Synthesis and antiprotozoal activity of 2,5-bis[amidinoaryl]thiazoles

Article abstract of DOI:10.1016/j.bmc.2010.03.058

Seven novel diamidino 2,5-bis(aryl)thiazoles (5a-g) were synthesized and evaluated against Trypanosoma brucei rhodensiense (T. b. r.) and Plasmodium falciparum (P. f.). The diamidines were obtained directly from the corresponding bis-nitriles (4a-g) by th

Full text of DOI:10.1016/j.bmc.2010.03.058

Bioorganic & Medicinal Chemistry 18 (2010) 3551–3558
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiprotozoal activity of 2,5-bis[amidinoaryl]thiazoles
Danuta Branowska a, , Abdelbasset A. Farahat a, Arvind Kumar a, Tanja Wenzler b, Reto Brun b, Yang Liu a,
W. David Wilson a, David W. Boykin a,
*
a Department of Chemistry, Georgia State University, Atlanta, GA 30303-3083, USA
b University of Basel and Parasite Chemotherapy Swiss Tropical and Public Health Institute, Basel CH4002, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven novel diamidino 2,5-bis(aryl)thiazoles (5a–g) were synthesized and evaluated against Trypano-
soma brucei rhodensiense (T. b. r.) and Plasmodium falciparum (P. f.). The diamidines were obtained directly
from the corresponding bis-nitriles (4a–g) by the action of lithium bis(trimethylsilyl)amide. The bis-
nitriles 4a–f were synthesized in four steps starting with the Stille coupling of 2-tributyltinthiazole with
the appropriate cyanoaryl halide. The bis-nitrile 5g was obtained by the palladium facilitated coupling of
the mixed tin-silyl reagent 2-trimethylsilyl-5-trimethyltinthiazole with 2-bromo-5-cyanopyridine. The
amidoxime potential prodrugs 6a–e, 6g were obtained by the reaction of hydroxylamine with the bis-
nitriles. O-Methylation of the amidoximes gave the corresponding N-methoxyamidines 7a–c, 7e, 7g.
Received 3 February 2010
Revised 23 March 2010
Accepted 24 March 2010
Available online 27 March 2010
Keywords:
Diamidines
Antiprotozoals
Minor groove binders
The diamidines showed strong DNA binding affinity as reflected by
DTm measurements. Four of the
diamidines 5a, 5b, 5d and 5e were highly active in vitro against P. f. giving IC50 values between 1.1
and 2.5 nM. The same four diamidines showed IC50 values between 4 and 6 nM against T. b. r. The selec-
tivity indices ranged from 233 to 9175. One diamidine 5a produced one of four cures at an ip dose of
4 Â 5 mg/kg in the STIB900 mouse model for acute African trypanosomiasis. The amidoxime and N-meth-
oxyamidine of 5a were the only produgs to provide cures (1/4 cures) in the same mouse model on oral
dosage at 4 Â 25 mg/kg.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
zoal agents for nearly eight decades.6 Despite broad spectrum anti-
microbial activity of this class, pentamidine remains the only
It has been estimated that approximately 2.2 billion people are
at risk for the related protozoan diseases malaria and trypanosomi-
asis.1 Available drugs for treatment of both of these diseases, exhi-
bit significant shortcomings. The growth of multi-drug resistance
strains of Plasmodium falciparum, the species which causes the
most severe cases of malaria, has created an urgent need for new
antimalarials with different modes of action.2,3 Presently, only
three such compounds are noted as undergoing advanced evalua-
tion.3 Currently four compounds are registered for use against hu-
man African trypanosomiasis (HAT); two are used for first stage
disease [suramin and pentamidine (I)] and two are used against
second stage disease [melarsoprol and eflornithine].4,5 All of these
drugs are administered by injection and have undesirable side ef-
fects. Thus, the need for new drugs to treat diseases caused by
these two parasites is evident.
member of this class of compounds to see significant human use.
Pafuramidine (IIb), a prodrug of furamidine (IIa) (Fig. 1), was sub-
jected to extensive clinical evaluation advancing to Phase III trials
against both HAT and AIDS-related Pneumocystsis jiroveci pneumo-
nia and to Phase II trials against malaria.7,8 Unfortunately, in an
additional safety study which paralleled the phase III studies some
volunteers exhibited liver and renal toxicities and trials with pafu-
NH
NH
NH2
H2N
O
O
I
Aromatic diamidines, pentamidine (I) being the preeminent
member of the class, have been studied extensively as antiproto-
NR
NH2
X
RN
H2N
M
IIa = X = O; M = CH; R = H
IIb = X = O; M = CH; R = OMe
IIc = X = O; M = N; R = H
IId = X = O; M = N; R = OMe
IIe = X = S; M = CH; R = H
* Corresponding author. Tel.: +1 04 413 5498; fax: +1 404 413 5505.
E-mail address: dboykin@gsu.edu (D.W. Boykin).
On leave of absence from Department of Chemistry, University of Podlasie,
08-110 Siedlce, Poland.
Figure 1. Important antiparasitic compounds.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.058

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D. Branowska et al. / Bioorg. Med. Chem. 18 (2010) 3551–3558
ramidine were discontinued.7 Thus it is important to continue to
search for effective and safe members of this highly active class
of compounds. We have shown that replacement of the phenyl
rings of furamidine with pyridyls leads to compounds which are
active in a model for CNS stage HAT.9 Both the azafuramidine IIc
and its prodrug IId were effective in mouse models for both stages
of the disease.9,10 A number of studies have focused upon replace-
ment of the central furan ring of IIa with other 5-ring heterocyclic
systems including pyrrole,11,12 thiophene (IIe),11,12 imidazole,11
oxazole,11,13 thiadiazole,13 oxadiazole,13 as well as several with
several 6-ring heterocyclic units.14,15 Despite these extensive stud-
ies it appears that use of a thiazole central linker has not been
investigated. This report describes the synthesis and evaluation
of diamidino 2,5-diarylthiazoles including several analogues in
which the aryl units are pyridyls. Such compounds are expected
to have different absorption and distribution properties than IIa
and IIe and thus may exhibit different toxicity profiles. Given the
locations where these diseases occur it is highly desirable to devel-
op orally effective antiprotozoal drugs.4,16 Due to the dicationic
nature of diamidines, they typically exhibit quite poor oral bio-
availability. This problem has been overcome in several cases by
use of amidoxime prodrugs.17,18 Consequently, we also report the
synthesis and evaluation of several amidoxime prodrugs in the thi-
azole series.
Subsequent Suzuki coupling of 3a–c with 4-cyanophenyl boronic
acid gave 4a–c in good yield (Scheme 1). In a similar way, the bis-ni-
triles 4d–f were prepared from 2a–c however in this case 5-(5,5-di-
methyl-[1,3,2]dioxaborinan-2-yl)-2-cyanopyridine was used to
couple with the bromothiazoles 3a–c. The target bis-amidines 5a–
f were obtained from the bis-nitriles 4a–f by the action of lithium
bis(trimethylsilyl)amide in THF.22
As a part of this study, we also prepared a bis-amidine with
both pyridyl nitrogen atoms adjacent to thiazole core (Scheme
2). The synthesis of the bis-amidine 5g required the corresponding
bis-nitrile 4g (Scheme 2). The preparation of 4g was achieved by
using the mixed tin and silyl reagent 2-trimethylsilyl-5-trimethyl-
tinthiazole23 in a palladium catalyzed coupling reaction with 2-
bromo-5-cyanopyridine to give 4g in a modest yield. The bis-ami-
dine 5g was obtained in good yield employing lithium bis(trimeth-
ylsilyl)amide as previously described.
The potential prodrugs of the bis-amidines were prepared as
outlined in Scheme 3. The bis-amidoximes 6a–e,g were readily ob-
tained by the reaction of the corresponding bis-nitriles 4 with
hydroxylamine. Methylation of the diamidoximes 6 with dimeth-
ylsulfate in the presence of aqueous lithium hydroxide gave the
N-methoxyamidines 7a–c,e,g in reasonable yield.
2.2. Biology
Table 1 summarizes the DNA binding affinities for the thiazole
diamidines and the in vitro evaluation of the dications against P.
falciparum (P. f.) and Trypanosoma brucei rhodensiense ( T. b. r.).
