Discovery of inhibitors of Trypanosoma brucei by phenotypic screening of a focused protein kinase library

Article abstract of DOI:10.1002/cmdc.201500300

A screen of a focused kinase inhibitor library against Trypanosoma brucei rhodesiense led to the identification of seven series, totaling 121 compounds, which showed >50 % inhibition at 5 μm. Screening of these hits in a T. b. brucei proliferation assay highlighted three compounds with a 1H-imidazo[4,5-b]pyrazin-2(3H)-one scaffold that showed sub-micromolar activity and excellent selectivity against the MRC5 cell line. Subsequent rounds of optimisation led to the identification of compounds that exhibited good in vitro drug metabolism and pharmacokinetics (DMPK) properties, although in general this series suffered from poor solubility. A scaffold-hopping exercise led to the identification of a 1H-pyrazolo[3,4-b]pyridine scaffold, which retained potency. A number of examples were assessed in a T. b. brucei growth assay, which could differentiate static and cidal action. Compounds from the 1H-imidazo[4,5-b]pyrazin-2(3H)-one series were found to be either static or growth-slowing and not cidal. Compounds with the 1H-pyrazolo[3,4-b]pyridine scaffold were found to be cidal and showed an unusual biphasic nature in this assay, suggesting they act by at least two mechanisms. Focused on tipping the HAT: We report a phenotypic screen of a focused kinase library against Trypanosoma brucei and subsequent optimisation of a hit, with sub-micromolar activity, based on a 1H-imidazo[4,5-b]pyrazin-2(3H)-one scaffold. Scaffold hopping gave a second series based on a 1H-pyrazolo[3,4-b]pyridine scaffold, also with sub-micromolar activity. The first series of compounds were static or growth-slowing and not cidal, whilst those from the second series were cidal, but showed an unusual biphasic growth curve, suggestive of several mechanisms of action.

Full text of DOI:10.1002/cmdc.201500300

DOI: 10.1002/cmdc.201500300
Full Papers
Discovery of Inhibitors of Trypanosoma brucei by
Phenotypic Screening of a Focused Protein Kinase Library
Andrew Woodland,[a] Stephen Thompson,[a] Laura A. T. Cleghorn,[a] Neil Norcross,[a]
Manu De Rycker,[a] Raffaella Grimaldi,[a] Irene Hallyburton,[a] Bhavya Rao,[a] Suzanne Norval,[a]
Laste Stojanovski,[a] Reto Brun,[b] Marcel Kaiser,[b] Julie A. Frearson,[a] David W. Gray,[a]
Paul G. Wyatt,[a] Kevin D. Read,[a] and Ian H. Gilbert*[a]
A screen of a focused kinase inhibitor library against Trypano-
soma brucei rhodesiense led to the identification of seven
series, totaling 121 compounds, which showed >50% inhibi-
tion at 5 mm. Screening of these hits in a T. b. brucei prolifera-
tion assay highlighted three compounds with a 1H-imida-
zo[4,5-b]pyrazin-2(3H)-one scaffold that showed sub-micromo-
lar activity and excellent selectivity against the MRC5 cell line.
Subsequent rounds of optimisation led to the identification of
compounds that exhibited good in vitro drug metabolism and
pharmacokinetics (DMPK) properties, although in general this
series suffered from poor solubility. A scaffold-hopping exercise
led to the identification of a 1H-pyrazolo[3,4-b]pyridine scaf-
fold, which retained potency. A number of examples were as-
sessed in a T. b. brucei growth assay, which could differentiate
static and cidal action. Compounds from the 1H-imidazo[4,5-
b]pyrazin-2(3H)-one series were found to be either static or
growth-slowing and not cidal. Compounds with the 1H-pyrazo-
lo[3,4-b]pyridine scaffold were found to be cidal and showed
an unusual biphasic nature in this assay, suggesting they act
by at least two mechanisms.
Introduction
Human African trypanosomiasis (HAT), or sleeping sickness, as
it is more commonly known, is an often fatal infection endemic
in sub-Saharan Africa. Current treatments for HAT are inade-
quate due to toxicity and inappropriate means of administra-
tion for remote rural settings, where the disease is predomi-
nantly found. Fortunately, the number of cases of HAT appears
to be decreasing; however, there is an urgent need for new ef-
fective treatments for HAT, to improve treatment and to facili-
tate the eventual eradication of this disease. HAT is caused by
the protozoan parasites Trypanosoma brucei gambiense and
T. b. rhodesiense. The parasites have a complex life cycle, involv-
ing the human host and the tsetse fly, which is the vector. Ini-
tially there is a peripheral infection in which the parasite is pre-
dominantly found in the blood. However, the parasites eventu-
ally invade the central nervous system, which can lead to
coma and death.[1,2]
Protein kinases are a major drug target for human diseases,
particularly oncology.[3] Analysis of the T. brucei genome indi-
cates that there are 156 protein kinases in the trypanosome
kinome.[4–6] Whilst parasite protein kinases have been relatively
poorly investigated, there appear to be significant differences
from human kinases. In particular:
·
A relatively large number of protein kinases associated with
cell-cycle control, which may be a consequence of the com-
plex life cycle of the parasite;
·
·
A lack of tyrosine kinases (except dual-specificity kinases);
A number of unique kinases.
[a] Dr. A. Woodland,+ Dr. S. Thompson,+ Dr. L. A. T. Cleghorn, Dr. N. Norcross,
Dr. M. De Rycker, Dr. R. Grimaldi, I. Hallyburton, Dr. B. Rao, S. Norval,
L. Stojanovski, Prof. J. A. Frearson, Dr. D. W. Gray, Prof. P. G. Wyatt,
Dr. K. D. Read, Prof. I. H. Gilbert
In addition, a number of protein kinases have been geneti-
cally validated as potential drug targets in T. brucei using RNA
interference (RNAi) or gene knockouts.[4,5,7–9] Given that protein
kinases are generally known to be druggable, they represent
promising drug targets in trypanosomatids. The Drug Discov-
ery Unit (DDU) in Dundee has recently carried out screens and
chemical optimisation programmes against several parasite
protein kinases. Although we have prepared compounds with
significant (low-nanomolar) activity against the proteins, this
did not always translate into potent cellular activity.[10]
Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery
University of Dundee, Dundee, DD1 5EH (UK)
E-mail: i.h.gilbert@dundee.ac.uk
[b] Prof. R. Brun, Dr. M. Kaiser
Swiss Tropical and Public Health Institute
Socinstrasse 57, P.O. Box, 4002 Basel (Switzerland)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500300: synthesis details for all com-
pounds and additional biological data.
A complementary method to target-based drug discovery is
phenotypic drug discovery,[11] which has several advantages.
Firstly, all possible drug discovery targets are present in their
natural environment, allowing an unbiased and more physio-
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logically relevant screening platform; this may give rise to
compounds that inhibit more than one target. Indeed it has
been found in the oncology field that compounds that inhibit
more than one protein kinase are often required for activity.
