Patent Application
20240279174
Gram-negative bacterial pathogens cause many life-threatening illnesses, including pneumonia and bloodstream, wound, and surgical site infections in healthcare and community settings. Antibiotics are essential medications for the treatment of Gram-negative bacterial infection. Unfortunately, as more and more Gram-negative bacteria are becoming resistant to nearly all antibiotics, patients are left with fewer effective treatment options. According to the report by Centers for Disease Control and Prevention on antibiotic resistance threats in the United States, over half of the eighteen antibiotic-resistant threats are from Gram-negative bacteria. In spite of these looming public health concerns, there has been little activity in the antibiotic development pipeline, with no antibiotic with a novel mode of action against Gram-negative bacteria discovered during the past five decades. Hence, there is an urgent need for new antibiotics to treat bacterial infections caused by multidrug-resistant Gram-negative bacteria.
Since the outer membrane of Gram-negative bacteria is required for bacterial viability and serves as a permeability barrier to prevent toxic compounds from entering the cell, disrupting its biogenesis presents an attractive strategy for antibacterial drug discovery. A distinctive feature of the outer membrane of Gram-negative pathogens is the presence of lipopolysaccharide (LPS) or lipooligosaccharide (LOS) extruding from the outer monolayer of the outer membrane. Lipid A is the hydrophobic membrane anchor of LPS/LOS and the active component of the bacterial endotoxin that causes Gram-negative septic shock during bacterial infection. Its constitutive biosynthesis via the Raetz pathway is required for bacterial viability and fitness in the human host (
LpxH is a uniquely attractive antibiotic target for multidrug-resistant Gram-negative bacteria, as inhibition of LpxH not only disrupts lipid A biosynthesis, but also accumulates toxic lipid A intermediates in the bacterial inner membrane. In 2015, AstraZeneca reported a sulfonyl piperazine compound, dubbed AZ1 (1,
The accompanying Figures and Examples are provided by way of illustration and not by way of limitation. The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the accompanying example figures (also “FIG.”) relating to one or more embodiments, in which:
Other aspects and iterations of the present disclosure are detailed below.
In some aspects, the present disclosure is directed to a compound of formula (I), or a pharmaceutically acceptable salt thereof:
In some aspects, the substituted aryl is substituted with one or more of halogen, hydroxyl, carboxylic acid, C1-C2 alkyl substituted carboxylic acid, cycloheteroalkenyl, aryl, C1-C3 alkyl, or C1 alkyl substituted with a halogen.
In some aspects, the C1 alkyl is substituted with a halogen is —CF3.
In some aspects, the substituted C5-C6 heteroaryl is substituted with one or more of —CF3 or Cl.
In some aspects, R1 is Cl, F, or hydrogen.
In some aspects, R2 is hydrogen.
In some aspects, R5 is hydrogen or carboxylic acid.
In some aspects, R3 and R4 are interconnected to form an indoline or indole.
In other aspects, the present disclosure is directed to a compound of formulae (IIa) or (IIb), or a pharmaceutically acceptable salt thereof:
In some aspects, the C1-C6 substituted alkyl is substituted with —CF3, carboxylic acid, methyl substituted carboxylic acid, heterocycl, amine, methyl substituted amine, substituted ether, hydroxyamide, or methanesulfonamide.
In some aspects, the substituted amine is substituted with C1-C6 alkyl hydroxyamide, C1-C6 alkyl carboxylic acid, C1-C6 alkyl carboxylic acid, C1-C6 amine, or substituted C1-C6 amine.
In some aspects, the halogen is F, Cl, or Br.
In some aspects, in formula (IIa) (R1)1 and (R1)3 are hydrogen, (R1)2 is —CF3, (R1)4 is chlorine, (R1)5 is absent, X1-X2 are carbon, X3 is nitrogen, R2, R3, and R6 are hydrogen; and Z is —C(O)R7. In some aspects, R7 is substituted aryl. In some aspects, the substituted aryl is substituted with —NS(O)(O)CH3.
In some aspects, in formula (IIb) (R1)1, (R1)3, and (R1)5 are hydrogen, (R1)2 is chlorine, (R1)4 is —CF3, X1-X5 are carbon, R2, R3, R5, and R6 are hydrogen, and Z is —C(O)R7. In some aspects, R7 is substituted amine with formula —NHC4H8C(O)NHOH.
In other aspects, the present disclosure is directed to a compound of
wherein (R1)1 and (R1)2 are each independently hydrogen or —CF3.
In yet other aspects, the present disclosure is directed a compound of formulae (IId) or (IIe), or a pharmaceutically acceptable salt thereof:
In some aspects, the C1-C6 substituted alkyl is —CF3.
In some aspects, the halogen is F, Cl, or Br, or I.
