Discovery of a selective small-molecule melanocortin-4 receptor agonist with efficacy in a pilot study of sexual dysfunction in humans

Article abstract of DOI:10.1021/jm9017866

The relevance of the melanocortin system to sexual activity is well established, and nonselective peptide agonists of the melanocortin receptors have shown evidence of efficacy in human sexual dysfunction. The role of the MC4 receptor subtype has received particular scrutiny, but the sufficiency of its selective activation in potentiating sexual response has remained uncertain owing to conflicting data from studies in preclinical species. We describe here the discovery of a novel series of small-molecule MC4 receptor agonists derived from library hit 2. The addition of methyl substituents at C3 and C5 of the 4-phenylpiperidin-4-ol ring was found to be markedly potency-enhancing, enabling the combination of low nanomolar potencies with full rule-of-five compliance. In general, the series shows only micromolar activity at other melanocortin receptors. Our preferred compound 40a provided significant systemic exposure in humans on both sublingual and oral administration and was safe and well tolerated up to the maximum tested dose. In a pilot clinical study of male erectile dysfunction, the highest dose of 40a tested (200 mg) provided a similar level of efficacy to sildenafil.

Full text of DOI:10.1021/jm9017866

J. Med. Chem. 2010, 53, 3183–3197 3183
DOI: 10.1021/jm9017866
Discovery of a Selective Small-Molecule Melanocortin-4 Receptor Agonist with Efficacy in a Pilot
Study of Sexual Dysfunction in Humans
Mark I. Lansdell,* David Hepworth, Andrew Calabrese, Alan D. Brown, Julian Blagg,† Denise J. Burring, Peter Wilson,
David Fradet, T. Bruce Brown, Faye Quinton, Neela Mistry, Kim Tang, Natalie Mount, Peter Stacey, Nick Edmunds,
Cathryn Adams, Samantha Gaboardi, Stevie Neal-Morgan, Chris Wayman, Susan Cole, Joanne Phipps, Mark Lewis,
Hugh Verrier, Val Gillon, Neil Feeder, Anne Heatherington, Stefan Sultana, Scott Haughie, Steven W. Martin,
Maria Sudworth, and Sarah Tweedy
Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, U.K.
†Current address: Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories,
15 Cotswold Road, Sutton SM2 5NG, UK.
Received December 2, 2009
The relevance of the melanocortin system to sexual activity is well established, and nonselective peptide
agonists of the melanocortin receptors have shown evidence of efficacy in human sexual dysfunction.
The role of the MC4 receptor subtype has received particular scrutiny, but the sufficiency of its selective
activation in potentiating sexual response has remained uncertain owing to conflicting data from
studies in preclinical species. We describe here the discovery of a novel series of small-molecule MC4
receptor agonists derived from library hit 2. The addition of methyl substituents at C3 and C5 of the
4-phenylpiperidin-4-ol ring was found to be markedly potency-enhancing, enabling the combination of
low nanomolar potencies with full rule-of-five compliance. In general, the series shows only micromolar
activity at other melanocortin receptors. Our preferred compound 40a provided significant systemic
exposure in humans on both sublingual and oral administration and was safe and well tolerated up to the
maximum tested dose. In a pilot clinical study of male erectile dysfunction, the highest dose of 40a tested
(200 mg) provided a similar level of efficacy to sildenafil.
Introduction
receptors are also subject to regulation by two endogenous
inverse agonists, namely agouti and agouti-related protein
(AGRP).2,7 The MC1 receptor is expressed mainly on cutane-
ous melanocytes and is involved in skin and hair pigmenta-
tion.1,2,4 The MC2 receptor is activated only by ACTH,
differentiating it pharmacologically from the other melano-
cortin receptors, and expression is localized in the adrenal
cortex, where it controls steroidoneogenesis. In contrast, both
the MC3 and MC4 receptors are extensively expressed in the
central nervous system (CNS), where they have been shown to
have major roles in energy homeostasis and feeding behavior,
and they are present only to a much lesser extent in the
periphery. The MC5 receptor is widely distributed in periph-
eral tissues but has only very limited expression in the CNS,
and, beyond control of sebaceous secretion and immuno-
regulation, its function is not well established. These disparate
expression patterns are reflected by the wide range of disease
pathologies in which the melanocortin receptors are consid-
ered to have relevance.3
The involvement of the melanocortin system in regulating
sexual response was first indicated several decades ago after
observation of the effects of administration of melanocortin
peptides in male rabbits8 and female rats.9 More recently,
studies designed to reveal the relative importance of the
melanocortin receptor subtypes in promoting erectile activity
in male rodents have reached conflicting conclusions. The use
of a selective peptidic MC4 receptor antagonist to inhibit the
action of R-MSH and ACTH failed to block penile erection
The melanocortin-4 (MC4)a receptor is a member of a
family of five G-protein coupled receptors (GPCRs) activated
by the melanocortin peptides R-, β-, and γ-melanocyte stimu-
lating hormone (MSH) and adrenocorticotropin hormone
(ACTH).1-4 The MSH and ACTH peptides are products
derived from post-translational processing of a single pro-
opiomelanocortin (POMC) precursor5,6 and share a common
His-Phe-Arg-Trp motif considered crucial to binding. Uniquely
among seven-transmembrane GPCRs, the melanocortin
*To whom correspondence should be addressed. Phone: þ44 1304
645037. Fax: þ44 1304 651819. E-mail: mark.i.lansdell@googlemail.
com.
a Abbreviations: MC, melanocortin; GPCR, G-protein coupled
receptor; ACTH, adrenocorticotropin hormone; MSH, melanocyte
stimulating hormone; POMC, pro-opiomelanocortin; AGRP, agouti-
related protein; CNS, central nervous system; BBB, blood-brain bar-
rier; MED, male erectile dysfunction; PDE, phosphodiesterase; FSAD,
female sexual arousal disorder; DMF, N,N-dimethylformamide; TFA,
trifluoroacetic acid; THF, tetrahydrofuran; NMP, N-methyl pyrrolidi-
none; KHMDS, potassium bis(trimethylsilyl)amide; PMB, para-meth-
oxybenzyl; ACE-Cl, 1-chloroethylchloroformate; NMR, nuclear
magnetic resonance; BINAP, 2,20-bis(diphenylphosphino)-1,10-bi-
naphthalene; CHO, Chinese hamster ovary; SAR, structure-activity
ꢀ
relationship; hERG, human ether-a-go-go related gene; SEM, standard
error of the mean; HEK, human embryonic kidney cells; HLM, human
liver microsomes; RLM, rat liver microsomes; DLM, dog liver micro-
somes; MDCK, Madin-Darby canine kidney cells; MDR, multidrug
resistance protein; CSF, cerebrospinal fluid; AUC, area under the curve;
VSS, visual sexual stimulation; MTBE, methyl tert-butyl ether; SPA,
scintillation proximity assay.
r
2010 American Chemical Society
Published on Web 03/24/2010
pubs.acs.org/jmc

3184 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Lansdell et al.
Figure 1. Structures of some important melanocortin receptor agonists.
even though other melanocortin-induced behavioral effects
such as grooming, stretching, and yawning were abolished.10,11
In contrast, observation of the effects of the selective nonpep-
tide MC4-receptor agonist THIQ (Figure 1) on wild-type
versus MC4 receptor knockout mice,12 and in rats with and
without competing peptide antagonists,13 has implicated the
MC4 receptor in promoting erectile activity.
This unresolved discrepancy in preclinical species notwith-
standing, the therapeutic potential of melanocortin receptor
activation for the treatment of human sexual dysfunction4,14-16
had already been demonstrated by the fortuitous discovery17,18
of the pro-erectile side effect of melanotan II (MT-II), a cyclic
peptide analogue of R-MSH exhibiting agonist activity at the
MC1, MC3, MC4, and MC5 receptors and developed origin-
ally to enhance skin pigmentation.19,20 Subsequent trials
confirmed the efficacy of subcutaneously administered MT-
II in producing erections in men both with psychogenic21 and
organic22 erectile dysfunction. This efficacy has been attrib-
uted to a centrally mediated effect on the basis of a long
latency to MT-II-induced erections and an absence of erecto-
genic activity observed on subcutaneous administration of
melanotan I (MT-I), a related potential tanning agent which
lacks penetration of the blood-brain barrier (BBB).23 Further
development was focused on the intranasal delivery of
bremelanotide (PT-141),24,25 an active metabolite of MT-II,
for which efficacy has been demonstrated both in male
erectile dysfunction (MED)26,27 (including PDE5-inhibitor
nonresponders28,29) and in female sexual arousal disorder
(FSAD).30,31 Despite these very encouraging results, the
development of intranasal bremelanotide for sexual dysfunc-
tion has since been halted prior to phase III due to concerns
regarding increases in blood pressure.32
Figure 2. Identification of compound 2 as a lead from a library of
diverse carboxamides.
erectogenic activity has recently been reported for MK-
0493,33 which is described as a selective small-molecule MC4
receptor agonist. Unfortunately, detailed descriptions of the
pharmacological and pharmacokinetic profiles of MK-0493
are not available, precluding deeper analysis of the features
which may be necessary to elicit clinical efficacy via activation
of the melanocortin system. This situation prompts us to
report here in detail our own efforts toward the design and
study of a small molecule with selectivity for MC4.
In view of the anticipated challenges of designing an orally
available, CNS-penetrant small molecule capable of agoniz-
ing a peptidic GPCR, we were attracted by the trans-4-
phenylpyrrolidine-3-carboxamide template first disclosed by
Goulet and co-workers34 (and subsequently also explored by
others35-39) due to its combination of relatively low basicity,
relatively high rigidity, and nonpeptide-like features com-
pared to most other reported MC4 receptor agonist chemo-
types.40,41 Synthesis and screening of a library of diverse
amides based on template 1 provided very few compounds
with sufficient potency to represent attractive leads aside from
a simple 4-phenylpiperidin-4-ol 2 (Figure 2). During optimiz-
ation of an earlier phenylalanine-based MC4 receptor agonist
chemotype also containing a 4-phenylpiperidin-4-ol subunit,
In contrast to the clinical efficacy observed with the non-
selective agonists MT-II and PT-141, an absence of clinical

