Development of o-chlorophenyl substituted pyrimidines as exceptionally potent aurora kinase inhibitors

Article abstract of DOI:10.1021/jm300334d

The o-carboxylic acid substituted bisanilinopyrimidine 1 was identified as a potent hit (Aurora A IC₅₀ = 6.1 ± 1.0 nM) from in-house screening. Detailed structure-activity relationship (SAR) studies indicated that polar substituents at the para position of the B-ring are critical for potent activity. X-ray crystallography studies revealed that compound 1 is a type I inhibitor that binds the Aurora kinase active site in a DFG-in conformation. Structure-activity guided replacement of the A-ring carboxylic acid with halogens and incorporation of fluorine at the pyrimidine 5-position led to highly potent inhibitors of Aurora A that bind in a DFG-out conformation. B-Ring modifications were undertaken to improve the solubility and cell permeability. Compounds such as 9m with water-solubilizing moieties at the para position of the B-ring inhibited the autophosphorylation of Aurora A in MDA-MB-468 breast cancer cells.

Full text of DOI:10.1021/jm300334d

Article
pubs.acs.org/jmc
Development of o‑Chlorophenyl Substituted Pyrimidines as
Exceptionally Potent Aurora Kinase Inhibitors
Harshani R. Lawrence,*,†,§,∥ Mathew P. Martin,†,# Yunting Luo,§,# Roberta Pireddu,†,# Hua Yang,†,#
Harsukh Gevariya,†,# Sevil Ozcan,† Jin-Yi Zhu,† Robert Kendig,§ Mercedes Rodriguez,§,† Roy Elias,†
Jin Q. Cheng,‡,∥ Saïd M. Sebti,†,∥,⊥ Ernst Schonbrunn,†,§,∥ and Nicholas J. Lawrence*,†,∥
‡
§
†Department of Drug Discovery, Department of Molecular Oncology, and Chemical Biology Core, Moffitt Cancer Center, 12902
Magnolia Drive, Tampa, Florida 33612, United States
⊥
∥Department of Oncologic Sciences and Department of Molecular Medicine, University of South Florida, Tampa, Florida 33620,
United States
S
* Supporting Information
ABSTRACT: The o-carboxylic acid substituted bisanilinopyr-
imidine 1 was identified as a potent hit (Aurora A IC50 = 6.1
1.0 nM) from in-house screening. Detailed structure−activity
relationship (SAR) studies indicated that polar substituents at
the para position of the B-ring are critical for potent activity. X-
ray crystallography studies revealed that compound 1 is a type
I inhibitor that binds the Aurora kinase active site in a DFG-in
conformation. Structure−activity guided replacement of the A-
ring carboxylic acid with halogens and incorporation of
fluorine at the pyrimidine 5-position led to highly potent
inhibitors of Aurora A that bind in a DFG-out conformation.
B-Ring modifications were undertaken to improve the solubility and cell permeability. Compounds such as 9m with water-
solubilizing moieties at the para position of the B-ring inhibited the autophosphorylation of Aurora A in MDA-MB-468 breast
cancer cells.
developed by Vertex and Merck is a potent pan-Aurora
inhibitor.23 Clinical development was halted after QTC
prolongation was observed. As one of the first reported Aurora
inhibitors, VX-680 has been an important tool for studying
Aurora biology. Other compounds remain in clinical trials
including the orally bioavailable Aurora A selective inhibitor
MLN805424−26 and its second generation analogue
MLN823727−30 (Figure 1). The AstraZeneca quinazoline
AZD-1152 (barasertib)31,32 in phase II trials is a potent and
selective Aurora B inhibitor administered via intravenous
infusion. The 3-aminopyrazole PHA-73935833−36 (Figure 1)
developed by Nerviano and Pfizer is in phase II trials for CML
and metastatic prostate cancer. The development of Aurora
inhibitors continues to attract attention37−41 and ultimately will
lead to therapeutic benefit in the clinic. The bisanilinopyr-
imidines are a class of compounds that has shown unusual high
selectivity for Aurora A over Aurora B.42 The pyrimidine
scaffold43−45 has been used by many groups to develop novel
Aurora kinase inhibitors.
INTRODUCTION
■
The Aurora kinases are a family of serine−threonine kinases that
play an important role in the regulation of cell division. They are
overexpressed in a variety of cancer types and are implicated in
many aspects of tumor development. The three members of the
Aurora family Aurora A, B, and C share a high degree of structural
homology in their kinase domain, but each kinase plays a
different role in the control of mitosis. Aurora A and B have
received the most attention to date as anticancer targets.1 Aurora
A regulates the cell cycle and is associated with late S phase and
entry into the M phase. It is essential for many processes
including centrosome maturation and separation, chromosome
alignment, and mitotic spindle formation.2 Aurora A is frequently
overexpressed in tumors and has characteristics of an
oncogene.3,4 Aurora B plays important roles in M phase to
ensure correct chromosome-microtubule alignment and attach-
ment and chromosomal cytokinesis. Aurora B is also overex-
pressed5 in many cancer types but does not have oncogenic
properties. Both Aurora A6−10 and Aurora B11−14 have been
validated as targets for anticancer drugs in preclinical models.
Intense activity has led to the development of numerous
inhibitors15−17 that display selectivity to either Aurora A or
Aurora B or that are dual inhibitors. Many have been or are now
in clinical development.18−22 Selected examples are shown in
Figure 1. The pyrimidine derivative VX-680 (tozasertib)
In this report we describe our efforts in identifying novel and
highly potent Aurora A inhibitors using the bisanilinopyrimidine
scaffold. To this end, we screened our in-house 20 000
Received: March 9, 2012
Published: July 17, 2012
© 2012 American Chemical Society
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Figure 1. Structures of selected Aurora inhibitors evaluated in the clinic. Inhibition data are shown for Aurora A and B.
Figure 2. (a) Compound 1 (hit) identified from in-house ChemDiv library as an Aurora A inhibitor. (b) Synthetic modifications for library synthesis.
membered ChemDiv library using a Z-lyte assay and identified
the bisanilinopyrimidine bis-carboxylic acid 1 (HLM008598,
Figure 2) as a hit. Optimization of compound 1 was undertaken
initially via SAR guided focused library synthesis followed by
rational design based on co-crystal structures of 1 and related
analogues bound to Aurora A.
dichloropyrimidine was initially reacted with the requisite
commercially available anilines using literature reported
protocols46,49,50 to obtain the intermediate library 2 (Scheme
1) with A-ring substituents such as carboxylic acid, carboxylic
acid ester, amide groups, and chlorine functional groups at the
ortho position. In this step, aqueous hydrochloric acid was used
predominantly as the solvent with microwave assisted heating
(method a) to obtain the required analogues 2. The o-chloro
analogues 2k and 2l were synthesized in good yields using
aqueous acid at room temperature (method b) as shown in
Scheme 1.46 The 5- and 6-aminopyrimidine building blocks 2h
and 2i were obtained from commercially available 5- and 6-
amino-2,4-dichloropyrimidine starting materials, respectively, in
moderate yields. The library 2 members were further function-
alized under acid catalysis (methods d−g) to provide the library 3
in good yields.46 Although the pyrimidine scaffold is featured in
many kinase inhibitors, only one member of library 3, i.e.,
compound 3c, has been reported as a kinase inhibitor.50
Intermediates 2h and 2i were converted to 3u and 3v using
methods f and e, respectively, to obtain ethyl esters as
intermediates. Similarly, ethyl esters 3s and 3t were obtained
from 2m and 2n, respectively, using method f. Intermediates 3s−
v were hydrolyzed (method h) to obtain 4a−d as final
compounds.
CHEMISTRY
■
The bisanilinopyrimidine carboxylic acid 1 was resynthesized in-
house to confirm the structure and activity against Aurora A and
B. It has not previously been reported as an Aurora kinase A
inhibitor. The general synthetic route used for preparation of
compound 1 (Figure 2a) and 2,4-bisanilinopyrimidine focused
libraries 3 and 4 from readily available building blocks is shown in
Scheme 1. The displacement of the 4-chloro group of 2,4-
dichloropyrimidine by various anilines is already widely reported
in the literature.42,46−48 The hit 1 was resynthesized by hydrolysis
(method h, Scheme 1) of the methyl ester 3w, which was
prepared from 2a and methyl p-aminobenzoate (method m,
Scheme 2). Attempts to directly synthesize 1 from 2a and p-
aminobenzoic acid via method a resulted in formation of
decarboxylated byproduct 3b (1:3b, ∼5:1, observed by NMR
and HPLC−MS analysis). Attempts to purify the mixture were
not successful. For the library synthesis of analogues of 1 the 2,4-
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a
Scheme 1. Synthetic Route to Bisanilinopyrimidine Carboxylic Acid Library
a
Reagents and conditions. Method a: HCl (0.1 M, aq, 1-3 mL/mmol), microwave, 100 °C, 30 min. Method b: HCl (0.1 M, aq, 3 mL/mmol), rt, 3−5
days. Method c: HCl (0.1 M, aq, 1.5 mL/mmol), sealed tube, 100 °C, 24 h. Method d: HCl (0.1 M, aq, 3−6 mL/mmol), microwave, 160 °C, 15
min. Method e: EtOH: HCl (1 M, aq, 1:1, 4 mL/mmol), microwave, 160 °C, 15 min to 1 h. Method f: (i) HCl (4 M in dioxane, 0.5 mL/mmol), 2-
butanol (3 mL/mmol), sealed tube, 120 °C, overnight (24 h), 72% or (ii) EtOH, sealed tube, 120 °C, overnight to 4 days. Method g: THF/HCl (1
M, aq) (1:2, 6 mL/mmol), microwave, 160 °C, 15 min. Method h: THF/NaOH (2 M, aq) (1:2, 4−7 mL/mmol), sealed tube, 85−100 °C, 0.5−16
h.
The high in vitro potency (Table 1) observed with compounds
3n (with 5-fluoropyrimidine moiety), 3l, and 3o (o-chloro
analogues, Scheme 1) prompted us to further investigate this
series. The synthesis of library 6 with hydrophobic groups (Cl,
Br, I, CF3, phenyl) and hydrogen-bond acceptor groups (OMe,
CN, OCF3) on the A-ring is described in Scheme 2. Synthesis of
the key intermediates 5a−i with F, Cl, Br, I, OCF3, CF3, OMe,
CN, and phenyl (Scheme 2) was achieved from commercially
available 2,4-dichloropyrimidine building blocks using literature
reported protocols.50−52 Intermediates 5j and 5k were
synthesized via alkylation of 2l using Cs2CO3 in acetonitrile
(Scheme 2).51,52 These intermediates were directly reacted via
nucleophilic aromatic substitution with anilines to obtain the
final library 6 possessing halogens (F, Cl, Br, and I), polar groups
(CN), nonpolar groups (Ph, H), and polar hydrophobic groups
(OCF3, CF3, OMe) in the ortho position of the A-ring in
moderate to high yields (method l, m, or n in Scheme 2). The
majority of the library members 6 also readily precipitated under
the reaction conditions, and the purity of final compounds tested
against Aurora A inhibitory activity was determined as >95% by
HPLC (high performance liquid chromatography). The
analogue 6k with p-carboxylic acid and m-OH (i.e., salicylic
acid moiety) was obtained from its precursor methyl ester 6u via
basic hydrolysis (method o in Scheme 2).
Using the synthetic routes and protocols shown in Schemes 1
and 2, we were able to explore detailed in vitro SAR toward
Aurora A inhibition. Furthermore, we designed and synthesized
new molecules, exploiting the structures of compounds 1, 3l, 3n,
and 3o complexed with Aurora A to develop potent Aurora A
inhibitors with desirable druglike properties for in vitro and in
vivo studies.53 Compounds 3l and 3o (Scheme 1), with o-chloro
and p-carboxylic acid groups were further modified via
introduction of water-solubilizing groups to improve solubility
and cell permeability (Scheme 4). The solubilizing group was
attached via an amide of the B-ring p-carboxylic acid, as this part
of the molecule is solvent exposed in the co-crystal complexes53
of 1 (see Figure 3) and 3o with Aurora A. The building blocks
8a−c were synthesized as shown in the Scheme 3 via acylation of
commercially available p-nitrobenzoyl chloride with amines
possessing water-solubilizing groups (morpholine, N,N-dime-
thylamine, and methyl ether) followed by the hydrogenation of
the nitro group in high yields.54 The final bisanilinopyrimidines
with water-solubilizing groups 9 (Scheme 4) were prepared by
reaction of intermediates 2k and 2l (Scheme 1) with appropriate
anilines in moderate yields and >95% HPLC purity. The library 9
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Scheme 2. Synthetic Route to Bisanilinopyrimidine Library 6 with Halogens (F, Cl, Br, and I), CN, Nonpolar Groups (H, Ph), and
a
Polar Hydrophobic Groups (OCF3, CF3, and OMe) on the A-Ring
a
Reagents and conditions. Method i: n-BuOH, DIPEA, 125 °C. Method j: n-BuOH, Na2CO3, 100 °C. Method k: DMF, NaH, rt, overnight (14−16
h). Method l: EtOH (1 drop of 1M HCl), microwave, 160 °C, 15 min, 56−75%. Method m: EtOH or MeOH, 150 °C, 20 min, microwave. Method
n: cat. HCl, THF, reflux 14 h. Method o: THF, NaOH (1.8 M, aq, 5 equiv), THF, reflux, 14 h. Method b: HCl (0.1 M, aq, 3 mL/mmol), rt, 3−5
days.
with water-solubilizing moieties in the B-ring and an o-chloro A-
ring comprises novel compounds that have not previously been
reported as kinase inhibitors.
highly selective inhibitor for Aurora A (in vitro IC50 = 0.075
0.039 μM) over Aurora B (in vitro IC50 = 5.4 1.8 μM) using the
Z-LYTE assay with LRRASLG as an Aurora substrate.56,57 We
verified the dose−response curve of the hit (compound 1) using
a coupled enzyme assay58 (DiscoveRx) that measures ADP
formation from the Aurora A phosphorylation of the same
synthetic peptide LRRASLG, as described under methods. The
determination of dose−response curve and IC50 value of the
compound 1 using this coupled assay revealed in vitro Aurora A
potency in the range of 6.1 1.0 nM, and we used this assay to
establish the SAR described in this study.
The bis-anilinopyrimidine scaffold, but not specifically
compound 1, has previously been reported for inhibitors of
Aurora kinase42,44 as well as other kinases such as JNK1,50
FAK,59 ephrin type B receptor 4 kinase,60 CDK2, and CDK4.61
For an HTS hit, compound 1 displayed an unusually high
potency in the range of the most active Aurora A inhibitors
reported to date. The suitability of this scaffold to focused library
synthesis and availability of crystallization-grade protein
Synthesis of the p-tetrazole aniline building block 11 was
achieved as shown in the Scheme 5 and used to generate
compounds 12 (derivatives of 3l, 3n, and 3o shown in Scheme
1). The starting material 4-nitrobenzonitrile was reacted with
sodium azide (condition b, Scheme 5) to obtain intermediate 10
in high yield.55 Hydrogenation of intermediate 10 using Pd−C as
the catalyst provided the required building block 11 in good yield
(condition c). Compound 11 was next coupled with
intermediate 2k, 2l (Scheme 1), or 2o to provide the final
compounds 12a−d (Scheme 5) with >95% purity (determined
by HPLC). The final compounds 12 with a tetrazole moiety in
the B-ring are reported for the first time.
RESULTS AND DISCUSSION
■
The high throughput screening (HTS) hit 1 was identified from
our in-house 20 000 compound ChemDiv library as a potent and
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Table 1. Synthetic Modifications, Structure−Activity Relationship Studies, and in Vitro Activities of Bisanilinopyrimidine
Libraries 3 and 4 against Aurora A
entry
compound
R1
R2
R3
R4
in vitro IC50
1
1
H
H
o-CO2H
o-CO2H
o-CO2H
o-CO2H
o-CO2H
o-CO2Me
H
p-CO2H
o-CONH2
H
6.1 1.0 nM
9.0 6.8 μM
79.4 18 nM
57.6 5.4 nM
31.3 5.9 μM
24.6 6.0 μM
423 66 nM
38.2 8.8 nM
10 1.6 nM
256 38 nM
281 59 nM
>50 μM
2
3a
3b
3c
3d
3e
3f
H
H
H
H
H
H
H
H
H
CH3
H
H
F
H
3
H
4
H
p-morpholine
o-CO2H
p-CO2Me
H
5
H
6
H
7
H
8
3g
3h
3i
H
o-CONH2
H
p-CONH2
p-CO2H
p-CO2H
p-CO2H
p-CO2H
p-CO2H
all H
9
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
H
p-CO2H
o-CO2H
o-CO2H
o-Cl
3j
H
3k
3l
CH3
H
2.5 0.3 nM
11.3 1.7 nM
3.9 0.5 nM
0.8 0.16 nM
19.9 2.2 nM
18.3 3.4 nM
5.1 1.1 nM
1.49 0.196 μM
3.17 0.51 nM
376 64 nM
>50 μM
3m
3n
3o
3p
3q
3r
4c
4d
4a
4b
H
o-CO2H
o-CO2H
o-Cl
F
H
p-CO2H
p-CO2H
H
F
H
F
H
o-Cl
H
F
H
o-CO2H
o-CO2H
o-CO2H
o-CO2H
o-CO2H
o-CO2H
m-CO2H
m-CO2H
p-CO2H
p-CO2H
p-CO2H
p-CO2H
H
Cl
Cl
NH2
H
Cl
H
H
NH2
Figure 3. (a) Cartoon representing key binding interactions of hit (compound 1) with Aurora A. (b) X-ray structure of compound 1 bound to Aurora A
active site (compound 1; cyan, carbon; red, oxygen; blue, nitrogen; PDB entry 3UP7). (c) Compound 1 co-crystallized with Aurora A shows hinge
region (green), DFG-in (magenta), activation loop (blue) open conformation (compound 1; yellow, carbon; red, oxygen; blue, nitrogen). (d) Overlay
of ADP (carbons shown green) and compound 1 (carbons shown cyan). (e) ADP bound to Aurora A active site (PDB entry 4DEE).
prompted us to pursue the improvement of 1 by SAR studies and
In the beginning of this work, SAR studies were initiated while
structure-based design.
attempts were being made to co-crystallize compound 1 with
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a
Scheme 3. Synthesis of Building Blocks with Water-Solubilizing Groups
a
Reagents and conditions. Method p: 10% Pd/C, EtOH, H2, rt, overnight. Method q: H-Cube, 10% Pd/C, MeOH, 30 bar, four loops, rt.
a
Scheme 4. Synthesis of Bisanilinopyrimidines with Water-Solubilizing Groups in the B-Ring
a
Reagents and conditions. Method r: coonc HCl, i-PrOH, 170 °C, microwave, 20 min. Method s: EtOH, 150 °C, microwave, 20 min. Method t:
EtOH, cat. HCl, 180 or 160 or 140 °C, microwave, 15 min. Method u: X-Phos (10 mmol %), bis(dibenzylideneacetone)palladium(0) (10 mmol %),
K2CO3, t-BuOH, reflux,18 h. Method v: MeOH, 100 °C, sealed tube, 6 h.
Aurora A. Focused library synthesis based on 1 (Figure 2) was
first undertaken varying four points of molecular diversity (R1,
R2, R3, and R4; see Figure 2b) by systematically replacing or
introducing the functional groups in the A and B rings.
Replacement of the B-ring p-carboxylic acid in 1 by hydrogen
or a morpholino as in 3b or 3c resulted in 13- or 10-fold loss of
inhibitory activity, respectively, suggesting that the carboxylic
acid is critical (entries 3 and 4, Table 1). Replacing both
carboxylic acid moieties in 1 by hydrogens as in 3f (entry 7, Table
1) resulted in 70-fold loss of potency, further emphasizing the
importance of the carboxylic acid moieties. In addition,
replacement of both carboxylic acid moieties by corresponding
methyl esters as shown in 3e (entry 6, Table 1) was detrimental
and resulted in over 4000-fold loss of potency, demonstrating the
importance of the negative charge of the acid moieties in the
binding region. However, compound 3g (entry 8, Table 1) with
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position as in 3i (entry 10, Table 1) resulted in 42-fold loss of
potency. Replacement of the B-ring p-carboxylic acid by o-amide
(compound 3a) was also detrimental for Aurora A inhibitory
activity, resulting in greater than 1000-fold loss of inhibitory
activity (entry 2, Table 1). These observations suggested that the
ortho position of the A-ring and para position of the B-ring are
critical for inhibition of enzymatic activity and focused library
synthesis.
