Assessment of a pretomanid analogue library for African trypanosomiasis: Hit-to-lead studies on 6-substituted 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides

Article abstract of DOI:10.1016/j.bmcl.2017.10.067

A 900 compound nitroimidazole-based library derived from our pretomanid backup program with TB Alliance was screened for utility against human African trypanosomiasis (HAT) by the Drugs for Neglected Diseases initiative. Potent hits included 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides, which surprisingly displayed good metabolic stability and excellent cell permeability. Following comprehensive mouse pharmacokinetic assessments on four hits and determination of the most active chiral form, a thiazine oxide counterpart of pretomanid (24) was identified as the best lead. With once daily oral dosing, this compound delivered complete cures in an acute infection mouse model of HAT and increased survival times in a stage 2 model, implying the need for more prolonged CNS exposure. In preliminary SAR findings, antitrypanosomal activity was reduced by removal of the benzylic methylene but enhanced through a phenylpyridine-based side chain, providing important direction for future studies.

Full text of DOI:10.1016/j.bmcl.2017.10.067

Accepted Manuscript
Assessment of a pretomanid analogue library for African trypanosomiasis: Hit-
to-lead studies on 6-substituted 2-nitro-6,7-dihydro-5H-imidazo[2,1-b]
[1,3]thiazine 8-oxides
Andrew M. Thompson, Andrew J. Marshall, Louis Maes, Nigel Yarlett, Cyrus
J. Bacchi, Eric Gaukel, Stephen A. Wring, Delphine Launay, Stephanie
Braillard, Eric Chatelain, Charles E. Mowbray, William A. Denny
PII:
S0960-894X(17)31067-3
https://doi.org/10.1016/j.bmcl.2017.10.067
BMCL 25396
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 August 2017
18 October 2017
26 October 2017
Please cite this article as: Thompson, A.M., Marshall, A.J., Maes, L., Yarlett, N., Bacchi, C.J., Gaukel, E., Wring,
S.A., Launay, D., Braillard, S., Chatelain, E., Mowbray, C.E., Denny, W.A., Assessment of a pretomanid analogue
library for African trypanosomiasis: Hit-to-lead studies on 6-substituted 2-nitro-6,7-dihydro-5H-imidazo[2,1-b]
[1,3]thiazine 8-oxides, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.
2017.10.067
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Assessment of a pretomanid analogue library for African trypanosomiasis:
Hit-to-lead studies on 6-substituted 2-nitro-6,7-dihydro-5H-imidazo[2,1-
b][1,3]thiazine 8-oxides
Andrew M. Thompsona,*, Andrew J. Marshalla, Louis Maesb, Nigel Yarlettc, Cyrus J.
Bacchic, Eric Gaukeld, Stephen A. Wringd, Delphine Launaye, Stephanie Braillarde, Eric
Chatelaine, Charles E. Mowbraye, and William A. Dennya
aAuckland Cancer Society Research Centre, School of Medical Sciences, The University of
Auckland, Private Bag 92019, Auckland 1142, New Zealand; bLaboratory for Microbiology,
Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences,
University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium; cHaskins
Laboratories, Pace University, NY 10038, USA; dScynexis, Inc., Research Triangle Park, NC
27713, USA; eDrugs for Neglected Diseases initiative, 15 Chemin Louis Dunant, 1202
Geneva, Switzerland.
*Corresponding author: Dr Andrew M. Thompson, Auckland Cancer Society Research
Centre, School of Medical Sciences, The University of Auckland. Ph: (+649) 923 6145. Fax:
(+649) 373 7502. Email: am.thompson@auckland.ac.nz
Keywords: African trypanosomiasis, pretomanid, library screening, in vivo efficacy,
pharmacokinetics, nitroimidazole
1

A 900 compound nitroimidazole-based library derived from our pretomanid backup program
with TB Alliance was screened for utility against human African trypanosomiasis (HAT) by
the Drugs for Neglected Diseases initiative. Potent hits included 2-nitro-6,7-dihydro-5H-
imidazo[2,1-b][1,3]thiazine 8-oxides, which surprisingly displayed good metabolic stability
and excellent cell permeability. Following comprehensive mouse pharmacokinetic
assessments on four hits and determination of the most active chiral form, a thiazine oxide
counterpart of pretomanid (24) was identified as the best lead. With once daily oral dosing,
this compound delivered complete cures in an acute infection mouse model of HAT and
increased survival times in a stage 2 model, implying the need for more prolonged CNS
exposure. In preliminary SAR findings, antitrypanosomal activity was reduced by removal of
the benzylic methylene but enhanced through a phenylpyridine-based side chain, providing
important direction for future studies.
2

