Pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides as novel anti-proliferative agents: Parallel synthesis for lead optimization of amide region

Article abstract of DOI:10.1016/j.bmcl.2005.01.066

A novel series of p21 chemoselective agents containing a pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides were identified by high throughput screening. Optimization of the amide region by parallel synthesis and the iterative design toward understanding structu

Full text of DOI:10.1016/j.bmcl.2005.01.066

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1591–1594
Pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides as novel
anti-proliferative agents: parallel synthesis for
lead optimization of amide region
Ariamala Gopalsamy,a,* Hui Yang,a John W. Ellingboe,a Hwei-Ru Tsou,a
Nan Zhang,a Erick Honores,a Dennis Powell,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 7 December 2004; revised 25 January 2005; accepted 27 January 2005
Abstract—A novel series of p21 chemoselective agents containing a pyrazolo[1,5-a]pyrimidin-7-yl phenyl amides were identified by
high throughput screening. Optimization of the amide region by parallel synthesis and the iterative design toward understanding
structure–activity relationship to improve potency are described. The isopropyl carbamate derivative 34 was identified as a highly
chemoselective agent displaying a potency of 51 nM in the p21 deficient cell line.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Deregulation of cell proliferation, or a lack of appropri-
ate cell death, has a wide range of clinical implications,
including cancers, restenosis, angiogenesis, hyperplasia,
endometriosis, lymphoproliferative disorders, graft
rejection and the like. Such cells may lack the normal
regulatory control of cell division, therefore fail to un-
dergo appropriate cell death. Progression from one
phase of the cell division cycle to the next phase is con-
trolled by a series of sensors and arresting mechanisms
called checkpoints.1 Loss of checkpoint control is a hall-
mark of tumor cells, as it increases the mutation rate
and allows a more rapid progression to the tumorigenic
state.1,2 However, inactivation of these checkpoints re-
sults in aberrant responses to cellular damage. This fail-
ure of checkpoint responses in malignant cells can be
exploited in cancer drug discovery. Identification of
compounds that selectively kill checkpoint-deficient cells
compared with checkpoint-proficient cells can be ex-
pected to preferentially target tumor cells, while sparing
normal cells.3,4
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-
dependent kinases (CDKs) and arrests cell cycle pro-
gression in response to DNA damage.7,8 Disruption of
this checkpoint by deletion of the p21 protein, leads to
the failure of the cell to arrest in response to DNA dam-
age, endoreduplication, and ultimately, apoptosis.9
These p21-deficient cells show increased chemosensiti-
vity10 compared with the isogenic p21-proficient paren-
tal cells to a variety of DNA damaging agents,
including clinically used anti-neoplastic drugs, validat-
ing the role of checkpoints in determining chemosensi-
tivity. We have used this isogenic pair of cell lines
(p21+/+ and p21À/À) to identify novel molecules that
preferentially induce apoptosis in the p21-deficient cells.
High throughput screening11 using the above cell lines
lead to the identification of pyrazolo[1,5-a]pyrimidin-7-
yl phenyl amide 1 (Fig. 1), which had an IC50 of
0.4 lM in a p21 deficient cell line (80S14)12 and an
IC50 of 12 lM in a p21 proficient cell line (HCT116)12
with a selectivity index of 30 (ratio of IC50s of p21-pro-
ficient and p21-deficient cells). The parallel synthesis
effort carried out to understand the structure–activity
Keywords: Pyrazolopyrimidine; Anti-proliferative agents; Anti-cancer
agents; Parallel synthesis.
*
Corresponding author. Fax: +1 845 602 3045; e-mail:
gopalsa@wyeth.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.066

