Discovery of novel pyrrolopyridazine scaffolds as transient receptor potential vanilloid (TRPV1) antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.09.059

A novel indolizine class of compounds was identified as TRPV1 antagonist from an HTS campaign. However, this indolizine class proved to be unstable and reacted readily with glutathione when exposed to light and oxygen. Reactivity was reduced by the introduction of a nitrogen atom alpha to the indolizine nitrogen. The pyrrolopyridazine core obtained proved to be inert to the action of light and oxygen. The synthesis route followed the one used for the indolizine compounds, and the potency and ADMET profile proved to be similar.

Full text of DOI:10.1016/j.bmcl.2012.09.059

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6888–6895
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel pyrrolopyridazine scaffolds as transient receptor potential
vanilloid (TRPV1) antagonists
Ismet Dorange a, , Rickard Forsblom a, Istvan Macsari a, Mats Svensson a, Johan Bylund b,
⇑
Yevgeni Besidski a, Jan Blid a, Daniel Sohn a, Ylva Gravenfors a
a Medicinal Chemistry, CNSP iMed Science, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden
b DMPK, CNSP iMed Science, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel indolizine class of compounds was identified as TRPV1 antagonist from an HTS campaign. How-
ever, this indolizine class proved to be unstable and reacted readily with glutathione when exposed to
light and oxygen. Reactivity was reduced by the introduction of a nitrogen atom alpha to the indolizine
nitrogen. The pyrrolopyridazine core obtained proved to be inert to the action of light and oxygen. The
synthesis route followed the one used for the indolizine compounds, and the potency and ADMET profile
proved to be similar.
Received 23 August 2012
Revised 6 September 2012
Accepted 10 September 2012
Available online 25 September 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Vanilloid receptor
Ligand-gated ion channel
TRPV1
Pain
Indolizine
Pyrrolopyridazine
Transient receptor potential vanilloid 1 (TRPV1)1 is a non-
selective ligand-gated cation channel which upon activation by a
range of stimuli such as heat,2 acid,3 voltage,4 exogenous capsai-
cin5 and inflammatory mediators such as anandamine6 and lipoxy-
genase products7 allows the flow of sodium and calcium ions
through the nerve cell. The increased intracellular Ca2+ level causes
an excitation of the primary sensory neurons and ultimately leads
to the central perception of pain. A TRPV1 knockout mice model
provided evidence that thermal hyperalgesia depends on TRPV1
activation and sensitization, making this receptor a very attractive
target for the treatment of chronic and neuropathic pain.8–11
The pharmaceutical industry responded by undertaking signifi-
cant efforts in finding small molecule TRPV1 antagonist acting as
analgesics and several compounds entered clinical trials.12–15
Further successful development of these compounds was unfortu-
nately hindered by diverse adverse effects.12–19 Perhaps the most
troublesome is the indication that the TRPV1 channel blockade
can lead to hyperthermia as well as an impaired noxious heat
perception; in other words that this ion channel plays a role in
the body temperature regulation and in setting the heat pain
threshold.20,21 It is however important to put these findings into
perspective and to realize that the data available as of today is still
limited. Meanwhile, the search for a potent small molecule TRPV1
antagonist with the adequate ADMET (absorption, distribution,
metabolism, excretion and toxicity) profile continues.22
Previous effort in identifying TRPV1 antagonists relied on the
traditional Ca2+ influx induced by capsaicin assay and led to the
identification of several small molecule TRPV1 antagonists with
the progression of few of them to clinical trial. Unfortunately, as
aforementioned the presence of side effects put a stop to the
further development of many of these compounds.
We hypothesized that these adverse effects could be caused by
the inherent properties of the molecule and/or by the TRPV1 acti-
vation mode profile. In order to identify new hit compounds having
a different phenotypic profile we proceeded with a new HTS cam-
paign wherein the Ca2+ influx assay was replaced by a Rb+ atomic
absorption spectroscopy assay.23 Because one of the aims was to
reduce the lipophilicity of the new hit relative to the clinical
O
OH
N
N
1
⇑
Corresponding author.
