Discovery of 2-(phenoxypyridine)-3-phenylureas as small molecule P2Y 1 antagonists

Article abstract of DOI:10.1021/jm301708u

Two distinct G protein-coupled purinergic receptors, P2Y₁ and P2Y₁₂, mediate ADP-driven platelet activation. The clinical effectiveness of P2Y₁₂ blockade is well established. Recent preclinical data suggest that P2Y_1<

Full text of DOI:10.1021/jm301708u

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Article
Discovery of 2-(Phenoxypyridine)-3-
phenylureas as Small Molecule P2Y1 Antagonists
Hannguang Chao, Huji Turdi, Timothy F Herpin, Jacques Y Roberge, Yalei Liu, Dora M Schnur, Michael
A Poss, Robert Rehfuss, Ji Hua, Qimin Wu, Laura A Price, Lynn M Abell, William A Schumacher,
Jeffrey S Bostwick, Thomas E Steinbacher, Anne B Stewart, Martin L Ogletree, Christine S Huang, Ming
Chang, Angela M Cacace, Maredith J Arcuri, Deborah Celani, Ruth R. Wexler, and R. Michael Lawrence
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301708u • Publication Date (Web): 31 Jan 2013
Downloaded from http://pubs.acs.org on February 18, 2013
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Discovery of 2-(Phenoxypyridine)-3-phenylureas as
Small Molecule P2Y1 Antagonists
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Hannguang Chao,* Huji Turdi, Timothy F. Herpin,
Jacques Y. Roberge, Yalei Liu, Dora M. Schnur,
Michael A. Poss, Robert Rehfuss, Ji Hua, Qimin Wu,
Laura A. Price, Lynn M. Abell , William A.
Schumacher, Jeffrey S. Bostwick, Thomas E.
Steinbacher, Anne B. Stewart, Martin L. Ogletree,
Christine S. Huang, Ming Chang, Angela M. Cacace,
Maredith J. Arcuri, Deborah Celani, Ruth R. Wexler,
and R. Michael Lawrence
BristolꢀMyers Squibb Research and Development, P.O. Box 5400, Princeton NJ 08543
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RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
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Abstract
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Two distinct G proteinꢀcoupled purinergic receptors, P2Y1 and P2Y12, mediate ADP driven platelet
activation. The clinical effectiveness of P2Y12 blockade is well established. Recent preclinical data
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suggest that P2Y1 and P2Y12 inhibition provide equivalent antithrombotic efficacy, while targeting P2Y1
has the potential for reduced bleeding liability. In this account, the discovery of a 2ꢀ(phenoxypyridine)ꢀ
3ꢀ(phenyl)urea chemotype that inhibited ADPꢀmediated platelet aggregation in human blood samples is
described. Optimization of this series led to the identification of compound 16, 1ꢀ(2ꢀ(2ꢀtertꢀ
butylphenoxy)pyridinꢀ3ꢀyl)ꢀ3ꢀ4ꢀ(trifluoromethoxy)phenylurea, which demonstrated a 68 ± 7% thrombus
weight reduction in an established rat arterial thrombosis model (10 mg/kg plus 10 mg/kg/h) while only
prolonging cuticle and mesenteric bleeding times by 3.3ꢀ and 3.1ꢀfold, respectively in provoked rat
bleeding time models. These results suggest that a P2Y1 antagonist could potentially provide a safe and
efficacious antithrombotic profile.
Introduction
The P2Y1 receptor, a Gꢀproteinꢀcoupled receptor, expressed in the heart, skeletal, and various smooth
muscles as well as the prostate, brain, and circulating blood cells,1 is a member of the P2Y family of
receptors. The P2Y receptor family is generally considered to consist of eight members, P2Y1, P2Y2,
P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14, encoded by distinct genes, which can be subdivided into
two groups based on their coupling to specific Gꢀproteins.2 Several studies have suggested that
modulators of specific members of the P2Y family could have therapeutic potential for the treatment of
a variety of disorders, 3 such as diabetes, cancer, and ischemiaꢀreperfusion injury.4 Among the P2Y
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family, P2Y1 and P2Y12 are of particular interest because they play a major physiological role in ADPꢀ
mediated platelet aggregation, an important component of thrombosis.5
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ADP is a key activator of platelets, and platelet activation is known to play a pivotal role in thrombus
formation. ADP activates platelets by simultaneously stimulating P2Y1 and P2Y12 to produce two
separate intracellular signals, which synergize to produce complete platelet activation. The
antithrombotic effect of antagonizing the P2Y12 receptor alone is very well validated clinically with the
thienopyridines6 and other drugs7 (Figure 1), but the same level of proof of principle in humans for the
P2Y1 receptor has yet to be demonstrated. In 2001, Léon8 first reported that inhibition of P2Y1 activity
alone could lead to an antiꢀthrombotic effect in vivo in a model of thromboplastinꢀinduced
thromboembolism using both a P2Y1 knockꢀout mouse and the P2Y1 antagonist MRS2179.9
Subsequently, Jacobson and others described a series of adenine nucleotideꢀbased P2Y1 receptor
antagonists that inhibit ADPꢀinduced platelet aggregation.10 In general, those adenine nucleotide
analogs exhibited good in vitro potency. However, their chemical and enzymatic stabilities are less than
desirable, which would be expected to limit their usefulness as oral drug candidates. Thus, the
discovery of novel P2Y1 antagonists with improved pharmaceutical characteristics could have
significant utility in the treatment of a variety of thromboembolic disorders.11
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Figure 1. Known P2Y12 and P2Y1 antagonists
Results and Discussion
Given the promising evidence that P2Y1 receptor antagonists may represent a novel antithrombotic
strategy that might complement current therapy, a discovery effort to identify small molecule P2Y1
antagonists was carried out. A high throughput screening effort using the full BMS screening deck (>1
million compounds) against the human P2Y1 receptor employing a binding assay with [βꢀ33P]ꢀ2ꢀ
methylthioadenosine diphosphate as the ligand revealed the diaryl urea 1 (Figure 2) with good affinity
and binding selectivity toward the P2Y1 receptor versus P2Y12 (P2Y1 Ki = 75 nM, P2Y12 Ki > 70 ꢀM).12
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F
H
N
H
1
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N
3
4
O
O
F
N
5
6
1
P2Y1 Ki = 75 nM
P2Y12 Ki > 70 M
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CF3
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14
Figure 2. P2Y1 lead compound
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The favorable profile of this compound was validated by resynthesis and it was selected as the starting
point for further structural optimization. The modular nature of this compound made it particularly well
suited for utilization of array synthesis to efficiently explore the SAR of the chemotype. Optimization
efforts began with exploration of the phenyl ring of the urea moiety. Scheme 1 represents the general
synthetic route to the targeted compounds. First, reacting 3-CF3PhOH 2 with 2ꢀchloroꢀ3ꢀnitropyridine 3
in DMF at 80 oC afforded the 3ꢀnitroꢀ2ꢀphenoxypyridine 4. Subsequent reduction of the nitro group
with Zn/NH4Cl gave the corresponing 3ꢀaminopyridine, which was then reacted with diphosgene in the
presence of N1,N1,N8,N8ꢀtetramethylnaphthaleneꢀ1,8ꢀdiamine to provide the key intermediate isocyanate
5.13 In general, the isocyanate derivatives were purified by silica gel chromatography and were stable
up to a year when stored at 0 oC. For subsequent array synthesis, the isocyanate was distributed into 96
vessels and reacted with 96 diverse amines in THF at 60 oC in a parallel format to afford urea analogs 6.
Scheme 1 Reagents and conditions: a) Cs2CO3, DMF, 80 oC, 14 h, 60%; b) Zn, NH4Cl, MeOH, 92%; c) diphosgene, N1,N1,N8,N8ꢀtetramethylnaphthaleneꢀ
1,8ꢀdiamine, CH2Cl2, 100%; d) 96 R1R2NH, THF, 60 oC, yields in range of 12ꢀ75%
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All analogs of type 6 were initially screened in the human P2Y1 receptor binding assay. This
exploration of the distal urea substitution demonstrated monosubstitution was required for activity.
Additionally, aliphatic substituents were not tolerated. Shown in Table 1 are selected examples from
the evaluation of the urea phenyl ring. The SAR indicated that substitution of the phenyl ring and the
positions of the substitution were critical for P2Y1 activity. The unsubstituted analog (6a) showed a 3ꢀ
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fold drop in activity compared to the lead 1. Replacement of the ortho fluorine (6b) with a larger group
such as chlorine was not tolerated (6c). Substitution at the metaꢀposition was acceptable but, in
general, provided less active analogs (6d and 6e). Substitution at the paraꢀposition with flourine or
chlorine showed comparable activity (6f and 6g) but methyl and methoxy, as well as, larger lipophilic
groups like tꢀBu, OCF3 or Ph provided a modest improvement in activity (6h-6l). Various other
functional groups such as dimethylamine, nitrile, and methyl ester were acceptable at this position but
resulted in analogs with weaker binding (6m-6o). Bisꢀsubstitution was also explored with analogs
showing no improvement in activity (6p and 6q). Insertion of one or two methylene groups between the
phenyl ring and distal nitrogen atom of the urea resulted in a dramatic loss of activity (6r and 6s).
