Synthesis and Antiobesity Properties of 6-(4-Chlorophenyl)-3-(4-((3,3-difluoro-1-hydroxycyclobutyl)methoxy)-3-methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (BMS-814580): A Highly Efficacious Melanin Concentrating Hormone Receptor 1 (MCHR1) Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.6b00676

The potent MCHR1 in vitro and in vivo antagonist activity of a series of cyclic tertiary alcohols derived from compound 2b is described. Subsequent pharmacokinetic and pharmacodynamic studies identified BMS-814580 (compound 10) as a highly efficacious antiobesity agent with a relatively clean in vitro and in vivo safety profile.

Full text of DOI:10.1021/acs.jmedchem.6b00676

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Article
Synthesis and Anti-obesity Properties of 6-(4-Chlorophenyl)-3-(4-((3,3-
difluoro-1-hydroxycyclobutyl)methoxy)-3-methoxyphenyl)thieno[3,2-
d]pyrimidin-4(3H)-one (BMS-814580): A Highly Efficacious
Melanin Concentrating Hormone Receptor-1 (MCHR1) Inhibitor
Saleem Ahmad, William N. Washburn, Andres S. Hernandez, Sharon Bisaha, Khehyong Ngu, Wei Wang,
Mary Ann Pelleymounter, Daniel Longhi, Neil Flynn, Anthony V. Azzara, Kenneth Rohrbach, James
Devenny, Suzanne Rooney, Michael A Thomas, Susan Glick, Helen Godonis, Susan Harvey, Hongwei
Zhang, Brian Gemzik, Evan B. Janovitz, Christine Huang, Lisa Zhang, Jeffrey A. Robl, and Brian J. Murphy
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00676 • Publication Date (Web): 26 Aug 2016
Downloaded from http://pubs.acs.org on August 27, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Page 1 of 35
Journal of Medicinal Chemistry
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Synthesis and Anti-obesity Properties of 6-(4-Chlorophenyl)-3-
(4-((3,3-difluoro-1-hydroxycyclobutyl)methoxy)-3-
methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-one (BMS-
814580): A Highly Efficacious Melanin Concentrating
Hormone Receptor-1 (MCHR1) Inhibitor
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Saleem Ahmad#*, William N. Washburn#, Andres Hernandez#, Sharon Bisaha#, Khehyong Ngu#, Wei Wang#,
Mary Ann Pelleymounter+, Daniel Longhi+, Neil Flynn+, Anthony V. Azzara+, Kenneth Rohrbach+, James
Devenny+, Suzanne Rooney+, Michael Thomas+, Susan Glick+, Helen Godonis+, Susan Harvey+, Hongwei
Zhangε, Brian Gemzik β, Evan B. Janovitz β, Christine Huang°, Lisa Zhang°, Jeffrey A. Robl# and Brian J.
Murphy+
# Metabolic Diseases Chemistry, + Metabolic Diseases Biology, °PCO MAP, β PCO Discovery Toxicology,
εPCO Bioanalytical Research
BristolꢀMyers Squibb Research & Development, P.O. Box 4000, Princeton, New Jersey 08543
Abstract. The potent MCHR1 in vitro and in vivo antagonist activity of a series of cyclic tertiary alcohols
derived from compound 2b is described. Subsequent pharmacokinetic and pharmacodynamic studies identified
BMSꢀ814580 (compound 10) as a highly efficacious antiꢀobesity agent with a relatively clean in vitro and in
vivo safety profile.
Introduction.
Melanin concentrating hormone (MCH) is a 19 amino acid cyclic peptide which has been implicated in a
variety of physiological conditions including feeding and energy homeostasis in mammals.1 It is synthesized
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primarily in cells within the lateral hypothalamus and zona incerta of the central nervous system and is identical
in all mammals tested. MCH binds to two class A G proteinꢀcoupled receptors (GPCRs), MCHR1 and MCHR2
with a 38% sequence homology between the two isoforms. MCHR1, a 353 amino acid peptide receptor, is
found in rodents and higher mammals including humans where it is expressed in the central nervous system
including hypothalamus. MCHR2 is expressed in higher mammals such as humans, rhesus monkeys and dogs;
it is not expressed in rodents. The overall expression pattern of MCHR2 in the brain is similar to MCHR1. The
pharmacological role of MCHR2 has not yet been clearly established.
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The prominent role of MCH in feeding and energy homeostasis has been thoroughly reviewed. MCH
messenger RNA is upꢀregulated in the hypothalamus of genetically obese mice.2 Intracerebroventricular
injection of the MCH peptide in mice results in significantly increased food intake.3 Mice overexpressing MCH
are obese and display insulin resistance.4 MCH knockout mice are lean, hypophagic, resistant to diet induced
obesity and exhibit an increased metabolic rate.5 The observed weight loss in the MCH knockout mice has thus
been attributed to a combination of both hypophagia and increased metabolism firmly establishing the potential
utility of MCHR1 antagonists as antiꢀobesity agents.
Pharmacological proof of principle is supported by published studies with several small molecule MCHR1
antagonists demonstrating reduction in feeding and body weight in rats and mice. Several compounds have
been advanced into clinical trials.6 However, to date there are no literature reports of demonstrated weight loss
in humans with an inhibitor of MCHR1.7,8
A major challenge to the development of many MCHR1 inhibitors is the potential cardiovascular risk often
stemming from inhibition of various cardiac ion channels including the human etherꢀaꢀgoꢀgo related gene
(hERG) potassium channel. The ion channel activity is presumably caused by the hydrophobic nature and the
amine functionalities present in most compounds described in the literature. Efforts to modulate the basicity or
replace the amino groups to avoid offꢀtarget cardiovascular effects while maintaining potency against MCHR1
and ensuring acceptable pharmacokinetic properties have met with mixed success.9
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Journal of Medicinal Chemistry
1
2
3
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5
OH
R
Me
OH
O
O
O
N
N
O
O
O
N
S
N
OMe
6
Cl
7
8
S
S
N
OMe
N
OMe
Cl
Cl
N
2a
1
GW-803430 (compound , ref. 9c)
MCHR1 Ki = 3.8 2.4 nM; n = 15
R = H, Compound
2c
BMS-819881 (Compound , ref. 10)
MCHR1 Ki = 10.4 nM 0.6 nM; n = 37
hERG patch clamp = 48% inhibition at 3
9
MCHR1 Ki = 9.5 nM 1.4 nM; n = 6
hERG patch clamp = 43.8% inhibition at 1
R = Me, Compound 2b
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hERG patch clamp = 86% inhibition at 1
M
M
M
MCHR1 Ki = 7.9 0.7 nM, n = 7
hERG patch clamp = 17% inhibition at 10
M
Figure 1. Comparison of MCHR1 binding and hERG activity of selected MCHR1 inhibitors.
Figure 2. In vivo oxidative metabolism of compounds 2a and 2b in rats.
We have previously disclosed the discovery of a series of potent nonꢀbasic MCHR1 inhibitors with significantly
reduced activity against hERG and other cardiac ion channels.10 The design of the previously disclosed series
represented by compounds 2a, 2b and 2c was based on the observation that a screening hit lacking an amino
group exhibited MCHR1 activity, suggesting that incorporation of a basic functionality in a MCHR1 inhibitor
was not an absolute requirement. This finding, coupled with extensive structureꢀactivity relationship (SAR)
studies with numerous series of MCHR1 inhibitors reported in the medicinal chemistry literature, including the
well investigated thienopyrimidinones,11 provided a framework for the execution of our program. A detailed
discussion of SAR and in vitro and in vivo studies have been described leading to the discovery of potent
anorectic agents 2a and 2b.
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While substantial reduction in body weight was seen with select analogs, chronic oral administration of 2a and
2b to rats caused biliary lesions in several animals. In vivo exposure data of parent compounds and various
metabolites established a strong correlation between incidents of the occurrence of lesions and plasma
concentrations of the corresponding αꢀhydroxyacid metabolites (4a, 4b).10 The apparent involvement of these
metabolites in the induction of biliary lesions necessitated designing inhibitors of MCHR1 with more
metabolically robust groups adjacent to the alcohol functionality. To that end, a series of alcohols with reduced
propensity to produce a carboxyl group in close proximity to the hydroxy functionality were synthesized and
evaluated for activity against MCHR1. The previously published efforts were focused on blocking metabolic
oxidation of the terminal methyl groups of compounds 2a and 2b via direct substitution at these sites. Herein
we summarize the synthesis and biological evaluation of a series of cyclic tertiary carbinols with particular
emphasis on the excellent potency, oral efficacy, and good in vivo safety profile of compound 10.
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Figure 3. Tertiary alcohols designed to block in vivo formation of an αꢀhydroxy acid metabolite.
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Journal of Medicinal Chemistry
Scheme 1.a
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Scheme 2.a
Figure 4.
