CB2 selective sulfamoyl benzamides: Optimization of the amide functionality

Article abstract of DOI:10.1016/j.bmcl.2008.11.091

Previous research within our laboratories identified sulfamoyl benzamides as novel cannabinoid receptor ligands. Optimization of the amide linkage led to the reverse amide 40. The compound exhibited robust antiallodynic activity in a rodent pain model whe

Full text of DOI:10.1016/j.bmcl.2008.11.091

Bioorganic & Medicinal Chemistry Letters 19 (2009) 309–313
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
CB2 selective sulfamoyl benzamides: Optimization of the amide functionality
a
a
a
Allan J. Goodman a, , Christopher W. Ajello , Karin Worm , Bertrand Le Bourdonnec ,
Markku A. Savolainen a, Heather O’Hare a, Joel A. Cassel b, Gabriel J. Stabley b, Robert N. DeHaven b,
Christopher J. LaBuda b, Michael Koblish b, Patrick J. Little b, Bernice L. Brogdon c, Steven A. Smith c,
Roland E. Dolle a
*
a Department of Chemistry, Adolor Corporation, 700 Pennsylvania Drive, Exton, PA 19341, USA
b Department of Pharmacology, Adolor Corporation, 700 Pennsylvania Drive, Exton, PA 19341, USA
c Department of DMPK, Adolor Corporation, 700 Pennsylvania Drive, Exton, PA 19341, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Previous research within our laboratories identified sulfamoyl benzamides as novel cannabinoid receptor
ligands. Optimization of the amide linkage led to the reverse amide 40. The compound exhibited robust
antiallodynic activity in a rodent pain model when administered intraperitoneally. Efficacy after oral
administration was observed only when ABT, a cytochrome P450 suicide inhibitor, was coadministered.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 7 October 2008
Revised 21 November 2008
Accepted 24 November 2008
Available online 27 November 2008
Keywords:
Cannabinoid
CB1
CB2
Sulfamoyl benzamides
Antiallodynia
Activation of the endogenous cannabinoid receptors by com-
pounds such as
9-tetrahydrocannbinol (THC) (1) (Fig. 1), the ac-
tive antagonist, but not by a CB1 selective antagonist, thus demon-
strating a CB2 receptor mediated effect.4 Additionally, GW405833
(3) exhibited antihyperalgesic activity in rodent models of inci-
sional, neuropathic and chronic inflammatory pain, but was devoid
of significant activity in similar models in CB2 knock-out mice.5
It is perhaps not surprising then that development of CB2 selec-
tive agonists for the treatment of chronic and inflammatory pain
has recently received a large amount of interest. From this research
several structurally diverse classes of compounds have been iden-
tified. These include indoles (2, 3),4,5 benzimidazoles (4),6 iminopy-
razoles (5),7 oxadiazoles (6)8 and aryl sulfonamides (7) (Fig. 2).9
Independently, through high-throughput screening, research
within our organization identified the sulfamoyl benzamide 8 dis-
playing weak affinity for the CB2 receptor (Fig. 3).10
D
tive component of Cannabis sativa, has long been used in a
variety of medical applications. These include appetite stimulation
as well as treatments for emesis, cramps, fever, rheumatism and
pain.1 Two cannabinoid receptors, designated CB1 and CB2, have
been characterized from the superfamily of G protein-coupled
receptors (GPCRs). Sharing approximately 44% amino acid se-
quence homology, the two receptors differ in anatomical distribu-
tion with CB1 receptors found mainly in the CNS, while CB2
receptor expression occurs primarily in peripheral tissues associ-
ated with immune functions, including B and T cells and macro-
phages.2 CB2 receptors have also been isolated from peripheral
nerve endings and mast cells.3
The clinically undesired effects associated with cannabis use,
such as euphoria, are believed to be predominantly centrally med-
iated through activation of the CB1 receptors. The use of CB2 selec-
tive agonists is, therefore, one approach that could be adopted for
the use of cannabinoid receptor activation in the treatment of pain.
The antihyperalgesia produced by the selective CB2 agonist
AM1241 (2) in the rat carrageenan induced inflammatory thermal
hyperalgesia assay was reversed by pretreatment with a CB2 selec-
Optimization of the scaffold led to compound 9 which exhibited
vastly improved affinity and selectivity for the CB2 receptor. To fur-
ther explore the SAR around the phenylsulfonamide core we first
* Corresponding author. Tel.: +1 484 595 1939; fax: +1 484 595 1551.
E-mail address: agoodman@adolor.com (A.J. Goodman).
Figure 1. Structure of THC.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.091

