Patent Application
20060189576
The present invention relates to novel vitamin D analogues, more specifically to 2-substituted-19-nor-14-epi-1,25(OH)2D3 and 2-substituted-19-nor-1,25(OH)2D3 analogues, to their use as a medicine and to pharmaceutical preparations containing the vitamin D analogues of the invention. The present invention also relates to methods of preparation of these 2-substituted-19-nor-14-epi-1,25(OH)2D3 and 2-substituted-19-nor-1,25(OH)2D3 analogues.
Vitamin D of either nutritional (vitamin D2 or D3) origin or produced in the skin under the influence of ultraviolet light is metabolized in several tissues to produce firstly 25-hydroxyvitamin D3 [25OHD3] and later 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and numerous other vitamin D metabolites. Several hydroxylases present in different tissues (e.g. liver, kidney, placenta, keratinocytes, fibroblasts, monocytes, bone cells, . . . ) are responsible for both the activating and inactivating pathways of the parent vitamin D molecules. 1α,25(OH)2D3 behaves as a classical steroid hormone as its synthesis is feedback controlled by several hormones, ions and humoral factors to maintain a normal body homeostasis of plasma and bone minerals. Moreover the vitamin D hormone(s) act(s) via binding and activation of specific vitamin D receptors, present in most tissues and cells. High affinity binding of 1α,25(OH)2D3 to the nuclear vitamin D receptor (VDR), followed by dimerization of the liganded VDR with the retinoid X receptor (RXR) and binding of the VDR-RXR heterodimer to specific vitamin D responsive elements (VDREs) in the promoter region of vitamin D target genes regulates the transcription of a large and diverse set of genes. Moreover there is some evidence for vitamin D, its metabolites and analogues to act via nongenomic mechanisms, either by activating ion channels or other membrane related or second messenger signals. Vitamin D, its metabolites and analogues have potent effects on calcium and phosphate metabolism, and therefore they can be used for prevention and therapy of vitamin D deficiency and other disorders of plasma and bone mineral homeostasis (e.g. osteomalacia, osteoporosis, renal osteodystrophy, disorders of the parathyroid function). Moreover vitamin D receptors are found in numerous tissues and cells that do not belong to the target tissues responsible for the above mentioned calcium homeostasis. Vitamin D receptors and vitamin D activity have also been documented in calcium transporting tissues other than the intestine and bone (e.g. placenta and mammary glands). In addition vitamin D receptors and vitamin D action have been observed in most other cells (e.g. cells of the immune system, skin cells, colon, brain, endocrine glands, muscle cells). These cells or tissues can be of a benign, adenomatous or of a malignant type. The hormone was found to be capable of regulating proliferation and differentiation of a variety of immunological and malignant cells. 1α,25(OH)2D3 appears to determine the transition from a state of proliferation to a state of differentiation. It is involved in the stimulation of non-specific immunicty by monocytes and the inhibition of lymphocytic specific immunity as well as in the regulation of the growth and differentiation of normal cells (embryogenesis) and cancer cells (induction of differentiation of melanoma, breast cancer, myeloid leukaemia, lymphoma and osteosarcoma cells). These so-called non-calcemic effects of vitamin D, its metabolites and analogues create the possibility to use such compounds for various therapeutic applications such as modululation of the immune system, modification of hormone secretion, altering calcium transport in several tissues, influencing intercellular calcium concentration, induction of cell differentiation or inhibition of cell proliferation. In particular such compounds have been considered to be potentially useful in the therapy of hyperproliferative disorders [e.g. psoriasis, cancer, (auto)immune diseases].
At the present time, the pathological conditions associated with vitamin D are classified as vitamin D deficiencies or excesses. Vitamin D deficiencies are due either to insufficient exposure to sunlight combined with an inadequate exogenous provision in the food, or to abnormalities of vitamin D metabolism. Genetic abnormalities are described in respect of the renal hydroxylase (1α-hydroxylase) or in respect of the vitamin D receptor (vitamin D resistance). Interference with the metabolism can also occur in the course of various pathological conditions, and especially renal insufficiency and hypoparathyroidism, or as a result of pharmacological interactions, for example with antiepileptic drugs and corticoids.
The clinical manifestations of these vitamin D deficiencies are most clearly apparent at bone level: rickets, osteomalacia and possibly participation in the phenomena of osteoporosis. There are other, less obvious associated disorders, for example an immune deficiency and a higher incidence of certain cancers and of vascular and endocrine disorders. Most deficiencies are readily corrected by the exogenous provision of vitamin D. An active form, for example 1α,25(OH)2D3, must be administered if the metabolism is abnormal, in particular in case of renal insufficiency. 1α,25(OH)2D3 however has a short half-life, which often justifies the taking of two doses daily.
Vitamin D excesses are encountered essentially during vitamin D poisoning, or during an ectopic production of active metabolites, for example during granulomatous diseases (sarcoidosis). Hyperparathyroidism stimulates the excessive production of 1,25-(OH)2-vitamin D, which also appears to accompany familial idiopathic hypercalciuria. The active derivatives of vitamin D employed therapeutically, and especially 1,25-(OH)2)-vitamin D used, in particular, in renal insufficiency, have a very narrow therapeutic index, so that vitamin poisoning is common during their administration. Thus, the treatment of a vitamin D poisoning is often the result of the treatment of a vitamin D deficiency;
The major drawback regarding the use of 1α,25(OH)2D3 is its toxicity associated with its calcemic effect, which prevents the application of pharmaceutical doses. Current research is therefore aimed at the synthesis of analogues with superagonistic potentcy but, in particular at the decoupling of the advantageous effects from the calcemic effects.
A large number of analogues incorporating modifications in the A-ring, in the CD-ring fragment and, especially, in the side chain have been synthesized and tested biologically. It is now well established that removal of the 19-exomethylene function is beneficial: 19-nor-1α,25-dihydroxyvitamin D3 displays a smaller calcemic effect (10% of that of 1α,25(OH)2D3) while retaining good cell-differentiating properties. It was further observed that, among other modifications, 14-epimers (first disclosed in U.S. Pat. No. 6,017,907) are among the best analogues known to date in that they show still smaller hypercalcemic effects (circa 0.1% of that of 1α,25(OH)2D3).
Recently a new class of vitamin D analogues have been discovered namely 19-nor-14-epi-1,25(OH)2D3 analogues (Novel structural analogues of vitamin D” (EP 0 972 762 A2, U.S. Pat. No. 6,017,907 Bouillon R., De Clercq P., Vandewalle M). Biological testing of such analogues revealed a selective activity profile with strong antiproliferative activity and very low calcemic effects. These compounds could be used as therapeutic agents for the treatment of cancer or various skin disorders.
2β-hydroxy and alkoxy analogues of 1,25(OH)2D3 have been described by Chugai Company in the U.S. Pat. No. 4,666,634. 2-alkyl and 2-hydroxyalkyl analogues of 1,25(OH)2D3 have been described by Nikagawa et al. Biochem Pharmacol 60, 1937-1947, 2000; Nakagawa et al., Biochem Pharmacol 59, 691-702, 2000; Takayama et al., Steroids 66, 277-285 (2001); Fujishima et al. Bioorg Med Chem Lett 8, 2145-2148, 1998; Suhara et al. J Org Chem 66, 8760-8771, 2001; Konno et al. J Med Chem 43, 4247-4265, 2000. Synthesis and biological activity of 2-hydroxy and 2-alkoxy analogues of 19-nor-1,25(OH)2D3 have been described in J Med Chem 37, 3730-3738, 1994. 2-substituted A-ring 19-nor-analogues have been described and examined such as 2-alkyl (U.S. Pat. No. 6,127,559) and 2-alkylidene (U.S. Pat. No. 5,936,133) vitamin D compounds. See also Sicinski et al. J Med Chem 45, 3366-338, 2002; Sicinski et al. J Med Chem 41, 4662-4674, 1998. Only one 14-epi-19-nor-1,25(OH)2D3 analogue with a substitution on carbon 2 has already been described, namely 2-methylene-14-epi-19-nor-1,25(OH)2D3 in U.S. Pat. No. 5,936,105.
The present invention relates to novel vitamin D analogues and the use of vitamin D analogues with improved properties in the treatment and prevention of particular conditions and diseases.
Thus, a first aspect of the invention relates to vitamin D derivatives having a different pharmaco-kinetic profile and a more favourable therapeutic index.
According to a particular aspect, the analogues of the present invention enable the different biological activities with respect to the target cells to be dissociated; more particularly a dissociation of the beneficial effects of vitamin D from the calcemic effects is envisaged.
It was surprisingly found that the compounds described herein have a selective activity on cell function, such as inhibition of cell proliferation (non-malignant cells such as keratinocytes as well as malignant cell such as breast carcinoma, osteo-sarcoma and leukemia cells) and/or have a high potency for induction of cell differentiation (e.g. cell types as just mentioned) but on the other hand have strikingly lower effect on calcium and bone homeostasis as evaluated in rachitic chicks (by measuring serum and bone calcium, and by measurement of two vitamin D-dependent proteins, serum osteocalcin and duodenal calbindin D) as well as in vitamin D repleted normal mice (using similar end points). Thus, unlike the classical vitamin D compounds, the new drugs do not have the same toxic effect on calcium and bone homeostasis. Specifically the new drugs can generally be used for the therapy or prevention of:
The amount of the new compounds necessary for their therapeutic effect can vary according to its indication, route of administration and species (animal/man) treated. The compounds can be administered by enteral, parenteral or local topical route. In the treatment of dermatological disorders a topical application as ointment, cream or lotion is to be preferred over systemic treatment, preferably in a dose of 0.1 to 500 μg/g. The systemic administration as tablets, capsules, liquid or as sterile preparation in an appropriate carrier, diluent and/or solvent for parenteral injection will use microgram quantities of the compounds per day depending on the indication and the clinical/veterinary situation.
According to a particular embodiment of the invention analogues are described with increased activity on bone forming cells without a simultaneous potency on bone resorbing cells or vice versa. Such analogues are usefull in the treatment of bone disorders such as osteoporosis.
According to another particular embodiment of the invention analogues are described which have an increased potency to inhibit proliferation and/or increase differentiation of cancer cells (e.g. leukemia or breast cancer cells) and at the same time have a reduced potency to influence serum, urinary or bone calcium or phosphate homeostasis. Such analogues are of interest in cancer treatment.
According to yet a further aspect of the invention, enhanced effects on proliferation and differentiation are obtained by combining analogues of 1α,25(OH)2D3 with other drugs (e.g. growth factors or cytokines, other steroid or antisteroid hormones or retinoic acids or related compounds, chemotherapeutics). Similarly, analogues are envisaged with increased activity on specific hormone secretion (e.g. parathyroid hormone, insulin) without the same relative potency for the other activities of the natural vitamin D hormone(s). Analogues with increased activity on of the immune system (activated T-cells, antigen presenting cells) are envisaged for the treatment of immune disorders. Indeed, analgues of vitamin D have proven to be effective in experimental models of type I diabetes, graft rejection without major effects on calcium and phosphate metabolism.
According to a further aspect of the invention, the use of inactive precursors is envisaged, which has the advantage of limiting direct activity on the intestine when an oral dose is taken. Some precursors can then be activated by pathways independent of the normal metabolism of vitamin D (the so-called “prodrugs”). Such derivatives display a special biodistribution capable of imparting a selective biological effect in vivo. Some of them are especially well suited for other administration routes such as transcutaneous administration which constitutes, for example, an effective treatment for psoriasis.
The present invention relates to vitamin D compounds and more particularly to certain stereoisomers of 14-epi-19-nor-1,25(OH)2D3 and 19-nor-1,25(OH)2D3 analogues with one or more lower alkyl substituents at carbon 2, the said lower alkyl substituents being optionally substituted with functional atoms or groups, with or without modification of the side chain at carbons 20 and higher. In particular it was found surprisingly found that certain combinations of stereoisomeric configurations at carbon 1, carbon 2 and carbon 3 provide unexpected biological activity profile, toxicity profile and pharmaco-kinetic profile to the corresponding vitamin D analogues. Thus, according to a particular embodiment, the present invention relates to to certain stereoisomers of 19-nor-1,25(OH)2D3 of the invention, more particularly of the 14-epi-19-nor-1,25(OH)2D3 with only one lower alkyl substituent present at carbon 2 and preferably when said lower alkyl substituent has no more than 1 carbon atom, whereby the selection of a configuration α at said carbon 2 is able to provide a significant and useful decrease of side effects with respect to the corresponding stereoisomer having a configuration β at said carbon 2. It is demonstrated that such compounds are capable of increasing the level of calcium in bone, without resulting in an elevation in urine and/or serum calcium. Consequently the selected stereoisomer (having a configuration α at said carbon 2) is of preferred interest in the treatment of bone diseases such as osteoporosis.
According to another particular embodiment of the invention, 19-nor-1,25(OH)2D3, more particularly of the 14-epi-19-nor-1,25(OH)2D3 having only one lower alkyl substituent present at carbon 2, whereby the lower alkyl substituent has 2 to 5 carbon atoms are provided which are able to provide a significantly higher specificity ratio than the corresponding analogues with an alkyl substituent having at least five carbon atoms at position 2. Consequently the selected analogues (having an alkyl substituent with 2 to 5 carbon atoms, particularly having an alkyl substituent with 2 carbon atoms, with either a configuration α or a configuration β at said carbon 2) are of preferred interest in the treatment of cancer.
