Structure-Activity Relationship and in Vitro Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) Studies of N-aryl 3-Trifluoromethyl Pyrido[1,2- a]benzimidazoles That Are Efficacious in a Mouse Model of Schistosomiasis

Article abstract of DOI:10.1021/acsinfecdis.8b00313

We have previously reported on the antischistosomal activity of pyrido[1,2-a]benzimidazole (PBI) derivatives. As a follow-up, we designed and prosecuted further structure-activity relationship (SAR) studies that incorporate N-aryl substitutions on the PBI scaffold. Investigations into the in vitro antischistosomal activity against newly transformed schistosomula (NTS) and adult worms revealed several leads with promising potency. Active compounds with a good cytotoxicity profile were tested in vivo whereby 6 and 44 induced noteworthy reduction (62-69%) in the worm load in the Schistosoma mansoni mouse model. Pharmacokinetic analysis on 44 pointed to slow absorption, low volume of distribution, and low plasma clearance indicating the potential of these compounds to achieve a long duration of action. Overall, our work demonstrates that PBI chemotype is a promising scaffold in the discovery of new antischistosomal leads.

Full text of DOI:10.1021/acsinfecdis.8b00313

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Article
Structure-activity relationship and in vitro ADMET studies
of N-aryl 3-trifluoromethyl pyrido [1,2-a] benzimidazoles
that are efficacious in a mouse model of schistosomiasis.
Godfrey Mayoka, Jennifer Keiser, Cecile Haeberli, and Kelly Chibale
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00313 • Publication Date (Web): 23 Dec 2018
Downloaded from http://pubs.acs.org on December 24, 2018
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Structure-activity relationship and in vitro ADMET studies of N-
aryl 3-trifluoromethyl pyrido[1,2-a]benzimidazoles that are
efficacious in a mouse model of schistosomiasis.
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,
,
Godfrey Mayoka†, Jennifer Keiser# ∞, Cécile Häberli# ∞ and Kelly Chibale*,†, §, T
†Department of Chemistry, University of Cape Town, Rondebosch, 7701, South Africa
,
# ∞Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland and
University of Basel, Basel, Switzerland
§Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Rondebosch,
7701, South Africa
TSouth African Medical Research Council Drug Discovery Unit, University of Cape Town,
Rondebosch, 7701, South Africa
*Corresponding Author: Kelly Chibale
Email: Kelly.Chibale@uct.ac.za: Phone: +27-21-6502553. Fax: +27-6505195
We have previously reported on the antischistosomal activity of pyrido [1,2-a] benzimidazole
(PBI) derivatives. As a follow-up, we designed and prosecuted further structure-activity
relationship (SAR) studies that incorporate N-aryl substitutions on the PBI scaffold. Investigations
into the in vitro antischistosomal activity against newly transformed schistosomula (NTS) and
adult worms revealed several leads with promising potency. Active compounds with a good
cytotoxicity profile were tested in vivo whereby 6 and 44 induced noteworthy reduction (62-69%)
in the worm load in the Schistosoma mansoni mouse model. Pharmacokinetic analysis on 44
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pointed to slow absorption, low volume of distribution and low plasma clearance indicating the
potential of these compounds to achieve a long duration of action. Overall, our work demonstrates
that PBI chemotype is a promising scaffold in the discovery of new antischistosomal leads.
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KEYWORDS: Schistosoma mansoni, Newly transformed schistosomula, Pyrido[1,2-
a]benzimidazole, antischistosomal lead
Flatworms of the genus Schistosoma cause schistosomiasis (also known as Bilharzia) with
S. haematobium, S. japonicum and S. mansoni being largely responsible for most of the human
cases of schistosomiasis. The latest estimates reveal that the disease is endemic in 78 countries
where 780 million people are at risk and over 200 million infected with schistosmiasis.1–4
Persistently, schistosomiasis has been rife in the tropical and sub-tropical regions of sub-Saharan
Africa, Middle East, South East Asia, South America and the Caribbean and is associated with
poor hygiene and sanitation that promotes completion of the parasite’s life cycle and hence
propagation of infection.4,5 The chronic and insidious nature of the disease has been blamed for
the under-estimation of its public health impact with the generalised non-specific symptoms,
frequent in other disease states, being a common cause for either wrong or late diagnosis.6,7
Chemotherapy has played a pivotal role in the control of schistosomiasis. Praziquantel is currently
the only drug of choice with pan-activity across the species whereas previously useful agents like
oxamniquine and metrifonate that display activity singly against S. mansoni and S. haematobium
respectively, are no longer in use.7,8 Although praziquantel is effective, safe and affordable, its
over-reliance especially in use during mass drug administration programs, so called preventive
chemotherapy, has sparked concerns of resistance emerging with decreased clinical efficacy
having been reported previously.9 Additionally, praziquantel displays poor activity towards the
immature stages of the worms, making repeated administration necessary.10,11
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That a disease with public health significance of such magnitude should depend on a single drug
is startling and raises discomforting concerns regarding emergence of resistance in the absence of
effective alternatives. This scenario has provoked the scientific community into offering greater
commitment to drug discovery and development towards schistosome infections. In contributing
towards this goal, our research group has been interested in studying the antischistosomal activities
of pyridobenzimidazole (PBI) analogues. We previously reported on the antischistosomal activity
of a PBI series arising from SAR work that generated mainly aliphatic amine derivatives with
substituted 3-phenyl groups on the PBI scaffold as exemplified by compound A in Figure
1.12Moreover, enhanced in vitro potency was noticed among a small subset of aromatic amine
derivatives bearing a 3-trifluoromethyl substitution on the PBI core such as compound B. The
aromatic analogues also displayed an improved cytotoxicity profile as suggested by higher
selectivity indices with compound B. (Figure 1).
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A
B
SO2Me
NHEt
HN
HN
N
N
N
N
CF3
CN
CN
Adult worm IC50 = 0.97
Cl
Adult worm IC50 = 2.44
M

M

CHO IC50 = 148.1
SI=152.7
M

CHO IC50 = 9.60
SI=3.9
% total worm reduction (400mg/kg, po):58
M

%worm total reduction (400mg/kg, po): 38
Figure 1. Exemplar compounds from previous SAR studies
As a follow-up, we set out an expanded SAR program involving modifications of the appendages
to the PBI core as depicted in Figure 2. Presently, we report the in vitro potency, selectivity,
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metabolic stability as well as in vivo efficacy and pharmacokinetics of compounds produced in the
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current work.
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.
.
.
Remove linker
Introduce chirality
Replace with other groups e.g. sulfonyl; NH, NHCO
2° or 3° Nitrogen
.
.
