Oligoamides of 2-amino-5-alkylthiazole 4-carboxylic acids: Anti-trypanosomal compounds

Article abstract of DOI:10.1007/s00044-013-0723-0

A range of minor groove binders (MGBs) and the intermediates formed in their synthesis was screened against infectious agents including bacteria and parasites. Activity was found against Trypanosoma brucei. Of particular interest is a molecule which does not contain a typical basic flexible tail group found in MGBs, but instead has an unprotected carboxylic acid group at that position (compound 4), which has activity of 63 nM against T. brucei.

Full text of DOI:10.1007/s00044-013-0723-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1170–1179
DOI 10.1007/s00044-013-0723-0
ORIGINAL RESEARCH
Oligoamides of 2-amino-5-alkylthiazole 4-carboxylic acids:
anti-trypanosomal compounds
•
Stuart Lang Abedawn I. Khalaf David Breen
•
•
•
Judith K. Huggan Carol J. Clements
•
•
Simon P. MacKay Colin J. Suckling
Received: 17 April 2013 / Accepted: 14 August 2013 / Published online: 28 August 2013
Ó Springer Science+Business Media New York 2013
Abstract A range of minor groove binders (MGBs) and the
intermediates formed in their synthesis was screened against
infectious agents including bacteria and parasites. Activity
was found against Trypanosoma brucei. Of particular
interest is a molecule which does not contain a typical basic
flexible tail group found in MGBs, but instead has an
unprotected carboxylic acid group at that position (com-
pound 4), which has activity of 63 nM against T. brucei.
are oligoamides constructed from N-alkylpyrroles and
C-alkylthiazoles usually with flexible basic C-terminal
groups. From this collection of about 150 compounds, some
oligoamides of 2-amino-5-alkylthiazole 4-carboxylic acids
were hits in a primary kinase screen but on further investi-
gation, no significant improvement in activity was found.
Nevertheless MGBs are known to be widely active as anti-
infective compounds (Barrett et al., 2013) and further
screening was carried out against a range of routinely
available infectious species, including Gram-positive bac-
teria (represented by Staphylococcus aureus, both methi-
cillin resistant and sensitive, and Mycobacterium aureum),
Gram-negative bacteria (Escherischia coli and Klebsiella
pneumoniae), fungi (Aspergillus niger and Candida albi-
cans). No significant activity was found against the bacteria
and fungi but the series showed consistent activity against
Trypanosoma brucei. 2-Amino-5-alkylthiazole 4-carboxylic
acid derivatives were found to be common components of
the active compounds. It was of interest, therefore, to probe
the structure–activity relationship in this series in particular
by reducing the size of the compounds from the original
tetracyclic minor groove binders so that compounds more
typical of drugs were obtained. This paper describes the
synthesis and biological activity of a number of oligoamides
containing two 2-aminothiazole 4-carboxylic acids coupled
with C-terminal aminoalkyl groups, 3-amino-N-methylpyr-
role 2-carboxylic acid, and N-terminal acetamides. The best
compounds showed sub-micromolar activity in a cell-based
assay of the blood form of T. brucei.
Keywords DNA Á Minor groove binder Á Amide Á
Thiazole Á Anti-parasitic activity
Introduction
Anti-infective drugs are important goals for medicinal
chemistry research and new chemotypes with specific
activities are always of interest. Whilst establishing a pro-
gramme of research seeking new inhibitors for a number of
kinase including Aurora kinases and IK kinases, a library of
minor groove binders for DNA based upon the structures of
the natural products, distamycin and netropsin was screened
(Khalaf et al., 2004; Anthony et al., 2007); these molecules
S. Lang (&) Á A. I. Khalaf Á D. Breen Á C. J. Suckling (&)
WestCHEM, Department of Pure and Applied Chemistry,
University of Strathclyde, 295 Cathedral Street,
Glasgow G1 1XL, UK
e-mail: stuart.lang@strath.ac.uk
C. J. Suckling
e-mail: c.j.suckling@strath.ac.uk
Results and discussion
J. K. Huggan Á C. J. Clements Á S. P. MacKay
Strathclyde Institute of Pharmacy and Biomedical Sciences,
University of Strathclyde, 161 Cathedral Street,
Glasgow G4 0NR, UK
The synthetic strategy involved hydrogenation of previ-
ously reported nitropyrrole (1) using Pd/C as the catalyst
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1171
(Khalaf et al., 2004). The intermediate amine formed in
this reaction was subsequently coupled, without isolation
due to its air sensitive nature, with acid (2) using T3P and
Et3N in THF to form compound (3) in 71 % yield over the
two steps. Hydrolysis of ester (3) gave acid (4), which was
coupled with a range of amines using our standard condi-
tions amide bond forming conditions gave compounds (5b–
d) (Scheme 1). These amide-forming reactions were rou-
tine but the coupling of 2-aminothiazoles often was more
difficult, especially with a second, sterically hindered,
thiazole as partner. However, coupling of aminothiazole
(6) with the thiazole carboxylic acid (2) using T3P amide
bond forming conditions gave the thiazole dimer (7) in
79 % yield (Scheme 2). This was a particularly satisfying
yield as this is the first reported example of two thiazole
units, bearing an isopropyl group, being coupled. Previous
attempts to form this motif have failed even with standard
acylating agents, acid chlorides and mixed anhydrides, but
also with direct coupling agents such as HBTU and HATU.
Hydrolysis of ester (7) led to the formation of acid (8) as
the only product that could be isolated in a yield of 39 %
yield, which was followed by formation of compounds
(9a–c). This three step procedure again used in order to
form ester (11), leading to acid (12) and amide (13)
(Scheme 3).
