Antikinetoplastid antimitotic activity and metabolic stability of dinitroaniline sulfonamides and benzamides

Article abstract of DOI:10.1016/j.bmc.2006.04.017

N¹-Phenyl-3,5-dinitro-N⁴,N⁴-di-n-propylsulfanilamide (1) and N¹-phenyl-3,5-dinitro-N⁴,N⁴-di-n-butylsulfanilamide (2) show potent in vitro antimitotic activity against kinetoplastid parasites but display poor in vivo activity. Seventeen new dinitroaniline sulfonamide and eleven new benzamide analogs of these leads are reported here. Nine of the sulfonamides display in vitro IC₅₀ values under 500 nM against African trypanosomes, and the most active antikinetoplastid compounds also inhibit the in vitro assembly of purified leishmanial tubulin with potencies similar to that of 2. While several of the potent compounds are rapidly degraded by rat liver S9 fractions in vitro, N¹-(3-hydroxy)phenyl-3,5-dinitro-N⁴,N⁴-di-n-butylsulfanilamide (21) displays an IC₅₀ value of 260 nM against African trypanosomes in vitro and is more stable than 2 in the in vitro metabolism assay.

Full text of DOI:10.1016/j.bmc.2006.04.017

Bioorganic & Medicinal Chemistry 14 (2006) 5699–5710
Antikinetoplastid antimitotic activity and metabolic stability
of dinitroaniline sulfonamides and benzamides
a
a
a
a
´
Tesmol G. George, Jayaseharan Johnsamuel, Dawn A. Delfın, Adam Yakovich,
Mitali Mukherjee,a Mitch A. Phelps,b James T. Dalton,b Dan L. Sackett,c
Marcel Kaiser,d Reto Brund and Karl A. Werbovetza,*
aDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University,
500 West 12th Avenue, Columbus, OH 43210, USA
bDivision of Pharmaceutics, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210, USA
cLaboratory of Integrative and Medical Biophysics, National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, MD 20892, USA
dSwiss Tropical Institute, Basel CH4002, Switzerland
Received 24 March 2006; accepted 6 April 2006
Available online 3 May 2006
Abstract—N1-Phenyl-3,5-dinitro-N4,N4-di-n-propylsulfanilamide (1) and N1-phenyl-3,5-dinitro-N4,N4-di-n-butylsulfanilamide (2)
show potent in vitro antimitotic activity against kinetoplastid parasites but display poor in vivo activity. Seventeen new dinitroan-
iline sulfonamide and eleven new benzamide analogs of these leads are reported here. Nine of the sulfonamides display in vitro IC50
values under 500 nM against African trypanosomes, and the most active antikinetoplastid compounds also inhibit the in vitro
assembly of purified leishmanial tubulin with potencies similar to that of 2. While several of the potent compounds are rapidly
degraded by rat liver S9 fractions in vitro, N1-(3-hydroxy)phenyl-3,5-dinitro-N4,N4-di-n-butylsulfanilamide (21) displays an IC50
value of 260 nM against African trypanosomes in vitro and is more stable than 2 in the in vitro metabolism assay.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
mation regarding the geographical distribution, preva-
lence, transmission, and symptoms of these diseases is
available online from the World Health Organization
(see <http://www.who.int/leishmaniasis/disease_epide-
miology/en/index.html/>, <http://www.who.int/media-
centre/factsheets/fs259/en//>, and <http://www.who.int/
ctd/chagas/disease.htm/>). Despite the morbidity and
mortality caused by these diseases, their chemotherapy
remains unsatisfactory (see Croft et al.1 for a recent re-
view), underscoring the urgent need for improved anti-
trypanosomal and antileishmanial drugs.
Leishmaniasis, African trypanosomiasis, and Chagas
disease are vector-borne diseases affecting millions in
the developing world that are caused by parasites of
the order kinetoplastida. The clinical manifestations of
leishmaniasis, which range from limited, self-healing
cutaneous lesions to fatal visceral disease, depend on
the identity of the Leishmania species responsible for
infection. African trypanosomiasis is always fatal in
the absence of treatment; infection with Trypanosoma
brucei rhodesiense results in death within a few months,
while Trypanosoma brucei gambiense infections follow a
longer course. The parasite responsible for Chagas dis-
ease, Trypanosoma cruzi, causes severe chronic disease
affecting the heart, esophagus, and colon. Further infor-
Dinitroaniline compoundshavedisplayed activityagainst
various parasites,2–4 including Leishmania5–8 and trypan-
osomes.6,9 Tubulin has been proposed to be the antipara-
sitic target of these molecules.10–12 We previously showed
that both N1-phenyl-3,5-dinitro-N4,N4-di-n-propylsulfa-
nilamide (GB-II-5, compound 1)13 and N1-phenyl-
3,5-dinitro-N4,N4-di-n-butylsulfanilamide (GB-II-150,
compound 2)14 possess selective antimicrotubule activity
against Leishmania and African trypanosomes in vitro.
A decrease in alkyl chain length at N4 and substitution
Keywords: Leishmania; Trypanosome; Chemotherapy; Tubulin;
Dinitroaniline.
*
Corresponding author. Tel.: +1 614 292 5499; fax: +1 614 292
2435; e-mail: werbovetz.1@osu.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.017

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T. G. George et al. / Bioorg. Med. Chem. 14 (2006) 5699–5710
of N1-alkyl chains for the N1 aromatic ring diminished
antiparasitic activity, as did large substituents on the N1
aromatic ring.14 Despite their in vitro activity, 1 and 2
showed no significant in vivo activity when administered
ip at 20 mg/kg/day for four days in a murine model of
African trypanosomiasis.14
2. Results and discussion
2.1. Chemistry
Reaction of primary amines with cyclopropanecarbonyl-
chloride (3) followed by amide reduction, methanolysis,
trapping with di-tert-butyl dicarbonate, and deprotec-
tion with HCl (Scheme 1) as described by Dubowchik
et al.16 provided secondary amine hydrochlorides 6a–c
containing a cyclopropylmethyl group. Dinitroaniline
sulfonamides were synthesized as outlined in Scheme
2 by the general methods reported earlier.14 Reaction
between 3,5-dinitro-4-chloro-benzenesulfonate 717 and
the desired secondary amines afforded intermediates
8a–i in high yield, which were converted to sulfonyl
chlorides 9a–i by treatment with PCl5 in dichlorometh-
ane as solvent. The target sulfonamides 10–26 were
prepared from 9a–i by reaction of these sulfonyl chlo-
rides with aromatic amines in pyridine at 40 ꢁC. Dinit-
roaniline benzamides were prepared as shown in
Scheme 3. Commercially available 4-chloro-3,5-dinitro-
benzoic acid 27 was reacted with either dipropylamine
or dibutylamine (15 equiv) in methanol at reflux to
provide benzoic acids 28a and 28b in high yield. Ben-
zamides 29–39 were synthesized by coupling 28a or
28b with 1.5 equiv of the desired aromatic amines in
dichloromethane at room temperature in the presence
of DCC and catalytic DMAP.
N
N
O N
2
NO
O N
2
NO
2
2
SO
SO
2
2
HN
HN
1
2
To provide a rationale for the lack of in vivo antitrypan-
osomal efficacy of these compounds, pharmacokinetic
and metabolism studies were conducted with 2.15 As
expected, the compound was extensively metabolized
in vitro and in vivo, with the major products resulting
from N1 ring oxidation, N4 alkane oxidation, and N4
oxidation. Based on the results of these studies, we have
prepared analogs of 1 and 2 in an effort to achieve
greater metabolic stability while maintaining selective
antiparasitic activity. The synthesis and biological eval-
uation of these compounds is reported here.
O
O
i
v
ii, iii, iv
a R = nPr
b R = CH2cPr
c R = CH2Ph
HCl
HN R
NH
Cl
R
N
Boc
R
4a-c
5a-c
6a-c
3
Scheme 1. Reagents and conditions: (i) RNH2, Et3N, CH2Cl2, 0 ꢁC to rt; (ii) BH3–THF (3 equiv), reflux, 14 h; (iii) MeOH, reflux, 2 h; (iv) Boc2O
(1.4 equiv), CH2Cl2, rt, 14 h; (v) 4 M HCl/dioxane (1.2 equiv), CH2Cl2, rt, 14 h.
a: R1 = R2 = nPr
b: R1 = R2 = nBu
c: R1 = R2 = iBu
d: R1 = R2 = CH2Ph
e: R1 = R2 = -(CH2)4-
f: R1 = R2 = H
R1
R2
R1
R2
Cl
N
N
O2N
NO2
O2N
NO2
O2N
NO2
i
ii
g: R1 = CH2cPr, R2 = nPr
h: R1 = R2 = CH2cPr
i: R1 = CH2cPr, R2 = CH2Ph
-
SO3 K+
SO3 K+
-
SO2Cl
9a-i
8a-i
7
R1
R2
10: R1 = R2 = nPr, R3 = R4 = H, R5 = F
11: R1 = R2 = nPr, R3 = R5 = H, R4 = F
12: R1 = R2 = nBu, R3 = R4 = H, R5 = F
13: R1 = R2 = nBu, R3 = R5 = H, R4 = F
14: R1 = R2 = iBu, R3 = R4 = H, R5 = F
15: R1 = R2 = iBu, R3 = R5 = H, R4 = F
16: R1 = R2 = CH2Ph, R3 = R4 = H, R5 = F
17: R1 = R2 = CH2Ph, R3 = R5 = H, R4 = F
18: R1 = R2 = CH2Ph, R3 = R4 = R5 = H
19: R1 = R2 = -(CH2)4-, R3 = R4 = H, R5 = F
20: R1 = R2 = nBu, R3 = R4 = H, R5 = OH
21: R1 = R2 = nBu, R3 = R5 = H, R4 = OH
22: R1 = R2 = nBu, R3 = OH R4 = R5 = H
23: R1 = R2 = H, R3 = R4 = R5 = H
24: R1 = CH2cPr, R2 = nPr, R3 = R4 = H, R5 = F
25: R1 = R2 = CH2cPr, R3 = R4 = H, R5 = F
26: R1 = CH2cPr, R2 = CH2Ph, R3 = R4 = H, R5 = F
N
O2N
NO2
iii
SO2 R3
HN
R4
R5
10-26
Scheme 2. Reagents and conditions: (i) secondary amine/reflux, secondary amine/MeOH/reflux, or NH3/H2O/reflux; (ii) PCl5/CH2Cl2; (iii) NH2Ar/
pyridine/40 ꢁC.

