Synthesis, SAR, and antitumor properties of diamino-C,N-diarylpyrimidine positional isomers: Inhibitors of lysophosphatidic acid acyltransferase-β

Article abstract of DOI:10.1016/j.bmcl.2004.01.104

2,4-Diamino-N⁴,6-diarylpyrimidines were identified as potent, isoform specific inhibitors of lysophosphatidic acid acyltransferase-β (LPAAT-β). Active inhibitors also blocked proliferation of tumor cell lines in vitro. The effect of 2j in an in

Full text of DOI:10.1016/j.bmcl.2004.01.104

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2303–2308
Synthesis, SAR, and antitumor properties of diamino-C,N-
diarylpyrimidine positional isomers: inhibitors of lysophosphatidic
acid acyltransferase-b
Baoqing Gong, Feng Hong, Cory Kohm, Scott Jenkins, John Tulinsky, Rama Bhatt,
Peter de Vries, Jack W. Singer and Peter Klein*
Cell Therapeutics, Inc., 201 Elliott Ave. W., Suite 400, Seattle, WA 98119, USA
Received 21 December 2003; accepted 30 January 2004
Abstract—2,4-Diamino-N4,6-diarylpyrimidines were identified as potent, isoform specific inhibitors of lysophosphatidic acid acyl-
transferase-b (LPAAT-b). Active inhibitors also blocked proliferation of tumor cell lines in vitro. The effect of 2j in an in vivo tumor
model was investigated.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Lysophosphatidic acid acyltransferases (LPAATs)
catalyze the sn-2 acylation of 1-acyl-sn-glycerol-3-
phosphate (lysophosphatidic acid, LPA) to produce
1,2-diacyl-sn-glycerol-3-phosphate (phosphatidic acid,
PA). The majority of LPAAT activity in mammalian
cells has been attributed to two isoforms, LPAAT-a
and LPAAT-b.1 Both isoforms are integral membrane
proteins. While LPAAT-a is uniformly expressed in
all human tissues tested, LPAAT-b displays distinct
tissue distribution and is highly expressed in a wide
variety of tumor cells and their surrounding stroma.1–5
This group is exemplified by 1 (CT-32228, LPAAT-b
IC50 ¼ 0:06 lM) an effective antiproliferative agent to-
ward a variety of tumor cell lines in vitro with IC50s
ranging from 0.025 to 0.5 lM.5;10
In our search for structural variants of active triazines
we synthesized bioisosteric diamino-C,N-diarylpyrim-
idine analogs. A pyrimidine, possessing one less ring
nitrogen than a triazine, provides the opportunity to
examine preferences for LPAAT-b inhibition by three
possible pyrimidine positional isomers 2, 3, and 4. This
report summarizes the synthesis and elaboration of the
SAR for LPAAT-b inhibition by all three structural
variants. Also, we investigated the effects of these pyr-
imidines on proliferation of tumor cell lines in vitro.
PA is an important lipid cofactor that has been impli-
cated in cell signaling events including Raf translocation
to membranes, mTOR activation, epidermal growth
factor receptor (EGFR) internalization, and activation
of PKCf.6–9 Ectopic overexpression of LPAAT-b
cooperates in activation of Ras/Raf/Erk pathway in
Xenopus oocytes and PA produced by LPAAT-b
appears to play an important role in signaling pathways
involved in tumor cell survival.10 RNAi knockdown of
LPAAT-b blocked tumor cell proliferation.5 Accord-
ingly, LPAAT-b may provide a novel target for cancer
therapy.
