Synthesis and SAR of 6-chloro-4-fluoroalkylamino-2-heteroaryl-5-(substituted)phenylpyrimidines as anti-cancer agents

Article abstract of DOI:10.1016/j.bmc.2008.11.016

The synthesis and SAR of a series of 6-chloro-4-fluoroalkylamino-2-heteroaryl-5-(substituted)phenylpyrimidines as anti-cancer agents are described. This series of 2-heteroarylpyrimidines was developed by modifying a series of anti-tumor [1,2,4]triazolo[1,5-a]pyrimidines and 2-cyanoaminopyrimidines we reported earlier. For the 2-heteroaryl group, the best activity is obtained when the heteroaryl group has a nitrogen atom at the ortho-position to the pyrimidyl core. The structure-activity relationship for the rest of the molecule in this 2-heteroarylpyrimidine series mimics that of the [1,2,4]triazolo[1,5-a]pyrimidine series. Like triazolopyrimidines and 2-cyanoaminopyrimidines, the 2-heteroarylpyrimidines retain the capability to overcome multidrug resistance due to Pgp. Mechanism of action studies showed that the lead compounds behaved in the same manner as triazolopyrimidines and 2-cyanoaminopyrimidines. The lead compounds in this series are more potent than the corresponding triazolopyrimidines in vitro and in vivo. Compound 21 (PTI-868) showed tumor growth inhibition in several nude mouse xenograft models, and was selected to advance to preclinical development.

Full text of DOI:10.1016/j.bmc.2008.11.016

Bioorganic & Medicinal Chemistry 17 (2009) 111–118
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and SAR of 6-chloro-4-fluoroalkylamino-2-heteroaryl-
5-(substituted)phenylpyrimidines as anti-cancer agents
a
a
b
b
Nan Zhang a, , Semiramis Ayral-Kaloustian , Thai Nguyen , Richard Hernandez , Judy Lucas ,
*
Carolyn Discafani b, Carl Beyer b
a Chemical and Screening Sciences, Wyeth Research, 401 North Middletown Road, Pearl River, New York 10965, USA
b Discovery Oncology, Wyeth Research, 401 North Middletown Road, Pearl River, New York 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and SAR of a series of 6-chloro-4-fluoroalkylamino-2-heteroaryl-5-(substituted)phenylpyr-
imidines as anti-cancer agents are described. This series of 2-heteroarylpyrimidines was developed by
modifying a series of anti-tumor [1,2,4]triazolo[1,5-a]pyrimidines and 2-cyanoaminopyrimidines we
reported earlier. For the 2-heteroaryl group, the best activity is obtained when the heteroaryl group
has a nitrogen atom at the ortho-position to the pyrimidyl core. The structure–activity relationship for
the rest of the molecule in this 2-heteroarylpyrimidine series mimics that of the [1,2,4]triazolo[1,5-
a]pyrimidine series. Like triazolopyrimidines and 2-cyanoaminopyrimidines, the 2-heteroarylpyrimi-
dines retain the capability to overcome multidrug resistance due to Pgp. Mechanism of action studies
showed that the lead compounds behaved in the same manner as triazolopyrimidines and 2-cyanoami-
nopyrimidines. The lead compounds in this series are more potent than the corresponding triazolopyrim-
idines in vitro and in vivo. Compound 21 (PTI-868) showed tumor growth inhibition in several nude
mouse xenograft models, and was selected to advance to preclinical development.
Received 6 October 2008
Revised 6 November 2008
Accepted 7 November 2008
Available online 14 November 2008
Keywords:
6-Chloro-4-fluoroalkylamino-2-heteroaryl-
5-(substituted)phenylpyrimidines
Anti-cancer agents
Microtubule-stabilizing activity
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
poxy]phenyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazol-
o[1,5-a]pyrimidin-7-amine (TTI-237),18,19 the lead compound in
Anti-cancer agents that bind tubulin have shown great promise
in cancer treatments and have attracted much interest in chemo-
therapy development.1,2 These agents are categorized depending
on their mechanism of action on the microtubule dynamics and
on their binding sites on tubulin. Most of these agents inhibit poly-
merization of tubulin to microtubules, and they are further catego-
rized as vinca alkaloid site binders and colchicines site binders.3
Taxoids, on the other hand, promote tubulin polymerization and
stabilize microtubules.4 Later, several other natural products, for
example, epothilones,5 discodermolide,6 and eleutherobin,7 were
discovered that bind at the taxoid site and show taxoid-like micro-
tubule-stabilizing activity.8–10 Other natural products, for example,
laulimalide11 and peloruside,12 however, show taxoid-like micro-
tubule-stabilizing activity, but do not bind at the taxoid site.13,14
Laulimalide and peloruside are synergistic with paclitaxel, but
not with each other.15–17
this series, entered Phase I clinical trials. We then modified the triaz-
olopyrimidine core 1 by N1-methylation, followed by opening of the
five-membered triazolo ring (e.g., 2). A series of 2-cyanamidopyrim-
idines20 (e.g., 3) was thus obtained and shown to retain in vitro po-
tency observed with the triazolopyrimidines. We then further
modified the series by mimicking the 2-cyanamido group with more
stable and pharmaceutically acceptable heteroaryls (4, Scheme 1).
We now report synthesis and structure–activity relationship of this
series of 6-chloro-4-fluoroalkylamino-2-heteroaryl-5-(substi-
tuted)phenylpyrimidines as anti-cancer agents.21
CF3
H
N
H
CH3
F
O
NH
N
N
N
F
N
Cl
TTI-237
Recently, we reported the synthesis and structure–activity rela-
tionship of a series of triazolopyrimidines 1, which act as anti-cancer
agents by interfering with the microtubule dynamics in a unique
manner.18,19
5-Chloro-6-{2,6-difluoro-4-[3-(methylamino)pro-
2. Chemistry
Compounds 8–26 were prepared,22 as shown in Scheme 2.
Cyclization of diethyl 2,4,6-trifluorophenylmalonate 523 with car-
boxamidine 6, followed by chlorination with POCl3 provided
* Corresponding author. Tel.: +1 845 602 3425; fax: +1 845 602 5561.
E-mail address: zhangn@wyeth.com (N. Zhang).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.016

112
N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 111–118
CF3
CF3
CF3
CF3
H
R
F
F
F
F
H
R
H
R
H
R
F
F
F
F
NH
NH
N
NH
1-Methylation
New Core
Base
N
N
N
N
N
N
N
N
F
Cl
F
F
F
Cl
N
Het
N
N
N
Cl
N
Cl
N
Het = Heteroaryl
1
CN
2
3
4
Scheme 1. From [1,2,4]triazolo[1,5-a]pyrimidines to 2-heteroarylpyrimidines.
Table 2
F
EtO2C CO2Et
F
Inhibition of COLO 205 cell proliferation by compounds 21–26, 32, and 33
Cl
N
NH
F
F
a
+
CF3
F
N
H2N Het
H
OR'
F
NH
R
Het
H
Cl
6
F
5
7
N
F
Het
N
Cl
CF3
CF3
F
F
H
F
OR'
21-26,32, and 33
NH
R
NH
R
c
b
N
Compounda
R
Het
R0
IC50 (nM)b
N
F
F
Het
N
Cl
Het
N
Cl
21
CH3
CH3
CH3
H
H
H
H
H
H
CH3
2-Pyrazinyl
2-Pyrazinyl
2-Pyridinyl
2-Pyrimidinyl
2-Pyrazinyl
2-Pyrazinyl
2-Pyrazinyl
2-Pyrazinyl
1-Imidazolyl
1-Pyrazolyl
—(CH2)3NHCH3
—(CH2)3NHCH3
—(CH2)3NHCH3
—(CH2)3N(CH3)2
—(CH2)3N(CH3)2
—(CH2)3N(CH3)2
—(CH2)2N(CH3)2
—(CH2)4N(CH3)2
—(CH2)3N(CH3)2
—CH2)3NHCH3
6.9 2.2
8.9 0.7
5.2 1.3
49 (n = 1)
21c
8-20
21-26
22c
8,9: R = CH3
10-20: R = H
21,22: R = CH3
23-26: R = H
23
24
22
18
5
3
24c
Scheme 2. Synthesis of 8–26. Reagents and conditions: (a) i—tributylamine, 180 °C;
25
26
128 47
32 14
682 232
2.4 0.3
ii—POCl3, reflux; (b) CF3CH(R)NH2, DMF; (c) R0OH, NaH, DMSO.