E-mail address: i.dorange@hotmail.com (I. Dorange).
Figure 1. Novel indolizine TRPV1 antagonist.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.059

I. Dorange et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6888–6895
6889
candidates, we placed a special emphasis on the ligand lipophilic
efficiency (LLE) parameter with the expectation to obtain a better
promiscuity profile.
Screening our corporate library led to the identification of ind-
olizine 1 (Fig. 1). This starting point was viewed as attractive
because the structure of compound 1 shared few common features
with the reported TRPV1 antagonists and has relatively good phys-
ical properties (molecular weight 322, eLogD 3.3). The activity of
the hit was subsequently confirmed in a more physiologically rel-
evant assay where an IonWorksTM Quattro (QT) apparatus was
used to determine the inhibitory effects of the compound in a
human-TRPV1 cell assay as an 8-points concentration–
response-curve.24
The structure of 1, an indolizine flanked with a carboxamide
and an aryl moiety respectively at the C-6 and C-7 position, pro-
vided an opportunity for efficient synthesis of analogues. We have
O
O
N
O
O
O
i
ii
iii
Br
O
R
R
R
R
+
Et
N
N
N
N
R1
O
Et
Et
A
A
A
A
O
R2
Ar
Ar
A = N, C
Scheme 1. Reagents and conditions: (i) NaHCO3, ethanol, 120 °C; (ii) ArX, bis(triphenylphosphine)palladium(II)chloride, KOAc, dioxane, 90–110 °C; (iii) aminoalcohol,
bis(trimethylaluminum)-1,4diazabicyclo[2.2.2]octane adduct, THF, 40 °C.
Table 1
Early SAR of the indolizine series
Compound Structure
MW eLogD TRPV1b
LLEc Solubilityd hERGe
Caco-2f Papp
(10À6 cm/s)
RLM/HLMg Clint (
min/mg)
l
L/ Rat/human GSH
trappingh (%)
a
pIC50
(l
M)
pIC50
O
N
OH
OH
N
1
322.4 3.3
322.4 3.2
5.7
6.8
2.4 280
ND
60
>500/220
30/NV
O
N
N
2
3.6 260
ND
ND
170/89
ND/ND
O
N
OH
OH
OH
N
3
338.4 2.8
6.0
3.2 150
<4.48
54
65/54
15/31
O
N
O
N
4
375.4 2.5
5.9
3.4 190
<4.48
56
31/31
25/22
N
O
N
O
N
5
377.3 3.6
6.3
2.7 360
ND
ND
48/62
ND/ND
N
F
F
F
O
N
OH
N
6
364.4 3.3
6.3
3.0 260
<4.48
58
93/160
9/15
O
(continued on next page)

6890
I. Dorange et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6888–6895
Table 1 (continued)
Compound Structure
MW eLogD TRPV1b
LLEc Solubilityd hERGe
Caco-2f Papp
(10À6 cm/s)
RLM/HLMg Clint (
min/mg)
l
L/ Rat/human GSH
trappingh (%)
a
pIC50
(l
M)
pIC50
O
N
OH
N
7
359.4 2.5
5.4
2.9 250
4.61
ND
79/77
51/34
NC
a
Determined by reversed phase liquid chromatography.
b
Inhibitory effect against the human transient receptor potential vanilloid 1 (hTRPV1) ligand-gated ion channel expressed in CHO cells measured with an IonWorksTM
Quattro (QT) apparatus.
c
LLE calculated as pIC50–eLogD.
Dried DMSO solubility, Ref. 25.
Measured in hERG-expressing CHO cells using an IonWorks assay setting, Ref. 26.
Measured permeability (A–B) through Caco-2 cells.
Rat and human liver microsome intrinsic apparent clearance. It is likely that the Clint values are a combination of intrinsic clearance and light chemical decomposition.
