Activity-Directed Synthesis: A Flexible Approach for Lead Generation.

Activity-directed synthesis (ADS) is a structure-blind, functional-driven molecular discovery approach. In this Concept, four case studies highlight the general applicability of ADS and showcase its flexibility to support different medicinal chemistry strategies. ADS deliberately harnesses reactions with multiple possible outcomes, and allows many chemotypes to be evaluated in parallel. Resources are focused on bioactive molecules, which emerge in tandem with associated synthetic routes. Some of the future challenges for ADS are highlighted, including the realisation of an autonomous molecular discovery platform. The prospects for ADS to become a mainstream lead generation approach are discussed.

Synthesis and biological activity of 5-aryliden-2-thiohydantoin S-aryl derivatives.

Three new and complementary approaches to S-arylation of 2-thiohydantoins have been developed: copper-catalyzed cross coupling with either arylboronic acids or aryl iodides under mild conditions, or direct nucleophilic substitution in activated aryl halides. For 38 diverse compounds, reaction yields for all three methods have been determined. Selected by molecular docking, they have been tested on androgen receptor activation, and p53-Mdm2 regulation, and A549, MCF7, VA13, HEK293T, PC3, LnCAP cell lines for cytotoxicity, Two of them turned out to be promising as androgen receptor activators (likely by allosteric regulation), and another one is shown to activate the p53 cascade. It is hoped that 2-thiohydantoin S-arylidenes are worth further studies as biologically active compounds.

Chemical synthesis of N-glycosylated insulin-like androgenic gland factor from the freshwater prawn Macrobrachium rosenbergii.

Crustacean insulin-like androgenic gland factor (IAG) of Macrobrachium rosenbergii, a heterodimeric peptide having both four disulfide bonds and an N-linked glycan, was synthesized by the combination of solid-phase peptide synthesis and the regioselective disulfide formation reactions. The disulfide isomer of IAG could also be synthesized by the same manner. The conformational analysis of these peptides by circular dichroism (CD) spectral measurement indicated that the disulfide bond arrangement affected the peptide conformation in IAG. On the other hand, the N-linked glycan attached at A chain showed no effect on CD spectra of IAG. This is the first report for the chemical synthesis of insulin-like heterodimeric glycopeptide having three interchain disulfides, and the synthetic strategy shown here might be useful for the synthesis of other glycosylated four-disulfide insulin-like peptides.

Evidence that cytochrome b5 acts as a redox donor in CYP17A1 mediated androgen synthesis.

Cytochrome P450 17A1 (CYP17A1) is an important drug target for castration resistant prostate cancer. It is a bi-functional enzyme, catalyzing production of glucocorticoid precursors by hydroxylation of pregnene-nucleus, and androgen biosynthesis by a second CC lyase step, at the expense of glucocorticoid production. Cytochrome b5 (cyt b5) is known to be a key regulator of the androgen synthesis reaction in vivo, by a mechanism that is not well understood. Two hypotheses have been proposed for the mechanism by which cyt b5 increases androgen biosynthesis. Cyt b5 could act as an allosteric effector, binding to CYP17A1 and either changing its selective substrate affinity or altering the conformation of the P450 to increase the catalytic rate or decrease unproductive uncoupling channels. Alternatively, cyt b5 could act as a redox donor for supply of the second electron in the P450 cycle, reducing the oxyferrous complex to form the reactive peroxo-intermediate. To understand the mechanism of lyase enhancement by cyt b5, we generated a redox-inactive form of cyt b5, in which the heme is replaced with a Manganese-protoporphyrin IX (Mn-b5), and investigated enhancement of androgen producing lyase reaction by CYP17A1. Given the critical significance of a stable membrane anchor for all of the proteins involved and the need for controlled stoichiometric ratios, we employed the Nanodisc system for this study. The redox inactive form was observed to have no effect on the lyase reaction, while reactions with the normal heme-iron containing cyt b5 were enhanced ∼5 fold as compared to reactions in the absence of cyt b5. We also performed resonance Raman measurements on ferric CYP17A1 bound to Mn-b5. Upon addition of Mn-b5 to Nanodisc reconstituted CYP17A1, we observed clear evidence for the formation of a b5-CYP17A1 complex, as noted by changes in the porphyrin modes and alteration in the proximal FeS vibrational frequency. Thus, although Mn-b5 binds to CYP17A1, it is unable to enhance the lyase reaction, strongly suggesting that cyt b5 has a redox effector role in enhancement of the CYP17A1 mediated lyase reaction necessary for androgen synthesis.

