Identification and optimization of small molecule antagonists of vasoactive intestinal peptide receptor-1 (VIPR1)

Article abstract of DOI:10.1016/j.bmcl.2012.01.082

Identification, synthesis and structure-activity relationship of small-molecule VIPR1 antagonists encompassing two chemical series are described.

Full text of DOI:10.1016/j.bmcl.2012.01.082

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2287–2290
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and optimization of small molecule antagonists of vasoactive
intestinal peptide receptor-1 (VIPR1)
Lalgudi S. Harikrishnan a, , Neelam Srivastava b, Lauren E. Kayser c, David S. Nirschl c,
⇑
Kumaragurubaran K d, Amrita Roy d, Anuradha Gupta d, Sukhen Karmakar d, Tajudheen Karatt d,
Arvind Mathur e, Neil T. Burford f, Jing Chen g, Yan Kong g, MaryEllen Cvijic g, Christopher B. Cooper a,
Michael A. Poss a, George L. Trainor a, Tai W. Wong b
a Early Discovery Chemistry, Bristol-Myers Squibb Co., PO Box 4000, Princeton, NJ 08543-4000, USA
b Discovery Biology, Bristol-Myers Squibb Co., PO Box 4000, Princeton, NJ 08543-4000, USA
c Synthesis and Analysis Technology Team, Bristol-Myers Squibb Co., PO Box 4000, Princeton, NJ 08543-4000, USA
d Bristol-Myers Squibb Biocon Research Center, Syngene International Ltd, Plot 2&3 Bommasandra IV Phase, Jigani Link Road, Bangalore 560 100, India
e Discovery Chemistry Synthesis, Bristol-Myers Squibb Co., PO Box 4000, Princeton, NJ 08543-4000, USA
f Lead Discovery, Bristol-Myers Squibb Co., 5 Research Parkway, Wallingford, CT 06492, USA
g Lead Evaluation, Bristol-Myers Squibb Co., PO Box 4000, Princeton, NJ 08543-4000, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Identification, synthesis and structure–activity relationship of small-molecule VIPR1 antagonists encom-
passing two chemical series are described.
Received 19 December 2011
Revised 17 January 2012
Accepted 20 January 2012
Available online 2 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Vasoactive intestinal peptide receptor
(VIPR)
Vasoactive intestinal peptide (VIP)
Vasoactive intestinal peptide receptor-1 (VIPR1) is a class B
GPCR expressed predominantly in lung, small intestine, thymus
and brain. VIPR1 and its endogenous agonist VIP (28 AA polypep-
tide) have been reported to be overexpressed in several tumors.1,
VIPR1 antagonists are peptide-based with poor drug-like proper-
ties. To our knowledge, there are no literature reports on small
molecule antagonists of VIPR1. In this communication, identifica-
tion and initial optimization of two chemical series of VIPR1 antag-
onists are described.
2
The density of VIPR1 in certain tumors is so high that labeled
ligands like (125IVIP) are used in tumor imaging and diagnostic pro-
cedures.3 Thus expression data alone suggests a possible role for
VIPR1 in cancer. Consequently, VIPR1 is viewed as a potential ther-
apeutic intervention point for drug discovery efforts. In fact,
several peptidic VIP-based VIPR modulators have been reported
to exhibit antiproliferative effects on various cancer cell lines4
including breast,5 colon,6 glioblastoma,7 pancreatic8 and NSCLC.9
In addition to inhibition of cancer cell proliferation, the VIPR1
antagonist SNH has been reported to potentiate the effect of vari-
ous chemotherapeutic agents on the NSCLC NCI-H727 cell line.6b
Further, the VIP hybrid antagonist (neurotensin6–11VIP7–28) has
been shown to reduce tumor volume in vivo in mice.6a Encouraged
by the literature data as well as in-house siRNA experiments,10 we
decided to investigate VIPR1 as an oncology target. All the reported
High throughput screening (HTS) of the Bristol-Myers Squibb
compound collection was carried out using a VIPR1 HTRF cAMP
assay in 1536 well format.11 Active compounds were retested in
F
CN
NH
OH
S
O
2
1
VIPR1 cAMP IC50 = 12µM
VIPR1 cAMP IC50 = 0.74 µM
⇑
Corresponding author.
