Activities of quinoxaline, nitroquinoxaline, and [1,2,4]triazolo [4,3-a]quinoxaline analogs of mmv007204 against schistosoma mansoni

Article abstract of DOI:10.1128/AAC.01370-20

The reliance on one drug, praziquantel, to treat the parasitic disease schistosomiasis in millions of people a year shows the need to further develop a pipeline of new drugs to treat this disease. Recently, an antimalarial quinoxaline derivative (MMV007204) from the Medicines for Malaria Venture (MMV) Malaria Box demonstrated promise against Schistosoma mansoni. In this study, 47 synthesized compounds containing quinoxaline moieties were first assayed against the larval stage of this parasite, newly transformed schistosomula (NTS); of these, 16 killed over 70% NTS at 10mM. Further testing against NTS and adult S. mansoni yielded three compounds with 50% inhibitory concentrations (IC50s) of#0.31mM against adult S. mansoni and selectivity indices of$8.9. Administration of these compounds as a single oral dose of 400mg/kg of body weight to S. mansoni-infected mice yielded only moderate worm burden reduction (WBR) (9.3% to 46.3%). The discrepancy between these compounds' good in vitro activities and their poor in vivo activities indicates that optimization of their pharmacokinetic properties may yield compounds with greater bioavailabilities and better antischistosomiasis activities in vivo.

Full text of DOI:10.1128/AAC.01370-20

AAC Accepted Manuscript Posted Online 30 November 2020
Antimicrob Agents Chemother doi:10.1128/AAC.01370-20
Copyright © 2020 American Society for Microbiology. All Rights Reserved.
1
2
Activities of Quinoxaline, Nitroquinoxaline and [1,2,4]Triazolo[4,3-a]quinoxaline
Analogs of MMV007204 against Schistosoma mansoni
3
4
Stefan L. Debbert,a# Mikaela J. Hintz,a Christian J. Bell,a Kenya R. Earl,a Grant E.
Forsythe,a Cécile Häberli,b,c Jennifer Keiserb,c#
5
6
7
aDepartment of Chemistry, Lawrence University, Appleton, Wisconsin, USA.
bDepartment of Medical Parasitology and Infection Biology, Swiss Tropical and Public
Health Institute, Basel, Switzerland.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
cUniversity of Basel, Basel, Switzerland.
Running head: Quinoxalines active against S. mansoni
#Address correspondence to Stefan L. Debbert, stefan.debbert@lawrence.edu, or
Jennifer Keiser, jennifer.keiser@swisstph.ch.
ABSTRACT:
The reliance on one drug, praziquantel, to treat the parasitic disease schistosomiasis in
millions of people a year shows the need to further develop a pipeline of new drugs to
treat this disease. Recently, an antimalarial quinoxaline derivative (MMV007204) from
the Medicines for Malaria Venture (MMV) Malaria Box demonstrated promise against
Schistosoma mansoni. In this study, 47 synthesized compounds containing quinoxaline
moieties were first assayed against the larval stage of this parasite, newly transformed
schistosomula (NTS); of these, 16 killed over 70% NTS at 10 µM. Further testing
against NTS and adult S. mansoni yielded three compounds with 50% inhibitory
1

24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
concentrations (IC50s) of ≤ 0.31 µM against adult S. mansoni and selectivity indices of ≥
8.9. Administration of these compounds as a single oral dose of 400 mg/kg of body
weight to S. mansoni-infected mice yielded only moderate worm burden reduction
(WBR) (9.3% – 46.3%). The discrepancy between these compounds’ good in vitro
activities and their poor in vivo activities indicates that optimization of their
pharmacokinetic properties may yield compounds with greater bioavailabilities and
better antischistosomiasis activities in vivo.
INTRODUCTION
Schistosomiasis is a parasitic disease affecting over 200 million people, mostly in the
developing world, caused by parasites of the Schistosoma genus, primarily S. mansoni,
S. haematobium, and S. japonicum. Though this disease is responsible for a
considerable health burden (1), its treatment thus far has relied on only one drug, the
tetrahydroisoquinoline praziquantel. While this drug has proven safe, inexpensive at
scale and efficacious, the sheer scale of its use as an antiparasitic suggests that drug
resistance may eventually become a concern (2-4).
For this reason, the development of new drugs effective against the parasite is a clear
and pressing need. One of us has looked to antimalarial drugs for possible new leads
(5), and screened the Medicines for Malaria Venture (MMV) Malaria Box (6) of 400
commercially available compounds for antischistosomal activity against both newly
transformed schistosomula (NTS) and adult worms (7). In that work, two of the most
active compounds, both in vitro and in vivo in a mouse model, were the N,N’-diarylurea
2