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A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1591–1594
Figure 1. High throughput screen lead for identification of p21
chemoselective agent.
relationship of the amide region of this novel lead is de-
scribed below.
Scheme 2. Reagents and conditions: (a) Me2NCH(OMe)2, reflux, 18 h,
92%; (b) NH2OHÆHCl, reflux, 3 h, 65%; (c) Me2NCH(OMe)2, reflux,
18 h, 74%; (d) NH2NH2ÆH2O, 60%.
The compounds for this series were readily synthesized13
utilizing parallel solution phase synthesis as shown in
Schemes 1–3. The common intermediate amine 6 re-
quired for varying the amide region of the molecule
was synthesized starting from m-nitro acetophenone 2.
Conversion of the ketone 2 to enamine 3 followed by
condensation with amino pyrazole derivative 4 (synthe-
sis shown in Scheme 2) afforded the nitro compound 5.
Reduction of 5 afforded the common intermediate
amine 6. Reaction of the amine with cyclopropyl carb-
onyl chloride gave the lead compound 1.14
A number of amides 11–32 were synthesized using vari-
ous acid chlorides or acids with the appropriate activa-
tion. To vary the amide region of the molecule to
sulfonamide, carbamate, or urea, intermediate amine 6
was reacted with the required sulfonyl chlorides, chloro-
formates, or isocyanates (Scheme 3). Converting the
Scheme 3. Parallel synthesis. Reagents and conditions: (a) R1COCl,
pyridine, 0 ꢁC to rt, 6 h; (b) R1COOH, EDCI, HOBT, Et3N, DMF, rt,
12 h; (c) R1SO2Cl, pyridine, 0 ꢁC to rt, 6 h; (d) R1OCOCl, pyridine,
0 ꢁC to rt, 6 h; (e) 1: (4-NO2Ph)OCOCl, pyridine, 0 ꢁC to rt, 3 h, 2:
R1OH, rt, 18 h; (f) R1NCO, pyridine, 0 ꢁC to rt, 6 h; (g) 1: (4-
NO2Ph)OCOCl, pyridine, 0 ꢁC to rt, 3 h, 2: R1NH2, rt, 18 h.
amine 6 into the 4-nitrophenyl carbamate derivative
and reacting it with various amines also afforded ureas.
Similarly, carbamates were also synthesized by reacting
the 4-nitrophenyl carbamate of 6 with various alcohols.
N-Alkylation of 1 using sodium hydride and ethyl iodide
afforded 40.
Our initial effort toward understanding the SAR of the
lead molecule was focused on replacing the cyclopropyl
group with other cycloalkyl groups. As seen from the
data on Table 1, going to larger rings was very well tol-
erated and compounds 11–13 displayed similar activity
and selectivity. At this point we decided to incorporate
simple alkyl groups in the place of cyclopropyl to ex-
plore the spatial requirements for this region and also
to see if flexible alkyl groups can take the place of the
rigid cycloalkyl rings. The smaller methyl group 14 aboli-
shed the activity and the ethyl derivative 15 showed
slightly weaker activity. The activity was regained as
we went to the larger n-propyl and n-butyl groups (16–
17). However, the longer and flexible pentyl and hexyl
groups (18–19) showed a loss of potency, indicating
the spatial constraint for this region of the molecule.
Scheme 1. Reagents and conditions: (a) Me2NCH(OMe)2, reflux, 18 h,
89%; (b) 4, acetic acid, reflux, 82%; (c) Fe, NH4Cl, MeOH, H2O, 61%;
(d) cyclopropyl carbonyl chloride, pyridine, 0 ꢁC to rt, 6 h, 82%.

