Synthesis and Antitubulin Activity of N1- and N 4-Substituted 3,5-Dinitro Sulfanilamides against African Trypanosomes and Leishmania

Article abstract of DOI:10.1021/jm0304461

Thirty analogues of N¹-phenyl-3,5-dinitro-N⁴,N ⁴-di-n-propylsulfanilamide (GB-II-5, compound 3), a new antikinetoplastid antimitotic agent, have been synthesized and evaluated. The addition of simple functional groups to the N1 aromatic ring generally decreases antiparasitic and antimitotic potency, but placement of a dibutyl substituent at the N4 nitrogen to give N¹-phenyl-3,5-dinitro-N ⁴,N⁴-di-n-butylsulfanilamide (compound 35) augments antitrypanosomal and antileishmanial activity. Compound 35 possesses IC ₅₀ values of 0.12 and 2.6 μM against cultured T. brucei and L. donovani amastigote-like forms, surpassing the activity of compound 3 against these parasites by 3.4- and 1.9-fold, respectively. Compound 35 inhibits the assembly of leishmanial tubulin with an IC₅₀ of 6.9 μM and displays antimitotic effects in cultured T. brucei as assessed by flow cytometry, but shows little effect on purified mammalian tubulin, and displays 100-fold selectivity for trypanosomes over two mammalian cell lines. Although 3 and 35 were not effective in initial in vivo antitrypanosomal assays, the in vitro potency and selectivity of these compounds make N ¹-aryl-3,5-dinitro-N⁴,N⁴-dialkylsulfanilamides a promising new class of antikinetoplastid agents that act on parasite tubulin.

Full text of DOI:10.1021/jm0304461

J . Med. Chem. 2004, 47, 1823-1832
1823
Syn th esis a n d An titu bu lin Activity of N1- a n d N4-Su bstitu ted 3,5-Din itr o
Su lfa n ila m id es a ga in st Afr ica n Tr yp a n osom es a n d Leish m a n ia
Gautam Bhattacharya,† J ohnathan Herman,† Dawn Delf´ın,† Manar M. Salem,† Todd Barszcz,† Mike Mollet,‡
Guy Riccio,# Reto Brun,# and Karl A. Werbovetz*,†
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue,
Columbus, Ohio 43210, University Cell Analysis and Sorting Core, Heart and Lung Research Institute, The Ohio State
University, Columbus, Ohio 43210, and Swiss Tropical Institute, Basel, CH4002, Switzerland
Received September 9, 2003
Thirty analogues of N1-phenyl-3,5-dinitro-N4,N4-di-n-propylsulfanilamide (GB-II-5, compound
3), a new antikinetoplastid antimitotic agent, have been synthesized and evaluated. The addition
of simple functional groups to the N1 aromatic ring generally decreases antiparasitic and
antimitotic potency, but placement of a dibutyl substituent at the N4 nitrogen to give N1-
phenyl-3,5-dinitro-N4,N4-di-n-butylsulfanilamide (compound 35) augments antitrypanosomal
and antileishmanial activity. Compound 35 possesses IC50 values of 0.12 and 2.6 µM against
cultured T. brucei and L. donovani amastigote-like forms, surpassing the activity of compound
3 against these parasites by 3.4- and 1.9-fold, respectively. Compound 35 inhibits the assembly
of leishmanial tubulin with an IC50 of 6.9 µM and displays antimitotic effects in cultured
T. brucei as assessed by flow cytometry, but shows little effect on purified mammalian tubulin,
and displays 100-fold selectivity for trypanosomes over two mammalian cell lines. Although 3
and 35 were not effective in initial in vivo antitrypanosomal assays, the in vitro potency and
selectivity of these compounds make N1-aryl-3,5-dinitro-N4,N4-dialkylsulfanilamides a promising
new class of antikinetoplastid agents that act on parasite tubulin.
In tr od u ction
anticancer chemotherapy5 and in the treatment of
helminth infections.6 Clearly, selective ligands for ki-
netoplastid tubulin would be of great interest as drug
candidates against leishmaniasis and African trypano-
somiasis. Previous work from our group and another
laboratory has shown that classical mammalian colchi-
cine-site antimicrotubule agents possess little activity
against purified leishmanial tubulin7 and trypanosomal
tubulin,8 indicating that differences in drug susceptibil-
ity exist between the kinetoplastid and mammalian
proteins. Over 10 years ago, the antimitotic herbicides
trifluralin 19 and oryzalin 210 were suggested as selec-
tive lead compounds against leishmanial tubulin. How-
ever, no reports of related molecules with improved
activity against leishmanial tubulin or with heightened
efficacy against the parasite have appeared until re-
cently. In 2002, we showed that simple synthetic
modifications to the N1 and N4 positions of oryzalin’s
sulfonamide core augmented activity compared to the
parent compound against purified leishmanial tubulin
and against Leishmania donovani parasite growth.11
Subsequent work from our lab indicated that one of
these compounds, N1-phenyl-3,5-dinitro-N4,N4-di-n-pro-
pylsulfanilamide (GB-II-5, compound 3 in this paper),
possessed selectivity for leishmanial tubulin compared
to mammalian tubulin in vitro and affected cell division
in Leishmania parasites. Compound 3 also blocked the
growth of African trypanosomes in vitro and caused a
striking accumulation of T. brucei parasites in the G2M
cell cycle phases at 0.5 µM.12 Compound 3 is thus
an exciting lead molecule against kinetoplastid para-
sites that selectively targets the tubulin of these
organisms (see Figure 1 for the structures of compounds
1-3).
The need for new drugs against leishmaniasis and
African trypanosomiasis is clear. These diseases, both
caused by protozoan parasites of the order Kinetoplas-
tida, are major public health threats in many developing
countries. It is estimated that 1.5-2.0 million new cases
of leishmaniasis occur each year, half a million of which
are of the potentially fatal visceral manifestation (http://
www.who.int/inf-fs/en/fact116.html). Although only about
45 000 cases of African trypanosomiasis are reported
annually, the World Health Organization estimates that
300 000 to 500 000 cases of the disease actually occur
each year (http://www.who.int/inf-fs/en/fact259.html).
Both visceral leishmaniasis and African trypanosomia-
sis are fatal in the absence of treatment. Unfortunately,
the chemotherapeutic mainstays against these diseases
are administered by injection, are moderately to ex-
tremely toxic, and in some cases are compromised by
the development of resistance (see refs 1 and 2 for recent
reviews). Rays of hope are provided by the registration
of miltefosine in India as the first oral treatment for
visceral leishmaniasis3 and clinical trials of the orally
available pentamidine analogue DB289 as a candidate
for the treatment of early-stage African trypanosomia-
sis.4 The widespread utility of these agents is not yet
established, however, and the development of new drug
candidates with oral activity against these diseases
remains critical.
Tubulin, which plays an indispensable role in eukary-
otic cell division, is an established drug target in
* To whom correspondence should be addressed. Phone: 614-292-
5499. Fax: 614-292-2435. E-mail: werbovetz.1@osu.edu.
† College of Pharmacy, The Ohio State University.
‡ Heart and Lung Research Institute, The Ohio State University.
#
Swiss Tropical Institute.
10.1021/jm0304461 CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/14/2004

1824 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Bhattacharya et al.
Sch em e 2a
a
Reagents and conditions: (i) ClSO3H, 120 °C; (ii) PhNH2/
pyridine, 45 °C; (iii) dipropylamine, reflux.
F igu r e 1. Structures of compounds 1-3.
(method B). Alkylamines reacted with 6a in the amine
as solvent (method C), in THF (method D), or in
dichloromethane (method E) to form sulfonamides 18-
33 in good to excellent yield.
Sch em e 1a
The mononitrosulfonamide derivative 36 was synthe-
sized starting from 1-chloro-2-nitrobenzene 37 as shown
in Scheme 2. Thus 37, when reacted with chlorosulfonic
acid at 120 °C, produces the sulfonyl chloride derivative
38, which when treated with aniline in the presence of
pyridine at 45 °C affords sulfonamide 39. Compound 39
was converted to mononitro N1-substituted sulfonamide
36 through the Ullmann condensation of 39 with
dipropylamine.
Biologica l Resu lts
Table 1 shows the structures of the compounds
synthesized, and Table 2 displays the in vitro activities
of these agents against axenic amastigote-like L. dono-
vani, T. brucei variant 221 bloodstream forms, and two
mammalian cancer cell lines. Most of these compounds
are more active against T. brucei than against L.
donovani, perhaps because of the rapid proliferation
rate of trypanosomes in culture. In general, an aryl
substituent placed on the sulfonamide nitrogen (the N1
position of the sulfanilamide core) increases activity
against kinetoplastid parasites compared to oryzalin 2.
The meta-substituted compounds were more active
against Leishmania and T. brucei than those molecules
containing a para substituent, indicating a steric bias
against para-substituted compounds. The m-chloro com-
pound 12 possesses an activity similar to that of
compound 3 against Leishmania, while 10 is the most
active meta-substituted compound against T. brucei.
When a dipropyl substituent is present at the N4
position, as is the case with most of the analogues, the
3,5-dichloro-substituted 14 possesses the strongest an-
tileishmanial activity, being slightly more active against
L. donovani than the mono-chlorinated 12 and the
unsubstituted 3. In vitro antileishmanial activity gener-
ally correlates with the inhibition of leishmanial tubulin
assembly shown in Table 3, where the activity of the
five most potent analogues against the proposed target
follows the order 14 > 3 ∼ 35 > 12 > 10. Only compound
13, which is a comparatively weak leishmanial tubulin
inhibitor, joins the five compounds mentioned above as
agents with IC50 values below 10 µM against L. dono-
vani in vitro. All of the N1-alkyl compounds are consid-
erably less active than the most potent N1-aryl
analogues, although analogues with N1-monoalkyl sub-
stituents bearing less than three carbons are more
a
Reagents and conditions: (i) dialkylamine or dialkylamine/
MeOH, reflux; (ii) PCl5/CH2Cl2; (iii) method A: 7 N ammonia in
MeOH, 40 °C; method B: YH/pyridine, 45 °C; method C: YH;
method D, YH in THF; method E, YH in CH2Cl2. Note: Y ) NHR
or NR1R2 (see Table 1).