47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
1 (MMV665852) and the 2,3-dianilinoquinoxaline 2 (MMV007204) (Figure 1). Further
exploration of the former led to the development of several analogs, including N-
phenylbenzamides and N-arylphenylcarbamates, with excellent in vitro activity against
S. mansoni but only moderate effect in a mouse model (8).
In this work, we have developed analogs of the latter of those lead compounds,
dianilinoquinoxaline 2. Quinoxaline compounds have shown promising anticancer (9-
11), antiprotozoal (12) and antimycobacterial activities (13, 14). Among this set are
several 6-nitroquinoxaline analogs; nitroaromatic antiparasitic compounds have shown
activity against malaria (15), giardiasis (16, 17), trypanosomiasis (18, 19), amoebiasis
(20), and trichomoniasis (21). Nitroquinoxaline compounds in particular have
demonstrated potent activity against Gram-positive bacteria (22).
We have also prepared and tested a small set of other compounds that include a
quinoxaline moiety within a more complex polyheterocyclic system, including a series of
[1,2,4]triazolo[4,3-a]quinoxalines. Similar triazolopyrazines have shown antimalarial
potential
in
work
done
in
the
Open
Source
Malaria
program.
(https://openwetware.org/wiki/OpenSourceMalaria:Triazolopyrazine_(TP)_Series)
Moreover, in previous studies, triazolopyrazines have exhibited a range of biological
activities (11, 23, 24), including broad antimicrobial activity (25).
MATERIALS AND METHODS
3

69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
Synthesis. Disubstituted quinoxaline and nitroquinoxaline compounds were
synthesized from the nucleophilic aromatic substitution (SNAr) reactions of 2,3-
dichloroquinoxaline (3-23) and 6-nitro-2,3-dichloroquinoxaline (24-32), respectively
(Figure 2). While substitution reactions with aliphatic amines generally proceeded
smoothly at moderate temperatures, those involving anilines required more robust
heating. When 2-(dimethylamino)ethylamine was used as the amine nucleophile, the
major product isolated was 1-methyl-1,2,3,4-tetrahydropyrazino[2,3-b]quinoxaline (18),
the unexpected result of an intramolecular SNAr reaction by the tertiary amine followed
by demethylation. Two nitroquinoxaline products, 29 and 30, were subjected to nitro
reduction and acetylation to give the analogous acetamides 33 and 34.
Tetracyclic compounds 35 and 36 were synthesized by the condensation of o-
phenylenediamine with isatin and ninhydrin, respectively; (26) the latter was then
subjected to palladium catalyzed transfer hydrogenolysis (27) to yield 11H-indeno[1,2-
b]quinoxaline (37) (Figure 3). [1,2,4]triazolo[4,3-a]quinoxalines (38-49) were
synthesized from 2-hydrazino-3-chloroquinoxaline by acid-mediated condensation with
an aldehyde or orthoester, followed by nucleophilic aromatic substitution at the 4-
position with a secondary amine heterocycle (24).
1
Experimental details and H NMR spectral characterization data for all synthesized
compounds can be found in the Supporting Information.
4

