Identification and synthesis of [1,2,4]triazolo[3,4-a]phthalazine derivatives as high-affinity ligands to the α2δ -1 subunit of voltage gated calcium channel

Article abstract of DOI:10.1016/j.bmcl.2004.03.008

We have identified and synthesized a series of [1,2,4]triazolo[3,4-a] phthalazine derivatives as high-affinity ligands to α₂δ-1 subunit of voltage gated calcium channels. Structure-activity relationship studies directed toward improving the potency and physical properties of 2 lead to the discovery of 20 (IC₅₀=15nM) and (S)-22 (IC₅₀=30nM). A potent and selective radioligand, [³H]-(S)-22 was also synthesized to demonstrate that this ligand binds to the same site as gabapentin.

Full text of DOI:10.1016/j.bmcl.2004.03.008

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2463–2467
Identification and synthesis of [1,2,4]triazolo[3,4-a]phthalazine
derivatives as high-affinity ligands to the a2d-1 subunit of voltage
gated calcium channel
Alec D. Lebsack,a,*,ꢀ Janet Gunzner,a,ꢀ Bowei Wang,a Richard Pracitto,a
b
b
b
b
ꢀ
Herve Schaffhauser, Angelina Santini, Jayashree Aiyar, Robert Bezverkov,
Benito Munoz,a Wensheng Liuc and Shankar Venkatramana
aDepartment of Chemistry, Merck Research Laboratories, MRLSDB2, 3535 General Atomics Court, San Diego, CA 92121, USA
bDepartment of Neurobiology, Merck Research Laboratories, MRLSDB1, 3535 General Atomics Court, San Diego, CA 92121, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, RY80R, PO Box 2000, Rahway, NJ 07065, USA
Received 3 February 2004; revised 26 February 2004; accepted 3 March 2004
Abstract—We have identified and synthesized a series of [1,2,4]triazolo[3,4-a]phthalazine derivatives as high-affinity ligands to a2d-1
subunit of voltage gated calcium channels. Structure–activity relationship studies directed toward improving the potency and
physical properties of 2 lead to the discovery of 20 (IC50 ¼ 15 nM) and (S)-22 (IC50 ¼ 30 nM). A potent and selective radioligand,
[3H]-(S)-22 was also synthesized to demonstrate that this ligand binds to the same site as gabapentin.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
hypothesized to be mediated through this high affinity
binding site.
Gabapentin (1-(aminomethyl)cyclohexane acetic acid;
Neurontinâ) 1 is an anticonvulsant1 that is also effica-
cious for the treatment of neuropathic pain in human
and animal models.2 Recently, gabapentin has been
shown to bind with high affinity to the a2d-1 subunit
of voltage gated calcium channel3 (IC50 ¼ 30 nM).4
Although the mechanism of action is not well under-
stood, gabapentin’s pharmacological actions have been
We sought to discover novel small molecule lead struc-
tures with high affinity binding to a2d-1 subunit. The
initial lead, [1,2,4]triazolo[3,4-a]phthalazine 2 with a
binding affinity for a2d-1 of IC50 ¼ 220 nM was identi-
fied by a high throughput screen. Herein, we report
the identification and synthesis of [1,2,4]triazolo[3,4-
a]phthalazine derivatives as high-affinity ligands to the
a2d-1 subunit of voltage gated calcium channel. Our
primary goal was to optimize the binding affinity of
these ligands through structure–activity relationships
(SAR) focusing mainly on modifications to the methyl
isoxazole and phenethylamine moieties.
N
N
N
O
N
N
NH2 COOH
O
2. Biological results
N
H
1
Meager physical properties such as poor solubility led to
the immediate replacement of the methyl isoxazole unit
in 2 with more soluble groups (Table 1). In general,
replacement of the methyl isoxazole with a variety of
alkyl groups resulted in only modest changes in binding
affinity for the a2d-1 subunit. However, replacement of
methyl isoxazole by trifluoromethyl 5 produced a 3-fold
increase in potency (IC50 ¼ 72 vs 220 nM) and also
N
2
* Corresponding author. Tel.: +1-858-202-5588; fax: +1-858-202-5753;
e-mail: alec_lebsack@merck.com
ꢀ
Both authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.008

