Discovery and synthesis of 6,7,8,9-tetrahydro-5H-pyrimido-[4,5-d]azepines as novel TRPV1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.09.023

Utilization of a tetrahydro-pyrimdoazepine core as a bioisosteric replacement for a piperazine-urea resulted in the discovery a novel series of potent antagonists of TRPV1. The tetrahydro-pyrimdoazepines have been identified as having good in vitro and in

Full text of DOI:10.1016/j.bmcl.2010.09.023

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7137–7141
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and synthesis of 6,7,8,9-tetrahydro-5H-pyrimido-[4,5-d]azepines as
novel TRPV1 antagonists
⇑
Natalie A. Hawryluk , Jeffrey E. Merit, Alec D. Lebsack, Bryan J. Branstetter, Michael D. Hack,
Nadia Swanson, Hong Ao, Michael P. Maher, Anindya Bhattacharya, Qi Wang, Jamie M. Freedman,
Brian P. Scott, Alan D. Wickenden, Sandra R. Chaplan, J. Guy Breitenbucher
Johnson & Johnson Pharmaceutical Research and Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2010
Revised 3 September 2010
Accepted 7 September 2010
Available online 17 September 2010
Utilization of a tetrahydro-pyrimdoazepine core as a bioisosteric replacement for a piperazine-urea
resulted in the discovery a novel series of potent antagonists of TRPV1. The tetrahydro-pyrimdoazepines
have been identified as having good in vitro and in vivo potency and acceptable physical properties.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
TRPV1
Tetrahydro-pyrimdoazepine
Vanilloid
The transient receptor potential vanilloid subtype I (TRPV1 or
VR1) receptor is a ligand-gated cation channel, that is, activated
or modulated by a variety of mediators, such as capsaicin, noxious
heat, low pH, polyamines and lipids such as endogenous ananda-
mide.1 In addition, various endogenous substances such as brady-
kinin, substance P, glutamate, prostaglandins and ATP sensitize
TRPV1.2 As an integrator and mediator of nociceptive and/or
inflammatory stimuli, TRPV1 is an attractive therapeutic target
for the treatment of various neuro-inflammatory disorders. Recent
reviews on the discovery of TRPV1 antagonists highlight efforts to
develop novel therapeutics in this area.3
Tetrahydro-pyridopyrimidines were prepared from condensa-
tion of commercially available b-ketoesters 4 with various amidines
providing pyrimidinol 5 (Scheme 1). Hydrogenolysis of the benzyl
group followed by SN-aryl displacement under microwave
conditions with 2-fluoro-3-trifluoromethyl-pyridine provided the
pyridinyl-tetrahydro-pyridopyrimidinol 8. The pyrimidinol was
chlorinated using POCl3. The desired tetrahydro-pyridopyrimidines
11–16 were prepared via SN-aryl displacement with a variety of aryl
amines.
Utilizing the previously disclosed SAR from the piperazine-urea
series,4 the 3-trifluoromethyl-pyridine was chosen as the preferred
We previously characterized a series of piperazine-ureas as po-
tent TRPV1 antagonists.4 From that series, JNJ-17203212 (hTRPV1
IC50 = 160 nM) was identified as a lead compound, showing good
in vitro activity, PK properties, and in vivo efficacy. In an effort to de-
velop a new series of compoundsas well as improve the in vivo prop-
erties of JNJ-17203212, an analogous scaffold to the piperazine-urea
series was developed. We envisioned that we could use a fused
pyrimidine ring to mimic the urea moiety of JNJ-17203212, leading
to a novel series of TRPV1 antagonists. A flexible molecular align-
ment of the new fused-ring scaffold using a low energy conforma-
tion search demonstrated that it overlaid well with JNJ-17203212
(Fig. 1).5 It was suggested that the newly formed tetrahydro-pyrido-
pyrimidine core would serve well as a bioisosteric replacement for
the piperazine-urea.6
CF3
CF3
N
N
O
CF3
N
H
CF3
N
N
H
N
N
N
N
N
JNJ-17203212 (1)
hTRPV1 IC50 = 160 170 nM
2
⇑
Corresponding author. Tel.: +1 8587843035.
