Synthesis and evaluation of oryzalin analogs against Toxoplasma gondii

Article abstract of DOI:10.1016/j.bmcl.2010.07.003

The synthesis and evaluation of 20 dinitroanilines and related compounds against the obligate intracellular parasite Toxoplasma gondii is reported. Using in vitro cultures of parasites in human fibroblasts, we determined that most of these compounds selectively disrupted Toxoplasma microtubules, and several displayed sub-micromolar potency against the parasite. The most potent compound was N¹,N¹-dipropyl-2,6-dinitro-4-(trifluoromethyl)-1,3- benzenediamine (18b), which displayed an IC₅₀ value of 36 nM against intracellular T. gondii. Based on these data and another recent report [Ma, C.; Tran, J.; Gu, F.; Ochoa, R.; Li, C.; Sept, D.; Werbovetz, K.; Morrissette, N. Antimicrob. Agents Chemother. 2010, 54, 1453], an antimitotic structure-activity relationship for dinitroanilines versus Toxoplasma is presented.

Full text of DOI:10.1016/j.bmcl.2010.07.003

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5179–5183
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of oryzalin analogs against Toxoplasma gondii
Molla M. Endeshaw a, Catherine Li b, Jessica de Leon b, Ni Yao b, Kirk Latibeaudiere a, Kokku Premalatha a,
Naomi Morrissette b, Karl A. Werbovetz a,
*
a Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210-1291, USA
b Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, 92697, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and evaluation of 20 dinitroanilines and related compounds against the obligate intracel-
lular parasite Toxoplasma gondii is reported. Using in vitro cultures of parasites in human fibroblasts, we
determined that most of these compounds selectively disrupted Toxoplasma microtubules, and several
displayed sub-micromolar potency against the parasite. The most potent compound was N1,N1-dipro-
pyl-2,6-dinitro-4-(trifluoromethyl)-1,3-benzenediamine (18b), which displayed an IC50 value of 36 nM
against intracellular T. gondii. Based on these data and another recent report [Ma, C.; Tran, J.; Gu, F.;
Ochoa, R.; Li, C.; Sept, D.; Werbovetz, K.; Morrissette, N. Antimicrob. Agents Chemother. 2010, 54, 1453],
an antimitotic structure–activity relationship for dinitroanilines versus Toxoplasma is presented.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 14 April 2010
Revised 30 June 2010
Accepted 2 July 2010
Available online 8 July 2010
Keywords:
Toxoplasma
Tubulin
Oryzalin
Trifluralin
Dinitroaniline
Infection by the apicomplexan parasite Toxoplasma gondii can
result in miscarriage, birth defects in infants, vision disturbances
in immunocompetent hosts, and toxoplasmic encephalitis in the
immunocompromised.1 Acute toxoplasmosis in otherwise healthy
individuals is typically treated with the antifolate combination of
pyrimethamine and sulfadiazine together with folinic acid.1 Pyri-
methamine has potential side effects, including bone marrow sup-
pression2 and teratogenicity.3,4 Resistance to pyrimethamine in
malaria chemotherapy can occur through mutations in the dihydro-
folate reductase target protein,5 although no evidence for clinical
resistance to pyrimethamine in Toxoplasma exists to date.6 Sulfadi-
azine use is also complicated by the occurrence of kidney stones,7
allergic reactions,8 and the development of resistance.6 Other alter-
natives to pyrimethamine–sulfadiazine treatment of toxoplasmosis
include clindamycin, atovaquone, and spiramycin,1 but these drugs
each possess their own limitations.9–11 An ideal drug against human
toxoplasmosis would kill the different stages ofT. gondii, be well dis-
tributed in the main sites of infection, including the brain and eyes,
and lack the potential for teratogenicity.