NH2
N
NH2
Y
Br
CH3
Cl
N
R1
R2
X
R3
N
N
H
Z
N
H
1
2 (X = Y = N, Z = CH)
3 (X = Z = N, Y = CH)
4 (Y = Z = N, X = CH)
A group of 2,4-diamino-N4,6-diaryltriazines have been
reported as isoform specific inhibitors of LPAAT-b.11
2,4-Diamino-N4,6-diarylpyrimidine 2 was synthesized
in two to four steps involving Suzuki coupling for
arylation of the pyrimidine ring (Scheme 1).12;13 Aryl
* Corresponding author. Fax: +1-206-284-6206; e-mail: jpeterklein@
yahoo.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.104

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B. Gong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2303–2308
NH2
R1
NH2
N
N
R1
R2
X
b
R3
N
N
+
Cl
+
H2N
Cl
Cl
R2
9
8
7
5 (X = I)
a
c
6 (X = B(OH)2)
NH2
N
N
R1
R2
R3
N
H
2
Scheme 1. Reagents and conditions: (a) i-PrMgCl, B(OMe)3, THF; (b) Pd(OAc)2, PPh3, Na2CO3, H2O, monoglyme; (c) HCl–dioxane, EtOH, reflux,
1 h.
boronic acid 6 was either commercially available or
synthesized from the corresponding aryl iodide 5 by
iodine–magnesium exchange using i-PrMgCl followed by
magnesium–boron exchange with B(OMe)3.14 To obtain
aryl iodide 5 (R1 ¼ Me, R2 ¼ Br), 4-bromotoluene was
iodinated using NaIO4–H2SO4–Ac2O–AcOH.15 Palla-
dium catalyzed Suzuki coupling of 6 with 2-amino-4,6-
dichloropyrimidine 7 yielded the requisite intermediate
2-amino-6-aryl-4-chloropyrimidine 8. Phenolic pyrim-
idines 8 (R1 ¼ Me, R2 ¼ OH), 8 (R1 ¼ OH, R2 ¼ Cl),
and 8 (R1 ¼ OH, R2 ¼ Br), used in preparing 2m, 2n,
and 2o, respectively, were produced by O-demethylation
of their corresponding methyl ethers using BBr3–DCM.
Displacement of the remaining chloro group in 8 with
aniline 9 under acidic conditions provided pyrimidine
isomer 2. Apparently, activation of the pyrimidine ring
in 8 by protonation is essential for this displacement to
proceed since basic conditions failed to yield 2.16 The
synthesis of 2r was completed by LiAlH4–THF reduc-
tion of 2 (R1 ¼ OEt, R2 ¼ Cl, R3 ¼ CO2Et).
Treatment of benzonitrile 10 with HCl–EtOH provided
a benzimidic acid ethyl ester, which upon treatment
with NH3–THF yielded benzamidine 11 as its HCl
salt.17Condensation of 11 with ethyl malonate in the
presence of sodium methoxide as base provided aryl-
dihydroxypyrimidine 12. Conversion of 12 to the sym-
metrical dichloropyrimidine 13 was accomplished by
heating 12 with neat POCl3.18 Monoamination of 13
with NH3–THF in a pressure tube provided amino-
chloropyrimidine 14. Displacement of the remaining
chloro group in 14 with aniline 9 under acidic conditions
generated pyrimidine positional isomer 3 albeit at a
slower rate than comparable reaction conditions to
produce 2.
Synthesis of 2,4-diamino-N2,6-diarylpyrimidine 4 pro-
vided additional evidence to support our structure
assignment for 3 (Scheme 3). Treatment of trichloro-
pyrimidine 15 with aniline 9 in dioxane under reflux
yielded a 1:1 mixture of two dichloropyrimidine posi-
tional isomers 16 and 17, which were efficiently sepa-
rated by silica gel column chromatography.19 Purified
symmetrical dichloropyrimidine 17 was treated with
4,6-Diamino-2,N4-diarylpyrimidine 3 was synthesized in
six steps involving pyrimidine ring construction (Scheme 2).
R1
OMe
OMe NH
HCl
N
OMe
Cl
CN
EtO2C
EtO2C
a,b
c
+
NH2
R3
+
R2
N
H2N
9
Cl
Cl
10
11
12 (R1 = R2 = OH)
d
b
e
13 (R1 = R2 = Cl)
14 (R1 = NH2, R2 = Cl)
NH2
N
OMe
R3
N
H
N
Cl
3a-c
Scheme 2. Reagents and conditions: (a) HCl, EtOH; (b) NH3, THF; (c) NaOMe, MeOH, reflux; (d) POCl3, reflux; (e) HCl, dioxane, EtOH, reflux.