Scheme 5. Synthesis of Bisanilinopyrimidine Tetrazole
Derivatives
a
The X-ray co-crystal structure of 1 bound to Aurora A supports
the above findings (Figure 3). Analysis of this structure shows the
p-carboxylic acid group of the B-ring forming key H-bond
interactions with the solvent exposed residues Arg137 and
Arg220 (Figure 3a and Figure 3b). As expected of a
bisanilinopyrimidine, compound 1 is a type I62 kinase inhibitor
that targets the ATP binding site (Figure 3c). Compound 1 binds
to Aurora A in an active DFG-in conformation, as shown in
Figure 3c (DFG motif shown in magenta). The activation loop
(shown blue in Figure 3c) is oriented away from the ATP binding
site. The pyrimidine scaffold and the amine moiety of the B-ring
establish H-bonding with the hinge region (residues 211−213,
Figure 3a and Figure 3b). Analysis of ADP/ATP bound to
Aurora A indicated highly conserved residues Lys162, Asp274,
and Glu181 undergoing electrostatic interactions with ADP
phosphate moieties in the active site (Figure 3e). Compound 1
bound to Aurora A shows that these key residues in the active site
are now in contact with the o-COOH moiety of the A-ring
(Figure 3b). The o-carboxylic acid of the A-ring in compound 1 is
in the vicinity (3.5 Å) to form an electrostatic interaction with
Lys162 (Figure 3b). The carboxylic acid moiety of the Asp274 of
the DFG motif (Figure 3a and Figure 3b) is also in close distance
(3.2 Å) with Lys162. These key interactions contribute to the
high in vitro potency of the compound 1. Clearly the apparent
disfavored interaction between the o-COOH moiety of 1 and the
side chain carboxylic acid moiety of the Asp274 must be
compensated by the presence of the Lys162. This is consistent
a
Reagents and conditions. (a) 12a: ethanol, microwave, 150 °C, 40
min, 37%. 12b: ethanol, microwave, 170 °C, 40 min, 33%. 12c:
ethanol, microwave, 160 °C, 40 min, 36%. 12d: ethanol, microwave,
150 °C, 40 min, 25%. (b) 10a and 10b: NaN3, Et3N·HCl, toluene, 100
°C, 15 h, 92% and 95%. (c) 11a and 11b: H2, Pd/C, methanol, rt, 20
h, 93% and 98%.
carboxamide in ortho and para positions of the A and B rings,
respectively, was only 6-fold less potent than the compound 1.
Our SAR data showed that the positions of both carboxylic acid
moieties in A and B rings are critical for activity. Moving the B-
ring p-carboxylic acid moiety to the ortho position as in 3d
demonstrated 5000-fold loss of potency (entry 5, Table 1, IC50 =
31 300 nM). Compound 3q (entry 18, Table 1, IC50 = 18.3 nM)
with m-carboxylic acid in the B-ring was less detrimental with 3-
fold loss of in vitro potency. Furthermore, moving the A-ring
carboxylic acid moiety in compound 1 from the ortho to para
Figure 4. X-ray structures of compounds 3i (A), 3g (B), and 13 (C) (see Supporting Information) bound to Aurora A active site (PDB entries 4DEA,
4DED, and 4DEB, respectively). Compound 1 bound to Aurora A (D). The R1 hydrogen is shown in yellow and Leu194, Leu210, and Ala160 are shown
as spheres to show the narrow space where R1 is occupying in the binding region.
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Table 2. Structure−Activity Relationship Studies and in Vitro Inhibitory Activities of Library 6 against Aurora A
a
entry
compound
R1
R2
R3
o-F
R4
p-CO2H
R5
IC50 (nM)
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
6a
6b
6c
6d
6e
6f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3.7 0.7
1773 142
2.0 0.2
28 4.8
o-CF3
2-Cl-4-F
o-OCF3
o-OMe
o-OMe
o-CN
o-CF3
o-Br
all H
p-CO2H
p-CO2H
p-CO2H
all H
4.0 0.2
46.6 8.4
560 70.3
35.1 4.0
2.1 0.4
3.3 1.5
6.6 0.6
284 11.3
35 3.3
6g
6h
6i
all H
p-CO2H
p-CO2H
p-CH2-CO2H
p-CO2H, m-OH
all H
6j
o-Cl
6k
6l
o-Cl
o-F
6m
6n
6o
6p
6q
6r
6s
6t
o-I
p-CO2H
p-CO2H
m-CO2H
p-CONH2
p-CO2H
p-CO2H
p-CO2H
m-CO2H
o-CN
o-Cl
43 8.0
18.7 1.5
30.2 1.4
149 23
8.5 1.2
50.2 2.7
4.5 1.1
o-Cl
o-phenyl
o-Cl
CH3
o-Cl
CH3-CH2
H
o-Cl
a
Determined in-house using the DiscoveRx format.
with the fact that compound 3f (entry 7, Table 1) where both A
and B rings contain unsubstituted phenyl was 70-fold less active
than parent compound 1, and the dimethyl ester 3e lost the in
vitro inhibitory activity (IC50 = 24.6 μM, entry 6, Table 1). These
observations further confirm that the key interactions observed
from the X-ray structure are important for inhibitory activity
(Figure 3b). The loss of inhibitory activity observed with 3d
(entry 5, Table 1) in our SAR studies is consistent with the X-ray
structure of compound 1 bound to Aurora A where ortho-
substituted B-ring causes steric clash with the main chain residues
Ala213 and Pro214 (Figure 3a and Figure 3b). However, the
activity of compound 3q is retained with carboxylic acid in the
meta position and improved when fluorine is added as in 3r
(entries 18 and 19, Table 1). It is also likely that the m-COOH is
able interact with Arg220 and Arg137.
During further investigation of this series, several X-ray
structures were obtained in order to improve our understanding
of the binding modes of this class of compound with Aurora A
(Figure 4). Substitution of both carboxylic acid groups in
compound 1 by primary amides (Figure 4B) reduced the
inhibitory activity 6-fold (entry 8, 3g, Table 1). The biscarboxylic
acid 3i (Figure 4A) where the A-ring o-carboxylic acid is moved
to the para position was 42-fold less potent than parent molecule
(Table 1, entry 10, IC50 = 256 nM). This decrease in inhibitory
activity is probably due to lack of key binding interactions of the
p-carboxyl or o-carboxamide groups in the A-ring with the
Lys162 active site residue (Figure 4A and Figure 4B, compounds
3i and 3g bound to Aurora A). In contrast, removal of the o-
carboxylic group as in 3h (entry 9, Table 1) retains activity,
reflecting both the loss of positive interaction with Lys162 and
negative interaction with Asp274. The co-crystal structure of 3i
(Figure 4) does not explain the differences in activities of 3h and
3i. Compound 13 (Figure 4C), where A-ring has a CF3 in the
meta position, is a weaker binder (IC50 = 371 nM; see Supporting
Information Table S3 for IC50 data of additional compounds)
and has the trifluoromethyl group positioned in the region that
binds the diphosphate group of ADP (see Figure 3e). Thus, the
presence of a hydrophobic group close to Asn261 and Glu260
does not contribute to binding affinity of Aurora A. Overall meta
substituents (not reported here) in the A-ring did not improve
the activity.
The surprising observation that replacement of the o-
carboxylic acid of the A-ring in compound 1 with hydrogen as
in 3h did not result in great loss of potency prompted us to
further probe SAR at this position. Therefore, library 6 (Scheme
2) was synthesized to improve our understanding of the binding
modes of Aurora A to bisanilinopyrimidines with different groups
in the ortho position of the A-ring. To this end we synthesized
analogues of 1 by replacing the carboxylic acid in the ortho
position of the A-ring by fluoro, chloro, bromo, iodo,
trifluoromethyl, trifluoromethoxy, methoxy, cyano, and phenyl
as ortho substituents (6a−q, Scheme 2). Compounds with
halogens (Cl, F, Br) at ortho position of the A-ring and p-COOH
moiety in the B-ring [compounds 3l (Table 1) and 6a and 6i
(Table 2), respectively] improved potency by 1.5- to more than
3-fold, whereas compounds with bulky halogenated groups such
as OCF3 and CF3 (entries 27 and 31, compounds 6d and 6h,
respectively, in Table 2) were much less active at inhibiting
Aurora A. Compound 6q (entry 40, Table 2) with an o-phenyl
group was 24-fold less potent than compound 1, further
indicating that bulky groups are not tolerated in this region.
Compound 6n (IC50 = 43 nM, entry 37, Table 2) that possess an
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Table 3. In Vivo and in Vitro Aurora A Activities of Bisanilinopyrimidines with Water-Solubilizing Moieties
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Table 3. continued
a
b
c
Determined in-house using the DiscoveRx format. Measured by Reaction Biology using a 33P assay. The IC50 of 9m in the Reaction Biology 33
assay for 9m, generated at the same time, was 5.7 nM for Aurora A and 15.6 nM for Aurora B.
P
o-CN group is more than 17-fold less active than 3l. The in vitro
activity of 3l (IC50 = 2.5 0.3 nM, entry 13, Table 1) with o-Cl
and a p-COOH in B-ring was further improved when R1 is
fluorine in compound 3o (IC50 = 0.8 0.16 nM, entry 16, Table
1). The exceptional potency of this compound is thought to be
derived from its novel binding mode, recently described by us.53
Compound 3o binds to Aurora A in an inactive DFG-out
conformation with the activation loop folded back over the ATP
binding site, effectively encapsulating the inhibitor. This binding
mode is a consequence of the presence of the o-chloro group,
which we postulate induces a dipole in the N-flanking alanine
adjacent to the DFG motif.53 The contrasting binding modes of 1
and 3o is striking given their high structural similarity and
provides chemical tools for studying the phenotypical con-
sequences of these two binding modes. The p-COOH moiety in
the B-ring was important to maintain the Aurora A inhibitory
activity in library 6 (Scheme 2). The removal of the COOH
moiety resulted in loss of in vitro potency as observed with 6b, 6f,
6g, and 6l (entries 25, 29, 30, and 35, Table 2). The loss of in
vitro inhibitory activity in compounds lacking a p-COOH moiety
was a general trend observed with libraries 3 and 6 and highlights
the importance of H-bond interactions associated with Arg137
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Figure 5. Inhibition of phosphorylation of Thr288 on Aurora A in MDA-MB-468 cells by bisanilinopyrimidines with water-solubilizing moieties.
Inhibition at three concentrations (1, 3, and 10 μM) for each compound is shown. Alisertib (0.3 μM) was used as a positive control.
and Arg220. Switching the position of B-ring COOH moiety
from para to meta position as in compound 6o (entry 38, Table
2) reduced the inhibitory activity compared to 3o (entry 16,
Table 1). Modification of B-ring p-COOH to p-CONH2 in
compound 6p reduced the in vitro activity by 5-fold (entry 39,
Table 2). Compounds 6r and 6s (entries 41 and 42, Table 2)
with N-alkylated moieties retained the activity; the N-methyl
derivative was highly potent (IC50 = 8.5 1.2 nM), while the N-
Alignment of the Aurora A−ADP complex with compound 1
indicated R1 and R2 in compound 1 as potential sites for synthetic
modifications (Figure 3a,d,e). The R1 position (Figure 2b) of the
pyrimidine ring is in proximity (∼4 Å) to the gatekeeper residue
Leu210, and R2 is close to Glu211 (∼3.5 Å) (Figure 3a and
Figure 3b). To exploit this narrow space, we first introduced
small groups such as methyl (entry 11, 3j, Table 1), amine (entry
22, 4a, Table 1), chlorine (entries 20 and 21, 3s and 3t,
respectively, Table 1), and fluorine (entries 15 and 17, 3n−p) as
R1. The methyl and NH2 derivatives (compounds 3j and 4a,
respectively (entries 11 and 22, Table 1)) did not contribute to
increased activity most probably because of the steric effect of
these groups, making them unable to establish desired
interactions. In contrast, compounds with fluorine (3n, 3o
ethyl derivative was less active (IC50 = 50.2
2.7 nM).
Compound 6t (entry 43, Table 2) with m-COOH and fluorine as
R1 was 4-fold more potent compared to compound 6o where R1
is hydrogen. Overall our SAR indicated that fluorine or chlorine
as R1 in this series is beneficial and improves in vitro and in vivo
Aurora A activity (see Table 3).
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(entries 15−16, Table 1)) and chlorine (4d (entry 21, Table 1))
were among the most potent inhibitors with IC50 values between
0.8 and 4 nM (entries 15, 16, and 21, Table 1). It is likely that
fluorine and chlorine as R1 (Figure 2b) undergo van der Waals
interactions with the side chains of the hydrophobic pocket of
Ala160, Leu194, and Leu210 (Figure 4D) as observed in the
structures of fluorobisanilinopyrimidine inhibitors from Gen-
entech.42 Substitution of the R2 position (Figure 2b) of the
pyrimidine moiety with methyl, chloro, and amine as shown in
compounds 3k, 4c, and 4b (entries 12, 20, and 23, respectively,
Table 1) was detrimental for inhibitory activity probably because
of steric clash with the backbone carbonyl of Glu211. By contrast,
as mentioned above, synthetic modifications at R3 (Figure 3a and
Figure 3b) are largely tolerated, since the region around the DFG
is less confined than that opposite the hinge region.
We examined the isoform selectivity of the most potent
compound 3o. The inhibition of Aurora B activity was
determined using the Reaction Biology 33P kinase Hotspot
assay. The IC50 of 3o for Aurora A and Aurora B, using this assay
format, was 0.2 and 42.9 nM, respectively. Similarly the IC50 of 1
for Aurora A and Aurora B was 5.1 and 31 nM, respectively.
Significant levels of Aurora A selectivity was also observed for the
potent compound 3h (IC50(Aurora A) = 8.9 nM and
IC50(Aurora B) = 193 nM).
Having several potent analogues of bisanilinopyrimidines in
hand, we next focused on our attention to obtaining potent
Aurora inhibitors that are cell permeable and able to inhibit
Aurora A kinase in intact cells. However, the potent compounds
3l, 3n, 3o, 4d, 6a, 6j, 6k, and 6r that showed low nanomolar
activities in the enzymatic assay showed poor aqueous solubility
and poor activity in intact cells. Introduction of water-solubilizing
groups at the para position of the B-ring was explored to improve
both the solubility and cell permeability. Therefore, substitution
of the p-carboxylic acid in the B-ring with groups that contain a
variety of neutral polar moieties (Scheme 4) was employed to
exploit H-bond interactions with Arg137 and Arg220. Direct
replacement of the p-COOH by the carboxylic acid isostere
tetrazole moiety63 provided compounds 12a−d, which retained
in vitro potency similar to that of 3l (entries 59−62, Table 3 and
Scheme 5) with good aqueous solubility (49 μg/mL in DMEM
buffer at pH 7.4). We were able to obtain the X-ray structure of
compound 12a (Scheme 5) bound to Aurora A, and compound
12a adopted a binding conformation similar to that of compound
3l.53 Our primary aim with B-ring modifications described in
Schemes 4 and 5 was to obtain a compound with chlorine at the
ortho position of the A-ring and a water-solubilizing moiety at
the para position of the B-ring.
Determination of the Effects of Aurora Kinase
Inhibitors on Autophosphorylation of Aurora A on
Thr288 and Phosphohistone H3 (Ser10) Levels in Breast
Cancer Cells. We next determined the activity of the most
potent Aurora kinase inhibitors in intact cells by assessing the
inhibition of phosphorylation of Ser10 on Histone H3 and of
Aurora A Thr 288 as surrogates for Aurora B32 and A,24,30
respectively. MDA-MB-468 breast cancer cells were treated with
the inhibitors at various concentrations for 2 h and processed for
immunoblotting as described in the Experimental Section. For
Aurora A the cells were first synchronized by pretreatment with
nocodazole prior to treatment with inhibitors. The hit 1 (Aurora
A and B IC50 values 6.1 and 31 nM, respectively), which contains
a carboxylic acid moiety on each of the A-ring and the B-ring as
well as the most potent compound 3o (in vitro Aurora A and B
IC50 values 0.8 and 42.9 nM, respectively), had little effect on
histone H3 Ser10 phosphorylation levels (Supporting Informa-
tion Figure S3). This suggested that the carboxylic acid moiety
may hinder the ability of these series of compounds to be taken
up by cells. We therefore set out to improve the ability of these
compounds to inhibit Aurora activity in intact cells by varying the
substituents on the B-ring. The inhibition of Aurora A and B in
vitro and cellular inhibition of Aurora A Thr288 and histone H3
phosphorylation levels are summarized in Figure 5. Replacement
of the B-ring carboxylate with the acid bioisostere63 tetrazole
such as in 12a, 12b, 12c, and 12d did not improve cellular
activity. The tetrazole series retained greater than 30-fold Aurora
A/B in vitro selectivity observed for carboxylic acids 3o and 3h.
In contrast, compounds with chlorine on the A-ring, substituting
the B-ring carboxylate with a carboxamide (6p, Table 3),
sulfonamide (9i, Table 3), or morpholino (9h, Table 3), greatly
improved their activity and resulted in suppression of histone H3
and Aurora A Thr288 phosphorylation with concentrations as
low as 1 μM. This is consistent with the observed lower
selectivity of these compounds that significantly inhibit Aurora B
in the in vitro assay. Similarly, compounds with o-chlorine on the
A-ring and fluorine as R1 on the pyrimidine ring and substitutions
of the carboxylic acid moiety with carboxamide (9m, Table 3),
morpholino (9j, Table 3), methylene morpholino (9k, Table 3),
sulfonamide (9n, Table 3) also greatly improved the ability to
suppress histone H3 phosphorylation and Aurora A autophos-
phorylation. Other substitutions of the B-ring p-carboxylic acid
that resulted in improvements in cellular activity included 9a, 9b,
9c, 9l, and 9f (Table 3). Taken together, these results
demonstrate that in vitro Aurora A inhibitors that contain
carboxylic groups are inactive in intact cells but that those where
the B-ring carboxylic moiety was replaced by a carboxamide,
sulfonamide, or morpholino group are highly potent at
inhibiting, in intact cells, the phosphorylation of the Aurora A
and B kinase substrates.
Compound 9m was subjected to limited profiling using the
Reaction Biology 33P kinase Hotspot kinase profiling service
(Table 4).64 At a test concentration of 1 μM, 9m was most potent
Table 4. Selectivity Profile of Compound 9m and Control VX-
680 against a Panel of Kinases at 1 μM
% inhibition at 1 μM
kinase
9m
VX-680
ABL2/ARG
AKT1
75
8
98
3
Aurora A
Aurora B
Aurora C
CDK1/cyclin B
CDK2/cyclin A
CHK1
99
93
84
90
94
8
100
98
86
5
7
16
4
CHK2
30
95
94
21
3
FLT3
102
81
11
−3
−5
0
JAK2
NEK11
NEK2
NEK6
11
64
70
−5
39
16
PLK1
RET
96
−2
95
2
ROCK1
TRKA
WEE1
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where A is the remaining activity, [I] is the concentration of the
inhibitor, and n is the Hill slope coefficient. The VX-680 compound was
used as a standard for IC50 determinations.23
against Aurora A (99% inhibition). Aurora B, CDK1, CDK2,
FLT3, and JAK2 were also inhibited by >85%. JAK2 and FLT3
are often cross-inhibited by Aurora inhibitors, including VX-680
which was included as a control. The Aurora kinases while not
strictly members of the AGC kinase class65,66 are structurally
related67 to this group. The AGC kinases ROCK1 and AKT1
were not significantly inhibited by 9m. The IC50 of the most
potent compounds in the in vitro assay, 3l and 3o, were also
tested in the Hotspot assay (see Supporting Information) and
had IC50 of 0.74 and 0.2 nM, respectively (VX-680 had an IC50 of
0.24 nM) (see Figure S1, Supporting Information). The IC50 of
3o against JAK2 in this assay was 91 nM, showing that the high
potency against Aurora A is matched by high selectivity (over
450-fold vs JAK2) (see Figure S2, Supporting Information). The
selectivity of 3o was high when tested against the University of
Dundee panel of 131 kinases (see Table S2, Supporting
Information). Aurora A showed the highest inhibition (97%)
when tested at 0.5 μM. Only 7 of the 131 kinases (including
Aurora A and B) were inhibited by greater than 80%. These
include the tyrosine kinase JAK2, in agreement with the Reaction
Biology data, and the serine/threonine kinases EIF2AK3,
GSK3β, NUAK1, and PIM3. The 12 members of the AGC
kinase class tested showed no greater than 35% inhibition.