Plasmodium, and Toxoplasma. We have previously characterized the
sensitivity of Toxoplasma to dinitroanilines and have found that
IC50 values for commercially available dinitroanilines range from
45 nM to 6.7 l
M.15–17 Toxoplasma parasites only replicate inside
of host cells, and extracellular parasites do not have dynamic
microtubules. Intracellular Toxoplasma parasites are sensitive to
disruption by dinitroanilines: dinitroaniline-treated parasites lack
all microtubules and cannot carry out microtubule-dependent
functions including mitosis and cytokinesis.15,18,19 Following lysis
of the original host cell, round, non-polar dinitroaniline-treated
parasites are unable to invade new host cells and rapidly die. Thus,
dinitroanilines could serve as excellent leads for the discovery of
new drugs against toxoplasmosis since they disrupt protozoan par-
asite microtubules at nanomolar concentrations while showing lit-
tle or no effect on host cell microtubules.16,20–23 Computational
methods have been used to identify a binding site for the dinitro-
anilines on parasite tubulin.16,24 These studies predict that protofil-
ament contacts in the microtubule lattice are disrupted when
dinitroanilines selectively bind to protozoan
a-tubulin beneath
Tubulin, which assembles into microtubules, is an essential pro-
tein for formation of the mitotic spindle. It continues to be a prime
target for established and investigational anticancer agents12,13
and is also believed to be the molecular target of anthelmintic
benzimidazoles.14 Dinitroanilines are tubulin-binding herbicides
which display microtubule selective toxicity for many different
classes of protozoan parasites, including Leishmania, Trypanosoma,
the H1-S2 loop. Analysis of vertebrate -tubulin through computa-
a
tional methods indicates that oryzalin has non-specific, low affin-
ity interactions with this protein and no consensus binding site,
consistent with in vivo and in vitro observations that dinitroani-
lines do not bind to vertebrate tubulin or disrupt vertebrate micro-
tubules.20,25–28
Previous work in our laboratories has shown that structural
modification of the commercial dinitroaniline oryzalin leads to
increased potency against kinetoplastid parasites.23,29,30 However,
* Corresponding author. Tel.: +1 614 292 5499; fax: +1 614 292 2435.
E-mail address: werbovetz.1@osu.edu (K.A. Werbovetz).
a
detailed structure–activity study of dinitroanilines against
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.003

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M. M. Endeshaw et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5179–5183
Toxoplasma has not been reported. We were therefore motivated to
synthesize analogs of oryzalin (6i) and other dinitroaniline herbi-
cides such as trifluralin (14a) and dinitramine (18a) for testing
against T. gondii. In the present manuscript, we describe the syn-
thesis and biological activity of such derivatives.
To synthesize several of the desired target compounds, unsym-
metrical secondary amines were needed. The required secondary
amine salts were synthesized using the method reported previ-
ously.29,31 Hence, the standard coupling of the acid chlorides
1a–d with amines gave the respective amides 2a–e, followed by
reduction with borane–THF solution, treatment with methanol,
protection with Boc, and deprotection using HCl in dioxane to af-
ford the secondary amine salts 3a–e in good yields (see Scheme 1).
Dinitroaniline sulfonamide target compounds were prepared as
shown in Scheme 2. The desired amines reacted with the
potassium salt of 4-chloro-3,5-dinitrobenzenesulfonate 4 in the
presence of triethylamine to give N4,N4-disubstituted-3,5-dinitro-
benzenesulfonates 5a–e, 5f–h,31 and 5i–j30,31 in good yields. These
sulfonates contained traces of the secondary amine salts as deter-
mined from their 1H NMR spectra, but these salts did not interfere
with further reactions. Sulfonyl chlorides were synthesized from
benzenesulfonates 5a–j using PCl5 in dichloromethane. After brief
work up, the resulting sulfonyl chlorides were treated with a meth-
anolic solution of NH3 or the desired amines in dry THF to afford
the target compounds 6a–h, 6i–j,29 6k–l, and 6m–n30 in good to
excellent yields. Mono-N4-alkylated and free N4-amino sulfona-
mides were isolated as bi-products, especially for bulky N4,N4-
disubstituted analogs such as 6a–g.
O
O
a
b
HCl
R1 Cl
R1
N
H
R2
R1
N
R2
H
3a-e
1a-d
2a-e
1a: R1 = Ph
2a, 3a: R1 = Ph,R2 = CH2CH2OH
1
1 2
1b:
1c:
2b 3b
, : R = Ph, R = cPr
R = 4-Tolyl
1
1
2
2c 3c
: R = R = 4-Tolyl
R = cHexane
,
The synthesis of 12, where the nitro moieties present in 6i were
replaced with nitrile groups, was accomplished according to
Scheme 3. The sulfonyl chloride corresponding to sulfonate 732
was prepared in situ from 7 using PCl5/POCl3 in dry chlorobenzene;
treatment of this sulfonyl chloride with methanolic NH3 afforded 8.
