AZT derivatives exhibiting spermicidal and anti-viral activity

FIELD OF THE INVENTION

[0002] The present invention is directed to the use of AZT derivatives and its analogs in providing contraceptive, e.g. spermicidal, effects. In one particular embodiment, the present invention is directed to novel dual-function derivatives of AZT that exhibit spermicidal activity and maintain potent anti-HIV activity.

BACKGROUND OF THE INVENTION

[0003] The known spermicidal agents, nonoxynol-9 (N9) and gramicidin, exert their effects via a detergent-like ability to damage the sperm plasma membrane, perturb its conformation and destroy its semi-permeable nature thereby impairing the sperm motility and egg fertilizing functions (Wilborn, et al., Fertil Steril 1983; 39:717-719; Bourinbaiar, et al., Life Sci 1994; 54:PL 5-9). Because of their non-specific membrane disruptive properties, such vaginal spermicides have been shown to damage the cervicovaginal epithelium, as well, which may lead to a lower degree of protection from sexually transmitted diseases (Niruthisard, et al., Sex Transm Dis 1991; 18:176-179). A vaginal contraceptive that does not function with the non-specific membrane toxicity mediated by detergent-type action of the currently available vaginal contraceptives would be desirable.

[0004] In addition, the dangers of sexually-transmitted diseases have been widely recognized. However, despite the educational and preventive measures taken to date, the spread of diseases such as HIV remains a serious health problem. It would be desirable to provide a contraceptive that also provides some protection against sexually-transmitted diseases such as HIV, especially to reduce the risks for women who otherwise would be at high risk of acquiring such diseases by heterosexual transmission.

SUMMARY OF THE INVENTION

[0005] While AZT (3′-azido-3′-deoxythymidine; also known as zidovudine, or ZDV) has been widely studied for its activity against HIV infection, AZT itself has not been recognized as having any contraceptive, for example spermicidal and/or sperm-immobilizing, activity. The present invention provides AZT derivatives that are useful as contraceptive agents and products and methods using them. Examples of such products include vaginal foams, creams, lotions or gels, sponges or other vaginal inserts, and condom lubricating compositions. The present invention also is directed to certain AZT derivatives that exhibit the contraceptive properties while maintaining activity against HIV.

[0006] One aspect of the present invention is directed to AZT derivatives that include 5-halo and 6-alkoxy substitution on the thymine ring of AZT and exhibit contraceptive properties.

[0007] Some embodiments of the present invention are directed to providing a contraceptive effect using novel dual-function AZT derivatives with both anti-HIV and spermicidal activity that include 5-halo and 6-alkoxy substitution on the thymine ring of AZT and substitution on the pentose ring of AZT that facilitates entry of the compound into a cell. For example, among others, the substitution on the pentose ring can be with a phosphate group, which may be further substituted with an aryl-containing group, and the aryl group itself can be further substituted. In the above aspects of the invention, the azide group on the pentose ring of AZT can be replaced with NH2, which optionally can be substituted, or the azide group could be replaced with halo, CN, or COOH.

[0008] Some embodiments are directed to contraceptive products that use a spermicidal AZT derivative as an active agent, and to the production of such contraceptive products.

[0009] Some embodiments are directed to a method of contraception that includes a step of contacting sperm with a spermicidal AZT derivative, for example by means of a contraceptive product of this invention as discussed above.

[0010] Some embodiments include a compound of the following formula:

[0011] where:

[0012] R1 is N3, NH2, NH—CH3, NH—COCH3, NH—Ph, NH—COPh, or NH—CH2—Ph;

[0013] R2 is halogen;

[0014] R3 is C1-3 alkoxy;

[0015] R4, R5, and R6 are the same or different and are selected from hydrogen, alkyl, aryl, alkoxy, halo, haloalkyl, arylalkyl, aralkoxy, haloalkoxy, nitro, cyano, and animo; and

[0016] R″ is an amino acid residue;

[0017] or a pharmaceutically acceptable salt or ester thereof.

[0018] Some embodiments include a method of inhibiting conception in a mammal, comprising contacting mammalian sperm with an effective spermicidal amount of a compound of the above formula. Other embodiments include a method of inhibiting virus replication in a cell infected with virus, the method comprising administering to the infected cell a virus replication inhibiting amount of a compound of the above formula. Yet other embodiments include a pharmaceutical composition, comprising an effective spermicidal amount or antiviral amount of a compound of the above formula.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]
FIGS. 1A and 1B illustrate the chemical structures of AZT and examples of certain AZT derivatives of the present invention and comparative derivatives.

[0020]
FIGS. 2A and 2B are graphs showing the effects of AZT and various AZT derivatives on sperm motility.

[0021]
FIG. 3 shows the effects of various AZT derivatives on sperm plasma membrane and acrosomal membrane integrity.

[0022]
FIG. 4A shows scanning laser confocal microscopy images of sperm-zona binding for control sperm treated with various AZT derivatives of the present invention.

[0023]
FIG. 4B is a table showing the dose dependent effect of some aryl phosphate derivatives of AZT on the binding and penetration ability of sperm.

[0024] FIGS. 5A-D illustrate the reduction in inflammation of cervicovaginal tissues from an AZT derivative of the present invention versus N9.

[0025]
FIG. 5E is a table showing the results of experiments showing the reduction in inflamation of cervicovaginal tissue and disruption of the epithelial lining from an AZT derivative of the present invention versed N9.

[0026] FIGS. 6A-C are graphs showing the concentration dependent inhibition of sperm motility by Ala (A), Trp (B), and Pro (C)-containing amino acid esters of aryl phosphate derivatives of bromo-methoxy ZDV with (Br, Cl, F, OMe or NO2) or without (H) substitution on the C-5 position of the phenyl moiety. Highly motile fractions of sperm were incubated with increasing concentrations (1.9 μM to 1000 μM) of listed compounds or 0.5% DMSO in assay medium, and the percentage of motile sperm was evaluated by CASA. Each data point represent the mean ±SD from three independent experiments.

[0027] FIGS. 7A-C are graphs showing the concentration-dependent effect of Ala (A), Trp (B), and Pro (C) containing unsubstituted analogues of WHI-07 on sperm motion parameters. Highly motile fraction of sperm were incubated with increasing concentrations of WHI-07 analogues or 0.5% DMSO in the assay medium, and their effects on progressive motility (PRG MOT), curvilinear velocity (VCL), average path velocity (VAP), and straight line velocity (VSL) were evaluated by CASA. Each data point represents the mean ±SD from two independent experiments. Values: percentage for progressive motility; lm/sec for VCL, VAP, and VSL.

[0028]
FIGS. 8A and B are Scatter diagrams showing the absence of correlation between EC50 values (μM) of 14 spermicidal WHI-07 analogues determined by CASA and their cytotoxicity IC50 values (μM) determined by MTT assays against normal human ectocervical (A) and endocervical (B) epithelial cells.

DETAILED DESCRIPTION

[0029] Terms and Definitions:

[0030] The term “aryl” refers to monovalent unsaturated aromatic carbocyclic radicals having a single ring, such as phenyl, or multiple condensed rings, such as naphthyl or anthryl, which can be optionally substituted by substituents such as halogen, alkyl, arylalkyl, alkoxy, aralkoxy, and the like.

[0031] The term “alkyl’ refers to straight or branched hydrocarbon radicals, such as methyl, ethyl, propyl, butyl, octyl, isopropyl, tert-butyl, sec-pentyl, and the like. Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g., halogen, alkoxy, aryl, arylalkyl, aralkoxy, NO2, CN, OCN, SeCN, SCN, N3, OH, SH, amine and the like. Typically, alkyl groups include 1 to 8 carbon atoms, preferably 1 to 5, and more preferably 1 to 3 carbon atoms.

[0032] The term “alkoxy” refers to an oxygen atom substituted with an alkyl radical as defined above. Typical alkoxy groups include methoxy, ethoxy, propoxy, iopropoxy, and the like. Preferable alkoxy groups include methoxy and ethoxy. Alkoxy can be substituted with one or more substituents, e.g., halogen, alkyl, aryl, arylalkyl, aralkoxy, NO2, CN, OCN, SeCN, SCN, N3, OH, SH, amine and the like.

[0033] The term “halo” refers to fluoride, chloride, bromide, and iodide radicals.

[0034] The term “haloalkyl” refers to an alkyl group substituted with a halo radical as defined above.

[0035] The term “arylalkyl” refers to an alkyl radical defined as above substituted with an aryl radical as defined above. Typical arylalkyl groups include phenethyl, benzyl, and naphthethyl. Preferable alylalkyl groups include phenethyl and benzyl.

[0036] The term “aralkoxy” refers to an alkoxy group as defined above where the alkoxy group is substituted with an aryl radical as defined above.

[0037] The term “haloalkoxy” refers to an alkoxy group as defined above where the alkoxy group is substituted with a halo radical as defined above.

[0038] “Amino acid” refers to any of the naturally occurring amino acids, as well as optical isomers (enantiomers and diasteriomers), synthetic analogs and derivatives thereof. α-Amino acids include a carbon atom to which is bonded an amino group, a carbonyl group, a hydrogen atom, and a distinctive side chain. The side chains of naturally occurring amino acids are well known in the art and include, for example, hydrogen (e.g., as in glycine), alkyl (e.g. as in alanine, valine, leucine, isoleucine, proline), substituted alkyl (e.g., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginie, and lysine), arylalkyl (e.g., as in phenylalanine and tryptophan), substituted arylalkyl (e.g., as in tyrosine), and heteroalkyl (e.g., as in histidine). One of skill in the art will appreciate that the term amino acid also includes β-, γ-, δ-, and Ω-amino acids, and the like. Synthetic, or unnatural, amino acids such as, for example, trifluoroleucine, p-fluorophenylalanine, and 3-triethylalanine are also known in the art. The term amino acid also includes carboxy protected amino acids. Typically, the carboxy protected amino acids are esters of natural and synthetic amino acids. Preferably, the carboxy protected amino acid is an alanine ester. Most preferably the carboxy protected amino acid is alanine methyl ester.

[0039] “Carboxy-protected amino acids” can be prepared from a natural or synthetic amino acid and a carboxy-protecting group using methods known to those skilled in the art. Typical methods for preparing carboxy-protected amino acids include, for example, the preparation of the methyl ester, ethyl ester, benzyl ester, methoxymethyl ester, and benzyloxymethyl ester. Suitable carboxy protective groups and methods for the preparation of carboxy-protected amino acids are described in Protective Groups in Organic Synthesis, Greene, ed., John Wiley & Sons, New York (1981) and The Peptides: Analysis, Synthesis, Biology: Vol.3: Protections of Functional Groups in Peptide Synthesis, E. Gross and J. Meinenhofer, eds., Academic Press, New York (1981), the disclosures of which are incorporated herein by reference. Suitable carboxy-protected amino acids include carboxy-protected natural and synthetic amino acids.

[0040] The following is a list of abbreviations and their corresponding meanings which are used throughout the specification:

[0041] AZT: 3′-azido-3′-deoxythymidine, also known as zidovudine or ZDV

[0042] Ph: phenyl;

[0043] Ala: alanine;

[0044] Trp: tryptophan;

[0045] Pro: proline;

[0046] Phe: phenylalanine;

[0047] Leu: leucine;

[0048] Met methionine;

[0049] Val: valine;

[0050] Gly: glycine;

[0051] WHI-07: 5-Bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-(p-Bromophenyl Methoxyalaninyl Phosphate), which has the following chemical structure:

[0052] In the course of pursuing active agents having improved activity against HIV, it was unexpectedly discovered that derivatives of AZT possess spermicidal and/or sperm-immobilizing activity and thus are useful as active agents for contraceptive products and methods. These derivatives are particularly useful in forming contraceptive products that can reduce the spread of sexually-transmitted diseases as well as inhibit conception.

[0053] In particular, AZT derivatives having contraceptive activity are provided by substituting the thymine ring of AZT with halo at the 5-position and alkoxy at the 6-position, particularly C 1-3 alkoxy. The derivatives of this aspect of the present invention have the chemical structure illustrated below:

[0054] where R1 is H, N3, halo, CN, COOH or NH2, R2 is halo (in some embodiments, for example, Cl, Br or I, and more preferably Br) and R3 is alkoxy (in some embodiments C1-3 alkoxy, and more preferably methoxy (—OCH3)). The NH2 group can be functionalized, for example with —CH3, —COCH3, —Ph, —COPh, and —CH2Ph. Pharmaceutically acceptable salt or ester forms also can be used, such as sodium, potassium or ammonium salts.

[0055] In a further aspect of the present invention, the derivatives of formula (I) above include substitution on the AZT pentose ring member. The derivatives of this aspect of the present invention have the chemical structure illustrated below:

[0056] where R1 is H, N3, halo, CN, COOH or NH2, R2 is halo (particularly Cl, Br or I, and more particularly Br), R3 is alkoxy (particularly C1-3 alkoxy, and more particularly methoxy (—OCH3)) and R′ is a group that facilitates the passage of the compound into a cell. As in Formula (I), the NH2 group can be converted, for example to NHCH3, NHCOCH3, NHPh, NHCOPh, and NHCH2Ph. The R′ group can be, for example, a substituted or unsubstituted phosphate, lipid or fatty acid group. Alternatively, sperm-reactive antibodies or cytokines could be used to derivatize these compounds (as well as those of Formula (I)) at the R′ or R1 positions for targeted delivery. Pharmaceutically acceptable salt or ester forms, such as the sodium, potassium or ammonium salts, can be used as well.

[0057] In a further aspect of the invention, the R′ group forms a substituted or unsubstituted phosphate group. The H of an —OH member of the phosphate can be replaced with C1-4 alkyl or aryl substituents (e.g. phenyl-, naphthyl- or anthracinyl-substitution), which optionally may be substituted, and SH or NH2 groups can replace the OH of the phosphate, and in each of these cases a H of the NH2 or SH can be replaced in the same manner as the H of the OH group discussed previously. For example, in such embodiments, R′ is a group of the formula:

[0058] wherein R10 and R11 are the same or different and are selected from OH, SH, NH2, OR12, SR12, and NHR12, wherein R12 is a substituted or unsubstituted C1-4 alkyl or a substituted or unsubstituted aryl.