T. G. George et al. / Bioorg. Med. Chem. 14 (2006) 5699–5710
5701
R1
R1
R1
R1
Cl
N
N
29: R1 = nPr, R2 = R3 = R4 = R5 = H
30: R1 = nPr, R2 = R3 = R5 = H, R4 = F
31: R1 = nPr, R2 = R3 = R4 = H, R5 = F
32: R1 = nBu, R2 = R3 = R4 = R5 = H
33: R1 = nBu, R2 = R3 = R5 = H, R4 = F
34: R1 = nBu, R2 = R3 = R4 = H, R5 = F
35: R1 = nBu, R2 = R3 = R4 = H, R5 = Cl
36: R1 = nBu, R2 = R3 = R4 = H, R5 = OCH3
37: R1 = nBu, R2 = R3 = R4 = H, R5 = CH3
38: R1 = nBu, R2 = R3 = H, R4 = R5 = Cl
39: R1 = nBu, R2 = R4 = H, R3 = R5 = Cl
O2N
NO2
O2N
NO2
O2N
NO2
i
ii
COOH
27
COOH
O
NH
28a: R1 = nPr
28b: R1 = nBu
R2
R3
R5
R4
29 - 39
Scheme 3. Reagents and conditions: (i) dipropylamine/MeOH/reflux or dibutylamine/MeOH/reflux; (ii) NH2Ar/DCC/DMAP/CH2Cl2.
2.2. Biological evaluation
activity against mammalian tubulin was unanticipated,
it is likely that subtle distinctions between tubulins
account for the differences in susceptibilities between
the leishmanial and mammalian proteins in binding to
dinitroanilines. In the protozoan parasite Toxoplasma
gondii, organisms resistant to the dinitroaniline herbi-
cide oryzalin generated by chemical mutagenesis pos-
sessed numerous point mutations in a-tubulin.10 The
majority of these point mutations occurred within the
core of a-tubulin and not at the proposed binding site
for dinitroanilines determined by docking simulations.10
Also, the proposed binding site residues identified in
these docking studies are not specific to protozoa, sug-
gesting that minor differences between mammalian and
protozoal tubulin account for dinitroaniline specificity.
In addition, our mammalian tubulin assay, in which
the protein does not assemble as robustly as does the
parasite protein, may be more sensitive to assembly inhi-
bition than our leishmanial tubulin assay, in which the
protein assembles rapidly.13 Supporting this idea, Hamel
has shown that differing conditions employed in tubulin
assembly assays have a profound effect on experimental-
ly determined IC50 values.18
Since earlier studies indicated that the N4-dialkyl groups
and the N1-phenyl ring of 2 were extensively metabo-
lized,15 synthetic modifications to lead compounds 1
and 2 aiming to increase metabolic stability focused on
these two regions of the molecules. Substitution with a
fluorine atom was attempted on the N1-phenyl ring be-
cause the presence of larger groups led to a significant
loss of activity.14 Potent antikinetoplastid activity was
maintained when a fluorine atom was placed either para
or meta to the sulfonamide group in both N4,N4-di-n-
propyl compounds 10 and 11 and N4,N4-di-n-butyl com-
pounds 12 and 13, with the para-substituted compounds
being more active against T. b. rhodesiense in vitro
(Table 1). Compounds 10 and 12 also maintained selec-
tive activity for leishmanial tubulin (Table 2). However,
substantial losses in antiparasitic activity were observed
when the N4 position was disubstituted with branched
chains (compounds 14 and 15) or benzyl groups (com-
pounds 16–18), particularly against African trypano-
somes. For example, N4,N4-diisobutyl analog 14 is 19-
and 11-fold less potent against T. b. rhodesiense than
the corresponding N4,N4-di-n-propyl compound 10
and N4,N4-di-n-butyl analog 12, respectively. Interest-
ingly, such substitutions did not lead to a decrease in
toxicity to mammalian L6 cells, suggesting that this class
of compounds exerts antiparasitic and toxic effects
through different mechanisms. While these bulky substi-
tutions at N4 in 14–18 led to a loss of antikinetoplastid
activity, so did the presence of a pyrrolidine ring (com-
pound 19), and the absence of an N4 dialkyl group (com-
pound 23) led to decreased antileishmanial activity
compared to 1, 2, and 10–13.
Compound 26, which possessed the mixed benzyl-cyclo-
propylmethyl substitution pattern at N4, showed antile-
ishmanial activity that was slightly lower than 1, 2, and
10–13, but was as potent as 2 against T. b. rhodesiense
and also displayed selective activity for leishmanial
tubulin. The activity of 26 against leishmanial tubulin
demonstrates that a single substituent at N4 with added
bulk compared to an alkyl chain is permitted for binding
to this parasite protein. Considering the poor activity of
dibenzyl compounds 16–18 against T. b. rhodesiense, the
potency of 26 against these organisms is surprising.
Because large quantities of assembly-competent tubulin
from T. brucei subspecies are not currently available, an
assessment of the cellular antimitotic effects of several of
these compounds on African trypanosomes at their IC50
concentrations will be required in order to provide fur-
ther evidence that these new dinitroaniline sulfonamides
exert their trypanocidal effects through a tubulin
mechanism.
Compounds 24 and 25, which contained either one or
two cyclopropylmethyl groups at N4, possessed activity
against Leishmania donovani and T. b. rhodesiense that
was comparable to that of 1, 2, and 10–13. Surprisingly,
the presence of these cyclopropyl groups at N4 led to a
decrease in selectivity for parasite tubulin. While 24
and 25 retained strong activity in blocking the assembly
of purified leishmanial tubulin, these compounds were
only 2.4- and 2.8-fold more potent against parasite tubu-
lin assembly compared to porcine tubulin assembly (Ta-
ble 2). Although the observation that 24 and 25 possess
The activity of N1-aryl-3,5-dinitrosulfanilamides against
intracellular T. cruzi is presented for the first time in

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T. G. George et al. / Bioorg. Med. Chem. 14 (2006) 5699–5710
Table 1. Activities of dinitroanilines (lM) against parasites and mammalian cellsa
Compound
IC50 versus L. donovani
axenic amastigotesa
IC50 versus
T. b. rhodesienseb
IC50 versus T. cruzib
IC50 versus L6 cellsb
IC50 versus Vero cellsa
1
5.8
3.5
5.7
4.9
5.1
4.5
9.3
10
0.18
0.12
0.17
0.48
0.28
1.4
3.2
2.6
19
1.9
0.59
7.5
7.0
3.0
1.8
9.6
11
17
12
26
19
2
10
20
22
6.3
9.3
11
12
10
2.9
13
14
6.2
11
3.3
NTd
NT
NT
NT
NT
NT
NT
24
15
16
14
18
12
10
17
18
6.9
14
19
8.9
8.6
51
15
21
14.5
9.9
21
19
20
73
15
70
12
2.6
1.3
9.6
NT
7.3
11
21
22
2.8
8.6
19
0.26
22
13
13
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
>100
NT
NT
NT
23
24
NT
0.37
0.31
0.13
29
NT
14
4.5
3.7
8.4
>100
>100
22
25
26
15
20
13
21
29
30
>100
64
17
99
30
>50
12
31
32
>100
>100
17
24
33
6.5
>50
5.1
5.1
3.8
3.5
7.8
23
23
33
34
>50
16
21
33
35
36
22
20
14
41
23
15
37
38
56
22
20
22
34
6.0
14
4.2c
NT
NT
0.01
39
39
Pentamidine
Melarsoprol
Benznidazole
Podophyllotoxin
2.4
NT
NT
NT
NT
0.015
NT
NT
NT
NT
3.4
NT
a Activities represent the mean of three independent experiments; the standard error is less than 30% of the mean in all cases.
b Activities represent the mean of at least two independent experiments; IC50 values used to calculate the average for a given compound are within a
factor of two.
c Value obtained from Ref. 26.
d NT, not tested.
Table 2. Antimicrotubule activity of selected compoundsa
However, compounds 10, 24, and 25 also show potent
activity against leishmanial tubulin but are less active
against intracellular T. cruzi in vitro. While subtle differ-
ences in susceptibility between leishmanial tubulin and
T. cruzi tubulin could help to account for these differenc-
es, the presence of a host cell membrane may also have a
profound influence on the accumulation of the com-
pounds within the American trypanosome, interfering
with correlations between antitubulin activity and
potency against intracellular T. cruzi.
Compound
IC50 (lM) versus
L. tarentolae tubulin
IC50 (lM) versus
porcine tubulin
2
10
12
20
21
24
25
26
29
6.5 0.3
5.3 0.3b
5.1 1.4
17 3b
>40
>40
>40
9.7 1.0
8.2 0.2
5.6 0.7
4.5 0.2
3.9 1.1
5.4 0.8
>40
11
11
1
4
>40
>40
Compounds 20–23 were prepared as potential metabo-
lites of 2. Through comparison of HPLC retention times
and fragmentation patterns with synthetic standards,
compounds 20, 22, and 23 were identified as metabolites
of this lead compound.15 Interestingly, the three metab-
olites in general show low antikinetoplastid activity,
while 21 displays in vitro antileishmanial and antitry-
panosomal activity comparable to that of 1 and 2. The
in vitro activity of 21 against kinetoplastid parasites
and leishmanial tubulin assembly suggests that the meta
a Unless otherwise noted, values represent means range of two
duplicate experiments.
b Values represent the mean standard error of at least three duplicate
experiments.
