Discovery and optimization of a new class of pyruvate kinase inhibitors as potential therapeutics for the treatment of methicillin-resistant Staphylococcus aureus infections

Article abstract of DOI:10.1016/j.bmc.2014.01.020

A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK is not only critical for bacterial survival which

Full text of DOI:10.1016/j.bmc.2014.01.020

Bioorganic & Medicinal Chemistry 22 (2014) 1708–1725
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and optimization of a new class of pyruvate kinase
inhibitors as potential therapeutics for the treatment
of methicillin-resistant Staphylococcus aureus infections
Nag S. Kumar a, Edie M. Dullaghan b, B. Brett Finlay c,d, Huansheng Gong d, Neil E. Reiner d,e
,
J. Jon Paul Selvam a, Lisa M. Thorson d, Sara Campbell b, Nicholas Vitko f, Anthony R. Richardson f,
Roya Zoraghi d, Robert N. Young a,
⇑
a Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada
b Centre for Drug Research and Development (CDRD), Vancouver, BC, Canada
c Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada
d Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, BC, Canada
e Department of Microbiology and Immunology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada
f Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized and
evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK is
not only critical for bacterial survival which would make it a target for development of novel antibiotics,
but it is reported to be one of the most highly connected ‘hub proteins’ in MRSA, and thus should be very
sensitive to mutations and making it difficult for the bacteria to develop resistance. From the co-crystal
structure of cis-3-4-dihydrohamacanthin B (4) bound to S. aureus PK we were able to identify the pharma-
cophore needed for activity. Consequently, we prepared simple direct linked bis-indoles such as 10b that
have similar anti-MRSA activity as compound 4. Structure–activity relationship (SAR) studies were carried
out on 10b and led us to discover more potent compounds such as 10c, 10d, 10k and 10m with enzyme
inhibiting activities in the low nanomolar range that effectively inhibited the bacteria growth in culture
Received 23 August 2013
Revised 7 January 2014
Accepted 15 January 2014
Available online 24 January 2014
Keywords:
Antibacterial
MRSA
Pyruvate kinase
Bis-indole
with minimum inhibitory concentrations (MIC) for MRSA as low as 0.5 lg/ml. Some potent PK inhibitors,
such as 10b, exhibited attenuated antibacterial activity and were found to be substrates for an efflux
mechanism in S. aureus. Studies comparing a wild type S. aureus with a construct (S. aureus LAC
D
pyk::ErmR) that lacks PK activity confirmed that bactericidal activity of 10d was PK-dependant.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
to avoid mechanism based toxicity. This has severely limited the
available targets for drug design.
Multidrug-resistant bacteria, such as methicillin-resistant
Staphylococcus aureus (MRSA), have developed numerous resis-
tance mechanisms in response to antibiotic pressure.1 There is an
increasing incidence of MRSA infections in hospitals worldwide
and they have begun to penetrate into the general community.
The vast majority of current antibiotics in use are directed to crit-
ical proteins unique to the bacteria and without human homologs
Recently, pyruvate kinase (PK) was identified as a highly inter-
connected essential hub protein in MRSA, with structural features
distinct from the human homologs, as a novel drug target.2–4 This
was based on the supposition that hub proteins are not only critical
for bacterial survival but should be very sensitive to mutations5
and targeting them should reduce the potential for development
of resistance strains and species. In silico library screening, initially
directed to putative binding sites unique to MRSA PK, combined
with enzyme assays identified several active MRSA PK inhibitors
including compound 16 (Fig. 1). Compound 1 was very selective
for the bacterial enzyme compared to four human PK isoforms
(M1, M2, R and L) and it did not inhibit growth of HeLa cells indi-
cating a lack of overt toxicity to mammalian cells. Structure–activ-
ity relationship (SAR) studies were initiated which led to the
Abbreviations: BHI, brain–heart infusion broth; MRSA, methicillin-resistant
Staphylococcus aureus; PK, pyruvate kinase; MIC, minimal inhibitory concentration;
TSA, tryptic soy agar; TSB, tryptic soy broth.
⇑
Corresponding author. Tel.: +1 (778) 782 3351; fax: +1 (778) 782 3765.
E-mail address: robert_young@sfu.ca (R.N. Young).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2014.01.020

N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
1709
Br
Br
Br
O
O
O
H
N
H
N
NH
HO
Br
N
NH
N
N
NH
HO
N
HO
N
1
2
3
H
N
O
Br
Br
N
H
HN
NH
4
Figure 1. MRSA PK inhibitors 1–4.
identification of more potent enzyme inhibitors (such as 2) and
which showed effective inhibition of a wide panel of gram positive
bacterial growth, with potencies comparable to standard antibiot-
ics such as vancomycin.3,7 In addition, the MIC was not signifi-
cantly increased even after 25 bacterial passages in culture with
compound 2 at the highest sub-lethal concentration,4 which con-
firmed that MRSA PK is an essential target less prone to developing
resistance. X-ray crystal structures for 1 and 3 bound to MRSA282
PK were obtained which revealed that both compounds bind to a
flat lipophilic pocket at the minor interfaces in the homo-tetra-
meric enzyme structure. This pocket was found to be modified
and not accessible in the human PK enzymes.
pseudo-symmetrical properties of the ligand. Hence it appears that
both the indoles in the scaffold are critical for binding and the
structure suggested that by linking the two indole at the C-2 posi-
tion and removing the lactam ring of 4, one might derive com-
pounds such as compound 10b (Fig. 2b) having all the necessary
elements to bind tightly to MRSA PK.
In this paper, we present a detailed account of SAR for enzyme
inhibitory and optimization of antibacterial activity for such an
extensive series of bis-indoles.
2. Results
More recently, Zoraghi et al.8 screened the inhibitory potential of
a natural marine product library of 968 crude benthic invertebrate
extracts and identified cis-3-4-dihydrohamacanthin B (4) as a
potent inhibitor of MRSA PK with an IC50 of 16 nM. Compound 4
2.1. Chemistry
The syntheses of all target compounds were carried out as
described in Schemes 1–6. The indole NH was first protected with
a phenylsulfonyl group to give intermediate 6 which was subse-
quently iodinated at the 2-position to give 2-iodoindole 7 by treating
6 with LDA followed by the addition of diiodoethane (Scheme 1). We
did attempt to couple 7 with the boronic acid 9 under standard
Suzuki–Miyura conditions but no desired product was isolated.
Fortunately, the coupling reaction of boronic acid 9 with the
unprotected indole 8, (obtained by hydrolysis of compound 7),
proceeded smoothly to give the desired adduct. Finally, removing
the Boc protecting group with TFA gave the desired compound 10.
In order to prepare the alkylated bis-indole 12, 8 was first alkylated
with alkyl halide to give intermediate 11, which was subsequently
coupled with boronic acid 9. Compound 22 was prepared in a similar
manner where an appropriate 2-iodo-hetrocycle 21 was coupled
with boronic acid 9 under standard Suzuki–Miyura conditions and
finally the Boc protecting group was removed with TFA (Scheme 2).
also exhibited anti-bacterial activity with MIC of 12.5 lg/ml (tested
against S. aureus stains RN4229 and MRSA252). They were able to
derive an X-ray crystal structure of 4 bound to MRSA PK and found
that it binds to the same site as the hydrazone compounds 1 and 3.
From examination of the crystal structure of cis-3-4-dihydro-
hamacanthin B (4) bound to S. aureus PK, it is apparent that the
two indole moieties lie in a linear relationship to each other and
are essentially in the same plane. The compound is anchored by
symmetric hydrogen bonds between Ser362 and Ser365 from
chains A and B, respectively, and the indole nitrogens (Fig. 2a).
The indole phenyl rings have prominent hydrophobic interactions
with Ile361 and His365. The two bromine atoms are oriented to-
wards the interior of the binding site in the deep hydrophobic
pocket formed by Thr353, Ser354, Ala358, and Leu370. The
symmetrical nature of the binding pocket was mirrored by the
Figure 2. Binding mode of compound 4 at the MRSA PK tetramer interface binding site; (a) a two-dimensional map (MOE software) of the binding interactions between 1 and
the interface site based on its co-crystallization with MRSA PK. Green arrows depict hydrogen-accepting interactions between 4 and MRSA PK residues from the interface
(left). (b) Modeled overlay of compound 10b with 4 in the MRSA binding site (right) (ICM software from PDB data file 3t07).

1710
N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
R1'
R2'
B(OH)2
N
Boc
9
R1
R2
R1
R1
R2
R1'
R2'
LDA, I2(CH2)2
THF,-78 oC,
R1
R2
TBAF, THF
I
I
N
N
H
N
H
N
H
R2
N
1) Pd(PPh3)2Cl2, Na2CO3
SO2Ph
ACN-H2O, μwave,100 o
C
SO2Ph
8
7
6
10
2) TFA,DCM,RT,1-2 h
NaH /THF
PhSO2Cl
R3X/K2CO3
R1'
B(OH)2
N
R1
R2
R2'
Boc
9
R1
R2
R1'
R2'
N
H
R1
R2
I
5
N
R3
N
R3
N
1) Pd(PPh3)2Cl2, Na2CO3
H
ACN-H2O, wave,100 oC
μ
11
12
2) TFA,DCM,RT,1-2 h
Scheme 1. General synthesis of bis-indoles 10 and 12.
1) Pd(PPh3)2Cl2, Na2CO3
R1
R1
R2
R1'
R2'
Z
Y
R1'
R2'
Z
Y
ACN-H2O, wave,100 oC
μ
I
B(OH)2
N
H
N
R2
2) TFA,DCM,RT,1-2 h
Boc
21
9
22
Y = O, Z = CH
Y = S, Z = CH
Y = S, Z = N
Scheme 2. General synthesis of mono-indole coupled with heterocycles.
B(OH)2
N
Br
Boc
9a
Br
1)BrCH2CO2Et/
NaH/THF 2 h
Br
R'R"NH, HBTU, DIPEA
DCM, RT, 12 h,
Br
Br
I
I
N
N
N
N
H
N
Br
Br
N
H
1) Pd(PPh3)2Cl2, Na2CO3
ACN-H2O, μwave,100 o
2) LiOH, THF
H
C
O
8b
HO2C
HO2C
2) TFA,DCM,RT,1-2 h
N
14
13
Bromoethanol, K2CO3
O
DMF, μwave,180o C, 20 min
15
9a
1) Pd(PPh3)2Cl2, Na2CO3
ACN-H2O, μwave,100 o
Br
MsCl, Et3N,
DCM
Br
Br
Br
C
R'R"NH,K2CO3
I
I
I
N
N
Br
N
N
DMF, 50 oC, 6 h
N
H
2) TFA,DCM,RT,1-2 h
NR'R"
NR'R"
OMs
16 OH
20
18
19
H
N
H
N
1) Pd(PPh3)2Cl2, Na2CO3
ACN-H2O, μwave,100 o
R'R"NH=
C
9a
N
O
Me
2) TFA,DCM,RT,1-2 h
Br
N
N
H
Br
17
OH
Scheme 3. General synthesis of bis-indoles 14, 15, 17 and 20.
Compound 14 was prepared from 8b where it was first treated
with alkyl bromide and then the ester was hydrolyzed with LiOH to
give the corresponding carboxylic acid derivative 13 which was
then coupled with 9a (Scheme 3). The carboxylic acid on 14 was
then reacted with morpholine and HBTU to give compound 15.
Treating intermediate 8b with 2-bromoethanol gave alcohol 12
that was then coupled with boronic acid 9a and subsequent
removal of Boc protecting group with TFA gave compound 17.
Compound 20 was prepared from alcohol 12 which was first con-
verted to the mesylate and then displaced by an amine to give
intermediate 19 which was subsequently coupled with 9a followed
by the removal of the Boc protecting group.
2-Acetylene-indole 24 was prepared by coupling 2-iodoindole 7
with TIPS-acetylene using Sonogashira coupling condition with

N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
1711
7
1) , PdCl2(PPh3)2,
1) TIPS-acetylene, PdCl2(PPh3)2,
R1
R2
R1
R2
R1
R2
R1'
R2'
CuI, ACN, μwave, 100 oC
CuI, ACN, μwave, 100 o
C
I
N
N
H
2) TBAF, THF, 2 h
N
H
N
H
2) TBAF, THF, 2 h
SO2Ph
7
24
25
MeI / K2CO3
R1
R2
R1'
R2'
N
Me
N
(Me or H)
26
Scheme 4. General synthesis of bis-indoles with acetylene linker.
H
N
Pd/C/Et3N/HCO2H
N
H
N
H
THF
Br
Br
N
H
Br
25
27a
H
N
H
Br
Br
N
TiCl4 / Zn
THF
CHO
N
H
N
H
N
H
Br
Br
Br
28a
27b
Br
CHO
SO2Ph
N
PhO2S
N
PPh3Br
OH
1) PBr3, DCM, RT
Br
DBU, DCM
RT,12 h
N
N
N
2) TPP,Benzene,
Reflux,12 h
Br
Br
Br
SO2Ph
SO2Ph
31
SO2Ph
30
29
Cs2CO3,MeOH-THF
90 οC,30 min.
H
N
H
N
Pt/C, MeOH-EtOAc
RT
Br
Br
N
H
N
H
Br
Br
27c
28b
Scheme 5. General synthesis of bis-indoles 27 and 28.
1) Pd(PPh3)2Cl2, Na2CO3
ACN-H2O, mwave, 100 o
C
B(OH)2
Ar
Y
Ar
Y
N
Boc
N
H
N
H
Br
Br
Br
2) TFA, DCM, rt, 1-2 h
32
9a
33a,b
B(OH)2
N
Br
1) Pd(PPh3)2Cl2, Na2CO3
ACN-H2O, mwave, 100 o
Boc
9a
C
Ar
B(OH)2
Ar Y
Y
Ar
Y
N
H
N
H
N
N
Br
1) Pd(PPh3)2Cl2, Na2CO3
ACN-H2O, mwave, 100 o
2) TFA, DCM, rt, 1-2 h
32
H
Boc
C
34
9
33c-e
2) TFA, DCM, rt, 1-2 h
Scheme 6. General synthesis of bis-indoles with an aryl linker.