Peaks of glutathione adducts are integrated (in the total ion current trace) and the area is divided with the integrated area of the major glutathione adduct of clozapine.
d
e
f
g
h
The value is reported as GSH conjugate ratio*100, Ref. 27; ND: Not determined; NV: No value.
Small lipophilic substituents not allowed
No aromatic N
O
Small substituents
OH
Donor important
Linker size Important
III amide Important
allowed; No aromatic N
N
N
A
R
Small EWG or EDG tolerated
meta-position best
Para-position allowed
A = C indolizine series
A = N pyrrolopyridazine series
4-aromatic N not tolerated
Figure 2. SAR analysis of the indolizine and pyrrolopyridazine series.
SG
O
O
SG
O
O
O
R
O
R
R
R
N
Light
O2
GSH
+ e-
N
N
N
N
R
O
HO
m/z M + 16
m/z M + 323
A
B
C
Scheme 2. Putative mechanism for the light and oxygen promoted addition of GSH to the indolizine scaffold.
developed the synthetic route outlined in Scheme 1 that allowed
us to quickly evaluate the structure–activity relationship and the
ADMET profile within the series. Starting from 2-methylpyridine
and ethyl bromopyruvate the indolizine core was first prepared.
Then using synthetic conditions that utilize readily available
reagent classes (Heck and amide couplings) a number of analogues
were readily synthesized (see examples in Table 1). Noteworthy,
the sequence order between the Heck reaction and the amide for-
mation could be interchanged.
the program, the activity and LLE were judged to be in a decent
range (pIC50 above 5.4 and LLE above 3; up to 6.8 for 2 with an
LLE of 3.6), and while the solubility and the permeability data
proved to be in a reasonable range, the clearance was less than
optimal. More importantly all the tested compounds showed a pro-
pensity to react with glutathione (GSH) when they were incubated
with human liver microsome and GSH (see Table 1 GSH trapping).
This phenomenon was found to be dependent on the presence of
light and oxygen. A mass spectroscopy study, where GSH was
added to the compound in the presence of light and oxygen, led
us to rationalize the GSH addition could occur as shown in Scheme
2. A single electron transfer oxidation would lead to the reactive
A selection of the analogues synthesized is outlined in Table 1
as well as some of their associated ADMET data. The SAR will be
discussed at a later point (vide infra, see Fig. 2). At this stage of

I. Dorange et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6888–6895
6891
Table 2
Evaluation of diverse scaffolds using the indolizine core as foundation
Compound
Structure
MW
eLogDa
TRPV1b pIC50
LLEc
3.8
Solubilityd
(lM)
RLM/HLMe Clint (
lL/min/mg)
O
N
OH
OH
OH
OH
OH
OH
N
N
8
377.4
3.2
7.0
200
130/91
F
F
F
O
N
O
N
F
9
378.4
377.4
377.4
377.4
378.4
3.2
2.8
2.4
2.0
3.8
6.3
6.0
NV
NV
NV
3.1
3.2
NV
NV
NV
>400
>300
270
240
24
13/43
F
F
O
N
N
N
N
F
10
11
12
13
<10/32
<10/14
29/30
F
F
O
N
N
F
F
F
O
N
N
N
F
F
F
O
N
N
N
N
<10/<10
F
F
F
a
Determined by reversed phase liquid chromatography.
Inhibitory effect against the human transient receptor potential vanilloid 1 (hTRPV1) ligand-gated ion channel expressed in CHO cells measured with an IonWorksTM
b
Quattro (QT) apparatus.
c
LLE calculated as pIC50–eLogD.
Dried DMSO solubility, Ref. 25.
Rat and human liver microsome intrinsic apparent clearance; NV: No value.
d
e
intermediate B which in the presence of oxygen would give rise to
the Michael acceptor C, which in turn would readily react with GSH
to form the M+323 adduct detected in the LCMS experiment. When
tested against TRPV1 compound C was shown to lose all activity.