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Scheme 3.a
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6
F
F
F
F
F
F
F
F
F
F
f
e
d
a, b, c
O
OH
N
N
O
7
8
24
23
O
21
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20
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F
F
F
F
O
O
NH2
i
j, k
or l, m
O
N
O
g, h
O
10
OH
S
N
OMe
Cl
F
F
X
OMe
25a X = NO2
25b
10a
O
OR
P
O
X = NH2
O
OR
10b, R = Bn
10c, R = H
aReagents and conditions: a) oxalyl chloride/CH2Cl2; b) dimethylamine/THF, 77% from 20; c) LAH/THF, 55%; d) H2O2/ MeOH; e) 165 oC neat,
o
66% from amine 22; f) m-CPBA/CH2Cl2, quantitative yield; g) potassium 2-methoxy-4-nitrophenolate/NaH2PO4/CH3CN/water/CH2Cl2/135 C,
steel bomb, 64%; h) 10% Pd-C/MeOH/H2, 100%; i) compound /phenol/130 oC, 75%; j) EDCI/boc-GlyOH/DMAP/CH2Cl2/reflux; k)
16
TFA/CH2Cl2/RT, 86% 2 steps; l) dibenzyl diisopropylphosphoramidite/triazole then H2O2; m) TFA (neat, 3h at RT), 84%.
Chemistry:
The cyclopropyl alcohol 5 was synthesized via Mitsunobu coupling of the readily available alcohol 12 with the
known intermediate 11 (Scheme 1).12 Synthesis of the oxetane analog 6 originated with methylene oxetane 1313
and involved osmium tetroxide mediated dihydroxylation prior to coupling with 1ꢀchloroꢀ2ꢀmethoxyꢀ4ꢀ
nitrobenzene (Scheme 2). Preparation of the tertiary alcohols 7, 8 and 9 involved synthesis of the
corresponding readily accessible nitrobenzene derivatives outlined in Figure 4.
Synthesis of compound 10 required preparation of the previously unknown spirocyclic epoxide 24 which
originated from the readily available difluorocyclobutane carboxylic acid 20.14 Conversion of 20 to the
corresponding amide followed by lithium aluminum hydride mediated reduction afforded the tertiary amine 21.
Oxidation of 21 using hydrogen peroxide in methanol afforded the Nꢀoxide 22. Pyrolysis of crude 22 (neat) via
the Cope elimination reaction at 165 oC afforded the methylene cyclobutane derivative 23 under slightly
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Journal of Medicinal Chemistry
reduced pressure.15 Finally, oxidation of 23 using mꢀCPBA in dichloromethane gave the desired spirocyclic
epoxide 24 as a low boiling colorless liquid in quantitative yield.16 The epoxide 24 was treated with potassium
2ꢀmethoxyꢀ4ꢀnitrophenolate followed by reduction and condensation with 16 as described previously to afford
10.10 It is noteworthy that efforts to prepare compound 10 from the corresponding vicinal diol, similar to the
ones described for compound 6, failed presumably due to lack of stability of the relatively strained and electron
deficient difluorocyclobutane ring under strongly basic conditions.
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Results and Discussion.
Our interest in compound 2b was based on the excellent potency and robust reduction in body weight seen in
rats (Tables 1, 2) as well as its relatively clean in vitro profile.10 A multiꢀpronged investigation was initiated to
design analogs of 2b that would preclude formation of the putative hepatotoxic metabolites. In a previously
reported approach, replacement of these labile methyls with groups not prone to oxidation led to the discovery
of 2c. The discussion here is limited to the design, synthesis and in vitro and in vivo evaluation of a series of
cyclic tertiary alcohols directly derived from cyclization of the methyls of compound 2b. Our finding that a
3,3ꢀdiflurocyclobutanol can be employed as metabolically more robust isosteric counterpart of the gem
dimethylcarbinol moiety of 2b culminated the discovery of compound 10 as a highly efficacious anorectic agent
with an excellent safety profile.
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Table 1. In vitro data and physical properties.
1
2
3
4
5
Compound stability in liver
microsomes
Solubility (ꢀg/mL)c
6
Ki
7
8
9
Compd
LogPd
% Freee
(% remaining)b
(nM)a
10
11
12
13
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16
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20
21
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Parent
Human
100
Rat
98
Mouse
90
Glycinate Phosphate
compd
7.9 ±
0.7; (n =
7)
<1
<1
ND
<1
<1
4.5
4.1
3.8
4.7
4.7
0.8
ND
ND
ND
ND
2b
5
17.5 ±
7.0; (n =
3)
94
100
ND
73
61
47
24
14
99
<1
<1
<1
<1
NA
NA
NA
NA
8.7 ±
0.8; (n =
3)
100
ND
ND
6
9.8 ±
1.8; (n =
3)
<1
7
11.9 ±
0.8; (n =
3)
NA
8
11.2 ±
2.8; (n =
3)
87
90
27
95
98
<1
<1
NA
<1
NA
4
4.2
4.8
ND
0.2
9
16.9 ±
100
10
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Journal of Medicinal Chemistry
5.9; (n =
12)
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6
a Human MCHR1 binding affinities were measured as described in reference 10. b Compounds were incubated
7
8
9
c
with liver microsomes at 3.0 ꢀM for 10 minutes. Solubility of parent compounds and prodrugs at pH 6.4 in 50
10
11
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e
mM phosphate buffer. d HPLC logP at pH 7.0. Plasma protein binding in rat. ND = not determined. NA =
not available (prodrug not prepared).
SAR studies had revealed that the binding site tolerated a wide range of polar and nonꢀpolar functionalities
adjacent to the hydroxyl group. Thus, all tertiary alcohols described in this report displayed potent antagonism
at the human MCHR1 receptor with Ki values ranging between 8 and 18 nM (Table 1). Binding affinities
approximated that of 2b but were 3 to 6 times lower than the 2.9 nM value measured for the tertiary amine
derivative 1 (GWꢀ803430). Affinity towards rat MCHR1 (data not shown) was comparable to the human
receptor presumably due to the high homology between the two. Evaluation of select analogs, including the
lead compound 10, against MCHR2 showed no inhibitory activity at concentrations up to 10 ꢀM.
Although the discovery that a hydroxy group could replace the tertiary amine functionality was critical in
addressing ion channel related issues with MCHR1 inhibitors, this modification induced such a severe reduction
in aqueous solubility that in vivo evaluation required administration of these alcohols as their corresponding
proꢀdrug. Extensive screening of a variety of acidic, basic and sugar based derivatives led to the identification
of glycine and phosphate esters as suitable prodrugs for the various tertiary alcohols.10 Plasma samples from
animals in all in vivo studies were analyzed to determine exposures of parent compounds as well as the
corresponding prodrugs. The amounts of the unconverted prodrugs found in the plasma were generally very
low and often did not reach detection limits even at the earliest measurement time points.
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1
2
3
4
5
Table 2. Pharmacokinetics (PK) and pharmacodynamics (PD) data: Plasma and brain concentrations of cyclic
6
7
tertiary alcohols and the effect on body weights in SpragueꢀDawley rats.
8
9
4 Days PD
studyc
Day 4 parent compd
concentrations at 20 h
Compd
Dosed asa
8 h Rat PK studyb
10
11
12
13
14
15
16
17
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19
20
21
22
23
24
25
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60
AUC
Plasma
(nM)
% Weight
loss
Plasma
Brain (nM)
Brain (nM)
(nM)
(ꢀM•h)
45
glycinate
glycinate (5a)
glycinate (6a)
parent (7)
7800
<3
3530
113
1164
6
6.1%d
NT
1116
ꢀ
682
ꢀ
2b
5
5.9
34.9
0.2
1130
<3
0%e
NT
<3
37
6
ꢀ
ꢀ
7
glycinate (7a)
parent (9)
1.3
0.4
<3
<3
15
29
NT
NT
ꢀ
ꢀ
9
glycinate (10a)
38.1
11330
4500
6.4% d
7955
12801
10
a Rats were dosed orally with parent compound or a prodrug; prodrugs doses were adjusted to parent
compounds. Vehicle = 0.5% Methocel/0.1% Tween 80/99.4% distilled water. b Dose = 10 mg/kg, plasma and
c
brain concentrations are reported 8 h post dose, n = 2. Compounds were dosed at 3, 10 and 30 mg/kg orally
once a day for 4 days; weight loss data is reported only for the 10 mg/kg dose, n = 8; reduction in body weight
is reported as % change from baseline body weight compared to vehicle treated animals. d No biliary lesions
were seen with compounds 2b or 10 in this model; lesions were seen with 2b only after 28 days in an efficacy
study in DIO rats, see reference 10 for details. e Biliary lesions were seen in 40% of the animals at 30 mg/kg
dose; no reduction in body weight seen at any of the doses used. NT = not tested.
Brain and plasma exposures of alcohols 5-10 in SpragueꢀDawley (SD) rats were determined following 10
mg/kg oral dosing as the glycine proꢀdrug prior to in vivo efficacy evaluation in a 4 days sub chronic rat weight
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Journal of Medicinal Chemistry
loss model (Table 2). It is noteworthy that over the course of the program we had realized that prolonged
plasma and brain exposures to the drug were required in order to achieve significant (>5%) reduction in body
weight in our primary 4 day in vivo efficacy model. Achieving in vivo efficacy was further complicated by the
fact that most compounds in this series displayed very high plasma protein binding (>98%) necessitating
exposures exceeding the nanomolar range and reaching micromolar concentrations in vivo.
