Hit-to-lead optimization of pyrrolo[1,2-a]quinoxalines as novel cannabinoid type 1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.05.010

Hit-to-lead optimization of a novel series of N-alkyl-N-[2-oxo-2-(4-aryl-4H-pyrrolo[1,2-a]quinoxaline-5-yl)-ethyl]-car boxylic acid amides, derived from a high throughput screening (HTS) hit, are described. Subsequent optimization led to identification of

Full text of DOI:10.1016/j.bmcl.2009.05.010

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3471–3475
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hit-to-lead optimization of pyrrolo[1,2-a]quinoxalines as novel cannabinoid
type 1 receptor antagonists
György Szabó a, , Róbert Kiss a,b, Dóra Páyer-Lengyel a, Krisztina Vukics a, Judit Szikra a, Andrea Baki a,
*
a
a
a
}
László Molnár , János Fischer , György M. Keseru
a Gedeon Richter Plc, Budapest 10, PO Box 27, H-1475, Hungary
Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hogyes Endre u. 9., H-1092, Hungary
b
}
a r t i c l e i n f o
a b s t r a c t
Article history:
Hit-to-lead optimization of a novel series of N-alkyl-N-[2-oxo-2-(4-aryl-4H-pyrrolo[1,2-a]quinoxaline-5-
yl)-ethyl]-carboxylic acid amides, derived from a high throughput screening (HTS) hit, are described. Sub-
sequent optimization led to identification of in vitro potent cannabinoid 1 receptor (CB1R) antagonists
representing a new class of compounds in this area.
Received 10 April 2009
Revised 4 May 2009
Accepted 5 May 2009
Available online 7 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
CB1 receptor
Rimonabant
Hit-to-lead
SAR
Pyrrolo[1,2-a]quinoxaline
According to the WHO Global InfoBase, 78% of the population of
United States and 73% of the United Kingdom is classified as obese
or overweight. Moreover, it is not only western society that is
affected as the prevalence of obesity has increased worldwide by
75%since1980andtheWHOhaveconsequentlydefineditasaglobal
epidemic.1 The discovery of the endocannabinoid system, in the 90s,
has provided a novel opportunity to develop and introduce efficient
pharmaceuticals for the treatment of obesity.2 The endocannabinoid
system, and the CB1 receptor (cloned in 1990)3 in particular, plays a
special role in energy homeostasis. Blockade of CB1 receptors
decreases food intake leading to a reduction in body weight. There-
fore, it was hoped that CB1 receptor antagonists/inverse agonists
could provide effective therapies for the treatment of obesity.4,5
Unfortunately, rimonabant6 (SR141716A), the first potent and selec-
tive CB1 receptor inverse agonist (Fig. 1) was recently reported to
cause serious psychiatric side-effects.7 Consequently, several CB1
antagonists (including taranabant8 (MK-0364) and otenabant9
(CP-945,598)) were withdrawn from the market and/or clinical
development. Whether these side effects arise from the mechanism
of action itself or they are compound specific remains uncertain. It is
therefore important to identify and develop novel structures that
may provide a distinct therapeutic profile.
4H-pyrrolo[1,2-a]quinoxaline-5-yl)-ethyl]-carboxylic acid amides,
are described.
An in-house high throughput screening (HTS) campaign using a
functional Ca2+ assay10 yielded several hits featuring a pyrrolo[1,2-
a]quinoxaline core, as represented by compound 11 (Fig. 2).
O
H3C
N
N
Cl
N
H
N
Cl
Cl
Figure 1. Structure of rimonabant 1.
O
Cl
Cl
N
N
N
Herein, the discovery and hit-to-lead optimization of a novel
series of CB1 receptor antagonists, N-alkyl-N-[2-oxo-2-(4-aryl-
O
O
CH3
* Corresponding author. Tel.: +36 1 481 5180; fax: +36 1 432 6979.
E-mail address: gy.szabo@richter.hu (G. Szabó).
