Selective inhibition of bacterial topoisomerase i by alkynyl- bisbenzimidazoles

Article abstract of DOI:10.1039/c4md00140k

Hoechst dyes are well known DNA binders that non-selectively inhibit the function of mammalian topoisomerase I and II. Herein, we show that Hoechst 33258 based bisbenzimidazoles (DPA 151-154), containing a terminal alkyne, are effective and selective inhibitors of E. coli topoisomerase I. These bisbenzimidazoles displayed topoisomerase I inhibition much better than Hoechst 33342 or Hoechst 33258 with IC50 values in the range of 2.47-6.63 μM. Bisbenzimidazoles DPA 151-154 also display selective inhibition of E. coli topoisomerase I over DNA gyrase and human topoisomerases I and II, and effectively inhibit bacterial growth.

Full text of DOI:10.1039/c4md00140k

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Selective inhibition of bacterial topoisomerase I by
alkynyl-bisbenzimidazoles†
Cite this: Med. Chem. Commun., 2014,
5, 816
Nihar Ranjan,a Geraldine Fulcrand,b Ada King,c Joseph Brown,d Xiuping Jiang,d
b
ac
*
Fenfei Leng and Dev P. Arya
Hoechst dyes are well known DNA binders that non-selectively inhibit the function of mammalian
topoisomerase I and II. Herein, we show that Hoechst 33258 based bisbenzimidazoles (DPA 151–154),
containing a terminal alkyne, are effective and selective inhibitors of E. coli topoisomerase I. These
bisbenzimidazoles displayed topoisomerase I inhibition much better than Hoechst 33342 or Hoechst
33258 with IC50 values in the range of 2.47–6.63 mM. Bisbenzimidazoles DPA 151–154 also display
selective inhibition of E. coli topoisomerase I over DNA gyrase and human topoisomerases I and II, and
effectively inhibit bacterial growth.
Received 25th March 2014
Accepted 24th April 2014
DOI: 10.1039/c4md00140k
www.rsc.org/medchemcomm
Introduction
New approaches for the discovery of antibacterial drugs are
paramount to our efforts in the continuing ght against
bacterial resistance. In this regard, enzyme inhibitors that
selectively target a bacterial enzyme over their human coun-
terpart offer unique opportunities for such selective inhibition
approaches. Bacterial DNA topoisomerases1,2 are one such class
of enzymes that help in regulating DNA topology. The cellular
functions of topoisomerases include relaxing (+) and (ꢀ)
supercoil in DNA as well as introducing supercoils to their DNA
substrates.3 These functions of DNA topoisomerases can be
used to develop anticancer or antibacterial agents.2,4 The ther-
apeutic interest in the development of small molecules as
inhibitors of DNA topoisomerase lies in their ability to act as
both cleavable complex stabilizing agents as well as in their
ability to bind at the ATP binding site.2
Chart 1 Structures of compounds used in the study.
activities. Halogenated monobenzimidazoles have shown anti-
mycobacterial activity better than isoniazid.13 Similarly, triazolyl
derivatized monobenzimidazoles have displayed antimicrobial
properties.14 In comparison to abundant literature reports on
the biological properties of monobenzimidazoles, studies on
the antimicrobial properties of bisbenzimidazoles (particularly
those modeled from Hoechst 33258) are very limited.12,15
Hoechst 33258 is a bisbenzimidazole compound that has been a
subject of intense study for over three decades due to its
binding to AT rich duplex DNA structures.16–18
In this report we present the synthesis, nucleic acid binding,
topoisomerase I activity, and antimicrobial activity of Hoechst
33258 functionalized bisbenzimidazoles (Chart 1). We show that
the addition of alkyne functionalized alkyl chain converts
Hoechst 33258 from a non-selective topoisomerase (bacterial and
human) inhibitor to a highly selective bacterial topoisomerase I
inhibitor. The results obtained opens up a new approach to
targeting bacterial topoisomerases and the potential role of a
hydrophobic pocket in the DNA–E. coli topoisomerase I complex.
A number of small molecules have been discovered that
poison the functions of DNA topoisomerases. These have
included camptothecin5 and its derivatives, intercalators and
compounds that interact with the minor groove of B-DNA such
as bisbenzimidazoles.6–12 Benzimidazoles are important class of
compounds that display a widespread range of biological
aDepartment of Chemistry, Clemson University, 461 H.L. Hunter Chemistry Labs,
Clemson, South Carolina 29634, USA. E-mail: dparya@clemson.edu; Fax: +1-864-
656-6613; Tel: +1-864-656-1106
bDepartment of Chemistry and Biochemistry, Florida International University, Miami,
Florida 33199, USA
cNUBAD, LLC, 900 B West Faris Road, Greenville, South Carolina 29605, USA
Results and discussion
Synthesis of ligands DPA 151–154
dDepartment of Biological Sciences, Clemson University, Clemson, South Carolina
29634, USA
† Electronic supplementary information (ESI) available: Images of spectroscopic
characterization of all newly synthesized compounds as well additional images
for topoisomerase inhibition experiments. See DOI: 10.1039/c4md00140k
The synthesis of the ligands (DPA 151–154) was performed
using a divergent strategy19,20 to construct the alkyl linkers
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Fig. 1 Inhibitory activities of Hoechst 33258 and DPA 151 against
E. coli DNA topoisomerase I. Inhibition assays against E. coli DNA
topoisomerase I were performed as described under Materials and
methods. The plasmid DNA molecules were isolated and subjected to
1% agarose gel electrophoresis in the absence of chloroquine. (A) The
inhibition activities of Hoechst 33258 against E. coli DNA top-
oisomerase I. Lanes 1 to 11 contain 0, 5, 7.5, 10, 12.5, 15, 20, 22.5, 25, 25,
30 mM of Hoechst 33258, respectively. (B) The inhibition activities of
DPA 151 against E. coli DNA topoisomerase I. Lanes 1 to 11 contain 0,
1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10 mM of DPA151 respectively. (C)
The quantification analyses of the inhibitory activities of Hoechst
33258 (open circles) and DPA 151 (closed squares) against E. coli DNA
topoisomerase I. The values of IC50 (the half maximal inhibitory
concentration) were obtained from these analyses. Standard deviation
was obtained from three independent determinations. scDNA repre-
sents supercoiled DNA.
Scheme
1 Reagent and conditions (i) PPh3, DIAD, 1,4-dioxane,
dichloromethane, rt, overnight, 50–85%, (ii) Pd–C, H2, ethanol, rt, 5 h,
qaunt, (iii) DPA 151a–DPA 154a, ethanol, Na2S2O5, H2O, reflux, 12–14
h, 61–85% (for two steps), (iv) THF–ether, LAH, ꢀ78 ꢂC to 0 ꢂC, 6–12 h,
55–73%, (v) DPA 151c–DPA 154c, ethanol, Na2S2O5, H2O, reflux,
overnight, 50–72% (for two steps).
(Scheme 1). To introduce the linkers, we carried out Mitsunobu
reactions of 4-hydroxy benzaldehyde with aliphatic alcohols (1–
4) that terminated in the requisite alkyne functionality. The
aliphatic alcohols were obtained commercially or prepared in
one step from a corresponding diol. The 4-substituted benzal-
dehydes (DPA 151a–DPA 154a) were coupled with 3,4-diamino-
N-methoxy-N-methylbenzamide in the presence of an oxidant to
yield the corresponding benzimidazoles (DPA 151b–DPA 154b).
These benzimidazoles containing the weinreb amide function-
ality were then easily reduced to their corresponding aldehydes
(DPA 151c–DPA 154c) using lithium aluminum hydride.
Coupling of these aldehydes with 4-(4-methylpiperazin-1-yl)-
benzene-1,2-diamine,21 in the presence of an oxidant resulted in
the synthesis of target bisbenzimidazoles DPA 151–DPA 154 in
good yields. The presence of a rather inert functional group
alkyne also makes these molecules useful for further modi-
cations using click chemistry applications. All compounds were
characterized by spectroscopic techniques (NMR, IR and HRMS/
MALDI-TOF, see ESI, Fig. S1–S16†).
topoisomerase I. For Hoechst 33258, approximately 20 mM was
needed to completely inhibit the activities of E. coli DNA top-
oisomerase I (lane 10 of Fig. 1A). In contrast, it took only
approximately 5 mM of DPA151 to fully suppress the activities of
E. coli DNA topoisomerase I (lane 9 of Fig. 1B). The IC50 of
Hoechst 33258 and DPA151 were determined to be 19.50 ꢁ 1.32
and 5.50 ꢁ 0.50 mM, respectively (Fig. 1C and Table 1). These
results demonstrated that the addition of a hydrophobic group
to the hydroxyl tail of Hoechst 33258 dramatically increased the
Table 1 IC50 values of the newly synthesized bisbenzimidazoles
against E. coli DNA topoisomerase I, human DNA topoisomerase I, and
human DNA topoisomerase II
Inhibition of bacterial DNA topoisomerase I
IC50a (mM)
We tested the inhibitory activities of the newly synthesized
bisbenzimidazoles against a few DNA topoisomerases, i.e.,
E. coli DNA topoisomerase I, E. coli DNA gyrase, human DNA
topoisomerase I, and human DNA topoisomerase II. To our
surprise, these newly synthesized compounds showed a selec-
tive and enhanced inhibition against E. coli DNA topoisomerase
I. Fig. 1 shows the results of inhibitory assays of Hoechst 33258
and its derivative, DPA151, against E. coli DNA topoisomerase I.
