Virtual screening, selection and development of a benzindolone structural scaffold for inhibition of lumazine synthase

Article abstract of DOI:10.1016/j.bmc.2010.03.072

Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a K_i of 70 μM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (K_i 38 μM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve π-π stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.

Full text of DOI:10.1016/j.bmc.2010.03.072

Bioorganic & Medicinal Chemistry 18 (2010) 3518–3534
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Virtual screening, selection and development of a benzindolone
structural scaffold for inhibition of lumazine synthase
Arindam Talukdar a, Ekaterina Morgunova b, Jianxin Duan d, Winfried Meining e, Nicolas Foloppe b,
Lennart Nilsson b, Adelbert Bacher c, Boris Illarionov c, Markus Fischer c, Rudolf Ladenstein b,
Mark Cushman a,
*
a Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and The Purdue Center for Cancer Research,
Purdue University, West Lafayette, IN 47907, USA
b Karolinska Institute, Department of Bioscience, Hälsovägen 7-9, S-14157 Huddinge, Sweden
c Institute of Biochemistry and Food Chemistry, Food Chemistry Division, University of Hamburg, Grindelallee 117, D-20146 Hamburg, Germany
d Anterio Consult & Research GmbH, Augustaanlage 23, 68165 Mannheim, Germany
e Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium
tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic
acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis
Received 8 January 2010
Revised 24 March 2010
Accepted 25 March 2010
Available online 8 April 2010
lumazine synthase with a Ki of 70
onamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate
38 that displayed a moderate increase in enzyme inhibitory activity (Ki 38 M). Molecular modeling
based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing
the present benzindolone/enzyme complexes involve stacking interactions with Trp27 and hydrogen
lM. Lead optimization through replacement of the carboxymethylsulf-
l
Keywords:
Mycobacterium tuberculosis
Lumazine synthase
Inhibitor
p–p
bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain
hydroxyl of Thr87.
Virtual screening
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
(1H,3H)pyrimidinedione (1) yielding 6,7-dimethyl-8-D-ribityllum-
azine (3).11–25 The final process in the biosynthesis of riboflavin (4)
Riboflavin (vitamin B2) plays a crucial role in many biological
processes, including photosynthesis and mitochondrial electron
transport. Riboflavin is biosynthesized by plants and numerous
microorganisms but not by animals. Whereas animals obtain
riboflavin from dietary sources, certain microorganisms such as
Gram-negative pathogenic bacteria and yeasts lack efficient ribofla-
vin uptake systems and are therefore absolutely dependent on
endogenous riboflavin biosynthesis.1–4 Riboflavin biosynthesis is
therefore an attractive target for the design and synthesis of new
antibiotics, which are urgently needed because pathogens are
becoming drug resistant at an alarming rate. Lumazine synthase
and riboflavin synthase catalyze the last two steps in the biosynthe-
sis of riboflavin (4) (Scheme 1). The biosynthetic pathway starts off
from one molecule of GTP,5–7 which is converted to 5-amino-6-
ribitylamino-2,4(1H,3H)-pyrimidinedione (1) by a sequence of ring
opening, deamination, reduction, and dephosphorylation.8–10
Lumazine synthase catalyzes the condensation of 3,4-dihydroxy-
2-butanone 4-phosphate (2) with 5-amino-6-ribitylamino-2,4-
involves a mechanistically unusual dismutation of two molecules
of 6,7-dimethyl-8-D-ribityllumazine (3) that results in the formation
of one molecule of riboflavin and one molecule of the pyrimidinedi-
one derivative 1.26–30
Although the precise details remain to be established, the lum-
azine synthase-catalyzed reaction most likely proceeds along a
mechanistic pathway involving formation of the Schiff base 5,
phosphate elimination affording 6, tautomerization to 7, ring clo-
sure, and dehydration to yield the lumazine 3 (Scheme 2).31 Varia-
tions of this mechanism are possible depending on the Schiff base
geometry and possible isomerization, conformational changes, and
the timing of phosphate elimination.
Screening drug-like compounds by computational docking
against the structure of a known target has become a promising
avenue in lead discovery.32–34 This approach is arguably much
cheaper than experimental high-throughput screening and has
been validated in other lead discovery projects.35,36 The successful
history of lumazine synthase structure determination and inhibitor
development provides a sound basis for the selection of lead com-
pounds from commercial chemical libraries and possible structural
modification in order to improve the binding properties. The
* Corresponding author. Tel.: +1 765 494 1465; fax: +1 765 494 6790.
E-mail address: cushman@pharmacy.purdue.edu (M. Cushman).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.072

A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
3519
-2
OH
OPO3
OH
HO
OH
OH
-
OPO32
O
Pi
H
OH
2
HN
N
O
+
O
NH
Lumazine
Synthase
H2N
O
2
OH
OH
OH
OH
1
HO
HO
N
OH
OH
OH
OH
OH
OH
HO
HO
H
N
OH
HN
O
H3C
H3C
N
O
OH
H
N
H
N
NH
NH
HN
N
O
HN
N
O
H2N
N
O
O
NH
NH
3
H
Pi
1
O
Riboflavin
Synthase
O
O
HO
H
6
_
O
_
5
O
O
OH
P
HO
OH
OH
P
O
O
HO
N
OH
OH
OH
OH
OH
H3C
H3C
N
O
HO
HO
OH
OH
OH
NH
H
N
N
H
N
O
HN
N
O
O
N
N
O
3
4
HO
NH
NH
Scheme 1.
O
O
7
design of most of the known inhibitors of lumazine synthase has
been based on the structures of both substrates of lumazine syn-
thase and the putative Schiff base intermediate in the enzymatic
reaction.37,38
8
Scheme 2.
The ZINC database,39 maintained by the Shoichet group at
UCSF, was subjected to virtual screening for the discovery of
new lumazine synthase inhibitors. Docking the structures into
the lumazine synthase structure led to an interesting new scaf-
fold, compound 9 (Fig. 1), which has ‘drug-like’ features, such
as a sulfonamide moiety, and therefore has more potential for
drug development than mechanism-based probes containing po-
lar ribityl side chains. The lead compound 9 displayed a Ki of
OH
OH
HO
O
H
N
NH
OH
H
Cl
O
N
N
O
NH
O
NH
O
N
O S O
NH
70 lM versus Mycobacterium tuberculosis lumazine synthase. It
O
O
consists of an aromatic oxobenzindole ring system and a carb-
oxymethylsulfonamide side chain. The inhibitor 9 lacks any close
analogy to the pyrimidinedione ring or the ribityl side chain of
the substrate 1, which explains its relatively low affinity for the
enzyme. However, it has been recently demonstrated that the
ribityl chain present in the substrate, which was thought to be
necessary for binding to the enzyme, can be replaced by a simple
hydroxybenzylidene moiety (compound 12) with retention of
lumazine synthase inhibitory activity.40 Moreover, it has recently
been shown that the attachment of aliphatic chains bearing
phosphate or phosphonate groups on the heterocyclic core
dramatically increases binding affinity of the inhibitors.41–45 An
alkylphosphate chain consisting of 4–5 carbon atoms produces
the strongest inhibitory effect. It therefore seemed logical to re-
place the carboxymethyl group of the lead compound 9 with alkyl
phosphate groups and test the influence of the length of aliphatic
chains with 2–6 carbon atoms. The rationale behind this design is
that the phosphate moieties in compounds of general structure
O P OH
OH
HO
O
O
9
10
11
O P OH
OH
O
NH
OH
H
N
O
O S O
NH
NH
O2N
O
n
O
12
HO P
OH
O
13
Figure 1. Representative M. tuberculosis lumazine synthase Inhibitors.

3520
A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
113 are expected to bind in the same phosphate binding pocket
as the inhibitors 10 and 11.
O
O
O
NH
NH
O
n
P
OH
H2N
a
OH
2. Results and discussion
2.1. Virtual screening
O
S O
Cl
14
Virtual screening aims to discover inhibitors with novel scaf-
folds. In the present case, more than one million compounds were
screened from the ZINC database,39 which has been prepared for
docking screening. The M. tuberculosis lumazine synthase crystal
15
O
NH
structure in complex with the inhibitor 3-(1,3,7-trihydro-9-D-ribi-
tyl-2,6,8-purinetrione-7-yl) propane 1-phosphate (PDB: 1w19)
was prepared using Protein Preparation tools in Maestro 7.5
(Schrödinger LLC, Portland, Oregon), removing all crystal water
molecules. The compounds were first docked with Glide 4.0
(Schrödinger LLC, Portland, Oregon) High Throughput Virtual
Screening mode without any constraints.46 A multi-stage screening
workflow was applied, increasing the ligand conformational search
and applying different scoring functions in order to rapidly reduce
the number of possible hits. During the latter two stages of the
screening, the following pharmacophore constraints were applied:
three backbone hydrogen bonds to Ile83, Val81, Ala59 and the
phosphate interaction to Arg128. At least three of these constraints
had to be satisfied. The backbone hydrogen bonds are desirable be-
cause they could prevent future drug resistance issues. Based on
solubility criteria posed by the ITC measurement, a solubility filter
was also applied in the workflow. The cutoff score of ꢀ10 was cho-
sen based on docking of several known active compounds41 under
the same conditions. The resulting 44 ligands were manually in-
spected and based on the interactions and their chemical scaffold,
12 compounds with different chemical scaffolds in total were se-
lected for assay. Of these compounds, compound 9 demonstrated
inhibition in the subsequent kinetic assay (Table 1). The principal
of docking/pharmacophore based filtering is that it selects com-
pounds complementary to the targeted site. Therefore, statistically,
there is a much higher probability that the compounds selected by
such a screening protocol will bind at the targeted site.
O
O
S
O
NH
n
O
P
HO
OH
13
Scheme 3. Reagent and condition: (a) ClSO3H, 0 °C.
Various procedures were tried unsuccessfully to produce the fi-
nal compound 13. The solvent system, base and temperature were
varied without any improvement in results. The phosphate group
was therefore protected before performing the coupling reaction.
The syntheses of compounds 26 and 27 were both carried out
the same way starting with the appropriate amino alcohol. Accord-
ingly, the synthesis was started from ethanolamine (16), which on
Cbz protection provided compound 18 (Scheme 4). The phosphate
functionality in compound 20 was introduced by treatment of 18
with di-tert-butyl diisopropyl phosphoramidite under 1-H tetra-
zole catalysis, followed by in situ oxidation with hydrogen perox-
ide.48,49 The Cbz group of compound 20 was removed via
hydrogenolysis with Pd/C to afford the free amine compound 22.
Reaction of compound 15 with the free amine 22 in the presence
of triethylamine provided the sulfonamide 24. Subsequent depro-
tection of the phosphate with trifluoroacetic acid provided the de-
sired product 26.50
A straightforward synthesis (Scheme 3) was proposed for com-
pounds having general structure 13. The synthesis started from
commercially available benz[cd]indol-2(1H)-one (14), which on
treatment with chlorosulfonic acid yielded compound 15.47
Scheme 4 was shortened by removing the Cbz protection step
and the subsequent deprotection by hydrogenation. The improved
Table 1
Inhibition of recombinant riboflavin synthase (RS) and/or lumazine synthase (LS) of M. tuberculosis by selected compoundsa
Kcat (mꢀ1
)
Kid
(l
M)
Kis
(lM)
Mechanism
b
c
e
Compd
Enzyme
Ks
(lM)
9
26
LS
LS
RS
LS
RS
LS
RS
LS
RS
LS
RS
LS
RS
LS
RS
70
260 109
58
125 57
>1000
434 115
>1000
572 210
>1000
563 56
971 179
723 91
898 187
518 94
>1000
9.8 0.5
5.7 0.04
0.41 0.01
0.30 0.01
0.45 0.01
0.28 0.01
0.44 0.01
0.29 0.01
0.45 0.01
0.56 0.01
0.45 0.01
0.56 0.01
0.57 0.01
0.15 0.01
0.61 0.02
0.34 0.01
Partial
Mixed
Mixed
27
38
39
40
72
77
15
5.6 0.04
11
6.5 0.3
19
9.8 0.6
18
9.8 0.6
16
4.1 0.3
21
7.8 0.3
2
99 30
1
38
8
1
Uncompetitive
Uncompetitive
Uncompetitive
Uncompetitive
Mixed
1
1
832 418
832 418
2
>1000
897 104
Mixed
Uncompetitive
Compounds 31–33, 54–55, 71, 73, 79 are not listed in this table since they did not significantly inhibit either lumazine synthase or riboflavin synthase.
a
The assays with lumazine synthase were performed with dihydroxybutanone phosphate substrate held constant, while the concentration of the pyrimidinedione
substrate was varied.
b
Ks is the substrate dissociation constant for the equilibrium E þ S ꢀ ES.
c
Kcat is the rate constant for the process ES ꢀ E þ P.
d
Ki is the inhibitor dissociation constant for the process E þ I ꢀ EI.
e
Kis is the inhibitor dissociation constant process ES þ I ꢀ ESI.