Protein Crystallography. Aurora A (123−390) was crystallized in
the presence of 2 mM inhibitor compound using the precipitant
PEG3350 as described previously.53 X-ray diffraction data were recorded
at −180 °C using Cu Kα radiation generated by a Rigaku Micro-Max
007-HF rotating anode (MSC, The Woodlands, TX) using a CCD
Saturn 944+ in the Moffitt Structural Biology Core facility. Data were
reduced with XDS.68 The structures were solved by molecular
replacement using the MolRep program from CCP469 with PDB
entry 3FDN as the search model. CNS70 was employed for refinement,
and model building was performed using Coot.71 Figures were prepared
using PYMOL.72 The data and refinement statistics for the co-crystal
structures are provided in the Supporting Information (Table S1). The
atomic coordinates and structure factors for Aurora A in complex with
compounds 1, 3i, 3g, 13, and ATP have been deposited under accession
numbers 3UP7, 4DEA, 4DED, 4DEB, and 4DEE, respectively.
Determination of the Effects of Aurora Kinase Inhibitors on
Autophosphorylation of Aurora A on Thr288 and Phosphohi-
stone H3 (Ser10) Levels in Breast Cancer Cells. MDA-MB-468 cells
(American Type Culture Collection) were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) plus 10% fetal bovine serum (FBS)
(Invitrogen, U.S.) at 37 °C and 5% CO2. The cells were plated in 6 cm
dishes at a density of 2 × 105 cells/dish. Aurora A activity was
determined by measuring autophosphorylation of Aurora A on Thr288
in cells that were synchronized by treatment with nocodazole (100 ng/
mL) (Sigma) for 20 h prior to the treatment with Aurora inhibitors,
whereas Aurora B activity was determined by measuring phosphor-
ylation of histone H3 on Ser10 (pHisH3-Ser10). Cells were then treated
with the compounds (0−10 μM). DMSO was used as a vehicle control,
and MLN8237 (alisertib) (0.3 μM) or VX-680 (tozasertib) (0.5 μM)
(Selleck Chemical LLC) was used as a positive control. Cells were
harvested after 2 h of treatment and processed for SDS−PAGE and
Western blotting as described previously.73 Briefly, following electro-
transfer, the membranes were blocked at room temperature for 1 h with
TBS containing 5% (w/v) milk and then washed with a mixture of TBS
containing 0.2% Tween 20 (Sigma). The membranes were then gently
shaken at 4 °C overnight with anti-phospho Aurora A (Thr288)
antibody (3079, Cell Signaling), anti-phospho-histone H3 (Ser-10)
antibody (9701, Cell Signaling), anti-Aurora A antibody (4718, Cell
Signaling), or anti-GAPDH monoclonal antibody (E10086CF,
Covance) diluted in TBS containing 5% BSA. The membranes were
then incubated with HRP conjugated anti-rabbit or anti-mouse IgG
antibody (Jackson ImmunoResearch Lab) at room temperature for 1 h
followed by washing with Tween 20−PBS. The membranes were
washed again with PBS and developed with the ECL system (Perkin-
Elmer) as described previously.74
Chemistry. General. All reagents were purchased from commercial
suppliers and used without further purification. Melting points were
determined using a Barnstead international melting point apparatus and
remain uncorrected. Proton NMR spectra were recorded on an Agilent-
Varian Mercury 400 MHz spectrometer with CDCl3 or DMSO-d6 as the
solvent. 13C NMR spectra are recorded at 100 MHz. The 13C spectrum
of 1 was recorded at 150 MHz, using an Agilent VNMRS 600
spectrometer with cold probe (University of South Florida Center for
Drug Discovery and Innovation). All coupling constants are measured in
hertz (Hz), and the chemical shifts (δH and δC) are quoted in parts per
million (ppm) relative to TMS (δ 0), which was used as the internal
standard. High resolution mass spectrometry was carried out on an
Agilent 6210 LC−MS (ESI-TOF). Low resolution mass spectrometry
(LRMS) was performed on an Agilent single quad G1956A (Depart-
ment of Chemistry, University of South Florida). Microwave reactions
were performed in CEM 908005 model and Biotage initiator 8
machines. All final compounds were purified to ≥95% purity as
determined HPLC analysis using a JASCO HPLC system equipped with
a PU-2089 Plus quaternary gradient pump and a UV-2075 Plus UV−vis
detector, using an Alltech Kromasil C-18 column (150 mm × 4.6 mm, 5
μm) and Agilent Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm).
Melting points were recorded on an Optimelt automated melting point
CONCLUSIONS
■
The screening hit 1 has provided an excellent starting point for
the design and synthesis of highly potent cell permeable Aurora A
and B inhibitors. The X-ray structure of 1 bound to Aurora A
revealed key interactions that were exploited for focused library
synthesis and rational design. As expected of a bisanilinopyr-
imidine, 1 is a type 1 kinase inhibitor that targets the ATP binding
site and binds in a DFG-in conformation. In the course of our
study, several X-ray structures were obtained and a novel DFG-
out binding mode was discovered with compounds, such as 3o,
that contain a halogen at the ortho position of the A-ring.53
Through SAR, X-ray structures, and focused library synthesis, we
found that fluorine on the 5-position of the pyrimidine ring in
combination with chlorine at the ortho position of the A-ring
greatly increases the in vitro potency. The carboxylic acid in the
para position of the B-ring is important for in vitro potency as
exemplified by the DFG-out inhibitor 3o; several compounds
9g−m with water-solubilizing moieties at the para position of the
B-ring inhibited the auto-phosphorylation of Aurora A and the
phosphorylation of the Aurora B substrate histone H3 in MDA-
MB-468 breast cancer cells. The carboxamide 9m displayed
selectivity against a small panel of kinases. This selectivity was
also observed for the carboxylic acid 3o when screened against a
larger panel of kinases.
EXPERIMENTAL SECTION
■
Protein Expression and Purification. Aurora A kinase (123−390)
was subcloned into a modified pET28 plasmid, overexpressed in E. coli,
and purified through a combination of affinity, ion exchange, and size
exclusion chromatography as described previously.53
In Vitro Enzyme Activity Assay. The synthetic peptide LRRASLG
served as a substrate for Aurora A. Formation of ADP from ATP was
quantified using a coupled enzyme assay (DiscoveRx, Fremont, CA) as
described previously.53 Inhibitor was added to the assay solution, and
the reaction was initiated through the addition of 75 μM ATP and 2 mM
peptide substrate. IC50 values were obtained by fitting the data to eq 1,
1
A =
n
[I]
1 +
(IC )
(1)
50
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system (Stanford Research Systems). Thin layer chromatography was
performed using silica gel 60 F254 plates (Fisher), with observation
under UV when necessary. Anhydrous solvents (acetonitrile,
dimethylformamide, ethanol, isopropanol, methanol, and tetrahydrofur-
an) were used as purchased from Aldrich. Burdick and Jackson HPLC
grade solvents (methanol, acetonitrile, and water) were purchased from
VWR for HPLC and high resolution mass analysis. HPLC grade TFA
was purchased from Fisher. Synthetic protocols and analytical data for
compounds 2a−o, 5a−k, 7a−c, 8a−c, 10a,b, and 11a,b are reported in
the Supporting Information.
124.49, 123.42, 119.53, 100.29, 65.36, 52.16; LC−MS (ESI−) m/z
390.15 (M − H − HCl)−; HRMS (ESI−) m/z calculated for
C21H20N5O3 (M − H − HCl)− 390.1572, found 390.1577.
N4-(2-Carboxyphenyl)-N2-(2-carboxyphenyl)pyrimidine-2,4-
diamine Hydrochloride (3d, Method d in Scheme 1). A
suspension of 2,4-dichloropyrimidine (0.149 g, 1.00 mmol) and 2-
aminobenzoic acid (0.274 g, 2.00 mmol) in HCl (3.0 mL, 0.1 M) was
heated in a microwave reactor at 160 °C for 15 min. The precipitate
obtained was filtered and washed with water (10 mL), followed by
washing with acetone (5 mL × 2) to obtain the desired compound 3d
(0.232 g, 60%) as a white solid, mp 254 °C (dec). HPLC 99% (tR = 3.53
min, 55% MeOH in 0.1% TFA in water, 20 min); 1H NMR (400 MHz,
DMSO-d6) δ 11.80 (s, 1H), 8.91 (d, J = 8.0 Hz, 1H), 8.22 (dd, J = 8.0, 1.2
Hz, 1H), 8.04 (dd, J = 8.0, 1.2 Hz, 1H), 7.97−7.93 (m, 2H), 7.76 (td, J =
7.6, 1.6 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.54−7.47 (m, 2H), 7.11
(appd, J = 8.0 Hz, 1H); LRMS (ESI−) m/z 331.0 (M−OH − HCl)−;
HRMS (ESI+) m/z calculated for C18H13N4O3 (M − HO − HCl)+
333.0982, found 333.0997.
N4-(2-Carboxyphenyl)-N2-(4-carboxyphenyl)pyrimidine-2,4-
diamine Hydrochloride (1, Method h in Scheme 1). A suspension
of 3w (0.040 mg, 0.100 mmol) in NaOH (0.2 mL, 4 M) and THF (0.5
mL) was refluxed in a sealed tube at 85 °C for 2 h. When the mixture was
cooled, THF was removed and water (2 mL) was added to the mixture,
followed by addition of HCl (1M) to acidify (pH 2) the mixture. The
solid obtained was filtered and washed with water (3 mL × 2) and
MeOH (3 mL × 2) and dried under high vacuum to afford 1 as a white
solid (0.033 mg, 85%), mp 256−258 °C. HPLC 98% (tR = 7.80 min,
N4-(2-Carboxymethylphenyl)-N2-(4-carboxymethylphenyl)-
pyrimidine-2,4-diamine Hydrochloride (3e, Method d in
Scheme 1). To a mixture of methyl 4-aminobenzoate (0.166 g, 1.099
mmol) and 2e (0.263 g, 0.876 mmol) was added HCl (3.0 mL, 0.1 M).
The reaction mixture was heated in a microwave reactor at 160 °C for 15
min. The crude product that was precipitated was filtered, dried under
vacuum, and purified using SiO2 chromatography (gradient elution 0−
20% EtOAc in hexane) to obtain the desired product 3e (0.116 g, 32%)
as a white solid, mp 196−198 °C. HPLC 99% (tR = 5.82 min, 60%
MeOH in 0.1% TFA in water, 20 min); 1H NMR (400 MHz, DMSO-d6)
δ 10.08 (s, 1H), 9.73 (s, 1H), 8.37 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 6.0 Hz,
1H), 7.94 (dd, J = 8.0, 1.6 Hz, 1H), 7.84 (d, J = 9.2 Hz, 2H), 7.80 (d, J =
9.2 Hz, 2H), 7.64−7.60 (m, 1H), 7.18 (appt, J = 7.2 Hz, 1H), 6.45 (d, J =
6.0 Hz, 1H), 3.81 (s, 3H), 3.79 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 168.25, 166.69, 160.74, 159.58, 157.54, 146.04, 141.41, 134.30,
131.39, 130.62, 123.02, 122.94, 121.99, 118.58, 118.39, 101.12, 52.97,
52.35; LRMS (ESI+) m/z 379.1 (M − Cl)+; HRMS (ESI+) m/z
calculated for C20H19N4O4 (M − Cl)+ 379.1401, found 379.1402.
N4,N2-Diphenylpyrimidine-2,4-diamine Hydrochloride (3f,
Method d in Scheme 1). A mixture of 2,4-dichloropyrimidine
(0.050 g, 0.336 mmol) and aniline (0.063 g, 0.677 mmol) in HCl (1.0
mL, 0.1 M) was heated in a microwave reactor at 160 °C for 15 min. The
product obtained was purified using SiO2 chromatography (gradient
elution 0−20% EtOAc in hexane) to afford the desired product 3f (0.048
g, 49%) as a white solid, mp 144 °C (dec). HPLC 96% (tR = 5.33 min,
1
45% MeOH in 0.1% TFA in water, 20 min); H NMR (400 MHz,
DMSO-d6) δ 12.57 (br s, 1H), 10.68 (s, 1H), 9.93 (s, 1H), 8.44 (appt, J =
5.6 Hz, 1H), 8.15 (d, J = 6.0 Hz, 1H), 7.98 (dd, J = 8.0 Hz, 1H), 7.84−
7.79 (m, 4 H), 7.60 (t, J = 7.6 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 6.46 (d, J
= 5.6 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ 169.85, 167.77,
160.93, 158.28, 155.08, 144.94, 141.45, 134.18, 131.90, 130.81, 123.92,
123.22, 122.95, 119.22, 118.83, 101.18; LRMS (ESI−) m/z 351.11 (M
− Cl)+; HRMS (ESI+) m/z calculated for C18H15N4O4 (M − Cl)+
351.1088, found 351.1092.
N4-(2-Carboxyphenyl)-N2-(4-carbamoylphenyl)pyrimidine-
2,4-diamine Hydrochloride (3a, Method d in Scheme 1). A
mixture of 2a (0.050 g, 0.175 mmol) and 2-aminobenzamide (0.027 g,
0.199 mmol) in HCl (1.0 mL, 0.1 M) was heated in a microwave reactor
at 160 °C for 15 min. The precipitate obtained upon cooling the mixture
was filtered and washed with water (5 mL) and acetone (5 mL × 2) to
obtain pure compound 3a (0.036 g, 53%) as a light yellow solid, mp 226
°C (dec). HPLC 96% (tR = 3.55 min, 55% MeOH in 0.1% TFA in water,
20 min); 1H NMR (400 MHz, DMSO-d6) δ 8.82 (d, J = 7.6 Hz, 1H),
8.63 (appd, J = 7.6 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.03 (dd, J = 8.0, 1.4
Hz, 1H), 7.84 (td, J = 7.6, 1.2 Hz, 1H), 7.72−7.69 (m, 1H), 7.59 (d, J =
8.4 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 6.83 (d, J =
8.0 Hz, 1H); LRMS (ESI+) m/z 333.1 (M − NH2 − HCl)+; HRMS
(ESI+) m/z calculated for C18H15N5O2 (M − NH2 − HCl)+ 333.0982,
found 333.1002.
1
N4-(2-Carboxyphenyl)-N2-(phenyl)pyrimidine-2,4-diamine
Hydrochloride (3b, Method d in Scheme 1). A mixture of 2a (0.100
g, 0.349 mmol) and aniline (0.038 g, 0.409 mmol) in HCl (1.0 mL, 0.1
M) was heated in a microwave reactor at 160 °C for 15 min. The
precipitate formed upon cooling the mixture was filtered and washed
with water (5 mL) and hot methanol (5 mL × 2) to obtain 3b (0.065 g,
54%) as a white solid, mp 233 °C (dec). HPLC 91% (tR = 17.27 min,
60% MeOH in 0.1% TFA in water, 40 min); H NMR (400 MHz,
CD3OD) δ 7.85 (d, J = 6.0 Hz, 1H), 7.59 (d, J = 7.6 Hz, 2H), 7.55 (dd, J
= 7.6 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 7.23 (d, J = 7.6 Hz, 2H), 7.05−
6.98 (m, 2H), 6.20 (d, J = 6.0 Hz, 1H); LRMS (ESI+) m/z 263.1 (M −
Cl)+; HRMS (ESI+) m/z calculated for C16H15N4 (M − Cl)+ 263.1291,
found 263.1293.
N4-(2-Carbamoylphenyl)-N2-(4-carbamoylphenyl)-
pyrimidine-2,4-diamine Hydrochloride (3g, Method d in
Scheme 1). A mixture of 2b (0.248 g, 0.871 mmol) and 4-
aminobenzamide (0.136 g, 1.00 mmol) in HCl (3.0 mL, 0.1 M) was
heated in a microwave reactor at 160 °C for 15 min. The precipitate was
filtered and washed with water (10 mL), hot MeOH (10 mL), hot THF
(10 mL), dioxane (5 mL), DMF (5 mL), and MeOH (10 mL)
sequentially to obtain pure 3g (0.160 g, 48%) as a light yellow solid, mp
257−260 °C. HPLC 97% (tR = 3.32 min, 50% MeOH in 0.1% TFA in
water, 20 min); 1H NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H
disappeared on D2O shake), 9.65 (s, 1H disappeared on D2O shake),
8.57 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 5.2 Hz, 1H), 7.98 (dd, J = 7.6, 1.6
Hz, 1H), 7.83−7.73 (m, 5H becomes 4H on D2O shake), 7.63−7.58 (m,
1H), 7.17 (brs, 1H disappeared on D2O shake), 7.12 (t, J = 8.4 Hz, 1H),
6.42 (d, J = 6.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 170.31,
168.30, 160.55, 159.67, 157.36, 144.07, 142.41, 134.35, 131.96, 128.81,
127.22, 122.25, 121.83, 118.50, 117.57, 101.05; LRMS (ESI+) m/z
349.1 (M − Cl)+; HRMS (ESI+) m/z calculated for C18H17N6O2 (M −
Cl)+ 349.1408, found 349.1407.
1
50% MeOH in 0.1% TFA in water, 20 min); H NMR (400 MHz,
DMSO-d6) δ 10.67 (s, 1H), 9.34 (s, 1H), 8.65 (d, J = 8.0 Hz, 1H), 8.11
(d, J = 5.6 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H),
7.55 (t, J = 6.8 Hz, 1H), 7.25 (t, J = 8.0 Hz, 2H), 7.08 (t, J = 7.6 Hz, 1H),
6.93 (t, J = 6.8 Hz, 1H), 6.35 (d, J = 5.6 Hz, 1H); LRMS (ESI−) m/z
305.1 (M − H − HCl)−; HRMS (ESI+) m/z calculated for C17H15N4O2
(M − Cl)+ 307.1189, found 307.1187.
N4-(2-Carboxyphenyl)-N2-(4-morpholinophenyl)pyrimidine-
2,4-diamine Hydrochloride (3c, Method e in Scheme 1). A
mixture of 2a (0.125 g, 0.437 mmol) and 4-morpholinoaniline (0.089 g,
0.500 mmol) in a solution of EtOH/1 M HCl (1:1, 2 mL) was heated in
a microwave reactor at 160 °C for 20 min. Addition of EtOAc (0.5 mL)
gave a precipitate. The precipitate was filtered and dried under vacuum
to afford the desired compound 3c (0.060 g, 32%) as a light yellow solid,
mp 162 °C (dec). HPLC 97% (tR = 2.99 min, 50% MeOH in 0.1% DEA
in water, 20 min); 1H NMR (400 MHz, CD3OD) δ 8.22 (brs, 1H), 8.14
(dd, J = 8.0, 1.6 Hz, 1H), 7.88 (d, J = 7.2 Hz, 1H), 7.64−7.54 (m, 5H),
7.35 (t, J = 7.6 Hz, 1H), 6.56 (d, J = 7.2 Hz, 1H), 4.06 (appt, J = 4.6 Hz,
4H), 3.14 (appt, J = 4.7 Hz, 4H); 13C NMR (100 MHz, DMSO-d6) δ
168.36, 162.30, 152.53, 144.03, 137.78, 133.70, 131.71, 126.68, 126.62,
N4-(Phenyl)-N2-(4-carboxyphenyl)pyrimidine-2,4-diamine
Hydrochloride (3h, Method d in Scheme 1). A mixture of 2c (0.020
7405
dx.doi.org/10.1021/jm300334d | J. Med. Chem. 2012, 55, 7392−7416

Journal of Medicinal Chemistry
Article
g, 0.082 mmol) and 4-aminobenzoic acid (0.013 g, 0.097 mmol) in HCl
(1.5 mL, 0.1 M) was heated in a microwave reactor at 160 °C for 15 min.
