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bstract
This report describes the discovery of RAD140, a potent, orally bioavailable, nonsteroidal selective androgen receptor modulator (SARM). The characterization of RAD140 in several preclinical models of anabolic androgen action is also described.
Keywords: Androgen, SARM, cachexia, oxadiazole, Herschberger assay, primate
The androgen receptor (AR) is a member of the steroid hormone nuclear receptor superfamily that includes estrogen, progestin, glucocorticoid and mineralocorticoid receptors.1 The binding of the prototypical, endogeneously produced androgen testosterone (1) and the important active metabolite dihydrotestosterone (2) to AR initiates a remarkably diverse array of biological activities that can vary according to a subject’s sex, age and hormonal status. The activity of AR is critical to normal human sexual development and function, but beyond this signature role, AR activation also has important effects on diverse targets such as bone, liver, muscle and the central nervous system.2,3 The therapeutic potential of androgen signaling is well-appreciated in the medicinal chemistry community, and for quite some time, chemists have sought compounds that selectively stimulate muscle and bone growth while minimizing the proliferative and/or hypertrophic effects on sex tissues such as the prostate in males and clitoris in females.4,5 Such compounds have been termed selective androgen receptor modulators or SARMs. In this regard, the prototypical and endogenous androgen, testosterone, is considered to be a logical benchmark comparator. Compound 3 is the GTx SARM S-22 and compound 4 is the BMS SARM 562929, both of which have been reported in the literature as being orally active compounds with selectivity for muscle over prostate relative to testosterone in various preclinical models.6,7
The possibility of obtaining compounds having tissue-selective activities that are different from that of the endogenous benchmark testosterone might derive from the fact that typical AR receptor activation, which is initiated by the binding of a molecule with affinity for the AR to the AR ligand binding domain, is then followed by a rather remarkable, coordinated series of interactions: These may include a change in receptor topology, dissociation of heat shock proteins, receptor dimerization, receptor phosphorylation, rapid-signaling events, translocation to the nucleus (AR), association with many different coregulatory proteins to form a transcriptional complex that results in the activation or suppression of RNA synthesis from AR-modulated genes, and finally receptor degradation.8 Since each receptor−ligand complex topology is unique to that ligand structure, one can appreciate that the interaction of any particular ligand−receptor complex with coregulatory proteins is likely to be unique to that ligand as well. Furthermore, because the expression level of AR, the constellation and expression level of coregulatory proteins, and the patterns of post-transcriptional regulatory events differ in each type of androgen target cell, and the topography of AR regulatory sites in the genome differs at each gene, this remarkable choreography of events and interactions provides a rich environment within which one might search for SARMs having a desirable pattern of tissue-selective pharmacology, such as high anabolic but limited androgenic activity.
Further complicating our understanding of the origin of SARM selectivity is the “bio-amplification” of the primary endogenous androgen testosterone. Interestingly, the endogenously produced and very important androgen testosterone serves as a type of “anti-SARM” or “inverse SARM” because its androgenic activity is increased by conversion to the more potent 5α-dihydrotestosterone by the 5α-reductase enzyme in certain tissues including the scalp and prostate (but not in muscle or bone). As a result, androgens that do not undergo such bioamplification in the prostate will demonstrate improved selectivity regarding muscle vs prostate when compared to a testosterone-treated control or an intact animal whose primary endogenous androgen is testosterone.9 More broadly put, one might appreciate that metabolic differences between endogenous androgens such as testosterone or dihydrotestosterone and SARMs can also vouch for at least some selectivity differences.
Our work in the SARM area resulted in the synthesis and evaluation of a large number of candidate templates. While we found it relatively easy to obtain compounds with high affinity for AR, we struggled to achieve compounds that demonstrated good oral efficacy and high in vivo tolerability. After scanning many potential leads for oral, in vivo activity, we arrived at high affinity compound 5 through a combination of synthetic intermediate testing, literature evaluation and fragment combination. We were delighted when 5 demonstrated oral activity in rats.
However, when we performed a pharmacokinetic analysis in rats, we could detect only very low levels of 5 after oral dosing (F < 5%). Further analysis revealed that 5 was efficiently converted to 6in vivo, presumably by cytochromes P450 in the rat liver.10 Compound 6 had similar activity to compound 5in vivo, suggesting that 6 was largely responsible for the activity of compound 5.11 An in vitro screen with human microsomes revealed rapid metabolism of compound 5, thus indicating this transformation as a potential human metabolic liability and prompting us to prepare compounds in which the 4′-position of the pendant phenyl was blocked from P450-induced hydroxylation.12 We looked at several analogues containing a 4′-blocking group, and in the course of our efforts we identified compound 7 (RAD140; Figure Figure1)1) as our preclinical development candidate.