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The cyclopropyl alcohol 5, designed to block metabolic oxidation of methyl groups of 2b that would produce a
carboxyl group, was a potent inhibitor of MCHR1 (Ki 17.5 ± 7.0 nM). While modest overall plasma exposures
were achieved with 5 after a 10 mg/kg oral dose (0ꢀ8 h AUC = 5.9 ꢀM•h, dosed as the glycine prodrug 5a), the
plasma concentration of 5 at the 8 hour time point was rather low (113 nM). In addition, there was no
detectable amount of compound 5 present in the rat brain after 8 hours. Compound 5 was thus deemed
unsuitable for further in vivo studies. The cyclobutyl and tetrahydropyranyl alcohols (7 and 9 respectively) also
designed to block formation of αꢀhydroxy acid metabolites, failed to exhibit sufficient plasma and brain
exposures as well, when evaluated both as parent compounds and/or glycine prodrugs (Table 2).
The potential use of an oxetane ring to block in vivo metabolism has been described previously.17 Incorporation
of this functionality afforded potent MCHR1 inhibitor 6 (Ki 8.7 ± 0.8 nM). Compound 6 displayed high
plasma exposures in the rat PK study (0ꢀ8 hours AUC = 34.9 ꢀM•h) and over 1 ꢀM plasma and brain
concentrations after 8 hours when dosed as the glycine prodrug 6a. However, 6 failed to produce a significant
reduction in body weight in the 4 days efficacy study in rat. Analysis of the brain and plasma samples from this
study revealed exceedingly low plasma concentrations (37 nM) with no detectable amount of 6 in the brain at
the 20 hour time point. The plasma concentrations of 6 had dropped by >95% during the 8 to 20 h time course.
The lower plasma and brain concentrations seen with 6 at a later time point can be rationalized by the relatively
faster in vitro clearance of the parent compounds in a rat liver microsomes based assay (only 47% remaining
after incubation for 10 minutes vs. 98% remaining for 2b, Table 1). The failure to produce a reduction in body
weight in rats by compounds with fast clearance perhaps underscores the significance of the requirement for a
prolonged and sustained inhibition of MCHR1 in this model.
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Efforts to design and prepare analogs with more metabolically robust alcohols continued resulting in the
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generation of a wide range of potent inhibitors of MCHR1. The list of analogs ranged from highly nonꢀpolar
polyfluorinated compounds to analogs incorporating more polar functionalities such as hydroxyl and sulfone
groups (analogs not shown). However, most of these compounds proved inadequate in reducing body weight in
vivo in the rat model presumably due to higher molecular weight, poor aqueous solubility or high polar surface
area. The focus on designing CNS penetrant analogs with low molecular weight prompted a reexamination of
the failure of the oxetane (compound 6) and the cyclobutane (compound 7) derivatives. Although oxidative
metabolites had not been identified, we hypothesized that fluorination of compound 7 would diminish the
susceptibility of the four membered ring towards metabolic oxidation.
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Synthesis of a fluorocyclobutanol analog proved initially cumbersome as the requisite precursors were not
readily available. Nonetheless, the efforts succeeded resulting in the generation of the potent
difluorocyclobutane derived MCHR1 inhibitor 10 (Ki 16.9 ± 5.9 nM). In vitro evaluation of compound 10 in rat
liver microsomes (Table 1) revealed adequate metabolic stability with 95% remaining after incubation for 10
minutes. Upon oral dosing of the corresponding glycine prodrug 10a at 10 mg/kg, high in vivo exposure of the
parent compound (0ꢀ8 h AUC = 38 ꢀM•h) as well as high 8 h brain (11.3 ꢀM) and plasma (4.5 ꢀM)
concentrations were attained. The distribution of 10 in to the brain was high with a brainꢀtoꢀplasma
concentration ratio of 2.5 at 8 h time point (Table 2). Further assessment of compound 10 at 10 mg/kg in the
sub chronic 4 day in vivo efficacy model demonstrated a significant 6.4% reduction in body weight in rats. The
degree of reduction in body weight with compound 10 was comparable to the lead tertiary alcohol 2b in this
model (Table 2).
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Journal of Medicinal Chemistry
1
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A
6.0
3
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9
Vehicle
0.03 mpk
0.1 mpk
0.3 mpk
1 mpk
4.0
2.0
0.0
-2.0
-4.0
-6.0
-8.0
10
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3 mpk
10 mpk
rimonabant
P < 0.05
0
5
10
15
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25
30
Day
B
6.0
4.0
Vehicle
2.0
1 mpk
3 mpk
0.0
-2.0
-4.0
-6.0
-8.0
Pair-fed to1
mpk
Pair-fed to 3
mpk
P < 0.05
0
5
10
15
20
25
30
Day
Figure 5. A. Effect of compound 10 (dosed as 10c) on body weights after once daily oral dosing in DIO rats
for 28 days (data plotted for 26 days); n = 8; vehicle: 14% propylene glycol, 1% tween, 85% water, v/v/v. Body
weight and consumption of food and water were measured daily, see reference 18 for details. B. Comparison of
weight reduction seen in pairꢀfed animals to the corresponding animals dosed with compound 10 at 1 and 3
mg/kg.
Encouraged by the efficacy data from the sub chronic study, compound 10 was further evaluated in a chronic
dietꢀinduced obese (DIO) rat model for 28 days (Figure 5) using obese male SD rats (8 rats/group).18 Animal
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were dosed once daily at doses ranging from 0.03 to 3 mg/kg with compound 10 administered as the phosphate
prodrug 10c.19 Body weight and food consumption were measured daily. In addition to the vehicle and positive
control (10 mg/kg rimonabant)20 animal groups, two pairꢀfed groups matching the food intake of animals
administered 1 and 3 mg/kg doses of the drug were also included in this study in order to assess contribution of
reduction in food intake to overall weight loss. Compound 10 was well tolerated; moreover, histopathology
revealed no signs of biliary toxicity at the end of the study. Compound 10 dose dependently reduced body
weight in DIO rats compared to the vehicle treated animals giving a linear dose response (R = 0.998). The
benchmark requirement for efficacy in obesity (5% reduction in bodyweight) was achieved at a 0.3 mg/kg dose
of 10 dosed as the phosphate prodrug 10c on day 25. The reduction in body weight at the 3 mg/kg dose (7.8%)
was roughly comparable to the positive control rimonabant (8.5% at 10 mg/kg) group.
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Figures 5B shows comparisons of changes in body weight for the 1 and 3 mg/kg pairꢀfed groups vs. the
corresponding drug treated animals. While the untreated rats on limited diet lost slightly over 4% of body
weight (vs. vehicle) during the course of this study, matched rats treated with 1 and 3 mg/kg doses lost
significantly more body weight (6.6 and 7.8% respectively). Table 3 outlines food calories consumed by the
various cohorts including the pairꢀfed animals in addition to the body weight changes. The data shows that
animals treated with 10 dose dependently consumed fewer calories compared to the vehicle control group. The
plasma concentrations at 2 and 24 h time points (Table 3) for 0.3 and 3 mg/kg doses suggests that the prolonged
exposure to 10 and the resulting sustained inhibition of MCHR1 may be contributing to the robust weight loss
seen in this model. The pairꢀfed data suggests that the reduction in consumed calories only partially accounts
for the observed weight loss seen in DIO rats, perhaps suggesting a role for MCHR1 in modulating metabolic
rate.5
Compound 10 exhibits potent binding affinity (Ki 17 nM) for human MCHR1. It is a potent and selective
functional antagonist (Kb 117 nM) of human MCHR1 in a FLIPRꢀbased assay10 exhibiting no activity against
MCHR2 at 10 ꢀM. Compound 10 also exhibits potent binding affinities for cynomolgus monkey and rat
MCHR1 (Ki 4.9 and 11.5 nM respectively). The phosphate prodrug 10c displays weak binding affinity for
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Journal of Medicinal Chemistry
MCHR1 (Ki = 2.92 ꢀM). Compound 10 displayed high plasma protein binding in all species tested with 0.2,
0.1, 0.3, 0.4, 0.2 and 0.3 % free drug respectively in rat, DIO rat, dog, cynomolgus monkey, rabbit and human.
The potential for compound 10 to cause drugꢀdrug interactions appears low since 10 is neither a substrate nor an
inhibitor (>40 ꢀM) of cytochrome P450 enzymes (CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4), nor an inducer of
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CYP3A4. There was no evidence of significant receptor binding or enzyme inhibition at 10 ꢀM in a broad
panel of Panlab assays.21 Results of Ames and in vitro micronucleus assays were negative, suggesting a low
potential for mutagenicity.
Compound 10 displayed modest in vitro ion channel inhibition of 43%, 21% and 15% respectively for hERG,
Na (4 Hz) and Lꢀtype Ca channels at 10 ꢀM. However, subsequent evaluation in an anesthetized rabbit model
revealed no compound related effects on QT or QRS duration up to plasma concentrations of 38 ꢀM suggesting
no hERG or sodium channel related effects. In addition, there was no compound related effect on mean arterial
blood pressure or heart rate in this model.
Full PK studies were carried out with 10 in three distinct animal species (rat, dog and cynomolgus monkey)
using the phosphate prodrug 10c at a 10 mg/kg oral dose while using the parent compound 10 at an intravenous
dose of 1 mg/kg (Table 4). Compound 10 displayed modest absolute oral bioavailability in all three species;
54% (rat), 33% (dog) and 46% (monkey). The volume of distribution of 10 was 4.1, 7.2, and 5.4 L/kg in rats,
dogs, and monkeys, respectively. Extended toxicity studies with compound 10 (dosed as 10c) in rat
demonstrated adequate safety, producing no hepatobiliary lesions at up to 100 mg/kg daily dose for 30 days.
