Synthesis, SAR study and biological evaluation of novel pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides as anti-proliferative agents

Article abstract of DOI:10.1016/j.bmc.2008.12.046

Checkpoint deficiency of malignant cells can be exploited in cancer drug discovery. Compounds that selectively kill checkpoint-deficient cells versus checkpoint-proficient cells can be utilized to preferentially target tumor cells, while sparing normal ce

Full text of DOI:10.1016/j.bmc.2008.12.046

Bioorganic & Medicinal Chemistry 17 (2009) 2091–2100
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, SAR study and biological evaluation of novel
pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides as anti-proliferative agents
a
a
a
a
b
Yanong D. Wang a, , Erick Honores , Biqi Wu , Steve Johnson , Dennis Powell , Miriam Miranda ,
*
John P. McGinnis b, Carolyn Discafani b, Sridhar K. Rabindran b, Wendy Cheng a, Girija Krishnamurthy a
a Chemical and Screening Sciences, Wyeth Research, Pearl River, NY 10965, USA
b Oncology, Wyeth Research, Pearl River, NY 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Checkpoint deficiency of malignant cells can be exploited in cancer drug discovery. Compounds that
selectively kill checkpoint-deficient cells versus checkpoint-proficient cells can be utilized to preferen-
tially target tumor cells, while sparing normal cells. The protein p21Wafl/Cipl/Sdi1 (hereafter referred to
as p21) inhibits progression of the cell cycle by inhibiting the activity of G1 kinases (cyclin D/cdk4 and
cyclin E-cdk2) and the G2 kinase (cyclin B/cdkl) in response to DNA damage or abnormal DNA content.
The expression of p21 is often low in human cancer cells due to frequent loss of the upstream activator,
p53, and is associated with poor prognosis in some cancer patients. Using an isogenic pair of cell lines,
HCT116 (p21+/+) and 80S14 (p21ꢀ/ꢀ), we have disclosed previously a novel series of pyrazolo[1,5-
a]pyrimidines that preferentially kill the p21-deficient cells. We will present the synthesis, biological
activities and SAR study of a series of pyrazolo[1,5-a]pyrimidines with an optimized phenyl amide moiety
at the C-7 position. The mechanism of action of these compounds will also be discussed.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 14 October 2008
Revised 15 December 2008
Accepted 17 December 2008
Available online 25 December 2008
Keywords:
Pyrazolo[1,5-a]pyrimidine
Checkpoint deficiency
p21 Deficient cells
Microtubulin binding
Anti-proliferative activity
1. Introduction
of the cells to arrest in response to DNA damage and other environ-
mental stressors, followed by endoreduplication and apoptosis.9,10
Cell cycle checkpoints are signal transduction pathways that
monitor the orderly progression of the cell division cycle. Loss of
checkpoint control is a hallmark of tumor cells, as it increases
the mutation rate and allows a more rapid progression to the
tumorigenic state. Checkpoint deficiency in malignant cells can
be exploited in cancer drug discovery.1 Compounds that selectively
kill checkpoint-deficient cells versus checkpoint-proficient cells
can be in principle utilized to preferentially target tumor cells.2,3
The p53 tumor suppressor gene is the major regulator of the G1/
S checkpoint and one of the most commonly mutated genes in hu-
man cancer (50–70%). As an important downstream effector of p53,
p21 inhibits the cyclin-dependent kinases (CDKs) and arrests cell
cycle progression in response to genotoxic stress. The protein
p21 blocks progression of the cell cycle by inhibiting the activity
of G1 (cyclin D/cdk4 and cyclin E-cdk2) and G2 kinases (cyclin B/
cdkl) in response to DNA damage or abnormal DNA content.4–6
The expression of p21 is often low in human cancer cells due to fre-
quent loss of the upstream activator p53, and this low expression is
associated with poor prognosis in some cancer patients.7,8 Disrup-
tion of the checkpoint by deletion of the p21 gene results in failure
It was shown that the absence of p21 renders cells remarkably sen-
sitive to apoptosis following treatment with several cancer thera-
peutics. Vogelstein et al reported1–3 a human colorectal cancer
cell line, 80S14, in which the p21 genes have been inactivated by
targeted gene deletion. These cells show increased chemosensitiv-
ity compared with the isogenic p21-proficient parental cells (HCT
116), to various antineoplastic drugs and gamma irradiation
in vitro and in vivo, validating the role of checkpoints in determin-
ing chemosensitivity.
O
X
N
HN
N
O
1a: X=CH2
1b: X=NH
1c: X=O
S
N
C-7
Using an isogenic pair of cell lines, HCT116 (p21+/+) and 80S14
novel series of pyrazolo[1,5-
(p21ꢀ/ꢀ), we have identified
a
a]pyrimidines, including 1a–1c, that preferentially inhibit the
p21ꢀ/ꢀ cells, as described previously.11–13 For example, pyrazol-
o[1,5-a]-pyrimidin-7-yl phenyl amide 1a exhibited excellent activ-
ity in p21ꢀ/ꢀ cells (IC50 = 0.14
(IC50 = 0.021 M). It was also found that the aryl ketone moiety
and the pyrazolo[1,5-a]pyrimidine core are the essential compo-
lM) and LoVo cells
l
* Corresponding author. Tel.: +1 845 602 5253; fax: +1 845 602 5561.
E-mail address: wangd@wyeth.com (Y.D. Wang).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.046

2092
Y. D. Wang et al. / Bioorg. Med. Chem. 17 (2009) 2091–2100
nents for their anti-proliferative activities. We present here the syn-
thesis and SAR study of a series of pyrazolo[1,5-a]pyrimidines with
an optimized phenyl amide moiety at the C-7 position along with a
group of derivatives with reversed amide and amine side chains.
As illustrated in Scheme 3, pyrazolo[1,5-a]pyrimidines with
substituted phenyl amides at C-7 (compounds 22–30) were pre-
pared starting from substituted m-nitroacetophenone (18a–18e).
In general, substituted m-nitroacetophenones were converted to
enamines (19a–19d) by reaction with DMF–DMA. However, the
reaction between 4-fluoro-3-nitroacetophenone (18e) and DMF–
DMA resulted in a substantial amount of by-product due to the
replacement of fluoro by dimethylamine. To circumvent this prob-
lem, 4-fluoro-3-nitroacetophenone was treated with N,N-dimeth-
ylformamide di-tert-butyl acetal at ambient temperature to
produce enamine 19e cleanly.
All enaminones (19a–19e) reacted smoothly with 5 in HOAc to
give 20a–20e. Reduction of most nitro intermediates (20a–20d)
proceeded readily using Fe in HOAc/MeOH, while a milder reaction
condition (SnCl2/THF) was required for the reduction of 20e in or-
der to minimize the undesired substitution of fluoro by methanol.
Anilines 21a–21e were thus converted to amide 22–28
(RCH2COCl/Et3N/THF), urea 29 (RNCO) or carbamate 30 (p-nitro-
phenyl chloroformate/ROH) analogs.
2. Chemistry
To investigate whether the amide moiety is part of the essential
pharmacophore, compounds 8–12, pyrazolopyrimidines with re-
verse phenyl amide groups, were prepared as shown in Scheme
1. Esterification of m-acetylbenzoic acid provided 3. Treatment of
3 with N,N-dimethylformamide dimethyl acetal (DMF–DMA) gave
enamine 5 which was readily coupled with 414 in HOAc to afford 6.
Compound 6 was hydrolyzed in aqueous KOH to give acid 7, which
was subsequently treated with various amines to provide reverse
amide products 8–12.
To further explore the SAR of the amide side chain, compounds
14–17, wherein various alkyl groups replaced the alkyl carbonyl
group of 1a, were prepared by reductive amination of compound
1311 with a variety of aldehydes in the presence of NaBH(OAc)3
(Scheme 2).
Using an analogous approach, pyrazolo[1,5-a]pyrimidines with
a pyridine or a thiophene moiety (35a–35c, Scheme 4) at C-7 were
prepared starting from the appropriate nitro-substituted hetero-
O
N
NH
CO2Me
CO2Me
CO2H
5
S
O
(a)
H2N
(b)
99%
94%
(c)
84%
O
O
3
2
N
4
O
O
O
O
NR1R2
N
N
N
N
CO2H
N
N
CO2Me
(d)
(e)
47-98%
S
S
S
90%
N
N
N
8-12
6
7
Scheme 1. Reagents and conditions: (a) MeOH/H2SO4, reflux; (b) Me2NCH(OMe)2, reflux; (c) 514, acetic acid, reflux; (d) KOH (aq), heat; (e) R1R2NH/Py-BOP/CH2C.
O
O
NaBH(OAc)3/RCHO/
DMF-CH2Cl2
N
N
NH2
N
N
HN
R
14. R=CH2CH(CH3)2
15, R=CH2C(CH3)3
16, R=n-butyl
S
S
N
N
17, R=CH=CHCH3
14-17
13
13-72%
Scheme 2.
O
NO2
3
NO2
(b)
2
6
O
N
N
NO2
1
(c)
28-53%
4
(a)
36-94%
S
X
X
N
O
74-97%
5
X
18a-18e (X: a, 4-Me; b,
N
4-OMe; c, 4-Cl; d, 6-Cl;
20a-20e
19a-19e
e, 4-F)
O
O
Y
R
N
N
NH2
X
N
N
(d)
HN
O
S
N
16-52%
S
N
X
21a-21e
22-30
Scheme 3. Reagents and conditions: (a) Me2NCH(OMe)2, reflux; (b) 5, acetic acid, reflux; (c) Fe, NH4Cl, MeOH or SnCl2/THF; (d) RCOCl/THF, RNCO, or p-nitrophenyl
chloroformate/ROH.