1712
N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
PdCl2(PPh3)2 and CuI, and then the phenylsulfonyl protecting
group was removed with TBAF (Scheme 4). A second Sonogashira
coupling of intermediate 24 with 7 followed by removal of the
phenylsulfonyl group gave compound 25. Treating 25 with MeI
gave a mixture of mono-methylated compound 26a and dimethy-
lated compound 26b. Attempts were made to reduce the acetylene
linker of compound 25 as a route to synthesize 27b; however, we
were only able to isolate the mono-brominated compound 27a,
which was nonetheless useful for our SAR study (Scheme 5).
Compound 27b was finally synthesized by cross-coupling two
molecules of 6-bromo-1H-indole-2-carbaldehyde using titanium
tetrachloride and zinc dust, which also gave 28a as a bi-product.
Compound 27c was prepared from alcohol 29 where it was first
converted to Wittig salt 30 (Scheme 5). Compound 30 was then
coupled with the corresponding aldehyde to give 31 and finally
the phenylsulfonyl protecting groups were removed with Cs2CO3
to give the desired adduct 27c. The double bond of compound
27c was reduced by hydrogenation over Pt/C to give compound
28b.
Symmetrical bis-indoles 33a and 33b were prepared by double
Suzuki–Miyura reaction of boronic acid 9 with aryl di-halide 32
followed by the removal of the Boc protecting group with TFA
(Scheme 6). In order to prepare unsymmetrical bis-indoles 33c–e,
aryl di-halide was first coupled with 1 equiv of boronic acid 9 to
give intermediate 34 which was consequently coupled with a dif-
ferent boronic acid 9a and finally the Boc group was cleaved with
TFA to give the desired compounds.
antimicrobial activity was evaluated for ability to fully inhibit the
growth of S. aureus ATCC 29213 in culture (determining minimum
fully inhibitory concentration (MIC) values) (Tables 1–3). Selectiv-
ity for the MRSA PK over the four human isoforms was confirmed
for a selected series of compounds (Table 4). Cytotoxicity was eval-
uated for selected compounds with HEK 293 cells and they were
found not to be significantly cytotoxic at concentration up to
100 lg/ml (Table 5).
2.2.1. SAR for in vitro inhibition of MRSA PK
To further improve and optimize biological activity we set out
to develop SAR for both MRSA PK enzyme and the cellular antibac-
terial activity in parallel. For the purpose of clarity we will discuss
the SAR derived for inhibition of the MRSA PK enzyme separately
from the cellular antibacterial activity.
We systematically evaluated the effect of substitution on bis-in-
dole scaffold (Table 1), replacing one indole ring with a number of
heterocycles (Table 2) and modifying the central linking moiety
(Table 3).
The directly linked 6,60-dibromo-1H,10H-2,20-biindole (10b) was
prepared and we were pleased to note that it was a potent inhibi-
tor of the MRSA PK enzyme with an IC50 of 7.0 nM and that it also
gave an MIC against S. aureus of 16 lg/ml (Table 1). To evaluate the
role of the two bromines, the mono-brominated compound 10a
was prepared and was found to be about threefold less active.
However, the asymmetrically substituted 6,50-dibrominated com-
pound 10c was more potent with IC50 of 2.2 nM. It was found that
the 50-bromine could be substituted with chloro (10d), fluoro
(10e), methoxy (10f) or even with a relatively bulky group such
as phenyl (10g) without significant loss of potency suggesting that
there are still some room in the binding pocket which might be fur-
ther exploited to improve activity.
2.2. Biological results and SAR studies
All compounds prepared were screened against in an in vitro
enzyme assay to assess MRSA PK inhibitory activity. Cellular
Table 1
PK inhibitory activity and antimicrobial activity of substituted bis-indoles
R1 R2
7
7'
N
N
6'
5'
6
5
1
2
1'
2'
3
3'
4
4'
MICb
(l
g/ml)
R1
R2
Substitutions
a
Analogs
IC50 (nM)
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
12a
12b
12c
14
21.4
7.0
2.2
2.0
2.8
2.5
2.2
36% @ 10
3.0
39% @ 10
1.5
2.0
1.6
1.0
25% @ 10
1.9
2.0
16.0
0.3 (3)
0.3
0.3
>64
>64
ND
>64 (2)
ND
0.5
>64
1.0
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
6-Bromo
6,60-Dibromo
6,50-Dibromo
6-Bromo-50-chloro
6-Bromo-50-fluoro
6-Bromo-50-methoxy
6-Bromo-50-phenyl
5-Bromo
l
M
M
5,50-Dibromo
5,6-Dibromo
l
5,6,60-Tribromo
5,50,6,60-Tetrabromo
5,6,50-Tribromo
6,50-Dibromo
>64 (2)
ND
>64
>64
l
M
6,50-Dibromo
CH2OCH3
CH2COOH
6,50-Dibromo
11.1
6,50-Dibromo
N
O
15
6.0
>64
H
6,50-Dibromo
O
17
1.3
2.0
2.0
4.0
H
H
CH2CH2OH
6,50-Dibromo
6,50-Dibromo
N
N
20a
N
20b
1.2
>64
H
6,50-Dibromo
O
a
IC50 values are calculated from a triplicate 15-point titration. Alternatively the % inhibition at the highest concentration tested is presented.
Minimum concentration to give >98% inhibition of growth of S. aureus ATCC 29213 (single determination or average of (n) determinations). Control MIC (vancomycin) is
b
1
l
g/ml.

N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
1713
10a and comparable in potency to 5,50-dibromo-bis-indole 10i, and
hence might have a similar mode of binding.
Table 2
PK inhibitory activity and antimicrobial activity of compounds 22a–g
7
Next we investigated whether a spacer moiety could be placed
between the two indoles. Compound 25a, with an acetylene linker,
was found to be threefold more potent than direct linked com-
pound (10a) indicating that the binding pocket has more linear
space. Next we made the 6,60-dibromo analog (25b) and 6,50-dibro-
mo analog (25c) with an acetylene linker, but there was no signif-
icant improvement in potency. One indole NH could be methylated
(26a) without significant loss in activity, but as expected, methyl-
ation of both NH (26b) led to a compound that was essentially
inactive. Surprisingly 27a and 27b with an ethylene linker was al-
most equipotent as the corresponding acetylene linker. We had
anticipated a loss in potency as the two indoles should adopt a lin-
ear staggered conformation with ethylene linker that should dis-
place one of the indoles within the binding pocket but evidently
this is not detrimental. More interestingly, compounds (28a and
28b) with the fully saturated ethane linker moiety were still very
potent, both with IC50 of 6 nM. Compounds 28a and 28b presum-
ably are able to orient in a staggered linear and planer conforma-
tion similar to 27a and 27b to bind in the flat lipophilic pocket
similar to the natural product 4. In order to make the spacer
slightly longer we synthesized 1,4-phenyl linked compound 33a.
There was a significant drop in activity observed for 33a (as com-
pared to 25a) and modelling suggests that it might be now too long
to fit in the binding pocket. Subsequently, we made a slightly
shorter analog 33c (by removing one bromine atom) and this com-
pound was found to be very potent (IC50 of 5.6 nM). Hence it ap-
pears that 33c is occupying most of the binding pocket and it
defines the breadth of the site.
7'
H
1
N
X
6'
5'
6
5
2
1'
2'
Y
3
3'
4
4'
MICb
(
l
g/ml)
X
Y
Substitutions
a
Analogs
IC50 (nM)
10a
22a
22b
22c
22d
22e
22f
21.4
2.0
151.7
107.6 (2)
14.0
>64
>64
>64
>64
ND
S
N
6-Bromo
6-Bromo
6-Bromo
5-Bromo
5-Bromo
5-Bromo
5,60-Dibromo
O
S
CH
CH
N
CH
CH
N
358.1
S
45% @ 10
42% @ 1
4.1
l
l
M
M
O
S
>64
>64
22g
S
a
IC50 values are calculated from a triplicate 15-point titration or are an average
of (n) such determinations as indicated. Alternatively the % inhibition at the highest
concentration tested is presented.
Minimum concentration to give >98% inhibition of growth of S. aureus ATCC
29213 (single determination). Control MIC (vancomycin) is 1 lg/ml.
b
We next prepared the 50-monobrominated bis-indole analog
(10h) and noted a significant drop in activity. This suggested that
at least one bromine atom in the 6-position of one of the indole
moieties was critical for activity. On the other hand, however,
the 5,50 dibromo bis-indole (10i) was found to be very potent with
an IC50 of 3 nM. It is not clear why 10h was not active whereas 10i
is potent but one possible explanation could be that one of the bro-
mines of 10i is oriented towards the interior of the binding pocket
and the other bromine is facing outwards, thus placing the indole
NH towards the interior to provide a necessary hydrogen bonding
with Ser362. Mysteriously, the 5,6 dibromo compound 10j was not
potent whereas the 5,6,60- and 5,6,50-tribromo compounds 10k and
10m and tetrabromo bis-indole 10l were found to be very potent.
The activity of these relatively large molecules was in keeping with
there being still more space in the binding pocket to be exploited
(discussed later, Table 3).
Next we investigated the effect of substitution on the NH of in-
dole. The mono-N-methyl bis-indole 12a was very active with an
IC50 of 1.0 nM whereas the isomeric mono-N-methyl bis-indole
12b was almost inactive. This further confirmed that the presence
of a bromine atom at the 6-position relative to the indole NH is
important for activity. It appears that the bromine and NH of
6-bromo-indole fragment binds very tightly to the pocket and
there is no room for further substitution in that region of the
molecule. The methyl group in 12a is most likely oriented towards
the outside (water side) of the binding site and that explains why
compounds 12c, 14, 15, 17, 20a and 20b with bulky groups
attached to the nitrogen atom of the 5-bromo indole are still very
potent. There was no further improvement in activity by either
introducing polar (12c, 15, 17 and 20b), acidic (14) or basic (20a)
groups at NH of the second indole.
It was noted that if 33 was modelled into the binding site, the
central ring was flanked above and below by the His-365 residues.
Considering that these interactions might lead to some potential
for charge transfer interaction, we investigated the effect of placing
an electron-withdrawing group (33d) or an electron-releasing
group (33e) on the phenyl linker. However both 33d and 33e were
about 2–3 fold more potent than 33c so it is not clear if such an
interaction exists. 1,3-Phenyl-linked (33f) and 2,5-thiophene
linked (33b) were essentially inactive which demonstrates that
both indoles have to be able to adopt an essentially linear attitude
for significant binding.
2.2.2. SAR for anti-S. aureus activity
For those tested, compounds with low potency against MRSA PK
(IC50 >100 nM) were also poorly effective (MIC >64 lg/ml) against
S. aureus ATCC95923. However while many potent inhibitors of
MRSA PK gave low MICs (e.g., compound 10d), other potent ana-
logs had high MICs. This suggested that issues such as solubility,
cellular penetration or active export from the bacterial cells might
impair full translation of activity. The MIC of the initial lead com-
pound 10b was similar to that reported for cis-3-4-dihydroham-
acanthin B (4). However, compound 10a with one less bromine
was about eightfold more potent than 10b despite it being three-
fold less potent in MRSA PK enzyme assay. Direct linked bis-indole
compounds 6,50-dibromo (10c), 6-bromo-5-chloro (10d) and
Our next step was to investigate whether both indoles are crit-
ical for binding or if one can be replaced with other heterocycles.
Keeping the 6-bromoindole element constant and replacing the
second indole with benzothiazole (22a) or benzofuran (22b) led
to a 5–8-fold decrease in activity (compared to 10a). However,
the benzothiophene derivative (22c) is slightly more potent than
10a suggesting that one indole can indeed be replaced with benzo-
thiophene. In the cases where we kept one 5-bromoindole constant
and replaced the other indole with benzothiazole (22d), benzofu-
ran (22e) or benzothiophene (22f), there was a significant drop
in activity as expected from earlier observation. Nevertheless,
6-bromothiazole analog (22g) was about fivefold more potent than
6-bromo-5-fluoro (10e) were most potent with MICs of 0.3 lg/
ml. However, although 5,50-dibromo derivative 10i and tetrabromo
10l were equipotent in vitro compared to 10b–10e, they had much
higher MICs (>64 lg/ml). Both tribromo substituted compounds
10k and 10m showed excellent MICs.
Many of these compounds had limited solubility that might
limit ability to access and to penetrate cells and therefore a num-
ber of potentially solubilizing groups were installed on one of the
indole NHs (see compounds 12c, 14, 15, 17, 20a and 20b).
However, although all these compounds were potent inhibitors
of MRSA PK, only the hydroxyethyl analog 17 and piperazinylethyl

1714
N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
Table 3
PK inhibitory activity and antimicrobial activity of compounds with modification on the linker
R1
R2
N
7
7'
4'
N
6
6'
5'
1
2
1'
2'
Linker
5
3
3'
4
a
Analogs
10a
IC50 (nM)
MICb
(lg/ml)
Linker
R1
R2
Substitutions
21.4
7.1
2.0
25a
>64
H
H
6-Bromo
25b
25c
26a
26b
2.4
>64
16
H
H
6,60-Dibromo
6,50-Dibromo
6,60-Dibromo
6,60-Dibromo
4.7
H
H
13.7
>64
ND
CH3
CH3
H
27% @ 10
4.5
l
M
CH3
27a
27b
27c
28a
>64
>64
>64
>64
H
H
H
H
H
H
H
H
6-Bromo
3.9
4.1
6.5
6.3
6,60-Dibromo
6,50-Dibromo
6,60-Dibromo
28b
33a
33c
>64
>64
>64
H
H
H
H
H
H
6,50-Dibromo
6,60-Dibromo
6-Bromo
54% @ 1
5.6
lM
O2N
33d
33e
1.8
2.0
H
H
H
H
6-Bromo
6-Bromo
MeO
2.1
>64
33f
36% @ 10
l
M
ND
H
H
H
H
6-Bromo
S
33b
28% @ 1
l
M
>64
6,60-Dibromo
a
IC50 values are calculated from a triplicate 15-point titration. Alternatively the % inhibition at the highest concentration tested is presented.
Minimum concentration to give >98% inhibition of growth of S. aureus ATCC 29213 (single determination). Control MIC (vancomycin) is 1 lg/ml.
b
derivative 20a showed reasonable MICs. Hence, it appears that
there was no clear correlation between the MIC and polarity. In
the cases where one indole was replaced with a heterocycle
(22a–g), none gave significant MIC despite being potent in the
enzyme assay (e.g., compound 22g). Of all the compounds made
with a spacer between the two indoles only 25c and 33d gave
significant MIC values further exemplifying the lack of correlation
of enzymatic activity and anti-bacterial activity. Of all the phenyl-
linked compounds made, only 33d bearing an electron-withdraw-
ing group was MIC active.
antibiotics.9 The antihypertensive drug, verapamil has been shown
to be an inhibitor the ABC class of pumps in Gram-positive bacteria
such as Staphylococci and verapamil has been demonstrated to
inhibit efflux pumps in S. aureus.10,11 We therefore evaluated the
effect of verapamil on the observed MIC for several key compounds.
MICs were determined for S. aureus ATCC92953 in the absence and
presence of verapamil (200 mg/L,
reported as an optimal sub-inhibitory concentration11). Vancomycin
was used as control and gave the same MIC (2 g/ml) in presence
and absence of verapamil as did compound 10d (MIC = 0.5 g/ml).
However, the MIC of 10l was shifted at least fourfold (from >64 to
16 g/ml) by verapamil while the MIC of 10b was dramatically
shifted, 32-fold (from 16 to 0.5 g/ml) (Fig. 3).
a concentration previously
l
l
2.2.3. Evaluation of the effect of verapamil on the MIC of
selected PK inhibitors
l
l
Considering the divergent anti-S. aureus activity observed
among very close structural analogs such as 10b and 10l compared
to 10c, 10d or 10e, in spite of similar IC50s for inhibition of MRSA
PK, it seemed unlikely that cell penetrability or solubility could
explain these discrepancy. One possibility is that compounds such
as 10b may be substrates for putative bacterial efflux systems, such
as has been proposed to impart resistance of many bacteria to
2.2.4. Evaluation of efficacy of selected MRSA PK inhibitors
against MRSA
To further investigate the antibacterial properties of this class of
PK inhibitors, the three of the most potent compounds (10c, 10d
and 10e) from the SAR study were tested in vitro for their antibac-
terial activities against MRSA strain MW2 (USA400) with

