Dihydropyrrolopyrazol-6-one MCHR1 antagonists for the treatment of obesity: Insights on in vivo efficacy from a novel FLIPR assay setup

Article abstract of DOI:10.1016/j.bmcl.2015.05.008

Our investigation of the structure-activity and structure-liability relationships for dihydropyrrolopyrazol-6-one MCHR1 antagonists revealed that off-rate characteristics, inferred from potencies in a FLIPR assay following a 2 h incubation, can impact in

Full text of DOI:10.1016/j.bmcl.2015.05.008

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2793–2799
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dihydropyrrolopyrazol-6-one MCHR1 antagonists for the treatment
of obesity: Insights on in vivo efficacy from a novel FLIPR assay setup
a
a
a
b
Pratik Devasthale a, , Wei Wang , Andres S. Hernandez , Fang Moore , Kishore Renduchintala ,
Radhakrishnan Sridhar b, Mary Ann Pelleymounter c, Daniel Longhi c, Ning Huang c, Neil Flynn c,
Anthony V. Azzara c, Kenneth Rohrbach c, James Devenny c, Suzanne Rooney c, Michael Thomas c,
Susan Glick c, Helen Godonis c, Susan Harvey c, Mary Jane Cullen c, Hongwei Zhang d,
Christian Caporuscio d, Paul Stetsko d, Mary Grubb d, Christine Huang d, Lisa Zhang d,
⇑
Chris Freeden d, Yi-Xin Li d, Brian J. Murphy c, Jeffrey A. Robl a, William N. Washburn a,
⇑
a Metabolic Diseases Chemistry, Bristol-Myers Squibb Research and Development, Princeton, NJ 08543-5400, United States
b BMS-Biocon Research Center, Biocon, 20th KM, Hosur Road, Electronic City, Bangalore 560 100, India
c Metabolic Diseases Biology, Bristol-Myers Squibb Research and Development, Princeton, NJ 08543-5400, United States
d Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research and Development, Princeton, NJ 08543-5400, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Our investigation of the structure–activity and structure–liability relationships for dihydropyrrolopyra-
zol-6-one MCHR1 antagonists revealed that off-rate characteristics, inferred from potencies in a FLIPR
assay following a 2 h incubation, can impact in vivo efficacy. The in vitro and exposure profiles of dihy-
dropyrrolopyrazol-6-ones 1b and 1e were comparable to that of the thienopyrimidinone counterparts 41
and 43 except for a much faster MCHR1 apparent off-rate. The greatly diminished dihydropyrrolopyrazol-
6-one anti-obesity response may be the consequence of this rapid off-rate.
Received 2 March 2015
Revised 30 April 2015
Accepted 6 May 2015
Available online 15 May 2015
Keywords:
Ó 2015 Elsevier Ltd. All rights reserved.
MCHR1 receptor antagonists
Dihydropyrrolopyrazolones
Off-rates
Obesity
An increasing awareness and recognition of obesity as a serious
health hazard across the world1 has garnered significant attention
from the pharmaceutical industry leading to several recently
approved treatments, such as BelviqÒ, QsymiaÒ, ContraveÒ, and
SaxendaÒ over a decade after XenicalÒ became available.2 Among
a number of targets being pursued for the treatment of obesity,
antagonism of the MCHR1 receptor was actively sought after by
the pharmaceutical industry.3 MCHR1 and MCHR2 are two class
A GPCRs with the latter predominantly expressed in the brain of
higher mammals such as humans, monkeys, and dogs but not in
rodents such as mice and rats. Antagonism of MCHR1, which is
expressed in most species examined, including rodents and
humans, results in a reduction in feeding that translates to a reduc-
tion in weight gain in rodents. The vast majority of MCHR1 recep-
tor antagonists include a basic functionality that is likely to be
associated with the hERG liability that often accompanies them.
A non-basic MCHR1 antagonist from our laboratories, BMS-
830216 (a phosphate ester prodrug of BMS-819881), that is devoid
of the hERG liability, was advanced into clinical trials (Fig. 1).4
A projected human efficacious dose of 400 mg qd and a long
half-life led to a follow-up focus toward identifying more potent
analogues that would lower the projected human dose and possess
a shorter t1/2. In an effort to expand upon our discovery that non-
basic entities containing a thienopyrimidine core were potent
MCHR1 antagonists, multiple non-basic counterparts containing
bicyclo[3.3.0] cores were explored.5 This report details our findings
with dihydropyrrolopyrazol-6-one-(1), dihydropyrrolopyrazol-4-
one-(2) and dihydropyrrolotriazolone-based (3) antagonists
(Fig. 2).
