Selective Inhibition of Escherichia coli RNA and DNA Topoisomerase i by Hoechst 33258 Derived Mono-and Bisbenzimidazoles

Article abstract of DOI:10.1021/acs.jmedchem.7b00191

A series of Hoechst 33258 based mono-and bisbenzimidazoles have been synthesized and their Escherichia coli DNA topoisomerase I inhibition, binding to B-DNA duplex, and antibacterial activity has been evaluated. Bisbenzimidazoles with alkynyl side chains display excellent E. coli DNA topoisomerase I inhibition properties with IC₅₀ values <5.0 μM. Several bisbenzimidazoles (3, 6, 7, 8) also inhibit RNA topoisomerase activity of E. coli DNA topoisomerase I. Bisbenzimidazoles inhibit bacterial growth much better than monobenzimidazoles for Gram-positive strains. The minimum inhibitory concentration (MIC) was much lower for Gram positive bacteria (Enterococcus spp. and Staphylococcus spp., including two MRSA strains 0.3-8 μg/mL) than for the majority of Gram negative bacteria (Pseudomonas aeruginosa, 16-32 μg/mL, Klebsiella pneumoniae > 32 μg/mL). Bisbenzimidazoles showed varied stabilization of B-DNA duplex (1.2?ê'23.4 °C), and cytotoxicity studies show similar variation dependent upon the side chain length. Modeling studies suggest critical interactions between the inhibitor side chain and amino acids of the active site of DNA topoisomerase I.

Full text of DOI:10.1021/acs.jmedchem.7b00191

Subscriber access provided by NEW YORK UNIV
Article
Selective Inhibition of E. coli RNA and DNA Topoisomerase
I by Hoechst 33258 Derived Mono and Bisbenzimidazoles
Nihar Ranjan, Sandra Story, Geraldine Fulcrand, Fenfei Leng, Muzammil Ahmad,
Ada King, Souvik Sur, Weidong Wang, Yuk-Ching Tse-Dinh, and Dev Priya Arya
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 17 May 2017
Downloaded from http://pubs.acs.org on May 17, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Selective Inhibition of E. coli RNA and DNA
Topoisomerase I by Hoechst 33258 Derived Mono
and Bisbenzimidazoles
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
2
3,4
3,4
5
Nihar Ranjan , Sandra Story , Geraldine Fulcrand , Fenfei Leng , Muzammil Ahmad , Ada
2
1
5
3,4
1, 2
King, Souvik Sur, Weidong Wang , Yuk-Ching Tse-Dinh and Dev P. Arya *
1
Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University, Clemson,
South Carolina, United States 29634
2
3
NUBAD, LLC 900B West Faris Road, Greenville, South Carolina, United States 29605
Department of Chemistry and Biochemistry, Florida International University, Miami, Florida,
United States 33199
4
Biomolecular Sciences Institute, Florida International University, Miami, Florida, United States
33199
5
Genome Instability and Chromatin Remodeling Section, Lab of Genetics, National Institute on
Aging, National Institutes of Health, 251 Bayview Boulevard, Baltimore, Maryland, United
States 21224
Corresponding author email address: dparya@clemson.edu
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT:
A series of Hoechst 33258 based mono and bisbenzimidazoles have been synthesized and their
E. coli DNA topoisomerase I inhibition, binding to BꢀDNA duplex and antibacterial activity has
been evaluated. Bisbenzimidazoles with alkynyl side chains display excellent E. coli DNA
topoisomerase I inhibition properties with IC values < 5.0 ꢀM. Several bisbenzimidazoles (3, 6,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
7, 8) also inhibit RNA topoisomerase activity of E. coli DNA topoisomerase I.
Bisbenzimidazoles inhibit antibacterial growth much better than monoꢀbenzimidazoles for gram
positive strains. The minimum inhibitory concentration (MIC) was much lower for Gram
positive bacteria (Enterococcus spp. and Staphylococcus spp including two MRSA strains 0.3ꢀ8
ꢀg/mL) than for the majority of Gram negative bacteria (P.aeruginosa, 16ꢀ32 ꢀg/mL, K.
pneumoniae >32µg/mL). Bisbenzimidazoles showed varied stabilization of BꢀDNA duplex (8.0ꢀ
ο
2
2.9 C), and cytotoxicity studies show similar variation, dependent upon the side chain length.
Modeling studies suggest critical interactions between the side chain and active site amino acids.
KEYWORDS: Topoisomerase, Hoechst 33258, bisbenzimidazole, antibacterial, DNA binding
2
ACS Paragon Plus Environment

Page 3 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
DNA topoisomerases are important class of enzymes that help in regulating DNA
1
topology. They are involved in several cellular functions such as removing supercoils, strand
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
breakage during recombination, chromosome condensation as well as disentangling of
2
ꢀ5
intertwined DNA. Eukaryotic DNA topoisomerases I and II have gained significant attention
6
ꢀ8
as drug targets particularly in cancer treatment. On the other hand, bacterial DNA gyrase and
9ꢀ11
topoisomerase IV have been targets of some established antibiotics.
Therefore, controlling
DNA topoisomerase functions has been envisioned for developing new anticancer and
1
2,13
antibacterial agents.
A number of small molecules have been tested for their ability as
14
poisons of DNA topoisomerase functions. The therapeutic interest in the development of small
molecules as inhibitors of DNA topoisomerase lies in their ability to act as DNA cleavage
12
15
complex stabilizing agents and to recognize ATP binding site. The emergence of resistance to
antiꢀbacterials has necessitated the search of novel molecules that could help tackle these issues.
Small molecules that are both DNA binders and nonꢀbinders have been known to poison the
14
functions of DNA topoisomerases. A classic DNA topoisomerase poison camptothecin is
1
6
believed to act by forming a ternary complex with the topoisomerase and DNA. Several other
planar small molecules (both DNA intercalators and nonꢀintercalators) have been shown to
17ꢀ24
inhibit the functions of DNA topoisomerases.
Both symmetric and asymmetric
bisbenzimidazoles have recently been reported as effective antibacterial agents and DNA
2
5, 26
topoisomerase poisons.
Although bisbenzimidazoles derived from Hoechst 33258 have long
2
7, 28
29ꢀ33
been known for their BꢀDNA binding
and topoisomerase I poisoning,
recent reports by
us and others have identified some of them for their selectivity towards bacterial DNA
34, 35
topoisomerase I inhibition.
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Chemical structures of compounds used in this study.
Of other features such as the presence of bulky groups on terminal phenyl rings, linkers
2
6, 35
containing long alkyl groups add to the DNA poisoning abilities of these compounds.
We
4
ACS Paragon Plus Environment

