Synthesis and pharmacological evaluation of N -(3-(1 H -Indol-4-yl)-5-(2- methoxyisonicotinoyl)phenyl)methanesulfonamide (LP-261), a potent antimitotic agent

Article abstract of DOI:10.1021/jm100659v

The synthesis and optimization of a series of orally bioavailable 1-(1H-indol-4-yl)-3,5-disubstituted benzene analogues as antimitotic agents are described. A functionalized dibromobenzene intermediate was used as a key scaffold, which when modified by sequential Suzuki coupling and Buchwald-Hartwig amination provided a flexible entry to 1,3,5-trisubstituted phenyl compounds. A 1H-indol-4-yl moiety at the 1-position was determined to be a critical feature for optimal potency. The compounds have been shown to induce cell cycle arrest at the G2/M phase and demonstrate efficacy in both cell viability and cell proliferation assays. The primary site of action for these agents is revealed by their colchicine competitive inhibition of tubulin polymerization, and a computational model has been developed for the association of these compounds to tubulin. An optimized lead LP-261 significantly inhibits growth of a human non-small-cell lung tumor (NCI-H522) in a mouse xenograft model.

Full text of DOI:10.1021/jm100659v

J. Med. Chem. 2011, 54, 179–200 179
DOI: 10.1021/jm100659v
Synthesis and Pharmacological Evaluation of N-(3-(1H-Indol-4-yl)-5-
(2-methoxyisonicotinoyl)phenyl)methanesulfonamide (LP-261), a Potent Antimitotic Agent
Rupa S. Shetty,* Younghee Lee, Bin Liu, Arifa Husain, Rhoda W. Joseph, Yixin Lu, David Nelson, John Mihelcic,
Wenchun Chao, Kristofer K. Moffett, Andreas Schumacher,† Dietmar Flubacher,† Aleksandar Stojanovic,†
Marina Bukhtiyarova, Ken Williams, Kyoung-Jin Lee, Alexander R. Ochman, Michael S. Saporito,
William R. Moore, Gary A. Flynn, Bruce D. Dorsey, Eric B. Springman, Ted Fujimoto, and Martha J. Kelly
Ansaris, Four Valley Square, 512 East Township Line Road, Blue Bell, Pennsylvania 19401, United States.
†Solvias AG, Klybeckstrasse 191, 4002 Basel, Switzerland. Phone: 41-61-686-61-61. Fax: 41-61-686-65-65.
Received June 1, 2010
The synthesis and optimization of a series of orally bioavailable 1-(1H-indol-4-yl)-3,5-disubstituted
benzene analogues as antimitotic agents are described. A functionalized dibromobenzene intermediate
was used as a key scaffold, which when modified by sequential Suzuki coupling and Buchwald-Hartwig
amination provided a flexible entry to 1,3,5-trisubstituted phenyl compounds. A 1H-indol-4-yl moiety
at the 1-position was determined to be a critical feature for optimal potency. The compounds have been
shown to induce cell cycle arrest at the G2/M phase and demonstrate efficacy in both cell viability and
cell proliferation assays. The primary site of action for these agents is revealed by their colchicine
competitive inhibition of tubulin polymerization, and a computational model has been developed for
the association of these compounds to tubulin. An optimized lead LP-261 significantly inhibits growth
of a human non-small-cell lung tumor (NCI-H522) in a mouse xenograft model.
Introduction
Another natural product, 1 (colchicine),6 destabilizes micro-
tubules by binding to β-tubulin at the interface with R-tubulin
within the heterodimer, a site distinct from the Vinca alkaloid
binding site. A number of structurally diverse natural products
and small molecules have been found to inhibit tubulin poly-
merization competitively with colchicine.7 A pharmacophore
model for colchicine site inhibitors has been proposed based on
these ligands.8 The crystal structures of tubulin complexed with
a colchicine analogue,9 2 (ABT-751),10 and two other col-
chicine competitive inhibitors have been reported.11 The bind-
ing sites of these inhibitors overlap, although there are some
differences in the pockets targeted by each compound. On the
basis of these structures, it has been hypothesized that colchi-
cine site inhibitors lock the tubulin complexes into a curved
conformation and interfere with the adoption of a straight
conformation that allows for microtubule assembly.9,11
The combretastatins, especially 3a (CA4), represent another
class of natural products that are colchicine competitive inhibi-
tors of tubulin polymerization.12 The combretastatins, and many
of the other colchicine binding site inhibitors, exhibit potent
vascular disrupting activity.13 Colchicine, which is approved for
the treatment of gout, has not been used as an anticancer agent,
presumably because of its toxicity with prolonged exposures and
high doses. There are no currently marketed colchicine compe-
titive drugs, but a number of inhibitors are in clinical develop-
ment, including 2, the combretastatins 3b (CA-4P)14 and 3c
(AVE 8062),15 2-methoxyestradiol (2ME2),16 and the more
metabolically stable 4 (ENMD-1198),16 5 (NPI 2358),17 and 6
(MPC-6827)18 (Figure 1). Unlike the taxanes and Vinca alka-
loids which in general require intravenous dosing, many of the
colchicine competitive small molecules have the potential for
good oral bioavailability.
Microtubules are highly dynamic noncovalent polymers
formed by tubulin heterodimers consisting of R- and β-tubulin.
They are major components of the cytoskeleton of all eukary-
otic cells and play a vital role in maintenance of cell shape and
cell signaling.1 The elongation and shortening of the micro-
tubules are critical to mitosis. Interference with the dynamics of
microtubules blocks the cell cycle at the G2/M stage and leads
to apoptosis. For this reason, targeting microtubule dynamics
has been a successful strategy for chemotherapeutic agents.2
Antimitotic agents are often divided into two classes: one
class that stabilizes microtubules and increases the mass of
microtubules in the cells; the other class that destabilizes micro-
tubules.3 The well-characterized and clinically used taxanes and
epothilones are examples of compounds that stabilize micro-
tubules.
The majority of compounds that destabilize microtubules
are characterized based on their binding site as those that bind
˚
at the Vinca or the colchicine site. A 4.1 A resolution crystal
structure of vinblastine, a Vinca alkaloid, bound to tubulin in
complex with RB3 protein stathmin-like domain (RB3-SLDa)
shows the molecule bound between the tubulin Rβ heterodi-
mers near the R-tubulin GTP binding site (1z2b.pdb).4 Many
other natural products such as the halichondrins and dolasta-
tins have been found to bind at or near the Vinca binding site.5
*To whom correspondence should be addressed. Phone: 215-358-
2037. Fax: 215-358-2030. E-mail: rshetty@ansarisbio.com.
a Abbreviations: WST-1, (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-
5-tetrazolino]-1,3-benzene disulfonate); MDR1, multidrug resistance 1;
SLD, stathmin-like domain; NCI-ADR, National Cancer Institute
adriamycin-resistant cell line; TIPS, triisopropylsilyl; TBAF, tetrabuty-
lammonium fluoride; PMB, 4-methoxybenzyl.
r
2010 American Chemical Society
Published on Web 12/02/2010
pubs.acs.org/jmc

180 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shetty et al.
Figure 1
Scheme 1a
a Reagents and conditions: (a) KOH, benzyl alcohol, 4-methoxylbenzyl alcohol or methanol, Bu4NBr, TMU; (b) HBr, Bu4NBr; (c) NaH, DMF,
TIPSCl or TBDPSCl; (d) pyridine-3-amine, Pd2(dba)3, BINAP, t-BuONa, toluene, 90 °C; (e) bis(pinacolato)diborane, PdCl2(dppf), KOAc, DMSO, 80 °C;
(f) Pd(PPh3)4, 2 M aqueous Na2CO3, DME, 85 °C, or PdCl2(dppf), 2 M K3PO4, THF, 65 °C, or chloro(di-2-norbornylphosphino)(2-dimethylamino-1,
10-biphenyl-2-yl)palladium, 2 M K3PO4, 1,4-dioxane, 100 °C; (g) H2, Pd/C or TFA, thioanisole, DIEA or BF3 OEt2, DMS, DCM, 0 °C.
3
The discovery that compound 7, designed in house for a
nuclear hormone receptor program, exhibited potent inhibi-
tion of mitosis at the G2/M stage led us to initiate an opti-
mization effort around this structure. The results of that work
are reported here, along with a proposed binding mode for the
compounds on β-tubulin and the in vitro and in vivo activity
of these compounds.
3-aminopyridine to give the protected 3-bromo-5-(pyridine-3-
ylamino)phenoxy compounds 10a-d.20 These reactions pro-
ceeded in about 60% yield for the benzyoxyl, 4-methoxybenzy-
loxy, and methoxy protected analogues. For analogue 10b, the
yield could be increased to 75% by heating the reaction mixture
to 90 °C prior to the addition of the catalyst. The lower yield
(45%) of the amination with the triisopropyl silyl (TIPS) pro-
tected phenol 9e was due to the instability of the protecting group
under the reaction conditions. No product 10e was obtained
when analogue 9f was subjected to the same reaction conditions.
The tert-butyldiphenylsilyl protected analogue 10e was synthe-
sized from 10b by deprotection of the 4-methoxybenzyloxy
group using trifluoroacetic acid in thiaanisole, followed by
protection of the phenol using tert-butyldiphenylsilyl chloride
in DMF. Suzuki coupling21 of the bromides 10a-d with an
arylboronic acid or pinacol boronate followed by deprotection
gave the 1,3,5-trisubstituted phenols. Alternatively, 10a,b, and
10e were converted to the pinacol boronates22 11a-cwhich were
Chemistry
The 3-aryl substituted 5-(pyridine-3-ylamino)phenols 7,
12-20 were synthesized as outlined in Scheme 1. The benzyloxy
9a, 4-methoxybenzyloxy 9b, and methoxyphenyl dibromide 9c
were synthesized from 3,5-dibromonitrobenzene according to
the procedure reported by Effenberger et al.19 The silyl ether
analogues 9e and 9f were synthesized by cleavage of the methyl
ether 9c, followed by silylation of the resulting phenol as shown
in Scheme 1. The symmetrical dibromides 9 were subjected to
Buchwald-Hartwig amination conditions using 1 equiv of

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 181
Scheme 2a
a Reagents and conditions: (a) Pd(PPh3)4, 2 M aqueous Na2CO3, DME, 85 °C; (b) 21a, 2 M aqueous K3PO4, 22, 1,4-dioxane, 100 °C (50%); (c) 21b,
2 M aqueous K3PO4, 22, 1, 4-dioxane, microwave, 130 °C (∼75%); (d) H2, Pd/C; (e) Pd/C, NH4CO2H, HCOOH, MeOH; (f) DDQ, DCM, 0 °C;
(g) BF3 OEt2, DMS, DCM, 0 °C .
3
coupled with aryl bromides and deprotected to give the corre-
sponding phenols.
Methylation of the Buchwald-Hartwig amination product
obtained from reacting key intermediate 9b with 3-aminopyr-
idine gave the N-methylated product 25. Copper-catalyzed
Ullmann ether synthesis with 3-hydroxypyridine afforded
the ether linked bromide 27.27 The ketone 33 was synthesized
by monolithiation of dibromide 9b followed by reaction
with 3-cyanopyridine, analogous to the method of Dickinson
et al.28 The bromides 25, 27, and 33 were then subjected to
Suzuki coupling to introduce the TIPS protected indole.
Deprotection of the 4-methoxybenzyl (PMB) group with
The Suzuki coupling of 4-bromoindole to the pinacol bo-
ronate 11a using catalytic amount of Pd(PPh3)4 as shown in
Scheme 2 yielded only 35% of the coupled product. Better yields
(50%) were obtained by coupling 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-indole (21a) with bromide 10b using
chloro(di-2-norbornylphosphino)(2-dimethylamino-1,10-biphe-
nyl-2-yl)palladium (22) as a catalyst.23 A 75% yield of 23c along
with small amounts of the deprotected indole 23b was obtained
by using 4-(4,4,5,5-tetramethyl-1,3,2-dioxoborolan-2-yl)-1-(triiso-
propylsilyl)-1H-indole (21b) in the reaction with 10b and 22 under
microwave conditions.
Unfortunately, no single method of deprotection worked for
all the compounds of Schemes 1 and 2. While deprotection of the
TIPS protected phenols proceeded to give the phenol cleanly, the
lability of the TIPS group limited its use to reaction sequences
with mild coupling conditions. Deprotection of compounds con-
taining the sensitive indole moiety was especially challenging,
and the results are summarized in Scheme 2. Hydrogenolysis of
23b using Pd/C gave a low yield of 7 and required multiple addi-
tions of fresh catalyst, probably due to catalyst poisoning by the
pyridine or by trace amounts of phosphines from previous steps.
Transfer hydrogenolysis24 of 23a using ammonium formate and
Pd/C proceeded in only 35% yield. Oxidative deprotection25 of
23b or 23c using 2,3-dichloro-5,6-dicyanobenzoquinone failed to
BF3 OEt2/dimethyl sulfide followed by treatment with tetra-
3
butylammonium fluoride (TBAF) yielded analogues 26, 28,
and 34. Suzuki reaction of the 1-bromo-3-iodo-5-(4-methoxy-
benzyloxy)benzene 9g with 21b gave intermediate 29 which
was subjected to a second Suzuki reaction followed by deprotec-
tion to give analogue 32. Lithiation of 29 followed by quenching
with 3-pyridinecarboxaldehyde and subsequent deprotection
provided methyl alcohol analogue 30.
The des-hydroxylphenyl analogue 38 was synthesized from
dibromobenzene as shown in Scheme 4. Introduction of 3-(N-
acetylamino)pyridine via a Buchwald-Hartwig amination
reaction using catalytic copper iodide29 provided bromide
37. Suzukicouplingfollowed by removal of thesilylprotecting
group and subsequent hydrolysis of the N-acetyl group under
acidic conditions yielded 38. Other phenol replacement ana-
logues were synthesized (Scheme 4) from commercially available
substituted phenyldibromides 39a-e which under Buchwald-
Hartwig amination conditions gave the aminopyridine inter-
mediates 40a-e. The bromide analogue 40e was subjected to
palladium catalyzed amidation conditions with acetamide to
give 40f. Installation of the TIPS protected indole group via
the Suzuki reaction followed by desilylation using TBAF gave
analogues 41-45.
Tribromobenzene 39e was used as the starting material for
the synthesis of ketone analogues where the phenolic hydroxy
moiety was replaced with a substituted amine. Scheme 5 shows
the synthesis of the 3,5-dibromophenyl ketone intermediates.
Tribromobenzene 39e was monolithiated with n-BuLi and then
reacted with either a nitrile or Weinreb amide 46a-d to yield
give the desired product. The BF3 OEt2/dimethyl sulfide sys-
tem26 proved to be the best condition for deprotection of the
4-methoxybenzyl group. A side product observed in the BF3
3
3
OEt2 deprotections of 23a was 24, which was formed by the acid
catalyzed addition of the benzyl cation to the electron rich
phenol. Addition of the cation scavenger to the reaction mixtures
minimized formation of this side product. Thus, the best general
method of making these compounds involved the use of the
4-methoxylbenzyl (PMB) protected phenols, which gave high
yields in both the Buchwald-Hartwig amination and the BF3
OEt2/dimethyl sulfide mediated deprotection steps.
3
Compounds with replacements of the NH group linking the
pyridine to the phenol were made as shown in Scheme 3.

182 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shetty et al.
Scheme 3a
a Reagents and conditions: (a) Pd2(dba)3, BINAP, t-BuONa, toluene, 90 °C; (b) MeI, NaH, DMF; (c) 21b, 22, 2 M aqueous K3PO4, 1,4-dioxane, 100 °C;
(d) BF3 OEt2, DMS, DCM, 0 °C or TFA, DCM, DMS; (e) n-Bu4NF, THF; (f) 3-hydroxypyridine, NaH, Cu2O; (g) n-BuLi, ether,-76 °C, 3-pyridinecarbox-
3
aldehyde; (h) 3-pyridylboronic acid, Pd(PPh3)4, 2 M aqueous Na2CO3, DME, 85 °C; (i) n-BuLi, ether, -76 °C, 3-cyanopyridine.
Scheme 4a
a Reagents and conditions: (a) Pd2(dba)3, BINAP, NaO-t-Bu, toluene, 90 °C; (b) 21b, 22, 2 M aqueous K3PO4, 1,4-dioxane, 100 °C; (c) TFA, DMS,
6 N HCl; (d) acetamide, Pd2(dba)3, XantPhos, Cs2CO3, 1, 4-dioxane, 110 °C; (e) n-Bu4NF, THF.
ketones 47a,b,d,f.28 The reaction yields with nitriles varied from
very poor to good depending on the pyridine substituents, while
the yields with the Weinreb amide were generally good. Func-
tionalization of the chloro group on the pyridine of 47b, 47d, and
47f by nucleophilic substitution with sodium methoxide or
morpholine gave the substituted pyridines 47c, 47e, and 47g,
respectively.
Buchwaldreactionofdibromide 47a withbenzyl carbamate
provided the protected amine 48a (Scheme 6), which on treat-
ment with 12 M HCl gave the aniline 48b. The amino group of
48b was reacted with acetyl chloride, methyl chloroformate, or
methylsulfonyl chloride to give the acetamide 48d, methylcar-
bamate 48c, and methyl sulfonamide 48e, respectively. The
N,N-dimethylurea 48f was synthesized by first forming the

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 183
Scheme 5a
a Reagents and conditions: (a) n-BuLi, ether, -76 °C; (b) NaOMe, MeOH, 80 °C; (c) morpholine, 1,4-dioxane, 100 °C.
Scheme 6a
a Reagents and conditions: (a) benzyl carbamate, Pd2(dba)3, XantPhos, t-BuONa, 1,4-dioxane, 110 °C; (b) 12 N HCl, 80 °C, MeOH; (c) MeCOCl or
ClCO2Me or MeSO2Cl or 4-nitrophenyl chloroformate and NHMe2, pyridine or DIEA, DCM, 0 °C; (d) 21b, 22, 2 M aqueous K3PO4, 1,4-dioxane,
100 °C; (e) KOH, MeOH, reflux; (f) n-Bu4NF, THF; (g) acetamide or methylcarbamate or methylsulfonamide, Pd(PPh3)4, XantPhos, t-BuONa, 1,4-
dioxane, 110 °C; (h) acetamide or methylcarbamate or methylsulfonamide, CuI, Cs2CO3, N,N0-dimethylethylenediamine, 1,4-dioxane, 100 °C.
4-nitrophenylcarbamate, followed by reaction with dimethy-
lamine to give the urea. Reaction of the bromides 48a,c-f
under Suzuki conditions with the TIPS protected indole pinacol
boronic ester yielded 49a,c-f. Treatment of 49a with refluxing
KOH in methanol resulted in hydrolysis of the benzylcarba-
mate as well as desilylation to give analogue 50. Analogues
49c-f were deprotected with TBAF in THF to give analogues
51-54. Alternatively, the amine derivatives 55 can also be
synthesized via a direct palladium or copper catalyzed reaction
with acetamide, methyl carbamate, or methylsulfonamide to
install the desired moiety. Installation of the amine derivative
could be performed before or after the indole is in place as
illustrated for compounds 57-62.
The benzo[d]oxazole and oxazolo[4,5-b]pyridine analogues
were synthesized as shown in Scheme 7 by reacting the 3,5-
dibromobenzoic acid 63 with either 3-hydroxyaniline 64a or
2-amino-3-hydroxypyridine 64b in polyphosphoric acid to give
the desired dibromo compounds 65a and 65b.30 Buchwald
coupling of 65a with acetamide and 65b with methylcarbamate
gave intermediates 66a-b respectively. The subsequent Suzuki