The initial route used for synthesis of dihydropyrrolopyrazol-6-
one analogues 1 is described in Scheme 1. Conversion of 4-
chloroaniline to its diazonium salt with NaNO2/HCl followed by
treatment with benzyl 2-chloro-3-oxobutanoate (5), gave hydra-
zonoyl chloride 6. Following a known protocol,6 pyrazole diester
⇑
7
was obtained by condensation of (E)-ethyl 3-(dimethy-
Corresponding authors. Tel.: +1 609 818 4974 (P.D.).
E-mail address: pratik.devasthale@bms.com (P. Devasthale).
lamino)acrylate with 6. Pyrazole diester 7 was converted to acid
http://dx.doi.org/10.1016/j.bmcl.2015.05.008
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

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P. Devasthale et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2793–2799
HO
OH
P
OH
O
O
O
O
O
O
O
S
N
O
Cl
S
N
N
Cl
N
BMS-819881
BMS-830216
Figure 1. Parent (BMS-819881) and prodrug (BMS-830216) structures of the BMS clinical lead.
O
In the case of 11, the chlorinating conditions (MsCl/Et3N/DCM) also
O
X
removed the SEM protecting group to reveal the phenol which was
cyclized in the presence of a base to yield 11. Alkylation of 11 with
1-(2-chloroethyl)pyrrolidine yielded 1e.
N
Cl
N
N
R1
OMe
Cl
N
N
R1
N
Dihyropyrrolopyrazol-6-ones
Dihydroyrrolopyrazol-4-ones
An alternative method to access dihydropyrrolopyrazol-6-one
analogues 1 is shown in Scheme 2. Starting from 17 and 18, pyra-
zole aldehyde 20 was prepared using known procedures.7
Subsequent reduction and hydrolysis of 20 yielded acid 21. Acid
21 was coupled with appropriate anilines under standard condi-
tions. The resulting alcohol was converted to chloride and then
cyclized to yield 1j, 1m, 1n, and 22. Substitution of 22 with suitable
amines yielded 1h and 1i.
2
1
O
R2
OMe
R1
=
N
Cl
N
N
R1
OMe
X = CH or N
N
Dihydropyrrolotriazolones
3
Dihydropyrrolopyrazolon-6-ones 1k and 1l were prepared as
outlined in Scheme 3. Reductive amination of ester 20 with 4-
methoxy benzylamine, followed by hydrolysis of the ester yielded
acid 26 (Scheme 3). Cyclization using standard amide coupling
conditions followed by deprotection afforded lactam 27. Lactam
Figure 2. Non-basic dihydroazolopyrrolidinone-containing MCHR1 antagonists.
8 by hydrogenation, and was coupled with the appropriate anilines
to afford ester 9. Conversion of ester 9 to chloride 10 followed by
subsequent cyclization and deprotection yielded 1a–c, 1f, and 1g.
CO2Bn
N
O
O
b
a
O
O
c
Cl
NH
Cl
OBn
OBn
Cl
4
6
5
CO2 Bn
CO2 Et
CO2H
CO2 Et
N
N
e
d
Cl
N
Cl
N
7
8
O
O
R1
R1
f
N
N
N
H
N
H
Cl
N
Cl
N
CO2 Et
OH
10
9
g-i
j, h
O
O
N
OMe
OH
k
N
N
R 1
Cl
N
1e
Cl
N
N
11
1a-c, 1f, 1g
OSEM
OTBDMS
OSEM
N
O
O
O
N
Boc
N
OMe
OMe
OMe
H2N
H 2
N
H2N
H2
N
15 (S ); 16 (R )
12
13
14
Scheme 1. Reagents and conditions: (a) SOCl2, chloroform, 0 °C–rt, 4 h, 99%; (b) 4-chloroaniline, NaNO2, 12 N HCl, H2O, NaOAc, 0 °C–rt, 3 d, 87%; (c) (E)-ethyl 3-
(dimethylamino)acrylate, TEA, i-PrOH, 80 °C, 2 h, 46%; (d) 10% Pd/C, H2, EtOAc, rt, 5 h, 47%; (e) 12/13/14/15/16 or 3-methoxy-4-((2-(trimethylsilyl)ethoxy)methoxy)aniline,
BOP, DIPEA, CH2Cl2, rt, 4.5 h, 80–100%; (f) LiBH4, THF, rt, 2 h, 82%; (g) MsCl, DIPEA or Et3N, CH2Cl2, rt, 16 h, 51–80%; (h) K2CO3, DMF, rt, 16 h, 42–95%; (i) deprotection, 1a, 1b,
and 11, –OSEM to –OH: TFA, CH2Cl2, 0 °C, 45 min–2.5 h, 21-62%; 1c, –OTBS to –OH: TBAF, AcOH, THF, rt, 3 h, 31%; 1f and 1g, –NBocMe to –NHMe: 4 M HCl in dioxane, rt, 2 h,
90–100%; (j) MsCl, Et3N, CH2Cl2, rt, 1.5 h, 44%; SEM group removed under these conditions (k) 1-(2-chloroethyl)pyrrolidine, Cs2CO3, MeCN, 80 °C, 5 h, 11%.

