Synthesis and antiparasitic evaluation of bis-2,5-[4-guanidinophenyl]thiophenes

Article abstract of DOI:10.1016/j.ejmech.2006.11.006

A series of bis-2,5-[4-guanidinophenyl]thiophenes were prepared in a five step process starting from 2,5-bis[trimethylstannyl]thiophene. The compounds were evaluated in vitro against Trypanosoma brucei rhodesiense (T. b. r.), Plasmodium falciparum (P. f.)

Full text of DOI:10.1016/j.ejmech.2006.11.006

European Journal of Medicinal Chemistry 42 (2007) 552e557
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antiparasitic evaluation of
bis-2,5-[4-guanidinophenyl]thiophenes
Jose L. Gonzalez a, Chad E. Stephens a, Tanja Wenzler b, Reto Brun b, Farial A. Tanious a,
W. David Wilson a, Todd Barszcz c, Karl A. Werbovetz c, David W. Boykin a,
*
a Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, University Plaza, Atlanta, GA 30303-3083, USA
b Parasite Chemotherapy, Swiss Tropical Institute, Basel CH4002, Switzerland
c Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
Received 14 August 2006; received in revised form 12 November 2006; accepted 16 November 2006
Available online 22 November 2006
Abstract
A series of bis-2,5-[4-guanidinophenyl]thiophenes were prepared in a five step process starting from 2,5-bis[trimethylstannyl]thiophene. The
compounds were evaluated in vitro against Trypanosoma brucei rhodesiense (T. b. r.), Plasmodium falciparum (P. f.), Leshmania donovani (L.
d.) and Trypanasoma cruzi (T. c.), and in vivo against T. b. r. Certain compounds show promising in vitro activity against T. b. r. and P. f. and
have superior in vivo activity against T. b. r. to that of pentamidine and furamidine.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Diguanidino thiophenes; Antiparasitic agents
1. Introduction
gambiense, is fatal if not treated and impacts the socioeco-
nomic well being of millions of people in central Africa [1].
Chagas disease, caused by Trypanosoma cruzi (T. c.), is found
in much of South America, all of Central America and in Mex-
ico. The Leishmania parasite is broadly distributed in humans
and animals. It is found in the Far East, southern Europe and
now even in the United States [1]. Many of the drugs currently
in use for the treatment of these parasitic infections were de-
veloped over 50 years ago and have major limitations includ-
ing significant toxicity, variable efficacy, lack of oral
bioavailability, extensive courses of parenteral administration,
and problems of cost and supply. Furthermore, there is consid-
erable evidence that extended use of these drugs is leading to
the development of resistance [5]. The need for the discovery
of new drugs to treat these diseases is clear.
Over three billion people are estimated to be at risk for the
parasitic diseases such as malaria, African (sleeping sickness)
and American (Chagas disease) trypanosomiasis and leish-
maniasis [1]. Furthermore, the combined number of cases of
these diseases is estimated to be approximately 300 million
[1,2]. A relatively new development further increases the pub-
lic health burden, through transmission of Chagas disease by
travelers with blood transfusions in countries not normally af-
fected by the diseases [3]. Malaria is widely distributed
throughout the tropics and it is estimated that between 350
and 500 million clinical episodes occur annually in areas
where some 3.2 billion people are at risk [4]. Plasmodium fal-
ciparum (P. f.) and Plasmodium vivax cause the majority of
human cases. Sleeping sickness caused by the parasites Trypa-
nosoma brucei rhodesiense (T. b. r.) and Trypanosoma brucei
Dicationic molecules were first reported to have significant
antiprotozoal activity in the 1930’s [5]. Despite numerous
studies of various classes of dications, the diamidine pentam-
idine (I) is the only compound from this class which has seen
significant human use. Pentamidine is used to treat early stage
* Corresponding author. Tel.: þ1 404 651 3798; fax: þ1 404 651 1416.
E-mail address: dboykin@gsu.edu (D.W. Boykin).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.11.006

J.L. Gonzalez et al. / European Journal of Medicinal Chemistry 42 (2007) 552e557
553
T. b. g. human African trypanosomiasis (HAT), antimony-
2. Chemistry
resistant leishmaniasis and AIDS-related P. jiroveci pneumo-
nia [9]. The diamidine furamidine and its analogues have
shown significant activity against various parasites, and pafur-
amidine, an orally effective prodrug of furamidine, was in
Phase II clinical trials against malaria, and is in Phase III trials
against HAT and pneumocystis pneumonia [5e9]. The mode
of action of this class of compounds is still being elucidated.
