Urea Derivatives of 2-Aryl-benzothiazol-5-amines: A New Class of Potential Drugs for Human African Trypanosomiasis

Article abstract of DOI:10.1021/acs.jmedchem.6b01163

A previous publication from this lab (Patrick, et al. Bioorg. Med. Chem. 2016, 24, 2451-2465) explored the antitrypanosomal activities of novel derivatives of 2-(2-benzamido)ethyl-4-phenylthiazole (1), which had been identified as a hit against Trypanosoma brucei, the causative agent of human African trypanosomiasis. While a number of these compounds, particularly the urea analogues, were quite potent, these molecules as a whole exhibited poor metabolic stability. The present work describes the synthesis of 65 new analogues arising from medicinal chemistry optimization at different sites on the molecule. The most promising compounds were the urea derivatives of 2-aryl-benzothiazol-5-amines. One such analogue, (S)-2-(3,4-difluorophenyl)-5-(3-fluoro-N-pyrrolidylamido)benzothiazole (57) was chosen for in vivo efficacy studies based upon in vitro activity, metabolic stability, and brain penetration. This compound attained 5/5 cures in murine models of both early and late stage human African trypanosomiasis, representing a new lead for the development of drugs to combat this neglected disease.

Full text of DOI:10.1021/acs.jmedchem.6b01163

Article
pubs.acs.org/jmc
Urea Derivatives of 2‑Aryl-benzothiazol-5-amines: A New Class of
Potential Drugs for Human African Trypanosomiasis
Donald A. Patrick,† J. Robert Gillespie,§ Joshua McQueen,§ Matthew A. Hulverson,§
Ranae M. Ranade,§ Sharon A. Creason,§ Zackary M. Herbst,§ Michael H. Gelb,‡,∥ Frederick S. Buckner,§
and Richard R. Tidwell*,†
†Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, United States
§
∥
‡Departments of Chemistry, Medicine, and Biochemistry, University of Washington, Seattle, Washington 98195, United States
S
* Supporting Information
ABSTRACT: A previous publication from this lab (Patrick, et al. Bioorg. Med. Chem. 2016, 24, 2451−2465) explored the
antitrypanosomal activities of novel derivatives of 2-(2-benzamido)ethyl-4-phenylthiazole (1), which had been identified as a hit
against Trypanosoma brucei, the causative agent of human African trypanosomiasis. While a number of these compounds,
particularly the urea analogues, were quite potent, these molecules as a whole exhibited poor metabolic stability. The present
work describes the synthesis of 65 new analogues arising from medicinal chemistry optimization at different sites on the
molecule. The most promising compounds were the urea derivatives of 2-aryl-benzothiazol-5-amines. One such analogue, (S)-2-
(3,4-difluorophenyl)-5-(3-fluoro-N-pyrrolidylamido)benzothiazole (57) was chosen for in vivo efficacy studies based upon in
vitro activity, metabolic stability, and brain penetration. This compound attained 5/5 cures in murine models of both early and
late stage human African trypanosomiasis, representing a new lead for the development of drugs to combat this neglected disease.
ea) for early stage and melarsoprol (an organoarsenical) for late
stage disease. First line treatments for T. b. gambiense infections
include pentamidine (an aromatic diamidine) for early stage
and nifurtimox−eflornithine combination therapy (NECT) for
late stage disease. Neither drug currently used against early
stage disease is able to cross the BBB.2,4,5
INTRODUCTION
■
Human African trypanosomiasis (HAT) is an insect vectored
disease caused by infection of either Trypanosoma brucei
gambiense or Trypanosoma brucei rhodesiense. Despite the
decreased incidence of the disease during the first decade of
this century,1 the World Health Organization (WHO) currently
estimates 20000 actual cases with 65 million people at risk and
that over 98% of cases are due to T. b. gambiense [http://www.
who.int/mediacentre/factsheets/fs259/en/]. The disease pro-
gresses from a first stage (hemolymphatic) infection to second
stage (CNS) infection after the parasites have crossed the
blood−brain barrier (BBB). The T. b. gambiense infection is
characterized by a slow progression of months or even years
from the early to late stage infection, while the T. b. rhodesiense
infection progresses more rapidly. Late stage HAT is always
fatal in untreated patients regardless of the causative parasite.2,3
The need for new anti-HAT drugs persists, as current drugs
are few, antiquated, toxic, prone to resistance, and require
parenteral administration. Treatments for T. b. rhodesiense
infections are limited to suramin (a polysulfonated naphthylur-
Pafuramidine,6,7 an oral prodrug of 2,5-bis(4-
amidinophenyl)furan (furamidine),8,9 advanced to phase III
clinical trials against early stage HAT but was abandoned after
the observation of nephrotoxicity in an expanded phase I trial.10
The nitrated imidazole derivative fexinidazole11 and the
benzoxaborole SCYX-715812 have also entered clinical trials
in recent years. The high attrition rate of clinical trial candidates
(around 90% of those entering phase I)13 underscores the need
for a pipeline of new trypanocidal compounds.
Following the failure of pafuramidine in clinical trials, the
emphasis of this lab has shifted from amidines to other classes
Received: August 1, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.6b01163
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Article
Table 1. Antitrypanosomal Activities of Thiazole Derivatives 1−28
T. brucei
a
,
b
c,d
compd
R1
R2
EC50 (μM)
0.191 (0.632)
EC90 (μM)
1
phenyl
phenyl
phenyl
0.340
0.188
2
pyrrolidin-1-yl
pyrrolidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
piperidin-1-yl
azepan-1-yl
phenyl
0.0848 (0.125)
0.0322 (0.0495)
0.0194 (0.0204)
0.0113 (0.0090)
0.0094 (0.0099)
0.0105 (0.0097)
0.110 (0.0516)
1.44
3
2-fluorophenyl
phenyl
0.0868
0.0336
0.0163
0.0117
0.0203
0.236
4
5
3-fluorophenyl
2,4-difluorophenyl
3,4-difluorophenyl
phenyl
6
7
8
9
phenyl
2.63
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
piperidin-1-yl
phenyl
0.0243
0.0424
0.0586
0.564
2-methylpiperidin-1-yl
cis-2,6-dimethylpiperidin-1-yl
4-oxopiperidin-1-yl
3-fluoropiperidin-1-yl
3,3-difluoropiperidin-1-yl
4-fluoropiperidin-1-yl
4,4-difluoropiperidin-1-yl
1,2,3,4-tetrahydroquinolin-1-yl
1,2,3,4-tetrahydroisoquinolin-2-yl
phenyl
3-fluorophenyl
3-fluorophenyl
3-fluorophenyl
3-fluorophenyl
3-fluorophenyl
3-fluorophenyl
3-fluorophenyl
3-fluorophenyl
3-fluorophenyl
phenyl
0.0370
0.399
0.667
0.813
0.0285
0.0557
0.168
0.108
0.0366
0.0575
0.230
0.164
5.95
14.0
13.8
>10
9.76
>10
phenyl
3-fluorophenyl
phenyl
>10
>10
>10
phenyl
>10
phenyl
phenyl
1.30
2.4
phenyl
phenyl
>10
>10
>10
pyrrolidin-1-yl
phenyl
>10
piperidin-1-yl
phenyl
7.84
9.9
>10
0.216
azepan-1-yl
phenyl
>10
piperidin-1-yl
3-fluorophenyl
0.0946
a
Concentration of compound required to inhibit growth by 50% (EC50) against T. b. brucei BF427 or T. b. rhodesiense STIB900 (numbers in
parentheses, data reported previously14). Control for T. b. brucei BF427 EC50 assay average standard error of the mean (SEM): pentamidine
(1.05 0.12 nM, n = 30). Concentration of compound required to inhibit growth by 90% (EC90) against T. b. brucei BF427. Control for T. b.
brucei BF427 EC90 average SEM: pentamidine (3.54 0.36 nM (n = 30).
b
c
d
of compounds. A phenotypic high-throughput screen (HTS) of
a 700000 compound library performed by the Genomics
Institute of the Novartis Research Foundation (GNF)3 led to
the identification of over 1000 compounds (grouped into over
100 distinct scaffolds) that inhibited growth of T. brucei in vitro
at concentrations below 3.6 μM and were nontoxic to
mammalian cells (Huh7). Promising results based on the
synthesis and in vitro activity of analogues of one of these
scaffolds, an oxazolopyridine derivative, have been reported.3
Another hit from this HTS was 2-(2-benzamido)ethyl-4-
phenylthiazole (1, Table 1). A number of analogues of this
compound were highly potent against the parasite but had poor
metabolic stability.14 This paper describes continued work on
the latter scaffold.
8).14 Traditional medicinal chemistry approaches for hit to lead
optimization were followed as the biochemical targets of these
compounds were unknown. These derivatives had alterations
primarily at either of the two terminal rings, but the SAR of the
internal portion of the molecule remained largely unexplored.
The present work explores modification of other sites of the
molecule with the syntheses of 65 novel compounds. The new
compounds 9 and 10 are thiocarbonyl analogues of existing
compounds 1 and 4. Analogues 11−19 are derivatives of the
highly potent urea 5 bearing various substituents on the
piperidine ring. Modification of the ethylene linker, either by
alkylation or unsaturation, gave rise to compounds 20−28.
Further alteration of the internal portion of the molecule led to
fused ring analogues including benzimidazole 29, benzoxazoles
30−32, and benzothiazoles (33−73, Table 2). In all cases
except analogues 71−73, the three-carbon bridge between the
amide and thiazole nitrogen atoms of 1 is retained.