184 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shetty et al.
Scheme 7a
a Reagents and conditions: (a) polyphosphoric acid, 185 °C; (b) methyl carbamateor acetamide, Pd2(dba)3, XantPhos, t-BuONa, 1,4-dioxane, 110 °C;
(c) 21b, 22, 2 M aqueous K3PO4, 1,4-dioxane, 100 °C; (d) n-Bu4NF, THF.
Scheme 8a
a Reagents and conditions: (a) DPPA, DIEA, t-BuOH, toluene 100 °C; (b) HCl, 1,4-dioxane; (c) methanesulfonyl chloride or acetic anhydride,
pyridine, DCM, 0 °C; (d) arylboronic acid, PdCl2(dppf), K2CO3, 1,4-dioxane-H2O, 65 °C; (e) 1H-indol-4-ylboronic acid, Pd(PPh3)4, 2 M aqueous
Na2CO3, toluene/ethanol, 100 °C or 21b, 22, 2 M aqueous K3PO4, 1,4-dioxane, 100 °C; (f) n-Bu4NF, THF.
reaction with 21b followed by deprotection of the TIPS group
gave analogues 67 and 68.
4-quinoline 15 and the 4-benzothiaxole 14 were inactive in both
assays. While retaining some activity, the oxindole 17 was still
less potent than 7, and the ring-opened analogue 18 was
inactive in both cell viability and G2/M block assays. N-Methy-
lation of the 4-indole, analogue 16, resulted in complete loss of
activity in both the cell viability and G2/M cell cycle inhibition
assays. These results clearly indicate that the indole with an
unsubstituted N1 is a critical feature for potent activity. Many
inhibitors of tubulin polymerization described in the literature
contain an indole,31 but with the exception of the complex
marine natural product diazonamide A, none of these com-
pounds have a 4-substituted indole. Methyl substitution in the
2-position of the 4-indolyl moiety was well tolerated as illus-
trated by analogue 20, but substitution of a 3-chloro resulted in
loss of activity as demonstrated by analogue 19.
Analogues 72-74, where the aromatic group is directly
attached to the phenyl core, were synthesized as described in
Scheme 8. The Curtius rearrangement of 5-bromo-3-iodoben-
zoic acid 69 followed by quenching with tert-butanol gave
tert-butyl 3-bromo-5-iodophenylcarbamate, which on depro-
tection with HCl gave 3-bromo-5-iodoaniline 70. Derivatiza-
tion of the amine with acetyl anhydride or methanesulfonyl
chloride in the presence of base provided intermediates 71a,b.
Via two sequential Suzuki reactions, the aryl group and indole
moiety were introduced to give compounds 72-74.
Results and Discussion
Biological Evaluation of Compounds in Cell Cycle Inhibition
and Cell Viability Assays. The 3-(1H-indol-4-yl)-5-(pyridine-3-
ylamino)phenol 7 was shown to be a potent inhibitor of cell
viability in Jurkat and HeLa human cancer cell lines. Cell
viability was determined using the 4-[3-(4-iodophenyl)-2-(4-
nitrophenyl)-2H-5-tetrazolino]-1,3-benzene disulfonate (WST-1)
colorimetric mitochondrial reduction assay. Flow cytometric
analysis of cell-cycle status showed that MCF-7 cells treated
with 7 became arrested in the G2/M phase of the cell cycle
growth in a dose-dependent manner. Our initial efforts to
optimize the activity of compound 7 focused on understanding
the contributions of the indole moiety. As replacements to the
indole, various small aryl groups were explored, including
phenyl and various 5,6 and 6,6 bicyclic systems. The explora-
tion of this region of the molecule is summarized in Table 1. The
phenyl analogue 12 showed an approximately 140- to 20-fold
loss in potency in Jurkat and HeLa cell viability assays and was
inactive in the MCF-7 G2/M cell cycle block assay. The
4-substituted indazole 13 lost potency in both assays, while the
Next, we evaluated the effect of substitution on the amine
linking the pyridine to the phenol, and also the effect of changing
this amine group to other one atom linkers or eliminating it
altogether (Table 2). Methylation of the amine resulted in
compound 26 which retained activity in both cell viability and
G2/M cell cycle inhibition assays. Ketone 34 and the directly
linked pyridine 32 also maintained potency, with analogue 34
being more potent in the cell viability assays than in the cell cycle
inhibition assay. The reduction of the ketone to the alcohol 30
resulted in loss of activity, as did changing to the ether linker in
28. The central phenol of this series is very electron rich, which
could be a potential metabolic liability.32 The discovery that the
amine can be replaced by the more electron withdrawing carbon-
yl or directly bound heteroaromatic group allowed us to reduce
the electron density of this core. Much of the subsequent work
focused on the analogues of these two subseries.
Modifications to the R5 position, which are summarized in
Table 3, were made in order to understand the importance of

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 185
Table 1. Effect of Replacing or Substituting the Indole on Cell Viability and G2/M Block Cell Cycle Inhibition
Table 2. Effect of Varying the Pyridyl Linker on Cell Viability and G2/
M Block Cell Cycle Inhibition
Table 3. Effect of Varying R5 Substituents on the G2/M Block Cell
Cycle Inhibition in Jurkat and MCF-7 Cell Lines
cell viability assay (WST-1),
IC50 ( μM)
cell cycle inhibition G2/M block IC50 ( μM)
compd
R5
X
Jurkat
MCF-7
cell cycle inhibition G2/M
block IC50 ( μM)
MCF-7
7
OH
H
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-NH-
-C(O)-
-C(O)-
-C(O)-
-C(O)-
-C(O)-
-C(O)-
0.02
2.0
0.29
>3.0
>1.0
>10.0
>1
38
41
42
43
44
45
34
50
51
52
53
54
compd
X
Jurkat
HeLa
Cl
0.20
>0.1
>3
7
-NH-
0.02
0.06
NDa
>3
0.13
0.33
0.89
NDa
NDa
0.05
0.29
0.10
>1.0
>1.0
0.02
0.74
OMe
Me
CN
26 -N(Me)-
28 -O-
30 -CH(OH)-
32
0.16
0.12
0.03
1.30
0.01
0.02
0.02
1.60
0.67
0.15
0.74
0.86
0.08
0.12
0.07
0.99
NHCOMe
OH
NH2
0.04
0.03
34 -C(O)-
a ND: not determined.
NHCO2Me
NHCOMe
NHSO2Me
NHCONMe2
the phenol group for activity and to find replacements for the
phenol, which was susceptible to glucoronidation. Flow
cytometry was used to evaluate the potency and effectiveness
of the compounds to block mitosis by locking MCF-7 and
Jurkat human tumor cells in the G2/M phase of cell cycle
growth. The des-hydroxyl analogue 38 lost potency against
both cell lines, as did the chloro 41 and methyl 43 analogues.
Methylation of the hydroxyl group to give the methoxy
analogue 42 resulted in complete loss in activity, indicating
that the phenol was most likely acting as a hydrogen bond
donor rather than an acceptor. The cyano analogue 44
showed a decrease in activity of 3- to 5-fold. Acetamide 45
lost some potency in the Jurkat cell line but was potent
against the MCF-7 cell line. Aniline 50 was less potent than
phenol 34 on the Jurkat cell line. Derivatization of the amine
to the methylcarbamate 51, acetamide 52, and methylsulfo-
namide 53 resulted in improved potency in both Jurkat and
MCF-7 cell lines. Increased size of the amine substituent
resulted in loss of activity as evidenced by urea 54.

186 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shetty et al.
Table 4. Effect of Various Aromatic Groups on Cell Cycle Inhibition in Multiple Cell Lines
cell cycle inhibition G2/M block IC50 (μM)
compd
R
R5
X
MCF-7
U266
H522
Jurkat
SW- 620
H23
BXPC-3
PC-3
57
58
59
60
61
62
67
68
72
73
74
1
2-methoxy-3-pyridyl
3-methoxy-4-pyridyl
3-morpholino-4-pyridyl
3-chloro-5-methyl-4-pyridyl
3,5-di-methyl-4-pyridyl
3-methyl-5-morpholino-4-pyridyl
2- benzo[d]oxazole
NHCOMe
NHSO2Me
NHCO2Me
NHCO2Me
NHCO2Me
NHCOMe
NHCOMe
NHCO2Me
NHSO2Me
NHCOMe
NHCOMe
CO
CO
CO
CO
CO
CO
0.03
0.01
0.03
0.11
0.05
0.01
0.02
0.18
0.02
0.03
0.03
NDa
0.02
0.12
0.12
0.02
0.03
0.02
0.16
0.03
0.01
0.02
0.01
0.01
0.03
0.01
0.01
0.12
0.06
0.03
0.01
0.13
0.01
0.03
0.01
0.014
0.03
0.02
0.05
0.05
0.03
0.13
0.04
0.06
0.30
0.03
0.02
0.36
0.04
0.014
0.05
0.38
0.03
0.10
0.01
0.04
0.35
0.10
0.01
0.03
0.02
0.013
0.03
0.05
0.01
0.06
0.04
0.07
0.11
0.06
0.01
0.02
0.02
0.016
0.06
0.07
0.02
0.08
0.03
0.05
3.00
0.05
0.02
0.05
0.10
0.033
0.01
0.03
1.28
0.02
0.55
0.03
2-oxazolyl [4,5-b] pyridine
4-pyridyl
0.01
0.02
4-methoxy-3-pyridyl
3,4-difluorophenyl
<0.01
0.017
a ND: not determined.
Table 5. Cell Proliferation Data
cell proliferation IC50 ( μM)
Jurkat SW-620 A2780 HeLa NCI-ADR MDR1
Table 6. Inhibition of in Vitro Tubulin Polymerization (Microtubule
Formation) and Colchicine Competition
colchicine competition
compd
58
59
anti-tubulin activity,
EC50 ( μM)
% inhibition
at 30 μM
0.09
0.10
0.15
0.21
0.16
0.01
0.60
0.20
0.25
0.45
0.55
0.19
0.03
13.00
0.20
0.22
0.35
0.48
0.17
0.01
0.65
0.21
0.28
0.49
0.64
0.27
0.02
4.40
0.30
0.30
1.10
0.34
0.29
13.00
6.90
compd
EC50 ( μM)
7
16
4.1
17
62
68
51
52
4.5
6.1
2
paclitaxel
cisplatin
58
59
5.0 ( 1.2
4.60
3.2
79
77
3.2 ( 0.78
61
72
Substituted 3- and 4-pyridyl ring systems were explored to
improve potency and modulate physical properties. The
results for the most potent compounds are summarized in
Table 4. The 2-methoxy-3-pyridyl analogue 57 was more
potent in the MCF-7 cell line than the unsubstituted analogue
52. For the compounds with a ketone linker, it was found that
3-substituted 4-pyridyl analogues such as 58 (LP-261) and 59
and 3,5-disubstituted 4-pyridyl analogues such as 60, 61, and
62 were highly potent. Compounds were tested for G2/M cell
cycle inhibition against a panel of eight tumor cell lines and
exhibited a range of activity from 10 nM to 1.3 μM across the
tested cell lines.
The carbonyl linker was replaced by a fused oxazole in the
benzo[d]oxazole 67 and the oxazolo[4,5-b]pyridine 68 ana-
logues. While retaining some activity, 67 was less potent in
most cell lines, but 68 was reasonably potent.
Simple aryl substituents such as 4-pyridyl 72, 4-methoxy-3-
pyridyl 73, and the 3,4-difluorophenyl 74 directly connected to
the phenyl core resulted in highly potent compounds with low
nanomolar activity across the majority of cell lines.
Selected analogues that showed potent G2/M block activ-
ity in multiple cell lines were tested in cell proliferation assays
(Table 5). The compounds were less active in the cell prolifera-
tion assay when compared to the G2/M block. Across all cell
lines the compounds showed comparable activity to 2 but were
less active than paclitaxel. In contrast all selected compounds
were substantially more active including 2 when compared to
cisplatin. These compounds were also active against the pacli-
taxel resistant National Cancer Institute adriamycin-resistant
2.2
4.6
73
74
2.1
2.0 ( 0.50
2.50 ( 0.14
81
79
1
vincristine
3.70 ( 0
inactive
multidrug resistance 1 (NCI-ADR MDR1) cell line which over-
expresses P-glycoprotein, suggesting that the compounds will
not be subject to multidrug resistant mechanisms.
In Vitro Tubulin Polymerization Assay. To further explore
the mechanism of cell cycle inhibition, these compounds
were evaluated in an in vitro tubulin polymerization assay.
The results are summarized in Table 6. The initial lead com-
pound 7 was shown to be a potent inhibitor of tubulin poly-
merization with an IC50 of 4 μM. Analogue 16, which was signi-
ficantly less active in the cell viability and G2/M cell cycle block
assays, was also less potent at inhibiting tubulin polymerization.
Analogues 51 and 52 and the substituted 4-pyridyl analogues 58,
59, and 61 all showed low micromolar potency in the tubulin
polymerization assay. Analogues 72, 73 and 74 with the aro-
matic group directly connected to the phenyl core were also
found to be potent inhibitors of tubulin polymerization with
IC50 values in the 2-4 μM range and thus comparable to the
inhibitory activity of reference drugs colchicine and vincristine
(2.0 and 2.5 μM, respectively). Overall, there is a good correla-
tion between inhibition of tubulin polymerization and inhibition
of tumor cell growth for all tested analogues; however, the IC50
values for the inhibition of tubulin formation are significantly
higher compared to the IC50 values for inhibition of tumor cell

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 187
Figure 2. Proposed binding mode of 58 in tubulin structure 1SA0 generated by the Ansaris technology platform. Image was generated with the
program PyMol.
growth. This appears to be a common characteristic of anti-
mitotic drugs.33 Various factors may contribute to this discrep-
ancy including the fact that the in vitro tubulin polymerization
assays are typically performed using a very high concentration
of isolated tubulin.
from the previously identified indole poses which involved
hydrogen bonding to Glu71 and Asp68. Compounds 7, 61, and
74 were assembled in a similar manner. The putative bind-
ing pose for 58, which spans both the GTP and the colchicines
binding site, is depicted in Figure 2.
Analogue 58 was assessed for its ability to compete with
colchicine for binding to tubulin using [3H]colchicine com-
petition binding assay and was found to inhibit the binding
with a potency similar to that of colchicine itself (Table 6). In
addition analogues 72 and 74 were assessed for their ability
to compete with [3H]colchicine at 30 μM and showed similar
inhibition as compound 58 and colchicine itself. In contrast,
the Vinca alkaloid vincristine did not inhibit [3H]colchicine
binding to biotinylated tubulin. These results suggest that
analogues 58, 72, and 74 inhibit tubulin polymerization by
binding at or near the colchicine binding site.
During the molecule build, a distribution of molecular
poses were generated, from which one was selected as a
representative for depiction in Figure 2. Figure 3 shows the
actual distribution of molecule poses obtained for 58.
Confirmation of this binding mode requires further bio-
physical studies; however, this proposed binding mode is
consistent with the observed SAR. In this binding mode, the NH
of the sulfonamide makes a hydrogen bond with Glu182 and
explains the fact that hydrogen bond donors in the aryl ring 5
position increase potency. Methylation of the indole would pre-
vent the hydrogen bond to Glu71 which accounts for the sub-
stantial loss in activity of the N-methylated analogue 26. The
molecule positioning in front of the GTP site would certainly
prevent its binding, and while the compound only has a small
overlap with colchicine, it appears large enough to prevent
colchicine from occupying its binding site.
Oral Efficacy. This class of 4-aroylindole compounds dis-
plays modest to excellent oral bioavailability in rat PK studies.
The original phenolic lead 7 was only 24% bioavailable in rats
when dosed orally in a solution of ethanol/propylene glycol/
10% Tween 80 in phosphate buffered saline (1:4:5) at a dose of
20 mg/kg. Not surprisingly, LC-MS analysis of the plasma
showed extensive glucoronidation. However, when compounds
59, 61,68, 72,and74, in which the phenol function is replaced by
amide surrogates, were evaluated in rat PK in cassettes of three
compounds each at 4 mg/kg, improved oral bioavailability
ranging from 46% to 66% was observed. As a single compound
dosed by oral gavage at 4 mg/kg in rat, administered as a solu-
tion formulation composed of 3:1 polyethylene glycol 400 and
100 mM citrate buffer, pH 3, compound 58 was 80% bioavail-
able (Table 7). Insignificant hepatic extraction in rats was
indicated by a systemic clearance rate of 0.67 ( 0.43 (L/h)/kg,
which is approximately 5-fold lower than the hepatic blood flow
in rats (approximately 3.3 (L/h)/kg). The volume of distribution
Computational Model. We utilized our proprietary
fragment-based screening technology platform34 to develop a
binding hypothesis for 58 bound to tubulin. Unlike docking
programs such as GLIDE35 that probe a specific region of the
target protein with various orientations (poses) and conforma-
tions of the entire molecule of interest, our approach first
identifies the high affinity poses of molecular fragments. We
then connect these fragment poses together to construct a
9
˚
complete molecule. The tubulin structure 1SA0.pdb, a 3.5 A
resolved X-ray structure, was used as the protein model for our
simulations. Since the indole ring was established as a critical
moiety for tubulin binding in this series, our initial focus was to
find the high affinity sites for this fragment. The highest affinity
indole poses involved a hydrogen bonding interaction between
the indole NH and Glu71. A second high affinity pose with an
interaction to Asp68 was also identified and only a minor
change in the orientation of the indole ring is needed to flip the
interacting NH between the two residues. The binding site
identified by this method, which is a key region in the GTP site,
appears to be the best location for the indole fragment. By use
of the poses created by the Ansaris technology platform for
other key fragments (acetophenone, methoxypyridine, and N-
methylsulfonamide), compound 58 was readily constructed

188 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shetty et al.
Figure 3. Distribution of molecule poses of 58 from molecule builds. Image was generated with the program PyMol.
Table 7. Pharmacokinetic Parameters of 58 after iv and po Adminis-
tration (Mean ( SD, N = 4)
route
iv
po
dose (mg/kg)
AUC ( μg h/mL)
C0 ( μg/mL)
2
4
3.7 ( 1.6
4.9 ( 3.4
5.9 ( 1.9
3
T
max (h)
2.0
Cmax ( μg/mL)
half-life (h)
CL ((L/h)/kg)
1.14 ( 0.43
1.4 ( 0.2
0.73 ( 0.31
1.4 ( 0.2
0.67 ( 0.43
1.25 ( 1.13
Vss (L/kg)
oral F (%)
80
(Vss) was 1.25 ( 1.13 L/kg, which is approximately twice the
body water of the rat (0.6 L/kg), suggesting that compound 58 is
bound to tissues in the body. Finally, compound 58 displayed
rapid adsorption by the oral route (Tmax = 2.0 h) and the
terminal half-life of 1.4 ( 0.2 h indicated a moderate rate of
elimination.
In Vivo Efficacy of Compound 58 in NCI-H522 Xenografts.
On the basis of its efficacy in a broad array of tumor cell lines
and excellent oral bioavailability, 58 was selected for evalua-
tion in a human tumor xenograft model in mice. Cultured
NCI-H522 human non-small-cell lung tumors were im-
planted subcutaneously in NCr-nu mice with 10 animals
per group, and the animals were treated with vehicle, 58, or
cisplatin, a drug known to inhibit NCI-H522 tumors in this
model. Compound 58 was administered by oral gavage twice
daily at doses of 15 and 50 mg/kg for 28 days. The results are
shown in the Figure 4.
Significant inhibition of tumor growth was observed in the
groups treated with 58 at doses of 50 mg/kg b.i.d. and cisplatin.
Treatment with 58 at 50 mg/kg b.i.d. resulted in a mean tumor
volume of 130 ( 190 mm3 versus 3769 ( 1636 mm3 in the
vehicle treated group. This represents a 96% reduction in mean
tumor volume, and the difference is statistically significant
compared to the vehicle treated group (P < 0.001). Compara-
tively, cisplatin treatment reduced mean tumor volume by 98%
to 63 ( 1 mm3 (P < 0.001 compared to vehicle), which is the
smallest measurable tumor size, and all tumors diminished in
size during the study. Apparent partial inhibition of tumor
growth was observed in the group treated with 58 at 15 mg/kg
twice daily for 28 days (41% inhibition).
Figure 4. Inhibition of human H522 NSCL xenograft growth in
vivo by 58.
Overall, there were no significant changes in body weights
for the 58 treated groups compared to the vehicle treated
group. The cisplatin treated group initially exhibited weight
loss that fully recovered by the end of the study.
Conclusion
In conclusion a novel series of 1-(1H-indol-4-yl)-3,5-disub-
stituted benzene analogues with potent antimitotic activity has
been discovered. While the 4-indolyl substituent at the 1-posi-
tion was found to be critical for potent activity against tumor
cells in culture, the heteroaryl substituent at the 3-position can
be either directly attached or attached via a linker such as the
carbonyl group or the amino group. Placement of a hydrogen
bond donor, such as hydroxyl, at the 5-position afforded potent
analogues. Replacement of this phenolic hydroxyl group,
which suffered from rapid glucoronidation, with an acylated
or sulfonylated amine gave highly potent compounds with
significantly improved oral bioavailability.
These compounds arrest cell division at the G2/M check-
point in multiple cell linesand are highly potent in cell viability
assays. The mechanism by which these agents induce G2/M
block was shown to be colchicine-competitive inhibition of
tubulin polymerization. Our fragment-based technology plat-
form was used to develop a possible binding hypothesis that is
consistent with the SAR of this series. The optimized lead
compound 58 inhibits multiple cancer cell lines including an