A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
3521
tetrazole catalysis, followed by in situ oxidation with hydrogen
peroxide to provide compounds 24 and 35–37. The final deprotec-
tion of the tert-butyl groups with trifluoroacetic acid produced the
required substances 26, 38, 39 and 40 with different linker chain
lengths between the phosphate group and the sulfonamide moiety.
Enzyme assays were performed with recombinant lumazine
synthase and riboflavin synthase from M. tuberculosis. Assays with
serial dilutions of each respective inhibitor were conducted in
microtiter plates that were monitored photometrically over a per-
iod of 15 min. Data analysis was performed using Dynafit soft-
ware.51 Compound 38, with a C-4 chain length, showed the best
NHCbz
n
NH2.HCl
a
b
HO
HO
n
18 n = 1
19 n = 2
16 n = 1
17 n = 2
O
O
c
P
Ot-Bu
O
P
Ot-Bu
O
CbzHN
H2N
n
Ot-Bu
n
Ot-Bu
20 n = 1
21 n = 2
22 n = 1
23 n = 2
inhibitory activity (Ki of 38 lM) against M. tuberculosis lumazine
O
O
NH
synthase (Table 1). The inhibitory activity increases significantly
as the connector chain length between the sulfonamide and phos-
phate groups is increased from two to four carbons.
NH
d
e
The compounds studied had been designed to fit the active site
of lumazine synthase. Since the product of lumazine synthase is
the substrate of riboflavin synthase and there may be some simi-
larity of the ligand recognition pattern of lumazine synthase and
riboflavin synthase, the compounds under study were assayed ver-
sus the riboflavin synthase of M. tuberculosis. In fact, compounds
39, 40, and 77 were found to be weak, uncompetitive inhibitors
O
S
O
n
O
S O
HN
HN
n
O
O
O
P
OH
O P Ot-Bu
Ot-Bu
OH
of the enzyme with Ki values in the upper lM range (Table 1).
24 n = 1
25 n = 2
26 n = 1
27 n = 2
2.2. Molecular modeling of ligand binding to lumazine synthase
Scheme 4. Reagents and conditions: (a) benzylchloroformate, Et3N, CH2Cl2, rt; (b)
iPr2NP(OtBu)2, H2O2, tetrazole, rt; (c) H2, Pd/C, MeOH; (d) 15, Et3N, THF, rt; (e) TFA,
CH2Cl2, rt.
The X-ray crystal structure of the complex of 4-(6-chloro-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)butyl dihydrogen phos-
phate (11)42 with lumazine synthase [PDB code: 2C97] with
resolution 2.00 Å allowed the rational docking and energy minimi-
zation of newly synthesized compounds in the active site of
M. tuberculosis lumazine synthase. Docking of the lead and synthe-
sized compounds in the active site of M. tuberculosis lumazine
synthase was performed with GOLD (BST, version 3.0, 2005).
Energy minimization was performed with the MMFF94s force field
in order to reduce steric clashes between the GOLD-docked
compounds and the protein.
synthesis of oxobenzindole derivatives is outlined in Scheme 5.
Simply mixing compound 15 with the respective amino alcohols
in THF at room temperature provided the required intermediates
31–34 (Scheme 5). The phosphate group was introduced by
treatment with di-tert-butyl diisopropyl phosphoramidite under
O
Figure 2 was constructed by displaying the amino acid residues
of the enzyme involved in hydrogen bonding with the inhibitors 27
and 38 in the hypothetical model derived from docking and energy
minimization. The maximum distance between the heavy atoms
participating in the hydrogen bonds shown in Figure 2 was set at
3.8 Å. The binding patterns of both compounds are rather similar
to each other and are comparable to that observed in the crystal
structure of 11 in M. tuberculosis lumazine synthase. In general,
the binding most likely reflects the contributions of the charged
residue Arg128 and several polar residues that interact with the li-
gands directly via hydrogen bonding. The phosphate moiety of the
ligands is extensively hydrogen bonded to the side chain nitrogens
of Arg128, as well as the backbone nitrogens of Gly85 and Gln86
and the side chain hydroxyl of Thr87. Besides hydrogen bonding,
the interaction of M. tuberculosis lumazine synthase and the inhib-
itor is predominantly hydrophobic. As expected, the calculated
structural model positions the benzindolone aromatic rings of the
ligands stacked with the indole ring of Trp27, consistent with the
experimental structures of different lumazine synthase inhibitor
complexes.7,42,43,51–53 The carbonyl oxygen of the benzindolone
moiety of both compounds hydrogen bonds with the backbone
nitrogen of Ala59. Because of the longer aliphatic chain of 38, the
hydrophobic moiety of this compound is slightly shifted to the pro-
tein compared to the position of this moiety in the model with com-
pound 27. This shift results in formation of two additional hydrogen
bonds by the carbonyl oxygen of the benzindolone moiety with the
main chain nitrogen of Ile60 and with side chain oxygen of Glu61.
Apparently, the formation of the additional hydrogen bonds is re-
flected in the better binding constant found for compound 38.
NH
a
NH2
n
b
HO
16 n = 1
28 n = 3
29 n = 4
30 n = 5
O
S O
HN
n
31 n = 1
32 n = 3
33 n = 4
34 n = 5
OH
O
O
NH
NH
c
O
S O
O S O
HN
HN
n
n
O
O
O P OH
OH
O P Ot-Bu
Ot-Bu
26 n = 1
38 n = 3
39 n = 4
40 n = 5
24 n = 1
35 n = 3
36 n = 4
37 n = 5
Scheme 5. Reagents and conditions: (a) 15, Et3N, THF, rt; (b) iPr2NP(OtBu)2, H2O2,
tetrazole, rt; (c) TFA, CH2Cl2, rt.

3522
A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
Figure 2. Structural representation of the hypothetical structure of compounds 27 and 38 bound in the active site of M. tuberculosis lumazine synthase. The amino acid
residues involved in the active site are presented in ball and sticks and colored differently for two different subunits. The carbonyl atoms are shown in yellow and green, and
nitrogen and oxygen atoms are shown in blue and red, respectively. The respective inhibitor molecules are colored in magenta (C3-alkylphosphate 27) and cyan (C4-
alkylphosphate 38). The black dashed lines represent the putative interactions between compounds 38 and enzyme molecules. This figure was generated by PyMol [DeLano,
W. L. (2002), the PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA, USA] and programmed for wall-eyed (relaxed) viewing.
Comparison of these models with the known structures of
M. tuberculosis lumazine synthase in complex with different sub-
strate 1 analogues showed the lack of five hydrogen bonds involv-
ing the ribityl chains of the substrate 1 analogues,43,54 and at least
three hydrogen bonds formed between oxygen/nitrogen atoms of
heterocyclic rings of the substrate 1 analogues and main/side chain
atoms of Val81, Ile83 and Trp27 (Fig. 3). Trp27 is involved in bind-
or C4-alkylphosphate chain (38). Earlier crystallographic studies
of lumazine synthase from various organisms ( Bacillus subtilis,
Schizosaccharomyces pombe and Aquifex aeolicus)21,51,55 suggested
that orthophosphate ions are sufficient for the stability of the pen-
tameric assemblies and consequently for the activity of the protein.
Moreover, all attempts to crystallize any lumazine synthase in a
phosphate-free environment and to obtain the thermodynamic
characteristics have ended unsuccessfully. Thus, we have been
forced to run our experiments in phosphate buffer and would like
to emphasize that we are dealing with a tertiary binding reaction
involving a phosphate ion, an inhibitor molecule, and free enzyme.
As a result, the association constants and the binding free energy
derived from our ITC experiments should be considered as ‘appar-
ent’ thermodynamic parameters. Fitting of the binding isotherm
was achieved with a model using a ‘single set of identical sites’
assuming that each of the active sites in the pentameric assembly
is occupied by one inhibitor molecule. Binding of both compounds
is exothermic with negative changes in binding enthalpy, although
the titration of compounds to the reference phosphate buffer
showed endothermic changes in the system. In order to correct
for compound dilution, the compound was injected into phosphate
buffer and the dilution heat was subtracted from the reaction heat.
The measured negative enthalpy changes of ꢀ7.6 1.3 kcal molꢀ1
for C4-alkylphosphate afforded an apparent association constant
of 7.7 ꢁ 103 0.5 ꢁ 103 Mꢀ1 with stoichiometry 1 for the binding
reaction, in agreement with the expected binding of one inhibitor
molecule in each of the five active sites of the pentameric protein.
ing through p–p stacking interactions. The hydrogen bonds involv-
ing the heterocyclic rings of the substrate 1 analogues seem to be
particularly important for the affinities of the substrate analogues.
For instance, the earlier discovered inhibitor 11,42,44 which also
lacks the ribityl chain but contains oxygen/nitrogen substitutions
in the heterocyclic ring, demonstrated very high inhibitory activity
with an inhibition constant in the nanomolar range. The lumazine
synthase inhibitory activities displayed by the benzindolone deriv-
atives, which lack both a ribityl side chain and a pyrimidinedione
system, are therefore surprising.
2.3. Thermodynamic characterization of binding
The apparent thermodynamic binding characteristics of com-
pounds 26, 27, 38, 39 and 40 were obtained by Isothermal Titration
Calorimetry (ITC). Analysis of the changes in heat accompanying
the binding reaction allowed the binding enthalpy of the processes
(D
H) to be derived, the stoichiometry (n) and association constants
(Ka) to be estimated, and the entropy ( S) and free energy ( G) of
D
D
the binding reactions to be calculated. Figure 4 shows a typical
calorimetric titration of M. tuberculosis lumazine synthase in
100 mM potassium phosphate buffer at pH 7.0 and 30 °C with
the compound containing the benzindolone group and a C3- (27)
The calculated entropy
D
S and free energy of binding
D
G were
ꢀ7.2 cal molꢀ1 degꢀ1 and ꢀ5.4 kcal molꢀ1
, respectively, which
showed that the binding reaction is favored enthalpically. The
Figure 3. Crystal structure of 10 [PDB Code: 1W29] shown in green and 11 [PDB Code: 2C97] shown in pink bound to M. tuberculosis lumazine synthase. Hydrogen bonds
formed by protein with the ribityl group, heterocyclic ring and phosphate group are shown for the compound 10. The diagram is programmed for wall-eyed (relaxed)
viewing.

A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
3523
2
0
2
0
-2
0
20 40 60 80 100 120 140
-10 0 10 20 30 40 50 60 70 80 90 100 110
Time (min)
Time (min)
Figure 4. Calorimetric titration profiles of M. tuberculosis lumazine synthase with C4-alkylphosphate (38) (a) and C3-alkylphosphate (27) (b) at 30 °C. Top panels. Raw data
obtained for 35 injections 8 L each. The differential power signal recorded in the control titration of compound to the potassium phosphate buffer is presented on the
l
insertions of top panels (a). Bottom panels. Integrated heat of binding reaction after subtraction of integrated heat of compound dilution plotted against molar ratio of total
ligand concentration to total pentameric protein concentration. The solid line shows the best fit to the data, according to the model that assumes a single set of identical sites.
For comparison, the titration calorimetry curves for lumazine synthase from Bacillus anthracis and Schizosaccharomyces pombe, respectively, titrated with C4-alkylphosphate
compound 38 presented on panels (c) and (d) do not indicate a binding reaction.
measured enthalpy change for C3-alkylphosphate compound 27
was ꢀ5.2 0.7 kcal molꢀ1, which is less favorable than for C4-
alkylphosphate compound 38, but the loss in favorable enthalpy
the same experimental limits (Fig. 4). A structural comparison of
the active sites of the enzymes showed a single mutation in
M. tuberculosis lumazine synthase, G136E, which could be
responsible for a different affinity to the ligands. Thus, the com-
pounds produced in this study can be considered as specific li-
gands for M. tuberculosis enzyme and as leads for further
development.
To determine the structural motif required for the activity, the
intermediates 31, 32 and 33 (Fig. 5) without the phosphate group
were tested. They were found to be totally inactive. Although in the
molecular modeling study the oxybenzindolone moieties were in-
was compensated by a favorable positive entropy change
D
G and
S of
0.98 cal molꢀ1 degꢀ1. The resulting free energy of binding
D
apparent association constant Ka were ꢀ5.5 kcal molꢀ1 and
8.9 ꢁ 103 3.5 ꢁ 103 Mꢀ1, respectively. A similar enthalpy–entro-
py compensation effect was observed for the binding reaction of
M. tuberculosis lumazine synthase 1043 and for the reaction of
Candida albicans LS with 11.44
Notably, the titration calorimetry experiments with 38 versus
icosahedral lumazine synthase from Bacillus anthracis and the
pentameric enzyme from S. pombe did not detect binding within
volved in
p–p stacking interactions with Trp27, the compounds do
not have any binding affinity in the active site. The evidence clearly

3524
A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
indicates that the phosphate group is necessary for the inhibitory
activity.
O
P
O
a
I
I
+
EtO
H
I
EtO
P
n
To find metabolically stable inhibitors of M. tuberculosis
lumazine synthase, compounds with phosphonate 41 and fluor-
ophosphonate 42 moieties ( Fig. 6) were synthesized with C-3,
C-4 and C-5 chain length. Phosphonate and fluorophosphonate
groups are stable to hydrolysis by phosphatases. The fluoro deriv-
atives were synthesized because they more closely resemble the
phosphate group electronically than the phosphonates do. The
n
OEt
OEt
43 n = 2
44 n = 3
46 n = 2
47 n = 3
45
O
O
b
c
pKa values of a,a-difluorophosphonates are closer to those of phos-
EtO
P
N
phates, while phosphonates are less acidic than phosphates. On the
other hand, fluorophosphonates resemble phosphonates sterically
(the van der Waals radii for fluorine and hydrogen are 1.35 and
1.20 Å, respectively).56
The synthesis (Scheme 6) started from diethylphosphite (45),
which on treatment with 1,4-diiodobutane (43) and 1,5-diiodo-
pentane (44) provided compounds 46 and 47.57,58 The iodo deriv-
atives were converted to amines 50 and 51 via phthalimide
derivatives 48 and 49.59,60 Treatment of benzindole sulfonyl chlo-
ride (Scheme 6) derivative 15 with amines 50 and 51 in THF pro-
vided the phosphonate derivatives 52 and 53. The deprotection
of the ethyl groups with TMSBr furnished the desired phosphonate
derivatives 54 and 55.
n
OEt
O
48 n = 2
49 n = 3
O
NH
O
d
EtO
P
NH2
n
OEt
O
S O
O
50 n = 2
51 n = 3
HN
P OEt
OEt
n
O
NH
52 n = 2
53 n = 3
e
The phosphonate derivatives 54 and 55 showed absolutely no
inhibitory activity. At physiological pH, the difference between
the phosphate and phosphonate groups is that a phosphate group
is mainly diionic and a phosphonate group is mainly monoionic.
The synthesis of the difluorophosphonates 64–66 is outlined in
Scheme 7. After much effort, reagent 56 was converted to the re-
quired compound 57.61–64 Reaction of 56 with activated zinc dust65
O
S O
O
HN
P OH
OH
n
54 n = 2
55 n = 3
Scheme 6. Reagents and conditions: (a) NaH, THF, ꢀ20 °C 1 h then ꢀ10 °C 18h; (b)
DMF, 100 °C; (c) NH2NH2ꢂH2O, EtOH, reflux; (d) 15, Et3N, THF, rt; (e) TMSBr, CH2Cl2,
rt.
gave the stable [(diethoxyphosphinyl)difluoromethyl]zinc bro-
mide, which on hydrolysis provided compound 57.
O
O
O
NH
NH
NH
Compound 57 when reacted at ꢀ78 °C with LDA in THF in the
presence of hexamethylphosphoramide (HMPA) afforded a lithium
anion, which was treated with diiodo compounds to provide
the desired products 61, 62 and 63 in approximately 20% yield.
The poor yields observed in these reactions are probably due to the
poor nucleophilicity of the highly stabilized lithium anion and its
poor stability above ꢀ25 °C. Without HMPA, no alkylation was
observed. An attempt was made to convert the iodo derivatives
61, 62 and 63 to amines via the standard Gabriel procedure by
making the phthalimide derivatives 64, 65 and 66, but mixtures
of products were obtained that were very hard to separate and
characterize. Thus, the reverse strategy was considered, and
O
S O
O
S O
O
S O
HN
HN
HN
OH
OH
31
HO
32
33
Figure 5.
O
P
O
P
b
a
EtO
CF2Br
EtO
CF2H
I
O
OEt
56
O
OEt
I
n
NH
NH
58 n = 2
59 n = 3
60 n = 4
57
O
KN
O
S O
O
S O
O
N
F
F
O
HN
O
F
F
HN
n
O
n
EtO
P
I
EtO
P
n
n
F
F
c
OEt
OEt
O
HO
P O
HO P
O
61 n = 2
62 n = 3
63 n = 4
64 n = 2
65 n = 3
66 n = 4
OH
OH
42
41
Scheme 7. Reagents and conditions: (a) (i) Zn, Et2O, reflux; (ii) H2O; (b) LDA, HMPA,
ꢀ78 °C; (c) DMF, 100 °C.
Figure 6. Compounds with phosphonate and fluorophosphonate moieties.