Trypanosomes have characteristic structures, known as
kinetoplasts, in which circular DNA is packaged densely
within a large mitochondrion (Lanteri et al., 2008; Shapiro
and Englund, 1990). Since the origin of these thiazole
oligoamides was in minor groove binders for DNA, it was
appropriate to include an organism with a specialized and
unusual handling of DNA in the assays undertaken. Rou-
tinely, primary anti-infective screening at Strathclyde
includes strains of Staphylococcus aureus, including
resistant strains, E. coli, and M. marinum, a less pathogenic
relative of the organism that causes tuberculosis, M.
Scheme 1 Synthesis of heterocyclic oligoamide trimers
Scheme 2 Synthesis of C-isopropyl-substituted thiazole amide dimers
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Med Chem Res (2014) 23:1170–1179
Scheme 3 Synthesis of C-phenylethyl-substituted thiazole amide dimers
tuberculosis, together with T. brucei. Only in the T. brucei
brucei screen was consistently significant activity observed
for this series of compounds.
activity than the trimers, the greatest activity being found
in compounds with the flexible dimethylaminopropyl
C-terminal amide (9a, 13). This group is common in anti-
bacterial minor groove binders (Khalaf et al., 2004;
Anthony et al., 2007; Bu¨rli et al., 2004). The phenylethyl
group in (13) and the corresponding isopropyl group in (9a)
seem to have little difference in their effects on anti-try-
panosomal activity. However overall it does appear that an
ionized C-terminal group, most commonly a protonated
amine at physiological pH, is important to give significant
activity. This suggests that these compounds may possibly
be acting by disrupting the kinetoplast, a well known lethal
event for trypanosomes (Suckling, 2008). The exception-
ally high activity of the carboxylic acid trimer (4) is unu-
sual in terms of DNA binding but the ionic group may also
be important in transport of the active compound to its
target, a factor known to be important in anti-trypanosomal
compounds (Barrett et al., 2013). Clearly, the total struc-
ture of (4) is required because neither of the two dimer
carboxylic acids (8) or (12) is significantly active.
The original design features of the minor groove binders
included thiazoles to provide hydrogen bond acceptors but
without increasing the polarity greatly, as would be the
case if an imidazole were used (Suckling, 2008). Also, the
introduction of the isopropyl group provides a good bal-
ance for the high polarity of the amide links. These features
are illustrated by (5a), a compound that came from the
original library; (5a) and close relatives are significantly
active against Gram-positive bacteria with minimum
inhibitory concentration values (MICs) in the range 5 lM
(Table 1). In SAR terms, compounds were selected for
synthesis based upon their composition in terms of the
heterocyclic ring building blocks rather than the atomic
level of detail in developing a typical medicinal chemical
structural template. Additional structural components used
in this study were N-methylpyrroles and a variety of
C-terminal esters, acids, and alkyl amides. As a structural
variation within the thiazole, a 5-phenylethyl thiazole was
included in place of the 5-isopropyl group. A further
question was whether it would be possible to obtain sig-
nificant activity from a dimer instead of the trimer such as
(5a) which is typical of the minor groove binder. The
results of these studies are shown in Tables 1, 2, and 3.
Assays for activity against T. brucei were carried out
using established methods in our laboratories (Gibson
et al., 2009) and Table 1 lists the trimers with significant
activity. The trimer (5a) is typical of an anti-bacterial and
anti-fungal minor groove binder (MIC vs. S. aureus
4.3 lM, vs. A. niger 4.3 lM) but significantly, it was
essentially inactive in this study. On the other hand, the
C-terminal carboxylic acid (4) stands out with an activity
of 63 nM. This was significantly the highest activity found
in any of the compounds investigated. The corresponding
ester (3) and piperazinyl amide (5c) were 20–30-fold
weaker in activity and the morpholino amides (4) and (5d)
3–10-fold weaker in activity whilst retaining the same
basic structures. In Tables 2 and 3, the results for the
dimers are presented and these show consistently weaker
The class of heterocyclic oligoamide carboxylic acid
represented by (4) is a new type of potential anti-trypan-
osomal compound. Whether its action is directly associated
with DNA binding remains to be established. If it is indeed
a DNA minor groove binder, it would share a mode of
action base with the well-established anti-trypanosomal di-
amidines (Barrett et al., 2013), which has been studied up
to clinical trials for many years. Most other recent studies
in this field leading to compounds with similar activity to
(4), including those from this laboratory (Gibson et al.,
2009; Tulloch et al., 2010), are built upon an exceptionally
wide range of heterocyclic templates such as furans (Lan-
golf et al., 2011), isoxazoles (Conti et al., 2011), pyrrolo-
pyridines (Gibson et al., 2009; Tulloch et al., 2010),
pyrazolines (Orrling et al., 2012), pyrroles (Langolf et al.,
2011), quinolines (Reid et al., 2011), and quinolones
(Hiltensperger et al., 2012), which also have a wide variety
of biological molecular targets, both enzymes and nucleic
acids. Notably, anti-microbial peptides, usually bearing
significant charge, are known to have anti-trypanosomal
activity typically working through plasma membrane
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Table 1 Anti-trypanosomal
activity of heterocyclic
oligoamide trimers
Compound
Structure
Activity
MIC (lM)
3
1.56
4
0.0634
5a
[25
5b
0.78
5c
1.92
5d
0.815
permeabilisation or endosomal disruption (Harrington,
2011). It is also possible that such mechanisms may be
relevant to the action of (4), a possibility that is reinforced
by the observation that some anti-bacterial minor groove
binders act at least in part by membrane disruption (Voo-
turi et al., 2011).