Stereocontrolled synthesis of the four 16-hydroxymethyl-19-nortestosterone isomers and their antiproliferative activities.

Novel 16-hydroxymethyl-19-nortestosterone diastereomers were prepared by Birch reduction from the corresponding 3-methoxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol isomers with known configurations. The synthesized compounds are 16α- and 16ß-hydroxymethyl-substituted 19-nortestosterone and their 17α-epimers. To prepare 17α-19-nortestosterone, the Mitsunobu inversion reaction of 19-nortestosterone with different alkyl and aryl carboxylic acids was chosen. Deacylation of the new compounds by the Zemplén method yielded the required 17α-19-nortestosterone. The antiproliferative activities of the structurally related compounds were determined in vitro through microculture tetrazolium assays on a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780) cell lines. The 17α epimer of 19-nortestosterone demonstrated considerable activity, selectively for HeLa cells, with a calculated IC50 of 0.65 µM. The reference compound, cisplatin, displayed an order of magnitude higher IC50 (12.4 µM). The cancer selectivity of 17α-19-nortestosterone was tested by MTT assay performed with noncancerous human fibroblast cell line MRC-5. The results indicated that 17α-19-nortestosterone selectively disturbs the viability of HeLa cells without greatly affecting other cancer cell types and intact fibroblasts.

Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs). Part I.

To develop effective drugs for hypogonadism, sarcopenia, and cachexia, we designed, synthesized, and evaluated selective androgen receptor modulators (SARMs) that exhibit not only anabolic effects on organs such as muscles and the central nervous system (CNS) but also neutral or antagonistic effects on the prostate. Based on the information obtained from a docking model with androgen receptor (AR), we modified a hit compound A identified through high-throughput screening. Among the prepared compounds, 1-(4-cyano-1-naphthyl)-2,3-disubstituted pyrrolidine derivatives 17h, 17m, and 17j had highly potent AR agonistic activities in vitro and good tissue selectivity in vivo. These derivatives increased the weight of the levator ani muscle without influencing the prostate and seminal vesicle. In addition, these compounds induced sexual behavior in castrated rats, indicating that the compounds could also act as agonists on the CNS.

Discovery of aryloxy tetramethylcyclobutanes as novel androgen receptor antagonists.

An aryloxy tetramethylcyclobutane was identified as a novel template for androgen receptor (AR) antagonists via cell-based high-throughput screening. Follow-up to the initial "hit" established 5 as a viable lead. Further optimization to achieve full AR antagonism led to the discovery of 26 and 30, both of which demonstrated excellent in vivo tumor growth inhibition upon oral administration in a castration-resistant prostate cancer (CRPC) animal model.

(17α,20E)-17,20-[(1-methoxyethylidene)bis(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11) is a partial agonist of the androgen receptor.

A novel steroid compound, (17α,20E)-17,20-[(1-methoxyethylidene)bis(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), was found to be a partial agonist of the androgen receptor (AR) in an androgen responsive element (ARE)-luciferase reporter assay. YK11 accelerates nuclear translocation of AR. Furthermore, YK11 does not induce amino/carboxyl-terminal (N/C) interaction and prevents 5-α-dihydrotestosterone (DHT)-mediated N/C interaction. Thus, YK11 activates AR without causing N/C interaction, which may in turn be responsible for the partially agonistic nature of YK11 observed in the ARE-luciferase reporter system. YK11 acts as a gene-selective agonist of AR in MDA-MB 453 cells. The effect of YK11 on gene expression relative to that of androgen agonist varies depending on the gene context. YK11 activated the reporter gene by inducing the translocation of the AR into the nuclear compartment, where its amino-terminal domain (NTD) functions as a constitutive activator of AR target genes. Our results suggest that YK11 might act as selective androgen receptor modulator (SARM).