Figure 2. The original HTS hit, compound 11.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.010

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G. Szabó et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3471–3475
Compound 11 displayed submicromolar affinity for the CB1
of TEA. Alkaline hydrolysis of ester 30 readily produced the
expected intermediate 31. Compounds 32–38 were obtained in a
similar method as described for compound 3 in Scheme 1. Target
compounds 39–45 were obtained by parallel synthesis via amida-
tions of 32–38 with intermediate 31 using polymer-supported DCC
in dry DCM. The IR, 1H NMR, 13C NMR and MS spectra for all the
synthesized intermediates and final compounds were consistent
with the assigned structures. Moreover, the purity of compounds
was checked by HPLC and LCMS.
receptor (Ki = 831 nM), our efforts were therefore focused on the
hit-to-lead optimization and structure–activity relationship (SAR)
development of the pyrrolo[1,2-a]quinoxaline template to identify
compounds with improved in vitro affinity.
In order to improve the potency of compound 11, three areas
were addressed: (R) the effect of the alkyl side chain, (R1) the effect
of acyl groups, and (R2) the effect of groups of the pyrrolo-quinox-
aline ring.
A general synthetic strategy of N-alkyl-N-{2-oxo-2-[4-(4-meth-
oxy-phenyl)-4H-pyrrolo[1,2-a]quinoxaline-5-yl]-ethyl}-carboxylic
acid amide analogues is illustrated in Scheme 1. Starting from com-
mercially available 1-(2-aminophenyl)-pyrrole 2, cyclization with
p-methoxy-benzaldehyde in the presence of a catalytic amount
of acetic acid in ethanol gave dihydro-pyrrolo[1,2-a]quinoxaline
3, which could be easily converted to the acetylated product 4
using triethylamine (TEA) and dichloromethane (DCM) as solvent.
A facile two-step protocol involving an alkylation followed by an
acetylating step (using the appropriate carboxylic acid chloride in
the presence of TEA or using the carboxylic acid in the presence
of dicyclohexylcarbodiimide (DCC)) provided rapid access to the
desired products 11–27 bearing structural variations in the R and
R1 positions.
We began with modification of R1 to reduce the molecular
weight (MW), therefore, initial efforts focused on replacing the
halogen substituents and/or removing the phenyl ring. As shown
in Table 1 replacing the halogen substituents increased the potency
as the substituents of the phenyl ring were in the ortho-position
(compounds 16, 17, 18). In order to further reduce the MW we
prepared a series of cycloalkyl and alkyl derivatives. As shown,
lower cycloalkyl ring size afforded better potency, while replacing
the phenyl ring by a methyl group led to a total loss of potency
(24). Interestingly, introducing a chloroethyl substituent led to an
18-fold increase in efficacy (compound 27, Ki = 46 nM).
With the result of compound 27 in hand, we next focused on the
R substituent. Removal of the butyl group in 27, potency is lost. The
same results were obtained when smaller alkyl, i-alkyl or cyclo-
alkyl groups were used, the potency lost (13, 15) or resulted in a
drastic drop in efficacy (14). To further optimize the potency of
27, we investigated the role of R1 substituents. Simple methyl
(41) or ethyl (42) derivative of 27 led to a complete loss of activity,
indicating the importance of bulky substituents at this position