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Med Chem Res (2014) 23:1170–1179
Table 2 Anti-trypanosomal
activity of C-isopropyl-
substituted thiazole amide
dimers
Compound
Structure
Activity
MIC (lM)
7
2.20
8.74
0.715
3.12
8
9a
9b
9c
25
Summary
were recorded either at 400.13 or 500.13 MHz for the
proton, and 100.61 or 125.76 MHz for the carbon. The
chemical shifts are quoted in part per million (ppm) and the
coupling constant in Hertz (Hz). They are referenced on
tetra methyl silane but calibrated on the solvent residual
signal.
We have demonstrated that the thiazole carboxylic acid (4),
a heterocylic triamide related to anti-infective minor
groove binders, has significant activity (MIC = 63 nM)
against T. brucei. Activity was retained, although to a
lesser extent, in compounds containing simply two thia-
zoles directly coupled through an amide bond (9a, 13).
These compounds have potential for further development
in the anti-parasitic drug field.
The normal resolution mass spectra of the compounds
were recorded on a Thermofinnigan LCQ DUO LD mass
spectrometer using electro spray ionization (ESI) at the
University of Strathclyde Mass Spectrometry Service. The
high resolution mass spectra were recorded at the Uni-
versity of Strathclyde on Thermo Exactive Orbitrap Mass
Analyzer. The IR spectra were obtained using an ADR
crystal or from pressed disks using KBr as a matrix.
A Perkin-Elmer Spectrum One FT-IR spectrometer was
employed to record those spectra. Melting points were
obtained using Gallencamp Griffin SG94/05/530
apparatus.
Experimental
General
The proton (1H) and carbon (13C) NMR were recorded on
a Bruker AV400 or a Bruker AV500 spectrometer. They
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1175
Table 3 Anti-trypanosomal
Entry
Compound
Activity
MIC (lM)
activity of C-phenylethyl-
substituted thiazole amide
dimers
11
12
13
25
3.38
0.342
Synthesis of products
(1H, sep, J = 6.8 Hz), 4.00 (1H, sep, J = 6.8 Hz), 3.89 (3H,
s), 3.82 (3H, s), 2.27 (3H, s), 1.33–1.31 (12H, m). 13C NMR
(100 MHz, DMSO) d ppm 168.4, 163.1, 159.8, 158.5, 154.6,
153.4, 152.6, 149.2, 134.9, 133.3, 122.2, 122.0, 120.0, 105.5,
52.0, 37.1, 27.7, 27.1, 24.8, 24.8, 22.9; MS (ESI) m/z 533
[(M?H)? 100 %,].
Methyl 2-(4-2-acetamido-5-isopropylthiazole-4-
carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-5-
isopropylthiazole-4-carboxylate (3)
Methyl 2-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-5-
isopropylthiazole-4-carboxylate (2 g, 5.68 mmols) was
dissolved in methanol (250 ml). 10 % Pd/C (605 mg,
0.57 mmols) was added under an argon atmosphere, which
was changed to a hydrogen atmosphere and stirred for 5 h.
The hydrogen was removed and the palladium removed by
filtration through Celite and the solvent removed under
vacuum. The residue was dissolved in THF (15 ml) along
with 2-acetamido-5-isopropylthiazole-4-carboxylic acid
(1.94 g, 8.52 mmols). Triethylamine (4.7 ml, 34.1 mmols)
was added followed by T3P (5.4 ml, 8.52 mmols) at 0 °C
and the mixture stirred overnight. The volatiles were
removed and the residue added to a saturated sodium
bicarbonate solution (30 ml). This was stirred overnight and
the solid collected by filtration. This solid was then sus-
pended in diethyl ether (30 ml) and stirred overnight and
collected by filtration to give the desired compound (3)
(2.15 g, 71 %) as a brown powder (99 % pure HPLC),
mp = 156–158 °C. HRMS (ESI) calcd for C23H29N6O5S2
[M?H]?: 533.1635, found: 533.1630; mmax (ATR)/cm-1
3390, 3198, 2965, 2922, 2868, 1718, 1653, 1539, 1463; 1H
NMR (400 MHz, CDCl3) d ppm 10.27 (1H, br s), 9.04 (1H,
s), 7.52 (1H, d, J = 1.5 Hz), 6.75 (1H, d, J = 1.5 Hz), 4.32
2-(4-2-Acetamido-5-isopropylthiazole-4-carboxamido)-1-
methyl-1H-pyrrole-2-carboxamido)-5-isopropylthiazole-4-
carboxylic acid (4)
Methyl
2-(4-2-acetamido-5-isopropylthiazole-4-carbox-
amido)-1-methyl-1H-pyrrole-2-carboxamido)-5-iso-
propylthiazole-4-carboxylate (1.8 g, 3.38 mmols) was
suspended in a water (15 ml) methanol (15 ml) mixture and
lithium hydroxide (0.81 g, 33.8 mmols) added. The mixture
was stirred for 2 days then added to water (30 ml) and
washed with dichloromethane (30 ml). The aqueous layer
was acidified with concentrated hydrochloric acid and
extracted with dichloromethane (3 9 20 ml). The solvent
was removed and the residue stirred in brine (20 ml). This
was filtered to give the desired compound (4) (1.22 g, 70 %)
as a yellow powder (88 % pure HPLC), mp = 268–271 °C.
HRMS (ESI) calcd for C22H27N6O5S2 [M?H]?: 519.1479,
found: 519.1472; max (ATR)/cm-1 3142, 2965, 2932, 1668,
1
1660, 1539, 1463; H NMR (400 MHz, DMSO) d ppm
12.32 (1H, br s), 12.12 (1H, s), 9.56 (1H, s), 7.56 (1H, d,
J = 1.6 Hz), 7.35 (1H, d, J = 1.6 Hz), 4.16 (1H, sep,
J = 6.9 Hz), 4.03 (1H, sep, J = 6.9 Hz), 3.89 (3H, s), 2.15
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Med Chem Res (2014) 23:1170–1179
(3H, s), 1.29–1.27 (12H, m); 13C NMR (100 MHz, DMSO)
d ppm 168.8, 163.6, 159.3, 159.0, 153.8, 153.5, 149.5, 145.6,
135.9, 134.7, 121.8, 121.4, 120.3, 107.0, 36.7, 26.9, 26.3,
24.7, 24.6, 22.4; MS (ESI) m/z 519 [(M?H)? 100 %,], 477
(33).