Table 1. Inhibition data for compounds 1 and 6a-6s
Cpds
1
R1
hP2Y1 Kia (nM)
75 ± 5
171 ± 105
47 ± 27
2, 4ꢀdiꢀFꢀPh
Ph
2ꢀFꢀPh
2ꢀClꢀPh
3ꢀFꢀPh
3ꢀClꢀPh
4ꢀFꢀPh
4ꢀClꢀPh
4ꢀMePh
4ꢀtꢀBuꢀPh
4ꢀOCF3ꢀPh
4ꢀPhꢀPh
4ꢀOMeꢀPh
6a
6b
6c
6d
6e
6f
6g
6h
6i
>5000
607 ± 105
128 ± 45
145 ± 28
69 ± 23
48 ± 28
31 ± 4
313 ± 198
28 ± 4
21 ± 11
6j
6k
6l
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6m
6n
6o
6p
6q
6r
4ꢀNMe2ꢀPh
4ꢀCNꢀPh
4ꢀCO2MeꢀPh
3,4ꢀdiꢀClꢀPh
3,5ꢀdiꢀClꢀPh
CH2Ph
506 ± 85
571 ± 150
644 ± 296
84 ± 16
169 ± 38
>5000
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6
6s
CH2CH2Ph
>5000
a
Data represent the mean ± SEM, n = 2.
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Optimization efforts on the phenoxy ring and the linker were carried out utilizing the 2,4ꢀdifluorophenyl
urea present in the HTS hit 1. The same general synthetic scheme was utilized in an array format
(Scheme 2) and several analogs were thus prepared. Introduction of the variable group in the first step
necessitated all the subsequent steps be carried out in parallel for all vials. Instead of generating an
isocyanate of type 5 of each core, 2,4ꢀdifluorophenyl isocyanate was condensed with each of the 3ꢀ
aminopyridine.
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Scheme 2 Reagents and conditions: a) Cs2CO3, DMF, 80 oC b) Zn, NH4Cl, EtOH/EtOAc (4:1) c) toluene, 80 oC, 1.5 h, yields in range of 22ꢀ44%
Shown in Table 2 are selected representatives from the exploration of the phenyl ether. As seen with
the phenyl urea, substituents and their position were pivotal to P2Y1 activity. The unsubstituted phenyl
analog 11a displayed more than a 30ꢀfold drop in activity compared to compound 1. On the contrary,
substitution with a tꢀbutyl group at the orthoꢀposition (11b) improved potency by 4ꢀfold, and at the
metaꢀposition (11c) by 2.5ꢀfold, respectively. Additionally, an iꢀpropyl group (11d) at the orthoꢀ
position provided a modest improvement in activity, whereas the ethyl group (11e) displayed the same
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level of potency as compound 1. Combined, the data indicated lipophilicity at the orthoꢀposition was
important for activity. In the case of the CF3 substituent, while substituting at the orthoꢀposition
maintained activity (11f) compared to compound 1, moving the CF3 substituent to the paraꢀposition
(11g) resulted in a substantial loss of activity. This was further confirmed using a methyl group as the
para substituent (11h).
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Table 2. Inhibition data for compounds 1 and 11a-11h
Cpds
1
R2
3ꢀCF3
hP2Y1 Kia (nM)
75 ± 5
11a
H
2000 ± 1158
18 ± 4
2ꢀtꢀBu
3ꢀtꢀBu
11b
11c
33 ± 7
11d
11e
2ꢀiꢀPr
28 ± 2
77 ± 6
2ꢀEt
2ꢀCF3
4ꢀCF3
4ꢀMe
172 ± 71
>5000
>5000
11f
11g
11h
a
Data represent the mean ± SEM, n = 2.
The linker between the phenyl ring and pyridyl ring was explored by replacing the oxygen atom with
sulfur or nitrogen. Synthesis of these analogs utilized the same chemistry sequence except the initial
starting phenol was replaced by the correspondingly substituted thiophenol or aniline. When the oxygen
atom in compound 12 was replaced by sulfur (13) a modest drop in activity was observed (Table 3).
However, replacing with nitrogen (14) resulted in a 20ꢀfold drop in activity. Furthermore, methylation
of the linking nitrogen of 14 provided a more significant loss in activity (15).
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Table 3. Inhibition data for compounds 12-15
Cpds
12
X
O
hP2Y1 Kia (nM)
7 ± 0.1
13
S
20 ± 8
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NH
NMe
175 ± 40
>5000
a
Data represent the mean ± SEM, n = 2.
Following the exploration of the two terminal phenyl rings, a focused library was executed based on the
discovered SAR, in which preferred substituents at each position were combined. This soꢀcalled "bestꢀ
best" array included 8 preferred substituents each from the phenoxy moiety and the phenyl ring of the
urea moiety. This array was prepared following the same chemistry outlined in Scheme 1. This "bestꢀ
best" array produced several potent antagonists with a clear trend that preferred the combination of
substitution of the phenoxy ring with a lipophilic group, such as tꢀbutyl or iꢀpropyl at the ortho or meta
position and substitution of the urea phenyl ring at the para position. Listed in Table 4 are a few
selected examples from the array demonstrating that several compounds had single digit nanomolar
binding affinity. These compounds were also evaluated in a platelet aggregation assay to assess their
ability to block ADPꢀinduced platelet aggregation in human platelet rich plasma14 with several of these
showing single digit micromolar potency. To insure this antiplatelet activity was due exclusively to
P2Y1 antagonism all compounds were tested for their P2Y12 activity. As shown all compounds had
excellent selectivity over P2Y12. Additionally, these compounds also showed good selectivity over
other P2Y receptors such as P2Y2, P2Y6, P2Y11 and P2Y14 (Table 4).
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Table 4. Inhibition, platelet aggregation, and selectivity data for compounds 1, 12, and 16-19
Cpds R2
R3
hP2Y1
hPRPb
hP2Y12
hP2Y2
hP2Y6
hP2Y11
hP2Y14
Met Stab
% remaining
(h/r)d
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Kia (nM)
IC50 (ꢀM)
Kia (nM) Kic (nM) Kic (nM) Kic (nM)
Kia (nM)
1
3ꢀCF3
2ꢀtꢀBu
2ꢀtꢀBu
2ꢀtꢀBu
2ꢀiꢀPr
2ꢀiꢀPr
2,4ꢀDiF
4ꢀMe
75 ± 5
7 ± 0.1
6 ± 0.6
8 ± 0.4
7 ± 0.3
16 ± 2
53 ± 28
(n = 5)
> 70000
> 70000
ꢀ
ꢀ
ꢀ
ꢀ
75/50
33/38
80/86
58/33
73/35
97/80
12
16
17
18
19
7.2 ± 2.1
(n = 2)
2700
> 15000 > 15000 2350 ± 950
4ꢀOCF3
4ꢀtꢀBu
4ꢀtꢀBu
4ꢀOCF3
2.1 ± 0.28
(n = 7)
> 70000 > 15000 > 15000 > 15000 3500 ± 1000
> 70000 > 15000 > 15000 > 15000 5950 ± 950
1.2 ± 0.2
(n = 2)
4.4 ± 3.8
(n = 4)
> 70000 > 15000 > 15000 > 15000
> 15000
9.4 ± 4
(n = 2)
> 70000 6400 > 15000 > 15000 3300 ± 600
a
b
c
d
Data represent the mean ± SEM, n = 2.
PRP run in human platelet rich plasma using 2.5 uM ADP.
n = 1
h: human; r: rat. Compounds (3 ꢀM) were incubated with human or rat liver microsomes (1.0 mg/mL) for 10 min.
Compounds with good in vitro P2Y1 binding affinity and functional activity were evaluated to
determine their metabolic stability. Since the in vivo studies were carried out in rats, the metabolic
stability in rat became an important factor for compound selection. Among those compounds tested,
only compounds 16 and 19 showed reasonable metabolic stability in rats, 86% and 80% remaining after
incubation with rat liver microsomes for 10 min, respectively. Although compound 19 was reasonably
stable in rats, its functional activity was not quite potent enough for further consideration. The Cacoꢀ2
permeability for compound 16 was found to be low (21 nm sꢀ1) as was the aqueous solubility of the
hydrochloride salt ( < 1 ꢀg/mL at pH 6.5). Solubilizing vehicles that were compatible with the in vivo
models were used for solution dosing for pharmacokinetic evaluation. The pharmacokinetic profile of
compound 16 (Table 5) revealed this compound had reasonable plasma exposure, clearance and halfꢀ
life with moderate oral bioavailability (F = 18%) when dosed at 30 mg/kg using 10%EtOH/10%
cremophor/80% H2O as vehicle.
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Table 5. Pharmacokinetic properties for compound 16
Cpd
number
16a
16b
Dosage
mg/kg
Cmax
(nM)
Tmax
(h)
AUC total CL
Vss
L/kg
Thalf
(h)
F(%)
1
2
3
4
(nM *h)
mL/min/kg
1
160
0.8
2.0
169
16
13
5.6
0.8
15.4
1.43
7
30
5830
15900
18
5
6
7
8
a dose: IV: 1 mg/kg (n = 3), PO: 1 mg/kg (n = 2 ), vehicle: 10% DMAC/10%EtOH/10% cremophor/70% H2O.
b dose: IV: 1 mg/kg (n = 3), PO: 30 mg/kg (n = 2 ), vehicle: 10%EtOH/10% cremophor/80% H2O.