In some aspects, in formula (IId) (R1)1 and (R1)3 are hydrogen, (R1)2 is —CF3, (R1)4 is chlorine, (R1)5 is absent, X1-X4 are carbon, X5 is nitrogen, R2, R3, R5, and R6 are hydrogen; and R4 is —NHR7C(O)NHOH. In some aspects, R7 is pentyl.
In some aspects, in formula (IId) (R1)1, (R1)3, and (R1)5 are hydrogen, (R1)2 and (R1)4 are chlorine, X1-X5 are carbon, R2, R3, R5, and R6 are hydrogen, and R4 is substituted aryl. In some aspects, the substituted aryl is substituted with methanesulfonamide.
In some aspects, in formula (IId) (R1)1, (R1)3, and (R1)5 are hydrogen, (R1)2 is chlorinde, (R1)4 is —CF3, X1-X4 are carbon, X5 is nitrogen, R2, R3, R5, and R6 are hydrogen, and R4 is NHR7C(O)NHOH. In some aspects, the R7 is pentyl.
In some aspects, the substituted sugar is
In some aspects, the substituted heterocyclyl is selected from the group consisting of
In yet other aspects, the present disclosure is directed a compound of formulae (IIIa), (IIIb), (IV), (V), (VIa), or (VIb) or a pharmaceutically acceptable salt thereof:
In yet other aspects, the present disclosure is directed a compound of formula (VII), or a pharmaceutically acceptable salt thereof:
In some aspects, the compound of formula (VII) or a pharmaceutically acceptable salt thereof has a formula (VIIa)
In yet other aspects, the present disclosure is directed a compound of formula (VIII), or a pharmaceutically acceptable salt thereof:
In yet other aspects, the present disclosure is directed a compound of formula (IX), or a pharmaceutically acceptable salt thereof:
In yet other aspects, the present disclosure is directed a compound of formula (X), or a pharmaceutically acceptable salt thereof:
In yet other aspects, the present disclosure is directed a compound of formula (XI), or a pharmaceutically acceptable salt thereof:
In yet other aspects, the present disclosure is directed to a pharmaceutical composition comprising the compound of any one of the claims above and a pharmaceutically acceptable carrier, diluent, and/or an excipient.
The present disclosure describes formulations, compounded compositions, kits, capsules, containers, and/or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.
This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.
As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
The phrase ‘consisting essentially of’ limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase ‘consisting of’ excludes any component, step, or element that is not recited in the claim. The phrase ‘comprising’ is synonymous with ‘including’, ‘containing’, or ‘characterized by’, and is inclusive or open-ended. ‘Comprising’ does not exclude additional, unrecited components or steps.
As used herein, when referring to any numerical value, the term ‘about’ means a value falling within a range that is ±10% of the stated value.
Ranges can be expressed herein as from ‘about’ one particular value, and/or to ‘about’ another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent ‘about,’ it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as ‘about’ that particular value in addition to the value itself. For example, if the value ‘10’ is disclosed, then ‘about 10’ is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
As used herein, the terms ‘optional’ or ‘optionally’ means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.
As used herein, the term ‘subject’ refers to the target of administration, e.g., a human being. The term ‘subject’ also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered. In an aspect, a subject can be a human patient. In an aspect, a subject can have cancer, be suspected of having cancer, or be at risk of developing cancer.
The term “disease” as used herein includes, but is not limited to, any abnormal condition and/or disorder of a structure or a function that affects a part of an organism. It may be caused by an external factor, such as an infectious disease (e.g., a bacterial infection), or by internal dysfunctions, such as cancer, cancer metastasis, and the like.
A “patient” refers to a subject afflicted with a disease or disorder (e.g., a hematological cancer). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder such as a hematological cancer. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease or disorder and is seeking treatment or receiving treatment for a disease or disorder (such as hematological cancer).
The term “biological sample” as used herein includes, but is not limited to, a sample containing tissues, cells, and/or biological fluids isolated from a subject. Examples of biological samples include, but are not limited to, tissues, cells, biopsies, blood, lymph, serum, plasma, urine, saliva, mucus and tears. A biological sample may be obtained directly from a subject (e.g., by blood or tissue sampling) or from a third party (e.g., received from an intermediary, such as a healthcare provider or lab technician).
As used herein, “inhibit,” “inhibiting”, and “inhibition” mean to diminish or decrease an activity, level, response, condition, severity, disease, or other biological parameter. This can include, but is not limited to, the complete ablation of the activity, level, response, condition, severity, disease, or other biological parameter. This can also include, for example, a 10% inhibition or reduction in the activity, level, response, condition, severity, disease, or other biological parameter as compared to the native or control level (e.g., a subject not having received one or more of the disclosed chimeric fusion proteins, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed cells, the disclosed pharmaceutical formulations, or a combination thereof). Thus, in an aspect, the inhibition or reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between as compared to native or control levels. In an aspect, the inhibition or reduction can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% as compared to a native or control level (e.g., a subject not having received one or more of the disclosed chimeric fusion proteins, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed cells, the disclosed pharmaceutical formulations, or a combination thereof). In an aspect, the inhibition or reduction can be 0-25%, 25-50%, 50-75%, or 75-100% as compared to native or control levels. In an aspect, a native or control level can be a pre-disease or pre-disorder level (such as a pre-cancer state).