We now report the synthesis and in vitro antileish-
manial and antitrypanosomal activity of 30 compounds
based on 3, as well as the antitubulin effects and
selectivity of the molecules with the most potent anti-
kinetoplastid activity. Most of these agents possess
variations at the N1 position of oryzalin’s sulfanilamide
core, although we also show that modification at the N4
position can augment antiparasitic potency. In general,
the compounds with the highest activity against purified
leishmanial tubulin are also the most active against
L. donovani and T. brucei parasites in vitro.
Ch em istr y
Dinitroanilines were synthesized as outlined in Scheme
1. A modified Ullman condensation of 3,5-dinitro-4-
chloro-benzenesulfonate 413 provided intermediates 5a-c
in high yield. These N4,N4-disubstituted anilinosul-
fonate derivatives were then converted to sulfonyl
chlorides 6a-c by reaction with PCl5 in dichloromethane
as solvent. Transformation of compounds 6a -c to the
target sulfonamides 2, 3, and 7-35 was accomplished
by reaction of the sulfonyl chlorides with ammonia,
aliphatic amines, or aromatic amines in pyridine at 45
°C. Treatment of 6a with methanolic ammonia (method
A) provided oryzalin 2 in excellent yield. Conversion of
the sulfonyl chlorides to the N1-aryl compounds 3, 7-17,
and 34-36 was accomplished in good yield by heating
the aromatic amines with 6a -6c in pyridine at 45 °C

Sulfanilamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1825
Ta ble 1. N1- and N4-Substituted 3,5-Dinitrosulfanilamides Synthesized
potent against T. brucei than oryzalin 2. The compound
with the most potent antikinetoplastid activity in this
series is 35, which contains a phenyl ring at N1 and a
dibutyl substitution at N4. In agreement with our
previous observation that 3 was over an order of
magnitude more potent against T. brucei bloodstream
forms than against L. donovani axenic amastigotes,12
35 is 48 times more potent against African trypano-
somes than against L. donovani in vitro.
Although the structure-activity relationships for the
oryzalin analogues are similar for L. donovani and T.
brucei, there are some intriguing distinctions. In addi-
tion to differences in the order of activity for the meta-
substituted N1-aryl compounds mentioned earlier, the
3,5-dichloro compound 14 is substantially less active
against trypanosomes than compounds 3, 10, 11, and
12. Also, the compounds with short N1-monoalkyl chains
(18, 20, 29, and 30) are more active than 14 against T.
brucei. This is despite the fact that compound 14
inhibits leishmanial tubulin assembly with an IC50 of
5.8 µM, while compounds 18 and 29 show little activity
in this assay when tested at a concentration of 10 µM
(see Table 3). These results suggest that differences in
drug susceptibility exist between leishmanial and try-
panosomal tubulin. Given that leishmanial R- and
â-tubulins share 94% and 93% amino acid sequence
identity with tubulin from African trypanosomes, these
data are a bit surprising. However, even our most potent
compounds show little activity against porcine brain
tubulin, despite the fact that mammalian tubulins share
approximately 80% amino acid identity with leishmanial
tubulins. When tested at a concentration of 40 µM,
compounds 3, 7-14, 18, 29, and 34-36 inhibit the
assembly of porcine brain tubulin by less than 20% (in
five separate experiments, compound 3 inhibits the
assembly of the mammalian protein by 14 ( 10%).
Substitutions of one or two amino acids in the target
protein are thus likely to have a dramatic influence on
the binding affinities of these compounds for tubulins
from different species.
Other structural modifications decrease antikineto-
plastid activity. Compared to compound 3, replacement
of the N1 hydrogen with a methyl group as in 16
decreases antikinetoplastid activity 2.5- to 4-fold, while
exchange of the N1 hydrogen with a phenyl group as in
17 causes a more drastic diminution in activity. Re-
placement of a nitro group on the sulfanilamide aro-
matic ring with a hydrogen also had an adverse effect

1826 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Bhattacharya et al.
Ta ble 2. IC50 Values for N1- and N4-Substituted 3,5-Dinitrosulfanilamides (µM) against Protozoan Parasites and Mammalian Cell
Lines
compd
L. donovani axenic amastigotes
T. b. brucei variant 221
J 774 macrophages
PC3 prostate
56 ( 20
2
3
7
8
65 ( 4
5.0 ( 1.1
32 ( 15
32 ( 13
13 ( 3
8.1 ( 3.3
23 ( 1
5.5 ( 0.1
5.0 ( 0.8
3.7 ( 0.9
60 ( 6
21 ( 3
>100
12 ( 0
44 ( 8
28 ( 4
15 ( 7
11 ( 2
10 ( 3
25 ( 4
16 ( 2
8.7 ( 0.2
7.2 ( 0.6
5.1 ( 0.4
45 ( 16
11 ( 3
>100
0.41 ( 0.05
19 ( 3
31 ( 8
31 ( 5
21 ( 2
16 ( 5
36 ( 10
25 ( 0
18 ( 1
15 ( 4
11 ( 5
NDa
13 ( 0
13 ( 3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
1.3 ( 0.2
1.9 ( 0.2
2.1 ( 0.2
7.9 ( 0.6
8.0 ( 0.6
9.2 ( 1.7
1.0 ( 0.2
7.0 ( 0.5
5.3 ( 0.4
5.8 ( 1.0
5.4 ( 0.5
8.3 ( 1.8
8.9 ( 0.8
17 ( 1
>100
>100
NDa
>100
NDa
>100
NDa
NDa
>100
NDa
NDa
27 ( 0
54 ( 9
55 ( 3
50 ( 24
48 ( 7
43 ( 11
26 ( 5
>100
NDa
NDa
NDa
NDa
NDa
NDa
12 ( 1
NDa
NDa
38 ( 9
NDa
NDa
17 ( 3
NDa
NDa
15 ( 1
NDa
NDa
>50
NDa
NDa
>100
4.1 ( 0.3
3.4 ( 0.5
11 ( 1
NDa
NDa
43 ( 2
50 ( 7
>100
NDa
NDa
NDa
NDa
>100
NDa
NDa
47 ( 4
11 ( 3
2.6 ( 0.3
43 ( 3
2.0 ( 0.1
NDa
6.5 ( 0.6
2.8 ( 0.3
0.12 ( 0.07
20( 2
NDa
NDa
48 ( 2
12 ( 1
31 ( 5
NDa
56 ( 14
13 ( 2
48 ( 15
NDa
pentamidine
suramin
ansamitocin P3
0.013 ( 0.003
0.21 ( 0.10
NDa
NDa
NDa
NDa
0.00047 ( 0.00022
0.000060 ( 0.000042
a
ND: not determined.
Ta ble 3. Antimicrotubule Activity of Selected Compounds
compounds for kinetoplastid parasites, these agents
were examined for their effects on the proliferation of
murine J 774 macrophages and PC3 prostate cancer
cells. Many of the compounds, including the lead
compound oryzalin 2, showed little selectivity for kine-
toplastid parasites over the tumor cell lines. This is not
surprising, given that oryzalin has been investigated as
an anticancer candidate.14 However, striking antitry-
panosomal selectivity is observed for compounds 3 and
35. The selective action of compound 3 against African
trypanosomes was recently demonstrated,12 and com-
pound 35 is 100-fold and 110-fold more active against
T. brucei than J 774 macrophages and PC3 prostate
cancer cells in vitro, respectively. Although compounds
3, 10, and 35 are clearly more active against L. donovani
in vitro than against the mammalian cell lines, the
selectivity in this case is much less dramatic. These
agents are 5.6-fold, 3.1-fold, and 4.6-fold more active
against L. donovani than against the J 774 cells. It is
likely that antitubulin activity plays a major factor in
selectivity, since compounds 3, 10, and 35 all inhibit
leishmanial tubulin assembly at low micromolar con-
centrations (Table 3) and are much less potent against
porcine brain tubulin. Given the results in Table 3 and
the known effect of oryzalin 2 against intracellular
calcium signaling in mammalian cells,14 the mild to
moderate toxicity against the two mammalian cell lines
displayed by the N1-aryl compounds may be due to
mechanisms other than interference with microtubule-
mediated processes.
% inhibition of
leishmanial tubulin
compd
assembly at 10 µM compounda
IC50 (µM)b
3
7
8
9
89 ( 10c
<20
6.7 ( 0.7
NDd
<20
NDd
<20
NDd
10
11
12
13
14
16
18
22
29
31
34
35
36
39 ( 3
<20
NDd
NDd
57 ( 3
<20
NDd
NDd
102 ( 3
<20
5.8 ( 0.7
NDd
<20
NDd
<20
NDd
<20
NDd
<20
NDd
<20
NDd
104 ( 1
<20
6.9 ( 0.0
NDd
a
IC50 value ( standard deviation of one duplicate experiment
b
unless otherwise noted. IC50 value ( standard deviation of two
duplicate experiments. c IC50 value ( standard deviation of five
d
duplicate experiments. ND: not determined.
on antiparasitic activity and potency against leishma-
nial tubulin (note the 49-fold loss in antitrypanosomal
activity in 36 compared to 3). We earlier reported that
restricting the conformation of the N4-alkylamino chain
and changing the sulfonamide group at N1 to another
functional group decreased antileishmanial and anti-
tubulin activity.11
To assess the selectivity of the more potent N1-aryl

Sulfanilamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1827
F igu r e 2. Flow cytometry analysis of T. brucei treated with compound 35. Bloodstream-form T. brucei parasites (MITat 1.2,
variant 221) were incubated for 24 h at 37 °C in HMI-9 medium in the presence of 1% DMSO (A), 0.2 µM compound 35 (B), or 0.4
µM compound 35 (C). Trypanosomes were fixed, stained with propidium iodide, and analyzed by flow cytometry as described in
the Experimental Section. The results shown are from a representative experiment performed on three separate occasions. FL2-
A, the x-axis label, refers to the fluorescence intensity of a given particle as measured by the flow cytometer.