Page 5 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
have recently reported that a Hoechst 33258 derived bisbenzimidazole with a linker containing a
3
5
long alkyl group (twelve carbon atoms) showed excellent DNA topoisomerase I inhibition.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In this article, we investigate the bacterial DNA and RNA topoisomerase inhibition of
3
6
bisbenzimidazoles (Figure 1) by systematically a] varying the alkyl chain length b] comparing
the antibacterial and topoisomerase activity of bisbenzimdiazoles with monoꢀbenzimidazoles
derived from Hoechst 33258. We then explore the relationships between the topoisomerase I
inhibition, antibacterial activity and DNA binding of these compounds.
RESULTS AND DISCUSSION
Synthesis of Novel Mono and Bisbenzimidazoles.
The alkynyl bisbenzimidazoles (1-9) were synthesized using literature procedures reported
35, 37ꢀ40
earlier.
As shown in Scheme 1, a divergent strategy was employed. To introduce the
4
1
linkers, we performed Mitsunobu reactions of 4ꢀhydroxy benzaldehyde with aliphatic alcohols
(
1a-9a) that terminated in an alkyne functionality (the aliphatic alcohols were obtained
42
commercially or prepared in one step from corresponding diols). This reaction afforded 4ꢀ
substituted benzaldehydes (1b-9b) which were coupled with 3,4ꢀdiaminoꢀNꢀmethoxyꢀNꢀ
methylbenzamide in the presence of an oxidant to yield corresponding benzimidazoles (1c-9c)
3
7, 38
derivatives.
The benzimidazoles (1c-9c) which contained Wienreb amide on one of the
phenyl rings were easily reduced to corresponding aldehydes (1d-9d) using lithium aluminum
38
hydride.
Coupling of aldehydes (1d-9d) with 4ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)benzeneꢀ1,2ꢀ
4
3
diamine , in the presence of Na S O resulted in the synthesis of alkynyl bisbenzimidazoles 1-9
2
2
5
in good yields (55ꢀ72%).
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Reagent and conditions (i) PPh , DIAD, dioxane, dichloromethane, rt, overnight,
3
5
0ꢀ85 %, (ii) PdꢀC, H , ethanol,rt, 5h, qaunt, (iii) 1b-9b, ethanol, Na S O , H O, reflux, 12ꢀ14
2
2
2
5
2
ο
h, 61ꢀ85 % (for two steps), (iv) THFꢀ ether, LAH , ꢀ78 C to rt., 6ꢀ12 h, 42ꢀ73 %, (v) 1d-9d,
ethanol, Na S O , H O, reflux, overnight, 55ꢀ72 % (for two steps).
2
2
5
2
To achieve the synthesis of the monobenzimidazoles, three different strategies were employed.
For the synthesis of monobenzimidazoles that bear no triazolyl functionalities (compounds 10-
1
4, 16, 18), the desired compounds were obtained via condensation of an orthoꢀdiamine with an
4
4, 45
37
appropriate aldehyde
(Scheme 2a) in the presence of an oxidant (sodium metabisulfite).
6
ACS Paragon Plus Environment

Page 7 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Reagent and conditions (a) (i) PdꢀC, H , ethanol, rt, 5 h, quant. (ii) Na S O , H O,
2
2
2
5
2
ο
ethanol, reflux, overnight, 47ꢀ84 % (b) (i) 1N NaOH (aq), 80 C, 58 % (ii) Trifluoroacetic acid
(TFA), dichloromethane (DCM), rt, quant.
Monobenzimidazole derivative 15 was synthesized by the basic hydrolysis of 14, while 17 was
obtained by the removal of the Boc protecting group of 16 using trifluoroacetic acid (Scheme
2b). To achieve the synthesis of triazolyl containing monobenzimidazoles (19-22), we exploited
the chemical nature of the functionalities present at the ends of 10 (a terminal alkyne) and 11 (an
azide). Alkynes and azides are key components for the copper catalyzed 1, 3 dipolar
4
6, 47
cylcoaddition, which results in the regioselective formation of 1,4 substituted 1,2,3 triazoles.
Compound 10 was reacted with excess bisazides to afford triazole bearing monoꢀbenzimidazoles
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
that terminate with an azide end (19, 20). Similarly, compound 11 was reacted with excess
bisalkynes to yield triazole bearing monoꢀbenzimidazoles (21, 22) that terminate with an alkyne
4
4,48
end (Scheme 3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Reagent and conditions (i) ethanol, sodium ascorbate, CuSO , rt, 24 h, 30ꢀ68 % (ii)
4
1, 4 diethynyl benzene, ethanol, sodium ascorbate, CuSO rt, 24 h, 57% (iii) 1,9 decadiyene,
4,
ethanol, sodium ascorbate, CuSO , 24 h, 79% .
4
Enhanced and Selective Inhibition of E. coli DNA Topoisomerase I. Enzymes, such as
1
2, 49
topoisomerase I and gyrase are potential targets of antibacterial compounds.
Bisbenzimidazoles and their derivatives are well known to inhibit the activity of eukaryotic DNA
5
0
topoisomerase I and promote anti helicase activity. Related bisbenzimidazole derivatives have
also been shown to poison eukaryotic DNA topoisomerase I activity which is believed to elicit
3
3
its effect in a manner similar to camptothecin which involves DNA and protein binding. To
probe the role of terminal alkyl chain length, we have synthesized a small library of
bisbenzimidazoles (1-9) where the length of terminal alkyl chain ranges from 3ꢀ21 atoms.
8
ACS Paragon Plus Environment