For comparative purposes similar data for furamidine (IIa), azafu-
ramidine (IIc) and the thiophene analogue (IIe) are also provided
in Table 1. The former two compounds are included since promis-
ing antiprotzoan data has been reported for them and the latter is
included due to the close structural analogy with the thiazoles. We
have found that the determination of melting temperature in-
2. Results and discussion
2.1. Chemistry
The key step(s) in the synthesis of the various diarylthiazoles
employs palladium catalyzed coupling reaction which we have pre-
viously extensively used.10,19–21 The 2,5-bis-(amidinophenyl)thi-
azoles5a–c were synthesizedfrom2,5-bis(4-cyanophenyl)thiazoles
4a–c which were obtained in four steps (Scheme 1). The first step
involves a Stille coupling reaction between 2-tributylstanyl thiazole
and 4-bromocyanophenyl (1a), 5-bromo-2-cyanopyridine (1b), and
6-chloronicotinonitrile (1c), respectively, to form the mono-aryl thi-
azoles 2a–c. Bromination of 2a–c with N-bromosuccinimide in DMF
solution, furnished 3a–c in good yield in the second step.
creases
DTm (Tm of complex—Tm of free DNA) provides a rapid
and accurate method for ranking the binding affinities for a variety
of aryldiamidines.6a,24,25 Typical results are illustrated in Figure 2.
The
poly (dAÁdT) range from 10 to 22.9 °C. The
thiazole diamidine 5a is 21.1 °C. This value is approximately 20%
less than that for both IIa and IIe. The differences in Tm values
D
Tm values of the thiazole diamidines for their complexes with
D
Tm value for the parent
D
for the furamidine and IIe and 5a reflects the introduction of the
nitrogen atom into the five-membered ring heterocycle and is con-
sistent with the ‘N-effect’ we have noted for a number of cases of
replacement of phenyl rings with pyridyl rings in other triaryl
diamidine systems.26 On binding to DNA, transfer of the aza-ana-
logues from aqueous solution into the less polar environment of
the minor groove is less favorable than that for furamidine and
IIe.26 The thiazole analogues which contain pyridyl rings replacing
one or both of the phenyl rings of 5a generally show a decline in
N
N
ii
i
X
S
Br
S
NC
b
NC
b
a
a
NC
b
a
1a-c
2a-c
3a-c
a
b
X
a
b
a
b
1a CH CH Br
3a CH CH
2a CH CH
1b
N
CH Br
Cl
3b
N
CH
N
2b
N
CH
N
iii
1c CH
N
3c CH
2c CH
N
N
S
D
Tm values. However, there is not an obvious systematic change
iv
in
D
Tm with structure which suggests that small effects from
NH
S
HN
H2N
CN
c
NC
b
a
c
b
d
d
a
NH2
5a-f
4a-f
N
a
b
c
d
N
5a, 4a CH CH CH CH
5b, 4b CH CH CH
5c, 4c CH CH CH
5d, 4d CH CH CH
5e, 4e CH CH
5f, 4f CH CH
S
i
NC
b
CN
c
a
4g
d
Me3Si
SnMe3
N
S
N
ii
N
N
N
N
N
N
HN
S
a
b
c
d
NH
NH2
b
c
a
d
5g, 4g CH
N
N
CH
5g
H2N
Scheme 1. Synthesis of bis-amidines 5a–f. Reagents and conditions: (i) 2-tributyltin
thiazole, Pd(PPh3)4; (ii) NBS, DMF; (iii) 4-cyanophenyl boronic acid or 5-(5,
5-dimethyl-[1,3,2]dioxaborinan-2-yl)-2-cyanopyridine Pd(PPh3)4; (iv) (1) LiN
[Si(CH3)3]2, THF; (2) NaOH/H2O; (3) EtOH/HCl.
Scheme 2. Synthesis of bis-amidine 5g. Reagents and conditions: (i) 2-bromo-5-
cyanopyridine, Pd(PPh3)4; (ii) (1) LiN[Si(CH3)3]2, THF; (2) NaOH/H2O; (3) EtOH/HCl.

D. Branowska et al. / Bioorg. Med. Chem. 18 (2010) 3551–3558
3553
NOH
NH2
HON
H2N
1
0.8
0.6
0.4
0.2
0
NOMe
NH2
MeON
H2N
i
ii
NC
CN
4a-g
6
7
polydA polydT
polydApolydT + 0.3 5g
polydApolydT + 0.3 5d
Scheme 3. Synthesis of bis-amidoxime prodrugs. Reagents and conditions: (i)
NH2OH–HCl, KO-t-Bu, rt; (ii) Me2SO4, LiOH–H2O
subtle differences in van der Waals, H-bonding interactions and
molecular geometry are likely contributors to the variation in val-
ues. Consistent with other series of aryl diamidines26 generally the
compounds with higher DNA affinities exhibit greater antiprotozo-
an activity.
In order to determine if these thiazole analogues are binding in
the DNA minor groove, we acquired CD spectra for 5a along with
IIa and IIe, for comparison, on binding to DNA (see Fig. 3). Minor
groove binding compounds give a large positive induced CD signal
on binding to AT DNA sequences and cause only small changes in
the pattern of the DNA CD spectrum.27 Such a pattern is observed
on adding IIa and IIe to polydAÁpolydT (Fig. 3) with a strong, posi-
tive induced signal between 350 to 430 nm smaller changes in the
DNA spectral region below 300 nm. As can be seen in Figure 2, 5a
exhibits a similar CD spectra with that of IIa and IIe. These results
clearly support a minor groove binding mode for the thiazole 5a.27
A detailed analysis of the effect of structure induced variation by
thiazoles analogues awaits the results of the biophysical studies
which are in progress.
The four thiazoles 5a, 5b, 5d, 5e were highly active against P. f.
exhibiting IC50 values between 1.1 and 2.5 nM which are about
nearly a factor of ten more active than IIa and IIe. These low nano-
molar values meet our group’s criteria of IC50 values of less than
100 nM to enter animal studies and the properties of these com-
pounds are in general agreement with criteria suggested by oth-
ers.16 The thiazoles 5a–f are highly active in vitro versus T. b. r.
giving IC50 values ranging between 4 and 38 nM. Four (5a, 5b,
5d, 5e) of the seven new diamidines gave IC50 values (4–6 nM)
comparable to furamidne (3.2 nM) and IIe(3 nM) and pentamidine
(2.8 nM). The selectivity indices for the four most active com-
pounds against T. b. r. are reasonable ranging from 233 to 9175,
however they are not as large as that of pentamidine (16,643).
20
30
40
50
60
70
80
90
Temperature
Figure 2. Thermal melting curves (Absorbance at 260 nm vs temperature in °C) are
Tm values were
shown for poly dAÁdT and its complexes with 5g and 5d.
D
determined from the peak in first derivative plots (dA260/dT) and are collected in
Table 1.
Based upon activity and selectivity the compounds were advanced
to the rigorous T. b. r. STIB900 mouse model of infection.
Data for the parent diamidines 5a–f as well as data for the pro-
drugs 6a–e, 6g and 7a–c, 7e, 7g obtained from the STIB900 mouse
model for the acute phase of African trypanosomiasis are pre-
sented in Table 2. The parent diamidines were administered by
intraperitoneal injection at the screening dose of 4 Â 5 mg/kg
whereas the potential prodrugs were given orally at the screening
dose of 4 Â 25 mg/kg. At this low dose level only the parent diami-
dine 5a cured one of four infected test animals. This result is com-
parable to that for IIa and inferior to that for IIc and IIe which gave
3/4 and 2/4 cures, respectively, at this dose level. Only two thiazole
prodrugs 6e and 7e, prodrugs of 5e, cured; 1/4 infected mice. The
results for 6e and 7e are inferior to that for pafuramidine and sig-
nificantly less effective than those for IId which gave 4/4 cures at
both 25 mg/kg and the quite low dose of 5 mg/kg. This relatively
Table 1
DNA affinities and in vitro antiprotozoan activity for thiazole analogs
N
HN
S
NH
b
c
a
d
H2N
NH2
Code
a
b
c
d
DNA affinity
T. b. r.
P. f.
cytotox
a
b
b
c
D
Tm (°C)
IC50 (nM)
IC50 (nM)
IC50
(l
M)
5a
5b
5c
5d
5e
5f
5g
IIa
IIc
IIe
I
CH
N
CH
CH
N
CH
CH
N
CH
CH
N
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
N
N
N
21.1
19.2
13.9
22.9
18.2
12.8
10.0
25
4
4
16
6
2.0
2.5
12.5
2.4
1.1
13
24.6
13.8
6.5
13.8
46.4
36.7
1.8
102.5
40.2
1.4
37.4
>195.0
6.5
77.9
51.7
46.6
6
CH
CH
38
176
3.2
7.0
3
N
CH
19.3
25
12.6
2.8
a
b
c
Increase in thermal melting of poly(dAÁdT)n.21
The T. b. r. (Trypanosoma brucei rhodesiense) strain was STIB900 and the P. f. (P. falciparum) strain was K1. The values are the average of duplicate determinations.10,28
Cytotoxicity was evaluated using cultured L6 rat myoblast cells.28

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D. Branowska et al. / Bioorg. Med. Chem. 18 (2010) 3551–3558
Table 2
Antitrypanosomal evaluation of the thiazole analogues in the STIB900 mouse model
Code
Dosage routeb
Dosage (4Â mg/kg)
5
25
25
5
25
25
5
25
25
5
25
5
25
25
5
Curesc
Survival (daysd)
10
5
5a
6a
7a
5b
6b
7b
5c
6c
7c
5d
6d
5e
6e
7e
5f
Ip
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
1/4
0/4
0/4
0/4
0/4
2/4
1/4
4/4
0/4
3/4
4/4
4/4
2/4
>44.5
35
41
44.5
37.5
42.75
30.25
24
Po
Po
Ip
Po
Po
Ip
Po
Po
Ip
Po
Ip
35
>36
36.75
50.5
>37.5
>47.25
29.25
21.5
9
9
.45
>46
>60
30
0
Po
Po
Ip
5g
6g
7g
I
IIa
IIb
Ip
5
DNA
IIa
Po
Po
Ip
Ip
Po
25
25
5
5
25
5
5
25
5
-5
Ie
IIc
IId
Ip
Po
>54.5
>60
>60
5a
-10
-15
IIe
Ip
5
>48.75
a
See Refs. 10 and 29 for details of STIB900 model.
