Design, synthesis, and preclinical characterization of the selective androgen receptor modulator (SARM) RAD140

Article abstract of DOI:10.1021/ml1002508

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.

Full text of DOI:10.1021/ml1002508

pubs.acs.org/acsmedchemlett
Design, Synthesis, and Preclinical Characterization of the
Selective Androgen Receptor Modulator (SARM) RAD140
Chris P. Miller,*,† Maysoun Shomali,† C. Richard Lyttle,† Louis St. L. O'Dea,†
Hillary Herendeen,† Kyla Gallacher,† Dottie Paquin,† Dennis R. Compton,‡
Bishwabhusan Sahoo,‡ Sean A. Kerrigan,‡ Matthew S. Burge,‡ Michael Nickels,‡
Jennifer L. Green,‡ John A. Katzenellenbogen,§ Alexei Tchesnokov, and Gary Hattersley†
†Radius Health, Inc., 300 Technology Square, Cambridge, Massachusetts 02139, United States, ‡Obiter Research, 2809 Gemini
Court, Champaign, Illinois 61822-9647, United States, §Department of Chemistry, University of Illinois at Urbana-Champaign,
600 South Mathews Avenue, Urbana, Illinois 61801, United States, and Cambridge Major Laboratories, Inc., W130 N10497
Washington Drive, Germantown, Wisconsin 53022, United States
ABSTRACT 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
he androgen receptor (AR) is a member of the steroid
hormone nuclear receptor superfamily that includes
estrogen, progestin, glucocorticoid and mineralocorti-
topology, dissociation of heat shock proteins, receptor di-
merization, 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 synthesisfromAR-modulated genes, and finallyreceptor
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 en-
dogenously produced and very important androgen testos-
terone serves as a type of “anti-SARM” or “inverse SARM”
because its androgenic activity is increased by conversion
tothemorepotent5R-dihydrotestosterone by the 5R-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
T
coid receptors.1 The binding of the prototypical, endogen-
eously 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. Com-
pound 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-
selectiveactivitiesthataredifferentfromthatoftheendogenous
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
Received Date: August 17, 2010
Accepted Date: November 15, 2010
Published on Web Date: December 02, 2010
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Figure 1. Structures of testosterone (1), 5R-dihydrotestosterone (2), GTx S-22 (3), BMS 562929 (4), initial lead 5, active metabolite 6, and 7
(RAD140).
Scheme 1. Synthesis of Compound 7 (RAD140)
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 andro-
gens such as testosterone or dihydrotestosterone and
SARMs can also vouch for at least some selectivity differ-
ences.
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 demon-
strated 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 6 in vivo, presumably by cyto-
chromes P450 in the rat liver.10 Compound 6 had similar
activity to compound 5 in vivo, suggesting that 6 was largely
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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
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 < 0.05 vs
TP for all tissues.
subcutaneously daily in corn oil. Five rats were included in each
§
treatment group. *p < 0.05 vs vehicle for prostate. p < 0.05 vs
vehicle for LABC.
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 40-position of the pendant
phenyl was blocked from P450-induced hydroxylation.12 We
looked at several analogues containing a 40-blocking group,
and in the course of our efforts we identified compound 7
(RAD140; Figure 1) as our preclinical development candidate.
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 cou-
pling 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.
The stability of RAD140 was high (t1/2 > 2 h) in incuba-
tions with rat, monkey, and human microsomes, and it also
had good bioavailability in rats (F = 27-63%) and mon-
keys (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 hormonenuclear receptors, withtheclosest
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 ex-
ogenously administered androgen is superimposed on an
essentially blank background.20 In Figure 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 subcuta-
neously 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 equivalent to
the sham-operated animal at 0.3 mg/kg.
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 antag-
onize the effect of TP on rat prostate and seminal vesicles
and, at the same time, determine what effect the coadminis-
tration of RAD140 and TP might have on the levator ani
muscle. From the results shown in Figure 3, it is apparent that
a high dose of RAD140 (10 mg/kg, po) actuallyantagonizes the
effect of TPat 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
off target receptor being the progesterone receptor (IC50
=
750 nM vs 0.2 nM for progesterone).18 In vitro functional
androgen agonist activity was confirmed in the C2C12 osteo-
blast 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
The goal of most preclinical, in vivo models is to best
predict how a drug will perform in the drug target population.
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When considering the issue of how stimulatory an andro-
gen is on any given tissue in a preclinical model, one should
keep in mind that the background level of androgen signal-
ing can affect the response observed in an animal. The
castrated rat model has limitations because the very low
endogenous androgen level in this model is an artificial
situation, not reflected in the target adult human male
population.23 In particular, the target male population will
have an androgenic background well above a castrate,
although the androgen levels will likely be lower than the
norm for their group.