[0059] The aryl phosphate group is surprisingly effective in maintaining excellent anti-HIV activity. A general structure of an exemplary aryl phosphate for use at the R′O group of formula II is illustrated below:

[0060] where R4, R5, and R6 are the same or different and are selected from hydrogen, alkyl, aryl, alkoxy, halo, haloalkyl, arylalkyl, aralkoxy, haloalkoxy, nitro, cyano, animo and the like. In some embodiments, R4, R5, and R6 are the same or different and are selected from hydrogen, C1-3 alkyl, C1-3 alkoxy, halo, haloalkyl, and haloalkoxy. In some particular embodiments, R4, R5, and R6 are the same or different and are selected from hydrogen, methyl, ethyl, fluoro, chloro, bromo, iodo, dichloro, dibromo, difluoro, trifluoromethyl, nitro, cyano, methoxy, trifluromethoxy and ethoxy, particularly hydrogen, fluoro, bromo, nitro, and methoxy.

[0061] R″ is an amino acid residue. The amino acid residue may optionally be substituted and/or esterified. In some embodiments, the amino acid residues are residues of amino acids including non-polar side chains. In some embodiments, the amino acid residue is a carboxy protected amino acid residue. Preferably the carboxy protected amino acid residue includes a methyl ester. Some examples of suitable amino acid residues include alanine, tryptophan, proline, phenylalanine, leucine, methionine, valine, and glycine. In some embodiments, the amino acid residue is selected from alanine, tryptophan, proline, methionine, and glycine. The alanine amino acid residue is preferred.

[0062] In the derivatives of the above formulae (I) and (II), when R1 is NH2, improved spermicidal (i.e. contraceptive) activity is seen versus the cases where R1 is N3. However, anti-HIV activity is decreased, although still remaining at a useful level. Of the R4 substituents, bromo substitution provided the best spermicidal activity, followed by fluoro, methoxy and hydrogen in that order. The bromo R4 substitution also showed the best anti-HIV activity, followed by hydrogen, methoxy and fluoro in that order.

[0063] All of the derivatives discussed herein are believed to yield useful metabolites in sperm cells. It will be readily apparent to those skilled in the art that other structures yielding the same metabolites as the AZT derivatives mentioned above, and structures functioning as precursors to the present structures, can be developed. The present invention should be considered to encompass these other structures. Examples of other structures that will yield the same metabolites are illustrated below:

[0064] where R2 is Cl, Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl

[0065] where R2 is Cl, Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl

[0066] where R2 is Cl, Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl

[0067] where R2 is Cl, Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl

[0068] where R2 is Cl, Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl

[0069] where R2 is Cl, Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl

[0070] where R2 is Cl, Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl

[0071] where R2 is Cl, Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl

[0072] where R2 is Cl , Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl

[0073] where R2 is Cl, Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl

[0074] where R2 is Cl, Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl

[0075] where R2 is Cl , Br or I, R3 is methoxy, ethoxy, or isopropoxy, R6 is H, methyl, ethyl, propyl, butyl or CH2Ph and R7 is methyl, ethyl or propyl.

[0076] While the present invention has been illustrated with respect to the thymine-containing nucleoside AZT and its derivatives, the 5-halogen and 6-alkoxy substitution providing contraceptive effects also will be effective for other nucleosides and nucleotides based on the pyrimidine ring system, of which thymine is one example. These other nucleosides can include ring members such as uracil or cytosine rings, for example, which are suitable for substitution in the same manner as thymine.

[0077] The AZT derivatives with 5-bromo and 6-methoxy substitution on the thymine ring can be prepared using known techniques. See R. Kumar et al. J. Med. Chem. 37, 4297 (1994); L. Wang et al. J. Med. Chem. 39, 826 (1996). Similarly, the aryl phosphate derivatives can be prepared using known phosphorochloridate chemistry techniques. See C. McGuigan et al. J. Med. Chem. 36, 1048 (1993); J. Med. Chem. 39, 1748 (1996); Biorg. Med. Chem. Lett. 6, 1183 (1996). The conversion of the azide group, for example to the NH2 group, can be accomplished by known techniques. See J. Med. Chem., 21, 109 (1978).

[0078] An exemplary synthetic scheme is illustrated below.

[0079] Synthetic Scheme I

[0080] Compound iii(a) was prepared according to known literature procedures. Compound iii(a) was treated with BrOMe to afford a diastereomeric mixture (approximately 2:1) of (5R, 6R)-5-bromo-6-methoxy-5,6-dihydro-3′-azidothyrnidine 5′-(p-methoxyphenyl methoxyalaninyl phosphate) and (5S, 6S)-5-bromo-6-methoxy-5,6-dihydro-3 ′-azidothymidine 5′-(p-methoxyphenyl methoxyalaninyl phosphate) iv(a). The preparation of iii(b) was started with commercially available p-bromophenol and phosphorus oxychloride in ethyl ether, to obtain intermediate i(b). Intermediate i(b) is coupled with alanine, to afford intermediate ii(b). Intermediate ii(b) is further coupled with AZT under catalysis of N-methylimidazole, to obtain compound iii(b). Compound iii(b) was treated with BrOMe to obtain the desired product iv(b). The (R, R) diastereomer can be achieved by coupling intermediate ii(b) with compound namely trans-(5R,6R) 5-bromo-6- methoxy-5,6 dihydro-3′-azido-3′-deoxythymidine.

[0081] Another exemplary synthetic scheme showing the production of aryl phosphate derivatives of AZT having different amino acid residues is illustrated below in Synthetic Scheme II of Example 9.

[0082] The nucleosides used as spermicidal active agents in the present invention can be formulated into contraceptive compositions for use. Such compositions are intended particularly for use with mammals, i.e. any class of higher vertebrates that nourish their young with milk secreted from mammary glands, for example humans, rabbits and monkeys. It is expected that the present invention will be used by humans in most practical applications.

[0083] The contraceptive compositions of the present invention contain one or more of the spermicidal nucleosides. The total amount of spermicide will typically range from about 0.025 to 0.5 weight percent based on the total weight of the contraceptive composition. The amount of spermicide employed generally will be that amount necessary to achieve the desired spermicidal and anti-viral protective results. The amounts can be varied as needed for specific compositions. Preferably, the amount of the spermicidal AZT derivative employed will be from about 0.05 to 0.5 weight percent, and more preferably from about 0.05 to 0.25 weight percent, based on the total weight of the contraceptive composition.

[0084] The contraceptive compositions of the present invention contain not only the spermicidal nucleoside, but also pharmaceutically acceptable carriers, diluents or vehicles as needed, i.e., materials for appropriately delivering and/or maintaining the spermicidal nucleosides to a site for contact with sperm and so as to provide the desired spermicidal and anti-viral protective activity.

[0085] One advantageous component in the pharmaceutical composition for administration of a spermicide is a polymeric delivery component as described in U.S. Pat. No. 5,595,980, which patent is incorporated herein by reference. It has been found that such polymeric delivery component enhances the effectiveness of a spermicide and reduces vaginal irritation on administration.

[0086] In addition to the polymeric component, the balance of the contraceptive compositions, i.e., typically from about 0.1 to 99.8% and often about 50 to 99.8 weight percent, may optionally comprise one or more cosmetic ingredients. Such cosmetic ingredients are known to those skilled in the art and are often referred to in the art as diluents, solvents and adjuvants. Typically cosmetic ingredients include, for example; water, ethyl alcohol, isopropyl alcohol, glycerin, glycerol propylene glycol, sorbitol and other high molecular weight alcohols. In addition, contraceptive compositions may contain minor amounts, e.g. from about 0.1 to 5% weight based on the weight of the contraceptive compositions, of other additives, such as, for example; stabilizers, surfactants, menthol, eucalyptus oil, other essential oils, fragrances, and the like. Polyoxyethylene 20 sorbitan monolaurate is a preferred stabilizer for use in the compositions. The selection and amounts of cosmetic ingredients, other additives, and blending procedures can be carried out in accordance with techniques well-known in the art.

[0087] The spermicidal active ingredients, and contraceptive compositions containing the same, of the present invention may be delivered to the vagina of a mammal by any means known to those skilled in the art. Typical forms for delivery of the compositions include, for example; creams, lotions, gels, foams, intervaginal devices such as sponges and suppositories, and films. In addition, the contraceptive compounds and compositions of the present invention may be used as personal care products, such as, for example, condom lubricants, and the like. Such lubricants may comprise commonly known ingredients such as, for example: humectants; e.g., glycerin, sorbitol, mannitol, glycols and glycol ethers; buffers, e.g., glucono-d-lactone; germicides or bactericides; e.g., chlorhexidine gluconate; preservatives, e.g., methylparaben; viscosifiers; e.g., hydroxyethyl cellulose, etc.; other adjuvants; e.g., colors and fragrances; in addition to the compositions of the present invention. Those skilled in the art will recognize that the physical properties, e.g., viscosity, of such delivery forms may vary widely. For example, the viscosity of a gel form of the composition of the present invention, e.g., 150,000 centipoise, may be substantially higher than the viscosity of lotion form of the composition of the present invention, e.g., 100 centipoise. Further details concerning the materials, ingredients, proportions and procedures of such delivery forms can be selected in accordance with techniques well-known in the art.

[0088] The contraceptive compositions of the present invention are preferably administered to the vagina of the mammal in a dosage which is effective to immobilize sperm present in the vagina and/or to inhibit their penetration in cervical mucus. Typical dosages range between about 0.0001 to 0.001 grams of the composition per kilogram of body weight of the mammal.

[0089] Intervaginal devices also may be used to aid in the administration of the spermicidal active ingredients or contraceptive compositions containing the same as described in U.S. Pat. No. 5,069,906, the disclosure of which is incorporated herein by reference.

[0090] In administering the spermical active ingredients in the form of the above compositions, the compositions also may be formulated to release the spermicide both rapidly and with a prolonged release of the drug. Such a formulation providing both rapid and prolonged release has been described in U.S. Pat. No. 4,707,362, which also is incorporated herein by reference.

[0091] The invention will be explained further with reference to the following examples, which should not be considered to limit the invention.

EXAMPLES

[0092] The effect of AZT derivatives of the present invention was evaluated. Several AZT derivatives were prepared in accordance with the synthesis techniques discussed above. Examples of some of the specific derivatives are shown in the following examples, and in Tables 1, 2, 3 and 4 below.

Synthetic Examples

[0093] 5-Bromo-6-methoxy-5,6-dihydro-3′-amino-3 ′-deoxythymidine

[0094] A freshly prepared solution of methyl hypobromite (bromine in anhydrous methanol) was added dropwise into a solution of 3′-amino-3′-deoxythymidine (0.10 g, 0.41 mmol) in anhydrous methanol (5 mL) with stirring until the light yellow color of the reaction mixture persisted. The reaction was allowed to proceed for 20 minutes. Removal of the solvent in vacuo afforded 5-bromo-6-methoxy-5,6-dihydro-3′-amino-3′-deoxythymidine (0.2 g, 100%) and as yellow foam solids; m.pt. 188-190° C. (d), UV (MeOH): λmax 202,235 nm. IR(KBr): 3440, 2979, 2358, 2107, 1699, 1469, 1249 and 1085 Cm−1. 1H NMR (DMSO) δ 8.25 (s, 1H, NHC═O), 6.20 (dd, J=8.0, 5.9 Hz, 1H, H-1′), 5.08 (s, 1H, H-6), 3.99 (m, 1H, H-3′), 3.69 (br s, 2H, NH2), 3.40 (s, 2H, H-5′), 3.21 (s, 3H, CH30), 2.49-2.15 (m, 2H, CH2-2′), 1.83 (s, 3H, CH3).MS (CI, m/e) 353.9(M+1, 23), 351.6 (21), 339.8(4), 338 (5), 321.8 (10), 319.8(13), 304.8 (1), 230(1).

[0095] (5R,6R)-5-Bromo-6-(d3)methoxy-5,6-dihydro-3 ′-azido-3 ′-deoxythymidine

[0096] A freshly prepared solution of deuterated methyl hypobromite (bromine in deuterated methanol) was added dropwise into a solution of AZT (0.50 g, 1.87 mmol) in deuterated methanol (5 mL) with stirring until the light yellow color of the reaction mixture persisted. The reaction was allowed to proceed for 20 minutes. Removal of the solvent in vacuo and application of the residue to preparative thin-layer chromatography (PTLC) afforded both (5R, 6R)-5-bromo-6-(d3)methoxy-5,6-dihydro-3′-amino-3′-deoxythymidine (0.791 g, 44%) and (5S, 6S)-5-bromo-6-(d3)methoxy-5,6-dihydro-3′-amino-3′-deoxythymidine (0.527 g, 28%) as white foam solids; UV (MeOH): λmax, 237, 305 nm. IR (Neat liquid,KBr disc): 3396, 3097, 2942, 2836, 2096, 1712, 1459, 1380, 1247, 1080 Cm−1 1H NMR (CDCl3) (5R, 6R) δ 1.95 (s, CH3), 2.2 (m), 2.85 (q), 3.7-3.78 (dd), 4.2-4.44 (m), 4.90 (s), 5.90 (t), 8.6 (bs, NH); 13C NMR (CDCl3): δ 22.82 (CH3), 36.98 (C-2′), 53.15 (C-5), 57.53 (OCH3), 60.02 (C-3′), 61.97 (C-5′), 83.94 (C-4′),86.36 (C-1′), 89.10 (C-6), 150.65 (C-2,CO), 167.33 (C-4,CO). GC/MS: M+NH3, 398 (100.0), M +2, 383, (62.0), M +1, 382 (9.9), M+, 381 (65.5), 365 (8.0), 363 (8.0), 348 (61.0), 346 (62.0), 287 (4.9), 285 (10.5), 268 (23.6), 211 (6.8), 159 (6.8), 127 (7.8), 116 (8.7), 114 (17.8), 99 (26.7), 89.1 (12.6), 86.1 (9.6), 35 (4.5).

