Novel progestogenic androgens for male contraception: design, synthesis, and activity of C7 α-substituted testosterone†

Abstract

Male contraceptive development has included use of testosterone (T) with or without a progestin or the use of a single molecule such as progestogenic androgens (PA) for suppression of testicular T production. Expanding upon the vast amount of data accumulated from nortestosterone (NT), NT analogs, and their prodrugs, a new series of PA, the C7 methyl, and ethyl α-substituted T analogs 7α-Methyltestosterone (7α-MT) and 7α-Ethyltestosterone (7α-ET), respectively, were hypothesized and designed to have superior androgenic and progestogenic activities when compared with parent T. Results from androgen receptor and progesterone receptor competitive binding and transcriptional activation assays showed favorable activities for these T analogs. Additionally, 7α-MT and 7α-ET were shown to be active substrates for aromatase in vitro, mitigating a potential negative impact on bone mineral density with long-term use. In conjunction with this observation, the diminished metabolism of these T analogs by 5α-reductase may reduce potential concerns for prostatic growth. In the Hershberger in vivo rat bioassay, 7α-MT and 7α-ET showed superior androgenic and anabolic activities as compared with T. These C7 α-substituted T analogs also showed clear progestogenic activity in the McPhail bioassay which evaluated endometrial glandular arborization in a rabbit model. The discovery of aromatizable molecules with reduced metabolism by 5α-reductase that have androgenic, anabolic, and progestogenic properties indicates that the core and/or prodrugs of 7α-MT and 7α-ET are promising molecules for further development as male contraceptive PAs.

Keywords: male contraception; metabolism; progestogenic androgen; testosterone analogs.

Graphical Abstract

Scheme 1

Synthesis of C7-Substituted T Analogs^a, ^aReagents and conditions: (a) Isopropenyl acetate, p-toluene sulfonic acid, DME, reflux, 4 h, 99%; (b) DMF, H2O, NBS, 0°C, 30 min;(c) LiBr, Li2CO3, DMF, 90°C, 1 h; (d) crude product, MeOH, NaOMe/MeOH, RT, 3 h, 81%; (e) TBDMSCl, imidazole, dry DMF, RT, overnight, 91%; (f) RMgCl, LiCl, CuI, dry THF, –75°C, 3 h, H2O, RT, overnight; (g) Column purification and crystallization.

Figure 1

Human Aromatase Assay Results. (A) T and test articles % remaining depicted in solid lines when incubated with human CYP19 + P450 and co-factor NADPH at times 0, 15, 30, and 60 min. In dashed lines, T and test articles incubated in the absence of co-factor NADPH at times 0 and 60 min. (B) Calculated AUC (% remaining * min) of T and test articles (in the presence of NADPH, as presented in panel A) converted to % of total AUC. Low % of total AUC indicates rapid metabolism, while a high % of total AUC indicates slow to no metabolism by human aromatase CYP19.

Figure 2

Human 5α-Reductase Assay Results. (A) In solid lines, T and test articles in the absence of 5α-reductase type 2 specific inhibitor finasteride at times 0, 15, 30, 60, 90, and 120 min. In dashed lines, T and test articles incubated in the presence of finasteride at times 0, 15, 30, 60, 90, and 120 min. (B) The difference between the AUC with finasteride and the AUC without finasteride was calculated for T and the test articles and then graphed as a % difference in AUC. The larger the % AUC difference the faster the metabolism, suggesting the test articles are substrates for 5α-reductase, while small to no % AUC difference suggests that the test articles are not substrates for 5α-reductase.

Figure 3

Hershberger assay results evaluating androgenic and anabolic potencies in castrated weanling rats. Mean ventral prostate (A) and seminal vesicle (B) organ weights to evaluate androgenicity and levator ani muscle (C) organ weights to evaluate anabolic potency were determined on Day 8 after seven previous days of once daily SC administrations. (A–C) X-axis depicts total dose received per rat (μmol). Mean ± standard deviation. N = 8 rats/dose/test article. N = 20 rats for 6.9 μmol T condition and for vehicle (0 μmol). N = 8 rats for the other T dose levels. Test article symbols shown in panel A also apply to panels B and C. (D) Dose level versus organ weight AUC from panels A–C graphed ± standard error per test article per organ weight. Test articles with organ weight AUCs that differed with statistical significance from that of T are indicated with asterisks (P ≤ 0.01; MENT and DMA for ventral prostate and seminal vesicles and 7α-ET and DMA for levator ani).

Figure 4

H&E-stained uteri cross sections representing each of the four possible McPhail Index scores for evaluation of endometrial glandular arborization. (A) Grade 0, vehicle control. (B) Grade 1, 7α-MT, 0.8 μmol total dose. (C) Grade 2, 7α-MT, 7.2 μmol total dose. (D) Grade 3, 7α-MT, 21.6 μmol total dose. (E) Grade 4, progesterone, 2.4 μmol total dose. Scale bar = 1000 μm.

Figure 5

Progestogenic effect of test articles on rabbit uteri after 17β-estradiol priming followed by 5 once daily SC administrations of test article. (A) Average McPhail Index of rabbit uteri per treatment. Uteri were collected on Day 12 (the day after the last test article dose), fixed, sectioned, stained, and scored based on the five-step scoring system (0–4) of McPhail to generate an average endometrial glandular arborization score for each rabbit. Data presented as average McPhail index ± standard deviation per treatment group (four sections scored per rabbit, N = 9 rabbits for vehicle and progesterone, N = 6 rabbits for each test article dose level, n = 3 rabbits for T that was tested only at 2.4 μmol/rabbit). (B) Mean uterus to body weight ratio (%) in the same rabbits depicted in panel A. (C) AUC for dose level versus McPhail score is plotted against the left y-axis in black bars ± standard error using the data graphed in panel A. AUC for dose level versus uterus body weight ratio is plotted against the right y-axis in white bars ± standard error using the data graphed in panel B. AUC values different with statistical significance (P ≤ 0.01) from that of 7α-MT are indicated by asterisks. None of the other pairwise AUC comparisons differed with statistical significance.