E-mail address: nhawrylu@its.jnj.com (N.A. Hawryluk).
Figure 1. JNJ-17203212-aqua, compound 2-green.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.023

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N. A. Hawryluk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7137–7141
R1
R1 = H, CH3, iPr
N
N
N
N
N
H2N
O
NH
R1
3
R1
N
H
N
H
N
F
7
+
N
N
7
O
N
N
N
N
6
6
CF3
CF3
(a)
(b)
7
OEt
OH
CF3
N
OH
R1
N
N
HN
(c)
4
5
6
Bn
Bn
(d)
18
hTRPV1 IC50 = >10 µM
13
hTRPV1 IC50 = 9 5 nM
R1
R1
N
N
N
N
N
N
R2
(e)
OH
Cl
N
H
N
N
N
N
N
N
CF3
8
9
10
CF3
CF3
Scheme 1. Reagents and conditions: (a) NaOEt, EtOH, reflux, 75%; (b) Pd(OH)2,
1,4-cyclohexadiene, EtOH, 86%; (c) t-amyl-OH, 180 °C W, 5 h, 60%; (d) POCl3,
l
CH3CN, reflux, 70%; (e) ArNH2, n-BuOH, 120 °C, 2 h, 60–90%.
Figure 2. Compound 18-magenta, JNJ-17203212-aqua.
substituent on the piperidine ring nitrogen. The tetrahydro-pyrido-
pyrimidine SAR paralleled the SAR from the piperazine-urea series
in showing a preference for para-substitution on the aryl amine por-
tion (R2). Additionally, it was demonstrated that substitution at the
2-position on the pyrimidine 16–17 was tolerated (Table 1), which
added an additional point of diversity as compared to the urea
series.7
Movement of the nitrogen in the piperidine ring from the 7-po-
sition to the 6-position resulted in a loss of potency. The lack of
potency suggested a conformational distinction between the two
regio-isomers. Molecular modeling provided a rationale for this
hypothesis by showing poor overlap of 18 with piperazine-urea
JNJ-17203212 (Fig. 2).
N
N
N
H
N
8
N
7
CF3
hTRPV1 IC50 = 2040 1180 nM
19
N
N
N
H
N
N
N
N
N
H
CF3
hTRPV1 IC50 = 9 5 nM
13
8
N
7
N
We were concerned that the tetrahydro-pyridopyrimidine core
could undergo oxidation, resulting in potentially toxic pyridinium
CF3
20
hTRPV1 IC = 25 10 nM
50
Table 1
In vitro FLIPR data for recombinant human TRPV1 activated by capsaicin8
R1
N
N
R2
N
H
N
N
Figure 3. Compound 13-green, compound 19-blue.
CF3
Compound
R1
H
R2
hTRPV1 IC50 (nM)a
15 09
CF3
CF3
11
12
13
H
H
1450 605
9
5
Figure 4. Compound 13-green, compound 20-orange.
14
15
16
17
H
520 355
formation.9 To circumvent this issue, we chose to evaluate the tet-
rahydro-pyrimidoazepine scaffold as well. In the tetrahydro-pyri-
dopyrimidine scaffold, a loss of activity was observed when the
position of the piperidine nitrogen was moved from the 7-position
13 to the 6-position 18. It was unclear which azepine, the 8-posi-
tion 19 or the 7-position 20, would be most analogous to the 7-po-
sition tetrahydro-pyridopyrimidine 13; therefore, both were
synthesized. Based on the data from pyridopyrimidines 13 and
18 our expectation was that the 8-position azepine would be the
more potent isomer. Unexpectedly, in vitro data showed that the
H
>20,000
CH3
i-Pr
80 28
18
4
a
Values are means of three experiments SEM.

N. A. Hawryluk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7137–7141
7139
R1
7-position azepine 20 was the more potent isomer. This result was
rationalized via molecular alignment. Molecular alignments of 13
together with each of the azepines are shown in Figures 3 and 4.