Y. D. Wang et al. / Bioorg. Med. Chem. 17 (2009) 2091–2100
2093
O
O
N
N
N
N
c
Het
NO2
b
a
NO2
15-55%
Het
Het
NO2
N
NH2
S
S
72-87%
60-81%
N
Het
N
O
O
31a-31c
32a-32c
34a-34c
33a-33c
d
35a-35c
Structures of 35a-35c:
53-84%
O
O
O
N
N
N
N
H
N
N
N
HN
N
H
N
O
S
S
S
S
N
N
N
O
O
S
35c
35b
35a
Scheme 4. Reagents and conditions: (a) Me2NCH(OMe)2, reflux; (b) 4, HOAc, reflux; (c) Fe, NH4Cl, MeOH; (d) (CH3)2CHCH2CO2H/isobutyl chloroformate/N-methylmor-
pholine/THF.
aryl methyl ketones, via a reaction sequence of enamine formation,
cyclocondensation, reduction and amidation.
Table 2
Anti-proliferative activity: substituted phenyls and heteroarylsa
O
3. Results and discussion
N
N
H
Y
N
3.1. Antiproliferative activity in P21 isogenic cells
S
R
N
Ar/Het
O
All pyrazolo[1,5-a]pyrimidines were evaluated for their inhibi-
tory activity in the p21 isogenic cells. Their activities and the selec-
tivity ratios between p21ꢀ/ꢀ (80s14) and p21+/+ (HCT116) were
shown in Tables 1 and 2. Pyrazolo[1,5-a]pyrimidine 8, which has
a reversed isobutyl amide was about 3 fold less active in p21ꢀ/ꢀ
cells than 1a. As seen from the data in Table 1, small alkyl groups
in the amide moiety were well tolerated (9, 10 and 11), while a re-
versed phenyl amide analog showed good inhibitory activity
against p21ꢀ/ꢀ cells (12). This is similar to what we observed with
the amide series (compound 1a and its analogs).11 All reversed
amide analogs showed selectivity ratios of 12–19 fold for p21ꢀ/ꢀ
cells over p21+/+ cells.
compd Ar/Het
R
Y
p21ꢀ/ꢀ
p21+/+
Ratio
IC50
(l
M)b IC50 M)c
(l
1a9
1b9
1c9
22
1,3-Ph
1,3-Ph
1,3-Ph
1,3-Ph-4-Me
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
isopropyl
CH2
NH
O
CH2
CH2
CH2
CH2
0.14
6.4
2.7
1.1
9.2
19
46
30
22
9.2
3.0
20
7.1
36
0.089
0.051
1.0
6.4
0.43
2.8
23
24
25
26
27
28
29
30
1,3-Ph-4-OMe
1,3-Ph-4-Cl
1,3-Ph-6-Cl
1,3-Ph-4-F
1,3-Ph-4-F
1,3-Ph-4-F
1,3-Ph-4-F
1,3-Ph-4-F
8.8
20
CH2 0.089
3.2
2.5
3.7
5.3
2.3
>20
>20
>2.6
CH(CH3)(CF3) CH2
tert-butyl
i-Pr
0.12
0.19
0.041
0.035
2.0
21
19
CH2
NH
O
CH2
CH2
CH2
130
65
>10
>2.6
>3.8
i-Pr
It was also observed that the replacement of amide carbonyl
group with methylene (–CH2–) resulted in loss of activity. For
example, 14, the corresponding analog of 1a, had a IC50 of
35a
35b
35c
2,5-Thiophene i-Pr
2,4-Thiophene i-Pr
3,5-Pyridine
7.8
7.6
i-Pr
0.77
l
M in p21ꢀ/ꢀ cells, comparing to 0.14
lM of 1a. Small alkyl
a
b
c
The IC50 values are average of multiple determinations.
The standard deviations, on average, were 33% of the IC50 value.
The standard deviations, on average, were 25% of the IC50 value.
Table 1
Anti-proliferative activity (IC50
,
l
M): reverse amide and amines derivativesa
O
N
N
R
or alkenyl groups are generally tolerated (15, 16, and 17), showing
IC50 range from 0.43 to 0.84
logs (compounds 14–17) were in general less selective than 1a
l
M in p21ꢀ/ꢀ cells. The amine ana-
S
N
and reversed amide analogs (compounds 8–12).
compd
R
80s14
HCT116
Selectivity
ratio
As shown in Table 2, introducing substitution onto the phenyl
ring has substantial impact on the activity of pyrazol[1,5-a]pyrim-
idine compounds. Compared to 1a, reduced activity in p21ꢀ/ꢀ
cells was seen with 4-methyl derivative 22 and the 4-methoxy
derivatives 23 as well as lessened selectivity over P21+/+ cells.
While a chloro substitution at C-4 position (24) of the phenyl led
to better activity than methyl and methoxy substitutions, a chloro
substitution at-C-6 (25) was unfavorable to the activity in p21ꢀ/ꢀ
cells and its selectivity ratio over P21+/+ cells.
(p21ꢀ/ꢀ)b
(p21+/+)c
1a
8
9
10
11
12
14
15
16
17
NHCO–isobutyl
CONH–isobutyl
CONH–butyl
CONH–cyclopentyl
CONH–CH(CH3)2
CONH–Ph
NH(CH2)2CH(CH3)2
NH(CH2)2C(CH3)3
NH–pentyl
0.14
0.40
0.34
0.39
0.68
0.31
0.77
0.55
0.84
0.43
6.4
5.7
5.3
6.8
>13
5.1
5.7
4.8
7.9
3.8
46
12
16
17
>19
16
7.6
8.9
9.5
8.7
NHCH2CH@CHCH3
Interestingly, 4-fluoro derivative 26 showed improved activity,
a
with an IC50 of 0.089
l
M in p21ꢀ/ꢀ cells. The fluorinated deriva-
The IC50 values are average of multiple determinations except compound 14.
The standard deviations, on average, were 25% of the IC50 value.
The standard deviations, on average, were 36% of the IC50 value.
b
c
tive 27 and tert-butyl analog 28 were comparably active to 1a. Both
urea and carbamate analogs (29 and 30) of 26 were more potent in
p21ꢀ/ꢀ cells compared to the corresponding non-fluorinated com-

2094
Y. D. Wang et al. / Bioorg. Med. Chem. 17 (2009) 2091–2100
pounds (1b and 1c). We also observed a significant improvement
of selectivity ratio against p21+/+ cells for both 29 and 30 (Table 2).
Compounds 35a–35c, having heteroaryl groups (pyridine or
thiophenes) instead of the phenyl group in 1a, were much less ac-
tive in p21ꢀ/ꢀ and less selective against p21+/+ cells, indicating
that the phenyl ring is a crucial component of the anti-proliferative
pharmacophore.
3.4. Pharmacokinetic assessment
To select a compound for in vivo study, the plasma levels of 3
most potent compounds (26, 29, 30) were evaluated. An oral dose
of 50 mg/kg of 26 and 30 was administered to nude mice, and
blood samples were taken after 1 and 4 h. The plasma levels of
compound 30 were determined in a cassette-dosing format at
20 mg/kg po at 1 and 4 h time points. As shown in Table 4, the plas-
ma levels at both 1 h and 4 h time points for amide analog 26 were
substantially higher than those of 29 and 30, even with the adjust-
ment from the fact that 30 was dosed lower than 26.
3.2. Antiproliferative activity in colon cells
To study the activity of the compounds against non-engi-
neered colon tumor cell lines, a group of selected compounds
were further evaluated against a panel of 4 human colon cell
lines (DLD1, HT29, SW620 and LoVo)15,16 and their activities
are shown in Table 3. In general, IC50 values observed for the
pyrazolo[1,5-a]pyrimidine derivatives in the colon cell lines are
fairly correlated with their activities in p21ꢀ/ꢀ cells. The re-
3.5. In vivo xenograft studies
Based on its plasma exposure level, compound 26 was chosen
for evaluation in an in vivo LoVo xenograft model. Tumors were
implanted 3 days prior to dosing and a 30 mg/kg dose of 26 admin-
istered po for 20 days produced a T/C of 57% at day 21 (Fig. 1). A
more effective inhibition of tumour growth was achieved at
50 mg/kg (T/C value: 24%) with statistic significance.
versed amide derivative 8 had an IC50 of 0.06–0.15
the panel, while the amine analog 14 showed IC50 values of
0.081–0.77 M compared to 0.021–0.14 M for compound 1a.
lM across
l
l
All fluoro derivatives (26, 29 and 30), which showed improved
In conclusion, replacement of C-7 phenyl amide group with re-
versed amides or amines led to a reduction in cell growth inhibi-
tion. Fluoro substitution at C-4 position improved the potency
and selectivity while a larger group (Cl, Me or OMe) reduced activ-
ity. Replacement of the phenyl moiety with a heteroaryl group
(phenyl or thiophene) led to a major reduction in activity. Tubulin
binding activity was measured for 26 and may be the underlying
mechanism or a contributing mechanism for anti-proliferative
activity. Compound 26 effectively inhibit the tumor growth in an
in vivo LoVo xenograft model.
potency in p21ꢀ/ꢀ cells over compounds 1a, had IC50 values
ranging from 0.011 to 0.015 lM in LoVo cells compared to
0.021
lM for 1a. Pyridine derivative 35a showed moderate activ-
ity in the colon cell lines.
3.3. Microtubulin binding studies
We have previously determined that pyrazolo[1,5-a]pyrimi-
dines, exemplified by 1a, bind to the colchicines site of tubu-
lin,12,13 suggesting that tubulin binding may be the underlying
mechanism or a contributing mechanism for their potent anti-
proliferative cell activity. Several selected compounds were eval-
uated both for their binding affinity and effect on the oligomer-
4. Experimental
4.1. General methods
ization-state of tubulin. Their binding affinities (KD
were
values)
determined based on the fluorescence change of compound
and tryptophan residues in tubulin upon binding.17 In the bind-
ing assays, unlabeled tubulin was used as a substrate, in which
the intrinsic fluorescence of the protein due to tryptophan resi-
dues served as a reporter group for interactions of inhibitor with
tubulin. Upon binding to compounds, the fluorescence of trypto-
phan residues is quenched significantly due to either electronic
interactions or local conformation/domain changes in the protein
such that the quantum yield of the fluorescence is decreased.
The changes in the fluorescence were used to estimate the bind-
ing affinity.
