Synthesis and SAR of [1,2,4]triazolo[1,5-a]pyrimidines, a class of anticancer agents with a unique mechanism of tubulin inhibition

Article abstract of DOI:10.1021/jm060717i

The synthesis and SAR of a series of triazolopyrimidines as anticancer agents are described. Treatment of 5-chloro-6-(trifluorophenyl)-N-fluoroalkyl[1, 2,4]triazolo[1,5-a]pyrimidin-7-amine with an alcohol, a thiol, or an alkylamine provided the corresponding final compounds. A clear SAR requirement has been established for optimal activity. A (1S)-2,2,2-trifluoro-1-methylethylamino group or an achiral 2,2,2-trifluoroethylamino group is required at the 5-position to achieve high potency. On the phenyl ring, both fluoro atoms, at the positions ortho to the triazolopyrimidine core, are needed for optimal activity. At the position para to the triazolopyrimidine core, on the phenyl ring, the best activity is achieved with an oxygen linkage followed by a three-methylene unit, and an alkylamino or a hydroxy group. The mechanism of action for this series of triazolopyrimidines was shown to be unique in that they promoted tubulin polymerization in vitro, but did not bind competitively with paclitaxel. Instead, they inhibit the binding of vincas to tubulin. Selected compounds were studied further, and it was shown that these compounds were able to overcome resistance attributed to several multidrug resistance transporter proteins. Lead compounds were shown to inhibit tumor growth in several nude mouse xenograft models, with high potency and efficacy, when dosed either orally or intravenously.

Full text of DOI:10.1021/jm060717i

J. Med. Chem. 2007, 50, 319-327
319
Synthesis and SAR of [1,2,4]Triazolo[1,5-a]pyrimidines, a Class of Anticancer Agents with a
Unique Mechanism of Tubulin Inhibition
Nan Zhang,*,† Semiramis Ayral-Kaloustian,† Thai Nguyen,† Jay Afragola,† Richard Hernandez,‡ Judy Lucas,‡
James Gibbons,‡ and Carl Beyer‡
Chemical and Screening Sciences, and DiscoVery Oncology, Wyeth Research, 401 North Middletown Road, Pearl RiVer, New York 10965
ReceiVed June 14, 2006
The synthesis and SAR of a series of triazolopyrimidines as anticancer agents are described. Treatment of
5-chloro-6-(trifluorophenyl)-N-fluoroalkyl [1,2,4]triazolo[1,5-a]pyrimidin-7-amine with an alcohol, a thiol,
or an alkylamine provided the corresponding final compounds. A clear SAR requirement has been established
for optimal activity. A (1S)-2,2,2-trifluoro-1-methylethylamino group or an achiral 2,2,2-trifluoroethylamino
group is required at the 5-position to achieve high potency. On the phenyl ring, both fluoro atoms, at the
positions ortho to the triazolopyrimidine core, are needed for optimal activity. At the position para to the
triazolopyrimidine core, on the phenyl ring, the best activity is achieved with an oxygen linkage followed
by a three-methylene unit, and an alkylamino or a hydroxy group. The mechanism of action for this series
of triazolopyrimidines was shown to be unique in that they promoted tubulin polymerization in Vitro, but
did not bind competitively with paclitaxel.1 Instead, they inhibit the binding of Vincas to tubulin. Selected
compounds were studied further, and it was shown that these compounds were able to overcome resistance
attributed to several multidrug resistance transporter proteins. Lead compounds were shown to inhibit tumor
growth in several nude mouse xenograft models, with high potency and efficacy, when dosed either orally
or intravenously.
Introduction
interfere with the microtubule dynamics in a manner unique
among all microtubule-active compounds. The initial lead
compound 517 came through selective screening of our Agri-
cultural Research Division compound libraries based on evi-
dence for antimicrotubule-like mechanism of action against
fungi. Our subsequent study in human tumor cells showed that
this compound inhibits tumor cell growth by interacting directly
with tubulin with a novel mechanism of action. Additional
analogues were then prepared to optimize potency and other
pharmaceutical properties.
Microtubules are dynamic protein fibers formed by poly-
merization of R,â-tubulin heterodimers. They are vital compo-
nents of the mitotic spindle and serve multiple functions,
including maintenance of cell shape, vesicle and protein
transport, and chromosome segregation during mitosis. Anti-
microtubule drugs are a major category of anticancer agents.2
These agents work by interfering with the microtubule dynamics
and thus disrupting normal spindle function.3 The most potent
compounds and those of clinical relevance are natural products
or semisynthetic derivatives of natural products.
Most antimicrotubule agents work by inhibiting polymeri-
zation of tubulin to microtubules.4 These agents are categorized
into two major classes depending on their distinctive binding
sites on â-tubulin. The first class is the Vinca alkaloid site
binders, as represented by vinblastine and vincristine. The other
class is the colchicine site binders, which have attracted
considerable synthetic efforts. So far, no colchicine site binders
have proven successful in clinical trials. Taxoids are the first
class of antimicrotubules that were reported to promote tubulin
polymerization and stabilize microtubules.5 Since then, a number
of natural products have been discovered that have taxoid-like
activity, including epothilones,6 discodermolide,7 eleutherobin,8
laulimalide,9 and peloruside.10 While epothilones, discoder-
molide, and eleutherobin bind to microtubules competitively
with paclitaxel,11-13 laulimalide and peloruside do not compete
with paclitaxel and thus do not bind in the taxoid site.14,15 The
clinical success of the taxanes has encouraged efforts to develop
other microtubule-stabilizing agents for cancer chemotherapy.16
Now, we report the synthesis and structure-activity relation-
ship of a series of triazolopyrimidines as anticancer agents that
Chemistry. Compounds 5-30 and 32-39 were prepared as
shown in Scheme 1. Coupling of 1-bromo-2,4,6-trifluorobenzene
1a with diethyl malonate gave diethyl 2,4,6-trifluorophenylma-
lonate 2.18 Cyclization of 2 with 3-amino-1,2,4-triazole provided
5,7-dihydroxy-6-(2,4,6-trifluorophenyl)[1,2,4]triazolo[1,5-a]py-
rimidine 319 as a single regioisomer. Bis-chlorination was
achieved by refluxing 3 in excess POCl3, affording compound
4.19 Treatment with a fluoroalkylamine [CF3C(R1R2)NH2] in
DMF replaced the 7-chloro exclusively,17 yielding the desired
7-amino compounds 5-7. Compounds 8-30 (Table 1) were
prepared by further treatment of compounds 5-7 with the
corresponding alcohols (for compounds 8-27) or a thiol (for
compound 28), in the presence of sodium hydride in DMSO,
or with neat amines in the presence of sodium hydride (for
compounds 29 and 30).
Compounds 32-39 (Tables 1 and 2) were prepared analo-
gously to compounds 5-30. Compound 32 was prepared by
treating dihydroxy compound 3 with POBr3 instead of POCl3,
followed by replacement of the 7-bromo with an amine.
Subsequent treatment with 3-dimethylamino-1-propanol gave
compound 33. Compounds 34 and 36 were prepared in the same
manner as compound 5, starting from 1-bromo-2,4-difluoroben-
zene (1b) and 1-bromo-2,3,6-trifluorobenzene (1c), respectively.
Reaction of compounds 34 and 36 with amino alcohols provided
compounds 35 and 37-39, respectively.
* To whom correspondence should be addressed. Phone: (845)602-3425.
Fax: (845) 602-5561. E-mail: zhangn@wyeth.com
† Chemical and Screening Sciences.
‡ Discovery Oncology.
10.1021/jm060717i CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/21/2006

320 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Scheme 1. Synthesis of 5-30 and 32-39a
Zhang et al.
comparison of 9 to 8 or 12 to 11 (also 6 to 5). In contrast,
removal of the chirality by introduction of an additional 1-methyl
group to the 2,2,2-trifluoro-1-methylethylamino group (i.e., gem-
1,1-dimethyl-2,2,2-trifluoroethylamino group) led to much
diminished activity (data to be published separately), illustrating
space limitations in the binding region.