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The antithrombotic efficacy of compound 16 was assessed in established arterial thrombosis and
bleeding models in 86 male anesthetized rats.12 As shown in Figure 3, compound 16 clearly improved
blood flow and reduced thrombus weight in a doseꢀdependent manner. A maximum of 68 ± 7%
thrombus weight reduction compared to vehicle was observed using a 10 mg/kg bolus followed by 10
mg/kg/h infusion dosing paradigm. Furthermore, vascular occlusion was prevented in all drugꢀtreated
rats at this dose. Ex vivo platelet aggregation responses to ADP were also significantly inhibited at all
antithrombotic doses. Compound 16 was also tested in provoked bleeding time models. As shown in
Figure 4, the highest tested dose of 10 mg/kg plus 10 mg/kg/h prolonged cuticle and mesenteric
bleeding times by 3.3ꢀ and 3.1ꢀfold over control, respectively. In previous studies using these same rat
models, the P2Y12 antagonist clopidogrel administered as an oral pretreatment dose of 20 mg/kg,
reduced arterial thrombus weight by 67 ± 5% and prolonged cuticle and mesenteric bleeding times by
4.1ꢀ and 8.2ꢀfold over control, respectively.12
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Figure 3. Effect of compound 16 on arterial thrombosis and platelet function in anesthetized rats. Vehicle or compound 16 was dosed as a bolus plus
sustaining i.v. infusion starting 15 min prior to inducing thrombus formation by topical FeCl2. a) Carotid blood flow was measured during thrombus
formation. b) Thrombus weight, c) integrated blood flow and d) ex vivo platelet aggregation responses to ADP were determined at the end of the experiment
when platelet counts were sufficient for the assay (resulted in fewer replicates in some groups). Percent reductions in thrombus weight and platelet
aggregation are indicated for responses that were significantly different from vehicle. The number of rats in the thrombosis model is indicated in the
“proportion of total number that occluded”. Compound 16 doses (mg/kg + mg/kg/h) of 0.1 + 0.1, 0.5 + 0.5, 1 + 1, 5 + 5 and 10 + 10 resulted in plasma
concentrations (μM) of 0.4 ± 0.1, 1.8 ± 0.7, 4.0 ± 0.2, 16.5 ± 1.6, and 36.5 ± 2.5, respectively.
.
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time limit
time limit
Pre
Post
900
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600
500
400
300
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100
0
600
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p<0.05 to Vehicle
*
Pre
Post
=SEM
500
400
Mesenteric
Artery
Bleeding
Time
(sec)
Cuticle
Bleeding
Time
2.2-fold
3.3-fold
300
*
3.1-fold
*
*
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(sec)
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100
0
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1.9-fold
1.7-fold
*
*
Vehicle 0.5+0.5 1+1 5+5 10+10
(n=6) (n=6) (n=6) (n=7) (n=9)
Vehicle 1+1 5+5 10+10
(n=7) (n=5) (n=5) (n=5)
Compound 11
16
(mg/kg + mg/kg/hr)
16
Compound 11
(mg/kg + mg/kg/hr)
Figure 4. Effect of compound 16 on provoked bleeding times in anesthetized rats. Vehicle or compound 16 was dosed as a bolus plus sustaining i.v.
infusion. Bleeding times were determined before and 15 min after test article administration. Fold increases over control bleeding times are indicated for
responses that were significantly different from vehicle.
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Conclusions
Novel P2Y1 antagonists have been identified by optimization of a high throughput screening hit. These
urea analogs have demonstrated excellent in vitro P2Y1 antagonist activity. Compound 16 showed
inhibition of platelet aggregation in human platelet rich plasma with no activity towards the P2Y12
receptor. Furthermore, compound 16 provided significant reduction in thrombus weight in a rat arterial
thrombosis model with a limited effect on bleeding. The antithrombotic and bleeding profiles of
compound 16 were favorable in comparison to the established P2Y12 antagonist clopidogrel,15
suggesting that the current series of P2Y1 antagonists may have potential as novel antithrombotic
agents.
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Experimental Section
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General Methods. Starting materials, reagents, and solvents were obtained from commercial sources
and used as received. All reactions were carried out with continuous stirring under an atmosphere of
dry nitrogen. The resonance frequency for 1H (13C) on a Jeol JNMꢀECPꢀ500 is 500 MHz 125(MHz) and
Brucker Variance 400 is 400 MHz (100 MHz). Chemical shifts (δ) are reported in ppm downfield from
internal tetramethylsilane (TMS), and coupling constants (J) are in hertz (Hz). Peak multiplicities are
expressed as follows: singlet (s), doublet (d), doublet of doublets (dd), triplet (t), quartet (q), multiplet
(m), broad singlet (br s). TLC was performed on EMD silica gel 60 F254 glass plate (2.5 × 7.5 cm).
Microwave reactions were run in a Biotage initiator instrument. Products were analyzed by reverse
phase analytical HPLC carried out on a Shimadzu Analytical HPLC system running DiscoveryVP
software using Method A: Phenominex Luna C18 column (4.6 × 50 mm) eluted at 4 mL/min with a 4
min gradient from 100% A to 100% B (A: 10% methanol, 89.9% water, 0.1% TFA; B: 10% water,
89.9% methanol, 0.1% TFA, UV 220 nm), Method B: Phenominex Luna C18 column (4.6 × 50 mm)
eluted at 4 mL/min with a 4 min gradient from 100% A to 100% B (A: 10% acetonitrile, 89.9% water,
0.1% TFA; B: 10% water, 89.9% acetonitrile, 0.1% TFA, UV 220 nm), or Method C: Zorbax SB C18
column (4.6 × 75 mm) eluted at 2.5 mL/min with methanol/water with 0.2% H3PO4 as a gradient of
10% to 90% methanol over 8 min followed by holding at 90% methanol for 3 min (UV 220 nm).
Purification of intermediates and final products was carried out via either normal or reverse phase
chromatography. Normal phase chromatography was carried out on an ISCO CombiFlash™ System
Sq16x using prepacked SiO2 cartridges eluted with gradients of hexanes and ethyl acetate. Reverse
phase preparative HPLC was carried out using a Shimadzu Preparative HPLC system running
DiscoveryVP software on a ShimꢀPackVPꢀODS column (50 × 20 mm) at 20 mL/min, 6 min gradient
100% A to 100% B with the solvent systems used for the analytical. LCMS were obtained on a
Shimadzu HPLC system running DiscoveryVP software, coupled with a Waters Model Platform LC
mass spectrometer running MassLynx version 3.5 software using the same column and conditions as
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utilized for analytical described above. Compounds of interest were greater than 95% pure as
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determined by orthogonal HPLC methods. The analysis was carried out on a Shimadzu HPLC system,
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equipped with orthogonal columns; Waters sun fire column (C18, 3.5 ꢀm, 3.0 × 150 mm) and Waters
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7
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Xbridge phenyl (C18, 3.5 ꢀm, 4.6 × 150 mm); eluted at 2 mL/min with a 12 min gradient from 100% A
to 100% B. For low pH; solvent A: 95% H2O/5% CH3CN/0.05% TFA; solvent B: 5% H2O/95%
CH3CN/0.05% TFA. For high pH; solventA: 95% H2O/5% CH3CN/0.01M NH4HCO3; solvent B: 5%
H2O/95% CH3CN/0.01MNH4HCO3.
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1-(2,4-difluorophenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (1). A solution of 2ꢀ
chloroꢀ3ꢀnitropyridine 3 (20 g, 125 mmol) in DMF (120 mL) was treated with 3ꢀtrifluoromethylphenol
2 (21 g, 129 mmol) and cesium carbonate (50 g, 154 mmol). The mixture was heated at 70 °C for 14 h.
The reaction was cooled to room temperature and DMF was evaporated. The residue was taken up in
EtOAc which was washed with 5% LiCl (3 × 100 mL) and then brine (3 × 100 mL). Drying (MgSO4)
and removal of solvent afforded a brown solid which was recrystallized from ethanol to afford
1
compound 4 as an offꢀwhite solid (21 g, 60 %). (M+H+) = 285.04; H NMR (400 MHz, CD3OD) δ 7.2
(dd, J = 7.96, 1.39 Hz, 1H), 7.4 (m, 2H), 7.45 (m, 1H), 7.11 (m, 2H), 7.5ꢀ7.54 (td, J = 4.93, 1.64 Hz,
1H), 8.48 (dd, J = 7.83, 1.77 Hz, 1H), 8.52 (dd, J = 7.83, 1.77 Hz, 1H).
Compound 4 (21 g, 74 mmol) was dissolved in a 1:4 mixture of methanol and THF (160 mL).