Thus, the present invention relates to novel vitamin D analogues and to their use as a medicine. The present invention relates more in particular to the use of the specific compounds of the invention for the preparation of a medicament for the treatment of cancer or other diseases characterised by a cellular hyperproliferation, for induction of cell differentiation, for the treatment or prevention of immunological or inflammatory disorders and for the improvement of the function of cells in which calcium is an essential regulating agent, such as for the treatment and prevention of osteoporosis. The present invention furthermore relates to a suitable method of preparation of the specific compounds of the invention in high yield and purity.
As used herein with respect to a substituting group and unless otherwise stated, the terms “C1-5 alkyl” and “alkyl groups having from 1 to 5 carbon atoms” mean straight and branched chain saturated acyclic hydrocarbon monovalent radicals or groups having from 1 to 5 carbon atoms such as, for example, methyl, ethyl, propyl, n-butyl, 1-methylethyl (isopropyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (ter-butyl), 2-methylbutyl, n-pentyl, dimethylpropyl, and the like;
As used herein with respect to a substituting group and unless otherwise stated, the term “aryl” designate any mono- or polyaromatic monovalent hydrocarbon radical having from 6 up to 30 carbon atoms such as but not limited to phenyl, naphthyl, anthracenyl, phenantracyl, fluoranthenyl, chrysenyl, pyrenyl, biphenylyl, terphenyl, picenyl, indenyl, indacenyl, benzocyclobutenyl, benzocyclooctenyl and the like, including fused benzo-C5-8 cycloalkyl radicals (the latter being as defined above) such as, for instance, indanyl, tetrahydronaphtyl, fluorenyl and the like;
As used herein, the term “unsaturated” refers to aliphatic unsaturated hydrocarbon radical, i.e. straight or branched acyclic hydrocarbon monovalent radicals having one or more ethylenic or acetylenic unsaturations.
As used herein and unless otherwise stated, the term “cycloalkyl” means a monocyclic saturated hydrocarbon monovalent radical having from 3 to 10 carbon atoms, such as for instance cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like, or a C7-10 polycyclic saturated hydrocarbon monovalent radical having from 7 to 10 carbon atoms such as, for instance, norbornyl, fenchyl, trimethyltricycloheptyl or adamantyl.
As used herein with respect to a substituting group and unless otherwise stated, the term “heterocyclic” means a mono- or polycyclic, saturated or mono-unsaturated or polyunsaturated monovalent hydrocarbon radical having from 2 up to 15 carbon atoms and including one or more heteroatoms in one or more heterocyclic rings, each of said rings having from 3 to 10 atoms (and optionally further including one or more heteroatoms attached to one or more carbon atoms of said ring, for instance in the form of a carbonyl or thiocarbonyl or selenocarbonyl group and/or to one or more heteroatoms of said ring, for instance in the form of a sulfone, sulfoxide, N-oxide, phosphate, phosphonate or selenium oxide group), each of said heteroatoms being independently selected from the group consisting of nitrogen, oxygen, sulfur, selenium and phosphorus, also including radicals wherein a heterocyclic ring is fused to one or more aromatic hydrocarbon rings for instance in the form of benzo-fused, dibenzo-fused and naphto-fused heterocyclic radicals;
As used herein and unless otherwise stated, the term “enantiomer” means each individual optically active form of a compound or an intermediate of the invention, having an optical purity or enantiomeric excess (as determined by methods standard in the art) of at least 80% (i.e. at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.
A first aspect of the invention thus relates to novel 2-alkylated-19-nor-14-epi-1,25(OH)2D3 compounds and 2-alkylated-19-nor-1,25(OH)2D3 compounds. The invention relates to the synthesis and biological evaluation of said novel compounds which still maintain some of the essential characteristics of vitamin D action but with a more selective pattern (i.e. not all the actions of the physiological vitamin D hormone are maintained with the same relative potency) and with a structure being modified in the A-ring and in the side-chain and preferably but not exclusively with cis-fused CD-ring systems (i.e. 14-epi analogues) as represented in the following general formula (I). The invention thus relates to 2-alkylated-19-nor-14-epi-1,25(OH)2D3 compounds and 2-alkylated-19-nor-1,25(OH)2D3 compounds, which according to the general embodiment of the invention correspond to the general formula (I), pharmaceutically acceptable salts and/or solvates thereof,
wherein:
In a particular embodiment, the invention relates to preferred vitamin D analogues wherein the hydrogen atom at carbon 14 is in a configuration β (the so-called 14-epi). In another particular embodiment, the invention relates to less preferred vitamin D analogues wherein the hydrogen atom at carbon 14 is in a configuration α. In another particular embodiment, the invention relates to vitamin D analogues wherein none of R and R′ is hydrogen. In another particular embodiment, the invention relates to vitamin D analogues wherein one of R and R′ is hydrogen and the other one of R and R′ is an alkyl group with only one carbon atom, and further wherein the OP group is in a configuration α at carbon 1. In another particular embodiment, the invention relates to vitamin D analogues wherein one of R and R′ is hydrogen and the other one of R and R′ is an alkyl group with only one carbon atom, and further wherein the OP group is in a configuration α at carbon 3. In another particular embodiment, the invention relates to vitamin D analogues wherein one of R and R′ is hydrogen and the other one of R and R′ is an alkyl group with only one carbon atom, and further wherein the OP group is in a configuration β at carbon 3. In another particular embodiment, the invention relates to vitamin D analogues wherein one of R and R′ is hydrogen and the other one of R and R′ is an alkyl group with two carbon atoms.
In a specific embodiment the invention relates to compounds with the following structure:
In another specific embodiment the invention relates to compounds with the following structure:
In a specific embodiment the invention relates to compounds with the following structure:
In a specific embodiment the invention relates to compounds with the following structure:
Another particular embodiment of the present invention relates to compounds with the structure (20S configuration)
A second aspect of the invention relates to the use of the compounds according to formula I as a medicine. The invention also relates to the use of the compounds according to formula I for the treatment of hyperproliferative disorders or for the preparation of a medicament for the prevention or treatment of hyperproliferative disorders such as cancer and psoriasis and for the induction of cell differentiation. The invention also relates to the use of the compounds according to formula I for the treatment of immunological disorders (such as allergy, asthma, auto-immune disorders, transplant rejection, etc.) or for the preparation of a medicament for the prevention or treatment of immunological disorders optionally in combination with an immune system interfering drug, inflammatory diseases (i.e. rheumatoid arthritis), skin disorders such as psoriasis and hyperproliferative disorders such as cancer. The invention further relates to use of a compound of formula for the preparation of a medicament for improvement of the function of cells in which calcium is an essential regulating agent, for instance hormone secretion by endocrine glands, muscle cells and bone cells such as necessary in osteoporosis.
The invention also relates to the use of a compound of formula I, as a pharmaceutically active ingredient, especially as an inhibitor of cell proliferation and/or an inductor of cell proliferation. Therefore, the invention also relates to the use of a compound of formula I for the manufacture of a medicine or a pharmaceutical composition having an inhibitory effect on cell proliferation and/or an inductor effect on cell differentiation for the prevention and/or treatment of hyperproliferative disorders such as cancer and psoriasis, immunological disorders, inflammatory disorders, calcium related diseases such as osteoporosis and for the induction of cell differentiation in humans and mammals. The invention also relates to a pharmaceutical preparation comprising a therapeutically effective amount of a compound of the invention and a pharmaceutically and/or veterinarily acceptable carrier or diluent. The present invention further relates to a method of treatment of hyperproliferative disorders in a mammal, including a human, comprising administering to the mammal in need of such treatment a therapeutically effective amount of a compound of formula I, optionally in a mixture with at least a pharmaceutically acceptable carrier.
Another aspect of the invention relates to methods for the preparation of compounds of formula I, more particularly to methods for the preparation of the compounds specifically disclosed herein, to pharmaceutical compositions comprising them in admixture with at least a pharmaceutically acceptable carrier, the active ingredient optionally being in a concentration range of about 0.1-100% by weight, and to the use of these derivatives;
The compounds of the invention are employed for the treatment or prophylaxis of hyperproliferative disorders, immunological, inflammatory and calcium metabolism related disorders. The compounds of the invention have an inhibitory effect on cell proliferation such as in disorders like cancer and psoriasis. The compounds have also a positive effect on immunological and/or inflammatory diseases such as auto-immune disorders. The compounds of the invention have furthermore an activity on the calcium metabolism and are useful in disorders like osteoporosis.
The compounds may be present in a composition in an amount from about 0.1 μg/gm to about 100 μg/gm of the composition.
The dosages envisaged within the context of the invention are in the range of 0.1 μg/kg/day to 500 μg/kg/day, particularly 0.5 μg/kg/day to 100 μg/kg/day, more particularly 1.0 μg/kg/day to 10 μg/kg/day. Depending upon the % pathologic condition to be treated and the patient's condition, the said effective amount may be divided into one or more sub-units per day or may be administered at more than one day intervals.
According to a particular embodiment, the vitamin D analogues of the present invention are characterized by a higher specificity, thus allowing the treatment of a patient in need thereof with higher doses and/or more frequent administration and/or for a prolonged period of time without the occurrence of side-effects which, when are referred to as vitamin D toxicity. Vitamin D toxicity, mainly associated to its calcemic effect can cause symptoms such as but not limited to nausea, vomiting, poor appetite, constipation, weakness and weight loss. High levels of calcium can also cause changes in mental status (e.g. confusion) and heart rhythm abnormalities. Calcinosis, i.e. the deposition of calcium and phosphate in soft tissues like the kidney, can also be caused by vitamin D toxicity.
Administration of vitamin D analogues at a higher dose than currently being used in clinical settings (e.g. paricalcitol, calcitriol etc) are of interest because the calcemic effects of these analogues are dose-limiting, often resulting in a sub-optimal effect.
Prolonged administration (i.e. several months up to lifelong) of the analogues of the present invention are of particular interest in the treatment of metabolic vitamin D deficiencies, the treatment of persistent or structural (?) diseases such as cancer, autoimmune diseases, Alzheimer's and osteoporosis, or the administration to persons subjected to reduced sunlight for longer periods of time.
Moreover the analogues of the present invention are of particular use in the treatment of patients in need thereof which are susceptible to the calcemic side-effects of vitD, such as patients that are underweight or with metabolic deficiencies or in situations where administration of vitamin D is contra-indicated such as in patients with severe renal failure, patients receiving therapy with cardiac glycosides (eg digoxin, digitalis) or patients with sarcoidosis.
As is conventional in the art, the evaluation of a synergistic effect in a drug combination may be made by analyzing the quantification of the interactions between individual drugs, using the median effect principle described by Chou et al. in Adv. Enzyme Reg. (1984) 22:27. Briefly, this principle states that interactions (synergism, additivity, antagonism) between two drugs can be quantified using the combination index (hereinafter referred as CI) defined by the following equation:
wherein EDx is the dose of the first or respectively second drug used alone (1a, 2a), or in combination with the second or respectively first drug (1c, 2c), which is needed to produce a given effect. The said first and second drug have synergistic or additive or antagonistic effects depending upon CI<1, CI=1, or CI>1, respectively.
This principle may be applied to a combination of different drugs of the invention or to a combination of the drugs of the invention with other drugs that exhibit therapeutic effects on hyperproliferative disorders, immunological, inflammatory and calcium metabolism related disorders. The invention thus relates to a pharmaceutical composition or combined preparation having synergistic effects on hyperproliferative disorders, immunological, inflammatory and calcium metabolism related disorders and containing either:
(A) a combination of:
The pharmaceutical composition or combined preparation with synergistic activity against hyperproliferative disorders, immunological, inflammatory and calcium metabolism related disorders according to this invention may contain the 19-nor-14-epi-1,25(OH)2D3 derivatives of the present invention over a broad content range depending on the contemplated use and the expected effect of the preparation. Generally, the content of the 19-nor-14-epi-1,25(OH)2D3 derivatives of the present invention of the combined preparation is within the range of 0.1 to 99.9% by weight, preferably from 1 to 99% by weight, more preferably from 5 to 95% by weight.
The present invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefor. Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.
More generally, the invention relates to the compounds of formula I being useful as agents having biological activity or as diagnostic agents. Any of the uses mentioned with respect to the present invention may be restricted to a non-medical use, a non-therapeutic use, a non-diagnostic use, or exclusively an in vitro use, or a use related to cells remote from an animal.
Those of skill in the art will also recognize that some compounds of the invention may exist in different protonation states, depending on, among other things, the pH of their environment. While the structural formulae provided herein depict the compounds in only one of several possible protonation states, it will be understood that these structures are illustrative only, and that the invention is not limited to any particular protonation state, any and all protonated forms of the compounds are intended to fall within the scope of the invention.
