Design, green one-pot synthesis and molecular docking study of novel N,N-bis(cyanoacetyl)hydrazines and bis-coumarins as effective inhibitors of DNA gyrase and topoisomerase IV

Article abstract of DOI:10.1016/j.bioorg.2020.103672

A novel, quick, environmentally safe, and one-pot synthesis of a series of N,N-bis(cyanoacetyl)hydrazine derivatives, bis-imino-2H-chromenes and bis-2-oxo-2H-chromene derivatives have been designed. Some selected newly synthesized compounds were investigated in vitro for their antibacterial activity. Compound 5j is the most toxic compound against Staphylococcus aureus with activity index 171%, followed by compound 15b with activity index 136% compared to standard drug ampicillin. Moreover, compound 15a is the most toxic compound against Escherichia coli with activity index 111% compared to standard drug gentamicin. Minimum inhibitory concentration (MIC) was carried out for compounds with high antibacterial activity. Compound 5j has good MIC (7.8 μg/ml) against Staphylococcus aureus while 15a has good MIC (31.25 μg/ml) against Streptococcus mutans which is better than MIC of the standard drug ampicillin (MIC = 62.5 μg/ml). Compounds 5j, 5k, 15a, 15b and 15e which have good MIC values were introduced to enzyme assay against DNA gyrase and topoisomerase IV. The results showed that compound 15a can strongly inhibit DNA gyrase and topoisomerase IV (IC₅₀ = 27.30 and 25.52 μM respectively), compared to methotrexate as the standard drug (IC₅₀ = 29.01 and 23.55 μM respectively). Structure-activity relationships were also discussed based on the biological and docking simulation results.

Full text of DOI:10.1016/j.bioorg.2020.103672

Journal Pre-proofs
Design, green one-pot synthesis and molecular docking study of novel N,N-
bis(cyanoacetyl)hydrazines and bis-coumarins as effective inhibitors of DNA
gyrase and topoisomerase IV
Nadia Hanafy Metwally, Sanaa Osman Abdallah, Marwa Maher Abdel
Mohsen
PII:
S0045-2068(19)32129-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.103672
YBIOO 103672
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Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
12 December 2019
24 January 2020
14 February 2020
Please cite this article as: N. Hanafy Metwally, S. Osman Abdallah, M. Maher Abdel Mohsen, Design, green one-
pot synthesis and molecular docking study of novel N,N-bis(cyanoacetyl)hydrazines and bis-coumarins as
effective inhibitors of DNA gyrase and topoisomerase IV, Bioorganic Chemistry (2020), doi: https://doi.org/
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Design, green one-pot synthesis and molecular docking study of novel
N,N-bis(cyanoacetyl)hydrazines and bis-coumarins as effective
inhibitors of DNA gyrase and topoisomerase IV
Nadia Hanafy Metwally*, Sanaa Osman Abdallah and Marwa Maher Abdel
Mohsen
Cairo University, Faculty of Science, Chemistry Department, Giza, Egypt
E-mail: nhmmohamed@yahoo.com
Abstract: A novel, quick, environmentally safe, and one-pot synthesis of a series of N,N-
bis(cyanoacetyl)hydrazine derivatives, bis-imino-2H-chromenes and bis-2-oxo-2H-chromene
derivatives have been designed. Some selected newly synthesized compounds were
investigated in vitro for their antibacterial activity. Compound 5j is the most toxic
compound against Staphylococcus aureus with activity index 171 %, followed by
compound 15b with activity index 136% compared to standard drug ampicillin.
Moreover, compound 15a is the most toxic compound against Escherichia coli with
activity index 111% compared to standard drug gentamicin. Minimum inhibitory
concentration (MIC) was carried out for compounds with high antibacterial activity.
Compound 5j has good MIC (7.8 μg/ml) against Staphylococcus aureus while 15a has
good MIC (31.25 μg/ml) against Streptococcus mutans which is better than MIC of
the standard drug ampicillin (MIC = 62.5 μg/ml). Compounds 5j, 5k, 15a, 15b and
15e which have good MIC values were introduced to enzyme assay against DNA
gyrase and topoisomerase IV. The results showed that compound 15a can strongly
inhibit DNA gyrase and topoisomerase IV (IC50 = 27.30 and 25.52 μM respectively),
compared to methotrexate as the standard drug (IC50 = 29.01 and 23.55 μM
respectively). Structure-activity relationships were also discussed based on the
biological and docking simulation results.
Keywords: N,N-bis(cyanoacetyl)hydrazines; bis-coumarins; antibacterial; DNA gyrase
and topoisomerase IV; molecular docking study; enzyme inhibition.
1