Page 9 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Additionally, to gauge the effect of bisbenzimidazole vs. monobenzimdiazole moiety, we have
synthesized monoꢀbenzimidazoles related to Hoechst 33258 (10-22) to compare their DNA
topoisomerase and bacterial inhibition properties with bisbenzimidazoles (1-9). The inhibitory
effects of the compounds (5-22) were tested against four DNA topoisomerases (E. coli DNA
topoisomerase I, E. coli DNA gyrase, human DNA topoisomerase I, human DNA topoisomerase
II).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. A representative image showing the inhibitory activity of bisbenzimidazole 7 against
E. coli DNA topoisomerase I. (A) The inhibition effects of bisbenzimidazole 7 against E. coli
DNA topoisomerase I. The E. coli DNA topoisomerase I inhibition assays were performed as
described in the experimental section. The plasmid DNA was isolated and subjected to 1%
agarose gel electrophoresis. Lanes 1 to 13 contain 0, 2, 4, 6 8, 10, 12, 14, 16, 18, 20, 30, and 50
µM of bisbenzimidazole 7 respectively. (B) The quantification analysis of the inhibitory
activities of bisbenzimidazole 7 against E. coli DNA topoisomerase I. The values of IC were
5
0
obtained from these analyses. Standard deviation was obtained from three independent
determinations. (scDNA = Supercoiled DNA).
A representative inhibition of E. coli DNA topoisomerase I is displayed in Figure 2 (also see
supporting information, Figures S37ꢀ S40). In this assay, the relaxation of supercoiled plasmid
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
pBADꢀGFPuv was observed in the presence of various inhibitors. In the absence of inhibitor, E.
coli DNA topoisomerase I fully relaxed the supercoiled plasmid DNA pBADꢀGFPuv (Fig. 2A,
lane 1). Lanes 2ꢀ13 show the inhibitory effect of compound 7 on the relaxation. As increasing
amounts of compound 7 were added, the percentage of supercoiled DNA increased which
attained saturation level at inhibitor concentration 20 ꢀM and above. The IC values of effective
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
topoisomerase inhibitors are summarized in Table 1. All monoꢀbenzimidazoles, except
compounds 9 and 20, showed enhanced inhibition of E. coli DNA topoisomerase I. While
compound 9 at 50 ꢀM did not significantly inhibit the activity of E. coli DNA topoisomerase I,
compound 20 showed an IC of 21.5 ꢀM, which is comparable to the IC value of Hoechst
50
50
33258. We also tested the inhibitory effects of these compounds against E. coli DNA gyrase and
found that none of the tested bisbenzimidazoles (1-4, 8) showed any inhibitory activities against
E. coli DNA gyrase. Nevertheless, results in Table 1 reveal that all of the bisbenzimidazoles
except compounds 9 and 20 have much more enhanced inhibition (requiring <13 ꢀM inhibitor)
of E. coli DNA topoisomerase I than Hoechst 33258 or Hoechst 33342 does.
Table 1. IC values of the bisbenzimidazoles (1-9) and monobenzimidazole
5
0
(20) against E. coli DNA topoisomerase I, human DNA topoisomerase I, and
human DNA topoisomerase II
a
IC (ꢀM)
5
0
10
ACS Paragon Plus Environment

Page 11 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
b
b
b
Compound
ecTopoI
hTopoI
hTopoII
c
c
Hoechst 33258
Hoechst 33342
19.50±1.32
29.83±2.75
5.50±0.50
22.86±1.55
>50µM
28.92±4.45
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ
c
1
25.41±2.20
>50µM
51.6±2.5
ꢀ
c
2
4
2
6
.57±0.81
.47±0.06
.63±0.47
c
3
>50µM
ꢀ
ꢀ
c
4
>50µM
5
6
7
8
9
3.20±0.26
>50µM
ꢀ
2
1
4
.80±0.26
2.24±0.63
.00±1.32
>50µM
ꢀ
>50µM
>50 µM
ꢀ
>50µM
>50
ꢀ
ꢀ
ꢀ
20
21.50±2.18
>50µM
a
IC was determined as described in the experimental section. The values are the average of at least three
50
b
independent determinations. ecTopo I, hTopo I, and hTopo II represent E. coli DNA topoisomerase I,
c
human DNA topoisomerase I, and human DNA topoisomerase II, respectively. The values have been
reproduced from reference 35.
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Decreased inhibitions of bisbenzimidazoles 5-8 against human DNA topoisomerase
I. Inhibition assays against human topoisomerase I were performed as described in the
experimental section 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. Lane 1 represents the relaxed plasmid DNA pBADꢀGFPuv. Lanes 2ꢀ8 represent
the two different concentrations tested for each compound (10 and 50 ꢀM) in these assays.
To determine their selectivity against E. coli DNA topoisomerase, we tested the inhibitory
effects of bisbenzimidazoles 1-9 and the lone monobenzimidazole 20 (which had shown
comparable E. coli DNA topoisomerase I IC values to Hoechst 33258) against human DNA
5
0
topoisomerase I and II. Figure 3 shows the human DNA topoisomerase I inhibition of
compounds 5-8 at two tested concentrations 10 and 50 ꢀM. Our results summarized in Table 1
clearly show the selectivity of bisbenzimidazoles towards the inhibition of bacterial DNA
topoisomerase I. The IC values of these compounds against human topoisomerase I and II are
5
0
much higher than that against E. coli topoisomerase I (Table 1).
Inhibition of RNA Topoisomerase: We and others have recently shown that E.coli
5
1,
topoisomerase I (EcoTop1) possesses not only topoisomerase activity for DNA, but also RNA.
52
We, therefore, examined whether the compounds that inhibit the DNA topoisomerase activity
12
ACS Paragon Plus Environment

