1,2-Diamino-ethane-substituted-6,7,8,9-tetrahydro-5H-pyrimido[4,5-d] azepines as TRPV1 antagonists with improved properties

Article abstract of DOI:10.1016/j.bmcl.2010.09.006

Based upon a previously reported lead compound 1, a series of 1,2-diamino-ethane-substituted-6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepines were synthesized and evaluated for improved physiochemical and pharmacokinetic properties while maintaining TRPV1 antagonist activity. Structure-activity relationship studies directed toward improving the aqueous solubility (pH 2 and fasted-state simulated intestinal fluid (SIF)) and rat pharmacokinetics led to the discovery of compound 13. Aqueous solubility of compound 13 (pH 2 = >237 μg/mL and SIF = 11 μg/mL) was significantly improved over compound 1 (pH 2 = 5 μg/mL and SIF = 0.5 μg/mL). In addition, compound 13 afforded improved rat pharmacokinetics (CL = 0.7 L/kg/h) compared to compound 1 (CL = 3.1 L/kg/h). Compound 13 was orally bioavailable and afforded a significant reversal of carrageenan-induced thermal hyperalgesia at 5 and 30 mg/kg in rats.

Full text of DOI:10.1016/j.bmcl.2010.09.006

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7142–7146
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1,2-Diamino-ethane-substituted-6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepines
as TRPV1 antagonists with improved properties
⇑
Alec D. Lebsack , Jason C. Rech, Bryan J. Branstetter, Natalie A. Hawryluk, Jeffrey E. Merit,
Brett Allison, Raymond Rynberg, Johnathan Buma, Michele Rizzolio, Nadia Swanson, Hong Ao,
Michael P. Maher, Michelle Herrmann, Jamie Freedman, Brian P. Scott, Lin Luo, Anindya Bhattacharya,
Qi Wang, Sandra R. Chaplan, Alan D. Wickenden, J. Guy Breitenbucher
Johnson & Johnson Pharmaceutical Research and Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Based upon a previously reported lead compound 1, a series of 1,2-diamino-ethane-substituted-6,7,8,9-
tetrahydro-5H-pyrimido[4,5-d]azepines were synthesized and evaluated for improved physiochemical
and pharmacokinetic properties while maintaining TRPV1 antagonist activity. Structure–activity rela-
tionship studies directed toward improving the aqueous solubility (pH 2 and fasted-state simulated
intestinal fluid (SIF)) and rat pharmacokinetics led to the discovery of compound 13. Aqueous solubility
Received 30 June 2010
Revised 31 August 2010
Accepted 1 September 2010
Available online 7 September 2010
of compound 13 (pH 2 = >237
l
g/mL and SIF = 11
lg/mL) was significantly improved over compound 1
Keywords:
TRPV1
Antagonists
Azepine
(pH 2 = 5 g/mL and SIF = 0.5
l
lg/mL). In addition, compound 13 afforded improved rat pharmacokinetics
(CL = 0.7 L/kg/h) compared to compound 1 (CL = 3.1 L/kg/h). Compound 13 was orally bioavailable and
afforded a significant reversal of carrageenan-induced thermal hyperalgesia at 5 and 30 mg/kg in rats.
Ó 2010 Elsevier Ltd. All rights reserved.
The vanilloid receptor 1 (VR1 or TRPV1) is the best characterized
member of the transient receptor potential family of ion channels.1,2
TRPV1 is a ligand-gated, non-selective cation channel that is primar-
ily expressed in nociceptive C- and Ad fibers. The TRPV1 receptor is
activated by a wide range of stimuli including heat (>43 °C), low
pH, vanilloid ligands such as capsaicin and resiniferatoxin (RTX),
and a wide range of endogenous mediators such as bradykinin and
anandamide.3 In general, activation of the TRPV1 channel results
in depolarization, neuronal hyper-excitability, and ultimately the
sensation of pain.4 TRPV1 knockout mice demonstrate an impaired
ability to develop inflammatory thermal hyperalgesia, suggesting
that TRPV1 has an important role in transmitting inflammatory pain
signals.5 For this reason, efforts to discover small molecule TRPV1
antagonists have received considerable attention from many pain
research groups.6 Despite the abundance of structurally distinct
TRPV1 antagonist scaffolds, two of the challenges facing a number
of molecules reported are their relatively high lipophilicity and cor-
respondingly poor aqueous solubility.6 Recently, several approaches
toward improving the aqueous solubility of TRPV1 antagonists have
been reported.7 Herein, we report our efforts to improve the solubil-
ity of a promising TRPV1 antagonist scaffold.