Ta ble 4. In Vivo Assays with Compounds 3 and 35 against T.
b. brucei STIB 795
tubulin and cultured organisms indicate that these
compounds act by interfering with microtubules in
dose
(mg/kg)
average survival
time (day)
kinetoplastid parasites. Future studies will seek to
augment the potency of the N1-aryl-N4-dialkyl-3,5-
dinitrosulfanilamides against trypanosomes and Leish-
mania in vitro and to determine what pharmacokinetic
barriers must be overcome to attain in vivo antikine-
toplastid activity with these agents. Since the synthesis
of these compounds is straightforward, a wide variety
of analogues can be prepared to optimize in vitro and
in vivo activity. In addition, the inexpensive large-scale
synthesis of appropriate candidates should be possible.
Given the economic realities of chemotherapy for try-
panosomiasis and leishmaniasis, the simplicity of the
N1-aryl-N4-dialkyl-3,5-dinitrosulfanilamides bodes well
for their future development if in vivo activity can be
achieved.
group
cured/infected
control
3
35
0/4
0/4
0/4
6.75
8.0
8.0
4 × 20
4 × 20
Past work showed that exposure of L. donovani and
T. brucei to compound 3 caused clear, dose-dependent
effects on the parasite cell cycle, consistent with the
hypothesis that this agent exerts its antiparasitic effects
through a tubulin mechanism.12 Similar effects are
observed when African trypanosomes are cultured with
compound 35 (Figure 2). Trypanosomes incubated for
24 h in the presence of 200 nM compound 35 show an
increase in the percentage of cells in the G2M cell cycle
phases compared to those in the G1 phase. When the
appropriate peak areas were quantitated in four inde-
pendent experiments, the G2M/G1 ratio was 0.68 ( 0.08
for control samples and 1.14 ( 0.09 for cultures treated
with 200 nM compound 35 (values represent mean (
standard deviation). Treatment with 400 nM compound
35 causes the appearance of cell types possessing 4
times the amount of DNA present in G1 parasites,
indicating a dramatic effect on the trypanosome cell
cycle under these conditions. These results, taken
together with the effect of compound 35 on purified
leishmanial tubulin, provide support for the hypothesis
that compound 35 also acts through a tubulin mecha-
nism against kinetoplastid parasites.
Exp er im en ta l Section
Gen er a l Meth od s. Unless otherwise indicated, all reagents
and solvents were from Aldrich and were used without further
purification. The melting points were measured on Thomas-
Hoover capillary melting point apparatus and are uncorrected.
Nuclear magnetic resonance (NMR) spectra were recorded on
Bruker 250, 400, and 600 MHz NMR spectrometers. Elemental
analyses were performed by Atlantic Microlabs, Atlanta, GA.
Thin-layer chromatography was conducted on precoated TLC
plates from E. Merck. The yield of sulfonamides is given as
the yield of the products after two steps, i.e., after the
formation of the sulfonyl chloride and after the subsequent
synthesis of the sulfonamide target by reaction of the sulfonyl
chloride with the appropriate amine.
3,5-Din itr o-N4,N4-d i-n -p r op ylsu lfon a te (5a ). A suspen-
sion of 3,5-dinitro-4-chlorosulfonate13 (4, 2.82 g, 8.8 mmol) in
dipropylamine (50 mL) was refluxed for 3 h. By that time
almost all the solid had dissolved. After 3 h, the solvent was
evaporated, and water was added. The resulting yellow
precipitate was filtered and collected. The product was purified
by silica gel column chromatography, eluting with MeOH/
To examine the in vivo antitrypanosomal potential of
compounds 3 and 35, these agents were tested for
activity against T. b. brucei strain STIB 795 in mice
(Table 4). When the compounds were given an intra-
peritoneal dose of 20 mg kg-1 day-1 for 4 days, no
significant increase in survival time was observed
compared to control animals. No signs of toxicity were
observed when either of these compounds were given
at the 20 mg/kg dose.
1
EtOAc (1:8): yield 2.6 g, 77%; H NMR (DMSO-d6) δ 0.88 (t,
3H, J ) 7.25 Hz), 0.90 (t, 3H, J ) 7.75 Hz), 1.51 (m, 2H), 1.61
(m, 2H), 2.88 (q, 4H, J ) 8.50 Hz), 8.17 (s, 2H); MS m/z 348
(M + H)+.
3,5-Din itr o-N4,N4-d i-n -eth ylsu lfon a te (5b). A suspension
of 3,5-dinitro-4-chlorosulfonate (4, 1.41 g, 4.4 mmol) in diethyl-
amine (30 mL) was refluxed for 3 h. After 3 h, the solvent was
evaporated, then water was added to the solid. The resulting
yellow precipitate was filtered out and collected. The product
was purified by silica gel column chromatography, eluting with
Con clu sion s
N1-Aryl-N4-dialkyl-3,5-dinitrosulfanilamides are ex-
citing new antimitotic agents with activity against
kinetoplastid parasites. Dramatic selectivity has been
achieved against African trypanosomes with compounds
3 and 35, and experiments with purified leishmanial
1
MeOH/EtOAc (1:8): yield 781 mg, 49%; H NMR (DMSO-d6)

1828 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Bhattacharya et al.
δ 0.96 (t, 6H, J ) 7.25 Hz), 2.95 (q, 4H, J ) 7.25 Hz), 8.10 (d,
2H, J ) 1 Hz).
NMR (CDCl3) δ 11.08, 20.65, 20.85, 53.99, 122.87, 128.61,
129.33, 130.27, 132.44, 136.76, 141.61, 144.39; MS (FAB) m/z
calcd for C19H24N4O6S (M + Na)+ 459.1314, measured (M +
Na)+ 459.1316. Anal. (C16H24N4O6S) C, H, N.
3,5-Din itr o-N4,N4-d i-n -bu tylsu lfon a te (5c). A suspension
of 3,5-dinitro-4-chlorosulfonate (4, 0.70 g, 2.2 mmol) in dibut-
ylamine (1 mL, 5.98 mmol) in 30 mL of methanol was refluxed
for 3 h. After 3 h, the solvent was evaporated. The product
was purified by silica gel column chromatography, eluting with
MeOH/EtOAc (1:8): yield 689 mg, 76%; 1H NMR (CDCl3) 0.84
(m, 6H), 1.30 (m, 8H), 2.89 (m, 4H), 8.24 (s, 2H).
N1-(4-Ch lor o)p h en yl-3,5-d in itr o-N4,N4-d i-n -p r op ylsu lf-
a n ila m id e (9). 9 was synthesized by the reaction of sulfonyl
chloride 6a (prepared from 5a , 347 mg, 0.90 mmol) and 351
mg (3 mmol) of 4-chloroaniline in 20 mL of pyridine. The
product was isolated by chromatography (hexane/EtOAc (6:
1)) and crystallized using dichloromethane/hexane to obtain
yellow crystals: yield 258 mg, 63%; mp ) 130 °C; 1H NMR
(CDCl3) δ 0.86 (t, 6H, J ) 7.50 Hz), 1.61 (m, 4H), 2.94 (m,
4H), 6.95 (bs, 1H), 7.09 (d, 2H, J ) 7.25 Hz), 7.30 (d, 2H, J )
7.25 Hz), 8.14 (d, 2H, J ) 0.15 Hz); 13C NMR (CDCl3) δ 11.09,
20.65, 54.03, 123.37, 128.65, 128.77, 129.92, 132.21, 133.75,
141.85, 144.38; MS (FAB) m/z calcd for C18H21ClN4O6S (M +
3,5-Din itr o-N4,N4-d i-n -p r op ylsu lfa n ila m id e (2). To a
suspension of 3,5-dinitro-N4,N4-di-n-propylsulfonate (5a , 347
mg, 0.90 mmol) in dichloromethane (10 mL), was added PCl5
(624 mg, 3 mmol), and the reaction mixture was stirred for 2
h. The dichloromethane layer was collected after washing with
water and was dried with Na2SO4. The product 3,5-dinitro-
N4,N4-di-n-propylsulfonyl chloride (6a ) was used without
further purification. To this product was added 10 mL of 7 N
ammonia in methanol (method A), and the mixture was stirred
for 3 h at 40 °C. The solvent was evaporated and the product
was purified by silica gel column chromatography, eluting with
hexane/ethyl acetate (2:1) and crystallized in dichloromethane/
hexane: yield 286 mg, 92%; mp ) 138-140 °C (lit. 141 °C 15);
1H NMR (DMSO-d6) δ 0.78 (t, 6H, J ) 7.25 Hz), 1.52 (m, 4H),
2.91 (t, 4H, J ) 7.25 Hz), 7.62 (bs, 2H), 8.38 (s, 2H); 13C NMR
(DMSO-d6) δ 11.26, 20.78, 53.80, 127.55, 135.69, 140.24,
145.04; MS (FAB) m/z calcd 369.0826, measured 369.0830 (M
+ Na)+. Anal. (C12H18N4O6S) C, H, N.
Na)+ 479.0768, measured (M + Na)+ 479.0767. Anal. (C18H21
ClN4O6S) C, H, N.