The words “treat” or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder (e.g., a hematological cancer); preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder (e.g., a hematological cancer); and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder (e.g., a hematological cancer). In an aspect, the terms cover any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating a disease or disorder can reduce the severity of an established a disease or disorder in a subject by 1%-100% as compared to a control (such as, for example, an individual not having cancer). In an aspect, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of a disease or disorder (e.g., a hematological cancer). For example, treating a disease or disorder can reduce one or more symptoms of a disease or disorder in a subject by 1%-100% as compared to a control (such as, for example, an individual not having cancer). In an aspect, treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms of an established a disease or disorder (e.g., a hematological cancer). It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of a disease or disorder. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a disease or disorder (such as a hematological cancer).
As used herein, the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease or disorder having chromatin deregulation and/or chromatin dysregulation is intended. The words “prevent”, “preventing”, and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given a disease or disorder (such as a hematological cancer) or related complication from progressing to that complication. In an aspect, preventing metastasis is intended.
As used herein, the terms “administering” and “administration” refer to any method of providing one or more of the disclosed chimeric fusion proteins, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed cells, the disclosed pharmaceutical formulations, or a combination thereof, or by one or more of the disclosed methods to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, the following: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, in utero administration, intratumoral administration, intrahepatic administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can also include hepatic intra-arterial administration or administration through the hepatic portal vein (HPV). Administration of one or more of the disclosed chimeric fusion proteins, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed cells, the disclosed pharmaceutical formulations, or a combination thereof can comprise administration directly into the CNS or the PNS. Administration can be continuous or intermittent. Administration can comprise a combination of one or more routes.
As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. In an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.
As used herein, the term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, for reference, Remington's Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.
As used herein, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.
As used herein, “determining” can refer to measuring or ascertaining the presence and severity of a disease or disorder, such as, for example, a hematologic cancer. Methods and techniques used to determine the presence and/or severity of a disease or disorder are typically known to the medical arts. For example, the art is familiar with the ways to identify and/or diagnose the presence, severity, or both of a disease or disorder (such as, for example, cancer).
As used herein, “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and/or prevention of a disease or disorder (e.g., a hematological cancer) or a suspected disease or disorder (e.g., a hematological cancer). As used herein, the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition (e.g., a hematological cancer). For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. In an aspect, “therapeutically effective amount” means an amount the disclosed chimeric fusion proteins, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed cells, the disclosed pharmaceutical formulations, the disclosed anti-chemokines, the disclosed anti-cancer agents, the disclosed chemotherapeutics, or any combination thereof that (i) treats the particular disease, condition, or disorder (e.g., a hematological cancer), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., a hematological cancer), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein (e.g., a hematological cancer). The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; one or more of the disclosed chimeric fusion proteins, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed cells, the disclosed pharmaceutical formulations, or any combination thereof employed; the disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of one or more of the disclosed chimeric fusion proteins, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed cells, the disclosed pharmaceutical formulations, or any combination thereof employed; the duration of the treatment; drugs used in combination or coincidental with the one or more of the disclosed chimeric fusion proteins, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed cells, the disclosed pharmaceutical formulations, or any combination thereof employed, and other like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the disclosed chimeric fusion proteins, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed cells, the disclosed pharmaceutical formulations, the disclosed anti-chemokines, the disclosed anti-cancer agents, the disclosed chemotherapeutics, or any combination thereof at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of the disclosed chimeric fusion proteins, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed cells, the disclosed pharmaceutical formulations, the disclosed anti-chemokines, the disclosed anti-cancer agents, the disclosed chemotherapeutics, or any combination thereof can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition, such as, for example, a hematological cancer.
As used herein, “conjugate” or “conjugated” can be used to define the operative association of one disclosed component to another disclosed component. In an aspect, conjugated does not intend to refer solely to any type of operative association and is not particularly limited to chemical “conjugation”.
A “target” or “target antigen” is any molecule bound by a binding motif (e.g., a PS-expressing hematological cancer cell). A disclosed target can be cells and/or tissues in a subject.
As used herein, the term “in combination” in the context of the administration of other therapies (e.g., other agents) includes the use of more than one therapy (e.g., drug therapy). Administration “in combination with” one or more further therapeutic agents includes simultaneous (e.g., concurrent) and consecutive administration in any order. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. By way of non-limiting example, a first therapy (e.g., one or more of the disclosed chimeric fusion proteins, the disclosed isolated nucleic acid molecules, the disclosed vectors, the disclosed cells, the disclosed pharmaceutical formulations, or a combination thereof) can be administered prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer) the administration of a second therapy to a subject having or diagnosed with hematological cancer.