Substitution of the 4-fluoro group on the phenyl ring in
compounds 5-7 led to compounds 8-31. A clear trend of SAR
was manifested from activity shown by these compounds. High
potency was achieved with an oxygen linkage and a three-
methylene tether followed by an alkylamino group or a hydroxy
group, as in compounds 8, 9, 11-16, and 19. Compound 15,
bearing a six-membered piperidine ring at the terminal, was less
potent than compound 14, with a five-membered pyrrolidine
ring at the terminal. With a more hydrophilic morpholino group,
compound 16 was comparable to compound 14 in potency.
Surprisingly, the four-membered azetidine ring, as in compound
17, led to much decreased potency. When the terminal amino
group was unsubstituted, as in compound 18, lower potency
was observed. Lower potency was also observed when the
terminal hydroxy group was capped by an alkyl group (com-
pound 20). Activity was further diminished when the terminal
group was a straight alkyl group, as in compound 21. These
results suggest that the potency is at its best with a moderately
hydrophilic group at the terminal position.
Compounds 22-24, with a two-methylene tether instead of
the three-methylene tether, showed much lower potency, sug-
gesting strict spatial requirements for the functional groups at
the binding site. This hypothesis is further supported by activity
of compounds 25-27. With a four- or a five-methylene tether,
compounds 25-27 showed to be less potent than their three-
methylene analogues, though twice as potent as their two-
methylene analogues.
Replacement of the oxygen linkage with a sulfur linkage
(compound 28), an amino group (compounds 29 and 30), or a
methylene unit (compound 31) invariably led to reduced activity.
Among these analogues, compound 28 exhibited about 4-fold
reduction in potency. Other analogues were even less potent.
Compounds 32 and 33, with a 5-bromo substituent instead of a
5-chloro group, were comparable with or slightly less potent
than their 5-chloro analogues.
As shown in Table 2, compounds 37-39, with the ami-
noalkoxy group moved from the 4- to the 3-position on the
phenyl ring, proved to be less potent than their corresponding
regioisomers 22, 11, and 25, even though the 2,3,6-trifluorophe-
nyl compound 36 showed potency comparable to that of the
2,4,6-trifluorophenyl analogue 5. Interestingly, compound 39,
with a four-methylene tether, showed the highest activity among
37-39. Modeling suggests that, from the 3-position on the
phenyl ring, it takes a four-methylene tether (as in 39) to reach
the same space and putative interactions, as reached by a three-
methylene tether from the 4-position (as in 11).
a (a)18 Diethyl malonate, CuI, NaH, dioxane, 60 °C; (b)19 3-Amino-1,2,4-
triazole, tributylamine, 160-180 °C; (c)19 POCl3, reflux; (d) CF3(CR1R2)NH2,
N,N-diisopropylethylamine, DMF; (e) For 8-27, 33, 35, 37-39: ROH,
NaH, DMSO; for 28: RSH, NaH, DMSO; for 29: RN(CH3)H, NaH, 100
°C; for 30: RNH2, NaH, 100 °C; (f) POBr3, 120 °C.
We found that the 4-fluoro group on the phenyl ring (in 5-7,
32, and 34) could be replaced cleanly with no replacement of
the 2-fluoro group detected. Replacement of the 3-fluoro group
on the phenyl ring (in 36), however, was less selective, with
some replacement of the 2-fluoro group detected.
As depicted in Scheme 2, synthesis of compound 31 started
from diethyl 2-(2,6-difluoro-4-methoxyphenyl)malonate 40,20
which was prepared from 1-bromo-2,6-difluoro-4-methoxyben-
zene 1d. Demethylation followed by triflate formation set the
stage for palladium-catalyzed coupling, to introduce the alkyl
chain. Conversion of the hydroxy group of the resultant
compound 43 to the dimethylamino group was accomplished
by mesylation and subsequent nucleophilic displacement. Cy-
clization of 44 with 3-amino-1,2,4-triazole followed by chlorina-
tion in excess POCl3 and replacement of the 7-chloro with (1S)-
2,2,2-trifluoro-1-methylethylamine in DMF, as before, yielded
the desired compound 31.
Compounds 8-9, 11-12, 16, and 33 were further studied in
order to elucidate the mechanism of action for this series of
compounds.22 Tubulin polymerization studies using either MAP-
rich tubulin or pure tubulin showed these compounds promoted
tubulin polymerization in Vitro, a phenomenon similar to the
effect of paclitaxel. Competitive binding studies with [3H]-
vinblastine, [3H]colchicine, and [3H]paclitaxel showed that these
compounds did not bind competitively with [3H]colchicine or
[3H]paclitaxel. Instead, they prevented binding of [3H]vinblastine
to tubulin.22 These results established the unique mechanism
of action for these compounds, in that they promote tubulin
polymerization like paclitaxel does, but through a binding site
Results and Discussion
Compounds 5-39 were evaluated in the COLO 205 cyto-
toxicity assay for their ability to inhibit cancer cell proliferation.
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 1, the chirality in the 2,2,2-trifluoro-1-
methylethylamino group is important for activity, as the
S-enantiomers 5 and 8 were 3-4 fold more potent than their
corresponding R-enantiomers 7 and 10.21 Removal of the
chirality by removal of the 1-methyl group in the chiral amino
group resulted in analogues that retained potency, as shown by

Anticancer [1,2,4]Triazolo[1,5-a]pyrimidines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 321
Table 1. Inhibition of COLO 205 Cell Proliferation by Compounds 5-35
compda
R1
R2
X
L
Y
R
IC50 (nM)b
5
6
7
CH3
H
H
CH3
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Cl
Cl
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
H
H
F
F
F
267 ( 73
372 ( 68
705 ( 21
31 ( 10
H
CH3
H
H
CH3
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
8c
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
(CH2)3NH(CH3)
(CH2)3NH(CH3)
(CH2)3NH(CH3)
(CH2)3N(CH3)2
(CH2)3N(CH3)2
(CH2)3N(CH3)(CH2CH3)
(CH2)3-pyrrolidine
(CH2)3-piperidine
(CH2)3-morpholine
(CH2)3-azetidine
(CH2)3NH2
(CH2)3OH
(CH2)3OCH2CH3
(CH2)3CH3
(CH2)2N(CH3)2
(CH2)2-morpholine
(CH2)2OH
(CH2)4N(CH3)2
(CH2)4OH
9c
29 ( 7
10
126 ( 1
11c
12c
13
CH3
H
56 ( 9
53 ( 16
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
48 ( 6
14
44 ( 13
15
113 ( 12
51 ( 30
16c
17
326 ( 116
452 ( 71
67 ( 12
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
269 ( 45
1898 ( 258
581 ( 157
245 ( 118
422 ( 175
276 ( 53
151 ( 19
214 ( 31
192 ( 72
1671 ( 34
719 ( 185
665 ( 103
232 (n ) 1)
96 ( 21
(CH2)5OH
(CH2)3N(CH3)2
(CH2)3N(CH3)2
(CH2)3N(CH3)2
(CH2)3N(CH3)2
N(CH3)
NH
CH2
F
O
F
(CH2)3N(CH3)2
(CH2)3N(CH3)2
34
35
953 ( 54
262 ( 18
3.3 ( 1
O
paclitaxel
vincristine
2.6 ( 0.5
a Unless indicated otherwise, compounds were tested as the free base. b Concentration needed to inhibit cell growth by 50% as determined from the
dose-response curve. Determinations were made at 10 concentrations, in triplicate, and repeat values agreed, on average, within 40%. c Compounds were
tested as the HCl salts.
gp) is shown in Table 3. The KB lines24 express different
amounts of the P-gp (MDR1) membrane pump which causes
Table 2. Inhibition of COLO 205 Cell Proliferation by Compounds
36-39
resistance to the action of many cytotoxic compounds, including
paclitaxel and vincristine, via enhanced efflux from the cell.