N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
1715
Table 4
Inhibition of enzymatic activities of MRSA PK and human PK isoforms at 10
l
M
Analogs
% Inhibition of MRSA PK
% Inhibition of human M1
% Inhibition of human M2
% Inhibition of human R
% Inhibition of human L
10b
10c
10i
10k
10l
84.9 (2)
97.8
71
10.3 (2)
6.9
12.3
8.6
11.1
13.9
14.6
3.2
13.1
12.4
5.3
19.5
16.1
9.9
9.5
7.3
0.1 (2)
8.7
0.5
15.8
2.3
9.1
12.1
1.7
3.5
3.0
0.4
18.3 (2)
13.6
24.4
33.4
20.7
2.7
25.2
26.1
50.7
21.6
ꢀ5.5
27.8
23.8
24.1
0.0
7.9 (2)
6.1
9.8
23.7
8.2
2.8
94.1
97.6
99.0
100.9
100.1
100.6
89.8
95.9
86.3
93.4
93.4
86.2
62.2
12a
17
6.8
20a
20b
22a
22b
25c
27a
28a
33d
33e
16.8
32.1
7.8
ꢀ6.0
14.0
12.9
9.7
8.7
ꢀ0.9
1.3
6.8
14.6
ꢀ8.9
13.5
4.3
Table 5
Mammalian cell cytotoxicity of selected compounds
work has described PK as an essential enzyme required for
growth.2,3 In each of these previous studies however, the growth
medium used contained primarily glycolytic carbon sources such
as glucose (G). By designing media lacking carbohydrates but re-
plete with pyruvate (P), PK becomes dispensable and a mutant
can therefore be made that has relatively no growth defect under
Analogues
% Cell viability found on incubation with HEK 293 cell line
1
lg/ml
10
lg/ml
100 lg/ml
10b
10f
103
107
106
88
118
72
63
73
115
these specific conditions (Fig. 5 shows that in TSB-G+P, the
Dpyk
10k
12c
27a
28a
102
123
107
104 (2)
71
102
114
and WT grow identically in the absence of 10d). Initial character-
ization of the mutant revealed that it exhibits an aerobic growth
defect when grown on Brain–Heart Infusion Broth (BHI) but not
on Tryptic Soy Broth (TSB) without glucose, provided the medium
is supplemented with 1% sodium pyruvate (TSB-G+P) (Fig. 5). The
92 (2)
85 (2)
growth defect exhibited by S. aureus
D
pyk::ErmR on BHI was
vancomycin as positive control. In each case an MIC of 0.5
lg/ml
was observed while vancomycin gave an MIC of 2 g/ml (Fig. 4).
l
reversible via complementation using a plasmid expressing pyk
from a constitutive promoter (data not shown). These data indicate
that the metabolic activity of PK is required for aerobic growth of S.
aureus on glycolytic carbon sources (e.g., BHI) but not certain
combinations of gluconeogenic carbon sources (e.g., TSB-G+P).
We next examined the bacteriostatic and bactericidal effects of
2.2.5. Evaluation of MRSA PK inhibitors in bacteria pyruvate
kinase deficient S. aureus cells
To implicate 10d-mediated inhibition of S. aureus PK as the di-
rect cause of bacterial killing, we constructed a S. aureus LAC
pyk::ErmR mutant. Essentially, we replaced the pyk gene on the
10d on wild type (WT) S. aureus as well as the
D
pyk::ErmR. We
D
chromosome of S. aureus with an ErmR cassette using a standard
found that, while 10d was highly bactericidal to WT S. aureus,
allelic exchange procedure as previously described.12 Previous
the drug merely acted statically on the
D
pyk::ErmR mutant in
Vancomycin
10d
MHCAB
MHCAB
MHCAB + Verapamil
120
100
80
60
40
20
0
120
100
80
60
40
20
0
MHCAB+Verapamil
MIC
MIC
MHCAB = 2µg/ml
MHCAB = 0.5µg/ml
MHCAB + Verapamil = 2µg/ml
MHCAB + Verapamil = 0.5µg/ml
0.1
1
10
100
0.1
1
10
100
-20
-20
Concentration (µg/mL)
Concentration (µg/mL)
10b
10l
MHCAB
MHCAB
120
MHCAB + Verapamil
120
100
80
60
40
20
0
MHCAB + Verapamil
100
80
60
40
20
0
MIC
MIC
MHCAB > 64µg/ml
MHCAB > 16µg/ml
MHCAB + Verapamil = 16µg/ml
MHCAB + Verapamil = 0.5µg/ml
0.1
1
10
100
0.1
1
10
100
-20
-20
Concentration (µg/mL)
Concentration (µg/mL)
Figure 3. The MIC determinations of 10d, 10b and 10l in the presence and absence of efflux inhibitor Verapamil with S. aureus (ATCC29213). Assay of vancomycin, 10d and
10b was performed in triplicate. Compound 10l was performed in duplicate.

1716
N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
Vancomycin
10d
140
120
100
80
60
40
20
0
-20
-40
140
120
MIC = 0.5 µg/ml
MIC = 2 µg/ml
100
80
60
40
20
0
0.1
1
10
100
0.1
1
10
100
-20
-40
Concentration (µg/mL)
Concentration (µg/mL)
10e
10c
140
120
100
80
140
120
100
80
MIC = 0.5 µg/ml
MIC = 0.5 µg/ml
60
60
40
20
40
20
0
-20
-40
0
-20
-40
0.1
1
10
100
0.1
1
10
100
Concentration (µg/mL)
Concentration (µg/mL)
Figure 4. MIC determinations of 10c, 10d and 10e with MRSA strain MW2 (USA 400).
BHI medium (Fig. 5A). The
D
pyk::ErmR mutant grows slower than
3. Conclusions
WT in BHI medium and to test whether this had any effect on 10d
toxicity, we tested the compound’s effect in media in which the
mutant has no growth defect. As in BHI, 10d was unable to kill
Based on observations from the published crystal structure of a
naturally occurring marine alkaloid 4 bound to the tetrameric
MRSA PK enzyme, a novel series of bis-indoles were [stat] designed
and prepared and shown to be very potent inhibitors of MRSA
pyruvate kinase. Compounds such as 10c, 10d, 10e, 10k, 10m
and 12a are among the most potent inhibitors of this enzyme iden-
tified to this date with IC50s of 1–3 nM. Numerous analogs were
synthesized in order to define structure–activity relationships
and to further optimize the physiochemical properties for effective
inhibition of bacterial growth. Lead compounds were very selective
for the bacterial PK over the human isoforms. Although there was,
overall, a poor correlation between the in vitro enzyme inhibitory
activity and anti-bacterial activity against S. aureus ATCC 29213,
some compounds showed excellent MIC values of as low as
the
(Fig. 5B). Thus, while 10d efficiently eliminated growth of both
WT and
pyk::ErmR S. aureus (no significant difference in 10d
D
pyk::ErmR mutant in TSB-G+P as it did WT S. aureus
D
MIC for either strain), the bactericidal activity of this compound
is completely dependent on PK activity (Fig. 5).
2.2.6. Selectivity and cytotoxicity
For selected compounds selectivity for MRSA PK relative to the
mammalian PK isoforms M1, M2, R and L (Table 4) was determined
using enzymes and methods as previously described.4,8 Those
compounds tested for selectivity generally showed no significant
inhibition at the highest concentration tested (10 lM) and only
20b showed inhibition of a mammalian PK isoform of greater than
50% (51% for the R isoform). Cytotoxicity was evaluated for
selected compounds with HEK 293 cells as previously described4,8
and they were found not to be significantly cytotoxic at concentra-
0.3
l
g/ml while lacking significant mammalian cytotoxicity.
Considering that this lack of correlation might be due (at least
in part) to selective active efflux from the bacteria, studies were
carried out on the effect of the known efflux inhibitor, verapamil,
on the MIC values of selected close structural analogs, 10a, 10d
and 10l. These studies revealed a pronounced enhancement of
tion up to 100 lg/ml (Table 5).
Figure 5. Resistance of S. aureus LAC
D
pyk::ErmR to killing by compound 10d. (A) Resistance of S. aureus LAC
pyk::ErmR to killing by 1.6
M 10d during aerobic growth in TSB without glucose supplemented with 1% sodium pyruvate. Significance
pyk::ErmR LAC treated with 10d at 8 and 24 h using a Students two-sided t-test (⁄p-value 60.05, ⁄⁄p-value 60.0001). Error bars = SD.
D lM 10d during aerobic growth in
pyk::ErmR to killing by 1.6
BHI. (B) Resistance of S. aureus LAC
was assessed between WT LAC and
(n = 3).
D
D
l

N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
1717
MIC values for 10a and 10l but not for 10d (or vancomycin control)
indicating that at least for these key compounds the attenuated
antibacterial activity is due to active efflux mechanisms.
Compounds 10c, 10d and 10e were further evaluated for effi-
cacy against MRSA strain MW2 where MICs (0.5 lg/ml) similar to
EtOAc (2 ꢁ 50 ml). The combined organic phases were washed
with brine, dried over anhydrous Na2SO4, filtered and concentrated
in vacuo. The crude product was purified by automated flash chro-
matography using EtOAc and hexanes as eluents to give the desired
product.
those obtained for S. aureus ATCC 29213 were observed. In previ-
ous studies on MRSA pyruvate kinase inhibitors4,8 potency against
S. aureus ATCC 29213 was found to be representative of potency
against MRSA strains and thus we infer that this will also be the
case for this class of compounds.
In order to gain further insight into the mechanism of action of
these compounds on S. aureus we evaluated the effects of the
compound 10d on a S. aureus mutant where the gene for pyruvate
kinase (Pyk) is knocked out. These cells are viable when cultured in
4.1.1.2. General procedure for the synthesis of 2-iodo-(phenyl-
sulfonyl)-1H-indole (7).
To a stirred solution of 6 (1 mmol) in
anhydrous THF (10 ml) at ꢀ78 °C was added a solution of LDA
(1.5 mmol) in THF (5 ml). The mixture was stirred for at ꢀ78 °C
for 100 min and then warmed to 0 °C for 30 min. The solution
was re-cooled to ꢀ78 °C and then either a solution of 1,2-diiodo
ethane or molecular iodine (1.5 mmol) in THF (10 ml) was added.
The reaction mixture was stirred at 0 °C for 15 min and then al-
lowed for warm to room temperature for 1 h. The reaction was
quenched with saturated NH4Cl solution and extracted with EtOAc
(2 ꢁ 50 ml). The combined organic phases were washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
The crude product was purified by automated flash chromatogra-
phy using EtOAc and hexanes as eluents to give the desired
product.
4.1.1.3. 6-Bromo-2-iodo-1-(phenylsulfonyl)-1H-indole (7a).
Yield = 53%, white solid. 1H NMR (CDCl3, 400 MHz): d 8.16 (d,
J = 9.0 Hz, 1H), 7.87 (d, J = 7.6 Hz, 2H), 7.58 (t, J = 7.5 Hz, 1H), 7.5
(d, J = 1.6 Hz, 1H), 7.46 (t, J = 7.8 Hz, 2H), 7.37 (dd, J = 9.0, 1.9 Hz,
1H), 6.93 (s, 1H). HRMS calcd for (C14H9BrINO2SꢀH)ꢀ 460.8582;
found 460.85998.
a
medium lacking usable carbohydrate and additionally
supplemented with pyruvate. These studies showed that the
bacteriostatic and bactericidal effects of 10d on S. aureus occur
by different mechanisms. The bacteriostatic effects of 10d are
pyk-independent while the bactericidal effects of 10d are
pyk-dependant. The bactericidal mechanism of 10d does not
require PK metabolic activity (10d is bactericidal to S. aureus on
TSB-G+P, a growth media where pyk::ErmR exhibits no growth
defects). This supports the model of pyruvate kinase as a highly
interactive protein and the expectation that pyruvate kinase inhib-
itors of this class may yield effective anti-MRSA compounds with
low potential for development of resistance.
The lead compounds (10c, 10d, 10e, 10k and 10m) with best
MIC values are currently being evaluated to determine in vivo
absorption and tissue exposure in preparation for evaluation in
in vivo animal infection models. These studies will be reported
elsewhere.
4.1.1.4. 5-Bromo-2-iodo-1-(phenylsulfonyl)-1H-indole (7b).
Yield = 54%, white solid. 1H NMR (CDCl3, 400 MHz): d 8.15 (d,
J = 9.0 Hz, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.56 (t, J = 8.1 Hz, 1H), 7.53
(d, J = 1.7 Hz, 1H), 7.44 (t, J = 7.5 Hz, 2H), 7.37 (dd, J = 2.0, 9.0 Hz,
1H), 6.91 (s, 1H).
4. Experimental section
4.1. Chemistry
4.1.1.5. 5-Chloro-2-iodo-1-(phenylsulfonyl)-1H-indole (7c).
Yield = 75%, white Solid. 1H NMR (CDCl3, 400 MHz): d 8.21 (d,
J = 9.0 Hz, 1H), 7.84–7.90 (m, 2H), 7.55–7.61 (m, 1H), 7.46 (dt,
J = 7.4, 1.8 Hz, 2H), 7.38 (d, J = 1.9 Hz, 1H), 7.24 (dd, J = 9.0,
2.2 Hz, 1H), 6.93 (d, J = 0.7 Hz, 1H). 13C NMR (CDCl3, 125 MHz):
138.12, 136.95, 134.44, 132.87, 129.82, 129.42, 127.31, 126.80,
125.21, 123.43, 119.36, 116.55 ppm. HRMS calcd for (C14H9ClINO2-
SꢀH)ꢀ 416.9087; found 416.9094.
4.1.1. General synthesis methods
1H and 13C NMR spectra were recorded with either Bruker
Avance II™ 600 MHz, Bruker Avance III™ 500 MHz or Bruker
Avance III™ 400 MHz. Processing of the spectra was performed
with MestRec™ software. The high-resolution mass spectra were
recorded either in positive or negative ion-mode with an ESI or
multimode ESI/APCI ion source on an Agilent™ 6210 Time-
of-Flight LC/MS mass spectrometer. Analytical thin-layer chroma-
tography (TLC) was performed on aluminum plates pre-coated
with silica gel 60F-254 as the absorbent. The developed plates
were air-dried, exposed to UV light and/or dipped in KMnO4 solu-
tion and heated. Column chromatography was performed with sil-
ica gel 60 (230–400 mesh). Automated flash chromatography was
carried out on Biotage Isolera Flash Purification Systems using
4.1.1.6. 5-Fluoro-2-iodo-1-(phenylsulfonyl)-1H-indole (7d).
Yield = 94%, white solid. 1H NMR (CDCl3, 400 MHz): d 8.23 (dd,
J = 9.2, 4.4 Hz, 1H), 7.84–7.89 (m, 2H), 7.54–7.61 (m, 1H), 7.42–
7.48 (m, 2H), 6.97–7.08 (m, 2H), 6.95 (d, J = 0.5 Hz, 1H). HRMS
calcd for (C14H9FINO2SꢀH)ꢀ 400.9383; found 400.9392.
4.1.1.7. General procedure for the synthesis of substituted 2-
iodo-1H-indole (8).
To a stirred solution of compound 7
commercial 50 lm silica gel cartridges. Purity (>95%) for all final
(1 mmol) in THF (20 ml) at room temperature was added a solution
of TBAF (1 ml, 1 M in THF, 1 mmol). The mixture was stirred at
ambient temperature for 5 h and then partitioned between EtOAc
(100 ml) and H2O (50 ml). The organic phase was washed with
brine (50 ml), dried over anhydrous Na2SO4 and concentrated.
The residue was purified by automated flash chromatography
using EtOAc and hexanes as eluents to give compound 8.
compounds was confirmed by analytical reverse-phase HPLC uti-
lizing a Dikma Technologies™ InspireÒ C18 reverse-phase analyti-
cal column (4.6 ꢁ 150 mm) using ACN and H2O with 0.1% formic
acid as eluent. All HPLC purifications were carried out using an Agi-
lent™ C18 reverse-phase preparatory column (21.2 ꢁ 250 mm)
using ACN and H2O with 0.1% formic acid as eluent.
4.1.1.1. General procedure for the synthesis of 1-(phenylsulfo-
4.1.1.8. 6-Bromo-2-iodo-1H-indole (8a).
Yield = 48%, white
nyl)-1H-indole (6).
To a stirred solution of an appropriate in-
solid. 1H NMR (CDCl3, 400 MHz): d 8.07 (br s, 1H), 7.46–7.50 (m,
1H), 7.39 (d, J = 8.4 Hz, 1H), 7.19 (dd, J = 8.4, 1.7 Hz, 1H), 6.69 (dd,
J = 2.0, 0.9 Hz, 1H). 13C NMR (DMSO-d6, 125 MHz): 139.38,
128.33, 122.17, 120.23, 114.01, 112.90, 110.84, 79.73. HRMS calcd
for (C8H5BrINꢀH)ꢀ 320.8650; found 320.8658.
dole (1 mmol) in THF (25 ml) at 0 °C was added NaH (60% in oil,
1.2 mmol) gradually. After stirring at room temperature for
10 min benzenesulphonyl chloride (1.2 mmol) was added and the
mixture was further stirred for 2 h. The reaction was quenched
with saturated ammonium chloride solution and extracted with