The term “pharmaceutically acceptable salts” as used herein means the therapeutically active non-toxic salt forms which some of the compounds of formula I are able to form. Therefore, the compounds of this invention optionally comprise salts of the compounds herein, especially pharmaceutically acceptable non-toxic salts containing, for example, Na+, Li+, K+, Ca+2 and Mg+2. Such salts may include those derived by combination of appropriate cations such as alkali and alkaline earth metal ions or ammonium and quaternary amino ions with an acid anion moiety, typically a carboxylic acid. The compounds of the invention may bear multiple positive or negative charges. The net charge of the compounds of the invention may be either positive or negative. Any associated counter ions are typically dictated by the synthesis and/or isolation methods by which the compounds are obtained. Typical counter ions include, but are not limited to ammonium, sodium, potassium, lithium, halides, acetate, trifluoroacetate, etc., and mixtures thereof. It will be understood that the identity of any associated counter ion is not a critical feature of the invention, and that the invention encompasses the compounds in association with any type of counter ion. Moreover, as the compounds can exist in a variety of different forms, the invention is intended to encompass not only forms of the compounds that are in association with counter ions (e.g., dry salts), but also forms that are not in association with counter ions (e.g., aqueous or organic solutions). Furthermore, this term also includes the solvates which the compounds of formula I as well as their salts are able to form, such as for example hydrates, alcoholates and the like. Finally, it is to be understood that the compositions herein comprise compounds of the invention in their unionized, as well as zwitterionic form, and combinations with stoichiometric amounts of water as in hydrates. Also included within the scope of this invention are the salts of some of the parental compounds with one or more amino acids, especially the naturally-occurring amino acids found as protein components. The amino acid typically is one bearing a side chain with a basic or acidic group, e.g., lysine, arginine or glutamic acid, or a neutral group such as glycine, serine, threonine, alanine, isoleucine, or leucine.
The compounds of the invention also include physiologically acceptable salts thereof. Examples of physiologically acceptable salts of the compounds of the invention include salts derived from an appropriate base, such as an alkali metal (for example, sodium), an alkaline earth (for example, magnesium), ammonium and NX4+ (wherein X is C1-C4 alkyl). Physiologically acceptable salts of a compound containing a hydroxy group include the anion of said compound in combination with a suitable cation such as Na+ and NX4+ (wherein X typically is independently selected from H or a C1-C4 alkyl group). However, salts of acids or bases which are not physiologically acceptable may also find use, for example, in the preparation or purification of a physiologically acceptable compound. All salts, whether or not derived form a physiologically acceptable acid or base, are within the scope of the present invention.
The terms cis and trans are used herein in accordance with Chemical Abstracts nomenclature and include reference to the position of the substituents on a ring moiety. The absolute stereochemical configuration of the compounds of formula (I) may easily be determined by those skilled in the art while using well-known methods such as, for example, X-ray diffraction.
The compounds of the invention may be formulated with conventional carriers and excipients, which will be selected in accord with ordinary practice. Tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic. Formulations optionally contain excipients such as those set forth in the “Handbook of Pharmaceutical Excipients” (1986) and include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like.
Subsequently, the term “pharmaceutically acceptable carrier” as used herein means any material or substance with which the active ingredient is formulated in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing the said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness. The pharmaceutically acceptable carrier may be a solid or a liquid or a gas which has been compressed to form a liquid, i.e. the compositions of this invention can suitably be used as concentrates, emulsions, solutions, granulates, dusts, sprays, aerosols, suspensions, ointments, creams, tablets, pellets or powders.
Suitable pharmaceutical carriers for use in the said pharmaceutical compositions and their formulation are well known to those skilled in the art, and there is no particular restriction to their selection within the present invention. They may also include additives such as wetting agents, dispersing agents, stickers, adhesives, emulsifying agents, solvents, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided the same are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to mammals. The pharmaceutical compositions of the present invention may be prepared in any known manner, for instance by homogeneously mixing, coating and/or grinding the active ingredients, in a one-step or multi-steps procedure, with the selected carrier material and, where appropriate, the other additives such as surface-active agents may also be prepared by inicronisation, for instance in view to obtain them in the form of microspheres usually having a diameter of about 1 to 10 gm, namely for the manufacture of microcapsules for controlled or sustained release of the active ingredients.
Suitable surface-active agents, also known as emulgent or emulsifier, to be used in the pharmaceutical compositions of the present invention are non-ionic, cationic and/or anionic materials having good emulsifying, dispersing and/or wetting properties. Suitable anionic surfactants include both water-soluble soaps and water-soluble synthetic surface-active agents. Suitable soaps are alkaline or alkaline-earth metal salts, unsubstituted or substituted ammonium salts of higher fatty acids (C10-C22), e.g. the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures obtainable form coconut oil or tallow oil. Synthetic surfactants include sodium or calcium salts of polyacrylic acids; fatty sulphonates and sulphates; sulphonated benzimidazole derivatives and alkylarylsulphonates. Fatty sulphonates or sulphates are usually in the form of alkaline or alkaline-earth metal salts, unsubstituted ammonium salts or ammonium salts substituted with an alkyl or acyl radical having from 8 to 22 carbon atoms, e.g. the sodium or calcium salt of lignosulphonic acid or dodecylsulphonic acid or a mixture of fatty alcohol sulphates obtained from natural fatty acids, alkaline or alkaline-earth metal salts of sulphuric or sulphonic acid esters (such as sodium lauryl sulphate) and sulphonic acids of fatty alcohol/ethylene oxide adducts. Suitable sulphonated benzimidazole derivatives preferably contain 8 to 22 carbon atoms. Examples of alkylarylsulphonates are the sodium, calcium or alcanolamine salts of dodecylbenzene sulphonic acid or dibutyl-naphtalenesulphonic acid or a naphtalene-sulphonic acid/formaldehyde condensation product. Also suitable are the corresponding phosphates, e.g. salts of phosphoric acid ester and an adduct of p-nonylphenol with ethylene and/or propylene oxide, or phospholipids. Suitable phospholipids for this purpose are the natural (originating from animal or plant cells) or synthetic phospholipids of the cephalin or lecithin type such as e.g. phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, lysolecithin, cardiolipin, dioctanylphosphatidyl-choline, dipalmitoylphoshatidyl-choline and their mixtures.
Suitable non-ionic surfactants include polyethoxylated and polypropoxylated derivatives of alkylphenols, fatty alcohols, fatty acids, aliphatic amines or amides containing at least 12 carbon atoms in the molecule, alkylarenesulphonates and dialkylsulphosuccinates, such as polyglycol ether derivatives of aliphatic and cycloaliphatic alcohols, saturated and unsaturated fatty acids and alkylphenols, said derivatives preferably containing 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenol. Further suitable non-ionic surfactants are water-soluble adducts of polyethylene oxide with poylypropylene glycol, ethylenediaminopolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ether groups. Such compounds usually contain from 1 to 5 ethyleneglycol units per propyleneglycol unit. Representative examples of non-ionic surfactants are nonylphenol-polyethoxyethanol, castor oil polyglycolic ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethyleneglycol and octylphenoxypoly-ethoxyethanol. Fatty acid esters of polyethylene sorbitan (such as polyoxyethylene sorbitan trioleate), glycerol, sorbitan, sucrose and pentaerythritol are also suitable non-ionic surfactants.
Suitable cationic surfactants include quaternary ammonium salts, particularly halides, having 4 hydrocarbon radicals optionally substituted with halo, phenyl, substituted phenyl or hydroxy; for instance quaternary ammonium salts containing as N-substituent at least one C8C22 alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl, oleyl and the like) and, as further substituents, unsubstituted or halogenated lower alkyl, benzyl and/or hydroxy-lower alkyl radicals.
A more detailed description of surface-active agents suitable for this purpose may be found for instance in “McCutcheon's Detergents and Emulsifiers Annual” (MC Publishing Crop., Ridgewood, N.J., 1981), “Tensid-Taschenbucw’, 2 d ed. (Hanser Verlag, Vienna, 1981) and “Encyclopaedia of Surfactants, (Chemical Publishing Co., New York, 1981).
Compounds of the invention and their physiologically acceptable salts (hereafter collectively referred to as the active ingredients) may be administered by any route appropriate to the condition to be treated, suitable routes including, oral, rectal, nasal, topical (including transdermally, ocular, buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intra-arterially, intradermal, intrathecal and epidural). The preferred route of administration may vary with for example the condition of the recipient.
While it is possible for the active ingredients to be administered alone it is preferable to present them as pharmaceutical formulations. The formulations, both for veterinary and for human use, of the present invention comprise at least one active ingredient, as above described, together with one or more pharmaceutically acceptable carriers therefore and optionally other therapeutic ingredients. The carrier(s) optimally are “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. The formulations of the invention are optionally applied as a topical ointment or cream (i.e. for psoriasis) containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ingredient(s) in a range between 0.1% and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc), preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG400) and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogs.
The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Optionally, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus the cream should optionally be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate. Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns (including particle sizes in a range between 20 and 500 microns in increments of 5 microns such as 30 microns, 35 microns, etc), which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as for example a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol administration may be prepared according to conventional methods and may be delivered with other therapeutic agents.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
Compounds of the invention can be used to provide controlled release pharmaceutical formulations containing as active ingredient one or more compounds of the invention (“controlled release formulations”) in which the release of the active ingredient can be controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given invention compound. Controlled release formulations adapted for oral administration in which discrete units comprising one or more compounds of the invention can be prepared according to conventional methods.
Additional ingredients may be included in order to control the duration of action of the active ingredient in the composition. Control release compositions may thus be achieved by selecting appropriate polymer carriers such as for example polyesters, polyamino acids, polyvinyl pyrrolidone, ethylene-vinyl acetate copolymers, methylcellulose, carboxymethylcellulose, protamine sulfate and the like. The rate of drug release and duration of action may also be controlled by incorporating the active ingredient into particles, e.g. microcapsules, of a polymeric substance such as hydrogels, polylactic acid, hydroxymethylcellulose, polyniethyl methacrylate and the other above-described polymers. Such methods include colloid drug delivery systems like liposomes, microspheres, microemulsions, nanoparticles, nanocapsules and so on. Depending on the route of administration, the pharmaceutical composition may require protective coatings. Pharmaceutical forms suitable for injectionable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation thereof. Typical carriers for this purpose therefore include biocompatible aqueous buffers, ethanol, glycerol, propylene glycol, polyethylene glycol and the like and mixtures thereof.
In view of the fact that, when several active ingredients are used in combination, they do not necessarily bring out their joint therapeutic effect directly at the same time in the mammal to be treated, the corresponding composition may also be in the form of a medical kit or package containing the two ingredients in separate but adjacent repositories or compartments. In the latter context, each active ingredient may therefore be formulated in a way suitable for an administration route different from that of the other ingredient, e.g. one of them may be in the form of an oral or parenteral formulation whereas the other is in the form of an ampoule for intravenous injection or an aerosol.
The vitamin D analogues of the present invention can be prepared by several methods involving a series of chemical reactions, each of them being well known to those skilled in the art, altogether making up the process for preparing said compounds and exemplified further. The processes described further are only meant as examples and by no means are meant to limit the scope of the present invention.
A first method of synthesis of these analogues is very schematically shown in
More specifically, a typical example of the synthesis of 14-epi-2β-methyl-1α,25-dihydroxyvitamin D3 (compound 101) is described in the
The other isomers (102 to 114) were obtained by the same general procedure, starting either from intermediates 15 or 16 or their respective 2-ethyl homologues and the appropriate diastereoisomer of the bicyclic A-ring precursor (IV in
The following 20-epi analogues were obtained similarly starting from 20-epi □.
In the 19-nor series of vitamin D analogues, as shown in
We herein provide a convergent synthesis of the compounds of the invention via the so-called cyclovitamin approach as an alternative to the more widely used Lythgoe coupling process. Again referring to
When the hydroxy function is flanked by a cis-vicinal alkyl substituent, facile anti-elimination occurs in high yield, as is shown in
In the next step (d), the easy transformation of cyclohexene intermediates was exploited. Hydroboration of intermediates 33a,b in tetrahydrofuran as a solvent, at 0° C. during 2-3 hours was non-selective and afforded a separable circa 1:1 mixture of intermediates 34a,b and 35a,b. Both diastereoisomers are suitable for further transformation into the corresponding mesylates 36a,b and 37a,b in step (e) through a reaction with mesityl chloride preferably in the presence of a catalyst such as triethylamine, in methylene chloride as a solvent, at 0° C. during about 3 hours. The mesylate 37a,b led, upon base-mediated cyclopropane formation in step (f) and subsequent conversion of the ester function of intermediate 38a,b into a formyl group in step (g), to intermediates 39a,b which are the precursors for the 2α-alkyl-19-nor-1α,25-dihydroxyvitamin D3 analogues 102, 104, 106, 108, 110 112, 114 and 116. Preferred reaction conditions for performing step (f) include the presence of potassium tert-butoxide, in a solvent such as a mixture of THF and tert-butanol, during about 30 minutes at about 50° C. Preferred reaction conditions for performing step (g) include for instance a reaction with lithium aluminum hydride in a solvent such as THF, during about 3 hours at about 0° C. On the other hand Mitsunobu inversion of the hydroxy group of intermediates 34a,b is performed in step (h) and provides intermediates 41a,b in high yield. Preferred reaction conditions for performing step (h) include for instance a reaction with p-nitrobenzoic acid during about 24 hours in the presence of a phosphine such as triphenylphosphine, a diazo compound such as DIAD, in a solvent such as THF at room temperature. This opens, successively via intermediates 41a,b through step (b), then intermediates 42a,b through step (e), and then intermediates 43a,b through step (f), a route to intermediates 44a,b which are the precursors for the epimeric 2β-alkyl vitamin D3 analogues 101, 103, 105, 107, 109, 111, 113 and 115. Finally, mesylation of intermediates 34a,b into intermediates 36a,b, followed by cyclopropane formation in intermediates 45a,b in step (f) and ester reduction in step (g) afforded intermediates 46a,b which are the precursors for vitamin D analogues with a 1α, 2β, 3α configuration.
