Optimization and structure-activity relationships of a series of potent inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as novel antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2012.10.002

A novel series of hydrazones were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK has been identified as one of the most highly connected 'hub proteins' in MRSA. PK has been shown to b

Full text of DOI:10.1016/j.bmc.2012.10.002

Bioorganic & Medicinal Chemistry 20 (2012) 7069–7082
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Optimization and structure–activity relationships of a series
of potent inhibitors of methicillin-resistant Staphylococcus aureus (MRSA)
pyruvate kinase as novel antimicrobial agents
Nag S. Kumar a, Emily A. Amandoron b, Artem Cherkasov b,c, B. Brett Finlay d,e, Huansheng Gong b,
Linda Jackson e, Sukhbir Kaur b, Tian Lian b, Anne Moreau a, , Christophe Labrière a, Neil E. Reiner b,e
Raymond H. See b,f, Natalie C. Strynadka d, Lisa Thorson b, Edwin W. Y. Wong a, Liam Worrall d,
,
Roya Zoraghi b, Robert N. Young a,
⇑
a Department of Chemistry, Simon Fraser University, Burnaby, Canada
b Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, BC, Canada
c Vancouver Prostate Centre, Vancouver, BC, Canada
d Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada
e Department of Microbiology and Immunology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada
f University of British Columbia Centre for Disease Control, Vancouver, BC, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2012
Revised 25 September 2012
Accepted 3 October 2012
Available online 17 October 2012
A novel series of hydrazones were synthesized and evaluated as inhibitors of methicillin-resistant Staph-
ylococcus aureus (MRSA) pyruvate kinase (PK). PK has been identified as one of the most highly connected
‘hub proteins’ in MRSA. PK has been shown to be critical for bacterial survival which makes it a potential
target for development of novel antibiotics and the high degree of connectivity implies it should be very
sensitive to mutations and thus less able to develop resistance. PK is not unique to bacteria and thus a
critical requirement for such a PK inhibitor would be that it does not inhibit the homologous human
enzyme(s) at therapeutic concentrations. Several MRSA PK inhibitors (including 8d) were identified using
in silico screening combined with enzyme assays and were found to be selective for bacterial enzyme
compared to four human PK isoforms (M1, M2, R and L). However these lead compounds did not show
significant inhibitory activity for MRSA growth presumably due to poor bacterial cell penetration. Struc-
ture–activity relationship (SAR) studies were carried out on 8d and led us to discover more potent com-
pounds with enzyme inhibiting activities in the low nanomolar range and some were found to effectively
Keywords:
Methicillin-resistant Staphylococcus aureus
Antimicrobial
Pyruvate kinase inhibitor
Structure–activity relationships
inhibit bacteria growth in culture with minimum inhibitory concentrations (MIC) as low as 1 lg/mL.
These inhibitors bind in two elongated flat clefts found at the minor interfaces in the homo-tetrameric
enzyme complex and the observed SAR is in keeping with the size and electronic constraints of these
binding sites. Access to the corresponding sites in the human enzyme is blocked.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
these infections have begun to find their ways out into the general
community and this growing phenomenon only offers to worsen
The rapid increase in antibiotic-resistant hospital-borne infec-
tions such as methicillin-resistant Staphylococcus aureus (MRSA)
and vancomycin-resistant Staphylococcus aureus (VRSA) is creating
a crisis in public health systems in the developed world. Recently,
the crisis. Little progress has been made in the past several decades
to identify new antibacterial targets that could be less prone to the
development of resistance. A general principle of antibiotic target
selection has been to target critical proteins and processes unique
to the bacteria and without human homologs so as to avoid mech-
anism-based toxicity. This has severely limited the target options.
We have recently described preliminary results of an alterna-
tive effort wherein a highly interconnected ‘hub protein’, bacterial
pyruvate kinase (PK), was selected for evaluation1 based on consid-
erations that; (1) the essentiality of the enzyme should render
inhibition lethal, (2) the high connectivity should confound the
Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; PK, pyruvate
kinase; MIC, minimal inhibitory concentration; VRSA, vancomycin-resistant Staph-
ylococcus aureus.
⇑
Corresponding author. Tel.: +1 778 782 3351; fax: +1 778 782 3765.
E-mail addresses: roberty@sfu.ca, robert_young@sfu.ca (R.N. Young).
Current address: 186 bis, Avenue du Marechal Joffre, 1 Villa Leseur, 94170 Le
Perreux-sur-Marne, France.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.002

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N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
Table 1
viability of mutations that might seek to produce resistant strains,
(3) there is no known available ‘escape’ paralog enzyme in the bac-
terial genome, (4) there is a wealth of available structural informa-
tion on both the bacterial and human homologous enzymes and (5)
there are unique peptide sequence insertion and deletion differ-
ences between the bacterial and human enzymes that appear to
produce druggable lipophylic pockets on the bacterial enzyme that
are absent on the human forms. One of these pockets was probed
in an in silico screening effort2 which led to the identification of a
number of compounds with MRSA PK inhibiting activities in the
low nanomolar range (e.g. IS-63) that were selective over the four
known human homologs (M1, M2, R and L). A subsequent round of
similarity searching and screening identified compound IS-130
(8d) (Fig. 1) that was 10-fold more potent (IC50 91 nM) and selec-
tive (>1000-fold) over the human PK. However, neither compound
showed efficacy in inhibiting growth of intact bacteria suggesting
that they were unable to pass through the bacterial cell wall (see
Table 1). Subsequent exploration of structure–activity relation-
ships and optimization of physico-chemical properties identified
analogs (such as compounds 7d and 6/) which were both potent
enzyme inhibitors and which showed effective inhibition of a wide
panel of gram positive bacterial growth comparable to standard
antibiotics such as vancomycin.3 We eventually were able to ob-
tain an X-ray structure for 8d and 6/ bound to MRSA282 PK which
revealed that the compounds do not bind to the originally targeted
lipophilic pocket but rather in a flat lipophilic pocket at the minor
interfaces in the homo-tetrameric enzyme structure (see Fig. 2).2
Subsequently some dimeric indole natural products have also been
shown to bind to the same site.4 Differing sequences for the bacte-
rial versus human enzymes meant that this binding site was not
accessible in the human PK enzymes (see Discussion). PK is
thought to be significantly more active in its tetrameric form and
it is not entirely clear how this binding inhibits activity. It has been
hypothesized that allosteric binding at this site may prevent the
enzyme tetramer from adopting a more compressed and rigidized
confirmation required for effective enzymatic activity.4 Notably,
MRSA passaged 25 consecutive times in the presence of sub-lethal
concentration of 7d did not develop significant resistance suggest-
ing that inhibition of MRSA PK may indeed yield anti-bacterial
agents that are less prone to develop any resistance.3
PK inhibitory activity and antimicrobial activity of compounds with modification on
the indole moiety
Br
Br
O
O
R1
7
N
NH
HO
N
6
5
1
2
N
NH
HO
X
Y
3
4
6a-n,7a-j
8a-d
Analogues
IC50 (nM)a
MICb
>186
(
lg/mL)
R1
Substitutions
6a
6b
6c
6d
6e
6f
6g
6h
6i
85 (2)
49 (2)
52 (2)
49 (3)
43 (2)
46% @ 10
214
178 (2)
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
—
>195 (2)
>81(2)
>226
>64 (2)
ND
5-Fluoro
5-Chloro
5-Bromo
5-Iodo
lM
5-Trifluoromethyl
5-Hydroxy
5-Methoxy
5-Phenyl
⁄>64
>80
0% @ 1
24
20
l
M
>90
6j
6k
6l
⁄>64
6-Bromo
>82
5,6-Difluoro
4,5-Difluro
7-Fluoro
24 (2)
41
6m
6n
7a
7b
7c
7d
7e
7f
39% @ 0.5
15
l
M
>156
⁄⁄>85
⁄⁄7.7 (3)
3.3 (3)
1.3 (5)
1.4 (2)
⁄>64
4,5,6-Trifluoro
381 (2)
165 (2)
228
146 (3)
54% @ 1
794
17
61
145 (2)
55
286 (2)
224
5-Fluoro
5-Chloro
5-Bromo
5-Hydroxy
5-Methoxy
6-Bromo
lM
10.4
7g
7h
7i
1.0
1.6 (2)
2.0 (2)
⁄⁄2.0
>195
>78 (3)
⁄⁄>150
>187
>194(2)
5,6-Difluoro
4,5-Difluoro
4,5,6-Trifluoro
X = S, Y = CH
X = S, Y = N
X = O, Y = N
X = NH, Y = N
X = NCH3, Y = N
7j
8a
8b
8c
8d
8e
—
—
—
—
26% @ 0.5
91 (2)
227
l
M
a
IC50 values are calculated from triplicate 15 point titrations or are an average of
(n) such determinations as indicated. Alternatively the % inhibition at the highest
concentration tested is presented.
b
Minimum concentration to give >98% inhibition of growth of S. aureus RN4220,
⁄S. aureus ATCC 25923 or ⁄⁄ATCC 29213 (single determination or average of (n)
determinations). Control MIC (vancomycin) is 1 lg/ml.
In this paper we present a detailed account of structure–activity
relationships for enzyme inhibitory and optimization of antibacte-
rial activity for an extensive series of indole hydrazones.
subsequently treated with either Grignard or organolithium re-
agent to give ketones 3l–n. To make the aldehyde derivatives (R5
is H), compound 1 was reduced to an alcohol with LiAlH4 and then
re-oxidized with MnO2 to give aldehyde 3o. Subsequently, com-
pound 3 was coupled to the hydrazide 5 (which was prepared from
the corresponding ester and hydrazine) by a simple condensation
reaction either in neutral or mild acidic conditions to give com-
pound 6. In order to prepare compound 7, intermediate 3 was first
alkylated with alkyl halide and then condensed with hydrazide 5.
Compounds 8 and 10 were prepared in a similar manner where
an appropriate ketone was condensed with phenyl, pyridine or
naphthalene hydrazides (Scheme 2).
2. Results
2.1. Chemistry
The syntheses of all target compounds were carried out as de-
scribed in Schemes 1 through 4. Palladium-catalyzed annulation
of ortho-iodoanilines and 2-oxopropanoic acid gave 2-carboxylic
acid indoles (1) (Scheme 1). Treatment of 1 with MeLi furnished
the methyl ketones (3a–f). In the case where R5 is ethyl, t-butyl
or phenyl, the 2-carboxylic acid indole (1) was first converted to
Weinreb amide (2) (in order to avoid double addition) which was
N-methyl hydrazide 9 was synthesized from 1-(1H-indol-2-
yl)ethanone (3p) where it was first treated with methylhydrazine
to form a Schiff’s base and subsequently reacted with an activated
salicylic acid derivative to give compound 9 (Scheme 3). Com-
pound 12 with an oxime linking moiety was synthesized by react-
ing appropriate acid chloride with oxime 11, which was itself
prepared from the corresponding ketone 3 by condensation with
hydroxylamine hydrochloride in presence of pyridine. Finally the
sulfonohydrazide 13 was made by coupling appropriate methyl ke-
tone with 5-bromo-2-methoxybenzenesulphonohydrazide which
Br
H
O
HO
O
N
N
H
N
N
N
NH
N
HO
O
IS-63
8d
Figure 1. MRSA PK inhibitors identified through in silico screening.