In the absence of Hoechst 33258 or DPA151, 6 nM of E. coli DNA
Compound
ecTopo Ib
hTopo Ib
hTopo IIb
Hoechst 33258
Hoechst 33342
DPA151
DPA152
DPA153
19.50 ꢁ 1.32
29.83 ꢁ 2.75
5.50 ꢁ 0.50
4.57 ꢁ 0.81
2.47 ꢁ 0.06
6.63 ꢁ 0.47
22.86 ꢁ 1.55
>50 mM
28.92 ꢁ 4.45
—
25.41 ꢁ 2.20
51.6 ꢁ 2.5
>50 mM
—
—
—
>50 mM
>50 mM
DPA154
a
IC50 was determined as described under Materials and methods. The
topoisomerase I was sufficient to relax the supercoiled plasmid values are the average of at least three independent determinations.
b
ecTopo I, hTopo I, and hTopo II represent E. coli DNA
DNA template, pBAD-GFPuv (lanes 1 of Fig. 1A and C). The
topoisomerase I, human DNA topoisomerase I, and human DNA
topoisomerase II, respectively.
titration of increasing amounts of either compounds into the
reaction mixtures resulted in the inhibition of E. coli DNA
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These results suggest that the addition of a hydrophobic tail to
the hydroxyl group of Hoechst 33258 signicantly reduced the
inhibitory activities against human DNA topoisomerase I. In
this study, we also tested the inhibitory activities of these newly
synthesized compounds against two type II topoisomerases,
E. coli DNA gyrase and human DNA topoisomerase II. Our
results showed that Hoechst 33258 and the newly synthesized
bisbenzimidazoles did not inhibit DNA gyrase's activities under
our experimental conditions (Fig. S21†). For human DNA top-
oisomerase II, 50 mM of Hoechst 33258 was sufficient to
completely inhibit its activities (lane 10 of Fig. 3A). In contrast,
the addition of a hydrophobic moiety to the hydroxyl group of
Hoechst 33258 completely eliminated the inhibition of 50 mM of
these newly synthesized compounds against human DNA top-
Fig. 2 Decreased inhibitions of the newly synthesized bisbenzimida-
zoles against human DNA topoisomerase I. Inhibition assays against
human topoisomerase I were performed as described in Materials and
methods in the presence of one of the bisbenzimidazoles. Following
the inhibition assays, the plasmid DNA molecules were isolated and
subjected to 1% agarose gel electrophoresis in the absence of chlo- oisomerase II except DPA151 (Fig. 3; 50 mM of DPA151 partially
roquine. Lane 1 represents the relaxed plasmid DNA pBAD-GFPuv.
Two different concentrations for each compound (10 and 50 mM) were
used in these assays.
inhibit the activities of human DNA topoisomerase II). The IC50
values of Hoechst 33258 and DPA151 against human top-
oisomerases I and II are summarized in Table 1, and Fig. S22.†
inhibitory capacity against E. coli DNA topoisomerase I. Similar
results were also obtained for DPA152, 153 and 154 in which a
hydrophobic group with different lengths were added to the
hydroxyl group of Hoechst 33258. Our results are summarized
UV thermal denaturation studies
Bisbenzimidazoles are known to bind to the minor groove of AT
rich DNA. The UV thermal denaturation experiments of the
synthesized ligands were carried out with an AT rich DNA
duplex. A 60 mer B-DNA duplex that was prepared by mixing
equimolar amounts of a 60 mer homoadenine with a 60 mer
in Table 1 and Fig. S17–19.†
Inhibition of human DNA topoisomerase I
homothymine polymer. The results obtained from these
experiments are shown in Table 2 and the denaturation curves
in Fig. S23.† The thermal denaturation experiments show some
dependence of thermal stabilization on the length and
composition of the linker present on the Hoechst 332258
derivatives DPA 151–DPA 154. As depicted in Fig. S23,† in the
absence of ligand, the duplex dA60$dT60 exhibited a sharp
hyperchromism at 62.5 ꢂC indicating the dissociation of the
duplex into single strands. The thermal denaturation of
dA60$dT60 was then carried out in the presence of DPA 151
(10 mM). At this concentration, the DNA was saturated with
ligand, and lower concentrations (1–5 mM) of ligand resulted in
a biphasic thermal denaturation proles. In the presence of
Next we examined the inhibitory activities of these newly
synthesized bisbenzimidazoles against human DNA top-
oisomerase I, a type IB topoisomerase. Fig. 2 shows our results.
For Hoechst 33258 and DPA151, 50 mM of these two compounds
was able to prevent about 70% (Fig. S20†) of supercoiled DNA
template from relaxation by human DNA topoisomerase I
(compare lanes 3 and 5 to lane 1 of Fig. 2). However, 50 mM of
other newly synthesized bisbenzimidazoles was only capable of
preventing 5–15% of supercoiled DNA from relaxation (Fig. 2).
ꢂ
DPA 151, a 22.9 C thermal stabilization of DNA was observed.
The thermal stabilization afforded by DPA 151 (22.9 ꢂC) was
similar to the thermal stabilization afforded by Hoechst 33258
and Hoechst 33342 (ꢃ24 ꢂC). This thermal stabilization was,
Table 2 A table showing the thermal denaturation temperatures of
duplex DNA (dA60$dT60) in the presence of all studied ligands (10 mM
each) in buffer 10 mM sodium cacodylate, 0.1 mM EDTA and 100 mM
NaCl at pH 7.0
Ligand
Tm (ꢂC)
DTm (ꢂC)
Fig. 3 Inhibitory activities of DPA151, DPA153, and Hoechst 33258
against human DNA topoisomerase II. Inhibition assays against human
DNA topoisomerase II were performed as described in Materials and
methods. The plasmid DNA molecules were isolated and subjected to
1% agarose gel electrophoresis in the absence of chloroquine. Lanes
1 and 2 are the supercoiled and relaxed plasmid DNA template pBAD-
GFPuv, respectively. Two different concentrations for each compound
(10 and 50 mM) were used in these assays.
None
62.5
87.1
86.6
85.4
83.4
70.5
85.9
—
Hoechst 33258
Hoechst 33242
DPA 151
DPA 152
DPA 153
24.6
24.1
22.9
20.9
8.0
DPA 154
23.4
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however found to be dependent on the linker length and 1, all four compounds are effective antibacterial compounds
composition of the linker. As the linker length increases to a against a variety of strains, including the two E. coli strains. DPA
very long carbon chain (eleven atoms) in DPA 153, a signicant 152 and DPA 154 show markedly improved activity against
drop in DTm was observed (23.9 ꢂC thermal stabilization for DPA E. faecalis 29212. Both Hoechst dyes (33258 and 33342) are not
151 and 8 ꢂC thermal stabilization for DPA 153). Control good inhibitors of E. faecalis 29212. Of note also is that the
experiments with a GC rich calf thymus DNA showed very poor molecular mass of DPA 151–154 is considerably higher than
ꢂ
(1–2 C) thermal stabilization of the DNA by DPA 151–154 that Hoechst 33342 (10–35% higher mass), implying that the anti-
conrmed the preference of these ligands for AT rich DNA bacterial activities listed here (in mg mLꢀ1) are even better on a
sequences (data not shown). Surprisingly, DPA 153 is the most per mole basis for all newly synthesized compounds.
effective inhibitor of E. coli topoisomerase I. The addition of a
long hydrophobic linker, capable of aggregation, limits the DNA
binding of the dye. However, in the presence of the top-
Conclusions
oisomerase, it is possible that DNA binding is restored as the
hydrophobic pocket in the enzyme acts to free the ligand
aggregation. An alternative explanation is that duplex DNA
binding is not required for enzyme activity. The inhibitory
activities of small molecules against these enzymes are believed
to mainly stem from the binding of these ligands to the minor
groove of the DNA double helix.12,22 However, duplex DNA
binding is not a sole criterion for effective topoisomerase
inhibitions as a DNA non-binder, camptothecin,23 is a well-
known DNA topoisomerase I poison.5 We also tested cytotoxicity
of these compounds against a prostate cancer cell line DU-145.
Considerable variation in toxicity was observed with changes in
linker length. DPA 153, the most potent Topo I inhibitor, dis-
played much lower cytotoxicity (IC50 > 10 mM) compared to
Hoechst 33242 (IC50 ¼ 4.25 mM), whereas DPA 152 was nearly
twice as toxic as Hoechst 33242. These results clearly warrant
further studies to understand the role of hydrophobic linkers in
modulating the activity/cytotoxicity ratios (Table S1, ESI†).