However, transport mediated uptake appears to be a key
step. It has been suggested that these dicationic molecules
act by binding in the minor groove of DNA at AT rich sites
[5]. Binding to DNA does not directly kill the parasite, but
there is considerable evidence that minor groove binding leads
to inhibition of DNA dependant enzymes or possibly direct in-
hibition of transcription [5,10e13].
The structurally related dicationic diguanidines have re-
ceived relatively little attention compared to the extensive
studies of diamidines. However, a few reports have recently
appeared which show that these types of molecules exhibit sig-
nificant antiprotozoal activity. For example, 4,40-bis[guanidi-
no]diphenylamine has shown promising in vitro and in vivo
activity against T. b. r. [14,15]. In a study of fused ring digua-
nidino compounds, several bisguanidino indenes have been
found to exhibit potent in vitro activity against both P. f.
and T. b. r. and significant in vivo activity was found in
a mouse model for African trypanosomiasis [16]. Bis-2,5-[4-
guanidinophenyl]furans [17] and bis-2,5-[3- or 4-guanidino-
phenyl]-N-methylpyrroles [18] have shown promising in vitro
antifungal activity. Also, the bis-2,5-[4-guanidinophenyl]fur-
ans [19] were active in vitro against L. d. and T. c. Collec-
tively, these results for various aryl diguanidino compounds
led us to synthesize and evaluate the antiprotozoal activity
of bis-2,5-[4-guanidinophenyl]thiophenes.
The synthesis of the various bis-2,5-[4-guanidinophenyl]th-
iophenes is outlined in Scheme 1 and is patterned after the ap-
proach we developed to make the corresponding furan analogs
[17]. Stille coupling of bis-2,5-[trimethylstannyl]thiophene
and 4-bromonitrobenzene gave bis-2,5-[4-nitrophenyl]thio-
phene (1) in high yield. Stannous chloride reduction of 1
gave bis-2,5-[4-aminophenyl]thiophene (2) in very good yield.
The diamine 2 was used to prepare bis-2,5-[4-guanidinophe-
nyl]thiophene (3a) and the various bis-2,5-[4-(N-alkyl and
N-aryl)guanidinophenyl]thiophenes (3be3g). The parent mole-
cule 3a was made in a two-step process in which 2 was allowed
to react with Boc-protected S-methylthiourea in the presence of
mercuric chloride. The Boc-protected analogue of 3a was ob-
tained in 70% yield and it was subsequently deprotected using
anhydrous HCl in ethanol/dichloromethane solution. The N-
alkyl and N-aryl diguanidines 3be3g were obtained first by
the reaction of 2 with ethyl isothiocyanatoformate to yield the
carbethoxy thiourea 4 [20]. The reaction of 4 with the appropri-
ate amine in the presence of carbodiimide EDCI yielded the cor-
responding carbethoxy diguanidines 5be5g [21]. The action of
KOH/EtOH provide the diguandine free bases 3be3g which
were readily converted to dihydrochloride salts.
3. Biology
The DNA affinity as measured by DTm for their complexes
with polydA.polydT and the in vitro efficacy of 3ae3g against
four different parasites T. b. r., P. f., Leishmania donovani (L.
d.) and T. c. are presented in Table 1. The DNA affinity of the
parent thiophene 3a is slightly less than that of its furan coun-
terpart [17]. The smaller N-alkyl substituted compounds 3b
i)
+
Br
NO2
(H3C)3Sn
Sn(CH3)3
S
S
O
2N
NO2
1
ii)
O
S
S
O
v)
S
S
H2N
NH2
N
N
N
H
N
EtO
OEt
H
H
H
4
2
iii) iv)
vi)
vii)
NH
NH
NH
O
NR
NR O
S
S
RHN
N
N
N
N
N
N
EtO
OEt
H
H
R
H
H
H
H
5b-g
3a-g
Legend for compounds 3 and 5
a R = H e R = c-hexyl
b R = methyl f R = phenyl
c R = i-Pr
d R = c-pentyl
g R = p-tolyl
Scheme 1. (i) Pd(PPh3)4, 1,4-dioxane; (ii) SnCl2 dihydrate, EtOH; (iii) S-methyl-di-Bocthiourea, HgCl2, TEA, DMF; (iv) HCl, CH2Cl2, EtOH; (v) ethyl isothio-
cyanatoformate, CH2Cl2; (vi) NH2R, DIPEA, EDCI, CH2Cl2; (vii) KOH, EtOH.