E-mail address: lalgudi.harikrishnan@bms.com (L.S. Harikrishnan).
Figure 1. Representative compounds from biaryl and cyanothiophene series.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.082

2288
L. S. Harikrishnan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2287–2290
EtO2OC
COO2Et
O
HOOC
O
O
a, b
c, d
H
4
5
3
O
O
O
f
COOEt
g
e
6
7
OH
OH
OTf
COOEt
CHO
CHO
h, i
j
8
9
10
Ar
Ar
CHO
k
l
OH
11
12
Scheme 1. Preparation of biaryl compounds. Reagents and conditions: (a) Ispropylmethylketone, NaOEt, EtOH, rt, 5 h, 40% yield; (b) Diethylmalonate, NaOEt, EtOH, rt, 12 h,
60% yield; (c) KOH, EtOH, reflux, 1 h; (d) Cu2O, MeCN, reflux, 1.5 h, 90% yield (two steps); (e) HClO4, Ac2O, EtOAc, rt, 5 h, 91% yield; (f) cyclohexyl(iPr)NLi, EtOAc, ꢁ78 °C, 1.5 h,
79% yield; (g) DDQ, dioxane, reflux, 17 h, 51% yield; (h) LAH, THF, rt, 1 h, 78% yield; (i) DDQ, DCM, rt, 1.5 h, 65% yield; (j) Tf2O, pyridine, DCM, 0 °C-rt, 55% yield; (k) ArB(OH)2,
[Pd(dppf)Cl2], K3PO4, dioxane, 95 °C, 24 h; (l) NaBH4, EtOH, rt, 4 h, 10–36% yield (two steps).
Table 1
dose–response mode in 384 well format.12 This resulted in the iden-
VIPR1 cAMP IC50 data for biaryl series
tification of a biaryl series and a cyanothiophene series as exempli-
Z
4
fied by compounds 1 and 2, respectively (Fig. 1).13 Both series were
pursued to explore SAR and improve potency against VIPR1.
A variation of a reported synthetic route was utilized to access
compounds in the biaryl series having various 2-aryl rings (Scheme
1).14 Condensation of isopropylmethylketone with aldehyde 3 fol-
lowed by Michael addition of diethylmalonate gave ketone 4.
Saponification and subsequent decarboxylation of diester 4 gave
keto acid 5. Phenol 8 was then obtained via dehydrative cyclization
of acid 5, followed by ring opening of lactone 6 with the anion of
ethyl acetate and subsequent oxidation/aromatization. Reduction
of ester 8 to the corresponding primary alcohol followed by Swern
oxidation gave aldehyde 9. Sulfonylation of phenol 9 using triflic
anhydride yielded the corresponding aryl triflate 10. Suzuki cou-
pling of aldehyde 10 with various aryl boronic acids followed by
reduction of aldehyde 11 to alcohol 12 was carried out in parallel.15
All final compounds were purified by reverse phase HPLC.
The SAR of various substitutions on the 2-phenyl ring are sum-
marized in Table 1. Mesomeric or hyperconjugative electron
releasing substituents at the 3- and 4-positions of the 2-aryl ring
were preferred over substitution at 2-position (3 > 4 ꢀ 2). Overall,
3-chloro and 3,4-dimethyl compounds exhibited the best potency
against VIPR1.
3
2
OH
Compd.