Faced with this light stability issue, we then embarked on the
search of a more stable scaffold that uses the indolizine core as
the foundation. We hypothesized that the addition of a heteroatom
onto the bicyclic core structure would serve two purposes, improv-
ing the light stability by modifying the electron density of the ring
system and improving the metabolic stability by reducing the
lipophilic character of the compound which was also an issue asso-
ciated with the indolizine series. In order to check this hypothesis a
selected set of compounds was synthesized.28 The associated set of
data is outlined in Table 2. Only few new scaffolds proved to retain

6892
I. Dorange et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6888–6895
Table 3
SAR of the pyrrolopyridazine series
Compound Structure
MW
eLogDa TRPV1b
LLEc Solubilityd
M)
hERGe
pIC50
Caco-2f Papp
(10À6 cm/s)
RLM/HLMg Clint (
min/mg)
lL/
pIC50
(l
O
OH
N
N
N
F
8
377.4 3.2
7.0
3.8
200
4.63
42
130/91
F
F
O
N
OH
OH
N
N
N
14
15
323.4 2.3
6.6
5.9
4.3
4.0
330
340
<4.5
ND
ND
130/23
130/19
O
N
N
339.4 1.9
ND
O
N
O
N
OH
N
16
377.4 3.3
5.6
2.3
22
ND
ND
47/78
F
F
F
O
N
OH
N
N
17
391.4 3.8
5.1
1.3
170
ND
ND
>500/>500
F
F
F
O
OH
N
N
N
18
428.5 2.4
NV
NV
25
ND
ND
75/55
H
N
O
O
N
N
OH
N
F
19
389.4 3.7
6.7
3.0
360
ND
ND
100/99
F
F
N
O
N
NH
N
N
20
413.4 3.5
NV
NV
150
ND
ND
500/360
F
F
F

I. Dorange et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6888–6895
6893
Table 3 (continued)
Compound Structure
MW
eLogDa TRPV1b
LLEc Solubilityd
M)
hERGe
pIC50
Caco-2f Papp
(10À6 cm/s)
RLM/HLMg Clint (
min/mg)
lL/
pIC50
(l
OH
N
N
N
O
21
403.4 3.7
5.8
2.1
220
ND
ND
140/150
F
F
F
a
Determined by reversed phase liquid chromatography.
b
Inhibitory effect against the human transient receptor potential vanilloid 1 (hTRPV1) ligand-gated ion channel expressed in CHO cells measured with an IonWorksTM
Quattro (QT) apparatus.
c
LLE calculated as pIC50–eLogD.
Dried DMSO solubility, Ref. 25.
Measured in hERG-expressing CHO cells using an IonWorks assay setting, Ref. 26.
Measured permeability (A–B) through Caco-2 cells.
d
e
f
g
Rat and human liver microsome intrinsic apparent clearance; ND: Not determined; NV: No value.
Table 4
Attempts to improve metabolic stability
Compound
Structure
MW
eLogDa
TRPV1b pIC50
LLEc
3.8
Solubilityd
(l
M)
RLM/HLMe Clint(
lL/min/mg)
O
N
OH
N
N
8
377.4
3.2
7.0
200
130/91
F
F
F
O
N
OH
N
N
22
391.4
3.7
6.2
2.5
32
84/87
F
F
F
O
N
OH
OH
N
N
23
24
25
391.4
391.4
407.4
3.8
3.6
3.7
6.1
5.3
5.3
2.3
1.7
1.6
28
130/170
320/120
110/91
F
F
F
O
N
N
N
190
F
F
F
O
O
N
OH
N
N
F
h
25
F
F
(continued on next page)

6894
I. Dorange et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6888–6895
Table 4 (continued)
Compound
Structure
MW
eLogDa
TRPV1b pIC50
LLEc
3.3
Solubilityd
(lM)
RLM/HLMe Clint(
lL/min/mg)
O
N
N
N
OH
26
351.4
2.2
5.5
>400
33/10
O
a
Determined by reversed phase liquid chromatography.
b
Inhibitory effect against the human transient receptor potential vanilloid 1 (hTRPV1) ligand-gated ion channel expressed in CHO cells measured with an IonWorksTM
Quattro (QT) apparatus.
c
LLE calculated as pIC50–eLogD.