Aryl group with Craig Plot substituents
R1
R2
N
R
N
Heteroaryls such as pyridyl, pyrimidyl and pyraziny
Introduce halogen substituents
N
CF3
CN
(R3)
Replace with other groups such as
H, COOMe, CONH2
Figure 2. SAR approach to target compounds
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RESULTS AND DISCUSSION
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Chemistry. Target compounds were synthesised using methods adapted from literature13 and as
shown in Scheme 1.
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Scheme 1. General reaction scheme for synthesis of pyridobenzimidazoles.
R=CN
III
II
IIIa
IIIb
Y
: X,Y,Z=H
: X, Z=Cl, Y=H
X
OH
R
X
H
N
IIIc:
X=H, Y, Z=Cl
Z
Y
Z
R
N
IIId
: X,Y,Z=H; R=COOMe
ii
N
R=CN
N
CF3
R=CN
IIa
IIb
: X,Y,Z=H
: X, Z=Cl, Y=H
Va
Vb
: X,Y,Z=H
: X, Z=Cl, Y=H
IIc:
X=H, Y, Z=Cl
i
Vc:
X=H, Y, Z=Cl
iii
IId
: X,Y,Z=H; R=COOMe
Vd
: X,Y,Z=H; R=COOMe
: X,Y,Z=H; R=CONH2
Ve
X
Y
Z
Y
Y
NH2
NH2
X
X
NHR1
Cl
Z
Z
iv
N
N
Ia
: X, Z=Cl, Y=H
I
N
N
CF3
CF3
R=CN
Ib:
X=H, Y, Z=Cl
R
R
V
IVa
IVb
: X,Y,Z=H
: X, Z=Cl, Y=H
X=H, Y, Z=Cl
IV
IVc:
IVd
IVe
: X,Y,Z=H; R=COOMe
: X,Y,Z=H; R=CONH2
Reagents and conditions: (i) Ethyl cyanoacetate, DMF, 160˚C, 2 h, 61-76%; (ii) Ethyl 4,4,4-trifluoro-3-oxobutanoate,
NH4OAc, 145˚C, 2h, 27-66%; (iii) POCl3, (20eq), 130˚C, 3 h, 63-94%; (iv) Relevant amine, Et3N, 80˚C, 150 W,
20-60 min (9-86%) or appropriate amine, Pd2(dba)3, BINAP or Brettphos, K2CO3 or Cs2CO3, toluene or 1,4-Dioxane
or tert-butanol, 100-120˚C, 12-16 h, 6-54% . X, Y and Z are as described in Table 1.
Compounds with no substitution on the left-hand side (LHS) of the PBI scaffold (X, Y, Z = H;
Table 1) were synthesised from the commercially available 2-benzimidazole acetonitrile IIa. For
other targets, the appropriately substituted diamino benzene starting material I was subjected to a
condensation reaction with ethyl-2-cyanoacetate to form the appropriately substituted
benzimidazole acetonitrile derivative II.
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Thereafter, the condensation of the benzimidazole acetonitrile and ethyl 4,4,4-trifluoro-3-
oxobutanoate occurs in the presence of ammonium acetate to give the tricyclic hydroxyl
intermediate III, which is converted to the penultimate intermediate IV in a reaction involving
substitution of the hydroxyl with a chloro, a process mediated by phosphoryl chloride (POCl3).
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Finally, amination via nucleophilic aromatic substitution of IV using the relevant amine in the
presence of triethylamine in a microwave reactor or the palladium -catalysed Buchwald-Hartwig
reaction in the presence of a suitable ligand and base were employed to give the final compounds
1 - 4, 6, 7, 9 - 13, 17 - 25, 27 - 35, 37 - 45, 48, 49, 53 - 55 and 5, 8, 14 - 16, 26, 36, 46, 47, 50 -
52 respectively. To obtain 8, the benzimidazole methyl ester IId was used as the starting material.
For other nitrile replacement analogues 53 – 55, intermediate IVa was converted to the amide IVe
using concentrated sulphuric acid before amination as in step iv. The final compound 7 was
accessed via ester decarboxylation of 8.
In vitro antischistosomal activity against NTS and adult S. mansoni. With the aim of
prioritising compounds capable of targeting newly transformed schistosomula (NTS), compounds
were first screened for activity against these immature worms at an initial concentration of 10 µM.
Thereafter, compounds that displayed significant activity against NTS were subsequently exposed
to adult S. mansoni at the same concentration. Compounds with significant activity (≥70%) in the
adult worms at 10 µM were progressed for further dose-response experiments from which IC50
values were determined. The activity progression criteria adopted was intended to guide the
progression of compounds with dual life cycle stage activity as this profile is a requisite for both
the treatment and prevention of re-infection with schistosomiasis. Ensuing SAR trends were
analysed based on the observed activities as presented in Table 1.
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Table 1. In vitro activity against S. mansoni NTS and adult worms.
8
9
Y
X
R
Z
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N
N
CF3
R1
% S. mansoni
mortality at
Adult
Compound
Identity
X
Y
Z
R1
R
S. mansoni
10 µM
NTS
Adult
33
IC50 (µM)
H
N
1
2
3
H
H
H
H
H
CN
CN
CN
74
100
43
H
N
H
H
H
H
50
OPr
H
N
NMe2
H
N
NHMe
4
H
H
H
CN
30
O
H
N
NMe2
5
6
7
8
H
H
H
H
H
H
H
H
H
H
H
H
CN
CN
H
30
100
100
67
O
H
N
100
77
0.210
3.37
OCF3
OCF3
OCF3
H
N
H
N
COOMe
56
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X
N
R
Z
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CF3
R1
9
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% S. mansoni
mortality at
Adult
Compound
Identity
X
Y
Z
R1
R
S. mansoni
10 µM
NTS
Adult
100
IC50 (µM)
1.05
H
OCF3
N
9
H
H
H
H
H
CN
CN
100
OCF3
H
N
10
H
H
H
100
100
1.65
1.49
1.38
2.01
H
N
11
12
13
H
H
H
CN
CN
CN
96
100
26
63
COOMe
H
N
Cl Cl
100
OCF3
N
H
H
H
H
H
H
OMe
OMe
N
H
N
OMe
14
15
H
H
CN
CN
100
30
100
N
H
N
N
N
H
N
N
16
17
H
H
H
H
H
H
CN
CN
91
17
92
N
HN
NMe2
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% S. mansoni
mortality at
Adult
Compound
Identity
X
Y
Z
R1
R
S. mansoni
10 µM
NTS
Adult
IC50 (µM)
HN
18
19
20
21
22
23
24
H
H
H
H
H
H
H
H
H
CN
CN
CN
CN
CN
CN
CN
22
4
N
HN
H
H
H
H
H
H
H
H
H
H
H
H
F
N
9
11
0
N
HN
0
HN
4
OMe
HN
9
OCF3
HN
HN
13
SO2Me
25
H
H
H
CN
48
SO2Me
HN
26
27
28
H
H
H
H
H
H
H
H
H
CN
CN
CN
30
4
N
HN
HN
N
N
30
N
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Z
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R1
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% S. mansoni
mortality at
Adult
Compound
Identity
X
Y
Z
R1
R
S. mansoni
10 µM
NTS
Adult
