Synthesis, biological evaluation, and molecular modeling of 3,5-substituted-N1-phenyl-N4,N4-di-n-butylsulfanilamides as antikinetoplastid antimicrotubule agents

Article abstract of DOI:10.1016/j.bmc.2007.06.042

Dinitroanilines are of interest as antiprotozoal lead compounds because of their selective activity against the tubulin of these organisms, but concern has been raised due to the potentially mutagenic nitro groups. Analogues of N¹-phenyl-3,5-di

Full text of DOI:10.1016/j.bmc.2007.06.042

Bioorganic & Medicinal Chemistry 15 (2007) 6071–6079
Synthesis, biological evaluation, and molecular modeling
of 3,5-substituted-N1-phenyl-N4,N4-di-n-butylsulfanilamides
as antikinetoplastid antimicrotubule agents
Tesmol G. George, Molla M. Endeshaw, Rachel E. Morgan, Kiran V. Mahasenan,
´
Dawn A. Delfın, Mitali S. Mukherjee, Adam J. Yakovich, Jean Fotie,
Chenglong Li and Karl A. Werbovetz*
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University,
500 West 12th Avenue, Columbus, OH 43210, USA
Received 8 May 2007; revised 18 June 2007; accepted 19 June 2007
Available online 27 June 2007
Abstract—Dinitroanilines are of interest as antiprotozoal lead compounds because of their selective activity against the tubulin of
these organisms, but concern has been raised due to the potentially mutagenic nitro groups. Analogues of N1-phenyl-3,5-dinitro-
N4,N4-di-n-butylsulfanilamide (GB-II-150, compound 2b), a selective antimitotic agent against African trypanosomes and Leish-
mania, have been prepared where the nitro groups are replaced with amino, chloro, cyano, carboxylate, methyl ester, amide, and
methyl ketone moieties. Dicyano compound 5 displays IC50 values that are comparable to 2b against purified leishmanial tubulin
assembly (6.6 vs 7.4 lM), Trypanosoma brucei brucei growth in vitro (0.26 vs 0.18 lM), Leishmania donovani axenic amastigote
growth in vitro (4.4 vs 2.3 lM), and in vitro toxicity against Vero cells (16 vs 9.7 lM). Computational studies provide a rationale
for the antiparasitic order of activity of these analogues and further insight into the role of the substituents at the 3 and 5 positions
of the sulfanilamide ring.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
be desirable, however, since several nitroaromatic com-
pounds have been shown to be mutagenic.12–14
Dinitroaniline herbicides are well known for their
activity against protozoan parasites. Chan and Fong
first reported the activity of trifluralin (1a) against the
pathogenic protozoan Leishmania in 1990 and showed
that this compound bound selectively to partially puri-
fied leishmanial tubulin compared to rat brain tubulin.1
Another dinitroaniline herbicide, oryzalin (1b), also dis-
played antimicrotubule activity against Leishmania.2
These compounds were later shown to possess antipara-
sitic activity against trypanosomes,3,4 Plasmodium,5,6
and Toxoplasma.7–9 Given the need for new drug
candidates against diseases caused by protozoan para-
sites,10,11 the dinitroaniline herbicides are intriguing lead
compounds. Replacement of the nitro moieties present
in these molecules with other functional groups would
Our laboratory previously demonstrated that N1-phe-
nyl-3,5-dinitro-N4,N4-di-n-propylsulfanilamide (GB-II-
5, compound 2a), N1-phenyl-3,5-dinitro-N4,N4-di-n-
butylsulfanilamide (GB-II-150, compound 2b), and
other N1-aryl-3,5-dinitroaniline sulfanilamides possess
selective antimicrotubule activity against Leishmania
and African trypanosomes in vitro and are far superior
to 1a and 1b in their potency against these parasites.15–18
Replacement of one of the nitro groups present in 2a
with a hydrogen resulted in a profound loss in antipar-
asitic and antitubulin activity.17 A recent computational
study by Mitra and Sept suggested that these nitro
groups in 2a serve as hydrogen bond acceptors at a
unique binding site occurring on the a-subunit of leish-
manial tubulin.19 These authors also predicted that cya-
no and methyl ketone groups could act as hydrogen
bond acceptors if they were substituted for the nitro
groups present in the dinitroaniline sulfanilamides. In
the present manuscript, we describe the synthesis and
biological activity of analogues of 2b where the nitro
Keywords: Leishmania; Trypanosome; Chemotherapy; Tubulin;
Dinitroaniline.
*
Corresponding author. Tel.: +1 614 292 5499; fax: +1 614 292
2435; e-mail: werbovetz.1@osu.edu
These authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.042

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T. G. George et al. / Bioorg. Med. Chem. 15 (2007) 6071–6079
moieties are replaced by several groups with chemical
and physical similarities.
to complex the potassium ion were unsuccessful. The
presence of the bulky dibutylamino groups in the sub-
strate may interfere with the Sandmeyer reaction in this
case and may be the cause of the poor reaction yield.
N((CH2)2CH3)2
NO2
N((CH2)nCH3)2
NO2
O2N
O2N
In order to provide a more efficient and reproducible
alternative for the preparation of 5 and additional target
compounds, another synthetic route was envisioned (see
Scheme 2). Permanganate oxidation of commercially
available 2-bromo-m-xylene (6) gave 2-bromoisophtha-
lic acid (7),21 which was sulfonylated with fuming sulfu-
ric acid and esterified using methanol/HCl to afford the
sulfonic acid 8. Treatment of 8 with POCl3 provided the
sulfonyl chloride 9, which was then converted to sulfon-
amide 10 by reaction with aniline in the presence of pyr-
idine in acetone. An Ullmann reaction22 between
sulfonamide 10 and dibutylamine afforded the desired
diester analogue 11, which was hydrolyzed to yield dia-
cid 12. The formation of bis-amide 13 was carried out in
a one-pot reaction between ammonia and the intermedi-
ate bis-acid chloride derivative of 12, which in turn was
prepared by the reaction of 12 with hexachloroacetone
in the presence of PPh3.23 Attempts to synthesize the
bis-amide directly from diester 11 were unsuccessful,
resulting in quantitative recovery of the starting mate-
rial. Bis-amide 13 was found to be an excellent precursor
for the target dicyano compound 5. Dehydration of 13
using PdCl2 in an acetonitrile/water mixture24 afforded
5 in good and reproducible yield. The synthesis of dicy-
ano compound 5 from bis-amide 13 is an equilibrium
process in which the presence of excess acetonitrile fa-
vors the formation of 5.
