Pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides as novel antiproliferative agents: Exploration of core and headpiece structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2006.12.116

A novel series of antiproliferative agents containing pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides, selective for p21-deficient cells, were identified by high-throughput screening. Exploration of the SAR relationships in the headpiece, core, and tailpiece

Full text of DOI:10.1016/j.bmcl.2006.12.116

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1641–1645
Pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides as novel
antiproliferative agents: Exploration of core and headpiece
structure–activity relationships
Dennis Powell,a,* Ariamala Gopalsamy,a Yanong D. Wang,a Nan Zhang,a
Miriam Miranda,b John P. McGinnisb and Sridhar K. Rabindranb
aChemical and Screening Sciences, Wyeth Research, Pearl River, NY 10965, USA
bOncology, Wyeth Research, Pearl River, NY 10965, USA
Received 4 December 2006; accepted 22 December 2006
Available online 13 January 2007
Abstract—A novel series of antiproliferative agents containing pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides, selective for p21-defi-
cient cells, were identified by high-throughput screening. Exploration of the SAR relationships in the headpiece, core, and tailpiece is
described. Strict steric, positional, and electronic requirements were observed, with a clear preference for both core nitrogens, a thie-
noyl headpiece, and meta substituted tailpiece.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Progression from one phase of the cell division cycle to
the next phase is controlled by a series of sensors and
arresting mechanisms called checkpoints.1 Loss of
checkpoint control is a hallmark of tumor cells, as it
increases the mutation rate and allows a more rapid pro-
gression to the tumorigenic state.1,2 However, inactiva-
tion of these checkpoints results in aberrant responses
to cellular damage. This failure of checkpoint responses
in malignant cells can be exploited in cancer drug dis-
covery. Identification of compounds that selectively kill
checkpoint-deficient cells compared with checkpoint-
proficient cells can be expected to preferentially target
tumor cells, while sparing normal cells.3,4
cation, and ultimately, apoptosis.9 These p21-deficient
cells show increased chemosensitivity,10 compared with
the isogenic p21-proficient parental cells, to a variety
of DNA damaging agents, including clinically used anti-
neoplastic drugs. We have used this isogenic pair of cell
lines [HCT116 (p21+/+) and 80S14 (p21À/À)12] to iden-
tify novel molecules that preferentially induce apoptosis
in the p21-deficient cells.
High-throughput screening11 led to the identification of
pyrazolo[1,5-a]pyrimidin-7-yl phenyl amide 1 (Fig. 1)
which had an IC50 of 0.45 lM in the p21-deficient cell
line and an IC50 of 11 lM in the p21-proficient cell line
The p53 tumor suppressor gene is the major regulator of
the DNA damage checkpoint and one of the most com-
monly mutated genes in human cancer (50–70%).5,6 As a
downstream effector of p53, p21 inhibits the cyclin-de-
pendent kinases (CDKs) and arrests cell cycle progres-
sion in response to DNA damage.7,8 Disruption of this
checkpoint by deletion of the p21 gene in the HCT116
colorectal carcinoma cell line leads to the failure of the
cell to arrest in response to DNA damage, endoredupli-
H
N
H
N
O
O
N
N
N
N
N
N
O
O
S
S
Keywords: P21 deficient; Antiproliferative; Structure–activity relation-
ship; Checkpoint.
1
2
*
Figure 1. High-throughput screening lead, 1, for identification of p21
chemoselective agent and optimized amide analog, 2.
Corresponding author. Fax: +1 845 602 5561; e-mail:
powelld@wyeth.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.116

1642
D. Powell et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1641–1645
giving a selectivity ratio11 of 23. Subsequent efforts ex-
plored the structure–activity requirements for the amide
region of the molecule.13 These efforts led to the identi-
fication of the isobutyl amide 2 with improved potency
as well as selectivity (Table 1). The corresponding iso-
propyl carbamate and urea of 2 (not shown) also
showed improved properties compared to 1.13 Our ef-
forts to probe the steric requirements of the core and
aryl ketone headpiece, attachment position for the tail-
piece as well as the requirement for both core nitrogens
are described below.