IC50 (µM)
O
HN
29
30
31
H
H
H
H
H
CN
CN
CN
9
4
0
N
HN
H
H
H
H
OH
HN
0
HN
NHMe
32
H
H
H
CN
17
O
HN
HN
NMe2
33
34
35
H
H
H
H
H
H
H
H
H
CN
CN
CN
26
22
26
O
SO2NH2
HN
N
HN
36
37
38
H
H
H
H
CN
CN
CN
26
N
HN
Cl
H
Cl
100
100
100
98
0.770
1.69
F
F
HN
Cl Cl
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X
N
R
Z
N
CF3
R1
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% S. mansoni
mortality at
Adult
Compound
Identity
X
Y
Z
R1
R
S. mansoni
10 µM
NTS
Adult
100
IC50 (µM)
1.58
HN
39
40
41
42
H
Cl Cl
Cl Cl
Cl Cl
Cl Cl
CN
CN
CN
CN
100
100
100
100
OCF3
HN
H
H
H
99
1.74
1.17
N
HN
100
100
O
HN
0.470
S
HN
HN
43
44
H
H
Cl Cl
CN
CN
100
100
100
100
0.430
F
Cl Cl
Cl Cl
0.380
0.400
F
HN
45
46
H
H
CN
CN
100
89
100
29
H
N
HN
H
H
H
H
O
H
N
N
47
48
H
H
CN
CN
9
H
Cl
HN
Cl Cl
23
66
SO2Me
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R
Z
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CF3
R1
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mortality at
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Compound
Identity
X
Y
Z
R1
R
S. mansoni
10 µM
NTS
Adult
100
IC50 (µM)
0.970
HN
49
50
51
52
H
Cl Cl
Cl Cl
Cl Cl
Cl Cl
CN
CN
CN
CN
100
44
93
9
SO2Me
O
O
S
HN
H
H
H
84
87
1.11
1.09
CN
O
O
S
HN
OCF3
O
O
S
HN
N
HN
CONH2
CONH2
CONH2
53
54
H
H
H
H
H
H
H
H
H
4
21
8
34
53
15
F
HN
OCF3
HN
55
N
Praziquantel
0.100
Values are a mean of n≥2 determinations. Generally, IC50 values from individual experiments differed less than 2-
fold; values that differed more than 2-fold were excluded from analysis.
SAR plan and analysis. The SAR program pursued resulted in compounds varying in the type of
linkage between the side group and the PBI core by either being connected directly or via a
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methylene group (or its isosteres) as depicted in Figure 2. Other alterations on the side group
included the introduction of chirality at the methylene linker via installation of methyl
substitutions. Similar substitutions were introduced on the nitrogen attached to the PBI core (R1,
Figure 2) to make some analogues with a tertiary nitrogen. Additionally, small lipophilic halogen
groups (R, Figure 2) were introduced on the left-hand side (LHS) of the PBI core and other small
groups were introduced to replace the nitrile at R3 (Figure 2).
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Overall, 19 compounds produced over >70% activity against NTS and adult worms at a
concentration of 10 µM. Out of these, 18 compounds achieved an IC50 of ≈2 µM and below (as
low as 0.21 µM), similar to praziquantel, characterized by an IC50 of 0.10 µM against adult worms.
In the sub-series where the aromatic side group is directly linked to the PBI core by a nitrogen and
with no changes on neither the LHS nor R1 (compounds 1 - 16), the analogues 6, 9, 10, 12, 14 and
16 were most active with 6 (IC50 = 0.21 µM), bearing a lipophilic electron withdrawing
trifluoromethoxy (-OCF3) group, being the most potent compound in this category. On the
contrary, compounds with hydrophilic groups on the aromatic side group (R, Table 1) abrogated
activity as seen, for example, with 3, 4 and 5 which showed low activity against NTS at 10 µM.
Additional compounds with good antischistosomal activity were identified among compounds
incorporating heteroaromatic side groups instead of a phenyl moiety as exemplified by pyrimidyl
and pyrazinyl groups in 14 (IC50 = 1.38 µM) and 16 (IC50 = 2.01 µM), respectively. The positioning
of the nitrogen atoms in the aromatic side group seem crucial for antischistosomal activity since
15, in which the nitrogen atoms have a 2,6 relationship, showed poor activity towards NTS at 10
µM compared to its regio-isomer 16 (NTS, 91% at 10 µM; adult worm IC50=2.01 µM) where the
nitrogen atoms occur in a 2,5 relationship.
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The next SAR analysis concerns a series of analogues comprising the aromatic side group attached
to the PBI core via a methylamine bridge (17 - 45, 48 and 49, Table 1). In the absence of
modifications elsewhere in the scaffold, these compounds displayed poor antischistosomal
activity. Most of these compounds were weakly active against NTS at the screening concentration
of 10 µM rendering them ineligible for additional screening in adult worms according to the
workflow adopted in the assay. For example, the potent activity observed with 6 (IC50 = 0.21 µM)
is lost in its methylene-linked analogue 23. A similar pattern of inactivity is noticed with 28, the
analogue of the active aniline-type series compound 16 (IC50 = 2.01 µM).
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Notably, when the methylene linker in 21 (0% NTS and adult; 10 µM) was substituted by an amino
group to produce 46, better activity in both the immature and adult worms was observed. Other
side groups such as the more polar hydrazide found in 47 were detrimental to activity (9% NTS,
10 µM). Subsequent SAR iterations involved introducing changes on the LHS of the PBI core
through installation of chloro- substitutions. Interestingly, for target compounds in the
benzylamine series which were previously inactive, this modification seemed to restore their
activity. For example, 19 has poor activity with only 4% reduction against the larval stage at 10
µM. In contrast, the analogues 37 and 38 (Table 1) with a dichloro substitution pattern on the
phenyl portion of the PBI scaffold have potent activity at 10 µM against both juvenile and adult
worms (98-100% mortality) with promising adult worm IC50 values of 0.77 µM and 1.69 µM,
respectively. Consistent observations are seen with 31 vs 41; 23 vs 39 and 18 vs 40 in which
enhanced mortality is observed with the di-chlorinated analogues, in both NTS and adult worms
at 10 µM.