Table 1. Compounds 2, 12, and 21 are among the most
active of these agents against the American trypano-
some, and all of these compounds also display strong
activity against purified leishmanial tubulin in vitro.

T. G. George et al. / Bioorg. Med. Chem. 14 (2006) 5699–5710
5703
hydroxyl group present in this molecule may participate
in hydrogen bonding with the tubulin target, compen-
sating for the otherwise unfavorable steric interactions
that were observed in most other N1-phenyl substituted
compounds in this series.14 As with 24 and 25, a decrease
in selectivity for parasite tubulin was also observed with
compounds 20 and 21, and 20 showed higher activity
against porcine tubulin than against leishmanial tubulin.
Despite this, mitotic arrest was not observed in MCF-7
breast cancer cells incubated for 24 h with 3, 10, and
30 lM 20 (Dan Sackett, data not shown). We hypothe-
size that other cellular targets are affected by 20 in mam-
malian cells prior to tubulin. It is worth noting that
compounds 21, 24, and 25 maintain selectivity for kine-
toplastid parasites over mammalian cells, particularly
against African trypanosomes.
16 and 18 displaying 2.2- and 2.1-fold greater in vitro
stability compared with 2 when incubated with the S9
fraction for 10 min. In addition, the pyrrolidine-contain-
ing compound 19 also displayed greater in vitro stability
than 2. N4-Dibutyl, N4-isobutyl, and N4-cyclopropyl-
methyl containing compounds 12, 14, and 25, which
all possess a para-fluoro group on the N1 ring, were less
stable than compound 2. Taken together, these data sug-
gest that sufficiently bulky substituents at the N4 posi-
tion stabilize the compounds to metabolism compared
with 2, while the addition of a para-fluoro group on
the N1 ring does not prevent degradation, perhaps due
to the greater influence of the N4 groups on metabolic
oxidation. The role played by the N4 dialkyl groups in
rapid metabolism is further highlighted by the stability
of 23, which contains an unsubstituted anilino nitrogen
atom. Unfortunately, 16, 18, and 23 display low activity
against kinetoplastid parasites. Interestingly, 26 shows
the lowest metabolic stability, suggesting that dibenzyl
groups are required at N4 to introduce sufficient steric
bulk to slow in vitro metabolism. Compounds 20 and
21, which possess para and meta hydroxyl groups on
the N1 phenyl ring, respectively, display greater meta-
bolic stability compared to 2. These data suggest that
introduction of polar functional groups may increase
metabolic stability in this series of molecules. Com-
pound 21 displays similar antiparasitic and antitubulin
activity compared to 2 and retains its selectivity for kine-
toplastid parasites compared to mammalian cell lines,
particularly for African trypanosomes.
We also prepared dinitroaniline benzamides 29–39 to
examine the effect of changing the linker between the
two aromatic rings in this series of molecules. In general,
substitution of the amide for the sulfonamide group
leads to significant losses in antikinetoplastid activity.
For example, benzamide 29 shows IC50 values of >100
and 29 lM versus L. donovani and T. b. rhodesiense,
respectively, while the corresponding sulfonamide 1 dis-
plays IC50 values of 5.8 and 0.18 lM against these
parasites. While amides that contained either an unsub-
stituted aniline ring or that possessed a para-fluoro atom
on this ring were inactive, meta-fluorinated compounds
31 and 34 showed moderate activity against L. donovani
and T. cruzi, encouraging us to prepare meta-substituted
compounds 35–39. Although 36 and 37 showed
reasonable activity against T. cruzi, sulfonamides 1, 2,
13, and 21 remained more potent against this parasite.
Thus the dinitroaniline benzamides were not investigat-
ed further.
3. Conclusion
Based on in vitro antikinetoplastid activity, selectivity,
and metabolic stability, compound 21 emerges as a can-
didate for further evaluation against kinetoplastid para-
sites. This compound also provides a possible starting
point for the synthesis of further analogs with improved
metabolic characteristics and for the preparation of
dinitroaniline sulfanilamide prodrug candidates against
kinetoplastid parasites.
Given the extensive metabolism of 2,15 a major goal of
this work was to prepare active antimitotic antikineto-
plastid agents with improved metabolic stability. We
thus examined the rate at which selected compounds
were converted to other molecules upon incubation with
the S9 fraction of rat liver microsomes (Table 3). The
presence of an N4-dibenzyl substitution stabilized com-
pounds 16 and 18 to metabolism compared to 2, with
Several new dinitroaniline sulfonamides have been iden-
tified that possess selective activity against leishmanial
tubulin, while others also display activity against the
corresponding mammalian protein. Such observations
should not dampen enthusiasm for the investigation of
tubulin as an antiprotozoal drug target. For example,
while the dihydrofolate reductase (DHFR) inhibitor
methotrexate is highly toxic to mammalian cells and
bacteria alike, the success of the DHFR inhibitor tri-
methoprim as antibacterial agent cannot be disputed.
While the structural basis of the preference of trimetho-
prim for prokaryotic dihydrofolate reductase was not
immediately appreciated,19 further study of the ligand
binding properties of prokaryotic and eukaryotic DHFR
shed light on the basis of antimicrobial selectivity and led
to the identification of new, selective ligands.20,21 Future
studies focused on providing a model for the interaction
between kinetoplastid tubulin and the dinitroanilines
could be useful in identifying compounds with increased
potency and selectivity for the parasite protein. Given
Table 3. In vitro metabolism of selected compounds in the presence of
rat liver S9 enzymes
Compound
Percent remaining at 10 mina
2
12
14
16
18
19
20
21
23
25
26
31
20
21
69
64
49
48
45
3
1
3
3
4
9
1
5
115 12
6
1
0
0
a Values represent means standard error of two triplicate experi-
ments (N = 6).

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T. G. George et al. / Bioorg. Med. Chem. 14 (2006) 5699–5710
the vital cellular role of tubulin, the potency of dinitroan-
iline sulfonamides against kinetoplastid parasites, and
their selectivity profile against leishmanial and mamma-
lian tubulins, further exploration of this target protein in
protozoans is warranted.
(972 mg, 9.3 mmol) and cyclopropylmethylamine hydro-
chloride (1 g, 9.3 mmol); yield 369 mg, 25% (after five
steps); mp = 275 ꢁC; 1H NMR (CDCl3) d 0.46–0.51
(m, 4H), 0.69–0.75 (m, 4H), 1.26–1.39 (m, 2H), 2.92–
2.96 (m, 4H), 9.69 (br s, 2H).
4.2.3. N-Benzyl-N-(cyclopropylmethyl)amine hydrochlo-
ride (6c). Reaction of cyclopropanecarbonyl chloride
(3.0 g, 28.7 mmol) and benzylamine (3.1 g, 28.7 mmol)
afforded cyclopropionyl benzylamide (5.02 g, quantita-
tive). The reduction of cyclopropionyl benzylamide
(3 g, 17.1 mmol) afforded 6c: yield 1.6 g, 47% (after four
steps); mp = 184 ꢁC; H NMR (CDCl3) d 0.36–0.41 (m,
2H), 0.58–0.69 (m, 2H), 1.21–1.27 (m, 1H), 2.67–2.73
(m, 2H), 4.09–4.13 (m, 2H), 7.38–7.64 (m, 5H), 9.98
(br s, 2H).
4. Experimental
4.1. General Methods
All reagents were obtained from commercial vendors
and were used without further purification unless other-
wise indicated. Nuclear magnetic resonance spectra were
obtained at 250 or 300 MHz for 1H and 62.5 or 75 MHz
for 13C using instruments from Bruker. Elemental anal-
yses were performed by Atlantic Microlabs (Norcross,
GA) and were within 0.4% of the calculated values un-
less otherwise noted. Thin layer chromatography was
conducted on precoated TLC plates from E. Merck.
Compounds 1 and 2 were prepared as described
previously.14
1
4.2.4. 3,5-Dinitro-N4,N4-diisobutylbenzenesulfonate (8c).
A suspension of 3,5-dinitro-4-chlorobenzenesulfonate
(7, 3 g, 9.3 mmol) and diisobutylamine (4.9 mL,
28.1 mmol) in 60 mL methanol was refluxed for 3 h.
After 3 h the solvent was evaporated. The product was
purified by silica gel column chromatography, eluting
1
4.2. General procedure for the synthesis of secondary
amine hydrochlorides 6a–c16
with MeOH/EtOAc 1:25; yield 2.74 g, 70%; H NMR
(DMSO-d6): d 0.81 (d, J = 6.6 Hz, 12H), 1.72–1.87 (m,
2H), 2.72 (d, J = 6.9 Hz, 4H), 8.05 (s, 2H). Note: some
traces of the dialkylamine salts were often associated
with dinitrosulfonates 8a–i as indicated by their 1H
NMR spectra which did not interfere with the further
reactions.
To a solution of the primary amine or primary amine
hydrochloride (1 equiv) in dry CH2Cl2 at 0 ꢁC, triethyl-
amine (1.5–3 equiv) was added followed by cyclopropan-
ecarbonyl chloride (1 equiv). After 10 min, the ice bath
was removed and stirring was continued at rt overnight.
Dichloromethane was removed in vacuo and the residue
was partitioned between EtOAc and saturated NaHCO3.