The parental KB line expresses little P-gp, KB 8.5 expresses
moderate, clinically relevant, levels of the protein, and KB-V1
expresses artificially high levels. The ability of P-gp to recognize
and export a potential cytotoxic agent can be inferred from the
change in IC50 values in these lines. Compared with paclitaxel
and vincristine, compounds 8, 9, 11, 12, and 26 showed much
lower levels of recognition by P-gp, as evidenced by the
relatively low resistance ratios.
compda
Y
R
IC50 (nM)b
36
37
38
39
F
183 ( 23
1568 ( 81
785 ( 64
433 ( 74
O
O
O
(CH2)2N(CH3)2
(CH2)3N(CH3)2
(CH2)4N(CH3)2
Selected lead compounds were evaluated in U87-MG human
glioblastoma, using both intravenous and oral administration.
Significant inhibition of tumor growth in ViVo was observed
with compounds 8, 9, 11, 12, 14, and 16. Representative testing
results (compound 9 in U87-MG human glioblastoma and
compound 8 in A549 non-small cell lung carcinoma) are shown
in Figure 1 and Figure 2, respectively.
a Compounds were tested as the free base. b Concentration needed to
inhibit cell growth by 50% as determined from the dose-response curve.
Determinations were made at 10 concentrations, in triplicate, and repeat
values agreed, on average, within 40%.
that may overlap with the binding site of agents which inhibit
tubulin polymerization. Cell cycle analysis of compounds 8, 9,
11, and 12 showed induction of apoptosis at low compound
concentration and G2/M block at high compound concentration,
a pattern that is similar to what was observed with paclitaxel.23
Compounds 8, 9, 11, 12, and 26 were shown to be poor
substrates for multidrug resistance (MDR) transporter proteins.
Their ability to overcome resistance due to P-glycoprotein (P-
Compound 8 has excellent pharmaceutical properties. It is
very stable metabolically and has high solubility in water and
saline. It is minimally metabolized in female nude mouse, rat
and human liver microsomes, with t1/2 values at 433, 88, and
133 min, respectively. Its solubility in water is 0.89 mg/mL at
pH 6.74 and 7.99 mg/mL at pH 5.48. Thus, no special

322 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Scheme 2. Synthesis of 31a
Zhang et al.
Figure 1. In ViVo antitumor activity with compound 9 against U87-
MG human glioblastoma. 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.
a (a) Diethyl malonate, CuI, NaH, dioxane, 60 °C; (b) BBr3, CH2Cl2;
(c) (CF3SO2)2O, Et3N, CH2Cl2; (d) (i) 9-BBN, 3-buten-1-ol, THF, (ii) 42,
Pd(PPh3)4, K3PO4, dioxane, 90 °C, (iii) trimethylamine-N-oxide, 180 °C;
(e) (i) CH3SO2Cl, Et3N, CH2Cl2, 0 °C, (ii) (CH3)2NH, THF; (f) 3-amino-
1,2,4-triazole, tributylamine, 160-180 °C; (g) POCl3, reflux; (h) (1S)-
CF3CH(CH3)NH2, N,N-diisopropylethylamine, DMF.
Table 3. Inhibition of KB, KB 8.5, and KBV1 Cell Proliferation by
Compounds 8, 9, 11, 12, and 26
IC50 (nM)b
KB 8.5
ratioc
compda
KB
KB V1
KB8.5/KB KB V1/KB
8
9
11
12
26
23 ( 1.4
67 ( 11 953 ( 480
2.9
3.0
1.8
2.0
1.6
11
41.5
118
6.1
14.0
1.6
19 ( 4.7 56 ( 17 2237 ( 880
18.7 ( 0.2 33 ( 13 114 ( 74
18.7 ( 0.9 38 ( 13 263 ( 133
42 ( 5.6 70 ( 6.4 67 ( 6.4
paclitaxel 2.45 ( 0.1 26 ( 0.1 2013 ( 108
vincristine 2.2 ( 0.6 58 ( 16 2035 ( 25
822
925
26
a Compounds 8, 9, 11, and 12 were tested as the HCl salts; compound
26 was tested as a free base. b Concentration needed to inhibit cell growth
by 50% as determined from the dose-response curve. Determinations were
made at 10 concentrations, in duplicate, and repeat values 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.
formulation is required. PK studies in rat showed high volume
of distribution (14 L/kg), low clearance (14 mL/min/kg), high
half-life (13 h), and good oral bioavailability (61%). Compound
8 showed comparable inhibition of tumor growth by either
intravenous or oral administration in U87-MG human glioblas-
toma.22 It also exhibited good tumor inhibition in other tumor
models, e.g., in A549 non-small cell lung carcinoma (NSCLC,
Figure 2). On the basis of its superb pharmaceutical properties,
in ViVo activity, and more favorable toxicity profile in ViVo,
compound 8 was chosen for further development.
Conclusion. A series of triazolopyrimidine analogues was
systematically prepared and tested as anticancer agents. A clear
SAR requirement was established for optimal activity. A (1S)-
2,2,2-trifluoro-1-methylethylamino group or an achiral 2,2,2-
trifluoroethylamino group was required at the 5-position to
achieve high potency. Both fluoro atoms at the ortho positions
on the phenyl ring were needed for optimal activity. On the
para position on the phenyl ring, the best activity was achieved
with an oxygen linkage followed by a three-methylene unit, and
an alkylamino or a hydroxy terminal. The mechanism of action
Figure 2. In ViVo antitumor activity with compound 8 against A549
human non-small cell lung carcinoma (NSCLC). Tumors were initiated
in the flanks of nude mice by subcutaneous injection of 107 cells per
animal in 100% Matrigel and staged at about 300 mm3. The compound
was administered to tumor-bearing mice on days 0, 7, 14, and 21 after
staging (indicated by arrows) by intravenous injection. Vehicle was
0.9% saline. Each group contained nine animals.
for this series of triazolopyrimidines was shown to be unique
in that they promoted tubulin polymerization in Vitro, but did
not bind competitively with paclitaxel. As far as we know, these
compounds represent the only series of small, synthetic mol-
ecules to promote tubulin polymerization. Selected compounds
were shown to inhibit tumor growth in several nude mouse
xenograph models with high potency and efficacy. Compound
8 (TTI-237) is currently in phase I clinical trials.
Experimental Section
Chemistry. General. All reactions were conducted under
nitrogen atmosphere with magnetic stirring. Chromatographic

Anticancer [1,2,4]Triazolo[1,5-a]pyrimidines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 323
purifications were performed using Baker 40-µm silical gel. Melting
points were determined in open capillary tubes on a Meltemp
melting point apparatus and are uncorrected. 1H NMR spectra were
recorded with a Bruker 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.
5-Chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-
N-[(1R)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (10). According to the procedure used to prepare
8, reaction of 7 with 3-(methylamino)propan-1-ol25 provided 10 in
51% yield as a light tan solid: mp 139-141 °C; 1H NMR (DMSO-
d6) δ 1.18 (m, 3H), 2.03 (t, J ) 7 Hz, 2H), 2.55 (s, 3H), 2.99 (t, J
) 7 Hz, 2H), 4.10 (t, J ) 6 Hz, 2H), 5.76 (m, 1H), 6.72 (brs, 2H),
8.06 (brs, 1H); MS (ES+): m/z 465.2 (M + 1). Anal. (C18H18-
ClF5N6O) C, H, N.