Secondly, as the primary screening platform is a functional effi-
cacy screen, the relationship between target and phenotype
does not need to be established. Finally, compounds must be
able to penetrate cells and have a sufficient free fraction in the
assay to elicit their response, eliminating compounds with in-
appropriate properties.[12–14] We therefore decided to conduct
a phenotypic screen of a focused kinase compound library
against whole parasites. A similar exercise was recently report-
ed by Diaz et al., in which a phenotypic screen of a kinase-tar-
geted library from GlaxoSmithKline (GSK) was reported and
gave rise to a number of actives.[15] There is also a recent
report of a large screen against kinetoplastids with 1.8 million
compounds from GSK.[16] The ideal target product profile to
treat HAT requires a compound that can treat both stage 1
(peripheral) and stage 2 (CNS) infection;[8] thus the compound
should have blood–brain barrier (BBB) permeability.
The physicochemical properties were calculated in StarDrop
(www.optibrium.com). It has been proposed that for a com-
pound to have BBB permeability, it should have a topological
2
polar surface area (tPSA) of <90 Š and a molecular weight
(Mr) less than 450 Da.[18] Wager’s research group have proposed
more sophisticated models.[19–21] The Mr and tPSA values were
all within the preferred range for a potential CNS-penetrant
compound. The calculated logP values were also in the range
of CNS-penetrant compounds.[19] Based on the initial data we
decided to progress the project into hits-to-leads develop-
ment.
Hit validation
Medicinal chemistry was initiated with the aim of validating
and profiling the hit series and increasing the potency of the
compounds. The desired 1H-imidazopyrazinones were synthes-
ised in four steps from 2-aminopyrazine 4. Bromination with
NBS gave the versatile scaffold 2,5-dibromo-4-aminopyrazine 5.
Regioselective displacement of the 5-bromine group with
amines could be conducted using microwave heating to give
intermediates 6a–f (Scheme 1). Compounds 8–11, 13–15, 17,
Results and Discussion
The focused screen
The Dundee focused protein kinase library,[17] which at that
point contained 3885 compounds, was assayed by the Swiss
Tropical and Public Health Institute (STPH) against T. b. rhode-
siense at 1 and 5 mm. From this original triage, seven series, to-
taling 121 compounds, were identified which showed >50%
inhibition of parasite growth at 5 mm. These were progressed
into EC50 determination in a T. b. brucei proliferation assay and
assessed in a MRC5 proliferation assay to provide an early indi-
cator of toxicity to mammalian cells. From this, seven com-
pounds showed EC50 values <1 mm against T. b. brucei. The
most active hit compound was a 1H-imidazo[4,5-b]pyrazin-
2(3H)-one (1), which had an EC50 value of 80 nm against
T. b. brucei and was nontoxic to the mammalian MRC5 cell line
(EC50 >50 mm). Two additional 1H-imidazopyrazinone ana-
logues had EC50 values of <1 mm against T. b. brucei (Table 1).
Scheme 1. Reagents and conditions: a) NBS, MeCN, 18 h, RT, 12%; b) amine,
DIPEA, EtOH, 1808C (microwave), 30 min, 27–64%; c) cyclohexylamine,
DIPEA, nBuOH, 2208C (microwave), 30 min, 95%; d) CDI, 1,4-dioxane, 808C,
16 h, 31%; e) for 8–11, 13–15, 17, and 18: 1) Pd(PPh3)4 (3 mol%), K2CO3
(1.5m aqueous), boronic acid, DMF, 1408C, 5 min; 2) CDI, 1,4-dioxane, 1408C,
20 min, 3–34%; f) for 12 and 19–30: Pd(PPh3)4 (3 mol%), K2CO3 (1.5m aque-
ous), boronic acid, DMF, 1408C, 5 min, 7–67%; g) for 16: 7, H2, 5% Pd/C,
MeOH, 15%.
Table 1. Potency of hits 1–3.
and 18 were prepared by Suzuki reaction with the
appropriate diamine precursor followed by cyclisa-
tion with CDI. Cyclisation of the appropriate diamine
(6a or b) with CDI gave 7a or 7b, which were then
reacted under Suzuki conditions to give compounds
12 and 19–30. Treatment of 7a under hydrogenation
conditions afforded compound 16.
Enantiomers 1 and 8 were equipotent, whilst re-
moval of the benzylic methyl group (compound 9)
ID
clogP
tPSA [Š2]
Mr [Da]
EC50 [mm][a]
T. b. brucei
MRC5
resulted in a fivefold decrease in activity (Table 2). Re-
placement of the benzyl group with methylcyclopro-
pane (compound 10) had little effect on the activity,
whilst further truncation of the amine to a methyl
group (in 11) resulted in a 100-fold drop in activity
relative to methyl cyclopropane (in 10). Replacement
1
2
3
3.3
2.8
2.9
57
75
77
334
370
334
0.08
0.5
0.9
>50
15
ND
[a] Values are the geometric mean of two or more determinations; standard deviation
is typically within 2-fold of the EC50 value. ND: not determined.
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Table 2. Variation of the R1 group of compound 1.
Table 3. Variation of the R2 group of compound 1.
ID
1
R1
R2
clogP
3.6
EC50 [mm][a]
T. b. brucei MRC5
Aq. Sol.
ID
R1
R2
clogP
EC50 [mm][a]
T. b. brucei MRC5 [mm][b]
Aq. Sol.
[mm][b]
0.08
0.09
>50
>50
>50
<12
ND
1
3.6
3.5
4.0
2.0
3.6
2.5
0.08
0.1
1
>50
26
<12
<12
ND
14
15
16
17
18
8
3.6
39
9
3.3
0.5
0.3
ND
36
>50
44
ND
10
11
2.7
1.9
>50
>50
ND
ND
40
0.3
3
ND
12
13
3.9
2.0
0.03
0.59
>50
>50
55
46
ND
[a] Values are the geometric mean of two or more determinations; stan-
dard deviation is typically within 2-fold of the EC50 value. [b] Aqueous ki-
netic solubility. ND: not determined.
ND
[a] Values are the geometric mean of two or more determinations; stan-
dard deviation is typically within 2-fold of the EC50 value. [b] Aqueous ki-
netic solubility. ND: not determined.
pounds further for potential inhibition of human kinases and
to study their DMPK properties to ensure that there were no
major issues which may impact further development.
of the methylbenzyl group of 1 with a methoxypropyl group
(in 13) resulted in a sevenfold loss in activity. Interestingly, re-
placement of the methylbenzyl group of 1 with cyclohexane
(compound 12) resulted in a threefold improvement in activity.
Compounds showed good selectivity for the parasite over
MRC5 cells.
The DDU kinase-focused compound set contains lead-like
scaffolds that are designed to target protein kinases; they have
kinase hinge binding motifs. Four of the 1H-imidazopyrazi-
nones were screened against a human/mammalian kinase
panel at the University of Dundee (www.kinase-screen.mrc.a-
c.uk) at a concentration of 10 mm. At the time of testing the
panel contained 79 mammalian kinases selected to provide
a broad coverage of the mammalian kinome (~500 kinases in
the human genome).[3] None of the compounds were active
against any of the kinases at 10 mm. Although the results did
not provide any clues as to the molecular target in the para-
site, they did show the 1H-imidazopyrazinones to have promis-
ing selectivity over mammalian kinases.
The 4-fluorophenyl group of 1 could be replaced with
a phenyl ring (in 14) with no loss of activity (Table 3). The
larger 4-trifluoromethyloxy analogue 15 was 10-fold less active
than 1, but was comparable with the hit compound 2, further
demonstrating that changes to the R1 group were tolerated.
Replacement of the phenyl ring of 14 with a hydrogen atom
in 16 resulted in a >300-fold loss of activity relative to 14.