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3185
Scheme 1a
a Reagents and conditions: (a) 200 °C; (b) CH2O, MeOH, K2CO3.
Scheme 2a
a Reagents and conditions: (a) (i) (COCl)2, DMF, CH2Cl2, (ii) (S)-(-)-4-benzyl-2-oxazolidinone, LiCl, CH2Cl2; (b) 6 or 7, TFA, CH2Cl2;
(c) formation of 1 from 10a or 12: LiOH, H2O, THF; (d) formation of 12 from 10a: NaOMe, dimethyl carbonate, CH2Cl2; (e) formation of 13 from
11a: MeOH, Sm(OTf)3.
we had observed42 the beneficial effects on potency of intro-
ducing small flanking substituents at C3 and C5 of the
piperidine ring, and thus we chose to begin our follow-up of
lead 2 by examining the addition of a methyl group at these
positions.
acetate and pentane. Removal of the chiral auxiliary was
initially achieved by exposure of 10a to lithium hydroxide
followed by neutralization with a stoichiometric quantity of
aqueous hydrochloric acid, providing carboxylic acid1, which
we isolated as an amorphous foam and used without complete
removal of the residual lithium chloride. On larger scales, a
convenient alternative was to use a two-step process involving
generation of the methyl ester 12 by cleavage of the oxazoli-
dinone auxiliary with sodium methoxide in the presence of
dimethyl carbonate,44 followed by hydrolysis with lithium
hydroxide and acidification to provide the hydrochloride salt
of 1, which could be crystallized from isopropyl alcohol.
To access different pyrrolidine N-substituents, a similar
route was employed using the commercially available N-benzyl
azomethine ylide precursor 7, leading to the formation of
cycloadducts 11a/b, again readily separable by flash chroma-
tography. The only substantial difference in the route was that
the chiral auxiliary was removed from the cycloadduct by
reaction with methanol in the presence of samarium triflate,45
forming ester 13. This key intermediate allowed two alter-
native paths forward, which were chosen according to con-
venience or the demands of other substituents elsewhere in the
target compounds. Hydrogenolysis of 13 provided the sec-
ondary amine 14 (Scheme 3), which was then alkylated to
provide tertiary amines 15-17 or heteroarylated to 18, and
these esters in turn were hydrolyzed to acids 19-22. Alter-
natively, 13 was subjected to transfer hydrogenation in the
presence of di-tert-butyl dicarbonate to generate carba-
mate 23, which similarly was hydrolyzed to the corresponding
acid 24.
Chemistry
Enantiopure pyrrolidine 1 was prepared by a slightly modi-
fied version of literature [3 þ 2] cycloaddition routes.34,39,43
Thus, reaction of chloromethyltrimethylsilane 4 with an ex-
cess of tert-butylamine 3 in a sealed bomb at high temperature
provided secondary amine 5 (Scheme 1), the excess 4 being
readily removed by washing with water. Azomethine ylide
precursor 6 was then obtained by reaction of 5 with formal-
dehyde and methanol in the presence of potassium carbonate.
In our hands, the grade of potassium carbonate (325 mesh)
proved crucial to the success of the reaction, and we found it
most convenient to isolate 6 as a 4:1 mixture with unreacted 5
because purification procedures led to significant loss of 6.
The presence of 5 did not influence subsequent cycloaddition
reactions.
The dipolarophile 9 was prepared by conversion of 2,4-
difluorocinnamic acid 8 to the corresponding acid chloride
and subsequent reaction with the Evans auxiliary (S)-(-)-4-
benzyl-2-oxazolidinone in the presence of lithium chloride
(Scheme2). Exposure of a solution of6 and 9 totrifluoroacetic
acidprovided 3,4-trans disubstituted pyrrolidine cycloadducts
10a/b with negligible diastereoselectivity. However, separa-
tion was readily achieved via flash column chromatography,
thereby enabling selective access to both 1 and ent-1. On
multigram scales, we found that 10a could be obtained
selectively in good yield and high purity from the crude
mixture of cycloadducts 10a/b by crystallization from ethyl
A racemic cis-3,4-disubstituted pyrrolidine 28 was obtained
by cycloaddition of 6 with Z-olefin 27, itself prepared via a Still-
Gennari modification46 of the Horner-Emmons olefination

3186 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Lansdell et al.
Scheme 3a
a Reagents and conditions: (a) Pd(OH)2/C, 50 psi H2, EtOH; (b) CH2O (for 15), or acetone (for 17), NaBH(OAc)3, AcOH, CH2Cl2; (c) EtOTs, K2CO3,
MeCN, 70 °C (for 16); (d) 3-chloropyridazine, i-Pr2NEt, NMP, 120 °C; (e) LiOH, THF, then HCl(aq) (for 19, 21, 22, and 24); (f) HCl(conc) (for 20);
(g) Pd(OH)2/C, 1-methylcyclohexa-1,4-diene, (Boc)2O, EtOH.
Scheme 4a
pounds 40-45 or 49 (Scheme 6), generally after conversion to
their hydrochloride salts. Alternatively, coupling to car-
boxylic acid 24 produced 46, which was N-deprotected and
capped by subsequent N-alkylation or heteroarylation, fol-
lowed again by conversion to the hydrochloride salts. The
products 40-45 and 47-49 exist as mixtures of amide rota-
mers partly resolved on the NMR time-scale, leading to very
complex spectra. However, the full relative and absolute
stereochemistry of a representative compound, 40a, was con-
a Reagents and conditions: (a) (CF3CH2O)2P(O)CH2CO2Me, KHMDS,
firmed by X-ray crystallography (Figure 3).48
18-crown-6, THF, -78 °C; (b) LiOH, H2O, THF; (c) 6, TFA, CH2Cl2.
Results and Discussion
(Scheme 4). Use of the acrylic acid as the dipolarophile avoided
issues of stereochemical lability which we had encountered
during base-mediated hydrolysis of the 3,4-cis isomer of methyl
ester 12.
The structure-activity relationship (SAR) of our com-
pounds at the MC4 receptor is shown in Table 1. Addition
of methyl substituents in a cis-relationship at C3 and C5 of the
piperidine ring was found to provide a marked increase in
potency at the MC4 receptor (compare compounds 2 and
40a). Comparison of the potencies for enantiomeric pair 40a/
44 and racemic cis-diastereomer 45 clearly established that
attractive levels of activity reside only in the trans-3S,4R-
pyrrolidine, and subsequent efforts were focused on this
stereoisomer.
Exploration of the R2 group revealed that various small
substituents are broadly tolerated on the phenyl ring (40a-k),
although no significant potency advantage was found versus
unsubstituted 40a. Notably, the least successful substituent of
these, nitrile, is also the most polar (40f/h). Replacement of the
phenyl ring by a 2-pyridyl (40l) occurred with only minor
reduction in activity, but moving to a 3-pyridyl or 5-pyrimi-
dinyl led to a progressively more pronounced loss of potency
(40 m/n). Replacement of the piperidine’s aromatic substitu-
ent by small alkyl groups at C4 was also briefly examined
(40o-q) in an effort to temper lipophilicity, but the extent of
potency loss outweighed the modest gain in physicochemical
properties (vide infra).
Synthesis of 3,5-dimethyl-4-arylpiperidin-4-ols47 and 3,5-
dimethyl-4-alkylpiperidin-4-ols began with dimethylation of
dimethyl 3-oxopentanedioate 29, the use of 325-mesh potas-
sium carbonate once again proving crucial for successful
reaction (Scheme 5). The double Mannich product 31, ob-
tained on exposure of 30 to benzylamine and formaldehyde in
methanol, was decarboxylated to generate a 6:1 mixture of
meso 33a and racemic 33b. The ratio could be improved to
24:1 favoring the meso-isomer by epimerization with sodium
methoxide in methanol, and the mixture was readily separated
by chromatography on silica. Addition of Grignard reagents
or aryl and alkyl lithium species with subsequent hydrogeno-
lytic benzyl removal provided piperidines 39. Itwasfoundthat
some aryl groups (e.g., para-chlorophenyl) were insufficiently
stable to hydrogenolysis, and therefore an alternative N-PMB
protection was employed(32), leading toa similar24:1ratio of
stereoisomers 34a/b. The PMB protection was also advanta-
geous for larger scale work because 34a could be purified by
crystallization. Removal of the PMB group occurred rapidly
in the presence of 1-chloroethylchloroformate, and this pro-
cess was found to be significantly more facile than for benzyl
deprotection under the same conditions. Similarly, N-Boc
protection was found to be preferable for the addition of
some nucleophiles to the piperidinone, and therefore 33a was
debenzylated in the presence of di-tert-butyl dicarbonate to
provide 35.
The pyrrolidine N-substituent was also subjected to sys-
tematic variation. The SAR in progressing from one- to four-
carbon alkyl groups was found to be rather flat, for example
N-ethyl 42a is essentially equipotent with N-butyl isomers
48c-e, and yet tert-butyl remained the optimum alkyl group
throughout this study, with only n-propyl, cyclopropyl, and
cyclobutyl substituents maintaining potency within 5-fold.
Introduction of polarity into the alkyl chain by means of an
Finally, the secondary amines 39 were then coupled to
carboxylic acids 1, ent-1, and 19-22 to provide test com-

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3187
Scheme 5a
a Reagents and conditions: (a) MeI, K2CO3, THF, 60 °C; (b) benzylamine, HCl(aq), MeOH, CH2O; (c) 1 M HCl(aq), reflux; (d) NaOMe, MeOH;
(e) Pd(OH)2/C, H2, EtOH, Boc2O; (f) R2Li, or R2MgBr, THF; (g) from 36: Pd/C, dihydrotoluene, EtOH, or Pd(OH)2/C, H2, EtOH, or Pd/C, H2,
MeOH; (h) from 37: ACE-Cl, Et3N, CH2Cl2, then MeOH reflux; (i) from 38: 4 M HCl in dioxane, CH2Cl2.
Scheme 6a
a Reagents and conditions: (a) 1, ent-1, 19-22, or 24, 1-propylphosphonic acid cyclic anhydride, Et3N, CH2Cl2; (b) 4 M HCl in dioxane, CH2Cl2;
(c) aldehydes or ketones, NaBH(OAc)3, CH2Cl2; (d) Het-Br or Het-Cl, Et3N, EtOH, reflux; or Het-Cl, Et3N, DMF, 80-100 °C; or Het-Br, sodium
2-methylpropan-2-olate, Pd2(dba)3, (()-BINAP, PhMe, reflux.
etherfunctiondiminishedpotency significantly (48g), whereas
within a cyclic framework the ether was well tolerated (48h).
The absence of a pyrrolidine N-substituent altogether led to a
more dramatic loss of potency (47a-d), although these com-
pounds nonetheless maintained superiority to the original
lead 2.
Wealsocappedthe pyrrolidine nitrogen withsix-membered
heterocycles, which can be organized into two groups on
potency terms: those that maintained or even slightly en-
hanced potency versus a tert-butyl substituent (49a-c), and
those that suffered a 10- to 20-fold loss (49j-l). The results
obtained with these heterocyclic members demonstrate that a
strongly basic center is not essential for achieving high
potency and full efficacy at the MC4 receptor. Of particular
note is the impact that switching to a heterocycle can have on
the tolerance for otherwise less ideal R1 substituents, as
demonstrated by 49e-h in comparison with 40l/o-q. Along-
side the crucial 3,5-dimethyl substituents on the piperidine
ring, the use of a heterocycle at R1 was the only markedly
potency-enhancing SAR feature that we encountered during
this study.
To assess further the potential of this series, a selection of
compounds was screened for selectivity over activity at other
melanocortinreceptorsand thehERG potassium ion channel.
Figure 3. X-ray crystal structure of compound 40a.