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Table 3. Caloric intake vs. reduction in body weights in DIO rats after once daily oral dosing of compound 10
(dosed as 10c).
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Plasma conc. of 10 (ꢀM)a
Treatment
% Weight
change
% Reduction in
consumed calories
2 h
24 h
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Vehicle
10 (0.03 mg/kg)
0
0
ꢀ
ꢀ
ꢀ1.7
ꢀ3.2
ꢀ5.0
ꢀ6.6
ꢀ7.8
ꢀ4.2
ꢀ4.1
ꢀ8.5
ꢀ6.5
ND
ND
10 (0.1 mg/kg)
ꢀ10.5
ꢀ12.8
ꢀ14.5
ꢀ14.7
ꢀ14.3
ꢀ14.7
ꢀ27
ND
ND
10 (0.3 mg/kg)
2.0 ± 0.6
1.2 ± 0.1
10 (1 mg/kg)
ND
ND
10 (3 mg/kg)
13.5 ± 0.5
14.4 ± 0.4
pairꢀfed to 1 mg/kg of 10
pairꢀfed to 3 mg/kg of 10
rimonabant (10 mg/kg)
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
a Plasma concentrations of the phosphate prodrug 10c were undetectable (<2 nM). ND = not determined.
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Journal of Medicinal Chemistry
Table 4. Pharmacokinetics data for compound 10 in rat, dog and cynomolgus monkey.a
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Route
of
CL
T1/2 (h) (mL/min/
kg)
AUC0ꢀ∞
Cmax
Compd
(mg/kg)
Vss
F
Species
Tmax (h)
(ꢀM)
(L/kg)
(%)
µM•h
admin
i.v.
10 (1)
10c
ꢀ
4.0
ꢀ
ꢀ
6.7
ꢀ
11.5
>24
0.9
ꢀ
4.1
ꢀ
ꢀ
54
ꢀ
10
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rat
p.o.
i.v.
107
5.2
ꢀ
16.0
ꢀ
(10)
10 (1)
10c
6.9
ꢀ
7.2
ꢀ
dog
p.o.
i.v.
1.2
ꢀ
9.3b
ꢀ
15.3
22.1
87.0
33
ꢀ
(10)
10 (1)
10c
37.0
ꢀ
1.7
ꢀ
5.4
ꢀ
monkey
p.o.
1.2
10.7c
46
(10)
a Vehicles: IV, PEG400:DMAC:water (70:5:25); PO, Methocel:Tween80:water (0.5:0.1:99.4). b n = 3;
individual animal data, 2 h, 2 h, 24 h; one dog had Tmax at 2 hours followed by a second peak at 24 hours; Tmax
for all 3 dogs before the 24 h time point was 2 h.22 c n = 3, individual animal data, 4 h, 4 h, 24 h; one monkey
displayed delayed oral absorption.22
In summary, the improved metabolic stability conferred upon replacement of the dimethylcarbinol of 2b with a
3,3ꢀdifluorocyclobutanol moiety without incurring any loss of favorable properties illustrates the advantages
conferred by this novel structural modification. The data presented here as well as data from additional in vivo
studies in rats and higher species support further consideration of 10 as an antiꢀobesity agent. The worldwide
prevalence of obesity and the scarcity of safe and effective antiꢀobesity drugs clearly emphasizes a vital need
for new antiꢀobesity treatments with improved therapeutic margins.23 Although the potential involvement of
MCHR2 in weight management in humans is a possible explanation for the current inability of MCHR1
selective molecules to exhibit weight loss in humans, the challenge to demonstrating efficacy in humans with
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MCHR1 inhibitors, as has been suggested by Souers at al.9, may be discovering a molecule such as 10 (BMSꢀ
814580) with prolonged and sustained in vivo exposure and a safe therapeutic index.
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Experimental Section
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General. All reagents and solvents used were of commercial quality and were primarily obtained from Aldrich
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Chemical Co., Sigma Chemical Co., Lancaster Chemical Co., Acros Chemical Co. or EM Science Chemical
1
Co. H NMR (400 or 500 MHz) and 13C NMR (100 or 125 MHz) spectra were recorded on JEOL GSX400 and
JEOL JNMꢀECP500 spectrometers using tetramethylsilane as an internal standard. Chemical shifts are given in
parts per million (ppm) in δ units. All flash chromatographic separations were performed using E. Merck silica
gel (particle size, 0.040−0.063 mm). Reactions were monitored by TLC using 0.25 mm E. Merck silica gel
plates (60 F254). LCꢀMS data were recorded on a Shimadzu LCꢀ10AT equipped with a SILꢀ10A injector and a
SPDꢀ10AV detector, normally operating at 220 nm and interfaced with a Micromass ZMD mass spectrometer.
Analytical HPLC and LCꢀMS analyses were carried out using the following methods: Method A; Phenomenex
Luna S5 C18 4.6x30 mm column/waterꢀMeOHꢀTFA 90:10:0.1 to 10:90:0.1 gradient over 2 min at 4 mL/min
with 1 min hold at the end of the gradient. Method B; SunFire C18 3.5 micron 4.6x150 mm column/waterꢀ
CH3CNꢀTFA 95:5:0.05 to 5:95:0.05 gradient over 15 min, 1 mL/min, 3 min hold. Method C; XBridge Phenyl
3.5 micron 4.6x150 mm column/waterꢀCH3CNꢀTFA 95:5:0.05 to 5:95:0.05 gradient over 15 min, 1 mL/min, 3
min hold. Method D; YMC S5 C18 4.6x50 mm column/waterꢀMeOHꢀH3PO4 90:10:0.2 to 10:90:0.2 gradient
over 4 min, 4 mL/min, 1 min hold. Method E; Phenomenex Synergi 4ꢁ ODS 4.6x50 mm column/waterꢀMeOHꢀ
H3PO4 90:10:0.2 to 10:90:0.2 gradient over 4 min, 4 mL/min, 1 min hold. Method F (used with LC/MS); YMC
S5 C18 4.6x50 mm column/waterꢀMeOHꢀTFA 90:10:0.1 to 10:90:0.1 gradient over 4 min, 4 mL/min, 1 min
hold. Method G; Zorbax SB C18, 4.6x75 mm column/waterꢀMeOHꢀH3PO4 90:10:0.2 to 10:90:0.2 gradient
over 30 min, 1 mL/min. Method H; Phenomenex Luna S5 C18 4.6x30 mm column/10 mM NH4OAc in waterꢀ
CH3CN 95:5 to 5:95 gradient over 2 min, 4 mL/min, 1 min hold. Method I; Phenomenex Luna S5 C18 4.6x30
mm column/waterꢀCH3CNꢀTFA 95:5:0.05 to 5:95:0.05 gradient over 4 min, 4 mL/min, 1 min hold. Method J;
Zorbax SB C18, 4.6x75 mm column/waterꢀMeOHꢀH3PO4 90:10:0.2 to 10:90:0.2 gradient over 8 min, 1
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Journal of Medicinal Chemistry
mL/min. Method K (used with LC/MS); Phenomenex Luna C18 4.6x50 mm column/waterꢀMeOHꢀTFA
90:10:0.1 to 10:90:0.1 gradient over 4 min, 4 mL/min.
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All final compounds were of ≥ 95% purity by the LC/MS and analytical HPLC systems and were characterized
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by NMR analysis. Samples prepared for in vivo studies were of ≥ 98% purity.
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6-(4-Chlorophenyl)-3-(4-((1-hydroxycyclopropyl)methoxy)-3-methoxyphenyl)thieno[3,2-d]pyrimidin-
4(3H)-one (5): A solution of methyl 1ꢀhydroxycyclopropanecarboxylate (465.2 mg, 3.61 mmol), tꢀ
butyldimethylsilyl chloride (752 mg, 4.89 mmol) and imidazole (644 mg, 9.37 mmol) in DMF (4.5 mL) was
stirred at 35 °C for 15 h. After cooling to RT, the solution was diluted with ether, washed with brine, dried
(Na2SO4), concentrated and the residue was subjected to flash chromatograph (silica gel/hexaneꢀethyl acetate
95:5) to give methyl 1ꢀ((tertꢀbutyldimethylsilyl)oxy)cyclopropanecarboxylate (658 mg, 79 % yield) as a
colorless oil.
To a solution of the above ester in toluene (6.8 mL) was added 1M DIBALH in toluene (6.3 mL, 6.30 mmol)
dropwise at ꢀ78 °C. The mixture was stirred at ꢀ78 to ꢀ70 °C for 2 h followed by the addition of methanol (2.5
mL). The mixture was allowed to come to RT. Stirring was continued at RT for 15 h and then the resulting
gelatinous mixture was filtered through a sintered glass funnel. The funnel contents were rinsed with DCM and
the combined filtrates were concentrated to provide crude alcohol 12 (501 mg, 87% crude yield).