R
SO2NHPh
1a: R = CF3
1b
2a: n = 2
2b: n = 3
: R = SO2NH2
2. Results
2.1. Chemistry
It was proposed that target compounds containing Cl
atoms and cyano moieties in place of the nitro groups
present in 2b could be introduced through a strategy cul-
minating with a Sandmeyer reaction. Reduction of 2b
was carried out using H2 in the presence of 10% Pd–C,
which afforded the diamino compound 3 in high yield
(Scheme 1). Treatment of 3 with NaNO2 in HCl fol-
lowed by reaction of the resultant bis-diazonium salt
with CuCl afforded the dichloro analogue 4, albeit in
low yield (Scheme 1). All efforts to improve the yield
of 4 by changing the reaction conditions were unsuccess-
ful. Target compound 5 was also prepared by this strat-
egy, but in extremely low yield (Scheme 1). The
diazotization reaction was carried out using NaNO2
and HCl in a mixture of H2O/AcOH containing 3 at
0 ꢁC. This solution was then treated with an excess of
CuCN and KCN in a mixture of CHCl3/H2O at 0 ꢁC20
followed by heating to 50 ꢁC. These conditions did not
reproducibly provide the desired product. Other related
approaches, such as preparing the bis-diazonium salt
using either tert-butyl nitrite or tert-butyl nitrite in
BF3 etherate in situ, isolating the bis-diazonium salt
prior to reaction with cyanide, or adding 18-crown-6
With target compounds 3–5 and 11–13 in hand, atten-
tion was focused on the preparation of bis-ketone 14.
Initial attempts to synthesize 14 by treating the bis-acid
chloride derivative of 12 with dimethyl malonate in the
presence of NaH followed by decarboxylation25 were
unsuccessful. Reaction between methyl magnesium bro-
mide and the bis-acid chloride gave compound 14 along
with other unidentified side products. The formation of
these side products could not be avoided by varying the
N
Cl
Cl
b
N
N
SO2NHPh
O2N
NO2
H2N
NH2
4
a
N
SO2NHPh
SO2NHPh
NC
CN
2b
3
c
SO2NHPh
5
Scheme 1. Reagents and conditions: (a) H2, 10% Pd–C, EtOAc, rt, 98%; (b) 1—NaNO2, concd HCl, H2O, 0 ꢁC; 2—CuCl, HCl, 0 ꢁC to rt, 23%;
(c) 1—NaNO2, concd HCl, H2O/AcOH, 0 ꢁC; 2—CuCN, KCN, CHCl3/H2O, 0–50 ꢁC, 12%.

T. G. George et al. / Bioorg. Med. Chem. 15 (2007) 6071–6079
6073
Br
Br
Br
H3C
CH3
HOOC
COOH
H3COOC
COOCH3
a
b
c
SO3H
6
7
8
Br
Br
N
H3COOC
COOCH3
H3COOC
COOCH3
H3COOC
COOCH3
d
e
f
SO2Cl
SO2NHPh
10
SO2NHPh
11
9
N
N
N
HOOC
COOH
H2NOC
CONH2
NC
CN
g
h
SO2NHPh
12
SO2NHPh
13
SO2NHPh
5
i
N
H3COC
COCH3
SO2NHPh
14
Scheme 2. Reagents and conditions: (a) KMnO4, H2O, reflux, 66%; (b) 1—fuming H2SO4, 170 ꢁC; 2—HCl, MeOH, reflux, 79% after two steps; (c)
POCl3, reflux, 70%; (d) aniline, pyridine, acetone, 0 ꢁC–rt, 66%; (e) dibutylamine, K2CO3, Cu, dioxane, reflux, 79%; (f) 3 N NaOH, MeOH, reflux,
88%; (g) 1—CCl3COCCl3, PPh3, THF, 0 ꢁC; 2—NH3/dioxane, rt, 68% after two steps; (h) PdCl2, CH3CN/H2O, 70 ꢁC, 71%; (i) 1—CCl3COCCl3,
PPh3, THF, 0 ꢁC; 2—MeMgBr/THF, CuBr, THF, 0 ꢁC, 55% after two steps.
reaction temperature, time, and the amount of Grignard
reagent. We hypothesize that further reaction of the bis-
ketone with methyl magnesium bromide was responsible
for the occurrence of the side products. Therefore, it was
reasoned that a soft nucleophile such as an organocup-
rate might lead to more efficient production of the
diketone. In accord with this expectation, reaction of
the acid chloride with a mixture of MeMgBr and CuBr
in THF at 0 ꢁC afforded the target bis-ketone 14 in mod-
erate yield (Scheme 2).
3 and 11–14 displayed IC50 values >100 lM according
to the previously defined conditions of the assembly as-
say, which measures the change in absorbance due to
tubulin polymerization at 351 nm after 20-min incuba-
tion at 30 ꢁC for samples containing the compound of
interest compared to controls.17,18 However, both 3
and 14 caused a pronounced lag in the assembly of leish-
manial tubulin at concentrations P20 lM and thus
show a significant effect on the kinetics of polymeriza-
tion. The effects of 3 and 14 on the kinetics of leishman-
ial tubulin assembly are dose-dependent, with higher
concentrations producing a greater lag in assembly. Sim-
ilar effects of other antimitotic compounds have been
observed previously in our laboratory16 and by oth-
ers.27,28 Leishmanial tubulin assembly was relatively
insensitive to 11–13 at concentrations up to 100 lM.