N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
7071
Figure 2. Resolved structure of MRSA252 pyruvate kinase tetramer showing the domain boundaries and tetramer architecture. Each monomer has been coloured to facilitate
the identification of the small and large interface shown as lines. The binding site located at the small interface sits in between two alpha helices formed by two PK monomers
shown as circles.
R1
CO2R
R2
R3
O
N
R5MgX or R5Li
R10
R7
R8
THF
N
H
R5 = Me, Et, t-Bu, Ph
MeO
R4
R9
2
H2N-NH2 . H2O
EtOH
Me
NH . HCl
MeO
CONHNH2
HBTU
N-Methylmorpholine
R10
R7
R8
R8
DMF
O
CO2H
DABCO
O
R7
R9
R1
R1
R4
R1
R4
R1
R4
R9
R10
5
R2
R3
R2
R2
R3
R2
R3
I
O
N
NH
Pd(OAc)2
Procedure A, B,C or D
MeLi
CO2H
DMF
Et2O
R5 = Me
N
H
N
H
N
H
R5
R5
NH2
R3
R4
1
6
3
R6X
K2CO3
DMF
1) LiAlH4, Et2O
2) MnO2, CH3CN
R8
R
5 = H
O
R7
R1
R4
R1
R4
5
R9
R10
R2
R3
R2
R3
O
N
NH
Procedure A, B,C or D
N
R6
N
R6
R5
R5
4
7
Scheme 1. General syntheses of the hydrazones 6 and 7. Procedure: (A) nPrOH, reflux. (B) EtOH, reflux. (C) AcOH, nPrOH, reflux. (D) AcOH, EtOH, reflux.

7072
N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
R8
O
R7
R1
R4
R1
R4
5
R9
R10
R2
R3
R2
R3
O
N
R5
8
NH
Z
Y
Z
Y
Procedure A, B,C or D
R5
Y = NMe, Z = N
Y = S, Z = N
Y = O, Z = N
Y = S, Z = CH
CONHNH2
O
R1
N
R1
N
OH
R4
R3
R4
R3
O
N
NH
HO
Procedure C or D
R5
R5
10
R2
R2
Scheme 2. Preparation of hydrazones 8 and 10. Procedure: (A) nPrOH, reflux. (B) EtOH, reflux. (C) AcOH, nPrOH, reflux. (D) AcOH, EtOH, reflux. (E) EtOH, MW at 100 °C.
HO
H
N
H
N
1) H2N-NHMe . H2SO4 / DIPEA / EtOH
O
N
O
N
O
OH
2)
Br
PFPO
9
3
Pyr. / DCM
Br
Scheme 3. Preparation of N-methyl hydrazones 9.
was prepared by condensing 5-bromo-2-methoxyphenyl-1-sulfo-
nyl chloride with hydrazine (Scheme 4).
at the highest concentration tested (10 lM) (See Tables 6–9 Sup-
plementary data). Cellular antimicrobial activity was evaluated
for ability to fully inhibit the growth of MRSA in culture (determin-
ing minimum inhibitory concentration (MIC) values). Cytotoxicity
was evaluated for selected compounds using mammalian cells
(THP-1 or Hela 293).
2.2. Biological results and SAR studies
Compounds were evaluated to determine their ability to inhibit
MRSA pyruvate kinase (PK) activity and for selected compounds
selectivity was determined using the mammalian PK isoforms
M1, M2, R and L. Of those compounds tested for selectivity none
showed inhibition of the mammalian PK isoforms greater than
50% and they generally showed no significant inhibition (<20%)
2.2.1. SAR for in vitro inhibition of MRSA PK
The lead structure 8d showed interesting potency on the MRSA
PK enzyme but not in the cellular anti-MRSA assay and so we set
out to develop SAR for both enzyme inhibition and antibacterial
O
Cl
OR
RO
O
O
N
N
Br
Et3N / DCM
N
NH2OH.HCl / Pyr. / EtOH
N
N
OH
O
Br
11
12
3
R3
R3
R2
R2
O
OCH3
OCH3
Br
OCH3
SO2NHNH2
N
R1
N
R1
N
NH
S
O
H2N-NH2 . H2O
EtOH
SO2Cl
O
Br
13
AcOH / EtOH
Scheme 4. Preparation of oxime linker 12 and sulfonohydrazide linker 13.
Br

N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
7073
Table 2
of the molecule could not tolerate substitution. Equally, bulky sub-
stituents at position 5 (6f and 6i) lost most or all activity. Polar
substitutions such as OH or OCH3 (6g or 6h) were tolerated but
trended to lesser potency.
PK inhibitory activity and antimicrobial activity of phenol modified derivatives
2'
3'
O
1'
O
4'
R1
7
4
N
NH
N
We also investigated the effects of substitutions on the N-methyl
indole series and found that (relative to 7a), 5-halogen substituted
compounds (7b, 7c and 7d) appeared to be slightly more potent while
the 6-bromo analog 7g was most potent (IC50 17 nM). Polyhalogenat-
ed analogs (7h, 7i and 7j) showed either no advantage or lost potency.
The 5-hydroxy (7e) or 5-methoxy (7f) analogs were much less potent.
We next turned our attentions to the phenolic element in these
hydrazones (Table 2). Removal of either the hydroxyl- or bromo-
substituents (or both) led to a dramatic drop in activity (6o, 6p,
6r) while placing the bromine at the 40 position led to a less dra-
matic (ca. 10-fold) loss in activity. Notably, the phenolic hydroxyl
function could not be replaced by a pyridine nitrogen (6s) nor
could the 50 bromine be replaced by chlorine (6t). However the cor-
responding iodo-analog (6u) had similar potency compared to 6a
suggesting these substituents occupied an important lipophilic
binding pocket. Compound 6v, with phenyl substitution at the 50
position, was essentially inactive indicating that this pocket was
of limited size. It was interesting to note that the hydroxyl function
in 6a could be substituted (6w, 6x, 6y, 6z) with little or no loss in
activity, which strongly suggested that the oxygen in all these
compounds was acting as a hydrogen bond acceptor and not a do-
nor. Simple modelling energy calculations (MM2; ChemDraw) sug-
gested that the hydrazone NH forms a strong hydrogen bond with
these adjacent oxygen atoms and that this serves to flatten and ori-
ent the critical bromine atom in a specifically defined direction.
Subsequently, X-ray diffraction of 6/ (Fig. 3) as well as X-ray struc-
tural analysis on 8d2 and 6/ (unpublished) bound to MRSA PK
(Fig. 4) confirmed this orientation. We also prepared several ana-
logs with multiple substitutions on the ‘phenol’ ring and found that
H
N
6
5
6'
5'
1
2
N
NH
X
3
6s
Analogues IC50
MICb
(lg/mL)
R1
Substitutions
(nM)a
6o
6p
6q
6r
8150 (2)
8615
1312
>178
59 (3)
ND
H
H
H
H
50-Bromo
20-Hydroxy
20-Hydroxy-40-bromo
28% @
>139
10
15% @
10
36% @
lM
6s
6t
>139
>64
—
H
X = N
lM
20-Hydroxy-50-chloro
1 lM
151
20% @
10 lM
182
63
251
6u
6v
>84 (2)
ND
H
H
20-Hydroxy-50-iodo
20-Hydroxy-50-phenyl
6w
6x
6y
>193
⁄⁄>64
⁄⁄>64
H
H
H
20-Methoxy-50-bromo
20-Ethoxy-50-bromo
20-Ethoxymethoxy-5-
bromo
6z
64 (2)
74
>205
64
H
H
H
H
H
20-(Prop-2-yn-1-
yloxy)-5-bromo
20-Hydroxy-30,50-
dibromo
6a
6b
57% @
1 lM
⁄>93
20-Methoxy-30,50-
dibromo
6v
40% @ 10 ND
20-Hydroxy-30,50-
diisopropyl
uM
324
6d
>161
20-Hydroxy-40-
methoxy-50-bromo
7k
7l
483
61% @
5.4
4.8
CH3 20-Hydroxy-50-iodo
CH3 20-Hydroxy-40-bromo
the 30,50-dibromo analog (6
a) was essentially equipotent when
0.5
lM
compared with 6a while interestingly, the corresponding methoxy
analog 6b was less active when compared to its analogous mono-
bromo analog 6w. This likely reflects the steric compression that
the methoxyl methyl experiences from the adjacent bromine atom
as it tries to orient itself to achieve the important hydrogen bond
7m
841
>167
CH3 20-Hydroxy-40-
methoxy-50-bromo
a
IC50 values are calculated from triplicate 15 point titrations or are an average of
(n) such determinations as indicated. Alternatively the % inhibition at the highest
concentration tested is presented.
with the hydrazone NH. A 30,50-diisopropyl analog 6
v was not ac-
b
Minimum concentration to give >98% inhibition of growth of S. aureus RN4220
or ⁄S. aureus ATCC 25923 or ⁄⁄ATCC 29213 (single determination or average of (n)
tive while the 40-methoxy-50-bromo analog 6d showed reduced but
still significant activity. 40,50-disubstitution was shown to be toler-
ated as reinforced by the potent enzyme inhibitory activity we ob-
serve for some naphthalene analogs (10a, 10b; Table 5).
determinations).
activity in parallel. However, for purpose of clarity we will discuss
the SAR derived for the in vitro enzyme inhibition separately from
the cellular antibacterial activity. We systematically evaluated
structural changes in the three regions of the lead molecule (8d);
the ‘left hand’ heteroaryl moiety (Table 1), the ‘right hand’ phenolic
moiety (Tables 2 and 5) and central linking moiety (Tables 3 and 4)
To evaluate the role of the benzimidazole nitrogen functions in
compound 8d we prepared a series of heterocyclic analogs. Thus
the corresponding indole (6a), benzothiophene (8a), benzothiazole
(8b), and benzoxazole (8c) analogs were prepared and notably 6a
retained potency and selectivity (compared to 8d) while 8a and
8b were about 3-fold less potent and 8c was much less potent
(Table 1). Similarly the N-methylated benzimidazole (8e) lost
about 3-fold potency compared to 8d and the N-methyl indole
7a, was about 4-fold less active (than the parent 6a) thus suggest-
ing that neither a basic nitrogen nor an NH were critical of enzyme
inhibitory activity. At this point, observation of anti-MRSA activity
in the analog 7a caused us to focus efforts on the indole series. We
thus evaluated the effect of substitution(s) on the indole ring. Hal-
ogen substitution at positions 5 (compounds 6b–e) or 6 (6j) led to
2 to 4-fold increased potency (relative to 6a) as did multi-substitu-
tion at positions 4,5 (6l), positions 5,6 (6k) or 4,5,6 (6n). Notably,
the 7-fluoro analog 6m was much less potent suggesting this face
Our next step was to investigate the structural requirements for
enzyme inhibitory activity as it related to the linking hydrazone
element (Tables 3 and 4). Replacing the R2 methyl group with
hydrogen led to a loss of activity (6
ethyl group (6/) retained activity. The phenyl-substituted analog
(6 ) showed a 4 to 5-fold loss of activity on the enzyme. Finally,
the t-butyl analog 6 was prepared and found to be essentially
inactive on the enzyme. NMR analysis and the X-ray diffraction
structure (Fig. 3) indicated 6 was completely present as the
e) while replacing it with an
u
i
i
Z-isomer and presumably this dramatic shape change is not
accommodated by the enzyme binding site.
We next examined the NH and carbonyl functions of the linker
element and found that the N-methyl hydrazone (9) and the acyl-
oxylamines (12a, 12b) were essentially without activity. This is in
keeping with the crucial role the NH plays via a hydrogen bond in
orienting the phenol ring and the bromine atom. Notably the sul-
phonamides (13a and 13b) were also inactive. This may relate to
the need for absolute planarity in this part of the binding site.
2.2.2. Structural and molecular modelling studies
The SAR observations for inhibition of MRSA PK were found to
agree very well with the binding site revealed in the X-ray