310
A. J. Goodman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 309–313
Figure 3. Optimization of high-throughput screening hit.
looked at the isosteric replacement of the amide functionality of 9
with various heterocycles.11 These included thiazoles, oxadiazoles,
pyrazoles, imidazoles, triazoles and tetrazoles. Examples are
shown in Table 1.12 Synthesis of compounds 10–27 is described
in Scheme 1. Coupling of the previously described sulfonamide
benzoic acid 2810 with ammonium chloride gave the primary
amide 29. Thiazoles 10–12 were then prepared from 29 in a two
step process, that is, thioamide formation using Lawesson’s reagent
followed by cyclization with the respective bromoacetate. Dehy-
dration of intermediate 29 with phosphorus oxychloride gave the
nitrile 30 which was then used as a common intermediate for
the synthesis of the tetrazoles 13–15, the triazoles 16–18, the
Figure 2. Structures of CB2 selective agonists.
Table 1
Bioisosteric replacement of amide functionality—in vitro resultsa,b,c,d
a,c
a,c
Compound
R
CB1Kia,b
(nM)
CB2Kia,b
(nM)
Ratio
CB1/CB2
CB2EC50
(nM)
Compound
R
CB1Kia,b
(nM)
CB1Kia,b
(nM)
Ratio
CB1/CB2
CB2EC50
10
200
390
5.2
23
38
17
23
19
1100
1100
36
23
31
48
47
11
12
13
14
15
16
17
120
20
21
22
23
24
25
26
27
58
130
70
260
130
5.7
17
47
21
49
770
270
81
59
9.4
4.5
5.9
6.8
10
7.6
6.9
n.d.d
1.6
11
560
82
78
740
130
75
110
77
>5000
140
>5000
85
n.d.d
64
510
750
75
120
190
130
310
27
57
1400
6990
87
16
18
530
100
5.3
130
160
6.1
a
Values are the geometric means computed from at least three separate determinations.
For assay description see Ref. 13a.
b
c
EC50 values determined through [35S]GTP
Not determined.
cS stimulation. For assay description see Ref. 13b.
d

A. J. Goodman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 309–313
311
Figure 4. Reversal of amide linkage.
Table 2
In vitro binding of ‘reverse amides’ for select compoundsa,b,c
a,c
Compound
R
X
Y
CB1Kia,b CB2Kia,b Ratio
CB2EC50
(nM)
(nM)
CB1/
CB2
(nM)
9
32
C@O NH
130
640
3.9
1.7
31
380
4.6
9.6
NH
C@O
33
34
C–0
NH
NH
C@0 1200
540
48
9.4
11
130
25
49
35
36
C@0 NH
NH C@O 1200
310
34
11
9.0
110
21
31
a
Values are the geometric means computed from at least three separate
determinations.
b
For assay description see Ref. 13a.
c
EC50 values determined through [35S]GTP
cS stimulation. For assay description
see Ref. 13b.
From this research the thiazole substituted compounds (10, 12)
showed the best overall profile for the CB2 receptor. However, as
can be seen from Table 1, replacement of the amide moiety with
various heterocycles did not significantly improve affinity or selec-
tivity for the CB2 receptor in comparison to the lead compound 9.
Continuing our focus on optimization of the amide moiety we then
turned our attention to reversal of the amide linkage (Fig. 4).14
Three compounds from our original study were initially selected
for comparison and their reverse amide analogs prepared (Table 2).
Reversal of the amide linkage of compound 9, to give 32, led to a
compound with superior selectivity over the CB1 receptor (CB1/CB2
9 = 30-fold; CB1/CB2 32 = 380-fold) while retaining good affinity for
CB2. Similarly, compounds 34 and 36, when compared to their
respective analogs 33, 35, exhibited superior affinities and selectiv-
ities for the CB2 receptor in in vitro testing. Following the identifi-
cation of these novel and selective CB2 agonists, we decided to
extend the SAR around the amide functionality. Compounds 37–
48 were prepared as shown in Scheme 2.
Scheme 1. Reagents and conditions12: (a) NH4Cl, TBTU, DIEA, CH3CN, 89%; (b)
Lawesson’s Reagent, toluene, 66%; (c) RCOCH2Br, DBU, EtOH, 11–70%; (d) POCl3,
DMF, 75%; (e) NaN3, ZnBr, H2O, 52%; (f) RX, Et3N, DMF, 51–81%; (g) RCONHNH2,
K2CO3, CH3(CH2)3OH, 10–74%; (h) NH2OHꢀHCl, K2CO3, EtOH, 32–65%; (i) Raney
Nickel, CH3COOH, MeOH, H2, 28–91%; (j) RCOCH2Br, DBU, EtOH, 11–14%; (k)
NH2OHꢀHCl, K2CO3, EtOH, 32%; (l) RCOCl, pyridine, 2–22%; (m) SOCl2, DMF, 94%; (n)
CH3NHOCH3, Et3N,ꢁ CH2Cl2, 76%; (o) CH3MgBr, Et2O, THF, 92%; (p)
C6H5CH2N(CH3)3+Br3
,
CH2Cl2, MeOH, 47%; (q) (CH3)3CC@(NH)NH2ꢀHCl, K2CO3,
DMF, 83%; (r) CDI, RCOOH, LAH, THF, 56–89%; (s) NH2NH2 H2O, EtOH, 8–21%.
imidazoles 19–20 and the oxadiazoles 22–24. Compounds 21 and
25–27 were generated from the ketone intermediate 31. Com-
pound 31 was prepared in a three step procedure from the acid
28, i.e. acid chloride formation using thionyl chloride, Weinreb
amide synthesis followed by reaction with methylmagnesium
bromide.
The synthesis of the morpholinosulfonamide intermediate 50
was achieved by addition of morpholine to the commercially avail-
able nitrophenylsulfonyl chloride 49. After reduction of the nitro
group to give 51, the target compounds (32, 34, 36–48) were gen-
erated using a variety of coupling methods.
In vitro binding data for compounds 37–48 at the CB1 and CB2
receptors is shown in Table 3.