The effect of 2b, 5, 13, and 14 on parasite tubulin assem-
bly is illustrated in Figure 1. Compounds 2b, 3–5, and 14
were also examined for their effects on the assembly of
purified porcine brain tubulin (Table 1), with 2b, 4, 5,
and 14 displaying little influence on the kinetics or ex-
tent of polymerization at the highest concentrations
tested. Approximately 50% inhibition of porcine tubulin
2.2. Biological results
Target compounds 3–5 and 11–14 were judged against
2b for their ability to block the assembly of purified
tubulin isolated from Leishmania tarentolae (see Table
1). Compound 2b displayed an IC50 value of 7.4 lM,
which compares favorably with the previously reported
IC50 values for this compound (6.9 lM17 and 5.8 lM26)
in the same assay. Of the new compounds, the two most
potent inhibitors of leishmanial tubulin assembly were 5
and 4, whose IC50 values of 6.6 and 9.1 lM are compa-
rable to and slightly higher than that of 2b. Compounds

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T. G. George et al. / Bioorg. Med. Chem. 15 (2007) 6071–6079
Table 1. Biological activity of target compoundsa (lM)
Compound
IC50 versus
L. tarentolae
tubulin assembly
IC50 versus
porcine brain
tubulin assembly
IC50 versus
L. donovani
axenic amastigotesa
IC50 versus
T. b. brucei
IC50 versus
Vero cells
bloodstream forms
2b
3
7.4 0.2
>100b
9.1 4.5
6.6 0.7
>100
>40e
>50
>50e
>50e
NT
2.3 0.5
0.18 0.01
4.0 0.1
1.6 0.1
0.26 0.03
>50
9.7 1.1
12
11
2
3
27
19
4
1
1
4
5
11
4.4 0.9
>50
>50
16
NT
NT
NT
26
12
13
>100
>100
>100b
NTc
NT
NT
NT
>50
>50
>50
30
14
Pentamidine
Suramin
Podophyllotoxind
>100
NT
NT
7
1.6 0.3
NT
1
2.3 0.9
NT
NT
NT
NT
0.077 0.001
NT
NT
NT
0.016 0.006
a Activities represent the mean standard deviation of at least three independent experiments.
b Produces a lag in tubulin assembly; see text and Figure 1.
c NT, not tested.
d Podophyllotoxin is used here solely as a standard for the cytotoxicity assay. Colchicine-site antimicrotubule agents such as podophyllotoxin have
little effect on Leishmania tubulin assembly.37
e Compounds not soluble at higher concentrations.
0.05
assembly was observed in assays containing 3 at a con-
centration of 100 lM.
0.04
0.03
0.02
0.01
0.00
-0.01
Compounds 2b, 3–5, and 11–14 were docked into the
proposed dinitroaniline binding pocket on leishmanial
a-tubulin19 using AutoDock29 and Glide.30 These mole-
cules bind similarly as shown in Figure 2 and in a man-
ner consistent with the model proposed by Mitra and
Sept.19 Both docking methods show that 4 has the best
binding clustering, 2b and 5 follow, and the remaining
compounds display worse binding clustering (see Table
2). While there is some variability between the values
determined by AutoDock and Glide, the calculated
binding energies of 2b, 3–5, 11, 13, and 14 are compara-
ble. Interestingly, compound 12, where isosteric replace-
ment of the nitro moieties with carboxylate groups has
occurred, shows intermediate binding clustering with
0
5
10
15
20
Time (m)
Figure 1. The assembly of 1.5 mg/mL purified tubulin from Leish-
mania tarentolae was assessed as described previously26 in the absence
(open squares) or presence of 5 (closed circles), 13 (closed squares), or
14 (triangles) at 20 lM or 2b (open circles) at 10 lM. Data shown are
from a representative experiment performed on three separate
occasions.
Figure 2. The docked positions of compounds 2b and 3–5 are shown as stick models (2b, blue; 3, green; 4, red; 5, cyan), while the tubulin protein is
shown as ribbons. Stick models are also shown for Ala4 (which takes part in hydrogen bonding to a nitro group oxygen of compound 2b), Thr239/
Ala240 (which participate in electrostatic interactions with the other oxygen atom of this nitro moiety), and Arg64/Asp47 (which form a salt bridge
and interact with the other nitro group of 2b through the solvent network). This figure was created with the aid of AutoDock.

T. G. George et al. / Bioorg. Med. Chem. 15 (2007) 6071–6079
Table 2. In silico binding energies and binding clustering of target compounds
6075
Compound
Percent binding
clustering (AutoDock)
Binding energy
(AutoDock, in kcal/mol)
Percent binding
clustering (Glide)
Binding energy
(Glide, in kcal/mol)
2b
3
93
4
À11.0
À9.8
44
3
À7.9
À8.9
À9.9
À8.5
À7.6
À3.2
À8.7
À7.5
4
96
92
2
À10.2
À10.7
À11.2
À7.0
76
44
2
5
11
12
13
14
33
2
2
À10.3
À10.6
2
2
2
AutoDock but displays poor binding clustering with
Glide and shows binding energies that are inferior to
all of the other target compounds.
slightly superior to 2a in in vitro antiparasitic activity
and differs in structure from 2a only in the presence of
an additional methylene group in the N4 dialkyl
chains.17 Replacement of the nitro moieties of 2b with
carboxylate groups (12) represents an isosteric substitu-
tion. Substitutions of the nitro groups with methyl ester
(11), amide (13), and acetyl (14) groups were envisioned
as non-classical bioisosteric replacements.31 Nitrile,
methyl ester, amide, and acetyl functional groups pres-
ent in compounds 5, 11, 13, and 14, respectively, have
similar lipophilic and electronic properties as compared
to the nitro group.32 Evaluation of 3–5 and 11–14 for
their effects on purified leishmanial tubulin assembly
now permits refinement of the hypotheses advanced by
Mitra and Sept and provides greater insight into the role
of these groups in tubulin-ligand interactions.