RESULTS AND DISCUSSION
■
Chemistry. The previous report describes the syntheses of
the initial hit (1, Table 1) and over 70 analogues (including 2−
B
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a
Table 2. Antitrypanosomal Activities of Fused Ring Derivatives 29−73
T. b. brucei
a,
b
c,d
compd
R1
R2
EC50 (μM)
EC90 (μM)
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
61
62
63
64
65
66
67
68
69
70
71
72
73
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
>10
>10
phenyl
>10
>10
pyrrolidin-1-yl
piperidin-1-yl
phenyl
5.25
>10
2.80
9.30
7.74
2.13
2.49
>10
2.38
thiophen-2-yl
thiophen-3-yl
thiazol-2-yl
thiazol-4-yl
pyrrolidin-1-yl
thiazolidin-3-yl
piperidin-1-yl
0.882
1.57
5.86
>10
>10
0.325
1.24
0.817
2.38
0.809
1.35
3.00
0.895
0.737
3.62
0.391
0.602
7.55
>20
0.366
1.12
1,2,3,6-tetrahydropyridin-1-yl
phenyl
4-fluorophenyl
1.92
pyrroldin-1-yl
4-fluorophenyl
0.375
0.427
1.78
piperidin-1-yl
4-fluorophenyl
phenyl
3-fluorophenyl
pyrrolidin-1-yl
3-fluorophenyl
0.158
0.174
3.15
piperidin-1-yl
3-fluorophenyl
4-methylpiperazin1-yl
piperazin-1,4-diyl
3-fluorophenyl
3-fluorophenyl
>20
pyrrolidin-1-yl
2-fluorophenyl
0.983
0.935
0.183
0.929
0.0918
0.0519
0.326
0.0348
0.934
0.114
1.69
1.24
1.14
0.394
1.48
0.181
0.0919
0.491
0.0548
1.66
0.747
4.88
4.67
2.72
1.95
13.6
4.59
0.282
0.596
4.17
>20
piperidin-1-yl
2-fluorophenyl
pyrrolidin-1-yl
2,3-difluorophenyl
2,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
H
pyrrolidin-1-yl
pyrrolidin-1-yl
3-fluoropyrrolidin-1-yl
(R)-3-fluoropyrrolidin-1-yl
(S)-3-fluoropyrrolidin-1-yl
3,3-difluoropyrrolidin-1-yl
(3R,4R)-3,4-difluoropyrrolidin-1-yl
3-(trifluoromethyl)pyrrolidin-1-yl
3-cyanopyrrolidin-1-yl
3-aminopyrrolidin-1-yl
3-(dimethylamino)pyrrolidin-1-yl
3-hydroxypyrrolidin-1-yl
3-methoxypyrrolidin-1-yl
piperidin-1-yl
2.85
2.32
1.70
7.68
3.05
0.223
0.341
2.98
4-fluoropiperidin-1-yl
4,4-difluoropiperidin-1-yl
pyrrolidin-1-yl
>20
pyrrolidin-1-yl
Me
>20
>20
3-fluoropyrrolidin-1-yl
3-fluoropyrrolidin-1-yl
3-fluoropyrrolidin-1-yl
3,4-difluorophenyl
3,4-difluorophenyl
3,4-difluorophenyl
>20
>20
2.23
>20
2.64
5.28
a
Concentration of compound required to inhibit growth by 50% (EC50) against T. b. brucei BF427 or T. b. rhodesiense STIB900 (numbers in
parentheses, data reported previously14). Control for T. b. brucei BF427 EC50 assay average standard error of the mean (SEM): pentamidine
(1.05 0.12 nM, n = 30). Concentration of compound required to inhibit growth by 90% (EC90) against T. b. brucei BF427. Control for T. b.
b
c
d
brucei BF427 EC90 average SEM: pentamidine (3.54 0.36 nM (n = 30).
C
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a
The treatment of amide 1 with Lawesson’s reagent in THF
gave thioamide 9 (Scheme 1). The reaction of amine 74,14
Scheme 2. Synthesis of Compounds 20 and 21
a
Scheme 1. Synthesis of Thiazole Derivatives 11−19
a
Reagents and conditions: (a) NaN3, concd HCl, Et3N, DCM; (b)
NH2OH·HCl, aq NaHCO3; (c) Ac2O, reflux; (d) H2, 10% Pd/C,
Boc2O, MeOH; (e) NaHS·xH2O, MgCl2, 15-crown-5, DMF,
appropriate bromomethyl ketone, EtOH, reflux; (g) benzoyl chloride,
Et3N, THF.
hydroxylamine, and the resulting oxime was dehydrated to
give 3-azido-3-methylbutanenitrile using acetic anhydride. The
azide underwent catalytic hydrogenation (60 psi, 10% Pd/C,
MeOH) in the presence of di-tert-butyl dicarbonate to afford 3-
boc-amino-3-methylbutanenitrile. The nitrile was converted to
thioamide 78 using sodium hydrosulfide hydrate, magnesium
chloride, and 15-crown-5 in DMF.14,18 This intermediate was
reacted with 2-bromoacetophenone or 2-bromo-3′-fluoroace-
tophenone in refluxing ethanol (effecting thiazole ring closure
and nitrogen deprotection) to give amines 79 and 80, which
precipitated from the reaction mixture as their hydrobromide
salts. Benzoylation of the amines (benzoyl chloride, Et3N,
THF) gave target compounds 20 and 21.
Ethyl cyanoacetate (81, Scheme 3) underwent methylation
(CH3I, Ce2CO3, DMF) to its 2,2-dimethyl derivative19
followed by treatment with ammonia in methanol to give
cyano-amide 82.20 Catalytic hydrogenation of the nitrile (H2,
60 psi, 5% Rh/Al2O3, concd NH4OH, EtOH) to the amine
(avoiding the use of Raney nickel21 or circuitous pathways22)
followed by protection with di-tert-butyl dicarbonate gave the
amido-carbamate 83, which was reacted with Lawesson’s
reagent to afford thioamide 84. This intermediate was reacted
with 2-bromoacetophenone as above to give amine 85 as the
hydrobromide salt, which underwent acylation as above to
target compounds 22 and 23.
The synthesis of target compounds 24−27 (Scheme 4)
began with the conjugate addition of hydrazoic acid (NaN3,
concd HCl, DCM)16 to mesityl oxide (86) to give 4-azido-4-
methylpentan-2-one.23 Bromination of the α-methyl carbon
followed by a Hantzsch thiazole synthesis involving thiobenza-
mide and then catalytic hydrogenation of the azido group gave
amine 87, isolated as its HCl salt. The reaction of 87 with the
appropriate acyl halide as above gave benzamide 24, and ureas
25−26. The decreased reactivity of the sterically hindered
amine 87 allowed the use of triphosgene for selective formation
of its isocyanate derivative, which was then quenched with
azepane to prepare urea 27 in the absence of the less readily
available azepane-1-carbonyl chloride.
The preparation of compound 28 (Scheme 5) began with a
Hantzsch thiazole synthesis involving thioamide 88 (prepared
from the corresponding amide and P2S5)24 and α-bromo
ketone 89 followed by acidic hydrolysis of the acetal. The
resulting aldehyde was reacted with triethyl phosphonoacetate
and excess sodium hydride in THF by a modification of a
known procedure25 to give the vinyl acid 90. The acid was
treated with ethyl chloroformate in acetone in the presence of
Et3N followed by the addition of sodium azide26 to give acyl
a
Reagents and conditions: (a) Lawesson’s reagent, THF; (b) CS2,
Et3N, DCM, rt, and then Boc2O, DMAP, 0 °C; (c) piperidine, Et3N,
DCM; (d) carbonyl 1,1′-diimidazole, Et3N, DCM, 0 °C and then
appropriate 2° amine, 0 °C to rt; (e) appropriate 2° amine,
triphosgene, Et3N, DCM, 0 °C and then 69 (for 14 and 16).
Structures 9−17 are defined in Table 1.
carbon disulfide, and trimethylamine in DCM overnight,
followed by the addition of di-tert-butyl dicarbonate and
DMAP at 0 °C,15 give the crude isocyanate derivative, which
was reacted with piperidine to give thiourea 10, isolated as its
hydrochloride salt.
While piperidyl urea 5 was readily prepared by the reaction
of the primary amine 75 and piperidine-1-carbonyl chloride,14
alternate strategy was required for substituted piperidine ureas
11−19 where the corresponding N-carbonyl chlorides were not
readily available. The use of triphosgene to generate these
derivatives from substituted piperidines was generally unreliable
as the reactions proceeded very slowly and were difficult to
monitor in the absence of UV chromophores. For example, 4-
fluoropiperidine was reacted with triphosgene for 4 days before
the addition of 75 to afford urea 16. Attempts at preparing the
isocyanate derivative of 75 using triphosgene resulted in varying
amounts of the undesired symmetric urea, regardless of the
reaction conditions employed, due to the high reactivity of the
primary amine. The reaction of 69 with 1,1′-carbonyldiimida-
zole (CDI) in DCM in the presence of Et3N at 0 °C resulted in
the selective formation of the desired carbonylimidazolide
derivative in situ. Addition of the appropriate secondary amine
to the reaction mixture gave piperidine ureas 11−15, 17, and
tetrahydroisoquinoline urea 19. However, this method was not
amenable to analogue 18 (presumably due to steric hindrance),
which was successfully prepared from of 75 and the N-carbonyl
chloride of 1,2,3,4-tetrahydroquinoline generated in situ using
triphosgene.
The analogues with modified linkers include thiazole
analogues having geminal dimethyl groups on the carbon
adjacent to the amide nitrogen (20−21) or on the carbon
attached to the thiazole ring (22−23). Analogues 24−27 have
the methyl groups on the carbon next to the amide nitrogen as
well as a reversed substitution pattern on the thiazole ring.
Compound 28 incorporates a vinyl linker.
The syntheses of compounds 20 and 21 (Scheme 2) began
with the conjugate addition of hydrazoic acid (NaN3, concd
HCl, DCM)16 to 3-methylbut-2-enal (76) to obtain 3-azido-3-
methylbutanal (77).17 The aldehyde was reacted with
D
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a
Scheme 3. Synthesis of Compounds 22 and 23
a
Reagents and conditions: (a) CH3I, Cs2CO3, DMF; (b) NH3, MeOH; (c) H2, 60 psi, 5% Rh/Al2O3, concd NH4OH, EtOH; (d) Boc2O,
montmorillonite K10 (from base) or aq NaOH, DCM (from HCl salt); (e) Lawesson’s reagent, THF; (f) 2-bromoacetophenone, EtOH, reflux; (g)
appropriate acyl chloride, Et3N, DCM
a
Scheme 4. Synthesis of Compounds 24−27
a
Reagents and conditions: (a) NaN3, concd HCl, Et3N, DCM; (b) Br2, MeOH; (c) thiobenzamide, EtOH; (d) H2, 10% Pd/C, EtOH and then
EtOH/HCl; (e) appropriate acyl halide, Et3N, THF or DCM; (f) triphosgene, Et3N, DCE, −5 °C to rt and then azepane (for 27). Structures 24−27
are defined in Table 1.
a
Scheme 5. Synthesis of Compound 28
a
Reagents and conditions: (a) EtOH, rt, overnight; (b) aq HCl, acetone, reflux 1.5 h; (c) triethyl phosphonoacetate, NaH (excess), THF; (d)
ClCO2Et, Et3N, acetone, 0 °C and then NaN3; (e) toluene, reflux 1.5 h and then piperidine, Et3N, rt.
a
Scheme 6. Synthesis of Benzimidazole and Benzoxazole Derivatives 29−32
a
Reagents and conditions: (a) benzoyl chloride, Et3N, THF, −10 °C; (b) BF3·Et2O, dioxane, reflux, 3 h; (c) SnCl2·2H2O, concd HCl, reflux, 2 h; (d)
benzoyl chloride, Et3N, DCM, rt, overnight; (e) benzoic acid, PPA, 110−180 °C, 4 h; ( f) benzoyl chloride, Et3N, DCM, rt, overnight; (g)
triphosgene, Et3N, DCM, 0 °C and then appropriate 2° amine, 0 °C to rt, overnight. Structures 28−30 are defined in Table 2.
azide 91. A Curtius rearrangement of the azide to the vinyl
isocyanate was effected in refluxing toluene (in place of
benzene)26 followed by the addition of piperidine to give the
vinyl urea 28.
