Synthesis and biological evaluation of 6-aryl-6H-pyrrolo[3,4-d]pyridazine derivatives as high-affinity ligands of the α2δ subunit of voltage-gated calcium channels

Article abstract of DOI:10.1016/j.bmcl.2004.02.070

A novel class of 2H-pyrrolo[3,4-c]pyridazine ligands of the α₂δ subunit of voltage-gated calcium channels is described. Compound 4a with high affinity toward α₂δ was identified through structure-activity relationship studies of the l

Full text of DOI:10.1016/j.bmcl.2004.02.070

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2031–2034
Synthesis and biological evaluation of 6-aryl-6H-pyrrolo[3,4-d]-
pyridazine derivatives as high-affinity ligands of the a2d subunit
of voltage-gated calcium channels
Tao Hu,a,* Brian A. Stearns,a Brian T. Campbell,a Jeannie M. Arruda,a Chixu Chen,a
b
b
b
b
ꢀ
Jayashree Aiyar, Robert E. Bezverkov, Angelina Santini, Herve Schaffhauser,
Wensheng Liu,c Shankar Venkatramana and Benito Munoza
aDepartment of Medicinal Chemistry, Merck Research Laboratories, MRLSDB2, 3535 General Atomics Court, San Diego,
CA 92121, USA
bDepartment of Neuropharmacology, Merck Research Laboratories, MRLSDB1, 3535 General Atomics Court, San Diego,
CA 92121, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 22 January 2004; accepted 13 February 2004
Abstract—A novel class of 2H-pyrrolo[3,4-c]pyridazine ligands of the a2d subunit of voltage-gated calcium channels is described.
Compound 4a with high affinity toward a2d was identified through structure–activity relationship studies of the lead compound.
Tritiated ligand [3H]-4b was synthesized to demonstrate that this ligand binds to the same site as Gabapentin toward a2d subunit of
voltage-gated calcium channels.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
posed that this site may be involved in the mediation of
the pharmacological actions of Gabapentin.5
Gabapentin (1-(aminomethyl)cyclohexane acetic acid;
Neurontin), 1, is a novel anticonvulsant agent, which is
useful as add-on therapy in the treatment of epileptic
seizures (Fig. 1).1 Several clinical reports suggest that
Gabapentin has therapeutically beneficial effects against
chronic pain states and anxiety.2 Originally, Gabapentin
was designed as a lipophilic c-amino butyric acid
(GABA) analogue; however, it has since been shown not
to interact directly with any of the enzymes in the meta-
bolic pathways of glutamate and GABA at any sig-
nificant concentration, nor does it bind to any of the
GABA receptors.3 Recently, it has been shown that
Gabapentin binds with high affinity to a novel site on
the a2d subunit of a calcium channel;4 it has been pro-
Herein, we report the synthesis and evaluation of
nonamino acid a2d ligands identified during our
screening campaign. The initial lead, pyrrolopyridazine
(2), identified through a high-throughput [3H]-Gaba-
pentin binding assay, exhibited an IC50 of 180 nM in
A710 cells.6 In a previous communication,7 we docu-
mented the SAR result primarily on the pyrrolopyrid-
azine portion of the lead structure 2. In this paper, we
report our continued SAR studies of 2, with emphasis
on the phenyl ring.
H2N
CO2H
N
N
N
OEt
2
Keywords: Gabapentin; Affinity; SAR.
* Corresponding author. Tel.: +1-858-202-5587; fax: +1-858-202-5752;
e-mail: tao_hu@merck.com
Figure 1. Structure of Gabapentin 1 and lead compound 2.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.070