While the 7-position azepine 20 shows good overlap with 13, the
8-position azepine 19 shows significant deviation. In particular,
the azepine ring is somewhat bulkier than the analogous pipera-
zine ring, and in 19 it occupies a region of space thought be sensi-
tive to steric bulk, based on the SAR described in the piperazine-
urea series.4
R1
R1
H2N
O
NH
O
3
O
N
N
N
N
O
+
N2
OEt
OH
(b)
OH (c)
24
22
(a)
OEt
N
H
N
N
23
25
N
Boc
R1
Boc
Boc
R1
R1
21
N
F
N
N
N
N
N
N
R2
(f, g,
or h)
The tetrahydro-pyrimidoazepine were synthesized in
a
R3
OH
Cl
(e)
N
H
28
straightforward fashion (Scheme 2).10 Ring expansion of 4-N-Boc-
piperidone 21 using ethyl diazoacetate and BF3ÁOEt2 followed by
condensation using various amidines 3 afforded the pyrimidinol
24. Deprotection of the azepine nitrogen and SNAr displacement
under microwave conditions with a variety of 2-chloropyridines
resulted in the appropriate N-substituted azepine 26. Chlorination
of the pyrimidinol with POCl3 provided the chloropyrimidine 27,
which could be displaced with a variety of aryl amines via SNAr dis-
placement or with heteroaryl amines using palladium-mediated
coupling to provide the desired tetrahydro-pyrimidoazepine.
Alternatively, the hydroxy-pyrimidine can be accessed by
installing the pyridine onto the piperidone prior to ring expansion
and condensation (Scheme 3).11
7
(d)
N
N
N
26
27
N
N
N
R3
R3
R3
Scheme 2. Reagents and conditions: (a) BF3ÁOEt2, ether, 0 °C, 1 h 58%; (b) NaOEt,
EtOH, reflux, 24 h, 70%; (c) HCl/dioxane, CH2Cl2, RT, 12 h then PL-HCO3 MP-resin,
MeOH, RT, 30 min., quant.; (d) t-amyl-OH, 180 °C
CH3CN, reflux, 70%; (f) R2NH2, n-BuOH, 120 °C W, 30 min., 60–90%; (g) R2NH2,
TsOH, toluene, 130 °C, 1 h, 50–80%; (h) R2NH2, Pd(OAc)2, DCPB, NaOt-Bu, toluene,
130 °C W, 30 min., 70–85%.
lW, 3 h, 40–60%; (e) POCl3,
l
l
O
N
Utilizing the SAR from our previous work, the 4-CF3-aniline was
chosen as the optimal substituent. Maintaining the 4-CF3-aniline
constant, the 2-position of the pyrimidine was evaluated. It was
shown that the 2-position tolerated a variety of groups, some
showing a substantial increase in potency when compared to the
unsubstituted analog 32 (Table 2). Compounds 38–43 suggested
that a lipophilic basic amine at the 2-position of the pyrimidine
Cl
O
O
(a, b)
CF3
+
N
CF3
N
N
H
Scheme 3. Reagents and conditions: (a) K2CO3, DMSO, 100 °C; (b) conc. HCl, 70%
over two-steps.
Table 3
In vitro FLIPR data for recombinant human TRPV1 activated by capsaicin
Table 2
R1
In vitro FLIPR data for recombinant human TRPV1activated by capsaicin
R1
N
N
2
R2
CF3
N
N
N
H
N
H
N
N
N
CF3
N
CF3
R1 = i-Pr
compound
hTRPV1 IC50
(nM)a
R1
Compound
=
R1
=
R
1
1
N
N
O
R
=
=
Compound
R1
hTRPV1 IC50 (nM)a
R2
Compound
hTRPV1 IC50 (nM)a hTRPV1 IC50 (nM)a
32
33
H
CH3
188 76
97 69
CF3
CF3
45
46
47
34
35
31 13
51 12
130 48
43 13
38
2
N
N
48
70
49
21
ND
5
4
N
36
37 24
CF3
O
50
51
>6670
ND
2460 780
N
37
38
NH2
1058 480
56 37
N
52
180 110
53
361 391
54
56 10
OH
N
39
40
87 58
61 59
N
55
580 76
56
18
57
37 27
CN
7
N
O
S
41
42
6
2
7
OH
58
59
60
N
N
N
19
1450 920
58 31
230 170
O
O
O
O
O
43
44
S
>20,000
S
61
62
25
63
52 27
N
2520 2550
5
NH
3350 351
a
a
Values are means of three experiments SEM. ND, not determined.
