Using 'biased-privileged' scaffolds to identify lysine methyltransferase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.02.024

Methylation of histones by lysine methyltransferases (KMTases) plays important roles in regulating chromatin function. It is also now clear that improper KMTases activity is linked to human diseases, such as cancer. We report an approach that employs drug

Full text of DOI:10.1016/j.bmc.2014.02.024

Bioorganic & Medicinal Chemistry 22 (2014) 2253–2260
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Using ‘biased-privileged’ scaffolds to identify lysine
methyltransferase inhibitors
a
a
a
a,
Sudhir Kashyap a,b, , Joel Sandler , Ulf Peters , Eduardo J. Martinez , Tarun M. Kapoor
⇑
⇑
a Laboratory of Chemistry and Cell Biology, Rockefeller University, New York, NY 10065, USA
b Organic & Biomolecular Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, AP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Methylation of histones by lysine methyltransferases (KMTases) plays important roles in regulating chro-
matin function. It is also now clear that improper KMTases activity is linked to human diseases, such as
cancer. We report an approach that employs drug-like ‘privileged’ scaffolds biased with motifs present in
S-adenosyl methionine, the cofactor used by KMTases, to efficiently generate inhibitors for Set7, a bio-
chemically well-characterized KMTase. Setin-1, the most potent inhibitor of Set7 we have developed also
inhibits the KMTase G9a. Together these data suggest that these inhibitors should provide good starting
points to generate useful probes for KMTase biology and guide the design of KMTase inhibitors with drug-
like properties.
Received 14 December 2013
Revised 10 February 2014
Accepted 10 February 2014
Available online 28 February 2014
Keywords:
Post-translational modifications
KMTases
Ó 2014 Elsevier Ltd. All rights reserved.
SET7
‘Privileged’ scaffolds
Inhibitors
1. Introduction
developing small molecule inhibitors of KMTases to probe their
functions and to properly validate these enzymes as targets for
Covalent and reversible post-translational modifications (PTMs)
of histones, proteins that assemble into octamers around which
DNA is ‘spooled’, play key roles in regulating gene expression in
eukaryotes.1–3 The histone-based structures, called nucleosomes,
are the basic building blocks of chromatin.4 In current models,
PTMs of histone, such as acetylation, methylation, phosphoryla-
tion, glycosylation, sumoylation or ubiquitination, modulate pro-
tein recruitment to nucleosomes and can regulate chromatin
organization.5–8 For example, methylation of lysine-4 at the N-ter-
minus of histone H3 recruits different proteins to chromatin and is
believed to ‘mark’ a transcriptionally active ‘on’-state of chroma-
tin.9,10 Like other dynamic PTMs, the level of histone lysine meth-
chemotherapy has become an important goal.
It has been estimated that there are over 50 lysine methyltrans-
ferases in humans.13,14 Currently, selective inhibitors for a handful
of KMTases (e.g., G9a and Dot1L) have been reported.15–17 How-
ever, when compared to other important enzyme targets (e.g.,
kinases),18 the chemical diversity of available KMTase inhibitors
and structural information on the modes of inhibition is restricted
to a few examples. Therefore, designing probes for different
KMTases remains challenging.
KMTases use S-adenosyl methionine (SAM) as the source of the
methyl group in the reaction they catalyze (Fig. 1A). Structural
analyses of KMTases have provided insight into how these en-
zymes bind this cofactor.19,20 Unlike kinases, in which the ATP’s
phosphate groups are polar and the key hydrophobic contacts are
made with the adenine, KMTases make numerous contacts with
most of the atoms in SAM. Consistent with these observations
SAM-related compounds, such as sinefungin and S-adenosylhomo-
cysteine, inhibit KMTases and have been useful in studies analyz-
ing their activities (Fig. 1B).21–25 In addition, systematic
modifications of SAM have led to the development of inhibitors
of KMTases.16,17 Encouraged by these findings, we developed a
strategy that uses features of SAM to develop drug-like inhibitors
for KMTases. We reasoned that ‘privileged’ chemical scaffolds,
which have provided good starting points for developing inhibitors
for different enzymes (e.g., kinases and myosins),26,27 may be
ylation is regulated by
a balance in the activity of lysine
methyltransferases (KMTases), which add the methyl group, and
demethylases, which reverse the modification.11 While important
advances have been made in identifying KMTases, our understand-
ing of the dynamic regulation and function of the different histone
methylations remains incomplete. In line with critical roles of
these enzymes in basic cellular processes, their dysfunction has
been linked to human diseases, such as cancer.12 Therefore,
⇑
Corresponding authors. Tel.: +91 402 716 1649; fax: +91 402 716 0387 (S.K.);
tel.: +1 212 327 8173; fax: +1 212 327 7358 (T.M.K.).
E-mail addresses: skashyap@iict.res.in (S. Kashyap), kapoor@rockefeller.edu
(T.M. Kapoor).
http://dx.doi.org/10.1016/j.bmc.2014.02.024
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2254
S. Kashyap et al. / Bioorg. Med. Chem. 22 (2014) 2253–2260
2. Results and discussion
A
Histone -Lys
Histone -Lys
We generated a handful of compounds in which homocysteine
KMTase
was appended to ‘privileged’ chemical scaffolds, which are com-
monly found in drugs (e.g., kinase inhibitors) (Fig. 2B). Briefly, N-
alkylation, palladium-catalyzed Suzuki coupling and copper cata-
lyzed Buchwald couplings were used to obtain compounds 5–9.28
We tested their activities against the KMTase, SET7, a well-stud-
ied member of this enzyme family. Recombinant SET7 (residues
52–366) was expressed in bacteria as a GST-fusion and purified
as previously described.20,29 Compounds were tested using an ELI-
SA assay in which biotin-conjugated histone H3 peptide (H31-20-
cys-biotin) was immobilized on multi-well plates and methylation
detected using an antibody that recognizes the monomethylated
lysine-4 of histone H3. This assay was based on the method re-
ported by Kubicek et al.15 Compound 7 was the only active com-
H(3-n)
NH3
HN
n = 1-3
AdoMet
AdoHcy
(CH3)n
NH2
N
NHR2
B
I
N
N
N
N
N
N
NH2
N
N
R1
O
O
R1
OH
OH OH
OH
1 Sinefungin
2
DOT1L Inhibitors
NH2
N
R2 = H, Me, Bn
R2HN
N
pound, revealing modest activity (ꢀ12% inhibition at 40
lM),
while all others tested were inactive (Fig. 3). Encouraged by these
N
N
N
R1
data, we generated analogs of 7.
O
NH2
As a first step, we focused on the homocysteine portion of com-
R1
=
HO2C
pound 7, with the goal to reduce the
a-amino acid character. We
OH OH
Set7/9 Inhibitors
2 = alkyl
3
R
generated -butyric acid and meta-benzoic acid substituted ver-
c
sions of 7 using a Buchwald coupling and substitution reactions
(Scheme 1A). We found that compounds 10 and 11 were more po-
tent inhibitors of SET7 than 7. As compound 11 was the most po-
tent, we retained the meta-benzoic acid moiety for subsequent
analysis. We next examined how changes in the substitutions of
the indole impacted activity. We found that Br-substitution at
the 4-position (12) of the indole increased potency and replacing
Figure 1. (A) Schematic for the KMTase catalyzed reaction. (B) SAM-based
inhibitors of KMTases.
coupled to homocysteine to yield broad specificity KMTase inhibi-
tors (Fig. 2A). In particular, the heterocycles (e.g., diaminopyrimi-
dine and indoles) common in many kinase inhibitors could
mimic the adenosine and a benzyl linkage to the homocysteine
could position the key functional groups in correct spacing and ori-
entations. Here we report the design, development and validation
of SETin-1, an inhibitor of histone KMTases based on ‘biased-
privileged’ scaffolds.
the Br with
a phenyl group (13) was even more effective
(Scheme 1B). We then examined whether the thioether functional-
ity could be replaced by a secondary amine (14), as it would reduce
molecular weight and lipophilicity. In addition, this replacement
would decrease the likelihood of inhibitor decomposition via oxi-
dation. Gratifyingly, compounds 13 and 14 had similar potencies,
yielding ꢀ50–60% inhibition of SET7 at 40
lM.
