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

Article abstract of DOI:10.1016/j.bmcl.2003.12.036

A novel class of 6-aryl-6H-pyrrolo[3,4-d]pyridazine ligands for the α₂δ subunit of voltage-gated calcium channels has been described. Substitutions in the aryl ring of the molecule were generally not tolerated, and resulted in diminished bindin

Full text of DOI:10.1016/j.bmcl.2003.12.036

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1295–1298
Synthesis and biological evaluation of
6-aryl-6H-pyrrolo[3,4-d]pyridazine derivatives: high-affinity
ligands to the ꢀ2ꢁ subunit of voltage gated calciumchannels
Brian A. Stearns,a,* Naomi Anker,a Jeannie M. Arruda,a Brian T. Campbell,a
Chixu Chen,a Merryl Cramer,a Tao Hu,a Xiaohui Jiang,a Kenneth Park,b Kun Kun Ren,b
Marciano Sablad,b Angelina Santini,c Herve Schaffhauser,c Mark O. Urbanb
and Benito Munoza
aDepartment of Medicinal Chemistry, Merck Research Laboratories, MRLSDB2, 3535 General Atomics Court,
San Diego, CA 92121, USA
bDepartment of Behavioral Pharmacology, Merck Research Laboratories, MRLSDB1, 3535 General Atomics Court,
San Diego, CA 92121, USA
cDepartment of Neuropharmacology, Merck Research Laboratories, MRLSDB1, 3535 General Atomics Court,
San Diego, CA 92121, USA
Received 11 November 2003; revised 4 December 2003; accepted 5 December 2003
Abstract—A novel class of 6-aryl-6H-pyrrolo[3,4-d]pyridazine ligands for the a2d subunit of voltage-gated calcium channels has
been described. Substitutions in the aryl ringof the molecule were generally not tolerated, and resulted in diminished bindingto the
a2d subunit. Modifications to the pyridazine ringrevealed numerous permissive substitutions, and detailed SAR studies were car-
ried out in this portion of the molecule. Replacement of the pyridazine ringmethyl group with an aminomethyl functionality pro-
vided greatly improved potency over the initial lead. The initial lead compound displayed good rat pharmacokinetic properties, and
was shown to be efficacious in the Chungmodel for neuropathic pain in rats.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
subunit of voltage gated calcium channels (VGCCs).6
Although various hypotheses have been put forth for
the in vitro functional consequences of bindingto the
a2d subunit of VGCCs, neither a signal transduction
pathway nor a functional consequence of a2d binding
have been unambiguously defined. Despite this fact, it
has been postulated that the efficacy of gabapentin in
neuropathic pain models may be a result of interactions
with the a2d subunit.7
Gabapentin (1, Neurontin) is a clinically prescribed
anticonvulsant agent employed for the treatment of
epilepsy.1 Recent studies have suggested that gaba-
pentin and related GABA analogue pregabalin (2) may
represent promisingnew therapeutic aegnts for treat-
ment of both neuropathic pain2 and anxiety.3 Further-
more, efficacy for both Gabapentin and pregabalin (2)
has been demonstrated in clinical neuropathic pain in
humans (Fig. 1).4
A number of publications have disclosed extensive SAR
studies around the general gabapentin structure.8 Our
Although originally designed as a constrained GABA
analogue, gabapentin was found to have no activity at
3
GABA receptors.5 Exposure of H-gabapentin to por-
cine cerebral cortex membranes resulted in the identifi-
cation of a single high affinity binding site, the a2d
* Correspondingauthor. Tel.: +1-858-202-5307; fax: +1-858-202-
5752; e-mail: brian_stearns@merck.com
Figure 1. Gabapentin (1) and Pregabalin (2).
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.036

