated under reduced pressure. The solid was dried in vacuo over the weekends. The solid was then suspended in acetone and heated under reflux for 30 minutes. The mixture was cooled to room temperature and the solid was collected by filtration, washed with acetone and dried overnight in vacuo. Compound AD was obtained as a white fine solid (0.7610 g, 2.32 mmol, 78%). The 1H NMR was consistent with the structure.
3-Amino-1-propanesulfonic acid sodium salt (1.09 g, 6.76 mmol) was dissolved in water for a final concentration of 0.5 M. A solution of CBZ-OSuc in CH3CN (2 M, 3.55 mL, 7.1 mmol, 1.05 eq.) was added. The reagent precipitated. A mixture of 1,4-dioxane and ethanol was added until almost all the solid was dissolved. After 3.75 hours, the solvent was evaporated under reduced pressure. The solid was dried in vacuo over the weekends. The solid was then suspended in acetone and heated under reflux for 30 minutes. The mixture was cooled to room temperature and the solid was collected by filtration, washed with acetone and dried overnight in vacuo. Compound AE was obtained as a white fine solid (1.58 g, 5.06 mmol, 75%). The 1H NMR was consistent with the structure. 90% pure (10% mol of homotaurine).
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A solution of (Boc)2O (800 mg, 3.5 mmol) in 1,4-dioxane (5 mL) was added to a cold (0° C.) solution of di-L-Phe-Phe (1 g, 3.20 mmol) in 1,4-dioxane (6 mL) and 1N NaOH (3.3 mL). The mixture was stirred at 0-5° C. for 2 hours. Another portion of (BOC)2O (100 mg) was added and the mixture was stirred for an additional 60 minutes at 0-5° C. then at room temperature for 30 minutes. The mixture was then evaporated to dryness. The solid was taken in a mixture of water/EtOAc and pH was adjusted to 2 with 2N HCl. The aqueous layer was extracted 3 times with EtOAc. The combined organic layers were dried with brine and the solvent was evaporated. Some solid was insoluble in a mixture of EtOAc/CHCl3: it was removed by filtration. The desired N-Boc-L-Phe-L-Phe 1 was obtained as a white foamy solid (913.7 mg, 2.215 mmol, 71% yield).
Component 2: 3-amino1-propanesulfonic acid isobutyl ester
A solution of 1,3-propane sultone 4 (6.12 g, 49.1 mmol) in acetone (30 mL) was added to a mixture of sodium azide (3.22 g, 49.1 mmol) in water/acetone (70 mL, 20 to 50). The clear solution was stirred at room temperature. The reaction was completed within 1 hour. The solvent was removed by evaporation under reduced pressure. The solid obtained was rinsed with hot ether (50 mL) and then with ether at room temperature (150 ml). The solid was then dried in the vacuum oven at 40° C. for overnight. The title compound 5 was obtained as a white solid (8.69 g, 46.4 mmol, 95% yield).
3-Azidopropanesulfonic acid, sodium salt 5 (1.87 g, 10.0 mmol) was suspended in dry benzene (20 mL), and PCl5 (2.3 g, 10.5 mmol) was added to the suspension. The mixture was stirred at room temperature for 30 minutes, then at gentle reflux for about 1 hour. The benzene and P(O)Cl3 were removed by evaporation under reduced pressure. Benzene was added to the crude mixture and the solvent was removed again under reduced pressure. The residue was dried in vacuo. The dried residue was dissolved in dichloromethane (anhydrous, 15 mL) and cooled at −10° C. using an ice/acetone bath.
A solution of isobutanol (1.00 mL, 10.8 mmol) and 2,6-lutidine (1.3 mL, 11.2 mmol) in dichloromethane (10 mL) was added slowly to the cold sulfonyl chloride solution. The mixture was stirred at −10° C. for 5 minutes then at room temperature for 2 hours. The reaction was quenched with water and dichloromethane was added to extract the product. The organic layer was washed once with water, aqueous saturated NaHCO3, brine, and then dried over magnesium sulfate. The solvent was removed by evaporation under reduced pressure and the residue was dried in vacuo. The remaining 2,6-lutidine hydrochloride was removed by washing the residue with ether. The resulting oil (1.78 g) was then applied on flash column (silica gel, EtOAc in Hexanes from 15% to 20%) to give afford the desired ester as an oil (790 mg, 35%).
A solution of isobutyl 3-azidopropanesulfonate (1.13 g, 5.11 mmol) in isobutanol (10 mL) was added under H2, via a cannula, to a suspension of Pd/C (10%, 200 mg) in isobutanol (4 mL) which had been saturated with H2. The mixture was then stirred under H2 (40 psi) at room temperature overnight. The solid was then removed by filtration. The filtrate was evaporated to dryness. And the residue was dried in vacuo. The title compound 2, homotaurine isobutyl ester, was obtained as brown oil (808.7 mg, 81%).
Reaction of Component 1 with Component 2
HOBT (340 mg, 2.215 mmol) was added to a cold (0-5° C.) solution of N-Boc-L-Phe-L-Phe 1 (913.7 mg, 2.215 mmol) and homotaurine isobutyl ester 2 (423 mg, 2.21 mmol) in dichloromethane (anhydrous, 30 mL). After 5 minutes, a solution of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (982 mg, 2.215 mmol) in dichloromethane (10 mL) was added dropwise. The solution was stirred overnight at room temperature. The mixture was diluted with dichloromethane (50 mL) and the organic layer was washed sequentially with 1N NaHSO4, aqueous saturated NaHCO3, and brine, and dried over sodium sulfate. The solvent was removed by evaporation under reduced pressure. Three compounds in the mixture were shown on TLC. Since the impurities were less soluble in methanol, repeated treatment with methanol followed by filtration removed most of the impurities. Column chromatography on silica gel (2% MeOH in CHCl3) afforded the tripeptide 3 as amber glassy solid (156.1 mg, 12%).
Concentrated HCl (0.7 mL) was added to a cold (0° C.) solution of N-Boc-L-Phe-L-Phe-homotaurine isobutyl ester (202 mg, 0.343 mmol) in methanol. The mixture was stirred at room temperature for 2 hours and then left standing in the refrigerator overnight. The solvent was removed under reduced pressure and the residual solid was dried in vacuo to afford the L-Phe-L-Phe-homotaurine isobutyl ester as a white solid (171.8 mg, 95%).
A solution of N—BOC-L-phenylalanine-N-hydroxusuccinimide ester (400 mg, 1.1 mmol) in a mixture of ethanol (6 mL) and 1,4-dioxane (4 mL) was added to a solution of L-Phe-Homotaurine (273 mg, 1.0 mmol) in 1N NaOH (1.05 mL), water (3 mL) and ethanol (4 mL). The mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the solid (601.9 mg) was suspended in a mixture of acetone (8 mL) and isopropanol (0.2 mL) and stirred overnight at room temperature. The mixture was heated under refluxed for 30 minutes and then was cooled to room temperature. The white solid was collected by filtration, washed with ether, then dried in the vacuum oven for 45 minutes. The resulting solid (423.1 mg) was dissolved in a mixture of water/tert-butanol (7:3, 5 mL) and treated with Amberlite IR-120 plus (washed, 15 g dry weight) for 2 minutes at room temperature. The resin was removed by filtration and washed 3 times with the mixed solvents of water and tert-butanol (10 mL). Concentrated HCl (4 mL) was added. The solvents were removed under reduced pressure, and the resulting solid was dried in vacuum. The compound was purified by recrystallization from a mixed solvent of THF and MeOH. The resulting solid was heated under reflux in methanol (about 3 mL) to remove the yellowish color. The solid was dried in vacuo. Compound X was obtained as white solid (84.4 mg, 20%). The 1H and 13C NMR were consistent with the structure.
The 3-chloropropane-1-sulfonyl chloride (10 mmol, 1.21 mL) was added dropwise to a cold (−10° C.) solution of L-phenylalanine ethyl ester (10 mmol, 2.3 g) and 4-methylmorpholine (20 mmol, 2.2 mL) in dichloromethane (30 mL). The mixture was stirred for 30 minutes at −10° C. and for 2 hours at room temperature. The mixture was diluted with dichloromethane (40 mL) and washed twice with water, once with brine, dried over sodium sulfate. Evaporation of the solvent under reduced pressure gave rise to almost pure 3-chloropropyl sulfonamide as a yellow-oil in quantitative yield. The 1H and 13C NMR were consistent with the structure.
A mixture of the 3-chloropropyl sulfonamide (10 mmol), sodium azide (20 mmol) and a catalytic amount of Bu4NI in DMF (40 ml) was heated at 60° C. for 24 hours. The mixture was diluted with ethyl acetate, washed with water three times, and once with brine, dried over sodium sulfate. Evaporation of the solvent gave rise to the azide as a brown-oil (3.0489 g, 8.96 mmol, 90%). The 1H and 13C NMR were consistent with the structure.
The azide (2.70 g, 7.94 mmol) was stirred under H2 (40 psi) with 10% Pd/C (348 mg) in ethanol (16 mL) at room temperature overnight. The solid was removed by filtration over Celite. The filtrate was treated with TMSCl/EtOH in attempt to obtain a crystalline hydrochloride salt of the product. The solvent was evaporated to give a thick residue (2.2 g, 6.27 mmol, 79%) that crystallized under none of the conditions tried. The crude hydrochloric acid salt was dissolved in dichloromethane and washed once with saturated sodium bicarbonate. The organic layer was recovered and dried over sodium sulfate. The solvent was removed by evaporation under reduced pressure. The residue was dissolved in methanol and treated with activated charcoal. The solid was removed by filtration over Celite and the filtrate was evaporated to dryness. The residue was dried in vacuo to afford a tan oil (1.3969 g, 56% from the azide). The 1H and 13C NMR were consistent with the structure of Compound CL.
A solution of N-t-BOC-L-Phe N-hydroxysuccinimide ester (2.80 mmol, 1.01 g) in dichloromethane (12 mL) was added to a cold (0° C.) solution of 3-aminopropane-1-sulfonyl-L-Phe-OEt (2.66 mmol, 839 mg) in dichloromethane (10 mL). The mixture was stirred overnight at room temperature. The mixture was diluted with dichloromethane, and washed with 2N HCl, aqueous saturated NaHCO3, and brine. The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure. The residue was applied on flash column chromatography on silica gel (2% MeOH in CHCl3). A portion of the pure desired material (600 mg) was isolated. The remaining product was mixed with 40% of the succinimide Some 3-aminopropanol (70 μL) was added to the mixture that was dissolved in dichloromethane (8 mL) and cooled to 0° C. The mixture was stirred for 1 hour at room temperature. The mixture was diluted with dichloromethane, and washed with 2N HCl, aqueous saturated NaHCO3, brine. The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure. The product was purified by flash column chromatography on silica gel (2% MeOH in CHCl3). Another portion of the pure desired material (432.4 mg) was isolated along with the adduct of the succinimide and 3-aminopropanol (220.6 mg, 0.684 mmol). Compound Y was obtained as a white crystalline foamy solid (1030 mg, 1.83 mmol, 65%). The 1H and 13C NMR were consistent with the structure.
Concentrated HCl (0.8 mL) was added to a cold (0° C.) solution of the N-Boc-L-Phe-(3-Aminopropane-1-Sulfonyl)-L-Phe, Ethyl Ester (197 mg, 0.350 mmol) in ethanol (8 mL). The mixture was cooled using an ice/water bath and stirred for 45 min and for 3 hours at room temperature. And the mixture was kept in freezer (−20° C.) over the weekend. The solvent was removed under reduced pressure. The residual ethanol was removed by coevaporation 3 times with chloroform under reduced pressure. The residue was dried in vacuo to give an off-white crystalline solid (175.3 mg) in quantitative yield. The 1H and 13C NMR were consistent with the structure of Compound Z.
One equivalent of 1N NaOH (202 μL) was added to a solution of L-(N-Boc)-Phe-(3-aminopropane-1-sulfonyl)-L-Phe, ethyl ester (110 mg, 0.197 mmol) in methanol (2 mL). The mixture was stirred overnight at room temperature. A white suspension was observed after overnight stirring. MeOH (1 mL), water (1 mL) and 1N NaOH (10 μL) were added. The mixture was stirred in a warm water bath for about 2 hours. The solvent methanol was removed under reduced pressure. The wet residue was then freeze-dried to give compound AA as a white powder in quantitative yield (110.2 mg). The 1H and 13C NMR were consistent with the structure.
The hydrolysis was done by the conventional LiOH/MeOH method. The product was purified by recrystallization from EtOAc and Hexanes.
The L-(N-Boc)-Phe-(3-aminopropane-1-sulfonyl)-L-Phe-OH (203 mg, 0.382 mmol) was dissolved in methanol (4 mL) and the solution was cooled to 0° C. Concentrated HCl (0.35 mL) was added and the mixture was stirred for 2 hours at 0° C. and for 2.5 h at room temperature. The volatile solvents were removed under reduced pressure. The aqueous residue was freeze-dried to give the product as a white solid (171.4 mg). The NMR and MS showed to product to be a mixture of the free acid and the methyl ester. The MS showed also a strong association of the peptide. The dimer was the major species on the MS. The solid was dissolved into methanol and treated with HCl overnight at room temperature. The solvent was evaporated and the residue was dried in vacuo. The product was obtained as a white foamy solid (180.4 mg, 97%). The 1H and 13C NMR were consistent with the structure of compound AB.
Thionyl chloride (8.2 mL, 112.5 mmol) was added to a cold MeOH (60 mL, in an ice-bath). The ice bath was removed and 4-iodo-L-phenylalanine (6.55 g, 22.4 mmol) was added to the mixture. The solution was stirred at reflux for 2 h. The solvent was removed under reduced pressure. The residual solid was dissolved in MeOH (40 mL) and the solution was poured into Et2O (300 mL). The solid was collected by filtration, washed with Et2O (2×50 mL) and dried in vacuo.
The solid (1.96 g, 5.8 mmol) was dissolved in a minimum amount of water. To the solution was added aqueous NH4OH (28-30%, 15 mL). The reaction mixture was stirred at room temperature over weekend. The solvent was removed under reduced pressure and EtOAc (15 mL) was added. The mixture was heated under reflux. The hot solution was filtered. The filtrate was cooled to room temperature and was stored in the fridge. The solid was collected by filtration, washed with EtOAc, to give 4-iodophenylalanine amide. The amide (1.3 g, 4.4 mmol) was dissolved in 15 mL of 2-butanone with a few drops of DMF before 1,3-propane sultone (560 mg, 4.9 mmol) was added. The reaction mixture was stirred at reflux for 2 hours. The mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×20 mL) and dried in vacuo. The solid was suspended in MeOH (25 mL) and a small amount of water (1 mL). The suspension was stirred at reflux. The solid material was collected by filtration while the mixture was still hot. The solid was washed with hot MeOH (2×10 mL). Compound CO was obtained as a white solid (320 mg).
The 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine hydrochloride (2.58 g, 14.5 mmol) was treated with 1N NaOH (20 mL). The aqueous mixture was extracted with CH2Cl2 (20 mL). The organic layer was separated and dried over MgSO4. The solvents were removed by evaporation under reduced pressure.
To a solution of 4-(4-fluorophenyl)-1,2,3,6-tetrahydropyridine (1.96 g, 13.7 mmol) in acetone (30 mL) was added 1,3-propane sultone (1.74 g, 14.5 mmol). The mixture was stirred at reflux overnight. Only a small amount of compound precipitated. The resulting suspension was cooled to room temperature with stiffing and a larger amount of solid precipitated. The suspension was heated with the addition of a small amount of MeOH until complete dissolution of the solid. The resulting solution was stirred at reflux for a few minutes and was cooled to room temperature with stiffing. The solid was collected by filtration, washed with MeOH and dried in vacuo. This allowed the isolation of compound F, 1.33 g (32%).
To a solution of 4-(4-bromophenyl)-4-piperidinol (2.51 g, 9.8 mmol) in MeOH (25 mL) was added 1,3-propane sultone (1.28 g, 10.7 mmol). The mixture was stirred at reflux for 2 h. Only a small amount of compound precipitated. The resulting suspension was cooled to room temperature with stirring and a solution of 50% MeOH/Acetone was added to precipitate a maximum of compound. The solid was collected by filtration, washed with 50% MeOH/Acetone (2×25 mL) and dried in vacuo. This allowed the isolation of compound G, 2.11 g (57%).
To a solution of 4-(4-chlorophenyl)-4-piperidinol (2.5 g, 11.8 mmol) in acetone (25 mL) was added 1,3-propane sultone (1.56 g, 13.0 mmol). The mixture was stirred at reflux for 2 h. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×20 mL) and dried in vacuo. This allowed the isolation of compound H, 2.83 g (72%).
4-Acetyl-4-phenylpiperidine hydrochloride (3.32 g, 12.5 mmol) was treated with 1N NaOH (20 mL). The aqueous mixture was extracted with CH2Cl2 (20 mL). The organic layer was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure.
To a solution of 4-acetylphenylpiperidine (1.83 g, 9.0 mmol) in acetone (22 mL) was added 1,3-propane sultone (1.20 g, 10.0 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×20 mL) and dried in vacuo. This allowed the isolation of compound I, 2.65 g (90%).
The 4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine hydrochloride (2.52 g, 10.9 mmol) was treated with 1N NaOH (20 mL); and aqueous mixture was extracted with CH2Cl2 (20 mL). The organic layer was separated, dried over Na2SO4, and filtered. The solvent was removed under reduced pressure.
To a solution of 4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine (2.07 g, 10.7 mmol) in acetone (25 mL) was added 1,3-propane sultone (1.41 g, 11.8 mmol). The mixture was stirred at reflux for 2 h. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×20 mL) and dried in vacuo. The product was suspended in 50% MeOH/acetone (75 mL). The suspension was stirres at reflux for 5 minutes before 25 mL of cold acetone was added. The solid material was filtered and washed with acetone (2×25 mL). This allowed the isolation of compound J, 1.48 g (44%).
To a solution of 1-phenylpiperazine (2.0 g, 1.9 mL, 12.3 mmol) in acetone (20 mL) was added 1,3-propane sultone (1.53 g, 12.9 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. This allowed the isolation of compound K, 3.04 g (87%).
The 1-(4-chlorophenyl)piperazine dihydrochloride (2.5 g, 9.3 mmol) was treated with 1N NaOH (40 mL); and the aqueous mixture was extracted with CH2Cl2 (40 mL). The organic layer was separated, dried over Na2SO4, filtered and solvent was removed under reduced pressure.
To a solution of 1-(4-chlorophenyl)piperazine (1.62 g, 8.2 mmol) in acetone (20 mL) was added 1,3-propane sultone (1.06 g, 8.6 mmol). The mixture was stirred at reflux for 2 h. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. This allowed the isolation of compound L, 2.11 g (81%).
To a solution of 1-(2-fluorophenyl)piperazine (2.5 g, 2.2 mL, 13.9 mmol) in acetone (25 mL) was added 1,3-propane sultone (1.73 g, 14.6 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. This allowed the isolation of compound M, 3.56 g (85%).
To a solution of 1-(4-nitrophenyl)piperazine (2.58 g, 12.1 mmol) in acetone (25 mL) was added 1,3-propane sultone (1.06 g, 8.6 mmol). The mixture was stirred at reflux for 2 h. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. This allowed the isolation of compound N, 2.85 g (71%).
To a solution of 1-(4-fluorophenyl)piperazine (2.0 g, 11.1 mmol) in acetone (20 mL) was added 1,3-propane sultone (1.46 g, 11.7 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. This allowed the isolation of compound P, 2.62 g (78%).
The 4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (1.5 g, 7.8 mmol) suspended in 16 mL of CH2Cl2. To this suspension was added triethylamine (2.1 mL, 15.3 mmol) followed by methyl 3-bromopropionate (1.0 mL, 9.2 mmol). The reaction was stirred at room temperature for 4 h and at reflux for 2 h. The reaction mixture was washed with water, 1N HCl (2×20 mL), 1N NaOH (2×20 mL) and Brine (1×20 mL). The organic layer was separated, dried over Na2SO4, filtered; and solvent was removed under reduced pressure. To the crude product was added 2N NaOH (15 mL). The mixture was stirred at reflux for 1 h. The reaction mixture was washed with CH2Cl2 (3×20 mL) and neutralized with concentrated HCl. The aqueous solution was concentrated to dryness under reduced pressure, to give a solid residue. Sodium chloride in the residue was removed in the following way (three repeats): dissolving the residue in a minimum amount of water, treating the aqueous solution with acetone, removing the resultant solid material by filtration, and concentrate the filtrate to dryness under reduced pressure. This allowed the isolation of compound Q (159.4 mg).
To a solution of dibenzylamine (9.8 mL, 50.8 mmol) in toluene (50 mL) was added 1,3-propane sultone (6.50 g, 53.3 mmol). The mixture was stirred at reflux for 3 h. A sticky paste was formed at the bottom of the flask. The reaction mixture was cooled to room temperature. The top layer was decanted; and the paste was partially dissolved in EtOAc with heating. The mixture was poured in a 10% EtOAc/Hexanes (200 mL). The mixture was heated and the paste was spread on the walls of the conical flask. The solvent was removed. This process was repeated twice. MeOH (75 mL) was added to the paste. The mixture was heated until a white solid appeared. The solid was collected by filtration, washed with cold MeOH, and dried in vacuo, affording compound AV, 7.03 g (43%).
The 4-cyano-4-phenylpiperidine hydrochloride (2.0 g, 9.0 mmol) was treated with 1N NaOH (20 mL) and the aqueous phase was extracted with CH2Cl2 (20 mL). The organic layer was separated, dried over MgSO4, filtered and solvent was removed under reduced pressure.
To a solution of piperidine (1.43 g, 7.7 mmol) in acetone (20 mL) was added 1,3-propane sultone (1.02 g, 8.5 mmol). The mixture was stirred at reflux for 2 h. The resulting suspension was cooled to room temperature. The solid was collected by filtration, washed with acetone and dried in vacuo. The solid was recrystallized from MeOH (and traces of water) to afford compound E, 800 mg (34%).
The 4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (2.01 g, 10.2 mmol) was treated with 1N NaOH (20 mL) and the aqueous phase wa extracted with CH2Cl2 (20 mL). The organic layer was separated, dried over MgSO4, filtered and solvent was removed under reduced pressure.
The resulting 4-phenyl-1,2,3,6-tetrahydropyridine (1.55 g, 9.7 mmol) was dissolved in 20 mL of 2-butanone. To this solution was added potassium carbonate (2.02 g, 14.6 mmol). The mixture was stirred 30 minutes at room temperature; ethyl 4-bromobutyrate (1.46 mL, 10.1 mmol) was added. The reaction mixture was stirred at reflux for 5 hours. After cooling to room temperature, inorganic salts were filtered. The solvent was evaporated under reduced pressure. The residue was dissolved in CH2Cl2 (30 mL). The organic phase was washed with water (2×30 mL), 2N HCl (2×30 mL) and Brine (2×30 mL). The organic layer was dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo. This allowed the isolation of 1.55 g (58%) of the desired ester.
The ester (5.7 mmol) was dissolved in 6N HCl (40 mL). The reaction mixture was stirred at room temperature for 5 hours and at reflux for 1 hour before it was cooled to room temperature. The reaction mixture was extracted with CH2Cl2 (3×30 mL). The aqueous phase was evaporated under reduced pressure. The residue was dissolved in water (20 mL); and the aqueous solution was concentrated to dryness under reduced pressure. The resultant material was further dried in vacuo, affording compound R, 973 mg (61%).
To a solution of piperonylamine (2.5 mL, 19.8 mmol) in acetone (30 mL) was added 1,3-propane sultone (2.52 g, 20.8 mmol). The mixture stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. The product was suspended in 90% Acetone/MeOH (75 mL). The suspension was stirred at reflux for 30 sec., the solid was collected by filtration and dried in vacuo. This allowed the isolation of compound AW, 2.56 g (45%).
To a solution of 3,4,5-trimethoxybenzylamine (2.2 mL, 12.7 mmol) in 2-butanone (20 mL) was added 1,3-propane sultone (1.66 g, 13.3 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. The product was suspended in 90% Acetone/MeOH (75 mL). The suspension was stirred at reflux for 30 sec., the solid was collected by filtration, and dried in vacuo; affording compound AY, 2.61 g (64%).
To a solution of 2,3-dimethoxybenzylamine (2.2 mL, 15.0 mmol) in 2-butanone (20 mL) was added 1,3-propane sultone (1.97 g, 15.8 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. The crude product was suspended in 90% Acetone/MeOH (75 mL). The suspension was stirred at reflux for 30 seconds, the solid was collected by filtration, and dried in vacuo; affording compound AZ, 1.95 g (45%).