Ip = intraperitoneal; po = oral.
Number of mice that survive and are parasite free for 60 days.
Average days of mice survival; untreated control animals expire between day 7
b
c
d
240
280
320
360
400
440
480
and 10 post-infection.
Wavelength
Figure 3. Circular dichroism plots (CD versus wavelength) are shown for poly
dAÁdT (DNA) and its complexes with IIa, IIe and 5a. DNA without bound compound
does not have any CD signal above 300 nm while the unbound compounds are not
chiral. On binding to DNA the compounds exhibit a large positive CD spectrum
above 300 nm that is characteristic of minor groove binding. The changes below
300 are due to induced changes in the compound CD as well as slight changes in the
DNA spectrum.
3.1.2. Circular dichroism (CD)
CD spectra were collected with a Jasco J-810 spectrometer at
different ratios of compound to DNA at 25 °C in MES 10 buffer. A
DNA solution in a 1 cm quartz cuvette was first scanned over a de-
sired wavelength range. Compounds IIa, IIc and 5a at increasing ra-
tios were then titrated into the same cuvette and the complexes
rescanned under same conditions.31
modest in vivo activity may be due to poor PK. Currently, we are
using IId as the lead compound that new compounds must match
or surpass in efficacy to be taken into preclinical evaluations for
second stage HAT, none of the thiazoles meet this standard and
consequently they will not be pursued further.
3.2. Chemistry experimental section
Melting points were recorded using a Mel-Temp capillary melt-
ing apparatus and are uncorrected. TLC analysis was carried out on
Silica Gel 60 F254 precoated aluminum sheets and detected under
UV light. 1H and 13C NMR spectra were recorded employing a Bru-
ker 400 Ultrashield™ or Varian Unity Plus™ 300 spectrometer and
chemical shifts (d) are in ppm relative to TMS as internal standard.
Mass spectra were recorded on an Applied Biosystems MALDI-TOF-
TOF MS (4800) spectrometer. Elemental analyses were obtained
from Atlantic Microlab Inc. (Norcross, GA) and are within 0.4 of
the theoretical values. The compounds reported as salts frequently
analyzed correctly for fractional moles of water and/or ethanol of
solvation. In each case proton NMR showed the presence of indi-
cated solvent (s). All chemicals and solvents were purchased from
Aldrich Chemical Co., VWR, Fischer Scientific, or Frontier and were
used as received. The synthesis of 2-trimethylsilyl-5-trimethyltin-
thiazole has been previously reported.23
3. Experimental
3.1. Biology
3.1.1. Efficacy evaluations
In vitro assays28 with T. b. r. STIB 900 and P. f. K1 strain as well
as the efficacy study in an acute mouse model for T. b. r. STIB 90029
were carried out as previously reported.
3.1.2. Tm measurements
Thermal melting experiments were conducted with a Cary 300
spectrophotometer. Cuvettes for the experiment are mounted in a
thermal block and the solution temperatures are monitored by a
thermistor in the reference cuvette. Temperatures were main-
tained under computer control and are increased at 0.5 °C/min.
The experiments were conducted in 1 cm path length quartz cuv-
ettes in CAC 10 buffer (cacodylic acid 10 mM, EDTA 1 mM, NaCl
100 mM with NaOH added to give pH 7.0). The concentrations of
DNA were determined by measuring the absorbance at 260 nM.
A ratio of 0.3 mol compound per mol of DNA was used for the com-
3.2.1. 5-(5,5-Dimethyl-[1,3,2]dioxaborinan-2-yl)-2-cyanopyridine
To a round-bottom flask were added 2.35 g (12.8 mmol) 5-bro-
mo-2-cyanopyridine in 45 mL DMSO, 3.76 g (38.4 mmol) potas-
sium acetate, 3.5 g (15.4 mmol) bis(neopentylglycolato)diboron
and 0.282 g (3 mol %) PdCl2(dppf). The mixture was heated at
80 °C for 2 days. The reaction solution was cooled to room temper-
ature and poured into ice-water. The mixture was extracted with
ethyl acetate, washed with saturated brine, dried over MgSO4,
plex and DNA with no compound was used as a control.30
ues are determined by the peak in first derivative curves (dA/dT).
DTm val-

D. Branowska et al. / Bioorg. Med. Chem. 18 (2010) 3551–3558
3555
and concentrated under reduced pressure. The residue was puri-
fied by crystallization from a mixture of EtOAc/hexane to give
0.98 g (36%),white solid, mp 126 °C. 1H NMR (300 MHz, CDCl3) d:
9.05(s, 1H), 8.20(d, 1H, J = 7.5 Hz), 7.68(d, 1H, J = 7.5 Hz), 3.83(s,
4H), 1.07(s, 6H). 13C NMR (75 MHz, CDCl3) d: 22.1, 31.2, 32.3,
72.7, 117.7, 127.8, 135.4, 142.6, 156.1. Anal. Calcd for C11H13N2BO2
C, 61.15; H, 6.07; N, 12.97. Found: C, 61.39; H, 6.07; N, 12.93.
8.86(s, 1H). 13C NMR (75 MHz, CDCl3) d: 110.4, 114.8, 116.7, 119.1,
140.7, 146.4, 152.6, 153.6, 168.3. Anal. Calcd for C9H4BrN3S: C,
40.62; H, 1.52; N, 15.79. Found: C, 40.67; H, 1.61; N, 15.63.
3.5. General procedure for Suzuki coupling of 3a–c
A mixture of 3a (2.65 g, 10 mmol), 4-cyanophenyl boronic acid
or
5-(5,5-dimethyl-[1,3,2]dioxaborinan-2-yl)-2-cyanopyridine
3.3. General procedure for Stille coupling reaction
(1.65 g, 12 mmol), Pd (PPh3)4 (0.23 g, 2 mol %) and K2CO3 (4.14 g,
30 mmol) in 50 mL anhydrous 1,4-dioxane was allowed to reflux
overnight. After cooling to room temperature, the mixture was
poured into water and extracted with ethyl acetate. The organic
layer was washed with brine, dried over MgSO4, and concentrated.
The crude product 4a was purified by crystallization from a mix-
ture of EtOAc/hexane for 4a and from DMF for 4b–g.
To a round-bottom flask were added 5.49 g (30 mmol) 4-bro-
mo-2-cyanopyridine in 70 mL 1,4-dioxane, 0.69 g (2 mol %)
Pd(PPh3)4 and 11.22 g (30 mmol) 2-tributylstannanylthiazole. The
mixture was allowed to reflux overnight. The solvent was removed
under reduced pressure. Hexane was added to the residue. The pre-
cipitate was filtered. The solid was dissolved in EtOAc and washed
with 5% NaF. The organic layer was dried over Na2SO4. The solvent
was evaporated, and the crude product was purified by column
chromatography using hexane/EtOAc (20:1).
3.5.1. 2,5-Bis(4-cyanophenyl)thiazole (4a)
Yield (77%), yellow solid, mp 276–277 °C. 1H NMR (300 MHz,
CDCl3) d: 7.76(s, 4H), 7.80(d, 2H, J = 8.4 Hz), 8.11(d, 2H,
J = 8.4 Hz), 8.22(s, 1H). 13C NMR (75 MHz, CDCl3) d: 118.6, 127.2,
127.4, 133.2, 133.3, 137.2, 139.2, 141.8. Anal. Calcd for
C17H9N3SÁ0.1H2O: C, 70.62; H, 3.21; N, 14.53. Found: C, 70.63; H,
3.03; N, 14.25.