5-Chloro-6-(2,4,6-trifluorophenyl)-N-[(1S)-2,2,2-trifluoro-1-
methylethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (5). A mix-
ture of 5,7-dichloro-6-(2,4,6-trifluorophenyl)[1,2,4]triazolo[1,5-
a]pyrimidine 419 (3.0 g, 9.4 mmol), (1S)-2,2,2-trifluoro-1-
methylethylamine hydrogen chloride17 (4.2 g, 28.2 mmol), and N,N-
diisopropylethylamine (4.9 mL, 28.2 mmol) in 100 mL of N,N-
dimethylformamide was stirred at room temperature under nitrogen
atmosphere for 13 h. The reaction mixture was diluted with ethyl
acetate. The organic layer was washed with 1 N hydrochloric acid
(2×) and saturated sodium chloride (2×), dried over magnesium
sulfate, and concentrated. The residue was chromatographed over
silica gel, eluting with 20% ethyl acetate in hexanes to provide
5-Chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophe-
nyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (11). According to the procedure used to
prepare 8, reaction of 5 with 3-dimethylamino-1-propanol provided
hydrogen chloride salt of 11 in 84% yield as a white solid: mp
1
65-67 °C; H NMR (CDCl3) δ 1.41 (d, J ) 7 Hz, 3H), 2.46 (m,
2H), 2.90 (d, J ) 5 Hz, 6H), 3.28 (m, 2H), 4.23 (t, J ) 6 Hz, 2H),
4.81 (brs, 1H), 5.91 (d, J ) 10 Hz, 1H), 6.66 (d, J ) 9 Hz, 2H),
8.39 (s, 1H); MS (ES+): m/z 479.1 (M + 1). Anal. (C19H20-
ClF5N6O‚2HCl) C, H, N.
1
3.56 g (96%) of 5 as a light yellow solid: mp 124-126 °C; H
5-Chloro-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophe-
nyl}-N-(2,2,2-trifluoroethyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (12). According to the procedure used to prepare 8, reaction
of 6 with 3-dimethylamino-1-propanol provided hydrogen chloride
salt of 12 in 44% yield as a white solid: mp 130 °C (decomposed);
1H NMR (CDCl3) δ 2.47 (m, 2H), 2.87 (d, J ) 5 Hz, 6H), 3.23
(m, 2H), 4.26 (t, J ) 6 Hz, 2H), 4.42 (m, 2H), 6.47 (m, 1H), 6.66
(d, J ) 8 Hz, 2H), 8.39 (s, 1H); MS (ES+): m/z 465.1 (M + 1).
Anal. (C18H18ClF5N6O‚1.95HCl) C, H, N.
5-Chloro-6-(4-{3-[ethyl(methyl)amino]propoxy}-2,6-difluo-
rophenyl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (13). According to the procedure used
to prepare 8, reaction of 5 with 3-[ethyl(methyl)amino]propan-1-
ol26 provided 13 in 23% yield as colorless oil: 1H NMR (CDCl3)
δ 1.07 (t, J ) 7 Hz, 3H), 1.40 (d, J ) 7 Hz, 3H), 2.02 (m, 2H),
2.26 (s, 3H), 2.46 (q, J ) 7 Hz, 2H), 2.54 (t, J ) 7 Hz, 2H), 4.11
(t, J ) 6 Hz, 2H), 4.76 (brs, 1H), 5.88 (brs, 1H), 6.66 (m, 2H),
8.39 (s, 1H); MS (ES+): m/z 493.1 (M + 1). Anal. (C20H22-
ClF5N6O) C, H, N.
5-Chloro-6-[2,6-difluoro-4-(3-pyrrolidin-1-ylpropoxy)phenyl]-
N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (14). According to the procedure used to prepare
8, reaction of 5 with 3-pyrrolidin-1-ylpropan-1-ol27 provided 14 in
60% yield as a tan solid: mp 35-38 °C; 1H NMR (CDCl3) δ 1.41
(d, J ) 7 Hz, 3H), 1.83 (m, 4H), 2.08 (m, 2H), 2.59 (m, 4H), 2.69
(t, J ) 7 Hz, 2H), 4.12 (t, J ) 6 Hz, 2H), 4.78 (brs, 1H), 5.89 (brs,
1H), 6.67 (m, 2H), 8.39 (s, 1H); MS (ES+): m/z 505.2 (M + 1).
Anal. (C21H22ClF5N6O‚0.3EtOAc) C, H, N.
5-Chloro-6-[2,6-difluoro-4-(3-piperidin-1-ylpropoxy)phenyl]-
N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (15). According to the procedure used to prepare
8, reaction of 5 with 3-piperidin-1-ylpropan-1-ol27 provided 15 in
65% yield as a tan solid: mp 40-43 °C; 1H NMR (CDCl3) δ 1.40
(d, J ) 7 Hz, 3H), 1.46 (m, 2H), 1.61 (m, 4H), 2.03 (m, 2H), 2.43
(m, 4H), 2.49 (t, J ) 7 Hz, 2H), 4.10 (t, J ) 6 Hz, 2H), 4.78 (brs,
1H), 5.87 (brs, 1H), 6.67 (m, 2H), 8.39 (s, 1H); MS (ES+): m/z
519.3 (M + 1). Anal. (C22H24ClF5N6O) C, H, N.
NMR (DMSO-d6) δ 1.44 (d, J ) 7 Hz, 3H), 6.17 (m, 1H), 7.48
(m, 2H), 8.03 (d, J ) 8 Hz, 1H), 8.70 (s, 1H); MS (ES+): m/z
396.0 (M + 1). Anal. (C14H8ClF6N5) C, H, N.
Chiral HPLC showed that the compound had a 99.6% ee.
5-Chloro-6-(2,4,6-trifluorophenyl)-N-(2,2,2-trifluoroethyl)-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (6). According to the
procedure used to prepare 5, reaction of 419 with 2,2,2-trifluoro-
ethylamine provided 6 in 95% yield as a light yellow solid: mp
1
168-170 °C; H NMR (CDCl3) δ 4.19 (m, 2H), 6.17 (m, 1H),
6.93 (m, 2H), 8.42 (s, 1H); MS (ES+): m/z 382.0 (M + 1). Anal.
(C13H6ClF6N5) C, H, N.
5-Chloro-6-(2,4,6-trifluorophenyl)-N-[(1R)-2,2,2-trifluoro-1-
methylethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (7). Accord-
ing to the procedure used to prepare 5, reaction of 419 with (1R)-
2,2,2-trifluoro-1-methylethylamine hydrogen chloride21 provided 7
1
in 83% yield as a light yellow solid: mp 98-101 °C; H NMR
(CDCl3) δ 1.43 (d, J ) 7 Hz, 3H), 4.69 (m, 1H), 5.87 (d, J ) 10
Hz, 1H), 6.93 (m, 2H), 8.41 (s, 1H); MS (ES+): m/z 396.1 (M +
1). Anal. (C14H8ClF6N5) C, H, N.
5-Chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-
N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (8). To a suspension of sodium hydride (60% in
mineral oil, 2.3 g, 57.6 mmol) in 20 mL of dimethylsulfoxide at
room temperature was added a solution of 3-(methylamino)propan-
1-ol25 (5.14 g, 57.6 mmol) in 10 mL of dimethyl sulfoxide. The
solution was stirred at room temperature for 1 h, and 5 (5.7 g, 14.4
mmol) was added. The mixture was heated at 60 °C for 3 h and
cooled to room temperature. The reaction mixture was diluted with
ethyl acetate and washed with water and saturated sodium chloride.
The organic layer was dried over magnesium sulfate, and concen-
trated. The residue was triturated with small amount of acetone
and then hexanes and chromatographed over silica gel, eluting with
a gradient of 100% ethyl acetate to 100% methyl alcohol to provide
2.7 g (40%) of 8 as a white solid. This product was dissolved in
150 mL of 10% methyl alcohol in methylene chloride and filtered.
To the filtrate was bubbled hydrogen chloride gas. Concentration
provided 2.92 g of hydrogen chloride salt of 8 as a light yellow
5-Chloro-6-[2,6-difluoro-4-(3-morpholin-4-ylpropoxy)phenyl]-
N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (16). According to the procedure used to prepare
8, reaction of 5 with 3-morpholin-4-ylpropan-1-ol27 provided
hydrogen chloride salt of 16 in 65% yield as a white solid: mp
1
solid: mp 42-44 °C; H NMR (DMSO-d6) δ 1.44 (d, J ) 7 Hz,
3H), 2.14 (m, 2H), 2.57 (t, J ) 6 Hz, 3H), 3.05 (m, 2H), 4.20 (t,
J ) 6 Hz, 2H), 5.90 (m, 1H), 6.99 (d, J ) 9 Hz, 2H), 8.09 (d, J )
9 Hz, 1H), 9.01 (m, 2H); MS (ES+): m/z 465.2 (M + 1). Anal.
(C18H18ClF5N6O‚2HCl) C, H, N.