The solid obtained upon cooling the mixture was filtered, washed with
water (5 mL), and dried under vacuum to obtain the desired compound
3h (0.020 g, 71%) as a white solid, mp 215 °C (dec). HPLC 99% (tR =
7.28 min, 50% MeOH in 0.1% TFA in water, 20 min); 1H NMR (400
MHz, CD3OD) δ 8.00 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 7.2 Hz, 1H), 7.63
(d, J = 8.4 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.28
(t, J = 7.6 Hz, 1H), 6.46 (d, J = 7.2 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 167.44, 161.79, 152.70, 144.17, 141.82, 137.81, 130.87,
129.50, 126.80, 126.15, 123.30, 121.17, 100.58. LRMS (ESI−) m/z
305.0 (M − Cl)−; HRMS (ESI+) m/z calculated for C17H15N4O2 (M −
Cl)+ 307.1190, found 307.1187.
MHz, DMSO-d6) δ 167.36, 163.18, 152.30, 145.07, 141.91, 134.61,
130.72, 130.64, 130.47, 129.67, 129.52, 128.56, 126.30, 120.01, 95.58;
LC−MS (ESI+) m/z 341.09 (M − Cl)+; HRMS (ESI+) m/z calculated
for C17H14ClN4O2 (M − Cl)+ 341.0800, found 341.0810.
N4-(2-Carboxyphenyl)-N2-phenyl-5-fluoropyrimidine-2,4-di-
amine Hydrochloride (3m, Method e in Scheme 1). A mixture of
2g (0.134 g, 0.441 mmol) and aniline (0.140 g, 1.50 mmol) in 1:1 ratio
of EtOH/1 M HCl (2.0 mL) was heated in a microwave reactor at 160
°C for 30 min. The precipitate formed upon cooling the mixture was
filtered and washed with MeOH (5 mL), acetone (5 mL) and dried
under vacuum to afford the desired compound 3m (0.150 g, 94%) as a
white solid, mp 240 °C (dec). HPLC 99% (tR = 4.21 min, 70% MeOH in
0.1% TFA in water, 20 min); 1H NMR (400 MHz, DMSO-d6) δ 11.50
(s, 1H), 9.41 (s, 1H), 8.96 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 3.1 Hz, 1H),
8.04 (dd, J = 7.9, 1.5 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.60 (t, J = 8.4 Hz,
1H), 7.27 (t, J = 8.4 Hz, 2H), 7.12 (t, J = 7.6 Hz, 1H), 6.94 (t, J = 7.3 Hz,
1H); 19F NMR (376 MHz, DMSO-d6) δ −166.03 (s); 13C NMR (100
MHz, DMSO-d6) δ 170.83, 156.07 (d, J = 3.0 Hz), 149.60 (d, J = 10.0
Hz), 142.26, 141.68 (d, J = 245 Hz), 141.52, 141.33, 141.25, 134.72,
132.02, 129.05, 122.15 (d, J = 21.0 Hz), 120.74, 119.80, 116.24; LC−MS
(ESI+) m/z 325.12 (M − Cl)+; HRMS (ESI+) m/z calculated for
C17H14FN4O2 (M − Cl)+ 325.1095, found 325.1093.
N4,N2-Di(4-carboxyphenyl)pyrimidine-2,4-diamine Hydro-
chloride (3i, Method d in Scheme 1). A mixture of 2d (0.100 g,
0.350 mmol) and 4-aminobenzoic acid (0.055 g, 0.401 mmol) in HCl
(1.5 mL, 0.1 M) was heated in a microwave reactor at 160 °C for 15 min.
The precipitate obtained upon cooling the mixture was filtered and
washed with water (10 mL), followed by quick wash with acetone (5 mL
× 2) to give pure 3i (0.136 g, 99%) as a white solid, mp 281−283 °C.
HPLC 98% (tR = 4.97 min, 50% MeOH in 0.1% TFA in water, 20 min);
1H NMR (400 MHz, DMSO-d6) δ 10.71 (brs, 1H), 10.46 (brs, 1H),
N4-(2-Carboxyphenyl)-N2-(4-carboxyphenyl)-5-fluoropyrimi-
dine-2,4-diamine Acid (3n, Method e in Scheme 1). A mixture of
2g (0.134 g, 0.44 mmol) and 4-aminobenzoic acid (0.206 g, 1.50 mmol)
in 1:1 ratio of EtOH/HCl (2.0 mL, 1 M) was heated in a microwave
reactor at 160 °C for 30 min. The precipitate formed upon cooling the
mixture was filtered and washed with saturated NaHCO3 (3 mL) and
water (5 mL). The solid obtained was slurried in hot DMF (3 mL),
filtered, washed with MeOH (5 mL), and dried under vacuum to afford
the desired compound 3n (0.096 g, 59%) as a white solid, mp 287−290
°C. HPLC 99% (tR = 14.73 min, 55% MeOH in 0.1% TFA in water, 20
min); 1H NMR (400 MHz, DMSO-d6) δ 11.42 (s, 1H disappeared on
D2O shake), 9.83 (s, 1H disappeared on D2O shake), 8.94 (d, J = 8.6 Hz,
1H), 8.29 (appd, J = 2.7 Hz, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.83 (d, J = 8.4
Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.63 (t, J = 7.2 Hz, 1H), 7.16 (t, J = 7.5
Hz, 1H); 19F NMR (376 MHz, DMSO-d6) δ −164.42 (s); 13C NMR
(100 MHz, DMSO-d6) δ 170.76, 167.82, 155.42 (d, J = 3.5 Hz), 149.76
(d, J = 10 Hz), 145.58, 142.09 (d, J = 247 Hz), 142.00, 141.25 (d, J = 18
Hz), 134.70, 132.03, 130.87, 123.39, 122.57, 120.93, 118.13, 116.65;
LC−MS (ESI+) m/z 369.10 (M + H)+; HRMS (ESI+) m/z calculated
for C18H14FN4O4 (M + H)+ 369.0994, found 369.0994.
8.09 (d, J = 6.8 Hz), 7.90 (d, J = 6.0 Hz, 2H partially overlapping) 7.88
(d, J = 6.0 Hz, 2H partially overlapping), 7.79 (d, J = 8.8 Hz, 2H), 7.71
(d, J = 8.4 Hz, 2H), 6.50 (d, J = 6.4 Hz, 1H); LRMS (ESI−) m/z 349.0
(M − H − HCl)−; HRMS (ESI+) m/z calculated for C18H15N4O4 (M −
Cl)+ 351.1088, found 351.1082.
N4-(2-Carboxyphenyl)-N2-(4-carboxyphenyl)-5-methylpyri-
midine-2,4-diamine Hydrochloride (3j, Method g in Scheme 1).
A mixture of 2j (0.115 g, 0.383 mmol) and 4-aminobenzoic acid (0.179
g, 1.307 mmol) in HCl (1 M) /THF (2:1, 3.0 mL) was heated in a
microwave reactor at 160 °C for 15 min. The precipitate obtained upon
cooling the mixture was filtered and washed with water (5 mL) and
MeOH (5 mL × 2). The solid obtained was slurried in DMF (2 mL),
filtered, and washed with MeOH (5 mL), acetone (5 mL), and DCM (5
mL) sequentially to obtain the desired compound 3j (0.055 g, 36%) as a
white solid, mp 213 °C (dec). HPLC 97% (tR = 5.17 min, 55% MeOH in
0.1% TFA in water, 20 min); 1H NMR (400 MHz, DMSO-d6) δ 12.50
(brs, 1H disappeared on D2O shake), 11.00 (s, 1H disappeared on D2O
shake), 9.68 (s, 1H disappeared on D2O shake), 9.01 (d, J = 8.4 Hz, 1H),
8.08 (s, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.85−7.80 (m, 4H), 7.60 (t, J = 7.9
Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 2.16 (s, 3H); LC−MS (ESI+) m/z
365.13 (M − Cl)+; HRMS (ESI+) m/z calculated for C19H17N4O4 (M −
Cl)+ 365.1244, found 365.1240.
N4-(2-Chlorophenyl)-N2-(4-carboxyphenyl)-5-fluoropyrimi-
dine-2,4-diamine Hydrochloride (3o, Method d in Scheme 1). A
mixture of 2k (0.300 g, 1.12 mmol) and 4-aminobenzoic acid (0.153 g,
1.12 mmol) in ethanol (1.5 mL) was heated in a microwave reactor at
150 °C for 20 min. The reaction mixture was cooled and stirred at room
temperature for 48 h. The white precipitate was isolated by filtration and
washed with ethyl acetate (5 mL). The product was suspended in ethyl
acetate (5 mL) and sonicated for 5 min and filtered to provide 3o (0.232
g, 52%) as a white powder, mp 308 °C (dec). HPLC 99% (tR = 7.57 min,
N4-(2-Carboxyphenyl)-N2-(4-carboxyphenyl)-6-methylpyri-
midine-2,4-diamine Hydrochloride (3k, Method d in Scheme 1).
A mixture of 2f (0.263 g, 0.877 mmol) and 4-aminobenzoic acid (0.137
g, 1.00 mmol) in HCl (3.0 mL, 0.1 M) was heated in a microwave reactor
at 160 °C for 15 min. The precipitate obtained was filtered and washed
with water (10 mL), MeOH (10 mL × 2), DMSO (1 mL, quick wash),
and acetone (5 mL) to obtain the desired compound 3k (0.187 g, 53%)
as a white solid, mp 243 °C (dec). HPLC 90% (tR = 5.11 min, 55%
MeOH in 0.1% TFA in water, 20 min); 1H NMR (400 MHz, DMSO-d6)
δ 10.56 (s, 1H), 9.73 (s, 1H), 8.55 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 6.8 Hz,
1H), 7.86 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 8.8 Hz, 2H), 7.58 (t, J = 7.2 Hz,
1H), 7.11 (t, J = 7.2 Hz, 1H), 6.33 (s, 1H), 2.28 (s, 3H); LC−MS (ESI−)
m/z 363.11 (M − H − HCl)−; HRMS (ESI−) m/z calculated for
C19H15N4O4 (M − H − HCl)− 363.1099, found 363.1107.
1
55% MeOH in 0.1% TFA in water, 20 min); H NMR (400 MHz,
DMSO-d6) δ 12.44 (s, 1H), 9.59 (s, 1H), 9.38 (s, 1H), 8.15 (d, J = 3.4
Hz, 1H), 7.62−7.52 (m, 6H), 7.45−7.36 (m, 2H); 19F NMR (376 MHz,
DMSO-d6) δ −164.92; 13C NMR (100 MHz, DMSO-d6) δ 167.77,
155.67 (d, J = 3.0 Hz), 151.48 (d, J = 12.0 Hz), 145.80, 141.60 (d, J =
20.0 Hz), 141.46 (d, J = 245.0 Hz), 136.04, 131.63, 130.55, 130.40,
130.19, 128.44, 128.38, 122.66, 117.37; LC−MS (ESI+) m/z 359.07 (M
− Cl)+; HRMS (ESI+) m/z calculated for C17H13ClFN4O2 (M − Cl)+
359.0706, found 359.0709.
N4-(2-Chlorophenyl)-N2-(4-carboxyphenyl)pyrimidine-2,4-
diamine Hydrochloride (3l, Method e in Scheme 1). A mixture of
2l (0.094 g, 0.340 mmol) and 4-aminobenzoic acid (0.107 g, 0.781
mmol) in EtOH/1 M HCl (2.0 mL, 1:1) was heated in a microwave
reactor at 160 °C for 15 min. The mixture was cooled to room
temperature. The precipitate obtained was filtered, washed with water (2
mL), and MeOH (2 mL) sequentially to afford the desired compound 3l
(0.055 g, 43%) as a white solid, mp 234 °C (dec). HPLC 99% (tR = 4.32
min, 55% MeOH in 0.1% TFA in water, 20 min); 1H NMR (400 MHz,
DMSO-d6) δ 12.59 (s, 1H), 10.00 (s, 1H), 9.76 (s, 1H), 8.06 (d, J = 6.0
Hz, 1H), 7.70−7.68 (m, 3H), 7.63−7.58 (m, 3H), 7.43 (t, J = 7.8 Hz,
1H), 7.33 (t, J = 7.4 Hz, 1H), 6.40 (d, J = 6.1 Hz, 1H); 13C NMR (100
N4-(2-Chlorophenyl)-N2-phenyl-5-fluoropyrimidine-2,4-dia-
mine Hydrochloride (3p, Method m in Scheme 2). A mixture of 2k
(0.061 g, 0.207 mmol) and aniline (0.020 g, 0.207 mmol) in EtOH (2.0
mL) was heated in a microwave reactor at 150 °C for 20 min. The
precipitate formed upon cooling the mixture was filtered and quickly
washed with MeOH (1 mL) to afford the desired product 3p (0.037 g,
51%) as a white solid, mp 145−148 °C. HPLC 99% (tR = 9.43 min, 55%
MeOH in 0.1% TFA in water, 20 min); 1H NMR (400 MHz, DMSO-d6)
δ 9.18 (s, 1H disappeared on D2O shake), 9.12 (s, 1H disappeared on
D2O shake), 8.08 (d, J = 4.0 Hz, 1H), 7.59−7.56 (m, 2H), 7.44 (d, J = 8.4
7406
dx.doi.org/10.1021/jm300334d | J. Med. Chem. 2012, 55, 7392−7416

Journal of Medicinal Chemistry
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Hz, 2H), 7.39 (td, J = 7.6, 1.6 Hz, 1H), 7.31 (td, J = 7.6, 1.6 Hz, 1H), 7.02
(t, J = 7.2 Hz, 2H), 6.77 (t, J = 7.6 Hz, 1H); 19F NMR (376 MHz,
DMSO-d6) δ −166.49; 13C NMR (100 MHz, DMSO-d6) δ 155.97,
151.42 (d, J = 12 Hz), 141.40, 141.24 (d, J = 20 Hz), 141.11 (d, J = 244
Hz), 136.05, 131.18, 130.35, 129.80, 128.76, 128.29, 128.13, 121.33,
118.80; LC−MS (ESI+) m/z 315.08 (M − Cl)+; HRMS (ESI+) m/z
calculated for C16H13ClFN4 (M − Cl)+ 315.0807, found 315.0812.
N4-(2-Carboxyphenyl)-N2-(3-carboxyphenyl)pyrimidine-2,4-
diamine Hydrochloride (3q Method d in Scheme 1). A mixture of
2a (0.050 g, 0.175 mmol) and 3-aminobenzoic acid (0.024 g, 0.175
mmol) in HCl (0.1 mL, 0.1 M) was heated in a microwave reactor at 160
°C for 15 min. The solution was cooled and the precipitate obtained was
filtered and washed quickly with hot methanol (3 mL) to obtain the
desired product 3q (0.056 g, 72%) as a white solid, mp 275 °C (dec).
HPLC 99% (tR = 6.31 min, 50% MeOH in 0.1% TFA in water, 20 min);
1H NMR (400 MHz, DMSO-d6) δ 12.85 (brs, 1H), 10.73 (s, 1H), 9.52
5 - A m i n o - N 4 - ( 2 - c a r b o x y p h e n y l ) - N 2 - ( 4 -
ethoxycarbonylphenyl)pyrimidine-2,4-diamine Hydrochloride
(3u, Method f(i) in Scheme 1). This was prepared by using a method
described Gray and co-workers.47 A mixture of 2h (0.080 g, 0.266
mmol), ethyl 4-aminobenzoate (0.099 g, 0.600 mmol), and HCl (0.15
mL, 4 M in dioxane) in 2-butanol (1.0 mL) was heated in a sealed tube at
120 °C (oil bath temperature) for 24 h. After the mixture was cooled to
room temperature, the suspension obtained was filtered and washed
with water (5 mL), MeOH (3 mL) to afford the desired compound 3u
(0.082 g, 72%) as a yellow solid, mp 262 °C (dec). HPLC 99% (tR =
15.48 min, 50% MeOH in 0.1% TFA in water, 20 min); 1H NMR (400
MHz, DMSO-d6) δ 9.88 (s, 1H), 9.87 (s, 1H), 9.18 (s, 1H), 7.94−7.84
(m, 5H), 7.80 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 8.4 Hz, 1H), 7.19 (d, J = 8.0
Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 4.26 (q, J = 7.0 Hz, 2H), 1.30 (t, J = 7.0
Hz, 3H); LC−MS (ESI+) m/z 376.15 (M − OH − HCl)+; HRMS (ESI
+) m/z calculated for C20H18N5O3 (M − OH − HCl)+ 376.1404, found
376.1405.
(s, 1H), 8.67 (d, J = 8.4 Hz, 1H), 8.27 (s, 1H), 8.12 (d, J = 5.7 Hz, 1H),
7.96 (d, J = 7.9 Hz, 2H), 7.54−7.47 (m, 2H), 7.51 (t, J = 7.7 Hz, 2H),
7.05 (t, J = 7.6 Hz, 1H), 6.36 (d, J = 5.7 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 170.49, 168.16, 160.41, 159.99, 157.52, 142.75, 141.52,
134.46, 131.93, 131.72, 129.22, 124.08, 122.79, 121.79, 121.25, 120.71,
116.79, 100.98; LC−MS (ESI−) m/z 349.10 (M − H − HCl)−; HRMS
(ESI−) m/z calculated for C18H14N4O4 (M − H − HCl)− 349.0942,
found 397.0940.
6 - A m i n o - N 4 - ( 2 - c a r b o x y p h e n y l ) - N 2 - ( 4 -
ethoxycarbonylphenyl)pyrimidine-2,4-diamine Hydrochloride
(3v, Method e in Scheme 1). A mixture of 2i (0.265 g, 0.880 mmol)
and ethyl 4-aminobenzoate (1.65 g, 10.00 mmol) in EtOH/1 M HCl
(1:1, 12 mL) was heated in a microwave reactor at 160 °C for 1 h. The
mixture was cooled to room temperature, and the solid obtained was
filtered, washed with MeOH (5 mL), and slurried in acetone (10 × 5
mL) until no impurity was shown by NMR, affording the desired
1
N4-(2-Carboxyphenyl)-N2-(3-carboxyphenyl)-5-fluoropyrimi-
dine-2,4-diamine Hydrochloride (3r Method m Scheme 2). A
mixture of 2g (0.100 g, 0.329 mmol) and 3-aminobenzoic acid (0.048 g,
0.350 mmol) in HCl (1.0 mL, 0.1 M) was heated in a microwave reactor
at 150 °C for 20 min. The mixture was cooled to room temperature and
the precipitate obtained was filtered and washed quickly with ethanol (3
mL) to obtain the desired product 3r (0.089 g, 66%) as a white solid, mp
272 °C (dec). HPLC 99% (tR = 14.34 min, 50% MeOH in 0.1% TFA in
compound 3v (0.125 g, 33%) as a beige solid, mp 280 °C (dec). H
NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H disappeared on D2O
shake), 9.36 (s, 1H disappeared on D2O shake), 8.85 (appd, J = 7.6 Hz,
1H), 7.94 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.4
Hz, 2H), 7.47 (t, J = 8.4 Hz, 1H), 6.94 (t, J = 8.0 Hz, 1H), 6.54 (brs, 2H
disappeared on D2O shake), 5.54 (s, 1H), 4.26 (q, J = 7.0 Hz, 2H), 1.29
(t, J = 7.0 Hz, 3H); LC−MS (ESI+) m/z 394.15 (M − Cl)+; HRMS (ESI
+) m/z calculated for C20H20N5O4 (M − Cl)+ 394.1510, found
394.1509.