Values are means of three experiments SEM.

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N. A. Hawryluk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7137–7141
Table 4
150
Vehicle
In vitro FLIPR data for recombinant human TRPV1 activated by capsaicin
30mg/kg po
R1
CF3
N
N
100
50
0
N
H
N
R3
Compound
R1
H
R3
hTRPV1 IC50 (nM)a
6160 622
N
N
64
CF3
Cl
2
3
4
5
N
N
32
65
66
H
H
H
190 76
907 507
2330 887
Time (hr)
Figure 5. Representative data showing the effect of 41 on carrageenan-induced
thermal hyperalgesia in rats (dosed p.o. in 5% pharmasolve/20% RH-40 cremophor/
75% dextrose (5%) in water, administered 1 h prior to carrageenan injection). %
Maximal possible effect (%MPE).14
O
O
S
N
N
O
O
S
Further exploration of the aniline substituent yielded a limited
set of groups that maintained good activity (Table 3). Initially, good
activity was seen when R1 was an isopropyl group. However, these
compounds had limited oral absorption in vivo due to poor aque-
ous solubility. Introduction of an amine in the 2-position of the
pyrimidine not only resulted in an increase in potency but also pro-
vided an opportunity for improvement in developability via salt
formation.
Analysis of the SAR suggested that substitution in the 3-position
of the pendant pyridine on the azepine was required for activity,
preferably 3-CF3 32. It was proposed that a substituent in the 3-po-
sition imparts an orthogonal arrangement between the pyridine and
the azepine core. This hypothesis may be confirmed with the lack of
activity observed with compound 64 in which a co-planar arrange-
mentis predicted. A variety of compoundscontaining pyridylgroups
with substituents in the 3-position that might be expected to give an
orthogonal arrangement were prepared (Table 4). However, most of
these substituents resulted in decreased activity with respect to
compound 32, indicating that orthogonal geometry was not the only
requirement for activity. Additionally, a combination of substituents
in the 2-position of the pyrimidine as well as di-substitution in the
3- and 5-positions of the pyridine led to active compounds 72, 73.
Introduction of polar substituents in the 5-position of the pyridine
also resulted in compounds with improved solubility (72,
NH2
67
68
69
H
2780 2390
2360 2150
1940 1180
O
H
N
NH2
O
O
S
i-Pr
N
N
Cl
70
71
72
i-Pr
N
4220 1630
449 89
CO2H
O
O
S
NH2
N
N
N
Cl
462 65
CO2H
N
O
Cl
N
73
176 163
CO2CH3
SIF12 = 218
lg/mL) at the expense of hTRPV1 activity.
Upon identifying a novel series of functional antagonists of
TRPV1, compound 41 was chosen for further profiling both
in vitro and in vivo (Table 5).
The rat pharmacokinetic profile of 41 displayed moderate clear-
ance and volume of distribution (Table 6). Limited absorption due
to poor solubility (pH 2 = 5 lg/mL and SIF = 0.5 lg/mL) was ob-
served for 41. Although 41 did not exhibit ideal PK properties, it
was examined in an in vivo model of inflammatory pain, carra-
geenan-induced thermal hyperalgesia13 of the paw. Compound
41 significantly attenuated thermal hyperalgesia, expressed as %
maximal possible effect (% MPE), when dosed orally at 30 mg/kg
(Fig. 5). Despite the low oral bioavailability, the compound concen-
trations were adequate in order to achieve efficacy in vivo. Termi-
nal plasma concentrations, which were taken approximately 5 h
post-carrageenan injection, were determined to be 146 35 nM
for compound 41.
a
Values are means of three experiments SEM.