3188 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Lansdell et al.
Table 1. Functional Activity of R-MSH, MT-II, 2, 40-45, and 47-49 at the MC4 Receptor
pyrrolidine
stereochemistry
MC4 EC50
(nM)a,b
compd
R2
R1
R
Emax (%)b
R-MSH
MT-II
2
2.2 ( 0.2
0.11 ( 0.05
1166 ( 211
12 ( 1
53 ( 14
30 ( 4
99 ( 1
109 ( 3
87 ( 6
97 ( 2
100 ( 0.3
95 ( 3
97 ( 1
83 ( 8
100 ( 0
90 ( 3
97 ( 4
92 ( 4
95 ( 5
93 ( 3
100 ( 2
95 ( 5
97 ( 3
89 ( 3
100 ( 0
101 ( 2
93 ( 3
77 ( 2
86 ( 4
100 ( 0
100 ( 0
79 ( 4
81 ( 2
92 ( 8
90 ( 4
100 ( 0
100 ( 0
86 ( 4
88 ( 5
100 ( 0.5
Ph
Ph
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
Me
H
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3R,4S
3RS,4RS
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
3S,4R
40a
40b
40c
40d
40e
40f
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
4-MeO-Ph
4-Cl-Ph
4-F-Ph
4-CF3-Ph
4-CN-Ph
3-F-Ph
3-CN-Ph
2,6-diF-Ph
2,4-diF-Ph
3,4-diF-Ph
pyridin-2-yl
pyridin-3-yl
pyrimidin-5-yl
n-Pr
18 ( 3
108 ( 11
347 ( 117c
6 ( 0.7
644 ( 414
45 ( 8
62 ( 28
15 ( 3
46 ( 16
40g
40h
40i
40j
40k
40l
40m
40n
40o
40p
40q
41a
41b
41c
42a
42b
42c
42d
42e
43a
43b
43c
43d
43e
43f
3630 ( 1630
11100 ( 2900c
126 ( 18
626 ( 112c
962 ( 205
717 ( 241
130 ( 21
365 ( 75
85 ( 11c
147 ( 12c
61 ( 9
i-Pr
c-Pr
Ph
4-Cl-Ph
4-F-Ph
Ph
Me
Me
Et
Et
4-F-Ph
3-F-Ph
2,4-diF-Ph
3,4-diF-Ph
Ph
Et
Et
51 ( 7c
Et
69 ( 12c
69 ( 17
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
t-Bu
t-Bu
H
4-Cl-Ph
4-F-Ph
3-F-Ph
2,4-diF-Ph
3,4-diF-Ph
Ph
91 ( 9
52 ( 2c
40 ( 5
220 ( 124
257 ( 216
44
45
>10000c
Ph
Ph
3900 ( 1940
462 ( 115
255 ( 59
159 ( 65
288 ( 50c
19 ( 5
55 ( 20
105 ( 74
73 ( 38
81 ( 23c
32 ( 9
86 ( 0.5
90 ( 4
84 ( 5
79 ( 3
108 ( 15
93 ( 7
47a
47b
47c
47d
48a
48b
48c
48d
48e
48f
4-F-Ph
3-F-Ph
3,4-diF-Ph
Ph
H
H
H
n-Pr
c-Pr
n-Bu
i-Bu
Ph
Ph
100 ( 0
100 ( 1
100 ( 0
100 ( 0.3
78 ( 4
82 ( 4
100 ( 1
90 ( 2
92 ( 4
97 ( 7
94 ( 7
95 ( 4
89 ( 7
98 ( 6
88 ( 4
73 ( 2
Ph
Ph
c-PrCH2
c-Bu
Ph
Ph
48g
48h
49a
49b
49c
49d
49e
49f
(CH2)2OMe
THP-4-yl
374 ( 106
18 ( 5
15 ( 8
Ph
Ph
pyridin-2-yl
pyridin-3-yl
pyridazin-3-yl
pyridazin-3-yl
pyridazin-3-yl
pyridazin-3-yl
pyridazin-3-yl
pyridazin-3-yl
pyrimidin-4-yl
pyrimidin-5-yl
Ph
Ph
5 ( 0.6
6 ( 3
4-F-Ph
pyridin-2-yl
n-Pr
4 ( 0.8c
7 ( 7
21 ( 4
89 ( 8
49g
49h
49i
i-Pr
c-Pr
82 ( 20
4 ( 1
Ph
Ph
49j
117 ( 21c

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3189
Table 1. Continued
pyrrolidine
stereochemistry
MC4 EC50
(nM)a,b
compd
R2
R1
R
Emax (%)b
49k
49l
Ph
Ph
pyrimidin-2-yl
pyrazin-2-yl
Me
Me
3S,4R
3S,4R
243 ( 37
111 ( 19
75 ( 4
87 ( 4
a Human recombinant MC4 receptor expressed in CHO cells, β-lactamase reporter gene format, with the maximal response defined by MT-II (see the
Supporting Information for full details). b Except where noted, values are mean of n g 3 experiments ( SEM. c n = 2.
Table 2. Activities of Selected Compounds at the MC1, MC3, and MC5 Receptors, the hERG Potassium Ion Channel, and in Binding at the MC4 Receptor
MC3 EC50 (μM)
[Emax (%)]a,f
MC1 EC50 (μM)
[Emax (%)]b,f
MC5 EC50 (μM)
[Emax (%)]c,f
hERG Ki
(μM)d,f
MC4 binding
Ki (nM)e,f
compd
R2/R1
R-MSH
MT-II
40a
40c
40d
40g
40i
0.0027 ( 0.0004 [99 ( 4] 0.0009 ( 0.0001 [96 ( 2]
0.0054 ( 0.0008 [87 ( 7] 0.0001 ( 0.00002 [98 ( 6] 0.069 ( 0.016 [61 ( 12] NT
0.36 ( 0.07 [105 ( 4]
NT
21 ( 2
NT
Ph/t-Bu
1.16 ( 0.35 [99 ( 5]
3.68 ( 2.89 [75 ( 21]g
3.05 ( 1.25 [79 ( 3]
0.57 ( 0.09 [81 ( 4]g
4.57 ( 2.79 [76 ( 34]
10.7 ( 5.1
1.02 ( 0.30 [103 ( 6]
>5.88
1.98 ( 0.20 [100 ( 5]
>10.0
>10.0h
7.91 ( 0.34g 36 ( 4
NT
5.00 ( 0.7g
NT
5.84h
NT
27 ( 4g
48 ( 12
4-Cl-Ph/t-Bu
4-F-Ph/t-Bu
3-F-Ph/t-Bu
2,6-diF-Ph/t-Bu
2,4-di-F-Ph/t-Bu
pyridin-2-yl/t-Bu
n-Pr/t-Bu
5.19 ( 3.00 [107 ( 5]
3.60 ( 1.22 [116 ( 14]
1.80 [74]h
>5.81
1.12 ( 0.36 [99 ( 3]
>6.47
97 ( 22
99 ( 14
>5.01
40j
40l
6.57 ( 2.00 [93 ( 12]g
1.17 ( 0.10 [97 ( 10]
3.61 ( 0.26 [95 ( 9]
>7.81
3.50 [84]h
17.8 [100]h
NT
176 ( 24
350 ( 66
844 ( 4g
1070 ( 598g
346 ( 66g
355 ( 62
114 ( 15
>33.3
>24.7
40o
41a
41c
42c
43a
47a
48b
48c
48f
Ph/Me
>19.1
>33.4
>20.0h
NT
NT
4-F-Ph/Me
3-F-Ph/Et
Ph/i-Pr
>6.15
>8.25
15.0 [100]h
6.41 [92]h
3.64 ( 1.33 [103 ( 21]
>20.0g
>13.5
11.6 ( 7.44 [72 ( 5]
>25.2
>33.3h
>5.72h
0.50 ( 0.06 [81 ( 1]g
>14.5
NT
>7.88h
0.75 ( 0.26 [100 ( 8]
>8.74
>4.91
Ph/H
Ph/c-Pr
6.65 ( 1.19g 264 ( 94g
NT
1.43 [72]h
NT
>20.0h
0.96 [29]h
NT
>20.0
>9.17h
NT
290 ( 106
233 ( 30
Ph/n-Bu
Ph/c-Bu
1.10 ( 0.16 [72 ( 7]
0.35 ( 0.06 [79 ( 5]g
>11.9
5.87 ( 0.28g 62 ( 3
48h
49a
49b
49c
49e
49f
Ph/THP-4-yl
Ph/pyridin-2-yl
Ph/pyridin-3-yl
Ph/pyridazin-3-yl
>5.27g
36 ( 5
0.071 ( 0.006 [78 ( 6]g
0.065 ( 0.024 [90 ( 1]g
0.49 ( 0.21 [89 ( 6]
>16.7
>8.22
>3.07
4.65 ( 0.36g 79 ( 2
5.07 ( 0.71g 50 ( 5
>10.0g
NT
10 ( 3
52 ( 4
56 ( 4
172 ( 29
64 ( 19
300 ( 41g
Pyridin-2-yl/pyridazin-3-yl 3.66 ( 2.50 [75 ( 8]
1.85 ( 0.38 [67 ( 3]
3.90 ( 0.55 [104 ( 6]g
>16.7
NT
NT
n-Pr/pyridazin-3-yl
c-Pr/pyridazin-3-yl
Ph/pyrimidin-4-yl
Ph/pyrimidin-2-yl
7.48 ( 6.40 [79 ( 14]
4.13 ( 0.5 [42 ( 6]g
0.60 ( 0.28 [75 ( 4]
>26.7
>10.0g
>10.0g
>10.0g
NT
49h
49i
NT
>14.0
>11.1h
>9.00
>10.00
49k
a Human recombinant MC3 receptor expressed in CHO cells, measuring adenylyl cyclase activation. b Human recombinant MC1 receptor expressed
in HEK-293 cells, measuring adenylyl cyclase activation. c Human recombinant MC5 receptor expressed in CHO-cells, β-lactamase reporter gene
format. d Competitive binding versus [3H]-dofetilide at the hERG product expressed in HEK-293 cells. e Competitive binding versus [125I]-MT-II at the
human recombinant MC4 receptor expressed in CHO cells. f Except where noted, values are the mean of g3 experiments ( SEM. g n = 2. h n = 1.
As can be seen from Table 2, compounds in the R1 = alkyl or
H series (40-43, 47, 48) were found in general to have only
relatively weak activity at the MC1, MC3, and MC5 recep-
tors, providing good selectivity margins in many cases.
Among the R1 = alkyl subset, the presence of a cyclobutyl
substituent at R1 (48f) stands out as providing the highest
absolute potency at both the MC3 and MC1 receptors,
although, given the high MC4 receptor potency of this
compound, significant selectivity still exists.
In contrast, the presence of a heteroaryl substituent at R1
was found to confer high potency at the MC3 receptor in some
instances, especially the pyridyls 49a and 49b. However, it is
clear that the R2 substituent can be varied to selectively
attenuate MC3 receptor activity in preference to MC4 activity
(compare 49c with 49e/f, Tables 1 and 2). Interestingly, the
heterocycles which were found to be less active at the MC4
receptor were also correspondingly weaker atMC3 (e.g., 49k).
No significant activity at the MC1 and MC5 receptors was
observed within the heteroaryl subset.
(Table 2), their affinities very approximately tracking with the
agonist potencies.
A further selection of compounds was assessed for meta-
bolic stability in human liver microsomes (HLM), and repre-
sentative examples are shown in Table 3. The presence of a
metabolically inert substituent at the 4-position of the R2 aryl
ring consistently provided increased microsomal stability
(compare 40c-e versus 40a, or 40g versus 40d/k, and similarly
42f versus 42d and 43b/d/f versus 43a), suggesting this as a
location of oxidation on the unsubstituted parents (vide
infra). Replacement of the aryl ring by a small alkyl chain
was also beneficial to metabolic stability (40q versus 40a),
although here the reduction in lipophilicity may also play a
role alongside the removal of a potentially labile aryl site.
Complete removal of the pyrrolidine N-substituent con-
ferred high metabolic stability (47b/d), whereas variation of
the R1 alkyl group isomers and homologues showed little
benefit to be derived from minimization of the lipophilicity in
this region, and all changes explored were actually detrimental
compared to tert-butyl (compare for example compound 40c
versus 43b, and compound 40k versus 42f and 43f). Themarked
deterioration in microsomal stability for the cyclopropyl
analogue 48b may result in part from its significantly increased
Only weak micromolar activity was encountered at the
hERG potassium ion channel across the whole series, regard-
less of basicity and despite the high lipophilicities. Finally, all
compounds were confirmed as binders of the MC4 receptor