Figure 1
Structures of testosterone (1), 5α-dihydrotestosterone (2), GTx S-22 (3), BMS 562929 (4), initial lead 5, active metabolite 6, and 7 (RAD140).
The synthesis of compound 7 is shown in Scheme 1.13,14 We relied on an expeditious, ipso-fluorine substitution of the left-hand side precursor, piece 8, with d-threonine in the presence of K2CO3 in DMSO to give the desired product 9 in workable yields (typically >50%). The d-Thr adduct 9 was coupled with 4-cyanobenzohydrazide under standard coupling conditions using EDCI and HOBt. The resultant product 10 was silylated with TBDMS-Cl, subjected to dehydrative cyclization conditions in the presence of TPP/I2, and then desilyated for the final step.15−17 Overall, this has proven to be a reliable and efficient synthesis using a fairly inexpensive, albeit nonproteinogenic amino acid as the chirality source.
Scheme 1
Synthesis of Compound 7 (RAD140)
The stability of RAD140 was high (t1/2 > 2 h) in incubations with rat, monkey, and human microsomes, and it also had good bioavailability in rats (F = 27−63%) and monkeys (65−75%). RAD140 demonstrated excellent affinity for the androgen receptor (Ki = 7 nM vs 29 nM for testosterone and 10 nM for DHT) as well as good selectivity over other steroid hormone nuclear receptors, with the closest off target receptor being the progesterone receptor (IC50 = 750 nM vs 0.2 nM for progesterone).18In vitro functional androgen agonist activity was confirmed in the C2C12 osteoblast differentiation assay, where an EC50 of 0.1 nM was shown (DHT = 0.05 nM).19
RAD140 was characterized in a number of in vivo assays to determine its oral efficacy on a number of parameters associated with androgenic activity in preclinical models. For example, RAD140 was dosed in both young castrated and intact male rats in order to assess its effects through a range of endogenous androgenic signaling backgrounds. The young castrated rat provides a very sensitive in vivo assay for androgenic activity because the animal is relatively androgen-naïve; thus, any signaling activity from an exogenously administered androgen is superimposed on an essentially blank background.20 In Figure Figure2,2, the effect of increasing doses of orally administered RAD140 (0.5% methylcellulose) on levator ani bulbocavernosus muscle (“levator ani” or “LABC”) weight and prostate weight is shown relative to vehicle (castrated control), sham (noncastrated control), and testosterone propionate (TP) dosed subcutaneously at 1 mg/kg in corn oil.21 As can be seen, RAD140 stimulates the levator ani muscle beginning at a dose of 0.03 mg/kg (po) and reaches a level of efficacy %yequivalent to the sham-operated animal at 0.3 mg/kg.
Figure 2
Tissue-selective agonist activity of RAD140 in castrated immature rats. The muscle (levator ani) and prostate weights from animals treated for 11 days are plotted with sham and vehicle controls together with the SD. TP is testosterone propionate dosed ...
Because we consistently observed that RAD140 failed to achieve a level of prostate or seminal vesicle stimulation equal to TP at 1 mg/kg (no matter how high the dose of RAD140), we decided to test whether RAD140 could antagonize the effect of TP on rat prostate and seminal vesicles and, at the same time, determine what effect the coadministration of RAD140 and TP might have on the levator ani muscle. From the results shown in Figure Figure3,3, it is apparent that a high dose of RAD140 (10 mg/kg, po) actually antagonizes the effect of TP at 1 mg/kg on the seminal vesicles but adds to the effect of TP on the levator ani muscle. We were able to ascertain that the effective dose for achieving antagonism by RAD140 is 0.3−1 mg/kg (po) for 1 mg/kg TP (sc) (data not shown). In the prostate, RAD140 also caused a downward trend in the stimulation by TP, but the change did not reach statistical significance. Thus, in the young castrate male rat model, RAD140 appears to be a potent and complete androgen agonist on the levator ani, but a weaker, partial antagonist on the seminal vesicle and possibly the prostate.22
Figure 3
Tissue-selective antagonist activity of RAD140. The muscle (levator ani), seminal vesicles, and prostate weights from castrated immature rats treated for 11 days are plotted as a percent of testosterone propionate (TP) together with the SD. *p < ...