1
68-70 °C; H NMR (CDCl3) δ 1.43 (d, J ) 7 Hz, 3H), 2.57 (m,
2H), 2.94 (m, 2H), 3.24 (m, 2H), 3.52 (d, J ) 12 Hz, 2H), 4.02 (d,
J ) 10 Hz, 2H), 4.22 (t, J ) 7 Hz, 2H), 4.35 (t, J ) 12 Hz, 2H),
4.78 (brs, 1H), 5.90 (d, J ) 10 Hz, 1H), 6.65 (d, J ) 8 Hz, 2H),
8.39 (s, 1H); MS (ES+): m/z 521.1 (M + 1). Anal. (C21H22-
ClF5N6O2‚2HCl) C, H, N.
6-[4-(3-Azetidin-1-ylpropoxy)-2,6-difluorophenyl]-5-chloro-N-
[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyrimi-
din-7-amine (17). According to the procedure used to prepare 8,
reaction of 5 with 3-azetidin-1-ylpropan-1-ol27 provided 17 in 45%
5-Chloro-6-{2,6-difluoro-4-[3-(methylamino)propoxy]phenyl}-
N-(2,2,2-trifluoroethyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine
(9). According to the procedure used to prepare 8, reaction of 6
with 3-(methylamino)propan-1-ol25 provided hydrogen chloride salt
1
of 9 in 50% yield as a yellow solid: mp 78-80 °C; H NMR
(DMSO-d6) δ 2.11 (m, 2H), 2.58 (t, J ) 5 Hz, 3H), 3.05 (m, 2H),
4.20 (t, J ) 6 Hz, 2H), 4.72 (m, 2H), 7.00 (d, J ) 9 Hz, 2H), 8.48
(t, J ) 7 Hz, 1H), 8.68 (s, 1H), 8.37 (brs, 2H); MS (ES+): m/z
451.3 (M + 1). Anal. (C17H16ClF5N6O‚1.3HCl) C, H, N.

324 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Zhang et al.
1
yield as a tan solid: mp 48-50 °C; H NMR (CDCl3) δ 1.41 (d,
(t, J ) 7 Hz, 2H), 4.06 (t, J ) 6 Hz, 2H), 4.77 (m, 1H), 5.90 (brs,
1H), 6.65 (m, 2H), 8.39 (s, 1H); MS (ES+): m/z 493.1 (M + 1).
Anal. (C20H22ClF5N6O‚0.6 H2O) C, H, N.
J ) 7 Hz, 3H), 1.88 (m, 2H), 2.11 (m, 2H), 2.46 (m, 2H), 2.62 (t,
J ) 7 Hz, 2H), 3.25 (m, 2H), 4.08 (t, J ) 6 Hz, 2H), 4.77 (brs,
1H), 5.88 (brs, 1H), 6.66 (m, 2H), 8.39 (s, 1H); MS (ES+): m/z
491.1 (M + 1). Anal. (C20H20ClF5N6O‚0.3EtOAc) C, H, N.
6-[4-(3-Aminopropoxy)-2,6-difluorophenyl]-5-chloro-N-[(1S)-
2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (18). According to the procedure used to prepare 8, reaction
of 5 with 3-amino-1-propanol provided 18 in 42% yield as a yellow
4-[4-(5-Chloro-7-{[(1S)-2,2,2-trifluoro-1-methylethyl]amino}-
[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenoxy]butan-
1-ol (26). According to the procedure used to prepare 8, reaction
of 5 with 1,4-butanediol provided 26 in 23% yield as a light yellow
oil: 1H NMR (CDCl3) δ 1.37 (t, J ) 5 Hz, 1H), 1.41 (d, J ) 7 Hz,
3H), 1.79 (m, 2H), 1.96 (m, 2H), 3.77 (q, J ) 7 Hz, 2H), 4.09 (t,
J ) 6 Hz, 2H), 4.77 (brs, 1H), 5.88 (d, J ) 10 Hz, 1H), 6.65 (m,
2H), 8.39 (s, 1H); MS (ES-): m/z 463.9 (M-1). Anal. (C18H17-
ClF5N5O2‚0.4EtOAc) C, H, N.
5-[4-(5-Chloro-7-{[(1S)-2,2,2-trifluoro-1-methylethyl]amino}-
[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenoxy]pentan-
1-ol (27). According to the procedure used to prepare 8, reaction
of 5 with 1,5-pentanediol provided 27 in 63% yield as a light yellow
oil: 1H NMR (CDCl3) δ 1.32 (t, J ) 5 Hz, 1H), 1.41 (d, J ) 7 Hz,
3H), 1.60 (m, 2H), 1.67 (m, 2H), 1.89 (m, 2H), 3.71 (q, J ) 6 Hz,
2H), 4.04 (t, J ) 6 Hz, 2H), 4.76 (brs, 1H), 5.89 (d, J ) 10 Hz,
1H), 6.65 (m, 2H), 8.39 (s, 1H); MS (ES-): m/z 477.9 (M-1). Anal.
(C19H19ClF5N5O2‚0.3EtOAc) C, H, N.
5-Chloro-6-(4-{[3-(dimethylamino)propyl]thio}-2,6-difluo-
rophenyl)-N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (28). According to the procedure used
to prepare 8, reaction of 5 with 3-(dimethylamino)propan-1-thiol28
provided 28 in 48% yield as a white solid: mp 38-40 °C; 1H NMR
(CDCl3) δ 1.41 (d, J ) 7 Hz, 3H), 1.90 (m, 2H), 2.26 (s, 6H), 2.44
(t, J ) 7 Hz, 2H), 3.09 (t, J ) 7 Hz, 2H), 4.80 (brs, 1H), 5.86 (d,
J ) 10 Hz, 1H), 7.03 (m, 2H), 8.40 (s, 1H); MS (ES+): m/z 495.2
(M + 1). Anal. (C19H20ClF5N6S) C, H, N.
1
solid: mp 170-180 °C; H NMR (CDCl3) δ 1.41 (d, J ) 7 Hz,
3H), 2.05 (m, 2H), 3.04 (t, J ) 7 Hz, 2H), 4.16 (t, J ) 6 Hz, 2H),
4.82 (m, 1H), 6.68 (m, 2H), 8.38 (s, 1H); MS (ES-): m/z 448.9
(M-1). Anal. (C17H16ClF5N6O‚1.0CH3OH) C, H, N.
3-[4-(5-Chloro-7-{[(1S)-2,2,2-trifluoro-1-methylethyl]amino}-
[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenoxy]propan-
1-ol (19). According to the procedure used to prepare 8, reaction
of 5 with 1,3-propanediol provided 19 in 66% yield as a tan solid:
1
mp 86-88 °C; H NMR (CDCl3) δ 1.41 (d, J ) 7 Hz, 3H), 1.52
(t, J ) 4 Hz, 1H), 2.11 (m, 2H), 3.90 (q, J ) 4 Hz, 2H), 4.13 (t,
J ) 7 Hz, 2H), 4.77 (m, 1H), 5.89 (d, J ) 6 Hz, 1H), 6.68 (m,
2H), 8.39 (s, 1H); MS (ES+): m/z 452.1 (M + 1). Anal. (C17H15-
ClF5N5O2‚0.25EtOAc) C, H, N.
5-Chloro-6-[4-(3-ethoxypropoxy)-2,6-difluorophenyl]-N-[(1S)-
2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (20). According to the procedure used to prepare 8, reaction
of 5 with 3-ethoxy-1-propanol provided 20 in 52% yield as a light
yellow oil: 1H NMR (CDCl3) δ 1.22 (t, J ) 7 Hz, 3H), 1.41 (d,
J ) 7 Hz, 3H), 2.11 (m, 2H), 3.52 (q, J ) 7 Hz, 2H), 3.61 (t, J )
6 Hz, 2H), 4.15 (t, J ) 6 Hz, 2H), 4.76 (m, 1H), 5.89 (d, J ) 11
Hz, 1H), 6.67 (m, 2H), 8.39 (s, 1H); MS (ES-): m/z 477.9 (M-1).