Palladium on activated charcoal (10%, 2.1 g, 1.93 mmol) was added, and the mixture was stirred
overnight under 1 atm hydrogen pressure. The reaction mixture was filtered over celite and concentrated
to afford a white solid which was recrystallized from EtOAc to afford the 2ꢀ(3ꢀ
(trifluoromethyl)phenoxy)pyridinꢀ3ꢀamine (17.5 g, 92%) as a white powder. (M+H+) = 254.3. To a
solution of the amine (8.2 g, 34.3 mmol) and diphosgene (5.5 g, 28 mmol) in CH2Cl2 (175 mL) at 0 oC
was added dropwise a solution of CH2Cl2 (125 mL) containing N1,N1,N8,N8ꢀtetramethylnaphthaleneꢀ1,8ꢀ
diamine (14.8 g, 67 mmol). After addition completed, the resulting mixture was stirred at 0 oC for an
additional 45 min, then washed with 0.5 N HCl (3 × 150 mL), 1N NaOH (2 × 100 mL) and brine.
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Drying (MgSO4) and removal of solvent afforded compound 5 as a solid (9.6 g, 100%). This material
was used for the subsequent reaction without further purification.
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To a solution of isocyanate (5) (28 mg, 1.0 mmol) in toluene (1 mL) in a 2ꢀdram vial was added 2,4ꢀ
difluoroaniline (16.1 mg, 0.125 mmol). The resulting mixture was heated with shaking at 80 °C for 1.5
h. The solvent was removed by vacuum centrifugation in a speedvac and the crude product was purified
by a reverse phase preparative HPLC using CH3CN/H2O/TFA solvent system. After removal of solvent
in vacuo the TFA salt of compound 1 was obtained as a white solid (13.0 mg, 32%). 1H NMR (500
MHz, CD3OD) δ 6.94 (t, J = 8.80 Hz, 1 H) 7.02 (ddd, J = 11.27, 8.52, 2.75 Hz, 1 H) 7.13 (dd, J = 7.70,
4.95 Hz, 1 H) 7.44 (d, J = 8.25 Hz, 1 H) 7.50 (s, 1 H) 7.53 (d, J = 7.70 Hz, 1 H) 7.62 (t, J = 7.97 Hz, 1
H) 7.73 (d, J = 4.95 Hz, 1 H) 8.09 (td, J = 9.07, 6.05 Hz, 1 H) 8.63 (d, J = 8.25 Hz, 1 H).
LCꢀMS (ESI) m/z 410.1 Orthogonal HPLC column 1 retention time = 12.12 min, column 2 =
10.29 min.
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Compounds 6a-6s, 11a-11h, 12, and 16-19 were prepared according to the procedures described for 1.
1-phenyl-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6a). Yield: 23.9 mg (49%). 1H NMR
(400 MHz, CD3OD) δ 7.03 (t, J = 7.42 Hz, 1 H) 7.13 (dd, J = 7.70, 4.95 Hz, 1 H) 7.26 ꢀ 7.32 (m, 2 H)
7.44 (d, J = 8.25 Hz, 3 H) 7.50 (s, 1 H) 7.54 (d, J = 7.70 Hz, 1 H) 7.63 (t, J = 7.97 Hz, 1 H) 7.72 (d, J =
3.30 Hz, 1 H) 8.61 ꢀ 8.66 (m, 1 H).
LCꢀMS (ESI) m/z 374.0 (M+H+); Orthogonal HPLC column 1 retention time = 12.04 min, column 2 =
10.56 min.
1-(2-fluorophenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6b). Yield: 17.2 mg (44%).
1H NMR (400 MHz, CD3OD) δ 7.00 ꢀ 7.06 (m, 1 H) 7.09 ꢀ 7.17 (m, 3 H) 7.44 (d, J = 8.25 Hz, 1 H) 7.48
ꢀ 7.55 (m, 2 H) 7.63 (t, J = 7.97 Hz, 1 H) 7.73 (d, J = 4.95 Hz, 1 H) 8.11 ꢀ 8.18 (m, 1 H) 8.65 (d, J =
8.25 Hz, 1 H).
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LCꢀMS (ESI) m/z 392.0 (M+H+); Orthogonal HPLC column 1 retention time = 12.40 min, column 2 =
10.79 min.
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1-(2-chlorophenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6c). Yield: 19.5 mg (48%).
1H NMR (400 MHz, CD3OD) δ 7.02 ꢀ 7.08 (m, 1 H) 7.14 (dd, J = 7.70, 4.95 Hz, 1 H) 7.26 ꢀ 7.31 (m, 1
H) 7.40 (d, J = 9.34 Hz, 1 H) 7.44 (d, J = 7.70 Hz, 1 H) 7.50 (s, 1 H) 7.54 (d, J = 8.25 Hz, 1 H) 7.63 (t, J
= 7.97 Hz, 1 H) 7.73 (d, J = 4.95 Hz, 1 H) 8.12 (d, J = 8.24 Hz, 1 H) 8.63 ꢀ 8.68 (m, 1 H).
LCꢀMS (ESI) m/z 408.0 (M+H+); Orthogonal HPLC column 1 retention time = 12.81 min, column 2 =
11.05 min.
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1-(3-fluorophenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6d). Yield: 9.8 mg (25%).
1H NMR (400 MHz, CD3OD) δ 6.71 ꢀ 6.79 (m, 1 H) 7.07 (d, J = 7.70 Hz, 1 H) 7.14 (dd, J = 8.24, 4.95
Hz, 1 H) 7.23 ꢀ 7.31 (m, 1 H) 7.41 ꢀ 7.51 (m, 3 H) 7.54 (d, J = 7.70 Hz, 1 H) 7.63 (t, J = 7.97 Hz, 1 H)
7.72 (d, J = 4.40 Hz, 1 H) 8.64 (d, J = 7.70 Hz, 1 H).
LCꢀMS (ESI) m/z 392.0 (M+H+); Orthogonal HPLC column 1 retention time = 12.44 min, column 2 =
10.85 min.
1-(3-chlorophenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6e). Yield: 15.5 mg (38%).
1H NMR (400 MHz, CD3OD) δ 6.99 ꢀ 7.03 (m, 1 H) 7.14 (dd, J = 8.25, 4.95 Hz, 1 H) 7.26 (d, J = 5.50
Hz, 2 H) 7.44 (d, J = 8.24 Hz, 1 H) 7.49 ꢀ 7.56 (m, 2 H) 7.62 (d, J = 7.70 Hz, 1 H) 7.67 (s, 1 H) 7.73 (d,
J = 3.30 Hz, 1 H) 8.63 (d, J = 6.05 Hz, 1 H).
LCꢀMS (ESI) m/z 408.0 (M+H+); Orthogonal HPLC column 1 retention time = 13.07 min, column 2 =
11.27 min.
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1-(4-fluorophenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6f). Yield: 14.1 mg (36%).
1H NMR (400 MHz, CD3OD) δ 7.04 (t, J = 8.79 Hz, 2 H) 7.13 (dd, J = 7.70, 4.95 Hz, 1 H) 7.44 (dd, J =
9.07, 4.67 Hz, 3 H) 7.50 (s, 1 H) 7.54 (d, J = 7.70 Hz, 1 H) 7.63 (t, J = 7.70 Hz, 1 H) 7.72 (d, J = 3.30
Hz, 1 H) 8.62 (d, J = 6.60 Hz, 1 H).
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LCꢀMS (ESI) m/z 392.0 (M+H+); Orthogonal HPLC column 1 retention time = 10.12 min, column 2 =
9.72 min.
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1-(4-chlorophenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6g). Yield: 21.6 mg (53%).
1H NMR (400 MHz, CD3OD) δ 7.13 (dd, J = 8.25, 4.95 Hz, 1 H) 7.25 ꢀ 7.31 (m, 2 H) 7.40 ꢀ 7.48 (m, 3
H) 7.50 (s, 1 H) 7.54 (d, J = 7.70 Hz, 1 H) 7.63 (t, J = 7.97 Hz, 1 H) 7.72 (d, J = 4.95 Hz, 1 H) 8.62 (d, J
= 8.25 Hz, 1 H).
LCꢀMS (ESI) m/z 408.0 (M+H+); Orthogonal HPLC column 1 retention time = 12.95 min, column 2 =
11.19 min.
1-(4-methylphenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6h). Yield: 18.06 mg
(47%). 1H NMR (400MHz, CD3OD) δ 2.29 (s, 3H) 7.08 ꢀ 7.15 (m, 3H) 7.29 ꢀ 7.34 (m, 2H) 7.40 ꢀ 7.46
(m, 1H) 7.48 ꢀ 7.56 (m, 2H) 7.61 (d, J = 7.90 Hz, 1H) 7.71 (dd, J = 4.80, 1.80 Hz, 1H) 8.61 (dd, J =
8.10, 1.80 Hz, 1H).
LCꢀMS (ESI) m/z 388.1 (M+H+); Orthogonal HPLC column 1 retention time = 12.09 min, column 2 =
9.98 min.
1-(4-t-butylphenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6i). Yield: 15.9 mg (37%).