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B. A. Stearns et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1295–1298
goal was to probe the a2d hypothesis further by search-
ingfor non amino acid small molecules with binding
affinity to the a2d subunit in an effort to further under-
stand the pharmacology of gabapentin. Pyrrolopyr-
idazine (3) was identified through a high-throughput
bindingassay in a program directed at the discovery of
non amino acid gabapentin mimetics. Utilizing 3H-
gabapentin as the radioligand, compound (3) displayed
an IC50 of 180 nM in rat a2d membranes (Fig. 2).
Condensation of 2,5-hexanedione (7) with p-phenetidine
(8) afforded dimethylpyrrole 9. Exposure of 9 to the
action of acetic anhydride smoothly provided mono-
acetyl pyrrole 10.10 Acetylpyrrole 10 was reacted with a
variety of acid chlorides in the presence of SnCl4. This
afforded the intermediate diacyl pyrroles, which were
condensed with hyrdazine in EtOH to afford substituted
pyrrolopyridazines 11.
Removal of one or both of the methyl groups adjacent
to the pyridazine was accomplished as shown in Scheme
3. Exposure of 9 to the action of the Vilsmeier reagent
afforded a 9:1 mixture of mono and diformyl pyrroles
12 and 13. Intermediate 12 was reacted with acetyl
chloride and SnCl4, followed by hydrazine to afford 14.
Intermediate 13 was reacted with hydrazine to afford 15.
This compound was an attractive lead from a medicinal
chemistry point of view, and in this communication, we
report the synthesis and biological evaluation of vari-
ants of this structure.
2. Chemistry
Installation of heteroatoms adjacent to the pyridazine
was accomplished utilizingthe chemistry outlined in
Scheme 4. Condensation of p-phenetidine with ethyl 2-
acetyl-4-oxovalerate11 (16) afforded ester 17. Acylation
with acetyl chloride and SnCl4, followed by conden-
The general synthetic approach towards the synthesis of
analogues of 3 is shown in Scheme 1. Condensation of a
variety of substituted anilines (5) with commercially
available tetraketone (4) provided correspondingdiace-
tyl pyrroles.9 These crude pyrroles were exposed to
excess hydrazine in ethanol at room temperature. The
resultingpyrrolopyridazines were isolated in good yield
by filtration after dilution of the reaction mixtures with
water.
Modifications in the pyridazine ringof the molecule
were accomplished with the chemistry shown in
Scheme 2.
Scheme 3. Reagents: (i) POCl3, DMF, 90%; (ii) AcCl, SnCl4, CH2Cl2,
85%; (iii) H2NNH2, EtOH (99%).
Figure 2. Pyrrolopyridazine (3).
Scheme 1. Reagents: (i) AcOH, PhMe, Á; (ii) H2NNH2, EtOH, (60–
99%).
Scheme 4. Reagents: (i) AcOH, PhMe, Á; (ii) AcCl, SnCl4, CH2Cl2;
(iii) H2NNH2, EtOH (75%); (iv) POCl3, Á, 95%; (v) NaH, MeI,
DMF, 49%; (vi) NHRR0 or NaOR, DMF (30–65%).
Scheme 2. Reagents: (i) AcOH, PhMe, Á; (ii) Ac2O (80%); (iii)
RCOCl, SnCl4, CH2Cl2; (iv) H2NNH2, EtOH (65–90%).