The syntheses of the benzimidazole and benzoxazole
derivatives are depicted in Scheme 6. The reaction of 4-
nitrobenzene-1,2-diamine (92) with benzoyl chloride (THF at
−10 °C) resulted in the selective formation of amide 93, which
underwent ring closure to benzimidazole 94 in the presence of
boron trifluoride etherate in refluxing dioxane. Reduction of the
nitro group using tin(II) dichloride in refluxing concentrated
HCl gave amine 95, which underwent benzoylation in DCM to
E
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a
Scheme 7. Synthesis of Benzothiazole Derivatives 33−70
a
Reagents and conditions: (a) appropriate benzoyl chloride, Py, rt, overnight; (b) HCO2H, reflux (for 99h); (c) Na2S·9H2O, S8, EtOH, reflux; (d)
Fe, NH4Cl, aq EtOH, reflux; (e) appropriate acyl halide or active ester, Et3N, DCM, rt, overnight (for amides); (f) triphosgene, Et3N, DCM, −5 °C
and then appropriate 2° amine, 0 °C to rt, overnight (for ureas).
a
Scheme 8. Synthesis of Compound 71
a
Reagents and conditions: (a) NaNO2, CuBr, aq HBr, 0 °C to rt; (b) 3,4-difluorophenylboronic acid, PdCl2(PPh3)2, PPh3, aq Na2CO3, DME, reflux
1.5 h; (c) Fe, NH4Cl, aq EtOH, reflux; (d) triphosgene, Et3N, DCM, −5 °C and then appropriate 2° amine, 0 °C to rt, overnight).
a
Scheme 9. Synthesis of Compounds 72 and 73
a
Reagents and conditions: (a) 3,4-difluorophenylboronic acid, Pd(dppf)Cl2·CH2Cl2, aq K2CO3, dioxane, reflux; (b) carbonyl 1,1′-diimidazole,
CH3CN rt; (c) 3-fluoropyrrolidine HCl, Et3N, DCM, rt; (d) phenyl chloroformate, Py, THF, 0 °C to rt; (e) 3-fluoropyrrolidine HCl, Py, reflux.
target compound 29, the HCl salt of the known free base.27 4-
Hydroxybenzene-1,3-diamine dihydrochloride (96) was reacted
with benzoic acid in PPA to give 2-phenyl-5-aminobenzoxazole
(97). Amine 97 underwent benzoylation to give amide 30 or
reaction with triphosgene and triethylamine in DCM followed
by addition of the appropriate secondary amine to obtain ureas
31 and 32.
The syntheses of benzothiazoles 33−70 (Scheme 7) began
with the acylation of 2-chloro-5-nitroaniline (98a) with the
appropriate benzoyl chloride in pyridine to obtain N-phenyl-
benzamides 99a−g. Formamide 99h28,29 was prepared from
bromoaniline 98b in refluxing formic acid. Amides 99a−h were
reacted with sodium sulfide nonahydrate and sulfur in refluxing
ethanol30 to give 5-nitrobenzothiazoles 100a−h. These
intermediates plus commercially available 100i were reduced
to the corresponding amines 101a−i using iron powder and
ammonium chloride in refluxing aqueous ethanol30 after the
failure to obtain 101a either by catalytic hydrogenation or
stannous chloride reduction of 100a. The target benzothiazole
amides 33−35, 37, 42, and 45 were prepared from amines
101a−c and the appropriate acyl chloride in the presence of
Et3N in DCM. Thiazole 2-carboxylic acid was treated with 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate) (HATU) in the
presence of Et3N in THF followed by the addition of amine
101a to obtain amide 36. Urea analogues 38−41, 43−44, and
46−70 were prepared by reaction the isocyanate derivatives of
primary amines 101a−i (generated in situ using triphosgene)
with the appropriate secondary amine. Initially (for 38, 40, 41,
and 43), the isocyantes were prepared by dropwise addition of
a solution of triphosgene (in DCM) to a solution of the
primary amine and triethylamine in DCM at −5 °C in order to
avoid formation of the undesired symmetric urea. In
subsequent reactions, solid triphosgene was added to the
reaction mixture in a single portion, producing the same result
provided the concentration of the primary amine was below 20
mM. The 3-aminopyrrolidinyl urea 62 was prepared from 3-
Boc-aminopyrrolidine and 101g, followed by amine depro-
tection using TFA. The hydrochloride salt of (3R,4R)-3,4-
difluoropyrrolidine,31 the secondary amine precursor to
F
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Article
analogue 59, was prepared in five steps from L-tartaric acid by
modification of known procedures.32−37
on the ethylene linker (20−27) generally resulted in no
detectable activity, regardless of the position of the methyl
groups or thiazole geometry. The introduction of a trans-double
bond in the linker resulted in an 8-fold loss of potency (5 vs
28).
Compound 71 (Scheme 8) is a regioisomer of 55 in which
the urea function is attached to the 6-position of the
benzothiazole system. A pathway to intermediate 103
analogous to that of 100g beginning with 2-chloro-4-nitroana-
line was unsuccessful with difficulties encountered at the ring
closure step. Intermediate 103 was prepared by diazotization−
bromination of 2-amino-6-nitro-benzothiazole (102) followed
by a Suzuki coupling with 3,4-difluorophenylboronic acid
catalyzed by tetrakis(triphenylphosphine)palladium(0). The
two remaining steps in the pathway are analogous to the
preparation of 55.
Compounds 72 and 73 (Scheme 9) are regioisomers of 71
and 55, respectively, in which the substitution patterns on the
benzothiazole system are reversed. A Suzuki coupling between
2-amino-6-bromobenzothiazole (105) and 3.4-difluorophenyl-
boronic acid catalyzed by Pd(dppf)Cl2 gave intermediate 106,
after the failure of reaction conditions using Pd(PPh3)4
analogous to those employed to prepare 103. Amine 106 was
reacted with CDI, followed by treatment of the resulting
intermediate with 3-fluoropyrrolidine hydrochloride and
trimethylamine in DCM to give urea 72. While the preparation
of intermediate 108 from 107 was analogous to that of 106
from 105, different conditions were required for the formation
of urea 73. Amine 108 was treated with phenyl chloroformate,
and the resulting carbamate was reacted with 3-fluoropyrroli-
dine hydrochloride in refluxing pyridine to give the desired
product 73.
In Vitro Antitrypanosomal Activity. Compound 1 (Table
1) and over 70 analogues (including 2−8) were tested
previously against T. b. rhodesiense.14 The potency of 1 was
enhanced 30-fold by replacement of the aromatic ring attached
to the carbonyl group (R1) with piperidine (urea analogue 4).
Smaller enhancements of activity were observed upon
fluorination of the opposite aromatic ring (R2) at any position
or in the 2,4- and 3,4-difluorophenyl derivatives. Incremental
enhancements were achieved by combining favorable mod-
ifications at both sites (e.g., 5, the most potent overall). Little
change in activity resulted when the substitution pattern on the
thiazole ring was reversed, as observed in multiple pairs of
regioisomers.
The new compounds were tested against T. b. brucei (a
different parasite). To provide a more accurate comparison of
activities of the new and existing compounds, a small number of
the existing compounds (1−8) were also tested against T. b.
brucei. A greater than 3-fold disparity between the EC50 values
against the two different parasites was observed for 1, but these
differences were less than 2-fold for 2−8.
Activities of the fused ring analogues 29−73 are shown in
Table 2. Neither the 5-benzamidobenzimidazole (29) nor
-benzoxazole (30) derivatives showed detectable activity, while
the corresponding benzothiazole 33 exhibited an EC50 value of
2.38 μM. The benzothiazole urea derivatives 38 (EC50 = 325
nM) and 40 (EC50 = 366 nM) were around seven times more
potent than amide 33 as well as 8−16 times more potent than
the corresponding benzoxazoles 31 and 32 (EC50 ≥ 2.8 μM).
After these findings, all further efforts were focused on the
benzothiazole derivatives. The thiophenyl amides 34 and 35
were slightly more potent than benzamide 33, but the thiazolyl
amides 36 and 37 were less potent, Of the amides 33−37, only
34 exhibited submicromolar potency but was still less potent
than urea 38. Insertion of a sulfur atom into the pyrrolidine ring
or a double bond into the piperidine ring resulted in 3−4-fold
losses of potency (38 vs 39, 40 vs 41).
The effect of fluorination of the aromatic ring attached to the
2-position of benzothiazole (R2) upon activity depended upon
the position of the fluorine atom(s). The potency of amide 33
was only slightly enhanced (less than 2-fold) by introduction of
either a 3-fluoro (42) or 4-fluoro (45) substituent. The potency
of pyrrolidyl urea 38 was either slightly diminished, enhanced
2-fold, or diminished 3-fold by introduction of a 4-, 3-, or 2-
fluoro substituent (43, 46 (EC50 = 158 nM), and 50,
respectively). A similar pattern was observed for piperidyl
urea 40, as seen in analogues 44, 47, and 51. The potency of
pyrrolidyl urea 46 bearing a 3-fluorophenyl group was
enhanced by the addition of a second fluorine atom at the 4-
position (54, EC50 = 91.8 nM). However, the 2,3-difluoro
analogue 52 was similar in potency to 46, while the 2,4-difluoro
isomer 53 was nearly six times less potent.
Further modifications were made to the urea portion of the
molecule. The replacement of piperidine with N-methylpiper-
azine resulted in an 18-fold loss of activity (47 vs 48). No
activity was detected with the piperazine-1,4-dicarboxamide
derivative 49.
The potency of pyrrolidyl urea 54 was slightly enhanced by
the introduction of a 3-fluoro substituent on the pyrrolidine
ring (55, EC50 = 51.9 nM). The (S)-enantiomer 57 (EC50
=
34.8 nM) proved to be the active stereoisomer, being nearly 10
times more potent than (R)-enantiomer 56 and the most
potent overall in this study. The 3,3-difluoro analogue 58 (EC50
= 934 nM was over 25 times less active than 57 and over 10
times less active than 54. The trans-3,4-difluoro derivative 59
(in which either of the two aliphatic fluorines has the same
spatial orientation as the aliphatic fluorine of 57) had an EC50
value of 114 nM, being about three times less potent than 57
but about eight times more potent than 58.
Replacement of the 3-pyrrolidyl fluorine atom of 55 with
other substituents led to decreased activity in all cases. The
trifluoromethyl analogue 60 was over 30 times less active. The
electron withdrawing cyano derivative 61 as well as the electron
donating amino, dimethylamino, hydroxyl, and methoxy
analogues (62−65) were all substantially less potent than 55,
with EC50 values between 1.5 and 8 μM.