Z
VIPR1 cAMP IC50 (lM)
13
14
15
16
17
18
1
19
20
21
22
23
24
25
26
27
28
29
30
31
2-Me
3-CF3
3-CN
2-Ph
4-OMe
2-OMe
4-F
>50
>50
>50
3.2
1.3
0.82
0.74
0.68
0.39
0.31
0.30
0.29
0.24
0.21
0.20
0.17
0.18
0.12
0.10
0.081
4-Ph
H
3-NMe2
4-Me
4-Cl
3-Me
3-Ph
3-OMe
3-SMe
Naphthyl (2,3-fused)
Naphthyl (3,4-fused)
3-Cl
To determine the potency of compounds on cell proliferation,
three cell lines, namely H727, H1299 and Calu6 were chosen based
on high levels of VIPR1 expression. Further, siRNA mediated knock
down of VIPR1 in these three cell lines resulted in robust reduction
of intracellular cAMP and significant inhibition of cell proliferation
(data not shown). In spite of the improvement in potency in the
cAMP assay, no significant inhibition of cell proliferation was ob-
served in any of the three cell lines at concentrations of compounds
3,4-Dimethyl
The SAR for various amides in the cyanothiophenes is summa-
rized in Table 2. Larger acyl groups led to improvement in VIPR1
antagonism both in acyclic as well as cyclic examples (see methyl
compound 32 to isobutyl compound 34, as well as cyclopropyl
compound 35 to cyclohexyl compound 38). In addition, moving
the phenyl ring farther from the acyl carbon led to significant
improvements in potency. Phenethyl compound 41, the most po-
tent compound in this series, was explored further by substituting
up to 10 lM. In order to minimize the impact of compounds being
potentially sequestered by albumin, the cell proliferation assays
were carried out in as low as 2% serum.16
The synthetic approach employed to access various amides in
the cyanothiophene series is shown in Scheme 2.17

L. S. Harikrishnan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2287–2290
2289
Table 3
N
N
VIPR1 cAMP IC50 data for various substituted and constrained analogs of the
phenethyl amide compound 41
HO
O
a or b
+
R
CN
O
NH
O
NH2
S
R
S
R
N
H
S
Scheme 2. Preparation of cyanothiophene analogs. Reagents and conditions: (a)
T3P, Et3N, DCM, 0 °C to rt, 5–69% yield; (b) CDI, DIPEA, DCM, rt, 15–30% yield.
Compd.
R=
VIPR1 cAMP IC50
11
(lM)
Me
Table 2
42
VIPR1 cAMP IC50 data for various amides in the cyanothiophene series
CN
O
Me
Me
43
44
45
46
0.75
0.48
0.61
0.41
R
N
H
S
Compd.
R=
VIPR1 cAMP IC50 (lM)
32
2
13
12
OMe
33
3.4
34
35
1.9
2.7
OMe
Cl
47
1.2
36
37
1.8
S
O
48
49
50
51
1.9
3.8
50
0.47
38
0.47
HN
39
40
41
15
12
1.6
0.29
52
53
17
O
9.1
the phenyl ring as well as by constraining the ethylene linker. The
results are summarized in Table 3. Unfortunately, none of the com-
pounds showed improved potency over the phenethyl compound
41. Furthermore, despite the improvements in potency, none of
the compounds showed significant antiproliferative effects in the
cell lines tested (H727, H1299 and Calu6).
for distribution of compounds to Lead Discovery and Lead Evalua-
tion groups.
In summary, the Bristol-Myers Squibb compound collection was
screened, and series of biaryl alcohols and cyanothiophenes were
identified as VIPR1 antagonists. SAR studies using parallel synthe-
sis led to modest improvements in potencies (in both series) in the
cAMP assay. However, none of the compounds showed significant
antiproliferative effects in the cell lines tested. Further improve-
ments in potency may be required to elicit the desired antiprolifer-
ative response.
References and notes
1. Reubi, J. C.; Laderach, U.; Waser, B.; Gebbers, J.; Robberecht, P.; Laissue, J. A.
Cancer Res. 2000, 60, 3105.
2. Schulz, S.; Rocken, C.; Mawrin, C.; Weise, W.; Hollt, V.; Schulz, S. Clin. Cancer
Res. 2004, 10, 8235.
3. Virgolini, I.; Raderer, M.; Kurtaran, A.; Angelberger, P.; Banyai, S.; Yang, Q.; Li,
S.; Banyai, M.; Pidlich, J.; Niederle, B.; Scheithauer, W.; Valent, P. NEJM 1994,
331, 1116.
4. (SN)VH is reported to have inhibited 51 of 56 cancer cell lines tested. Moody, T.