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T. Huet al. / Bioorg. Med. Chem. Lett. 14 (2004) 2031–2034
2. Results and discussion
Electron donating groups such as methoxy (4a), ethyl
(4b), vinyl (4c), thiomethyl (4e), and ethoxy (4f) retained
Structure–activity studies were focused around modify-
ing the phenyl ring system and directed toward
improving the potency of the series. Our initial attempt
was to replace the right phenyl ring with various het-
erocycles such as pyridine, pyridazine, or pyrimidine,
afforded analogues with diminished binding affinities to
the a2d subunit relative to 2 (Table 1).
Table 2. a2d Binding affinity of compounds 2 and 4a–m
R
N
N
OEt
N
Compounds
R
a2d Binding IC50 (nM)
As the heterocyclic replacements did not improve
potency, we focused on introducing one or more methyl,
alkoxy, or halogen substitutions around the phenyl ring
as shown in Table 2. Incorporating substituents on the
phenyl ring revealed dramatic stereo-electronic effects in
the binding affinity of the pyrrolopyridazine lead to a2d
subunit. A highly potent compound 4d (a2d IC50 27 nM)
with an ortho-methyl substituent was identified. In
contrast, the corresponding meta-methyl substituted
analogue (4e, a2d IC50 298 nM) was 10-fold less potent
as compared to compound 4a (Table 2). The presence of
the ortho-methyl moiety most likely changes the con-
formation of the molecule by placing the two rings
perpendicular to each other such that the phenyl group
rotates with respect to the pyrrolopyridazine ring thus
4a
4
OEt
MeO
4b
4c
13
25
OEt
OEt
4d
4e
27
28
OEt
providing
a comparable nonplanar conformation,
resulting in improved binding affinity. However, incor-
porating additional methyl group, leading to dimethyl
analogues, either in the 3-, 4-, or 5-position (4g,h and 4j)
failed to significantly improve potency.
OEt
MeS
EtO
To further optimize binding affinity of 4e, SAR was
focused around various ortho-substituents on the phenyl
ring while retaining ethoxy group at the para-position.
4f
OEt
OEt
OEt
33
54
4g
Table 1. a2d Binding affinity of compounds 2 and 3a–f
N
N
Ar
N
4h
94
Compounds
Ar¼
a2d Binding IC50 (nM)
Cl
2
180
OEt
OEt
OEt
4i
4j
100
282
N
3a
3b
3c
2100
5500
6100
OEt
OEt
OEt
N
OH
4k
4l
292
298
OEt
N
N
N
N
OEt
OEt
3d
3f
7600
OEt
OEt
N
N
4m
313
>10000
F

T. Huet al. / Bioorg. Med. Chem. Lett. 14 (2004) 2031–2034
2033
or improved binding affinity significantly; the methoxy
group provided the most potent compound (4a, IC50
4 nM) in this series. Ligand 4a showed a 45-fold increase
in potency over the initial lead 2. Various electron
withdrawing groups at ortho-position, exemplified by
4m, led to significant decrease in potency. In general,
analogues with electron donating groups were preferred,
and ortho-substituents were preferred relative to meta-
substituents.
and the resulting intermediates were treated with
hydrazine to afford compounds 6a and 6e in 90% yield
over two steps.
To establish that binding affinity of the compounds is
specific for a2d subunit, the radio ligand [3H]-4b was
prepared from the commercially available (2-amino-5-
ethoxyphenyl)-methanol 10 (Scheme 3). Compound 10
was ethylated, followed by reduction (H2/Pd) of the
nitro group to afford the free amine 11. This crude
material was then condensed with tetraketone 5, fol-
lowed by subsequent treatment with hydrazine to yield
3. Chemistry
1. EtBr, K2CO3
O2N
OH
H2N
OEt
The general synthetic approach for the synthesis of
analogues of 3 is shown in Scheme 1. Condensation of a
variety of anilines 6 with commercially available tetra-
ketone 5 provided corresponding diacetyl pyrroles.8
Treatment of diacetylpyrroles with excess of hydrazine
in ethanol at room temperature over 30 min, followed by
quenching with water afforded the desired pyrrolo-
pyridazines 3 after simple filtration.
2. H2/Pd
90 %
OH
OH
10
11
1. AcOH/EtOH, 3
2. NH2NH2
83 %
MnO2, rt;
N
O
N
O
The chemical synthesis of analogues 4a and 4f is out-
lined in Scheme 2. Condensation of 2,4-dimethoxy ani-
N
OEt
OEt
N
86 %
line
7
with tetraketone
3
afforded pyrrole 8.
O
H
OH
Regioselective removal of para-methoxy group of the
phenyl ring was achieved by treating compound 8 with
boron tribromide (2.0 equiv, 0 °C), to afford 9a and 9b in
80% and 15% yield, respectively.9 Compounds 9a and 9b
were mono and bis-ethylated under standard procedure,
13
12
Ph3P+CH3Br-,
nBuLi,THF, rt
90 %
NH2NH2
100%
N
N
N
OEt
OEt
N
N
4b
N
OEt
O
O
O
O
T2/Pd
100%
N
N
N
1. AcOH, EtOH
N
N
+
4c
H2N Ar
N Ar
2. NH2NH2
(5-65% over all)
H3
[3H]-4b
H3
5
6
3
Scheme 1.
Scheme 3.
AcOH, EtOH,
reflux, 12 h
O
O
O
O
O
O
N
OMe
H2N
MeO
OMe
O
8, 95%
7
5
BBr3, CH2Cl2,
0 oC to rt;
1. EtBr, DMF,
K2CO3
O
O
N
N
N
N
OR1
OR1
2. NH2NH2
(92%, two steps)
R2O
R2O
4a; R1 =Et, R2 = Me
4f; R1 = R2 = Et
9a; R1 = H, R2 = Me; 80 %
9b; R1 = R2 = H; 15 %
Scheme 2.

