Discovery and SAR of a novel series of 2,4,5,6-tetrahydrocyclopenta[c] pyrazoles as N-type calcium channel inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.03.063

A novel series of substituted 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles were investigated as N-type calcium channel blockers (Ca_v2.2 channels), a chronic pain target. One compound was active in vivo in the rat CFA pain model.

Full text of DOI:10.1016/j.bmcl.2014.03.063

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2057–2061
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR of a novel series
of 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles
as N-type calcium channel inhibitors
⇑
Michael P. Winters , Nalin Subasinghe, Mark Wall, Edward Beck, Michael R. Brandt, Michael F. A. Finley,
Yi Liu, Mary Lou Lubin, Michael P. Neeper, Ning Qin, Christopher M. Flores, Zhihua Sui
Janssen Research and Development, LLC, 1400 McKean Rd., Spring House, PA 19477, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 28 March 2014
A novel series of substituted 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles were investigated as N-type cal-
cium channel blockers (Cav2.2 channels), a chronic pain target. One compound was active in vivo in
the rat CFA pain model.
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
2,4,5,6-Tetrahydrocyclopenta[c]pyrazoles
N-type calcium channel
Cav2.2 blockers
Pain
Rat CFA model
N-type calcium channels (Cav2.2 channels) have been impli-
cated in the neurotransmission of pain.1 They are highly expressed
in the dorsal horn of the spinal cord and are frequently up-regu-
lated in pain syndromes.2 In addition, N-type knockout mice have
reduced nociceptive sensitivity.3 There is also a marketed drug, the
cone snail toxin ziconotide (PrialtÒ), that is a potent blocker of
N-type calcium channels. Ziconotide is prescribed for severe
chronic pain and is limited to intrathecal administration.4 In
addition, it has a side-effect profile that gives it a very narrow
therapeutic window.4 Due to these limitations, many groups have
tried to develop novel oral N-type calcium channel inhibitors for
the treatment of chronic pain.5
One hit from a screen of our compound collection was pyrazole
1, a 100 nM inhibitor of the N-type calcium channel (Fig. 1). It was
hypothesized that rigidifying the sidechain of 1 by forming a fused
cyclopentane ring on the pyrazole scaffold might improve potency
by lessening the rotational degrees of freedom of the sidechain.
This novel ring system also gives a favorable IP position. Pharmaco-
phore modeling of the 2 scaffolds showed that the 2,3,5-substitued
tetrahydrocyclopenta[c]pyrazole 2 filled very similar space to 1
and should be a good substitute for the straight chain scaffold.
Compound 3 was one of a few compounds initially made to assess
the activity and selectivity of the series and was found to be 26 nM
for N-type in the Functional Drug Screening System (FDSS) assay
but only have 17 and 62% inhibition of L-type calcium channel at
1 and 5
M, respectively.6,7 Rat liver microsome (RLM) and human
liver microsome stability (HLM) of 3 was low, and metabolic ID in
both species indicated that loss of the methyl from the 2-methoxy
was the major metabolite. This encouraging activity and selectivity
led to the start of this series.
l
The synthesis of the cyclopenta[c]pyrazole nucleus begins with
alkyne 4, which is available in 4 steps from TMS-acetylene and
ethyl 2-(oxiran-2-yl)acetate (Scheme 1).8,9 Treatment of 4 with
NBS and AgNO3 gives the alkynyl bromide 5.10 Conversion to the
hydrazide and then treatment with PS-PPh3 and CCl4 gives the
hydrazonyl chloride 6.8 Subsequent generation of the nitrilimine
and intramolecular 3+2 dipolar cycloaddition on the alkyne gives
the cyclopenta[c]pyrazole 7.11 Installation of the 3-aryl substituent
by Suzuki coupling and deprotection of the TIPS gives the key
5-hydroxycyclopenta[c]pyrazole 8.12
A great deal of synthetic work was done functionalizing the
5-position of the cyclopenta[c]pyrazole ring system from the
5-hydroxy intermediate 8 (Scheme 2). Treatment with NaHMDS
and MeI or iPrI gives the ether 9; however, more substituted ethers
were not accessible using this method. Treatment with NaHMDS
and cyclohexanecarbonyl chloride gives the ester, which can be
reduced with Et3SiH in the presence of InBr3 to give the ether
10.13 The cyclopentyl ether 11 was accessed by reacting with
cyclopentene and PhSe-phthalimide in the presence of BF3-OEt2
to give the selenide intermediate, which was reduced with Bu3SnH
and AIBN.14 The phenyl ether 12 was made by Mitsunobu reaction
⇑
Corresponding author. Tel.: +1 215 628 7843; fax: +1 215 540 4611.