A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
3525
accordingly, the sulfonyl chloride compound 15 was converted to
the sulfonamide derivative 67 with ammonia in THF (Scheme 8).
Treatment of the sulfonamide derivative 67 with the iodo com-
pounds 61, 62 and 63 in the presence of K2CO3 (Scheme 8) pro-
vided the protected benzindolone fluorophosphonate derivatives
68, 69 and 70. Finally, the removal of the ethyl groups with TMSBr
furnished the desired products 71, 72 and 73 with fluorophospho-
nate groups. Surprisingly, the fluorophosphonate derivatives 71, 72
and 73 turned out to be inactive against M. tuberculosis lumazine
synthase and M. tuberculosis riboflavin synthase, except for
compound 72, which showed poor inhibitory activity against
M. tuberculosis lumazine synthase (Table 1). Compound 72 corre-
sponds to compound 38, bearing an alkylphosphate chain consist-
ing of 4 carbon atoms, which exhibited the strongest enzyme
inhibitory effect. Evidently, in this particular case, phosphonates
and fluorophosphonates proved to be very poor mimics of the
phosphate group of the inhibitors, although in other cases involv-
ing lumazine synthase, they were effective.41,66,67
The mechanism outlined in Scheme 2 is certainly reasonable,
but questions still remain about the timing of phosphate elimina-
tion relative to the conformational reorganization of the side chain.
Nevertheless, it is clear that the inorganic phosphate must eventu-
ally be released at some point to make way for new substrate. As
mentioned above, the benzindolone moiety by itself does not have
significant binding affinity considering that compounds 31, 32 and
33 are totally inactive. On the other hand, the active phosphates
must be able to displace inorganic phosphate from the phosphate
binding pocket for any inhibitory activity and therefore the non-
phosphate part of their structures must contribute positively in
this regard. Compound 11, with a C-4 chain length, showed strong
Compounds 27 and 38, with C-3 and C-4 alkyl phosphate
chains, showed better inhibitory activity against M. tuberculosis
lumazine synthase. The inhibitory activity increases significantly
with an increase in the alkyl phosphate chain length connector be-
tween sulfonamide and phosphate groups from two carbons to
four carbons, but then the inhibitory activity decreases as the chain
length increases. The expected pKa of the phosphonate derivatives
54 and 55 suggest that these derivatives are mainly monoionic,
thus, they are unable to displace the inorganic phosphate.
Although the fluorophosphonate derivatives 71, 72 and 73 have
favorable pKa values, it seems they are also unable to displace
the inorganic phosphate from the phosphate binding pocket. Fluor-
ophosphonate derivative 72, which corresponds to compound 38
bearing an alkyl phosphate chain consisting of four carbon atoms,
showed some inhibitory activity against M. tuberculosis lumazine
synthase, with Ki values of 832 lM and Kis 518 lM. Molecular
modeling of fluorophosphonate–lumazine synthase complex did
not reveal a structural basis for these unexpected findings.
To delve deeper into the nature of the phosphate binding site,
information obtained by REDOR NMR spectroscopy of complexes
of lumazine synthase from Saccharomyces cerevisiae with phospho-
nate reaction intermediate analogues 80 was used (Fig. 7).45 The
15N{31P} REDOR NMR spectra of those complexes indicated that
mobility of the Lys92 side chain could facilitate the required active
site release of phosphate during the enzyme catalysis. Although
the exact nature of the phosphate binding pocket still remains elu-
sive, it is clear that the inorganic phosphate must be displaced by
organic phosphate from the phosphate binding pocket for any en-
zyme inhibitory activity. These compounds have a pyrimidinedi-
one ring and a ribityl side chain, which contribute to their
affinity for lumazine synthase. The binding of oxybenzindolone
compounds in the active site of lumazine synthase depends on
their abilities to bind at the phosphate binding site. The metaboli-
cally stable fluorophosphonate derivative 72, which showed mod-
erate activity, could possibly be used as a 19F NMR mechanistic
probe to study the structure of the occupied phosphate binding
sites of lumazine synthase.
inhibitory activity (Ki 0.88 lM) against M. tuberculosis lumazine
synthase. Compounds 77 and 79, which lack the phosphate group,
were synthesized (Scheme 9). As expected, compounds 77 and 79
were inactive. This substantiates the point that although the
pyrimidinedione ring alone does not have any binding affinity; it
contributes positively to the binding of compound 11, since its
phosphate group displaces inorganic phosphate from the phos-
phate binding pocket. The highlights of the synthesis of com-
pounds 77 and 79 were the selective reduction of the nitro group
to an amino group with sodium dithionate, and the removal of
benzyl protecting groups under controlled hydrogenolysis using
1,4-cyclohexediene as a source of hydrogen.
In virtual screening, finding novel active scaffolds is the most
important success criterion. The oxybenzindolone derivatives pro-
vide unique structural probes of the active site of lumazine syn-
Cl
Cl
N
OBn
N
OBn
a
N
N
O2N
H2N
O
NH
OBn
OBn
F
O
P
F
a
b
74
75
15
+
EtO
I
n
H
N
OEt
Cl
O
S O
NH2
Cl
O
N
OBn
b
d
61 n = 2
62 n = 3
63 n = 4
NH
N
HN
HN
67
O
OBn
O
O
O
O
O
O
OEt
NH
OEt
NH
77
76
75
c
Cl
N
OBn
H
Cl
N
O
F
c
O
S O
F
F
F
O S O
HN
O
O
P
N
d
HN
HN
NH
P
OEt
n
OH
HN
n
OBn
OEt
OH
O
O
71 n = 2
72 n = 3
73 n = 4
68 n = 2
69 n = 3
70 n = 4
O
78
79
Scheme 9. Reagents and conditions: (a) Na2S2O4, 1,4-dioxane, MeOH, buffer, reflux;
(b) ClCOCO2Et, Et3N, THF, rt; (c) C2H5COCl, Et3N, THF, rt; (d) Lindlar catalyst, 1,4-
cyclohexadiene, ethanol, r.t.
Scheme 8. Reagents and conditions: (a) NH3, THF, rt; (b) K2CO3, THF, rt; (c) TMSBr,
CH2Cl2, rt.

3526
A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
100), 774 (2M+, 94), 410 (MNa+, 48), 388 (MH+, 80); HRMS m/z calcd
OH
OH
for C18H30NO6P (MH+) 388.1889, found 388.1880.
HO
3.3. Benzyl-2-(di-tert-butoxyphosphoryloxy)propylcarbamate (21)
OH
H
N
Tetrazole (3 wt % solution in CH3CN, 4.5 mL, 1.91 mmol) and di-tert-
butyl-N,N-diisopropylphosphoramidite (95%, 0.45 mL, 1.43 mmol)
were added to a solution of benzyl-2-hydroxypropylcarbamate
(19) (200 mg, 0.96 mmol) in THF (5 mL) and the mixture was stirred
at room temperature for 16 h. The mixture was cooled to 0 °C and
H2O2 (70% aqueous, 0.4 mL, 24 mmol) was added. After 15 min, the
cooling bath was removed and the mixture was stirred for a further
6 h, and then aqueous Na2SO3 (10%, 50 mL) was added with water
bath cooling. After 25 min, the organic solvents were removed under
reduced pressure and the aqueous residue was extracted with EtOAc
(3 ꢁ 10 mL). The combined extracts were washed with brine, dried,
and evaporated. The residue was purified by silica gel column chro-
matography, eluting with EtOAc–petroleum ether (1:1), to provide
the product 21 (250 mg, 65%) as a colorless oil. 1H NMR (300 MHz,
CDCl3) d 7.24 (m, 5H), 5.76 (br s, 1H), 5.00 (s, 2H), 3.92 (m, 2H),
3.23 (m, 2H), 1.76 (m, 2H), 1.38 (s, 18H); 13C NMR (75 MHz, CDCl3)
d 156.2, 136.3, 128.2, 127.7, 82.5, 82.4, 66.3, 65.5, 41.1, 30.1, 29.51,
29.47; EIMS m/z (rel intensity) 825 (2MNa+, 2), 424 (MNa+, 100);
HRMS m/z calcd for C19H32NO6P (MNa+) 424.1865, found 424.1864.
HN
O
NH
HO
O
P
- O
O
80
Figure 7. Phosphonate reaction intermediate analogues.
thase. The lead compound has ‘drug-like’ features and therefore
has more potential for drug development than the mechanism
probes containing polar ribityl side chains. Although the oxybenz-
indolone moiety has poor affinity to the active site, the enzyme
inhibitory activity could possibly be optimized by improving the
binding affinity of the oxybenzindolone moiety through incorpora-
tion of oxygen- or nitrogen-containing functional groups. Optimi-
zation of the enzyme inhibitory activity through logical structure
manipulation of the lead compound 9 already provided compound
38 with moderately improved inhibitory activity (Ki 38
against M. tuberculosis lumazine synthase.
lM)
3.4. Di-tert-butyl-2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamido)ethyl phosphate (24)
3. Experimental
Method 1. A solution of 20 (1.17 g, 3.02 mmol) in MeOH
(30 mL) with Pd/C (5%, 0.21 g) was hydrogenated at 50 psi for
4 h. The mixture was filtered through Celite, washed with MeOH
(2 ꢁ 10 mL), and the filtrate was evaporated to give 2-amino-
ethyl di-tert-butyl phosphate (22) (604 mg, 79%) as colorless
oil, which was used directly for the next reaction. A solution
of amine 22 (100 mg, 0.39 mmol) and Et3N (0.11 mL, 0.79 mmol)
in THF (2 mL) was added to a solution of compound 15 (106 mg,
0.39 mmol) in THF (3 mL) at 0 °C. After 5 min, the cooling bath
was removed and the reaction mixture was stirred overnight.
The mixture was diluted with water and extracted with CH2Cl2
(3 ꢁ 15 mL). The combined extracts were dried and evaporated
and the residue was purified by silica gel column chromatogra-
phy, eluting with ethyl acetate–petroleum ether (9:1), to provide
compound 24 (135 mg, 71%) as yellow solid: mp 200–203 °C.
Method 2. Tetrazole (3 wt % solution in CH3CN, 1.6 mL,
0.68 mmol) and di-tert-butyl-N,N-diisopropylphosphoramidite
3.1. 2-Oxo-1,2-dihydrobenzo[cd]indole-6-sulfonyl chloride (15)
Chlorosulfonic acid (3.2 mL) was added slowly to compound 14
(1.0 g, 5.9 mmol). The reaction mixture was stirred at 0 °C for 1 h
and at room temperature for 2 h. The mixture was then poured
into ice water (20 mL). The precipitate was washed with water
(2 ꢁ 10 mL) and dried to give compound 15 as yellow solid
(0.76 mg, 44%): mp 143–146 °C. 1H NMR (300 MHz, MeOH-d4) d
10.34 (br s, 1H), 8.68 (d, J = 8.3 Hz, 1H), 8.38 (d, J = 7.9 Hz, 1H),
8.20 (d, J = 7.0 Hz, 1H), 8.12 (dd, J = 7.0 Hz, 8.3 Hz, 1H), 7.22 (d,
J = 7.9 Hz, 1H); 13C NMR (75 MHz, MeOH-d4) d 169.0, 147.3,
135.5, 133.0, 132.5, 129.1, 128.0, 127.6, 126.5, 124.7, 105.2; EIMS
m/z (rel intensity) 267 (M+, 4), 232 (5), 155 (38), 91 (C5H7+, 100);
CIMS m/z (rel intensity) 268 (MH+, 76), 232 (53), 197 (100). Anal.
Calcd for C11H6ClNO3S: C, 49.36; H, 2.26; N, 5.23; S, 11.98. Found:
C, 49.53; H, 2.08; N, 5.47; S, 12.21.
(95%, 0.16 mL, 0.51 mmol) were added to
a solution of 31
3.2. Benzyl-2-(di-tert-butoxyphosphoryloxy)ethylcarbamate (20)
(100 mg, 0.34 mmol) in THF (4 mL) and the mixture was stirred
at room temperature for 16 h. The mixture was cooled to 0 °C
and H2O2 (70% aqueous, 0.3 mL, 1.71 mmol) was added. After
15 min, the cooling bath was removed and the mixture was stir-
red for a further 6 h, and then aqueous Na2SO3 (10%, 5 mL) was
added with water bath cooling. After 25 min, the organic sol-
vents were removed under reduced pressure and the aqueous
residue was extracted with EtOAc (3 ꢁ 10 mL). The combined ex-
tracts were washed with brine, dried, and evaporated. The resi-
due was purified by silica gel column chromatography, eluting
with EtOAc–petroleum ether (9:1), to give 24 (110 mg, 67%) as
a as yellow solid: mp 200–203 °C. 1H NMR (300 MHz, CDCl3) d
9.55 (br s, 1H), 8.68 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 7.5 Hz, 1H),
8.03 (d, J = 7.0 Hz, 1H), 7.79 (m, 1H), 6.96 (d, J = 7.5 Hz, 1H),
6.34 (br s, 1H), 3.99 (m, 2H), 3.12 (m, 2H), 1.38 (s, 18H); 13C
Tetrazole (3 wt % solution in CH3CN, 32 mL, 11.0 mmol) and di-
tert-butyl-N,N-diisopropylphosphoramidite (95%, 2.73 mL, 8.2 mmol)
were added to a solution of benzyl-2-hydroxyethylcarbamate (18)
(1.07 g, 5.48 mmol) in THF (20 mL) and the mixture was stirred at
room temperature for 16 h. The mixture was cooled to 0 °C and
H2O2 (70% aqueous, 1.0 mL, 24 mmol) was added. After 15 min,
the cooling bath was removed and the mixture was stirred for a fur-
ther 6 h, and then aqueous Na2SO3 (10%, 50 mL) was added with
water bath cooling. After 25 min, the organic solvents were removed
under reduced pressure and the aqueous residue was extracted with
EtOAc (3 ꢁ 30 mL). The combined extracts were washed with brine,
dried, and evaporated. The residue was purified by silica gel column
chromatography, eluting with EtOAc–petroleum ether (1:1), to af-
ford the product 20 (1.36 g, 64%) as a colorless oil. 1H NMR
(300 MHz, CDCl3) d 7.37–7.25 (m, 5H), 5.64 (br s, 1H), 5.03 (s, 2H),
3.96 (m, 2H), 3.38 (q, J = 5.1 Hz, 2H), 1.41 (s, 18H); 13C NMR
(75 MHz, CDCl3) d 156.3, 136.5, 128.1, 127.7, 82.2, 82.1, 66.1, 64.1,
37.3, 30.1, 29.9, 29.8, 29.5; EIMS m/z (rel intensity) 796 (2MNa+,
NMR (75 MHz, CDCl3)
d 169.8, 142.2, 132.7, 130.5, 130.0,
128.6, 126.8, 125.3, 124.9, 104.9, 83.4, 83.3, 65.6, 43.4, 29.7; neg-
ative ion EIMS m/z (rel intensity) 483 [(MꢀH+)ꢀ, 78], 464 (20),
276 (50); HRMS m/z calcd for C21H29N2O7PS (MꢀH+)ꢀ
483.1355, found 483.1348.