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A. D. Lebsack et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2463–2467
Table 2. a2d-1 Binding affinities for substitutions on phenyl ring
Table 1. a2d-1 Binding affinities for isoxazole replacements
N
N
N
N
CF3
X
N
N
N
N
O
O
N
O
H
N
N
H
N
Y
Compds
X
H
a2d-1 binding IC50 (nM)
Compds
Y
a2d-1 binding IC50 (nM)
N
13
14
15
16
17
18
19
20
21
2-OMe
3-OMe
4-OMe
2-F
3163
116
829
291
161
630
448
15
2
220
3
4
5
6
7
8
230
145
72
3-F
4-F
Me
CF3
SMe
Et
2-Me
3-Me
4-Me
194
241
131
260
CH2OCH3
9
272
342
10
i-Pr
Table 3. a2d-1 Binding affinities for modifications to linking tether
N
N
11
12
145
195
CF
3
N
N
O
N
Z
provided improved solubility. Therefore our strategy
consisted of maintaining the 3-trifluoromethyl func-
tionality in the [1,2,4]triazolo[3,4-a]phthalazine, while
SAR was conducted on the phenethylamine portion of
the molecule.
Compds
Z
a2d-1 binding IC50 (nM)
H
N
rac-22
36
Having discovered the trifluoromethyl group as a
replacement for methyl isoxazole, we turned our atten-
tion to substituting methoxy, fluorine, and methyl
groups around the phenyl ring (Table 2). From the
structure–activity trends found in compounds 13–21, it
was apparent that the 3-substituted phenyl rings were
consistently more potent than the corresponding 2- or
4-substitutions. The most potent compound in this series
was 3-methyl-phenyl-[1,2,4]triazolo[3,4-a]phthalazine 20
(IC50 ¼ 15 nM) showing a 5-fold increase in potency
over unsubstituted phenyl 5 and a 14-fold increase over
the initial methyl isoxazole lead 2.
H
N
(S)-22
30
H
N
(R)-22
rac-23
rac-24
10,000
183
H
N
H
N
As part of our efforts to improve metabolic stability of
the amine in the scaffold, we incorporated alkyl groups
on the linking tether between the pyridine and phenyl
moiety (Table 3). These changes resulted in the identi-
fication of another high affinity analogue, rac-22
(IC50 ¼ 36 nM). To determine the influence of the chiral
center on the biological activity, the enantiomers of rac-
22 were synthesized. As shown in Table 3, only the (S)-
22 (IC50 ¼ 30 nM) showed binding affinity while (R)-22
was essentially inactive. Exchanging the methyl group in
rac-22 for other alkyl groups such as rac-23 and rac-24
174
H
N
rac-25
(S)-25
1369
700
H
N