Melting points were determined in open capillary tubes on a
Meltemp melting point apparatus and are uncorrected. 1H NMR
Table 4
Plasma concentration of 26, 29, 30
Compd
Dosage
(PO)
Plasma concn at 1 h
g/mL)
Plasma concn at 4 h
(lg/mL)
(
l
26
29
30
50 mg/kg
50 mg/kg
20 mg/kg
0.11
0.014
0.038
0.04
0.008
0.006
It was found that compounds 1a and 26 bind to tubulin stoi-
chiometrically, with KD values of 4.3 lM and 2.5 lM, respectively.
In a separate experiment, the oligomerization state of tubulin in
the absence and presence of the p21 inhibitor (26) was evaluated
using static multi-angle light scattering.17 It was shown that both
compound 1a and 26 did not induce oligomerization of tubulin
even though they bind tubulin saturably. We also found that the
compounds’ binding affinities are correlated with their cellular
activities in p21ꢀ/ꢀ cells and colon cell lines.
Table 3
Anti-proliferative activity (IC50, lM) of selected compounds in colon cell lines
compd
p21ꢀ/ꢀ
LoVo
SW620
DLD1
HT-29
1a9
8
14
26
29
30
35a
0.14
0.40
0.77
0.089
0.041
0.045
2.0
0.021
0.15
0.045
0.15
0.11
0.015
0.012
0.015
0.76
0.042
0.06
0.12
0.016
0.019
0.015
0.62
0.036
0.15
0.45
0.016
0.017
0.015
0.67
0.081
0.015
0.011
0.012
0.68
Figure 1. Antitumor activity of 26 in LoVo xenografts.

Y. D. Wang et al. / Bioorg. Med. Chem. 17 (2009) 2091–2100
2095
spectra were recorded using a NT-300 WB spectrometer. Chemical
shifts (d) are in parts per million referenced to Me4Si. Electrospray
(ES) mass spectra were recorded in positive mode on a Micromass
Platfor spectrometer. Electron impact (EI) and high-resolution
mass spectra were obtained on a Finnigan MAT-90 spectrometer.
Flash chromatography was performed with Baker 40 lM silica
gel. Reactions were carried out under an inert atmosphere, either
nitrogen or argon, if not otherwise specified.
nium hexafluorophosphate (290 mg, 0.56 mmol) and isobutyl-
amine (31 mg, 0.43 mmol) in methylene chloride (4 mL) was
stirred at room temperature for 5 h. The reaction mixture was par-
titioned between saturated aqueous sodium bicarbonate and
methylene chloride. The combined organics were dried over so-
dium sulfate, concentrated and purified by flash column chroma-
tography (eluting with a gradient of methanol and methylene
chloride) to give 170 mg (98%) of the title compound as a white so-
lid; 1H NMR (DMSO-d6) d 8.93 (d, J = 3 Hz, 1H), 8.84 (s, 1H), 8.64 (t,
J = 4 Hz, 1H), 8.51 (t, J = 1 Hz, 1H), 8.27 (dd, J = 1, 6 Hz, 1H), 8.23
(dd, J = 1, 3 Hz, 1H), 8.10 (dd, J = 1, 6 Hz, 1H), 8.06 (d, J = 4 Hz,
1H), 7.79 (t, J = 6 Hz, 1H), 7.59 (d, J = 3 Hz, 1H), 7.31 (dd, J = 4,
3 Hz, 1H), 3.13 (t, J = 5 Hz, 2H), 1.91–1.82 (m, 1H), 0.91 (d,
J = 5 Hz, 6H); MS (ES) m/e 405.2 (M+H).
4.2. Methyl 3-acetylbenzoate (3)
3-Acetylbenzoic acid (5.1 g, 31 mmol) was dissolved in anhy-
drous methanol (250 mL) and concentrated sulfuric acid (1.0 mL)
was added. The reaction mixture was heated at reflux overnight,
concentrated and partitioned between water and methylene chlo-
ride. The combined organics were dried over MgSO4, concentrated
to yield 5.25 g (94%) of the title compound as a light yellow liquid,
which was used in next step without further purification, mp 43–
45°C; 1H NMR (CDCl3) d 8.60 (S, 1H), 8.23 (d, J = 6 Hz, 1H), 8.16 (d,
J = 6 Hz, 1H), 7.56 (t, J = 6 Hz, 1H), 3.96 (s, 3H), 2.66 (S, 3h); MS (ES)
m/z 179.1 (M+H).
Anal. (C22H20N4O2S–1.0H2O) C, H, N.
4.6. N-Butyl-3-[3-(2-thienylcarbonyl)pyrazolo[1,5-a]pyrimidin-
7-yl]benzamide (9)
According to the procedure used to prepare 8, reaction of 7 and
n-butylamine provided 9 in 55% yield as an off-white solid: mp
172–174 °C; 1H NMR (DMSO-d6) d 8.93 (d, J = 3 Hz, 1H), 8.84 (s,
1H), 8.62 (t, J = 4 Hz, 1H), 8.50 (s, 1H), 8.27 (d, J = 6 Hz, 1H), 8.23
(d, J = 3 Hz, 1H), 8.10 (d, J = 6 Hz, 1H), 8.06 (dd, J = 1, 4 Hz, 1H),
7.73 (t, J = 6 Hz, 1H), 7.59 (d, J = 3 Hz, 1H), 7.31 (dt, J = 1, 3 Hz,
1H), 3.32–3.28 (m, 2H), 1.57–1.50 (m, 2H), 1.40–1.31 (m, 2H),
0.91 (t, J = 5 Hz, 3H); MS (ES) m/e 405.2 (M+H).
Anal. (C10H10O3) C, H.
4.3. Methyl 3-[3-(2-thienylcarbonyl)pyrazolo[1,5-a]pyrimidin-
7-yl]benzoate (6)
A mixture of 3 (5.28 g, 29.6 mmol) and DMF–DMA (75 mL) was
heated at reflux for 2 h and evaporated to remove volatiles to give a
yellow oil, which crystallized on standing. The crude solid was dis-
solved in methylene chloride. The solution was passed through a
short pad of magnesol, washed with 5% MeOH/methylene chloride.
The filtrate was concentrated to give 6.8 g (99%) of enamine 4
intermediate as a yellow solid, which was used for next step with-
out further purification.
A mixture of enamine intermediate (131 mg, 0.6 mmol) and 5
(120 mg, 0.62 mmol) in HOAc (2.0 mL) was heated at reflux for
5 h, cooled to room temperature and diluted with hexane. The pre-
cipitates were collected by filtration, washed thoroughly with
water and ether, and dried to give 177 mg (87%) of 6 as a tan solid;
1H NMR (DMSO-d6) d 8.91 (d, J = 3 Hz, 1H), 8.85 (s, 1H), 8.69 (t,
J = 1 Hz, 1H), 8.33 (d, J = 6 Hz, 1H), 8.24–8.21 (m, 2H), 8.06 (d,
J = 4 Hz, 1H), 7.81 (t, J = 6 Hz, 1H), 7.59 (d, J = 3 Hz, 1H), 7.31 (dd,
J = 4, 3 Hz, 1H), 3.92 (s, 3H); MS (ES) m/z 364.2 (M+H).
Anal. (C22H20N4O2S–0.3H2O) C, H, N.
4.7. N-Cyclopentyl-3-[3-(2-thienylcarbonyl)pyrazolo[1,5-
a]pyrimidin-7-yl]benzamide (10)
According to the procedure used to prepare 8, reaction of 7 and
cyclopentylamine provided 10 in 58% yield as an off-white solid:
mp 207–209 °C; 1H NMR (DMSO-d6) d 8.93 (d, J = 3 Hz, 1H), 8.84
(s, 1H), 8.48 (t, J = 1 Hz, 1H), 8.45 (d, J = 5 Hz, 1H), 8.25 (dd, J = 1,
6 Hz, 1H), 8.23 (dd, J = 1, 3 Hz, 1H), 8.10 (d, J = 6 Hz, 1H), 8.06 (dd,
J = 1, 4 Hz, 1H), 7.73 (t, J=6 Hz, 1H), 7.59 (d, J = 3 Hz, 1H), 7.31 (dd,
J = 3, 4 Hz, 1H), 4.29–4.24 (m, 1H), 1.93–1.90 (m, 2H), 1.71–1.68
(m, 2H), 1.65–1.50 (m, 4H); MS (ES) m/e 461.1 (M+HCOOꢀ).
Anal. (C23H20N4O2S–0.2H2O) C, H, N.
4.8. N-isoPropyl-3-[3-(2-thienylcarbonyl)pyrazolo[1,5-
a]pyrimidin-7-yl]benzamide (11)
Anal. (C10H10O3-0.5H2O) C, H, N.
4.4. 3-[3-(2-Thienylcarbonyl)pyrazolo[1,5-a]pyrimidin-7-
yl]benzoic acid (7)
According to the procedure used to prepare 8, reaction of 7 and
isopropylamine provided 11 in 47% yield as a white solid: mp 218–
220 °C; 1H NMR (DMSO-d6) d 8.93 (d, J = 3 Hz, 1H), 8.84 (s, 1H),
8.48 (t, J = 1 Hz, 1H), 8.40 (d, J = 5 Hz, 1H), 8.26 (dd, J = 1, 6 Hz,
1H), 8.23 (dd, J = 1, 3 Hz, 1H), 8.10 (d, J = 6 Hz, 1H), 8.06 (dd, J = 1,
4 Hz, 1H), 7.73 (t, J = 6 Hz, 1H), 7.59 (d, J = 3 Hz, 1H), 7.31 (dd,
J = 3, 4 Hz, 1H), 4.17–4.10 (m, 1H), 1.20 (d, J = 5 Hz, 4H); MS (ES)
m/e 391.1 (M+H).
A mixture of 6 (177 mg, 0.49 mmol), potassium hydroxide
(1.0 M, 5.0 mL) and methanol (0.3 mL) was stirred at room temper-
ature overnight and then heated at 50 °C for 15 min. The reaction
mixture was cooled to room temperature and solid precipitated.