E-mail address: mwinter4@its.jnj.com (M.P. Winters).
http://dx.doi.org/10.1016/j.bmcl.2014.03.063
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2058
M. P. Winters et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2057–2061
R1
N
OMe
OMe
OMe
R2
N
N
N
N
N
N
N
Cl
Cl
Cl
N
c
SO2Me
R1
19
HO
HO
R1
N
R1
N
R1
N
OCONMe2
2
1
3
R2
N
R2
R2
R2
N
2,3,5-substitued
2,3,5-substitued
N
N
a
N
b
d
e
tetrahydrocyclopenta[c]
pyrazole
pyrazole
N
O
O
N3
g
OH
NH2
f
Figure 1. Initial pyrazole hit and proposed bicyclic pyrazoles.
17
18
8
20
R1
R2
N
N
R1
R1
Br
Br
R1
HN
N
O
OH
R2
N
O
OH
R2
a
Cl
N
b
N
N
N
O
OTIPS
OTIPS
5
21
OTIPS
6
4
N
N
N
N
N
TMS
R1
N
R1
N
22
23
R2
Br
N
N
c
d
Scheme 3. Reagents and conditions: (a) (i) TEA, MsCl, (ii) NaN3, DMF, 100 °C; (b)
H2, 10% Pd/C; (c) (i) TEA, MsCl, (ii) NaHMDS, MeI; (d) succinimide, TEA, toluene,
110 °C; (e) (i) 4-chlorobutanoyl chloride, K2CO3, (ii) NaHMDS; (f) propionaldehyde,
glyoxal, NH4OAc; (g) TMS-acetylene, CuI, DIEA.
OTIPS
OH
7
8
Scheme 1. Reagents and conditions: (a) NBS, AgNO3; (b) (i) i-BuOCOCl, NMM,
R1NHNH2, DCM, (ii) PS-Ph3P, CCl4, ACN, 50 °C; (c) TEA, toluene, 100 °C; (d) (i)
R2B(OH)2, Pd(OAc)2, (o-tol)3P, Na2CO3, 80 °C, (ii) TBAF.
of 17 with a sulfonyl chloride gives the sulfonamide, which can be
alkylated on nitrogen giving 19. Reaction with succinic anhydride
gives the succinimide 20, whereas condensation with 4-chlorobu-
tanoyl chloride gives the lactam 21. Imidazoles 22 can be made by
reaction with aldehydes in the presence of glyoxal and ammonia.
Click chemistry on the intermediate azide 17 gives the triazole 23.
From the straight-chain pyrazole hits from the HTS campaign,
ortho-substitution on the aryl ring at the 2-position of the pyrazole
was critical for activity, and the o-methoxy 1 was particularly po-
tent (Table 1). Removal of the methoxy 24, or moving the methoxy
to the m- or p-positions 25 and 26 lost potency, as did the 2-CF3 27,
2-OH 28 and 2-OiPr 29. 4-Chloro was one of the best substituents
on the phenyl ring at the 3-position of the pyrazole; removal re-
sulted in a loss of potency 30. The acid functionality on the side-
chain abolished activity 31. This SAR led us to attempt to
enhance the potency by making dramatic changes at the 5-position
of the cyclopentylpyrazole wherein many different substituents
were tolerated. It was hoped that these changes would also
with phenol. Oxidation of the alcohol with Dess–Martin period-
inane gives the ketone 13, a versatile intermediate. Treatment with
the appropriate diol and BF3-OEt2 gives the ketal 14. The aryl and
heteroaryl derivatives 15 were made from the ketone, by first mak-
ing the vinyl triflate followed by Suzuki coupling and reduction of
the double bond. Reductive amination of the ketone can give cer-
tain amine derivatives 16.