3190 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Lansdell et al.
logD, presumably driven by diminished pyrrolidine basicity.
The placement of polarity directly within the R2 alkyl group
was also unsuccessful in improving metabolic stability (48g/h
versus 40a).
The N-heteroaryl subseries showed very poor HLM stabi-
lity (e.g., 49c), and even the use of a 4-fluoro substituent on the
aryl ring (49d) or reduction in logD (49e/f) provided no
improvement. Nonetheless, the high MC4 receptor potency
of these compounds remained attractive to us, and the
examples shown above ultimately became the progenitors of
a promising neutral/weakly basic series that will be described
in more detail elsewhere.
compounds showed somewhat higher metabolic vulnerability
in rat liver microsomes (RLM) than in HLM. In dog liver
microsomes (DLM), however, 40d has high stability, whereas
40a is metabolized very rapidly. Unsurprisingly, both com-
pounds are similar to each other in their levels of plasma
protein binding across the three species, the slightly higher
figures for 40d no doubt reflecting its slightly higher lipophi-
licity. Both compounds showed a slight preference for parti-
tioning into blood versus plasma across all three species. Their
good flux across MDR1-overexpressing MDCK monolayers,
with minor (40a) or negligible (40d) efflux ratios, provided
encouragement that CNS penetration would be high.
On the basis of the combined considerations of potency at
the MC4 receptor, selectivity over the MC1/3/5 receptors and
the hERG ion channel, and in vitro metabolic stability, we
selected compounds 40a and 40d for further evaluation owing
to the superior balance of their profiles across these various
parameters. Table 4 shows additional in vitro data that were
generated prior to in vivo profiling in preclinical species. Both
The results from rat and dog pharmacokinetic studies with
40a and 40d are shown in Table 5. In keeping with the rapid in
vitro metabolism of 40a in rat and dog liver microsomes, 40a
was found to be subject to a high rate of metabolism by both
species in vivo, with oral bioavailability being limited by the
high first-pass extraction. The high volumes of distribution
measured for 40a and 40d are consistent with the physico-
chemistry of a lipophilic base (measured pKa 8.95 and 8.74,
respectively) and provide for moderate half-lives in both rat
and dog. As anticipated by the good MDCK-MDR1 flux
values, 40a and 40d partitioned freely into the cerebrospinal
fluid (CSF) of the rat, suggesting unimpaired blood-brain
barrier penetration.
Analysis of rat urine following iv administration revealed
that <3% of the dose was renally excreted as the parent
compound. Furthermore, in an isolated perfused rat liver
experiment, <1% of 40a was eliminated unchanged in the
bile. The identification of metabolites generated from 40a in
vitro was carried out after incubation in human, rat, and dog
hepatocyte preparations. In human hepatocytes, the major
metabolites detected were a mono-oxidized species consistent
with oxidation of the tert-butyl substituent (50) and N-de-
alkylated product 47a (Figure 4). Several less abundant
metabolites were also identified, including at least two species
resulting from oxidation localized within the phenylpiperidine
moiety (51) and an M þ 34 species (52) presumed to be a
dihydrodiol. Metabolites 50-52 and 47a were all major
metabolites in dog hepatocytes, whereas in rat 50, 47a, and
several mono-oxidized species 51 were predominant. A pyr-
rolidine N-oxide was alsodetected as a majorcomponent in all
hepatocyte experiments but was likewise found in buffer
control, so is not necessarily enzymatic in origin.
Table 3. Experimentally Measured logD and Human Liver Microsome
Stability
HLM Clint
logD(pH7.4) (μL/min/mg protein)a
compd
R2/R1
40a Ph/t-Bu
2.8
3.9
3.1
4.1
3.3
3.5
2.2
2.9
3.2
3.2
3.5
3.2
3.3
1.8
2.0
45
16
40c 4-Cl-Ph/t-Bu
40d 4-F-Ph/t-Bu
40e 4-CF3-Ph/t-Bu
40g 3-F-Ph/t-Bu
40k 3,4-diF-Ph/t-Bu
40q c-Pr/t-Bu
28
<7
52
22
14
42c 3-F-Ph/Et
3,4-diF/Et
73
28
42f
43a Ph/i-Pr
128
34
43b 4-Cl-Ph/i-Pr
43d 3-F-Ph/i-Pr
70
28
43f 3,4-diF-Ph/i-Pr
47b 4-F-Ph/H
13
7
47d 3,4-diF-Ph/H
48b Ph/c-Pr
>4
342
84
48g Ph/(CH2)2OMe
48h Ph/THP-4-yl
3.1
3.1
3.3
3.70
3.0
2.8
151
243
310
>440
>440
49c Ph/pyridazin-3-yl
49d 4-F-Ph/pyridazin-3-yl
49e pyridin-2-yl/pyridazin-3-yl
49f
n-Pr/pyridazin-3-yl
a The volume of in vitro incubation cleared of test compound per unit
time, normalized to 1 mg of human liver microsomal protein.
In comparison with 40a, the improved rat and dog liver
microsome stability of 40d translated accordingly to lower
Table 4. In Vitro Pharmacokinetic Measurements for Compounds 40a and 40d
RLM Clint
(μL/min/mg
protein)a
DLM Clint
(μL/min/mg
protein)a
human
plasma
rat
plasma
dog
plasma
human/rat/dog
blood:plasma
ratio
MDCK-MDR1
AB/BA Papp
(ꢀ 106 cm s-1 c
18/41
22/26
)
b
b
b
compd
fu
fu
fu
3
40a
40d
60
47
242
<8
0.13
0.07
0.07
0.04
0.15
0.09
1.1/1.1/1.1
1.7/1.3/1.6
a The volume of in vitro incubation cleared of test compound per unit time, normalized to 1 mg of rat or dog liver microsomal protein. b The free
fraction of test compound in human, rat, or dog plasma. c The apparent permeability of the test compound across cell monolayers as measured in the
apical to basolateral (AB) and basolateral to apical (BA) directions.
Table 5. In Vivo Rat and Dog Pharmacokinetic Measurements for Compounds 40a and 40d
rata Clplasma
(mL/min/kg)
rata Vdplasma
(L/kg)
rata T1/2
(h)
ratb CSF:free
plasma
ratc F
(%)
dogd Clplasma
(mL/min/kg)
dogd Vdplasma
(L/kg)
dogd T1/2
(h)
doge F
(%)
compd
40a
40d
89
34
19
11
2.4
3.9
0.9
0.8
20
74
46
12
17
11
4.2
10.4
1
28
a 40a/d 1 mg/kg iv. b iv infusion to steady state. c 40a 1 mg/kg po, 40d 0.1 mg/kg po. d 40a 0.5 mg/kg iv, 40d 0.05 mg/kg iv. e 40a 0.5 mg/kg po, 40d
0.1 mg/kg po.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3191
in vivo plasma clearances in these species, leading to signifi-
cantly enhanced oral bioavailabilities (Table 5). Metabolite
identification after incubation in liver microsomes revealed a
similar array of metabolites to those observed with 40a across
allthreespecies, with the exception thatinthiscase two further
oxidized products, corresponding to 53 and 54, were detected
in HLM and DLM (Figure 5).
The broad off-target profiles of 40a and 40d were assessed
in the CEREP Bioprint wide ligand screening panel and were
both found to be similar and generally very clean, with the
most potent binding activities being at the σ receptor (40a
Ki = 330 nM; 40d not tested), the sodium ion channel (40a
Ki = 690 nM; 40d Ki = 520 nM), and the muscarinic M2
receptor (40a Ki = 730 nM; 40d Ki = 1200 nM). Thus, judged
by the results of the preclinical pharmacokinetic studies, 40d
appears to potentially offer advantages over 40a. However,
althoughthedataarepresentedhereside-by-side, inrealitythe
chronology of compound synthesis meant that40a progressed
through our screen sequence far in advance of 40d. Conse-
quently, the potential utility of 40a for testing the mechanism
in humans was considered in isolation from much of the data
shown herein for 40d, with the latter eventually being posi-
tioned as a possible back-up to 40a.
be compromised by very low bioavailability owing to exten-
sive first-pass extraction. To mitigate this risk, we chose to
examine alternative routes of administration, specifically in-
tranasal and sublingual, deemed to be appropriate for on-
demand dosing in sexual health indications. In dog pharma-
cokinetic studies, bothintranasal andsublingual dosing of40a
produced significant improvement in AUC and bioavailabil-
ity over oral administration (Table 6). Nonetheless, it was
considered important to be able to demonstrate higher Cmax
levels than that achieved by the 0.14 mg/kg doses (equating
to ∼1 nM unbound). The prospects for achieving this via the
intranasal route seemed poor owing to limitations in the
maximum solution volume which can be administered,
whereas sublingual dosing offered greater flexibility. Two
higher sublingual doses were therefore studied, and we were
pleased to observe dose-dependent, approximately propor-
tional increases in Cmax and AUC.
With our increased confidence that adequate bioavailabil-
ity in humans would be enabled by use of sublingual dosing
should oral bioavailability prove poor, compound 40a, desig-
nated internally as PF-00446687,49 was progressed through
the remaining preclinical in vitro and in vivo toxicology
studies with satisfactory outcomes. In phase I trials in healthy
male volunteers, 40a was found to be safe and generally well
tolerated via both oral and sublingual administration up to
the maximum tested dose. The most common treatment-
related systemic adverse event was restlessness (6.3%).
Stretching and yawning were other events recorded which
could be indicative of centrally mediated effects. Nausea
(6.1%) was predominantly associated with sublingual dosing,
and its lack of prevalence is notable when compared with the
human data from MT-II and PT-141, for which nausea is
dose-limiting.22,23 Sublingual dosing of 40a also gave rise to
reports of application-site anesthesia, irritation, and hypothe-
sia. There were no serious adverse events and no discontinua-
tions.
Because 40a suffers essentially liver-blood-flow clearance in
rat and dog, we were concerned that, on oral dosing in
humans, the compound’s otherwise attractive profile could
Following sublingual doses ranging from 3 to 195 mg, peak
plasma concentrations were achieved within 4 h at the lower
doses and up to 8 h at the higher doses. Systemic exposures
increased supraproportionally with dose (Cmax increasing
162-fold for a 65-fold increase in dose across the entire range),
and the estimated mean terminal half-life was 12 h for all
doses. In the event, oral dosing provided very similar expo-
sures at the comparable dose levels (32.5 and 195 mg). Peak
plasma concentrations were uniformly found at 4 h post oral
dose, with a terminal half-life again of 12 h. Reflecting the
preclinical pharmacokinetics in rat (vide supra), unchanged
40a excreted in urine was <2.5% of the 195 mg doses and
similar for both routes of administration. The mean peak
concentration from the 195 mg oral dose was 30 ng/mL
(compared to 31 ng/mL from the 195 mg sublingual dose),
equating to 8.3 nM unbound.
Figure 4. Metabolites of 40a identified from incubation with human,
rat, and dog liver microsomes.
The effect of 40a at oral doses of up to 200 mg in men with
moderate to severe MED was assessed versus 100 mg of sil-
denafil50,51 by the penile plethysmography technique in a pilot
placebo-controlled trial utilizing a randomized, double-blind,
Figure 5. Additional oxidized species identified from 40d in human
and dog liver microsomes.
Table 6. Comparison of Oral, Intranasal, and Sublingual Dog Pharmacokinetic Parameters for 40a
dose (mg/kg)
route
Cmax (ng/mL)
AUCinf (ng.h/ml)
Tmax (h)
T1/2 (h)
F (%)
0.5
oral
0.9
2.6
2.6
15
1.8
12
1.0
1.6
0.7
0.3
0.3
1.9
3.3
3.7
3.6
4
1
24
35
26
20
0.14
0.14
0.67
2.04
intranasal
sublingual
sublingual
sublingual
17
64
41
146