The activities of the target compounds against
Leishmania donovani axenic amastigotes, Trypanosoma
brucei brucei bloodstream forms, and African Green
Monkey kidney (Vero) cells are shown in Table 1. Con-
sistent with previous reports, 2b possesses potent
in vitro activity against these organisms and displays
excellent selectivity against African trypanosomes
compared to the mammalian cell line.17,18 Compound
5 was only 1.4-fold less active than 2b against T. b. brucei
and twofold less active than this compound against
L. donovani while showing 1.6-fold lower toxicity
against Vero cells. Compounds 4 and 14 were sixfold
less potent than 5 against T. b. brucei, while 3 displayed
15-fold lower activity than 5 against this parasite. In
terms of antileishmanial activity, 3 and 4 were nearly
threefold less active than 5 against L. donovani, while
14 was sevenfold less potent in this assay. Compounds
11–13 failed to display measurable activity against either
Leishmania or African trypanosomes at the highest
concentrations tested.
In accord with previous expectations,19 dicyano com-
pound 5displays activity in the leishmanial tubulinassem-
bly assay comparable to that of dinitro lead 2b. Dichloro
compound 4 shows potency similar to 5 in this assay,
while 3 and 14 display weaker activity and 11–13 are
essentially inactive. Refinements to the binding site mod-
el19 through the present docking studies provide a ratio-
nale for these observations. Compounds 2b, 4, and 5,
the most effective inhibitors of leishmanial tubulin assem-
bly described in this work, also exhibited the best Auto-
Dock and Glide scores of the group. At the positions
where nitro groups occur in 2b, each of these compounds
possesses functional groups that can serve as a hydrogen
bond acceptor. Figure 2 clearly shows the proposed
hydrogen bond formed between a nitro group oxygen
atom of 2b and the amide group of Ala4. Similarly, a
Cl atom of 4 and particularly a cyano group of 5 can also
serve as hydrogen bond acceptors as pictured in Figure 2.
While a hydrogen bond acceptor is required where one of
the nitro moieties is located in 2b, close inspection of the
binding site model suggests that activity should be main-
tained when a different functional group is placed at the
other position. Although the second nitro group of 2b is
proposed to interact with the solvent network as men-
tioned earlier, placement of a bulky hydrophobic group
at this subpocket consisting of Trp21, Phe24, Met36,
Ile42, and His61 may also permit favorable interactions
at the binding site. Individual molecular dynamics simula-
tions will be required to determine whether the salt bridge
between Asp47 and Arg64 interacts with the newer ana-
logues through the solvent network.
3. Discussion
Nitro groups have thus far proven essential for the anti-
parasitic antimitotic activity of dinitroaniline sulfon-
amide analogues. Mitra and Sept’s binding site model
for dinitroaniline sulfonamide 2a on leishmanial a-tubu-
lin permitted formulation of the first hypothesis regard-
ing the role of these nitro moieties in tubulin binding.19
According to this model, one of the nitro groups makes
several direct interactions with the protein. Electrostatic
interactions are observed between one of the oxygen
atoms in this moiety and a partially positively charged
structural motif consisting of Thr239 and Ala240, while
the other oxygen atom of this group accepts a hydrogen
bond from the amide NH of Ala4 (see Fig. 2). The sec-
ond nitro group does not interact directly with a-tubu-
lin; instead, a solvent network connects this moiety to
the salt bridge between Asp47 and Arg64. Through
docking experiments, Mitra and Sept also showed that
an analogue of 2a bearing cyano groups in place of
the nitro moieties displays in silico binding affinity to
leishmanial tubulin similar to 2a, while an analogue pos-
sessing acetyl groups in these positions binds more
strongly than 2a to the parasite protein. In this work,
we have prepared a series of analogues of 2b, which is
Inactive compounds 11–13 show poor scores in molecular
docking studies. Steric considerations are likely to be

6076
T. G. George et al. / Bioorg. Med. Chem. 15 (2007) 6071–6079
responsible in part for the lack of activity of diester 11,
while amide bond resonance limits the availability of
the lone pair electrons present on the amino group of
13. Although the substitution of nitro moieties with car-
boxylate groups in 12 is an isosteric replacement, the ni-
tro moieties lack a net charge while the carboxylate
groups are expected to possess a negative charge at phys-
iological pH. The difference in net charge between 2b and
12 is likely to be responsible for the lack of activity of the
latter molecule. Compounds 3 and 14 also display poor
AutoDock and Glide scores but have measurable effects
on leishmanial tubulin assembly (albeit weaker than 2b,
4, and 5). As an arylamine, 3 is potentially carcinogenic,33
but this compound was prepared as a necessary interme-
diate in the synthesis of 4 and was tested because of the
contribution that could be made to the structure–activity
binding site model for 2a and its analogues19 indicates
that sufficient space is available for the design of mole-
cules containing substituents at N4 that may be less sus-
ceptible to metabolism. The ability to substitute the
cyano groups for the potentially mutagenic nitro moiety
while retaining activity provides further flexibility in the
design of antimitotic antiparasitic molecules. Given the
current knowledge of the SAR for this group of mole-
cules and the availability of a binding site model for
the generation of new target compounds, the design of
new structural classes of antimitotic antikinetoplastid
agents with increased metabolic stability may now be
possible.