A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
3527
3.5. Di-tert-butyl-3-(2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamido)propyl phosphate (25)
3.8. N-(2-Hydroxyethyl)-2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamide (31)
A solution of 21 (0.3 g, 0.75 mmol) in MeOH (30 mL) with Pd/C
(5%, 20 mg) was hydrogenated at 50 psi for 4 h. The mixture was
filtered through Celite, washed with MeOH (2 ꢁ 10 mL), and the fil-
trate was evaporated to give 2-aminopropyl di-tert-butyl phos-
phate (23) (140 mg, 70%) as colorless oil, which was used directly
for the next reaction. A solution of amine 23 (100 mg, 0.37 mmol)
and Et3N (0.1 mL, 0.75 mmol) in THF (2 mL) was added to a solu-
tion of compound 15 (100 mg, 0.37 mmol) in THF (3 mL) at 0 °C.
After 5 min, the cooling bath was removed and the reaction mix-
ture was stirred overnight. The mixture was diluted with water
and extracted with CH2Cl2 (3 ꢁ 15 mL). The combined extracts
were dried and evaporated and the residue was purified by silica
gel column chromatography, eluting with ethyl acetate–petroleum
ether (9:1), to give compound 25 (112 mg, 60%) as yellow solid: mp
196–198 °C. 1H NMR (300 MHz, acetone-d6) d 8.98 (d, J = 8.4 Hz,
1H), 8.39 (d, J = 7.5 Hz, 1H), 8.34 (d, J = 7.0 Hz, 1H), 8.18 (m, 1H),
7.35 (d, J = 7.5 Hz, 1H), 4.16 (dd, J = 6.3, 12.8 Hz, 2H), 3.26 (t,
J = 6.9 Hz, 2H), 2.03 (m, 2H), 1.62 (s, 18H); 13C NMR (75 MHz, ace-
tone-d6) d 169.2, 142.4, 132.4, 130.1, 129.4, 128.6, 126.8, 126.5,
124.7, 124.4, 104.2, 81.8, 81.7, 63.8, 63.7, 39.1, 29.9; EIMS m/z
(rel intensity) 521 (MNa+, 92), 499 (MH+, 100), 387 (62); negative
ion EIMS m/z (rel intensity) 995 [(2MꢀH+)ꢀ, 34], 533 (100), 497
[(MꢀH+)ꢀ, 91]; HRMS m/z calcd for C22H31N2O7PS 521.1487
(MNa+), found 521.1492.
Ethanolamine (0.05 mL, 0.84 mmol) was added to solution of
compound 15 (150 mg, 0.56 mmol) in THF (5 mL) and the reaction
mixture was stirred at room temperature for 12 h. THF was re-
moved under vacuum and the residue was purified by silica gel
column chromatography, eluting with EtOAc–petroleum ether
(4:6), to give compound 31 (115 mg, 70%) as yellow solid: mp
192–195 °C. 1H NMR (300 MHz, MeOH-d4) d 8.72 (d, J = 8.4 Hz,
1H), 8.12 (d, J = 7.5 Hz, 1H), 8.10 (d, J = 6.8 Hz, 1H), 7.90 (m, 1H),
7.04 (d, J = 7.5 Hz, 1H), 3.48 (t, J = 6.0 Hz, 1H), 2.95 (d, J = 6.0 Hz,
1H); 13C NMR (125 MHz, MeOH-d4) d 171.1, 144.2, 134.1, 132.3,
132.2, 131.0, 128.1, 128.0, 126.5, 106.4, 61.8, 45.9; EIMS m/z (rel
intensity) 315 (MNa+, 85), 293 (MH+, 42); negative ion EIMS m/z
(rel intensity) 291 [(MꢀH+)ꢀ, 10], 248 (100); HRMS m/z calcd for
C13H12N2O4S (MNa+) 315.0416, found 315.0420.
3.9. N-(2-Hydroxybutyl)-2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamide (32)
4-Amino-1-butanol (28) (0.08 mL, 0.84 mmol) was added to
solution of compound 15 (150 mg, 0.56 mmol) in THF (5 mL)
and the reaction mixture was stirred at room temperature for
12 h. THF was removed under vacuum and the residue was puri-
fied by silica gel column chromatography, eluting with EtOAc–
petroleum ether (4:6), to give compound 32 (120 mg, 67%) as
yellow solid: mp 238–240 °C. 1H NMR (300 MHz, MeOH-d4) d
8.73 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 7.6 Hz, 1H), 8.11 (d,
J = 6.9 Hz, 1H), 7.90 (dd, J = 7.1, 8.4 Hz, 1H), 7.04 (d, J = 7.6 Hz,
1H), 3.39 (t, J = 6.0 Hz, 1H), 2.87 (d, J = 6.6 Hz, 1H), 1.42 (m,
4H); 13C NMR (75 MHz, MeOH-d4) d 171.5, 143.9, 133.8, 131.5,
131.1, 130.7, 128.1, 126.3, 126.2, 106.0, 62.2, 43.7, 30.6, 27.1;
EIMS m/z (rel intensity) 321 (MH+, 83), 283 (34); negative ion
EIMS m/z (rel intensity) 639 [(2MꢀH+)ꢀ, 66], 319 [(MꢀH+)ꢀ,
100], 248 (79); HRMS m/z calcd for C15H16N2O4S (MH+)
321.0909, found 321.0911.
3.6. 2-(2-Oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)ethyl
dihydrogen phosphate (26)
TFA (0.06 mL, 0.8 mmol) was added to a solution of 24 (40 mg,
0.083 mmol) in CH2Cl2 (5 mL) and the solution was allowed to
stand at room temperature for 16 h. The mixture was evaporated,
and the residue was washed with CH2Cl2 (3 ꢁ 10 mL) and dried
to yield 26 (23 mg, 74%) as a light-yellow solid: mp 185–188 °C.
1H NMR (300 MHz, MeOH-d4) d 8.72 (d, J = 8.4 Hz, 1H), 8.14 (d,
J = 7.5 Hz, 1H), 8.10 (d, J = 7.0 Hz, 1H), 7.92 (m, 1H), 7.05 (d,
J = 7.5 Hz, 1H), 3.89 (dd, J = 6.0, 13.0 Hz, 2H), 3.13 (t, J = 5.8 Hz,
2H); 13C NMR (75 MHz, MeOH-d4 and DMSO-d6) d 169.1, 142.7,
132.7, 130.8, 129.9, 128.6, 127.1, 126.5, 125.1, 124.7, 104.9, 64.0,
42.9; EIMS m/z (rel intensity) 395 (MNa+, 25), 373 (MH+, 100),
338 (37), 293 (26), 274 (21); negative ion EIMS m/z (rel intensity)
743 [(2MꢀH+)ꢀ, 100], 371 [(MꢀH+)ꢀ, 51], 236 (10); HRMS m/z
calcd for C13H13N2O7PS (MH+) 373.0259, found 373.0252. Anal.
Calcd for C13H13N2O7PSꢂ1.5H2O: C, 39.10; H, 4.04; N, 7.02; P,
7.76. Found: C, 39.15; H, 3.73; N, 7.02; P, 7.89.
3.10. N-(5-Hydroxypentyl)-2-oxo-1,2-dihydrobenzo[cd]indole-
6-sulfonamide (33)
5-Amino-1-pentanol (29) (0.3 g, 2.9 mmol) was added to a solu-
tion of compound 15 (200 mg, 0.75 mmol) in THF (5 mL) and the
reaction mixture was stirred at room temperature for 24 h under
argon gas. THF was removed under vacuum and yellow solid was
purified using silica gel column chromatography, eluting with
MeOH–EtOAc (8:92) to provide compound 33 (180 mg, 70%) as a
yellow solid: mp 186–189 °C. 1H NMR (300 MHz, MeOH-d4) d
8.71 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 7.5 Hz, 1H), 8.09 (d, J = 6.9 Hz,
1H), 7.89 (t, J = 8.3 Hz, 1H), 7.04 (d, J = 7.6 Hz, 1H), 3.36 (t,
J = 6.4 Hz, 2H), 2.85 (t, J = 6.8 Hz, 2H), 1.35 (m, 4H), 1.22 (m, 2H);
13C NMR (75 MHz, MeOH-d4) d 171.3, 143.9, 133.8, 131.4, 131.1,
130.7, 128.3, 128.0, 126.1, 105.9, 62.6, 43.7, 32.9, 30.3, 23.8; nega-
tive ion ESIMS m/z (rel intensity) 667 ([2MꢀH+]ꢀ, 100), 333
([MꢀH+]ꢀ, 45); HRMS m/z calcd for C16H18N2O4S (MH+) 335.1066,
found 335.1063.
3.7. 3-(2-Oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamido)propyl dihydrogen phosphate (27)
TFA (0.07 mL, 0.8 mmol) was added to a solution of 25 (40 mg,
0.081 mmol) in CH2Cl2 (5 mL) and the solution was allowed to stand
at room temperature for 16 h. The mixture was evaporated, the res-
idue was washed with CH2Cl2 (3 ꢁ 10 mL), and the product was
dried to provide 27 (21 mg, 68%) as a light-yellow solid: mp 192–
195 °C. 1H NMR (300 MHz, MeOH-d4) d 8.65 (d, J = 8.4 Hz, 1H), 8.07
(d, J = 7.6 Hz, 1H), 8.01 (d, J = 7.0 Hz, 1H), 7.84 (m, 1H), 7.00 (d,
J = 7.6 Hz, 1H), 3.91 (dd, J = 6.3, 12.8 Hz, 2H), 2.97 (t, J = 6.8 Hz, 2H),
1.75 (m, 2H); 13C NMR (75 MHz, MeOH-d4) d 171.4, 143.8, 133.8,
131.5, 131.0, 130.1, 128.1, 127.8, 126.1, 106.1, 64.8, 40.4, 31.7; EIMS
m/z(relintensity)387(MH+, 100), 338(3), 311(3); negativeionEIMS
m/z (rel intensity) 771 [(2MꢀH+)ꢀ, 100], 385 [(MꢀH+)ꢀ, 36]; HRMS
m/z calcd for C14H15N2O7PS (MH+) 387.0416, found 387.0419. Anal.
Calcd for C14H15N2O7PSꢂH2O: C, 41.59; H, 4.24; N, 6.93. Found: C,
41.35; H, 4.31; N, 6.99.
3.11. N-(6-Hydroxyhexyl)-2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamide (34)
6-Amino-1-hexanol (30) (315 mg, 2.69 mmol) was added to a
solution of compound 15 (180 mg, 0.672 mmol) in THF (7 mL)
and the mixture was allowed to stir overnight. THF was removed
under vacuum and the residue was purified using silica gel column
chromatography, eluting with EtOAc–hexane (8:2) and then
MeOH–EtOAc (5:95), to result in starting material 15 (72 mg)