The 3-amino-1-propanesulfonic acid (1.0 g, 7.2 mmol) was dissolved in 3N NaOH (370 mg, 9.4 mmol in 3 mL of water). After the solution was cooled to 0° C., diphenylmethyl isocyanate (1.4 mL, 7.2 mmol) was added. The reaction mixture was allowed to warm up to room temperature, stirred for 8 h (r.t.), and followed by addition of 3N NaOH (3 mL). The reaction mixture was stirred for 18 h. The pH of the reaction mixture was brought to 3 with 5N HCl. The solvent was evaporated under reduced pressure. EtOH (15 mL) was added and the mixture was stirred at reflux for 30 sec. The hot mixture was filtered. The filtrate was evaporated to dryness. This process was repeated 2 more times. The final product was dried in vacuo, affording compound AF, 837 mg (34%).
To a solution of 3,5-dimethoxybenzylamine (2.5 g, 15.0 mmol) in 2-butanone (22 mL) was added 1,3-propane sultone (1.95 g, 15.8 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL), and dried in vacuo. This allowed the isolation of compound BA, 2.89 g (67%).
The 2,4-dimethoxybenzylamine hydrochloride (2.51 g, 12.3 mmol) was treated with 1N NaOH (20 mL) and the aqueous phase was extracted with CH2Cl2 (20 mL). The organic layer was separated, dried over MgSO4, and filtered. Solvent was removed under reduced pressure to give the amine (free base form).
To a solution of 2,4-dimethoxybenzylamine (1.71 g, 10.3 mmol) in 2-butanone (15 mL) was added 1,3-propane sultone (1.31 g, 10.7 mmol). The mixture was stirred at reflux for 2.5 h. The reaction mixture was cooled to room temperature. The supernatant was decanted; and the paste was washed with acetone (2×30 mL), and dissolved in MeOH with heating. Acetone addition to the methanolic solution caused precipitation. The solid material was collected by filtration, washed with acetone (2×25 mL), and dried in vacuo; affording compound BB, 1.14 g (38%).
3-amino-1-propanesulfonic acid (1.0 g, 7.2 mmol) was dissolved in solution of 3M NaOH (7.2 mL). The mixture was cooled to 0° C. before phenylacetyl chloride (1.4 mL, 10.8 mmol) was added. The reaction mixture was allowed to warm up to room temperature and it was stirred for 22 h. The solvent was evaporated under reduced pressure. The residue was suspended in 50% EtOH/Acetone. The mixture was stirred at reflux for 30 sec. The solid material was collected by filtration and dried in vacuo. The product was recrystallized from 95% EtOH/H2O and dried in vacuo. This allowed the isolation of compound AG, 880 mg (44%).
3-amino-1-propanesulfonic acid (1.06 g, 7.7 mmol) was dissolved in 1.5N NaOH (5.3 mL). To this solution was added benzyl isocyanate (927 μL, 7.7 mmol). The reaction mixture was stirred at 70° C. for 30 min, followed by addition to the mixture one-equivalent of benzyl isocyanate (927 μL, 7.7 mmol). The reaction mixture was stirred for 1 hour. The solvent was evaporated under reduced pressure. The residue was suspended in hot acetone. The solid material was collected by filtration, washed with hot acetone, and dried in vacuo; affording compound AH, 2.07 g (92%).
3-amino-1-propanesulfonic acid (1.06 g, 7.7 mmol) was dissolved in 1.5N NaOH (5.3 mL). To this solution was added n-dodecyl isocyanate (1.7 mL, 7.7 mmol). The reaction mixture was stirred at 70° C. for 30 min followed by addition to the mixture one-equivalent of n-dodecyl isocyanate (1.7 mL, 7.7 mmol). The reaction mixture was stirred for 1 h. The solvent was removed under reduced pressure. The residual material was suspended in hot acetone. The solid material was collected by filtration, washed with hot acetone, and dried in vacuo; affording compound AJ, 2.47 g (86%).
3-amino-1-propanesulfonic acid (1.06 g, 7.7 mmol) was dissolved in 1.5N NaOH (5.3 mL). To this solution was added 1-adamantyl isocyanate (1.36 g 7.7 mmol) in hot EtOH (5 mL). The reaction mixture was stirred at 70° C. for 30 min followed by addition (to the mixture) of one-equivalent of 1-adamantyl isocyanate (1.37, 7.7 mmol) in hot EtOH (5 mL). The reaction mixture was stirred for 1 h. The solvent was removed under reduced pressure. The residue was suspended in hot acetone. The solid material was collected by filtration, washed with hot acetone, and dried in vacuo. The solid was recrystallized from EtOH, affording compound AK, 519.4 mg (20%).
Thionyl chloride (1.6 mL, 21.1 mmol) was added to ibuprofen (1.02 g, 4.9 mmol). The reaction mixture was heated to reflux for 4 h. The solvent was evaporated, dried in vacuo, giving corresponding acid chloride.
3-amino-1-propanesulfonic acid (308 mg, 2.2 mmol) was dissolved in 1.5N NaOH (3 mL). To this solution was added dropwise the acid chloride (500.8 mg, 4.4 mmol, prepared above). The reaction mixture was stirred at 70° C. overnight. The solvent was removed under reduced pressure. The residue was suspended in acetone. The suspension was stirred at reflux for 30 seconds. The solid material was removed by filtration. The filtrate was evaporated to dryness under reduced pressure. The residual material was subjected to separation by flash chromatography (80% CH2Cl2/MeOH). This allowed the isolation of compound AL, 237 mg (14%).
3-amino-1-propanesulfonic acid (1.07 g, 7.7 mmol) was dissolved in 1.5N NaOH (5.3 mL). To this solution was added benzyl isothiocyanate (1.02 mL, 7.7 mmol). The reaction mixture was stirred at 70° C. for 0.5 h; a second-equivalent of benzyl isocyanate (1.02 mL, 7.7 mmol) was added. The reaction mixture was stirred for 1 h. The solvent was evaporated under reduced pressure. The residue was suspended in hot acetone. The solid material was collected by filtration, washed with hot acetone, and dried in vacuo. The residual material was recrystallized from MeOH (traces of water), affording compound AM, 1.00 g (42%).
To a solution of 3,4-dimethoxybenzylamine (2.2 mL, 15.0 mmol) in 2-butanone (20 mL) was added 1,3-propane sultone (1.98 g, 15.8 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. The crude product was suspended in 75% Acetone/MeOH (75 mL). The suspension was stirred at reflux for 30 sec.; the solid material was collected by filtration, washed with acetone (2×25 mL), and dried in vacuo. The solid (1.94 g, 6.7 mmol) was dissolved hydrobromic acid (48%, 27 mL). The solution was stirred at 100° C. for 4 h. The solvent was removed under reduced pressure. The residue was dissolved in water (20 mL). The aqueous phase was washed with CH2Cl2 (3×20 mL), and evaporated under educed pressure. The solid residue was suspended in hot MeOH (75 mL). The suspension was stirred at reflux for 30 sec.; the solid was collected by filtration, washed with 50% MeOH/acetone, and dried in vacuo. This allowed the isolation of compound S, 1.22 g (70%).
To a solution of 4-(3-phenylpropyl)pyridine (14.5 mL, 76 mmol) in 2-butanone (150 mL) was added 1,3-propane sultone (10.0 g, 83.6 mmol). The mixture was stirred at reflux for 1.5 h. Once the reaction mixture was cooled to room temperature, the precipitate was collected by filtration and washed with acetone. The solid material was recrystallized from EtOH (traces of Et2O), affording compound C, 15.8 g (66%).
4-(3-phenylpropyl)-1-sulfopropylpyridine (10.7 g, 33.5 mmol) was dissolved in 60 mL of MeOH. The solution was cooled to 0° C. before sodium borohydride (2.55 g, 67.0 mmol) was added portionwise. The reaction mixture was stirred for 0.5 hour at room temperature. Water (10 mL) and concentrated HCl (5 mL) were successively added to the mixture. The inorganic material was removed by filtration. The filtrate was concentrated to dryness under reduced pressure and dried in vacuo. The resultant viscous residue was dissolved in MeOH (60 mL). The solution was stirred with Amberlite IR-120 ion exchange resin (8.3 g) for 15 min. The resin was removed by filtration and washed with MeOH. The filtrate and washing was combined and concentrated to dryness under reduced pressure. The residual material was recrystallized from water, affording compound D (8.05 g, 75%) as white crystals.
Tetrahydrofuran (THF, 800 mL) was placed a 3 neck 2-L flask (equipped with a condenser) and cooled to 5° C. with an ice-bath. To the cold THF was added aqueous ethylamine (70 wt. % solution in water, 85 mL, 1.07 mol), followed by addition of a cold solution of 1,3-propane sultone (25.08 g, 201 mmol) in THF (100 mL) over 24-min period. The mixture was stirred, while cooled with an ice-bath, for 1 h. The ice-bath was removed and the mixture was stirred at room temperature overnight. It was then heated under reflux for 1 h to distill off the ethylamine. The hot mixture was biphasic. Upon cooling, a solid crystallized at the bottom of the flask. Ether (400 mL) was added and the mixture was cooled to −20° C. The supernatant was decanted. Methanol (about 120 mL) was added to the residue. The mixture was heated to reflux; and complete dissolution of the solid material was achieved. After the solution was cooled to room temperature, precipitates formed. The mixture was cooled in an ice-bath; and the solid material was collected by filtration, rinsed with cold methanol and dried in vacuo (20.66 g, pure by NMR analysis). The solid material was recrystallized from methanol (100 mL). After the mixture was cooled using an ice-bath, the solid was collected by filtration, rinsed with cold methanol, and dried in a vacuum oven at 40° C. Compound CV was obtained as white fine needles (19.12 g, 57%). The 1H and 13C NMR were consistent with the structure.
The 1-adamantanamine hydrochloride (80 g, 0.426 mol) was treated with NaOH (10%, 400 mL) in water. The aqueous mixture was extracted with dichloromethane (1×400 mL, 2×100 mL). The combined organic layers were washed with brine (50 mL) and dried over sodium sulfate (10 g). Solvent was removed under reduced pressure. The resulting white waxy solid was co-evaporated with acetonitrile (50 mL). The wet solid was suspended in acetonitrile (200 mL). The suspension mixture was added dropwise over 20 min to a solution of 1,3-propane sultone (53 g, 0.426 mol) in acetonitrile (300 mL) and THF (200 mL). The thick mixture was stirred for 2 hours under reflux with a mechanical stirrer. The suspension was then cooled to 13° C. The solid was collected by suction-filtration, rinsed with acetonitrile (2×100 mL) and ether (1×100 mL), air-dried for 30 min, and further dried in vacuo at 60° C. overnight (104.17 g for crop 1). Another crop was collected from the filtrate and dried in vacuo in the same manner (3.39 g for crop 2). Both crops gave identical proton NMR spectrum. The two batches were combined for further purification. The solid was suspended in methanol (720 mL) and the mixture was heated to reflux. Water (490 mL) was added dropwise over 45 min, while maintaining reflux. After complete dissolution of the solid, the solution was kept under reflux for 30 minute. The mixture was left in the power-off heating mantle and allowed to cool slowly. After 90 min, the temperature reached 40° C. The heating mantle was replaced by a thermostated water bath. The mixture was cooled to 5° C. and stirred overnight at this temperature. The white flaky solid was collected by filtration, rinsed with cold (0° C.) methanol (2×125 mL), air-dried for 60 minutes, and then dried in the vacuum oven at 60° C. overnight. Compound BW was obtained as a white flaky solid (white plates, 88.48 g, 76% yield for the first crop). The 1H NMR and MS were consistent with the structure. A second crop (8.62 g) of compound BW was obtained from the mother liquid. The 1H NMR was identical to that of the first crop. This made the reaction a total yield of 83% from the hydrochloride.
To a solution of 2-aminonorbornane (7.3 g, 65.7 mmol) in 2-butanone (50 mL) was added dropwise a solution of 1,3-propane sulfone (8.1 g, 65.7 mmol) in 2-butanone (10 mL). The mixture was stirred at 60° C. for 1 h. The suspension was cooled to room temperature. The solid material was collected by filtration and washed with ethanol (2×20 mL). The crude material was recrystallized from 95% EtOH to afford compound BY as a white crystalline solid (8.2 g, 53% yield).
The 2-aminoadamantane hydrochloride (2×5 g) was treated with NaOH in water. The aqueous mixture was extracted with dichloromethane. The organic layer was dried over magnesium sulfate. The solvent was removed under reduced pressure. The resulting white solid was dried 30 minutes at room temperature under vacuum. A solution of 1,3-propane sultone (7.4 g, 60 mmol) in THF was added to a solution of the free amine (7.98 g, 52 mmol) in THF (70 mL, total). The mixture was heated under reflux for 4 h, cooled into an ice bath. The solid was collected by filtration, air-dried for 15 min, and further dried in vacuo (11.2 g). Recrystallization was done with methanol/water (60 mL/35 mL). After cooled in a fridge, the solid was collected by filtration, rinsed with methanol, dried in the vacuum oven at 60° C. overnight. A white crystalline sandy solid (small plates, 10.45 g, 74% yield) was obtained. The 1H and 13C NMR were consistent with the structure of Compound BZ.
To a solution of 3,4-dimethoxybenzylamine (2.2 mL, 15.0 mmol) in acetone (20 mL) was added 1,3-propane sultone (1.97 g, 15.8 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. The crude product was suspended in 90% acetone/MeOH (75 mL). The suspension was stirred at reflux for 30 sec., the solid material was collected by filtration, and dried in vacuo. Compound S (1.84 g, 43%) was isolated as a white solid. 1H NMR (D2O, 500 MHz) 8 ppm 6.96 (m, 3H), 4.06 (s, 2H), 3.74 (s, 6H), 3.07 (t, 1H, J=7.8 Hz), 2.86 (t, 1H, J=7.8 Hz), 2.01 (m, 2H). 13C NMR (D2O, 125 MHz) 8 ppm 149.23, 148.50, 123.63, 123.37, 55.90, 55.86, 50.96, 48.06, 45.62, 21.39. ES_MS 290 (M+1).
To a solution of 1,2-diphenylethylamine (2.49 g, 12.7 mmol) in 2-butanone (15 mL) was added 1,3-propane sultone (1.67 g, 13.3 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL), and dried in vacuo, to give compound ET: 3.02 g (74%). 1H NMR (DMSO, 500 MHz) 8 ppm 8.58 (s (broad), 1H), 7.34 (m, 8H), 7.23 (t, 2H, J=7.3 Hz), 4.32 (t, 1H, J=7.8 Hz), 3.68 (d, 2H, J=7.3 Hz), 3.08 (t, 2H, J=6.1 Hz), 2.57 (t, 2H, J=7.3 Hz), 1.92 (m, 2H). 13C NMR (DMSO, 125 MHz) 8 ppm 141.63, 129.46, 128.47, 127.76, 50.85, 49.91, 48.53, 22.07. ES-MS 318 (M−1).
To a solution of tert-butylamine (1.0 mL, 9.5 mmol) in tetrahydrofuran (15 mL) was added 2,4-butane sultone (1.33 g, 10.0 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid product was collected by filtration, washed with THF (2×20 mL), and dried in vacuo; affording compound ES: 1H NMR (DMSO, 500 MHz) 8 ppm 2.97 (t, 2H, J=6.6 Hz), 2.62 (m, 1H), 1.95 (m, 0.5H), 1.750 (m, 0.5H), 1.22 (s, 9H), 1.12 (d, 3H, J=6.8 Hz). 13C NMR (DMSO, 125 MHz) 8 ppm 56.17, 53.15, 29.87, 25.87, 17.05. ES-MS 207 (M−1).
To a solution of tert-butylamine (1.0 mL, 9.5 mmol) in tetrahydrofuran (4 mL) was added 1,4-butane sultone (1.36 g, 10.0 mmol) at room temperature. The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid product was collected by filtration, washed with acetone (2×20 mL), and dried in vacuo; affording compound ER 690 mg, (34%); 1H NMR (D2O, 500 MHz) 8 ppm 2.92 (t, 2H, J=7.1 Hz), 2.82 (t, 2H, J=7.1 Hz), 1.68 (m, 4H), 1.22 (s, 9H). 13C NMR (D2O, 125 MHz) 8 ppm 57.07, 50.30, 40.95, 25.28, 24.96, 21.62. ES-MS 210 (M−1).
To a solution of 1-ethylpropylamine (10.0 g, 115 mmol) in tetrahydrofuran (80 mL) was added a solution of 1,3-propane sultone (13.7 g, 110 mmol) in 20 mL of THF. The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid product was collected by filtration, washed with acetone (2×50 mL), and dried in vacuo., to afford compound DD (18.1, 80%): 1H NMR (D2O, 500 MHz) 8 ppm 3.08 (t, 2H, J=7.3 Hz), 3.01 (m, 1H), 2.87 (t, 2H, J=7.3 Hz) 2.00 (m, 2H), 1.59 (m, 4H), 0.82 (t, 6H, J=7.3 Hz). 13C NMR (D2O, 125 MHz) 8 ppm 60.87, 48.14, 43.68, 21.81. 21.60, 8.25. ES-MS 208 (M−1).
To a solution of tert-amylamine (2.0 g, 23.3 mmol) in tetrahydrofuran (15 mL) was added 1,3-propane sultone (2.76 g, 22.2 mmol). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid product was collected by filtration, washed with acetone (2×25 mL), and dried in vacuo, to afford compound DG (3.3 g, 73%): 1H NMR (D2O, 500 MHz) 8 ppm 3.04 (t, 2H, J=7.8 Hz), 2.89 (t, 2H, J=7.8 Hz), 2.87 (t, 2H, J=7.3 Hz), 1.97 (m, 2H), 1.55 (m, 2H), 1.18 (s, 6H), 0.82 (t, 6H, J=7.3 Hz). 13C NMR (D2O, 125 MHz) 8 ppm 60.42, 48.17, 39.88, 30.81, 22.23, 21.98, 7.25. ES-MS 208 (M−1).
To a solution of 2-amino-2-methyl-1-propanol (2.0 g, 21.4 mmol) in tetrahydrofuran (15 mL) was added 1,3-propane sultone (2.66 g, 21.4 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The crude product was collected by filtration, washed with acetone (2×25 mL). The solid was suspended in EtOH (50 mL). The suspension was stirred at reflux for 5 minutes. The solid was collected by filtration and dried in a vacuum oven (50° C.), affording compound DH (2.5 g, 58%): 1H NMR (D2O, 500 MHz) 8 ppm 3.48 (s, 2H), 3.04 (t, 2H, J=7.8 Hz), 2.90 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.18 (s, 6H). 13C NMR (D2O, 125 MHz) 8 ppm 64.88, 60.27, 48.19, 40.10, 21.92, 20.02. ES-MS 210 (M−1).
To a cold (5° C.) mixture of 2-aminoisobutyric acid (2.0 g, 19.4 mmol), NaOH (776 mg, 19.4 mmol) in 1,4-dioxane (10 mL) and water (4 mL) was added via syringe pump (over a 4-hour period), a solution 1,3-propane sultone (2.02 g, 16.2 mmol) in 1,4-dioxane (total: 4 mL). The solution was stirred at room temperature for 2 hours before it was allowed to warm up to room temperature. The reaction mixture was stirred under these conditions overnight. The solvent was evaporated under reduced pressure. The resultant solid material was recrystallized from 5% water/EtOH. The resulting solid was dissolved in water; and the aqueous solution was passed through an ion exchange column (Dowex 50WX 8, 100 g, solvent: water). The solvent was evaporated under reduced pressure. The product was lyophilized to afford Compound DI (880 mg, 28%). 1H NMR (D2O, 500 MHz) 8 ppm 3.00 (t, 2H, J=7.6 Hz), 2.91 (t, 2H, J=7.3 Hz), 2.01 (m, 2H), 1.34 (s, 6H). 13C NMR (D2O, 125 MHz) 8 ppm 176.93, 63.89, 48.13, 42.04, 22.15, 21.86. ES-MS 224 (M−1).
To a solution of 2-methylcyclohexylamine (98% cis and trans isomers, 10.0 g, 88.3 mmol) in tetrahydrofuran (60 mL) was slowly added a solution of 1,3-propane sultone (10.5 g, 84.1 mmol) in THF (20 mL). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×50 mL). The solid was dissolved in 50% EtOH/water (200 mL); and solution was treated with Dowex 50WX8 resin (15 g). The suspension stirred at room temperature for 15 min. The resin was removed by filtration. The filtrate was concentrated to half of the original volume on a rotary evaporator. The solid product slowly crystallized. The product was collected by filtration, washed with acetone (2×50 mL) and dried in a vacuum oven (50° C.), to afford compound DJ (10.4 g, 53%): 1H NMR (D2O, 500 MHz) δ ppm 3.15 (m, 1H), 3.03 (m, 1H), 2.88 (t, 2H, J=7.3 Hz), 2.76 (m, 1H), 1.98 (m, 3H), 1.68 (m, 2H), 1.51 (m, 2H), 1.18 (m, 3H), 1.01 (m, 1H), 0.92 (m, 3H). 13C NMR (D2O, 125 MHz) δ ppm 62.97, 48.16, 42.72, 34.77, 33.50, 27.57, 24.51, 24.23, 21.51, 17.80. ES-MS 234 (M−1).
To a solution of 2,3-dimethylcyclohexylamine (10.0 g, 79.0 mmol) in tetrahydrofuran (60 mL) was slowly added a solution of 1,3-propane sultone (9.3 g, 75.0 mmol) in THF (20 mL). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with THF (50 mL) and acetone (50 mL). The solid was dissolved in 25% EtOH/water (150 mL), and treated with Dowex 50WX8 resin (15 g). The suspension stirred at room temperature for 5 minutes. The resin was removed by filtration. The filtrate was concentrated to dryness under reduced pressure; and the solid residue was suspended in acetone (100 mL). The solid material was collected by filtration, and dried in vacuo; affording compound DK (7.4 g, 43%). 1H NMR (D2O, 500 MHz) 8 ppm 3.29 (m, 0.5H), 3.09 (m, 2H), 2.88 (t, 2H, J=7.3 Hz), 2.80 (m, 0.5H), 1.99 (m, 3H), 1.40 (m, 7H), 0.81 (m, 6H). 13C NMR (D2O, 125 MHz) 8 ppm 62.85, 61.56, 59.88, 58.22, 48.26, 48.15, 44.29, 43.84, 43.59, 42.65, 42.01, 41.03, 37.34, 36.12, 34.73, 34.45, 33.88, 33.64, 29.39, 28.00, 26.50, 24.25, 24.02, 23.64, 22.42, 21.55, 21.45, 21.35, 19.38, 19.13, 18.82, 18.40, 14.38, 13.39, 4.59. ES-MS 248 (M−1).
To a solution of neopentylamine (8.5 g, 98 mmol) in tetrahydrofuran (75 mL) was slowly added a solution of 1,3-propane sultone (11.5 g, 93 mmol) in THF (20 mL). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×50 mL). The solid was suspended in EtOH (150 mL). The suspension was stirred at reflux for 15 minutes. The solid product was collected by filtration, washed with acetone (2×50 mL), and dried in vacuo, to afford compound DL (13.3 g, 69%). 1H NMR (D2O, 500 MHz) 8 ppm 3.09 (t, 2H, J=7.3 Hz), 2.89 (t, 2H, J=7.3 Hz), 2.78 (s, 2H), 2.04 (m, 2H), 0.901 (s, 9H). 13C NMR (D2O, 125 MHz) 8 ppm 59.44, 48.31, 47.83, 29.91, 26.42, 21.06. ES-MS 208 (M−1).
To a solution of cumylamine (10.5 g, 78 mmol) in tetrahydrofuran (75 mL) was slowly added a solution of 1,3-propane sultone (9.2 g, 74 mmol) in THF (20 mL). The mixture was stirred at reflux for 4 h. The reaction mixture was cooled to room temperature. The solid was collected by filtration, and washed with THF (2×35 mL). The solid was suspended in EtOH (80 mL). The suspension was stirred at reflux for 15 minutes. The solid product was collected by filtration, washed with EtOH (35 mL) and acetone (35 mL). The resulting solid was dried in vacuo, affording compound DM (5.6 g, 30%). 1H NMR (D2O, 500 MHz) 8 ppm 7.41 (m, 5H), 2.74 (m, 4H), 1.88 (m, 2H), 1.66 (s, 6H). 13C NMR (D2O, 125 MHz) 8 ppm 138.36, 129.44, 129.41, 126.52, 61.56, 48.04, 41.21, 24.80, 21.81. ES-MS 256 (M−1).
To a solution of (R)-(+)-1-(4-methoxyphenyl)ethylamine (5.83 g, 38.6 mmol) in tetrahydrofuran (25 mL) was slowly added 1,3-propanesultone (4.56 g, 36.8 mmol). The solution was stirred at reflux for 4 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with THF (25 mL) and acetone (25 mL). The solid was suspended in EtOH (200 mL). The suspension was stirred at reflux for 15 min. The solid was collected by filtration, washed with cold EtOH (50 mL), and dried in a vacuum oven (50° C.), to afford compound FN (5.6 g, 56%). 1H NMR (D2O, 500 MHz) δ ppm 7.26 (d, 2H, J=8.3 Hz), 6.90 (d, 2H, J=8.3 Hz), 4.22 (m, 1H), 3.68 (s, 3H), 2.92 (m, 1H), 2.76 (m, 3H), 1.90 (m, 2H), 1.49 (d, 3H, J=6.8 Hz). 13C NMR (D2O, 125 MHz) 8 ppm 159.87, 129.34, 128.18, 114.85, 57.99, 55.58, 48.05, 44.21, 21.45, 18.21. ES-MS 272 (M−1).