This report describes the discovery of RAD140, a potent, orally bioavailable, nonsteroidal selective androgen receptor modulator (SARM). The characterization of RAD140 in several preclinical models of anabolic androgen action is also described.
Keywords: Androgen, SARM, cachexia, oxadiazole, Herschberger assay, primate
The androgen receptor (AR) is a member of the steroid hormone nuclear receptor superfamily that includes estrogen, progestin, glucocorticoid and mineralocorticoid receptors.1 The binding of the prototypical, endogeneously produced androgen testosterone (1) and the important active metabolite dihydrotestosterone (2) to AR initiates a remarkably diverse array of biological activities that can vary according to a subject’s sex, age and hormonal status. The activity of AR is critical to normal human sexual development and function, but beyond this signature role, AR activation also has important effects on diverse targets such as bone, liver, muscle and the central nervous system.2,3 The therapeutic potential of androgen signaling is well-appreciated in the medicinal chemistry community, and for quite some time, chemists have sought compounds that selectively stimulate muscle and bone growth while minimizing the proliferative and/or hypertrophic effects on sex tissues such as the prostate in males and clitoris in females.4,5 Such compounds have been termed selective androgen receptor modulators or SARMs. In this regard, the prototypical and endogenous androgen, testosterone, is considered to be a logical benchmark comparator. Compound 3 is the GTx SARM S-22 and compound 4 is the BMS SARM 562929, both of which have been reported in the literature as being orally active compounds with selectivity for muscle over prostate relative to testosterone in various preclinical models.6,7
The possibility of obtaining compounds having tissue-selective activities that are different from that of the endogenous benchmark testosterone might derive from the fact that typical AR receptor activation, which is initiated by the binding of a molecule with affinity for the AR to the AR ligand binding domain, is then followed by a rather remarkable, coordinated series of interactions: These may include a change in receptor topology, dissociation of heat shock proteins, receptor dimerization, receptor phosphorylation, rapid-signaling events, translocation to the nucleus (AR), association with many different coregulatory proteins to form a transcriptional complex that results in the activation or suppression of RNA synthesis from AR-modulated genes, and finally receptor degradation.8 Since each receptor−ligand complex topology is unique to that ligand structure, one can appreciate that the interaction of any particular ligand−receptor complex with coregulatory proteins is likely to be unique to that ligand as well. Furthermore, because the expression level of AR, the constellation and expression level of coregulatory proteins, and the patterns of post-transcriptional regulatory events differ in each type of androgen target cell, and the topography of AR regulatory sites in the genome differs at each gene, this remarkable choreography of events and interactions provides a rich environment within which one might search for SARMs having a desirable pattern of tissue-selective pharmacology, such as high anabolic but limited androgenic activity.
Further complicating our understanding of the origin of SARM selectivity is the “bio-amplification” of the primary endogenous androgen testosterone. Interestingly, the endogenously produced and very important androgen testosterone serves as a type of “anti-SARM” or “inverse SARM” because its androgenic activity is increased by conversion to the more potent 5α-dihydrotestosterone by the 5α-reductase enzyme in certain tissues including the scalp and prostate (but not in muscle or bone). As a result, androgens that do not undergo such bioamplification in the prostate will demonstrate improved selectivity regarding muscle vs prostate when compared to a testosterone-treated control or an intact animal whose primary endogenous androgen is testosterone.9 More broadly put, one might appreciate that metabolic differences between endogenous androgens such as testosterone or dihydrotestosterone and SARMs can also vouch for at least some selectivity differences.
Our work in the SARM area resulted in the synthesis and evaluation of a large number of candidate templates. While we found it relatively easy to obtain compounds with high affinity for AR, we struggled to achieve compounds that demonstrated good oral efficacy and high in vivo tolerability. After scanning many potential leads for oral, in vivo activity, we arrived at high affinity compound 5 through a combination of synthetic intermediate testing, literature evaluation and fragment combination. We were delighted when 5 demonstrated oral activity in rats.
However, when we performed a pharmacokinetic analysis in rats, we could detect only very low levels of 5 after oral dosing (F < 5%). Further analysis revealed that 5 was efficiently converted to 6in vivo, presumably by cytochromes P450 in the rat liver.10 Compound 6 had similar activity to compound 5in vivo, suggesting that 6 was largely responsible for the activity of compound 5.11 An in vitro screen with human microsomes revealed rapid metabolism of compound 5, thus indicating this transformation as a potential human metabolic liability and prompting us to prepare compounds in which the 4′-position of the pendant phenyl was blocked from P450-induced hydroxylation.12 We looked at several analogues containing a 4′-blocking group, and in the course of our efforts we identified compound 7 (RAD140; Figure Figure1)1) as our preclinical development candidate.