To a stirred suspension of 6ꢀ(4ꢀchlorophenyl)ꢀ3ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)thieno[3,2ꢀd]pyrimidinꢀ4(3H)ꢀ
one (11) (72 mg, 0.187 mmol),12 crude alcohol 12 from above (38 mg, 0.16 mmol) and triphenylphosphine (55
mg, 0.21 mmol) in DCM (1.0 mL) at RT was added diethyl azodicarboxylate (34 µL, 0.209 mmol) dropwise.
Stirring was continued at RT for 13 h and the reaction mixture was passed through a silica gel column eluting
with DCMꢀEther (95:5) to obtain 3ꢀ(4ꢀ((1ꢀ((tꢀButyldimethylsilyl)oxy)cyclopropyl)methoxy)ꢀ3ꢀmethoxyphenyl)ꢀ
6ꢀ(4ꢀchlorophenyl)thieno[3,2ꢀd]pyrimidinꢀ4(3H)ꢀone (24 mg, 26 % yield) as a white solid. This material was
dissolved in THF (0.9 mL) followed by the addition of tetrabutylammonium fluoride in THF (1M, 50 µL, 0.05
mmol). The mixture was stirred at RT for 2 h, diluted with water (2.0 mL) and extracted with DCM. The
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organic phase was dried (Na2SO4) and concentrated. The residue was subjected to flash chromatography (silica
gel/DCMꢀEtOAc 30:70 to 20:80 gradient) to afford compound 5 (17 mg, 0.037 mmol) as a white solid. 1H
NMR (400 MHz, CD2Cl2) δ 8.15 (1H, s), 7.71 (2H, d, J=8.3 Hz), 7.57 (1H, s), 7.47 (2H, d, J=8.3 Hz), 7.07 –
7.00 (1H, m), 6.99 – 6.92 (2H, m), 4.09 (2H, s), 3.89 (3H, s), 0.96 – 0.87 (2H, m), 0.69 (2H, dd, J=6.6, 5.3 Hz);
LC/MS (ESI) m/z 455 [M+H]+; analytical HPLC tR 3.55 min (method K), 6.54 min (method J); HRMS (ESI)
m/z 455.08262 [M+H]+, calcd for C23H19O4N2SClH+ 455.08268.
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1-((4-(6-(4-Chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)methyl)cyclopropyl
glycinate (5a). To a stirred suspension of 6ꢀ(4ꢀChlorophenyl)ꢀ3ꢀ(4ꢀ((1ꢀhydroxycyclopropyl)methoxy)ꢀ3ꢀ
methoxyphenyl)thieno[3,2ꢀd]pyrimidinꢀ4(3H)ꢀone (5) (25.5 mg, 0.056 mmol), 4ꢀpyrrolidinopyridine (13.0 mg,
0.088 mmol) and bocꢀGlyOH (34.0 mg, 0.194 mmol) at 42 °C was added N,N'ꢀdiisopropylcarbodiimide (26 µL,
0.168 mmol) over a 15 min period. After the addition was complete, the mixture was stirred at 42 °C for 1.3 h
and then at RT for 3h. The final mixture was diluted with EtOAc (30 mL) and washed with cold phosphate
buffer solution (0.5M KH2PO4 + H3PO4 added to pH 3, 2x15 mL) and cold 2% NaHCO3 (3x15 mL). The
organic layer was dried (Na2SO4) and evaporated. The residue was subjected to flash chromatography (two
consecutive columns: silica gel/hexaneꢀEtOAc 30:70 followed by silica gel/DCMꢀEther 60:40) to afford 1ꢀ((4ꢀ
(6ꢀ(4ꢀchlorophenyl)ꢀ4ꢀoxothieno[3,2ꢀd]pyrimidinꢀ3(4H)ꢀyl)ꢀ2ꢀmethoxyphenoxy)methyl)cyclopropyl (tertꢀ
butoxycarbonyl)glycinate (31.2 mg, 91 % yield) as a white solid. The last material was treated with 1:2 TFAꢀ
DCM (0.9 mL) for 30 min. The volatiles were evaporated and the residue was taken up in DCM (20 mL) and
washed with cold 2% NaHCO3. The organic solution was dried (Na2SO4) and evaporated to afford the tittle
1
compound (24.6 mg, 94 % yield) as a yellowish solid. H NMR (400 MHz, CDCl3) δ 8.14 (1H, s), 7.66 (2H, br
d, J=8.3 Hz), 7.54 (1H, s), 7.45 (2H, br d, J=8.3 Hz), 7.03 (1H, br d, J=8.3 Hz), 6.96 (1H, br s), 6.92 (1H, d,
J=8.3 Hz), 4.36 (2H, s), 3.89 (3H, s), 3.43 (2H, br s), 1.06 (4H, br d, J=13.2 Hz); LC/MS (ESI) m/z 512
[M+H]+; analytical HPLC tR 3.14 min (method K), 5.55 min (method J).
6-(4-Chlorophenyl)-3-(4-((3-hydroxyoxetan-3-yl)methoxy)-3-methoxyphenyl)thieno[3,2-d]pyrimidin-
4(3H)-one (6). A mixture of 15b (280 mg, 1.243 mmol), 16 (401 mg, 1.243 mmol)12 and phenol (3.0 g) was
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Journal of Medicinal Chemistry
heated at 135 ºC for 20 min, diluted with methanol, cooled to 0 ºC and filtered to obtain the crude product.
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2
3
1
Trituration of the solid with methanol yielded 6 (190 mg, 0.4 mmol, 33 % yield) as a white solid. H NMR (400
4
5
6
7
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MHz, DMSOꢀD6) δ 8.40 (1 H, s), 7.98 (1 H, s), 7.93 (2 H, d, J=8.6 Hz), 7.58 (2 H, d, J=8.6 Hz), 7.22 (1 H, d,
J=2.3 Hz), 7.18 (1 H, d, J=8.8 Hz), 7.06 (1 H, dd, J=8.6, 2.3 Hz), 6.04 (1 H, s), 4.53 (2 H, d, J=6.5 Hz), 4.49 (2
H, d, J=6.5 Hz), 4.16 (2 H, s), 3.79 (3 H, s); LC/MS (ESI) m/z 471 [M+H]+; analytical HPLC tR 2.34 min
(method A), 3.92 min (method D), 4.3 min (method E).
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3-((4-(6-(4-Chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)methyl)oxetan-3-yl
glycinate (6a). N,N'ꢀDiisopropylcarbodiimide (70 µL, 0.45 mmol) was added in small portions over 1 h to a
refluxing mixture of 6 (71 mg, 0.15 mmol), bocꢀGlyOH (79 mg, 0.45 mmol) and 4ꢀ(pyrrolidinꢀ1ꢀyl)pyridine (22
mg, 0.15 mmol) in DCM (3 mL). The mixture was refluxed for an additional 0.5 h. The mixture was diluted
with DCM, washed sequentially with cold 5% sulfuric acid and saturated sodium bicarbonate solution. The
organic phase was dried (MgSO4) and concentrated. Subsequently the crude product was subjected to flash
chromatography (silica gel/hexaneꢀEtOAc 100:0 to 0:100 gradient) to afford bocꢀglycine ester of compound 6
(150 mg) as a white solid (contaminated with diisopropylurea as determined by proton NMR). After dissolution
of the crude product in DCM (1 mL) and addition of TFA (1 mL), the reaction was allowed to stand at RT for
10 min. After concentration, the residue was partitioned between DCM and saturated sodium bicarbonate
solution. The organic phase was dried (MgSO4) and concentrated. The crude product was subjected to flash
chromatography (silica gel/methylene chlorideꢀmethanol 100:0 to 90:10 gradient) to afford 6a (60 mg, 48 %
1
yield) as a white solid; TLC Rf = 0.28 (silica gel/methylene chlorideꢀmethanol 93:7). H NMR (400 MHz,
CDCl3) δ 8.12 (1 H, s), 7.64 (2 H, d, J=8.6 Hz), 7.52 (1 H, s), 7.43 (2 H, d, J=8.6 Hz), 7.04 (1 H, d, J=8.3 Hz),
6.96 (1 H, d, J=2.3 Hz), 6.92 (1 H, dd, J=8.3, 2.3 Hz), 4.83 (2 H, d, J=7.8 Hz), 4.79 (2 H, d, J=8.1 Hz), 4.60 (2
H, s), 3.86 (3 H, s), 3.47 (2 H, s).
Proꢀdrug 6a underwent slow decomposition when stored as a free base in methanol solution at RT to generate
the parent alcohol. Therefore conc. HCl (15 ꢁL) was added to a ꢀ70 ºC solution of 6a which had been prepared
by dissolution of 6a in DCM (0.5 mL) followed by addition of methanol (0.3 mL) and rapid cooling to ꢀ70 ºC.
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The volatiles were removed at RT yielding compound 6a as a white solid (64 mg, HCl salt). MS (ESI) m/z 528
[M+H]+; Analytical HPLC tR = 2.17 min (method A), 3.89 min (method F), 3.5 min (method D).
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6-(4-Chlorophenyl)-3-(4-((1-hydroxycyclobutyl)methoxy)-3-methoxyphenyl)thieno[3,2-d]pyrimidin-
4(3H)-one (7). 17b was converted to 7 following the procedure used to prepare 6. 1H NMR (400MHz, CDCl3)
δ 8.07 (s, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.46 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 7.6 Hz, 1H), 6.89 (d,
J = 2.2 Hz, 1H), 6.85 (dd, J = 2.2, 7.6 Hz, 1 H), 4.01 (s, 2H), 3.80 (s, 3H), 3.09 (br s, 1H), 2.15 (m, 4H), 1.80
(m, 1H), 1.53 (m, 1H); analytical HPLC (method I) 4.32 min; 28.32 min (method G); MS m/z 469 [M+H]+.
