Innovative Disulfide Esters of Dithiocarbamic Acid as Women-Controlled Contraceptive Microbicides: A Bioisosterism Approach.

In an ongoing effort to discover an effective, topical, dual-function, non-surfactant contraceptive vaginal microbicide, a novel series of 2,2'-disulfanediylbis(3-(substituted-1-yl)propane-2,1-diyl) disubstituted-1-carbodithioates were designed by using a bioisosterism approach. Thirty-three compounds were synthesized, and interestingly, most demonstrated multiple activities: they were found to be spermicidal at a minimal effective concentration of 1-0.001 %, trichomonacidal against drug-susceptible and resistant Trichomonas strains at minimal inhibitory concentration (MIC) ranges of 10.81-377.64 and 10.81-754.14 µM, respectively, and fungicidal at MIC 7.93-86.50 µM. These compounds were also found to be non-cytotoxic to human cervical (HeLa) epithelial cells and vaginal microflora (Lactobacilli) in vitro. The most promising compound, 2,2'-disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl)dipyrrolidine-1-carbodithioate (5), exhibited spermicidal activity 15-fold higher than that of the marketed spermicide Nonoxynol-9 (N-9) and also demonstrated microbicidal potency. To identify common structural features required for spermicidal activity, a 3D-QSAR analysis was carried out, as well as in vivo efficacy studies and fluorescent labeling studies to determine the biological targets of compound 5.

Synthesis, Cytotoxic and Contraceptive Activity of 6,8,9-Trihydroxy-2-methyl-2H-naphtho[2,3-b]pyran-5,10-dione, a Pigment of Echinothrix diadema, and its Analogs.

6,8,9-Trihydroxy-2-methyl-2H-naphtho[2,3-b]pyran-5,10-dion, a pigment of the sea urchin Echinothrix diadema, and six analogs were synthesized. The cytotoxic activity and contraceptive properties of the synthesized pyranonaphthazarins have been investigated using the sperm and eggs of the sea urchin Strongylocentrotus intermedius.

Synthesis and acrosin inhibitory activities of substituted ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives.

A series of novel ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives were designed and synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds exhibited acrosin inhibitory activities. Among them, three compounds (5l, 5n, and 5v) were more potent than that of the control TLCK. These provide a new structural type for the development of novel contraceptive acrosin inhibitory agents.

Total synthesis of (+)-(2'S,3'R)-zoapatanol exploiting the B-alkyl Suzuki reaction and the nucleophilic potential of the sulfinyl group.

A stereoselective synthesis of the diterpenoid oxepane (+)-zoapatanol is described. The key steps include a B-alkyl Suzuki cross-coupling reaction for the stereoselective synthesis of trisubstituted alkenes, creation of the two stereogenic centers on the oxepane ring by heterofunctionalization of an alkene through substrate control exploiting the nucleophilic potential of an intramolecular sulfinyl group, and transformation of a ß-hydroxy sulfoxide into a terminal alkene.

Synthesis, antiviral and contraceptive activities of nucleoside-sodium cellulose sulfate acetate and succinate conjugates.

Chemical conjugates between sodium cellulose sulfate (CS), displaying contraceptive and HIV-entry inhibiting properties, and nucleoside reverse transcriptase inhibitors (NRTIs) (3'-azido-2',3'-dideoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine (FLT), or 2',3'-dideoxy-3'-thiacytidine (3TC)) were designed to simultaneously provide contraceptive and anti-HIV activity. Two linkers, acetate and succinate, were used to conjugate the nucleoside analogs with CS. The conjugates containing cellulose sulfate-acetate (CSA) (e.g., AZT-CSA and FLT-CSA) were found to be more potent than CS and other conjugates (e.g., AZT-succinate-CS, and FLT-succinate-CS). The presence of both sulfate and the acetate groups on cellulose were critical for generating maximum anti-HIV activity. In addition to showing equal potency against wild-type and multidrug resistant HIV-1, the AZT-CSA conjugate displayed significant contraceptive activity in an animal model, providing the initial proof-of-concept for the design and synthesis of dual-activity compounds based on these combinations.

Nitric oxide-dependent human acrosomal loss induced by PPCM (SAMMA) and by nitric oxide donors occurs by independent pathways: basis for synthesis of an improved contraceptive microbicide.