NO2
NO2
NO2
(b)
(a)
R3
R2
N
O
R3
N
N
O
R4
R1
R3
O
N
R2
3
4
6
H
N
NH2
N
R5
O
(d)
(c)
The majority of the compounds for this series were read-
ily prepared13,14 utilizing the syntheses shown in
Schemes 1 and 2. In Scheme 1 ortho, meta or para-nitro
substituted aryl ketones 3 were converted into the corre-
sponding enamines 4 with either N,N-dimethyl formam-
ide dimethyl acetal [DMFDMA] (R2 = H) or N,
N-dimethylacetamide dimethyl acetal [DMADMA]
(R2 = CH3). Condensation of 4 with the amino pyrraz-
ole 5 (synthesis shown in Scheme 2) afforded the cyclized
nitro compound 6. The reduction of 6 afforded the
amine 7. The reaction of 7 with cyclopropyl carbonyl
chloride, isobutyl carbonyl chloride, isopropyl chloro-
formate or isopropyl isocyanate gave compounds 12–
26, 36 and 37.15
R3
R2
R3
R2
N
N
N
R4
R1
R4
R1
N
N
O
O
7
12-26, 36-37
Scheme 1. Reagents and conditions: (a) DMFDMA or DMADMA,
reflux, 18 h; (b) 5, acetic acid, reflux; (c) Fe, NH4Cl, MeOH, H2O; (d)
for 12–17, 21 R5COCl, pyridine, 0 ꢁC to rt; for 18, 22, 25, 36, 37
R5OCOCl, pyridine, 0 ꢁC to rt; for 20, 24 R5NCO, pyridine, 0 ꢁC to rt.
R4
N
R4
N
O
(e)
(f)
O
O
R1
Scheme 2 outlines the synthesis of the amino pyrrazoles
5. Reaction of aryl ketones 8 with either DMFDMA
(R4 = H) or DMADMA (R4 = CH3) afforded the corre-
sponding enamines 9. Condensation of 9 with hydroxyl-
amine followed by ring opening with DMFDMA
afforded the enamines 11 which were condensed with
hydrazine to afford the pyrrazoles 5.
R1
10
R1
9
8
NH2
N
R4
R1
HN
N
(g)
(h)
O
O
NC
R4
R1
In order to explore whether both pyrimidine nitrogens
were necessary to provide good activity, the pyrazolo
pyridines, 32–35, were synthesized as shown in Scheme
3. Amination of pyridine 27 afforded the amine salt
28. Condensation of 28 with the alkyne 29 gave the cyc-
lized compound 30. Reduction of 30 afforded the inter-
mediate amine 31, which was acylated in the same
manner as 7 to afford compounds 32–35.
5
11
Scheme 2. Reagents and conditions: (e) DMFDMA or DMADMA,
reflux, 18 h; (f) NH2OH–HCl, reflux, 3 h; (g) DMFDMA, reflux, 18 h;
(h) NH2NH2–H2O.
Our initial efforts were focused on understanding the
steric and electronic requirements of the aryl ketone,
Table 1. Activity comparison of aryl ketone derivatives in p21-proficient (p21+/+) and -deficient (p21À/À) cell lines
H
N
R5
O
N
N
N
O
R1
Compound
R1
R5
p21+/+ (IC50 lM)
p21À/À (IC50 lM)
Ratio
1
2
2-Thienyl
2-Thienyl
Phenyl
Cyclopropyl
iso-Butyl
11
6.4
0.45
0.14
8.1
23
46
12
13
14
15
16
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
Cyclopropyl
>20
>20
>20
16.9
>20
>2.5
>5.6
>2.8
15
2-Furyl
3.6
7.2
5-Methyl-2-thienyl
2-Thiazole
2-Pyridyl
1.1
>20
—

D. Powell et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1641–1645
1643
We then focused our attention on exploring the steric
requirements in the pyrazolo[1,5-a]pyrimidine core by
introducing a methyl probe at all of the open positions
(Table 2). For comparison purposes we prepared each
methyl analog with the corresponding most potent R5
substituents described earlier.13 Introduction of a methyl
group adjacent to the biaryl linkage, R3 = CH3 (21–24),
led to an almost complete loss in potency in both cell
lines. In addition, introduction of a methyl group adja-
cent to the aryl ketone, R4 = CH3 (25 and 26), also led to
a decrease in potency. Both of these substitutions
probed the steric requirements in their respective por-
tions of the core, also causing an increase in the dihedral
angles of the adjacent flanking substituents to avoid a
steric clash with the methyl group. Either of these fac-
tors could be responsible for the change in potency.
