Synthesis and biological evaluation of novel pyrimidine derivatives as sub-micromolar affinity ligands of GalR2

Article abstract of DOI:10.1016/j.bmcl.2011.09.033

GalR1 and GalR2 represent unique pharmacological targets for treatment of seizures and epilepsy. A novel series of 2,4,6-triaminopyrimidine derivatives were synthesized and found to have sub-micromolar affinity for GalR2. Optimization of a series of 2,4,6

Full text of DOI:10.1016/j.bmcl.2011.09.033

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7210–7215
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel pyrimidine derivatives
as sub-micromolar affinity ligands of GalR2
Vasudeva Naidu Sagi a, Tianyu Liu a, Xiaoying Lu b, Tamas Bartfai b, Edward Roberts a,
⇑
a Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, United States
b Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, CA 92037, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2011
Revised 7 September 2011
Accepted 12 September 2011
Available online 25 September 2011
GalR1 and GalR2 represent unique pharmacological targets for treatment of seizures and epilepsy. A
novel series of 2,4,6-triaminopyrimidine derivatives were synthesized and found to have sub-micromolar
affinity for GalR2. Optimization of a series of 2,4,6-triaminopyrimidines led to the discovery of several
analogs with IC50 values ranging from 0.3 to 1 lM.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Galanin
G protein-coupled receptor
Seizure
Binding affinities
The neuropeptide galanin1 is widely expressed in the central
nervous system (CNS) and peripheral nervous system (PNS) of
many different species, including humans.2–4 Galanin regulates a
variety of physiological and pathological processes, including pain,
learning, memory, mood, addiction, and food intake.5 In addition, a
critical role for galanin in seizure control has been suggested.
Galanin has been shown to exhibit potent anticonvulsant effect
in both acute and chronic seizure models in rodents. Intrahippo-
campal injected galanin strongly and irreversibly attenuated the
status epilepticus induced by perforant path stimulation in rats.6
Mice with null mutation of galanin were more susceptible to
develop status epilepticus after perforant path stimulation or sys-
temic kainic acid injection, and exhibited more severe seizures fol-
lowing pentylenetetrazol injection.7 In contrast, mice that
overexpress galanin under a dopamine-b-hydroxylase promoter
developed increased resistance to seizure induction in these three
models.7
(IP) accumulation.8 Both GalR1 and GalR2 are G protein-coupled
receptors that are expressed at high levels in the hippocampus.9
Much effort has been made in recent years toward development
of systemically active agonists/ modulators for GalR1 and GalR2
receptors. So far, two nonpeptide, systemically active agonists,
Galnon6 and Galmic11 (Fig. 1), and a CNS-penetrating peptide gal-
anin analog Gal-B210,12 have been synthesized and characterized.
While exhibiting micromolar to submicromolar affinities to GalR1
and GalR2, Galnon and Galmic are not selective between the two
receptors and thus raise concerns of possible side effects.13 A new-
ly synthesized peptide analog B-25 has been tested as a galanin
analog and shown impressive in vivo efficacy.10 Peptides typically
have short in vivo half lives but the half life of B-25 is greater than
10 h.10 Moreover, it may be cost-inefficient to produce peptides in
sufficient quantities needed for in vivo evaluation. Therefore, there
is a continuous need for selective as well as systemically active,
nonpeptide, agonist or modulator of GalR1/R2.
The physiological effects of Galanin are known to be mediated
by the activation of one or more of the three known G-protein-cou-
pled galanin receptor subtypes designated as GalR1, GalR2 and
GalR3. GalR1 and/or GalR2 are particularly relevant to seizures
and epilepsy. GalR1 receptor is predominantly coupled to Gi-medi-
ating suppression of forskolin-stimulated cAMP accumulation. In
addition to being weakly coupled to Gi, GalR2 is found to couple
to Gq, resulting in membrane lipid turnover and inositol phosphate
As part of our research efforts to discover and develop nonpep-
tide agonists for the treatment of seizures and epilepsy, we re-
cently reported the discovery of compound CYM2503 (3), a
peptidomimetic, positive allosteric modulator of GalR2. CYM2503
induces anticonvulsant effects in animal models.14 However, find-
ing a small-molecule agonist for the GalR2 is particularly challeng-
ing. In this report, we describe the discovery of small molecular
novel pyrimidine derivatives as sub-micromolar affinity ligands
of GalR2.
