Molecular modeling study and synthesis of novel dicationic flexible triaryl guanidines and imidamides as antiprotozoal agents

Article abstract of DOI:10.1016/j.ejmech.2011.09.047

A new series of fourteen dicationic flexible triaryl bis-guanidines 3a,b, bis-N-substituted guanidines 7a,b and 8a,b as well as bis-imidamides 9-12a,b having a 1,3- or 1,4-diphenoxybenzene scaffold backbone were synthesized. The in vitro activity of the novel dications as antiprotozoal agents against Trypanosoma brucei rhodesiense (T.b.r.) and Plasmodium falciparum (P.f.) was assessed. Interestingly, six of the newly synthesized dications viz 3a,b, 7a,b and 8a,b were more active against P.f. than the reference drug pentamidine. Also, some of the dications showed moderate antitrypanosomal activity. Thermal melting analysis of the novel dications was performed to determine their ligand-DNA relative binding affinities. Finally, docking of the dications with an AT rich DNA oligonucleotide was executed to understand their binding mode with the minor groove.

Full text of DOI:10.1016/j.ejmech.2011.09.047

European Journal of Medicinal Chemistry 46 (2011) 5852e5860
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Molecular modeling study and synthesis of novel dicationic flexible triaryl
guanidines and imidamides as antiprotozoal agents
,
b
,
,
Reem K. Arafa a , Tanja Wenzler c, Reto Brun b c, Yun Chai d, W. David Wilson d
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini st., 11562, Cairo, Egypt
b Swiss Tropical and Public Health Institute, Basel CH4002, Switzerland
c University of Basel, Basel CH-4002, Switzerland
d Department of Chemistry, Georgia State University, Atlanta, GA 30303-3083, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of fourteen dicationic flexible triaryl bis-guanidines 3a,b, bis-N-substituted guanidines 7a,b
and 8a,b as well as bis-imidamides 9e12a,b having a 1,3- or 1,4-diphenoxybenzene scaffold backbone
were synthesized. The in vitro activity of the novel dications as antiprotozoal agents against Trypanosoma
brucei rhodesiense (T.b.r.) and Plasmodium falciparum (P.f.) was assessed. Interestingly, six of the newly
synthesized dications viz 3a,b, 7a,b and 8a,b were more active against P.f. than the reference drug
pentamidine. Also, some of the dications showed moderate antitrypanosomal activity. Thermal melting
analysis of the novel dications was performed to determine their ligand-DNA relative binding affinities.
Finally, docking of the dications with an AT rich DNA oligonucleotide was executed to understand their
binding mode with the minor groove.
Received 5 August 2011
Received in revised form
19 September 2011
Accepted 26 September 2011
Available online 2 October 2011
Keywords:
Dicationic flexible triaryl guanidines
Dicationic flexible triaryl imidamides
Antiprotozoal agents
Ó 2011 Elsevier Masson SAS. All rights reserved.
DNA minor groove binders
Thermal melting analysis
Molecular modeling and docking
1. Introduction
fused heteroaryl linkers have displayed promising antiprotozoal
activity [6,7]. We have recently reported linear triaryl diguanidi-
Protozoal manifestations caused by pathogenic parasites, and
more specifically by those hemoflagellates predisposing to human
African trypanosomiasis (HAT), leishmaniasis and malaria have
always afflicted mankind. For the management of such infections,
many therapeutic strategies have been explored including the use of
aryl or heteroaryl dicationic minor groove binders. Up till now,
pentamidine (I) Fig. 1), currently designated by FDA as an orphan
drug, is the only aryl dicationic minor groove binder clinically
available for control of first stage HAT, antimony-resistant leish-
maniasis and also as a secondary drug for AIDS-related Pneumocystis
jirovecii pneumonia (PCP) [1,2]. Furamidine (II) (Fig. 1), a diphenyl
furan diamidinium analogue of pentamidine, has demonstrated
better potency and toxicity profiles in murine models of trypano-
somiasis [3]. On the other hand, many investigations have shown
that introduction of other cationic centers viz guanidine and imi-
damides, formerly known as “reversed amidines”, to classical groove
binding frameworks, furamidine like, can lead to effective antimi-
crobial agents [4,5]. Also, benzguanidines connected by alkyl or
niums and N-substituted guanidiniums (III) Fig. 1) as promising
antiprotozoal agents against Trypanosoma brucei rhodesiense (T.b.r.)
and Plasmodium falciparum (P. f.) [8]. The mode of action of such
compounds, though still not certain, is believed to involve binding to
DNA minor groove at AT rich sites with subsequent inhibition of DNA
dependent enzymes and direct inhibition of transcription [9,10].
It was earlier believed that activity resides in crescent shaped
dications that match the curvature of the minor groove [1,11]. Yet, it
has been proven that linear analogues like CGP40215 (IV) (Fig.1) and
linear triaryl dications (e.g. amidines, guanidines and imidamides)
exhibit strong DNA minor groove binding by virtue of integration of
water molecule(s) with the drug-DNA complex [12e14].
Based on the previous findings, it was deemed of interest to
explore the effect of introducing some flexibility to the triaryl
spacer moiety between the dicationic centers on the DNA binding
affinity and the antiprotozoal activity of such potential drug
molecules. For that, fourteen novel dications comprising bis-
guanidines 3a,b, bis-N-substituted guanidines 7a,b and 8a,b
as well as bis-imidamides 9e12a,b having
a 1,3- or 1,4-
diphenoxybenzene backbone were synthesized. The novel
compounds were screened against T.b.r. and P. f. to determine their
in vitro antiprotozoal activity. Furthermore, a thermal melting study
* Corresponding author. Tel.: þ2 (010) 207 4028; fax: þ2 (023) 362 8426.
E-mail address: rkarafa@cu.edu.eg (R.K. Arafa).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.047

R.K. Arafa et al. / European Journal of Medicinal Chemistry 46 (2011) 5852e5860
5853
O
O
NH
HN
H2N
O
HN
NH
NH2
NH2
NH2
furamidine (II)
pentamidine (I)
NR
HN
RN
NH
NH2
H2N
NH2
HN
N
N
NH
NH2
N
N
H
NH2
X
X
CGP 40215 (IV)
III
X = CH, N
R = H, Me, i-Pr
Fig. 1. Structures of key dicationic antiprotozoan agents.
of all the new compounds was performed to assess their ligand-
DNA relative binding affinities. Finally, minor groove binding abil-
ities of the synthesized compounds was assessed by evaluation of
their binding modes and energies with a short AT rich DNA olig-
omer by utilizing docking techniques.
Finally, flexible triaryl imidamides were prepared as depicted in
Scheme 3 where the diamines (1a or 1b) were reacted with cyclo-
hexyl-, phenyl-, 4-t-butylphenyl- or biphenyl-S-(2-naphthylmethyl)
thioimidate to provide the target compounds (9e12a,b), respectively
[5,6,18].
2. Results and discussion
2.2. Biology
2.1. Chemistry
In vitro activities. The in vitro antiprotozoal activity against T.b.r.
and P.f. as well as the cytotoxicity of all the newly synthesized
flexible triaryl dications was assessed (data listed in Table 1). For
comparative purposes the analogous data for pentamidine are also
included.
The target flexible triaryl unsubstituted bis-guanidines 3a,b
were synthesized in two steps as depicted in Scheme 1 starting
from the corresponding commercially available 4,40-(1,3- or 1,4-
phenylenebis(oxy))dianiline derivatives 1a and 1b, respectively.
The first step comprises the preparation of the intermediate bis-
Boc-protected guanidines 2a,b by the use of bis-Boc-protected
N-methylpseudothiourea and HgCl2. Subsequent treatment of
2a,b with ethanolic HCl provided the twofold purpose of guanidine
deprotection, as well as formation of the bis-guanidine dihydro-
chloride salts 3a,b [4e7].
Scheme 2 outlines the synthesis of flexible triaryl N-substituted
bis-guanidines 7a,b and 8a,b. Starting with the appropriate
diamine (1a or 1b), reaction with ethyl isothiocyanatoformate gave
the carbamoyl thioureas (4a, b), which were further reacted with
methylamine or i-propylamine in the presence of EDCI to furnish
the carbamoyl guanidines 5a,b and 6a,b, respectively [15e17].
These synthetic intermediates were decarbamoylated through base
catalyzed hydrolysis of the ester moiety with simultaneous decar-
boxylation to obtain the N-alkylguanidines 7a,b and 8a,b.
Even as none of the new dications displayed better anti-
trypanosomal activity against T.b.r. compared to the reference drug
pentamidine (IC50 ¼ 2.2 nM), most of these novel compounds
exhibited much better antimalarial potencies against P.f. compared to
pentamidine (IC50 ¼ 46.4 nM). All the guanidines 3a,b, N-methyl
guanidines 7a,b and N-isopropyl guanidines 8a,b displayed the
highest potency in this series with IC50 values between 14 and 38 nM
being 3.2 to 1.2 times as active than pentamidine. In general, the 1,4-
phenylenebis(oxy)dianiline derivatives showed higher potencies
against P.f. than the corresponding 1,3- isomers. Also, the
N-substituted guanidines were more potent than the parent unsub-
stituted ones with the N-methyl analogues displaying higher anti-
malarial activity than the N-isopropyl counterparts. The
bis(N-methyl)guanidine (1,4-phenylenebis(oxy)dianiline derivative)
7b not only exhibited the highest antimalarial activity in the series
with an IC50 value of 14 nM but also demonstrated the second highest
O
O
(a)
NBoc
NHBoc
O
O
NH2
N
H
BocN
1a,b
2a,b
(b)
H2N
NH
BocHN
O
Legend for Compounds 1-3a,b
NH
NH2.HCl
O
= 1,3- isomers
= 1,4- isomers
a
b
N
H
HN
NH
3a,b
HCl.H2N
(a) Bis-Boc protected S-methylpseudothiourea, HgCl , TEA, DMF, r.t., overnight;
(b) HCl (g), EtOH, CH2Cl2, r.t., 3 days
Reagents and condtions:
2
Scheme 1. Synthesis of flexible triaryl unsubstituted bis-guanidines.