To a solution of (R)-(−)-1-aminoindan (1.0 g, 7.5 mmol) in tetrahydrofuran (10 mL) was slowly added 1,3-propane sultone (890 mg, 7.1 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, and washed with THF (20 mL) and acetone (20 mL). The solid was suspended in 80% acetone/EtOH (40 mL). The suspension was stirred at reflux for 30 sec. The solid product was collected by filtration, washed with acetone (2×20 mL), and dried in vacuo, to afford compound DO (1.1 g, 61%). 1H NMR (D2O, 500 MHz) 8 ppm 7.41 (d, 1H, J=7.3 Hz), 7.30 (m, 2H), 7.24 (m, 1H), 4.72 (m, 1H), 3.14 (t, 2H, J=7.8 Hz), 3.02 (m, 1H), 2.88 (m, 3H), 2.43 (m, 1H), 2.12 (m, 1H), 2.01 (m, 2H). 13C NMR (D2O, 125 MHz) δ ppm 145.38, 136.39, 130.31, 127.20, 125.72, 125.54, 62.98, 48.10, 43.93, 29.84, 28.62, 21.64. [α]D=−1.3° (c=0.00515 in water). ES-MS 254 (M−1).
3-amino-1-propanesulfonic acid (2.0 g, 14.3 mmol) was dissolved in 1.6M NaOH (10 mL). To this solution was added tert-butyl isocyanate (1.1 g, 14.3 mmol). The reaction mixture was stirred at 70° C. for 1 h, followed by addition of one equivalent of tert-butyl isocyanate (1.1 g, 14.3 mmol). The reaction mixture was stirred for 1 h. The solvent was evaporated under reduced pressure. The residue was suspended in EtOH (30 mL). The solid product was collected by filtration, washed with EtOH (20 mL) and acetone (20 mL). The resulting solid was dried in vacuo, to afford the sodium salt of compound DP (2.1 g, 66%). 1H NMR (D2O, 500 MHz) δ ppm 3.03 (t, 2H, J=6.6 Hz), 2.76 (t, 2H, J=7.6 Hz), 1.72 (m, 2H), 1.12 (s, 9H). 13C NMR (D2O, 125 MHz) δ ppm 160.35, 50.26, 48.74, 38.31, 28.86, 25.24. ES-MS 280 (M+Na).
To a solution of 1,2-dimethylpropylamine (10.0 g, 115 mmol) in tetrahydrofuran (80 mL) was slowly added a solution of 1,3-propane sultone (13.7 g, 110 mmol) in THF (20 mL). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid product was collected by filtration, washed with THF (50 mL) and EtOH (50 mL). The resulting solid was dried in vacuo, to afford compound DQ (17.5 g, 76%). 1H NMR (D2O, 500 MHz) δ ppm 3.07 (m, 3H), 2.88 (t, 2H, J=7.3 Hz), 1.97 (m, 3H), 1.10 (d, 3H, J=6.8 Hz), 0.85 (d, 3H, J=6.8 Hz), 0.81 (d, 3H, J=6.8 Hz). 13C NMR (D2O, 125 MHz) δ ppm 59.79, 48.19, 44.18, 29.72, 21.51, 18.44, 15.02, 10.71. ES-MS 232 (M+Na).
To a solution of 4-methylcyclohexylamine (97% cis and trans isomers, 11.0 g, 97.4 mmol) in tetrahydrofuran (70 mL) was slowly added a solution of 1,3-propane sultone (11.5 g, 92.8 mmol) in THF (20 mL). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with THF (50 mL) and acetone (50 mL). The solid was suspended EtOH. The suspension was stirred at room temperature for 5 minutes. The solid product was collected by filtration, washed with EtOH (50 mL), and dried in a vacuum oven (50° C.), to afford compound DR (16.1 g, 75%). 1H NMR (D2O, 500 MHz) δ ppm 3.08 (m, 2.5H), 2.93 (m, 0.5H), 2.87 (m, 2H), 1.99 (m, 4H), 1.64 (m, 3H), 1.47 (m, 1H), 1.24 (m, 2H), 0.88 (m, 1H), 0.78 (m, 3H). 13C NMR (D2O, 125 MHz) δ ppm 57.35, 56.52, 48.16, 18.05, 43.73, 43.30, 32.45, 31.13, 28.92, 28.69, 27.77, 24.55, 21.65, 21.53, 21.20, 18.38. ES-MS 236 (M+1).
To a solution of (+/−)-2-methylbutylamine (10 g, 115 mmol) in tetrahydrofuran (80 mL) was slowly added a solution of 1,3-propane sultone (13.5 g, 109 mmol) in THF (20 mL). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×30 mL). The solid was suspended 95% Acetone/EtOH (200 mL). The suspension was stirred at room temperature for 5 minutes. The solid product was collected by filtration, and dried in a vacuum oven (50° C.), to afford compound DS (17.6 g, 78%). 1H NMR (D2O, 500 MHz) δ ppm 3.07 (t, 2H, J=7.8 Hz), 2.93 (m, 3H), 2.76 (m, 1H), 2.01 (m, 2H), 1.67 (m, 1H), 1.30 (m, 1H), 1.09 (m, 1H), 0.81 (d, 3H, J=6.8 Hz), 0.77 (t, 3H, J=6.8 Hz). 13C NMR (D2O, 125 MHz) δ ppm 53.48, 48.13, 46.92, 31.96, 26.38, 21.29, 16.11, 10.19. ES-MS 210 (M+1).
3-amino-1-propanesulfonic acid (2.0 g, 14.4 mmol) was dissolved a NaOH (1.2 g, 30.2 mmol) solution in a mixture of 1,4-dioxane (5 mL) and water (15 mL). The mixture was cooled to 0° C. before pivaloyl chloride (2.8 mL, 21.6 mmol) in 1,4-dioxane (5 mL) was added dropwise. The reaction mixture was allowed to warm up to room temperature and it was stirred at 65° C. for 4 h. The solvent was evaporated under reduced pressure. The resulting solid was dissolved in water (30 mL), and treated with Dowex 50WX8 resin. The suspension was stirred for 5 minutes and the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The residual material was suspended in 20% EtOH/Acetone. The mixture was stirres at reflux for 30 seconds. The solid product was collected by filtration, and dried in vacuo, to afford compound DT (1.3 g, 41%). 1H NMR (D2O, 500 MHz) δ ppm 3.16 (t, 2H, J=6.8 Hz), 2.75 (t, 2H, J=7.8 Hz), 1.78 (m, 2H), 1.1 (s, 9H). 13C NMR (D2O, 125 MHz) δ ppm 182.75, 48.70, 38.57, 38.18, 26.65, 24.27. ES-MS 222 (M−1).
To a solution of 3,3,5-trimethylcyclohexylamine (5.0 g, 35.4 mmol) in tetrahydrofuran (35 mL) was slowly added a solution of 1,3-propane sultone (4.17 g, 33.7 mmol) in THF. The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL). The solid was suspended 90% Acetone/EtOH (100 mL) The suspension was stirred at room temperature for 5 min. The solid product was collected by filtration, and dried in a vacuum oven (50° C.), affording compound ED (5.9 g, 67%). 1H NMR (D2O, 500 MHz) δ ppm 3.20 (m, 1H), 3.08 (m, 2H), 2.87 (t, 2H, J=6.8 Hz), 1.96 (m, 3H), 1.60 (m, 2H), 1.29 (m, 1H), 1.01 (m, 1H), 0.84 (s, 3H), 0.73 (m, 8H). 13C NMR (D2O, 125 MHz) δ ppm 54.98, 48.06, 46.51, 43.28, 40.96, 37.02, 32.07, 31.23, 26.68, 24.28, 21.67, 21.52. ES-MS 262 (M−1).
To a solution of 2-aminoindan (2.50 g, 18.8 mmol) in tetrahydrofuran (25 mL) was slowly added 1,3-propane sultone (2.24 g, 17.9 mmol). The mixture was stirred at reflux for 2.5 h. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL). The crude product was suspended in 90% Acetone/EtOH (100 mL). The suspension was stirred at room temperature for 5 minutes. The solid product was collected by filtration, and dried in a vacuum oven (50° C.), to afford compound EE (3.1 g, 67%). 1H NMR (DMSO, 500 MHz) δ ppm 7.25 (m, 2H), 7.20 (m, 2H), 4.00 (m, 1H), 3.30 (m, 2H), 3.13 (m, 2H), 3.02 (m, 2H), 2.64 (m, 2H), 1.96 (m, 2H). 13C NMR (DMSO, 125 MHz) δ ppm 130.98, 127.80, 125.25, 57.70, 49.24, 45.87, 36.32, 22.66. ES-MS 254 (M−1).
To a solution of 4-aminobiphenyl (3.0 g, 17.8 mmol) in tetrahydrofuran (25 mL) was slowly added 1,3-propane sultone (2.11 g, 16.9 mmol). The solution was stirred at reflux for 3 h. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL). The crude product was dissolved in a hot solution of 80% MeOH/H2O (120 mL). To this warm solution was added Dowex 50WX8 ion exchange resin (10 g). The hot suspension was stirred for 5 minutes and the resin was removed by filtration. The filtrate was concentrated to dryness under reduced pressure. The residual solid was dried in a vacuum oven (50° C.), affording compound EF (283 mg, 6%). 1H NMR (DMSO, 500 MHz) δ ppm 7.77 (d, 2H, J=7.8 Hz), 7.66 (d, 2H, J=7.8 Hz), 7.46 (m, 3H), 7.37 (m, 1H), 3.44 (m, 2H), 2.68 (m, 2H), 1.98 (m, 2H). 13C NMR (DMSO, 125 MHz) δ ppm 139.74, 129.69, 128.74, 128.33, 127.31, 122.23, 49.70, 22.93. ES-MS 290 (M−1).
To a solution of (1S,2S)-2-amino-3-methoxy-1-phenyl-1-propanol (1.0 g, 5.5 mmol) in tetrahydrofuran (10 mL) was slowly added 1,3-propane sultone (662 mg, 5.3 mmol). The mixture was stirred at reflux for 2.5 h. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL). The crude product was suspended 80% Acetone/EtOH. The suspension was stirred at reflux for 30 seconds. The solid product was collected by filtration and dried in a vacuum oven (50° C.), to afford compound EG (1.0 g, 63%). 1H NMR (D2O, 500 MHz) δ ppm 7.32 (m, 5H), 4.77 (d, 1H, J=9.8 Hz), 3.53 (m, 1H), 3.37 (m, 1H). 3.26 (m, 1H), 3.17 (m, 6H), 2.91 (t, 2H, J=7.3 Hz), 2.07 (m, 2H). 13C NMR (D2O, 125 MHz) δ ppm 139.09, 129.33, 129.27, 127.11, 70.85, 66.29, 62.62, 58.76, 48.14, 44.24, 21.47. [α]D=+42.6° (c=0.00091 in water), ES-MS 302 (M−1).
To a solution of (1R,2R,3R,5S)-(−)-isopinocampheylamine (2.0 g, 13.0 mmol) in tetrahydrofuran (20 mL) was slowly added 1,3-propanesultone (1.56 g, 12.5 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid product was collected by filtration, washed with acetone (2×25 mL), and dried in a vacuum oven (50° C.), to afford compound EH (2.7 g, 80%). 1H NMR (DMSO, 500 MHz) δ ppm 3.32 (m, 2H), 3.09 (d, 2H), 2.67 (m, 2H), 2.30 (m, 2H), 1.96 (m, 4H), 1.75 (m, 2H), 1.18 (s, 3H), 1.11 (m, 4H), 0.90 (s, 3H). 13C NMR (DMSO, 125 MHz) δ ppm 55.97, 50.11, 47.55, 45.86, 41.15, 40.91, 38.94, 32.81, 31.61, 27.96, 23.85, 22.59, 21.21, [α]D=−17.2° (c=0.00083 in water), ES-MS 274 (M−1).
To a solution of 2-amino-1-methoxypropane (5.0 g, 17.8 mmol) in tetrahydrofuran (25 mL) was slowly added 1,3-propane sultone (2.12 g, 17.0 mmol). The mixture was stirred at reflux for 4 h. The reaction mixture was cooled to room temperature. The solid product was collected by filtration, washed with acetone (2×25 mL), and dried in a vacuum oven (50° C.), to afford compound EI (3.1 g, 86%). 1H NMR (D2O, 500 MHz) δ ppm 3.53 (m, 1H), 3.41 (m, 2H), 3.27 (s, 3H), 3.11 (m, 2H), 2.89 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.18 (d, 3H, J=5.9 Hz). 13C NMR (D2O, 125 MHz) δ ppm 71.73, 58.80, 53.79, 48.08, 43.55, 21.52, 12.79. ES-MS 210 (M−1).
To a solution of (R)-(+)-2-amino-3-phenyl-1-propanol (1.0 g, 6.6 mmol) in tetrahydrofuran (10 mL) was slowly added 1,3-propane sultone (785 mg, 6.3 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL). The crude product was suspended 80% acetone/EtOH (100 mL). The suspension was stirred at reflux for 30 seconds. The solid product was collected by filtration, washed with acetone (2×25 mL), and dried in a vacuum oven (50° C.), to afford compound EJ (890 mg, 52%). 1H NMR (DMSO, 500 MHz) δ ppm 8.58 (s (broad), 1H), 7.26 (m, 5H), 5.30 (s (broad), 1H), 3.53 (m, 1H), 3.31 (m, 2H). 3.14 (t, 2H), 2.98 (m, 1H), 2.80 (m, 1H), 2.62 (t, 2H), 1.98 (m, 2H). 13C NMR (DMSO, 125 MHz) δ ppm 137.31, 130.00, 129.26, 127.49, 60.16, 57.78, 49.90, 45.34, 33.78, 22.49. [α]D=+9.7° (c=0.00118 in water). ES-MS 272 (M−1).
To a solution of (S)-(−)-2-amino-3-phenyl-1-propanol (2.0 g, 13.2 mmol) in tetrahydrofuran (20 mL) was slowly added 1,3-propane sultone (1.57, 12.6 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL). The crude product was suspended 80% Acetone/EtOH. The suspension was stirred at reflux for 30 seconds. The solid product was collected by filtration, and dried in a vacuum oven (50° C.), to afford compound EK (1.9 g, 56%). 1H NMR (DMSO, 500 MHz) δ ppm 8.63 (s (broad), 1H), 7.27 (m, 5H), 5.31 (s (broad), 1H), 3.53 (m, 1H), 3.25 (m, 2H). 3.15 (t, 2H), 2.98 (m, 1H), 2.80 (m, 1H), 2.61 (t, 2H), 1.99 (m, 2H). 13C NMR (DMSO, 125 MHz) δ ppm 137.31, 130.01, 129.27, 127.49, 60.18, 57.77, 49.88, 45.32, 33.78, 22.49. [α]D=−7.5° (c=0.00118, H2O). ES-MS 272 (M−1).
To a solution of N-methyl-tert-butylamine (2.0 g, 22.9 mmol) in acetone (25 mL) was slowly added 1,3-propane sultone (2.72 g, 21.8 mmol). The mixture was stirred at reflux for 3 h. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL). The crude product was suspended in 80% Acetone/EtOH. The suspension was stirred at reflux for 30 seconds. The solid product was collected by filtration, and dried in a vacuum oven (50° C.), to afford compound EN (2.9 g, 65%). 1H NMR (DMSO, 500 MHz) δ ppm 9.40 (s (broad), 1H), 3.45 (m, 1H), 2.85 (m, 1H), 2.59 (m, 5H), 1.99 (m, 2H), 1.28 (s, 9H). 13C NMR (DMSO, 125 MHz) δ ppm 63.23, 51.12, 49.73, 34.79, 25.50, 21.72. ES-MS 208 (M−1).
To a solution of (1R,2S)-1-amino-2-indanol (2.37 g, 15.9 mmol) in tetrahydrofuran (25 mL) was slowly added 1,3-propane sultone (1.89 g, 15.1 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, and washed with acetone (2×25 mL). The crude product was suspended in 80% acetone/ethanol (75 mL). The suspension was stirred at reflux for 30 seconds. The solid product was collected by filtration, and dried in a vacuum oven (50° C.), to afford the compound EO (2.7 g, 65%). 1H NMR (D2O, 500 MHz) δ ppm 7.36 (d, 1H, J=7.4 Hz), 7.24 (m, 3H), 4.70 (q, 1H, J=5.5 Hz), 4.53 (d, 1H, J=5.4 Hz), 3.23 (t, 2H, J=7.8 Hz), 3.10 (m, 1H), 2.89 (m, 3H), (m, 1H), 2.08 (m, 2H). 13C NMR (D2O, 125 MHz) δ ppm 141.60, 134.30, 130.53, 127.61, 126.10, 125.69, 70.42, 64.08, 48.25, 44.73, 38.33, 21.50. [α]D=+3.0° (c=0.0018, water) ES-MS 272 (M+1).
To a solution of (S)-(−)-2-amino-3-methyl-1-butanol (2.50 g, 24.2 mmol) in tetrahydrofuran (35 mL) was slowly added 1,3-propane sultone (2.89 g, 23.0 mmol). The mixture was stirred at reflux for 3 h. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL). The crude product was suspended in 80% acetone/ethanol (75 mL). The suspension was stirred at reflux for 30 seconds. The solid product was collected by filtration, and dried in a vacuum oven (50° C.), to afford compound EP (2.9 g, 56%). 1H NMR (D2O, 500 MHz) δ ppm 3.78 (dd, 1H), 3.62 (dd, 1H), 3.13 (m, 2H), 2.90 (m, 1H), 2.88 (t, 3H), 1.90 (m, 3H), 0.90 (d, 3H), 0.84 (d, 3H). 13C NMR (D2O, 125 MHz) δ ppm 64.74, 57.24, 48.20, 44.44, 26.98, 21.49, 18.53, 17.00. [α]D=+4.1° (c=0.0017 in water), ES-MS 224 (M−1).
L-valinamide hydrochloride (2.50 g, 16.4 mmol) was treated with a saturated solution of K2CO3 (75 mL). The mixture was extracted with EtOAc (3×75 mL). The organic extracts were combined, dried over Na2SO4. The solid material was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure. The residual material was dried in vacuo.
To a solution of L-valinamide (1.57 g, 13.5 mmol) in tetrahydrofuran (20 mL) was slowly added 1,3-propane sultone (1.61 g, 12.9 mmol). The mixture was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, washed with acetone (2×25 mL). The crude product was dissolved in water (60 mL) and treated with ion exchange resin Dowex Marathon C (strongly acidic, 15 g). The mixture was stirred for 15 minutes. The resin was removed by filtration. The filtrate was poured into EtOH (250 mL). The solid product, after the completion of the precipitation, was collected by filtration, and dried in vacuo, to afford compound EQ (1.6 g, 51%). 1H NMR (D2O, 500 MHz) δ ppm 3.63 (d, 1H), 3.05 (m, 2H), 2.85 (m, 2H), 2.05 (m, 4H), 0.93 (d, 3H), 0.88 (d, 3H). 13C NMR (D2O, 125 MHz) δ ppm 170.24, 65.92, 48.16, 46.31, 29.59, 21.31, 18.02, 17.07. [α]D=+10.5° (c=0.0027 in water), ES-MS 237 (M−1).
Isobutylamine (2.4 mL, 24 mmol) was added to a solution of 1,3-propane sultone (3.04 g, 24.5 mmol) in 2-butanone (20 mL). The mixture was heated to reflux. After about 10 minutes, the mixture had turned into a lump. It was cooled to room temperature. Acetone was added and the lump was crushed. The solid was collected by filtration and dried in-vacuo (2.2 g). The white solid was suspended in ethanol (10 mL) and the mixture brought to reflux. A significant amount of the solid dissolved. Water was added slowly until a clear pink solution was obtained. The solution was left at room temperature overnight. The flask was placed in a fridge for 2 hours. The solid product was collected by filtration, rinsed with ethanol (5 mL), washed with ether (10 mL), and dried in-vacuo. Compound CE was obtained as long, fine white needles (1.87 g, 40% yield). m.p. 255-57° C. 1H NMR (500 MHz, D2O) δ 0.88 (d, J=6.8 Hz, 6H), 1.85-1.93 (m, J=6.8 Hz, 1H), 2.03 (qt, J=7.6 Hz, 2H), 2.80 (d, J=7.3 Hz, 2H), 2.90 (t, J=7.3 Hz, 2H), 3.08 (t, J=8.1 Hz, 2H). 13C NMR (125 MHz, D2O) δ 19.2, 21.2, 25.8, 46.9, 48.1, 54.9 ES-MS 196 (M+1). FT-IR (KBr) νmax 3566, 2973, 2021, 1719.
Isoamylamine (4 mL, 34.5 mmol) was added to a solution of 1,3-propane sultone (4.7 g, 38 mmol) in 2-butanone (70 mL). The mixture was warmed to reflux. After 30 minutes, the mixture was too thick to stir. Acetone (15 mL) was added. The reflux was maintained for a total of 4 hours. The suspension was cooled at room temperature. The white solid was collected by filtration, rinsed with acetone (10 mL), then with ether (10 mL). Compound CH was obtained as a very light, fluffy white solid (4.48 g, 62% yield). m.p. 220° C.: decomposed. 1H NMR (500 MHz, D2O) δ 0.65 (d, J=6.3 Hz, 6H), 1.29 (q, J=7.7 Hz, 2H), 1.35-1.42 (m, J=6.6 Hz, 1H), 1.86 (qt, J=7.6 Hz, 2H), 2.74 (t, J=7.3 Hz, 2H), 2.81 (t, J=8.1 Hz, 2H), 2.93 (t, J=7.8 Hz, 2H). 13C NMR (125 MHz, D2O) δ 21.3, 21.4, 25.2, 34.2, 46.1, 46.2, 47.9
A solution of 2-bromoethanesulfonic acid, sodium salt (4.2 g, 20 mmol) in water (total 12 mL) was added over 6 hours to a 42° C. solution of t-butylamine (10 mL, 94 mmol) in a mixture of water (10 mL) and 1,4-dioxane (10 mL). The mixture was stirred at 42° for 18 hours. The mixture was then heated to 60° C. for 24 h. By proton NMR, 30% of elimination product (vinylsulfonic acid) was observed. The mixture was concentrated to dryness and treated with ethanol at refluxing temperature. The solid material was collected (crop 1). The mother liquor was concentrated to dryness and the solid was again treated with ethanol at refluxing temperature, and the solid material was collected (crop 2). Both crops of the solid material were dissolved in water, and the resultant aqueous solutions passed in sequence through a Dowex 50 W X 8 ion-exchange column (100 g resin). The fractions containing the title compound were collected and concentrated to dryness. The solid material obtained was recrystallized from a mixture of ethanol (20 mL) and water (2 mL). The crystals were collected by filtration, dried in a vacuum oven at 60° C. for 18 hours. Compound DU was obtained as fine white needles (860 mg, 24% yield). 1H NMR (500 MHz, D2O) δ 1.16 (s, 9H), 3.02 (t, J=6.8 Hz, 2H), 3.19 (t, J=6.8 Hz, 2H). 13C NMR (125 MHz, D2O) δ 24.8, 37.3, 47.0, 57.8. ES-MS 182 (M+1)
A mixture of cyclohexanemethylamine (11.12 mL, 0.085 mol) and 1,3-propane sultone (11.00 g, 0.090 mol) in acetonitrile (120 mL) was heated at reflux for 2 hours. The mixture was cooled to room temperature. The solid was collected by filtration, air-dried for 20 minutes (19 g). The solid was suspended in methanol (100 mL) and the suspension was heat to reflux. Water (4 mL) was added dropwise until a clear solution was obtained at refluxing temperature. The mixture was then cooled to 5° C. with stirring. The solid was collected by suction filtration, air-dried for 45 minutes, and further dried in a vacuum oven at 60° C. for over 3 days. Compound DV was obtained as white flakes, (16.23 g, 81% yield.). 1H NMR (500 MHz, D2O) δ 0.82 (br q, J=11 Hz, 2H), 0.91-1.09 (m, 3H), 1.43-1.53 (m, 6H), 1.93 (qt, J=7.3 Hz, 2H), 2.71 (d, J=6.3 Hz, 2H), 2.80 (t, J=7.3 Hz, 2H), 2.71 (t, J=7.8 Hz, 2H). 13C NMR (125 MHz, D2O) δ 21.2, 25.0, 25.5, 29.9, 34.7, 46.8, 48.1, 53.7. ES-MS 236 (M+1)
A mixture of 1,1-diethylpropargylamine (5 g, 45 mmol) and 1,3-propane sultone (6.05 g, 49.5 mmol) in THF (25 mL) was heated at reflux for 5 hours. The mixture was cooled to room temperature. The solid was collected by filtration, rinsed with diisopropylether (2×10 mL) then dried overnight in the vacuum oven (7.16 g). The solid was suspended in ethanol (30 mL) and the suspension was heated at reflux for 1 hour. The mixture was then cooled to room temperature and the solid was collected by suction filtration, air-dried for 5 m, and further dried in a vacuum oven at 60° C. overnight (5.86 g). There was still a significant amount of ethanol present. The solid was further dried in the vacuum oven for 40 hours. Compound DW was obtained as a fine white solid, (5.66 g, 81% yield). 1H NMR (500 MHz, D2O) δ 0.92 (t, J=7.6 Hz, 2H), 1.71 (q, J=7.3 Hz, 3H), 1.93 (qt, J=7.3 Hz, 2H), 2.81 (t, J=7.3 Hz, 2H), 2.94 (s, 1H), 3.13 (t, J=7.6 Hz, 2H). 13C NMR (125 MHz, D2O) δ 7.2, 21.6, 27.8, 41.4, 48.1, 62.2, 78.6, 78.9. ES-MS 234 (M+1)
A mixture of 1-ethynylcyclohexylamine (6 g, 48.7 mmol) and 1,3-propane sultone (6.55 g, 53.6 mmol) in THF (35 mL) was heated at reflux for 2 hours (thick paste). The mixture was cooled to room temperature. The solid was collected by filtration, rinsed with THF (3×5 mL), air-dried 15 minutes (7.3 g). The solid was suspended in ethanol (30 mL) and the suspension was heated at reflux for 1 hour. The mixture was then cooled to room temperature and the solid was collected by suction filtration, rinsed with ethanol (2×5 mL), air-dried for 10 min, and further dried in a vacuum oven at 60° C. overnight (cropl, 7.10 g). The combined mother liquors were stirred overnight at room temperature. There was a lot of solid. The solid was collected by filtration, rinsed with acetone (3×5 mL), air-dried for 30 minutes, and then suspended in ethanol (12 mL). The suspension was heated at reflux for 1 hour. The mixture was then cooled to room temperature and the solid was collected by suction filtration, rinsed with ethanol (2×5 mL), air-dried for 2 minutes, and further dried in a vacuum oven at 60° C. overnight (crop 2: 1.85 g). Compound DX was obtained as a fine white solid (two crops in total 8.95 g, 75% yield). 1H NMR (500 MHz, D2O) δ 0.95-1.05 (m, 1H), 1.38-1.54 (m, 5H), 1.60-1.64 (m, 2H), 1.94 (qt, J=7.8 Hz, 2H), 2.85 (t, J=7.3 Hz, 2H), 3.01 (s, 1H), 3.22 (t, J=7.8 Hz, 2H). 13C NMR (125 MHz, D2O) δ 21.8, 22.3, 24.2, 34.4, 40.9, 48.0, 59.2, 78.6, 79.3. ES-MS 243.0 (M−1).
