Discovery of N-(3-(morpholinomethyl)-phenyl)-amides as potent and selective CB2 agonists

Article abstract of DOI:10.1016/j.bmcl.2009.07.057

Recently sulfamoyl benzamides were identified as a novel series of cannabinoid receptor ligands. Replacing the sulfonamide functionality and reversing the original carboxamide bond led to the discovery of N-(3-(morpholinomethyl)-phenyl)-amides as potent and selective CB₂ agonists. Selective CB₂ agonist 31 (K_i = 2.7; CB₁/CB₂ = 190) displayed robust activity in a rodent model of postoperative pain.

Full text of DOI:10.1016/j.bmcl.2009.07.057

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5004–5008
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of N-(3-(morpholinomethyl)-phenyl)-amides as potent
and selective CB2 agonists
a
a
a
a
Karin Worm a, , Damian G. Weaver , Rosalyn C. Green , Christopher T. Saeui , Doreen-Marie S. Dulay ,
William M. Barker a, Joel A. Cassel b, Gabriel J. Stabley b, Robert N. DeHaven b, Christopher J. LaBuda b,
Michael Koblish 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:
Recently sulfamoyl benzamides were identified as a novel series of cannabinoid receptor ligands. Replac-
ing the sulfonamide functionality and reversing the original carboxamide bond led to the discovery of N-
(3-(morpholinomethyl)-phenyl)-amides as potent and selective CB2 agonists. Selective CB2 agonist 31
(Ki = 2.7; CB1/CB2 = 190) displayed robust activity in a rodent model of postoperative pain.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 15 May 2009
Revised 8 July 2009
Accepted 9 July 2009
Available online 12 July 2009
Keywords:
Cannabinoid CB2 receptor agonist
Selectivity
Structure–activity relationship
Metabolic stability
Antiallodynic activity
D
9-Tetrahydro-cannabinol (THC, 1), the main active compo-
We recently discovered a series of novel sulfamoyl benzamides
with modest potencies at the CB1 and CB2 receptors. Subsequently,
the affinity and selectivity of the series for the CB2 receptor could
be increased as represented by agonist 8 with a binding affinity
for CB2 of 3.9 nM and 31-fold selectivity over the CB1 receptor.9
Herein, we describe optimization efforts focused on further
improving selectivity and physico-chemical properties.
We targeted the replacement of the sulfonamide moiety with a
carboxamide and a methylene amine, optionally linked by addi-
tional methylene spacers (8?9?10; Fig. 2). These modifications
were designed to reduce molecular weight, alter ring electronics
and modify the conformational flexibility of the molecules. For
example, replacing the sulfonyl bridge with a methylene group
introduces a basic nitrogen moiety, permitting salt formation and
thereby potentially improving water solubility.
Scheme 1 describes the synthesis of various sulfonamide
replacement analogs 9 starting from central intermediate methyl
3-iodo-4-methylbenzoate 11. Reaction with vinyltributyl tin or
allyltributyl tin followed by standard transformations introduced
two (16a, 19a) and three carbon (16b, 19b) spacers. A Buch-
wald–Hartwig amination attached the morpholine directly to the
core (17).
nent of Cannabis sativa, and other classical cannabinoids display
a wide range of physiological effects including analgesic, anti-
inflammatory, anti-convulsive and immuno-suppressive activi-
ties.1 Two G-protein coupled cannabinoid receptors, CB1 and
CB2, are well characterized. The two receptors share less than half
of their amino acid sequence. CB1 receptors are located primarily
in the CNS and CB2 receptors are located primarily in peripheral
tissues.2 Cannabinoid receptor agonists also induce a number of
side effects, which seem to be mediated predominantly by CB1
receptors in the CNS.3
A separation between therapeutic effects and undesirable CNS
effects may be accomplished either by preventing the cannabinoid
from crossing the blood-brain barrier4 or by increasing the selec-
tivity for the CB2 receptor over the CB1 receptor.5 There is consid-
erable interest in developing new cannabimimetic compounds
possessing preferentially high affinity for the CB2 receptor as po-
tential novel therapeutics for the treatment of inflammation and
chronic pain.6 Several structural classes displaying selectivity for
the CB2 receptor have already been described and recent examples
(2–7) are shown in Figure 1.7,8
The reaction of isopropyl magnesium chloride and DMF with 11
followed by reductive amination or oxidation and amide coupling
yielded one carbon spacers (21, 23).
* Corresponding author. Tel.: +1 14845951944; fax: +1 14845951551.
E-mail address: kworm@adolor.com (K. Worm).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.057