91
92
Drugs and culture media. Compounds were prepared as 10 mM stock solutions in
dimethyl sulfoxide (DMSO) (Sigma-Aldrich). The culture media were prepared from
medium 199 (NTS testing) or RPMI 1640 (adult testing) (Life Technologies) with L-
glutamine (Sigma-Aldrich), 5% heat-inactivated fetal calf serum (FCS), 1% penicillin,
and streptomycin mix, which were purchased from LuBioScience.
93
94
95
96
97
Mice and parasites. Animal studies were carried out following Swiss national and
cantonal regulations on animal welfare at the Swiss Tropical and Public Health Institute
(Basel, Switzerland (Swiss TPH) (permission no. 2070). The S. mansoni life cycle
(Liberian strain) is maintained at Swiss TPH. For the in vitro and in vivo studies female
mice (NMRI strain; age 3 weeks; weight ca. 20-22 g) were purchased from Charles
River, Germany. Mice were kept under environmentally controlled conditions
(temperature ∼25 °C; humidity ∼70 %; 12 h light and 12 h dark cycle) with free access
to water and rodent diet, and acclimatized for 1 week before infection.
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
Newly transformed schistosomula (NTS) drug assay. S. mansoni cercariae were
gathered from infected snails and mechanically transformed to newly transformed
schistosomula (NTS). 30-40 NTS/well were incubated with 0.01-10 µM of the drugs for
72 h at 37°C, 5 % CO2 in a final well volume of 200-250 µl. Compounds were tested in
triplicate and the highest concentration of DMSO (<1%) served as control. Evaluation
was done by microscopic readout (Carl Zeiss, Germany, magnification 80x) using a
viability scale as described recently (3 = motile, no changes to morphology or
transparency; 2 = reduced motility and/or some damage to tegument noted, as well as
5

114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
reduced transparency and granularity; 1 = severe reduction of motility and/or damage to
tegument observed, with high opacity and high granularity; 0 = dead) (28).
Adult S. mansoni drug assay. Adult schistosomes were collected by mechanical
picking from the hepatic portal system and mesenteric veins 49 day post-infection with
100 S. mansoni cercariae. Worms were incubated with 0.1 and 1 µM of the compounds
for 72 h. Wells with 1% DMSO served as negative controls. Phenotypes were monitored
under an inverted microscope and viability scores calculated (28). Each compound was
tested twice in duplicate. To calculate IC50 values, viability scores were converted into
effect scores using CompuSyn2® (ComboSyn Inc., 2007).
Rat skeletal myoblast L6 cytotoxicity. Rat skeletal myoblast L6 cells were seeded in
96 well plates (2 x 103 cells/ well) using supplemented RPMI 1640 medium as
described above. Following adhesion of the cells for 24 h at 37 °C and 5% CO2, the IC50
of the compounds was determined using concentrations of 0.12, 0.37, 1.11, 3.33, 10,
30, and 90 µM. Podophyllotoxin served as positive control. After 70 h post-incubation,
10 µL resazurin dye (Sigma) was added and the plates incubated for another 2 h.
Analysis was done at 72 h using a SpectraMax M2 (Molecular Devices) plate reader
with an excitation wavelength of 530 nm and emission wavelength of 590 nm.
Calculation of physicochemical properties. The in silico prediction tool ALOGPS 2.1
was used to calculate log P (clogP) and log S values for all compounds
((29); http://www.vcclab.org).
6

137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
In vivo studies. Mice were infected with 100 S. mansoni cercariae subcutaneously.
Single oral doses of 400 mg/kg of the three lead compounds were administered to
groups of four mice 49 days (adult infection) post-infection, respectively. A 400 mg/kg
dose is often used as starting dose in S. mansoni in vivo experiments (30), as it is the
efficacious dose of praziquantel in S. mansoni infected mice. Untreated mice (31)
served as controls. Mice were euthanized using CO2 three weeks post treatment, and
worms were picked, sexed and counted and the worm burden reduction was calculated.
A Kruskal Wallis test was employed to determine statistical significance.
RESULTS
In vitro activity against NTS. 47 compounds were tested at 10 μM for activity against
NTS. Tetracycles 35-37 and triazoloquinoxalines 38-49 showed marginal activity
(<35%) at this concentration after 72 h (data in Supporting Information). Among the
other quinoxaline test compounds, the best activity was found with
dianilinoquinoxalines, with 16 of these showing an activity of more than 70% after 72 h
(Table 1). At a lower, 1 µM concentration, nine of those sixteen compounds revealed an
activity above 70% against NTS after 72 h. The most active compounds,
nitroquinoxalines 29 and 30, affected NTS with an activity of over 70% at 0.1 µM, and
showed low activity (21.9% for each) even at 0.01 µM, the lowest concentration tested.
For comparison, praziquantel shows an IC50 of 2.2 µM against NTS (30).
7