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 189
1
(14.23 g, 97%) was obtained as pale brown needles. H NMR
MDR resistant line and inhibits tumor cell growth in a human
non-small-cell lung mouse xenograft model. On the basis of
these encouraging results, compound 58 was selected for
further preclinical evaluation as a promising novel oral anti-
cancer therapy.
(400 MHz, acetone-d6): δ 9.20 (s, 1H), 7.20 (s, 1H), 7.04 (s, 3H).
(3,5-Dibromophenoxy)triisopropylsilane (9e). Representative
conditions for silyl protection of phenol were used. 3,5-Dibro-
mophenol 9d (23.07 g, 56.5 mmol) was dissolved in DMF
(100 mL) under dry argon and cooled to 0 °C. Then 60%
NaH (2.49 g, 62.3 mmol) was added in small portions over a
period of 15 min. Stirring was continued for 15 min, followed by
dropwise addition of triisopropylsilyl chloride (12.1 mL, 56.5
mmol). The mixture was warmed to room temperature and
stirred for 20 h. The reaction mixture was diluted with tert-butyl
methyl ether and washed with water and brine (2 ꢀ 50 mL each).
The organic layer was dried (MgSO4) and concentrated to give
the crude product which was purified by flash chromatography
on silica gel (500 g, EtOAc/heptane 1:1).to give (23.1 g, quant) of
9e as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.23 (s, 1H),
6.96 (s, 2H), 1.30 - 1.18 (m, 3H), 1.09 (d, J = 7.2 Hz, 18H).
N-(3-Bromo-5-(4-methoxybenzyloxy)phenyl)pyridin-3-amine
(10b). Representative coupling conditions for Buchwald-Hartwig
amination of protected 3,5-dibromophenols with 3-aminopyridine
were used. An oven-dried flask was charged with (()-BINAP
Experimental Section
General Methods and Materials. All commercially available
solvents and reagents were used without further purification.
Anhydrous solvents were purchased from Sigma-Aldrich. Re-
actions that were air or moisture sensitive were carried out under
an atmosphere of nitrogen. Microwave reactions were carried
out in a CEM Discover unit. Flash chromatography was carried
out using Selecto silica gel (30-63 μm) or with silica gel Flashþ
cartridges on a Biotage Flashþ system. Thin layer chromatog-
raphy (TLC) was performed on EMD silica gel 60 F254 plates
using reagent grade solvents. Preparative TLC was performed
on a 1000 μM Whatman Partisil PK6F silica gel 60 plate. 1H and
13C NMR spectra were obtained on a Varian Inova 400 MHz
spectrometer. Compounds were analyzed for purity and molec-
ular mass by analytical SFC using a Berger analytical SFC
system (equipped with an automated column switching valve)
with detection using an HP/Agilent DAD (190-300 nm) and a
Waters Micromass ZQ (APCI mode). All compounds were
g95% pure unless otherwise noted. Elution was by gradient
using 5-55% MeOH in supercritical carbon dioxide over 5 min
using Berger silica, amino, diol, pyridine, or cyano columns
(column dimensions, 150 mm ꢀ 4.2 mm) at 2.5 mL/min.
Purifications by preparative supercritical fluid chromatography
were performed on a Berger preparative SFC system. Elution
was by gradient using 5-55% MeOH in supercritical carbon
dioxide over 5 min using Berger silica, amino, diol, pyridine, or
cyano columns (all are 150 mm ꢀ 40 mm) at 50 mL/min.
3,5-Dibromo-1-benzyloxybenzene (9a). 1,3-Dibromo-5-nitro-
benzene 8 (2.81 g, 10.0 mmol), freshly powdered KOH (1.00 g,
17.8 mmol), and n-Bu4NBr (0.32 g, 1.00 mmol) were dissolved in
tetramethylurea (TMU, 8 mL). Oxygen was bubbled through
the reaction mixture for 5 min, and a solution of benzyl alcohol
(1.30 g, 12.02 mmol) in TMU (2 mL) was added dropwise at
room temperature over a period of 1 h. The mixture was stirred
for 6 h at room temperature during which oxygen was bubbled
through. The reaction mixture was poured on ice (30 g) and was
extracted with tert-butyl methyl ether (2 ꢀ 50 mL). The com-
bined organics were dried (MgSO4) and concentrated to give the
crude product which was purified by flash chromatography
(120 g silica gel, EtOAc/heptane 1:4) to provide 3.15 g (92%)
of 9a. 1H NMR (300 MHz, CDCl3): δ 8.29 (d, J=2.8 Hz, 1H),
8.15 (dd, J=4.7, 1.4 Hz, 1H), 7.36-7.22 (m, 6H), 7.11 (dd, J=
8.3, 4.7 Hz, 1H), 6.68 (dt, J=3.8, 1.7 Hz, 2H), 6.48 (t, J=2.1 Hz,
1H), 5.85 (s, 1H), 4.93 (s, 2H).
(50 mg, 0.08 mmol), Pd2(dba)3 CHCl3 complex (28 mg, 0.027
3
mmol) and flushed with argon. Degassed toluene (2 mL) was added
and the solution stirred for 10 min. The catalyst solution was added
to a mixture of 9b (2.00 g, 5.38 mmol), 3-aminopyridine (252 mg,
2.68 mmol), and t-BuONa (259 mg, 3.75 mmol) in degassed toluene
(10 mL), and the mixture was heated to 90 °C for 24 h. The reaction
mixture was cooled to room temperature. Brine (30 mL) and EtOAc
(30 mL) were added, and the mixture was filtered over Hyflo. The
aqueous layer was extracted with EtOAc (2 ꢀ 20 mL), and the
organic layers were washed with brine (2 ꢀ 20 mL), dried (Na2SO4)
and concentrated to give the crude product. Flash chromatography
(silica gel, EtOAc/heptane 1:2 f 2:1) gave 10b (636 mg, 62%) as a
yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.37 (d, J = 2.6 Hz,
1H), 8.21 (dd, J = 4.7, 1.1 Hz, 1H), 7.39 (ddd, J = 8.3, 2.6, 1.3 Hz,
1H),7.30(d,J= 8.6 Hz, 2H), 7.18 (dd, J= 8.3, 4.7 Hz, 1H), 6.90 (d,
J = 8.6 Hz, 2H), 6.77 (t, J = 1.7 Hz, 1H), 6.72 (t, J = 1.8 Hz, 1H),
6.54 (t, J = 2.0 Hz, 1H), 5.88 (s, 1H), 4.91 (s, 2H), 3.80 (s, 3H).
N-(3-(4-Methoxybenzyloxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)phenyl)pyridin-3-amine (11b). Representative Suzuki-
Miyura coupling conditions for synthesis of pinacol boronates
were used. A round-bottom flask was charged with 10b (623 mg,
1.62 mmol), bis(pinacolato)diboron (453 mg, 1.78 mmol), KOAc
(477 mg, 4.86 mmol), and PdCl2(dppf) CH2Cl2 complex (65 mg,
3
0.08 mmol). DMSO (10 mL) was added under argon, and the
mixture was heated to 80 °C for 2 h. After completion of the
reaction, the mixture was cooled to room temperature, filtered over
Hyflo and the filter cake washed with EtOAc (2 ꢀ 5 mL). The
filtrate was diluted with brine (100 mL) and the mixture extracted
with EtOAc (3 ꢀ 15 mL). The combined organics were dried
(Na2SO4) and concentrated under reduced pressure to give the
crude material. Flash chromatography on silica gel (40 g, EtOAc/
heptane 2:1 f 3:1) provided pure 11b (640 mg, 91%) as an off
white solid. 1H NMR (400 MHz, CDCl3): δ 8.34 (d, J = 2.3 Hz,
1H), 8.13 (d, J = 4.2 Hz, 1H), 7.39-7.30 (m, 3H), 7.14 (dd, J =
8.3, 4.7 Hz, 1H), 7.07 (dd, J = 8.7, 1.9 Hz, 2H), 6.89 (d, J = 8.6Hz,
2H), 6.79 (t, J= 2.2 Hz, 1H), 5.83 (s, 1H), 4.98 (s, 2H), 3.80 (s, 3H),
1.32 (s, 12H). SFC-MS (APCIþ), M þ H found 433.3.
1,3-Dibromo-5-(4-methoxybenzyloxy)benzene (9b). Com-
pound 9b was synthesized from compound 8 in a similar manner
as described for the preparation of 9a using p-methoxybenzyl
1
alcohol in 81% yield. H NMR (400 MHz, CDCl3): δ 7.31 (d,
J = 8.6 Hz, 2H), 7.24 (s, 1H), 7.05 (d, J = 1.5 Hz, 2H), 6.91 (d,
J = 8.6 Hz, 2H), 4.93 (s, 2H), 3.81 (s, 3H).
3,5-Dibromo-1-methoxybenzene (9c). Compound 9c was
synthesized from compound 8 in a similar manner as described
for the preparation of 9a using methanol in 84% yield. 1H NMR
(400 MHz, CDCl3): δ 7.23 (t, J=1.4 Hz, 1H), 6.98 (d, J=1.5 Hz,
2H), 3.77 (s, 3H).
3,5-Dibromophenol (9d). 3,5-Dibromo-1-methoxybenzene 9c
(15.56 g, 58.5 mmol) and n-Bu4NBr (1.0 g, 3.1 mmol) were
suspended in 48% HBr (100 mL) and refluxed for 3 days. After
cooling to room temperature, the reaction mixture was ex-
tracted with DCM (3 ꢀ 60 mL). The combined organic layers
were washed with water (2 ꢀ 50 mL), dried (MgSO4), and
evaporated. The crude product was filtered over a pad of silica
gel (EtOAc/heptane 10:1). After removal of the solvent, 9d
General Procedure A for the Suzuki Couplings Using Chloro-
(di-2-norbornylphosphino)(20-dimethylamino-1,10-biphenyl-2-yl)-
palladium (22) as Catalyst. N-(3-(1H-Indol-4-yl)-5-(4-methoxy-
benzyloxy)phenyl)pyridin-3-amine (23b). A 25 mL Schlenk flask
was charged with 10b (190 mg, 0.49 mmol) and 22 (11 mg, 0.02 mmol)
in 1,4-dioxane (10 mL) under argon. A solution of 4-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)-1H-indole (21b, 92 mg, 0.38 mmol)
in 1,4-dioxane (10 mL) followed by aqueous 2 M K3PO4 (0.57 mL,
1.14 mmol) was added, and the mixture was heated at reflux for 12 h.
The reaction mixture was cooled, filtered over Hyflo and the filter
cake washed with 1,4-dioxane (2 ꢀ 5 mL). The filtrate was concen-
trated in vacuo and the residue purified by flash chromatography

190 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shetty et al.
(silica gel, EtOAc/heptane 6:1 f 1:2) to provide N-(3-(1H-indol-
4-yl)-5-(4-methoxybenzyloxy)phenyl)pyridine-3-amine 23b (80 mg,
50%) as light brown solid. 1H NMR (400 MHz, CDCl3): δ 8.60 (s,
1H), 8.40 (d, J = 2.6 Hz, 1H), 8.14 (d, J = 4.4 Hz, 1H), 7.45 (dd,
J = 8.3, 1.2 Hz, 1H), 7.35 (dd, J = 8.2, 3.4 Hz, 3H), 7.26-7.07 (m,
4H), 7.00-6.80 (m, 4H), 6.68 (dd, J = 11.0, 9.2 Hz, 2H), 5.99
(s, 1H), 5.01 (s, 2H), 3.80 (s, 3H). SFC-MS (APCIþ), Mþ H found
422.7.
3.73-3.49 (m, 2H), 0.95 (dd, J = 8.8, 7.7 Hz, 2H), -0.01 (d, J =
3.3 Hz, 9H).
3-(1H-Indazol-4-yl)-5-(pyridin-3-ylamino)phenol (13). N-(3-(4-
Methoxybenzyloxy)-5-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-
indazol-4-yl)phenyl)pyridin-3-amine was prepared from 11b
(472 mg, 1.09 mmol) and 4-bromo-1-((2-(trimethylsilyl)ethoxy)-
methyl)-1H-indazole (370 mg, 0.84 mmol) using general Suzuki
condition A. The Suzuki product (550 mg, 1.00 mmol) was dissolved
in THF (3 mL) and ethylene diamine (0.67 mL, 10.0 mmol), and
1 M solution of TBAF in THF (3.0 mL, 3.0 mmol) was added. The
reaction solution was heated at reflux for 7 days during which more
TBAF solution (3xl.0 mL) was added. The reaction mixture was
cooled, diluted with EtOAc (30 mL), and washed with 0.1 M HCl
(20 mL). The layers were separated, and the aqueous layer was
washed with EtOAc (2 ꢀ 29 mL). The combined organics were
washed with saturated NaHCO3 (20 mL), water (20 mL) and dried
(Na2SO4). Concentration and filtration of the residue through a
short plug of silica gel (EtOAc) provided the intermediate product
(265 mg).
3-(1H-Indol-4-yl)-5-(pyridin-3-ylamino)phenol (7). N-(3-(1H-
Indol-4-yl)-5-(4-methoxybenzyloxy)phenyl)pyridine-3-amine 23b
(35 mg, 0.083 mmol) was dissolved in dimethylsulfide (5 mL) and
cooled to 0-5 °C, and BF3 OEt2(0.25 mL, 7.9 mmol) was added
3
dropwise. The mixture was stirred for 10 min at 0-5 °C, quenched
with saturated NaHCO3 (5 mL), and extracted with EtOAc
(10 mL). The layers were separated, and the aqueous layer was
extracted with EtOAc (2 ꢀ 10 mL). The combined organics were
dried (Na2SO4), filtered, and concentrated. The residue was purified
by flash chromatography (silica gel, DCM/MeOH 10:1) to give 7
(15 mg, 60%). 1H NMR (400 MHz, DMSO-d6): δ 11.18 (s, 1H),
9.36 (s, 1H), 8.36 (d, J= 2.7 Hz, 1H), 8.32 (s, 1H), 8.00 (dd, J=4.6,
1.3 Hz, 1H), 7.48 (ddd, J = 8.4, 2.7, 1.4 Hz, 1H), 7.38-7.33 (m,
2H), 7.22 (dd, J=8.3, 4.6Hz, 1H), 7.11(t,J= 7.7 Hz, 1H), 7.00 (d,
J = 6.8 Hz, 1H), 6.78 (d, J = 1.6 Hz, 1H), 6.58 (t, J = 1.6 Hz, 1H),
6.55-6.51 (m, 2H). SFC-MS (APCIþ), M þ H found 302.7.
General Procedure B for the Suzuki Couplings Using Pd(PPh3)4 as
Catalyst. 5-(Pyridin-3-ylamino)biphenyl-3-ol (12). A round-bottom
flask was charged with 10a (400 mg, 1.12 mmol) and Pd(PPh3)4
(38 mg, 0.034 mmol). 1,2-Dimethoxyethane (10 mL) was added
under argon, and the mixture was stirred for 10 min at room
temperature. A solution of phenylboronic acid (189 mg, 1.46 mmol)
in ethanol (1.0 mL) and 2 M aqueous Na2CO3 (1.12 mL, 2.24
mmol) were added, and the mixture was heated at reflux overnight.
After completion of the reaction, the mixture was cooled to room
temperature, filtered over Hyflo and the filter cake washed with
EtOAc (2 ꢀ 5 mL). The filtrate was concentrated under reduced
pressure, and the oily residue was taken up in brine/EtOAc 1:1
(20 mL). The layers were separated, and the aqueous layer was
extracted with EtOAc (10 mL). The combined organics were dried
(Na2SO4) and concentrated under reduced pressure to give the
crude material (450 mg) which was flash chromatographed (silica
gel 30 g, EtOAc/heptane 2:1) to give pure N-(5-(benzyloxy)biphe-
nyl-3-yl)pyridin-3-amine (337 mg, 85%) as a yellow solid.
This intermediate was dissolved in dimethylsulfide (3.5 mL), and
the solution was cooled to 0 °C. BF3 OEt2 (0.294 mL, 2.34 mmol)
3
was added, and the mixture was stirred for 2 h at 0 °C and 2 h at
room temperature. The reaction mixture was quenched with
saturated NaHCO3 solution (10 mL) and extracted with EtOAc
(2 ꢀ 10 mL). The combined organics were dried (Na2SO4) and
evaporated under reduced pressure. Silica gel chromatography of
the crude material using THF/MeOH/NH3 (97:2:1) gave product
contaminated with BHT from the THF. The beige solid was taken
up in TBME/heptane 1:1 (4 mL) and the suspension stirred at
room temperature for 15 min. Filtration of the solid provided 13
(87 mg, 29%). 1H NMR (400 MHz, acetone-d6): δ 12.33 (s, 1H),
8.48 (d, J = 2.3 Hz, 1H), 8.46 (s, 1H), 8.19 (s, 1H), 8.10 (d, J = 4.7
Hz, 1H), 7.68 (s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.4 Hz,
1H), 7.42 (t, J = 7.7 Hz, 1H), 7.28-7.22 (m, 2H), 7.00 (s, 1H), 6.82
(s, 1H), 6.75 (s, 1H). LC-MS (ESþ) M þ H found 303.
Benzo[b]thiophen-4-yl Trifluoromethanesulfonate. Benzo[b]thio-
phen-4-ol (1.00 g, 6.66 mmol) was dissolved in dry CH2Cl2 (10 mL)
under argon. Triethylamine (1.40 mL, 9.99 mmol) was added,
and the reaction solution was cooled to 0-5 °C. A solution of
trifluoromethanesulfonic acid anhydride (2.07 g, 7.32 mmol) in
CH2Cl2 (2 mL) was added dropwise and the mixture was stirred
at0 °C for 10 min. Then10%Na2CO3 solution(5 mL) was added,
and the mixture was diluted with CH2Cl2 (10 mL) and water
(10 mL). The layers were separated, and the aqueous layer was
extracted with CH2Cl2 (2 ꢀ 10 mL). The combined organics were
washed with brine (10 mL), dried (Na2SO4), filtered, and con-
centrated. Flash chromatography (silica gel, EtOAc/heptane 1:6)
gave pure benzo[b]thiophen-4-yl trifluoromethanesulfonate (1.72 g,
87%) as a yellow oil. 1H NMR (300 MHz, CDCl3): δ 7.91 (d,
J = 7.9 Hz, 1H), 7.61 (d, J = 5.6 Hz, 1H), 7.49 (d, J = 5.6 Hz,
1H), 7.41 (t, J = 7.9 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H)
N-(5-(Benzyloxy)biphenyl-3-yl)pyridin-3-amine (170 mg,
0.48 mmol) was dissolved in THF (4 mL), and the flask was
flushed with argon. Pd/C (50 mg) was added, and the argon was
replaced by hydrogen (H2 balloon). The mixture was stirred for 2 h,
more Pd/C (150 mg) was added, and the mixture was stirred under
H2 for 48 h. The catalyst was filtered over Hyflo, the filter cake was
washed with THF (2 ꢀ 5 mL). The filtrate was concentrated and
flash chromatographed (silica gel 10 g, EtOAc/heptane 3:1) to give
pure 12 (75 mg, 60%). 1H NMR (400 MHz, acetone-d6): δ 8.45 (d,
J = 2.5 Hz, 1H), 8.41 (s, 1H), 8.09 (d, J = 4.5 Hz, 1H), 7.59 (d, J =
7.4 Hz, 4H), 7.43 (t, J = 7.6 Hz, 2H), 7.34 (t, J = 7.3 Hz, 1H), 7.24
(dd, J = 8.2, 4.7 Hz, 1H), 6.89 (s, 1H), 6.70 (s, 1H), 6.67 (s, 1H).
LC-MS (ESþ), M þ H found 263.11.
General Procedure C for the Suzuki Couplings Using PdCl2-
(dppf) CH2Cl2 as Catalyst. N-(3-(Benzo[b]thiophen-4-yl)-5-(4-
3
methoxybenzyloxy)phenyl)pyridine-3-amine. A 10 mL Schlenk
flask was charged with 11b (200 mg, 0.46 mmol), benzo[b]thio-
phen-4-yl trifluoromethanesulfonate (155 mg, 0.55 mmol), K3PO4
(195 mg, 0.92 mmol), and dry THF (4 mL). The mixture was
4-Bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazole. A
solution of 4-bromo-1H-indazole (585 mg, 2.97 mmol) in dry
DMF (5 mL) was cooled to 0 °C under argon. NaH (60%
dispersion, 142 mg, 3.56 mmol) was added, and the suspension was
stirred for 2 h at 0-5 °C. SEM chloride (265 μL, 3.86 mmol) was
added at 0-5 °C, and the reaction mixture was left to warm to
room temperature and stirred for 1 h. The mixture was cooled to
0 °C and quenched with water (15 mL) and was then extracted with
isobutyl acetate (3 ꢀ 20 mL). The combined organics were dried
(Na2SO4) and concentrated to provide the crude product as a
mixture of isomers. Flash chromatography (silica gel, EtOAc/
heptane 6:1) provided pure 4-bromo-1-((2-(trimethylsilyl)ethoxy)-
methyl)-1H-indazole (770 mg, 75%) as a yellow oil. 1H NMR (300
MHz, CDCl3):δ8.10 (d, J= 0.8 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H),
7.42 (dd, J = 7.4, 0.7 Hz, 1H), 7.36-7.30 (m, 1H), 5.79 (s, 2H),
degassed for 10 min with argon, and PdCl2(dppf) CH2Cl2 com-
3
plex (19 mg, 0.023 mmol) was added. The dark red mixture was
heated at reflux for 6 h, cooled to room temperature, and diluted
with EtOAc (20 mL) and saturated NaHCO3 (20 mL). The layers
were separated, and the aqueous layer was extracted with EtOAc
(2 ꢀ 20 mL). The combined organics were washed with brine
(20 mL), dried (Na2SO4), and concentrated to provide the crude
product. Flash chromatography (silica gel, EtOAc/heptane 2:1)
gave N-(3-(benzo[b]thiophen-4-yl)-5-(4-methoxybenzyloxy)phenyl)-
pyridine-3-amine (198 mg, 95%). 1H NMR (400 MHz, CDCl3): δ
8.45 (d, J=2.1 Hz, 1H), 8.16 (d, J=4.4 Hz, 1H), 7.86 (d, J=7.8 Hz,
1H), 7.50-7.44(m,1H),7.42-7.31 (m, 6H), 7.17 (dd, J=8.2, 4.7 Hz,
1H), 6.92 (d, J=8.6 Hz, 2H), 6.81 (d, J=12.5 Hz, 2H), 6.73 (d,