K. Worm et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5004–5008
5005
Figure 1. THC (1), and recently described selective CB2 ligands.
Replacing the oxygen in the morpholine with a basic nitrogen
was not tolerated (data not shown).
Different substituents in R1, R2 and R3 were investigated using
the reverse amide core10 and the results are summarized in Table
2. In general the addition of a methyl group in the 3-position (R1)
increased binding affinity (31, 32 compared to 28, 29), a methyl
group in the 2-position (R2) led to a loss of affinity (36, 37 com-
pared to 29, 30). Replacing the methyl in R1 with a CF3 caused a
slight decrease in binding and a significant decrease in selectivity
(33). A fluorine atom in the 2-position (R2) did not notably
change binding affinity (34, 35 vs 29, 30). Modifying the substit-
uents in R3 by adding fluorine substituents significantly increased
both binding and selectivity (e.g., 28 compared to 30). A combina-
tion of a 3-methyl at R2 and 3,3,3-trifluoro-2-methyl-2-(trifluoro-
methyl)propyl at R3 yielded compound 31 with a Ki CB2 of 2.7 nM,
the compound with the best binding affinity observed within this
series and almost 200-fold CB2 selective. The selectivity is even
more pronounced when comparing functional activities of EC50
Figure 2. Design rationale.
Within the sulfonamide series we previously observed that
reversing the amide group led to an increase in CB2 selectivity.10
Therefore further optimization of substituents R1, R2 and R3 was
conducted on the reverse amide methyl amine analog 10
(Fig. 2). This synthesis is shown in Scheme 2. Appropriately
substituted methyl benzoic acids 24 were nitrated. The nitro
acids were reduced to alcohols 25 using borane–THF, followed
by mesylation and reaction with morpholine to intermediate
26. The nitro group was then reduced with iron and the final
products 27 were obtained under standard amide coupling
conditions.
CB2 = 5.5 nM and EC50 CB1 = 3.6 lM.
Since rapid in vitro metabolism posed an issue within the sul-
fonamide series,10 compounds were tested for metabolic stability
in rat and human liver microsomes (RLM, HLM).13 The results are
also presented in Table 2. Methyl and fluoro substituents eluci-
dated the structure-metabolism-relationship. Substitution in the
2-position (R2) increased metabolic stability, especially in rat liver
microsomes. Unfortunately, substituents other than fluorine de-
creased binding affinities significantly.
Constraining a molecular system can influence activity as well
as metabolic stability. Therefore R1 was tethered to the methy-
lene group yielding a six membered ring as in 43. This modifica-
tion was tolerated by the receptor. The synthesis is depicted in
Scheme 3.
Starting from 7-nitro-1-tetralone 38, the key transformation
was a one pot, three step conversion of alcohol 39 to amine
42 via mesylation and displacement with morpholine. A com-
peting dehydration reaction began before complete mesylation
had been achieved, resulting in a 4:1 mixture of chloride 40
to alkene 41 (by LC–MS). In order to get the morpholine dis-
placement to go to completion, it was necessary to add aceto-
nitrile, then concentrate to remove DCM, so the reaction could
be refluxed at a higher temperature. The nitro group was re-
duced with iron and amide formation was performed under
standard conditions.
In vitro binding to the CB1 and CB2 receptors was determined on
the basis of [3H]-CP-55940 binding to membranes of CHO cells sep-
arately expressing human CB1 and CB2 receptors. Functional activ-
ity at the CB2 receptor was determined by [35S]GTP
cS binding
using membranes from the transfected CHO cells.11 All molecules
described are agonists in this assay. The results are shown in Tables
1 and 2.12 When compared to sulfonamide 8, the sulfonamide
replacements with methylene (21), ethylene (19a) and propylene
(19b) showed a 5-, 10- and 15-fold decrease in binding affinity
to the CB2 receptor. Replacing the sulfonamide with a carboxamide
was also poorly tolerated. The optimum number of carbon atoms
between the amine portion and the phenyl ring was three for the
carboxamide (16b) as compared to one in the methylene series
(21). There was a fourfold decrease in activity in 16b compared
to the best methylene core (21) and a 20-fold decrease in CB2 bind-
ing compared to the sulfonamide 8. Attaching the morpholine di-
rectly to the aromatic core (17) led to a 100-fold loss in affinity.

