Discovery and structure-activity relationship of 2-phenyl-oxazole-4-carboxamide derivatives as potent apoptosis inducers

Article abstract of DOI:10.1016/j.bmcl.2006.06.018

As a continuation of our efforts to discover novel apoptosis inducers as anticancer agents using a cell-based caspase HTS assay, 2-phenyl-oxazole-4-carboxamide derivatives were identified. The structure-activity relationships of this class of molecules were explored. Compound 1k, with EC₅₀ of 270 nM and GI₅₀ of 229 nM in human colorectal DLD-1 cells, was selected and demonstrated the ability to cleave PARP and displayed DNA laddering, the hallmarks of apoptosis. Compound 1k showed 63% tumor growth inhibition in human colorectal DLD-1 xenograft mouse model at 50 mpk, bid.

Full text of DOI:10.1016/j.bmcl.2006.06.018

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4554–4558
Discovery and structure–activity relationship of 2-phenyl-oxazole-4-
carboxamide derivatives as potent apoptosis inducers
Vincent W.-F. Tai,a,* David Sperandio,a Emma J. Shelton,a Joane Litvak,a
Keith Pararajasingham,a Ben Cebon,a Julia Lohman,a John Eksterowicz,a Seema Kantak,a
Peter Sabbatini,a Cindy Brown,a Jennifer Zeitz,a Chris Reed,a Bill Maske,a Doris Graupe,a
Alberto Estevez,a Jason Oeh,a Darren Wong,a Yong Ni,a Paul Sprengeler,a Robert Yee,a
Catherine Magill,a Anthony Neri,a Sui Xiong Cai,b John Drewe,b Ling Qiu,b John Herich,b
Ben Tseng,b Shailaja Kasibhatlab and Jeffrey R. Spencera,*
aCelera Genomics, 180 Kimball Way, South San Francisco, CA 94080, USA
bEpiCept Corporation, 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
Received 16 May 2006; revised 5 June 2006; accepted 6 June 2006
Available online 19 June 2006
Abstract—As a continuation of our efforts to discover novel apoptosis inducers as anticancer agents using a cell-based caspase HTS
assay, 2-phenyl-oxazole-4-carboxamide derivatives were identified. The structure–activity relationships of this class of molecules
were explored. Compound 1k, with EC50 of 270 nM and GI50 of 229 nM in human colorectal DLD-1 cells, was selected and dem-
onstrated the ability to cleave PARP and displayed DNA laddering, the hallmarks of apoptosis. Compound 1k showed 63% tumor
growth inhibition in human colorectal DLD-1 xenograft mouse model at 50 mpk, bid.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Apoptosis, or programmed cell death, is central to a
number of physiological processes and is essential for
animal development as well as tissue homeostasis.1,2 Re-
cent efforts in this field have identified multiple signaling
cascades leading to cell death. Two major mechanisms
are involved in the induction of apoptosis: agents that
activate a family of death receptors leading to the activa-
tion of the apoptotic cascade; or agents that trigger the
release of cytochrome C from mitochondria followed by
apoptosis. Both mechanisms lead to the activation of
caspases, a group of cysteine proteases, which carry
out the cleavage of both structural and functional ele-
ments of the cell.3 This results in morphological changes
such as cellular shrinkage, chromatin condensation,
membrane blebbing, and fragmentation into mem-
brane-enclosed vesicles.
identification of structurally diverse apoptosis inducers
offers a novel approach in the discovery of new antican-
cer agents. A cell-based high-throughput screen tracking
the activation of endogenous caspase-3 was previously
reported6–8 and employed for this purpose. Herein, we
report the discovery and structure–activity relationships
(SAR) of a new class of 2-aryl-oxazole-4-carboxamide-
containing apoptosis inducers and their in vivo
activities.
A panel of three different human cancer cell lines was
used for the HTS assays. Briefly, human breast cancer
cell lines T47D, ZR751 and human colorectal cancer cell
line DLD-1 were plated in 384-well microtiter plates
containing various concentrations of test compound
and incubated at 37 ꢁC for 24 h. After incubation, the
samples were treated with fluorogenic substrate N-(Ac-
DEVD)-N-ethoxycarbonyl-R1109 and incubated for
3 h. The caspase activation activity (EC50) was deter-
mined by sigmoidal dose–response calculation.10 Com-
pound 1a was found to activate caspases and induce
apoptosis with EC50 values of 0.55, 0.29, and 0.92 lM
in T47D, ZR751, and DLD-1 cells, respectively. In this
report, we will focus on the colorectal DLD-1 cell line
It has been demonstrated that various anticancer agents
cause apoptosis in cancer cells;4,5 therefore, the rapid
Keywords: Apoptosis; Oxazole; Apoptosis activators; Antitumor.
*
Corresponding authors. Tel.: +1 650 829 1000 (V.W.-F.T.); e-mail:
vince.tai@gmail.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.018