P. Devasthale et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2793–2799
2795
O
N
NH2
NH
b
a
O
O
Cl
Cl
NH
OEt
O
17
18
19
CO2 Et
CHO
CO2H
OH
e-h
N
N
c,d
Cl
N
Cl
N
21
20
O
N
Cl
N
N
R 1
1j, 1m, 1n
22 (R1 =
i
Z
1h, 1i
)
N
Z = Br, F
OSEM
OSEM
OSEM
N
N
N
Boc
N
N
OMe
H2
N
H2N
OMe
H
2N
(rac)
24
23
25
Scheme 2. Reagents and conditions: (a) NaOAc, H2O, rt, 0.5 h, 80%; (b) POCl3, DMF, 0 °C–rt, 17 h, 66%; (c) NaBH4, THF, 0 °C, 2 h, 77%; (d) NaOH, H2O, reflux, 2 h, 91%; (e) 23/24/
25, HOBt, EDC, rt, 16 h, 56–100%; (f) MsCl, DIEA, CH2Cl2, rt, 16 h, 23–99%; (g) K2CO3, DMF, rt, 12–40 h, 25–100%; (h) deprotection, 1j, –NBocMe to –NHMe: 4 M HCl in dioxane,
rt, 2 h, 100%; 1m and 1n, –OSEM to –OH: TFA, CH2Cl2, 0 °C, 3 h, 43%; (i) 1h, from 23 (Z = Br), (S)-N,N-dimethylpyrrolidin-3-amine, DMSO, microwave reactor, 120 °C, 1 h,
100%; 1i, from 23 (Z = F), (1) (S)-tert-butyl pyrrolidin-3-ylcarbamate, DMSO, microwave reactor, 120 °C, 0.5 h; (2) TFA, CH2Cl2, 2 h, 96% for two steps.
O
CO2
H
a,b
c,d
N
N
OMe
20
Cl
N
NH
H
N
Cl
N
26
27
O
e
N
Cl
N
N
R1
1k, 1l
Scheme 3. Reagents and conditions: (a) 4-methoxy benzyl amine, NaBH(OAc)3, CH2Cl2, rt, 1.5 h, 88%; (b) NaOH, THF/EtOH/H2O, rt, 12 h, 75%; (c) EDCI, HOBt, TEA, CH2Cl2,
5 °C–rt, 12 h, 60%; (d) ceric ammonium nitrite, MeCN/H2O (3:1), rt, 3 h, 50%; (e) aryl bromide intermediate, N1,N2-dimethylethane-1,2-diamine, K3PO4, CuI, dioxane, 115 °C,
12 h, ꢀ10%.
27 was coupled with appropriate bromides using Buchwald condi-
tions to yield 1k and 1l.
arylpyrrolidinedione 37. Conversion of 37 to oxime with
NaNO2/H2SO4/NaOH followed by reaction with (4-chlorophenyl)
hydrazine yielded 38. Subsequent cyclization with PCl5 and depro-
tection afforded phenol 39. Alkylation of 39 with 2,2-dimethyloxi-
rane yielded 3a. Compound 3b was obtained by alkylation of 39
followed by reduction of the resulting ketone and 3c by alkylation
with the corresponding alkyl chloride.
A comparative analysis of equivalent compounds from each of
the three cores (X = CH), 1, 2 and 3, shown in Table 1 suggested ser-
ies 1 to be most promising from an MCHR1 potency perspective.