A mixture of (±)-2-Amino-1-phenylethanol (9.9 g, 72 mmol) and 1,3-propane sultone (9.3 g, 76 mmol) in acetonitrile (70 mL) and ethanol (2 mL) was heated at reflux for 1.5 hours. The mixture was cooled to room temperature. The solid was collected by filtration, rinsed with acetonitrile (2×25 mL) air-dried for 20 minutes (21.3 g). The solid was suspended in methanol (110 mL) and the suspension was heated to reflux. Water (4 mL) was added dropwise until a clear solution was obtained. The mixture was then cooled to room temperature. The solid was collected by suction filtration, air-dried for 30 minutes, and further dried in a vacuum oven at 60° C. for 40 hours (crop 1, 4.47 g). The combined mother liquor was stored at −20° C. for 40 hours. A second crop of the solid was collected by filtration, rinsed with acetone (2×15 mL), air-dried (1 hour), and further dried in a vacuum oven at 60° C. for 24 hours. Compound DY was obtained in two crops (total 7.82 g, 42% yield). 1H NMR (500 MHz, D2O) 8, 2.03-2.06 (m, 2H), 2.90 (t, J=7.3 Hz, 2H), 3.16 (t, J=7.3 Hz, 2H), 3.20-3.24 (m, 2H), 4.92-4.95 (m, 1H), 7.30-7.37 (m, 5H). 13C NMR (125 MHz, D2O) δ 21.3, 46.6, 78.1, 53.2, 69.0, 126.1, 129.0, 129.2, 139.6. ES-MS 260 (M+1).
A mixture of (S)-(−)-(4-methoxyphenyl)ethylamine (1.83 g, 12.1 mmol) and 1,3-propane sultone (1.6 g, 13 mmol) in acetonitrile (25 mL) was heated at reflux for 2.5 hours. The mixture was cooled to room temperature. The solid was collected by filtration, rinsed with acetonitrile (2×5 mL) air-dried for 15 minutes (3.07 g). The solid was suspended in ethanol (15 mL) and the suspension was heated at reflux for 1 hour. The mixture was then cooled to room temperature. The solid was collected by suction filtration, rinsed with ethanol (2×10 mL), air-dried for 15 minutes, and further dried in a vacuum oven at 60° C. for 18 hours. Compound DZ was obtained as a white solid (2.95 g, 10.8 mmol, 89% yield). 1H NMR (500 MHz, D2O) δ 1.52 (d, J=6.8 Hz, 3H), 1.88-1.98 (m, 2H), 2.76-2.79 (m, 3H), 2.80-2.98 (m, 1H), 3.71 (s, 3H), 4.25 (qt, J=6.7 Hz, 2H), 6.93 (d, J=8.3 Hz, 2H), 7.29 (d, J=8.3 Hz, 2H). 13C NMR (125 MHz, D2O) δ 18.3, 21.5, 44.2, 48.1, 55.6, 58.0, 114.9, 128.2, 129.4, 159.9. ES-MS 274. (M+1). [α]D=−28.8° (c=0.0038 in water)
A mixture of 4-Bromophenethylamine (4 g, 20 mmol) and 1,3-propane sultone (2.56 g, 21 mmol) in acetonitrile (30 mL) was heated at reflux for 2.5 hours. The mixture was cooled to room temperature. The solid was collected by filtration, rinsed with acetonitrile (2×5 mL) air-dried for 15 minutes (9.57 g), and further dried for 15 minutes in vacuo (8.02 g). The solid was suspended in ethanol (40 mL) and the suspension was heated at reflux for 1 hour. The mixture was then cooled to room temperature. The solid was collected by suction filtration, rinsed with ethanol (2×5 mL), air-dried for 15 minutes, and further dried in a vacuum oven at 60° C. for 18 hours. Compound EA was obtained as a white solid (6.04 g, 18.8 mmol, 94% yield). 1H NMR (500 MHz, DMSO) δ 1.95 (t, J=6.3 Hz, 3H), 2.63 (t, J=6.1 Hz, 2H), 2.88 (t, J=7.6 Hz, 2H), 3.09 (t, J=6.3 Hz, 2H), 3.15 (t, J=7.8 Hz, 2H), 7.25 (2, J=7.8 Hz, 2H), 7.53 (2, J=7.8 Hz, 2H), 8.63 (br s, 2H). 13C NMR (125 MHz, DMSO) δ 21.8, 31.0, 46.7, 47.2, 48.8, 119.9, 131.0, 131.4, 136.5. ES-MS 324 (M+1).
A mixture of (S)-(−)-1-aminoindan (0.92 g, 6.9 mmol) and 1,3-propane sultone (0.93 g, 7.6 mmol) in acetonitrile (15 mL) was heated at reflux for 2.5 hours. The mixture was cooled to room temperature. The solid was collected by filtration, rinsed with acetonitrile (2×4 mL), air-dried for 15 minutes. The solid was suspended in ethanol (12 mL) and the suspension was heated at reflux for 1 hour. The mixture was then cooled to room temperature. The solid was collected by suction filtration, rinsed with ethanol (2×4 mL), air-dried for 15 minutes, and further dried in a vacuum oven at 60° C. over the weekend. Compound EB was obtained as a light pink solid (1.54 g, 87% yield). 1H NMR (500 MHz, D2O) δ 2.00 (qt, J=7.3 Hz, 2H), 2.09-2.13 (m, 1H), 2.40-2.45 (m, 1H), 2.84-2.87 (m, 3H), 2.98-3.04 (m, 1H), 3.12 (t, J=7.8 Hz, 2H), 4.67-4.70 (m, 1H), 7.22 (m, 2H), 7.29-7.32 (m, 2H), 7.40 (d, J=7.8 Hz, 1H). 13C NMR (125 MHz, D2O) δ 21.6, 28.6, 29.8, 43.9, 48.1, 63.0, 125.5, 125.7, 127.2, 130.3, 136.3, 145.4. ES-MS 256 (M+1). [α]D=−1.0° (c=0.003095 in water).
A mixture of cyclobutylamine (1.11 g, 15.6 mmol) and 1,3-propane sultone (2 g, 17 mmol) in acetonitrile (18 mL) was heated at reflux. The mixture turned to a lump within 15 minutes. THF (10 mL) was added. The reflux was maintained for 1 hour. The mixture was cooled to room temperature. The solid was collected by filtration, rinsed with acetonitrile (2×4 mL), air-dried for 60 minutes (2.41 g). The solid was suspended in methanol (20 mL) and the suspension was heated at reflux until all the solid material was dissolved. The mixture was then cooled to room temperature. The solid thus formed was collected by suction filtration, rinsed with methanol (2×4 mL), air-dried for 20 minutes, and further dried in a vacuum oven at 40° C. for 18 hours. Compound EC was obtained as a white solid (1.81 g, 60% yield). 1H NMR (500 MHz, D2O) δ 1.70-1.77 (m, 2H), 1.94-2.03 (m, 4H), 2.18 (br s, 2H), 2.85 (t, J=6.8 Hz, 1H), 2.95 (t, J=7.3 Hz, 1H), 3.63-3.66 (m, 1H). 13C NMR (125 MHz, D2O) δ 14.5, 21.5, 26.1, 43.6, 48.0, 51.8. ES-MS 194 (M+1).
Mexiletine hydrochloride (2.45 g, 11.3 mmol) was freed with 1N NaOH (50 mL), extracted with ethyl acetate (2×50 mL). The combined extract was dried over sodium sulfate. The solvent was evaporated. A solution of 1,3-propane sultone (1.46 g, 11.9 mmol) in THF (35 mL) was added to the free amine. The mixture was heated at reflux for 4 hours. The mixture was cooled to room temperature; and the resultant solid material was collected by filtration, rinsed with THF (5 mL). The solid was dried overnight at 40° C. The filtrated dried in air overnight to afford a brownish solid (1.45 g) it was less pure than the first crop thus discarded. Compound EV was obtained as a white solid (1.19 g, 56% (99% crude) yield). 1H NMR (500 MHz, DMSO) δ 1.39 (d, J=6.3 Hz, 3H), 2.02 (t, J=5.9 Hz, 2H), 2.26 (s, 6H), 2.67 (t, J=5.9 Hz, 2H), 3.21 (br d, J=19.5 Hz, 2H), 3.61 (br s, 1H), 3.83-3.91 (m, 2H), 6.95 (t, J=7.3 Hz, 1H), 7.04 (m, 2H), 8.91 (br s, 2H). 13C NMR (125 MHz, DMSO) δ 13.3, 16.0, 21.8, 44.6, 49.2, 52.9, 70.8, 124.3, 128.9, 130.4, 154.2. ES-MS 302 (M+1).
S-(+)-2-Amino-1-methoxy-3-phenylpropane hydrochloride (2.06 g, 10.0 mmol) was freed with saturated potassium carbonate (20 mL). The aqueous mixture was extracted with ethyl acetate (3×15 mL); and the combined extract was dried over sodium sulfate. The solvent was evaporated. A solution of 1,3-propane sultone (1.29 g, 10.5 mmol) in THF (15 mL) was added to the free amine. The mixture was heated at reflux for 4 hours. The mixture was cooled to room temperature and stirred for 1 hour. The solid was collected by filtration, rinsed with acetone (5 mL). The solid was dried overnight at 40° C. Compound EW was obtained as a white solid (2.48 g, 83% yield). 1H NMR (500 MHz, DMSO) δ 1.99 (m, 2H), 2.65 (t, J=6.1 Hz, 2H), 2.80 (t, J=12.0 Hz, 1H), 3.05 (m, 2H), 3.17 (m, 3H), 3.22 (dd, J=3.4 Hz, 10.7 Hz, 2H), 3.33 (m, 1H), 3.43 (d, J=10.7 Hz, 2H), 3.50 (m, 1H), 7.27 (m, 3H), 7.35 (t, J=7.1 Hz, 2H), 8.86 (br d, 1H). 13C NMR (125 MHz, DMSO) δ 21.8, 33.4, 45.0, 49.2, 57.8, 58.5, 68.1, 126.9, 128.7, 129.2, 136.3. ES-MS 310 (M+1). [α]=−0.8° (c=0.0025 in water).
A solution of hydroxylamine 50% in water (wt./wt, 7 mL) was added to a solution of L-N-(3-sulfopropyl)phenylalanine ethyl ester (1.00 g, 3.17 mmol in water (5 mL). The mixture was stirred at room temperature for 24 hours. The mixture was concentrated to dryness. The resulting solid was dissolved in a mixture of hot methanol (10 mL) and water; and the mixture was stored at 5° C. for 3 days. Only a very small amount of solid had formed. Upon addition of acetone (3 mL), a large amount of solid formed. The solid was collected by suction-filtration, rinsed with acetone (2×5 mL) then dried overnight at 40° C. in a vacuum oven. Compound FO was obtained as a white solid (700 mg, 73%). 1H NMR (500 MHz, D2O) δ 2.03-2.07 (m, 2H), 2.88-2.90 (M, 2H), 2.99-3.03 (M, 1H), 3.07-3.12 (M, 1H), 3.18-3.23 (m, 1H), 3.82-3.85 (m, 1H), 7.15 (d, J=6.8 Hz, 2H), 7.26-7.31 (m, 3H). 13C (125 MHz, D2O) δ 19.3, 33.8, 43.2, 45.8, 57.9, 126.0, 127.1, 127.3, 131.4, 162.2. ES-MS 303 (M+Na). [α]D=40° (c=0.001983 in water).
L-Phenylalanine benzylester hydrochloride (2.0 g, 6.9 mmol) was treated with a saturated aqueous solution of K2CO3 (50 mL) and EtOAc (3×50 mL) was added. The organic extracts were separated, combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of L-Phenylalanine benzylester (1.8 g, 6.8 mmol) in 1,4-dioxane (10 mL) was added 1,3-propanesultone (708 mg, 6.5 mmol). The solution was stirred at reflux. After 1 hour, 20 mL of 1,4-dioxane was added to allow for good stiffing. The reaction was stirred at reflux for an additional 1 hour. It was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL). The product was suspended 80% Acetone/EtOH. The suspension was stirred at reflux for 30 seconds. The solid was filtered and dried in the vacuum oven (50° C.) affording the title compound (1.14 g, 46%). 1H NMR (DMSO, 500 MHz) δ ppm 7.27 (m, 6H), 7.12 (m, 4H), 5.05 (dd, 2H, J=12.3 Hz), 4.49 (m, 1H). 3.29 (m, 1H), 3.00 (m, 1H), 2.98 (m, 1H), 2.61 (t, 2H, J=6.5 Hz), 1.97 (m, 2H). 13C (DMSO, 125 MHz) δ ppm 168.88, 135.21, 134.90, 130.00, 129.31, 129.07, 128.01, 67.98, 60.50, 49.77, 46.72, 35.87, 22.43. [α]D=+4.8° (c=0.00073 in water), ES-MS 378 (M+1).
L-valine methylester hydrochloride (5.0 g, 29.8 mmol) was treated with a saturated solution of K2CO3 (75 mL) and EtOAc (3×75 mL) was added. The organic extracts were separated, combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of L-valine methylester in THF (25 mL) was slowly added 1,3-propanesultone (2.49 g, 19.9 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×30 mL). It was dried in the vacuum oven (50° C.) affording the title compound (2.52 g, 50%). 1H NMR (D2O, 500 MHz) δ ppm 3.92 (m, 1H), 3.75 (s, 3H), 3.13 (t, 2H, J=6.8 Hz), 2.88 (t, 2H, J=6.8 Hz), 2.24 (m, 1H), 2.06 (m, 2H)), 0.96 (d, 3H, J=6.8 Hz), 0.88 (d, 3H, J=6.8 Hz). 13C (D2O, 125 MHz) δ ppm 169.35, 65.85, 55.61, 48.14, 45.59, 29.48, 21.32, 18.25, 16.57. [α]D=+9.6° (c=0.0014 in water), ES-MS 254 (M+1).
To a solution of (R)-(−)-cyclohexylethylamine (2.5 g, 19.7 mmol) in tetrahydrofuran (25 mL) was slowly added 1,3-propanesultone (2.33 g, 18.7 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo, affording the title compound (3.47 g, 74%). 1H NMR (D2O, 500 MHz) δ ppm 3.09 (m, 3H), 2.88 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.58 (m, 6H), 1.13 (m, 5H), 1.03 (m, 3H). 13C (D2O, 125 MHz) δ ppm 58.37, 48.17, 44.00, 39.84, 29.00, 26.01, 25.82, 25.73, 25.47, 21.51, 11.79. [α]D=+4.5° (c=0.0022 in water), ES-MS 250 (M−1).
To a solution of (S)-(+)-cyclohexylethylamine (5.0 g, 39.3 mmol) in tetrahydrofuran (50 mL) was slowly added 1,3-propanesultone (4.66 g, 37.4 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and washed with acetone (2×25 mL). The solid was suspended in 80% acetone/EtOH (200 mL). The suspension was stirred at reflux for 30 seconds before the solid was filtered and dried in vacuo, affording the title compound (6.13 g, 66%). 1H NMR (D2O, 500 MHz) δ ppm 3.09 (m, 3H), 2.88 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.55 (m, 6H), 1.13 (m, 5H), 1.03 (m, 3H). 13C (D2O, 125 MHz) δ ppm 59.37, 48.17, 44.00, 39.84, 29.00, 26.01, 25.82, 25.73, 25.47, 21.51, 11.78. [α]D=−2.8° (c=0.0014 in water), ES-MS 250 (M−1).
To a solution of 4-tert-butylcyclohexylethylamine (mixture of cis and trans isomers, 2.5 g, 16.1 mmol) in tetrahydrofuran (30 mL) was slowly added 1,3-propanesultone (1.84 g, 15.3 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and washed with acetone (2×35 mL). The solid was suspended in 80% acetone/EtOH (200 mL). The suspension was stirred at reflux for 30 seconds before the solid was filtered and dried in vacuo, affording the title compound (3.07 g, 72%). 1H NMR (DMSO, 500 MHz) δ ppm 3.21 (m, 0.5H), 3.04 (m, 2H), 2.89 (m, 1H), 2.67 (m, 0.5H), 1.97 (m, 4H), 1.77 (m, 1H), 1.52 (m, 1H), 1.19 (m, 2H), 0.96 (m, 2H), 0.81 (s, 9H). 13C (DMSO, 125 MHz) δ ppm 56.47, 53.62, 50.44, 49.77, 47.56, 47.04, 46.28, 44.49, 32.98, 32.74, 29.61, 28.10, 28.03, 25.57, 22.68, 22.48, 21.01. ES-MS 276 (M−1).
To a solution of (1S,2S)-2-benzyloxycyclopentylamine (1.0 g, 5.2 mmol) in tetrahydrofuran (12 mL) was slowly added 1,3-propanesultone (601 mg, 5.0 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo, affording the title compound (1.36 g, 87%). 1H NMR (D2O, 500 MHz) δ ppm 7.32 (m, 5H), 4.53 (d, 1H, J=11.2 Hz), 4.41 (d, 1H, J=11.2 Hz), 4.01 (m, 1H), 3.36 (m, 1H), 3.00 (t, 2H, J=7.8 Hz), 2.80 (t, 2H, J=7.8 Hz), 2.00 (m, 4H), 1.64 (m, 3H), 1.49 (m, 1H). 13C NMR (D2O, 125 MHz) δ ppm 136.99, 129.06, 129.01, 128.77, 81.78, 71.81, 63.88, 48.01, 45.33, 29.91, 27.43, 21.60, 20.93. [α]D=+31.1° (c=0.0064 in water). ES-MS 314 (M+1).
To a solution of (1R,2R)-2-benzyloxycyclopentylamine (1.0 g, 5.2 mmol) in tetrahydrofuran (12 mL) was slowly added 1,3-propanesultone (601 mg, 5.0 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and washed with acetone (2×15 mL). The product was suspended in EtOH and the solvent was evaporated (to remove THF residue). The solid was dried in vacuo, affording the title compound (717 mg, 46%). 1H NMR (D2O, 500 MHz) δ ppm 7.32 (m, 5H), 4.53 (d, 1H, J=11.2 Hz), 4.42 (d, 1H, J=11.2 Hz), 4.02 (m, 1H), 3.36 (m, 1H), 3.01 (t, 2H, J=7.8 Hz), 2.81 (t, 2H, J=7.8 Hz), 2.01 (m, 4H), 1.65 (m, 3H), 1.49 (m, 1H). 13C NMR (D2O, 125 MHz) δ ppm 137.00, 129.07, 129.01, 128.77, 81.78, 71.81, 63.89, 48.02, 45.34, 29.93, 27.43, 21.61, 20.94. [α]D=−38.8° (c=0.00122 in water). ES-MS 314 (M+1).
To a solution of L-Phenylalanine cyclohexylamide (2.5 g, 10.1 mmol) in tetrahydrofuran (25 mL) was added 1,3-propanesultone (1.17 g, 9.7 mmol). The solution was stirred at reflux for 2 hours. It was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in the vacuum oven (50° C.), affording the title compound (1.39 g, 39%). 1H NMR (D2O, 500 MHz) δ ppm 7.21 (m, 3H), 7.08 (m, 2H), 4.42 (m, 0.5H), 3.83 (m, 1H). 3.29 (m, 1H), 3.15 (m, 1H), 3.02 (m, 2H), 2.86 (m, 3H), 2.49 (m, 0.5H), 2.01 (m, 2H), 1.54 (m, 1H), 1.45 (m, 1H), 1.33 (m, 2H), 1.02 (m, 4H), 0.55 (m, 1H). 13C (D2O, 125 MHz) δ ppm 133.71, 129.54, 129.18, 128.03, 62.08, 49.25, 47.97, 45.41, 36.29, 31.73, 31.46, 24.86, 24.28, 24.20, 21.39. [α]D=+36.4° (c=0.0019 in water), ES-MS 369 (M+1).
To a solution of (1S,2S)-2-benzyloxycyclohexylamine (1.0 g, 5.2 mmol) in tetrahydrofuran (12 mL) was slowly added 1,3-propanesultone (601 mg, 5.0 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×20 mL) and dried in the vacuum oven (50° C.), affording the title compound (1.15 g, 75%). 1H NMR (D2O, 500 MHz) δ ppm 7.34 (m, 5H), 4.62 (d, 1H, J=11.2 Hz), 4.42 (d, 1H, J=11.2 Hz), 3.40 (m, 1H), 2.97 (m, 2H), 2.90 (m, 1H), 2.76 (t, 2H, J=6.5 Hz), 2.26 (m, 1H), 1.92 (m, 3H), 1.66 (m, 2H), 1.18 (m, 4H). 13C NMR (D2O, 125 MHz) δ ppm 137.22, 129.34, 129.11, 128.84, 76.74, 70.26, 60.84, 48.02, 42.74, 29.49, 26.43, 23.57, 23.02, 21.53. [α]D=+74.8° (c=0.00207 in water). ES-MS 326 (M−1).
To a solution of (1R,2R)-2-benzyloxycyclohexylamine (1.0 g, 5.2 mmol) in tetrahydrofuran (12 mL) was slowly added 1,3-propanesultone (601 mg, 5.0 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and washed with acetone (2×35 mL). The solid was suspended in 80% acetone/EtOH (200 mL). The suspension was stirred at reflux for 30 seconds before the solid was filtered and dried in the vacuum oven (50° C.), affording the title compound (844 mg. 55%). 1H NMR (D2O, 500 MHz) δ ppm 7.32 (m, 5H), 4.60 (d, 1H, J=11.2 Hz), 4.40 (d, 1H, J=11.2 Hz), 3.39 (m, 1H), 2.94 (m, 2H), 2.85 (m, 1H), 2.74 (t, 2H, J=6.5 Hz), 2.24 (m, 1H), 1.90 (m, 3H), 1.64 (m, 2H), 1.14 (m, 4H). 13C NMR (D2O, 125 MHz) δ ppm 137.35, 129.28, 129.19, 128.90, 76.90, 70.35, 60.97, 48.12, 42.90, 29.59, 26.53, 23.64, 23.09, 21.63. [α]D=−68.9° (c=0.0026 in water). ES-MS 326 (M−1).