Human African trypanosomiasis (HAT, also known as sleeping sickness) is a particularly
lethal neglected tropical disease that is endemic in remote sub-Saharan Africa.1 HAT arises
from infection by two subspecies of the kinetoplastid parasite Trypanosoma brucei (T. b.
gambiense and T. b. rhodesiense), which are transmitted through the bite of tsetse flies.2
Because symptoms of the initial bloodstream stage are fairly mild and non-specific (e.g.,
headache, fever, weakness), the disease often progresses to the potentially fatal CNS stage
characterised by neurological and psychiatric disorders before treatment is sought.1,3
However, there are pitifully few available drugs for late stage HAT, and all require
hospitalization.3,4 The antiquated first-line remedy melarsoprol (1, see Fig. 1) is highly toxic,
causing death in ~5% of patients, and is increasingly less effective due to drug resistance.4,5
Eflornithine (2) is less toxic but more costly and cumbersome to administer and is ineffective
against T. b. rhodesiense (<5% of total cases).6 Combination of 2 with nifurtimox (3) (NECT)
has recently led to reduced cost and workload without compromising efficacy,7 but similar
issues (adverse effects and parenteral administration) plus a lack of CNS penetration limit the
two early stage drugs, pentamidine and suramin.3-5 Thus, there is a compelling need for more
universally effective, safe and affordable oral therapies. Two promising new agents are now
in phase II/III clinical trials; fexinidazole (4) and oxaborole SCYX-7158 (5).8,9 Nevertheless,
in order to mitigate development risks and minimise the emergence of drug resistance, it
remains essential to develop a pipeline of novel agents with unique mechanisms of action.4
Figure 1. Various antitrypanosomal, antitubercular, or antileishmanial agents
The nitroimidazooxazine pretomanid (PA-824, 6) has demonstrated excellent bactericidal
efficacy in phase II clinical studies for tuberculosis (TB), stimulating its further appraisal in
new drug combination trials.10 Within a comprehensive backup program, in collaboration
with the TB Alliance, we generated a library of more than 1000 compounds, whose
assessment led to the advancement of a second generation TB candidate (TBA-354, 7) into
phase I clinical evaluation.11 We recently disclosed that phenotypic screening of some early
examples against kinetoplastid diseases by the Drugs for Neglected Diseases initiative
(DNDi) unexpectedly enabled the discovery of DNDI-VL-2098 (8) as a preclinical lead for
visceral leishmaniasis.12 Unfortunately, 8 exhibited poor potency against T. b. brucei (IC50 53
µM) and it was reported13 that 6 also had weak activity versus this parasite (IC50 38 µM).
However, unlike fexinidazole (4), 6 did not display cross-resistance to nifurtimox (3),13
indicating that it is not activated by the same type I nitroreductase employed by 3 and 4
3