As shown at bottom of
As shown in
As already mentioned above, an alternative for the Mitsunobu reaction when the hydroxy function is flanked by a cis-vicinal 4-alkyl substituent consists of inversion of the corresponding p-nitrophenylsulfonate with cesium propionate. Thus intermediate 54 is able, via intermediate 55, to provide 57a, an intermediate for the A-ring precursor 67a.
The synthesis of the stereoisomers 69a,b and 71a,b is shown in
With the A-ring precursors in hand we turned our attention to the construction of the vitamin D3 skeleton by the cyclovitamin D strategy described in
The structure of intermediate 72a was proven by nuclear Overhauser effect (hereinafter NOE) and 2D correlation spectroscopy (hereinafter COSY-2D) experiments. The vinylic protons 6-H and 7-H (AB system; δ=6.14 and β=5.46; J=11.3 Hz) gave a NOE enhancement with respectively 10α-H and 4α,β-H. The assignment of these protons followed from the observation of a NOE enhancement with respectively 1-H (δ=3.72) and 3-H (δ=3.94). Localization of the latter protons follows from the trichloroacetate of intermediate 72a. For 3-H a chemical shift from δ=3.94 to δ=5.17 was observed and COSY-2D experiments and NOE enhancements corroborated with those obtained for intermediate 72a.
It is noteworthy that for the 1-OTBDPS ether series, 1-H is downfield relative to 3-H for the isomers with a 2α-substituent while a reversed situation is observed for the 20-substituted isomers. Identical observations have been made for the stereoisomers with varying carbon 1, carbon 2 and carbon 3 configurations in both the 2-methyl and 2-ethyl substituted series.
In a few cases in the 14-epi series a NOE enhancement between 7-H and an aromatic proton of the 1-OTBDPS substituent was also observed, which by itself is a structural proof. The most simple tool for structural identification in these series is provided by a consistent upfield chemical shift of 7-H in 1-OTBDPS ethers for which 6-H and 7-H give a Δδ=0.5-0.7. This upfield shift is probably due to the anisotropy of a phenyl group of the DBDPS ether, which is in fact confirmed by the observed NOE enhancement (7-H, H—Ar). The regioisomers (3-OTBDPS) show the signals for 6-H and 7-H closer together, (Δδ=0.1-0.3) with a small upfield shift for 6-H compared to the 6-values observed for the unprotected title compounds.
Finally, TBDPS ether cleavage led to the desired 2-alkyl-substituted vitamin D analogues. In conclusion the above disclosed cyclovitamin route of synthesis provides good stereoselectivity.
The following examples are provided as an illustration of the invention and should in no way interpreted as limiting its scope.
All synthesis reactions were carried out under argon or nitrogen atmosphere with magnetic stirring. All solvents were purified or dried according to standard procedures. Solutions were dried over MgSO4. The solvent was removed from the filtered solutions on a rotary evaporator.
Column chromatography separations were performed on silica gel, eluents are given between brackets. HPLC separations were performed on a Knauer 64, a Waters 6000 A or a Kontron 420 delivery system with RI detection, eluents are given between brackets. Optical rotations were measured with a Perkin Elmer 421 polarimeter. IR spectra were recorded on a Perkin Elmer FTIR-1600 spectrometer and mass-spectra on a HP-5988 spectrometer. The 1H NMR spectra were recorded at 500 MHz (WH-Bruker) and 13C NMR spectra at 200 MHz (Varian-Gemini), the chemical shifts are expressed in ppm relative to TMS and coupling constants are in Hz.
Reference is made to
Reference is made to
Hydroboration of the intermediate of example 1 was effected as follows. To a stirred solution of 33a (130 mg, 0.319 mmol), in THF (12 mL) was added dropwise a BH3 solution (380 uL, 1M in THF, 0.38 mmol) at 0° C. and stirring was continued at this temperature for 2.5 hours. Then H2O2 (0.5 mL) and saturated NaHCO3 (3 mL) were added. After stirring for 0.5 hour the reaction solution was poured into a H2O-EtOAc mixture and the organic layer was separated. The aqueous layer was extracted by means of EtOAc. The combined organic extracts were washed, dried and concentrated. The residue was purified by chromatography (using isooctane/EtOAc mixtures ranging from 9:1 to 4:1), thus affording intermediate 34a (48 mg, 35%) which was characterized as follows: Rf=0.21 (isooctane/EtOAc, 4:1).—[α]Dr.t=−51.8 (c=0.55, CHCl3).—IR (film): ν=3361, 2932, 2858, 1737, 1589, 1403, 1428, 1363, 1282, 1250, 1173, 1111 cm−1.—1H NMR (CDCl3): δ=7.70-7.36 (m, 10H), −3.59 (s, 3H), 3.25 (m, 1H), 3.09 (m, 1H), 2.12-2.05 (m, 2H), 1.91 (dt, J=12.7, 4.3 Hz, 1H), 1.62 (br.s, 1H), 1.54-1.38 (m, 3H), 1.07 (d, J=6.4 Hz, 3H), 1.05 (s, 9H).—13C NMR (CDCl3): δ=174.6, 135.9, 134.3, 133.6, 129.7, 127.5, 74.8, 73.0, 51.8, 47.9, 37.9, 37.1, 36.8, 27.1, 19.5, 14.5.—MS (m/z, %): 409 (M+−H2O−H, 1), 369 (M+−57, 10), 337 (25), 309 (5), 199 (75), 153 (25), 121 (15), 93 (100). Elemental analysis: C25H34O4Si (426.63): calcd. C, 70.38; H, 8.03. found C, 70.16; H, 8.14.
Starting from the intermediate of example 2, intermediate 35a (61 mg, 45%) was prepared following the same hydroboration procedure as described in example 3, and characterized as follows: Rf=0.19 (isooctane/EtOAc, 4:1).—[α]Dr.t=−38.5 (c=0.69, CHCl3).—IR (film): ν=3442, 2954, 2893, 2857, 1735, 1715, 1589, 1463, 1427, 1378, 1273, 1196, 1111 cm−1. 1H NMR (CDCl3): δ=7.68-7.36 (m, 10H), 4.17 (dt, J=10.8, 4.7 Hz, 1H), 3.89 (d, J=3.1 Hz, 1H), 3.63 (s, 3H), 2.62 (tt, J=11.8, 4.4 Hz, 1H), 1.82-1.66 (m, 5H), 1.59 (br.s, 1H), 1.06 (s, 9H), 0.96 (d, J=7.2 Hz, 3H).—13C NMR (CDCl3): δ=175.7, 135.8, 134.3, 129.6, 127.5, 72.0, 68.9, 51.7, 41.3, 36.6, 31.4, 29.6, 27.0, 19.3, 10.7.—MS (m/z, %): 369 (M+−57, 100), 339 (5), 319 (4), 273 (6), 253 (10), 199 (85), 153 (65), 135 (40).—Elemental analysis: C25H34O4Si (426.63): calcd. C, 70.38; H, 8.03. found C, 70.21; H, 8.20.
The ethyl homologue (34b) of intermediate (34a) was prepared in a similar manner and characterized as follows: Rf=0.12 (isooctane/EtOAc, 4:1).—[α]Dr.t=−51.1 (c=0.52, CHCl3).—IR (film): ν=3421, 2956, 2857, 1736, 1508, 1458, 1428, 1363, 1272, 1242 cm−1. 1H NMR (CDCl3): δ=7.69-7.36 (m, 10H), 3.59 (s, 3H), 3.43 (dt, J=10.6, 4.3 Hz, 1H), 3.33 (dt, J=10.6, 4.3 Hz, 1H), 2.05 (m, 2H), 1.92 (d, J=12.4 Hz, 1H), 1.78 (m, 1H), 1.70 (m, 1H) 1.54-1.41 (m, 4H), 1.09 (s, 9H), 0.69 (t, J=7.5 Hz, 3H).—13C NMR (CDCl3): δ=175.2, 136.5, 134.8, 33.9, 130.3, 130.1, 128.2, 127.9, 71.3, 69.8, 52.8, 52.3, 38.3, 37.5, 37.3, 27.6, 19.9, 19.1, 9.4.—MS (m/z, %): 383 (M+−57, 2), 351 (8), 305 (24), 273 (5), 213 (9), 199 (70), 183 (15), 153 (20), 135 (28), 107 (100), 79 (30). Elemental analysis: C26H36O4Si (440.65): calcd. C, 70.87; H, 8.23. found: C, 70.50; H, 8.33.
The ethyl homologue (35b) of intermediate (35a) was prepared in a similar manner and characterized as follows: Rf=0.17 (isooctane/EtOAc, 4:1). [α]Dr.t=−38.9 (c=0.81, CHCl3).—IR (film): ν=3453, 2958, 2858, 1736, 1589, 1460, 1428, 1382, 1255, 1172, 1195, 1110 cm−1. 1H NMR (CDCl3): δ=7.67-7.37 (m, 10H), 4.18 (dt, J=11.7, 4.6 Hz, 1H), 4.02 (d, J=2.8 Hz, 1H), 3.63 (s, 3H), 2.60 (m, 1H), 1.95 (m, 1H), 1.80 (dt, J=12.6, 4.1 Hz, 1H), 1.65-1.45 (m, 5H), 1.05 (s, 9H), 0.87 (m, 3H).—13C NMR (CDCl3): δ=176.2, 136.3, 134.8, 134.7, 130.1, 130.0, 128.0, 69.3, 52.1, 49.1, 36.8, 32.6, 30.0, 27.4, 19.7, 17.6, 13.3.—MS (m/z, %): 383 (M+−57, 90), 351 (6), 333 (5), 305 (4), 273 (10), 213 (50), 199 (100), 183 (58), 153 (60), 135 (65), 107 (48).—Elemental analysis: C26H36O4Si (440.65): calcd. C, 70.87; H, 8.23. found C, 70.75; H, 8.33.
Reference is made again to
The ethyl homologue 41b of intermediate 41a was prepared starting from intermediate 34b via 40b as described in example 7 and characterized as follows: Rf=0.22 (isooctane/EtOAc, 4:1), -[α]Dr.t=−63.4 (c=0.71, CHCl3).—IR (film): ν=3506, 3071, 2956, 2858, 1736, 1472, 1428, 1361, 1266, 1176, 1110 cm−1. 1H NMR (CDCl3): δ=7.67-7.35 (m, 10H), 4.2 (d, J=3.2 Hz, 1H), 3.78 (m, 1H), 3.58 (s, 3H), 2.57 (m, 1H), 1.94 (dt, J=13.1, 3.6 Hz, 2H), 1.60-1.50 (m, 2H), 1.43 (br.s, 1H), 1.33 (m, 1H), 1.13 (m, 2H), 1.05 (s, 9H), 0.86 (t, J=7.5 Hz, 3H).—13C NMR (CDCl3): δ=175.6, 136.0, 129.6, 129.5, 127.5, 127.4, 71.5, 66.4, 51.6, 50.2, 37.2, 36.2, 35.0, 27.1, 19.7, 19.5, 11.3.—MS (m/z, %): 383 (M+−57, 100), 351 (6), 305 (4), 273 (8), 199 (90), 153 (45), 135 (50), 77 (65), 57 (68).—Elemental analysis: C26H36O4Si (440.65): calcd. C, 70.87; H, 8.23. found C, 71.02; H, 8.20.
Reference is made to
This synthesis is effected through the hydroboration procedure of example 3 and achieves, with a yield of 42%, colourless crystals from n-hexane/acetone which were characterized as follows: M.p=128-129° C.; Rf=0.21 (isooctane/EtOAc 1:1); [α]Dr.t=−109.3 (c=0.97, CHCl3).—IR (film): ν=3448, 2933, 1766, 1359, 1273, 1227 cm−1. 1H NMR (CDCl3): δ=4.56 (d, J=5.9 Hz, 1H), 3.57 (m, 1H), 2.70 (br.s, 1H), 2.41 (m, 1H), 2.32 (m, 1H), 2.16 (br.s, 1H), 1.84 (d, J=11.7 Hz, 1H), 1.65 (m, 1H), 1.58 (m, 1H), 1.19 (d, J=6.9 Hz, 3H). 13C NMR (CDCl3): δ=178.3, 82.8, 71.5, 42.4, 37.9, 37.6, 35.5, 16.1.—MS (m/z, %): 182 (M+, 1), 161 (5), 154 (4), 128 (14), 113 (48), 97 (54), 67 (50), 55 (100). Elemental analysis: C8H12O3 (156.18): calcd. C, 61.52; H, 7.74. found C, 59.92; H, 7.78.