L-valine benzylester p-tosylate (2.5 g, 6.6 mmol) was treated with a saturated solution of K2CO3 (50 mL) and EtOAc (3×50 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of L-valine benzylester in MeOH (12 mL) was slowly added 1,3-propanesultone (604 mg, 5.0 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with cold MeOH and acetone (2×25 mL). It was dried in the vacuum oven (50° C.), affording the title compound (649 mg, 39%). 1H NMR (D2O, 500 MHz) δ ppm 7.47 (m, 5H), 5.41 (d, 1H, J=11.7 Hz), 5.31 (d, 3H, J=11.7 Hz), 4.04 (m, 1H), 3.19 (m, 2H), 2.95 (t, 2H, J=6.8 Hz), 2.35 (m, 1H), 2.14 (m, 2H), 1.04 (d, 3H, J=6.3 Hz), 0.95 (d, 3H, J=6.3 Hz). 13C (D2O, 125 MHz) δ ppm 168.75, 134.81, 129.38, 129.31, 129.17, 69.00, 65.96, 48.24, 46.71, 29.67, 21.40, 18.39, 16.65. [α]D=−7.2° (c=0.0015 in water), ES-MS 330 (M+1).
L-alanine ethyl ester hydrochloride (2.5 g, 16.3 mmol) was treated with a saturated solution of K2CO3 (50 mL) and EtOAc (3×50 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of L-alanine ethyl ester (1.67 g, 14.3 mmol) in tetrahydrofuran (25 mL) was slowly added 1,3-propanesultone (1.42 g, 11.9 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo, affording the title compound (1.19 g, 42%). 1H NMR (D2O, 500 MHz) δ ppm 4.16 (m, 2H), 4.01 (m, 1H), 3.12 (m, 2H), 2.87 (t, 2H, J=7.3 Hz), 2.01 (m, 2H), 1.43 (d, 3H, J=7.3 Hz), 1.14 (t, 3H, J=6.8 Hz). 13C (D2O, 125 MHz) δ ppm 170.22, 63.84, 55.69, 47.94, 44.73, 21.51, 14.05, 13.25. [α]D=−2.4° (c=0.0022 in water), ES-MS 240 (M+1).
L-valine methylester hydrochloride (5.0 g, 29.8 mmol) was treated with a saturated solution of K2CO3 (75 mL) and EtOAc (3×75 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of L-valine methylester in tetrahydrofuran (25 mL) was slowly added 1,3-propanesultone (2.49 g, 19.9 mmol). The solution was stirred at reflux for 2.5 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×30 mL) and dried in vacuo affording the desired ester.
The ester (860 mg, 3.4 mmol) was dissolved in 2M NaOH (1.20 g of NaOH and 15 mL of water). The reaction was stirred at room temperature overnight. Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure and dried in vacuo, affording the title compound (645 mg, 79%). 1H NMR (D2O, 500 MHz) δ ppm 3.66 (m, 1H), 3.09 (t, 2H, J=6.3 Hz), 2.86 (t, 2H, J=7.3 Hz), 2.17 (m, 1H), 2.07 (m, 2H)), 0.93 (d, 3H, J=6.8 Hz), 0.87 (d, 3H, J=6.8 Hz). 13C (D2O, 125 MHz) δ ppm 171.24, 66.83, 48.28, 46.77, 29.33, 21.44, 18.30, 16.98. [α]D=−16.5° (c=0.0020 in water), ES-MS 238 (M−1).
L-Leucine methylester hydrochloride (5.0 g, 27.5 mmol) was treated with a saturated solution of K2CO3 (50 mL) and EtOAc (3×50 mL) was added. The organic extracts were separated, combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of L-valine methylester (3.74 g, 25.6 mmol) in tetrahydrofuran (35 mL) was slowly added 1,3-propanesultone (2.04 g, 17.2 mmol). The solution was stirred at reflux for 3 hours. The reaction was cooled to room temperature. The solid was collected by filtration. Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The solid was suspended in acetone (50 mL), filtered and dried in vacuo, affording the title compound (1.80 g, 39%). 1H NMR (D2O, 500 MHz) δ ppm 3.99 (m, 1H), 3.72 (s, 3H), 3.12 (m, 2H), 2.87 (t, 2H, J=7.3 Hz), 2.02 (m, 2H), 1.74 (m, 1H), 1.60 (m, 2H), 0.81 (d, 3H, J=5.4 Hz), 0.87 (d, 3H, J=6.8 Hz). 13C (D2O, 125 MHz) δ ppm 170.60, 58.91, 53.81, 48.08, 45.50, 38.17, 24.44, 22.15, 21.59, 20.93. [α]D=+13.8° (c=0.0016 in water), ES-MS 268 (M+1).
L-valine tert-butylamide hydrochloride (2.5 g, 12.0 mmol) was treated with a saturated solution of K2CO3 (50 mL) and EtOAc (3×50 mL) was added. The organic extracts were separated, combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of L-valine tert-butylamide (1.87 g, 11.0 mmol) in 1,4-dioxane (20 mL) was added 1,3-propanesultone (1.07 g, 9.0 mmol). The solution was stirred at reflux for 5 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×30 mL) and dried in vacuo, affording the title compound (801 mg, 25%). 1H NMR (D2O, 500 MHz) δ ppm 3.43 (m, 1H), 3.00 (m, 2H), 2.85 (m, 2H), 2.03 (m, 3H), 1.21 (m, 9H), 0.92 (d, 3H, J=6.3 Hz), 0.85 (d, 3H, J=6.8 Hz). 13C (D2O, 125 MHz) δ ppm 166.47, 66.55, 52.56, 48.23, 46.11, 29.91, 27.81, 21.29, 18.30, 17.44. [α]D=−11.6° (c=0.0023 in water), ES-MS 293 (M−1).
To a solution of L-(+)-leucinol (5.0 g, 42.8 mmol) in THF (65 mL) was slowly added 1,3-propanesultone (4.85 g, 40.7 mmol in THF (10 mL)). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and washed with acetone (2×50 mL). The solid was dissolved in 50% water/EtOH (400 mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The solid was suspended in acetone (150 mL), filtered and dried in the vacuum oven (50° C.), affording the title compound (6.11 g, 63%). 1H NMR (D2O, 500 MHz) δ ppm 3.77 (m, 1H), 3.59 (m, 1H), 3.23 (m, 1H), 3.13 (m, 2H), 2.90 (m, 2H), 2.02 (m, 2H), 1.53 (m, 2H), 1.35 (m, 1H), 0.81 (d, 3H, J=16.1 Hz). 13C (D2O, 125 MHz) δ ppm 58.72, 58.00, 48.17, 43.54, 35.96, 24.34, 22.53, 21.62, 20.89. [α]D=+16.6° (c=0.0022 in water), ES-MS 240 (M+1).
To a solution of L-(+)-isoleucinol (2.0 g, 17.1 mmol) in THF (30 mL) was slowly added 1,3-propanesultone (1.94 g, 16.3 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and washed with acetone (2×20 mL). The solid was dissolved in 70% water/EtOH (240 mL). Dowex Marathon C ion exchange resin (strongly acidic, 15 g) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The solid was suspended in acetone (60 mL), filtered and dried in the vacuum oven (50° C.), affording the title compound (1.70 g, 44%). 1H NMR (D2O, 500 MHz) δ ppm 3.78 (d, 1H, J=13.1 Hz), 3.64 (m, 1H), 3.14 (m, 3H), 2.03 (m, 2H), 1.75 (m, 1H), 1.32 (m, 1H), 1.17 (m, 1H), 0.79 (m, 6H). 13C (D2O, 125 MHz) δ ppm 63.42, 57.38, 48.27, 44.77, 33.64, 25.91, 21.52, 13.31, 10.94. [α]D=+20.4° (c=0.00212 in water), ES-MS 240 (M+1).
To a solution of D-(−)-leucinol (2.0 g, 17.1 mmol) in THF (30 mL) was slowly added 1,3-propanesultone (1.94 g, 16.3 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and washed with acetone (2×20 mL). The solid was dissolved in 50% water/EtOH (240 mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The solid was suspended in acetone (50 mL), filtered and dried in the vacuum oven (50° C.), affording the title compound (2.55 g, (65%). 1H NMR (D2O, 500 MHz) δ ppm 3.74 (d, 1H, J=12.7 Hz), 3.56 (d, 1H, J=12.7 Hz), 3.20 (m, 1H), 3.10 (t, 2H, J=7.3 Hz), 2.87 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.49 (m, 2H), 1.31 (m, 1H), 0.80 (d, 3H, J=6.3 Hz), 0.76 (d, 3H, J=6.3 Hz). 13C (D2O, 125 MHz) δ ppm 58.74, 58.01, 48.19, 43.56, 35.98, 24.34, 22.54, 21.63, 20.90. [α]D=−16.3° (c=0.0019 in water), ES-MS 238 (M−1).
L-Phenylglycine amide hydrochloride (1.0 g, 6.7 mmol) was treated with a solution of K2CO3 (20 mL). The resultant mixture was extracted with EtOAc (3×20 mL). The organic extracts were separated, combined, dried over Na2SO4. The solid material was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure.
To a solution of L-Phenylglycine amide (670 mg, 5.9 mmol) in tetrahydrofuran (10 mL) and 1,4-dioxane (4 mL) was added 1,3-propanesultone (674 mg, 5.6 mmol). The solution stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and washed with acetone (2×20 mL). The solid was dissolved in 50% water/EtOH mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The solid was suspended in acetone (50 mL), filtered and dried in the vacuum oven (50° C.), affording the title compound (743 mg, 50%). 1H NMR (D2O, 500 MHz) δ ppm 7.38 (m, 5H), 4.92, (s, 1H), 3.01 (m, 1H), 2.91 (m, 1H), 2.78 (t, 2H, J=7.3 Hz), 2.0 (m, 2H). 13C (D2O, 125 MHz) δ ppm 170.15, 130.95, 130.24, 129.94, 128.74, 63.40, 47.99, 44.92, 21.27. [α]D=−124° (c=0.0041 in water), ES-MS 271 (M−1).
L-Alanine tert-butylester hydrochloride (2.61 g, 14.4 mmol) was treated with a solution of K2CO3 (75 mL). The resultant mixture was extracted with EtOAc (3×75 mL). The organic extracts were separated, combined, dried over Na2SO4. The solid material was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure.
To a solution of L-Alanine tert-butylester (1.53 g, 10.5 mmol) in tetrahydrofuran (20 mL) was added 1,3-propanesultone (1.16 g, 9.6 mmol). The solution stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and washed with acetone (2×20 mL). The solid was dissolved in water (80 mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The solid was suspended in acetone (80 mL), filtered and dried in the vacuum oven (50° C.), affording the title compound (1.37 g, 54%). 1H NMR (D2O, 500 MHz) δ ppm 3.88 (m, 1H), 3.09 (m, 2H), 2.86 (t, 2H, J=7.3 Hz), 2.00 (m, 2H), 1.39 (d, 3H, J=7.3 Hz), 1.35 (s, 9H). 13C (D2O, 125 MHz) δ ppm 169.13, 86.12, 56.24, 47.94, 44.71, 27.11, 21.52, 14.17. [α]D=−1.1° (c=0.0027 in water), ES-MS 266 (M−1).
D-Phenylglycine amide hydrochloride (1.0 g, 6.7 mmol) was treated with a solution of K2CO3 (20 mL). The resultant mixture was extracted with EtOAc (3×20 mL). The organic extracts were separated, combined, dried over Na2SO4. The solid material was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure.
To a solution of D-Phenylglycine amide (850 mg, 7.5 mmol) in tetrahydrofuran (10 mL) and 1,4-dioxane (4 mL) was added 1,3-propanesultone (818 mg, 6.8 mmol). The solution stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and washed with acetone (2×20 mL). The solid was dissolved in 50% water/EtOH mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The solid was suspended in acetone (50 mL), filtered and dried in the vacuum oven (50° C.), affording the title compound (720 mg, 34%). 1H NMR (D2O, 500 MHz) δ ppm 7.38 (m, 5H), 4.92, (s, 1H), 3.00 (m, 1H), 2.90 (m, 1H), 2.78 (m, 2H), 1.97 (m, 2H). 13C (D2O, 125 MHz) δ ppm 170.14, 130.95, 130.24, 129.94, 128.74, 63.40, 47.99, 44.92, 21.27. [α]D=+106° (c=0.0016 in water), ES-MS 273 (M+1).
L-alanine methylester hydrochloride (5.0 g, 35.8 mmol) was treated with a saturated solution of K2CO3 (75 mL). The mixture was extracted with EtOAc (3×75 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of L-alanine methylester (2.37 g, 23.3 mmol) in tetrahydrofuran (35 mL) was added 1,3-propanesultone (2.41 g, 20.0 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×30 mL) and dried in vacuo.
The ester (2.21 g, 9.8 mmol) was dissolved in 2M NaOH (2.40 g of NaOH and 30 mL of water). The reaction was stirred at room temperature overnight. Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure and lyophilized, affording the title compound (1.81 g, 87%). 1H NMR (D2O, 500 MHz) δ ppm 3.76 (m, 1H), 3.07 (m, 2H), 2.85 (t, 2H, J=7.3 Hz), 1.99 (m, 2H), 1.38 (d, 3H, J=7.3 Hz), 0.87 (d, 3H, J=6.8 Hz). 13C (D2O, 125 MHz) δ ppm 173.31, 56.66, 47.97, 44.76, 21.56, 14.51. [α]D=+3.5° (c=0.0023 in water), ES-MS 210 (M−1).
The N-(3-sulfo-propyl)-phenylalanine ethyl ester (DM-258-069, 860 mg, 2.7 mmol) was dissolved in 2N NaOH (1.2 g of NaOH and 15 mL of water). The reaction was stirred at room temperature overnight. Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure and lyophilized, affording the title compound (654 mg, 84%). 1H NMR (D2O, 500 MHz) δ ppm 7.20 (m, 5H), 3.96 (t, 1H, J=6.3 Hz), 3.11 (m, 4H), 2.80 (t, 2H, J=7.3 Hz), 1.95 (m, 2H). 13C (D2O, 125 MHz) δ ppm 171.46, 134.03, 129.50, 129.28, 128.10, 62.02, 47.97, 45.64, 35.23, 21.39. [α]D=+14.9° (c=0.0013 in water), ES-MS 286 (M−1).
L-Valine allylester p-tosylate (3.0 g, 9.1 mmol) was treated with a saturated solution of K2CO3 (30 mL). The mixture was extracted with EtOAc (3×30 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered and evaporated under reduced pressure.
To a solution of L-valine allylester (1.30 g, 8.3 mmol) in tetrahydrofuran (6 mL), 1,4-dioxane (6 mL) and MeOH (0.5 mL) was added 1,3-propanesultone (910 mg, 7.5 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solvent was evaporated under reduced pressure. The sticky paste was suspended in 20% acetone/ether. The solid was filtered and dissolved EtOH (75 mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated to dryness under reduced pressure, affording the title compound (605 mg, 26%). 1H NMR (D2O, 500 MHz) δ ppm 5.84 (m, 1H), 5.27 (d, 1H, J=17.1 Hz), 5.19 (m, 1H, J=10.3 Hz), 3.91 (d, 1H, J=3.9 Hz), 3.10 (t, 2H, J=7.3 Hz), 2.85 (t, 2H, J=7.3 Hz), 2.22 (m, 1H), 2.03 (m, 2H), 0.93 (d, 3H, J=6.8 Hz), 0.85 (d, 3H, J=6.8 Hz). 13C (D2O, 125 MHz) δ ppm 168.55, 130.90, 120.31, 67.58, 65.82, 48.09, 46.57, 29.53, 21.29, 18.25, 16.57. [α]D=+5.0° (c=0.0011 in water), ES-MS 278 (M−1).
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L-Leucinamide hydrochloride (5.0 g, 30.0 mmol) was treated with a saturated solution of K2CO3 (100 mL). The mixture was extracted with EtOAc (3×100 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered and evaporated under reduced pressure.
To a solution of L-Leucinamide (3.20 g, 24.5 mmol) in tetrahydrofuran (35 mL) was added 1,3-propanesultone (2.82 g, 23.3 mmol). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was filtered and washed with acetone (2×25 mL). The solid was dissolved in 50% EtOH/water (200 mL). Dowex Marathon C ion exchange resin (strongly acidic, 25 g) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated to dryness under reduced pressure. The solid was suspended in acetone (75 mL), and it was then filtered and dried in vacuo, affording the title compound (3.13 g, 53%). 1H NMR (D2O, 500 MHz) δ ppm 3.79 (m, 1H), 3.04 (m, 2H), 2.85 (m, 2H), 2.02 (m, 2H), 1.65 (m, 1H), 1.54 (m, 2H), 0.80 (m, 6H). 13C (D2O, 125 MHz) δ ppm 171.46, 59.42, 48.04, 45.46, 39.04, 24.27, 22.24, 21.49, 21.17. [α]D=+13.5° (c=0.0026 in water), ES-MS 251 (M−1).
L-Leucine benzylester p-tosylate (5.0 g, 12.7 mmol) was treated with a saturated solution of K2CO3 (100 mL). The mixture was extracted with EtOAc (3×100 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered and evaporated under reduced pressure.
To a solution of L-Leucine benzylester (2.81 g, 12.7 mmol) in tetrahydrofuran (6 mL), 1,4-dioxane (6 mL) and MeOH (6 mL) was added 1,3-propane sultone (1.40 g, 11.5 mmol). The solution was stirred at reflux for 2.5 hours. The reaction mixture was cooled to room temperature. The solid was filtered and washed with acetone (2×20 mL). The filtrate was evaporated under reduced pressure. The residue was dissolved in acetone (20 mL). The product was precipitated with Et2O (200 mL). The solid material was filtered. Both solids were combined and dissolved in 50% EtOH/water (200 mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure and lyophilized, affording the title compound (1.87 g, 47%). 1H NMR (DMSO, 500 MHz) δ ppm 9.34 (s (broad), 1H), 7.39 (m, 5H), 5.25 (s, 2H), 4.10 (m, 1H), 3.09 (m, 2H), 2.60 (m, 2H), 1.95 (m, 2H), 1.64 (m, 3H), 0.86 (m, 6H). 13C (DMSO, 125 MHz) δ ppm 168.90, 134.91, 128.53, 128.50, 128.41, 67.38, 57.37, 49.17, 45.79, 38.06, 24.07, 22.79, 21.78, 21.33. [α]D=+1.8° (c=0.0017 in water), ES-MS 344 (M+1).
L-Isoleucine methylester hydrochloride (5.0 g, 27.5 mmol) was treated with a saturated solution of K2CO3 (100 mL). The mixture was extracted with EtOAc (3×100 mL). The organic layers were separated, combined, dried with Na2SO4, filtered and evaporated under reduced pressure.
To a solution of L-Isoleucine methlylester (3.43 g, 23.6 mmol) in acetone (30 mL) was added 1,3-propane sultone (2.62 g, 21.5 mmol). The solution was stirred at reflux for 2 h. The reaction mixture was cooled to room temperature. The solid was filtered and washed with acetone (2×20 mL). The filtrate was evaporated under reduced pressure. The residue was suspended in acetone (50 mL). The solid was filtered. The solid materials were combined and dissolved in water (100 mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The solid product was suspended in acetone (100 mL), filtered and dried in vacuo, affording the title compound (3.23 g, 56%). 1H NMR (D2O, 500 MHz) δ ppm 4.00 (m, 1H), 3.74 (s, 3H), 3.14 (t, 2H, J=7.8 Hz), 2.89 (t, 2H, J=7.3 Hz), 2.05 (m, 2H), 1.97 (m, 1H), 1.41 (m. 1H), 1.23 (m, 1H), 0.83 (m, 6H). 13C (D2O, 125 MHz) δ ppm 169.29, 64.51, 53.55, 48.14, 46.52, 36.07, 25.92, 21.34, 13.76, 11.09. [α]D=+22.6° (c=0.0023 in water), ES-MS 266 (M−1).
L-Isoleucine methylester hydrochloride (5.0 g, 27.5 mmol) was treated with a saturated solution of K2CO3 (100 mL). The mixture was extracted with EtOAc (3×100 mL). The organic layers were separated, combined, dried with Na2SO4, filtered and evaporated under reduced pressure.
To a solution of L-Isoleucine methlylester (3.43 g, 23.6 mmol) in acetone (30 mL) was added 1,3-propane sultone (2.62 g, 21.5 mmol). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was filtered and washed with acetone (2×20 mL). The filtrate was evaporated under reduced pressure. The residue was suspended in acetone (50 mL). The solid was filtered. The solid materials were combined and dissolved in water (100 mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The solid product was suspended in acetone (100 mL), filtered and dried in vacuo (3.23 g, 56%).
The solid (1.0 g, 3.7 mmol) was dissolved in 2M NaOH (30 mL). The reaction mixture was stirred at room temperature overnight. Dowex Marathon C ion exchange resin (strongly acidic, 15 g) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was coevaporated with EtOH and lyophilized, affording the title compound (740 mg, 83%). 1H NMR (D2O, 500 MHz) δ ppm 4.00 (m, 1H), 3.59 (m, 3H), 3.07 (t, 2H, J=7.3 Hz), 2.86 (m, 2H), 2.02 (m, 2H), 1.84 (m, 1H), 1.39 (m. 1H), 1.19 (m, 1H), 0.81 (m, 6H). 13C (D2O, 125 MHz) δ ppm 169.29, 64.51, 53.55, 48.14, 46.52, 36.07, 25.92, 21.34, 13.76, 11.09. [α]D=+30.4° (c=0.0031 in water), ES-MS 252 (M−1).
L-Phenylalaninamide hydrochloride (5.0 g, 24.9 mmol) was treated with a saturated solution of K2CO3 (75 mL). The mixture was extracted with EtOAc (3×75 mL). The organic layers were separated, combined, dried with Na2SO4, filtered and evaporated under reduced pressure.
To a solution of L-Phenylalaninamide (3.93 g, 23.9 mmol) in acetonitrile (25 mL) was added 1,3-propane sultone (2.70 g, 21.8 mmol). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was filtered and washed with acetonitrile (2×25 mL). The solid product was suspended in EtOH (100 mL). The suspension was stirred at reflux for 1 hour. The solid material was filtered and dried in vacuo, affording the title compound (5.18 g, 83%). 1H NMR (D2O, 500 MHz) δ ppm 7.24 (m, 3H), 7.16 (m, 2H), 4.02 (m, 1H), 3.15 (m, 1H), 3.01 (m, 3H), 2.83 (m, 2H), 2.02 (m, 2H), 1.98 (m, 2H). 13C (D2O, 125 MHz) δ ppm 170.48, 133.72, 129.58, 129.21, 128.11, 61.55, 47.95, 45.52, 36.21, 21.44. [α]D=+23.1° (c=0.0021 in water), ES-MS 285 (M−1).
D-Leucine methylester hydrochloride (2.63 g, 14.5 mmol) was treated with a saturated solution of K2CO3 (50 mL). The aqueous mixture was extracted with EtOAc (3×50 mL). The organic layers were separated, combined, dried with Na2SO4, filtered and evaporated under reduced pressure.
To a solution of D-Leucine methylester (1.58 g, 10.9 mmol) in acetonitrile (35 mL) was added 1,3-propanesultone (1.21 g, 9.9 mmol). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, recrystallized from EtOH and dried in vacuo, affording the title compound (1.59 g, 60%). 1H NMR (D2O, 500 MHz) δ ppm 3.98 (m, 1H), 3.70 (s, 3H), 3.11 (m, 2H), 2.85 (m, 2H), 2.00 (m, 2H), 1.72 (m, 1H), 1.60 (m, 2H), 0.81 (m, 6H). 13C (D2O, 125 MHz) δ ppm 170.51, 58.78, 53.69, 47.97, 45.36, 38.09, 24.34, 22.07, 21.51, 20.82. [α]D=+13.1° (c=0.0019 in water), ES-MS 266 (M−1).
D-Valinamide hydrochloride (2.49 g, 14.7 mmol) was treated with a solution of K2CO3 (50 mL). The organic mixture was extracted with EtOAc (3×50 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of D-valinamide (1.76 g, 14.7 mmol) in acetonitrile (30 mL) was slowly added 1,3-propanesultone (1.75 g, 14.4 mmol). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, washed with acetonitrile (2×25 mL). The solid product was dissolved in water (75 mL). Dowex Marathon C resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The solid material was suspended in acetone (50 mL), filtered and dried in vacuo, affording the title compound (1.57 g, 51%). 1H NMR (D2O, 500 MHz) δ ppm 3.80 (m, 1H), 3.19 (m, 2H), 3.00 (m, 2H), 2.25 (m, 1H), 2.16 (m, 2H), 1.08 (d, 3H, J=6.8 Hz), 1.02 (d, 3H, J=6.8 Hz). 13C (D2O, 125 MHz) δ ppm 169.94, 65.86, 48.10, 46.27, 29.54, 21.23, 17.99, 17.02. [α]D=−12.4° (c=0.0037 in water), ES-MS 237 (M−1).
D-Phenylalaninamide hydrochloride (2.53 g, 12.6 mmol) was treated with a saturated solution of K2CO3 (50 mL). The mixture was extracted with EtOAc (3×50 mL). The organic layers were separated, combined, dried with Na2SO4, filtered and evaporated under reduced pressure.