An Ullmann reaction33 between sulfonamide 8 and dipropylamine
in the presence of potassium carbonate and catalytic copper gave
1d: R1 = CH2Ph
2d, 3d: R1 = R2 = cHexane
2e, 3e: R1 = R2 = CH2Ph
Scheme 1. Reagents and conditions: (a) RNH2, Et3N, CH2Cl2, 0 °C to rt, overnight;
(b) (i) BH3–THF (3 equiv), reflux, overnight; (ii) MeOH, reflux, 5 h; (iii) Boc2O
(1.5 equiv) in CH2Cl2, rt, overnight; (iv) 4 M HCl in 1,4-dioxane (1.3 equiv), CH2Cl2,
rt, overnight.
Cl
R1
O2N
N
R2
NO2
R1
O2N
N
R2
NO2
O2N
NO2
a
b
O S O
O-K+
4
O S O
O-K+
5a-j
O S O
NHR3
6a-n
6a: R1 = Ph, R2 = CH2Cl, R3 = H
6b: R1 = Ph, R2 = (CH2)2Cl, R3 = H
6c: R1 = R2 = 4-Tolyl, R3 = H
6d: R1 = R2 = cHexane, R3 = H
6e: R1 = R2 = CH2Ph, R3 = H
6f: R1 = R2 = Ph, R3 = H
5a: R1 = Ph, R2 = CH2OH
5b: R1 = Ph, R2 = (CH2)2OH
5c: R1 = R2 = 4-Tolyl
5d: R1 = R2 = cHexane
5e: R1 = R2 = CH2Ph
5f:
R
1 = R2 = Ph
6g: R1 = Ph, R2 = cPr, R3 = H
6h: R1 = R2 = CH(CH3)2, R3 = H
5g: R1 = Ph, R2 = cPr
5h: R1 = R2 = CH(CH3)2
R
1 = R2 = CH2CH3
6i:
R
1 = R2 = CH2CH3, R3 = H
5i:
5j:
R
1 = R2 = (CH2)2CH3
6j: R1 = R2 = (CH2)2CH3, R3 = H
6k: R1 = R2 = CH2CH3, R3 = (CH2)2OH
6l: R1 = R2 = CH2CH3, R3 = 4-phenol
6m:R1 = R2 = CH2CH3, R3 = Ph
6n: R1 = R2 = CH2CH3, NHR3 = N(CH3)2
Scheme 2. Reagents and conditions: (a) secondary amine, reflux, or secondary amine hydrochloride, Et3N, MeOH, reflux, 4 h; (b) (i) PCl5, CH2Cl2, rt, 3 h; (ii) NH3 (7 N in
MeOH), or amine, Et3N, THF, 0 °C to rt, 2 h.
Br
Br
N
MeO2C
CO2Me
MeO2C
CO2Me
MeO2C
CO2Me
a
b
c
O S O
O S O
NH2
O S O
NH2
O-Na+
7
8
9
N
N
N
HOOC
COOH
H2NOC
CONH2
NC
CN
d
e
O S O
NH2
O S O
NH2
O S O
NH2
10
11
12
Scheme 3. Reagents and conditions: (a) (i) PCl5/POCl3, chlorobenzene, reflux, 24; (ii) NH3 (0.5 M in 1,4-dioxane), 0 °C, 1 h, rt, 1 h; (b) dipropylamine, K2CO3, Cu, dioxane,
reflux, 5 h; (c) 3 N NaOH, MeOH, reflux, 2 days; (d) (i) CCl3COCCl3, PPh3, THF, 0 °C, 1 h; (ii) NH3/dioxane, rt, 1 h; (e) PdCl2, CH3CN/H2O, 24 h.

M. M. Endeshaw et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5179–5183
5181
9. Hydrolysis of this ester using aqueous NaOH in methanol affor-
ded 10. Treatment of dicarboxylate 10 with hexachloroacetone and
PPh3 in THF followed by the addition of NH3 in 1,4-dioxane pro-
vided bis-amide 11. Dehydration of 11 using PdCl2 in aqueous ace-
tonitrile34 afforded the target compound 12 in excellent and
reproducible yield.