In some aspects, the present disclosure is directed to the use of AZ1 as a novel antibiotic targeting LpxH. In some aspects, the detailed structure-activity relationship of AZ1 (1) may be analyzed and to elucidate the molecular details of the LpxH-AZ1 interaction by X-ray crystallography and NMR. By harnessing the ligand dynamics information and structural insights, sulfonyl piperazine LpXH inhibitors may be designed. Examples of the LpXH inhibitors include but is not limited to JH-LPH-33 (2,
The present disclosure is directed to sulfonyl piperazine LpxH inhibitors.
The present disclosure is directed to a compound of formula (I), or a pharmaceutically acceptable salt thereof:
In some embodiments, the substituted aryl may be substituted with one or more of halogen, hydroxyl, carboxylic acid, C1-C2 alkyl substituted carboxylic acid, cycloheteroalkenyl, aryl, C1-C3 alkyl, or C1 alkyl substituted with a halogen.
In some embodiments, the C1 alkyl substituted with a halogen may be —CF3.
In some embodiments, the substituted C5-C6 heteroaryl may be substituted with one or more of —CF3 or Cl.
In some embodiments, R1 may be Cl, F, or hydrogen.
In some embodiments, R2 may be hydrogen.
In some embodiments, R5 may be hydrogen or carboxylic acid.
In some embodiments, R3 and R4 may be interconnected to form an indoline or indole.
The present disclosure is also directed to a compound of formulae (IIa) or (IIb), or a pharmaceutically acceptable salt thereof:
In some embodiments, the C1-C6 substituted alkyl may be substituted with —CF3, carboxylic acid, methyl substituted carboxylic acid, heterocycl, amine, methyl substituted amine, substituted ether, hydroxyamide, or methanesulfonamide.
In some embodiments, the substituted amine may be substituted with C1-C6 alkyl hydroxyamide, C1-C6 alkyl carboxylic acid, C1-C6 alkyl carboxylic acid, C1-C6 amine, or substituted C1-C6 amine.
The halogen may be F, Cl, or Br.
In some embodiments of formula (IIa),
In some embodiments of formula (IIb),
The present disclosure is also directed to a compound of formula (IIc), or a pharmaceutically acceptable salt thereof:
The present disclosure is also directed to a compound of formulae (IId) or (IIe), or a pharmaceutically acceptable salt thereof:
In some embodiments, the C1-C6 substituted alkyl may be —CF3.
In some embodiments, the halogen is F, Cl, or Br, or I.
In some embodiments of formula (IId),
In some embodiments of formula (IId),
In some embodiments of formula (IId),
In some embodiments the substituted sugar may be
In some embodiments the substituted heterocyclyl is selected from the group consisting of
The present disclosure is further directed to a compound of formulae (IIIa), (IIIb), (IV), (V), (VIa), or (VIb) or a pharmaceutically acceptable salt thereof:
The present disclosure is further directed to a compound of formula (VII), or a pharmaceutically acceptable salt thereof:
In some embodiments, the compound or a pharmaceutically acceptable salt thereof of formula (VI) may have a formula (VIIa):
The present disclosure is further directed to a compound of formula (VIII), or a pharmaceutically acceptable salt thereof:
The present disclosure is further directed to a compound of formula (IX), or a pharmaceutically acceptable salt thereof:
The present disclosure is further directed to a compound of formula (X), or a pharmaceutically acceptable salt thereof:
The present disclosure is further directed to a compound of formula (XI), or a pharmaceutically acceptable salt thereof:
The compounds of the present disclosure are listed in Table 1.
In one aspect, the present disclosure provides sulfonyl piperazine LpxH inhibitors with the potential of chelating the di-manganese cluster in the active site of LpxH.
In another aspect, the present disclosure provides di-manganese-chelating LpxH inhibitors with significantly enhanced potency in suppressing LpxH activity in vitro.
In one embodiment, the present disclosure provides a sulfonyl piperazine LpxH inhibitor comprising, consisting of, or consisting essentially of JH-LPH-45 or a variant, salt or ester thereof.
Another embodiment of the present disclosure provides a sulfonyl piperazine LpxH inhibitor comprising, consisting of, or consisting essentially of JH-LPH-50 or a variant, salt, or ester thereof.
In one embodiment, the present disclosure provides a sulfonyl piperazine LpxH inhibitor comprising, consisting of, or consisting essentially of JH-LPH-104 or a variant, salt or ester thereof.
In one embodiment, the present disclosure provides a sulfonyl piperazine LpxH inhibitor comprising, consisting of, or consisting essentially of JH-LPH-105 or a variant, salt or ester thereof.