Page 13 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of EcoTop1, can also inhibit its RNA topoisomerase activity. We found that ethacridine and
Hoechst 33342, which are known DNA binders and can inhibit DNA topoisomerase activity nonꢀ
specifically, had little effect on the RNA topoisomerase activity of EcoTop1, even at 80 ꢀM
concentrations (Fig. 4A). In addition, six inhibitors 1, 2, 4, 5, 9 and 20 had no discernible
inhibitory effect on the RNA topoisomerase activity of EcoTop1 at concentrations up to 50 ꢀM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(Fig. 4B). Notably, four inhibitors 3, 6, 7 and 8 inhibited the RNA topoisomerase activity at
about 10 ꢀM concentration. Our data thus suggest that only a selected group of inhibitors of
DNA topoisomerase activity can also inhibit the RNA topoisomerase activity. The mechanism of
why only these four inhibitors can inhibit the RNA topoisomerase activity remains to be
investigated. However, close inspection of their chemical structure reveals that they all share one
similarity: the long alkyl chain at their ends. Therefore, it is possible that these alkyl chains at the
ends have a role in the RNA topoisomerase inhibition.
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4: Effect of inhibitors towards inhibiting RNA topoisomerase activity of EcoTop1. (A)
Autoradiographs show that ethacridine and Hoechst 33342 do not have any effect on RNA
topoisomerase activity of EcoTop1. The inhibitor concentrations are 2.5, 5, 10, 20, 40 and 80 ꢀM
(lane 3ꢀ8) (B) Autoradiographs of inhibitors 1-9 and 20 show that out of ten inhibitors tested,
only 3 , 6, 7 and 8 inhibit RNA topoisomerase activity of EcoTop1 at 50 ꢀM concentration
14
ACS Paragon Plus Environment

Page 15 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
while others 1, 2, 4 , 5 and 20 did not inhibit RNA topoisomerase activity of EcoTop1 at 50 ꢀM
concentration (C) Autoradiographs show concentrationꢀdependent inhibition of EcoTop1 RNA
topoisomerase activity by inhibitors 3, 6, 7 and 8. In all the reactions, 10 nM EcoTop1 was
used. Since all inhibitors were diluted in DMSO, a control reaction (Lane 2 of every
autoradiograph) with DMSO only also performed. The final inhibitor concentrations were 2.5, 5,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10, 20, and 40 ꢀM (lane 3ꢀ7). The autoradiography was done using Phosphoimager (Molecular
Dynamics).
UV thermal denaturation studies show the requirement of a bisbenzimidazole unit for B-
DNA binding. DNA associated with topoisomerases plays an important role in the inhibitor’s
activities and usually forms a DNAꢀinhibitorꢀtopoisomerase ternary complex. Therefore, the
DNA binding studies could provide information about any relationship between inhibitor ꢀDNA
interaction and the topoisomerase inhibitor activity. Bisbenzimidazoles are known topoisomerase
inhibitors whose inhibitory activity has been proposed to stem from ternary complex formation
32
involving AT rich DNA sites, the topoisomerase enzyme and the inhibitor as well as its binding
5
3
at AT rich distal site on DNA. These studies have revealed that such inhibition does not have
strong DNA binding dependence and that their minor groove binding may not be the sole
determinant of topoisomerase inhibition. Bisbenzimidazole Hoechst 33258 is an established AT
5
4ꢀ58
rich BꢀDNA binder known to bind in the DNA minor groove with nanomolar affinities.
Table 2. A table showing the thermal denaturation temperatures of
duplex DNA (dA .dT ) in the presence of all studied compounds (10
6
0
60
ꢀM each) in buffer 10 mM sodium cacodylate, 0.1 mM EDTA and 100
mM NaCl at pH 7.0.
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compound
Tm(°C)
ꢁT (°C)
m
None
62.5
87.1
86.6
85.4
83.4
70.5
85.9
80.8
75.6
68.1
63.7
75.6
65.1
64.4
64.2
62.5
63.9
63.5
62.5
62.5
65.8
ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Hoechst 33258
Hoechst 33342
24.6
24.1
22.9
20.9
8.0
a
a
1
a
2
a
3
a
4
23.4
18.3
13.1
5.6
5
6
7
8
9
1.2
13.1
2.6
1
1
1
0
1
2
1.9
1.7
13
0.0
1
1
4
5
1.4
1.0
16
0.0
1
1
7
8
0.0
3.3
16
ACS Paragon Plus Environment

Page 17 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
9
63.7
63.0
64.4
63.0
1.2
0.5
1.9
0.5
20
2
2
1
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
The values have been taken from reference 35.
To further evaluate the DNA binding ability of these compounds, we performed thermal
denaturation studies with a BꢀDNA duplex dA .dT . The formation of the duplex was
6
0
60
ο
confirmed by the sharp melting transition at 62.5 C in the presence of 100 mM NaCl. The
compounds (10 ꢀM) were mixed with the DNA (1 ꢀM/duplex) and heated further to determine
the thermal stability. At this ratio, the DNA was saturated with the compound as using lower
concentrations (< 5 ꢀM) of the compound resulted in biphasic transitions (data not shown). The
results obtained from the thermal denaturation studies are shown in Table 2 (for melting profiles
see supporting information, Figure S36). Of the bisbenzimidazoles studied (1-9), few compounds
ο
(1, 2, 5, 6) displayed thermal stabilization greater than 18.0 C which were comparable to the
change in melting temperature afforded by Hoechst 33258 and 33242. However, compounds 3, 7
ο
ο
ο
and 8 showed much lower thermal stabilization (8.0 C, 5.6 C, and 1.2 C respectively). The
thermal stabilization afforded by bisbenzimidazole 8 is comparable to the thermal stabilization
obtained with monoꢀbenzimidazoles (10-18) and triazole bearing monoꢀbenzimidazoles (19-22).
The differential thermal stabilization by bisbenzimidazoles indicates the role of linker length in
perturbing the DNA minor groove binding of the benzimidazole moeity.
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ο
All monoꢀbenzimidazoles (10-18) showed only up to a 3 C increase in the thermal
stabilization of dA .dT duplex. Similar thermal stabilization was also found with the triazolylꢀ
60
60
benzimidazoles (19-22). The thermal stabilization afforded by monoꢀbenzimidazoles was much
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ο
smaller in comparison to most of the bisbenzimidazoles which showed up to ~ 25 C increases in
the thermal denaturation temperatures of dA .dT duplex. These results confirm that monoꢀ
6
0
60
benzimidazoles are much weaker DNA binders than bisbenzimidazoles. Wilson and coworkers
have previously shown that Hoechst 33258 like compounds with two benzimidazoles units (but
lack the piperazine unit) afford similar thermal stabilizations as Hoechst 33258 when bound in
2
2
the minor groove of BꢀDNA. However, Hoechst 33258 derived monoꢀbenzimidazoles, which
terminate with a guanidinium group or an imidazole (in place of piperazine), can produce much
2
2
higher thermal stabilization in comparison with compounds containing a piperazine. This
observation, coupled with our findings, suggest that key requirements of the Hoechst 33258
derived compounds for DNA binding are the presence of a linked bisbenzimidazole skeleton and
moieties capable of making significant electrostatic interactions. Our data presented in Table 2
similarly highlight the requirement of a bisbenzimidazole moiety for effective BꢀDNA
recognition. These results are important in the context of converting these DNA binding
compounds into enzyme inhibitors with reduced background genotoxicity. Our data clearly
show that with appropriate modifications, DNA binding and topoisomerase enzyme inhibition
activities can be decoupled.
Antibacterial Studies. Compounds belonging to the bisbenzimidazole class have shown
significant antibacterial effect against a variety of strains, which include methicillinꢀresistant
5
9, 60
Staphylococcus aureus (MRSA) and vancomycinꢀresistant Entercoccus faecalis.
Hoechst
18
ACS Paragon Plus Environment