5854
R.K. Arafa et al. / European Journal of Medicinal Chemistry 46 (2011) 5852e5860
O
O
(a)
S
O
O
NH2
N
H
NHCO2Et
S
1a,b
4a,b
H2N
NH
EtO2CHN
(b)
O
O
NR
NH2
NR
NHCO2Et
O
O
N
H
(c)
N
H
RN
H2N
RN
NH
7,8a,b
NH
5,6a,b
EtO2CHN
Legend for Compounds 1a,b and 4-8a,b
R = Me
R = i-Pr
5,7
6,8
= 1,3- isomers
a
= 1,4- isomers
b
(a) ethyl isothiocyanatoformate, CH Cl , r.t., overnight; (b) RNH , DIPEA, EDCI,
Reagents and condtions:
2
2
2
CH2Cl2, r.t., overnight; (c) aq. alc KOH, 55 oC, 10 h
Scheme 2. Synthesis of flexible triaryl n-substituted bis-guanidines.
P.f. compared to T.b.r. selectivity amongst the tested guanidines
(SI ¼ 11) with compound 7a possessing the highest selectivity
(SI ¼ 14.8).
the imidamides displayed similar to better cytotoxicity profiles
when compared to pentamidine with the biphenyl derivative 12b
being almost 60 fold less cytotoxic.
On the other hand, three of the imidamides class of dications
showed moderate potency against P.f. with IC50 values 60e75 nM.
Interestingly, it was the 1,4-phenylenebis(oxy)dianiline derivatives
that demonstrated antimalarial activity while the 1,3- isomers were
devoid of such activity potentially due to their unfavorable geom-
etry and lack of complementarity with the minor groove curvature
causing steric clashing with the minor groove floor. Also, whilst the
arylimidamides 10b, 11b and 12b displayed moderate antimalarial
potency, the cyclohexylimidamide 9b displayed none. This may
suggest that the presence of an aromatic ring, even if large in size
(c.f. biphenyl in 12b), is well tolerated and probably required due to
2.3. Relative binding affinity:
DTm measurements
Dicationic compounds have been known to target the DNA minor
groove. Tm increases for ligand-DNA complexes relative to uncom-
plexed DNA (DTm) at specific DNA sequences provide an excellent
method for ranking compound binding affinities. In this study, Tm
values for the prepared flexible dications were determined by their
addition to an AT polymer and data obtained are listed in Table 1.
Unexpectedly, our charged compounds displayed DTm values in the
the potential formation of
p
e
p
stacking interactions with the
range of 5 to 0 ꢀC reflecting poor to absolutely no minor groove
binding affinities (Table 1). These surprising findings are suggested
to be a consequence of an over-acute curvature of the dicationic
linker 1,3- or 1,4-diphenoxyphenyl backbone which reduces
complementarity with the curvature of the DNA minor groove and
hence reducing the possibility of formation of the non-covalent
binding interactions necessary for formation of the ligand-DNA
complex.
minor groove. However, the presence of the non-aromatic non-
planar cycloalkyl (c.f. 9b) does not help the biological activity due to
lack of such an interaction.
Furthermore, all the tested guanidines and N-substituted
guanidines were 5 to 39 fold less cytotoxic when compared to
pentamidine with the most active analogue against P.f., 7b, having
27 folds lower cytotoxicity. Also, the biologically active members of
O
O
(a)
NH
R1
O
O
NH2
N
H
HN
R1
1a,b
9-12a,b
H2N
NH
Legend for Compounds 1a,b and 9-12a,b
R = cyclohexyl
R = phenyl
1
1
9
10
R = biphenyl
12
1
R = 4-t-butylphenyl
11
1
= 1,3- isomers
= 1,4- isomers
a
b
NH.HBr
R1
S
(a)
, EtOH, MeCN, r.t., overnight;
Reagents and condtions:
Scheme 3. Synthesis of flexible triaryl bis-imidamides.

R.K. Arafa et al. / European Journal of Medicinal Chemistry 46 (2011) 5852e5860
5855
Table 1
In vitro antiprotozoal activity and cytotoxicity for flexible dicationic triaryl guanidines and imidamides.a
Code
Substitution
R
R1
D
Tma poly dA-dT
T.b.r.
P.f.
SIc
Cytotoxicity
IC50 (nM)b
IC50 (nM)b
IC50 (m
M)d
Pentamidine I
3a
3b
7a
7b
8a
8b
9a
e
e
e
12.6
1.1
2.1
0.1
0.8
0.0
0.0
0.0
2.1
2.0
5.0
0.0
0.0
0.0
0.1
2.2
171
151
318
155
155
160
395
427
1042
863
2779
400
82,300
612
46.4
27.0
26.5
21.5
14.1
38.2
17.2
307
0.05
2.1
10.6
11.6
45.7
57.3
82.1
19.6
3.9
3.1
0.9
2.8
81.4
1.5
1,3- (meta)
1,4- (para)
1,3- (meta)
1,4- (para)
1,3- (meta)
1,4- (para)
1,3- (meta)
1,4- (para)
1,3- (meta)
1,4- (para)
1,3- (meta)
1,4- (para)
1,3- (meta)
1,4- (para)
H
H
NH2
NH2
NH2
NH2
NH2
NH2
cyclohexyl
cyclohexyl
Phenyl
Phenyl
4-t-butylphenyl
4-t-butylphenyl
Biphenyl
Biphenyl
6.3
5.7
14.8
11
4.1
9.3
1.3
1.6
4.2
12.1
4.4
6.7
17
Me
Me
i-Pr
i-Pr
H
H
H
H
H
9b
259
251
10a
10b
11a
11b
12a
12b
71.5
627
60.0
4698
63.3
H
H
H
9.1
>120
9.6
a
Buffer: MES10; ratio (compound/DNA): 0.3.
T.b.r. strain used was STIB900 and the P. f. strain was K1, values are of duplicate determinations; see Ref. [21].
Selectivity Index (IC50 T.b.r./IC50 P.f.).
b
c
d
Cytotoxicity was evaluated using cultured L-6 rat myoblast cells using an Alamar Blue assay; see Ref. [21].
2.4. Molecular modeling
the possible reason behind the poor DNA binding of this class of
dications with the DNA minor groove, docking study was performed
using MOE software Version 2008.10. The X-ray crystal structure of
the selective antiprotozoal agent pentamidine bound to the minor
groove of DNA dodecamer d(CGCGAATTCGCG)2 was obtained from
the RCSB Protein Data Bank (PDB ID: 1d64) [20] and employed for
this study. The docking study was initiated by a validation step
through docking of pentamidine in its active site, and comparing the
results with the binding mode determined experimentally through
X-ray crystallography with the docked ligand. MOE was successful in
reproducing the binding position for pentamidine, showing a RMS
deviation of 0.51Å (Fig. 2). According to the crystal structure [20],
pentamidine establishes several hydrogen bonding interactions with
neighboring A and T base-pairs on both strands of the DNA minor
groove a couple being mediated through a water bridge as shown in
Fig. 2.
Dications are well reported to bind to DNA minor groove and in-
silico screening has proved to be efficient in displaying and
explaining such interactions [19]. In an attempt to better understand
After removing pentamidine from the complex, virtual
screening of each of the new dications was done through docking
with the short DNA dodecamer d(CGCGAATTCGCG)2 to predict the
mode of drug-DNA binding using MOE default parameters for all
the variables adopting flexible-ligand rigid-receptor approach. The
most stable docking solutions of the flexible guanidines and imi-
damides complexed with the DNA oligonucleotide along were
determined.
The binding profile of the synthesized dications was compared
with that of pentamidine molecule. Unexpectedly, the ligands were
found to mainly interact with the DNA oligonucleotide through the
formation of hydrogen bond(s) between only one of the dicationic
centres and the DNA base pair(s) while the other dicationic group
points out of the minor groove. An exemplary illustration for this
binding mode is displayed for the dication with the highest anti-
plasmodial activity in this study, 7b, in Fig. 3 showing the 3D (panel
a) and 2D (panel b) ligand binding interactions of this compound
with the receptor DNA oligomer in comparison with the X-ray
Fig. 2. Docking validation and binding mode of pentamidine in the minor groove of
the polydA.polydT DNA oligonucleotide: crystal structure ligand (blue) docked ligand
(yellow); RMSD 0.51 Å. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

5856
R.K. Arafa et al. / European Journal of Medicinal Chemistry 46 (2011) 5852e5860
Fig. 3. Panel (a): 3D Binding interaction of 7b (yellow) in the minor groove of the polydA.polydT DNA compared to that of pentamidine (blue); panel (b): overlay of 7b (green) and
pentamidine (red) showing their 2D binding interaction in the minor groove of the polydA.polydT DNA. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
crystal ligand pentamidine. This behaviour of this class of dications
can be possibly explained in terms of the comparatively over-acute
curvature of these compounds, especially the 1,3-analogues,
compared to pentamidine. Docking studies showed that for these
derivatives to engage both dicationic moieties in their binding
interactions they have to over arch and twist in an energetically
unfavourable conformation with the triaryl backbone protruding
out of the DNA minor groove (data not shown). This positioning is
apparently not thermodynamically preferred by the molecules as
reflected in the high conformational energy of such orientations
(Econf in MOE) leading to overall instability of the ligand-DNA
complex.