We next examined whether additional modifications of the 4-
phenyl appended to the indole in 14 improved potency. A carbox-
ylic acid at the para-position (15) of the phenyl ring greatly sup-
pressed activity, while a trifluoromethyl group (19) enhanced
efficacy. Other substitutions did not lead to further improvements
(Schemes 1C and 2A), making compound 19 the best compound in
this series. We then examined whether modification of the indole
moiety itself impacted the activity of compounds in this series. To
this end, we generated compounds 20–24 (Scheme 2B), in which
the indole in compound 19 was replaced by pyrrolo-pyrimidine
(20) and benzimidazole (21). As we could not readily access
7-aza-indole and 2-indazole analogs for the precise substitution
pattern in compound 19, we generated analogs (22–24) in which
A
NH2
N
N
H2N
CO2H
S
N
N
O
OH OH
Homocysteine
Surrogate
Drug-like Scaffolds
4
AdoHcy (S-adenosylhomocysteine)
B
N
N
N
N
R1
NH2
R1
N
H
S
S
N
N
5
6
8
H
N
N
R1
R1
H
N
H
R1
S
N
N
S
N
9
N
S
NH2
Br
R1 =
HO2C
7
Figure 3. Potency comparison of Indole scaffolds based inhibitors against SET7 in
Figure 2. (A) Schematic for the ‘biased-privileged’ scaffold based strategy to
ELISA; inhibition studies were performed with 40 lM inhibitors and DMSO as
develop KMTase inhibitors. (B) Compounds designed and tested.
control (n P2).

S. Kashyap et al. / Bioorg. Med. Chem. 22 (2014) 2253–2260
2255
the IC50 was similar under these different conditions (Fig. 6A), we
can exclude the possibility SETin-1 inhibits KMTases via such a
non-specific mechanism.
A
B
Br
a)
OH
Br
N
N
b)
Br
Br
N
H
10a
11a
12a
3. Conclusion
R1
1 = g-butyric acid
1 = 3-CO2H-Ph
10:
11:
R
R
Our data suggests that our approach, in which we bias drug-like
‘privileged’ scaffolds with a fragment of an enzyme’s cofactor that
is known to make important contacts in the binding pocket, can be
effective in developing inhibitors. SETin-1 was obtained by testing
a handful of compounds and a focused SAR analysis. Improving the
potency of this compound will likely require structural studies that
should reveal how this compound binds the enzyme. We predict
that inhibitors in this series should inhibit KMTases, other that
Set7 and G9a. It is also possible that once structural data becomes
available, the inhibitors can be modified (or ‘bumped’) so that they
inhibit KMTases with compensatory mutations (or ‘holes’). It is
likely that these compounds would serve as useful tools to exam-
ine KMTase function and the contributions on histone methylation
to chromosome biology.
N
c)
S
Br
R2
R2
OH
Br
R2
N
N
a)
b)
HN
10b: R2 = Br
12b: R2 = Br
11b: R2 = Br
d)
R
2 = Ph
2 = Ph
2 = Ph
11c:
R
12c:
10c:
R
R2
2 = Br
12:
R
c)
N
13: R2 = Ph
HO2C
S
C
R2
R2
N
e, f, g)
HO
d)
a)
10b
4. Experimental
HN
11c-11g
10c, 11c, 14: R2 = Ph
10c-10g
4.1. General synthesis information
R2
R
R
2 = 4-CO2H-Ph
10d, 11d, 15:
10e, 11e, 16:
Reactions were run in capped 1 dram vials (4 mL) stirred with
TeflonÒ-coated magnetic stir bars. Moisture- and air-sensitive
reactions were performed in flame-dried round bottom flasks, fit-
ted with rubber septa or glass gas adapters, under a positive pres-
sure of nitrogen. Concentration of solvents was accomplished by
rotary evaporation using a Büchi rotary evaporator, equipped
with a dry ice-acetone condenser, at 5–75 mm Hg at tempera-
tures between 35 and 50 °C. Analytical TLC was performed using
Whatman 250 micron aluminum backed UV F254 pre-coated silica
gel flexible plates. Proton nuclear magnetic resonance spectra (1H
NMR) were recorded on Bruker DPX 400 or 600 MHz nuclear
magnetic resonance spectrometers. Chemical shifts for 1H NMR
spectra are reported as d in units of parts per million (ppm) rel-
ative to tetramethylsilane (d 0.0) using the residual solvent signal
as an internal standard or tetramethylsilane itself: chloroform-d
(d 7.26, singlet), dimethylsulfoxide-d6 (d 2.50, quintet), metha-
nol-d4 (d 3.30, quintet), and deuterium oxide-d2 (d 4.80, singlet).
Liquid chromatography mass spectral analyses were obtained
using a Waters MicroMassZQ mass spectrometer, with an electron
spray ionization (ESI) probe, connected to a Waters 2795 HT
Separation Module Alliance HT HPLC system running MassLynx
(V4.0).
2 = 4-NHAc-Ph
N
HO2C
N
H
14-18
10f, 11f, 17: R2 = 4-CH3-Ph
10g, 11g, 18: R2 = 2-Naphthyl
Scheme 1. Synthesis scheme for compounds 10–18. Reagents and conditions: (a)
(3-iodophenyl)methanol, 1,4-dioxane, CuI, K3PO4, trans-N,N0-dimethylcyclohexane-
1,2-diamine, 100 °C, 24 h; (b) CBr4, PPh3, DMF, 25 °C, 24 h; (c) R1SH, NaOH, 100 °C,
5 min, THF, 25 °C, 16 h; (d) ArB(OH)2, 1,4-dioxane, Pd-catalyst, K2CO3, 100 °C, 24 h;
(e) pyridinium dichromate, CH2Cl2, 23 °C, 24 h; (f) 3-aminobenzoic acid, isopropa-
nol, reflux, 4 h; and (g) NaBH3CN, AcOH, acetonitrile, 23 °C, 24 h.
the indole was substituted at the 5-position. Testing revealed that
compound 19 remained the most potent compound in the series,
with indole being the favoured heterocycle (Fig. 4).
We next focused on the other aromatic rings in compound 20.
Altering the position of the secondary amine (25) linking the ben-
zoic acid to the central phenyl ring, or replacing the central phenyl
ring with a thiophene (26), reduced inhibitor potency. Finally,
changes in the benzoic acid moiety (27–35) also did not yield com-
pounds that were more potent than 19 (Fig. 4).
Figure 5 summarizes our SAR data, with the modifications that
improved (+), reduced (ꢁ) or did not significantly alter (=) inhibitor
activity are indicated. As compound 19 was the best inhibitor of
SET7 based on ‘biased-privileged’ scaffolds, we named it SETin-1.