The resulting suspension was diluted with water until a solution
and acidified with concentrated hydrochloric acid. The precipitates
were collected, washed with water and dried to give 154 mg (90%)
of 7 as a light yellow solid: mp 174–177 °C; 1H NMR (DMSO-d6) d
13.3 (s, br, 1H), 8.91 (d, J = 3 Hz, 1H), 8.85 (s, 1H), 8.69 (t, J = 1 Hz,
1H), 8.32 (d, J = 6 Hz, 1H), 8.23 (dd, J = 1, 3 Hz, 1H), 8.20 (d, J = 6 Hz,
1H), 8.06 (d, J = 4 Hz, 1H), 7.79 (t, J = 6 Hz, 1H), 7.59 (d, J = 3 Hz, 1H),
7.31 (dd, J = 3, 4 Hz, 1H); MS (ES) m/z 348.0 (M+H).
Anal. (C21H18N4O2S–0.1H2O) C, H, N.
4.9. N-Phenyl-3-[3-(2-thienylcarbonyl)pyrazolo[1,5-
a]pyrimidin-7-yl]benzamide (12)
According to the procedure used to prepare 8, reaction of 7 and
aniline provided 12 in 51% yield as a white solid: mp 211–212 °C;
1H NMR (DMSO-d6) d 10.42 (s, 1H), 8.95 (d, J = 3 Hz, 1H), 8.86 (s,
1H), 8.62 (t, J = 1 Hz, 1H), 8.34 (dd, J = 1, 5 Hz, 1H), 8.24 (dd, J = 1,
3 Hz, 1H), 8.22 (d, J = 6 Hz, 1H), 8.06 (dd, J = 1, 4 Hz, 1H), 7.83–
7.80 (m, 3H), 7.65 (d, J = 3 Hz, 1H), 7.39 (d, J = 5 Hz, 1H), 7.38 (d,
J = 6 Hz, 1H), 7.13 (t, J = 5 Hz, 1H); MS (ES) m/e 425.1 (M+H).
Anal. (C24H16N4O2S–0.5H2O) C, H, N.
4.5. N-isobutyl-3-[3-(2-thienylcarbonyl)pyrazolo[1,5-
a]pyrimidin-7-yl]benzamide (8)
A mixture of 7 (150 mg, 0.43 mmol), diisopropylethylamine
(167 mg, 1.3 mmol), benzotriazole-1-yloxy-tripyrrolidinophospho-

2096
Y. D. Wang et al. / Bioorg. Med. Chem. 17 (2009) 2091–2100
4.10. {7-[3-(isoPentylamino)phenyl]pyrazolo[1,5-a]pyrimidin-
4.15. (2E)-3-(Dimethylaino)-1-(4-methoxy-3-nitrophenyl)prop-
3-yl}(thien-2-yl)methanone (14)
2-en-1-one (19b)
The mixture of 13 (100 mg, 0.312 mmol) and 3-metylbutyralde-
hyde (35 mg, 0.328 mmol) in methylene chloride (5.0 mL and DMF
(1.0 mL) was stirred at room temperature for 15 min and
Na(OAc)3BH (330 mg, 1.56 mmol) was added in portions. The reac-
tion mixture was stirred at room temperature for 3 h, concentrated
to dryness, dissolved in methanol (4.0 mL) and purified by HPLC
(eluting with a gradient of acetonitrile/H2O) to give 86 mg (72%)
of 14 as a yellow solid: mp 75–77 °C; 1H NMR (DMSO-d6) d 8.85
(d, J = 4 Hz, 1H), 8.80 (s, 1H), 8.24 (t, J = 1 Hz, 1H), 8.04 (d,
J = 3 Hz, 1H), 7.43 (d, J = 4 Hz, 1H), 7.33–7.29 (m, 2H), 7.25 (s,
1H), 7.20 (d, J = 6 Hz, 1H), 6.83 (d, J = 5 Hz, 1H), 6.1–5.7 (s, br,
1H), 3.08 (t, J = 5 Hz, 2H), 1.75–1.67 (m, 1H), 1.49 (q, J = 5 Hz,
2H), 0.92 (d, J = 5 Hz, 6H); MS (ES) m/e 391.2 (M+H).
According to the procedure used to prepare 4, reaction of 4-
methoxy-3-nitroacetophenone and DMF–DMA provided 19b in
84% yield as an orange solid: mp 165–167 °C; 1H NMR (CDCl3) d
8.38 (d, J = 2 Hz, 1H), 8.18 (dd, J = 2, 7 Hz, 1H), 7.84 (d, J = 9 Hz,
1H), 7.11 (d, J = 7 Hz, 1H), 5.66 (d, J = 9 Hz, 1H), 4.01 (s, 3H), 3.18
(s, 3H), 2.96 (s, 3H); MS (ES) m/e 251.2 (M+H).
Anal. (C12H14N2O4) C, H, N.
4.16. (2E)-3-(Dimethylaino)-1-(4-chloro-3-nitrophenyl)prop-2-
en-1-one (19c)
According to the procedure used to prepare 4, reaction of 4-
chloro-3-nitroacetophenone and DMF–DMA provided 19c in 36%
yield as an orange solid: mp 150–152 °C; 1H NMR (DMSO-d6) d
8.51 (s, 1H, 8.19 (d, J = 6 Hz, 1H), 7.85–7.81 (m, 2H), 5.75 (d,
J = 9 Hz, 1H), 3.19 (s, 3H), 2.96 (s, 3H); MS (ES) m/e 255.1 (M+H).
4.11. (7-{3-[(3,3-Dimethylbutyl)amino]phenyl}pyrazolo[1,5-
a]pyrimidin-3-yl)(thien-2- yl)methanone (15)
Anal. (C11H11ClN2O3–0.1H2O) C, H, N.
According to the procedure used to prepare 14, reaction of 13
and 3, 3-dimethylbutyraldehyde provided 15 in 45% yield as a yel-
low solid: mp 127–130 °C; 1H NMR (DMSO-d6) d 8.85 (d, J = 3 Hz,
1H), 8.79 (s, 1H), 8.24 (dd, J = 1, 3 Hz, 1H), 8.05 (dd, J = 1, 4 Hz,
1H), 7.33–7.29 (m, 2H), 7.23 (d, J = 1 Hz, 1H), 7.18 (d, J = 3 Hz,
1H), 6.81 (dd, J = 1, 6 Hz, 1H), 5.86 (t, J = 4 Hz, 1H), 3.11–3.05 (m,
2H), 1.55–1.49 (m, 2H), 0.95 (s, 9H); MS (ES) m/e 405.2 (M+H).
Anal. (C21H20N4OS–0.25H2O) C, H, N.
4.17. (2E)-3-(Dimethylaino)-1-(2-chloro-5-nitrophenyl)prop-2-
en-1-one (19d)
According to the procedure used to prepare 4, reaction of 2-
chloro-5-nitroacetophenone and DMF–DMA provided 19d in 65%
yield as an orange solid: mp 123–125 °C; 1H NMR (DMSO-d6) d 8.21
(dd, J = 6, 9 Hz, 1H), 8.20–8.00 (m, 2H), 7.76 (d, J = 9 Hz, 1H), 5.29
(d, J = 9 Hz, 1H), 3.09 (s, 3), 2.86 (s, 3H); MS (ES) m/e 255.1 (M+H).
4.12. {7-[3-(Butylamino)phenyl]pyrazolo[1,5-a]pyrimidin-3-
yl}(thien-2-yl)methanone (16)
4.18. (2E)-3-(Dimethylamino)-1-(4-fluoro-3-nitrophenyl)prop-
2-en-1-one (19e)
According to the procedure used to prepare 14, reaction of 13
and n-butyraldehyde provided 16 in 13% yield as a yellow solid:
mp 87–91 °C; 1H NMR (DMSO-d6) d 8.85 (d, J = 3 Hz, 1H), 8.80 (s,
1H), 8.24 (dd, J = 1, 3 Hz, 1H), 8.04 (dd, J = 1, 4 Hz, 1H), 7.43 (d,
J = 3 Hz, 1H), 7.32–7.28 (m, 2H), 7.23 (d, J = 1 Hz, 1H), 7.18 (d,
J = 3 Hz, 1H), 6.82 (dd, J = 1, 6 Hz, 1H), 5.94 (t, J = 4 Hz, 1H), 3.07
(q, J = 5 Hz, 2H), 1.61–1.53 (m, 2H), 1.45–1.36 (m, 2H), 0.92 (t,
J = 5 Hz, 3H); MS (ES) m/e 377.1 (M+H).
To 4-Acetyl-2-aminofluorobenzene18 (15.0 g, 81.0 mmol) in THF
(300 mL) was added N,N-dimethylformamide di-tert-butyl acetal
(29 mL, 243 mmol) in portions. The reaction mixture was stirred
at room temperature overnight and evaporated. The residual solids
were collected by filtration, washed with water and ether to give
17.8 g (94%) of 19e as an orange solid: mp 131–136 °C; 1H NMR
(DMSO-d6) d 8.57 (dd, J = 2, 5 Hz, 1H), 8.36–8.32 (m, 1H), 7.82 (d,
J = 9 Hz, 1H), 7.65 (dd, J = 7, 8 Hz, 1H), 5.96 (d, J = 9 Hz, 1H), 3.18
(s, 3H), 2.97 (s, 3H); MS (ES) m/e 239.2 (M+H).
Anal. (C21H20N4OS–0.95H2O) C, H, N.
Anal. (C11H11FN2O3) C, H, N.