Overall, our results clearly show that bisbenzimidazoles (DPA
151–DPA 154) which are excellent inhibitors of E. coli DNA
topoisomerase I, also display good antibacterial activity. Addi-
tionally, and more importantly, the E. coli topoisomerase I
inhibition is extremely selective as DNA gyrase and mammalian
topoisomerases are not inhibited. The E. coli. Topo I IC50
(2.47ꢁ0.06 mM) for DPA 153 is even better than recently repor-
ted bisbenzimidazole derivative DMA (3.8 mM).26 It is plausible
that alkynyl linkers present in our benzimidiazoles interact with
the bacterial topoisomerase I enzyme leading to a stabilization
of the cleavable complex. The alkynyl chains likely interact with
the ternary complex as the bisbenzimidazole binds in the minor
groove of DNA. Our ndings suggest that the ternary complex
formed by the bacterial topoisomerase I has distinct sites for
small molecule recognition, as compared to those found in DNA
gyrase and mammalian topoisomerases, and these differences
could be further exploited for antibacterial drug development.
Further studies to investigate the mechanism of antibacterial
activity and enzyme inhibition are being investigated and will
be reported in due course.
Antibacterial activity
Compounds belonging to the bisbenzimidazole class of ligands
have shown profound antibacterial effect against a variety of
strains, which include methicillin-resistant Staphylococcus
aureus (MRSA) and vancomycin-resistant Entercoccus faeca-
lis.24,25 To discern if these inhibitors of E. coli topoisomerase I
are effective at inhibiting bacterial growth, we evaluated the
antibacterial effect of these compounds against both gram
positive and gram negative strains, as listed in Table 3. In cases
where a sharp inection in the bacterial growth was not
observed, the MIC is given as a range of values. As seen in Table
Experimental section
General methods
Unless otherwise specied, chemicals were purchased from
Aldrich (St. Louis, MO) or Fisher Scientic (Pittsburgh, PA) and
used without further purication. Hoechst 33258 and Hoechst
33242 were obtained as their hydrochloride salts and used
without further purication. All solvents were purchased from
VWR (West Chester, PA). Silica gel (32–65 mM mesh size) was
purchased from Sorbtech (Atlanta, GA). H NMR and 13C NMR
1
spectra were recorded on a Bruker Avance (300/500 MHz)
spectrometer. Chemical shi are given in ppm and are refer-
enced to residual solvent peaks (1H and 13C NMR). Mass
(MALDI-TOF) spectra were collected using a Bruker Microex
mass spectrometer. Ultra Violet (UV) spectra were collected on a
Varian (Walnut Creek CA) Cary 100 Bio UV-Vis spectropho-
tometer equipped with a thermoelectrically controlled 12 cell
holder.
Table 3 Minimal inhibitory concentrations (MIC) of the studied ligands
against various bacterial strains by microbroth dilution. S1 ¼ S. aureus
29213; S2 ¼ S. aureus 33591; S3 ¼ E. coli 25922; S4 ¼ P. aeruginosa
27853; S5 ¼ E. coli K 12; S6 ¼ E. faecalis 29212
MIC (mg mLꢀ1
)
Sample
S1
S2
S3
S4
S5
S6
Hoechst 33258
Hoechst 33342
DPA 151
DPA 152
DPA 153
$32
2–4
2–4
2–4
16
16–32
2–4
2–4
2–4
16
16
8–16
8
8–16
16
8–16
16
16
16
16
16
16
16
8
8
8–16
8–16
8–16
16–32
16–32
16–32
4
16–32
8
Synthesis
Synthesis of 4-(prop-2-ynyloxy)benzaldehyde (DPA 151a). To
a solution of p-hydroxybenzaldehyde (2.00 g, 16.3 mmol) in dry
dichloromethane (30.0 mL) and 1,4 dioxane (5.00 mL), triphenyl
DPA 154
2–4
2–4
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phosphine (6.30 g, 24.2 mmol) and propargyl alcohol (0.91 g, ethyl acetate (3 ꢄ 50 mL). Organic layers were combined and
16.3 mmol) were dissolved under argon and the solution was ice then dried over sodium sulfate. Volatiles were removed under
cooled. To this mixture, diisopropyl azodicarboxylate (DIAD) reduced pressure. The crude product was puried by column
(4.80 mL, 24.2 mmol) was added dropwise over a period of 15 chromatography on silica gel using hexanes–ethyl acetate (1 : 1–
min at 0 ꢂC. The contents were initially stirred at 0 ꢂC for 30 min 2 : 1 v/v) to yield the desired compound as a light yellow solid
and then allowed to slowly warm up to room temperature and (0.18 g, 73%): Rf ¼ 0.76 (in ethyl acetate); mp > 255 ꢂC (dec); IR
stirred overnight. Progress of the reaction was monitored using (neat, cmꢀ1) 3278, 2919, 2120, 1669; 1H NMR (500 MHz, DMSO-
thin layer chromatography (TLC) on silica gel. The volatiles were d6) d 13.25 (s, br, 1H), 10.05 (s, 1H), 8.18 (d, J ¼ 8.50 Hz, 2H),
removed under reduced pressure and the gummy residue was 8.10 (br, 1H), 7.77 (br, 1H) 7.68 (br, 1H) 7.20 (d, J ¼ 9.0 Hz, 2H),
redissolved in ethyl acetate–hexane (80.0 mL, 1 : 1 v/v). The 4.92 (d, J ¼ 2.66 Hz, 2H, –OCH2CCH), 3.65 (t, J ¼ 2.28 Hz, 1H,
reaction mixture was allowed to stand overnight in the refrig- –OCH2CCH); 13C NMR (125 MHz, DMSO-d6) d 193.0, 159.6,
erator. The precipitated solid was vacuum ltered and the 155.4, 154.1, 149.0, 144.2, 135.5, 131.5, 129.0, 123.4, 119.3,
ltrate was concentrated under reduced pressure. The crude 115.8, 114.2, 112.2, 79.4, 60.2, 56.1; MS (MALDI-TOF) m/z calcd
product was puried by column chromatography on silica gel for C17H12N2O2 [M]+ 276.09, found 277.66 ([M + H]+).
(hexanes–ethyl acetate, 100 : 0–70 : 30) to afford the desired
Synthesis of 6-(4-methylpiperazin-1-yl)-20-(4-(prop-2-ynyloxy)-
compound as a white solid (1.3 g, 50%): Rf ¼ 0.42 (hexanes– phenyl)-1H,30H-2,50-bibenzo[d]imidazole (DPA 151). To a solu-
ethyl acetate 7 : 3 v/v); mp 80–81 ꢂC; IR (neat, cmꢀ1) 3419, 2112, tion of 5-(4-methylpiperazin-1-yl)-2-nitroaniline (0.06 g, 0.27
1
1654; H NMR (500 MHz, CDCl3) d 9.91 (s, 1H), 7.87 (dd, J1 ¼ mmol) in ethanol (8.0 mL), 10% Pd–C (40.0 mg) was added and
8.82 Hz, J2 ¼ 1.94 Hz, 2H), 7.10 (dd, J1 ¼ 8.74 Hz, J2 ¼ 1.70 Hz, then it was hydrogenated for 6 h at the atmospheric pressure.
2H), 4.77 (d, J ¼ 2.36 Hz, 2H, –OCH2CCH), 2.60 (t, J ¼ 2.43 Hz, TLC on silica gel (ethyl acetate–methanol 8 : 2 v/v) showed
1H, –OCH2CCH); 13C NMR (125 MHz, CDCl3) d 190.7, 162.3, complete reduction of the starting material. Aer ltering the
131.8, 130.6, 115.1, 77.8, 76.3, 55.9.
catalyst over a bed of celite, 2-(4-(prop-2-ynyloxy)phenyl)-3H-
N-Methoxy-N-methyl-2-(4-(prop-2-ynyloxy)phenyl)-1H-benzo- benzoimidazole-5-carbaldehyde (0.08 g, 0.31 mmol) was added.