Other amine derivatives at the 5-position were made by con-
verting the alcohol to the amine and derivatizing from there
(Scheme 3). Formation of the mesylate and displacement with
azide gives 17, which is hydrogenated to give amine 18. Treatment
R1
R2
N
R1
N
N
R1
N
R2
R2
N
N
Table 1
O
O
SAR of pyrazoles from HTS campaign
O
14
f
OMe
a
9
d
R1
12
R1
R1
N
R1
N
N
R2
N
R2
e
R2
R2
N
g
h
N
N
N
c
O
OH
R3
N
13
R1
R1
15
b
8
R2
N
R2
R1
N
N
N
Compd No.
R1
R2
R3
FDSS IC50 (nm)
N
R2
N
1
24
25
26
27
28
29
30
31
2-OMe
H
3-OMe
4-OMe
2-CF3
2-OH
2-OiPr
2-OMe
2-OMe
Cl
Cl
Cl
F
Cl
Cl
Cl
H
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
COOH
100
1200
690
2370
790
O
O
11
N
O
10
16
2300
260
16%b
29%a
Scheme 2. Reagents and conditions: (a) NaH, MeI; (b) (i) NaHMDS, cyclohexane-
COCl, (ii) InBr3, Et3SiH; (c) (i) cyclopentene, PhSe-phthalimide, BF3-OEt2, (ii)
Bu3SnH, AIBN; (d) phenol, Ph3P, DIAD; (e) Dess–Martin periodinane; (f) 1,3-
propanediol, BF3-OEt2; (g) (i) KHMDS, PhN(SO2CF3)2, (ii) 3-pyridyl pinacolboronate,
(Ph3P)4Pd, (iii) H2, PtO2; (h) morpholine, NaBH(OAc)3, HOAc.
Cl
a
% Inhibition at 5
% Inhibition at 1
l
l
M.
M.
b

M. P. Winters et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2057–2061
2059
Table 2
remaining after 10 min incubation with RLM). It also was much
more soluble in aqueous media than previous compounds. At-
tempts to build the potency back by adding alkyl groups to the
imidazole were unsuccessful, as shown by 40 and 41. Other N-het-
erocyles, such as morpholine 42, pyrrolidone 43, triazole 44 and
succinimide 45, were more potent but lacked RLM stability.
Two series of compounds that were initially promising were the
5-spirocyclic ketals and the sulfonamides (Table 3). Unsubstituted
propyl ketal 46 was 50 nM. Adding bulk to the ketal ring by making
the 2,2-dimethylpropyl ketal 47 and pinacol ketal 48 improved po-
tency. An attempt to increase solubility by replacing the 4-chloro-
phenyl with 4-dimethylaminophenyl 49 maintained potency but
reduced RLM stability. Introduction of pyridyl at R3 50 lowered
the potency but maintained stability. In the sulfonamide series,
excellent potency was achieved by sulfamide 51 and sulfonamide
52, whereas demethylation of these functional groups resulted in
a loss in potency. Attempts to increase the aqueous solubility of
the series by introducing a pyridyl at R3 54 lowered the potency.
Most of the sulfamides and sulfonamides lacked appreciable RLM
stability.
A set of some of the more active compounds were tested in sec-
ondary patch clamp assays16 and a more physiologically relevant
ex vivo calcitonin gene-related peptide (CGRP) release assay17
using rat spinal cord slices (Table 4). In general, there was good
correlation between the FDSS assay and the QPatch assay. It has
been postulated that relatively greater inhibition at high versus
low frequency may result in an improved therapeutic index, and
many of the compounds exhibited this characteristic.18 Of the
compounds tested in the CGRP release assay, compound 47 stands
out as being more active than predicted by its FDSS and QPatch
potencies.
SAR of 5-ethers and 5-heterocycles
R2
OR3
N
N
R1
Compd
No.