1H NMR (400 MHz, CD3OD) δ 1.30 (s, 9 H) 7.13 (dd, J = 7.70, 4.95 Hz, 1 H) 7.30 ꢀ 7.39 (m, 4 H) 7.44
(d, J = 8.25 Hz, 1 H) 7.50 (s, 1 H) 7.54 (d, J = 7.70 Hz, 1 H) 7.62 (d, J = 8.24 Hz, 1 H) 7.71 (d, J = 3.30
Hz, 1 H) 8.62 (d, J = 6.05 Hz, 1 H).
LCꢀMS (ESI) m/z 430.1 (M+H+); Orthogonal HPLC column 1 retention time = 13.80 min, column 2 =
11.76 min.
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1-(4-trifluoromethoxyphenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6j). Yield: 18.7
mg (41%). 1H NMR (400 MHz, CD3OD) δ 7.13 (dd, J = 7.97, 4.67 Hz, 1 H) 7.21 (d, J = 8.25 Hz, 2 H)
7.44 (d, J = 8.25 Hz, 1 H) 7.50 (s, 1 H) 7.52 ꢀ 7.58 (m, 3 H) 7.63 (t, J = 7.97 Hz, 1 H) 7.71 ꢀ 7.75 (m, 1
H) 8.63 (d, J = 9.89 Hz, 1 H).
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LCꢀMS (ESI) m/z 458.0 (M+H); Orthogonal HPLC column 1 retention time = 13.25 min, column 2 =
11.34 min.
1-(biphenyl-4-yl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6k). Yield: 5.4 mg (12%). 1H
NMR (400 MHz, CD3OD) δ 7.14 (dd, J = 8.24, 4.95 Hz, 1 H) 7.29 (t, J = 7.42 Hz, 1 H) 7.38 ꢀ 7.48 (m,
3 H) 7.50 ꢀ 7.67 (m, 9 H) 7.72 (d, J = 4.95 Hz, 1 H) 8.65 (d, J = 8.25 Hz, 1 H).
LCꢀMS (ESI) m/z 450.0 (M+H+); Orthogonal HPLC column 1 retention time = 13.45 min, column 2 =
11.68 min.
1-(4-methoxyphenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6l). Yield: 17.3 mg
(43%). 1H NMR (400 MHz, CD3OD) δ 3.77 (s, 3 H) 6.88 (d, J = 8.79 Hz, 2 H) 7.12 (dd, J = 8.24, 4.95
Hz, 1 H) 7.33 (d, J = 9.34 Hz, 2 H) 7.43 (d, J = 7.70 Hz, 1 H) 7.49 (s, 1 H) 7.52 ꢀ 7.56 (m, 1 H) 7.62 (t,
J = 7.97 Hz, 1 H) 7.71 (d, J = 4.95 Hz, 1 H) 8.60 (d, J = 8.25 Hz, 1 H).
LCꢀMS (ESI) m/z 404.1 (M+H+); Orthogonal HPLC column 1 retention time = 11.68 min, column 2 =
10.32 min
1-(4-dimethylaminophenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6m). Yield: 11.2
1
mg (27%). H NMR (400 MHz, CD3OD) δ 3.06 (s, 6 H) 7.13 (dd, J = 8.25, 4.95 Hz, 2 H) 7.29 ꢀ 7.37
(m, 1 H) 7.41 ꢀ 7.51 (m, 4 H) 7.52 ꢀ 7.57 (m, 1 H) 7.63 (t, J = 7.97 Hz, 1 H) 7.72 (d, J = 3.30 Hz, 1 H)
8.61 (d, J = 6.05 Hz, 1 H).
LCꢀMS (ESI) m/z 417.0 (M+H+); Orthogonal HPLC column 1 retention time = 6.98 min, column 2 =
7.65 min.
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1-(4-cyanophenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6n). Yield: 14.3 mg (36%).
1H NMR (400 MHz, CD3OD) δ 7.14 (dd, J = 7.70, 4.95 Hz, 1 H) 7.45 (d, J = 8.24 Hz, 1 H) 7.51 (s, 1 H)
7.53 ꢀ 7.57 (m, 1 H) 7.60 ꢀ 7.64 (m, 1 H) 7.66 (s, 4 H) 7.74 (d, J = 4.95 Hz, 1 H) 8.62 ꢀ 8.68 (m, 1 H).
LCꢀMS (ESI) m/z 399.0 (M+H+); Orthogonal HPLC column 1 retention time = 11.82 min, column 2 =
10.41 min.
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Methyl 4-(3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)ureido)benzoate (6o). Yield: 22.8 mg
(53%). 1H NMR (400 MHz, CD3OD) δ 3.87 (s, 3 H) 7.14 (dd, J = 7.70, 4.95 Hz, 1 H) 7.45 (d, J = 8.25
Hz, 1 H) 7.49 ꢀ 7.66 (m, 5 H) 7.74 (d, J = 4.95 Hz, 1 H) 7.96 (d, J = 8.79 Hz, 2 H) 8.65 (d, J = 6.05 Hz,
1 H).
LCꢀMS (ESI) m/z 432.0 (M+H+); Orthogonal HPLC column 1 retention time = 12.09 min, column 2 =
10.48 min.
1-(3,4-chlorophenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6p). Yield: 17.26 mg
(39%). 1H NMR (400MHz, CD3OD) δ 7.13 (dd, J = 8.00, 5.00 Hz, 1H) 7.29 (dd, J = 8.80, 2.60 Hz, 1H)
7.38 ꢀ 7.46 (m, 2H) 7.48 ꢀ 7.56 (m, 2H) 7.62 (d, J = 8.10 Hz, 1H) 7.73 (dd, J = 5.00, 1.70 Hz, 1H) 7.83
(d, J = 2.40 Hz, 1H) 8.62 (dd, J = 8.00, 1.70 Hz, 1H).
LCꢀMS (ESI) m/z 442.0 (M+H+); Orthogonal HPLC column 1 retention time = 14.20 min, column 2 =
11.92 min.
1-(3,5-chlorophenyl)-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6q). Yield: 33.1mg
(75%). 1H NMR (400 MHz, CD3OD) δ 7.06 (s, 1 H) 7.14 (dd, J = 8.25, 4.95 Hz, 1 H) 7.44 (d, J = 8.25
Hz, 1 H) 7.47 ꢀ 7.52 (m, 3 H) 7.54 (d, J = 7.70 Hz, 1 H) 7.63 (t, J = 7.97 Hz, 1 H) 7.73 (d, J = 4.95 Hz, 1
H) 8.63 (d, J = 6.05 Hz, 1 H).
LCꢀMS (ESI) m/z 442.0 (M+H+); Orthogonal HPLC column 1 retention time = 14.28 min, column 2 =
11.99 min.
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Journal of Medicinal Chemistry
1
1-benzyl-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6r). Yield: 10.0 mg (26%). H NMR
(400 MHz, CD3OD) δ 4.41 (s, 2 H) 7.10 (dd, J = 8.24, 4.95 Hz, 1 H) 7.21 ꢀ 7.28 (m, 1 H) 7.32 (d, J =
4.40 Hz, 4 H) 7.39 (d, J = 8.25 Hz, 1 H) 7.45 (s, 1 H) 7.48 ꢀ 7.54 (m, 1 H) 7.60 (t, J = 7.97 Hz, 1 H)
7.66 ꢀ 7.71 (m, 1 H) 8.55 (d, J = 8.25 Hz, 1 H).
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LCꢀMS (ESI) m/z 388.1 (M+H+); Orthogonal HPLC column 1 retention time = 11.46 min, column 2 =
10.24 min.
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1-phenethyl-3-(2-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (6s). Yield: 8.1 mg (20%). H
NMR (400 MHz, CD3OD) δ 2.83 (t, J = 7.15 Hz, 2 H) 3.46 (t, J = 7.15 Hz, 2 H) 7.09 (dd, J = 7.97, 4.67
Hz, 1 H) 7.18 (t, J = 6.87 Hz, 1 H) 7.22 ꢀ 7.31 (m, 4 H) 7.37 (d, J = 8.25 Hz, 1 H) 7.43 (s, 1 H) 7.51 (d,
J = 7.70 Hz, 1 H) 7.60 (t, J = 7.97 Hz, 1 H) 7.67 (d, J = 4.95 Hz, 1 H) 8.52 (d, J = 8.25 Hz, 1 H).
LCꢀMS (ESI) m/z 402.1 (M+H+); Orthogonal HPLC column 1 retention time = 11.79 min, column 2 =
10.46 min.
3-Nitro-2-phenoxypyridines (8). 48 phenols 7 (1.06 mmol each in 1 mL DMF) were dispensed into
24ꢀwell miniblocks. To these solutions Cs2CO3 (360 mg, 1.1 mmol) was added to each well followed
by 2ꢀchloroꢀ3ꢀnitropyridine (3) (158 mg, 1.0 mmol) in DMF (1 mL). The reactions were heated with
shaking at 80 oC for 18 h. The reaction mixtures were filtered and collected into 16 × 100 mm test tubes.
EtOAc (2 mL) was added to each well and shaken for 2ꢀ3 minutes, then combined with the DMF
filtrate. The combined solutions were dried in speedvac. The crude mixtures were dissolved in EtOAc
(2.5 ꢀ 4 mL) and washed with H2O (2 × 0.6 mL), saturated NaHCO3 (1 × 0.5 mL), and H2O (2 × 0.5
mL). Solvents were removed by vacuum centrifugation in a speedvac to afford crude products which
were used for the subsequent reaction without further purification.