pound 1 as a potent TRPV1 antagonist.8 During the course of that re-
port, the structure–activity relationships for the 6,7,8,9-tetrahydro-
5H-pyrimido[4,5-d]azepine series were elucidated. These studies
suggested that in order to maintain TRPV1 potency, the 3-trifluoro-
methyl-pyridin-2-yl and 4-trifluoromethylphenyl amine substitu-
ents were preferred, whereas a variety of substituents at the C2-
position of the pyrimidine were tolerated. Unfortunately, further
profiling of compound 1 revealed low aqueous solubility (pH
2 = 5
6.8) = 0.5
l
g/mL and fasted-state simulated intestinal fluid (SIF; pH
l
g/mL) and high clearance in rats (CL = 3.1 L/h/kg).8 To
gain insight into the clearance issue, we conducted in vitro metabo-
lism studies in the presence of both rat and human liver microsomes.
These studies demonstrated that the morpholine ring in compound
1wasoxidizedandcouldpotentiallycontributetothehighclearance
observed in rats. Therefore, our strategy was to replace the morpho-
line ring with substituents that could enhance aqueous solubility
and rat pharmacokinetics while maintaining TRPV1 potency. To that
end, our efforts were directed toward improving aqueous solubility
through the incorporation of 1,2-diamino-ethane analogues at the
C2-position of the pyrimidine (e.g., compound 2) (Fig. 1). We rea-
soned that incorporation of 1,2-diamino-ethane analogues could
provide an additional group for ionization and enhanced polarity.
In addition to the potential improvements in aqueous solubility,
we also hoped these efforts would afford opportunities for salt for-
mation providing greater ease of development.
In a previous report, we described a series of 6,7,8,9-tetrahydro-
5H-pyrimido[4,5-d]azepines that led to the identification of com-
The general synthesis of 1,2-diamino-ethane substituted-
6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepines is outlined in
⇑
Corresponding author.
E-mail address: alebsack@its.jnj.com (A.D. Lebsack).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.006

A. D. Lebsack et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7142–7146
7143
2R3RN
R
N
O
N
O
NHR
R1N
N
2
N
2
N
N
N
CF3
CF3
N
CF3
CF3
N
N
N
N
N
H
N
H
4
a
N
N
H
H
N
N
N
F3C
N
N
N
F3C
N
N
CF3
CF3
1
2
23: R = H
25: R = CH3
22: R = H
24: R = CH3
hTRPV1 IC50 = 6 2 nM
rTRPV1 IC50 = 19 4 nM
Scheme 2. Reagents and conditions: (a) 2 N HCl (aq), reflux, 12 h, 85–89%.
Figure 1. 6,7,8,9-Tetrahydro-5H-pyrimido[4,5-d]azepines 1 and 2.
Scheme 1.8 The synthesis began from commercially available 1,4-
dioxa-8-aza-spiro[4.5]decane (3) and 2-chloro-3-trifluoromethyl-
pyridine (4) which were transformed to piperidin-4-one 5 in two
steps.9 Ring expansion of piperidin-4-one 5 in the presence of dia-
zo-acetic acid ethyl ester and BF3ꢀOEt2 afforded b-keto ester 6 in
92% yield. Condensation of b-keto ester 6 with thiourea followed
by chlorination in the presence of POCl3 provided 4-chloro-2-
methylsulfanyl-pyrimidine 7 in 55% yield over two steps. Amina-
tion of compound 7 by reaction with 4-trifluoromethylphenyl
amine and hydrochloric acid afforded compound 8 in 77% yield.