[d]imidazole-6-carboxamide (DPA 151-b). To a solution of N- To this solution, Na2S2O5 (30.0 mg, 0.16 mmol) in water
methoxy-N-methyl-3,4-dinitrobenzamide (0.90 g, 3.52 mmol) in (0.2 mL) was added and the mixture was reuxed for 14 h. The
ethanol (20.0 mL), 150 mg of 10% Pd–C was added. Hydroge- reaction mixture was allowed to come to the room temperature.
nation for 5 h at the atmospheric pressure afforded corre- The volatiles were evaporated under reduced pressure. The
sponding diamine. The diamine was used immediately aer crude product was puried by column chromatography on silica
ltration of the catalyst without further purication. 4-(Prop-2- gel using dichloromethane–methanol as eluent (0–18% meth-
ynyloxy)benzaldehyde (0.62 g, 3.87 mmol) and sodium meta- anol in dichloromethane) to afford the desired product as
bisulte (0.37 g, 1.93 mmol) in water (1.00 mL) were added into yellow solid (85 mg, 65%): Rf ¼ 0.23 (ethyl acetate–methanol
1
the diamine and the reaction mixture was reuxed for 12 h. The 8 : 2 v/v); mp 252–256 C; IR (neat, cmꢀ1) 3235, 2919, 2109; H
ꢂ
volatiles were evaporated under reduced pressure. The crude NMR (300 MHz, methanol-d4) d 8.25 (br, 1H), 8.06 (d, J ¼ 8.84
product was puried by column chromatography on silica gel Hz, 2H), 7.95 (dd, J1 ¼ 9.97 Hz, J2 ¼1.30 Hz, 1H), 7.69 (d, J ¼ 8.32
using dichloromethane–methanol (0–10% methanol in Hz, 1H), 7.51 (d, J ¼ 8.77 Hz, 1H), 7.15 (3H), 7.05 (s, 1H), 4.82 (d,
dichloromethane) as eluent to afford the desired product as a J ¼ 2.29 Hz, 2H), 3.24 (t, J ¼ 4.55 Hz, 4H), 3.04 (t, J ¼ 2.33 Hz,
pale yellow solid (0.55 g, 46%): Rf ¼ 0.53 (dichloromethane– 1H), 2.72 (t, J ¼ 4.64 Hz, 4 Hz), 2.42 (s, 3H) (some proton peaks
methanol 9 : 1, v/v); mp 245–246 ꢂC; IR (neat, cmꢀ1) 2974, 2124, are masked with the solvent peaks); 13C NMR (75 MHz, DMSO-
1
1617; H NMR (500 MHz, DMSO-d6) d 13.1 (s, 1H), 8.15 (d, J ¼ d6) d 159.1, 153.2, 152.1, 145.3, 144.5, 138.6, 136.4, 135.7, 128.6,
8.5 Hz, 2H), 7.91–7.79 (m, 1H), 7.67 (d, J ¼ 8.50 Hz, 1H), 7.55 (d, 123.5, 122.0, 120.7, 119.1, 116.5, 115.7, 111.9, 97.6, 79.4, 79.0,
J ¼ 8.00 Hz, two sets of doublets, 1H), 7.50–7.46 (m, 1H), 7.18 56.0, 55.3, 50.7 50.2, 46.2; ESI-HRMS (m/z) calcd for C28H27N6O
(dd, J1 ¼ 2.50 Hz, J2 ¼ 9.00 Hz, 2H), 4.92 (d, J ¼ 2.53 Hz, 2H, 462.2246, found 463.2237.
–OCH2CCH), 4.36 (d, J ¼ 2.12 Hz, 1H, –OCH2CCH), 3.58 (s, 3H),
Synthesis of 4-(hex-5-ynyloxy)benzaldehyde (DPA 152-a). To
3.35 (s, 3H); 13C NMR (125 MHz, DMSO-d6) d 170.1, 169.9, 159.4, an ice cold solution of p-hydroxy benzaldehyde (1.00 g, 8.18
159.3, 153.6, 153.1, 145.9, 137.0, 128.6, 123.1, 119.0, 115.8, mmol) in dry dichloromethane (15.0 mL) and dioxane (5.0 mL),
111.0, 79.0, 62.5, 56.1, 25.9; MS (MALDI-TOF) m/z calcd for 5-hexyn-1-ol (0.80 g, 8.18 mmol) and triphenyl phosphine
C
19H17N3O3 335.13, found 336.38 [M + H]+.
(3.17 g, 12.1 mmol) were added under argon. To this solution,
2-(4-(Prop-2-ynyloxy)phenyl)-1H-benzo[d]imidazole-6-carbal- DIAD (2.40 mL, 12.1 mmol) was added dropwise. The reaction
dehyde (DPA 151-c). To a solution of N-methoxy-N-methyl-2-(4-(prop- mixture was stirred at 0 ꢂC for 1 h followed by stirring at room
2-ynyloxy)phenyl)-1H-benzo[d]imidazole-6-carboxamide (0.30 temperature was 6 h. Volatiles were evaporated and the crude
g, 0.89 mmol) in tetrahydrofuran (THF)–ether (40 mL, 3 : 1 v/v), mixture was redissolved in ethyl acetate–hexanes (80.0 mL, 1 : 1
lithium aluminum hydride (0.13 g, 3.57 mmol) was added in v/v). The mixture was allowed to stand in the refrigerator for a
small portions at ꢀ70 ꢂC under argon and then the stirring was day and the precipitated solid was vacuum ltered. The ltrate
continued for 6 h while allowing the slush bath to warm up to containing the crude product was concentrated under reduced
ꢀ20 ꢂC. The reaction mixture was quenched by the addition of pressure. The crude mixture was puried on a silica gel column
saturated ammonium chloride solution (50 mL). The precipi- using hexanes–ethyl acetate (0–25% ethyl acetate in hexanes) as
tated grey solid was ltered off. The ltrate was extracted with eluent to yield the desired compound as colorless oil (1.4 g,
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85%): Rf ¼ 0.7 (hexanes–ethyl acetate 7 : 3); 1H NMR (300 MHz,
CDCl3) d 9.81 (s, 1H), 7.82 (dd, J1 ¼ 8.82 Hz, J2 ¼ 1.98 Hz, 2H),
7.00 (dd, J1 ¼ 8.71 Hz, J2 ¼ 1.75 Hz, 2H), 4.08 (d, J ¼ 6.25 Hz, 2H,
–OCH2CCH), 2.32–2.27 (m, 2H), 2.00–1.91 (m, 3H), 1.79–1.69
(m, 2H); 13C NMR (125 MHz, CDCl3) d 190.8, 164.1, 132.0, 130.7,
114.7, 84.8, 68.8, 67.7, 28.0, 24.8, 18.1.
Synthesis of 20-(4-(hex-5-ynyloxy)phenyl)-6-(4-methylpiper-
azin-1-yl)-1H,30H-2,50-bibenzo[d]imidazole (DPA 152). To
a
solution of 5-(4-methylpiperazin-1-yl)-2-nitroaniline (0.28 g,
1.00 mmol) in ethanol (40.0 mL), Pd–C (0.10 g) was added which
was followed by hydrogenation at atmospheric pressure for 5 h.
Charcoal was ltered off over a bed of celite. To this solution,
2-(4-(hex-5-ynyloxy)phenyl)-3H-benzimidazole-5-carbaldehyde
(0.35 g, 1.10 mmol) and a solution of Na2S2O5 (0.10 g, 0.55
mmol) in water (0.20 mL) were added and the mixture was
reuxed for 23 h. The reaction mixture was allowed to come to
room temperature. The ltrate was evaporated under reduced
pressure. Column chromatography on silica gel using
dichloromethane–methanol as eluent (0–15% methanol in
dichloromethane) afforded the desired product as yellow solid
(0.36 g, 72%): Rf ¼ 0.15 (ethyl acetate–methanol 8 : 2 with two
Synthesis of 2-(4-(hex-5-ynyloxy)phenyl)-N-methoxy-N-methyl-
3H-benzoimidazole-5-carboxamide (DPA 152-b). To a solution
of N-methoxy-N-methyl-3,4-dinitrobenzamide (1.00 g, 3.91
mmol) in ethanol (30.0 mL), 10% Pd–C (0.10 g) was added.
Hydrogenation for 5 h at atmospheric pressure yielded corre-
sponding diamine which was used immediately aer ltration
of the catalyst. 4-(Hex-5-ynyloxy)benzaldehyde (0.82 g, 4.10
mmol) and sodium metabisulte (0.39 g, 2.05 mmol) in water
(0.50 mL) were added into it. The reaction mixture was reuxed
for 8 h. Volatiles were evaporated under reduced pressure.
Column chromatography on silica gel using dichloromethane–
methanol (0–8% methanol in dichloromethane) as eluent
afforded the desired product as pale brown oil (1.1 g, 74%): Rf ¼
drops of triethylamine); mp 210–220 C; IR (neat, cmꢀ1) 3293
ꢂ
1
(alkyne C–H stretch), 2116 (alkyne C–C stretch), 1618; H NMR
(500 MHz, methanol-d4) d 8.21 (s, 1H), 8.00 (dd, J1 ¼ 8.60 Hz,
J2 ¼ 2.06 Hz, 2H), 7.92 (dd, J1 ¼ 8.64 Hz, J2 ¼ 2.02 Hz, 1H) 7.66
(d, J ¼ 8.30 Hz, 1H), 7.51 (d, J ¼ 9.00 Hz, 1H), 7.12 (d, J ¼ 8.80 Hz,
1H), 7.04 (d, J ¼ 8.54 Hz, 1H), 7.02 (dd, J1 ¼ 8.84 Hz, J2 ¼ 2.06 Hz,
2H), 4.01 (t, J ¼ 2.26 Hz, 2H, –OCH2CCH), 3.24 (t, J ¼ 4.62 Hz,
4H), 2.75 (t, J ¼ 4.66 Hz, 4H), 2.53 (s, br, 1H), 2.44 (s, 3H), 2.28–
2.24 (m, br, 2H), 1.93–1.86 (m, br, 2H), 1.70–1.77 (m, br, 2H)
(imino protons were not observed because of exchange with the
NMR solvent); ESI-HRMS (m/z) calcd for C31H33N6O 505.2716,
found 505.2701; HPLC: tR 2.87 min, purity 96.1% (see procedure
for method details).