A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1591–1594
1593
Table 1. Activity comparison of amide derivatives in p21 proficient
and deficient cell lines
Table 2. Activity comparison for derivatives with various linkers in
p21 proficient and deficient cell lines
Ex R1
W
HCT116
80S14
Ratio
Ex R1
HCT116
80S14
Ratio
IC50 (lM) IC50 lM
IC50(lM) IC50 (lM)
33 i-Propyl
34 i-Propyl
35 n-Butyl
36 i-Propyl
37 n-Butyl
38 Neopentyl
NHSO2
12
1.1
14
2.7
2.1
6
22
93
30
12
28
8
1
Cyclopropyl
12
11
0.40
0.19
0.2
30
58
35
34
>2
26
20
15
9
NHCOO
NHCOO
NHCONH
NHCONH
NHCONH
0.051
0.15
0.089
0.36
0.14
1.6
11 Cyclobutyl
12 Cyclopentyl
13 Cyclohexyl
14 Methyl
15 Ethyl
6.9
8.4
>20
29
8.0
0.25
4.4
3.9
12
1.1
0.4
39 Cyclopropyl NHCONH
NH
40 Cyclopropyl N(Et)CO
13
16 n-Propyl
17 n-Butyl
18 n-Pentyl
19 n-Hexyl
20 i-Propyl
21 i-Butyl
6
H
>20
>20
>20
>20
1
5.4
9.1
0.35
1.0
1
13
16
6.4
4.3
0.6
3
27
46
14
27
21
32
40
23
34
32
1
0.14
0.33
0.29
0.19
0.25
0.21
0.27
0.44
0.47
22 t-Butyl
4.7
7.7
4.0
7.9
8.3
6.1
was used as the linker, as in the case of amide and car-
bamate series, the isopropyl analog 36 was found to be
the most potent analog. The intermediate amine 6,
which lacks the lipophilic capping group, was com-
pletely inactive. Similarly, removing the hydrogen bond
donor from the amide by alkylating the amide NH, as in
the case of compound 40, rendered the molecule inac-
tive. This observation lead to the conclusion that a
hydrogen bond donor–hydrogen bond acceptor se-
quence is preferred for the linker region.
23 Neopentyl
24 Homoallyl
25 Buty-3-nyl
26 Phenyl
27 Benzyl
28 2-Furyl
29 2-Thiophenyl
30 –CH2NH2
31 –CH2N(Me)2
15
15
>20
>20
>20
11.5
>20
>2
1
32 –CH2NHCO–cyclopropyl >20
The most potent compounds with the three linkers of
interest, amide (21), carbamate (34), and urea (36) were
selected for further characterization in the colon cell line
panel (Table 3). These compounds displayed good po-
tency across the panel and with no significant difference
in potency based on the variation in the linker.
Branched alkyls (20–23) performed on par or slightly
better when compared to the corresponding straight
chain analogs. In particular, the isobutyl derivative 21
showed improved potency as well as selectivity. Unsatu-
ration (24–25) was well tolerated in this region of the
molecule but did not significantly improve the activity
or selectivity. Similarly, replacing the cyclopropyl
group with aryl groups (26–27) or heteroaryl groups like
furan 28 or thiophene 29 maintained the biological
activity. A dramatic drop in activity was seen as we
introduced polar amino groups (30–31) at this region
of the molecule. Also separating the cyclopropyl moiety
with a glycine spacer (32) decreased the potency signifi-
cantly (Table 1).
In conclusion, we have identified pyrazolo[1,5-a]pyrim-
idine derivatives as a novel class of p21 chemoselective
anti-proliferative agents. We have explored the struc-
ture–activity requirement for the amide region of the
molecule. For the lipophilic region of the molecule a
small branched aliphatic group like isopropyl or isobu-
tyryl group was found to be ideal to achieve potency
as well as selectivity. Of the linkers explored, carbamate
and urea linkers were slightly more potent than an
amide linker. This effort has defined a promising
strategy to discover new agents with anti-proliferative
Although we optimized the lipophilic requirements
using the amide linker, it was of interest to see if any
other linkers would have an impact on improving the
biological profile of the lead. The results of this study
are summarized in Table 2. Replacing the amide with
sulfonamide decreased the potency (33). However, a carb-
amate linker was more favorable, leading to a several
fold improvement in potency while retaining the selec-
tivity in the case of isopropyl carbamate 34. When urea
Table 3. Activity comparison in the colon cell line panel
Ex
LoVo
IC50 (lM)
SW620
IC50 (lM)
DLD1
IC50 (lM)
HT29
IC50 (lM)
21
34
36
0.021
0.017
0.024
0.045
0.022
0.052
0.042
0.028
0.046
0.036
0.023
0.052