B. Gong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2303–2308
2305
Cl
Cl
N
Cl
a
N
N
N
N
N
+
+
R
R
R
H2N
Cl
N
H
Cl
N
H
Cl
Cl
9
15
16
17
b
NH2
N
OMe
Cl
NH2
N
OMe
Cl
B(OH)2
c
R
+
N
N
H
R
Cl
N
N
H
4a-c
18
Scheme 3. Reagents and conditions: (a) dioxane, reflux; (b) NH3, THF; (c) Pd(OAc)2, Ph3P, Na2CO3, H2O, monoglyme.
NH3–THF to produce aminochloropyrimidine 18.
Suzuki coupling of 18 with 5-chloro-2-methoxyphenyl
boronic acid generated positional isomer 4.
aniline 9 under acidic conditions provided 4. TLC
1
behavior (Rf ) and the H NMR spectrum of 4a synthe-
sized by this method were identical to material produced
by the method outlined in Scheme 3 confirming that the
Suzuki arylation of 4-amino-2,6-dichloropyrimidine
with 5-chloro-2-methoxyphenyl boronic acid occurred
at the pyrimidine 6-position to produce 19 rather than at
the 2-position to produce 14 (Scheme 4).
Compound 4 was synthesized more conveniently and in
higher yield by the method described in Scheme 4.
Suzuki coupling of 5-chloro-2-methoxyphenyl boronic
acid with 4-amino-2,6-dichloropyrimidine yielded
aminochloropyrimidine 19. Although we anticipated a
mixture of two isomers (14 and 19) from this Suzuki
coupling, only 19 was observed. Treatment of 19 with
The compounds listed in Tables 1 and 2 were tested for
their ability to inhibit human LPAAT-b overexpressed
NH2
OMe
NH2
B(OH)2
a
OMe
Cl
N
b
+
N
+
R
4a-c
N
Cl
H2N
Cl
N
Cl
Cl
19
a
14
Scheme 4. Reagents and conditions: (a) Pd(OAc)2, Ph3P, Na2CO3, H2O, monoglyme; (b) HCl, dioxane, EtOH, reflux.
Table 1. LPAAT-b inhibition and antiproliferative activity of diamino-C,N-diarylpyrimidine positional isomers
NH2
OMe X
Cl
Y
R
Z
N
H
Compd
X
Y
Z
R
LPAAT-b (IC50, lM)
Tumor cell proliferation (IC50, lM)
K-562
DU145
2a
2b
2c
3a
3b
3c
4a
4b
4c
N
N
CH
CH
CH
N
4-Cl
4-Br
0.054
0.028
0.080
7
0.15
0.15
0.25
0.75
0.15
5.5
0.5
0.25
1
N
N
N
N
4-CH@N–NH-5
4-Cl
4-Br
N
CH
CH
CH
N
5
0.8
N
N
8
N
N
4-CH@N–NH-5
4-Cl
4-Br
2.1
7
>10
>10
>10
>10
CH
CH
CH
N
3
N
N
2.3
0.8
3
>10
N
N
4-CH@N–NH-5

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B. Gong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2303–2308
Table 2. LPAAT-b inhibition and antiproliferative activity by 2,4-diamino-N4,6-diarylpyrimidines
NH2
R1
R2
N
N
R3
N
H
Compd
R1
R2
R3
LPAAT-b (IC50, lM)
Tumor cell proliferation(IC50, lM)
K-562
DU145
2d
2e
H
Cl
H
4-Cl
4-Cl
1.1
0.02
0.15
0.022
0.034
0.61
0.14
0.031
0.018
8.3
Cl
F
<0.1
0.25
0.06
0.06
0.2
0.19
>1
2f
2g
Me
Me
4-Cl
4-Cl
Cl
Cl
Cl
Cl
Br
Br
OH
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
0.12
0.13
0.4
2h
2i
Me
Me
4-Br
3-Br
4-CF3
4-Cl
2j
Me
Me
0.14
0.07
0.07
0.23
0.1
2k
2l
Me
Me
4-Br
4-Cl
0.11
2m
2n
OH
OH
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
4-Cl
4-Cl
0.45
10
>1
>1
2o
2p
4-Cl
2-Cl
0.017
4.9
<0.012
0.07
2q
2r
4-CH2OH
0.075
0.028