1
water, 20 min); H NMR (400 MHz, DMSO-d6) δ 13.07 (brs, 1H),
N4-(2-Carboxyphenyl)-N2-(4-methoxycarbonylphenyl)-
pyrimidine-2,4-diamine Hydrochloride (3w, Method m in
Scheme 2). A suspension of 2a (0.060 g, 0.210 mmol) and methyl 4-
aminobenzoate (0.032 g, 0.210 mmol) in MeOH (1 mL) was heated in a
microwave reactor at 150 °C for 20 min. When the mixture was cooled,
the resulting precipitate was filtered and washed with MeOH (2 mL) to
afford 3w (0.064 mg, 76%) as a yellow solid which was used without
further purification. 1H NMR (400 MHz, DMSO-d6) δ 13.40 (br s, 1H),
10.74 (s, 1H), 10.11 (s, 1H), 8.33 (br d, J = 5.6 Hz, 1H), 8.14 (d, J = 6.0
Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.84−7.76 (m, 4 H), 7.63 (t, J = 7.6
Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 6.50 (d, J = 6.4 Hz, 1H), 3.80 (s, 3H);
LC−MS (ESI+) m/z 365.13 (M − Cl)+; HRMS (ESI+) m/z calculated
for C19H17N4O4 (M − Cl)+ 365.1244, found 365.1243.
11.48 (s, 1H), 9.61 (s, 1H), 8.97 (d, J = 8.4 Hz, 1H), 8.31−8.23 (m, 2H),
8.03 (d, J = 7.9 Hz, 1H), 7.60−7.48 (m, 2H), 7.39 (t, J = 7.9 Hz, 1H),
7.12 (t, J = 7.6 Hz, 1H); 13C NMR (101 MHz, DMSO-d6) δ 170.87,
168.29, 155.90, 149.69 (d, J = 9.7 Hz), 151.37 (d, J = 11.5 Hz), 141.91
(d, J = 235.3 Hz), 142.22, 141.54, 134.89, 132.04, 131.77, 129.32,
123.78, 122.87, 122.29, 120.62, 120.37, 116.19; 19F NMR (376 MHz,
DMSO) δ −165.34. Anal. Calcd for C18H14ClFN4O4: C, 53.41; H, 3.49;
N, 13.84. Found: C, 53.22; H, 3.37; N, 13.57.
N4-(2-Carboxyphenyl)-N2-(4-ethoxycarbonylphenyl)-5-chlor-
opyrimidine-2,4-diamine Hydrochloride (3s, Method f(ii) in
Scheme 1). A mixture of 2m (0.063 g, 0.193 mmol) and ethyl 4-
aminobenzoate (0.043 g, 0.260 mmol) in ethanol (0.8 mL) was heated
in a sealed tube at 120 °C (oil bath temperature) overnight. The
resulting precipitate was filtered, washed with ethanol (1 mL × 2), Et2O
(3 mL), and hexane (2 mL) sequentially, and dried under vacuum to
afford the title compound 3s (0.079 g, 90%) as a white solid, mp 221 °C
(dec). 1H NMR (400 MHz, DMSO-d6) δ 13.77 (s, 1H), 11.46 (s, 1H),
9.97 (s, 1H), 8.93 (d, J = 8.3 Hz, 1H), 8.34 (s, 1H), 8.04 (dd, J = 1.5, 7.9
Hz, 1H), 7.86 (d, J = 9.1 Hz, 2H), 7.83 (d, J = 9.1 Hz, 2H), 7.64−7.60
(m, 1H), 7.19−7.15 (m, 1H), 4.26 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.1 Hz,
3H); LC−MS (ESI−) m/z 412.09 (M + H)+; HRMS (ESI−) m/z
calculated for C20H18ClN4O4 (M + H)+ 413.1011, found 413.0989.
N4-(2-Carboxyphenyl)-N2-(4-ethoxycarbonylphenyl)-5,6-di-
chloropyrimidine-2,4-diamine Hydrochloride (3t, Method f(ii)
in Scheme 1). A mixture of 2n (0.091 g, 0.257 mmol) and ethyl 4-
aminobenzoate (0.044 g, 0.266 mmol) in ethanol (1.0 mL) was heated
in a sealed tube at 110 °C (oil bath temperature) for 4 days. The
resulting precipitate was filtered, washed with ethanol (1 mL × 2), and
suspended in methanol (1 mL). The mixture was sonicated and the solid
was filtered and dried under vacuum to afford the title compound 3t
(0.022 g, 17%) as a white solid, mp 206 °C (dec). 1H NMR (400 MHz,
DMSO-d6) δ 11.52 (s, 1H), 10.25 (s, 1H), 8.74 (s, 1H), 8.04 (dd, J = 1.5,
7.9 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.9 Hz, 2H), 7.64−7.60
(m, 1H), 7.23−7.20 (m, 1H), 4.27 (q, J = 7.1 Hz, 2H), 1.29 (t, J = 7.1 Hz,
3H); LC−MS (ESI−) m/z 447.07 (M − Cl)+; HRMS (ESI−) m/z
calculated for C20H17Cl2N4O4 (M − Cl)+ 447.0621, found 447.005 98.
5-Amino-N4-(2-carboxyphenyl)-N2-(4-carboxyphenyl)-
pyrimidine-2,4-diamine (4a, Method h in Scheme 1). A
suspension of 3u (0.069 g, 0.161 mmol) in NaOH/THF (2 M, 0.44
mL/0.2 mL) was heated at 100 °C (oil bath temperature) in a sealed
tube for 30 min. A solution of HCl (1 M) was added to acidify the
solution to pH 1−2 after removing THF. The solid obtained was filtered
and washed with water (5 mL), saturated NaHCO3 (3 mL), water (5
mL), acetone (3 mL), and MeOH (3 mL) sequentially to afford the
desired compound 4a (0.038 g, 65%) as a brown solid, mp 160 °C (dec).
HPLC 96% (tR = 4.19 min, 55% MeOH in 0.1% TFA in water, 20 min);
1H NMR (400 MHz, DMSO-d6) δ 9.33 (brs, 1H disappeared on D2O
shake), 8.86 (brs, 1H), 7.99 (brs, 1H), 7.88−7.78 (m, 5H), 7.50 (brs,
1H), 7.01 (brs, 1H); LC−MS (ESI+) m/z 366.12 (M + H)+; HRMS
(ESI+) m/z calculated for C18H16N5O4 (M + H)+ 366.1197, found
366.1198.
6-Amino-N4-(2-carboxyphenyl)-N2-(4-carboxyphenyl)-
pyrimidine-2,4-diamine (4b, Method h in Scheme 1). A
suspension of 3v (0.070 g, 0.163 mmol) in NaOH (0.45 mL, 2 M)
and THF (0.25 mL) was heated at 100 °C (oil bath temperature) in a
sealed tube for 16 h. The THF in the mixture was evaporated, and HCl
(1 M) was added to acidify (pH 1−2) the mixture. The solid obtained
was filtered, washed with water (3 mL), saturated aqueous NaHCO3 (3
mL), and water (3 mL) sequentially, and dried to afford the desired
compound 4b (0.047 g, 79%) as a yellow solid, mp 220 °C (dec). HPLC
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1
95% (tR = 5.20 min, 55% MeOH in 0.1% TFA in water, 20 min); H
NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H disappeared on D2O
shake), 9.78 (s, 1H disappeared on D2O shake), 8.54 (brs, 1H), 7.96
(appd, J = 5.2 Hz, 1H), 7.82−7.80 (m, 2H), 7.59−7.53 (m, 3H), 7.34−
7.10 (m, 3H), 5.60 (s, 1H); LC−MS (ESI+) 366.13 m/z (M + H)+;
HRMS (ESI+) m/z calculated for C18H16N5O4 (M + H)+ 366.1197,
found 366.1194.
mL) to afford 6b (0.085 g, 84%) as a white solid, mp 207 °C (dec).
HPLC 100% (tR = 9.1 min, 55% MeOH in 0.1% TFA in water, 20 min);
1H NMR (400 MHz, DMSO-d6) δ 10.62 (s, 1H), 10.43 (s, 1H), 8.01
(apparent s, 1H), 7.86−7.80 (m, 2H), 7.71−7.48 (m, 2H), 7.27 (d, J =
7.2 Hz, 2H), 7.14 (t, J = 7.5 Hz, 2H), 7.03 (t, J = 7.3 Hz, 1H), 6.46 (s,
1H); LC−MS (ESI+) m/z 331.13 (M − Cl)+; HRMS (ESI+) m/z
calculated for C17H14F3N4 (M − Cl)+ 331.1165, found 331.1170.
N4-(2-Chloro-4-fluorophenyl)-N2-(4-carboxyphenyl)-
pyrimidine-2,4-diamine Hydrochloride (6c, Method m in
Scheme 2). A mixture of chloropyrimidine 5b (0.096 g, 0.372 mmol)
and 4-aminobenzoic acid (0.058 g, 0.422 mmol) in EtOH (0.5 mL) was
heated in a microwave reactor at 150 °C for 20 min. The resulting
precipitate was isolated by filtration and washed with EtOH (0.5 mL ×
2), diethyl ether (2 mL), and hexane (2 mL) sequentially to provide the
title compound 6c (0.102 g, 69%) as a white solid, mp 268 °C (dec).
HPLC 97.7% (tR = 4.8 min, 55% MeOH in 0.1% TFA in water, 20 min);
1H NMR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 10.72 (s, 1H), 8.12 (d,
N4-(2-Carboxyphenyl)-N2-(4-carboxyphenyl)-5,6-dichloro-
pyrimidine-2,4-diamine Hydrochloride (4c, Method h in
Scheme 1). A mixture of 3t (0.018 g, 0.034 mmol) in THF (0.3 mL)
and NaOH (0.1 mL, 2 M) was heated in a sealed tube at 110 °C (oil bath
temperature) overnight. The THF was then removed under reduced
pressure, and HCl (1 M aqueous, 0.5 mL) was added to the residue. The
resulting precipitate was filtered and washed with water (2 mL) and
dried under vacuum. The solid obtained was then slurried in methanol
(1 mL), filtered, and dried under vacuum to afford the title compound
4c (0.005 g, 32%) as a white solid, mp 230 °C (dec). HPLC 84% (tR =
1
10.84 min, 20% MeOH, 80% water (with 0.1% DEA), 20 min); H
J = 7.0 Hz, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.72−7.63 (m, 2H), 7.50 (d, J =
8.6 Hz, 2H), 7.40 (td, J = 8.5, 2.9 Hz, 1H), 6.53 (d, J = 7.0 Hz, 1H); 19
F
NMR (400 MHz, DMSO-d6) δ 12.62 (s, 1H), 11.47 (s, 1H), 10.21 (s,
1H), 8.72 (s, 1H), 8.04 (dd, J = 7.9, 1.4 Hz, 1H), 7.84 (d, J = 8.8 Hz, 2H),
7.72 (d, J = 8.7 Hz, 2H), 7.65−7.54 (m, 1H), 7.23−7.20 (m, 1H); LC−
MS (ESI−) m/z 417.02 (M − H − HCl)−; HRMS (ESI−) m/z
calculated for C18H13Cl2N4O4 (M − H − HCl)− 417.0163, found
417.0160.
NMR (400 MHz, DMSO-d6) δ 112.66; 13C NMR (100 MHz, DMSO-
d6) δ 167.37 163.39, 161.22 (d, J = 246.3 Hz), 152.39, 145.37, 141.91,
131.87 (d, J = 11.12 Hz); LC−MS (ESI−) m/z 357.06 (M − H −
HCl)−; HRMS (ESI−) m/z calculated for C17H11ClFN4O2 (M − H −
HCl)− 357.0560, found 357.0521.
N4-(2-Carboxyphenyl)-N2-(4-carboxyphenyl)-5-chloropyri-
midine-2,4-diamine Hydrochloride (4d, Method h in Scheme 1).
A mixture of 3s (0.058 g, 0.119 mmol) in THF (0.4 mL) and NaOH (2
M aqueous, 0.2 mL) was heated in a sealed tube at 110 °C (oil bath
temperature) overnight. The THF was then removed under reduced
pressure, and HCl (1 M aqueous, 0.6 mL) was added to the residue. The
resulting precipitate was filtered, washed with water (2 mL × 3), and
dried under vacuum. The solid obtained was then slurried in DMF (2
mL), filtered, washed with DMF (5 mL), methanol (1 mL), and Et2O (3
mL) sequentially, and dried under vacuum to afford the title compound
N4-[2-(Trifluoromethoxy)phenyl]-N2-(4-carboxyphenyl)-
pyrimidine-2,4-diamine Hydrochloride (6d, Method m in
Scheme 2). A mixture of chloropyrimidine 5c (0.074 g, 0.255 mmol)
and 4-aminobenzoic acid (0.042 g, 0.306 mmol) in EtOH (0.5 mL) was
heated with a microwave reactor at 150 °C for 20 min. The resulting
precipitate was isolated by filtration and washed with EtOH (0.5 mL ×
2), diethyl ether (2 mL), and hexane (2 mL) sequentially to provide the
title compound 6d (0.63 g, 58%) as a white solid, mp 228 °C (dec).
HPLC 96.4% (tR = 5.8 min, 55% MeOH in 0.1% TFA in water, 20 min);
1H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 10.75 (s, 1H), 8.12 (d,
J = 7.0 Hz, 1H), 7.77−7.73 (m, 3H), 7.63−7.39 (m, 5H), 6.58 (d, J = 6.7
Hz, 1H); 19F NMR (400 MHz, DMSO-d6) δ 57.09; 13C NMR (100
MHz, DMSO-d6) δ 167.40, 163.23, 152.77, 145.96, 143.26, 142.04,
130.77, 130.16, 129.31, 129.10, 128.65, 126.30, 122.51, 120.22, 118.10
(q, J = 256 Hz), 99.76; LC−MS (ESI−) m/z 389.07 (M − H − HCl)−;
HRMS (ESI−) m/z calculated for C18H12F3N4O3 (M − H − HCl)−
389.0867, found 389.0818.
1
4d (0.036 g, 72%) as a white solid, mp 261 °C (dec). H NMR (400
MHz, DMSO-d6) δ 12.59 (s, 1H), 11.45 (s, 1H), 9.90 (s, 1H), 8.87 (s,
1H), 8.30 (s, 1H), 8.01−7.55 (m, 6H), 7.13 (s, 1H); 13C NMR (100
MHz, DMSO-d6) δ 170.57 167.72, 157.79, 155.85, 155.36, 145.16,
141.90, 134.51, 131.90, 130.84, 123.84, 122.82, 121.54, 118.76, 117.26,
107.11; HPLC 97% (tR = 8.74 min, 15% MeOH, 85% water (with 0.1%
DEA), 20 min); LC−MS (ESI−) m/z 383.04 (M − H − HCl)−; HRMS
(ESI−) m/z calculated for C18H14ClN4O4 (M − H − HCl)− 383.0553,
found 383.0552.
N4-(2-Methoxyphenyl)-N2-(4-carboxyphenyl)pyrimidine-2,4-
diamine Hydrochloride (6e, Method m in Scheme 2). A mixture of
chloropyrimidine 5d (0.075 g, 0.317 mmol) and 4-aminobenzoic acid
(0.046 g, 0.328 mmol) in EtOH (0.5 mL) was heated in a microwave
reactor at 150 °C for 20 min. Ethanol (0.5 mL) was added to the reaction
mixture. The resulting precipitate was isolated by filtration and washed
with EtOH (0.5 mL) and hexane (5 mL) to provide the title compound
6e (0.8 g, 68%) as a white solid, mp 247.5−249.3 °C. HPLC 99.6% (tR =
N4-(2-Fluorophenyl)-N2-(4-carboxyphenyl)pyrimidine-2,4-di-
amine Hydrochloride (6a, Method m in Scheme 2). A mixture of
2-chloro-N-(2-fluorophenyl)pyrimidin-4-amine (5a) (0.096 g, 0.428
mmol) and 4-aminobenzoic acid (0.069 g, 0.503 mmol) in EtOH (0.5
mL) was heated in a microwave reactor at 150 °C for 20 min. The
mixture was filtered and the resulting precipitate was washed with EtOH
(0.5 mL × 2) to provide the title compound 6a (0.107 g, 69%) as a white
solid, mp 264 °C (dec). HPLC 98.7% (tR = 3.8 min, 55% MeOH in 0.1%
1
6.0 min, 50% MeOH in 0.1% TFA in water, 20 min); H NMR (400
MHz, DMSO-d6) δ 12.75 (s, 1H), 10.85 (s, 1H), 10.41 (s, 1H), 8.03 (d, J
= 7.1 Hz, 1H), 7.77 (d, J = 8.6 Hz, 2H), 7.59−7.56 (m, 3H), 7.33 (t, J =
7.9 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.50
(apparent s, 1H), 3.80 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ
167.42, 162.78, 153.64, 152.19, 144.09, 142.02, 130.81, 128.73, 127.42,
126.30, 125.47, 120.89, 120.17, 112.66, 99.85, 56.32; LC−MS (ESI−)
m/z 335.11 (M − H − HCl)−; HRMS (ESI−) m/z calculated for
C18H15N4O3 (M − H − HCl)− 335.1150, found 335.1153.
1
TFA in water, 20 min); H NMR (400 MHz, DMSO-d6) δ 12.79 (s,
1H), 11.04 (s, 1H), 10.87 (s, 1H), 8.13 (d, J = 7.0 Hz, 1H), 7.77 (d, J =
8.7 Hz, 2H), 7.66 (t, J = 7.9 Hz, 1H), 7.56 (d, J = 8.5 Hz, 2H), 7.46−7.36
(m, 2H), 7.33−7.25 (m, 1H), 6.58 (d, J = 6.7 Hz, 1H); 19F NMR (400
MHz, DMSO-d6) δ 121.12; 13C NMR (100 MHz, DMSO-d6) δ 167.40,
163.00, 156.59 (d, J = 245 Hz), 152.55, 145.08, 141.92, 130.79, 129.14,
(d, J = 9.27 Hz), 128.30, 126.43, 125.39, 125.04 (d, J = 12.15 Hz),
120.29, 116.88 (d, J = 18.72 Hz), 99.84; LC−MS (ESI−) m/z 323.10
(M − H − HCl)−; HRMS (ESI−) m/z calculated for C17H12FN4O2 (M
− H − HCl)− 323.0950, found 323.0974.
N4-(2-Methoxyphenyl)-N2-(phenyl)pyrimidine-2,4-diamine
(6f, Method m in Scheme 2). A mixture of chloropyrimidine 5d
(0.105 g, 0.444 mmol) and aniline (0.05 mL, 0.548 mmol) in EtOH (0.5
mL) was heated in a microwave reactor at 150 °C for 20 min. The
solvent was removed under reduced pressure. Aqueous saturated
NaHCO3 (10 mL) was added to the residue and extracted with ethyl
acetate (10 mL × 2). The combined organic phase was dried (Na2SO4),
filtered, and concentrated to dryness. The residue was purified by flash
chromatography (10 g silica gel, hexane/EtOAc) to afford the title
compound 6f (0.100 g, 76%) as an off-white solid, mp 142.3−144.5 °C.