32
33c
Paclitaxel
Vincristine
3.3
2.6 0.5
1
dichloropyrimidine 7. Replacement of one of the two equivalent
chloro groups with fluoroalkylamines yielded the desired com-
pounds 8–20 (Table 1). Compounds 21–26 (Table 2) were prepared
by further treatment of compounds 8–11 with the corresponding
alcohols, in the presence of sodium hydride in DMSO. As in the
triazolopyrimidine series, the 4-fluoro group on the phenyl ring
a
Unless indicated otherwise, compounds were tested as the free base.
Concentration needed to inhibit cell growth by 50% as determined from the
b
dose–response curve. Determinations of each IC50 value were conducted at 10
concentrations, and in triplicate. Triplicate values at each concentration agreed, on
average, within 10%.
c
Compounds were tested as the HCl salts.
Table 1
could be replaced cleanly in the presence of the 2- and 6-fluoro
groups.
Inhibition of COLO 205 cell proliferation by compounds 8–20 and 29–31
CF3
F
Compounds 29–33 (Tables 1 and 2), in which the five-mem-
bered heteroaryl groups are connected to the pyrimidine core
through a C–N bond, were prepared as shown in Scheme 3. Cycli-
zation of diethyl 2,4,6-trifluorophenylmalonate 5 with urea, fol-
lowed by chlorination with POCl3, provided trichloropyrimidine
27. Replacement of the 2-chloro group with the N–H containing
five-membered heteroaryl groups yielded compound 28. Further
H
F
NH
R
N
F
Het
N
Cl
8-20 and 29-31
Compound
R
Het
IC50 (nM)a
8
9
10
11
12
13
14
15
16
17
18
19
20
29
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
2-Pyrazinyl
2-Pyridinyl
2-Pyrazinyl
2-Pyrimidinyl
4-Pyridinyl
3-Pyridinyl
2-Pyridinyl
2-Quinolinyl
1-Isoquinolinyl
3-Isoquinolinyl
2-Thienyl
51
29
84 11
265 (n = 1)
>5000 (n = 2)
3930 373
39
250 23
4680 251
119
2
6
F
F
EtO2C CO2Et
F
F
Cl
N
Cl
N
F
F
b
a
N
N
F
F
8
Het
F
Cl
Cl
Cl
d
F
5
28
27
CF3
4
CF3
F
H
R
H
OR'
3465 (n = 1)
232 (n = 1)
77 22
F
NH
NH
R
2-Furyl
2-(1-Methylimidazolyl)
1-Imidazolyl
c
N
N
H
3030 111
F
F
Het
N
Cl
32, 33
32: R = H
33: R = CH3
Het
N
Cl
30
31
H
CH3
1-Pyrazolyl
1-Pyrazolyl
53
19
3.3
5
6
1
29-31
Paclitaxel
Vincristine
29, 30: R = H
31: R = CH3
2.6 0.5
a
Concentration needed to inhibit cell growth by 50% as determined from the
dose–response curve. Determinations of each IC50 value were conducted at 10
concentrations, and in triplicate. Triplicate values at each concentration agreed, on
average, within 10%.
Scheme 3. Synthesis of 29–33. Reagents and conditions: (a) i—urea, NaH, EtOH,
80 °C, ii—POCl3, reflux; (b) Het-H, K2CO3, DMF; (c) CF3CH(R)NH2, N,N-diisopropyl-
ethylamine, DMF; (d) R0OH, NaH, DMSO.

N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 111–118
113
replacement of one of the two remaining equivalent chloro groups
with the fluoroalkylamines yielded the desired compounds 29–31.
Compounds 32 and 33 (Table 2) were prepared by further treat-
ment of compounds 29 and 31 with the corresponding alcohols,
in the presence of sodium hydride in DMSO.
To confirm the regiochemistry from 27 to 28, an alternative
synthesis22 of 29 was carried out (Scheme 4). Cyclization of
diethyl 2,4,6-trifluorophenylmalonate 5 with thiourea provided
34. Selective methylation of the mercapto group, followed by
chlorination with POCl3 yielded 36. Replacement of one of the
two equivalent chloro groups with 2,2,2-trifluoroethylamine gave
37. Oxidation of the thiomethyl group with mCPBA led to a meth-
ylsulfonyl group (in 38) that was replaced by imidazole to get 29,
which was spectroscopically identical with the material obtained
from Scheme 3.
ophenyl analogs (in Table 1). A 4- to 8-fold increase in potency
is achieved with a three-methylene unit tether (21 vs 8, 22 vs
9, 23 vs 11, 24 vs 10, 32 vs 29, and 33 vs 31). A three-methylene
unit is optimal for potency. Compound 26, with a four-methylene
unit, is less potent than compound 24, while compound 25, with
a two-methylene unit, shows further decreased potency. This
observation is in agreement with SAR observed in the
[1,2,4]triazolo[1,5-a]pyrimidine18 and the 2-cyanoaminopyrimi-
dine series.20
Compounds 21, 22, and 33 were further studied in the mecha-
nism of action studies18–20 and were shown to behave in the same
manner as was observed with the [1,2,4]triazolo[1,5-a]pyrimi-
dines and 2-cyanoaminopyrimidines.18–20 They promoted tubulin
polymerization using either MAP-rich tubulin or pure tubulin,
and did not cause microtubule depolymerization in vitro, a phe-
nomenon similar to the effect of paclitaxel. Competitive binding
studies with [H]vinblastine, [3H]colchicine, and [3H]paclitaxel
showed that, like their [1,2,4]triazolo[1,5-a]pyrimidine counter-
parts,18,19 these phenylpyrimidines did not inhibit the binding of
[3H]colchicine or [3H]paclitaxel effectively. Instead, they inhibited
the binding of [3H]vinblastine.19 These findings strongly suggest
that 2-heteropyrimidines and [1,2,4]triazolo[1,5-a]pyrimidines
bind at the same site of tubulin with the same specific binding
mode. Cell cycle analysis of compounds 21, 22, and 33 showed
apoptosis induction at low compound concentration and G2/M
block at high compound concentration, a pattern that is similar
to what was observed with [1,2,4]triazolo[1,5-a]pyrimidines19
and paclitaxel.24
3. Results and discussion
Compounds 8–26 and 29–33 were evaluated in the COLO 205
cytotoxicity assay18,19 for their ability to inhibit cancer cell prolif-
eration. As shown in Table 1, for the heteroaryl group attached to
the 2-position of the pyrimidine ring, the best activity is obtained
when the heteroaryl group has a nitrogen atom at the position
ortho- to the pyrimidyl core. With such a nitrogen atom, both a
five-membered ring and a six-membered ring can render high po-
tency. Thus, the highest potency was observed with a 2-pyrazinyl
(8 and 10), a 2-pyridinyl (9 and 14), a 1-pyrazolyl (30 and 31), or
a 2-(1-methylimidazolyl) (20). Interestingly, compound 11, with
a 2-pyrimidinyl ring and thus two nitrogen atoms ortho- to the
pyrimidyl core (at the 2- and 6-positions), was less potent than
compound 10. Compounds that lack a nitrogen atom at the posi-
tion ortho- to the pyrimidyl core (12, 13, 18, 19, and 29) showed
much lower potency. We further modified the 2-pyridyl group by
fusing an additional phenyl ring in all three possible orientations
(15–17). The potency for these isomeric analogs varies greatly.