Introduction of a methyl group next to the pyrimidine
nitrogen, R2 = CH3 (17–20), had relatively little effect
on potency and selectivity with the biggest loss seen
for the iso-butyl analog, 19, when compared to its corre-
sponding analog 2. This would indicate that not only is
there more space to introduce substituents in this por-
tion of the core, but that access to the pyrimide nitrogen
may not be crucial to activity.
O
NO2
NO2
NH2
S
(i)
(j)
(l)
29
N
N+
27
28
H
N
NO2
NH2
R5
O
(k)
N
N
N
N
N
N
O
O
O
S
S
S
30
31
32-35
Scheme 3. Reagents and conditions: (i) O-mesitylsulfonylhydroxyl-
amine, CH2Cl2, rt, 20 h, 67%; (j) K2CO3, DMF, rt, 12 h, 41%; (k) Fe,
NH4Cl, MeOH, H2O, 60%; (l) for 32, 34 R5COCl, pyridine, 0 ꢁC to rt;
for 33 R5OCOCl, pyridine, 0 ꢁC to rt; for 35 R5NCO, pyridine, 0 ꢁC to
rt.
R1 (Table 1). Conservative replacement of the 2-thienyl
group with phenyl (12) resulted in a large decrease in cell
potency. Heterocyclic replacements for 2-thienyl includ-
ing five-membered ring isosteres 2-furyl (13), 2-thiazole
(15) as well as 2-pyridyl (16) also led to a reduction in
potency in both cell lines with 2-thiazole retaining the
most potency and selectivity for p21À/À cells. Introduc-
tion of a 5-methyl onto the thiophene (14) led to a
reduction of potency and selectivity. Replacement of
the entire aryl ketone with a nitrile or ethyl ester (data
not shown) led to a complete loss of activity in both cell
lines. These initial studies indicated a strict electronic
and steric requirement for good activity in this portion
of the molecule.
With this information in hand we explored the require-
ment of the non-bridgehead pyrimidine nitrogen for
activity (Table 3). The de-aza compounds (32–35)
showed a >10-fold loss in potency in the p21À/À line
and a corresponding loss in selectivity. Although the
methyl probe results suggested only a minor role for a
direct binding interaction of the pyrimidine nitrogen in
activity, there appears to be a significant electronic effect
of this pyrimidine nitrogen on the rest of the core that
influences activity.
We also explored the positional requirements of the at-
tached amide by utilizing the potent isobutyl amide
Table 2. Activity comparison for methyl substituted derivatives in p21-proficient and -deficient cell lines
H
N
R5
O
R3
R2
N
N
R4
N
O
S
Compound
R2
R3
R4
R5
p21+/+ (IC50 lM)
p21À/À (IC50 lM)
Ratio
17
18
19
20
21
22
23
24
25
26
CH3
CH3
CH3
CH3
H
H
H
Cyclopropyl
O-iso-Propyl
iso-Butyl
9.2
3.9
0.64
0.28
0.48
0.62
>20
14.4
14
H
H
H
H
3.7
7.7
18
H
H
NH-iso-propyl
Cyclopropyl
O-iso-Propyl
iso-Butyl
11
CH3
CH3
CH3
CH3
H
H
>20
>20
>20
>20
>20
>20
—
H
H
17.3
>20
>20
>1
—
H
H
H
H
NH-iso-propyl
O-iso-Propyl
iso-Butyl
—
—
H
CH3
CH3
>20
>20
H
H
—

1644
D. Powell et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1641–1645
Table 3. Activity comparison for de-aza derivatives in p21-proficient and -deficient cell lines
H
N
R5
O
N
N
O
S
Compound
R5
p21+/+ (IC50 lM)
p21À/À (IC50 lM)
Ratio
32
33
34
35
Cyclopropyl
O-iso-Propyl
iso-Butyl
12.8
>20
>20
6.4
2.4
4.2
5.6
2
>8.3
>4.8
>3.8
NH-iso-propyl
>20
Table 4. Activity comparison for phenyl positional isomers in p21-proficient and -deficient cell lines
H
N
O
N
N
N
O
S
Compound
Position
p21+/+ (IC50 lM)
p21À/À (IC50 lM)
Ratio
36
37
2
Para
Ortho
Meta
>50
>50
6.4
>50
>50
0.14
—
—
46
Table 5. Activity comparison in Colon carcinoma cell line panel
Compound
LoVo (IC50 lM)
SW620 (IC50 lM)
DLD1 (IC50 lM)
HT29 (IC50 lM)
1
2
0.068
0.021
0.076
0.70
0.11
0.045
0.11
0.65
0.085
0.042
0.084
0.85
0.078
0.036
0.078
0.62
19
33
group (Table 4). Movement of the amide to the para and
ortho positions led to loss in activity.