In one embodiment, the present disclosure provides a sulfonyl piperazine LpxH inhibitor comprising, consisting of, or consisting essentially of JH-LPH-106 or a variant, salt or ester thereof.
In one embodiment, the present disclosure provides a sulfonyl piperazine LpxH inhibitor comprising, consisting of, or consisting essentially of JH-LPH-107 or a variant, salt or ester thereof.
In another aspect, the present disclosure provides compositions comprising one or more of sulfonyl piperazine LpxH inhibitors as described herein and an appropriate carrier, excipient or diluent. The exact nature of the carrier, excipient or diluent will depend upon the desired use for the composition, and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use. The composition may optionally include one or more additional compounds.
When used to treat or prevent a disease, such as a bacterial infection, the compounds described herein may be administered singly, as mixtures of one or more compounds or in mixture or combination with other agents (e.g., therapeutic agents) useful for treating such diseases and/or the symptoms associated with such diseases. Such agents may include, but are not limited to, antibiotics, NSAIDS, anti-inflammatory compounds, hemoperfusion devices, quorum sensing inhibitors, lytic bacteriophage, polyclonal or monoclonal antibodies, non-immune tolerizing approaches, liposome-based cytotoxin inhibitors, to name a few. The compounds may be administered in the form of compounds per se, or as pharmaceutical compositions comprising a compound.
Pharmaceutical compositions comprising the compound(s) may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically.
The compounds may be formulated in the pharmaceutical composition per se, or in the form of a hydrate, solvate, N-oxide or pharmaceutically acceptable salt, as previously described. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.
Pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.
For topical administration, the compound(s) may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.
Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives. Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use. To this end, the active compound(s) may be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.
For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.
For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art with, for example, sugars, films or enteric coatings.
Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, Cremophore™ or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give controlled release of the compound, as is well known. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For rectal and vaginal routes of administration, the compound(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.
For nasal administration or administration by inhalation or insufflation, the compound(s) can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
For ocular administration, the compound(s) may be formulated as a solution, emulsion, suspension, etc. suitable for administration to the eye. A variety of vehicles suitable for administering compounds to the eye are known in the art.
For prolonged delivery, the compound(s) can be formulated as a depot preparation for administration by implantation or intramuscular injection. The compound(s) may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the compound(s) for percutaneous absorption may be used. To this end, permeation enhancers may be used to facilitate transdermal penetration of the compound(s).
Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver compound(s). Certain organic solvents such as dimethyl sulfoxide (DMSO) may also be employed, although usually at the cost of greater toxicity.
The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the compound(s). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
The compound(s) described herein, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. Therapeutic benefit also generally includes halting or slowing the progression of the disease, regardless of whether improvement is realized.
The amount of compound(s) administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular compound(s) the conversation rate and efficiency into active drug compound under the selected route of administration, etc.
Determination of an effective dosage of compound(s) for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of compound for use in animals may be formulated to achieve a circulating blood or serum concentration of the metabolite active compound that is at or above an IC50 of the particular compound as measured in as in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound via the desired route of administration is well within the capabilities of skilled artisans. Initial dosages of compound can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavailability and/or metabolism of compounds into active metabolites are also well-known. Ordinarily skilled artisans can routinely adapt such information to determine dosages of particular compounds suitable for human administration.
Dosage amounts will typically be in the range of from about 0.0001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the active compound, the bioavailability of the compound, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the compound(s) and/or active metabolite compound(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the compounds may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of compound(s) and/or active metabolite compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation.
Disclosed herein are cells transformed or transfected by one or more disclosed isolated nucleic acid molecules. Disclosed herein are cells transformed or transfected by an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric fusion protein. Disclosed herein are cells transformed or transfected by an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed chimeric fusion protein specific for phosphatidylserine (PS). Disclosed herein are cells transformed or transfected by an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric fusion protein comprising a phosphatidylserine (PS) binding domain. Disclosed herein are cells transformed or transfected by an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric fusion protein comprising a phosphatidylserine (PS) binding domain and an immunostimulatory domain. Disclosed herein are cells transformed or transfected by an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric fusion protein comprising a phosphatidylserine (PS) binding domain operably linked an immunostimulatory domain. Disclosed herein are cells transformed or transfected by an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a phosphatidylserine (PS) binding domain, a linker, and an immunostimulatory domain. Disclosed herein are cells transformed or transfected by an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a chimeric fusion protein comprising a phosphatidylserine (PS) binding domain operably linked to an immunostimulatory domain, wherein the immunostimulatory domain further comprise a conjugated small molecule.
The present disclosure further provides kits comprising the compositions provided herein and for carrying out the subject methods as provided herein. For example, in one embodiment, a subject kit may comprise, consist of, or consist essentially of a sulfonylpiperazine LpxH inhibitor, or a salt, ester, thereof and/or pharmaceutical composition as provided herein.