4. Conclusion
relationship. The van der Waals radius of the nitrogen
34
˚
˚
atom (1.55 A) is 0.2 A smaller than that of chlorine,
suggesting that the lone pair electrons of the amino group
of 3 may not be positioned ideally to serve as a hydrogen
bond acceptor. In addition, the amino group is most of-
ten a hydrogen bond acceptor rather than a donor. One
of the oxygen atoms of the nitro group in 2b is proposed
to provide electrostatic interactions with Thr239 and
Ala240 at the binding site, which would not be possible
for the methyl group of diketone 14.19
Replacement of the nitro groups present in antimitotic
antikinetoplastid molecule 2b with cyano moieties
provides a compound (5) which displays potent and
selective activity against African trypanosomes and
Leishmania. Activity is also observed when other groups
are placed at these positions, although such molecules
are less potent than the corresponding nitro- and cya-
no-containing compounds. This knowledge, together
with refinement of the tubulin binding site model for
such ligands, represents a point of departure from the
dinitroaniline scaffold in the design of selective antitub-
ulin antiparasitics and provides renewed hope for the
development of tubulin-targeted drugs against African
trypanosomes, Leishmania, and perhaps other parasitic
protozoans as well.
Data shown in Table 1 and Figure 1 provide support for
the hypothesis that the disruption of microtubule assem-
bly plays a critical role in the antikinetoplastid action of
the target molecules. The rank order of these com-
pounds for activity against leishmanial tubulin assembly
(2b, 5, 4 > 3, 14 > 11–13) is very similar to the rank or-
der for activity against L. donovani axenic amastigotes
in vitro (2b > 5 > 3, 4 > 14 > 11–13) and T. b. brucei
in vitro (2b, 5 > 4, 14 > 3 > 11–13). Minor variation
between antiparasitic potency and activity against leish-
manial tubulin could be due to the ability of the com-
pounds to reach their cellular target or, in the case of
antitrypanosomal activity, to subtle differences between
the ligand susceptibility of leishmanial and trypanoso-
mal tubulin (the amino acid identity between L. tarento-
lae and T. brucei tubulin is 94%).26 Given the potent
antitrypanosomal activity and selectivity of compounds
2b and 5, further investigation of the effects of these
molecules on purified trypanosomal tubulin and the
trypanosomal cell cycle is warranted.
5. Experimental
5.1. General methods
All reagents and solvents were from the Sigma–Aldrich
Corporation (St. Louis, MO) unless otherwise indicated.
Nuclear magnetic resonance spectra were obtained at
1
250 or 300 MHz for H and 62.5 or 75 MHz for 13C
using instruments from Bruker. Thin layer chromatora-
phy was conducted on precoated TLC plates from E.
Merck (Darmstadt, Germany). HPLC analysis was car-
ried out using a System Gold Model 127 pump equipped
with a Model 166 UV detector (Beckman, Fullerton,
CA) and a 10 · 250 mm Polaris C18-A column (Varian,
Palo Alto, CA). Melting points were recorded on a Tho-
mas–Hoover capillary melting point apparatus and are
uncorrected.
In addition to the presence of nitro groups, another bar-
rier to the development of 2a and 2b as antikinetoplastid
drug candidates is metabolic instability. Neither
compound showed significant activity against murine
African trypanosomiasis when administered intraperito-
17
5.2. N1-Phenyl-3,5-diamino-N4,N4-di-n-butylsulfanila-
mide (3)
neally at 4 · 20 mg kgÀ1 dayÀ1
.
Rapid and extensive
metabolism of 2b was later observed, primarily through
N4-alkane oxidation, providing a rationale for the lack
of activity of these dinitroaniline sulfanilamides.35 While
the metabolic stability of 5 remains to be investigated,
this molecule retains the N4-dibutyl chain that was the
site of extensive metabolism in 2b. However, a recent
SAR investigation revealed that a range of hydrophobic
substituents was permitted at the N4 position in terms of
antiparasitic and antitubulin activity,18 and the tubulin
To a degassed solution of 2b (1 g, 2.22 mmol) in EtOAc
(44 mL), 10 wt% Pd on activated carbon (133 mg) was
added and stirred at rt under an atmosphere of H2 for
1 day. The reaction mixture was then filtered through
Celite and evaporated to afford 3 (850 mg, 98%) as a col-
orless solid, mp = 85–86 ꢁC. 1H NMR (CDCl3) d 0.88 (t,
J = 7.1 Hz, 6H), 1.19–1.44 (m, 8H), 2.95 (t, J = 7.7 Hz,
4H), 4.01 (bs, 4H), 6.51 (s, 2H), 6.61 (bs, 1H), 7.06–

T. G. George et al. / Bioorg. Med. Chem. 15 (2007) 6071–6079
6077
7.31 (m, 5H); 13C NMR (CDCl3) d 14.01, 20.48, 31.81,
52.68, 104.24, 121.61, 125.09, 125.15, 129.15, 136.85,
136.88, 146.75; HRMS (ESI) calcd for C20H30N4NaO2S
(M+Na)+ 413.1987, measured (M+Na)+ 413.1994.
Anal. (C20H30N4O2S) calcd C, 61.51; H, 7.74, N,
14.35; found C, 61.53; H, 7.77; N, 14.15.
(s, 6H), 7.94 (s, 2H); HRMS (ESI) calcd for C10H9BrO7S
(M)+ 351.9252, measured (M)+ 351.9255.
5.6. Dimethyl 2-bromo-5-(chlorosulfonyl)isophthalate (9)
A suspension of 8 (2.08 g, 5.89 mmol) in POCl3 (6.3 mL,
from Acros Organics, Morris Plains, NJ) was refluxed
for 24 h. This mixture was then cooled to rt, added to
ice water, and stirred for 20 min. The crystals formed
were filtered, washed with ice water, and dried to afford
9 (1.52 g, 70%) as colorless solid, mp = 98–99 ꢁC.1H
NMR (CDCl3) d 4.03 (s, 6H), 8.35 (s, 2H); 13C NMR
(CDCl3) d 53.51, 127.68, 129.93, 137.06, 142.98, 164.64.