[0097] 5-Bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-(p-Methoxyphenyl Methoxyalaninyl Phosphate):

[0098] A freshly prepared solution of methyl hypobromite (bromine in anhydrous methanol) was added dropwise into a solution of 3′-azidothymidine 5′-(p-methoxyphenyl methoxyalaninyl phosphate) (0.11 g, 0.2 mmol) in anhydrous methanol (5 mL) with stirring until the light yellow color of the reaction mixture persisted. The reaction was allowed to proceed for 20 min. Removal of the solvent in vacuo and application of this material to the top of a silica gel column followed by elution with chloroform-methanol (95:5, v/v) afforded 5-bromo-6-methoxy-5,6-dihydro-3′-azidothymidine 5′-(p-methoxyphenyl methoxyalaninyl phosphate) (0.085 g, 0.13 mmol, 64%) as a yellow viscous oil; UV (MeOH) λmax 329, 280, 241 nm. IR (neat in KBr disc): 3282, 2954, 2838, 2358, 2103, 1739, 1635, 1506, 1456, 1378, 1209, 1103 Cm−1. 1H NMR (CDCl3) δ 7.66 (s, 1H, NHC═O), 7.26-7.10 (m, 2H, o-H), 6.87-6.76 (m, 2H, m-H), 6.04 (d, J=6.2, 2.3 Hz, 1H, H-1′), 4.88 (s, 1H, H-6), 4.33-4.22 (m, 1H, H-3′), 4.07-3.98 (m, 3H, H-4′, 5′), 3.77 (s, 3H, CH3OPh), 3.72 (s, 3H, CH3C═O), 3.48 (s, 3H, CH30-6), 3.44 (d, J=3.5 Hz, 1H, NH), 2.46-2.31 (m, 2H, CH2-2′), 1.96 (s, 3H, CH3-5), 1.36 (d, J=6.8 Hz, 3H, CH3-Ala).13C NMR (CDCl3) δ 173.7 (COOMe), 167.30 (C2), 156.6 (Ph para), 150.7 (C4), 149.0(Ph ipso), 121.6 (Ph ortho), 114.6(Ph meta), 87.4(C6), 84.7 (C1′), 81.44 (C4′), 61.3 (C5′), 60.1(C3′), 57.9(OMe), 55.6 (Ph OMe), 52.5 (C5), 52.4 (COOMe), 50.5 (Ala CH), 36.61 (C2′), 22.74 (Me), 20.93 (Ala Me). Additional peaks were observed for the isomers as shown below: 150.2, 120.8, 114.44), 93.3, 89.82, 65.3, 65.6, 65.9, 56.9, 55.58, 50.37, 35.2 etc.,

[0099]

31
P NMR (CDCl3) δ: 3.51 & 3.38 ppm, relative to phosphoric acid as standard at 0 ppm. MS/Electron spray: 651, 649 (M+1). GC/mass: 645 (0.2), 643 (2.3), 572 (0.8), 559 (20.4), 558 (100), 501 (2.3), 446 (4.6), 435(3.5), 416 (1.4), 409 (7.9), 328 (2.7), 272(6.1), 245 (1.6), 244 (8.8), 204 (3.2), 200 (0.1), 139 (6.1), 138 (4.7), 126 (10.1), 124 (29.0), 123 (3.3), 109 (14.01), 91 (11.5), 81 (11.9), 77 (13.1), 60 (13.6), 55 (9.0), 44 (26.2), 43 (54.9).

[0100] p-Bromophenyl Phosphorodichloridate.

[0101] A solution of p-Bromophenol and triethylamine in anhydrous diethyl ether was added dropwise to a vigorously stirred solution of phosphoryl chloride in diethyl ether at 0° C. The mixture was allowed to warm to ambient temperature, with stirring for 15 hours, and then heated under reflux for 2 hours. The mixture was filtered, and the precipitate was washed with diethyl ether. The combined filtrate and washings were evaporated to dryness under reduced pressure to yield a colored oil. The oil was subjected to vacuum distillation to give the product as a colorless oil; B.Pt 97° C./0.1 mm, Yield 90%, UV (MeOH) λmax 271, 242 nm, IR (KBr disc) 3878, 3095, 2358, 1888, 1712, 1483, 1189, 831 Cm−1, 1H NMR(CDCl3) δ 7.50 (d, 2H, aryl H, J=9.0 Hz), 7.15 (d, 2H, aryl H, J=9.0 Hz, 13C NMR (CDCl3) 6: 148.5, 133.30, 122.38, 120.48. 31P NMR (CDCl3.) δ 3.12.

[0102] p-Bromophenyl Methoxyalaninyl Phosphorochloridate.

[0103] A solution of triethylamine in anhydrous dichloromethane was added dropwise with vigorous stirring to a solution of L-alanine methyl ester hydrochloride and p-bromophenyl phosphorodichloridate in dichloromethane at −70° C. The reaction mixture was slowly warmed to ambient temperature with stirring over 6 hours, and the solvent was then removed in vacuo. The residue was treated with diethyl ether, the mixture was filtered, and the filtrate was concentrated to give the product as a colorless Viscous oil; Yield 80%, UV (MeOH): λmax 272, 231, 1H NMR (CDCl3) δ 8.70 (s, br, 1H, Ala-NH), 7.16 (d, 2H, aryl H, J=9.0 Hz), 7.48 (d, 2H, aryl H, J=9.0 Hz), 3.793 (s, 1H, —OCH3), 3.77 (s, 1H, —OCH3), 1.51 (d, 3H, Ala-CH3), 1.40 (d, 3H, Ala-CH3); 13C NMR (CDCl3) δ: 172.78 & 172.54, 148.46 & 148.50, 132.61 & 132.49, 122.14& 122.05, 52.70 & 52.60, 50.31 & 50.51, 20.20 & 20.11 (two isomers). 31P NMR (CDCl3) δ: 8.48& 8.35. IR (neat): 3212 (s, br), 2989 & 2952 (w-m), 1747 (s), 1483 (s), 1270, 1209 & 1147 (s), 1010 & 927 (s), 831 (m), 757 (m) cm−1; MS (CI, m/e): 357.9 (M++2), 355.9 (M+), 322.0 (M++2-Cl), 320 (M+ —Cl), 295.9 (M+ —COOCH3), 186.0(M++2-Br—Ph—O), 184.0 (M+ —Br—Ph—O).

[0104] 3′-Azidothymidine 5′-(p-Bromophenyl Methoxyalaninyl Phosphate)

[0105] AZT was dissolved in THF, and p-bromophenyl methoxyalaninyl phosphorochloridate and N-methylimidazole was added with vigorous stirring. After 12 hours at ambient temperature the solvent was removed under vacuum. The residue was dissolved in chloroform and washed with 1 M hydrochloric acid, saturated sodium bicarbonate solution, and then water. The organic phase was dried and evaporated under vacuum, and the residue was purified by chromatography on silica gel by elution with 5% methanol in chloroform. Concentration led to the desired product; Yield 83%. 1H NMR (CDCl3) δ 8.69 (s, br, 1H, 3-NH), 7.45 (d, 2H, aryl H, J=9.0 Hz), 7.33 (s, 1H, vinyl H at C6), 7.11 (d, 2H, aryl H, J=9.0 Hz), 6.18 (t, 1H, J=6.6 Hz, H at C1) 6.13 (t, 1H, J=6.6 Hz, H at C1), 4.44-3.77 (m, 6H, H's at C3′, 4′, 5′, Ala-NH and Ala-CH), 3.73 (s, 3H, -COOCH3), 3.72 (s, 3H, —COOCH3), 2.18 (s, 3H, —CH3 at C5), 1.90 (s, br, 3H, Ala-CH3).13C NMR (CDCl3) 6:173.6 (COOMe, weak), 163.2, 160.7 (C2, weak), 149.9 (C4),144.1,144.3(d, Ph ipso, J=6 Hz), 135.4(C6), 121.79,121.72 (m,Ph ortho),119 (Ph meta), 111.36 (C5), 85.41,85.05 (C1′), 82.20(C4′), 65.73 (C5′), 60.31, 60.17 (C3′), 52.75 (Ala OMe), 50.37, 50.22 (Ala CH), 37.28 (C2′), 21.07 (Ala Me), 12.54 (C5- Me ). IR (KBr): 3205 (m, br), 3066 (w), 2954 9w), 2109 (s), 1475 (s), 1691 (vs), 1484 (m), 1270 & 1216 (m), 1153 (w-m), 1010 & 926 (m), 833 & 757 (w) cm−1. MS (CI, m/e): 589.1 (M++2), 587.1 (M+), 418.0 (M++2-Br—Ph—O), 416.0 (M+ —Br—Ph—O), 340.0 [M++2-(AZT-O)], 338.0 [M+-(AZT-O)], 250.1 [(AZT-O)+], 81 (Br).

[0106] 5-Bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-(p-Bromophenyl Methoxyalaninyl Phosphate).

[0107] A freshly prepared solution of methyl hypobromite (bromine in anhydrous methanol) was added dropwise into a solution of 3′-azidothymidine 5′-(p-bromophenyl methoxyalaninyl phosphate) in anhydrous methanol (5 mL) with stirring until the light yellow color of the reaction mixture persisted. The reaction was allowed to proceed for 20 minutes. Removal of the solvent in vacuo and application of this material to the top of a silica gel column followed by elution with chloroform-methanol (95:5, v/v) afforded 5-bromo-6-methoxy-5,6-dihydro-3′-azidothymidine 5′-(p-bromophenyl methoxyalaninyl phosphate) as a yellow viscous oil; Yield 73%.IR (Neat,KBr Disc):3218, 3093, 2925, 2850, 2105.9, 1712, 1484, 1456, 1378, 1241, 1153, 1010, 929 Cm−1. 1H NMR (CDCl3) δ 7.70 (br s, 1H, 3-NH ), 7.43 (2H, d, Aryl H, J=9.0 Hz), 6.01 (H, t, —CH at C-1′), 4.87 (1H, s, —CH at C-6), 4.35-3.96 (6H, m, —CH at C-3′, 4′, 5′ & Ala-NH, a —CH), 3.74 (3H, s, —COOCH3), 3.44 (3H, s, —OCH3 at C-6), 2.56-2.47 & 2.41-2.30 (1H & 1H, m & m, —CH at C-2′),1.93 (3H, s, —CH3 at C-5), 1.39 & 1.37 (3H, d, a —CH3 of Ala); 13C NMR (CDCl3) δ: 173.6(COOMe), 166.7 (C2), 150.1 (C4), 148.9 (d,Ph ipso),132.7 & 132.5 (C6), 121.9 (Ph, ortho), 121.7 (Ph, meta), 85.14 (C1′), 81.46 & 81.57 & 81.7(m,C4′), 65.48 & 65.60 (m,C5′), 60.0(C3′), 57.9 & 57.8(C5), 52.77 (al Ome),50.25 & 50.37 (Ala CH), 36.96 & 36.91 (C2′), 22.87 & 22.77 (Me), 21.13 & 21.19 (d, Ala Me) (double peaks are due to several isomers) 31P NMR (CDCl3, Phosphoric acid as internal reference at 0 ppm) δ: 2.854, 2.754 (1:1) & 2.7 & 2.46 (3:1) due to four isomers present in the system. MS (CI, m/e): 700.5 (M++4), 698.5 (M++2), 696.5 (M+), 588.8 (M++2-Br—OCH3), 586.8 (M+—Br—OCH3).MALDI-TOF 721.8 (M+ +Na).

[0108] Physical Data of other Synthesized Compounds

[0109] (5S, 6S)-5-Bromo-6-(d3)-methoxy-5,6-dihydro-3′-azidothymidine

[0110]

1
H NMR (CDCl3) δ 8.00 (br s, 1H), 5.28-5.20 (t, 1H), 4.58 (s, 1H), 4.52-4.48 (m, 2H), 3.75-3.70 (dd, 1H), 2.90 (q, 1H), 2.20 (m, 2H), 2.00 (s, 3H); GC/mass: 383 (M+2, 97.0), 382 (M+1, 13.0), 381 (M+, 100), 377 (5.0), 365 (6.0), 363 (6.0), 351 (4.0), 349 (3.0), 348 (45.0), 347 (5.0), 346 (41.0), 310 (22.0), 309 (2.0), 308 (21.0), 268 (23.0), 267 (9.0), 186 (4.0), 159 (4.0), 127 (10.0), 116 (5.0), 115 (1.0), 114 (7.0), 102 (7.0), 99 (4.0), 89 (15.0), 81 (6.0); UV (MeOH): λmax 237 and 305 nm; IR (neat): 3396, 3097, 2942, 2836, 2096, 1712 cm−1.

[0111] trans-(5R,6R)-5-Bromo-6-methoxy-5,6-dihydro-3′-azidothymidine 5′-[p-Bromophenyl methoxyalaninyl phosphate] (WHI-07a)

[0112] Pale yellow viscous oil; yield (73%); 1H NMR (CDCl3) δ 7.75 (br s ,1H), 7.44 (d, 2H), 7.14 (d, 2H), 6.03 (t, 1H), 4.87 (s, 1H), 4.22-4.37, 3.93-4.08, 3.62-3.78 (m, 6H), 3.74 (s, 3H), 3.44 (s, 3H), 2.31-2.58 (m, 2H), 1.93 (s, 3H), 1.38 (d, 3H); 13C NMR (CDCl3) δ 174.0, 167.1, 150.6, 133.2, 122.2, 122.1, 88.2, 85.6, 81.9, 65.9, 60.4, 58.3, 54.1, 53.2, 50.7, 37.3, 23.3, 21.6; MS (MALDI-TOF) m/z 721.5 (M+Na); HPLC retention time 14.4 minutes; UV (MeOH): λmax 217, 226 and 270 nm; IR (Neat): 3218, 3093, 2850, 2106, 1712 cm−1.

[0113] trans-(5S,6S)-5-Bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-[p-Bromophenyl methoxyalaninyl phosphate] (WHI-07b)

[0114] Colorless viscous oil; yield (42%); 1H NMR (CDCl3) δ 8.37 (br, 1H), 7.44 (dd, 2H), 7.13 (d, 2H), 5.34 (t, 1H,), 5.01 (s, 1H), 3.92-4.54 (m, 6H), 3.73 (s, 3H), 3.43 (s, 3H), 2.77-2.90 (m, 1H), 2.25-2.37 (m, 1H), 1.93 (s, 3H), 1.38 (dd, 3H); 13C NMR (CDCl3) δ 176.0, 166.7, 149.6, 132.5, 122.9, 94.2, 82.38, 66.2, 61.4, 57.1, 53.3, 52.6, 50.2, 35.4, 29.8, 22.8, 21.1; UV: (MeOH): λmax 217, 226 and 270 nm; IR (Neat): 3218, 3093, 2850, 2106, 1712 cm−1.

[0115] 5-Bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-[(p-Fluorophenyl methoxyalaninyl phosphate] (WHI-06)

[0116] Viscous liquid; yield (71%); 1H NMR (CDCl3) δ 8.41, 8.21 (br s, 1H), 7.19 (dd, 2H), 7.03 (dd, 2H), 6.13 (t, 0.7H, 5R, 6R isomer), 5.38 (m, 0.3H, 5S, 6S isomer), 4.88 & 4.61 (d&d, 0.7H & 0.3H), 4.46-3.80 (m, 6H), 3.73 & 3.71 (s, 3H), 3.49 & 3.44 (s, 3H), 2.83-2.22 (m, 2H), 1.95 (s, 3H), 1.36 (dd, 3H); MS (MALDI-TOF) m/z 660.1 (M+Na); IR (neat): 3230,3081, 2107,1736, 1712, 1504, cm−1.