In other embodiments, a kit may further include other components. Such components may be provided individually or in combinations, and may provide in any suitable container such as a vial, a bottle, or a tube. Examples of such components include, but are not limited to, one or more additional reagents, such as one or more dilution buffers; one or more reconstitution solutions; one or more wash buffers; one or more storage buffers, one or more control reagents and the like. Components (e.g., reagents) may also be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). Suitable buffers include, but are not limited to, phosphate buffered saline, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof.
In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
The sulfonyl piperazine LpxH inhibitors provided herein may be used to prevent and/or treat a disease, such as a bacterial infection, in a subject. Accordingly, another aspect of the present disclosure provides a method for preventing and/or treating a disease in a subject, the method comprising, consisting of, or consisting essentially of administering to the subject a therapeutically effective amount of a sulfonyl piperazine LpxH inhibitor as provided in Table 1 such that the disease is prevented and/or treated in the subject.
In one embodiment, the sulfonyl piperazine LpxH inhibitor comprises JH-LPH-45 or a salt, ester and/or pharmaceutical composition thereof.
In another embodiment, the sulfonyl piperazine LpxH inhibitor comprises JH-LPH-50 or a salt, ester and/or pharmaceutical composition thereof.
In another embodiment, the disease comprises a bacterial infection.
Another aspect of the present disclosure provides all that is described and illustrated herein.
The Examples that follow are illustrative of specific aspects of the invention, and various uses thereof. They set forth for explanatory purposes only and are not to be taken as limiting the invention.
Design and synthesis of LoxH inhibitors: As metalloenzymes depend critically on their catalytic metal ion for activity, incorporation of a metal chelating group has been an effective strategy for enhancing the potency of metalloenzyme inhibitors. For example, the FDA-approved histone deacetylase (HDAC) inhibitor Vorinostat has a hydroxamic acid coordinating the catalytic Zn2+ in the HDAC active site as a monoanionic ligand through the carbonyl and hydroxyl donor atoms. Elvitegravir employs a quinolone carboxylic acid metal-binding pharmacophore to inhibit human immunodeficiency virus (HIV) integrase.
As LpxH harbors an active site di-manganese cluster that is required for structural integrity and catalysis, it was hypothesized that incorporation of a metal-binding group for the di-manganese cluster into LpxH inhibitors would similarly enhance their potency. It was noticed that the most recent LpxH inhibitor JH-LPH-41 (3), containing an aniline core with an N-acyl hydroxamate group extension, unexpectedly occupies the space of the hexose ring shared between the substrate and product as observed in the crystal structures of the lipid X-bound and JH-LPH-41-bound LpxH complexes (
Indoline and aniline analogs: To develop a di-manganese-chelating LpxH inhibitor, evaluation began looking at the effect of acyl chain extension of JH-LPH-33 (2,
To test this hypothesis, efforts were made on the synthesis of the indoline urea analogs 7 and 8 (Scheme 1A). Treatment of the known piperazinyl sulfonyl indoline 6 with triphosgene, coupling with ethyl 5-aminopentanoate, and basic hydrolysis afforded the corresponding carboxylic acid 7. Compound 7 was converted to the TBS-protected hydroxamic acid by treatment with ClCO2Et/Et3N/NH2OTBS. Final TBS deprotection by TFA gave the indoline urea analog 8 in 78%. To investigate the effect of the urea linkage of 8 on the orientation of the N-acyl linker, we also prepared the corresponding amide analog 10. Acylation of 6 with ethyl 7-chloro-7-oxoheptanoate followed by hydrolysis proceeded smoothly to give carboxylic acid 9 (Scheme 1A). Treatment of 9 with ClCO2Et/Et3N/NH2OTBS and final TBS deprotection by TFA completed the synthesis of the amide analog 10.
After the successful incorporation of a metal-binding pharmacophore for the di-manganese cluster into the indoline core of JH-LPH-33 (2), attention was turned to re-examining the design of aniline-based inhibitors such as JH-LPH-41 (3,
Analogs with a cyclic metal-binding group or a rigid linker: Antibiotics against Gram-negative bacteria need to find their way through the negatively charged LPS layer present on the surface of the outer membrane or translocate through porins to penetrate the hydrophobic outer membrane. Then, they need to cross the hydrophobic lipid bilayer of the inner membrane before they find their cytosolic targets such as LpxH. The flexible acyl chains of the above-mentioned compounds may restrict the membrane permeability of the compounds. To investigate the effect of structural rigidification of the flexible acyl chain on of 13 on activity, the acyclic hydroxamic acid moiety of 13 was replaced with a more rigid cyclic metal binding group. The 1-hydroxy-2(1H)-pyridinethione metal binding group was originally developed by Cohen and co-workers and has been used in matrix metalloproteinase inhibitors. CDI coupling of 11 to 14 (see the Supporting Information for details) completed the synthesis of the hydroxy pyridinethione analog 15 (Scheme 2).