Substitution of the phenyl ring with a 3-fluoro group was toler-
ated in 17, while the larger and more polar 3-methanesulfona-
mide group (in 18) resulted in a 30-fold loss of activity.
Two key compounds, 1 and 12, showed low in vitro mouse
hepatic microsomal intrinsic clearance (CLint <0.5 and
0.7 mLminÀ1 gÀ1, respectively), consistent with good metabolic
stability. Compound 1, with promising in vitro anti-parasitic ac-
tivity, low toxicity to human cells, and promising in vitro meta-
bolic stability, was progressed into in vivo pharmacokinetic
(PK) determination. After a single oral dose at 5 mgkgÀ1 to
female NMRI mice, the compound showed a low Cmax value
and took approximately 2 h to reach Cmax, both of which are
suggestive of solubility-limited absorption (Table 4). Further-
more, 1 showed high protein binding (0.3% unbound). Conse-
Kinase panel and DMPK data
These data validated the series, as there was a definite struc-
ture activity relationship, and several examples demonstrated
sub-100 nm EC50 values in the T. b. brucei proliferation assay.
The compounds also showed excellent selectivity over human
MRC5 cells. Consequently, it was decided to profile the com-
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introduction of polar groups was poorly tolerated, with hy-
droxymethyl compound 21 being 10-fold less active, while
amide 22 and dimethylaminomethyl compound 23 were 30-
and 50-fold less active, respectively. Substituents at the para
position generally caused a drop in activity, relative to those in
the ortho or meta positions. Encouragingly, the hydroxymethyl
substituents retained activity in the ortho and meta positions
(28 and 24, respectively) and gave rise to good solubility.
Further work was undertaken in which the phenyl ring of 19
was replaced with a heterocycle or saturated ring system,
which should increase solubility. For the introduction of
amines we used a Buchwald reaction on intermediate 7b
(Scheme 2). For the introduction of aromatic heterocycles we
used the chemistry described above. Both 4-pyridyl 29 and 3-
pyridyl 30 compounds were equipotent to phenyl 19, demon-
strating a tolerance for a hydrogen bond acceptor at the 3-
and 4-positions (Table 6). Both piperidine 31 and the more
polar morpholine 32 were tolerated, but the basic piperazine
33 was 50-fold less active than the phenyl compound 19.
Table 4. PK data for compound 1.
Route
Dose
[mgkgÀ1
Cmax
[ngmLÀ1
tmax [h][b]
AUC(0–8)
t1/2 [h][d]
[a]
[c]
]
]
[ngminmLÀ1
]
p.o.
5
110
4
36000
3
[a] Maximum concentration reached. [b] Time after initial dose at which
Cmax was reached. [c] Area under the curve. [d] Half-life of compound in
the blood.
quently, the observed free blood concentration was likely to
be too low for the compound to be efficacious in vivo even at
high dose, so further development was required to improve
free oral exposure for 1. Common strategies to address the key
issues of poor solubility and/or high protein binding include
decreasing the lipophilicity (logP), addition of solubilising
groups, and reducing the planarity of the molecule to mini-
mise crystal stacking.[22–25]
Hit to leads
The R2 position
A round of optimisation for the R2 position was commenced.
During this process R1 was retained as a cyclohexyl moiety, as
this substituent gave the greatest activity and, due to its non-
planar structure, should also increase the solubility of the com-
pounds. A variety of substituents were investigated at the
para, meta, and ortho positions of the phenyl group at the R2
position. The substituents were designed either to disrupt the
packing and/or to reduce lipophilicity. Compounds were pre-
pared using the route described in Scheme 1 (Table 5).
Scheme 2. Reagents and conditions: a) Pd2dba3 (5 mol%), amine (2 equiv),
rac-binap (10 mol%), tBuOK (3 equiv), 1,4-dioxane, 1608C (microwave),
20 min, 5–24%.
The R1 position
The initial rounds of medicinal chemistry demonstrated that
changes to the R1 group were tolerated and that the cyclohex-
yl ring of 12 was slightly more active than the initial a-methyl-
benzyl group of 1. To complete the exploration of
the R1 group, an optimisation array was conducted,
maintaining a phenyl ring at the R2 position for refer-
ence. 3-Bromo-5-phenylpyrazine-2-amine 35 was pre-
pared from 3-amino-6-bromopyrazine-2-carboxylic
acid 34 in two steps (Scheme 3). Displacement of the
3-bromo group followed by cyclisation with CDI gave
compounds 36–42. Truncation of 1 to an isopropyl
For the para-substituted compounds, the electron-donating
methoxy group in 20 showed an eightfold drop in activity. The
Table 5. Variation of the R3, R4, and R5 groups of compound 19.
group (in 36) resulted in a 10-fold drop in activity
ID
R3
R4
R5
PPB
[%free]
clogP
EC50 [mm][a]
Aq. Sol.
while rigidification by replacement of cyclohexyl with
an aromatic ring (in 37) resulted in a complete loss
of activity (Table 7). The majority of changes were de-
signed to introduce polar solubilising groups. Alkyl
ethers 38 and 39 showed only two- and fivefold
losses of activity, respectively. Alkoxy compound 40
was ~10-fold less active than the cyclohexyl, whereas
the basic compounds 41 and 42 showed poor anti-
trypanosomal activity.
T. b. brucei MRC5 [mm][b]
19
20
21
H
OMe
CH2OH
H
H
H
H
H
H
H
H
H
H
H
0.8
0.7
4.7
ND
ND
ND
ND
0.7
3.9
3.8
2.9
2.9
3.8
2.9
3.2
3.9
3.9
2.9
0.04
0.33
0.4
>50
24
110
ND
ND
ND
ND
>250
ND
20
ND
>50
>50
>50
>50
>50
>50
>50
>50
22 C(O)NHMe
23 CH2NMe2
1.7
5
H
24
25
26
27
28
H
H
H
H
H
CH2OH
NHCOMe
0.03
0.9
H
H
H
F
0.06
0.9
OMe
CH2OH
ND
7.2
0.05
>250
Both 28 and 38 showed decreased protein binding
relative to 1 (7.2 and 15% unbound, respectively,
versus 0.3% unbound) and improved solubility (>
250 and 55 mm, respectively, compared with
[a] Values are the geometric mean of two or more determinations; standard deviation
is typically within 2-fold of the EC50 value. [b] Aqueous kinetic solubility. ND: not de-
termined.
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Table 6. Variation of the R2 group of compound 19.
Table 7. Variation of the R1 group of compound 19.
ID
R1
clogP
EC50 [mm][a]
T. b. brucei
Aq. Sol.
MRC5
>50
>50
>50
[mm][b]
ID
R2
clogP
EC50 [mm][a]
T. b. brucei
Aq. Sol.
[mm][b]
MRC5
19
36
37
3.9
2.5
2.9
0.04
0.3
110
ND
ND
19
29
30
31
32
33
3.9
3.0
3.0
3.5
2.3
2.3
0.04
0.07
0.1
>50
110
ND
ND
ND
220
ND
>50
>50
>50
>50
>50
>50
38
39
2.3
2.0
0.1
0.2
>50
>50
55
0.08
0.05
2
ND
40
1.7
0.5
>50
ND
[a] Values are the geometric mean of two or more determinations; stan-
dard deviation is typically within 2-fold of the EC50 value. [b] Aqueous ki-
netic solubility. ND: not determined.