Thus, docking studies were useful in showing that some of these
dications prefer to bind to DNA as monocations and attain a more
thermodynamically relaxed conformation which rationalizes the
unexpected experimental findings of the thermal melting analysis
that revealed poor to no DNA interactions.
backbone against P.f., some being even more active than the
reference drug pentamidine, suggests that antiplasmodial activity
may be only partly dependent on minor groove binding abilities
and there may exist another mode of action through which these
cations display their activity that is yet unknown and needs to be
further explored.
4. Experimental
4.1. Synthetic protocols
Melting points were recorded using a Thomas-Hoover (Uni-Melt)
capillary melting point 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 Varian Unity Plus 300 spectrometer, and chemical shifts
(d) are in ppm relative toTMS as internal standard. Mass spectra were
recorded on a VG analytical 70-SE spectrometer (EI) or a Thermo-
Finningan LCQ MSD (ESI). Elemental analyses were obtained from
Atlantic Microlab Inc. (Norcross, GA). Some compounds were
analyzed correctly for fractional moles of water and/or ethanol of
solvation. In each case 1H NMR showed the presence of the indicated
solvent(s). All chemicals and solvents (including anhydrous solvents)
were purchased from Aldrich Chemical Co. or Lancaster Synthesis
and used as purchased. Acetonitrile and triethylamine were distilled
from CaH2. Substituted S-(2-naphthylmethyl)thioimidates were
prepared adopting the reported procedure [4].
3. Conclusion
The current study portrays the synthesis of fourteen novel
dicationic flexible triaryl guanidines and imidamides. The in vitro
antiprotozoal testing of the compounds against T.b.r. and P.f.
showed that the new compounds had poor antitrypanosomal
activity. However, the dicationic guanidiniums 3a,b; 7a,b and 8a,b
showed high antiprotozoal activity profiles against P.f. Also, the
imdamides with a 1,4-diphenoxyphenyl dicationic groups bearing
scaffold displayed reasonable antiplasmodial activity. On the other
hand, thermal melting analysis experiments surprisingly showed
that this series of dications bind poorly to the minor groove of
DNA. For that, docking techniques were adopted in a trial to
predict the binding mode of the tested compounds with the DNA
minor groove. When the results of the thermal melting and
docking studies are considered together with the biological
screening data, it can be suggested that: (i) the dications especially
those with the 1,3-diphenoxyphenyl backbone bind very poorly to
4.1.1. Preparation of Bis(N0,N00-di-bocguanidino) derivatives
(general procedure)
1,10-(4,40-(1,3-phenylenebis(oxy))bis(4,1-phenylene))-N0,N00-di-
Bocguanidine (2a). To a solution of 4,40-(1,3-phenylenebis(oxy))
dianiline (1a) (0.5 g, 1.71 mmol) in anhydrous DMF (10 mL) was
added 1,3-bis(tert-butoxycarbonyl)-2-methylthiopseudourea (1.05 g,
3.62 mmol), triethylamine (1.08 g, 10.74 mmol) and mercury(II)
chloride (1.07 g, 3.96 mmol). The suspension was kept stirring at
room-temperature overnight. The reaction mixture was diluted with
CH2Cl2, washed with Na2CO3 solution, and filtered through a pad of
Celite. The organic layer was washed with water (3ꢁ) followed by
brine and then dried over anhydrous Na2SO4. After evaporating the
solvent the obtained residue was crystallized from CH2Cl2/MeOH
DNA, as reflected in their
DTm values and docking contours,
probably due to over-acute curvature and hence inadequate
complementarity with the minor groove; (ii) poor activity of these
dications against T.b.r. can possibly be explained in terms of lack of
minor groove binding affinity; finally (iii) the activity of the gua-
nidiniums and those imidamides with a 1,4-diphenoxyphenyl
giving a white solid (1.19 g, 89%), mp >300 ꢀC; 1H NMR (CDCl3)
d 1.50,

R.K. Arafa et al. / European Journal of Medicinal Chemistry 46 (2011) 5852e5860
5857
1.54 (2s, 36H, 4ꢁ C(CH3)3), 6.67e6.70 (m, 3H, Ar-H), 6.98 (d, J ¼ 9 Hz,
4H, Ar-H), 7.22 (t, J ¼ 8.1 Hz, 1H, Ar-H), 7.57 (d, J ¼ 9 Hz, 4H, Ar-H),
10.29 (br s, 2H, 2ꢁ NH), 11.64 (br s, 2H, 2ꢁ NH). MS (EI) m/z (rel.
int.) 777 (Mþ þ 1, 92), 277 (100). Anal. Calcd. for C40H52N6O10: C,
61.82; H, 6.75; N, 10.82. Found: C, 61.66; H, 6.66; N, 10.73.
(calcd. for C26H27N4O6S2: 555.1372). Anal. Calcd. for C26H26N4O6S2: C,
56.30; H, 4.72; N, 10.10. Found: C, 56.08; H, 4.54; N, 9.78.
4.1.6. 1,10-(4,40-(1,4-Phenylenebis(oxy))bis(4,1-phenylene))bis(N0-
ethoxycarbonylthiourea) (4b)
The procedure described for 4a was adopted starting with 1b
(1 g, 3.42 mmol) to furnish the bis-carbamoylthiourea as a yellow
solid in quantitative yield; m.p. 236-8 ꢀC; 1H NMR (DMSO-d6):
4.1.2. 1,10-(4,40-(1,4-phenylenebis(oxy))bis(4,1-phenylene))-N0,N00-di-
Bocguanidine (2b)
The general procedure was adopted using 4,40-(1,4-
phenylenebis(oxy))dianiline (1b) to furnish 2b as a white solid
d
1.27 (t, J ¼ 7.2 Hz, 6H, 2 X CH2CH3), 4.22 (q, J ¼ 7.2 Hz, 4H, 2ꢁ
CH2CH3), 7.02 (d, J ¼ 9 Hz, 4H, Ar-H), 7.07 (s, 4H, Ar-H), 7.56 (d,
J ¼ 9 Hz, 4H, Ar-H), 10.98 (br s, 2H, 2ꢁ NH), 11.40 (br s, 2H, 2ꢁ NH).
(1.27 g, 95%), mp >300 ꢀC; 1H NMR (CDCl3)
d
1.50, 1.55 (2s, 36H, 4ꢁ
C(CH3)3), 6.95e6.98 (m, 8H, Ar-H), 7.56 (d, J ¼ 8.7 Hz, 4H, Ar-H),
13C NMR (DMSO-d6)
d 178.58, 154.89, 153.26, 152.05, 133.13, 126.01,
10.28 (br s, 2H, 2ꢁ NH), 11.65 (br s, 2H, 2ꢁ NH). 13C NMR (CDCl3)
120.31, 117.73, 61.74, 13.80. MS (ESI) m/e (rel. int.): 555 (Mþþ1, 41),
509 (100). Anal. Calcd. for C26H26N4O6S2: C, 56.30; H, 4.72; N, 10.10.
Found: C, 55.91; H, 4.52; N, 9.86.
d
163.78, 154.72, 153.79, 153.56, 153.06, 132.21, 124.01, 120.36,
118.98, 83.90, 79.81, 28.36. MS (EI) m/z (rel. int.) 777 (Mþ þ 1, 28),
677 (100). Anal. Calcd. for C40H52N6O10: C, 61.82; H, 6.75; N, 10.82.
Found: C, 61.53; H, 6.80; N, 10.76.
4.1.7. 1,10-(4,40-(1,3-phenylenebis(oxy))bis(4,1-phenylene))bis(N0-
ethoxycarbonyl-N00-methylguanidine) (5a)
A stirred solution of carbamoyl-thiourea 4a (1.25 g, 2.25 mmol),
methylamine hydrochloride (0.73 mgl, 9 mmol), and diisopropy-
lethylamine (1.73 g, 15 mmol) in anhydrous DMF (10 mL) was
cooled to 0 ꢀC. EDCI (1.73 g, 9 mmol) was added, and the solution
was stirred at room-temperature over night. The reaction mixture
was poured onto ice/water. The solid obtained was filtered, washed
with water (3 ꢁ 100 mL) and dried. The residue remaining after
removal of the solvent was crystallized from DMF/water, yield 86%;
4.1.3. Deprotection of N0,N00-di-bocguanidines (general procedure)
(Scheme 1)
1,10-(4,40-(1,3-phenylenebis(oxy))bis(4,1-phenylene))diguani-
dine (3a). The N0,N00-di-Bocguanidine (2a) (0.75 g, 0.96 mmol) was
dissolved in CH2Cl2 (10 mL), diluted with dry EtOH (15 mL) and the
chilled solution was saturated with dry HCl. The reaction mixture
was then kept stirring at room temperature for 3 days (drying
tube), where a precipitate of the product started forming overtime.