[M?H]?: 601.2374, found: 601.2460; mmax (ATR)/cm-1
3390, 3265, 3215, 2957, 2860, 2804, 1655, 1608, 1545,
1462; 1H NMR (400 MHz, CDCl3) d ppm 10.78 (1H, br s),
9.11 (1H, br s), 7.51 (1H, s), 6.75 (1H, s), 4.30 (1H, sep,
J = 6.8 Hz), 3.94 (3H, s), 3.65 (2H, br s), 3.31 (2H, br s),
3.23 (1H, sep, J = 6.5 Hz), 2.78 (2H, br s), 2.36 (2H, br s),
2.27 (3H, s) 2.23 (3H, s), 1.31 (6H, d, J = 6.8 Hz), 1.16
(6H, d, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3) d ppm
168.8, 165.5, 159.9, 158.4, 156.0, 153.4, 149.3, 141.5,
137.4, 135.0, 122.4, 121.8, 120.6, 105.8, 55.3, 54.6, 47.0,
46.0 42.0, 37.3, 27.3, 27.2, 25.1, 25.0, 24.0, 23.1; MS (ESI)
m/z 601 [(M?H)? 100 %,], 559 (27).
2-Acetamido-5-isopropyl-N-(5-((isopropyl-4-((2-
morpholinoethyl)carbamoyl)thiazol-2-yl)carbamoyl)-1-
methyl-1H-pyrrole-3-yl)thiazole-4-carboxamide (5b)
2-(4-2-Acetamido-5-isopropylthiazole-4-carboxamido)-1-
methyl-1H-pyrrole-2-carboxamido)-5-isopropylthiazole-4-
carboxylic acid (100 mg, 0.19 mmols) and 4-(2-amino-
ethyl)morpholine (39 ll, 0.3 mmols) were dissolved in
THF (10 ml). Triethylamine (0.16 ml, 1.16 mmols) was
added followed by T3P (0.19 ml, 0.3 mmols) at 0 °C and
the mixture stirred overnight. The volatiles were removed
and the residue added to a saturated sodium bicarbonate
solution (30 ml). This was stirred overnight and the solid
collected by filtration. This solid was then suspended in
diethyl ether (30 ml) and stirred overnight and collected by
filtration to give the desired compound (5b) (102 mg,
85 %) as a light brown powder (98 % pure HPLC),
mp = 178–180 °C. HRMS (ESI) calcd for C28H39N8O5S2
[M?H]?: 631.2479, found: 631.2463; mmax (ATR)/cm-1
3370, 3186, 2963, 2932, 2864, 1645, 1539, 1504, 1456; 1H
NMR (400 MHz, CDCl3) d ppm 10.85 (1H, br s), 8.98
(1H, s), 7.91 (1H, s), 7.67 (1H, s), 6.83 (1H, s), 4.39–4.26
(2H, m), 3.96 (3H, s), 3.68–3.61 (6H, m), 2.58–2.45 (6H,
m), 2.36 (3H, s), 1.35–1.28 (12H, m); 13C NMR (100 MHz,
CDCl3) d ppm 168.6, 163.6, 159.8, 158.4, 154.6, 153.6,
149.3, 144.0, 135.2, 134.7, 122.2, 122.1, 120.3, 104.6,
66.4, 58.0, 53.5, 37.3, 35.6, 27.2, 27.2, 24.9, 24.9, 22.9; MS
(ESI) m/z 631 [(M?H)? 100 %,], 589 (33), 546 (4).
2-Acetamido-5-isopropyl-N-(5-((isopropyl-4-(morpholine-
4-carbamoyl)thiazol-2-yl)carbamoyl)-1-methyl-1H-
pyrrole-3-yl)thiazole-4-carboxamide (5d)
2-(4-2-Acetamido-5-isopropylthiazole-4-carboxamido)-1-
methyl-1H-pyrrole-2-carboxamido)-5-isopropylthiazole-4-
carboxylic acid (100 mg, 0.19 mmols) and 4-(2-amino-
ethyl)morpholine (26 ll, 0.3 mmols) were dissolved in
THF (10 ml). Triethylamine (0.16 ml, 1.16 mmols) was
added followed by T3P (0.19 ml, 0.3 mmols) at 0 °C and
the mixture stirred overnight. The volatiles were removed
and the residue added to a saturated sodium bicarbonate
solution (30 ml). This was stirred overnight and the solid
collected by filtration. This solid was then suspended in
diethyl ether (30 ml) and stirred overnight and collected by
filtration to give the desired compound (5d) (95 mg, 85 %)
as a white powder (99 % pure HPLC), mp = 174–176 °C.
HRMS (ESI) calcd for C26H34N7O5S2 [M?H]?: 588.2057,
found: 588.2054; mmax (ATR)/cm-1 3397, 3260, 3213,
2967, 2926, 2866, 1668, 1654, 1616, 1541, 1522, 1458; 1H
NMR (400 MHz, CDCl3) d ppm 10.60 (1H, br s), 9.88
(1H, br s), 9.03, (1H, s), 7.43 (1H, s), 6.76 (1H, s), 4.30
(1H, sep, J = 6.8 Hz), 3.94 (3H, s), 3.72–3.30 (9H, m),
2.31 (3H, s), 1.31 (6H, d, J = 6.8 Hz), 1.22 (6H, d,
J = 6.7 Hz); 13C NMR (100 MHz, CDCl3) d ppm 168.6,
165.4, 159.7, 158.1, 155.5, 153.2, 149.3, 142.2, 137.0,
134.3, 122.2, 121.5, 120.6, 105.5, 66.9, 66.7, 47.5, 42.6,
37.1, 27.2, 25.0, 25.0, 24.9, 24.8, 23.0; MS (ESI) m/z 588
[(M?H)? 100 %,], 546 (20).