Installation of the 1,2-diamino-ethane analogues at the C2-posi-
tion of the pyrimidine was accomplished through a two-step oxi-
dation and displacement sequence to furnish 1,2-diamino-ethane
substituted-6,7,8,9-tetrahydro-5H-pyrimido[4,5-d]azepines 9–22,
24, 26–28, 30, 31, 33, 34, 36–38.
Boc
N
H
N
NR1
N
NR1
N
CF3
CF3
N
N
a
N
H
N
H
N
N
F3C
F3C
N
N
28: R1 = H
29
: R1 = H
32: R1 = CH3
31
: R1 = CH3
Scheme 3. Reagents and conditions: (a) 4 N HCl in dioxane, rt, 2 h, 38–62%.
In cases where displacement of the 2-methylsulfonyl substitu-
ent with the desired 1,2-diamino-ethane analogue had the poten-
tial to afford mixtures of regioisomeric products, incorporation of
acetyl or a tert-butoxycarbonyl (Boc) group on the distal nitrogen
suppressed unwanted regioisomers. Removal of acetyl or Boc pro-
tecting groups in compounds 22, 24, 28, and 31 using acidic condi-
Compounds were evaluated for their ability to inhibit capsaicin-
induced influx of Ca2+ in cells (HEK293) expressing human and rat
TRPV1.10 Inhibition is reported as IC50 SEM (nM) and the results
are the average of at least three independent experiments. In addi-
tion, none of the compounds reported herein displayed agonist
activity. Aqueous equilibrium solubility of each compound was
determined in (SIF; pH 6.8) and pH 2 using 30 mM phosphate
buffer.11
tions is summarized in Schemes
2 and 3. Consistent with
conditions shown in Scheme 3, the Boc group in compound 34
was removed to provide compound 35 in 56% yield.
SCH3
O
O
O
N
N
Cl
O
O
OEt
Cl
CF3
c
a, b
F3C
d, e
N
N
F3C
N
N
N
N
F3C
N
H
N
3
4
6
5
7
SCH3
N
NR1R2
N
CF3
CF3
N
N
g, h
f
N
N
H
H
N
N
N
N
F3C
F3C
8
9-22, 24, 26-28, 30-31, 33-34, 36-38
Scheme 1. Reagents and conditions: (a) K2CO3, DMSO, 100 °C, 12 h (quant); (b) concd HCl, 24 h (70%); (c) diazo-acetic acid ethyl ester, BF3ꢀOEt2 (1.3 equiv) Et2O/CH2Cl2, 0 °C
to rt, 3 h (92%); (d) 1.5 equiv thiourea, 3 equiv sodium ethoxide, EtOH, 90 °C, 12 h, followed by 1.1 equiv MeI at rt, 1 h (98%); (e) 2 equiv POCl3, CH3CN, 90 °C (55% over two
steps); (f) 4-trifluoromethyl-phenylamine, 2.2 equiv HCl, IPA, 90 °C (77%); (g) 2 equiv m-CPBA, CH2Cl2, rt (93%); (h) HNR1R2, t-amyl-OH, 120 °C (60–90%).

7144
A. D. Lebsack et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7142–7146
Table 2
As part of our effort to improve aqueous solubility while main-
TRPV1 activity and solubility for compounds 21–38
taining TRPV1 potency, we first evaluated a variety of piperazines
as 1,2-diamino-ethane analogues (Table 1). In general, piperazines
12–18 with bulky alkyl substituents provided excellent human
TRPV1 potency. On the other hand, piperazines 9–11 that con-
tained smaller substituents were at least eightfold less potent at
human TRPV1 when compared to compound 13. The addition of
polar functionality to the substituted piperazine in compounds
19 and 20 also significantly decreased TRPV1 potency. Compounds
shown in Table 1 improved on both pH 2 solubility (>43-fold) and
SIF solubility (>4-fold) when compared to the aqueous solubility of
compound 1. Unfortunately, in the many instances where SIF and
pH 2 solubility were dramatically improved, such as in compounds
9, 10 and 20, TRPV1 potency of each compound was significantly
reduced. A balance was achieved in compounds 12–18 where po-
tent human TRPV1 activity was observed while aqueous solubility
in both pH 2 and SIF was enhanced compared to compound 1.