With regards to regio-isomerism in the benzylamine side group, 38 (IC50 = 1.69 µM), was four
times less potent compared to its ortho- [44 (IC50 = 0.38 µM)] and meta- [45 (IC50 = 0.40 µM)]
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analogues. On the other hand, incorporating a methyl substitution at the benzylic position of 48
(23% NTS and 66% adult) resulted in the improvement of potency of the analogous compound 49
(100%, NTS and adult) at 10 µM. It may be postulated that the methyl group offers additional
binding interactions or influences compound orientation for interactions with biological targets in
the parasite.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
When the methylene linker in the side group R was replaced with a sulfonyl group, there were
mixed observations since activity was lost with 52 (9% NTS, 10 µM), an analogue of 40 (100%
NTS, 99% adult at 10 µM and adult worm IC50 = 1.74 µM) whereas similar changes conserved the
activity of 51 (93% NTS, 87% adult at 10 µM and adult IC50 = 1.09 µM), the analogue of 39 (100%
NTS and adult; 10 µM; IC50 = 1.58 µM).
Towards interrogating the relevance of the nitrile substitution on the pyridyl portion of the PBI
scaffold, some compounds were synthesised wherein this group was replaced by other small
groups. Of note, this modification led to a decrease in antischistosomal activity as seen with 7 and
8, the analogues of active compound 6 which contain a hydrogen and a methyl ester, respectively,
instead of a nitrile. Other compounds (53 - 55) in which the nitrile was replaced with an amide did
not produce potent activity (Table 1). Taken together, these results suggest that the nitrile at this
position is important for antischistosomal activity.
In vitro hepatic microsomal metabolic stability and cytotoxicity evaluation. To gain insight
into the anticipated pharmacokinetic behaviour, selective toxicity and to prioritise compounds for
in vivo efficacy studies, in vitro metabolic stability and cytotoxicity experiments were conducted
on prioritised compounds based on in vitro potency. Metabolic stability was carried out using
mouse and human liver microsomes whereas cytotoxicity was evaluated using the Chinese
Hamster Ovarian cell lines. The percentage of compound remaining after 30 minutes incubation
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was determined and the projected half-life calculated as previously described.14 The data from
5
6
these assays is presented in Table 2.
7
8
9
Table 2. Physicochemical, in vitro microsomal metabolic stability and cytotoxicity profiles.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
% rem. after 30
aCHO
IC50
(µM)
a
Chemical
Structure
min (t
)
Compound
Code
½ min
Log D
Solubility
bSelectivity Index
HL
M
MLM
OCF3
>99
>99
HN
N
6
5.34
<5
17.58
84
17
N
CF3
(>150)
(>150)
CN
O
O
N
28
69
HN
N
14
3.91
6.4
23.99
N
(17)
(57)
N
CF3
CN
Cl
HN
76
(75)
82
(105)
Cl
N
F
37
42
43
44
45
4.29
3.77
4.13
4.16
4.11
<5
<5
<5
<5
5
1.66
4.84
8.48
4.30
7.03
2
N
CF3
CN
Cl
O
HN
66
(50)
100
(>150)
Cl
N
10
20
11
18
N
CF3
CN
Cl
S
HN
Cl
N
N
CF3
CN
F
Cl
HN
84
(119)
97
(>150)
Cl
N
N
CF3
CN
Cl
F
HN
84
(119)
100
(>150)
Cl
N
N
CF3
CN
Midazolam
MMV390048
Emetine
1.3
94
0.1
93
0.095
LogD: logarithm of distribution co-efficient, computed using StarDrop™ version 4.0. Solubility was determined as previously described15.
MLM: Mouse liver microsomes; HLM: Human liver microsomes; t : projected half-life in minutes; CHO: Chinese hamster ovarian cell
½
lines. aValue is a mean of n≥2 independent determinations; bSelectivity Index being the ration of IC50 CHO: IC50 adult S. mansoni.
Overall, the most stable compounds were 6, 44 and 45 which contain lipophilic electron-
withdrawing substituents. For these compounds, over 80% of the compound was remaining after
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incubating for 30 minutes with an estimated half-life of over 100 minutes in both MLM and HLM.
Introducing heteroatoms in the side chain decreased metabolic stability as evident with 14. Species
variation in the metabolic stability of this series was evident. Metabolism was more efficient in
MLM for 14, 42, 44 and 45 whereas compound 6 displayed equal stability in both species.
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37
38
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41
42
43
44
45
46
47
48
49
50
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52
53
54
55
56
57
58
59
60
Most of the compounds had a good cytotoxicity profile (SI>10) with only 37, with a selectivity
index of 2, showing high potential for causing cytotoxicity. Evidently, although the introduction
of chloro substitutions on the LHS of the molecules improved their antischistosomal potency, this
change also increased their risk for inducing cytotoxicity as reflected in their reduced selectivity
indices of most compounds bearing the LHS modifications (Table 2).
In vivo oral efficacy. Compounds were selected for in vivo proof-of-concept studies, using the
oral route of administration, in a mouse model of schistosomiasis based on their in vitro potency
and acceptable cytotoxicity profile. In vivo potency was measured based on the percent worm
burden reduction achieved in infected and treated NMRI mice and comparing with infected but
untreated mice which served as controls. The results are shown in Table 3.
Table 3. In vivo antischistosomal oral efficacy for prioritised compounds.a
Compound
tested
Number of mice
Mean number Per cent worm
cured/investigated
of worms
(SD)
burden
reduction
Control 1
6
0/8
41.0 (16.2)
15.5 (0.7)
0/2*
62.2
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14
0/4
0/4
0/8
0/4
0/5
0/4
0/5
52.3 (45.2)
0
16
25.3 (3.3)
47.0 (36.2)
32.0 (19.8)
27.6 (11.1)
14.5 (2.5)
15.4 (3.8)
38.8
--
Control 2
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12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
42
31.9
41.3
69.1
67.3
94.116
43
44
45
Praziquantel
aCompounds were dissolved in 7% Tween 80 and 3% ethanol in water (v/v/v) prior to
dosing. SD: standard deviation *One mouse was not infected and excluded from
analysis, and one mouse died prematurely.
Of the compounds tested, 14 failed to induce any in vivo effect, 16, 42 and 43 produced slight (30-
42%) reduction in worm burden while moderate (>50%) reduction (yet statistically not significant
p > 0.05) in worms was realised with 6, 44 and 45. It is notable that compounds bearing
heteroaromatic side chains (14, 16, 42 and 43) generally produced lower in vivo efficacy.
Expectedly, there was a trend towards poor activity for compounds that had shown low metabolic
stability and higher activity for those with better metabolic stability. Biotransformation of these
compounds to metabolites with decreased or no antischistosomal activity is likely. Furthermore, it
is also probable that physicochemical hurdles are responsible, at least in part, for the low or lack
of in vivo activity of some compounds such as 14, is likely poorly absorbed, a hypothesis supported
by a negative human intestinal absorption (HIA) index score predicted by StarDrop™17,18
software; all other tested compounds were predicted to have a positive HIA score (Table S1,
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Supporting Information). Moreover, suboptimal bioavailability may be the reason for the lower
efficacy of otherwise in vitro potent compounds with outstanding metabolic stability, such as 6,
44 and 45. Pharmacokinetic profiling was undertaken using compound 44 to understand its in vivo
disposition.