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N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
4.1.1.9. 5-Bromo-2-iodo-1H-indole (8b).
Yield = 92%, white
anhydrous Na2SO4 and concentrated. The residue was dissolved in
solid. 1H NMR (DMSO-d6, 400 MHz): d 11.9 (br s, 1H), 7.65 (d,
J = 1.6 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H), 7.15 (dd, J = 1.9, 8.6 Hz,
1H), 6.6 (s, 1H).
a mixture of THF and LiOH solution at 0 °C and stirred at rt until the
completion of the reaction as indicated by TLC. The reaction mix-
ture was acidified and extracted with EtOAc. The organic phase
was washed with brine, dried over anhydrous Na2SO4 and concen-
trated. The crude product was purified by automated flash chroma-
tography to give white solid (1.01 g, 89%). 1H NMR (DMSO-d6,
500 MHz): d 13.17 (br s, 1H), 7.69 (d, J = 1.8 Hz, 1H), 7.50 (d,
J = 8.8 Hz, 1H), 7.21 (dd, J = 8.7, 2.0 Hz, 1H), 6.81 (s, 1H), 5.0 (s,
2H). 13C NMR (DMSO-d6, 125 MHz): 169.46, 136.74, 130.82,
123.92, 121.11, 112.36, 112.26, 111.22, 89.24, 48.26. HRMS calcd
for (C10H7BrINO2ꢀH)ꢀ 378.8705; found 378.8691.
4.1.1.10. 5-Chloro-2-iodo-1H-indole (8c).
Yield = 96%, pale
brown solid. 1H NMR (CDCl3, 400 MHz): d 8.12 (br s, 1H), 7.51 (d,
J = 2.0 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.09 (dd, J = 8.6, 2.0, 1H),
6.67 (d, J = 1.2 Hz, 1H). 13C NMR (DMSO-d6, 125 MHz): d 137.16,
130.31, 123.94, 121.09, 117.68, 111.88, 110.33, 80.64. HRMS calcd
for (C8H5ClINꢀH)ꢀ 276.9155; found 276.9158.
4.1.1.11. 5-Fluoro-2-iodo-1H-indole (8d).
Yield = 85%, white
solid. 1H NMR (CDCl3, 400 MHz): d 8.08 (br s, 1H), 7.22–7.25 (m,
1H), 7.19 (dd, J = 9.4, 2.5 Hz, 1H), 6.89 (td, J = 9.1, 2.5 Hz, 1H),
6.68 (dd, J = 2.0, 0.8 Hz, 1H).
4.1.1.18. Synthesis of 2-(5-bromo-2-iodo-1H-indol-1-yl)ethanol
(16).
was added bromoethanol (14 mg, 0.12 mmol) and K2CO3 (49 mg,
0.36 mmol). The reaction heated at 180 °C with wave for 30 min
To a solution of 8b (38 mg, 0.11 mmol) in DMF (1 ml)
l
4.1.1.12. 5,6-Dibromo-2-iodo-1H-indole.
Yield = 89%, white
and then partitioned between EtOAc and H2O. The organic phase
was washed with brine, dried over anhydrous Na2SO4 and concen-
trated. The residue was purified by automated flash chromatogra-
phy using EtOAc and hexanes as eluents to afford 16 (24 mg, 59%)
as white solid. 1H NMR (CDCl3, 500 MHz): d 7.65 (s, 1H), 7.28 (d,
J = 7.7 Hz, 1H), 7.23 (dd, J = 8.7, 1.8 Hz, 1H), 6.75 (s, 1H), 4.32 (t,
J = 5.6 Hz, 2H), 3.9–3.97 (m, 2H).
solid. 1H NMR (CDCl3, 400 MHz): d 8.09 (br s, 1H), 7.80 (s, 1H),
7.69 (d, J = 0.7 Hz, 1H), 6.64 (dd, J = 0.8, 2.3 Hz, 1H).
4.1.1.13. General procedure for the synthesis of substituted 2-
iodo-1-methyl-1H-indole (11).
A mixture of an appropriate
2-iodo-1H-indole 8 (1 mmol), K2CO3 (2 mmol) and methyl iodide
(1.5 mmol) in DMF was stirred at room temperature for 3 d. The
reaction mixture was partitioned between EtOAc (100 ml) and
H2O (50 ml). The organic phase was washed with brine (50 ml),
dried over anhydrous Na2SO4 and concentrated. The residue was
purified by automated flash chromatography using EtOAc and hex-
anes as eluents to afford compound 11.
4.1.1.19. Synthesis of 2-(5-bromo-2-iodo-1H-indol-1-yl)ethyl-
methanesulfonate (18).
To a stirred solution of 16 (3.0 g,
8.2 mmol) in DCM (20 ml) at 0 °C was added Et3N (1.8 ml,
12.4 mmol) and methanesulfonyl chloride (0.64 ml, 8.2 mmol).
The reaction mixture was stirred at rt for 1 h and then quenched
by the addition of ice. The reaction mixture was extracted into
DCM (3 ꢁ 10 ml) and the combined extract was washed with brine,
dried over anhydrous Na2SO4 and concentrated to give essentially
pure compound 14 as brown solid (2.98 g, 82%). 1H NMR (CDCl3,
500 MHz): d 7.66 (m, 1H), 7.24–7.28 (m, 2H), 6.78 (s, 1H), 4.45–
4.55 (m, 4H), 2.71 (s, 3H). HRMS calcd for (C11H11BrINO3S)ꢀ
442.8688; found 442.8686.
4.1.1.14.
(11a).
6-Bromo-2-iodo-1-methyl-1H-indole
Yield = 97%, white solid. 1H NMR (CDCl3, 400 MHz): d
7.46–7.47 (m, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.17 (dd, J = 8.4,
1.7 Hz, 1H), 6.76 (d, J = 0.8 Hz, 1H), 3.72 (s, 3H).
4.1.1.15. 5-Bromo-2-iodo-1-methyl-1H-indole (11b).
Yield = 99%,
white solid. 1H NMR (CDCl3, 500 MHz): d 7.63 (d, J = 1.6 Hz, 1H), 7.22
(dd, J = 8.7, 1.8 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 6.72 (s, 1H), 3.73 (s,
3H). HRMS calcd for (C9H7BrIN)ꢀ 334.8807; found 334.8780.
4.1.1.20. General procedure for the synthesis of 2-iodoindole
derivative (19).
To a stirred solution of the intermediate 18
(1 mmol) in DMF (3 ml) was added K2CO3 (1.5 mmol) and the cor-
responding amine (3 ml). The mixture was heated at 50 °C for 12–
20 h and then partitioned between EtOAc and H2O. The organic
phase was washed with saturated NH4Cl, brine, dried over anhy-
drous Na2SO4 and concentrated. The residue was purified by auto-
mated flash chromatography using EtOAc and hexanes as eluents
to afford the desired product 19.
4.1.1.16. Synthesis of 5-bromo-2-iodo-1-(methoxymethyl)-1H-
indole (11c).
To a stirred slurry of NaH (22 mg, 60% in oil,
0.94 mmol) in THF (5 ml) and DMF (1 ml) at 0 °C was added a solu-
tion of 8b (200 mg, 0.63 mmol) in THF (5 ml). After stirring for 1 h
methoxymethyliodide (64 lL, 0.75 mmol) was added and the mix-
ture was further stirred for 2 h. The reaction mixture was parti-
tioned between Et2O (100 ml) and H2O (50 ml). The organic
phase was washed with brine (50 ml), dried over anhydrous Na2-
SO4 and concentrated. The residue was purified by automated flash
chromatography using EtOAc and hexanes as eluents to afford 11c
(178 mg, 77%) as yellow solid. 1H NMR (DMSO-d6, 500 MHz): d 7.70
(d, J = 1.8 Hz, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.26 (dd, J = 8.7, 1.9 Hz,
1H), 6.85 (s, 1H), 5.52 (s, 2H), 3.19 (s, 3H). 13C NMR (DMSO-d6,
125 MHz): 136.66, 131.25, 124.32, 121.35, 112.96, 112.51,
112.31, 88.25, 76.62, 55.46. HRMS calcd for (C10H9BrINO)ꢀ
364.8192; found 364.8923.
4.1.1.21. 5-Bromo-2-iodo-1-(2-(4-methylpiperazin-1-yl)ethyl)-
1H-indole (19a).
Yield = 89%, white solid. 1H NMR (CDCl3,
500 MHz): d 7.61–7.64 (m, 1H), 7.19–7.21 (m, 2H), 6.69 (s, 1H),
4.20–4.30 (m, 2H), 2.35–2.65 (m, 10H), 2.29 (s, 3H). 13C NMR
(125 MHz): 136.02, 131.22, 124.66, 122.06, 113.30, 111.53,
111.01, 103.91, 57.18, 55.01, 53.46, 46.14, 45.21. HRMS calcd for
(C15H19BrIN3)ꢀ 446.9807; found 446.9814.
4.1.1.22. 4-(2-(5-Bromo-2-iodo-1H-indol-1-yl)ethyl)morpholine
(19b).
Yield = 55%, brown solid. 1H NMR (CDCl3, 400 MHz): d
4.1.1.17. Synthesis of 2-(5-bromo-2-iodo-1H-indol-1yl)acetic
7.65–7.64 (m, 1H), 7.26–7.22 (m, 2H), 6.72 (s, 1H), 4.27 (t,
acid (13).
To a stirred slurry of NaH (144 mg, 60% in oil,
J = 7.2 Hz, 2H), 3.70 (br s, 4H), 2.63 (t, J = 7.0 Hz, 2H), 2.53 (br s, 4H).
6.0 mmol) in DMF (5 ml) at 0 °C was added a solution of 8b
(960 mg, 3.0 mmol) in DMF (15 ml). The mixture was stirred at
0 °C for 30 min and then at rt for an additional 30 min. Ethyl
4.1.1.23. General procedure for the synthesis of compounds 10,
12, 14, 17, 20, 22, 33 and 24.
A solution of boronic acid 9
2-iodoacetate (225
was stirred at rt for 16 h. The reaction mixture was diluted with
EtOAc and then washed with 1 M HCl followed by brine, dried over
l
L, 3.6 mmol) was added and the mixture
(1 mmol), iodo-heterocycle (8, 11, 21, 32 or 34) (1 mmol), Na2CO3
(1 M aqueous solution, 3.5 mmol) in ACN (5 ml) was purged with
argon for 10 min followed by the addition of Pd(PPh3)2Cl2 catalyst