Screening of our internal compound collection for modulators
of the GalR2 receptor resulted in the identification of 4-bromo-
N2,N6-dicyclohexylpyridine-2,6-diamine (4, CYM2024). Although
⇑
Corresponding author. Tel.: +1 858 784 7770; fax: +1 858 784 7745.
E-mail address: eroberts@scripps.edu (E. Roberts).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.033

V. N. Sagi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7210–7215
7211
O
NH
O
O
N
H
O
N
N
O
NH
HN
O
O
NH
N
O
NH
CO2Me
O
HN
3TFA
Galmic (1)
H2N
O
BocHN
O
H
N
H
O
H
N
H
N
H
O
FmocHN
O
O
FmocHN
O
N
H
N
H
H
H
(2)
Galnon
(3)
CYM2503
Figure 1. Structures of nonpeptide ligands of the Galanin receptor.
X1
compound 4 has moderate binding affinity to GalR2, we were not
able to increase affinity by further chemical modifications with dif-
ferent substitutions on the pyridine core. We then turned our
attention to the pyrimidine and triazine system. Specifically, we
focused on 2,4,6-triaminopyrimidines and/or triazines, as exempli-
fied by general structures 5 and 6 (Fig. 2).
To develop a structure–activity relationship (SAR) around 5 and
6, series of pyrimidines and triazines were first synthesized. The
triaminopyrimidines were synthesized via routes depicted in
Scheme 1. 2,4,6-trichloropyrimidine (7) was treated with an appro-
priate aliphatic amine at 0 °C to give 4-aminopyrimidine 8 in
75–85% yield. The second amino substitution at position 2 was
effected by treating compound 8 with an arylamine in the presence
of diisopropylethylamine in refluxing n-BuOH for 1–4 days to give
compound 10. To shorten the reaction time, compound 8 was trea-
ted with an arylamine under microwave conditions at 160 °C to
give the diaminopyrimidine 10 in 85–95% yield. Compound 10
was further treated with another arylamine under Buchwald–Har-
twig coupling conditions15 to give triaminopyrimidine derivative 6
in 80–90% yield.
Cl
N
Cl
4
N
N
N
a
6
2
X1
Cl
Cl
N
N
Cl
Cl
N
7
Cl
8 (major)
9 (minor)
X1= NHAr1 or NR1R2
X1
X1
X1
N
N
c
b
Cl
N
8
Cl
X2
N
10
Cl
X2
N
6
X3
X2= NHAr2 or NR3R4
X3 = NHAr3 or NR5R6
[Ar1, Ar2, and Ar3 = Aryl
R1, R2, R3, R4, R5 and R6 = alkyl]
Scheme 1. General strategy for the synthesis of triaminopyrimidine derivatives.
Reagents and conditions: (a) R1R2NH2 (1.0 equiv), Et3N (1.1 equiv), 0 °C–rt, 30 min,
75–85%; Ar1NH2 (2.0 equiv), Na2CO3 (2.0 equiv), EtOH, reflux, 2–4 h, 85–95%; (b)
Ar2NH2 (1.5 equiv), DIPEA (5.0 equiv), n-BuOH, reflux, 1–4 days or n-BuOH, micro-
wave, 160 °C, 4–7 h, 85–90%; R3R4NH2 (1.5 equiv), DIPEA (2.0 equiv), n-BuOH, rt,
overnight, 90–95%; (c) Ar3NH2, Pd2(dba)3, XPhos, t-BuOK, dioxane, 85 °C, overnight,
80-90%; R5R6NH2, n-BuOH, microwave, 160 °C, 2–5 h, 90–95%.
In an alternative method, 2,4,6-trichloropyrimidine (7) was first
treated with an arylamine in the presence of Na2CO3 in refluxing
EtOH for 2–4 h to give 4-aminopyrimidine 8 in 85–95% yield.16
Compound 8 was further treated with another arylamine using
diisopropylethylamine followed by final substitution with an
aliphatic amine under microwave conditions to afford triamino-
pyrimidine derivative 6 in 90–95% yield.17
X1
X1
Br
N
X1
N
N
X1
X1
Cl
N
N
N
a
N
N
b
c
N
N
X2
X3
N
6
X3
X2
N
5
N
N
N
H
N
H
X2
N
13
Cl
X2
N
5
X3
Cl
N
11
Cl
Cl
N
12
Cl
X1 = NHAr1 or NR1R2
X2 = NHAr2 or NR3R4
X3 = NHAr3 or NR5R6
4
(CYM2024)
X1 = NHAr1
X2 = NHAr2
or NR3R4
X3 = NHAr3
or NR5R6
or NR1R2
[Ar1, Ar2, and Ar3 = Aryl
R1, R2, R3, R4, R5 and R6 = alkyl]
Scheme 2. General strategy for the synthesis of triaminotriazine derivatives.