The aqueous layer was further extracted with EtOAc, the
combined organic layers were washed with H2O and
brine and concentrated in vacuo. This residue was dried
under high vacuum, then treated with 1 M BH3 in THF
(3 equiv). The mixture was heated at reflux for 14 h and
then cooled to 0 ꢁC. MeOH was carefully added and the
solution was heated to reflux for 5 h. Upon recooling to
0 ꢁC, a solution of di-tert-butyl dicarbonate (1.4 equiv)
in CH2Cl2 was added, the resulting mixture was stirred
at rt overnight, solvent was removed in vacuo, then the
residue was partitioned between EtOAc and water. The
organic phase was washed with water, brine, dried over
MgSO4, and concentrated in vacuo to give the crude
Boc protected amine. This protected amine was dissolved
in CH2Cl2 and treated carefully with 4 M HCl in dioxane
(1.2 equiv). The mixture was stirred at rt overnight and
concentrated in vacuo. The resulting white solid was trit-
urated with ether, collected by filtration, washed with
ether, and dried in vacuo.
4.2.5. 3,5-Dinitro-N4,N4-dibenzylbenzenesulfonate (8d).
A suspension of 7 (2 g, 6.23 mmol) and dibenzylamine
(3.6 mL, 18.7 mmol) in 100 mL methanol was refluxed
for 3 h. After 3 h the solvent was evaporated. The prod-
uct was purified by silica gel column chromatography,
1
eluting with MeOH/EtOAc 1:15; yield 2.5 g, 83%; H
NMR (DMSO-d6) d 4.13 (s, 4H), 7.14–7.36 (m, 10H);
8.11 (s, 2H).
4.2.6. 3,5-Dinitro-4-pyrrolidin-1-yl-benzenesulfonate (8e).
A suspension of 7 (1 g, 3.12 mmol) and pyrrolidine
(0.5 mL, 6.1 mmol) in 20 mL methanol was refluxed
for 3 h. The solvent was removed under reduced pres-
sure and the resulting residue was purified by silica gel
chromatography, eluting with MeOH/EtOAc 1:25; yield
1
1.1 g, 97%; H NMR (MeOH-d4) d 3.28 (m, 4H), 4.83
(m, 4H), 8.25 (s, 2H).
4.2.7. 4-Amino-3,5-dinitrobenzenesulfonate (8f). A sus-
pension of 7 (1 g, 3.12 mmol) in a mixture of aq NH3
(4 mL) and H2O (8 mL) was refluxed for 2 h, then
cooled to rt and kept for crystallization at 0 ꢁC. The
crystals were filtered and dried to afford 8f: 624 mg,
67%; 1H NMR (DMSO-d6) d 8.51 (br s, 2H, NH2),
8.55 (s, 2H, ArH).
4.2.1. N-(Cyclopropylmethyl)-N-propylamine hydrochlo-
ride (6a). Prepared from cyclopropanecarbonyl chloride
(1.5 g, 14.3 mmol)andpropylamine (848 mg, 14.3 mmol);
yield 651 mg, 31% (after five steps); mp = 276 ꢁC; H
NMR (CDCl3) d 0.44–0.50 (m, 2H), 0.68–0.76 (m, 2H),
1.03 (t, 3H, J = 7.6 Hz), 1.23–1.36 (m, 1H), 1.89–2.04
(m, 2H), 2.86–3.02 (m, 4H), 9.56 (br s, 2H).
1
4.3. General procedure for the synthesis of N4,N4-di-n-
substituted sulfonates 8g–i
To a suspension of 7 (1 equiv) and N,N-dialkylamine
hydrochloride (1.1 equiv) in methanol, triethylamine
(6 equiv) was added and refluxed for 3 h, after which
4.2.2. N,N-Di(cyclopropylmethyl)amine hydrochloride
(6b). Prepared from cyclopropanecarbonyl chloride

T. G. George et al. / Bioorg. Med. Chem. 14 (2006) 5699–5710
5705
the solvent was evaporated. The residue was extracted
with EtOAc, washed with water and saturated brine,
respectively, and dried using anhydrous MgSO4 and
evaporated. The product was purified by silica gel col-
umn chromatography using a solvent mixture of
EtOAc/MeOH.
125.21 (d, JCF = 8.3 Hz), 128.66, 128.81, 130.99 (d,
JCF = 3.0 Hz), 141.78, 144.40, 161.24 (d, JCF
246.0 Hz); HRMS (ESI) calcd for C18H21FN4NaO6S
(M+Na)+ 463.1064, measured (M+Na)+ 463.1077.
Anal. (C18H21FN4O6S) C, H, N.
=
4.4.2. N1-(3-Fluoro)phenyl-3,5-dinitro-N4,N4-di-n-propyl-
sulfanilamide (11). The reaction of sulfonylchloride 9a
(730 mg, 1.99 mmol, prepared from 8a) and 3-fluoroan-
iline (576 lL, 5.98 mmol) in pyridine (32 mL) at 40 ꢁC
afforded 11 as yellow crystals: yield 474 mg, 54%;
4.3.1. 3,5-Dinitro-N4-(cyclopropylmethyl)-N4-propylben-
zenesulfonate (8g). Reaction of 7 (487 mg, 1.52 mmol)
and 6a (250 mg, 1.67 mmol) in the presence of triethyl-
amine (1.26 mL, 9.12 mmol) in MeOH (45 mL) afforded
8g as a yellow solid: 480 mg, 79%; 1H NMR (DMSO-d6)
d 0.05–0.11 (m, 2H), 0.39–0.45 (m, 2H), 0.73–0.92 (m,
4H), 1.38–1.48 (m, 2H), 2.83 (d, J = 6.8 Hz, 2H), 2.92–
2.97 (m, 2H), 8.14 (s, 2H).
mp = 117 ꢁC; 1H NMR (CDCl3)
d 0.88 (t, 6H,
J = 7.2 Hz), 1.53–1.68 (m, 4H), 2.94–2.99 (m, 4H), 6.60
(br s, 1H), 6.83–6.96 (m, 3H), 7.21–7.35 (m, 1H), 8.17
(s, 2H); 13C NMR (CDCl3) d 11.07, 20.63, 54.00,
108.80 (d, JCF = 25.5 Hz), 113.03 (d, JCF = 21.0 Hz),
116.74, 128.48, 128.85, 131.05 (d, JCF = 9 Hz), 136.97
(d, JCF = 9.8 Hz), 142.01, 144.32, 163.11 (d,
4.3.2. 3,5-Dinitro-N4,N4-di(cyclopropylmethyl)benzene-
sulfonate (8h). Reaction of 7 (472 mg, 1.47 mmol) and
6b (250 mg, 1.55 mmol) in the presence of triethylamine
(1.2 mL, 8.6 mmol) in MeOH (40 mL) afforded 8h as a
yellow solid: 514 mg, 85%; 1H NMR (DMSO-d6) d
0.03–0.09 (m, 4H), 0.36–0.43 (m, 4H), 0.77–0.88 (m,
2H), 2.89 (d, J = 7.0 Hz, 4H), 8.16 (s, 2H).
JCF = 246.8 Hz);
HRMS
(ESI)
calcd
for
C18H21FN4NaO6S (M+Na)+ 463.1064, measured
(M+Na)+ 463.1070 . Anal. (C18H21FN4O6S) C, H, N.
4.4.3. N1-(4-Fluoro)phenyl-3,5-dinitro-N4,N4-di-n-butyl-
sulfanilamide (12). The reaction of sulfonylchloride 9b
(900 mg, 2.3 mmol, prepared from 8b) and 4-fluoroani-
line (770 lL, 8.01 mmol) in pyridine (38 mL) at 40 ꢁC
afforded 12 as yellow crystals: yield 450 mg, 42%;
4.3.3. 3,5-Dinitro-N4-benzyl-N4-(cyclopropylmethyl)ben-
zenesulfonate (8i). Reaction of 7 (200 mg, 0.62 mmol)
and 6c (135 mg, 0.69 mmol) in the presence of triethyl-
amine (0.5 mL, 3.6 mmol) in MeOH (20 mL) afforded
mp = 137 ꢁC; 1H NMR (CDCl3)
d 0.89 (t, 6H,
1
8i as a yellow solid: 190 mg, 69%; H NMR (DMSO-
J = 7.4 Hz), 1.22–1.35 (m, 4H), 1.52–1.62 (m, 4H),
2.97–3.06 (m, 4H), 6.49 (br s, 1H), 7.02–7.15 (m, 4H),
8.09 (s, 2H); 13C NMR (CDCl3) d 13.65, 19.86, 29.37,
52.10, 116.68 (d, JCF = 22.5 Hz), 125.31 (d,
JCF = 8.3 Hz), 128.58, 128.85, 130.98 (d, JCF = 3.8 Hz),
141.69, 144.47, 161.28 (d, JCF = 246.8 Hz); HRMS
(ESI) calcd for C20H25FN4NaO6S (M+Na)+ 491.1377,
measured (M+Na)+ 491.1393. Anal. (C20H25FN4O6S)
C, H, N.
d6) d À0.05 to 0.01 (m, 2H), 0.33–0.40 (m, 2H), 0.78–
0.86 (m, 1H), 2.76 (d, J = 6.6 Hz, 2H), 4.23 (s, 2H),
7.22–7.59 (m, 5H), 8.13 (d, J = 1.2 Hz, 2H).
4.4. General procedure for the synthesis of N1-aryl-3,5-
dinitro-N4,N4-di-n-substituted sufanilamide derivatives
(10–26)
To a suspension of sulfonic acids (8a–i) in dichloro-
methane, PCl5 (2.5 equiv) was added and the reaction
mixture was stirred for 2 h for 8a–e and 8g–i and
overnight for 8f. The reaction mixture was washed
with water and dried using anhydrous Na2SO4. The
product 3,5-dinitro-N4,N4-di-n-substituted sulfonyl-
chlorides 9a–i were used without further purification.