0.75 (in dichloromethane–isopropanol 9 : 1 v/v); IR (neat, cmꢀ1
)
3297 (alkyne C–H stretch), 2938 (aromatic C–H stretch), 2116
(alkyne C–C stretch), 1723; 1H NMR (300 MHz, DMSO-d6) d 8.13
(d, br, J ¼ 13.7 Hz, 2H), 7.93–7.80 (1H), 7.67 (dd, J1 ¼ 8.27 Hz,
J2 ¼ 8.36 Hz, 1H), 7.48 (d, J ¼ 8.62 Hz, 1H), 7.11 (d, br, J ¼ 8.87
Hz, 2H), 4.07 (t, J ¼ 4.64 Hz, 2H, –OCH2CCH), 3.57 (s, 3H), 3.30
(s, 3H), 2.80 (t, J ¼ 2.64 Hz, 1H, –OCH2CCH), 2.28–2.22 (m, 2H),
1.88–1.79 (m, 2H), 1.67–1.57 (m, 2H) (imino proton was not
observed); 13C NMR (75 MHz, DMSO-d6) d 170.2, 160.7, 153.8,
146.0, 143.4, 137.0, 128.7, 123.0, 119.0, 115.3, 111.8, 84.7, 71.8,
67.6, 60.9, 34.1, 28.2, 25.0, 21.1, 17.9; MS (MALDI-TOF) m/z calcd
for C22H23N3O3 [M]+ 377.17, found 378.34 [M + H]+.
Synthesis of 4-(undec-10-ynyloxy)benzaldehyde (DPA153-a).
To an ice cold solution of p-hydroxybenzaldehyde (0.50 g, 4.09
mmol) in dry dichloromethane–dioxane mixture (15.0 mL 2 : 1
v/v), triphenyl phosphine (1.60 g, 6.05 mmol) and 10-undecyn-1-
ol (0.70 g, 6.05 mmol) were dissolved and kept at 0 ꢂC. To this,
diisopropyl azodicarboxylate (1.22 g, 6.05 mmol) was added
dropwise over a period of 15 min. The contents were initially
stirred at 0 ꢂC for 30 min and then allowed to warm up to room
temperature and stirred overnight. The crude mixture was
concentrated and redissolved in ethyl acetate–hexanes mixture
(50 mL, 1 : 1 v/v) and kept in the refrigerator for a day. The
precipitated solid was ltered off and the ltrate was concen-
trated under reduced pressure. Column chromatography on
silica gel using hexanes–ethyl acetate as eluent (0–50% ethyl
acetate in hexanes) afforded the desired compound as white
solid (0.58 mg, 52%): Rf ¼ 0.54 (hexanes–ethyl acetate 7 : 3 v/v);
Synthesis of 2-(4-(hex-5-ynyloxy)phenyl)-3H-benzoimidazole-
5-carbaldehyde (DPA 152-c). To a stirred suspension of 2-(4-
(hex-5-ynyloxy)phenyl)-N-methoxy-N-methyl-3H-benzoimidazole-
5-carboxamide (0.77 g, 2.04 mmol) in dry THF (40.0 mL),
lithium aluminum hydride (0.31 g, 8.17 mmol) was added in
small portions at ꢀ70 ꢂC under argon and the stirring was
ꢂ
continued for 12 h at 0 C. TLC was used to monitor the prog-
ress of the reaction. The reaction mixture was quenched by the
addition of saturated ammonium chloride solution (100 mL).
The resulting grey precipitate was ltered off. The ltrate was
extracted by ethyl acetate (3 ꢄ 100 mL). Organic layers were
combined and dried over sodium sulfate. Volatiles were
removed under reduced pressure. Column chromatography on
silica gel using hexanes–ethyl acetate (1 : 1–2 : 1) as eluent
afforded the desired compound as light yellow liquid (0.47 g,
72%): Rf ¼ 0.66 (ethyl acetate–hexanes 6 : 4 v/v); mp 156–158 ꢂC;
IR (neat, cmꢀ1) 3289 (alkyne C–H stretch 2116 (alkyne C–C
stretch), 1696; 1H NMR (300 MHz, methanol-d4) d 13.20 (br, 1H),
9.89 (s, 1H), 7.96 (br, 1H) 7.87 (dd, J1 ¼ 8.89 Hz, J2 ¼ 2.01 Hz,
2H), 7.69 (dd, J1 ¼ 9.8 Hz, J2 ¼ 1.44 Hz, 1H), 7.56 (d, J ¼ 8.32 Hz,
1H), 6.91 (dd, J1 ¼ 8.92 Hz, J2 ¼ 2.02 Hz, 2H), 3.93–3.89 (t, J ¼
5.91 Hz, 2H, –OCH2CCH), 2.25–2.20 (m, 3H), 1.88–1.79 (m, 2H),
1.69–1.59 (m, 2H); 13C NMR (75 MHz, DMSO-d6) d 192.4, 171.5,
161.5, 155.2, 131.7, 128.3, 123.7, 121.0, 114.6, 83.3, 68.5, 67.4,
60.1, 27.9, 24.8, 19.9, 17.4, 16.5; MS (MALDI-TOF) m/z calcd for
mp 65–68 C; IR (neat, cmꢀ1) 3421 (alkyne C–H stretch), 2097
ꢂ
(alkyne C–C stretch), 1684; 1H NMR (300 MHz, CDCl3) d 9.90 (s,
1H), 7.84 (dd, J1 ¼ 7.1 Hz, J2 ¼ 1.9 Hz, 2H), 7.00 (dd, J1 ¼ 7.1 Hz,
J2 ¼ 1.9 Hz, 2H), 4.06 (t, J ¼ 6.50 Hz, 2H, –OCH2CCH), 2.23–2.18
(m, 2H), 1.96 (t, J ¼ 2.71 Hz, 1H, –OCH2CCH), 1.88–1.79 (m, 2H),
1.58–1.27 (12H); 13C NMR (125 MHz, CDCl3) d 190.8, 164.2,
131.9, 129.7, 114.7, 84.7, 74.3 68.4, 68.1, 29.3, 29.1, 29.0, 28.7
28.4, 21.6, 18.4.
Synthesis of N-methoxy-N-methyl-2-(4-(undec-10-ynyloxy)-
phenyl)-1H-benzo[d]imidazole-6-carboxamide (DPA 153-b). To
solution of N-methoxy-N-methyl-3,4-dinitrobenzamide (0.40 g,
1.37 mmol) in ethanol–ethyl acetate mixture (15.0 mL), 10%
C
20H18N2O2 [M]+ 318.14, found 319.25 [M + H]+.
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Pd–C (0.09 g) was added. Hydrogenation at atmospheric pres- ltrate, 2-(4-(undec-10-ynyloxy)phenyl)-1H-benzo[d]imidazole-6-
sure for 5 h afforded corresponding diamine which was used carbaldehyde (0.40 g, 1.02 mmol) and a solution of Na2S2O5
without purication aer ltration of the catalyst. 4-(Undec-10- (0.19 g, 1.02 mmol) in water (1.0 mL) was added and the mixture
ynyloxy)benzaldehyde (0.49 g, 1.78 mmol) and sodium meta- was reuxed for 14 h. Volatiles were removed under reduced
bisulte (0.26 g, 1.37 mmol) in water (1.00 mL) were added into pressure. Column chromatography on silica gel in dichloro-
it and the reaction mixture was reuxed overnight. Volatiles methane–methanol (0–15% methanol in dichlromethane)
were evaporated under reduced pressure. The crude product afforded the pure product as yellow solid (0.29 g, 50%): Rf ¼ 0.32
was puried on a silica gel column using dichlromethane– (dichlromethane–methanol 8 : 2); mp 170–175 ꢂC; IR (neat,
1
methanol (0–10% methanol in dichloromethane) as eluent to cmꢀ1) 2919 (aromatic C–H stretch), 1614; H NMR (300 MHz,
afford the desired product as slightly yellow gummy solid (0.60 methanol-d4) d 8.20 (br, 1H), 7.95–7.89 (br, 3H), 7.63 (d, J ¼ 8.5
g, 85%): Rf ¼ 0.67 (dichloromethane–isopropanol 9 : 1 v/v); IR Hz, 1H), 7.50 (d, J ¼ 8.7 Hz, 1H), 7.10 (d, J ¼ 2.0 Hz, 1H), 7.01
(neat, cmꢀ1) 3305 (alkyne C–H stretch), 2112 (alkyne C–C (dd, J1 ¼ 8.8 Hz, J2 ¼ 2.2 Hz, 1H), 6.93 (d, J ¼ 8.5 Hz, 2H), 3.85 (t,
stretch), 1614; 1H NMR (300 MHz, acetone-d6) d 8.17 (d, J ¼ 8.4 J ¼ 6.2 Hz, 2H), 3.21 (t, J ¼ 4.5 Hz, 4H), 2.72 (t, J ¼ 4.8 Hz, 4H),
Hz, 2H), 8.03–7.87 (br, 1H), 7.68–7.50 (m, 2H), 7.11 (d, J ¼ 8.7 2.42 (s, 3H), 2.19–2.11 (m, br, 3H), 1.73–1.63 (m, 2H), 1.52–1.22
Hz, 2H), 4.14 (t, J ¼ 6.2 Hz, 2H, –OCH2CCH), 3.62 (s, 3H), 3.34 (s, (br, 12H) (imino protons were not observed because of exchange
3H), 2.32 (t, J ¼ 2.5 Hz, 1H, –OCH2CCH), 2.21–2.16 (m, 2H), with the NMR solvent); 13C NMR (75 MHz, DMSO-d6) d 160.7,
1.88–1.78 (m, 2H), 1.57–1.30 (m, 12H) (imino proton was not 153.1, 148.0, 145.4, 144.6, 139.3, 136.5, 135.7, 128.6, 124.7,
observed because of exchange with the NMR solvent); 13C NMR 122.6, 120.7, 119.0, 115.3, 115.1, 111.8, 85.02, 71.5, 71.5, 68.1,
(75 MHz, acetone-d6) d 170.1, 160.9, 153.4, 138.9, 137.2, 128.3, 61.5, 55.2, 46.0, 33.6, 29.3, 29.2, 29.1, 28.9, 28.5, 28.4, 25.9, 18.1;
128.2, 122.7, 122.4, 118.2, 117.5, 114.7, 113.8, 101.2, 84.1, 68.9, ESI-HRMS (m/z) calcd for C36H43N6O 575.3498, found 565.3497;
67.9, 60.2, 33.3, 29.7–28.2 (peaks masked with the acetone-d6 HPLC: tR 8.04 min, purity 98.4% (see procedure for method
peak), 25.8, 27.8; MS (MALDI-TOF) m/z calcd for C27H33N3O3 details).