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N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
Table 3
Table 5
PK inhibitory activity and antimicrobial activity of compounds with modification on
the linker
PK inhibitory activity and antimicrobial activity of naphthol derivatives
O
Br
R1
7
N
NH
N
6
5
R1
R4
N R3
1
2
7
4
HO
N
6
5
1
2
R2
3
O
4
R5
R2
3
Analogues
IC50 (nM)a
MIC (
l
g/mL)b
R1
R2
Substitutions
Analogues IC50 (nM)b
MICc
g/mL)
R1 R2
R3
R4
R5 Substitutions
10a
10b
10c
10d
10e
10f
42
26
31
18
115
79
59
52
>64
>64
>64
16
>64
1
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
(l
5-Bromo
4,5,6-Trifluoro
5-Bromo
5-Bromo
5-Bromo
C2H5
C2H5
CH3
C2H5
CH3
6
6/
e
863(3)
126 (2)
451
>179
9.7
H
H
H
H
H
NH
C2H5 NH
NH
C2H5 NH
C(O) H
C(O) H
C(O) CH3
C(O) H
C(O) H
C(O) H
C(O) H
6c
⁄⁄>64
⁄⁄4.0
H
6g
49
5-Bromo
5-Bromo
7n
168
⁄⁄2.0
CH3 C2H5 NH
CH3 NH
CH3 C2H5 NH
10g
10h
>64
32
4,5,6-Trifluoro
4,5,6-Trifluoro
C2H5
7o
3087 (2)
461
>186
5
H
7p
a
IC50 values are calculated from a triplicate 15 point titration unless otherwise
noted.
9a
33% @ 1
34% @ 10
33% @ 10
12% @ 10
11% @ 10
l
M
⁄>80
H
CH3 NCH3 C(O) CH3
12a
12b
13a
13b
l
l
l
l
M >155
M >161
M ND
M ND
CH3 CH3
CH3 CH3
O
O
C(O) H
C(O) CH3
SO2 CH3
b
Minimum concentration to give >98% inhibition of growth of S. aureus ATCC
29213 (single determination unless noted).
H
H
CH3 NH
CH3 NH
SO2 CH3 4,5-Difluoro
a
structure of 6/ gives us confidence in the modelled orientation
in the interface binding pocket. The ligand in the X-ray structure
was found to exist in the expected conformation with a hydrogen
bond between the hydrazone NH and the phenol oxygen. The 5-
bromo substituent was oriented toward the interior of the site in
a deep lipophilic pocket formed by Ala 358-A and Ile 351-A. The in-
dole NH, hydrazone C@N and carbonyl all appear to form hydrogen
bonds to serine 362-A and B which is also interior to the site (see
Fig. 4). The phenol and ethyl group of 6/ are oriented towards the
outside (water side) of the binding site in keeping with our obser-
vations that the phenol could be substituted with a variety of short
or long chains without significant loss of activity. Equally we ob-
served that the ethyl group can be replaced by a flat phenyl group
but not with the bulky t-butyl group that disfavours the elongated
E-isomer. The X-ray structure revealed some vacant space in the
vicinity of the indole phenyl ring to allow for small substitution
but indicated a tight fit adjacent to the indole NH (the 7-position)
that would be in keeping with the observed intolerance for substi-
tution at that position. It is not clear if the methyl group of the N-
methyl indole analogs can be accommodated oriented to the inte-
rior or would prefer the exterior of the site. The former appears to
also be a tight fit but may nonetheless be preferred as the ‘outside’
rotamer would produce a peri interaction between the ethyl group
(or methyl) of the hydrazone and the N-methyl of the indole and
thus it seems unlikely that strict planarity could be maintained.
Perhaps the tolerability of non-planarity is greater in this part of
the binding site. Nonetheless, N-methylation would remove the
putative H-bond between the indole NH and serines 362 (vide su-
pra) and thus would explain the 3 to 4-fold loss of potency for the
N-methyl versus the NH indole compound pairs.
Present in two isomeric forms, major isomer shown.
IC50 values are calculated from triplicate 15 point titrations or are an average of
b
(n) such determinations as indicated. Alternatively the % inhibition at the highest
concentration tested is presented.
c
Minimum concentration to give >98% inhibition of growth of S. aureus RN4220
or ⁄S. aureus ATCC 25923 or ⁄⁄ATCC 29213 (single determination or average of (n)
determinations).
Table 4
PK inhibitory activity antimicrobial activity and cytotoxicity in mammalian THP-1
cells for compounds which are either exclusively present in Z configuration (6i,7q) or
in two isomeric forms (6
u
, 7r)
R2
N
O
R1
N
HN
HO
Br
Analogues IC50 (nM)b
MICc
mL)
(lg/
R1
R2
Cytotoxicity (%
dead THP-1 cells)
25
C(CH3)3 50
l
M
6.25
42
lM
6
i
0% @
10
445 (2)
0% @10
6043
83
H
H
l
M
6
ua
1.4 (2)
10.7
1.4
Ph
83
43
25
19
7q
l
M
CH3 C(CH3)3 73
CH3 Ph 78
7ra
a
Present in two isomeric forms.
IC50 values are calculated from a triplicate 15 point titration or are an average of
b
(n) such determinations as indicated. Alternatively the % inhibition at the highest
concentration tested is presented.
c
Minimum concentration to give >98% inhibition of growth of S. aureus RN4220
(single determination or average of (n) determinations).
2.2.3. SAR for anti-MRSA activity
In spite of the relatively potent MRSA PK inhibitory activity ob-
served of the original benzimidazole lead (8d) and the analogous
benzoheterocycles (6a, 8a, 8b, 8d) these compounds did not show
significant anti-MRSA activity at concentrations 300 to 1000-fold
higher than enzyme IC50s (Table 1). Equally none of the series of
substituted NH-indoles tested showed measureable MIC values
structure obtained for compounds 8d2 and 6/ (unpublished)
bound to MRSA252 pyruvate kinase (Fig. 4). These two identical
binding sites are located at the minor interface between the paral-
lel domains of the enzyme tetramer (between monomers A and B
and between monomers C and D). The pockets are flat and elon-
gated and accessible in the MRSA PK but access in the human PK
isoforms is blocked by five amino acids (Glu 418-B, Arg 399-A
and B and Arg 400 A and B) (the corresponding amino acids in
MRSA PK are His 365-B, Lys-349 A and B and Thr 348-A and B)
(see Fig. 5). Despite the resolution of the data for the compounds
in complex with MRSA PK (3.1 Å for 8d and 3.3 Å for 6/), the agree-
ment with the independently determined small molecule X-ray
with the exception of 6/ and 6g (Table 3) which gave MIC values
of 9.7 and 4 g/ml respectively.
l
Considering that the anti-MRSA activity of these compounds
might be attributed to the added lipophilicity of the ethyl or the
methyl group (CLogP values of 4.73 for 6/ and 4.67 for 7a versus
4.20 for 6a) we investigated the N-methylated analogs in the in-
dole series which were predicted to be more lipophilic than their

N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
7075
Figure 3. X-ray crystal structure diagrams of 6/ (left) and 6i (right).
Figure 4. Binding mode of compounds 8d and 6/ at the interface binding site (a) a two-dimensional map of the binding interactions between 8d and the interface site based
on its co-crystallization with MRSA PK. Green arrows depict hydrogen-accepting interactions between 8d and MRSA PK residues from the interface (left). Binding orientation
of 8d within the interface-binding pocket based on the protein–ligand crystal structure (right). (b) The same view for 6/.
NH counterparts (Table 10, Supplementary data). We were grati-
fied to observe that many of these N-methylated analogs gave good
to excellent MIC values (see Table 1). Although the CLogP values for
N-methyl indole derivatives (7a–j) were calculated to be generally
0.3–0.6 higher than the corresponding NH derivatives, in an abso-
lute sense there was no clear correlation of CLogP and MIC. In
addition, we noted that while the N-methyl indole analogs (7k,
7l) showed reasonable MICs, surprisingly, the corresponding naph-
thalene analogs, 10e and 10g, did not (although the naphthalene
analog with R2 = ethyl (10f) had an excellent MIC of 1 lg/ml
(Table 5). Thus it was apparent that the SAR for antibacterial activ-
ity was not fully transferable from the phenyl to the naphthalene
series and in general the factors affecting translation of in vitro
enzyme inhibition potency to anti-MRSA activity are unclear.

7076
N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
While characterizing the compounds we noted that while the
enforced by suitably placed hydrogen bonds is also very important.
Correlation of in vitro enzyme inhibitory activity and anti-MRSA
activity was poor although some compounds, generally N-methyl
indole analogs, showed excellent MIC values and lack of mamma-
lian cytotoxicity. The lead compounds with best MIC values are
currently being formulated and evaluated to maximize in vivo
exposure in preparation for evaluation in in vivo animal infection
models. These studies will be reported elsewhere.
NH indoles or benzimidazoles exist as one isomer at room temper-
ature (as evidenced in their NMR spectra), the N-methyl analogs
showed evidence of variable ratio of isomers5 which interconvert-
ed rapidly at higher temperature. It is assumed these isomers are
rotamers about the 2-indole C–C bond. What effect this phenome-
non may have on the anti-MRSA activity is also unclear.
In investigating the effects of substitutions in the hydrazone
linking moiety we noted that some compounds (6i, 6u, 7q, 7r) with
bulky groups on the bridging hydrazone showed good MIC values
in the cell assay in spite of very weak potency as inhibitors of
MRSA PK (Table 4). This lack of correlation was surprising but nota-
4. Experimental Section
4.1. Chemistry
bly 6u was found (NMR and HPLC) to exist in two isomeric forms
about the hydrazone double bond (although the E-isomer predom-
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 in positive ion-mode with an ESI ion source on an Agi-
lent™ Time-of-Flight LC/MS mass spectrometer. Analytical thin-
layer chromatography (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
solution and heated. Column chromatography was performed with
silica gel 60 (230–400 mesh). Purity of >95% for all final compounds
was confirmed by analytical reverse-phase HPLC utilizing a Dikma
Technologies™ InspireÒ C18 reverse-phase analytical column (4.6
ꢀ 150 mm). All HPLC purifications were carried out using an Agi-
lent™ C18 reverse-phase preparatory column (21.2 ꢀ 250 mm).
The syntheses of all the intermediates (1, 3, 4, 5, 11) and N-acyl-
hydrazones (7, 8, 9, 10, 12, 13) are described in detail in Supple-
mentary data.
inated) and, as noted above, the corresponding t-butyl analog 6
i
was found by X-ray analysis to exist exclusively as the Z-isomer.
These ‘anomalous’ antibacterial activities suggested that these
isomeric compounds may exert antibacterial activity via a different
(unknown) mechanism and, as these compounds show significant
or predominant propensity to exist in the Z-configuration, this off-
target activity may relate to the Z-isomer rather than the E-isomer.
Notably these ‘anomalous’ compounds (6
significant cytotoxicity on mammalian THP-1 cells with LD50s be-
tween 6–25 g/ml while comparable compounds (e.g. 7a, 7b, 7d)
that existed as predominantly E-isomers did not show mammalian
i. 6u 7q, 7r), also showed
l
cell cytotoxicity at much higher concentrations (LD50 >500
(Table 4 and data not shown).
lg/ml)
3. Conclusion
In summary, a series of potent inhibitors of MRSA PK were syn-
thesized in order to understand the SAR and to further optimize
the physiochemical properties for effective inhibition of bacterial
growth in vitro and, potentially, in vivo. X-ray structures for com-
pounds 8d and 6/ bound to MRSA252 PK show that the active
compounds fit in a very flat and elongated binding site with an
important interior lipophilic binding pocket which is best fit by
an appropriately appended and oriented bromine atom or
naphthalene equivalent. The pre-organization and flat structure
4.1.1.1. General procedures for the synthesis of the N-acylhy-
drazones 6–8.
Procedure A: A mixture of the appropriate ke-
tone (0.25 mmol) and hydrazide (0.25 mmol) in propan-1-ol
(3 mL) was refluxed until completion (or good conversion) of the
reaction (monitored by TLC). If the product precipitated, it was col-
lected by filtration and the solid was washed with hot propan-1-ol
Figure 5. (a) Structures of the interface-binding site for MRSA and human PK. Orange spheres show the interface cavity in MRSA and human. The MRSA PK model shows an
accessible binding pocket located at the interface of two PK monomers. In contrast, the pocket in human PK is partially obstructed by five amino acid residues (Glu418-B,
Arg399-A, B and Arg400-A, B. (b) A sequence alignment showing the interface region (highlighted residues) for pyruvate kinase (PK) from Staphylococcus aureus and homo
sapiens. The poorly conserved residues between MRSA and human PK are highlighted purple. The small interface also encompasses an insertion region in human PK (shown in
yellow) and the corresponding deletion area in MRSA PK.