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Using a procedure similar to that of example 12, the ethyl homologue of (isooctane/EtOAc, 9:1). [α]Dr.t=+74.2 (c=1.09, CHCl3).—IR (film): ν=2959, 2857, 1738, 1652, 1589, 1456, 1428, 1388, 1246 cm−1. 1H NMR (CDCl3): =7.72-7.38 (m, 10H), 5.37 (m, 1H), 4.26 (br.s, 1H), 3.58 (s, 3H), 2.35 (m, 1H), 2.30-1.98 (m, 5H), 1.75 (m, 1H), 1.05 (s, 9H), 0.87 (t, J=7.4 Hz, 3H).—13C NMR (CDCl3): δ=175.1, 136.7, 134.4, 133.5, 129.5, 127.1, 119.7, 70.2, 51.5, 38.7, 35.3, 27.7, 27.0, 25.3, 19.4, 12.3.—MS (m/z, %): 422 (M+, 2), 401 (3), 365 (88), 333 (8), 287 (9), 255 (3), 227 (7), 213 (90), 183 (50), 137 (58), 107 (45), 79 (110).
Intermediate 58a was obtained by performing the hydroboration of the intermediate of example 12 under the same conditions as in example 3, and was characterized as follows: Rf=0.18 (isooctane/EtOAc, 4:1).—[α]Dr.t=+40.5 (c=0.66, CHCl3).—IR (film): ν=3452, 3070, 2953, 2857, 1735, 1717, 1427, 1379, 1272, 1195 cm−1.—1H NMR (CDCl3): δ=7.68-7.36 (m, 10H), 4.17 (dt, J=10.8, 4.7 Hz, 1H), 3.89 (d, J=2.7 Hz, 1H), 3.63 (s, 3H), 2.62 (m, 1H), 1.81-1.67 (m, 5H), 1.56 (br.s, 1H), 1.06 (s, 9H), 0.96 (d, J=7.2 Hz, 3H).—13C NMR (CDCl3): δ=175.7, 135.8, 134.4, 129.7, 127.6, 72.0, 68.9, 56.9, 51.7, 41.3, 36.6, 31.4, 29.6, 27.0, 19.3, 10.6.—MS (m/z, %): 425 (M+−1, 1), 385 (2), 369 (M+−57, 100), 337 (5), 291 (4), 259 (10), 221 (4), 199 (85), 153 (65).
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Using a procedure similar to that of example 14, the ethyl homologue of intermediate 58a was made and characterized as follows: Rf=0.17 (isooctane/EtOAc, 4:1).—[α]Dr.t=+38.0 (c=1.13, CHCl3).—IR (film): ν=3453, 2958, 2858, 1736, 1589, 1460, 1428, 1382, 1255 cm−1.—1H NMR (CDCl3): δ=7.67-7.37 (m, 10H), 4.18 (dt, J=11.7, 4.6 Hz, 1H), 4.02 (d, J=2.8 Hz, 1H), 3.63 (s, 3H), 2.60 (m, 1H), 1.95 (m, 1H), 1.80 (dt, J=12.6, 4.1 Hz, 1H), 1.65-1.45 (m, 6H), 1.05 (s, 9H), 0.87 (m, 3H).—13C NMR (CDCl3): δ=176.2, 136.3, 134.8, 134.7, 130.1, 130.0, 128.0, 69.3, 52.1, 49.1, 36.8, 32.6, 30.0, 27.4, 19.7, 17.6, 13.3.—MS (m/z, %): 383 (M+−57, 100), 351 (4), 287 (6), 273 (7), 213 (55), 199 (95), 183 (55), 153 (50), 107 (35), 55 (86).
Using a procedure similar to that of example 15, the ethyl homologue of intermediate 60a was made and characterized as follows: Rf=0.12 (isooctane/EtOAc, 4:1).—[α]Dr.t=+50.7 (c=0.52, CHCl3).—IR (film): ν=3421, 2956, 2857, 1736, 1508, 1458, 1428, 1363, 1272, 1242, 1169, 1110, 1040, 1007, 866, 822, 740, 703, 612 cm−1. 1H NMR (CDCl3): δ=7.69-7.36 (m, 10H), 3.59 (s, 3H), 3.43 (dt, J=10.6, 4.3 Hz, 1H), 3.33 (dt, J=10.6, 4.3 Hz, 1H), 2.05 (m, 2H), 1.92 (d, J=12.4 Hz, 1H), 1.78 (m, 1H), 1.70 (m, 1H) 1.54-1.41 (m, 3H), 1.09 (s, 9H), 0.89 (m, 1H), 0.69 (t, J=7.5 Hz, 3H). 13C NMR (CDCl3): δ=175.2, 136.5, 134.8, 133.9, 130.3, 130.1, 128.2, 127.9, 71.3, 69.8, 52.8, 52.3, 38.3, 37.5, 37.3, 27.6, 19.9, 19.1, 9.4.—MS (m/z, %): 383 (M+−57,3), 351 (9), 305 (32), 273 (6), 199 (64), 183 (15), 153 (18), 135 (35), 107 (100), 79 (35).
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To a stirred solution of intermediate 55 (0.22 g, 0.36 mmole) and 18-crown-6-ether (476 mg, 1.80 mmole) was added freshly prepared EtCO2Cs (371 mg, 1.80 mmole). The mixture was stirred at 110° C. for 2.5 hours and then cooled to room temperature. The mixture was diluted with EtOAc, washed and concentrated. Column chromatography (using a isooctane/EtOAc 100:4 mixture) afforded intermediate 57a (95 mg, 55%) characterized as follows: Rf=0.26 (isooctane:EtOAc, 4:1).—[α]Dr.t=+30.7 (c=0.79, CHCl3).—IR (film): ν=3049, 2954, 2858, 1738, 1463, 1428, 1274, 1189, 1112 cm−1. 1H NMR (CDCl3): δ=7.67-7.34 (m, 10H), 4.90 (d, J=2.9 Hz, 1H), 3.98 (dt, J=11.6, 4.7 Hz, 1H), 3.65 (s, 3H), 2.48 (m, 1H), 2.14-1.99 (m, 2H), 1.93 (m, 1H), 1.87 (dt, J=12.8, 4.2 Hz, 1H), 1.93-1.81 (m, 3H), 1.06 (s, 9H), 1.04 (d, J=7.3 Hz, 3H), 0.97 (t, J=7.6 Hz, 3H).—13C NMR (CDCl3): δ 175.1, 173.1, 135.8, 133.9, 129.7, 127.5, 74.1, 69.1, 51.8, 38.0, 37.3, 30.9, 27.7, 26.9, 26.7, 19.2, 9.9, 9.0.—MS (m/z): 451 (M+−31, 2), 425 (M+−57, 21), 386 (1), 351 (15), 291 (3), 255 (27), 199 (29), 183 (19), 135 (21), 93 (26). Elemental analysis: C28H38O5Si (482.69): calcd. C, 69.67; H, 7.94. found C, 69.82; H, 7.82.
Finally, the methanolysis of intermediate 57a provided intermediate 58a in a 98% yield.
The following general procedure was used for mesylate formation: to a stirred solution of a hydroxy compound and Et3N (1.5 equivalent) in CH2Cl2 (0.04-0.05 mmol/mL) was added dropwise mesityl chloride (1.2 equivalent) at 0° C. and stirring was continued for 3 hours. The resulting solution was subjected to flash chromatography (using a isooctane/EtOAc 7:3 mixture). The residue was purified by HPLC (using a isooctane/EtOAc 9:1 mixture) to give the corresponding mesylate (yield circa 95%).
The following general procedure was used for the formation of 3a-carbomethoxy-bicyclo[3,1,0]hexane: to a stirred solution of a mesylate obtained for instance according to example 19 (0.03-0.05 mmol/mL) in a tBuOH-THF mixture (3:2) was added dropwise tBuOK (1 M in tBuOH, 1.2 equivalent) at 45-50° C. and stirring was continued for 0.5 hour. The solution was poured into H2O-EtOAc and then the organic layer was separated. The aqueous layer was extracted with EtOAc. The combined organic extracts were washed, dried and concentrated. The residue was purified by flash chromatography (using a isooctane/EtOAc 100:3 mixture) affording the bicyclic product with a yield of about 70%.
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The corresponding enantiomer 70a was also made and characterized as follows:—[α]Dr.t=+13.9 (c=0.65, CHCl3). Elemental analysis: C25H32O3Si (408.61): calcd. C, 73.49; H, 7.89. found C, 73.41; H, 8.13.
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The corresponding enantiomer 70b was also made and characterized as follows—[α]Dr.t=+28.4 (c=0.75, CHCl3).
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Intermediate 72a: Rf=0.22 (isooctane/EtOAc 4:1).—[α]Dr.t=−15.7 (c=0.76, CHCl3).—IR (film): ν=3394, 2942, 1616, 1471, 1428, 1377, 1110 cm−1.—1H NMR (CDCl3): δ=7.71-7.35 (m, 10H), 6.14 (d, J=11.3 Hz, 1H), 5.46 (d, J=11.3 Hz, 1H), 3.94 (m, 1H), 3.72 (td, J=8.9, 4.2 Hz, 1H), 2.72 (m, 1H), 2.66 (dd, J=13.3, 4.0 Hz, 1H), 2.37-2.44 (m, 2H), 2.25 (dd, J=13.6, 5.2 Hz, 1H), 2.05-1.85 (m, 4H), 1.80 (m, 1H), 1.70-1.60 (m, 3H), 1.50-1.24 (m, 13H), 1.23 (s, 6H), 1.07 (s, 9H), 1.03 (d, J=6.9 Hz, 3H), 9.93 (d, J=6.5 Hz, 3H), 0.45 (s, 3H).—13C NMR (CDCl3): δ=142.2, 135.9, 134.8, 134.2, 132.1, 129.6, 127.5, 122.9, 115.4, 73.9, 71.4, 71.1, 56.5, 56.2, 45.6, 44.4, 43.3, 40.4, 36.5, 36.4, 36.1, 30.1, 29.2, 28.8, 27.7, 27.1, 25.5, 22.5, 22.3, 20.8, 19.4, 18.8, 14.0, 11.9.—MS (m/z, %): 656 (M+, 1), 638 (M+−18, 1), 600 (M+−tBu+H, 1), 581 (3), 563 (2), 503 (3), 472 (1), 400 (4), 365 (6), 321 (9), 239 (11), 199 (100), 149 (19), 135 (52), 59 (88).
Intermediate 73a: Rf=0.19 (isooctane/EtOAc 4:1).—[α]Dr.t=+18.3 (c=0.31, CHCl3).—IR (film): ν=3405, 3071, 2958, 2879, 1459, 1429, 1376, 1217, 1147, 1076, 1053 cm−1.—1H NMR (CDCl3): δ=7.71-7.36 (m, 10H), 5.99 (d, J=11.2 Hz, 1H), 5.77 (d, J=11.2 Hz, 1H), 4.00 (m, 1H), 3.71 (td, J=8.1, 4.2 Hz, 1H), 2.68 (dd, J=13.8, 5.1 Hz, 1H), 2.58 (dd, J=13.7, 5.9 Hz, 1H), 2.29 (dd, J=13.7, 3.7 Hz, 1H), 2.23 (dd, J=13.1, 3.9 Hz, 1H), 2.07-1.82 (m, 5H), 1.68-1.23 (m, 17H), 1.22 (s, 6H), 1.05 (s, 9H), 1.03 (d, J=6.7 Hz, 3H), 0.93 (d, J=6.5 Hz, 3H), 0.51 (s, 3H).—13C NMR (CDCl3): δ=142.3, 135.9, 134.9, 133.0, 129.5, 127.5, 123.4, 115.4, 74.4, 71.3, 71.1, 56.5, 56.2, 45.7, 44.4, 44.0, 40.5, 36.4, 36.1, 34.8, 30.1, 29.4, 28.8, 27.6, 27.0, 25.4, 23.4, 22.2, 20.8, 19.4, 18.8, 13.6, 12.0.—MS (m/z, %): 656 (M+, 1), 599 (M+−tBu, 1), 581 (3), 521 (1), 468 (1), 400 (1), 365 (6), 325 (5), 245 (8), 199 (61), 183 (18), 135 (42), 59 (100).
A solution of intermediate 72a (27 mg, 0.041 mmol) in THF (0.5 mL) was treated with tetrabutylammonium fluoride (hereinafter TBAF) (3.5 mL, 1 M in THF). After stirring at room temperature in the dark for 72 hours, the solution was subjected to flash chromatography (using a isooctane/EtOAc 1:1 mixture). The residue was purified by HPLC (using a isooctane/EtOAc 3:2 mixture) affording the vitamin D compound 101 (11 mg, 82%) which was characterized as follows: Rf=0.21 (isooctane/EtOAc 1:1).—IR (KBr): ν=3422, 2946, 1618, 1452, 1376, 1350, 1150, 1056 cm−1.—1H NMR (CDCl3): δ=6.26 (d, J=11.2 Hz, 1H), 5.87 (d, J=11.2 Hz, 1H), 3.51 (td, J=10.1, 4.7 Hz, 1H), 3.08 (dd, J=12.9, 4.0 Hz, 1H), 2.79 (dd, J=12.9, 4.0 Hz, 1H), 2.44 (d, J=13.1 Hz, 1H), 2.37 (m, 1H), 2.04-1.98 (m, 3H), 1.90 (t, J=10.7 Hz, 2H), 1.80-1.23 (m, 18H), 1.22 (s, 6H), 1.14 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.5 Hz, 3H), 0.55 (s, 3H).—MS (m/z, %): 418 (M+, 9), 400 (6), 385 (4), 357 (5), 317 (2), 289 (6), 245 (8), 203 (4), 189 (6), 149 (27), 135 (41), 84 (58), 59 (100).