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1-((4-(6-(4-Chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)methyl)cyclobutyl
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glycinate (7a). 7 was converted to7a following the procedure used to prepare 6a. H NMR (500MHz, CDCl3)
δ 8.13 (s, 1H), 7.66 (d, J=8.8 Hz, 2H), 7.53 (s, 1H), 7.44 (d, J=8.2 Hz, 2H), 7.04 (d, J=8.2 Hz, 1H), 6.96 (d,
J=2.2 Hz, 1H), 6.92 (dd, J=8.8, 2.7 Hz, 1H), 4.43 (s, 2H), 3.88 (s, 3H), 3.38 (s, 2H), 2.59 ꢀ 2.47 (m, 2H), 2.46 ꢀ
2.38 (m, 2H), 2.01 ꢀ 1.91 (m, 1H), 1.87 ꢀ 1.74 (m, 1H); MS m/z 528 [M+H]+; analytical HPLC tR 2.28 min
(method A), 13.88 min (B), 3.52 min (method F), 25.92 min (method G).
6-(4-Chlorophenyl)-3-(4-((1-hydroxycyclopentyl)methoxy)-3-methoxyphenyl)thieno[3,2-d]pyrimidin-
4(3H)-one (8). 18b was converted to 8 following the procedure used to prepare 6. 1H NMR (CDCl3) 8.14 (s,
1H), 7.67 (d, J = 9.4 Hz, 2H), 7.52 (s, 1H), 7.46 (d, J = 9.4 Hz, 2H), 7.03 (d, J = 8.2 Hz, 1H), 6.96 (d, J = 2.2
Hz, 1H), 6.93 (dd, J = 2.2, 8.2 Hz, 1 H), 4.01 (s, 2H), 3.87 (s, 3H), 2.37 (s, 1H), 1.90ꢀ1.60 (m, 8H); MS m/z 483
[M+H]+; analytical HPLC tR 2.13 min (method A), 29.4 min (method G).
6-(4-Chlorophenyl)-3-(4-((4-hydroxytetrahydro-2H-pyran-4-yl)methoxy)-3-methoxyphenyl)thieno[3,2-
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d]pyrimidin-4(3H)-one (9). 19b was converted to 9 following the procedure used to prepare 6. H NMR
(CDCl3) δ ppm 1.70 ꢀ 1.87 (m, 4 H), 3.78 ꢀ 3.96 (m, 9 H), 6.91 ꢀ 6.96 (m, 1 H), 6.97 (d, 1 H), 7.03 (d, 1 H), 7.45
(d, 2 H), 7.51 ꢀ 7.57 (m, 1 H), 7.66 (d, 2 H); analytical HPLC tR 3.13 min (method F), 9.17 (method B), 8.44
min (method C); MS (ESI) m/z 499 [M+H]+.
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6-(4-Chlorophenyl)-3-(4-((3,3-difluoro-1-hydroxycyclobutyl)methoxy)-3-methoxyphenyl)thieno[3,2-
d]pyrimidin-4(3H)-one (10). A stirred mixture of 16 (33.9 g, 105 mmol) and 25b (27.2 g, 105 mmol) in
phenol (200 g) was heated at 135ꢀ140 ºC for 45 min.12 The mixture was diluted with methanol (700 mL) and
stirred at RT for 15 min. The mixture was allowed to stand at RT overnight. The precipitated product was
isolated by filtration, washed with dryꢀice chilled methanol and dried under vacuum giving compound 10 ( 37 g,
70% yield) as a white solid. The mother liquor was diluted with ether and hexane and the precipitated solid was
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triturated from MeOH to give a second batch of the desired product (1.8 g). H NMR (400 MHz, CDCl3) δ 8.14
(s, 1H), 7.66 (d, J=8.6 Hz, 2H), 7.54 (s, 1H), 7.45 (d, J=8.6 Hz, 2H), 7.08 (d, J=8.6 Hz, 1H), 6.99 (d, J=2.3 Hz,
1H), 6.95 (dd, J=8.3, 2.0 Hz, 1H), 4.14 (s, 2H), 3.89 (s, 3H), 2.87 ꢀ 2.76 (m, 4H); MS (ESI) m/z 505 [M+H]+;
analytical HPLC (method A) tR = 2.34 min, (method B) tR = 16.34 min, (method C) tR = 15.36 min.
1-((4-(6-(4-Chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)methyl)-3,3-
difluorocyclobutyl glycinate (10a). N,N'ꢀDiisopropylcarbodiimide (44 µL, 0.28 mmol) was added slowly to a
refluxing mixture of compound 10 (70 mg, 0.139 mmol), bocꢀGlyOH (73 mg, 0.416 mmol) and 4ꢀ(pyrrolidinꢀ1ꢀ
yl)pyridine (21 mg, 0.139 mmol) in DCM (3 mL). After the mixture was heated at reflux for 45 min, LC/MS
analysis indicated complete conversion to the desired product. The mixture was diluted with DCM, washed
sequentially with cold 5% sulfuric acid and saturated sodium bicarbonate solutions. The organic phase was
dried (MgSO4) and concentrated to give white solid. To the crude product dissolved in DCM (1 mL), was
added TFA (1 mL). After stirring at RT for 5 min, the mixture was concentrated and partitioned between DCM
and saturated sodium bicarbonate solution. The organic phase was dried (MgSO4) and concentrated. The crude
product was subjected to flash chromatography (silica gel/hexaneꢀEtOAc 100:0 to 0:100 gradient then DCMꢀ
methanol 100:0 to 85:15 gradient) to afford compound 10a (65 mg, 0.116 mmol, 83 % yield) as a white solid,
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TLC Rf = 0.5 (silica gel/DCMꢀmethanol 85:15). H NMR of HCl salt (400MHz, DMSOꢀD6) δ 8.43 (br. s., 3H),
8.41 (s, 1H), 7.99 (s, 1H), 7.93 (d, J=8.8 Hz, 2H), 7.59 (d, J=8.6 Hz, 2H), 7.26 (d, J=2.5 Hz, 1H), 7.17 (d, J=8.6
Hz, 1H), 7.08 (dd, J=8.4, 2.4 Hz, 1H), 4.42 (s, 2H), 3.86 ꢀ 3.77 (m, 5H), 3.30 ꢀ 3.05 (m, 4H); 19F NMR
(376MHz, DMSOꢀD6) δ ꢀ84.80 (d, J=199.3 Hz, 1F), ꢀ93.89 (d, J=198.0 Hz, 1F); MS (ESI) m/z 562 [M+H]+;
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analytical HPLC tR = 2.04 min (method A), 7.61 min (method B), 8.87 min (method C). The product from
above was dissolved in DCM (2 mL), cooled to ꢀ30 ºC, prior to addition of a solution of conc. HCl (25 ꢀL) in
0.5 mL MeOH. The mixture was stirred at RT for 5 min and concentrated to give HCl salt of the title
compound 10a (66 mg).
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Dibenzyl 1-((4-(6-(4-chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)methyl)-
3,3-difluorocyclobutyl phosphate (10b). A mixture of compound 10 (50 g, 99 mmol), dibenzyl
diisopropylphosphoramidite (113 g, 327 mmol) and 1Hꢀ1,2,4ꢀtriazole (22.57 g, 327 mmol) in 1,2ꢀ
dichloroethane (2 L) was heated at reflux for 1 hr. After cooling to RT, 30% hydrogen peroxide (60 mL, 979
mmol) was added over 10 min at while maintaining the reaction temperature between 15ꢀ45 ºC (exothermic,
used iceꢀwater cooling bath). After stirring for 15 min. at RT, the mixture was diluted with DCM, washed
sequentially with water and 5% aq. sodium thiosulfate, dried (MgSO4) and concentrated. The crude residue was
subjected to flash chromatography (2 Kg silica gel/DCMꢀEtOAc 100:0 to 0:100 gradient) to obtain 10b (70.5 g,
93% yield) TLC Rf = 0.6, silica gel/1:1 DCMꢀEtOAc; 1H NMR (400MHz, CDCl3) δ 8.10 (s, 1H), 7.67 (d,
J=8.6 Hz, 2H), 7.54 (s, 1H), 7.45 (d, J=8.6 Hz, 2H), 7.40 ꢀ 7.29 (m, 10H), 6.99 ꢀ 6.84 (m, 3H), 5.08 (dd, J=7.9,
1.6 Hz, 4H), 4.32 (s, 2H), 3.76 (s, 3H), 3.27 ꢀ 2.94 (m, 4H); MS (ESI) m/z 765 [M+H]+; analytical HPLC tR =
2.54 min (method A), 13.52 min (method B), 12.22 min (method C).
1-((4-(6-(4-Chlorophenyl)-4-oxothieno[3,2-d]pyrimidin-3(4H)-yl)-2-methoxyphenoxy)methyl)-3,3-
difluorocyclobutyl dihydrogen phosphate (10c). Compound 10b (70.5 g) was dissolved in TFA (500 mL)
and stirred at RT for 8 h. The mixture was concentrated, reꢀconcentrated twice from DCM prior to
recrystallization of the residue from refluxing EtOH (ca. 10 L) to give compound 10c as a light yellow solid.