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 191
J=1.8 Hz, 1H), 5.99 (s, 1H), 5.01 (s, 2H), 3.81 (d, J = 3.6 Hz, 3H).
SFC-MS (APCIþ), M þ H found 439.1.
The contents of the vial are stirred at room temperature overnight.
The DMF was reduced in volume in vacuo and the residue is taken
up in EtOAc, washed with H2O, brine, dried over MgSO4, filtered,
and concentrated to opaque foam (108 mg). 1H NMR (400 MHz,
CDCl3):δ7.51 (d, J= 8.1 Hz, 1H), 7.43 (d, J= 6.9 Hz, 1H), 7.22 (s,
1H), 7.15 (dd, J = 8.3, 7.1 Hz, 1H), 1.65 (dt, J = 15.0, 7.5 Hz, 3H),
1.42 (s, 12H), 1.11 (d, J = 7.5 Hz, 18H).
3-Benzo[b]thiophen-4-yl-5-(pyridine-3-ylamino)phenol (14). N-(3-
(Benzo[b]thiophen-4-yl)-5-(4-methoxybenzyloxy)phenyl)pyridine-
3-amine (35 mg, 0.08 mmol) was dissolved in CH2Cl2 (1 mL), and
thioanisole (188 μL, 1.60 mmol) was added. The solution was cooled
to 0 °C, and TFA (0.1 mL) was added. The reaction mixture was
stirred at room temperature for 5 h and quenched with water (3 mL).
Saturated NaHCO3 solution was added until pH 7, and the mixture
was extracted with CH2Cl2 (4 ꢀ 10 mL). The combined organics
were washed with brine (5 mL), dried (Na2SO4), and concentrated.
Flash chromatography (silica gel, DCM/MeOH 95:5) provided 14
(17 mg, 58%). 1H NMR (300 MHz, CD3OD): δ 8.2 (d, 1H), 7.8 (d,
1H),7.7(d,1H),7.45(d,1H),7.4(2d,2H),7.2(m,2H),7.1(dd,1H),
6.4 (s, 1H), 6.35 (s, 1H), 6.3 (s, 1H). LC-MS (ESI) M þ H found
319. LC-MS indicated 87% purity.
3-(3-Chloro-1H-indol-4-yl)-5-(pyridin-3-ylamino)phenol (19).
3-Chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(triiso-
propylsilyl)-1H-indole was coupled with 10d using general Suzuki
condition A, and the silyl groups were deprotected with 1 M TBAF
1
to give 19 in 31% yield over two steps. H NMR (400 MHz,
CD3OD): δ8.31 (br s, 1H), 7.90 (br s, 1H), 7.59 (d, J=8.2Hz, 1H),
7.35 (d, J = 8.2 Hz, 1H), 7.24-7.18 (m, 2H), 7.15 (t, J = 7.7 Hz,
1H), 6.92(d, J= 7.2 Hz, 1H), 6.66 (s, 1H), 6.59 (s, 1H), 6.50 (s, 1H).
SFC-MS (APCIþ), M þ H found 336.7.
3-(Pyridin-3-ylamino)-5-(quinolin-4-yl)phenol (15). N-(3-(4-
Methoxybenzyloxy)-5-(quinolin-4-yl)phenyl)pyridin-3-amine
was prepared from 11b and 4-bromoquinoline using general
Suzuki condition A in 98% yield. The PMB group was deprotected
3-(2-Methyl-1H-indol-4-yl)-5-(pyridin-3-ylamino)phenol (20).
4-Hydroxy-2-methylindole (1.00 g, 6.79 mmol) and TEA (1.40 mL,
12.2 mmol) was taken up in dry CH2Cl2 (10 mL), and the solution
was cooled to 0 °C. A solution of trifluoromethanesulfonic acid
anhydride (1.23 mL, 7.47 mmol) in CH2Cl2 (2 mL) was added
dropwise. The reaction mixture was stirred for 10 min at 0 °C and
was diluted with CHCl3 and extracted with saturated K2CO3. The
organic layer was dried over K2CO3, filtered, and concentrated
under reduced pressure. The residue was taken up in dry THF
(3 mL), and NaH (60% dispersion, 360 mg, 9.00 mmol) was added
portionwise. After the hydrogen evolution had ceased a solution of
TBDMSCl (1.13 g, 7.50 mmol) in dry THF (2 mL) was added and
the mixture was stirred overnight at room temperature. The reac-
tion mixture was diluted with CH2Cl2 (20 mL), washed with
saturated NH4Cl solution (10 mL), dried (Na2SO4), filtered, and
concentrated to provide the crude product. Flash chromatography
(silica gel, EtOAc/heptane 1:50) gave pure 1-(tert-butyldimethyl-
silyl)-2-methyl-1H-indol-4-yl trifluoromethanesulfonate (1.70 g,
64%). LC-MS (ESþ) M þ H found 394.
using the BF3 OEt2 conditions described for compound 7 to give 15
3
in 20% yield. 1H NMR (400 MHz, DMSO-d6): δ 9.61 (s, 1H), 8.89
(d, J = 4.4 Hz, 1H), 8.44 (s, 1H), 8.36 (d, J = 2.3 Hz, 1H), 7.90-
8.04 (m, 3H), 7.75 (dd, J = 8.4, 1.4 Hz, 1H), 7.60 (dd, J = 8.4, 1.4
Hz, 1H), 7.50 (d, J= 1.4 Hz, 1H), 7.42 (d, J= 4.4 Hz, 1H), 7.22 (dd,
J = 8.2, 4.7 Hz, 1H), 6.65 (s, 1H), 6.59 (s, 1H), 6.43-6.34 (m, 1H).
SFC-MS (APCIþ), M þ H found 314.8.
3-(1-Methyl-1H-indol-4-yl)-5-(pyridin-3-ylamino)phenol (16).
N-(3-(4-Methoxybenzyloxy)-5-(1-methyl-1H-indol-4-yl)phenyl)-
pyridin-3-amine was prepared from 10b and 1-methyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole using general
Suzuki condition A in 53% yield. The PMB group was depro-
tected using the BF3 OEt2 conditions described for compound 7
3
to give 16 in 11.9% yield. 1H NMR (400 MHz, CD3OD): δ 8.32
(br s, 1H), 7.98 (br s, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.42-7.36 (m,
1H), 7.34 (d, J = 8.2 Hz, 1H), 7.21 (d, J = 7.2 Hz, 1H), 7.19-7.17
(m, 1H), 7.08 (dd, J = 7.2, 0.8 Hz, 1H), 6.91 (t, J = 1.7 Hz, 1H),
6.79-6.77 (m, 1H), 6.62 (t, J = 2.0 Hz, 1H), 6.59 (d, J = 3.1 Hz,
1H), 3.81 (s, 3H). SFC-MS (APCIþ), M þ H found 316.2.
4-(3-Hydroxy-5-(pyridin-3-ylamino)phenyl)-1,3-dihydroindol-
2-one (17). 4-(3-(4-Methoxybenzyloxy)-5-(pyridin-3-ylamino)-
phenyl)indolin-2-one was prepared from 11b and 4-bromoox-
indole using general Suzuki condition A in 40% yield. The PMB
[3-[l-(tert-Butyldimethylsilanyl)-2-methyl-1H-indol-4-yl]-5-(4-
methoxybenzyloxy)phenyl]pyridin-3-ylamine was prepared from
11b and 1-(tert-butyldimethylsilyl)-2-methyl-1H-indol-4-yl tri-
fluoromethanesulfonate using general procedure C for Suzuki
coupling in 40% yield. The product (133 mg, 0.24 mmol) was
deprotected using the BF3 OEt2 condition described for compound
3
7 to give 20 (57 mg, 59%) as a beige solid. 1H NMR (400 MHz,
DMSO-d6): δ 10.99 (s, 1H), 9.32 (s, 1H), 8.35 (d, J = 2.5 Hz, 1H),
8.31 (s, 1H), 8.00 (dd, J = 4.5, 1.5 Hz, 1H), 7.48 (ddd, J = 8.4,
2.7,1.4 Hz, 1H), 7.24-7.19 (m, 2H), 7.01 (t, J = 7.6 Hz, 1H), 6.94
(d, J = 6.4 Hz, 1H), 6.76 (s, 1H), 6.56 (d, J = 1.4 Hz, 1H), 6.50 (t,
J = 2.0 Hz, 1H), 6.25 (s, 1H), 2.36 (s, 3H). LC-MS (ESþ) M þ H
found 316. LC-MS indicated 86% purity.
group was deprotected using the BF3 OEt2 conditions de-
3
scribed for compound 7 to give 17 in 30% yield. 1H NMR
(400 MHz, acetone-d6): δ 9.47 (s, 1H), 8.60 (b s, 1H), 8.45 (d, J =
2.7 Hz, 1H), 8.09 (dd, J = 4.7, 1.4 Hz, 1H), 7.67 (s, 1H), 7.58
(ddd, J = 8.2, 2.8,1.4 Hz, 1H), 7.29-7.21 (m, 2H), 7.03 (dd, J =
7.8, 0.6 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 6.78 (t, J = 1.7 Hz,
1H), 6.69 (t, J = 2.0 Hz, 1H), 6.61 (t, J = 1.8 Hz, 1H), 3.56 (s,
2H). SFC-MS (APCIþ), M þ H found 318.8.
N-(3-Bromo-5-(4-methoxybenzyloxy)phenyl)-N-methylpyridin-
3-amine (25). To a suspension of KH (30% in mineral oil, 2.0 mmol,
mineral oil washed with ether) in dry ether (20 mL) at 0 °C was
added 10b (385 mg, 1.0 mmol) in THF/ether (1:1, 10 mL), and the
reaction mixture was stirred for 10 min at 0 °C. Iodomethane (74 μL,
1.2 mmol) was added, and the reaction mixture was stirred for 1 h at
0 °C. The reaction mixture was quenched with water and concen-
trated. The residue was dissolved in EtOAc, and the organic layer
was washed with brine, dried, and concentrated. The crude product
was passed through a short pad of silica gel using EtOAc/DCM (1:5)
to give pure 25 (340 mg, 85%). 1H NMR (400 MHz, CD3OD): δ
8.39 (d, J = 2.8 Hz, 1H), 8.28 (dd, J = 4.8, 1.2 Hz, 1H), 7.36-7.32
(m, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.20 (dd, J = 8.4, 4.8 Hz, 1H),
6.89 (d, J = 8.4 Hz, 2H), 6.72 (t, J = 2.0 Hz, 1H), 6.68 (t, J = 2.0
Hz, 1H), 6.44 (t, J = 2.0 Hz, 1H), 4.89 (s, 2H), 3.80 (s, 3H), 3.28 (s,
3H). SFC-MS (APCIþ), M þ H found 399.0.
N-(30-Hydroxy-50-(pyridin-3-ylamino)biphenyl-3-yl)acetamide
(18). N-(30-(4-Methoxybenzyloxy)-50-(pyridine-3-ylamino)biphe-
nyl-3-yl)acetamide was prepared from 10d and 3-acetamidophe-
nylboronic acid using general Suzuki condition B. To the Suzuki
product (166 mg, 0.35 mmol) in THF (5 mL) at 0 °C was added
1 M TBAF in THF (0.39 mL, 0.39 mmol), and the mixture was
stirred at room temperature for 30 min. The mixture was concen-
trated and crude was purified on a preparative SFC system to give
18 (31 mg, 27%). 1H NMR (400 MHz, CD3OD): δ 8.29 (dd, J =
2.8, 0.6 Hz, 1H), 7.96 (dd, J = 4.8, 1.4 Hz, 1H), 7.78 (t, J = 1.8 Hz,
1H), 7.58 (ddd, J = 8.4, 2.8, 1.4 Hz, 1H), 7.51-7.44 (m, 1H), 7.33
(t, J = 7.8 Hz, 1H), 7.30-7.23 (m, 2H), 6.83-6.77 (m, 1H),
6.65-6.60 (m, 1H), 6.58 (t, J = 2.1 Hz, 1H), 2.12 (s, 3H). SFC-MS
(APCIþ), M þ H found 320.8.
3-(1H-Indol-4-yl)-5-(methyl(pyridin-3-yl)amino)phenol (26). N-(3-
(4-Methoxybenzyloxy)-5-(1-(triisopropylsilyl)-1H-indol-4-yl)phe-
nyl)-N-methylpyridine-3-amine was prepared from 25 and 21b
using general Suzuki condition A in 57% yield. The crude Suzuki
product was deprotected via a two-step protocol described for com-
pound 13 to give 26 as a solid in 28% yield. 1H NMR (400 MHz,
3-Chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(triiso-
propylsilyl)-1H-indole. A 20 mL scintillation vial was charged with
21b (100 mg, 0.250 mmol), N-chlorosuccinimde (35 mg, 0.262 mmol,
1.05 equiv), and anhydrous DMF (1.0 mL) under nitrogen. To the
resulting solution is added 1 drop of TFA/DMF (10:1) via syringe.