After evaporating the solvent to dryness, the residue was washed
with ether multiple times and was dried under reduced pressure at
50e60 ꢀC overnight to give white solid of the bis-guanidine dihy-
mp 97-8 ꢀC; 1H NMR (DMSO-d6):
d
1.13 (t, J ¼ 7.2 Hz, 6H, 2ꢁ
CH2CH3), 2.81 (s, 6H, 2ꢁ CH3), 3.92 (q, J ¼ 7.2 Hz, 4H, 2ꢁ CH2CH3),
6.62 (t, J ¼ 2.4 Hz, 1H, Ar-H), 6.71 (dd, J ¼ 2.4, 8.4 Hz, 2H, Ar-H), 7.03
(d, J ¼ 8.7 Hz, 4H, Ar-H), 7.32e7.37 (m, 5H, Ar-H), 11.20 (br s, 2H, 2ꢁ
NH), 11.40 (br s, 2H, 2ꢁ NH). MS (ESI) m/e (rel. int.): 459 (Mþþ1,
100), 275 (34). Anal. Calcd. for C28H32N6O6: C, 61.28; H, 5.88; N,
15.32. Found: C, 61.07; H, 5.95; N, 15.10.
drochloride (0.13 g), mp 146e8 ꢀC; 1H NMR (DMSO-d6)
d 6.68 (t,
J ¼ 2.4 Hz, 1H, Ar-H), 6.69 (dd, J ¼ 2.4, 8.1 Hz, 2H, Ar-H), 7.11 (d,
J ¼ 8.7 Hz, 4H, Ar-H), 7.25 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.38 (t, J ¼ 8.1 Hz,
1H, Ar-H), 7.51 (br s, 8H, 2ꢁ NH2 þ 4ꢁ NH), 10.01 (br s, 2H, 2ꢁ HCl);
13C NMR (DMSO-d6)
d 157.97, 156.43, 154.35, 130.95, 130.63, 126.89,
4.1.8. 1,10-(4,40-(1,4-phenylenebis(oxy))bis(4,1-phenylene))bis(N0-
ethoxycarbonyl-N00-methylguanidine) (5b)
119.96, 113.05, 108.46. HRMS m/z 377.1730 (Mþ þ 1) (calcd. for
C
20H21N6O2: 377.1726). Anal. Calcd. for C20H20N6O2-2HCl-0.5H2O-
The procedure used for preparation of 5a was adopted starting
0.25C2H5OH: C, 52.40; H, 5.25; N, 17.88. Found: C, 52.41; H, 5.24; N,
17.60.
with 4b (1 g, 1.80 mmol), yield 83%; mp 91-2 ꢀC; 1H NMR (DMSO-
d6):
d
1.13 (t, J ¼ 7.2 Hz, 6H, 2ꢁ CH2CH3), 2.81 (s, 6H, 2ꢁ CH3), 3.92
(q, J ¼ 7.2 Hz, 4H, 2ꢁ CH2CH3), 6.97 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.05 (s,
4H, Ar-H), 7.31 (d, J ¼ 8.7 Hz, 4H, Ar-H),10.82 (br s, 2H, 2ꢁ NH),11.33
(br s, 2H, 2ꢁ NH). MS (ESI) m/e (rel. int.): 459 (Mþþ1, 100), 275 (30).
HRMS m/z 549.2455 (Mþ þ 1) (calcd. for C28H33N6O6: 549.2462).
Anal. Calcd. for C28H32N6O6: C, 61.28; H, 5.88; N, 15.32. Found: C,
61.08; H, 5.61; N, 15.54.
4.1.4. 1,10-(4,40-(1,4-phenylenebis(oxy))bis(4,1-phenylene))
diguanidine (3b)
The general procedure was adopted starting with 2b, mp 250-
2
ꢀC; 1H NMR (DMSO-d6)
d 7.04e7.09 (m, 8H, Ar-H), 7.25
(d, J ¼ 8.7 Hz, 4H, Ar-H), 7.58 (br s, 8H, 2ꢁ NH2 þ 4ꢁ NH), 10.02
(br s, 2H, 2ꢁ HCl); 13C NMR (DMSO-d6)
d 156.36, 155.68, 152.26,
130.36, 127.23, 120.60, 119.18. HRMS m/z 377.1720 (Mþ þ 1)
(calcd. for C20H21N6O2: 377.1726). Anal. Calcd. for C20H20N6O2-
2HCl-0.4H2O: C, 52.61; H, 5.03; N, 18.40. Found: C, 52.64; H, 5.03;
N, 18.11.
4.1.9. 1,10-(4,40-(1,3-phenylenebis(oxy))bis(4,1-phenylene))bis(N0-
ethoxycarbonyl-N00-isopropylguanidine) (6a)
The procedure used for preparation of 5a was adopted starting
with 4a and using isoprpylamine, yield 88%; mp 98-9 ꢀC; 1H NMR
(DMSO-d6):
d
1.09e1.15 (m, 18H, 2ꢁ CH2CH3 þ 2ꢁ CH(CH3)2), 3.90
4.1.5. Preparation of Bis(N00-Substitutedguanidino)Derivatives
(general procedure)
(q, J ¼ 7.2 Hz, 4H, 2ꢁ CH2CH3), 4.01e4.09 (m, 2H, 2ꢁ CH(CH3)2),
6.60 (t, J ¼ 2.4 Hz, 1H, Ar-H), 6.71 (dd, J ¼ 2.4, 8.4 Hz, 2H, Ar-H), 7.02
(d, J ¼ 8.7 Hz, 4H, Ar-H), 7.32e7.34 (m, 5H, Ar-H), 11.20 (br s, 2H, 2ꢁ
NH), 11.40 (br s, 2H, 2ꢁ NH). MS (ESI) m/e (rel. int.): 605 (Mþþ1,
100). HRMS m/z 605.3080 (Mþ þ 1) (calcd. for C32H41N6O6:
605.3088). Anal. Calcd. for C32H40N6O6: C, 63.54; H, 6.67; N, 13.90.
Found: C, 63.14; H, 6.57; N, 13.61.
1,10-(4,40-(1,3-Phenylenebis(oxy))bis(4,1-phenylene))bis(N0-
ethoxycarbonylthiourea) (4a). Ethyl isothiocyanatoformate (1.86 g,
15.04 mmol) was added to a solution of 1a (2 g, 6.84 mmol) in CH2Cl2
(10 mL) and the mixture was stirred at room-temperature for 24 h.
The reactionwas diluted with hexane and the precipitate formed was
collected and dried to yield the bis-carbamoylthiourea as a white
solid in quantitative yield; m.p. 174-6 ꢀC; 1H NMR (DMSO-d6):
d 1.25
4.1.10. 1,10-(4,40-(1,4-phenylenebis(oxy))bis(4,1-phenylene))bis(N0-
ethoxycarbonyl-N’00-isopropylguanidine) (6b)
(t, J ¼ 7.2 Hz, 6H, 2ꢁ CH2CH3), 4.20 (q, J ¼ 7.2 Hz, 4H, 2ꢁ CH2CH3), 6.66
(t, J ¼ 2.4 Hz, 1H, Ar-H), 6.75 (dd, J ¼ 2.4, 8.4 Hz, 2H, Ar-H), 7.06 (d,
J ¼ 8.7 Hz, 4H, Ar-H), 7.38 (t, J ¼ 8.4 Hz,1H, Ar-H), 7.57 (d, J ¼ 8.7 Hz, 4H,
Ar-H), 11.22 (br s, 2H, 2ꢁ NH), 11.45 (br s, 2H, 2ꢁ NH). MS (ESI) m/e
(rel. int.): 555 (Mþþ1, 100), 360 (17). HRMS m/z 555.1352 (Mþ þ 1)
The procedure used for preparation of 5a was adopted starting
with 4b and using isoprpylamine, yield 89%; mp 93-5 ꢀC; 1H NMR
(DMSO-d6):
d
1.09e1.15 (m, 18H, 2ꢁ CH2CH3 þ 2ꢁ CH(CH3)2), 3.90
(q, J ¼ 7.2 Hz, 4H, 2ꢁ CH2CH3), 4.01e4.09 (m, 2H, Ar-H), 6.97 (d,

5858
R.K. Arafa et al. / European Journal of Medicinal Chemistry 46 (2011) 5852e5860
J ¼ 8.4 Hz, 4H, Ar-H), 7.04 (s, 4H, Ar-H), 7.31 (d, J ¼ 8.4 Hz, 4H, Ar-H),
10.84 (br s, 2H, 2ꢁ NH), 11.30 (br s, 2H, 2ꢁ NH). MS (ESI) m/e
(rel. int.): 605 (Mþþ1, 100). HRMS m/z 605.3097 (Mþ þ 1) (calcd. for
4.1.14. 1,10-(4,40-(1,4-phenylenebis(oxy))bis(4,1-phenylene))bis(N’-
isopropylguanidine) (8b)
The procedure described for 7a was adopted starting with 6b,
C
32H41N6O6: 605.3088). Anal. Calcd. for C32H40N6O6: C, 63.54; H,
yield 96%; mp 203-5 ꢀC; 1H NMR (DMSO-d6):
d
1.08 (d, J ¼ 6.3 Hz,
6.67; N, 13.90. Found: C, 63.48; H, 6.41; N, 13.54.