554
J.L. Gonzalez et al. / European Journal of Medicinal Chemistry 42 (2007) 552e557
Table 1
DNA affinity and in vitro antiparasitic activities of diguanidino dications
NH
NH
S
N
N
H
RHN
NHR
H
Code
R
DTm
IC50 (nM)
IC50 (mM)
b,c
poly dA.dT a
T. b. r.b
P. f.
L. d.b,d
T. c.b,e
L-6b,f
Chloroquine
Pentamidine
Furamidine
NA
NA
e
e
201
e
e
188.5
46.6
6.4
12. 6
25.0
19.9
16.2
15.4
19.4
17.8
19.5
16.8
2.8
4.3
28
58.4
1.9
2.8
6.7
6.2
11
5.35
23.3
154.0
30.5
28.2
8.4
NA
15.5
3.6
23
3a
3b
3c
3d
3e
3f
3g
a
H
Me
100.3
54.3
69.4
15.9
4.3
6.2
116
306
354
1900
573
i-Pr
107
c-pent
c-hex
Phenyl
4-Mephenyl
134
160
154
1.5
2.5
35
1.7
9.9
2.5
1.1
42.7
16
3.7
DNA: polydA.polydT; buffer MES 10; compound/DNA ratio ¼ 0.3 see Ref. [24].
b
Activities represent the mean of at least two independent experiments; IC50 values used to calculate the average for a given compound are within a factor of
two.
c
The T. b. r. strain used was STIB900 and the P. f. strain was K1; see Refs. [15,22e24].
See Refs. [26,27] for details of this amastigote screen.
d
e
f
Benznidazole in this screen IC50 ¼ 1.5 mM; see Ref. [25] for details of this screen.
Cytotoxicity was evaluated using cultured L-6 rat myoblast cells using the alamar blue assay, see Ref. [23].
and 3c exhibited DTm values which are reduced by about 20e
30% compared to the parent 3a, whereas the larger N-alkyl
and N-aryl compounds (3de3g) only show a modest drop in
affinity. Generally, the DTm values of guanidines 3ae3g are
20e40% smaller than that of furamidine, the prototype diami-
dine. Nevertheless, these diguanidino analogues show strong
DNA affinity, superior to that of pentamidine [22].
4. Conclusions
In summary, we report an efficient synthetic approach to
bis-2,5-[4-guanidinophenyl]thiophenes and their promising
antiparasitic activity, especially against T. b. r. and P. f. The
in vivo studies in the STIB900 mouse model show that the di-
guandino compounds are well tolerated and efficacious against
African trypanosomes. These and other diguanidino com-
pounds clearly merit further study as antiparasitic agents.
The IC50 values of this series of diguanidines against T. b.
r. range from 6.2 to 1900 nM. The most active compounds are
the parent 3a and its N-methyl analog 3b; progressively larger
substituents than methyl lead to significant reduction in activ-
ity. The activity of these compounds against P. f. shows a sim-
ilar structureeactivity relationship but with a less dramatic
drop in activity with large substituents. The IC50 values range
from 3.6 to 160 nM against P. f. with the most active antima-
larials being 3a and 3b. The IC50 values of these compounds
against L. d. and T. c. are modest and range from 2 to
35 mM versus L. d. and from 1 to 154 mM against T. c. With
the exception of 3ee3g, the compounds do not show cytotox-
icity and thus are reasonably selective for T. b. r. and P. f.
Given the good in vitro activity against T. b. r. of several of
these diguanidines and the availability of the well-established
T. b. r STIB900 mouse model, we decided to evaluate the in
vivo activity of the 4 most active compounds (Table 2). The mouse
model used is very stringent and the extended survival indicates
improved in vivo activity for all the four tested diguanidines
over the current drug pentamidine. The three N-alkyl analogues
tested (3be3d) gave 1/4 or 2/4 cures in this model, activity which
is superior to that of both pentamidine and furamidine. Interest-
ingly, both 3c and 3d, whose intrinsic antitrypanosomal activity
in vitro is significantly less than that of 3a and 3b is more active
in vivo, suggesting superior pharmacodynamic properties.
5. Experimental section
5.1. Biology
In vitro assays with T. b. r. STIB900, P. f. K1 and T. cruzi
Tulahuen Lac Z C4 strain as well as the efficacy study in an
Table 2
In vivo antitrypanosomal activity of dicationic compounds in the STIB900
mouse modela
Code
Dosageb mg/kg
Curesc
Survivald (days)
Pentamidine
Furamidine
4 ꢀ 20
4 ꢀ 20
4 ꢀ 20
4 ꢀ 20
4 ꢀ 5
0/4
0/4
0/4
2/4
1/4
2/4
40.8
>52.5
43
3a
3b
3c
3d
a
>40.25
>34
>39.5
4 ꢀ 20
See Refs. [15,22e24] for details of the T. b. r. mouse model STIB900 strain.
b
c
d
Dosage ¼ intraperitoneal for four days.