3.3.1. 2-(Benzonitrile-4-yl)thiazole (2a)
The physical properties observed are consistent with those pre-
viously reported.32
3.3.2. 2-(2-Cyanopyridin-5-yl)thiazole (2b)
3.5.2. 5-(4-Cyanophenyl)-2-(2-cyanopyridin-5-yl)thiazole (4b)
Yield (87%), yellow solid, mp 308 °C. 1H NMR (300 MHz, DMSO)
d: 8.0(s, 4H), 8.22(d, 1H, J = 8.1 Hz), 8.60(d, 1H, J = 8.1 Hz), 8.74(s,
1H), 9.36(s, 1H). Anal. Calcd for C16H8N4SÁ0.3H2O: C, 65.42; H,
2.95; N, 19.07. Found: C, 65.57; H, 2.73; N, 19.09.
Yield (82%), white solid, mp 167 °C. 1H NMR (400 MHz, CDCl3)
d: 7.51(d, 1H, J = 3.2 Hz), 7.8(dd, 1H, J1 = 0.8, J2 = 8 Hz), 8.01(d, 1H,
J = 3.2 Hz), 8.40(dd, 1H, J1 = 2.4, J2 = 5.6 Hz), 9.29(d, 1H, J = 2.4 Hz).
13C NMR (100 MHz, CDCl3) d: 117.3, 121.8, 128.9, 132.5, 134.2,
134.4, 145.2, 149.0, 162.8. Anal. Calcd for C9H5N3S: C, 57.74; H,
2.69; N, 22.44. Found: C, 57.64; H, 2.73; N, 22.31.
3.5.3. 5-(4-Cyanophenyl)-2-(3-cyanopyridin-6-yl)thiazole (4c)
Yield (87%), yellow solid, mp 357 °C. 1H NMR (300 MHz, DMSO)
d: 9.14(s, 1H), 8.72(d, 1H, J = 6.8 Hz), 8.33(d, 1H, J = 9.6 Hz), 8.31(d,
1H, J = 9.6 Hz), 8.00(dd, 4H, J1 = 4.4 Hz, J2 = 2.0 Hz). Anal. Calcd for
C16H8N4SÁ0.3H2O: C, 65.42; H, 2.95; N, 19.07. Found: C, 65.65; H,
2.79; N, 19.13.
3.3.3. 2-(3-Cyanopyridin-6-yl)thiazole (2c)
Yield (74%), white solid, mp 167 °C. 1H NMR (300 MHz, CDCl3)
d: 7.60(d, 1H, J = 3 Hz), 8.03(d, 1H, J = 3 Hz), 8.09(dd, 1H, J1 = 2.1,
J2 = 8.4 Hz), 8.35(d, 1H, J = 8.1 Hz), 8.89(d, 1H, J = 2.1 Hz). 13C
NMR (75 MHz, CDCl3) d: 110.2, 116.8, 119.7, 123.8, 140.6, 145.2,
152.5, 154.3, 167.2. Anal. Calcd for C9H5N3S: C, 57.74; H, 2.69; N,
22.44. Found: C, 57.92; H, 2.70; N, 22.55.
3.5.4. 2-(4-Cyanophenyl)-5-(2-cyanopyridin-5-yl)thiazole (4d)
Yield (27%), yellow solid, mp 292–293 °C. 1H NMR (400 MHz,
DMSO) d: 8.02(d, 2H, J = 8.4 Hz), 8.16(d, 1H, J = 8 Hz), 8.18(d, 2H,
J = 8.4 Hz), 8.43(dd, 1H, J1 = 2.4, J2 = 7.6 Hz), 8.75(s, 1H), 9.19(d,
1H, J = 2 Hz). 13C NMR (100 MHz, DMSO) d: 113.6, 118.8, 119.2,
127.4, 130.0, 130.8, 132.1, 133.9, 135.4, 135.8, 136.7, 144.4,
149.0, 165.5. Anal. Calcd for C16H8N4SÁ0.2H2O: C, 65.83; H, 2.90;
N, 19.19. Found: C, 65.70; H, 2.85, N, 19.03.
3.4. General procedure for bromination of (2a–c)
To a round-bottom flask were added 2a (3.55 g, 19 mmol,
1 equiv) in anhydrous DMF (45 mL) and (4.1 g, 23 mmol, 1.2 equiv)
NBS. The mixture was stirred overnight at room temperature and
was poured into ice-water. The precipitate was filtered and washed
with water. The crude product was crystallized from a mixture of
EtOAc/hexane.
3.5.5. 2,5–Bis(2-cyanopyridin-5-yl)thiazole (4e)
Yield (63%), yellow solid, mp 333 °C. 1H NMR (400 MHz, DMSO)
d: 7.96(d, 1H, J = 6.8 Hz), 8.16(d, 1H, J = 8.0 Hz), 8.40(dd, 1H,
J1 = 2.4, J2 = 8.4 Hz), 8.57(dd, 1H, J1 = 2.4, J2 = 8.4 Hz), 8.72(s, 1H),
9.17(d, 1H, J = 2.4 Hz), 9.33(d, 1H, J = 2.0 Hz). ESI-MS m/z: (MÀH)
288. Anal. Calcd for C15H7N5SÁ0.6H2O: C, 60.03; H, 2.75; N, 23.33.
Found: C, 60.33; H, 2.49; N, 22.98.
3.4.1. 5-Bromo-2-(4-cyanophenyl)thiazole (3a)
Yield (89%), white solid, mp 149 °C. 1H NMR (300 MHz, CDCl3)
d: 7.77(d, 2H, J = 8.1 Hz), 7.85(s, 1H), 8.00(d, 2H, J = 8.1 Hz). 13C
NMR (75 MHz, CDCl3) d: 110.7, 113.6, 118.2, 126.6, 132.9, 136.7,
145.6, 166.8. Anal. Calcd for C10H5BrN2SÁ0.1H2O: C, 44.99; H,
1.96; N, 10.49. Found: C, 44.89; H, 1.79; N, 10.26.
3.5.6. 5–(2-Cyanopyridin-5-yl)-2-(3-cyanopyridin-6-yl)thiazole
(4f)
3.4.2. 5-Bromo-2-(2-cyanopyridin-5-yl)thiazole (3b)
Yield (61%), yellow solid, mp 368 °C. 1H NMR (400 MHz, DMSO)
d: 9.26(d, 1H, J = 2.4 Hz), 9.15(d, 1H, J = 2.0 Hz), 8.82(s, 1H),
8.52(dd, 1H, J1 = 2.0, J2 = 8.0 Hz), 8.48(dd, 1H, J1 = 2.4, J2 = 8.4 Hz),
8.33(dd, 1H, J1 = 0.8, J2 = 8.4 Hz), 8.18(dd, 1H, J1 = 0.8, J2 = 8.0 Hz).
ESI-MS m/z: (MÀH) 288. Anal. Calcd for C15H7N5SÁ0.2H2O: C,
61.51; H, 2.55; N, 23.91. Found: C, 61.42; H, 2.43; N, 23.71.
Yield (78%), white solid, mp 159–160 °C. 1H NMR (300 MHz,
CDCl3) d: 7.77(d, 1H, J = 8.1 Hz), 7.85(s, 1H), 8.00(d, 2H, J = 8.1 Hz).
13C NMR (75 MHz, CDCl3) d: 112.1, 117.1, 128.9, 131.9, 134.1,
134.6, 146.4, 148.6, 163.8. Anal. Calcd for C9H4BrN3SÁ0.1H2O: C,
40.35; H, 1.58; N, 15.68. Found: C, 40.49; H, 1.52; N, 15.53.
3.4.3. 5-Bromo-2-(3-cyanopyridin-6-yl)thiazole (3c)
3.5.7. Synthesis of 2,5-bis(3-cyanopyridin-6-yl)thiazole (4g)
To a round-bottom flask were added 2-trimethylsilyl-5-trim-
ethyltinthiazole1 (1.5 g, 4.73 mmol) in dioxane (50 mL), 2-bromo-
Yield(78%), whitesolid,mp208–209 °C. 1H NMR (300 MHz, CDCl3)
d: 7.90(s, 1H), 8.09(dd, 1H, J1 = 2.1 J2 = 8.4 Hz), 8.26(d, 1H, J = 8.1 Hz),

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D. Branowska et al. / Bioorg. Med. Chem. 18 (2010) 3551–3558
5-cyanopyridine (1.7 g, 9.45 mmol) and Pd(PPh3)4 (2 mol %). The
reaction mixture was allowed to reflux overnight. The solvent
was evaporated and the crude product was crystallized from
DMF to give 0.38 g (28%), yellow solid. mp 366 °C. 1H NMR
(400 MHz, DMSO) d: 8.33(s, 1H), 8.35(s, 1H), 8.46(dd, 1H, J1 = 1.2,
J2 = 8.4 Hz), 8.50(dd, 1H, J1 = 2.0, J2 = 8.4 Hz), 8.95(s, 1H), 9.08(d,
1H, J = 2 Hz), 9.15(d, 1H, J = 1.6 Hz). Anal. Calcd for
C15H7N5SÁ0.3H2O: C, 61.13; H, 2.60; N, 23.76. Found: C, 61.10; H,
2.54; N, 23.65.