This material was reꢀdissolved in refluxing EtOH (ca. 10 L), treated with charcoal and stirred for 10 min. The
mixture was filtered through celite while hot and allowed to stand at RT for 24 h and then at 4 ºC for 48 h to
afford 10c as a white crystalline solid (37 g, 1st crop). The celiteꢀcharcoal filter cake was extracted with hot
DMF (2 X 1.5L), the DMF extract was concentrated and the residue was triturated alternately with EtOH and
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1,2ꢀdichloroethane affording additional 10c (8.4 g); 78.4% combined yield. H NMR (400 MHz, DMSOꢀD6) δ
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Journal of Medicinal Chemistry
8.40 (1 H, s), 7.98 (1 H, s), 7.92 (2 H, d, J=8.3 Hz), 7.57 (2 H, d, J=8.3 Hz), 7.23 (1 H, d, J=1.8 Hz), 7.15 (1 H,
d, J=8.8 Hz), 7.07 (1 H, d, J=8.1 Hz), 4.27 (2 H, s), 3.79 (3 H, s), 3.21 (2 H, q, J=14.4 Hz), 2.94 ꢀ 3.10 (2 H, m);
19F NMR (376MHz, DMSOꢀd6) δ ꢀ84.76 (d, J=196.6 Hz, 1F), ꢀ94.38 (d, J=193.8 Hz, 1F). MS (ESI) m/z 585
[M+H]+; analytical HPLC tR = 2.2 min (method A), 9.34 min (method B), 8.56 min (method C).
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3-((2-Methoxy-4-nitrophenoxy)methyl)oxetan-3-ol (15a). A solution of aqueous osmium tetroxide (4%, 0.1
mL) and hydrogen peroxide (50%, 1 mL) were sequentially added to a stirred solution of 13 (0.5g, 4.8 mmol)13
in 1:1 THFꢀwater (2 mL) at 4 oC. The mixture was allowed to come to RT and stirred for 1 h. After dilution of
the reaction mixture with water (3 mL) and filtration through a pad of celite and charcoal, the filtrate was
concentrated in vacuo at 40ꢀ50 ºC (ca. 30 mm vacuum) to afford 3ꢀ(Hydroxymethyl)oxetanꢀ3ꢀol (14) as a thick
brown oil (0.6 g, 49 % crude yield).
Sodium hydride (0.384 g, 9.6 mmol) was added in small portions to a stirred solution of 1ꢀchloroꢀ2ꢀmethoxyꢀ4ꢀ
nitrobenzene (0.9 g, 4.80 mmol) and crude 3ꢀ(hydroxymethyl)oxetanꢀ3ꢀol from above (0.5 g, 4.80 mmol) in
DMSO (20 mL) at RT. After stirring at RT for 6 h, the reaction mixture was diluted with ether and washed with
saturated NH4Cl solution. The organic phase was dried (MgSO4), concentrated and the crude product was
subjected to flash chromatography (silica gel/hexaneꢀEtOAc 100:0 to 0:100 gradient) to afford the title
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compound (340 mg, 28 % yield) as a yellow gummy solid (TLC Rf = 0.15; silica gel/hexaneꢀEtOAc 1:1). H
NMR (500 MHz, CDCl3) δ 7.83 (1 H, dd, J=8.9, 2.6 Hz), 7.69 (1 H, d, J=2.7 Hz), 6.95 (1 H, d, J=8.8 Hz), 4.69
(2 H, d, J=7.4 Hz), 4.57 (2 H, d, J=7.4 Hz), 4.31 (2 H, s), 3.88 (3 H, s); analytical HPLC (method A) tR = 1.66
min.
3-((4-Amino-2-methoxyphenoxy)methyl)oxetan-3-ol (15b). A mixture of 15a (340 mg, 1.33 mmol) and 10%
palladium on carbon (50 mg) was stirred under hydrogen at 50 psi for 3 h, filtered and concentrated to give 15b
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(280 mg, 93 % crude yield) as a thick brown oil. H NMR (500 MHz, CD3OD) δ 6.85 (1 H, d, J=8.5 Hz), 6.51
(1 H, d, J=2.5 Hz), 6.34 (1 H, dd, J=8.5, 2.5 Hz), 4.66 (2 H, d, J=7.1 Hz), 4.58 (2 H, d, J=6.9 Hz), 4.05 (2 H, s),
3.80 (3 H, s); MS (ESI) m/z 248 [M+Na]+; analytical HPLC tR = 0.29 min (method A).
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1-((2-Methoxy-4-nitrophenoxy)methyl)cyclobutanol (17a). 1ꢀ(Hydroxymethyl)cyclobutanol (1.27g, 12.5
mmol)24 was added to a suspension of NaH (60% mineral oil dispersion, 0.50g, 12.5 mmol) in DMSO (14.6
mL). Subsequently, 30 min after cessation of gas evolution, 2ꢀbromoꢀ4ꢀnitroanisole (1.95g, 8.4 mmol) in
DMSO (5.9 mL) was added to the stirred solution. The reaction was stirred for 2.5 h before being poured into
0.1N HCl (300 mL). After extraction of the mixture with ether, the combined organic layers were washed with
brine, dried over MgSO4 and concentrated. The residue was subjected to flash chromatography (silica
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gel/DCMꢀEtOAc 0:100 to 50:50 gradient) to yield 0.65 g of 17a. H NMR (400MHz, CDCl3) δ 7.89 (dd, J=8.9,
2.8 Hz, 1H), 7.74 (d, J=2.5 Hz, 1H), 6.97 (d, J=9.2 Hz, 1H), 4.11 (s, 2H), 3.93 (s, 3H), 3.05 (br. s., 1H), 2.22
(dd, J=9.2, 6.6 Hz, 4H), 1.95 ꢀ 1.81 (m, 1H), 1.74 ꢀ 1.55 (m, 1H); 13C NMR (101MHz, CDCl3) δ 154.0, 149.3,
141.7, 117.6, 111.8, 106.7, 74.5, 73.3, 56.1, 32.6, 12.2; analytical HPLC (method A) tR1.56 min; MS m/z 254
[M+H]+.
1-((4-Amino-2-methoxyphenoxy)methyl)cyclobutan-1-ol (17b). 17a was converted to17b following the
procedure used to prepare 15b. 1H NMR (400MHz, CDCl3) δ 6.76 (d, J=8.1 Hz, 1H), 6.28 (d, J=2.5 Hz, 1H),
6.20 (dd, J=8.6, 2.5 Hz, 1H), 3.90 (s, 2H), 3.73 (s, 3H), 2.19 ꢀ 2.04 (m, 4H), 1.84 ꢀ 1.70 (m, 1H), 1.62 ꢀ 1.45 (m,
1H); analytical HPLC tR 0.78 min (method A).
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1-((2-Methoxy-4-nitrophenoxy)methyl)cyclopentan-1-ol (18a). 18a was prepared as described for 17a. H
NMR (500MHz, CDCl3) δ 7.84 (dd, J=8.7, 2.7 Hz, 1H), 7.70 (d, J=2.7 Hz, 1H), 6.89 (d, J=9.4 Hz, 1H), 4.01 (s,
2H), 3.91 (s, 3H), 1.89 ꢀ 1.64 (m, 8H); 13C NMR (126MHz, CDCl3) δ 154.0, 149.1, 141.5, 117.5, 111.6, 106.7,
80.9, 76.2, 56.1, 37.1, 24.0; analytical HPLC tR 3.55 min (method D), 22.3 min (method G).
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1-((4-Amino-2-methoxyphenoxy)methyl)cyclopentan-1-ol (18b). 18b was prepared as described for 15b. H
NMR (400 MHz, CD3OD) δ 6.79 (d, J=8.65 Hz, 1H), 6.51 (d, J=2.03 Hz, 1H), 6.35 (dd, J=2.54, 8.14 Hz, 1H),
3.81 (s, 2H), 3.79 (s, 3H), 1.48ꢀ1.93 (m, 8H); MS m/z 238 [M+H]+; analytical HPLC tR 1.02 min (method A).
4-((2-Methoxy-4-nitrophenoxy)methyl)tetrahydro-2H-pyran-4-ol (19a). 1,6ꢀDioxaspiro[2.5]octane25 was
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converted to 19a as described for the preparation of 25a. H NMR (400MHz, CDCl3) δ 7.89 (dd, J=9.2, 2.5 Hz,
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Journal of Medicinal Chemistry
1H), 7.75 (d, J=2.5 Hz, 1H), 6.92 (d, J=9.2 Hz, 1H), 3.93 (s, 5H), 3.91 ꢀ 3.79 (m, 4H), 1.87 ꢀ 1.69 (m, 4H); MS
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m/z 301 [M+H]+; analytical HPLC tR 1.18 min (method H).
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4-((4-Amino-2-methoxyphenoxy)methyl)tetrahydro-2H-pyran-4-ol (19b). 19b was prepared as described
for 15b. 1H NMR (400MHz, CDCl3) δ 6.78 (d, J=8.1 Hz, 1H), 6.29 (d, J=2.5 Hz, 1H), 6.21 (dd, J=8.1, 2.5 Hz,
1H), 3.92 ꢀ 3.73 (m, 9H), 1.80 ꢀ 1.61 (m, 4H); MS m/z 276 [M+Na]+; analytical HPLC tR 0.23 min (method H).