that can be accommodated at the binding site. The para-position
of the phenyl ring is preferred, as the ortho- (39) and disubstituted
The synthesis of compounds 39–45 containing different R2
substituents were accomplished according to the procedures
depicted in Scheme 2. The key intermediate 31 required for the
synthesis was obtained in three steps. Ethyl-bromoacetate 28
was treated with n-butylamine in toluene at room temperature
to afford amine 29 in a yield of 90%. Compound 30 was synthesized
via acetylating of 29 using 2-chloro-acetyl chloride in the presence
O
N
N
b
a
N
NH
Cl
N
NH2
O
CH3
O
CH3
2
3
4
O
O
O
O
d or e
c
N
N
R
N
N
R
NH
N
R1
O
CH3
CH3
5
R = (CH2)3CH3
R = H
R1 = 3,5-diCl-Ph
11 R = (CH2)3CH3,
12 R = H,
R1
R1
R1
R1
R1
R1
R1
R1
R1
R1
R1
R1
R1
R1
R1
R1
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
=
CH(CH3)Cl
CH(CH3)Cl
CH(CH3)Cl
CH(CH3)Cl
2-Cl-Ph
2-Me-Ph
2-F-Ph
CH2CH(CH3)2
cyclopentyl
cyclobutyl
cyclopropyl
CH(CH3)2
Me
6
7
8
9
R = Me
13 R = Me,
R = Et
R = i-propyl
14 R = i-propyl,
15 R = cyclopropyl,
16 R = (CH2)3CH3,
17 R = (CH2)3CH3,
18 R = (CH2)3CH3,
19 R = (CH2)3CH3,
20 R = (CH2)3CH3,
21 R = (CH2)3CH3,
22 R = (CH2)3CH3,
23 R = (CH2)3CH3,
24 R = (CH2)3CH3,
25 R = (CH2)3CH3,
26 R.= (CH2)3CH3,
27 R = (CH2)3CH3,
10 R = cyclopropyl
Et
CH2OMe
CH(CH3)Cl
Scheme 1. Reagents and conditions: (a) p-methoxy-benzaldehyde, CH3COOH, EtOH, 50 °C; (b) ClCH2COCl, Et3N, CH2Cl2, 0–5 °C; (c) R1NH2, CH3CN, K2CO3, 60 °C; (d) RCOCl,
Et3N, CH2Cl2, 0–5 °C; (e) RCOOH, DCC, CH2Cl2.

G. Szabó et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3471–3475
3473
O
a
O
b
(CH2)3CH3
NH
EtO
EtO
EtO
Br
28
29
O
O
(CH2)3CH3
(CH2)3CH3
c
HO
+
N
Cl
N
Cl
N
NH
R2
O
CH3
O
CH3
30
31
32-38
8
7
6
9
d
32,39 R2 = 2-MeO-Ph
33,40 R2 = 2,5-diMeO-Ph
34,41 R2 = Me
5a
9a
O
5
(CH2)3CH3
10 N
N
1
2
4
Cl
N
1
2
3a
1'
R2
2'
35,42 R2 = Et
3
36,43 R2 = Ph
CH3
O
3'
37,44 R2 = 4-F-Ph
38,45 R2 = 4-Cl-Ph
39-45
Scheme 2. Reagents and conditions: (a) n-butylamine, toluene, rt; (b) CH3CH(Cl)COCl, Et3N, CH2Cl2, 0–5 °C; (c) KOH, MeOH, rt; (d) PS-DCC, CH2Cl2, rt.
Table 1
Biological assay results for compounds 11–27 and 39–45
O
R
N
N
N
R1
R2
O
Cmpds
R
R1
R2
n
CB1 assaya Inh (%)b, Ki (nM)
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
39
40
41
42
43
44
45
1
Butyl
H
CH3
3,5-DiCl-phenyl
CH(CH3)Cl
CH(CH3)Cl
CH(CH3)Cl
CH(CH3)Cl
2-Cl-phenyl
2-Me-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
4-MeO-Ph
2-MeO-Ph
2,5-DiMeO-Ph
CH3
1
1
1
3
1
1
1
3
3
3
3
3
3
1
3
3
3
1
1
1
1
3
3
3
5
831
25%
46%
220 32
40%
i-Propyl
Cyclo-propyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
Butyl
137
503
2-F-Ph
i-Butyl
349 94
236 0.9
602 4.7
214 8.0
78 10
124 24
31%
539 118
461 83
46 1.0
36%
45%
15%
14%
260 9.4
162 13
45 7.1
6.5 0.8
Cyclopentyl
Cyclobutyl
Cyclopropyl
CH(CH3)2
CH3
CH2CH3
CH2OMe
CH(CH3)Cl
CH(CH3)Cl
CH(CH3)Cl
CH(CH3)Cl
CH(CH3)Cl
CH(CH3)Cl
CH(CH3)Cl
CH(CH3)Cl
CH2CH3
Ph
4-F-Ph
4-Cl-Ph
a
CB1 receptor affinity of the compounds was determined using rat cerebellum membrane preparation and [3H]SR141716A.11 Values represent means S.E.M. The number
of experiments (n) is indicated.
b
Inh % was obtained using 1 lM concentration of compounds.
analogues (40) were inactive. Simple substituents such as halogens
were well tolerated at this position, yielding compound 45 with
comparable in vitro potency to that of compound 27.