5006
K. Worm et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5004–5008
Scheme 1. Reagents and conditions: (a) vinylSn(Bu)3 or allylSn(Bu)3,Pd(PPh3)4, DMF, 95 °C, 24 h; (b) KOH, H2O, THF, dioxane, 25 °C, 16 h; (c) fenchylamine, TBTU, i-Pr2EtN,
acetonitrile, 0–25 °C, 16 h; (d) 9-BBN, THF, NaOH, H2O2, THF, 25–50 °C, 16 h; (e) tosyl chloride, pyridine, i-Pr2EtN, CH2Cl2; (f) morpholine, DCM; (g) NaOCl, NaClO2, Na2HPO4,
TEMPO, ACN, H2O, 42 °C, 16 h; (h) morpholine, TBTU, i-Pr2EtN, ACN, 0–25 °C, 16 h; (i) [Pd2(dba)3], BINAP, KOt-Bu, morpholine, microwave, 50 min. 95 °C; (j) i-PrMgCl, THF,
DMF, À35–25 °C, 4 h; (k) morpholine, AcOH, NaCNBH3, EtOH, 5–25 °C, 24 h; (l) NaOH, EtOH, H2O 25 °C, 5 h; (m) fenchylamine, i-Pr2EtN, ACN, TBTU, 5–25 °C, 24 h.
Table 1a,b
Compd
X
Ki CB1 (nM) Ki CB2 (nM) Ratio CB1/CB2 EC50 CB2 (nM)
8
21
SO2
CH2
130
1100
1400
2100
9000
1700
3900
>5000
3.9
19
45
62
300
83
700
1200
31
58
31
34
30
20
5.6
ND
6.6
42
64
110
ND
ND
ND
ND
19a
19b
17
16b
16a
23
(CH2)2
(CH2)3
Bond
(CH2)2CO
CH2CO
CO
Scheme 2. Reagents and conditions: (a) HNO3, H2SO4, HOAc; (b) BH3THF, THF, 5–
25 °C, 16 h; (c) MsCl, i-Pr2EtN, DCM, 5–25 °C, 16 h; (d) morpholine, DCM 5–25 °C,
16 h; (e) Fe, EtOH, NH4Cl, H2O 70 °C, 4 h; (f) R3-acid, BOP-Cl, i-Pr2EtN, ACN, 5–25 °C,
16 h.
a
Values are the geometric means computed from at least three separate
determinations.
For assay description see Ref. 11.
b
The resulting racemic product 43 was chirally separated and the
more active isomer 44 showed an affinity of 2.3 nM and 570-fold
selectivity for the CB2 receptor (Table 3). Unfortunately, introduc-
ing this constraint had detrimental consequences on the metabolic
stability of the system (<5% remaining after 30 min in either the
RLM or HLM assay).
Compound 31, displaying the best profile within this series, was
evaluated for efficacy in the hindpaw incision model14 of postoper-