B. A. Stearns et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1295–1298
1297
sation with hydrazine afforded pyridazinone 18.12 This
intermediate was N-methylated by exposure to sodium
hydride and methyl iodide to afford 22. Alternatively,
exposure of 18 to POCl3 provided chloropyridazine 19.
The corresponding2-amino ( 20) or 2-alkoxypyridazines
(21) were prepared by reactingchloride 19 with either
amines or sodium alkoxides in DMF.
the molecule. Replacement of one (14) or both (15)
pyridazine ring methyl groups with hydrogens abolished
a2d binding. Installation of groups larger than methyl in
this position was tolerated. Table 2 shows representative
SAR information on the pyridazine methyl group
replacements made in 3. Replacement of the methyl
group in 3 with a phenyl group afforded 11a (a2d IC50
382 nM), suggesting the existence of a lipophilic pocket
adjacent to the pyridazine nitrogen. Scanning sub-
stituents on the newly introduced phenyl ringdemon-
strated significant stereoelectronic effects in the binding
affinities of the pyrrolopyridazine ligands to the a2d
subunit. In general, analogues with electron donating
groups were preferred relative to electron withdrawing
groups, and para substituents were preferred relative to
meta and ortho substituents. Potency tracked well with
this trend: 4-OMe>3-OMe>2-OMe4-Cl>3-Cl>2-Cl.
The optimal substituent identified in the phenyl ringwas
a 4-methyl group (11b) with a bindingaffinity of 86 nM.
Substituents as large as a phenyl group (11k) were tol-
erated (a2d IC50 1200 nM). A variety of alkyl and
cycloalkyl groups were installed in this position (11o–x)
and displayed good binding affinity to a2d.
3. Biological results
Initial exploration into the primary lead 3 focused on
the right-hand ethoxyphenyl ring. Representative SAR
information from the right-hand portion of the mole-
cule is shown in Table 1. Compounds were profiled in a
bindingassay utilizing 3H-gabapentin as the radio-
ligand.13 In general, replacement of the ethoxyphenyl
group of 3 provided analogues with diminished a2d
potency relative to 3. Shiftingthe ethoxy group from
the 4 position to the 3 position (6a) or replacement of the
ethoxy group with a methoxy group (6d) abolished a2d
binding. However, substituents in the 2-position appeared
to be tolerated, as 2-ethoxy derivative 6b showed only a
10-fold loss in potency at the a2d protein relative to 3.
Replacement of the pyridazine methyl group of 3 with
heteroatoms provided more promisingimprovements in
bindingaffinity. Compound 20a in which the pyridazine
methyl group of 3 was replaced with an N-methyl group
showed a significant increase in potency. The corres-
pondingring-alkylated isomer 21 was significantly less
potent, and the correspondingketo tautomers 22 and 18
did not bind to a2d. Dimethylamino derivative 20b was
significantly less potent than the corresponding mono-
methyl amine 20a (Table 3).
Given the tight SAR revealed in the ethoxyphenyl ring,
efforts were shifted to the pyrrolopyridazine portion of
Table 1. a2d Bindingaffinities for compounds 3 and 6a–e
Compd
R
a2d IC50, nMa
3
4-OEt
3-OEt
2-OEt
4-OMe
4-O-n-Pr
180
>10,000
1850
>10,000
5180
6a
6b
6d
6e
Groups as large as aryl were tolerated on the nitrogen
(20c, 20e, and 20f). Introduction of polar substituents
on the aryl ringsuch as amino groups ( 20b) or pyridyl
groups (20g) diminished a2d binding.
a Values are means of three experiments.
Table 2. a2d Bindingaffinities for compounds 11a–x
a2d IC50, nMa
Table 3. a2d Bindingaffinities for compounds 20a–g and 21
Compd
R
Compd
R
R0
a2d IC50, nMa
11a
11b
11c
11d
11e
11f
11g
11h
11i
Ph
4-Methylphenyl
4-Methoxyphenyl
3-Methoxyphenyl
2-Methoxyphenyl
4-Chlorophenyl
3-Chlorophenyl
2-Chlorophenyl
2,4-Dimethoxyphenyl
4-Ethylphenyl
p-Biphenyl
382
86
20a
20b
20c
20d
20e
20f
20g
21
Me
Me
Ph
H
Me
H
H
H
H
H
n/a
40
170
75
154
335
737
701
1597
4367
597
107
1263
153
1729
528
250
148
360
263
171
577
211
185
235
170
4-NH2-Ph
4-OMe-Ph
4-Me-Ph
4-Pyridyl
Me
>10,000
440
240
>10,000
2181
11j
11k
11l
a Values are means of three experiments (n/a=not applicable).
4-Ethoxyphenyl
4-Trifluoromethoxyphenyl
p-Fluorophenyl
Et
11m
11n
11o
11p
11q
11r
11s
11t
11u
1v
Table 4. Selected rat pharmacokinetic parameters for compound 3
2-Cyclopentylethyl
n-Pentyl
Dose/Route
2 mg/kg iv
10 mg/kg po
20 mg/kg ip
3-Methylbutyl
2-Methylpropyl
2,4,4-Trimethylpentyl
Cyclopentylmethyl
Cyclopentyl
%F
n/a
6.4
n/a
n/a
0.91
18.5
1.3
81%
27
n/a
141
101
0.3
1.2
n/a
n/a
AUC (mM*h)
Cmax (mM)
Tmax (h)
T1/2 (h)
Clp (mL/min/kg)
Vdss (L/kg)
3.6
2.8
4.5
n/a
n/a
11w
11x
Cyclohexyl
Cyclopropyl
a Values are means of three experiments.
n/a, not applicable.

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B. A. Stearns et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1295–1298
4. Conclusion
In conclusion, a novel series of non-amino acid a2d
ligands has been disclosed. Like gabapentin and prega-
balin, compound 3 displays efficacy in a preclinical
model for neuropathic pain. Further profilingof these
novel compounds is currently underway.
References and notes
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Figure 3. Data for compound 3 in the rat Chungmodel.
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Figure 4. Data for gabapentin (1) in the rat Chungmodel.
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Compound 3 was examined in rat pharmacokinetic
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parameters.14 Doses of 20 mg/kg ip resulted in plasma
concentrations of 100 mM and an AUC of 141 mM*h.
The compound displayed excellent oral bioavailability,
and low clearance (Table 4).
12. Lapinski, L.; Fulara, J.; Czerminski, R.; Nowak, M. J.
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13. [3H]-gabapentin binding assay was performed with A710
cell membranes and was adopted from the method pre-
viously described by Sunam-Chauhan et al., 1993: Suman-
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In light of the favorable pharmacokinetic profile of 3,
we were compelled to examine the performance of the
compound in the rat Chungmodel 15 (neuropathic pain).
Figures 3 and 4 shows Chungmodel data for compound
3 (n=6 rats per treatment group). In a dose-dependent
fashion, 3 provided full reversal of tactile allodynia in
rats. An ID50 of 9.4 mg/kg was obtained after intrapar-
itoneal dosing. This compound was slightly more potent
than gabapentin (1, ID50=39 mg/kg ip, n=6 per treat-
ment group) (shown in Fig. 3) in the Chungmodel.
14. For rat pharmacokinetic studies, male Sprague–Dawley
rats (300–400 g) were dosed at 2, 10, and 20 mg/kg iv,
p.o., and ip respectively. Vehicle for the studies was saline.
Heparinized blood samples were collected at 0.083, 0.25,
0.5, 1, 2, 4, and 8 h. Plasma was obtained by centrifuga-
tion and stored at ꢀ80ꢁC until analysis.
15. Kim, S. H.; Chung, J. M. Pain 1992, 50, 355.