Dried DMSO solubility, Ref. 25.
Rat and human liver microsome intrinsic apparent clearance.
d
e
the indolizine hTRPV1 activity and while the variation of the TRPV1
activity across the set of compounds could be attributed to a spe-
cific interaction between the bicyclic core and the target, a rational
overall structure relationship activity remains elusive. The light
sensitivity of the relevant scaffolds (8–10) was then assessed and
these compounds proved to be inert to UV-light (254 nM) exposure
when tested as a 1 mM DMSO solution. Under the same conditions
the initial indolizine scaffold was shown to be unstable and was
fully converted to the Michael acceptor C (Scheme 2).
Among thesecompounds(see Table 2), pyrrolopyridazine 8 exhib-
ited good potency, a relatively good ADME profile and an increased
LLE as compared to the initial indolizine scaffold, in other words the
best overall profile (all data not shown). Encouraged by these data,
we then set out to evaluate the potential of this new series. The syn-
thesis of the pyrrolopyridazine compounds follows the same steps as
the indolizine series (see Scheme 1). In general, the yields of the cycli-
zation reaction proved to be lower, which could be attributed to the
weaker nucleophilic character of the pyridazine ring.
A first iteration of compounds indicated that the early SAR
established for the indolizine compounds correlated well with
the pyrrolopyridazine SAR. Guided by the SAR analysis of the ind-
olizine series, a subsequent iteration of synthesis led to a quick
evaluation of the SAR and ADME properties for this series (Table
3). Substitution at the meta-position of the phenyl moiety such
as in 8 led to the most beneficial effect, while substitution at
para-position such as in 16 was tolerated, albeit with a potency
drop. Small lipophilic substituents at the meta-position of the
phenyl group, such as a methyl (14) or a trifluoromethyl group
(8) proved to be the most beneficial for the potency and the
LLE; more hydrophilic substituent such as the methoxy group in
15 was tolerated but was accompanied with a substantial drop
in potency. Cumulating two small substituents did not lead to
any beneficial effect (data not shown). The replacement of the
phenyl ring with a benzylic moiety (17) or the extension of the
phenyl ring with a phenylcarboxamide moiety (18) was detrimen-
tal to the activity.
On the amide side, the H-bond donor group proved to be crucial
for the potency as well as the two carbons linker between the
amide and the H-bond donor atom. Only tertiary amides showed
activity while secondary amide exhibited no activity (data not
shown). The fact that the size of the linker between the hydroxyl
group and the amide moiety and that only tertiary amides were
active led us to hypothesize the formation of a hydrogen bond
between the hydroxyl moiety and the amide heteroatoms placing
the compound into a more favorable conformation (closer to the
bioactive conformation). To quickly evaluate this hypothesis,
cyclopentyl 19 was synthesized but while it demonstrated similar
level of activity as compare to 8, the anticipated boost of potency
was not achieved. Noteworthy the other enantiomer was not
active. The integration of the H-bond donor group onto an aromatic
ring such as pyrazole 20 was also plagued with a total loss of activ-
ity. Introduction of a carbon atom spacer between the pyrrolopy-
ridazine and the amide in 21 was also received with very little
success (the enantiomer showed no activity). A summary of the
SAR findings is depicted in Figure 2.
The overall profile of this series was relatively good as indicated
in Table 3. However as in the case of the indolizine series the met-
abolic stability proved to be less than optimal for the pyrrolopyrid-
azine series. We investigated the use of small substituents on the
pyridazine ring such as the methyl in compounds 22–24 and the
methoxy group in 25, hypothesizing that the pyridazine ring could
be the primary site of metabolism (see Table 4). However none of
the compounds showed any improved metabolic stability and even
worse, the potency and the LLE was lower.