Having identified several piperazines that improved aqueous
solubility and retained potency for TRPV1, we turned our attention
to the evaluation of other 1,2-diamino-ethane analogues (Table 2).
Replacement of a N-methylated-piperazine in compound 10 with
an N,N,N0-trimethyl-ethane-1,2-diamine in compound 21 signifi-
cantly reduced TRPV1 potency. This result suggested that a more
rigid orientation of the distal nitrogen of the piperazine was impor-
tant for TRPV1 potency. Overall, the structure–activity trends for
compounds 22–38 suggested that while aqueous solubility (pH 2
and SIF) could be improved, the TRPV1 potency could not be main-
tained when compared to compound 1. For example, compounds
23, 25–27, 29, 30, 32, and 35–37 improved aqueous solubility to
a significant extent, but human TRPV1 potency was reduced by
as much as 833-fold for compound 29 when compared to com-
pound 1. In contrast, synthetic intermediates containing tert-
butoxycarbonyl groups such as compounds 28 and 31 afforded
poor aqueous solubility and human TRPV1 IC50’s of 140 and
53 nM, respectively.
R
CF3
N
N
N
H
N
N
CF3
Compounda
R
hTRPV1
rTRPV1
pH 2
solubility solubility
g/mL) g/mL)
SIF
IC50 SEM
(nM)
IC50 SEM
(nM)
(l
(l
N
N
21
2667 367 4449 1444 >221
210
H
N
O
22
23
24
150 62
410 397
202
>215
21
19
N
N
NH2
3700 234 >5000
>215
O
N
62 20
460 341
2
N
N
H
N
N
N
25
2790 35
243 124
>5000
>220
233
57
Compounds 12, 13, and 15 were selected for pharmacokinetic
assessment in Sprague–Dawley rats based on their excellent TRPV1
(R)-26
1042 402 >226
N
N
Table 1
TRPV1 activity and solubility for compounds 9–20
(S)-27
28
185 104
140 99
>5000
586 231
239 15
>5000
>226
57
14
R
N
Boc
N
N
1
CF3
N
N
NH
NH
NH
N
N
H
H
N
N
29
>235
204
1
203
226
4
N
CF3
Me
N
Compound
R
hTRPV1
IC50 SEM IC50 SEM
(nM) (nM)
rTRPV1
pH 2
solubility solubility
g/mL) g/mL)
SIF
30
3080 370 >5000
(
l
(l
9
10
11
12
13
14
15
16
17
18
19
20
–H
–Me
–Et
3350 490 >5000
367 102 506 81
215 158
Boc
N
>221
>226
>232
>237
243
193
6
2
11
109
9
132 92
91 37
506 295
386 279
89 43
185 120
45 11
161 81
123 13
207 107
31
53 39
97
9
–iso-Propyl
–CH2CH(CH3)2
–CH(CH2CH3)2
–Cyclopropyl
–Cyclobutyl
–Cyclopentyl
–CH2CH2OCH3
–CH2CH2OH
11
41 22
11
24 13
15
65 45
6
H
N
5
230
>237
221
>238
2
11
6
32
3600 480 1540 470 >241
211
7
N
498 265 1161 343 >233
4
>243
–CH2CH2N(CH3)2 2000 740 2600 1650 >243

A. D. Lebsack et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7142–7146
7145
Table 2 (continued)
Vehicle
Compounda
R
hTRPV1
IC50 SEM
(nM)
rTRPV1
IC50 SEM
(nM)
pH 2
solubility solubility
g/mL) g/mL)
SIF
13 5 mg/kg
13 30 mg/kg
(l
(l
140
120
100
80
60
40
N
33
34
>5000
>5000
>232
>232
N
NBoc
NH
250 80
230 180
3
3
20
0
-20
2
3
4
5
NH
NH
35
36
37
38
100 34
>5000
>272
237
254
>283
1
Time (h)
Figure 2. Representative data showing the effect of 13 on carrageenan-induced
thermal hyperalgesia in rats, n = 6/group (dosed po in 5% pharmasolve/20% RH-40
cremophor/75% dextrose (5%) in water, administered 1 h prior to carrageenan
injection).
