1H-pyrazolo-[3,4-c]cyclophepta[1,2-c]thiophenes: A unique structural class of dopamine D4 selective ligands

Article abstract of DOI:10.1016/S0960-894X(03)00587-0

A series of novel 1H-pyrazolo-[3,4-c]cyclophepta[1,2-c]thiophenes was prepared and screened at selected dopamine receptor subtypes. Compound 4 (NGB 4420) displayed high affinity and selectivity (>100-fold) for the D ₄ over D₂ and other CNS receptors. This compound was identified as a D₄ antagonist via its attenuation of dopamine agonist-induced GTPγ³⁵S binding at D₄ receptor.

Full text of DOI:10.1016/S0960-894X(03)00587-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2921–2924
1
H-Pyrazolo-[3,4-c]cyclophepta[1,2-c]thiophenes: A Unique
Structural Class of Dopamine D Selective Ligands
4
a
a
a
a
a
Andrew Thurkauf, Xi Chen, Suoming Zhang, Yang Gao, Andrzej Kieltyka,
b
a
a
b
Jan W.F.Wasley, Robbin Brodbeck, William Greenlee, Ashit Ganguly
a,
and He Zhao *
aNeurogen Corporation, 35 Northeast Industrial Road, Branford, CT 06405, USA
bSchering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
Received 15 December 2002; accepted 25 April 2003
Abstract—A series of novel 1H-pyrazolo-[3,4-c]cyclophepta[1,2-c]thiophenes was prepared and screened at selected dopamine
receptor subtypes.Compound 4 (NGB 4420) displayed high affinity and selectivity (>100-fold) for the D over D and other CNS
4
2
3
5
receptors.This compound was identified as a D 4 antagonist via its attenuation of dopamine agonist-induced GTPg S binding at
D receptor.
#
4
2003 Elsevier Ltd.All rights reserved.
Most classical neuroleptics are generally believed to
exert their antipychotic actions through interaction with
dopamine receptors and the antipsychotic efficacy of
these agents has been shown to have a strong corre-
in the following years to identify specific D antago-
4
3
nists. The observation that clozapine, unlike typical
neuroleptics, displayed a selectivity for the D receptor
4
over the D2 sparked the theory that a selective D4
antagonist might share its atypical neuroleptic profile.
lation with their affinity for the dopamine D receptor
2
subtype.The neuroleptic agent clozapine has been
referred to as an atypical antipsychotic agent because it
does not induce the extrapyramidal motor side effects
characteristic as many other ‘typical’ antipsychotic
medications such as haloperidol and chlorpromazine.A
large body of research has been carried out in an effort
to identify the properties of clozapine which leave it
devoid of this liability.The broad spectrum of CNS
receptors for which clozapine has significant affinity has
The medicinal chemistry efforts in the D area lead to
4
the identification of a wide array of chemical entities
with selectivity for this receptor subtype over that of
4ꢀ19
D .
2
Although the search for D selective agents has
4
been fruitful, interest in this area was dampened by the
results of a number of clinical trials, notably those of L-
745,870, CP-293,019 and U-101,387 (Fig.1 ), which
failed to show significant efficacy against either positive
20ꢀ22
1
made this a daunting task.
or negative symptoms of schizophrenia.
These trials
bring into question the relevance of this receptor
subtype in the etiology of psychosis.
In 1991, Seeman characterized a new dopamine receptor
subtype.This new receptor was found to be related to
the D receptor in that stimulation of the receptor by
As part of our efforts within the area, screening of a
Schering-Plough compound library for D activity led
2
dopamine leads to an inhibition of c-AMP production
2
4
by adenylate cyclase. The localization of this new
receptor subtype, termed D , in the limbic areas of the
to the identification of SCH 26682 (1) (Fig.1 ) as a
compound with significant D4 activity.Although, in
4
central nervous system, coupled with the upregulation
of this receptor found in postmortem autoradiography
study of the schizophrenic brain, led to an intense effort
retrospect, identification of a new D selective entity
4
seems unremarkable, SCH 26682 stands out from other
known D selective compounds in a number of ways.In
4
terms of basicity, the presence of piperazine, piperidine
or pyrrolidine subunits lead that many D selective
4
3,24
2
compounds show the pK values around 7.