Realizing that the lipophilic character of the compound could
play an important role in the metabolic stability of the series, we
focus our attention to the small subset of compounds having an
eLogD < 3. For most compounds, keeping an eLogD < 3 proved to
be unfruitful for improving the rat microsome stability, however,
the human microsome stability was impacted positively (see for
example compounds 14, 15 and 18, Table 3). Looking back into
the clearance activity relationship of the indolizine series (Table
1), we noticed that a more rigid/bulky moiety alpha to the hydro-
xyl group, such as the cyclohexyl ring in compounds 6 and 7, dis-
played a positive effect on the rat clearance data. Gratifyingly, by
combining both factors, eLogD kept below 3 and a less flexible
hydroxylamide moiety such as in compound 26 (Table 4), a sub-
stantial metabolic stability improvement was achieved.
In summary, a new class of TRPV1 antagonist was identified
from an HTS campaign that used an Rb+ atomic absorption read
out. This indolizine class of compounds proved to be unstable in
the presence of light and oxygen and we have demonstrated that
the pyrrolopyridazine scaffold can be used to address this issue.
This class of compounds exhibited the same level of potency, no
light stability issue and a similar DMPK profile. The metabolic
stability liability inherent to the indolizine class remained a prob-
lem for the pyrrolopyridazine scaffold. However, the appropriate
selection of small substituents on the aromatic pendant (keeping
the eLogD below 3) combined with the use of a more bulky amide
alleviated this issue and still provided potent TRPV1 antagonists.
Acknowledgements
The authors wish to thank their colleagues from the Physical
Chemistry Characterization Team for providing solubility data, their
colleagues from the Safety Screening Centre for providing hERG
data, Kim Dekermendjian and Stephen Zicha for their input in the
HTS strategy and Shideh O’Brien for providing the IonWorks data.

I. Dorange et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6888–6895
6895
L-A.; Palin, R.; Tracey, H.; Walker, G. Bioorg. Med. Chem. Lett. 2011, 21, 2559; (c)
Saku, O.; Ishida, H.; Atsumi, E.; Sugimoto, Y.; Kodaira, H.; Kato, Y.; Shirakura, S.;
Nakasato, Y. J. Med. Chem. 2012, 55, 3436; (d) Kim, Y. S.; Kil, M-J.; Kang, S-U.;
Ryu, H.; Kim, M. S.; Cho, Y.; Bhondwe, R. S.; Thorat, S. A.; Sun, W.; Liu, K.; Lee, J.
H.; Choi, S.; Pearce, L. V.; Pavlyukovets, V. A.; Morgan, M. A.; Tran, R.; Lazar, J.;
Blumberg, P. M.; Lee, J. Bioorg. Med. Chem. 2012, 20, 215; (e) Sun, W.; Liu, K.;
Ryu, H.; Kang, D. W.; Kim, Y. S.; Kim, M. S.; Cho, Y.; Bhondwe, R. S.; Shivaji, A.;
Thorat, S. A.; Kim, H. S.; Pearce, L. V.; Pavlyukovets, V. A.; Tran, R.; Morgan, M.
A.; Lazar, J.; Ryder, C. B.; Toth, A.; Blumberg, P. M.; Lee, J. Bioorg. Med. Chem.
2012, 20, 1310; (f) Urbahns, K.; Yura, T.; Gupta, J. B.; Tajimi, M.; Fujishima, H.;
Masuda, T.; Yamamoto, M.; Ikegami, Y.; Marumo, M.; Yasoshima, K.; Yoshida,
N.; Moriwaki, T.; Madge, D.; Chan, F.; Mogi, M. Bioorg. Med. Chem. Lett. 2012, 22,
3408.
References and notes
1. Szallasi, A.; Blumberg, P. M. Pharmacol. Rev. 1999, 51, 159.
2. Caterina, M. J.; Schumacher, M. A.; Tominaga, M.; Rosen, T. A.; Levine, J. D.;
Julius, D. Nature 1997, 389, 816.