Page 19 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
3258 has been reported to have antimicrobial activity against Pneumocystis cariniif. sp. muris,
6
1
Candida albicans and Candida dubliniensis. We have probed the antibacterial activity of all
compounds described in the study. The antibacterial effect evaluation of compounds (1-22)
included both gram positive and gram negative strains (Table 3). In cases where a sharp
inflection in the bacterial growth was not observed, the MIC is given as a range of values.
Nearly all monoꢀbenzimidazoles (10-22) displayed similar MIC values (16ꢀ32 ꢀg/mL) against
gram positive bacterial strains S. aureus ATCC 29213, S. aureus ATCC 33591, S. epidermidis,
the two MRSA strains, E. faecium, and E. faecalis. A number of bisbenzimidazoles (Hoechst
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
3258, Hoechst 33342, 1, 2, and 4) showed much lower MIC values against most of the strains
(0.3 ꢀ 4 ꢀg/mL). Bisbenzimidazoles 3 and 5-9 showed relatively higher MIC values (16ꢀ32
g/mL respectively) than other bisbenzimidazoles. As described in the aforementioned sections,
ꢀ
3 and 5-9 also showed poor/decreased DNA binding as compared to Hoechst 33258 as evident
from thermal denaturation experiments. Bisbenzimidazoles 3 and 5-9 have a common feature in
their chemical structure, which is the presence of long alkyl linker. The presence of the long
aliphatic group at the terminal renders the molecule to be more hydrophobic. The alkyl chains
can impact the BꢀDNA binding of these molecules possibly due to selfꢀaggregation of alkyl
groups in aqueous solutions. It is unclear if the alkyl chain length play a similar role in affecting
the antibacterial activity of these compounds.
Table 3. Minimal inhibitory concentrations (MIC) of the studied compounds against various
bacterial strains by microbroth dilution.
MIC (µg/mL)
Compound
MRSA
33591
MRSA
A960649
S. aureus
29213
S. aureus
33591
S. epidermidis
12384
______________________________________________________________________________
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Hoechst
3342
Hoechst
1
0.5
2ꢀ4
2ꢀ4
1
3
ND
ND
≥32
16ꢀ32
ND
33258
1
2
3
4
5
6
7
8
9
8
4
>32
1
1ꢀ2
4
>32
1ꢀ2
2ꢀ4
2ꢀ4
16
2ꢀ4
16ꢀ32
>32
>32
16ꢀ32
16ꢀ32
≥32
2ꢀ4
2ꢀ4
16
2ꢀ4
16ꢀ32
16ꢀ32
>32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
>32
>32
16ꢀ32
16
16
16ꢀ32
1ꢀ2
1ꢀ2
>32
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
ND
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
ND
>32
>32
>32
>32
>32
>32
>32
>32
>32
16ꢀ32
>32
>32
>32
>32
>32
>32
>32
ND
>32
>32
>32
>32
>32
10
11
≥32
12
13
14
15
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
>32
16
17
18
19
20
21
>32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
22
MIC (µg/mL) continued
Compound
E. faecium
BM4105RF 29212
E. faecalis
S. flexneri
2457NR517 25922
E. coli
E. coli
K12
______________________________________________________________________________
Hoechst
33342
Hoechst
0.3
16ꢀ32
16ꢀ32
1ꢀ2
ND
16
16
8
ND
8ꢀ16
3
1
2
3
4
5
6
7
8
9
1
1
3258
1ꢀ2
1ꢀ2
>32
1ꢀ2
>32
>32
>32
≥32
≥32
>32
>32
16ꢀ32
4
16ꢀ32
8
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
1ꢀ2
8
>32
4
>32
>38
>32
>32
≥32
>32
>32
8
8ꢀ16
16
8ꢀ16
16ꢀ32
16ꢀ32
>32
16
8
8ꢀ16
8ꢀ16
8ꢀ16
8
2ꢀ4
2ꢀ4
16
2ꢀ4
16
16
16ꢀ32
16
16
0
1
20
ACS Paragon Plus Environment