41
42
1.7
2.4
13
>50
>50
ND
ND
2
[a] Values are the geometric mean of two or more determinations; stan-
dard deviation is typically within 2-fold of the EC50 value. [b] Aqueous ki-
netic solubility. ND: not determined.
<12 mm). However, both compounds were less metabolically
stabile when incubated with mouse hepatic microsomes
(CLint =2.4
and
1.6 mLminÀ1 gÀ1,
respectively,
versus
<0.5 mLminÀ1 gÀ1) but still progressable. Given these results,
both 28 and 38 were progressed into in vivo PK studies. Com-
pound 28 showed significantly higher levels of exposure rela-
tive to 38. Compound 28 also exhibited an improvement in
the time to reach Cmax compared to 1 (15 min versus 2 h) when
dosed orally (Table 8), presumably a result of its improved solu-
bility. However, 28 was cleared quite rapidly after both oral
and i.p. administration, likely due to the higher metabolic in-
stability of the compound; it would be unlikely to deliver suffi-
cient duration of efficacious free drug concentration at a toler-
ated dose. We decided to investigate alternative scaffolds with
the aim of improving metabolic stability and solubility.
Table 8. PK data for compounds 1, 28, and 38.
ID Route
Dose
[mgkgÀ1
Cmax
[ngmLÀ1
tmax [h][b]
AUC(0–8)
t1/2 [h][d]
[a]
[c]
]
]
[ngminmLÀ1
]
1
p.o.
i.p.
p.o.
i.p.
5
20
20
20
110
5200
910
83
4
36000
230000
69000
22000
3
28
28
38
0.083
0.25
2
1.2
1.7
2.2
[a] Maximum concentration reached. [b] Time after initial dose at which
Cmax was reached. [c] Area under the curve. [d] Half-life of compound in
the blood.
Scheme 4). Thus, 1-bromo-3-fluoro-4-nitrophenyl 43
was reacted with a-methylbenzyl amine to give inter-
mediate 44 (Scheme 4). The nitro group was reduced,
then 45 was cyclised with CDI to give (R)-6-bromo-1-
(1-phenylethyl)-1H-benzo[d]imidazol-2(3H)-one (46).
Finally a cross-coupling reaction gave the desired
compound 47. Removal of both ring nitrogen atoms
of 1 (EC50: 0.08 mm) to give 47 (EC50: 10 mm) resulted
in a 100-fold loss of activity, indicating that one or
both of the pyrazine nitrogen atoms are important.
Capping the imidazo NH group of 1 with a methyl
Scheme 3. Reagents and conditions: a) phenylboronic acid, Pd(PPh3)4, K2CO3 (2m aque-
ous), DMF, 1408C (microwave), 10 min, 100%; b) NaOAc, AcOH, Br2, RT, 16 h, 63%;
c) amine, DIPEA, nBuOH, 2208C (microwave), 1 h; d) CDI, dioxane, 1308C (microwave),
10 min, 2–71% over two steps.
Scaffold hopping: the core group
group gave 48, leading to a 100-fold loss of activity (EC50:
8 mm versus 0.08 mm for 48 and 1, respectively), suggesting the
importance of the imidazole NH (Scheme 5). To probe the
Initial work was conducted to investigate the importance of
the pyrazine nitrogen atoms (by removal of both of them,
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Table 9. Replacement of N7 nitrogen.
ID
51
52
R1
R2
clogP
EC50 [mm][a]
Aq. Sol.
T. b. brucei
MRC5
[mm][b]
3.9
3.1
0.054
0.79
>50
36
23
>50
>50
ND
53
54
2.8
3.2
0.67
ND
ND
Scheme 4. Reagents and conditions: a) (R)-methylbenzylamine, Cs2CO3, DMF,
1208C; b) charcoal (200% w/w), NaBH4, THF, H2O, RT, 16 h, 34% over two
steps; c) CDI, 1,4-dioxane, 1408C, 20 min, 61%; d) Pd(PPh3)4 (5 mol%), K2CO3
(1.5m aqueous), boronic acid, DMF, 1408C, 5 min, 44%.
15
55
56
57
2.8
3.3
2.2
0.23
0.093
0.33
>50
>50
>50
180
>250
ND
[a] Values are the geometric mean of two or more determinations; stan-
dard deviation is typically within 2-fold of the EC50 value. [b] Aqueous ki-
netic solubility. ND: not determined.
Scheme 5. Reagents and conditions: a) NaH, MeI, DMF, RT, 3 h, 38%.
effect of the N7 nitrogen, a series of compounds were made in
which the N7 nitrogen atom was replaced with CH, along with
a variety of substituents at the N1- and 6-positions. 2,3-Diami-
no-5-bromopyridine 49 was subjected to either reductive ami-
nation or alkylation conditions to introduce the N3 substituent,
followed by cyclisation with CDI to afford the key 6-bromo-
tively). At the R1 position, replacement with 2-phenylpropan-2-
yl groups (52, 54) was less favourable.
It was hypothesised that the “urea” moiety may be a cause
of the poor solubility, possibly through its ability to cause anti-
parallel hydrogen bonded dimers. However, if this was to be
replaced, certain features would need to be retained: the car-
bonyl group could act as a hydrogen bond acceptor, and the
NH group as a hydrogen bond donor. As N1 was a tertiary ni-
trogen it was hypothesised that does not make any important
interactions and could be replaced by a carbon atom. This
would then allow the carbonyl to be replaced by a nitrogen,
which could also act as a hydrogen bond acceptor, giving an
aza-indazole scaffold: a known kinase scaffold.
1-(R)-1H-imidazo[4,5-b]pyrazin-2(3H)-one
scaffold
50a–b
(Scheme 6). Treatment of this under either Suzuki or Buchwald
conditions afforded desired 6-substituted analogues (51–57).
Removal of N7 was tolerated if R1 was retained as cyclohexyl
(51) and R2 was retained as a phenyl ring (Table 9). Replace-
ment of the phenyl group at R2 with a piperidine (56) was also
tolerated. However, introduction of either 3-pyridyl- (55), mor-
pholino- (57), or (2-hydroxymethyl)phenyl groups (53) gave
rise to compounds that were approximately 2-, 7- and 13-fold
less active than their N7 homologues (30, 32, and 28, respec-
The key intermediate 5-bromo-3-cyclohexyl-1H-pyrazolo[3,4-
b]pyridine 59 was synthesised in three steps from commercial-
ly available 5-bromo-2-fluoronicotinaldehyde 58
and was subsequently reacted under Suzuki condi-
tions to give the corresponding 5-aryl-/heteroaryl-
substituted compounds 60–63 (Scheme 7). Both
phenyl (60) and 3-pyridyl (63) were tolerated, with
the former showing similar levels of potency and se-
lectivity to the other core scaffolds bearing an unsub-
stituted phenyl group (51 EC50 =0.054 mm and 19
Scheme 6. Reagents and conditions: a) 1) cyclohexanone, AcOH, CH2Cl2; 2) Na(OAc)3BH,
63%; b) PhC(CH3)2Cl, Et3N, THF, 9%; c) CDI, MeCN, 69–90%; d) ArB(OR)2, K3PO4, Pd(PPh3)4,
DMF/H2O (3:1), 1308C (microwave), 3–69% (compounds 51–55); e) (S)-PHOS, tBuONa,
(R2)2NH, Pd2dba3, toluene, 1208C (microwave), 32–47% (compounds 56–57).