Hereditary angioedema: a current state-of-the-art review, V: attenuated androgens for the treatment of hereditary angioedema.

OBJECTIVE: To provide a summary of the literature regarding the use of attenuated androgens during the past 40 to 50 years for the treatment of hereditary angioedema (HAE). DATA SOURCES: MEDLINE and PubMed were searched to identify studies involving the treatment of HAE with androgens. STUDY SELECTION: Studies were selected based on their relevance to the use of androgens for the treatment of HAE. RESULTS: Attenuated androgens have proven successful for the short- and long-term treatment of HAE. Adverse effects are still concerning, and their use in children and pregnant women must be undertaken with great caution. Scheduled monitoring of liver function tests and lipid profiles in patients treated with these medications is critical. CONCLUSIONS: Attenuated androgens have been successful in the short- and long-term treatment of HAE, and they are still the most frequently used medications in the United States for the treatment of this disease. There is a lack of readily available options for the treatment of acute HAE attacks apart from the administration of fresh frozen plasma or safe prophylactic therapies; however, several appropriate agents currently in clinical trials in the United States appear promising.

7alpha,11beta-Dimethyl-19-nortestosterone: a potent and selective androgen response modulator with prostate-sparing properties.

7alpha,11beta-Dimethyl-19-nortestosterone, made by 1,6-methyl addition to 17beta-acetoxy-11beta-methylestra-4,6-dien-3-one, was a highly potent and selective androgen response modulator, with enhanced androgen receptor binding, androgenic activity and anabolic:androgenic ratio over its two monomethyl homologs.

Synthesis of the 7alpha-cyano-(17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones: potential radioligands for androgen and progesterone receptors.

We report the preparation of the 7alpha-cyano derivative of the isomeric (17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones (IVNT) together with their binding affinity for the androgen receptor (AR) and their biodistribution in two different animal models. The cyano group was introduced at the 7alpha-position by hydrocyanation of 4,6-estradien-17beta-ol-3-one with diethylaluminum cyanide. Selective protection of the A-ring enone system as the dienol ether followed by ethynylation and deprotection under base and acid hydrolysis condition gave 7alpha-cyano-17alpha-ethynyl-19-nortestosterone. The stannyl derivatives were prepared by addition of tri-n-butylstannyl hydride and converted stereospecifically to the corresponding [125I]iodovinyl analog using [125I]NaI and H2O2. The [125I]iodovinylsteroids were intravenously administered to male rats and estrogen-primed immature female rats and tissue uptake was measured up to 6h post-injection. Co-administration of NLP-004 or ORG-2058, highly selective ligands for the progesterone receptor, to the female rats did not affect uterus uptake of the 125I-ligands. However co-injection of testosterone to DES-primed male rats induced a marked increase in prostate uptake of the 20Z-isomer of 7alpha-cyano-[125I]-IVNT. The relative binding affinity (RBA) of either 7alpha-cyano-(17alpha,20E/Z)-IVNT isomer for the AR is low (RBA=4 and 3, respectively, versus 100 for 5alpha-dihydrotestosterone (DHT)), suggesting the absence of a possible role of the AR in the localization process. These findings contrast previously reported data for the analogous 7alpha-methyl-[125I]-IVNT where co-administration of testosterone was shown to result in a 50% drop in prostate uptake. These data indicate that the addition of an electron withdrawing 7alpha-cyano group to 123I-labeled nortestosterone derivatives does not improve their potential to serve as SPECT agents for the imaging of AR densities in the prostate.

3Beta-sulfamate derivatives of C19 and C21 steroids bearing a t-butylbenzyl or a benzyl group: synthesis and evaluation as non-estrogenic and non-androgenic steroid sulfatase inhibitors.