Page 21 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
2
3
4
>32
>32
>32
>32
>32
ND
>32
>32
>32
>32
>32
16
16
16ꢀ32
16ꢀ32
16ꢀ32
>32
>32
>32
>32
>32
>32
ND
>32
>32
>32
>32
>32
16
16
16
16
16
16
16
16
15
16
17
18
19
16
16
>32
>32
16
16
16
>32
>32
16
16
16
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
>32
16ꢀ32
16ꢀ32
16ꢀ32
16ꢀ32
20
21
22
16
16
MIC (µg/mL) continued
A. baumanni P. aeruginosa K. pneumoniae E. cloacae C. freundii
Compound
19606
27853
NR15410
13047
4747CFAA
______________________________________________________________________________
Hoechst
3
Hoechst
8
16
8
8
4
8
3342
ND
ND
ND
ND
3
1
2
3
4
5
6
7
8
9
1
1
3258
>32
16
>32
8
16
16
16
16
16ꢀ32
16ꢀ32
16ꢀ32
16
16ꢀ32
16
16
16
16
16
16
16
>32
>32
16
>32
>32*
>32
>32*
>32
>32*
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
ND
32
4
>32
>32
>32*
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
ND
>32
>32
>32
>32
>32
>32*
>32
16
>32
>32
>32
>32
>32
>32
>32
>32
16ꢀ32
>32
>32
>32
ND
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
ND
>32
>32
>32
>32
>32
0
1
12
1
1
1
1
3
4
5
6
17
1
1
2
2
22
*
8
9
0
1
>32
>32
>32
>32
>32
16
16
16
Indicates the %inhibition in K. pneumoniae, E. cloacae and C. freundii was the greatest for
these compounds (though with low values) as compared to the other compounds.
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Bisbenzimidazoles (1-6 and 8) that effectively inhibit E. coli DNA topoisomerase I and RNA
topoisomerase (3, 6, 7, 8) also display varying degrees of antibacterial activity, with compound 4
being the most potent overall. For gram negative strain Escherichia coli K 12, long alkyl chains
on some bisbenzimidazoles imparted better antibacterial activity. Typically all of the
bisbenzimidazole compounds that had shorter alkyl linkers displayed better antibacterial activity
than same compound with alkyl ends greater than ten atoms. Overall, these results show that
alkynyl bisbenzimidazoles derived from parent Hoechst 33258 are capable of inhibiting both
gram positive and gram negative bacteria.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cytotoxicity and Docking Studies.
Cytotoxicity of compounds 1-9 was determined against human prostate cancer cell line DUꢀ145.
Our results indicate that some of these compounds (1, 2, 4, 5, 6) are more cytotoxic to DUꢀ145
than parent compound Hoechst 33258 (IC >10 ꢀM) while others (compounds 3, 7, 8, 9) display
5
0
comparable inhibitory effects (IC50 >10 ꢀM) to Hoechst 33258 (Table S1, see supporting
information). A feature clearly discernible among compounds 1, 2, 4ꢀ6 (linker length up to 12
atoms) and 3, 7ꢀ9 (linker length up to 21 atoms) is the linker length dependent cytotoxicity of
these compounds. Revisiting the thermal stability results shown in table 3, it can be observed that
compounds 3, 7-9 display poorer binding to BꢀDNA than compounds 1, 2, 4ꢀ6. These results
indicate that the cytotoxicity of these compounds may have its origins in their DNA binding
abilities. One of the best DNA and RNA topoisomerase inhibitor (compound 8) also showed
similarly low cytotoxicity to a normal dermal fibroblast cell line PCS 201ꢀ010 with IC >10
5
0
ꢀM.
22
ACS Paragon Plus Environment

Page 23 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. (a) Docked pose of Hoechst 33258 (red) and compound 3 (magenta) at the active site
of E. coli. topoisomerase I covalent complex showing differences in the bisbenzimidazole unit
orientation of the two compounds. (b) A closer view compound 3 (magenta) bound at the active
site showing the amino acid residues that are involved in the ternary interaction.
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We have performed molecular docking experiments to gain insight into the inhibitor binding to
bacterial topoisomerase I. We (TseꢀDinh) have previously reported the crystal structures of the
active site of E. coli. topoisomerase I including a structure where the covalent intermediate is
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
62
trapped with a cleaved single stranded oligonucleotide substrate. Using the structure of this
enzymeꢀDNA complex (PDB ID: 3PX7), we performed docking experiments with one of the
most potent E. coli. topoisomerase I inhibitor: compound 3. As a control experiment, we also
docked Hoechst 33258 to observe any spatial differences in their binding at the active site.
Docking of compound 3 showed that the benzimidazole moiety is in the vicinity of the cleaved
oligonucleotide at the active site with the piperazine end being closest to it. Compound 3 makes
several interactions (hydrogen binding, π and van der Walls) at the active site, with additional
interactions with amino acids Arg493, Asp323, Pro494 residues. Both benzimidazole moieties of
compound 3 make interactions with the amino acid residues. The benzimidazole unit adjacent to
the piperazine unit makes πꢀinteractions with Asp113 while the other benzimidazole unit close to
the phenoxy group makes π and van der Walls interactions with Lys113 and Ptr319 residues. The
6
3
long alkyl chain is directed away from the active site towards helices J and K of domain III of
the enzyme where it makes key contacts with Val375, Asp372 and Tyr316. Contrary to this, the
Hoechst 33258 docking results showed a somewhat different view with major difference being
that the piperazine end of the molecule protrudes away from the active site in the proximity of
helices A and D of domain I and helix Q of domain IV. Overall, these results suggest that the
additional hydrophobic interactions made by the alkyl chain of compound 3 with helices J and K
of domain III provide critical interactions for binding of the inhibitor, leading to a stronger and
more selective inhibition than Hoechst 33258. For additional details of docking studies, please
see appendix I in the supporting information.
24
ACS Paragon Plus Environment