N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
1719
(10 mol %). The mixture was heated in a sealed tube with
l
wave at
(dd, J = 8.7, 4.6 Hz, 1H), 7.34 (dd, J = 9.9, 2.3 Hz, 1H), 7.14 (dd,
J = 8.4, 1.8 Hz, 1H), 6.90–6.97 (m, 3H). 13C NMR (DMSO-d6,
151 MHz): d 157.68 (d, J = 231.7 Hz), 138.26, 134.13, 133.10,
132.42, 129.06 (d, J = 10.6 Hz), 127.86, 122.87, 122.31, 112.48 (d,
J = 9.8 Hz), 114.82, 114.02, 110.46 (d, J = 26.1 Hz), 105.13 (d,
J = 23.4 Hz), 99.48 (d, J = 4.7 Hz), 99.27. HRMS calcd for (C16H10-
BrFN2ꢀH)ꢀ 328.0011; found 328.0019.
110 °C until all the staring material was consumed as indicated by
TLC (typically in about 40–60 min). The reaction mixture was par-
titioned between EtOAc (100 ml) and H2O (50 ml). The organic
phase was washed with brine (50 ml), dried over anhydrous Na2-
SO4 and concentrated. The residue was taken up in DCM (10 ml)
and then TFA (1 ml) was added. After stirring at room temperature
for 2 h, solvent was removed and the crude product was purified
by automated flash chromatography using either EtOAc and hex-
anes or MeOH and DCM as eluents to give the desired adduct.
4.1.1.29. 60-Bromo-5-methoxy-1H,10H-2,20-biindole (10f).
Compound 10f was prepared from (6-bromo-1-(tert-butoxycar-
bonyl)-1H-indol-2-yl)boronic acid (9a) and tert-butyl 2-iodo-5-
methoxy-1H-indole-1-carboxylate.
4.1.1.24. 6-Bromo-1H,10H-2,20-biindole (10a).
Compound
10a was prepared from (6-bromo-1-(tert-butoxycarbonyl)-1H-in-
dol-2-yl)boronic acid (9a) and tert-butyl 2-iodo-1H-indole-1-
carboxylate.
Yield = 16%, pale brown solid. Mp = 240–241 °C. 1H NMR
(DMSO-d6, 500 MHz): d 11.67 (s, 1H), 11.43 (s, 1H), 7.50–7.54 (m,
2H), 7.28 (d, J = 8.7 Hz, 1H), 7.12 (dd, J = 8.4, 1.7 Hz, 1H), 7.07 (d,
J = 2.4 Hz, 1H), 6.89 (s, 1H), 6.84 (s, 1H), 6.76 (dd, J = 8.7, 2.4 Hz,
1H), 3.77 (s, 3H). 13C NMR (DMSO-d6, 150 MHz): 153.68, 137.69,
132.51, 132.10, 131.19, 128.73, 127.50, 122.24, 121.61, 113.99,
113.40, 112.20, 111.75, 101.67, 98.80, 98.17, 55.30. HRMS calcd
for (C17H13BrN2OꢀH) 340.0211; found 340.0217.
Yield = 35%, pink solid. Mp = 199–201 °C. 1H NMR (DMSO-d6,
400 MHz): d 11.71 (s, 1H), 11.59 (s, 1H), 7.51–7.59 (m, 3H), 7.41
(d, J = 8.1 Hz, 1H), 7.09–7.16 (m, 2H), 7.02 (t, J = 7.4 Hz, 1H),
6.92–6.95 (m, 2H). 13C NMR (DMSO-d6, 150 MHz): 137.04,
136.85, 132.34, 130.69, 128.20, 127.42, 122.21, 121.92, 121.65,
120.15, 119.48, 114.14, 113.35, 111.13, 98.79, 98.37. HRMS calcd
for (C16H11BrN2ꢀH)ꢀ 310.0106; found 310.0107.
4.1.1.30.
60-Bromo-5-phenyl-1H,10H-2,20-biindole
(10g).
Compound 10g was prepared from (6-bromo-1-(tert-butoxycar-
bonyl)-1H-indol-2-yl)boronic acid (9a) and tert-butyl 2-iodo-5-
phenyl-1H-indole-1-carboxylate.
4.1.1.25. 6,60-Dibromo-1H,10H-2,20-biindole (10b).
Com-
pound 10b was prepared from (6-bromo-1-(tert-butoxycar-
bonyl)-1H-indol-2-yl)boronic acid (9a) and 6-bromo-2-iodo-1H-
indole (8a).
Yield = 17%, pale yellow solid. Mp = 237–239 °C. 1H NMR
(DMSO-d6, 600 MHz): d 11.78 (s, 1H), 11.69 (s, 1H), 7.85–7.86 (m,
1H), 7.68–7.70 (m, 2H), 7.51–7.56 (m, 2H), 7.42–7.50 (m, 3H),
7.31 (t, J = 7.4 Hz, 1H), 7.14 (dd, J = 8.4, 1.7 Hz, 1H), 7.0 (s, 1H),
6.96 (s, 1H). 13C NMR (DMSO-d6, 151 MHz): 141.62, 137.79,
136.60, 132.25, 132.00, 131.52, 128.95, 128.76 (2C), 127.46,
126.63 (2C), 126.27, 122.31, 121.74, 121.36, 118.17, 114.19,
113.45, 111.48, 99.30, 98.60. HRMS calcd for (C22H15BrN2ꢀH)ꢀ
386.0419; found 386.0429.
Yield = 63%, white solid. Mp = 266–267 °C. 1H NMR (500 MHz,
DMSO-d6): d 11.77 (s, 2H), 7.56 (s, 2H), 7.54 (d, J = 8.4 Hz, 2H),
7.15 (dd, J = 1.6 Hz, zH8.4 Hz, 2H), 6.95 (d, J = 1.1 Hz, 2H). 13C
NMR (125 MHz, DMSO-d6): d 137.8 (2C), 131.8 (2C), 127.4 (2C),
122.4 (2C), 121.9 (2C), 114.4 (2C), 113.5 (2C), 99.0 (2C). HRMS
calcd for (C16H10Br2N2ꢀH)ꢀ 388.9118, found 388.9123.
4.1.1.26. 5,6-Bibromo-1H,10H-2,20-biiindole (10c).
Com-
pound 10c was prepared from (6-bromo-1-(tert-butoxycarbonyl)-
1H-indol-2-yl)boronic acid (9a) and 5-bromo-2-iodo-1H-indole
(8b).
4.1.1.31. 5-Bromo-1H,10H-2,20-biindole (10h).
Compound
10h was prepared from (5-bromo-1-(tert-butoxycarbonyl)-1H-in-
dol-2-yl)boronic acid (9b) and tert-butyl 2-iodo-1H-indole-1-
carboxylate.
Yield = 45%, pale brown solid. Mp = 270–272 °C. 1H NMR
(DMSO-d6, 400 MHz): d 11.82 (s, 1H), 11.78 (s, 1H), 7.79 (s, 1H),
7.53–7.57 (m, 2H), 7.37 (d, J = 8.5 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H),
7.15 (d, J = 8.4 Hz, 1H), 6.97 (s, 1H), 6.93 (s, 1H). 13C NMR
(DMSO-d6, 100 MHz): 137.86, 135.68, 132.28, 131.74, 130.26,
127.38, 124.30, 122.43, 122.28, 121.88, 114.47, 113.58, 113.01,
111.98, 99.11, 98.43. HRMS calcd for (C16H10Br2N2ꢀH)ꢀ
387.9211; found 387.9227.
Yield = 50%, white solid. 1H NMR (DMSO-d6, 400 MHz): d 11.77
(s, 1H), 11.61 (s, 1H), 7.77 (d, J = 1.7 Hz, 1H), 7.57 (d, J = 7.8 Hz, 1H),
7.41 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.22 (dd, J = 8.6,
1.9 Hz, 1H), 7.10–7.15 (m, 1H), 7.0–7.04 (m, 1H), 6.95 (d,
J = 1.3 Hz, 1H), 6.92 (d, J = 1.4 Hz, 1H). 13C NMR (DMSO-d6,
100 MHz): 137.0, 135.60, 132.91, 130.73, 130.37, 128.30, 124.03,
122.12, 121.96, 120.18, 119.50, 112.92, 111.87, 111.14, 99.02,
97.94. HRMS calcd for (C16H11BrN2ꢀH)ꢀ 310.0106; found
310.0107.
4.1.1.27.
60-Bromo-5-chloro-1H,10H-2,20-biindole
(10d).
Compound 10d was prepared from (6-bromo-1-(tert-butoxycar-
bonyl)-1H-indol-2-yl)boronic acid (9a) and 5-chloro-2-iodo-1H-in-
dole (8c).
4.1.1.32. 5,50-Bibromo-1H,10H-2,20-biindole (10i).
pound 10i was prepared from (5-bromo-1-(tert-butoxycarbonyl)-
1H-indol-2-yl)boronic acid (9b) and 5-bromo-2-iodo-1H-indole
(8b).
Com-
Yield = 60%, white solid. Mp = 257–259 °C. 1H NMR (DMSO-d6,
500 MHz): d 11.80 (s, 1H), 11.77 (s, 1H), 7.64 (d, J = 1.8 Hz, 1H),
7.52–7.55 (m, 2H), 7.40 (d, J = 8.6 Hz, 1H), 7.14 (dd, J = 8.4, 1.7 Hz,
1H), 7.11 (dd, J = 8.6, 2.0 Hz, 1H), 6.95 (d, J = 1.5 Hz, 1H), 6.92 (d,
J = 1.5 Hz, 1H). 13C NMR (DMSO-d6, 100 MHz): 137.83, 135.44,
132.44, 131.77, 129.51, 127.36, 123.99, 122.41, 121.89, 121.77,
119.23, 114.43, 113.55, 112.54, 99.04, 98.53. HRMS calcd for (C16-
H10BrClN2ꢀH)ꢀ 343.9716; found 343.9727.
Yield = 65%, brown solid. Mp = 304–306 °C. 1H NMR (DMSO-d6,
400 MHz): d 11.83 (s, 2H), 7.79 (d, J = 1.7 Hz, 2H), 7.36 (d,
J = 8.6 Hz, 2H), 7.23 (dd, J = 8.6, 1.9 Hz, 2H), 6.94 (d, J = 1.4 Hz,
2H). 13C NMR (DMSO-d6, 100 MHz): 135.68, 132.22, 130.22,
124.33, 122.30, 113.03, 111.96, 98.52. HRMS calcd for (C16H11BrN2-
ꢀH)ꢀ 387.9211; found 387.9221.
4.1.1.28.
60-Bromo-5-fluoro-1H,10H-2,20-biindole
(10e).
4.1.1.33. 5,6-Dibromo-1H,10H-2,20-biindole (10j).
Com-
Compound 10e was prepared from (6-bromo-1-(tert-butoxycar-
bonyl)-1H-indol-2-yl)boronic acid (9a) and 5-fluoro-2-iodo-1H-in-
dole (8d).
pound 10j was prepared from (1-(tert-butoxycarbonyl)-1H-indol-
2-yl)boronic acid (9c) and 5,6-dibromo-2-iodo-1H-indole (8e).
Yield = 53%, white solid. Mp = 233 °C —(decomposition). 1H
NMR (400 MHz, DMSO-d6): d 11.86 (s, 1H), 11.66 (s, 1H), 8.00 (s,
1H), 7.74 (s, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H),
Yield = 61%, white solid. Mp = 246–248 °C. 1H NMR (DMSO-d6,
600 MHz): d 11.73 (s, 1H), 11.68 (s, 1H), 7.51–7.56 (m, 2H), 7.38

1720
N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
7.14 (t, J = 7.2 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 6.97 (s, 1H), 6.94 (s,
1H). 13C NMR (100 MHz, DMSO-d6): d 137.0, 136.7, 133.8, 130.2,
129.6, 128.2, 124.1, 122.1, 120.3, 119.5, 115.4, 115.3, 113.5,
111.2, 99.4, 97.8. HRMS calcd for (C16H10Br2N2ꢀH)ꢀ 388.9118;
found 388.9108.
4.1.1.39. 5,60-Dibromo-1-(methoxymethyl)-1H,10H-2,20-biindole
(12c). Compound 12c was prepared from (6-bromo-1-(tert-
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9a) and 5-bromo-2-
iodo-1-(methoxymethyl)-1H-indole (11c).
Yield = 65%, white solid. Mp = 199–201 °C. 1H NMR (DMSO-d6,
500 MHz): d 11.74 (s, 1H), 7.87 (d, J = 1.9 Hz, 1H), 7.74 (d,
J = 8.8 Hz, 1H), 7.55–7.61 (m, 2H), 7.36 (dd, J = 8.3, 2.4 Hz, 1H),
7.17 (dd, J = 8.4, 1.8 Hz, 1H), 6.98 (s, 1H), 6.95 (s, 1H), 5.71(s, 2H)
3.30 (s, 3H). 13C NMR (DMSO-d6, 125 MHz): 137.69, 137.46,
133.97, 129.50, 129.18, 127.40, 124.95, 122.55, 122.45, 122.26,
114.87, 113.67, 113.14, 112.51, 102.23, 101.93, 74.19, 55.55. HRMS
calcd for (C18H14Br2N2OꢀH)ꢀ 431.9473; found 431.9485.
4.1.1.34.
5,6,60-Tribromo-1H,10H-2,20-biindole
(10k).
Compound 10k was prepared from (6-bromo-1-(tert-butoxycar-
bonyl)-1H-indol-2-yl)boronic acid (9a) and 5,6-dibromo-2-iodo-
1H-indole (8e).
Yield = 24%, pale yellow solid. 1H NMR (400 MHz, DMSO-d6): d
11.95 (s, 1H), 11.87 (s, 1H), 8.02 (s, 1H), 7.75 (s, 1H), 7.75 (s, 1H),
7.56 (s, 1H), 7.55 (d, J = 8.6 Hz, 1H), 7.15 (dd, J = 1.7 Hz, 8.4 Hz,
1H), 6.98 (br s, 1H), 6.94 (s, 1H). 13C NMR (100 MHz, DMSO-d6):
d 137.9, 136.7, 133.2, 131.3, 129.5, 127.3, 124.3, 122.5, 122.0,
115.6, 115.5, 114.6, 113.64, 113.60, 99.5, 98.3. HRMS calcd for (C16-
H9Br3N2ꢀH)ꢀ 466.8223; found 466.8223.
4.1.1.40. 2-(5,60-Dibromo-1H,10H-[2,20-biindol]-1-yl)acetic acid
(14).
Compound 14 was prepared from (6-bromo-1-(tert-
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9a) and 2-(5-bro-
mo-2-iodo-1H-indol-1yl)acetic acid (13).
Yield = 33%, white solid. Mp = 321–323 °C. 1H NMR (DMSO-d6,
400 MHz): d 11.77 (s, 1H), 7.84 (d, J = 1.9 Hz, 1H), 7.52–7.60 (m,
3H), 7.31 (dd, J = 8.7, 2.0 Hz, 1H), 7.17 (dd, J = 8.5, 1.7 Hz, 1H),
6.91 (s, 1H), 6.69 (d, J = 1.5 Hz, 1H), 5.24 (s, 2H). 13C NMR
(DMSO-d6, 150 MHz): 170.07, 137.48, 137.29, 133.85, 129.69,
129.03, 127.34, 124.57, 122.51, 122.39, 122.13, 114.84, 113.67,
112.71, 112.34, 101.64, 100.79, 46.05. HRMS calcd for (C18H12Br2-
N2O2ꢀH)ꢀ 445.9266; found 445.9272.
4.1.1.35.
(10l).
5,50,6,60-Tetrabromo-1H,10H-2,20-biindole
Compound 10l was isolated as a minor bi-product dur-
ing the synthesis of 10k from 9a and 8e.
Yield = 7%, pale yellow solid. 1H NMR (400 MHz, DMSO-d6): d
11.98 (bd, J = 0.8 Hz, 2H), 8.04 (s, 2H), 7.75 (d, J = 0.6 Hz, 2H),
6.97 (d, J = 1.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): d 136.8
(2C), 132.6 (2C), 129.3 (2C), 124.5 (2C), 115.9 (2C), 115.6 (2C),
113.8 (2C), 98.9 (2C). HRMS calcd for (C16H8Br4N2ꢀH)ꢀ 546.7308;
found 546.7292.
4.1.1.41.
(17).
2-(5,60-Dibromo-1H,10H-[2,20-biindol]-1-yl)ethanol
Compound 17 was prepared from (6-bromo-1-(tert-
4.1.1.36.
5,50,6-Tribromo-1H,10H-2,20-biindole
(10m).
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9a) and 2-(5-bro-
mo-2-iodo-1H-indol-1-yl)ethanol (16).
Compound 10m was prepared from (5-bromo-1-(tert-butoxycar-
bonyl)-1H-indol-2-yl)boronic acid (9b) and 5,6-dibromo-2-iodo-
1H-indole (8e).
Yield = 8%, pale white solid. 1H NMR (DMSO-d6, 500 MHz): d
11.71 (s, 1H), 7.81 (d, J = 1.9 Hz, 1H), 7.54–7.59 (m, 3H), 7.30 (dd,
J = 8.7, 2.0 Hz, 1H), 7.18 (dd, J = 8.4, 1.8 Hz, 1H), 6.91 (s, 1H), 6.86
(s, 1H), 5.17 (t, J = 5.1 Hz, 1H), 4.47 (t, J = 5.9 Hz, 2H), 3.78 (q,
J = 5.6 Hz, 2H). 13C NMR (DMSO-d6, 100 MHz): 137.48, 136.82,
133.84, 130, 129.06, 127.36, 124.22, 122.42, 122.26, 122.07,
114.66, 113.70, 112.74, 112.39, 101.74, 101.32, 59.97, 46.44. HRMS
calcd for (C18H14Br2N2OꢀH)ꢀ 431.9473; found 431.9483.
Yield = 49%, white solid. 1H NMR (400 MHz, DMSO-d6): d 12.04
(s, 1H), 11.97 (s, 1H), 8.02 (s, 1H), 7.79 (d, J = 1.8 Hz, 1H), 7.75 (s,
1H), 7.37 (d, J = 8.6 Hz, 1H), 7.23 (dd, J = 1.9 Hz, 8.6 Hz, 1H), 6.95
(br s, 2H). 13C NMR (100 MHz, DMSO-d6): d 136.8, 135.7, 133.2,
131.8, 130.1, 129.5, 124.5, 124.3, 122.4, 115.58, 115.55, 113.6,
113.1, 112.0, 98.9, 98.4. HRMS calcd for (C16H9Br3N2ꢀH)ꢀ
466.8223; found 466.8211.
4.1.1.42.
1H,10H-2,20-biindole (20a).
5,60-Dibromo-1-(2-(4-methylpiperazin-1-yl)ethyl)-
Compound 20a was prepared from
4.1.1.37.
(12a).
5,60-Dibromo-1-methyl-1H,10H-2,20-biindole
Compound 12a was prepared from (6-bromo-1-(tert-
(6-bromo-1-(tert-butoxycarbonyl)-1H-indol-2-yl)boronic acid (9a)
and 5-bromo-2-iodo-1-(2-(4-methylpiperazin-1-yl)ethyl)-1H-indole
(19a).
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9a) and 5-bromo-2-
iodo-1-methyl-1H-indole (11b).
Yield = 53%, white solid. Mp = 180–182 °C. 1H NMR (DMSO-d6,
400 MHz): d 11.78 (s, 1H), 7.82 (d, J = 1.9 Hz, 1H), 7.51–7.61 (m,
3H), 7.31 (dd, J = 8.7, 2.0 Hz, 1H), 7.18 (dd, J = 8.4, 1.8 Hz, 1H),
6.91 (s, 1H), 6.89 (s, 1H), 3.96 (s, 3H). 13C NMR (DMSO-d6,
125 MHz): 137.58, 137.0, 133.90, 130.03, 128.85, 127.41, 124.25,
122.45, 122.31, 122.06, 114.75, 113.68, 112.35, 112.14, 101.76,
100.61, 31.78. HRMS calcd for (C17H12Br2N2ꢀH)ꢀ 401.9367; found
401.9384.
Yield = 57%, pale white solid. Mp: 140 °C. 1H NMR (CDCl3,
500 MHz): d 11.94 (s, 1H), 7.81 (s, 1H), 7.52–7.61 (m, 3H), 7.31
(d, J = 8.7 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 6.89 (s, 1H), 6.84 (s,
1H), 4.51 (t, J = 6.4 Hz, 2H), 2.62 (t, J = 6.4 Hz, 2H), 2.15–2.45 (m,
8H), 2.11 (s, 3H). 13C NMR (150 MHz): 137.47, 136.46, 133.66,
130.04, 129.12, 127.38, 124.34, 122.45, 122.41, 122.10, 114.65,
113.72, 112.54, 112.49, 101.71, 101.50, 56.77, 54.39, 52.79, 45.38,
42.41. HRMS calcd for (C23H24Br2N4ꢀH)ꢀ 514.0368; found
514.0372.
4.1.1.38.
(12b).
50,6-Dibromo-1-methyl-1H,20H-2,20-biindole
Compound 12b was prepared from (5-bromo-1-(tert-
4.1.1.43.
yl)ethyl)morpholine (20b).
4-(2-(5,60-Dibromo-1H,10H-[2,20-biindol]-1-
Compound 20b was prepared
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9b) and 6-bromo-2-
iodo-1-methyl-1H-indole (8a).
from (6-bromo-1-(tert-butoxycarbonyl)-1H-indol-2-yl)boronic acid
(9a) and 4-(2-(5-bromo-2-iodo-1H-indol-1-yl)ethyl)morpholine
(19b).
Yield = 48%, pale yellow solid. Mp = 192–194 °C. 1H NMR
(DMSO-d6, 400 MHz): d 11.76 (s, 1H), 7.84 (s, 1H), 7.78 (d,
J = 1.9 Hz, 1H), 7.57 (d, J = 8.4, 1H), 7.39 (d, J = 8.6 Hz, 1H), 7.26
(dd, J = 8.6, 1.9 Hz, 1H), 7.21 (dd, J = 8.4, 1.7 Hz, 1H), 6.92 (d,
J = 0.6 Hz, 1H), 6.86 (d, J = 1.5 Hz, 1H). 13C NMR (DMSO-d6,
100 MHz): 139.13, 135.39, 133.41, 130.59, 130.25, 126.04,
124.57, 122.71, 122.37, 121.92, 114.76, 113.16, 112.97, 111.92,
101.48, 101.06, 31.78. HRMS calcd for (C17H12Br2N2ꢀH)ꢀ
401.9367; found 401.9376.
Yield = 22%, white form. 1H NMR (400 MHz, DMSO-d6): d 11.89
(s, 1H), 7.81 (d, J = 1.3 Hz, 1H), 7.59–7.56 (m, 3H), 7.31 (dd,
J = 1.4 Hz, 8.7 Hz, 1H), 7.18 (dd, J = 1.2 Hz, 8.4 Hz, 1H), 6.89 (s,
1H), 6.85 (s, 1H), 4.54 (t, J = 6.4 Hz, 2H), 3.43 (br s, 4H), 2.60 (t,
J = 6.3 Hz, 2H), 2.30 (br s, 4H). 13C NMR (100 MHz, DMSO-d6): d
137.5, 136.5, 133.7, 130.0, 129.1, 127.3, 124.3, 122.4, 122.3,
122.1, 114.6, 113.7, 122.5, 122.4, 101.7, 101.4, 66.0, 57.3, 53.5,