131.5, 135.6, 135.8, 137.3, 143.2, 144.4, 147.5, 161.7, 165.4,
167.1. Anal. Calcd for C16H14N6SÁ3HClÁ0.5EtOHÁ1.1H2O: C, 43.02;
H, 4.71; N, 17.70. Found: C, 43.24; H, 4.33; N, 17.92.
3.6.5. 2,5-Bis(2-amidinopyridin-5-yl)thiazole dihydrochloride
(5e)
Yield 71% pale yellow, mp >300 °C. 1H NMR (400 MHz, DMSO) d:
8.51(d, 1H, J = 8.4 Hz), 8.55(d, 1H, J = 8.0 Hz), 8.59(dd, 1H, J1 = 2 Hz,
J2 = 7.6 Hz), 8.72(dd, 1H, J1 = 2.0 Hz, J2 = 8.0 Hz), 8.88(s, 1H), 9.23(d,
1H, J = 2 Hz), 9.39(d, 1H, J = 2 Hz), 9.50(br s, 2H), 9.60(br s, 2H),
9.73(br s, 2H), 9.78(br s, 2H). 13C NMR (100 MHz, DMSO) d: 124.3,
124. 5, 131.2, 132.5, 135.8, 136, 136.1, 136.6, 143.5, 143.8, 144.6,
145.5, 147.3, 161.8, 164.2. Anal. Calcd for C15H13N7SÁ2HClÁ0.5EtOHÁ
1.2H2O: C, 43.58; H, 4.66; N, 22.23. Found: C, 43.94; H, 4.26; N, 21.85.
3.6. General procedure for conversion of dinitriles 4a–g to
diamidines 5a–g
3.6.1. 2,5-Bis(4-amidinophenyl)thiazole dihydrochloride (5a)
The bis-nitrile 4a (0.28 g, 1 mmol) was suspended in anhydrous
THF (8 mL) and LiN[(CH3)3Si]2 solution 1.0 M in THF (5 mL) was
added to the flask and the mixture was stirred overnight at room
temperature, saturated ethanolic HCl (10 mL) was carefully added,
and the mixture was stirred overnight. The precipitate was filtered,
washed with ether, and dried. The salt of 5a was put into water,
basified with aqueous NaOH 10%, and stirred vigorously for 24 h.
The precipitate was then filtered, washed with water, ether, and
dried to afford 5a (free base) as a yellow solid, which was placed
in a 250 mL flask, EtOH was added to the flask, and the solution
was chilled in an ice-bath. After passing dry HCl gas for 15 minutes
the mixture was stirred overnight. The precipitate was filtered,
washed with ether to give a pale yellow solid in 84% yield. mp
>330 °C. 1H NMR (300 MHz, CDCl3) d: 7.97(d, 2H, J1 = 8.4 Hz),
8.02(d, 2H, J = 8.4 Hz), 8.03(d, 2H, J = 8.4 Hz), 8.23(d, 2H,
J = 8.4 Hz), 8.67(s, 1H), 9.26(br s, 2H), 9.30(br s, 2H), 9.50(br s,
2H), 9.55(br s, 2H). 13C NMR (100 MHz, CDCl3) d: 126.5, 126.8,
126.9, 127.1, 127.2, 129.1, 129.8, 135.9, 137.5, 142, 143.1, 165.5,
166. ESI-MS m/z (M++H) 322. Anal. Calcd for C17H15N5S-
2HClÁ1.0H2OÁ0.2EtOH: C, 49.57; H, 4.83; N, 16.61. Found: C,
49.58; H, 4.52; N, 16.36.
3.6.6. 2-(3-Amidinopyridin-6-yl)-5-(2-amidinopyridin-5-yl)thi-
azole dihydrochloride (5f)
Yield 70%, pale yellow solid, mp >350 °C. 1H NMR (400 MHz,
DMSO) d: 8.35(d, 1H, J = 8.4 Hz), 8.43(dd, 1H, J1 = 2 Hz, J2 = 7.6 Hz),
8.49(d, 1H, J = 8.4 Hz), 8.61(dd, 1H, J1 = 2.4 Hz, J2 = 8.4 Hz), 8.86(s,
1H), 9.00(d, 1H, J = 2 Hz), 9.27(d, 1H, J = 2 Hz), 9.47(br s, 2H),
9.51(br s, 2H), 9.73(br s, 2H), 9.76(br s, 2H). 13C NMR (100 MHz,
DMSO) d: 124.5, 126.9, 127.3, 129.6, 131.3, 135, 135.7, 139.3,
140.3, 141.9, 142.8, 147.3, 154.4, 165.8, 167.7. HRMS: m/z calcd.
C15H14N7S: 324.1031: found 324.1044. Anal. Calcd for
C15H13N7SÁ2HClÁ0.2EtOHÁ1.6H2O: C, 42.59; H, 4.50; N, 22.59. Found:
C, 42.64; H, 4.05; N, 22.20.
3.6.7. 2,5-Bis-(3-amidinopyridin-6-yl)thiazole dihydrochloride
(5g)
Yield 82%, pale yellow solid, mp >350 °C. 1H NMR (400 MHz,
DMSO) d: 8.34(d, 1H, J = 8.4 Hz), 8.37(d, 1H, J = 8.8 Hz), 8.42(dd,
1H, J1 = 2.0, J2 = 8.8 Hz), 8.44(dd, 1H, J1 = 2.4, J2 = 8.4 Hz), 8.95(s,
1H), 9.07(d, 1H, J = 1.6 Hz), 9.11(d, 1H, J = 2 Hz), 9.49(br s, 2H),
9.54(br s, 2H), 9.77(br s, 2H), 9.82(br s, 2H). 13C NMR (100 MHz,
DMSO) d: 119.3, 120, 123.7, 125.8, 138.0, 138.6, 142.5, 145.1,
145.2, 149.9, 154, 154.1, 163.8, 163.9, 169.4. Anal. Calcd for
C15H13N7SÁ3HClÁ0.8H2OÁ0.3C2H5OH: C, 40.64; H, 4.24; N, 21.26.
Found: C, 40.94; H, 4.15; N, 20.88.
3.6.2. 5-(4-Amidinophenyl)-2-(2-amidinopyridin-5-yl)thiazole
dihydrochloride (5b)
Yield 73% yellow solid, mp >320 °C. 1H NMR (300 MHz, DMSO)
d: 7.98(d, 2H, J = 8.4 Hz), 8.05(d, 2H, J = 8.4 Hz), 8.48(d, 1H,
J = 8.5 Hz), 8.72(dd, 1H, J1 = 2.2, J2 = 8.0 Hz), 8.75(s, 1H), 9.21(br s,
2H), 9.38(d, 1H, J = 2.4 Hz), 9.48(br s, 4H), 9.71(br s, 2H). 13C
NMR (100 MHz, DMSO) d: 124.4, 127.4, 128.4, 129.7, 133, 135.8,
135.9, 140.2, 143.1, 145.4, 147.2 162.3, 163.2, 165.7. ESI-MS m/z:
(M++H) 323. Anal. Calcd for C16H14N6SÁ2HClÁ0.2EtOHÁ1.3H2O: C,
46.02; H, 4.66; N, 19.63. Found: C, 45.97; H, 4.27; N, 19.31.
3.7. General procedure for the synthesis of N-hydroxyamidines
To
a solution of hydroxylamine hydrochloride (0.695 g,
10 mmol) in anhydrous DMSO (15 mL) was added t-BuOK (1.12 g,
10 mmol) in a few portions at 5 °C and stirred for 30 min. To this
solution was added the bis-nitrile (1 mmol) and the reaction mix-
ture was stirred overnight. The reaction mixture was poured into
ice-water (100 mL) and the light yellow precipitate was filtered,
washed with water and dried under reduced pressure. The free
base was converted to the hydrochloride salt by treatment with
ethanol saturated with HCl gas.
3.6.3. 5-(4-Amidinophenyl)-2-(3-amidinopyridin-6-yl)thiazole
dihydrochloride (5c)
Yield 74% pale yellow solid, mp >350 °C. 1H NMR (400 MHz,
DMSO) d: 7.95(d, 2H, J = 6.3 Hz), 8.08(d, 2H, J = 8.8 Hz), 8.35(d,
1H, J = 8.4 Hz), 8.41(dd, 1H, J1 = 2.4, J2 = 8.4 Hz), 8.73(s, 1H),
9.06(d, 1H, J = 2.0 Hz), 9.14(br s, 2H), 9.32(br s, 2H), 9.45(s, 2H),
9.65(s, 2H). 13C NMR (100 MHz, DMSO) d: 119.1, 126.0, 127.3
128.3, 129.7, 136.0, 138.6, 141.1, 143.6, 149.9, 154, 163.9, 165.2,
167.4. HRMS: m/z calcd. C16H15N6S: 323.1079: found 323.1082.
Anal. Calcd for C16H14N6SÁ3.5HClÁ0.4H2OÁ0.2EtOH: C, 42.23; H,
4.21; N, 18.02. Found: C, 42.53; H, 4.06; N, 17.63.