Reagents and conditions: (a) R1R2NH2 or Ar1NH2, NaHCO3, acetone, H2O, 0–5 °C,
80–85%; (b) R3R4NH2 or Ar2NH2, K2CO3, THF, rt, 2–4 h, 80–85%; (c) R5R6NH2 or
Ar3NH2, K2CO3, dioxane, microwave, 120 °C, 1–4 h, 90–95%.
Figure 2. Structures of
pyrimidines and triazines.
4
(CYM2024) and general structures of 2,4,6-triamino

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V. N. Sagi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7210–7215
Table 1
GalR1 and GalR2 binding affinitiesa of compounds 6a–6t
X1
4
N
2
6
X2
N
X3
6
Compdb
X1
X2
X3
GalR1 IC50
(
lM)
GalR2 IC50 (lM)
6a
6b
2,5-Dimethylaniline
4-OCH3-Aniline
NA
8.59
4.57
1.09
6c
6d
6e
6f
6g
6h
3- OCF3-Aniline
4- OCF3-Aniline
4-(OCF2H)Aniline
3-(OCF2H)Aniline
Piperonylamine
3-F,4-OCH3-Aniline
34.86
NA
NA
6.9
NA
6.62
NA
3.38
4.15
3.69
8.65
N
NH
6.77
6i
6j
Pyrrolidine
Pyrrolidine
1-Phenylpiperazine
3-OCH3-Aniline
12.8
4.6
3.4
0.93
OCH3
6k
6l
6m
6n
6o
6p
Pyrrolidine
4-OCH3-Aniline
4-(OCF2H)Aniline
3-(OCF2H)Aniline
4-OCF3-Aniline
3-OCH3-Aniline
3-OCF3-Aniline
NA
8.04
12.5
0.77
1.06
1.75
0.55
Cyclohexanamine
Cyclohexanamine
Cyclohexanamine
Cyclohexanamine
Cyclohexanamine
27.5
5.03
5.93
5.62
6.82
NH
6q
6r
6s
6t
4-OCH3-Aniline
2,5-Dimethylaniline
3-F,4-OCH3-Aniline
3-F,2-CH3-Aniline
3.35
3.90
6.32
1.30
1.92
2.08
2.03
0.56
N
N
N
N
CF3
a
Binding affinity was determined using [125I] galanin displacement assay.19 NA: not active; up to 100
l
M.
b
All compounds were at least 95% pure with the majority being greater than 98% pure by LC–MS20
,
1H- and 13C NMR.
The 2,4,6-triaminotriazine series was prepared by the consecu-
6j, 6m, 6n and 6p. However, this series of compounds did not
exhibit selectivity between GalR1 and GalR2. Interestingly, when
2- and 4-positions of pyrimidine were substituted with piperidine
and 1-(3-(trifluoromethyl)pyridin-2-yl)-1,4-diazepane, the result-
ing triaminopyrimidines were showed even higher binding affinity
with nonselectivity between GalR1 and GalR2 (Table 1).
After many modifications on the pyrimidine core, we found that
p-OCH3-aniline at 2-position and piperidine at 4-position were nec-
essary for selective binding affinity for GalR2. We then maintained
the two optimal substituents at 2- and 4- positions and varied the
substitutions at 6-position (structure 6A). Most of the compounds
from this series show selective binding affinity for GalR2 with
tive nucleophilic substitution of cyanuric chloride (11) with differ-
ent nucleophiles as shown in Scheme 2. Commercially available
cyanuric chloride was first treated with an aliphatic or aromatic
amine at 0 °C to yield the monoaminotriazine 12 in 80–85% yield.