N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
1721
42.0. HRMS calcd for (C22H21Br2ON3ꢀH)ꢀ 504.0105, found
102.41, 99.81. HRMS calcd for C16H10BrNO is 310.9946; found
310.9954.
504.0116.
4.1.1.44.
(22a).
2-(6-Bromo-1H-indol-2-yl)benzo[d]thiazole
Compound 22a was prepared from (6-bromo-1-(tert-
4.1.1.49.
(22f).
2-(Benzo[b]thiophen-2-yl)-5-bromo-1H-indole
Compound 22f was prepared from (5-bromo-1-(tert-
butoxycarbonyl)-1H-indol-2-yl)boronic
iodobenzo[d]thiazole (21a).
acid
(9a)
and
2-
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9b) and 2-
iodobenzo[b]thiophene (21c).
Yield = 43%, pink solid. 1H NMR (DMSO-d6, 600 MHz): d 12.35 (s,
1H), 8.16 (ddd, J = 8.0, 1.1, 0.6 Hz, 1H), 8.04 (ddd, J = 8.2, 1.1, 0.6 Hz,
1H), 7.63–7.64 (m, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.57 (ddd, J = 8.2,
7.2, 1.2 Hz, 1H), 7.48 (ddd, J = 8.2, 7.2, 1.2 Hz, 1H), 7.29 (dd,
J = 2.2, 0.8 Hz, 1H), 7.21 (dd, J = 8.4, 1.8 Hz, 1H). 13C NMR (DMSO-
d6, 150 MHz): 159.40, 153.21, 150.64, 138.43, 134.23, 131.92,
126.85, 126.78, 125.60, 123.21, 123.10, 122.50, 122.41, 116.61,
114.63, 105.08. HRMS calcd for (C15H9BrN2SꢀH)ꢀ 327.9670; found
327.9674.
Yield = 57%, white solid. Mp = 269–271 °C. 1H NMR (DMSO-d6,
400 MHz): d 12.0 (s, 1H), 7.98–8.01 (m, 1H), 7.86–7.90 (m, 1H),
7.85 (s, 1H), 7.76 (d, J = 1.8 Hz, 1H), 7.35–7.44 (m, 3H), 7.25 (dd,
J = 8.6, 1.9 Hz, 1H), 6.82 (d, J = 1.6 Hz, 1H). 13C NMR (DMSO-d6,
100 MHz): 139.97, 138.37, 135.90, 134.64, 133.38, 130.18,
124.97, 124.89, 124.83, 123.75, 122.48, 122.38, 120.03, 113.24,
112.15, 100.26. HRMS calcd for (C16H10BrNSꢀH)ꢀ 326.9717; found
326.9722.
4.1.1.50. 6-Bromo-2-(5-bromo-1H-indol-2-yl)benzo[d]thiazole
4.1.1.45.
(22b).
2-(Benzofuran-2-yl)-6-bromo-1H-indole
Compound 22b was prepared from (6-bromo-1-(tert-
(22g).
Compound 22g was prepared from (5-bromo-1-(tert-
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9b) and 6-bromo-2-
iodobenzo[d]thiazole (21c).
butoxycarbonyl)-1H-indol-2-yl)boronic
iodobenzofuran (21b).
acid
(9a)
and
2-
Yield = 48%, yellow solid. Mp = 246–248 °C. 1H NMR (DMSO-d6,
400 MHz): d 12.45 (s, 1H), 8.48 (d, J = 2.0 Hz, 1H), 7.97 (d, J = 8.7 Hz,
1H), 7.87 (d, J = 1.8 Hz, 1H), 7.72 (dd, J = 8.7, 2.0 Hz, 1H), 7.44 (d,
J = 8.7 Hz, 1H), 7.36 (dd, J = 8.7, 1.9 Hz, 1H), 7.28 (d, J = 1.5 Hz,
1H). 13C NMR (DMSO-d6, 150 MHz): 160.26, 152.30, 136.42,
136.23, 131.87, 129.93, 129.45, 126.64, 125.01, 123.85, 123.38,
118.00, 114.26, 112.66, 104.67. HRMS calcd for (C15H8Br2N2SꢀH)ꢀ
405.8775; found 405.8765.
Yield = 40%, pale white solid. Mp = 196–198 °C. 1H NMR (DMSO-
d6, 400 MHz): d 12.02 (s, 1H), 7.71 (d, J = 7.1 Hz, 1H), 7.64 (d,
J = 8.1 Hz, 1H), 7.60 (s, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.25–7.37 (m,
3H), 7.18 (dd, J = 8.4, 1.7 Hz, 1H), 7.0 (s, 1H). 13C NMR (DMSO-d6,
100 MHz): 154.0, 149.12, 137.97, 129.34, 128.55, 127.11, 124.75,
123.44, 122.75, 122.26, 121.55, 115.11, 113.92, 111.0, 102.28,
100.45. HRMS calcd for (C16H10BrNOꢀH)ꢀ is 310.9946; found
310.9948.
4.1.1.51. 2-(4-Bromo-3-nitrophenyl)-1H-indole (34a).
Com-
4.1.1.46.
(22c).
2-(Benzo[b]thiophen-2-yl)-6-bromo-1H-indole
Compound 22c was prepared from (6-bromo-1-(tert-
pound 34a was prepared from (1-(tert-butoxycarbonyl)-1H-indol-
2-yl)boronic acid and 1,4-dibromo-2-nitrobenzene.
butoxycarbonyl)-1H-indol-2-yl)boronic
iodobenzo[b]thiophene (21c).
acid
(9a)
and
2-
Yield = 38%, brown form. 1H NMR (400 MHz, CDCl3): d 8.45 (br s,
1H), 7.96 (d, J = 2.0 Hz, 1H), 7.76 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 7.65
(dd, J = 0.9 Hz, 7.9 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H) 7.42 (dd,
J = 0.8 Hz, 8.2 Hz, 1H), 7.28–7.24 (m, 1H), 7.17–7.13 (m, 1H),
6.73–6.72 (m, 1H).
Yield = 57%, white solid. Mp = 250–252 °C. 1H NMR (DMSO-d6,
400 MHz): d 11.91 (s, 1H), 7.94–7.98 (m, 1H), 7.86 (dd, J = 7.1,
1.5 Hz, 1H), 7.82 (s, 1H), 7.54–7.57 (m, 1H), 7.50 (d, J = 8.4 Hz,
1H), 7.33–7.43 (m, 2H), 7.14 (dd, J = 8.4, 1.8 Hz, 1H), 6.83 (d,
J = 1.4 Hz). 13C NMR (DMSO-d6, 150 MHz): 139.98, 138.35,
138.03, 135.14, 132.93, 127.38, 124.99, 124.86, 123.75, 122.67,
122.47, 122.01, 119.87, 114.87, 113.67, 100.86. HRMS calcd for
(C16H10BrNSꢀH)ꢀ 326.9717, found 326.9708.
4.1.1.52.
(34b).
2-(4-Bromo-3-methoxyphenyl)-1H-indole
Compound 34b was prepared from (1-(tert-butox-
ycarbonyl)-1H-indol-2-yl)boronic
acid
and
1,4-dibromo-
2-methoxybenzene.
Yield = 57%, white solid. 1H NMR (DMSO-d6, 600 MHz): d 11.59
(s, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.54 (d,
J = 8.0 Hz, 1H), 7.42–7.39 (m, 2H), 7.14–7.11 (m, 1H), 7.02–7.00
(m, 2H). 13C NMR (DMSO-d6, 150 MHz): 156.2, 137.6, 137.2,
133.7, 133.6, 129.0, 122.4, 120.7, 120.0, 119.0, 111.8, 109.8,
109.5, 100.1, 56.9.
4.1.1.47.
(22d).
2-(5-Bromo-1H-indol-2-yl)benzo[d]thiazole
Compound 22d was prepared from (5-bromo-1-(tert-
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9b) and 2-
iodobenzo[d]thiazole (21a).
Yield = 35%, colorless solid. Mp = 208–210 °C. 1H NMR (DMSO-
d6, 400 MHz): d 12.43 (s, 1H), 8.15 (d, J = 7.9 Hz, 1H), 8.05 (d,
J = 8.1 Hz, 1H), 7.84 (d, J = 1.5 Hz, 1H), 7.57 (t, J = 7.7 Hz, 1H),
7.42–7.50 (m, 2H), 7.34 (dd, J = 8.7, 1.8 Hz, 1H), 7.22 (s, 1H). 13C
NMR (DMSO-d6, 100 MHz): 159.35, 153.22, 136.32, 134.22,
132.31, 129.50, 126.80, 126.44, 125.58, 123.30, 122.44 (2C),
114.22, 112.59, 104.24. HRMS calcd for (C15H9BrN2SꢀH)ꢀ
327.9670; found 327.9676.
4.1.1.53.
(33a).
1,4-Bis-(6-bromo-1H-indol-2-yl)benzene
Compound 33a was prepared from (6-bromo-1-(tert-
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9a) (2 equiv) and
1,4-diiodobenzene.
Yield = 67%, white solid. 1H NMR (600 MHz, DMSO-d6): d 11.75
(s, 2H), 7.96 (s, 4H), 7.55 (s, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.14 (dd,
J = 1.7 Hz, 8.4 Hz, 2H), 7.02 (d, J = 1.5 Hz, 2H). 13C NMR (150 MHz,
DMSO-d6): d 138.2 (2C), 138.0 (2C), 130.8 (2C), 127.7 (2C), 125.5
(4C), 122.3 (2C), 121.8 (2C), 114.2 (2C), 113.6 (2C), 99.2 (2C). HRMS
calcd for (C22H14Br2N2ꢀH)ꢀ 464.9432; found 464.9441.
4.1.1.48.
(22e).
2-(Benzofuran-2-yl)-5-bromo-1H-indole
Compound 22e was prepared from (5-bromo-1-(tert-
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9b) and 2-
iodobenzofuran (21b).
Yield = 38%, white solid. Mp = 236–238 °C. 1H NMR (DMSO-d6,
400 MHz): d 12.06 (s, 1H), 7.80 (d, J = 1.9 Hz, 1H), 7.70–7.73 (m,
1H), 7.61–7.66 (m, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.25–7.38 (m,
4H), 6.97 (d, J = 1.5 Hz, 1H). 13C NMR (DMSO-d6, 150 MHz):
154.09, 154.08, 149.06, 149.04, 135.82, 129.91, 129.81, 128.50,
124.98, 124.80, 123.44, 122.64, 121.29, 113.42, 112.20, 111.01,
4.1.1.54.
(33b).
2,5-Bis-(6-bromo-1H-indol-2-yl)thiophene
Compound 33b was prepared from (6-bromo-1-(tert-
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9a) (2 equiv) and
2,5-diiodothiophene.
Yield = 88%, yellow solid. Decomposes >228 °C. 1H NMR
(600 MHz, DMSO-d6): d 11.81 (s, 2H), 7.55 (s, 2H), 7.53 (br s, 2H),