3192 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Lansdell et al.
in contrast to the observed dog pharmacokinetics, exposure
after oral dosing of 40a in humans compared favorably with
that achieved by sublingual administration. At the highest
dose tested (200 mg), compound 40a has shown a level of
efficacy that is similar to sildenafil and differentiated from
placebo in a study of erectile activity in patients with moderate
to severe MED, although lower doses were not efficacious.
The mean peak unbound plasma concentration achieved at
the highest dose is approximately equal to the EC50 of 40a
measured at the MC4 receptor in vitro and markedly lower
than its potency at other melanocortin receptors, thus provid-
ing support for the hypothesis that MC4 receptor activation is
indeed sufficient to elicit erectogenic effects in humans.
Experimental Section
All commercially available chemical reagents were used as
received without further purification unless otherwise noted.
Proton NMR data were recorded at 400 MHz on a Varian
Mercury 400BB or a Varian Inova 400 instrument. Mass spectro-
scopic data were recorded using a Thermo Scientific Finnigan
aQa APCI instrument, a Thermo Scientific Navigator ESI in-
strument, a Waters ZQ APCI LCMS instrument, or a Waters ZQ
ESI LCMS instrument. Elemental analyses were performed by
Exeter Analytical (UK) Ltd., and optical rotations were mea-
sured by Warwick Analytical Service. All test compounds were
confirmed to be of g95% purity by either elemental analysis or
HPLC (see Supporting Information).
Figure 6. Mean and 80% confidence interval of rigidity response
(%) by treatment over viewing segments (PD = predose; PVSS1 =
previsual sexual stimulation period 1; VSS1 = visual sexual stimu-
lation period 1; N1 = neutral period 1; VSS2 = visual sexual
stimulation period 2; N2 = neutral period 2). The two doses of 40a
were separated by a minimum 7-day washout between each study
period.
crossover design.52 Subjects were shown sequences of visual
sexual stimulation (VSS) and neutral (non-VSS) material
beginning 2 h postdose. The 200 mg dose of 40a (n = 15)
provided similar efficacy to sildenafil and was superior to
placebo for both base-rigidity (Figure 6) and questionnaire
(data not shown) end-points during periods of VSS. However,
below 200 mg (n = 24), only a weak dose-response relation-
ship (not considered clinically meaningful) was observed.
The syntheses ofcompounds 40a, 40c-d, 40f-g, 40i-l, 40o-q,
41b-c, 42c, 43a-c, 43e-f, 45, 47a-d, 48a-h, 49a-c, 49e,
and 49i-l have been described previously.49 Compo-
unds 40a, 40d, 40k, and 45 serve as representative procedures
for the final-step synthesis of the remainder of the compounds.
Method A. (3R,4R,5S)-1-{[(3S,4R)-1-tert-Butyl-4-(2,4-difluo-
rophenyl)pyrrolidin-3-yl]carbonyl}-3,5-dimethyl-4-phenylpiperidin-
4-ol Hydrochloride (40a). (3S,4R)-1-tert-Butyl-4-(2,4-difluoro-
phenyl)pyrrolidine-3-carboxylic acid49 (1) (26.0 g, 92 mmol)
and (3R,4s,5S)-3,5-dimethyl-4-phenylpiperidin-4-ol49 (39, R2 =
Ph) (17.4 g, 85 mmol) were suspended in dichloromethane (1000 mL).
Triethylamine (14.2 mL, 102 mmol) was added, and the mixture
was cooled to 0 °C with stirring under nitrogen. 1-Propylpho-
sphonic acid cyclic anhydride (50% in ethyl acetate, 54.5 mL,
92 mmol) was added dropwise, maintaining the temperature below
5 °C. The mixture was then allowed to warm to room temperature
with continuous stirring, and after 1 h acetic acid (5 mL) was
added to remove the last traces of the (3R,4s,5S)-3,5-dimethyl-4-
phenylpiperidin-4-ol. The reaction mixture was stirred for a further
1 h at room temperature. A 10% aqueous potassium carbonate
solution (500 mL) was added, and the mixture was stirred vigor-
ously at room temperature for 2 h. The organic layer was separated
and then stirred with 10% aqueous potassium carbonate solution
(500 mL) for 1 h. The dichloromethane layer was then separated,
washed with water (3 ꢀ 300 mL), dried over sodium sulfate, and
filtered. A solution of 4 M hydrogen chloride in dioxane (50 mL)
was then added to the dichloromethane solution. The solvent was
then evaporated to give the crude hydrochloride as a white powder.
Acetone (500 mL) was added to the crude hydrochloride, and the
mixture was boiled for 30 min and then allowed to cool to room
temperature. The hydrochloride salt was filtered off and washed
with acetone (5 ꢀ 100 mL). Recrystallization of the product from
isopropyl alcohol gave analytically pure hydrochloride (39.5 g). 1H
NMR (400 MHz, CD3OD) δ (rotamers) 1.28-1.59 (4d, 6H),
0.64-1.70 (m, 10H), 1.97-12.80 (m, 2H), 3.18 (t, 1H),
3.43-3.62 (m, 2H), 3.70-3.98 (m, 4H), 4.02-4.15 (m, 1H), 4.36
(dd, 1H), 7.02-7.40 (m, 7H), 7.60-7.78 (2q, 1H). MS (APCI) m/z
471 [MH]þ. [R]D25 -51.9 (3.00 mg/mL, MeOH).
Conclusion
In summary, we have discovered a novel series of small-
molecule MC4 receptor agonists based on initial library hit 2.
The addition of methyl substituents at C3 and C5 of the 4-
phenylpiperidin-4-ol unit provided significant potency en-
hancement and allowed us to achieve potent agonism with
compounds which remained fully rule-of-five compliant,53 a
major challenge when targeting a peptidic GPCR.54,55 Ex-
ploration of the SAR atthe pyrrolidine N-substituent revealed
tert-butyl to provide the optimum balance of potency and
metabolic stability compared to other alkyl substituents. Use
of a six-membered heterocycle at this position can be advan-
tageous for potency, especially with regard to allowing greater
flexibility in the substituents at C4 of the piperidine, but in
some cases significant MC3 receptor activity is encountered,
and in all cases shown herein high metabolic vulnerability was
a major flaw. With regard to variation at C4 of the piperidine
ring, efforts to diminish metabolic vulnerability by introduc-
tion of polarity or reduction in lipophilic bulk did not lead to
overall superiority because of the counterbalancing detrimen-
tal impact on MC4 receptor potency. However, the presence
of a lipophilic substituent in the para-position of the aryl ring
enhanced in vitro metabolic stability in several cases. The
translation of this effect to a reduction in in vivo clearance and
a corresponding increase in oral bioavailability was demon-
strated with 40d. Compound 40a was deemed to provide the
best opportunity for most rapidly testing the MC4-selective
hypothesis in the clinic, although alternative routes of administ-
ration were explored in an effort to mitigate the risk that oral
dosing would provide inadequate bioavailability. Ultimately,
Method B. (3R,4R,5S)-1-{[(3S,4R)-1-tert-Butyl-4-(2,4-difluo-
rophenyl)pyrrolidin-3-yl]carbonyl}-4-(4-fluorophenyl)-3,5-dimethyl-
piperidin-4-ol Hydrochloride (40d). 1-Propylphosphonic acid cyclic