(implying a different mechanism of action). Therefore, as part of a wider search for improved
development candidates for HAT, ~900 analogues of 6 were screened by DNDi and several
promising hits were unearthed. Herein, we reveal initial in vitro/in vivo profiling data on
these hits, and findings from a preliminary SAR study of a nitroimidazothiazine oxide lead.
Table 1. Inhibitory potency, metabolic stability, aqueous solubility, and MDCK-MDR1 cell
permeability for 15 screening hits against T. b. brucei
Compd IC50 (µg/mL)a
Selectivity Mouse S9b Solubilityc Permeability (nm/s)d
T. b. brucei L929 Index
t1/2 (min)
173
ND
50
>350
ND
ND
67
ND
>350
ND
298
31
239
146
38
(µg/mL) Papp Papp+918 AQ
>10 >667
>10 >303
>10 >77
>10 >63
>10 >43
>10 >36
>10 >22
>10 >19
>10 >13
>10 >12
>10 >11
>10 >4.5
>10 >3.7
>10 >3.6
>10 >3.3
5.4
ND
1.3
39
ND
ND
1.2
ND
>72
ND
10
771 799
36.9 254
651 637
798 848
<0.8 <0.8
117 71
0.035
0.85
-0.022
0.059
ND
-0.64
-0.016
0.29
-0.019
-1
9
0.015  0.005
0.033  0.018
0.13  0.04
0.16  0.07
0.23  0.04
0.28  0.11
0.46  0.21
0.53  0.23
0.78  0.01
0.83  0.41
0.90  0.40
2.2  0.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
583 574
35
804 789
74 37
49
624 656
465 465
769 767
793 758
363 332
0.049
0
1.2
18
10
-0.003
-0.046
-0.093
2.7  0.5
2.8  0.2
3.0  0.9
2.4
aIC50 values for inhibition of the growth of T. b. brucei 427 or for cytotoxicity toward L929
mouse fibroblasts. Each value is the mean of ≥2 independent determinations  standard
deviation. bHalf life in mouse liver S9 fraction (ND: not determined). cKinetic aqueous
solubility in pH 7.4 PBS. dPermeability of compounds (at 3 µM) in an MDCK-MDR1 cell
monolayer assay (A to B) in the presence or absence of the P-gp inhibitor GF120918 (2 µM);
AQ is the absorption quotient, as defined by the equation: AQ = (Papp+918 - Papp) / Papp+918.
In this assay, the CNS positive drug propranolol gave Papp 556 nm/s and 4 had Papp 732 nm/s.
4

Medium-throughput screening and follow-up IC50 testing at Scynexis14 identified 48 active
hits (IC50 <3 µg/mL), of which 19 were initially considered to be of potential interest (mean
IC50 <1 µg/mL, with selectivity index >10). Intriguingly, the most active compounds (9-12;
Table 1) were either 2-nitroimidazothiazine oxides15 or 6-nitroimidazothiazole oxides,16 but a
wide variety of other structures, including extended side chain analogues of 6, featured in this
set. Since good CNS penetration is a critical requirement for the effective treatment of stage 2
HAT,9 the 10 most potent hits were first evaluated for cell permeability in the MDCK-MDR1
assay. In this test system, apparent permeability (Papp) values ≥150 nm/s are indicative of
high brain penetration potential provided that the transport is not affected by P-gp inhibition17
(necessitating an absorption quotient in the range -0.1 to 0.1). Unsurprisingly, the compound
with a triaryl side chain (13, MW>500) lacked any significant permeability (Papp <0.8 nm/s),
while four others (10, 14, 16, and 18)15,18,19 gave only modest permeability values (Papp <150
nm/s) and were suggested to be P-gp substrates (absolute AQs ≥0.3).20 On the basis of results
from this training set, additional hits were selected for assessment (19-23)15,16,19 and,
pleasingly, all of these demonstrated a high propensity to cross the blood-brain barrier.
In order to determine the suitability of the more permeable hits for in vivo efficacy studies,
we first measured their aqueous solubilities, and their tendencies to metabolise, following a 1
h incubation with CD-1 mouse liver S9 subcellular fractions.14 Here, the most poorly soluble
compounds (11, 15, 20, and 23) were also found to be the least stable, displaying half-lives of
less than 70 min. Overall, the 2-nitroimidazothiazine oxides 9, 12 and 19,15 together with the
6-amino-linked analogue of 6 (17),21 provided the best balance of potency, stability, aqueous
solubility and CNS penetration potential. This led us to probe their in vivo pharmacokinetic
(PK) profiles in mice, examining concentration levels in plasma, whole blood and brain tissue
following both intravenous and oral administration (Table 2; for further experimental details,
see the Supporting Information).
Table 2. Mouse pharmacokinetic parameters for selected compounds
Compd Intravenous (0.5-3 mg/kg)a
Oral (50-80 mg/kg)a
Cmax Tmax t1/2
(µg/mL) (h)
CL
Vdss
t1/2
Fb
(%)
AUClast
AUClast
(µgh/mL)
(L/h/kg) (L/kg) (h)
(h)
(µgh/mL)
Plasma
0.97
0.52
6.8
0.59
1.6
4.7
3.6
0.43 0.505
2.5 2.09
0.48 0.418
2.5 1.80
0.20
9.3
12
2
4
1
2
3.7
6.9
1.2
2.7
0.869
51.9
20.5
18.7
1.4
55
100
42
9
12
17
19
1.0
2.3
Whole blood
0.87
0.39
2.4
0.33
1.7
8.1
2.8
0.42 0.489
0.11
8.6
26
2
4
0.5
2
5.3
5.8
1.5
3.3
0.494
63.5
42.2
23.7
0.8
52
100
37
9
9.3
9.3
5.9
2.70
1.10
2.60
12
17
19
Brain
9
12
17
19
0.69
3.0
1.5
0.40
2.9
1.2
1.3
2.3
9.1
1.6
2.3
0.304
2.85
0.06
14
35
2
4
0.5
4
3.2
6.9
1.3
3.0
0.290
71.2
50.9
18.8
0.8
55
100
43
0.26 0.962
14 1.77
0.51
2.4
aThe corrected intravenous doses for 9, 12, 17 and 19 were 0.5, 1.1, 2.9 and 2.0 mg/kg,
respectively, and the corresponding oral doses were 62, 50, 78 and 49 mg/kg, respectively.
bOral bioavailability, determined using dose normalised AUClast values.
5