A. D. Lebsack et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2463–2467
2465
Table 3 (continued)
3. Chemistry
Compds
Z
a2d-1 binding IC50 (nM)
The synthesis began with commercially available 1,4-di-
chlorophthalazine 32,6 which was transformed to
6-chloro-3-trifluoromethyl-[1,2,4]triazolo[3,4-a]phthal-
azine 33 using an improved version of a procedure
developed earlier (Scheme 1).7 Exposure of 6-chloro-3-
trifluoromethyl-[1,2,4]triazolo[3,4-a]phthalazine 33 and
pyridine 348 to lithium bis(trimethylsilyl)amide in DMF
at )78 °C delivered the coupled pyridal-[1,2,4]triaz-
olo[3,4-a]phthalazine 35 in 69% yield.9 Cleavage of the
silyl ether in the presence of TBAF and oxidation of the
resulting alcohol with Dess–Martin periodinane10 pro-
vided aldehyde 36 in 75% yield over two steps. Reductive
amination of the aldehyde 36 in the presence of the
corresponding amine (R2HNR3) and NaBH(AcO)3
provided 5, 13–21, and 25–29 in 70–80% yield.11 A sim-
ilar reductive amination protocol was used to prepare
rac-22–rac-24 and rac-30 from ketones 37–39.
H
N
26
10,000
H
N
rac-27
rac-28
198
229
H
N
N
29
10,000
H
N
The synthetic approach to enantiomerically enriched
[1,2,4]triazolo[3,4-a]phthalazine (S)-22 is detailed in
Scheme 2. Reduction of ketone 4012 by reaction with
0.7 equiv of (S)-oxazaborolidine 41 and 1.1 equiv of
BH3ÆTHF at )20 °C provided enantioenriched alcohol
(R)-42.13;14 Mitsunobu reaction of the alcohol (R)-42
rac-30
29
28
30
H
N
Br
(S)-31
H
N
3H
[3H]-(S)-22
O
N
Cl
N
H2N
N
H
CF3
CF3
N
N
N
N
1,4-dioxane, 80 °C
(65%)
led to significant decreases in binding affinity. Unfor-
tunately, synthesis of hybrid structure rac-30
Cl
Cl
a
32
33
(IC50 ¼ 29 nM), afforded similar potency to that of the
parent structures 20 and rac-22.
N
N
CF3
N
N
To rigorously establish that binding affinities of the
compounds presented were specific for a2d-1, we pre-
pared [3H]-(S)-22 from the corresponding bromide (S)-
31 by hydrogenation (Table 3). [3H]-(S)-22 showed high
specific binding to A710 membranes4 and soluble human
a2d-1 ([3H]-(S)-22, IC50 ¼ 30 nM) when using both
gabapentin and (S)-22 as a cold displacer (Fig. 1). This
result supports the use of [3H]-(S)-22 as a potent radio-
ligand for in vitro binding assays and demonstrated that
this ligand binds to the same site as gabapentin.
N
TBSO
OH
O
(1) TBAF, THF
34
N
LiHMDS, DMF, -78 °C
(69%)
(2) Dess-Martin
periodinane,
CH2Cl2
OTBS
35
(75% over two steps)
N
N
N
N
CF3
CF3
N
N
N
N
O
O
R2HNR3
NaBH(AcO)3,DCE
(70-80%)
N
N
NR2R3
O
R1
R1
36; R1 = H
(1) R1MgBr, THF
(2) Dess-Martin
periodinane,
CH2Cl2
37; R1= Me
38; R1= Et
39; R1= i-Pr
(50-70%)
Figure 1. Specific binding of [3H]-(S)-22 to a2d-1 subunit of human
voltage gated calcium channels.5
Scheme 1. General synthesis of [1,2,4]triazolo[3,4-a]phthalazine
derivatives.

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A. D. Lebsack et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2463–2467
Ph
H
Ph
O
TBSO
TBSO
TBSO
O
N
H
(1) DPPA, PPh3,
DEAD, THF
B
N
N
N
44
41
NaBH(AcO)3,
AcOH,DCE
BH3THF,
(2) Pd/C, H2,
MeOH
O
OH
(R)-42
NH2
THF, -20 °C
40
(S)-43
(90%)
(90%)
(65% over two steps)
N
N
N
N
CF3
CF3
N
N
N
N
TBSO
HO
(1) Boc2O, Et3N,
Cl
33
N
N
THF (80%)
O
H
Boc
N
N
(1) LiHMDS, DMF, -78 °C
(2) TBAF, THF
(72%)
N
H
N
(2) 4N HCl in dioxane
(66% over two steps)
(S)-45
(S)-46; >95% ee
(S)-22
Scheme 2. Synthesis of enantioenriched [1,2,4]triazolo[3,4-a]phthalazine derivative (S)-22.
followed by reduction of the resulting azide afforded
amine (S)-43 in 65% yield over two steps.15 Reductive
References and notes
1. Taylor, C. P. The Role of Gabapentin. In New Trends in
Epilepsy Management; Chadwick, D., Ed.; Royal Society
of Medicine Services Ltd: London, 1993; pp 13–40.
2. For recent review, see: Backonja, M.; Glanzman, R. L.
Clin. Ther. 2003, 25, 81; (a) Singh, L.; Field, M. J.; Ferris,
P.; Hunter, J. C.; Oles, R. J.; Williams, R. G.; Woodruff,
G. N. Psychopharmacol. Berl. 1996, 127, 1; (b) Rosner, H.;
Rubin, L.; Kestenbaum, A. Clin. J. Pain 1996, 12, 56; (c)
Zapp, J. J. Am. Fam. Phys. 1996, 53, 2442.
amination of (S)-43 and phenylacetylaldehyde 44 by
treatment of NaBH(AcO)3 in dichloroethane provided
(S)-45 in 90% yield.9 The secondary amine of (S)-45 was
protected with di-tert-butoxycarbonate ((Boc)2O) to
afford the carbamate in 80% yield. Cleavage of the tert-
butyldimethylsilyl ether in the presence of TBAF pro-
vided alcohol (S)-46 in 72% yield, >95% ee by chiral
HPLC analysis.16 Coupling of (S)-46 and 33 in the
presence of lithium bis(trimethylsilyl)amide in DMF at
)78 °C followed by exposure of the product to 4 N HCl
in dioxane cleaved the tert-butoxycarbonyl group to
provide (S)-22 in 66% yield over two steps. Synthesis of
(R)-22 was achieved by replacing (S)-oxazaborolidine 41
with (R)-oxazaborolidine in the initial reduction step of
Scheme 2. In addition, (S)-31 could be easily accessed by
replacing phenylacetylaldehyde 44 with 3-bromo-
phenylacetylaldehyde in Scheme 2.17
3. Gee, N. S.; Brown, J. P.; Dissanayake, V. U. K.; Offord,
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4. [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.
5. Methods: 100 lg/well of A710 membranes or 250 ng of
purified soluble human a2d-1b was used in the assay.
Binding was performed with 16 nM [3H]-(S)-22 using
protocols similar to those described in Ref. 3 (above).
Physiological saline containing 50 mM Tris pH 7.5 was
used as the assay buffer and membranes were incubated
for 1–2 h at rt.
6. 1,4-Dichlorophthalazine may also be synthesized from
1,4-phthalazinedione, see: (a) Amberg, W.; Bennani, Y. L.;
Chadha, R. K.; Crispino, G. A.; Davis, W. D.; Hartung,
J.; Jeong, K. S.; Ogino, Y.; Shibata, T.; Sharpless, K. B.
J. Org. Chem. 1993, 58, 844; (b) Becker, H.; King, S. B.;
Taniguchi, M.; Vanhessche, K. P. M.; Sharpless, K. B.
J. Org. Chem. 1995, 60, 3940.
7. 1,2,4-Triazolo[3,4,a]phthalazines 2–4 and 6–12 presented
in Table 1 were synthesized by way of the 1-chloro-4-
hydrazinophthalazine hydrochloride followed by treat-
ment of triethylamine, appropriate acid chloride, and
dioxane at reflux for 12 h. The resulting products were
elaborated using the general scheme illustrated in Scheme
1. For references, see: (a) Druey, J.; Ringier, B. H. Helv.
Chim. Acta 1951, 34, 195; (b) Blurton, P.; Burkamp, F.;
4. Conclusion
In summary, we have discovered several [1,2,4]triaz-
olo[3,4-a]phthalazine derivatives such as (S)-22 and 20
that are high-affinity ligands to a2d-1 and are structur-
ally distinct from gabapentin. Use of the [3H]-(S)-22 in
binding studies revealed it binds with high affinity to
soluble human a2d-1 and is displaced by cold gabapen-
tin and cold (S)-22. The ligands described herein will be
used as tools to better understand the role of a2d-1 in
various gabapentin mediated mechanisms.
Acknowledgements
The authors would like to thank William Bray for
analytical support.

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