PPCM (previously designated sulfuric acid-modified mandelic acid [SAMMA]) is a contraceptive microbicide in preclinical development. Its contraceptive activity is attributable in part to its ability to promote premature acrosomal loss. Prior studies showed that PPCM-induced human acrosomal loss (PAL) is Ca(2+)-dependent. This study was carried out to determine transduction elements downstream from Ca(2+) entry. PAL is inhibited by inhibitors selective for endothelial-type nitric oxide synthase. PAL is completely inhibited by 0.1 microM ODQ (soluble guanylate cyclase inhibitor). PAL is inhibited by protein kinase G inhibitors with selectivity for the type II isotype. Several inhibitors of the nitric oxide/cyclic guanosine monophosphate (cGMP)/protein kinase G pathway induce Ca(2+)-dependent acrosomal loss when added alone. These responses are inhibited by nifedipine, a blocker of Ca(v1.x) voltage-dependent channels. Acrosomal loss induced by the nitric oxide donor SNAP (SNAL) does not require added Ca(2+). Sperm production of nitric oxide is increased by PPCM, an effect inhibited by nitro-L-arginine (nitric oxide synthase inhibitor). Although inhibited by ODQ, SNAL and acrosomal loss induced by other nitric oxide donors are unaffected by KT5823 (protein kinase G inhibitor). Unlike PAL, SNAL is partially inhibited by KT5720 (protein kinase A inhibitor) and genistein (protein tyrosine kinase inhibitor). Acrosomal loss response to PPCM and SNAP added in combination suggests that these agents act by independent mechanisms. A PPCM derivative was synthesized, in which a nitric oxide donor was esterified to PPCM (NOSPPA-23). NOSPPA-23 induces acrosomal loss with or without added Ca(2+). The ED(50) of NOSPPA-23 (4.8 nM) in the presence of Ca(2+) is 35-fold less than that of PPCM. These findings suggest the following: 1) elements responsible for PAL include endothelial nitric oxide synthase, soluble guanylate cyclase, and type II protein kinase G; 2) the resting state of the nitric oxide/cGMP/protein kinase G pathway is a determinant of acrosomal status; 3) PPCM and nitric oxide donors induce acrosomal loss via nitric oxide, but through independent pathways; and 4) covalent attachment of a nitric oxide donor to PPCM provides synergistic efficacy as a stimulus of acrosomal loss. Further studies with this novel prototype as an improved contraceptive microbicide are warranted.

Synthesis of toll-like receptor-2 targeting lipopeptides as self-adjuvanting vaccines.

Effective Th1- and Th2-type immune responses that result in protective immunity against pathogens can be induced by self-adjuvanting lipopeptides containing the lipid moiety dipalmitoyl-S-glyceryl cysteine (Pam2Cys). The potent immunogenicity of these lipopeptides is due to their ability to activate dendritic cells by targeting and signaling through Toll-like receptor-2 (TLR-2). In addition, the simplicity and flexibility in their design as well as their ease of chemical definition and characterisation makes them highly attractive vaccine candidates for humans and animals. We describe in this chapter the techniques involved in the synthesis of an immunocontraceptive lipopeptide vaccine as well as the experimental assays carried out to evaluate its efficiency.

Synthesis and in vivo evaluation of 11-substituted estradiol derivatives as anti-implantation agents.

Synthesis of 11-substituted estradiol derivatives (12-17) has been carried out by the Grignard reaction with alkyl, allyl, and benzyl halides on 17beta-hydroxy-3-methoxy-11-oxo-estra-1,3,5(10),8(9)-tetraene (10). The novel compounds (10 and 12-17) were evaluated for their preliminary post-coital contraceptive (anti-implantation) activity in Sprague-Dawley rats. The tested compounds were administered orally and showed significant anti-implantation activity. Compound 13 is the most potent compound in the series which showed 100% contraceptive efficacy at 1.25 mg kg(-1).

Strategies to facilitate the development of new contraceptives.

Contraception is not readily accessible to much of the world's population and, in any case, no method is 100% effective or appropriate for all users. There is a pressing need for new methods to address the diverse requirements of the global community of men and women at all stages of their reproductive lives. This article will look at some of the new opportunities in contraceptive research and highlight strategies to overcome old challenges for the development of novel contraceptives.