The most potent compound among the three, 17, is slightly less po-
tent than 10, suggesting there is space in the binding pocket to
accommodate substituents at the 4- and 5-positions on the pyridyl
ring. As a head-piece, (S)-2,2,2-trifluoro-1-methylethylamine (as in
the triazolopyrimidines) seems to provide higher potency than ob-
served with 2,2,2-trifluoroethylamine (8 vs 10, 9 vs 14, and 31 vs
30).
Like the [1,2,4]triazolo[1,5-a]pyrimidines18,19 and 2-cyanoami-
nopyrimidines,20 compounds 21, 22, 24, and 33 were able to over-
come resistance due to P-glycoprotein (Pgp). As shown in Table 3,
Table 3
Inhibition of KB, KB 8.5 and KB V1 cell proliferation by compounds 21, 22, 24, and 33
Compounda
IC50 (nM)
KB 8.5
Ratioc
KB 8.5/KB
b
KB
KB V1
KB V1/KB
21
22
24
33
7.0 0.5
6.2 0.4
24
21
24
32
26 0.1
58 16
1
3
2
4
405 27
423 19
3.4
3.4
1.2
13
10
26
58
68
5.0
237
805
925
20
3
99
4
2.4 0.1
2.5 0.1
2.2 0.6
569 36
2013 108
2035 25
Paclitaxel
Vincristine
Selected compounds in Table 1 were further modified by
replacing the 4-fluoro group with methylamino alkylalcohols, a
strategy proven successful in the triazolopyrimidine series.18
Comparable IC50 values were obtained when a given compound
was tested either as a free base or as a hydrogen chloride salt.
As shown in Table 2, compounds with proper methylamino alkyl-
alcohol tails are more potent than their corresponding trifluor-
a
Compounds were tested as the HCl salts.
Concentration needed to inhibit cell growth by 50% as determined from the
b
dose–response curve. Determinations of each IC50 value were conducted at 10
concentrations, and in duplicate. Duplicate values at each concentration agreed, on
average, within 10%.
c
Ratio = IC50 on KB 8.5 or KB V1 cells/IC50 on KB cells. A ratio of about 1 indicates
no resistance.
F
F
F
F
EtO2C CO2Et
F
F
OH
N
OH
N
Cl
F
F
a
b
c
N
N
N
F
F
f
F
HS
OH
e
S
OH
S
F
N
Cl
F
5
34
35
36
CF3
CF3
CF3
F
F
H
H
H
H
H
F
F
F
NH
NH
NH
H
d
N
N
N
F
F
F
S
N
N
Cl
N
Cl
S
N
Cl
N
O2
37
38
29
Scheme 4. Alternative synthesis of 29. Reagents and conditions: (a) thiourea, n-Bu3N, 150 °C; (b) CH3I, NaOH; (c) POCl3, reflux; (d) CF3CH2NH2, N,N-diisopropylethylamine,
DMF; (e) mCPBA; (f) Imidazole, K2CO3, DMF.

114
N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 111–118
these compounds showed relatively low IC50 ratios between the KB
line and KB 8.5 and KB V1 lines. The KB lines25 express different
amounts of the Pgp membrane pump which causes resistance to
the action of many cytotoxic compounds, including paclitaxel
and vincristine, via enhanced efflux from the cell. The parental
KB line has very little or no expression of Pgp, KB 8.5 expresses
moderate levels of the protein, and KB V1 expresses artificially
high levels. The change in IC50 values in these lines reflects the
ability of Pgp to recognize and export a potential drug agent. Com-
pounds 21, 22, 24, and 33 showed much lower resistance ratios,
and thus much lower levels of recognition by Pgp than paclitaxel
and vincristine.
25
20
15
10
5
The lead compound in this series, compound 21, has very desir-
able pharmaceutical properties. Its water solubility at pH 7.4 is
l
g/mL (measured by shake flask method26), enough for dosing
0
540
directly in saline with no special formulation needed. The meta-
bolic stability of PTI-868 (1 M) was determined using liver micro-
0
5
10
15
20
25
Day
l
somes obtained from female nude mice, male CD-1 mice, male and
female Sprague–Dawley rats, male beagle dogs, male cynomolgus
monkeys and male humans (1 mg/mL). Incubations were con-
ducted for 30 min at 37 °C in the presence of NADPH. It is metabol-
ically stable in rat, dog, monkey, human and female nude mouse
liver microsomes, with t1/2 values greater than 60 min. The phar-
macokinetic parameters were determined using a non-compart-
mental analysis module (Model 201 and 202) of the
pharmacokinetic software package WinNonlin, ver. 3.1 (Pharsight,
CA). The program applies a model-independent approach and the
standard methods described by Gibaldi and Perrier.27 In PK studies
in rat, the compound showed a high volume of distribution (14 L/kg),
moderate clearance (21 mL/min/kg), long half-life (8.6 h), and high
bioavailability (ꢀ100%).
Vehicle Control
6 mg/kg
5 mg/kg
3 mg/kg
2 mg/kg
1 mg/kg
4 mg/kg
Figure 2. In vivo anti-tumor activity with Compound 21 against Lox melanoma.
The compound was administered to tumor-bearing mice on days 1, 5 and 9 after
staging (indicated by arrows) by intravenous injection. The control group received
vehicle only by iv injection. Vehicle was 0.9% saline. Each group contained five
animals. Relative tumor growth = mean tumor mass on day d/mean tumor mass of
that group on day 0. Three animals died in the 6 mg/kg group, and two died in each
of the 5 mg/kg and 4 mg/kg groups.
In in vivo xenograft studies, compound 21 showed good efficacy
against several human tumor models, including H460 non-small
cell lung carcinoma (Fig. 1) and human Lox melanoma (Fig. 2).
The compound was selected to advance to preclinical develop-
ment, based on its pharmaceutical properties and efficacies in
in vivo xenograft studies.
4. Conclusion
By modifying a series of [1,2,4]triazolo[1,5-a]pyrimidine anti-
tumor agents, we developed a series of 2-heteroarylpyrimidines
as anti-cancer agents. For the 2-heteroaryl group, the best activ-
ity is obtained when the heteroaryl group has a nitrogen atom
at the position ortho- to the pyrimidyl core. The structure–activ-
ity relationship for the rest of the molecule in this 2-heteroaryl-
pyrimidine series parallels that of the [1,2,4]triazolo
[1,5-a]pyrimidine and 2-cyanoaminopyrimidine series. Like
triazolopyrimidines and 2-cyanoaminopyrimidines, the 2-hetero-
arylpyrimidines have the capability to overcome multidrug
resistance due to Pgp. The lead compounds in this 2-heteroaryl-
pyrimidine series promote tubulin polymerization and inhibit
microtubule depolymerization in vitro. They inhibit binding of
[3H]vinblastine, but not [3H]colchicine or [3H]paclitaxel, as was
observed with the triazolopyrimidines and 2-cyanoaminopyrim-
idines. These findings strongly suggest that this series of 2-het-
eroarylpyrimidines binds at the same site and in the same
binding mode as the triazolopyrimidines and 2-cyanoaminopyr-
imidines do. The lead compounds in this series are more potent
than the corresponding triazolopyrimidines in vitro and in vivo.
Compound 21 showed tumor growth inhibition in several nude
mouse xenograft models, and was selected to advance to pre-
clinical development.