Page 25 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CONCLUSIONS
Benzimidazoles derived from Hoechst 33258 are an important class of molecules that are
effective against wide range of biological targets. Since molecules belonging to
bisbenzimidazole class are known inhibitors of mammalian topoisomerases as well, they have
been widely used in targeting cancer growth. The results obtained from this study show that
Hoechst 33258 derived alkynyl bisbenzimidazoles are much more effective E. coli
topoisomerase I inhibitors than monoꢀbenzimidazoles derivatives and are much more selective in
their inhibition properties when compared to Human topoisomerases I and II. The DNA binding
ability and topoisomerase I inhibitions of these compounds are unrelated suggesting that novel
compounds that inhibit these enzymes but show reduced binding to background DNA can be
identified using our approach. Since some bisbenzimidazoles with longer alkynyl linkers showed
improved IC values than the compounds with shorter linkers, it is plausible that alkyl linkers
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
0
interact with the enzyme leading to greater disruption of the DNAꢀtopoisomerase I complex. Our
modeling results lend credence to such an interaction between the enzyme and the ligand side
chain. There is an increasing interest in developing small molecules that selectively target
bacterial topoisomerases over mammalian topoisomerases. These compounds present a new
structural feature (long alkyl ends terminating in an alkyne) that can lead to selective inhibition
of bacterial topoisomerase I over human topoisomerases I and II as well. The same structural
feature also appears to be responsible for its RNA topoisomerase inhibition activity. Structural
studies are needed to gain a deeper insight in the interaction of these bisbenzimidazoles with
topoisomerases, and to better elucidate the preferential binding site of long alkyl chains.
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EXPERIMENTAL SECTION
General Methods. Chemicals were purchased from Sigma Aldrich (St. Louis, MO) or Fisher
Scientific (Pittsburgh, PA) and used as obtained from the supplier. Hoechst 33258 and Hoechst
33342 were obtained as their hydrochloride salts and used without further purification. Diꢀtertꢀ
butyl dicarbonate (Boc anhydride) was purchased from Advanced Chem Tech (Louisville, KY).
All solvents were purchased from VWR (Atlanta, GA). Silica gel (32ꢀ65 ꢀM mesh size) was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
13
purchased from Sorbtech (Atlanta, GA). H NMR and C NMR spectra were recorded on a
Bruker Avance (300/500 MHz) spectrometer. Chemical shift are given in ppm and are referenced
1
13
to residual solvent peaks ( H and C NMR). Mass (MALDIꢀTOF) spectra were collected using
a Bruker Microflex mass spectrometer. Ultra Violet (UV) spectra were collected on a Varian
(Walnut Creek CA) Cary 100 Bio UVꢀVis spectrophotometer equipped with a thermoelectrically
controlled 12ꢀcell holder. HPLC analysis of all new compounds reported in this article was done
on HP1100 series analytical HPLC instrument. The purity of all tested compounds was > 95%.
The autoradiography was done using Phosphoimager (Molecular Dynamics).
Nucleic Acids. Nucleic acids were purchased from Eurofins MWG Operon (Huntsville, AL).
The nucleic acids were used without further purification and their concentration was determined
using the extinction coefficients provided by the supplier. Nucleic acid solutions were prepared
in buffer 10 mM sodium cacodylate, 0.1 mM EDTA and 100 mM NaCl at pH 7.0 by heating at
ο
9
5 C for 15 minutes and then slowly allowing it to cool back to room temperature. After two
days of incubation, the duplex formation was checked by UV thermal denaturation experiments.
ο
The stock solution was stored at 4 C and diluted to desired concentrations as required.
26
ACS Paragon Plus Environment

Page 27 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis of compounds 1b-4b, 1c-4c, 1d-4d, 4a, 1-4, 10, 11 and 20 have been reported
3
5,48
elsewhere.
6-(prop-2-ynyloxy) hexan-1-ol (5a). To a solution of 1, 6 hexanediol (7.00 g, 59.2 mmol) in dry
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ο
THF (60.0 mL), sodium hydride (1.08 g, 45.0 mmol) was added under argon at 0 C and stirred
42
ο
for 30 min. To this, propargyl bromide (5.00 g, 42.0 mmol) was added and stirred at 0 C for
another 30 minutes. The mixture was allowed to warm to room temperature and stirred for 24 h.
The progress of reaction was monitored using TLC (silica gel). The reaction was quenched by
pouring the reaction mixture into water. The mixture was extracted with diethyl ether (3 × 100
mL). The organic layers were combined and dried with sodium sulfate. The volatiles were
evaporated and the crude product was purified by column chromatography on a silica gel column
using hexanes–ethyl acetate as eluent to afford the desired product as pale yellow oil (2.80 g, 42
ꢀ1
%
): R = 0.31 (hexanesꢀethyl acetate 7:3 v/v); IR (neat, cm ) 3399 (ꢀOH Hꢀbonded), 3297
f
(
alkyne CꢀH stretch), 2937 (aromatic CꢀH stretch), 2868, 2120 (alkyne CꢀC stretch), 1711, 1462,
1
1
360, 1241, 1090, 669; H NMR (300 MHz, CDCl ) δ 4.07 (d, J = 2.3 Hz, 2H, ꢀCH OCH CCH),
3
2
2
3.54 (t, J = 6.7 Hz, 2H, ꢀOCH CH ꢀ), 3.46 (t, J = 6.6 Hz, 2H), 2.56 (br, 1H), 2.41 (t, J = 2.3 Hz,
2
2
13
1
H, ꢀCH OCH CCH), 1.58ꢀ1.48 (m, 4H), 1.35ꢀ1.28 (br, m, 4H); C NMR (75 MHz, CDCl )
2
2
3
δ 79.8, 74.2, 70.0, 62.4, 57.9, 32.5, 29.3, 25.8, 25.5.
-(6-(prop-2-ynyloxy)hexyloxy) benzaldehyde (5b). To a solution of pꢀhydroxybenzaldehyde
1.50 g, 12.2 mmol) in dry dichloromethaneꢀdioxane (30.0 mL, dichloromethane: dioxane 4:1,
4
(
v/v), triphenyl phosphine (4.75 g, 18.2 mmol) and 6ꢀ(propꢀ2ꢀynyloxy) hexanꢀ1ꢀol (1.87 g, 12.2
mmol) were added and the solution was ice cooled. To this, diisopropylazodicarboxylate (DIAD)
ο
(
3.66 g, 18.2 mmol) was added at 0 C slowly over 15 min. The contents were initially stirred at
0 οC for 30 min and then at room temperature overnight. Progress of the reaction was monitored
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
by thin layer chromatography on silica gel. The solvents were removed under reduced pressure.
The gummy residue was dissolved in 100 mL (1:1 v/v) ethyl acetate:hexanes and was allowed to
stand in the refrigerator for a day. The precipitated solid was filtered off and the filtrate was
concentrated under reduced pressure. The crude product was purified by column chromatography
on silica gel using hexanesꢀethyl acetate as eluent which afforded the desired compound as pale
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
yellow liquid (1.10 g, 32 %): R = 0.73 (hexanes: ethyl acetate 6.5:3.5, v/v); IR (neat, cm ) 3288
f
(alkyne CꢀH stretch), 2945 (aromatic CꢀH stretch), 2859, 2160 (alkyne CꢀC stretch), 2060, 1683,
1
1593, 1503, 1315, 1254, 1164, 1106, 1025, 841, 661, 620; H NMR (500 MHz, CDCl ) δ 9.88
3
(s, 1H), 7.83 (d, J = 8.77 Hz, 2H), 6.99 (d, J = 8.77 Hz, 2H), 4.15 (d, J = 2.29 Hz, 2H, ꢀ
CH OCH CCH), 4.05 (t, J = 6.30 Hz, 2H, ꢀOCH CH ꢀ), 3.54 (t, J = 6.67 Hz, 2H), 2.44 (t, J =
2
2
2
2
1
3
2
.48 Hz, 1H, ꢀCH OCH CCH), 1.86ꢀ1.81 (m, 2H), 1.68ꢀ1.62 (m, 2H), 1.54ꢀ1.43 (m, 4H); C
2 2
NMR (125 MHz, CDCl ) δ 190.7, 164.2, 131.9, 129.8, 114.7, 80.0, 74.1, 70.0, 68.2, 58.0, 29.4,
3
+
2
9.9, 25.8, 25.7; MS MALDIꢀTOF m/z calcd for C H O 260.32, found 261.87 [M+H] .
16
20
3
N-methoxy-N-methyl-2-(4-(6-(prop-2-ynyloxy)hexyloxy)phenyl)-1H-benzo[d]imidazole-6-
carboxamide (5c). To solution of Nꢀ Methoxy, Nꢀmethyl 3, 4 dinitrobenzamide (0.93 g, 3.65
mmol) in ethanolꢀethyl acetate mixture (40.0 mL, 3:1 v/v), 10% PdꢀC (0.30 g) was added.
Hydrogenation for 5 h at atmospheric pressure yielded corresponding diamine which was, after
filtration of the catalyst, used without further characterization (Rf = 0.46 in
dichloromethane:methanol 9:1, v/v). 4ꢀ(6ꢀ(propꢀ2ꢀynyloxy) hexyloxy) benzaldehyde (0.95 g,
3.65 mmol) and sodium metabisulfite (0.69 g, 3.65 mmol) in water (1.00 mL) were added into
it and the reaction mixture was refluxed for 6 h. Volatiles were evaporated under reduced
pressure. The crude product was purified on a silica gel column using dichloromethaneꢀ
28
ACS Paragon Plus Environment