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3193
anhydride (50 wt % solution in ethyl acetate, 0.67 mL, 2.0 mmol)
was added dropwise to a mixture of (3R,4s,5S)-4-(4-fluorophenyl)-
3,5-dimethylpiperidin-4-ol49 (39, R2 = 4-F-Ph) (267 mg, 1.2
mmol), (3S,4R)-1-tert-butyl-4-(2,4-difluorophenyl)pyrrolidine-3-
carboxylic acid49 (1) (450 mg, 1.4 mmol), and triethylamine
(0.48 mL, 3.6 mmol) in dichloromethane (25 mL) at 0 °C under
nitrogen. On completion of addition, the resulting homogeneous
solution was stirred for 6 h at room temperature. The solution
was washed with 10% aqueous potassium carbonate solution
(3 ꢀ 20 mL) and then dried over sodium sulfate and filtered. The
solvent was removed invacuo, andthe crude productwas purified
by column chromatography (reverse phase C-18, 40 g Redisep
cartridge, utilizing an ISCO Companion autopurification system.
Mobile phase gradient over 20 min: MeCN/H2O/TFA(5%/95%/
0.1%) 95%:MeCN (100%) 5% eluting to MeCN/H2O/TFA-
(5%/95%/0.1%) 5%:MeCN(100%) 95%). The purified product
was then dissolved in 1,4-dioxane (100 mL), and a 4 M solution of
hydrogen chloride in dioxane (20 mL) was added. The solution
was then concentrated to dryness, and the residue was redissolved
in a solution of 4 M hydrogen chloride in dioxane (100 mL) and
concentrated to dryness once more. The residue was then dried in
vacuo at 50 °C to give the product hydrochloride (391 mg) as a
white amorphous solid. 1H NMR (CD3OD 400 MHz) δ
(rotamers) 0.31-0.57 (3d, 6H), 0.83-2.08 (3m, 2H), 1.55 (s,
9H), 1.60-2.07 (3m, 2H), 2.68-3.20 (2m, 2H), 3.20-4.12 (m,
5H), 4.29-4.35 (m, 1H), 6.95-7.19 (m, 5H), 7.38-7.85 (m, 2H).
MS (APCI) m/z 489 [MH]þ. [R]D 25 -42.7 (3.10 mg/mL, MeOH).
Method C. (3R,4R,5S)-1-{[(3S,4R)-1-tert-Butyl-4-(2,4-difluo-
rophenyl)pyrrolidin-3-yl]carbonyl}-4-(3,4-difluorophenyl)-3,5-dim-
ethylpiperidin-4-ol Hydrochloride (40k). A solution of (3S,4R)-1-
tert-butyl-4-(2,4-difluorophenyl)pyrrolidine-3-carboxylic acid49
(1) (159 mg, 0.49 mmol), (3R,4s,5S)-4-(3,4-difluorophenyl)-3,5-
dimethylpiperidin-4-ol49 (39, R2 = 3,4-diF-Ph) (100 mg,
0.41 mmol), 1-propylphosphonic acid cyclic anhydride (50% in
ethyl acetate, 244 μL, 0.41 mmol), and triethylamine (120 μL,
0.41 mmol) in dichloromethane (2.5 mL) was stirred for 3 days at
room temperature. The reaction was then diluted with dichlor-
omethane (20 mL) and partitioned with 10% aqueous potassium
carbonate solution (20 mL). The phases were separated, and the
organic phase was washed with brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. Purification by
column chromatography on silica gel using dichloromethane:
methanol:0.88 ammonia (99:1:0.1 to 96:4:0.4) as eluent gave the
free base as a colorless oil 117 mg. The oil was dissolved in
dichloromethane (1 mL) and treated with 2 M hydrogen chloride
in diethyl ether (3 mL). The solvent was then removed in vacuo,
and the residue was azeotroped with diethyl ether to afford the
phases were separated, and the organic phase was washed with brine
(30 mL), dried over magnesium sulfate, filtered, and concentrated in
vacuo to give a crude residue. Purification by column chromatog-
raphy on silica gel using dichloromethane:methanol:0.88 ammonia
(99:1:0.1 to 98:2:0.2) gave the desired product as a white foam,
497 mg. 1H NMR (400 MHz, CD3OD) δ (rotamers) 0.43-0.55 (m,
6H), 0.75-0.79 (m, 1H), 1.25 (s, 9H), 1.87-1.97(m, 1H), 2.16-2.66
(m, 2H), 3.09 (t, 2H), 3.18-3.30 (m, 2H), 3.41-3.61 (m, 2H), 3.80-
4.17 (m, 3H), 6.91-7.09 (m, 3H), 7.28-7.35 (m, 3H), 7.47 (q, 1H).
MS (APCI) m/z 471 [MH]þ.
1-{[(3S,4R)-1-tert-Butyl-4-(2,4-difluorophenyl)pyrrolidin-3-yl]-
carbonyl}-4-phenylpiperidin-4-ol (2). (3S,4R)-1-tert-Butyl-4-(2,4-
difluorophenyl)pyrrolidine-3-carboxylic acid49 (1) (670 mg,
2.3 mmol) and 4-phenylpiperidin-4-ol (350 mg, 2.0 mmol) were
suspended in dichloromethane (50 mL). Triethylamine (0.61 mL,
4.4 mmol) was added, and the mixture was cooled to 0 °C with
stirring under nitrogen. 1-Propylphosphonic acid cyclic anhy-
dride (50% in ethyl acetate, 1.46 mL, 2.3 mmol) was added
dropwise while the reaction temperature was maintained below
5 °C. The mixture was allowed to warm to room temperature
with continuous stirring and was then stirred for 1 h at room
temperature. The mixture was then washed with 10% aqueous
potassium carbonate solution (3 ꢀ 50 mL), dried over sodium
sulfate, filtered, and then concentrated in vacuo. The crude
residue was purified by column chromatography on silica gel
eluting from 100% dichloromethane to dichloromethane:metha-
nol:0.880 ammonia (90:10:0.5) to give the title compound as a
1
white amorphous solid (690 mg, 68%). H NMR (400 MHz,
CD3OD) δ 1.25 (s, 9H), 1.87-1.97 (m, 2H), 2.16-2.66 (m, 2H),
3.09 (t, 2H), 3.18-3.30 (m, 2H), 3.41-3.61 (m, 2H), 3.80-4.17
(m, 4H), 6.91-7.09(m, 3H), 7.28-7.35(m, 4H), 7.47(q, 1H). MS
(APCI) m/z 443 [MH]þ. [R]D25 -21.6 (1.61 mg/mL, MeOH).
(3R,4R,5S)-1-{[(3S,4R)-1-tert-Butyl-4-(2,4-difluorophenyl)pyr-
rolidin-3-yl]carbonyl}-4-(4-methoxyphenyl)-3,5-dimethylpiperidin-
4-ol Hydrochloride (40b). Prepared according method B from
(3S,4R)-1-tert-butyl-4-(2,4-difluorophenyl)pyrrolidine-3-carboxylic
acid49 (1) and (3R,4s,5S)-4-(4-methoxyphenyl)-3,5-dimethylpi-
peridin-4-ol49 (39, R2 = 4-MeO-Ph). 1H NMR (400 MHz,
CD3OD) δ (rotamers) 0.31-0.59 (3d, 6H), 1.58 (s, 9H), 0.79-
1.99 (m, 4H), 1.60-2.07 (m, 3H), 2.68-3.20 (m, 2H), 3.21-4.12
(m, 5H), 4.29 (m, 2H), 6.81-7.29 (m, 5H), 7.60-7.74 (m, 2H).
25
MS (APCI) m/z 501 [MH]þ. [R]D -18.53 (1.11 mg/mL,
MeOH).
(3R,4R,5S)-1-{[(3S,4R)-1-tert-Butyl-4-(2,4-difluorophenyl)pyr-
rolidin-3-yl]carbonyl}-3,5-dimethyl-4-[4-(trifluoromethyl)phenyl]-
piperidin-4-ol Hydrochloride (40e). Prepared according to method
C from (3S,4R)-1-tert-butyl-4-(2,4-difluorophenyl)pyrrolidine-3-
carboxylic acid49 (1) and (3R,4s,5S)-3,5-dimethyl-4,4-(trifluoro-
methyl)phenyl-piperidin-4-ol49 (39, R2 = 4-CF3-Ph). 1H NMR
(400 MHz, CD3OD) δ (rotamers) 0.29-0.58 (4d, 6H), 1.50 (s,
9H), 1.60-2.15 (m, 2H), 2.70-2.81 (m, 1H), 3.11-3.21 (m, 1H),
3.38-3.72 (m, 3H), 3.79-4.15 (m, 4H), 4.36 (d, 1H), 7.02-7.24
1
title compound as a white solid, 108 mg. H NMR (400 MHz,
CD3OD) δ (rotamers) 0.29-0.60 (4d, 6H), 1.49 (s, 9H), 1.58-
2.08 (m, 2H), 2.75-3.19 (2t, 2H), 3.38-3.64 (m, 2H), 3.70-3.99
(m, 4H), 4.01-4.17 (m, 1H), 4.36 (dd, 1H), 7.02-7.37 (m, 5H),
7.58-7.73 (m, 1H). MS (APCI) m/z 507 [MH]þ. [R]D25 -34.89
(2.30 mg/mL, MeOH).
25
(m, 3H), 7.58-7.70 (m, 4H). MS (APCI) m/z 539 [MH]þ. [R]D
-49.53 (2.15 mg/mL, MeOH).
Method D. (3R,4S,5S)-1-{[(3RS,4RS)-1-tert-Butyl-4-(2,4-di-
fluorophenyl)pyrrolidin-3-yl]carbonyl}-3,5-dimethyl-4-phenylpiper-
idin-4-ol (45). To a cooled solution of (3RS,4RS)-1-tert-butyl-
4-(2,4-difluorophenyl)pyrrolidine-3-carboxylic acid49 (28) (500 mg,
1.76 mmol) in dichloromethane (20 mL) was added a catalytic
amount of N,N-dimethylformamide followed by oxalyl chloride
(309 μL, 3.53 mmol). The reaction mixture was stirred for 2 h at
room temperature, and the solvent was then removed in vacuo. The
residual white powder was azeotroped with dichloromethane (2 ꢀ
10 mL). The white powder was then redissolved in dichloromethane
(10 mL), and the resulting solution was added dropwise to a solution
of (3R,4s,5S)-3,5-dimethyl-4-phenylpiperidin-4-ol49 (39, R2 = Ph)
(362 mg, 1.76 mmol) and triethylamine (246 μL, 1.76 mmol) in
dichloromethane (10 mL) over 10 min at room temperature. The
resulting mixture was stirred for 24 h at room temperature, diluted
with dichloromethane (10 mL), and partitioned with saturated
aqueous sodium hydrogen carbonate solution (2 ꢀ 30 mL). The
(3R,4s,5S)-4-(3-Bromophenyl)-1-(4-methoxybenzyl)-3,5-dimet-
hylpiperidin-4-ol (37, R2 = 3-Br-Ph). A solution of 3-bromoio-
dobenzene (4.6 g, 25 mmol) in anhydrous diethyl ether (200 mL)
was cooled to -78 °C under nitrogen. n-Butyl lithium (2.5 M in
hexanes, 15.2 mL, 20 mmol) was added dropwise while the
reaction temperature was maintained below -65 °C. The mix-
ture was stirred at -78 °C for 2 h and then allowed to warm to
room temperature. The resulting solution of 3-bromophenyl
lithium was then added dropwise to a solution of (3R,5S)-1-(4-
methoxybenzyl)-3,5-dimethylpiperidin-4-one49 (34a) (5.0 g,
20 mmol) in diethyl ether (25 mL) at -78 °C. The mixture was
stirred at -78 °C for a further 2 h and then allowed to warm to
room temperature. The mixture was quenched with saturated
aqueous ammonium chloride (50 mL). The organic phase was
separated, washed with water (3 ꢀ 50 mL), dried over sodium
sulfate, and then concentrated in vacuo. Recrystallization of the