Preparation and characterization of starch/cyclodextrin bioadhesive microspheres as platform for nasal administration of Gabexate Mesylate (Foy) in allergic rhinitis treatment.

Bioadhesive and biodegradable microspheres were obtained by chemical cross-linking with epichlorohydrin of an alkaline solution of a mixture of starch and alpha-, beta-, or gamma-cyclodextrin (CyD). Microspheres were characterized by scanning electron microscopy, swelling degree, and water retention. The percentage of the effective CyD in microspheres was estimated by measuring the amount of iodine and typical organic compounds (TOCs) retained in the hydrophobic cavity of CyD. Gabexate Mesylate (trade name Foy); GM), an antiallergic drug, was included in microspheres by soaking in an aqueous solution containing the drug, followed by solvent evaporation or lyophilization. UV, IR, and DSC data indicated that despite the fact that GM is a hydrophilic drug, its hydrophobic moiety close to the benzene ring is able to penetrate the CyD cavity and to form stable inclusion complexes. Values of the association equilibrium constant for GM binding to CyD, obtained by UV differential spectroscopy, indicated that the affinity of the drug for alpha- and gamma-CyD is higher than that for beta-CyD. In vitro, GM was gradually released during 1h. Even if the release rate of the drug is relatively fast, the microspheres might actually provide the best platform since the material adheres to the nasal mucosa which was proved by adhesion tests. The GM integrity was checked by comparing its anti-trypsin activity before and after release.

Evaluation of novel biodegradable cyclic carbonate polyester copolymers for cytocompatibility using MRC-5 cells.

The objective of this work was to synthesize and characterize a novel series of biodegradable cyclic carbonate polyester copolymers based on lactide and 5-methyl-5-benzyloxy-carbonyl-1,3-dioxan-2-one (MBC). Two compositions were selected for characterization. One copolymer was based on a racemic mixture of 1-lactide with 15.4 mole % MBC and the other was based on 1-lactide with 8.2 mole % MBC. These polymers contain carboxylic acid moieties along the backbone that may be used for tethering bioactive agents, forming ionic crosslinks or be reacted with vinyl containing monomers to allow free radical crosslinking. The initial materials evaluated have the carboxylic acid functionalities blocked with benzene. These polymers and the de-blocked versions may have potential applications for hard and soft tissue scaffolds, control drug delivery matrixes or a variety of other applications in medicine. The copolymer samples were pressed into 7.0-mm diameter disk using a KBr press. The disks were then sterilized using U.V radiation under a laminar flow hood. After sterilization, the copolymer disks were submerged in 2 ml of media and placed in a CO2 regulated incubator at 37 degrees C. A total of six groups per phase (n = 7 test tubes per group) were used in this study. Test tubes in groups I and III were plated with MRC-5 and subsequently treated with media alone (controls). Test tubes in groups II and IV were plated with MRC-5 and subsequently treated with media before being introduced to copolymer samples. Cell number, as well as, biochemical markers such as protein and malondialdehyde (MDA) were determined at the end of the 24, 48 and 72-hour time periods. Representative test tubes were subjected to an H&E staining procedure for microscopic morphological evaluation. The results of this evaluation suggest that the exposure of both copolymers produced a non-cytotoxic environment with the MRC-5 cell line. Although both copolymers are non-cytotoxic, the sample having the higher MBC content is the preferred composition based upon MDA levels and morphological evaluations.

Oxidized cellulose esters: I. Preparation and characterization of oxidized cellulose acetates--a new class of biodegradable polymers.

Oxidized cellulose acetates (OCA), with a degree of substitution (DS) value ranging between 1.1 and 2.3 and a free carboxylic acid group content of 20% (w/w), have been prepared by reacting oxidized cellulose (OC, COOH content 20% w/w) with a mixture of acetic acid and acetic anhydride in the presence of sulfuric acid as a catalyst. The DS of OCA, in general, increased with increasing reaction temperature, reaction time, and concentration of acetic anhydride in the reaction mixture. The yield of OCA, in contrast, increased with increasing concentration of acetic anhydride and decreased with increasing reaction time and temperature. The intrinsic viscosity of OCA varied between 0.100 and 0.275, depending on the reaction conditions used during its preparation. In general, an increase in reaction temperature and the use of a prolonged reaction time decreased the intrinsic viscosity of OCA. No correlation was found between DS and intrinsic viscosity of OCA. The apparent pKa of OCA is 3.7-3.9. The new OCA polymers are practically insoluble in water and slowly dissolve in pH 7.4 phosphate buffer solution. They are, however, soluble in a range of organic solvents (e.g. ethyl acetate, acetone, acetone/water, chloroform/methylene chloride, dimethylsulfoxide, dimethylformamide, and/or chloroform/methanol).