192 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shetty et al.
CD3OD): δ8.20 (d, J= 2.7 Hz, 1H), 7.95 (dd, J= 4.8, 1.3 Hz, 1H),
7.40 (ddd, J = 8.4, 2.9, 1.4 Hz, 1H), 7.34 (dt, J = 4.5, 2.7 Hz, 1H),
7.28-7.22 (m, 2H), 7.12 (t, J = 7.7 Hz, 1H), 7.03 (dd, J = 7.4, 1.0
Hz, 1H), 6.92 (dd, J = 2.3, 1.4 Hz, 1H), 6.90-6.89 (m, 1H), 6.57 (t,
J = 2.1 Hz, 1H), 6.54 (dd, J = 3.3, 1.0 Hz, 1H), 3.33 (s, 3H). SFC-
MS (APCIþ), M þ H found 315.4.
quenched with saturated NH4Cl and extracted with EtOAc. The
organic phase was dried (Na2SO4) and concentrated. The crude
product was purified by flash chromatography on silica gel to
afford (3-(4-methoxybenzyloxy)-5-(1-(triisopropylsilyl)-1H-indol-
4-yl)phenyl)(pyridin-3-yl)methanol (70 mg, 20%). This product
was deprotected via a two-step protocol described for compound
13 to give 30 (7.5 mg, 20%). 1H NMR (400 MHz, CD3OD): δ 8.61
(s, 1H), 8.40 (d, J = 4.3 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.39 (dd,
J = 7.9, 4.9 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.22 (d, J = 3.2 Hz,
1H), 7.17 (s, 1H), 7.12 (t, J = 7.7 Hz, 1H), 7.05-6.99 (m, 2H), 6.82
(s, 1H), 6.51 (d, J = 3.1 Hz, 1H), 5.84 (s, 1H). SFC-MS (APCIþ),
M þ H found 317.1.
3-(3-Bromo-5-(4-methoxybenzyloxy)phenoxy)pyridine (27).
A mixture of 9b (250 mg, 0.672 mmol), 3-hydroxypyridine
(128 mg, 1.34 mmol), and copper oxide (96 mg, 0.67 mmol) in
collidine (3 mL) was treated with NaH (27 mg, 0.67 mmol) in a
pressure tube. After 10 min the tube was sealed and the reac-
tion mixture was heated to 210 °C overnight. The mixture was
cooled and treated with EtOAc, aqueous NH4OH and filtered
through Celite. The phases were separated, and the aqueous
phase was extracted with additional EtOAc. The combined
organics were washed with brine, dried over Na2SO4, and
concentrated. The product was chromatographed (silica gel,
3-(1H-Indol-4-yl)-5-(pyridin-3-yl)phenol (32). To a 30 mL vial
equipped with stir bar was added 29 (0.400 g, 0.71 mmol),
3-pyridinylboronic acid (0.096 g, 0.78 mmol), toluene (3 mL),
ethanol (3 mL), 2 M Na2CO3 (0.71 mL), and n-Bu4NBr (0.011 g,
0.035 mmol). The starting indole did not dissolve, so ethylene
glycol dimethyl ether (3 mL) was added. Nitrogen was bubbled
through the reaction mixture for 30 min, followed by addition of
Pd(PPh3)4 (0.018 g, 0.022 mmol), and the mixture was heated to
100 °C for 18 h. The reaction mixture was cooled and filtered
through Celite and washed with EtOAc. The solvent was
removed under vacuum and the crude product was purified by
preparative TLC (EtOAc/hexane, 30:70) to give 4-(3-(4-meth-
oxybenzyloxy)-5-(pyridin-3-yl)phenyl)-1-(triisopropylsilyl)-
1H-indole (0.184 g). This compound was deprotected using
similar condition as described for compound 13 to give 32 in
20% yield. 1H NMR (400 MHz, CDCl3): δ 10.07 (d, J = 2.1 Hz,
1H), 9.74 (dd, J = 4.9, 1.3 Hz, 1H), 9.30 (dt, J = 4.9, 2.6 Hz,
1H), 8.70 (q, J = 4.3 Hz, 1H), 8.65-8.61 (m, 2H), 8.53 (d, J =
3.3 Hz, 1H), 8.45-8.36 (m, 3H), 8.29 (t, J = 1.9 Hz, 1H), 7.89 (d,
J = 3.1 Hz, 1H). SFC-MS (APCIþ), M þ H found 287.1. SFC-
MS indicated 94% purity.
1
33% EtOAc/hexane) to give 27 (66 mg, 25%). H NMR (400
MHz, CDCl3): δ 8.47 (s, 2H), 7.33-7.25 (m, 4H), 6.98-6.82 (m,
3H), 6.80-6.66 (m, 1H), 6.53 (t, J = 2.2 Hz, 1H), 4.92 (s, 2H),
3.80 (s, 3H).
3-(1H-Indol-4-yl)-5-(pyridin-3-yloxy)phenol (28). Compound
28 was prepared from 27 in a similar manner as described for the
preparation of 26 in 15% yield over three steps. 1H NMR (400
MHz, CD3OD): δ 11.22 (s, 1H), 9.77 (s, 1H), 8.43 (d, J = 2.5 Hz,
1H), 8.36 (d, J = 3.5 Hz, 1H), 7.53 (ddd, J = 8.4, 2.7,1.3 Hz,
1H), 7.42 (dd, J = 8.4, 4.7 Hz, 1H), 7.36 (dd, J = 5.4, 2.4 Hz,
2H), 7.11 (t, J = 7.7 Hz, 1H), 7.00 (d, J = 7.0 Hz, 1H), 6.86 (s,
1H), 6.65 (s, 1H), 6.45 (s, 1H), 6.40 (t, J = 2.0 Hz, 1H). SFC-MS
(APCIþ), M þ H found 303.26.
4-(3-Bromo-5-(4-methoxybenzyloxy)phenyl)-1-(triisopropylsilyl)-
1H-indole (29). 3-Bromo-5-iodophenol (8.48 g, 28.37 mmol) was
dissolved in DMF (50 mL) under dry argon and cooled to 0 °C.
Then K2CO3 (3.92 g, 28.37 mmol) was added, followed by dropwise
addition of 4-methoxybenzyl chloride (3.7 mL, 27.0 mmol). The
mixture was slowly warmed to room temperature and stirred for
20 h. Additional 4-methoxybenzyl chloride (0.19 mL, 1.4 mmol)
was added and stirred for another 24 h. The reaction mixture was
diluted with tert-butyl methyl ether (300 mL) and water (150 mL).
The layers were separated, and the aqueous one was extracted with
additional tert-butylmethyl ether (2 ꢀ 150 mL). The combined
organics were washed with water and brine (150 mL), dried
(Na2SO4), and concentrated to give the crude product which was
purified by flash chromatography (silica gel 330 g, EtOAc/heptane
1:9) to give 1-bromo-3-iodo-5-(4-methoxybenzyloxy)benzene (9g,
8.80 g, 59%) as a yellow oil.
(3-Bromo-5-(4-methoxybenzyloxy)phenyl)(pyridin-3-yl)metha-
none (33). In a flame-driedthree-neck flask, 9b (37.2 g, 100 mmol)
was dissolved in 1 L of diethyl ether, and the solution was cooled
to -75 °C. To the suspension was added n-BuLi (1.6 M in
hexane, 62.5 mL, 100 mmol) in a fashion such that the tempera-
ture did not rise above -71 °C. The mixture was stirred for 30 min
at -75 °C, and a solution of 3-cyanopyridine (10.4 g, 100 mmol) in
diethyl ether (100 mL) was added slowly to maintain temperature
below -70 °C. The mixture was stirred at -75 °C for 2 h and then
slowly allowed to warm to -25 °C, when 2 N HCl (110 mL) was
added. The reaction mixture was stirred for 20 min at room
temperature, basified by addition of 1 N NaOH, and extracted
with EtOAc. The combined organic layers were dried over Na2SO4
and concentrated. The product was purified by chromatography
(silica gel 700 g, CH2Cl2, to CH2Cl2/EtOAc 10:1, followed by
neutral Alox A3 1000 g, heptane/EtOAc 10:1) to yield 33 (22.4 g,
A 1 L flask was charged with 1-bromo-3-iodo-5-(4-methoxy-
benzyloxy)benzene (17.44 g, 43.67 mmol), 21b (18.30 g, 43.67
mmol) in EtOH (180 mL), toluene (180 mL), and under argon
was added Pd(PPh3)4 (1.51 g, 1.31 mmol). A solution of Na2CO3
(9.26 g, 87.34 mmol) in water (92.6 mL) was added and the
mixture heated to 90 °C and stirred for 14 h. The reaction
mixture was cooled to room temperature, stirred with EtOAc
(300 mL) and brine (200 mL), and filtered through a pad of
Hyflo. The aqueous layer was extracted with additional EtOAc
(2 ꢀ 300 mL). The combined organic layers were washed with
water, brine (150 mL), dried over Na2SO4, filtered, and evapo-
rated to dryness. Flash chromatography (silica gel 1 kg, EtOAc/
heptane 1:19) gave 29 (22.5 g, 91%) as a colorless oil. 1H NMR
(400 MHz, CDCl3): δ 7.50 (d, J = 8.2 Hz, 1H), 7.43 (s, 1H), 7.36
(d, J = 8.6 Hz, 2H), 7.27 (d, J = 3.3 Hz, 1H), 7.23 (s, 1H), 7.19 (t,
J = 7.8 Hz, 1H), 7.14-7.11 (m, 2H), 6.94-6.90 (m, 2H), 6.70 (d,
J = 3.0 Hz, 1H), 5.02 (s, 2H), 3.82 (s, 3H), 1.71 (dt, J = 15.0, 7.5
Hz, 3H), 1.14 (dd, J = 10.6, 8.0 Hz, 18H).
1
56%). H NMR (400 MHz, CDCl3): δ 9.03-8.92 (m, 1H), 8.81
(dd, J = 4.9, 1.7 Hz, 1H), 8.13-7.94 (m, 1H), 7.47 (t, J = 1.5 Hz,
1H), 7.43 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H), 7.36 (dd, J = 2.4, 1.7 Hz,
1H), 7.31 (ddd, J = 4.5, 3.9, 1.8 Hz, 3H), 6.94-6.87 (m, 2H), 5.01
(s, 2H), 3.81 (s, 3H).
(3-Hydroxy-5-(1H-indol-4-yl)phenyl)(pyridin-3-yl)methanone
(34). (3-(4-Methoxybenzyloxy)-5-(1-(triisopropylsilyl)-1H-in-
dol-4-yl)phenyl)(pyridin-3-yl)methanone was prepared from
33 and 21b using general Suzuki condition A in 64% yield.
The Suzuki product (80 mg, 0.14 mmol) was dissolved in DCM
(1 mL), dimethylsulfide (1 mL), and TFA (2 mL). The resulting
solution was stirred at room temperature for 2 h. DCM was
added to the reaction mixture, followed by saturated NaHCO3.
The aqueous phase was adjusted to a pH 6 and then extracted
with DCM twice. The organic phase was dried over Na2SO4,
filtered, and concentrated to provide the crude product. The
crude product was further purified by preparative TLC to give
34 (13 mg, 31%). 1H NMR (400 MHz, CD3OD): δ 8.97 (d, J =
2.0 Hz, 1H), 8.70 (dd, J = 4.8, 1.6 Hz, 1H), 8.21 (dt, J = 8.0, 2.0
Hz, 1H), 7.54-7.50 (m, 2H), 7.46-7.45 (m, 1H), 7.41 (d, J = 8.0
Hz, 1H), 7.27 (d, J = 3.2 Hz, 1H), 7.22 (t, J = 2.0 Hz, 1H), 7.17
3-(Hydroxy(pyridin-3-yl)methyl)-5-(1H-indol-4-yl)phenol (30).
To 0.298 g (0.529 mmol) of 29 in dry ether at -76 °C was added
n-BuLi in hexane (1 M, 0.530 mL), and the resulting reaction
mixture was stirred at -76 °C for 30 min. After slow addition of
nicotinaldehyde (40 mg, 0.529 mmol) in ether, the reaction mixture
was allowed to warm to room temperature. The reaction was

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 193
(t, J = 8.0 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 6.61 (d, J = 3.2 Hz,
1H). SFC-MS (APCIþ), M þ H found 315.3.
N-(3-Chloro-5-(1H-indol-4-yl)phenyl)pyridin-3-amine (41). N-
(3-Bromo-5-chlorophenyl)pyridin-3-amine was prepared from
1,3-dibromo-5-chlorobenzene 39a and pyridin-3-amine as de-
scribed for preparation of 10b in 30% yield. This material was
reacted with 21b using general Suzuki condition A (54%). The
Suzuki product (54 mg, 0.11 mmol) in THF (3 mL) at 0 °C was
treated with 1 M TBAF in THF (0.13 mL, 0.13 mmol) and
stirred at room temperature for 30 min, the mixture was con-
centrated, and the crude product was purified on a preparative
SFC system to give 41 (11 mg, 31%). 1H NMR (400 MHz,
CD3OD): δ 8.35 (br s, 1H), 8.03 (d, J = 4.3 Hz, 1H), 7.63 (ddd,
J = 8.3, 2.7,1.3 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.33-7.28 (m,
3H), 7.20-7.14 (m, 2H), 7.08-7.04 (m, 2H), 6.57 (d, J = 3.2 Hz,
1H). SFC-MS (APCIþ), M þ H found 320.1.
N-(3-Bromophenyl)-N-(pyridin-3-yl)acetamide (37). To a mix-
ture of N-(3-pyridyl)acetamide 36 (272 mg, 2 mmol), CuI
(powdered, 190 mg, 1.0 mmol), Cs2CO3 (651 mg, 2.0 mmol), 1,3-
dibromobenzene 35 (2.3 g, 10.0 mmol) in 1,4-dioxane (10 mL) was
added N,N0-dimethylethylenediamine (176 mg, 2 mmol). The
reaction mixture was heated to 110 °C for 16 h. After cooling to
room temperature, the reaction mixture was filtered through a pad
of Celite, and the filtrate was concentrated. The residue was
extracted with DCM (5 mL), and the organic layer was washed
with water (15 mL). The organic layer was dried, concentrated, and
the residue was passed through a short pad of silica gel using
DCM/MeOH (10:1) as eluent. The combined solution was con-
centrated under vacuum to give N-(3-bromophenyl)-N-(pyridin-3-
N-(3-(1H-Indol-4-yl)-5-methoxyphenyl)pyridin-3-amine (42).
N-(3-Bromo-5-methoxyphenyl)pyridin-3-amine was prepared
from 1,3-dibromo-5-methoxybenzene 39b and pyridin-3-amine
as described for the preparation of 10b in 50% yield. This
material was converted to 42 as described for compound 41 in
19.4% yield over two steps. 1H NMR (400 MHz, CDCl3): δ 8.44
(d, J = 2.7 Hz, 1H), 8.34 (br s, 1H), 8.17 (dd, J = 4.7, 1.2 Hz,
1H), 7.50 (ddd, J = 8.2, 2.7, 1.4 Hz, 1H), 7.39 (d, J = 8.2 Hz,
1H), 7.27-7.23 (m, 2H), 7.19-7.15 (m, 2H), 6.98 (t, J = 1.7 Hz,
1H), 6.89 (dd, J = 2.2, 1.4 Hz, 1H), 6.73 (t, J = 2.1 Hz, 1H), 6.64
(t, J = 2.2 Hz, 1H), 5.81 (s, 1H), 3.84 (s, 3H). SFC-MS (APCIþ),
M þ H found 316.4.
1
yl)acetamide 37 (97 mg) in 16.7% yield. H NMR (400 MHz,
CDCl3): δ 8.53-8.44 (m, 2H), 7.64-7.59 (m, 1H), 7.48-7.39 (m,
2H), 7.35-7.24 (m, 2H), 7.21-7.19 (m, 1H), 2.07 (s, 3H). SFC-MS
(APCIþ), M þ H found 291.0.
N-(3-(1H-Indol-4-yl)phenyl)pyridin-3-amine (38). N-(Pyridin-
3-yl)-N-(3-(1-(triisopropylsilyl)-1H-indol-4-yl)phenyl)acetamide
was prepared from N-(3-bromophenyl)-N-(pyridin-3-yl)aceta-
mide 37 and 21b using general Suzuki procedure A. The Suzuki
product (15 mg, 0.03 mmol) was treated with 10% dimethylsul-
fide and 50% TFA in DCM (1 mL) for 10 min. The volatiles were
evaporated with a stream of nitrogen and mild heating. The
residue was purified by preparative SFC to obtain N-(3-(1H-
indol-4-yl)phenyl)-N-(pyridin-3-yl)acetamide as an oil in 30%
yield. To the oil (15 mg, 0.045 mmol) was added 6 N HCl (1 mL),
and the solution was heated to 110 °C for 1 h in a sealed reaction
tube. Volatiles were evaporated by blowing a gentle stream of
nitrogen, and the residue was dissolved in H2O and passed through
a short pad of cotton filter. The clear solution was concentrated to
give 38 (12.5 mg, 0.038 mmol) as the hydrochloride salt in 84.4%
yield. 1H NMR (400 MHz, D2O): δ8.15 (t, J= 4.6 Hz, 1H), 7.66 (s,
1H), 7.60 (s, 1H), 7.56-7.52 (2H), 7.46 (d, 4.8 Hz, 1H), 7.43-7.38
(4 H), 7.31 (d, J = 8.8 Hz, 1H), 7.07 (t, J = 7.0 Hz, 1H), 7.00 (d,
J = 8.4 Hz, 1H). SFC-MS (APCIþ), M þ H found 286.3.
N-(3,5-Dibromophenyl)pyridin-3-amine (40e). A vial with a
septum cap was charged with 1,3,5-tribromobenzene 39e (337 mg,
1.2 mmol), pyridin-3-amine (96 mg, 1.02 mmol), t-BuONa (112 mg,
2.02 mmol), and XantPhos (57 mg, 0.098 mmol). The atmosphere
was flushed with nitrogen. Then 1,4-dioxane (6 mL) was added
followed by Pd2(dba)3 (45 mg, 0.05 mmol). The atmosphere was
again flushed with nitrogen and the mixture was heated to 110 °C.
At 23 h, the reaction mixture was cooled, filtered through Celite,
and the filter cake was eluted with EtOAc. Solvent was removed
in vacuo and the residue was purified by flash column (3.8 cm ꢀ
12 cm, silica gel) using DCM:/MeOH (98:2) to give of N-(3,5-
dibromophenyl)pyridin-3-amine (206.1 mg, 62%). 1H NMR (400
MHz, CDCl3): δ 8.38 (d, J = 2.0 Hz, 1H), 8.23 (d, J = 4.1 Hz, 1H),
7.44 (dd, J = 8.2, 1.0 Hz, 1H), 7.21 (dd, J = 8.0, 4.8 Hz, 1H), 7.16
(t, J = 1.4 Hz, 1H), 7.07 (d, J = 1.3 Hz, 2H), 6.45 (s, 1H). SFC-MS
(APCIþ), M þ H found 306.1.
N-(3-(1H-Indol-4-yl)-5-methylphenyl)pyridin-3-amine (43). N-
(3-Bromo-5-methylphenyl)pyridin-3-amine was prepared from
1,3-dibromo-5-methylbenzene 39c and pyridin-3-amine as de-
scribed for preparation of 10b in 31% yield. This material was
converted to compound 43 as described for compound 41 in 24%
yield. 1H NMR (400 MHz, CD3OD): δ 8.31 (s, 1H), 7.93 (d, J =
4.1 Hz, 1H), 7.56 (ddd, J = 8.4, 2.8, 1.3 Hz, 1H), 7.35 (d, J = 8.1
Hz, 1H), 7.29-7.19(m, 3H), 7.09 (ddd, J = 11.3, 8.2, 4.2Hz, 3H),
6.93 (s, 1H), 6.59 (dd, J = 3.2, 0.9 Hz, 1H), 2.38 (s, 3H). SFC-MS
(APCIþ), M þ H found 300.8.
3-(1H-Indol-4-yl)-5-(pyridin-3-ylamino)benzonitrile (44). 3-Bromo-
5-(pyridin-3-ylamino)benzonitrile was prepared from 3,5-dibromo-
benzonitrile 39d and pyridin-3-amine as described for the prepara-
tion of 10b in 62% yield. This material was converted to compound
44 as described for compound 41 in 9.7% yield over two steps. 1H
NMR (400 MHz, CDCl3): δ 8.47 (s, 2H), 8.28 (d, J = 4.3 Hz, 1H),
7.55-7.49 (m, 3H), 7.44 (d, J = 8.2 Hz, 1H), 7.30-7.23 (m, 4H),
7.12 (dd, J =7.3, 0.9 Hz, 1H), 6.63(t, J = 2.5 Hz, 1H), 6.05 (s, 1H).
SFC-MS (APCIþ), M þ H found 311.1.
N-(3-(1H-Indol-4-yl)-5-(pyridin-3-ylamino)phenyl)acetamide
(45). To a vial containing 40f (0.109 g, 0.36 mmol) in toluene
(0.7 mL), EtOH (0.7 mL), and H2O (0.27 mL) was added Na2CO3
(0.076 g, 0.719 mmol) followed by n-Bu4NBr (0.005 g, 0.02 mmol)
and 21b (0.158 g, 0.39 mmol). The vial was purged with nitrogen
before and after addition of Pd(Ph3P)4 (0.020 g, 0.017 mol), capped,
and heated at 110 °C for 18 h. The mixture was cooled, filtered
through Celite, washed with MeOH. The solvent was removed in
vacuo, giving a mixture of N-[3-(pyridin-3-ylamino)-5-(1-(triiso-
propylsilyl)-1H-indol-4-yl)phenyl)acetamide and N-(3-(1H-indol-
4-yl)-5-(pyridin-3-ylamino)phenyl)acetamide. The mixture of Su-
zuki products (177 mg, 0.35 mmol) in THF (2 mL) at 0 °C was
treated with 1 M TBAF in THF (0.5 mL, 0.5 mmol) and stirred at
room temperature for 30 min The mixture was concentrated, and
the crude product was purified on preparative SFC to give 45
(0.071 g, 58.7%). 1H NMR (400 MHz, acetone-d6): δ 10.41 (s, 1H),
9.28 (s, 1H), 8.51 (d, J= 2.5 Hz, 1H), 8.09 (dd, J= 4.6, 1.1 Hz, 1H),
7.77 (d, J = 17.0 Hz, 1H), 7.69 (s, 1H), 7.62 (ddd, J = 8.2, 2.7,1.3
Hz, 1H), 7.53 (s, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.38 (t, J = 2.8 Hz,
1H), 7.23 (q, J = 4.3 Hz, 1H), 7.20-7.17 (m, 2H), 7.16-7.11 (m,
1H), 6.76-6.72 (m, 1H), 2.11 (s, 3H). SFC-MS (APCIþ), M þ H
found 343.2.
N-(3-Bromo-5-(pyridin-3-ylamino)phenyl)acetamide (40f). The
N-(3,5-dibromophenyl)pyridin-3-amine 40e (150 mg, 0.44 mmol)),
acetamide (36 mg, 0.62 mmol), and Cs2CO3 (204 mg, 0.44 mmol)
were stirred together with 1,4-dioxane (2.5 mL), and the flask was
flushed with argon. In a separate vial under argon was added
Pd2(dba)3 (0.02 mmol) and XantPhos (0.03 mmol) to 1,4-dioxane
(0.5 mL). The catalyst mixture was stirred at room temperature for
15 min, and this was added via syringe to the amine mixture which
was heated at 110 °C for 4.5 h. The mixture was cooled and filtered
through Celite, eluting with EtOAc. Then solvent was removed in
vacuo. Purification by flash chromatography on silica gel using
DCM/MeOH (95:5) gave 40f (76.3 mg, 53%). 1HNMR(400MHz,
acetone-d6):δ9.35 (s, 1H), 8.45 (d, J= 1.8 Hz, 1H), 8.15 (d, J=4.2
Hz, 1H), 7.84 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.46 (s, 2H), 7.26
(dd, J = 8.1, 4.6 Hz, 1H), 6.93 (s, 1H), 2.07 (s, 3H). SFC-MS
(APCIþ), M þ H found 306.1.
(3,5-Dibromophenyl)(pyridin-3-yl)methanone (47a). In a flame-
dried three-neck flask under argon, 1,3,5-tribromobenzene 39e
(31.4 g, 100 mmol) was dissolved in 1 L of diethyl ether, and the