4.13. (7-{3-[(2E)-But-2-enylamino]phenyl}pyrazolo[1,5-
a]pyrimidin-3-yl)(thien-2- yl)methanone (17)
4.19. [7-(4-Methyl-3-nitrophenyl)pyrazolo[1,5-a]pyrimidin-3-
According to the procedure used to prepare 14, reaction of 13
and crotonaldehyde provided 17 in 32% yield as a yellow solid:
mp 110–113 °C; 1H NMR (DMSO-d6) d 8.85 (d, J = 3 Hz, 1H), 8.79
(s, 1H), 8.23 (dd, J = 1, 3 Hz, 1H), 8.05 (dd, J = 1, 4 Hz, 1H), 7.41 (d,
J = 4 Hz, 1H), 7.32–7.28 (m, 2H), 7.23 (t, J = 1 Hz, 1H), 7.20 (d,
J = 6 Hz, 1H), 6.83 (dd, J = 1, 6 Hz, 1H), 6.12 (t, J = 4 Hz, 1H), 5.75–
5.50 (m, 2H), 3.68 (t, J = 5 Hz, 2H), 1.67 (d, J = 4 Hz, 3H); MS (ES)
m/e 375.1 (M+H).
yl](thien-2-yl)methanone (20a)
According to the procedure used to prepare 6, reaction of 19a and
5 provided 20a in 86% yield as an orange solid: mp 217–219 °C; 1H
NMR (DMSO-d6) d 8.93 (d, J = 3 Hz, 1H), 8.87 (s, 1H), 8.82 (d,
J = 1 Hz, 1H), 8.36 (dd, J = 1, 6 Hz, 1h), 8.21 (dd, J = 1, 3 Hz, 1H), 8.06
(dd, J = 1, 4 Hz, 1H), 7.79 (d, J = 6 Hz, 1H), 7.65 (d, J = 3 Hz, 1H), 7.30
(dd, J = 1, 6 Hz, 1H), 2.66 (s, 3H); MS (ES) m/e 365.2 (M+H).
Anal. (C18H12N4O3S) C, H, N.
Anal. (C21H18N4OS) C, H, NꢁH, N.
4.14. (2E)-3-(Dimethylamino)-1-(4-methyl-3-nitrophenyl)prop-
4.20. [7-(4-Methoxy-3-nitrophenyl)pyrazolo[1,5-a]pyrimidin-
2-en-1-one (19a)
3-yl](thien-2-yl)methanone (20b)
According to the procedure used to prepare 4, reaction of 4-
methyl-3-nitroacetophenone and DMF–DMA provided 19a in 73%
yield as a brown solid: mp 123–125 °C; 1H NMR (DMSO-d6) d
8.41 (d, J = 1 Hz, 1H), 8.14 (dd, J = 1, 6 Hz, 1H), 7.79 (d, J = 9 Hz,
1H), 7.56 (d, J = 6 Hz, 1H), 5.90 (d, J = 9 Hz, 1H), 3.17 (s, 3H), 2.95
(s, 3H), 2.55 (s, 3H); MS (ES) m/e 235.1 (M+H).
According to the procedure used to prepare 6, reaction of 19b
and 5 provided 20b in 97% yield as a light yellow solid: mp
>250 °C; 1H NMR (DMSO-d6) d 8.90 (d, J = 3 Hz, 1H), 8.87 (s, 1H),
8.80 (d, J = 2 Hz, 1H), 8.48 (dd, J = 2, 7 Hz, 1H), 8.22 (d, J = 5 Hz,
1H), 8.06 (d, J = 4 Hz, 1H), 7.65 (d, J = 5 Hz, 1H), 7.63 (s, 1H), 7.30
(t, J = 3 Hz, 1H), 4.07 (s, 3H); MS (ES) m/e 351.1 (M+H).
Anal. (C18H14N4O4S–0.2H2O) C, H, N.
Anal. (C12H14N2O3–0.2H2O) C, H, N.

Y. D. Wang et al. / Bioorg. Med. Chem. 17 (2009) 2091–2100
2097
4.21. [7-(4-Chloro-3-nitrophenyl)pyrazolo[1,5-a]pyrimidin-3-
yl](thien-2-yl)methanone (20c)
NMR (DMSO-d6) d 8.86 (d, J = 4 Hz, 1H), 8.82 (s, 1H), 8.22 (dd,
J = 1, 3 Hz, 1H), 8.05 (dd, J = 1, 4 Hz, 1H), 7.53 (d, J = 2 Hz, 1H),
7.44 (d, J = 6 Hz, 1H), 7.42 (d, J = 3 Hz, 1H), 7.30 (dd, J = 1, 4 Hz,
1H), 7.22 (dd, J = 2, 6 Hz, 1H), 5.72 (s, 2H); MS (ES) m/e 355.2 (M+H).
Anal. (C17H11ClN4OS–0.4H2O) C, H, N.
According to the procedure used to prepare 6, reaction of 19c
and 5 provided 20c in 90% yield as a yellow solid: mp 220–
222 °C; 1H NMR (DMSO-d6) d 8.90 (d, J = 3 Hz, 1H), 8.87 (s, 1H),
8.86 (d, J = 1 Hz, 1H), 8.44 (dd, J = 2, 6 Hz, 1H), 8.20 (dd, J = 1,
3 Hz, 1H), 8.09 (d, J = 6 Hz, 1H), 8.07 (dd, J = 1, 4 Hz, 1H), 7.67 (d,
J = 3 Hz, 1H), 7.31 (dd, J = 1, 4 Hz, 1H); MS (ES) m/e 385.2 (M+H).
Anal. (C17H9ClN4O3S–0.2H2O) C, H, N.
4.27. [7-(5-Amino-2-chlorophenyl)pyrazolo[1,5-a]pyrimidin-3-
yl](thien-2-yl)methanone (21d)
According to the procedure used to prepare 21a, reaction of 20d
provided 21d in 46% yield as a reddish solid: mp 162–168 °C; 1H
NMR (DMSO-d6) d 8.91 (d, J = 3 Hz, 1H), 8.76 (s, 1H), 8.25 (d,
J = 1 Hz, 1H), 8.05 (d, J = 3 Hz, 1H), 7.38 (d, J = 3 Hz, 1H), 7.30–
7.26 (m, 2H), 6.79 (d, J = 6 Hz, 1H). 6.78 (s, 1H), 5.59 (s, 2H); MS
(ES) m/e 355.2 (M+H).
4.22. [7-(2-Chloro-5-nitrophenyl)pyrazolo[1,5-a]pyrimidin-3-
yl](thien-2-yl)methanone (20d)
According to the procedure used to prepare 6, reaction of 19d
and 5 provided 20d in 75% yield as a reddish solid: mp 162–
166 °C; 1H NMR (DMSO-d6) d 9.00 (d, J = 3 Hz, 1H), 8.79 (s, 1H),
8.69 (d, J = 2 Hz, 1H), 8.50 (dd, J = 2, 6 Hz, 1H), 8.23 (dd, J = 1,
3 Hz, 1H), 8.07 (dd, J = 2, 2 Hz, 1H), 8.05 (s, 1H), 7.57 (d, J = 3 Hz,
1H), 7.31 (dd, J = 1, 4 Hz, 1H); MS (ES) m/e 385.2 (M+H).
Anal. (C17H9ClN4O3S–1.0H2O) C, H, N.
Anal. (C17H11ClN4OS–3.2H2O) C, H, N.
4.28. [7-(3-Amino-4-fluorophenyl)pyrazolo[1,5-a]pyrimidin-3-
yl](thien-2-yl)methanone (21e)
According to the procedure used to prepare 21a, reaction of 20e
provided 21e in 52% yield as a yellow solid: mp 241–242 °C; 1H
NMR (DMSO-d6) d 8.84 (d, J = 4 Hz, 1H), 8.81 (s, 1H), 8.23 (dd,
J = 2, 3 Hz, 1H), 8.05 (dd, J = 1, 4 Hz, 1H), 7.54 (dd, J = 1, 6 Hz, 1H),
7.40 (d, J = 3 Hz, 1H), 7.30 (dd, J = 1, 4 Hz, 1H), 7.24 (dd, J = 4,
6 Hz, 1H), 5.52 (s, 2H); MS (ES) m/e 339.1 (M+H).
4.23. [7-(4-Fluoro-3-nitrophenyl)pyrazolo[1,5-a]pyrimidin-3-
yl](thien-2-yl)methanone (20e)
According to the procedure used to prepare 6, reaction of 19e
and 5 provided 20e in 74% yield as an orange solid: mp 205–
208 °C; 1H NMR (DMSO-d6) d 8.99 (dd, J = 2, 5 Hz, 1H), 8.94 (d,
J = 4 Hz, 1H), 8.87 (s, 1H), 8.60–8.55 (m, 1H), 8.21 (dd, J = 3, 3 Hz,
1H), 8.07 (dd, J = 3, 4 Hz, 1H), 7.90 (dd, J = 2, 9 Hz, 1H), 7.66 (d,
J = 3 Hz, 1H), 7.31 (dd, J = 3, 4 Hz, 1H); MS (ES) m/e 369.2 (M+H).
Anal. (C17H9FN4O3S) C, H, N.
Anal. (C17H11FN4OS–0.1H2O) C, H, N.
4.29. 3-Methyl-N-{2-methyl-5-[3-(2-thienylcarbonyl)-
pyrazolo[1,5-a]pyrimidin-7-yl]phenyl}butanamide (22)
To a solution of isovalaeric acid (80 mg, 0.783 mmol) and N-
methylmorpholine (118 mg, 1.18 mmol) in THF (2.0 mL) was
added isobutyl chloroformate (107 mg, 0.783 mmol) dropwise at
0–5 °C. The above mixture was stirred at 0–5 °C for 5 min; 21a
(130 mg, 0.389 mmol) in THF (2.0 mL) was added. The resulting
mixture was stirred at room temperature for 22 h and diluted with
methylene chloride. The organic phase was washed with saturated
aqueous sodium bicarbonate and brine, dried over sodium sulfate,
concentrated and purified by preparative TLC (eluting with 90%
EtOAc/Hexanes) to give 75 mg (46%) of 22 as a yellow solid: mp
185–187 °C; 1H NMR (DMSO-d6) d 9.51 (s, 1H), 8.86 (d, J = 3 Hz,
1H), 8.81 (s, 1H), 8.23 (dd, J = 1, 3 Hz, 1H), 8.16 (d, J = 1 Hz, 1H),
8.05 (dd, J = 1, 4 Hz, 1H), 7.86 (dd, J = 1, 6 Hz, 1H), 7.48 (d,
J = 6 Hz, 1H), 7.46 (d, J = 3 Hz, 1H), 7.30 (dd, J = 1, 6 Hz, 1H), 2.33
(s, 3H), 2.26 (d, J = 5 Hz, 2H), 2.17–2.05 (m, 1H), 0.97 (d, J = 5 Hz,
6H); MS (ES) m/e 419.2 (M+H).