Anal. (C19H19ClF5N5O2‚0.2CH3OH) C, H, N.
6-(4-Butoxy-2,6-difluorophenyl)-5-chloro-N-[(1S)-2,2,2-trifluoro-
1-methylethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21). Ac-
cording to the procedure used to prepare 8, reaction of 5 with
1-butanol provided 21 in 34% yield as a light yellow oil: 1H NMR
(CDCl3) δ 1.01 (t, J ) 6 Hz, 3H), 1.40 (d, J ) 7 Hz, 3H), 1.52 (m,
2H), 1.84 (m, 2H), 4.03 (q, J ) 6 Hz, 2H), 4.76 (m, 1H), 5.89 (d,
J ) 10 Hz, 1H), 6.65 (m, 2H), 8.39 (s, 1H); MS (ES-): m/z 447.9
(M-1). Anal. (C18H17ClF5N5O‚0.2EtOAc) C, H, N.
5-Chloro-6-{4-[2-(dimethylamino)ethoxy]-2,6-difluorophenyl}-
N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (22). According to the procedure used to prepare
8, reaction of 5 with N,N-dimethylethanolamine provided 22 in 46%
yield as a light yellow oil: 1H NMR (CDCl3) δ 1.41 (d, J ) 7 Hz,
3H), 2.40 (s, 6H), 2.82 (t, J ) 6 Hz, 2H), 4.15 (t, J ) 6 Hz, 2H),
4.72 (m, 1H), 5.90 (d, J ) 9 Hz, 1H), 6.69 (m, 2H), 8.39 (s, 1H);
MS (ES+): m/z 465.1 (M + 1). Anal. (C18H18ClF5N6O‚1.0CH3-
OH) C, H, N.
5-Chloro-6-[2,6-difluoro-4-(2-morpholin-4-ylethoxy)phenyl]-
N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (23). According to the procedure used to prepare
8, reaction of 5 with 4-(2-hydroxyethyl)morpholine provided 23
N1-[4-(5-Chloro-7-{[(1S)-2,2,2-trifluoro-1-methylethyl]amino}-
[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl]-N1,N3,N3-
trimethylpropane-1,3-diamine (29). To a mixture of 5 (200 mg,
0.51 mmol) in N,N,N′-trimethyl-1,3-propanediamine (3.0 g, 25.8
mmol) was added sodium hydride (100 mg, 2.5 mmol). The
resulting mixture was heated at 100 °C for 16 h. The reaction was
then quenched with water and extracted with ethyl acetate (×2).
The combined organic extracts were washed with saturated sodium
chloride, dried over magnesium sulfate, and concentrated. The
residue was chromatographed over silica gel, eluting with a gradient
of 100% ethyl acetate to 50% methyl alcohol in ethyl acetate to
100% methyl alcohol to provide 20 mg (8%) of 29 as a light yellow
solid: mp 50-55 °C; 1H NMR (CDCl3) δ 1.41 (d, J ) 7 Hz, 3H),
1.78 (m, 2H), 2.24 (s, 6H), 2.29 (t, J ) 6 Hz, 2H), 3.02 (s, 3H),
3.43 (t, J ) 7 Hz, 2H), 4.90 (brs, 1H), 5.86 (brs, 1H), 6.38 (m,
2H), 8.37 (s, 1H); MS (ES+): m/z 492.1 (M + 1). Anal. (C20H23-
ClF5N7) C, H, N.
N1-[4-(5-Chloro-7-{[(1S)-2,2,2-trifluoro-1-methylethyl]amino}-
[1,2,4]triazol o[1,5-a]pyrimidin-6-yl)-3,5-difluorophenyl]-N3,N3-
dimethylpropane-1,3-diamine (30). According to the procedure
used to prepare 29, reaction of 5 with 3-(dimethylamino)propy-
lamine provided 30 in 17% yield as yellow oil: 1H NMR (CDCl3)
δ 1.41 (d, J ) 6 Hz, 3H), 1.81 (m, 2H), 2.27 (s, 6H), 2.46 (t, J )
6 Hz, 2H), 3.24 (m, 2H), 4.91 (brs, 1H), 5.94 (brs, 2H), 6.25 (m,
2H), 8.36 (s, 1H); MS (ES+): m/z 478.2 (M + 1). Anal. (C19H21-
ClF5N7‚0.5CH3OH) C, H, N.
5-Chloro-6-{4-[4-(dimethylamino)butyl]-2,6-difluorophenyl}-
N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (31). To a solution of 4020 (2.11 g, 7.0 mmol) in
60 mL of methylene chloride at -78 °C was added boron tribromide
(2.65 mL, 28 mmol) dropwise. The mixture was then stirred at
-78 °C for 10 min, warmed to 0 °C, and stirred at 0 °C for 1 h. A
5% aqueous solution of sodium bicarbonate was added slowly to
quench the reaction. The product was extracted with ethyl acetate.
The combined organic extracts were washed with saturated sodium
chloride, dried over magnesium sulfate, and concentrated. The
residue was chromatographed over silica gel, eluting with a gradient
of 10% ethyl acetate in hexanes to 30% ethyl acetate in hexanes,
to provide 1.91 g (95%) of 41 as a colorless oil: 1H NMR (CDCl3)
δ 1.23 (t, J ) 7 Hz, 6H), 4.26 (q, J ) 7 Hz, 4H), 4.86 (s, 1H), 6.01
(brs, 1H), 6.34 (d, J ) 9 Hz, 2H); MS (ES-): m/z 287.2 (M-1).
1
in 64% yield as a light yellow solid: mp 36-38 °C; H NMR
(CDCl3) δ 1.41 (d, J ) 7 Hz, 3H), 2.61 (t, J ) 5 Hz, 4H), 2.86 (t,
J ) 6 Hz, 2H), 3.76 (t, J ) 5 Hz, 4H), 4.17 (t, J ) 6 Hz, 2H), 4.75
(brs, 1H), 5.89 (d, J ) 10 Hz, 1H), 6.68 (m, 2H), 8.39 (s, 1H); MS
(ES-): m/z 504.9 (M-1). Anal. (C20H20ClF5N6O2) C, H, N.
2-[4-(5-Chloro-7-{[(1S)-2,2,2-trifluoro-1-methylethyl]amino}-
[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenoxy]etha-
nol (24). According to the procedure used to prepare 8, reaction of
5 with ethylene glycol provided 24 in 16% yield as a light tan
solid: mp 39-41 °C; 1H NMR (CDCl3) δ 1.41 (d, J ) 7 Hz, 3H),
4.05 (t, J ) 5 Hz, 2H), 4.16 (t, J ) 5 Hz, 2H), 4.73 (m, 1H), 5.90
(d, J ) 6 Hz, 1H), 6.71 (m, 2H), 8.39 (s, 1H); MS (ES+): m/z
438.1 (M + 1). Anal. (C16H13ClF5N5O2) C, H, N.
5-Chloro-6-{4-[4-(dimethylamino)butoxy]-2,6-difluorophenyl}-
N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (25). According to the procedure used to prepare
8, reaction of 5 with 4-dimethylamino-1-butanol provided 25 in
56% yield as a tan solid: mp 91-92 °C; 1H NMR (CDCl3) δ 1.41
(d, J ) 7 Hz, 3H), 1.67 (m, 2H), 1.88 (m, 2H), 2.25 (s, 6H), 2.35

Anticancer [1,2,4]Triazolo[1,5-a]pyrimidines
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 325
To a solution of 41 (288 mg, 1.0 mmol) and triethylamine (505
mg, 5.0 mmol) in 5 mL of methylene chloride at room temperature
was added trifluoromethanesulfonic anhydride (1.41 g, 5.0 mmol).