-
N1-(3-Met h oxy)p h en yl-3,5-d in it r o-N4,N4-d i-n -p r op yl-
su lfa n ila m id e (10). 10 was synthesized by the reaction of
sulfonyl chloride 6a (prepared from 5a , 201 mg, 0.52 mmol)
and 123 mg (1 mmol) of 3-methoxyaniline in 7 mL of pyridine.
The product was isolated by chromatography (hexane/EtOAc
(6:1)) and crystallized using dichloromethane/hexane to obtain
a yellow crystalline solid: yield 137 mg, 58%; mp ) 133 °C;
1H NMR (CDCl3) δ 0.86 (t, 6H, J ) 7.33 Hz), 1.58 (m, 4H),
2.93 (t, 4H, J ) 7.40 Hz), 3.78 (s, 3H), 6.49 (bs, 1H), 6.75 (m,
3H), 7.21 (m, 1H), 8.12 (s, 2H); 13C NMR (CDCl3) δ 11.00, 20.55,
53.89, 55.39, 107.45, 111.70, 113.62, 128.73, 128.89, 130.38,
136.51, 141.72, 144.26, 160.46; MS m/z 451.1 (M - H)-. Anal.
(C19H24N4O7S) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of N1-Ar yl-3,5-
d in itr o-N4,N4-d i-n -p r op ylsu lfa n ila m id e Der iva tives (3,
7-17). To a suspension of 3,5-dinitro-N4,N4-di-n-propylsulfonic
acid 5a in dichloromethane was added PCl5 (2 equiv), and the
reaction mixture was stirred for 2 h. The dichloromethane
layer was extracted after washing with water and drying with
Na2SO4. The product 3,5-dinitro-N4,N4-di-n-propylsulfonyl chlo-
ride 6a was used without further purification. Pyridine was
added, the temperature was adjusted to 40 °C, 2-6 equiv of
the aniline derivative was added, and the reaction mixture
was stirred for 3 h at 40 °C (method B). Pyridine was then
evaporated in vacuo, the dark residue was washed with water
and extracted with ethyl acetate, and the product was purified
by silica gel column chromatography.
N 1 -P h e n y l-3,5-d in it r o -N 4 ,N 4 -d i-n -p r o p y ls u lfa n il-
a m id e (3). 3 was synthesized by the reaction of sulfonyl
chloride 6a (prepared from 5a , 347 mg, 0.9 mmol) and 547 µL
(6 mmol) of aniline in 20 mL of pyridine. The product was
isolated by chromatography (hexane/EtOAc (3:1)) and crystal-
lized with dichloromethane/hexane to afford a yellow crystal-
line solid: yield 342 mg, 90%. Melting point, 1H NMR, 13C
NMR, and HRMS data have been reported previously.11 Anal.
(C18H22N4O6S) C, H, N.
N1-(4-Met h oxy)p h en yl-3,5-d in it r o-N4,N4-d i-n -p r op yl-
su lfa n ila m id e (7). 7 was synthesized by the reaction of
sulfonyl chloride 6a (prepared from 5a , 347 mg, 0.90 mmol)
and 369 mg (3 mmol) of 4-methoxyaniline in 20 mL of pyridine.
The product was isolated by chromatography (hexane/EtOAc
(6:1)) and crystallized using dichloromethane/hexane to obtain
a yellow crystalline solid: yield 321 mg, 81%; mp ) 121 °C;
1H NMR (CDCl3) δ 0.86 (t, 6H, J ) 7.25 Hz), 1.61 (m, 4H),
2.91 (m, 4H), 3.79 (s, 3H), 6.58 (bs, 1H), 6.83 (dd, 2H, J ) 2.00,
7.00 Hz), 7.30 (dd, 2H, J ) 2.00, 7.00 Hz), 8.04 (d, 2H, J )
0.75 Hz); 13C NMR (CDCl3) δ 11.08, 20.65, 53.99, 55.49, 114.85,
125.92, 127.42, 128.60, 129.31, 141.53, 144.40, 158.71; MS
(FAB) m/z calcd for C19H24N4O7S (M + Na)+ 475.1263, mea-
sured (M + Na)+ 475.1275. Anal. (C19H24N4O7S) C, H, N.
N1-(3-Meth yl)p h en yl-3,5-d in itr o-N4,N4-d i-n -p r op ylsu lf-
a n ila m id e (11). 11 was synthesized by the reaction of sulfonyl
chloride 6a (prepared from 5a , 202 mg, 0.52 mmol) and 107
mg (1 mmol) of 3-methylaniline in 7 mL of pyridine. The
product was isolated by chromatography (hexane/EtOAc (6:
1)) and crystallized using dichloromethane/hexane to obtain
a yellow crystalline solid: yield 125 mg, 55%; mp ) 103 °C;
1H NMR (CDCl3) δ 0.86 (m, 6H), 1.57 (m, 4H), 2.31 (s, 3H),
2.93 (t, 4H, J ) 7.44 Hz), 6.67 (bs, 1H), 6.90 (m, 2H), 7.03 (d,
1H, J ) 7.08 Hz), 7.19 (m, 1H), 8.11 (s, 2H); 13C NMR (CDCl3)
δ 11.03, 20.58, 21.28, 53.93, 119.08, 122.79, 127.25, 128.56,
129.16, 129.47, 135.04, 139.92, 141.57, 144.28; MS m/z 435.1
(M - H)-. Anal. (C16H24N4O6S) C, H, N.
N1-(3-Ch lor o)p h en yl-3,5-d in itr o-N4,N4-d i-n -p r op ylsu lf-
a n ila m id e (12). 12 was synthesized by the reaction of sulfonyl
chloride 6a (prepared from 5a , 201 mg, 0.52 mmol) and 117
mg (1 mmol) of 3-chloroaniline in 7 mL of pyridine. The product
was isolated by chromatography (hexane/EtOAc (6:1)) and
crystallized by dichloromethane/hexane to obtain yellow crys-
tals: yield 125 mg, 52%; mp ) 108 °C; 1H NMR (CDCl3) δ 0.87
(t, 6H, J ) 7.25 Hz), 1.59 (m, 4H), 2.95 (t, 4H, J ) 7.43 Hz),
6.74 (s, 1H), 7.17 (m, 4H), 8.16 (s, 2H); 13C NMR (CDCl3) δ
11.52, 21.06, 54.43, 119.96, 122.17, 126.89, 128.96, 129.15,
131.24, 135.83, 136.90, 142.36, 144.71; MS m/z 455.1 (M - H)-.
Anal. (C18H21ClN4O6S) C, H, N.
N1-(3,4-Dich lor o)p h en yl-3,5-d in itr o-N4,N4-d i-n -p r op yl-
su lfa n ila m id e (13). 13 was synthesized by the reaction of
sulfonyl chloride 6a (prepared from 5a , 347 mg, 0.90 mmol)
and 486 mg (3 mmol) of 3,4-dichloroaniline in 20 mL of
pyridine. The product was isolated by chromatography (hex-
ane/EtOAc (6:1)) and crystallized using dichloromethane/
hexane to obtain a yellow crystalline solid: yield 264 mg, 60%;
1
mp ) 111 °C; H NMR (CDCl3) δ 0.88 (m, 6H), 1.61 (m, 4H),
N1-(4-Meth yl)p h en yl-3,5-d in itr o-N4,N4-d i-n -p r op ylsu l-
fa n ila m id e (8). 8 was synthesized by the reaction of sulfonyl
chloride 6a (prepared from 5a , 347 mg, 0.90 mmol) and 321
mg (3 mmol) of 4-methylaniline in 20 mL of pyridine. The
product was isolated by chromatography (hexane/EtOAc (6:
1)) and crystallized using dichloromethane/hexane to obtain
yellow crystals: yield 338 mg, 88%; mp ) 107 °C; 1H NMR
(CDCl3) δ 0.85 (t, 6H, J ) 7.25 Hz), 1.60 (m, 4H), 2.31 (s, 3H),
2.91 (t, 4H, J ) 7.75 Hz), 6.77 (bs, 1H), 7.10 (d, 2H, J ) 8.25
2.95 (m, 4H), 6.84 (bs, 1H), 7.01 (dd, 1H, J ) 2.50, 8.75 Hz)),
7.23 (d, 1H, J ) 2.50 Hz), 7.40 (d, 1H, J ) 8.75 Hz), 8.16 (s,
2H); 13C NMR (CDCl3) δ 11.06, 20.63, 54.02, 120.64, 123.27,
128.27, 128.77, 130.16, 131.33, 133.61, 134.84, 142.04, 144.34;
MS (FAB) m/z calcd for C18H20ClN4O6S (M + Na)+ 513.0378,
measured (M + Na)+ 513.0388. Anal. (C18H22N4O6S) C, H, N.
N1-(3,5-Dich lor o)p h en yl-3,5-d in itr o-N4,N4-d i-n -p r op yl-
su lfa n ila m id e (14). 14 was synthesized by the reaction of
sulfonyl chloride 6a (prepared from 5a , 347 mg, 0.90 mmol)
and 486 mg (3 mmol) of 3,5-dichloroaniline in 20 mL of
Hz), 7.20 (d, 2H, J ) 8.25 Hz), 8.10 (d, 2H, J ) 0.75 Hz)); 13
C

Sulfanilamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1829
pyridine. The product was isolated by chromatography (hex-
ane/EtOAc (6:1)) and crystallized by dichloromethane/hexane
to obtain yellow crystals: yield 356 mg, 81%; mp ) 144 °C;
1H NMR (CDCl3) δ 0.87 (t, 6H, J ) 7.50 Hz), 1.63 (m, 4H),
2.95 (q, 4H, J ) 6.63 Hz), 6.69 (bs, 1H), 7.06 (d, 1H, J ) 0.17
Hz), 7.20 (d, 2H, J ) 0.17 Hz), 8.17 (d, 2H, J ) 0.25 Hz); 13C
NMR (CDCl3) δ 11.01, 20.58, 53.99, 119.28, 126.13, 128.14,
128.64, 136.02, 137.27, 142.02, 144.30; MS m/z 489.1 (M - H)-.