The binding mode of one of the most potent compound of the
pyrrolo-quinoxaline series (compound 45 in Table 1) was analyzed
by docking calculations. A novel human CB1 receptor (hCB1R)
homology model was developed by using the recently solved crys-
tal structure of the human b2 adrenergic receptor (hb2AR) (PDB ID:
2RH1).12 Automated sequence alignment was carried out by
ClustalW 2.0.10.13 with the amino acid sequences of hCB1R and
hb2AR. Manual adjustments were applied by using the information
available on conserved GPCR residues. 100 preliminary homology

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G. Szabó et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3471–3475
models were generated by Modeller 9v6.14 The best model indi-
cated by the Modeller Objective Function was selected for further
studies. Next, validation tests were carried out to verify the quality
of this model. Procheck15 and Whatif16 tests indicated that the
model is of acceptable quality, no problematic residues were
observed at the binding site. Moreover, all residues reported to
be important for ligand binding faced to the interior of the binding
site.
The best preliminary model was prepared for docking using the
Protein Preparation Wizard available in MAESTRO.
17 Rimonabant was
18
prepared for docking by LIGPREP
,
and it was subsequently docked
to the binding site of our hCB1R homology model by GLIDE19 in stan-
dard precision (SP) mode. The best docking pose represented a con-
formation where rimonabant formed strong lipophilic interactions
with aromatic residues at the binding site.
Furthermore, rimonabant was in a favorable orientation to form
an H-bond with Lys192 (3.28). On the other hand, the conforma-
tion of the side chain of Lys192 (3.28) was suboptimal for a strong
interaction.
Therefore, 5000 steps Polak-Ribiere-type conjugate gradient
energy optimization was carried out with the OPLS_2005 force
17
field20 by MacroModel available in MAESTRO
.
Since the role of
Lys192 (3.28) in rimonabant binding was already proved by site-
directed mutagenesis,21 a distance constraint between the side
chain N atom of Lys192 (3.28) and the carbonyl O atom was
applied with
a harmonic potential (force constant = 100 kJ/
mol Å2). As a result, an optimized hCB1R-rimonabant complex
was obtained including a strong H-bond between the carbonyl
oxygen atom of rimonabant and Lys192 (3.28) (Fig. 3A).
In addition, rimonabant formed several lipophilic interactions
with the binding site. The two phenol rings and the pyrazole ring
of rimonabant accommodated favorably in a lipophil pocket com-
posed by Phe170 (2.57), Leu193 (3.29), Val196 (3.32), Phe200
(3.36), Pro269 (5.33), Ile271 (5.35), Tyr275 (5.39), Trp279 (5.43),
Trp356 (6.48), Leu359 (6.51) and Phe379 (7.35). The piperidine
ring was surrounded by Ile175 (2.62), His178 (2.65), Val179
(2.66), Pro269 (5.33) and Phe379 (7.35). These results are in line
with the mutagenesis data reported by McAllister et al.22 These
authors found that mouse CB1 binding affinity of rimonabant
was significantly reduced by Phe201(3.36)Ala, Trp280(5.43)Ala
and Trp357(6.48)Ala mutations. On the other hand, no significant
affinity drop was observed in the case of Phe190(3.25)Ala muta-
tion. The optimized complex is also in agreement with the muta-
genesis data published by Kapur et al.,23 that is, rimonabant did
not form interaction with either Ser383 (7.39) or Ser173 (2.60).