Table 5
In vitro data for 418
Human TRPV1 (nM)
Rat TRPV1 (nM)
180 80
RTX binding Ki
Capsaicin IC50
pH IC50
14
6
2
2
7
19
4
6
ND
Table 6
Pharmacokinetic profile for 41 (1 mg/kg iv and 5 mg/kg p.o) in fasted Sprague–
Dawley rats
T1/2 (h) Cl (L/h/kg) Vss (L/kg) AUCinf (h ng/mL)
Cmax
(lM) %F
41 1.1 3.1 4.7 322 (iv) 236 (p.o.) 0.160
14
In conclusion, utilizing a pyrimidine-azepine core as a bioisoster-
ic replacement for the piperazine-ureas resulted in the discovery a
novel series of potent antagonists of TRPV1. The tetrahydro-pyr-
imdoazepines have been identified as having good in vitro activity
provided improved activity. Additionally, simple alkyl substituents
33–36 also showed an increase in activity.

N. A. Hawryluk et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7137–7141
7141
6. (a) Zheng, X.; Hodgets, K. J.; Brielmann, H.; Hutchinson, A.; Burkamp, F.; Jones,
A. B.; Blurton, P.; Clarkson, R.; Chandrasekhar, J.; Bakthavatchalam, R.; De
Lombaert, S.; Crandall, M.; Cortright, D.; Blum, C. A. Bioorg. Med. Chem. Lett.
2006, 16, 5217; (b) Blum, C. A.; Caldwell, T.; Zheng, X.; Bakthavatchalam, R.;
Capitosti, S.; Brielmann, H.; De Lombaert, S.; Kershaw, M. T.; Matson, D.;
Krause, J. E.; Cortright, D.; Crandall, M.; Martin, W. J.; Murphy, B. A.; Boyce, S.;
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across species and acceptable physical properties for evaluation in
vivo. The examination of 41 aids in the further evaluation of the ther-
apeutic potential of TRPV1 antagonists.15
Acknowledgment
7. During the course of this work the following patent applications were
published: WO 200,50,66,171 A1, 2005; WO 200,60,62,981 A2, 2006; U.S.
200,51,65,032 A1, 2005.
We are grateful to Michele Rizzolio and Raymond Rynberg for
obtaining the solubility data.
8. For experimental methods see: Bhattacharya, A.; Scott, B. P.; Nasser, N.; Ao, H.;
Maher, M. P.; Dubin, A. E.; Swanson, D. M.; Shankley, N. P.; Wickenden, A. D.;
Chaplan, S. R. J. Pharmacol. Exp. Ther. 2007, 323, 665.
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subcutaneously into the plantar surface of the hind paw. For methods and
references on the thermal hyperalgesia testing see Ref. 4a.
14. The paw withdrawal latency (PWL), in seconds, for each group is expressed as
the mean standard error of the mean (SEM) at each time point. PWL of the
injected paw is compared between the vehicle and the treated group. A % MPE
value was generated by taking the area under curve (AUC) of PWL in seconds
over the time course of the experiment in hours for the drug-treated group and
normalized against the vehicle-treated group. The 100% MPE mark was defined
as the pre-test latencies of each animal multiplied by the duration of the
experiment and the baseline 0% MPE mark was defined as the mean vehicle
AUC. Statistical analysis was performed using two-way ANOVA with
Bonferroni’s multiple comparisons with a significance level of p <0.05.
15. For TRPV1 anatogonists with improved properties see: Lebsack, A. D.; Rech, J.
C.; Branstetter, B. J.; Hawryluk, N. A.; Merit, J. E.; Allison, B.; Rynberg, R.; Buma,
J.; Rizzolio, M.; Swanson, N.; Ao, H.; Maher, M. P.; Herrmann, M.; Freedman, J.
M.; Scott, B. P.; Luo, L.; Bhattacharya, A.; Wang, Q.; Wickenden, A. D.; Chaplan,
S. R.; Breitenbucher, J. G. Bioorg. Med. Chem. Lett 2010, 20, 7142.
5. MOE (the molecular operating environment) version 2006.08, software
available from Chemical Computing Group, 1010 Sherbrooke Street West,
Suite 910, Montreal, Canada H3A 2R7. http://www.chemcomp.com.