Analysis of dose-dependent inhibition of SET7 activity using an
4.2. Synthetic procedures and characterization of compounds
ELISA-based assay yielded an IC50 of 10 lM (see Supporting infor-
4.2.1. (S)-4-((3-((6-Amino-9H-purin-9-
mation, Fig. S1). In addition, we analyzed SETin-1’s potency using a
scintillation proximity assay (SPA), which was designed based on
published work,19,30 as it allowed for homogenous reaction condi-
yl)methyl)phenyl)methylthio)-2-aminobutanoic acid (5)
Alkylation of adenine using
a,
a0-dichloro-m-xylene (195.0 mg,
1.10 mmol) and purification by crystallization (methanol-ethyl
acetate) afforded 9-(3-(chloromethyl)benzyl)-9H-purin-6-amine
(178.5 mg, 56% yield) as white powder; 1H NMR (400 MHz,
DMSO-d6): d 8.63 (s, 1H), 7.41–7.23 (m, 4H), 5.39 (s, 2H), 4.73
(s, 2H). Bromine displacement by L-homocysteine thiolate
using 9-(3-(chloromethyl)benzyl)-9H-purin-6-amine (66.9 mg,
0.244 mmol), ethanol (0.20 mL), sodium iodide (36.6 mg,
0.244 mmol), stirred at 110 °C for 15 min and purification by re-
verse phase HPLC (gradient run: 5% B for 3 min then ramp to
75% B over 30 min) afforded compound 5, (15.8 mg, 17% yield)
as clear film and as trifluoroacetate salt; 1H NMR (400 MHz,
tions. This assay yielded a comparable IC50 of ꢂ22
lM (Fig. 6A). As
we expect these compounds to be active against other KMTases,
we generated recombinant G9a, based on literature precedent.31
As shown in Figure 6B, SETin-1 inhibited this KMTase with compa-
rable potency (IC50: 26.4 4.9 lM) (Fig 6B).
To exclude the possibility that SETin-1 inhibited KMTase activ-
ity via an aggregation-type mechanism, we analyzed inhibitor po-
tency at different enzyme concentrations. An unchanged IC50 has
been recently reported to most reliably indicate that an inhibitor
that is not an ‘aggregator’.32 Therefore, we determined IC50 values
at three different enzyme concentrations (10, 50, and 100 nM). As

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S. Kashyap et al. / Bioorg. Med. Chem. 22 (2014) 2253–2260
A
B
CF3
R1
a) 3-iodo
N
benzaldehyde
d)
MeO2C
N
H
MeO2C
methyl 3-amino
benzoate
NH2
N
b) 10b
HO2C
N
H
10h:
11h:
R
R
1 = Br
SETin-1 (19)
c)
CF3
1 = 4-CF3-Ph
CF3
Cl
X
X
X
Y
OHC
N
c, b)
N
a)
HO2C
N
H
HN
Z
Y
Y
Z
Z
10i: X = CH, Y = Z = N
11i: X = CH, Y = Z = N
20
21
: X = CH, Y = Z = N
: X =N, Y = Z = CH
10j
11j
: X = N, Y = Z = CH
: X = N, Y = Z = CH
CF3
X
N
Br
X
N
c, b)
a)
X
CF3
N
H
Y
Y
Y
N
H
HO2C
OHC
10k
11k
: X = Y = CH
: X = Y = CH
22:
23:
24:
X = Y = CH
X = N, Y = CH
X = CH, Y = N
10l: X = N, Y = CH
11l: X = N, Y = CH
10m:
11m:
X = CH, Y = N
X = CH, Y = N
CF3
CF3
H2N
a)
N
H
N
N
N
HO2C
N
N
N
c, b)
25
10i
11n
CF3
CF3
S
OHC
a)
N
S
N
N
H
HO2C
N
N
N
N
11o
26
C
CF3
CF3
2 = 3-CO2H, 4-OH,
6-CH3-Ph
27:
28:
29:
R
R
R
2 = 2-CO2H-Ph
33:
34:
R
R
2 = 3-CO2H-Ph
2 = 4-CO2H-Ph
H
2 = 4-SO2CH3-Ph
H2N
N
R2
N
N
b)
e)
S
30: R2 = 2-NO2-Ph
10h
N
O2
35: R2
=
AcHN
31:
32:
R
R
2 = 3-NO2-Ph
2 = 4-NO2-Ph
S
11p
27-35
Scheme 2. General Synthesis scheme for compounds 19–35: (A) 4-CF3-phenyl substitution on indole. (B) Inhibitors containing similar drug-like heterocycles. (C) General
synthetic scheme for sulfonamide compounds. Reagents and conditions: (a) ArNH2 (CO2H) or ArCHO (CO2H), NaBH3CN, p-TSA, MeOH: CH2Cl2 (1:1), 25 °C, 24 h; (b) Het-Br or
ArI, 1,4-dioxane, CuI, K3PO4, trans-N,N0-dimethylcyclohexane-1,2-diamine, 100 °C, 24 h; (c) 4-(trifluoromethyl)phenylboronic acid, 1,4-dioxane, Pd-catalyst, K2CO3, 100 °C,
24 h; (d) LiOHꢃH2O, THF/MeOH/H2O (1:1:1), 25 °C, 24 h; and (e) ArSO2Cl, pyridine, CH2Cl2, 25 °C, 24 h.
R = 4-CO2H or OH, X = NH, Y = CH2 (=)
R = 4-CO2H or NO2, X = SO2, Y = NH (-)
R = 3-CO2H, X = NH, Y = CH2 (+)
R = 3-CO2H or NO2, X = SO2, Y = NH (-)
R = 3-CO2H, X = CO or CH2, Y = NH (-)
R = 3-CO2H, X = O, Y = CH2 (-)
CF3, tert-butyl (+)
CO2H, Cl, Me (-)
R = 3-OH, X = NH, Y = CH2 (-)
5
4
6
1
3
R
Y
N
X
2
Indazole, azaindole,
pyazole-pyrimidine
benzimidazole, (=)
R = 2-CO2H or OH, X = NH, Y = CH2 (-)
R = 2-CO2H, X = SO2, Y = NH (-)
R = 2-NO2, X = SO2, Y = NH (=)
thiophene (=)
Figure 4. Inhibitory effect of CF3-substituted heteroaryl groups and sulfonamides
on SET7 activity in SPA, 50 and 15 M of inhibitors and DMSO as control (n P2).
Figure 5. SAR summary for drug-like scaffold inhibitors of KMTases.
l
4.2.2. (S)-4-(3-(Isoquinolin-5-ylamino)benzylthio)-2-
aminobutanoic acid (6)
Buchwald coupling using 5-aminoisoquinoline (175.8 mg,
1.22 mmol), stirred at 100 °C for 14 h and purified by chromatogra-
phy (12 g silica gel, 60% ethyl acetate–petroleum ether to 100%
D2O): d 8.47 (s, 1H), 8.44 (s, 1H), 7.45–7.30 (m, 4H), 5.57 (s, 2H),
4.13 (t, J = 6.4 Hz, 1H), 3.82 (s, 2H), 2.62 (t, J = 7.4 Hz, 2H), 2.21–
2.09 (m, 2H). Calcd mass for C17H20N6O2S: 372.14; LRMS (ESI)
m/z [M+H]+ = 373.43.

S. Kashyap et al. / Bioorg. Med. Chem. 22 (2014) 2253–2260
2257
1H). Calcd mass for C19H19BrN2O2S: 418.04; LRMS (ESI) m/z
[M+H]+ = 419.24/421.22 (bromine pattern).