HPLC 99.4% (tR = 4.90 min, 60% MeOH in 0.1% TFA in water, 20
min); 1H NMR (400 MHz, DMSO-d6) δ 9.06 (s, 1H), 8.54 (s, 1H), 7.99
N4-[2-(Trifluoromethyl)phenyl]-N2-phenylpyrimidine-2,4-di-
amine Hydrochloride (6b, Method m in Scheme 2). A mixture of
chloropyrimidine 5f (0.076 g, 0.277 mmol) and aniline (0.03 mL, 0.328
mmol) in EtOH (0.4 mL) was heated in a microwave reactor at 150 °C
for 20 min. The solvent was removed under reduced pressure to provide
an off-white solid. Ethyl acetate (3 mL) was added to the reaction
mixture. The mixture was left at room temperature for 30 min and
sonicated occasionally. The resulting precipitate was isolated by
filtration and washed with ethyl acetate (1 mL × 5) and hexane (3
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(d, J = 8.0 Hz, 1H), 7.96 (d, J = 5.7 Hz, 1H), 7.69 (d, J = 7.6 Hz, 2H),
7.18−7.14 (m, 2H), 7.11−7.02 (m, 2H), 6.97−6.89 (m, 1H), 6.85 (t, J =
7.3 Hz, 1H), 6.32 (d, J = 5.8 Hz, 1H), 3.82 (s, 3H); 13C NMR (100 MHz,
CDCl3) 161.00, 160.08, 156.52, 149.55, 140.01, 128.97, 128.31, 123.69,
122.60, 121.50, 120.91, 120.33, 110.62, 98.24; LC−MS (ESI+) m/z
293.15 (M + H)+; HRMS (ESI+) m/z calculated for C17H17N4O (M +
H)+ 293.1397, found 293.1393.
for 16 h. The THF was evaporated from the mixture, and HCl (1 M) was
added to acidify the mixture (pH 1−2). The precipitate obtained was
filtered, washed with water (5 mL), and dried under high vacuum to
afford 6j (0.137 g, 70%) as a beige solid, mp 132 °C (dec). HPLC 97%
(tR = 3.77 min, 50% MeOH in 0.1% TFA in water, 20 min); 1H NMR
(400 MHz, DMSO-d6) δ 12.19 (s, 1H disappeared on D2O shake), 9.30
(s, 1H disappeared on D2O shake), 9.21 (s, 1H disappeared on D2O
shake), 8.01 (d, J = 6.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.54 (dd, J =
8.0, 1.4 Hz, 1H partially overlapping), 7.51 (d, J = 8.4 Hz, 2H partially
overlapping with dd), 7.38 (apptd, J = 8.0, 1.4 Hz, 1H), 7.24 (apptd, J =
8.0, 1.4 Hz, 1H), 7.03 (d, J = 8.4 Hz, 2H), 6.30 (d, J = 6.0 Hz, 1H), 3.45
(s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 173.65, 162.04, 158.90,
155.15, 139.43, 136.41, 130.31, 129.87, 129.85, 128.56, 128.44, 128.10,
126.90, 119.66, 119.63, 98.71; LC−MS (ESI+) m/z 355.11 (M − Cl)+;
HRMS (ESI+) m/z calculated for C18H16ClN4O2(M − Cl)+ 355.0956,
found 355.0971.
N4-(2-Cyanophenyl)-N2-(phenyl)pyrimidine-2,4-diamine (6g,
Method m in Scheme 2). A mixture of chloropyrimidine 5e (0.092 g,
0.398 mmol) and aniline (0.037 mL, 0.398 mmol) in EtOH (0.5 mL)
was heated in a microwave reactor at 150 °C for 20 min. The solvent was
removed under reduced pressure. The solid obtained was slurried in
ethyl acetate (3 mL), filtered, and washed with ethyl acetate (5 mL) and
hexane (5 mL). The dried solid was dissolved in methanol (3 mL).
Diisopropylethylamine (1 mL) was added, and the solvent was removed
under reduced pressure. The solid obtained was slurried with water (5
mL), filtered, washed with water (10 mL × 2), ether (5 mL), slurried
with methanol (1 mL), filtered, and dried under vacuum to provide the
title compound 6g (0.027 g, 24%) as an off-white solid, mp 170.0.2−
172.7 °C. HPLC 100% (tR = 5.1 min, 50% MeOH, 50% water (with 0.1%
TFA), 20 min); 1H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 9.15 (s,
1H), 8.07 (d, J = 5.7 Hz, 1H), 7.82 (d, J = 8.8 Hz, 1H), 7.77−7.66 (m,
2H), 7.59 (d, J = 8.8 Hz, 2H), 7.32 (t, J = 8.0 Hz, 1H), 7.11 (t, J = 7.9 Hz,
2H), 6.83 (t, J = 7.3 Hz, 1H), 6.28 (d, J = 5.7 Hz, 1H); LC−MS (ESI+)
m/z 288.13 (M + H)+; HRMS (ESI+) m/z calculated for C17H14N5 (M
+ H)+ 288.1244, found 288.1241.
N 4 - ( 2 - C h l o r o m e t h y l ) p h e n y l ] - N 2 - ( 4 - c a r b o x y- 3 -
hydroxyphenyl)pyrimidine-2,4-diamine Hydrochloride (6k,
Methods n and o in Scheme 2). A mixture of chloropyrimidine 2l
(0.100 g, 0.416 mmol) and methyl 5-aminosalicylate (0.070 g, 0.416
mmol) in THF (10 mL) was stirred at room temperature for 10 min.
Two drops of concentrated HCl were added, and the mixture was heated
at reflux for 14 h. The solvent was removed under reduced pressure to
provide a gray solid which was suspended in sodium bicarbonate
(saturated aqueous 50 mL), sonicated for 10 min, filtered, and washed
with water (10 mL) to provide a white powder. The crude material was
washed with methanol (2 × 10 mL) and finally with ethyl acetate (20
mL) to give methyl 4-(4-(2-chlorophenylamino)pyrimidin-2-ylamino)-
2-hydroxybenzoate (6u) (0.081 g, 56%). 1H NMR (400 MHz, DMSO-
d6) δ 10.61 (s, 1H), 9.63 (s, 1H), 9.15 (s, 1H), 8.07 (s, 1H), 7.76 (s, 1H),
7.59−7.33 (3 × broad s, 3H), 7.25−7.10 (2 × broad s, 2H), 6.35 (s, 1H),
3.83 (s, 3H); LC−MS (ESI+) 371.09, m/z calculated for C18H16ClN4O3
(M + H)+ 371.0905, found 371.0914.
To a stirred solution of ester 6u (200 mg, 0.540 mmol) in THF (15
mL) was added sodium hydroxide (108 mg, 2.70 mmol) in water (1.5
mL). The mixture was heated under reflux for 14 h. The solvent was
removed under reduced pressure to provide a white solid which was
then dissolved in water (20 mL) and acidified to pH ∼6−7 by addition
of HCl (1 M). The colorless precipitate was filtered, washed with water,
and dried under vacuum. The crude solid was suspended in methanol
(20 mL), sonicated for 10 min, filtered, washed with methanol (5 mL),
and dried under vacuum to provide the title compound 6k (118 mg,
62%) as an off-white powder, mp 202−204 °C. HPLC 100% (tR = 8.3
min, 45% MeOH, 65% water (with 0.1% TFA), 20 min); 1H NMR (400
MHz, DMSO-d6) δ 13.55 (brs, 1H), 11.29 (brs, 1H), 10.14 (brs, 1H),
9.93 (s, 1H), 8.07 (d, J = 6.7 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.57 (d, J
= 7.6 Hz, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.30 (t, J
= 8.0 Hz, 1H), 7.25 (s, 1H), 7.02 (d, J = 8.5 Hz, 1H), 6.43 (d, J = 5.7 Hz,
1H); 13C NMR (100 MHz, DMSO-d6), δ 172.34, 162.82, 162.58,
155.69, 146.06, 135.35, 131.18, 130.58, 129.32, 128.64, 128.53, 128.25,
111.11, 107.27, 106.54, 99.78; LC−MS (ESI+) m/z 357.08 (M − Cl)+;
HRMS (ESI+) m/z calculated for C17H14ClN4O3 (M − Cl)+ 357.0749,
found 357.0751.
N4-[2-(Trifluoromethyl)phenyl]-N2-(4-carboxyphenyl)-
pyrimidine-2,4-diamine Hydrochloride (6h, Method m in
Scheme 2). A mixture of chloropyrimidine 5f (0.055 g, 0.2 mmol)
and 4-aminobenzoic acid (0.03 g, 0.218 mmol) in EtOH (0.3 mL) was
heated in a microwave reactor at 150 °C for 20 min. Ethanol (0.5 mL)
was added to the reaction mixture and sonicated at room temperature
for 5 min. The resulting precipitate was isolated by filtration and washed
with EtOH (0.5 mL × 2), and hexane (3 mL × 2) to afford the title
compound 6h (0.067 g, 82%) as a white solid, mp 240.0−242.6 °C.
HPLC 99.6% (tR = 7.7 min, 50% MeOH in 0.1% TFA in water, 30 min);
1H NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 10.76 (s, 1H), 8.11 (d,
J = 7.0 Hz, 1H), 7.90−7.85 (m, 2H), 7.69−7.64 (m, 4H), 7.38 (d, J = 8.5
Hz, 2H), 6.53 (d, J = 6.7 Hz, 1H); 19F NMR (400 MHz, DMSO-d6) δ
59.99 (s); 13C NMR (100 MHz, DMSO-d6) δ 167.35, 164.21, 152.47,
145.71, 142.05, 135.37, 134.40, 131.77, 130.65, 129.09, 127. (q, J = 4.6
Hz), 126.62 (q, J = 29.5 Hz), 126.10, 124.03 (q, J = 271.5 Hz), 119.77;
LC−MS (ESI−) m/z 373.08 (M + H)+; LC−MS (ESI+) m/z 375.11
(M + H)+; HRMS (ESI+) m/z calculated for C18H14F3N4O2 (M + H)+
375.1063, found 375.1068.
N4-(2-Bromophenyl)-N2-(4-carboxyphenyl)pyrimidine-2,4-
diamine Hydrochloride (6i, Method m in Scheme 2). A mixture of
chloropyrimidine 5g (0.100 g, 0.350 mmol) and 4-aminobenzoic acid
(0.048 g, 0.350 mmol) in EtOH (0.5 mL) was heated in a microwave
reactor at 150 °C for 20 min. The resulting precipitate was isolated by
filtration and washed with EtOH (0.5 mL × 2) and dried under vacuum
to provide the title compound 6i (0.069 g, 47%) as a white solid, mp 240
°C (dec). HPLC 98% (tR = 7.40 min, 50% MeOH, 50% water (with
0.1% TFA), 20 min); 1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 2H),
8.06 (d, J = 6.8 Hz, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.68 (d, J = 8.5 Hz,
2H), 7.59 (d, J = 8.3 Hz, 1H), 7.51−7.47 (m, 3H), 7.32 (t, J = 8.3 Hz,
1H), 6.45 (s, 1H); 13C NMR (100 MHz, d6-DMSO) 167.43, 163,07,
153.17, 146.60, 142.37, 136.36, 133.80, 130.71, 129.95, 129.71, 129.19,
125.91, 121.27, 119.83, 99.45; LC−MS (ESI+) m/z 385.02 (M − Cl)+;
HRMS (ESI+) m/z calculated for C17H14BrN4O2 (M − Cl)+ 385.0295,
found 385.0292.
N4-(2-Fluorophenyl)-N2-4-phenylpyrimidine-2,4-diamine (6l,
Method m in Scheme 2). A mixture of chloropyrimidine 5a (0.073 g,
0.325 mmol) and aniline (0.04 mL, 0.438 mmol) in EtOH (0.5 mL) was
heated in a microwave reactor at 150 °C for 20 min. The solvent was
removed under reduced pressure. Aqueous saturated NaHCO3 (10 mL)
was added to the residue and extracted with ethyl acetate (10 mL × 2).
The combined organic phase was dried (Na2SO4), filtered, and
concentrated to dryness. The residue was purified by flash
chromatography (10 g silica gel, hexane/EtOAc) to afford the title
compound 6l (0.050 g, 55%) as an off-white solid, mp 133.2−134.6 °C.
HPLC 99.5% (tR = 4.4 min, 60% MeOH in 0.1% TFA in water, 20 min);
1H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 1H), 9.07 (s, 1H), 8.02 (d, J =
N4-(2-Chlorophenyl)-N2-(4-methylcarboxyphenyl)-
pyrimidine-2,4-diamine Hydrochloride (6j, Method l in Scheme
2). A mixture of chloropyrimidine 2l (0.120 g, 0.500 mmol) and 4-
aminophenylacetic acid (0.076 g, 0.5 mmol) in EtOH (2 mL with 1 drop
of 1 M hydrochloric acid) was heated in a microwave reactor at 160 °C
for 15 min. A clear solution was obtained. Ethanol was removed from the
mixture under vacuum, and the analysis of the crude NMR showed
formation of 25% ethyl ester of 6j. The crude material was stirred in
THF (0.5 mL) and NaOH solution (1 mL, 2 M) at room temperature
5.7 Hz, 1H), 7.97 (t, J = 7.9 Hz, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.32−7.23
(m, 1H), 7.22−7.07 (m, 4H), 6.86 (t, J = 7.3 Hz, 1H), 6.30 (d, J = 5.7 Hz,
1H); LC−MS (ESI+) m/z 281.11 (M − Cl)+; HRMS (ESI+) m/z
calculated for C16H14FN4 (M − Cl)+ 281.1197, found 281.1209.
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N4-(2-Iodophenyl)-N2-(4-carboxyphenyl)pyrimidine-2,4-dia-
mine Hydrochloride (6m, Method m in Scheme 2). A mixture of
chloropyrimidine 5h (0.119 g, 0.358 mmol) and 4-aminobenzoic acid
(0.048 g, 0.350 mmol) in EtOH (0.5 mL) was heated in a microwave
reactor at 150 °C for 20 min. The solvent was then removed under
reduced pressure to provide an off-white solid. The solid was slurried in
methanol (2 mL), filtered, washed with methanol (2 mL), and dried
under vacuum to provide the title compound 6m (0.053 g, 32%) as a
white solid, mp 253 °C (dec). HPLC 99.7% (tR = 7.80 min, 50% MeOH,
50% water (with 0.1% TFA), 20 min); 1H NMR (400 MHz, DMSO-d6)
δ 12.76 (bs, 1H), 10.72 (s, 1H), 10.62 (s, 1H), 8.07 (d, J = 7.0 Hz, 1H),
8.01 (dd, J = 1.2, 7.9 Hz, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.54−7.45 (m,
4H), 7.19−7.15 (m, 1H), 6.44 (s, 1H); 13C NMR (100 MHz, DMSO-
d6) δ 167.36, 163.09, 152.32, 145.44, 142.04, 139.99, 139.67, 130.68,
130.04, 129.94, 129.45, 126.12, 119.90, 99.24; LC−MS (ESI+) m/z
433.02 (M − Cl)+; HRMS (ESI+) m/z calculated for C17H14IN4O2 (M
− Cl)+ 433.0156, found 433.0150.
(ESI+) m/z calculated for C17H15ClN5O (M − Cl)+ 340.0960, found
340.0971.
N4-(2-Biphenyl)-N2-(4-carboxyphenyl)pyrimidine-2,4-dia-
mine Hydrochloride (6q, Method m in Scheme 2). A mixture of
chloropyrimidine 5i (0.096 g, 0.34 mmol) and 4-aminobenzoic acid
(0.054 g, 0.393 mmol) in EtOH (0.4 mL) was heated in a microwave
reactor at 150 °C for 20 min. The resulting precipitate was filtered and
washed with EtOH (0.5 mL × 3) to provide the title compound 6q
(0.098 g, 67%) as a white solid, mp 259 °C (dec). HPLC 100% (tR = 8.7
min, 55% MeOH in 0.1% TFA in water, 20 min); 1H NMR (400 MHz,
DMSO-d6) δ 12.83 (s, 1H), 10.96 (s, 1H), 10.71 (s, 1H), 7.96 (d, J = 7.2
Hz, 1H), 7.73 (d, J = 8.3 Hz, 2H), 7.61−7.42 (m, 6H), 7.42−7.17 (m,
5H), 6.31 (apparent s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 167.41,
163.26, 152.00, 144.30, 142.07, 138.96, 134.37, 131.35, 130.75, 129.35,
129.09, 128.74, 128.17, 126.18, 119.89, 99.22; LC−MS (ESI−) m/z
381.13 (M − H − HCl)−; HRMS (ESI−) m/z calculated for
C23H17N4O2 (M − H − HCl)− 381.1357, found 381.113 66.
N4-(2-Chlorophenyl)-N2-(4-carboxyphenyl)-N4-methylpyri-
midine-2,4-diamine Hydrochloride (6r, Method m in Scheme 2).
This was obtained as a white solid (0.127 g, 0.36 mmol, 80%) from 5j
(0.115 g, 0.452 mmol) and 4-aminobenzoic acid (0.062 g, 0.452 mmol)
in the same manner as described for 6s, mp 288 °C (dec). HPLC 98%
(tR = 8.39 min, 50% MeOH, 50% water (with 0.1% TFA), 20 min); 1H
NMR (400 MHz, CD3OD) δ 8.17−8.04 (m, 2.57 H), 7.77−7.74 (m,
3H), 7.70−7.67 (m, 2H), 7.58−7.54 (m, 4H), 7.49−7.41 (m, 0.42 H),
7.17 (d, J = 8.8 Hz, 1H, major), 6.83 (d, J = 7.5 Hz, 0.41 H, minor), 5.80
(d, J = 7.4 Hz, 1H, major), 3.55 (s, 4H); LC−MS (ESI+) m/z 355.10 (M
− Cl)+; HRMS (ESI+) m/z calculated for C18H16ClN4O2 (M − Cl)+
355.0956, found 355.0962. Anal. Calcd for C18H16Cl2N4O2: C, 55.26%;
H, 4.12; N, 14.32. Found: C, 54.98%; H, 4.12; N, 14.18. .
N4-(2-Cyanophenyl)-N2-(4-carboxyphenyl)pyrimidine-2,4-di-
amine Hydrochloride (6n, Method m in Scheme 2). A mixture of
chloropyrimidine 5e (0.094 g, 0.406 mmol) and 4-aminobenzoic acid
(0.037 mg, 0.408 mmol) in EtOH (0.5 mL) was heated in a microwave
reactor at 150 °C for 20 min. The solvent was removed under reduced
pressure. The compound was purified by reverse phase C-18 preparative
HPLC (Eclipse XDB-C18 PrepHT 21.2 mm × 250 mm, 7 μm; 40%
MeOH, 60% water (with 0.1% TFA), 20 min, 20 mL/min) to provide
the title compound as its TFA salt. The solid obtained was suspended in
methanol (9 mL) followed by the addition of HCl (4 M in dioxane, 1
mL). The solvent was then removed in a Genevac evaporator. The dried
solid was slurried with acetonitrile (1 mL), filtered, washed with
acetonitrile (1 mL), and dried under vacuum to provide the title
compound 6n (0.022 g, 15%) as an off-white solid, mp 222 °C (dec).
HPLC 97.6% (tR = 5.5 min, 40% MeOH, 60% water (with 0.1% TFA),
N4-(2-Chlorophenyl)-N2-(4-carboxyphenyl)-N4-ethylpyrimi-
dine-2,4-diamine Hydrochloride (6s, Method m in Scheme 2). A
solution of 5k (0.151 g, 0.673 mmol) and 4-aminobenzoic acid (0.092 g,
0.674 mmol) in anhydrous ethanol (0.673 mL) was heated in a
microwave reactor at 150 °C for 20 min. The resulting precipitate was
isolated by filtration and washed with EtOH (1 mL × 2) and dried under
vacuum to provide the title compound 6s (0.172 g, 71%) as a white solid,
mp 284 °C (dec). HPLC 100% (tR = 13.96 min, 50% MeOH, 50% water
(with 0.1% TFA), 20 min). Two rotamers are present: 1H NMR (400
MHz, CD3OD) δ 8.11 (d, J = 8.8 Hz, 2H, major), 8.07 (d, J = 7.6 Hz, 0.4
H, minor), 7.75−7.66 (m, 5H), 7.63−7.54 (m, 5H), 7.15 (d, J = 8.8 Hz,
1H, major), 6.87 (d, J = 7.5 Hz, 0.5 H, minor), 5.74 (d, J = 7.4 Hz, 1H,
major), 4.30−4.21 (m, 1H), 4.09−4.02 (m, 0.6 H, minor), 3.93−3.83
(m, 2H), 1.35 (t, J = 7.2 Hz, 1.5 H, minor), 1.29 (t, J = 7.2 Hz, 3H,
major); LC−MS (ESI+) m/z 369.12 (M − Cl)+; HRMS (ESI+) m/z
calculated for C19H18ClN4O2 (M − Cl)+ 369.1113, found 369.1123.
Anal. Calcd for C19H18Cl2N4O2: C, 56.31%; H, 4.48; N, 13.82. Found:
C, 56.30%; H, 4.45; N, 13.76.