EC50 =0.04 mm; Table 10). Phenyl groups (61 and 62)
bearing a substituent at the 2-position were less fa-
vourable.
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Removal of one the nitrogen atoms from the pyrazine ring
was predicted to lead to improvements in metabolic stability,
protein binding, and solubility (Table 11). However, replace-
ment of the core scaffold with the aza-indazole had a negative
effect on the in vitro DMPK properties of the compounds
(compare 51 and 60; 55 and 63).
Compound 56 was selected for in vivo PK studies, as it had
the most favourable in vitro profile (potency, metabolic stabili-
ty, protein binding, and solubility). However, 56 had lower ex-
posure levels than 28 and was also cleared rapidly, in line with
its higher CLint value (Table 12).
Scheme 7. Reagents and conditions: a) cyclohexylmagnesium bromide, THF,
À788C!RT, 55%; b) PDC, CH2Cl2, 08C!RT, 91%; c) NH2NH2·H2O, EtOH,
reflux, 95%; d) RB(OH)2, 2m Na2CO3, Pd(PPh3)4, DME/H2O/EtOH (7:3:2), 1408C,
microwave, 16–69%.
Table 10. SAR for aza-indazoles.
Table 12. PK data for compounds 28 and 56.
ID Route
Dose
[mgkgÀ1
Cmax
[ngmLÀ1
tmax [h][b]
AUC(0–8)
[ngminmLÀ1
]
t1/2 [h][d]
[a]
[c]
]
]
28
56
i.p.
i.p.
20
10
5200
580
0.083
0.25
230000
53000
1.2
0.7
ID
R
clogP
EC50 [mm][a]
T. b. brucei
Aq. Sol.
[mm][b]
[a] Maximum concentration reached. [b] Time after initial dose at which
Cmax was reached. [c] Area under the curve. [d] Half-life of compound in
the blood.
MRC5
60
4.7
0.059
1.3
>50
20
Determination of the mode of the antiparasitic effect: static
or cidal action
61
3.6
>50
ND
During the course of these studies, work from parallel series in-
dicated that compounds that are cytostatic against T. brucei
are not effective in animal models of infection. Therefore com-
pounds that only exert a static effect (i.e., stopping the growth
of cells, whilst not killing them) or a decrease in growth rate
are unlikely to clear the parasites completely and should be re-
moved from drug discovery pipelines as early as possible. To
provide information regarding the mode of action of the com-
pounds discussed, we selected a number of examples to run in
a static–cidal assay.[22,26] Compounds with either an imida-
zo[4,5-b]pyrazin-2(3H)-one (24, 26, 28, 30, and 39) or
imidazo[4,5-b]pyridin-2(3H)-one (51, 55, and 56) core
showed growth-slowing or static behaviour at all
concentrations tested, whereas compounds contain-
ing a 1H-pyrazolo[3,4-b]pyridine core (60 and 63)
showed cidal behaviour at a minimum cidal concen-
tration of 17 mm (Table 13). With the exception of
pentamidine and melarsoprol, all compounds
62
63
4.7
3.5
2.6
>50
>50
ND
0.26
250
[a] Values are the geometric mean of two or more determinations; stan-
dard deviation is typically within 2-fold of the EC50 value. [b] Aqueous ki-
netic solubility. ND: not determined.
Table 11. In vitro ADMET data.
showed either biphasic dose–response curves or par-
tial curves. This suggested these compounds may act
on at least two targets, the first of which caused
a growth-slowing or static mode of action, whilst in-
hibition of the subsequent target(s) had a cytocidal
effect (Figure 1). Only pentamidine, melarsoprol, 60,
and 63 reached a well-defined plateau at 100% in-
hibition, which explains why only these compounds
showed clear cidal activity.
ID
R1
X
EC50 [mm][a]
T. b. brucei MRC5 [mLminÀ1 gÀ1
CLint
PPB
Aq. Sol. tPSA
]
[%free] [mm][b]
[Š2]
28 (2-CH2OH)Ph
N
N
CH
N
CH
N
CH
CH
N
0.046
0.044
0.054
0.12
>50
>50
>50
>50
>50
>50
>50
>50
>50
2.4
3.6
5.8
2.8
2.7
7.2
0.8
2.9
2.3
11
0
11
1.4
5.2
>250
ND
83
64
51
76
64
67
54
42
54
19
51
30
55
31
56
60
63
Ph
Ph
36
3-pyridyl
3-pyridyl
piperidine
piperidine
–
ND
0.23
180
ND
0.077
0.093
0.059
0.26
15
3.6
15
6.6
>250
20
–
250
The mode(s) of action of these compounds are not
known. Analysis of the literature suggests that these
chemotypes have been investigated against many
families of enzyme in humans, including protein kin-
[a] Values are the geometric mean of two or more determinations; standard deviation
is typically within 2-fold of the EC50 value. [b] Aqueous kinetic solubility. ND: not de-
termined.
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Given our experience from other programmes, in order for
a compound to show efficacy in a mouse model of HAT, it is
important that the compound is cidal, rather than static, in its
activity against T. brucei. Compounds that are consistent with
this are 60 and 63. However, given their metabolic instability
and poor potency (minimum cidal concentrations of 17 mm), it
is highly unlikely that either would deliver efficacy in a mouse
model of HAT. Further work is needed to improve the potency
and pharmacokinetic properties of the molecules.
Table 13. Static/cidal data.
ID
T. b. brucei
EC50 [mm][a]
MCC
SCEC50(1)
SCEC50(2)
Mode[c]
[mm][b]
[mm][b]
[mm][b]
24
26
28
30
39
51
55
56
60
63
0.03
0.06
0.05
0.12
0.20
0.05
0.23
0.09
0.06
0.26
0.004
0.009
>50
>50
>50
>50
>50
>50
>50
>50
17
0.07
0.09
0.10
0.23
0.41
0.02
0.33
0.07
0.08
0.28
0.019
0.031
30
–
>50
–
32
41
–
>50
7.9
6.1
–
S
GS
GS
GS
S
S[d]
GS
GS
C
Conclusions
17
0.1
0.1
C
C
C
Through our phenotypic screening approach with a relatively
small library, we have developed a series of compounds that
exhibit good levels of potency in the T. b. brucei growth inhibi-
tion assay, and that are nontoxic to MRC5 mammalian cells.
Despite the fact that the screening library was a focused pro-
tein kinase library, it was possible to obtain compounds with
selectivity over the mammalian protein kinases assayed. These
compounds possess reasonable physicochemical properties
and show oral exposure, but have modest half-lives. By chang-
ing the substituents R1 and R2 it was possible to optimise the
compounds for potency and to alter key physico-
chemical and DMPK properties, in the absence of
knowledge of the molecular target. It was also possi-
ble to scaffold-hop, although it is possible that the
new scaffold works on a different molecular target.
The ultimate aim would be to devise a compound
that has oral bioavailability and the capacity to pene-
trate the BBB. The compounds prepared in this study
generally have physicochemical properties consistent
with BBB penetration as reported by Wager,[19] in
terms of Mr, tPSA, clogP, and number of hydrogen
bond donors. Although of course for compounds
taken forward, BBB permeability would have to be
determined experimentally.
pentamidine
melarsoprol
–
[a] Values determined by the standard T. brucei cell viability assay and are
the geometric mean of two or more determinations; standard deviation
is typically within 2-fold of the EC50 value. [b] Compounds were also
tested in the static–cidal assay carried out in triplicate to determine mini-
mal cidal concentration (MCC) and static cidal EC50 values for the first
[SCEC50(1)] and second [SCEC50(2)] sites. EC50 and SCEC50(1) are expected
to represent the same mode of action of the compounds and are there-
fore very similar.[26] [c] GS: growth-slowing, S: static, C: cidal. [d] Indication
of cidality at 50 mm.