312
A. J. Goodman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 309–313
Scheme 2. Reagents and conditions:12 (a) Morpholine, EtOAc, 100%; (b) Fe, NH4Cl, EtOH, H2O, 100%; (c) RCOOH, Bop-Cl, Et3N, THF, 20–81%; (d) RCOOH, TBTU, DIEA, CH3CN, 3–
18%; (e) RCOOH, BEP, DIEA, CH2Cl2, 20%; (f) RCOCl, Et3N, THF, 20–28%.
Table 3
In vitro binding results for reverse amides at the CB1 and CB2 receptorsa,b,c,d
Ma,b CB2Kia,b
RatioCB1/ CB2EC50
a,c
a,c
Compound
R
CB1Kia,b
CB2Kia,b
RatioCB1/ CB2EC50
Compound
R
CB1Ki or % inh. @ 10
l
(nM)
(nM)
CB2
(nM)
(nM)
(nM)
CB2
(nM)
37
790
11
72
8400
43
4.7%
540
n.d.d
n.d.d
38
39
1800
470
14
23
130
20
42
29
44
45
270
120
170
2.3
6.5
220
390
1100
40
41
3400
23
150
11
46
25%
2900
n.d.d
n.d.d
1200
1900
35
34
16
33
47
48
2900
44%
660
4.4
n.d.d
n.d.d
42
120
n.d.d
2000
n.d.d
a
Values are the geometric means computed from at least three separate determinations.
For assay description see Ref. 13a.
b
c
EC50 values determined through [35S]GTP
Not determined.
cS stimulation. For assay description see Ref. 13b.
d
Attempts to replace the lipophilic amide group of 32 generally
To determine whether this lack of efficacy after oral dosing was
due to poor absorption or rapid metabolism, a second study was
performed. In this study, rats were pretreated with the cytochrome
P450 suicide inhibitor aminobenzotriazole (ABT) before adminis-
tration of compound 40 (300 mg/kg po) (Fig. 6).
The robust activity of the ABT pretreated animals with com-
pound 40 supported our assertion that the compound was rapidly
metabolized. In vitro metabolism studies of compound 40 in the
presence of human and rat liver microsomes confirmed these find-
ings (RLM = 2%; HLM = 1% remaining at 30 min).16
In summary, the SAR of the phenylsulfonamide series of CB2 li-
gands was expanded. Replacement of the amide substituent with
various heterocycles offered no improvement in potency or selec-
tivity for the CB2 receptor over the CB1 receptor. However, reversal
of the amide linkage led to a series of sulfamoyl benzamides with
improved affinity and selectivity for the CB2 receptor. Optimization
led to a decrease in affinity for the CB2 receptor. For example, in
the aryl chloride series of compounds, 44–46, the affinity for the
receptor was decreased by ꢂ100- to 2000-fold compared to com-
pound 32. Likewise, incorporation of heteroatoms into the amide
substituent (47, 48) was not tolerated, leading to substantial loss
in receptor affinity. However, other lipophilic substituents (37–
41) were well tolerated and retained good activity for the CB2
receptor. From this subset of ligands, the tetramethylcyclopropyl
compound (40) (EC50 = 11 nM, Emax = 84%) was determined to pos-
sess the best overall profile and was selected for in vivo evaluation
in the rodent hindpaw incisional model (Fig. 5).15
As can be seen from Figure 5, compound 40 exhibited robust
antiallodynic activity when administered at a dose of 30 mg/kg
ip. However, the compound did not produce a significant antiallo-
dynic effect following oral administration.