core nitrogen significantly reduced activity and the
attachment of the amide side in the meta position was
found to be optimal. This effort has helped to define
the SAR for this interesting new series of antiprolifera-
tive agents which will be explored further in subsequent
publications.
The most potent compounds from the p21À/À cell line
were tested in a panel of colon carcinoma cells (Table
5). The overall potency trends seen in the p21À/À cell
line were also seen in the colon panel, with the de-aza
analog, 33, being the least potent. The underlying mech-
anism for the preferential killing of p21À/À cells is not
currently understood. However representative com-
pounds in this series have shown an ability to bind to
the colchicine binding site of tubulin.16
Acknowledgments
The authors thank Dr. Dan Berger for helpful discus-
sions and the discovery analytical chemistry group at
Wyeth Research, Pearl River, NY, for spectral data.
In conclusion, we have explored some of the steric, elec-
tronic, and positional requirements of the lead series.
For the aryl ketone region of the molecule a strict
requirement for an unsubstituted 2-thienyl ketone was
found. A methyl probe of the core revealed an opportu-
nity for substituents at the R2 position. Removal of a
References and notes
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11. Cell proliferation assays. p21+/+ and p21À/À cells were
plated in 96-well tissue culture plates. The following day,
dilutions of compounds were added and cells were
cultured for 5 days. Cell survival was determined using
sulforhodamine B, a protein binding dye. The concentra-
tion of the compound that inhibits cell proliferation by
50% (IC50) was estimated from inhibition curves. The
selectivity ratio is the ratio of the IC50 in the p21-proficient
to the IC50 in the p21-deficient cells.
15. Compounds were purified by HPLC and the purity was
>90%. LC Conditions: HP 1100, 23 ꢁC, 10 lL injected;
Column: YMC-ODS-A 4.6 · 5.0 5 lm; Gradient A: 0.05%
TFA/water, B: 0.05% TFA/acetonitrile; time 0 and 1 min:
98% A and 2% B; 7 min: 10% A and 90% B; 8 min: 10% A
and 90% B; 8.9 min: 98% A and 2% B; Post time 1 min;
Flow rate 2.5 mL/min; Detection: 215 and 254 nm, DAD.
Semi-Prep HPLC: Gilson with Unipoint software; Col-
umn: Phenomenex C18 Luna 21.6 mm · 60 mm, 5 lM;
solvent A: water (0.02% TFA buffer); Solvent B: acetoni-
trile (0.02% TFA buffer); Solvent gradient: time 0: 5% B;
2.5 min: 5% B; 12 min: 95% B; hold 95% B 3 min; flow
rate: 22.5 mL/min; detection: 215 and 254 nm.
16. Rabindran, S. K.; Beyer, C.; Discafani, C. M.; McGinnis,
M. P.; Miranda, M.; Brown, G.; Goplasamy, A.; Krish-
namurthy, G.; Martinez, R.; Powell, D.; Wang, D.;
Zhang, N. 96th Annual Meeting of the American Asso-
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CA, Apr 16–20, 2005.
12. HCT116 is a human colon carcinoma cell line containing
an intact p21 checkpoint (p21+/+). 80S14 cells are