R1
R2
R3
FDSS IC50
(nm)
RLMa
(%)
32
33
34
35
36
37
38
OMe
OiPr
O-Cyclopentyl
OPh
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Me
Me
Me
Me 130
Me 130
52
5.7
22
2
3
19
54
25
14
98
OCH2-cyclohexyl
Pyrid-3-yl
4-Cyclopropylpyrid-3-
yl
Me
Me 280
8.6
39
40
41
42
43
44
45
Imidazole
Cl
Et
38
37
86
2
21
1
3
8
4,5-Di-Et-imidazole
2-Et-imidazole
Morpholine
Pyrrolidone
Triazole
CN Me
CN Me 200
Cl
Cl
Cl
Cl
Me
Et
Me
Et
16
19
19
Succinimide
7.5
2
a
% Remaining after 10 min incubation with rat liver microsomes.
enhance the RLM stability because the HTS hits and the initial SAR
on a very similar series, the 2,3,5-tetrahydropyrrolo[3,4-c]pyra-
zoles (N in the ring at the 5-position of the bicycle), indicated it
would be difficult to replace the o-methoxy and maintain
potency.15
One series of compounds evaluated were ethers and heteroaryls
at the 5-position (Table 2). Methyl ether 32 was modestly potent
against N-type in the FDSS assay, and adding some bulk gave the
most potent compound in this series, the isopropyl 33.7 Increasing
the size further gave less potent compounds, such as cyclopentyl
34. Phenyl 35 was particularly poor as were the compounds with
methylene spacers, such as 36. Potency and RLM stability in this
series appear to be inversely correlated. We attempted to improve
solubility in the 5-heteroaryl series by making pyridyl 37; how-
ever, this compound suffered from poor metabolic stability.
Increasing bulk at the 4-position of the pyridyl 38 decreased the
potency. One attempt to increase solubility, N-linked imidazole
39, had moderate potency and excellent RLM stability (86%
Following the secondary assays, compounds 47, 48 and 51 were
evaluated in the rat Complete Freund’s Adjuvant (CFA) radiant heat
model of inflammatory pain due to their potency and relative sta-
bility.19 Only ketal 47 at 30 mpk PO showed effects in lengthening
paw withdrawal latencies while the others had no effect (Fig. 2).20
An IV only pharmacokinetic study in rats showed that after a
0.4 mg/kg dose of 47, the initial concentration c0 was 199 ng/mL,
the AUClast was 98 h ng/mL, the volume of distribution at steady
state was 4.8 L/kg indicating extensive tissue distribution, clear-
ance was high at 62.7 mL/min/kg, and the t1/2 was 1.3 h. A tissue
distribution study in rats 1 h after an oral dose of 30 mg/kg 47
showed 133 ng/g in brain (tissue/plasma ratio 3.95), 109 ng/g in
spinal cord (tissue/plasma ratio 3.24), and below levels of
Table 3
SAR of 5-ketals and 5-sulfonamides
OMe
R3
N
N
R1 R2
Compd No.
R1
R2
R3
FDSS IC50 (nm)
RLMa (%)
46
47
48
49
50
51
52
53
54
55
–O(CH2)3O–
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Me2N-Ph
4-Me-pyrid-3-yl
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
50
22
6.6
3.0
19
3.1
6.4
14
26
13
21
18
2
4
18
6
8
2
18
1
–OCH2C(Me)2CH2O–
–OC(Me)2C(Me)2O–
–OC(Me)2C(Me)2O–
–OC(Me)2C(Me)2O–
NMeSO2NMe2
NMeSO2Me
NHSO2NMe2
NHSO2Me
NMeSO2NMe2
H
H
H
H
H
4-Cl-Ph
4-MeO-pyrid-3-yl
a
% Remaining after 10 min incubation with rat liver microsomes.

2060
M. P. Winters et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2057–2061
Table 4
Patch clamp and CGRP release data for selected compounds
Compd No.
FDSS IC50 (nm)
QPatch Lo freqa
QPatch Hi freqa
QPatch ratio Lo/Hi (%)
CGRP releaseb
RLMc (%)
39
48
47
51
52
33
45
42
38
6.6
22
3.1
6.4
5.7
7.5
16
13
37
21
37
44
48
47
33
29
48
37
59
46
61
53
31
45
77
57
63
96
79
89
106
21
77
63
54
NT
NT
NT
NT
86
21
18
6
8
3
2
1
a
b
c
% Inhibition at 0.1
% Inhibition at 1.0
% Remaining after 10 min incubation with rat liver microsomes.
l
l
M.
M.