3528
A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
and pure compound 34 as a yellow solid (100 mg, 45%): mp 190–
192 °C. 1H NMR (300 MHz, MeOH-d4) d 8.73 (d, J = 8.4 Hz, 1H),
8.12 (d, J = 7.6 Hz, 1H), 8.11 (d, J = 6.9 Hz, 1H), 7.91 (dd, J = 7.2,
8.3 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 3.39 (t, J = 6.5 Hz, 2H), 3.34 (s,
1H), 2.85 (t, J = 6.9 Hz, 2H), 1.33 (m, 4H), 1.13 (m, 4H); 13C NMR
(125 MHz, MeOH-d4) d 171.2, 143.5, 133.8, 131.2, 131.1, 130.0,
128.0, 127.9, 126.0, 105.5, 62.3, 43.7, 33.1, 30.5, 27.0, 26.5; ESIMS
m/z (rel intensity) 371 (MNa+, 51), 349 (MH+, 100); negative ion
ESIMS m/z (rel intensity) 695 ([2MꢀH+]ꢀ, 100), 347 ([MꢀH+]ꢀ,
47); HRMS m/z calcd for C17H20N2O4S (MH+) 349.1222, found
349.1227
3.14. Di-tert-butyl-5-(2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamido)hexyl phosphate (37)
Tetrazole(3 wt %solutioninCH3CN, 2.1 mL, 1.0 mmol)anddi-tert-
butyl-N,N-diisopropyl-phosphoramidite (95%, 0.20 mL, 0.8 mmol)
were added to a solution of 34 (172 mg, 0.5 mmol) in THF (5 mL)
and the reaction mixture was stirred at room temperature under ar-
gon for 16 h. The mixture was cooled to 0 °C and H2O2 (70% aqueous,
1.2 mL) was added. After 15 min, the cooling bath was removed and
the mixture was stirred for a further 6 h, and then aqueous Na2SO3
(10%, 5 mL) was added with water bath cooling. After 25 min, the or-
ganic solvents were evaporated and the aqueous residue was ex-
tracted with EtOAc (3 ꢁ 10 mL). The extracts were washed with
brine, dried with Na2SO4, and evaporated. The resulting yellow res-
idue was purified by silica gel column chromatography, eluting with
EtOAc–hexane (8:1), to result in purecompound 37(95 mg, 18%) as a
yellowoil. 1HNMR (300 MHz, CDCl3) d 9.91 (s, 1H), 8.66 (d, J = 8.4 Hz,
1H), 8.06 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 7.0 Hz, 1H), 7.73 (t, J = 7.3 Hz,
1H), 6.99 (d, J = 7.6 Hz, 1H), 6.90 (s, 1H), 3.84 (q, J = 6.5 Hz, 2H), 2.87
(q, J = 4.2 Hz, 2H), 1.47 (m, 22H), 1.16 (m, 4H); 13C NMR (75 MHz,
CDCl3) d 169.8, 142.9, 142.2, 132.5, 130.3, 129.7, 128.6, 126.6,
125.1, 124.8, 105.1, 82.9, 82.8, 66.9, 66.8, 42.7. 29.6, 29.3, 25.8,
24.8; ESIMS m/z (rel intensity) 541 (M+, 9), 429 (100); negative ion
ESIMS m/z (rel intensity) 539 ([MꢀH+]ꢀ, 3), 483 ([MꢀtBu+]ꢀ, 100);
HRMS m/z calcd for C25H37N2O7PS (MH+) 541.2137, found 541.2136.
3.12. Di-tert-butyl-4-(2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamido)butyl phosphate (35)
Tetrazole(3 wt % solution in CH3CN, 0.4 mL, 0.16 mmol)anddi-tert-
butyl-N,N-diisopropylphosphoramidite (95%, 0.24 mL, 0.12 mmol)
were added to a solution of 32 (0.25 mg, 0.078 mmol) in THF
(3 mL) and the reaction mixture was stirred at room temperature
under argon for 16 h. The mixture was cooled to 0 °C and H2O2
(70% aqueous, 0.1 mL) was added. After 15 min, the cooling bath
was removed and the mixture was stirred for a further 6 h, and then
aqueous Na2SO3 (10%, 2 mL) was added with water bath cooling.
After 25 min, the organic solvents were evaporated and the aqueous
residue was extracted with EtOAc (3 ꢁ 10 mL). The extracts were
washedwith brine, driedwith Na2SO4, and evaporated. Theresulting
residue was purified by silica gel column chromatography, eluting
with 90:10 EtOH–hexane, to yield pure compound 35 (24 mg, 60%)
as a yellow residue. 1H NMR (300 MHz, MeOH-d4) d 8.72 (d,
J = 8.4 Hz, 1H), 8.11 (d, J = 7.6 Hz, 1H), 8.09 (d, J = 7.0 Hz, 1H), 7.90
(dd, J = 7.1, 8.3 Hz, 1H), 7.04 (d, J = 7.6 Hz, 1H), 3.79 (dd, J = 5.9,
12.0 Hz, 2H), 2.89 (t, J = 6.3 Hz, 2H), 1.55–1.40 (22H); 13C NMR
(75 MHz, MeOH-d4) d 171.4, 143.9, 133.8, 131.5, 131.1, 130.6,
128.3, 128.0, 126.2, 106.0, 84.1, 84.0, 67.7, 43.2, 30.1, 30.08, 28.3,
26.9; EIMS m/z (rel intensity) 1024 (2MH+, 45), 513 (MH+, 100),
457 (8); negative ion EIMS m/z (rel intensity) 556 (MꢀHCOOꢀ, 38],
511 [(MꢀH+)ꢀ, 100]; HRMS m/z calcd for C23H33N2O7PS 513.1824
(MH+), found 513.1822.
3.15. 4-(2-Oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)-
butyl dihydrogen phosphate (38)
TFA (0.03 mL, 0.35 mmol) was added to a solution of 35 (18 mg,
0.035 mmol) in CH2Cl2 (2 mL) and the solution was stirred at room
temperature for 16 h. The mixture was evaporated, and the residue
was washed with CH2Cl2 (3 ꢁ 10 mL) and dried under vacuum to
yield compound 38 (35 mg, 69%) as a light-yellow solid: mp
194–196 °C. 1H NMR (300 MHz, MeOH-d4) d 8.72 (d, J = 8.1 Hz,
1H), 8.12 (d, J = 6.3 Hz, 1H), 8.09 (d, J = 5.4 Hz, 1H), 7.90 (dd,
J = 7.2, 8.4 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 3.83 (dd, J = 6.0,
12.3 Hz, 2H), 2.88 (t, J = 6.6 Hz, 2H), 1.56 (t, J = 6.3 Hz, 2H), 1.52
(t, J = 5.1 Hz, 2H); 13C NMR (75 MHz, MeOH-d4) d 171.5, 143.9,
133.8, 131.5, 130.6, 128.3, 128.0, 126.2, 106.0, 67.0, 43.3, 28.5,
26.9; EIMS m/z (rel intensity) 801 (2MH+, 55), 401 (MH+, 100),
303 (19); negative ion EIMS m/z (rel intensity) 799 [(2MꢀH+)ꢀ,
100], 399 [(MꢀH+)ꢀ, 21]; HRMS m/z calcd for C15H17N2O7PS
3.13. Di-tert-butyl-5-(2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamido)pentyl phosphate (36)
(MH+)
401.0572,
found
401.0580.
Anal.
Calcd
for
Tetrazole(3 wt %solutioninCH3CN,3.0 mL, 1.3 mmol)anddi-tert-
butyl-N,N-diisopropylphosphoramidite (95%, 0.26 mL, 0.75 mmol)
were added to a solution of 33 (180 mg, 0.54 mmol) in THF (5 mL)
and the reaction mixture was stirred at room temperature under ar-
gon for 16 h. The mixture was cooled to 0 °C and H2O2 (70% aqueous,
1.5 mL) was added. After 15 min, the cooling bath was removed and
the mixture was stirred for a further 6 h, and then aqueous Na2SO3
(10%, 5 mL) was added with water bath cooling. After 25 min, the or-
ganic solvents were evaporated and the aqueous residue was ex-
tracted with EtOAc (3 ꢁ 10 mL). The extracts were washed with
brine, dried with Na2SO4, and evaporated. The resulting yellow res-
idue was purified on a silica gel column made with EtOAc–hexane
(7:3), eluting with EtOAc–hexane (9:1), to result in pure compound
36 (125 mg, 44%). 1H NMR (300 MHz, acetone-d6) d 10.22 (s, 1H),
8.66 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 7.5 Hz, 1H), 7.98 (d, J = 6.9 Hz,
1H), 7.82 (t, J = 6.9 Hz, 1H), 7.01 (d, J = 7.6 Hz, 1H), 6.78 (t,
J = 4.8 Hz, 1H), 3.73 (dd, J = 5.1, 6.3 Hz, 2H), 2.82 (q, J = 5.7 Hz, 2H),
1.36 (m, 22H), 1.22 (m, 2H); 13C NMR (75 MHz, acetone-d6) d
206.1, 169.1, 143.2, 133.0, 130.7, 129.7, 127.6, 127.3, 125.51,
125.0, 104.858, 82.0, 66.8, 66.7, 43.1. 29.6, 22.9; ESIMSm/z(relinten-
sity) 549 (MNa+, 100); negative ion ESIMS m/z (rel intensity) 531
(92), 525 ([MꢀH+]ꢀ, 100); HRMS m/z calcd for C24H35N2O7PS
(MNa+) 549.1800, found 549.1802.
C15H17N2O7PSꢂ1H2O: C, 43.06; H, 4.58; N, 6.70. Found: C, 43.35;
H, 4.25; N, 6.56.
3.16. 5-(2-Oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)-
pentyl dihydrogen phosphate (39)
TFA (0.1 mL, 1.27 mmol) was added to a solution of 36 (120 mg,
0.23 mmol) in CH2Cl2 (3 mL) and the solution was allowed to stir at
room temperature for 16 h overnight. The mixture was evaporated,
and the residue was washed with CH2Cl2 (3 ꢁ 10 mL) and dried to
provide compound 39 (65 mg, 62%): mp 209–212 °C. 1H NMR
(300 MHz, MeOH-d4) d 8.71 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 7.5 Hz,
1H), 8.09 (d, J = 6.9 Hz, 1H), 7.91 (t, J = 7.3 Hz, 1H), 7.04 (d,
J = 7.6 Hz, 1H), 3.73 (q, J = 6.6 Hz, 2H), 2.82 (t, J = 6.6 Hz, 2H), 1.40
(m, 6H); 13C NMR (75 MHz, MeOH-d4) d 171.5, 143.9, 133.8,
131.5, 131.1, 130.6, 128.3, 128.0, 126.2, 106.0, 67.4, 43.6, 30.9,
30.8, 30.1, 23.6; ESIMS m/z (rel intensity) 829, ([2M+H]+, 7), 415
(MH+, 100), 317 (10); negative ion ESIMS m/z (rel intensity) 827
([2MꢀH+]ꢀ, 100), 413 ([MꢀH+]ꢀ, 19); HRMS m/z calcd for
C16H19N2O7PS (MH+) 415.0729, found 415.0725. Anal. Calcd for
C16H19N2O7PS: C, 46.38; H, 4.62; N, 6.67; S, 7.74. Found: C,
45.99; H, 4.67; N, 6.63; S, 7.78.