10
1
Plasma - PO
Blood - PO
Brain - PO
Plasma - IV
Blood - IV
Brain - IV
0.1
0.01
0
5
10
15
20
25
Time (h)
Figure 2. Time vs concentration curves for 12, following administration to male CD-1 mice
(at 50 mg/kg po and 1.1 mg/kg iv). The horizontal line represents the MIC for complete
inhibition of visible parasite growth in vitro.
The most potent hit (9) exhibited an unacceptable PK profile, giving inadequate oral exposure
and poor oral bioavailability (<1.5%), consistent with both its low solubility (causing
unsatisfactory absorption) and more rapid metabolism. This was unsurprising, as the 4-
benzyloxybenzyl analogue of 6 was known to be markedly inferior to 6 against
Mycobacterium tuberculosis in vivo, despite being an order of magnitude more potent than 6
in vitro, due to similar PK issues.22 In contrast, the 4-trifluoromethoxybenzyl congener of 9
(12) demonstrated the slowest rate of clearance of the four, and a prolonged, high exposure
level above the MIC following oral dosing (Fig. 2), with good oral bioavailability (52-55%)
at all three sampling sites. Moreover, the high brain:plasma concentration ratio (~3:2)
presented by 12 was encouraging for CNS uptake, as required in the treatment of stage 2
HAT.14 The sulfone derivative of 12 (19), which was produced to a significant extent in PK
samples from the analysis of 12, showed reduced oral exposure, in accordance with its
inferior solubility and faster rate of clearance. Given its weaker potency (5.6-fold vs 12),
these results for 19 were not predictive of good in vivo activity, thus in situ oxidation of 12
should have a minimal contribution to efficacy. Finally, the most soluble hit 17 (the 6-amino
analogue of 6) was notable for having the best oral bioavailability, with excellent
concentration levels observed in brain tissue (2- to 3-fold higher than in plasma). However,
this compound also suffered from a high rate of clearance and a rather short oral half-life
(1.2-1.5 h), leading to inadequate exposure above the MIC beyond ~2 h. These latter results
mirrored findings from a recently reported PK-PD study of analogues of 6 against TB, in
which 17 displayed a 1.3 h oral half-life in mouse lung tissue (in comparison to 4.8 h for 6),23
effectively precluding useful in vivo activity. Hence, of the four most promising hits, only the
2-nitroimidazothiazine oxide 12 proved to be suitable for efficacy assessment in the acute
infection mouse model of HAT.
One remaining matter to resolve with racemic hit 12 was which one of the four possible
stereoisomers was the most active chiral form. This issue was partially clarified through a
better optimised resynthesis of 12 (Scheme 1). Following side chain attachment to the
racemic alcohol 4215 (93% yield), careful oxidation of thiazine 30 with fresh m-CPBA (1.01
equiv) led to a separable mixture of 12 (75%) and a previously unidentified more polar
racemic diastereomer 38 (20%) (for experimental details, see the Supporting Information).
6