447.57, found 448.69 ([M + H]+).
Synthesis 4-(4-(prop-2-ynyloxy)butoxy)benzaldehyde (DPA
Synthesis of 2-(4-(undec-10-ynyloxy)phenyl)-1H-benzo[d]- 154-a). To a solution of p-hydroxybenzaldehyde (4.00 g, 32.7
imidazole-6-carbaldehyde (DPA 153-c). To a solution of N- mmol) in dry dichloromethane–dioxane (40.0 mL, 3 : 1 v/v),
methoxy-N-methyl-2-(4-(undec-10-ynyloxy)phenyl)-1H-benzo[d]- triphenyl phosphine (12.6 g, 48.0 mmol) and 4-(prop-2-ynyloxy)
imidazole-6-carboxamide (0.80 g, 1.78 mmol) in THF–ether butan-1-ol (4.20 g, 32.7 mmol) were dissolved and the solution
(80.0 mL, 3 : 1), lithium aluminum hydride (0.20 g, 5.28 mmol) was ice cooled. To this mixture, diisopropyl azodicarboxylate
ꢂ
was added at ꢀ78 C under argon and then allowed to stir at (9.70 g, 48.0 mmol) was added drop wise over a period of 15 min
0 ꢂC for 8 h. The reaction mixture was quenched by the addition at 0 ꢂC. The contents were initially stirred at 0 ꢂC for 30 min and
of saturated ammonium chloride solution (75.0 mL). The then at room temperature overnight. The solvents were
resulting grey precipitate was ltered off. The ltrate was removed under reduced pressure and the mixture was redis-
extracted with ethyl acetate (3 ꢄ 100 mL). Organic layers were solved in ethyl acetate–hexanes (100 mL, 1 : 1 v/v) and allowed
combined and dried over sodium sulfate. Volatiles were to stand in the refrigerator overnight. The precipitated solid was
removed under reduced pressure. Column chromatography on ltered off and the ltrate was concentrated under reduced
silica gel using hexanes–ethyl acetate as eluent afforded the pressure. Column chromatography on silica gel using hexanes–
desired compound as a light yellow solid (0.42 g, 60%): Rf ¼ 0.78 ethyl acetate as eluent, afforded the desired compound as pale
(ethyl acetate–hexanes 6 : 4); mp 126–128 ꢂC; IR (neat, cmꢀ1
)
yellow oil (6.2 g, 82%): Rf ¼ 0.55 (hexanes–ethyl acetate 7 : 3 v/v);
1
3305 (alkyne C–H stretch), 2108 (alkyne C–H stretch), 1503; H IR (neat, cmꢀ1) 3289 (alkyne C–H stretch), 2116 (alkyne C–C
1
NMR (300 MHz, acetone-d6) d 10.08 (s, 1H), 8.20 (d, J ¼ 8.8 Hz, stretch), 1688; H NMR (500 MHz, CDCl3) d 9.84 (s, 1H), 7.79
2H), 8.14 (1H), 7.80 (dd, J1 ¼ 8.3 Hz, J2 ¼ 1.3 Hz, 1H), 7.72 (d, J ¼ (dd, J1 ¼ 1.79 Hz, J2 ¼ 8.81 Hz, 2H), 6.96 (d, J ¼ 8.81 Hz, 2H), 4.13
8.3 Hz, 1H), 7.11 (d, J ¼ 8.9 Hz, 2H), 4.10 (t, J ¼ 6.5 Hz, 2H, (d, J ¼ 2.39 Hz, 2H), 4.04 (t, J ¼ 6.27 Hz, 2H), 3.57 (t, J ¼ 6.42 Hz,
–OCH2CCH), 2.32 (t, J ¼ 2.7 Hz, 1H, –OCH2CCH), 2.21–2.15 (m, 2H), 2.45 (t, J ¼ 2.38 Hz, 1H), 1.92–1.87 (m, 2H), 1.79–1.74 (m,
2H), 1.87–1.78 (m, 2H), 1.56–1.37 (m, 12H) (imino proton was 2H); 13C NMR (125 MHz, CDCl3) 190.7, 164.1, 131.9, 129.8,
not observed because of exchange with the NMR solvent); 13C 114.7, 79.9, 74.3, 69.4, 67.9, 58.0, 26.0, 25.8.
NMR (125 MHz, acetone-d6) d 191.5, 161.3, 155.3, 154.0, 149.3,
Synthesis of N-methoxy-N-methyl-2-(4-(4-(prop-2-ynyloxy)-
144.5, 139.7, 138.9, 135.3, 131.8, 128.4, 127.9, 123.3, 122.1, butoxy)phenyl)-1H-benzo[d]imidazole-6-carboxamide (DPA 154-
119.0, 114.9, 113.7, 112.9, 111.3, 84.0, 68.8, 67.9, 33.5, 28.1; MS b). To solution of N-methoxy-N-methyl-3,4-dinitrobenzamide
(MALDI-TOF) m/z calcd for C25H28N2O2 388.50, found 390.12 (0.73 g, 2.86 mmol) in ethanol–ethyl acetate mixture (40.0 mL,
([M + 2H]+).