The N-acyl linker of 13 was also made more rigid by incorporating a double bond or a triazole ring. For the synthesis of the (E)-olefinic linker analog 18, 11 was reacted with tert-butyl (E)-6-aminohex-2-enoate (16) (see Appendix A for details), CDI, and DIPEA to give the corresponding urea intermediate (Scheme 2). Treatment of the urea intermediate with TFA afforded carboxylic acid 17. Carboxylic acid 17 was converted to the final olefinic linker analog 18 following the same procedure used for 13. Another rigid analog that was designed was the triazole linker analog 20 (Scheme 2). To synthesize 20, the process started with coupling 11 with 1-chloro-2-isocyanatoethane (89%) followed by treating with NaN3 and TBAl to give azide 19 (59%). The click chemistry of azide 19 and N-hydroxypropiolamide provided the desired triazole analog 20, albeit in low yield (28%).
Evaluation of LpxH inhibition by analogs: After completing the synthesis of sulfonyl piperazine compounds with various forms of linkers, the KpLpxH inhibition was biochemically characterized by these analogs at 0.1 μM using the nonradioactive, colorimetric malachite green assay that we had previously reported. It was found that compound 8 was more potent in inhibiting KpLpxH (vi/v0=18±1%) than 2 (vi/v0=21%) (Table 1). Such an enhanced potency requires the presence of the hydroxamate group, as its substitution with a carboxylate group in compound 7 reduced the compound activity (vi/v0=30±6%). This result suggests that the superior inhibitory effect of 8 is likely derived from chelation of the di-manganese cluster.
It was also found that compound 13, with a one-carbon extension compared to the acyl hydroxamate group of 3, nearly completely inhibited KpLpxH at 0.1 μM, leaving 3±2% of the enzymatic activity (vi/v0), whereas KpLpxH retained 36% activity in the presence of 0.1 μM of 3 (Table 2). Compound 13 is also a more potent inhibitor than 2 at 0.1 μM (vi/v0=21%). Such a significant improvement in potency was lost when the hydroxamate group was substituted with the carboxylate group (12; vi/v0±57±3%), suggesting that the inhibitory effect similarly requires the hydroxamate group and its ability to chelate the di-manganese cluster.
Unfortunately, neither substituting the hydroxamate group with a cyclic metal binding group as in 15 (vi/v0=39±6% inhibition) nor introducing a double bond as in 18 (vi/v0=34±7% inhibition) or a cyclic ring as in 20 (vi/v0=59±7% inhibition) next to the hydroxamate group enhanced the compound activity when tested at 0.1 μM concentration (Table 2). It is likely due to the loss of the di-manganese chelation in these analogs.
[a]Percentage activity was normalized against the specific activity of KpLpxH measured in the presence of 10% DMSO (309 ± 12 μmol/min/mg). Errors represent the standard percent error.
Structures of K. pneumoniae LpxH in complex with 8 (JH-LPH-45) and 13 (JH-LPH-50); In order to gain a better understanding of the nature of the interaction of 8 (JH-LPH-45) with K. pneumoniae LpxH (KpLpxH), the co-crystal structure of the KpLpxH/8 complex at 1.74 Å (
To verify that the gain of activity of 13 (JH-LPH-50) was due to chelation of the di-manganese cluster, the crystal structure of the KpLpxH/13 complex resolved at 1.73 Å resolution was also analyzed (
Evaluation of antibiotic activity of compounds 8 (JH-LPH-45) and 13 (JH-LPH-50); To evaluate the effect of di-manganese chelation on antibiotic activity, compounds 8 and 13 were tested against K. pneumoniae (ATCC 10031) in vitro (Table 3). Despite the significant reduction of KI values of 8 (7.3 nM) and 13 (3.1 nM) in comparison with 2 (10 nM) in enzymatic assays, compounds 8 and 13 displayed lower antibiotic activity (MIC=18.7 μg/mL for 8 and MIC-3.3 μg/mL for 13) than the non-metal chelating compound 2 (MIC=0.83 μg/mL). Such a discrepancy was presumably caused by the flexible acyl chain that hindered the membrane permeability of the compounds as suggested by the ENTRY rule.
Both JH-LPH-45 and JH-LPH-50 leverage a terminal hydroxamate group to chelate the di-manganese cluster and enhance ligand-LpxH interaction. The hydroxamate group has been used as a metal chelating group in FDA approved histone deacetylase inhibitors, such as Belinostat, Panobinostat, and Vorinostat. Although hydroxamate-containing LpxH inhibitors may inhibitor human metalloenzymes, the unique lipid binding feature of LpxH could be leveraged to ensure the specificity of LpxH inhibitor and minimize off-target effects. Furthermore, replacing the hydroxamate group with alternative metal-chelating groups may further enhance the selectivity of LpxH inhibitors containing a metal chelating group, thus mitigating the potential off-target consequence.