Dinitrobenzotrifluoride 16 was treated with 2.2 equiv of each of
the desired secondary amines and heated in a sealed flask at
100 °C.36,37 After a brief workup, the crude products 17a–c were
dissolved in 3 equiv of ammonia in dioxane and heated once again
in a sealed flask at 100 °C, which gave the target compounds 18a–c
in 60–80% yield for two steps. The use of excess amine and sealed
flask conditions were critical for the success of the latter two
reactions.
Structures for the proposed target compounds were confirmed
with the aid of 1H and 13C NMR spectroscopy and mass spectrom-
etry analysis, while the purity of the compounds was verified by
elemental analysis. Synthetic procedures and spectral data for rep-
resentative target compounds 6h, 12, and 18b are provided in Sup-
plementary data.
Trifluralin (14a) and its regioisomer 14b35 were prepared
according to Scheme 4. Heating 13a and 13b to reflux in dipropyl-
amine gave target compounds 14a and 14b, respectively, in excel-
lent yield.
Dinitramine (18a) and its analogs were synthesized as shown in
Scheme 5. 2,4-Dichloro-3,5-dinitrobenzotrifluoride (16) was
prepared by nitration of 2,4-dichlorobenzotrifluoride (15) using
fuming nitric acid in the presence of fuming sulfuric acid.36
The growth inhibitory activity of compounds of interest against
T. gondii in vitro was measured by a plaque assay,17 with the re-
sults shown in Table 1. The IC50 values for 6i, 14a, and 18a are con-
sistent with those obtained previously in this assay.17 Toxoplasma
microtubules were examined by an in vitro immunofluorescence
assay (Fig. 1). Figure 1A and B shows the effect of a selective anti-
mitotic compound (18b), while Figure 1D and E illustrates the ef-
fect of a non-selective agent (6e). Experimental methods for both
the plaque assay and the immunofluorescence assay are available
in Supplementary data.
Cl
N
O2N
R1
O2N
R1
a
R2
R2
13a,b
14a,b
13a, 14a: R1 = NO2, R2 = CF3
13b, 14b: R1 = CF3, R2 = NO2
Among the compounds lacking a meta-amino group (6a–n, 12,
14a, and 14b), the dinitroaniline oryzalin (6i) was the most effec-
Scheme 4. Reagent and condition: (a) dipropylamine, reflux, overnight.
R
R
R
R
Cl
Cl
N
N
17a, 18a:R = CH2CH3
17b, 18b:
O2N
H2N
NO2
O2N
Cl
NO2
O2N
Cl
NO2
R = (CH2)2CH3
17c, 18c:R = (CH2)3CH3
a
b
c
Cl
CF3
15
CF3
16
CF3
17a-c
CF3
18a-c
Scheme 5. Reagents and conditions: (a) fuming sulfuric acid containing 30–33% SO3/fuming 90% nitric acid; (b) secondary amine, absolute ethanol, 100 °C, 72 h; (c) NH3 in
1,4-dioxane, 100 °C, 40 h.
Table 1
Activity of target compounds against Toxoplasma gondii in vitro
R1
X
N
Y
R2
X
Z
Compound
R1
R2
X
Y
Z
IC50 (lM) versus
Toxoplasma gondiia
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
12
14a
14b
18a
18b
18c
Ph
Ph
4-Tolyl
cHexyl
CH2Ph
Ph
CH2Cl
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
CN
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH(CH2)2OH
SO2NH(4-phenol)
SO2NHPh
SO2N(CH3)2
SO2NH2
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.85 0.03
2.6 0.1
(CH2)2Cl
4-Tolyl
cHexyl
CH2Ph
Ph
cPr
CH(CH3)2
CH2CH3
(CH2)2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH3
Non-specific
Non-specific
Non-specific
Non-specific
2.1 0.1
0.35 0.02
0.25 0.01
1.1 0.2
Ph
CH(CH3)2
CH2CH3
(CH2)2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH3
26
2
2.6 0.3
6.7 0.7
3.4 0.2
4.8 0.2
0.65 0.06
7.8 1.0
0.070 0.004
0.036 0.002
0.51 0.02
NO2
NO2, CF3
NO2
NO2
NO2
NO2
CF3
CF3
CF3
H
NH2
NH2
NH2
CH2CH3
(CH2)2CH3
CH2CH3
(CH2)2CH3
a
Mean standard deviation of three independent measurements, experimental methods are given in Supplementary data.