A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
3529
3.17. 5-(2-Oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamido)hexyl dihydrogen phosphate (40)
trated in vacuo and the residue taken up in water (50 mL). After
extraction with diethyl ether (3 ꢁ 30 mL), the organic phase was
dried (Na2SO4) and evaporated, and the residue purified by flash
silica gel column chromatography using CH2C12–EtOAc (1:9) as
eluant to afford 48 (0.9 g, 85%) as white solid: mp 52–54 °C. 1H
NMR (300 MHz, CDCl3) d 7.75 (m, 4H), 4.13–4.02 (m, 4H), 3.64 (t,
J = 6.7 Hz, 2H), 1.91–1.72 (m, 4H), 1.63–1.57 (m, 2H), 1.34 (t,
J = 7.0 Hz, 6H); 13C NMR (75 MHz, CDCl3) d 169.4, 135.2, 133.0,
123.9, 62.94, 62.86, 37.9, 30.1, 29.9, 26.1, 24.2, 20.7, 20.6, 16.8,
16.7; EIMS m/z (rel intensity) 340 (MH+, 2), 362 (MNa+, 100); HRMS
m/z calcd for C16H22NO5P (MNa+) 362.1133, found 362.1139.
TFA (0.12 mL, 1.7 mmol) was added to a solution of 37 (90 mg,
0.17 mmol) in CH2Cl2 (5 mL) and the solution was allowed to stir at
room temperature for 16 h overnight. The mixture was evaporated,
and the residue was washed with CH2Cl2 (3 ꢁ 10 mL) and dried to
give compound 40 (22 mg, 31%): mp 224–228 °C. 1H NMR
(300 MHz, MeOH-d4) d 8.73 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 7.6 Hz,
1H), 8.11 (d, J = 6.9 Hz, 1H), 7.91 (dd, J = 7.2, 8.3 Hz, 1H), 7.05 (d,
J = 7.6 Hz, 1H), 3.83 (q, J = 6.6 Hz, 2H), 2.82 (t, J = 6.9 Hz, 2H), 1.45
(m, 2H), 1.35 (m, 2H), 1.18 (m, 4H); 13C NMR (75 MHz, MeOH-d4)
d 171.5, 143.9, 133.8, 131.5, 131.2, 130.8, 128.3, 128.1, 126.3,
106.0, 67.6, 67.5, 43.7, 31.3, 31.2, 30.4, 27.1, 26.0; ESIMS m/z (rel
intensity) 451, (MNa+, 10), 429 (MH+, 100); HRMS m/z calcd for
C17H21N2O7PS (MH+) 429.0885, found 429.0890. Anal. Calcd for
C17H21N2O7PS: C, 47.66; H, 4.94; N, 6.54; S, 7.45. Found: C,
47.31; H, 4.70; N, 6.47; S, 7.45.
3.21. Diethyl 4-(1,3-dioxoisoindolin-2-yl)pentylphosphonate
(49)
A DMF (5 mL) solution of iodopentylphosphonate 47 (1.0 g,
3.0 mmol) and potassium phthalimide (1.11 g, 6.0 mmol) was stir-
red at 100 °C for 8 h. The mixture was cooled to 25 °C and the pre-
cipitated material filtered off. The filtrate was concentrated in
vacuo and the residue taken up in water (50 mL). After extraction
with diethyl ether (3 ꢁ 30 mL), the organic phase was dried
(Na2SO4), evaporated and the residue purified by flash silica gel
column chromatography using silica gel and CH2C12–EtOAc (1:9)
as eluant to afford 49 (0.91 g, 86%) as yellowish semisolid. 1H
NMR (300 MHz, MeOH-d4) d 7.76 (m, 4H), 4.09–4.03 (m, 4H),
3.62 (t, J = 7.0 Hz, 2H), 1.85–1.60 (m, 6H), 1.42 (m, 2H), 1.30 (t,
J = 7.0 Hz, 6H); 13C NMR (75 MHz, MeOH-d4) d 169.6, 135.2,
133.2, 124.0, 63.04, 62.95, 38.4, 28.9, 28.6, 28.4, 26.5, 24.6, 23.0,
22.9, 16.8, 16.7; EIMS m/z (rel intensity) 354 (MH+, 87), 376
(MNa+, 100); HRMS m/z calcd for C17H24NO5P (MNa+) 376.1290,
found 376.1296.
3.18. Diethyl 4-iodobutylphosphonate (46)
Sodium hydride (60%) in mineral oil (0.75 g, 18.8 mmol) was
suspended in anhydrous THF (70 mL) and the reaction mixture
cooled to ꢀ20 °C.
A solution of diethyl phosphate (2.0 g,
14.5 mmol) in THF (10 mL) was introduced under argon. After stir-
ring at ꢀ20 °C for 1 h, 1,4-diiodobutane (43) (3.8 mL, 29.0 mol) was
added and the mixture was stirred at ꢀ10 °C for 18 h. The mixture
was then concentrated under vacuum, taken up in EtOAc (30 mL)
and water (30 mL), and the pH adjusted to 8 with concentrated
HCl. The EtOAc was washed with brine, dried over Na2SO4, and
evaporated to a colorless oil, which was purified by silica gel flash
column chromatography with CH2C12–EtOAc (3:7) as eluant to af-
ford 46 (2.6 g, 57%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d
4.09–4.01 (m, 4H), 3.20 (t, J = 6.6 Hz, 2H), 1.86 (m, 2H), 1.70 (m,
4H), 1.27 (t, J = 7.0 Hz, 6H); 13C NMR (75 MHz, CDCl3) d 61.5,
61.41, 33.8, 33.6, 25.4, 23.5, 23.4, 16.4, 16.4, 5.5; EIMS m/z (rel
intensity) 321 (MH+, 15), 343 (MNa+, 100); HRMS m/z calcd for
C8H18IO3P (MNa+) 342.9936, found 342.9944.
3.22. Diethyl 4-(2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamido)butylphosphonate (52)
Compound 48 (0.4 g, 1.3 mmol) was dissolved in absolute etha-
nol (8 mL) and 98% hydrazine hydrate (0.18 mL, 3.9 mmol) was
added. The reaction mixture was stirred at reflux for 3 h. The eth-
anol was removed in vacuo and the residue purified by flash col-
umn chromatography with silica gel, eluting with 5% MeOH in
ethyl acetate, to afford compound 50 (0.2 g, 81%) as a colorless
oil. Amine 50 (117 mg, 0.56 mmol) and derivative 15 (100 mg,
0.37 mmol) were dissolved in THF (5 mL). The reaction mixture
was stirred at room temperature for 12 h. The THF was removed
in vacuo and the residue purified by flash silica gel column chro-
matography, eluting with 1% MeOH in ethyl acetate, to afford com-
pound 52 (0.1 g, 61%) as yellow oil. 1H NMR (300 MHz, MeOH-d4) d
8.68 (d, J = 8.3 Hz, 1H), 8.08 (d, J = 7.6 Hz, 1H), 8.04 (d, J = 7.2 Hz,
1H), 7.93 (dd, J = 7.2, 8.3 Hz, 1H), 7.02 (d, J = 7.5 Hz, 1H), 3.98 (m,
4H), 2.86 (m, 2H), 1.61 (m, 2H), 1.46 (m, 4H), 1.24 (t, J = 7.1 Hz,
6H); 13C NMR (75 MHz, MeOH-d4) d 171.3, 143.8, 133.9, 133.7,
131.5, 131.0, 130.5, 128.2, 127.9, 126.1, 106.0, 63.1, 63.0, 43.1,
31.1, 30.9, 26.0, 24.1, 20.4, 20.3, 16.73, 16.65; EIMS m/z (rel inten-
sity) 441 (MH+, 100), 463 (MNa+, 69); negative ion EIMS m/z (rel
intensity) 439 [(MꢀH+)ꢀ, 100]; HRMS m/z calcd for C19H25N2O6PS
(MH+) 441.1249, found 441.1251.
3.19. Diethyl 4-iodopentylphosphonate (47)
Sodium hydride (60%) in mineral oil (0.75 g, 18.8 mmol) was
suspended in anhydrous THF (70 mL) and the reaction mixture
cooled to ꢀ20 °C.
A solution of diethyl phosphate (2.0 g,
14.5 mmol) in THF (10 mL) was introduced under argon. After stir-
ring at ꢀ20 °C for 1 h, 1,5-diiodopantane (44) (2.6 mL, 17.4 mol)
was added and the mixture was stirred at ꢀ10 °C for 18 h. The mix-
ture was then concentrated in vacuo, taken up in EtOAc (30 mL)
and water (30 mL), and the pH adjusted to 8 with concentrated
HCl. The EtOAc was washed with brine, dried over Na2SO4, and
evaporated to a colorless oil, which was purified by flash silica
gel column chromatography using CH2C12 as eluant to yield 47
(2.5 g, 52%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 3.78–
3.70 (m, 4H), 2.84 (t, J = 6.9 Hz, 2H), 1.53–1.20 (m, 6H), 1.16 (m,
2H), 0.96 (t, J = 7.0 Hz, 6H); 13C NMR (75 MHz, CDCl3) d 60.8,
60.7, 32.3, 30.8, 30.5, 25.8, 23.9, 20.9, 20.8, 15.9, 15.86, 5.9; EIMS
m/z (rel intensity) 335 (MH+, 100), 207 (14); HRMS m/z calcd for
C9H20IO3P (MH+) 335.0273, found 335.0283.
3.23. Diethyl 4-(2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamido)pentylphosphonate (53)
3.20. Diethyl 4-(1,3-dioxoisoindolin-2-yl)butylphosphonate
(48)
Compound 49 (0.25 g, 0.7 mmol) was dissolved in absolute eth-
anol (5 mL) and 98% hydrazine hydrate (0.1 mL, 2.1 mmol) was
added. The reaction mixture was stirred at reflux for 3 h. The eth-
anol was removed in vacuo and the residue purified by silica gel
flash column chromatography, eluting with 5% MeOH in ethyl ace-
tate, to afford compound 51 (0.13 g, 82%) as a colorless oil. Amine
A DMF solution (5 mL) of iodobutylphosphonate 46 (1.0 g,
3.13 mmol) and of potassium phthalimide (1.16 g, 6.26 mmol)
was stirred at 100 °C for 8 h. The mixture was cooled to 25 °C
and the precipitated material filtered off. The filtrate was concen-

3530
A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
51 (125 mg, 0.56 mmol) and derivative 15 (100 mg, 0.37 mmol)
were dissolved in THF (5 mL). The reaction mixture was stirred
at room temperature for 12 h. The THF was removed in vacuo
and the residue purified by silica gel flash column chromatography
with silica gel, eluting with 1% MeOH in ethyl acetate, to afford
compound 53 (110 mg, 67%) as a yellow oil. 1H NMR (300 MHz,
MeOH-d4) d 8.67 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 7.6 Hz, 1H), 8.04
(d, J = 7.0 Hz, 1H), 7.86 (dd, J = 7.0, 8.4 Hz, 1H), 7.01 (d, J = 7.6 Hz,
1H), 4.01 (m, 4H), 2.84 (t, J = 6.5 Hz, 2H), 1.49 (m, 2H), 1.46 (m,
6H), 1.25 (t, J = 7.1 Hz, 6H); 13C NMR (75 MHz, MeOH-d4) d 171.3,
143.8, 133.9, 133.7, 131.4, 131.1, 130.6, 128.2, 127.9, 126.5,
126.1, 106.0, 63.1, 63.0, 43.4, 29.8, 28.3, 28.1, 26.3, 24.5, 22.7,
22.6, 16.74; EIMS m/z (rel intensity) 455 (MH+, 100), 477 (MNa+,
44); negative ion EIMS m/z (rel intensity) 453 [(MꢀH+)ꢀ, 100];
HRMS m/z calcd for C20H27N2O6PS (MH+) 455.1406, found
455.1411.
aqueous layer was extracted with ether (20 mL ꢁ 2). The com-
bined ether extracts were washed with brine, dried over Na2SO4,
and concentrated under vacuum. The residual oil was purified by
silica gel column chromatography using CH2Cl2-30% EtOAc as
eluent to give 61 (0.35 g, 19%) as
(300 MHz, CDC13)
a
yellow oil: 1H NMR
4.23 (dt, J = 7.2, 14.9 Hz, 4H), 3.18 (t,
d
J = 6.6 Hz, 2H), 2.09 (m, 4H), 1.34 (t, J = 6.9 Hz, 6H); 13C NMR
(75 MHz, CDCl3) d 121.5 (dt), 64.4, 64.3, 34.6 (m), 24.8, 24.8,
16.3, 16.2, 4.9; EIMS m/z (rel intensity) 357 (MH+, 100), 379
(MNa+, 78); HRMS m/z calcd for C8H16F2IO3P (MH+) 356.9928,
found 356.9927.
3.27. Diethyl 1,1-difluoro-5-iodopentylphosphonate (62)
A solution of LDA (2.5 mL, 4.97 mmol, 2M solution) in dry THF
(10 mL) was added at ꢀ78 °C to a solution of diethyl (difluorome-
thyl)phosphonate (57) (0.85 g, 4.52 mmol) in HMPA (2 mL). After
stirring for 1 h at ꢀ78 °C, a solution of diiodobutane (59) (1.3 mL,
9.94 mmol) in THF (10 mL) was added and the mixture was al-
lowed to stir for 6 h at ꢀ78 °C. The reaction mixture was then
poured into ether (50 mL)-20% H3PO4 (10 mL) and the aqueous
layer was extracted with ether (20 mL ꢁ 2). The combined ether
extracts were washed with brine, dried over Na2SO4, and concen-
trated under vacuum. The residual oil was purified by silica gel col-
umn chromatography using CH2Cl2-30% EtOAc as eluent to give 62
(0.28 g, 17%) as a yellow oil: 1H NMR (300 MHz, CDC13) d 4.22 (dt,
J = 7.2, 14.9 Hz, 4H), 3.13 (t, J = 6.8 Hz, 2H), 2.03 (m, 2H), 1.80 (m,
2H), 1.66 (m, 2H), 1.34 (t, J = 7.0 Hz, 6H); 13C NMR (75 MHz, CDCl3)
d 112.3 (dt), 63.8, 63.7, 32.2 (m), 21.3, 21.2, 15.8, 15.7, 5.2; EIMS
m/z (rel intensity) 371 (MH+, 100), 343 (29); HRMS m/z calcd for
C9H18F2IO3P (MH+) 371.0085, found 371.0082.
3.24. 4-(2-Oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)-
butylphosphonic acid (54)
Bromotrimethylsilane (0.6 mL, 0.45 mmol) was added to a solu-
tion of compound 52 (50 mg, 0.12 mmol) in CH2Cl2 (4 mL). After
stirring for 36 h at 25 °C, the volatiles were removed in vacuo,
and the residue was purified by passing it through lipophilic
Sephadex (LH20), using CH2Cl2–MeOH (8:2) as eluant, to produce
54 (28 mg, 64%) as a yellowish solid: mp 259–261 °C (dec). 1H
NMR (300 MHz, DMSO-d6) d 11.13 (s, 1H), 8.65 (d, J = 8.1 Hz, 1H),
8.09 (d, J = 6.9 Hz, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.93 (dd, J = 7.2,
8.1 Hz, 1H), 7.78 (t, J = 7.2 Hz, 1H), 7.04 (d, J = 7.5 Hz, 1H), 2.70
(m, 2H), 1.39 (m, 6H); 13C NMR (125 MHz, MeOH-d4 and DMSO-
d6) d 169.4, 135.2, 133.0, 123.9, 62.94, 62.86, 37.9, 30.1, 29.9,
26.1, 24.2, 20.7, 20.6, 16.8, 16.7; EIMS m/z (rel intensity) 791
(2MNa+, 100), 384 (MNa+, 27), 179 (38); negative ion EIMS m/z
(rel intensity) 767 [(2MꢀH+)ꢀ, 100], 383 [(MꢀH+)ꢀ, 75]; HRMS
m/z calcd for C15H17N2O6PS (MNa+) 407.0443, found 407.0441.
Anal. Calcd for C15H17N2O6PSꢂCH2Cl2: C, 40.95; H, 4.08; N, 5.97.
Found: C, 41.18; H, 4.16, N, 6.30.
3.28. Diethyl 1,1-difluoro-5-iodohexylphosphonate (63)
A solution of LDA (2.5 mL, 4.97 mmol, 2M solution) in dry THF
(10 mL) was added at ꢀ78 °C to a solution of diethyl (difluorome-
thyl)phosphonate (57) (0.85 g, 4.52 mmol) in HMPA (2 mL). After
stirring for 1 h at ꢀ78 °C,
a solution of diiodopentane (60)
3.25. 4-(2-Oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)-
pentylphosphonic acid (55)
(1.3 mL, 9.94 mmol) in THF (10 mL) was added and the mixture
was allowed to stir for 6 h at ꢀ78 °C. The reaction mixture was
then poured into ether (50 mL)-20% H3PO4 (10 mL) and the aque-
ous layer was extracted with ether (20 mL ꢁ 2). The combined
ether extracts were washed with brine, dried over Na2SO4, and
concentrated under vacuum. The residual oil was purified by silica
gel column chromatography using CH2Cl2-30% EtOAc as eluent to
give 63 (0.4 g, 23%) as a yellow oil: 1H NMR (300 MHz, CDC13) d
4.13 (dt, J = 7.1, 15.0 Hz, 4H), 3.06 (t, J = 6.9 Hz, 2H), 1.90 (m, 2H),
1.7.2 (m, 2H), 1.49 (m, 2H), 1.34 (m, 2H), 1.24 (t, J = 7.0 Hz, 6H);
13C NMR (75 MHz, CDCl3) d 119.0 (dt), 64.1, 64.0, 33.5 (m), 32.8,
29.8, 19.4, 19.3, 16.2, 16.1, 6.2; EIMS m/z (rel intensity) 385
(MH+, 55), 329(MH+ꢀC4H8, 100); HRMS m/z calcd for C10H20F2IO3P
(MH+) 385.0241, found 385.0243.
Bromotrimethylsilane (0.6 mL, 0.44 mmol) was added to the
solution of compound 53 (50 mg, 0.11 mmol) in CH2Cl2 (4 mL).
After stirring for 36 h at 25 °C, the volatiles were removed in vacuo,
and the residue was purified by lipophilic Sephadex column
(LH20), using CH2Cl2–MeOH (8:2) as eluant, to result in 55
(34 mg, 61%) as a yellowish solid: mp 294–296 °C (dec). 1H NMR
(500 MHz, DMSO-d6) d 11.13 (s, 1H), 8.66 (d, J = 8.3 Hz, 1H), 8.09
(d, J = 6.9 Hz, 1H), 8.01 (d, J = 7.5 Hz, 1H), 7.94 (dd, J = 7.2, 8.1 Hz,
1H), 7.76 (t, J = 7.2 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 2.72 (m, 2H),
1.30 (m, 6H), 1.18 (m, 2H); 13C NMR (125 MHz, DMSO-d6) d
169.0, 142.6, 132.5, 130.4, 129.8, 128.9, 127.0, 126.2, 125.0,
124.5, 104.9, 42.2, 28.7, 27.9, 27.2, 27.0, 26.9, 22.4; negative ion
EIMS m/z (rel intensity) 795 [(2MꢀH+)ꢀ, 100], 398 [(MꢀH+)ꢀ, 55].
Anal. Calcd for C16H19N2O6PSꢂH2O: C, 46.15; H, 5.08; N, 6.73.
Found: C, 46.22; H, 4.93, N, 6.8.
3.29. 2-Oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide (67)
Compound 15 (300 mg, 1.12 mmol) was dissolved in dry THF
(10 mL). Ammonia gas was dissolved in the reaction mixture at
ꢀ78 °C for 20 min. The reaction mixture was stirred at room tem-
perature for 6 h. The yellow precipitate was filtered and dried to
give compound 67 (0.22 g, 79%) as yellow solid: mp 272–275 °C
(dec). 1H NMR (300 MHz, DMSO-d6) d 8.62 (d, J = 8.3 Hz, 1H),
8.09 (d, J = 7.0 Hz, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.92 (dd, J = 7.3 Hz,
8.0 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H); 13C NMR (75 MHz, DMSO-d6)
d 169.4, 142.2, 132.8, 130.7, 130.09, 130.08, 127.1, 126.3, 125.2,
124.5, 105.1; EIMS m/z (rel intensity) 531 [(2M++Clꢀ), 60], 496
3.26. Diethyl 1,1-difluoro-4-iodobutylphosphonate (61)
A solution of LDA (2.9 mL, 5.85 mmol, 2M solution) in dry
THF (10 mL) was added at ꢀ78 °C to a solution of diethyl (diflu-
oromethyl)phosphonate (57) (1.0 g, 5.32 mmol) in HMPA (2 mL).
After stirring for 1 h at ꢀ78 °C, a solution of diiodopropane (58)
(1.35 mL, 11.69 mmol) in THF (10 mL) was added and the mix-
ture was allowed to stir for 6 h at ꢀ78 °C. The reaction mixture
was then poured into ether (50 mL)-20% H3PO4 (10 mL) and the
(2M+ 100), 283 [(M++Clꢀ), 33], 248 (M+, 85). Anal. Calcd for
,