To better understand how this group might respond, we
decided to look at young intact male rats, since they have
endogenous testosterone but at somewhat reduced levels.
Therefore, they retain prostate sensitivity to an androgenic
compound but at the same time have a baseline stimulation
that is more similar to the target population than castrated
animals. As shown in Figure 4, RAD140 increased the weight
of the levator ani muscle above that of the intact control starting
with the lowest tested dose (0.1 mg/kg). Interestingly, RAD140
demonstrated no stimulation of the prostate above the intact
animal control level until the highest dose tested, 30 mg/kg. At
0.3 mg/kg, RAD140 demonstrated muscle efficacy similar to
TP at 0.5 mg/kg, but a dose of 30 mg/kg of RAD140 was
required to approximate the prostate efficacy of 0.5 mg/kg
TP.24 From this study it is apparent that in young intact male
rats RAD140 has avery wide range of selectivity relative to both
TP-treated rats as well as sham-control rats.
Finally, we were interested in evaluating the effect of
RAD140 in young, male cynomolgous monkeys to establish
efficacious dosing levels in what we considered to be a more
relevant preclinical species. We performed a relatively sim-
ple, nonterminal study that still allowed us to evaluate
anabolic as well as lipid and other clinical chemistry param-
eters. To assess anabolic activity, we first looked at gross
body weight, which we knew to be a sensitive marker of
anabolic androgen action in young nonhuman primates. The
results on animal body weight of 28-day dosing with
RAD140 at 0.01 mg/kg, 0.1 mg/kg, and 1 mg/kg are shown
in Figure 5.
Due to the small group size (n = 3 for each dosing group),
we used each animal's background weight change for the
weeks prior to the experiment to establish the baseline as
control. Since the mean body weight for each group of three
monkeys converged to an almost identical number (day -1),
with the absolute body weight range between groups of only
4.26-4.29kg,weplottedtheabsolutebodyweightinFigure5.
In this study, a mean weight gain of greater than 10% in just
28 days of dosing was achieved at a dose of just 0.1 mg/kg,
with a similareffectobserved atthe 1.0 mg/kg dosinggroup.26
Dual energy X-ray absorptiometry (“DEXA”) scans of all
monkeys were taken two days before dosing began and one
day after the final dose (day -2 and day 29) in order to
determine the effects of RAD140 on lean tissue and fat; the
results are shown in Figure 6. As can be seen, there was no
consistent effect on absolute fat mass, whereas muscle
showed a qualitative trend that increases with dose.
Figure 4. Tissue-selective agonist activity of RAD140 in young
intact male rats. The muscle (levator ani) and prostate weights
from intact immature rats treated for 11 days are plotted with
sham and vehicle controls together with the SD. Eight rats were
included in each treatment group. *p < 0.05 vs vehicle for
prostate. §p < 0.05 vs vehicle for LABC.
Figure 5. Primate body weight from day -21, through 28 days dosing and 21 days postdosing with RAD140 (0.01, 0.1, and 1 mg/kg, po).25
Three monkeys were included for each treatment group. The change in baseline subtracted body weight from day -1 to day 29 was
statistically significant for the 0.1 mg/kg (p < 0.01) and 1.0 mg/kg (p < 0.05) groups only. The change in body weight at day 29 between the
0.1 mg/kg group and the 0.01 mg/kg group was statistically significant (p < 0.05) but not for 1.0 mg/kg and the 0.01 mg/kg group (p < 0.1).
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family: glucocorticoid, mineralocorticoid, progesterone,
and androgen receptors. Pharmacol. Rev. 2007, 58 (4),
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(2)
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Testosterone Action Deficiency Substitution, 3rd ed.; Nieschlag,
E., Behre, H., Eds.; Cambridge University Press: 2004.
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Mohler, M. L.; Bohl, C. E.; Narayanan, R.; He, Y.; Hwang, D. J.;
Dalton, J. T.; Miller, D. D. Nonsteroidal tissue-selective andro-
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2008, 39, 249–304 (Nuclear Receptors as Drug Targets).
Kim, J.; Wu, D.; Hwang, D. J.; Miller, D. D.; Dalton, J. T. The
para substituent of S-3-(phenoxy)-2-hydroxy-2-methyl-N-(4-
nitro-3-trifluoromethyl-phenyl)-propionamides is a major
structural determinant of in vivo disposition and activity of
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Ostrowski, J.; Kuhns, J. E.; L, J. A.; M, M. C.; Beehler, B. C.;
Krystek, S. R., Jr.; B, Y.; Sun, C.; Seethala, R.; Golla, R.; Sleph,
P. G.; Fura, A.; An, Y.; Kish, K. F.; Sack, J. S.; Mookhtiar, K. A.;
Grover, G. J.; Hamann, L. G. Pharmacological and X-ray
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receptor modulator: potent hyperanabolic stimulation of
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Endocrinology 2007, 148 (1), 4–12.