3 : 1 v/v), 10% Pd–C (0.30 g) was added. Hydrogenation at the
6-(4-Methylpiperazin-1-yl)-20-(4-(undec-10-ynyloxy)phenyl)- atmospheric pressure for 5 h yielded corresponding diamine
1H,30H-2,50-bibenzo[d]imidazole (DPA 153). To a solution of 5- which was used without further characterization aer ltration
(4-methylpiperazin-1-yl)-2-nitroaniline (0.24 g, 1.02 mmol) in of the catalyst (Rf ¼ 0.46 in dichloromethane–methanol 9 : 1, v/
ethanol–ethyl acetate mixture (30.0 mL), 10% Pd–C (0.10 g) was v). 4-(4-(Prop-2-ynyloxy)butoxy)benzaldehyde (0.70 g, 2.86
added followed by hydrogenation at atmospheric pressure for 5 mmol) and sodium metabisulte (0.54 g, 2.86 mmol) in water
h. TLC on silica gel showed complete reduction of the starting (1.00 mL) were added into it and the reaction mixture was
material. Charcoal was ltered over a bed of celite. To the reuxed for 6 h. Volatiles were evaporated under reduced
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pressure. The crude product was puried by column chroma- come to room temperature. Volatiles were removed under
tography on a silica gel using dichloromethane–methanol as reduced pressure. The crude mixture was puried by column
eluent to afford the desired product as pale yellow gummy solid chromatography on a silica gel column using dichloromethane–
(0.70 g, 61%): Rf ¼ 0.50 (in dichloromethane–methanol 9 : 1 v/ methanol as eluent (0–15% methanol in dichloromethane)
v); IR (neat, cmꢀ1) 3231 (alkyne C–H stretch), 2230 (alkyne C–C which afforded the desired product as yellow solid (0.29 g, 60%):
stretch), 1608; 1H NMR (500 MHz, methanol-d4) d 7.98 (dd, J1 ¼ Rf ¼ 0.40 (dichloromethane–methanol 8 : 2 v/v); mp 150–155 ꢂC;
8.90 Hz, J2 ¼ 2.00 Hz, 2H), 7.93 (br, 1H), 7.59–7.55 (m, 2H), 6.99 IR (neat, cmꢀ1) 2236 (alkyne C–C stretch), 1630, 1433; 1H NMR
(dd, J1 ¼ 8.65, J2 ¼ 2.00, 2H), 4.13 (d, J ¼ 2.39 Hz, 2H, (300 MHz, methanol-d4) d 8.21 (d, J ¼ 1.11 Hz, 1H) 7.98 (dd, J1 ¼
–CH2OCH2CCH), 3.97 (t, J ¼ 6.27 Hz, 2H, –OCH2CH2–), 3.61 (s, 8.86 Hz, J2 ¼ 2.00 Hz, 2H), 7.91 (dd, J1 ¼ 8.46 Hz, J2 ¼ 1.75 Hz,
3H), 3.55 (t, J ¼ 6.40 Hz, 2H), 3.39 (s, 3H), 2.85 (t, J ¼ 2.26 Hz, 1H), 7.65 (d, J ¼ 8.46 Hz, 1H), 7.50 (d, J ¼ 8.86 Hz, 1H), 7.14 (d,
1H, –CH2OCH2CCH), 1.84–1.78 (m, 2H), 1.74–1.68 (m, 2H) J ¼ 2.00 Hz, 1H), 7.04–7.00 (m, 3H), 4.17 (d, J ¼ 2.47 Hz, 2H),
(imino proton was not observed because of exchange with the 4.01 (t, J ¼ 6.15 Hz, 2H), 3.59 (t, J ¼ 6.14 Hz, 2H), 3.34–3.30
NMR solvent); 13C NMR (75 MHz, methanol-d4) d 170.5, 161.1, (some peaks are masked with the NMR solvent signal) 3.03 (t, J
154.1, 140.5, 138.4, 128.1, 127.6, 122.7, 121.2, 114.6, 113.6, 79.5, ¼ 4.63 Hz, 4H), 2.86 (t, J ¼ 2.32 Hz, 1H), 2.66 (s, 3H), 1.88–1.72
74.3, 69.4, 69.1, 67.5, 60.1, 57.3, 33.4, 25.74, 25.70; MS (MALDI- (m, 4H) (imino protons were not observed because of exchange
TOF) m/z calcd for C23H25N3O4 [M]+, 407.18, found 407.10 with the NMR solvent); 13C NMR (75 MHz, methanol-d4) d 161.1,
([M]+).
Synthesis
153.9, 152.4, 147.4, 138.9, 134.5, 129.9, 128.1 (two peaks), 124.2,
of 2-(4-(4-(prop-2-ynyloxy)butoxy)phenyl)-1H- 121.3, 121.0, 115. 1, 115.0, 114.5, 112.1, 101.3, 79.4, 74.2, 71.2,
benzo[d]imidazole-6-carbaldehyde (DPA 154-c). To a solution of 69.1, 67.5, 63.5, 57.3, 54.2, 49.3, 43.5, 25.7; ESI-HRMS (m/z) m/z
N-methoxy-N-methyl-2-(4-(4-(prop-2-ynyloxy)butoxy)phenyl)-1H- calcd for C32H35N6O2 535.2821, found 535.2814; HPLC: tR 2.46
benzo[d]imidazole-6-carboxamide (0.70 g, 1.71 mmol) in THF– min, purity 98.6% (see procedure for method details).
ether (80.0 mL, 3 : 1), lithium aluminum hydride (0.26 g, 6.58
mmol) was added at ꢀ78 ꢂC and then allowed to warm up and HPLC analysis
ꢂ
stir at 0 C for 6 h. The reaction mixture was quenched by the
HPLC analysis of compounds DPA 151–154 was performed on
addition of saturated ammonium chloride solution (100 mL).
HP1100 series analytical HPLC instrument. The experiments
The grey solid that precipitated out was ltered off. The ltrate
were performed on a Supelcosil LC-18S column using the
following gradient conditions.
was extracted with ethyl acetate (3 ꢄ 75 mL). Organic layers
were combined and dried over sodium sulfate. Volatiles were
DPA 151: 40% B in A with initial hold for 2 minutes and then
removed under reduced pressure. The crude product was puri-
ed by column chromatography on silica gel using hexanes–
ethyl acetate (0–80% ethyl acetate in hexanes) as eluent to yield
equilibrate at 40% B in A to 100% B over 8 minutes at a ow rate
of 2.0 mL minꢀ1; DPA 152: 60% B in A with initial hold for
2 minutes and then equilibrate at 60% B in A to 100% B over
the desired compound as a light yellow liquid (0.33 g, 55%); Rf ¼
0.65 (ethyl acetate–hexane 8 : 2 v/v); IR (neat, cmꢀ1) 2945
(aromatic C–H stretch), 2066 (alkyne C–C stretch), 1686; 1H
NMR (500 MHz, methanol-d4) d 9.99 (s, 1H), 8.08 (s, br, 1H), 8.00
(dd, J1 ¼ 8.82 Hz, J2 ¼ 1.95 Hz, 2H), 7.80 (dd, J1 ¼ 8.28 Hz, J2 ¼
1.46 Hz, 1H), 7.67 (s, br, 1H), 7.05 (dd, J1 ¼ 9.06 Hz, J2 ¼ 2.01 Hz,
2H), 4.18 (d, J ¼ 2.32 Hz, 2H, –CH2OCH2CCH), 4.05 (t, J ¼ 6.32
Hz, 2H, –OCH2CH2–), 3.61 (t, J ¼ 6.27 Hz, 2H), 2.87 (t, J ¼ 2.56
Hz, 1H, –CH2OCH2CCH), 1.91–1.85 (m, 2H), 1.80–1.75 (m, 2H)
(imino proton was not observed because of exchange with the
NMR solvent); 13C NMR (75 MHz, methanol-d4) d 192.4, 161.4,
131.7, 128.3, 123.7, 120.0, 114.7, 79.4, 74.2, 69.1, 67.5, 57.3,
29.13, 25.73, 25.70 (four aromatic carbon peaks were not
observed likely because of aggregation of the molecule); MS
(MALDI-TOF) m/z for C21H20N2O3 [M]+ calcd 348.15, found
349.48 [M + H]+.
8 minutes at a ow rate of 2.0 mL minꢀ1; DPA 153: 60% B in A
with initial hold for 2 minutes and then equilibrate at 60% B in A
to 100% B over 8 minutes. This was followed by 100% B over next
ve minutes at a ow rate of 2.0 mL minꢀ1; DPA 154: 60% B in A
with initial hold for 2 minutes and then equilibrate at 60% B in A
to 100% B over 8 minutes. This was followed by 100% B over next
ve minutes at a ow rate of 2.0 mL minꢀ1. Where, A – H2O
containing 0.1% triuoroacetic acid and B – 95 : 5 CH3CN–H2O.
Nucleic acids
Nucleic acids were purchased from Integrated DNA Technolo-
gies (Coraville, IA). The concentration of the nucleic acid was
determined using the extinction coefficient provided by the
supplier. The DNA duplex was prepared by heating the dA60 and
dT60 in equimolar ratio in buffer 10 mM sodium cacodylate, 0.1
mM EDTA and 100 mM NaCl at pH 7.0 at 95 ꢂC for 15 minutes
and then slowly allowing it to cool back to room temperature.
Aer two days of incubation, the duplex formation was checked
by UV thermal denaturation experiments. The stock solution
was stored at 4 ꢂC and diluted to desired concentrations as
required.
Synthesis of 6-(4-methylpiperazin-1-yl)-20-(4-(4-(prop-2-ynyloxy)-
butoxy)phenyl)-1H,30H-2,50-bibenzo[d]imidazole (DPA 154). To
a solution of 5-(4-methylpiperazin-1-yl)-2-nitroaniline (0.21 g,
0.91 mmol) in ethanol–ethyl acetate mixture (20.0 mL), Pd–C
(0.15 g) was added and the mixture was hydrogenated for 5 h at
the atmospheric pressure. Charcoal was ltered off. To this
solution,
2-(4-(4-(prop-2-ynyloxy)butoxy)phenyl)-1H-benzo[d]-
Ultra violet (UV) thermal denaturation experiments
imidazole-6-carbaldehyde (0.32 g, 0.91 mmol) and a solution of
Na2S2O5 (0.17 g, 0.91 mmol) in water (1.00 mL) were added. The All UV spectra were obtained on a 12 cell holder Cary 1E UV-Vis
mixture was reuxed for 12 h following which it was allowed to spectrophotometer equipped with temperature controller.
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Quartz cells with 1 cm path length were used for all the exper- calculated as the amount of the drug for which 50% of the DNA
iments. Spectrophotometer stability and wavelength alignment was still in a (ꢀ) supercoiled state.
were checked prior to initiation of each thermal denaturation
experiment. For all experiments, the samples were prepared by Inhibition assays against E. coli DNA gyrase
diluting a stock sample. The melting of DNA with and without
The reaction mixture (30 mL) contained 35 mM Tris–HCl at pH
the ligand was performed at a heating rate of 0.2 ꢂC minꢀ1
.