Anal. (C18H20ClN4O6S) C, H, N.
N1-(2-P yr id in yl)-3,5-d in itr o-N4,N4-d i-n -p r op ylsu lfa n il-
a m id e (15). 15 was synthesized by the reaction of sulfonyl
chloride 6a (prepared from 5a , 347 mg, 0.90 mmol) and 564
mg (6 mmol) of 2-aminopyridine in 20 mL of pyridine. The
product was isolated by chromatography (hexane/EtOAc (1:
1)) and crystallized using dichloromethane/hexane to afford
yellow crystals: yield 326 mg, 86%; mp ) 204 °C; 1H NMR
(CDCl3) δ 0.86 (t, 6H, J ) 7.50 Hz), 1.62 (m, 4H), 2.94 (m,
4H), 6.90 (m, 1H), 7.47 (d, 1H, J ) 8.00 Hz), 7.85 (m, 1H),
8.24 (m, 1H), 8.31 (s, 2H), 8.65 (s, 1H); 13C NMR (CDCl3) δ
11.17, 11.25, 20.65, 20.75, 53.67, 54.03, 114.02, 116.12, 127.94,
133.61, 138.62, 141.11, 144.12, 144.83, 155.69; MS m/z 424.0
(M + H)+. Anal. (C18H22N4O6S) C, H, N.
to obtain yellow crystals: yield 85 mg, 91%; mp ) 138 °C; 1H
NMR (CDCl3) δ 0.89 (t, 6H, J ) 7.25 Hz), 1.63 (m, 4H), 2.81
(s, 6H), 2.98 (m, 4H), 8.15 (s, 2H); 13C NMR (CDCl3) δ 11.05,
20.62, 37.76, 53.89, 126.88, 128.64, 141.47, 144.65; MS m/z
397.00 (M + Na)+. Anal. (C14H22N4O6S) C, H, N.
N1-Eth yl-3,5-din itr o-N4,N4-di-n -pr opylsu lfan ilam ide (20).
20 was synthesized by the reaction of sulfonyl chloride 6a and
10 mL of 2 M ethylamine in THF (method D). The product
was isolated by chromatography (hexane/EtOAc ) 3:1) and
crystallized using dichloromethane/hexane to obtain yellow
1
crystals: yield 78 mg, 83%; mp ) 131 °C; H NMR (CDCl3) δ
0.88 (t, 6H, J ) 7.25 Hz), 1.20 (t, 3H, J ) 7.25 Hz), 1.62 (m,
4H), 3.08 (m, 6H), 4.44 (bt, 1H), 8.24 (s, 2H); 13C NMR (CDCl3)
δ 11.06, 15.13, 20.63, 38.41, 53.94, 128.32, 130.64, 141.39,
144.61; MS m/z 372.9 (M - H)-. Anal. (C14H22N4O6S) C, H, N.
N1,N1-Diet h yl-3,5-d in it r o-N4,N4-d i-n -p r op ylsu lfa n il-
a m id e (21). 21 was synthesized by the reaction of sulfonyl
chloride 6a and 10 mL (0.79 mol) of diethylamine (method C).
The product was isolated by chromatography (hexane/EtOAc
(6:1)) and crystallized using dichloromethane/hexane to obtain
yellow crystals: yield 73 mg, 73%; mp ) 102 °C; 1H NMR
(CDCl3) δ 0.88 (m, 6H), 1.20 (t, 6H, J ) 7.25 Hz), 1.62 (m,
4H), 2.97 (m, 4H), 3.28 (q, 4H, J ) 7.00 Hz), 8.18 (s, 2H); 13C
NMR (CDCl3) δ 11.05, 14.27, 20.63, 42.38, 53.92, 128.03,
131.35, 141.07, 144.73; MS m/z 403.1 (M + H)+. Anal.
(C16H26N4O6S) C, H, N.
N1,N1-P h en ylm eth yl-3,5-d in itr o-N4,N4-d i-n -p r op ylsu lf-
a n ila m id e (16). 16 was synthesized by the reaction of sulfonyl
chloride 6a (prepared from 5a , 347 mg, 0.90 mmol) and 547
µL (6 mmol) of N-methylaniline in 20 mL of pyridine. The
product was isolated by chromatography (hexane/EtOAc
(8:1)) and crystallized by dichloromethane/hexane to afford a
N 1 -P r o p y l-3,5-d i n i t r o -N 4 ,N 4 -d i -n -p r o p y ls u lfa n i l-
a m id e (22). 22 was synthesized by the reaction of sulfonyl
chloride 6a and 10 mL (0.12 mol) of propylamine (method C).
The product was isolated by chromatography (hexane/EtOAc
(3:1)) and crystallized using dichloromethane/hexane to obtain
yellow crystals: yield 86 mg, 88%; mp ) 114 °C; 1H NMR
(DMSO-d6) δ 0.82 (m, 9H), 1.55 (m, 6H), 2.97 (m, 6H), 7.88
(bs, 1H), 8.37 (s, 2H); 13C NMR (DMSO-d6) δ 11.24, 11.38,
20.74, 22.77, 44.64, 53.71, 128.27, 132.28, 140.59, 145.11; MS
m/z 389.3 (M + H)+. Anal. (C15H24N4O6S) C, H, N.
1
yellow crystalline solid: yield 241 mg, 61%; mp ) 96 °C; H
NMR (CDCl3) δ 0.87 (m, 6H), 1.63 (m, 4H), 2.96 (q, 4H, J )
7.00 Hz), 3.24 (s, 3H), 7.15 (m, 2H), 7.35 (m, 3H), 7.87 (d, 2H,
J ) 0.75 Hz); 13C NMR (CDCl3) δ 11.17, 20.74, 38.46, 54.01,
126.65, 127.37, 128.31, 128.81, 129.41, 140.57, 141.42, 144.52;
MS m/z 437.1 (M + H)+. Anal. (C18H22N4O6S) C, H, N.
N 1-Dip h e n yl-3,5-d in it r o-N 4,N 4-d i-n -p r op ylsu lfa n il-
a m id e (17). 17 was synthesized by the reaction of sulfonyl
chloride 6a (prepared from 5a , 347 mg, 0.90 mmol) and 547
µL (6 mmol) of diphenylamine in 20 mL of pyridine. The
product was isolated by chromatography (hexane/EtOAc (16:
1)) and crystallized by dichloromethane/hexane to afford a
N1,N1-Dip r op yl-3,5-d in it r o-N4,N4-d i-n -p r op ylsu lfa n il-
a m id e (23). 23 was synthesized by the reaction of sulfonyl
chloride 6a and 10 mL (0.73 mol) of dipropylamine (method
C). The product was isolated by chromatography (hexane/
EtOAc (6:1)) to obtain a yellow oil: yield 97 mg, 90%; 1H NMR
(CDCl3) δ 0.91 (m, 12H), 1.61 (m, 8H), 2.98 (m, 4H), 3.17 (m,
4H), 8.19 (s, 2H); 13C NMR (CDCl3) δ 11.01, 20.62, 22.03, 29.56,
50.09, 53.78, 53.91, 128.02, 131.21, 141.00, 144.74; MS m/z
453.2 (M + Na)+.
1
yellow crystalline solid: yield 248 mg, 55%; mp ) 132 °C; H
NMR (CDCl3) δ 0.88 (m, 6H), 1.63 (m, 4H), 2.98 (m, 4H), 7.35
(m, 10H), 8.03 (s, 2H); 13C NMR (CDCl3) δ 11.11, 20.67, 53.96,
117.76, 120.93, 128.17, 128.21, 128.78, 129.28, 129.66, 130.66,
140.57, 141.37, 143.10, 144.45; MS m/z 499.5 (M + H)+. Anal.
(C18H22N4O6S) C, H, N.
N1-Bu tyl-3,5-din itr o-N4,N4-di-n -pr opylsu lfan ilam ide (24).
24 was synthesized by the reaction of sulfonyl chloride 6a and
10 mL (0.10 mol) of butylamine (method C). The product was
isolated by chromatography (hexane/EtOAc (5:1)) and crystal-
lized using dichloromethane/hexane to obtain yellow crystals:
Syn th esis of N1-Alk yl-3,5-d in itr o-N4,N4-d i-n -p r opylsu lf-
a n ila m id e Der iva tives (18-33). To a suspension of 3,5-
dinitro-N4,N4-di-n-propylsulfonate (5a , 86 mg, 0.22 mmol) in
dichloromethane (5 mL) was added PCl5 (208 mg, 1 mmol),
and the reaction mixture was stirred for 2 h. The dichlo-
romethane layer was collected after washing with water, then
this layer was dried with Na2SO4 and solvent was removed in
vacuo. The product 3,5-dinitro-N4,N4-di-n-propylsulfonyl chlo-
ride (6a ) was used without further purification. The sulfona-
mides were prepared by method C, D, or E. Solvent was
evaporated, and the product was purified by silica gel column
chromatography.