Number of mice that survive and are parasite free for 60 days post infection.
Average days of survival; untreated control animals expire between day 7
and 8 post infection. The > symbol reflects the fact that animals are alive at
the end of the experiment (60 days) and so an absolute value for the survival
time cannot be given.

J.L. Gonzalez et al. / European Journal of Medicinal Chemistry 42 (2007) 552e557
555
acute mouse model for T. b. r. STIB900 were carried out as
previously reported [15,22e25]. Assays against L. donovani
axenic amastigotes were performed as outlined earlier [26,27].
brine and dried over anhydrous MgSO4. The solvent was re-
moved under reduced pressure and the solid obtained was
washed with hexanes and crystallized from acetone to yield
0.63 g (91%, mp 169.2e170.8 ꢁC, lit. [26] mp 168e169 ꢁC)
of 2,5-bis(4-aminophenyl)thiophene. 1H NMR (300 MHz,
DMSO-d6) 7.29 (d, 4H, J ¼ 8.4 Hz), 7.10 (s, 2H), 6.58 (d,
4H, J ¼ 8.4 Hz), 5.39 (br s, 4H); 13C NMR (300 MHz,
DMSO-d6) 148.4, 141.7. 126.3, 122.3, 121.8, 114.6.
5.2. Tm measurements
Thermal melting experiments were conducted with a Cary
300 spectrophotometer. For these measurements cuvettes are
mounted in a thermal block and the solution temperatures
are monitored by a thermistor in a reference cuvette. Temper-
atures are maintained under computer control and are in-
creased at 0.5 ꢁC/min. The experiments were MES 10 buffer
(MES 10 mM, EDTA 1 mM, NaCl 100 mM) are conducted
in 1 cm path length quartz cuvettes. The concentrations of
DNA were determined by measuring the absorbance at
260 nm. A ratio of 0.3 compound per base was used for the
complex and DNA with no compound was used as a control.
5.3.3. 2,5-Bis(4-guanidinophenyl)thiophene (3a)
2,5-Bis(4-guanidinophenyl)thiophene was prepared accord-
ing to the general two-step literature procedure used for the
synthesis of the analogous furan [17]. Bis-Boc derivative: yel-
1
low solid. Yield: 70%; H NMR (CDCl3) 10.41 (br s, 2H),
7.67 (d, 4H), 7.59 (d, 4H), 7.28 (s, 2H), 1.54e1.57 (2s,
36H). Dihydrochloride: yellow/pale green hygroscopic solid.
1H NMR (DMSO-d6) 10.14 (br s, 2H), 7.74 (d, 4H), 7.59
(br s, 8H), 7.56 (s, 2H), 7.28 (d, 4H). MS (FAB, thioglycerol):
m/z 351 (MHþ). Anal. Calcd. for C18H18N6S$2.0HCl$
0.5H2O$0.2EtOH: C, 50.04; H, 4.84; N, 19.03. Found: C,
50.28; H, 4.94; N, 18.9.
5.3. Chemistry
Melting points were determined in open capillary tubes
with a Mel-Temp 3.0 melting point apparatus and are uncor-
1
rected. H and 13C NMR spectra were recorded on Varian
5.3.4. 2,5-Bis[4-(N-ethoxycarbonylthiourea)-
phenyl]thiophene (4)
Unity 300 and Varian VRX 400 instruments and chemical
shifts are reported in parts per million relative to TMS.
Mass spectra (MS) were performed by the Georgia Tech
Mass Spectra Laboratory at the Georgia Institute of Technol-
ogy in Atlanta, GA. Elemental analyses were performed by
Atlantic Microlab in Norcross, GA. The compounds reported
as salts frequently analyzed correctly for fractional moles of
water and/or ethanol of solvation; in all such cases proton
NMR confirmed the presence of solvent (s). All chemicals
and solvents were purchased from Aldrich Chemical Co. or
Fisher Scientific.