Key compounds in a static–cidal assay showed an
unusual biphasic nature, giving two points of inflec-
tion on the dose–response curve. This suggests at
least two modes of action (or, less likely due to the
use of a clonal line, differential susceptibility of cell
sub-populations). Compounds from the 1H-imida-
zo[4,5-b]pyrazin-2(3H)-one scaffold appeared to be
either static or growth-slowing, whilst compounds
from the 1H-pyrazolo[3,4-b]pyridine series were cidal.
It is possible that compounds from the 1H-imida-
Figure 1. Static/cidal dose–response curves for pentamidine, 24, 28, and 60. Data are the
average of triplicate measurements after 48 h incubation of T. b. brucei cells with a) pen-
tamidine (cidal), b) compound 24 (static at concentration tested), c) compound 28
(growth-slowing), and d) compound 60 (cidal). Inhibitors were tested in a 10-point dose–
response curve at a 1:3 dilution factor; data were analysed with either a monophasic or
a biphasic fit. Static and cidal are defined by analysis of the growth curves as shown in
the Supporting Information (Figure S4), where cidal compounds show a decrease in the
numbers of parasites.
zo[4,5-b]pyrazin-2(3H)-one scaffold will be cidal at
higher concentrations, and that the key difference
between the two scaffolds is not a difference in
mode-of-action but a difference in potency against
the target(s) represented by the second phase of the
biphasic curves.
A way forward would be to identify the molecular
target(s) of the compounds, specifically which tar-
ases,[27–32] hydrolases,[33] and transferases.[34] Our work described
in Figure 1 suggests that there are likely to be several modes
of action in trypanosomes.
get(s) causes the cytocidal activity, and to focus efforts on
potent inhibition of this target. Subsequent progression of the
series will require identification of compounds that are cytoci-
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(414 mg, 3.0 mmol) in H2O (5 mL), and DMF (5 mL) were combined
and heated under microwave irradiation at 1608C for 5 min. The
reaction was then cooled to room temperature, and then diluted
with a 10:1 mixture of CH2Cl2/MeOH (10 mL). The mixture was
poured onto an SCX-2 column and allowed to drip through; it was
then washed with a 10:1 mixture of CH2Cl2/MeOH (10 mL). The
SCX-2 column was then eluted with NH3 in MeOH (7n, 20 mL) and
the eluates were concentrated in vacuo. The resultant brown solid
was dissolved in 1,4-dioxane (2 mL), and CDI (140 mg, 0.86 mmol)
was added. The mixture was heated under microwave irradiation
at 1408C for 20 min, cooled to room temperature and concentrat-
ed in vacuo. The resultant crude residue was purified by column
chromatography, eluting with 0–100% EtOAc/petroleum ether (PE)
40–608C to give the title compound 9 (47 mg, 15%) as an off-
dal. It will also be necessary to optimise the key properties of
the molecule (microsomal stability, solubility, and protein bind-
ing). Although it has proved possible to optimise the com-
pound in terms of potency and to some extent at least the
pharmacokinetic parameters, we have reached a biological
issue which has proved difficult to resolve in the absence of
knowledge of the cidal target. In this case, a combination of
phenotypic and target-based approaches would probably be
optimal.
Experimental Section
1
Chemistry
white solid. H NMR (500 MHz, [D6]DMSO): d=12.19 (s, 1H), 8.53 (s,
1H), 8.09–8.06 (brs, 2H), 7.43–7.27 (m, 7H), 5.08 ppm (s, 2H); LRMS
(ES+): m/z (%) 321.0 [M+H]+ (100); HRMS (m/z): [M+H]+ calcd
for C18H14FN4O: 321.1146, found: 321.1131.
Compounds 1, 2, 3 and 13 were purchased from BioFocus. Chemi-
cals and solvents were purchased from Aldrich Chemical Co.,
Fluka, ABCR, VWR, Acros, Fisher Chemicals, and Alfa Aesar and
were used as received unless otherwise stated. Air- and moisture-
sensitive reactions were carried out under an inert atmosphere of
argon in oven-dried glassware. Analytical thin-layer chromatogra-
phy (TLC) was performed on pre-coated TLC plates (layer 0.20 mm
silica gel 60 with fluorescent indicator UV254, Merck). Developed
plates were air dried and analyzed under a UV lamp (l 254/
365 nm). Flash column chromatography was performed using pre-
packed silica gel cartridges (230–400 mesh, 40–63 mm, SiliCycle)
(unless otherwise stated) using a Teledyne ISCO Combiflash Com-
panion or Combiflash Retrieve. 1H and 13C NMR spectra were re-
corded on a Bruker Avance II 500 spectrometer (1H at 500.1 MHz,
13C at 125.8 MHz), or a Bruker DPX300 spectrometer (1H at
300.1 MHz). Chemical shifts (d) are expressed in ppm recorded
using the residual solvent as internal reference in all cases. Signal
splitting patterns are described as singlet (s), doublet (d), triplet (t),
quartet (q), pentet (p), multiplet (m), broad (br), or a combination
thereof. Coupling constants (J) are quoted to the nearest 0.1 Hz.
LC–MS analyses were performed with either an Agilent HPLC 1100
series instrument connected to a Bruker Daltonics MicrOTOF or an
Agilent Technologies 1200 series HPLC connected to an Agilent
Technologies 6130 quadrupole spectrometer, where both instru-
ments were connected to an Agilent diode array detector. LC–MS
(R)-6-Phenyl-1-(1-phenylethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-
one (14): (R)-6-bromo-N2-(1-phenylethyl)pyrazine-2,3-diamine (6a;
100 mg, 0.35 mmol), phenyl boronic acid (44 mg, 0.35 mmol), tetra-
kis(triphenylphosphine)palladium(0) (20 mg, 0.018 mmol), potassi-
um carbonate (700 mL of a 1.5m solution in H2O, 1.05 mmol), and
DMF (2 mL) were combined and heated under microwave irradia-
tion at 1608C for 5 min. The reaction was then cooled to room
temperature, and then diluted with a 10:1 mixture of CH2Cl2/MeOH
(10 mL). The mixture was poured onto an SCX-2 column and al-
lowed to drip through, then washed with a 10:1 mixture of CH2Cl2/
MeOH (10 mL). The SCX-2 column was then eluted with NH3 in
MeOH (7n, 20 mL), and the eluates were concentrated in vacuo.