None of the new thiazoles 9−28 showed enhanced potencies
relative to their existing counterparts. The replacement of the
carbonyl oxygen with sulfur resulted in an 8-fold loss of activity
(1 vs 9) or similar potency (4 vs 10). The potency of 5 was not
enhanced by the introduction of substituents on the piperidine
ring (11−19). Small losses of activity (around 3-fold) were
observed in compounds having of 2-methyl (11), 3-fluoro (14),
and 4-fluoro substituents (16) on the piperidine ring, all having
EC50 values below 50 nM. Greater losses of activity were
observed with the 2,6-dimethyl (12), 4-oxo (13), 3,3- and 4,4-
difluoro (15 and 17) analogues, with EC50 values between 100
and 700 nM. The tetrahydroquinoline (18) and -isoquinoline
(19) derivatives were the least active of this subset, with EC50
values above 5 μM. The presence of geminal dimethyl groups
The activity of piperidyl urea 66 was slightly diminished by
the introduction of a fluorine atom at the 4-position of the
piperidine ring (67), in contrast to the results noted above with
G
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Journal of Medicinal Chemistry
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Table 3. Cytotoxicity and Selectivity of Select Compounds
a
,
b
c,d
T. b. brucei
CRL-8155
HepG2
e
f
g
e
f
g
compd
EC50 (μM)
EC50 (μM)
EC90 (μM)
SI
EC50 (μM)
EC90 (μM)
SI
10
11
12
13
14
15
16
17
28
34
38
40
43
44
46
47
50
51
52
54
55
56
57
58
59
66
67
0.0243
0.037
0.399
0.667
0.0285
0.108
0.0366
0.164
0.0946
0.882
0.325
0.366
0.375
0.427
0.158
0.174
0.983
0.935
0.183
0.0918
0.0519
0.326
0.0348
0.934
0.114
0.223
0.341
0.00105
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
33.9
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>100
>2058
>1351
>125
>75
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>100
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>100
>2058
>1351
>125
>75
>1754
>463
>1366
>305
>529
>57
>1754
>463
>1366
>305
>529
>57
>154
>137
>133
>117
>316
>287
>51
>154
>137
>133
>117
>316
>287
>51
>53
>53
>273
369
>273
>545
>963
>153
>1437
>54
>963
>153
>1437
>54
h
27.1
>238
>224
>147
>43200
>439
>224
>147
>93000
>50
>50
45.4
i
pent
a
b
Human lymphoblasts (CRL-8155). Control for CRL-8155 EC50 assay average SEM: quinacrine (4.23 0.97 μM (n = 7); EC90 assay average
c
d
SEM: quinacrine (9.94 2.41 μM (n = 7). Human hepatocytes (HepG2). Control for HepG2 EC50 assay average SEM: quinacrine (10.44
1.39 μM (n = 7); EC90 assay average SEM: quinacrine (18.23 2.34 μM (n = 7). . Concentration of compound required to inhibit growth by
50% (EC50) of mammalian cell lines. Concentration of compound required to inhibit growth by 90% (EC90) of mammalian cell lines. Selectivity
index expressed as the ratio EC50 (cell line)/EC50 (T. b. brucei), rounded to the nearest integer. Average of three assays. Pentamidine.
e
f
g
h
i
the pyrrolidyl ureas (54 vs 55). The 4,4-difluoro derivative 68
was over 10 times less active than 66, similar to the disparity
between pyrrolidyl urea 54 and its 3,3-difluoro analogue 58.
The presence of an aromatic substituent on the benzothia-
zole system, as well as the spatial orientation of both
substituents, proved to be essential to activity. Removal of
the aromatic ring from the 2-position (69) or replacement by a
methyl group (70) resulted in loss of activity. The relocation of
the urea moiety from the 5- to the 6-position also resulted in no
detectable activity (55 vs 71). The reversal of the substitution
patterns on the benzothiazole system (71 vs 72, 55 vs 73)
resulted in weak activity (EC50 > 2 μM). Thus, the spatial
orientation of the thiazole and amide nitrogens proved to be
essential to activity.
Similarities and differences existed between the SAR of the
thiazole14 and benzothiazole derivatives. In both groups, the
urea derivatives were more potent than the corresponding
benzamides. Piperidyl ureas were more potent than the
corresponding pyrrolidyl ureas among the thiazoles, but the
opposite was true for the benzothiazoles. Monofluorination of
the aromatic ring at any position was generally beneficial in the
thiazoles, but monofluorination was beneficial only at the 3-
position for the benzothiazoles. Aromatic 3,4-difluorination was
beneficial in both groups, while 2,4-difluorination was beneficial
only to the thiazoles. The introduction of any substituents on
the piperidine ring resulted in losses of activity regardless of the
internal core of the molecule.
Cytotoxicity and Selectivity. Cytotoxicity data for
compounds 1−8 were reported previously.14 All new
compounds with EC50 values below 1 μM (except 53) were
tested for toxicity against human lymphocytes CRL-8155 and
human hepatocytes HepG2 (Table 3). Most of the compounds
(including 57) exhibited no detectable toxicity to either cell
line. Only compounds 54 and 59 had detectable toxicity to the
CRL-8155 cells (EC50 = 33.9 and 27.1 μM respectively), and
none had detectable toxicity to the HepG2 cells. The selectivity
of each compound for the parasite over each of the two cell
lines was calculated. Compounds 10, 11, 14, 16, and 57
exhibited selectivity indices above 1000 against either cell line.
Seventeen analogues (12, 15, 17, 28, 38, 40, 43, 44, 46, 47, 52,
54, 55, 56, 59, 66, and 67) had selectivity indices above 100
against either cell line. Within this group, compound 28 had a
selectivity index above 500 in the CRL-8155 assay, while 54
and 55 had selectivity indices above 500 in the HepG2 assay.
Metabolic Stability. Select compounds were assayed for
stability to mouse and human liver microsomes (Table 4). The
short half-lives of compounds 4 and 5 are consistent with
previous results obtained using a different protocol.14 The
introduction of an additional aromatic fluorine atom (5 vs 6, 5
vs 7) offered no advantage regarding metabolic stability.
H
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Journal of Medicinal Chemistry
Article
although more potent had 33, had shorter half-lives (>20 min)
in in mouse microsomes, and 38 had a shorter half-live (30
min) in human microsomes. Fluorination of the phenyl ring at
the 3-position (38 vs 46, 40 vs 47) led to enhanced potency of
both ureas but not enhanced stability, as both fluorinated
derivatives 46 and 47 were less stable to mouse microsomes
and only 46 was at least as stable to human microsomes. The
pyrrolidyl urea 46 (R2 = 3-fluorophenyl, t1/2 = 37 min) was four
times more stable to human microsomes than corresponding
piperidyl urea 47. Urea 54, the (more highly potent) 3,4-
Table 4. Stability of Select Compounds to Mouse and
Human Liver Microsomes
T. b. brucei EC50 mouse microsomes t1/2 human microsomes t1/2
a,
b
a,c
compd
(μM)
(min)
(min)
7.6
5.3
4
0.0194
0.0113
0.0094
0.0105
1.44
<5 (2.4%)
d
5
<5 (0%)
d
6
<5 (0%)
<5 (46%)
<5 (22%)
d
7
<5 (0%)
d
9
<5 (0%)
10
11
14
15
16
17
23
26
28
30
32
33
38
40
46
47
54
55
57
59
0.0243
0.0370
0.0285
0.108
0.0366
0.164
1.30
<5 (1.8%)
<5 (21%)
<5 (18%)
<5 (14%)
<5 (5.5%)
<5 (20%)
8.2
difluorophenyl analogue of 46, showed similar stability (t1/2
=
<5 (3.9%)
14 min) to mouse microsomes but enhanced stability (t1/2 > 60
min) to human microsomes. The addition of a 3-fluoro
substituent on the pyrrolidine ring (55) resulted in greater
potency, greater than 4-fold increased stability to mouse
microsomes, and similar stability to human microsomes
compared to 54. High metabolic stability (t1/2 > 60 min in
both mouse and human microsomes) was also observed in the
(S)-enantiomer 57 and the trans-3,4-difluoropyrrolidine de-
rivative 59.
Mouse Oral PK. Compounds 54, 55, and 57, and 59 were
administered to groups of three mice in single doses of 50 mg/
kg by oral gavage in a dosing vehicle consisting of Tween 80
(7%), EtOH (3%), and DMSO (5%) in 0.9% sodium chloride
solution (Table 5). Compound 57 (the most potent in vitro)
10
5.1
5.6
5.8
d
<5 (0%)
d
7.84
<5 (0%)
0.0946
13
<5 (28%)
60
>10
17
2.80
11
>60 (77%)
>60 (53%)
30
2.38
50
0.325
0.366
0.158
0.174
0.0918
0.0519
0.0348
0.114
18
17
>60 (62%)
37
12
11
8.9
14
>60 (66%)
>60 (99%)
>60 (53%)
>60 (96%)
Table 5. Oral Pharmacokinetics of Compounds 54, 55, 57,
and 59 in Mice
>60 (52%)
>60 (73%)
>60 (67%)
a
a
compd
Cmax (μM)
AUC0 min−∞ (min × μM)
a
b
Microsome reactions were incubated at 37 °C with KH2P04 (0.16
M), NADPH (1 mM), of microsomes (0.5 mg/mL), and test
compounds (1.5 μM). Numbers in parentheses are percentages of
compound remaining after 5 min (where t1/2 < 5 min) or after 60 min
54
2.01 0.46
10.04 0.65
23.34 3.02
18.55 5.54
8.54 0.41
10.96 0.58
443.3 43.07
6459.4 481.8
13254 1237
14673 2833
6352 469
b
55
b
57
c
57
b
(where t1/2 > 60 min). Control for mouse microsome assay average
d
57
SEM: dextromethorphan (7.79 1.40 min, n = 7) and testosterone
b
c
59
13515 224.6
(5.26 min. 0.637 min, n = 7). Control for human microsome assay
a
average SEM: dextromethorphan (39.15 5.13 min, n = 7) and
Average values SEM of three mice each given a single dose at 50
d
b
testosterone (22.7 5.89 min, n = 6). Below limit of detection.
mg/kg by oral gavage. Dosing vehicle consisted of Tween 80 (7%),
EtOH (3%), and DMSO (5%) in 0.9% sodium chloride solution.
c
Dosing vehicle consisted of Phosal 53 MCT(60%), PEG400 (30%),
d
None of the new thiazole derivatives (9−28) that were tested
were metabolically stable. Only analogue 28 (t1/2 = 13 min) had
a half-life above 10 min in the mouse microsomes, and all
others in this group had half-lives below 5 min with 0−21% of
the test compounds remaining after the 5 min time point.
These compounds were also unstable to the human micro-
somes, where only analogue 14 had a half-life as high as 10 min.
All four compounds in this group having potencies below 50
nM (10, 11, 14, and 16) were unstable to both mouse and
human microsomes. No modifications of the urea portion of
the molecule or of the ethylene bridge were found to improve
metabolic stability. Thus, none of the compounds 9−28 offered
any advantage regarding either potency or metabolic stability.
The metabolic stabilities of the fused ring analogues 29−73
were more promising. All compounds in this group that were
tested had half-lives greater than 10 min in the mouse
microsomes and at least 30 min (except for 47) in the human
microsomes. The inactive benzoxazole amide 30 and its weakly
active piperidyl urea analogue 32 had half-lives of between 10
and 20 min in mouse microsomes and at least 60 min in human
microsomes. The benzothiazole amide 33, although only
weakly active against the parasite, had half-lives of 50 and
>60 min in the mouse and human microsomes, respectively.