5.3. N1-Phenyl-3,5-dichloro-N4,N4-di-n-butylsulfanil-
amide (4)
To a suspension of diamine 3 (200 mg, 0.51 mmol) in a
mixture of concd HCl (1.5 mL) and H2O (0.5 mL), an
ice-cold solution of NaNO2 in H2O (1 mL) was added
very slowly over 15 min at 0 ꢁC. To this an ice-cold solu-
tion of CuCl (126 mg, 1.26 mmol) in HCl (1 mL) was
added dropwise with stirring. The reaction mixture
was stirred for 3 h while warming to rt, then was neu-
tralized with 6 N NH4OH. This mixture was extracted
with dichloromethane and washed successively with
water, brine and dried. The crude product was then
purified by column chromatography using hexanes/
EtOAc (10:1) to afford 4 (51 mg, 23%) as a colorless
5.7. Dimethyl 2-bromo-5-(phenylsulfamoyl)isophthalate (10)
To a solution of 9 (3.0 g, 8.07 mmol) in acetone (60 mL),
pyridine (616 mg, 7.79 mmol, from Mallinckrodt Chem-
icals, Phillipsburg, NJ) was added at rt, and the solution
was cooled to 0 ꢁC. Aniline (725 mg, 7.79 mmol) was
then added and the solution was stirred at 0 ꢁC and al-
lowed to warm to rt overnight. Solvent was evaporated
and the remaining mixture was extracted using EtOAc.
The organic layer was washed with sat. NH4Cl, dried
over anhydrous Na2SO4, and evaporated. Purification
by silica gel flash column chromatography using hex-
ane/ethyl acetate 5:1 to 4:1 afforded 10 (2.29 g, 69%),
mp = 106–107 ꢁC. 1H NMR (CDCl3) d 3.95 (s, 6H),
6.68 (s, 1H), 7.07–7.34 (m, 5H), 8.04 (s, 2H); 13C
NMR (CDCl3) d 53.22, 122.58, 124.76, 126.45, 129.67,
130.49, 135.32, 136.16, 138.58, 165.35.
solid, mp = 90–91 ꢁC. 1H NMR (CDCl3)
d 0.86
(t, J = 7.2 Hz, 6H), 1.16–1.42 (m, 8H), 3.17 (t, J =
7.2 Hz, 4H), 6.62 (s, 1H), 7.09–7.34 (m, 5H), 7.65 (d,
J = 0.9 Hz, 2H); 13C NMR (CDCl3) d 13.93, 20.16,
30.79, 52.22, 122.00, 125.99, 127.94, 129.52, 134.82,
135.74, 135.83, 149.69. HRMS (ESI) calcd for
C20H26Cl2N2O2S (M+Na)+ 451.0990, measured
(M+Na)+ 451.0970. Anal. (C20H26Cl2N2O2S) calcd C,
55.94; H, 6.10, N, 6.52; found C, 56.11; H, 6.17; N, 6.48.
5.4. 2-Bromoisophthalic acid (7)21
5.8. Dimethyl 2-(di-n-butylamino)-5-(phenylsulfamoyl)iso-
phthalate (11)
To
a suspension of 2-bromo-m-xylene 6 (13.9 g,
75 mmol) in water (260 mL), KMnO4 (18 g, 113 mmol)
was added and refluxed overnight. Another portion of
KMnO4 (18 g, 113 mmol) was added and again refluxed
for 24 h. A third portion of KMnO4 (18 g, 113 mmol)
was added and refluxed for an additional day. The reac-
tion mixture was filtered while hot through Celite and
washed with hot water, then the combined aqueous
phase was evaporated to about 75 mL and neutralized
with 20% HCl. The reaction mixture was then kept at
0 ꢁC overnight and the crystals formed were filtered
and dried to afford 7 (12.1 g, 66%) as a white solid,
mp = 209–210 ꢁC (literature 218 ꢁC36). 1H NMR
(DMSO-d6) d 7.50–7.71 (m, 3H). 13.61 (s, 2H).
To a solution of 10 (2.26 g, 5.27 mmol) in dioxane
(40 mL), K2CO3 (870 mg, 6.3 mmol) was added followed
by Cu (50 mg, 0.79 mmol) and dibutylamine (2.07 g,
16.0 mmol). The reaction mixture was refluxed overnight.
Solvent was evaporated, then the residue was extracted
with EtOAc, and washed with sat. NH4Cl followed by
brine and dried using anhydrous MgSO4. Purification
by silica gel flash column chromatography (hexane/ethyl
acetate 10:1) afforded 11 (1.99 g, 79%) as a yellow viscous
liquid. 1H NMR (CDCl3) d 0.86 (t, J = 7.3 Hz, 6H), 1.19–
1.31 (m, 4H), 1.47–1.57 (m, 4H), 3.04 (t, J = 7.4 Hz, 4H),
3.87 (s, 6H), 6.76 (s, 1H), 7.08–7.30 (m, 5H), 8.01 (s, 2H);
13C NMR (CDCl3) d 13.81, 19.95, 29.63, 52.58, 52.86,
121.82, 125.54, 126.23, 128.36, 129.41, 133.25, 136.25,
153.36, 167.25; HRMS (ESI) calcd for C24H33N2O6S
(M+H)+ 477.2059, measured (M+H)+ 477.2061. Anal.
(C24H32N2O6S) calcd C, 60.48; H, 6.77, N, 5.88; found
C, 60.65; H, 7.02; N, 5.50.