The second chloride of compound 12 was substituted by various
amines at room temperature to provide diaminotriazine 13 in
80–85% yield. Compound 13 was further treated with an aliphatic
or aromatic amine under microwave conditions at 120 °C to give
triaminotriazine 5 in 90–95% yield. All intermediates and final
compounds provided satisfactory 1H, 13C NMR, LCMS, and mass
spectral data.18
With the initial libraries of pyrimidines and triazines in hand,
we then evaluated their affinities to GalR1 and GalR2. All com-
pounds were first screened at 1 and 10 lM in duplicate for affinity
to GalR1 and GalR2. The affinity screening was performed twice for
each receptor subtype. Compounds that exhibited greater than 50%
binding inhibition for GalR2 or 60% binding inhibition for GalR1 at
IC50s ranging from 0.33 to 1.0 lM. Small structural changes of
the substituents on the aromatic ring at 6-position of pyrimidine
core influenced the binding affinities of these compounds. Com-
pounds with 4-OCF3 and 3-OCF3 anilines 6w (CYM2229) and 6x
(CYM2235) have same binding affinities. In a similar manner, com-
pound 6v with 2,5-dimethyl substituted aniline has more binding
affinity (twofold) than its regio-isomer 2,4-dimethyl derivative
(6u). Though, dimethyl anilines showed reasonable selective bind-
ing affinities against GalR2, it is worthwhile to evaluate the binding
affinity of the mono-methyl substituent anilines. We then prepared
all 3 possible regio-isomers (o-, m- and p-) of mono-methyl aniline
derivatives, but none of the derivatives showed binding affinity for
both GalR1 and GalR2. In the most of the cases, 2-methyl-fluoro ani-
lines at the 6-position of pyrimidine core shows selective and good
binding affinity for GalR2 [6dd and 6ee (CYM2248)]. Surprisingly,
compound with 4-OCF2H substituted aniline (6aa) has no binding
affinity, where as compound with 3-OCF2H substituent 6z
either 1 or 10 lM concentration were progressed to full curve
binding assay for the corresponding receptor subtype.
Unexpectedly, none of the triazine series exhibited significant
binding affinities to GalR1 and GalR2. Fortunately, we were able
to produce meaningful SAR for the 2,4,6-triaminopyrimidines
(ꢀ250) that were synthesized and screened for binding affinity.
Based on these results, we made additional optimization efforts
on substitution patterns at the pyrimidine nucleus to improve
the binding affinity to GalR1 and GalR2. The SAR of a focused li-
brary of 2,4,6-triaminopyrimidines is shown in Tables 1–3.
When the 2- and 4-position of pyrimidine were substituted,
respectively, with aniline and piperidine, the resulting triamino-
pyrimidines (6a–6h) were nonselective between GalR1 and GalR2
with moderate binding affinity. When the 4-position was substi-
tuted with p-OCH3-aniline and the 2- and 6- positions of the
pyrimidine were modified with other aromatic/aliphatic amines,
the best binding affinity of this series was obtained for compounds
(CYM223) has good binding affinity with IC50 = 0.54 lM (Table 2).
Anilines with electron withdrawing groups (NO2, CN) at the 6-posi-
tion did not show significant binding affinity for either receptors
(not listed in the table).
Next SAR studies (Table 3) at the 4-position were performed
while maintaining p-OCH3-aniline at 2-position and 3-F-4-OCH3

V. N. Sagi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7210–7215
7213
Table 2
Table 3
GalR1 and GalR2 binding affinitiesa of compound series 6A (6u–6ff)
GalR1 and GalR2 binding affinitiesa of compound series 6B (6gg–6qq)
X1
F
4
H3CO
OCH3
N
2
N
6
4
H3CO
N
H
N
N
H
N
6
N
H
N
X3
6B
2
6A
Compdb
X1
GalR1 IC50
NA
(l
M)
GalR2 IC50 (lM)
Compdb
X3
GalR1 IC50
NA
(
lM)
GalR2 IC50
1.18
(lM)
6gg
NA
N
CH3
H
N
6hh
6ii
5.48
NA
0.77
N
6u
CH3
2.85
NA
N
CH3
CH3
H
N
CH3
6v
NA
NA
0.44
0.38
6jj
NA
N
H
N
CH3
6w
6kk
6.78
1.34
N
N
OCF3
H
N
6x
6y
NA
NA
0.33
0.78
6ll
NA
NA
NA
CH3
OCF3
CF3
H
N
6mm
3.84
NH
N
N
H
N
N
N
6nn
6oo
6pp
6qq
3.84
6.82
16.01
2.82
NA
6z
NA
NA
0.54
OCF2H
H
N
6aa
NA
N
OCF2H
24.85
H
N
F
N
6bb
6cc
NA
NA
0.43
1.93
OCH3
CF3
N
N
N
H
NA
CH3
CH3
H
N
6dd
NA
0.98
F
F
O
N
O
H
N
9.07
6ee
6ff
NA
NA
0.38
4.18
N
H
N
Cl
F
Binding affinity was determined using [125I] galanin displacement assay.19 NA:
not active; up to 100 M.
All compounds were at least 95% pure with the majority being greater than 98%
pure by LCMS20 1H- and 13C NMR.
a
l
b
Binding affinity was determined using [125I] galanin displacement assay.19 NA:
not active; up to 100 M.
All compounds were at least 95% pure with the majority being greater than 98%
pure by LC–MS20 1H- and 13C NMR.
a
,
l
b
,
pane (6hh) or substituted piperidine (6jj, 6kk, 6ll and 6pp) re-
sulted in a loss of selectivity and/or binding affinity for GalR2.
SAR around the position-2 underlined the unique role of piperidine
aniline at 6-position (structure 6B). When piperidine was replaced
with lower membered pyrrolidine (6gg), higher membered aze-

7214
V. N. Sagi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7210–7215
in binding. The replacement of carbon at 4th position of piperidine
with O, S, SO2, NH, substituted (alkyl or aryl) NH, or removal of cyc-
lic constraint were in fact not tolerated and caused significant loss
of GalR2 binding affinity. Replacement of p-OCH3-aniline at 2-posi-
tion with p-OC2H5, p-isopropoxy or p-Cl aniline also caused loss of
affinity. In fact, while keeping piperidine at the 4-position and
interchanging of 3-F-4-OCH3 and p-OCH3-aniline from 6-position
to 2-position also caused significant loss of GalR2 binding affinity
(not listed in the table). Among all pyrimidine derivatives,
compounds 6mm and 6nn shows selective binding affinity for
the reaction. The reaction progress was followed by TLC. After completion of
the reaction, an equal volume of water was added with cooling. The resulting
white precipitate was filtered, washed with water, and dried in vacuum over
night to yield 4-substituted 2,6-dichloro pyrimidine. In case of no
precipitation, ethanol was removed by rota vap., and the residue was
dissolved in CH2Cl2. The organic layer was washed twice with water, brine,
dried (Na2SO4), filtered, and concentrated. The resulting crude was purified by
column chromatography to afford the 4-amino-2,6-dichloro pyrimidines in
85–95% yield. Step-2: 2,4-diamino-6-chloropyrimidine: 4-amino-2,6-dichloro
pyrimidine (1.0 mmol) prepared from the above procedure was treated with
another aliphatic amine or aromatic amine (2.0 mmol) in the presence of
DIEPA (5.0 mmol) in n-BuOH (5 mL) at rt. For an aliphatic amine the reaction
mixture was stirred at rt for overnight. For an aromatic amine the reaction
mixture was refluxed for 24–72 h or placed in microwave (150 °C, 2–7 h) until
completion of the reaction. The reaction progress was followed by TLC. After
completion of the reaction, solvents were removed by rota vap., and the
residue was dissolved in CH2Cl2. The organic layer was washed twice with
water, brine, dried (Na2SO4), filtered, and concentrated. The resulting
crude was purified by column chromatography (EtOAc/hexane) to afford the
2,4-diammino-6-chloropyrimidines in 85–90% yield. Step-3: 2,4,6-
GalR1 with IC50 3.8 lM each.
In summary, small molecular novel 2,4,6-triaminopyrimidine
derivatives were designed, synthesized, and subsequently tested
for affinities for GalR1 and GalR2. Eight compounds of the series
of structure 6A, where p-OCH3-aniline at the 4-position and piper-
idine at the 2-position of the pyrimidine core exhibited the highest
triaminopyrimidine:
2,4-diamino-6-chloropyrimidine
(1.0 mmol)
prepared from the above procedure was treated with another
suitable aliphatic amine or aromatic amine (3.0 mmol). For an
aliphatic amine, 2,4-diamino-6-chloropyrimidine (1.0 mmol) was
treated with aliphatic amine (3.0 mmol) and DIPEA (5.0 mmol) in n-
BuOH (5 mL) and placed in microwave (150 °C) for 3–7 h. After the
completion of the reaction (monitored by TLC), solvents were removed
and the residue was dissolved in EtOAc. The organic layer was washed
twice with water, brine, dried (Na2SO4), filtered, and concentrated. The
resulting crude was purified by column chromatography to afford the
2,4,6-triaminopyrimidines 90–95% yield. For aromatic amine; 2,4-
diamino-6-chloropyrimidine (1.0 equiv) was dissolved in dioxane
under argon and to that were added Pd2(dba)3 (10 mol %), Xantphos
(10 mol %), aromatic amine (1.2 mmol), t-BuOK (1.2 mmol). The
resulting solution was degassed with argon for 5 min and heated to
85 °C for overnight. The reaction mixture was filtered through a pad of
celite, washed with CH2Cl2 (2 Â 10 mL) and the resulting filtrate was
concentrated. The resulting crude was purified by flash column
chromatography to yield 2,4,6-triaminopyrimidines in 90–95% yield.