[0117] trans-(5R, 6R)-5-Bromo-6-ethoxy-5,6-dihydro-3′-azidothymidine 5′-[P-Methoxyphenyl methoxyalaninyl phosphate]

[0118] Viscous oil; yield (40%); 1HNMR (CDCl3) δ 8.20 (br s, 1H), 7.15-7.11 (d, 2H), 6.86-6.82 (d, 2H), 6.10 (t, 3H), 4.94 (s, 1H), 4.33-4.00 (m, 6H), 4.03-4.02 (m, 2H), 3.80 (m, 3H), 3.70 (m, 3H), 2.50-2.40 (m, 1H), 2.40-2.20 (m, 1H), 1.92(s, 3H), 1.39-1.38 (d, 3H), 1.36 (t, 3H); 13C NMR (CDCl3) δ 180.1, 166.9, 150.3, 148.1, 121.1, 121.0, 120.8, 114.6, 114.5, 114.5, 86.3, 84.9, 81.7, 81.5, 66.3, 66.2, 65.5, 65.3, 60.1, 55.9, 52.6, 52.4, 50.4, 50.2, 36.9, 22.93, 22.9, 21.2, 15.2; MS (MALDI-TOF) m/z 687.9 (M+Na); HPLC retention time 1.74 & 1.82 minutes; UV (MeOH): λmax 208, 221, 278 nm; IR (neat): 3242, 2852, 2106, 1732, 1506 cm−1.

[0119] trans-(5R,6R)-5-Bromo-6-ethoxy-5,6-dihydro-3′-azidothymidine

[0120] Yield (60%); 1HNMR (CDCl3) δ 7.70 (s, 1H), 5.82 (t, 1H), 4.98 (s, 1H), 4.40-4.30 (m, 1H), 4.00-3.72 (m, 4H), 3.60-3.50 (m, 1H), 2.77-2.68 (q, 1H), 2.37-2.29 (dd, 1H), 1.97 (s, 3H), 1.17 (t, 3H); MS (MALDI-TOF) m/z 415.7 (M+Na); UV (MeOH): λmax 210 and 214 nm; IR (neat): 3485, 3217, 3093, 2104, 1685 cm−1.

[0121] trans-(5S,6S)-5-Bromo-6-ethoxy-5,6-dihydro-3′-azidothymidine

[0122] Viscous oil; yield (24%); 1HNMR (CDCl3) δ 7.40 (s, 1H), 5.22 (t, 1H), 4.60 (s, 1H), 4.02-3.68 (m, 4H) 3.60-3.50 (m, 1H), 2.98 (q, 1H), 2.22 (dd, 1H), 1.97 (s, 3H), 1.25-1.22 (t, 3H); MS (MALDI-TOF) m/z 415.7 (M+Na); UV (MeOH): λmax 210 and 214 nm; IR (neat): 3485, 3217, 3093, 2929, 2104, 1685 cm−1.

[0123] 5-Bromo-6-methoxy-5,6-dihydro-3′-deoxythymidine 5′-[p-Methoxyphenyl methoxyalaninyl phosphate]

[0124] Viscous oil; yield (86%); 1HNMR (CDCl3) δ 8.24 (br s, 1H), 7.36-7.26 (m, 2H), 6.94-6.92 (m, 2H), 6.03-5.99 (m, 1H), 5.1-5.0 (s, 1H), 4.40-4.0 (m, 5H), 3.87-3.86 (s, 3H), 3.80-3.79 (s, 3H), 3.55-3.54 (m, 3H), 2.34-2.03 (m, 7H), 1.46-1.43 (d, 3H); 13C NMR (CDCl3) δ 173.7, 167.9, 156.3, 150.3*, 143.7*, 120.7*, 114.3*, 86.7, 86.2*, 78.4*, 67.6*, 57.8*, 55.6, 52.4*, 50.1*, 31.8*, 25.7*, 22.8, 21.1; 31P NMR (CDCl3) δ 3.11, 3.22 (1:1); MS (MALDI-TOF) m/z 631.8 (M+Na); HPLC retention time 14.54, 15.18 minutes; UV (MeOH): λmax 226 and 279 nm; IR (neat): 3419, 2850, 1645, 1506 cm−1.

[0125] trans-(5R,6R)-5-Bromo-6-methoxy-5,6-dihydro-3′-azidothymidine 5′-[o,o-Dimethoxyphenyl methoxyalaninyl phosphate]

[0126] Colourless viscous liquid; 1H NMR (CDCl3) δ 7.44 (s, 1H), 7.05 (t, 1H), 6.59 (d, 1H), 6.12* (t, 1H), 5.90* (t, 1H), 4.98 (s, 1H), 4.5-4.3 (m, 1H), 4.20-4.05 (m, 1H), 3.88* (s, 3H), 3.86* (s, 3H), 3.90-3.84 (m, 2H), 3.74* (s, 3H), 3.73* (s, 3H), 3.73 (q, 1H), 3.48 (s, 6H), 2.70-2.30 (m, 2H), 1.97 (s, 3H), 1.43* (d, 3H), 1.38* (d, 3H); 13C NMR (CDCl3) δ 151.9, 137.5, 129.1, 125.3, 124.9, 124.7, 88.2, 87.1, 85.8, 84.4*, 84.3*, 84.1, 81.8*, 81.7*, 61.7, 60.1, 56.1, 49.8, 37.0*, 36.8*, 22.9, 21.3, 21.2; 31P NMR (CDCl3) δ 5.70, 5.27; MS (MALDI-TOF) m/z 702.4 (M+Na); IR (neat): 3340 (br), 2096, 1741, 1710 cm−1.

[0127] (5S,6S )-5-Bromo-6-methoxy-5,6-dihydrothymidine

[0128] White foam, yield (20%); 1H NMR (DMSO-d6) δ 10.87 (s, 1H), 5.76 (t, J=6.3 Hz, 1H), 5.18 (s, 1H), 5.17 (s, 1H), 4.80 (s, 1H), 4.24 (m, 1H), 3.72 (m, 1H,), 3.52 (m, 1H), 3.44 (s, 3H), 2.2 (m, 1H), 2.0 (m, 1H), 1.84 (s, 3H); 13C NMR (DMSO-d6) δ 167.9, 150.4, 88.9, 86.9, 86.7, 71.1, 62.2, 57.3, 53.8, 37.7, 22.7; MS (MALDI-TOF) m/z 375.3 (M+Na), 377.3 (M +2 +Na); IR (neat): 3371 (br), 3214 (shoulder), 1706 cm−1.

[0129] (5R,6R)-5-Bromo-6-methoxy-5,6-dihydrothymidine

[0130] White foam; yield (80%); 1H NMR (DMSO-d6) δ 10.84 (br s, 1H), 6.04 (dd, 1H, J=8.4, 5.4 Hz), 5.18 (s, 1H), 5.13 (s, 1H), 4.89 (s, 1H), 4.18 (m, IH), 3.74 (m, 1H), 3.53 (m, 1H), 3.42 (s, 3H), 2.20 (m, 1H), 1.80 (m, 1H), 1.84 (s, 3H); 13C NMR (DMSO-d6) δ 167.9, 150.9, 86.7, 86.2, 83.4, 70.5, 61.8, 57.9, 54.5, 38.2, 22.6; MS (MALDI-TOF) m/z 375.8 (M+Na) & 377.4 (M+2+Na); IR (neat): 3366 (br), 3214 (shoulder), 1701 cm−1.

[0131] 5-Bromo-6-methoxy-5,6-dihydro-3′-deoxythymidine 5′-[p-Bromo phenyl methoxyalaninyl phosphate] (WHI-11)

[0132] Viscous oil; yield (86%); 1H NMR (CDCl3) δ 8.74 (br s, 1H), 7.46-7.44 (m, 2H), 7.16-7.10 (m, 2H), 5.91 (t, 1H), 5.1-5.0 (s, 1H), 4.40-4.0 (m, 5H), 3.79* (s, 3H), 3.54* (s, 3H), 2.29-1.93 (m, 7H), 1.39-1.37 (d, 3H); 13C NMR (CDCl3) δ 173.5, 167.1, 150.5,148.5* 132.5*, 121.9*, 117.7, 86.8, 86.2*, 78.3*, 67.6*, 57.8*, 53.8*, 50.1*, 31.6*, 25.7*, 22.8*, and 20.9*; 31P NMR (CDCl3) δ 2.65 and 2.74 (1:1), MS (MALDI-TOF) m/z 679.1 (M−1+Na); HPLC retention time 23.87, 24.49 minutes; UV (MeOH): λmax 226 and 274 nm; IR (neat): 3417, 1705, 1614 cm−1.

[0133] 3′-Amino-3′-deoxythymidine 5′-[p-Methoxyphenyl methoxyalaninyl phosphate]

[0134] Colourless liquid; yield (70%); 1H NMR (CDCl3) δ 7.44 (br d, 1H), 7.15-7.11 (d, 2H), 6.82-6.80 (d, 2H), 6.28-6.20 (t, 1H), 4.42-3.80 (m, 6H), 3.75 (s, 3H), 3.71 (s, 3H,), 2.40-2.28 (m, 2H), 1.88 (br s, 3H), 1.39 & 1.35 (dd, 3H); 13C NMR (CDCl3) δ 173.8, 163.8, 156.4, 150.3, 135.2, 121.0, 120.8, 114.4, 113.8, 110.8, 84.9, 84.2, 66.0, 58.0, 55.5, 55.3, 51.1, 34.2, 21.1, 20.8, 20.1; MS (MALDI-TOF) m/z 535.5 (M+Na); HPLC retention time 10.48, 10.49 minutes; UV (MeOH): λmax 218, 225 and 273 mn; IR ( Neat): 3261, 2839, 2096, 1741, 1693, 1506 cm−1.

[0135] 5-Bromo-6-methoxy-5,6-dihydro-3′-azidothymidine 5′-[Phenyl methoxyalaninyl phosphate] (WHI-04)

[0136] Viscous oil; yield (92%); 1HNMR (CDCl3) 6 7.90 (br s, 1H), 7.37-7.15 (m, SH), 6.06-6.02 (t, 1H), 4.87* (s, 1H), 4.40-4.0 (m, 5H), 3.72 (s, 3H), 3.44* (s, 3H), 2.29-1.93 (m, 6H), 1.39-1.37 (d&d, 3H); 13C NMR (CDCl3) δ 173.5, 167.1, 152.1*, 150.5, 129.8*, 125.2*, 120.1*, 87.3, 84.7,81.5, 60.0, 57.9*, 52.6*, 50.1*, 36.7,35.2, 22.7, 21.0; 31P NMR (CDCl3) δ 2.32, 2.51 (minor peaks) and 2.58, 2.71 (major peaks) (1:1); MS (MALDI-TOF) m/z 679.1 (M−1+Na); HPLC retention time 17.60, 20.90 minutes; UV (MeOH): λmax 218 and 262 nm; IR (neat): 3265, 2852, 2104, 1713, 1591 cm−1.

[0137] 5-Bromo-6-methoxy-5,6-dihydro-3 ′-deoxythymidine 5′-[Phenyl methoxyalaninyl phosphate] (WHI-12)

[0138] Viscous oil; yield (86%); 1H NMR (CDCl3) 6 7.94 (br s, 1H), 7.46-7.10 (m, 5H), 5.93-5.90 (t, 1H), 5.02* (s, 1H), 4.40-4.0 (m, 5H), 3.70* (s, 3H), 3.45* (s, 3H), 2.29-1.93 (m, 7H), 1.39-1.37 (d&d, 3H); 13C NMR (CDCl3) δ 173.5, 167.2, 150.5*, 129.8, 129.5*, 125.0, 120.0, 86.7*, 78.4*, 67.6*, 57.9*, 53.7, 52.6, 50.1*, 31.7*, 25.6, 22.8, 21.0; 31P NMR (CDCl3) δ 2.51, 2.58 (minor peaks) and 2.65, 2.74 (major peaks) (1:1); MS (MALDI-TOF) m/z 642.1(M+Na); HPLC retention time 6.97, 7.41 minutes; UV (MeOH): λmax: 230 and 241 nm; IR (neat): 3226, 2850, 1728,1591 cm−1.

Example 1

Methods

[0139] To evaluate anti-HIV-1 activity of AZT and various derivatives, normal peripheral blood mononuclear cells were cultured for 72 hours in RPMI 1640 medium (Gibco-BRL) supplemented with 20% (v/v) heat-inactivated fetal calf serum, 3% interleukin-2, 2 mM glutamine, 25 mM HEPES, 2 g/l NaHCO3, 50 μg/ml gentamicin, and 4 μg/ml phytohemagglutinin prior to exposure to HIV-1 at a multiplicity of infection of 0.1 during a 1 hour adsorption period. Subsequently, cells were cultured in 96-well microtiter plates (100 μl/well; 2×106/ml, triplicate wells) in the presence and absence of various concentrations (0.00 μM to 100 μM) of drugs for 7 days. Aliquots of culture supernatants were removed from the wells on the 7th day after infection for p24 antigen enzyme immunoassay as an indication of viral replication. Erice et al. Antimicrob. Agents and Chemother. 37, 835 (1993); J. M. Zarling et al. Nature 347, 92 (1990) F. M. Uckun et al., Antimicrob. Agents and Chemother. 42, 383 (1998). Percent inhibition of virus replication was calculated by comparing the p24 antigen values from the test drug-treated infected cells with p24 antigen values from untreated infected cells. The anti-HIV activity of tested compounds was expressed as the IC50 (the final concentration of the compound in culture medium that decreases the replication of HIV-1, by 50%). Cell viability was quantified by a colorimetric MTA assay. F. M. Uckun et al., Antimicrob. Agents and Chemother. 42, 383 (1998).