The corresponding pyrrolidyl and piperidyl ureas 38 and 40,
and EtOH (10%). Dosing vehicle consisted of methylcellulose cP 400
(0.5%) and Tween 80 (0.5%) in water.
clearly showed the highest blood levels of the four compounds
tested in the same vehicle, with a maximum blood
concentration of 23.3 μM and an AUC of 13216 min·μM.
Compound 59 had a lower Cmax but a similar AUC compared
to 57. Less than 1% of the Cmax of 57 remained at the 24 h time
point compared to 34% of the Cmax of 59. More than the 17%
of the Cmax of 59 remained at the 32 h time point. Compound
57 gave a PK profile similar to that obtained using the original
vehicle when administered in a vehicle consisting of Phosal 53
MCT (60%), PEG400 (30%), and EtOH (10%); however, the
use of a vehicle consisting of methylcellulose cP 400 (0.5%)
and Tween 80 (0.5%) in water resulted in a lower Cmax and
AUC values (lines 3−5 of Table 5).
Brain Penetration. Brain penetration studies (Table 6)
were initially performed upon compounds 54, 55, and 57 but
were expanded to include other compounds in order to explore
the SAR of brain penetration. Groups of three mice were given
single 5 mg/kg ip doses of the test compounds and were
sacrificed 1 h postdose. The benzothiazole pyrrolidyl urea 38
exhibited a mean brain concentration of 4.2 μM (over 10 times
I
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Journal of Medicinal Chemistry
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Table 6. Brain Penetration of Select Compounds in Mice at
One Hour Post-Dose
Table 7. Brain Time-Course Study of Compound 57
[compd]plasma
[compd]brain
brain/plasma
ratio
a
a
a
a
time (h)
(μM)
(μM)
compd [compd]plasma (μM)
[compd]brain (μM)
brain/plasma ratio
1
27.14 9.91
10.25 2.21
8.97 1.05
38.23 13.51
45.2 10.4
1.48 0.16
4.43 0.3
5.48 0.6
1
0.507 0.087
0.533 0.328
2.00 0.885
0.270 0.036
3.403 0.328
2.22 0.461
1.3 0.406
0.153 0.033
0.244 0.109
0.187 0.089
0.397 0.077
4.24 0.698
2.28 0.532
2.31 0.746
4.25 0.693
0.300 0.040
0.613 0.128
0.089 0.006
1.58 0.484
1.24 0.157
1.01 0.037
1.76 0.032
1.81 0.458
2.11 0.669
4.86 1.055
4.00 0.361
7.92 0.693
7.71 1.63
4
4
8
48.08 2.47
0.28 0.13
10
31
38
46
52
54
55
56
57
58
59
60
63
66
67
b
24
<LLQ
Cmax (μM)
27.39 9.67
54.84 4.64
43116.8 2304
AUC0 min−∞ (min ×
μM)
10798 1975
a
Average concentration SEM of 3 mice at the stated time point after
2.61 0.727
3.26 0.630
1.20 0.270
2.27 0.888
2.81 0.452
2.19 0.424
4.15 1.368
0.780 0.202
2.52 0.185
2.51 0.203
each mouse received a single oral dose at 50 mg/kg in vehicle
consisting of Tween 80 (7%), EtOH (3%), and DMSO (5%) in 0.9%
sodium chloride solution. LLQ (lower limit of quantitation) is 0.010
μM.
b
b
5.48 0.683
6.35 2.257
9.07 3.973
21.7 1.96
16.2 2.88
1.97 0.612
3.00 0.796
1.29 0.156
2.52 0.248
b
In Vivo Efficacy. Upon the basis of its promising in vitro
activity, cytotoxicity, metabolic stability, PK, and brain
penetration data, compound 57 was selected as a candidate
for in vivo efficacy studies. An acute model (requiring 60 days
to complete) was employed, followed by a chronic model
(requiring 180 days to complete).
In a model of the acute phase of HAT, five mice infected
with T. b. rhodesiense STIB900 were given the test compound at
50 mg/kg po b.i.d. × 4 days, beginning 2 days postinfection.
Efficacy PK blood samples were collected from three of the five
mice prior to dose 7, and at 1 and 6 h postdose. Average plasma
levels of the test compound were 9.1 7.6 μM (predose), 18.6
0.477 0.052
3.80 0.331
0.510 0.023
1.01 0.081
a
Average values SEM of three mice each given a single ip dose at 5
mg/kg in vehicle consisting of Tween 80 (7%), EtOH (3%), and
DMSO (5%) in 0.9% sodium chloride solution. Average values
SEM of six mice.
b
greater than that of the corresponding benzoxazole analogue
31) and a mean brain to plasma ratio (BPR) of 1.2.
Fluorination of the terminal aromatic ring had relatively little
effect as 38, and the 3,4-difluorophenyl derivative 54 had nearly
identical mean brain concentrations, although the latter did
have a slightly higher BPR. By contrast, the mean brain
concentrations of the 3-fluoro (46) and 2,3-difluoro (52)
analogues were somewhat lower than that of 38. The brain
levels of 54 were enhanced by the introduction of a 3-fluoro
substituent on the pyrrolidine ring (55−57). Of these
compounds, the greatest enhancement was seen in the (S)-
enantiomer 57, with a mean brain concentration of 9.1 μM and
a BPR of 4.0. The (R)-enantiomer 56 also exhibited the ability
to cross the BBB. The highest brain levels were seen in the
difluoropyrrolidyl derivatives 58 and 59, each having a mean
brain concentration exceeding 16 μM and a BPR above 7.5.
Replacement of the fluorine atom with a trifluoromethyl group
(55 vs 60) resulted in lower brain levels. Similar decreases also
resulted when the pyrrolidine ring was replaced with piperidine
(54 vs 66). Introduction of a 4-fluoro substituent on the
piperidine ring also resulted in enhanced brain levels (66 vs
67), consistent with the results of fluorination of the pyrrolidine
ring.
The brain and plasma concentrations of compound 57 were
studied in a time-course experiment following oral dosing at 50
mg/kg (Table 7). The data show that 57 partitions to the brain
compartment with BPRs of 1.48, 4.43, and 5.48 at 1, 4, and 8 h
postdose, respectively. At 24 h, the plasma concentrations are
undetectable and brain levels are down to submicromolar
concentrations. Compared to the previous experiment (Table
6), the BPR ratio for 57 is lower at 1 h postdose (1.48 vs 4.0),
probably due to the oral route of administration (as opposed to
the ip route used before), leading to slower systemic absorption
and distribution. The time-course experiment demonstrates
that brain concentrations of 57 are sustained at high levels for
at least 8 h postdose and help account for the successful results
in the late-stage efficacy model discussed below.
7.2 μM (1 h), and 15.0
4.2 μM (6 h). Compound 57
attained 5/5 cures as determined by the absence of detectable
parasitemia in any of the treated mice 60 days postinfection
(Figure 1). All mice receiving only vehicle showed high
parasitemia on the last day of dosing, with all concentrations
>1.5 × 107 parasites/mL of blood, and were euthanized.
Figure 1. Mouse efficacy model of acute T. brucei infection. All mice
were infected with T. b. rhodesiense STIB900 on day 0. Groups of five
mice were treated with compound 57 (50 mg/kg by oral gavage b.i.d.)
or vehicle from day 2−5 (gray-shaded area). Mice were monitored for
parasitemia in tail blood samples through day 60 postinfection.
In a model of the chronic phase of HAT, compound 57 was
administered to five mice infected with T. b. brucei TREU667 at
50 mg/kg/po b.i.d. × 10 days, beginning 21 days postinfection.
This dose was chosen because earlier PK studies demonstrated
good plasma exposure at 50 mg/kg (Table 5). Efficacy PK
blood samples were collected from three of the five mice prior
to dose 15, and at 1 and 6 h postdose. Average plasma levels of
the test compound were 15.8 8.7 μM (predose), 23.1 603
μM (1 h), and 26.7 7.1 μM (6 h). Compound 57 attained 5/
5 cures as determined by the absence of detectable parasitemia
in any of the treated mice through 180 days postinfection
J
DOI: 10.1021/acs.jmedchem.6b01163
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Journal of Medicinal Chemistry
Article
(Figure 2). No mice receiving vehicle alone had spontaneous
cures. The mice receiving diminazene aceturate (which does
experiments in both studies demonstrated that the compound
accumulated in plasma after dosing. Synthesis of additional
analogues, as well as further testing of compound 57 (and 59),
including dose optimization and comprehensive pharmacoki-
netic studies, is either planned or currently in progress.
Experiments to identify the biochemical target(s) of action in T.
brucei are also underway.
EXPERIMENTAL SECTION
■
T. brucei Growth Inhibition Assay. Compounds were tested for
antitrypanosomal activity against T. b. brucei (strain BF427) in HMI-9
media as previously described.39 Cells were tested in triplicate against
serial dilutions of compounds and quantified with Alamar Blue
(ThermoFisher Scientific, Waltham, MA) at 48 h.40 EC50 and EC90
values were calculated by nonlinear regression using software by the
Collaborative Drug Database (Burlingame, CA. www.
collaborativedrug.com). Growth assays for T. b. brucei included the
control compound, pentamidine isethionate (Sigma-Aldrich, St. Louis,
MO.
Figure 2. Mouse efficacy model of chronic T. brucei infection. All mice
were infected with T. b. brucei TREU667 strain on day 0. Groups of
five mice were treated with compound 57 (50 mg/kg by oral gavage
b.i.d.) or vehicle from day 21−30 (gray-shaded area). A control group
received a single dose of diminazene on day 21. Mice were monitored
for parasitemia in tail blood samples through day 180 postinfection.
Cytotoxicity in Mammalian Cells. Compounds were assayed for
cytotoxicity against CRL-8155 (human lymphoblasts) and HepG2
cells (human hepatocellular carcinoma) as previously described.41
Cells were exposed to serial dilutions of compounds for 48 h, and
cytotoxicity was quantified using Alamar Blue41 (ThermoFisher
Scientific, Waltham, MA). Compounds were assayed in quadruplicate,
and CC50 and CC90 values were calculated by nonlinear regression
using software by the Collaborative Drug Database (Burlingame, CA.
www.collaborativedrug.com). Growth assays for CRL-8155 and
HepG2 included the control compound, quinacrine (Sigma-Aldrich.
In Vitro Liver Microsome Assays. Pooled liver microsomes from
mouse and human sources were purchased from BD Biosciences (San
Jose, CA). Microsome reactions were preincubated for 5 min at 37 °C
with 0.16 M KH2PO4, 1 mM NADPH, and 0.5 mg/mL of microsomes.
At t = 0, 1.5 μM of test compounds were added and the t = 0 time
point was immediately quenched with the addition of 4× the sample
volume of 100% acetonitrile. Subsequent time points were quenched
at 5, 10, 15, 30, and 60 min post addition of test compounds, then
processed using liquid chromatography/tandem mass spectrometry
analysis. Assays were performed with control compounds, testoster-
one, and dextromethorphan (purchased from Sigma-Aldrich).