4',17-dioxo-5'H-estra-1(10),4-dieno[3,2-b]furan: synthesis, binding affinity to the estrogen receptor, uterotrophic and antiimplantation activities.

4',17-Dioxo-5'H-estra-1(10),4-dieno[3,2-b]furan (3) has been prepared by several routes starting from 2-bromoacetylestrone (2). Performance of the reaction with thiourea at elevated temperature provided compound 3 in good yield. When other reagents such as thiosemicarbazide, morpholine, sodium hydroxide or sodium hydride were treated with 2-bromoacetylestrone at room temperature, the furano derivative 3 was also obtained as the sole product. This new type of structural modification provided an estrogen nucleus deprived of the 3-hydroxyl function which was previously thought to be an essential requisite for binding to the estrogen receptor (ER). When evaluated in vitro for binding to the ER and in vivo for uterotrophic and antifertility activities, the furano derivative 3 was capable of inhibiting [3H]E2 binding by 16% while still eliciting high uterotrophic (99%) and postcoital antiimplantation (100%) activities relative to estradiol.

Political constraints on contraceptive development in the United States.

PIP: In the US there is a great need for new contraceptives because the current available choices are too limited. Many groups of women, teenagers, women over 40 and lactating mothers have even greater need because of their special requirement. There are 6 million annual pregnancies in the US, 50% of them are unintended. This is the highest percentage of unintended pregnancies in the developed world with Canada having only 39%, the UK 32%, and the Netherlands 17%. 46% of women can expect to have at least 1 unintended pregnancy in their lifetime. Almost half of these unintended pregnancies end in abortion. Of those seeking abortion, 26% are under 20 and 81% are under 30. 69% are white and 82% are single. 49% of these women reported not using contraception when they conceived. Even when a woman uses contraceptives she is still exposed to the risk of contraceptive failure. These failure rates vary from 5-30% for the pill to spermicides. Over a 10-year period the rate climbs to 25-50% for the pill or the IUD. In the US, contraceptives are the most expensive with the pill selling for 60 times what is costs for similar formulations in other countries. Norplant and IUDs, the most reliable reversible methods cost hundreds of dollars and thus make them unavailable for teenagers and poor women who need them most. The primary benefit of increased contraceptive prevalence (CP) is a reduction in the number of unwanted pregnancies and thus abortions. The CP rate for married women of reproductive age in the us is 66%, compared to 73% in Canada, 83% in the UK, 78% in Sweden, and 72% in the Netherlands. The reason new methods are not being developed are multiple: fear of product liability litigation; fear of poor product sales due to public fear; regulation and market pressures that simply do not make them profitable. It can take 12 years and $200 million to develop a new drug and US patents only last 17 years. Thus in order to make a profit the company must have a high rate of sales. Changes in the approval process and financial incentives similar to those for orphan drugs could bring new methods to market.^ieng

Design of potent cyclic gonadotropin releasing hormone antagonists.

In order to improve the biological potency of cyclic gonadotropin releasing hormone (GnRH) antagonists, we have synthesized analogues, the conformations of which were restrained through internal side chain/side chain amide bridges linking aspartic acid or glutamic acid and L-2,3-diaminopropionic acid or L-ornithine. A disulfide bridge linking L-cysteine residues was also introduced. Residues belonging to the bridge spanned from position 4 to positions 9 or 10. Two series of analogues were synthesized and are characterized by residues at positions 1 [Ac-D-3-(2'-naphthyl)alanine], 2 [D-(4-chlorophenyl)alanine or D-(4-fluorophenyl)alanine], 3 [D-3-(3'-pyridyl)alanine or D-tryptophan], 5 (arginine or tyrosine), and 6 [D-3-(3'-pyridyl)alanine or D-arginine], respectively. These substitutions were selected in an effort to optimize high biopotency for inhibition of luteinizing hormone secretion, minimization of histamine release activity, and high (relative) hydrophilicity. The most potent analogues in the antiovulatory assay were cyclo(4-10) [Ac-DNal1,DCpa2,DPal3,(Asp4 or Glu4),Arg5,DPal]6,Dpr10]GnRH (compounds 5 and 7), which were fully active at ca. 12.5 micrograms/rat in the first series, and cyclo(4-10)[Ac-DNal1,DFpa2,DTrp3,Asp4,DArg6++ +,Dpr10]GnRH (compound 12), which was fully active at 2.5 micrograms/rat in the second.