A
solution of ethyl isothiocyanatoformate (0.17 g,
1.30 mmol) in 20 mL of dichloromethane was cooled to
0 ꢁC (ice bath) before adding 2 (0.17 g, 0.65 mmol). The ice
bath was removed and the solution was stirred at rt overnight
under nitrogen. The mixture was poured into 50 mL of pentane
and the precipitate was collected by filtration and dried under
reduced pressure to yield 0.26 g (75%, mp above 350 ꢁC) of
1
2,5-bis[4-(N-ethoxycarbonylthiourea)phenyl]thiophene. H NMR
(300 MHz, DMSO-d6) 11.61 (s, 2H), 11.30 (s, 2H), 7.69
(m, 8H), 7.54 (s, 2H), 4.21 (q, 4H, J ¼ 6.9 Hz), 1.25 (t, 6H,
J ¼ 6.9 Hz). Anal. Calcd. for C24H24N4O4S3: C, 54.52; H,
4.58; N, 10.60. Found: C, 54.80; H, 4.60; N, 10.55.
5.3.1. 2,5-Bis(4-nitrophenyl)thiophene (1)
To a solution of 4-bromonitrobenzene (1.39 g, 6.90 mmol)
and tetrakis(triphenylphosphine)palladium(0) (0.15 g) in anhy-
drous 1,4-dioxane (20 mL) was added 1.00 g (3.40 mmol) of
2,5-bis[trimethylstannyl]thiophene [28] and the mixture was
heated at reflux overnight under nitrogen. The resulting orange
suspension was diluted with hexanes, cooled to rt, and filtered.
The obtained solid was crystallized from acetone to yield
1.09 g (99%, mp 261.9e263.1 ꢁC, lit. [29] mp 257e258 ꢁC)
of 2,5-bis(4-nitrophenyl)thiophene as a yellow crystalline
solid. 1H NMR (300 MHz, DMSO-d6) 8.30 (d, 4H,
J ¼ 8.1 Hz), 8.02 (d, 4H, J ¼ 8.1 Hz), 7.94 (s, 2H); 13C
NMR (300 MHz, DMSO-d6) 146.5, 142.5, 139.1, 128.5,
126.1, 124.4.
5.3.5. 2,5-Bis[4-(N0-ethoxycarbonyl-N00-methylguanidino)-
phenyl]thiophene (5b)
To a 2.0 M dioxane solution of N-methylamine (1.89 mL,
3.78 mmol) in 20 mL of anhydrous CH2Cl2, 0.73 g
(5.65 mmol) of DIPEA and 0.05 g (0.95 mmol) of 4 were
added. The suspension was cooled to 0 ꢁC using an ice bath
and 0.73 g (3.78 mmol) of 1-(3-dimethylaminopropyl)-3-eth-
ylcarbodiimide hydrochloride was added and the mixture
was stirred for l h at 0 ꢁC. The mixture was allowed to
warm to rt and stirred overnight. The solvent was removed un-
der reduced pressure to yield a yellow solid that was washed
several times with water. The solid was suspended in EtOH
and heated at reflux for 1 h, allowed to cool to rt, filtered
and dried under reduced pressure to yield 0.34 g (69%, mp
181.2e183.4 ꢁC) of 2,5-bis[4-(N0-ethoxycarbonyl-N00-methyl-
5.3.2. 2,5-Bis(4-aminophenyl)thiophene (2)
A mixture of 0.85 g (2.60 mmol) of 1, 5.86 g (10.0 mmol)
of stannous chloride dihydrate and 40 mL of ethanol was
heated at 70 ꢁC under nitrogen for 5 h. The mixture was al-
lowed to cool, treated with an aqueous sodium hydroxide so-
lution until a pH of 8e9 was reached and then extracted with
ethyl acetate. The organic extract was washed with water,
1
guanidino)phenyl]thiophene. H NMR (300 MHz, DMSO-d6/
D2O) 7.64 (d, 4H, J ¼ 8.6 Hz), 7.48 (s, 2H), 7.42 (br d, 4H,
J ¼ 8.6 Hz), 3.95 (q, 4H, J ¼ 7.2 Hz), 2.85 (s, 6H), 1.15 (t,
6H, J ¼ 7.2 Hz); 13C NMR (300 MHz, DMSO-d6/D2O)
163.6, 159.0, 142.3, 130.0, 125.8, 124.9, 124.6, 60.2, 28.4,

556
J.L. Gonzalez et al. / European Journal of Medicinal Chemistry 42 (2007) 552e557
14.9. Anal. Calcd. for C26H30N6O4S: C, 59.75; H, 5.79; N,
16.08. Found: C, 59.72; H, 5.77; N, 16.09.
[4-(N-isopropylguanidino)phenyl]thiophene dihydrochloride.