For example, tertiary alcohol 1b was the most potent analogue
among 1b, 2b, and 3a. Conversion of the chlorophenyl moiety of
1b to a more polar 5-chloro-2-pyridyl (X = N) 1d diminished
MCHR1 affinity 6-fold.
Arylation of 318 with 2-bromo-5-chloropyridine yielded pyra-
zole 32, which was converted to acid 33 via reduction and hydrol-
ysis (Scheme 4). Acid 33 was coupled with the corresponding SEM-
protected substituted aniline (14) under standard conditions. The
resulting alcohol was converted to chloride. Subsequent cycliza-
tion and SEM-deprotection yielded 1d.
The synthesis of regioisomeric dihydropyrrolopyrazol-4-one
analogues 2 is described in Scheme 5. Selective reduction of 7 fol-
lowed by hydrolysis afforded acid 34, which was coupled with
appropriate anilines 12, 13, or 14 under standard conditions.
Transformation of the resulting alcohol to the corresponding chlo-
ride followed by subsequent cyclization and deprotection gave
dihydropyrrolopyrazol-4-one 2a, 2b, and 2c.
Scheme 6 describes the synthesis of dihydropyrrolotriazinones
3. The copper-mediated amidation of bromide 36 with 4-meth-
oxy-1H-pyrrol-2(5H)-one followed by demethylation yielded
Among all alcohol-containing analogs from this chemotype (all
data not shown), dosed as prodrugs, only compound 1b (rat
Ki = 3.8 nM; rat plasma free fraction = 1%), dosed at 30 mpk as a
glycine prodrug, exhibited promising brain exposures of 1060 nM

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P. Devasthale et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2793–2799
O
O
O
a
b
O
N
CO2 Et
H2
N
NHNH2
H2
N
N
H
O
28
29
30
CO2Et
CO2 Et
CHO
d,e
N
c
N
N
Cl
N
HN
CHO
32
31
O
N
OH
CO2H
OH
N
N
N
N
f-i
N
N
Cl
O
Cl
1d
33
Scheme 4. Reagents and conditions: (a) NaOAc, H2O, rt, 12 h, 90%; (b) POCl3, DMF, 0 °C–rt, 17 h, 55%; (c) 2-bromo-5-chloropyridine, K2CO3, DMF, 120 °C, 2 h, 90%; (d) NaBH4,
THF, 0 °C, 1.5 h, 70%; (e) 1 N NaOH, MeOH, rt, 2 h, 100%; (f) 3-methoxy-4-(2-methyl-2-((2-(trimethylsilyl)ethoxy)methoxy)propoxy)aniline, EDC, HOBt, CH2Cl2, rt, 2 h, 100%;
(g) MsCl, (i-Pr)2NEt, CH2Cl2, rt, 4 h, 24%; (h) K2CO3, DMF, 95 °C, 2 h; (i) TFA, CH2Cl2, 0 °C, 1 h, 24% two steps.
a,b
N
c-f
OH
CO2
Cl
N
7
H
34
N
Cl
N
N
R2
OMe
O
2a-c
Scheme 5. Reagents and conditions: (a) LiBH4, THF, rt, 5 h, 58%; (b) NaOH, H2O, reflux, 2 h, 90%; (c) 12, 13, or 14, HOBt, EDC, CH2Cl2, rt, 12 h, 61–72%; (d) MsCl, (i-Pr)2NH,
CH2Cl2, rt, 12 h–3 d, 52–76%; (e) K2CO3, DMF, 95 °C, 2-3.5 h, 48–70%; (f) 2a and 2b: TFA, CH2Cl2, 0 °C, 45 min–2.5 h, 32–62%; 2c: TBAF, AcOH, THF, rt–63 °C, 3 h, 31%.