Analogues of [(triethylsilyl)ethynyl]estradiol as potential antifertility agents.

Various 17 alpha-ethynylsteroids were prepared and derivatized as the corresponding triethylsilyl compounds 2-35, which were examined for a ratio of antifertility to estrogenic activity that would be more beneficial than that of the presently used agent. Among the triethylsilyl compounds evaluated, only 23 displayed this desired ratio, although two other compounds without the triethylsilyl moiety, 18 and 26, shared similar characteristics.

PIP: In a previous study of ethynyl estradiol derivatives by the authors, it was found that even a small presence of silicon was beneficial. The silyl derivatives showed a reduction in estrogenic activity along with a retention of the level of oral antifertility activity. In relation to endocrine disorders and the undesired side effects of prescribed contraceptives, the finding is positive. In the present study, the effects of structural changes in the A, B, C, and D rings of the ethynyl estradiol steroidal nucleus were examined in conjunction with C-21 triethylsilyl moiety. The general method of Ethynylation and Triethylsilylation are described in detail and the oral antifertility and oral estrogenic potencies of the compounds are described and compared. Of the various triethylsilyl compounds examined for an antifertility to estrogenic activity ration that would be more beneficial than that of a present agent, only 23 manifested the desired ratio. Compound 18, which was 66% as effective as ethynyl estradiol as an antifertility agent, had 0.1% of the estrogenic activity of ethynyl estradiol and was singled out for the greatest separation between antifertility activity and estrogenic activity. 2 groups of rats, 1 immature females and the other adult, female, cycling rats were tested for oral estrogenic activity and oral antifertility activity, respectively.

Synthesis and antifertility activity of some new fluorine containing 2-([2-(fluoroaryl)-1H-indol-3- yl]methylene)hydrazinecarbothioamides and 2-(fluoroaryl)-([5-(substituted benzylidene)-4-oxo-2-thiazolidinylidene]hydrazone)- 1H-indole-3-carboxaldehydes.

New fluorine containing 2-(fluoroaryl)-1H-indole-3-carboxaldehydes have been synthesized and subjected to reaction with thiosemicarbazide to give corresponding 2-([2- (fluoroaryl)-1H-indol-3-yl]methylene)hydrazinecarbothiamides which were cyclized in the presence of chloroacetic acid, sodium acetate and substituted benzaldehydes to 2-(fluoroaryl)-([5- (substituted benzylidene)-4-oxo-2-thiazolidinylidene]hydrazone)- 1H-indole-3-carboxaldehydes as potential antifertility agents. In preliminary screening, 2-(4'-fluorophenyl)- ([5-(methylene-3,4-dioxyphenyl)-4-oxo-2- thiazolidinylidene]hydrazone)-1H-indole-3-carbocaldehyde exhibited pronounced antifertility activity. All these new compounds have been characterized by analytical and spectral (IR, PMR, MS) studies.

Steroid antifertility agents. Ionic complexes of basic derivatives for prolonged action.

Ethynylestradiol 3-dimethylaminopropionate (1), norethindrone 3-(O-dimethylaminopropyl)oxime (syn and anti isomers, 2a and 2b), and testosterone 3-(O-dimethylaminopropyl)oxime (3) have been prepared and converted to zinc and aluminum tannate complexes as potentially long-acting prodrug forms of the parent steroids. The basic derivatives and the complexes showed the appropriate hormonal activities although they were less active in acute tests than the respective parents. The complexes of 1 showed prolonged activities and, in particular, the zinc tannate showed a prolonged duration of antifertility activity in the rat on subcutaneous administration in an aluminum monostearate gel.