Pyridine was then added, the temperature was adjust-
ed to 40 ꢁC, 3–5 equiv of aniline derivative was added
and the reaction mixture was stirred for 3 h at 40 ꢁC.
Pyridine was then evaporated in vacuo, the dark resi-
due was washed with water and extracted with ethyl
acetate and the product was purified by silica gel col-
umn chromatography. Recrystallization was performed
using a solvent mixture of dichloromethane and
hexane.
4.4.4. N1-(3-Fluoro)phenyl-3,5-dinitro-N4,N4-di-n-butyl-
sulfanilamide (13). The reaction of sulfonylchloride 9b
(1.22 g, 3.1 mmol, prepared from 8b) and 3-fluoroaniline
(1.04 mL, 10.8 mmol) in pyridine (50 mL) at 40 ꢁC affor-
ded 13 as yellow crystals: yield 676 mg, 48%;
mp = 139 ꢁC; 1H NMR (CDCl3)
d 0.89 (t, 6H,
J = 7.4 Hz), 1.22–1.34 (m, 4H), 1.52–1.62 (m, 4H), 2.99
(t, 4H, J = 7.5 Hz), 6.71 (br s, 1H), 6.89–6.96 (m, 3H),
7.27–7.35 (m, 1H), 8.16 (s, 2H); 13C NMR (CDCl3) d
13.65, 19.86, 29.35, 52.12, 108.97 (d, JCF = 24.8 Hz),
113.16 (d, JCF = 21.0 Hz), 116.88 (d, JCF = 3.0 Hz),
128.60, 128.69, 131.07 (d, JCF = 9.8 Hz), 136.92 (d,
JCF = 10.5 Hz),
JCF = 246 Hz);
141.91,
HRMS
144.44,
(ESI)
163.14
calcd
(d,
for
C20H25FN4NaO6S (M+Na)+ 491.1377, measured
(M+Na)+ 491.1383. Anal. (C20H25FN4O6S) C, H, N.
4.4.1. N1-(4-Fluoro)phenyl-3,5-dinitro-N4,N4-di-n-propyl-
sulfanilamide (10). Reaction of the sulfonyl chloride 9a
(670 mg, 1.83 mmol, prepared from 8a) and 4-fluoroan-
iline (527 lL, 5.49 mmol) in pyridine (30 mL) at 40 ꢁC
afforded 10 as yellow crystals, yield 552 mg, 68%;
4.4.5.
N1-(4-Fluoro)phenyl-3,5-dinitro-N4,N4-diisobutylsulfa-
nilamide (14). The reaction of sulfonylchloride 9c
(900 mg, 2.29 mmol, prepared from 8c) and 4-fluoroan-
iline (770 lL, 8.01 mmol) in pyridine (38 mL) at 40 ꢁC
afforded 14 as yellow crystals: yield 672 mg, 63%;
mp = 56–57 ꢁC; 1H NMR (CDCl3) d 0.89 (d, 12H,
J = 6.6 Hz), 1.86–1.99 (m, 2H), 2.79 (d, 4H,
J = 6.9 Hz), 6.45 (br s, 1H), 7.01–7.13 (m, 4H), 8.05 (s,
mp = 91 ꢁC; 1H NMR (CDCl3)
d 0.88 (t, 6H,
J = 7.4 Hz), 1.52–1.68 (m, 4H), 2.94–3.03 (m, 4H), 6.49
(br s, 1H), 6.99–7.15 (m, 4H), 8.09 (s, 2H); 13C NMR
(CDCl3) d 11.10, 20.67, 54.02, 116.68 (d, JCF = 22.5 Hz),

5706
T. G. George et al. / Bioorg. Med. Chem. 14 (2006) 5699–5710
2H); 13C NMR (CDCl3) d 20.17, 26.66, 60.75, 116.54 (d,
JCF = 22.5 Hz), 125.26 (d, JCF = 8.3 Hz), 127.66,
129.18, 131.25, (d, JCF = 3.0 Hz), 141.22, 143.43,
161.16 (d, JCF = 245.3 Hz); HRMS (ESI) calcd for
C20H25FN4NaO6S (M+Na)+ 491.1377, measured
(M+Na)+ 491.1360. Anal. (C20H25FN4O6S) C, H, N.
C26H22N4NaO6S (M+Na)+ 541.1158, measured
(M+Na)+ 541.1161. Anal. (C26H22N4O6S) H, N, C.
Calcd: 60.22; found: 60.95.
4.4.10. N1-(4-Fluoro)phenyl-3,5-dinitro-N4-pyrrolidin-1-yl-
sulfanilamide (19). The reaction of sulfonyl chloride
9e (0.95 g, 2.82 mmol, prepared from 8e) and 4-fluo-
roaniline (0.8 g, 7.2 mmol) in pyridine (20 mL) at
40 ꢁC afforded 19 as yellow crystals, yield 842 mg,
73%; mp = 201 ꢁC; 1H NMR (MeOH-d4) d 2.05 (m,
4H), 3.28 (m, 4H), 7.08 (m, 2H), 7.29 (m, 2H),
8.24 (s, 2H), 9.1 (s, 1H); 13C NMR (CDCl3) d
25.64, 54.59, 116.67 (d, JCF = 22.5 Hz), 122.74,
4.4.6. N1-(3-Fluoro)phenyl-3,5-dinitro-N4,N4-diiso-
butylsulfanilamide (15). The reaction of sulfonylchloride
9c (1.03 g, 2.62 mmol, prepared from 8c) and 3-fluoro-
aniline (880 lL, 9.17 mmol) in pyridine (43 mL) at
40 ꢁC afforded 15 as yellow crystals: yield 682 mg,
1
56%; mp = 113–114 ꢁC; H NMR (CDCl3) d 0.89 (d,
12H, J = 6.3 Hz), 1.84–1.97 (m, 2H), 2.78 (d, 4H,
J = 6.9 Hz), 6.67 (br s, 1H), 6.88–6.99 (m, 3H), 7.26–
7.34 (m, 1H), 8.12 (s, 2H); 13C NMR (CDCl3) d 20.19,
26.67, 60.79, 109.19 (d, JCF = 25.5 Hz), 113.25 (d,
JCF = 20.3 Hz), 117.10 (d, JCF = 3.0 Hz), 127.71,
124.96 (d, JCF = 8.3 Hz), 129.15, 134.21 (d, JCF =
3 Hz), 138.87, 141.13, 159.61 (d, JCF = 241.5 Hz);
HRMS (ESI) calcd for C16H15FN4O6S (M+Na)+
433.0594, measured (M+Na)+ 433.0584. Anal.
(C16H15FN4O6S) C, H, N.
129.09, 131.03 (d, JCF = 9.0 Hz), 136.94 (d, JCF
=
9.8 Hz), 141.34, 143.46, 163.14 (d, JCF = 246.8 Hz);
HRMS (ESI) calcd for C20H25FN4NaO6S (M+Na)+
491.1377, measured (M+Na)+ 491.1401. Anal.
(C20H25FN4O6S) C, H, N.
4.4.11. N1-(4-Hydroxy)phenyl-3,5-dinitro-N4,N4-di-n-butyl-
sulfanilamide (20). The reaction of sulfonylchloride 9b
(prepared from 8b, 600 mg, 1.45 mmol) and 4-hydrox-
yaniline (632 mg, 5.8 mmol) in pyridine (20 mL) at
40 ꢁC afforded 20 as yellow crystals: yield 443 mg,
66%; mp 180 ꢁC; 1H NMR (CDCl3) d 0.86 (t,
J = 7.4 Hz, 6H), 1.19–1.31 (m, 4H); 1.45–1.55 (m, 4H),
2.95 (t, J = 7.5, 4H), 4.86 (s, 1H), 6.35 (br s, 1H), 6.76
(d, J = 8.9, 2H), 6.97 (d, J = 8.9, 2H), 8.02 (s, 2H); 13C
NMR (CDCl3) d 13.65, 19.86, 29.41, 52.10, 116.43,
126.35, 127.59, 128.54, 129.32, 141.52, 144.55, 154.90;
HRMS (ESI) calcd for C20H26N4NaO7S (M+Na)+
489.1420, measured (M+Na)+ 489.1416. Anal.
(C20H26N4O7S) C, H, N.
4.4.7. N1-(4-Fluoro)phenyl-3,5-dinitro-N4,N4-di-
benzylsulfanilamide (16). The reaction of sulfonylchlo-
ride 9d (prepared from 8d, 540 mg, 1.121 mmol) and
4-fluoroaniline (537 lL, 5.60 mmol) in pyridine
(38 mL) at 40 ꢁC afforded 16 as yellow crystals: yield
1
287 mg, 48%; mp = 139 ꢁC; H NMR (CDCl3) d 4.15
(s, 4H), 6.41 (s, 1H), 6.92–7.33 (m, 14H), 8.09 (s, 2H);
13C NMR (CDCl3) d 56.96, 116.75 (d, JCF = 23.3 Hz),
125.71 (d, JCF = 8.3 Hz), 128.43, 128.61, 129.16,
130.35, 130.74 (d, JCF = 3.0 Hz), 134.17, 141.21,
145.04, 161.40 (d, JCF = 246.8 Hz); HRMS (ESI) calcd
for C26H21FN4NaO6S (M+Na)+ 559.1064, measured
(M+Na)+ 559.1090. Anal. (C26H21FN4O6S) C, H, N.
4.4.12. N1-(3-Hydroxy)phenyl-3,5-dinitro-N4,N4-di-n-butyl-
sulfanilamide (21). The reaction of sulfonylchloride 9b
(prepared from 8b, 600 mg, 1.45 mmol) and 3-hydrox-
yaniline (632 mg, 5.8 mmol) in pyridine (20 mL) at
40 ꢁC afforded 21 as yellow crystals: yield 432 mg,
64%; mp 152 ꢁC; 1H NMR (CDCl3) d 0.89 (t,
J = 7.5 Hz, 6H), 1.24–1.34 (m, 4H), 1.51–1.61 (m, 4H),
2.99 (t, J = 7.5 Hz, 4H), 4.95 (s, 1H), 6.56 (s, 1H),
6.66–6.70 (m, 3H), 7.19 (m, 1H), 8.15 (s, 2H); 13C
NMR (CDCl3) d 13.65, 19.87, 29.37, 52.11, 108.92,
113.45, 113.78, 128.67, 128.94, 130.79, 136.53, 141.80,
144.47, 156.66; HRMS (ESI) calcd for C20H26N4NaO7S
(M+Na)+ 489.1420, measured (M+Na)+ 489.1431.
Anal. (C20H26N4O7S) C, H, N.
4.4.8. N1-(3-Fluoro)phenyl-3,5-dinitro-N4,N4-dibenzyl-
sulfanilamide (17). The reaction of sulfonylchloride 9d
(prepared from 8d, 600 mg, 1.24 mmol) and 3-fluoroani-
line (550 lL, 5.7 mmol) in pyridine (40 mL) at 40 ꢁC affor-
ded 17 as yellow crystals: yield 250 mg, 38%; mp = 66–
67 ꢁC; 1H NMR (CDCl3) d 4.10 (s, 4H), 6.74 (br s, 1H),
6.80–7.31 (m, 14H), 8.17 (d, J = 0.9 Hz, 2H); 13C NMR
(CDCl3) d 56.93, 109.39 (d, JCF = 25.5 Hz), 113.48
(d, JCF = 21.0 Hz), 117.28 (d, JCF = 3.0 Hz), 128.46,
128.56, 128.60, 129.18, 130.01, 131.13 (d, JCF = 9.0 Hz),
134.02, 136.68 (d, JCF = 10.5 Hz), 141.47, 144.82,
163.13 (d, JCF = 246.8 Hz); HRMS (ESI) calcd for
C26H21FN4NaO6S (M+Na)+ 559.1064, measured
(M+Na)+ 559.1048. Anal. (C26H21FN4O6S) H, N, C.
Calcd: 58.20; found: 57.70.
4.4.13. N1-(2-Hydroxy)phenyl-3,5-dinitro-N4,N4-di-n-butyl-
sulfanilamide (22). The reaction of sulfonylchloride 9b
(prepared from 8b, 600 mg, 1.45 mmol) and 2-hydrox-
yaniline (632 mg, 5.8 mmol) in pyridine (20 mL) at
40 ꢁC afforded 22 as yellow crystals: yield 481 mg,
71%; mp 128 ꢁC; 1H NMR (CDCl3) d 0.89 (t,
J = 7.4 Hz, 6H), 1.28 (m, 4H), 1.51–1.60 (m, 4H), 2.99
(t, J = 7.5 Hz, 4H), 5.52 (s, 1H), 6.65 (s, 1H), 6.86–
6.94 (m, 2H), 7.12–7.19 (m, 2H), 8.12 (s, 2H); 13C
NMR (CDCl3) d 13.66, 19.85, 29.38, 52.09, 116.52,
121.74, 122.34, 125.10, 128.37, 128.58, 128.80, 141.66,
144.39, 148.92; HRMS (ESI) calcd for C20H26N4NaO7S
(M+Na)+ 489.1420, measured (M+Na)+ 489.1414.
Anal. (C20H26N4O7S) C, H, N.
4.4.9. N1-Phenyl-3,5-dinitro-N4,N4-dibenzylsulfanilamide
(18). The reaction of sulfonylchloride 9d (prepared from
8d, 600 mg, 1.24 mmol) and aniline (550 lL, 6 mmol) in
pyridine (40 mL) at 40 ꢁC afforded 18 as yellow crystals:
yield 250 mg, 40%; mp = 64–65 ꢁC; 1H NMR (CDCl3) d
4.11 (s, 4H), 6.49 (s, 1H), 7.09–7.38 (m, 15H), 8.10 (s,
2H); 13C NMR (CDCl3) d 56.87, 122.69, 126.80,
128.39, 128.45, 128.58, 129.17, 129.82, 130.65, 134.20,
135.06, 141.21, 144.98; HRMS (ESI) calcd for

T. G. George et al. / Bioorg. Med. Chem. 14 (2006) 5699–5710
5707
4.4.14. N1-Phenyl-3,5-dinitrosulfanilamide (23). The
reaction of 4-amino-3,5-dinitrosulfonylchloride 9f (pre-
pared from 8f, 50 mg, 0.16 mmol) and aniline (54 lL,
0.58 mmol) in pyridine (2 mL) at 40 ꢁC afforded 23 as
yellow crystals: yield 30 mg, 54%; mp 197 ꢁC; 1H
NMR (DMSO-d6) d 7.07–7.13 (m, 3H), 7.26–7.31
(m, 2H), 8.60 (s, 2H), 8.75 (br s, 2H), 10.40 (br s,
1H); 13C NMR (DMSO-d6) d 121.67, 123.81, 125.47,
129.89, 131.46, 135.07, 137.33, 143.01; HRMS (ESI)
calcd for C12H10N4NaO6S (M+Na)+ 361.0219, mea-
sured (M+Na)+ 361.0231. Anal. (C12H10N4O6S) C,
H, N.
4.4.18. 4-Dipropylamino-3,5-dinitrobenzoic acid (28a). A
suspension of 4-chloro-3,5-dinitrobenzoic acid 27
(5.0 g, 20.0 mmol) and dipropylamine (30 mL) in
150 mL methanol was refluxed for 3 h until the solution
become clear. This solution was evaporated to dryness,
the residue was dissolved in EtOAc, washed with 5%
HCl, then washed with water. The organic layer was
dried over anhydrous Na2SO4 and the solvent was evap-
orated to obtain a yellow solid, which was purified by
silica column chromatography to obtain 6.1 g (96%) of
28a. Rf1 0.33 (MeOH/dichloromethane 1:8); mp =
133 ꢁC; H NMR (CDCl3) d 0.90 (t, 6H, J = 7.32 Hz),
1.59–1.74 (m, 4H), 2.80–3.35 (m, 4H), 8.51 (s, 2H); 13C
NMR (CDCl3) d 11.16, 20.74, 54.06, 119.35, 131.35,
142.43, 144.70, 167.56; MS (HR) C13H18N3O6 (M+H)+
calcd: 312.1195, found: 312.1187.
4.4.15. N1-(4-Fluoro)phenyl-3,5-dinitro-N4-(cyclopropyl-
methyl)-N4-propylsulfanilamide (24). The reaction of sul-
fonylchloride 9g (prepared from 8g, 475 mg, 1.19 mmol)
and 4-fluoroaniline (460 lL, 4.8 mmol) in pyridine
(18 mL) at 40 ꢁC afforded 24 as yellow crystals: yield
4.4.19. 4-Dibutylamino-3,5-dinitrobenzoic acid (28b).
Experimental conditions for the synthesis of 28b were
identical to those described for compound 28a. Reaction
between 27 (4.0 g, 16 mmol) and 40 mL of dibutylamine
(30.7 g, 240 mmol) yielded 4.5 g (82%) of compound 28b
as a yellow solid. Rf 0.50 (MeOH/dichloromethane 1:8);
1
268 mg, 50%; mp = 80 ꢁC; H NMR (CDCl3) d 0.14–
0.20 (m, 2H), 0.56–0.64 (m, 2H), 0.89 (t, 3H, J = 7.3
Hz), 0.99–1.05 (m, 1H), 1.58–1.67 (m, 2H), 2.92 (d,
2H, J = 7.0 Hz), 3.07–3.13 (m, 2H), 6.65 (s, 1H), 7.02–
7.16 (m, 4H), 8.13 (s, 2H); 13C NMR (CDCl3) d 4.39,
9.63, 11.52, 21.20, 54.60, 57.84, 117.03 (d,
JCF = 22.8 Hz), 125.51 (d, JCF = 8.5 Hz), 129.09,
129.47, 131.51 (d, JCF = 3.1 Hz), 142.33, 145.11, 161.58
(d, JCF = 245.6 Hz); HRMS (ESI) calcd for
C19H21FN4NaO6S (M+Na)+ 475.1064, measured
(M+Na)+ 475.1066. Anal. (C19H21FN4O6S) C, H, N.
mp 139 ꢁC; 1H NMR (CDCl3)
d 0.85 (t, 6H,
J = 7.32 Hz), 1.21–1.36 (m, 4H), 1.48–1.62 (m, 4H),
3.01 (t, 4H, J = 7.5 Hz), 8.45 (s, 2H); 13C NMR
(DMSO-d6) d 13.58, 19.80, 29.37, 52.00, 119.56,
131.30, 142.36, 144.79, 168.19; MS (HR) C15H22N3O6
(M+H)+ calcd: 340.1508, found: 340.1510.
4.4.16. N1-(4-Fluoro)phenyl-3,5-dinitro-N4,N4-di-(cyclo-
propylmethyl)sulfanilamide (25). The reaction of sulfo-
nylchloride 9h (prepared from 8h, 500 mg, 1.22 mmol)
and 4-fluoroaniline (470 lL, 4.9 mmol) in pyridine
(18 mL) at 40 ꢁC afforded 25 as yellow crystals: yield
4.5. General procedure for the synthesis of amide
derivatives (29–39)
A mixture of compound 28a or 28b (1 equiv), substitut-
ed aniline (1.5 equiv), DCC (1.3 equiv), and DMAP
(0.3 equiv) in 5 mL of dichloromethane were reacted at
room temperature for 3 h. The reaction was quenched
by adding 1 mL water, then the organic layer was
extracted with EtOAc, washed with water and dried
over anhydrous Na2SO4. The solid material obtained
after evaporation of the organic layer was purified by sil-
ica gel column chromatography to obtain target com-
pounds 29–39. Recrystallization of the target
compounds was carried out using a solvent mixture of
dichloromethane and hexane.
1
250 mg, 44%; mp = 114 ꢁC; H NMR (CDCl3) d 0.13–
0.19 (m, 4H), 0.53–0.61 (m, 4H), 0.95–1.06 (m, 2H),
3.03 (d, 4H, J = 6.6 Hz), 6.60 (s, 1H), 7.02–7.16 (m,
4H); 8.14 (s, 2H); 13C NMR (CDCl3) d 4.60, 9.82,
58.14, 117.11 (d, JCF = 22.8 Hz), 125.78 (d, JCF
=
8.4 Hz), 128.81, 130.14, 131.33 (d, JCF = 2.9 Hz),
142.25, 145.73, 161.71 (d, JCF = 246.31 Hz); HRMS
(ESI) calcd for C20H21FN4NaO6S (M+Na)+ 487.1064,
measured (M+Na)+ 487.1042. Anal. (C20H21FN4O6S)
C, H, N.
4.4.17. N1-(4-Fluoro)phenyl-3,5-dinitro-N4-(cyclopropyl-
methyl)-N4-benzylsulfanilamide (26). The reaction of
sulfonylchloride 9i (prepared from 8i, 161 mg,
0.37 mmol) and 4-fluoroaniline (143 lL, 1.50 mmol)
in pyridine (6 mL) at 40 ꢁC afforded 26 as yellow
crystals: yield 59 mg, 32%; mp = 160 ꢁC; 1H NMR
(CDCl3) d 0.02–0.08 (m, 2H), 0.53–0.59 (m, 2H),
0.99–1.10 (m, 1H), 2.89 (d, J = 6.6 Hz, 2H), 4.36
(s, 2H), 6.66 (s, 1H), 7.01–7.10 (m, 4H), 7.31–7.33
(m, 5H), 8.11 (s, 2H); 13C NMR (CDCl3) d 4.08,
9.21, 56.12, 58.64, 116.73 (d, JCF = 22.5 Hz), 125.65
(d, JCF = 9.0 Hz), 128.21, 128.35, 128.65, 128.71,
130.20, 130.78 (d, JCF = 3.0 Hz), 134.93, 141.62,
145.20, 161.4 (d, JCF = 246.7 Hz); HRMS (ESI)
calcd for C23H21FN4NaO6S (M+Na)+ 523.1064,
measured (M+Na)+ 523.1072. Anal. (C23H21FN4O6S)
C, H, N.
4.5.1. N-Phenyl-4-dipropylamino-3,5-dinitrobenzamide (29).
Reaction between 28a (0.50 g, 1.6 mmol) and aniline
(0.26 g, 2.8 mmol) yielded 0.46 g (74%) of 29 as orange
crystals; Rf 0.53 (EtOAc/hexane 1:4); mp 178 ꢁC; 1H
NMR (CDCl3) d 0.85 (t, 6H, J = 7.32 Hz), 1.50–1.66
(m, 4H), 2.94 (t, 4H, J = 7.5 Hz), 7.08–7.16 (m, 1H),
7.22–7.33 (m, 2H), 7.48–7.58 (m, 2H), 8.24 (s, 1H),
8.33 (s, 2H); 13C NMR (CDCl3) d 11.08, 20.78, 54.10,
120.76, 125.30, 125.57, 128.45, 129.10, 137.07, 140.99,
145.10, 161.66; MS (HR) C19H22N4O5Na (M+Na)+
calcd: 409.1488, found: 409.1520; Anal. (C19H22N4O5)
C, H, N.
4.5.2. N-(4-Fluorophenyl)-4-dipropylamino-3,5-dinitro-
benzamide (30). Reaction between 28a (0.50 g,
1.6 mmol) and 4-fluoroaniline (0.26 g, 2.3 mmol) yielded
0.43 g (66%) of 30 as orange crystals. Rf 0.42 (EtOAc/

5708
T. G. George et al. / Bioorg. Med. Chem. 14 (2006) 5699–5710
hexane 1:4); mp 201 ꢁC; 1H NMR (DMSO-d6) d 0.80 (t,
6H, J = 7.32 Hz), 1.46–1.55 (m, 4H), 2.94 (t, 4H, J = 7.5
Hz), 7.10–7.22 (m, 2H), 7.65–7.75 (m, 2H), 8.60 (s, 2H),
10.48 (s, 1H); 13C NMR (acetone-d6) d 11.41, 20.69,
53.89, 115.26 (d, JCF = 22.5 Hz), 122.11 (d,
JCF = 7.64 Hz), 127.04, 128.24, 134.96 (d, JCF = 2.4 Hz),
140.06, 145.82, 161.07, 161.26 (d, JCF = 241.8 Hz); MS
(HR) C19H21FN4O5Na (M+Na)+ calcd: 427.1394,
found: 427.1418; Anal. (C19H21FN4O5) C, H, N.
(d, JCF = 25.9 Hz), 112.11 (d, JCF = 21.6 Hz), 115.71 (d,
JCF = 2.9 Hz), 124.87, 128.32, 130.29 (d, JCF = 9.1 Hz),
138.57 (d, JCF = 11.04 Hz), 141.09, 145.05, 161.41,
164.83 (d, JCF = 245.7 Hz); MS (HR) C21H25FN4O5Na
(M+Na)+ calcd: 455.1707, found: 455.1692; Anal.
(C21H25FN4O5) C, H, N.
4.5.7. N-(3-Chlorophenyl)-4-dibutylamino-3,5-dinitroben-
zamide (35). Reaction between 28b (0.50 g, 1.5 mmol)
and 3-chloroaniline (0.37 g, 3.0 mmol) yielded 0.36 g
(53%) of 35 as yellow crystals; Rf0.47 (EtOAc/hexane
1:4); mp 145 ꢁC; 1H NMR (CDCl3) d 0.86 (t, 6H,
J = 7.32 Hz), 1.19–1.32 (m, 4H), 1.49–1.61 (m, 4H),
3.00 (t, 4H, J = 7.5 Hz), 6.95–6.98 (m, 1H), 7.16–7.22
(m, 1H), 7.32–7.42 (m, 2H), 7.87 (s, 1H), 8.32 (s, 2H);
13C NMR (CDCl3) d 13.71, 19.92, 29.52, 52.15,
118.54, 120.69, 124.83, 125.36, 128.48, 130.17,134.87,
4.5.3. N-(3-Fluorophenyl)-4-dipropylamino-3,5-dinitroben-
zamide (31). Reaction between 28a (0.50 g, 1.6 mmol)
and 3-fluoroaniline (0.26 g, 2.3 mmol) yielded 0.42 g
(65%) of 31 as orange crystals; Rf 0.60 (EtOAc/hexane
1:4); mp 160 ꢁC; 1H NMR (CDCl3) d 0.99 (t, 6H,
J = 7.32 Hz), 1.58–1.70 (m, 4H), 3.01 (t, 4H, J = 7 Hz),
6.89–6.95 (m, 1H), 7.26–7.29 (m, 1H), 7.30–7.40 (m,
1H), 7.57–7.62 (m, 1H), 7.81 (s, 1H), 8.34 (s, 2H); 13C
NMR (CDCl3) d 11.06, 20.69, 54.04, 108.24 (d,
JCF = 26.4 Hz), 112.15 (d, JCF = 21.1 Hz), 115.78 (d,
JCF = 2.9 Hz), 124.87, 128.41, 130.27 (d, JCF = 9.1 Hz),
138.40 (d, JCF = 11.04 Hz), 141.13, 144.96, 161.50,
164.80 (d,JCF = 246.16 Hz); MS (HR) C19H21FN4O5Na
(M+Na)+ calcd: 427.1394, found: 427.1385; Anal.
(C19H21FN4O5) C, H, N.
138.20,
141.22,
145.08,
161.52;
MS
(HR)
C21H25ClN4O5Na (M+Na)+ calcd: 470.1411, found:
470.1408; Anal. (C21H25ClN4O5) C, H, N.
4.5.8. N-(3-Methoxyphenyl)-4-dibutylamino-3,5-dinitro-
benzamide (36). Reaction between 28b (0.50 g,
1.5 mmol) and m-anisidine (0.36 g, 3.0 mmol) yielded
0.37 g (55%) of 36 as orange crystals; Rf 0.45 (EtOAc/
1
hexane 1:4); mp 125 ꢁC; H NMR (CDCl3) d 0.85 (t,
4.5.4. N-Phenyl-4-dibutylamino-3,5-dinitrobenzamide (32).
Reaction of 28b (0.60 g, 1.8 mmol) and aniline (0.29 g,
3.0 mmol) yielded 0.60 g (80%) of 32 as orange crystals;
Rf 0.50 (EtOAc/hexane 1:4); mp 152 ꢁC; 1H NMR
(CDCl3) d 0.86 (t, 6H, J = 7.32 Hz), 1.19–1.32 (m,
4H), 1.53–1.64 (m, 4H), 2.98 (t, 4H, J = 7.5 Hz), 7.10–
7.15 (m, 1H), 7.24–7.32 (m, 2H), 7.52–7.55 (m, 2H),
8.17 (s, 1H), 8.33 (s, 2H); 13C NMR (CDCl3) d 13.70,
19.91, 29.54, 52.11, 120.75, 125.30, 125.53, 128.42,
129.12, 137.05, 140.97, 145.17, 161.66; MS (HR)
C21H26N4O5Na (M+Na)+ calcd: 437.1801, found:
437.1830; Anal. (C21H26N4O5) C, H, N.
6H, J = 7.32 Hz), 1.19–1.32 (m, 4H), 1.49–1.62 (m,
4H), 3.02 (t, 4H, J = 7.5 Hz), 3.79 (s, 3H), 6.70 (dd,
1H, J = 7.80 Hz, J = 2.47 Hz), 7.04–7.10 (m, 1H),
7.20–7.27 (m, 1H), 7.28–7.31 (m, 1H), 7.90 (s, 1H),
8.32 (s, 2H); 13C NMR (CDCl3) d 13.72, 19.93, 29.55,
52.14, 55.34, 106.44, 110.98, 112.69, 125.50, 128.36,
129.87, 138.25, 141.02, 145.18, 160.23, 161.49; MS
(HR) C22H28N4O6Na (M+Na)+ calcd: 467.1907, found:
467.1905; Anal. (C22H28N4O6) C, H, N.
4.5.9. N-(m-Tolyl)-4-dibutylamino-3,5-dinitrobenzamide
(37). Reaction between 28b (0.60 g, 1.8 mmol) and m-to-
luidine (0.37 g, 3.5 mmol) yielded 0.40 g (52%) of 37 as
yellow crystals; Rf 0.47 (EtOAc/hexane 1:4); mp
4.5.5. N-(4-Fluorophenyl)-4-dibutylamino-3,5-dinitroben-
zamide (33). Reaction between 28b (0.60 g, 1.8 mmol)
and 4-fluoroaniline (0.30 g, 2.7 mmol) yielded 0.52 g
(67%) of 33 as red crystals; Rf 0.30 (EtOAc/hexane
1:4); mp 164 ꢁC; 1H NMR (CDCl3) d 0.85 (t, 6H,
J = 7.32 Hz), 1.26–1.36 (m, 4H), 1.51–1.64 (m, 4H),
3.03 (t, 4H, J = 7.5 Hz), 7.00–7.10 (m, 2H), 7.50–7.60
(m, 2H), 7.97 (s, 1H), 8.26 (s, 2H); 13C NMR (CDCl3)
d 13.60, 19.83, 29.45, 52.06, 116.02 (d, JCF = 22.5 Hz),
122.58 (d, JCF = 7.67 Hz), 125.09, 128.24, 132.91 (d,
JCF = 2.9 Hz), 140.98, 145.12, 161.45, 161.81(d,
1
125 ꢁC; H NMR (CDCl3) d 0.88 (t, 6H, J = 7.32 Hz),
1.20–1.35 (m, 4H), 1.48–1.62 (m, 4H), 2.25 (s, 3H),
3.01 (t, 4H, J = 7.5 Hz), 6.93–6.99 (m, 1H), 7.16–7.24
(m, 1H), 7.32–7.35 (m, 1H), 7.43 (s, 1H), 8.30 (s, 1H),
8.32 (s, 2H); 13C NMR (CDCl3) d 13.72, 19.93, 21.44,
29.55, 52.12, 117.72, 121.30, 125.66, 126.10, 128.34,
128.96, 136.99, 139.17, 140.95, 145.20, 161.49; MS
(HR) C22H28N4O5Na (M+Na)+ calcd: 451.1957, found:
451.1953; Anal. (C22H28N4O5) C, H, N.
JCF = 244.72 Hz);
MS
(HR)
C21H25FN4O5Na
4.5.10. N-(3,4-Dichlorophenyl)-4-dibutylamino-3,5-di-nitro-
benzamide (38). Reaction between 28b (0.60 g,
1.8 mmol) and 3,4-dichloroaniline (0.47 g, 3.0 mmol)
yielded 0.56 g (64%) of 37 as yellow crystals; Rf0.55
(M+Na)+ calcd: 455.1707, found: 455.1693; Anal.
(C21H25FN4O5) C, H, N.
1
4.5.6. N-(3-Fluorophenyl)-4-dibutylamino-3,5-dinitroben-
zamide (34). Reaction between 28b (0.60 g, 1.8 mmol) and
3-fluoroaniline (0.30 g, 2.7 mmol) yielded 0.53 g (68%) of
compound 34 as orange crystals; Rf 0.33 (EtOAc/hexane
1:4); mp 154 ꢁC; 1H NMR (CDCl3) d 0.83 (t, 6H,
J = 7.32 Hz), 1.20–1.32 (m, 4H), 1.52–1.64 (m, 4H),
3.24 (t, 4H, J = 7.5 Hz), 6.80–6.90 (m, 1H), 7.20–7.33
(m, 2H), 7.49–7.57 (m, 1H), 7.97 (s, 1H), 8.32 (s, 2H);
13C NMR (CDCl3) d 13.60, 19.82, 29.43, 52.07, 108.19
(EtOAc/hexane 1:4); mp 150 ꢁC; H NMR (CDCl3) d
0.86 (t, 6H, J= 7.32 Hz), 1.19–1.32 (m, 4H), 1.54–1.65
(m, 4H), 2.99 (t, 4H, J = 7.5 Hz), 7.35–7.40 (m, 1H),
7.42–7.47 (m, 1H), 7.78–7.80 (m, 1H), 8.23 (s, 1H),
8.34 (s, 2H); 13C NMR (acetone-d6) d 13.07, 19.62,
29.62, 51.97, 119.99, 121.68, 126.49, 126.52, 128.47,
130.54, 131.77, 138.74, 140.31, 145.92, 161.68; MS
(HR) C21H24Cl2N4O5Na (M+Na)+ calcd: 505.1021,
found: 505.1006; Anal. (C21H24Cl2N4O5) C, H, N.

T. G. George et al. / Bioorg. Med. Chem. 14 (2006) 5699–5710
5709
4.5.11. N-(3,5-Dichlorophenyl)-4-dibutylamino-3,5-dinit-
robenzamide (39). Reaction between 28b (0.50 g,
1.5 mmol) and 3,5-dichloroaniline (0.48 g, 3.0 mmol)
yielded 0.41 g (57%) of 37 as red crystals; Rf 0.45
tion mixtures (with BSA) for each LC/MS/MS run. The
linear range for each compound covered 2–3 orders of
magnitude of concentration with lower limits of quanti-
tation between 3 and 10 nM. Triplicate experiments
were completed twice for a total of six replicates per
compound.
1
(EtOAc/hexane 1:4); mp 170 ꢁC; H NMR (CDCl3) d
0.91 (t, 6H, J = 7.32 Hz), 1.23–1.38 (m, 4H), 1.48–1.63
(m, 4H), 3.05 (t, 4H, J = 7.5 Hz), 7.20–7.22 (m, 1H),
7.62 (s, 1H), 7.63 (s, 1H), 7.80 (s, 1H), 8.35 (s, 2H);
13C NMR (CDCl3) d 13.71, 19.92, 29.51, 52.18,
118.67, 124.28, 125.25, 128.46, 135.53, 138.88, 141.40,
145.06, 161.39; MS (HR) C21H24Cl2N4O5Na (M+Na)+
Acknowledgments
This work was funded by NIH Grant AI061021 (to
K.A.W.). D.A.D. was supported by a Procter and Gam-
ble graduate fellowship administered through the
National Organization of Black Chemists and Chemical
Engineers and a United Negro College Fund/Merck Co.
Science Education Initiatives Graduate Fellowship. We
would also like to thank John Swayze and Rich Green
of Dow Agrosciences for the donation of 3,5-dinitro-4-
chlorobenzenesulfonate, which was used to prepare
some of the compounds.
calcd:
(C21H24Cl2N4O5) C, H, N.
505.1021,
found:
505.1028;
Anal.
4.6. Cell based assays
Assays used to measure the activity of the target com-
pounds against L. donovani axenic amastigotes,13,22 T.
b. rhodesiense bloodstream forms23,24 and intracellular
T. cruzi23,24 have been described previously. Methods
for measuring the toxicity of the compounds against
Vero cells25 and L6 rat myoblasts23,24 have also been
outlined earlier.
Supplementary data
4.7. Tubulin assays
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2006.04.017
Tubulin from Leishmania tarentolae was purified
according to the general method of Werbovetz et al.22
with some modifications (A. Yakovich et al., submitted).
The purification of mammalian tubulin and methods to
assay the effects of target compounds against the assem-
bly of leishmanial and mammalian tubulin have been de-
scribed previously.14
References and notes
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4.8. In vitro metabolism
Triplicate rat liver S9 (BD Biosciences, San Jose, CA)
reaction mixtures (0.1 M phosphate, 1 mg/mL S9,
1 mM NADPH, pH 7.4) were incubated at 37 ꢁC for
5 min before adding the test compound at a final con-
centration of 300 nM (final reaction volume = 330 lL).
Triplicate control reactions were produced by replacing
S9 with bovine serum albumin (BSA). Reactions were
mixed by pipetting up and down for 10 s. Aliquots
(100 lL) were withdrawn immediately at 10 min and
added directly to 1 mL acetonitrile at 4 ꢁC containing
10 nM N1-(3,5-dichloro)phenyl-3,5-dinitro-N4,N4-di-n-
propylsulfanilamide14 internal standard (IS). Samples
were then centrifuged at 16,000g for 10 min and the
supernatant was recovered and dried in a vacuum con-
centrator with vapor trap (Thermo Electron, Waltham,
MA). Samples were resuspended in 150 lL of 50% ace-
tonitrile, centrifuged as above, and the supernatant was
loaded for analysis. An LC/MS/MS method was created
for each compound using an Agilent 1100 LC system
and a ThermoFinnigan TSQ Quantum Discovery Max
Mass Spectrometer. Samples were separated on a re-
versed-phase gradient of water and acetonitrile, each
containing 0.1% acetic acid using an Agilent Zorbax
extended C18 column (2.1 · 50 mm, 3.5 lm) and ana-
lyzed via electrospray ionization in negative ion mode
with single reaction monitoring of the parent com-
pound. Standard curves were generated in control reac-
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