[0140] To evaluate the potential toxic effects of AZT and various derivatives and to compare with the detergent spermicide nonoxynol-9 (N-9; IGEPAL CO-630; Rhone Poulenc, Cranbury, N.J.), on sperm motility, a highly motile fraction of pooled donor sperm (n=10) was prepared by discontinuous (90-45%) Percoll gradient (Conception Technologies, San Diego, Calif.) centrifugation and “swim-up” method as previously described [O. J. D'Cruz and G. G. Haas Jr., Biol. Reprod. 53, 1118 (1995)]. All donor specimens were obtained after informed consent and in compliance with the guidelines of the Wayne Hughes Institute Institutional Review Board. Motile sperm (≧10×106/ml), were suspended in 0.5 ml of Biggers, Whitten, and Whittingam's medium (BWW) containing 25 mM HEPES (Irvine Scientific, Santa Ana, Calif.), and 0.3% bovine serum albumin (BSA; Fraction V, Sigma Chemical Co, St. Louis, Mo.) in the presence and absence of serial two-fold dilutions of test substance (300 μM to 4.6 μm) in 1% DMSO. The stock solutions of synthetic WHI compounds were prepared in DMSO (10 mg/ml) and diluted in DMSO to yield the desired concentrations. Corresponding volume of DMSO (1%) was added to control tubes. N-9 was diluted in BWW-0.3% BSA (pH 7.4) to yield the desired concentrations (4.6 to 300 μM). After 3 hours of incubation at 37° C., the percentage of motile sperm was evaluated by computer-assisted sperm analysis (CASA) as described [L. J. Burkman, Fertil. Steril. 55, 363 (1991), O. D'Cruz et al., in press, Biol. Reprod. 1998]. The percent motilities were compared with sham-treated control suspensions of motile sperm. The spermicidal activity of test compounds was expressed as the EC50 (the final concentration of the compound in medium that decreases the proportion of motile sperm by 50%). Non-linear regression analyses were used to determine the EC50 values from the concentration effect curves using GraphPad PRISM software (San Diego, Calif.).

[0141] For evaluating sperm motion kinematics using CASA, 4 μl each of sperm suspension was loaded into two 20-μm Microcell chambers (Conception Technologies) that was placed onto a counting chamber at 37° C. At least 5-8 fields per chamber were scanned for analysis using a Hamilton Thorne Integrated Visual Optical System (IVOS), version 10 instrument (Hamilton Thorne Research Inc., Danvers, Mass.). Each field was recorded for 30 seconds. The Hamilton Thorne computer calibrations were set at 30 frames at a frame rate of 30/second; microscope stage temperature maintained at 37° C. with stage warmer; minimum contrast 8; minimum size 6; low-size gate, 1.0; high-size gate, 2.9; low-intensity gate, 0.6; high-intensity gate, 1.4; phase-contrast illumination; low path velocity at 10 μm/second, and threshold straightness at 80%; HTM magnification factor, 1.95. The performance of the analyzer was periodically checked using the play-back function. The attributes of sperm kinematic parameters evaluated included numbers of motile (MOT) and progressively (PRG) motile sperm; curvilinear velocity (VCL; a measure of the total distance traveled by a given sperm during the acquisition divided by the time elapsed); average path velocity (VAP; the spatially averaged path that eliminates the wobble of the sperm head), straight line velocity (VSL; the straight-line distance from beginning to end of track divided by time taken), beat cross frequency (BCF, frequency of sperm head crossing sperm average path), the amplitude of lateral head displacement (ALH; the mean width of sperm head oscillation) and the derivatives, straightness (STR=100×VAP/VCL); linearity (LIN=100×VSL/VCL; departure of sperm track from a straight line). Data from each individual cell track were recorded and analyzed. At least 200 motile sperm were analyzed for each aliquot sampled.

Results

[0142] Physiological fertilization depends on the ability of the ejaculated sperm to swim, bind the zona pellucida, and penetrate the egg. These processes are primarily dependent on sperm motility. Exposure of the highly motile fraction of human sperm to AZT, which inhibited HIV-1 replication in human peripheral blood mononuclear cells in vitro with an IC50 value of 0.006 μm, did not affect sperm motility even at concentrations as high as 300 μm (FIG. 1A). Further, sperm motion kinematics using computer-assisted sperm analysis (CASA) confirmed that AZT treatment did not alter the sperm motion parameters, such as the progressive motility, track speed, path velocity, straight line velocity, straightness of the swimming pattern, linearity of the sperm tracks, beat-cross frequency, and the amplitude of lateral sperm-head displacement. Surprisingly, introduction of a [5-bromo-6-methoxy] substituent on the thymine base ring of AZT to yield 5-bromo-6-methoxy-5,6-dihydro-AZT (compound WHI-01) resulted in gain of significant spermicidal function (EC50=104 μm) without significantly decreasing the anti-HIV activity (FIG. 1A, FIG. 2A). Replacement of the azide group in the pentose ring with an NH2 group (compound WHI-03) enhanced the spermicidal activity (EC50=12 μM), but with a substantially reduced anti-HIV activity.

[0143] The spermicidal activity of the aryl-phosphate substituted derivatives also was evaluated by CASA (FIG. 1B). Compound WHI-04 with the unsubstituted aryl moiety exhibited improved spermicidal activity over WHI-01. Introduction of a p-methoxy (compound WHI-05, EC50 29 μM), p-fluoro (compound WHI-06, EC50=15 μM), or p-bromo (compound WHI-07, EC50 6 μM) substituent in the aryl moiety showed further increases in the spermicidal activity with an order of potentiation p-bromo >p-fluoro >p-methoxy. The p-bromo substitution of the aryl moiety also unexpectedly showed a significant gain in anti-HIV function. The p-methoxy substituted aryl phosphate derivative WHI-05 (5-bromo-6-methoxy-5,6-dihydro-3 ′-azidothymidine-5 ′-(p-methoxyphenyl) methoxyalaninyl phosphate) had an EC50 value of 29 μM in sperm motility assays and an IC50 value of 0.05 μM in HIV replication assays. The compound WHI-07 with a p-bromo substitution (5-bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-(p-bromophenyl)-methoxyalaninyl phosphate) had an EC50 value of 6 μM in sperm motility assays (FIG. 1B, FIG. 2A), which is consistent with a one-log higher potency than that of the detergent spermicide N-9 (EC50=81 μM). WHI-07 displayed a potent anti-HIV activity with an IC50 value of 0.005 μM in HIV replication assays, which was virtually identical to that of AZT (IC50=0.006 μM) and 439-fold more potent than that of N-9 (IC50=2.195 μM) (FIG. 2B).

[0144] The removal of the azido group of the pentose ring (WHI-11 and WHI-12) was associated with a reduction in the anti-HIV activity but an improvement of the spermicidal activity for these AZT derivatives (FIG. 1B).

Example 2

Methods

[0145] To test the effect of duration of incubation on sperm immobilizing activity (SIA) in the presence of AZT derivatives of the present invention (WHI compounds 01, 03, 05, and 07—see FIGS. 1A and B), motile fractions of sperm (107/ml) were incubated at 37° C. in 0.5 ml of BWW-0.3% BSA in the presence of 200 μM WHI-01, or 100 μM each of WHI-03, WHI-05, and WHI-07 or 1% DMSO alone. At timed intervals, duplicate aliquots (4-μl) were transferred to two 20 μm Microcell chambers and sperm motility was assessed by CASA. For WHI-01, CASA were performed every 20 minutes for 180 minutes, for WHI-05, every 10 minutes for 70 minutes, and for WHI-03 and WHI-07 CASA was performed every 5 minutes for 40 minutes.

Results

[0146] The kinetics of sperm immobilization were fast with the present AZT derivatives. See FIG. 1B. The corresponding times required for 50% motility loss of progressively motile sperm exposed to WHI-05 and WHI-07 (at 100 μM concentration) were 17 minutes (95% CI: 12-26 minutes) and 6 minutes (95% CI: 4-11 minutes), respectively. Complete sperm immobilization was achieved after 60 minutes exposure to WHI-05 and 30 minutes of exposure to WHI-07. By comparison, the sperm motility in control samples remained stable (96±2.5% compared to baseline) during the 180 minutes monitoring period.

Example 3

Methods

[0147] The possible effects of seminal plasma on SIA of WHI compounds were studied either in the presence of 10% cell-free seminal plasma in the assay medium or by direct addition (200 μM) of WHI-03, WHI-05, and WHI-07 to dilutions (1:2-1:6) of liquefied donor semen (n=3) in phosphate-buffered saline (PBS) and incubation at 37° C. Following 3 hours of incubation, duplicate aliquots (4-μl) were used for CASA.

[0148] To assess the persistence of SIA after removal of WHI compounds, pooled motile sperm (5×106) were added to 0.5 ml of assay medium in the presence (100 μm) or absence of WHI-01, WHI-05, and WHI-07 in 1% DMSO. After incubation for 30 and 60 minutes at 37° C., duplicate aliquots were used for sperm motility assessment using CASA. The remaining sperm suspension was washed by the addition of fresh assay medium and centrifugation (500× g for 5 minutes). This supernatant was discarded, and the pellet was resuspended in fresh medium (without WHI compounds) to the original volume and reincubated. Following 30 minutes at 37° C., duplicate aliquots were reassessed for sperm motion parameters by CASA. The results were expressed as the mean of two assessments and were compared to the sperm motion parameters of similarly processed sperm suspensions of motile sperm suspended in medium containing DMSO-only controls.

Results

[0149] Direct addition of compounds WHI-05 and WHI-07 to semen diluted in phosphate-buffered saline resulted in complete loss of motility assessed by CASA. Thus, the spermicidal activity was unaffected by the presence of seminal plasma. In order to determine whether the SIA of WHI-05 or WHI-07 was reversible, sperm exposed to 100 μM of either compound for 30 minutes were washed and resuspended in fresh sperm motility assay medium and sperm motility was reassessed by CASA. No recovery in sperm motility was observed, indicating that the drug-induced sperm immobilization was irreversible. The dose- and time-dependent sperm motility loss induced by WHI-05 and WHI-07 was associated with significant changes in the movement characteristics of the surviving sperm, including markedly decreased track speed, path velocity, and straight line velocity.

Example 4

Methods

[0150] Because the great majority of spermicidal compounds are believed to immobilize sperm as a result of a detergent-type action on the sperm plasma membrane, the effects of WHI-05 and WHI-07 on sperm plasma membrane permeability were tested by flow cytometric analysis of propidium iodide (PI)-stained sperm and acrosome integrity by examination of FITC-lectin, TOTO-3 iodide, and Nile red-stained sperm using confocal laser scanning microscopy.

[0151] The percentages of sperm with an intact acrosome were evaluated by fluorescence microscopy (Olympus BX-60) following fixation and ethanol permeabilization of the sperm pellets and subsequent staining with fluorescein (FITC)-conjugated Pisum sativum lectin (Sigma). In positive control sperm, the acrosome reaction was induced by incubating (3 hours at 37° C.) the sperm suspension with 10 μm calcium ionophore (Cal) A23187 (Sigma). Motile sperm (5×106/ml) were incubated for 4 hours at 37° C. in the presence and absence of 100 μM each of WHI-01, WHI-03, WHI-05, WHI-07 or N-9 followed by the addition of PI (10 μg/ml) and analyzed by flow cytometry. The percentage of sperm positive for PI were determined by flow cytometry using a FACS Vantage flow cytometer (Becton Dickinson and Co., Mountain View, Calif.). The sperm in the swim-up fractions were identified by their characteristic forward and 90° angle light scattering properties [O. J. D'Cruz and G. G. Haas, Jr. Fertil. Steril. 58, 633 (1992)]. All analyses were done using the 488-nm excitation from an krypton/argon laser with a 635-nm band pass filter for PI emission. The percentage of sperm positive for PI staining were determined using cutoff signals for membrane-intact motile sperm.

[0152] Ethanol permeabilized and air-dried sperm smears were stained sequentially with the three fluorescent markers, FITC-Pisum sativum, TOTO-3 iodide, and Nile red, each marking a different target (acrosome, nucleus, and plasma membrane of permeabilized sperm respectively). Samples were examined under a BioRad MRC-1024 Laser Scanning Confocal Microscope (BioRad Laboratories, Hercules, Calif.) equipped with a krypton/argon mixed gas laser (excitation lines 488, 568, and 647 nm) and mounted on a Nikon Eclipse E800 series upright microscope. The fluorescence emission of fluorescein, TOTO-3 iodide, and Nile red from the acrosomal region, nucleus, and the plasma membrane of sperm after ethanol permeabilization were simultaneously detected using the 598/40 nm, 522 DF32, and 680 DF32 emission/filter respectively. Confocal images were obtained using a Nikon 60× (NA 1.35) objective lens and Kalman collection filter. Digitized images were processed with the Adobe Photoshop software (Adobe Systems, Mountain View, Calif.). Final images were printed using a Fujix Pictography 3000 (Fuji Photo Film Co., Tokyo, Japan) color printer.

[0153] High-resolution low-voltage scanning electron microscopy was utilized for topographical imaging of different membrane domains over the sperm head as described [M. H. Stoffel et al. Molecular. Reprod. Develop. 34, 175 (1993); S. L. Erlandsen et al. Scanning 11, 169 (1989)]. Aliquots (20×106) of motile sperm were incubated with DMSO alone (1%), or 100 μm each of WHI-05, WHI-07 or 10 μM Cal in DMSO for 3 hours at 37° C. Washed sperm suspensions were placed on 0.1% poly-L-lysine-coated glass chips and allowed to adhere to the glass over a 60 minute incubation period on ice. The supernatants were decanted, and adherent cells were fixed in 1% paraformaldehyde and 1% glutaraldehyde in 0.14 M sodium cacodylate buffer for 3 hours. To preserve plasma membrane integrity, sperm were post-fixed in 1% osmium tetroxide (OSO4) containing 0.1% ruthenium red in 0.14 M cacodylate buffer for 1 hour at 4° C. All samples were dehydrated through an ascending ethanol series, critical point dried, and coated with approximately 2 nm of platinum using ion beam sputtering with argon (4 mA at 10 ke V; Ion Tech Ltd., Middlesex, England). All samples were examined in a Hitachi S-900 SEM at an accelerating voltage of 2 keV. Sperm were observed under low magnification (×2,000-5000) and representative sperm were photographed under intermediate magnification (×18,000-25,000). In each specimen evaluated, at least 200 sperm were scanned for the intactness of sperm acrosomal region.

[0154] For transmission electron microscopy, treated sperm were fixed in 3% glutaraldehyde in 5% sucrose in 0.2 M cacodylate buffer for 3 hours at room temperature. The samples were post-fixed in 1% OSO4, 5% sucrose and 0.1% ruthenium red for 2 hours at 4° C. The samples were dehydrated in ascending ethanol series, and embedded in Spurr's epoxy. Thin sections (90 nm) were prepared on a Reichert-Jung Ultracut E and stained with uranyl acetate and lead citrate. The grids were examined using JEOL-1200 EX II electron microscope (JEOL, Paris) at a accelerating voltage of 60 kV. Sperm were observed under low (×25, 000) and high (×100, 000) magnification, and representative sperm were photographed. In each specimen evaluated, 100-200 sperm were evaluated for the intactness of sperm head membranes.