3-Amino-2-phenoxypyridines (9). The nitro compounds 8 were dissolved in a 4:1 mixed solution
containing absolute EtOH (4 mL) and EtOAc (1 mL). Zn dust (1.3 g, 20 mmol) and NH4Cl (300 mg,
5.4 mmol) were added to these solutions sequentially. The resulting mixtures were shaken at room
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temperature overnight. The solutions were filtered through a bed of celite and collected into 16 × 100
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mm test tubes. Solvents were removed by vacuum centrifugation in a speedvac to afford crude products
which were used for the subsequent reaction without further purification.
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1-(2,4-Difluoro-phenyl)-3-(2-phenoxy-pyridin-3-yl)ureas (11). To these 3ꢀAminoꢀ2ꢀ
phenoxypyridines 9 (0.085 mmol) in 2ꢀdram vials were added 2,4ꢀdifluorophenylisocyanate 10 (0.127
mmol) in anhydrous toluene (1 mL). The reaction mixtures were heated with shaking at 80 °C for 1.5 h.
Solvents were removed by vacuum centrifugation in a speedvac, and the crude products were purified
by a reverse phase preparative HPLC.
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1-(2,4-difluorophenyl)-3-(2-phenoxypyridin-3-yl)urea (11a). Yield: 11.9 mg (35%). H NMR (500
MHz, CD3OD) δ 6.94 (t, J = 8.80 Hz, 1 H) 7.01 (ddd, J = 11.41, 8.66, 3.02 Hz, 1 H) 7.09 (dd, J = 8.25,
4.95 Hz, 1 H) 7.15 (d, J = 8.25 Hz, 2 H) 7.22 (t, J = 7.42 Hz, 1 H) 7.42 (t, J = 7.97 Hz, 2 H) 7.69 (d, J =
4.95 Hz, 1 H) 8.08 (td, J = 9.07, 6.05 Hz, 1 H) 8.61 (d, J = 7.70 Hz, 1 H).
LCꢀMS (ESI) m/z 342.2 (M+H+); Orthogonal HPLC column 1 retention time = 11.05 min, column 2 =
9.47 min.
1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-(2,4-difluorophenyl)urea (11b). Yield: 17.5 mg (44%).
1H NMR (500 MHz, CD3OD) δ 1.40 (s, 9 H) 6.86 (d, J = 8.25 Hz, 1 H) 6.94 (t, J = 8.80 Hz, 1 H) 7.01
(ddd, J = 11.27, 8.52, 3.30 Hz, 1 H) 7.06 (dd, J = 7.97, 4.67 Hz, 1 H) 7.13 ꢀ 7.19 (m, 1 H) 7.20 ꢀ 7.24
(m, 1 H) 7.48 (d, J = 7.70 Hz, 1 H) 7.68 (d, J = 4.95 Hz, 1 H) 8.07 (td, J = 9.07, 5.50 Hz, 1 H) 8.56 (d, J
= 7.70 Hz, 1 H)
LCꢀMS (ESI) m/z 398.2 (M+H+); Orthogonal HPLC column 1 retention time = 12.78 min, column 2 =
10.66 min.
1-(2-(3-tert-butylphenoxy)pyridin-3-yl)-3-(2,4-difluorophenyl)urea (11c). Yield: 15.9 mg (40%).
1H NMR (500 MHz, CD3OD) δ 1.33 (s, 9 H) 6.91 ꢀ 6.96 (m, 2 H) 7.01 (ddd, J = 11.27, 8.52, 3.30 Hz, 1
H) 7.08 (dd, J = 7.70, 4.95 Hz, 1 H) 7.19 (s, 1 H) 7.26 ꢀ 7.30 (m, 1 H) 7.34 (t, J = 7.97 Hz, 1 H) 7.69 (d,
J = 3.30 Hz, 1 H) 8.08 (td, J = 9.21, 5.77 Hz, 1 H) 8.61 (d, J = 8.25 Hz, 1 H).
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Journal of Medicinal Chemistry
LCꢀMS (ESI) m/z 398.2 (M+H+); Orthogonal HPLC column 1 retention time = 13.03 min, column 2 =
10.92 min.
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1-(2,4-difluorophenyl)-3-(2-(2-isopropylphenoxy)pyridin-3-yl)urea (11d). Yield: 16.8 mg (44%).
1H NMR (500 MHz, CD3OD) δ 1.20 (d, J = 6.60 Hz, 6 H) 3.12 (t, J = 7.15 Hz, 1 H) 6.94 (t, J = 8.52
Hz, 1 H) 6.99 ꢀ 7.07 (m, 3 H) 7.19 ꢀ 7.29 (m, 2 H) 7.41 (dd, J = 5.77, 3.57 Hz, 1 H) 7.62 (d, J = 4.95
Hz, 1 H) 8.09 (td, J = 9.07, 6.05 Hz, 1 H) 8.57 (d, J = 7.70 Hz, 1 H).
LCꢀMS (ESI) m/z 384.2 (M+H+); Orthogonal HPLC column 1 retention time = 12.55 min, column 2 =
10.55 min.
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1-(2,4-difluorophenyl)-3-(2-(2-ethylphenoxy)pyridin-3-yl)urea (11e). Yield: 15.7 mg (42%). H
NMR (500 MHz, CD3OD) δ 1.17 (t, J = 7.42 Hz, 3 H) 2.59 (q, J = 7.70 Hz, 2 H) 6.94 (t, J = 8.80 Hz, 1
H) 6.99 ꢀ 7.09 (m, 3 H) 7.16 ꢀ 7.28 (m, 2 H) 7.34 (d, J = 7.70 Hz, 1 H) 7.62 (d, J = 4.95 Hz, 1 H) 8.09
(td, J = 9.07, 6.05 Hz, 1 H) 8.58 (d, J = 7.70 Hz, 1 H).
LCꢀMS (ESI) m/z 370.2 (M+H+); Orthogonal HPLC column 1 retention time = 12.10 min, column 2 =
10.22 min.
1-(2,4-difluorophenyl)-3-(2-(2-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (11f). Yield: 13.9 mg
(34%). 1H NMR (400MHz, CD3OD) δ 6.99 ꢀ 6.91 (m, 1H), 6.99 ꢀ 6.91 (m, 1H), 7.03 (s, 1H), 7.11 (dd,
J = 8.00, 4.90 Hz, 1H), 7.27 (d, J = 8.30 Hz, 1H), 7.44 (s, 1H), 7.74 ꢀ 7.65 (m, 2H), 7.79 (d, J = 7.80 Hz,
1H), 8.09 (d, J = 5.80 Hz, 1H), 8.61 (dd, J = 8.10, 1.80 Hz, 1H).
LCꢀMS (ESI) m/z 410.1 (M+H+); Orthogonal HPLC column 1 retention time = 12.19 min, column 2 =
10.36 min.
1-(2,4-difluorophenyl)-3-(2-(4-(trifluoromethyl)phenoxy)pyridin-3-yl)urea (11g). Yield: 13.9 mg
1
(34%). H NMR (500 MHz, CD3OD) δ 6.94 (t, J = 8.80 Hz, 1 H) 6.98 ꢀ 7.05 (m, 1 H) 7.15 (dd, J =
8.25, 4.95 Hz, 1 H) 7.34 (d, J = 8.80 Hz, 2 H) 7.71 ꢀ 7.77 (m, 3 H) 8.08 (td, J = 9.07, 6.05 Hz, 1 H) 8.65
(d, J = 7.70 Hz, 1 H).
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LCꢀMS (ESI) m/z 410.0 (M+H+); Orthogonal HPLC column 1 retention time = 12.46 min, column 2 =
10.81 min.
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1-(2, 4-difluorophenyl)-3-(2-(4-(methyl)phenoxy)pyridin-3-yl)urea (11h). Yield: 15.3 mg (43%).
1H NMR (400 MHz, CD3OD) δ 2.36 (s, 3 H) 6.90 ꢀ 6.97 (m, 1 H) 6.98 ꢀ 7.04 (m, 3 H) 7.06 (dd, J =
8.25, 4.95 Hz, 1 H) 7.23 (d, J = 8.25 Hz, 2 H) 7.66 (d, J = 4.95 Hz, 1 H) 8.08 (td, J = 9.21, 5.77 Hz, 1
H) 8.59 (d, J = 6.05 Hz, 1 H).
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LCꢀMS (ESI) m/z 356.2 (M+H+); Orthogonal HPLC column 1 retention time = 11.67 min, column 2 =
9.91 min.
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1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-p-tolylurea (12). Yield: 8.6 mg (23%). H NMR (500
MHz, CD3OD) δ 1.39 (s, 9 H) 2.29 (s, 3 H) 6.86 (d, J = 8.25 Hz, 1 H) 7.06 (dd, J = 8.25, 4.95 Hz, 1 H)
7.11 (d, J = 8.80 Hz, 2 H) 7.14 ꢀ 7.19 (m, 1 H) 7.20 ꢀ 7.24 (m, 1 H) 7.32 (d, J = 8.25 Hz, 2 H) 7.48 (d, J
= 7.70 Hz, 1 H) 7.66 (d, J = 4.95 Hz, 1 H) 8.56 (d, J = 6.60 Hz, 1 H).