3000
2500
2000
1500
1000
500
0
0
2
4
6
8
10
12
14
Day
Vehicle Control
10 mg/kg
5 mg/kg
2.5 mg/kg
5. Experimental
7.5 mg/kg
5.1. Chemistry: general
Figure 1. In vivo anti-tumor activity with Compound 21 against H460 human non-
small cell lung carcinoma (NSCLC). The compound was administered to tumor-
bearing mice on days 0 and 7 after staging (indicated by arrows) by oral gavage. The
control group received vehicle only (Klucel) by iv injection. Each group contained
nine animals. One animal died in the 10 mg/kg group.
All reactions were conducted under nitrogen atmosphere with
magnetic stirring. Chromatographic purifications were performed
using Baker 40-lm silical gel. Melting points were determined in
open capillary tubes on a Meltemp melting point apparatus and

N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 111–118
115
are uncorrected. 1H NMR spectra were recorded with a Bruker
5.5. 6-Chloro-2-pyridin-2-yl-N-[(1S)-2,2,2-trifluoro-1-methy
Avance 400 spectrometer at 400 MHz. Chemical shifts are quoted
in parts per million from internal standard tetramethylsilane. Mass
spectra were obtained using a Micromass LCT mass spectrometer
operating at 20 eV. Elemental analyses were performed on a Per-
kin-Elmer 2400 CHN analyzer instrument.
lethyl]-5-(2,4,6-trifluorophenyl)pyrimidin-4-amine (9)
According to the procedure used to prepare 8, starting from 2-
cyanopyridine, 9 was obtained as a white solid: mp 230–232 °C;
1H NMR (CDCl3) d 1.41 (d, J = 7 Hz, 3H), 4.53 (d, J = 10 Hz, 1H),
5.34 (m, 1H), 6.89 (m, 2H), 7.43 (m, 1H), 7.87 (m, 1H), 8.42 (d,
J = 8 Hz, 1H), 8.85 (m, 1H); MS (ES+): m/z 433.0 (M+1). Anal. Calcd
for C18H11ClF6N4: C, 49.96, H, 2.56, N, 12.95. Found: C, 50.01, H,
2.57, N, 12.77.
5.2. 2-Pyrazinecarboxamidine hydrochloride (6)
To methyl alcohol (20 mL) was added sodium (109 mg,
4.74 mmol) with stirring. After disappearance of the solid, 2-pyr-
azinecarbonitrile (5.0 g, 47.6 mmol) was added. The mixture was
stirred at room temperature for 6 h, and ammonium chloride
(2.8 g, 52.3 mmol) was added. The mixture is then stirred at room
temperature for 18 h. Diethyl ether (50 mL) was added to the reac-
tion mixture, and the precipitates were collected by filtration. The
solid was washed with diethyl ether (2ꢁ) and dried in vacuum
oven to give 6.5 g (86%) of 6 as a hydrochloride salt as a white solid.
MS: m/z 123.1 (M+H).
5.6. 6-Chloro-2-pyrazin-2-yl-N-(2,2,2-trifluoroethyl)-5-(2,4,6-
trifluorophenyl)pyrimidin-4-amine (10)
According to the procedure used to prepare 8, reaction of 7 with
2,2,2-trifluoroethylamine provided 10 in 94% yield as a white solid:
mp 189–190 °C; 1H NMR (DMSO-d6) d 4.35 (m, 2H), 7.48 (t, J = 9 Hz,
2H), 7.97 (t, J = 6 Hz, 1H), 8.84 (m, 2H), 9.57 (s, 1H); MS (ES+): m/z
419.9 (M+1). Anal. Calcd for C16H8ClF6N5: C, 45.79, H, 1.92, N,
16.69. Found: C, 46.07, H, 2.23, N, 16.29.
5.3. 4,6-Dichloro-2-pyrazin-2-yl-5-(2,4,6-trifluorophenyl)pyri-
midine (7)
5.7. 6-Chloro-N-(2,2,2-trifluoroethyl)-5-(2,4,6-trifluorophenyl)-
2,20-bipyrimidin-4-amine (11)
A
mixture of diethyl 2-(2,4,6-trifluorophenyl)malonate23
(870 mg, 3.0 mmol), 2-pyrazinecarboxamidine (6) hydrochloride
(500 mg, 3.15 mmol), and 600 mg of tributylamine was stirred un-
der nitrogen atmosphere at 180 °C for 1 h and cooled to room tem-
perature. The mixture was cooled to room temperature and treated
with 1.0 N hydrochloric acid. The precipitates were collected by fil-
tration, washed with water and dried to give 2-pyrazin-2-yl-5-
(2,4,6-trifluorophenyl)pyrimidine-4,6-diol as a tan solid (401 mg),
which was used directly in the next step.
According to the procedure used to prepare 10, starting from 2-
cyanopyrimidine, 11 was obtained as a tan solid: mp 245–250 °C;
1H NMR (DMSO-d6) d 4.28 (m, 2H), 7.49 (m, 2H), 7.69 (t, J = 5 Hz,
1H), 7.95 (t, J = 6 Hz, 1H), 9.03 (d, J = 5 Hz, 2H); MS (ES+): m/z
420.0 (M+1). Anal. Calcd for C16H8ClF6N5: C, 45.79, H, 1.92, N,
16.69. Found: C, 46.00, H, 1.91, N, 16.59.
5.8. 6-Chloro-2-pyridin-4-yl-N-(2,2,2-trifluoroethyl)-5-(2,4,6-
A mixture of 2-pyrazin-2-yl-5-(2,4,6-trifluorophenyl)pyrimi-
dine-4,6-diol (401 mg) in phosphorous oxychloride (5 mL) and
2,6-lutidine (1 mL) was heated at 110 °C for 16 h. The excess phos-
phorous oxychloride was removed in vacuo, and the resulting res-
idue was dissolved in ethyl acetate. The organic layer was washed
with water and saturated sodium chloride, dried over magnesium
sulfate, and concentrated. The residue was purified by flash chro-
matography over silica gel, eluting with a gradient of 20% ethyl
acetate in hexanes to 33% ethyl acetate in hexanes. Concentration
provided 201 mg (19%) of 7 as a red solid: mp 73–75 °C; 1H NMR
(CDCl3) d 6.89 (m, 2H), 8.78 (d, J = 2 Hz, 1H), 8.85(dd, J = 2, 1 Hz,
1H), 9.76 (d, J = 1 Hz, 1H); MS (ES+): m/z 356.9 (M+1). Anal. Calcd
for C14H5Cl2F3N4: C, 47.09, H, 1.41, N, 15.69. Found: C, 47.48, H,
1.62, N, 15.29.
trifluorophenyl)pyrimidin-4-amine (12)
According to the procedure used to prepare 10, starting from 4-
cyanopyridine, 12 was obtained as a tan solid: mp 180–183 °C; 1H
NMR (CDCl3) d 4.33 (m, 2H), 4.90 (t, J = 6 Hz, 1H), 6.93 (m, 2H), 8.25
(d, J = 4 Hz, 2H), 8.79 (d, J = 4 Hz, 2H); MS (ES+): m/z 419.0 (M+1).
Anal. Calcd for C17H9ClF6N4): C, 48.76, H, 2.17, N, 13.38. Found:
C, 49.03, H, 2.21, N, 13.26.
5.9. 6-Chloro-2-pyridin-3-yl-N-(2,2,2-trifluoroethyl)-5-(2,4,6-
trifluorophenyl)pyrimidin-4-amine (13)
According to the procedure used to prepare 10, starting from 3-
cyanopyridine, 13 was obtained as a dark oil: 1H NMR (CDCl3) d
4.33 (m, 2H), 4.91 (t, J = 6 Hz, 1H), 6.91 (m, 2H), 7.43 (m, 1H),
8.66 (m, 1H), 8.74 (m, 1H), 9.61 (m, 1H); MS (ES+): m/z 419.1
(M+1). Anal. Calcd for C17H9ClF6N4ꢂ0.25EtOAc: C, 49.05, H, 2.52,
N, 12.71. Found: C, 49.18, H, 2.68, N, 12.68.