N 1 -Me t h y l-3,5-d in it r o -N 4 ,N 4 -d i-n -p r o p y ls u lfa n il-
a m id e (18). 18 was synthesized by the reaction of sulfonyl
chloride 6a and 10 mL of 2 M methylamine in THF (method
D). The product was isolated by chromatography using hexane/
EtOAc (2:1) as eluent and crystallized using dichloromethane/
hexane to obtain yellow crystals: yield 88 mg, 97%; mp ) 148
°C; 1H NMR (CDCl3) δ 0.86 (t, 6H, J ) 7.25 Hz), 1.62 (m, 4H),
2.76 (d, 3H, J ) 5.25 Hz), 2.97 (m, 4H), 4.38 (bq, 1H), 8.25 (s,
2H); 13C NMR (CDCl3) δ 11.06, 20.63, 29.31, 53.94, 128.47,
129.55, 141.48, 144.61; MS m/z 361.00 (M + H)+. Anal.
(C13H20N4O6S) C, H, N.
1
yield 42 mg, 41%; mp ) 118 °C; H NMR (CDCl3) δ 0.91 (m,
9H), 1.28 (m, 4H), 1.60 (m, 4H), 2.98 (m, 6H), 4.32 (bt, 1H),
8.23 (s, 2H); 13C NMR (CDCl3) δ 11.06, 13.41, 19.55, 20.63,
31.61, 43.04, 53.95, 128.28, 130.72, 141.35, 144.62; MS m/z
401.0 (M - H)-. Anal. (C16H26N4O6S) C, H, N.
N1,N1-Dib u t yl-3,5-d in it r o-N4,N4-d i-n -p r op ylsu lfa n il-
a m id e (25). 25 was synthesized by the reaction of sulfonyl
chloride 6a and 510 µL (3 mmol) of dibutylamine in 10 mL of
dichloromethane (method E). The product was isolated by
chromatography (hexane/EtOAc (15:1)) to afford a yellow oil
that solidified upon refrigeration: yield 81 mg, 71%; mp ) 48
1
°C; H NMR (CDCl3) δ 0.90 (m, 12H), 1.29 (m, 4H), 1.61 (m,
8H), 2.97 (q, 4H, J ) 6.85 Hz), 3.15 (t, 4H, J ) 7.50 Hz), 8.16
(d, 2H, J ) 1.25 Hz); 13C NMR (CDCl3) δ 11.06, 13.56, 19.78,
20.65, 30.70, 47.96, 53.95, 128.04, 131.22, 140.99, 144.73; MS
m/z 459.1 (M + H)+.
N1-P en t yl-3,5-d in it r o-N4,N4-d i-n -p r op ylsu lfa n ila m id e
(26). 26 was synthesized by the reaction of sulfonyl chloride
6a and 450 µL (3 mmol) of amylamine in 10 mL of dichlo-
romethane (method E). The product was isolated by chroma-
tography (hexane/EtOAc (6:1)) and crystallized using dichlo-
romethane/hexane to obtain yellow crystals: yield 91 mg, 87%;
N1,N1-Dim eth yl-3,5-d in itr o-N4,N4-d i-n -p r op ylsu lfa n il-
a m id e (19). 19 was synthesized by the reaction of sulfonyl
chloride 6a and 10 mL of 2 M dimethylamine in THF (method
D). The product was isolated by chromatography (hexane/
EtOAc (2:1)) and crystallized using dichloromethane/hexane
1
mp ) 110 °C; H NMR (CDCl3) δ 0.88 (m, 9H), 1.29 (m, 6H),
1.62 (m, 4H), 2.98 (m, 6H), 4.38 (bt, 1H), 8.23 (d, 2H, J ) 0.75

1830 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Bhattacharya et al.
Hz); 13C NMR (CDCl3) δ 11.04, 13.73, 20.63, 22.02, 28.49,
29.21, 43.31, 53.94, 128.29, 130.79, 141.33, 144.63; MS m/z
431.1 (M + H)+. Anal. (C17H28N4O6S) C, H, N.
lized by hexane/dichloromethane to obtain yellow crystals:
yield 84 mg, 84%; mp ) 120 °C; H NMR (CDCl3) δ 0.88 (t,
6H, J ) 7.5 Hz), 1.64 (m, 4H), 1.89 (m, 4H), 2.97 (m, 4H), 3.30
(t, 4H, J ) 6.5 Hz), 8.20 (s, 2H); 13C NMR (CDCl3) δ 11.11,
20.69, 25.36, 48.07, 53.96, 128.43, 128.60, 141.34, 144.80; MS
m/z 401.0 (M + H)+. Anal. (C16H24N4O6S) C, H, N.
1
N1-H exyl-3,5-d in it r o-N4,N4-d i-n -p r op ylsu lfa n ila m id e
(27). 27 was synthesized by the reaction of sulfonyl chloride
6a and 400 µL (3 mmol) of hexylamine in 10 mL of dichlo-
romethane (method E). The product was isolated by chroma-
tography (hexane/EtOAc (15:1)) and crystallized using dichlo-
romethane/hexane to obtain a yellow crystalline solid: yield
N1-P h en yl-3,5-d in it r o-N4,N4-d i-n -et h ylsu lfa n ila m id e
(34). The sulfonyl chloride derivative 6b was prepared as
before from the sulfonate 5b and PCl5. Then 6b was treated
with 547 µL (6 mmol) of aniline in 20 mL of pyridine. The
product was isolated by chromatography (hexane/EtOAc (6:
1)) and crystallized using dichloromethane/hexane to afford a
1
77 mg, 72%; mp ) 123 °C; H NMR (CDCl3) δ 0.91 (m, 9H),
1.26 (bm, 6H), 1.65 (m, 6H), 3.02 (m, 6H), 4.46 (bt, 1H), 8.23
(s, 2H); 13C NMR (CDCl3) δ 11.20, 13.88, 20.71, 22.40, 26.12,
29.60, 31.18, 43.42, 54.03, 128.33, 130.91, 141.40, 144.74; MS
m/z 429.1 (M - H)-. Anal. (C18H30N4O6S) C, H, N.
1
yellow crystalline solid: yield 262 mg, 67%; mp ) 157 °C. H
NMR (DMSO-d6) δ 0.99 (t, 6H, J ) 7.00 Hz), 2.99 (q, 4H, J )
7.00 Hz), 7.17 (m, 5H), 8.28 (s, 2H), 10.45 (bs, 1H); 13C NMR
(DMSO-d6) δ 12.54, 45.60, 120.82, 124.95, 127.92, 129.43,
130.69, 136.73, 140.63, 144.96; MS m/z 392.9 (M - H)-. Anal.
(C16H18N4O6S) H, N. C: calcd, 48.72; found, 48.13.
N1,N1-Dih exyl-3,5-d in it r o-N4,N4-d i-n -p r op ylsu lfa n il-
a m id e (28). 28 was synthesized by the reaction of sulfonyl
chloride 6a and 700 µL of 3 mmol of dihexylamine in 10 mL
of dichloromethane (method E). The product was isolated by
chromatography (hexane/EtOAc (12:1)) to provide a yellow
oil: yield 89 mg, 69%; 1H NMR (CDCl3) δ 0.90 (m, 12H), 1.38
(m, 12H), 1.68 (m, 8H), 2.96 (m, 4H), 3.15 (t, 4H, J ) 7.5 Hz),
8.16 (d, 2H, J ) 1.5 Hz); 13C NMR (CDCl3) δ 11.03, 13.84,
20.65, 22.39, 26.27, 28.50, 31.27, 48.10, 53.96, 128.00, 131.43,
140.97, 144.77; MS m/z 515.3 (M + H)+. Anal. (C24H42N4O6S)
C, H, N.
N1-P h en yl-3,5-d in it r o-N4,N4-d i-n -b u t ylsu lfa n ila m id e
(35). The sulfonyl chloride derivative 6c was prepared as
before from sulfonate 5c and PCl5. It was then treated with
547 µL (6 mmol) of aniline in 20 mL of pyridine. The product
was isolated by chromatography (hexane/EtOAc (6:1)) and
crystallized by dichloromethane/hexane to afford a yellow
crystalline solid. Yield 286 mg, 64%; mp ) 156 °C. 1H NMR
(CDCl3) δ 0.87 (t, 6H, J ) 7.50 Hz), 1.28 (m, 4H), 1.55 (m,
4H), 2.96 (t, 4H, J ) 7.50 Hz), 6.57 (bs, 1H), 7.25 (m, 5H),
8.10 (s, 2H). 13C NMR (CDCl3) δ 13.66, 19.85, 29.35, 52.07,
122.18, 126.52, 128.61, 129.09, 129.78, 135.24, 141.66, 144.42.
N 1-Isop r op yl-3,5-d in it r o-N 4,N 4-d i-n -p r op ylsu lfa n il-
a m id e (29). 29 was synthesized by the reaction of sulfonyl
chloride 6a and 10 mL (0.12 mol) of isopropylamine (method
C). The product was isolated by chromatography (hexane/
EtOAc (6:1)) and crystallized using dichloromethane/hexane
to give a yellow crystalline solid: yield 77 mg, 79%; mp ) 150
MS m/z calcd for
(C20H26N4O6S) C, H, N.
C
20H26N4O6S (M - H)- 449.0. Anal.
1
°C; H NMR (CDCl3) δ 0.88 (t, 6H, J ) 7.25 Hz), 1.27 (d, 6H,
3-Nitr o-4-ch lor osu lfon yl Ch lor id e (38). A solution of
1-chloro-2-nitrobenzene (37, 5 g, 31.84 mmol) in 25 mL of
chlorosulfonic acid was heated at 120 °C for 16 h. The red
solution was poured slowly into a container containing ice. The
product was extracted from the aqueous solution using EtOAc,
the ethyl acetate layer was dried with sodium sulfate and
filtered, and then the ethyl acetate was evaporated to afford
a brown viscous liquid which upon refrigeration became
J ) 7.25 Hz), 1.63 (m, 4H), 2.97 (m, 4H), 3.57 (m, 1H), 4.41
(bs, 1H), 8.25 (s, 2H); 13C NMR (CDCl3) δ 11.11, 20.70, 23.85,
46.68, 54.07, 128.22, 131.99, 141.33, 144.71; MS m/z 389.0 (M
+ H)+. Anal. (C15H24N4O6S) C, H, N.