3.8. General procedure for the synthesis of N-methoxyamidines
A mixture of N-dihydroxyamidine (1 mmol) in DMF (10 mL)
was stirred at room temp. A solution of lithium hydroxide mono-
hydrate [LiOHÁH2O] (6 mmol) was dissolved in H2O (3 mL) and
added dropwise to the reaction mixture and stirring was continued
for 30 min. Dimethylsulfate (5 mmol) was added dropwise and the
mixture was allowed to stir overnight. The reaction mixture was
poured slowly into ice-water (100 mL). The precipitate formed
was filtered washed with water and dried under reduced pressure.
The crude product was purified by column chromatography using a
mixture EtOAc/hexane (1:1), (3:1), and (4:1) to give a pale yellow
compound. The free base was converted to the hydrochloride salt
by treatment with ethanol saturated with HCl gas.
3.6.4. 2-(4-Amidinophenyl)-5-(2-amidinopyridin-5-yl)thiazole
dihydrochloride (5d)
Yield 67% pale yellow solid, mp >350 °C. 1H NMR (400 MHz,
DMSO) d: 8.02(d, 2H, J = 8.4 Hz), 8.24(d, 2H, J = 8.4 Hz), 8.46(d,
1H, J = 8.4 Hz), 8.56(d, 1H, J = 8.4 Hz), 8.81(s, 1H), 9.23(d, 1H,
J = 2 Hz), 9.28(br s, 2H), 9.46(br s, 2H), 9.55(br s, 2H), 9.67(br s,
2H). 13C NMR (100 MHz, DMSO) d: 124.3, 127, 129.9, 130.3,

D. Branowska et al. / Bioorg. Med. Chem. 18 (2010) 3551–3558
3557
3.8.1. 2,5-Bis-(4-N-hydroxyamidinophenyl)thiazole dihydro-
chloride (6a)
1H, J = 8.0 Hz), 8.20(dd, 1H, J1 = 2.0, J2 = 8.4 Hz), 8.45(s, 1H), 8.92(d,
1H, J = 2.0 Hz), 9.77(s, 1H), 10.03(s, 1H). 13C NMR (100 MHz, DMSO)
Freebase:yield(97%), mp191–192 °C. 1HNMR(400 MHz, DMSO)
d: 5.89(s, 2H), 5.92(s, 2H), 7.74(d, 2H, J = 8.4 Hz), 7.78(d, 2H,
J = 8.4 Hz), 7.83(d, 2H, J = 8.4 Hz), 7.97(d, 2H, J = 8.4 Hz), 8.39(s,
1H), 9.76(s, 1H), 9.83(s, 1H). 13C NMR (100 MHz, DMSO) d: 126.3,
126.6, 126.6, 131.5, 133.6, 133.7, 135.5, 139.0, 140.9, 150.7, 166.0.
Salt: Yield (89%), mp>260 °C. 1H NMR (DMSO, 400 MHz) d:
7.85(m, 6H), 8.16(d, 2H, J = 8.4 Hz), 8.63(s, 1H), 9.25(bs, 2H). 13C
NMR (DMSO, 100 MHz) d: 125.8, 126.8, 126.9, 127.6, 129.5,
129.7, 137.2, 139.1, 141.8, 142.8, 159, 166.1, 167.7. Anal. Calcd
for C17H15N5O2SÁ2HClÁ1.4H2OÁ0.2C2H5OH: C, 45.36; H, 4.59; N,
15.19. Found: C, 45.49; H, 4.40; N, 15.01.
d: 119.0, 126.6, 129.7, 130.5, 131.6, 134.7, 141.0, 141.3, 147.2, 149.0,
150.7, 150.8, 167.3
Salt: yield (86%), yellow solid, mp>220 °C. 1H NMR (400 MHz,
DMSO) d: 7.89(d, 2H, J = 8.4 Hz), 8.04(d, 2H, J = 8.4 Hz), 8.32(d, 1H,
J = 8.4 Hz), 8.37(dd, 1H, J1 = 2.4, J2 = 8.4 Hz), 8.70(s, 1H), 9.02(d, 1H,
J = 1.2 Hz), 9.33(br s, 2H). 13C NMR (100 MHz, DMSO) d: 123.2, 126,
127.3, 129.6, 131.5, 135.2, 135.3, 139.7, 142.8, 146.9, 154.2, 159,
163.5, 167.3. Anal. Calcd for C16H14N6O2SÁ2HClÁ2H2O: C, 41.47; H,
4.35; N, 18.13. Found: C, 41.79; H, 3.95; N, 17.74.
3.8.6. 5-(4-N-Methoxyamidinophenyl)-2-(3-N-methoxyamidino-
pyridin-6-yl)thiazole dihydrochloride (7c)
3.8.2. 2,5-Bis-(4-N-methoxyamidinophenyl)thiazole dihydro-
chloride (7a)
Free base: yield (25%), yellow solid, mp 203 °C. 1H NMR
(400 MHz, DMSO) d: 3.77(s, 3H), 3.80(s, 3H), 6.14(s, 2H), 6.35(s,
2H), 7.79(q, 4H, J1 = 6.4, J2 = 8.4 Hz), 8,16(d, 1H, J = 8.4 Hz),
8.19(dd, 1H, J1 = 2, J2 = 6.4 Hz), 8.48(s, 1H), 8.90(d, 1H, J = 2 Hz).
13C NMR (100 MHz, DMSO) d: 61.1, 61.2, 119.0, 126.7, 127.0,
129.9, 132.0, 132. 9, 135.2, 141.1, 141.5, 147.4, 149.2.
Free base: yield, (41%), pale yellow solid, mp 186 °C. 1H NMR
(400 MHz, DMSO) d: 3.77(s, 3H), 3.78(s, 3H), 6.14(s, 2H), 6.17(s,
2H), 7.76(s, 4H), 7.82(d, 2H, J = 8.8 Hz), 7.98(d, 2H, J = 8.4 Hz),
8.41(s, 1H). 13C NMR (400 MHz, DMSO) d: 61.2, 61.3, 126.3,
126.6, 127.0, 134.0, 141.0, 150.8.
Salt: yield (87%), yellow solid, mp >200 °C. 1H NMR (400 MHz,
DMSO) d: 3.81(s, 3H), 3.99(s, 3H), 7.81(d, 2H, J = 8.4 Hz), 7.95(d,
2H, J = 8 Hz), 8.19(d, 1H, J = 8 Hz), 8.22(dd, 1H, J1 = 2, J2 = 8.4 Hz),
8.57(s, 1H), 8.92(s, 1H). 13C NMR (100 MHz, DMSO) d: 61.9, 64.3,
119, 122.9, 124.4, 128.2, 128.6, 129.9, 135.8, 141.1, 146.4, 147.7,
150.2, 155.4, 164.2, 165.8. Anal. Calcd for C18H18N6O2SÁ2HClÁ
1.8H2OÁ0.2C2H5OH: C, 44.46; H, 5.03; N, 16.90. Found: C, 44.55;
H, 4.73; N, 16.59.
Salt: Yield, (90%), pale yellow solid, mp >230 °C.1H NMR
(400 MHz, DMSO) d: 3.83(s, 3H), 3.84(s, 3H), 7.83(d, 2H,
J = 8.4 Hz), 7.87(d, 2H, J = 8.0 Hz), 7.89(d, 2H, J = 7.2 Hz), 8.07(d,
2H, J = 8.4 Hz), 8.53(s, 1H). 13C NMR (100 MHz, DMSO) d: 61.9,
62.8, 119.1, 127.8, 129, 130.5, 134.4, 135.7, 140.7, 142.1, 147.9,
150.9, 155.5, 166.2, 168. Anal. Calcd for C19H19N5O2SÁ2HClÁ1.2H2O:
C, 47.94; H, 4.95; N, 14.71. Found: C, 48.26; H, 4.85; N, 14.33.