4.2.4. (S)-4-(3-(6-(Phenylamino)pyrazin-2-yl)benzylthio)-2-
aminobutanoic acid (8)
Aniline (265.7 mg, 2.85 mmol) and 2,6-dichloropyrazine
(427.2 mg, 2.87 mmol) were dissolve in n-butanol (2.0 mL), and
4.0 M hydrochloric acid in 1,4-dioxane (2 mL) was added. The mix-
ture was heated to 120 °C for 96 h, poured in water (30 mL), ex-
tracted with ethyl acetate (30 mL), the organic layer was washed
with saturated aqueous sodium bicarbonate solution (30 mL) and
brine (25 mL), concentrated and purified by chromatography
(30 g silica gel, 0.5–1.0% ethyl acetate–dichloromethane) to afford
6-chloro-N-phenylpyrazin-2-amine (225.9 mg, 38% yield) as a dark
yellow semi-solid; 1H NMR (400 MHz, CDCl3): d 8.10 (s, 1H), 7.97
(s, 1H), 7.40–7.35 (m, 4H), 7.17–7.12 (m, 1H), 6.74 (s, 1H); Calcd
mass for C10H8ClN4: 205.04; LRMS (ESI) m/z [M+H]+ = 206.20.
Figure 6. (A) Dose response curve of compound 19 at increasing concentrations of
SET7 in SPA (IC50: 10 nM SET7 = 19.0 1.0 M; 50 nM SET7 = 22.5 1.2 M, 100 nM
M). (B) Dose–response curves for 19 against SET7 or G9a, IC50’s:
M; G9a = 26.4 4.9 M.
l
l
SET7 = 19.5 1.5
SET7 = 22.5 1.2
l
l
l
Suzuki
coupling
using
6-chloro-N-phenylpyrazin-2-amine
(47.7 mg, 0.232 mmol), 3-hydroxymethylphenylboronic acid
(39.0 mg, 0.257 mmol) and purified by chromatography (4 g silica
gel, 0–10% methanol–dichloromethane) to afford (3-(6-(phenyla-
ethyl acetate) to afford (3-(isoquinolin-5-ylamino)phenyl)metha-
nol (18.3 mg, 6% yield) as pale orange film; 1H NMR (400 MHz,
CDCl3): d 9.18 (s, 1H), 8.42 (d, J = 5.9 Hz, 1H), 7.72 (d, J = 5.9 Hz,
1H), 7.60 (d, J = 7.7 Hz, 1H), 7.52–7.47 (m, 2H), 7.25 (t, J = 7.5 Hz,
1H), 7.05 (s, 1H), 6.94 (t, J = 8.2 Hz, 2H), 6.11 (s, 1H), 4.65 (s, 2H).
Bromination using (3-(isoquinolin-5-ylamino)phenyl)methanol
(18.3 mg, 0.073 mmol), and purified very quickly by chromatogra-
phy (10 g silica gel, 0–2% methanol–dichloromethane) to afford
N-(3-(bromomethyl)phenyl)isoquinolin-5-amine as pale yellow
film, which was not fully concentrated to avoid the intermolecular
reation. Bromine displacement by L-homocysteine thiolate using
N-(3-(bromomethyl)phenyl) isoquinolin-5-amine, EtOH (0.4 mL),
stirred at room temperature for 16 h, and purification by reverse
phase HPLC (gradient run: 5% B for 3 min then ramp to 75% B over
30 min) afforded compound 6, (8.6 mg, 32% yield over two steps)
as bright yellow film and as trifluoroacetate salt; 1H NMR
(400 MHz, D2O): d 9.60 (s, 1H), 8.54 (d, J = 6.8 Hz, 1H), 8.46 (d,
J = 6.8 Hz, 1H), 8.07 (d, J = 7.0 Hz, 1H), 7.95–7.90 (m, 2H), 7.37
(t, J = 7.9 Hz, 1H), 7.11 (s, 1H), 7.06 (d, J = 6.8 Hz, 2H), 4.14 (t,
J = 6.1 Hz, 1H), 3.78 (s, 1H), 2.69 (t, J = 7.4 Hz, 2H), 2.29–2.17 (m,
mino)pyrazin-2-yl)phenyl)methanol (48.6 mg, 76% yield) as
yellow solid; Calcd mass for C17H15N4O: 277.12; LRMS (ESI) m/z
[M+H]+ = 278.29.
Bromination using (3-(6-(phenylamino)
a
pyrazin-2-yl)phenyl)methanol (48.6 mg, 0.175 mmol), 1:1 dichlo-
romethane–tetrahydrofuran (2.0 mL) as solvent, and purified by
chromatography (12 g silica gel, 25–80% ethyl acetate–petroleum
ether) to afford 6-(3-(bromomethyl)phenyl)-N-phenylpyrazin-
2-amine which was used quickly for the next step. Bromine
displacement by L-homocysteine thiolate using 6-(3-(bromo-
methyl)phenyl)-N-phenylpyrazin-2-amine (16.6 mg, 0.49 mmol),
1:1 tetrahydrofuran–water (0.20 mL), stirred at 55 °C for 13 h,
and purification by reverse phase HPLC (gradient run: 50% B for
3 min then ramp to 70% B over 30 min) afforded compound 8,
(8.5 mg, 34% yield) as a pale yellow’ solid and as the trifluoroace-
tate salt; 1H NMR (400 MHz, DMSO-d6): d 9.60 (s, 1H), 8.54 (s,
1H), 8.20 (s, 1H), 8.08 (s, 1H), 7.96 (d, J = 7.3 Hz, 1H), 7.81 (d,
J = 7.9 Hz, 2H), 7.50–7.34 (m, 4H), 6.99 (t, J = 7.3 Hz, 1H), 3.84 (s,
2H), 2.58 (t, J = 7.3 Hz, 2H), 2.10–2.01 (m, 1H), 1.90–1.83 (m, 1H).
2H). Calcd mass for
C20H21N3O2S: 367.14; LRMS (ESI) m/z
Calcd mass for
C21H22N4O2S: 394.15; LRMS (ESI) m/z
[M+H]+ = 368.40.
[M+H]+ = 395.42.