5.4. 6-Chloro-2-pyrazin-2-yl-N-[(1S)-2,2,2-trifluoro-1-methyleth-
yl]-5-(2,4,6-trifluorophenyl)pyrimidin-4-amine (8)
A mixture of 7 (205 mg, 0.57 mmol), (1 S)-2,2,2-trifluoro-1-
methylethylamine hydrogen chloride28 (298 mg, 2 mmol), and
N,N-diisopropylethylamine (258 mg, 2 mmol) in N,N-dimethyl-
formamide (5 mL) was stirred at 90 °C in a sealed tube for
18 h. The reaction mixture was partitioned between ethyl acetate
and saturated sodium chloride. The organic layer was washed
with saturated sodium chloride (3ꢁ), dried over magnesium sul-
fate, and concentrated. The residue was purified by flash chroma-
tography over silica gel, eluting with a gradient of 20% ethyl
acetate in hexanes to 50% ethyl acetate in hexanes. Concentration
provided 111 mg (45%) of 8 as a light yellow solid: mp 168–
172 °C; 1H NMR (DMSO-d6) d 1.35 (d, J = 7 Hz, 3H), 5.52 (m,
1H), 7.47 (m, 2H), 7.71 (d, J = 9 Hz, 1H), 8.85 (m, 2H), 9.58 (d,
J = 1 Hz, 1H); MS (ES+): m/z 434.1 (M+1). Anal. Calcd for
C17H10ClF6N5: C, 47.08, H, 2.32, N, 16.15. Found: C, 47.14, H,
2.32, N, 16.08.
5.10. 6-Chloro-2-pyridin-2-yl-N-(2,2,2-trifluoroethyl)-5-(2,4,6-
trifluorophenyl)pyrimidin-4-amine (14)
According to the procedure used to prepare 10, starting from 2-
cyanopyridine, 14 was obtained as a white solid: mp > 200 °C; 1H
NMR (CDCl3) d 4.36 (m, 2H), 4.95 (t, J = 6 Hz, 1 H), 6.88 (m, 2H),
7.44 (m, 1H), 7.88 (m, 1H), 8.43 (d, J = 8 Hz, 1H), 8.85 (m, 1H);
MS (ES+): m/z 419.0 (M+1). Anal. Calcd for C17H9ClF6N4: C, 48.76,
H, 2.17, N, 13.38. Found: C, 48.91, H, 2.21, N, 13.23.
5.11. 6-Chloro-2-quinolin-2-yl-N-(2,2,2-trifluoroethyl)-5-(2,4,6-
trifluorophenyl)pyrimidin-4-amine (15)
According to the procedure used to prepare 10, starting from 2-
quinolinecarbonitrile, 15 was obtained as a white solid: mp 260–

116
N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 111–118
264 °C; 1H NMR (CDCl3) d 4.41 (m, 2H), 5.07 (t, J = 6 Hz, 1H), 6.86
(m, 2H), 7.64 (m, 1H), 7.78 (m, 1H), 7.89 (d, J = 8 Hz, 1H), 8.35 (t,
J = 8 Hz, 2H), 8.51 (m, 1H); MS (ES+): m/z 469.0 (M+1). Anal. Calcd
for C21H11ClF6N4ꢂ0.2C6H14: C, 54.86, H, 2.86, N, 11.53. Found: C,
54.81, H, 2.67, N, 11.48.
carbonitrile (1.18 g, 11 mmol) was added. The mixture was stirred
at room temperature for 20 h, and ammonium chloride (588 mg,
11 mmol) was added. The tube was then sealed and stirred at
80 °C for 8 h, and cooled to room temperature. The mixture was fil-
tered, and the filtrate was concentrated. The residue was treated
with 1% methyl alcohol in diethyl ether, and the precipitates were
collected by filtration and dried to give 1.6 g of 1-methyl-2-imidaz-
ole carboxamidine hydrochloride as a gray solid. MS: m/z 125.2
(M+H).
5.12. 6-Chloro-2-isoquinolin-1-yl-N-(2,2,2-trifluoroethyl)-5-
(2,4,6-trifluorophenyl)pyrimidin-4-amine (16)
According to the procedure used to prepare 10, starting from 1-
isoquinolinecarbonitrile, 16 was obtained as a light yellow solid:
mp 225–226 °C; 1H NMR (CDCl3) d 4.28 (m, 2H), 4.98 (t, J = 6 Hz,
1H), 6.92 (m, 2H), 7.62 (m, 1H), 7.74 (m, 1H), 7.80 (d, J = 6 Hz,
1H), 7.92 (d, J = 8 Hz, 1H), 8.49 (d, J = 9 Hz, 1H), 8.70 (d, J = 6 Hz,
1H); MS (ES+): m/z 469.0 (M+1). Anal. Calcd for C21H11ClF6N4: C,
53.80, H, 2.37, N, 11.95. Found: C, 54.17, H, 2.10, N, 11.66.
According to the procedure used to prepare 10, starting from 1-
methyl-2-imidazole carboxamidine hydrochloride, 20 was ob-
tained as a light yellow solid: mp >230 °C; 1H NMR (DMSO-d6) d
4.04 (s, 3H), 4.25 (m, 2H), 7.10 (s, 1H), 7.42 (s, 1H), 7.46 (m, 2H),
7.82 (t, J = 6 Hz, 1H); MS (ES+): m/z 422.0 (M+1). Anal. Calcd for
C10H16ClF6N5ꢂ0.25EtOAcꢂ0.15C6H14: C, 47.08, H, 3.11, N, 15.34.
Found: C, 47.44, H, 2.75, N, 15.04.
5.13. 6-Chloro-2-isoquinolin-3-yl-N-(2,2,2-trifluoroethyl)-5-
(2,4,6-trifluorophenyl)pyrimidin-4-amine (17)
5.17. 6-Chloro-5-{2,6-difluoro-4-[3-(methylamino)propoxy]-
phenyl}-2-pyrazin-2-yl-N-[(1S)-2,2,2-trifluoro-1-methylethyl]-
pyrimidin-4-amine (21)
According to the procedure used to prepare 10, starting from 3-
isoquinolinecarbonitrile, 17 was obtained as a white solid: mp
275–277 °C; 1H NMR (CDCl3) d 4.44 (m, 2H), 4.93 (t, J = 6 Hz, 1H),
6.88 (m, 2H), 7.71 (m, 1H), 7.77 (m, 1H), 8.03 (d, J = 8 Hz, 1H),
8.05 (t, J = 8 Hz, 1H), 8.87 (s, 1H), 9.45 (s, 1H); MS (ES+): m/z
469.0 (M+1). Anal. Calcd for C21H11ClF6N4ꢂ0.1EtOAcꢂ0.1C6H14: C,
54.34, H, 2.74, N, 11.52. Found: C, 54.41, H, 2.71, N, 11.22.