Results

[0155] Despite complete immobilization of sperm in the presence of WHI-05 and WHI-07, less than 3% of sperm were permeable to PI following 4 hours of incubation with these compounds. In acrosome integrity studies, WHI-05 and WHI-07-treated sperm (similar to sham-treated sperm) remained acrosome-intact (97.0±2.0 and 93.0±5.6 respectively) after 3 hours of incubation at a concentration of 100 μFIG. M, despite a complete loss of motility. Examination of FITC-lectin, TOTO-3, and Nile red-stained sperm by confocal microscopy revealed an intense acrosomal staining with FITC-lectin (green), nuclear staining with TOTO-3 (blue), and membrane staining (red) with Nile red, respectively (FIGS. 3A-3D). In non-acrosome-reacted sperm, the acrosomal regions of the sperm head exhibited a uniform, bright green fluorescence in sperm exposed to vehicle (i.e., 1% DMSO) alone (FIG. 3A), 100 μM WHI-05 (FIG. 3B), and 100 μm WHI-07 (FIG. 3C) for 3 hours. By comparison, sperm exposed to 100 μM of N-9 for 3 hours under identical conditions revealed only acrosome-reacted sperm due to disruption of membrane integrity (FIG. 3D), consistent with previous observations. W. H. Wilborn, D. W. Hahn, J. J. McGuire, Fertil. Steril. 39, 717 (1983); A. S. Bourinbaiar, S. Lee-Huang, Contraception 49, 131 (1994). Thus, the spermicidal activity of the dual-function AZT analogs was not accompanied by a loss of membrane integrity.

[0156] Topographical imaging of drug-treated sperm head by high-resolution low-voltage scanning electron microscopy revealed intact acrosomes (FIGS. 3A′-3D′) with smooth contiguous surfaces in sperm exposed to vehicle (FIG. 3A′) or 100 μM WHI-05 (FIG. 3B′), whereas sperm treated with WHI-07 (FIG. 3C′) revealed signs of a mild acrosomal membrane ruffling. By comparison, sperm exposed to 10 μM Cal A23187 revealed characteristic blebbing or vesiculation, fenestration, and loss of plasma and acrosomal membranes (FIG. 3D′). Transmission electron microscopy of the tangential sections of sperm head confirmed the intactness of the plasma membrane, outer, and inner acrosomal membranes of sperm exposed to vehicle (FIG. 3″), WHI-05 (FIG. 3B″), and WHI-07 (FIG. 3C″) but not to the CaI A23187 (FIG. 3D″) for 3 hours (FIG. 3 bottom panels). Taken together, these results demonstrate that the spermicidal effects of WHI-05 or WHI-07 were not caused by a detergent-type action resulting in disruption of the sperm plasma membrane within the acrosomal region of the sperm head. These features of bromo-methoxy-substituted aryl phosphate derivatives of AZT differ from those of N-9, the most widely used prototype detergent spermicide.

Example 5

Methods

[0157] The human sperm-zona recognition and binding assay appears to predict the in vitro and in vivo fertilization outcome. Therefore, the inhibitory effects of WHI-05 and WHI-07 on sperm-egg interaction were next assessed by laser scanning confocal microscopy using intact human zona and two-colored sperm separately labeled with cell permeant DNA-specific dyes, SYBR 14 (green) and SYTO 17 (blue) in a homologous sperm-zona binding assay. Due to the variable number of sperm binding to human eggs, the sperm-zona binding ratio was used to assess sperm-zona binding capacity.

[0158] Specifically, human eggs that failed to fertilize in vitro were separately inseminated with a mixture of equal numbers of SYBR 14 (green)- and SYTO 17 (blue)-labeled control sperm or test sperm containing equal numbers of SYBR 14- and SYTO 17-labeled sperm, the latter pretreated with three concentrations (25, 50, and 100 μM) of the tested compounds. Following coincubation, the number of tightly bound green (SYBR 14) and blue (SYTO 17) sperm were assessed by laser scanning confocal microscopy. Frozen human eggs (n=46) were thawed and rinsed three times in BWW-0.3% BSA, and in BWW-3.5% BSA, before addition of sperm suspension. The process of egg recovery, freezing, thawing, and manipulation in vitro invariably resulted in cumulus-free, non viable eggs. To investigate the effect of WHI-05 and WHI-07 on the binding of human sperm to human zona, motile fractions of capacitated sperm were divided into two aliquots of 107/ml and labeled with two cell permeant DNA-specific dyes (SYBR 14 and SYTO 17). The first aliquot was incubated with 2.5 μM SYBR 14 (Molecular Probes) and the second aliquot was incubated with 5 μM SYTO 17. Following 30 minutes of incubation, sperm motility was assessed by CASA. Motile sperm with intensely stained green or blue nuclei were washed twice in BWW and one million motile sperm labeled with SYBR 14 (green) and SYTO 17 (blue) were added to dish containing human zona (n=2) under mineral oil and coincubated for 4 hours. Following extensive washing of eggs to dislodge any loosely adherent sperm, the eggs were mounted on glass slides and examined under a BioRad MRC-1024 Laser Scanning Confocal Microscope as described above. Using fluorescence imaging, the fluorescence emission of SYBR-14 and SYTO-17 localized on the sperm head was simultaneously recorded using 598/40 nm, and 680 DF32 emission/filter respectively. Confocal images of Z-sections were obtained using a Nikon 20× (NA 1.4) objective and Kalman collection filter. Digitized images were processed with the Adobe Photoshop software and final images were printed using a Fujix Pictography 3000 color printer. The number of tightly bound green and blue-colored sperm nuclei adherent to each human egg were counted from the Z-sections of fluorescence images of each egg on all focal planes and expressed as a binding ratio.

Results

[0159] The ratio of the number of green and blue sperm bound to human eggs in control and test showed marked differences. The mean sperm-zona binding ratio for control eggs coincubated with SYBR 14 and SYTO 17-labeled control sperm was 1.07. In contrast, mean sperm-zona binding ratios for eggs coincubated with control (SYBR 14) and 25, 50, and 100 μM WHI-07-treated sperm (SYTO 17) were 0.088, 0.039, and 0.009, respectively. The corresponding mean ratios for sperm treated with 25, 50 and 100 μm of WHI-05 were 0.18, 0.07, and 0.10 respectively. FIG. 4A shows the representative sperm-egg binding patterns of green and blue sperm to control egg (A.1) and the test eggs in which the blue sperm were pretreated with either 100 μM of WHI-05 (A.2) or 25 μM of WHI-07 (A.3) prior to coincubation with green-labeled sperm. Despite the variable number of sperm binding to individual zona, a clear reduction in the number of blue colored sperm bound to test eggs is apparent when compared with control egg.

Example 6

Methods

[0160] The heterologous zona-free hamster egg penetration assay appears to correlate with the in vivo fertilizing capacity of human sperm as well as the ability of human sperm to fertilize intact human eggs in vitro. Therefore, the inhibitory effects of WHI-05 and WHI-07 on sperm-egg interaction were further confirmed by zona-free hamster egg penetration assay.

[0161] The fertilizing capacity of human sperm exposed to WHI-05, and WHI-07 was evaluated using zona-free hamster egg penetration assay [Yanagimachi et al. Biol. Reprod. 15, 471 (1976)]. Motile fractions of sperm (n=4) obtained by 90-45% Percoll gradient centrifugation and wash were resuspended in BWW-3.5% BSA medium (pH 7.4) and divided into aliquots of 20×106 sperm/ml and allowed to capacitate for 24 hours. Subsequently, sperm were treated at 37° C. for 3 hours with 25 μM or 100 μm concentrations of WHI-05 and WHI-07 (in 0.5% DMSO) under capacitating conditions. After exposure to drugs, the control and WHI-treated sperm suspensions were washed in BWW-3.5% BSA medium. Sperm motility following capacitation and washing in BWW-3.5% BSA medium was assessed by CASA.

[0162] Frozen hamster eggs (Charles River Laboratories, Wilmington, Mass.) were thawed and transferred to 35 mm glass bottom microwells (Mat Tek Corporation, Ashland, Mass.) in BWW medium. To remove zona, the washed eggs were treated with 0.05% trypsin (Irvine Scientific) in BWW medium for 20 min and then transferred to BWW-3.5% BSA medium and washed three times. Fifteen to 45 zona-free eggs per test were transferred in 100 μl of BWW-3.5% BSA medium under mineral oil. Control and test sperm suspensions (2×106) in <100 Pl of BWW-3.5% BSA medium were added and the gametes were coincubated for 4 hours at 37° C. in 5% CO2 in air. Following incubation, the eggs were washed three times with BWW medium to remove the loosely associated sperm. The eggs were fixed and stained, as previously described [EI-Danasouri et al. Fertil. Steril. 59, 470 (1993)]. Bound and decondensed fluorescent sperm were viewed with a Olympus BX 60 epifluorescent microscope equipped with an excitation filter BP 360-370 and DM 400 barrier filter BA 420. The number of sperm bound per egg, the number of swollen sperm heads per egg, and the percentage of penetrated eggs were determined for control and test sperm. An egg was considered penetrated when it contained at least one swollen sperm head in the cytoplasm.

Results

[0163] Preincubation of capacitated sperm with either compound resulted in significant inhibition (P<0.001) of sperm binding to zona-free eggs (FIG. 4B). Similarly, the penetration rate of human sperm to zona-free hamster eggs following exposure to increasing doses of WHI-05 or WHI-07 showed marked inhibition (41% to 100% inhibition when compared with control). Thus, pretreatment of human sperm with spermicidal aryl phosphate derivatives of AZT resulted in a dose-dependent loss of the ability to bind and penetrate zona-free hamster eggs as well as inhibition of binding to human eggs.

Example 7

Methods

[0164] Because fertilization in vivo is dependent on successful sperm transport through the female genital tract, we next determined whether exposure of sperm to the WHI-07 in vivo affected the subsequent fertility outcome. Hormonally primed adult female Swiss (CD-1) mice were artificially inseminated with motile epididymal sperm via the cervix with and without prior intravaginal application of WHI-07 in a cream base. Females were examined 8 days later for the presence or absence of embryos in uteri.

[0165] More particularly, adult (5-6 months old) female CD-1 mice were superovulated by an intraperitoneal injection (i. p.) of 5 IU of pregnant mare's serum gonadotropin followed by an i.p. injection of 5 IU of human chorionic gonadotropin 46-48 hours later. Mice were randomly assigned to one of the two treatment groups (27 or 24/group); vehicle controls [cream base (Taro Pharmaceuticals, Hawthorne, N.J.) with 1% DMSO], or test group (cream base with 1% WHI-07 in 1% DMSO) and given intravaginally (50 μl) prior to artificial insemination. Caudae epididymal sperm were obtained from proven breeder adult CD-1 male (5-6 months old) [B. Hogan, R. Beddington, F. Costantini, E. Lacy, Eds., Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Plainview, N.Y., 1994] and suspended in a modified Krebs Ringer-bicarbonate medium (M2; Gibco-BRL) supplemented with pyruvate, lactate, and glucose. Sperm were capacitated in M2 medium-5% BSA for 1 hour prior to use. For each experiment cauda epididymal sperm pooled from 25 male mice and adjusted to 1-2×106 motile sperm/50 μl were used for insemination. A 50 μl volume/mouse was ejected through a 1 ml syringe with a blunted 18 gauge needle. Preparations were analyzed for sperm concentration, motility, and sperm motion parameters by CASA using software designed for oval sperm head morphology (Hamilton Thorne). On day 8, individual females representing the control and test group were killed and their uteri were examined for the presence or absence of embryos. A total of three independent fertility experiments were performed.

Results

[0166] The results of three fertility trials are summarized in Table 1. In mice given WHI-07 intravaginally prior to artificial insemination, the percentage of fertility rate was drastically reduced when compared to controls (7.6% vs 38.4%; P<0.001) indicative of the inability of WHI-07-exposed sperm to reach the site of fertilization.

1TABLE 1Fertility of female mice after artificial insemination of sperm via cervixwith and without intravaginal application of WHI-07.No. of control miceNo. of WHI-07-Exper-No. of micefertile†treated mice fertileimentinseminated/group*(%)(%)12710 (37.0)2 (7.4)22711 (40.7)1 (3.7)324 9 (37.5) 3 (12.5)Total7830 (38.4)6 (7.6)‡*Animals were sacrificed on day 8 of the expected pregnancy. †Fertility was determined by examining the uteri for the presence or absence of embryos. ‡Significantly different from control group (p < 0.001).

Example 8

Methods

[0167] Any proposed improvement in vaginal contraceptives should take into account the non-specific membrane toxicity caused by the detergent-type action of the currently available vaginal contraceptives. This can damage the vaginal tissues, rendering the subject more susceptible to infection by sexually-transmitted diseases such as HIV. Therefore, the local tissue alterations and inflammatory response to repetitive intravaginal application of N-9 versus WHI-07 in mice was compared. Two groups of 15 adult female Swiss (CD-1) mice were treated for 5 (group A) or 20 (group B) consecutive days with either 5% N-9, 5% WHI-07, or control vehicle in a cream base, and the cervicovaginal tissue sections were examined for histopathological changes and influx of inflammatory cells (FIG. 5). The cervicovaginal region consisted of a stratified squamous epithelium overlying a vascular submucosa. The thickness of the epithelium varied depending on the four stages of the estrous cycle.

[0168] In particular, 30 female CD-1 mice, 6 months old were randomly divided into two groups (A and B). Each group was further divided into three subgroups of 5 mice. Mice from group A were treated for 5 days and mice from group B for 20 days via daily intravaginal application of a cream base (Taro Pharmaceuticals) supplemented with 1% DMSO alone (control), 5% WHI-07 in 1% DMSO or 5% N-9. After 5 and 20 days respectively, mice were killed by cervical dislocation, and tissues from the genitourinary tract were fixed in 10% buffered formalin. To determine the degree of inflammation and membrane integrity of squamous epithelia, conventional paraffin-embedded sections (6-pm) were prepared and stained with hematoxylin and eosin and observed under 300× magnification with a Leica microscope interfaced with an image analysis system (Scion), and images were transferred to Adobe Photoshop software for printout. The four stages of the estrous cycle were determined histologically.