2-Acetamido-5-isopropyl-N-(5-((isopropyl-4-(4-
(methylpiperazine-1-carbamoyl)thiazol-2-yl)carbamoyl)-1-
methyl-1H-pyrrole-3-yl)thiazole-4-carboxamide (5c)
2-(4-2-Acetamido-5-isopropylthiazole-4-carboxamido)-1-
methyl-1H-pyrrole-2-carboxamido)-5-isopropylthiazole-4-
carboxylic acid (100 mg, 0.19 mmols) and 1-methylpi-
perazine (33 ll, 0.3 mmols) were dissolved in THF
(10 ml). Triethylamine (0.16 ml, 1.16 mmols) was added
followed by T3P (0.19 ml, 0.3 mmols) at 0 °C and the
mixture stirred overnight. The volatiles were removed and
the residue added to a saturated sodium bicarbonate solu-
tion (30 ml). This was stirred overnight and the solid col-
lected by filtration. This solid was then suspended in
diethyl ether (30 ml) and stirred overnight and collected by
filtration to give the desired compound (5c) (118 mg,
Methyl 2-(2-acetamido-5-isopropylthiazole-4-
carboxamido)-5-isopropylthiazole-4-carboxylate (7)
A mixture of 2-acetamido-5-isopropylthiazole-4-carbox-
ylate (6 g, 26.3 mmols) and methyl 2-amino-5-iso-
propylthiazole-4-carboxylate (5.26 g, 26.3 mmols) were
dissolved in THF (50 ml). Triethylamine (21.9 ml,
157.9 mmols) was added followed by T3P (25.1 ml,
39.5 mmols) at 0 °C and the mixture stirred overnight. The
volatiles were removed and the residue added to a saturated
99 %) as
a white powder ([99 % pure HPLC),
mp = 168–170 °C. HRMS (ESI) calcd for C27H37N8O4S2
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1177
sodium bicarbonate solution (125 ml). This was stirred
overnight and the solid collected by filtration. This solid
was then suspended in diethyl ether (50 ml) and stirred
overnight and collected by filtration to give the desired
compound (7) (8.56 g, 79 %) as a white powder ([99 %
pure HPLC), mp = 120–122 °C. HRMS (ESI) calcd for
C17H23N4O4S2 [M?H]?: 411.1155, found: 411.1151; mmax
(ATR)/cm-1 3549, 3351, 2967, 2932, 1703, 1672, 1539,
1440; 1H NMR (400 MHz, CDCl3) d ppm 10.63 (1H, br s),
9.93 (1H, br s), 4.31 (1H, sep, J = 6.8 Hz), 4.10 (1H, sep,
J = 6.8 Hz), 3.91 (3H, s), 1.35 (12H, d, J = 6.6 Hz). 13C
NMR (100 MHz, DMSO) d ppm 168.2, 162.9, 160.2,
153.8, 153.8, 153.0, 152.4, 133.7, 133.5, 52.1, 27.7, 27.4,
24.8, 24.8, 23.2. MS (ESI) m/z 411 [(M?H)? 100 %,].
calcd for C21H33N6O3S2 [M?H]?: 481.2050, found:
481.2042; mmax (ATR)/cm-1 3333, 3242, 2969, 1680, 1664,
1620, 1557, 1530, 1462, 1437; 1H NMR (400 MHz,
CDCl3) d ppm 11.27 (1H, br s), 10.54 (1H, br s), 8.49 (1H,
br s), 4.40 (1H, sep, J = 6.8 Hz) 4.25 (1H, sep,
J = 6.8 Hz), 3.55 (2H, td, J = 6.8 and 6.2 Hz), 2.38 (3H,
s) 2.33 (2H, t, J = 6.8 Hz), 2.12 (6H, s), 1.79 (2H, tt,
J = 6.8 and 6.8 Hz) 1.32 (12H, d, J = 6.8 Hz); 13C NMR
(100 MHz, CDCl3) d ppm 169.0, 165.2, 159.9, 154.4,
153.9, 152.3, 149.2, 135.8, 133.3, 57.0, 45.3, 37.9, 27.6,
27.5, 27.4, 25.3, 24.9, 23.0; MS (ESI) m/z 481 [(M?H)?
100 %,], 339 (19).