3194 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Lansdell et al.
residue from cyclohexane/ethyl acetate afforded the pure pro-
duct as light-yellow needles (6.5 g, 56%). 1H NMR (400 MHz,
CD3OD) δ 0.48 (d, 6H), 2.19-2.27 (m, 2H), 2.31-2.39 (m, 2H),
2.79-2.83 (m, 4H), 3.65 (s, 3H), 7.15-7.20 (d, 2H), 7.26-
7.35 (d, 2H), 7.46-7.51 (m, 1H), 7.94-8.25 (m, 3H). MS
(APCI) m/z 405 [MH]þ.
2.46 mmol) and 5-bromopyrimidine (1.17 g, 7.33 mmol) were
dissolved in anhydrous THF (5 mL) with stirring, and the resulting
solution was cooled to -78 °C. n-Butyl lithium (2.5 M in hexanes)
(1.08 mL, 2.7 mmol) was then added dropwise over 25 min. The
reaction mixture was then allowed to warm to ambient temperature
over 16 h. The solvents were removed in vacuo, and the residual
orange oil was dissolved in EtOAc (25 mL) and partitioned with
water (25 mL). The organic layer was separated, washed with brine
(25 mL), and then dried over anhydrous MgSO4 before being filtered
and concentrated in vacuo to give the crude product as an orange oil.
Purification by cartridge chromatography (Redisep 20 g cartridge)
eluting with CH2Cl2:MeOH:NH3 98:2:0.2 gave the title compound
as a yellow oil (252 mg, 33%). 1H NMR (400 MHz, CDCl3) δ=0.62
(d, 6H), 1.49 (s, 9H), 1.90 (s, 1H), 2.08 (bs, 2H), 2.82 (bs, 2H),
3.90-4.10 (m, 2H), 8.76-8.79(m,2H), 9.15(s, 1H).MS(APCI) m/z
308 [MH]þ.
(3R,4s,5S)-3,5-Dimethyl-4-pyrimidin-5-ylpiperidin-4-ol Hydro-
chloride (39, R2 = pyrimidin-5-yl). To a room temperature
solution of tert-butyl (3R,4s,5S)-4-hydroxy-3,5-dimethyl-4-pyr-
imidin-5-ylpiperidine-1-carboxylate (38, R2 = pyrimidin-5-yl)
(251 mg, 0.82 mmol) in CH2Cl2 (1 mL) was added HCl in
dioxane (4 M solution, 5 mL, 20 mmol), and the reaction
mixture was stirred at room temperature for 16 h. The solvents
were evaporated in vacuo, and the residue was azeotroped with
CH2Cl2 (2 ꢀ 20 mL) to yield the title compound as a white solid
(182 mg, 80%). 1H NMR (400 MHz, CD3OD) δ 1.70 (d, 6H),
2.36-2.40 (m, 2H), 3.15 (t, 2H), 3.24 (dd, 2H), 8.82-9.10 (m,
2H), 9.17 (s, 1H). MS (APCI) m/z 208 [MH]þ.
3-[(3R,4s,5S)-4-Hydroxy-1-(4-methoxybenzyl)-3,5-dimethyl-
piperidin-4-yl]benzonitrile (37, R2 = 3-CN-Ph). A solution of
(3R,4s,5S)-4-(3-bromophenyl)-1-(4-methoxybenzyl)-3,5-dimethyl-
piperidin-4-ol (37, R2 = 3-Br-Ph) (3.50 g, 8 mmol), potassium
cyanide (1.05 g, 16 mmol), tetrakis(triphenylphosphine)pallad-
ium(0) (0.462 g, 0.4 mmol), and copper iodide (1.52 g, 8 mmol) in
propionitrile (30 mL) was heated at reflux for 1 h. The mixture
was cooled to room temperature, diluted with ethyl acetate
(30 mL), and filtered through celite. The filtrate was washed
with water (3 ꢀ 50 mL), brine (1 ꢀ 50 mL), dried over sodium
sulfate, and filtered. Concentration in vacuo gave a residue
which was purified by column chromatography on silica gel
using ethyl acetate:hexane (3:97 to 15:85, gradient elution) to
1
afford the title compound as a yellow solid (2.51 g, 59%). H
NMR (400 MHz, CD3OD) δ 0.51 (d, 6H), 2.25-2.31 (m, 2H),
2.42-2.53 (m, 2H), 2.79-2.83 (m, 4H), 3.61 (s, 3H), 7.22 (d, 2H),
7.25-7.35, (d, 2H) 7.66-7.79 (m, 1H), 8.12-8.35 (m, 3H). MS
(APCI) m/z 351 [MH]þ.
3-[(3R,4s,5S)-4-Hydroxy-3,5-dimethylpiperidin-4-yl]benzonitrile
(39, R2 = 3-CN-Ph). 1-Chloroethylchloroformate (1.50 mL,
14 mmol) was added dropwise to a stirred solution of 3-[(3R,4s,
5S)-4-hydroxy-1-(4-methoxybenzyl)-3,5-dimethylpiperidin-4-yl]-
benzonitrile (37, R2 = 3-CN-Ph) (2.50 g, 7.1 mmol) and triethy-
lamine (2.0 mL, 14 mmol) in dichloromethane (50 mL) at -15 °C.
The mixture was then stirred for 30 min at -15 °C. The solvent
was removed in vacuo, and the crude residue was heated in
methanol (100 mL) at reflux for 3 h. After cooling to room
temperature, the solvent was removed in vacuo, and the
residue was dissolved in dichloromethane (100 mL). Potassium
carbonate (325 mesh, 5 g) was added, and the mixture was
stirred at room temperature for 1 h. The mixture was then
filtered, and the filtrate was concentrated in vacuo. The crude
residue was recrystallized from acetonitrile to afford the pure
product as fine white needles (1.23 g, 61%). 1H NMR (400 MHz,
CD3OD) δ 0.60 (d, 6H), 2.25-2.45 (m, 2H), 3.1-3.36 (m, 4H),
7.62-7.75 (m, 1H), 8.14-8.28 (m, 3H). MS (APCI) m/z 231
[MH]þ.
(3R,4R,5S)-1-{[(3S,4R)-1-tert-Butyl-4-(2,4-difluorophenyl)-
pyrrolidin-3-yl]carbonyl}-3,5-dimethyl-4-pyrimidin-5-ylpiperidin-
4-ol Dihydrochloride (40n). Prepared according to method C
from (3S,4R)-1-tert-butyl-4-(2,4-difluorophenyl)pyrrolidine-
3-carboxylic acid49 (1) and (3R,4s,5S)-3,5-dimethyl-4-pyrimi-
din-5-ylpiperidin-4-ol hydrochloride (39, R2 = pyrimidin-5-yl).
1H NMR (400 MHz, CD3OD) δ (rotamers) 0.36-0.64 (4d, 6H),
1.51 (s, 9H), 1.70-2.21 (m, 1H), 2.65-2.81 (m, 1H), 3.18 (q, 1H),
3.42-3.72 (m, 3H), 3.80-4.16 (m, 4H), 4.36-4.42 (m, 1H),
7.02-7.20 (m, 2H), 7.58-7.76 (m, 1H), 8.70 (bs, 1H), 8.96 (d,
1H), 9.16-9.21 (m, 1H). MS (APCI) m/z 473 [MH]þ. [R]D
-46.8 (2.1 mg/mL, MeOH).
25
(3R,4R,5S)-1-{[(3S,4R)-4-(2,4-Difluorophenyl)-1-methylpyrr-
olidin-3-yl]carbonyl}-3,5-dimethyl-4-phenylpiperidin-4-ol Hydro-
chloride (41a). Prepared according to method C from (3S,4R)-
4-(2,4-difluorophenyl)-1-methylpyrrolidine-3-carboxylic acid49
(19) and (3R,4s,5S)-3,5-dimethyl-4-phenylpiperidin-4-ol49 (39,
3-[(3R,4R,5S)-1-{[(3S,4R)-1-tert-Butyl-4-(2,4-difluorophenyl)-
pyrrolidin-3-yl]carbonyl}-4-hydroxy-3,5-dimethylpiperidin-4-yl]-
benzonitrile Hydrochloride (40h). Prepared according to method
B from (3S,4R)-1-tert-butyl-4-(2,4-difluorophenyl)pyrrolidine-
3-carboxylic acid49 (1) and 3-[(3R,4s,5S)-4-hydroxy-3,5-dimet-
1
R2 = Ph). H NMR (400 MHz, CD3OD) δ (rotamers) 0.21-
0.58 (4d, 6H), 0.90-1.05 (m, 1H), 1.71-2.07 (m, 1H), 2.75-2.81
(m, 1H), 3.10 (s, 3H), 3.11-3.19 (m, 1H), 3.32-3.38 (m, 1H),
3.43-3.79 (m, 2H), 3.85-4.20 (m, 4H), 4.36 (d, 1H), 7.05-7.39
1
hylpiperidin-4-yl]benzonitrile (39, R2 = 3-CN-Ph). H NMR
(m, 7H), 7.58-7.76 (m, 1H). MS (ESI) m/z = 429 [MH]þ. [R]D
-49.56 (4.10 mg/mL, MeOH).
25
(400 MHz, CD3OD) δ (rotamers), 0.36-0.57 (m, 6H), 0.83-
2.08 (m, 2H), 1.55 (s, 9H), 1.60-2.07 (m, 2H), 2.68-3.20 (m,
2H), 3.20-4.12 (m, 7H), 4.29-4.32 (m, 1H), 6.95-7.19 (m, 5H),
7.78-7.99 (m, 2H). MS (APCI) m/z 496 [MH]þ. [R]D25 -40.53
(2.10 mg/mL, MeOH).
(3R,4R,5S)-1-{[(3S,4R)-4-(2,4-Difluorophenyl)-1-ethylpyrro-
lidin-3-yl]carbonyl}-3,5-dimethyl-4-phenylpiperidin-4-ol Hydro-
chloride (42a). Prepared according to method C from (3S,4R)-
4-(2,4-difluorophenyl)-1-ethylpyrrolidine-3-carboxylic acid49 (20)
and (3R,4s,5S)-3,5-dimethyl-4-phenylpiperidin-4-ol49 (39, R2 =
Ph). 1H NMR (400 MHz, CD3OD) δ (rotamers) 0.23-0.56 (4d,
6H), 0.61-1.01 (m, 1H), 1.28-1.48 (m, 3H), 1.68-2.04 (m, 1H),
2.71-2.86 (m, 1H), 3.10-3.22 (m, 1H), 3.35-3.50 (m, 3H),
3.57-4.24 (m, 6H), 4.31-4.47 (m, 1H), 7.01-7.73 (m, 8H). MS
(ESI) m/z = 443 [MH]þ. [R]D25 -49.06 (1.60 mg/mL, MeOH).
(3R,4R,5S)-1-{[(3S,4R)-4-(2,4-Difluorophenyl)-1-ethylpyrro-
lidin-3-yl]carbonyl}-4-(4-fluorophenyl)-3,5-dimethylpiperidin-4-ol
Hydrochloride (42b). Prepared according to method B from
(3S,4R)-4-(2,4-difluorophenyl)-1-ethylpyrrolidine-3-carboxylic
acid49 (20) and (3R,4s,5S)-4-(4-fluorophenyl)-3,5-dimethylpi-
peridin-4-ol49 (39, R2 = 4-F-Ph). 1H NMR (400 MHz, CD3OD)
δ (rotamers) 0.30-0.59 (3d, 6H), 0.89-2.01 (m, 2H), 1.51-1.60
(m, 4H), 1.54-2.09 (m, 2H), 2.61-2.81 (m, 1H), 3.16-4.26 (m,
8H), 4.39-4.53 (m, 1H), 6.80-7.27 (m, 4H), 7.39-7.46 (m, 2H),
(3R,4R,5S)-1-{[(3S,4R)-1-tert-Butyl-4-(2,4-difluorophenyl)pyr-
rolidin-3-yl]carbonyl}-3,5-dimethyl-4-pyridin-3-ylpiperidin-4-ol
Dihydrochloride (40m). Prepared according to method C from
(3S,4R)-1-tert-butyl-(2,4-difluorophenyl)pyrrolidine-3-carboxy-
lic acid49 (1) and (3R,4s,5S)-3,5-dimethyl-4-pyridin-3-ylpiper-
idin-4-ol49 (39, R2 = pyridin-3-yl). 1H NMR (400 MHz,
CD3OD) δ (rotamers) 0.37-0.61 (4d, 6H), 0.70-0.99 (m, 1H),
1.50 (s, 9H), 1.80-1.98 (m, 1H), 2.09-2.29 (m, 1H), 2.63-2.81
(m, 2H), 3.19 (q, 1H), 3.45-3.57 (m, 1H), 3.59-3.70 (m, 1H),
3.82-4.17 (m, 4H), 4.38-4.45 (m, 1H), 7.01-7.13 (m, 2H),
7.60-7.83 (m, 1H), 8.09-8.17 (m, 1H), 8.79 (dd, 1H), 8.82-8.92
(m, 1H). MS (ESI) m/z = 472 [MH]þ. [R]D25 -35.95 (4.20 mg/
mL, MeOH).
tert-Butyl (3R,4s,5S)-4-Hydroxy-3,5-dimethyl-4-pyrimidin-5-
ylpiperidine-1-carboxylate (38, R2 = pyrimidin-5-yl). tert-Butyl
(3R,5S)-3,5-dimethyl-4-oxopiperidine-1-carboxylate49 (35) (560 mg,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3195
7.81-7.97 (m, 1H). MS (APCI) m/z 461 [MH]þ. [R]D25 -21.07
(1.45 mg/mL, MeOH).
solutionoflithium hydroxide (129 mg, 5.4 mmol) inwater (3 mL).
The resulting solution was then stirred at room temperature for
5 h. The solvents were removed in vacuo, and the residual orange
oil was dissolved in water (10 mL) and HCl (1N aq solution)
(5.4 mL) was added to give an orange precipitate which had a