Mouse Oral Pharmacokinetics. Compounds were administered
orally to mice at a concentration of 50 mg/kg in a vehicle consisting of
either: (1) DMSO (5%), Tween 80 (7%), and EtOH (3%) in
physiological saline (0.9%) solution, (2) Phosal 53 MCT (60%),
PEG400 (30%), and EtOH (10%), or (3) methylcellulose cP400
(0.5%) and Tween 80 (0.5%) in water. Phosal 53 MCT is a lecithin
emulsifier purchased from American Lecithin Co. Three mice were
used per group, and 40 μL of blood was collected from the tail at time
points extending out to as much as 32 h postdose into heparinized
capillary tubes. Whole blood was then spotted onto FTA DMPK-C
cards (GE Healthcare, Boston, MA) and was measured by liquid
chromatography/tandem mass spectrometry. For the efficacy PK
experiments, samples were collected from experimental mice (n = 3)
before the stated day of dose, then at 1 and 6 h postdose using the
same collection method as above.
not cross the BBB)38 had temporary clearance of parasitemia
but subsequently relapsed, most likely from parasites leaving
the brain and returning to the hemolymphatic system. By
observing mice for 180 days, more than ample time was allowed
for mice to show signs of illness or parasites to become visible
on blood films (as was observed with the diminazene-treated
mice), thus providing assurance that the 57-treated mice were
cured. Experiments evaluating lower doses of 57 are underway
and will be published at a later time.
CONCLUSIONS
■
While a number of the initial analogues14 were highly potent
against the parasite in vitro, they exhibited poor metabolic
stability, which precluded their further advancement as drug
candidates. The goal of the present study was to improve
metabolic stability while maintaining potency. Neither replacing
the carbonyl oxygen with sulfur, the introduction of various
substituents at the urea end of the molecule, nor the alkylation
or unsaturation of the ethylene linker provided any advantage
regarding either antitrypanosomal activity or metabolic stability.
Enhanced metabolic stability was observed only in the fused
ring analogues where thiazol-2-ethylamino fragment was
replaced with a benzoxazol- or benzothiazol-5-amino moiety,
but antitrypanosomal activity was retained only in the
benzothiazole derivatives. These results strongly suggest that
the ethylene linker played a role in the poor metabolic stability
of the initial thiazole analogues. Brain penetration studies
demonstrated the ability of the benzoxazole and benzothiazole
derivatives, as a whole, to cross the BBB and even concentrate
in the brain.
The most significant findings in this paper were the
promising biological data for compound 57. This molecule
exhibited good activity in vitro (EC50 = 34.8 nM), no detectable
toxicity to two human cell lines (CRL-8155 and HepG2), and
half-lives of at least 60 min in both mouse and human liver
Brain: Plasma Compound Concentration Measurements in
Mice. Test compounds were administered at 5 mg/kg ip to three mice
in a vehicle consisting of DMSO (5%), Tween 80 (7%), and EtOH
(3%) in physiological saline (0.9%) solution.3 At 1 h post injection,
blood was collected, plasma was separated by brief centrifugation, and
the brain was removed and homogenized in acetonitrile. Levels of
compound in the plasma and brain were determined via liquid
chromatography/tandem mass spectrometry. Calculations of brain
concentrations accounted for 3% volume/weight of blood in the brain.
For the brain time-course experiment of compound 57, compounds
were administered at 50 mg/kg po in the same vehicle listed above.
Mice were sacrificed in groups of three at 1, 4, 8, and 24 h postdosing
for collection of brains and plasma for analysis as mentioned above.
microsomes. It also demonstrated promising oral PK (Cmax
=
23.3 μM and AUC = 13216 min × μM) and excellent brain
penetration (brain concentration = 9.07 μM and BPR = 4.00)
at 1 h after administration of a 5 mg/kg ip dose in mice. These
results led to the selection of compound 57 for in vivo efficacy
studies. Oral dosing of this molecule achieved 5/5 cures in both
acute and chronic murine models of HAT. The efficacy PK
K
DOI: 10.1021/acs.jmedchem.6b01163
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Acute Efficacy Studies in Mice. Experiments were carried out
using the standard operating procedure used by WHO screening
centers42 and done in compliance with the University of Washington
Institutional Animal Care and Use Committee (IACUC) approved
protocol. Groups of five female Swiss-Webster mice (ND4 outbred,
ages 6−8 weeks) were infected on day 0 with 1 × 104 T. b. rhodesiense
(strain STIB900) parasites. The test compound was administered by
oral gavage at 50 mg/kg every 12 h from day 2 to day 5, for a total of
eight doses in a 200 μL volume of a vehicle consisting of DMSO (5%),
Tween 80 (7%), and EtOH (3%) in physiological saline (0.9%)
solution. Parasitemia was monitored via microscopic analysis of tail
blood for 60 d postinfection or until parasites were detected. Mice
were removed from the experiment once parasites were detected in the
blood, at which point the mouse was euthanized.
Chronic Efficacy. Again, experiments were done in compliance
with the University of Washington IACUC approved protocol.
According to published procedures,38 groups of five mice were
infected with 1 × 104 T. b. brucei (strain TREU667) at day 0 to
establish a chronic infection. Treatment began on day 21 postinfection,
and mice received 50 mg/kg test compound orally b.i.d. for 10 d (total
of 20 doses) in a 200 μL volume of a vehicle composed of Phosal 53
MCT (60%), PEG400 (30%), and EtOH (10%). A control group
received vehicle with no compound and another control group
received a single intraperitoneal dose of diminazene aceturate at 10
mg/kg in water on day 21. The diminazene aceturate temporarily
clears parasites from the blood, but because it does not cross the BBB,
the blood is later repopulated from parasites in the CNS.38 Post
dosing, parasitemia was monitored via microscopic examination of tail
blood slides until 180 days postinfection. Mice were removed from the
experiment once parasites were detected in the blood.
was recrystallized from MeOH as light-yellow crystals (184 mg, 50%):
mp 124−126 °C. H NMR (400 MHz, DMSO-d6) δ 10.43 (t, J = 5.5
1
Hz, 1H), 8.00 (d, J = 0.7 Hz, 1H), 7.99−7.92 (m, 2H), 7.80−7.72 (m,
2H), 7.54−7.46 (m, 1H), 7.46−7.37 (m, 4H), 7.37−7.29 (m, 1H),
4.12 (td, J = 6.9, 5.2 Hz, 2H), 3.49 (t, J = 6.9 Hz, 2H). EIMS m/z
324.9 (M + 1)+. HPLC 100 area % (265 nm). Anal. Calcd for
C18H16N2S2: C, 66.63; H, 4.97; N, 8.63; S, 10.40. Found: C, 66.36; H,
5.16; N, 8.38.
2-(2-Piperidin-1-yl-thioamido)ethyl-4-phenylthiazole Hy-
drochloride (10). A suspension of 4-phenylthiazol-2-ethylamine
hydrobromide (74,14 500 mg, 1.75 mL) in DCM (8 mL) was treated
with Et3N (0.6 mL, 4.30 mmol) to give a homogeneous solution.
Carbon disulfide (1.2 mL, 19.3 mmol) was added, and the mixture was
stirred overnight. After the mixture cooled in an ice bath, DMAP (10
mg, 4.8 mol %) and a solution of di-tert-butyl dicarbonate (383 mg,
1.75 mmol) in DCM (1 mL) were added.15 The ice bath was removed,
and the mixture was stirred for 2 h. The mixture was concentrated
under reduced pressure, diluted with ether, and filtered. The filtrate
was evaporated to give 2-(2-isothiocyanatoethyl)-4-phenylthiazole as
an oil. 1H NMR (400 MHz, DMSO-d6) δ 8.04 (s, 1H), 8.02−7.91 (m,
2H), 7.49−7.39 (m, 2H), 7.42−7.29 (m, 1H), 4.11 (dd, J = 6.7, 5.9
Hz, 2H), 3.44 (dd, J = 6.7, 5.8 Hz, 2H).
A solution of the 2-(2-isothiocyanatoethyl)-4-phenylthiazole,
piperidine (200 μL, 2.02 mmol), and Et3N (300 μL, 2.15 mmol) in
DCM (10 mL) was stirred overnight at room temperature. After
aqueous workup, the crude product was converted to the HCl salt
using EtOH and concentrated HCl, followed by recrystallization from
EtOH/ether to give white crystals (249 mg, 39%): mp 138−140 °C.
1H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 0.6 Hz, 1H), 7.96−7.92
(m, 2H), 7.80 (s, 1H), 7.48−7.39 (m, 2H), 7.37−7.30 (m, 1H), 3.88
(td, J = 6.8, 4.3 Hz, 2H), 3.79−3.72 (m, 4H), 3.34 (t, J = 7.0 Hz, 2H),
1.63−1.55 (m, 2H), 1.52−1.41 (m, 4H). EIMS m/z 332.0 (M + 1)+.
HPLC 96.9 area % (254 nm). Anal. Calcd for C17H21N3S2·HCl: C,
55.49; H, 6.03; N, 11.42. Found: C, 55.59; H, 6.12; N, 11.31.
General Procedure for Compounds 11−19. A mixture of 4-(3-
fluorophenyl)thiazol-2-ethylamine hydrobromide (75,14 0.4−0.7
mmol) and Et3N (min of 3 equiv) in DCM was cooled to −5 °C
before the addition of 1,1′-carbonyldiimidazole (CDI, 1.2−1.3 equiv).
After 1 h, the secondary amine or its hydrochloride salt was added.
The mixture was stirred at room temperature overnight or until
complete by HPLC before being diluted with water and extracted into
DCM. The crude products were then recrystallized from appropriate
solvents
General Chemistry Experimental. Uncorrected melting points
were measured on a Thermo Scientific 9200 melting point apparatus.
1H NMR spectra were recorded on a Varian Inova 400 MHz, a Bruker
AVANCE 400 MHz, or a Varian Inova 600 MHz spectrometer.
Anhydrous solvents were purchased from Aldrich Chemical Co.,
Milwaukee, WI, or from Fisher Scientific, Waltham, MA, in Sure-Seal
or AcroSeal containers and were used without further purification.
Organic starting materials were purchased from the same sources or
were prepared by published procedures as noted. Reaction mixtures
were monitored by TLC on silica gel or by reverse phase HPLC.
Organic layers of extraction mixtures were neutralized as necessary
with acidic or basic washes, washed with saturated NaCl solution, and
dried over MgSO4 before being evaporated under reduced pressure.