2-Acetamido-5-isopropyl-N-(5-isopropyl-4-((2-
morpholinoethyl)carbamoyl)thiazol-2-yl)thiazole-4-
carboxamide (9b)
2-(2-Acetamido-5-isopropylthiazole-4-carboxamido)-5-
isopropylthiazole-4-carboxylic acid (8)
2-(2-Acetamido-5-isopropylthiazole-4-carboxamido)-5-
isopropylthiazole-4-carboxylic acid (100 mg, 0.25 mmols)
and 4-(2-aminoethyl)morpholine (50 ll, 0.38 mmols) were
dissolved in THF (10 ml). Triethylamine (0.21 ml,
1.5 mmols) was added followed by T3P (0.24 ml,
0.38 mmols) at 0 °C and the mixture stirred overnight. The
volatiles were removed and the residue added to a saturated
sodium bicarbonate solution (30 ml). This was stirred
overnight and the solid collected by filtration to give the
desired compound (9b) (102 mg, 85 %) as a white powder
(94 % purity HPLC), mp = 113–116 °C. HRMS (ESI)
calcd for C22H33N6O4S2 [M?H]?: 509.1999, found:
509.1989; mmax (ATR)/cm-1 3333, 3231, 2967, 2932, 2851,
2801, 1620, 1560, 1531, 1439; 1H NMR (400 MHz,
CDCl3) d ppm 11.24 (1H, br s), 10.56 (1H, br s), 8.50 (1H,
t, J = 4.9 Hz), 4.44 (1H, sep, J = 6.9 Hz), 4.25 (1H, sep,
J = 6.8 Hz), 3.65 (2H, td, J = 5.9 and 4.9 Hz), 3.43–3.41
(4H, m), 2.54 (2H, t, J = 5.9 Hz), 2.39 (3H, s), 2.31–2.29
(4H, m), 1.35–1.31 (12H, m); 13C NMR (100 MHz,
CDCl3) d ppm 168.9, 165.4, 160.1, 154.6, 153.9, 152.6,
149.8, 135.7, 133.3, 66.7, 58.0, 53.9, 36.1, 27.5, 27.4, 25.4,
24.9, 23.0; MS (ESI) m/z 509 [(M?H)? 100 %,].
Methyl
2-(2-acetamido-5-isopropylthiazole-4-carbox-
(8 g,
amido)-5-isopropylthiazole-4-carboxylate
19.5 mmols) was suspended in water (75 ml) methanol
(75 ml) mixture and lithium hydroxide (4.67 g,
195 mmols) added. The mixture was stirred overnight then
added to water (400 ml) and washed with dichloromethane
(400 ml). The aqueous layer was acidified with concen-
trated hydrochloric acid and extracted with dichlorometh-
ane (3 9 300 ml). The solvent was removed and the
residue stirred in brine (250 ml). This was filtered to give
the desired compound (8) (3 g, 39 %) as a white powder
(92 % purity HPLC), mp = 240–242 °C. HRMS (ESI)
calcd for C16H21N4O4S2 [M?H]?: 397.0999, found:
397.0990; mmax (ATR)/cm-1 3331, 3183, 2965, 2928, 2868,
1
1672, 1534, 1437; H NMR (400 MHz, DMSO) d ppm
4.11–4.00 (2H, m), 2.17 (3H, s), 1.31–1.28 (12H, m); 13C
NMR (100 MHz, DMSO) d ppm 168.9, 163.4, 160.0,
154.0, 152.4, 150.3, 148.9, 135.1, 133.9, 27.0, 26.6, 24.7,
24.5, 22.5; MS (ESI) m/z 397 [(M?H)? 100 %,], 355 (40).
2-Acetamido-N-(4-((3-(dimethylamino)propyl)carbamoyl)-
5-isopropylthiazol-2-yl)-5-isopropylthiazole-4-
carboxamide (9a)
2-Acetamido-5-isopropyl-N-(5-isopropyl-4-(morpholine-4-
carbonyl)thiazol-2-yl)-thiazole-4-carboxamide (9c)
2-(2-Acetamido-5-isopropylthiazole-4-carboxamido)-5-
isopropylthiazole-4-carboxylic acid (100 mg, 0.25 mmols)
and 3-dimethylaminopropyl amine (48 ll, 0.38 mmols)
were dissolved in THF (10 ml). Triethylamine (0.21 ml,
1.5 mmols) was added followed by T3P (0.24 ml,
0.38 mmols) at 0 °C and the mixture stirred overnight. The
volatiles were removed and the residue added to a saturated
sodium bicarbonate solution (30 ml). This was stirred
overnight and the solid collected by filtration to give the
desired compound (9a) (97 mg, 80 %) as a white powder
(93 % purity HPLC), mp = 101–103 °C. HRMS (ESI)
2-(2-Acetamido-5-isopropylthiazole-4-carboxamido)-5-
isopropylthiazole-4-carboxylic acid (100 mg, 0.25 mmols)
and morpholine (33 ll, 0.38 mmols) were dissolved in
THF (10 ml). Triethylamine (0.21 ml, 1.5 mmols) was
added followed by T3P (0.24 ml, 0.38 mmols) at 0 °C and
the mixture stirred overnight. The volatiles were removed
and the residue added to a saturated sodium bicarbonate
solution (30 ml). This was stirred overnight and the solid
collected by filtration to give the desired compound (9c)
(98 mg, 84 %) as a white powder (90 % purity HPLC),
123

1178
Med Chem Res (2014) 23:1170–1179
mp = 84–86 °C. HRMS (ESI) calcd for C20H28N5O4S2
[M?H]?: 466.1577, found: 466.1571; mmax (ATR)/cm-1
3323, 3256, 3200, 2961, 2924, 2866, 1686, 1668, 1608,
1554, 1521, 1431; 1H NMR (400 MHz, CDCl3) d ppm
11.41 (1H, s), 11.27 (1H, s), 4.20 (1H, sep, J = 6.9 Hz),
3.78–3.73 (4H, m), 3.68 (1H, sep, J = 6.8 Hz) 3.57–3.56
(4H, m), 2.30 (3H, s), 1.36 (6H, d, J = 6.9 Hz), 1.31 (6H,
d, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) d ppm
168.9, 166.1, 161.1, 155.8, 154.5, 152.3, 145.9, 137.2,
133.8, 67.1, 67.0, 47.8, 43.1, 27.6, 27.5, 25.6, 25.1, 23.0;
MS (ESI) m/z 466 [(M?H)? 100 %,].