5.5. 4-Bromo-3,5-bis(methoxycarbonyl)benzenesulfonic
acid (8)
2-Bromoisophthalicacid7 (5.0 g, 20.4 mmol)was added to
fuming sulfuric acid (26–29%, 12 mL) at rt and heated to
170 ꢁC for 4 h. The reaction mixture was cooled to rt,
added to ice, and neutralized with sodium bicarbonate.
The crystals formed were filtered, co-evaporated with tolu-
ene, and dried. This crude material was then suspended in
MeOH (150 mL) and concd HCl (6 mL) was added and
refluxedfor24 h. Solventwasremovedunderreducedpres-
sure, then the residue was co-evaporated with toluene (3·)
and dried under vacuum to afford 8 (5.7 g, 79% after two
5.9. 2-(Di-n-butylamino)-5-(phenylsulfamoyl)isophthalic
acid (12)
To a solution of 11 (518 mg, 1.09 mmol) in MeOH
(30 mL), 3 N NaOH (5 mL) was added at rt and the reac-
tion mixture was refluxed for 2 days. Solvent was evapo-
rated; the residue was dissolved in water (5 mL) and
neutralizedwith2 N HCl. The crystalsformedwerefiltered
and dried to afford 12 (430 mg, 88%) as a colorless solid,
1
steps), mp = 139–140 ꢁC. H NMR (DMSO-d6) d 3.88

6078
T. G. George et al. / Bioorg. Med. Chem. 15 (2007) 6071–6079
mp = 237–238 ꢁC. 1H NMR (DMSO-d6) d 0.77 (t,
J = 7.2 Hz, 6H), 1.12–1.24 (m, 4H), 1.33–1.42 (m, 4H),
3.10–3.15 (m, 4H), 7.03–7.27 (m, 5H), 7.99 (s, 2H), 10.28
(s, 1H), 14.19 (bs, 2H); 13C NMR (DMSO-d6) d 14.07,
19.88, 29.50, 53.28, 120.93, 124.95, 129.35, 129.69,
131.65, 132.36, 137.81, 150.72, 168.05; HRMS (ESI) calcd
for C22H28N2NaO6S (M+Na)+ 471.1566, measured
(M+Na)+ 471.1560. Anal. (C22H28N2O6S) calcd C, 58.91;
H, 6.29, N, 6.25; found C, 58.31; H, 6.09; N, 5.99.
4H), 6.50 (s, 1H), 7.07–7.28 (m, 5H), 7.72 (s, 2H); 13C
NMR (CDCl3) d 13.76, 20.11, 29.03, 29.58, 54.22,
122.17, 125.71, 129.36, 129.54, 130.30, 135.83, 136.34,
151.13, 200.95; HRMS (ESI) calcd for C24H32N2NaO4S
(M+Na)+ 467.1980, measured (M+Na)+ 467.1984.
5.12. N1-Phenyl-3,5-dicyano-N4,N4-di-n-butylsulfanil-
amide (5)
The title compound was synthesized with slight modifica-
tion of the literature procedure.24 To a solution of bis-
amide 13 (65 mg, 0.146 mmol) in a mixture of acetonitrile
and water (2:1 ratio, 15 mL), PdCl2 (12.5 mg,
0.073 mmol) was added. The resulting yellow solution
was stirred at 70 ꢁC for 24 h. Acetonitrile was removed
in vacuo and the residue was extracted with CH2Cl2 (3·
10 mL) and dried over anhydrous Na2SO4. Silica gel flash
column chromatography [hexane/EtOAc (10:1)] of the
crude material gave compound 5 (42.5 mg, 71%) as white
solid, mp = 145–146 ꢁC. 1H NMR (CDCl3)d 0.91 (t,
J = 7.2 Hz, 6H), 1.23–1.39 (m, 4H), 1.55–1.65 (m, 4H),
3.62 (m, 4H), 6.64 (s, 1H), 7.09–7.37 (m, 5H), 7.99 (m,
2H); 13C NMR (CDCl3) d 13.64, 19.69, 30.09, 52.77,
107.56, 116.15, 122.15, 126.51, 129.76, 130.80, 135.33,
138.11, 158.89; HRMS (ESI) calcd for C22H26N4NaO2S
(M+Na)+ 433.1674, measured (M+Na)+ 433.1666. Anal.
(C22H26N4O2S) calcd C, 64.36; H, 6.38, N, 13.65; found
C, 64.00; H, 6.40; N, 13.13.
5.10. 2-(Di-n-butylamino)-5-(phenylsulfamoyl)isophthal-
amide (13)
To a suspension of the diacid 12 (50 mg, 0.11 mmol) and tri-
phenylphosphine (58 mg, 0.22 mmol, from Acros Organics,
Morris Plains, NJ) in THF (3 mL), hexachloroacetone
(30 lL, 0.20 mmol) was added at 0 ꢁC and stirred for 1 h.
Then, 0.5 M NH3 in dioxane (1.32 mL, 0.67 mmol) was
added, the reaction mixture was allowed to warm to rt
and was stirred for 1 h. Solvent was evaporated and water
was added. The product was then extracted with EtOAc,
washed with brine, and dried over anhydrous Na2SO4.
Evaporation of the solvent followed by column chromatog-
raphy using hexane/EtOAc 3:1 to 2:1 afforded 13 (33.7 mg,
68%) as a colorless solid, mp = 161–162 ꢁC. HPLC (MeOH/
H2O 65:35, 1.0 mL/min, 254 nm) >98%, tR = 4.09 min. 1H
NMR (acetone-d6) d 0.84 (t, J = 7.4 Hz, 6H), 1.19–1.32
(m, 4H), 1.45–1.56 (m, 4H), 3.17–3.22 (m, 4H), 6.89 (bs,
2H), 7.07–7.10 (m, 1H), 7.21–7.29 (m, 4H), 7.89 (bs, 2H),
8.01 (s, 2H), 9.02 (s, 1H); 13C NMR (acetone-d6) 14.27,
21.18, 31.05, 54.47, 121.71, 125.50, 130.15, 130.87, 134.44,
135.32, 138.75, 152.20, 169.61. HRMS (ESI) calcd for
C22H30N4O4S (M+H)+ 447.2066, measured (M+H)+
447.2046.