NMe
NH
1756 650 2277 415 >277
displace [3H]-RTX in HEK293 cells expressing rat TRPV1 (Table 4).
Compound 13 blocked proton activation to an extent similar to its
blockade of capsaicin activation.
N
N
3330 1424 >5000
>283
224
When dosed orally, compound 13 was efficacious in a carra-
geenan-induced thermal hyperalgesia model in rats (Fig. 2).12
Compound 13 significantly prevented the development of thermal
hyperalgesia at 5 and 30 mg/kg compared with vehicle (p <0.05).
When expressed as % Maximal Possible Effect (% MPE), the maxi-
mal degree of inhibition was approximately 100% in rats treated
with 30 mg/kg at 3 h post-carrageenan injection. Compound 13
did not affect basal paw withdrawal latency of the contralateral
hind paws. Terminal plasma concentrations (approximately 5 h
post-carrageenan injection) were determined to be 277 68 nM
(n = 6) at 5 mg/kg and 1054 442 nM (n = 4) at 30 mg/kg for com-
pound 13.
In conclusion, we have synthesized and evaluated a series of
1,2-diamino-ethane-substituted-6,7,8,9-tetrahydro-5H-pyrimido
[4,5-d]azepines in an effort to improve the aqueous solubility
and rat pharmacokinetics. We identified compound 13 as a potent
TRPV1 antagonist with improved aqueous solubility (pH
N
476 271
1342 779
NH
a
Compounds 22–25 and 28–37 described in Table 2 were racemates.
Table 3
Mean pharmacokinetic parameters for compounds 12, 13, and 15 following 0.5 mpk
iv and 2 mpk po administration in fasted Sprague–Dawley rats using 5% pharmasolve/
20% RH-40 cremophor/75% dextrose (5%) in water as a vehicle
Compound CL
Vss
T1/2 po Cmax po AUCinf
po
%F
(L/h/kg) (L/kg) (h)
(
l
M) (h ng/mL) Tmax (h)
12
13
15
4.8
0.7
1.3
5.1
0.5
1.0
1.5
1.8
1.6
—
—
598
—
—
2.3
—
—
21
—
0.3
2 = >237
l
g/mL and SIF = 11
lg/mL) compared to compound 1
—
(pH 2 = 5
l
g/mL and SIF = 0.5
lg/mL). In addition, compound 13
provided improved rat pharmacokinetics (CL = 0.7 L/h/kg) com-
pared to compound 1 (CL = 3.1 L/h/kg). Compound 13 was orally
bioavailable and afforded a significant reversal of carrageenan-in-
duced thermal hyperalgesia at 5 and 30 mg/kg in rats.
Table 4
Additional TRPV1 activity for compound 13
Compound hTRPV1(pH)
IC50 SEM (nM)
rTRPV1(pH)
IC50 SEM (nM)
rRTX Binding
Ki SEM (nM)
References and notes
13
67 39
63 42
330 160
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11. The solubility assay was conducted in a 96-well format using DMSO stock
solutions (10 mM of compound). DMSO was evaporated and residual solids
were re-suspended in fasted-state simulated intestinal fluid (SIF; pH 6.8) or pH
2.0 for 3 days. The resulting mixtures were filtered and analyzed by HPLC
against external standards.
12. Under anesthesia, 100 lL of 1% carrageenan (Sigma) in saline was injected
subcutaneously into the plantar surface of the hind paw. For methods and
references on the thermal hyperalgesia testing see Ref. 6d.