[0169] Indirect immunofluorescence assay and confocal microscopy were used to identify the neutrophil infiltrates in the squamous epithelia of cervicovaginal sections of control, WHI-07, and N-9-treated mice. Cervicovaginal tissue sections were deparaffinized, hydrated through graded ethanol, and immunostained with a rat mAb (Cederlane Laboratories Ltd., Westbury, N.Y.) directed against mouse neutrophils (clone 5120-26.1-110). Following antigen retrieval by heating (93° C. for 10 min in 10 mM sodium citrate buffer, pH 6.0), sections were preincubated with 10% normal goat serum in PBS, treated with optimal dilution (5 μg/ml) of the mAb followed by FITC-conjugated goat anti-rat IgG (Pierce Chemical Co., Rockford, IL). Each incubation step lasted 60 min with 5 min PBS washes between each step. The slides were counterstained with PI (1 μg/ml), washed in deionized water, mounted in anti-fade (BioRad), and visualized by laser confocal microscopy as described above. Negative controls were carried out by replacing the primary mAb with PBS or using rat IgG2 as control mAb

Results

[0170] Similar to the 10 vehicle alone-treated control mice, none of the 10 mice treated with WHI-07 for 5 days or 20 days exhibited any significant inflammatory response or membrane disruption of the squamous epithelia (FIG. 5 A and E). By contrast, disruption of the epithelial lining and an inflammatory response with influx of neutrophils in the squamous epithelia of cervicovaginal crypts were evident in 9 of 10 mice given N-9 intravaginally (FIGS. 5B and E), consistent with previously published observations in rats. See Tryphonas and Buttar, Toxicol. Lett., 20, 289 (1984). Two color laser scanning confocal fluorescence images of the cervicovaginal epithelia of WHI-07 and N-9-treated sections with a monoclonal antibody specific to mouse neutrophils revealed absence of neutrophils in the stratified squamous epithelial crypts of WHI-07-treated specimens (FIG. 5C) and intense positive staining (green color) in the squamous epithelia of N-9-treated tissue section (FIG. 5D). These studies demonstrate that, unlike N-9 treatment, intravaginal application of WHI-07 in a cream base does not cause any membrane disruption or an acute inflammatory response in the cervicovaginal epithelial crypts.

Example 9

[0171] This Example presents a study that reports the structural as well as functional characteristics of novel analogues of WHI-07 which we synthesized by replacing the Ala side chain of WHI-07 with different amino acids containing nonpolar side chains.

Methods and Materials

[0172] Reagents and Instrumentation

[0173] All of the anhydrous solvents and chemical reagents were purchased from Aldrich Chemical Co. (Milwaukee, Wis.) except for AZT (also called ZDV) which was obtained from Toronto Research Chemicals Co. (Ontario, Canada). Proton nuclear magnetic resonance (1H NMR), carbon (13C), phosphorous (31P), and fluorine (19F) NMR spectra were recorded on a Varian Oxford 300 MHz spectrometer (Varian Associates, Palo Alto, Calif.) using an automatic broad band probe. All NMR spectra were recorded in CDCl3 at room temperature. The chemical shifts were recorded as 6 values in parts per million (ppm) downfield from tetramethyl silane (δ=0.0 ppm) as internal standard or from the residual chloroform signal (δ=7.24 ppm for 1H NMR or δ=77.0 ppm for 13C NMR). For 31P NMR, a solution of 1% phosphoric acid contained in a sealed capillary served as a internal standard and the chemical shifts were recorded relative to this standard. In the case of 19F NMR, a fused capillary having 1% solution of trifluoroacetic acid in water was used as an internal standard. The multiplicity of the signals were designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad peak. Ultraviolet (UV) spectra were recorded from a Beckmann Model DU 7400 UV/visible spectrophotometer (Beckman Instruments, Fullerton, Calif.) using a cell path length of 1 cm. Fourier Transform Infra Red (FT-IR) spectra were recorded using an FT-Nicolet model Protege 460 instrument (Nicolet Instrument Corp., Madison, Wis.). The IR spectra of the liquid samples were run as neat liquids using KBr discs. Mass spectra analysis were performed using two different instruments. Some samples were analyzed using a Finnigan MAT 95 instrument (Madison, Wis.) after dissolving in chloroform. The source temperature was maintained at 200° C. The accelerating voltage was set at 5000 V and the number of scans were approximately 70 for each sample. In addition, a Hewlett-Packard Matrix Assisted Laser Desorption (MALDI) spectrometer model G2025A (Hewlett Packard Co., Wilmington, Del.) was used in the molecular ion detection mode as a quick reference. The matrix used was cyano hydroxy cinnamic acid. Analytical HPLC was performed using a Hewlett Packard 1100 series system consisting of a diode array detector, a quarternary pump, an automatic degasser, and a thermostat controlled column compartment along with a Chem station software. In addition, an automatic injection assembly was used for repetitive analysis. A reverse-phase LiChrospher column (250×4 mm, Hewlett-Packard, RP-18) was used and the analysis was performed using a isocratic flow consisting of water and acetonitrile (70%:30%). The flow rate was maintained at 1 ml/min throughout the analysis. Column chromatography was performed using silica gel.

[0174] Synthesis of WHI-07 and its Analogues

[0175] WHI-07 was synthesized as shown in Scheme 2 in four steps by sequentially replacing the three chlorides in phosphorus oxychloride 1 with 4-bromophenol 2 (R2=Br), L-alanine-methyl ester 3 (R1=CH3) and ZDV to produce the 5′-phosphorylated-ZDV 4, followed by the addition of bromo and methoxy groups across the double bond in the thymidine moiety of 4. [Jan S-T, et al., Antivir Chem Chem 1999; 10:39-46; D'Cruz O J, et al., Mol Hum Reprod 1999; 5:421-432; and D'Cruz O J, et al., Contraception 1999; 59 (in press)]. Accordingly, the new WHI-07 analogues were synthesized by (i) replacing L-alanine-methyl ester in the second step with the methyl ester derivatives of various amino acids such as Trp, Pro, Phe, Leu, Met, Val or Gly (R1=the side chain of Trp, Pro, Phe, Leu, Met, Val or Gly) (ii) and/or replacing 4-bromophenol in the first step with different phenols (R2=H, F, Br, Cl, OCH3 or NO2).

[0176] For the synthesis of Trp substituted phosphoramidate derivatives, a modification was introduced in which after condensing the amino acid in methylene chloride solution, the resulting residue was solubilized in anhydrous THF instead of ether. All the 23 new WHI-07 analogues synthesized as shown in Scheme 2 were characterized by 1H, 13C and 31P NMR, FT-IR, UV-visible, mass spectra, and HPLC analyses. For each compound, the purity was >98%.

[0177] Examples of compounds synthesized in this example are shown in tables 2, 3, and 4, below. As shown in the Tables, the compounds are correspondingly labled compounds 1a-e, 2a-d, 3a-d, 4a-d, 5a-c, 6a-b, 7a-b, and 8a, and will be referred to as such in the text.

[0178] In Vitro Sperm Immobilizing Activity (SIA)

[0179] All donor specimens were obtained after informed consent and in compliance with the guidelines of the Hughes Institute Institutional Review Board. To evaluate the spermicidal effects of ZDV derivatives, highly motile fraction of pooled donor sperm (n 8) were prepared from liquefied normospermic semen by discontinuous (90-45%) gradient centrifugation using Enhance-S-Plus (Conception technologies, San Diego, Calif.) cell isolation medium as previously described. [D'Cruz O J, et al., Biol Reprod 1999; 60:1419-1428; D'Cruz O J, et al., Biol Reprod 1995; 53:118-1130; and D'Cruz O J, et al., Biol Reprod 1998; 58:1515-1526]. The resulting pellet was washed twice and resuspended in 1-ml of Biggers, Whitten, and Whittingam's medium (BWW) (Irvine Scientific, Santa Ana, Calif.) containing 3% BSA (fraction V; Sigma Chemical Co., St. Louis, Mo.). One-ml aliquots of the sperm suspension were centrifuged (500× g, 5 min) and incubated at 45° for 90 min at 37° C. in 5% CO2 atmosphere. The supernatant containing primarily motile sperm was aspirated carefully, washed once in BWW medium containing 25 mM HEPES and 0.3% BSA and resuspended in the same medium. Pooled motile sperm (=10×106/ml) prepared from 3-5 donors were suspended in 1-ml of BWW-0.3% BSA in the presence and absence of serial 2-fold dilutions of test substance (1.9 μm to 500 μM) in 0.5% dimethyl sulfoxide (DMSO). The stock solutions of synthetic compounds were prepared in DMSO (100 mM) and diluted in medium to yield the desired concentrations. A corresponding volume of DMSO (0.5%) was added to control tubes. After 3 hour incubation at 37° C., the percentage of motile sperm was evaluated by computer-assisted sperm motion analysis (CASA) [D'Cruz O J, et al., Biol Reprod 1998; 58:1515-1526; D'Cruz O J, et al., Mol Hum Reprod 1998; 4:683-693; D'Cruz O J, et al., Biol Reprod 1999; 60:435-444]. The percentage motilities were compared with those of sham-treated control suspensions of motile sperm. The spermicidal activity of test compounds was expressed as the mean EC50 values (the final concentration of the compound in the medium that decreases the proportion of motile sperm by 50%) calculated from three independent experiments.

[0180] Sperm Kinematic Parameters

[0181] For CASA, 4 μl of each sperm suspension were loaded into a 20-μm Microcell chamber at 37° C. Eight to ten fields per chamber were scanned for analysis using a Hamilton Thome Integrated Visual Optical System (IVOS), version 10 instrument (Hamilton Thome Research Inc., Danvers, Mass.) [see, D'Cruz O J, et al. Biol Reprod 1998; 58:1515-1526; and D'Cruz O J, et al., Mol Hum Reprod 1998; 4:683-693; and D'Cruz O J, et al., Biol Reprod 1999; 60:435-444]. Each field was recorded for 30 sec. The computer calibrations were set at 30 frames at a frame rate of 30/s. Other settings were as follows: minimum contrast 8; minimum size 6; low-size gate, 1.0; high-size gate, 2.9; low-intensity gate, 0.6; high-intensity gate, 1.4; phase-contrast illumination; low path velocity at 10 μm/s, and threshold straightness at 80%; magnification factor, 1.95.

[0182] The sperm kinematic parameters that were determined included numbers of motile (MOT) and progressively (PRG) motile sperm; curvilinear velocity (VCL; a measure of the total distance traveled by a given sperm during the acquisition divided by the time elapsed); average path velocity (VAP; the spatially averaged path that eliminates the wobble of the sperm head), straight line velocity (VSL; the straight-line distance from beginning to end of track divided by time taken), beat cross frequency (BCF, frequency of sperm head crossing sperm average path), the amplitude of lateral head displacement (ALH; the mean width of sperm head oscillation), straightness (STR=VSL/VAP×100); linearity (LIN=VSL/VCL×100; departure of sperm track from a straight line). Data from each individual cell track were recorded and analyzed. At least 200 motile sperm were analyzed for each aliquot sampled.

[0183] In Vitro Assay of Anti-HIV-1 Activity

[0184] The HIV-1 strain, HTLVIIIB, which was propagated in CCRF-CEM cells, was used for in vitro assays of the anti-HIV-1 activity of selected novel WHI-07 analogues. Cell-free supernatants of HTLVIIIB-infected CCRF-CEM cells were harvested, dispensed into 1 ml aliquots, and frozen at −70° C. Titration of stock virus was performed by examining its cytopathic effects in MT-2 cells. [see, Uckun FM, et al., Antimicrob Agents Chemther 1998; 42:383-388]. Normal human peripheral blood mononuclear cells (PBMC) from HIV-negative donors were cultured for 72 h in RPMI 1640 supplemented with 20%(v/v) heat-inactivated fetal bovine serum (FBS), 3% interleukin-2, 2 mM L-glutamine, 25 mM HEPES, 2 g/L NaHCO3, 50 μg/ml gentamicin, and 4 μg/ml phytohemagglutinin prior to exposure to HIV-1 at a multiplicity of infection of 0.1 during a 1 h adsorption period at 37° C. in a humidified 5% CO2 atmosphere. Subsequently, cells were cultured in 96-well microtiter plates (100 μl/well; 2×106 cells/ml) in the presence of various concentrations (0.001 to 100 μM) of spermicidal WHI-07 and its Trp-containing analogues (2a, 2b, and 2d). Aliquots of culture supernatants were removed from the wells on the seventh day after infection for p24 antigen assays, as previously described. [Uckun F M, et al., Antimicrob Agents Chemther 1998; 42:383-388; and Zarling J M, et al., Nature 1990; 347:92-95]. The p24 enzyme immunoassay applied was the unmodified kinetic assay commercially available from Coulter Corporation/Immunotech, Inc. (Westbrook, Me.). This assay utilizes a murine monoclonal antibody to HIV core protein coated onto microwell strips to which the antigen present in the test culture supernatant binds. Percent viral inhibition was calculated by comparing the p24 values from the drug-treated infected cells with p24 values from untreated infected cells (i.e., virus controls). The effects of various treatments on cell viability were also examined. Non-infected PBMNC were treated with WHI-07 analogues for 7 days under identical experimental conditions. A microculture tetrazolium assay, using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide, was performed to quantitate cellular proliferation.

[0185] In Vitro Cytotoxicity Assay

[0186] The potential cytotoxicity of aryl phosphate derivatives of bromo-methoxy ZDV in comparison to N-9, against normal human ectocervical and endocervical epithelial cells (Clonetics Corporation, San Diego, Calif.), was measured using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell viability assays (Boehringer Mannheim Corp., Indianapolis, Ind.) [see, D'Cruz O J, et al., Mol Hum Reprod 1999; 5:421-432; D'Cruz O J, et al., Contraception 1999; 59 (in press); and Uckun F M, et al., Antimicrob Agents Chemther 1998; 42:383-388]. Briefly, exponentially growing ectocervical and endocervical epithelial cells were seeded into 96-well plate at a density of 2×105 cells/well and incubated for 24 h at 37° C. prior to drug exposure. On the day of treatment, culture medium was carefully aspirated from the wells and replaced with fresh medium containing drug concentrations ranging from 3.9 μM to 1000 μm. Triplicate wells were used for each treatment. The cells were incubated with the various compounds for 24 hours at 37° C. in a humidified 5% CO2 atmosphere. To each well, 10 μl of MTT (0.5 mg/ml final concentration) was added and the plates were incubated at 37° C. for 4 hours to allow MTT to form formazan crystals by reacting with metabolically active cells. The formazan crystals were solubilized overnight at 37° C. in a solution containing 10% SDS in 0.01 M HCl. The absorbance of each well was measured in a microtiter reader at 540 nm and a reference wavelength of 690 nm. To translate the OD540 values into the number of live cells in each well, the OD540 values were compared to those of standard OD540 versus cell number curves generated for each cell line. The percent cell survival was calculated using the formula: % survival=live cell number[test]/live cell number[control]×100. The results were expressed as IC50 values. The IC50 was defined as the concentration required for 50% reduction in cell survival. Two separate experiments were performed in triplicate to assess the potential cytotoxicity of spermicidal WHI-07 analogues against ectocervical and endocervical epithelial cells.