N1-Cyclop r op yl-3,5-d in itr o-N4,N4-d i-n -p r op ylsu lfa n il-
a m id e (30). 30 was synthesized by the reaction of sulfonyl
chloride 6a and 10 mL (0.14 mol) of cyclopropylamine (method
C). The product was isolated by chromatography (hexane/
EtOAc (4:1)) and crystallized by dichloromethane/hexane to
afford a yellow crystalline solid: yield 89 mg, 90%; mp ) 138
1
solid: yield 6.2 g, 77%; H NMR (CDCl3) δ 7.90 (d, 1H, J )
8.50 Hz), 8.21 (dd, 1H, J ) 2.25, 8.75 Hz), 8.56 (d, 1H, J ) 2
Hz).
1
Syn th esis of N1-P h en yl-3-n itr o-4-ch lor osu lfon a m id e
(39). Aniline (1.08 mL, 12 mmol) was added in a stirred
solution of 3-nitro-4-chlorosulfonyl chloride (38, 762 mg, 3
mmol) in 20 mL of pyridine at 45 °C. Stirring was continued
for another 3 h. Then pyridine was evaporated out, and the
product was extracted using dichloromethane. It was purified
as an oil using silica gel column chromatography eluting with
hexane/ethyl acetate (2:1): yield 636 mg, 90%; 1H NMR
(CDCl3) δ 6.89 (bs, 1H), 7.10 (m, 1H), 7.27 (m, 4H), 7.81 (dd,
1H, J ) 2.00, 8.25 Hz), 7.62 (d, 1H, J ) 8.00 Hz), 8.24 (d, 1H,
J ) 2 Hz), 8.68 (s, 1H).
°C; H NMR (CDCl3) δ 0.72 (m, 4H), 0.88 (t, 6H, J ) 7.5 Hz),
1.63 (m, 4H), 2.55 (m, 1H), 2.98 (m, 4H), 4.94 (bs, 1H), 8.28 (s,
2H); 13C NMR (CDCl3) δ 6.62, 11.04, 20.63, 24.30, 53.97,
128.61, 130.13, 141.47, 144.59; MS m/z 387.0 (M + H)+. Anal.
(C15H22N4O6S) C, H, N.
N1-Cyclop en t yl-3,5-d in it r o-N4,N4-d i-n -p r op ylsu lfa n il-
a m id e (31). 31 was synthesized by the reaction of sulfonyl
chloride 6a and 300 µL (3 mmol) of cyclopentylamine in 10
mL of dichloromethane (method E). The product was isolated
by chromatography (hexane/EtOAc (5:1)) and crystallized
using dichloromethane/hexane to obtain a yellow crystalline
solid: yield 81 mg, 78%; mp ) 116 °C; 1H NMR (CDCl3) δ 0.88
(t, 6H, J ) 7.5 Hz), 1.50 (m, 2H), 1.68 (m, 8H), 1.94 (m, 2H),
2.97 (m, 4H), 3.57 (m, 1H), 4.58 (bd, 1H), 8.25 (s, 2H); 13C NMR
(CDCl3) δ 11.07, 20.62, 23.08, 35.57, 53.93, 55.38, 128.31,
131.47, 141.31, 144.35; MS m/z 415.11 (M + H)+. Anal.
(C17H26N4O6S) C, H, N.
N1-P h en yl-3-n itr o-N4,N4-d i-n -p r op ylsu lfa n ila m id e (36).
A solution of N1-phenyl-3-nitro-4-chlorosulfonamide (39, 636
mg, 2.69 mg) in 20 mL of dipropylamine was refluxed for 3 h.
After 3 h the solvent was evaporated under vacuum. The crude
product was purified by column chromatography eluting with
hexane/EtOAc (3:1) to afford a viscous red oil that was further
purified by distillation at 80 °C and 5 mbar pressure: yield
3,5-Din itr o-N4,N4-d i-n -p r op ylsu lfon ylm or p h olin e (32).
32 was synthesized by the reaction of sulfonyl chloride 6a and
261 µL (3 mmol) of morpholine in 10 mL of dichloromethane
(method E). The product was isolated by chromatography
(hexane/EtOAc (5:1)) and crystallized by dichloromethane/
hexane to obtain yellow crystals: yield 76 mg, 73%; mp ) 193
°C; 1H NMR (CDCl3) δ 0.89 (t, 6H, J ) 7.25 Hz), 1.65 (m, 4H),
2.98 (t, 4H, J ) 7.5 Hz), 3.08 (t, 4H, J ) 4.75 Hz), 3.78 (t, 4H,
J ) 4.75 Hz), 8.12 (s, 2H); 13C NMR (CDCl3) δ 11.05, 20.61,
45.82, 53.89, 65.86, 125.62, 128.89, 141.68, 144.52; MS m/z
417.1 (M + Na)+. Anal. (C15H23N4O6S) C, H, N.
1
616 mg, 61%; H NMR (CDCl3) δ 0.82 (m, 6H), 1.58 (m, 4H),
3.13 (t, 4H, J ) 7.25 Hz), 6.68 (bs, 1H), 6.99 (d, 1H, J ) 9.25
Hz), 7.20 (m, 5H), 7.61 (dd, 1H, J ) 2.75, 9.50 Hz), 8.13 (d,
1H, J ) 2.25 Hz); 13C NMR (CDCl3) δ 10.94, 20.35, 53.38,
119.79, 121.23, 125.11, 126.14, 126.96, 129.22, 130.78, 136.29,
137.94, 147.56; MS m/z 377.0 (M - H)-. Anal. (C18H22N3O6S)
H, N. C: calcd, 57.28; found, 57.69.
Su scep tibility Testin g of P a r a sites a n d Ma m m a lia n
Cells. The susceptibility of Leishmania donovani amastigote-
like parasites (WHO designation: MHOM/SD/62/1S-CL2D) to
growth inhibition by compounds of interest was measured in
a 3-day assay using the tetrazolium dye-based CellTiter
reagent (Promega).7,16 Compounds were tested for their activity
against bloodstream-form Trypanosoma brucei brucei (MITat
3,5-Din itr o-N4,N4-d i-n -p r op ylsu lfon ylp yr r olid in e (33).
33 was synthesized by the reaction of sulfonyl chloride 6a and
10 mL (0.12 mol) of pyrrolidine (method C). The product was
isolated by chromatography (hexane/EtOAc (4:1)) and crystal-

Sulfanilamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1831
1.2, variant 221) axenically cultured in HMI-9 medium17 by
the procedure of Ellis et al.18 with some modifications. Briefly,
an amount of 100 µL of T. brucei in late log phase was
incubated in 96-well plates (Costar) at an initial concentration
of 105 cells/mL with or without test compounds at 37 °C in a
humidified 5% CO2 atmosphere for 72 h. To each well was then
added 25 µL of a 5 mg/mL solution of MTT (prepared in
phosphate-buffered saline and filter-sterilized), and plates
were reincubated at 37 °C as before for 2 h. One hundred
microliters of 10% SDS lysis buffer (prepared in 50% aqueous
DMF) was added to each well, and plates were incubated as
before for an additional 3-4 h. A SpectraMax Plus microplate
reader (Molecular Devices) was then used to measure optical
densities at 570 nm. IC50 values, the concentration of the
compound that inhibited cell growth by 50% compared to
untreated control, were determined with the aid of the
software program SoftMax Pro (Molecular Devices). This
program uses the dose-response equation y ) ((a - d)/(1 +
(x/c)b)) + d, where x is the drug concentration, y is absorbance
tubulin was stored at -80 °C in 80 µL aliquots. Leishmanial
tubulin was purified by the method of Werbovetz et al.7 except
that the elution of parasite tubulin from the DEAE-Sepharose
Fast Flow column was accomplished using PME containing
0.75 M glutamate and 0.3 M KCl as above for mammalian
tubulin rather than with PME containing 0.7 M KCl as
reported earlier.
Tu bu lin Assem bly Assa ys. Assembly reactions (final
volume of 50 µL) were conducted in 96-well half-area micro-
plates (Costar). Reactions examining either leishmanial or
brain tubulin contained final concentrations of 1.5 mg/mL (15
µM) tubulin, 0.1 mM PIPES (pH 6.9), 1 mM EGTA, 1 mM
MgCl2, 10% (v/v) DMSO, and 1 mM GTP with and without
compounds of interest. Components of the reaction mixtures
were added to the microplate on ice, then assembly was
initiated, and components were brought to the final concentra-
tions indicated above by the simultaneous addition of a cold
GTP solution (10 µL) to all sample wells with a multichannel
pipet. The change in turbidity was measured at 351 nm using
the SpectraMax Plus microplate reader immediately after GTP
addition, with reaction mixtures allowed to warm to either 30
°C (for leishmanial tubulin) or 37 °C (for brain tubulin) within
the plate reader. To ensure maximal solubility of the dinitroa-
nilines at a concentration of 40 µM, compounds were added to
reactions containing brain tubulin in 5 µL volumes containing
100% DMSO, and assembly was initiated by adding 10 µL of
5 mM GTP in water. The assembly of leishmanial tubulin
occurred too rapidly for accurate measurement under the
conditions described for brain tubulin. Since the dinitroaniline
sulfonamides were tested at 10 µM concentrations against
leishmanial tubulin, solubility was not as much of a concern.
Compounds were thus added to reactions containing parasite
tubulin in 5 µL volumes containing 50% DMSO, and then
assembly was initiated by adding 10 µL of 5 mM GTP in 25%
DMSO. Because of the expense of growing large numbers of
Leishmania parasites for the tubulin isolation, a single
duplicate measurement was made to screen compounds of
interest for activity against this parasite protein.
at 570 nm, a is the upper asymptote, b is the slope, c is IC50
and d is the lower asymptote.