Next, we docked the most potent compound of the newly dis-
covered pyrrolo-quinoxaline series (compound 45) to the hCB1R
binding site. Since compound 45 contains two chiral centers, and
CB1 binding affinity data were obtained only for the racemic form,
Figure 3. Binding mode of rimonabant (A) and compound 45 (B). Important
residues and the ligands are represented as ‘ball and sticks’. Van der Waals surfaces
of the ligands are shown in light blue. H-bonds are shown as yellow dotted lines.
Some residues are omitted for clarity.
of Lys192 (3.28). This resulted in a complex with two strong H-
bonds with Ser383 (7.39) and Lys192 (3.28) (Fig. 3B). The 4-chloro-
phenyl group of compound 45 formed lipophil interactions with
Ile271 (5.35), Phe268 (5.32), Pro269 (5.33) and Phe379 (7.35).
The pyrrolo-quinoxaline ring system fitted favorably to the lipophil
pocket comprised by Ile271 (5.35), Leu193 (3.29), Phe170 (2.57),
Val196 (3.32), Tyr275 (5.39), Trp279 (5.43), Phe200 (3.36),
Trp356 (6.48) and Leu359 (6.51). The butyl side chain was sur-
rounded by Phe268 (5.32), Phe379 (7.35) and Ala380 (7.36), while
the 1-chloroethyl group formed lipophil interactions with Ile175
(2.62), His178 (2.65), Val179 (2.66). In summary, compound 45
was found to possess a similar binding conformation to that of
rimonabant, but an additional H-bond with Ser383 (7.39) was also
observed.
In conclusion, a new class of CB1 antagonist compounds has
been described and tested for CB1 receptor binding affinities. Pre-
liminary hit-to-lead optimization of the series has led to com-
pounds such as 45, which shows significant in vitro activity. The
investigation of the binding mode of 45 revealed similar binding
mode to that of rimonabant. Further optimization and application
of this series of compounds will be reported in due course.
all four possible stereoisomers were prepared for docking by
18
LIGPREP
.
All stereoisomers were docked to the binding site, but
GLIDE has found potential docking conformation for one stereoiso-
mer only (20S,4R). Consequently, we suggest that this stereoisomer
is responsible for the high CB1 affinity. GLIDE yielded a reasonable
binding conformation for compound 45, where the ring system
occupied a similar position as rimonabant. Energy optimization
of the complex was carried out as described above without any
constraints. In the resulting optimized structure, one of the
carbonyl oxo groups formed an H-bond with Ser383 (7.39). This
residue was previously reported to be crucial for taranabant bind-
ing.24 Moreover, the other carbonyl oxygen got into a favorable
orientation to form an H-bond with Lys192 (3.28), but similarly
to the case of rimonabant, the conformation of Lys192 (3.28) was
suboptimal for a strong interaction. Therefore a second energy
optimization was carried using a distance constraint between the
appropriate oxygen of compound 45 and the side chain nitrogen
References and notes
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(Euroscreen s.a., Belgium, Cat. No: ES-110-F). Cells grown in ultraCHO medium
without antibiotics were loaded with equal volume of loading buffer (20 l)
and were incubated for 1 h at 37 °C. 10 l of test compounds were added and
a
16 protein
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l
l
the plates were incubated for 10 min at room temperature. The intracellular
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l
l of agonist (CP 55,940). The concentration–effect relationship for
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l
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and the final DMSO concentration was set to be 0.9%.
11. In vitro [3H]SR141716A ligand binding at CB1 receptors. Rat cerebellum was
removed and homogenized and a membrane preparation was stored at ꢁ80 °C
until use. A solution of 0.4 nM [3H]SR-141716A in 50 mM Tris–HCl buffer
complemented with 5 mM MgCl2, 1 mM EDTA and containing 20% ethanol was
prepared freshly each day from a stock solution (0.002 mCi/ml, in ethanol).
Incubation was performed in the above-mentioned buffer complemented with
1 mg/ml bovine serum albumin (BSA) and 1 mM dithiothreitol (DTT) for
60 min at 30 °C in a thermostated shaker. Incubation mixture (total volume of