N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
7077
(10 mL). If no precipitation was observed, the solvent was evapo-
rated in vacuo and the compound was purified by flash column
chromatography.
Procedure B: A mixture of the appropriate ketone (0.35 mmol)
and hydrazide (0.35 mmol) in EtOH (5 mL) was refluxed until com-
pletion (or good conversion) of the reaction (monitored by TLC). If
the product precipitated, it was collected by filtration and the solid
was washed with hot EtOH (2 ꢀ 2 mL). If no precipitation was ob-
served, the solvent was evaporated in vacuo and the compound
was purified by flash column chromatography.
Procedure C: A mixture of the appropriate ketone (0.41 mmol),
hydrazide (0.41 mmol) and AcOH (1 drop) in propan-1-ol (4 mL)
was refluxed until completion (or good conversion) of the reaction
(monitored by TLC). If the product precipitated, it was collected by
filtration and the solid was washed with hot propan-1-ol
(2 ꢀ 2 mL). If no precipitation was observed, the solvent was evap-
orated in vacuo and the compound was purified either by flash col-
umn chromatography or by reverse-phase HPLC.
Procedure D: A mixture of the appropriate ketone (0.41 mmol),
hydrazide (0.41 mmol) and AcOH (1 drop) in ethanol (4 mL) was
refluxed until completion (or good conversion) of the reaction
(monitored by TLC). If the product precipitated, it was collected
by filtration and the solid was washed with hot ethanol
(2 ꢀ 2 mL). If no precipitation was observed, the solvent was evap-
orated in vacuo and the compound was purified by flash column
chromatography.
Procedure E: A mixture of the appropriate ester (1.0 equiv) and
hydrazine hydrate (>3.0 equiv) in ethanol was irradiated with
microwaves for 60 min at 100 °C. If the hydrazide precipitated, it
was collected by filtration. If no precipitation was observed, the
mixture was partitioned between ethyl acetate and water, the or-
ganic layer dried over Na2SO4 and evaporated in vacuo. To the
hydrazide taken up in ethanol was added the appropriate ketone
and AcOH (1 drop). The mixture was refluxed—classical heating—
until completion (or good conversion) of the reaction (monitored
by TLC). If the product precipitated, it was collected by filtration
and the solid was washed with hot ethanol (2 ꢀ 2 mL). If no precip-
itation was observed, the solvent was evaporated in vacuo and the
compound was purified by flash column chromatography.
13.7. HRMS calcd for (C17H1379BrFN3O2+H)+ 390.0248, found
390.0247.
4.1.1.4. (E)-5-Bromo-N0-(1-(5-chloro-1H-indol-2-yl)ethylidene)-
2-hydroxybenzohydrazide (6c).
Compound 6c was prepared
from 1-(5-chloro-1H-indol-2-yl)ethanone (3q) and 5c using gen-
eral Procedure C.
Yield = 77% (filtration), pale yellow solid. Mp = 282–285 °C. 1H
NMR (600 MHz, DMSO-d6): d 12.11 (s, 1H), 11.52 (s, 1H), 11.34
(s, 1H), 8.09 (d, J = 2.6 Hz, 1H), 7.61 (d, J = 2.2 Hz, 1H), 7.60 (dd,
J = 2.7 Hz, 8.7 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.15 (dd, J = 2.1 Hz,
8.7 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 1.5 Hz, 1H), 2.37
(s, 3H). 13C NMR (150 MHz, DMSO-d6): d 160.8, 156.1, 147.3,
137.8, 136.6, 136.2, 133.1, 129.1, 124.3, 123.6, 120.8, 120.2,
119.8,
114.2,
111.3,
104.7,
14.2.
HRMS
calcd
for
(C17H1381BrClN3O2+H)+ 407.9931, found 407.9931.
4.1.1.5. (E)-5-Bromo-N0-(1-(5-bromo-1H-indol-2-yl)ethylidene)-
2-hydroxybenzohydrazide (6d). Compound 6d was prepared
from 1-(5-bromo-1H-indol-2-yl)ethanone (3r) and 5c using gen-
eral Procedure A.
Yield = 61% (filtration), pale yellow solid. Mp = 295–299 °C. 1H
NMR (600 MHz, DMSO-d6): d 12.12 (br s, 1H), 11.53 (s, 1H),
11.35 (br s, 1H), 8.08 (d, J = 2.6 Hz, 1H), 7.75 (d, J = 1.8 Hz, 1H),
7.59 (dd, J = 2.6 Hz, 8.7 Hz, 1H), 7.45 (d, J = 8.7 Hz, 1H), 7.26 (dd,
J = 1.9 Hz, 8.6 Hz, 1H), 7.02 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 1.5 Hz,
1H), 2.37 (s, 3H). 13C NMR (150 MHz, DMSO-d6): d 161.2, 156.6,
147.6, 138.1, 137.3, 136.6, 133.5, 130.3, 126.5, 123.7, 121.2,
120.3, 115.1, 112.7, 111.7, 105.0, 14.7. HRMS calcd for
(C17H1379Br2N3O2+H)+ 451.9428, found 451.9428.
4.1.1.6.
yl)ethylidene]benzohydrazide (6e).
5-Bromo-2-hydroxy-N0-[(1E)-1-(5-iodo-1H-indol-2-
Compound 6e was pre-
pared from 1-(5-iodo-1H-indol-2-yl)ethanone (3m) and 5c using
general Procedure D.
Yield: 64%, light brown solid. Mp = 272–274 °C. 1H NMR
(500 MHz, DMSO-d6): d 12.12 (s, 1H), 11.52 (s, 1H), 11.37 (s, 1H),
8.09 (d, J = 2.5 Hz, 1H), 7.94 (m, 1H), 7.60 (dd, J = 8.8 Hz,
J = 2.5 Hz, 1H), 7.41 (dd, J = 8.6 Hz, J = 1.4 Hz, 1H), 7.34 (d,
J = 8.6 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.94 (s, 1H), 2.38 (s, 3H).
13C NMR (100 MHz, DMSO-d6): d 160.8, 156.2, 147.2, 137.2,
137.1, 136.2, 133.0, 131.5, 130.8, 129.4, 120.6, 119.8, 115.1,
111.3, 104.2, 83.4, 14.3. HRMS calcd for (C17H1379BrIN3O2-H)-
495.9163, found 495.9143.
4.1.1.2. (E)-N0-(1-(1H-indol-2-yl)ethylidene)-5-bromo-2-hydrox-
ybenzohydrazide (6a).
Compound 6a was prepared from 1-
(1H-indol-2-yl)ethanone (3p) and 5-bromo-2-hydroxybenzohyd-
razide (5c) using general Procedure B.
Yield = 53% (after flash chromatography 20/1; DCM/MeOH),
pale yellow solid. Mp = 276–279 °C. 1H NMR (600 MHz, DMSO-
d6): d 12.12 (s, 1H), 11.31 (s, 2H), 8.09 (s, 1H), 7.59 (d, J = 8.7 Hz,
1H), 7.56 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.14 (t,
J = 7.9 Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 6.98
(s, 1H), 2.38 (s, 3H). 13C NMR (150 MHz, DMSO-d6): d 160.3,
155.7, 147.3, 137.7, 135.74, 135.65, 132.5, 127.6, 123.2, 120.7,
120.3, 119.33, 119.31, 112.1, 110.8, 104.9, 13.8. HRMS calcd for
(C17H1479BrN3O2+H)+ 372.0342, found 372.0333.
4.1.1.7. 5-Bromo-2-hydroxy-N0-{(1E)-1-[5-(trifluoromethyl)-1H-
indol-2-yl]ethylidene}benzohydrazide (6f).
Compound 6f
was prepared from 1-[5-(trifluoromethyl)-1H-indol-2-yl]ethanone
(3f) and 5c using general Procedure D.
Yield: 90%, beige solid. Mp = 268–270 °C. 1H NMR (500 MHz,
DMSO-d6): d 12.14 (s, 1H), 11.79 (s, 1H), 11.42 (s, 1H), 8.09 (d,
J = 2.7 Hz, s), 7.97 (d, J = 1.4 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.60
(dd, J = 8.7 Hz, J = 2.7 Hz, 1H), 7.44 (dd, J = 8.7 Hz, J = 1.4 Hz, 1H),
7.14 (m, 1H), 7.02 (d, J = 8.7 Hz, 1H), 2.41 (s, 3H). 13C NMR
(100 MHz, DMSO-d6): d 160.9, 156.3, 147.0, 139.6, 138.4, 136.2,
133.1, 127.4, 125.9 (q, J = 271 Hz, 1C), 120.6 (m, 1C), 119.8 (m,
1C), 118.8 (m, 1C), 113.3, 111.2, 105.8, 14.2. HRMS calcd for
(C18H1379BrF3N3O-H)ꢁ 438.0070, found 438.0089.
4.1.1.3. (E)-5-Bromo-N0-(1-(5-fluoro-1H-indol-2-yl)ethylidene)-
2-hydroxybenzohydrazide (6b).
Compound 6b was prepared
from 1-(5-fluoro-1H-indol-2-yl)ethanone (3a) and 5c using general
Procedure A.
Yield = 46% (filtration), white powder. Mp = 262–263 °C. 1H
NMR (400 MHz, DMSO-d6): d 12.11 (br s, 1H), 11.42 (s, 1H),
11.32 (s, 1H), 8.09 (d, J = 2.4 Hz, 1H), 7.59 (dd, J = 8.6 Hz, 2.6 Hz,
1H), 7.48 (dd, J = 8.8 Hz, 4.8 Hz, 1H), 7.32 (dd, J = 10.0 Hz, 2.4 Hz,
1H), 7.03–6.96 (m, 3H), 2.37 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): d 160.3, 157.0 (d, J = 230 Hz), 155.7, 147.0, 137.5, 135.7,
134.4, 132.5, 127.6 (d, J = 10 Hz), 120.3, 119.3, 113.2 (d, J = 9 Hz),
111.5 (d, J = 26 Hz), 110.8, 104.9 (d, J = 23 Hz), 104.6 (d, J = 5 Hz),
4.1.1.8. (E)-5-Bromo-2-hydroxy-N0-(1-(5-hydroxy-1H-indol-2-
yl)ethylidene)benzohydrazide (6g).
Compound 6g was pre-
pared from 1-(5-hydroxy-1H-indol-2-yl)ethanone (3j) and 5c
using general Procedure C.
Yield = 65% (filtration), pale yellow solid. Mp = 190–192 °C. 1H
NMR (600 MHz, DMSO-d6): d 12.10 (br s, 1H), 11.27 (s, 1H),
11.01 (s, 1H), 8.71 (s, 1H), 8.09 (d, J = 2.3 Hz, 1H), 7.59 (dd,