3. Tominaga, M.; Caterina, M. J.; Malmberg, A. B.; Rosen, T. A.; Gilbert, H.; Skinner,
K.; Raumann, B. E.; Basbaum, A. I.; Julius, D. Neuron 1998, 21, 531.
4. (a) Gunthorpe, M. J.; Harries, M. H.; Prinjha, R. K.; Davis, J. B.; Randall, A. J.
Physiol. 2000, 3, 747; (b) Piper, A. S.; Yeats, J. C.; Bevan, S.; Docherty, R. J. J.
Physiol. 1999, 518, 721.
5. Walpole, C. S. J.; Wrigglesworth, R. Capsaicin in the Study of Pain; Academic
Press, 1993. 63.
6. Zygmunt, P. M.; Petersson, J.; Andersson, D. A.; Chuang, H.-H.; Sorgard, M.; Di
Marzo, V.; Julius, D.; Hogestatt, E. D. Nature 1999, 400, 452.
7. Hwang, S. W.; Cho, H.; Kwak, J.; Lee, S. Y.; Kang, C. J.; Jung, J.; Cho, S.; Min, K. H.;
Suh, Y. G.; Kim, D.; Oh, U. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6155.
8. Chuang, H. H.; Prescott, E. D.; Kong, H.; Shields, S.; Jordt, S. E.; Basbaum, A. I.;
Chao, M. V.; Julius, D. Nature 2001, 411, 957.
9. Moriyama, T.; Higashi, T.; Togashi, K.; Iida, T.; Segi, E.; Sugimoto, Y.; Tominaga,
T.; Narumiya, S.; Tominaga, M. Mol. Pain 2005, 1, 3.
10. Negri, L.; Lattanzi, R.; Giannini, E.; Colucci, M.; Margheriti, F.; Melchiorri, P.;
Vellani, V.; Tian, H.; De Felice, M.; Porreca, F. J. Neurosci. 2006, 26, 6716.
11. Palazzo, E.; Luongo, L.; de Novellis, V.; Rossi, F.; Marabese, I.; Maione, S. Curr.
Opin. Pharmacol. 2012, 12, 9.
12. Gunthorpe, M.; Chizh, B. Drug Discovery Today 2009, 14, 56.
13. Khairatkar-Joshi, N.; Szallasi, A. Trends Mol. Med. 2009, 15, 14.
14. Kort, M. E.; Kym, P. R. Prog. Med. Chem. 2012, 57, 51.
23. Chinese Hamster Ovary cells, stably expressing TRex repressor and transfected
with hVR1, were used to determine Rubidium efflux under different
depolarization conditions. Rubidium levels were measured using the ICR8000
single channel atomic absorption flame spectrometer.
24. An assay was developed to determine the antagonistic effect of compounds on
the human transient receptor potential vanilloid 1 ligand-gated ion channel
(hTRPV1) expressed in CHO cells. An IonWorksTM Quattro (QT) apparatus was
used and the IC50 is calculated from an 8 points dose–response curve. The
protocol consists of a depolarizing pulse from 0 mV to +100 mV, immediately
followed by a hyperpolarizing pulse to À100 mV (200 ms each pulse). This
protocol is applied once before and once after compound addition.
25. Alelyunas, Y. W.; Liu, R.; Pelosi-Kilby, L.; Shen, C. Eur. J. Pharm. Sci. 2009, 37,
172. The method was modified by a pre-incubation step for 4 h at a fixed
temperature of 1 °C with magnetic stirrers at 500-rpm in order to generate the
most stable solid form.
26. Bridgland-Taylor, M. H.; Hargreaves, A. C.; Easter, A.; Orme, A.; Henthorn, D. C.;
Ding, M.; Davis, A. M.; Small, B. G.; Heapy, C. G.; Abi-Gerges, N.; Persson, F.;
Jacobson, I.; Sullivan, M.; Albertson, N. J. Pharmacol. Toxicol. Methods 2006, 54,
189.