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Using the same procedure as in example 33, the vitamin D analogue 110 was made and characterized as follows: Rf=0.18 (isooctane:EtOAc, 3:2); [α]Dr.t=+28.0 (c=0.21, CHCl3).—IR (film): ν=3389, 2958, 1454, 1188, 1045c m−1.—1H NMR (CDCl3): δ=6.38 (d, J=11.3 Hz, 1H), 5.83 (d, J=11.3 Hz, 1H), 4.14 (m, 1H), 3.63 (m, 1H), 2.87 (dd, J=13.9, 4.0 Hz, 1H), 2.80 (dd, J=12.7, 4.5 Hz, 1H), 2.61 (dd, J=12.5, 4.1 Hz, 1H), 2.20-1.22 (m, 24H), 1.21 (s, 6H), 1.00 (t, J=7.4 Hz, 3H), 0.91 (d, J=6.7 Hz, 3H), 0.53 (s, 3H).—13C NMR (CDCl3): δ=143.3, 131.5, 124.1, 115.4, 71.7, 71.3, 68.0, 56.7, 56.5, 50.9, 45.9, 45.5, 44.6, 40.6, 36.5, 36.2, 35.7, 30.2, 29.5, 29.1, 27.8, 25.6, 23.6, 22.4, 20.9, 18.9, 12.2, 11.8.—MS (m/z, %): 432 (M+, 10), 414 (6), 371 (2), 303 (5), 267 (6), 245 (8), 208 (6), 173 (10), 149 (30), 133 (40), 81 (65), 55 (100).
Using the same procedure as in example 33, the vitamin D analogue 109 was made and characterized as follows: Rf=0.22 (isooctane/EtOAc 3:2); [α]Dr.t=+42.1 (c=0.32, CHCl3).—IR (film): ν=3378, 2959, 1454, 1378 cm−1.—1H NMR (CDCl3): δ=6.26 (d, J=11.3 Hz, 1H), 5.87 (d, J=11.3 Hz, 1H), 4.09 (m, 1H), 3.54 (m, 1H), 3.10 (dd, J=12.7, 4.3 Hz, 1H), 2.80 (dd, J=12.8, 4.5 Hz, 1H), 2.38 (m, 2H), 2.05-1.25 (m, 25H), 1.22 (S, 6H), 1.00 (t, J=7.4 Hz, 3H), 0.94 (d, J=6.5 Hz, 3H), 0.54 (s, 3H).—13C NMR (CDCl3): δ=143.1, 131.4, 123.5, 115.4, 71.2, 67.7, 56.6, 56.4, 51.5, 45.9, 44.5, 44.2, 40.6, 37.9, 36.5, 36.2, 31.0, 29.5, 29.3, 29.1, 27.8, 23.6, 22.4, 20.9, 20.3, 18.9, 12.2, 11.7.—MS (m/z, %): 432 (M+, 10), 414 (5), 303 (8), 267 (8), 245 (15), 208 (5), 173 (5), 135 (30), 105 (35), 81 (70), 59 (100).
Using the same procedure as in example 33, the vitamin D analogue 106 Was made and characterized as follows: Rf=0.20 (isooctane/EtOAc 1:1); [α]Dr.t=+61.1 (c=0.34, CHCl3).—IR (film): ν=3384, 2960, 1455, 1379, 1147, 1043 cm−1.—1H NMR (CDCl3): δ=6.31 (d, J=11.2 Hz, 1H), 6.01 (d, J=11.2 Hz, 1H), 3.97 (dd, J=5.6, 2.4 Hz, 1H), 3.59 (td, J=9.5, 4.6 Hz, 1H), 2.84 (dd, J=13.9, 4.6 Hz, 1H), 2.59 (dd, J=12.8, 4.4 Hz, 1H), 2.47(dt, J=14.6, 5.1 Hz, 1H), 2.21 (d, J=13.7 Hz, 1H), 2.15-2.08 (m, 2H), 1.88-1.21 (m, 22H), 1.22 (s, 6H), 1.14 (d, J=6.9 Hz, 3H), 0.92 (s, 3H), 0.88 (d, J=6.7 Hz, 3H).—13C NMR (CDCl3): δ=143.4, 131.6, 124.2, 118.5, 72.4, 71.6, 71.1, 57.8, 54.6, 45.3, 44.9, 44.4, 43.9, 37.9, 35.5, 34.5, 34.0, 29.9, 29.4, 29.2, 26.8, 24.8, 22.3, 21.9, 21.7, 19.8, 13.6.—MS (m/z, %): 418 (M+, 1), 400 (M+−H2O, 22), 387 (7), 357 (4), 340 (5), 289 (14), 271 (21), 245 (19), 191 (17), 147 (29), 133 (38), 81 (59), 59 (100).
Using the same procedure as in example 33, the vitamin D analogue 114 was made and characterized as follows: Rf=0.20 (isooctane/EtOAc, 3:2); [α]Dr.t=+34.4 (c=0.48, CHCl3).—IR (film): ν=3372, 2958, 1464, 1378, 1190, 1044 cm−1.—1H NMR (CDCl3): δ=6.32 (d, J=11.3 Hz, 1H), 6.01 (d, J=11.3 Hz, 1H), 4.15 (br.s, 1H), 3.64 (m, 1H), 2.92 (m, 1H), 2.59 (m, 1H), 2.47 (m, 1H), 2.18-2.05 (m, 3H), 1.86-1.24 (m, 23H), 1.22 (s, 6H), 1.00 (t, J=7.4 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H), 0.86 (s, 3H).—13C NMR (CDCl3): δ=143.5, 131.6, 124.2, 118.7, 71.6, 71.2, 68.0, 58.0, 54.7, 51.0, 45.6, 45.4, 44.5, 38.0, 35.7, 34.7, 34.1, 30.0, 29.5, 29.3, 27.0, 24.9, 22.3, 22.0, 21.8, 20.0, 19.8, 11.8.—MS (m/z, %): 432 (M+, 2), 414 (15), 386 (4), 265 (5), 245 (10), 199 (20), 161 (15), 135 (30), 81 (50), 55 (100).
Using the same procedure as in example 33, the vitamin D analogue 105 was made and characterized as follows: Rf=0.18 (isooctane/EtOAc 1:1); [α]Dr.t=+38.7 (c=0.40, CHCl3).—IR (film): ν=3382, 2958, 1455, 1377, 1212, 1045 cm−1.—1H NMR (CDCl3): δ=6.19 (d, J=11.2 Hz, 1H), 6.05 (d, J=11.2 Hz, 1H), 3.90 (dd, J=6.2, 3.0 Hz, 1H), 3.52 (td, J=10.1, 4.6 Hz, 1H), 3.08 (dd, J=12.9, 4.4 Hz, 1H), 2.41-2.48 (m, 2H), 2.33 (dd, J=13.2, 4.2 Hz, 1H), 2.15-2.03 (m, 2H), 1.94-1.80 (m, 2H), 1.72-1.23 (m, 19H), 1.22 (s, 6H), 1.13 (d, J=6.9 Hz, 3H), 0.92 (s, 3H), 0.88 (d, J=6.7 Hz, 3H).—13C NMR (CDCl3): δ=143.2, 131.6, 123.7, 118.6, 71.9, 71.7, 71.1, 57.9, 54.6, 45.4, 44.4, 44.1, 43.9, 37.9, 37.4, 34.5, 34.0, 29.8, 29.4, 29.2, 26.8, 24.8, 22.4, 21.8, 21.6, 19.8, 14.0.—MS (m/z, %): 418 (M+, 1), 401 (M+−H2O+H, 1), 387 (2), 357 (4), 370 (1), 293 (1), 292 (5), 260 (2), 199 (35), 183 (11), 153 (25), 111(28), 93 (100).
Using the same procedure as in example 33, the vitamin D analogue 113 was made and characterized as follows: Rf=0.21 (isooctane/EtOAc 3:2); [α]Dr.t=+17.0 (c=0.15, CHCl3).—IR (film): ν=3369, 2958, 1455, 1378, 1190, 1044 cm−1.—1H NMR (CDCl3): δ=6.19 (1H, d, J=11.2 Hz), 6.06 (d, J=11.3 Hz, 1H), 4.09 (m, 1H), 3.50 (m, 1H), 3.11 (dd, J=12.7, 4.2 Hz, 1H), 2.97 (m, 1H), 2.47 (m, 1H), 2.38 (m, 1H), 2.18-1.25 (m, 25H), 1.22 (s, 6H), 0.99 (t, J=7.4 Hz, 3H), 0.90 (d, J=6.7 Hz, 3H), 0.88 (s, 3H).—MS (m/z, %): 432 (M+, 2), 414 (28), 381 (4), 301 (4), 267 (8), 245 (10), 199 (30), 149 (30), 105 (50), 81 (70), 59 (100).
Using the same procedure as in example 33, the vitamin D analogue 113 was made and characterized as follows: Rf=0.18 (isooctane/EtOAc 1:1); [α]Dr.t=+42.7 (c=0.11, CHCl3).—IR (film): 3368, 2929, 1614, 1454, 1377, 1261, 1166, 1024 cm−1.—1H NMR (CDCl3): δ=6.36 (d, J=11.1 Hz, 1H), 5.82 (d, J=11.1 Hz, 1H), 3.96 (br.s, 1H), 3.61 (m, 1H), 2.80 (d, J=14.1 Hz, 2H), 2.60 (d, J=12.8 Hz, 1H), 2.28-1.52 (m, 17H), 1.51 (s, 6H), 1.38-1.25 (m, 3H), 1.13 (d, J=6.7 Hz, 3H), 1.06 (d, J=6.3 Hz, 3H), 0.54 (s, 3H).—MS (m/z, %): 414 (M+, 14), 396 (M+−H2O, 8), 381 (7), 353 (4), 317 (12), 267 (3), 241 (9), 199 (13), 185 (16), 161 (21), 105 (37), 84 (52), 43 (100).
Using the same procedure as in example 33, the vitamin D analogue 112 was made and characterized as follows: Rf=0.18 (isooctane/EtOAc 3:2); [α]Dr.t=+23.1 (c=0.26, CHCl3).—IR (film): ν=3367, 2958, 2238, 1455, 1378, 1167 cm−1.—1H NMR (CDCl3): δ=6.38 (d, J=11.2 Hz, 1H), 5.83 (d, J=11.1 Hz, 1H), 4.14 (m, 1H), 3.64 (td, J=9.8, 4.8 Hz, 1H), 2.97 (m, 1H), 2.87 (dd, J=13.9, 4.3 Hz, 1H), 2.80 (dd, J=12.8, 4.5 Hz, 1H), 2.30-1.51 (m, 17H), −1.50 (s, 6H), 1.46-1.25 (m, 5H), 1.06 (d, J=6.6 Hz, 3H), 0.98 (t, J=7.4 Hz, 3H), 0.54 (s, 3H).—MS (m/z, %): 428 (M+, 2), 410 (10), 370 (8), 331 (5), 313 (8), 295 (4), 241 (3), 199 (8), 161 (20), 149 (40), 91 (40), 43 (100).
Using the same procedure as in example 33, the vitamin 0 analogue 113 was made and characterized as follows: Rf=0.18 (isooctane/EtOAc 1:1); [α]Dr.t=+28.2 (c=0.37, CHCl3).—IR (film): ν=3380, 2930, 1455, 1377, 1346, 1166, 1041 cm−1.—1H NMR (CDCl3): δ=6.44 (d, J=11.3 Hz, 1H), 6.04 (d. J=11.3 Hz, 1H), 4.07 (dd, J=4.1, 3.0 Hz, 1H), 3.68 (td, J=10.1, 4.8 Hz, 1H), 3.24 (dd, J=12.9, 3.8 Hz, 1H), 2.97 (dd, J=12.9, 4.4 Hz, 1H), 2.60 (d, J=3.6 Hz, 1H), 2.51 (dd, J=13.8, 3.4 Hz, 1H), 2.44 (dd, J=16.6, 3.4 Hz, 1H), 2.26-2.16 (m, 5H), 2.07 (m, 2H), 1.68 (s, 6H), 1.88-1.66 (m, 8H), 1.52-1.41 (m, 3H), 1.31 (d, J=6.8 Hz, 3H), 1.24 (d, J=6.5 Hz, 3H), 0.72 (s, 3H).—MS (m/z, %): 414 (M+, 18), 396 (M+−H2O, 8), 376 (7), 356 (4), 353 (1), 317 (15), 267 (4), 241 (9), 199 (2.1), 173 (23), 161 (25), 105 (42), 91 (53), 43 (100).
Using the same procedure as in example 33, the vitamin D analogue 112 was made and characterized as follows: Rf=0.19 (isooctane/EtOAc 3:2); [α]Dr.t=+24.9 (c=0.54, CHCl3).—IR (film): 3378, 2930, 1454, 1166, 1039 cm−1.—1H NMR (CDCl3): δ=6.26 (d, J=11.2 Hz, 1H), 5.87 (d, J=11.2 Hz, 1H), 4.10 (m, 1H), 3.55 (m, 1H), 3.10 (dd, J=12.9, 4.1 Hz, 1H), 2.80 (dd, J=12.5, 4.2 Hz, 1H), 2.40-1.53 (m, 18H), 1.52 (s, 6H), 1.50-1.24 (m, 5H), 1.07 (d, J=6.5 Hz, 3H), 1.00 (t, J=7.4 Hz, 3H), 0.56 (s, 3H).—13C NMR (CDCl3): δ=142.6, 131.6, 123.4, 115.5, 86.1, 81.3, 71.2, 67.6, 65.4, 56.3, 55.7, 51.0, 45.7, 44.1, 40.4, 37.9, 36.0, 31.9, 30.1, 29.0, 27.7, 25.7, 23.5, 22.3, 20.2, 19.2, 12.2, 11.6.—MS (m/z, %): 428 (M+, 10), 410 (2), 370 (4), 331 (3), 267 (3), 241 (4), 199 (8), 173 (8), 149 (20), 105 (30), 91 (45), 43 (100).
Using the same procedure as in example 33, the vitamin D analogue 108 was made and characterized as follows: Rf=0.19 (isooctane/EtOAc 1:1); [α]Dr.t=+55.1 (c=0.11, CHCl3).—IR (film): ν=3362, 2959, 2929, 1450, 1376, 1329, 1243, 1175, 1127 cm−1.—1H NMR (CDCl3): δ=6.30 (d, J=11.3 Hz, 1H), 6.02 (d, J=11.3 Hz, 1H), 3.98 (m, 1H), 3.60 (td, J=9.4, 4.6 Hz, 1H), 2.83 (dd, J=13.9, 4.6 Hz, 1H), 2.59 (dd, J=12.8, 4.3 Hz, 1H), 2.41 (dt, J=12.5, 3.8 Hz, 1H), 2.27-1.99 (m, 5H), 1.87 (m, 1H), 1.76-1.52 (m, 11H), 1.49 (s, 6H), 1.33-1.25 (m, 3H), 1.13 (d, J=6.7 Hz, 3H), 1.02 (d, J=6.5 Hz, 3H), 0.95 (s, 3H).—13C NMR (CDCl3): δ=142.9, 131.9, 124.1, 118.5, 86.1, 81.7, 72.5, 71.6, 65.4, 57.7, 52.6, 44.9, 44.9, 43.9, 37.7, 35.5, 33.9, 31.7, 31.7, 29.4, 27.9, 25.2, 24.5, 22.5, 21.8, 20.0, 13.6.—MS (m/z, %): 653 (M++H, 1), 634 (M+−H2O, 5), 597 (M+−57+H, 2), 459 (1), 385 (4), 361 (3), 3.35 (3), 267 (4), 199 (90), 183 (38), 135 (75), 43 (100).
Using the same procedure as in example 33, the vitamin D analogue 116 was made and characterized as follows: Rf=0.24 (isooctane/EtOAc 3:2); [α]Dr.t=+22.3 (c=0.38, CHCl3).—IR (film): ν=3370, 2958, 2874, 2233, 1731, 1614, 1462, 1378, 1337, 1240, 1167 cm−1.—1H NMR (CDCl3): δ=6.31 (d, J=11.3 Hz, 1H), 6.03 (d, J=11.3 Hz, 1H), 4.15 (m, 1H), 3.64 (m, 1H), 2.89 (m, 1H), 2.60 (dd, J=12.8, 4.3 Hz, 1H), 2.45-1.51 (m, 18H), 1.50 (s, 6H), 1.40-1.25 (m, 5H), 1.02 (t, J=7.4 Hz, 3H), 0.91 (d, J=6.9 Hz, 3H), 0.88 (s, 3H).—13C NMR (CDCl3): δ=143.1, 132.1, 124.2, 118.7, 86.2, 81.8, 71.7, 68.0, 57.9, 53.3, 52.8, 51.0, 45.6, 45.1, 37.9, 35.8, 34.2, 31.9, 30.2, 29.5, 28.2, 25.6, 25.3, 24.7, 22.7, 21.9, 20.2, 11.9.—MS (m/z, %): 428 (M+, 2), 410 (M+−18, 8), 370 (5), 313 (5), 277 (6), 199 (30), 149 (35), 142 (30), 91 (50), 43 (100).
Using the same procedure as in example 33, the vitamin D analogue 107 was made and characterized as follows: Rf=0.18 (isooctane/EtOAc 1:1); [α]Dr.t=+43.8 (c=0.21, CHCl3).—IR (film): ν=3358, 2929, 1455, 1377, 1338, 1239, 1166 cm−1.—1H NMR (CDCl3): δ=6.19 (d, J=11.4 Hz, 1H), 6.06 (d, J=11.4 Hz, 1H), 3.89 (dd, J=6.3, 3.2 Hz, 1H), 3.54 (td, J=10.1, 4.6 Hz, 1H), 3.07 (dd, J=12.9, 4.2 Hz, 1H), ), 3.01 (m, 1H), 2.46-2.31 (m, 3H), 2.23 (dd, J=16.7, 3.5 Hz, 2H), 2.15-2.01 (m, 3H), 1.94-1.86 (m, 2H), 1.78-1.50 (m, 8H), 1.49 (s, 6H), 1.35-1.25 (m, 3H), 1.14 (d, J=6.7 Hz, 3H), 1.03 (d, J=6.6 Hz, 3H), 0.88 (s, 3H).—MS (m/z, %): 652 (M+, 1), 634 (M+−H2O+1, 6), 594 (M+−57+H, 2), 537 (3), 459 (2), 396 (1), 378 (4), 321 (5), 261 (6), 199 (100), 183 (27), 135 (72).
Using the same procedure as in example 33, the vitamin D analogue 115 was made and characterized as follows: Rf=0.22 (isooctane/EtOAc 3:2); [α]Dr.t=+8.9 (c=0.69, CHCl3).—IR (film): ν=3381, 2958, 2233, 1454, 1383, 1166 cm−1.—1H NMR (CDCl3): δ=6.19 (d, J=11.3 Hz, 1H), 6.07 (d, J=11.3 Hz, 1H), 4.10 (m, 1H), 3.57 (m, 1H), 3.10 (dd, J=12.8, 4.3 Hz, 1H), 2.97 (m, 1H), 2.38 (m, 2H), 2.22 (m, 2H), 2.15-2.00 (m, 3H), 1.94-1.86 (m, 2H), 1.84-1.51 (m, 9H), 1.50 (s, 6H), 1.45-1.24 (m, 5H), 0.98 (t, J=7.5 Hz, 3H), 0.90 (d, J=6.7 Hz, 3H), 0.88 (s, 3H).—13C NMR (CDCl3): δ=142.8, 132.1, 123.5, 118.3, 86.3, 81.6, 71.0, 67.7, 57.8, 52.0, 51.0, 45.1, 44.1, 37.9, 37.8, 34.0, 31.9, 31.7, 30.1, 28.8, 28.2, 25.5, 24.8, 22.6, 22.0, 20.2, 19.9, 11.6.—MS (m/z, %): 428 (M+, 2), 410 (6), 313 (4), 277 (4), 241 (4), 199 (15), 173 (10), 149 (30), 105 (25), 91 (40), 43 (100).
Using the same procedure as in example 33, this vitamin D analogue was made and characterized as follows: Rf=0.21 (isooctane/EtOAc 3:2).—[α]Dr.t+53.11 (c=0.31, CHCl3).—IR (film)=3345, 2928, 1455, 1362, 1232, 1167, 1103, 1067, 1038, 944, 874, 750 cm−1.—1H NMR (500 MHz, CDCl3): □=6.31 (d, J=11.3 Hz, 1H), 6.08 (d, J=11.3 Hz, 1H), 3.95 (d, J=2.1 Hz, 1H), 3.90 (d, J=2.9 Hz, 1H), 3.05 (d, J=14.1 Hz, 1H), 2.49-2.40 (m, 4H), 2.26-1.50 (m, 15H), 1.49 (s, 6H), 1.31-1.23 m, 3H), 1.19 (d, J=7.2 Hz, 3H), 1.01 (d, J=6.7 Hz, 3H), 0.96 (s, 3H).—13C NMR (50 Mhz, CDCl3): □=142.2, 129.8, 125.0, 118.7, 86.0, 81.8, 73.3, 73.1, 65.4, 57.4, 52.6, 44.9, 44.9, 38.9, 37.6, 36.5, 33.9, 31.7, 31.7, 29.3, 27.8, 25.1, 24.4, 22.4, 21.8, 20.1, 14.5.—MS (m/z, %): 414 (M+, 1), 396, (M+−H2O, 9), 381 (5), 363 (7), 356 (4), 299 (7), 267 (8), 241 (9), 213 (12), 185 (14), 147 (21), 107 (21), 107 (26), 91 (45), 43 (100).
Using the same procedure as in example 33, this vitamin D analogue was made and characterized as follows: Rf=0.21 (isooctane/EtOAc 4:1); [α]Dr.t+20.42 (c=0.31, CHCl3).—IR (film)=3353, 2930, 1611, 1455, 1376, 1169, 1070, 1028, 988, 948, 882, 729 cm−1.—1H NMR (500 MHz, CDCl3): □=6.32 (d, J=11.3 Hz, 1H), 6.08 (d, J=11.3 Hz, 1H), 3.96 (br.s, 1H), 3.90 (br.s, 1H), 3.06 (dd, J=14.1, 3.2 Hz, 1H), 2.49-2.38 (m, 3H), 2.25-1.50(m, 16H), 1.49 (s, 6H), 1.32-1.24 (m, 3H), 1.18 (d, J=7.2 Hz, 3H), 1.02 (d, J=6.6 Hz, 3H), 0.95 (s, 3H).—MS (m/z, %): 396 (M+−H2O, 24), 378 (8), 335 (2), 299 (13), 267 (12), 241 (13), 199 (18), 185 (20), 145 (21), 105 (51), 91 (74), 43 (100).
a) Affinity for Vitamin D Receptor (VDR)
The methods used to evaluate the binding properties of the new analogues are examples of the state of the art techniques used for steroid hormone (including vitamin D) binding assays as described previously (Verstuyf A. et al. J Bone Mineral Res 13: 549-558, 1998).
The affinity of the C2-substituted analogues of 1α,25(OH)2D3 to the vitamin D receptor was evaluated by their ability to compete with [3H]1α,25(OH)2D3 for binding to high speed supernatant from intestinal mucosa homogenates obtained from normal pigs. The incubation was performed at 4° C. for 20 h and phase separation was obtained by addition of dextran-coated charcoal. The relative affinity of the analogues was calculated from their concentration needed to displace 50% of [3H]1α,25(OH)2D3 from its receptor compared with the activity of 1α,25(OH)2D3 (assigned a value of 100%).
Results
All the 19-nor-1α,25(OH)2D3 analogues (with the natural or 23-yne side chain; with or without 14-epimerisation) with a 2α-methyl (102, 104, 106, 108, 117) or 2α-ethyl (110, 112, 114, 116) substituted A-ring possess higher affinity for the vitamin D receptor compared to their 2β-methyl (101, 103, 105, 107, 118) or 2β-ethyl (109, 111, 113, 115) counterparts (Table 1 and 2).
The C2-methyl substituted 19-nor-1α,25(OH)2D3 analogues (101-108, 141) have mostly a higher binding affinity for the VDR than their C2-ethyl substituted 19-nor-1α,25(OH)2D3 counterparts (109-116, 142).
The binding affinity for the VDR is always higher for the 2α-methyl analogues with the natural side chain of 1α,25(OH)2D3 (102,106) compared to their 2α-methyl counterparts with the 23-yne side chain (104,108). This observation is even more pronounced for the 2β-methyl analogues with the natural side chain of 1α,25(OH)2D3 (101,105) compared to the 2β-methyl analogues with the 23-yne side chain (103,107). The introduction of the 23-yne side chain in the 2α-ethyl (112) or 2β-ethyl (111) analogues also decreased the affinity of VDR compared to the 2α-ethyl (110) or 2β-ethyl (109) analogues with the natural side chain of 1α,25(OH)2D3.
The 2α-methyl-1α,25(OH)2D3 analogue with (106) or without 14-epimerisation (102) together with the 2α-ethyl-1α,25(OH)2D3 analogue (110) displayed the highest affinity for the vitamin D receptor [90% compared to 1α,25(OH)2D3 (=100% binding)].
b) Affinity for Human DBP
Binding of 1α,25(OH)2D3 analogues to hDBP was performed at 4° C. essentially as described previously [18]. [3H]1α,25(OH)2D3 and 1α,25(OH)2D3 or its analogues were added in 5 μl ethanol into glass tubes and incubated with hDBP (0.18 μM) in a final volume of 1 ml (0.01 M Tris-HCl buffer and 0.154 M NaCl, pH 7.4) for 3 h at 4° C. Phase separation was then obtained by the addition of 0.5 ml of cold dextran-coated charcoal.
Results (Table 1 and 2)
All the investigated C2 substituted analogues have a binding affinity for DBP equivalent or lower than 10% compared to 1α,25(OH)2D3 (=100% affinity), except for compounds 114, 102 and 110 demonstrating 40, 50 and 20% to affinity, respectively.
The 2α-methyl (102, 104, 106, 108) or 2α-ethyl (110, 112, 114) 19-nor-1α,25(OH)2D3 analogues have higher affinity for DBP compared to their 20-methyl (101, 103, 105, 107) or 2β-ethyl (109, 111, 113) counterparts.
The C2-methyl substituted 19-nor-1α,25(OH)2D3 analogues have always a higher binding affinity for DBP than their C2-ethyl substituted 19-nor-1α,25(OH)2D3 counterparts, except for 2α-methyl-14-epi-19-nor-1α,25(OH)2D3 [9% affinity compared to 1α,25(OH)2D3 (=100% binding), compound 106] versus 2α-ethyl-14-epi-19-nor-1α,25(OH)2D3 [40% affinity compared to 1α,25(OH)2D3 (=100% binding), compound 114].
The binding affinity for DBP is always higher for the 2α-methyl or 2α-ethyl analogues with the natural side chain of 1α,25(OH)2D3 compared to their 2α-methyl respectively 2α-ethyl counterparts with the 23-yne side chain.
When the trans oriented C14 hydrogen of the analogues is changed into a cis oriented C14 hydrogen the affinity for DBP decreased except for compound 114.
a) Breast Carcinoma Cells MCF-7
To evaluate the effect of on cell proliferation, malignant MCF-7 cells were cultured in DMEM/nut. mix. F12 (HAM) medium supplemented with 10% heat inactivated FCS, glutamine (2 mM), penicillin (100 units/ml) and streptomycin (0.1 mg/ml). Cultures were maintained at 37° C. in a humidified atmosphere of 5% CO2 in air. MCF-7 cells were seeded at 5×103 cells/well in the above described medium in 96-well microtiter plates in a final volume of 0.2 ml per well. Triplicate cultures were performed. After 24 h, 1α,25(OH)2D3 or analogues were added in the appropriate concentrations for an incubation period of 72 h. Then, 1 μCi [3H]thymidine was added to each well and cells were harvested after 4 h incubation with a Packard harvester and measured by the Packard Topcount System (Packard, Meriden, USA).
b) Promyelocytic Leukemia Cells HL-60
To evaluate the effect on cell differentiation HL-60 cells were seeded at 4×104 cells/ml in 25 cm2 Falcon tissue chambers using RPMI 1640 medium supplemented with 20% FCS and gentamycin (50 μg/ml) in a final volume of 5 ml. Cultures were maintained at 37° C. in a humidified atmosphere of 5% CO2 in air. One day later, 1α,25(OH)2D3 or analogues were added to the cell culture in ethanol (final concentration<0.2%). After 4 days of culture, the dishes were shaken to lose adherent cells. Cells were washed twice in RPMI, counted and assayed for differentiation markers (NBT reduction assay). Superoxide production was measured as NBT reducing activity as described previously (Ostrem V. K et al. Proc Natl Acad Sci USA 84: 2610-2614, 1987). HL-60 cells at 1×106/ml were mixed with an equal volume of freshly prepared solution of phorbol 12-myristate 13-acetate (200 ng/ml) and NBT (2 μg/ml) and incubated for 30 min at 37° C. The percentage of cells containing black formazan deposits was determined using a hemacytometer.
Results (Table 1 and 2;
Of all the investigated analogues compound 108 was the most potent analogue to induce cell differentiation (40 times that of 1α,25(OH)2D3) and to inhibit cell proliferation [120 times that of 1α,25(OH)2D3]. As described above the intrinsic activity of the 19-nor-14-epi-23-yne-1α,25(OH)2D3 analogue (SDB 112/TX522) increased strongly when it was substituted with a 2α-methyl A-ring (compound 108) but when TX 522 was substituted with a 2α-ethyl A-ring (116) the biological profile was not further enhanced. On the other hand the introduction of a 2β-methyl (107) or 2β-ethyl (115) A-ring in TX 522 decreased its in vitro activity. 3-Epimerisation (117) of compound 108 markedly decreased the prodifferentiative activity and 20-epimerisation (141) of compound 108 strongly decreased the inhibition of MCF-7 cell proliferation. 1-Epimerisation (118) of compound 107 was not active at all.
The 2β-methyl-19-nor analogue with the natural side chain of 1α,25(OH)2D3 (101) was less potent than 1α,25(OH)2D3 to induce differentiation or inhibit proliferation. The introduction of 2β-methyl (105) in the 14-epi-19-nor-1α,25(OH)2D3 analog increased the prodifferentiating effects on HL 60 cells but decreased the antiproliferative effects on MCF-7 cells. The opposite was seen when 2β-ethyl (113) was introduced in 14-epi-19-nor-1α,25(OH)2D3. The 2β-methyl-19-nor analogue with the 23-yne side chain (103) and its 14-epi counterpart (107) were 3 to 9 times more potent than 1α,25(OH)2D3.
The substitution of a 2α-ethyl group in place of a 2α-methyl group on the A-ring of 19-nor-1α,25(OH)2D3 (110) decreased its prodifferentiating activity on HL60 cells. Nevertheless its activity was still 2 to 6 times more potent than 1α,25(OH)2D3. The 2β-ethyl counterpart of compound 24 (109) lost potency and was less active than 1α,25(OH)2D3. The introduction of the 23-yne side chain in the analogue 2α-ethyl-19-nor-1α,25(OH)2D3 (112) enhanced the antiproliferative (3-fold) effects on MCF-7 cells. The 2β-ethyl-19-nor-23-yne-1α,25(OH)2D3 (111) was again less active than 2α-ethyl-19-nor-23-yne-1α,25(OH)2D3 (112).
Eight weeks old, male NMRI mice were obtained from the Proefdierencentrum of Leuven (Belgium) and fed a vitamin D-replete diet (0.2% calcium, 1% phosphate, 2000 U vitamin D/kg; Hope Farms, Woerden, The Netherlands). Groups of six mice were subcutaneously injected daily during 7 consecutive days with different doses of 1α,25(OH)2D3 (0.1, 0.2 and 0.4 μg/kg/day) or analogues. The control group was injected with vehicle (arachis oil). The average weight of each group of 6 mice was determined at the beginning and at the end of the experiment. The following parameters were evaluated: serum calcium, serum osteocalcin, femur calcium and urinary calcium. Serum and urinary calcium were measured by a microcolorimetric assay (Sigma, St. Louis, Mo.). Femurs were removed and femur calcium content was measured in HCl-dissolved bone ash (obtained by heating for 24 h in an oven at 100° C.), using the same technique as for serum and urinary calcium. Serum osteocalcin was determined by an in-house radioimmunoassay that used mouse osteocalcin as standard and a polyclonal guinea pig anti-mouse osteocalcin antiserum (Bouillon R. et al. Clin Chem 38:2055-60, 1992).
Results (Table 13)
The 2α-methyl-19-nor analogue with the natural side chain of 1α,25(OH)2D3 (102) was 2.5 times less calcemic than 1α,25(OH)2D3. Further reduction of calcemic activity (80-fold) could be obtained by introduction of a 2β-methyl A-ring (101). The introduction of the 23-yne side chain (104) or 14-epimerisation (106) or a combination of both (108) reduced the calcemic activity of 2α-methyl-19-nor-1α,25(OH)2D3 (102). The calcemic effects of 2β-methyl-19-nor-1α,25(OH)2D3 was not further reduced by introduction of unsaturation into the side chain (23-yne, 103), 14-epimerisation (105) or a combination of both (107).
The substitution of a 2α-ethyl group in the A-ring of 19-nor-1α,25(OH)2D3 (110) was 7.6 times less calcemic than 1α,25(OH)2D3 and its 2β counterpart (109) further decreased the calcemic activity 16-fold. Again the introduction of the 23-yne side chain (112) or 14-epimerisation (114) or a combination of both (116) reduced the calcemic effects of 2α-ethyl-19-nor-1α,25(OH)2D3 (110). Only the combination of 14-epimerisation with the 23-yne side chain (115) reduced the calcemic effects of 2β-ethyl-19-nor-1α,25(OH)2D3 (109). Some of these novel compounds exhibited a very interesting pattern of biological activity with low effects on serum calcium levels together with anabolic actions on bone. For example, the mice treated during 7 consecutive days (i.p. 1 μg/kg/d) with the analogue 2α-methyl-14-epi-19-nor-23-yne-1α,25(OH)2D3 (108) have low serum calcium levels compared to the mice treated with 1,25(OH)2D3 (0.1; 0.2; 0.4 μg/kg/d; Table 3). Beside little or no effect on serum calcium levels this compound at a dose of 1 μg/kg/d increased calcium levels in bone (10% increase compared to vehicle treated mice and 18%, 14%, 20% increase compared to 0.1, 0.2 and 0.4 μg/kg/d 1,25(OH)2D3 respectively; Table 3). Because of their preferential activity on bone; these compounds are ideal candidates for the treatment of bone disorders such as osteoporosis, osteomalacia and renal osteodystrophy.
Table 4 represents the selectivity profile of the most potent C2-substituted 19-nor-1α,25(OH)2D3 analogues based on data obtained in vitro on MCF-7 cells compared with their actual in vivo calcemic effects in mice (serum calcium levels after 7 days treatment). All activities are calculated as percent activity compared with 1α,25(OH)2D3. The selectivity profile of is therefore 1. Although the C2α-methyl substituted 14-epi-19-nor-1α,25(OH)2D3 analogue with the 23 yne side chain (108) has the most potent intrinsic effect on cell differentiation and proliferation, the C2-ethyl substituted (2α as well as 2β) 14-epi-19-nor-23-yne-1α,25(OH)2D3 analogues (116 and 115) show the best dissociation ratio between antiproliferative and calcemic effects. The selectivity profile of the analogues 116, 115 and 108′ exceeds several fold that of the best analogues of 1α,25(OH)2D3 yet published when measured with the same methods in the same laboratory (Table 4) and such analogues might be useful in the treatment of hyperproliferative disorders (cancer, psoriasis) and autoimmune diseases.
A) Primary Prevention of Osteoporosis by Vitamin D Analogues
12 week old C3H female mice are subjected to bilateral ovariectomy or sham surgery. The animals are treated with the analog or vehicle by oral gavage or intraperitoneally. Dosing is started 3 days after sugery and continued for 8-9 weeks.
Before the first treatment in vivo measurements are performed to determine bone mineral density (BMD), bone mineral content (BMC) of total body and spine by dual-energy X-ray absorptiometry (DXA). Urine and serum is collected to measure calcium levels together with collagen cross-links in urine and osteocalcin in serum. The animals are weighed regularly during the experimental period. After 4 weeks treatment urine and serum is again collected and biochemical parameters are determined. At the end of the experiment (8-9 weeks) urine is collected and DXA measurement is performed in vivo to determine BMD and BMC. After killing the animals tibiae and femora are dissected The following biochemical parameters are investigated: serum calcium, serum osteocalcin, urine calcium, urine collagen cross-links, femur calcium.
Tibiae are used for histomorphometric analysis and femurs for measurement of cortical and trabecular volumetric density and geometry by peripheral quantitative computed tomography (pQCT) ex vivo.
In vivo administration of the analogues of the present invention, more particularly of compound 108 results in an increase in BMD, BMC, and femur calcium while serum calcium or urinary calcium levels are not significantly increased after at least four weeks, more particularly after 8 weeks of treatment.
B) Secondary Prevention or Treatment of Osteoporosis by Vitamin D Analogues
12 week old C3H female mice are subjected to bilateral ovariectomy or sham surgery. The animals are treated with the analog or vehicle by oral gavage or intraperitoneally. Dosing is started 4 weeks after sugery and continued for 4-10 weeks.
Before the first treatment in vivo measurements are performed to determine bone mineral density (BMD), bone mineral content (BMC) of total body and spine by dual-energy, X-ray absorptiometry (DXA). Urine and serum is collected to measure calcium levels together with collagen cross-links in urine and osteocalcin in serum. The animals are weighed regularly during the experimental period. After 4 weeks treatment urine and serum is again collected and biochemical parameters are determined and urine is collected and DXA measurement is performed in vivo to determine BMD and BMC. After killing the animals tibiae and femora are dissected The following biochemical parameters are investigated: serum calcium, serum osteocalcin, urine calcium, urine collagen cross-links, femur calcium.
Tibiae are used for histomorphometric analysis and femurs for measurement of cortical and trabecular volumetric density and geometry by peripheral quantitative computed tomography (pQCT) ex vivo.
In vivo administration of the analogues of the present invention, more particularly of compound 108 results in an increase in BMD, BMC and femur calcium concentration as compared to the levels observed prior to treatment while serum calcium or urinary calcium levels are not significantly increased after four weeks, more particularly after 8 weeks of treatment. More particularly it is observed that the levels of BMD, BMC, and femur calcium are higher than the levels of sham operated control animals while serum calcium or urinary calcium levels are not significantly higher.
Significantly different from vehicle treated group (p < 0.001)*
Significantly different from 1,25(OH)2D3 treated group at different doses (p < 0.001)$
All results are expressed as percentage activity (at 50% of the dose response) with 1α,25(OH)2D3 (=100% activity). The antiproliferative action of 1α,25(OH)2D3 on MCF-7 breast cancer cells was measured by [3H]thymidine incorporation. The calcemic effects (serum calcium levels) were determined in mice by a daily intraperitoneal injection of 1α,25(OH)2D3 or analogues during 7 consecutive days.
*Data published in J Bone Mineral Res 15 (2): 237-252 (2000).
| Number | Date | Country | Kind |
|---|---|---|---|
| 03053329 | Mar 2003 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/BE04/00037 | 3/10/2004 | WO | 9/9/2005 |