194 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shetty et al.
solution was cooled to -72 °C. To the resulting suspension was
added a solution of n-BuLi (1.6 M in hexane, 62.5 mL, 100 mmol)
slowly so that the temperature did not rise above -70 °C. The
mixture was stirred for 30 min at -75 °C, and a solution of
3-cyanopyridine 46a (10.4 g, 100 mmol) in diethyl ether (100 mL)
was added slowly so that the temperature did not rise above -71 °C.
The mixture was stirred at -75 °C for 60 min and slowly allowed to
warm to -25 °C, when 2 N HCl (250 mL) was added and the
mixture was stirred for 20 min at room temperature. The mixture
was made basic by addition of 1 N NaOH. The product was
extracted with EtOAc, and the combined organic layers were dried
over Na2SO4. The product was purified by chromatography (silica
gel,700 g) to yield 47a (27.7 g, 81%). 1H NMR (400 MHz, CDCl3):
δ 8.97 (s, 1H), 8.85 (d, J = 4.0 Hz, 1H), 8.10 (dt, J = 7.9, 1.8 Hz,
1H), 7.92 (t, J = 1.6 Hz, 1H), 7.84 (d, J = 1.6 Hz, 2H), 7.49 (dd,
J = 7.8, 4.9 Hz, 1H). SFC-MS (APCIþ), M þ H found 339.9.
(2-Chloropyridin-3-yl)(3,5-dibromophenyl)methanone (47b).
Compound 47b was prepared from 1,3,5-tribromobenzene 39e
and 2-chloronicotinonitrile 46b in a similar manner as described
(s, 1H), 3.53 (s, 3H), 3.32 (s, 3H), 2.54 (s, 3H). SFC-MS (APCIþ),
M þ H found 215.0
(2-Chloro-6-methylpyridin-4-yl)(3,5-dibromophenyl)methanone
(47f). To 1,3,5-tribromobenzene 39e (0.45 g, 1.4 mmol) in anhy-
drous ether (25.0 mL) at -78 °C was slowly added n-BuLi (0.57 mL,
1.4 mmol) so that the internal temperature was maintained
below -70 °C. After the addition was complete, the mixture was
stirred for 1 h at -78 °C. Then 2-chloro-N-methoxy-6,N-dimethy-
lisonicotinamide 46d (0.220 g, 1.1 mmol) in THF (2 mL) was added,
again keeping the temperature below -70 °C. After the addition was
complete, the mixture was allowed to stir at -25 °C for 45 min. Then
saturated NaHCO3 (100 mL) was added and the mixture was stirred
at room temperature for 30 min. The phases were separated, and the
aqueous phase was extracted with ether. The combined organic
phases were dried with Na2SO4, the volatiles were removed in vacuo,
and the crude product was chromatographed on silica gel using
hexane/EtOAc (5:1) to give 47f (330 mg, 77% yield). 1H NMR (400
MHz, CDCl3): δ 7.92 (t, J = 1.6 Hz, 1H), 7.81 (d, J = 1.6 Hz, 2H),
7.35 (s, 1H), 7.29 (s, 1H), 2.62 (s, 3H). SFC-MS (APCIþ), M þ H
found 387.8.
1
for the preparation of compound 47a in 20% yield. H NMR
(400 MHz, CDCl3): δ 8.05 (d, J = 1.6 Hz, 2H), 8.00 (d, J = 8.1
Hz, 1H), 7.73 (d, J = 1.4 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 6.88
(d, J = 1.4 Hz, 1H). SFC-MS (APCIþ), M þ H found 376.
(3,5-Dibromophenyl)(2-methoxypyridin-3-yl)methanone (47c).
To a 25 mL flask charged with anhydrous MeOH (4 mL) at
0 °C was added sodium (0.1 g, 5 mmol), and the reaction mixture
was stirred for 30 min. To the solution of sodium methoxide was
added 47b (0.374 g, 1.0 mmol) in DMF (0.8 mL) and the reaction
was heated to 80 °C for 5 h. The reaction mixture was cooled in an
ice bath, slowly quenched with 4 N HCl, and concentrated under
vacuum. The crude product was extracted with DCM (25 mL).
The DCM layer was washed with saturated NaHCO3, water,
brine, anddried over Na2SO4. The organic layer was filtered, con-
centrated, and chromatographed on silica gel using 10% EtOAc
in hexanes to give 47c (0.16 g, 43%). 1H NMR (400 MHz, CDCl3):
δ 8.35 (dd, J = 5.0, 1.9 Hz, 1H), 7.84 (t, J = 1.6 Hz, 1H), 7.79 (d,
J = 1.6 Hz, 2H), 7.75 (dd, J = 7.3, 1.9 Hz, 1H), 7.03 (dd, J = 7.3,
5.0 Hz, 1H), 3.88 (s, 3H). SFC-MS (APCIþ), M þ H found 372.1.
(2-Chloropyridin-4-yl)(3,5-dibromophenyl)methanone (47d).
Compound 47d was prepared from 1,3,5-tribromobenzene 39e
and 2-chloroisonicotinonitrile 46c in a similar manner as described
for the preparation of compound 47a in 75% yield. 1H NMR (400
MHz, CDCl3): δ 8.61 (d, J = 5.0 Hz, 1H), 7.94 (d, J = 1.5 Hz, 1H),
7.82 (d, J = 1.6 Hz, 2H), 7.58 (s, 1H), 7.45 (d, J = 5.0 Hz, 1H), 1.57
(s, 1H). SFC-MS (APCIþ), M þ H found 376.1.
Benzyl 3-Bromo-5-nicotinoylphenylcarbamate (48a). A 1 L four-
neck flask equipped with a reflux condenser, a mechanical stirrer,
and a thermometer was charged with 47a (15.35 g, 45.0 mmol) and
benzyl carbamate (16.33 g, 108.0 mmol). The flask was flushed
with argon. Then Cs2CO3 (20.50 g, 63.0 mmol) and 1,4-dioxane
(270 mL) were added, and the mixture was heated to an internal
temperature of 80 °C. In a 100 mL Schlenk flask were placed
together Pd2(dba)3 CHCl3 complex (0.465 g, 0.9 mmol) and
3
XantPhos (0.780 g, 1.35 mmol) in a toluene/1,4-dioxane mixture
(1:4, 60 mL). The orange Pd catalyst suspension was stirred at room
temperature for 20 min and then (10 mL) was added to the reaction
mixture at 80 °C. The temperature was then raised to 105 °C, and a
10 mL portion of the catalyst suspension was added in 1 h intervals.
After refluxing overnight the reaction mixture afforded a yellow
solution and a brown precipitate. The reaction mixture was cooled
to room temperature and the precipitate filtered and washed with
EtOAc (3 ꢀ 50 mL). The yellow solution was evaporated to afford
yellow brown oil. The crude was purified by chromatography (silica
gel 900 g, toluene/DCM/EtOAc/HCOOH, 15/20/10/2) to yield a
pale yellow oil. A further purification involved the precipitation of
the product by dissolving the oil in a minimum of EtOAc and
addition of hexane to yield 48a (8.51 g, 46%) as a white solid. 1H
NMR (400 MHz, CDCl3): δ 8.98 (s, 1H), 8.82 (d, J = 4.0 Hz, 1H),
8.13 (dt, J =7.9, 1.7Hz, 1H), 7.99(s, 1H), 7.67(s, 1H), 7.58(s, 1H),
7.47 (dd, J=7.8, 4.9 Hz, 1H), 7.40-7.30 (m, 5H), 6.97 (s, 1H), 5.19
(s, 2H). LC-MS (ESþ), M þ H found 412.0.
(3,5-Dibromophenyl)(2-morpholinopyridin-4-yl)methanone (47e).
In a pressure tube 47d (0.200 g, 0.53 mmol) was dissolved in 1,4-
dioxane (1.0 mL), and morpholine (0.070 g, 0.80 mmol) was added.
The mixture was subjected to microwave irradiation at 120 °C for
60 min. The reaction mixture was cooled and concentrated. The
crude product was chromatographed on silica gel using hexane/
(3-Amino-5-bromophenyl)(pyridin-3-yl)methanone (48b).
A
stirred slurry of 48a (1 g, 2.43 mmol) in 12 N HCl (50 mL) was
heated at 80 °C for 20 min. Methanol (10 mL) was added until
the material went into solution. Starting material was consumed
within 10 min of the addition of methanol. The material was
neutralized with NaHCO3 and extracted thrice with EtOAc (3 ꢀ
50 mL). The organics were combined, dried over Na2SO4,
filtered and the solution was concentrated under reduced pres-
sure, affording 48b (0.63 g, 93%). 1H NMR (400 MHz, DMSO-
d6): δ 10.30 (s, 1H), 8.86 (d, J = 1.7 Hz, 1H), 8.82 (dd, J = 4.7,
1.2 Hz, 1H), 8.21 (s, 1H), 8.10 (d, J = 7.9 Hz, 1H), 7.83 (s, 1H),
7.58 (dd, J = 7.8, 4.9 Hz, 1H), 7.53 (s, 1H). LC-MS (ESþ), M þ
CH3CN found 317.9.
1
EtOAc (1:1) to give 47e (0.118 g, 52%). H NMR (400 MHz,
CDCl3):δ8.34 (d, J = 5.0 Hz, 1H), 7.89 (t, J = 1.6 Hz, 1H), 7.85 (d,
J=1.6 Hz, 2H), 6.86 (s, 1H), 6.78 (d, J=5.0 Hz, 1H), 3.88-3.78 (m,
4H), 3.62-3.52 (m, 4H). SFC-MS (APCIþ), M þ H found 427.0.
2-Chloro-N-methoxy-6,N-dimethylisonicotinamide (46d). To
2-chloro-6-methylisonicotinic acid (0.500 g, 2.91 mmol) in a
mixture of DCM (5.0 mL) and DMF (0.5 mL) was added HOBT
(0.47 g, 3.5 mmol). The reaction mixture was stirred for 20 min at
0 °C followed by addition of EDCI (0.67 g, 3.5 mmol). The
reaction mixture was slowly warmed to room temperature and
stirred for 1 h. The reaction mixture was recooled to 0 °C. DIEA
(0.750 g, 5.8 mmol) and N,O-dimethylhydroxylamine (0.340 g,
3.5 mmol) were added. The reaction mixture was stirred at room
temperature for 16 h, then diluted with water and extracted with
DCM (50 mL). The organic phase was washed with water, brine,
dried over Na2SO4, filtered, and concentrated. The crude prod-
uct was chromatographed on silica gel using 5:1 hexane/EtOAc
to give 2-chloro-N-methoxy-6,N-dimethylisonicotinamide 46d
Methyl 3-Bromo-5-nicotinoylphenylcarbamate (48c). To a
stirred solution of 48b (0.20 g, 0.72 mmol) in DCM (5 mL)
was added pyridine (0.58 mL) and methyl chloroformate (0.55
mL). The reaction mixture was heated to 60 °C for 20 min and
diluted with DCM. The DCM layer was washed with saturated
NaHCO3, separated, dried over Na2SO4, and the volatiles were
removed under reduced pressure to give 48c (208 mg, 86%
1
yield). H NMR (400 MHz, CDCl3): δ 8.99 (s, 1H), 8.84 (s,
1H), 8.17-8.05 (m, 1H), 7.98 (s, 1H), 7.68 (d, J = 5.4 Hz, 1H),
7.59 (d, J = 1.5 Hz, 1H), 7.47 (d, J = 5.0 Hz, 1H), 6.80 (s, 1H),
3.79 (s, 3H). SFC-MS (APCIþ), M þ H found 335.0.
1
(360 mg). H NMR (400 MHz, CDCl3): δ 7.32 (s, 1H), 7.25

Article
N-(3-Bromo-5-nicotinoylphenyl)acetamide (48d). To 48b (1.32 g,
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 195
similar manner as described for the preparation of 51 in 21%
1
3.6 mmol) in DCE (10 mL) and DIEA (1.0 mL) was added acetyl
chloride (0.35 mL). The reaction mixture was stirred for 10 min,
then poured into water and extracted twice with DCM. The organic
phase was dried over Na2SO4, filtered, and concentrated to give 154
mg of 48d, which was used without further purification. 1H NMR
(400 MHz, DMSO-d6): δ 10.30 (s, 1H), 8.86 (d, J = 1.7 Hz, 1H),
8.82 (dd, J= 4.7, 1.2 Hz, 1H), 8.21 (s, 1H), 8.10 (d, J= 7.9 Hz, 1H),
7.83 (s, 1H), 7.58 (dd, J = 7.8, 4.9 Hz, 1H), 7.53 (s, 1H), 2.03 (s,
3H). SFC-MS (APCIþ), M þ H found 319.1.
yield for two steps. H NMR (400 MHz, CD3OD): δ 10.61 (s,
1H), 8.94 (s, 1H), 8.70 (s, 1H), 8.13-8.19 (m, 2H), 7.99 (s, 1H),
7.74 (s, 1H), 7.49 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.25 (s, 1H),
7.13 (t, J = 7.7 Hz, 1H), 7.06 (d, J = 7.0 Hz, 1H), 6.62 (s, 1H),
2.12 (s, 3H). SFC-MS (APCIþ), M þ H found 356.9.
N-[3-(1H-Indol-4-yl)-5-(pyridine-3-carbonyl)phenyl]methanesul-
fonamide (53). Compound 53 was synthesized from compound 48e
in a similar manner as described for the preparation of 51 in 13%
yield for two steps. 1H NMR (400 MHz, CD3OD/CDCl3): δ 8.97
(s, 1H), 8.71 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.87 (t, J = 1.8 Hz,
1H), 7.78 (t, J = 1.5 Hz, 1H), 7.56 (t, J = 1.9 Hz, 1H), 7.44 (dd,
J = 7.8, 4.9 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 3.1 Hz,
1H), 7.17 (t, J = 7.7 Hz, 1H), 7.09 (d, J = 7.3 Hz, 1H), 6.60 (d, J =
3.3 Hz, 1H), 3.00 (s, 3H). SFC-MS (APCIþ), M þ H found 392.2.
3-[3-(1H-Indol-4-yl)-5-(pyridine-3-carbonyl)phenyl]-1,1-dime-
thylurea (54). Compound 54 was synthesized from compound
48f in a similar manner as described for the preparation of 51 in
31% yield for two steps. 1H NMR (400 MHz, CD3OD): δ 8.97 (s,
1H), 8.75 (d, J= 3.7 Hz, 1H), 8.25 (td, J= 7.9, 1.9 Hz, 1H), 8.09 (t,
J = 1.9 Hz, 1H), 7.84 (t, J = 1.9 Hz, 1H), 7.73 (t, J = 1.6 Hz, 1H),
7.58 (dd, J = 7.8, 4.9 Hz, 1H), 7.39 (td, J = 8.0, 1.0 Hz, 1H), 7.28
(d, J = 3.1 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.12 (dd, J = 7.3, 1.1
Hz, 1H), 6.67 (dd, J = 3.1, 1.0 Hz, 1H), 3.02 (s, 6H). SFC-MS
(APCIþ), M þ H found 385.2.
N-(3-Bromo-5-nicotinoylphenyl)methanesulfonamide (48e). To a
stirred solution of 48b (0.200 g, 0.72 mmol) in DCE (10 mL) was
added pyridine (0.87 mL, 10.82 mmol), followed by methanesulfo-
nyl chloride (0.56 mL, 7.2 mmol). The mixture was stirred at 80 °C
for 2 h. The volatiles were removed under reduced pressure,
affording material which was flash chromatographed using a
gradient of EtOAc/hexane (25-100%) to give 48e (0.150 g,
1
59%). H NMR (400 MHz, CD3OD): δ 8.89 (d, J = 1.4 Hz,
1H), 8.75 (dd, J = 4.8, 1.2 Hz, 1H), 8.15-8.11 (m, 1H), 7.65 (t, J =
1.7 Hz, 1H), 7.58 (s, 1H), 7.56-7.51 (m, 2H), 4.43 (s, 1H), 3.01 (s,
3H). SFC-MS (APCIþ), M þ H found 355.1.
3-(3-Bromo-5-nicotinoylphenyl)-1,1-dimethylurea (48f). To 48b
(0.2 g, 0.72 mmol) in DCM (5 mL) were added 4-nitrophenyl
chloroformate (0.16 g, 0.79 mmol) and pyridine (0.12 mL). Addi-
tional DCM (10 mL) was added, and the mixture was stirred for 3 h
at room temperature, at which time DIEA (0.15 mL, 0.866 mmol)
and 2 N dimethylamine in THF (0.873 mL, 1.75 mmol) were added.
The mixture was stirred at ambient temperature for 18 h and then
diluted with DCM. The organic phase was washed with saturated
NaHCO3, driedoverNa2SO4, filtered, and concentrated. The crude
product was chromatographed to give 48f (quantitative). 1H NMR
(400 MHz, CD3OD): δ 8.90 (d, J = 1.5 Hz, 1H), 8.77 (dd, J = 4.9,
1.3 Hz, 1H), 8.19 (dt, J= 7.9, 1.8 Hz, 1H), 7.98 (dd, J= 3.9, 1.9 Hz,
1H), 7.83-7.77 (m, 1H), 7.60 (dd, J= 7.8, 5.0 Hz, 1H), 7.54 (d, J=
1.4 Hz, 1H), 2.98 (d, J = 11.2 Hz, 6H). SFC-MS (APCIþ), M þ H
found 348.1.
N-(3-(1H-Indol-4-yl)-5-(2-methoxypyridine-3-carbonyl)phenyl)-
ethanamide (57). In a pressure tube 47c (204 mg, 0.55 mmol) and
1H-indol-4-ylboronic acid (0.092 g,) were treated with toluene
(2.4 mL), EtOH (1.7 mL), H2O (0.72 mL), and Na2CO3 (176 mg).
The solution was degassed with a stream of nitrogen for 3 min,
followed by addition of catalytic Pd(PPh3)4 and heating at 70 °C
for 16 h. The mixture was filtered through Celite and washed with
CHCl3. The solution was concentrated, diluted with EtOAc
(10 mL), and washed with water (7 mL). The organic layer was
dried over Na2SO4, concentrated until only toluene was present,
loaded on a silica gel column (1.4 cm i.d. ꢀ 3.4 cm), and eluted with
EtOAC/hexane (1/3) to give (3-bromo-5-(1H-indol-4-yl)phenyl)-
(2-methoxypyridin-3-yl)methanone (89 mg, 40% yield).
To the bromide (65 mg, 0.16 mmol), 1,4-dioxane (2 mL) in a
pressure tube at 100 °C was added N,N0-dimethylethylenediamine
(0.28 mmol) and copper(I) iodide (0.27 mmol). The mixture was
cooled and treated with Cs2CO3 (0.38 mmol) and acetamide (3.1
mmol). The tube was sealed, and the mixture was stirred extremely
vigorously and heated at 112 °C for 16 h. The reaction mixture was
allowed to cool and was diluted with CHCl3 (20 mL). The organic
phase was washed with NH4OH (3 ꢀ 10 mL, 10%). The combined
aqueous layer was extracted with 20 mL of EtOAc. The crude
product was chromatographed on silica gel using DCM/MeOH
(10:1) to give to give 57 (16 mg, 25% yield). 1H NMR (400 MHz,
CDCl3): δ 8.56 (s, 1H), 8.30-8.02 (m, 3H), 7.82 (s, 1H), 7.69 (s,
1H), 7.60 (d, J = 6.9 Hz, 1H), 7.24 (d, J = 7.9 Hz, 1H), 7.05 (dt,
J= 20.8, 7.3 Hz, 3H), 6.92-6.72 (m, 1H), 6.56 (s, 1H), 3.81 (s, 3H),
2.03 (s, 3H). SFC-MS (APCIþ), M þ H found 386.1.
(3-Amino-5-(1H-indol-4-yl)phenyl)(pyridin-3-yl)methanone (50).
Benzyl 3-nicotinoyl-5-(1-(triisopropylsilyl)-1H-indol-4-yl)phenyl-
carbamate was prepared by Suzuki coupling of 48a with 21b using
general procedure A to give 49a in 74% yield. The Suzuki product
49a (40 mg, 0.066 mmol) in 40% KOH in MeOH/H2O (5 mL/5
mL) was brought to reflux for 2 h. After cooling to room
temperature, the mixture was diluted with water and extracted with
EtOAc. The organic layer was separated, dried, and concentrated.
Purification on silica gel afforded 50 (11.9 mg, 57%) as a yellow
solid. 1H NMR (400 MHz, CDCl3):δ9.01 (d, J= 1.6 Hz, 1H), 8.71
(dd, J = 4.8, 1.7 Hz, 1H), 8.29 (s, 1H), 8.09 (dt, J = 4.9, 2.6 Hz,
1H), 7.38-7.31 (m, 3H), 7.20-7.15 (m, 4H), 7.11-7.07 (m, 2H),
6.64-6.60 (m, 1H). SFC-MS (APCIþ), M þ H found 314.2.
[3-(1H-Indol-4-yl)-5-(pyridine-3-carbonyl)phenyl]carbamic Acid
Methyl Ester (51). In a pressure tube 48c (316 mg, 0.94 mmol),
21b (0.377 mg, 0.94 mmol), and 22 (53 mg, 0.094 mmol) were
dissolved in 1,4-dioxane (6 mL) under nitrogen. Aqueous 2 M
K3PO4 (1 mL, 1.2 mmol) was added, and the mixture was heated at
reflux overnight. The reaction mixture was filtered through Celite,
and the solvent was removed in vacuo. The product was chroma-
tographed to give 49c (0.350 g, 70% yield).
N-[3-Bromo-5-(2-chloropyridine-4-carbonyl)phenyl]methane-
sulfonamide. To a microwave tube was added 47d (0.250 g,
0.663 mmol), methanesulfonamide (0.063 g, 0.66 mmol), Cs2CO3
(0.304 g, 0.93 mmol), XantPhos (Strem, 0.007 g, 0.013 mmol), and
anhydrous 1,4-dioxane (5.0 mL). The reaction mixture was flushed
with argon. Then Pd(PPh3)4 (0.015 g, 0.013 mmol) was added. The
vial was capped and heated at 110 °C for 18 h. The mixture was
cooled, filtered through Celite, and eluted with EtOAc, and the
filtrate was concentrated in vacuo. The crude product was redis-
solved in EtOAc, and the organic phase was washed with water,
brine and dried over Na2SO4. The solution was filtered and
concentrated. The crude product was chromatographed on silica
gel using DCM/MeOH (10:1) to give N-[3-bromo-5-(2-chloro-
pyridine-4-carbonyl)phenyl]methanesulfonamide (0.168 mg, 65%
yield). 1H NMR (400 MHz, CDCl3): δ 8.61 (d, J = 5.0 Hz, 1H),
7.67 (dd, J = 13.6, 1.5 Hz, 2H), 7.59 (s, 1H), 7.50 (dd, J = 19.3, 3.2
Hz, 2H), 3.10 (s, 3H).
To a stirred solution of 49c (0.285 g, 0.54 mmol) in THF (10 mL)
was added 1 M TBAF in THF (1.0 mL). The mixture was stirred for
10 min, poured into water, and was extracted with EtOAc. The
organic phase was washed with saturated NaHCO3, brine and dried
over Na2SO4. The volatiles were removed under reduced pressure
and the crude product was chromatographed using methanol/chloro-
1
form (1-5%) to give 51 in 78.1% yield. H NMR (400 MHz,
CD3OD): δ 9.08 (s, 1H), 8.79 (d, J = 3.9 Hz, 1H), 8.43 (s, 1H), 8.16
(d,J= 7.8 Hz, 1H), 8.08 (s, 1H), 7.81 (s, 1H), 7.78 (s, 1H), 7.38-7.46
(m, 2H), 7.22-7.27 (m, 2H), 7.19 (d, J = 7.0 Hz, 1H), 7.01 (s, 1H),
6.72 (s, 1H), 3.79 (s, 3H). SFC-MS (APCIþ), M þ H found 372.3.
N-[3-(1H-Indol-4-yl)-5-(pyridine-3-carbonyl)phenyl]acetamide
(52). Compound 52 was synthesized from compound 48d in a