A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
3531
C11H8N2O3S: C, 53.22; H, 3.25; N, 11.28. Found: C, 53.24; H, 3.33, N,
11.42.
125.3, 120.4 (dt), 104.3, 64.8, 64.7, 40.5, 34.3 (m), 27.1, 21.2,
21.1, 16.7, 16.6; EIMS m/z (rel intensity) 505 (MH+, 100), 447
(45); HRMS m/z calcd for C21H27F2N2O6PS (MH+) 505.1374, found
505.1375
3.30. Diethyl 1,1-difluoro-4-(2-oxo-1,2-dihydrobenzo[cd
]indole-6-sulfonamido)butyl-phosphonate (68)
3.33. 1,1-Difluoro-4-(2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamido)butylphosphonic acid (71)
K2CO3 (83 mg, 0.6 mmol) was added to the solution of iodo
derivative 61 (79 mg, 0.22 mmol) and amine derivative 67
(50 mg, 0.20 mmol) in DMF (3 mL). The reaction mixture was stir-
red at room temperature for 12 h. Water (25 mL) was added to the
reaction mixture and the mixture was extracted with diethyl ether
(20 mL ꢁ 3). The combined organic extracts were washed with
brine, dried (Na2SO4), and evaporated. The residue was purified
by flash silica gel column chromatography, eluting with EtOAc–
petroleum ether (7:3), to afford compound 68 (60 mg, 63%) as a
yellow oil. 1H NMR (300 MHz, acetone-d6) d 8.73 (d, J = 8.4 Hz,
1H), 8.18 (d, J = 7.5 Hz, 1H), 8.10 (d, J = 7.0 Hz, 1H), 7.94 (t,
J = 8.1 Hz, 1H), 7.28 (d, J = 7.5 Hz, 1H), 6.80 (s, 2H), 4.24–4.14 (m,
4H), 4.07 (t, J = 6.7 Hz, 2H), 2.24–2.09 (m, 4H), 1.27 (t, J = 7.0 Hz,
6H); 13C NMR (75 MHz, acetone-d6) d 168.2, 144.1, 134.0, 131.5,
131.0, 130.8, 127.3, 126.5, 125.6, 120.6, 104.4, 64.9, 64.8, 40.1,
32.0 (m), 21.2, 16.6, 16.5; EIMS m/z (rel intensity) 499 (MNa+,
100), 477 (MH+, 23), 444 (29); HRMS m/z calcd for C19H23F2N2O6PS
(MNa+) 499.0880, found 499.0889.
Bromotrimethylsilane (0.6 mL, 0.42 mmol) was added to the
solution of compound 68 (50 mg, 0.10 mmol) in CH2Cl2 (4 mL).
After stirring for 36 h at 25 °C, the volatiles were removed in vacuo,
and the residue was purified by passing it through lipophilic
Sephadex (LH20) using CH2Cl2–MeOH (8:2) as eluant to give 71
(32 mg, 73%) as a yellowish solid: mp 127–130 °C. 1H NMR
(500 MHz, MeOH-d4) d 8.66 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 7.5 Hz,
1H), 8.00 (d, J = 6.6 Hz, 1H), 7.84 (t, J = 7.5 Hz, 1H), 7.11 (d,
J = 7.5 Hz, 1H), 3.98 (t, J = 6.6 Hz, 2H), 2.15 (m, 2H), 2.07 (m, 2H);
13C NMR (125 MHz, MeOH-d4) d 169.7, 144.0, 134.3, 131.7, 131.3,
127.2, 126.6, 126.2, 125.7, 124.9, 122.2 (dt), 105.3, 40.8, 32.4 (m),
21.6; EIMS m/z (rel intensity) 841 (2MH+, 63), 421 (MH+, 100); neg-
ative ion ESIMS m/z (rel intensity) 839 ([2MꢀH+]ꢀ, 100), 419
([MꢀH+]ꢀ, 33); HRMS m/z calcd for C15H15F2N2O6PS (MH+)
421.0435, found 421.0431. Anal. Calcd for C15H15F2N2O6PSꢂH2O:
C, 41.10; H, 3.91; N, 6.39; P, 7.07. Found: C, 41.32; H, 4.09; N,
6.58; P, 6.97.
3.31. Diethyl 1,1-difluoro-4-(2-oxo-1,2-dihydrobenzo[cd]-
indole-6-sulfonamido)pentyl-phosphonate (69)
3.34. 1,1-Difluoro-4-(2-oxo-1,2-dihydrobenzo[cd]indole-6-
sulfonamido)pentylphosphonic acid (72)
K2CO3 (83 mg, 0.6 mmol) was added to a solution of iodo deriv-
ative 62 (82 mg, 0.22 mmol) and amine derivative 67 (50 mg,
0.20 mmol) in DMF (3 mL). The reaction mixture was stirred at
room temperature for 12 h. Water (25 mL) was added to the reac-
tion mixture and the mixture was extracted with diethyl ether
(20 mL ꢁ 3). The combined organic extracts were washed with
brine, dried (Na2SO4), and evaporated. The residue was purified
by flash silica gel column chromatography, eluting with EtOAc–
petroleum ether (7:3), to afford compound 69 (65 mg, 66%) as a
yellow oil. 1H NMR (300 MHz, acetone-d6) d 8.87 (d, J = 8.4 Hz,
1H), 8.31 (d, J = 7.6 Hz, 1H), 8.23 (d, J = 7.0 Hz, 1H), 8.07 (t,
J = 8.1 Hz, 1H), 7.38 (d, J = 7.3 Hz, 1H), 6.94 (s, 2H), 4.38–4.28 (m,
4H), 4.15 (t, J = 6.8 Hz, 2H), 2.29–2.18 (m, 4H), 1.83 (m, 2H), 1.43
(t, J = 7.0 Hz, 6H); 13C NMR (75 MHz, acetone-d6) d 168.2, 144.2,
133.8, 131.5, 130.9, 130.7, 127.3, 126.4, 125.5, 125.4, 120.6 (dt),
104.4, 64.8, 64.7, 40.3, 34.2 (m), 19.02, 18.91, 16.7, 16.6; EIMS
m/z (rel intensity) 491 (MH+, 100), 471 (10); negative ion EIMS
m/z (rel intensity) 489 [(MꢀH+)ꢀ, 100], 457 (34); HRMS m/z calcd
for C20H25F2N2O6PS (MH+) 491.1217, found 491.1216.
Bromotrimethylsilane (0.6 mL, 0.42 mmol) was added to the
solution of compound 69 (50 mg, 0.10 mmol) in CH2Cl2 (4 mL).
After stirring for 36 h at 25 °C, the volatiles were removed in vacuo,
and the residue was purified by passing it through lipophilic
Sephadex (LH20) using CH2Cl2–MeOH (8:2) as eluant to give 72
(27 mg, 61%) as a yellowish solid: mp 170–173 °C. 1H NMR
(500 MHz, MeOH-d4) d 8.70 (d, J = 8.1 Hz, 1H), 8.17 (d, J = 7.5 Hz,
1H), 8.07 (d, J = 6.6 Hz, 1H), 7.87 (t, J = 7.5 Hz, 1H), 7.14 (d,
J = 7.5 Hz, 1H), 3.97 (t, J = 6.6 Hz, 2H), 2.12 (m, 2H), 1.86 (m, 2H),
1.68 (m, 2H); 13C NMR (125 MHz, MeOH-d4) d 169.7, 144.3,
134.4, 131.8, 131.3, 127.4, 126.8, 126.2, 121.0 (dt), 105.4, 40.9,
34.5 (m), 29.4, 19.6; EIMS m/z (rel intensity) 869 (2MH+, 100),
435 (MH+, 74); negative ion ESIMS m/z (rel intensity) 867
([2MꢀH+]ꢀ, 100), 433 ([MꢀH+]ꢀ, 25); HRMS m/z calcd for
C16H17F2N2O6PS (MH+) 435.0591, found 435.0593. Anal. Calcd for
C16H17F2N2O6PS: C, 44.24; H, 3.94; N, 6.45; P, 7.13. Found: C,
44.39; H, 4.25; N, 6.62; P, 6.92.
3.35. 1,1-Difluoro-4-(2-oxo-1,2-dihydrobenzo[cd]indole-6-
3.32. Diethyl 1,1-difluoro-4-(2-oxo-1,2-dihydrobenzo[cd
sulfonamido)hexylphosphonic acid (73)
]indole-6-sulfonamido)-hexyl-phosphonate (70)
Bromotrimethylsilane (0.5 mL, 0.42 mmol) was added to the
solution of compound 70 (50 mg, 0.10 mmol) in CH2Cl2 (4 mL).
After stirring for 36 h at 25 °C, the volatiles were removed in vacuo,
and the residue was purified by passing it through lipophilic
Sephadex (LH20) using CH2Cl2–MeOH (8:2) as eluant to give 73
K2CO3 (83 mg, 0.6 mmol) was added to the solution of iodo
derivative 63 (84 mg, 0.22 mmol) and amine derivative 67
(50 mg, 0.20 mmol) in DMF (3 mL). The reaction mixture was stir-
red at room temperature for 12 h. Water (25 mL) was added to the
reaction mixture and the mixture was extracted with diethyl ether
(20 mL ꢁ 3). The combined organic extracts were washed with
brine, dried (Na2SO4), and evaporated. The residue was purified
by flash silica gel column chromatography, eluting with EtOAc–
petroleum ether (7:3), to afford compound 70 (60 mg, 59%) as a
yellow oil. 1H NMR (300 MHz, acetone-d6) d 8.71 (d, J = 8.4 Hz,
1H), 8.16 (d, J = 7.5 Hz, 1H), 8.06 (d, J = 6.9 Hz, 1H), 7.90 (t,
J = 8.2 Hz, 1H), 7.20 (d, J = 7.7 Hz, 1H), 6.78 (s, 2H), 4.21 (m, 4H),
3.96 (t, J = 6.8 Hz, 2H), 2.03 (m, 2H), 1.86 (m, 2H), 1.60 (m, 2H),
1.48 (m, 2H), 1.30 (t, J = 7.0 Hz, 6H); 13C NMR (75 MHz, acetone-
d6) d 168.1, 144.2, 133.7, 131.5, 130.9, 130.6, 127.3, 126.3, 125.4,
(30 mg, 68%) as
a
yellowish solid: mp 90–93 °C. 1H NMR
(300 MHz, MeOH-d4) d 8.65 (d, J = 8.1 Hz, 1H), 8.13 (d, J = 7.5 Hz,
1H), 8.00 (d, J = 6.6 Hz, 1H), 7.83 (t, J = 7.5 Hz, 1H), 7.07 (d,
J = 7.5 Hz, 1H), 3.90 (t, J = 6.6 Hz, 2H), 2.02 (m, 2H), 1.84 (m, 2H),
1.78 (m, 2H), 1.42 (m, 2H); 13C NMR (125 MHz, MeOH-d4) d
169.7, 144.2, 134.3, 131.7, 131.23, 131.18, 127.3, 126.1, 125.7,
122.5 (dt), 105.3, 41.0, 34.8 (m), 29.3, 27.7, 21.7; EIMS m/z (rel
intensity) 897 (2MH+, 100), 449 (MH+, 86); negative ion ESIMS
m/z (rel intensity) 895 ([2MꢀH+]ꢀ, 100), 447 ([MꢀH+]ꢀ, 12); HRMS
m/z calcd for C17H19F2N2O6PS (MH+) 449.0748, found 449.0758.