The resultant brown solid was dissolved in 1,4-dioxane (2 mL), and
CDI (140 mg, 0.86 mmol) was added. The mixture was heated
under microwave irradiation at 1408C for 20 min, cooled to room
temperature and concentrated in vacuo. The resultant crude resi-
due was purified by column chromatography, eluting with 0–100%
EtOAc/PE 40–608C to give the title compound 14 (21 mg, 18%) as
1
a pale-yellow solid. H NMR (500 MHz, [D6]DMSO): d=12.12 (s, 1H),
8.51 (s, 1H), 8.00 (d, J=7.3 Hz, 2H), 7.55–7.47 (m, 4H), 7.42–7.34
(m, 3H), 7.27 (t, J=7.3 Hz, 1H), 5.74 (q, J=7.2 Hz, 1H), 2.03 ppm
(d, J=7.3 Hz, 3H); LRMS (ES+): m/z (%) 317.1 [M+H]+ (97); HRMS
(m/z): [M+H]+ calcd for C19H17N4O: 317.1397, found: 317.1394.
chromatographic separations were conducted with
a Waters
Xbridge C18 column, 50 mm”2.1 mm, 3.5 mm particle size; mobile
phase: H2O/MeCN+0.1% HCOOH, or H2O/MeCN+0.1% NH3;
linear gradient from 80:20 to 5:95 over 3.5 min and then held for
1.5 min; flow rate: 0.5 mLminÀ1. All tested compounds had a mea-
sured purity of ꢀ95% (by TIC [total ion current] and UV) as deter-
mined by this analytical LC–MS system. High-resolution electro-
spray MS measurements were performed on a Bruker Daltonics Mi-
crOTOF mass spectrometer. Microwave-assisted chemistry was per-
formed using a Biotage initiator microwave synthesiser.
1-Cyclohexyl-6-(4-fluorophenyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-
one (12): 6-Bromo-1-cyclohexyl-1H-imidazo[4,5-b]pyrazin-2(3H)-one
7b (100 mg, 0.33 mmol), tetrakis(triphenylphosphine)palladium(0)
(12 mg, 0.01 mmol), potassium carbonate (0.65 mL of a 1.5m solu-
tion in H2O, 1.0 mmol), 4-fluorophenyl boronic acid (46 mg,
0.33 mmol), in DMF (2.0 mL) were combined and the mixture was
heated under microwave irradiation at 1808C for 5 min. The reac-
tion mixture was absorbed onto silica and purified by chromatog-
raphy eluting with 50% EtOAc/PE 40–608C to give the title com-
pound 12 (17 mg, 16%) as a yellow solid. 1H NMR (500 MHz,
[D6]DMSO): d=12.04 (s, 1H), 8.49 (s, 1H), 8.10–8.07 (m, 2H), 7.34 (t,
J=8.8 Hz, 2H), 4.28–4.22 (m, 1H), 2.37–2.29 (m, 2H), 1.88–1.80 (m,
4H), 1.71 (d, J=12.8 Hz, 1H), 1.44–1.36 (m, 2H), 1.29–1.22 ppm (m,
1H); LRMS (ES+): m/z (%) 313.2 [M+H]+ (100); HRMS (m/z): [M+
H]+ calcd for C17H18FN4O: 313.1459, found: 313.1458.
1-Benzyl-6-(4-fluorophenyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one
(9): A mixture of 3,5-dibromopyrazin-2-amine (400 mg, 1.58 mmol),
benzylamine (189 mL, 1.74 mmol) and DIPEA (302 mL, 1.74 mmol) in
EtOH (2 mL) was heated under microwave irradiation at 1808C for
90 min. The reaction was cooled to room temperature and concen-
trated in vacuo. The resulting slurry was partitioned between
CH2Cl2 (20 mL) and H2O (10 mL), the organic layer was washed
with brine (10 mL) and concentrated in vacuo. The resultant crude
residue was purified by column chromatography, eluting with 20%
EtOAc/hexane to give N2-benzyl-6-bromopyrazine-2,3-diamine (6c;
281 mg, 64%) as a brown oil. Compound 6c (281 mg, 1.10 mmol),
4-fluorophenylboronic acid (154 mg, 1.10 mmol), tetrakis(triphenyl-
phosphine)palladium(0) (58 mg, 0.05 mmol), potassium carbonate
1-Cyclohexyl-6-(piperidin-1-yl)-1H-imidazo[4,5-b]pyrazin-2(3H)-
one (31): 6-Bromo-1-cyclohexyl-1H-imidazo[4,5-b]pyrazin-2(3H)-one
(100 mg,
(16 mg, 0.017 mmol), potassium tert-butoxide (114 mg, 1.0 mmol),
(22 mg,
0.33 mmol),
tris(dibenzylideneacetone)dipalladium
(Æ)-2,2’-bis(diphenylphosphino)-1,1’-binaphthyl
0.034 mmol), piperidine (67 mL, 0.68 mmol) and 1,4-dioxane
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(2.0 mL) were combined and heated under microwave irradiation
at 1808C for 5 min. The reaction was cooled to room temperature,
absorbed onto silica and purified by column chromatography, elut-
ing with 50% EtOAc/hexane to give the title compound 31
over time. For dose–response curves from this assay either a mono-
phasic or a biphasic equation was used, depending on which one
provided the best fit. For monophasic fits, the following four-para-
metric equation [Eq. (1)] was used:
1
(25 mg, 24%) as a yellow solid. H NMR (500 MHz, [D6]DMSO): d=
BÀA
11.36 (s, 1H), 7.45 (s, 1H), 4.16–4.09 (m, 1H), 3.38–3.32 (m, 2H),
2.52–2.50 (m, 2H), 2.27–2.19 (m, 2H), 1.83 (d, J=13.0 Hz, 2H),
1.73–1.67 (m, 3H), 1.59 (brs, 6H), 1.39–1.31 (m, 2H), 1.21–1.13 ppm
(m, 1H); LRMS (ES+): m/z (%) 302.2 [M+H]+ (100); HRMS (m/z):
[M+H]+ calcd for C16H24N5O: 302.1975, found: 302.1973.
y ¼ A þ
ð1Þ
D
1 þ ðC=xÞ
in which A=percent inhibition at bottom, B=percent inhibition at
top, C=EC50, D=slope, x=inhibitor concentration, and y=percent
inhibition. Equation (2) was used for biphasic fits:
1-(1-Methoxypropan-2-yl)-6-phenyl-1H-imidazo[4,5-b]pyrazin-
2(3H)-one (39): 3-Bromo-5-phenylpyrazine-2-amine (125 mg,
0.5 mmol), 1-methoxybutan-2-amine (77 mg, 0.75 mmol), DIPEA
(0.1 mL), and nBuOH (0.5 mL) were heated under microwave irradi-
ation at 2208C for 1 h. The reaction was diluted with EtOAc
(10 mL), washed with H20 (3”10 mL), the organic layer dried over
MgSO4, filtered, and the solvent was removed in vacuo. Column
chromatography eluting with CH2Cl2 to CH2Cl2/MeOH (95:5) afford-
ed the desired substituted 6-phenylpyrazine-2,3-diamine, which
was used for subsequent reaction without further purification. Sub-
stituted 6-phenylpyrazine-2,3-diamine (0.5 mmol), CDI (97 mg,
0.6 mmol), and 1,4-dioxane (1 mL) were heated under microwave
irradiation at 1308C for 10 min. The crude reaction mixture was
partitioned between EtOAc and H2O, the organic layer was dried
over MgSO4, filtered, and the solvent removed in vacuo. The resul-
tant crude residue was purified by column chromatography, elut-
ing with EtOAc to give the title compound 39 (13 mg, 10%) as
A
100ÀA
ð2Þ
þ
½CÀlogðxފÂB
½DÀlogðxފÂB
1 þ 10
1 þ 10
for which A=percent inhibition at mid-plateau, B=slope, C=
log[SCEC50(1)], D=log[SCEC50(2)]. Thus SCEC50(1) is the EC50 for
the first phase of the curve, and SCEC50(2) is that for the second
phase. Inhibition at the bottom of the curve is fixed at 0% and at
the top, 100%.