159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
In vitro activity against adult S. mansoni. The 16 quinoxaline compounds that
showed good in vitro activity against NTS at 10 μM were also generally active against
adult S. mansoni at the same concentration, with 15 of the 16 showing an activity of at
least 70% against the worms after 72 h (Table 1). Of these, 8 compounds were active
(>70%) at 1 µM. The three most active compounds, 27, 29, and 30, showed also
moderate activity against adult worms at 0.1 µM (16-41% after 72 h). The IC50 values
against adult worms for these compounds were all under 0.3 µM, and were comparable
to that of praziquantel (0.1 µM) (30).
Calculated physicochemical properties and solubility. Log P and log S values were
calculated for all 47 compounds in this study. In this set, the 19 compounds that showed
the greatest antiparasitic activity against NTS all had clog P values over 4.98,
essentially the “Lipinski limit” (32), and low calculated aqueous solubilities, ranging from
1.8 to 25.7 µM (clog S -5.74 to -4.59) (Table 1). Although these clog P values
contravene Lipinski’s “rule of five” (32), the use of those heuristics in antiparasitic drug
development has been cautioned against (33).
Antischistosomal selectivity. Nitroquinoxaline compounds 24-32 were tested for
cytotoxicity against an L6 rat skeletal muscle cell line. Compounds that showed good
activity against adult worms (>70% at 10 µM) were moderately cytotoxic to L6 cells
(IC50’s of 1.7-27.5 µM) (Table 2). Among our three most active compounds, 29 showed
the highest antischistosomal selectivity (44.6), significantly higher than those of 27 and
30 (18.3 and 8.9, respectively), due largely to the compounds’ differential in cytotoxicity.
8

181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
In vivo studies. The three most active compounds progressed to in vivo studies, where
they were tested in mice harboring adult S. mansoni. Compound 29 was the most active
compound, with a worm burden reduction (WBR) of 46.4% (P < 0.05) at 400 mg/kg
(Table 2). This is roughly half of the antiparasitic activity of praziquantel at the same
dosage (94%) (34). WBR values for compounds 27 and 30 were considerably lower
(9.3% and 12.5%, respectively).
DISCUSSION
The overwhelming dependence on praziquantel to treat schistosomiasis worldwide
demonstrates the potential danger that praziquantel resistance poses. There is
therefore a clear need for the development of antischistosomal drugs with novel
pharmacophores and modes of action. The dianilinoquinoxaline 2 was identified in
previous work as a promising lead for further antischistomiasis drug development (8).
In this follow-up study we have synthesized 47 analogs of this lead compound by
varying both the amine/aniline substituents on the quinoxaline scaffold and the scaffold
itself. Eleven of these analogs (3-13) were dianilinoquinoxalines like 2, while ten others
(14-23) were synthesized from 2,3-dichloroquinoxaline and non-aniline amines;
comparing these two groups, the aniline-containing quinoxalines showed better
antischistosomal activity than the second group did. Nitroquinoxaline analogs (24-32)
similar to the first, more active group were also synthesized, as were a series of
triazoloquinoxalines (38-49) and a small set of tetracyclic compounds (35-37)
incorporating the quinoxaline moiety.
9