4.24. [7-(3-Amino-4-methylphenyl)pyrazolo[1,5-a]pyrimidin-
3-yl](thien-2-yl)methanone (21a)
A mixture of 20a (1.95 g, 5.32 mmol), acetic acid (17.8 mL), iron
powder (2.97 g, 53.2 mmol) in methanol (54 mL) was heated at re-
flux for 1 h, cooled to room temperature, filtered through a pad of
celite. The filtrate was concentrated and the residue was parti-
tioned between EtOAc and aqueous sodium carbonate. The com-
bined organics were dried over sodium sulfate and filtered
through a pad of magnesol. The filtrate was concentrated to give
845 mg (48%) of 21a as a yellow solid: mp 193–195 °C; 1H NMR
(DMSO-d6) d 8.83 (d, J = 4 Hz, 1H), 8.80 (s, 1H), 8.24 (dd, J = 1,
3 Hz, 1H), 8.04 (dd, J = 1, 4 Hz, 1H), 7.38 (d, J = 1 Hz, 1H), 7.37 (s,
1H), 7.31 (dd, J = 1, 4 Hz, 1H), 7.21–7.15 (m, 2H), 5.20 (S, 2H),
2.17 (S, 3h); MS (ES) m/e 335.2 (M+H).
Anal. (C23H22N4O2S–0.3H2O) C, H, N.
Anal. (C18H14N4OS–0.05EtOAc) C, H, N.
4.30. N-{2-Methoxy-5-[3-(2-thienylcarbonyl)pyrazolo[1,5-
a]pyrimidin-7-yl]phenyl}-3-methylbutanamide (23)
4.25. [7-(3-Amino-4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-
3-yl](thien-2-yl)methanone (21b)
According to the procedure used to prepare 22, reaction of 21b
and isovalaeric acid provided 23 in 44% yield as a yellow solid: mp
192–194 °C; 1H NMR (DMSO-d6) d9.30 (s, 1H), 8.83 (d, J = 3 Hz, 1H),
8.81 (s, 1H), 8.72 (s, 1H), 8.24 (d, J = 3 Hz, 1H), 8.04 (d, J = 4 Hz, 1H),
7.96 (d, J = 6 Hz, 1H), 7.43 (d, J = 3 Hz, 1H), 7.30 (d, J = 4 Hz, 1H),
7.28 (s, 1H), 3.96 (s, 3H), 2.31 (d, J = 5 Hz, 2H), 2.12–2.03 (m, 1H),
0.94 (d, J = 5 Hz, 6H); MS (ES) m/e 435.1 (M+H).
According to the procedure used to prepare 21a, reaction of 20b
provided 21b in 53% yield as a yellow solid: mp 210–213 °C; 1H
NMR (DMSO-d6) d 8.81 (s, 1H), 8.82 (d, J = 3 Hz, 1H), 8.25 (dd, J = 1,
4 Hz, 1H), 8.04 (dd, J = 1, 3 Hz, 1H), 7.48 (d, J = 2 Hz, 1H), 7.40 (dd,
J = 2, 6 Hz, 1H), 7.38 (d, J = 3 Hz, 1H), 7.30 (dd, J = 1, 4 Hz, 1H), 7.03
(d, J = 6 Hz, 1H), 5.06 (s, 2H), 3.89 (s, 3H); MS (ES) m/e 351.1 (M+H).
Anal. (C18H14N4O2S–0.2H2O) C, H, N.
Anal. (C23H22N4O3S–0.3H2O) C, H, N.
4.26. [7-(3-Amino-4-chlorophenyl)pyrazolo[1,5-a]pyrimidin-3-
4.31. N-{2-Chloro-5-[3-(2-thienylcarbonyl)pyrazolo[1,5-a]-
yl](thien-2-yl)methanone (21c)
pyrimidin-7-yl]phenyl}-3-methylbutanamide (24)
According to the procedure used to prepare 21a, reaction of 20c
provided 21c in 28% yield as a reddish solid: mp 231–233 °C; 1H
According to the procedure used to prepare 22, reaction of 21c
and isovalaeric acid provided 24 in 16% yield as a yellow solid: mp

2098
Y. D. Wang et al. / Bioorg. Med. Chem. 17 (2009) 2091–2100
172–175 °C; 1H NMR (DMSO-d6) d 9.75 (s, 1H), 8.88 (d, J = 3 Hz, 1H),
8.82 (s, 1H), 8.39 (d, J = 2 Hz, 1H), 8.22 (dd, J = 1, 3 Hz, 1H), 8.05 (dd,
J = 1, 4 Hz, 1H), 7.93 (dd, J = 2, 6 Hz, 1H), 7.77 (d, J = 6 Hz, 1H), 7.51 (d,
J = 3 Hz, 1H), 7.30 (dd, J = 1, 4 Hz, 1H), 2.31(d, J = 5 Hz, 2H), 2.12–2.05
(m, 1H), 0.97 (d, J = 5 Hz, 6H); MS (ES) m/e 439.2 (M+H).
Anal. (C22H19ClN4O2S–0.7H2O) C, H, N.
4.36. N-{2-Fluoro-5-[3-(thien-2-ylcarbonyl)pyrazolo[1,5-a]-
pyrimidin-7-yl]phenyl}-N0-isopropylurea (29)
To a mixture of 21e (100 mg, 0.0.296 mmol) and 4-dimethyl-
aminopyridine (catalytic amount) in 1,4-dioxane (10 mL) was
added isopropyl isocyanate (50 mg, 0.588 mmol). The resulting
mixture was heated at reflux for 24 h and cooled to room temper-
ature. Additional isopropyl isocyanate (250 mg, 2.94 mmol) was
added and the reaction mixture was stirred for 6 days at room tem-
perature and partitioned between ethyl acetate and water. The
combined organics were dried over sodium sulfate, concentrated
and purified by column chromatographyl (eluting with 1–5% meth-
anol/methylene chloride) to give 24 mg (19%) of 29 as a yellow so-
lid: mp >200 °C; 1H NMR (DMSO-d6) d 8.88–8.86 (m, 2H), 8.81 (t,
J = 2 Hz, 1H), 8.41 (s, 1H), 8.23 (t, J = 2 Hz, 1H), 8.04 (d, J = 3 Hz,
1H), 7.67 (d, J = 3 Hz, 1), 7.45 (dd, J = 1, 8 Hz, 1H), 7.42 (d,
J = 3 Hz, 1H), 7.30 (d, J = 2 Hz, 1H), 6.62 (s, 1H), 5.47 (s, 1H),
3.85–3.60 (m, 2H), 1.11 (d J = 5 Hz, 3H), 1.00 (d, J = 5 Hz, 3H); MS
(ES) m/e 424.0 (M+H).
4.32. N-{4-Chloro-3-[3-(2-thienylcarbonyl)pyrazolo[1,5-a]-
pyrimidin-7-yl]phenyl}-3-methylbutanamide (25)
According to the procedure used to prepare 22, reaction of 21d
and isovalaeric acid provided 25 in 50% yield as a beige solid: mp
162–165 °C; 1H NMR (DMSO-d6) d 10.25 (s, 1H), 8.95 (d, J = 3 Hz,
1H), 8.77 (s, 1H), 8.25 (d, J = 1 Hz, 1H), 8.06 (d, J = 3 Hz, 1H), 8.01
(s, 1H), 7.79 (d, J = 3 Hz, 1H), 7.64 (d, J = 6 Hz, 1H), 7.47 (d,
J = 3 Hz, 1H), 7.31 (t, J = 3 Hz, 1H), 2.11 (d, J = 5 Hz, 2H), 2.09–2.05
(m, 1H), 0.93 (d, J = 5 Hz, 6H); MS (ES) m/e 439.1 (M+H).
Anal. (C22H19ClN4O2S–0.8H2O) C, H, N.
4.33. N-{2-Fluoro-5-[3-(2-thienylcarbonyl)pyrazolo[1,5-a]-
Anal. (C21H18FN5O2S–0.40H2O) C, H, N.
pyrimidin-7-yl]phenyl}-3-methylbutanamide (26)
4.37. isoPropyl 2-fluoro-5-[3-(thien-2-ylcarbonyl)pyrazolo[1,5-
a]pyrimidin-7- yl]phenylcarbamate (30)
To a mixture of 21e (1.0 g, 2.96 mmol) and 4-dimethylamino-
pyridine (36 mg, 0.294 mmol) in methylene chloride (20 mL) was
added isovaleryl chloride (71 mg, 0.591 mmol) in methylene
chloride (5 mL) dropwise followed by triethylamine (450 mg,
4.45 mmol). The resulting mixture was stirred at room tem-
perature for 90 min and filtered. The filtrate was washed with
aqueous sodium bicarbonate, dried over sodium sulfate and con-
centrated. The residue was purified by column chromatography
(eluting with 1–5% methanol in methylene chloride) to give
619 mg (52%) of 246 as a yellow solid: mp 192–194 °C; 1H
NMR (DMSO-d6) d 9.93 (s, 1H), 8.88 (d, J = 3 Hz, 1H), 8.82 (s,
1H), 8.59 (dd, J = 2, 6 Hz, 1H), 8.23 (dd, J = 2, 3 Hz, 1H), 8.05 (dd,
J = 3, 4 Hz, 1H), 7.54 (dd, J = 2, 8 Hz, 1H), 7.48 (d, J = 3 Hz, 1H),
7.30 (dd, J = 1, 4 Hz, 1H), 2.30 (d, J = 5 Hz, 2H), 2.12–2.03 (m,
1H), 0.95 (d, J = 5 Hz, 6H); MS (ES) m/e 423.1 (M+H).
To 21e (300 mg, 0.887 mmol) in THF (20 mL) was added tri-
ethylamine (97 mg, 0.958 mmol). After stirring for 20 min at
room temperature, isopropyl chloroformate (1.0 M in methylene
chloride, 0.97 mL, 0.97 mmol) was added dropwise. The reaction
mixture was stirred at room temperature overnight and addi-
tional isopropyl chloroformate (0.97 mL, 0.97 mmol) and triethyl-
amine (97 mg, 0.958 mmol) were added. The resulting mixture
was heated at reflux for 2 h, cooled to room temperature, filtered
to collect the solids. The crude solids were washed with hexanes
and further purified by HPLC (eluting with a gradient of MeCN/
water) to give 112 mg (30%) of 30 as a yellow solid: mp 172–
175 °C; 1H NMR (DMSO-d6) d 9.54 (s, 1H), 8.88 (d, J = 3 Hz,
1H), 8.82 (s, 1H), 8.42 (dd, J = 2, 6 Hz, 1H), 8.23 (dd, J = 2, 3 Hz,
1H), 8.06 (dd, J = 3, 4 Hz, 1H), 7.95–7.88 (m, 1H), 7.50 (dd,
J = 2, 8 Hz, 1H), 7.49 (d, J = 3 Hz, 1H), 7.30 (dd, J = 1, 4 Hz, 1H),
4.94–4.87 (m, 1H), 1.27 (d, J = 5 Hz, 6H); MS (ES) m/e 425.1
(M+H).
Anal. (C22H19FN4O2S–0.1H2O) C, H, N.
4.34. 4,4,4-Trifluoro-N-{2-fluoro-5-[3-(thien-2-ylcarbonyl)-
pyrazolo[1,5-a]pyrimidin-7- yl]phenyl}-3-methylbutanamide
(27)
Anal. (C21H17FN4O3S) C, H, N.
According to the procedure used to prepare 22, reaction of 21e
and 4,4,4-trifluoro-3-methylbutanoic acid provided 27 in 33%
yield as an off-white solid: mp 185–186 °C; 1H NMR (DMSO-d6)
d 10.21 (s, 1H), 8.89 (d, J = 3 Hz, 1H), 8.82 (s, 1H), 8.63 (dd,
J = 1, 6 Hz, 1H), 8.23 (dd, J = 1, 3 Hz, 1H), 8.06 (dd, J = 3, 6 Hz,
1H), 8.00–7.95 (m, 1H), 7.57 (dd, J = 3, 9 Hz, 1H), 7.48 (d,
J = 3 Hz, 1H), 7.31 (dd, J = 3, 6 Hz, 1H), 2.93–2.86 (m, 1H), 2.80
(dd, J = 4, 11 Hz, 1H), 2.57 (dd, J = 5, 11 Hz, 1H), 1.14 (d, J = 5 Hz,
3H); MS (ES) m/e 477.1 (M+H).
4.38. (2E)-3-(Dimethylamino)-1-(5-nitro-3-pyridinyl)-2-propen-
1-one (32a)
According to the procedure used to prepare 4, reaction of 3-
acetyl-5-nitropyridine19 and DMF–DMA provided 32a in 81% yield
as an orange solid: mp 170–172 °C; 1H NMR (CDCl3) d 9.48 (d,
J = 2 Hz, 1H), 9.36 (d, J = 1 Hz, 1H), 8.91 (t, J = J = 2 Hz, 1H), 7.94
(d, J = 9 Hz, 1H), 5.68 (d, J = 9 Hz, 1H), 3.24 (s, 3H), 3.02 (s, 3H);
MS (ES) m/e 222.1 (M+H).
Anal. (C22H16FN4O2S–0.05CH2Cl2) C, H, N.
Anal. (C10H11N3O3–0.15H2O) C, H, N.
4.35. N-{2-Fluoro-5-[3-(thien-2-ylcarbonyl)pyrazolo[1,5-
a]pyrim-idin-7-yl]phenyl}-3,3-dimethylbutanamide (28)
4.39. [7-(5-Nitro-3-pyridinyl)pyrazolo[1,5-a]pyrimidin-3-
yl](2-thienyl)methanone (33a)
According to the procedure used to prepare 24, reaction of 19e
and 3,3-dimethylbutanoyl chloride provided 26 in 27% yield as a
yellow solid: mp 180–181 °C; 1H NMR (DMSO-d6) d 9.86 (s, 1H),
8.88 (d, J = 3 Hz, 1H), 8.82 (s, 1H), 8.55 (dd, J = 2, 5 Hz, 1H), 8.23
(dd, J = 1, 3 Hz, 1H), 8.05 (dd, J = 1, 4 Hz, 1H), 7.95–7.90 (m, 1H),
7.54 (dd, J = 2, 8 Hz, 1H), 7.48 (d, J = 3 Hz, 1H), 7.30 (dd, J = 1,
4 Hz, 1H), 2.31 (s, 2H), 1.04 (s, 9H); MS (ES) m/e 437.2 (M+H).
Anal. (C23H21FN4O2S–0.15CH2Cl2) C, H, N.
According to the procedure used to prepare 6, reaction of 32a
and 5 provided 33a in 87% yield as a yellow solid: mp 170–
172 °C; 1H NMR (DMSO-d6) d 9.61 (t, J = 2 Hz, 1H), 9.39 (dd, J = 1,
2 Hz, 1H), 8.98 (d, J = 3 Hz, 1H), 8.90 (s, 1H), 8.21 (dd, J = 1, 3 Hz,
1H), 8.08 (dd, J = 1, 4 Hz, 1H), 7.81 (d, J = 3 Hz, 1H), 7.31 (dd, J = 1,
4Hz, 1H); MS (ES) m/e 352.1 (M+H).
Anal. (C16H9N5O3S) C, H, N.

Y. D. Wang et al. / Bioorg. Med. Chem. 17 (2009) 2091–2100
2099
4.40. [7-(5-Amino-3-pyridinyl)pyrazolo[1,5-a]pyrimidin-3-yl]-
4.46. (E)-3-(Dimethylamino)-1-(5-nitrothien-3-yl)prop-2-en-1-
(2-thienyl)methanone (34a)
one (32c)
According to the procedure used to prepare 21a, reaction of 33a
provided 34a in 55% yield as a yellow solid: mp >250 °C; 1H NMR
(DMSO-d6) d 8.87 (d, J = 3 Hz, 1H), 8.82 (s, 1H), 8.31 (d, J = 1,
3 Hz, 1H), 8.23 (dd, J = 1, 3 Hz, 1H), 8.15 (d, J = 2 Hz, 1H), 8.05
(dd, J = 1, 4 Hz, 1H), 7.65 (t, J = 2 Hz, 1H), 7.51 (d, J = 3 Hz, 1H),
7.30 (dd, J = 1, 4 Hz, 1H), 5.71 (s, 1H); MS (ES) m/e 322.1 (M+H).
Anal. (C16H11N5OS) C, H, N.
According to the procedure used to prepare 4, reaction of 4-
acetyl-2-nitrothiophene and DMF–DMA provided 32c in 63% yield
as a yellow solid: mp 165–166 °C; 1H NMR (DMSO-d6) d 8.53 (d,
J = 1 Hz, 1H), 8.46 (d, J = 1 Hz, 1H), 7.74 (d, J = 9 Hz, 1H), 5.82 (d,
J = 9 Hz, 1H), 3.16 (s, 3H), 2.94 (s, 3H); MS (ES) m/e 227.1 (M+H).
Anal. (C9H10N2O3S) C, H, N.
4.47. [7-(5-Nitro-3-thienyl)pyrazolo[1,5-a]pyrimidin-3-
yl](2-thienyl)methanone (33c)
4.41. 3-Methyl-N-{5-[3-(2-thienylcarbonyl)pyrazolo[1,5-
a]pyrimidin-7-yl}-3-pyridinyl}butanamide (35a)
According to the procedure used to prepare 6, reaction of 32c
and 5 provided 33c in 80% yield as an orange solid: mp 225–
226 °C; 1H NMR (DMSO-d6) d 9.44 (d, J = 1 Hz, 1H), 9.12 (d,
J = 1 Hz, 1H), 8.94 (s, 1H), 8.93 (d, J = 3 Hz, 1H), 8.21 (dd, J = 1,
3 Hz, 1H), 8.07 (dd, J = 1, 3 Hz, 1H), 7.99 (d, J = 3 Hz, 1H), 7.30
(dd, J = 1, 3 Hz, 1H); MS (ES) m/e 357.1 (M+H).
According to the procedure used to prepare 22, reaction of 33a
and isovalaerid acid provided 34a in 53% yield as a beige solid: mp
217–219 °C; 1H NMR (DMSO-d6) d 10.40 (s, 1H), 8.99 (d, J = 2 Hz,
1H), 8.92 (d, J = 3 Hz, 1H), 8.86 (d, J = 2 Hz, 1H), 8.84 (s, 1H), 8.80
(dd, J = 1, 3 Hz, 1H), 8.22 (d, J = 3 Hz, 1H), 7.31 (dd, J = 1, 3 Hz,
1H), 2.29 (d, J = 5 Hz, 2H), 2.16–2.06 (m, 1H), 0.96 (d, J = 5 Hz,
6H); MS (ES) m/e 406.1 (M+H).
Anal. (C15H8N4O3S2) C, H, N.
4.48. [7-(5-Aminothien-3-yl)pyrazolo[1,5-a]pyrimidin-3-yl]-
(thien-2-yl)methanone (34c)
Anal. (C21H19N5O2S0–0.4H2O–0.1Et2O) C, H, N.
4.42. (E)-3-(Dimethylamino)-1-(5-nitrothien-2-yl)prop-2-en-1-
one (32b)
According to the procedure used to prepare 21a, reaction of 33c
provided 34c in 15% yield as a light brown solid: mp 174–176 °C;
1H NMR (DMSO-d6) d 8.86 (s, 1H), 8.79 (d, J = 4 Hz, 1H), 8.23 (dd,
J = 1, 3 Hz, 1H), 8.21 (d, J = 1 Hz, 1H), 8.04 (dd, J = 1, 3 Hz, 1H),
7.58 (d, J = 4 Hz, 1H), 7.29 (dd, J = 1, 3 Hz, 1H), 6.76 (d, J = 1 Hz,
1H), 5.90 (s, br, 2H); MS (ES) m/e 327.1 (M+H).
According to the procedure used to prepare 4, reaction of 5-
acetyl-2-nitrothiophene and DMF–DMA provided 32b in 60% yield
as an orange solid: mp 202–203 °C; 1H NMR (DMSO-d6) d 8.12 (d,
J = 3 Hz, 1H), 7.85 (d, J = 3 Hz, 1H), 7.82 (d, J = 9 Hz, 1H), 5.90 (d,
J = 9 Hz, 1H), 3.19 (s, 3H), 2.98 (s, 3H); MS (ES) m/e 227.1 (M+H).