To
a solution of 8 (251 mg, 0.58 mmol) and 3-(methyl-
amino)propan-1-ol (267 mg, 3.0 mmol) in dimethylsulfoxide
(3 mL) at room temperature was added sodium hydride (60% in
mineral oil, 120 mg, 3.0 mmol). The mixture was stirred at 60 °C
for 2 h, cooled to room temperature, and partitioned between ethyl
acetate and saturated sodium chloride. The organic layer was
washed with saturated sodium chloride (3ꢁ), dried over magne-
sium sulfate, and concentrated. The residue was purified by flash
chromatography over silica gel, eluting with a gradient of ethyl
acetate to 50% methyl alcohol in ethyl acetate. Concentration pro-
vided 181 mg (62%) of 21 as a light tan solid: mp 48–50 °C. The
product thus obtained was dissolved in methylene chloride and fil-
tered. Into the filtrate was bubbled hydrogen chloride gas. Concen-
tration provided 210 mg of hydrogen chloride salt of 21 as a red
solid: mp 68–70 °C; 1H NMR (DMSO-d6) d 1.35 (d, J = 8 Hz, 3H),
2.11 (m, 2H), 2.59 (t, J = 5 Hz, 3H), 3.06 (m, 2H), 4.19 (t, J = 6 Hz,
2H), 5.53 (m, 1H), 6.97 (m, 2H), 7.67 (d, J = 8 Hz, 1H), 8.75 (br s,
2H), 8.83 (m, 2H), 9.57 (s, 1H); MS (ES+): m/z 503.0 (M+1). Anal.
Calcd for C21H20ClF5N6Oꢂ2.0HClꢂ0.45EtOAc: C, 44.50, H, 4.19, N,
13.66. Found: C, 44.83, H, 4.18, N, 14.00.
5.14. 6-Chloro-2-thien-2-yl-N-(2,2,2-trifluoroethyl)-5-(2,4,6-
trifluorophenyl)pyrimidin-4-amine (18)
According to the procedure used to prepare 10, starting from 2-
thiophenecarbonitrile, 18 was obtained as a white solid: mp 130–
135 °C; 1H NMR (CDCl3) d 4.28 (m, 2H), 4.73 (t, J = 6 Hz, 1H), 6.88
(m, 2H), 7.14 (dd, J = 5, 3 Hz, 1H), 7.51 (dd, J = 5, 1 Hz, 1H), 8.03
(dd, J = 3, 1 Hz, 1H); MS (ES+): m/z 424.0 (M+1). Anal. Calcd for
C16H8ClF6N3Sꢂ0.2C6H14: C, 46.84, H, 2.47, N, 9.53. Found: C, 46.94,
H, 2.18, N, 9.17.
5.15. 6-Chloro-2-(2-furyl)-N-(2,2,2-trifluoroethyl)-5-(2,4,6-trifluoro-
phenyl)pyrimidin-4-amine (19)
According to the procedure used to prepare 10, starting from 2-
furonitrile, 19 was obtained as a white solid: mp 175–177 °C; 1H
NMR (CDCl3) d 4.31 (m, 2H), 4.77 (t, J = 6 Hz, 1H), 6.57 (dd, J = 3,
2 Hz, 1H), 6.88 (m, 2H), 7.36 (dd, J = 3, 1 Hz, 1H), 7.64 (dd, J = 2,
1 Hz, 1H); MS (ES+): m/z 408.0 (M+1).
5.18. 6-Chloro-5-{2,6-difluoro-4-[3-(methylamino)propoxy]-
phenyl}-2-pyridin-2-yl-N-[(1S)-2,2,2-trifluoro-1-methylethyl]-
pyrimidin-4-amine (22)
According to the procedure used to prepare 21, starting from 9,
hydrogen chloride salt of 17 was obtained as a light yellow solid:
mp 55–60 °C; 1H NMR (DMSO-d6) d 1.36 (d, J = 7 Hz, 3H), 2.13
(m, 2H), 2.59 (t, J = 5 Hz, 3H), 3.04 (m, 2H), 4.20 (t, J = 6 Hz, 2H),
5.68 (m, 1H), 6.97 (m, 2H), 7.72 (d, J = 9 Hz, 1H), 7.83 (t, J = 6 Hz,
1H), 8.30 (t, J = 8 Hz, 1H), 8.58 (d, J = 8 Hz, 1H), 8.86 (d, J = 4 Hz,
1H), 8.92 (br s, 2H); MS (ES+): m/z 502.0 (M+1). Anal. Calcd for
C22H21ClF5N5Oꢂ2.8HClꢂ0.2EtOAc: C, 44.06, H, 4.12, N, 11.27. Found:
C, 44.32, H, 4.12, N, 11.37.
5.16. 6-Chloro-2-(1-methyl-1H-imidazol-2-yl)-N-(2,2,2-trifluo-
roethyl)-5-(2,4,6-trifluorophenyl)pyrimidin-4-amine (20)
1-Methyl-2-imidazole carboxaldehyde (1.7 g, 15.4 mmol) was
stirred in methyl alcohol (10 mL). N,N-Dimethylhydrazine (1.4 g,
23.3 mmol) was then added. The mixture was stirred for 5 h, and
the resulting hydrazone solution was added dropwise into a solu-
tion of magnesium monoperoxyphtalate hexahydrate (23.9 g, 80%,
38.6 mmol) in methyl alcohol (20 mL) at 0 °C. The resulting reac-
tion mixture was allowed to warm to room temperature overnight
and concentrated. The residue was diluted with water, then ex-
tracted with methylene chloride (3ꢁ). The combined organic was
washed with saturated sodium chloride, dried over magnesium
sulfate and concentrated. The residue was purified by flash chro-
matography, eluting with 40% ethyl acetate in hexanes to give
1-methyl-2-imidazole carbonitrile as a yellow oil.
5.19. 6-Chloro-5-{4-[3-(dimethylamino)propoxy]-2,6-difluoro-
phenyl}-N-(2,2,2-trifluoroethyl)-2,20-bipyrimidin-4-amine (23)
According to the procedure used to prepare 21, starting from 11,
23 was obtained as an off-white solid: mp 134–136 °C; 1H NMR
(DMSO-d6) d 1.91 (m, 2H), 2.20(s, 6H), 2.43 (m, 2H), 4.12 (t,
J = 6 Hz, 2H), 4.26 (m, 2H), 6.97 (d, J = 10 Hz, 2H), 7.68 (t, J = 5 Hz,
1H), 7.85 (t, J = 6 Hz, 1H), 9.02 (d, J = 5 Hz, 2H); MS (ES+): m/z
503.1 (M+1). Anal. Calcd for C21H20ClF5N6Oꢂ0.5EtOAc: C, 50.51, H,
4.42, N, 15.36. Found: C, 50.24, H, 4.44, N, 15.27.
To methyl alcohol (10 mL) in a sealable tube was added sodium
hydride (440 mg, 11 mmol) with stirring. 1-Methyl-2-imidazole

N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 111–118
117
5.20. 6-Chloro-5-{4-[3-(dimethylamino)propoxy]-2,6-difluoro-
phenyl}-2-pyrazin-2-yl-N-(2,2,2-trifluoroethyl)pyrimidin-4-
amine (24)
for 3 h. The mixture was partitioned between ethyl acetate and
saturated sodium chloride. The organic layer was washed with sat-
urated sodium chloride (4ꢁ), dried over magnesium sulfate, and
concentrated. The residue was purified by flash chromatography
over silica gel, eluting with a gradient of hexanes to 50% ethyl ace-
tate in hexanes. Concentration provided 71 mg (21%, two steps) of
28 as a tan solid: mp 72–74 °C; 1H NMR (CDCl3) d 6.88 (m, 2H), 7.20
(s, 1H), 7.87 (s, 1H), 8.61 (s, 1H); MS (ES+): m/z 345.2 (M+1). Anal.
Calcd for C13H5Cl2F3N4: C, 45.24, H, 1.46, N, 16.23. Found: C, 45.59,
H, 1.71, N, 15.92.