12H, 2ꢁ CH(CH3)2), 3.78e3.86 (m, 2H, 2ꢁ CH(CH3)2), 3.35 (br s, 2H,
2ꢁ NH), 5.15 (br s, 4H, 2ꢁ NH2), 6.73 (d, J ¼ 8.4 Hz, 4H, Ar-H), 6.83
(d, J ¼ 8.4 Hz, 4H, Ar-H), 6.92 (s, 4H, Ar-H).
4.1.11. 1,10-(4,40-(1,3-phenylenebis(oxy))bis(4,1-phenylene))bis(N0-
methylguanidine) (7a)
Hydrochloride salt of 7a. m.p. 158-60 ꢀC; 1H NMR (DMSO-d6):
The bis substituted carbamoyl-guanidine 5a (0.80 g,
1.45 mmol) was suspended in EtOH (20 mL). 1N KOH (5 mL,
11.66 mmol) was then added and the reaction mixture was kept
stirring over night 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 beige solid, yield 93%;
d
1.16 (d, J ¼ 6.3 Hz, 12H, 2ꢁ CH(CH3)2), 3.86e3.92 (m, 2H, 2ꢁ
CH(CH3)2), 7.05 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.08 (s, 4H, Ar-H), 7.23 (d,
J ¼ 8.7 Hz, 4H, Ar-H), 7.60 (br s, 4H, 2ꢁ NH2), 7.99 (br s, 2H, 2ꢁ NH),
9.72 (br s, 2H, 2ꢁ HCl). Anal. Calc. for C26H32N6O2-2.0HCl-
0.75C2H5OH: C, 58.14; H, 6.83; N, 14.79. Found: C, 58.24; H, 6.48; N,
14.59.
m.p. 209-11 ꢀC; 1H NMR (DMSO-d6):
d
2.64 (s, 6H, 2ꢁ CH3), 5.10 (br
4.1.15. Preparation ofarylimidamides (generalprocedure) (Scheme 3).
N,N’-(4,40-(1,3-phenylenebis(oxy))bis(4,1-phenylene))
dicyclohexanecarboximidamide (9a)
s, 6H, 2ꢁ NH2 þ 2ꢁ NH), 6.48 (t, J ¼ 1.5 Hz, 1H, Ar-H), 6.56 (dd,
J ¼ 1.5, 8.1 Hz, 2H, Ar-H), 6.75 (d, J ¼ 8.4 Hz, 4H, Ar-H), 6.87 (d,
J ¼ 8.4 Hz, 4H, Ar-H), 7.21 (t, J ¼ 8.1 Hz, 1H, Ar-H). 13C NMR (DMSO-
A chilled solution of the 1a (0.5 g,1.71 mmol) in dry MeCN (10 mL)
and dry EtOH (15 mL) was treated with S-(2-naphthylcyclohexyl)
thiobenzimidate hydrobromide (1.31 g, 3.59 mmol). After stirring at
r.t. for 24 h, the solvent was evaporated to dryness leaving an oily
residue. Treatment with ether gave the reversed amidine as the
hydrobromide salt, which was dissolved in EtOH, basified with 1N
NaOH, extractedwith EtOAc, dried overNa2SO4 and finally thesolvent
was evaporated to dryness giving the free base of the reversed ami-
dine in an analytically pure form, yield 57%, mp 156-8 ꢀC; 1H NMR
d6):
d 159.70, 152.39, 148.59, 147.67, 130.46, 124.05, 120.45, 110.49,
105.88, 27.69.
Hydrochloride salt of 7a. The free base was dissolved in dry
EtOH (20 mL) and the solution was chilled in an ice-bath. After
passing HCl gas for 10 min, the reaction was concentrated under
reduced pressure and then diluted with ether. The precipitate
formed was collected by filtration; m.p. 156-8 ꢀC; 1H NMR (DMSO-
d6):
d
2.79, 2.81 (2 s, 6H, 2ꢁ CH3), 6.68 (t, J ¼ 2.4 Hz, 1H, Ar-H), 6.76
(dd, J ¼ 2.4, 8.4 Hz, 2H, Ar-H), 7.11 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.25 (d,
J ¼ 8.7 Hz, 4H, Ar-H), 7.38 (t, J ¼ 8.4 Hz, 1H, Ar-H), 7.68 (br s, 4H, 2ꢁ
NH2), 7.80 (br s, 2H, 2ꢁ NH), 9.87 (br s, 2H, 2ꢁ HCl). HRMS m/z
405.2033 (Mþ þ 1) (calcd. for C22H25N6O2: 405.2039). Anal. Calc. for
(DMSO-d6):
d
1.13e1.30 (m, 6H, cyclohex.), 1.44 (q, J ¼ 11.4 Hz, 4H,
cyclohex.), 1.64 (t, J ¼ 11.4 Hz, 2H, cyclohex.), 1.72e1.84 (m, 8H,
cyclohex.), 2.11 (d, J ¼ 11.4 Hz, 2H, cyclohex.), 5.72 (br s, 4H, 4ꢁ NH),
6.48 (t, J ¼ 2.4 Hz,1H, Ar-H), 6.60 (dd, J ¼ 2.4, 8.1 Hz, 2H, Ar-H), 6.74 (d,
J¼ 8.7 Hz, 4H, Ar-H), 6.91 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.24(t, J ¼ 8.4 Hz,1H,
Ar-H).
C
22H24N6O2-2.0HCl-0.5H2O-0.25C2H5OH: C, 54.27; H, 5.76; N,16.87.
Found: C, 54.09; H, 5.73; N, 16.64.
Hydrochloride salt of 9a. An ice-bath cold solution of the free
base in dry EtOH was treated with HCl gas for 5e10 min, and the
reaction mixture was kept stirring for 5 h; mp 200-2 ꢀC; 1H NMR
4.1.12. 1,10-(4,40-(1,4-phenylenebis(oxy))bis(4,1-phenylene))bis(N’-
methylguanidine) (7b)
The procedure described for 7a was adopted starting with 5b,
(DMSO-d6): d 1.22e1.30 (m, 6H, cyclohex.), 1.68e1.90 (m, 14H,
yield 73%; mp 224-5 ꢀC; 1H NMR (DMSO-d6):
d
2.63 (s, 6H, 2ꢁ CH3),
cyclohex.), 2.74 (t, J ¼ 11.4 Hz, 2H, cyclohex.), 6.74 (t, J ¼ 2.1 Hz, 1H,
Ar-H), 6.84 (dd, J ¼ 2.1, 8.4 Hz, 2H, Ar-H), 7.17 (d, J ¼ 8.7 Hz, 4H, Ar-
H), 7.32 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.42 (t, J ¼ 8.4 Hz, 1H, Ar-H), 8.47
(br s, 2H, 2ꢁ NH), 9.37 (br s, 2H, 2ꢁ NH), 11.46 (br s, 2H, 2ꢁ HCl).
Anal. Calc. for C32H38N4O2-2.0HCl-H2O: C, 63.88; H, 7.03; N, 9.31.
Found: C, 63.82; H, 6.85; N, 9.17.
3.35 (br s, 2H, 2ꢁ NH), 5.15 (br s, 4H, 2ꢁ NH2), 6.74 (d, J ¼ 8.7 Hz, 4H,
Ar-H), 6.83 (d, J ¼ 8.7 Hz, 4H, Ar-H), 6.92 (s, 4H, Ar-H). MS (ESI) m/e
(rel. int.): 605 (Mþ þ 1, 100).
Hydrochloride salt of 7b. m.p. 162-4 ꢀC 1H NMR (DMSO-d6):
d
2.79, 2.81 (2 s, 6H, 2ꢁ CH3), 7.06 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.09 (s,
4H, Ar-H), 7.24 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.63 (br s, 4H, 2ꢁ NH2), 7.72
(br s, 2H, 2ꢁ NH), 9.70 (br s, 2H, 2ꢁ HCl). Anal. Calc. for C22H24N6O2-
2.0HCl-1.2H2O-0.5C2H5OH: C, 52.94; H, 6.06; N, 16.09. Found: C,
53.34; H, 5.92; N, 15.70.
4.1.16. N,N’-(4,40-(1,4-phenylenebis(oxy))bis(4,1-phenylene))
dicyclohexanecarboximidamide (9b)
The procedure used for 9a was adopted starting with 1b, yield
55%; mp 201-3 ꢀC; 1H NMR (DMSO-d6):
d 1.16e1.32 (m, 6H, cyclo-
4.1.13. 1,10-(4,40-(1,3-phenylenebis(oxy))bis(4,1-phenylene))bis(N’-
isopropylguanidine) (8a)
hex.), 1.47 (q, J ¼ 11.4 Hz, 4H, cyclohex.), 1.62e1.85 (m, 10H, cyclo-
hex.), 2.15 (t, J ¼ 11.4 Hz, 2H, cyclohex.), 5.65 (br s, 4H, 4ꢁ NH), 6.74
(d, J ¼ 8.4 Hz, 4H, Ar-H), 6.90 (d, J ¼ 8.4 Hz, 4H, Ar-H), 6.96 (s, 4H, Ar-
The procedure described for 7a was adopted starting with 6a,
yield 85%; mp 220-2 ꢀC; 1H NMR (DMSO-d6):
d
1.08 (d, J ¼ 6.3 Hz,
H). 13C NMR (DMSO-d6):
d 170.96, 156.58, 151.95, 128.85, 127.36,
12H, 2ꢁ CH(CH3)2), 3.78e3.86 (m, 2H, 2ꢁ CH(CH3)2), 4.82 (br s, 4H,
2ꢁ NH2), 5.22 (br s, 2H, 2ꢁ NH), 6.48 (t, J ¼ 2.4 Hz, 1H, Ar-H), 6.55
(dd, J ¼ 2.4, 8.1 Hz, 2H, Ar-H), 6.73 (d, J ¼ 8.4 Hz, 4H, Ar-H), 6.85
(d, J ¼ 8.4 Hz, 4H, Ar-H), 7.22 (t, J ¼ 8.1 Hz, 1H, Ar-H).