[M?H]?: 459.1155, found: 459.1150; mmax (ATR)/cm-1
3350, 2967, 2930, 1672, 1531, 1427; H NMR (400 MHz,
1
DMSO) d ppm 12.83 (1H, br s), 12.15 (1H, s), 11.1 (1H, br
s), 7.30-7.19 (5H, m), 4.05 (1H, sep, J = 6.7 Hz), 3.42
(2H, t, J = 7.6 Hz), 2.94 (2H, t, J = 7.6 Hz) 2.16 (3H, s),
1.29 (6H, d, J = 6.7 Hz); 13C NMR (100 MHz, DMSO)
d ppm 168.9, 163.3, 160.0, 154.0, 152.7, 149.0, 141.8,
140.6, 136.5, 133.9, 128.3, 128. 3, 126.1, 36.7, 28.1, 26.6,
24.5, 22.4; MS (ESI) m/z 459 [(M?H)? 100 %,], 417 (60),
100 (28).
2-Acetamido-N-(4-((3-(dimethylamino)propyl)carbamoyl)-
5-phenethylthiazol-2-yl)-5-isopropylthiazole-4-
carboxamide (13)
Methyl 2-(2-acetamido-5-isopropylthiazole-4-
carboxamido)-5-phenethylthiazole-4-carboxylate (11)
A mixture of 2-acetamido-5-isopropylthiazole-4-carbox-
ylate (913 mg, 4 mmols) and methyl 2-amino-5-iso-
propylthiazole-4-carboxylate (1.05 g, 4 mmols) were
dissolved in THF (15 ml). Triethylamine (3.34 ml,
24 mmols) was added followed by T3P (3.82 ml, 6 mmols)
at 0 °C and the mixture stirred overnight. The volatiles
were removed and the residue added to a saturated sodium
bicarbonate solution (50 ml). This was stirred overnight
and the solid collected by filtration. This solid was then
suspended in diethyl ether (30 ml) and stirred overnight
and collected by filtration to give the desired compound
(11) (982 mg, 52 %) as a white powder ([99 % purity
HPLC), mp = 129–131 °C. HRMS (ESI) calcd for
C22H25N4O4S2 [M?H]?: 473.1312, found: 473.1302; mmax
(ATR)/cm-1 2963, 1722, 1674, 1552; 1H NMR (400 MHz,
CDCl3) d ppm 10.41 (1H, br s), 9.48 (1H, br s), 7.31–7.19
(5H, m), 4.30 (1H, sep, J = 6.8 Hz), 3.92 (3H, s), 4.49
(2H, t, J = 8.0 Hz), 3.01 (2H, t, J = 8.0 Hz), 2.31 (3H, s),
1.36 (6H, d, J = 6.8 Hz). 13C NMR (100 MHz, CDCl3)
d ppm 168.4, 162.9, 160.3, 153.9, 153.9, 152.8, 143.9,
140.6, 135.4, 133.6, 128.7, 128.7, 126.6, 52.3, 37.7, 29.2,
27.6, 25.0, 23.4; MS (ESI) m/z 473 [(M?H)? 100 %,].
2-(2-Acetamido-5-isopropylthiazole-4-carboxamido)-5-
phenethylthiazole-4-carboxylic acid (100 mg, 0.22 mmols)
and 3-dimethylaminopropyl amine (55 ll, 0.44 mmols)
were dissolved in THF (10 ml). Triethylamine (0.18 ml,
1.32 mmols) was added followed by T3P (0.21 ml,
0.33 mmols) at 0 °C and the mixture stirred overnight. The
volatiles were removed and the residue added to a saturated
sodium bicarbonate solution (30 ml). This was stirred
overnight and the solid collected by filtration to give the
desired compound (13) (101 mg, 85 %) as a white powder
(96 % purity HPLC), mp = 72–74 °C. HRMS (ESI) calcd
for C26H35N6O3S2 [M?H]?: 543.2207, found: 543.2203;
mmax (ATR)/cm-1 3307, 3188, 2980, 2972, 1638, 1528,
1383; 1H NMR (400 MHz, CDCl3) d ppm 11.27 (1H, br s),
10.49 (1H, br s), 8.55 (1H, br s), 7.27–7.15 (5H, m), 4.26
(1H, sep, J = 6.7 Hz), 3.65–3.57 (4H, m), 3.00 (2H, t,
J = 7.9 Hz), 2.88 (2H, t, J = 7.0 Hz), 2.21 (3H, s), 2.13,
(6H, s), 1.85–1.76 (2H, m), 1.33 (6H, d, J = 6.7 Hz); 13C
NMR (100 MHz, CDCl3) d ppm 168.8, 164.8, 159.6,
154.2, 153.6, 152.1, 140.5, 140.1, 136.7, 133.0, 128.5,
128.1, 126.4, 56.9, 45.3, 37.8, 37.5, 28.4, 27.5, 27.3, 24.7,
22.8; MS (ESI) m/z 543 [(M?H)? 100 %,], 501 (12).
2-(2-Acetamido-5-isopropylthiazole-4-carboxamido)-5-
phenethylthiazole-4-carboxylic acid (12)
Biological assay
Methyl
2-(2-acetamido-5-isopropylthiazole-4-carbox-
(900 mg,
Compounds were assayed against compounds were assayed
against T. brucei as previously described (Gibson et al.,
2009) using a method based upon that of O’Brien et al.
(2000). Cell-based assays for trypanocidal activity were
carried out against the blood stream form of T. b. brucei.