196 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shetty et al.
N-[3-Bromo-5-(2-methoxypyridine-4-carbonyl)phenyl]methane-
sulfonamide. To a mixture of N-[3-bromo-5-(2-chloropyridine-4-
carbonyl)phenyl]methanesulfonamide (0.080 g, 0.21 mmol) and
anhydrous MeOH (10.0mL) in a pressure tubewas added sodium
(0.007 g, 0.21 mmol). The tube was sealed, and the mixture was
heated at 80 °C for 18 h. The methanol was removed under vacuum,
and water was added to the reaction mixture. The aqueous phase
was extracted with EtOAc. The EtOAc layer was washed with
water, brine and dried over Na2SO4. The solution was filtered and
concentrated to give 0.035 g of N-[3-bromo-5-(2-methoxy-pyridine-
4-carbonyl)phenyl]methanesulfonamide. 1H NMR (400 MHz, CD-
Cl3): δ 8.34 (d, J = 5.2 Hz, 1H), 7.68 (s, 2H), 7.52 (d, J = 1.5 Hz,
1H), 7.12 (d, J = 5.2 Hz, 1H), 6.96 (s, 1H), 6.68 (s, 1H), 3.99 (s, 3H),
3.09 (s, 3H).
N-[3-(1H-Indol-4-yl)-5-(2-methoxypyridine-4-carbonyl)phenyl]-
methanesulfonamide (58). Compound 58 was synthesized from
compound N-[3-bromo-5-(2-methoxypyridine-4-carbonyl)phe-
nyl]methanesulfonamide in a similar manner as described for the
preparation of 51 in 73% yield. 1H NMR (400 MHz, acetone-d6):
δ 10.49 (s, 1H), 8.96 (s, 1H), 8.38 (d, J = 5.1 Hz, 1H), 8.07 (t, J =
1.9 Hz, 1H), 7.90 (t, J = 1.6 Hz, 1H), 7.83 (t, J = 1.9 Hz, 1H), 7.51
(d, J = 7.8 Hz, 1H), 7.45 (t, J = 2.8 Hz, 1H), 7.30 (dd, J = 5.1, 1.3
Hz, 1H), 7.26-7.18 (m, 2H), 7.10 (s, 1H), 6.75-6.72 (m, 1H), 3.96
(s, 3H), 3.15 (s, 3H). HRMS calcd for C22H19N3O4S, 421.1096,
found 421.1093.
Methyl 3-Bromo-5-(2-morpholinoisonicotinoyl)phenylcarba-
mate. (3,5-Dibromophenyl)-(2-morpholin-4-ylpyridin-4-yl)metha-
none 47e was converted to methyl 3-bromo-5-(2-morpholinoisoni-
cotinoyl))phenylcarbamate using a similar procedure as for the
preparation of N-[3-bromo-5-(2-chloropyridine-4-carbonyl)phe-
nyl]methanesulfonamide using methyl carbamate in place of metha-
nesulfonamide in 56% yield. 1H NMR (400 MHz, CDCl3): δ 8.33
(d, J = 5.2 Hz, 1H), 7.94 (s, 1H), 7.70 (s, 1H), 7.62 (s, 1H), 6.93 (s,
1H), 6.85 (d, J = 4.8 Hz, 1H), 6.81-6.73 (m, 1H), 3.88-3.80 (m,
4H), 3.78 (s, 3H), 3.62 (bs, 4H).
[3-(1H-Indol-4-yl)-5-(2-morpholin-4-ylpyridine-4-carbonyl)-
phenyl]carbamic Acid Methyl Ester (59). Compound 59 was
synthesized from methyl 3-bromo-5-(2-morpholinoisonicoti-
noyl))phenylcarbamate in a similar manner as described for
the preparation of 51 in 4% yield. 1H NMR (400 MHz, CDCl3):
δ 8.28 (d, J = 5.3 Hz, 1H), 8.07 (t, J = 1.8 Hz, 1H), 7.95 (s, 1H),
7.76 (t, J = 1.6 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.29 (d, J =
3.3 Hz, 1H), 7.18 (t, J = 7.7 Hz, 1H), 7.10 (d, J = 7.4 Hz, 1H),
7.08 (s, 1H), 6.95 (dd, J = 5.1, 1.0 Hz, 1H), 6.62 (d, J = 3.1 Hz,
1H), 3.78 (t, J = 4.9 Hz, 4H), 3.75 (s, 3H), 3.54 (t, J = 4.9 Hz,
4H). SFC-MS (APCIþ), M þ H found 457.5.
was diluted with water and extracted with EtOAc. The organic
phase was washed with water, brine, dried over Na2SO4, filtered,
and concentrated. The crude product was chromatographed on
silica gel using DCM/MeOH (10:0.3) to give 60 (20 mg, 55%
yield). 1H NMR (400 MHz, acetone-d6): δ 10.12 (s, 1H), 8.75 (s,
1H), 8.26 (s, 1H), 7.99 (s, 1H), 7.78 (t, J = 1.5 Hz, 1H), 7.64 (s,
1H), 7.50 (d, J = 6.1 Hz, 2H), 7.45 (d, J = 7.8 Hz, 1H), 7.34 (t,
J = 2.8 Hz, 1H), 7.24-7.16 (m, 2H), 6.74-6.71 (m, 1H), 3.77 (s,
3H), 2.63 (s, 3H). SFC-MS (APCIþ), M þ H found 420.2.
Methyl 3-(2-Chloro-6-methylisonicotinoyl)-5-(1H-indol-4-yl)-
phenylcarbamate (61). To methyl 3-(2-chloro-6-methylisonico-
tinoyl)-5-(1-(triisopropylsilyl)-1H-indol-4-yl)phenylcarbamate
(0.100 g, 0.174 mmol) and anhydrous 1,4-dioxane (2.0 mL) in a
pressure tube were added K2CO3 (0.034 g, 0.24 mmol) and
methylboronic acid (0.015 g, 0.24 mmol). The reaction mixture
was degassed with argon, and then Pd(PPh3)4 (0.006 g, 0.005
mmol) was added. The pressure tube was capped and heated at
110 °C for 18 h. The reaction mixture was diluted with water and
extracted with EtOAc. The organic phase was washed with brine
and dried over Na2SO4. The solution was filtered, concentrated,
and the crude product was chromatographed on silica gel using
DCM/MeOH (10:1) to give methyl 3-(2,6-dimethylisonico-
tinoyl)-5-(1-(triisopropylsilyl)-1H-indol-4-yl)phenylcarbamate
(0.060 g, 60% yield). The silyl group was cleaved using 1 M
TBAF to give 61 in 63% yield. 1H NMR (400 MHz, CDCl3): δ
8.50 (s, 1H), 8.12 (s, 1H), 7.79-7.75 (m, 2H), 7.40 (d, J = 8.2 Hz,
1H), 7.29 (s, 2H), 7.27-7.22 (m, 2H), 7.17 (dd, J = 7.3, 0.9 Hz,
1H), 7.05 (s, 1H), 6.72 (s, 1H), 3.79 (s, 3H), 2.60 (s, 6H). SFC-MS
(APCIþ), M þ 1 found 400.8.
(3-Bromo-5-(1H-indol-6-yl)phenyl)(2-chloro-6-methylpyridin-
4-yl)methanone. In a pressure tube 47f (425 mg, 1.09 mmol) and
1H-indol-4-ylboronic acid (0.193 g, 1.2 mmol) were suspended
in toluene (5 mL), EtOH (3 mL), H2O (1 mL), and Na2CO3
(347 mg, 3.2 mmol). The solution was degassed with a stream of
nitrogen for 3 min followed by addition of Pd(PPh3)4 (0.063 g,
0.055 mmol) and heated at 70 °C for 16 h. The mixture was
filtered through Celite and washed with CHCl3. The filtrate was
concentrated, extracted with EtOAc, and washed with water.
The organic layer was dried over Na2SO4, concentrated, loaded
on a silica gel column, and eluted with EtOAc/hexane (1:3) to
give of (3-bromo-5-(1H-indol-6-yl)phenyl)(2-chloro-6-methyl-
pyridin-4-yl)methanone (250 mg, 54% yield). 1H NMR (400
MHz, CDCl3): δ 8.37 (s, 1H), 8.12 (d, J = 1.6 Hz, 1H), 7.99 (s,
1H), 7.94 (d, J = 1.6 Hz, 1H), 7.47 (t, J = 4.0 Hz, 2H), 7.39 (s,
1H), 7.31 (dd, J = 8.8, 5.4 Hz, 2H), 7.17 (d, J = 7.2 Hz, 1H), 6.66
(s, 1H), 2.64 (s, 3H).
(3-Bromo-5-(1H-indol-6-yl)phenyl)(2-methyl-6-morpholinopyri-
din-4-yl)methanone. To the (3-bromo-5-(1H-indol-6-yl)phenyl)(2-
chloro-6-methylpyridin-4-yl)methanone (0.1 g, 0.23 mmol) and
1,4-dioxane (1.0 mL) in a pressure tube was added the morpholine
(1.0 mmol), and the reaction mixture was subjected to microwave
at 120 °C for 3 h. The reaction mixture was cooled and concen-
trated. The crude product was chromatographed on silica gel
using hexane/EtOAc (1:1) to give (3-bromo-5-(1H-indol-6-yl)phe-
nyl)(2-methyl-6-morpholinopyridin-4-yl)methanone (45 mg, 40%
yield). 1H NMR (400 MHz, CDCl3): δ 8.51 (s, 1H), 7.93 (s, 3H),
7.42 (d, J = 6.9 Hz, 1H), 7.13 (s, 2H), 6.67 (d, J = 29.0 Hz, 4H),
3.78 (s, 4H), 3.53 (s, 4H), 2.46 (s, 3H).
N-(3-(1H-Indol-4-yl)-5-(2-methyl-6-morpholinoisonicotinoyl)-
phenyl)acetamide (62). The (3-bromo-5-(1H-indol-6-yl)phenyl)-
(2-methyl-6-morpholinopyridin-4-yl)methanone (35 mg, 0.07
mmol) was dissolved in dioxane (2 mL), and the solution was
transferred to a pressure tube. The mixture was heated at 100 °C
and treated with N,N0-dimethylethylenediamine (0.35 mmol)
and copper(I) iodide (0.12 mmol). The mixture was cooled and
treated with Cs2CO3 (0.15 mmol) and acetamide (1.5 mmol).
The tube was sealed, and the mixture was stirred extremely
vigorously and heated at 112 °C for 16 h. The reaction mixture
was allowed to cool, was diluted with CHCl3 (20 mL), was
washed with 10% NH4OH (3 ꢀ 10 mL). The combined aqueous
Methyl 3-Bromo-5-(2-chloro-6-methylisonicotinoyl)phenylcar-
bamate. (2-Chloro-6-methylpyridin-4-yl)-(3,5-dibromophenyl)-
methanone (47f) was converted to methyl 3-bromo-5-(2-chloro-
6-methylisonicotinoyl)phenylcarbamate under the palladium
catalyzed conditions described for N-[3-bromo-5-(2-chloropyr-
idine-4-carbonyl)phenyl]methanesulfonamide using methyl car-
bamate in place of methanesulfonamide in 45.8% yield. 1H NMR
(400 MHz, acetone-d6): δ 9.06 (s, 1H), 8.18 (d, J = 1.6 Hz, 1H),
7.92 (d, J = 1.3 Hz, 1H), 7.61 (d, J = 1.4 Hz, 1H), 7.51 (d, J = 5.7
Hz, 2H), 3.72 (d, J = 8.9 Hz, 3H), 2.59 (s, 3H).
Methyl 3-(2-Chloro-6-methylisonicotinoyl)-5-(1H-indol-4-yl)-
phenylcarbamate (60). In a pressure tube methyl 3-bromo-5-
(2-chloro-6-methylisonicotinoyl)phenylcarbamate (140 mg, 0.365
mmol), 21b (0.146 g, 0.365 mmol), and 22 (53 mg, 0.094 mmol)
were dissolved in dioxane (6 mL) under nitrogen. Aqueous 2 M
K3PO4 (0.18 mL) was added, and the mixture was heated at reflux
for 16 h. The reaction mixture was filtered through Celite, and the
solvent was removed under vacuum. The product was chromato-
graphed on silica gel using hexane/EtOAc (1:1) to give methyl 3-(2-
chloro-6-methylisonicotinoyl)-5-(1-(triisopropylsilyl)-1H-indol-4-yl)-
phenylcarbamate (0.120 g, 57% yield).
To the Suzuki product (0.05 g, 0.087 mmol) in THF (2.5 mL)
was added of 1 M TBAF (0.17 mL), and the reaction mixture
was stirred at room temperature for 3 h. The reaction mixture