Scheme 1. Reagents and conditions: (i) 4-OCF3BnBr, NaH, DMF, 20 C, 160 min (93%); (ii)
m-CPBA, Na2HPO4, CH2Cl2, -10 to 20 C, 19 h (12: 75%, 38: 20%); (iii) preparative chiral
SFC (see text).
The 1H NMR spectra of 12 and 38 showed pronounced chemical shift differences for the H-6
resonance in particular, which was ~0.4 ppm further downfield in the spectrum of 12. The
sulfoxide oxygen in six-membered rings is known to exhibit an axial preference, such that the
deshielding effect of the sulfoxide group on axial β-hydrogen atoms has been used to assign
relative stereochemistry.24 Hence, 12 is postulated to have the sulfoxide oxygen and H-6 in a
pseudo-diaxial orientation, placing the (4-OCF3)benzyloxy side chain at C-6 in a 1,3-trans
relationship to the sulfoxide oxygen. This assignment is supported by the diastereomer ratio
(3.5:1) in favour of 12, which might be rationalised by an expected preference for the C-6
side chain to adopt a pseudoaxial conformation in the thiazine precursor 30 (based on the
crystal structure of 6),25 providing a steric disincentive to formation of the cis sulfoxide 38.
Preparative chiral SFC separation of the enantiomers of 12 (24 and 26) and 38 (25 and 27)
facilitated the assessment of all four stereoisomers (Table 3). The C-6 configuration of 24 and
25 was later firmly established via a known26 chiral synthesis. The most active (6S) form 24
(IC50 0.07 µg/mL) was 40-fold more potent than cis isomer 25, and more than 70-fold more
potent than its (6R) enantiomer 26. This level of potency compared well with data reported
for 5 (IC50 0.29 µg/mL vs T. b. brucei 427) in the same Scynexis assay.9 Compound 24 also
displayed an improved selectivity index (>143), good aqueous solubility (106 µg/mL), and
excellent metabolic stability following a 1 h exposure to human and mouse liver microsomes
(respectively, 82% and 96% parent remaining).
Therefore, 24 was examined in a stage 1 HAT mouse model.14 Briefly, dosing was orally
once daily for four days, starting 24 h postinfection, and parasitemia was assessed weekly via
tail vein blood smears (see the Supporting Information). Excellent activity was observed
(Table 4), with 24 providing complete cures (i.e. parasite free blood smears after >30 days) to
all mice at doses as low as 5 mg/kg, similar to the control drug pentamidine (given i.p. at 2
mg/kg), whereas the vehicle only mice died on day 7. The efficacy seen with 24 in this model
was equivalent to the level of activity reported for 5 and ~20-fold superior to the results
described for fexinidazole (4),8,9 stimulating further evaluation of this lead in a stage 2 HAT
mouse model.14 Here, oral dosing of 24 (at 12.5 to 50 mg/kg once daily for seven days from
day 21 postinfection) led to significant increases in survival times in comparison to untreated
controls (66-70 days vs 31 days; Table 5), although cure rates were inadequate (0-20%). In
contrast, 5 was 100% curative in the same CNS model at a dosage of 25 mg/kg once daily for
7 days,9 while 4 gave an 88% cure rate in a comparable model when administered orally at
200 mg/kg once daily for 5 days.8
7

Table 3. In vitro potency and microsomal stability of the enantiomers of sulfoxides 12 and 38
(by convention,30 the sulfur-oxygen double bond has been depicted as a chiral single bond)
Microsomesb (%
Compd IC50 (µg/mL)a
remaining at 1 h)
T. b. brucei L929 Human
Mouse
96
93
26
89
>10
24
25
26
27
82
93
91
88
0.070  0.005
>10
>10
>10
2.8  0.4
>5
>5
aIC50 values for inhibition of the growth of T. b. brucei 427 or for cytotoxicity toward L929
mouse fibroblasts. Each value is the mean of 2 independent determinations  standard
deviation. bPooled human or CD-1 mouse liver microsomes.
Table 4. In vivo activity of 24 in a T. b. brucei (EATRO 110) acute infection mouse model
Dosagea Mean survival Cured/ Cured
Compd
(mg/kg) (days)
Total
5/5
5/5
5/5
4/4
1/4
0/4
3/3
0/3
(%)
100
100
100
100
25
0
100
0
50
25
12.5
5
2.5
1.25
>30
>30
>30
>30
13
7.5
>30
7
24
24
24
24
24
24
Pentamidine 2
Vehicleb
aDosing of 24 was orally, once daily for 4 days consecutively, while pentamidine was dosed
i.p. once daily for the same period. bVehicle for 24: 0.8% CMC, 0.1% SDS in water.
Table 5. In vivo activity of 24 in a T. b. brucei (TREU 667) CNS infection mouse model
Dosagea Mean relapse Cured/ Cured
Compd
(mg/kg)
50
25
time (days) Total
(%)
0
20
0
100
0
0
66
70
45
0/10
2/10
0/8
24
24
24
Berenil
Berenil
Vehiclec
12.5
10 (D4)b
5/5
0/5
0/5
10 (D21)b 41
31
aDosing of 24 was orally, once daily for 7 d consecutively, starting on day 21 post-infection.
bSingle i.p. dose on day 4 or day 21. cVehicle for 24: 0.8% CMC, 0.1% SDS in water.
8