7078
N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
J = 2.5 Hz, 8.7 Hz, 1H), 7.28 (d, J = 8.7 Hz, 1H), 7.01 (d, J = 8.7 Hz,
1H), 6.85 (d, J = 1.6 Hz, 1H), 6.79 (s, 1H), 6.68 (dd, J = 1.9 Hz,
8.7 Hz, 1H), 2.34 (s, 3H). 13C NMR (150 MHz, DMSO-d6): d 160.2,
155.7, 150.8, 147.6, 135.8, 135.6, 132.5, 132.4, 128.2, 120.3,
119.3, 114.0, 112.6, 110.8, 104.1, 103.9, 13.8. HRMS calcd for
(C17H1479BrN3O3+H)+ 388.0291, found 388.0284.
4.1.1.13. (E)-5-Bromo-N0-(1-(4,5-difluoro-1H-indol-2-yl)ethyli-
dene)-2-hydroxybenzohydrazide (6l). Compound 6l was
prepared from 1-(4,5-difluoro-1H-indol-2-yl)ethanone (3d) and
5c using general Procedure A.
Yield = 75% (filtration), beige powder. Mp = 275–276 °C. 1H
NMR (400 MHz, DMSO-d6): d 12.11 (br s, 1H), 11.71 (s, 1H),
11.35 (s, 1H), 8.08 (d, J = 2.8 Hz, 1H), 7.60 (dd, J = 8.8 Hz, 2.8 Hz,
1H), 7.29 (dd, J = 9.0 Hz, 3.4 Hz, 1H), 7.18 (td, J = 11.6 Hz, 8.0 Hz,
1H), 7.12 (d, J = 1.6 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 2.39 (s, 3H).
13C NMR (150 MHz, DMSO-d6): d 160.3, 155.6, 146.4, 143.1 (dd,
J = 231 Hz, 11 Hz), 141.9 (dd, J = 245 Hz, 14 Hz), 137.9, 135.8 (d,
J = 9 Hz), 135.7, 132.6, 120.2, 119.3, 117.5 (d, J = 18 Hz), 112.7 (d,
J = 21 Hz), 110.8, 108.4 (dd, J = 7 Hz, 4 Hz), 100.3 (d, J = 5 Hz),
13.6. HRMS calcd for (C17H1279BrF2N3O2+H)+ 408.0154, found
408.0161.
4.1.1.9. (E)-5-Bromo-2-hydroxy-N0-(1-(5-methoxy-1H-indol-2-
yl)ethylidene)benzohydrazide (6h).
Compound 6h was pre-
pared from 1-(5-methoxy-1H-indol-2-yl)ethanone (3s) and 5c
using general Procedure A.
Yield = 79% (filtration), pale yellow cotton. Mp = 250–251 °C. 1H
NMR (600 MHz, DMSO-d6): d 12.14 (br s, 1H), 11.30 (s, 1H), 11.20
(s, 1H), 8.09 (d, J = 2.4 Hz, 1H), 7.59 (dd, J = 8.7 Hz, 2.7 Hz, 1H), 7.37
(d, J = 9.0 Hz, 1H), 7.03 (d, J = 1.8 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H),
6.88 (d, J = 0.8 Hz, 1H), 6.80 (dd, J = 9.0 Hz, 2.4 Hz, 1H), 3.75 (s,
3H), 2.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6): d 160.2, 155.7,
153.5, 147.4, 136.0, 135.6, 133.0, 132.6, 127.9, 120.3, 119.3,
113.9, 112.9, 110.9, 104.6, 101.6, 55.2, 13.8. HRMS calcd for
(C18H1679BrN3O3+H)+ 402.0448, found 402.0450.
4.1.1.14.
dene)-2-hydroxybenzohydrazide (6m).
(E)-5-Bromo-N0-(1-(7-fluoro-1H-indol-2-yl)ethyli-
Compound 6m was
prepared from 1-(7-fluoro-1H-indol-2-yl)ethanone (3b) and 5c
using general Procedure A.
Yield = 86% (filtration), beige powder. Mp = 263–264 °C. 1H
NMR (400 MHz, DMSO-d6): d 12.11 (br s, 1H), 11.75 (s, 1H),
11.34 (s, 1H), 8.08 (d, J = 1.6 Hz, 1H), 7.60 (dd, J = 8.8 Hz, 2.4 Hz,
1H), 7.42–7.38 (m, 1H), 7.08 (br s, 1H), 7.03–6.97 (m, 3H), 2.40
(s, 3H). 13C NMR (100 MHz, DMSO-d6): d 160.5, 155.7, 149.0 (d,
J = 244 Hz), 147.1, 137.4, 135.7 (d, J = 4 Hz), 132.5 (d, J = 3 Hz),
131.5 (d, J = 6 Hz), 125.5 (d, J = 13 Hz), 120.2, 119.7, 119.3 (d,
J = 8 Hz), 116.9 (d, J = 8 Hz), 110.8, 108.0 (d, J = 16 Hz), 105.3 (d,
J = 8 Hz), 14.0. HRMS calcd for (C17H1379BrFN3O2+H)+ 390.0248,
found 390.0250.
4.1.1.10.
yl)ethylidene)benzohydrazide (6i).
pared from 1-(5-phenyl-1H-indol-2-yl)ethanone (3g) and 5c using
general Procedure A.
(E)-5-Bromo-2-hydroxy-N0-(1-(5-phenyl-1H-indol-2-
Compound 6i was pre-
Yield = 92% (filtration), beige solid. Mp = 275–276 °C. 1H NMR
(600 MHz, DMSO-d6): d 12.14 (br s, 1H), 11.42 (s, 1H), 11.33 (s,
1H), 8.10 (d, J = 2.4 Hz, 1H), 7.83 (s, 1H), 7.67 (d, J = 7.2 Hz, 2H),
7.60 (dd, J = 8.7 Hz, 2.7 Hz, 1H), 7.57 (d, J = 9.0 Hz, 1H), 7.48 (dd,
J = 8.4 Hz, 1.2 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.31 (t, J = 7.2 Hz,
1H), 7.05 (s, 1H), 7.02 (d, J = 9.0 Hz, 1H), 2.40 (s, 3H). 13C NMR
(150 MHz, DMSO-d6): d 160.3, 155.6, 147.2, 141.6, 137.4, 136.5,
135.7, 132.6, 131.9, 128.8 (2CH), 128.2, 126.7 (2CH), 126.3, 122.7,
120.3, 119.3, 118.6, 112.6, 110.9, 105.2, 13.8. HRMS calcd for
(C23H1879BrN3O2+Na)+ 470.0480, found 470.0455.
4.1.1.15.
indol-2-yl)ethylidene)benzohydrazide (6n).
(E)-5-Bromo-2-hydroxy-N0-(1-(4,5,6-trifluoro-1H-
Compound 6n
was prepared from 1-(4,5,6-trifluoro-1H-indol-2-yl)ethanone (3e)
and 5c using general Procedure A.
Yield = 46% (after column 50/50 hexanes/EtOAc then 90/10
DCM/MeOH), beige powder. 1H NMR (400 MHz, DMSO-d6): d
12.10 (br s, 1H), 11.78 (br s, 1H), 11.34 (s, 1H), 8.08 (d, J = 2.4 Hz,
1H), 7.60 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.28 (dd, J = 10.2 Hz, 6.2 Hz,
1H), 7.15 (d, J = 2.0 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 2.38 (s, 3H).
4.1.1.11.
dene)-2-hydroxybenzohydrazide (6j).
prepared from 1-(6-bromo-1H-indol-2-yl)ethanone (3i) and 5c
using general Procedure C.
(E)-5-Bromo-N0-(1-(6-bromo-1H-indol-2-yl)ethyli-
Compound 6j was
Yield = 50% (after reverse-phase HPLC (acetonitrile/H2O-0.1%
TFA, 80–100% for 15 min, 20 mL/min, 254 nm detection for
18 min), pale yellow solid. Mp = 338–340 °C. 1H NMR (600 MHz,
DMSO-d6): d 12.17 (br s, 1H), 11.58 (br s, 1H), 11.44 (s, 1H),
8.07 (d, J = 2.3 Hz, 1H), 7.66 (s, 1H), 7.57 (dd, J = 2.4 Hz, 8.6 Hz,
1H), 7.52 (d, J = 8.4 Hz, 1H), 7.13 (dd, J = 1.8 Hz, 8.4 Hz, 1H), 7.00
(s, 1H), 6.99 (d, J = 9.3 Hz, 1H), 2.37 (s, 3H). 13C NMR (150 MHz,
DMSO-d6): d 160.5, 156.3, 146.5, 138.5, 136.7, 135.6, 132.5,
126.6, 122.4, 122.2, 120.2, 119.5, 115.8, 114.5, 110.3, 104.8,
13.7. HRMS calcd for (C17H1379Br2N3O2+H)+ 451.9428, found
451.9410.
13C NMR (150 MHz, DMSO-d6):
d 160.3, 155.6, 148.0 (dd,
J = 239 Hz, 13 Hz), 146.0, 142.7 (ddd, J = 247 Hz, 11 Hz, 5 Hz),
137.8 (d, J = 3 Hz), 135.6, 133.7 (ddd, J = 235 Hz, 18 Hz, 14 Hz),
132.7 (t, J = 12 Hz), 132.5, 120.2, 119.2, 113.2 (d, J = 18 Hz), 110.8,
100.3, 95.5 (dd, J = 22 Hz, 3 Hz), 13.5. HRMS calcd for
(C17H1179BrF3N3O2+H)+ 426.0060, found 426.0070.
4.1.1.16.
mobenzohydrazide (6o).
(E)-N0-(1-(1H-indol-2-yl)ethylidene)-3-bro-
Compound 6o was prepared from
1-(1H-indol-2-yl)ethanone 3p and 5-bromobenzohydrazide (5d)
using general Procedure C.
Yield = 70% (after flash chromatography 3/1; hexanes/EtOAc),
pale yellow solid. Mp = 219–224 °C. 1H NMR (600 MHz, DMSO-
d6): d 11.31 (s, 1H), 10.89 (s, 1H), 8.09 (br s, 1H), 7.92 (d,
J = 7.6 Hz, 1H), 7.81 (d, J = 8.0 Hz,1H), 7.57 (d, J = 7.9 Hz, 1H), 7.51
(t, J = 7.9 Hz, 1H), 7.49 (d, J = 7.3 Hz, 1H), 7.16 (dt, J = 0.7 Hz,
7.2 Hz, 1H), 7.01 (dt, J = 0.9 Hz, 7.5 Hz, 1H), 6.99 (br s, 1H), 2.43
(s, 3H). 13C NMR (150 MHz, DMSO-d6): d 163.1, 151.9, 138.5,
137.2, 136.8, 135.1, 131.5, 131.3, 128.5, 128.0, 124.1, 122.5,
4.1.1.12. (E)-5-Bromo-N0-(1-(5,6-difluoro-1H-indol-2-yl)ethyli-
dene)-2-hydroxybenzohydrazide (6k).
Compound 6k was
prepared from 1-(5,6-difluoro-1H-indol-2-yl)ethanone (3c) and
5c using general Procedure A.
Yield = 55% (filtration), pale yellow powder. Mp = 271–272 °C.
1H NMR (400 MHz, DMSO-d6): d 12.11 (br s, 1H), 11.48 (s, 1H),
11.34 (br s, 1H), 8.08 (d, J = 2.4 Hz, 1H), 7.61–7.54 (m, 2H), 7.39
(dd, J = 11.0 Hz, 7.0 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.98 (d,
J = 1.6 Hz, 1H), 2.36 (s, 3H). 13C NMR (150 MHz, DMSO-d6): d
160.3, 155.7, 147.7 (dd, J = 238 Hz, 16 Hz), 146.5, 145.4 (dd,
J = 234 Hz, 15 Hz), 137.6 (d, J = 4 Hz), 135.7, 133.0 (d, J = 11 Hz),
132.6, 122.9 (d, J = 9 Hz), 120.2, 119.3, 110.8, 107.1 (d, J = 19 Hz),
121.6,
120.3,
113.1,
105.8,
15.4.
HRMS
calcd
for
(C17H1479BrN3O+H)+ 356.0393, found 356.0394.
4.1.1.17.
ybenzohydrazide (6p).
(E)-N0-(1-(1H-Indol-2-yl)ethylidene)-2-hydrox-
Compound 6p was prepared from 1-
104.8,
99.7
(d,
J = 22 Hz),
13.6.
HRMS
calcd
for
(1H-indol-2-yl)ethanone (3p) and 2-hydroxybenzohydrazide (5e)
using general Procedure C.
(C17H1279BrF2N3O2+H)+ 408.0159, found 408.0129.