4.2.3. (S)-4-(3-(5-Bromo-1H-indol-1-yl)benzylthio)-2-
4.2.5. (S)-4-(3-(2-(Phenylamino)pyrimidin-4-
aminobutanoic acid (7)
ylamino)benzylthio)-2-aminobutanoic acid (9)
Buchwald
coupling
using
5-bromoindole
(282.0 mg,
Amine displacement using ethyl 3-aminobenzoate (352.0 mg,
2.13 mmol), 2,4-dichloropyrimidine (318.0 mg, 2.13 mmol), diiso-
propylamine (0.742 mL, 4.26 mmol), and ethylene glycol (1.0 mL),
was stirred at 130 °C for 6 h. Instead of working up the reaction,
4.0 M hydrochloride acid in 1,4-dioxane (0.55 mL, 2.20 mmol),
and aniline (0.20 mL, 2.19 mmol) were added, and the mixture
was stirred at 120 °C for 11 h. The reaction was poured into water
(20 mL), extracted with 1:1 ethyl acetate–petroleum ether (2
ꢄ 30 mL), the organic layers were sequentially washed with water
(20 mL) and brine (20 mL), concentrated, and purified by chroma-
tography (12 g silica gel, 5–70% ethyl acetate–petroleum ether) to
1.438 mmol), stirred at 90 °C for 6.5 h (in order to avoid the poly-
merization and bromide to iodide exchange), and purified by chro-
matography (12 g silica gel, 0–40% ethylacetate–petroleum ether)
to afford (3-(5-bromo-1H-indol-1-yl)phenyl)methanol (284.7 mg,
67% yield) as clear film; 1H NMR (400 MHz, CDCl3): d 7.77 (s,
1H), 7.48–7.22 (m, 7H), 6.58 (d, J = 2.7 Hz, 1H), 4.75 (d, J = 5.3 Hz,
2H), 1.81 (t, J = 5.5 Hz, 1H). Bromination using (3-(5-bromo-1H-in-
dol-1-yl)phenyl)methanol (276.9 mg, 0.916 mmol), and purifica-
tion by chromatography (15 g silica gel, 0–5% ethyl acetate–
petroleum ether) afforded 5-bromo-1-(3-(bromomethyl)phenyl)-
1H-indole (261.4 mg, 78% yield) as pink yellow viscous oil; 1H
NMR (400 MHz, CDCl3): d 7.80 (s, 1H), 7.50–7.28 (m, 7H), 6.62 (d,
J = 2.9 Hz, 1H), 4.53 (s, 2H). Bromine displacement by L-homocys-
teine thiolate using 5-bromo-1-(3-(bromomethyl)phenyl)-1H-in-
dole (11.3 mg, 0.031 mmol), ethanol (0.4 mL), stirred at 110 °C
for 1 h, and purification by reverse phase HPLC (gradient run: 5%
B for 3 min then ramp to 75% B over 30 min) afforded compound
7, (5.3 mg, 32% yield) as clear film and as trifluoroacetate salt; 1H
NMR (400 MHz, D2O): d 7.77 (s, 1H), 7.52–7.37 (m, 6H), 7.27 (d,
J = 8.8 Hz, 1H), 6.64 (d, J = 2.7 Hz, 1H), 4.07 (t, J = 6.2 Hz, 1H), 3.88
(m, 2H), 2.66 (t, J = 7.5 Hz, 2H), 2.27–2.18 (m, 1H), 2.15–2.05 (m,
afford
ethyl-3-(2-(phenylamino)pyrimidin-4-ylamino)benzoate
(crude weight 260 mg) as a mixture of compounds used without
further purification. Crude ethyl-3-(2-(phenylamino)pyrimidin-4-
ylamino)benzoate was dissolved in dichloromethane (20 mL) and
cooled to 0 °C. A solution of 1.0 M diisobutylaluminum hydride in
toluene (3.0 mL, 3.00 mmol) was added slowly in three portions
every 20 min over 1 h. The reaction was deemed complete by
TLC (80% ethyl acetate–petroleum ether). The reaction was
quenched with methanol (1.2 mL), ground anhydrous sodium sul-
fate (8.5 g), water (10.8 mL), and CeliteÒ (75 mL), stirred vigorously
for 30 min, filtered, washed with dichloromethane, concentrated

2258
S. Kashyap et al. / Bioorg. Med. Chem. 22 (2014) 2253–2260
and purified by chromatography (12 g silica gel, 0–10% methanol–
dichloromethane) to afford (3-(2-(phenylamino)pyrimidin-4-yla-
mino)phenyl)methanol (58.5 mg, 9% yield) as a white foam; 1H
extracted with 1:1 ethyl acetate–petroleum ether (30 mL), the or-
ganic layer was washed with brine (10 mL), concentrated, and
purified by chromatography (4 g silica gel, 0–10% methanol–
dichloromethane). A portion of this material was filtered (PALL Life
Sciences, AcrodiscÒ Premium, 25 mm syringe filters with 0.45 mi-
cron GHP membrane, catalog# AP-4560T), purified by reverse
phase HPLC (VYDAC Cl8 , 11 ꢄ 250 mm column; flow rate 6 mL/
min; UV detection: 254 nm; solvent A: water with 0.1% ammo-
nium hydroxide, solvent B: acetonitrile; gradient run: 5% B for
3 min then ramp to 40% B over 25 min), and neutralized to afford
compound 11, (1.7 mg) as a clear film; 1H NMR (400 MHz, CDCl3):
d 7.88 (s, 1H), 7.86 (d, J = 12.99 Hz, 1H), 7.77 (d, J = 7.68 Hz, 1H),
7.60–7.65 (m, 2H), 7.49–7.54 (m, 2H), 7.40–7.47 (m, 3H), 6.68 (d,
NMR (400 MHz, CDCI3):
d 9.03 (d, J = 5.9 Hz, 1H), 7.56 (d,
J = 7.9 Hz, 2H), 7.40 (s, 1H), 7.35–7.26 (m, 4H), 7.13–7.10 (m, 2H),
7.02 (t, J = 7.3 Hz, 1H), 6.69 (s, 1H), 6.16 (d, J = 5.9 Hz, 1H), 4.68
(s, 2H); Calcd mass for C17H16N4O: 292.13; LRMS (ESI) m/z
[M+H]+ = 293.41. Bromination using (3-(2-(phenylamino)pyrimi-
din-4-ylamino)phenyl)methanol (58.5 mg, 0.20 mmol), 1:1 dichlo-
romethane–tetrahydrofuran (2.0 mL) as solvent, and purified by
chrornatography (4 g silica gel, 10% ethyl acetate–petroleum ether
to 100% ethyl acetate) to afford N4-(3-(bromomethyl)phenyl)-N2-
phenylpyrimidine-2,4-diamine (38.8 mg, 55% yield) as a clear film;
1H NMR (400 MHz, CDCI3): d 8.06 (d, J = 5.7 Hz, 1H), 7.56 (d,
J = 7.7 Hz, 2H), 7.46 (s, 1H), 7.38 (s, 1H), 7.33–7.25 (m, 4H), 7.14
(d, J = 6.6 Hz, 1lH), 7.03 (t, J = 7.3 Hz, 1H), 6.86 (s, 1H), 6.16 (d,
J = 5.9 Hz, 1H), 4.44 (s, 2H); Calcd mass for C17H15BrN4: 354.05;
LRMS (ESI) m/z [M+H]+ = 355.38/357.41 (bromine pattern). Bro-
mine displacement by L-homocysteine thiolate using N4-(3-(bro-
momethyl)phenyl)-N2-phenylpyrimidine-2,4-diamine (19.4 mg,
0.0546 mmol), 3:1 ethanol–water (0.40 mL), stirred at 55 °C for
1.5 h, and purification by reverse phase HPLC (gradient run: 15%
B for 3 min then ramp to 100% B over 30 min) afforded compound
9, (13.9 mg, 49% yield) as a clear film and as the trifluoroacetate
salt; 1H NMR (400 MHz, CD3OD): d 7.76 (d, J = 7.0 Hz, 1H), 7.56
(s, 1H), 7.50–7.40 (m, 5H), 7.30–7.25 (m, 2H), 7.18 (d, J = 7.5 Hz,
1H), 6.40 (d, J = 7.1 Hz, 1H), 3.79 (t, J = 6.2 Hz, 1H), 3.70 (s, 2H),
2.55 (t, J = 7.6 Hz, 2H), 2.18–2.03 (m, 1H). Calcd mass for C21H23N5-
O2S: 409.16; LRMS (ESI) m/z [M+H]+ = 410.42.