K. Worm et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5004–5008
5007
Table 2 a,b
Compd R1 R2 R3
Ki CB1 Ki CB2 Ratio
EC50 CB2 Stabilityc
(nM)
(nM)
CB1/CB2 (nM)
RLM HLM
28
29
30
31
32
33
34
35
36
H
H
6300
16
390
23
14
76
26
42
H
H
1600
4%
70
0.9 38
2.2 56
Scheme 3. Reagents and Conditions: (a) NaBH4, EtOH, rt, 1 h; (b) MsCl, TEA, DCM,
16 h; (c) morpholine, DCM, 16 h; (d) morpholine, ACN, reflux, 6 h; (e) Fe, NH4Cl,
EtOH, water, 70 °C, 2 h; (f) acid, BOP-Cl, i-Pr2EtN, DCM.
H
H
120
2.7
8.6
17
ND
190
63
110
Table 3 a,b
Me
Me
CF3
H
H
500
540
110
31%
10%
14%
18%
5.5
29
31
H
21
42
3.1 32
H
6.5
ND
ND
ND
ND
1.0
2.0
Compd
Ki CB1
(nM)
Ki CB2
(nM)
Ratio CB1/
CB2
EC50 CB2
(nM)
43
44
45
Racemic
Enantiomer 1
Enantiomer 2
3400
1300
>5000
10
2.3
120
340
570
ND
41
3.6
72
F
37
49
2.0 36
a
Values are the geometric means computed from at least three separate determi-
nations.
b
For assay description see Ref. 11.
H
F
120
440
530
200
590
500
15
57
49
61
parable to the antiallodynic effect produced by a dose of 3 mg/kg IP
of morphine with a longer duration of action. No behavioral side
effects were observed at these doses. Compound 31 did not show
activity when dosed orally.
H
Me
Me
20
In summary, N-(3-(morpholinomethyl)-phenyl)-amides 10
were discovered as potent and selective CB2 agonists by replacing
the sulfonamide in 8 with a methylene amine group. SAR studies
revealed that 3,4-dimethyl substitution and lipophilic reverse
branched or cyclic amides are preferred, leading to compound 31
with activity in an animal model of postoperative pain. Rapid
metabolism of these compounds still remains a concern. Additional
substitution in the 2-position seems to increase metabolic stability,
but substituents other than fluorine decrease binding affinity sig-
nificantly. A strategy to further improve metabolic stability and
bioavailability within this series while retaining potency and selec-
tivity is under investigation.
37
H
67
a
Values are the geometric means computed from at least three separate determi-
nations.
b
For assay description see Ref. 11.
% remaining after 30 min incubation, see Ref. 13.
c
ative pain. As shown in Figure 3, a dose of 10 mg/kg IP of com-
pound 31 produced a significant antiallodynic effect that was com-

5008
K. Worm et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5004–5008
References and notes
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D. J. Med. Chem. 2008, 51, 1904; (c) Kai, H.; Morioka, Y.; Koriyama, Y.; Okamoto, K.;
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11. Binding assays were performed by modification of the method of: (a) 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; The [35S]GTP
cS binding method is
a major modification of the method by (b) Selley, D. E.; Stark, S.; Sim, L. J.; Childers,
S. R. Life Sci. 1996, 59, 659. Additional details are given in Ref. 9.
Figure 3. (A) Mechanical paw withdrawal thresholds for the left hindpaw of the
hindpaw incision group, compound 31 and morphine treatment groups. Data are
plotted as the mean ( SEM) withdrawal threshold of the left hindpaw. All statistical
analyses were performed with one-way ANOVA followed by post-hoc comparisons
(protected t test) among groups. *p <0.05 compared to vehicle-treated, surgery
group, tested at 30 min post treatment. (B) Time course for compound 31 compared
to morphine.
12. All compounds exhibited physical and spectroscopic properties consistent with
their structure. For 31: 1H NMR (CDCl3), d 1.88 (s, 3H), 2.35 (s, 3H), 2.37 (s, 3H),
3.34 (m, 2H), 3.44 (d, J = 12 Hz, 2H), 3.67 (m, 2H), 3.80 (t, J = 12 Hz, 2H), 4.04 (d,
J = 12 Hz, 2H), 4.46 (s, 2H), 7.36 (s, 1H), 7.62 (s, 1H). MS m/z 413 (M+H+). The
compound was converted to the HCl salt. Anal. Calcd (C18H23ClF6N2O2): C,
48.17; H, 5.17; N, 6.24. Found: C, 48.10; H, 5.25; N, 6.09.
13. Obach, S. R. Drug Metab. Dispos. 1999, 27, 1350.
14. Brennan, T. J.; Vandermeulen, E. P.; Gebhart, G. F. Pain 1996, 64, 493.