The mixture was stirred at room temperature for 10 min. A 5%
aqueous solution of sodium bicarbonate was added slowly to quench
the reaction. The product was extracted with ethyl acetate. The
combined organic extracts were washed with saturated sodium
chloride, dried over magnesium sulfate, and concentrated. The
residue was chromatographed over silica gel, eluting with a gradient
of hexanes to 15% ethyl acetate in hexanes, to provide 361 mg
(86%) of 42 as a colorless oil: 1H NMR (CDCl3) δ 1.29 (t, J ) 7
Hz, 6H), 4.28 (q, J ) 7 Hz, 4H), 4.94 (s, 1H), 6.95 (d, J ) 7 Hz,
2H); MS (ES-): m/z 419.2 (M-1).
chromatographed over silica gel, eluting with a gradient of 100%
ethyl acetate to 50% methyl alcohol in ethyl acetate to 100% methyl
alcohol, to provide 44 mg of 31 as a light yellow oil: 1H NMR
(CDCl3) δ 1.39 (d, J ) 6 Hz, 3H), 1.52 (m, 2H), 1.73 (m, 2H),
2.22 (s, 6H), 2.31 (t, J ) 7 Hz, 2H), 2.77 (t, J ) 8 Hz, 2H), 4.66
(brs, 1H), 5.89 (brs, 1H), 6.95 (m, 2H), 8.39 (s, 1H); MS (ES+):
m/z 477.2 (M + 1). Anal. (C20H22ClF5N6‚0.7 CH3OH) C, H, N.
5-Bromo-6-(2,4,6-trifluorophenyl)-N-[(1S)-2,2,2-trifluoro-1-
methylethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (32). A mix-
ture of 5,7-dihydroxy-6-(2,4,6-trifluorophenyl)[1,2,4]triazolo[1,5-
a]pyrimidine19 (282 mg, 1.0 mmol) and phosphorus oxybromide
(2.0 g, 7.0 mmol) was heated at 120 °C for 4 h. Excess phosphorus
oxybromide was then removed in vacuo. The residue was dissolved
in methylene cholride and washed with water and saturated sodium
chloride (×3). The organic layer was dried over magnesium sulfate,
filtered through hydrous magnesium silicate, and concentrated to
provide 380 mg (93%) of crude 5,7-dibromo-6-(2,4,6-trifluorophenyl)-
[1,2,4]triazolo[1,5-a]pyrimidine as a tan semisolid. MS (ES+): m/z
408.9 (M + 1). The material was used directly in the next step
without further purification.
To a 0.5 M solution of 9-borabicyclo[3.3.1]nonane (9-BBN) in
tetrahydrofuran (95 mL, 47.6 mmol) was added dropwise 3-buten-
1-ol (4.1 mL, 47.6 mmol), and the mixture was stirred under
nitrogen atmosphere at room temperature for 6 h. The resulting
solution was then transferred by a double-ended needle into a
mixture of 42 (10.0 g, 23.8 mmol), potassium phosphate (10.1 g,
47.6 mmol), and tetrakis(triphenylphosphine)palladium(0) (825 mg,
0.714 mmol) in 40 mL of dioxane under nitrogen pressure. The
mixture was then heated at 90 °C for 8 h. The reaction was cooled
to room temperature, and trimethylamine-N-oxide (3.57 g, 47.6
mmol) was added. The reaction was heated at 80 °C for 1 h and
cooled to room temperature. Ethyl acetate was added to dilute the
mixture. The organic phase was washed with saturated sodium
chloride (×2), dried over magnesium sulfate, and concentrated. The
residue was chromatographed over silica gel, eluting with a gradient
of 10% ethyl acetate in hexanes to 50% ethyl acetate in hexanes,
to provide 3.5 g (43%) of 43 as a brown oil: 1H NMR (CDCl3) δ
1.28 (t, J ) 7 Hz, 6H), 1.60 (m, 2H), 1.69 (m, 2H), 2.63 (t, J ) 8
Hz, 2H), 3.66 (t, J ) 7 Hz, 2H), 4.25 (q, J ) 7 Hz, 4H), 4.91 (s,
1H), 6.76 (d, J ) 9 Hz, 2H); MS (ES+): m/z 345.2 (M + 1).
To a solution of 43 (2.0 g, 5.8 mmol) and triethylamine (2.43
mL, 17.4 mmol) in 15 mL of methylene chloride at 0 °C was added
methanesulfonyl chloride (0.898 mL, 11.6 mmol). The resulting
mixture was allowed to warm to room temperature in 1.5 h. The
mixture was washed with 10% hydrochloric acid, saturated sodium
bicarbonate, and saturated sodium chloride. The organic layer was
dried over magnesium sulfate and concentrated. The yellow oil thus
obtained was stirred with 2.0 M diethylamine in tetrahydrofuran
(56 mL, 112 mmol) at room temperature for 16 h, followed by
concentration. The residue was diluted with ethyl acetate. The
organic layer was washed with water and saturated sodium chloride,
dried over magnesium sulfate, and concentrated. The residue was
chromatographed over silica gel, eluting with a gradient of 100%
ethyl acetate to 50% methyl alcohol in ethyl acetate to 100% methyl
alcohol, to provide 1.2 g (56%) of 44 as a yellow oil: 1H NMR
(CDCl3) δ 1.27 (t, J ) 7 Hz, 6H), 1.51 (m, 2H), 1.62 (m, 2H),
2.20 (s, 6H), 2.26 (t, J ) 7 Hz, 2H), 2.61 (t, J ) 8 Hz, 2H), 4.25
(q, J ) 7 Hz, 4H), 4.91 (s, 1H), 6.75 (d, J ) 9 Hz, 2H); MS
(ES+): m/z 372.2 (M + 1).
A mixture of 5,7-dibromo-6-(2,4,6-trifluorophenyl)[1,2,4]triazolo-
[1,5-a]pyrimidine (320 mg, 0.78 mmol), (1S)-2,2,2-trifluoro-1-
methylethylamine hydrogen chloride17 (235 mg, 1.57 mmol), and
diisopropylethylamine (260 mg, 2.0 mmol) in 5 mL of N,N-
dimethylformamide was stirred at room temperature for 18 h. Water
was added to quench the reaction, and the product was extracted
with ethyl acetate. The combined organic extracts were washed
with saturated sodium chloride (×3), dried over magnesium sulfate,
and concentrated. The residue was chromatographed over silica gel,
eluting with a gradient of 9:1 hexanes/ethyl acetate to 2:1 hexanes/
ethyl acetate, to provide 60 mg (17%) of 32 as a light tan solid:
1
mp 95-97 °C; H NMR (CDCl3) δ 1.41 (d, J ) 7 Hz, 3H), 4.71
(brs, 1H), 5.79 (d, J ) 10 Hz, 1H), 6.94 (m, 2H), 8.40 (s, 1H); MS
(ES+): m/z 440.0, 442.0 (M + 1). Anal. (C14H8BrF6N5‚0.22EtOAc)
C, H, N.
5-Bromo-6-{4-[3-(dimethylamino)propoxy]-2,6-difluorophe-
nyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (33). To a solution of 32 (44 mg, 0.1 mmol)
and 3-dimethylamino-1-propanol (51 m g, 0.5 mmol) in 1 mL of
dimethylsulfoxide at room temperature was added sodium hydride
(60% in mineral oil, 20 mg, 0.5 mmol). The mixture was heated at
60 °C for 2h, and cooled to room temperature. Water is added to
quench the reaction, and the product is extracted with ethyl acetate.
The organic layer is washed with saturated sodium chloride (×3),
dried over magnesium sulfate, and concentrated. The residue is
chromatographed over silica gel, eluting with a gradient of ethyl
acetate to 30% methyl alcohol in ethyl acetate, to provide 41 mg
(78%) of 33 as a light tan solid: mp 40-42 °C; 1H NMR (CDCl3)
δ 1.41 (d, J ) 7 Hz, 3H), 2.03 (m, 2H), 2.31 (s, 6H), 2.52 (brs,
2H), 4.11 (t, J ) 6 Hz, 2H), 4.81 (brs, 1H), 5.84 (brs 1H), 6.66 (d,
J ) 9 Hz, 2H), 8.38 (s, 1H); MS (ES+): m/z 523.1, 525.1 (M +
1). Anal. (C19H20BrF5N6O‚0.25EtOAc) C, H, N.