O
OH
OBn
OMe
b,c
a
OBn
OMe
N
OMe
Br
Br
O
35
36
37
OH
O
O
d,e
f,g
N
N
N
N
Cl
N
N
OH
OMe
OBn
OMe
N
H
N
38
39
Cl
h
k
i,j
3b
3a
3c
Scheme 6. Reagents and conditions: (a) BnBr, K2CO3, MeCN, rt–70 °C, 14 h, 100%; (b) 4-methoxy-1H-pyrrol-2(5H)-one, CuI, K2CO3, N,N0-dimethylethylene-diamine, toluene,
18 h, 105 °C, 76%; (c) 5 M HCl, dioxane, 38 °C, 24 h, 94%; (d) NaNO2, H2SO4, NaOH, THF/H2O, 0 °C, 30 min; (e) (4-chlorophenyl)hydrazine, THF/H2O, rt, 12 h; (f) PCl5, DME, 0 °C–
rt, 3.5 h, 20% three steps; (g) thioanisole, TFA, CH2Cl2, rt, 17 h, 76%; (h) 2,2-dimethyloxirane, K2CO3, MeCN/H2O, microwave 120 °C, 1.5 h, 67%; (i) 2-cyclopropyl-2-oxoethyl 4-
methylbenzenesulfonate, Cs2CO3, MeCN, 80 °C, 5 h, 77%; (j) NaBH4, MeOH/THF, rt, 12 h, 86%; (k) 1-(2-chloroethyl)pyrrolidine, Cs2CO3, MeCN, 78 °C, 12 h, 62%.
at 20 h (Ctrough), corresponding to a free fraction coverage of 2.8-
fold over Ki (Table 4). Oral administration at 30 mg/kg qd of 1b
as the glycine pro-drug 40 for four days to young growing SD rats
produced a statistically insignificant 1.8% reduction in weight gain
whereas the structurally similar thienopyrimidinone standard 41
(rat Ki = 7.2 nM) (compound 19 in Ref. 4) caused a significant
6.2% decrease when administered as the glycine pro-drug 42 at
10 mg/kg (Fig. 3).
This lack of efficacy obtained with 1b was unexpected since
the free brain concentrations at trough, assuming free brain and
free plasma fractions are equivalent, for both 1b (at 30 mpk;
B/P = 0.42 at 20 h post-dose) and 41 (at 10 mpk; B/P = 1.7 at
20 h post-dose) were ꢀ2–3-fold greater than Ki. To probe
whether the weak in vivo activity of 1b was an inherent prop-
erty of this bicyclo[3.3.0] chemotype, we prepared and com-
pared pyrrolidine-containing dihydropyrrolopyrazolone 1e and

P. Devasthale et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2793–2799
2797
Table 1
Table 2
MCHR antagonist activities of dihydroazolopyrrolidinones
Comparative profiles of dihydroazolopyrrolidinones
O
O
O
Cl
N
X
N
R1
OMe
Cl
N
N
R1
OMe
CORE
Cl
N
N
N
Dihyropyrrolopyrazol-6-ones
Dihydroyrrolopyrazol-4-ones
O
O
N
2
O
N
1
N
N
S
N
N
N
N
O
R2
OMe
R1
=
N
Cl
N
N
R1
OMe
Compound
3c
1e
43
X = CH or N
N
Ki, nMa
HPLC logP
2.3
3.3
68
0.7
2.8
58
1
3.5
77
Dihydropyrrolotriazolones
3
hERG% inhib @ 0.3 lM
a
n = 3–5; versus human MCHR1.12b
Compound
X
R1
Ki, nMa
1a
1b
H
H
–OCH2CH(OH)CH3
–OCH2C(OH)(CH3)2
13
8
OH
enantiomers 1f and 1g. Nor was there any significant difference
between primary (1i), secondary (1f/1g) and tertiary (1h) amine
analogues. Pyridine (1f/1g) and benzene (1j) analogues also
appeared to be equivalent. Adding steric bulk on the amine (1l)
worsened the hERG liability. Although reduction of the basicity
of the amine (1k) produced a favorable impact on hERG inhibition,
it was accompanied by a 6-fold decrease in MCHR potency.
Addition of polar hydrogen-bonding functionalities is known to
generally mitigate the hERG liability.11 Hydroxylation or bishy-
droxylation to generate 1m and 1n respectively reduced hERG
activity such that for 1n the hERG electrophysiology Q-patch IC50
O
1c
1d
1e
H
N
H
9
–OCH2C(OH)(CH3)2
44
0.7
O
N
2a
2b
H
H
–OCH2CH(OH)CH3
–OCH2C(OH)(CH3)2
334
107
OH
O
2c
3a
H
H
41
34
–OCH2C(OH)(CH3)2
OH
(racemate)
O
O
3b
3c
H
H
112
2.3
N
was >30 lM. However this approach was abandoned due to the
accompanying decrease in MCHR1 affinity.
a
n = 3–5; versus human MCHR1.12b
Given the inability to improve significantly upon the profile of
amines such as 1e and 1f and before launching into a more exten-
sive effort for optimizing the MCHR1/hERG balance, a 7 day dose-
response study with 1f dosed orally qd at 1, 3 and 10 mg/kg was
conducted using young SD rats.12 One cohort was administered
10 mg/kg of 42 (prodrug of 41) as a positive control. Results are
summarized in Table 4 along with historical results for 43 in a sim-
ilar study. For the amine 1f to cause weight loss the free drug cov-
erage in the brain at 20 h (Ctrough) must be markedly higher than its
Ki value than that required for the thienopyrimidinones 41 and 43
just as was observed for the alcohol 1b. These findings suggest that
this weak efficacy is due to the dihydropyrrolopyrazolone core and
not the polar group.