3.8.3. 5-(4-N-Hydroxyamidinophenyl)-2(2-N-hydroxyamidino-
pyridin-5-yl)thiazole dihydrochloride (6b)
3.8.7. 2-(4-N-Hydroxyamidinophenyl)-5-(2-N-hydroxyamidino-
pyridin-5yl)thiazole dihydrochloride (6d)
Free base: yield, (90%), mp 242 °C. 1H NMR (400 MHz, DMSO) d:
5.95(s, 2H), 5.93(s, 2H), 7.78(q, 4H, J1 = 8.8, J2 = 14.8 Hz), 8.00(d, 1H,
J = 8.4 Hz), 8.33(dd, 1H, J1 = 2.4, J2 = 8.4 Hz), 8.47(s, 1H), 9.14(dd,
1H, J1 = 0.8, J2 = 2.4 Hz), 9.77(s, 1H), 10.14(s, 1H). 13C NMR
(100 MHz, DMSO) d: 120.3, 126.7, 129.4, 131.2, 133.9, 134.3,
139.8, 141.1, 146.0, 149.6, 150.6, 151.5, 163.1.
Free base: yield (82%), yellow solid, mp 249 °C. 1H NMR
(400 MHz, DMSO) d: 5.58(s, 2H), 5.93(s, 2H), 7.83(d, 2H, J = 8.4 Hz),
7.94(d, 1H, J = 8.4 Hz), 8.00(d, 1H, J = 8 Hz), 8.15(dd, 1H, J1 = 2.4
J2 = 8 Hz), 8.95(d, 1H, J = 2 Hz), 9.84(s, 1H), 10.05(s, 1H). 13C NMR
(100 MHz, DMSO) d: 120.2, 126.4, 126. 7, 127.5, 133.4, 134. 6,
135.5, 135.7, 142.0, 146.0, 149.6, 150.0, 150.6, 167.0.
Salt: yield, (93%), yellow solid, mp >220 °C. 1H NMR (400 MHz,
DMSO) d: 7.89(d, 2H, J = 8.8 Hz), 8.00(d, 2H, J = 8.4 Hz), 8.27(d, 1H,
J = 8.4 Hz), 8.57(dd, 1H, J1 = 2.4, J2 = 8.4 Hz), 8.69(s, 1H), 9.18(br s,
2H), 9.29(dd, 1H, J1 = 0.4, J2 = 2.0 Hz). 13C NMR (100 MHz, DMSO)
d: 119.3, 123.37, 124.3, 131.5, 135.9, 137.7, 138.7, 143.8, 144.9,
Salt: yield (81%), yellow solid, mp>350 °C. 1H NMR (400 MHz,
DMSO) d: 7.92(d, 2H, J = 8.4 Hz), 8.18(d, 1H, J = 8.4 Hz), 8.22(d,
1H, J = 8.4 Hz), 8.42(dd, 1H, J1 = 2.4, J2 = 8.4 Hz), 8.77(s, 1H),
8.73(s, 1H), 9.14(d, 1H, J = 1.6 Hz). 13C NMR (100 MHz, DMSO) d:
123.5, 127, 129.7, 130.4, 135.5, 135.8, 136.8, 143.8, 144, 147.2,
154.9, 158.9, 166.7, 167. Anal. Calcd for C16H14N6O2SÁ3HClÁ1.0H2O:
C, 39.88; H, 3.97; N, 17.44. Found: C, 40.06; H, 4.19; N, 17.04.
147.6,
149.9,
153.8,
163.9,
168.5.
Anal.
Calcd
for
C16H14N6O2SÁ2HClÁ1.95H2O: C, 41.55; H, 4.33; N, 18.17. Found: C,
41.88; H, 4.01; N, 17.83.
3.8.8. 2,5-Bis-(2-N-hydroxyamidinopyridin-5-yl)thiazole dihy-
drochloride (6e)
3.8.4. 5-(4-N-Methoxyamidinophenyl)-2-(2-N-methoxyamidino-
pyridin-5-yl)thiazole dihydrochlodide (7b)
Free base: yield (93%), yellow solid, mp >200 °C. 1H NMR
(400 MHz, DMSO) d: 5.90(s, 2H), 5.94(s, 2H), 7.94(d, 2H,
J = 8.4 Hz), 8.00(d, 2H, J = 8.4 Hz), 8.16(d, 1H, J = 8 Hz), 8.17(d, 1H,
J = 8 Hz), 8.58(s, 1H), 8.97(s, 1H), 9.15(s, 1H), 10.07(s, 1H),
10.16(s, 1H). 13C NMR (100 MHz, DMSO) d: 120.2, 120.4, 127.3,
129.2, 134.7, 136.3, 142.2, 146.1, 146.1, 149.6, 150.2, 151.7, 164.1.
Salt: yield (50%), yellow solid, mp >280 °C. 1H NMR (400 MHz,
DMSO) d: 8.19–8.21(m, 2H), 8.44(dd, 1H, J1 = 2.0, J2 = 8.0 Hz),
8.56(dd, 1H, J1 = 2.0, J2 = 8.4 Hz), 8.77(s, 1H), 9.15(s, 1H), 9.29(s,
1H). 13C NMR (100 MHz, DMSO) d: 119.5, 121.4, 122, 123.5,
130.3, 131.5, 136, 136.6, 137, 144, 147, 147.3, 155, 165.4, 166.8.
Anal. Calcd for C15H13N7O2SÁ4HClÁ0.7H2OÁ0.2C2H5OH: C, 35.36; H,
3.77; N, 18.76. Found: C, 35.64; H, 3.98; N, 18.47.
Free base: yield (62.5%), yellow solid, mp 211 °C. 1H NMR
(400 MHz, DMSO) d: 3.77(s, 3H), 3.84(s, 3H), 6.15(s, 2H), 6.19(s,
2H), 7.78(s, 4H), 7.99(d, 1H, J = 8 Hz), 8.35(d, 1H, J = 8 Hz), 8.48(s,
1H), 9.15(s, 1H). 13C NMR (100 MHz, DMSO) d: 61.2, 61.7, 120.7,
126.7, 127.0, 129.7, 131.6, 133.1, 134.5, 139.8, 141.3, 146.1,
149.45, 150.8, 163.0.
Salt: yield (93%), yellow solid, mp >240 °C. 1H NMR (400 MHz,
DMSO) d: 3.85(s, 3H), 3.87(s, 3H), 7.88(d, 2H, J = 8 Hz), 7.93(d,
2H, J = 7.2 Hz), 8.05(d, 1H, J = 8.4 Hz), 8.39(d, 1H, J = 8.4 Hz),
8.61(s, 1H), 9.18(s, 1H). 13C NMR (100 MHz, DMSO) d: 62.2, 63.4,
120, 125.2, 127.1, 129, 129.9, 135, 139.2, 142.1, 143.6, 148.4,
151.8, 167.9, 169.9. Anal. Calcd for C18H18N6O2SÁ2HClÁ0.4C2H5OH:
C, 47.66; H, 4.76; N, 17.74. Found: C, 47.94; H, 4.66; N, 18.02.
3.8.9. 2,5-Bis(2-N-methoxyamidinopyridin-5-yl)thiazole dihy-
drochloride (7e)
3.8.5. 5-(4-N-Hydroxyamidinophenyl)-2-(3-N-hydroxyamidino-
pyridin-6-yl)thiazole dihydrochloride (6c)
Free base: yield (39%), yellow solid, mp 191 °C. 1H NMR
(400 MHz, DMSO) d: 3.83(s, 3H), 3.84(s, 3H), 6.16(br s, 2H),
6.20(br s, 2H), 7.94(d, 1H, J = 8.4 Hz), 8.00(d, 1H, J = 8.4 Hz),
Free base: yield (88%), yellow solid, mp 225 °C. 1H NMR
(400 MHz, DMSO) d: 5.89(s, 2H), 6.08(s, 2H), 7.78(s, 4H), 8.15(d,

3558
D. Branowska et al. / Bioorg. Med. Chem. 18 (2010) 3551–3558
4. Barrett, M. P.; Boykin, D. W.; Brun, R.; Tidwell, R. R. Br. J. Pharmacol. 2007, 152,
1155.
8.18(dd, 1H, J1 = 2.4, J2 = 8.4 Hz), 8.36(dd, 1H, J1 = 2, J2 = 8.0 Hz),
8.60(s, 1H,), 8.98(d, 1H, J = 2.0 Hz), 9.16(d, 1H, J = 2 Hz). 13C NMR
(100 MHz, DMSO) d: 61.6, 61.7, 120.6, 120.8, 127.7, 129.5, 134.6,
134.8, 136.3, 142.4, 146.2, 149.2, 149.4, 150.8, 164.0.
5. Barrett, M. P.; Gilbert, I. H. Adv. Parasitol. 2006, 63, 125.
6. (a) Tidwell, R. R.; Boykin, D. W. Dicationic DNA Minor Groove Binders as
Antimicrobial Agents. In Small Molecule DNA and RNA Binders: From Synthesis to
Nucleic Acid Complexes; Demeunynck, M., Bailly, C., Wilson, W. D., Eds.; Wiley:
New York, 2003; Vol. 2, pp 414–460; (b) Wilson, W. D.; Nguyen, B.; Tanious, F.
A.; Mathis, A.; Hall, J. E.; Stephens, C. E.; Boykin, D. W. Curr. Med. Chem.