Normal phase flash column chromatography was performed using
Davisil grade 633, type 60A silica gel (200−425 mesh). Analytical
HPLC chromatograms were recorded on an Agilent 1100 or 1200
series chromatograph using a Zorbax Rx C8 column (4.6 mm × 75
mm, 3.5 μm) maintained at 40 °C and UV photodiode array detection
at 230, 254, 265, 290, and 320 nm. Area % values are reported at the
wavelengths where the strongest signals of the products were
observed. Mobile phases consisted of mixtures of MeOH (0−95%)
in water containing formic acid (80 mM), ammonium formate (20
mM), and Et3N (15 mM). Samples were eluted at appropriate
gradients at a flow rate of 1.5 mL/min. Low resolution ESI mass
spectra were recorded on an Agilent Technologies 1100 series LC/
MSD trap mass spectrometer or at the North Carolina State University
Mass Spectrometry Facility located in the Department of Chemistry.
In cases of hydrochloride salts, the m/z values reported are those of
the free bases. Elemental analyses were measured by Atlantic Microlab,
Norcross, GA, and unless stated otherwise, were within 0.4% of
calculated values. All target compounds are judged to be >95% pure by
elemental analysis and analytical HPLC. Representative syntheses for
all target compounds as well as for intermediates of benzothiazoles
33−70 are given herein. Synthetic details for all compounds are given
in Supporting Information.
2-[2-(2-Methyl)piperidin1-yl-amido]ethyl-4-(3-fluorophenyl)-
thiazole (11). 11 was prepared by the general procedure from 75 (153
mg, 0.506 mmol) and 2-methylpiperidine (100 μL, 0.851 mmol). The
crude product was recrystallized from DCM/hexanes as a white solid
1
(75.1 mg, 43%): mp 81−83 °C. H NMR (400 MHz, DMSO-d6) δ
8.09 (s, 1H), 7.80 (ddd, J = 7.8, 1.6, 0.9 Hz, 1H), 7.74 (ddd, J = 10.7,
2.6, 1.5 Hz, 1H), 7.48 (td, J = 8.0, 6.2 Hz, 1H), 7.16 (dddd, J = 9.0, 8.3,
2.7, 0.9 Hz, 1H), 6.57 (t, J = 5.4 Hz, 1H), 4.23 (d, J = 4.3 Hz, 1H),
3.79−3.70 (m, 1H), 3.41 (dt, J = 12.6, 6.5 Hz, 2H), 3.17 (t, J = 6.9 Hz,
2H), 2.73 (td, J = 13.1, 2.8 Hz, 1H), 1.60−1.40 (m, 5H), 1.30−1.10
(m, 1H), 1.04 (d, J = 6.8 Hz, 3H). EIMS m/z 348.0 (M + 1)+. HPLC
100 area % (254 nm). Anal. Calcd for C18H22FN3OS·0.25H2O: C,
61.43; H, 6.44; N, 11.94. Found: C, 61.22; H, 6.69; N, 11.82.
Representative Synthesis of Compounds 20−27. 2-(2-
Benzamido-2-methylpropyl)-4-phenylthiazole (20). Benzoyl chlor-
ide (200 μL, 1.72 mmol) was added to a stirred mixture of 2-(2-amino-
2-methylpropyl)-4-phenylthiazole hydrobromide (79, 258 mg, 0.824
mmol) and Et3N (500 μL, 3.59 mmol) in THF (10 mL). The mixture
was stirred overnight before being diluted with H2O and extracted into
EtOAc. The product was recrystallized from EtOAc/hexanes as white
crystals (191 mg, 69%): mp 107−108 °C. 1H NMR (400 MHz,
DMSO-d6) δ 7.94 (s, 1H), 7.93−7.88 (m, 3H), 7.83−7.79 (m, 2H),
7.54−7.48 (m, 1H), 7.46−7.37 (m, 4H), 7.34−7.28 (m, 1H), 3.60 (s,
2H), 1.45 (s, 6H). EIMS m/z 337.1 (M + 1)+. HPLC 98.4 area % (254
nm). Anal. Calcd for C20H20N2OS: C, 71.40; H, 5.99; N, 8.33. Found:
C, 71.49; H, 6.04; N, 8.22.
2-(2-Benzthioamido)ethyl-4-phenylthiazole (9). Lawesson’s
reagent (493 mg, 1.22 mmol) was added to a solution of 2-(2-
benzamido)ethyl-4-phenylthiazole (1,14 353 mg, 1.15 mmol) in THF
(20 mL). The mixture was stirred overnight before being diluted with
saturated NaHCO3 solution and extracted into DCM. The oily residue
L
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(E)-N-[2-(4-[3-Fluorophenyl]thiazol-2-yl)vinyl]piperidine-1-
carboxamide (28). A solution of (E)-3-[4-(3-fluorophenyl)thiazol-2-
yl]acryloyl azide (91, 140 mg, 0.514 mmol) in toluene (10 mL) was
refluxed for 1.5 h, resulting in a Curtius rearrangement to the vinyl
isocyanate.26 After the reaction mixture cooled to rt, piperidine (80 μL,
0.810 mmol) and Et3N (80 μL, 0.810 mmol) were added and the
mixture was stirred for 2 h before being diluted with water and
extracted into EtOAc. The product was purified on a column of silica
eluting with hexanes/EtOAc (2:1) followed by recrystallization from
EtOAc/hexanes to give a pale-yellow solid (56.1 mg, 33%): mp
which was filtered off and rinsed with acetone. This material was added
to a solution of 3-fluoropyrrolidine hydrochloride (87.0 mg, 0.693
mmol) and Et3N (250 μL, 1.79 mmol) in DCM (10 mL). The
suspension was stirred overnight with the dissolution of the newly
formed product. The resulting solution was diluted with water and
extracted into DCM. The crude product was recrystallized from
EtOH/H2O as a beige powder (177 mg, 62%): mp 171.5−173 °C. 1H
NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 8.26−8.20 (m, 1H),
7.81 (ddd, J = 12.4, 7.8, 2.2 Hz, 1H), 7.70 (dd, J = 8.4, 1.9 Hz, 1H),
7.65 (d, J = 8.6 Hz, 1H), 7.57 (dddd, J = 7.7, 4.7, 2.3, 0.9 Hz, 1H), 7.52
(dt, J = 10.6, 8.3 Hz, 1H), 5.39 (d, J = 53.1 Hz, 1H), 3.87−3.42 (m,
4H), 2.28−2.00 (m, 2H). EIMS m/z 378.1 (M + 1)+. HPLC 96.0 area
% (290 nm). Anal. Calcd for C18H14F3N3OS·0.4H2O: C, 56.21; H,
3.88; N, 10.93. Found: C, 56.17; H, 4.00; N, 10.75.
5-(3,4-Difluorophenyl)-2-(3-fluoro-N-pyrrolidylamido)-
benzothiazole (73). Phenyl chloroformate (200 μL, 1.59 mmol) was
added dropwise by syringe to a solution of 2-amino-5-(3,4-
difluorophenyl)benzothiazole (108, 132 mg, 0.505 mmol) and
pyridine (200 μL, 2.47 mmol) in THF (10 mL) maintained at −5
°C, accompanied by the formation of a precipitate. The reaction
mixture was diluted with water and extracted with EtOAc. The extract
was evaporated to near dryness followed by dilution with hexane to
give crude phenyl (5-(3,4-difluorophenyl)benzothiazol-2-yl)carbamate
(174 mg, 90%), which was used in the next step without further
purification.
1
101.5−103 °C. H NMR (400 MHz, DMSO-d6) δ 10.89 (d, J = 10.5
Hz, 1H), 8.04 (s, 1H), 7.75 (dt, J = 7.7, 1.2 Hz, 1H), 7.71 (ddd, J =
10.5, 2.6, 1.5 Hz, 1H), 7.52 (td, J = 8.0, 6.1 Hz, 1H), 7.29 (dd, J = 10.5,
8.7 Hz, 1H), 7.26−7.17 (m, 1H), 5.91 (d, J = 8.7 Hz, 1H), 3.50 (t, J =
5.4 Hz, 4H), 1.67−1.52 (m, 6H). EIMS m/z 332.1 (M + 1)+. HPLC
100 area % (290 nm). Anal. Calcd for C17H18FN3OS: C, 61.61; H,
5.47; N, 12.68. Found: C, 61.37; H, 5.48; N, 12.40.
Representative Synthesis of Fused Ring Amides 29−30, 33−
37, 42, and 45. 5-Benzamido-2-phenylbenzothiazole (33). Et3N
(0.5 mL, 3.59 mmol) was added to a solution of 5-amino-2-
phenylbenzothiazole (101a, 340.3 mg, 1.50 mmol) and benzoyl
chloride (250 μL, 2.16 mmol) in DCM. The mixture was stirred at rt
overnight before being diluted with water and extracted into DCM.
Recrystallization of the product from EtOH gave white crystals (406
mg, 82%): mp 221−222 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.50
(s, 1H), 8.62 (d, J = 2.0 Hz, 1H), 8.18−8.07 (m, 3H), 8.06−7.97 (m,
2H), 7.85 (dd, J = 8.7, 2.0 Hz, 1H), 7.68−7.52 (m, 6H). EIMS m/z
331.0 (M + 1)+. HPLC 99.2 area % (290 nm). Anal. Calcd for
C20H14N2OS: C, 72.69; H, 4.27; N, 8.48. Found: C, 72.45; H, 4.50; N,
8.42.
General Procedure for 5- and 6-Aminobenzothiazole Urea
Derivatives 38−41, 43−44, and 46−71. A stirred solution of the
appropriate primary amine (0.5−2 mmol) and Et3N (min of 2 equiv)
in DCM was chilled to maximum internal temperature of −5 °C (ice−
salt bath). Triphosgene (0.35−0.55 equiv) was added either dropwise
as a solution in DCM (for 38, 40, 41, and 43) or all at once in solid
form for all others. The method of introduction was noncritical
provided the final concentration of the primary amine was below 20
mM. The mixture was maintained at −5 °C until the starting material
was no longer detectable by HPLC (typically 1 h or less). The
secondary amine (min of 1 equiv) or its HCl salt (in which cases an
additional equivalent of Et3N was employed) was added, and the
solution was allowed to warm to room temperature overnight. The
reaction mixture was diluted with water and extracted into DCM.
Unless stated otherwise, the product was directly recrystallized from
the appropriate solvent(s).
3-Fluoropyrrolidine hydrochloride (56.8 mg, 0.524 mmol) was
added to a solution of crude (5-(3,4-difluorophenyl)benzothiazol-2-
yl)carbamate (153 mg, 0.401 mmol) in pyridine (10 mL). The mixture
was refluxed for 1 h. The cooled mixture was diluted with H2O (10
mL), and the resulting suspension was stored overnight at 4 °C. The
precipitate was filtered off and dried to give an off-white powder (95.7
1
mg, 57% overall): mp >250 °C (dec). H NMR (400 MHz, DMSO-
d6) δ 11.21 (s, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.91−7.73 (m, 2H),
7.64−7.45 (m, 3H), 5.39 (d, J = 53.0 Hz, 1H), 3.91−3.40 (m, 4H),
2.29−1.99 (m, 2H)). EIMS m/z 378.1 (M + 1)+. HPLC 96.0 area %
(290 nm). Anal. Calcd for C18H14F3N3OS: C, 57.29; H, 3.74; N, 11.13.