3532
A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
Anal. Calcd for C17H19F2N2O6PSꢂH2O: C, 43.78; H, 4.54; N, 6.01; P,
3.39. N-(6-Chloro-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-
yl)propanamide (79)
6.64. Found: C, 43.72; H, 4.69; N, 5.78; P, 6.41.
3.36. Ethyl 2-(2,4-bis(benzyloxy)-6-chloropyrimidin-5-
ylamino)-2-oxoacetate (76)
Lindlar catalyst (5 mg) was added to a solution of compound 78
(50 mg, 0.13 mmol) in anhydrous ethanol (4 mL). 1,4-Cyclohexedi-
ene (0.1 mL, 1.3 mmol) was added and argon was bubbled through
the reaction mixture for 10 min. The mixture was stirred at room
temperature for 12 h. The reaction mixture was filtered through
Celite, which was then washed with ethanol (2 ꢁ 5 mL). The solu-
tion was concentrated and the residue was washed several times
with CH2Cl2 and THF. Finally, compound 79 was precipitated out
by dissolving it in MeOH and adding excess diethyl ether to furnish
pure compound 79 (18 mg, 66%) as a white amorphous solid: mp
Triethylamine (0.3 mL, 2.2 mmol) was added to a solution of
compound 75 (80 mg, 0.23 mmol) in THF (5 mL). The solution
was cooled to 0 °C and ethyl 2-chloro-2-oxoacetate (31
lL,
0.28 mmol) was added dropwise. The reaction mixture was stir-
red at 0 °C for 12 h. The solvent was distilled off under reduced
pressure and the residue was dissolved in dichloromethane
(20 mL) and washed with water (2 ꢁ 15 mL). The organic layer
was dried and solvent was distilled off. The residue was flash
column chromatographed with silica gel, eluting with 35% ethyl
acetate in hexane, to afford compound 76 (78 mg, 76%) as a
208–211 °C (dec). 1H NMR (300 MHz, MeOH-d4)
d 2.38 (q,
J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H); 13C NMR (75 MHz, MeOH-
d4) d 177.1, 163.1, 152.0, 147.2, 109.8, 29.8, 10.1; EIMS m/z (rel
intensity) 262 (MꢀH++2Na+, 82), 240 (MNa+, 100), 130 (35); nega-
tive ion EIMS m/z (rel intensity) 216 [(MꢀH+)ꢀ, 100], 180 (25);
HRMS m/z calcd for C7H8ClN3O3 (MNa+) 240.0152, found
240.0153. Anal. Calcd for C7H8ClN3O3ꢂ1.4H2O: C, 34.62; H, 4.48;
N, 17.30. Found: C, 34.80; H, 4.18; N, 17.10.
white semisolid. 1H NMR (300 MHz, CDCl3)
d 8.35 (s, 1H),
7.44–7.30 (m, 10H), 5.41 (s, 2H), 5.36 (s, 2H), 4.37 (q, J = 7.13,
2H), 1.37 (t, J = 7.14, 3H); 13C NMR (75 MHz, CDCl3) d 166.4,
161.7, 159.7, 158.3, 154.5, 135.4, 135.0, 128.42, 128.37, 128.23,
128.18, 128.1, 127.8, 108.7, 70.1, 69.7, 63.6, 13.8; HRMS m/z
calcd for C22H20ClN3O5 (MNa+) 464.0989, found 464.0991. Anal.
Calcd for C22H20ClN3O5: C, 59.80; H, 4.56; N, 9.51. Found: C,
59.83; H, 4.31; N, 9.80.
3.40. Computational methods
3.40.1. Screening the database
3.37. Ethyl 2-(6-chloro-2,4,dioxo-1,2,3,4-tetrahydropyrimidin-
5-ylamino)-2-oxoethanoate (77)
The ZINC database maintained by the Shoichet group at UCSF
a pre-filtered comprehensive database of available com-
is
pounds.39 The ligands in this database have multiple protonation
and tautomeric states. Approximately 1.03 million compounds
were downloaded in SDF format with multiple tautomeric and
protonation states (pH range 5.75–8.25) selected from following
vendors: Asinex, Chembridge, Maybridge, Nanosyn and Sigma–
Aldrich. The M. tuberculosis lumazine synthase crystal structure
Lindlar catalyst (5 mg) was added to a solution of compound 76
(50 mg, 0.113 mmol) in anhydrous ethanol (4 mL). 1,4-Cyclohex-
ediene (90
lL, 1.13 mmol) was added and argon was bubbled
through the reaction mixture for 10 min. The mixture was stirred
at room temperature for 12 h. The reaction mixture was filtered
through Celite, which was then washed with ethanol (2 ꢁ 5 mL).
The solution was concentrated and the residue was washed several
times with CH2Cl2 and THF. Finally, compound 77 was precipitated
out by dissolving it in MeOH and adding excess diethyl ether to
furnish pure compound 77 (20 mg, 68%) as a white amorphous so-
lid: mp 174–176 °C. 1H NMR (300 MHz, MeOH-d4) d 4.37 (q,
J = 6.9 Hz, 2H), 1.37 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, MeOH-
d4) d 162.8, 160.8, 158.8, 149.6, 129,6, 107.9, 64.2, 14.2; negative
ion EIMS m/z (rel intensity) 260 [(MꢀH+)ꢀ, 100], 224 (43); HRMS
m/z calcd for C8H8ClN3O5 (MNa+) 284.0050, found 284.0053. Anal.
Calcd for C8H8ClN3O5ꢂ0.65H2O: C, 35.15; H, 3.43; N, 15.37. Found:
C, 34.8; H, 3.18; N, 15.10.
in complex with inhibitor 3-(1,3,7-trihydro-9-D-ribityl-2,6,8-purine-
trione-7-yl) propane 1-phosphate (PDB: 1w19) was prepared using
Protein Preparation tools in Maestro 7.5 (Schrödinger LLC, Portland,
Oregon) removing all crystal water molecules. The compounds
were first docked with Glide 4.0 (Schrödinger LLC, Portland, Ore-
gon) High Throughput Virtual Screening mode without any con-
straints. The top 5% of hits were then screened using Glide
Standard Precision (SP), applying the following pharmacophore
constraints: 3 backbone hydrogen bonds to Ile83, Val81, Ala59
and the phosphate interaction to Arg128. At least 3 of these con-
straints had to be satisfied. For all hits with Glide Score –5 or bet-
ter, log S and log P were calculated with QikProp 2.5 (Schrödinger
LLC, Portland, Oregon). Compounds that were predicted with rea-
sonable solubility (log P < 2.5 and log S > ꢀ3.6) were selected result-
ing in 6742 compounds. These selected compounds were docked
again with Glide Extra Precision (XP). Glide XP not only offers a
more extensive conformational search but also recognizes multiple
protein–ligand motifs such as enclosure of lipophilic ligand sub-
structures by hydrophobic protein residues and various hydrogen
bonds in hydrophobic environments. In addition, the scoring func-
tion has additional penalty terms including desolvation of polar
groups and solvent exposure of hydrophobic ligand group designed
to remove potential false positives.46 The backbone hydrogen bond
constraints were again applied. The final 44 compounds that had
Glide Score of ꢀ10 or better were inspected visually and 12 of
them were tested based on the chemical scaffolds and interaction
with the receptor for inhibition properties by enzymatic assay
and Isothermal Titration Calorimetry (ITC). The visual inspection
is a common practice which is important to avoid testing false pos-
itives due to assumptions and shortcomings in docking methods
and scoring functions.68 The score cutoff of ꢀ10 was chosen based
on docking of several known active compounds41 under the same
condition.
3.38. N-(6-Chloro-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-
yl)propionamide (78)
Triethylamine (0.3 mL, 2.2 mmol) was added to a solution of
compound 75 (80 mg, 0.23 mmol) in THF (5 mL). The solution
was cooled to 0 °C and propionyl chloride (25
lL, 0.28 mmol)
was added dropwise. The reaction mixture was stirred at 0 °C for
12 h. The solvent was distilled off under reduced pressure and
the residue was dissolved in dichloromethane (20 mL) and washed
with water (2 ꢁ 15 mL). The organic layer was dried and solvent
was distilled off. The residue was flash column chromatographed
with silica gel, eluting with 35% ethyl acetate in hexane, to afford
compound 78 (65 mg, 70%) as white solid: mp 122–124 °C. 1H
NMR (300 MHz, CDCl3) d 7.42–7.32 (m, 10H), 5.35 (s, 2H), 5.33
(s, 2H), 2.33 (q, J = 7.23, 2H), 1.37 (t, J = 7.1, 3H); 13C NMR
(75 MHz, CDCl3) d 172.8, 166.9, 161.2, 158.7, 135.5, 135.3, 128.4,
128.2, 127.7, 110.5, 70.0, 69.5, 29.3, 9.7; HRMS m/z calcd for
C21H20ClN3O3 (MNa+) 420.1091, found 420.1088. Anal. Calcd for
C21H20ClN3O3: C, 63.40; H, 5.07; N, 10.56. Found: C, 63.66; H,
4.82; N, 10.78.

A. Talukdar et al. / Bioorg. Med. Chem. 18 (2010) 3518–3534
3533
3.40.2. Molecular modeling
synthase, variable concentrations of 3 (3–60
200
follows. A solution (170
dithiothreitol, 1.2 M riboflavin synthase in 105 mM Tris hydro-
chloride, pH 7.0, was added to 4 L of inhibitor in 100% (v/v) DMSO
in a well of a 96-well microtiter plate. The reaction was started by
adding 20 L of a solution containing 105 mM NaCl, 5.3 mM dithi-
othreitol, and substrate 3 (30–600 M) in 105 mM Tris hydrochlo-
l
M) and inhibitor (0–
Using Sybyl (Tripos, Inc., version 7.1), the crystal structure of the
complex of compound 11 was clipped to include information within
a 12 Å sphere of one of the 5 equivalent ligand molecules. Docking of
the lead and synthesized compounds in the active site of M. tubercu-
losis lumazine synthase was performed with GOLD (BST, version 3.0,
2005). The programs were set to conduct 10,000 repeats for each li-
gand under identical conditions. The best fit result from GOLD dock-
ingwasaddedto thecomplex,MMFF94chargeswereloaded, andthe
energy of the complex was minimized using the Powell method to a
termination gradient of 0.05 kcal/mol employing the MMFF94s force
field, which was used to minimize steric clashes between the GOLD-
docked compounds and the protein.
l
M) in a volume of 0.2 mL. Assay mixtures were prepared as
l
L) containing 105 mM NaCl, 5.3 mM
l
l
l
l
ride, pH 7.0. The formation of riboflavin (4) was measured online
for a period of 30 min at 27 °C with a computer-controlled plate
reader at 470 nm (e
Riboflavin = 9600 Mꢀ1 cmꢀ1).
3.45. Evaluation of kinetic data
3.41. Isothermal titration calorimetry
The velocity-substrate data were fitted for all inhibitor concen-
trations with a non-linear regression method using the program
DYNAFIT.
69 Different inhibition models were considered for the calcu-
Calorimetric measurements for binding analysis were carried out
by using a VP-ITC MicroCalorimeter (MicroCal, Inc., Northampton,
MA, USA). The reference cell was filled with water and the instru-
ment was calibrated using standard electrical pulses. Binding iso-
therms were measured by direct titration. A solution of M.
tuberculosis lumazine synthase (0.05 mM) in 100 mM potassium
phosphate buffer at pH 7.1 in a total volume of 1.451 mL was titrated
lation. Ki and Kis values standard deviations were obtained from
the fit under consideration of the most likely inhibition model.
Acknowledgment
This research was made possible by NIH Grant Nos. RO1
GM51469 and R21 NS053634, the Hans Fischer Gesellschaft, and
the Swedish Research Council.
at 30 °C with 35 identical 8 lL injections at 3 min intervals. The syr-
inge was filled with 5 mM compound dissolved in the same buffer.
The heat evolved after each injection was obtained from the integral
of the calorimetric signal. All data were evaluated with the Microcal
Origin50 Software package (Microcal Software, INC., Northampton,
MA, USA) supplied with the calorimeter. The heat of dilution was
subtracted from the observed heat in the binding experiments. The
Supplementary data
Supplementary data (1H and 13C NMR spectra of compounds 20,
21, 24, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 46, 47, 48, 49,
52, 53, 54, 55, 61, 62, 63, 67, 68, 69, 70, 71, 72, 73, 76, 77, 78 and
79) associated with this article can be found, in the online version,
at doi:10.1016/j.bmc.2010.03.072.
association constants, Ka, binding enthalpy,
DH, and stoichiometry,
n, were obtained by fitting the data to standard equations for the
binding using a model for one set of independent and identical bind-
ing sites as implemented in the MicroCal Origin50 package. The
binding entropy
lated from the basic thermodynamic equations,
the Gibbs–Helmholtz equation G = S.
H ꢀ T
D
S and free energy
D
G of the binding were calcu-
D
G = ꢀRTln K and
References and notes
D
D
D
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Assay mixtures contained 100 mM Tris hydrochloride, pH 7.0,
100 mM NaCl, 2% (v/v) DMSO, 5 mM dithiothreitol, 100
lumazine synthase, variable concentrations of
M) and inhibitor (0–200
mixtures were prepared as follows. A solution (170
105 mM NaCl, 5.3 mM dithiothreitol, 118 M 2, 2.4
synthase in 105 mM Tris hydrochloride, pH 7.0, was added to
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Lumazine = 10,200 Mꢀ1 cmꢀ1).
lM 2,
2
l
M
1
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100 mM NaCl, 2% (v/v) DMSO, 5 mM dithiothreitol, 1 lM riboflavin

3534
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