Detailed in vivo studies in the benzoxaborole 6-carboxamide class have revealed that efficacy
in the CNS model is heavily dependent upon the maintenance of drug concentrations in the
brain for at least 15 h at levels above the MIC (defined as the lowest compound concentration
that completely inhibits visible parasite growth in vitro after a 72 h incubation).27,28 Thus, a
more potent analogue of 5 without the gem-dimethyl group (SCYX-6759) required an oral
dosing regimen of 50 mg/kg twice daily (b.i.d.) in order to obtain an 83% cure rate of the
CNS infection,14 due to the shorter time that its brain concentration level was at or above the
MIC (~12 h vs ~24 h for 5 at 25 mg/kg9). These findings imply that a similar oral dosing
regimen of 50 mg/kg b.i.d. might be required to achieve useful efficacy for 24 in the stage 2
HAT model (via more prolonged CNS exposure). Nevertheless, these initial in vivo results
with 24 were still regarded as encouraging, and indicated that 2-nitro-6,7-dihydro-5H-
imidazo[2,1-b][1,3]thiazine 8-oxides merited further investigation as potential treatments for
HAT. Specifically, as illustrated with benzoxaboroles, we considered the possibility of
designing new analogues of 24 having improved potency and extended CNS exposure. On
the basis of the results above and insights from previous SAR studies directed at developing a
backup TB candidate to the structurally related nitroimidazooxazine 6,19,29 we devised two
preliminary strategies for optimisation of the side chain of 24: a) removal of the benzylic
methylene group and b) insertion of a proximal pyridine ring (cf. 7). Notably, both strategies
had the potential to improve metabolic stability,19,29 leading to longer in vivo half-lives and
better exposure levels. Furthermore, to mitigate any reduction in solubility with the first
approach, we also proposed the preparation of a trifluoromethylpyridinyl ether analogue (cf.
21 and 22).
The synthetic methods employed to prepare the new nitroimidazothiazine derivatives (28, 29,
32-37, 39-41) are outlined in Scheme 2. Mitsunobu coupling of the orthogonally diprotected
triol 4431 with 4-(trifluoromethoxy)phenol and conversion of the product 45 to iodide 47 (via
successive hydrogenolysis of the benzyl ether and iodination using I2/PPh3/imidazole) set the
stage for the preparation of phenyl ether 28 (Scheme 2A). Thus, base-assisted alkylation of 2-
chloro-4-nitroimidazole with iodide 47, followed by desilylation (TBAF), provided the key
alcohol 49 (73%, 2 steps). Then, reaction of the tosylate derivative of 49 (50) with the lithium
salt of triisopropylsilanethiol and treatment of the crude product with TBAF enabled
cyclisation to thiazine 28 (31%). Finally, careful oxidation of 28 with fresh m-CPBA (1.2
equiv) led to a separable mixture of sulfone 40 (11%) and the diastereomeric sulfoxides 33
and 36 (82% and 2%), where the sizeable diastereomer ratio (dr ~34:1) was in accordance
with the greater steric hindrance induced by this phenoxy side chain. Thiazine pyridinyl ether
29 was more directly accessed via a sodium hydride-induced SNAr reaction of thiazine
alcohol 4215 with 2-chloro-5-trifluoromethylpyridine (52) (69%; Scheme 2B), while
alternative alkylation of 42 with 5-bromo-2-(bromomethyl)pyridine29 (53), followed by
Suzuki coupling with 4-(trifluoromethoxy)phenylboronic acid, furnished the extended side
chain thiazine 32 (35% over 2 steps; Scheme 2C). However, whereas m-CPBA oxidation of
29 proved straightforward, similar oxidation of 32 was complicated by the formation of
smaller amounts of pyridine N-oxide derivatives, such that only the sulfoxides 35 and 39
(55% and 8%) could be obtained. All new compounds (Table 6) were characterised by 1H
NMR, MS, melting point, and combustion analysis (or HRMS and HPLC); full synthetic
procedures and characterisation data have been provided in the Supporting Information.
9

