Di- and trisubstituted pyrazolo[1,5-a]pyridine derivatives: synthesis, dopamine receptor binding and ligand efficacy.

Article abstract of DOI:10.1016/S0960-894X(01)00814-9

Based on the lead molecule FAUC 113, a series of di- and trisubstituted pyrazolo[1,5-a]pyridine derivatives was synthesized and investigated for their dopamine receptor binding profile. The carbonitrile 11a (FAUC 327) showed excellent pharmacological properties combining high D4 affinity (K(i)=1.5 nM) and selectivity with significant intrinsic activity (31%) in low nanomolar concentrations (EC50=1.5 nM).

Full text of DOI:10.1016/S0960-894X(01)00814-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 633–636
Di- and Trisubstituted Pyrazolo[1,5-a]pyridine Derivatives:
Synthesis, Dopamine Receptor Binding and Ligand Efficacy
Stefan Lober, Tarek Aboul-Fadl, Harald Hubner and Peter Gmeiner*
Department of Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander University,
Schuhstraße 19, D-91052 Erlangen, Germany
Received 29 October 2001; revised 28 November 2001; accepted 30 November 2001
Abstract—Based on the lead molecule FAUC 113, a series of di- and trisubstituted pyrazolo[1,5-a]pyridine derivatives was synthe-
sized and investigated for their dopamine receptor binding profile. The carbonitrile 11a (FAUC 327) showed excellent pharmaco-
logical properties combining high D4 affinity (Ki=1.5 nM) and selectivity with significant intrinsic activity (31%) in low nanomolar
concentrations (EC50=1.5 nM). # 2002 Elsevier Science Ltd. All rights reserved.
The use of classical neuroleptics is frequently associated
with acute movement disorders, tardive dyskinesia and
hyperlactinemia, which are due to an unselective block-
ade of the D2-like dopamine receptor family.1 In con-
trast, the atypical neuroleptic clozapine, which does not
induce these side effects, reveals significant selectivity
for the D4 subtype.2ꢀ4 On the other hand, selective D4
receptor agonists or partial agonists might be of interest
for the treatment of attention deficit hyperactivity dis-
orders (ADHD), mood disorders, and Parkinson’s dis-
ease.5,6
we herein report on di- and trisubstituted analogues of
type A when an extension of the p-system of the pyr-
azolo[1,5-a]pyridine moiety turned out advantageous.
A number of SAR studies including piperazinylmethyl
substituted indoles and azaindoles led to the partial D4
agonists L-745,870, FAUC 299 and FAUC 113.7ꢀ9
According to molecular orbital calculations, their pref-
erence for the D4 subtype strongly depends on the size
and shape of the negative molecular electrostatic
potential (MEP) formed by the heteroaromatic moiety
when a large negative region extending the area of the
heteroarene obviously is not tolerated by the D1, D2
and D3 subtypes.
Taking into account the binding properties of our
recently described iodine labeled radioligand,10 our
synthetic investigations were initiated by site-directed
transformations facilitating the incorporation of sub-
stituents into position 7 of the pyrazolo[1,5-a]pyridine
moiety. Taking advantage of the ability of N1 to direct
metalation into position 7,11 the 7-iodo pyrazolo[1,5-
a]pyridine 1a and its 2-methyl derivative 1b were readily
synthesized by treatment of the corresponding lithiated
heterocycles with diiodoethane. Subsequent palladium
catalyzed coupling reactions resulted in formation of the
7-alkynyl, -aryl, -amino or -cyano derivatives 2a–6a and
2b–6b.12 Employing these valuable building blocks as
educts, we synthesized the piperazinylmethyl substituted
azaindoles 7a–11a and 7b–11b by Mannich amino-
methylation (Table 1).13,14
In order to further improve the pharmacological prop-
erties, we tried to develop structural derivatives com-
bining high D4 affinity and selectivity with significant
ligand efficacy in low nanomolar concentrations.
Employing the 7a-azaindole FAUC 113 as a major lead,
*Corresponding author. Tel.: +49-9131-852-9383; fax: +49-9131-
852-2585; e-mail: gmeiner@pharmazie.uni-erlangen.de
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00814-9

634
S. Lober et al. / Bioorg. Med. Chem. Lett. 12 (2002) 633–636
¨
Table 1. (a) See ref 12; (b) 4-chlorophenylpiperazine dihydrochloride,
Et(Me)2N, CH2O , CHCl2, CH3COOH, rt, 16 h
2
Scheme 2. (a) 4-Chlorophenylpiperazine dihydrochloride, Et(Me)2N,
CH2O, DCM, CH3COOH, rt, 4 h (54% for 16; 47% for 17); (b) (1)
MsCl, Et(Me)2N, THF, rt, 2 h; (2) LiAlH4, THF, rt, 30 min (47%).
R
R0
Yield (%)
113, the resulting 4-methylpyrazolo[1,5-a]pyridine 18 is
expected to be conformationally restricted when looking
at the rotation of the piperazinylmethyl side chain,
whereas the electrostatic properties of both molecules
should be very similar (Scheme 2).
2a
3a
4a
5a
6a
2b
3b
4b
5b
6b
7a
8a
9
10a
11a
7b
8b
9b
CCH
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
82
77
67
56
60
75
60
63
53
59
Phe
4-F-Phe
N(CH2CH2)2NBn
CN
CCH
Phe
4-F-Phe
N(CH2CH2)2NBn
CN
Receptor binding profiles of the test compounds 7a,b–
11a,b, 14a–c and 16–18 were determined in vitro by
measuring their ability to compete with [3H]spiperone
10b
11b
for the cloned human dopamine receptor subtypes
16
D2long, D2short
,
D317 and D4.418 stably expressed in
In order to extend the variety of substituents, introduc-
tion of electrophiles via an organolithium intermediate
was envisioned. Thus, lithiation of the pyrazolopyridine
12 followed by trapping with MeI resulted in formation
of the 7-methylazaindole 13 as outlined in Scheme 1.
Subsequent Mannich reaction furnished the corre-
sponding 3-aminomethyl analogue 14a. Regioselective
lithiation of the heteroarene also proved possible in the
presence of the piperazinylmethyl functionality. Thus,
treatment of FAUC 113 with n-BuLi at dry ice tem-
perature followed by addition of ethyl formate or
TMSCl resulted in the corresponding test compounds
14b and 14c, respectively.
Chinese hamster ovary cells (CHO).19 D1 receptor affi-
nities were measured employing bovine striatal mem-
branes and the D1 selective radioligand [3H]SCH
23390.19
The resulting Ki values of the test compounds are listed
in Table 2 in comparison to FAUC 113, L-745,870 and
the atypical neuroleptic clozapine. In fact, a variety of
saturated and unsaturated substituents in position 7 of
the heteroarene is tolerated by the D4 subtype when
incorporation of an alkynyl- (7a,b), cyano- (11a,b),
methyl- (14a), formyl- (14b) or TMS-substituent (14c)
provided Ki values between 1.2 and 2.8 nM. On the
other hand, introduction of an aryl- (8a,b, 9a,b) or
benzylpiperazine-substituent (10a,b) led to a strong
reduction of D4 affinity. Binding data of all the target
compounds investigated showed remarkable D4 select-
ivity, being superior to that of clozapine. Whereas the
test compound 7a displayed superior D4 affinity, the
2-methyl-7-alkynyl-derivative 7b as well as the 7-cyano-
pyrazolo[1,5-a]pyridines 11a,b revealed an outstanding
Finally, we envisioned synthesizing 4- and 6-substituted
analogues. Starting from the (hydroxymethyl)pyra-
zolo[1,5-a]pyridines 15a,b15 we obtained the amino-
methylation products 16 and 17 in 54 and 47% yield,
respectively. O-Activation of the regioisomer 16 fol-
lowed by LiAlH4 reduction gave access to the 4-methyl
derivative 18. In contrast to the lead structure FAUC
Scheme 1. (a) See ref 9; (b) (1) n-BuLi, THF, ꢀ78 ꢁC, 20 min; (2) MeI,
THF, ꢀ78 ꢁC, 30 min (84%); (c) (1) n-BuLi, THF, ꢀ78 to 0 ꢁC, 1 h; (2)
HCOOEt, THF, ꢀ78 ꢁC to rt, 1 h (14b: 78%); (2) TMSCl, THF,
ꢀ78 ꢁC to rt, 1 h (14c: 51%); (d) 4-chlorophenylpiperazine dihy-
drochloride, Et(Me)2N, CH2O, DCM, CH3COOH, rt, 4 h (14a: 78%).
Figure 1. Stimulation of mitogenesis as a functional assay to investi-
gate the agonist effect of 11a and 11b at the human dopamine D4.2
receptor in comparison with the full agonist quinpirole, the partial
agonists FAUC 113 and L-745,870 and the antagonist clozapine.

S. Lober et al. / Bioorg. Med. Chem. Lett. 12 (2002) 633–636
¨
635
Table 2. Binding data of target compounds to human and bovine dopamine receptor ligandsa
Compd
R
Pos. of R
R0
D1
810
15,000
930
3300
7000
1000
1500
1500
2500
11,000
2200
D2long
D2short
D3
4600
17,000
840
3100
5700
3800
410
D4
7a
7b
8a
8b
9a
9a
10a
10b
11a
11b
14a
14b
14c
16
17
18
CCH
CCH
Ph
7
7
7
7
7
7
7
7
7
H
CH3
H
CH3
H
CH3
H
CH3
H
3700
21,000
5400
28 000
15,000
14,000
640
6200
25,000
2800
24,000
15,000
16,000
400
1.2
1.5
7.6
37
29
87
180
26
1.5
2.3
2.7
Ph
4-F-Ph
4-F-Ph
N(CH2CH2)2NBn
N(CH2CH2)2NBn
CN
300
120
270
44,000
59,000
4900
45,000
58,000
5000
5900
2800
79,000
22,000
12,000
4300
38,000
28
26,000
21,000
3800
CN
CH3
CHO7
TMS
CH2OH
CH2OH
CH3
7
7
CH3
H
H
7
1200
9500
9200
2.6
H
H
H
H
H
7100
4800
1400
7000
12,000
9900
420
3100
56,000
28,000
13,000
3200
2000
>100,000
2600
2.8
110
4
6
4
—
20
50
3.1
0.62
16
9400
5000
22,000
960
FAUC 113
L-745,870
Clozapine
H
54,000
41
aKi values are the means of two to four independent experiments using eight different concentrations each in triplicate.
Table 3. Intrinsic activity of the azaindoles 11a,b in relation to the reference compounds FAUC 113, L-745,870, quinpirole and clozapine at the
human D4.2 receptor established by measuring the stimulation of mitogenesis
Test compounds
11a
11b
FAUC 113
L-745,870
Quinpirole
Clozapine
Intrinsic effecta (%)
EC50 (nM)
31
1.5
26
12
26
12
24
27
100
2.0
<3
nd
aRate of incorporation of [3H]thymidine as evidence for mitogenetic acivity related to the full agonist quinpirole as the means of quadruplicates from
four to 11 experiments. EC50 values in nM are derived from the mean curves of the experiments. nd, not determined.
D4 binding profile, including a 10,000–30,000-fold
selectivity over the D2 and D3 subtypes. It is interesting
to note that the 2-methyl substitution of 7b, 8b and 11b
significantly reduced D4 affinity. Hydroxymethyl or
methyl substituents in the positions 4 or 6 of the pyra-
zolo[1,5-a]pyridine moiety (16–18) caused substantially
reduced D4 receptor binding.
maximal effect to that of a full agonist. CHO10001 cells
stably expressing the human D4.2 receptor were estab-
lished for this mitogenesis assay.8,21 Comparative
experiments were performed with the full agonist quin-
pirole, the partial agonists FAUC 113 and L-745,870
and the antagonist clozapine. Figure 1 shows dose–
response curves clearly indicating partial agonist
effects of 11a and 11b (31 and 26% relative to quinpir-
ole, respectively) comparable to those of FAUC 113
and L-745,870.
In order to further characterize the binding properties
of the most potent and selective D4 ligands 11a,b, 5-
HT1A and 5-HT2 receptor recognition was evaluated.
Employing porcine brain homogenates and the radio-
ligands [3H]8-OH-DPAT and [3H]ketanserin, respect-
ively, the resulting Ki values indicated only moderate
5-HT1A (2200 nM for 11a and 4500 nM for 11b) and
5-HT2 affinities (180 nM for 11a and 1000 nM for 11b).
In order to investigate potential ligand efficacy, the
highly selective test compounds 11a,b were evaluated
for their mitogenetic activity. Agonist activation of
dopamine receptors is known to increase mitogenesis in
heterologously transfected cell lines. This can be deter-
mined by measuring the rate of [3H]thymidine incor-
poration into growing cells.20 Intrinsic activity can be
quantified by determination of the effective concentra-
tion (EC50) of a test compound and by comparing the
Interestingly, the dose–response curve and the EC50
value for the 7-cyanopyrazolopyridine 11a (FAUC 327)
demonstrate that the partial agonist effect is exerted at
substantially lower concentrations than for L-745,870
and FAUC 113 (Table 3), possibly indicating superior
properties in vivo.
In summary, SAR studies on analogues of the D4 par-
tial agonist FAUC 113 resulted in the finding that an
extension of the p-system of the pyrazolo[1,5-a]pyridine
moiety by the introduction of suitable substituents into
position 7 is tolerated well by the D4 receptor. Extra-
ordinary high selectivity was observed when a cyano
group was selected, leading to the partial agonists 11a

636
S. Lober et al. / Bioorg. Med. Chem. Lett. 12 (2002) 633–636
¨
(FAUC 327) and 11b. Functional studies of FAUC 327
showed an 8–18-fold lower EC50 value when compared
to L-745,870 and FAUC 113.
8. Hubner, H.; Kraxner, J.; Gmeiner, P. J. Med. Chem. 2000,
43, 4563.
9. Lober, S.; Hubner, H.; Gmeiner, P. Bioorg. Med. Chem.
Lett. 1999, 9, 97.
10. Prante, O.; Lober, S.; Hubner, H.; Gmeiner, P.; Kuwert,
T. J. Label. Compd. Radiopharm. 2001, 44, 849.
11. Finkelstein, B. L. J. Org. Chem. 1992, 57, 5539.
12. Aboul-Fadl, T.; Lober, S.; Gmeiner, P. Synthesis 2000,
1727.
Acknowledgements
The authors wish to thank Dr. H. H. M. Van Tol
(Clarke Institute of Psychiatry, Toronto), Dr. J.-C.
Schwartz and Dr. P. Sokoloff (INSERM, Paris) as well
as Dr. J. Shine (The Garvan Institute of Medical
Research, Sydney) for providing dopamine D4, D3 and
D2 receptor expressing cell lines, respectively. Dr. R.
Huff (Pharmacia & Upjohn, Inc., Kalamazoo, MI,
USA) is acknowledged for providing a D4 expressing
cell line employed for mitogenesis. Thanks are also due
to Mrs. H. Szczepanek, Mrs. P. Schmitt and Mrs. P.
Hubner for skillful technical assistance. This work was
supported by the Deutsche Forschungsgemeinschaft,
the BMBF, and the Fonds der Chemischen Industrie.
13. Typical procedure: A mixture of pyrazolo[1,5-a]pyridine
derivative (0.50 mmol), 4-chlorophenylpiperazine dihydro-
chloride (0.55 mmol), Et(Me)2N (1.1 mmol), formaldehyde
(37% solution in H2O; 2.0 mmol) and acetic acid (4 mL) was
stirred at room temp. for 16 h. The reaction mixture was neu-
tralized with 2 N NaOH and extracted with CH2Cl2. The
organic layer was dried (Na2SO4) and evaporated and the
residue was purified by flash chromatography (silica gel,
EtOAc).
14. Analytical data for 11a: IR (cmꢀ1) 3090 (arom. C–H),
2821 (aliph. C–H), 2239 (CꢂN), 1624 (C¼N), 1496 (C¼C),
1
1233 (C–N); H NMR (360 MHz) d 2.61 (br t, 4H, 2 ꢃ CH2–
N–CH2–CH2), 3.15 (br t, 4H, 2 ꢃ –CH2–CH2–N–Ph), 3.77 (s,
2H, Het–CH2–N), 6.79–6.82 (m, 2H, 4-Cl-Ph), 7.12–7.26 (m,
3H, 4-Cl–Ph and H-5), 7.33 (dd, J=7.0, 1.0 Hz, 1H, H-4), 7.96
(dd, J=9.0, 1.0 Hz, 1H, H-6), 8.06 (s, 1H, H-2); EI–MS: m/z
351 (M+). Anal. calcd for C19H18N5Cl: C 65.73; H 5.52; N
19.17. Found: C 65.57; H 5.60; N 18.97.
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