J = 3.29 Hz, 1H), 4.41 (s, 2H). Calcd mass for
C22H16BrNO2S:
437.01; LRMS (ESI) m/z [MꢁH]- = 436.21/438.28 (bromine pattern).
4.2.8. 3-(3-(4-Bromo-1H-indol-1-yl)benzylthio)benzoic acid (12)
Following the same procedure used to synthesize (7), but using
4-bromoindole in the Buchwald coupling step, affords compound
12; 1H NMR (400 MHz, DMSO-d6):
d 7.88 (s, 1H), 7.77 (d,
J = 8.27 Hz, 1H), 7.71 (d, J = 3.30 Hz, 1H), 7.64 (d, J = 7.78 Hz, 1H),
7.54–7.50 (m, 2H), 7.47–7.41 (m, 3H), 7.35 (d, J = 7.57 Hz, 1H),
7.30 (d, J = 8.41 Hz, 1H), 7.09 (t, J = 7.85 Hz, 1H), 6.65 (d,
J = 3.36 Hz, 1H), 4.41 (s, 2H). Calcd mass for
C22H16BrNO2S:
437.01; LRMS (ESI) m/z [MꢁH]- = 436/438 (bromine pattern).
4.2.9. 3-(3-(4-Phenyl-1H-indol-1-yl)benzylthio)benzoic acid
(13)
Suzuki coupling using 4-bromoindole (938.8 mg, 4.79 mmol),
phenylboronic acid (603.1 mg, 4.95 mmol), and purified by chro-
matography (40 g silica gel, 0–35% ethyl acetate–petroleum ether)
to afford 4-phenyl-1H-indole (622.7 mg, 67% yield) as a pale purple
viscous oil; Calcd mass for C14H11N: 193.09; LRMS (ESI) m/z
[M+H]+ = 194.28. Buchwald coupling using 4-phenyl-1H-indole
(104.4 mg, 0.54 mmol), and purification by chromatography (12 g
silica gel, 0–40% ethyl acetate–petroleum ether) afforded (3-(4-
phenyl-1H-indol-1-yl)phenyl)methanol (104.8 mg, 65% yield) as a
white crystalline solid; 1H NMR (400 MHz, CDCl3): d 7.71 (d,
J = 7.5 Hz, 2H} 7.55–7.44 (m, 6H), 7.40–7.35 (m, 3H), 7.29 (t,
J = 7.8 Hz, 1H), 7.23 (d, J = 7 .0 Hz, 1H), 6.84 (d, J = 3.1 Hz, 1H),
4.19 (s, 2H), 1.85 (s, 1H); Calcd mass for C21H17NO: 299.13; LRMS
(ESI) m/z [M+H]+ = 300.41. Bromination using (3-(4-phenyl-1H-in-
dol-1-yl)phenyl)methanol (104.8 mg, 0.35 mmol), and purification
by chromatography (4 g silica gel, 0–25% ethyl acetate–petroleum
ether) afforded 1-(3-(bromomethyl)phenyl)-4-phenyl-1H-indole
(112.5 mg, 89% yield) as a clear film; 1H NMR (400 MHz, CDCl3):
d 8.71 (d, J = 7.3 Hz, 2H), 7.56–7.45 (m, 6H), 7.40–7.36 (m, 3H),
1.31 (t, J = 7.7 Hz, 1H), 7.24 (d, J = 7.7 Hz, 1H), 6.85 (d, J = 2.9 Hz,
7H), 4.54 (s, 2H). A 1.03 M solution of 3-benzoate thiolate was pre-
pared from 3-mercaptobenzoic acid and 2.06 M aqueous sodium
hydroxide as above. The solution of 3-benzoate thiolate
(0.040 mL, 0.0412 mmol) was added to a solution of intermediate
4.2.6. 4-(3-(5-Bromo-1H-indol-1-yl)benzylthio)butanoic acid
(10)
A 1.5 M solution of
c-butyrate thiolate was prepared by dissolv-
ing -butyrothiolactone (30.0 mg, 0.296 mmol) into 2.96 M aque-
c
ous sodium hydroxide (0.195 mL, 0.577 mmol) at 100 °C for
5 min. A 0.18 M solution of intermediate 5-bromo-1-(3-(bromo-
methyl)phenyl)-1H-indole (37.3 mg, 0.102 mmol) in tetrahydrofu-
ran (0.57 mL) was added to the aqueous solution, and stirred at
room temperature for 16 h. Upon completion, the reaction was
poured into 1.0 M aqueous hydrochloric acid (20 mL), extracted
with 1:1 ethyl acetate–petroleum ether (30 mL), the organic layer
was washed with brine (10 mL), concentrated, and purified by
chromatography (4 g silica gel, 0–7% methanol–dichloromethane).
A portion of this material was filtered (PALL Life Sciences, Acro-
discÒ Premium, 25 mm syringe filters with 0.45 micron GHP mem-
brane, catalog# AP-4560T), purified by reverse phase HPLC (VYDAC
C18, 11 ꢄ 250 mm column; flow rate 6 mL/min; UV detection:
254 nm; solvent A: water with 0.1% ammonium hydroxide, solvent
B: acetonitrile; gradient run: 5% B for 3 min then ramp to 40% B
over 25 min), and neutralized to afford compound 10, (2.9 mg) as
a clear film; 1H NMR (400 MHz, CDCl3): d 7.80 (s, 1H), 7.52–7.37
(m, 7H), 6.61 (d, J = 2.9 Hz, 1H), 3.78 (s, 2H), 2.54 (t, J = 7.1 Hz,
2H), 2.47 (t, J = 7.1 Hz, 2H), 1.91 (quint, J = 7.1 Hz, 2H). Calcd mass
for C19H18BrNO2S: 403.02; LRMS (ESI) m/z [MꢁH]- = 402.22/404.22
(bromine pattern).
1-(3-(bromomethyl)phenyl)-4-phenyl-1H-indole
(14.1 mg,
0.0389 mmol) in tetrahydrofuran (0.5 mL), and the mixture was
stirred at room temperature for 16 h. Upon completion, the reac-
tion was diluted with 1.0 M aqueous hydrochloric acid (1 mL), ex-
tracted with ethyl acetate (2 mL), the organic layer was washed
with brine (10 mL), concentrated, and purified by chromatography
(4 g silica gel, 0–5% methanol–dichloromethane) to afford com-
4.2.7. 3-(3-(5-Bromo-1H-indol-1-yl)benzylthio)benzoic acid (11)
A 1.5 M solution of 3-benzoate thiolate was prepared by dis-
solving 3-mercaptobenzoic acid (31.3 mg, 0.203 mmol) into
2.96 M aqueous sodium hydroxide (0.135 mL, 0.40 mmol) at
100 °C for 5 min. A 0.18 M solution of intermediate 5-bromo-1-
(3-(bromomethyl)phenyl)-1H-indole (37.3 mg, 0.102 mmol) in tet-
rahydrofuran (0.57 mL) was added to the aqueous solution, and
stirred at room temperature for 16 h. Upon completion, the reac-
tion was poured into 1.0 M aqueous hydrochloric acid (20 mL),
pound 13, (16.6 mg, 98% yield) as
a
clear film; 1H NMR
(400 MHz, DMSO-d6): d 7.89 (s, 1H), 7.79 (d, J = 7.68 Hz, 1H),
7.69–7.62 (m, 4H), 7.58–7.50 (m, 4H), 7.48–7.38 (m, 4H), 7.31 (d,
J = 7.87 Hz, 1H), 7.24 (t, J = 8.05 Hz, 1H), 7.20 (d, J = 6.59 Hz, 1H),
6.76 (d, J = 3.29 Hz, 1H), 4.43 (s, 2H). Calcd mass for C28H21NO2S:
435.13; LRMS (ESI) m/z [MꢁH]- = 434.20.