(4)
(5)
Figure 6. Mean change in primate tissue weight as measured by
DEXA analysis at day -2 and day 29. Standard deviation for fat (36,
36, 40) and lean tissue (65, 205, 188) for 0.01 mg/kg, 0.1 mg/kg,
and 1.0 mg/kg, respectively. None of the changes were statistically
significant (p > 0.05).
(6)
(7)
Although itappears that the majority of mass increase shown
in Figure 5 was due to lean mass increase, none of the tissue
weight increases were quite statistically significant (p >
0.05), which might be due to the small group sizes (n = 3)
and relatively large standard deviations.27
Clinical chemistry indicated the expected lowering of
lipids (LDL, HDL, triglycerides).28 Despite the rather dra-
matic increases in body weight over such a short time, there
was no elevation of liver enzyme transaminase levels in any
animal at any dose >2 fold over its baseline value.29,30 Given
the well-established relationship between oral androgen use
and liver stress indicators, we were quite pleased that at a
dose 10-fold greater than the fully effective dose we saw
minimal liver enzyme elevations.31Taken in sum, RAD140
has all the hallmarks of a SARM. It is potency selective, since
it stimulates muscle weight increases at a lower dose than
that required to stimulate prostate weight increases. More-
over, it is also efficacy selective, because it is fully anabolic on
muscle but demonstrates less than complete efficacy on the
prostate and seminal vesicles and, in fact, can partially
antagonize the stimulation of the seminal vesicles induced
by testosterone. RAD140 has excellent pharmacokinetics
and is a potent anabolic in nonhuman primates as well. We
believe the overall preclinical profile of RAD140 is very good,
and the compound has completed preclinical toxicology in
both rats and monkeys. We are currently preparing RAD140
for phase I clinical studies in patients suffering from severe
weight loss due to cancer cachexia.
(8)
(9)
Gao, W.; Bohl, C. E.; Dalton, J. T. Chemistry and structural
biology of the androgen receptor. Chem. Rev. 2005, 105 (9),
3352–3370.
Gao, W.; Dalton, J. T. Ockham's razor and selective androgen
receptors (SARMs): Are we overlooking the role of 5R-reduc-
tase? Mol. Interventions 2007, 7 (1), 10–13.
(10) pK data in rats for compound 3 is provided in the Supporting
Information.
(11) Oral data for compounds 3 and 4 in the Herschberger assay is
shown in the Supporting Information.
(12) Human and rat microsome data are shown in the Supporting
Information.
(13) The left-hand side of the molecule as written is presumed to
overlay with the A-ring of testosterone. This particular left-
hand side equivalent has been utilized to good effect pre-
viously in nonsteroidal SARMs: (a) Li, J. J.; Sutton, J. C.;
Nirschl, A.; Zou, Y.; Wang, H.; Sun, C.; Pi, Z.; Johnson, R.;
Krystek, S. R., Jr.; Seethala, R.; Golla, R.; Sleph, P. G.; Beehler,
B. C.; Grover, G. J.; Fura, A.; Vyas, V. P.; Li, C. Y.; Gougoutas,
J. Z.; Galella, M. A.; Michael, A.; Zahler, R.; Ostrowski, J.;
Hamann, L. G. Discovery of Potent and Muscle Selective
Androgen Receptor Modulators through Scaffold Modifica-
tions. J. Med. Chem. 2007, 50 (13), 3015–3025. The precursor
fragment 6 has been described for the preparation of SARMs in: .
(b) Schlienger, N.; Lund, B. W.; Pawlas, J.; Badalassi, F.;
Bertozzi, F.;Lewinsky, R.;Fejzic, A.;Thygesen, M. B.;Tabatabaei,
A.; Bradley, S. R.; Gardell, L. R.; Piu, F.; Olsson, R. Synthesis,
structure-activity relationships, and characterization of novel
nonsteroidal and selective androgen receptor modulators.
J. Med. Chem. 2009, 52, 7186–7191.
SUPPORTING INFORMATION AVAILABLE Synthetic meth-
ods, NMR spectra, and biological assays. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: * E-mail: cmiller@radiuspharm.com.
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(17) The scheme shown was used to successfully produce ap-
proximately 2 kg of compound 5 in >99% purity under GMP
manufacturing conditions.