To a solution of D-Phenylalaninamide (1.83 g, 11.1 mmol) in acetonitrile (20 mL) was added 1,3-propane sultone (1.29 g, 10.6 mmol). The solution was stirred at reflux for 2.5 hours. The reaction mixture was cooled to room temperature. The solid was filtered and washed with acetonitrile (2×20 mL). The solid product was suspended in EtOH (75 mL). The suspension was stirred at reflux for 1 hours. The solid material was filtered, washed with acetone (1×25 mL) and dried in vacuo, affording the title compound (2.62 g, 89%). 1H NMR (D2O, 500 MHz) δ ppm 7.28 (m, 3H), 7.19 (m, 2H), 4.05 (m, 1H), 3.19 (dd, 1H, J=5.3 Hz, 14.2 Hz), 3.04 (m, 3H), 2.86 (t, 2H, J=5.8 Hz), 2.03 (m, 2H). 13C (D2O, 125 MHz) δ ppm 170.39, 133.73, 129.62, 129.26, 128.15, 61.57, 47.99, 45.57, 36.21, 21.45. [α]D=−20.7° (c=0.0038 in water), ES-MS 285 (M−1).
L-Isoleucine benzylester p-tosylate (2.50 g, 6.4 mmol) was treated with a saturated solution of K2CO3 (30 mL). The mixture was extracted with EtOAc (3×30 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered and evaporated under reduced pressure.
To a solution of L-isoleucine benzylester (1.41 g, 6.4 mmol) in acetonitrile (12 mL) was added 1,3-propane sultone (706 mg, 5.8 mmol). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was filtered and washed with acetone (2×20 mL). The solid material was dissolved in 50% EtOH/water (50 mL). Dowex Marathon C ion exchange resin (strongly acidic, 10 g) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure and lyophilized affording the title compound (778 mg, 39%). 1H NMR (D2O, 500 MHz) δ ppm 7.49 (m, 5H), 5.42 (d, 1H, J=11.7 Hz), 5.31 (d, 1H, J=11.7 Hz), 3.24 (m, 2H), 2.98 (m, 2H), 2.13 (m, 3H), 1.46 (m, 1H), 1.34 (m, 1H), 0.93 (m, 6H). 13C (D2O, 125 MHz) δ ppm 168.53, 134.69, 129.28, 129.22, 129.06, 68.86, 64.45, 48.12, 46.56, 36.21, 25.97, 21.31, 13.73, 11.05. [α]D=−1.5° (c=0.0031 in water), ES-MS 342 (M−1).
D-Leucinamide hydrochloride (1.0 g, 6.0 mmol) was treated with a saturated solution of K2CO3 (30 mL). The aqueous mixture was extracted with EtOAc (3×30 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered and evaporated under reduced pressure.
To a solution of D-Leucinamide (6.0 mmol) in acetonitrile (35 mL) was added 1,3-propanesultone (666 mg, 5.5 mmol). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid was filtered and washed with MeCN (2×20 mL). The solid was suspended in EtOH (50 mL). The suspension was stirred at reflux for 1 hour. The mixture was cooled to room temperature. The solid material was filtered, washed with acetone (1×20 mL) and dried in a vacuum oven (50° C.), affording the title compound (1.03 g, 74%): 1H NMR (D2O, 500 MHz) δ ppm 3.81 (m, 1H), 3.07 (m, 2H), 2.85 (t, 2H, J=7.3 Hz), 2.03 (m, 2H), 1.68 (t, 1H, J=7.8 Hz), 1.58 (m, 2H), 0.83 (m, 6H). 13C (D2O, 125 MHz) δ ppm 171.45, 59.39, 48.01, 45.41, 39.02, 24.24, 22.21, 21.47, 21.13. [α]D=−13.7° (c=0.0019 in water), ES-MS 251 (M−1).
For the Ritter reaction, the flask was closed with a septum and connected to a 20% NaOH scrubber. Sodium cyanide (powdered, 5.5 g, 112 mmol) was added to acetic acid (30 mL) in one portion. The mixture was stirred for 10 minutes at room temperature. A solution of sulfuric acid (16 mL) in acetic acid (15 mL) was added dropwise over a 20 minute period. Then, a solution of 1-methyl-1-cyclopentanol (10 g, 99.8 mmol) in acetic acid (5 mL) was added dropwise over a 5 minute period. The mixture was stirred at room temperature for 22 hours then poured over ice (approx. 100 g). The pH of the solution was adjusted to 9 with the addition of 50% NaOH (about 135 g). The layers were separated and the aqueous layer was extracted with ether (1×40 mL). The combined organic layers were washed with saturated sodium carbonate (1×10 mL) then dried over sodium sulfate. The ether was evaporated under reduced pressure to afford light brown oil (12.04 g, 94.7 mmol, 95%). The oil showed to be a mixture of cis and trans formamide but what otherwise pure enough to be used as such. 1H NMR (500 MHz, CDCl3) δ [1.40 and 1.45 (s, 3H)], 1.68-1.76 (m, 7H), 1.97-1.98 (m, 1H), [5.42 and 6.24 (br s, 1H)], [8.05 (s) and 8.24 (d, J=12.2 Hz) for 1H]; 13C NMR (125 MHz, CDCl3) δ 23.1, 23.6, 25.5, 28.2, 39.5, 40.7, 60.7, 61.3, 160.7, 163.9
A solution of NaOH (25%, 80 mL) was added to the crude 1-methyl-1-cyclopentylformamide (12.00 g, 94.7 mmol). The mixture was heated to reflux for 2.5 hours then cooled at room temperature. Some sodium chloride (20 g) was added to facilitate the phase separation. The layers were separated and the aqueous layer was extracted with toluene (1×15 mL). The combined organic layers were agitated. The addition of isopropyl ether (2.5 mL, chloroform (1 g) and cyclohexane (6.5 g) did not improved the seperation of the solution. The combined organic layers were washed with brine (1×10 mL) then dried over sodium sulfate and filtered. The filtrate was used as such in the next step. 1H NMR (500 MHz, CDCl3) δ 1.22 (s, 3H), 1.47-1.75 (4 m, 9H): 13C NMR (125 MHz, CDCl3) δ 24.1, 29.6, 42.2, 58.4
A solution of 1,3-propanesultone (9.4 g, 75 mmol) in 2-butanone (35 mL) was added dropwise to a the crude solution of 1-methyl-1-cyclopentylamine (mixture of solvent from previous step).
The mixture was heated to reflux for 20 hours then was cooled to room temperature. The solid was collected by suction-filtration and rinsed with acetone (2×10 mL). The solid was dried overnight at 45° C. in the vacuum oven. The title compound was obtained as a fine white solid (16.26 g, 73.47 mmol, 74% overall yield). 1H NMR (500 MHz, D2O) δ 1.31 (s, 3H), 1.591.6-1-85 (m, 8H), 2.03-2.06 (m, 2H), 2.96 (t, J=6.8 Hz, 2H), 3.15 (t, J=7.6 Hz, 2H); 13C NRM (125 MHz, D2O) δ 22.1, 22.5, 23.6, 36.5, 41.4, 48.1, 66.8.1; ES-MS 220 (M−H)
For the Ritter reaction, the flask was closed with a septum and connected to a 20% NaOH scrubber. Potassium cyanide (powdered, 3.3 g, 50 mmol) was added in portions to acetic acid (13 mL). The mixture was stirred for 10 minutes at room temperature. A solution of sulfuric acid (7 mL) in acetic acid (7 mL) was added drop-wise over a 10 minute period. Then, a solution of 1-methyl-1-cyclohexanol (5 g, 43.8 mmol) in acetic acid (4 mL) was added dropwise over a 5 minute period. The mixture was stirred at room temperature for 22 hours then poured over ice (approx. 50 g). The pH of the solution was adjusted to 9 with the addition of 50% NaOH (about 70 g). The layers were separated and the aqueous layer was extracted with ether (2×20 mL). The combined organic layers were washed with saturated sodium carbonate (1×10 mL) then dried over sodium sulfate. The ether was evaporated under reduced pressure to afford a clear yellow oil (6.56 g, quantitative). The oil showed to be a mixture of cis and trans formamide but what otherwise pure enough to be used as such. 1H NMR (500 MHz, CDCl3) δ 1.33-1.53 (3m, 11H), 1.67 (br s, 1H), 1.99 (m, 1H), [5.16 and 6.09 (br s, 1H)], [8.11 (s) and 8.25 (d, J=12.2 Hz) for 1H]; 13C NMR (125 MHz, CDCl3) δ 23.1, 23.6, 25.5, 28.2, 39.5, 40.7, 60.7, 61.3, 160.7, 163.9
A solution of NaOH (20%, 40 mL) was added to the crude 1-methyl-1-cyclohexylformamide (43.8 mmol). The mixture was heated to reflux for 3 hours then cooled at room temperature. Some sodium chloride (7.5 g) was added to facilitate the phase separation. The layers were separated and the aqueous layer was extracted with MTBK (1×10 mL). The combined organic layers were washed with brine (1×5 mL) the dried over sodium sulfate and filtered. The filtrate was used as such in the next step. 1H NMR (500 MHz, CDCl3) δ 1.08 (s, 3H), 1.33-1.51 (m, 10H); 13C NMR (125 MHz, CDCl3) δ 22.8, 25.8, 29.6, 40.8, 48.6
A solution of 1,3-propanesultone (5.00 g, 40 mmol) in toluene (10 mL) was added dropwise to a the crude solution of 1-methyl-1-cyclohexylamine in MTBK (total volume 30 mL). The mixture was heated to reflux for 18 hours then cooled to room temperature. The solid was collected by suction filtration, rinsed with acetone (2×8 mL). The solid was dried overnight at 45° C. in the vacuum oven. The title compound was obtained as a fine white solid (9.22 g). However, the proton NMR and the ES-MS were not clean. The solid was suspended in methanol (45 mL) and the suspension was warmed to reflux. Water (12 mL) was added dropwise until a clear yellow solution was obtained. The mixture was slowly cool to room temperature with stirring. The solid was collected by suction-filtration, rinsed with methanol (2×5 mL). Another crop was collected from the filtrate. Both crops were dried overnight at 45° C. in the vacuum oven. The title compound was obtained as a fine white solid (6.82 g, 29.0 mmol, 66% overall yield). Both crops were identical and were mixed for submitting the compound. 1H NMR (500 MHz, D2O) δ 1.04-1.11 (m, 1H), 1.19 (s, 3H), 1.31 (q, J=12.2 Hz, 2H), 1.40 (qt, J=12.2 Hz, 2H), 1.46-1.62 (m, 2H), 1.63 (br d, J=11.7 Hz, 2H), 1.94 (q, J=7.3 Hz, 2H), 2.86 (t, J=7.1 Hz, 2H), 3.03 (t, J=7.6 Hz, 2H); 13C NMR (125 MHz, D2O) δ 19.1, 21.5, 22.0, 24.6, 34.1, 39.1, 48.2, 60.2; ES-MS 236 (M+H).
For the Ritter reaction, the flask was closed with a septum and connected to a 20% NaOH scrubber. Potassium cyanide (powdered, 2.8 g, 43 mmol) was added in portions to acetic acid (10 mL). The mixture was stirred for 10 minutes at room temperature. A solution of sulfuric acid (7 mL) in acetic acid (7 mL) was added drop-wise over a 20 minute period. Then, the 1-methyl-1-cycloheptanol (5 g, 39.0 mmol) was added drop-wise over 5 minutes. The mixture was stirred at room temperature for 22 h then cooled to 0° C. with a ice/water bath. The pH of the solution was adjusted to 9 with the addition of 50% NaOH (about 70 g). The layers were separated and the aqueous layer was extracted with ether (1×20 mL). The combined organic layers were washed with saturated sodium carbonate (1×5 mL) then dried over sodium sulfate. The ether was evaporated under reduced pressure to afford a clear yellow oil (5.71 g, 94%). The oil showed to be a mixture of cis and trans formamide but what otherwise pure enough to be used as such. 1H NMR (500 MHz, CDCl3) δ 1.34 (s, 1.5H), 1.43 (s, 1.5H), 1.49-1.60 (m, 8H), 1.96-2.00 (m, 1H), [5.28 and 5.95 (br s, 1H)], [8.06 (s) and 8.28 (d, J=12.2 Hz) for 1H]; 13C NMR (125 MHz, CDCl3) δ 22.2, 22.4, 27.7, 29.3, 29.4, 30.7, 40.5, 42.5, 56.0, 57.4, 160.5, 163.3
A solution of NaOH (25%, 40 mL) was added to the crude 1-methyl-1-cycloheptylformamide (5.7 g). The mixture was heated to reflux for 3 hours then cooled at room temperature. Some sodium chloride (7.5 g) was added to facilitate the phase separation. The layers were separated and the aqueous layer was extracted with MTBK (1×10 mL). The combined organic layers were washed with brine (1×5 mL) the dried over sodium sulfate and filtered. The filtrate was used as such in the next step. 1H NMR (500 MHz, CD3OD) δ 1.10 (s, 3H), 1.40-1.48 (m, 2H), 1.5-1.65 (m, 10H); 13C NMR (125 MHz, CD3OD) δ 24.0, 31.2, 31.4, 44.4, 53.6
A solution of 1,3-propanesultone (4.3 g, 35 mmol) in toluene (10 mL) was added dropwise to a the crude solution of 1-methyl-1-cycloheptylamine in MTBK (total volume 30 mL). The mixture was heated to reflux for 18 hours then was cooled to room temperature. The solid was collected by suction filtration, rinsed with acetone (2×5 mL). The solid was dried overnight at 45° C. in the vacuum oven. The title compound was obtained as a fine white solid (7.77 g, 31.2 mmol, 80% overall yield). 1H NMR (500 MHz, D2O) δ 1.27 (s, 3H), 1.40-1.60 (m, 8H), 1.71-1.81 (m, 4H), 2.00-2.06 (m, 2H), 2.95 (t, J=6.3 Hz, 2H), 3.13 (t, J=7.1 Hz, 2H); 13C NMR (125 MHz, D2O) δ 22.0, 22.1, 23.3, 29.5, 37.1, 40.0, 48.3, 64.0; ES-MS 250(M+H)
D-Leucine benzylester p-tosylate (2.5 g, 6.3 mmol) was treated with an aqueous solution of K2CO3 (30 mL). The mixture was extracted with EtOAc (3×30 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of D-Leucine benzylester (6.3 mmol) in acetonitrile (9 mL) and MeOH (3 mL) was added 1,3-propane sultone (691 mg, 5.7 mmol). The solution was stirred at reflux for 2.5 hours. The reaction mixture was cooled to room temperature. The solid material was filtered and washed with aconitrile (2×20 mL). The solid was dissolved in 20% water/EtOH (75 mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure and dried in vacuo, affording the title compound (960 mg, 49%). 1H NMR (D2O, 500 MHz) δ ppm 7.52 (m, 5H), 5.41 (d, 1H, J=12.2 Hz), 5.35 (d, 1H, J=12.2 Hz), 4.16 (m, 1H), 3.22 (m, 2H), 2.97 (t, 2H, J=6.8 Hz), 2.16 (m, 2H), 1.88 (m, 1H), 1.79 (m, 1H), 1.76 (m, 1H), 0.94 (d, 6H, J=3.9 Hz). 13C (DMSO, 125 MHz) δ ppm 169.60, 135.62, 129.24, 129.21, 129.11, 68.08, 58.09, 49.87, 46.48, 24.77, 23.50, 22.50, 22.04. [α]D=−2.1° (c=0.00095 in water), ES-MS 344 (M+1).
To a solution of 6-amino-2-methyl-2-heptanol (2.5 g, 17.2 mmol) in acetonitrile (22 mL) was added 1,3-propane sultone (2.0 g, 16.4 mmol). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was filtered and washed with acetonitrile (2×20 mL). The solid was dissolved in 20% MeOH (75 mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure. The solid was suspended in acetone (150 mL), and then the solid material was filtered and dried in vacuo, affording the title compound (3.08 g, 70%). 1H NMR (D2O, 500 MHz) δ ppm 3.19 (m, 1H), 3.08 (m, 2H), 2.88 (t, 2H, J=7.3 Hz), 1.99 (m, 2H), 1.60 (m, 2H), 1.36 (m, 4H), 1.18 (d, 3H, J=6.8 Hz), 1.07 (s, 6H). 13C (D2O, 125 MHz) δ ppm 71.63, 54.73, 48.08, 43.46, 42.27, 32.97, 27.78, 27.73, 21.64, 19.67, 15.43. ES-MS 268 (M+1).
D-Alanine methylester hydrochloride (3.0 g, 21.5 mmol) was treated with a aqueous solution of K2CO3 (50 mL). The mixture was extracted with EtOAc (3×50 mL). The organic extracts were separated, combined, dried with Na2SO4, filtered and evaporated under reduced pressure.
To a solution of D-Alanine methylester (1.33 g, 12.9 mmol) in acetonitrile (15 mL was added 1,3-propane sultone (1.42 g, 11.7 mmol). The solution was stirred at reflux for 2 hours. The reaction mixture was cooled to room temperature. The solid material was filtered and washed with acetonitrile (2×15 mL). The solid was dissolved in water (30 mL). Dowex Marathon C ion exchange resin (strongly acidic) was added to the solution. The suspension was stirred for 15 minutes before the resin was removed by filtration. The filtrate was evaporated under reduced pressure and dried in vacuo, affording the title compound (1.52 g, 42%). 1H NMR (D2O, 500 MHz) δ ppm 4.07 (m, 1H), 3.72 (s, 3H), 3.14 (m, 2H), 2.89 (t, 2H, J=7.3 Hz), 2.03 (m, 2H), 1.46 (dd, 3H, J=1.95 Hz, 7.3 Hz). 13C (DMSO, 125 MHz) δ ppm 170.74, 55.62, 53.82, 47.96, 44.76, 21.53, 14.03. [α]D=+1.4° (c=0.0088 in water), ES-MS 224 (M−1).
To a solution of 1,2,3,4-tetrahydro-1-naphthylamine (2.01 g, 13.6 mmol) in 2-butanone (15 mL) was added 2,4-butane sultone (1.95 g, 14.3 mmol). The solution was stirred at for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. 1H NMR (DMSO, 500 MHz) δ ppm 8.56 (s (broad), 1H), 7.49 (dd, 1H, J=8.0 Hz, 11.9 Hz), 7.29 (m, 1H), 7.26 (m, 1H), 7.19 (d, 1H, J=8.0 Hz), 4.40 (d, 1H, J=13.7 Hz), 3.14 (m, 2H), 2.75 (m, 3H), 1.96 (m, 5H), 1.40 (m, 1H), 1.23 (m, 3H). 13C (DMSO, 125 MHz) δ ppm 138.81, 131.81, 130.40, 130.28, 130.16, 129.41, 126.76, 126.73, 54.97, 54.58, 54.08, 44.18, 43.23, 29.50, 28.84, 24.84, 24.76, 18.23, 18.20, 17.79, 17.01. ES-MS 284 (M+1).
To a solution of octylamine (2.00 g, 15.5 mmol) in 2-butanone (17 mL) was added 2,4-butane sultone (2.21 g, 16.2 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. It was suspended in a solution of 25% EtOH/acetone (50 mL). The suspension was stirred for 5 minutes. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. 1H NMR (DMSO, 500 MHz) δ ppm 8.45 (s (broad), 1H), 3.01 (m, 1H), 2.84 (m, 2H), 2.58 (m, 1H), 1.92 (m, 1H), 1.75 (m, 1H), 1.51 (m, 2H), 1.10 (d, 1H, J=6.8 Hz), 0.85 (t, 3H, J=6.8 Hz). 13C (DMSO, 125 MHz) δ ppm 53.05, 47.27, 46.15, 31.83, 29.42, 29.13, 26.51, 26.27, 22.75, 17.19, 14.64. ES-MS 266 (M+1).
1-adamantaneamine hydrochloride (2.51 g, 13.3 mmol) was treated with 1N NaOH (20 mL) and CH2Cl2 (3×20 mL). The organic extracts were combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of 2-adamantanamine (1.99 g, 13.1 mmol) in acetonitrile (15 mL) was added 2,4-butane sultone (1.87 g, 13.8 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetonitrile (3×25 mL) and dried in vacuo. 1H NMR (DMSO, 500 MHz) δ ppm 8.53 (s (broad), 1H), 3.33 (m, 2H), 2.61 (m, 1H), 2.10 (s, 3H), 1.93 (m, 1H), 1.77 (m, 7H), 1.61 (m, 6H), 1.12 (d, 1H, J=6.8 Hz). 13C (DMSO, 125 MHz) δ ppm 56.20, 53.34, 35.85, 29.75, 29.04, 17.206. ES-MS 288 (M+1).
The 2-adamantanamine hydrochloride (2.50 g, 13.3 mmol) was treated with 1N NaOH (2.0 mL) and CH2Cl2 (3×20 mL). The organic extracts were combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of 1-adamantanamine (1.99 g, 13.1 mmol) in acetonitrile (15 mL) was added 2,4-butane sultone (1.87 g, 13.8 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetonitrile (2×25 mL) and dried in vacuo. 1H NMR (DMSO, 500 MHz) δ ppm 3.20 (m, 1H), 3.05 (m, 2H), 2.67 (m, 1H), 2.07 (m, 2H), 2.00 (m, 1H), 1.95 (m, 4H), 1.82 (m, 4H), 1.69 (m, 4H), 1.55 (m, 4H), 1.12 (d, 1H, J=8.0 Hz). 13C (DMSO, 125 MHz) δ ppm 62.27, 53.91, 44.33, 37.30, 36.82, 36.77, 30.30, 30.20, 29.57, 29.50, 28.95, 17.12, 26.85, 17.44. ES-MS 288 (M+1).
To a solution of exo-2-aminonorbornane (1.0 g, 9.0 mmol) in tetrahydrofuran (THF, 10 mL) was added 2,4-butane sultone (1.28 g, 9.3 mmol). The solution was stirred at reflux for 3 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with THF (2×20 mL) and dried in vacuo. 1H NMR (DMSO, 500 MHz) δ ppm 8.43 (s (broad), 1H), 2.96 (m, 3H), 2.62 (m, 1H), 2.38 (m, 1H), 2.28, (m, 1H), 1.91 (m, 1H), 1.82 (m, 1H), 1.61 (m, 1H), 1.54 (m, 2H), 1.42 (m, 2H), 1.12 (m, 6H). 13C (DMSO, 125 MHz) δ ppm 60.92, 60.79, 53.61, 53.21, 44.55, 44.36, 39.80, 39.55, 36.27, 36.15, 36.11, 35.98, 35.19, 35.13, 29.62, 29.43, 28.07, 26.88, 17.56, 14.11. ES-MS 248 (M+1).
Quinuclidine hydrochloride (2.50 g, 16.9 mmol) was treated with 1N NaOH (20 mL) and CH2Cl2 (4×20 mL). The organic extracts were combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of quinuclidine (900 g, 8.2 mmol) in tetrahydrofuran (THF, 18 mL) and MeOH (0.5 mL) was added 2,4-butane sultone (1.16 g, 8.6 mmol). The solution was stirred at reflux overnight. The reaction was cooled to room temperature. The solid was collected by filtration, washed with THF (2×25 mL) and dried in vacuo. 1H NMR (DMSO, 500 MHz) δ ppm 3.40 (m, 7H), 3.20 (td, 1H, J=3.9 Hz, 12.7 Hz), 2.38 (m, 1H), 2.01 (m, 2H), 1.83, (m, 6H), 1.70 (m, 1H), 1.10 (d, 3H, J=12.7 Hz). 13C (DMSO, 125 MHz) δ ppm 62.63, 54.23, 52.18, 25.67, 24.07, 19.78, 17.04. ES-MS 208 (M+1).
To a solution of DL-2-aminopentanol (1.0 g, 9.7 mmol) in tetrahydrofuran (11 mL) was added 2,4-butane sultone (1.45 g, 10.2 mmol). The solution was stirred at reflux for 4 hours. The reaction was cooled to room temperature. The supernatant was removed and the solid was dried in vacuo. The product was suspended in 2-propanol (100 mL) and the mixture was stirred for 5 minutes. The white solid was filtered, washed with 2-propanol and dried in vacuo. 1H NMR (D2O, 500 MHz) δ ppm 3.74 (d, 1H, J=12.7 Hz), 3.59 (dd, 1H, J=5.4 Hz, 12.9 Hz), 3.13 (m, 3H), 2.89 (m, 1H), 2.07 (m, 1H), 1.82, (m, 1H), 1.52 (m, 2H), 1.28 (m, 2H), 1.18 (d, 3H, J=6.8 Hz). 13C (D2O, 125 MHz) δ ppm 59.43, 58.80, 53.47, 42.88, 29.30, 28.43, 18.40, 14.99, 13.23. ES-MS 240 (M+1).
To a solution of nonylamine (2.00 g, 14.0 mmol) in tetrahydrofuran (THF, 15 mL) was added 2,4-butane sultone (2.08 g, 14.7 mmol). The solution stirred at reflux for 5 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with THF (2×25 mL) and dried in vacuo.