We hypothesized that the weak in vivo potency of dihydropy-
rrolopyrazolones 1b and 1f may be due to a faster apparent off-rate
relative to the thienopyrimidinone counterparts, 41 and 43. A qual-
itative estimate of relative off-rates was gauged by measuring IC50
values in a novel FLIPR assay setup after incubation of test com-
pounds for 120 min (Table 5) followed by measurement of Ca2+
dihydropyrrolotriazolone 3c to the thienopyrimidinone counter-
part 43 (Table 2) as previous SAR within the group had sug-
gested that the pyrrolidine moiety would confer a significant
boost in potency.9
Since the dihydropyrrolopyrazolone 1e exhibited a three-fold
affinity differential over the pyrrolotriazolone 3c as had been pre-
viously observed for the alcohol counterparts, all subsequent work
focused on the dihydropyrrolopyrazolone series. Before embarking
on in vivo studies, we sought unsuccessfully to reduce the potent
hERG activity of 1e. A number of variations of basic functionalities
were prepared and assessed for MCHR1 antagonist activity and
hERG inhibition (Table 3). Initially N-pyridyl-3-amino pyrrolidines
1f–1i were explored as
a replacement for the N-(2-ary-
loxyethyl)pyrrolidine moiety of 1e.10
Several SAR observations emerge from Table 3. There was min-
imal difference in MCHR Ki and hERG inhibition between
NH2
O
O
O
O
N
Cl
N
N
O
40
OR3
O
O
O
O
O
N
N
O
N
S
N
S
N
Cl
Cl
41, R3 = H
42, R3 = -COCH2NH2
43 (GSK)
Figure 3. Thienopyrimidinone MCHR1 antagonists.

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P. Devasthale et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2793–2799
Table 3
3-Aminopyrrolidines
O
Y
N
Cl
N
N
R4
Compound
R4
Y
Ki, nMa
hERG@ 1
66%
l
Mb
N
N
N
1f
N
1.0
NHMe
NHMe
NMe2
1g
1h
1i
N
1.3
0.7
2.1
0.8
50%
50%
58%
51%c
N
N
N
NH2
N
N
1j
CH
NHMe
1k
N
N
6.5
23%
N
COCH3
N
N
1l
2.7
7.1
71%
NAd
NHt-Bu
1m
C-OMe
C-OMe
OH
OH
N
1n
11
<5%
OH
a
n = 3–5; versus human MCHR1.
b
An automated whole-cell patch clamp electrophysiology system (Q-patch) was used to directly measure hERG
currents in CHO cells stably expressing the cloned hERG potassium channel.
c
Percent inhibition @ 0.3
Not available; hERG flux IC50 >80
l
M.
d
l
M.
Table 4
Summary of findings from 7-day weight loss studies with 1f, 41 (dosed as 42) and 43 using SD rats12
Compd Rat Ki (nM) Dose
Wt. loss versus
Plasma free fraction (%) Cplasma at 20 h post-dose, nM Cbrain at 20 h post-dose, nM CNS free drugc/Ki
(mg/kg) vehicle controlb (%)
40a
1f
1f
1f
42
43
3.8
0.8
0.8
0.8
30
1
3
1.8
2.2
1.0
4.2
4.2
4.2
0.7 (for 41)
1.2
2557
131
574
1577
546
378
1060
<LLQ
739
2767
1610
654
2.8
—
39
145
1.6
23
3.8
10
7.5*
8.8*
8.7*
7.2 (for 41) 10
0.34 15
*
a
b
c
Statistically significant weight loss versus vehicle.
4-Day weight loss study.
Vehicle control: 0.5% methocel/0.1% tween 80/99.4% distilled H2O.