Figure 1Structures of testosterone (1), 5α-dihydrotestosterone (2), GTx S-22 (3), BMS 562929 (4), initial lead 5, active metabolite 6, and 7 (RAD140).
The synthesis of compound 7 is shown in Scheme 1.13,14 We relied on an expeditious, ipso-fluorine substitution of the left-hand side precursor, piece 8, with d-threonine in the presence of K2CO3 in DMSO to give the desired product 9 in workable yields (typically >50%). The d-Thr adduct 9 was coupled with 4-cyanobenzohydrazide under standard coupling conditions using EDCI and HOBt. The resultant product 10 was silylated with TBDMS-Cl, subjected to dehydrative cyclization conditions in the presence of TPP/I2, and then desilyated for the final step.15−17 Overall, this has proven to be a reliable and efficient synthesis using a fairly inexpensive, albeit nonproteinogenic amino acid as the chirality source.
Scheme 1Synthesis of Compound 7 (RAD140)
The stability of RAD140 was high (t1/2 > 2 h) in incubations with rat, monkey, and human microsomes, and it also had good bioavailability in rats (F = 27−63%) and monkeys (65−75%). RAD140 demonstrated excellent affinity for the androgen receptor (Ki = 7 nM vs 29 nM for testosterone and 10 nM for DHT) as well as good selectivity over other steroid hormone nuclear receptors, with the closest off target receptor being the progesterone receptor (IC50 = 750 nM vs 0.2 nM for progesterone).18In vitro functional androgen agonist activity was confirmed in the C2C12 osteoblast differentiation assay, where an EC50 of 0.1 nM was shown (DHT = 0.05 nM).19
RAD140 was characterized in a number of in vivo assays to determine its oral efficacy on a number of parameters associated with androgenic activity in preclinical models. For example, RAD140 was dosed in both young castrated and intact male rats in order to assess its effects through a range of endogenous androgenic signaling backgrounds. The young castrated rat provides a very sensitive in vivo assay for androgenic activity because the animal is relatively androgen-naïve; thus, any signaling activity from an exogenously administered androgen is superimposed on an essentially blank background.20 In Figure Figure2,2, the effect of increasing doses of orally administered RAD140 (0.5% methylcellulose) on levator ani bulbocavernosus muscle (“levator ani” or “LABC”) weight and prostate weight is shown relative to vehicle (castrated control), sham (noncastrated control), and testosterone propionate (TP) dosed subcutaneously at 1 mg/kg in corn oil.21 As can be seen, RAD140 stimulates the levator ani muscle beginning at a dose of 0.03 mg/kg (po) and reaches a level of efficacy %yequivalent to the sham-operated animal at 0.3 mg/kg.
Figure 2Tissue-selective agonist activity of RAD140 in castrated immature rats. The muscle (levator ani) and prostate weights from animals treated for 11 days are plotted with sham and vehicle controls together with the SD. TP is testosterone propionate dosed ...
Because we consistently observed that RAD140 failed to achieve a level of prostate or seminal vesicle stimulation equal to TP at 1 mg/kg (no matter how high the dose of RAD140), we decided to test whether RAD140 could antagonize the effect of TP on rat prostate and seminal vesicles and, at the same time, determine what effect the coadministration of RAD140 and TP might have on the levator ani muscle. From the results shown in Figure Figure3,3, it is apparent that a high dose of RAD140 (10 mg/kg, po) actually antagonizes the effect of TP at 1 mg/kg on the seminal vesicles but adds to the effect of TP on the levator ani muscle. We were able to ascertain that the effective dose for achieving antagonism by RAD140 is 0.3−1 mg/kg (po) for 1 mg/kg TP (sc) (data not shown). In the prostate, RAD140 also caused a downward trend in the stimulation by TP, but the change did not reach statistical significance. Thus, in the young castrate male rat model, RAD140 appears to be a potent and complete androgen agonist on the levator ani, but a weaker, partial antagonist on the seminal vesicle and possibly the prostate.22
Figure 3Tissue-selective antagonist activity of RAD140. The muscle (levator ani), seminal vesicles, and prostate weights from castrated immature rats treated for 11 days are plotted as a percent of testosterone propionate (TP) together with the SD. *p < ...