,
The effect of compounds on the proliferation of J 774 murine
macrophages and PC3 prostate cancer cells was measured in
a 3-day assay using the CellTiter reagent. J 774 macrophages
in DMEM medium supplemented with 10% heat-inactivated
fetal calf serum, 2.0 mM L-glutamine, 50 units/mL penicillin,
and 50 µg/mL streptomycin were added to individual wells of
a 96-well plate at a concentration of 104 cells/mL and a total
volume of 100 µL. Macrophages were allowed to adhere for 24
h, and then the medium was removed and replaced with serial
dilutions of the test compounds in the DMEM medium
mentioned above without phenol red. After 72 h of incubation
with the test compounds at 37 °C in a humidified 5% CO2
incubator, cell viability was determined using the CellTiter
reagent by adding 20 µL of assay solution to each well. After
a 5-7 h incubation at 37 °C to allow for color development,
absorbance readings in each well were measured at 490 nm
using the microplate reader. The effect of compounds on PC3
cells was assessed in the same way as for the macrophages
except that the medium used was RPMI 1640 supplemented
with 10% heat-inactivated fetal calf serum, 2.0 mM L-
glutamine, 50 units/mL penicillin, and 50 µg/mL streptomycin.
IC50 values for the J 774 and PC3 assays were determined with
the software program SoftMax Pro as described earlier.
F low Cytom etr y. T. b. brucei bloodstream forms were
incubated in T25 flasks (3 mL final volume) at 37 °C in a
humidified 5% CO2 atmosphere in HMI-9 medium for 24 h.
Cultures contained 1% DMSO (v/v) in the presence or absence
of test compounds. Trypanosomes were counted using a
hemacytometer and then were centrifuged at 1000g at 4 °C
for 10 min. Parasites were resuspended in 150 µL of PBS (0.01
M phosphate, 0.137 M NaCl, and 2.7 mM KCl), then 350 µL
of ice-cold methanol was added, and the samples were fixed
at -20 °C for 2-3 h. Cells were centrifuged as before and
resuspended in PBS containing 0.1% TX-100, 5 µg/mL RNase
A, and 10 µg/mL propidium iodide for 20-30 min at 37 °C.
Centrifugation was repeated, then cells were resuspended in
PBS to a final concentration of (5-10) × 105 parasites/mL and
stored at 4 °C until analysis. Fluorescence cell sorting was
conducted on a Becton-Dickinson FACSCalibur flow cytometer.
In each case, gating was performed to exclude doublets and
aggregates.
Tu bu lin P u r ifica tion . Porcine brains, freshly obtained
from a local slaughterhouse, were sliced into approximately 1
cm sections, frozen on dry ice, and stored at -80 °C until use.
Frozen brain (approximately 100 g) was broken into small
pieces and thawed in 100 mL PME + DTT buffer (0.1 M
PIPES, pH 6.9, 1 mM MgCl2, 1 mM EGTA, 1 mM DTT). This
brain tissue was homogenized in a blender at 4 °C using 3 ×
5 s bursts at high power. A 150 mL portion of the homogenate
was centrifuged in a Ti-70 rotor at 100000g for 40 min at 4
°C, and then the supernatant was loaded on an 11 mL column
containing DEAE-Sepharose Fast Flow matrix (Amersham
Pharmacia) equilibrated with PME + DTT. The DEAE-
Sepharose column was washed with two column volumes of
PME + DTT, four volumes of PME containing 0.3 M glutamate,
and four volumes of PME + DTT containing 1 M glutamate.
PME + DTT containing 0.75 M glutamate and 0.3 M KCl was
then used to elute tubulin from the column. Tubulin-rich
fractions (consisting of approximately 10 mL total) were
pooled, and then GTP and DMSO were added slowly with
gentle vortexing to bring the final concentrations of these
components to 1 mM and 8% (v/v), respectively. This solution
was incubated at 37 °C for 40 min. After this 37 °C incubation,
the solution became cloudy because of the formation of
microtubules, which were collected by centrifugation at 100000g
at 35 °C for 30 min. The pellet was resuspended in 2.3 mL of
PME on ice and sonicated once using a Microson XL2000
sonicator (Misonix, Farmingdale, NY) at an output power of
10 W to completely redissolve the pellet. Following an ad-
ditional 30 min of incubation on ice, this solution was spun at
80000g at 4 °C for 20 min. The supernatant containing porcine
In Vivo Scr een in g a ga in st Afr ica n Tr yp a n osom es. The
testing of antimitotic compounds against T. b. brucei in vivo
was carried out according to Kaminsky and Brun19 with minor
modifications. Female NMRI mice (20-25 g) were infected ip
with 104 T. b. brucei strain STIB 795 bloodstream forms, and
the animals were randomly assigned to groups of four.
Compounds were first dissolved in DMSO and then diluted
with water to provide the desired dose of 20 mg/kg (the
percentage of DMSO in the final solution was less than 10%).
The test compounds or vehicle was administered ip 4 days after
infection for 4 consecutive days. The day of death was then
recorded for each animal.
Ack n ow led gm en t. K.A.W. acknowledges the Col-
lege of Pharmacy at The Ohio State University and The
American Association of Colleges of Pharmacy New
Investigator Program for financial support. The flow

1832 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Bhattacharya et al.
(11) Bhattacharya, G.; Salem, M.; Werbovetz, K. Antileishmanial
dinitroaniline sulfonamides with activity against parasite tu-
bulin. Bioorg. Med. Chem. Lett. 2002, 12, 2395-2398.
(12) Werbovetz, K.; Sackett, D.; Delfin, D.; Bhattacharya, G.; Salem,
M.; et al. Selective antimicrotubule activity of N1-phenyl-3,5-
dinitro-N4,N4-di-n-propylsulfonilamide (GB-II-5) against kine-
toplastid parasites. Mol. Pharmacol. 2003, 64, 1325-1333.
(13) Schultz, H. 2,6-Dinitroaniline. In Organic Syntheses; Rabjohn,
N., Ed.; J ohn Wiley & Sons: New York, 1963; pp 364-366.
(14) Powis, G.; Gallegos, A.; Abraham, R.; Ashendel, C.; Zalkow, L.;
et al. Inhibition of intracellular Ca2+ signalling, cytotoxicity and
antitumor activity of the herbicide oryzalin and its analogs.
Cancer Chemother. Pharmacol. 1997, 41, 22-28.
(15) Zweig, G.; Sherma, J . Government regulations, pheromone
analysis, additional pesticides. In Analytical Methods for Pes-
ticides and Plant Growth Regulators; Zweig, G., Ed.; Academic
Press: New York, 1976; pp 433-442.
(16) Havens, C.; Bryant, N.; Asher, L.; Lamoreaux, L.; Perfetto, S.;
et al. Cellular effects of leishmanial tubulin inhibitors on L.
donovani. Mol. Biochem. Parasitol. 2000, 110, 223-236.
(17) Hirumi, H.; Hirumi, K. Continuous cultivation of Trypanosoma
brucei bloodstream forms in a medium containing a low concen-
tration of serum proteins without feeder layer cells. J . Parasitol.
1989, 75, 985-989.
cytometry work was funded in part by Grant P30
CA16058 from the National Cancer Institute.
Refer en ces
(1) Werbovetz, K. Promising therapeutic targets for antileishmanial
drugs. Expert Opin. Ther. Targets 2002, 6, 407-422.
(2) Burchmore, R.; Ogbunude, P.; Enanga, B.; Barrett, M. Chemo-
therapy of human African trypanosomiasis. Curr. Pharm. Des.
2002, 8, 256-267.
(3) Croft, S.; Seifert, K.; Duchene, M. Antiprotozoal activities of
phospholipid analogs. Mol. Biochem. Parasitol. 2003, 126, 165-
172.
(4) Legros, D.; Ollivier, G.; Gastellu-Etchegorry, M.; Paquet, C.;
Burri, C.; et al. Treatment of human African trypanosomiasiss
present situation and needs for research and development.
Lancet Infect. Dis. 2002, 2, 437-440.
(5) J ordan, A.; Hadfield, J .; Lawrence, N.; McGown, A. Tubulin as
a target for anticancer drugs: agents which interact with the
mitotic spindle. Med. Res. Rev. 1998, 18, 259-296.
(6) Lacey, E. Mode of action of benzimidazoles. Parasitol. Today
1990, 6, 112-115.
(7) Werbovetz, K.; Brendle, J .; Sackett, D. Purification, character-
ization, and drug susceptibility of tubulin from Leishmania. Mol.
Biochem. Parasitol. 1999, 98, 53-65.
(8) MacRae, T. H.; Gull, K. Purification and assembly in vitro of
tubulin from Trypanosoma brucei brucei. Biochem. J . 1990, 265,
87-93.
(18) Ellis, J .; Fish, W.; Sileghem, M.; McOdimba, F. A colorimetric
assay for trypanosome viability and metabolic function. Vet.
Parasitol. 1993, 50, 143-149.
(19) Kaminsky, R.; Brun, R. In vitro and in vivo activities of trybazine
hydrochloride against various pathogenic trypanosome species.
Antimicrob. Agents Chemother. 1998, 42, 2858-2862.
(9) Chan, M.; Fong, D. Inhibition of Leishmanias but not host
macrophages by the antitubulin herbicide trifluralin. Science
1990, 249, 924-926.
(10) Chan, M.; Triemer, R. E.; Fong, D. Effect of the anti-microtubule
drug oryzalin on growth and differentiation of the parasitic
protozoan Leishmania mexicana. Differentiation 1991, 46, 15-
21.
J M0304461