120.43, 119.04, 55.72, 41.88, 28.62, 24.92, 24.47, 18.17.
Hydrochloride salt of 9b. m.p. 276-8 ꢀC; 1H NMR (DMSO-d6):
d
1.22e1.33 (m, 6H, cyclohex.),1.67e1.90 (m,14H, cyclohex.), 2.71 (t,
J ¼ 11.7 Hz, 2H, cyclohex.), 7.09e7.13 (m, 8H, Ar-H), 7.31 (d,
J ¼ 8.7 Hz, 4H, Ar-H), 8.47 (br s, 2H, 2ꢁ NH), 9.31 (br s, 2H, 2ꢁ NH),
11.37 (br s, 2H, 2ꢁ HCl). Anal. Calc. for C32H38N4O2-2.0HCl-1.5H2O-
0.5C2H5OH: C, 62.55; H, 7.31; N, 8.84. Found: C, 62.91; H, 7.37; N,
8.26.
Hydrochloride salt of 7a. m.p. 131-3 ꢀC; 1H NMR (DMSO-d6):
d
1.16 (d, J ¼ 6.3 Hz, 12H, 2ꢁ CH(CH3)2), 3.89e3.97 (m, 2H, 2ꢁ
CH(CH3)2), 6.67 (t, J ¼ 2.4 Hz, 1H, Ar-H), 6.76 (dd, J ¼ 2.4, 8.4 Hz, 2H,
Ar-H), 7.10 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.25 (d, J ¼ 8.7 Hz, 4H, Ar-H),
7.38 (t, J ¼ 8.4 Hz, 1H, Ar-H), 7.68 (br s, 4H, 2ꢁ NH2), 8.13 (br s,
2H, 2ꢁ NH), 9.90 (br s, 2H, 2ꢁ HCl). HRMS m/z 461.2646 (Mþ þ 1)
(calcd. for C26H33N6O2: 461.2665). Anal. Calc. for C26H32N6O2-
2.0HCl-0.5H2O-C2H5OH: C, 57.13; H, 7.02; N, 14.27. Found: C, 57.13;
H, 7.11; N, 13.99.
4.1.17. N,N’-(4,40-(1,3-phenylenebis(oxy))bis(4,1-phenylene))
dibenzimidamide (10a)
The procedure used for 9a was adopted starting with 1a, yield
79%; mp 183-4 ꢀC; 1H NMR (DMSO-d6):
d
6.32 (br s, 4H, 4ꢁ NH),

R.K. Arafa et al. / European Journal of Medicinal Chemistry 46 (2011) 5852e5860
5859
6.56 (t, J ¼ 2.4 Hz, 1H, Ar-H), 6.65 (dd, J ¼ 2.4, 8.1 Hz, 2H, Ar-H), 6.87
4.1.21. N,N’-(4,40-(1,3-phenylenebis(oxy))bis(4,1-phenylene))
dibiphenyl-4-carboximidamide (12a)
The procedure used for 9a was adopted starting with 1a, yield
72%; mp 267-9 ꢀC; 1H NMR (DMSO-d6):
(d, J ¼ 8.7 Hz, 4H, Ar-H), 6.99 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.28
(t, J ¼ 8.4 Hz, 1H, Ar-H), 7.41e7.44 (m, 6H, Ar-H), 7.88 (d, J ¼ 8.4 Hz,
4H, Ar-H). 13C NMR (DMSO-d6):
d
159.53, 154.28, 150.08, 147.24,
d
6.20 (br s, 4H, 4ꢁ NH),
135.90, 130.65, 130.10, 128.03, 127.08, 123.00, 120.83, 11.04, 106.43.
6.59 (t, J ¼ 2.4 Hz, 1H, Ar-H), 6.70 (dd, J ¼ 2.4, 8.4 Hz, 2H, Ar-H), 6.93
(d, J ¼ 8.1 Hz, 4H, Ar-H), 7.00 (d, J ¼ 8.4 Hz, 4H, Ar-H), 7.318 (t,
J ¼ 8.4 Hz, 1H, Ar-H), 7.40 (t, J ¼ 7.2 Hz, 2H, Ar-H), 7.47 (t, J ¼ 7.2 Hz,
Hydrochloride salt of 10a. m.p. 198e201 ꢀC; 1H NMR (DMSO-
d6):
d
6.81 (t, J ¼ 2.4 Hz, 1H, Ar-H), 6.86 (dd, J ¼ 2.4, 8.4 Hz, 2H, Ar-
H), 7.24 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.42e7.52 (m, 5H, Ar-H), 7.66 (t,
J ¼ 7.5 Hz, 4H, Ar-H), 7.77 (t, J ¼ 7.5 Hz, 2H, Ar-H), 7.94 (d,
J ¼ 8.7 Hz, 4H, Ar-H), 8.97 (br s, 2H, 2ꢁ NH), 9.87 (br s, 2H, 2ꢁ
NH), 11.57 (br s, 2H, 2ꢁ HCl). HRMS m/z 499.2122 (Mþ þ 1) (calcd.
for C32H27N4O2: 499.2134). Anal. Calc. for C32H26N4O2-2.0HCl-
1.2H2O: C, 64.80; H, 5.16; N, 9.44. Found: C, 64.77; H, 5.07; N,
9.32.
4H, Ar-H), 7.69e7.71 (m, 8H, Ar-H), 8.00 (d, J ¼ 8.1 Hz, 4H, Ar-H). 13
C
NMR (DMSO-d6):
d 163.04, 157.97, 155.76, 145.17, 138.33, 131.29,
130.28, 129.55, 129.18, 128.71, 127.89, 127.18, 127.03, 126.88, 120.35,
113.55, 108.97.
Hydrochloride salt of 12a. m.p. 275-7 ꢀC; 1H NMR (DMSO-d6):
d
6.83 (t, J ¼ 2.4 Hz, 1H, Ar-H), 6.86 (dd, J ¼ 2.4, 8.4 Hz, 2H, Ar-H),
7.24 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.43e7.56 (m, 11H, Ar-H), 7.81 (d,
J ¼ 7.2 Hz, 4H, Ar-H), 7.98 (d, J ¼ 8.4 Hz, 4H, Ar-H), 8.06 (d, J ¼ 8.4 Hz,
4H, Ar-H), 8.98 (br s, 2H, 2ꢁ NH), 9.93 (br s, 2H, 2ꢁ NH), 11.65 (br s,
2H, 2ꢁ HCl). Anal. Calc. for C44H34N4O2-2.0HCl-0.75H2O: C, 71.68;
H, 5.12; N, 7.59. Found: C, 71.69; H, 5.05; N, 7.54.
4.1.18. N,N’-(4,40-(1,4-phenylenebis(oxy))bis(4,1-phenylene))
dibenzimidamide (10b)
The procedure used for 9a was adopted starting with 1b, yield
66%; mp 236-8 ꢀC; 1H NMR (DMSO-d6):
d
6.31 (br s, 4H, 4ꢁ NH),
6.85 (d, J ¼ 8.7 Hz, 4H, Ar-H), 6.98 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.02 (s,
4.1.22. N,N’-(4,40-(1,4-phenylenebis(oxy))bis(4,1-phenylene))
dibiphenyl-4-carboximidamide (12b)
4H, Ar-H), 7.39, 7.46 (m, 6H, Ar-H), 7.95 (d, J ¼ 7.5 Hz, 4H, Ar-H). 13
C
NMR (DMSO-d6):
d
154.15, 152.88, 151.50, 146.42, 135.90, 130.00,
The procedure used for 9a was adopted starting with 1b, yield
127.95, 127.00, 122.84, 119.67, 119.33.
82%; mp 298e300 ꢀC; 1H NMR (DMSO-d6):
d
6.23 (br s, 4H, 4ꢁ NH),
Hydrochloride salt of 10b. m.p. 287-9 ꢀC; 1H NMR (DMSO-d6):
7.17e7.20 (m, 8H, Ar-H), 7.49 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.65 (t,
J ¼ 7.5 Hz, 4H, Ar-H), 7.77 (t, J ¼ 7.5 Hz, 2H, Ar-H), 7.94 (d, J ¼ 7.5 Hz,
4H, Ar-H), 8.96 (br s, 2H, 2ꢁ NH), 9.87 (br s, 2H, 2ꢁ NH), 11.59 (br s,
2H, 2ꢁ HCl). HRMS m/z 499.2130 (Mþ þ 1) (calcd. for C32H27N4O2:
499.2134). Anal. Calc. for C32H26N4O2-2.0HCl-0.5H2O: C, 66.20; H,
5.03; N, 9.65. Found: C, 66.13; H, 5.00; N, 9.65.