1
20 min); H NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H), 10.43 (s,
1H), 8.14 (d, J = 6.8 Hz, 1H), 7.93 (d, J = 7.6 Hz, 1H), 7.80 (t, J = 7.6 Hz,
1H), 7.72−7.68 (m, 3H), 7.55 (d, J = 7.3 Hz, 2H), 7.50 (t, J = 7.6 Hz,
1H), 6.50 (d, J = 6.8 Hz, 1H); LC−MS (ESI+) m/z 332.11 (M − Cl)+;
HRMS (ESI+) m/z calculated for C18H14N5O2 (M − Cl)+ 332.1142,
found 332.1139.
N4-(2-Chlorophenyl)-N2-(3-carboxyphenyl)pyrimidine-2,4-
diamine Hydrochloride (6o, Method m in Scheme 2). A mixture of
2l (0.100 g, 0.417 mmol) and 3-aminobenzoic acid (0.057 g, 0.417
mmol) in EtOH (0.5 mL) was heated in a microwave reactor at 150 °C
for 20 min. The precipitate obtained upon cooling the reaction mixture
was filtered and washed quickly with ethanol (3 mL) to obtain the 6o
(0.097 g, 62%) as a white solid, mp 290−296 °C (dec). HPLC 100% (tR
= 7.58 min, 50% MeOH in 0.1% TFA in water, 20 min); 1H NMR (400
MHz, DMSO-d6) δ 13.03 (s, 1H), 10.49 (brs, 1H), 8.04 (d, J = 6.4 Hz,
1H), 7.88 (brs, 1H), 7.73 (appd, J = 7.6 Hz, 1H), 7.65 (d, J = 8.0 Hz,
1H), 7.60 (d, J = 7.6 Hz, 1H), 7.57 (dd, J = 7.9, 1.5 Hz, 1H), 7.37 (td, J =
7.6, 0.8 Hz, 1H), 7.32−7.27 (m, 2H), 6.49 (s, 1H); 13C NMR (101 MHz,
DMSO-d6) δ 167.5, 162.95, 153.3, 145.9, 138.08, 134.6, 132.1, 130.5,
129.6, 129.5, 129.1, 128.89, 128.36, 125.9, 125.5, 122.6, 99.5. Anal.
Calcd for C17H14Cl2N4O2: C, 54.13; H, 3.74; N, 14.85. Found: C, 53.78;
H, 3.64; N, 14.66.
N4-(2-Chlorophenyl)-N2-(3-carboxyphenyl)-5-fluoropyrimi-
dine-2,4-diamine Hydrochloride (6t, Method m in Scheme 2). A
mixture of 2k (0.100 g, 0.350 mmol) and 3-aminobenzoic acid (0.048 g,
0.350 mmol) in EtOH (0.5 mL) was heated in a microwave reactor at
150 °C for 20 min. The reaction mixture was cooled and the precipitate
obtained was filtered and washed quickly with ethanol (3 mL) to provide
6t (0.080 g, 57%) as a white solid, mp 257 °C (dec). HPLC 98% (tR =
9.45 min, 50% MeOH in 0.1% TFA in water, 20 min); 1H NMR (400
MHz, DMSO-d6) δ 12.74 (brs,1H), 9.32 (s, 1H), 9.18 (s, 1H), 8.12 (d, J
= 3.5 Hz, 1H), 8.02 (s, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 7.8 Hz,
1H), 7.56 (d, J = 7.9 Hz, 1H), 7.40−7.36 (m, 2H), 7.29 (t, J = 7.6 Hz,
1H), 7.11 (t, J = 7.9 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ
168.14, 156.0 (d, J = 2.9 Hz), 151.37 (d, J = 11.5 Hz), 141.75, 141.43 (d,
J = 246.44 Hz), 141.39 (d, J = 18.4 Hz), 135.92, 131.54, 130.88, 130.38,
129.55, 128.90, 128.32, 128.03, 122.84, 122.18, 119.87; 19F NMR (376
MHz, DMSO-d6) δ −165.76; LC−MS (ESI−) m/z 357.06 (M − H −
HCl)−; HRMS (ESI−) m/z calculated for C17H12ClFN4O2 (M − H −
HCl)− 357.0560, found 357.0554.
N4-(2-Chlorophenyl)-N2-(4-carbamoyl)pyrimidine-2,4-dia-
mine Hydrochloride (6p, Method l in Scheme 2). A mixture of
chloropyrimidine 2l (0.100 g, 0.417 mmol) and 4-aminobenzamide
(0.057 g, 0.417 mmol) in EtOH (2.0 mL with 1 drop of 1 M
hydrochloric acid) was heated with a microwave reactor at 160 °C for 15
min. The mixture was filtered and the solid obtained was washed with
MeOH (1 mL) and dried to afford the desired compound 6p (0.110 g,
70%) as a white solid, mp 227 °C (dec). HPLC 99% (tR = 3.70 min, 50%
MeOH in 0.1% TFA in water, 20 min); 1H NMR (400 MHz, DMSO-d6)
δ 10.75 (brs, 2H disappeared on D2O shake), 8.09 (dd, J = 6.1, 3.5 Hz,
1H), 7.91 (brs, 1H disappeared on D2O shake), 7.71−7.64 (m, 4H),
7.50−7.42 (m, 4H), 7.30 (brs, 1H disappeared on D2O shake), 6.52
(brs, 1H); 13C NMR (100 MHz, DMSO-d6) δ 167.76, 163.15, 152.33,
144.97, 140.39, 134.55, 130.62, 130.29, 130.02, 129.54, 129.49, 128.90,
128.55, 119.98, 99.43; LC−MS (ESI+) m/z 340.10 (M − Cl)+; HRMS
N4-(2-Chlorophenyl)-N2-[4-N-(2-morpholinoethyl)-
carbamoylphenyl]pyrimidine-2,4-diamine Dihydrochloride
(9a, Method r in Scheme 4). A solution of 2l (0.100 g, 0.42 mmol)
and 8a (0.105 g, 0.42 mmol) in isopropyl alcohol (8 mL) and
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concentrated HCl (0.315 mL) was heated in a microwave reactor at 170
°C for 20 min. The solvent was then removed under reduced pressure.
The resulting solid was dissolved in methanol (2 mL) followed by the
addition of diethyl ether (10 mL). The precipitate was filtered and dried
under vacuum to afford the title compound 9a (0.054 g, 29%) as white
crystals, mp 173.0−174.6 °C. HPLC 99.0% (tR = 5.53 min, 50% MeOH,
40% water (with 0.1% TFA), 20 min); 1H NMR (400 MHz, DMSO-d6)
δ 11.37−10.62 (m, 1H), 8.88 (s, 1H), 8.59−8.43 (m, 1H), 8.12 (s, 1H),
7.78 (s, 2H), 7.69−7.61 (m, 2H), 7.50 (s, 2H), 7.43 (s, 1H), 6.76−6.41
(m, 2H), 4.03−2.95 (m, 12H); 13C NMR (100 MHz, DMSO-d6) δ
166.39, 163.08, 152.86, 145.97, 140.91, 134.67, 130.67, 130.15, 129.42,
129.24, 128.82, 128.60, 119.81, 99.46, 63.80, 56.32, 51.87, 34.28; LC−
MS (ESI+) m/z 453.17 (M − HCl − Cl)+; HRMS (ESI+) m/z
calculated for C23H25ClN6O2 (M − HCl − Cl)+ 453.1800, found
453.1797.
(with 0.1% TFA), 20 min); 1H NMR (400 MHz, DMSO-d6) δ 10.63 (s,
1H), 10.45 (s, 1H), 10.00 (s, 1H), 8.00 (d, J = 5.0 Hz, 1H), 7.70−7.52
(m, 3H), 7.4−7.28 (m, 2H), 7.24−7.13 (m, 2H), 7.13−7.07 (m, 1H),
2.02 (s, 3H); LC−MS (ESI+) m/z 354.11 (M − Cl)+; HRMS (ESI+)
m/z calculated for C18H17ClN5O (M − Cl)+ 354.1116, found 354.1119.
N4-(2-Chlorophenyl)-N2-[6-(1H-benzo[d]imidazol-2(3H)-
one)]pyrimidine-2,4-diamine Hydrochloride (9f, Method s in
Scheme 4). A solution of 2l (0.105 g, 0.437 mmol) and 5-amino-1H-
benzo[d]imidazol-2(3H)-one (0.065 g, 0.437 mmol) and ethanol
(0.437 mL) was heated in a microwave reactor at 150 °C for 40 min. The
resulting precipitate was filtered, dried under vacuum, and suspended in
ethanol. The suspension was sonicated for 30 min, filtered, and dried
under vacuum to provide the title compound 9f (0.112 g, 66%) as an off-
white solid, mp 263 °C (dec). HPLC 98% (tR = 3.21 min, 50% MeOH,
50% water (with 0.1% TFA), 20 min); 1H NMR (400 MHz, CD3OD) δ
7.74 (s, 2H), 7.65−7.63 (m, 1H), 7.54−7.52 (m, 1H), 7.35−7.28 (m,
2H), 7.04 (d, J = 7.4 Hz, 3H), 6.42 (s, 1H); LC−MS (ESI+) m/z 353.10
(M − Cl)+; HRMS (ESI+) m/z calculated for C17H14ClN6O (M − Cl)+
353.0912, found 353.0913.
N4-(2-Chlorophenyl)-N2-[4-N-(2-dimethylaminoethyl)-
carbamoylphenyl]pyrimidine-2,4-diamine Dihydrochloride
(9b, Method r in Scheme 4). This was obtained as a white solid
(0.02 g, 0.04 mmol, 8%) from 2l (0.100 g, 0.48 mmol) and 8b (0.116 g,
0.48 mmol) in a similar manner as described for 9a, mp 143.1−145.8 °C.
HPLC 98% (tR = 5.10 min, 60% MeOH, 40% water (with 0.1% TFA), 20
min); 1H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H), 9.96 (s, 1H),
8.75 (s, 1H), 8.09 (s, 1H), 7.73 (s, 2H), 7.71−7.61 (m, 2H), 7.58−7.45
(m, 3H), 7.45−7.35 (m, 1H), 6.50 (s, 1H), 3.62−3.55 (m, 2H), 3.23 (d,
J = 5.7 Hz, 2H), 2.80 (d, J = 4.0 Hz, 6H); 13C NMR (100 MHz, DMSO-
d6) δ 166.50, 163.10, 152.72, 145.80, 140.87, 134.73, 130.67, 130.19,
129.45, 129.31, 128.61, 119.80, 99.50, 56.76, 43.01, 35.16; LC−MS (ESI
+) m/z 411.18 (M − HCl − Cl)+; HRMS (ESI+) m/z calculated for
C21H24ClN6O (M − HCl − Cl)+ 411.1695, found 411.1693.
N4-(2-Chlorophenyl)-N2-(4-carboxyl-3-methoxyphenyl)-
pyrimidine-2,4-diamine Hydrochloride (9g, Method s in
Scheme 4). A mixture of chloropyrimidine 2l (0.100 g, 0.416 mmol)
and 4-amino-2-methoxybenzoic acid (0.070 g, 0.418 mmol) in EtOH
(0.5 mL) was heated with a microwave reactor at 150 °C for 20 min. The
solvent was evaporated from the resulting thick mass under reduced
pressure. The residue was suspended in ethyl acetate (8 mL) and
sonicated for 10 min. The mixture was filtered and the solid was washed
with ethyl acetate (8 mL) and ethyl acetate/methanol (1:1, 1 mL) to
provide the title compound 9g (0.068 g, 57%) as a gray solid, mp 160−
162 °C. HPLC 95% (tR = 4.5 min, 50% MeOH, 50% water (with 0.1%
TFA), 20 min); 1H NMR (400 MHz, DMSO-d6), δ 12.69 (s, 1H), 10.42
(s, 1H), 10.24 (s, 1H), 7.95 (d, J = 6.2 Hz, 1H), 7.64−7.51 (m, 3H), 7.48
(d, J = 8.4 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 6.98
(d, J = 8.8 Hz, 1H), 6.45 (s, 1H), 3.76 (s, 3H); 13C NMR (100 MHz,
DMSO-d6), δ 167.37, 162.97, 155.67, 152.95, 144.61, 134.39, 130.51,
129.61, 129.00, 128.35, 127.11, 124.90, 122.22, 113.36, 99.26, 56.70;
LC−MS (ESI+) m/z 371.09 (M − Cl)+; HRMS (ESI+) m/z calculated
for C18H16ClN4O3 (M − Cl)+ 371.0905, found 371.0909.
N4 -(2-Chlorophenyl)-N2 -[4-N-(2-methoxyethyl)-
carbamoylphenyl]pyrimidine-2,4-diamine Hydrochloride (9c,
Method s in Scheme 4). This was obtained as a white solid (0.047 g,
0.12 mmol) from 2l (0.092 g, 0.47 mmol) and 8c (0.060 g, 0.47 mmol)
in a similar manner as described for compound 6s, mp 210.4−213.0 °C.
HPLC 97% (tR = 5.61 min, 50% MeOH, 50% water (with 0.1% TFA), 20
min); 1H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 2H), 8.44 (t, J = 5.6
Hz, 1H), 8.08 (d, J = 7.3 Hz, 1H), 7.68−7.62 (m, 4H), 7.49−7.38 (m,
3H), 6.52 (s, 1H), 3.41−3.37 (m, 4H), 3.24 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ 166.06, 163.12, 152.41, 145.17, 140.32, 134.55,
130.60, 130.23, 130.08, 129.50, 129.42, 128.56, 119.98, 99.48, 71.50,
58.58, 58.55; LC−MS (ESI+) m/z 398.14 (M − Cl)+; HRMS (ESI+)
m/z calculated for C20H21ClN5O2 (M − Cl)+ 398.1378, found 398.1370.
N4-(2-Chlorophenyl)-N2-[4-(methylsulfonyl)phenyl]-
pyrimidine-2,4-diamine (9d, Method t in Scheme 4). A solution of
2l (0.098 g, 0.41 mmol) and 4-(methylsulfonyl)aniline (0.070 g, 0.41
mmol) in ethanol (1 mL) and 1 M HCl (aqueous, 1.0 mL) was heated in
a microwave reactor at 180 °C for 15 min. The solvent was removed
under reduced pressure. The solid obtained was slurred in a saturated
solution of sodium bicarbonate, filtered, slurried with methanol (1 mL),
then filtered to yield the title compound 9d (0.077 g, 44%) as a white
solid, mp 169.3−171.3 °C. HPLC 99% (tR = 4.15 min, 50% MeOH, 50%
N4-(2-Chlorophenyl)-N2-[4-(N-morpholino)phenyl]-
pyrimidine-2,4-diamine Hydrochloride (9h, Method t in
Scheme 4). A mixture of chloropyrimidine 2l (0.120 g, 0.500 mmol)
and 4-morpholinoaniline (0.089 g, 0.500 mmol) in EtOH (2 mL with 1
drop of 1 M of hydrochloric acid) was heated in a microwave reactor at
160 °C for 15 min. The reaction mixture was cooled to room
temperature and the product precipitated. The mixture was filtered, and
the product obtained was quickly washed with MeOH (1 mL), DCM (1
mL) and dried to afford 9h (0.14 g, 67%) as a green solid, mp 136−138
°C. HPLC 99% (tR = 7.43 min, 50% MeOH in 0.1% TFA water, 20 min);
1H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H disappeared on D2O
shake), 10.63 (s, 1H disappeared on D2O shake), 10.37 (s, 1H
disappeared on D2O shake), 7.94 (brs, 1H), 7.65−.60 (m, 2H), 7.43
(appt, J = 7.6 Hz, 1H), 7.36 (appt, J = 7.6 Hz, 1H), 7.22 (d, J = 8.1 Hz,
2H), 6.84 (brs, 2H), 6.45 (brs, 1H), 3.73 (appt, J = 4.4 Hz, 4H), 3.05
(brt, 4H); 13C NMR (100 MHz, DMSO-d6) δ 163.03, 152.56, 148.63,
144.36, 134.58, 130.53, 129.99, 129.37, 129.12, 128.43, 122.90, 116.05,
98.78, 66.65, 49.38; LC−MS (ESI+) m/z 382.15 (M − Cl)+; HRMS
(ESI+) m/z calculated for C20H21ClN5O (M − Cl)+ 382.1429, found
382.1434.
1
water (with 0.1% TFA), 20 min); H NMR (400 MHz, DMSO-d6) δ
9.72 (s, 1H), 9.17 (s, 1H), 8.08 (d, J = 5.7 Hz, 1H), 7.84 (d, J = 8.7 Hz,
2H), 7.74 (d, J = 7.8, 1H), 7.65−7.51 (m, 3H), 7.41 (t, J= 7.6, 1H), 7.26
(t, J = 7.6 Hz, 1H), 6.35 (d, J = 5.7 Hz, 1H), 3.09 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ 162.03, 159.47, 156.92, 146.25, 136.52, 132.20,
130.40, 129.03, 128.38, 128.34, 128.21, 127.12, 118.38, 99.77, 44.70;
LC−MS (ESI+) m/z 375.06 (M + H)+; HRMS (ESI+) m/z calculated
for C17H16ClN4O2S (M + H)+ 375.0677, found 375.0670.
N4-(2-Chlorophenyl)-N2-[4-(aminosulfonyl)phenyl]-
pyrimidine-2,4-diamine Hydrochloride (9i, Method t in Scheme
4). A mixture of chloropyrimidine 2l (0.120 g, 0.500 mmol) and 4-
aminobenzenesulfonamide (0.086 g, 0.500 mmol) in EtOH (2 mL with
1 drop of 1 M HCl) was heated in a microwave reactor at 160 °C for 15
min. The precipitate formed upon cooling was filtered and washed with
MeOH (2 mL), then slurried and sonicated in DCM (5 mL) and
filtered. The product obtained was dried to afford 9i (0.155 g, 75%) as a
white solid, mp 215 °C (dec). HPLC 96% (tR = 3.23 min, 50% MeOH in
0.1% TFA in water, 20 min); 1H NMR (400 MHz, DMSO-d6) δ 10.81
(s, 1H), 10.67 (s, 1H), 8.10 (d, J = 6.9 Hz, 1H), 7.67−7.63 (m, 2H),
7.61−7.56 (m, 4H), 7.47 (td, J = 7.7, 1.6 Hz, 1H), 7.41 (td, J = 7.7, 1.6
Hz, 1H), 7.28 (s, 2H), 6.53 (appd, J = 5.2 Hz, 1H); 13C NMR (100 MHz,
N4-(2-Chlorophenyl)-N2-(3-acetamidophenyl)pyrimidine-
2,4-diamine Hydrochloride (9e, Method s in Scheme 4). This
compound was prepared from 2l (0.105 g, 0.44 mmol) and N-(3-
aminophenyl)acetamide (0.066 g, 0.44 mmol) in a similar manner as
described for 6s. The solvent was then removed under reduced pressure.
The resulting solid was dissolved in methanol (2 mL), followed by the
addition of ethyl acetate (10 mL). A precipitate formed, and the mixture
was sonicated for 30 min at room temperature. The precipitate was
filtered and washed with ethyl acetate (10 mL) and hexane (20 mL),
affording the pure product 9e (0.166 g, 95%) as an off-white solid, mp
150 °C (dec). HPLC 100% (tR = 5.30 min, 50% MeOH, 50% water
7411
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DMSO-d6) δ 163.15, 152.43, 145.28, 140.86, 139.52, 134.61, 130.64,
130.38, 129.60, 129.50, 128.57, 127.10, 120.32, 99.66; LC−MS (ESI+)
m/z 376.06 (M − Cl)+; HRMS (ESI+) m/z calculated for
C16H15ClN5O2S (M − Cl)+ 376.0629, found 376.0634.
N4-(2-Chlorophenyl)-N2-(4-carbamoyl)-5-fluoropyrimidine-
2,4-diamine (9m, Method v in Scheme 4). A mixture of
chloropyrimidine 2k (0.500 g, 1.935 mmol) and 4-aminobenzamide
(0.265 g, 1.935 mmol) in methanol (3 mL) was heated in a 5 mL sealed
pressure tube at 100 °C for 6 h. The white precipitate was isolated by
filtration and washed with methanol (2 mL). The white solid was
sonicated for 5 min in sodium bicarbonate (aqueous saturated, 10 mL).