1H NMR (400 MHz, DMSO-d6/D2O) 7.73 (d, 4H,
J ¼ 8.6 Hz), 7.55 (s, 2H), 7.26 (d, 4H, J ¼ 8.6 Hz), 3.88 (m,
2H, J ¼ 6.0 Hz), 1.18 (d, 12H, J ¼ 6.0 Hz); 13C NMR
(300 MHz, DMSO-d6/D2O) 153.6, 142.1, 135.6, 131.2,
126.6, 125.4, 124.7, 43.9, 22.4. Anal. Calcd. for
C24H30N6S$2HCl$1.0H2O: C, 54.85; H, 6.52; N, 16.00; O,
3.04; S, 6.10; Cl, 13.49. Found: C, 55.15; H, 6.31; N, 15.90;
Cl, 13.46.
5.3.6. 2,5-Bis[4-(N-methylguanidino)phenyl]-
thiophene dihydrochloride (3b)
The substituted carbethoxy guanidine 5b (0.11 g,
0.21 mmol) was suspended in 1 mL of EtOH. To this stirred
suspension, 1.7 mL (1.7 mmol) of 1 M aqueous potassium hy-
droxide solution was added. The temperature was maintained
at approximately 60 ꢁC for 20 h. The solvent was evaporated,
the resultant solid was washed with water several times and
then suspended in EtOH. This EtOH suspension was heated
at reflux, cooled in an ice bath and collected by filtration to
yield 0.07 g (89%, mp 138.5e141.0 ꢁC) of 2,5-bis[4-(N-meth-
5.3.9. 2,5-Bis[4-(N-cyclopentylguanidino)phenyl]-
thiophene dihydrochloride (3d)
The carbethoxy guanidine 5d obtained was an oil and was
treated directly with KOH as described above for 5b to yield
0.20 g (93%, mp 212.9 e214.9 ꢁC) of 2,5-bis[4-(N-cyclopen-
1
ylguanidino)phenyl]thiophene. H NMR (300 MHz, DMSO-
1
d6/D2O) 7.50 (d, 4H, J ¼ 7.8 Hz), 7.32 (s, 2H), 6.86 (br d,
4H, J ¼ 7.8 Hz), 2.69 (s, 6H). The above free base (0.31 g,
0.82 mmol) was suspended in 10 mL of ethanol and saturated
with HCl gas and then stirred at rt for 1 h. The solvent was re-
moved under reduced pressure and the solid obtained was col-
lected by filtration, washed with ether and dried in a vacuum
oven to yield 0.18 g (44%, mp 187.6e189.3 ꢁC) of 2,5-
bis[4-(N-methylguanidino)phenyl]thiophene dihydrochloride.
1H NMR (300 MHz, DMSO-d6/D2O) 7.73 (d, 4H,
J ¼ 8.0 Hz), 7.55 (s, 2H), 7.28 (d, 4H, J ¼ 8.0 Hz), 2.84 (s,
6H); 13C NMR (300 MHz, DMSO-d6/D2O) 155.4, 141.9,
135.2, 131.1, 126.3, 125.1, 124.7, 28.4. Anal. Calcd. for
C20H22N6S$2HCl$H2O$0.43C2H4OH: C, 51.21; H, 5.89; N,
17.18; O, 4.68; S, 6.55; Cl, 14.49. Found: C, 50.86; H, 5.54;
N, 16.82; S, 6.93; Cl, 14.74.
tylguanidino)phenyl]thiophene. H NMR (400 MHz, DMSO-
d6/D2O) w7.44 (d, 4H, J ¼ 7.60 Hz), 7.25 (s, 2H), 6.79 (br
d, 4H, J ¼ 7.60), 4.01 (m, 2H), 1.85 (br m, 4H), 1.05 (br m,
12H); 13C NMR (300 MHz, DMSO-d6/D2O) 152.5, 150.7,
142.6, 126.7, 126.4, 124.4, 123.5, 52.4, 33.4, 23.9. The free
base was converted into the salt as described above for 3b to
yield 0.17 g (73%, mp 231.8e234.7 ꢁC) of 2,5-bis[4-(N-cyclo-
1
pentylguanidino)phenyl]thiophene dihydrochloride. H NMR
(300 MHz, DMSO-d6/D2O) 7.73 (d, 4H, J ¼ 7.8 Hz), 7.55 (s,
2H), 7.27 (d, 4H, J ¼ 7.8 Hz), 4.03 (br m, 2H), 1.95 (br m,
4H), 1.61 (br m, 12H); 13C NMR (300 MHz, DMSO-d6/
D2O) 154.4, 142.6, 135.7, 131.9, 127.1, 125.9, 53.6, 32.7,
23.9. Anal. Calcd. for C28H34N6S$2HCl$1.05H2O: C, 58.13;
H, 6.64; N, 14.53; O, 2.90; S, 5.54; Cl, 12.26. Found: C,
58.49; H, 6.69; N, 14.13; Cl, 11.73.
5.3.7. 2,5-Bis[4-(N0-ethoxycarbonyl-N00-isopropylguanidino)-
phenyl]thiophene (5c)
5.3.10. 2,5-Bis[4-(N0-ethoxycarbonyl-N00-cyclohexylguanidino)-
phenyl]thiophene (5e)
The N-carbethoxy thiourea 4 was allowed to react with iso-
propyl amine as described above for 5b and yielded after
crystallization from EtOH 0.37 g (67%, mp 156.8e159.2 ꢁC)
of 2,5-bis[4-(N0-ethoxycarbonyl-N00-isopropylguanidino)phenyl]
thiophene as a yellow powder solid. 1H NMR (400 MHz,
DMSO-d6/D2O) 7.64 (d, 4H, J ¼ 7.8 Hz), 7.48 (s, 2H), 7.42
(br d, 4H, J ¼ 7.8 Hz), 4.10 (m, 2H), 3.94 (q, 4H, J ¼ 7.2 Hz),
1.16 (d, 12H, J ¼ 6.4 Hz), 1.13 (t, 6H, J ¼ 7.2 Hz); 13C NMR
(300 MHz, DMSO-d6/D2O) 163.4, 157.1, 129.4, 125.3, 124.4,
124.0, 59.6, 42.4, 22 6, 14.5. Anal. Calcd. for C30H38N6O4S:
C, 62.26; H, 6.62; N, 14.52; O, 11.06; S, 5.54. Found C,
62.18; H, 6.48; N, 14.50.
The N-ethoxycarbonylthiourea 4 was allowed to react with
cyclohexylamine as described above for 5b and yielded an oily
residue which was passed through a short column of silica gel
using a mixture of ethyl acetate/hexane (1:1) as the mobile
phase. After the removal of the solvent, 0.22 g (63%, mp
186.6e188.9 ꢁC) of 2,5-bis[4-(N0-ethoxycarbonyl-N00-cyclo-
hexylguanidino)phenyl]thiophene was obtained as a yellow
solid. 1H NMR (400 MHz, DMSO-d6/D2O) 7.63 (d, 4H,
J ¼ 7.6), 7.47 (s, 2H), 7.42 (br d, 4H), 3.92 (q, 4H,
J ¼ 6.53), 3.78 (br d, 2H), 1.88 (br m, 4H), 1.67 (br d, 4H),
1.55 (br d, 2H), 1.28 (m, 10H), 1.13 (t, 6H). Anal. Calcd.
for C36H46N6O4S: C, 65.63; H, 7.04; N, 12.76. Found: C,
65.37; H, 7.10; N, 12.73.
5.3.8. 2,5-Bis[4-(N-isopropylguanidino)phenyl]-
thiophene dihydrochloride (3c)
5.3.11. 2,5-Bis[4-(N-cyclohexylguanidino)phenyl]-
The carbethoxy guanidine 5c was treated with KOH as
described above for 5b to yield 0.11 g (100%, mp 215.9e
thiophene dihydrochloride (3e)
The carbethoxy guanidine 5e was treated with KOH as
described above for 5b to yield 0.15 g (77%, mp
252.4e254.8 ꢁC) of 2,5-bis[4-(N-cyclohexylguanidino)phenyl]
thiophene. 1H NMR (400 MHz, DMSO-d6) 7.45 (d, 4H,
J ¼ 8.2 Hz), 7.27 (s, 2H), 6.78 (d, 4H, J ¼ 8.2 Hz), 5.40 (br s,
2H), 4.95 (br s, 4H), 3.56 (br s, 2H), 1.91 (br d, 4H), 1.63 (m,
6H), 1.21 (m, 8H); 13C NMR (300 MHz, DMSO-d6) 151.1,
150.5, 142.1, 126.3, 125.8, 123.6, 122.8, 48.51, 33.0, 25.51, 34.5.
217.3 ꢁC)
of
2,5-bis[4-(N-isopropylguanidino)phenyl]-
thiophene. 1H NMR (400 MHz, DMSO-d6) 7.44 (d, 4H,
J ¼ 8.4 Hz), 7.25 (s, 2H), 6.78 (d, 4H, J ¼ 8.4 Hz), 5.34 (br
s, 2H), 4.94 (br s, 4H), 3.84 (m, 2H, J ¼ 6.4 Hz), 1.09 (d,
12H, J ¼ 6.4 Hz).
The free base was converted into the salt as described above
for 3b to yield 0.09 g (82%, mp 261.9e263.8 ꢁC) of 2,5-bis

J.L. Gonzalez et al. / European Journal of Medicinal Chemistry 42 (2007) 552e557
557
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Nucleic Acid Complexes, vol. 2, Wiley-VCH, New York, 2003, pp.
416e460;
The free base was converted into the salt as described above
for 3b to yield 0.11 g (66%, mp 209.5e210.8 ꢁC) of 2,5-bis[4-
(N-cyclohexylguanidino)phenyl]thiophene dihydrochloride.
1H NMR (300 MHz, DMSO-d6/D2O) 7.76 (d, 4H,
J ¼ 8.4 Hz), 7.55 (s, 2H), 7.29 (d, 4H, J ¼ 8.4 Hz); 13C
NMR (300 MHz, DMSO-d6) 154.0, 142.6, 135.6, 127.1,
126.0, 51.1, 32.6, 31.3, 25.3, 24.8. Anal. Calcd. for
C30H38N6S$2HCl$l.75H2O: C, 58.19; H, 7.08; N, 13.57.
Found: C, 58.38; H, 6.72; N, 13.26.
(b) W.D. Wilson, B. Nguyen, F.A. Tanious, A. Mathis, J.E. Hall,
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5.3.12. 2,5-Bis[4-(N-phenylguanidino)phenyl]-
thiophene dihydrochloride (3f)
[7] B. Bouteille, O. Oukem, S. Bisser, M. Dumas, Fundam. Clin. Pharmacol.
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The carbethoxy guanidine 5f obtained using aniline was an
oil and was treated directly with KOH as described above for
5b and the solid free base obtained was washed with water,
air dried and converted to the salt without further characteriza-
tion to yield 0.28 g (79%, mp 225.4e227.3 ꢁC, dec) of 2,5-
bis[4-(N-phenylguanidino)phenyl]thiophene dihydrochloride.
1H NMR (300 MHz, DMSO-d6/D2O) 7.72 (s, 2H), 7.2e7.65
(br m, 18H); 13C NMR (300 MHz, DMSO-d6/D2O) 154.3,
142.6, 135.8, 135.5, 132.1, 130.5, 127.4, 127.0, 126.0, 125.4,
124.8. Anal. Calcd. for C30H26N6S$2.1HCl$2.0H2O$0.4-
C2H5OH: C, 58.38; H, 5.49; N, 13.26; Cl, 11.75. Found: C,
58.40; H, 5.12; N, 12.94; Cl, 12.01.
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5.3.13. 2,5-Bis[4-(N-p-tolylguanidino)phenyl]-
thiophene dihydrochloride (3g)
The carbethoxy guanidine 5g obtained was an oil and was
treated directly with KOH as described above for 5b and the
solid free base obtained was washed with water, air dried and
converted to the salt without further characterization to yield
0.21 g (100%, mp 237.5e238.6 ꢁC, dec) of 2,5-bis[4-(N-p-tol-
ylguanidino)phenyl]thiophene dihydrochloride. 1H NMR
(300 MHz, DMSO-d6/D2O) 7.71 (d, 4H, J ¼ 8.4 Hz), 7.50 (s,
2H), 7.33 (d, 4H, J ¼ 8.4 Hz), 7.22 (br m, 8H), 2.33 (s, 6H);
13C NMR (400 MHz, DMSO-d6) 154.0, 141.8, 135.7, 135.1,
132.7, 131.1, 129.9, 126.1, 125.0, 124.4, 124.2, 20.4. Anal.
Calcd. for C32H30N6S$2.0HCl$1.6 H2O: C, 60.79; H, 5.61; N,
13.29. Found: C, 61.18; H, 5.39; N, 12.89.
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W.D. Wilson, D.W. Boykin, Bioorg. Med. Chem. Lett. 13 (2003)
2065.
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Acknowledgments
[22] M.A. Ismail, R. Brun, J.D. Easterbrook, F.A. Tanious, W.D. Wilson,
D.W. Boykin, J. Med. Chem. 46 (2003) 4761.
This work was supported by an award from the Bill and
Melinda Gates Foundation and NIH grant GM61587. The
technical assistance with the in vivo experiments by Guy
Riccio (Swiss Tropical Institute) is greatly appreciated.
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