(18) The AR binding assay was performed as specified from the
manufacturer. The assay is a fluorometric assay using a tracer
made from fluorescent tagged AR-ligand methyltrienolone
(R1881). Ki values were derived by the Cheng-Prushoff
equation (Ki = (IC50/(1 þ [S]/Km)), where Km was set equal
to Kd, Kd = 25 nM (fluorometric R1881), and [S] = 1.
(19) The C2C12 osteoblast differentiation assay procedure is
explained in the Supporting Information.
(20) Hershberger, L. G.; Shipley, E. G.; Meyer, R. K. Myotropic
activity of 19-nortestosterone and other steroids determined
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(p < 0.05). Although this measurement did not account for
possible diurnal variations in the animals and LH levels were
not definitive, since they were below the level of detection in
most pre- and postdose groups (LH < 0.8 ng/mL), one might
still consider the possibility that even the 0.01 mg/kg dose
was a fully effective, testosterone replacement dose, since
body weight and lean mass were at least maintained (if not
increased) in the low dose group despite significant testos-
terone suppression. Beyond this finding, we do not know
whether testosterone suppression is a proxy for other CNS-
related androgen effects beyond LH interference, such as
mood, libido, and cognition, but we do believe a SARM with
potent androgen agonist, CNS-type activity would be an
interesting tool for that sort of exploration.
(28) For a thorough examination of the effect of high dose,
injected dihydrotestosterone (DHT)on lipids in female, ovar-
iectomized cynomolgus monkeys, see:Nantermet, P.;Harada,
S.; Liu, Y.; Spring, C.; Johnson, C.; Yu, Y.; Kimme, D.; Holder, D.;
Phillips, R.; Ray, W. Gene expression analysis in cynomolgus
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(21) This dose of TP provides an approximate EC70 on prostate
and EC90 on muscle in our Herschberger assays.
(29) Lee, H.; Ha, M.-H.; Christian, D. C. Body weight, alcohol
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study. Int. J. Epidemiol. 2001, 30 (4), 766–770.
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male rats up through the 10 mg/kg po dose range, thereby
ruling out an exposure limited efficacyas opposed to compound-
limited efficacy. Antagonism of TP is further evidence of a
mechanism-specific, limited efficacy as opposed to a pharma-
cokinetic limitation of the compound.
(23) Testosterone levels in castrated rats are <0.5 ng/mL; i.e. see:
D'Souza, S. S.; Selmin, F.; Murty, S. B.; Qiu, W.; Thanoo, B. C.;
DeLuca, P. P. Assessment of fertility in male rats after
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(30) Mean triglyceride changes by group were (-26%, -36%,
-37%), LDL (þ8%, -24%, -53%), and HDL (-13%,
-42%, -64%) for 0.01 mg/kg, 0.1 mg/kg, and 1.0 mg/kg,
respectively. Mean liverenzyme (ALT)changes by group were
(ALT) (-15%, -2%, þ43%) and (ALP) (-9%, þ3%, þ31%)
for 0.01 mg/kg, 0.1 mg/kg, and 1.0 mg/kg, respectively.
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(24) For the intact animal dosing, we had originally planned not to
run testosterone as a comparator, since the intact animals
were relied on to provide their own controls and we were
primarily concerned with how RAD140 behaved within and
against the animal's endogeneous background. Neverthe-
less, we ultimately did include testosterone in one dose group
but used a slightly lower dose because the animals already
have endogeneous testosterone present. Nevertheless, we
did not run a separate testosterone curve in order to deter-
mine what the EC30, EC50, or EC70 for testosterone was in this
model.
(25) The monkeys were placed into groups of three at day-21, and
the weight of each monkey recorded at each time point and
the mean weight of each group are reflected on the graph.
Coincidentally, by day -1, the mean weight of each group
had each converged to a very similar value of 4.26 kg, 4.29 kg,
and 4.28 kg for the 0.01 mg/kg, 0.1 mg/kg, and 1.0 mg/kg
groups, respectively.
(26) pK analysis at various time points throughout this monkey
study indicated that significant increases in exposure were
seen with dose.
(27) Since these were young, intact male cynomolgous monkeys
(3 to 4 years of age), they had fairly high endogeneous total
plasma testosterone at day -1(approximately 600-800 ng/dL),
which is similar to the approximately 600 ng/mL that human
males have between the ages of 25 and 54 (the levels then
gradually decline with age). After 28 days of dosing with
RAD140, the testosterone levels in all three groups was
suppressed to approximately 200-300 ng/dL, with similar
suppression in all three groups, although testosterone levels
were significantly different for only the 0.01 mg/kg group
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