LCꢀMS (ESI) m/z 376.2 (M+H+); Orthogonal HPLC column 1 retention time = 12.82 min, column 2 =
10.75 min.
Synthesis of 1-(2-(2-tert-butylphenylthio)pyridin-3-yl)-3-p-tolylurea (13). To a solution of the 2ꢀ
chloroꢀ3ꢀnitropyridine 3 (158 mg, 1.0 mmol) and 2ꢀ(tertꢀbutyl)benzenethiol (166 mg, 1.0 mmol) in
DMF (3 mL) was added Cs2CO3 (383 mg, 1.2 mmol). The resulting mixture was stirred at 80 °C
overnight. The insoluble material was removed by filtration, the DMF solution was diluted with H2O (5
mL) and extracted with EtOAc (2 × 6 mL). The EtOAc solution was washed with 5% aq LiCl solution
(3 × 3 mL). Drying (MgSO4) and removal of solvent in vacuo afforded a yellow solid which was
triturated with MeOH to yield 2ꢀ((2ꢀ(tertꢀbutyl)phenyl)thio)ꢀ3ꢀnitropyridine as a yellow solid (115 mg,
40%). HPLC purity: 100%. This material was used for the next step without further purification.
To a solution of 2ꢀ((2ꢀ(tertꢀbutyl)phenyl)thio)ꢀ3ꢀnitropyridine (50 mg, 0.173 mmol) in 1:1
MeOH/EtOAc (3 mL) was added 10% palladium on activated charcoal (5 mg, 0.0047 mmol). The
resulting mixture was stirred at 3.5 atm hydrogen pressure for 1 h. The catalyst was removed by
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Journal of Medicinal Chemistry
filtration through a bed of celite and the solvent was removed by vacuum centrifugation in a speedvac to
afford 2ꢀ((2ꢀ(tertꢀbutyl)phenyl)thio)pyridinꢀ3ꢀamine as a yellow solid (33 mg, 74%). (M+H+) = 259.1.
To a solution of 2ꢀ((2ꢀ(tertꢀbutyl)phenyl)thio)pyridinꢀ3ꢀamine (33 mg, 0.12 mmol) in anhydrous THF (2
mL) was added pꢀtolylisocyanate (20 ꢁL, 0.15 mmol). The resulting mixture was stirred at 60 °C for
1.5 h. The solvent was removed by vacuum centrifugation in a speedvac and the crude mixture was
purified by a reverse phase preparative HPLC. After removal of solvent in vacuo the TFA salt of
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compound 13 was obtained as a yellow solid (12.5 mg, 27%). H NMR (500 MHz, CDCl3) δ 1.40 (s, 9
H) 2.28 (s, 3 H) 6.97 (d, J = 7.70 Hz, 1 H) 7.05 (d, J = 7.70 Hz, 2 H) 7.10 (t, J = 7.42 Hz, 1 H) 7.20 (d, J
= 7.70 Hz, 2 H) 7.25 ꢀ 7.37 (m, 2 H) 7.49 (d, J = 7.70 Hz, 1 H) 8.15 (d, J = 3.85 Hz, 1 H) 8.74 (d, J =
8.25 Hz, 1 H).
LCꢀMS (ESI) m/z 392.4 (M+H+); Orthogonal HPLC column 1 retention time = 13.12 min, column 2 =
10.95 min.
1-(2-(2-tert-butylphenylamino)pyridin-3-yl)-3-p-tolylurea (14). To a solution of 2ꢀchloroꢀ3ꢀ
nitropyridine 3 (1.58 g, 10 mmol) and 2ꢀ(tertꢀbutyl)aniline (1.79g, 12 mmol) in dioxane (50 mL) was
added NEt3 (1.21 g, 12 mmol). The resulting solution was reflux for 16 h, and solvent was evaporated
in vacuo to afford a yellow solid which was recrystallized from MeOH to give Nꢀ(2ꢀ(tertꢀbutyl)phenyl)ꢀ
3ꢀnitropyridinꢀ2ꢀamine (1.98 g, 73%) as a yellow solid. (M+H+) = 272.1.
The Nꢀ(2ꢀ(tertꢀbutyl)phenyl)ꢀ3ꢀnitropyridinꢀ2ꢀamine (272 mg, 1 mmol) was then dissolved in a 4:1
mixed solution containing absolute EtOH (4 mL) and EtOAc (1 mL). Zn dust (1.3 g, 20 mmol) and
NH4Cl (300 mg, 5.4 mmol) were added to this solution sequentially. The resulting mixture was stirred at
room temperature overnight. The solution was filtered through a bed of celite and the solvent was
evaporated in vacuo to afford the N2ꢀ(2ꢀ(tertꢀbutyl)phenyl)pyridineꢀ2,3ꢀdiamine (242 mg, 100%) as
solid. (M+H+) = 242.1. This material was used for the next step without further purification.
To a solution of N2ꢀ(2ꢀ(tertꢀbutyl)phenyl)pyridineꢀ2,3ꢀdiamine (24.2 mg, 0.1 mmol) in anhydrous THF
(1 mL) in a 1ꢀdram vial was added pꢀtolylisocyanate (20 ꢁL, 0.15 mmol). The resulting mixture was
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stirred at 60 °C for 1.5 h. The solvent was removed by vacuum centrifugation in a speedvac and the
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crude mixture was purified by a reverse phase preparative HPLC. After removal of solvent in vacuo the
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TFA salt of compound 14 was obtained as a white solid (15.3 mg, 41%). H NMR (500 MHz, CDCl3) δ
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1.26 (s, 9 H) 2.27 (s, 3 H) 6.70 ꢀ 6.76 (m, 1 H) 6.95 (d, J = 7.70 Hz, 1 H) 7.04 (d, J = 8.25 Hz, 2 H) 7.14
ꢀ 7.22 (m, 2 H) 7.24 ꢀ 7.30 (m, 1 H) 7.35 (d, J = 8.25 Hz, 2 H) 7.48 (d, J = 7.70 Hz, 1 H) 8.47 (d, J =
7.70 Hz, 1 H) 9.08 (br. s, 1 H) 9.94 (d, J = 6.60 Hz, 1 H) 10.05 (br. s, 1 H).
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LCꢀMS (ESI) m/z 375.2 (M+H+); Orthogonal HPLC column 1 retention time = 7.13 min, column 2 =
7.95 min.
1-(2-((2-tert-butylphenyl)(methyl)amino)pyridin-3-yl)-3-p-tolylurea (15). To a solution of Nꢀ(2ꢀ
(tertꢀbutyl)phenyl)ꢀ3ꢀnitropyridinꢀ2ꢀamine (816 mg, 3 mmol) in anhydrous THF (6 mL) was added 60%
NaH (360 mg, 9 mmol) followed by iodomethane (2.56 g, 18 mmol). After stirring at 60 oC for 24 h an
additional 3 equivalent of iodomethane (1.28 g, 9 mmo) was added. Stirring continued at 60 oC for an
additional 48 h. Reaction mixture was allowed to cool to room temperature, and solvent was evaporated
to afford a crude mixture which was purified by a reverse phase preparative HPLC. Removal of solvent
afforded a yellow oil which was dissolved in EtOAc (20 mL) and washed with NaHCO3. Drying
(MgSO4) and removal of solvent afforded the Nꢀ(2ꢀ(tertꢀbutyl)phenyl)ꢀNꢀmethylꢀ3ꢀnitropyridinꢀ2ꢀ
amine (427 mg, 50%) as a light yellow oil. (M+H+) = 286.1.
To a solution of Nꢀ(2ꢀ(tertꢀbutyl)phenyl)ꢀNꢀmethylꢀ3ꢀnitropyridinꢀ2ꢀamine (427 mg, 1.5 mmol) in
EtOH (10 mL) was added 10% palladium on activated charcoal (50 mg, 0.047 mmol). The resulting
solution was stirred under 1 atm hydrogen pressure for 48 h. The solution was filtered through a bed of
celite and the solvent was evaporated in vacuo to afford the N2ꢀ(2ꢀ(tertꢀbutyl)phenyl)ꢀN2ꢀ
methylpyridineꢀ2,3ꢀdiamine (267 mg, 70%) as a dark oil. (M+H+) = 256.1. This material was used for
the next step without further purification.
To a solution of N2ꢀ(2ꢀ(tertꢀbutyl)phenyl)ꢀN2ꢀmethylpyridineꢀ2,3ꢀdiamine (25.5 mg, 0.1 mmol) in
anhydrous THF (1 mL) in a 1ꢀdram vial was added pꢀtolylisocyanate (20 ꢁL, 0.15 mmol). The resulting
mixture was stirred at 60 °C for 1.5 h. The solvent was removed by vacuum centrifugation in a speedvac
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Journal of Medicinal Chemistry
and the crude mixture was purified by a reverse phase preparative HPLC. After removal of solvent in
vacuo TFA salt of compound 15 was obtained as a white solid (8.9 mg, 23%).