7.5, 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 1.75 mM ATP, 5 mM
spermidine, 0.1 mg mLꢀ1 BSA, 6.5% glycerol, 250 ng of the
relaxed plasmid pBAD-GFPuv, 0.5 units of E. coli DNA gyrase,
and one of the compounds at a nal concentration that ranges
from 1 to 50 mM. All components were assembled on ice and
Samples were brought back to 20 ꢂC aer the experiment. All UV
melting experiments were monitored at 260 nm. For the Tm
determinations, derivatives were used. Data points were recor-
ꢂ
ded every 1.0 C. The DNA concentration was 1 mM per duplex
while the ligand concentration was 10 mM. All ligand solutions
were prepared in dimethyl sulfoxide (DMSO) as concentrated
stock solution (6–10 mM) and diluted to desired concentrations
in buffer.
ꢂ
incubated for 30 min at 37 C. Aer the incubation, the reac-
tions were terminated by extraction with an equal volume of
phenol. The topological state of the DNA samples was analyzed
with 1% agarose gel electrophoresis in 1 ꢄ TAE buffer, pH 7.8.
Following electrophoresis, the agarose gel was stained with
ethidium bromide, destained, and photographed under UV
light. The intensity of DNA topoisomers was determined using
KODAK 1D Image Analysis Soware.
Minimum inhibitory concentration (MIC) determination
Bacteria used in this study were Staphylococcus aureus ATCC
29213, Escherichia coli ATCC 25922, Escherichia coli K12,
Staphylococcus aureus ATCC 33591, Pseudomonas aeruginosa
ATCC 27853, and Enterococcus faecalis ATCC 29212. All
compounds were tested by the microbroth dilution method
following Clinical and Laboratory Standards Institute guide-
lines.27 Briey, Mueller–Hinton broth (Difco Laboratories, Bec-
ton Dickinson) was inoculated with each organism and
incubated at 37 ꢂC with shaking to establish logarithmic
growth. Following incubation, each culture was pelleted by
centrifugation (3500 ꢄ g for 5 min) and resuspended in 0.85%
sterile saline solution to an optical density at 625 nm of 0.1.
Samples were tested in triplicate using 96 well microplates
(Corning Costar Corp. Cambridge, MA), yielding nal bacterial
concentrations of 5 ꢄ 105 CFU mLꢀ1, and incubated for 24 h at
37 ꢂC. Following incubation, optical densities of each well were
determined with a mQuant microplate spectrophotometer
(BioTek Instruments, Inc., Winooski, VT) at 625 nm. The MIC
was dened as the lowest concentration needed to completely
inhibit growth as compared to no treatment controls.
Inhibition assays against human DNA topoisomerase I
The inhibition assays were used to determine the activities of
the newly synthesized compounds against human DNA top-
oisomerase I. The reaction mixture (25 mL) contained 20 mM
Tris–HCl at pH 7.9, 50 mM KAc, 10 mM Mg(Ac)2, 1 mM DTT, 1
mg mLꢀ1 BSA, 250 ng of the supercoiled plasmid pBAD-GFPuv, 2
units of human DNA topoisomerase I, and one of the
compounds at a nal concentration that ranges from 5 to 50
mM. All components were assembled on ice and incubated for
30 min at 37 ꢂC. Aer the incubation, the reactions were
terminated by extraction with an equal volume of phenol. The
topological state of the DNA samples was analyzed with 1%
agarose gel electrophoresis in 1 ꢄ TAE buffer, pH 7.8. Following
electrophoresis, the agarose gel was stained with ethidium
bromide, destained, and photographed under UV light. The
intensity of DNA topoisomers was determined using KODAK 1D
Image Analysis Soware. The percentage of (ꢀ) supercoiled
DNA was calculated from a comparison of the intensity of the
(ꢀ) supercoiled band with the total intensity of all DNA top-
oisomers. The IC50 was calculated as the amount of the drug for
which 50% of the DNA was in a (ꢀ) supercoiled state.
Inhibition assays against E. coli DNA topoisomerase I
Inhibition assays were used to determine the activities of the
newly synthesized compounds against E. coli DNA top-
oisomerase I. The reaction mixture (30 mL) contained 20 mM
Tris–HCl at pH 7.9, 50 mM KAc, 10 mM Mg(Ac)2, 1 mM DTT, 1
Inhibition assays against human DNA topoisomerase II
mg mLꢀ1 BSA, 150 ng of the supercoiled plasmid pBAD-GFPuv, 6 The reaction mixture (25 mL) contained 20 mM Tris–HCl at pH
nM of E. coli topoisomerase I, and one of the compounds at a 7.9, 50 mM KAc, 10 mM Mg(Ac)2, 1 mM DTT, 1 mg mLꢀ1 BSA,
specied concentration that ranges from 0.5 to 45 mM. All 200 ng supercoiled plasmid pBAD-GFPuv, 4 units of human
components were assembled on ice and incubated for 15 min at topoisomerase II, and one of the compounds at a nal
37 ꢂC. Aer the incubation, the reactions were terminated by concentration that ranges from 5 to 50 mM. All components
extraction with an equal volume of phenol. The topological state were assembled on ice and incubated for 30 min at 37 ꢂC. Aer
of the DNA samples was analyzed with 1% agarose gel electro- the incubation, the reactions were terminated by extraction with
phoresis in 1 ꢄ TAE buffer, pH 7.8. Following electrophoresis, an equal volume of phenol. The topological state of the DNA
the agarose gel was stained with ethidium bromide, destained, samples was analyzed with 1% agarose gel electrophoresis in 1
and photographed under UV light. The intensity of DNA top- ꢄ TAE buffer, pH 7.8. Following electrophoresis, the agarose gel
oisomers was determined using KODAK 1D Image Analysis was stained with ethidium bromide, destained, and photo-
Soware. The percentage of (ꢀ) supercoiled DNA was calculated graphed under UV light. The intensity of DNA topoisomers was
from a comparison of the intensity of the (ꢀ) supercoiled band determined using KODAK 1D Image Analysis Soware. The
with the total intensity of all DNA topoisomers. The IC50 was percentage of (ꢀ) supercoiled DNA was calculated from a
824 | Med. Chem. Commun., 2014, 5, 816–825
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Concise Article
MedChemComm
comparison of the intensity of the (ꢀ) supercoiled band with the
total intensity of all DNA topoisomers. The IC50 was calculated
as the amount of the drug for which 50% of the DNA was in a (ꢀ)
supercoiled state.
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DU 145 cell line was cultured according to ATCC protocols. Cells
were harvested using trypsin–EDTA solution and counted using
trypan blue exclusion. Cells were seeded at a volume of 100 ml
per well in the wells of tissue culture treated 96 well plates at a
density of 10 ꢄ 105 cells per well. Seeded 96 well plates were
ꢂ
returned to incubator (37 C, 5% CO2) for twenty-four hours to
resume exponential growth.
Test compounds (Hoechst 33258, Hoechst 33342, DPA 151,
DPA 152, DPA 153 and DPA 154) and known drug control
(cisplatin) were diluted in appropriate culture media to the
following concentrations: 40, 20, 10, 5, 2.5, 1.25 and 0.125 mM.
Cell lines were then treated with 100 ml of each test compound
or cisplatin in triplicate. The nal concentrations of each
treatment were: 20, 10, 5, 2.5, 1.25, 0.625 and 0.0625 mM. Each
plate also contained wells containing untreated cells and media
only as controls. Aer receiving treatments, the 96 well plates
were returned to incubator (37 ꢂC, 5% CO2) for forty-eight
hours.
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xed with trichloroacetic acid and stained with sulforhodamine
B using a modied version of the protocol described by Skehan
et al.28 In short 50 ml of cold 80% TCA was added to each well, at
a nal concentration of 16% TCA, and plates were incubated at
¨
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ꢂ
4 C for one hour. The media and TCA solution was discarded
and plates were washed four times using room temperature tap
water. Plates were allowed to air dry overnight. Plates were
stained with the addition of 70 ml per well of 40% (w/v) SRB in
1% (v/v) acetic acid solution. Samples were stained for een
minutes and then stain was discarded. Plates were then washed
four times with 1% (v/v) acetic acid solution to remove unbound
stain and allowed to air dry overnight at room temperature.
Finally, SRB stain was solubilized by adding 150 ml of 10 mM
unbuffered Tris base to each well. The absorbance of each
samples at 560 nm was recorded using a Tecan plate reader and
the IC50 was determined using Origin 4.0 soware. Each study
was completed in duplicate.
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Acknowledgements
26 S. Bansal, D. Sinha, M. Singh, B. Cheng, Y. Tse-Dinh and
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27 National Committee for Clinical Laboratory Standards,
Performance standards for antimicrobial disk and dilution
susceptibility tests for bacteria isolated from animals (second
edition), 1997.
We thank the NIH for nancial support (CA125724 and
GM100607 to DPA and 5SC1HD063059-04 to FL). We also thank
Zhao Chen (Clemson University) for help with antibacterial
studies.
28 P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon,
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This journal is © The Royal Society of Chemistry 2014
Med. Chem. Commun., 2014, 5, 816–825 | 825