S. Kashyap et al. / Bioorg. Med. Chem. 22 (2014) 2253–2260
2259
4.2.10. 3-(3-(4-Phenyl-1H-indol-1-yl)benzylamino)benzoic acid
(14)
A solution of (3-(4-phenyl-1H-indol-1-yl)phenyl)methanol was
4.2.15. 3-(3-(4-(4-(Trifluoromethyl)phenyl)-1H-indol-1-
yl)benzylamino)benzoic acid (19)
To a preformed solution of methyl 3-aminobenzoate (500 mg,
3.30 mmol) and 3-iodobenzaldehyde (842 mg, 3.63 mmol) in
(1:1) MeOH/CH2Cl2 (1.0 M) was added p-TSA (624 mg, 3.63 mmol)
followed by NaBH3CN (228 mg, 3.63 mmol) over a period of 5 min
at room temperature. The reaction mixture was stirred at room
temperature under the nitrogen atmosphere. After completion of
the reaction (monitored by TLC), reaction mixture was diluted with
ethyl acetate (20 mL) and quenched with water (10 mL), extracted
with ethyl acetate (3 ꢄ 20 mL), the organic layer was washed with
water (20 mL), dried over Na2SO4. The solvent was removed under
reduced pressure and crude reaction mixture was purified by col-
umn chromatography (50 g silica gel, 0–15% ethyl acetate–petro-
leum ether) to afford methyl-3-(3-iodobenzylamino)benzoate
(640 mg, 53% yield) as off white powder; 1H NMR (400 MHz,
dissolved in dichloromethane and treated with pyridinium dichro-
mate to form the aldehyde (Swern conditions also work). The mix-
ture was filtered through Celite and the dark orange solution was
concentrated, and purified directly by chromatography (silica gel,
10–75% ethyl acetate–hexanes) to afford 3-(4-phenyl-1H-indol-1-
yl)benzaldehyde; 1H NMR (300 MHz, CDCl3): d 8 10.12 (s, 1H),
8.06 (d, 1H), 7.90–7.80 (m, 2H), 7.75–7.70 (m, 3H), 7.60–7.45 (m,
3H), 7.60–7.45 (m, 3H), 7.20–7.10 (m, 1H), 6.90 (d, 1H). 3-(4-phe-
nyl-1H-indol-1-yl)benzaldehyde (29.75 mg, 0.1 mmol) was dis-
solved in isopropanol (0.3 mL, 0.33 M) and 3-aminobenzoic acid
(13.7 mg, 0.1 mmol) was added. The reaction was heated at reflux
for 2–4 h and concentrated to remove isopropanol. The residue was
dissolved in acetonitrile (0.3 mL) then acetic acid (12 lL, 0.2 mmol)
and solid sodium cyanoborohydride (7 mg, 0.11 mmol) were added
at 23 °C. The reaction was stirred overnight at 23 °C and then
quenched with 0.5 M aqueous oxalic acid. The mixture was stirred
at 23 °C for 30 min, poured into water, extracted with 1:1 ethyl
acetate–hexane, filtered and concentrated. Purification by chroma-
tography (4 g silica gel, 0–5% methanol–dichloromethane with
0.5% acetic acid) afforded compound 14 (2 mg) as a clear film; 1H
NMR (400 MHz, CDCl3): d 7.70 (d, J = 7.68 Hz, 2H), 7.55 (s, 1H),
7.51–7.43 (m, 6H), 7.41–7.35 (m, 4H), 7.29–7.21 (m, 3H), 6.87
(dd, J = 5.85, 2.20 Hz, 1H), 6.83 (d, J = 3.29 Hz, 1H), 4.50 (s, 2H).
CDCl3): d 7.73 (s, 1H), 7.61 (d, J = 7.92 Hz, 1H), 7.40 (d,
J = 7.52 Hz, 1H), 7.34–7.29 (m, 2H), 7.22 (t, J = 7.88 Hz, 1H), 7.07
(t, J = 7.69 Hz, 1H), 6.77 (dd, J = 6.05, 2.17 Hz, 1H), 4.32 (s, 2H),
3.89 (s, 3H). Calcd mass for C15H14INO2: 367.01; LRMS (ESI) m/z
[M+H]+ = 368.74. To a solution of methyl-3-(3-iodobenzylami-
no)benzoate (200 mg, 0.544 mmol) and 4-bromoindole (117 mg,
0.599 mmol) in 1,4-dioxane (1.0 M) was added freshly ground
potassium phosphate tribasic (254 mg, 1.197 mmol), copper(I)
iodide (7.25 mg, 0.038 mmol) and racemic trans-N,N0-dimethylcy-
clohexane-1,2-diamine (12
lL, 0.0816 mmol). The suspension
Calcd mass for
C
28H22N2O2: 418.17; LRMS (ESI) m/z
was degassed for 5 min by bubbling nitrogen gas directly into
the solution using syringe needle. The reaction vessel was closed
tightly and stirred at 100 °C for overnight or until the completion
of reaction as judged by TLC. The reaction mixture was filtered
through a pad of celite, poured into water (25 mL) and extracted
with ethyl acetate (3 ꢄ 25 mL), the organic layer was washed with
water (20 mL), dried over Na2SO4. Excess of Solvent was evapo-
rated under reduced pressure and resulting crude mixture was
purified by chromatography (20 g silica gel, 0–15% ethylacetate–
petroleum ether) to afford methyl 3-(3-(4-bromo-1H-indol-1-
yl)benzylamino)benzoate (150 mg, 63% yield) as clear film; 1H
NMR (400 MHz, CDCl3): d 7.49 (t, J = 7.84 Hz, 1H), 7.47 (s, 1H),
7.41 (d, J = 7.66 Hz, 1H), 7.38–7.30 (m, 6H), 7.23 (t, J = 7.86 Hz,
1H), 7.01 (t, J = 7.87 Hz, 1H), 6.80 (dd, J = 6.74, 1.27 Hz, 1H), 6.71
(d, J = 2.94 Hz, 1H), 4.47 (s, 2H), 3.87 (s, 3H). Calcd mass for
[MꢁH]- = 417.74.
4.2.11. 3-(3-(4-(4-Carboxyphenyl)-1H-indol-1-
yl)benzylamino)benzoic acid (15)
Following the same procedure used to synthesize (14), but
using 4-carboxyphenylboronic acid in the Suzuki coupling step, af-
fords compound 15; 1H NMR (300 MHz , CDCl3+CD3OD): d 8.10 (d,
2H), 7.71 (d, 2H), 7.47–7.30 (m, 9H), 7.19–7.12 (m, 3H), 6.77–6.72
(m, 2H), 4.24 (s, 2H). Calcd mass for C29H22N2O4: 462.16; LRMS
(ESI) m/z [M+Na]+ = 485.56.