0.23
0.58
0.010
0.64
0.024
0.12
0.016
2.7
<0.012
<0.05
>1
0.04
0.07
>1
2s
4-(CH2)2OH
4-(CH2)3OH
4-CH2CO2H
4-NO2
2t
2u
>1
>1
2v
0.18
0.45
<0.1
0.26
0.05
0.35
>1
0.2
2w
2x
2y
3-NO2
3-NH@NCH-4
H
Cl
>1
2z
0.08
2aa
2ab
2ac
2ad
2ae
2af
2ag
2ah
2ai
2aj
2ak
2al
4-CO2H
4-CH2CO2H
4-B(OH)2
4-CN
0.21
0.091
0.017
0.72
0.75
0.088
0.4
>1
>1
0.5
0.4
>1
0.2
0.07
0.35
3-CN
4-C(@O)NH2
4-C(@O)NHOH
4-CH2C(@O)NHOH
4-C(@NH)NH2
4-C(C@O)Me
4-C(C@NHOH)Me
4-C(C@NHOMe)Me
0.7
>1
>1
>1
>5
0.034
0.084
0.97
0.05
<0.05
>1
0.09
<0.05
>1
in SF9 insect cell membranes.20 Also, compounds were
tested for their ability to inhibit similarly overexpressed
LPAAT-a. Only one of the listed compounds measur-
ably inhibited LPAAT-a activity up to a concentration
of 40 lM (2z, 50% inhibition at 40 lM). In addition,
most of the listed compounds were tested for their
ability to block proliferation of K-562 leukemia and
DU145 prostate carcinoma cell lines in vitro.21
N4,6-diarylpyrimidine positional isomers 2a–c sub-
sequent studies were focused on elaboration of the SAR
for this series. The 2,5-disubstitution pattern in the
6-aryl ring of 2 was essential for potent inhibition of
LPAAT-b. Analogs with altered substitution pattern in
this ring or lacking one or both (2d) of these substituents
were significantly weaker inhibitors (data not shown).
Compounds with nonpolar groups at the 2- and 5-
positions were active inhibitors, whereas, compounds
with polar groups at either of these sites (2m and 2n)
were significantly less potent. Compounds with the aryl
5-position substituted with chlorine or bromine and the
2-position substituted with a chloro, alkyl, or alkoxyl
group gave the highest LPAAT-b activity.
Comparing LPAAT-b inhibition by the pyrimidines
listed in Table 1 2a–c were the most potent. Comparison
with tumor cell proliferation data in Table 1 indicated
correlation between LPAAT-b inhibition and antipro-
liferative activity of the listed positional isomers. An
exception was 3b, although a weak LPAAT-b inhibitor
it was an effective antiproliferative agent.
Analysis of the data in Table 2 indicates that LPAAT-b
inhibition is dependent on location of substituents on
the N4-phenyl group. Comparing data for 2h with 2i, 2p
with 2q, 2v with 2w, and 2ad with 2ae it was evident that
Because of the exceptional LPAAT-b inhibition and
antitumor properties observed with the 2,4-diamino-

B. Gong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2303–2308
2307
1000
900
800
700
600
500
400
300
200
100
Vehicle; q1d x4
CT-32521: 100 mg/kg, q1d x4
0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Days Post Implantation
Figure 1. Effect of 2j (CT-32521) on growth of murine LL2 lung tumors in C57BL/6 mice.
the para-position provides more potent inhibitors than
compounds substituted at the meta- or ortho-positions.