6.60 (d, J ¼ 8.4 Hz, 2H, Ar-H), 6.75 (d J ¼ 8.4 Hz, 2H, Ar-H), 6.87e7.34
(m, 10H, Ar-H), 7.36e7.50 (m, 6H, Ar-H), 7.69e6.72 (m, 6H, Ar-H),
8.00 (d, J ¼ 7.8 Hz, 4H, Ar-H).
Hydrochloride salt of 12b. m.p. 222-4 ꢀC; 1H NMR (DMSO-d6):
7.19e7.21 (m, 8H, Ar-H), 7.46e7.56 (m,10H, Ar-H), 7.81 (d, J ¼ 7.8 Hz,
4H, Ar-H), 7.98 (d, J ¼ 8.4 Hz, 4H, Ar-H), 8.04 (d, J ¼ 8.4 Hz, 4H, Ar-H),
8.97 (br s, 2H, 2ꢁ NH), 9.89 (br s, 2H, 2ꢁ NH), 11.58 (br s, 2H, 2ꢁ
HCl). Anal. Calc. for C44H34N4O2-2.0HCl-1.25H2O: C, 70.82; H, 5.20;
N, 7.50. Found: C, 71.04; H, 5.22; N, 7.31.
4.1.19. N,N’-(4,40-(1,3-phenylenebis(oxy))bis(4,1-phenylene))bis(4-
tert-butylbenzimidamide) (11a)
The procedure used for 9a was adopted starting with 1a, yield
4.1.23. S-(2-Naphthylmethyl)-4-cyclohexylthioimidate.HBr
48%; mp 286-8 ꢀC; 1H NMR (DMSO-d6):
d
1.30 (s, 18H, 2ꢁ C(CH3)3),
Yield 73%, mp 187-9 ꢀC; 1H NMR (DMSO-d6)
d 1.15e1.31 (m, 3H,
6.24 (br s, 4H, 4ꢁ NH), 6.59 (t, J ¼ 2.4 Hz, 1H, Ar-H), 6.66 (dd, J ¼ 2.4,
8.4 Hz, 2H, Ar-H), 6.84 (d, J ¼ 8.1 Hz, 4H, Ar-H), 7.01 (d, J ¼ 8.4 Hz,
4H, Ar-H), 7.28 (t, J ¼ 8.4 Hz, 1H, Ar-H), 7.42 (d, J ¼ 8.4 Hz, 4H, Ar-H),
cyclohex.), 1.45 (q, J ¼ 11.4 Hz, 2H, cyclohex.), 1.63 (t, J ¼ 11.4 Hz, 1H,
cyclohex.), 1.72e1.84 (m, 4H, cyclohex.), 2.10 (d, J ¼ 11.4 Hz, 1H,
cyclohex.), 4.91 (s, 2H, CH2), 7.52e7.61 (m, 3H, Ar-H), 7.90e7.99 (m,
3H, Ar-H), 8.06 (s, 1H, Ar-H), 10.32 (br s, 1H, HBr). MS (ESI) m/e (rel.
int.): 365 (Mþ þ 2,10), 364 (Mþ þ 1,100). Anal. Calc. for C18H21NS.HBr:
C, 59.34; H, 6.09; N, 3.84. Found C, 59.29; H, 6.28; N, 3.49.
7.88 (d, J ¼ 8.1 Hz, 4H, Ar-H). 13C NMR (DMSO-d6):
d 163.22, 157.93,
157.06, 155.71, 131.27, 130.27, 128.68, 127.81, 125.74, 125.66, 120.31,
113.53, 108.96, 34.98, 30.75.
Hydrochloride salt of 11a. m.p. >300 ꢀC; 1H NMR (DMSO-d6):
d
1.33 (s, 18H, 2ꢁ C(CH3)3), 6.81 (t, J ¼ 2.4 Hz, 1H, Ar-H), 6.86 (dd,
4.2. Biology
J ¼ 2.4, 8.4 Hz, 2H, Ar-H), 7.24 (d, J ¼ 8.7 Hz, 4H, Ar-H), 7.42e7.50 (m,
5H, Ar-H), 7.68 (d, J ¼ 8.4 Hz, 4H, Ar-H), 7.89 (d, J ¼ 8.4 Hz, 4H, Ar-H),
8.92 (br s, 2H, 2ꢁ NH), 9.80 (br s, 2H, 2ꢁ NH), 11.47 (br s, 2H, 2ꢁ
HCl). HRMS m/z 611.3367 (Mþ þ 1) (calcd. for C40H43N4O2:
611.3386). Anal. Calc. for C40H42N4O2-2.0HCl-0.25H2O: C, 69.80; H,
6.51; N, 8.14. Found: C, 69.75; H, 6.44; N, 8.02.
4.2.1. In vitro assay for T.b.r.
Minimum essential medium (50 ml) supplemented according to
Baltz et al [21]. with 2-mercaptoethanol and 15% heat-inactivated
horse serum were added to each well of a 96-well microtiter plate.
Serial drug dilutions were added to the wells. Then 50
mL of
trypanosome suspension (T.b.r. STIB 900) was added to each well and
4.1.20. N,N’-(4,40-(1,4-phenylenebis(oxy))bis(4,1-phenylene))bis(4-
tert-butylbenzimidamide) (11b)
the plate incubated at 37 ꢀC under a 5% CO2 atmosphere for 72 h.
Alamar Blue (10 mL) was then added to each well and incubation
The procedure used for 9a was adopted starting with 1b, yield
continued for more 2ꢂ4 h. The plate was read in a microplate
fluorometer system (Spectramax Gemini by Molecular Devi-ces)
using an excitation wavelength of 536 nm and an emission wave-
length of 588 nm [21]. Fluorescence development was expre-ssed as
percentage of the control, and IC50 values determined. Assays were
carried out twice independently and in duplicates.
57%; mp 280-2 ꢀC; 1H NMR (DMSO-d6):
d
1.32 (s, 18H, 2ꢁ C(CH3)3),
6.23 (br s, 4H, 4ꢁ NH), 7.08e7.11 (m, 8H, Ar-H), 7.24 (d, J ¼ 8.4 Hz,
4H, Ar-H), 7.59 (d, J ¼ 8.4 Hz, 4H, Ar-H), 7.84 (d, J ¼ 7.8 Hz, 4H, Ar-H).
13C NMR (DMSO-d6):
d 154.15,152.88, 151.50,146.42, 135.90,130.00,
127.95, 127.00, 122.84, 119.67, 119.33.
Hydrochloride salt of 11b. m.p. >300 ꢀC; 1H NMR (DMSO-d6):
1.33 (s, 18H, 2ꢁ C(CH3)3), 7.17e7.19 (m, 8H, Ar-H), 7.47 (d, J ¼ 8.4 Hz,
4H, Ar-H), 7.67 (d, J ¼ 8.4 Hz, 4H, Ar-H), 7.89 (d, J ¼ 8.1 Hz, 4H, Ar-H),
8.91 (br s, 2H, 2ꢁ NH), 9.80 (br s, 2H, 2ꢁ NH), 11.48 (br s, 2H, 2ꢁ
HCl). HRMS m/z 611.3362 (Mþ þ 1) (calcd. for C40H43N4O2:
611.3386). Anal. Calc. for C40H42N4O2-2.0HCl-1.25H2O: C, 68.02; H,
6.63; N, 7.93. Found: C, 68.02; H, 6.27; N, 7.87.
4.2.2. In vitro assay for P. f.
Antiplasmodial activity was determined using the K1 strain of
P. falciparum (resistant to chloroquine and pyrimethamine). A
modification of the [3H]-hypoxanthine incorporation assay was
used [21]. Briefly, infected human red blood cells in RPMI 1640
medium with 5% Albumax were exposed to serial drug dilutions in

5860
R.K. Arafa et al. / European Journal of Medicinal Chemistry 46 (2011) 5852e5860
microtiter plates for 48 h. Viability was assessed by measuring the
incorporation of [3H]-hypoxanthine by liquid scintillation counting
24 h after the addition of the radiolabel. The counts were expressed
as percentage of the control cultures, sigmoidal inhibition curves
were drawn and IC50 values calculated. Assays were carried out
twice independently and in duplicates.
References
[1] (a) R.R. Tidwell, W.D. Boykin, in: M. Demeunynck, C. Bailly, W.D. Wilson (Eds.),
Small Molecule DNA and RNA Binders: From Synthesis to Nucleic Acid
Complexes, "Dicationic DNA Minor Groove Binders as Antimicrobial Agents,
vol. 2, Wiley-VCH, 2002, pp. 416e460;
(b) W.D. Wilson, B. Nguyen, F.A. Tanious, A. Mathis, J.E. Hall, C.E. Stephens,
D.W. Boykin, Curr. Med. Chem.-Anti-Cancer Agents 5 (2005) 389e408;
(c) M.N.C. Soeiro, E.M. de Souza, C.E. Stephens, D.W. Boykin, Expert Opin.
Invest. Drugs 14 (2005) 957e972;
4.2.3. Cytotoxicity studies
Cytotoxicity was evaluated using cultured L-6 rat myoblast cells
using the same assay procedure as for T.b.r. [21].
(d) C. Dardonville, Expert Ther. Pat. 15 (2005) 1241e1257;
(e) K.A. Werbovetz, Curr. Opin. Invest. Drugs 7 (2006) 147e157.
[2] J.N. Ashley, H.J. Barber, A.J. Ewins, G. Newbery, A.D.H. Self, J. Chem. Soc. (1942)
103e106.
[3] J.K. Thuita, S.M. Karanja, T. Wenzler, R.E. Mdachi, J.M. Ngotho, J.M. Kagira,
R. Tidwell, R. Brun, Acta Tropica. 108 (2008) 6e10.
4.3. Absorbance spectroscopy and thermal melting (Tm)
experiments
[4] C.E. Stephens, F. Tanious, S. Kim, W.D. Wilson, W.A. Schell, J.R. Perfect,
S.G. Franzblau, D.W. Boykin, J. Med. Chem. 44 (2001) 1741e1748.
[5] 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) 2065e2069.
[6] C. Dardonville, R.J. Brun, Med. Chem. 47 (2004) 2296e2307.
[7] R.K. Arafa, R. Brun, T. Wenzler, F.A. Tanious, W.D. Wilson, C.E. Stephens,
D.W. Boykin, J. Med. Chem. 48 (2005) 5480e5488.
[8] R.K. Arafa, M.A. Ismail, M. Munde, W.D. Wilson, T. Wenzler, R. Brun,
D.W. Boykin, Eur. J. Med. Chem. 43 (2008) 2901e2908.
[9] (a) D.W. Boykin, A. Kumar, J. Spychala, M. Zhou, R. Lombardy, W.D. Wilson,
C.C. Dykstra, S.K. Jones, J.E. Hall, R.R. Tidwell, C. Laughton, C.M. Nunn,
S.J. Neidle, Med. Chem. 38 (1995) 912e916;
Experiments were done in MES10 buffer (0.01M MES (2-(N-
morpholino)ethanesulfonic acid), 0.001M ethylenediaminetetra-
acetic acid (EDTA), 0.1M NaCl with the pH was adjusted to 6.25).
DNA polymers were purchased from Pharmacia and characterized
by their melting curves. Absorbance and thermal melting (Tm)
experiments were done using a Cary 300 Bio spectrophotometer
with the software supplied with the instrument. For absorbance
measurements, the buffer was scanned from 400 to 250 nm in 1 cm
quartz cuvettes and aliquots of concentrated stock solutions of the
compounds were titrated into the buffer and the solutions were
rescanned. Cuvettes were mounted in a thermal block, and the
solution temperatures were monitored by a thermistor in a refer-
ence cuvette with a computer-controlled heating rate of 0.5 ꢀC/min.
Experiments were conducted at a concentration of 2 ꢁ 10ꢂ5 M base
pair for polydA.polydT. For experiments with complexes a ratio of
0.3 compound per base pair for polydA.polydT was generally used.
(b) 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 (1998) 124e129;
(c) I. Francesconi, W.D. Wilson, F.A. Tanious, J.E. Hall, B.C. Bender, R.R. Tidwell,
D. McCurdy, D.W. Boykin, J. Med. Chem. 42 (1999) 2260e2265;
(d) C.A. Laughton, F.A. Tanious, C.M. Nunn, D.W. Boykin, W.D. Wilson,
S. Neidle, Biochemistry 35 (1996) 5655e5661;
(e) J.O. Trent, G.R. Clark, A. Kumar, W.D. Wilson, D.W. Boykin, J.E. Hall,
R.R. Tidwell, B.L. Blagburn, S. Neidle, J. Med. Chem. 39 (1996) 4554e4562.
[10] (a) 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) 10310e10321;
(b) C. Bailly, L. Dassonneville, C. Carrascol, D. Lucasl, A. Kumar, D.W. Boykin,
W.D. Wilson, Anti-Cancer Drug Des. 14 (1999) 47e60;
(c) D.J. Fitzgerald, J.N. Anderson, J. Biol. Chem. 274 (1999) 27128e27138;
(d) C.C. Dykstra, D.R. McClernon, L.P. Elwell, R.R. Tidwell, Antimicrob. Agents
Chemother. 38 (1994) 1890;
4.4. Molecular modeling and docking studies
(e) D. Henderson, L.H. Hurley, Nat. Med. 1 (1995) 525e527.
[11] D. Goodsell, R.E. Dickerson, J. Med. Chem. 29 (1986) 727e733.
[12] (a) B. Nguyen, M.P. Lee, D. Hamelberg, C. Bailly, R. Brun, S. Neidle, W.D. Wilson,
J. Am. Chem. Soc. 124 (2002) 13680e13681;
Docking studies were performed using MOE software Version
2008.10. The X-ray crystal structure of the selective antiprotozoal
agent pentamidine bound to the minor groove of DNA dodecamer
d(CGCGAATTCGCG)2 was obtained from the RCSB Protein Data Bank
(PDB ID: 1d64). The enzyme was prepared for docking as follows: (a)
pentamidine interactions were determined to reveal the different
types of interaction as a validation for the docking is to be per-
formed. (b) Pentamidine and solvent molecules were removed from
the binding site. (c) The hydrogens were added with their standard
geometry. (d) The newly synthesized dications were constructed
and were energy minimized for 1000 iterations reaching a conver-
gence of 0.01 kcal/mol. (e) Docking of the energy minimized ligand
molecules was carried out using the default MOE-dock parameters.
The docking aims at searching for favorable binding configuration
between the flexible dicationic ligands and the DNA minor groove
and to assess the strength of the binding interactions of the novel
dications as compared to the classical minor groove binder pent-
amidine. The quality of the docked structures was evaluated by
measuring the intermolecular energy of the ligand-DNA assembly
dG calculated in Kcal/mol (S value in MOE) [22].
(b) B. Nguyen, D. Hamelberg, C. Bailly, J. Colson, J. Stenek, R. Brun, S. Neidle,
W.D. Wilson, Biophys. J. 86 (2004) 1028e1041;
(c) Y. Miao, M.P.H. Lee, G.N. Parkinson, A. Batista-Parra, M.A. Ismail, S. Neidle,
D.W. Boykin, D.W. Wilson, Biochemistry 44 (2005) 14701e14708.
[13] M.A. Ismail, R.K. Arafa, R. Brun, T. Wenzler, Y. Miao, D.W. Wilson, C. Generaux,
A. Bridges, J.E. Hall, D.W. Boykin, J. Med. Chem. 49 (2006) 5324e5332.
[14] M. Munde, M.A. Ismail, R. Arafa, P. Peixoto, C.J. Collar, Y. Liu, L. Hu, M.-
H. David-Cordonnier, A. Lansiaux, C. Bailly, D.W. Boykin, W.D. Wilson, J. Am.
Chem. Soc. 129 (2007) 13732e13743.
[15] K.S. Atwal, S.Z. Ahmed, B.C. O’Reilly, Tetrahedron Lett. 30 (1989)
7313e7316.
[16] K.S. Atwal, G.J. Grover, F.N. Ferrara, S.Z. Ahmed, P.G. Sleph, S. Dzwonczyk,
D.E. Normandin, J. Med. Chem. 38 (1995) 1966e1973.
[17] B.R. Linton, A.J. Carr, B.P. Orner, A.D. Hamilton, J. Org. Chem. 65 (2000)
1566e1568.
[18] B.G. Shearer, J.A. Oplinger, S. Lee, Tetrahedron Lett. 38 (1997) 179e182.
[19] N.H. Campbell, D.A. Evans, M.P.H. Lee, G.N. Parkinsona, S. Neidle, Bioorg. Med.
Chem. Lett. 16 (2006) 15e19.
[20] K.J. Edwards, T.C. Jenkins, S. Neidle, Biochemistry 31 (1992) 7104e7109.
[21] (a) T. Baltz, D. Baltz, C. Giroud, J. Crockett, EMBO J. 4 (1985) 1273e1277;
(b) B. Räz, M. Iten, Y. Grether-Bühler, R. Kaminsky, R. Brun, Acta Trop. 68
(1997) 139e147;
(c) H. Matile, J.R.L. Pink, Plasmodium falciparum malaria parasite cultures and
their use in immunology. in: I. Lefkovits, B. Pernis (Eds.), Immunological
Methods. Academic Press, San Diego, 1990, pp. 221e234;
(d) M.A. Ismail, R. Brun, J.D. Easterbrook, F.A. Tanious, W.D. Wilson,
D.W. Boykin, J. Med. Chem. 46 (2003) 4761e4769;
Acknowledgements
(e) M.A. Ismail, A. Batista-Parra, Y. Miao, W.D. Wilson, T. Wenzler, R. Brun,
D.W. Boykin, Bioorg. Med. Chem. 13 (2005) 6718e6726.
[22] R.G. Kurumbail, A.M. Stevens, J.K. Gierse, J.J. McDonald, R.A. Stegeman, J.Y. Pak,
D. Gildehaus, J.M. Miyashiro, T.D. Penning, K. Seibert, P.C. Isakson,
W.C. Stallings, . Nature 384 (1996) 644e648.
The authors are highly indebted to Prof. D.W. Boykin for his
continuous guidance, never abating support and useful discussions.
We also thank the Department of Chemistry, Georgia State
University for use of the NMR facilities.