4.2.12. 3-(3-(4-(4-Acetamidophenyl)-1H-indol-1-
yl)benzylamino)benzoic acid (16)
C
23H19BrN2O2: 435.31; LRMS (ESI) m/z [M]+ = 435.55, 437.34 (bro-
Following the same procedure used to synthesize (14), but
using 4-acetamidophenylboronic acid in the Suzuki coupling step,
affords compound 16; 1H NMR (300 MHz , CDCl3): d 7.64 (d, 2H),
7.54–7.34 (m, 9H), 7.30–7.07 (m, 3H), 6.86 (dd, 1H), 6.81 (d, 2H),
4.52 (s, 2H), 2.23 (s, 3H). Calcd mass for C30H25N3O3: 475.19; LRMS
(ESI) m/z [M+Na]+ = 498.63.
mine pattern). To a solution of methyl 3-(3-(4-bromo-1H-indol-1-
yl)benzylamino)benzoate (40 mg, 0.092 mmol) and 4-triflurom-
ethylphenyl boronic acid (19.5 mg, 0.101 mmol) in 1,4-dioxane
(1.0 M) was added potassium carbonate (25 mg, 0.184 mmol,
2.0 M aqueous solution) followed by palladium catalyst [1,
10-bis(diphenylphoshphino)ferrocene]-dichloropalladium(II) 1:1
complex with dichloromethane (7.5 mg, 0.0092 mmol). The reac-
tion mixture was degassed for 5 min by bubbling nitrogen gas di-
rectly into the solution using syringe needle. The reaction vessel
was closed tightly and stirred at 100 °C for overnight or until the
completion of reaction as judged by TLC. The reaction mixture
was filtered through a pad of silica/celite to remove the catalyst,
poured into water (25 mL) and extracted with ethyl acetate
(3 ꢄ 25 mL), the organic layers were washed with water (20 mL),
dried over Na2SO4, concentrated under reduced pressure and puri-
fied by chromatography (5 g silica gel, 0–15% ethyl acetate–petro-
leum ether) to afford methyl 3-(3-(4-(4-(trifluoromethyl)phenyl)-
1H-indol-1-yl)benzylamino)benzoate (42 mg, 97% yield) as a pale
purple viscous oil; 1H NMR (400 MHz, CDCl3): d 7.81 (d, J =
8.20 Hz, 2H), 7.74 (d, J = 7.68 Hz, 2H), 7.55–7.32 (m, 8H), 7.28–
7.20 (m, 3H), 6.82 (dd, J = 5.12, 2.15 Hz, 1H), 6.79 (d, J = 3.26 Hz,
1H), 4.50 (s, 2H), 3.88 (s, 3H). Calcd mass for C30H23F3N2O2:
500.51; LRMS (ESI) m/z [M+H]+ = 501.35. To a solution of methyl
3-(3-(4-(4-(trifluoromethyl)phenyl)-1H-indol-1-yl)benzylamino)-
4.2.13. 3-(3-(4-p-Tolyl-1H-indol-1-yl)benzylamino)benzoic acid
(17)
Following the same procedure used to synthesize (14), but
using p-tolylboronic acid in the Suzuki coupling step, affords
compound 17; 1H NMR (300 MHz , CDCl3): d 7.61 (d, 2H),
7.46–7.32 (m, 8H), 7.30–7.22 (m, 8H), 6.85–6.84 (m, 2H), 4.50
(s, 2H). Calcd mass for C29H24N2O2: 432.18; LRMS (ESI) m/z
[M+Na]+ = 455.64.
4.2.14. 3-(3-(4-(Naphthalen-2-yl)-1H-indol-1-
yl)benzylamino)benzoic acid (18)
Following the same procedure used to synthesize (14), but
using naphthalen-2-ylboronic acid in the Suzuki coupling step, af-
fords compound 18; 1H NMR (300 MHz , CDCl3): d 8.17 (s, 1H), 7.80
(m, 4H), 7.55–7.50 (m, 6H), 7.42–7.31 (m, 7H), 6.92–6.89 (m, 2H),
4.52 (s, 2H). Calcd mass for C32H24N2O2: 468.18; LRMS (ESI) m/z
[M+Na]+ = 491.64.

2260
S. Kashyap et al. / Bioorg. Med. Chem. 22 (2014) 2253–2260
benzoate (40 mg, 0.080 mmol) in THF/MeOH/H2O (1:1:1) was
added 1.0 M aqueous solution of LiOH/H2O (33.5 mg, 0.80 mmol)
and the reaction mixture was allowed to stirred at room tempera-
ture until the completion of reaction as judged by TLC. After evap-
oration of THF, the solution was acidified with 1 N HCI and
extracted with ethyl acetate (3 ꢄ 25 mL), the organic layer was
washed with water (20 mL), dried over Na2SO4, concentrated un-
der reduced pressure and purified by chromatography (5 g silica
gel, 0–5% methanol–dichloromethane) to afford compound 19,
(33 mg, 0.068 mmol, 85 % yield) as white powder. 1H NMR
(400 MHz, CDCl3+CD3OD): d 7.81 (d, J = 7.39 Hz, 2H), 7.74 (d,
J = 7.95 Hz, 2H), 7.54 (s, 1H), 7.50 (dd, J = 13.64, 7.74 Hz, 2H),
7.45 (d, J = 9.28 Hz, 2H), 7.41–7.38 (m, 3H), 7.26–7.21 (m, 3H),
6.84 (dd, J = 7.75, 1.16 Hz, 1H), 6.79 (d, J = 3.05 Hz, 1H), 4.50 (s,
sodium acetate, pH 5.0, 1 mg/mL BSA, 1 mM EDTA, 10% glycerol,
1 mM unlabeled SAM) in white clear-bottomed 384 plates (BD Fal-
con). Counts per minute (CPM) for each sample were measured
using a Microbeta Trilux counter (Perkin Elmer). Testing was per-
formed twice (n P2). Background was subtracted and percent
activity was determined by dividing CPM for each sample by
CPM of the negative (DMSO only) control and multiplying by 100.
Acknowledgments
TMK is grateful to the NIH (GM98579) and the Leukemia and
Lymphoma Society for support.
Supplementary data
2H). Calcd mass for
C29H21F3N2O2: 486.16; LRMS (ESI) m/z
[M+H]+ = 487.40.
Supplementary data (dose–response curve of compound 19
against the Set7 in ELISA (Fig. S1); general experimental proce-
dures and characterization data for compounds 20–35) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmc.2014.02.024. These data include MOL
files and InChiKeys of the most important compounds described
in this article.
4.3. General inhibition studies in ELISA
Streptavidin-coated plates (PerkinElmer Life Sciences) were
washed one time with reaction buffer (50 mM Tris, 1 mM EDTA,
1 mg/mL BSA, 4% DMSO, 0.5 mM DTT, 0.1% Triton-X, pH 8.0).
SET7 at 2.8 nM and SAM at 300 nM were diluted in reaction buffer
and added in a volume of 16
lL. Control wells received only SET7.
References and notes
Test compounds (0.2 L, 5 mM) were added and plates were incu-
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For each sample, 12
lL of reaction buffer (50 mM Tris, pH 8.5,
1 mg/mL BSA, 1 mM EDTA, 1 mM DTT) containing SET7 (25 nM),
H3(1–20)-cys-biotin (900 nM), and test compound in DMSO (2% fi-
nal) were incubated at 30 °C for 5 min. Methyltransferase reactions
were initiated by addition of unlabeled 3
nine (SAM; 2
M) containing a trace amount of 3H-SAM (0.2
and samples were incubated at 30 °C. After 8 min, 10 L was re-
moved from each sample and added to 100 L Streptavidin-coated
SPA beads (Perkin Elmer; 0.9375 mg/mL) in quench buffer (50 mM
lL S-adenosyl methio-
l
l
M),
l
l