A. J. Goodman et al. / Bioorg. Med. Chem. Lett. 19 (2009) 309–313
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Figure 5. In vivo activity of compound 40 in the hindpaw incision model.
Mechanical paw-withdrawal thresholds for the left hindpaw of the hindpaw
incision group and compound 40 treated group. Data are plotted as the mean
( SEM) paw-withdrawal threshold of the left paw for each group. *p < 0.05
compared to vehicle-treated group. N = 8/group. Vehicle response 9.4 g. Drugs
were administered 30 min before testing.
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F.; Castelhano, A. L. Bioorg. Med. Chem. Lett. 1996, 6, 1601; (b) McBriar, M. D.;
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12. (a) Abbreviations: TBTU, O-(Benzotriazol-1-yl)-N, N, N0, N0-tetramethyluronium
tetrafluoroborate; BEP, 2-Bromo-1-ethylpyridinium tetrafluoroborate; Bop-Cl,
Bis(2-oxo-3-oxazolidinyl)phosphonic chloride; DIEA, diisopropylethylamine;
DBU, 1, 8-diazabicyclo-[5.4.0]undec-7-ene; (b) compounds were fully
characterized by 1H NMR and LC/MS.
13. (a) Binding assays were performed by modification of the method of Pinto, J. C.;
Potie, F.; Rice, K. C.; Boring, D.; Johnson, M. R.; Evans, D. M.; Wilken, G. H.;
Cantrell, C. H.; Howlett A. Mol. Pharmacol. 1994, 46, 516–522: Receptor binding
assays were performed by incubating 0.2–0.6 nM [3H]CP55940 with
membranes prepared from cells expressing cloned human CB1 or CB2
receptors in buffer consisting of 50 mM Tris–HCl, pH 7.0, 5.0 mM MgCl2,
1.0 mM ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0-tetraacetic acid
(EGTA), and 1.0 mg/ml fatty acid free bovine serum albumin. After
incubation for 60 min at room temperature for CB2 binding or 120 min at
30 °C for CB1 binding, the assay mixtures were filtered through GF/C filters that
had been pre-soaked overnight in 0.5% (w/v) poly(ethyleneimine) and 0.1% BSA
in water. The filters were rinsed 6 times with one mL each of cold assay buffer,
Figure 6. In vivo activity of orally administered compound 40 in the presence and
absence of ABT in the hindpaw incision model. Mechanical paw-withdrawal
thresholds for the left hindpaw of the hindpaw incision group, ABT, compound 40
and ABT/compound 40 treated groups. Data are plotted as the mean ( SEM) paw-
withdrawal threshold of the left paw for each group. All statistical analyses were
performed with one-way ANOVA followed by post-hoc comparisons (protected t-
test) among groups. *p < 0.01 compared to ABT-vehicle treated hindpaw incised
animals. N = 8/group. Vehicle response 8.9 g. Drugs were administered 120 min
before testing.
30 lL of MicroScint 20 (Perkin-Elmer) was added to each filter and the
radioactivity on the filters was determined by scintillation spectroscopy in a
TopCount (Perkin-Elmer). Nonspecific binding was determined in the presence
of 10
modification of the method by Selley, D. E.; Stark, S.; Sim, L. J.; Childers, S. R.
Life Sci. 1996, 59, 659–668: CB2-mediated stimulation of [35S]GTP
S binding
was measured in a mixture containing 100–150 pM [35S]GTP
S, 150 mM NaCl,
45 mM MgCl2, 3 M GDP, 0.4 mM dithiothreitol, 1.0 mM EGTA, 1.0 mg/mL fatty
acid free bovine serum albumin, 25 g of membrane protein, and agonist in a
total volume of 250 L of 50 mM Tris–HCl buffer, pH = 7.0 in 96-well Basic
lM WIN55212-2.(b) The [ cS binding method is a major
35S]GTP
c
c
of this scaffold afforded compound 40. In vivo efficacy of 40 was
demonstrated in the rodent hindpaw incisional model after intra-
peritoneal administration. Lack of activity after oral dosing of 40
was attributable to the rapid metabolism of the compound. At-
tempts to improve the metabolic stability of these CB2 selective
compounds will be reported in due course.
l
l
l
FlashPlates (PerkinElmer). After incubation at room temperature for 6 h, the
plates were centrifuged at 800g at 4 °C for 5 min and the radioactivity bound to
the membranes was determined by scintillation spectrometry using
TopCount (PerkinElmer). The extent of stimulation over basal
35S]GTP
percentage of the stimulation by 10
S binding was determined in the absence of
M WIN55212-2 was between 50%
a
[
c
S
binding was calculated as
WIN55212-2. Basal [35S]GTP
a
c
lM
References and notes
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l
and 100% over basal binding. Full agonists stimulate binding to the same
maximal extent as WIN55212-2.
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