27. Evans, D. C.; Watt, A. P.; Nicoll-Griffith, D. A.; Baillie, T. A. Chem. Res. Toxicol.
2004, 17, 3.
28. The central scaffold for compounds 9–13 was obtained following the following
references: azabenzofuran 9: (a) Trejo, A.; Arzeno, H.; Browner, M.; Chanda, S.;
Cheng, S.; Comer, D. D.; Dalrymple, S. A.; Dunten, P.; Lafargue, J. A.; Lovejoy, B.;
Freire-Moar, J.; Lim, J.; McIntosh, J.; Miller, J.; Papp, E.; Reuter, D.; Roberts, R.;
Sanpablo, F.; Saunders, J.; Song, K.; Villasenor, A.; Warren, S. D.; Welch, M.;
Weller, P.; Whiteley, P. E.; Zeng, L.; Goldstein, D. M. J. Med. Chem. 2003, 46,
4702; azaindole 10: (b) Hodgkinson, R. C.; Schulz, J.; Willis, M. C.;
imidazopyridine 11: (c) Kaizerman, J. A.; Gross, M. I.; Ge, Y.; White, S.; Hu,
W.; Duan, J. X.; Baird, E. E.; Johnson, K. W.; Tanaka, R. D.; Moser, H. E.; Buerli, R.
W. J. Med. Chem. 2003, 46, 3914; pyrrolopyrimidine 12: (d) Scheme 1.;
imidazopyridine 13: (e) Kurkin, A. V.; Bukhryakov, K. V.; Yurovskaya, M. A.
Chem. Heterocycl. Compd. 2009, 45, 188.
15. Eid, S. R. Curr. Top. Med. Chem. 2011, 11, 2118.
16. Voight, E. A.; Kort, M. E. Expert Opin. Ther. Pat. 2010, 20, 1107.
17. Lazar, J.; Gharat, L.; Khairathkar-Joshi, N.; Blumberg, P. M.; Szallasi, A. Expert
Opin. Drug Discov. 2009, 4, 159.
18. Wong, G. Y.; Gavva, N. R. Brain Res. Rev. 2009, 60, 267.
19. Kym, P. R.; Kort, M. E.; Hutchins, C. W. Biochem. Pharmacol. 2009, 78, 211.
20. (a) Caterina, M. J.; Leffler, A.; Malmberg, A. B.; Martin, W. J.; Trafton, J.;
Petersen-Zeitz, K. R.; Koltzenburg, M.; Basbaum, A. I.; Julius, D. Science 2000,
288, 306; (b) Gavva, N. R.; Treanor, J. J.; Garami, A.; Fang, L.; Surapaneni, S.;
Akrami, A.; Alvarez, F.; Bak, A.; Darling, M.; Gore, A.; Jang, J. R.; Kesslak, J. P.; Ni,
L.; Norman, M. H.; Palluconi, G.; Rose, M. J.; Salfi, M.; Tan, E.; Romanowsky, A.
A.; Banfield, C.; Davar, G. Pain 2008, 136, 202.
21. Szallasi, A.; Sheta, M. Expert Opin. Investig. Drugs 2012, 21, 1351.
22. Most recent TRPV1 antagonist publications: (a) Schmidt, R. G.; Bayburt, E. K.;
Latshaw, S. P.; Koenig, J. R.; Daanen, J. F.; McDonald, H. A.; Bianchi, B. R.; Zhong,
C.; Joshi, S.; Honore, P.; Marsh, K. C.; Lee, C-H.; Faltynek, C. R.; Gomtsyan, A.
Bioorg. Med. Chem. Lett. 2011, 21, 1338; (b) Ratcliffe, P.; Maclean, J.; Abernethy,
L.; Clarkson, T.; Dempster, M.; Easson, A.-M.; Edwards, D.; Everett, K.; Feilden,
H.; Littlewood, P.; McArthur, D.; McGregor, D.; McLuskey, D.; Nimz, O.; Nisbet,