Development of 2,4-diaminopyrimidine derivatives as novel SNSR4 antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.01.138

2,4-Diaminopyrimidines derivatives were developed as a novel class of SNSR4 antagonists. Structure activity relationship of the diamino pyrimidine core was explored and a tool compound suitable for target validation was identified.

Full text of DOI:10.1016/j.bmcl.2011.01.138

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2102–2105
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of 2,4-diaminopyrimidine derivatives as novel SNSR4 antagonists
Malken Bayrakdarian a, Joanne Butterworth b, Yun-Jin Hu a, Vijayaratnam Santhakumar a,
,
⇑
Mirosław J. Tomaszewski a
a Department of Medicinal Chemistry, AstraZeneca R&D Montreal, 7171 Frederick-Banting, Montréal, Québec, Canada H4S 1Z9
b Department of Invitro Biology and Drug Metabolism and Pharmaco Kinetic, AstraZeneca R&D Montreal, 7171 Frederick-Banting, Montréal, Québec, Canada H4S 1Z9
a r t i c l e i n f o
a b s t r a c t
Article history:
2,4-Diaminopyrimidines derivatives were developed as a novel class of SNSR4 antagonists. Structure
activity relationship of the diamino pyrimidine core was explored and a tool compound suitable for target
validation was identified.
Received 14 December 2010
Revised 27 January 2011
Accepted 31 January 2011
Available online 3 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
SNSR4
MrgX1
Sensory neuron specific receptor
Diamino pyrimidine
BAM 8-22
The G-protein-coupled receptor (GPCR) superfamily is the most
exploited protein family for drug discovery, yet, over 50% of the
members of the GPCR family remain classified as ‘orphan recep-
tors’, receptors whose regulatory ligands remain unknown. Given
that GPCRs are one of the most targeted protein families for ther-
apeutic intervention, understanding the physiological roles of
these receptors is of great importance.1a–d
Recently, the identification of a large family of GPCRs that are
related to the MAS oncogene and called Mas-related genes (Mrgs),
was reported.2 These genes, also known as the sensory neuron-spe-
cific receptors (SNSRs) or dorsal root receptors (DRRs), comprise a
large family of over 50 rodent and human orphan GPCRs. The re-
stricted expression of many members of the Mrg family to sensory
neurons of the dorsal root ganglion (DRG) suggests that these
members of MrgX receptors may play a role in nociception. In hu-
mans, the MrgX subset of Mrg receptors is selectively expressed in
DRG.3 Interestingly, the DRG-specific members of this family show
discrete patterns of expression that appear to be only partially
overlapping within the sensory neurons, implying that these
receptors may each play unique roles in pain sensation.3
therapeutic value as an analgesic.4a There is also a contradictory
evidence that SNSR4 agonist BAM 8-22 being analgesic.4b Due to
this contradictory report and because the homology between hu-
man and rat SNSR4 receptors are only 60%, we decided to test
the hypothesis that SNSR4 antagonists are analgesic in human
using an investigational compound.
To date, there has been just one report of SNSR antagonists, the
azabicyclooctanes.5 Herein, we describe the development and
structure activity relationship of 2,4-diaminopyrimidines as novel
small molecule SNSR4 antagonists.
In order to find a potent (IC50 <50 nM) and soluble (solubil-
ity >25 lM) SNSR4 tool compound to test the hypothesis that an
SNSR4 antagonist may be analgesic, the AstraZeneca compound
collection was screened using a functional (FLIPR) assay,6 which re-
sulted in the discovery of a number of hits with a quinazoline core
structure as exemplified by compound 1 (Fig. 1). Near neighbour
O
HN
N
4
O
S
5
N
N3
Human sensory neuron specific receptor 4 (SNSR4 or MrgX1) is
restricted to a subpopulation of sensory neurons in both humans
and rat. These neurons are known to express other nociceptive
markers such as IB4 and SNSR4 agonists such as BAM 8-22 are
pro-nociceptive in rodents. SNSR4 activation might have a pro-
nociceptive role in pain, and an antagonist may, therefore, have
O
N
2
NH
N
1
1
IC50 = 530 nM
Cl
⇑
Corresponding author. Tel.: +1 514 832 3200x22515; fax: +1 514 832 3232.
E-mail address: v.santhakumar@astrazeneca.com (V. Santhakumar).
Figure 1.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.138

M. Bayrakdarian et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2102–2105
2103
analysis of compound 1 revealed that vast majority of the near
neighbours screened from the compound collection contained
variations of the amino group of the sulfonamide group and con-
tained only limited variations at 2- and 4-positions of the quinaz-
oline core. In order to determine the significance of the
sulfonamide moiety, the corresponding truncated 2,4 disubstituted
quinazoline 3a was made by two consecutive SNAr reactions of
dichloroquiazoline with the corresponding amines as shown in
Scheme 1. Compound 3a was found to retain moderate micromolar
activity against SNSR4. Using the same route a small directed
library was prepared further to explore substitution at the 4-posi-
tion of the quinazoline core and so as to improve activity at SNSR4.
Compound 3b, identified from this library, showed comparable
activity to compound 1.
Interestingly, the simplified pyrimidine analogue, compound 8a
(Scheme 2), possessing only a 6-membered heterocyclic core
showed comparable activity and improved solubility as compared
with the quinazoline 3b. The pyrimidine analogues were made by
the route shown in Scheme 2 starting from 2-chloro-4-methoxy
pyrimidine 4. Displacement of the chloro group by p-chlorophenyl
ethylamine followed by demethylation with hydrochloric acid
gave the hydroxyl intermediate 6, which was treated with POCl3
to give the corresponding chloro intermediate 7. Displacement of
the chloro group with a variety of amines gave a set of 4-substi-
tuted pyrimidines 8.
Several variations were explored by synthesizing a library of
compounds varying at the 4-position of the pyrimidine core. The
library was prepared by treating the 4-chloro pyrimidine interme-
diate 7 with a selection of structurally diverse amines under the
conditions described in Scheme 2. Piperazine acetyl amide deriva-
tive 9, obtained from this library showed further improvement in
solubility and was half as potent as the sulfonamide 8a. In addition,
compound 9 provided further scope to explore SAR at the amide
position.
NR1R2
N
NR1R2
N
Cl
N
b
a
N
NR3R4
N
Cl
N
Cl
Several substitutions were explored at the C-2 position of
the pyrimidine core using similar chemistry to that shown in
Scheme 2. Analogues disubstituted at the nitrogen at the 2-posi-
tion of the pyrimidine were made by introducing alkyl or acetyl
3
2
O
S
NH2
O
N
HN
N
N
N
NH
N
NH
N
O
O
N
O
O
N
O
N
3a
IC50 = 8060 nM
N
N
N
N
3b
IC50 = 640 nM
Solubility = 4.6 µM
N
N
Cl
Cl
a OR b
c, d
N
Scheme 1. Reagents and conditions: (a) HNR1R2, NEt3, dichloromethane, rt; (b)
HNR3R4, NEt3, BuOH, 120 °C.
R
R
N
N
N
N
NH
OH
N
O
OMe
N
Cl
Cl
N
Cl
a
b
10
N
NH
11a. R = Me
11b. R = Acetyl
N
NH
12a. R = Me
12b. R = Acetyl
N
Cl
4
6
5
Scheme 3. Reagents and conditions: (a) NaH, MeI, DMF, rt; (b) NEt3, AcCl,
dichloromethane; (c) TFA, dichloromethane, rt; (d) NEt3, 1-(bromoacetyl)-piperi-
dine, DMF, rt.
Cl
Cl
NR1R2
N
Cl
N
d
c
N
NH
N
NH
N
O
Cl
N
Cl
N
N
7
8
a
b
N
O
N
Cl
Cl
NH
N
NH2
O
S
N
O
NH2
O
N
N
HN
13
N
NR1R2
=
N
NH
NR1R2
=
O
9
O
N
N
8a
14
IC50 = 758 nM
Solublity = 181 µM
N
IC50 = 301 nM
Solublity = 79 µM
Cl
H
15
Cl
Scheme 2. Reagents and conditions: (a) p-chlorophenylethyl amine, NEt3, BuOH,
120 °C; (b) 12 N HCl in AcOH; 100 °C; (c) POCl3, 80 °C; (d) HNR1R2, NEt3, BuOH,
120 °C.
Scheme 4. Reagents and conditions: (a) NEt3, 4-chlorophenylacetyl chloride, DMA,
rt; (b) NEt3, BuOH, 120 °C.

2104
M. Bayrakdarian et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2102–2105
groups into the intermediate 10, which was made by reacting
the chloro intermediate 7 with Boc protected piperazine. Re-
moval of the Boc group followed by alkylation with 1-(bromo-
acetyl)piperidine gave compounds 12a and 12b (Scheme 3).
Compound 15 was made by the acylation of 2-amino-4-chloro
pyrimidine 13 with 4-chlorophenylacetyl chloride followed by
displacement of the 4-chloro group with the corresponding
amine as shown in Scheme 4. However, the original 4-chloroph-
enyethylamino group, as in compound 9, could not be improved
upon. The N-acetyl amide 12b showed a ꢀ5-fold loss in potency
and 12a and 15 were completely inactive.
dichloropyrimidines with amines as shown in Scheme 5. A methyl
group is tolerated at the C-6 position of the pyrimidine ring as
shown by compound 18a, which showed comparable activity to
compound 9 while a methyl at the C-5 position (compound 18b)
resulted in a 5–10-fold drop in activity at SNSR4 compared to com-
pound 9.
The significance of the second nitrogen atom in the pyrimidine
ring was explored by making the corresponding pyridine deriva-
tives 21 and 24 (Scheme 6). 2,4-Di-substituted pyridine analogue
21 was made starting from a known intermediate 19.7 Palladium
catalyzed amination of the intermediate 19 with p-chlorophenyl-
ethylamine followed by removal of the Boc group and alkylation
with the corresponding bromo acetyl amide derivative gave 21.
2,6-Di-substituted pyridine analogue 24 was made by substitution
of one of the chloro groups by substituted piperazine followed
by palladium catalyzed amination at the second chloro group.
2,4-Di-substituted pyridine analogue 21 demonstrated a moderate
drop (ꢀ3-fold) in potency compared to compound 9 while the
2,6-di-substituted pyridine analogue 24 had no activity.
Substitution at the C-5 and C-6 positions of the pyrimidine ring
were then explored. These compounds were made by two consec-
utive substitution reactions of the appropriately substituted
N
N
N
Next, several amides on the C-4 position of the piperazine ring
of compound 9 were explored in an attempt to improve potency
O
O
O
N
N
N
N
N
Cl
N
R1
R2
N
H
R1
R2
R1
R2
4
N
N3
a
b
5
O
N
O
1
Cl
2
6
NR1R2
NH
N
OH
Cl
N
H
N
N
N
N
N
N
18a. R1 = H, R2 = Me
18b. R1 = Me, R2 = H
a, b
c
N
N
N
Cl
N
NH
N
NH
N
NH
Scheme 5. Reagents and conditions: (a) NEt3, dichloromethane, rt; (b) 4-chloro-
phenylethylamine, NEt3, BuOH, 120 °C.
25
26
27
Cl
Cl
Cl
O
O
O
Scheme 7. Reagents and conditions: (a) chloroacetyl ethyl ester, NEt3, DMF; (b)
N
TFA, dichloromethane, rt; (c) HNR1R2, HATU, DIPEA, DMF, rt.
O
O
N
N
N
N
N
N
a
b, c
Table 1
SAR at the top amide: variation of R2
N
NH
N
NH
R2
N
Cl
O
N
N
21
20
19
H
N
N
N
O
Cl
N
Cl
N
Cl
O
N
N
O
Compound
R2
IC50 (nM)
20
Solubility (
16
lm)
N
N
N
O
N
N
27a
N
N
Cl
N
N
N
27b
27c
22
24
N/D
6
d
H
e
NH
Cl
Cl
Cl
22
23
24
N
Cl
O
Cl
27d
27e
40
88
N/D
O
Scheme 6. Reagents and conditions: (a) 4-chlorophenylethylamine, 5 mol %-
Pd2(DBA)3, 5 mol % Xanphos, NaOtBu, toluene, 100 °C; (b) TFA, dichloromethane,
rt; (c) NEt3, 1-(bromoacetyl)-piperidine, DMF, rt; (d) NEt3, BuOH, 120 °C;
(e) 5 mol %Pd2(DBA)3, 5 mol % BINAP, NaOtBu, 4-chlorophenylethylamine, toluene,
80 °C.
>750
(CH2)3
Ethyl
Cyclopropyl methyl
27f
27g
244
295
1130
104

M. Bayrakdarian et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2102–2105
2105
Table 2
(compound 27i) at the pyridine ring improve activity but reduce
solubility. The 4-pyridyl group could also be replaced with a simple
phenyl group (compound 27j), but this results in a decrease in
solubility. Introduction of a morpholinopropyl group provided
compound 27k as a tool for target validation with good activity
and adequate solubility.
In conclusion, 2,4-diaminopyrimidines were developed as a no-
vel class of SNSR4 antagonists starting from a quinazoline core.
Systematic exploration of the SAR led to a potent and selective
SNSR4 antagonist tool compound 27k, suitable for target
validation.