A solution of 28 (35 mg, 0.10 mmol) and 2,2,2-trifluoroethyl-
amine (50 mg, 0.5 mmol) in 2 mL of N,N-dimethylformamide was
stirred at room temperature for 1 h. A saturated sodium chloride
solution is added, and the product was extracted with ethyl ace-
tate. The organic solution was washed with saturated sodium chlo-
ride (4ꢁ), dried over magnesium sulfate, and concentrated. The
residue was purified by flash chromatography over silica gel, elut-
ing with a gradient of 20% ethyl acetate in hexanes to 50% ethyl
acetate in hexanes. Concentration provided 36 mg (88%) of 29 as
a light tan solid: mp 168–170 °C; 1H NMR (CDCl3) d 4.23 (m, 2H),
5.11 (t, J = 6 Hz, 1H), 6.91 (m, 2H), 7.14 (s, 1H), 7.83 (s, 1H), 8.54
(s, 1H); MS (ES+): m/z 408.2 (M+1). Anal. Calcd for C15H8ClF6N5:
C, 44.19, H, 1.98, N, 17.18. Found: C, 44.15, H, 1.78, N, 16.83.
According to the procedure used to prepare 21, starting from 10,
hydrogen chloride salt of 24 was obtained as a yellow solid: mp
58–62 °C; 1H NMR (DMSO-d6) d 2.18 (m, 2H), 2.80 (d, J = 5 Hz,
6H), 3.20 (m, 2H), 4.19 (t, J = 6 Hz, 2H), 4.33 (m, 2H), 7.00 (d,
J = 9 Hz, 2H), 7.90 (t, J = 6 Hz, 1H), 8.86 (m, 2H), 9.57 (s, 1H),
10.37 (br s, 1H); MS (ES+): m/z 503.0 (M+1). Anal. Calcd for
C21H20ClF5N6Oꢂ1.8HClꢂ0.3EtOAc: C, 44.82, H, 4.10, N, 14.12. Found:
C, 44.80, H, 4.20, N, 14.22.
5.21. 6-Chloro-5-{4-[2-(dimethylamino)ethoxy]-2,6-difluoro-
phenyl}-2-pyrazin-2-yl-N-(2,2,2-trifluoroethyl)pyrimidin-4-
amine (25)
According to the procedure used to prepare 21, starting from 10,
25 was obtained as a white solid: mp 150–155 °C; 1H NMR (CDCl3)
d 2.34 (s, 6H), 2.78 (t, J = 6 Hz, 2H), 4.11 (t, J = 6 Hz, 2H), 4.37 (m,
2H), 5.11 (t, J = 6 Hz, 1H), 6.65 (m, 2H), 8.70 (d, J = 2 Hz, 1H), 8.78
(dd, J = 2, 1 Hz, 1H), 9.64 (d, J = 1 Hz, 1H); MS (ES+): m/z 489.2
(M+1). Anal. Calcd for C20H18ClF5N6Oꢂ0.2EtOAc: C, 49.32, H, 3.90,
N, 16.59. Found: C, 49.22, H, 3.56, N, 16.60.
5.24. 6-Chloro-2-(1H-pyrazol-1-yl)-N-(2,2,2-trifluoroethyl)-5-
(2,4,6-trifluorophenyl)pyrimidin-4-amine (30)
5.22. 6-Chloro-5-{4-[4-(dimethylamino)butoxy]-2,6-difluoro-
phenyl}-2-pyrazin-2-yl-N-(2,2,2-trifluoroethyl)pyrimidin-4-
amine (26)
According to the procedure used to prepare 29, starting from 27,
30 was obtained as a light tan solid: mp 150 °C (decomposed); 1H
NMR (CDCl3) d 4.29 (m, 2H), 4.93 (t, J = 6 Hz, 1H), 6.50 (dd, J = 3,
1 Hz, 1H), 6.91 (m, 2H), 7.85 (d, J = 1 Hz, 1H), 8.54 (d, J = 3 Hz,
1H); MS (ES+): m/z 407.9 (M+1). Anal. Calcd for C15H8ClF6N5: C,
44.19, H, 1.98, N, 17.18. Found: C, 44.20, H, 1.80, N, 16.81.
According to the procedure used to prepare 21, starting from 10,
26 was obtained as a white solid: mp 151–153 °C; 1H NMR (CDCl3)
d 1.70 (m, 2H), 1.87 (m, 2H), 2.29 (s, 6H), 2.38 (t, J = 7 Hz, 2H), 4.03
(t, J = 6 Hz, 2H), 4.35 (m, 2H), 5.18 (t, J = 6 Hz, 1H), 6.62 (m, 2H),
8.70 (d, J = 2 Hz, 1H), 8.78 (dd, J = 2, 1 Hz, 1H), 9.64 (d, J = 1 Hz,
1H); MS (ES+): m/z 517.2 (M+1). Anal. Calcd for
C22H22ClF5N6Oꢂ0.2EtOAc: C, 51.23, H, 4.45, N, 15.72. Found: C,
51.38, H, 4.21, N, 15.69.
5.25. 6-Chloro-2-(1H-pyrazol-1-yl)-N-[(1S)-2,2,2-trifluoro-1-
methylethyl)-5-(2,4,6-trifluorophenyl)pyrimidin-4-amine (31)
According to the procedure used to prepare 29, starting from 27,
31 was obtained as a light yellow solid: mp 200–205 °C; 1H NMR
(CDCl3) d 1.41 (d, J = 7 Hz, 3H), 4.679 (d, J = 9 Hz, 1H), 5.23 (m,
1H), 6.50 (dd, J = 3, 1 Hz, 1H), 6.90 (m, 2H), 7.85 (d, J = 1 Hz, 1H),
8.53 (d, J = 3 Hz, 1H); MS (ES+): m/z 422.0 (M+1). Anal. Calcd for
C16H10ClF6N5: C, 45.57, H, 2.39, N, 16.61. Found: C, 45.97, H, 2.27,
N, 16.40.
5.23. 6-Chloro-2-(1H-imidazol-1-yl)-N-(2,2,2-trifluoroethyl)-5-
(2,4,6-trifluorophenyl)pyrimidin-4-amine (29)
To
a mixture of diethyl 2-(2,4,6-trifluorophenyl)malonate
(580 mg, 2.0 mmol) and urea (360 mg, 6.0 mmol) in ethyl alcohol
(10 mL) at room temperature was added sodium hydride (60% in
mineral oil, 160 mg, 4.0 mmol). The mixture was then heated at
80 °C for 3 days, cooled to room temperature, and partitioned be-
tween ethyl acetate and 1 N hydrochloric acid. The aqueous layer
was extracted with ethyl acetate, and the combined organic ex-
tracts are dried over magnesium sulfate, and concentrated. The
residue was purified by flash chromatography over silica gel, elut-
ing with a gradient of ethyl acetate to 10% methyl alcohol in ethyl
acetate. Concentration provided 148 mg (29%) of 5-(2,4,6-trifluor-
ophenyl)pyrimidine-2,4,6-triol as a light tan solid. MS: m/z 257.0
(MꢃH).
5.26. 6-Chloro-5-{4-[3-(dimethylamino)propoxy]-2,6-difluoro-
phenyl}-2-(1H-imidazol-1-yl)-N-(2,2,2-trifluoroethyl)pyrimidin-
4-amine (32)
To a solution of 29 (20 mg, 0.05 mmol) and 3-dimethylamino-1-
propanol (103 m g, 1.0 mmol) in dimethylsulfoxide (3 mL) at room
temperature was added sodium hydride (60% in mineral oil, 40 mg,
1.0 mmol). The mixture was stirred at 60 °C for 2 h, cooled to room
temperature, and partitioned between ethyl acetate and saturated
sodium chloride. The organic layer was washed with saturated so-
dium chloride (3ꢁ), dried over magnesium sulfate, and concen-
trated. The residue was purified by flash chromatography over
silica gel, eluting with a gradient of ethyl acetate to 50% methyl
alcohol in ethyl acetate. Concentration provided 16 mg (65%) of
32 as a light tan solid: mp 47–49 °C; 1H NMR (DMSO-d6) d 1.87
(m, 2H), 2.16 (s, 6H), 2.37 (t, J = 7 Hz, 2H), 4.10 (t, J = 6 Hz, 2H),
4.30 (m, 2H), 6.94 (d, J = 10 Hz, 2H), 7.13 (s, 1H), 7.92 (s, 1H),
8.02 (t, J = 6 Hz, 1H), 8.61 (s, 1H); MS (ES+): m/z 491.1 (M+1). Anal.