Found: C, 57.13; H, 3.83; N, 11.15.
N-(2-Chloro-5-nitrophenyl)benzamides 99a−g. N-(2-Chloro-
5-nitrophenyl)benzamide (99a)..23,43,44 Benzoyl chloride (2.63 g,
18.71 mmol) was added dropwise by syringe to a solution of 2-chloro-
5-nitroaniline (98a, 3.06 g, 17.72 mmol) in dry pyridine (7.5 mL)
under argon, resulting in the formation of a precipitate. More pyridine
(7.5 mL) was added, and the mixture was stirred overnight. The
reaction mixture was poured into water. The precipitated product was
recrystallized from EtOH as white crystals (4.62 g, 95%): mp 171−172
°C. 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 8.58 (d, J = 2.8
Hz, 1H), 8.13 (dd, J = 8.9, 2.8 Hz, 1H), 8.08−7.97 (m, 2H), 7.88 (d, J
= 8.8 Hz, 1H), 7.69−7.61 (m, 1H), 7.61−7.52 (m, 2H). HPLC 100
area % (265 nm). Anal. Calcd for C13H9ClN2O3: C, 56.44; H, 3.28; N,
10.13. Found: C, 56.43; H, 3.19; N, 10.15.
(S)-2-(3,4-Difluorophenyl)-5-(3-fluoro-N-pyrrolidylamido)-
benzothiazole (57). 57 was prepared from 5-amino-2-(3,4-
difluorophenyl)benzothiazole (101g, 0.63 mmol) and (S)-3-fluorpyr-
rolidine hydrochloride. The product was purified on a silica gel column
eluting with hexanes/EtOAc (1:2) and recrystallized from EtOAc/
N-(2-Chloro-5-nitrophenyl)-3,4-difluorobenzamide (99g). 99g
was prepared analogously to 99a from 3,4-difluorobenzoyl chloride
(2.77 g, 15.69 mmol) and 2-chloro-5-nitroaniline (98a, 2.60 g, 15.07
mmol). The product was recrystallized from EtOH as white crystals
1
hexanes as white crystals (86 mg, 36%): mp 208−209 °C. H NMR
(400 MHz, DMSO-d6) δ 8.50 (s, 1H), 8.33 (d, J = 2.0 Hz, 1H), 8.13
(ddd, J = 11.3, 7.7, 2.2 Hz, 1H), 7.99 (d, J = 8.8 Hz, 1H), 7.96−7.89
(m, 1H), 7.69−7.58 (m, 2H), 5.39 (dm, J = 53.3 Hz, 1H), 3.80−3.52
(m, 3H), 3.47 (td, J = 10.3, 6.9 Hz, 1H), 2.27−2.00 (m, 2H). EIMS m/
z 378.0 (M + 1)+. HPLC 97.0 area % (254 nm). Anal. Calcd for
C18H14F3N3OS·0.3H2O: C, 56.48; H, 3.84; N, 10.98. Found: C, 56.21;
H, 3.77; N, 10.89.
1
(3.84 g, 82%): mp 149−51 °C. H NMR (400 MHz, DMSO-d6) δ
10.50 (s, 1H), 8.53 (d, J = 2.7 Hz, 1H), 8.15 (dd, J = 8.9, 2.8 Hz, 1H),
8.06 (ddd, J = 11.4, 7.8, 2.2 Hz, 1H), 7.95−7.89 (m, 1H), 7.89 (d, J =
8.9 Hz, 1H), 7.67 (dt, J = 10.5, 8.3 Hz, 1H). HPLC 99.1 area % (265
nm). Anal. Calcd for C13H7ClF7N2O3: C, 49.94; H, 2.26; N, 8.96.
Found: C, 49.87; H, 2.29; N, 8.98.
A scale-up synthesis from a total of 6.6 mmol of 101g was
performed in three batches. The combined product was chromato-
graphed as above and recrystallized from EtOH as white crystals (1.59
g, 64%): mp 210−211.55 °C. HPLC 97.8% (254 nm). Anal. Calcd for
C18H14F3N3OS: C, 57.29; H, 3.74; N, 11.13. Found: C, 57.32; H, 3.81;
N, 11.02.
6-(3,4-Difluorophenyl)-2-(3-fluoro-N-pyrrolidylamido)-
benzothiazole (72). 1,1′-Carbonyldiimidazole (163 mg, 1.01 mmol)
was added to a solution of 2-amino-6-(3,4-difluorophenyl)-
benzothiazole (106, 200 mg, 0.761 mmol) in CH3CN (10 mL).
The solution was stirred overnight with the formation of a precipitate,
2-Aryl-5-nitrobenzothiazoles 100a−g. 5-Nitro-2-phenylbenzo-
thiazole (100a). A mixture of N-(2-chloro-5-nitrophenyl)benzamide
(99a, 3.00 g, 10.85 mmol), sodium sulfide nonahydrate (2.96 g, 12.34
mmol), and sulfur (397 mg, 12.37 mmol) in EtOH (100 mL) was
refluxed for 2.5 h. The cooled reaction mixture was evaporated under
reduced pressure, diluted with 1 M HCl (100 mL), and extracted into
EtOAc. The product was purified on a column of silica gel eluting in
DCM, followed by recrystallization from EtOH to give white needles
1
(1.57 g, 57%): mp 195−196 °C. H NMR (400 MHz, DMSO-d6) δ
8.82 (d, J = 2.3 Hz, 1H), 8.45 (d, J = 8.8 Hz, 1H), 8.30 (dd, J = 8.8, 2.3
M
DOI: 10.1021/acs.jmedchem.6b01163
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Journal of Medicinal Chemistry
Article
Hz, 1H), 8.15 (ddt, J = 8.4, 3.1, 1.9 Hz, 2H), 7.74−7.57 (m, 3H).
HPLC 100 area % (290 nm). Anal. Calcd for C13H8N2O2S: C, 60.93;
H, 3.15; N, 10.93. Found: C, 90.95; H, 2.98; N, 11.03.
ABBREVIATIONS USED
■
BPR, brain to plasma ratio; CDI, 1,1′-carbonyldiimidazole;
HAT, human African trypanosomiasis; HATU, 1-[bis-
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate); IACUC, Institu-
tional Animal Care and Use Committee; LLQ, lower limit of
quantitation; SEM, standard error of the mean; SI, selectivity
index; WHO, World Health Organization
5-Nitro-2-(3,4-difluorophenyl)benzothiazole (100g). 100g was
prepared analogously to 100a from N-(2-chloro-5-nitrophenyl)-3,4-
difluorobenzamide (99g, 3.78 g, 12.09 mmol), but the crude product
was directly recrystallized from EtOH as a white solid (3.00 g, 85%):
1
mp 175−196 °C. H NMR (400 MHz, DMSO-d6) δ 8.84−8.79 (m,
1H), 8.48 (dt, J = 8.8, 0.4 Hz, 1H), 8.31 (dd, J = 8.8, 2.2 Hz, 1H), 8.20
(ddd, J = 11.3, 7.6, 2.3 Hz, 1H), 8.02 (dddd, J = 8.7, 4.3, 2.3, 1.4 Hz,
1H), 7.69 (dt, J = 10.5, 8.4 Hz, 1H). HPLC 97.6 area % (290 nm).
Anal. Calcd for C13H6F2N2O2S: C, 53.43; H, 2.07; N, 9.59. Found: C,
53.18; H, 2.09; N, 9.59.
REFERENCES
■
(1) Simarro, P. P.; Cecchi, G.; Paone, M.; Franco, J. R.; Diarra, A.;
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5-Amino-2-aryl-benzothiazoles 101a−g. 5-Amino-2-phenyl-
benzothiazole (101a).30 A mechanically stirred mixture of 5-nitro-2-
phenylbenzothiazole (100a, 1.87 g, 7.30 mmol), iron powder (2.21 g,
39.57 mmol), and ammonium chloride (818 mg, 15.30 mmol) in
EtOH (100 mL) and water (50 mL) was stirred at reflux for 2 h until
the reaction was complete by HPLC. The hot mixture was filtered
through Celite, and the filtrate was evaporated to near dryness under
reduced pressure. The residue was diluted with water and extracted
into DCM. The product was recrystallized from EtOH as yellow
crystals (1.47 g, 89%): mp 205−206 °C. 1H NMR (400 MHz, DMSO-
d6) δ 8.08−7.97 (m, 2H), 7.71 (dd, J = 8.6, 0.5 Hz, 1H), 7.61−7.49
(m, 3H), 7.17 (dd, J = 2.2, 0.5 Hz, 1H), 6.79 (dd, J = 8.6, 2.2 Hz, 1H),
5.33 (s, 2H). HPLC 100 area % (254 nm). Anal. Calcd for C13H10N2S:
C, 69.00; H, 4.45; N, 12.38. Found: C, 69.07; H, 4.59; N, 12.19.
5-Amino-2-(3,4-difluorophenyl)benzothiazole (101g). 101g was
prepared analogously to 101a from 5-nitro-2-(3,4-difluorophenyl)-
benzothiazole (100g, 2.96 g, 10.1 mmol) as a yellow solid (1.60 g,
60%): mp >174 °C dec. 1H NMR (400 MHz, DMSO-d6) δ 8.06 (ddd,
J = 11.4, 7.7, 2.2 Hz, 1H), 7.87 (dddd, J = 8.6, 4.3, 2.2, 1.4 Hz, 1H),
7.73 (dd, J = 8.6, 0.4 Hz, 1H), 7.61 (dt, J = 10.5, 8.4 Hz, 1H), 7.18 (dd,
J = 2.2, 0.5 Hz, 1H), 6.81 (dd, J = 8.6, 2.2 Hz, 1H), 5.37 (s, 2H).
HPLC 100 area % (254 nm). Anal. Calcd for C13H8F2N2S: C, 59.53;
H, 3.07; N, 10.68. Found: C, 59.29; H, 2.94; N, 10.62.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01163.
■
S
General experimental procedures, synthesis and charac-
terization of target compounds and intermediates (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: 919-966-4294. E-mail: tidwell@med.unc.edu.
ORCID
Donald A. Patrick: 0000-0001-7423-1379
J. Robert Gillespie: 0000-0002-5650-0333
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
■
Notes
The authors declare no competing financial interest.
(13) Pammolli, F.; Magazzini, L.; Riccaboni, M. The productivity
crisis in pharmaceutical r&d. Nat. Rev. Drug Discovery 2011, 10, 428−
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(14) Patrick, D. A.; Wenzler, T.; Yang, S.; Weiser, P. T.; Wang, M. Z.;
Brun, R.; Tidwell, R. R. Synthesis of novel amide and urea derivatives
of thiazol-2-ethylamines and their activity against trypanosoma brucei
rhodesiense. Bioorg. Med. Chem. 2016, 24, 2451−2465.
ACKNOWLEDGMENTS
■
This work was supported by the National Instituted of Health
(grant 5R01AI106850). We thank Patrick T. Weiser for his
constructive comments and assistance in editing this manu-
script.
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