18. Spectral data for selected compounds:N2-(4-methoxyphenyl)-6-(piperidin-1-yl)-
N4-(4-(trifluoromethoxy)phene-yl)pyrimidine-2,4-diamine[6x (CYM2235)]: 1H
NMR (300 MHz, CDCl3) d 7.44 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 7.14
(d, J = 8.3 Hz, 2H), 6.83 (d, J = 8.5 Hz, 2H), 6.61 (br s, 1H), 5.44 (s, 1H), 3.78 (s,
3H), 3.51 (br s, 4H), 1.60 (br s, 6H); 13C NMR (CDCl3, 75 MHz) d 164.01, 161.71,
159.87, 154.92, 144.44, 138.79, 133.70, 122.29, 121.99, 121.48, 113.99, 75.90,
55.64, 45.56, 25.66, 24.88. ESI-MS (m/z): calcd for C23H24F3N5O2 459.46, found
460.5 [M+H]+. LC–MS >98%, tR = 3.25 min, m/z 460 [M+H]+.
affinity for GalR2 receptor with IC50 ranging from 0.33 to 1.0 lM.
Further results from this lab regarding the activity of these and
similar compounds will be presented in due course.
Acknowledgment
This work was supported by National Institute of Health Grant
NS063560.
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Kuteeva, E.; Elvander-Tottie, E.; Hökfelt, T. Eur. J. Pharmacol. 2010, 626, 9; (i)
Picciotto, M. R.; Brabant, C.; Einstein, E. B.; Kamens, H. M.; Neugebauer, N. M.
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K.; Emery, M. S.; West, C. H. Ann. N. Y. Acad. Sci. 1998, 863, 364; (k) Lundström,
L.; Elmquist, A.; Bartfai, T.; Langel, U. Neuromolecular Med. 2005, 7, 157; (l)
Bartfai, T.; Hokfelt, T.; Langel, U. Crit. Rev. Neurobiol. 1993, 7, 229.
6. (a) Saar, K.; Mazarati, A. M.; Mahlapuu, R.; Hallnemo, G.; Soomets, U.; Kilk, K.;
Hellberg, S.; Pooga, M.; Tolf, B. –R.; Shi, T. S.; Hökfelt, T.; Wasterlain, C.; Bartfai,
T.; Langel, Ü. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7136; (b) Sollenberg, U.;
Bartfai, T.; Langel, U. Neuropeptides 2005, 39, 161.
N4-(3-fluoro-2-methylphenyl)-N2-(4-methoxyphenyl)-6-(piperidin-1-yl)pyrimidi
ne-2,4-diamine [6ee (CYM2248)]: 1H NMR (300 MHz, CDCl3) d 7.35 (d, J = 8.2 Hz,
2H), 7.07 (dt, J = 14.7, 7.7 Hz, 2H), 6.88 (br s, 1H), 6.76 (dd, J = 16.2, 8.3 Hz, 3H),
6.40 (br s, 1H), 5.13 (s, 1H), 3.69 (s, 3H), 3.38 (br s, 4H), 2.05 (s, 3H), 1.48 (br s,
6H); 13C NMR (CDCl3, 75 MHz)
d 164.03, 163.45, 162.55, 160.22, 159.78,
154.71, 139.58 (d, J = 6.3 Hz), 133.87, 126.78 (d, J = 10.1 Hz), 121.27, 120.27
(d, J = 3.0 Hz), 119.98 (d, J = 17.5 Hz), 113.96, 111.65 (d, J = 5.1 Hz), 75.33,
55.62, 45.51, 25.63, 24.86, 9.74 (d, J = 5.1 Hz). ESI-MS (m/z): calcd for
C
23H26FN5O 407.48, found 408.56 [M+H]+. LC–MS >98%, tR = 3.28 min, m/z
408 [M+H]+.