204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
All compounds were first tested in vitro on newly transformed schistosomula (NTS).
Test compounds with aniline substituents (3-13, 24-34) generally showed strong
antiparasitic activity against both NTS and adult worms at a concentration of 1 µM; the
remainder of our set showed only weak activity against NTS at 10 µM. The most active
were nitroquinoxalines 27, 29 and 30, which demonstrated sub-micromolar IC50 values
against adult worms, comparable to the published values for our lead compound 2 and
for praziquantel itself (7). Notably, compound 30 is similar to the diarylurea lead
compound 1 in that they both carry two 3,4-dichloroaniline moieties.
The addition of a nitro group to the quinoxaline scaffold increased the activity against
NTS in a few cases (4 vs. 29, 5 vs. 30), but this effect was not consistent across our set
of analogs. Like other nitroaromatic antiparasitic compounds, such as nitazoxanide and
metronidazole, this added activity upon nitration may be due to the targeting of parasitic
redox systems (16, 35).
Unfortunately, the highest WBR achieved here, with nitroquinoxaline 29, was only half
of that measured for praziquantel; two similar compounds with very good in vitro activity,
27 and 30, showed very little WBR at the same concentration. The in vitro/in
vivo discrepancy found here may be due to rapid metabolic reduction of these nitro
compound to anilines (36). However, acetamidoquinoxalines 33 and 34, which would
also ostensibly be metabolized to anilines within the parasite, showed markedly less
activity against both NTS and adult worms than their corresponding non-acetamido
10

227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
analogues in our compound set (4, 5, 29 and 30). More generally, the poor in vivo
activity of these compounds may simply be due to poor pharmacokinetic properties –
that is, high lipophilicities (clogP > 5) and low aqueous solubilities. Further structural
optimization may be able to improve the bioavailabilities of these compounds and
improve their activities in vivo.
In conclusion, several analogs of the antimalarial quinoxaline MMV007204 were
synthesized and shown to have high activities against NTS and adult S. mansoni worms
in in vitro experiments. While the in vivo activities of these compounds proved to be
moderate at best, further development of more hydrophilic derivatives may provide
more active compounds.
Acknowledgements:
S.L.D, M.J.H., K.R.E., C.J.B. and G.E.F. were supported in this work by the Lawrence
University Excellence in Science Fund. S.L.D. wishes to thank Judith Humphries for
helpful conversation, and Maxwell Stahl and the undergraduate students in Lawrence
University’s Organic Chemistry II classes in 2018 and 2019 for their contributions to our
synthetic efforts. J.K. is grateful to the Swiss National Science Foundation for financial
support (no. 320030_175585).
REFERENCES
11

249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
1. Colley, D, Bustinduy, A, Secor, W, King, C. 2014. Human schistosomiasis. The
Lancet. 383:2253-2264.
2. Wang, W, Wang, L, Liang, Y. 2012. Susceptibility or resistance of praziquantel in
human schistosomiasis: a review. Parasitol. Res. 111:1871-1877.
3. Doenhoff, MJ, Cioli, D, Utzinger, J. 2008. Praziquantel: mechanisms of action,
resistance and new derivatives for schistosomiasis. Curr. Opin. Infect. Dis. 21:659-667.
4. Bergquist, R, Utzinger, J, Keiser, J. 2017. Controlling schistosomiasis with
praziquantel: How much longer without a viable alternative? Infect. Dis. Poverty. 6:74-
017-0286-2.
5. Keiser, J, Utzinger, J. 2012. Antimalarials in the treatment of schistosomiasis. Curr.
Pharm. Des. 18:3531-3538.
6. Spangenberg, T, Burrows, JN, Kowalczyk, P, McDonald, S, Wells, TN, Willis, P.
2013. The open access malaria box: a drug discovery catalyst for neglected diseases.
PLoS One. 8:e62906.
7. Ingram-Sieber, K, Cowan, N, Panic, G, Vargas, M, Mansour, NR, Bickle, QD,
Wells, TN, Spangenberg, T, Keiser, J. 2014. Orally active antischistosomal early leads
identified from the open access malaria box. PLoS Negl Trop. Dis. 8:e2610.
8. Cowan, N, Dätwyler, P, Ernst, B, Wang, C, Vennerstrom, JL, Spangenberg, T,
Keiser, J. 2015. Activities of N,N′-Diarylurea MMV665852 Analogs against Schistosoma
mansoni. Antimicrob. Agents Chemother. 59:1935-1941.
9. Kaushal, T, Srivastava, G, Sharma, A, Negi, AS. 2019. An insight into medicinal
chemistry of anticancer quinoxalines. Bioorg. Med. Chem. 27:16-35.
12