Assumption: plasma free fraction = brain free fraction.
influx upon stimulation with MCH. Faster off-rates were inferred
from higher IC50 values following a 120-min incubation.
desirable MCHR1/hERG balance, and the inability to modulate
the apparent off-rate of this chemotype, further work on dihy-
dropyrrolopyrazolones was discontinued to pursue more promis-
ing opportunities in other chemotypes within our laboratories.
Compound 1b (Table 5) exhibited a significantly higher T120 IC50
than 32 which may account for the absence of in vivo efficacy with
1b despite free brain coverage over MCHR antagonist Ki similar to
that of 41. As in the case of 1b in Table 2, a significantly higher T120
IC50 for 1e relative to 43 suggested a faster off-rate and lower
intrinsic potency for the dihydropyrrolopyrazolone core. For 1f
itself, a high IC50 (241 nM) was observed which is consistent with
a lack of efficacy when free fraction exposure multiples in the brain
over Ki were less than 40.13
Table 5
Ki and T120 values
a
Compound
hMCHR1 Ki nM SEM
T120, IC50, nM
41
43
1b
1e
1f
7.9 1.8
2.2 1.1
8.7 5.3
0.42 0.32
0.96 0.19
13
16
1173
3057
241
Given the lower intrinsic potency of dihydropyrrolopyrazolones
such as 1b and 1f despite achieving excellent trough free drug
levels in the CNS well above Ki, the difficulty in achieving a
a
See note 13.

P. Devasthale et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2793–2799
2799
expressing a mutated (E4Q, A5T) hMCHR1 receptor were prepared by dounce
homogenization and differential centrifugation. Binding experiments were
Acknowledgments
carried out with 0.5–1.0 lg of membrane protein incubated in a total of 0.2 mL
We would like to thank Arvind Mathur and Richard Rampulla
for coordinating our collaboration with BMS-Biocon Research
Center, Bangalore, India.
in 25 mM HEPES (pH 7.4) with 10 mM MgCl2, 2 mM EGTA, and 0.1% BSA
(Binding Buffer) for 90 min. For competition binding assays, reactions were
carried out in the presence of with 0.06–0.1 nM [Phe13, [125I]Tyr19]-MCH and
increasing concentrations of unlabeled test molecules. Reactions were
terminated by rapid vacuum filtration over 96 well-GFC Unifilter plates pre-
coated with 0.075 mL binding buffer containing 1% BSA, and washed 3 times
with 0.4 mL of Phospho-buffered Saline (pH 7.4) containing 0.01% TX-100.
Filters were dried, 0.05 mL microscint 20 was added to each well and
References and notes
1. World Health Organization Global Health Observatory (GHO) website http://
www.who.int/mediacentre/factsheets/fs311/en/ (updated Aug 2014).
2. Kim, G. W.; Lin, J. E.; Blomain, E. S.; Waldman, S. A. Clin. Pharmacol. Ther. 2013,
95, 53.
3. Johansson, A.; Löfberg, C. Expert Opin. Ther. Patents 2015, 25, 193; Szalai, K. K.
Recent Pat. CNS Drug Discov. 2014, 9, 122; Rivera, G.; Monge, A.; Bocanegra-
Garcia, V. Anti-Obes. Drug Discov. Devel. 2011, 200.
4. Washburn, W. N.; Manfredi, M.; Devasthale, P. V.; Zhao, G.; Ahmad, S.;
Hernandez, A.; Robl, J.; Wang, W.; Mignone, J.; Wang, Z.; Ngu, K.;
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radioactivity was subsequently quantified by scintillation counting on
a
TopCount microplate scintillation counter (Packard). Inhibitory constants
were determined by nonlinear least squares analysis using a four parameter
logistic equation;
(c) In vivo rat efficacy studies: Male Sprague Dawley rats were dosed orally with
vehicle (0.5% methocel/0.1% tween 80/99.4% distilled H2O) or compounds
dissolved in vehicle, at
a volume of 5 mL/kg. Test compounds were
administered orally one hour before the onset of the dark cycle. Body weight
and food consumption were measured daily at the time of drug administration.
Upon completion of the study total caloric consumption, diet preference, and
change in body weight were tabulated. Drug effects on body weight were
analyzed using body weights converted to percent change from baseline and
evaluated across treatment groups using a one-way ANOVA.