gel-like consistency. The solution was filtered, and the residual
gel was dissolved in EtOAc:MeOH (1:1) (200 mL). The solvents
were then evaporated in vacuo to provide an orange solid. The
filtrate was extracted with EtOAc:MeOH (95:5) (2 ꢀ 60 mL),
and the combined organic fractions were dried over anhydrous
MgSO4 (s), filtered, combined with the previously collected
orange solid, and concentrated in vacuo to give an orange solid
(561 mg, 97%). 1H NMR (400 MHz, CD3OD) δ = 3.63 (q, 1H),
3.72 (t, 1H), 3.99 (t, 1H), 4.09-4.12 (m, 4H), 7.01 (t, 2H), 7.52 (q,
1H), 7.75 (d, 1H), 7.86 (dd, 1H), 8.55 (s, 1H). MS (APCI) m/z =
306 [MH]þ. [R]D25 -2.14 (2.80 mg/mL, MeOH).
(3R,4R,5S)-1-{[(3S,4R)-4-(2,4-Difluorophenyl)-1-pyridazin-3-
ylpyrrolidin-3-yl]carbonyl}-4-(4-fluorophenyl)-3,5-dimethylpiperi-
din-4-ol Hydrochloride (49d). (3R,4R,5S)-1-{[(3S,4R)-4-(2,4-Di-
fluorophenyl)pyrrolidin-3-yl]carbonyl}-4-(4-fluorophenyl)-3,5-
dimethylpiperidin-4-ol hydrochloride49 (47b) (250 mg, 0.53 mmol)
and 3-chloropyridazine (305 mg, 2.65 mmol) were treated as for
compound 18 above to give the title compound as an off-white
solid (24 mg, 6%). 1H NMR (400 MHz, CD3OD) δ (rotamers)
0.40-0.61 (4d, 6H), 0.72-0.81 (m, 1H), 1.61-1.69 (m, 1H),
2.70-2.80 (m, 1H), 3.10 (2t, 1H), 3.71 (2 ꢀ dd, 1H), 3.81-4.01
(m, 2H), 4.10-4.38 (m, 5H), 6.95-7.20 (m, 5H), 7.36 (q, 1H),
7.49-7.66 (m, 1H), 7.71-7.75 (m, 1H), 7.87 (dd, 1H), 8.52-8.57
(m, 1H). MS (ESI) m/z = 511 [MH]þ. [R]D25 -30.85 (2.35 mg/
mL, MeOH).
(3R,4R,5S)-1-{[(3S,4R)-4-(2,4-Difluorophenyl)-1-pyridazin-3-
ylpyrrolidin-3-yl]carbonyl}-3,5-dimethyl-4-propylpiperidin-4-ol
Hydrochloride (49f). Prepared according to method C from
(3S,4R)-4-(2,4-difluorophenyl)-1-pyridazin-3-ylpyrrolidin-3-yl-
carboxylic acid hydrochloride (22) and (3R,4s,5S)-3,5-dimethyl-
4-propylpiperidin-4-ol49 (39, R2 = n-Pr). 1H NMR (400 MHz,
CD3OD) δ (rotamers) 0.41-0.49 (m, 1H), 0.67-0.87 (4d, 6H),
0.87-0.96 (m, 4H), 1.16-1.71 (m, 4H), 2.58-2.67 (m, 1H), 2.96
(2t, 1H), 3.51 (2 ꢀ dd, 1H), 3.82-3.98 (m, 2H), 4.01-4.10 (m,
2H), 4.13-3.20 (m, 3H), 7.00-7.11 (m, 2H), 7.44-7.60 (m, 1H),
7.70-7.74 (m, 1H), 7.87 (dd, 1H), 8.53-8.55 (m, 1H). MS
(APCI) m/z = 459 [MH]þ. [R]D25 -7.30 (3.05 mg/mL, MeOH).
(3R,4R,5S)-1-{[(3S,4R)-4-(2,4-Difluorophenyl)-1-pyridazin-3-
ylpyrrolidin-3-yl]carbonyl}-4-isopropyl-3,5-dimethylpiperidin-4-ol
Hydrochloride (49g). Prepared according to method C from
(3S,4R)-4-(2,4-difluorophenyl)-1-pyridazin-3-ylpyrrolidin-3-yl-
carboxylic acid hydrochloride (22) and (3R,4s,5S)-4-isopropyl-
3,5-dimethylpiperidin-4-ol49 (39, R2=i-Pr).1H NMR(400MHz,
CD3OD) δ (rotamers) 0.58-0.67 (m, 1H), 0.72-1.01 (6d, 12H),
1.38-1.86 (m, 1H), 1.93-2.13 (m, 1H), 2.64-2,74 (m, 1H), 2.86-
3.13 (2t, 1H), 3.35-3.51 (m, 1H), 3.78-3.90 (m, 1H), 3.93-4.02
(m, 1H), 4.02-4.21 (m, 4H), 6.99-7.11 (m, 2H), 7.44-7.61 (m,
1H), 7.69-7.73 (m, 1H), 7.86 (dd, 1H), 8.53-8.55 (m, 1H). MS
(APCI) m/z = 459 [MH]þ. [R]D25 -7.80 (2.05 mg/mL, MeOH).
(3R,4R,5S)-4-Cyclopropyl-1-{[(3S,4R)-4-(2,4-difluorophenyl)-
1-pyridazin-3-ylpyrrolidin-3-yl]carbonyl}-3,5-dimethylpiper-
idin-4-ol Hydrochloride (49h). Prepared according to method C
from (3S,4R)-4-(2,4-difluorophenyl)-1-pyridazin-3-ylpyrrolidin-
3-yl-carboxylic acid hydrochloride (22) and (3R,4s,5S)-4-cyclo-
propyl-3,5-dimethylpiperidin-4-ol49 (39, R2 = c-Pr). 1H NMR
(400 MHz, CD3OD) δ (rotamers) 0.26-0.39 (m, 4H), 0.43 (q,
1H), 0.47-0.56 (m, 1H), 0.81-1.00 (4d, 6H), 1.31-1.80 (m, 1H),
2.56-2.65 (m, 1H), 2.94 (2t, 1H), 3.51 (2 ꢀ dd, 1H), 3.79-3.97
(m, 2H), 4.05-4.21 (m, 5H), 7.00-7.21 (m, 2H), 7.53 (2q, 1H),
7.71-7.75 (m, 1H), 7.87 (dd, 1H), 8.52-8.57 (m, 1H). MS (ESI)
m/z = 457 [MH]þ. [R]D25 -3.88 (2.90 mg/mL, MeOH).
(3R,4R,5S)-4-(2,4-Difluorophenyl)-1-{[(3S,4R)-4-(2,4-difluo-
rophenyl)-1-ethylpyrrolidin-3-yl]carbonyl}-3,5-dimethylpiperidin-
4-ol Hydrochloride (42d). Prepared according to method B from
(3S,4R)-4-(2,4-difluorophenyl)-1-ethylpyrrolidine-3-carboxylic
acid49 (20) and (3R,4s,5S)-4-(2,4-difluorophenyl)-3,5-dimethyl-
1
piperidin-4-ol49 (39, R2 = 2,4-diF-Ph). H NMR (400 MHz,
CD3OD) δ (rotamers), 0.32-0.59 (m, 6H), 0.85-1.98 (m, 2H),
1.47-1.59 (m, 4H), 1.55-2.05 (m, 2H), 2.55-2.61 (m, 2H),
3.11-4.23 (m, 7H), 4.31-4.48 (m, 1H), 6.85-7.20 (m, 2H),
7.35-7.42 (m, 2H), 7.76-7.99 (m, 2H). MS (APCI) m/z 479
[MH]þ. [R]D25 -29.53 (1.83 mg/mL, MeOH).
(3R,4R,5S)-4-(3,4-Difluorophenyl)-1-{[(3S,4R)-4-(2,4-difluo-
rophenyl)-1-ethylpyrrolidin-3-yl]carbonyl}-3,5-dimethylpiperidin-
4-ol Hydrochloride (42e). Prepared according to method C from
(3S,4R)-4-(2,4-difluorophenyl)-1-ethylpyrrolidine-3-carboxylic
acid49 (20) and (3R,4s,5S)-4-(3,4-difluorophenyl)-3,5-dimethyl-
1
piperidin-4-ol49 (39, R2 = 3,4-diF-Ph). H NMR (400 MHz,
CD3OD) δ (rotamers) 0.26-0.57 (4d, 6H), 0.62-0.92 (m, 1H),
1.39-1.46 (m, 3H), 1.69-2.00 (m, 1H), 2.69-2.82 (m, 1H), 3.13
(t, 1H), 3.35-4.14 (m, 8H), 4.33 (d, 1H), 6.78-7.34 (m, 5H),
25
7.55-7.72 (m, 1H). MS (APCI) m/z = 479 [MH]þ. [R]D
-56.17 (3.00 mg/mL, MeOH).
(3R,4R,5S)-1-{[(3S,4R)-4-(2,4-Difluorophenyl)-1-isopropylpyr-
rolidin-3-yl]carbonyl}-4-(3-fluorophenyl)-3,5-dimethylpiperidin-
4-ol Hydrochloride (43d). Prepared according to method B from
(3S,4R)-4-(2,4-difluorophenyl)-1-isopropylpyrrolidine-3-carbo-
xylic acid49 (21) and (3R,4s,5S)-4-(3-fluorophenyl)-3,5-di-
methylpiperidin-4-ol49 (39, R2 = 3-F-Ph). 1H NMR (400 MHz,
CD3OD) δ (rotamers), 0.34-0.52 (3d, 6H), 0.79-1.20 (m, 2H),
1.39-1.61 (m, 6H), 1.49-2.30 (m, 2H), 2.65-2.82 (m, 1H),
3.19-4.21 (m, 8H), 4.35-4.42 (m, 1H), 6.78-7.18 (m, 5H),
25
7.28-7.75 (m, 2H). MS (APCI) m/z 475 [MH]þ. [R]D -31.53
(1.05 mg/mL, MeOH).
(3R,4S,5S)-1-{[(3R,4S)-1-tert-Butyl-4-(2,4-difluorophenyl)pyr-
rolidin-3-yl]carbonyl}-3,5-dimethyl-4-phenylpiperidin-4-ol (44).
Prepared from (3R,4S)-1-tert-butyl-4-(2,4-difluorophenyl)pyr-
rolidine-3-carboxylic acid (ent-1) and (3R,4s,5S)-3,5-dimethyl-
4-phenylpiperidin-4-ol49 (39, R2 = Ph) according to method D.
1H NMR (400 MHz, CD3OD) δ (rotamers), 0.43-0.55 (m, 6H),
0.75-0.79 (m, 1H), 1.25 (s, 9H), 1.87-1.97 (m, 1H), 2.16-2.66
(m, 2H), 3.09 (t, 2H), 3.18-3.30 (m, 2H), 3.41-3.61 (m, 3H),
3.80-4.17 (m, 3H), 6.91-7.09 (m, 3H), 7.28-7.35 (m, 3H), 7.47
(q, 1H). MS (APCI) m/z 471 [MH]þ.
Methyl (3S,4R)-4-(2,4-Difluorophenyl)-1-pyridazin-3-ylpyr-
rolidine-3-carboxylate (18). To a room temperature solution of
methyl (3S,4R)-4-(2,4-difluorophenyl)pyrrolidine-3-carboxy-
late49 (14) (1.00 g, 4.14 mmol) in anhydrous NMP (10 mL)
was added N,N-diisopropylethylamine (2.15 mL, 12.42 mmol)
and 3-chloropyridazine (2.37 g, 20.7 mmol). The resulting
solution was then warmed to 120 °C under nitrogen with stirring
for 4 h. The reaction mixture was then cooled to room tempera-
ture and water (250 mL) and MTBE (150 mL) were added. The
aqueous layer was separated and extracted with MTBE (150 mL).
The organic fractions were combined and washed with brine
(150 mL) and then dried over anhydrous MgSO4 (s), filtered,
and evaporated in vacuo to give a crude red oil. Purification by
column chromatography on silica gel eluting with EtOAc:
pentane 20:80 to EtOAc 100% (gradient elution) gave the title
compound as an orange oil (581 mg, 44%). 1H NMR (400 MHz,
CDCl3) δ = 3.54 (q, 1H), 3.67 (s, 3H), 3.76-3.85 (m, 1H), 3.93-
3.98 (m, 1H), 4.00-4.06 (m, 1H), 4.12-4.24 (m, 2H), 6.79-6.88
(m, 2H), 7.28-7.31 (m, 2H), 7.81-7.84 (m, 1H), 8.65-8.66 (m,
1H). MS (APCI) m/z = 320 [MH]þ.
(3S,4R)-4-(2,4-Difluorophenyl)-1-pyridazin-3-ylpyrrolidine-3-
carboxylic Acid Hydrochloride (22). To a room temperature
solution of methyl (3S,4R)-4-(2,4-difluorophenyl)-1-pyridazin-
3-ylpyrrolidin-3-yl-carboxylate (18) (576 mg, 1.8 mmol) in
tetrahydrofuran (10 mL) was added dropwise a freshly prepared
Acknowledgment. We thank Hans Hockey for his assis-
tance with the statistical analyses of the clinical data pre-
sented, and we thank Cheryl Doherty, Sarah Kempshall,

3196 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Lansdell et al.
Katherine Fenner, and Anthony Harrison for assistance in
compiling data available in the Supporting Information.
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Supporting Information Available: Elemental and HPLC
analyses for test compounds, X-ray crystallographic data for
compounds 10a and 40a, proof of absolute stereochemistry of
compound 11a (via X-ray crystallography of a derivative), in
vitro biological assay protocols, in vitro and in vivo pharmaco-
kinetic protocols. This material is available free of charge via the
Internet at http://pubs.acs.org.
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the safety, pharmacokinetic properties and pharmacodynamic
effects of intranasal PT-141, a melanocortin receptor agonist, in
healthy males and patients with mild-to-moderate erectile dysfunc-
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