In con-
trast, the weakly basic pyrazole moiety of SCH 26682
*
4
Corresponding author.Te l: . +1-203-488-8201x3077; fax: +1-203-
83-7027; e-mail: hzhao@nrgn.com
a
0
960-894X/03/$ - see front matter # 2003 Elsevier Ltd.All rights reserved.
doi:10.1016/S0960-894X(03)00587-0

2922
A. Thurkauf et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2921–2924
through the condensation of the anion of 34 with ethyl
formate in toluene.The resulting aryl fused ketoalde-
hyde structure would serve as the common intermediate
in the preparation of both 1- and 2-substituted pyr-
azoles through condensation with the appropriate
hydrazine.For example, reaction of 35 with 1.3 equiv of
methylhydrazine in methanol provided compounds 5
and 21 in a ratio of 6:4 with an overall yield of 92%.
With sterically larger hydrazines, the isomer ratio was
more dramatically skewed toward the 1-substituted
pyrazoles.
Figure 1.
imparts the pK of only 3.06. In addition, SCH 26682
a
has a unique fused thiophene structure.Unfortunately,
a potentially poor pharmacokinetic profile for SCH
The imidazole 29 was prepared via the alpha bromin-
ation of 34 in acetic acid followed by heating in for-
mamide at 130 C as illustrated in Scheme 1.
ꢁ
2
6682 was indicated by a T1/2 of less than 1 min upon
exposure to human liver microsomes.In our exami-
nation of SCH 26682, we considered that replacement
of the sulfur in the thioether might prove beneficial to
the pharmacokinetic profile of the resulting derivatives.
In this paper, we disclose the details of structure–activ-
ity relationship (SAR) analysis of this unique structural
Imidazole 25 and triazoles 26 and 27 were prepared
from lactam 38, the Beckmann rearrangement product
of cyclohexanone 37 (Scheme 2).Treatment of 38 with
P S in dioxane gave thiolactam 39.Reaction of 39 with
formic or acetic hydrazide at 150 C provided triazole 26
or 27, respectively.Reaction of 39 with aminoacetalde-
hyde dimethyl acetal followed by cyclization in con-
centrated sulfuric acid gave imidazole 25.
2
5
ꢁ
class of D selective agents.
4
The synthetic route towards many of the described pyr-
azolocycloheptathiophenes (1–10, 20, and 21) is illu-
strated by the representative preparation depicted in
Scheme 1.The chlorinated ( 14–19), furanyl (11 and 12)
and phenyl (22–24 and 28) analogues were prepared in
similar fashion by use of appropriate starting materials
in the first step.The Friedel–Crafts acylation of 2,5-
dimethylthiophene 31 with ethyl glutaryl chloride pro-
duced ketoester 32.Reduction of the ketone carbonyl
using hydrazine and potassium hydroxide in triethylene
Aminomethylpyrazole 30 was prepared in five steps
from ketone 34 (Scheme 3).Condensation of the anion
of 34 with diethyloxalate followed by treatment of the
intermediate ketooxalate 41 with hydrazine and reduc-
tion gave the hydroxymethylpyrazole 42.Treatment of
ꢁ
glycol at 190 C yielded the 5-(2,5-dimethylthiophen-3-
yl)valeric acid 33 in 58% overall yield from 31.Cycli-
ꢁ
zation using polyphosphoric acid at 140 C afforded
cyclohepta[1,2-c]thiophene 34 in 80% yield.Hydroxy-
methylene ketone 35 was prepared in quantitative yield
.
OH HCl, EtOH/CH
Scheme 2. Reagents and conditions: (i) NH
rt, 16 h; (ii) PPA, EtOH/CHCl , rt, 16 h, 58% in two steps; (iii) P S
2
2
Cl
2
5
,
,
3
2
ꢁ
i
dioxane, 130 C, 1 h, 82%; (iv) H
2
NCH
2
CH(OMe
, rt, 3 h, 75%; (vi) RCONHNH
2
)
2
, PrOH, reflux,
, MeOH,
1
1
8 h, 82%; (v) concd H SO
2
40 C, 1 h, >80%.
4
2
ꢁ
Scheme 1. Reagents and conditions: (i) EtO C(CH
2
2
)
3
COCl, AlCl
, KOH, triethyl-
NH at
3
,
ꢁ
ꢁ
CH
2
Cl
2
, ꢀ10 C, 30 min, then 0 C, 1 h; (ii) NH
2
NH
2
ꢁ
ene glycol, 140 C, 10 min, then distillation of solvent and NH
2
ꢁ
2
ꢁ
ꢁ
1
80 C, then 205 C, 16 h, 58% in two steps; (iii) PPA, 140 C, 2 h,
ꢁ
Scheme 3. Reagents and conditions: (i) (CO
(ii) NH NH , MeOH, rt, 4 h; (iii) LAH, THF, rt, 2 h, 42% in three
2 2
Et) , NaH, THF, rt, 24 h;
2
NH
, MeOH, rt, 1 h, >50%; (vii) Br
2.
H
2
O,
2
2
80%; (iv) Na, EtOCHO, PhMe, 60 C, 16 h, 100%; (v) NH
MeOH, rt, 1 h, 44%; (vi) RNHNH
2
2
,
2 2 2 2
steps; (iv) SOCl , CH Cl , rt, 3 h; (v) aqueous Me NH, rt, 24 h, 82%
in two steps.
ꢁ
ꢁ
HOAc, 70 C, 0.5 h, 100%; (viii) HCONH
2
, 130 C, 7 h, 23%.

A. Thurkauf et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2921–2924
2923
4
2 with thionyl chloride and reaction of the chloro-
phene sulfur atom, whose oxidation products were pre-
viously determined to be inactive at the receptor (data
not shown).The gem-dimethyl 10 displayed low affinity
methyl intermediate with N,N-dimethylamine provided
the desired product 30.
for D .The 2,5-dichlorothiophene 14 displayed lower
4
Finally, cycloheptenimine 13 was prepared in eight steps
as depicted in Scheme 4.In this route, 2,5-dimethyl-
thiophene 31 was condensed with 2-cyanoacetyl chlor-
ide in the presence of aluminum chloride to give 43.
Hydroxymethylenation followed by treatment with
hydrazine gave the 4-cyanopyrazole 44.The cyano
function was reduced and formylated to provide 45
which was cyclized and reduced to the cyclohepten-
amine 46.Finally, reductive methylation of 46 using
formic acid and sodium cyanoborohydride afforded the
desired tertiary amine 13.
overall affinity for the receptors than the corresponding
2,5-dimethylthiophene 4 and the SAR of the corre-
sponding 2-alkylated pyrazole derivatives (15–19) was
also in agreement with what seen for 4, in that increas-
ing size of the N-substituent resulted largely in decreas-
ing affinity and selectivity for the D receptor.
4
Replacement of the thiophene ring by a simple phenyl
isostere largely eliminated D activity (22, 23, and 28)
4
although this loss was less prominent when the di-ortho-
methyl group of 4 was returned to the corresponding
aromatic positions as in compound 24.The furan deri-
vative 11 was also less active, although 2-methylation
(12) of this compound returned potent D4 activity
without much D /D selectivity.
Affinity at dopamine receptors was determined via
standard competitive displacement assays using recep-
2
5
tors cloned from human. The binding affinity data are
summarized in Table 1.
4
2
It can been seen from the binding results in Table 1 that,
while replacement of the sulfur in 1 by a methylene (3)
Table 1. Binding affinities
maintained D activity, the bioisosteric substituent eth-
4
ylene proved more successful (5) in this regard.Within
both the cyclohexyl and cycloheptyl series N-methyl-
ation of the pyrazole at the 2 position as in 3 and 5
improved the overall affinity for the D site over the
4
unsubstituted derivatives 2 and 4 although the D /D
4
2
selectivity was somewhat diminished.Methylation at
the N-1 position of the pyrazole (20 and 21) significantly
lowered D4 affinity.Larger N-2 substituents (6–9)
further diminished both D affinity and selectivity.
4
Although the sulfur-to-ethylene transformation was
successful in the maintaining receptor profile, it did not
lead to an appreciable modification of human micro-
somal T1/2.We postulated benzylic oxidation as a pri-
mary metabolic transformation.We felt that gem-
dimethyl substitution at the single non-benzylic methyl-
ene of 4 might block access by hepatic enzymes to both
benzylic carbons within the carbocycle (10).Replace-
ment of the ortho methyl groups of the thiophene by
chlorine might also negate oxidation at these positions
0
Compd Structure
R
R
X
Y
i
K (nM)
D
2
D
4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
Me Me
Me
Me Me
Me
S
S
S
S
S
S
S
S
S
S
CH
CH
111
4166
462
2066
176
402
3
H
2
170
26
12
2
22
97
138
1578
1297
275
11
2
H
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
Me Me
Me Et
Me n-Pr
Me i-Pr
Me Ph
885
555
>10,000
>10,000
ND
(14) and also lower the oxidation potential of the thio-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Me
Me
H
H
S
2 2
CH CMe
O
O
S
S
S
S
S
S
S
CH
CH
2
CH
CH
2
Me Me
2
2
28
Me
Cl
H
H
CH
CH
CH
CH
CH
CH
CH
2
NMe
ND >10,000
534
416
2
2
2
2
2
2
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
169
14
168
3548
1563
Cl Me
Cl Et
489
>10,000
>10,000
Cl n-Pr
Cl i-Pr
Cl Bn
Me Me
Me Me
I
I
>10,000 >10,000
II
II
III
III
III
IV
IV
IV
—
—
—
S
S
CH
2
ND
641
2866
228
CH
CH
CH
CH
2
2
2
2
CH
CH
CH
CH
2
2
2
2
H
H
Me
H
H
Me
H
—
—
—
—
—
—
—
—
—
N
N
N
—
—
—
>10,000 >10,000
>10,000 >10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
ND
425
301
4325
462
507
1293
2563
CH
N
N
—
—
—
Scheme 4. Reagents and conditions: (i) NCCH
ꢁ
2
COCl, AlCl , CH
3
2
Cl
2
,
H
ꢁ
2.
0
C, 1 h, 89%; (ii) Me
2
NCH(OMe)
2
, 85 C, 1 h; (iii) NH
2
NH
MeOH, rt, 1 h, 43% in two steps; (iv) LAH, THF, rt, 48 h; (v)
H
2
O,
Me
—
—
—
ꢁ
HCO
h; (vii) NaBH
HCHO, NaBH
2
Et, 0.5 N NaOH, rt, 48 h, 83% in two steps; (vi) PPA, 140 C, 2
4
, EtOH, rt, 2.5 h, 79% in two steps; (viii) 37% aqueous
ꢁ
3
CN, MeOH, 60 C, 2 h, 52%.

2
924
Table 2. Receptor-binding profile for compound 4 (NGB 4420)
Receptor D
5
A. Thurkauf et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2921–2924
3. Seeman, P.; Guan, H. C.; Van Tol, H. H. M. Nature 1993,
3
65, 441.
. Belliotti, T. R.; Brink, W. A.; Kesten, S. R.; Rubin, J. R.;
D
1
D
2
D
3
D
4
4
Wustrow, D. J.; Zoski, K. T.; Whetzel, S. Z.; Corbin, A. E.;
K
i
(nM)
>10,000
2067
>10,000
12
>10,000
Receptor
(nM)
5-HT1a
>1000
5-HT
2087
2
a1
>10,000
a2
>7800
Pugsley, T.A;. Heffner, T.G;. Wise, L.D.
Chem. Lett. 1998, 8, 1499.
Bioorg. Med.
K
i
5
. Arlt, M.; Bottcher, H.; Riethmuller, A.; Schneider, G.;
Bartoszyk, G. D.; Greiner, H.; Seyfried, C. A. Bioorg. Med.
Chem. Lett. 1998, 8, 2033.
6
. Moore, K. W.; Bonner, K.; Jones, E. A.; Emms, F.; Lee-
The loss of potency by replacement of pyrazole with
selected imidazoles and triazoles (25–27 and 29) indi-
cated that the pyrazole plays a key role as a pharmaco-
phore.Attempts to improve solubility via the amine 30
or insertion of a nitrogen into the cycloheptane ring (13)
were largely unsuccessful.
son, P. D.; Marwood, R.; Patel, S.; Patel, S.; Rowley, M.;
Thomas, S.; Carling, R. W. Bioorg. Med. Chem. Lett. 1999, 9,
1285.
7
. Haubmann, C.; Hubner, H.; Gmeiner, P. Bioorg. Med.
Chem. Lett. 1999, 9, 1969.
. Haubmann, C.; Hubner, H.; Gmeiner, P. Bioorg. Med.
Chem. Lett. 1999, 9, 3143.
. Carling, R. W.; Moore, K. W.; Moyes, C. R.; Jones, E. A.;
8
9
As compound 4 (NGB 4420) displayed a 100-fold
selectivity for the D over D receptor subtype, it was
Bonner, K.; Emms, F.; Marwood, R.; Patel, S.; Patel, S.;
Pletcher, A. E.; Beer, M.; Sohal, B.; Pike, A.; Leeson, P. D. J.
Med. Chem. 1999, 42, 2706.
10. Kesten, S. R.; Heffner, T. G.; Johnson, S. J.; Pugsley,
T. A.; Wright, J. L.; Wise, L. D. J. Med. Chem. 1999, 42, 3718.
11. Belliotti, T. R.; Wustrow, D. J.; Brink, W. A.; Zoski,
K. T.; Shih, Y.-H.; Whetzel, S. Z.; Georgic, L. M.; Corbin,
A. E.; Akunne, H. C.; Heffiner, T. G.; Pugsley, T. A.; Wise,
L.D. J. Med. Chem. 1999, 42, 5181.
4
2
chosen for further examination of its binding profile
against related CNS receptors (Table 2).Compound 4
displayed no appreciable affinity (>5000 nM) for the
D , D and D receptor subtypes.When tested against
1
3
5
selected cloned human serotonin and norepinephrine
receptors only micromolar affinities were observed.
Extended screening against a battery of 82 receptors,
ion channels and enzymes systems (Panlabs; Bothell,
WA, USA) revealed no affinity greater than 2 mM.
1
2. Perrone, R.; Berardi, F.; Colabufo, N. A.; Leopoldo, M.;
Tortorella, V. J. Med. Chem. 2000, 43, 270.
3. Hodgetts, K. J.; Kieltyka, A.; Brodbeck, R.; Tran, J. N.;
Wasley, J.W.F .; Thurkauf, A. Bioorg. Med. Chem. 2001, 9, 3207.
4. Lober, S.; Hubner, H.; Utz, W.; Gmeiner, P. J. Med.
1
The functional activity of 4 was assessed by measuring
its ability to block the agonist-induced binding of
1
Chem. 2001, 44, 2691.
15. Faraci, W. S.; Zorn, S. H.; Sanner, M. A.; Fliri, A. Cur.
Opini. Chem. Biol. 1998, 2, 535.
16. Thurkauf, A.; Yuan, J.; Chen, X.; Wasley, J. W. F.;
Meade, R.; Woodruff, K. H.; Huston, K.; Ross, P. C. J. Med.
Chem. 1996, 38, 4950.
3
5
35
GTPg S.The GTP g S binding functional assay was
used to demonstrate a dose dependent agonist stimula-
2
5
tion by a full agonist. Compound 4 and the reference
antagonist haloperidol demonstrated a baseline level of
activity when used alone suggesting that they possess no
agonist activity at the human D4.2 receptor.On the
other hand, 4 and haloperidol completely reversed the
1
7. Kulagowski, J. J.; Broughton, H. B.; Curtis, N. R.;
Mawer, I. M.; Ridgill, M. P.; Baker, R.; Emms, F.; Freedman,
S. B.; Marwood, R.; Patel, S.; Ragan, C. I.; Leeson, P. D. J.
Med. Chem. 1996, 39, 1941.
18. Ten Brink, R. E.; Bergh, C. L.; Duncan, J. N.; Harris,
D. W.; Huff, R. M.; Lahti, R. A.; Lawson, C. F.; Lutzke, B. S.;
Martin, I. J.; Rees, S. A.; Schlachter, S. K.; Sih, J. C.; Smith,
M.W. J. Med. Chem. 1996, 39, 2435.
3
5
agonist-stimulated GTPg S binding in a dose-depen-
dent fashion with EC values of 9 and 17 nM, respec-
5
0
3
5
tively.The GTP g S binding assay data suggest that
compound 4 functions as an pure antagonist at the
human D4.2 receptor.
1
9. Sanner, M. A.; Chppie, T. A.; Dunaiskis, A. R.; Fliri,
A. F.; Desai, K. A.; Zorn, S. H.; Jackson, C. G.; Faraci, W. S.;
Collins, J. L.; Duignan, D. B.; Di Prete, C. C.; Lee, J. S.;
Trozzi, A. Bioorg. Med. Chem. Lett. 1998, 8, 725.
In conclusion, using the lead compound 1 as reference, a
systematic SAR study has been carried out with the goal
of identifying compounds as selective dopamine D4
antagonists.Compound 4 (NGB 4420) displayed a good
2
0. Bristow, L. J.; Collinson, N.; Cook, J. P.; Curtis, N.;
Freedman, S. B.; Kulagowski, J. J.; Leeson, P. D.; Patel, S.;
Ragan, C. I.; Ridgill, M.; Saywell, K. L.; Trickleback, M. D.
J. Pharmacol. Exp. Ther. 1997, 283, 1256.
21. Mansbach, R. S.; Brooks, E. W.; Sanner, M. A.; Zorn,
S.H. Psychopharmacology 1998, 135, 194.
selectivity for the D over D and other related CNS
4
2
receptors.Its human microsomal stability ( T1/2=6 min)
is not ideal, but much better than that of compound 1
(
T1/2 <1 min).In addition, the unique structure of
compound 4 with different physicochemical properties
MW=218.2; mLogP=2.48; PSA=22.31) compared to
22. Bristow, L. T.; Kramer, M.; Kulagowski, J.; Patel, S.; Ragan,
C.; Seabrook, G. R. Trends Pharmacol. Sci. 1997, 18, 186.
(
2
3. Rowley, M.; Collins, H.; Broughton, H. B.; Davey, W. B.;
other D antagonists (Fig.1 ) may play a significant role
4
as for further biological evaluation.
Baker, R.; Emms, F.; Markwood, R.; Patel, S.; Patel, S.;
Ragan, I.; Freedman, S. B.; Ball, R.; Leeson, P. D. J. Med.
Chem. 1997, 40, 2374.
2
4. Chakrabarti, J. K.; Hotten, T. M.; Morgan, S. E.; Pullar,
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2
.Kulagowski, J .J; .Patel, S. Curr. Pharm. Des. 1997, 3, 355.
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6