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1-(3,3-Difluorocyclobutyl)-N,N-dimethylmethanamine (21): Oxalyl chloride (21.7 mL, 248 mmol) was
added dropwise to a stirred solution of 3,3ꢀdifluorocyclobutanecarboxylic acid (20)14 (26 g, 191 mmol) in
dichloromethane (500 mL) and DMF (0.5 mL) at 0 ºC. The reaction mixture was allowed to warm to RT,
stirred for 1 h and concentrated in vacuo at RT. The residue was dissolved in THF (300 mL) and treated at 0 ºC
with 2M dimethylamine (478 mL, 955 mmol) in THF. The reaction mixture was stirred at RT for 0.5 h and
partitioned between ether and 5% aq. sodium carbonate. The organic layer was dried over magnesium sulfate
and concentrated in vacuo. After portioning the residue between methylene chloride and water, the organic
layer was dried over magnesium sulfate and concentrated in vacuo to give 3,3ꢀdifluoroꢀN,Nꢀ
dimethylcyclobutanecarboxamide (24 g, 147 mmol, 77% yield) as a brown semiꢀsolid, used as such in the next
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step. H NMR (400 MHz, CDCl3) δ 2.99ꢀ3.12 (1H, m), 2.95 (3H, s), 2.94 (3H, s), 2.78ꢀ2.92 (2 H, m), 2.62ꢀ2.77
(2H, m). A solution of 3,3ꢀdifluoroꢀN,Nꢀdimethylcyclobutanecarboxamide (24 g, 147 mmol) in THF (500 mL)
was added dropwise to a stirred suspension of lithium aluminum hydride (7.5 g, 198 mmol) in THF (500 mL)
at 0 ºC. Upon completion of addition, the mixture was allowed to warm to RT. After stirring for 18 h, the
mixture was cooled to ca. 5 ºC and quenched by slowly adding 6N NaOH (10 mL) and water (5 mL) with
stirring. The mixture was allowed to warm to RT, then dried over sodium sulfate and filtered. The filtrate was
concentrated to ca. 30 mL by distilling off most of the THF using a vigreux column. The remaining material
was distilled under slightly reduced pressure (ca. 100ꢀ200 mm Hg) giving a fraction (20 mL, collected at 70ꢀ90
ºC) containing the title compound contaminated with THF. This material was carefully purged with a gentle
stream of nitrogen to remove the residual THF giving 1ꢀ(3,3ꢀdifluorocyclobutyl)ꢀN,Nꢀdimethylmethanamine (12
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g, 80 mmol, 55% yield) as a colorless liquid. H NMR (400 MHz, CDCl3) δ 2.60ꢀ2.75 (2H, m), 2.38 (2H, d, J =
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1-(3,3-Difluorocyclobutyl)-N,N-dimethylmethanamine oxide hydrate (22): Aqueous hydrogen peroxide
(30%, 18 mL) was added dropwise to a stirred solution of 1ꢀ(3,3ꢀdifluorocyclobutyl)ꢀN,Nꢀ
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dimethylmethanamine (12 g, 80 mmol) in methanol (100 mL) over 2 h, while maintaining the reaction
temperature between 5 and 22ºC. Upon completion of addition, the mixture was kept at RT for 20 h, then
additional 30% hydrogen peroxide (18 mL) was added and stirring continued for 3 h. A slurry of palladium
black (150 mg) in water (3mL) was then added in small portions carefully to the stirred reaction mixture,
maintaining the temperature between 5 to 25 ºC. The reaction mixture was stirred at RT until all of the excess
hydrogen peroxide (ca. 1 h) had decomposed. After filtration, the filtrate was concentrated in vacuo to give 1ꢀ
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(3,3ꢀdifluorocyclobutyl)ꢀN,Nꢀdimethylmethanamine oxide (15 g) as a semiꢀsolid hydrate. H NMR (400 MHz,
CD3OD) δ 3.47 (2H, d, J = 5.3 Hz), 3.16 (6H, s), 2.75ꢀ2.92 (3H, m), 2.42ꢀ2.58 (2H, m).
1,1-Difluoro-3-methylenecyclobutane (23): Crude 1ꢀ(3,3ꢀdifluorocyclobutyl)ꢀN,Nꢀdimethylmethanamine
oxide hydrate (15 g) was heated under vacuum (ca. 10 mm Hg) at 100 ºC using a distillation setup in order to
remove most of the water from the sample. The receiving flask was cooled to ꢀ78 ºC as the temperature was
gradually increased to 165 ºC. After ca. 1 h, most of the starting material had been pyrolized (a small amount of
dark brown residue remained in the distillation flask). Contents of the receiving flask were washed sequentially
with 5% aq. HCl (3X3 mL) and saturated sodium bicarbonate (5 mL). The organic layer (olefin 6) was filtered
through a small plug of sodium sulfate giving 1,1ꢀdifluoroꢀ3ꢀmethylenecyclobutane (5.5 g, 52.8 mmol, 66%
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yield from 21) as a colorless liquid. H NMR (400 MHz, CDCl3) δ 5.10 (2H, quin, J = 2.5 Hz), 2.77ꢀ3.57 (4H,
m). 19F NMR (376 MHz, chloroformꢀD) δ ꢀ95.16 (2F, s).
5,5-Difluoro-1-oxaspiro[2.3]hexane (24): mꢀCPBA (70%, 90.0 g, 365 mmol) was added in small portions to a
stirred solution of 1,1ꢀdifluoroꢀ3ꢀmethylenecyclobutane (21.0 g, 202 mmol) in methylene chloride (600 mL) at
RT. After ca. 1 h, a slight exotherm was noticed and the mixture was cooled using an ice bath. The reaction
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Journal of Medicinal Chemistry
mixture was allowed to warm to RT over 3 h and stirred for additional 18 h. Monitoring of the progress of the
reaction by 1H NMR revealed incomplete oxidation. After additional mꢀCPBA (10 g) was added, the mixture
was stirred at RT for additional 24 h. The mixture was filtered to remove solids prior to washing the filtrate
with 10% sodium carbonate. The organic layer was dried (sodium sulfate) and concentrated at atmospheric
pressure to ca. 250 mL using a vigreux column. The material was flash distilled at ca. 10 mm Hg employing
two sequential (in series) traps cooled to ꢀ78 ºC to collect the distillate. The combined distillate was further
concentrated at atmospheric pressure using a vigreux column to a volume of approximately 50 mL affording
5,5ꢀdifluoroꢀ1ꢀoxaspiro[2.3]hexane (71 g, contained ca. 2.5 equivalents of dichloromethane by NMR,
quantitative yield). While adequate for most chemical transformations, if desired, this material could be
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enriched by further distillation. H NMR (400 MHz, CDCl3) δ 2.91ꢀ3.16 (4H, m), 2.88 (2H, s). 19F NMR (376
MHz CDCl3) δ ꢀ90.84 (1F, m, J = 200.7 Hz), ꢀ99.53 (1F, dquin, J = 202.1, 12.37 Hz). HRMS (ESI) m/z
162.07237 [M+CH3CN+H]+, calcd for C7H10ONF2 162.07250.
3,3-Difluoro-1-((2-methoxy-4-nitrophenoxy)methyl)cyclobutanol (25a). A mixture of potassium 2ꢀ
methoxyꢀ4ꢀnitrophenolate (12.43 g, 0.06 mol), 5,5ꢀdifluoroꢀ1ꢀoxaspiro[2.3]hexane (contains 3 moles
DCM/mole of 24, 22.52 g, 0.06 mol) and sodium dihydrogen phosphate monohydrate (7.45 g, 0.054 mol) in
85:15 acetonitrileꢀwater (50 mL) was heated at 130 ºC in a steel bomb for 3.5 h. The reaction mixture was
diluted with EtOAc, washed with 5% sodium carbonate, dried (MgSO4) and concentrated. The crude product
was recrystallized from MTBE (150 mL) giving 25a (12.4 g, 73 % yield) as a light yellow solid. 1H NMR (400
MHz, CDCl3) δ 7.89 (1H, dd, J = 8.9, 2.6 Hz), 7.76 (1H, d, J = 2.8 Hz), 6.95 (1H, d, J = 9.1 Hz), 4.16 (2H, s),
3.94 (3H, s), 3.36 (1H, s), 2.73ꢀ2.92 (4H, m); MS (ESI) m/z 312 [M+Na]+; analytical HPLC (method A) tR =
1.71 min.
1-((4-Amino-2-methoxyphenoxy)methyl)-3,3-difluorocyclobutanol (25b). A mixture of 3,3ꢀdifluoroꢀ1ꢀ((2ꢀ
methoxyꢀ4ꢀnitrophenoxy)methyl)cyclobutanol (32.0 g, 111 mmol) and 10% palladium on carbon (2.0 g) in
methanol (700 mL) was stirred under hydrogen at 50 psi for 1.5 h, filtered and the filtrate was concentrated to
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give 25b (28.8 g, 111 mmol, 100 % yield) as a light purple solid. H NMR (400MHz, CD3OD) δ 6.78 (d, J=8.6
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