The product (1.10 g, 3.9 mmol) was dissolved with heating in a solution of EtOH (20 mL), water (600 uL) and NaOH (163 mg, 4.1 mmol). After a few minutes a white solid precipitated. The solid was collected by filtration, washed with acetone (2×25 mL) and dried in vacuo. 1H NMR (D2O, 500 MHz) δ ppm 2.70 (m, 1H), 2.52 (m, 2H), 2.37 (m, 1H), 1.95 (m, 1H), 1.46 (m, 1H), 1.34 (m, 1H), 1.14 (m, 17H), 0.70 (t, 3H, J=6.8 Hz). 13C (D2O, 125 MHz) δ ppm 54.31, 52.90, 50.44, 31.31, 28.80, 28.74, 28.57, 27.31, 26.95, 22.20, 14.60, 13.57. ES-MS 302 (M+1).
2,4-butanesultone (1.27 g, 8.9 mmol) was added to an ice-chilled solution of dimethylamine (40% w/w in water). The solution was stirred at 0° C. for 4 hours. The solvent was evaporated in vacuo until complete dryness. The solid was washed with acetone (50 mL), collected by filtration and dried in vacuo. 1H NMR (D2O, 500 MHz) δ ppm 3.18 (t, 2H, J=8.1 Hz), 2.85 (m, 1H), 2.76 (s, 6H), 2.09 (m, 1H), 1.81 (m, 1H), 1.17 (d, 3H, J=7.3 Hz). 13C (D2O, 125 MHz) δ ppm 55.65, 53.05, 42.88, 26.72, 14.81. ES-MS182a (M+1).
To a solution of benzylamine (1.50 g, 14.0 mmol) in tetrahydrofuran (THF, 18 mL) was added 2,4-butane sultone (1.98 g, 14.6 mmol). The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with THF (2×25 mL) and dried in vacuo.
The product (2.55 g, 10.5 mmol) was dissolved with heating in a solution of EtOH (25 mL), water (1.6 mL) and NaOH (440 mg, 11.0 mmol). Diethyl ether (150 mL) was added to the filtrate. The solid was filtered and dried in vacuo. Yield: 27%. 1H NMR (DMSO, 500 MHz) δ ppm 7.29 (m, 4H), 7.20 (m, 1H), 3.67 (m, 2H), 2.56 (m, 1H), 2.45 (m, 2H), 1.98 (m, 1H), 1.36 (m, 1H), 1.04 (d, 3H, J=6.8 Hz). 13C (DMSO, 125 MHz) δ ppm 141.49, 128.73, 128.61, 127.16, 53.49, 52.88, 47.31, 32.67, 16.53. ES-MS 266 (M+1).
A solution of 2,4-butanesultone (1.33 g, 9.3 mmol) in tetrahydrofuran (THF, 3.0 mL) was added via syringe pump over a 2 h period to ethylamine (70% w/w in water, 12.0 mL, 186.0 mmol) at 5° C. The solution was stirred at 5° C. for an additional 2 hours. The solvent was coevaporated with EtOH (3×25 mL). The solid was suspended in acetone (25 mL). The suspension was stirred for 5 minutes the solid was filtered, washed with acetone (2×25 mL) and dried in vacuo. Yield: 70%. 1H NMR (D2O, 500 MHz) δ ppm 3.06 (t, 2H, J=8.1 Hz), 2.97 (m, 2H), 2.87 (m, 2H), 2.06 (m, 1H), 1.77 (m, 1H), 1.18 (d, 3H, J=7.3 Hz), 1.14 (t, 3H, J=7.3 Hz). 13C (D2O, 125 MHz) δ ppm 53.16, 44.91, 43.03, 28.20, 14.76, 10.66. ES-MS 182 (M+1).
To a solution of tert-butylamine (1.0 mL, 9.5 mmol) in tetrahydrofuran (4 mL) was added 1,4-butane sultone (1.36 g, 10.0 mmol) at room temperature. The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×20 mL) and dried in vacuo. Yield: 690 mg (34%). 1H NMR (D2O, 500 MHz) δ ppm 2.92 (t, 2H, J=7.1 Hz), 2.82 (t, 2H, J=7.1 Hz), 1.68 (m, 4H), 1.22 (s, 9H). 13C (D2O, 125 MHz) δ ppm 57.07, 50.30, 40.95, 25.28, 24.96, 21.62. ES-MS 210 (M−1).
A solution of 2,4-butanesultone (1.0 g, 7 mmol) in tetrahydrofuran (THF, 4.0 mL) was added via syringe pump over a 4 h period to ammonium hydroxide (28-30% NH3, 43 mL, 350 mmol) at 5° C. The solution was stirred at 5° C. for an additional 30 minutes. The solvent was coevaporated with EtOH (3×25 mL). The solid was dried in vacuo. Yield: 94%. 1H NMR (D2O, 500 MHz) δ ppm 3.05 (m, 2H), 2.90 (m, 1H), 2.05 (m, 1H), 2.06 (m, 1H), 1.77 (m, 1H), 1.18 (d, 3H, J=6.8 Hz), 1.14 (t, 3H, J=7.3 Hz). 13C (DMSO, 125 MHz) δ ppm 52.75, 38.20, 30.87, 17.27. ES-MS 154 (M+1).
To a solution of piperidine (1.50 g, 17.6 mmol) in tetrahydrofuran (THF, 20 mL) was added 2,4-butanesultone (2.50 g, 18.5 mmol). The solution was stirred at reflux for 3 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with THF (2×20 mL) and dried in vacuo.
The product (3.53 g, 15.9 mmol) was dissolved with heating in a solution of EtOH (30 mL), water (1.3 mL) and NaOH (670 mg, 16.7 mmol). The solution was poured in a large excess of Et2O (500 mL). The solid was filtered, washed with Et2O (1×25 mL) and acetone (1×20 mL) and dried in vacuo. Yield: 64%. 1H NMR (D2O, 500 MHz) δ ppm 2.72 (m, 1H), 2.33 (m, 6H), 1.97 (m, 1H), 1.48 (m, 1H), 1.43 (m, 4H), 1.31 (m, 1H), 1.13 (d, 3H, J=6.8 Hz). 13C (D2O, 125 MHz) δ ppm 55.78, 54.42, 53.61, 27.73, 24.92, 23.59, 14.61. ES-MS 244 (M+1).
A solution of 1,4-butanesultone (2.66 g, 18.6 mmol) in tetrahydrofuran (total volume: 4 mL) was added via syringe pump over a 4 hour period to ethylamine (70% w/w in water, 24 mL, 372 mmol) at 5° C. The solution was stirred at 5° C. for an additional 3 hours before it was warm up to room temperature. The reaction was stirred in these conditions overnight. The solvent was coevaporated with EtOH (1×25 mL). The solid was suspended in 50% acetone/EtOH (50 mL). The suspension was stirred for 5 minutes, the solid was filtered and dried in vacuo. Yield: 75%. 1H NMR (D2O, 500 MHz) δ ppm 2.95 (m, 4H), 2.82 (m, 2H), 1.68 (m, 4H), 1.13 (t, 3H, J=7.3 Hz), 1.14 (t, 3H, J=7.3 Hz). 13C (D2O, 125 MHz) δ ppm 50.27, 46.68, 42.99, 24.66, 21.48, 10.64. ES-MS182 (M+1).
To a solution of quinuclidine (1.5 g, 13.5 mmol) in tetrahydrofuran (THF, 15 mL) was added 1,4-butanesultone (2.0 g, 14.4 mmol) at room temperature. The solution was stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with THF (2×25 mL) and dried in vacuo. 1H NMR (D2O, 500 MHz) δ ppm 3.26 (m, 6H), 3.02 (m, 2H), 2.82 (t, 2H, J=17.3 Hz), 2.04 (m, 1H), 1.84, (m, 6H), 1.75 (m, 2H), 1.64 (m, 2H). 13C (D2O, 125 MHz) δ ppm 63.68, 54.81, 50.14, 23.51, 21.45, 20.58, 19.19. ES-MS 248 (M+1).
A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (10 g, 48.3 mmol) in water (40 mL total volume) was added via a syringe pump over 4 hours to a cold (2.8-3.1° C.) solution of dimethylamine (40% wt in water, 300 mL) with stirring. The mixture was slowly warmed to room temperature overnight. The mixture was then coevaporated with absolute ethanol (20 mL) and concentrated to dryness. The solid was dried overnight at 60° C. in the vacuum oven. The solid was suspended in ethanol (40 mL) stirred at reflux for 2 hours. The suspension was cooled to 5° C. and the solid was collected by suction-filtration, aspirator-dried 5 minutes, then dried for the weekend at 60° C. in the vacuum oven (wet cake: 13.74 g). The desired material was obtained as an off-white solid (11.65 g, quantitative).
A solution of 1,4-butane sultone (7.5 mL, 73.6 mmol) in 1,4-dioxane (total volume: 10 mL) was added over 4 hours via a syringe pump to a cold (4.3° C.) solution of dimethylamine (40% wt in water, 275 mL). The mixture was stirred for 3 hours at 4° C. after the end of the addition, then overnight at room temperature. The mixture was concentrated to dryness. The solid was suspended in absolute ethanol (50 mL) and the mixture was heated to reflux for 90 minutes. The suspension was cooled to 5° C. and the solid was collected by suction-filtration, rinsed with ethanol (2×10 mL). The solid was dried for 18 h at 60° C. in the vacuum oven. The desired material was obtained as a fine white powder 13.21 g, 72.9 mmol, 99% yield. The 1H and 13C NMR and MS were consistent with the structure.
A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid sodium salt (10 g, 50.9 mmol) in water (total volume: 40 mL) was added over 5 hours, via a syringe pump, to a cold (4.7° C.) solution of ethyl amine in water. The mixture was stirred for an additional 2 hours at 4.7° C. then for 18 hours at room temperature. NMR: quantitative yield. The mixture was concentrated. A solid could not be obtained: the sodium salt was too hygroscopic. The solution was treated with Amberlite IR-120 Plus, acid form, ion-exchange resin to give the free acid. It was still too hygroscopic to be obtained as a solid form. Submitted as a solution: d=1.314 g/mL, 62.5% w/w of the free acid in water. The 1H and 13C NMR and MS were consistent with the structure.
A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (15 g, 25 mmol) in water (12 mL total volume) was added over 5 minutes to a mixture of tert-butylamine (12.5 mL), water (6 mL) and methanol (3 mL). The mixture was heated at 35° C. for 1 h, 40° C. for 1 h, 45° C. for 1.5 hours. The mixture was the concentrated to a thick oil. The crude reaction mixture was passed over a column of Dowex 50×8 (125 g). The fractions containing the product were concentrated to dryness. The solid was dried overnight at 60° C. in the vacuum oven. The solid was recrystallized in a mixture of methanol (25 mL) and water (7 mL). The mixture was cooled slowly to room temperature, then to 5° C. The solid was collected by suction-filtration, rinsed with ethanol (1×10 mL). The solid was then dried for 18 hours at 60° C. in the vacuum oven. The desired material was obtained as an off-white solid (3.11 g, 14.7 mmol, 59%). The 1H and 13C NMR and MS were consistent with the structure.
A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (4 g, 20 mmol) in water (17.5 mL total volume) was added over 2 hours to a mixture of octylamine (8 mL), water (20 mL) and 1,4-dioxane (11 mL) at 70-75° C. The mixture was stirred at this temperature for another 2 hours after the end of the addition. The 1,4-dioxane was removed under reduced pressure and the mixture was diluted with water (10 mL). The mixture was extracted with 40% ethyl acetate/hexane (3×40 mL). The aqueous layer was concentrated then the mixture was passed over a column of Dowex 50×8 (125 g). The fractions containing the pure product were concentrated to a thick oil then freeze-dried. The desired material was obtained as a white fluffy solid (150 mg, 0.56 mmol, 3%). The 1H and 13C NMR and MS were consistent with the structure.
A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (2 g, 10 mmol) in water (12 mL total volume) was added over 1 hour to a mixture of quinuclidine (1.63 g, 4.7 mmol), water (10 mL) and 1,4-dioxane (10 mL) at 80° C. The mixture was stirred at this temperature for another 2 hours after the end of the addition. The reaction mixture was concentrated then the mixture was passed over a column of Dowex 50×8 (125 g). The fractions containing the pure product were concentrated to a white solid. The solid was dried for 18 hours at 60° C. in the vacuum oven. The desired material was obtained as a white solid (1.92 g, 7.7 mmol, 77%). The 1H and 13C NMR and MS were consistent with the structure.
A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (4 g, 20 mmol) in water (12.5 mL total volume) was added over 2 hours to a mixture of benzylamine (4.28 g, 40 mmol), water (10 mL) and 1,4-dioxane (5 mL) at 80° C. The mixture was stirred at this temperature for another 2.5 hours after the end of the addition. The reaction mixture was extracted with chloroform (2×40 mL). It was then concentrated to dryness. The crude solid was recrystallized in a mixture of ethanol (30 mL) and water (4 mL). The mixture was left to cool to room temperature for the night. The solid was collected by suction-filtration, rinsed with ethanol (10 mL) and dried in the vacuum oven at 60° C. The desired material was obtained as a white solid (2.67 g, 10 mmol, 50%). The 1H and 13C NMR and MS were consistent with the structure.
A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (2 g, 10 mmol) in water (9.75 mL total volume) was added over 8 hours to a mixture of 1,2,3,4-tetrahydro-1-nahtylamine (2 g, 13.6 mmol), water (10 mL) and 1,4-dioxane (4 mL) at 40° C. The mixture was stirred at this temperature for another 18 hours after the end of the addition. The reaction was not completed. The mixture was heated for 2 hours at reflux. The mixture was diluted with water (10 mL) and 50% w/w NaOH (0.25 mL) was added. The reaction mixture was extracted with chloroform (2×25 mL). It was then concentrated to a thick oil. The solution was applied on a Dowex 50 W 8 column (100 g). The fractions containing the product were concentrated, treated with activated charcoal (no effect) and freeze-dried. The desired material was obtained as a glassy solid (0.85 g, 3 mmol, 30%). The 1H and 13C NMR and MS were consistent with the structure.
A solution of 3-chloro-2-hydroxy-1-propanesulfonic acid, sodium salt (4 g, 20 mmol) in water (13.35 mL total volume) was added over 5 hours to a solution of piperidine (8 mL g, 80 mmol), in water (15 mL) at 70° C. The mixture was stirred at 80° C. for 2 hours. The reaction was completed. The mixture was stirred at room temperature for the night. The mixture was diluted with water (10 mL) and was extracted with chloroform (3×30 mL). It was then concentrated to a thick oil. The solution was applied on a Dowex 50 W 8 column (100 g). The fractions containing the product were concentrated to dryness then recrystallized in a mixture of ethanol (30 mL) and water (2.1 mL). The mixture was cooled slowly at room temperature. The solid was collected by suction filtration, rinsed with ethanol (2×5 mL) air dried 5 minutes, then 18 hours at 60° C. in the vacuum oven. The desired material was obtained as a fine white solid (3.06 g, 13.7 mmol, 68%). The 1H and 13C NMR and MS were consistent with the structure.
1-adamantaneamine hydrochloride (2.67 g, 13.3 mmol) was treated with 1N NaOH (20 mL) and CH2Cl2 (3×20 mL). The biphasic solution was shaken. The organic extracts were combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of 2-adamantanamine (1.87 g, 12.4 mmol) in tetrahydrofuran (THF, 15 mL) was added 1,4-butane sultone (1.76 g, 13.0 mmol). The solution was stirred at reflux overnight. The reaction was cooled to room temperature. The solid was collected by filtration, washed with THF (1×15 mL) and dried in vacuo. A suspension of the solid in EtOH (25 mL) was stirred at reflux for 1 hour. The warm mixture was filtered. The solid was dried in vacuo. 1H NMR (D2O, 500 MHz) δ ppm 2.92 (m, 2H), 2.82 (m, 1H), 2.05 (s, 3H), 1.75 (s, 6H), 1.63 (m, 6H), 1.52 (m, 3H). 13C (D2O, 125 MHz) δ ppm 57.62, 50.30, 39.03, 38.14, 35.09, 28.98, 25.25, 21.63. ES-MS 288 (M+1).
To a solution of octylamine (2.20 g, 17.0 mmol) in tetrahydrofuran (11 mL) was added 1,4-butane sultone (2.30 g, 16.2 mmol). The solution was heated to reflux for 5 hours. The reaction was cooled to room temperature. The product formed a gel. A few drops of EtOH were added to dissolve the product. The solution was poured in a large excess of acetone (25 mL). After 5 minutes, a white solid precipitated. The solid was collected by filtration and dried in vacuo. The product was dissolved in EtOH and Dowex 50×8 resin (pre-washed, 6 g) was added to the solution. The suspension was stirred for 15 minutes and the resin was filtered. The filtrate was evaporated under educed pressure and the product was dried in vacuo. Yield: 31%. 1H NMR (DMSO, 500 MHz) δ ppm 8.24 (s (broad), 1H), 2.85 (m, 4H), 2.45 (m, 2H), 1.64 (m, 4H), 1.61 (m, 2H), 1.25 (m, 10H), (m, 2H), 0.85 (t, 3H, J=6.8 Hz). 13C (DMSO, 125 MHz) δ ppm 51.22, 47.54, 47.38, 31.82, 29.14, 26.59, 26.13, 25.51, 22.95, 22.75, 14.64. ES-MS 266 (M+1).
To a solution of cyclohexylamine (1.50 g, 15.1 mmol) in tetrahydrofuran (15 mL) was added 2,4-butane sultone (2.04 g, 14.4 mmol). The solution stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and dried in vacuo. Yield: 59%. 1H NMR (DMSO, 500 MHz) δ ppm 8.50 (s (broad), 1H), 3.02 (m, 2H), 2.93 (m, 1H), 2.60 (m, 1H), 1.93 (m, 3H), 1.75 (m, 3H), 1.57 (m, 1H), 1.21 (m, 4H), 1.11 (m, 4H). 13C (DMSO, 125 MHz) δ ppm 56.23, 53.20, 43.09, 29.54, 29.41, 29.39, 25.40, 24.42, 17.23. ES-MS 234 (M−1).
To a solution of DL-2-aminopentanol (1.0 g, 9.7 mmol) in tetrahydrofuran (6 mL) was added 1,4-butane sultone (1.31 g, 9.2 mmol) at room temperature. The solution was stirred at reflux for 5 hours. The reaction was cooled to room temperature. The supernatant was removed and the solid was dried in vacuo. The white solid was filtered, washed with acetone (2×25 mL) and dried in vacuo. Yield: 45%. 1H NMR (DMSO, 500 MHz) δ ppm 8.20 (s (broad), 1H), 5.23 (m, 1H), 3.66 (m, 1H), 3.49 (m, 1H), 3.02 (m, 1H), 2.91 (m, 2H), 2.46 (t, 2H, J=7.3 Hz), 1.65, (m, 4H), 1.54 (m, 2H), 1.38 (m, 2H), 0.88 (t, 3H, J=7.3 Hz). 13C (DMSO, 125 MHz) δ ppm 58.83, 58.54, 51.23, 44.77, 29.91, 25.56, 23.06, 18.95, 14.48. ES-MS 238 (M−1).
To a solution of veratrylamine (1.50 g, 9.0 mmol) in 1,4-dioxane (8 mL) was added 1,4-butane sultone (1.21 g, 8.5 mmol) at room temperature. The mixture was then heated at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×25 mL) and dried on pump. Yield: 18%. 1H NMR (D2O, 500 MHz) δ 6.96 (m, 3H), 4.04 (s, 2H), 3.74 (m, 6H), 2.95 (t, 2H, J=6.8 Hz), 2.80 (t, 2H, J=7.3 Hz), 1.68, (m, 4H). 13C (D2O, 125 MHz) δ ppm 149.19, 148.50, 123.82, 123.36, 113.25, 112.17, 55.91, 50.88, 50.24, 46.41, 24.55, 21.50. ES-MS 302 (M−1).
1-adamantaneamine hydrochloride (2.67 g, 14.2 mmol) was treated with 1N NaOH (20 mL) and CH2Cl2 (3×20 mL). The organic extracts were combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To an 80° C. solution of 1-adamantanamine (2.15 g, 14.2 mmol) in 1,4-dioxane (10 mL) and water (5 mL) was added via syringe pump (1 h addition) a solution of 3-chloro-2-hydroxy-propanesulfonic acid, sodium salt (1.93 g, 9.7 mmol) in 1,4-dioxane (0.5 mL) and water (10 mL). The solution was stirred at reflux overnight. The reaction was cooled to room temperature. The solvent was evaporated under reduced pressure. The solid was suspended in 25% acetone/EtOH. The mixture was heated to reflux for 1 minute. The solid was collected by filtration. The pure product crystallized in the filtrate. The product was filtered, washed with EtOH (2×10 mL), dissolved in water and lyophilized. Yield: 15%. 1H NMR (D2O, 500 MHz) δ ppm 4.18 (m, 1H), 3.22 (m, 1H), 3.01 (m, 2H), 2.94 (m, 1H), 2.06 (s, 3H), 1.77 (m, 7H), 1.61 (d, 3H), 1.53 (m, 3H). 13C (D2O, 125 MHz) δ ppm 64.23, 57.99, 55.05, 44.10, 38.10, 35.07, 29.03. ES-MS 288 (M−Na (23)).
2-adamantanamine hydrochloride (2.50 g, 13.3 mmol) was treated with 1N NaOH (20 mL) and CH2Cl2 (3×20 mL). The organic extracts were combined, dried with Na2SO4, filtered, evaporated under reduced pressure and dried in vacuo.
To a solution of 2-adamantanamine (1.06 g, 7.0 mmol) in 1,4-dioxane (6 mL) was added 1,4-butane sultone (955 mg, 6.7 mmol). The solution was stirred at reflux for 5 hours. The reaction was cooled to room temperature. The solid was collected by filtration. It was suspended in EtOH (25 mL) and the mixture was heated to reflux for 1 minute before the solid was filtered. It was washed with EtOH (1×15 mL) and dried in vacuo. Yield: 55%. 1H NMR (D2O, 500 MHz) δ ppm 3.29 (m, 1H), 2.97 (m, 2H), 2.83 (m, 2H), 2.02 (m, 2H), 1.83 (m, 2H), 1.68 (m, 14H). 13C NMR (D2O, 125 MHz) δ ppm 63.07, 50.25, 45.03, 36.55, 36.31, 29.85, 29.05, 26.68, 26.41, 24.47, 21.61. ES-MS 286 (M−1).
To an 80° C. solution of 2-adamantanamine hydrochloride (2.50 g, 13.3 mmol) and sodium hydroxide (586 mg, 14.6 mmol) in 1,4-dioxane (7 mL) and water (7 mL) was added via syringe pump (1 hour addition) a solution of 3-chloro-2-hydroxy-propane sulfonic acid, sodium salt (1.76 g, 8.9 mmol) in 1,4-dioxane (1 mL) and water (9 mL). The solution was stirred at 80° C. for an additional 4 hours. The reaction was cooled to room temperature. The solvent was evaporated under reduced pressure. The solid was suspended in EtOH (25 mL). The mixture was heated to reflux for 1 minute. The solid was removed by filtration. The pure product crystallized in the filtrate. The product was filtered, washed with EtOH (1×10 mL) and dried in vacuo. Yield: 30%. 1H NMR (D2O, 500 MHz) δ ppm 4.30 (m, 1H), 3.35 (m, 2H), 3.03 (m, 3H), 2.07 (m, 2H), 1.84 (m, 2H), 1.75 (m, 4H), 1.65 (d, 6H). 13C (D2O, 125 MHz) δ ppm 63.31, 55.03, 49.51, 36.51, 36.33, 36.26, 29.78, 29.23, 28.81, 26.63, 26.38. ES-MS 289 (M+1).
To an 80° C. solution of exo-2-aminonorbornane (910 mg, 8.2 mmol) and sodium hydroxide (242 mg, 6.1 mmol) in 1,4-dioxane (4 mL) and water (4 mL) was added via syringe pump (1 hour addition) a solution of 3-chloro-2-hydroxy-propane sulfonic acid, sodium salt (1.09 g, 5.5 mmol) in 1,4-dioxane (0.5 mL) and water (5.5 mL). The solution was stirred at 80° C. for an additional 5 hours. The reaction was cooled to room temperature. The solvent was evaporated under reduced pressure. The solid was suspended in EtOH (25 mL). The mixture was heated to reflux for 1 minute. The solid was recovered by filtration and it was passed through an ion exchange column (Dowex 50×8, 100 g, solvent: water). The product was recrystallized in EtOH/water (99/1). Yield: 17%. 1H NMR (D2O, 500 MHz) δ ppm 4.25 (m, 1H), 3.25 (m, 2H), 3.01 (m, 4H), 2.39 (m, 1H), 2.27 (m, 1H), 1.69 (m, 1H), 1.51 (m, 1H), 1.38 (m, 3H), 1.19 (m, 1H), 1.07 (m, 2H). 13C (D2O, 125 MHz) δ ppm 63.66, 63.50, 62.21, 61.98, 54.94, 50.11, 50.04, 39.26, 39.21, 36.02, 35.97, 35.91, 35.80, 34.70, 34.61, 27.11, 27.08, 26.50, 26.45. ES-MS 250 (M−1).