V. W.-F. Tai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4554–4558
4555
since we have successfully developed a DLD-1-implant-
ed xenograft mouse model.
To study the SAR of cyclic amines at position A, a more
potent phenylacetyl group was chosen at position D (see
below). As shown in Table 1, cyclic amino substitution
at position A showed an interesting trend as potency
peaks at the 7-membered homopiperidine ring. An inter-
esting correlation between the potency (EC50) of the
compounds and the van der Waals volume of the mole-
cules was observed (Fig. 1). The similar potency of 2,5-
dimethylpyrrolidine 1f and homopiperidine 1g can be
explained using this model as they have similar van
der Waals volumes. The piperidine and homopiperidine
rings were selected for further substitution and the
O
O
N
N
N
S
H
O
1a
EC50 T47D = 0.55 μM
EC50 ZR751 = 0.29 μM
EC50 DLD1 = 0.92 μM
Synthesis of 2-phenyloxazole scaffold was optimized as
shown in Scheme 1. Coupling of serine methyl ester
hydrochloride (2) and commercially available 4-{[(tert-
butoxycarbonyl)amino]methyl}benzoic acid (3) under
EDC/HOBt condition gave ester 4 in 90% yield.11 Treat-
ment of ester 4 with Burgess reagent induced cyclodehy-
dration to give oxazoline which was oxidized with
CBrCl3/DBU to give oxazole 5.12,13 Alternatively, con-
version of 4 to 5 can be carried out using Williams’ pro-
tocol [(diethylamino)sulfur trifluoride (DAST)]14
followed by oxidation. Ester 5, substituted with orthog-
onal protecting groups at the amino and carboxylic acid
groups, is a versatile intermediate for the SAR studies of
amides at positions A and D. In route A, the Boc pro-
tecting group was cleaved readily with 4 M hydrochloric
acid in dioxane/CH2Cl2 followed by coupling with an
acid to give amide 7. The methyl ester was then sapon-
ified with lithium hydroxide in THF/H2O mixture fol-
lowed by the coupling with various substituted cyclic
amines. This approach allowed rapid optimization of
cyclic amines at position A. Alternatively, optimization
of acyl group at position D can be carried out as illus-
trated in route B using similar reaction conditions.
Table 1. SAR studies at position A
O
O
R
N
N
H
O
Compound
Cyclic amines R
Ec50DLD-1 (nM)
553
1b
N
N
N
1c
1d
318
108
1e
1f
65
34
N
N
1g
1h
33
N
N
108
OH
OH
O
N
O
HO2C
a
b
O
O
+
NH2
.HCl
O
N
H
NHBoc
NHBoc
O
O
NHBoc
O
2
3
4
5
d
O
N
O
N
c
O
5
O
O
O
NH2.HCl
N
Route A
H
Ar
O
6
7
Route B
e
e, f
R
R
O
O
N
O
N
f
c, d
O
N
N
N
N
HO
O
NHBoc
NHBoc
H
Ar
O
O
9
1
8
C
A
B
D
i
Scheme 1. Reagents and conditions: (a) EDC, HOBt, Pr2NEt, DMF (90%); (b) i—Burgess reagent (MeO2CNÀSO2N+Et3), 60 ꢁC, 2 h; or DAST,
CH2Cl2, À78 ꢁC (80–90%); ii—CBrCl3, DBU, CH2Cl2 (73%); (c) HCl, dioxane, CH2Cl2 (90–100%); (d) acid, EDC, HOBt, iPr2NEt, DMF (70–85%);
i
(e) LiOH, THF, H2O (85–95%); (f) cyclic amine, PyBOP, Pr2NEt, DMF (65–80%).