A mixture of 44 (1.0 g, 2.7 mmol), 3-amino-1,2,4-triazole (250
mg, 3.0 mmol), and tributylamine (0.71 mL, 3.0 mmol) was stirred
under nitrogen atmosphere at 160 °C for 16 h and cooled to room
temperature. The mixture was stirred with 20 mL of hexanes. The
precipitates were collected by filtration, washed with hexanes, to
give 795 mg (81%) of 45 as a white solid: MS (ES-): m/z 362.1
(M-1).
A mixture of 45 (795 mg, 2.19 mmol) in 4 mL of phosphorus
oxychloride was heated at 115 °C for 4 h. The excess phosphorus
oxychloride was removed in vacuo, and the resulting residue was
dried further under high vacuum to give 1.13 g of 46 as a yellow
solid, which was used without further purification. A mixture of
46 (300 mg, 0.75 mmol), (1S)-2,2,2-trifluoro-1-methylethylamine
hydrogen chloride (675 mg, 4.51 mmol), and N,N-diisopropylethy-
lamine (0.787 mL, 4.51 mmol) in 4 mL of N,N-dimethylformamide
was stirred at room temperature for 18 h. The reaction mixture
was diluted with ethyl acetate. The organic layer was washed with
saturated sodium bicarbonate and saturated sodium chloride, dried
over magnesium sulfate, and concentrated. The residue was
5-Chloro-6-(2,4-difluorophenyl)-N-[(1S)-2,2,2-trifluoro-1-
methylethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (34). Ac-
cording to the procedure used to prepare 5, reaction of 5,7-dichloro-
6-(2,4-difluorophenyl)[1,2,4]triazolo[1,5-a]pyrimidine17 with (1S)-
2,2,2-trifluoro-1-methylethylamine hydrogen chloride17 provided 34
in 86% yield as a white solid: mp 35-37 °C; 1H NMR (CDCl3) δ
1.37 (d, J ) 7 Hz, 3H), 4.51-4.95 (m, 1H), 5.52-5.89 (m, 1H),
7.12 (m, 2H), 7.32 (m, 1H), 8.40 (s, 1H); MS (ES+): m/z 378.2
(M + 1). Anal. (C14H9ClF5N5) C, H, N.
5-Chloro-6-{4-[3-(dimethylamino)propoxy]-2-fluorophenyl}-
N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (35). According to the procedure used to prepare
8, reaction of 34 with 3-dimethylamino-1-propanol provided 35 in
1
52% yield as a light tan solid: mp 36-38 °C; H NMR (CDCl3)
δ 1.36 (d, J ) 7 Hz, 3H), 2.04 (m, 2H), 2.30 (s, 6H), 2.52 (t, J )
7 Hz, 2H), 4.11 (t, J ) 6 Hz, 2H), 4.59 (brs, 0.5H), 5.02 (brs 0.5H),
5.56 (brs 0.5H), 5.92(brs 0.5H), 6.86 (m, 2H), 7.18 (m, 1H), 8.38
(s, 1H); MS (ES+): m/z 461.2 (M + 1). Anal. (C19H21ClF4N6O)
C, H, N.

326 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Zhang et al.
5-Chloro-6-(2,3,6-trifluorophenyl)-N-[(1S)-2,2,2-trifluoro-1-
methylethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (36). Ac-
cording to the procedure used to prepare 5, reaction of 5,7-dichloro-
6-(2,3,6-trifluorophenyl)[1,2,4]triazolo[1,5-a]pyrimidine17 with (1S)-
2,2,2-trifluoro-1-methylethylamine hydrogen chloride17 provided 36
in 85% yield as a white solid: mp 145-150 °C; 1H NMR (CDCl3)
δ 1.44 (d, J ) 7 Hz, 3H), 4.70 (m, 1H), 5.97 (d, J ) 8 Hz, 1H),
7.10 (m, 1H), 7.42 (m, 1H), 8.41 (s, 1H); MS (ES+): m/z 396.2
(M + 1). Anal. (C14H8ClF6N5) C, H, N.
killed. The nine values for each compound were plotted against
concentration, and the concentration that produced a relative color
yield half way between the maximum and minimum was taken as
the IC50 value.
Tumor Xenograft Experiments. Athymic nu/nu female mice
were implanted subcutaneously in the flank with either 1 × 106
U87-MG human glioblastoma cells or 1 × 107 A549 human non-
small cell lung carcinoma (NSCLC) cells. Cells were suspended
in culture medium for injection. When tumors attained a mass of
between 80 and 120 mg for U87-MG or about 300 mg for NSCLC
(day 0), animals were randomized into treatment groups each
containing 5-10 animals. After staging, animals were treated
intravenously with the compound formulated in Klucel or 0.9%
saline according to the schedules given in the figure legend, or
with vehicle alone. Tumor volumes ([length × width2]/2) were
determined at regular intervals. The data were analyzed by a one-
sided Student’s t-test. A p-value e0.05 indicated a statistically
significant reduction in tumor growth of the treated group compared
to that of the vehicle control group. The protocols for KB, KB8.5,
and KB V1 cellular assays have been reported.24
5-Chloro-6-{3-[2-(dimethylamino)ethoxy]-2,6-difluorophenyl}-
N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (37). According to the procedure used to prepare
8, reaction of 36 with N,N-dimethylethanolamine provided 37 in
1
16% yield as a white solid: mp 48-58 °C; H NMR (CDCl3) δ
1.39 (m, 3H), 2.36 (s, 6H), 2.78 (t, J ) 6 Hz, 2H), 4.15 (t, J ) 6
Hz, 2H), 4.64 (brs, 0.5H), 4.74 (brs, 0.5H), 5.91 (brs, 1H), 7.04
(m, 1H), 7.21 (m, 1H), 8.40 (s, 1H); MS (ES+): m/z 465.1 (M +
1). Anal. (C18H18ClF5N6O) C, H, N.
5-Chloro-6-{3-[3-(dimethylamino)propoxy]-2,6-difluorophe-
nyl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (38). According to the procedure used to
prepare 8, reaction of 36 with 3-dimethylamino-1-propanol provided
38 in 10% yield as a white solid: mp 45-50 °C; 1H NMR (CDCl3)
δ 1.39 (m, 3H), 2.05 (m, 2H), 2.26 (s, 6H), 2.47 (m, 2H), 4.16 (m,
2H), 5.91 (brs, 1H), 7.02 (m, 1H), 7.20 (m, 1H), 8.41 (s, 1H); MS
(ES+): m/z 479.1 (M + 1). Anal. (C19H20ClF5N6O‚0.3EtOAc) C,
H, N.
Acknowledgment. The authors gratefully acknowledge the
Wyeth Research Chemical Technology Department for the
spectral data, elemental analysis, and chiral HPLC analysis. We
also thank Dr. Joseph Ashcroft for structural elucidations of
regioisomers through extensive NMR studies, Dr. Ramaswamy
Nilakantan for the molecular modeling support, and Dr. Frank
Loganzo for testing in the KB lines.24
5-Chloro-6-{3-[4-(dimethylamino)butoxy]-2,6-difluorophenyl}-
N-[(1S)-2,2,2-trifluoro-1-methylethyl][1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (39). According to the procedure used to prepare
8, reaction of 36 with 4-dimethylamino-1-butanol provided 37 in
Supporting Information Available: Elemental analysis data
for compounds 5-39 and description of the mechanism study
results. This material is available free of charge via the Internet at
http://pubs.acs.org.
1
15% yield as a white solid: mp 55-60 °C; H NMR (CDCl3) δ
1.41 (m, 3H), 1.69 (m, 2H), 1.81 (m, 2H), 2.23 (s, 6H), 2.33 (t,
J ) 7 Hz, 2H), 4.09 (m, 2H), 4.70 (brs, 1H), 5.90 (brs, 1H), 7.03
(m, 1H), 7.16 (m, 1H), 8.40 (s, 1H); MS (ES+): m/z 493.1 (M +
1). Anal. (C20H22ClF5N6O‚0.3EtOAc) C, H, N.
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JM060717I