Calcd for C20H20ClF5N6Oꢂ0.25EtOAc: C, 49.48, H, 4.60, N, 15.46.
Found: C, 49.14, H, 4.25, N, 15.26.
A
mixture of 5-(2,4,6-trifluorophenyl)pyrimidine-2,4,6-triol
(258 mg, 1.0 mmol) in phosphorous oxychloride (2.5 mL) and 2,6-
lutidine (0.5 mL) was heated at 110 °C for 16 h. The excess phos-
phorous oxychloride was removed in vacuo, and the resulting
residue was dissolved in a 1:1 mixture of methylene chloride and
hexanes. The organic layer was filtered through hydrous magne-
sium silicate, and the filtrate was concentrated to provide
104 mg of crude 2,4,6-trichloro-5-(2,4,6-trifluorophenyl)pyrimi-
dine (27) as a dark solid. A mixture of the above 2,4,6-trichloro-
5-(2,4,6-trifluorophenyl)pyrimidine (27, 104 mg), imidazole
(23 mg, 0.33 mmol), and potassium carbonate (92 mg, 0.66 mmol)
in N,N-dimethylformamide (2 mL) is stirred at room temperature

118
N. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 111–118
5. Höfle, G.; Bedorf, N.; Steinmetz, H.; Schomburg, D.; Gerth, K.; Reichenbach, H.
Angew. Chem. Int. Ed. Engl. 1996, 35, 1567.
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5.27. 6-Chloro-5-{2,6-difluoro-4-[3-(methylamino)propoxy]phen-
yl}-2-(1H-pyrazol-1-yl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl)-
pyrimidin-4-amine (33)
7. Lindel, T.; Jensen, P. R.; Fenical, W.; Long, B. H.; Casazza, A. M.; Carboni, J.;
Fairchild, C. R. J. Am. Chem. Soc. 1997, 119, 8744.
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Cancer Res. 1999, 59, 653.
According to the procedure used to prepare 32 and 21, starting
from 31, hydrogen chloride salt of 33 was obtained as a white so-
lid: mp 110–115 °C; 1H NMR (CDCl3) d 1.40 (d, J = 6 Hz, 3H), 2.45
(br s, 2H), 2.77 (br s, 3H), 3.24 (br s, 2H), 4.21 (br s, 2H), 4.99 (br
s, 1H), 5.19 (br s, 1H), 6.49 (br s, 1H), 6.65 (d, J = 8 Hz, 2H), 7.86
(s, 1H), 8.51 (d, J = 2 Hz, 1H), 9.75 (br s, 2H); MS (ES+): m/z 491.0
(M+1). Anal. Calcd for C20H20ClF5N6Oꢂ1.2HClꢂ0.35EtOAc: C, 45.46,
H, 4.2 8, N, 14.86. Found: C, 45.71, H, 4.32, N, 14.93.
12. Hood, K. A.; West, L. M.; Rouwe, B.; Northcote, P. T.; Berridge, M. V.; Wakefield,
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5.28. Biological evaluation
14. Gaitanos, T. N.; Buey, R. M.; Diaz, J. F.; Northcote, P. T.; Teesdale-Spittle, P.;
Andreu, J. M.; Miller, J. H. Cancer Res. 2004, 64, 5063.
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Pharm. 2006, 3, 457.
16. Wilmes, A.; Bargh, K.; Kelly, C.; Northcote, P. T.; Miller, J. H. Mol. Pharm. 2007, 4, 269.
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Pharmacol. 2006, 70, 1555.
The protocols for COLO 205 cytotoxicity assay,18,19 KB, KB 8.5
and KB V1 cellular assays,25 tubulin polymerization studies,18,19
competitive tubulin binding studies,18,19 and cell cycle analysis18,19
have been reported.
18. Zhang, N.; Ayral-Kaloustian, S.; Nguyen, T.; Afragola, J.; Hernandez, R.; Lucas, J.;
Gibbons, J.; Beyer, C. J. Med. Chem. 2007, 50, 319.
5.29. Xenograft assay
19. Beyer, C.; Zhang, N.; Hernandez, R.; Vitale, D.; Lucas, J.; Nguyen, T.; Discafani,
C.; Ayral-Kaloustian, S.; Gibbons, J. Cancer Res. 2008, 68, 2292.
20. Zhang, N.; Ayral-Kaloustian, S.; Nguyen, T.; Hernandez, R.; Beyer, C. Bioorg.
Med. Chem. Lett. 2007, 17, 3003.
21. Preliminary work was reported in Zhang, N.; Ayral-Kaloustian, S.; Nguyen,
T.; Hernandez, R.; Lucas, J.; Discafani, C.; Beyer, C. ACS Prospectives:
The procedure has been reported.18 To initiate tumors, cells
were injected subcutaneously into the flanks of female NU/NU-
Foxn1nu mice from Charles River Laboratories (1 ꢁ 106 cells/animal
for human H460 non-small cell lung carcinoma, 2 ꢁ 106 cells/ani-
mal for human LOX melanoma). When tumors attained a mass of
about 100 mg, mice were staged, that is, randomized into treat-
ment groups containing 5–10 animals each. Compound vehicles
are given in Figure legends.
Discovery
& Selection of Successful Drug Candidates, Boston, MA, April
29–May 2, 2007.
22. The synthetic route to 8–20 and 29–31 and the alternative synthesis of
synthetic route to 29 was disclosed in Grote, T.; Gypser, A.; Rheinheimer, J.;
Rose, I.; Schäfer, P.; Schieweck, F.; Sauter, H.; Gewehr, M.; Müller, B.; Blasco, J.
T.; Ammermann, E.; Strathmann, S.; Lorenz, G.; Stierl, R. US Patent 7,153,860
B2, 2006; Chem. Abstr. 2002, 137, 263046.
Acknowledgments
23. Meyer, O.; Eisenacht, R. US Patent 6,156,925, 2000; Chem. Abstr. 2000, 134,
17314.
24. Jordan, M. A.; Wendell, K.; Gardiner, S.; Derry, W. B.; Copp, H.; Wilson, L. Cancer
Res. 1996, 56, 816.
The authors thank the Wyeth Chemical Technologies Depart-
ment for analytical and spectral determinations, and Wyeth Re-
search management for support of this work.
25. Loganzo, F.; Discafani, C. M.; Annable, T.; Beyer, C.; Musto, S.; Hari, M.; Tan, X.;
Hardy, C.; Hernandez, R.; Baxter, M.; Singanallore, T.; Khafizova, G.;
Poruchynsky, M. S.; Fojo, T.; Nieman, J. A.; Ayral-Kaloustian, S.; Zask, A.;
Andersen, R. J.; Greenberger, L. M. Cancer Res. 2003, 63, 1838.
26. Baca, E.; Comer, J. E. A.; Takacs-Novak, K. J. Pharm. Biomed. Anal. 2008, 46, 335.
27. Gibaldi, M.; Perrier, D., 2nd ed.. In Drugs and the pharmaceutical sciences,
pharmacokinetics; Swarbrick, J., Ed.; Marcel-Dekker: New York, 1982; Vol. 15,
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