4556
V. W.-F. Tai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4554–4558
tween the amide and the aromatic group was first elab-
600
500
400
300
200
100
0
orated (Table 3). It is interesting to note that the optimal
spacer of a single methylene unit is the outcome from
the HTS campaign. Phenyl, 2-thienyl, 2-furanyl groups
gave similar potency (EC50 < 100 nM), while pyridines
and 3-methyl isoxazole ring substitutions were detrimen-
tal to caspase-3 activation activity. Substitution on the
phenyl ring in general does not improve potency com-
pared to the parent compound 1e but offers a handle
for optimizing other physical properties such as solubil-
ity. It is noteworthy that fluoro substitution is more tol-
erated at the 2- and 3-positions, while the hydroxyl
group is more tolerated at the 4-position.
1b
1c
1d
1h
1f, 1g
580
1e
Selected compounds were also tested by an in vitro
growth inhibition (GI50) assay to confirm that the active
compounds can inhibit tumor cell growth.10 As shown
in Table 4, compound 1i is one of the most potent com-
pounds in this scaffold. In general, compounds that are
more potent in the apoptosis induction assay, as mea-
sured by caspase activation, are also more potent in
the growth inhibition assay.
520
540
560
Vdw Vol (
600
620
3
)
˚
Figure 1. A plot of EC50 (DLD-1) versus van der Waals A volume that
˚
was calculated using a grid approximation (spacing 0.75 A).
15
results of the piperidine substitutions are reported in
Table 2.
Compound 1k was selected for further biological stud-
ies. Caspase-mediated cleavage of the DNA-repair en-
zyme PARP (poly-ADP-ribose polymerase) was
Substitutions at both the 3- and 4-positions of the piper-
idine ring resulted in loss of potency relative to 1e with
Table 3. SAR studies at position D
the
exception
of
3,3-difluoropiperidine
1i
(EC50 = 20 nM). Halogen substitutions in general are
more tolerated at the 3-position than at the 4-position.
On the other hand, compounds with oxygen-containing
substituents such as the hydroxyl, keto-, and hydroxy-
methyl- were more potent than the halo-substiutions at
the 4-position. Despite losing some potency compared
to 1e, these oxophilic compounds showed better water
solubility compared to the parent compound 1e.
O
O
N
N
N
H
R
O
Compound
R
EC50 DLD-1 (nM)
>10,000
2-Thienyl
1j
–CH2(2-Thienyl)
–(CH2)2(2-Thienyl)
–(CH2)3(2-Thienyl)
–CH2(2-Furanyl)
59
487
1080
90
Using the unsubstituted piperidine amide as in analog
1e, the SAR at position D was explored. The linker be-
–CH2(3-Methyl-isoxazol-5-yl) 2594
–CH2(3-Pyridyl)
–CH2(2-Pyridyl)
3315
3565
Table 2. SAR of substituted piperidine ring at position A
1e
–CH2Ph
65
65
O
–CH2(2-F)Ph
–CH2(2-OH)Ph
–CH2(3-F)Ph
–CH2(3-Cl)Ph
–CH2(3-OH)Ph
–CH2(4-F)Ph
–CH2(4-Cl)Ph
–CH2(4-OH)Ph
–CH2(4-Me)Ph
O
1006
72
R
N
N
N
H
O
533
139
233
274
73
Compound
R
EC50 DLD-1 (nM)
3-F
85
20
1i
3,3-F,F
3-Cl
3-Me
142
119
193
196
259
235
304
979
492
258
150
5229
115
196
1k
–CH2(4-OCF3)Ph
270
3-OH
3-OMe
3-(O)
4-F
Table 4. Comparison of caspase activation activity and growth
inhibition activity of selected compounds in DLD-1 cells
4,4-F,F
4-Cl
4-Me
Compound
EC50 DLD-1
(nM)
GI50 DLD-1
(nM)
Solubility
(lM)
4-OH
4-OMe
4-(O)
4-CH2OH
1e
1i
65
20
258
15
47
<10
29
1j
1k
59
270
75
229
44