N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
7079
Yield = 54% (after flash chromatography 40/1; DCM/MeOH),
pale yellow solid. Mp = 246–248 °C. 1H NMR (600 MHz, DMSO-
d6): d 11.86 (br s, 1H), 11.38 (br s, 1H), 11.31 (s, 1H), 8.03 (dd,
J = 1.4 Hz, 7.8 Hz, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 8.1 Hz,
1H), 7.43 (dt, J = 1.8 Hz, 7.2 Hz, 1H), 7.14 (dt, J = 1.1 Hz, 8.1 Hz,
1H), 7.04 (d, J = 7.6 Hz, 1H), 7.002 (t, J = 7.9 Hz, 1H), 7.001 (t,
J = 7.0 Hz, 1H), 6.97 (d, J = 1.5 Hz, 1H), 2.39 (s, 3H). 13C NMR
(150 MHz, DMSO-d6): d 162.3, 157.1, 147.2, 138.2, 136.4, 133.8,
131.0, 128.1, 123.6, 121.1, 120.1, 119.8, 118.4, 117.4, 112.6,
105.1, 14.3. HRMS calcd for (C17H15N3O2+H)+ 294.1237, found
294.1237.
4.1.1.22. 4-Hydroxy-N0-[(1E)-1-(1H-Indol-2-yl)ethylidene]-1,10-
biphenyl-3-carbohydrazide (6v).
Compound 6v was pre-
pared from 3p, hydrazine hydrate and methyl 4-hydroxy-1,10-
biphenyl-3-carboxylate using general Procedure E.
Yield: 22%, off-white solid. Mp = 274–276 °C. 1H NMR
(400 MHz, DMSO-d6): d 12.02 (br s, 1H), 11.48 (s, 1H), 11.36 (s,
1H), 8.28 (d, J = 2.3 Hz, 1H), 7.76 (dd, J = 8.6 Hz, J = 2.3 Hz, 1H),
7.66 (d, J = 7.3 Hz, 2H), 7.57 (d, J = 7.6 Hz, 1H), 7.50 (d, J = 7.6 Hz,
1H), 7.48 (d, J = 7.3 Hz, 2H), 7.35 (t, J = 7.3 Hz, 1H), 7.16 (t,
J = 7.6 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1H), 6.90
(br s, 1H), 2.42 (s, 3H). 13C NMR (100 MHz, DMSO-d6): d 162.3,
156.8, 147.9, 139.8, 138.2, 136.3, 132.2, 132.0, 129.4, 128.8,
128.1, 127.5, 126.7, 123.6, 121.1, 119.8, 118.7, 118.1, 112.6,
105.2, 14.4. HRMS calcd for (C23H19N3O2+H)+ 370.1550, found
370.1416.
4.1.1.18.
hydroxybenzohydrazide (6q).
(E)-N0-(1-(1H-Indol-2-yl)ethylidene)-4-bromo-2-
Compound 6q was prepared
from 3p and 4-bromo-2-hydroxy-benzohydrazide (5l) using gen-
eral Procedure C.
Yield = 62% (filtration), yellow solid. Mp = 281–284 °C. 1H NMR
(600 MHz, DMSO-d6): d 12.27 (br s, 1H), 11.31 (s, 1H), 11.25 (s,
1H), 7.94 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 7.9 Hz, 1H), 7.49 (d,
J = 8.2 Hz, 1H), 7.22 (d, J = 1.7 Hz, 1H), 7.20 (dd, J = 1.7 Hz, 8.4 Hz,
1H), 7.14 (dt J = 0.9 Hz, 7.6 Hz, 1H), 7.00 (dt, J = 0.9 Hz, 7.5 Hz,
8.7 Hz, 1H), 6.98 (d, J = 1.2 Hz, 1H), 2.38 (s, 3H). 13C NMR
(150 MHz, DMSO-d6): d 161.8, 158.1, 148.0, 138.7, 136.7, 133.4,
128.5, 126.8, 124.1, 123.6, 121.6, 120.3, 120.2, 118.7, 113.1,
105.7, 14.7. HRMS calcd for (C17H1479BrN3O2+H)+ 372.0342, found
372.0347.
4.1.1.23. (E)-N0-(1-(1H-Indol-2-yl)ethylidene)-5-bromo-2-meth-
oxybenzohydrazide (6w).
Compound 6w was prepared from
3p and 2-methoxy-4-bromobenzohydrazide (5i) using general
Procedure C.
Yield = 41% (filtration), pale yellow solid. Mp = 241–244 °C. 1H
NMR (600 MHz, DMSO-d6): d 11.31 (s, 1H), 10.90 (s, 1H), 7.96 (d,
J = 2.6 Hz, 1H), 7.73 (dd, J = 2.7 Hz, 8.8 Hz, 1H), 7.56 (d, J = 8.1 Hz,
1H), 7.49 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 8.9 Hz, 1H), 7.14 (dt,
J = 1.0 Hz, 7.6 Hz, 1H), 7.00 (dt, J = 0.8 Hz, 7.5 Hz, 1H), 7.23 (d,
J = 8.9 Hz, 1H), 6.98 (d, J = 1.5 Hz, 1H), 3.99 (s, 3H), 2.38 (s, 3H).
13C NMR (150 MHz, DMSO-d6): d 160.3, 156.84, 148.0, 138.2,
136.3, 135.5, 133.1, 128.1, 124.7, 123.6, 121.1, 119.8, 115.4,
4.1.1.19.
(6r).
(E)-N0-(1-(1H-Indol-2-yl)ethylidene)benzohydrazide
Compound 6r was prepared from 3p and benzohydraz-
112.8,
112.6,
105.3,
57.3,
14.2.
HRMS
calcd
for
ide (5f) using general Procedure C.
(C18H1679BrN3O2+H)+ 386.0499, found 386.0515.
Yield = 62%, pale yellow solid. Mp = 224–226 °C. 1H NMR (600
Mz, DMSO): d 11.29 (br s, 1H), 10.8 (s, 1H), 7.92 (d, J = 7.3 Hz,
4.1.1.24. (E)-N0-[1-(1H-indol-2-yl)ethylidene]-5-bromo-2-eth-
oxybenzohydrazide (6x). Compound 6x was prepared from
2H), 7.60 (t, J = 7.3 Hz, 1H), 7.58
– 7.52 (m, 3H), 7.48 (d,
J = 8.1 Hz, 1H), 7.14 (dt, J = 0.9 Hz, 7.6 Hz, 1H), 7.00 (dt, J = 0.8 Hz,
7.4 Hz, 1H), 6.96 (br s, 1H), 2.42 (s, 3H). 13C NMR (150 Mz, DMSO):
d 164.1, 150.8, 138.1, 136.5, 134.6, 132.0, 128.8, 128.3, 128.0,
123.6, 121.2, 119.8, 112.6, 105.1, 14.9. HRMS calcd for
(C17H15N3O+H)+ 278.1288, found 278.1278.
1-(1H-indol-2-yl)ethanone 3p, hydrazide hydrate and methyl 5-
bromo-2-ethoxybenzoate using general Procedure E.
Yield: 84%, beige solid. Mp = 260–262 °C. 1H NMR (400 MHz,
DMSO-d6): d 11.32 (br s, 1H), 10.81 (s, 1H), 8.01 (d, J = 2.3 Hz,
1H), 7.71 (dd, J = 8.9 Hz, J = 2.3 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H),
7.49 (d, J = 7.6 Hz, 1H), 7.22 (d, J = 8.9 Hz, 1H), 7.14 (d, J = 7.6 Hz,
1H), 6.99 (t, J = 7.6 Hz, 1H), 6.98 (s, 1H), 4.26 (q, J = 7.0 Hz, 2H),
2.39 (s, 3H), 1.47 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-
d6): d 159.8, 155.7, 147.1, 137.7, 135.8, 135.2, 133.0, 127.5, 123.8,
123.1, 120.6, 119.3, 115.5, 112.2, 112.1, 104.8, 65.2, 14.6, 13.9.
HRMS calcd for (C19H1879BrN3O +Na)+ 422.0475, found 422.0464.
4.1.1.20. (E)-N0-(1-(1H-Indol-2-yl)ethylidene)picolinohydrazide
(6s).
Compound 6s was prepared from 3p and picolinohyd-
razide (5g) using general Procedure C.
Yield = 87%, yellow solid. Mp = 219–221 °C. 1H NMR (600 MHz,
DMSO): d 11.36 (s, 1H), 11.12 (s, 1H), 8.74 (d, J = 4.7 Hz, 1H), 8.18
(d, J = 7.8 Hz,1H), 8.08 (dt, J = 1.7 Hz, 7.7 Hz, 1H), 7.70 (ddd,
J = 1.2 Hz, 4.7 Hz, 5.9 Hz, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.50 (dd,
J = 0.8 Hz, 8.2 Hz, 1H), 7.15 (dt, J = 1.1 Hz, 7.0 Hz, 1H), 7.01 – 6.99
(m, 2H), 2.45 (s, 3H). 13C NMR (150 MHz, DMSO): d 160.2, 150.3,
149.9, 149.6, 139.2, 138.7, 136.6, 128.5, 128.1, 124.2, 123.2,
121.7, 120.3, 113.1, 106.0, 14.3. HRMS calcd for (C16H14N4O+H)+
279.1240, found 279.1243.
4.1.1.25. (E)-N0-(1-(1H-indol-2-yl)ethylidene)-5-bromo-2-(prop-
2-ynyloxy)benzohydrazide (6z).
Compound 6z was prepared
from 3p and 5a using general Procedure A.
Yield = 54% (filtration), off-white solid. Mp = 246–251 °C. 1H
NMR (600 MHz, DMSO-d6): d 11.30 (s, 1H), 10.78 (s, 1H), 7.93 (d,
J = 2.6 Hz, 1H), 7.75 (dd, J = 2.6 Hz, 8.9 Hz, 1H), 7.56 (d, J = 7.9 Hz,
1H), 7.49 (d, J = 8.1 Hz, 1H), 7.26 (d, J = 8.9 Hz, 1H), 7.14 (dt,
J = 0.9 Hz, 8.0 Hz, 1H), 7.00 (dt, J = 0.7 Hz, 7.8 Hz, 1H), 6.97 (d,
J = 1.5 Hz, 1H), 5.04 (d, J = 2.3 Hz, 1H), 3.75 (t, J = 2.3 Hz, 1H), 2.39
(s, 3H). 13C NMR (150 MHz, DMSO-d6): d 160.8, 155.2, 148.7,
138.7, 136.7, 135.7, 133.6, 128.5, 126.2, 124.1, 121.6, 120.2,
117.0, 114.0, 113.1, 105.7, 80.5, 79.3, 58.2, 15.0. HRMS calcd for
(C20H1681BrN3O2+H)+ 412.0481, found 412.0467.
4.1.1.21.
iodobenzohydrazide (6u).
(E)-N0-(1-(1H-Indol-2-yl)ethylidene)-2-hydroxy-5-
Compound 6u was prepared from
3p and 2-hydroxy-5-iodobenzohydrazide (5h) using general Pro-
cedure C.
Yield = 32% (after flash chromatography 4/1 ? 1/1; hexanes/
EtOAc), pale yellow solid. Mp = 251–255 °C. 1H NMR (600 MHz,
DMSO-d6): d 12.10 (br s, 1H), 11.32 (s, 2H), 8.25 (d, J = 2.2 Hz,
1H), 7.72 (dd, J = 2.3 Hz, 8.5 Hz, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.49
(d, J = 8.0 Hz, 1H), 7.14 (dt, J = 1.0 Hz, 7.1 Hz, 1H), 7.00 (dt,
J = 0.9 Hz, 7.4 Hz, 1H), 6.98 (s, 1H), 6.89 (d, J = 8.6 Hz, 1H), 2.38 (s,
3H). 13C NMR (150 MHz, DMSO-d6): d 161.2, 157.3, 148.1, 142.2,
139.4, 138.7, 136.7, 128.5, 124.1, 121.6, 120.6, 120.3, 113.1,
105.7, 82.6, 14.7. HRMS calcd for (C17H14IN3O2+H)+ 420.0203,
found 420.0201.
4.1.1.26.
hydroxybenzohydrazide (6
(E)-N0-(1-(1H-indol-2-yl)ethylidene)-3,5-dibromo-2-
). Compound 6 was prepared
a
a
from 3p and 3,5-dibromo-2-hydroxybenzohydrazide (5j) using
general Procedure A.
Yield = 55% (filtration), pale yellow solid. Mp = 146–150 °C. 1H
NMR (600 MHz, DMSO-d6): d 11.64 (br s, 1H), 11.39 (s, 1H), 8.16
(d, J = 1.6 Hz, 1H), 7.99 (d, J = 1.8 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H),