5.13. Susceptibility testing of parasites and Vero cells
The susceptibility of L. donovani axenic amastigote-like
parasites,16,37 T. b. brucei bloodstream forms,38 and
Vero cells38 to growth inhibition by compounds of inter-
est was assayed as described previously.
5.11. N1-Phenyl-3,5-diacetyl-N4,N4-di-n-butylsulfanil-
amide (14)
5.14. Tubulin assembly assays
To a solution of diacid 12 (50 mg, 0.11 mmol) and tri-
phenylphosphine (58 mg, 0.22 mmol) in dry THF
(3 mL), hexachloroacetone (30 lL, 0.20 mmol) was
added at 0 ꢁC. The clear yellow reaction mixture was
stirred at 0 ꢁC for 1.5 h to give the corresponding diacyl
chloride, which was used as such for next reaction. To a
suspension of CuBr (157 mg, 1.11 mmol) in THF
(2 mL), methyl magnesium bromide (370 lL, 3.0 M in
THF, 1.11 mmol) was added at 0 ꢁC and stirred at room
temperature for 1 h, which gave a light yellow green sus-
pension. The reaction mixture was cooled once again to
0 ꢁC, and the solution of the diacyl chloride derivative of
12 in THF (3 mL) was transferred by cannula. The
resulting reaction mixture was stirred at 0 ꢁC for
40 min. Water (6 mL) was added, phases were sepa-
rated, and the aqueous phase was extracted with EtOAc
(3· 5 mL). The combined organic layers were washed
with aq NH4Cl (10 mL, 10% w/v), dried over anhydrous
Na2SO4, and concentrated. Silica gel flash column chro-
matography of the crude material [hexane/EtOAc (8:1 to
4:1)] gave compound 14 (26.7 mg, 55%) as light yellow
solid, mp = 99–100 ꢁC. HPLC (MeOH/H2O 80:20,
1.0 mL/min, 254 nm), >98%, tR = 5.24 min. 1H NMR
(CDCl3) d 0.85 (t, J = 7.2 Hz, 6H), 1.14–1.26 (m, 4H),
1.41–1.53 (m, 4H), 2.46 (s, 6H), 2.94 (t, J = 7.8 Hz,
Tubulin from L. tarentolae26 and from pig brain17 was
isolated as outlined earlier. Assembly reactions, used
to assess the effect of compounds on tubulin polymeriza-
tion, were carried out as described previously.17,26
5.15. AutoDock
AutoDock version 4.0.029 was used for docking simula-
tions. The Lamarckian genetic algorithm (LGA) was se-
lected for ligand conformational searching because it
has enhanced performance relative to simulated anneal-
ing or the simple genetic algorithm. For the ligands, all
hydrogens were added, Gasteiger charges39 were as-
signed, then non-polar hydrogens were merged.
80 · 80 · 70 3-D affinity grids centered on the 2b binding
˚
site with 0.375 A spacing were calculated for each of the
following atom types: (a) protein: A (aromatic C), C,
HD, N, NA, OA, SA; (b) ligand: C, A, OA, HD, S,
N, NA, e (electrostatic), and d (desolvation) using Auto-
grid4. The ligand’s translation, rotation, and internal
torsions are defined as its state variables and each gene
represents a state variable. LGA adds local minimiza-
tion to the genetic algorithm, enabling modification of
the gene population. The docking parameters were as
follows: trials of 100 dockings, population size of 150,

T. G. George et al. / Bioorg. Med. Chem. 15 (2007) 6071–6079
6079
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random starting position and conformation, translation
˚
step ranges of 2.0 A, rotation step ranges of 50ꢁ, elitism
of 1, mutation rate of 0.02, crossover rate of 0.8, local
search rate of 0.06, and 50 million energy evaluations.
Final docked conformations were clustered using a tol-
˚
erance of 1.5 A root-mean-square deviations (RMSD).
5.16. Glide30
Ligand preparation: the molecules were built in Maestro
Suite and were minimized using the OPLS-2005 force-
field. A maximum of 32 stereoisomers were allowed to
generate per ligand. Possible ionization states were
assigned at a target pH of 7.0 2.0. Target preparation:
the homology modeled structure of Leishmania tubulin19
was prepared for docking using the protein preparation
utility provided by Schrodinger LLC. This process opti-
mizes the protonation states and tautomers of His resi-
dues as well as the position of hydroxyl and thiol
hydrogens. The structure was then subjected to impact
´
18. George, T.; Johnsamuel, J.; Delfın, D.; Yakovich, A.;
Mukherjee, M.; Phelps, M.; Dalton, J.; Sackett, D.;
Kaiser, M.; Brun, R.; Werbovetz, K. Bioorg. Med. Chem.
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˚
minimization with a cut off RMSD of 0.3 A. Partial
atomic charges were assigned according to the OPLS-
2005 force field. Molecular Docking: the model was sub-
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˚
mitted to grid map calculations using a box of 12 A cube
centered on the bound ligand 2b. The consequent docking
has been conducted using the Glide program, with the
default settings using the previously prepared ligands.
The most stable Glide pose, one per ligand, was retained.
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Acknowledgments
25. Dai, Y.; Guo, Y.; Frey, R.; Ji, Z.; Curtin, M.; Ahmed,
A.; Albert, D.; Arnold, L.; Arries, S.; Barlozzari, T.;
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This work was funded by NIH Grant AI061021 (to
K.A.W.). We also thank Dr. David Sept for helpful
comments.
Supplementary data
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line version, at doi:10.1016/j.bmc.2007.06.042.
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