Page 29 of 72
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
methanol (0ꢀ10 % methanol in dichloromethane) as eluent to afford the desired product as thick
ꢀ1
brown liquid (1.15 g,73 %): R = 0.56 (in dichloromethaneꢀmethanol 9:1 v/v); IR (neat, cm )
f
3327 (alkyne CꢀH stretch), 2933 (aromatic CꢀH stretch), 2859, 2180 (alkyne CꢀH stretch), 1617,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
1
491, 1254, 1172, 1094, 1021, 849, 743, 661; H NMR (500 MHz, acetoneꢀd ) δ 12.23 (br, 1H),
6
8.18 (dd, J = 8.98 Hz, J = 2.05 Hz, 2H), 7.97 (br, 1H), 7.59 (br, 2H), 7.07 (d, J = 8.84, J =
1
2
1
2
1
3
1
1
.83, 2H), 4.14 (d, J = 2.15 Hz, 2H, ꢀCH OCH CCH), 4.06 (t, J = 6.47 Hz, 2H, ꢀOCH CH ꢀ),
2 2 2 2
.61 (s, 3H), 3.52 (t, J = 6.68 Hz, 2H), 3.35 (s, 3H), 2.95 (t, J = 2.37 Hz, 1H, ꢀCH OCH CCH),
2
2
1
3
.83ꢀ1.77 (m, 2H), 1.64ꢀ1.58 (m, 2H), 1.54ꢀ1.41 (m, 4H) ; C NMR (125 MHz, acetoneꢀd ) δ
6
70.1, 161.0, 153.4, 131.6, 128.3, 128.2, 122.7, 122.5, 114.8, 114.1, 80.3, 74.6, 71.1, 69.4, 67.9,
+
67.8, 60.2, 57.4, 33.3, 29.4, 25.7, 25.6; MS (MALDIꢀTOF) m/zcalcd for C H N O [M]
2
5
29
3
4
+
4
35.21, found 436.57 [M+H] .
-(4-(6-(prop-2-ynyloxy)hexyloxy)phenyl)-1H-benzo[d]imidazole-6-carbaldehyde (5d). To a
of NꢀmethoxyꢀNꢀmethylꢀ2ꢀ(4ꢀ(6ꢀ(propꢀ2ꢀynyloxy)hexyloxy)phenyl)ꢀ1Hꢀ
2
solution
benzo[d]imidazoleꢀ6ꢀcarboxamide (5c) (1.00 g, 2.37 mmol) in THFꢀether (80.0 mL, 3:1), lithium
ο
aluminum hydride (0.26 g, 6.93 mmol) was added at ꢀ78 C under argon and then allowed to
ο
stir at 0 C for 8 h . The reaction mixture was quenched by the addition of saturated ammonium
chloride solution (50.0 mL). The resulting grey precipitate was filtered off. The filtrate was
extracted with ethyl acetate (3 × 50 mL). Organic layers were combined and dried over sodium
sulfate. Volatiles were removed under reduced pressure. The crude product was purified by
column chromatography using hexanesꢀethyl acetate (0ꢀ80 % ethyl acetate in hexanes) as eluent
to give the desired compound as sticky light yellow solid (0.36 g, 42 %): R = 0.51 (ethyl
f
ꢀ1
acetateꢀhexanes 8:2 v/v); IR (neat, cm ) 2929 (aromatic CꢀH stretch), 2855, 1687 (C=O stretch),
1
1
605, 1262, 1184, 1094, 1012, 824, 743; H NMR (500 MHz, methanolꢀd ) δ 10.04 (s, 1H), 8.13
4
29
ACS Paragon Plus Environment