13. FLIPR assay protocol:
Inhibition of MCH-mediated Ca2+ was measured in a FLIPR in a hemiequilibria
based-format. Stable HEK-293 cells expressing human MCHR1 receptor were
plated at a density of 50,000 cells / well in 96 well poly-lysine coated plates
(BD #35-6640) and cultured overnight in DMEM (high glucose (4.5 g/mL),
25 mM HEPES, pH 7.4, 10% Fetal Bovine Serum, 1 mM Na Cl) at 37 °C, 5% CO2
conditions. For assay, media was replaced with 90 mL per well dye solution
consisting of 3.8 mM Fluo4 AM (Invitrogen #F14201), 0.04% Pluronic F-127
(Invitrogen #P3000MP), 2.5 mM Probencid (Sigma #P8761), in Base Buffer
(Hank’s balanced salt solution, 25 mM HEPES, 0.1% BSA). Dye solution was
allowed to ‘load’ for 1 h at room temperature in subdued light. Dye was
subsequently removed and replaced with 75 mL of Base Buffer and 75 mL
diluted test compound. Test compound dilution plates were prepared by serial
diluting test and reference compounds from 100% DMSO stocks first 1:50 in
Base Buffer and then serially (1:3.26) in Base Buffer containing 2% DMSO to
generate twelve half log test concentrations. Compounds were preincubated
with cells for 120 min prior to challenge with MCH. Unlike traditional FLIPR
inhibition assays where the compounds are challenged with an EC50 agonist
(MCH), in this hemiequilibria assay format compounds are challenged with an
EC100 agonist (MCH). Compounds with similar binding affinities but with
longer off-rates are better able to antagonize the EC100 challenge from MCH
due to the transient kinetic nature of the FLIPR assay. Compounds of similar
affinity but with slower off-rates will generate lower IC50 inhibition values
when compared to similar compounds that have faster off-rates.
5. A manuscript disclosing details regarding preparation and characterization of
bicyclo[3.3.0] cores not discussed in this report is in preparation. Preliminary
results were presented at 244th National Meeting of the American Chemical
Society, Philadelphia, PA, Aug. 19, 2012, Poster 379.
6. AL-Saleh, B.; Makhseed, S.; Hassaneen, H. M. E.; Elnagdi, M. H. Synthesis 2006, 1,
59.
7. Sridhar, R.; Perumal, P. T.; Etti, S.; Shanmugam, G.; Ponnuswamy, M. N.;
Prabavathy, V. R.; Mathivanan, N. Bioorg. Med. Chem. Lett. 2004, 14, 6035.
8. Baraldi, P. G.; Cacciari, B.; Spalluto, G.; Romagnoli, R.; Braccioli, G.; Zaid, A. N.;
Pineda de las Infantas, M. J. Synthesis 1997, 10, 1140.
9. Hertzog, D. L.; Al-Barazanji, K. A.; Bigham, E. C.; Bishop, M. J.; Britt, C. S.;
Carlton, D. L.; Cooper, J. P.; Daniels, A. J.; Garrido, D. M.; Goetz, A. S.; Grizzle, M.
K.; Guo, Y. C.; Handlon, A. L.; Ignar, D. M.; Morgan, R. O.; Peat, A. J.; Tavares, F.
X.; Zhou, H. Bioorg. Med. Chem. Lett. 2006, 16, 4723.
10. Huang, C. Q.; Baker, T.; Schwarz, D.; Fan, J.; Heise, C. E.; Zhang, M.; Goodfellow,
V. S.; Markison, S.; Gogas, K. R.; Chen, T.; Wang, X.-C.; Zhu, Y.-F. Bioorg. Med.
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11. For recent reviews on strategies for mitigation of hERG activity, see: (a) Bell, I.
M.; Bilodeau, M. T.; Lagrutta, A. A. In Polypharmacology in Drug Discovery;
Peters, J.-U., Ed.; Wiley: New York, 2012; pp 83–110; (b) Springer, C.;
Sokolnicki, K. L. Chem. Cent. J. 2013, 7, 167.
12. Experimental protocols:
(a) Chemistry: Experimental procedures and analytical data for
A
manuscript describing
a
more detailed decription of the FLIPR
dihydroazolopyrrolidones 1, 2, and
3 are described in Devasthale, P. V.;
hemiequilibrium assay and the association of slower apparent off-rates with
reduced weight loss efficacy across multiple MCHR1 antagonist chemotypes is
in preparation.
Washburn, W. N.; Wang, W.; Hernandez, A.; Ahmad, S.; Zhao, G. U S patent
8,415,386, 2013; Chem. Abstr. 2010, 152, 501369.
(b) Ki determination: Membranes from stably transfected HEK-293 cells