1H NMR (500 MHz, DMSOꢀd6) δ 1.28 (s, 9 H) 2.21 (s, 3 H) 3.19 (s, 3 H) 6.80 (dd, J = 7.70, 4.95 Hz, 1
H) 6.96 ꢀ 7.07 (m, 3 H) 7.12 ꢀ 7.23 (m, 4 H) 7.40 ꢀ 7.52 (m, 2 H) 7.98 (d, J = 4.40 Hz, 1 H) 8.59 (br. s, 1
H).
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LCꢀMS (ESI) m/z 389.3 (M+H+); Orthogonal HPLC column 1 retention time = 7.43 min, column 2 =
8.15 min.
1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-(4-(trifluoromethoxy)phenyl)urea (16). Yield: 15.1 mg
1
(34%). H NMR (500 MHz, CD3OD) δ 1.41 (s, 9 H) 7.05 ꢀ 7.12 (m, 1 H) 7.21 (d, J = 8.80 Hz, 2 H)
7.30 ꢀ 7.37 (m, 2 H) 7.43 (dd, J = 8.25, 5.50 Hz, 1 H) 7.54 ꢀ 7.66 (m, 3 H) 7.81 (d, J = 5.50 Hz, 1 H)
9.06 (d, J = 8.25 Hz, 1 H). 13C NMR (125 MHz, CD3OD) δ 29.81, 34.26, 118.68, 119.37, 119.80,
121.42, 121.90, 123.03, 124.64, 125.14, 126.96, 127.21, 127.81, 138.21, 139.10, 141.63, 144.14,
144.16, 152.82, 153.36, 153.42.
LCꢀMS (ESI) m/z 446.1 (M+H+); Orthogonal HPLC column 1 retention time = 12.37 min, column 2 =
12.78 min.
1
1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-(4-tert-butylphenyl)urea (17). Yield: 19.2 mg (46%). H
NMR (500 MHz, CD3OD) δ 1.30 (s, 9 H) 1.40 (s, 9 H) 6.91 (d, J = 7.70 Hz, 1 H) 7.14 (dd, J = 7.97,
5.22 Hz, 1 H) 7.18 ꢀ 7.22 (m, 1 H) 7.25 (t, J = 7.70 Hz, 1 H) 7.32 ꢀ 7.39 (m, 4 H) 7.51 (d, J = 7.70 Hz, 1
H) 7.69 (d, J = 5.50 Hz, 1 H) 8.68 (d, J = 7.70 Hz, 1 H).
LCꢀMS (ESI) m/z 418.2 (M+H+); Orthogonal HPLC column 1 retention time = 12.68 min, column 2 =
11.02 min.
1
1-(4-tert-butylphenyl)-3-(2-(2-isopropylphenoxy)pyridin-3-yl)urea (18). Yield: 25.4 mg (63%). H
NMR (500 MHz, CD3OD) δ 1.20 (d, J = 6.05 Hz, 6 H) 1.31 (s, 9 H) 3.06 ꢀ 3.17 (m, 1 H) 6.97 ꢀ 7.01 (m,
1 H) 7.03 (dd, J = 7.70, 4.95 Hz, 1 H) 7.24 (dd, J = 5.50, 3.30 Hz, 2 H) 7.32 ꢀ 7.39 (m, 4 H) 7.40 ꢀ 7.44
(m, 1 H) 7.60 (d, J = 4.95 Hz, 1 H) 8.57 (d, J = 7.70 Hz, 1 H).
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LCꢀMS (ESI) m/z 404.2 (M+H+); Orthogonal HPLC column 1 retention time = 13.52 min, column 2 =
12.00 min.
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1-(2-(2-isopropylphenoxy)pyridin-3-yl)-3-(4-(trifluoromethoxy)phenyl)urea (19). Yield: 24.1 mg
(57%). 1H NMR (500MHz, CDCl3) δ 1.25 ꢀ 1.16 (m, 6H), 3.07 (dt, J = 13.60, 6.70 Hz, 1H), 7.05 (d, J
= 8.20 Hz, 1H), 7.15 (s, 2H), 7.36 ꢀ 7.22 (m, 3H), 7.42 (d, J = 7.70 Hz, 1H), 7.55 (d, J = 3.30 Hz, 2H),
7.74 (d, J = 4.90 Hz, 1H), 9.11 (dd, J = 14.80, 8.20 Hz, 1H).
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LCꢀMS (ESI) m/z 424.3 (M+H+); Orthogonal HPLC column 1 retention time = 12.32 min, column 2 =
12.73 min.
P2Y1 binding assay
The P2Y1 binding assay was used to identify inhibitors of [βꢀ33P]ꢀ2MeSꢀADP binding to cloned human
P2Y1 receptors. The cDNA clone for human P2Y1 was obtained from Incyte Pharmaceuticals and its
sequence confirmed by established techniques (for a compendium of techniques used see Ausubel, F. et
al. Current Protocols in Molecular Biology, John Wiley and Sons, NY, NY (1995)). The essential
coding sequences were subcloned into pCDNA 3.1 (Invitrogen) to produce a P2Y1 expression construct.
This construct was then transfected into the human embryonic kidney cell line HEKꢀ293 and stable
transfectants selected in GENETICIN® (G418 sulfate; Life Technologies). Several lines were screened
for binding activity and one (HEK293 #49) selected for further characterization. Membranes were
prepared by growing HEK293 #49 in 150 mm dishes in DMEM/10% FBS in the presence of 1mg/mL
G418 until cells were 80ꢀ90% confluent. Plates were then washed with cold (4 ºC) DꢀPBS twice and
cells harvested by scraping into 10 mL DꢀPBS. Cells were pelleted by centrifugation (1,000 g, 10 min, 4
ºC) and the resulting pellet resuspended in Lysis Buffer (10 mM Tris (7.4), 5 mM MgCl2 containing
Complete protease inhibitor cocktail (Roche Cat #1873580). The suspension was then homogenized in a
Dounce homogenizer (10ꢀ15 strokes; B pestle, on ice) and the homogenate spun at 1,000 g, 4 ºC, 5 min
to pellet large debris. The supernatant was centrifuged at 150,000 g, 4 ºC, for 1 hour and the resulting
membrane pellet resuspended in 0.5ꢀ1 mL of Buffer B (15 mM HEPES (7.4), 145 mM NaCl, 0.1 mM
MgCl2, 5 mM EDTA, 5 mM KCl) and stored at ꢀ70 ºC until used. A typical procedure which was
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Journal of Medicinal Chemistry
adequate for solubilizing all urea compounds consisted of adding a 10 ꢀL aliquot of DMSO containing
the test compound diluted to the final assay concentrations of 0.01 nMꢀ1 mM in the bottom of a 96ꢀwell
WGA Flashplate® (NEN) and adding 190 ꢀL of Assay Buffer containing sufficient [βꢀ33P]ꢀ2MeSꢀADP
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and membrane preparation to produce final concentrations of 0.5 nM and 0.025 ꢀg/ꢀL respectively.
Binding reactions were allowed to proceed to completion at room temperature for 1 hour and then the
aqueous solution aspirated. Plates were sealed and the residual [βꢀ33P] bound to the plate determined by
scintillation counting. Doseꢀresponse curves (IC50) were fit by nonꢀlinear regression (XLFit, ID
Business Solutions Ltd.) and binding constants (Ki) calculated using the ChengꢀPrusoff relationship (Ki
= IC50/(1+L/Kd) in which a Kd for 2MeSꢀADP to the P2Y1 receptor was determined to be 14 nM).
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P2Y12 Binding Assay
The P2Y12 cell binding assay is designed for testing compound selectivity. The cDNA of P2Y12 was
obtained from Incyte Pharmaceticals its sequence confirmed by established technique. The essential
coding sequences were subcloned into pCDNA 3.1 (Invitrogen) to produce a P2Y12 expression
construct. This construct was then transfected into the human embryonic kidney cell line HEKꢀ293 and
transfected cells selected in Hygromycin B (Invitrogen Cat# 10687ꢀ010).
Plated 50K/well HEK293ꢀP2Y12 cells in 50 ꢁg/mL polyꢀDꢀlysin preꢀcoated 96ꢀwell assay plate (Costar
Cat# 3917) with selective medium (0.5 mg/ml Hygromycin, 10% FBS, DMEM) over night.
Aspirated cell culture medium and wash cells with 100 ꢀL of serumꢀfree DMEM once. Then added 98
ꢀL of serumꢀfree DMEM, 2 ꢀL of compound solution, 100 ꢀL of 1 nM [βꢀ33P]ꢀ2MeSꢀADP per well.
Incubated assay plate at 5% CO2, 37 ºC for 1 hour. In the end of incubation, aspirated aqueous medium
and washed the cells with 200 ꢀL of DPBS twice. Dried assay plate by inverting it on paper towel, then
added 100 ꢀL of scintillation fluid (Ultima Gold, PerkinꢀElmer Cat# 603329) per well and shaked the
assay plate for15 minutes. [βꢀ33P] bound to the cells was determined by scintillation counting in
TopCount. Doseꢀresponse curves (IC50) were fit by nonꢀlinear regression (XLFit, ID Business Solutions
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