N2-(4-methoxyphenyl)-6-(piperidin-1-yl)-N4-(3-(trifluoromethoxy)phenyl)pyrimi-
7. Mazarati, A. M.; Hohmann, J. G.; Bacon, A.; Liu, H.; Sankar, R.; Steiner, R. A.;
Wynick, D.; Wasterlain, C. G. J. Neurosci. 2000, 20, 6276.
dine-2,4-diamine 6w [(CYM2229)]: 1H NMR (300 MHz, CDCl3)
d 7.37 (d,
J = 8.3 Hz, 2H), 7.24 (s, 1H), 7.18 (d, J = 7.0 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H),
6.77 (d, J = 7.2 Hz, 3H), 6.61 (br s, 1H), 5.43 (s, 1H), 3.71 (s, 3H), 3.44 (br s, 4 H),
1.52 (br s, 6H), 1.18 (s, 1H); 13C NMR (CDCl3, 75 MHz) d 16 3.95, 161.28, 159.90,
155.02, 149.80, 141.67, 133.58, 130.19, 121.63, 118.85, 114.74, 114.04, 113.34,
100.18, 76.30, 55.63, 45.57, 25.64, 24.89. ESI-MS (m/z): calcd. for C23H24F3N5O2
459.46, found 469.56 [M+H]+. LC–MS >98%, tR = 2.07 min, m/z 470 [M+H]+.
N4-(3-fluoro-4-methoxyphenyl)-N2-(4-methoxyphenyl)-6-(piperidin-1-
8. Wang, S.; Hashemi, T.; Fried, S.; Clemmons, A. L.; Hawes, B. E. Biochemistry
1998, 37, 6711.
9. (a) O’Donnell, D.; Ahmad, S.; Wahlestedt, C.; Walker, P. J. Comp. Neurol. 1999,
409, 469; (b) Hohmann, J. G.; Jureus, A.; Teklemichael, D. N.; Matsumoto, A. M.;
Clifton, D. K.; Steiner, R. A. Neuroscience 2003, 117, 105.
10. Bulaj, G.; Green, B. R.; Lee, H. –K.; Robertson, C. R.; White, K.; Zhang, L.;
Sochanska, M.; Flynn, S. P.; Scholl, E. A.; Pruess, T. H.; Smith, M. D.; White, H. S.
J. Med. Chem. 2008, 51, 8038.
11. (a) Bartfai, T.; Lu, X.; Badie-Mahdavi, H.; Barr, A. M.; Mazarati, A.; Hua, X. –Y.;
Yaksh, T.; Haberhauer, G.; Ceide, S. C.; Trembleau, L.; Somogyi, L.; Kröck, L.;
Rebek, J., Jr. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10470; (b) Mazarati, A.; Lu, X.;
Kilk, K.; Langel, U.; Wasterlain, C.; Bartfai, T. Eur. J. Neurosci. 2004, 19, 3235.
12. (a) Robertson, C. R.; Scholl, E. A.; Pruess, T. H.; Green, B. R.; White, H. S.; Bulaj,
G. J. Med. Chem. 2010, 53, 1871; (b) White, H. S.; Scholl, E. A.; Klein, B. D.; Flynn,
S. P.; Pruess, T. H.; Green, B. R.; Zhang, L.; Bulaj, G. Neurotherapeutics 2009, 6,
372; (c) Zhang, L.; Robertson, C. R.; Green, B. R.; Pruess, T. H.; White, H. S.;
Bulaj, G. J. Med. Chem. 2009, 52, 1310.
13. Lu, X.; Lundström, L.; Langel, U.; Bartfai, T. Neuropeptides 2005, 39, 143.
14. Lu, X.; Roberts, E.; Xia, F.; Sanchez-Alavez, M.; Liu, T.; Baldwin, R.; Wu, S.;
Chang, J.; Wasterlain, C. G.; Bartfai, T. Proc. Natl. Acad. Sci. U.S.A. 2010, 107,
15229.