Intrinsic clearance experiments: Test compound (0.5 mm) was incu-
bated with female CD1 mouse liver microsomes (Xenotech LLC;
0.5 mgmLÀ1 50 mm potassium phosphate buffer, pH 7.4), and the
reaction started with the addition of excess NADPH (8 mgmLÀ1
50 mm potassium phosphate buffer, pH 7.4). Immediately, at t0,
then at 3, 6, 9, 15, and 30 min an aliquot (50 mL) of the incubation
mixture was removed and mixed with MeCN (100 mL) to stop the
reaction. Internal standard was added to all samples, the samples
were centrifuged to sediment precipitated protein, and the plates
were then sealed prior to UPLC–MS/MS analysis using a Quattro
Premier XE instrument (Waters Corp., USA). XLfit (IDBS, UK) was
used to calculate the exponential decay and consequently the rate
constant (k) from the ratio of peak area of test compound to inter-
nal standard at each time point. The rate of intrinsic clearance
[CLint, mLminÀ1 (g liver)À1] of each test compound was then calcu-
lated with Equation (3):
1
a colourless solid. H NMR (500 MHz, CDCl3): d=8.90 (brs, 1H), 8.41
(s, 1H), 7.99–7.97 (m, 2H), 7.51 (t, J=7.3 Hz, 2H), 7.44 (t, J=7.3 Hz,
1H), 4.98–4.94 (m, 1H), 4.27 (t, J=10.1 Hz, 1H), 3.68 (dd, J=10.1,
5.2 Hz, 1H), 3.37 (s, 3H), 1.65 ppm (d, J=7.1 Hz, 3H); LRMS (ES+):
m/z (%) 285.1 [M+H]+ (100); HRMS (m/z): [M+H]+ calcd for
C15H16N4O2: 285.1346; found, 285.1336.
Biological assays
Medium-throughput screen for African trypanosomes and cytotoxici-
ty: Measurement of inhibition of the proliferation of L-6 cells
(mouse muscle fibroblasts) and T. brucei bloodstream-stage cells
was performed as previously described.[35,36]
CLint ¼ k  V  ðmicrosomal protein yieldÞ
ð3Þ
in which V [mL(mg protein)À1] is the incubation volume per mg
protein added, and microsomal protein yield is taken as 52.5 mg
protein per gram of liver. Verapamil (0.5 mm) was used as a positive
control to confirm acceptable assay performance.
Trypanosome and MRC5 proliferation assay: Measurement of inhibi-
tion of the proliferation of MRC5 cells (human lung fibroblasts) and
T. brucei bloodstream-stage cells was performed using a modifica-
tion of a cell viability assay previously described.[35,36] Compounds
(50 mm to 0.5 nm) were incubated with 2”103 cells per well in
0.2 mL of the appropriate culture medium (MEM with 10% foetal
bovine serum for MRC5 cells) in clear 96-well plates. Plates were in-
cubated at 378C in the presence of 5% CO2 for 69 h. Resazurin
was then added to a final concentration of 50 mm, and plates were
incubated as above for a further 4 h before being read on a BioTek
Flx800 fluorescence plate reader.
Plasma protein binding experiments: In brief, a 96-well equilibrium
dialysis apparatus was used to determine the free fraction in
plasma for each compound (HT Dialysis LLC, Gales Ferry, CT, USA).
Membranes (12–14 kDa cutoff) were conditioned in deionised H2O
for 60 min, followed by conditioning in 80:20 deionised H2O/EtOH
for 20 min, and then rinsed in isotonic buffer before use. Female
CD1 mouse plasma was removed from the freezer and allowed to
thaw on the day of experiment. Thawed plasma was then centri-
fuged (Allegra X12-R, Beckman Coulter, USA), spiked with test com-
pound (10 mggÀ1), and 150 mL aliquots (n=6 replicate determina-
tions) loaded into the 96-well equilibrium dialysis plate. Dialysis
against isotonic buffer (150 mL) was carried out for 5 h in a temper-
ature-controlled incubator at ~378C (Barworld Scientific Ltd., UK)
using an orbital microplate shaker at 125 rpm (Barworld Scientific).
At the end of the incubation period, aliquots of plasma or buffer
were transferred to micronic tubes (Micronic B.V., the Netherlands),
and the composition in each tube was balanced with control fluid
such that the volume of buffer to plasma is the same. Sample ex-
traction was performed by the addition of 400 mL MeCN containing
Determination of cidality against T. brucei bloodstream-stage cells
was performed using a static–cidal (SC) assay as previously report-
ed.[26] Briefly, bloodstream-form trypanosomes were seeded into
384-well plates at 4”105 mLÀ1 (50 mL per well), followed by imme-
diate addition of resazurin (50 mm final) to one of the plates, and
all plates were incubated at 378C, 5% CO2. After 4 h the t0 plate
was read using a PerkinElmer Victor 3 plate reader (lex =528 nm,
lem =590 nm). The second plate was processed in the same
manner 20 h later, and at 44 h the last plate was processed. The
minimum cidal concentration (MCC) is defined as the lowest con-
centration of drug that results in a decrease of resorufin signal
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Full Papers
an appropriate internal standard. Samples were allowed to mix for
1 min and then centrifuged at 3000 rpm in 96-well blocks for
15 min (Allegra X12-R, Beckman Coulter, USA). All samples were an-
alysed by UPLC–MS/MS on a Quattro Premier XE Mass Spectrome-
ter (Waters Corp.). The unbound fraction was determined as the
ratio of the peak area in buffer to that in plasma.
HRMS analyses, Lars Sandberg (University of Dundee) for litera-
ture analysis, and Daniel James (University of Dundee) for data
management.
Keywords: cidal · kinases · phenotypic · static · Trypanosoma
brucei
Solubility experiments: The kinetic aqueous solubility of the test
compounds was measured using laser nephelometry. Compounds
were subject to serial dilution from 10 mm to 0.5 mm in DMSO. An
aliquot was then mixed with Milli-Q H2O to obtain an aqueous dilu-
tion plate with a final concentration range of 250–12 mm, with
a final DMSO concentration of 2.5%. Triplicate aliquots were trans-
ferred to a flat-bottomed polystyrene plate which was immediately
read on the NEPHELOstar (BMG Lab Technologies). The amount of
laser scatter caused by insoluble particulates (relative nephelome-
try units, RNU) was plotted against compound concentration using
a segmental regression fit, with the point of inflection being
quoted as the compounds’ aqueous solubility (in mm).
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Ethics: All regulated procedures on living animals were carried out
under the authority of a project license issued by the Home Office
under the Animals (Scientific Procedures) Act 1986, as amended in
2012 (and in compliance with EU Directive EU/2010/63). License
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Review Committee (ERC) before submission to the Home Office.
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aspects of the use of animals on University premises and is a sub-
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Acknowledgements
Funding for this work was provided by the Wellcome Trust (Grant
Ref. WT077705 and Strategic Awards WT083481 and WT100476).
We thank Gina McKay (University of Dundee) for performing
ChemMedChem 2015, 10, 1809 – 1820
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Full Papers
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Received: July 10, 2015
Published online on September 18, 2015
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1820 ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim