Novel linear triaryl guanidines, N-substituted guanidines and potential prodrugs as antiprotozoal agents

Article abstract of DOI:10.1016/j.ejmech.2008.02.008

A series of triaryl guanidines and N-substituted guanidines designed to target the minor groove of DNA were synthesized and evaluated as antiprotozoal agents. Selected carbamate prodrugs of these guanidines were assayed for their oral efficacy. The linear

Full text of DOI:10.1016/j.ejmech.2008.02.008

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European Journal of Medicinal Chemistry 43 (2008) 2901e2908
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Short communication
Novel linear triaryl guanidines, N-substituted guanidines and
potential prodrugs as antiprotozoal agents
Reem K. Arafa a, Mohamed A. Ismail a, Manoj Munde a, W. David Wilson a,
Tanja Wenzler b, Reto Brun b, David W. Boykin a,
*
a Department of Chemistry, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303-3083, USA
b Parasite Chemotherapy, Swiss Tropical Institute, Basel CH4002, Switzerland
Received 6 August 2007; received in revised form 2 November 2007; accepted 8 February 2008
Available online 29 February 2008
Abstract
A series of triaryl guanidines and N-substituted guanidines designed to target the minor groove of DNA were synthesized and evaluated as
antiprotozoal agents. Selected carbamate prodrugs of these guanidines were assayed for their oral efficacy. The linear triaryl bis-guanidines 6a,b
were prepared from their corresponding diamines 4a,b through the intermediate BOC protected bis-guanidines 5a,b followed by acid catalyzed
deprotection. The N-substituted guanidino analogues 9cef were obtained in three steps starting by reacting the diamines 4a,b with ethyl iso-
thiocyanatoformate to give the carbamoyl thioureas 7a,b. Subsequent condensation of 7a,b with various amines in the presence of EDCI pro-
vided the carbamoyl N-substituted guanidine intermediates 8aef which can also be regarded as potential prodrugs for the guanidino derivatives.
Compounds 9cef were obtained via the base catalyzed decarbamoylation of 8aef. The DNA binding affinities for the target dicationic bis-
guanidines were assessed by DTm values. In vitro antiprotozoal screening of the new compounds showed that derivatives 6a, 9c and 9e possess
high to moderate activity against Trypanosoma brucei rhodesiense (T.b.r.) and Plasmodium falciparum (P.f.). While the prodrugs did not yield
cures upon oral administration in the antitrypanosomal STIB900 mouse model, compounds 8a and 8c prolonged the survival of the treated mice.
Ó 2008 Published by Elsevier Masson SAS.
Keywords: Antiprotozoan; Bis-guanidines; DNA binding; Triaryl guanidines
1. Introduction
AT rich sites is essential for the antimicrobial activity of dica-
tions [1]. The consequent inhibition of transcription and/or
DNA dependent enzymes is postulated to be the cause of anti-
microbial action [1,8e11]. The belief that complementing ge-
ometry to that of the DNA minor groove is a structural
necessity for the binding affinity, lead to design and synthesis
of many dications with crescent shaped scaffold e.g. pentami-
dine, furamidine and other analogues [1,12e17].On the other
hand, current reports have indicated that linear dicationic diami-
dines can bind strongly to DNA and possess significant antipro-
tozoan activity. CGP40215A (IV, Fig. 1) is an example of these
type molecules with considerable antimicrobial activity [18e
20]. Moreover, the high binding affinity to AT rich sites of the
DNA minor groove was explained by analysis of the binding
data, crystal structure and molecular dynamic simulations
which lead to the finding that water-assisted interaction of IV
simulates the curved structure of DNA minor groove [19,20].
Aromatic dications have been long known to possess signif-
icant antimicrobial activity [1]. The aromatic diamidine pent-
amidine I, first introduced in the 1942, belongs to this group
of dicationic drugs and is the only one to see significant human
clinical use [1,2]. Currently, pentamidine is used in the manage-
ment of antimony-resistant leishmaniasis, initial stage human
African trypanosomiasis (HAT), and as a second line agent
for AIDS-related Pneumocystis jiroveci (formerly Pneumocys-
tis carinii) pneumonia [1]. Pafuramidine III (Fig. 1), an orally
bioavailable prodrug of furamidine II (Fig. 1), is currently in
phase III trials for pneumocystis pneumonia and HAT [1,3e
7]. It has been suggested that DNA minor groove binding at
* 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 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2008.02.008

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R.K. Arafa et al. / European Journal of Medicinal Chemistry 43 (2008) 2901e2908
We have most recently reported the marked antiprotozoan
through the intermediate BOC protected bis-guanidines 5a,b
by the use of bis-Boc-protected N-methylpseudothiourea and
HgCl2 as a Lewis acid. Treatment of 5a,b with ethanolic
HCl at room temperature served the dual purpose of guanidine
deprotection, as well as providing the hydrochloride salts of
the guanidine free bases 6a,b. While the key intermediate di-
amine 4a was commercially available, 4b was prepared in two
steps. The initial step was a suzuki coupling reaction between
benzene 1,4-bisboronic acid 1 and 2-chloro-5-nitropyridine 2 to
yield the dinitro derivative 3. Next was the palladium catalyzed
hydrogenation of 3 which furnished the desired diamine 4b.
As outlined in Scheme 2, the N-substituted alkyl-guanidino
analogues 9cef were obtained from the diamines 4a,b in three
steps. First, the diamines 4a,b were reacted with ethyl isothio-
cyanatoformate to give the carbamoyl thioureas 7a,b. Subse-
quently, condensation with variable aliphatic amines in the
presence of EDCI provided the carbamoyl N-substituted al-
kyl-guanidines intermediates 8aef which can also be regarded
as potential prodrugs for the guanidino derivatives. The target
compounds 9cef were finally obtained via the KOH catalyzed
decarbamoylation of 8aef. Hydrochloride salts of 9cef were
prepared by treating an ethanolic solution of the free bases
with HCl gas.
activity of the rigid-rod terphenyl diamidine (V, Fig. 1) and an-
alogues [21]. Also, the thermal melting data and circular di-
chroism (CD) studies of these linear derivatives reflect
a significant DNA minor groove binding affinity [21]. Struc-
tures with rigid frameworks of fused ring systems as linkers
for the dicationic groups (e.g. carbazole, benzofuran, benzo-
thiophene, 9H-fluorene, fluorenone, acridine and anthraqui-
none) were also reported as effective antimicrobial agents
[22e25] and had high DNA minor groove binding affinity
and postulated to involve water mediated interactions [26,27].
Previous studies from our laboratory have demonstrated
that guanidine dications display considerable affinity to AT
rich sites of the DNA minor groove and possess interesting
antiprotozoan activity [25,28e30]. In light of the previous re-
sults, we have designed and performed a study aiming at com-
bining the novelty of a rigid rod-like terphenyl framework
bearing a bisguanidine. We report here the DNA binding and
antiprotozoan activity of series of novel dicationic linear tri-
aryl bis-guanidines.
It is documented that aromatic amidines and guanidines are
charged at physiological pH since they have pK values of 10
or higher [31,32]. This accounts for the lack of oral bioavailabil-
ity of these dications [1]. Since oral administration is the pre-
ferred route of administration, especially for those diseases
requiring a prolonged course of treatment, then a prodrug ap-
proach is a plausible solution to circumvent the drawback of
limited oral absorption. Carbamates were introduced earlier as
potential prodrugs for guanidine derivatives [25,33e35]. In
this work a number of carbamate derivatives were prepared
and evaluated as potential prodrugs for the bis-guanidine series.
2.2. Biological activity
The DNA binding affinity of the dicationic bis-guanidines
was assessed by DTm values and the results from in vitro eval-
uation versus Trypanosoma brucei rhodesiense (T.b.r.) and
Plasmodium falciparum (P.f.) are shown in Table 1. The
DNA affinity of the bis-guanidine 6a is reduced by about
20% from that of the corresponding diamidine V. A similar re-
duction has also been observed recently in a diphenylthiophene
parent system and it seems that amidine dications are gener-
ally able to form slightly more favorable complexes with the
DNA minor groove than guanidines [36]. N-Methyl substitu-
tion (9c) has little effect on the DNA affinity; however, the
larger alkyl group of 9e results in a reduction of affinity by
2. Discussion
2.1. Chemistry
As depicted in Scheme 1, the linear triaryl bis-guanidines
6a,b were prepared from their corresponding diamines 4a,b
O
O
NH
HN
O
HN
NH
NH2
NH2
H2N
NH2
furamidine (II)
pentamidine (I)
NH2
NOMe
NH2
O
MeON
H2N
HN
N
N
NH
NH2
N
N
H
NH2
CGP 40215 (IV)
Pafuramidine (III)
HN
NH
H2N
NH2
V
Fig. 1. Structures of key dicationic antiprotozoan agents.

R.K. Arafa et al. / European Journal of Medicinal Chemistry 43 (2008) 2901e2908
2903
(i)
(ii)
O2N
Cl
O2N
NO2
(HO)2B
B(OH)2
+
N
N
N
X
1
2
3
BocN
NH
NBoc
HN
(iii)
NHBoc
BocHN
H2N
NH2
X
X
X
4a, b
5a, b
NH
HN
HN
NH
(iv)
H2N
NH2
Legends for compounds 2-6
a; X = CH
b; X = N
X
X
6a, b
Scheme 1. Reagents and conditions: (i) Pd(PPh3)4, Na2CO3, toluene, 80 ꢀC; (ii) H2/PdeC, EtOH, EtOAc; (iii) BocNH(MeS)C]NBoc, HgCl2, TEA, DMF, r.t.; (iv)
HCl (g), EtOH, CH2Cl2, r.t.
approximately one third. The introduction of nitrogen atoms in
the aryl ring (6b) also results in approximately a one third re-
duction in DNA affinity.
differences are related to the mechanism of action of these
molecules or their transport into the parasites. The potential
prodrugs (8bef) as expected show essentially no in vitro activ-
ity against T.b.r., however, they are active against P.f. suggest-
ing the presence of non-specific esterases in the parasite or the
culture media. Since the parent molecules 6a, 6b, 9c, 9d
showed promising in vitro activity they were advanced to
the STIB900 acute mouse model for African trypanosomiasis
and the results are shown in Table 2. The most effective com-
pounds in this model were 6a, 9c and 9d, all extended the av-
erage survival time of the animals by greater than 47 days,
however, none gave cures at intrapertoneal dose of 20 mg/kg
given for 4 days, and 9d showed acute toxicity. In this exper-
iment the cited three compounds were more effective than the
parent diamidine V. The two potential prodrugs 8a and 8c
were tested on oral administration and were found not to be
very effective as no cures were achieved and only 20 day sur-
vival times were noted. The amidoxime prodrugs of V were
also only moderately effective which was attributed, at least
The in vitro results against T.b.r. and P.f. also reflect a re-
duction in activity compared to that of V. Nevertheless, the
parent bis-guanidine 6a and the N-methyl analogue 9c show
potent activity against both T.b.r. and P.f. giving IC50 values
ranging from 7 to 27 nM. The activity of the N-isopropyl an-
alogue 9e is markedly reduced versus T.b.r.; however, its ac-
tivity versus P.f. is essentially the same as 6a and 9c. The
activity of the compounds with nitrogen atoms in the aryl rings
(6b, 9d, and 9f) in general is markedly reduced from their car-
bocycle counterparts. In summary, key SAR features noted are
that direct substitution on the guanidines with a methyl group
is tolerated whereas a moderately large alkyl group (isopropyl)
causes significant reduction in T.b.r. activity, however, the
same pattern is not found against P.f. Also, introduction of ni-
trogen atoms in the linear array results in loss of activity
against both parasites. It is unclear whether the origin of these
O
O
S
EtO
S
OEt
(i)
(ii)
4a, b
HN
NH
HN
NH
X
X
7a, b
O
O
EtO
RN
OEt
NR
RN
NH
NR
HN
NH2
NH
(iii)
H2N
HN
HN
NH
X
X
X
X
9c-f
8a-f
Legend for compounds 7-9
X
CH
N
CH
N
R
H
H
Me
Me
i-pro
i-pro
a;
b;
c;
d;
e;
f;
CH
N
Scheme 2. Reagents and conditions: (i) ethylisothiocyanatoformate, CH2Cl2, r.t.; (ii) RNH2, DIPEA, WSC, r.t.; (iii) KOH, EtOH, 55 ꢀC.

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R.K. Arafa et al. / European Journal of Medicinal Chemistry 43 (2008) 2901e2908
Table 1
DNA binding and in vitro antiprotozoan testing data for linear triaryl dications
Table 2
In vivo antitrypanosomal screening data for linear triaryl dications in STIB900
mouse modela
A
A
A
A
X
X
X
X
a
Code
A
X
DTm
(ꢀC)
T.b.r.b P.f.b
Cytotoxicityc
Code
A
X
Dose
Curesb Average survival
(days)c
IC50
(nM)
IC50
(nM)
IC50 (mM)
Vd
CH 20 mg/kg ip 0/4
29
NH
NH
V
CH 18.2
5
1
22.1
25.4
NH2
NH2
NH
NH
6a
8a
9c
CH 20 mg/kg ip 0/4
CH 25 mg/kg po 0/4
CH 20 mg/kg ip 0/4
CH 25 mg/kg po 0/4
>54.5
20.25
>56
HN
6a
8a
CH 14.2
CH NTd
18
27
HN
NH2
NH2
NCOOEt
NH2
NCOOEt
NH2
HN
HN
142K
1.7 >181
HN
NH
NH
9c
8c
CH 13.0
CH NT
18
7
65.1
5.7
HN
HN
HN
NHMe
NHMe
NCOOEt
NCOOEt
8c
20.25
20.25
8.5
7.5K
8.9
HN
HN
HN
NHMe
NH
NHMe
NH
6b
8b
9de
N
N
N
N
20 mg/kg ip 0/4
25 mg/kg po 0/4
20 mg/kg ip 0/2
25 mg/kg ip 0/4
9e
8e
CH 9.3
CH NT
530
14
21.9
5.3
NH2
NH
NCOOEt
NCOOEt
NH
NH2
NH
HN
14.2K
4.5
>47
NH
HN
HN
NHMe
6b
8b
9d
8d
9f
N
N
N
N
N
9.2
169
151
152
HN
HN
HN
NH2
NCOOEt
8d
7.75
NCOOEt
NHMe
NT
10.2
NT
8.1
61K
134
1.9 >181
NH2
NH
a
See Ref. [37] for details of the STIB900 mouse model. Dosage was for 4
days and was either intraperitoneal (ip) or oral (po) as noted.
b
54
>161
Number of mice that survived 60 days and are parasite-free.
Average days of survival; untreated controls died between day 7 and 9 post
c
NHMe
infection.
d
NCOOEt
17.8K
2.6 >169
The results for V in this experiment were moderately different from that
reported in Ref. [21].
HN
HN
NHMe
NH
e
Some acute toxicity was apparent as two of the four animals died within
one day after the first dose. Due to the modest IC50 values for 9e and 9f,
they were not tested in vivo, nor were their prodrugs 8e and 8f.
11.9K 393
89.7
NH
NCOOEt
NH
HN
8f
N
NT
8.2K
2.9 >135
than that of V. Unfortunately, both the active parent molecules
(6a and 6b) and their potential prodrugs (8a and 8b) were
only moderately effective in the STIB900 acute mouse model.
a
Poly(d(A-T))2 in MES10 buffer; ratio, compound/DNA is 0.3.
T.b.r. strain was STIB900, and the P.f. strain was K1. Values are duplicate
b
determinations, see Refs. [36,37].
c
Cytotoxicity was evaluated using cultered L6 rat myoblast cells using the
same assay procedure as for T.b.r.
3. Experimental section
d
NT ¼ not tested.
3.1. Efficacy evaluations
in part, to poor bioconversion [20]. It is unknown if the poor
efficacies of these carbamte prodrugs, which would be con-
verted by a different mechanism, are due to poor absorption/
distribution or poor bioconversion.
In conclusion, the linear bis-guanidines were found to bind to
DNA at a somewhat reduced level compared to V, yet they ex-
hibited very good in vitro activity against T.b.r. and P.f. al-
though reduced compared with that of V. However, the in vivo
efficacy of the most active compounds 6a, 9c and 9d was greater
In vitro activity over a 72 h drug exposure time was deter-
mined against the erythrocytic stages of the chloroquine and
pyrimethamine resistant P.f. strain K1 using the 3H-hypoxan-
thine incorporation assay and against the bloodstream form of
the T.b.r. strain STIB900 using the Alamar Blue assay. In vivo
efficacy was determined in the STIB900 acute mouse model.
Four infected mice were treated with the drugs either by the
intraperitoneal or for prodrugs by the oral route on days

R.K. Arafa et al. / European Journal of Medicinal Chemistry 43 (2008) 2901e2908
2905
3e6 post infection and parasitamia checked to day 60. The
experiments were carried out as previously reported [37].
3.3.2. 4,400-Bis-guanidino-[1,10;40,100]terphenyl (6a)
The N0,N00-di-BOCguanidine 5a (0.45 g, 0.60 mmol) was
dissolved in CH2Cl2 (10 ml), diluted with dry EtOH
(15 ml) and the chilled solution was saturated with dry
HCl. The reaction was then kept stirring at room tempera-
ture for 3 days (drying tube), where upon the product
started precipitating. After evaporating the solvent to dry-
ness, the residue was washed with ether multiple times
and was dried under vacuum at 50e60 ꢀC overnight to
give whitish yellow solid of the bis-guanidine dihydrochlor-
ide, m.p. >300 ꢀC. 1H NMR (DMSO-d6): d 7.85 (d,
J ¼ 8.4 Hz, 4H), 7.92 (s, 4H), 8.00 (d, J ¼ 8.4 Hz, 4H),
9.09 (br s, 4H), 11.25 (br s, 2H). 13C NMR (DMSO-d6):
d 155.98, 138.35, 137.32, 135.00, 127.79, 127.22, 124.79.
MS (ESI) m/e (rel. int.): 345 (Mþ þ 1, 6), 173 (100).
Anal. Calc. for C20H20N6e2.0HCleH2Oe0.2C2H5OH: C,
55.11; H, 5.71; N, 18.90. Found C, 55.39; H, 5.50; N,
18.71.
3.2. 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. Tem-
peratures were maintained under computer control and are in-
creased at 0.5 C/min. The experiments were conducted in
1 cm path length quartz curvettes in CAC 10 buffer (cacodylic
acid 10 mM, EDTA 1 mM, NaCl 100 mM with NaOH addi-
tion to give pH ¼ 7.0). The concentrations of DNA were deter-
mined by measuring the absorbance at 260 nm. A ratio of
0.3 mol compound per mole of DNA was used for the complex
and DNA with no compound was used as a control. The DTm
values obtained in this manner are reproducible within
ꢁ0.5 ꢀC.
3.3.3. 4,400-Bis(N0-ethoxycarbonylthiourea)-
[1,10;40,100]terphenyl (7a)
3.3. Synthetic protocols
A solution of 4a (0.50 g, 1.92 mmol) in CH2Cl2 (10 ml),
added to which ethyl isothiocyanatoformate (0.55 g,
4.22 mmol), was stirred at room temperature for 24 h. After
flash chromatography, the reaction was diluted with hexane
and the precipitate formed was collected and dried to yield
the bis-carbamoyl thiourea as a white solid, yield 89%, m.p.
Melting points were recorded using a ThomaseHoover
(Uni-Melt) capillary melting point apparatus and are uncor-
rected. TLC analysis was carried out on silica gel 60 F254 pre-
1
coated aluminum sheets and detected under UV light. H and
13C NMR spectra were recorded employing a Varian Unity
Plus 300 spectrometer (Varian, Inc., Palo Alto, California),
and chemical shifts (d) are in ppm relative to TMS as the in-
ternal standard. Mass spectra were recorded on a VG analyti-
cal 70-SE spectrometer (VG Analytical, Ltd., Manchester,
UK). Elemental analyses were obtained from Atlantic Micro-
lab 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 solva-
tion. In each case, proton NMR showed the presence of indi-
cated solvent(s). All chemicals and solvents were purchased
from Aldrich Chemical Co., Fisher Scientific, or Lancaster
Synthesis, Inc.
1
>300 ꢀC. H NMR (DMSO-d6): d 1.26 (t, J ¼ 7.2 Hz, 6H),
4.22 (q, J ¼ 7.2 Hz, 4H), 7.70e7.79 (m, 12H), 11.29 (br s,
2H), 11.61 (br s, 2H). MS (ESI) m/e (rel. int.): 522 (Mþ,
13), 344 (100), 328 (63). Anal. Calc. for C26H26N4O4S2: C,
59.75; H, 5.01. Found C, 59.58; H, 5.23.
3.3.4. 4,400-Bis(N0-ethoxycarbonyl)-guanidino-
[1,10;40,100]terphenyl (8a)
A stirred solution of the carbamoyl thiourea 7a (0.80 g,
1.53 mmol), 0.5 M ammonia solution in dioxane (11.7 ml,
6.12 mmol), and diisopropylethylamine (1.18 g, 9.18 mmol)
in anhydrous DMF (10 ml) was cooled to 0 ꢀC. EDCI
(1.17 g, 6.12 mmol) was added, and the solution was stirred
at room temperature overnight. The reaction mixture poured
onto ice/water, the solid collected by vacuum filtration. Fi-
nally, the carbamoyl-guanidine was crystallized from EtOH,
yield 67%, m.p. >300 ꢀC. 1H NMR (DMSO-d6): d 1.17
(t, J ¼ 6.9 Hz, 6H), 3.98 (q, J ¼ 6.9 Hz, 4H), 7.53e7.71 (m,
16H), 9.05 (br s, 2H). Anal. Calc. for C26H28N6O4e
0.2C2H5OH: C, 63.70; H, 5.91; N, 16.88. Found C, 63.66;
H, 5.71; N, 16.65.
3.3.1. 4,400-Bis(N0,N00-t-butoxycarbonyl)-
guanidino-[1,10;40,100]terphenyl (5a)
To a solution of 4,400-diamino-[1,10;40,100]terphenyl (4a)
(0.30 g, 1.15 mmol) in anhydrous DMF (10 ml) was added
1,3-bis(tert-butoxycarbonyl)-2-methylthiopseudourea (0.71 g,
2.4 mmol), triethylamine (0.74 g, 7.2 mmol) and finally mer-
cury(II) chloride (0.73 g, 2.6 mmol). The suspension was
kept stirring at room temperature for 24 h. The reaction, di-
luted with CH2Cl2 and Na2CO3 solution, was filtered through
a pad of Celite. The organic layer was washed with water (3ꢂ)
followed by brine and then dried over anhydrous Na2SO4. Af-
ter evaporating the solvent to dryness the obtained residue was
recrystallized from CH2Cl2/MeOH giving a creamy white
solid, yield 79%, m.p. >300 ꢀC. 1H NMR (DMSO-d6):
d 1.50, 1.59 (2s, 36H), 7.56e7.70 (m, 12H), 10.39 (br s,
2H), 11.65 (br s, 2H). Anal. Calc. for C40H52N6O8: C,
64.49; H, 7.03. Found C, 64.31; H, 6.85.
3.3.5. 4,400-Bis(N0-ethoxycarbonyl-N00-methyl)-
guanidino-[1,10;40,100]terphenyl (8c)
A stirred solution of carbamoyl thiourea 7a (1.00 g,
1.91 mmol),
methylamine
hydrochloride
(0.51 ml,
7.65 mmol), and diisopropylethylamine (1.48 g, 11.48 mmol)
in anhydrous CH2Cl2 (10 ml) was cooled to 0 ꢀC. EDCI
(1.46 g, 7.65 mmol) was added, and the solution was stirred
at room temperature overnight. The reaction mixture was

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R.K. Arafa et al. / European Journal of Medicinal Chemistry 43 (2008) 2901e2908
washed with water (3 ꢂ 100 mL), followed by brine and dried
over anhydrous Na2SO4. The residue remaining after removal
of the solvent was crystallized from EtOH/water, yield 82%,
126.69, 126.14, 123.36, 41.56, 22.84. MS (ESI) m/e (rel.
int.): 429 (Mþ, 8), 215 (100).
Hydrochloride salt of 9e. M.p. 275e277 ꢀC. 1H NMR
(DMSO-d6): d 1.19 (d, J ¼ 6.6 Hz, 12H), 3.85e3.96 (m,
2H), 5.38 (br s, 6H), 7.31 (d, J ¼ 8.4 Hz, 4H), 7.77e7.80
(m, 8H), 8.13 (br s, 2H), 9.91 (br s, 2H). Anal. Calc. for
C26H32N6e2.0HCle1.5H2Oe0.25C2H5OH: C, 58.93; H,
7.18; N, 15.56. Found C, 59.00; H, 6.82; N, 15.33.
m.p. 297e299 ꢀC. 1H NMR (DMSO-d6):
d 1.15 (t,
J ¼ 7.2 Hz, 6H), 3.32 (s, 6H), 3.95 (q, J ¼ 7.2 Hz, 4H), 7.45
(br s, 4H), 7.68e7.75 (m, 12H). 13C NMR (DMSO-d6):
d 163.01, 158.53, 138.25, 137.50, 135.41, 126.76, 126.66,
124.14, 59.60, 28.17, 14.54. MS (ESI) m/e (rel. int.): 517
(Mþ, 30), 259 (100). Anal. Calc. for C28H32N6O4e
0.25C2H5OH: C, 64.81; H, 6.39; N, 15.91. Found C, 64.84;
H, 6.20; N, 15.82.
3.3.9. 1,4-Bis-[50-nitropyridin-20-yl]phenylene (3)
To a stirred solution of 2-chloro-5-nitropyridine (3.16 g,
20 mmol), and tetrakis(triphenylphosphine) palladium
(800 mg) in toluene (40 mL) under a nitrogen atmosphere
was added 20 mL of a 2 M aqueous solution of Na2CO3 fol-
lowed by 1,4-phenylenebisboronic acid (1.64 g, 10 mmol) in
10 mL of methanol. The vigorously stirred mixture was
warmed to 80 ꢀC for 12 h. The solvent was evaporated, the
precipitate was filtered off, washed with ethanol, recrystallized
from DMF to afford 3 as a yellow solid in 89% yield, m.p.
3.3.6. 4,400-Bis(N0-methyl)-guanidino-
[1,10;40,100]terphenyl (9c)
The bis substituted carbamoyl-guanidine 8c (0.5 g,
0.96 mmol) was suspended in EtOH (10 ml). KOH (1 N,
9.6 ml, 9.6 mmol) was then added and the reaction mixture
was kept stirring overnight maintaining the temperature at
55e60 ꢀC. The reaction mixture was diluted with water and
the solid formed was collected by filtration, washed multiple
times with water and recrystallized from aqueous EtOH to
give a tan white solid, yield 75%, m.p. 243e244 ꢀC. 1H
NMR (DMSO-d6): d 2.67 (s, 6H), 5.35 (br s, 2H), 6.87 (d,
J ¼ 8.4 Hz, 4H), 7.53 (d, J ¼ 8.4 Hz, 4H), 7.64 (s, 4H). 13C
NMR (DMSO-d6): d 152.2, 149.8, 138.2, 131.3, 126.5,
126.0, 123.2, 27.5. MS (ESI) m/e (rel. int.): 373 (Mþ, 6),
187 (100).
1
>300 ꢀC. H NMR (DMSO-d6): d 8.35 (d, J ¼ 8.7 Hz, 2H),
8.39 (s, 4H), 8.67 (dd, J ¼ 2.1, 8.7 Hz, 2H), 9.47 (d,
J ¼ 2.1 Hz, 2H). MS (ESI) m/e (rel. int.): 323 (Mþ þ 1,
100), 307 (80), 287 (12).
3.3.10. 1,4-Bis-[50-aminopyridin-20-yl]phenylene (4b)
A suspension of the bis-nitro compound 3 (2.58 g,
8.0 mmol) in EtOAc (150 ml) and EtOH (50 ml) was hydroge-
nated with 10% Pd/C (1.0 g, Lancaster) at 60 psi until uptake
subsided (8 h). The mixture was then filtered over Celite and
concentrated in vacuo to afford a buff solid in 77% yield,
m.p. 250e251 ꢀC. 1H NMR (DMSO-d6): d 5.46 (s, 4H,
2NH2), 6.98 (dd, J ¼ 2.4, 8.4 Hz, 2H), 7.65 (d, J ¼ 8.4 Hz,
2H), 7.93 (s, 4H), 8.02 (d, J ¼ 2.4 Hz, 2H). 13C NMR
(DMSO-d6): d 143.4, 137.9, 136.0, 125.0, 120.5, 120.0. MS
(ESI) m/e (rel. int.): 263 (Mþ þ 1, 100).
Hydrochloride salt of 9c. The free base was dissolved in dry
EtOH (20 ml) and the solution was chilled in an ice-bath. Af-
ter passing HCl gas for 10 min, the reaction was concentrated
under reduced pressure and then diluted with ether. The pre-
cipitate formed was collected by filtration, m.p. 211e
1
214 ꢀC. H NMR (DMSO-d6): d 2.83, 2.85 (2 s, 6H), 7.33
(d, J ¼ 8.4 Hz, 4H), 7.77e7.80 (m, 8H), 7.96 (br s, 4H),
10.02 (br s, 4H). Anal. Calc. for C22H24N6e2.0HCle
1.25H2Oe0.3C2H5OH: C, 56.34; H, 6.33; N, 17.42. Found
C, 56.69; H, 6.07; N, 17.05.
3.3.7. 4,400-Bis(N0-ethoxycarbonyl-N00-isopropyl)-guanidino-
[1,10;40,100]terphenyl (8e)
The procedure described for 8a was adopted. Yield 89%,
3.3.11. 1,4-Bis-{50-[(N0,N00-t-butoxycarbonyl)-
guanidino]pyridin-20-yl}phenylene (5b)
The same procedure described for 5a was used starting with
4b. Yield 82%, m.p. >300 ꢀC. 1H NMR (CDCl3); d 1.51, 1.56
(2s, 36H), 7.79 (d, J ¼ 8.4 Hz, 2H), 8.07 (s, 4H), 8.28 (dd,
J ¼ 2.4, 8.4 Hz, 2H), 8.80 (d, J ¼ 2.4 Hz, 2H), 10.46 (s, 2H),
11.63 (s, 2H). Anal. Calc. for C38H50N8O8: C, 61.11; H,
6.75. Found C, 61.43; H, 6.69.
1
m.p. >300 ꢀC. H NMR (DMSO-d6): d 1.11e1.18 (m, 18H),
3.94 (q, J ¼ 7.2 Hz, 4H), 4.01e4.14 (m, 2H), 7.45 (d,
J ¼ 7.8 Hz, 4H), 7.68e7.75 (m, 8H), 9.24 (br s, 4H). 13C
NMR (DMSO-d6): d 163.40, 157.15, 138.22, 137.49, 135.21,
126.72, 126.57, 123.95, 59.58, 42.40, 22.54, 14.53. MS
(ESI) m/e (rel. int.): 573 (Mþ, 17), 287 (100). Anal. Calc.
for C32H40N6O4: C, 67.11; H, 7.03; N, 14.67. Found C,
66.95; H, 7.16; N, 14.37.
3.3.12. 1,4-Bis-[50-guanidinopyridin-20-yl]phenylene (6b)
The same procedure described for 6a was used starting with
5b. Yield 55%, m.p. 271e273 ꢀC. 1H NMR (D2O/DMSO-d6):
d 7.82 (s, 4H), 8.15e8.25 (m, 4H), 8.62 (s, 2H). 13C NMR
(DMSO-d6): d 156.4, 152.3, 144.7, 137.8, 133.7, 131.7,
126.9, 121.2. MS (ESI) m/e (rel. int.): 347 (Mþ þ 1, 25),
305 (40), 288 (5), 263 (7), 174 (100). Anal. Calc. for
C18H18N8e4.0HCle1.1H2Oe1.0C2H5OH: C, 43.04; H, 5.42;
N, 20.07. Found C, 43.03; H, 5.10; N, 19.78.
3.3.8. 4,400-Bis(N0-isopropyl)-guanidino-
[1,10;40,100]terphenyl (9e)
The procedure described for 9c was adopted. Yield 73%,
m.p. 246e247 ꢀC. 1H NMR (DMSO-d6):
d 1.11 (d,
J ¼ 6.3 Hz, 12H), 3.81e3.89 (m, 2H), 5.38 (br s, 6H), 6.87
(d, J ¼ 8.4 Hz, 4H), 7.53 (d, J ¼ 8.4 Hz, 4H), 7.74 (s, 4H).
13C NMR (DMSO-d6): d 150.95, 149.36, 138.23, 131.47,

R.K. Arafa et al. / European Journal of Medicinal Chemistry 43 (2008) 2901e2908
2907
3.3.13. 1,4-Bis-[50-(N0-ethoxycarbonylthiourea)pyridin-
20-yl]-phenylene (7b)
C30H38N8O4e0.5H2O: C, 61.73; H, 6.73; N, 19.19. Found
C, 61.75; H, 6.55; N, 18.87.
The same procedure described for 7a was used starting with
4a. Yield 89%, m.p. >300 ꢀC. 1H NMR (DMSO-d6); d 1.29 (t,
J ¼ 7.2 Hz, 6H), 4.25 (q, J ¼ 7.2 Hz, 4H), 8.02e8.20 (m, 8H),
8.80 (s, 2H), 11.12 (s, 2H), 11.51 (s, 2H). 13C NMR (DMSO-
d6); d 179.3, 153.0, 152.4, 145.4, 138.1, 133.9, 132.6, 126.4,
119.3, 61.7, 13.6. MS (ESI) m/e (rel. int.): 525 (Mþ, 10),
409 (25), 241 (40), 163 (100). Anal. Calc. for
C24H24N6O4S2: C, 54.95; H, 4.61. Found C, 54.76; H, 4.89.
3.3.18. 1,4-Bis-[50-(N0-isopropylguanidino)-pyridin-20-yl]-
phenylene (9f)
The procedure described for 9c was adopted. Yield 59%,
m.p. 259e260.5 ꢀC. 1H NMR (DMSO-d6): d 1.16 (d,
J ¼ 6.6 Hz, 12H), 3.88e3.96 (m, 2H), 6.30 (br s, 6H), 7.38
(dd, J ¼ 8.4, 2.1 Hz, 2H), 7.85 (d, J ¼ 8.4 Hz, 2H), 8.08 (s,
4H), 8.26 (d, J ¼ 2.1 Hz, 2H). 13C NMR (DMSO-d6):
d 152.3, 148.3, 144.7, 142.0, 138.1, 130.5, 125.7, 119.7,
42.0, 22.4. MS (ESI) m/e (rel. int.): 431 (Mþ, 100), 372 (5),
216 (60).
3.3.14. 1,4-Bis-{50-(N0-ethoxycarbonylguanidino)pyridin-20-
yl}phenylene (8b)
The procedure described for 8a was used. Yield 70%, m.p.
Hydrochloride salt of 9f. M.p. 278e280 ꢀC. Anal. Calc. for
C24H30N8e4.0HCle0.75H2O: C, 48.90; H, 6.06; N, 18.99.
Found C, 49.15; H, 6.09; N, 18.62.
1
>300 ꢀC. H NMR (DMSO-d6): d 1.19 (t, J ¼ 7.2 Hz, 6H),
4.03 (q, J ¼ 7.2 Hz, 4H), 7.53 (br s, 4H), 7.92e8.03 (m,
4H), 8.13 (s, 4H), 8.66 (s, 2H), 9.08 (br s, 2H). 13C NMR
(DMSO-d6): d 163.0, 155.4, 149.6, 142.3, 138.2, 135.4,
129.0, 126.2, 119.9, 59.8, 14.5. MS (ESI) m/e (rel. int.): 491
(Mþ þ 1, 40), 436 (5), 402 (10), 284 (10), 246 (100). Anal.
Calc. for C24H26N8O4e0.35H2O: C, 58.02; H, 5.41; N,
22.55. Found C, 58.05; H, 5.41; N, 22.20.
Acknowledgment
This work was supported by an award from the Bill and
Melinda Gates Foundation and NIH grants GM61587 and
AI46365.
3.3.15. 1,4-Bis-{50-[(N0-ethoxycarbonyl-N00-methyl)
guanidino]pyridin-20-yl}phenylene (8d)
The procedure used for 8a was adopted. Yield 65%, m.p.
References
1
>300 ꢀC; H NMR (DMSO-d6): d 1.14 (t, J ¼ 7.2 Hz, 6H),
[1] (a) R.R. Tidwell, D.W. Boykin, in: M. Demeunynck, C. Bailly,
W.D. Wilson (Eds.), Dicationic DNA Minor Groove Binders as Antimi-
crobial Agents, Small Molecule DNA and RNA Binders: From Synthesis
to Nucleic Acid Complexes, vol. 2, Wiley-VCH, New York, 2003, p. 414;
(b) W.D. Wilson, B. Nguyen, F.A. Tanious, A. Mathis, J.E. Hall,
C.E. Stephens, D.W. Boykin, Curr. Med. Chem. e Anti-Cancer Agents
5 (2005) 389;
2.92 (s, 6H), 3.95 (q, J ¼ 7.2 Hz, 4H), 7.93e8.18 (m, 12H),
8.71 (s, 2H). 13C NMR (DMSO-d6): d 163.2, 159.0, 144.7,
138.3, 134.2, 132.1, 126.4, 119.7, 59.6, 28.3, 14.6. MS (ESI)
m/e (rel. int.): 519 (Mþ, 40), 488 (5), 464 (10), 391 (145),
298 (20), 260 (100). Anal. Calc. for C26H30N8O4e
0.25C2H5OH: C, 60.04; H, 5.98; N, 21.13. Found C, 59.80;
H, 5.90; N, 20.91.
(c) M.N.C. Soeiro, E.M. de Souza, C.E. Stephens, D.W. Boykin, Expert
Opin. Invest. Drugs 14 (2005) 957;
(d) J.N. Ashley, H.J. Barber, A.J. Ewins, G. Newbery, A.D.H. Self,
J. Chem. Soc. (1942) 103.
[2] A.H. Fairlamb, Trends Parasitol. 19 (2003) 488.
3.3.16. 1,4-Bis-[50-(N0-methylguanidino)-pyridin-20-yl]-
phenylene (9d)
[3] B. Bouteille, O. Oukem, S. Bisser, M. Dumas, Fundam. Clin. Pharmacol.
17 (2003) 171.
The procedure described for 9c was used. Yield 58%, m.p.
262e264 ꢀC. 1H NMR (DMSO-d6): d 2.75 (s, 6H), 5.40 (br s,
6H), 7.21 (dd, J ¼ 8.4, 2.4 Hz, 2H), 7.80 (d, J ¼ 8.4 Hz, 2H),
8.06 (s, 4H), 8.12 (d, J ¼ 2.4 Hz, 2H). 13C NMR (DMSO-
d6): d 153.4, 146.7, 146.4, 144.8, 138.2, 130.2, 125.6, 119.9,
27.7. MS (ESI) m/e (rel. int.): 375 (Mþ þ 1, 10), 344 (10),
226 (25), 188 (100).
Hydrochloride salt of 9d. M.p. 292e294 ꢀC. Anal. Calc. for
C20H22N8e4.0HCle1.5H2Oe0.25C2H5OH: C, 44.06; H,
5.48; N, 20.05. Found C, 44.19; H, 5.18; N, 19.81.
[4] P.D. Yeramian, L.A. Castagnini, J.A. Allen, L. Umesh, E. Gotuzzo,
Efficacy and safety of DB289, a new oral drug for treatment of
pneumocystis carinii pneumonia (PCP) in AIDS patients, 43rd Annual
Interscience Conference on Antimicrobial Agents and Chemotherapy
Meeting, Chicago, IL, September 14e17, 2003.
[5] P. Yeramian, S. Krudsood, K. Chalermrut, U. Silachamroon, J. Allen, R.
Brun, S. Meshnick, R.R. Tidwell, S. Looareesuwan, Efficacy of DB289
in acute uncomplicated P. falciparum/P. vivax malaria, 52nd Annual
Meeting of the American Society of Tropical Medicine and Hygiene,
Philadelphia, PA, December 3e7, 2003.
[6] P. Yeramian, S.R. Meshnick, S. Krudood, K. Chalermurut,
U. Silachamroon, N. Tangukdee, J. Allen, R. Brun, J.J. Kweik,
R.R. Tidwell, S. Looareesuwan, J. Infect. Dis. 192 (2005) 319.
[7] C.C. Dykstra, D.R. McClernon, L.P. Elwell, R.R. Tidwell, Antimicrob.
Agents Chemother. 38 (1998) 1890.
3.3.17. 1,4-Bis-{50-[(N0-ethoxycarbonyl-N00-iso-propyl)
guanidino]pyridin-20-yl}-phenylene (8f)
1
Yield 64%, m.p. >300 ꢀC. H NMR (DMSO-d6): d 1.11e
[8] C. Bailly, L. Dassonneville, C. Carrascol, D. Lucasl, A. Kumar,
D.W. Boykin, W.D. Wilson, Anti-Cancer Drug Des. 14 (1999) 47.
[9] D.J. Fitzgerald, J.N. Anderson, J. Biol. Chem. 274 (1999) 27128.
[10] D. Henderson, L.H. Hurley, Nat. Med. 1 (1995) 525.
[11] (a) D. Goodsell, R.E. Dickerson, J. Med. Chem. 29 (1989) 245;
(b) M. Cory, R.R. Tidwell, T.A. Fairley, J. Med. Chem. 35 (1992) 431.
[12] A. Czarny, D.W. Boykin, A.A. Wood, C.M. Nunn, S. Neidle, M. Zhao,
W.D. Wilson, J. Am. Chem. Soc. 117 (1995) 4716.
1.21 (m, 18H), 3.95 (q, J ¼ 7.2 Hz, 4H), 4.05e4.12 (m, 2H),
7.86 (d, J ¼ 8.7 Hz, 2H), 8.01 (d, J ¼ 8.7 Hz, 2H), 8.17 (s,
4H), 8.63 (s, 2H), 9.25 (br s, 4H). 13C NMR (DMSO-d6):
d 162.7, 157.0, 150.2, 144.3, 138.1, 135.3, 131.2, 126.1,
119.4, 59.6, 42.4, 22.3, 14.2. MS (ESI) m/e (rel. int.): 575
(Mþ, 100), 529 (20), 503 (10). Anal. Calc. for

2908
R.K. Arafa et al. / European Journal of Medicinal Chemistry 43 (2008) 2901e2908
[13] S. Mazur, F.A. Tanious, D. Ding, A. Kumar, D.W. Boykin, I.J. Simpson,
S. Neidle, W.D. Wilson, J. Mol. Biol. 300 (2000) 321.
[25] F.A. Tanious, D. Ding, D.A. Patrick, R.R. Tidwell, W.D. Wilson,
Biochemistry 36 (1997) 15315.
[14] W.D. Wilson, F.A. Tanious, D. Ding, A. Kumar, D.W. Boykin, P. Colson,
C. Houssier, C. Bailly, J. Am. Chem. Soc. 120 (1998) 10310.
[15] D.W. Boykin, A. Kumar, G. Xiao, W.D. Wilson, B.C. Bender,
D.R. McCurdy, J.E. Hall, R.R. Tidwell, J. Med. Chem. 41 (1988) 124.
[16] D.W. Boykin, A. Kumar, J. Spychala, M. Zhou, R.L. Lombardy,
W.D. Wilson, C.C. Dykstra, S.K. Jones, J.E. Hall, R.R. Tidwell,
C. Laughton, C.M. Nunn, S. Neidle, J. Med. Chem. 38 (1995) 912.
[17] R. Brun, Y. Buhler, U. Sandmeier, R. Kaminsky, C.J. Bacchi, D. Rattendi,
S. Lane, S.L. Croft, D. Snowdon, V. Yardley, G. Caravatti, J. Frei,
J. Stanek, H. Mett, Antimicrob. Agents Chemother. 40 (1996) 1442.
[18] B. Nguyen, M.P.H. Lee, D. Hamelberg, A. Joubert, C. Bailly, R. Brun,
S. Neidle, W.D. Wilson, J. Am. Chem. Soc. 124 (2002) 13680.
[19] B. Nguyen, D. Hamelberg, C. Bailly, P. Colson, J. Stanek, R. Brun,
S. Neidle, D.W. Wilson, Biophys. J. 86 (2004) 1028.
[26] F.A. Tanious, D. Ding, D.A. Patrick, C. Bailly, R.R. Tidwell,
W.D. Wilson, Biochemistry 39 (2000) 12091.
[27] C.E. Stephens, F. Tanious, S. Kim, W.D. Wilson, W.A. Schell,
J.R. Perfect, S. Franzblau, D.W. Boykin, J. Med. Chem. 44 (2001)
1741.
[28] (a) C.E. Stephens, R. Brun, M.M. Salem, K.A. Werbovetz, F.A. Tanious,
W.D. Wilson, D.W. Boykin, Bioorg. Med. Chem. Lett. 13 (2003) 2065;
(b) R.K. Arafa, R. Brun, K.A. Werbovetz, F.A. Tanious, W.D. Wilson,
D.W. Boykin, Heterocycl. Commun. 10 (2004) 422.
[29] J. Oszczapowicz, in: S. Patai (Ed.), The Chemistry of Amidines and
Imidates, vol. 2, John Wiley & Sons, Chichester, 1991, pp. 623e688.
[30] Y. Yamamoto, S. Kojima, in: S. Patai (Ed.), The Chemistry of Amidines
and Imidates, vol. 2, John Wiley & Sons, Chichester, 1991, pp. 485e526.
[31] M.K. Saulner, D.B. Frennesson, M.S. Deshpande, S.B. Hansel,
D.M. Vyas, Bioorg. Med. Chem. Lett. 4 (1994) 1985.
[20] M.A. Ismail, R.K. Arafa, R. Brun, T. Wenzler, Y. Miao, W.D. Wilson,
C. Generaux, A. Bridges, J.E. Hall, D.W. Boykin, J. Med. Chem. 49
(2006) 5324.
[32] A. Schuster, G. Bernhardt, A. Buschauer, Eur. J. Pharm. Sci. 5 (1997) 79.
[33] W.G. Humphreys, M.T. Obermeier, S. Chong, S.D. Kimball, J. Das,
P. Chen, R. Moquin, W.-H. Han, R. Gedamke, R.E. White,
R.A. Morrison, Xenobiotica 33 (2003) 93.
[21] D.A. Patrick, D.W. Boykin, W.D. Wilson, F.A. Tanious, J. Spychala,
B.C. Bender, J.E. Hall, C.C. Dykstra, K.A. Ohemeng, R.R. Tidwell,
Eur. J. Med. Chem. 32 (1997) 781.
[34] J. Guan, Q. Zhang, G. Montip, J.M. Karle, C.A. Ditusa, W.K. Milhous,
D.R. Skillman, A.J. Lin, Bioorg. Med. Chem. 13 (2005) 699.
[35] J.L. Gonzalez, C.E. Stephens, T. Wenzler, R. Brun, F.A. Tanious,
W.D. Wilson, T. Barszez, K.A. Webovetz, D.W. Boykin, Eur. J. Med.
Chem. 42 (2007) 552.
[22] S.H. Wang, J.E. Hall, F.A. Tanious, W.D. Wilson, D.A. Patrick,
D.R. McCurdy, B.C. Bender, R.R. Tidwell, Eur. J. Med. Chem. 34
(1999) 215.
[23] D.A. Patrick, J.E. Hall, B.C. Bender, D.R. McCurdy, W.D. Wilson,
F.A. Tanious, S. Saha, R.R. Tidwell, Eur. J. Med. Chem. 34 (1999)
575.
[36] M.A. Ismail, A. Batista-Parra, Y. Miao, W.D. Wilson, T. Wenzler,
R. Brun, D.W. Boykin, Bioorg. Med. Chem. 13 (2005) 6718.
[37] M.A. Ismail, R. Brun, J.E. Easterbrook, F.A. Tanious, W.D. Wilson,
D.W. Boykin, J. Med. Chem. 46 (2003) 4761.
[24] R.K. Arafa, R. Brun, T. Wenzler, F.A. Tanious, W.D. Wilson,
C.E. Stephens, D.W. Boykin, J. Med. Chem. 48 (2005) 5480.