V. W.-F. Tai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4554–4558
4557
demonstrated as shown in Figure 2A. PARP cleavage of
compound 1k-treated cells was demonstrated at 48 h
using Western blot analysis, whereas untreated control
did not show PARP cleavage. Figure 2B shows the
DNA laddering of compound 1k-treated cells after 48 h.
determined to be superior at this position. At position
D, a single methylene unit was determined to be the best
spacer between the aryl/heteroaryl group and the amide
group. Replacement of the 2-thienyl group with 2-fura-
nyl or substituted phenyl groups is tolerated. Compound
1k was further shown to cleave PARP and displayed
DNA laddering pattern, the hallmarks for apoptosis.
This compound showed tumor growth inhibition in hu-
man colorectal DLD-1 xenograft models. Additional
biological characterization of this class of compounds
will help to understand the mechanism of action of these
compounds and improve the efficacy in the future.
Based on its good caspase activation potency, prolifera-
tion assay, solubility, and pharmacokinetic profile, com-
pound 1k was evaluated in human tumor xenograft
models. At once a day dosing, compound 1k showed
21% (p = 0.096) and 38% (p = 0.002) tumor growth inhi-
bition of colorectal DLD-1 tumor xenografts at 50 mpk
and 100 mpk, respectively. At 50 mg/kg twice a day,
compound 1k achieved 63% (p < 0.001) tumor growth
inhibition. No significant weight loss was observed in
the above doses (Fig. 3).
Acknowledgments
The authors thank Drs. Michael C. Venuti, Michael J.
Green, Peter Young, and Sriram Balasubramanian for
helpful discussions in the in vitro evaluation of these
analogs and in preparation of the manuscript.
In summary, a new class of apoptosis inducers with a
2-phenyl-oxazole-4-carboxamide scaffold was identified
through a cell-based caspase HTS assay. SAR studies
at position A revealed an interesting correlation between
the van der Waals volume of the molecule with caspase
activation in one subgroup of analogs. A strong prefer-
ence for 6- and 7-membered cyclic amides at position A
was found and 3,3-difluoropiperidine-substitution was
References and notes
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fragmentation
Figure 2. (A) Cleavage of PARP;16 The uncleaved PARP is approx-
imately 115 kDa and the caspase-mediated cleavage product (arrow)
migrates at approximately 85 kDa. (B) DNA fragmentation of DLD-1
cells.17 Lane 1, untreated control; lane 2, DLD-1 cells treated with 1k;
lane 3, DLD-1 cells treated with vinblastine.
11. All new compounds are characterized by 1H NMR and
HPLC-MS analyses. Compound 1k: 1H NMR (400 MHz,
DMSO-d6) d 1.51–1.68 (m, 6H), 3.55 (s, 2H), 3.58 (br s,
2H), 3.82 (br s, 2H), 4.35 (d, 2H, J = 5.6 Hz), 7.29 (br d,
2H, J = 8 Hz), 7.35–7.44 (m, 4H), 7.91 (dd, 2H, J = 1.6,
8.4 Hz), 8.56 (d, 1H, J = 2.4 Hz), 8.68 (t, 1H, J = 5.6 Hz);
MS (ES) m/z 488.1 (MH+); MS calcd: 487.2 (M).
12. Wipf, P.; Miller, C. P. Tetrahedron Lett. 1992, 33, 907.
13. Williams, D. R.; Lowder, P. D.; Gu, Y.-G.; Brooks, D. A.
Tetrahedron Lett. 1997, 38, 331.
Vehicle, ip, bid;
450
Vehicle, ip, qd;
50 mg/kg compound, 1k, ip, qd
100 mg/kg compound 1k, ip, qd
350
50 mg/kg compound 1k, ip, bid
250
14. Philips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D.
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50
0
2
4
6
8
10
12
14
16
16. PARP cleavage—DLD-1 cells were seeded at 106 cells per
10-cm dish and 24 h later were treated with compound 1k
(1 lM) or vinblastine (400 nM) (final concentration of
DMSO was 0.2%). After 48 h, cells were harvested and
whole-cell lysates prepared using standard procedures.
Cell extract (30 lg) was electrophoretically separated on a
Days post dosing
Figure 3. Anti-tumor activity of compound 1k-treated athymic female
mice bearing DLD-1 human colon tumors.18 Twelve days post implant
of DLD-1 cells, mice were treated with various doses of compound 1k
on days 1–5, 8–12, and 15.

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V. W.-F. Tai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4554–4558
4–20% Tris–glycine gel (Novex), blotted, and hybridized
then lyophilized, resuspended in Tris/EDTA containing
0.6 mg/mL RNASe, and incubated at 37 ꢁC for 30 min.
Samples were then electrophoresed on a 2% TAE gel
containing ethidium bromide and visualized under ultra-
violet light.
overnight with anti-PARP polyclonal antibody (cell
signaling, Inc.). After subsequent washing and hybridiza-
tion with HRP-anti-rabbit secondary antibody, proteins
were detected using enhanced chemiluminescence accord-
ing to the manufacturer’s instructions.
18. In vivo assays—female Balb/c nude mice (8–10 weeks old,
Jackson Lab) were implanted with 3 · 106 DLD-1 cells
resuspended in serum-free media containing 50% matrigel
(BD Biosciences). Twelve days postimplant, when tumors
reached 100–150 mm3, the mice were randomized into
treatment groups and treated with compound 1k. All
animals were weighted daily, and caliper measurements
were taken three times a week after tumors were calculated
by the Mann–Whitney rank sum test using SigmaStat
software. Two-sided p-values are given at a 95% confi-
dence level.
17. DNA fragmentation analysis—DLD-1 cells were seeded at
106 cells per 10-cm dish and 24 h later were treated with
compound 1k (1 lM) or vinblastine (400 nM). After 48 h,
the cells were harvested by trypsinization, washed with
PBS (pH 7.4), and lysed in Triton X buffer (0.2% Triton
X-100, 10 mM Tris, and 10 mM EDTA). After 10 min
incubation on ice, lysates were pelleted and supernatant
extracted twice with phenol/chloroform using standard
procedures. Small MW DNA was then precipitated with
300 mM NaCl and ethanol at À80 ꢁC. Precipitates were