A series of C19 and C21 steroids bearing one or two inhibiting groups (3beta-sulfamate and 17alpha- or 20(S)-t-butylbenzyl or benzyl) were synthesized and tested for inhibition of steroid sulfatase activity. When only a sulfamate group was added to dehydroepiandrosterone, androst-5-ene-3beta,17beta-diol, pregnenolone and 20-hydroxy-pregnenolone, no significant inhibition of steroid sulfatase occurred at concentrations of 0.3 and 3 microM. With only a t-butylbenzyl or a benzyl group, a stronger steroid sulfatase inhibition was obtained in the androst-5-ene than in the pregn-5-ene series. Comparative results from the screening tests and the IC50 values have shown that the effect of a sulfamate moiety as a second inhibiting group can be combined to the t-butylbenzyl or benzyl effect in the C19 and C21 steroid series. The 3beta-sulfamoyloxy-17alpha-t-butylbenzyl-5-androsten-17beta-ol (10) was thus found to be the most active compound with IC50 values of 46 +/- 8 and 14 +/- 1 nM, respectively for the transformations of E1S to E1 and DHEAS to DHEA. The IC50 values of compound 10 are similar to that of 17alpha-t-butylbenzyl-estradiol, which was previously reported by our group as a good steroid sulfatase reversible inhibitor, but remains higher than that of the potent inactivators estrone-3-O-sulfamate (EMATE) and 17alpha-t-butylbenzyl-EMATE. However, contrary to these two latter inhibitors, compound 10 did not induce any proliferative effect on estrogen-sensitive ZR-75-1 cells nor on androgen-sensitive Shionogi cells at concentrations tested, suggesting that this steroid sulfatase inhibitor is non estrogenic and non androgenic.

Danazol for the treatment of thrombocytopenia in patients with myelodysplastic syndrome.

Thrombocytopenia is a poor prognostic indicator in the myelodysplastic syndromes (MDS). Treatment options for patients with symptomatic thrombocytopenia are limited. Danazol, an attenuated androgen, may have some efficacy in increasing the platelet count of patients with MDS. We retrospectively reviewed 33 patients with primary MDS who were treated with danazol for 6 or more weeks. After 6 weeks on danazol, the mean platelet count increased from 42 x 10(9)/L to 60 x 10(9)/L (P < 0.015), and 25 out of 33 patients (76%) had an increase in their platelet counts. Following 12 weeks of treatment, the mean platelet count increased to 67 x 10(9)/L (P < 0.005), and 21 out of 29 patients (72%) had an increase in their platelet counts. Seven out of nine patients no longer required platelet transfusions because bleeding stopped after 6 weeks on danazol. Mean duration of response was 10 months (range 2-68 months). Responses were seen in all French-American-British (FAB) subtypes and in all International Prognostic Scoring System (IPSS) scores. Therapy was well tolerated. Danazol may be effective in MDS patients who are thrombocytopenic.

Synthesis of C-6 fluoroandrogens: evaluation of ligands for tumor receptor imaging.

Seven androgens, substituted with fluorine at C-6, were prepared as potential imaging agents for androgen receptor-positive prostate tumors and were evaluated in vitro in terms of their lipophilicity and their relative binding affinities (RBA, relative to R 1881 = 100) for the androgen receptor and for sex steroid binding protein. Introduction of a fluorine atom into the C-6 position of an androgen generally decreases binding affinity to the androgen receptor, except in the two cases: 6 alpha-fluoro-19-nor-testosterone (RBA = 41.6 versus 30.6 for the unsubstituted steroid) and 6 alpha-fluorotestosterone (RBA = 8.9 versus 6.6). Receptor binding of the C-6 fluoro-androgens is also stereospecific, showing higher binding affinities for the alpha-epimers compared to the corresponding beta-epimers (4:1-15:1). Binding affinity to sex steroid binding protein is the lowest with 19-nor-testosterone, which is also the least lipophilic androgen studied. Based on the binding properties of compounds in this series, 6 alpha-fluoro-19-nor-testosterone appears to have the most promise as a tumor imaging agent.

P450 inhibitors of use in medical treatment: focus on mechanisms of action.

A number of cytochrome P450s are targets for compounds that are clinically used or under clinical evaluation for treatment of patients with mycotic infections, such as dermatophytosis, superficial and systemic candidiasis, cryptococcosis and aspergillosis, with skin diseases, such as psoriasis or ichthyosis, and other retinoid-sensitive malignancies, e.g., neuro-ectodermal glioma. Some of the P450 inhibitors are candidates for the treatment of hirsutism or prostate cancer, others are potent inhibitors of the P450 isomerase involved in the synthesis of thromboxane A2, a potent platelet aggregation inducer and vasoconstrictor.