Anal. (C9H10N2O3S) C, H, N.
Anal. (C15H10N4OS2–0.3H2O) C, H, N.
4.493. -Methyl-N-{4-[3-(thien-2-ylcarbonyl)pyrazolo[1,5-a]pyrim-
idin-7-yl]thien-2-yl}butanamide (35c)
4.43. [7-(5-Nitro-2-thienyl)pyrazolo[1,5-a]pyrimidin-3-
yl](2-thienyl)methanone (33b)
According to the procedure used to prepare 22, reaction of 34c
and valaeryl chloride provided 35c in 84% yield as a light yellow
solid: mp 234–235 °C; 1H NMR (DMSO-d6) d 11.46 (s, 1H), 8.88
(d, J = 1 Hz, 1H), 8.85 (d, J = 1 Hz, 1H), 8.56 (d, J = 1 Hz, 1H), 8.24
(d, J = 1 Hz, 1H), 8.04 (dd, J = 1, 3 Hz, 1H), 7.68 (dd, J = 1, 3 Hz,
1H), 7.61 (dd, J = 1, 3 Hz, 1H), 7.23 (dd, J = 1, 3 Hz, 1H), 2.27 (t,
J = 5 Hz, 2H), 2.14–2.09 (m, 1H), 0.94 (d, J = 5 Hz, 6H); MS (ES) m/
e 411.1 (M+H).
According to the procedure used to prepare 6, reaction of 32b and
5 provided 33b in 72% yield as a yellow solid: mp 213–215 °C; 1H
NMR (DMSO-d6) d 9.02 (s, 1H), 8.97 (d, J = 5 Hz, 1H), 8.59 (d,
J = 5 Hz, 1H), 8.35 (d, J = 5 Hz, 1H), 8.30 (d, J = 5 Hz, 1H), 8.22 (dd,
J = 1, 4 Hz, 1H), 8.08 (dd, J = 1, 5 Hz, 1H), 7.31 (dd, J = 1, 5 Hz, 1H).
4.44. [7-(5-Aminothien-2-yl)pyrazolo[1,5-a]pyrimidin-3-yl]-
Anal. (C20H18N4O2S2–0.25H2O) C, H, N.
(thien-2-yl)methanone (34b)
4.50. Biological evaluation
According to the procedure used to prepare 21a, reaction of 33b
provided 34b in 55% yield as a reddish solid: mp 232–235 °C; 1H
NMR (DMSO-d6) d 8.77 (s, 1H), 8.48 (d, J = 4 Hz, 1H), 8.38 (dd,
J = 1, 3 Hz, 1H), 8.28 (d, J = 3 Hz, 1H), 8.01 (dd, J = 1, 4 Hz, 1H),
7.53 (d, J = 4 Hz, 1H), 7.28 (dd, J = 1, 4 Hz, 1H), 7.26 (s, 1H), 6.22
(d, J = 3 Hz, 1H), 5.71 (s, 2H); MS (ES) m/e 327.1 (M+H).
Anal. (C15H10N4OS2–0.5EtOAc) C, H, N.
The protocol for p21 isogenic cell and colon cell proliferation as-
says were previously reported.9
4.51. Microtubulin binding assay
The protocol for microtubulin binding assay was previously
reported.15
4.45. 3-Methyl-N-{5-[3-(thien-2-ylcarbonyl)pyrazolo[1,5-a]-
pyrim-idin-7-yl]thien-2-yl}butanamide (35b)
4.52. LoVo xenograft assay
According to the procedure used to prepare 22, reaction of 34b
and valaeryl chloride provided 35b in 59% yield as a light yellow
solid: mp 268–270 °C; 1H NMR (DMSO-d6) d 11.95 (s, 1H), 8.93 (s,
1H), 8.77 (d, J = 3 Hz, 1H), 8.43 (d, J = 3 Hz, 1H), 8.29 (dd, J = 1,
3 Hz, 1H), 8.04 (dd, J = 1, 3 Hz, 1H), 7.88 (d, J = 3 Hz, 1H), 7.30
(dd, J = 1, 3 Hz, 1H), 6.92 (d, J = 3 Hz, 1H), 2.35 (d, J = 5 Hz, 2H),
2.16–2.09 (m, 1H), 0.90 (d, J = 5 Hz, 6H); MS (ES) m/e 411.1
(M+H).
Compounds were evaluated in vivo using standard pharmaco-
logical test procedures which measure the ability to inhibit the
growth of human tumor xenografts. Human Colon Carcinoma LoVo
cells (American Type Culture Collection, Manassas, Maryland) were
grown in tissue culture in RPMI (Gibco/InVitrogen, Gaithersburg,
MD) supplemented with 10% FBS (Gemini Bio-Products Inc., Cala-
basas, CA). Athymic nu/nu female mice (Charles River, Wilmington,
MA) were injected SC in the flank area with 1 ꢂ 107 LoVo cells.
When tumors attained a mass of between 90 and 120 mg, the mice
Anal. (C20H18N4O2S2–0.3H2O) C, H, N.

2100
Y. D. Wang et al. / Bioorg. Med. Chem. 17 (2009) 2091–2100
4. El-Deiry, W. S.; Tokino, T.; Velculescu, V. E.; Levy, D. B.; Parsons, R.; Trent, J. M.;
Lin, D.; Mercer, W. E.; Kinzler, K. W.; Vogelstein, B. Cell 1993, 75, 817.
5. Harper, J. W.; Adami, G. R.; Wei, N.; Keyomarsi, K.; Elledge, S. J. Cell 1993, 75,
805.
6. Xiong, Y.; Hannon, G. J.; Zhang, H.; Casso, D.; Kobayashi, R.; Beach, D. Nature
1993, 366, 701.
7. Karjalainen, J. M.; Eskelinen, M. J.; Kellokoski, J. K.; Reinikainen, M.; Alhava, E.
M.; Kosma, V. M. Br. J. Cancer 1999, 79, 895.
8. Komiya, T.; Hosono, Y.; Hirashima, T.; Masuda, N.; Yasumitsu, T.; Nakagawa,
K.; Kikui, M.; Ohno, A.; Fukuoka, M.; Kawase, I. Clin. Cancer Res. 1997, 3,
1831.
9. Bunz, F.; Hwang, P. M.; Torrance, C.; Waldman, T.; Zhang, Y.; Dillehay, L.;
Williams, J.; Lengauer, C.; Kinzler, K. W.; Vogelstein, B. J. Clin. Invest. 1999, 1043,
263.
10. Bunz, F.; Dutriaux, A.; Lengauer, C.; Waldman, T.; Zhou, S.; Brown, J. P.; Sedivy,
J. M.; Kinzler, K. W.; Vogelstein, B. Science 1998, 282, 1497.
11. Gopalsamy, A.; Yang, H.; Ellingboe, J. W.; Tsou, H. R.; Zhang, N.; Honores, E.;
Powell, D.; Miranda, M.; McGinnis, J. P.; Rabindran, S. K. Bioorg. Med. Chem. Lett.
2005, 15, 1591.
were randomized into treatment groups (day zero), 5 animals per
group. Animals were treated PO once a day on days 1 through 20
post staging (day zero) with either compound prepared in 0.5%
Methocel/2.0% Tween 80 or 0.5% Methocel/0.4% Tween 80 or vehi-
cle alone. Tumor mass was determined every
7
days
[(length ꢂ width2)/2] for up to 28 days post staging. Relative tumor
growth (Mean tumor mass on days 7, 14, 21 and 28 divided by the
mean tumor mass on day zero) was determined for each treatment
group. Statistical analysis (Student-t-test) of log relative tumor
growth was used to compare treated verses control group in each
experiment. A p-value (p 6 0.05) indicates a statistically significant
reduction in relative tumor growth of treated group compared to
the vehicle control.
Acknowledgments
12. Powell, D.; Gopalsamy, A.; Wang, Y. D.; Zhang, N.; Miranda, M.; McGinnis, J. P.;
Rabindran, S. K. Bioorg. Med. Chem. Lett. 2007, 17, 1641.
13. Rabindran, S. K.; Beyer, C.; Discafani, C. M.; McGinnis, J. P.; Miranda, M.; Brown,
G.; Gopalsamy, A.; Krishnamurthy, G.; Martinez, R.; Powell, D.; Wang, D.;
Zhang, N. 96th Annual Meeting of the American Association for Cancer Research,
Anaheim, CA, April 16–20, 2005.
The authors thank the Chemical Technology department at
Wyeth Research, Pearl River, NY for the spectral data and elemen-
tal analyses. We also thank Drs. Diane H. Boschelli and Tarek Man-
sour for their support of this work.
14. Tomcufcik, A. S.; Dusza, J. P. U.S. Patent 4,900,836.
15. Rodrigues, N. R.; Rowan, A.; Smith, M. E.; Kerr, I. B.; Bodmer, W. F.; Gannon, I.
V.; Lane, D. P. Proc. Natl. Acad. Sci. U.S.A. 1990, 8719, 7555.
16. Hollstein, M.; Sidransky, D.; Vogelstein, B.; Harris, C. Science 1991, 253, 49.
17. Krishnamurthy, G.; Cheng, W.; Lo, M.-C.; Aulabaugh, A.; Razinkov, V.; Ding, W.;
Loganzo, F.; Zask, A.; Ellestad, G. Biochemistry 2003, 4246, 13484.
18. Hu, L.-Y.; Guyo, J.; Magar, S. S.; Fischer, J. B.; Burke-Howie, K. J.; Durant, G. J. J.
Med. Chem. 1997, 4026, 42.
References and notes
1. Waldman, T.; Kinzler, K.; Vogelstein, B. Cancer Res. 1995, 55, 5187.
2. Waldman, T.; Lengauer, C.; Kinzler, K. W.; Vogelstein, B. Nature 1996, 381, 713.
3. Torrance, C. J.; Agarwal, V.; Vogelstein, B.; Kinzler, K. W. Nature Biotechnol.
2001, 19, 940.
19. Hoffman, J. M.; Phillips, B. T.; Cochran, D. W. J. Org. Chem. 1984, 491, 193.