To a hot solution of isoamylamine (2.0 g, 22.9 mmol) in tetrahydrofuran (THF, 11 mL) was added via syringe pump (2 hour addition) a solution of 2,4-butane sultone (3.1 g, 21.8 mmol in THF (total of 5 mL)). The solution was stirred at reflux for an additional 2 hours. The reaction was cooled to room temperature. The solid was recovered by filtration and it was washed with THF (25 mL) and acetone (25 mL). The solid was dissolved in water (20 mL) and Dowex 50×8 (10 g) was suspended in the solution. The mixture was stirred for 15 minutes and the resin was filtered. The solvent was evaporated under reduced pressure. Yield: 28%. 1H NMR (H2O, 500 MHz) δ ppm 3.07 (t, 2H, J=7.8 Hz), 2.92 (t, 2H, J=7.8 Hz), 2.87 (m, 1H), 2.06 (m, 1H), 1.77 (m, 1H), 1.51 (m, 1H), 1.42 (m, 2H), 1.18 (d, 3H, J=6.8 Hz), 0.78 (d, 3H, J=6.3 Hz). 13C(H2O, 125 MHz) δ ppm 53.21, 46.32, 45.37, 34.36, 28.16, 25.35, 21.51, 14.79. ES-MS 224 (M+1).
To a 80° C. solution of isoamylamine (2.0 g, 22.9 mmol) in 1,4-dioxane (9 mL) and water (3 mL) was added via syringe pump (1 h addition) a solution of 3-chloro-2-hydroxy-propane sulfonic acid, sodium salt (3.04 g, 15.3 mmol) in 1,4-dioxane (9.5 mL) and water (0.5 mL). The solution was stirred overnight at 80° C. The solvent was evaporated. The product was passed through an ion exchange column (Dowex 50×8, 100 g, solvent: water). It was recrystallized in absolute EtOH and lyophilized. Yield: 27%. 1H NMR (H2O, 500 MHz) δ ppm 4.24 (m, 1H), 3.22 (m, 1H), 3.02 (m, 5H), 1.49 (m, 3H), 0.79 (d, 3H, J=6.3 Hz). 13C(H2O, 125 MHz) δ ppm 63.54, 54.89, 51.53, 46.48, 34.12, 25.46, 21.56, 21.46. ES-MS 226 (M+1).
To a 80° C. solution of DL-2-amino-1-pentanol (1.0 g, 9.7 mmol) in 1,4-dioxane (5 mL) and water (3 mL) was added via syringe pump (1 hour addition) a solution of 3-chloro-2-hydroxy-propane sulfonic acid, sodium salt (1.84 g, 9.2 mmol) in 1,4-dioxane (6 mL) and water (0.5 mL). The solution was stirred overnight at 80° C. The solvent was evaporated. The product was passed through an ion exchange column (Dowex 50×8, 100 g, solvent: water). The product was dissolved. It was recrystallized in absolute EtOH and lyophilized. Yield: 27%. 1H NMR (H2O, 500 MHz) δ ppm 4.26 (m, 1H), 3.77 (m, 1H), 3.32 (m, 1H), 3.24 (m, 1H), 3.03 (m, 3H), 1.54 (m, 2H), 1.29 (m, 2H), 0.81 (t, 3H, J=7.3 Hz). 13C(H2O, 125 MHz) δ ppm 63.69, 63.60, 59.49, 59.38, 58.81, 58.36, 54.98, 48.68, 48.27, 29.32, 28.85, 18.40, 18.38, 13.12. ES-MS 242 (M+1).
To a hot solution of 2-mercaptobenzimidazole (2.0 g, 13.3 mmol) in 1,4-dioxane (12 mL) and water (3 mL) was added via syringe pump (1 hour addition) a solution of 2,4-butane sultone (1.80 g, 12.7 mmol in 1,4-dioxane (total of 3 mL)). The solution was stirred at reflux for an additional 3 hours. The solid was collected by filtration. It was washed with acetone (2×20 mL) and dried in vacuo. Yield: 86%. 1H NMR (DMSO, 500 MHz) δ ppm 7.64(m, 2H), 7.44 (m, 2H), 3.63 (t, 2H, J=7.3 Hz), 2.68 (m, 1H), 2.10 (m, 1H), 1.90 (m, 1H), 1.15 (d, 3H, J=6.8 Hz). 13C (DMSO, 125 MHz) δ ppm 152.64, 133.32, 125.66, 113.67, 53, 10, 39.72, 33.48, 30.14, 16.85. ES-MS 287 (M+1).
To a solution of cyclohexylamine (2.0 g, 20.2 mmol) in 1,4-dioxane (13 mL) was added 1,4-butane sultone (2.61 g, 19.2 mmol). The solution was heated to reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2×20 mL) and dried in vacuo. Yield: 52%. 1H NMR (D2O, 500 MHz) δ ppm 2.95 (m, 3H), 2.81 (m, 2H), 1.92 (m, 2H), 1.67 (m, 6H), 1.52 (m, 1H), 1.18 (m, 4H), 1.02 (m, 1H). 13C (D2O, 125 MHz) δ ppm 57.32, 50.31, 44.01, 29.02, 24.84, 24.68, 24.07, 24.55. ES-MS 236 (M+1).
The flask was closed with a septum and connected to a 20% NaOH scrubber for the Ritter Reaction. Potassium cyanide (3.25 g, 50 mmol) was added to acetic acid (13 mL) and the mixture was stirred for 10 min at room temperature. A solution of sulfuric acid (7 mL) in acetic acid (6 mL) was added and the resulting suspension was stirred 10 minutes at room temperature. The 3-methyl-3-pentanol (5 g, 48.9 mmol) was added drop-wise over a 5 minute period. The mixture was stirred at room temperature for 4 hours, at which time some chunks of potassium cyanide were still visible.
Another portion of potassium cyanide (0.6 g, powdered) was added and the mixture was stirred for 18 hours at room temperature. The mixture was purged with nitrogen for 1 h then poured over ice (approx. 50 g). The pH of the solution was adjusted to 9 with the addition of 20% NaOH (use 50% next time to reduce the volume). The layers were separated and the aqueous layer was extracted with ether (1×20 mL). The combined organic layers were washed with saturated potassium carbonate (1×5 mL) then dried over magnesium sulfate. The ether was evaporated under reduced pressure to afford a yellow oil (4.11 g, 31.8 mmol, 64%). The oil showed to be a mixture of cis and trans formamide but what otherwise pure enough to be used as such. 1H NMR (500 MHz, DMSO-d6) δ 0.75-0.80 (m, 6H), 1.11-1.12 (m, 3H), 1.40-1.54 (m, 2H), 1.66-1.73 (m, 2H), 7.35-7.45 (br s and br d, 1H), [7.88 (s) and 8.08 (d, J=11.7 Hz) for 1H); 13C NMR (125 MHz, DMSO-d6) δ 7.7, 7.9, 23.4, 24.2, 30.6, 33.8, 55.6, 160.3, 163.3
The 1-ethyl-1-methyl-propylformamide (4.00 g, 31.1 mmol) was added to 20% NaOH (40 mL). The mixture was heated to reflux for 4 hours then was left overnight at room temperature. Toluene (10 mL) was added and the layers were separated. The organic layer was dried over sodium sulfate then filtered. The final volume of the filtrate was about 30 mL. It was used as such in the next step.
A solution of 1,3-propanesultone (2.5 g, 20 mmol) in 2-butanone (10 mL) was added to a solution of 3-methyl-3-ethyl-3-propylamine in toluene (total volume: 30 mL). The mixture was heated to reflux for 5 hours then was cooled to room temperature. The solid was collected by suction-filtration and rinsed with acetone (2×5 mL). The solid was dried overnight at 45° C. in the vacuum oven. The title compound was obtained as a fine white solid (3.63 g, 16.3 mmol, 33% overall yield). 1H NMR (500 MHz, DMSO-d6) δ 0.79 (t, J=7.3 Hz, 6H), 1.15 (s, 3H), 1.53-1.59 (m, 4H), 1.97-2.00 (m, 2H), 2.89 (t, J=7.1 Hz, 2H), 3.03 (t, J=7.6 Hz, 2H); 13C NMR (125 MHz, DMSO-d6) δ 6.8, 20.2, 21.9, 27.7, 39.7, 48.2, 63.3 ES-MS 224; (M+H)
To a cooled solution of sodium methoxide (0.5 M in MeOH, 25 mL1) was added via syringe over a 10 minutes period 2-nitropropane (5.0 g, 56 mmol). The reaction mixture was stirred at room temperature for 30 minutes and recooled before m-anisaldehyde (6.8 mL, 56 mmol) was added. The reaction mixture was stirred at room temperature overnight. The mixture was neutralized with Amberlite IR-120 (strongly acidic). The resin was removed by filtration and washed with MeOH (2×20 mL). The filtrate was evaporated. The resulting oil was purified by flash chromatography: 98% Hexanes/EtOAc to 90% Hexanes/EtOAc, affording the desired nitro compound (5.70 g, 45%).
To a solution of the nitro compound (5.70 g, 25.3 mmol)) in MeOH (25 mL) was added 6M HCl (25 mL). After cooling to 5° C., zinc powder (8.2 g, 125 mmol) was added. The suspension was stirred at 0-5° C. and at room temperature overnight. The mixture was filtered on a celite pad. The filter cake was washed with MeOH (2×20 mL). The combined filtrates were evaporated under reduced pressure. The residue was dissolved in EtOAc (40 mL). The mixture was exctracted with 5% NaOH (1×40 mL). The aqueous phase was exctracted with EtOAc (2×40 mL). The combined organic extracts were dried with Na2SO4, filtered, evaporated and dried in vacuo to afford the corresponding amine. The amine (2.15 g, 44%) was used without further purification.
To a solution of amine (2.15 g, 11.0 mmol) in Pinacolone (6 mL) and toluene (6 mL) was added 1,3-propane sultone (1.28 g, 10.5 mmol). The solution was stirred at reflux overnight. The reaction mixture was cooled to room temperature. The solid material was collected by filtration, was washed with acetone (2×20 mL). The solid was suspended in EtOH (30 mL). The suspension was stirred at reflux for 1 hour. The mixture was cooled to room temperature. The white solid was filtered, washed with acetone (2×15 mL) and dried in a vacuum oven at 50° C., affording the title compound, 2.26 g (66%). 1H NMR (DMSO, 500 MHz) δ ppm 8.45 (s (broad), 1H), 7.26 (t, 1H, J=7.9 Hz), 6.89 (m, 3H), 6.30 (d, 1H, J=3.2 Hz), 4.69 (d, 1H, J=3.8 Hz), 3.74 (s, 3H), 3.10 (m, 2H), 2.62 (t, 2H, J=6.7 Hz), 2.00 (m, 2H), 1.07 (m, 6H). 13C (DMSO, 125 MHz) δ ppm 159.24, 142.09, 129.45, 120.80, 114.30, 113.76, 74.16, 62.48, 55.92, 50.10, 41.57, 23.30, 21.18, 19.37. ES-MS 316 (M−1).
NaOMe (0.5M, 40 mL) was added to nitrocyclohexane (2.58 g, 20 mmol) and the solution was stirred for 30 minutes then concentrated to afford a white solid. To this solid was added 4-methylbenzylpyrridinium (6.6 g, 13 mmol) and DMSO (20 mL). The mixture was heated at 100° C. for 15 hours then cooled to rt and diluted with HCl (1M) and EtOAc. After separation of the two phases, the organic layer was washed twice with HCl (1M) then concentrated to obtain an oily crude. Methanol was added to precipitate the pyridinium byproduct which was filtered off, and the filtrate was concentrated and purified by column using Hex:EtOAc 90:10 to obtain the desired nitro (still contaminated with the pyridinium salt). 2 g, 66% yield.
To a stirred solution of the nitro (2.0 g, 8.58 mmol) in methanol (20 mL) was added a spatula of Raney-Ni in water. The suspension was hydrogenated under atmospheric pressure of hydrogen for 15 hours (TLC indicates complete consumption of the starting material) then filtered on celite and concentrated under reduced pressure. The crude was purified by column using CH2Cl2:MeOH 80:10 to afford 1.2 g of the corresponding amine.
To a stirred solution of the amine (800 mg, 3.93 mmol) in THF (8 mL) was added 1,3-propane sultone (480 mg, 3.93 mmol). The reaction mixture was stirred at reflux for 15 hours then cooled to room temperature. The solid was collected by filtration and was washed with THF. The solid was suspended in EtOH (10 mL) and stirred at reflux for 1 hour. The suspension was then cooled to room temperature. The solid was collected by filtration, washed with ethanol and dried under high vacuum to afford the title compound, 1.1 g (86%). 1H NMR (500 MHz, DMSO-d6) δ 1.18-1.78 (m, 10H), 2.00 (m, 2H), 2.29 (s, 3H), 2.65 (m, 2H), 2.92 (s, 2H), 3.12 (m, 2H), 7.10-7.16 (m, 2H), 8.39 (bs, 2H). 13NMR (125 MHz, DMSO-d6) δ 20.74, 21.35, 22.80, 25.05, 31.60, 41.04, 50.24, 61.63, 129.71, 131.38, 132.44, 136.80. ES-MS 324 (M−1).
To a stirred solution of the phenol (233 mg, 1 mmol) in DMF/THF (2.5 mL/2.5 mL) was added MeI (93 uL, 1.5 mmol) followed by K2CO3 (276 mg, 2 mmol). The suspension was heated at reflux for 15 hours then diluted with HCl (1M) and with EtOAc. The organic layer was washed with HCl(1M) then concentrated under high vacuum. The crude was purified by column using Hex:EtOAc 90:10 to obtain 215 mg of the methoxy (87% yield).
To a stirred solution of the nitro (300 mg, 1.2 mmol) in methanol (5 mL) was added a small spatula of Raney-Ni in water. The suspension was hydrogenated under atmospheric pressure of hydrogen for 3 hours (TLC indicates complete consumption of the starting material) then filtered on prewashed celite and concentrated under reduced pressure. The crude amine was used as such in the next step.
To the crude amine (240 mg, 1.34 mmol) in solution in THF (3 mL) was added 1,3-sultone (181 mg, 1.48 mmol) and the mixture was heated at reflux of THF for 12 hours. The suspension of was cooled down and filtered. The solid was dried to afford 270 mg of the homotaurin as a white solid (67% yield). 1H NMR (500 MHz, D2O) δ 1.11 (s, 6H), 2.00 (m, 2H), 2.67 (m, 2H), 2.80 (m, 2H), 3.12 (m, 2H), 3.74 (s, 3H), 6.90 (m, 2H), 7.14 (m, 2H), 8.61 (bs, 2H). ES-MS 272 (M−1). ES-MS 300 (M−1).
To a solution of 2-nitropropane (3.0 g, 34 mmol), p-tolualdehyde (4.0 mL, 34 mmol) and Tetrahydrofuran (30 mL) was added Amberlyst A-21 (7 g). The reaction mixture was stirred at room temperature for 40 hours. The resin was removed by filtration and washed with THF (2×20 mL). The filtrate was evaporated. The resulting oil was purified by flash chromatography: 98% Hexanes/EtOAc to 90% Hexanes/EtOAc, affording the desired nitro compound (820 mg, 12%).
A suspension of Pd/C and the nitro compound (820 mg, 3.9 mmol) in EtOAc (10 mL) was stirred under H2 (1 atm) overnight. The mixture was filtered on a celite pad. The celite was washed with EtOAc (2×15 mL). The combined filtrates were evaporated under reduced pressure to afford the corresponding amine. The amine (470 mg, 67%) was used without further purification.
To a solution of amine (470 mg, 2.6 mmol) in pinacolone (5 mL) and Toluene (5 mL) was added 1,3-propane sultone (310 mg, 2.5 mmol). The solution was stirred at reflux for 4 hours. The reaction mixture was cooled to room temperature. The solid was collected by filtration, was washed with acetone (2×10 mL) and dried in vacuo, affording the title compound, 196 mg (26%). 1H NMR (DMSO, 500 MHz) δ ppm 8.46 (s (broad), 1H), 7.24 (d, 2H, J=7.8 Hz), 7.16 (d, 2H, J=8.3 Hz), 6.23 (d, 1H, J=3.9 Hz), 4.68 (d, 1H, J=3.9 Hz), 3.11 (m, 2H), 2.63 (t, 2H, J=6.8 Hz), 2.29 (s, 3H), 2.00 (m, 2H), 1.04 (s, 6H). 13C (DMSO, 125 MHz) δ ppm 137.69, 137.56, 129.03, 128.45, 74.07, 62.38, 49.91, 41.35, 22.99, 21.39, 20.81, 18.76. ES-MS 300 (M−1).
NaOMe (0.5M, 20 mL) was added to 2-nitropropane (890 mg, 10 mmol) and the solution was stirred for 30 minutes then concentrated to afford a white solid. To this solid was added 4-methylbenzylpyrridinium (3.3 g, 15 mmol) and DMSO (15 mL). The mixture was heated at 100° C. for 15 hours then cooled to room temperature and diluted with HCl (1M) and EtOAc. After separation of the two phases, the organic layer was washed twice with HCl (1M) then concentrated to obtain an oily crude product. Methanol was added to precipitate the pyridinium byproduct which was filtered off, and the filtrate was concentrated and purified by column using Hex:EtOAc 90:10 to obtain the desired nitro but still contaminated with the pyridinium salt. 1.32 g, 66% yield.
To a stirred solution of the nitro (700 mg, 3.62 mmol) in methanol (10 mL) was added a small spatula of Raney-Ni in water. The suspension was hydrogenated under atmospheric pressure of hydrogen for 15 hours (TLC indicates complete consumption of the starting material) then filtered on celite and concentrated under reduced pressure. The crude amine was used as such in the next step.
To a stirred solution of the amine (550 mg, 3.39 mmol) in THF (8 mL) was added 1,3-propane sultone (414 mg, 3.39 mmol). The reaction mixture was stirred at reflux for 6 hours then cooled to room temperature. The solid was collected by filtration and was washed with THF. The solid was suspended in EtOH (5 mL) and stirred at reflux for 1 hour. The suspension was then cooled to room temperature. The solid was collected by filtration, washed with ethanol and dried under high vacuum to afford the title compound, 210 mg (22%). 1H NMR (500 MHz, DMSO-d6) δ 1.13 (s, 6H), 2.00 (m, 2H), 2.66 (dd, J=7.0 & 7.0 Hz, 2H), 2.75 (s, 2H), 3.10 (dd, J=7.0 & 7.0 Hz, 2H), 6.72 (d, J=8.3 Hz, 2H), 7.00 (d, J=8.3 Hz, 2H), 8.60 (bs, 2H), 9.36 (s, 1H). 13NMR (125 MHz, DMSO-d6) δ 23.1, 41.2, 43.2, 49.8, 59.4, 115.7, 125.8, 132.3, 157.1. ES-MS 284 (M−1).
14 g of washed Amberlyst A21 ion exchange resin were placed in a round bottom flask to which was added nitropropane (14 mL, 120 mmol) and 4F-fluorobenzaldehyde (7.45 g, 60 mmol). The reaction mixture was stirred overnight then diluted with Et2O and filtered. The filtrate was concentrated under rotavap vaccuo then pump vaccuo by heating at 120° C. to remove the excess of aldehyde. The crude was purified by column using Hex:EA 90:10 to afford 3.3 g (25%) of the Henry-aldol product as a colorless solid.
To a solution of the nitro compound (5 g, 23.5 mmol) in MeOH (100 mL) was added 6M HCl (25 mL). After cooling to 5° C., zinc powder (7.6 g, 117 mmol) was added. The suspension was stirred at room temperature for 3 hours then filtered on a celite pad. The filter cake was washed with MeOH (2×20 mL). The combined filtrates were evaporated under reduced pressure. The residue was dissolved in EtOAc (40 mL), then K2CO3 (1M) was added until basic pH. The organic phase was dried with Na2SO4, filtered, evaporated and dried in vacuo to afford 3.5 g (83% yield) of the corresponding amine. The amine was used without further purification.
To a stirred solution of the amine (3.3 g, 18.0 mmol) in THF (20 mL) was added 1,3-propane sultone (2.2 g, 18.0 mmol). The reaction mixture was stirred at reflux for 15 hours then cooled to room temperature. The solid was collected by filtration, washed with ethanol and with Et2O then dried under high vacuum to afford the title compound, 4.25 g (77% yield). 1H NMR (500 MHz, DMSO-d6) δ 1.13 (s, 6H), 2.00 (m, 2H), 2.66 (dd, J=7.0 & 7.0 Hz, 2H), 2.75 (s, 2H), 3.10 (dd, J=7.0 & 7.0 Hz, 2H), 6.72 (d, J=8.3 Hz, 2H), 7.00 (d, J=8.3 Hz, 2H), 8.60 (bs, 2H), 9.36 (s, 1H). 13NMR (125 MHz, DMSO-d6) δ 23.1, 41.2, 43.2, 49.8, 59.4, 115.7, 125.8, 132.3, 157.1. 19F (282 MHz, DMSO-d6) δ −115.15 (m, 1F). ES-MS 304 (M−1).
This application is a continuation application of U.S. patent application Ser. No. 11/316,694, filed Dec. 21, 2005, which, in turn, claims priority to U.S. Provisional Patent Application 60/638,819, filed on Dec. 22, 2004. U.S. patent application Ser. No. 11/316,694, filed Dec. 21, 2005 is also a continuation-in-part of U.S. patent application Ser. No. 10/871,514, filed Jun. 18, 2004, now U.S. Pat. No. 7,414,076, issued Aug. 19, 2008, which claims priority to U.S. Provisional Patent Application No. 60/512,047, filed Oct. 17, 2003, and U.S. Provisional Patent Application No. 60/480,906, filed Jun. 23, 2003, all entitled Methods and Compositions for Treating Amyloid-Related Diseases. This application is also related to U.S. Provisional Application No. 60/638,636, filed Dec. 22, 2004 and U.S. patent application Ser. No. 10/871,365, filed Jun. 18, 2004, now U.S. Pat. No. 7,244,764, issued Jul. 17, 2007. This application is also related to U.S. Provisional Patent Application No. 60/512,017, filed Oct. 17, 2003, U.S. Provisional Patent Application No. 60/480,918, filed Jun. 23, 2003, and U.S. patent application Ser. No. 10/871,613, filed Jun. 18, 2004, abandoned, all entitled Methods for Treating Protein Aggregation Disorders. This application is related to U.S. Provisional Patent Application No. 60/512,116, filed Oct. 17, 2003, U.S. Provisional Patent Application No. 60/480,984, filed Jun. 23, 2003, and U.S. application Ser. No. 10/871,549, filed Jun. 18, 2004, abandoned, all entitled Pharmaceutical Formulations of Amyloid-Inhibiting Compounds. This application is related to U.S. Provisional Patent Application No. 60/436,379, filed Dec. 24, 2002, U.S. Provisional Patent Application No. 60/482,214, filed Jun. 23, 2003, entitled Combination Therapy for the Treatment of Alzheimer's Disease, U.S. patent application Ser. No. 10/746,138, abandoned, filed Dec. 24, 2003, International Patent Application No. PCT/CA2003/002011, filed Dec. 24, 2003, and U.S. patent application Ser. No. 10/871,537, filed Jun. 18, 2004, pending, entitled Therapeutic Formulations for the Treatment of Beta-Amyloid Related Diseases. This application is related to U.S. Provisional Patent Application No. 60/512,135, filed Oct. 17, 2003, U.S. Provisional Patent Application No. 60/482,058, filed Jun. 23, 2003, both entitled Synthetic Process for Preparing Compounds for Treating Amyloidosis, and U.S. patent application Ser. No. 10/871,543, filed Jun. 18, 2004, now U.S. Pat. No. 7,253,306, issued Aug. 7, 2007, entitled Improved Pharmaceutical Drug Candidates and Method for Preparation Thereof. This application is related to U.S. Provisional Patent Application Ser. No. 60/512,018, filed on Oct. 17, 2003 and U.S. Provisional Patent Application Ser. No. 60/480,928, filed on Jun. 23, 2003, and U.S. patent application Ser. No. 10/871,512, filed Jun. 18, 2004, pending, all entitled Methods and Compositions for Treating Amyloid- and Epileptogenesis-Associated Diseases. This application is also related to Method for Treating Amyloidosis, U.S. patent application Ser. No. 08/463,548, now U.S. Pat. No. 5,972,328. The entire contents of each of these patent applications and patents are hereby expressly incorporated herein by reference including without limitation the specification, claims, and abstract, as well as any figures, tables, or drawings thereof.
| Number | Date | Country | |
|---|---|---|---|
| 60480906 | Jun 2003 | US | |
| 60512047 | Oct 2003 | US | |
| 60638819 | Dec 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11316694 | Dec 2005 | US |
| Child | 12613412 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10871514 | Jun 2004 | US |
| Child | 11316694 | US |