Analogs with a para-bromo or para-chloro substituent
were potent LPAAT-b inhibitors and effective inhibitors
of tumor cell proliferation in vitro. Of the compounds
incorporating short chain primary alcohols at the para-
position (2r–t), the 1- and 2-carbon alcohols 2r and 2s
were particularly noteworthy as potent inhibitors of
LPAAT-b and highly effective antiproliferative agents in
vitro. However, when tested in an in vivo mouse tumor
model 2r and 2s were inactive because of rapid meta-
bolic degradation of the primary alcohol groups (data
not shown). Compounds with electron withdrawing
substituents at the para-position (2v and 2ad) were also
potent LPAAT-b inhibitors and antiproliferative agents.
However, compound 2aa with an electron withdrawing
para-carboxyl group or its primary amide (2af) and
amidine (2ai) derivatives were only weak inhibitors.
Although the hydroxamic acid derivative (2ag) of car-
boxylic acid 2aa was an active LPAAT-b inhibitor it
displayed limited antiproliferative activity. Compound
2ab with a para-carboxyl group attached to the ring
through a methylene tether was an effective inhibitor.
Acetophenone 2aj and its oxime derivative 2ak also were
noteworthy for their potent LPAAT-b activity and
antiproliferative effects. Although meta-substitution was
generally not well tolerated, the 3,4-disubstituted ana-
logs 2c and 2x with the 3- and 4-positions fused in a
second ring were effective inhibitors and antiprolifera-
tive agents. For most of the compounds listed in Table 2
antiproliferative activity correlated quite well with
LPAAT-b inhibition.
Compound 2j (in vitro LL2 IC50 ¼ 0:16 lM), injected at
a dose of 100 mg/kg q1d · 4, displayed median tumor
growth delay (TGD) to 1000 mm3 compared to vehicle
alone of 6.0 days (Fig. 1). Because tumor growth
resumed at an exponential rate a few days following
withdrawal of this LPAAT-b inhibitor we surmised that
2j may function as a cytostatic agent.
In summary, the three diamino-C,N-diarylpyrimidine
positional isomers 2, 3, and 4 were synthesized and
tested for their ability to inhibit the enzymatic activity of
LPAAT-b and the proliferation of tumor cells in vitro.
The 2,4-diamino-N4,6-diarylpyrimidines positional iso-
mer 2 displayed more potent LPAAT-b inhibition
compared to positional isomers 3 and 4. Several exam-
ples of 2 were synthesized and found to be both potent
inhibitors of LPAAT-b and highly effective antiproli-
ferative agents in vitro. When tested in an in vivo tumor
model, the pattern of tumor growth delay displayed by
2j was consistent with it functioning as a cytostatic
agent. A compound like 2j used in combination with
a cytotoxic drug or radiation treatment may provide
a novel approach for cancer therapy.
Acknowledgements
We thank Lisa Romero for conducting LPAAT assays
and Tod Martin for conducting proliferation assays.
Many of the LPAAT-b inhibitors listed in Table 2 with
potent antiproliferative activity in vitro were highly
insoluble in aqueous media (data not shown). However,
2j (CT-32521) was sufficiently soluble in cremophor EL–
EtOH–PBS (1:1:10) as vehicle to warrant its testing in
an in vivo tumor model. Female C57BL/6 mice were
implanted with murine LL2 Lewis lung tumor cells.22
References and notes
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sizes averaged 50–100 mm3, groups of 10 mice each were
injected intravenously (20 mL/kg) with either vehicle (8.3%
cremophor EL and 8.3% EtOH in PBS) or 2j at 100 mg/kg
(5 mg/mL in vehicle) on four consecutive days. Tumor
volumes were determined by measuring three orthogo-
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formula: (length · width · height)/2. Mice bearing tumors
P1000 mm3 were euthanized. Antitumor efficacy was
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in the control group to reach the same volume.
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