The assays were conducted in 96 well microplates with
incubation at 37 °C under a humidified 5 % CO2 atmo-
sphere for 48 h. Fluorescence was detected using a Wallac
Victor 2 multilabel plate reader (excitation 560 nm, emis-
sion 590 nm) giving % of control values. MICs were
obtained for compounds showing less than 20 % of control
values against T. b. brucei.
amido)-5-phenethylthiazole-4-carboxylate
1.9 mmols) was suspended in water (7.5 ml) methanol
(7.5 ml) mixture and lithium hydroxide (456 mg,
19 mmols) added. The mixture was stirred for 2 days then
added to water (40 ml) and washed with dichloromethane
(40 ml). The aqueous layer was acidified with concentrated
hydrochloric acid and extracted with dichloromethane
(3 9 30 ml). The solvent was removed and the residue
stirred in brine (25 ml). This was filtered to give the
desired compound (12) (486 mg, 56 %) as a white powder,
mp = 158–160 °C. HRMS (ESI) calcd for C21H23N4O4S2
123

Med Chem Res (2014) 23:1170–1179
1179
Acknowledgments The authors would like to thank Cancer
Research UK for funding and Craig Irving and Patricia Keating for all
the help and support which they have provided during the course of
this research work.
Langolf S, Machon U, Ehlers M, Sicking W, Schirmeister T, Bu¨chhold
C, Gelhaus C, Rosenthal PJ, Schmuck C (2011) Development of
antitrypanosomal and antiplasmodial nonpeptidic cystine protease
inhibitors based on N-protected-guanidino-furan and -pyrrole
building blocks. Chem Med Chem 6:1581–1586
Lanteri CA, Tidwell RR, Meshnick SR (2008) The mitochondrion is a
site of trypanocidal action of the aromatic diamidine DB75 in
bloodstream forms of Trypanosomo Brucei. Antimicrob Agents
Chemother 52:875–882
References
Anthony NG, Breen D, Clark J, Donoghue G, Drummond AJ, Ellis
EM, Gemmell CG, Helsebeux JJ, Hunter IS, Khalaf AI, Mackay
SP, Parkinson JA, Suckling CJ, Waigh RD (2007) Antimicrobial
lexitropsins containing amide, amidine and alkene linking
groups. J Med Chem 50:6116–6125
O’Brian J, Wilson I, Orton T, Pognan F (2000) Investigation of the
Alamar Blue (resazurin) fluorescent dye for the assessment of
mammalian cell cytotoxicity. Eur J Biochem 267:5421–5426
Orrling KM, Jansen C, Vu XL, Balmer V, Bregy P, Shanmugham A,
England P, Bailey D, Cos P, Maes L, Adams E, van den Bogaart
E, Chatelain E, Ioset JR, van de Stolpe A, Zorg S, Veerman J,
Seebeck T, Sterk GJ, de Esch IJP, Leurs R (2012) Catechol
pyrazolinones as trypanocidals: fragment-based design, synthe-
sis, and pharmacological evaluation of nanomolar inhimitors of
trypanosomal phosphodiesterase B1. J Med Chem 55:8745–8756
Reid CS, Patrick DA, He S, Fotie J, Premalatha K, Tidwell RR, Wang
MZ, Liu Q, Gershkovich P, Wasan KM, Wenzler T, Brun R,
Werbovetz KA (2011) Synthesis and antitrypanosomal evalua-
tion of derivatives of N-benzyl-1,2-dihydroquinolin-6-ols: effect
of core substitutions and salt formations. Bioorg Med Chem
19:513–523
Shapiro TA, Englund PT (1990) Selective cleavage of kinetoplast
DNA minicircles promoted by antitrypanosomal drugs. Proc Natl
Acad Sci USA 87:950–954
Suckling CJ (2008) Molecular recognition and physicochemical
properties in the discovery of selective antibacterial minor
groove binders. J Phys Org Chem 21:575–583
Tulloch LB, Martini VP, Iulek J, Huggan JK, Lee JH, Gibson CL,
Smith TK, Suckling CJ, Hunter WN (2010) Structure based
design of pteridine reductase inhibitors targeting African sleep-
ing sickness and the leishmaniases. J Med Chem 53:221–229
Vooturi SK, Dewal MB, Firestine SM (2011) Examination of a
synthetic benzophenone membrane-targeted antibiotic. Org
Biomol Chem 9:6367–6372
Barrett MP, Gemmell CG, Suckling CJ (2013) Minor groove binders
as anti-infective agents. Pharmacol Ther 139:12–23
Bu¨rli RW, McMinn D, Kaizerman JA, Hu W, Ge Y, Pack Q, Jaing V,
Gross M, Garcia M, Tanaka R, Moser HE (2004) DNA binding
ligands targeting drug-resistant gram-positive bacteria. Part 1:
internal benzimidazole derivatives. Bioorg Med Chem Lett
14:1253–1257
Conti P, Pinto A, Wong PE, Major LL, Tamborini L, Iannuzzi MC,
DeMicheli C, Barrett MP, Smith TK (2011) Synthesis and
in vitro/in vivo evaluation of the antitrypanosomal activity of
3-bromoacivicin, a potent CTP synthetase inhibitor. Chem Med
Chem 6:329–333
Gibson CL, Huggan JK, Kennedy A, Kiefer L, Lee J-H, Suckling CJ,
Clements C, Harvey AL, Hunter WN, Tulloch LB (2009)
Diversity orientated synthesis of fused pyrimidines designed as
antifolates. Org Biomol Chem 7:1829–1842
Harrington JM (2011) Antimicrobial peptide killing of African
trypanosomes. Parasite Immunol 33:461–469
Hiltensperger G, Jones NG, Niedermeier S, Stich A, Kaiser M, Jung J,
Puhl S, Damme A, Braunschweig H, Meinel L, Engstler M,
Holzgrabe U (2012) Synthesis and structure–activity relation-
ships of new quinolone-type molecules against Trypansoma
Brucei. J Med Chem 55:2538–2548
Khalaf AI, Waigh RD, Drummond AJ, Pringle B, McGroarty I,
Skellern GG, Suckling CJ (2004) Distamycin analogues with
enhanced lipophicity: synthesis and antimicrobial activity. J Med
Chem 47:2133–2156
123