Bioorg. Med. Chem. Lett. 2011, 21, 3317; (b) Tyagarajan, S.; Chakravarty, P.; Park,
M.; Zhou, B.; Herrington, J.; Ratliff, K.; Bugianesi, R.; Williams, B.; Haedo, R.;
Swensen, A.; Warren, V.; Smith, M.; Garcia, M.; Kaczorowski, G.; McManus, O.;
Lyons, K.; Li, X.; Madeira, M.; Karanam, B.; Green, M.; Forrest, M.; Abbadie, C.;
McGowan, E.; Mistry, S.; Jochnowitz, N.; Duffy, J. Bioorg. Med. Chem. Lett. 2011,
21, 869; (c) Yamamoto, T.; Takahara, A. Curr. Top. Med. Chem. 2009, 9, 377; (d)
Pajouhesh, H.; Feng, Z.; Zhang, L.; Pajouhesh, H.; Jiang, X.; Hendricson, A.; Dong,
H.; Tringham, E.; Ding, Y.; Vanderah, T.; Porreca, F.; Belardetti, F.; Zamponi, G.;
Mitscher, L.; Snutch, T. Bioorg. Med. Chem. Lett. 2012, 22, 4153; (e) Beebe, X.;
Yeung, C.; Darczak, D.; Shekhar, S.; Vortherms, T.; Miller, L.; Milicic, I.;
Swensen, A.; Zhu, C.; Banfor, P.; Wetter, J.; Marsh, K.; Jarvis, M.; Scott, V.;
Schrimpf, M.; Lee, C.-H. Bioorg. Med. Chem. Lett. 2013, 23, 4857.
14
13
12
Vehicle
11
Compound 47
10
9
8
7
6
5
6. Only certain compounds were intermittently assayed for selectivity versus L-
type Calcium Channel during this program. No further L-type selectivity data is
available.
7. (a) Finley, M.; Lubin, M.; Neeper, M.; Beck, E.; Liu, Y.; Flores, C.; Qin, N. Assay
Drug Dev. Technol. 2010, 8, 685
(-24h)
0
30 60
120
180
300
(a) In the FDSS assay, functional activity of test compounds are determined by
their inhibitory effects on Ca2+ influx via N-type calcium channel after
depolarizing plasma membrane with 50 mM KCl. Calcium mobilization
responses to KCl depolarization are evaluated by measuring the intensity of
Ca2+ fluorescent signal in the presence of BD Calcium Assay Dye (BD
Time (min) aꢀer oral administraꢁon
Figure 2. Compound 47 in the rat CFA model of pain.
Biosciences), utilizing
Hamamatsu. The percent inhibition at a concentration of 1
values will be reported. For this screen, stable cell line (HEK parent)
expressing Cav2.2 (N-type calcium channel, Genbank accession number
AAO53230) subunits is used. Cav2.2 subunits were expressed in pcDNA3.1
a
Functional Drug Screening System (FDSS) by
lM as well as IC50
a
quantitation in both brain and spinal cord extracellular fluid. These
studies show that reasonable concentrations of 47 can be achieved
in the target tissues brain and spinal cord.
and pBudCE4.1 vectors under selection by 400 lg/ml of G418 and 200 lg/ml of
Zeocin. Cells were clonally isolated, expanded and screened by Western blot
analyses, and further tested for expression of characteristic N-type calcium
currents by whole-cell patch clamp. HEK293 cells stably expressing Cav2.2
subunits are routinely grown as monolayer in Dulbecco’s Modification of
Eagle’s Medium (DMEM; low glucose Cat # 12320-032) supplemented with
In summary, we developed a novel scaffold with improved po-
tency for N-type calcium channel from a very simple pyrazole hit.
Although identifying a compound in this series with relatively high
N-type potency and RLM stability was a challenge, ketal 47 none-
theless demonstrated robust efficacy in the rat CFA model of
inflammatory pain and showed efficacious concentrations could
be achieved in brain and spinal cord. While much work remains,
this novel series of N-type calcium channel inhibitors may lead
to candidates for analgesic drugs with improved therapeutic index
in the future.
10% FBS, 2 mM
L
-glutamine, 100 I.U./ml penicillin, 100
lg/ml streptomycin,
400 g/ml G418, and 200
l
lg/ml Zeocin (Split ratio: 1:5). Cells are maintained
in 5% CO2 at 37 °C. JNJ compounds are prepared as 10 mM stock in DMSO from
neat, if available. Otherwise, the 5 or 10 mM DMSO stock solutions provided in-
house are used. Calcium mobilization responses to KCl depolarization are
evaluated by measuring the intensity of Ca2+ fluorescent signal in the presence
of BD Calcium Assay Dye (BD Biosciences), utilizing
Screening System (FDSS) by Hamamatsu. 24 h prior to assay, cells are seeded
in clear-base poly- -lysine coated 384-well plates (BD Biosciences, NJ, USA) at a
a Functional Drug
D
density of 5000 cells per well in culture medium and grown overnight in 5%
CO2 at 37 °C. On assay day, growth media is removed and cells are loaded with
BD calcium assay dye (BD Bioscience) for 35 min at 37 °C, under 5% CO2 and
then for 25 min at room temp. Utilizing the FDSS, cells are challenged with test
compounds (at varying concentrations) and intracellular Ca2+ is measured for
5 min prior to the addition of 50 mM KCl for an additional 3 min of
measurement. IC50 values for compounds ran in this assay are determined
from six-point dose–response studies and represent the concentration of
compound required to inhibit 50% of the maximal response. Maximal
fluorescence intensity (FI) achieved upon addition of 50 mM KCl was
exported from the FDSS software and further analyzed using GraphPad Prism
3.02 (Graph Pad Software Inc., CA, U.S.A.). Data is normalized to the maximum
average counts of quadruplicate wells for each data points in the presence of
50 mM KCl and to the minimum average counts in the presence of buffer.
Theoretical curves are generated using nonlinear regression curve-fitting
analysis of either sigmoidal dose response or sigmoidal dose response
(variable slope) and the IC50 values with the best-fit dose curve determined
by GraphPad Prism are reported. Conotoxin was run as a positive control in the
assay and typically showed IC50’s of 10–20 nM.
Acknowledgments
The authors gratefully acknowledge the ADME and analytical
teams in Spring House for running the RLM assays and the PK
and tissue distribution studies, and Renee DesJarlais for initial
modeling discussions.
References and notes
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M. P. Winters et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2057–2061
2061
12. The same compounds can be synthesized using an arylalkyne in place of the
bromoalkyne if desired. See Ref. 8.
small-molecule evaluations were calculated using the following formula: %
Inhibition = (Total Release À Test Sample)/(Total Release À Conotoxin) Â 100
where Total Release = K+-evoked (50 mM KCl) release, Test Sample = release in
the presence of KCl plus test inhibitor, and Conotoxin = release in the presence
13. Sakai, N.; Moriya, T.; Konakahara, T. J. Org. Chem. 2007, 72, 5920.
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Lubin, M.; Neeper, M.; Qin, N.; Flores, C.; Sui, Z. Bioorg. Med. Chem. Lett. 2014,
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of KCl plus 1 lM conotoxin (full block of Cav2.2), all in the presence of 1 lM
nifedipine to block CGRP release due to L-type channel activity. Unless
otherwise indicated, statistics for comparing among CGRP results utilized a
one-way analysis of variance with Dunnett’s test (Ref. 7a).
16. Mathes, C.; Friis, S.; Finley, M.; Liu, Y. Comb. Chem. High Thr. Screen. 2009, 12,
78.
18. Winquist, R.; Pan, J.; Gribkoff, V. Biochem. Pharmacol. 2005, 70, 489.
19. Malmberg, A.; Gilbert, H.; McCabe, R.; Basbaum, A. Pain 2003, 101, 109. More
detail on the CFA model.
20. Compound 39 was below the potency threshold to be tested in the CFA model.
Perhaps the low RLM stability of 51 affected its in vivo activity. There is no
explanation for the lack of activity of 48; the compound has a very similar
profile to 47.
17. CGRP levels were evaluated using
a Spi Bio rat CGRP enzyme linked
immunoassay kit (Cayman Chemicals) as recommended by the
manufacturer. By including samples with known amounts of CGRP in each
enzyme immunoassay,
a standard curve was created that permitted the
conversion of absorbance to pg/mL CGRP for each of the unknowns, as
determined using GraphPad Prism software. Percentage of inhibitions for the