Synthesis of high affinity fluorine-substituted ligands for the androgen receptor. Potential agents for imaging prostatic cancer by positron emission tomography.

We have prepared nine androgens substituted with fluorine at C-16 or C-20 to evaluate their potential, as positron emission tomographic (PET) imaging agents for prostatic cancer when labeled with the positron emitting radionuclide fluorine-18 (t1/2 = 110 min). These compounds represent members from the following classes of androgens: testosterone (T), 5 alpha-dihydrotestosterone (DHT), 7 alpha-methyl-19-nortestosterone (MNT), mibolerone (Mib), and metribolone (R1881). All of these compounds were prepared by functionalization of suitable androgen precursors, and the synthetic routes were developed to allow the introduction of fluorine by a fluoride ion displacement reaction late in the synthesis, as is required for the preparation of these compounds in fluorine-18 labeled form. We have also prepared four androgens in which the C-3 carbonyl or 17 beta-hydroxyl groups are replaced by fluorine. Most of the fluorine-substituted androgens show high affinity for the androgen receptor (AR), although fluorine substitution lowers their affinity by a small factor. None of the androgens where fluorine replaces oxygen functions at C-3 or C-17 have substantial affinity for AR. Derivatives of the natural androgens (T and DHT) as well as MNT have little affinity for other steroid hormone receptors (progesterone and mineralocorticoid receptors), whereas the Mib and R1881 derivatives have somewhat greater heterologous binding. With sex steroid binding protein, a human serum binding protein, the pattern of binding affinities is nearly the reverse, with derivatives of Mib, R1881 and MNT having low affinity, and DHT and T, high affinity. From these fluorine-substituted compounds, we can select several whose preparation in fluorine-18 labeled form for further tissue distribution studies is merited.

Fluorinated androgens and progestins: molecular probes for androgen and progesterone receptors with potential use in positron emission tomography.

In order to develop imaging agents for receptor-positive tumors of the breast and prostate, we have investigated the binding affinity of several fluorine-substituted steroids in the testosterone and nortestosterone series for the androgen receptor and the progesterone receptor. The 6 alpha- and 11 beta-fluoro-, and 16 alpha-fluoroalkyl-substituted steroids were prepared by an olefin bromofluorination reaction followed by dehydrobromination or reductive debromination. The 17 alpha-fluoromethyl derivatives were prepared by fluoride ion attack on the 17-spiroepoxide or 17-spiro sulfate and the 17 alpha-fluoropropynyl derivative, by reaction of a propargyl alcohol precursor with diethylaminosulfur trifluoride. Of the compounds synthesized, 17 alpha-(3-fluoro-I-propynyl)nortestosterone was found to possess the highest binding affinity and selectivity for the progesterone receptor, and 11 beta-fluoronordihydrotestosterone had the greatest affinity for the androgen receptor. Both receptor systems seem to tolerate reasonably well the substitution of fluorine for hydrogen.

Synthesis of 16 alpha-3H androgen and estrogen substrates for 16 alpha-hydroxylase.

The synthesis of 16 alpha-3H androgens and estrogens is described. 1-(3H)-Acetic acid in the presence of zinc dust reacts with 16 alpha-bromo-17-ketosteroids to produce 16 alpha-3H-17-ketosteroids. This chemical reaction was used to prepare 16 alpha-3H-dehydroepiandrosterone (I) and 16 alpha-3H-estrone acetate (XI) from 16 alpha-bromo-dehydroepiandrosterone (X) and from 16 alpha-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16 alpha-3H-androstenedione (II) and 16 alpha-3H-estradiol-17 beta (VII). 16 alpha-3H-Estrone (VI) was obtained by the chemical hydrolysis of 16 alpha-3H-estrone acetate. The label distribution as determined by microbiological 16 alpha-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16 alpha position. These substrates can be used for measuring the 16 alpha hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX).