Syntheses and optimization of new GS39783 analogues as positive allosteric modulators of GABAB receptors

Article abstract of DOI:10.1016/j.bmcl.2007.09.023

The optimization of GS39783 into potent, selective, and safe positive allosteric modulators of GABA_B receptors is presented.

Full text of DOI:10.1016/j.bmcl.2007.09.023

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6206–6211
Syntheses and optimization of new GS39783 analogues
as positive allosteric modulators of GABAB receptors
,
,à
*
Sebastien Guery, Philipp Floersheim, Klemens Kaupmann* and Wolfgang Froestl*
´
Novartis Institutes for Biomedical Research, Neuroscience, Novartis Pharma A.G., CH-4002 Basel, Switzerland
Received 20 July 2007; revised 3 September 2007; accepted 5 September 2007
Available online 8 September 2007
Abstract—The optimization of GS39783 into potent, selective, and safe positive allosteric modulators of GABAB receptors is
presented.
Ó 2007 Elsevier Ltd. All rights reserved.
The receptors for the major inhibitory neurotransmitter
in the central nervous system, GABA, are subdivided
into ionotropic GABAA and GABAC receptors and
metabotropic GABAB receptors. Whereas GABAA
and GABAC receptors form chloride-permeable ion
channels, GABAB receptors are G-protein coupled
receptors (GPCRs). These receptors were discovered in
1980 by Norman G. Bowery1 and act post- and pre-syn-
aptically to inhibit neuronal excitability and neurotrans-
mitter release, respectively. A possible role of GABAB
receptors in a large number of CNS disorders such as
cognition deficits, anxiety, depression, epilepsy, pain,
and drug addiction has been discussed.2 Some of these
diseases like anxiety, pain, and drug addiction could
potentially be treated by activation of GABAB recep-
tors, which can be achieved by administration of either
agonists or positive allosteric modulators. Whereas ben-
zodiazepines are well-known positive allosteric modula-
tors of GABAA receptors, the first examples of allosteric
enhancers for GABAB receptors have been described
only recently.3 One of the most interesting compounds
found was GS39783 (Fig. 1).3b However, despite an
interesting in vitro and in vivo profile,3b,4 GS39783
was found to be genotoxic probably because of its aro-
matic nitro group (Fig. 1).5
This communication describes our efforts toward the
identification of a novel, drug-like class of compounds
acting as positive allosteric modulators for GABAB
receptors.
In order to introduce molecular diversity in position 5 of
the pyrimidine ring, a four steps procedure depicted in
Scheme 1 was optimized to obtain compounds with a
chlorine or with a hydrogen in position 6. 4,6-Di-
chloro-2-methylpyrimidine was first substituted by
cyclopentylamine and then iodinated to lead to com-
pound 6. This scaffold was then used in a Suzuki cross
coupling6 to give very efficiently a small focused library
of substituted 4-amino-6-chloro-5-phenylpyrimidines
(compounds 7a–15a) which were then hydrogenated un-
der standard conditions to give the desired 4-amino-5-
phenylpyrimidines (compounds 7b–15b). As a means
to introduce molecular diversity at the last step in posi-
tion 4 of the pyrimidine ring, a versatile way of synthesis
was designed (Scheme 2). Starting from the commercially
Keywords: GABAB positive allosteric modulators; GS39783; Drug
addiction.
*
S
Corresponding authors. Tel.: +39 045 821 9042; fax: +39 045 821
8196 (S.G.); tel.: +41 61 696 34 73 (K.K.); tel.: +41 21 693 91 26; fax:
+41 21 693 91 20 (W.F.); e-mail addresses: sebastien.2.guery@gsk.
N
O
N
O
com; klemens.kaupmann@novartis.com;
acimmune.com
wolfgang.froestl@
N
H
N
H
Present address: GlaxoSmithKline S.p.A., Via A. Fleming 4, 37135
Verona, Italy.
N+
à
Present address: AC Immune SA, PSE Building B, EPFL, CH-1015
Lausanne, Switzerland.
Figure 1. Structure of GS39783.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.023

S. Guery et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6206–6211
6207
S
F
S
S
N
F
N
F
N
F
N
F
N
N
N
N
Cl
N
H
N
H
N
H
Cl
N
H
N
H
N
H
I
I
F
Cpd 2 : 106 %
Cpd 3 : 100 %
C pd 4 : weakly active
Cpd 1 : 139 %
GS39783 (25 μM of Cpd, 20 μM of GABA) : 192 %
Figure 2. Biological activity of some GS39783 derivatives bearing nitro-mimetics in position 5. The potentiation of the GABA-induced stimulation of
GTP(c)35S binding was measured using membranes from GABAB(1b/2) expressing CHO-K1 cells as described.3a The activities of compounds (25 lM)
were determined in the presence of 20 lM GABA. The data are normalized to the maximal effect (100%) obtained with a saturating concentration of
GABA (1 mM). Twenty micromolars GABA, when applied alone, stimulates to approximately EC80 levels (80%).
available 5-bromo-2,4-dichloropyrimidine, a regioselec-
tive nucleophilic substitution was performed in position
4 exclusively to give the 4-methylthio derivative 177
which was then involved in a halogen exchange8 to give
2-iododerivative 18. The 2-methyl group and the 5-[(4-
trifluoromethyl)phenyl] substituents were introduced
by a Negishi cross coupling9 and a Suzuki cross cou-
pling,10 respectively, to afford 20. Our first attempt
was to oxidize the methylsulfide moiety of 20 to the
corresponding methylsulfonyl (21) by treatment with
N
N
N
N
c
N
N
b
a
63-98%
95 %
Cl
N
H
93 %
Cl
N
H
Cl
Cl
5
I
6
N
N
N
N
d
N
H
Cl
N
72-93%
H
R
R
7a-15a
7b-15b
Scheme 1. Reagents and conditions: (a) cyclopentylamine (4.0 equiv), dioxane, 0 °C–rt, 24 h; (b) NIS, DMF, 80 °C, 24 h; (c) arylboronic acid (1.05
equiv), Pd(OAc)2 (0.02 equiv), dppf (0.03 equiv), K3PO4 (2.0 equiv), DME, water, 85 °C; (d) Pd/C 10%, AcONa (1.1 equiv), EtOH, H2, rt.
Cl
Cl
S
S
N
N
N
N
N
N
N
N
b
d
c
a
Br
I
Br
54 %
56 %
91 %
66 %
S
Cl
Br
Br
19
16
17
18
O
S
S
NH
O
F
F
N
N
F
F
N
N
F
F
N
N
f
27 %
e
F
F
F
69 %
20
21
22
Scheme 2. Reagents and conditions: (a) MeSNa, THF, rt; (b) HI 57%, rt; (c) MeZnCl, Pd(PPh3)4, THF, rt–60 °C; (d) 4-trifluoromethylbenzen-
eboronic acid, Na2CO3, Pd(PPh3)4, EtOH, toluene, water, 110 °C; (e) m-CPBA, DCM, rt; (f) cyclohexylamine, DMF, 150 °C, microwaves, 20 min.

6208
S. Guery et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6206–6211
R
Cl
NH
S
HCl
OH
N
N
F
F
N
N
F
F
F
F
N
N
F
F
N
N
c
b
a
F
F
F
F
82 %
11-96 %
97 %
25-34
24
20
23
Scheme 3. Reagents and conditions: (a) HCl 37%, MeOH, reflux; (b) POCl3, one drop of DMF, 80 °C; (c) R–NH2, solvent, base, 80 °C.
NH
NH
Cl
NH
N
N
N
N
N
N
N
N
F
F
c
b
a
e
R1
S
Br
Cl
Br
Cl
Br
F
72 %
76 %
81 %
88 %
37: R1 = -SMe
39: R1 = H
16
35
36
d
40 %
NH
NH
O
N
N
F
F
F
f
R2
S
F
F
53-96 %
N
N
O
F
38
40-44
Scheme 4. Reagents and conditions: (a) exo-2-aminonorbornane, THF, 0 °C–rt; (b) MeSNa, THF, 0 °C–rt; (c) 4-trifluoromethylbenzeneboronic
acid, Na2CO3, Pd(PPh3)4, toluene, EtOH, water, 110 °C; (d) Ni-Raney, EtOH, 60 °C; (e) m-CPBA, DCM, rt, 1.5 h; (f) compound 40: KCN, DMSO,
80 °C. Compound 41: sodium methoxide, THF, rt. Compound 42: methylamine, EtOH, rt. Compound 43: dimethylamine, THF, 80 °C. Compound
44: N-methylpiperazine, THF, 80 °C.
m-CPBA11 and then to use 21 as a template. Unfortu-
nately, only few amines reacted cleanly with 21 probably
because of the steric hindrance near the leaving group.
Our second attempt was to hydrolyze the methylsulfide
moiety of 20 with hydrochloric acid12 to give 23 (Scheme
3). This compound was then reacted with POCl3 to give
the corresponding 4-chloropyrimidine 24. This useful
intermediate was submitted to nucleophilic substitutions
with a collection of amines to give compounds 25–34.
Finally, the third library with molecular diversity in
position 2 was synthesized starting from compound 16,
which was substituted first with exo-2-aminonorborn-
ane13 and then with MeSNa to give 36 (Scheme 4).
The phenyl moiety was introduced by a Suzuki cross
coupling to afford 37. On one hand, a desulfurization
by treatment with Ni-Raney in EtOH14 leads to 39
and, on the other hand, 37 can also be oxidized into
the corresponding 2-methylsulfonylpyrimidine 38 which
was then reacted with various nucleophiles.
4 of the pyrimidine ring (compare 1 with 2 and 3 with
4). Furthermore, it was also shown earlier that the
replacement of the 2-methylthio substituent by a 2-
methyl group is not detrimental for the activity.3b After
screening for GABAB receptor positive modulatory
activity of the first library of compounds (Table 1),
we found that the 6-chloro substituent was detrimental
to the efficacy at the receptor (compare, for example,
8a with 8b at 25 lM of compound, 12a with 12b and
15a with 15b). Moreover, the introduction of a second
cyclopentylamino substituent in position 6 led to only
weakly active or inactive compounds (data not shown),
confirming our hypothesis that the space in the recep-
tor is very limited and that a proton is the best substi-
tuent for the position 6 of the pyrimidine ring. On the
other hand, as postulated before, electron-withdrawing
groups on the phenyl ring such as a 4-trifluoromethyl
or a 4-trifluoromethoxy showed the best results (com-
pare 9b with 12b and 10b with 15b). Indeed, the
replacement of a 4-methylphenyl (9b) or 4-methoxy-
phenyl (10b) by a 4-trifluoromethylphenyl (12b) or a
4-trifluoromethoxyphenyl (15b) led to more efficacious
positive modulators (Table 1). Other electron-with-
drawing substituents were used on the phenyl ring
but none of them had increased activity at the receptor
compared to the trifluoromethyl or trifluoromethoxy
groups (data not shown). The introduction of a substi-
tuent in position 3 of the phenyl ring led to a decrease
of activity (compare 14b with 15b). To conclude, the
Thanks to a preliminary work with nitro-mimetics
(Fig. 2), we found that to replace the nitro group, we
should have a lipophilic substituent (see compound 3)
with an electron-withdrawing effect (see compounds 1
and 2). One way to combine both of these effects is
to substitute the position 5 of the pyrimidine ring by
a substituted phenyl. Moreover, substantial efficacy in
the biological assay15 was observed for compounds
with only one cyclopentylamine substituent in position

S. Guery et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6206–6211
Table 1. Biological activities obtained for compounds 7a–15b
6209
Compound
R
2.5 lM of compound,
1 lM of GABA
Effect (%) SEM
25 lM of compound,
1 lM of GABA
Effect (%) SEM
1 lM of GABA
pEC50 SEM
Emax
(%) SEM
GS39783
7a
7b
—
4-nButyl
132
27
25
23
29
23
23
21
21
25
21
17
62
14
26
20
23
19
48
4
1
1
2
1
2
2
1
1
1
0
1
3
1
0
1
1
1
3
153
39
18
39
61
34
41
29
38
36
28
26
114
5
2
1
3
2
1
1
2
1
2
1
2
1
2
3
0
1
1
1
3
6.13 0.06
—
146
—
—
—
—
—
—
—
—
—
—
—
127
—
—
—
—
—
117
5
—
—
8a
8b
4-Ethyl
—
—
9a
9b
4-Methyl
4-Methoxy
3-Methyl
4-CF3
—
—
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
—
—
—
—
5.30 0.03
—
3
7
4-COOEt
3-OCF3
4-OCF3
31
33
25
35
100
—
—
—
—
5.34 0.07
The compounds were assayed as described.3,15 When applied alone, 1 lM GABA stimulates to approximately EC20 levels (20% stimulation). The
effects (%) are normalized to the maximal effect of a saturating concentration of GABA (1 mM). Values were calculated from triplicate measure-
ments. pEC50 and Emax values were calculated from concentration–response curves (eight concentrations) in the presence of 1 lM of GABA.
best nitro-mimetic group identified in this series was a
4-(trifluoromethyl)phenyl substituent. Then we focused
our attention on position 4 of the pyrimidine ring
(Schemes 2 and 3).
these products were not considered for further evalua-
tion despite an increased selectivity profile because of
their high logPs (logP > 5.9) and low water solubilities
(<10 mg LÀ1). Finally, we focused our attention on 27
which bears a norbornyl group at its amino function.
Both the exo isomer (27) and the endo isomer (28) were
evaluated (Table 2). Compound 27 was more effica-
cious, and was of similar potency and efficacy com-
pared to GS39783. Genotoxicity16 and mutagenicity17
assays were performed and 27 was found to be safe
both in the micronucleus test and in the Ames
test. For that reason 27 was considered for further
in vitro and in vivo evaluations and the exo-2-norbor-
nyl substituent was kept for the optimization of the
position 2.
This collection of compounds shows interesting struc-
ture–activity relationships (Table 2). For this series it
became obvious that an increased size of the cycloalkyl
led to increased efficacy. This trend was first observed
with compound 29 in which the cyclopentyl substituent
was replaced by a cycloheptyl (compare 12b and 22
with 29 at 2.5 lM). Interestingly, the introduction of
an additional bulky substituent on the cyclohexyl ring
(30) led to a less active product (compare 30 with 22).
Moreover, the introduction of an aromatic ring onto
the amino group was not tolerated by the receptor
(compare 31, 32, 34 with 22); therefore, we thought that
a hydrophobic interaction with a spatially extended
substituent was necessary at this position. Surprisingly,
despite its 4-tert-butylamino substituent, 33 was found
to be a weak positive modulator. The needs of bulky,
cyclic aliphatic side chain was then confirmed when
cycloalkyl chains was used such as a norbornyl (27),
an adamantyl (25 and 26), or a cycloheptyl (29). A sub-
stantial increase in activity was observed when compar-
ing 29 to 12b and to 22. Despite its good efficacy, 29
was not investigated any further because of significant
binding activities to other GPCRs (data not shown).
We were then interested in compounds 25 and 26 bear-
ing an adamantyl substituent on the amino group. Both
compounds were potent positive allosteric modulators
(Table 2) which led to the hypothesis that the substitu-
ents in position 4 of the pyrimidine ring bind into a
large lipophilic pocket in the receptor. However, again
Finally, the screening of the collection of compounds
with molecular diversity in position 2 gave surprising re-
sults (Table 3). The replacement of the methyl moiety of
27 by a hydrogen led to a less active compound which
indicated that a substitution at this position is manda-
tory. The introduction of a 2-SMe group (37), a meth-
ylsulfonyl (38), or a methylamino (42) was detrimental
for activity (compare 37, 38 and 42 with 27). In contrast,
the replacement of a 2-methyl group by a cyano (40), a
methoxy (41), or a dimethylamino (43) gave compounds
with a similar potency than GS39783. Surprisingly, the
introduction of a N-methylpiperazin-1-yl (44) reduced
the biological activity suggesting that the space in the
receptor is limited and that only small substituents are
tolerated. Compounds 40, 41, and 43 are currently under
evaluation for their physicochemical and pharmacokinetics
properties as well as their full pharmacological
characterization.

6210
S. Guery et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6206–6211
Table 2. Biological activities obtained for compounds 22, 25–34
Compound
R
2.5 lM of compound,
1 lM of GABA
Effect (%) SEM
25 lM of compound,
1 lM of GABA
Effect (%) SEM
1 lM of GABA
pEC50 SEM Emax (%) SEM
22
25
80
90
1
1
93
80
5
2
6.06 0.05
5.56 0.13
83
4
N
H
H
N
132 14
26
125
4
141
6
5.46 0.10
185 15
N
H
27
28
29
30
31
32
33
34
122
82
3
5
4
1
2
1
3
110
52
70
32
69
39
21
59
0
4
4
2
6
2
7
4
5.78 0.03
183
—
4
N
H
—
N
H
128
28
5.78 0.03
137
—
3
N
H
—
—
—
—
—
N
H
36
—
N
H
N
H
29
—
H
N
40
—
58 11
—
N
H
The compounds were assayed as described in Table 1. Compounds 25–27, 29, and 34 have a relatively low water solubility due to their high
lipophilicity. This is reflected in this assay by a similar or reduced activity at 25 lM versus 2.5 lM. Concentration-dependent increase in activity,
however, was observed in full concentration–response curves, with maximal effects at 10 lM.
Positive allosteric modulators of GABAB receptors rep-
resent an interesting class of therapeutic agents for the
treatment of anxiety and drug addiction. Despite its
good in vitro and in vivo potency, GS39783 was only
useful as a pharmacological tool because of its genotox-
icity. We report herein a new class of positive allosteric
modulators of GABAB receptors derived from GS39783
with an increased drug-likeness, a decreased toxicity,
and an excellent selectivity profile. Some in vivo investi-
gations are currently ongoing in anxiety and drug addic-
tion models and the results of these studies will be
reported in due course.

S. Guery et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6206–6211
Table 3. Biological activities obtained for compounds 37–44
6211
Compound
R
2.5 lM of compound,
1 lM of GABA
Effect (%) SEM
25 lM of compound,
1lM of GABA
Effect (%) SEM
1 lM of GABA
pEC50 (lM) SEM Emax (%) SEM
37
38
39
40
41
42
43
44
–SMe
82
24
7
7
3
3
2
3
4
8
91
30 12
4
—
—
—
—
Me–SO2–
H
–CN
48
63
127
131
25
4
1
6
8
7
7
—
—
117
115
20
5.92 0.11
5.69 0.13
—
198 21
191 21
—
–OMe
–NHMe
–NMe2
N-Methylpiperazine
94
26
126
112
5.78 0.08
—
167 16
—
The compounds were assayed as described in Table 1.
10. Hannah, D. R.; Sherer, E. C.; Davies, R. C.; Titman, R.
B.; Laughton, C. A.; Stevens, M. F. G. Bioorg. Med.
Chem. 2000, 8, 739.
11. Herrera, A.; Martinez-Alvarez, R.; Chioua, R.; Benabd-
elouahab, F.; Chioua, M. Tetrahedron 2004, 60,
5475.
Acknowledgment
This work was supported by National Institutes of Men-
tal Health/National Institute on Drug Abuse Grant U01
MH69062.
12. Strekowski, L.; Harden, D.; Watson, R. A. Synthesis
1988, 70.
References and notes
13. Brumby, T.; Jautelat, R.; Prien, O.; Schaefer, M.; Siemei-
ster, G.; Luecking, U.; Huwe, C. WO 2002096888, 2002.
14. Morimoto, H.; Shimadzu, H.; Kushiyama, E.; Kawanishi,
H.; Hosaka, T.; Kawase, Y.; Yasuda, K.; Kikkawa, K.;
Yamauchi-Kohno, R.; Yamada, K. J. Med. Chem. 2001,
44, 3355.
1. Bowery, N. G.; Hill, D. R.; Hudson, A. L.; Doble, A.;
Middlemiss, D. N.; Shaw, J.; Turnbull, M. Nature 1980,
283, 92.
2. (a) Marshall, F. H. J. Mol. Neurosci. 2005, 26, 169; (b)
Cryan, J. F.; Kaupmann, K. Trends Pharmacol. Sci. 2005,
26, 36; (c) Bettler, B.; Kaupmann, K.; Mosbacher, J.;
Gassmann, M. Physiol. Rev. 2004, 84, 835.
3. (a) Urwyler, S.; Mosbacher, J.; Lingenhoehl, K.; Heid, J.;
Hofstetter, K.; Froestl, W.; Bettler, B.; Kaupmann, K.
Mol. Pharmacol. 2001, 60, 963; (b) Urwyler, S.; Pozza, M.
F.; Lingenhoehl, K.; Mosbacher, J.; Lampert, C.; Froestl,
W.; Koller, M.; Kaupmann, K. J. Pharmacol. Exp. Ther.
2003, 307, 322.
4. Cryan, J. F.; Kelly, P. H.; Chaperon, F.; Gentsch, C.;
Mombereau, C.; Lingenhoel, K.; Froestl, W.; Bettler, B.;
Kaupmann, K.; Spooren, W. P. J. M. J. Pharmacol. Exp.
Ther. 2004, 310, 952.
5. (a) Purohit, V.; Basu, A. K. Chem. Res. Toxicol. 2000, 13,
673; (b) Tocher, J. H. Gen. Pharmacol. 1997, 28, 485.
6. Richardson, M. L.; Stevens, M. F. G. J. Chem. Res.
Synop. 2002, 482.
7. Strekowski, L. Bull. Pol. Acad. Sci. Chem. 1976, 24, 17.
8. Vlad, G.; Horvath, I. T. J. Org. Chem. 2002, 67, 6550.
9. Hocek, M.; Votruba, I.; Dvorakova, H. Tetrahedron 2003,
59, 607.
15. GTP(c)35S binding was used as functional assay for
GABAB receptor activity (see Ref. 3b). To assay for
positive modulatory activity the compounds were co-
applied with GABA. One micromolar GABA stimu-
lates GABAB receptors to approximately EC20 values,
20 lM GABA stimulates to approximately EC80
values. If co-application of the test compounds with
GABA significantly increased the signal above EC20
(at 1 lM GABA) or EC80 (at 20 lM GABA), we
concluded that the compound positively modulated
the GABA response. In control experiments the test
compounds were assayed in the absence of GABA.
For
curves were generated and the EC50 values and
selected
compounds,
concentration–response
maximal stimulations at
1 lM determined.
a GABA concentration of
16. (a) Fenech, M. Mutat. Res. 2000, 455, 81; (b) Miller, B.;
Albertini, S.; Locher, F.; Thybaud, V.; Lorge, E. Mutat.
Res. 1997, 392, 45.
17. Flamand, N.; Meunier, J.-R.; Meunier, P.-A.; Agapakis-
´
Causse, C. Toxicol. In Vitro 2001, 15, 105.