The mixture was filtered and the solid washed with water (10 mL) and
finally with methanol (2 mL) to provide 9m (0.439 g, 63%) as an off-
white powder, mp 250−252 °C. HPLC 100% (tR = 7.8 min, 50%
N4-(2-Chlorophenyl)-N2-[4-(N-morpholino)phenyl]-5-fluoro-
pyrimidine-2,4-diamine (9j, Method s in Scheme 4). A mixture of
chloropyrimidine 2k (0.100 g, 0.387 mmol) and 4-morpholinoaniline
(0.069 g, 0.387 mmol) in ethanol (1 mL) was heated in a microwave
reactor at 150 °C for 20 min. The reaction mixture was cooled and
stirred at room temperature for 30 min. The solvent was removed in
vacuo. The residual solid was suspended in ethyl acetate (10 mL) and
sonicated for 10 min, filtered, and washed with ethyl acetate (5 mL). The
solid was suspended in sodium bicarbonate (saturated aqueous 50 mL),
filtered, and washed with water (10 mL) to provide the title compound
9k (0.92 g, 60%) as an off-white solid, mp 209−211 °C. HPLC 99% (tR =
7.2 min, 50% MeOH, 50% water (with 0.1% TFA), 20 min); 1H NMR
(400 MHz, DMSO-d6) δ 9.63 (s, 1H), 9.36 (s, 1H), 8.11 (d, JH−F = 4.2
Hz, 1H), 7.61−7.54 (m, 2H), 7.44−7.37 (m, 1H), 7.36−7.25 (m, 3H),
6.79 (s, 2H), 3.73 (s, 4H), 3.03 (s, 4H); 1H NMR (400 MHz, DMSO-d6
+ D2O) δ 8.06 (d, J = 4.1 Hz, 1H), 7.59−7.51 (m, 2H), 7.39 (td, J = 7.6,
1.5 Hz, 1H), 7.32 (td, J = 7.7, 1.7 Hz, 1H), 7.27 (d, J = 9.0 Hz, 2H), 6.79
(d, J = 8.9 Hz, 2H), 3.71 (t, J = 4.0 Hz, 4H), 3.03 (t, J = 4.4 Hz, 4H); 19F
NMR (376 MHz, DMSO-d6), δ −165.59; 13C NMR (100 MHz,
DMSO-d6) δ 154.38, 152.31 (d, J = 12.0 Hz), 141.63, 139.04, 139.19,
135.42, 134.20, 130.43, 129.76, 120.74, 117.11, 66.31, 50.71; LC−MS
(ESI+) m/z 400.13 (M − Cl)+; HRMS (ESI+) m/z calculated for
C20H20ClFN5O (M − Cl)+ 400.1335, found 400.1344.
1
MeOH, 50% water (with 0.1% formic acid), 20 min); H NMR (400
MHz, DMSO-d6) δ 9.42 (s, 1H), 9.31 (s, 2H), 8.12 (d, JH−F = 3.6 Hz,
1H), 7.70 (s, 1H), 7.59 (dd, J = 8.6, 1.3 Hz, 1H), 7.56 (dd, J = 8.6, 1.3
Hz) overlapping 7.54 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.42
(td, J = 7.7, 1.5 Hz, 1H), 7.35 (td, J = 7.6, 1.6 Hz, 1H), 7.08 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) δ 168.57, 155.66 (d, JC−F = 3.0 Hz),
151.35 (d, JC−F = 11.7 Hz), 144.27, 142.54, 141.66 (d, JC−F = 19.0 Hz),
140.09, 135.77, 131.27, 130.41, 129.79, 128.67, 128.41, 126.18, 117.31;
19F NMR (376 MHz, DMSO-d6), δ −165.45 (bd, JH−F = 3.4 Hz) LC−
MS (ESI+) m/z 358.09 (M + H)+; HRMS (ESI+) m/z calculated for
C17H14ClFN5O (M + H)+ 358.0865, found 358.0868.
N4-(2-Chlorophenyl)-N2-(4-aminosulfonyl)-5-fluoropyrimi-
dine-2,4-diamine (9n, Method u in Scheme 4). A mixture of
chloropyrimidine 2k (free base) (0.100 g, 0.387 mmol), 4-amino-
benzenesulfonamide (0.066 g, 0.0.387 mmol), X-Phos (2-dicyclohex-
ylphosphino-2′,4′,6′-triisopropylbiphenyl) (0.019 g, 0.0387 mmol),
bis(dibenzylideneacetone)palladium(0) (0.022 g, 0.0387 mmol), and
potassium carbonate (0117 g, 0.851 mmol) in t-BuOH (2.0 mL) was
heated in a 5 mL sealed pressure tube at 100 °C for 64 h under argon.
The solid was isolated by filtration and suspended in ethyl acetate (5
mL) and sonicated for 5 min. The mixture was filtered and the solid
washed with ethyl acetate (5 mL). The crude product was dissolved in
DMSO (2 mL) and filtered to remove undissolved material. The DMSO
was removed in vacuo at 50 °C to provide the title sulfonamide 9n
(0.080 g, 53%) as a brown solid, mp 253−255 °C. HPLC 99% (tR = 11.2
min, 40% MeOH, 60% water (with 0.1% TFA), 66 min); 1H NMR (400
MHz, DMSO-d6) δ 9.57 (s, 1H), 9.39 (s, 1H), 8.13 (d, JH−F = 3.7 Hz,
1H), 7.61−7.52 (m, 4H), 7.46−7.38 (m, 3H), 7.35 (t, J = 8.0 Hz, 1H),
4-(2-Chlorophenylamino)-2-(4-(morpholinomethyl)phenyl)-
5-fluoropyrimidine (9k, Method u in Scheme 4). A mixture of
chloropyrimidine 2k (0.100 g, 0.387 mmol), 4-(morpholinomethyl)-
aniline (0.087 g, 0.452 mmol), X-Phos (2-dicyclohexylphosphino-
2′,4′,6′-triisopropylbiphenyl) (0.019 g, 0.0387 mmol), bis-
(dibenzylideneacetone)palladium(0) (0.022 g, 0.0387 mmol), potas-
sium carbonate (0.117 g, 0.851 mmol) in t-BuOH (3.0 mL) was heated
under reflux for 18 h. The solvent was evaporated from the resulting dark
solution. The dark residue was purified by chromatography (SiO2, ethyl
acetate, and hexanes) to provide the title compound 9k (0.062 g, 39%)
as an off-white solid, mp 142−144 °C. HPLC 98% (tR = 23.4 min, 50%
1
7.07 (s, 1H); 1H NMR (400 MHz, DMSO-d6 + D2O) δ 8.09 (d, JH−F
=
MeOH, 50% water (with 0.1% TFA), 36 min); H NMR (400 MHz,
DMSO-d6) δ 9.16 (s, 1H), 9.10 (s, 1H), 8.07 (d, JH−F = 2.6 Hz, 1H), 7.58
(t, J = 6.8 Hz, 2H), 7.42−7.34 (m, 3H), 7.31 (t, J = 8.2 Hz, 1H), 6.94 (d, J
= 7.7 Hz, 2H), 3.52 (s, 4H), 2.26 (s, 4H); 13C NMR (100 MHz, DMSO-
3.5 Hz, 1H), 7.58 (dd, J = 7.9, 1.5 Hz, 1H), 7.54−7.48 (m, 3H), 7.44−
7.39 (m, 3H), 7.35 (dt, J = 7.4, 1.7 Hz, 1H); 19F NMR (376 MHz,
DMSO-d6 + D2O), δ −164.56 (d, J = 3.1 Hz); 13C NMR (100 MHz,
DMSO-d6) 155.67 (d, JC−F = 2.8 Hz), 151.57, 151.46, 144.66, 142.73,
141.79, 141.60, 140.27, 136.06, 135.87, 131.65, 130.42, 130.19, 128.42,
126.85, 117.46; LC−MS (ESI+) m/z 394.05 (M + H)+; HRMS (ESI+)
m/z calculated for C16H14ClFN5O2S (M + H)+ 394.0535, found
394.0537.
d6), δ 156.22 (d, JCF = 2.8 Hz), 151.27 (d, JCF = 11.4 Hz), 141.15 (d, JCF
=
245 Hz), 141.62 (d, JCF = 18.9 Hz), 140.44, 136.11, 131.08, 130.33,
130.26, 129.73, 129.52, 128.26, 128.01, 118.57, 66.84, 62.79, 53.74; 19F
NMR (376 MHz, DMSO-d6), δ −166.63 (s); LC−MS (ESI+) m/z
327.08 (M − morpholine); HRMS (ESI+) m/z calculated for
C21H22ClFN5O (M + H)+ 414.1491, found 414.1497 (M + H)+,
327.0819 (M − morpholine).
N4-(2-Chlorophenyl)-N2-[4-(2H-tetrazol-5-yl)phenyl]-
pyrimidine-2,4-diamine Hydrochloride (12a, Method a in
Scheme 5). A mixture of 2l (0.072 g, 0.299 mmol) and 4-(2H-
tetrazol-5-yl)phenylamine (11a) (0.050 g, 0.310 mmol) in EtOH (2.0
mL) was heated in a microwave reactor at 150 °C for 40 min. The
precipitate obtained upon cooling the reaction mixture was filtered and
washed with EtOH (5 mL) to provide 12a (0.095 g, 79%) as a yellow
solid (1H NMR analysis indicated the presence of trace impurities). The
product was further purified by washing with methanol (5 mL) to give
pure 12a (0.045 g, 37%) as a yellow solid, mp 250 °C (dec). HPLC
100% (tR = 6.21 min, 50% MeOH in 0.1% TFA in water, 20 min); 1H
NMR (400 MHz, DMSO-d6) δ 10.76 (s, 1H), 10.64 (s, 1H), 8.09 (d, J =
6.9 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.69−7.59 (m, 4H), 7.48 (t, J = 7.4
Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 6.52 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 163.07, 153.08, 146.47, 140.76, 134.74, 130.64, 130.17,
129.48, 129.32, 129.31, 128.57, 128.21, 121.13, 119.58. Anal. Calcd for
C17H14Cl2N8: C, 50.89; H, 3.52; N, 27.93. Found: C, 51.22; H, 3.50; N,
27.54. LC−MS (ESI−) m/z 363.08 (M − H − HCl)−; HRMS (ESI−)
m/z calculated for C17H12ClN8 (M − H − HCl)− 363.0879, found
363.0871.
N4 -(2-Chlorophenyl)-N2 -[4-N-(2-hydroxyethyl)-
carbamoylphenyl]pyrimidine-2,4-diamine (9l, Method t in
Scheme 4). A mixture of chloropyrimidine 2l (0.100 g, 0.416 mmol)
and 4-amino-N-(2-hydroxyethyl)benzamide (0.075 g, 0.416 mmol) in
HCl (1 mL of 0.1 N aqueous) was heated in a microwave reactor at 140
°C for 30 min. The contents were then stirred in the microwave vial at
room temperature for 30 min. The mixture was filtered, and the
obtained solid was washed with water (20 mL) and sodium bicarbonate
(saturated aqueous 50 mL). The crude product was purified by
chromatography (silica gel, hexane/ethyl acetate) to provide 9l as an off-
white solid (0.087 g, 54%), mp 171−173 °C. HPLC 100% (tR = 8.8 min,
1
40% MeOH, 60% water (with 0.1% TFA), 20 min); H NMR (400
MHz, MeOH-d4) δ 8.28 (t, J = 6.2 Hz, 1H, peak disappeared upon D2O
shake), 8.00 (d, J = 5.9 Hz, 1H), 7.84 (dd, J = 8.0, 1.5 Hz, 1H), 7.71−7.64
(m, 4H), 7.50 (dd, J = 8.0, 1.5 Hz, 1H), 7.37 (td, J = 8.0, 1.5 Hz, 1H),
7.22 (td, J = 8.0, 1.5 Hz, 1H), 6.30 (d, J = 5.9 Hz, 1H), 3.70 (t, J = 5.5 Hz,
2H), 3.49 (q, J = 5.5 Hz, 2H); 13C NMR (100 MHz, DMSO-d6), δ
172.68, 166.63, 161.90, 159.62, 156.84, 144.10, 136.56, 130.31, 128.61,
128.27, 128.07, 126.92, 126.82, 117.93, 99.18, 60.56, 42.68; LC−MS
(ESI+) m/z 384.12 (M + H)+; HRMS (ESI+) m/z calculated for
C19H19ClN5O2 (M + H)+ 384.1222, found 384.1219.
N4-(2-Chlorophenyl)-N2-[4-(2H-tetrazol-5-yl)phenyl]-5-fluo-
ropyrimidine-2,4-diamine Hydrochloride (12b, Method a in
Scheme 5). A mixture of 2k (free base) (0.076 g, 0.298 mmol) and 4-
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(2H-tetrazol-5-yl)phenylamine (11a) (0.050 g, 0.310 mmol) in EtOH
(2.0 mL) was heated in a microwave reactor at 170 °C for 40 min. The
precipitate obtained upon cooling the reaction mixture was filtered and
washed with EtOH (5.0 mL) to get 12b (0.090 g, 72%) as a brown-
yellow solid. The 1H NMR spectrum showed the presence of a baseline
impurity, and the product was further purified by washing with methanol
(5 mL) to provide pure 12b (0.041 g, 33%) as a brown-yellow solid, mp
223 °C (dec). HPLC 99% (tR = 10.37 min, 50% MeOH in 0.1% TFA in
water, 20 min); 1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.91 (s,
1H), 8.25 (d, J = 4.0 Hz, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.64 (d, J = 7.8
Hz, 1H), 7.62−7.56 (m, 3H), 7.50−7.38 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 155.74 (d, J = 3.0 Hz), 151.50 (d, J = 11.7 Hz), 144.37,
141.45 (d, J = 247.45 Hz), 141.68 (d, J = 17.6 Hz), 136.06, 131.58,
130.43, 130.15, 128.41, 127.85, 118.47; 19F NMR (376 MHz, DMSO) δ
−165.12 (s). Anal. Calcd for C17H13Cl2FN8: C, 48.70; H, 3.13; N, 26.73.
Found: C, 49.09; H, 3.13; N, 26.48. LC−MS (ESI−) m/z 381.1 (M −
H)−; HRMS (ESI−) m/z calculated for C17H11ClFN8 (M − H)−
381.0785, found 381.0784.
AUTHOR INFORMATION
■
Corresponding Author
*For H.R.L.: phone, +813-745-6076; +813-745-6748; e-mail,
Harshani.Lawrence@moffitt.org. For N.J.L.: phone, +813-745-
6037; fax, +813-745-6748; e-mail, Nicholas.Lawrence@moffitt.
org.
Author Contributions
#These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Chemical Biology Core at the Moffitt Cancer
Center and Research Institute for assistance with the Z-lyte
kinase assay (Dr. Richard Yip, HTS Unit), mass spectrometry,
and HPLC analysis (Chemistry Unit). We also thank the Moffitt
Structural Biology Core for use of the X-ray crystallography
facility. We thank Drs. Gary Daughdrill and Hongwei Wu for
access to the 600 MHz NMR facility and acknowledge that this
work has been supported in part by the Florida Center of
Excellence for Drug Discovery and Innovation at the University
of South Florida.
N4-(2-Chlorophenyl)-N2-[3-hydroxy-4-(2H-tetrazol-5-yl)-
phenyl]-5-fluoropyrimidine-2,4-diamine Hydrochloride (12c,
Method a in Scheme 5). A mixture of 2k (freebase) (0.072 g, 0.279
mmol) and tetrazole 11b (0.050 g, 0.282 mmol) in EtOH/HCl (1:1, 2.0
mL, 0.1 M HCl) was heated in a microwave reactor at 160 °C for 40 min.
The precipitate obtained upon cooling was filtered and rinsed with
EtOH (5 mL) to obtain the product with baseline impurity (0.096 g,
77%) as a yellow solid. This solid was further purified by washing with
methanol (5 mL) to provide pure 12c (0.044 g, 36%) as a yellow solid,
mp 214 °C (dec). HPLC 93% (tR = 12.99 min, 50% MeOH in 0.1% TFA
in water, 20 min); 1H NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H), 9.44
(s, 1H), 9.24 (s, 1H), 8.13 (d, J = 3.3 Hz, 1H), 7.66−7.54 (m, 4H), 7.41
(t, J = 7.5 Hz, 1H), 7.33 (t, J = 7.4 Hz, 1H), 7.29−7.23 (m, 2H); 13C
NMR (100 MHz, DMSO-d6) 156.34, 155.80 (d, J = 3.1 Hz), 151.46 (d, J
= 11.5 Hz), 145.58, 141.61 (d, J = 247.2 Hz), 141.23 (d, J = 19.1 Hz),
135.90, 131.04, 130.42, 129.71, 129.25, 128.38, 128.20, 110.49, 105.32,
103.39; 19F NMR (376 MHz, DMSO-d6) δ −165.94 (s); LC−MS
(ESI−) m/z 397.07 (M − H)−; HRMS (ESI−) m/z calculated for
C17H11ClFN8O (M − H)− 397.0734, found 397.0730.
ABBREVIATIONS USED
■
CML, chronic myeloid leukemia; DCM, dichloromethane; DEA,
diethylamine; DMEM, Dulbecco’s modified Eagle’s medium; EI,
electrospray ionization; ECL, enhanced chemiluminescence;
EIF2AK3, eukaryotic translation initiation factor 2α kinase 3;
GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GSK3β,
glycogen synthase kinase 3β; HPLC, high performance liquid
chromatography; HTS, high throughput screening; HRP,
horseradish peroxidase; JAK2, Janus kinase 2; LC−MS, liquid
chromatography−mass spectrometry; PBS, phosphate buffered
saline; PIM3, provirus integration site for Moloney murine
leukemia virus; NUAK1, SnF1-like kinase; SAR, structure−
activity relationship; TLC, thin layer chromatography; TFA,
trifluoroacetic acid
N4-(2-Chlorophenyl)-N2-[4-(2H-tetrazol-5-yl)phenyl]-5-chlor-
opyrimidine-2,4-diamine Hydrochloride (12d, Method a in
Scheme 5). A mixture of chloropyrimidine 2o (0.093 g, 0.3 mmol)
and 4-(2H-tetrazol-5-yl)phenylamine (11a) (50 mg, 0.3 mmol) in
EtOH (2 mL) was heated in a microwave reactor at 150 °C for 40 min.
The precipitate obtained upon cooling was filtered and washed with
EtOH (5 mL) to provide impure 12d (105 mg, 81%) as a yellow color
solid. Analysis of the 1H NMR spectrum indicated the presence of trace
impurities. The product was further purified by washing with hot ethyl
acetate (5 mL) and hot methanol (5 mL) to provide pure 12d (0.032 g,
25%) as a yellow solid, mp 238 °C (dec). HPLC 91% (tR = 5.71 min,
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1
70% MeOH in 0.1% TFA in water, 20 min); H NMR (400 MHz,
DMSO-d6) δ 9.72 (s, 1H), 9.05 (s, 1H), 8.19 (s, 1H), 7.71−7.56 (m,
6H), 7.52−7.38 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 157.73,
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(ESI−) m/z calculated for C17H11Cl2N8 (M − H)− 397.0489, found
397.0472.
ASSOCIATED CONTENT
■
S
* Supporting Information
(i) Synthetic protocols and characterization data for 2a−o, 5a−k,
7a,b, 8a,b, 10a,b and 11a,b; (ii) scanned NMR spectra and
HPLC and LC−MS reports for 1, 3l, 3n, 3o, 6c, 6i, 9h, 9m, 9n,
and 12a; (iii) determination of co-crystal structures of 1, 3i, 3g,
13, and ADP with Aurora A; (iv) kinase profiling of 3o; (v) IC50
data (Reaction Biology) for 9h, 3l, 3o against Aurora A and
JAK2; (vi) Aurora A IC50 data of compounds 13−24. This
material is available free of charge via the Internet at http://pubs.
acs.org.
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Journal of Medicinal Chemistry
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