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 197
layers were extracted with EtOAc (20 mL). The combined organic
phases were dried over Na2SO4, filtered, and concentrated. The
crude product was purified by preparative SFC to give 62 in (0.019
g, 57%). 1H NMR (400 MHz, CDCl3): δ 8.42 (s, 1H), 8.24 (s, 1H),
7.84 (s, 2H), 7.59 (s, 1H), 7.40 (d, J = 7.9 Hz, 1H), 7.22 (d, J = 8.4
Hz, 1H), 7.16 (d, J = 7.2 Hz, 1H), 6.76 (d, J = 19.6 Hz, 3H), 3.79
(d, J = 4.3 Hz, 4H), 3.55 (d, J = 4.3 Hz, 4H), 2.46 (s, 3H), 2.19
(s, 3H). SFC-MS (APCIþ), M þ H found 455.4.
To a stirred solution of 3-bromo-5-iodobenzoylazide (5.0 g,
14.0 mmol) in toluene (50 mL) was added tert-butanol (1.5 mL,
15.6 mmol). The reaction mixture was heated to reflux for 2 h,
cooled to room temperature, and evaporated to dryness to afford
5.6 g (99% yield) of (3-bromo-5-iodophenyl)carbamic acid tert-
butyl ester. The ester (5.6 g, 14 mmol) was dissolved in 1 M HCl in
dioxane (35 mL, 141 mmol), stirred at room temperature for 12 h,
and then cooled to 0 °C. Concentrated NaOH (50%) was added
to the reaction mixture to give a pH of 14. Then water (100 mL)
was added. The aqueous layer was extracted thrice with EtOAc
(20 mL). The organic layers were combined, dried over MgSO4,
filtered, and evaporated to dryness. The crude product was purified
by silica gel chromatography to give 70 (2.8 g, 67%). 1HNMR(400
MHz, CDCl3): δ 7.19 (m, 1H), 6.93 (m, 1H), 6.75 (m, 1H), 3.70 (bs,
2H). SFC-MS (APCIþ), M þ H found 297.9.
2-(3,5-Dibromophenyl)benzo[d]oxazole (65a). A mixture of
3,5-dibromobenzoic acid 63 (5.0 g, 17.3 mmol) and 2-amino-
phenol 64a (1.91 g, 17.3 mmol) in polyphosporic acid (50 g) was
heated at 185 °C for 16 h. The mixture was cooled, diluted with
water, and then extracted with EtOAc. The organic layer was
separated, and the aqueous layer was extracted twice with
EtOAc. The combined organic phase was dried over MgSO4,
filtered and the residue purified by flash chromatography (silica
gel, hexanes/EtOAc, 1:1) to give 65a (5.73 g, 94%) as a yellow
solid. 1H NMR (400 MHz, CDCl3): δ 8.32 (d, J = 2.0 Hz, 2H),
7.79 (t, J = 2.4 Hz, 1H), 7.78-7.75 (m, 1H), 7.59-7.56 (m, 1H),
7.41-7.35 (2H). SFC-MS (APCIþ), M þ H found 353.9.
N-(3-(Benzo[d]oxazol-2-yl)-5-bromophenyl)acetamide (66a).
To a pressure tube was added 65a (0.700 g, 2.0 mmol), acetamide
(0.10 g, 2.0 mmol), Cs2CO3 (1.0 g, 4.0 mmol), XantPhos (Strem,
0.08 g, 0.1 mmol), and anhydrous 1,4-dioxane (10.0 mL). The
tube was flushed with argon, and then Pd2(dba)3 (0.1 g, 0.1
mmol) was added. The tube was capped and heated at 110 °C
and monitored by SFC-MS. After 18 h, the mixture was cooled,
filtered through Celite and the filter cake washed with EtOAc.
The volatiles were removed in vacuo, and the crude product was
redissolved in EtOAc. The organic phase was washed with
water, brine and dried over Na2SO4. The solution was filtered,
concentrated and the crude product was chromatographed on
silica gel using DCM/MeOH (10:1) to give 66a (0.281 mg, 40%).
1H NMR (400 MHz, CD3OD): δ 8.10 (d, J = 2.0 Hz, 2H), 7.98
(t, J = 2.0 Hz, 1H), 7.63 (m, 1H), 7.53 (m, 1H), 7.34-7.28 (2H),
2.09 (s, 3H). SFC-MS (APCIþ), M þ H found 331.1.
N-(3-Bromo-5-iodophenyl)methanesulfonamide (71a). To a stirred
solution of 3-bromo-5-iodoaniline 70 (1.0 g, 3.4 mmol) and pyridine
(1.1 mL, 13.0 mmol) in CH2Cl2 (20 mL) at 0 °C was added methyl-
sulfonyl chloride (0.29 mL, 3.7 mmol). The reaction mixture was
allowed to warm to room temperature and was stirred for 12 h. The
reaction mixture was washed twice with 1 M HCl (5 mL) and brine
(5 mL). The organic layer was dried over Na2SO4, filtered, and
concentrated to give 71a (1.23 g, 97%). 1H NMR (400 MHz,
CDCl3): δ 7.66 (m, 1H), 7.47 (m, 1H), 7.35 (m, 1H), 6.52 (bs, 1H),
3.05 (s, 3H).
N-(3-Bromo-5-iodophenyl)acetamide (71b). To a stirred solu-
tion of 3-bromo-5-iodoaniline 70 (0.60 g, 2.01 mmol) and
pyridine (0.65 mL, 8.06 mmol) in CH2Cl2 (15 mL) at 0 °C was
added acetic anhydride (0.17 mL, 2.22 mmol). The reaction
mixture was allowed to warm to room temperature and was
stirred for 12 h. The reaction mixture was washed twice with 1 M
HCl (5 mL) and brine (5 mL). The organic layer was dried over
Na2SO4 and concentrated to give 71b (0.71 g, 99%). 1H NMR
(400 MHz, CDCl3): δ 7.76 (t, J = 1.6 Hz, 1H), 7.64 (m, 1H), 7.42
(t, J = 1.6 Hz, 1H), 1.99 (s, 3H).
N-(3-Bromo-5-(pyridin-4-yl)phenyl)methanesulfonamide. To a
sealed tube were added N-(3-bromo-5-iodophenyl)methanesul-
fonamide 71a (2.0 g, 5.30 mmol), pyridin-4-ylboronic acid
(0.92 g, 7.4 mmol), 2 M aqueous Na2CO3 (8.0 mL, 16 mmol),
and 1,4-dioxane (60 mL). A stream of nitrogen was passed
N-(3-(Benzo[d]oxazol-2-yl)-5-(1H-indol-4-yl)phenyl)acetamide
(67). N-(3-(Benzo[d]oxazol-2-yl)-5-bromophenyl)acetamide 66a
was converted to N-(3-(benzo[d]oxazol-2-yl)-5-(1H-indol-4-yl)-
phenyl)acetamide 67 by the method described for the synthesis
of compound 60 in 60.5% yield. 1H NMR (400 MHz, DMSO-d6):
δ 11.27 (s, 1H), 10.26 (s, 1H), 8.50 (s, 1H), 8.07 (s, 2H), 7.77-7.74
(2H), 7.41 (m, 1H), 7.39 (s, 1H), 7.38-7.33 (2H), 7.18-7.12 (2H),
6.59 (s, 1H), 2.06 (s, 3H). SFC-MS (APCIþ), M þ H found 368.6.
2-(3,5-Dibromophenyl)oxazolo[4,5-b]pyridine (65b). Compound
65b was prepared from 3,5-dibromobenzoic acid 63 and 2-amino-
pyridin-3-ol 64b in a similar manner as described for the prepara-
through the mixture for 30 min. Then PdCl2(dppf) CH2Cl2
3
(440 mg, 0.53 mmol) was added, and the tube was sealed and
heated to 60 °C for 16 h. The reaction mixture was filtered
through Celite, eluting with EtOAc. The filtrate was concen-
trated, and the residue was purified by silica gel column chro-
matography to give the desired product as an off white solid
1
(1.5 g, 86%). H NMR (400 MHz, CD3OD): δ 8.59 (m, 2H),
1
7.67-7.64 (3H), 7.53 (m, 2H), 3.03 (s, 3H).
tion of 65a in 47% yield. H NMR (400 MHz, CDCl3): δ 8.57
N-[3-(1H-Indol-4-yl)-5-pyridin-4-ylphenyl]methanesulfonamide
(72). To a sealed tube were added N-(3-bromo-5-(pyridin-4-
yl)phenyl)methanesulfonamide (400 g, 1.22 mmol), 1H-indol-4-
ylboronic acid (0.2 g, 1.22 mmol), 2 M aqueous Na2CO3 (2 mL,
4 mmol), toluene (4 mL), and EtOH (5 mL). A stream of nitrogen
was passed through the mixture for 3 min. Then Pd(PPh3)4 (100
mg, 0.12 mmol) was added, and the tube was sealed and heated to
110 °C for 24 h. The reaction mixture was filtered through Celite
and eluted with EtOAc. The filtrate was concentrated, and the
residue was purified by silica gel chromatography to provide 72
(0.24 g, 54%). 1H NMR (400 MHz, CD3OD): δ 8.60 (dd, J = 4.8,
1.6 Hz, 2H), 7.75 (dd, J = 4.5, 1.7 Hz, 2H), 7.72 (dt, J = 5.2, 2.6
Hz, 2H), 7.57 (t, J = 1.9 Hz, 1H), 7.42 (d, J = 7.2 Hz, 1H), 7.32
(d, J = 3.1 Hz, 1H), 7.23-7.16 (m, 2H), 6.67 (d, J = 3.1 Hz, 1H),
3.04 (s, 3H). SFC-MS (APCIþ), M þ H found 364.4.
(m, 1H), 8.33 (m, 2H), 7.84-7.80 (2H), 7.29 (m, 1H). SFC-MS
(APCIþ), M þ H found 352.9.
Methyl 3-Bromo-5-(oxazolo[4,5-b]pyridin-2-yl)phenylcarba-
mate (66b). Compound 66b was synthesized from compound
65b and methylcarbamate in a similar manner as described for
the preparation of 66a in 40% yield. SFC-MS (APCIþ), M þ H
found 348.1.
Methyl 3-(1H-Indol-4-yl)-5-(oxazolo[4,5-b]pyridin-2-yl)phenyl-
carbamate (68). Methyl 3-bromo-5-(oxazolo[4,5-b]pyridin-2-yl)-
phenylcarbamate 66b was converted to 68 by the method des-
cribed for the synthesis of compound 60 in 31.8% yield. 1H NMR
(400 MHz, DMSO-d6): δ 11.34 (s, 1H), 10.09 (s, 1H), 8.54 (dd, J =
5.2, 1.2 Hz, 1H), 8.43 (s, 1H), 8.26 (dd, J = 8.0, 1.2 Hz, 1H), 8.13
(d, J = 1.2 Hz, 1H), 8.11 (s, 1H), 7.48-7.45 (3H), 7.23-7.16 (2H),
6.65(m, 1H), 3.72 (s, 3H). SFC-MS(APCIþ), Mþ H found 384.4.
3-Bromo-5-iodoaniline (70). To a stirred solution of 3-bromo-
5-iodobenzoic acid 69 (5.0 g, 15 mmol) in methanol (30 mL) was
added DIEA (2.9 mL, 16.8 mmol) and diphenylphosphorylazide
(3.6 mL, 16.8 mmol). The reaction mixture was stirred for 12 h at
room temperature and then quenched by addition of water
(100 mL). The solid precipitate was filtered, washed with water,
and dried to give 3-bromo-5-iodobenzoylazide (5.0 g, 96%).
N-(3-Bromo-5-(6-methoxypyridin-3-yl)phenyl)acetamide. N-(3-
Bromo-5-(6-methoxypyridin-3-yl)phenyl)acetamide was synthe-
sized from 71b and 6-methoxypyridin-3-ylboronic acid in a similar
manner as described for the synthesis of N-(3-bromo-5-(pyridin-4-
yl)phenyl)methanesulfonamide in 87% yield. 1H NMR (400 MHz,
CDCl3): δ 8.25 (s, J=2.4 Hz, 1H), 8.09 (bs, 1H), 7.69 (s, 1H), 7.65
(dd, J=8.8, 2.4 Hz, 1H), 7.55 (s, 1H), 7.31 (s, 1H), 6.74 (s, J=8.8 Hz,

198 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Shetty et al.
1H), 3.93 (s, 3H), 2.16 (s, 3H). SFC-MS (APCIþ), M þ H found
321.2.
reagent was included in the culture well for a period of 1-4 h.
The WST-1 reaction was read on a multiwell spectrophotometer
as the difference in the absorbance between 450 and 600 nm
(baseline). Absorbance was proportional to the number of living
cells in the culture well. Concentration response curves and IC50
values were calculated using GraphPAD Prism graphing and
curve fitting program.
Cell Cycle Assay. MCF-7, Jurkat, U266, SW620, NCI-H522,
NCI-H23, BxPC-3, and PC3 cell lines were purchased from
American Type Culture Collection. Cells were maintained at
37 °C and 5% CO2 in MEM (MCF-7), RPMI-1640 (Jurkat,
U266, NCI-H522, NCI-H23, BxPC-3), L-15 (SW620), or F-12K
(PC3). All cell culture media were supplemented with 10% (v/v)
fetal bovine serum (FBS).
Cells were plated in 24-well plates at a volume of 1 mL/well at
a density of 2 ꢀ 105 cells/mL and allowed to incubate overnight.
FBS concentration was reduced to 0.5%, and cells were incubated
for an additional 30 min. Following serum reduction, cells were
incubated with various concentrations of test compound prepared
in culture media containing 0.5% FBS from a 10 mM DMSO stock.
After 24 h, cells were harvested for FACS analysis. Cells were fixed
in 80% ethanol for 30 min at room temperature, and the nuclear
DNA was stained with a solution of propidium iodide (PI)/RNase
staining buffer. FACS analysis was done on a FACScalibur system
with approximately 2000 events counted per condition. G1 and G2/
M cell populations were measured and reported as a percentage of
the total cell count.
Microtubule Polymerization Assay. The in vitro tubulin poly-
merization assay was conducted using tubulin polymerization
assay kit (Cytoskeleton, Denver, CO) according to manufac-
turer recommendations. Colchicine 1 (95% pure) and vincris-
tine (98% pure) were purchased from Sigma (St. Louis, MO).
The final DMSO concentration in each assay well was 1%, and
the final concentration series of each experimental tubulin
inhibitor was 10, 5, 2.5, 1.25, and 0.6 μM.
N-[3-(1H-Indol-4-yl)-5-(6-methoxypyridin-3-yl)phenyl]acetamide
(73). In a pressure tube N-(3-bromo-5-(6-methoxypyridin-3-yl)phe-
nyl)acetamide (206 mg, 0.641 mmol), 21b (0.384 g, 0.962 mmol),
and 22 (35 mg, 0.064 mmol) were dissolved in dioxane (7 mL) under
nitrogen. Aqueous 2 M K3PO4 (0.64 mL) was added, and the
mixture was heated at reflux for 16 h. The reaction mixture was
filtered through Celite and the solvent concentrated in vacuo. The
product was chromatographed on silica gel using hexane/EtOAc
(1:1) to give 0.298 g (90%) of coupled product. To the TIPS
protected product (0.298 g, 0.087 mmol) in THF (6.0 mL) was
added 1 M TBAF (0.64 mL), and the mixture was stirred at room
temperature for 3 h. The reaction mixture was diluted with water
and extracted with EtOAc. The EtOAc layer was washed with
water, brine, dried over Na2SO4, filtered, and concentrated. The
crude was chromatographed on silica gel using 60% EtOAc in
hexane to give 73 (132 mg, 63.7%). 1H NMR (400 MHz, CD3OD):
δ 8.31 (d, J = 2.5 Hz, 1H), 7.83 (t, J = 1.7 Hz, 1H), 7.75 (dd, J =
8.7, 2.6 Hz, 1H), 7.69 (t, J = 1.9 Hz, 1H), 7.47 (t, J = 1.5 Hz, 1H),
7.35 (d, J = 7.8 Hz, 1H), 7.22 (d, J = 3.1 Hz, 1H), 7.14-7.06 (m,
2H), 6.67 (d, J= 8.6 Hz, 1H), 6.65 (d, J =3.1Hz, 1H), 3.82(s, 3H),
2.09 (s, 3H). SFC-MS (APCIþ), M þ H found 358.4.
N-(5-Bromo-30,40-difluorobiphenyl-3-yl)acetamide. To a pres-
sure tube were added N-(3-bromo-5-iodophenyl)acetamide 71b
(2.5 g, 7.35 mmol), 3,4-difluorophenylboronic acid (1.22 g, 7.73
mmol), 2 M aqueous Na2CO3 (11 mL, 22 mmol), and dioxane
(45 mL). A stream of nitrogen was passed through the mixture
for 15 min. Then PdCl2(dppf) CH2Cl2 (600 mg, 0.735 mmol)
3
was added, and the tube was sealed and heated to 60 °C for 16 h.
The reaction mixture was filtered through Celite, eluting with
EtOAc. The filtrate was concentrated to a brown sludge. The
residue was taken up in EtOAc, washed with H2O, brine, dried
over MgSO4, filtered, and concentrated. The crude product was
purified by column chromatography (3:1 hexanes/EtOAc) to
1
Microtubule polymerization assays were performed using a
Spectra Max Gemini XS microplate reader (Molecular Devices
Corporation, Sunnyvale, CA) in a 96-well plate format. Tubulin
polymerization was detected by measuring the absorbance (OD)
of the solution at 340 nm once every minute over 60 min during
the incubation at 37 °C.
Animals. Treatment of animals for in vivo studies was in
accordance with study protocol and in adherence to regulations
outlined in the USDA Animal Welfare Act (9CFR Parts 1, 2,
and 3) and the conditions specified in the Guide for the Care and
Use of Laboratory Animals (LAR Publication, 1996, National
Academy Press).
give the desired product as a tan solid (88%). H NMR (400
MHz, CDCl3): δ 7.67 (m, 2H), 7.37-7.15 (4H), 2.20 (s, 3H).
N-(30,40-Difluoro-5-(1H-indol-4-yl)biphenyl-3-yl)acetamide (74).
Compound 74 was prepared from N-(5-bromo-30,40-difluorobiphe-
nyl-3-yl)acetamide in a similar manner as described for the prepara-
tion of 72 in 79% yield. 1H NMR (400 MHz, CD3OD): δ 7.87 (t,
J = 1.6 Hz, 1H), 7.79 (t, J = 1.6 Hz, 1H), 7.57 (t, J = 1.6 Hz, 1H),
7.56-7.51 (m, 1H), 7.46-7.42 (m, 1H), 7.40-7.37 (m, 1H),
7.34-7.27 (m, 1H), 7.28 (d, J = 3.2 Hz, 1H), 7.17 (t, J = 7.2 Hz,
1H), 7.13 (dd, J = 7.2, 1.6 Hz, 1H), 6.66 (dd, J = 3.2, 0.8 Hz, 1H),
2.16 (s, 3H). SFC-MS (APCIþ), M þ H found 363.4.
Computational Model. The protein from the tubulin cocrystal
X-ray structure PDB code 1SA0,9 resolved to 3.5 A, was
ꢀ
˚
In Vivo Pharmacokinetic Study. Sprague-Dawley rats were
used to determine oral bioavailability and PK parameters of the
free base form of 58 following oral and intravenous administra-
tion. Plasma concentration of 58 was determined by LC/MS/MS
analysis, and PK parameters were calculated using a noncompart-
mental model. A solution of 58 in polyethylene gycol 400 (PEG) at
1.33 mg/mL was prepared. One volume of 100 mM citrate buffer,
pH 3, was added to 3 volumes of 1.33 mg/mL solution of 58 in
PEG to generate a final concentration of 1 mg/mL. The solution
formulation of 58 was administered separately by bolus injection
into a lateral tail vein and by oral gavage. All animals were surgi-
cally implanted with a catheter in a jugular vein using polyure-
thane tubing and housed individually. Blood sample was collected
from four animals in each treatment group at 0 (predose), 0.0167,
0.083, 0.25, 0.5, 1, 2, 4, and 8 h for iv and 0 (predose), 0.083,
0.25, 0.5, 1, 2, 4, and 8 h for po. Calibration standards with 58
concentrations from 0.005 to 10 μg/mL were prepared by serial
dilution in naive rat plasma. A portion (60 μL) of each calibra-
tion standard and experimental sample was combined with two
volumes (120 μL) of acetonitrile containing an analytical internal
standard. The mixture was centrifuged at 2000g at 4 °C for 30 min
to extract the organic components. The supernatants containing
the organic component for each sample were used for analysis.
prepared by protonating histidines and correcting serine, tyrosine,
and threonine rotamers using an in-house algorithm.34 The structure
was then relaxed with 25-50 cycles of minimization using Macro-
Model.36 A set of fragments, which included indole, acetophenone,
2-methoxypyridine, phenol, 3-aminopyridine, acetanilide, 1,2-di-
fluorobenzene, benzaldehyde, 2,6-dimethylpyridine, and N-methyl-
sulfonamide, was simulated against the protein using the grand
canonical Monte Carlo simulation approach implemented using in-
house developed software.34 The fragment interaction positions
were rank-ordered by the calculated free energy values. The highest
affinity indole poses were identified, and the four inhibitors were
assembled by automatically linking fragments in their computed
positions, allowing some flexibility from standard bond lengths and
bond angles. The fragments were linked sequentially starting from
the distribution of the indole fragment.
Cell Viability Assay. Jurkat and HeLa cells were cultured in
RPMI and EMEM media, respectively, containing 0.5% fetal
calf serum. Cells were plated in a volume of 100 μL in a 96-well
format. Test compounds were included in the culture media at
concentrations ranging from 1 nM to 10 μM for a period of
48-72 h. Cell viability was determined using the WST-1 colori-
metric mitochondrial reduction assay. Briefly, 10 μL of WST-1

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 199
Recovery of 58 by this method was greater than 85%, and the
lower limit of quantitation was 0.005 μg/mL.
insight into the structural switch of tubulin. Proc. Natl. Acad. Sci.
U.S.A. 2009, 106, 13775–13779.
(12) Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani,
A. A. Medicinal chemistry of combretastatin A4: present and future
directions. J. Med. Chem. 2006, 49, 3033–3044.
In Vivo Antitumor Activity. Cultured NCI-H522 tumors
measuring 30-40 mm3 were implanted subcutaneously into
the thighs of athymic NCr-nu mice and allowed to grow to
approximately 150 mm3. Mice were assigned to groups by
matching median tumor volume that ranged from 120 to 162
mm3 in this study. The study lasted 28 days, and tumor was
measured every third day during treatment phase. The com-
pound was formulated as a free base, using 10 mg/mL 58 in
DMSO/PG/TW/PBS (5:15:2:18 by volume) and was adminis-
tered twice orally at 15 and 50 mg/kg. Vehicle was administered
as the untreated control. Animals with tumors exceeding 4000
mm3 or those exhibiting excessive morbidity were sacrificed
immediately. Tumor volume for each individual animal was
fitted to the exponential growth function using Prism software:
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Vt ¼ V0 eKt
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where Vt describes tumor volume at given time t, V0 is starting
tumor volume, and K is growth rate constant determined from
the fitting.
Acknowledgment. We thank Dr. Robert Schiksnis for
assistance with NMR studies. We thank Brandon Campbell
for performing preparative SFC purification and SFC-MS
studies.
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