271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
10. Montana, M, Mathias, F, Terme, T, Vanelle, P. 2019. Antitumoral activity of
quinoxaline derivatives: A systematic review. Eur. J. Med. Chem. 163:136-147.
11. Ibrahim, MK, Taghour, MS, Metwaly, AM, Belal, A, Mehany, ABM, Elhendawy,
MA, Radwan, MM, Yassin, AM, El-Deeb, NM, Hafez, EE, ElSohly, MA, Eissa, IH.
2018. Design, synthesis, molecular modeling and anti-proliferative evaluation of novel
quinoxaline derivatives as potential DNA intercalators and topoisomerase II inhibitors.
European Journal of Medicinal Chemistry. 155:117-134.
12. Hui, X, Desrivot, J, Bories, C, Loiseau, PM, Franck, X, Hocquemiller, R,
Figadere, B. 2006. Synthesis and antiprotozoal activity of some new synthetic
substituted quinoxalines. Bioorg. Med. Chem. Lett. 16:815-820.
13. Seitz, LE, Suling, WJ, Reynolds, RC. 2002. Synthesis and antimycobacterial
activity of pyrazine and quinoxaline derivatives. J. Med. Chem. 45:5604-5606.
14. Waisser, K, Beckert, R, Slosáfrek, M, Janota, J. 1997. Antimycobacterial activity
of some 2,3-dianilinoquinoxaline derivatives. Pharmazie. 52:797—798.
15. Roberts, BF, Zheng, Y, Cleaveleand, J, Lee, S, Lee, E, Ayong, L, Yuan, Y,
Chakrabarti, D. 2017. 4-Nitrostyrylquinoline is an antimalarial inhibiting multiple stages
of Plasmodium falciparum asexual life cycle. Int. J. Parasitol. Drugs Drug Resist. 7:120-
129.
16. Muller, J, Schildknecht, P, Muller, N. 2013. Metabolism of nitro drugs
metronidazole and nitazoxanide in Giardia lamblia: characterization of a novel
nitroreductase (GlNR2). J. Antimicrob. Chemother. 68:1781-1789.
13

292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
17. Adagu, IS, Nolder, D, Warhurst, DC, Rossignol, J. 2002. In vitro activity of
nitazoxanide and related compounds against isolates of Giardia intestinalis, Entamoeba
histolytica and Trichomonas vaginalis. J. Antimicrob. Chemother. 49:103-111.
18. Sokolova, AY, Wyllie, S, Patterson, S, Oza, SL, Read, KD, Fairlamb, AH. 2010.
Cross-resistance to nitro drugs and implications for treatment of human African
trypanosomiasis. Antimicrob. Agents Chemother. 54:2893-2900.
19. Trunz, BB, Jedrysiak, R, Tweats, D, Brun, R, Kaiser, M, Suwinski, J, Torreele,
E. 2011. 1-Aryl-4-nitro-1H-imidazoles, a new promising series for the treatment of
human African trypanosomiasis. Eur. J. Med. Chem. 46:1524-1535.
20. Mata-Cárdenas, B, Vargas-Villarreal, J, González-Garza, M, Said-Fernández, S.
1996. In-Vitro High Anti-amoebic Potency of Secnidazole and Dimetridazole. Pharmacy
and Pharmacology Communications. 2:513-514.
21. Hoffman, PS, Sisson, G, Croxen, MA, Welch, K, Harman, WD, Cremades, N,
Morash, MG. 2007. Antiparasitic drug nitazoxanide inhibits the pyruvate
oxidoreductases of Helicobacter pylori, selected anaerobic bacteria and parasites, and
Campylobacter jejuni. Antimicrob. Agents Chemother. 51:868-876.
22. Ahammed, KS, Pal, R, Chakraborty, J, Kanungo, A, Purnima, PS, Dutta, S.
2019. DNA Structural Alteration Leading to Antibacterial Properties of 6-Nitroquinoxaline
Derivatives. J. Med. Chem. 62:7840-7856.
23. Ali, I, Lee, J, Go, A, Choi, G, Lee, K. 2017. Discovery of novel [1,2,4]triazolo[4,3-
a]quinoxaline aminophenyl derivatives as BET inhibitors for cancer treatment.
Bioorganic & Medicinal Chemistry Letters. 27:4606-4613.
14

314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
24. Sarges, R, Howard, HR, Browne, RG, Lebel, LA, Seymour, PA, Koe, BK. 1990.
4-Amino-[1,2,4]triazolo[4,3-a]quinoxalines. A novel class of potent adenosine receptor
antagonists and potential rapid-onset antidepressants. J. Med. Chem. 33:2240-2254.
25. Omar, AM, Alswah, M, Ahmed, HEA, Bayoumi, AH, El-Gamal, KM, El-Morsy, A,
Ghiaty, A, Afifi, TH, Sherbiny, FF, Mohammed, AS, Mansour, BA. 2020.
Antimicrobial screening and pharmacokinetic profiling of novel phenyl-[1,2,4]triazolo[4,3-
a]quinoxaline analogues targeting DHFR and E. coli DNA gyrase B. Bioorganic
Chemistry. 96:103656.
26. Dowlatabadi, R, Khalaj, A, Rahimian, S, Montazeri, M, Amini, M, Shahverdi, A,
Mahjub, E. 2011. Impact of substituents on the isatin ring on the reaction between
isatins with ortho-phenylenediamine. Synthetic Communications. 41:1650-1658.
27. Ram, S, Spicer, LD. 1988. Reduction of aldehydes and ketones to methylen
derivatives using ammonium formate as a catalytic hydrogen transfer agent.
Tetrahedron Lett. 29:3741-3744.
28. Lombardo, FC, Pasche, V, Panic, G, Endriss, Y, Keiser, J. 2019. Life cycle
maintenance and drug-sensitivity assays for early drug discovery in Schistosoma
mansoni. Nature Protocols. 14:461-481.
29. Tetko, IV, Gasteiger, J, Todeschini, R, Mauri, A, Livingstone, D, Ertl, P,
Palyulin, VA, Radchenko, EV, Zefirov, NS, Makarenko, AS. 2005. Virtual
computational chemistry laboratory design and description. J. Comput. Aided Mol. Des.
19:453-463.
15

335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
30. Ingram, K, Yaremenko, IA, Krylov, IB, Hofer, L, Terent’ev, AO, Keiser, J. 2012.
Identification of antischistosomal leads by evaluating bridged 1,2,4,5-tetraoxanes,
alphaperoxides, and tricyclic monoperoxides. J. Med. Chem. 55:8700-8711.
31. Abla, N, Keiser, J, Vargas, M, Reimers, N, Haas, H, Spangenberg, T. 2017.
Evaluation of the pharmacokinetic-pharmacodynamic relationship of praziquantel in the
Schistosoma mansoni mouse model. PLoS Negl Trop Dis. 11:e0005942.
32. Lipinski, CA, Lombardo, F, Dominy, BW, Feeney, PJ. 1997. Experimental and
computational approaches to estimate solubility and permeability in drug discovery and
development settings. Advanced Drug Delivery Reviews. 23:3-25.
33. McKerrow, JH, Lipinski, CA. 2017. The rule of five should not impede anti-
parasitic drug development. International Journal for Parasitology: Drugs and Drug
Resistance. 7:248-249.
34. Meister, I, Ingram-Sieber, K, Cowan, N, Todd, M, Robertson, MN, Meli, C, Patra,
M, Gasser, G, Keiser, J. 2014. Activity of praziquantel enantiomers and main
metabolites against Schistosoma mansoni. Antimicrob. Agents Chemother. 58:5466-
5472.
35. Pal, C, Bandyopadhyay, U. 2012. Redox-Active Antiparasitic Drugs. Antioxidants &
Redox Signaling. 17:555-582.
36. Rickert, DE. 1987. Metabolism of nitroaromatic compounds. Drug Metab. Rev.
18:23-53.
16

356
% dead after 72 h
0.01 µM
NTS
Adult worms
X
H
H
H
H
H
H
H
R
1 µM
0.1 µM
1 µM
0.1 µM
clog P clog S
3
4
5
6
7
8
9
3-Cl-Ph
100 (0)
100 (0)
0 (0)
-
-
82.8 (9.8)
79.3 (4.9)
41.0 (5.6)
27.3 (2.8)
6.01
6.01
7.09
7.1
-5.41
-5.44
-5.6
12.5 (5.9)
4-Cl-Ph
3,4-diCl-Ph
3,5-diCl-Ph
3-4-Ph
100 (0)
100 (0)
12.5 (0)
-
-
88.5 (10.9)
84.6 (5.4)
37.1 (5.6)
37.1 (0)
18.8 (2.9)
-5.64
-4.94
-4.76
-4.78
-5.21
-4.77
-5.54
-5.51
-5.74
-4.61
-4.59
-5.19
-5.48
100 (0)
25.0 (0)
8.33 (0)
-
-
71.1 (2.7)
34.4 (0)
19.4 (2.8)
-
5.1
86.0 (2.8)
3-CH3O-Ph
4-CH3O-Ph
3-CF3-Ph
5.06
5.1
80.0 (5.7) 6.25 (8.8)
-
-
39.6 (2.4)
60.7 (0)
-
-
44.2 (8.2)
-
26 NO2
27 NO2
28 NO2
29 NO2
30 NO2
31 NO2
32 NO2
33 NHAc
34 NHAc
5.79
4.98
5.76
5.88
6.65
5.06
5.05
5.23
6.39
3-CH3O-Ph
4-Br-Ph
24.9 (2.7)
59.6 (2.7)
-
-
-
-
82.1 (0)
24.4 (2.9)
-
67.8 (5.1)
4-Cl-Ph
75 (2.7)
73.1 (5.4) 21.9 (2.8)
92.9 (0)
16.3 (2.9)
40.8 (8.7)
80.8 (5.4) 78.8 (2.7) 21.9 (8.5) 78.5 (10.1)
3,4-diCl-Ph
3,4-di(CH3O)-Ph
3,5-di(CH3O)-Ph
4-Cl-Ph
32.6 (2.7)
34.6 (5.4)
-
-
-
-
†
-
-
66 (2.5)
28.4 (6.8)
-
-
-
-
20.3 (2.9)
-
-
33.9 (11.8)
63.2 (11.6)
3,4-diCl-Ph
Only compounds with ≥70% activity against NTS at 10 µM shown. Numbers in parentheses are the standard
deviations of the data. For full results, see Supporting Information. †49.1% dead at 10 µM.
357
358
359
Table 1. In vitro activities of synthesized analogs.
IC50, adult IC50, L6
S. mansoni
(µM)
cells
(µM)
5.7
WBR
(%)
SI
27
29
30
0.31
18.3
44.6
8.9
9.3
0.28
12.5
1.7
46.4
12.5
0.19
17

praziquantel
0.1a
>96a
>960a 94.1b
360
361
362
363
SI = selectivity index (IC50,L6/IC50,S. mansoni). WBR = worm burden reduction. a) Ref. 30. b)
Ref. 34.
Table 2. IC50 and worm burden reduction values of synthesized analogs.
18