7080
N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
7.48 (d, J = 8.2 Hz, 1H), 7.17 (t, J = 7.9 Hz, 1H), 7.05 (s, 1H), 7.00 (t,
J = 7.8 Hz, 1H), 2.44 (s, 3H). 13C NMR (150 MHz, DMSO-d6): d 163.8,
156.6, 153.5, 138.6, 138.2, 135.8, 130.7, 128.0, 124.0, 121.4, 119.9,
119.2, 113.3, 112.7, 110.1, 106.2, 15.2. HRMS calcd for
(C17H1379Br2N3O2+H)+ 451.9428, found 451.9431.
4.1.1.31. (Z)-N0-(1-(1H-Indol-2-yl)-2,2-dimethylpropylidene)-5-
bromo-2-hydroxybenzohydrazide (6 ). Compound 6 was
prepared from 1-(1H-indol-2-yl)-2,2-dimethylpropan-1-one (3u)
(obtained by addition of t-BuLi to the Weinreb amide 2, cf. scheme)
and 5-bromo-2-hydroxybenzohydrazide (5c) using general Proce-
dure A.
Yield = 58% (after column 90/10 hexanes/EtOAc), white
powder. Mp = 217–218 °C. 1H NMR (600 MHz, DMSO-d6): d 11.35
(s, 1H), 11.01 (s, 2H), 7.98 (d, J = 1.8 Hz, 1H), 7.63 (d, J = 8.4 Hz,
1H), 7.45–7.41 (m, 2H), 7.16 (t, J = 7.8 Hz, 1H), 7.06 (t, J = 7.5 Hz,
1H), 6.77 (d, J = 8.4 Hz, 1H), 6.52 (s, 1H), 1.21 (s, 9H). 13C NMR
(150 MHz, DMSO-d6): d 159.8, 158.0, 155.0, 136.6, 135.6, 133.0,
127.7, 127.4, 122.1, 120.7, 120.0, 119.5, 119.1, 111.7, 110.8,
102.4, 38.5, 28.1. HRMS calcd for (C20H2079BrN3O2 Na)+ 436.0637,
found 436.0631.
i
i
4.1.1.27.
diisopropylbenzohydrazide (6
(E)-N0-[1-(1H-Indol-2-yl)ethylidene]-2-hydroxy-3,5-
). Compound 6 was pre-
v
v
pared from 3p, hydrazine hydrate and ethyl 2-hydroxy-3,5-dii-
sopropylbenzoate using general Procedure E.
Yield: 80%, yellow solid. Mp = 106–108 °C. 1H NMR (400 MHz,
DMSO-d6): d 12.35 (s, 1H), 11.40 (s, 1H), 11.12 (s, 1H), 7.67 (d,
J = 2.3 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H),
7.29 (1H, J = 2.3 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.04 (br s, 1H),
7.02 (t, J = 7.6 Hz, 1H), 3.30 (sept., J = 7.0 Hz, 1H), 2.89 (spet.,
J = 7.0 Hz, 1H), 1.24 (d, J = 7.0 Hz, 6H), 1.22 (d, J = 7.0 Hz, 6H). 13C
NMR (100 MHz, DMSO-d6): d 166.9, 156.4, 144.2, 138.8, 138.1,
136.6, 136.0, 129.1, 128.0, 123.9, 123.0, 121.3, 119.9, 114.1,
112.6, 105.8, 33.5, 26.7, 22.9, 19.0, 15.2. HRMS calcd for
(C19H1879BrN3O2+Na)+ 400.1995, found 400.1999.
4.1.1.32. (E/Z)-N0-((1H-indol-2-yl)(phenyl)methylene)-5-bromo-
2-hydroxybenzohydrazide (6
u
).
Compound 6u was pre-
pared from (1H-indol-2-yl)(phenyl)methanone (3v) (obtained by
addition of PhLi to the Weinreb amide 2, cf. scheme) and 5-bro-
mo-2-hydroxybenzohydrazide(5c) using general Procedure A.
Yield = 43% (after column 70/30 hexanes/EtOAc), pale yellow pow-
4.1.1.28.
hydroxy-4-methoxybenzohydrazide (6d).
(E)-N0-(1-(1H-indol-2-yl)ethylidene)-5-bromo-2-
Compound 6d
der. NMR analysis indicated that 6l is present in two isomeric
was prepared from 3p and 5-bromo-2-hydroxy-4-meth-
oxybenzohydrazide (5k) using general Procedure C.
forms (89:11).
4.1.1.32.1. Major isomer (E). 1H NMR (600 MHz, DMSO-d6): d
11.60 (s, 1H), 11.35 (s, 1H), 11.27 (s, 1H), 8.07 (s, 1H), 7.72–7.46
(m, 8H), 7.16 (t, J = 7.2 Hz, 1H), 6.97 (t, J = 6.9 Hz, 1H), 6.85 (d,
J = 8.4 Hz, 1H), 6.12 (s, 1H). 13C NMR (150 MHz, DMSO-d6): d
159.6, 155.0, 148.4, 137.9, 135.7, 135.5, 133.0, 132.0, 130.1, 129.4
(2CH), 128.3 (2CH), 127.5, 123.5, 120.8, 119.9, 119.5, 119.1,
112.2, 111.0, 107.4.
Yield = 82% (filtration), pale yellow solid. Mp = 242–244 °C. 1H
NMR (600 MHz, DMSO-d6): d 12.40 (br s, 1H), 11.31 (s, 1H),
11.17 (br s, 1H), 8.17 (s, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.48 (d,
J = 8.2 Hz, 1H), 7.16 (dt, J = 0.9 Hz, 7.6 Hz, 1H), 6.99 (t, J = 7.1 Hz,
1H), 6.97 (s, 1H), 6.69 (s, 1H), 3.88 (s, 3H), 2.38 (s, 3H). 13C NMR
(150 MHz, DMSO-d6): d 161.1, 158.8, 158.4, 147.4, 137.7, 135.8,
133.7, 127.6, 123.1, 120.6, 119.3, 112.1, 111.4, 104.7, 100.9,
100.6, 56.5, 13.8. HRMS calcd for (C18H1679BrN3O3+H)+ 402.0448,
found 402.0454.
4.1.1.32.2. Minor isomer (Z). 13C NMR (150 MHz, DMSO-d6): d
160.4, 155.7, 146.8, 137.1, 136.9, 135.8, 133.1, 129.9, 128.5
(2CH), 127.9 (2CH), 127.3, 127.2, 123.0, 121.2, 120.0, 119.9,
119.2, 112.1, 110.9, 105.2.
4.1.1.29. (E)-N0-((1H-Indol-2-yl)methylene)-5-bromo-2-hydrox-
HRMS (mixture) calcd for (C22H1679BrN3O2+Na)+ 456.0324,
found 456.0309.
ybenzohydrazide (6
e
).
Compound 6e was prepared from 1H-
indole-2-carbaldehyde 3o (obtained by reduction and oxidation of
1H-indole-2-carboxylic acid, cf. scheme) and 5-bromo-2-hydrox-
ybenzohydrazide (5c) using general Procedure A.
4.1.1.33.
dene)-2-hydroxybenzohydrazide (6
(E)-5-Bromo-N0-(1-(5-bromo-1H-indol-2-yl)propyli-
). Compound 6 was
c
c
Yield = 78% (filtration), pale yellow powder. Mp = 257–258 °C.
1H NMR (400 MHz, DMSO-d6): d 11.95 (br s, 1H), 11.84 (s, 1H),
11.62 (s, 1H), 8.48 (s, 1H), 8.05 (d, J = 2.8 Hz, 1H), 7.59 (dd,
J = 8.8 Hz, 2.4 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.17 (t, J = 7.4 Hz,
1H), 7.02 (t, J = 7.4 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 6.89 (d,
J = 1.2 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): d 163.0, 157.9,
141.8, 138.0, 136.0, 132.8, 130.8, 127.6, 123.5, 120.8, 119.6
(2CH), 112.1, 110.0, 107.6. HRMS calcd for (C16H1279BrN3O2+H)+
358.0186, found 358.0200.
prepared from 1-(5-bromo-1H-indol-2-yl)propan-1-one (3l) and
5-bromo-2-hydroxybenzohydrazide (5c) using general Procedure
C.
Yield = 41% (after flash chromatography 20/1; DCM/MeOH),
pale yellow solid. Mp = 252–254 °C. 1H NMR (600 MHz, DMSO-
d6): d 12.17 (br s, 1H), 11.60 (br s, 1H), 11.48 (s, 1H), 8.08 (d,
J = 2.5 Hz, 1H), 7.75 (d, J = 1.4 Hz, 1H), 7.58 (dd, J = 2.6 Hz, 8.7 Hz,
1H), 7.46 (d, J = 8.6 Hz, 1H), 7.26 (dd, J = 1.9 Hz, 8.6 Hz, 1H), 7.02
(d, J = 8.7 Hz, 1H), 6.95 (s, 1H), 2.81 (q, J = 7.7 Hz, 2H), 1.22 (t,
J = 7.6 Hz, 3H). 13C NMR (150 MHz, DMSO-d6): d 160.1, 155.7,
150.7, 136.4, 136.2, 135.6, 132.7, 129.5, 125.5, 122.7, 120.5,
119.4, 114.1, 111.7, 110.7, 103.6, 20.6, 10.7. HRMS calcd for
(C18H1579Br2N3O2+H)+ 465.9584, found 465.9588.
4.1.1.30.
hydroxybenzohydrazide (6/).
(E)-N0-(1-(1H-indol-2-yl)propylidene)-5-bromo-2-
Compound 6/ was prepared
from 1-(1H-indol-2-yl)propan-1-one 3t (obtained by addition of
ethylmagnesium bromide to the Weinreb amide 2, cf. scheme)
and 5-bromo-2-hydroxybenzohydrazide (5c) using general Proce-
dure A.
4.1.1.34. Specific procedures for the synthesis of the hydrazides
6.
5-Chloro-2-hydroxy-N0-((1E)-1-(1H-indol-2-yl)ethyli-
Yield = 73% (filtration), white powder. Mp = 255–256 °C. 1H
NMR (400 MHz, DMSO-d6): d 12.16 (br s, 1H), 11.48 (br d,
J = 0.8 Hz, 1H), 11.28 (s, 1H), 8.09 (d, J = 2.4 Hz, 1H), 7.59 (dd,
J = 8.6 Hz, 2.6 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz,
1H), 7.14 (td, J = 7.8 Hz, 0.6 Hz, 1H), 7.04–6.97 (m, 3H), 2.82 (q,
J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, DMSO-
d6): d 160.0, 155.4, 151.3, 137.8, 135.6, 134.8, 132.7, 127.6, 123.1,
120.6, 120.5, 119.3 (2CH), 112.1, 110.9, 104.4, 20.6, 10.8. HRMS
calcd for (C18H1679BrN3O2+H)+ 386.0499, found 386.0505.
dene)benzohydrazide (6t)
To a mixture of methyl 5-chloro-2-hydroxybenzoate (162 mg,
0.87 mmol) in isobutanol (0.5 mL) was added hydrazine hydrate
(42 lL, 0.87 mmol). The reaction mixture was irradiated with
microwaves for 15 min at 115 °C. Isobutanol was added and the
supernatant was removed. The residue was taken up in isobutanol
(0.5 mL) and 1-(1H-indol-2-yl)ethanone 3p (138 mg, 0.87 mmol)
was added. The reaction mixture was again irradiated with micro-
waves for 10 min at 110 °C. The solid product was filtered and
then, triturated with EtOH and Et2O to afford 6t as a yellow solid