1722
N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
7.50 (d, J = 8.4 Hz, 2H), 7.15 (dd, J = 1.7 Hz, 8.4 Hz, 2H), 6.76 (d,
J = 1.2 Hz, 2H). 13C NMR (150 MHz, DMSO-d6): d 137.8 (2C), 133.8
(2C), 132.8 (2C), 127.5 (2C), 124.9 (2C), 122.6 (2C), 121.7 (2C),
114.4 (2C), 113.5 (2C), 99.3 (2C). HRMS calcd for (C20H12Br2N2-
SꢀH)ꢀ 470.8995, found 470.9004.
Mp = 320–322 °C. 1H NMR (DMSO-d6, 400 MHz): d 11.17 (s, 1H),
7.65 (s, 1H), 7.50 (s, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 8.7 Hz,
1H), 7.11 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 8.5 Hz, 1H), 6.71 (s, 1H),
6.37 (s, 1H), 5.10 (s, 2H), 3.62 (br s, 2H), 3.54 (br s, 2H). HRMS calcd
for (C22H19Br2N3O2ꢀH)ꢀ 514.9844; found 514.9867.
4.1.1.55.
(33c).
2-(4-(1H-Indol-2-yl)phenyl)-6-bromo-1H-indole
Compound 33c was prepared from (6-bromo-1-(tert-
4.1.1.60. General procedure for the synthesis of substituted 2-
ethynyl-1H-indole (24) and substituted bis-indole (25).
A
butoxycarbonyl)-1H-indol-2-yl)boronic acid (9a) and tert-butyl 2-
(4-iodophenyl)-1H-indole-1-carboxylate.
solution of 2-iodoindole 7 (1 mmol) and acetylene derivative
(1 mmol) in THF (5 ml) was purged with argon for 10 min followed
by triethylamine (3.5 mmol), CuI (0.2 mmol) and Pd(Ph3P)2Cl2 as
Yield = 89%, Yellow solid. Mp P300 °C. 1H NMR (400 MHz,
DMSO-d6): d 11.74 (s, 1H), 11.57 (s, 1H), 7.97 (d, J = 8.7 Hz, 1H),
7.94 (d, J = 9.1 Hz, 1H), 7.55 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.50
(d, J = 8.4 Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.15–7.09 (m, 2H),
7.03–6.98 (m, 3H). 13C NMR (150 MHz, DMSO-d6): d 138.3, 138.0,
137.2, 137.1, 131.3, 130.4, 128.6, 127.7, 125.5, 125.3 (2C), 125.31
(2C), 122.3, 121.7, 120.0, 119.4, 114.1, 113.6, 111.2, 99.1, 99.0.
HRMS calcd for (C22H15BrN2ꢀH)ꢀ 387.0328; found 387.0333.
catalyst (10 mol %). The mixture was heated in
lwave at 100 °C
for 30 min. After completion of the reaction as monitored by TLC,
water was added and the mixture extracted with EtOAc
(2 ꢁ 20 ml). Combined organic layer was washed with brine, dried
over anhydrous Na2SO4 and evaporated in vacuo. The residue was
purified by automated flash chromatography and then taken up in
THF (5 ml) followed by the addition if TBAF (1 M in THF, 1 mmol).
The mixture was stirred at rt for 2 h and then partitioned between
EtOAc (50 ml) and H2O (50 ml). The organic phase was washed
with brine (50 ml), dried over anhydrous Na2SO4 and concentrated.
The crude product was purified by automated flash chromatogra-
phy using EtOAc and hexanes as eluents to give the desired
product.
4.1.1.56.
indole (33d).
2-(4-(1H-Indol-2-yl)-3-nitrophenyl)-6-bromo-1H-
Compound 33d was prepared from 9a and 34a.
Yield = 57%, brown solid. (Mixture of isomers ꢂ5:1) 1H NMR
(400 MHz, DMSO-d6):
d 11.97 (d, J = 1.3 Hz, 1H), 11.60 (d,
J = 1.3 Hz, 1H), 8.45 (d, J = 1.8 Hz, 1H), 8.25 (dd, J = 1.8 Hz, 8.2 Hz,
1H), 7.90 (d, J = 8.2 Hz, 1H), 7.60–7.55 (m, 3H), 7.43 (d, J = 8.1 Hz,
1H), 7.15–7.06 (m, 3H), 7.03 (dd, J = 7.0 Hz, 7.9 Hz, 1H), 6.59 (d,
J = 1.4 Hz, 1H). 13C NMR (150 MHz, DMSO-d6): d 148.9, 138.3,
137.1, 135.9, 132.1, 131.9, 131.4, 128.3, 128.1, 127.4, 124.4,
122.8, 122.4, 122.3, 120.5, 119.9, 119.6, 115.2, 113.9, 111.5,
101.7, 101.2. HRMS calcd for (C22H14BrN3O2ꢀH)ꢀ 432.0179; found
432.0195.
4.1.1.61. 6-Bromo-2-ethynyl-1H-indole (24a).
24a was prepared from 7a and TIPS-acetylene.
Compound
Yield = 52%, dark brown solid. 1H NMR (CDCl3, 400 MHz): d 8.20
(s, 1H), 7.48 (s, 1H), 7.44 (d, J = 8.3, 1.0 Hz, 2H), 7.23 (dd, J = 8.5,
1.6 MHz, 1H), 6.68 (s, 1H), 3.33 (s, 1H). 13C NMR (CDCl3,
100 MHz): 136.59, 126.23, 123.98, 122.17, 118.29, 117.47,
113.54, 109.61, 81.31, 75.55. HRMS calcd for (C10H6Br2NꢀH)ꢀ
218.9684; found 218.9687.
4.1.1.57. 2-(4-(1H-Indol-2-yl)-2-methoxyphenyl)-6-bromo-1H-
indole (33e).
Compound 33e was prepared from 9a and 34b.
Yield = 11%, yellow solid. 1H NMR (600 MHz, DMSO-d6): d 11.62
(d, J = 1.0 Hz, 1H), 11.37 (d, J = 1.0 Hz, 1H), 7.87 (d, J = 8.1 Hz, 1H),
7.65 (d, J = 1.3 Hz, 1H), 7.63 (s, 1H), 7.59 (dd, J = 1.6 Hz, 8.1 Hz,
1H), 7.56 (d, J = 7.8 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.44 (d,
J = 8.1 Hz, 1H), 7.14–7.11 (m, 2H), 7.06–7.01 (m, 3H), 4.09 (s, 3H).
13C NMR (150 MHz, DMSO-d6): d 156.4, 137.3, 137.2, 137.2,
135.4, 132.6, 128.6, 128.0, 127.2, 122.0, 121.8, 121.5, 120.1,
119.5, 118.9, 117.6, 113.8, 113.7, 111.2, 108.4, 101.7, 99.5, 55.9.
HRMS calcd for (C23H17BrN2OꢀH)ꢀ 417.0434; found 417.0442.
4.1.1.62. 5-Bromo-2-ethynyl-1H-indole (24b).
24b was prepared from 7b and TIPS-acetylene.
Compound
Yield = 63%, brown solid. 1H NMR (CDCl3, 400 MHz): d 8.23 (s,
1H), 7.72 (d, J = 1.32 Hz, 1H), 7.32 (dd, J = 8.7, 1.8 Hz, 1H), 7.19 (d,
J = 8.7 Hz, 1H), 6.75 (d, J = 1.5 Hz, 1H), 3.33 (s, 1H). 13C NMR
(126 MHz, CDCl3): 134.59, 129.20, 126.82, 123.52, 118.93, 113.96,
112.38, 109.15, 81.54, 75.77. HRMS calcd for (C10H6BrNꢀH)ꢀ
218.9684; found 218.9685.
4.1.1.63. 2-((1H-Indol-2-yl)ethynyl)-6-bromo-1H-indole (25a).
Compound 25a was prepared from 24a and 2-iodo-1-(phenylsulfo-
nyl)-1H-indole.
4.1.1.58. 2-(3-(1H-Indol-2-yl)phenyl)-6-bromo-1H-indole (33f).
Compound 33f was prepared from 9a and tert-butyl 2-(3-iodo-
phenyl)-1H-indole-1-carboxylate.
Yield = 64%, pale brown solid; Mp = 240–242 °C; 1H NMR
(DMSO-d6, 600 MHz): d 11.94 (s, 1H), 11.78 (s, 1H), 7.57 (dd,
J = 7.9, 0.6 Hz, 1H), 7.52–7.54 (m, 2H), 7.36 (dd, J = 8.2, 0.9 Hz,
1H), 7.17–7.22 (m, 2H), 7.06 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.89
(dd, J = 2.0, 0.7 Hz, 1H), 6.88 (dd, J = 2.0, 0.9 Hz, 1H). 13C NMR
(DMSO-d6, 151 MHz): d 150.63, 137.31, 136.61, 127.15, 126.22,
123.23, 122.91, 122.22, 120.50, 119.97, 118.80, 117.50, 115.92,
113.78, 113.39, 108.33, 108.16, 85.62, 84.50. HRMS calcd for (C18-
H11BrN2ꢀH)ꢀ 334.0106; found 334.0113.
Yield = 86%, white solid. Mp = 224–226 °C. 1H NMR (600 MHz,
DMSO-d6): d 11.78 (s, 1H), 11.60 (s, 1H), 8.38 (s, 1H), 7.82 (d,
J = 7.8 Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.58–7.54 (m, 4H), 7.44 (d,
J = 7.4 Hz, 1H), 7.17–7.01 (m, 5H). 13C NMR (150 MHz, DMSO-d6):
d 138.5, 138.0, 137.3, 137.1, 132.9, 132.3, 129.5, 128.6, 127.6,
124.2, 123.9, 122.3, 121.9, 121.8, 121.7, 120.1, 119.4, 114.2,
113.7, 111.3, 99.3, 99.2. HRMS calcd for (C22H15BrN2ꢀH)ꢀ
387.0328; found 387.0335.
4.1.1.59. Specific procedure for the synthesis of 2-(5,60-dibromo-
1H,10H-[2,20-biindol]-1-yl)-1-morpholinoethanone (15).
To
a stirred solution of compound 14 (50 mg, 0.11 mmol), morpholine
(10 L, 0.12 mmol) and HBTU (44 mg, 0.12 mmol) in DMF (1 ml) at
0 °C was added DIPEA (61 L, 0.35 mmol) and the resulting solution
4.1.1.64.
1,2-Bis-(6-bromo-1H-indol-2-yl)ethyne
(25b).
Compound 25b was prepared from coupling 7a with 24a.
Yield = 50%, yellow solid. 1H NMR (600 MHz, CDCl3): d 8.24 (br
s, 2H), 7.52 (s, 2H), 7.47 (d, J = 8.2 Hz, 2H), 7.26 (m, 2H), 6.84 (s,
2H). 13C NMR (150 MHz, CDCl3): d 137.0 (2C), 126.5 (2C), 124.3
(2C), 122.2 (2C), 118.6 (2C), 117.7 (2C), 113.8 (2C), 109.7 (2C),
85.1 (2C). HRMS calcd for (C18H10Br2N2ꢀH)ꢀ 412.9118; found
412.9126.
l
l
stirred at rt for 12 h. The mixture was diluted with DCM (10 ml) and
washed with 1 N HCl, saturated aqueous NaHCO3, brine and concen-
trated. The crude product was purified by automated flash chroma-
tography to give compound 15 as white solid (38 mg, 68%).

N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
1723
4.1.1.65.
indole (25c).
6-Bromo-2-((5-bromo-1H-indol-2-yl)ethynyl)-1H-
Compound 25c was prepared from 7b and 24a.
between EtOAc (50 ml) and H2O (20 ml). The organic phase was
washed with brine (20 ml), dried over anhydrous Na2SO4 and con-
centrated. The crude product was purified by automated flash
chromatography using EtOAc and hexanes as eluents to give com-
pounds 27b and 28a.
Yield = 32%, pale yellow solid; Mp = 236–237 °C; 1H NMR
(CDCl3, 600 MHz):
d 12.03 (s, 1H), 11.96 (s, 1H), 7.77 (d,
J = 1.8 Hz, 1H), 7.52–7.55 (m, 2H), 7.33 (d, J = 8.7 Hz, 1H), 7.30
(dd, J = 8.6, 1.9 Hz, 1H), 7.19 (dd, J = 8.5, 1.6 Hz, 1H), 6.93 (m, 1H),
6.86 (m, 1H). 13C NMR (CDCl3, 150 MHz): 137.78, 135.60, 129.35,
126.61, 126.12, 123.37, 122.97, 122.72, 119.45, 118.88, 116.50,
114.26, 113.75, 112.90, 108.93, 108.14, 85.50, 85.38. HRMS calcd
for (C18H10Br2N2ꢀH)ꢀ 412.9118; found 412.9102.
4.1.1.71.
(27b).
(E)-1,2-Bis-(6-bromo-1H-indol-2-yl)ethane
Yield = 20%, yellow solid. Decomposes >300 °C. 1H
NMR (600 MHz, DMSO-d6): d 11.59 (s, 2H), 7.49 (s, 2H), 7.46 (d,
J = 8.4 Hz, 2H), 7.20 (s, 2H), 7.11 (dd, J = 1.7 Hz, 8.4 Hz, 2H), 6.60
(d, J = 0.9 Hz, 2H). 13C NMR (150 MHz, DMSO-d6): d 138.2 (2C),
137.4 (2C), 127.5 (2C), 122.2 (2C), 121.7 (2C), 118.5 (2C), 114.6
(2C), 113.3 (2C), 103.0 (2C). HRMS calcd for (C18H12Br2N2ꢀH)ꢀ
414.9275; found 414.9280.
4.1.1.66. Synthesis of compounds 26a and 26b.
25b (70 mg, 0.17 mmol), K2CO3 (94 mg, 0.65 mmol) and methyl io-
dide (19 L, 0.30 mmol) in DMF (1.5 ml) was stirred at 30 °C for 3 d
A mixture of
l
and then partitioned between EtOAc (20 ml) and H2O (10 ml). The
organic phase was washed with brine (10 ml), dried over anhy-
drous Na2SO4 and concentrated. The crude product was purified
by automated flash chromatography using EtOAc and hexanes as
eluents to give compounds 26a and 26b.
4.1.1.72.
(28a).
1,2-Bis-(6-bromo-1H-indol-2-yl)ethane
Yield = 53%, yellow solid. 1H NMR (600 MHz, DMSO-
d6): d 11.18 (s, 2H), 7.46 (s, 2H), 7.46 (d, J = 8.3 Hz, 2H), 7.04 (d,
J = 8.3 Hz, 2H), 6.21 (s, 2H), 3.11 (s, 4H). 13C NMR (150 MHz,
DMSO-d6): d 140.4 (2C), 136.8 (2C), 127.3 (2C), 121.4 (2C), 120.8
(2C), 113.1 (2C), 112.7 (2C), 98.7 (2C), 27.0 (2C). HRMS calcd for
(C18H14Br2N2ꢀH)ꢀ 416.9431; found 416.9435.
4.1.1.67.
6-Bromo-2-((6-bromo-1H-indol-2-yl)ethynyl)-1-
Yield = 40%, yellow solid. 1H NMR
methyl-1H-indole (26a).
(600 MHz, DMSO-d6): d 11.99 (s, 1H), 7.79 (s, 2H), 7.56 (s, 1H),
7.54 (d, J = 8.4 Hz, 2H), 7.23 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 7.19 (dd,
J = 1.7 Hz, 8.5 Hz, 1H), 6.96 (s, 2H), 3.89 (s, 6H). 13C NMR
(150 MHz, DMSO-d6): d 138.0, 137.4, 126.2, 125.6, 123.2, 123.0,
122.4, 122.3, 121.7, 118.4, 116.4, 116.1, 113.8, 113.1, 108.5,
107.6, 88.4, 83.6, 30.9. HRMS calcd for (C19H12Br2N2ꢀH)ꢀ
426.9275; found 426.9279.
4.1.1.73. Synthesis of (6-bromo-1-(phenylsulfonyl)-1H-indol-2-
yl)methanol (29).
6-Bromo-N-benzenesulfonate (1.79 g,
5.3 mmol) in THF (20 ml) was cooled to ꢀ78 °C and LDA 1.8 M in
THF (4.46 ml, 8.0 mmol) was added. After 30 min at ꢀ78 °C and
30 min at rt the mixture was cooled to ꢀ78 °C and paraformalde-
hyde (206 mg, 6.89 mmol) was added all at once. The reaction
was warmed to rt and stirred for 12 h and then quenched by addi-
tion of saturated NH4Cl. The mixture was extracted with EtOAc, the
organics were washed with brine, dried over Na2SO4, concentrated
and purified by automated flash chromatography using EtOAc and
hexanes as eluents to give compound 29.
4.1.1.68.
(26b).
1,2-Bis-(6-bromo-1-methyl-1H-indol-2-yl)ethyne
Yield = 47%, pale brown solid. 1H NMR (600 MHz,
DMSO-d6):
d 7.82 (s, 2H), 7.55 (d, J = 8.3 Hz, 2H), 7.24 (d,
J = 8.3 Hz, 2H), 7.03 (s, 2H), 3.90 (s, 6H). 13C NMR (150 MHz,
DMSO-d6): d 138.0 (2C), 125.6 (2C), 123.2 (2C), 122.5 (2C), 121.5
(2C), 116.5 (2C), 113.1 (2C), 108.1 (2C), 86.9 (2C), 31.0 (2C). HRMS
calcd for (C20H14Br2N2+H)+ 442.9577; found 442.9562.
Yield = 72%, white solid; 1H NMR (CDCl3, 500 MHz): d 8.25 (d,
J = 0.6 Hz, 1H), 7.83 (dd, J = 1.1, 8.5 Hz, 1H), 7.61–7.56 (m, 1H),
7.47 (dd, J = 5.0, 10.8 Hz, 1H), 7.38–7.33 (m, 1H), 6.62 (s, 1H),
4.88 (s, 1H). 13C NMR (CDCl3, 126 MHz): 140.72, 138.27, 137.70,
134.47, 129.72, 128.01, 127.39, 126.51, 122.44, 118.88, 117.48,
111.12, 58.53.
4.1.1.69. Synthesis of (E)-2-(2-(1H-indol-2-yl)vinyl)-6-bromo-
1H-indole (27a).
0.17 mmol) and Et3N (0.34 ml, 2.4 mmol) in THF (2 ml) at 50 °C un-
der Ar was added formic acid (88%, 77 L, 1.8 mmol) followed by
To a stirred solution of 25b (72 mg,
l
4.1.1.74. Synthesis of (E)-6-bromo-2-(2-(5-bromo-1-(phenylsul-
fonyl)-1H-indol-2-yl)vinyl-1-(phenulsulfonyl)-1H-indole
addition of 10% Pd/C (8 mg). More 10% Pd/C (8 mg) was added
every 15 min to a total of 40 mg and the mixture was heated for
an additional 2 h, filtered through a pad of silica which was thor-
oughly washed with EtOAc (2 ꢁ 10 ml). The filtrate was washed
with H2O (10 ml), brine (10 ml), dried over anhydrous Na2SO4
and concentrated. The crude product was purified by automated
flash chromatography using EtOAc and hexanes as eluents to give
compounds 27a as yellow solid (4 mg, 7%). 1H NMR (600 MHz,
DMSO-d6): d 11.57 (s, 1H), 11.42 (s, 1H), 7.50 (d, J = 7.9 Hz, 1H),
7.49 (br s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H),
7.20 (d, J = 16.5 Hz, 1H), 7.18 (d, J = 16.5 Hz, 1H), 7.11–7.09 (m,
2H), 6.96 (dd, J = 7.1 Hz, 7.8 Hz), 6.57 (s, 2H). 13C NMR (150 MHz,
DMSO-d6): d 138.2, 137.7, 137.5, 136.5, 128.4, 127.6, 122.18,
122.17, 121.6, 120.0, 119.3, 119.1, 117.7, 114.5, 113.3, 110.9,
103.2, 102.6. HRMS calcd for (C18H13BrN2ꢀH)ꢀ 335.0189, found
335.0195.
(31).
Compound 29 (1.0 g, 2.54 mmol) was treated in dry
l, 2.4 mmol) added slowly
ether (15 ml) at 0 °C with PBr3 (240
l
and then the reaction was stirred at rt for 30 min. After addition
of aq KBr, the mixture was extracted with ether and the organics
were washed with brine, dried over Na2SO4, concentrated to pro-
vide the crude bromide as a brown solid which was dissolved in
DCM (20 ml) and triphenylphosphine (744 mg, 2.84 mmol) was
added and the mixture was stirred at rt overnight. The solvent
was removed in vacuo and the residue was suspended in EtOAc
(15 ml), sonicated and the solid collected by filtration. The crude
Wittig reagent (1.0 g, 1.49 mmol) was dissolved in 1:1, THF/MeOH
(20 ml) and DBU (285 ll, 2.02 mmol) was added followed by
5-bromo-N-benzenesulfonylindole (490 mg, 1.35 mmol). The mix-
ture was stirred at rt for 3 h and then the solvent was removed
under reduced pressure and the residue was partitioned between
water and EtOAc, and the aqueous layers were washed with EtOAc,
the organic extracts were combined, washed with brine dried over
Na2SO4, concentrated and purified by automated flash chromatog-
raphy using EtOAc and hexanes as eluents to give compound 31 as
a yellow solid.
4.1.1.70. Synthesis of compounds 27b and 28a.
solution of 6-bromo-1H-indole-2-carbaldehyde
0.36 mmol) and TiCl4 (58
To a stirred
(80 mg,
l, 0.53 mmol) in THF (5 ml) under Ar
l
was added Zn dust (70 mg, 1.1 mmol) gradually over 15 min and
the resulting mixture was refluxed for 3 h. After cooling to rt 10%
aqueous solution of K2CO3 (1 ml) was added and the reaction mix-
ture was stirred at rt for 16 h. The mixture was partitioned
Yield 36%; 1H NMR (DMSO-d6, 500 MHz): d 8.23 (s, 1H), 8.04 (d,
J = 8.9 Hz, 1H), 7.85 (d, J = 1.9 Hz, 1H), 7.83–7.48 (m, 15H), 7.35 (s,
1H), 7.27 (s, 1H). 13C NMR (DMSO-d6, 151 MHz): 139.40, 138.69,