15. Surry, D. S.; Buchwald, S. L. Angew. Chem., Int. Ed. 2008, 47, 6338.
16. Schomaker, J. M.; Delia, T. J. J. Heterocycl. Chem. 2000, 37, 1457.
17. General synthesis of 2,4,6-triaminopyrimidine compounds: Step-1: 4-amino-2,6-
dichloro pyrimidine: 2,4,6-trichloro pyrimidine (1.0 mmol) in ethanol (5 mL)
was treated with an aromatic amine (1.1 mmol) in the presence of Na2CO3
(1.1 mmol) at rt. The mixture was stirred at reflux for 2–4 h until completion of
yl)pyrimidine-2,4-diamine [6bb (CYM2233)]: 1H NMR (300 MHz, CDCl3) d 7.35 (d,
J = 8.0 Hz, 2H), 7.07 (d, J = 12.8 Hz, 1H), 6.88 (s, 1H), 6.85–6.67 (m, 4H), 5.57 (s,
1H), 5.26 (s, 1H), 3.77 (s, 3H), 3.68 (s, 3H), 3.39 (br s, 4H), 1.49 (br s, 6H); 13C
NMR (CDCl3, 75 MHz) d 163.98, 162.39, 159.86, 154.74, 153.96, 150.71, 144.08
(d), 133.83, 133.36 (d), 121.31, 118.10, 113.96, 111.56, 111.28, 75.19, 56.71,
55.61, 45.50, 25.61, 24.85. ESI-MS (m/z): calcd for C23H26FN5O2 423.48, found
424.56 [M+H]+. LC–MS >98%, tR = 1.94 min, m/z 425 [M+H]+.
N4-(3-(difluoromethoxy)phenyl)-N2-(4-methoxyphenyl)-6-(piperidin-1-yl)pyrim-
idine-2,4-diamine. [6z (CYM2231)]: 1H NMR (300 MHz, CDCl3)
d 7.35 (d,
J = 8.8 Hz, 2H), 7.24–7.10 (m, 2H), 6.96 (d, J = 8.0 Hz, 1H), 6.88 (s, 1H), 6.75 (d,
J = 8.8 Hz, 2H), 6.66 (d, J = 9.0 Hz, 1H), 6.39 (s, 1H), 5.43 (s, 1H), 3.70 (s, 3H), 3.43
(br s, 4H), 1.51 (br s, 6H); 13C NMR (CDCl3, 75 MHz) d 163.93, 161.38, 159.86,
154.92, 151.88, 141.72, 133.65, 130.18, 121.57, 117.51, 116.03, 113.98, 113.10,
111.84, 76.31, 55.60, 45.54, 25.62, 24.87. ESI-MS (m/z): calcd for C23H25F2N5O2
441.47, found 442.56 [M+H]+. LC–MS >98%, tR = 2.12 min, m/z 443 [M+H]+.
19. In the full curve binding, compounds were tested in concentrations between
10 nM and 100 lM, and a galanin control was always included in each plate.
An IC50 is calculated if the lowest portion of the nonlinear regression 1 site
competition curve is below 35% of the total binding. Ligand competition
binding of 125I porcine galanin to the membrane preparations was performed

V. N. Sagi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7210–7215
7215
in a volume of 150
l
L in a 96-well plate. Cell membranes were diluted in Hepes
three washes with cold PBS solution (pH 7.4) containing 0.01% (vol/vol) Triton
X-100, the filter was counted with Cobra II auto- -counting systems (Packard
Bioscience). All data were analyzed by nonlinear regression (Prism; GraphPad).
20. All compounds were characterized by LC–MS, and gave satisfactory results in
agreement with the proposed structure. Purity was determined by a C18
buffer containing 25 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM CaCl2, 0.5% BSA,
and 1Â protease inhibitor. The 125I porcine galanin was diluted in Tris buffer
(50 mM Tris–HCl, pH 7.4, 14 mM MgCl2, 2.45% BSA). Compounds and rat
c
galanin were diluted in 20% DMSO. Eighty microliters cell membrane (2.5
lg
for GalR1 and 5 g for GalR2 binding), 55
l
lL
125I porcine galanin, and 15
l
L
reverse phase HPLC column [PHENOMENEX–LUNA (50 Â 4.60 mm, 3 l)] in
compound/galanin preparations were combined and incubations were carried
out at room temperature for 1 h. The 125I porcine galanin was used at 0.1 nM
and 0.15 nM for GalR1 and GalR2 binding, respectively. The reactions were
terminated by rapid vacuum filtration through glass fiber filters (Packard
Bioscience), which had been pretreated with 0.3% polyethylenimine. After
10–90% CH3CN/H2O containing 0.02% AcOH. Flow rate 1 mL/min; (5 min
gradient) and monitored by a UV detector operating at 254 nm. Data collection
was in the positive ion mode. LC–MS M+H signals were consistent with
expected molecular weight for all reported products.