[0187] Statistical Analysis

[0188] Results are presented as the mean or mean ±SD values from independent measurements. Non-linear regression analysis was used to find EC50 and IC50 values from the concentration-effect curves using GraphPad Prism (version 2.0) software (San Diego, Calif.). Correlations between EC5o values versus IC50 values and sperm motility versus VCL, VAP, and VSL were calculated using the Pearson's correlation coefficient; significance was set at p<0.05.

Results

[0189] Contributions of the Ala Side Chain and Para-bromo Substituent of WHI-07 to the Potency of its SIA

[0190] WHI-07 is a potent spermicidal agent (EC50=5 μM). This novel agent is a phenyl phosphate derivative of ZDV and the presence of bromo-methoxy functional groups in the thymine ring of its ZDV moiety is preferred for its SIA. Although the phenyl phosphate moiety of WHI-07 is also preferred for its activity, very little is known about the importance of esterification of the phosphate group with an Ala residue. In order to determine the importance of the Ala side chain of its phenyl phosphate moiety, we have synthesized new analogues of WHI-07 by replacing the Ala side chain (R1) with different amino acids containing nonpolar side chains, namely, Val, Leu, Met, Pro, Trp, Phe, and Gly. The para substituents (R2) on the phenyl moieties of these analogues included Br, Cl, F, NO2, and OMe groups. The assessment of the SIA of these compounds using CASA demonstrated that the choice of the amino acid side chain is a factor for the SIA of the phenyl phosphate derivatives of ZDV. Notably, the replacement of the Ala side chain with Val, Leu, Pro or Phe led to a complete loss of SIA with EC5o values of >500 μm, whereas a replacement with Trp resulted in a 4-fold loss (EC50=20 μM; as seen in Table 2).

2TABLE 2

CompoundR1 (methyl ester)EC50(μM)1a

5 ± 2Ala2a

20 ± 6 Trp3a

>500Pro4a

>500Phe5a

>500Leu

[0191] Table 2 shows the effect of replacement of amino acid side chain on the spermicidal activity of aryl phosphate derivatives of bromo-methoxy ZDV. The SIA of the listed compounds was evaluated by CASA as described in the Methods and Materials. The EC50 values represent the concentration required to decrease sperm motility by 50% as measured from the concentration-response curves using CASA. EC50 values are mean ±SD of three separate experiments.

[0192] The Ala Side Chain and the Bromo Substituent on the Aryl Moiety Contribute to Maximal SIA of Aryl Phosphate Derivatives of Bromo-methoxy ZDV

[0193] No improvement in SIA could be achieved by replacement of the para-bromo substituent in the phenyl ring with other electron-withdrawing groups (F, Cl, NO2) regardless of the choice of the amino acid side chain (see Table 3). Table 3 shows the effect of replacement of amino acid side chain and addition of various substituents at the C-5 position of phenyl ring on the spermicidal activity of aryl phosphate derivatives of bromo-methoxy ZDV. The SIA of the listed compounds was evaluated by CASA as described in the Materials and Methods. The EC50 values calculated from the concentration-response curves are shown. EC50 values are mean +SD of three separate experiments.

3TABLE 3

CompoundR1 (methyl ester)R2EC50(μM)1b 1c 1d 1e

H F OMe NO2 35 ± 5 15 ± 3 24 ± 2 118 ± 11Ala2b 2c 2d

H Cl NO2 10 ± 1 >500 39 ± 3Trp3b 3c 3d

H OM3 NO2 61 ± 2 66 ± 6 80 ± 5Pro4b 4c 4d

H Cl NO2>500 >500 >500Phe5b 5c

Cl NO2>500 >500Leu6a 6b

H OM3 95 ± 8 71 ± 5Met7a 7b

H Cl>500 >500Val8a

NO2 116 ± 12Gly

[0194] However, many of the compounds with para substitution (R2) other than a bromine group exhibited SIA (see Table 2, and FIGS. 6A-C). Thus, both the Ala side chain and the para-bromo substitution of the phenyl phosphate moiety while neither essential for the SIA of WHI-07, uniquely contribute to its potency. Specifically, both the unsubstituted and substituted compounds (compounds 1a-e) exhibited SIA with EC50 values ranging from 5 to 118 μM (see Tables 2, 3, and FIG. 6A). This property was also observed with the unsubstituted (H) and/or substituted aryl phosphate derivatives of bromo-methoxy ZDV containing Trp, compounds 2a, 2b, and 2d. (see Tables 2, 3, and FIG. 6B), Pro, compounds 3b-d (see Tables 2, 3, and FIG. 6C), Meth, compounds 6a, 6b, and Gly, compound 8a, as the amino acid ester (see, Table 3). Whereas, both the unsubstituted and para substituted aryl phosphate derivatives ZDV containing Phe, compounds 4a-d, Leu, compounds 5a-c, Val, compounds 7a, 7b, and the chloro substituted derivative of Trp-containing side chain, compound 2c, lacked SIA (see, Table 3 and FIG. 6B). Interestingly, while the para-bromo substituted Ala compound WHI-07 was more potent than the unsubstituted Ala analogue (EC50: =5 μM versus 35 μM), the opposite was true for the Trp (EC50: =20 μm versus 10 μm) and Pro (EC50: =>500 μM versus 61 μM) containing compounds (see, Table 3). Among the Ala-containing WHI-07 analogues, the NO2 substituted compound was the least active. The potency order among the different compounds with various amino acid side chains and with or without para substituents on the phenyl ring was Br >F >OMe >H >NO2 for Ala-containing compounds, H >Br >NO2 >Cl for Trp-containing compounds, H >OMe >NO2 >Br for Pro-containing compounds, and OMe >H for Met-containing compounds.

[0195] The concentration-dependent SIA induced by WHI-07 analogues was associated with significant changes in the movement characteristics of the surviving sperm particularly with respect to track speed (VCL), path velocity (VAP), and straight line velocity (VSL). FIGS. 7A-C show the concentration-dependent changes in sperm kinematic parameters obtained for representative spermicidal Ala [A], Trp [B], and Pro [C] analogues of WHI-07 with unsubstituted phenyl moiety. All three spermicidal analogues of WHI-07 irrespective of their amino acid variation showed identical concentration-dependent changes in progressive motility which was accompanied by a parallel decline in VCL, VAP, and VCL (r2 0.889-0.936). The sperm motion parameters of control sperm showed no significant changes during the 180 min incubation period.

[0196] Diminished Anti-HIV Activity of Trp-Containing WHI-07 Analogues

[0197] Interestingly, while the para-bromo substituted Ala compound WHI-07 was the most potent (IC50: =0.005 μM), the opposite was true for the Trp-containing compounds (see, Table 4). The order of efficacy for Trp-containing WHI-07 analogues, compounds 2a, 2b, and 2d, was NO2>H>Br. In an attempt to enhance the anti-HIV activity of compound 2a, we replaced the para-bromo substituent in the phenyl ring with a stronger electron withdrawing para NO2 substituent, compound 2d. Although compound 2d had improved anti-HIV activity than compound 2a, it was 8-fold less potent than that of WHI-07.

[0198] Aryl Phosphate Derivatives of Bromo-methoxy ZDV are Selectively Spermicidal

[0199] The MTT assay measuring the cell proliferation and viability was used to test the in vitro cytotoxicity of novel spermicidal aryl phosphate derivatives of bromo-methoxy ZDV versus N-9 against confluent monolayers of normal human ectocervical and endocervical epithelial cells. Cells were exposed to these compounds at doses ranging from 3.9 μM to 1 mM for 24 h. Table 4 compares the mean IC50 (50% growth inhibitory calculated from the MTT assay-based concentration-response cell survival curves) values for these compounds with the mean EC50 values measured by CASA.

4TABLE 4Cytotoxic activity of spermicidal aryl phosphate derivatives of bromo-methoxy ZDV against normal human ectocervical and endocervical epithelial cells.SpermHTLVIIIBEctocervicalEndocervicalEC50IC50cellscellsCompdR1R2(μM)a[p24](μM)bIC50 (μM)c,dSIeIC50 (μM)SI1aAlaBr50.005>1000>200312 ± 3862.41bAlaH35ND>1000>28.5>1000>28.51cAlaF15ND304 ± 7920.2 939 ± 15762.61dAlaOMe24ND>1000>41.6388 ± 3816.11eAlaNO2118ND467 ± 5 3.9428 ± 783.62aTrpBr200.340430 ± 8 21.5>1000>50.02bTrpH100.140445 ± 3144.5934 ± 6293.42dTrpNO2390.040>1000>25.6>1000>25.63bProH61ND327 ± 8 5.3>1000>16.33cProOMe66ND481 ± 627.2 552 ± 2308.33dProNO280ND>100012.5 501 ± 1836.26aMetH95ND 534 ± 1045.6 856 ± 1539.06bMetOMe71ND307 ± 244.3418 ± 155.88aGlyNO2116ND>1000>8.6>1000>8.6N-9NAfNA81ND22 ± 80.2716 ± 50.19aEC50, Drug concentration inhibiting sperm motility by 50%; data are mean of three experiments. bIC50, Drug concentration inhibiting HIV-1 replication in PBMC by 50% as measured by the p24 antigen production assay. ND, not determined. cIC50, Drug concentration inhibiting cell growth by 50%; data are mean ± SD of two experiments. dConfluent monolayer cultures in multiwell plates were incubated in the absence and presence of increasing two-fold concentrations (3.9 μM to 1000 μM) of 14 spermicidal ZDV derivatives or N-9 for 24 h at 37° C. Cell proliferation was measured by the MTT assay. eSI, Selectivity index is equal to the ratio of IC50 to EC50. fNA, Not applicable.

[0200] In MTT assays, N-9 exhibited significant cytotoxicity to ectocervical epithelial and endocervical epithelial cells with mean IC50 values of 22±8 μM and 16±5 μM, respectively. By comparison, the IC50 values for spermicidal ZDV derivatives against normal human ectocervical and endocervical epithelial cells were >300 μM (see, Table 4). Thus, N-9 was spermicidal only at cytotoxic concentrations (EC50 value: 81 μM; selectivity indices [SI]: 0.27 and 0.19 for ectocervical and endocervical epithelial cells, respectively), whereas even the most potent spermicidal derivatives of ZDV showed very little toxicity to epithelial cells. None of the ZDV derivatives were cytotoxic to female reproductive tract epithelial cells at spermicidal EC50 concentrations. Thus, none of the amino acid side chain replacements orpara-position substituents in the phenyl ring resulted in increased cytotoxicity against ectocervical or endocervical epithelial cells. Also, there was no correlation between EC50 values and IC50 values for ectocervical epithelial cells (r2=0.0011; n=14) and endocervical epithelial cells (r2 0.0094; n=14). The absence of a linear relationship between the IC50 and EC50 values for the 14 spermicidal compounds tested in our study illustrates that the SIA of this class of compounds was not caused by a nonspecific cytotoxic function (FIG. 8).

Discussion

[0201] The present example extends our previous reports of spermicidal ZDV derivatives by incorporating the bromo-methoxy substituents on the double bond of the thymine ring of ZDV while varying the amino acid ester as well as the substituents on the phenyl ring of novel aryl phosphate derivatives of ZDV. We synthesized and tested a total of 23 novel aryl phosphoramidate derivatives of bromo-methoxy ZDV for SIA. Our structure-activity relationship studies revealed the following requirements for potent and selective SIA: (i) a C-5 bromo and C-6 methoxy functionalization on the thymine ring; (ii) an Ala side chain; and (iii) a bromo group on the C-5 position of phenyl ring.

[0202] We compared the influence of both aliphatic and aromatic amino acid side-chains on SIA of phosphoramidate derivatives of bromo-methoxy ZDV. Among the eight amino acid esters studied, Ala is the most effective phosphoramidate group. The hydrophobic methyl group on the Ala side chain may not confer advantage since methyl group-containing Leu and Val analogues [compounds 5a-c and 7a, 7b] were devoid of SIA. Because these amino acids with larger hydrophobic side chains than Ala lacked SIA suggests that the spatial bulk of the amino acid side chain may be one of the determinant for their SIA. The Met analogues [compounds 6a, 6b] were also less active than the Ala analogues with SIA rather similar to the Pro derivatives [compounds 3b-d]. Since the Pro analogues [compounds 3b-d] with blocked amino group retained SIA, although less active (12-16 fold) than the lead compound [compound 1a], the SIA appears to be not dependent on the amino group either. This is also supported by the variable potency of Trp analogues [compounds 2a, 2b, and 2d] with enhanced steric bulk in the side chain which were somewhat less active [2-8 fold] than the Ala lead compound [compound 1a]. The potency of SIA of Trp derivatives [compounds 2a, 2b, and 2d] can be attributed to the presence of a indole ring with an amino group which can facilitate faster intracellular entry as well as to the presence of two bromo-methoxy substitutions in Trp analogues. Comparative structure-activity relationship studies of Ala and Trp-containing analogues of WHI-07 unequivocally established the importance of Ala side chain and the contribution of a electron withdrawing para-bromo substituent on the phenyl ring in addition to the bromo-methoxy functionalization for the potent, dual-function anti-HIV and spermicidal activities.

[0203] Our finding that aryl phosphate derivatives of bromo-methoxy ZDV inhibit sperm motility without affecting the female reproductive tract epithelial cells suggests that these membrane permeable dual-function nucleoside analogues may provide the basis for a new strategy aimed at prevention of the sexual transmission of HIV while providing fertility control for women.

[0204] The substituted pyrimidine derivatives of the present invention unexpectedly exhibit potent spermicidal and anti-HIV activity profiles. As a result, they may be useful as contraceptives capable of preventing the sexual transmission of HIV, especially as vaginal contraceptives. As such, the AZT derivatives of the present invention will be particularly useful for providing fertility control for women who are at high risk for acquiring HIV by heterosexual transmission.

[0205] While a detailed description of the present invention has been provided above, the invention is not limited thereto. The invention described herein may be modified to include alternative embodiments, as will be apparent to those skilled in the art. All such alternatives should be considered within the spirit and scope of the invention, as claimed below.

[0206] The specification includes many references to patents and published literature, each of which is hereby incorporated by reference, for all purposes, as if fully set out herein.

	Number	Date	Country
Parent	09047609	Mar 1998	US
Child	09497297	Feb 2000	US

	Number	Date	Country
Parent	09497297	Feb 2000	US
Child	09728762	Dec 2000	US

AZT derivatives exhibiting spermicidal and anti-viral activity

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