Anticancer Agents 2005, 5, 389; (c) Soeiro, M. N. C.; de Souza, E. M.; Stephens, C.
E.; Boykin, D. W. Expert Opin. Invest. Drugs 2005, 14, 957; (d) Dardonville, C.
Expert Ther. Patents 2005, 15, 1241; (e) Werbovetz, K. A. Curr. Opin. Invest. Drugs
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7. (a) Fairlamb, A. H. Trends Parasitol. 2003, 19, 488; (b) Bouteille, B.; Oukem, O.;
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DB289, a New Oral Drug for Treatment of Pneumocystis carinii pneumonia (PCP)
in AIDS Patients. Presented at the 43rd Annual Interscience Conference on
Antimicrobial Agents and Chemotherapy Meeting, September 14–17, 2003,
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Brun, R. Antimicrob. Agents Chemother. 2009, 53, 4185.
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Infect. Dis. 2005, 192, 319.
9. Ansede, J. H.; Voyksner, R. D.; Ismail, M. A.; Boykin, D. W.; Tidwell, R. R.; Hall, J.
E. Xenobiotica 2005, 35, 211.
10. Ismail, M. A.; Brun, R.; Easterbrook, J. D.; Tanious, F. A.; Wilson, W. D.; Boykin,
D. W. J. Med. Chem. 2003, 46, 4761.
11. Dann, O.; Fick, H.; Pietzner, B.; Walkenhorst, E.; Fernbach, R.; Zeh, D. Liebigs
Ann. Chem. 1975, 160.
12. Das, B. P.; Boykin, D. W. J. Med.Chem. 1977, 20, 1219.
13. Das, B. P.; Wallace, R. A.; Boykin, D. W. J. Med.Chem. 1980, 23, 578.
14. Kumar, A.; Rhodes, R. A.; Spychala, J.; Wilson, W. D.; Boykin, D. W.; Tidwell, R.
R.; Dykstra, C. C.; Hall, J. E.; Jones, S. K.; Schinazi, R. K. Eur. J. Med. Chem. 1995,
30, 99.
15. (a) Kumar, A.; Boykin, D. W.; Wilson, W. D.; Jones, S. K.; Bender, B. K.; Dykstra,
C. C.; Hall, J. E.; Tidwell, R. R. Eur. J. Med. Chem. 1996, 31, 767; (b) Boykin, D. W.;
Kumar, A.; Bajic, M.; Wilson, W. D.; Bender, B. K.; Hall, J. E.; Tidwell, R. R. Eur. J.
Med. Chem. 1997, 32, 965.
16. Nwaka, S.; Ramirez, B.; Brun, R.; Maes, L.; Douglas, F.; Ridley, R. PLOS Neg. Trop.
Dis. 2009, 3, 1.
17. Boykin, D. W.; Kumar, A.; Bender, B. K.; Hall, J. E.; Tidwell, R. R. Bioorg. Med.
Chem. Lett. 1996, 6, 3017.
18. Fylaktaidou, K. C.; Hadjipavlou-Litina, D. J.; Litinas, K. E.; Varella, E. A.;
Nicolaides, D. N. Curr. Pharm. Des. 2008, 14, 1001.
19. Kumar, A.; Stephens, C. E.; Boykin, D. W. Heterocycl. Commun. 1999, 5, 301.
20. Batistia-Parra, A.; Venkitachalam, S.; Wilson, W. D.; Boykin, D. W. Heterocycles
2003, 60, 1367.
21. Ismail, M. A.; Arafa, R. K.; Brun, R.; Wenzler, T.; Miao, Y.; Wilson, W. D.; Boykin,
D. W. J. Med. Chem. 2006, 49, 5324.
22. Chackal-Catoen, S.; Miao, Y.; Wilson, W. D.; Wenzler, T.; Brun, R.; Boykin, D. W.
Bioorg. Med. Chem. 2006, 14, 7434.
Salt: yield, (84%), yellow solid, mp >280 °C. 1H NMR (400 MHz,
DMSO) d: 3.83(s, 3H), 3.84(s, 3H), 7.99(d, 1H, J = 8.0 Hz), 8.04(d, 1H,
J = 8.4 Hz), 8.22(dd, 1H, J1 = 2.4, J2 = 8.4 Hz), 8.40(dd, 1H, J1 = 2,
J2 = 8.4 Hz), 8.64(s, 1H), 9.01(d, 1H, J = 2.4 Hz), 9.18(d, 1H,
J = 2.4 Hz). 13C NMR (100 MHz, DMSO) d: 61.9, 62.7, 121, 127,
128.5, 129.3, 130, 133.8, 134.6, 139.4, 141.9, 146.2, 150, 150.4,
155, 163.7, 164.1. Anal. Calcd for C17H17N7O2SÁ2HClÁ1.0H2O: C,
43.04; H, 4.46; N, 20.67. Found: C, 43.27; H, 4.62; 20.67.
3.8.10. 2,5-Bis(3-N-hydroxyamidinopyridin-6-yl)thiazole dihy-
drochloride (6g)
Free base:yield, (97%), yellow solid, mp >250 °C. 1H NMR
(400 MHz, DMSO) d: 6.03(s, 2H), 6.07(s, 2H), 8.10–8.21(m, 4H),
8.90(d, 1H, J = 1.6 Hz), 8.94(d, 1H, J = 2 Hz), 9.94(s, 1H), 10.03(s,
1H). 13C NMR (100 MHz, DMSO) d: 111.3, 119.2, 119.8, 128.7,
130.6, 134.3, 134.7, 142.3, 142.8, 147.2, 149.0, 150.4, 150.9, 169.3.
Salt: yield, (78%), yellow solid, mp >300 °C. 1H NMR (400 MHz,
DMSO) d: 8.26(dd, 1H, J1 = 2.0, J2 = 8.4 Hz), 8.33(s, 2H), 8.91(s,
1H), 8.95(d, 1H, J = 2.0 Hz), 9.00(s, 1H). 13C NMR (100 MHz, DMSO)
d: 118.4, 120.2, 120.5, 122.8, 126.3, 128.6, 129.4, 131.6, 144.3,
144.5, 156.7, 160.3, 160.6, 165.8, 168. Anal. Calcd for
C15H13N7O2SÁ2HClÁ0.3H2OÁ0.2C2H5OH: C, 41.76; H, 3.82; N, 22.13.
Found: C, 41.86; H, 4.03; 22.01.
3.8.11. 2,5-Bis(3-N-methoxyamidinopyridin-6-yl)thiazole
dihydrochloride (7g)
Free base:yield (67%), yellow solid, mp>280 °C. 1H NMR
(400 MHz, DMSO) d: 3.79(s, 3H), 3.80(s, 3H), 6.30(s, 2H), 6.35(s,
2H), 8.12(d, 2H, J = 1.6 Hz), 8.19–8.22(m, 2H), 8.71(s, 1H), 8.87(s,
1H), 8.92(s, 1H). 13C NMR (100 MHz, DMSO) d: 61.3, 61.4, 119.2,
119.8, 127.9, 129.9, 134.7, 135.2, 142.3, 147.5, 149.2, 149.3,
150.7, 150.9, 151.3, 163.7, 169.4.
Salt: yield, (83%), yellow solid, mp >300 °C. 1H NMR (400 MHz,
DMSO) d: 3.80(s, 3H), 3.98(s, 3H), 8.14–8.21(m, 2H), 8.74(s, 1H),
8.86(s, 1H), 8.93(s, 1H). 13C NMR (100 MHz, DMSO) d: 62.9, 63.3,
119.3, 120, 123, 125.8, 135.6, 136.9, 140.5, 142.1, 143.7, 148.0,
148.3, 151.9, 152.8, 167.8, 169.6. Anal. Calcd for C17H17N7O2SÁ2HClÁ
0.8H2OÁ0.2C2H5OH: C, 43.54; H, 4.57; N, 20.43. Found: C, 43.26; H,
4.45; 20.25.
23. Dondoni, A.; Mastellari, A. R.; Medici, A.; Negrini, E.; Pedrini, P. Synthesis 1986,
757.
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D. J. Am. Chem. Soc. 1995, 117, 4716.
26. Wilson, W. D.; Tanious, F. A.; Mathis, A.; Tevis, D.; Hall, J. E.; Boykin, D. W.
Acknowledgments
Biochimie 2008, 90, 999.
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University Press: New York, 1997; (b) Lyng, R.; Rodger, A.; Norden, B.
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We thank Guy Riccio of STI for the technical assistance with the
T. b. r. animal model.
This work was supported by the Bill and Melinda Gates Founda-
tion and by NIH Grant AI46365.
29. Thuita, J. K.; Karanja, S. M.; Wenzler, T.; Mdachi, R. E.; Ngotho, J. M.; Kagira, J.
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