W.; Walters, J.; Casibang, M.; Zia, F.; Gozes, Y. Ann. N.Y. Acad. Sci. 2000, 921, 26.
5. (a) Zia, H.; Hida, T.; Jakowlew, S.; Birrer, M.; Gozes, Y.; Reubi, J. C.; Fridkin, M.;
Gozes, I.; Moody, T. W. Cancer Res. 1996, 56, 3486; (b) Moody, T. W.; Leyton, J.;
Chan, D.; Brenneman, D. C.; Fridkin, M.; Gelber, E.; Levy, A.; Gozes, I. Breast
Cancer Res. Treat. 2001, 68, 55.
Acknowledgments
We acknowledge Drs. Jeffrey A. Robl, Percy H. Carter, Samuel
W. Gerritz and Ashok V. Purandare for their advice and support.
We thank Preclinical Candidate Optimization group for protein
binding determination. We thank Compound Management group
6. (a) Levy, A.; Gal, R.; Granoth, R.; Dreznik, Z.; Fridkin, M.; Gozes, I. Regul. Pept.
2002, 109, 127; (b) Gelber, E.; Granoth, R.; Fridkin, M.; Dreznik, Z.; Brenneman,
D. E.; Moody, T. W.; Gozes, I. Cancer 2001, 92, 2172.

2290
L. S. Harikrishnan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2287–2290
7. Sharma, A.; Walters, J.; Gozes, Y.; Fridkin, M.; Brenneman, D.; Gozes, I.; Moody,
T. W. J. Mol. Neurosci. 2001, 17, 331.
binding studies, a final concentration of 5
lg/well of RKE-VIPR cell membrane
preparations were incubated with 200 g/well wheat germ agglutinin-coated
l
8. (a) Jiang, S.; Kopras, E.; McMichael, M.; Bell, R. H.; Ulrich, C. D. Cancer Res. 1997,
57, 1475; (b) Zia, H.; Leyton, J.; Casibang, M.; Hau, V.; Brenneman, D.; Fridkin,
M.; Gozes, I.; Moody, T. W. Life Sci. 1999, 1(66), 379.
PVT beads (Cat. 25007078, GE Healthcare) in 100 uL assay buffer [50 mM
HEPES, 5 mM MgCl2, 1 mM CaCl2, 0.1% BSA] for 10 min at room temperature.
This mixture was then added to 96-well assay plates (Cat. 3912, Costar)
9. (a) Moody, T. W.; Coelho, T.; Jakowlew, S.; Takahashi, K.; Jameison, F.; Koh, M.;
Fridkin, M.; Gozes, I.; Knight, M. Life Sci. 1997, 61, 1657; (b) Moody, T. W.; Zia,
F.; Draoui, M.; Brenneman, D. E.; Fridkin, M.; Davidson, A.; Gozes, I. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 4345.
containing 1.5
point serial dilutions) ranging from 10ꢁ10 to 10ꢁ5 M. The control for non-
specific binding was 1 M VIP (Cat. V6130, Sigma). A final concentration of
100 pM of [125I] VIP (Cat. NEX192050UC, Perkin Elmer) in 50
L assay buffer
lL test compounds at increasing concentrations (threefold, 11-
l
l
10. Srivastava et al. (Unpublished results).
11. High throughput screen (HTS) details: Compounds at 10 lM concentration were
was then added to the reaction. Sealed assay plates were incubated at room
temperature for 3 h then analyzed by TopCount. After correcting for non-
specific binding, IC50 values were determined. The concentration of test
compound that inhibited 50% of radioligand bound (IC50) was quantified using
the four parameter logistic equation to fit the data.
screened with 30 pM VIP (EC80) in HT29 or RKE cells using an HTRF cAMP assay
in 1536-well plates.