Scheme 2. Reagents and conditions: (i) TIPSCl, imidazole, DMF, 20 C, 3 d (92%); (ii) 4-
OCF3PhOH, PPh3, DEAD, THF, 0-20 C, 4.5 d (75%); (iii) H2, 10% Pd-C, EtOH, EtOAc, 2
d (98%); (iv) I2, PPh3, imidazole, CH2Cl2, 20 C, 15 h (98%); (v) 2-chloro-4-nitroimidazole,
K2CO3, DMF, 85 C, 64 h (88%); (vi) TBAF, THF, 0-5 C, 5 h (83%); (vii) TsCl, pyridine,
0-20 C, 25 h (50: 84%; 51: 9%); (viii) LiSTIPS, THF, -78 to 20 C, 2 d, then TBAF, THF,
20 C, 13 h (31%); (ix) m-CPBA, Na2HPO4, CH2Cl2, -10 to 20 C, 23-52 h (33: 82%, 36: 2%,
40: 11%; 34: 60%, 37: 11%, 41: 28%; 35: 55%, 39: 8%); (x) NaH, DMF, 0-20 C, 3-3.5 h
(29: 69%; 54: 79%); (xi) 4-OCF3PhB(OH)2, toluene, EtOH, DMF, 2M Na2CO3, Pd(dppf)Cl2
under N2, 84 C, 4.5 h (44%).
The new compounds and relevant comparators were screened at the University of Antwerp
against a panel of four protozoan parasites (T. b. brucei, T. b. rhodesiense, T. cruzi, and L.
infantum); cytotoxic effects on human lung fibroblasts (MRC-5 cells, the host for T. cruzi)
were also assessed.32 In all cases, recorded data (Table 6) are mean values derived from two
or more independent experiments. For the parent thiazines (28-32), antitrypanosomal potency
was enhanced by an order of magnitude with biaryl side chains (d and e), and this SAR
pattern was maintained for the considerably less lipophilic major sulfoxide disastereoisomers
(Ba-e), where 35 was the most impressive new HAT lead (T. b. brucei IC50 0.030 µM). This
lead was also highly effective against Chagas disease (T. cruzi IC50 0.067 µM) and was the
only compound to display submicromolar antileishmanial activity (L. inf IC50 0.41 µM). In
contrast, shorter linked aryl ether sulfoxides 33 and 34 were 4- to 6-fold less potent than the
initial hit 12 against T. b. brucei, while their sulfone derivatives (40 and 41) were an order of
magnitude inferior to sulfone 19, indicating that the original (OCH2) linkage was best. In
comparison to 12 (the racemic form of lead 24), racemic sulfoxide 35 displayed a 9-fold
greater potency against T. b. brucei, an 11-fold higher potency against T. b. rhodesiense, and
a 2.4-fold better selectivity index (MRC-5 IC50 >500 times larger than the HAT IC50).
Compound 35 also demonstrated acceptable aqueous solubility (9.9 µg/mL at pH 7 and 1260
µg/mL at pH 1), high permeability potential without P-gp mediated efflux (MDCK-MDR1
cell Papp A-B/B-A 117/182 nm/s cf. Papp A-B of 197 nm/s for the CNS positive drug
propranolol in the same assay) and very good stability toward human and mouse liver
microsomes (respectively, 78% and 72% parent remaining after 1 h). While we have not yet
had the opportunity to evaluate 35 beyond this stage, these promising results certainly point
to the viability of this SAR approach to provide useful new HAT leads.
10