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M. M. Endeshaw et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5179–5183
Figure 1. (A and B) Compound 18b selectively disrupts microtubules in intracellular Toxoplasma gondii but not host vertebrate cells. (A1) Immunofluorescence of DMSO
control treated parasite cultures illustrates that vehicle alone does not disrupt either vertebrate host cell (red) or Toxoplasma parasite (green) microtubules. Red: host cell
microtubules retain a filamentous pattern, which can be seen more clearly in the boxed enlargement. Green: parasite subpellicular microtubules are present in a typical linear
array. Blue: DAPI staining of host cell and parasite DNA. (A2) A phase contrast image of the same field; the scale bar represents 5
cultures treated for 28 h with 0.5 M 18b in DMSO show that parasite but not host cells are disrupted. Red: host cell microtubules retain a filamentous pattern, which can be
seen more clearly in the boxed enlargement. Green: parasite subpellicular microtubules are disrupted and free tubulin dimer staining is diffuse rather than organized into
linear arrays. Blue: host cell and parasite DNA. (B2) A matched phase contrast image; the scale bar is 5 m. (C) An MTS viability assay using the CellTiter reagent (Promega)
illustrates that 6c and 6e are toxic to host cells at 30 M, while 0.5
lm. (B1) Immunofluorescence of parasite
l
l
l
lM 18b (ꢀ10-fold greater than the antiparasitic IC50 value for this compound) does not have significant
toxicity. (D and E) Compound 6e inhibits replication of intracellular Toxoplasma gondii but does not disrupt parasite microtubules, indicating that the compound interacts
with target(s) other than tubulin. (D1) Immunofluorescence of a control culture: the rosette of eight daughter parasites indicates that the initial intracellular parasite has
undergone three cycles of replication 28 h after infection. Red: A parasite-specific surface antigen, P30/SAG1, labels the entire parasite surface. Green: parasite subpellicular
microtubules are present in a typical linear array. Blue: host cell and parasite DNA. (D2) A matched phase contrast image; the scale bar is 5 lm. (E1) Immunofluorescence of a
culture treated with 6e showing two single intracellular parasites which have not replicated 28 h post infection. This culture was set up at the same time as the control
culture, and parasite vacuoles should contain 4–16 parasites by 28 h. Note the intact microtubules in the parasites. Fluorochromes are the same as in the control sample. (E2)
A matched phase contrast image; the scale bar is 5 lm. Full experimental methods are given in Supplementary data.
tive against Toxoplasma in this study, possessing an IC50 value of
0.25 M. In other studies examining the efficacy of commercially
available dinitroanilines, 6i was one of the most efficacious com-
pounds to inhibit Toxoplasma growth as assessed by plaque as-
says.17 The other potent analogs of oryzalin are compounds 6h,
toxicity at ꢀ30
to quantify viable host HFF cells after 17 h of treatment with 30
of two of the non-specific compounds (6c and 6e) and with a par-
asite-specific compound (0.5 M 18b). Compounds 6c and 6e
cause host cell vacuolation and dramatically decrease the number
of viable host cells. Although 6c and 6e inhibit parasite replication,
parasite microtubules are not disrupted, suggesting that they have
a distinct target. In contrast, the specific compound 18b does not
compromise host cell viability at a concentration that selectively
disrupts parasite microtubules.
Substitutions on the sulfonamide nitrogen decrease activity
compared to 6i. Compounds with 4-hydroxyphenyl (6l), phenyl
(6m), dimethyl (6n), or 2-hydroxyethyl (6k) substituents at the
sulfonamide nitrogen are 10- to 100-fold less active than 6i. Inter-
estingly, replacement of the nitro groups with nitrile moieties (12)
results in a 19-fold drop in potency compared to 6i. This is in
contrast to the structure–activity relationship observed in kineto-
lM. We also used a tetrazolium dye assay (Fig. 1C)
l
lM
l
6a, and 6j, with IC50 values ranging from 0.35 to 1.1 lM. These re-
sults indicate that alkyl chains of three or four carbons in length at
the aniline nitrogen of the dinitroanilines confer strong activity
against intracellular T. gondii. Compound 6g, which possesses one
four-carbon group and one benzyl group at the aniline nitrogen
atom, is 8.5-fold less active than dipropyl derivative 6i and twofold
less active than dibutyl congener 6j. Interestingly, compounds with
two large substitutions at the aniline nitrogen (6c–f) do not appear
to be tubulin specific inhibitors in Toxoplasma. These compounds
inhibit parasite invasion, replication and growth at 10–25
without shortening the microtubules in the organism (Fig. 1D
and E, treatment with 30 M 6e shown) and display host HFF cell
lM
l

M. M. Endeshaw et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5179–5183
5183
N4-dialkyl groups with 4-carbon
Significant increases in the length
and bulk of the N4-dialkyl chains
decrease activity and specificity
branched chains maintain activity
Replacement of the nitro
groups with nitrile moieties
N
decreases activity
O2N
NO2
An amino substitution
increases activity
O S O
NH2
Replacement of the sulfonamide moiety
with a trifluoromethyl or isopropyl group
decreases activity slightly
Substitution at N1
decreases activity
Figure 2. Activity map for dinitroaniline analogs against Toxoplasma microtubules.
plastid parasites, where exchanging nitrile groups for the nitro
Supplementary data
moieties present in the potent antikinetoplastid compound 6m
results in only a 1.5- to 2-fold loss of potency.32
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.003.
Trifluralin (14a) is 2.6-fold less potent than 6i, indicating that
the sulfonamide group conveys greater potency against Toxo-
plasma than the trifluoromethyl group. A 12-fold decrease in po-
tency compared to 14a is observed for 14b, a compound where
the nitro and trifluoromethyl groups of 14a are interchanged. The
addition of the meta-amino group (compounds 18a–c), however,
causes a dramatic increase in activity compared to 14a. Ma et al.
recently reported that the meta-amino group present in 18a partic-
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ipates in two critical hydrogen bonds with the
a-tubulin target,
significantly increasing the activity of this compound compared
to both 14a and 6i.17 Our present study confirms this result for
18a and also shows that the dipropylamino group found in 18b im-
parts twofold greater potency against Toxoplasma compared to the
N,N-diethyl substitution present in 18a and 14-fold greater po-
tency against this parasite compared to the N,N-di-n-butyl substi-
tution occurring in 18c. The selective antimitotic action of 18b is
illustrated in Figure 1A and B. Figure 1A1 shows well-defined
microtubules present in both host cells and in Toxoplasma in the
absence of compound treatment. In the presence of 0.5 lM 18b,
host cell microtubules remain intact, while the diffuse staining of
parasite tubulin reveals microtubule disruption in the parasites
(Fig. 1B1). Figure 1B also indicates that parasites exposed to
0.5
l
M 18b are unable to replicate.
18. Morrissette, N.; Sibley, L. J. Cell Sci. 2002, 115, 1017.
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The results shown here, together with the data presented in Ma
et al.17 concerning the evaluation of commercial dinitroanilines
against T. gondii, permits us to develop an antimitotic structure–
activity relationship for dinitroanilines versus Toxoplasma. Isopro-
palin, a compound identical in structure to 6i and 14a except that
it contains an isopropyl group para to the alkylamino position,
reinforces the notion that sulfonamide-containing compounds
are slightly superior to congeners with other substitutions at this
position. As with compounds 6h and 6j, inclusion of a branched
chain four carbon alkyl group in ethalfluralin leads to better activ-
ity than with benfluralin, which possesses an n-butyl group at the
anilino nitrogen. The meta-amino derivatives 18a and 18b are
3.6- to 6.9-fold more potent than 6i against Toxoplasma in vitro,
and studies with 18a–c suggest that the optimal chain length of
the substitution on the aniline nitrogen is three carbons. These
structure–activity data are summarized in Figure 2. Future efforts
to optimize the anti-Toxoplasma potency and drug like properties
of the dinitroanilines will focus on meta-amino containing deriva-
tives 18a and 18b as lead compounds.
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This work was funded by NIH Grants AI061021 (to K.A.W.) and
AI067981 (to N.S.M.).