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T. Huet al. / Bioorg. Med. Chem. Lett. 14 (2004) 2031–2034
Tritiated analogue of 4b was successfully synthesized
and evaluated in the Gabapentin binding assay. This
allowed us to demonstrate that these ligands bind with
high affinity and are Gabapentin mimetic under the
described assay conditions.
References and notes
1. Taylor, C. P. In New Trends in Epilepsy Management;
Chadwick, D., Ed.; Royal Society of Medicine Service
Ltd: London, 1993; pp 13–40.
2. Bryans, J. S.; Wustrow, D. J. Med. Res. Rev. 1999, 19,
149–177.
Figure 2. Specific binding of [3H]-4b to a2d subunit.
3. (a) Satzinger, G. Arzneim. Forsch. 1994, 44, 262–266; (b)
Taylor, C. P. Neurology 1994, 44, S10–16.
4. Gee, N. S.; Brown, J. P.; Dissanayake, V. U. K.; Offord,
J.; Thurlow, R.; Woodruff, G. N. J. Biol. Chem. 1996, 271,
5768–5776.
5. Field, M. J.; Oles, R. J.; Lewis, A. S.; McCleary, S.;
Hughes, J.; Singh, L. Br. J. Pharmacol. 1997, 121, 1513–
1522.
6. [3H]-Gabapentin binding assay in human brain membrane
(A710 membrane) was adopted from the method in the
literature. A710 cells are defined as HEK 293 cells stably
transfected with human a1B, b3a, and a2d-1b subunits.
Suman-Chauhan, N.; Webdale, L.; Hill, D. R.; Woodruff,
G. N. Eur. J. Pharmacol., Mol. Pharmacol. Sect. 1993,
244, 293.
7. Stearns, B.; Anker, N.; Arruda, J.; Campbell, B.; Chen, C.;
Hu, T.; Sablad, M.; Park, K.; Ren, K.; Santini, A.;
Schaffhauser, H.; Urban, M.; Munoz, B. Bioorg. Med.
Chem. Lett., 2004, 14, 1295–1298.
8. All intermediates and target compounds provided satis-
1
factory H and LCMS spectra, and elemental analysis.
compound 12 (83% yield). This allylic alcohol was oxi-
dized with manganese dioxide at room temperature to
generate the corresponding aldehyde 13. The aldehyde
was then converted to olefin 4c under Wittig conditions
to yield 4c in 90% yield. Treatment of 4c with excess
hydrazine (30 equiv, EtOH, rt) for 2 h resulted in com-
plete reduction of the vinylic double bond to afford
ligand 4b (100%).10 Tritiated ligand [3H]-4b was then
prepared from 4c via standard tritiation procedure (T2/
Pd).
The ligand [3H]-4b was used to evaluate the specific
binding affinity of these compounds toward a2d subunit
of voltage-gated calcium channels and also in A710 cell
membrane (Fig. 2). [3H]-4b showed high specific binding
(Kd ¼ 4 nM) to soluble human a2d and in A710 cell.
Ligand [3H]-4b was completely displaced by Gabapentin
and cold 4b in soluble version of the protein and in
A710 cells membrane, suggesting that these nonamino
acid ligands are Gabapentin mimics in this in vitro
assay.
9. Interestingly, monodemethylation of the ortho-methoxy
group of compound 10 was effected using EtSNa/DMF
reaction conditions. For reference, see: Lal, K.; Ghosh, S.;
Salomon, R. J. Org. Chem. 1987, 52, 1072–1078.
10. We found that this metal-free double bond reduction
condition was general in other reaction systems. Detailed
discussions regarding the scope and limitation will be
reported in due course.
In summary, further SAR on the phenyl ring lead to
identification of ortho-substituents (4a,b) that improved
potency by 10- to 40-fold over the lead compound 2.