N. S. Kumar et al. / Bioorg. Med. Chem. 20 (2012) 7069–7082
7081
(90 mg, 33%). Mp = 198–200 °C. 1H NMR (400 MHz, DMSO-d6): d
4.2. PK enzymatic assays, cell-based assays and cytotoxicity
12.46 (s, 1H), 11.39 (s, 1H), 10.07 (s, 1H), 7.86 (d, J = 2.4 Hz, 1H),
7.60 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.41 (dd, J = 8.8 Hz,
2.4 Hz, 1H), 7.19 (tm, J = 7.6 Hz), 7.03 (m, 2H), 6.93 (d, J = 8.8 Hz,
1H), 2.48 (s, 3H). 13C NMR (100 MHz, DMSO-d6): d 158.0, 153.8,
137.5, 136.5, 132.8, 127.7, 126.8, 123.4, 122.3, 120.9, 119.3,
119.1, 111.8, 104.9, 14.5. HRMS calcd for (C17H1435ClN3O2+Na)+
350.0663, found 350.0667.
Measurements of pyruvate kinase activity, in vitro susceptibil-
ity testing and the determination of mammalian cytotoxicity were
performed using previously described procedures and statistical
analysis.3
4.3. X-ray crystallography
4.1.1.35. 5-Bromo-2-(ethoxymethoxy)-N0-((1E)-1-(1H-indol-2-
Suitable crystals of compounds 6/ and 6i were mounted on the
yl)ethylidene)benzohydrazide (6y).
To
a
solution of 6a
end of a glass fiber with epoxy. Single crystal X-ray crystallo-
graphic analysis was performed on a Bruker SMART diffractometer
equipped with an APEX II CCD area detector fixed at a distance of
(73 mg, 0.20 mmol) in anhydrous THF (30 mL) maintained at 0 °C
were added, under an argon atmosphere, NaH (8.1 mg, 60%,
0.24 mmol) and then, after 10 min stirring still at 0 °C, chlorometh-
6.0 cm from the crystal and a Mo K
a fine focus sealed tube
ylethyl ether (45
lL, 0.49 mmol). The mixture was stirred at room
(k = 0.71073 nm) operated at 1.5 kW (50 kV, 30 mA) and filtered
temperature for 4 h and then, quenched with a saturated aqueous
ammonium chloride solution. The aqueous layer was extracted
with EtOAc and the organic layer was dried over anhydrous
Na2SO4, filtered through a silica gel pad and evaporated under re-
duced pressure. The residue was triturated with Et2O to afford 6y
as a pale yellow solid (65 mg, 77%). Mp = 128–130 °C. 1H NMR
(400 MHz, DMSO-d6): d 11.32 (s, 1H), 10.90 (s, 1H), 7.94 (d,
J = 2.5 Hz, 1H), 7.71 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 7.57 (d, J = 7.6 Hz,
1H), 7.50 (d, J = 7.6 Hz, 1H), 7.29 (d, J = 9.0 Hz, 1H), 7.15 (t,
J = 7.6 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1H), 6.98 (s, 1H), 5.46 (s, 2H),
3.75 (q, J = 7.0 Hz, 2H), 2.39 (s, 3H), 1.16 (t, J = 7.0 Hz, 3H). 13C
NMR (100 MHz, DMSO-d6): d 160.0, 153.9, 147.6, 137.7, 135.8,
134.7, 132.5, 127.6, 125.6, 123.1, 120.6, 119.3, 117.6, 113.2,
112.1, 104.8, 93.9, 4.5, 14.9, 13.8. HRMS calcd for
(C20H2079BrN3O3+Na)+ 453.0562, found 453.0561.
with a graphite monochromator.
The structures were solved using direct methods (SIR92)6 and
refined by least-squares procedures using CRYSTALS.7 All hydrogen
atoms were placed in idealized geometric positions and linked to
their respective carbon atoms using a riding model during refine-
ment. The isotropic temperature factor of each hydrogen atom
was initially set to 1.2 times that of the atom it is bonded to and
then the temperature factors of groups of similar hydrogen atoms
were linked during refinement. Crystal structure diagrams were
generated using ORTEP-3 for Windows (v. 2.02).8
All crystals diffracted poorly and there was not enough data to
model all atoms anisotropically, but the identity and connectivity
of the atoms are clear. In 6/, the methyl carbon of the ethyl substi-
tuent was disordered and modeled accordingly (see CIF file for de-
tails). Only the carbon with higher occupancy is shown in Figure 3.
4.1.1.36.
methoxybenzohydrazide (6b).
(E)-N0-(1-(1H-Indol-2-yl)ethylidene)-3,5-dibromo-2-
A mixture of 1-(1H-indol-2-
Acknowledgments
yl)ethanone 3p (50 mg, 0.31 mmol) and 3,5-dibromo-2-meth-
oxybenzohydrazide (101 mg, 0.31 mmol) in EtOH (2.5 mL) was
irradiated with microwaves at 180 °C for 3 h. The precipitate was
filtered and was washed with hot EtOH (2 ꢀ 2 mL) to give pale yel-
low solid (75 mg, 51%). NMR analysis indicated that 6b is present in
two isomeric forms (15:1). 1H NMR (600 MHz, DMSO-d6): d 11.30
(br s, 1H), 10.97 (s, 1H), 8.06 (d, J = 2.4 Hz, 1H), 7.79 (d, J = 7.4 Hz,
1H), 7.56 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.14 (t,
J = 7.9 Hz, 1H), 7.00 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 1.9 Hz, 1H), 3.86
(s, 3H), 2.36 (s, 3H). 13C NMR (150 MHz, DMSO-d6): d 160.9,
154.1, 149.5, 138.2, 137.2, 136.2, 133.3, 132.1, 128.0, 123.7,
121.2, 119.8, 118.8, 117.0, 112.7, 105.4, 62.6, 14.7. HRMS calcd
for (C18H1579Br2N3O2+H)+ 465.9584, found 465.9597.
The authors acknowledge Genome British Columbia, Genome
Canada, the Natural Sciences and Engineering Council of Canada
(NSERC) and the Pfizer-CDRD Innovation Fund for financial
support.
The authors also acknowledge support of the Vancouver Gen-
eral Hospital and the University of British Columbia Hospital Foun-
dation and SARS Accelerated Vaccine Initiative through the
PRoteomics for Emerging PAthogen REsponse (PREPARE) Project.
RNY acknowledges the support of Simon Fraser University, the
British Columbia Government Leading Edge Endowment Fund
and Merck Frosst Canada Ltd.
Supplementary data
4.1.1.37. (E)-N0-[(1H-Indol-2-yl)methylene]-5-bromo-2-meth-
PDP ID Codes: Crystallographic data for compound 8d: ID 3T0T.
CCDC 873762–873763 contains the supplementary crystallo-
graphic data compounds 6/ and 6i. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.002.
oxybenzohydrazide (6
2-methoxybenzoate (1.35 g, 5.50 mmol, 1.0 equiv) in ethanol
(0.6 ml) was added hydrazine hydrate (535 L, 11.00 mmol,
g
).
To a mixture of methyl 5-bromo-
l
2.0 equiv). The reaction mixture was irradiated with microwaves
for 40 min at 100 °C. Ethanol was added and the supernatant was
removed. The residue was taken up in ethanol (5 mL) and 1H-in-
dole-2-carbaldehyde (0.80 g, 5.50 mmol) was added. The reaction
mixture was again irradiated with microwaves for 50 min at
100 °C. The solid product was filtered and then, triturated with
References and notes
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(s, 1H), 11.55 (s, 1H), 8.36 (s, 1H), 7.74 (d, J = 2.3 Hz, 1H), 7.68 (dd,
J = 8.9 Hz, J = 2.3 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 7.6 Hz,
1H), 7.16 (m, 1H), 7.16 (d, J = 8.9 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H),
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123.3, 120.7, 119.5, 114.4, 112.0, 111.8, 107.0, 56.2. HRMS calcd
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