12. VIPR1 cAMP Assay details: Dose-dependent stimulation of intracellular cAMP
accumulation upon treatment with ligand VIP (Cat. 064–16, Phoenix
Pharmaceuticals) was measured with an HTRF (high throughput time-
resolved fluorescence) cAMP kit (Cat. 62AM4PEC, Cisbio) according to the
manufacturer’s directions. 384-well assay plates containing test compounds at
increasing concentrations (threefold, 11-point serial dilutions) ranging from
10ꢁ10 to 10ꢁ5 M were prepared. RKE-VIPR1 cells were grown in DMEM media
(Cat. 11995, Gibco) containing 10% fetal bovine serum (Cat. 16000, Gibco) and
14. Robl, J. Tetrahedron Lett. 1990, 31, 3421.
15. Procedure for conversion of aryl triflate 10 to alcohol 12: To an array of vials
containing a solution of aryl triflate 10 (30 mg, 1 equiv) in 1,4-dioxane (1 mL)
was
added
various
aryl
boronic
acids
(1.1 equiv),
[1,10-
bis(diphenylphosphino)ferrocene] dichloropalladium(II) (0.03 equiv) and
aqueous K3PO4 (3 equiv). The reaction mixtures were degassed with nitrogen
and heated at 95 °C for 24 h. The reaction mixtures were cooled to room
temperature and were filtered through a short pad of silica. The filtrates were
evaporated in a Genevac™. To the resulting array of crude aldehyde was added
a suspension of sodium borohydride (1 equiv) in anhydrous ethanol (1 mL).
The reaction mixtures were stirred at room temperature for 4 h. The solvents
were evaporated in a Genevac™ and the residues were purified by reverse
phase preparative HPLC.
500 lg/mL G418 (Cat. 10131, Gibco). RK3E-VIPR1 cells were detached using
versene (Cat. 15040, Gibco) and, subsequently, harvested in assay buffer
[Hanks Buffer (Cat. 14025–092, Invitrogen), 20 mM HEPES (Cat. 15630, Gibco),
1 mM IBMX (Cat. I5879, Sigma), 0.1% BSA (Cat. A3059, Sigma)]. Cells were
added into compound plates for a final cell density of 4 K cells/well and
incubated at room temperature for 15 min. Ten nanometer VIP was then added
to the assay plates at the final concentration of the EC50 value; assay plates
were incubated for 30 min at room temperature. After VIP stimulation, cells
were lysed with lysis buffer (CisBio kit) for 30 min at room temperature. Next,
16. Plasma binding determination of a representative compound (compound 1)
revealed it was only 90% bound in mouse and human plasma.
17. (a) CDI method: To an array of carboxylic acids (1.5 equiv) was added a stock
solution of CDI (1.5 equiv) and DIPEA (3.0 equiv) in DCM (1 mL). After 1 h, a
solution of the primary amine (1.0 equiv) in DCM (1 mL) was added and the
reaction mixture was stirred for 18 h at room temperature. The reaction
mixture was concentrated, and purified by reverse phase preparative HPLC.
(b) T3P method: To an array of vials containing various carboxylic acids
(1.5 equiv) was added a stock solution of the scaffold primary amine (1 equiv)
and triethyamine (3.0 equiv) in DCM (1 mL). The reaction mixture was cooled
to 0 °C and 0.3 mL of T3P (propane phosphonic acid anhydride, 50% solution in
EtOAc) was added to each vial. The vials were warmed up to room temperature
and stirred overnight. The reaction mixture was then concentrated and the
compounds were purified by preparative HPLC.
10
l
L of cell lysate was transferred to 384-well proxyplates (Cat. 6008289,
L of D2 reagent (CisBio kit) and 5 L of cryptate
Perkin Elmer), mixed with 5
l
l
reagent (CisBio kit). Reactions were incubated for 1 h at room temperature. The
HTRF signal was read on an EnVision plate reader (Perkin Elmer) at emission
wavelengths of 665 nm and 620 nm. cAMP accumulation was normalized by a
cAMP standard curve; IC50s were quantified using the four parameter logistic
equation to fit the cAMP accumulation data. The cAMP IC50 values reported are
average readings from two wells, with individual readings reproducible within
twofold of each other.
13. Both series of compounds were found to bind to VIPR1 with IC50 values that
were comparable to the cAMP IC50 values. For VIPR radioligand competition