The LpxH enzyme in the lipid A biosynthetic pathway is a promising antibiotic target. Building on the sulfonyl piperazine scaffold of AZ1 (1), it was previously reported of more potent AZ1 analogs, such as JH-LPH-33 (2) with significant improvement of antibiotic activity and JH-LPH-41 (3) that reaches the polar binding pocket near the active site. After careful analyses of the structures of KpLpxH/JH-LPH-33 (2) and KpLpxH/JH-LPH-41 (3), the inventors designed JH-LPH-45 (8) and JH-LPH-50 (13) by switching the aniline core to the corresponding indoline core (8) or extending the linker by a methylene unit (13). Consistent with their prediction, both 8 and 13 chelate the di-manganese cluster in the active site of KpLpxH. Most notably, the chelation of the active site di-manganese cluster of KpLpxH significantly improves the potency of compounds 8 and 13, reducing the Kl values from 10 nM (JH-LPH-33) to 7.3 nM (8) and 3.1 nM (13). These compounds are the first-in-class LpxH inhibitors that achieve chelation of the di-manganese cluster in LpxH.
The MIC assay protocol was adapted from the broth microdilution methods described by the National Committee for Clinical Laboratory Standards using 96-well plates as described previously. In brief, overnight bacterial cultures were diluted to an OD600 of 0.006 in cation-adjusted Mueller-Hinton medium containing 7% DMSO and incubated at 37° C. for 22 h in the presence of varying concentrations of inhibitors. Following overnight incubation, each well is treated with 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL) for 3 h. Finally, the culture is solubilized by addition of 2-propanol, and the results are read by taking the difference of the UV reading at both 570 and 690 nm. MIC values were reported as the lowest compound concentration that inhibited bacterial growth. The baseline for inhibited cell growth was determined to be <4× the average control reading due to the baseline optical density of the initial starting bacterial culture.
MIC values of some of the compounds of Table 1 is shown in Table 4.
The LpxE-coupled LpxH activity assay was conducted as described previously using the GB1-K. pneumoniae LpxH-His10 fusion protein. Briefly, two reaction mixtures were prepared. Mixture 1 contains 20 mM Tris-HCl (pH 8.0), 0.5 mg/mL BSA, 0.02% Triton X-100, 1 mM MnCl2, 1 mM DTT, 10% DMSO, and 200 UM substrate (UDP-DAGn), and mixture 2 is comprised of the same buffer, but instead of substrate, contains both LpxH (20 ng/ml) and 2× inhibitor. These mixtures were then pre-incubated at 37° C. for 10 min. To initiate the reaction, an equal volume of the LpxH mixture (mixture 2) was added to the substrate mixture (mixture 1) at 37° C. The final reaction solution contains 100 μM substrate, 10 ng/ml enzyme, and 1× inhibitor. At the desired reaction time points, an aliquot of 20 μL reaction mixture was removed and added to a well in 96-well half-area plate containing 5 mM EDTA (final concentration) to quench the LpxH reaction. Purified Aquifex aeolicus LpxE was then added to a final concentration of 5 μg/mL. The plate was incubated at 37° C. for 30 min followed by addition of formic acid to a final concentration of 3.75 M to quench the LpxE reaction. The malachite green reagent (Sigma Aldrich, catalog MAK307) was diluted 5-fold into the solutions, and the plate was incubated for 30 min at room temperature before the absorbance at 620 nm was measured. All measurements were done in triplicates, and standard error (S.E.) was calculated.
IC50 of compounds of Table 1 is shown in Table 5.
The present application is a continuation in part of U.S. application Ser. No. 17/768,152, filed on Apr. 11, 2022, which is a U.S. National Phase Application under 35 U.S.C. § 371 of International Application No. PCT/US2020/055243, filed on Oct. 12, 2020, which claims the benefit of U.S. Provisional Application No. 62/913,784 filed on Oct. 11, 2019, entitled, “LPXH TARGETING COMPOUNDS, COMPOSITIONS THEREOF, AND METHODS OF MAKING AND USING THE SAME”, and claims priority to U.S. Provisional Application Nos. 63/456,606 and 63/524,037 filed on Apr. 3, 2023 and Jun. 29, 2023 respectively, entitled, “ANTIBIOTICS COMPRISING LPXH-TARGETING COMPOUNDS AND METHODS OF MAKING AND USING THE SAME”, the entire contents of which are entirely incorporated by reference herein.
This invention was made with Government support under Federal Grant nos. AI139216 and GM115355 awarded by the National Institutes of Health. The Federal Government has certain rights to this invention.
| Number | Date | Country | |
|---|---|---|---|
| 63456606 | Apr 2023 | US | |
| 63524037 | Jun 2023 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 62913784 | Oct 2019 | US |
| Child | 17768152 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17768152 | Apr 2022 | US |
| Child | 18626187 | US |