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A. Gopalsamy et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1591–1594
11. Cytotoxicity assays: p21+/+ and p21À/À cells isogenic
cells and other carcinoma cell lines (LoVo, SW620, DLD1,
HT29) were plated in 96-well tissue culture plates. The
following day, dilutions of compounds were added and
cells were cultured for an additional 4 days (colon cell
panel) or 5 days (isogenic cell lines). Cell survival was
determined using sulforhodamine B, a protein binding
dye. The concentration of the compound that inhibits cell
proliferation by 50% (IC50) was estimated from inhibition
curves. IC50s shown are the mean values compiled from
independent experiments (ranging from 2 to 67), except
for compounds 27, 33, and 40.
12. HCT116 is a human colon carcinoma cell line containing
an intact p21 checkpoint (p21+/+). 80S14 cells are
identical to HCT116 cells, except that the p21 checkpoint
was disrupted by targeted deletion of the p21 gene (p21À/À)
(Ref. 9a).
properties and led to the identification of several analogs
with sub-micromolar activity in a panel of colon cell
lines.
Acknowledgements
The authors would like to thank Dr. Tarek S. Mansour
and Yanong D. Wang for helpful discussions and dis-
covery analytical chemistry group at Wyeth Research,
Pearl River, NY for spectral data.
References and notes
1. Paulovich, A. G.; Toczyski, D. P.; Hartwell, L. H. Cell
1997, 88, 315–321.
13. Dusza, J. P.; Tomcufcik, A. S.; Albright, J. D.; Beer, B.
U.S. Patent 6,399,621, 2002.
2. Hanahan, D.; Weinberg, R. A. Cell 2000, 100, 57–70.
3. Torrance, C. J.; Agarwal, V.; Vogelstein, B.; Kinzler,
K. W. Nat. Biotechol. 2001, 19, 940–945.
4. Prendergast, G. C. Nat. Biotechnol. 2001, 19, 919–920.
5. Levine, A. J. Cell 1997, 88, 323–331.
6. Stewart, Z. A.; Pietenpol, J. A. Chem. Res. Toxicol. 2001,
14, 243–263.
7. Gartel, A. L.; Serfas, M. S.; Tyner, A. L. Proc. Soc. Exp.
Biol. Med. 1996, 213, 138–149.
14. Parallel solution phase synthesis was carried out in 8 mL
vials in 0.1 mmol scale using an orbital shaker. Reaction
completion was monitored by LC/MS and the solvent was
removed in Savant evaporator and compounds were
purified by HPLC and the purity was >90%. LC Condi-
tions: HP 1100, 23 ꢁC, 10 lL injected; Column: YMC-
ODS-A 4.6 · 50 mm, 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; Column: Pheno-
menex C18 Luna 21.6 mm · 60 mm, 5 lm; solvent A:
water (0.02% TFA buffer); solvent B: acetonitrile (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. Compounds integrity was
further determined by HRMS or CHN elemental analysis.
8. Sherr, C. J.; Roberts, J. M. Gene Dev. 1999, 13, 1501–
1512.
9. (a) Waldman, T.; Kinzler, K. W.; Vogelstein, B. Cancer
Res. 1995, 55, 5187–5190; (b) Waldman, T.; Lengauer, C.;
Kinzler, K. W.; Vogelstein, B. Nature 1996, 381, 713–716.
10. (a) Waldman, T.; Zhang, Y.; Dillehay, L.; Yu, J.; Kinzler,
K.; Vogelstein, B.; Williams, J. Nat. Med. 1997, 3, 1034–
1036; (b) Stewart, Z. A.; Mays, D.; Pietenpol, J. A. Cancer
Res. 1999, 59, 3831–3837.