Table 6. In vitro antiparasitic activities and calculated lipophilicities of 2-nitro-6,7-dihydro-
5H-imidazo[2,1-b][1,3]thiazine analogues
Compd Form CLogPa IC50 (µM)b
T. b. bruc T. b. rhod T. cruzi
L. inf
7.0
10
55
45
7.5
7.0
13
16
10
MRC-5
51
>64
>64
>64
21
23
>64
60
50
>64
64
16
>64
30
Aa
Ab
Ac
Ad
Ae
Ba
Bb
Bc
Bce
Bcf
Bd
Be
Ca
Cb
Cc
Cce
Ccf
Ce
Da
Db
Dc
Dd
2.83
2.68
3.05
3.67
3.39
1.19
1.05
1.42
1.42
1.42
2.04
1.76
1.19
1.05
1.42
1.42
1.42
1.76
1.50
1.36
1.73
2.35
40
59
2.2
5.3
1.1
0.49
1.4
2.1
4.1
1.5
1.4
6.5
0.12
0.067
7.7
12
28
29
30c
31c
32
33
34
12c
24d
26
9c
35
36
37
38
25
27
39
40
41
19c
10c
>64
44
>64
36
1.4
1.2
4.3
2.1
1.6
1.2
0.27
0.14
34
0.98
0.51
0.25
0.13
7.3
41
0.030
0.030
55
0.027
0.023
27
3.4
0.41
>64
41
17
9.9
5.6
5.6
16
0.075
15
19
1.1
0.097
5.1
1.9
4.3
0.10
13
11
0.94
0.027
9.1
13
13
0.14
3.5
2.6
1.6
0.35
>64
>64
48
2.0
32
30
>64
7.3
>64
>64
>64
54
>64
46
>64
>64
aCalculated lipophilicities derived from ACD LogP software (v 14.04). bIC50 values for
inhibition of growth of the parasites T. b. brucei 427, T. b. rhodesiense, Trypanosoma cruzi,
and Leishmania infantum, or for cytotoxicity toward human lung fibroblasts (MRC-5 cells).
Each value is the mean of 2 to 5 independent determinations. For complete results (mean 
SD), refer to the Supporting Information. cRef. 15. dRef. 26. e(6S)-Enantiomer. f(6R)-
Enantiomer.
In summary, this investigation set out to evaluate a nitroimidazole-based compound library
related to pretomanid for possible utility against HAT. Although the hit rate was low (~2%),
several compounds displayed good metabolic stability, adequate solubility, and excellent
CNS penetration potential. Comprehensive mouse pharmacokinetic studies of three oxidised
nitroimidazothiazines and a 6-amino-linked analogue of pretomanid identified the racemic
thiazine oxide 12 as a suitable candidate for in vivo efficacy studies. The most potent
11

stereoisomer of 12 (24) was indeed highly efficacious in the stage 1 HAT mouse model with
once daily oral dosing (similar to oxaborole 5), but was less effective in a stage 2 model.
While it seemed reasonable to speculate that more frequent dosing with 24 should achieve
better outcomes in this latter model, we also envisaged the generation of new analogues with
higher potency and longer half-lives. In preliminary SAR work, we noted that removal of the
benzylic methylene was disfavoured but that adding a proximal pyridine ring (35) enhanced
potency while broadly retaining other essential properties. These additional findings are very
encouraging and provide a rational foundation for further development of this interesting
class of antitrypanosomal agents.
Acknowledgments
The authors thank the Drugs for Neglected Diseases initiative for financial support through a
collaborative research agreement. For this project, DNDi received financial support from the
following donors: Department for International Development (DFID), UK; Federal Ministry
of Education and Research (BMBF), through KfW Germany; Directorate-General for
International Cooperation (DGIS), The Netherlands; Bill & Melinda Gates Foundation
(BMGF), USA; Médecins Sans Frontières (MSF), International. The donors had no role in
study design, data collection and analysis, decision to publish, or preparation of the
manuscript. The authors also thank Dr Bakela Nare and Ms Tana Bowling (Scynexis) for IC50
data, Sisira Kumara (ACSRC) for some solubility measurements, and Donna Sarno, Elena
Mejia and Wendy Becker (Pace University) for technical assistance.
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Graphical abstract
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