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16
17
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19
20
21
22
23
24
25
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29
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37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In vivo pharmacokinetic studies. The oral disposition of 44 was studied in mice to enable
investigation of its PK profile. In this regard, C57B1/6 mice (n=3) were dosed orally at 20 mg/kg
while in another set, the animals (n=2 mice) were dosed intra-venously at 2 mg/kg. Blood was
sampled between 0-24 hours at the intervals shown in Figure 3. Corresponding PK parameters
were calculated using non-compartmental analysis and are presented in Table 4.
15
0.5
a. 44 iv
b. 44 oral
12
9
In vitro IC50= 0.38 µM
0.4
0.3
0.2
0.1
6
3
0
0
4
8
12
16
20
24
0
4
8
12
Time (h)
16
20
24
Time (h)
Figure 3. Pharmacokinetics profile for 44 after a) intravenous and b) oral administration. Each data point
represents the mean from 2 (iv) and 3 (oral) mice with error bars indicating the standard deviation.
Table 4. Pharmacokinetic parameters for 44 in mice.
F
Cl
HN
Cl
N
N
CF3
CN
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Parameter
Cmax (µM)
i.v. (2mg/kg) a
p.o. (20mg/kg) b
-
0.3
3
Tmax (h)
-
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
t1/2 (h)
2.6
0.18
0.81
5426
0.3
-
Vd (L/kg)
CL (mL/min/kg)
AUC0-∞ (µM/L.min)
-
138
Oral bioavailability
(%)
-
<1%
aFor intravenous dosing (n=2 mice), compounds were formulated in a solution
of dimethylacetamide, polyethylene glycol and propylene glycol/ethanol
mixture 4:1 at a ratio 1:3:6. For oral dosing (n=3 mice), compounds were
b
formulated as suspension in 100% HPMC.
Compound 44 attained peak plasma concentrations (Cmax = 0.3 µM) 3h post oral administration,
displayed a short half-life (t = 0.3h), slow clearance (CL = 0.81 mL/min/kg) and low volume of
½
distribution (Vd = 0.18 L/kg) with low bioavailability (F < 1%). Solubility-limited absorption, as
further suggested by the delayed peaking of plasma compound concentrations, is a potential
phenomenon contributing to the suboptimal efficacy of these compounds. It is feasible that at the
400 mg/kg oral dose used in efficacy studies, saturation of absorption and compound precipitation
occurs thereby limiting systemic exposure. Of note, the 400 mg/kg dose is the efficacious dose for
praziquantel16 and therefore routinely used in the S. mansoni mouse model, however it might be
worthwhile to test lower doses of this compound series. On the other hand, improved
bioavailability through modulation of solubility has the potential to enhance the in vivo efficacy of
this series.
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Conclusion: Antischistosomal structure-activity relationship studies have been conducted around
5
6
a 3-trifluoromethyl PBI core using N-aryl appendages with SAR trends as summarised in Figure
7
8
9
4.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OCF3
N
X
H
0.21
Weak activity observed at 10 M
R2=H; R3=CN
R2=H; R3=CN
O
C
NMe2
O
C
NHMe
NH
NH
NH
NH
N
X
Me
COOMe
OPr
NMe2
OCF3
OCF3
H
F
HN
S
Aniline series
HN
NH
1.17
1.58
1.69
SO2Me
0.97
SO2NH2
F
OH
OMe
O
SO2Me
N
H
0.43
O
NH
O
O

0.38
F

F
S
X
HN
O
C
NMe2
N
1.74
NH
HN
NMe2
O
C
CN
Benzylamine series
NHMe
1.11
0.47
O
0.40
O
O
Me
N
H
N
O
S
NH
NH
N
N
NH
HN
HN
N
HN
N
N
HN
N
N
OCF3
N
1.09
2.01
1.38
R2=H; R3=CN
O
NH
N
NH
N
N
R2 7,8 Cl; R =CN
=
N
N
N
NH
3
H
H
Cl
N
Active
Heterocyclic and heteroaromatic appendages
Inactive
R1

N
H
R2
N
CF3
Ehanced potency, metabolic
stability but increased cytotoxicity
Cl
>
H
R3
;
CN 
COOCH3; CONH2
Nitrile group is optimal for activity
Figure 4: Observed antischistosomal SAR trends. Values are adult worm IC50 in µM.
Generally, electron-withdrawing groups as substituents on the aromatic appendage favoured
antiparasitic activity. The aniline series generally provided more active compounds in the absence
of changes on the LHS, compared to the benzylamine series. It was notable that changes on the
LHS of the PBI scaffold restored the activity of previously inactive benzylamine series analogues.
The changes explored on the LHS, however, diminished the selectivity of these compounds while
changing the nitrile on the pyridyl portion of the PBI core was detrimental to antischistosomal
activity. Evaluation of the in vitro microsomal metabolic stability indicated that heteroatoms in the
structures of target compounds rendered them more susceptible to metabolism. In vivo efficacy
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studies conducted on selected compounds produced good antischistosomal activities with 6, 44
and 45 which effected 62-69% worm burden reduction. Pharmacokinetics hurdles, especially
solubility-limited absorption was identified as likely a contributing factor for the sub-optimal
potency achieved by the lead compounds as was microsomal metabolic instability. Overall, this
work shows that, when duly optimized, N-aryl 3-trifluoromethyl PBI analogues are promising
leads for antischistosomal drug discovery.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental procedures. All commercially available chemicals were purchased from either
Sigma-Aldrich or Combi-Blocks. All solvents were dried by appropriate techniques. Unless
otherwise stated, all solvents used were anhydrous. 1H NMR spectra were recorded at 300 or 400
MHz and 13C NMR spectra at 100 or 151 MHz, on a Brucker Spectrometer. Analytical thin-layer
chromatography (TLC) was performed on aluminium-backed silica-gel 60 F254 (70-230 mesh)
plates. Column chromatography was performed with Merck silica-gel 60 (70-230 mesh). Chemical
shifts (δ) are given in ppm downfield from TMS as the internal standard. Coupling constants, J,
are recorded in Hertz (Hz). Purity was determined by HPLC and all compounds were confirmed
to have > 95% purity. The data that is not shown below is supplied in the Supporting Information
(Section A).
General procedures for the synthesis of compounds 1-4, 6, 7, 9 - 13, 17 - 25, 27 - 35, 37 – 45,
48, 49, 53 - 55: Method A. The appropriate amine (1.2 to 2 equiv.) was added to a stirred mixture
of the relevant chloro intermediate IV (1.015mmol, 1 equiv.), triethylamine (2.03mmol, 2 equiv.)
and tetrahydrofuran (THF) (4mL) and subjected to microwave irradiation at 150W, 80°C for 20-
40 minutes. The cooled reaction mixture was transferred to a round bottom flask and concentrated.
A minimum amount of either acetone or ethanol was added to precipitate the final product which
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was filtered off and dried. Occasionally, recrystallization in ethanol or column chromatography
was performed to improve purity.
7
8
9
1-((4-(trifluoromethoxy)phenyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-a]pyridine-4-
carbonitrile 6. Obtained from IVa (0.3g, 1.015mmol, 1 equiv.) using 4- (trifluoromethoxy)aniline
(0.36g, 2.03mmol, 2 equiv.). Yield: 0.071 g, 16%, as a light green fluffy powder. 1H NMR (400
MHz, DMSO) δ 8.82 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.64 – 7.54 (m, 1H), 7.46 –
7.42 (m, 1H), 7.39 (d, J = 8.3 Hz, 2H), 7.20 (d, J = 8.4 Hz, 2H), 6.24 (s, 1H). 13C NMR (151 MHz,
DMSO) δ 148.53, 144.88, 135.23, 133.74, 128.91, 127.21, 125.36, 123.71, 122.92, 122.71 (2C),
121.81, 121.58, 120.05, 119.68, 117.77 (2C), 115.20, 113.38, 95.93. LC-MS APCI+ m/z calcd for
C20H10F6N4O:436.08; found, 437.10 [M+H]+. ). HPLC purity: 99%
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1-((3-(trifluoromethoxy)phenyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-a]pyridine-4-
carbonitrile 9. Obtained from IVa (0.1g, 0.338 mmol, 1 equiv.) using 3-(trifluoromethoxy) aniline
1
(0.072g, 0.406 mmol, 1.2 equiv.). Yield: 0.072g, 10%, as a yellow solid. H NMR (300 MHz,
DMSO-d6) δ 8.81 (d, J = 8.3 Hz, 1H), 7.58 (m, 3H), 7.42 (t, J = 7.8 Hz, 1H), 7.19 – 6.96 (m, 3H),
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6.23 (s, 1H). C NMR (101 MHz, DMSO) δ 149.82, 148.61, 148.38, 135.72, 133.54, 131.74,
128.58, 127.31, 124.01, 123.11, 121.86, 121.27 (2C), 119.32, 117.88, 115.96, 115.17, 114.61,
112.64, 96.61. MS (EI+) m/z calcd for C20H10F6N4O:436.08; found, 437.10 (M + 1). HPLC purity:
98%.
1-((2-(trifluoromethoxy)phenyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-a]pyridine-4-
carbonitrile 10. Obtained from IVa (0.1g, 0.338 mmol, 1 equiv.) using 2-(trifluoromethoxy)
aniline (0.072g, 0.406 mmol, 1.2 equiv.). Yield: 0.043 g, 29%, as a yellow solid.1H NMR (300
MHz, DMSO-d6) δ 8.87 (d, J = 8.3 Hz, 1H), 7.75 – 7.52 (m, 2H), 7.52 – 7.34 (m, 3H), 7.22 (m,
2H), 6.10 (s, 1H). 13C NMR (101 MHz, DMSO) δ 148.65, 141.74, 140.85, 135.03, 134.72, 133.29,
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129.07, 128.59, 127.43, 124.84, 123.90, 123.24, 122.01, 121.27, 119.47, 117.73, 115.18, 112.16,
100.00, 97.15. MS (EI+) m/z calcd for C20H10F6N4O:436.08; found, 437.10 (M + 1). HPLC purity:
>99%.
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7,9-dichloro-1-((4-fluorobenzyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-a]pyridine-4-
carbonitrile 37. Obtained from IVb (0.1g, 0.274mmol,
1
equiv.) and (4-
fluorophenyl)methanamine (0.041g, 0.329mmol, 1.2 equiv.). Yield: 0.107 g, 86%, as a yellow
solid. 1H NMR (300 MHz, DMSO-d6) δ 8.82 (d, J = 4.1 Hz, 1H), 8.70 (s, 1H), 7.81 (d, J = 6.8 Hz,
1H), 7.56 (dd, J = 8.5, 5.5 Hz, 2H), 7.20 (t, J = 8.9 Hz, 2H), 6.32 (s, 1H), 4.89 (s, 2H). 13C NMR
(101 MHz, DMSO) δ 163.13, 160.71, 150.15 (2C), 141.56, 137.93, 133.95, 129.83, 129.50 (2C),
126.22, 125.36, 123.06, 121.27, 115.87 (2C), 114.78, 112.78, 88.01, 46.85. MS (EI+) m/z calcd
for C20H10Cl2F4N4:452.02; found, 453.00 (M + 1). HPLC purity: 99%.
7,8-dichloro-1-((4-fluorobenzyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-a]pyridine-4-
carbonitrile 38. Obtained from IVc (0.163g, 0.448mmol,
1
equiv.) and (4-
fluorophenyl)methanamine (0.0618g, 0.493mmol, 1.2 equiv.). Yield: 0.045 g, 22%, as a yellow
solid. 1H NMR (300 MHz, DMSO-d6) δ 9.03 (s, 1H), 7.69 (s, 1H), 7.52 (dd, J = 8.6, 5.6 Hz, 2H),
7.43 (dd, J = 8.4, 5.6 Hz, 2H), 6.00 (s, 1H), 4.59 (s, 2H). 13C NMR (101 MHz, DMSO) δ 162.97,
160.18, 154.24, 150.99, 145.67, 139.02, 135.82, 134.57, 130.64, 129.58, 125.88, 119.90, 117.50,
116.55, 115.63, 115.57, 115.18, 100.00, 88.62, 43.67. MS (EI+) m/z calcd for
C20H10Cl2F4N4:452.02; found, 453.00 (M + 1). HPLC purity: 97%.
7,8-dichloro-1-((4-(trifluoromethoxy)benzyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-
a]pyridine-4-carbonitrile 39. Obtained from IVc (0.15g, 0.411mmol, 1 equiv.) and (4-
(trifluoromethoxy)phenyl)methanamine (0.094g, 0.493mmol, 1.2 equiv.). Yield: 0.053 g, 25%, as
a brown solid. 1H NMR (300 MHz, DMSO-d6) δ 9.04 (s, 1H), 7.70 (s, 1H), 7.59 (dd, J = 14.0, 8.6
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Hz, 2H), 7.38 (t, J = 8.8 Hz, 2H), 6.01 (s, 1H), 4.64 (s, 2H). 13C NMR (101 MHz, DMSO) δ 151.42,
148.67, 148.48, 137.28, 134.58, 132.48, 130.43, 129.51, 128.51, 127.00 (2C), 124.13, 121.48 (2C),
119.22, 116.56, 115.75, 114.49, 112.94, 86.73, 51.67. MS (EI+) m/z calcd for
C21H10Cl2F6N4O:518.01; found, 519.00 (M + 1). HPLC purity: 99%.
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7,8-dichloro-1-((pyridin-2-ylmethyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-
a]pyridine-4- carbonitrile 40. Obtained from IVc (0.15g, 0.411mmol, 1 equiv.) and (pyridin-2-
ylmethanamine (0.0534g, 0.493mmol, 1.2 equiv.). Yield: 0.070 g, 39%, as a brown solid. 1H NMR
(300 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.66 – 8.52 (m, 1H), 8.10 (s, 1H), 7.83 (td, J = 7.6, 1.8 Hz,
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1H), 7.58 (d, J = 7.9 Hz), 7.42 – 7.26 (m, 1H), 6.39 (s, 1H), 4.94 (s, 2H). C NMR (101 MHz,
DMSO) δ 150.22, 149.36 (2C), 145.21, 137.52 (2C), 129.15, 128.31, 127.64, 123.18, 122.26 (2C),
119.39, 118.56, 117.09, 116.96, 115.08, 88.03, 49.95. MS (EI+) m/z calcd for
C19H10Cl2F3N5:435.03; found, 436.00 (M + 1). HPLC purity: 97%.
7,8-dichloro-1-((4-methylbenzyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-a]pyridine-4-
carbonitrile 41. Obtained from IVc (0.15g, 0.411mmol, 1 equiv.) and p-tolylmethanamine
(0.058g, 0.494mmol, 1.2 equiv.). Yield: 0.092 g, 50%, as a brown powder. 1H NMR (300 MHz,
DMSO-d6) δ 9.06 (s, 1H), 7.74 (s, 1H), 7.35 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 7.9 Hz, 2H), 6.03 (s,
1H), 4.59 (s, 2H), 2.31 (s, 3H). 13C NMR (101 MHz, DMSO) δ 160.73, 159.00, 155.06, 146.31,
135.62, 134.93, 129.58, 129.21 (2C), 127.62 (2C), 126.38, 121.69, 119.39, 117.52, 116.80, 115.61,
114.83, 88.39, 42.88, 21.08. MS (EI+) m/z calcd for C21H13Cl2F3N4:448.05; found, 449.00 (M +
1). HPLC purity: 99%.
7,8-dichloro-1-((furan-2-ylmethyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-a]pyridine-
4- carbonitrile 42. Obtained from IVc (0.15g, 0.411mmol, 1 equiv.) and furan-2- ylmethanamine
1
(0.048g, 0.494mmol, 1.2 equiv.). Yield: 0.098 g, 56%, as a brown solid. H NMR (300 MHz,
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DMSO-d6) δ 9.00 (s, 1H), 7.90 (s, 1H), 7.63 (dd, J = 1.8, 0.9 Hz, 1H), 6.43 (m, 2H), 6.27 (s, 1H),
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13
4.72 (s, 2H). C NMR (101 MHz, DMSO) δ 155.27, 153.52, 150.99, 145.30, 142.96, 142.30,
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134.97, 130.77, 128.78, 123.76, 117.67, 117.45, 114.20, 110.89, 107.15, 94.33, 93.02, 43.95. MS
(EI+) m/z calcd for C18H9Cl2F3N4:424.01; found, 425.00 (M + 1). HPLC purity: 99%.
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7,8-dichloro-1-((thiophen-2-ylmethyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-
a]pyridine- 4-carbonitrile 43. Obtained from IVc (0.15g, 0.411mmol, 1 equiv.) and thiophen-2-
ylmethanamine (0.056g, 0.494mmol, 1.2 equiv.). Yield: 0.051 g, 28%, as a brown solid. 1H NMR
(300 MHz, DMSO-d6) δ 9.16 (s, 1H), 7.77 (s, 1H), 7.42 (dd, J = 5.0, 1.3 Hz, 1H), 7.11 – 7.05 (m,
13
1H), 7.02 (dd, J = 5.0, 3.4 Hz, 1H), 6.05 (s, 1H), 4.80 (s, 2H). C NMR (101 MHz, DMSO) δ
150.91, 145.53, 138.51, 130.23, 129.21, 128.19, 127.02 (2C), 124.34 (2C), 123.52, 123.23, 117.75
(2C), 116.95 (2C), 88.34, 47.38. MS (EI+) m/z calcd for C18H9Cl2F3N4S:439.99; found, 441.00
(M + 1). HPLC purity: 98%.
7,8-dichloro-1-((2-fluorobenzyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-a]pyridine-4-
carbonitrile 44. Obtained from IVc (0.1g, 0.274mmol,
1
equiv.) and (2-
fluorophenyl)methanamine (0.069g, 0.548mmol, 2 equiv.). Yield: 0.072 g, 58%, as an orange
solid. 1H NMR (300 MHz, DMSO-d6) δ 9.00 (s, 1H), 7.94 (s, 1H), 7.59 (t, J = 7.5 Hz), 7.40 – 7.12
(m, 3H), 6.22 (s, 1H), 4.79 (s, 2H). 13C NMR (101 MHz, DMSO) δ 161.91, 159.49, 152.40, 150.75,
145.34, 136.58, 132.84, 131.42, 129.92, 129.22, 126.94, 127.87, 124.84, 121.78, 118.14, 117.37
(2C), 116.51, 115.46, 87.88. MS (EI+) m/z calcd for C20H10Cl2F4N4:452.02; found, 453.00 (M +
1). HPLC purity: 98%.
7,8-dichloro-1-((3-fluorobenzyl)amino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-a]pyridine-4-
carbonitrile 45. Obtained from IVc (0.1g, 0.274mmol,
1
equiv.) and (3-
fluorophenyl)methanamine (0.069g, 0.548mmol, 2 equiv.). Yield: 0.046 g, 37%, as a yellow solid.
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1H NMR (300 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.69 (bs, 1H), 8.20 (s, 1H), 7.51 – 7.29 (m, 3H),
7.18 – 7.06 (m, 1H), 6.32 (s, 1H), 4.96 (s, 2H). 13C NMR (101 MHz, DMSO) δ 161.91, 159.49,
152.40, 150.75, 145.34, 136.58, 131.42, 129.96, 129.22, 127.87, 125.10, 124.70, 121.78, 118.14,
117.37(2C), 116.51, 115.65, 115.46, 87.83. MS (EI+) m/z calcd for C20H10Cl2F4N4:452.02; found,
453.00 (M + 1). HPLC purity: 97%.
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General procedures for the synthesis of compounds 5, 8, 14 - 16, 26, 36, 46, 47, 50 - 52: Method
B. A mixture of the relevant chloro intermediate IV, the appropriate amine (1.2 equiv.),
Tris(dibenzylideneacetone) dipalladium(0), Pd2(dba)3 (0.1 equiv.), 2,2'-Bis(diphenylphosphino)-
1,1'-binaphthyl (BINAP) (0.06 equiv.) or BrettPhos (0.04 equiv.) or RuPhos (0.1 equiv.),
Potassium carbonate, K2CO3 or Caesium Carbonate, Cs2CO3 (3 equiv.) and toluene:tert-butanol
(1:1; 5ml) or 1,4-dioxane (5mL) were stirred in a sealed tube at 100-120˚C for 4-17 hours. The
cooled reaction mixture was stirred in ethylacetate (50ml) and water (100ml) for 10-20 minutes.
The separated organic layer was washed with water (2X50ml), followed by saturated NaCl
(2X50ml), dried over magnesium sulphate and filtered over celite. The organic fraction was
concentrated under reduced pressure and ethanol used to precipitate the crude product which was
filtered to furnish the final product. In some cases, compounds were purified by recrystallization
from ethanol or by column chromatography.
1-((5,6-dimethoxypyrimidin-4-yl)amino)-3-(trifluoromethyl) benzo[4,5] imidazo[1,2-a]pyridine-
4- carbonitrile 14. Obtained from IVa (0.3g, 1.015mmol, 1 equiv.) using 5,6-
dimethoxypyrimidin-4-amine (0.189g, 1.218mmol, 1.2 equiv.). Yield: 0.071 g, 17%, as a yellow
fluffy powder.1H NMR (300 MHz, DMSO) δ 9.08 (d, J = 10.1 Hz, 1H), 8.36 (s, 1H), 7.69 (d, J =
8.7 Hz, 1H), 7.67 – 7.59 (m, 1H), 7.52 – 7.43 (m, 1H), 7.40 (s, 1H), 4.00 (s, 3H), 3.80 (s, 3H). 13C
NMR (101 MHz, DMSO) δ 163.29, 162.70, 151.38, 147.54, 141.49, 135.42, 135.09, 128.73,
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127.71(2C), 124.15, 123.12 (2C), 121.42, 118.52, 114.91, 100.05, 60.64, 54.00. LC-MS APCI+:
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6
found m/z = 415.10 [M+H]+, (calculated for: C19H13F3N6O2:414.11). HPLC purity: 98%
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1-(pyrazin-2-ylamino)-3-(trifluoromethyl)benzo[4,5]imidazo[1,2-a]pyridine-4-carbonitrile 16.
Obtained from IVa (0.15g, 0.51mmol, 1 equiv.) using pyrazin-2-amine (0.058g, 0.61mmol, 1.2
equiv.). Yield: 0.041 g, 23%, as an orange powder.1H NMR (400 MHz, DMSO) δ 9.08 (d, J = 8.4
Hz, 1H), 8.54 (d, J = 1.4 Hz, 1H), 8.41 (dd, J = 2.7, 1.4 Hz, 1H), 8.25 (d, J = 2.7 Hz, 1H), 7.69
(ddd, J = 8.1, 1.2, 0.7 Hz, 1H), 7.63 (ddd, J = 8.2, 7.3, 1.1 Hz, 1H), 7.57 (s, 1H), 7.47 (ddd, J =
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8.5, 7.3, 1.3 Hz, 1H). C NMR (101 MHz, DMSO) δ 161.20, 156.43, 149.86, 147.99, 147.30,
142.50 (2C), 138.59, 135.58, 128.83, 127.54, 123.16, 118.91, 118.58, 114.80, 112.86, 99.37. LC-
MS APCI+: found m/z = 355.10 [M+H]+, (calculated for: C17H9F3N6:354.08). HPLC purity:98%
MATERIALS AND METHODS
In vitro antischistosomal assays. Screening on Newly Transformed Schistosomula.
S. mansoni cercariae were mechanically trans formed to newly transformed schistosomula as
described elsewhere.19 Briefly, snails were placed under light to stimulate cercarial shedding, and
cercarial suspension was collected. The tails were separated from the heads by rinsing three times
with cold HBSS. NTS were then incubated overnight in culture medium and used the next day.
Test compounds and controls were dissolved in DMSO (Fluka, Buchs, Switzerland) to a
concentration of 10 mM. One hundred NTS were then incubated in each well of a 96-well plate
with culture medium and the test compound for a final well volume of 250 μL. Culture medium
was composed of Medium 199 (Invitrogen, Carlsbad, CA) supplemented with 5% fetal calf serum
(Lucerne, Switzerland) and 1% penicillin/ streptomycin mixture (Lucerne, Switzerland).
Compounds were tested at 10 μM in triplicate with NTS incubated in no more than 1% DMSO
serving as control. NTS were kept in an incubator at 37 °C and 5% CO2 for up to 72 h, after which
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the condition of the NTS was microscopically evaluated using a scale from 3 (normal activity and
morphological alteration) to 0 (dead).
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Adult S. mansoni Worms. To obtain adult schistosomes, mice were infected subcutaneously with
80−100 cercariae. The mice were then euthanized after 7−8 weeks with CO2 and the worms
collected from the hepatic portal and mesenteric veins. Two pairs of adult worms were incubated
in each well of a 24- well plate with 2 mL culture medium and the test compound. Culture medium
was composed of RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 5% fetal calf serum
(Lucerne, Switzerland) and 1% penicillin/streptomycin mixture (Lucerne, Switzerland).
Compounds with >70% activity against NTS were evaluated against adult worms at 10 μM with
incubation at <1% DMSO used as control. Worms were kept in an incubator at 37 °C and 5% CO2
for up to 72 h, after which their condition was microscopically evaluated using a scale from 3
(normal activity and no morphological alterations) to 0 (dead). IC50 determination was conducted
for compounds with >60% activity. For the in vitro drug sensitivity assays, all viability scores were
averaged across replicates and normalized to the average viability scores of the control wells. IC50
values were calculated using CompuSyn2 (ComboSyn Inc., Paramus, NJ).
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Cytotoxicity Testing. Compounds were screened for in vitro cytotoxicity against CHO
mammalian cell lines, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. The reference standard, emetine, was prepared to 2 mg/mL in distilled water while
stock solutions of test compounds were prepared to 20 mg/mL in 100% DMSO with the highest
concentration of solvent to which the cells were exposed having no measurable effect on the cell
viability. The initial concentration of the drugs and control was 100 μg/mL, which was serially
diluted in complete medium with 10-fold dilutions to give six concentrations, the lowest being
0.001 μg/ mL. Plates were incubated for 48 h with 100 μL of drug and 100 μL of cell suspension
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