SAR at the top amide: variation of R1 and R2
R2
O
N
R1
N
H
N
N
N
N
Cl
Compound
R1
R2
IC50 (nM)
77
Solubility (
lm)
Cl
Br
27h
Ethyl
Ethyl
1.5
N
N
Reference and notes
Cl
1. (a) Marinissen, M. J.; Gutkind, J. S. Trends Pharmacol. Sci. 2001, 22, 368; (b)
Stadel, J. M.; Wilson, S.; Bergsma, D. J. Trends Pharmacol. Sci. 1997, 18, 430; (c)
Rohrer, D. K.; Kobilka, B. K. Physiol. Rev. 1998, 78, 35; (d) Hopkins, A. L.; Groom,
C. R. Nat. Rev. Drug Discov. 2002, 1, 727.
2. Dong, X.; Han, S.; Zylka, M. J.; Simon, M. I.; Anderson, D. J. Cell 2001, 106, 619.
3. Lembo, P. M.; Grazzini, E.; Groblewski, T.; O’Donnell, D.; Roy, M. O.; Zhang, J.;
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4. (a) Grazzini, E.; Puma, C.; Roy, M.; Yu, X.; O’Donnell, D.; Schmidt, R.; Dautrey, S.;
Ducharme, J.; Perkins, M.; Panetta, R.; Laird, J.; Ahmad, S.; Lembo, P. Proc. Natl.
Acad. Sci. 2004, 101, 7175; (b) Chen, T.; Cai, Q.; Hong, Y. Neuroscience 2006,
141(2), 965.
27i
27j
108
330
9
2
Ethyl
Cl
O
27k
14
43
N
N
Cl
(CH2)3
5. Kunapuli, P.; Lee, S.; Zheng, W.; Alberts, M.; Kornienko, O.; Mull, R.; Kreamer, A.;
Hwang, J.; Simon, M. I.; Strulovici, B. Anal. Biochem. 2006, 351, 50–61.
6. HEK293s cells (with or without co-expressed G proteins) were plated in a 384
and solubility. Synthesis of the amide analogues is outlined in
Scheme 7. Alkylation of intermediate 25 with chloroacetyl t-butyl
ester followed by deprotection of the tert butyl group gave the car-
boxylic acid 26. A library of amides was made by treating the por-
tions of the acid 26 with corresponding set of different amines in
the presence of a coupling agent.
N-Ethyl-N-(4-pyridyl methyl) amide 27f showed improved
activity and solubility compared with compound 9. A wide variety
of ethyl amide replacements were explored varying R2 group and
keeping the N-(4-pyridyl methyl) group constant and the results
are summarized in Table 1. Interestingly, a wide variety of groups
are tolerated. Bulkier substitutions improved potency but
decreased solubility. A moderate improvement in solubility was
observed with the furylmethyl amide 27a.
plate at 17,000 cells/well/25 ll for 24 h in a humidified incubator (5% CO2 and
37 °C) in DMEM supplemented with 10% FBS (also known as DMEM complete).
In general, receptors expressed in HEK 293s (with or without co-expressed G
proteins) cells will have to be grown at least 24 h in the absence of any selection
media, that is, DMEM complete. Prior to the experiment the cell culture medium
was removed from the plates by inversion. A loading solution of 30
balanced salt solution (without phenol red, Gibco, catalog #: 14065-056),
20 mM Hepes with 0.1% BSA at pH 7.4, with 2 M Fluo-3-AM (TEF labs, catalog #
0116, 1 mg/440 l low water content DMSO, 2 mM) and 0.02% Pluronic acid F-
ll of Hank’s
l
l
127 (Molecular Probes, catalog #P-3000-MP, stock: 20% solution in DMSO) was
added to each well. Plates were incubated at 37 °C (and 5% CO2) for 60 min prior
to start the experiment. The incubation was terminated by washing the cells
four times in assay buffer (Hank’s BSS, 20 mM HEPES 0.1% BSA, pH 7.4.),
leaving a residual 25 ll buffer per well. After the washing, the cells were
incubated at room temperature for 5–10 min and then transferred to the FLIPR,
ready for compound additions. The day of experiment, the reference agonist and
compounds were diluted in 3-fold concentration range (10 points serial
dilution) for addition by FLIPR instrument. For all calcium assays, a baseline
reading was taken for 30 s followed by the addition of 12.5
ll of compounds,
To improve activity and solubility further, replacements of the
N-(4-pyridylmethyl) group were synthesized by following the
route outlined in Scheme 7. The results are summarized in Table 2.
Substitutions such as chloro- (compound 27h) and bromo-
resulting in a total well volume of 37.5 l. Data were collected every 1.6 s for
l
300 s. The fluorescence emission was read using filter 1 (emission 520–545 nm)
by the FLIPR on board CCD camera.
7. Ji, J.; Li, T.; Bunnelle, W. Org. Lett. 2003, 5, 4611.