1724
N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
137.45, 137.14, 136.81, 136.72, 135.60, 135.10, 135.02, 131.62,
130.20, 130.09, 130.03, 129.92, 128.77, 128.05, 127.65, 126.45,
126.24, 126.21, 123.81, 123.25, 121.68, 121.58, 118.16, 117.13,
117.02, 116.50, 110.27, 109.61. HRMS calcd for (C30H20Br2N2O4S2-
ꢀH)ꢀ 692.9153; found 692.9158.
of 64–0.031
added, in duplicate, to either, 190
Hinton broth (CAMHB) or 190
lg/ml. 10 ll of the compound solution was then
l
l of cation adjusted Mueller
l
l CAMHB containing ꢂ2.5 ꢁ 105
CFU/ml of bacteria (final compound concentration 64–0.031 lg/
ml). Culture plates were then incubated for 18–24 h at 37 °C and
the optical density at 590 nm (OD590) was measured using a Tecan
M200 (Tecan). The absorbance control values for the series con-
taining CAMHB and inhibitor were subtracted as background from
the corresponding infected wells. The MIC was defined as the low-
est concentration of test compound leading to complete inhibition
of cell growth in relationship to compound-free control wells as
determined by optical density. Vancomycin was used as a refer-
ence compound. Experiments were replicated at least three times
to verify reproducibility using the above conditions.
4.1.1.75. Synthesis of (E)-6-bromo-2-(2-(5-bromo-1H-indol-2-
yl)vinyl)-1H-indole (27c).
was dissolved in 1 ml of THF/MeOH, (2:1) and Cs2CO3 (28 mg,
0.86 mmol) was added and the mixture has heated in a wave
Compound 31 (20 mg, 0.29 mmol)
l
reactor at 90 °C for 30 min. The reaction was cooled, solvents were
removed and the residue was stirred with H2O (1 ml) for 10 min
and then the mixture was extracted with DCM. The extracts were
dried (Na2SO4), concentrated to dryness and the residue was puri-
fied by automated flash chromatography using EtOAc and hexanes
as eluents to give compound 27c as a yellow solid.
4.2.4. The effect of efflux inhibitor verapamil on in vitro
susceptibility testing
Yield = 88%, yellow solid; 1H NMR (DMSO-d6, 500 MHz): d 11.65
(s, 1H), 11.60 (s, 1H), 7.68 (d, J = 1.6 Hz, 1H), 7.50 (s, 1H), 7.46 (d,
J = 8.4 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.22–7.17 (m, 3H), 7.11
(dd, J = 1.7, 8.4 Hz, 1H), 6.60 (s, 1H), 6.56 (s, 1H). 13C NMR
(DMSO-d6, 126 MHz): 138.27, 137.96, 137.38, 136.11, 130.33,
127.52, 124.44, 122.23, 122.05, 121.72, 118.86, 118.47, 114.69,
113.29, 112.84, 111.74, 103.12, 102.3. HRMS calcd for (C18H12Br2-
N2ꢀH)ꢀ 412.9289; found 412.9251.
MIC values for selected antimicrobial actives were determined
in the presence of efflux inhibitor verapamil with S. aureus
(ATCC29213) using the 96-well microtiter procedure described
above. A sub-inhibitory concentration of verapamil (200 mg/L)11
was added to CAMHB and all assays were preformed as described
above with either CAMHB or CAMHB containing verapamil
(vCAMHB). Culture plates were then incubated for 18–24 h at
37 °C and the optical density at 600 nm (OD600) was measured
using the PowerWave™ XS (BioTek). The absorbance control val-
ues for the series containing CAMHB or vCAMHB and inhibitor
were subtracted as background from the corresponding infected
wells. The MIC was defined as the lowest concentration of test
compound leading to complete inhibition of cell growth in rela-
tionship to compound-free control wells as determined by optical
density. Vancomycin was used as a reference compound. All com-
pounds with the exception of 10l, were replicated at least three
times to verify reproducibility using the above conditions. Com-
4.1.1.76. Synthesis of 6-bromo-2-(2-(5-bromo-1H-indol-2-
yl)ethyl)-1H-indole (28b).
Compound 27c (48 mg) was dis-
solved in 1:1 EtOAc/MeOH (2 ml) and 5 mg 10% Pt-C was added
and the mixture was stirred under a H2 atmosphere at rt following
the reaction by TLC. When complete, the mixture was filtered, con-
centrated to dryness and the residue was purified by automated
flash chromatography using EtOAc and hexanes as eluents to give
compound 28b as a white solid.
Yield = 81%. Mp = 208–210 °C; 1H NMR (DMSO-d6, 500 MHz): d
11.23 (s, 1H), 11.17 (s, 1H), 7.58 (d, J = 1.7 Hz, 1H), 7.45 (s, 1H), 7.35
(d, J = 8.4 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H), 7.10 (dd, J = 1.9, 8.5 Hz,
1H), 7.04 (dd, J = 1.8, 8.3 Hz, 1H), 6.20 (s, 1H), 6.18 (s, 1H), 3.14
(s, 4H). 13C NMR (DMSO-d6, 126 MHz): 141.00, 140.39, 136.83,
134.61, 130.18, 127.27, 122.51, 121.41, 121.32, 120.86, 113.14,
112.69, 112.53, 111.15, 98.68, 98.22, 27.04 (2C). HRMS calcd for
(C18H14Br2N2ꢀH)ꢀ 414.9445; found 414.9470.
pound 10l was replicated twice due to
compound.
a shortage of the
4.2.5. Construction of Pyk-deficient bacterial strains and
growth conditions
The S. aureus
D
pyk::ErmR LAC mutant (AR0891) was con-
structed by amplifying ꢂ1 kb 50 and 30 homology fragments sur-
rounding the pyk gene (SACOL1745) with the following primers:
pyk-5.1A (tggtagaattcGCAGTTTTAACTAGTGGTGG) pyk-5.1B (tggta-
gaattcGCTGGTCCAA-TTGTACATAC), pyk-3.1A (gtaggatccACGATTG
ATGCTGCTCAAGG), pyk-3.1B (gtagg-atccAGAAGGTGTTTGATCTTG
AGC). The resulting fragments were then cloned into the EcoRI
and BamHI sites flanking an ErmR cassette, which was cloned into
the SmaI site of the Escherichia coli/S. aureus shuttle vector pBT214
to yield pNV026. Following transformation of pNV026 into S. aur-
eus SF8300, allelic exchange was performed as previously
described12 using TSB without glucose supplemented with 1% so-
dium pyruvate (P). The pyk::ErmR mutation was then transduced
4.2. PK enzymatic assays, cell-based assays and cytotoxicity
4.2.1. Pyruvate kinase enzymes and inhibitory activity testing
The pyruvate kinase enzymes MRSA PK and human PK isoforms
were identical to and were sourced as His-tagged constructs as de-
scribed in Ref. 4. Synthetic candidate MRSA PK inhibitors were di-
luted in 100% DMSO and assayed for their abilities to inhibit
enzymatic activities of MRSA PK and (for selected compounds) hu-
man PKs as described in Ref. 4.
4.2.2. In vitro susceptibility testing Staphylococcus aureus
(ATCC29213)
into S. aureus LAC using bacteriophage
U80.
Determinations of in vitro susceptibility and MIC determina-
tions in S. aureus ATCC29213 were carried out as described in
Ref. 4 except that serial (twofold) dilutions of test compounds were
done in 100% DMSO and added to give a final concentration of
DMSO of 5% in brain heart infusion (BHI) broth (Difco).
4.2.6. Bacterial killing assay
Wild-type S. aureus LAC and S. aureus LAC
D
pyk::ErmR (AR0891)
were grown overnight in TSB without glucose supplemented with
1% sodium pyruvate with shaking at 37 °C. Overnight cultures were
washed 2ꢁ with sterile PBS and then diluted to an OD600 = 0.1
(ꢂ2.5 ꢁ 107 cfu/ml) in pre-warmed (37 °C) media (either BHI or
TSB without glucose supplemented with 1% sodium pyruvate)
4.2.3. In vitro susceptibility testing (MRSA)
MIC values for selected antimicrobial actives were determined
with MRSA strain MW2 (USA400) using the 96-well microtiter CLSI
recommended standard for twofold serial broth microdilution
method13 as described previously.4,7 Each compound was prepared
in DMSO with twofold serial dilutions to give a final concentration
1.6 lM 10d. Cultures were then grown at 37 °C with shaking at
250 rpm. Viable bacterial were enumerated by dilution plating at
0, 8, and 24 h post-inoculation using Tryptic Soy Agar (TSA) with-
out glucose supplemented with 1% sodium pyruvate.

N. S. Kumar et al. / Bioorg. Med. Chem. 22 (2014) 1708–1725
1725
L.; Jiang, J.; Thorson, L.; Labriere, C.; Foster, L.; Brunham, R. C.; McMaster, W. R.;
Finlay, B. B.; Strynadka, N. C.; Cherkasov, A.; Young, R. N.; Reiner, N. E.
Antimicrob. Agents Chemother. 2011, 55, 2042.
4.2.7. Mammalian cytotoxicity
Mammalian cytotoxicity was performed using procedures and
statistical analysis previously described.4
5. Fraser, H. B.; Hirsh, A. E.; Steinmetz, L. M.; Scharfe, C.; Feldman, M. W. Science
2002, 296, 750.
6. Axerio-Cilies, P.; See, R. H.; Zoraghi, R.; Worral, L.; Lian, T.; Stoynov, N.; Jiang, J.;
Kaur, S.; Jackson, L.; Gong, H.; Swayze, R.; Amandoron, E.; Kumar, N. S.; Moreau,
A.; Hsing, M.; Strynadka, N. C.; McMaster, W. R.; Finlay, B. B.; Foster, L. J.;
Young, R. N.; Reiner, N. E.; Cherkasov, A. ACS Chem. Biol. 2012, 7, 350.
7. Kumar, N. S.; Amandoron, E. A.; Cherkasov, A.; Finlay, B. B.; Gong, H.; Jackson,
L.; Kaur, S.; Lian, T.; Moreau, A.; Labrière, C.; Reiner, N. E.; See, R. H.; Strynadka,
N. C.; Thorson, L.; Wong, E. W. Y.; Zoraghi, R.; Young, R. N. Bioorg. Med. Chem.
2012, 20, 7069.
8. Zoraghi, R.; Worrall, L.; See, R. H.; Strangman, W.; Popplewell, W. L.; Gong, H.;
Samaai, T.; Swayze, R. D.; Kaur, S.; Vuckovic, M.; Finlay, B. B.; Brunham, R. C.;
McMaster, W. R.; Davies-Coleman, M. T.; Strynadka, N. C.; Andersen, R. J.;
Reiner, N. E. J. Biol. Chem. 2011, 286, 44716.
Acknowledgments
The authors thank Dr. Jianghong An of the Genome Sciences
Centre of Vancouver for valuable assistance in preparing Figure 2.
The authors acknowledge Genome British Columbia, the NaturalSci-
ences and Engineering Council of Canada (NSERC) and the Genome
BC-CDRD Innovation Fund for financial support. R.N.Y. acknowl-
edges the support of Simon Fraser University, the British Columbia
Government Leading Edge Endowment Fund and Merck Canada Inc.
9. Zechini, B.; Versace, L. Recent Patents Anti-Infect. Drug Discovery 2009, 4, 37.
10. Costa, S. S.; Falcao, C.; Viveiros, M.; Machado, D.; Martins, M.; Melo-Cristino, J.;
Amaral, L.; Couto, I. BMC Microbiol. 2011, 11, 241.
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