1. Introduction
Bactria resistance takes place in the presence of one or more antibiotics. When
this occurs, the resistant bacteria continue to cause infection. Bacteria are able to resist
antibiotics through many various ways [1-3]. The more diffusion of these resistant
organisms happens, the more they add to the resistance genes in all bacteria; as a
result, undefeatable strains of disease-causing microbes are generated. Therefore, the
continuous designing and developing of new antibacterial drugs is necessary.
DNA gyrase and topoisomerase IV (Topo IV) are both IIA topoisomerases
present in bacteria. They have similar structures and function as they exhibit 40% of
overall DNA sequence identity [4] and possess significantly similar active sites [5].
Therefore, they are considered as attractive targets for designing new antibiotics [6].
They are highly homologous enzymes that play essential roles in bacterial DNA
replication, chromosome segregation, and DNA compaction [7].
Both DNA gyrase and topoisomerase IV are heterotetramers consisting of two
A and two B subunits (A2B2). The A subunit contains the active-site tyrosine
involved in the DNA breakage-reunion activity while the B subunit catalyzes ATP
hydrolysis [8]. Though clearly related, based on amino acid sequence similarity, they
each play crucial, but distinct, roles in the cell.
DNA gyrase encoded by GyrA and GyrB; GyrA maps to 50.3 minutes on the
E. Coli chromosome and encodes a 97 KDa protein [875amino acids] [9,10] whereas
GyrB maps to 83.5 minutes and encodes a 89.9 KDa protein [804 amino acids] [9].
DNA gyrase has ability to introduce negative supercoils into DNA by looping the
template so as to form a crossing, then cutting one of the double helices and passing
the other through it before releasing the break, changing the linking number by two in
each enzymatic step. This process occurs in prokaryotes, whose single circular DNA
is cut by DNA gyrase and the two ends are then twisted around each other to form
supercoils.
In spite of, topoisomerase IV binds to DNA as a heterotetramer consisting two
parC and two parE genes [11]. Topoisomerase IV is a decatenating enzyme that
resolves interlinked daughter chromosomes following DNA replication (Fig. 1) [12].
Topoisomerase inhibitors act as an obstacle to the ligation step in both gram
positive and gram negative microbial cell cycle, generating single and double stranded
breaks that damage the integrity of the genome leading to apoptosis (cell death) in the
2

proliferating cell [13]. Its absence in the mammalian organism and its crucial role in
the bacterial DNA replication cycle make this enzyme a suitable target for the
development of antibacterial drugs with selective toxicity.
Fig. 1. Structure of type IIA topoisomerases (DNA gyrase and topoisomerase IV).
(a) Primary domain structure of type IIA topoisomerases, (b) 3D Structure of DNA
gyrase and topoisomerase IV with pdb entry [4Z2C for DNA gyrase] and
[3FOE for topoisomerase IV].
The best characterized inhibitor classes of type IIA topoisomerases are the
fluoroquinolone antibiotics which act by stabilizing the complex between a DNA
molecule and the ParC/GyrA active site of the enzyme [14,15]. However, the
3

increasing prevalence of fluoroquinolone resistant strains and problems with side
effects are beginning to limit the utility of this widely utilized antibiotic class [16].
On the other hand, aminocoumarins, catalytic inhibitors of DNA gyrase act via
competitive inhibition of GyrB ATPase activity [17]. Aminocoumarin antibiotics,
such as novobiocin, clorobiocin, and coumermycin A1 (Fig. 2) are effective at killing
Gram-positive bacteria, but were unsuccessful in the clinic. A number of structure-
based design efforts have been effective in generating ligand efficient inhibitors
superior to aminocoumarin antibiotics in potency, but to date, no synthetic or natural
product GyrB/ParE inhibitors have reached the clinic. Thus, the GyrB/ParE ATP-
binding subunits represent unexploited targets for the development of novel
antibiotics.
Derivatives of 2-cyanoacetohydrazide has been regarded as important
intermediates and starting materials in the synthesis of many remarkable heterocyclic
ring systems as they are highly reactive and polyfunctional compounds due to having
both electrophilic and nucleophilic centers. Furthermore, they have received a great
attention as pharmaceutical drugs [18, 19] and as antibacterial agents [20]. Moreover,
they are applicable in the industrial field in the production of herbicides [21] and dyes
[22]. 2-Cyanoacetohydrazide derivatives have been utilized in the synthesis of
coumarins [23,24], quinolines [25], pyrazoles [26], pyrazolopyrimidines [27],
thiazoles [28], pyridines [29], pyrimidines [30] and many other heterocyclic ring
systems [31]. In view of these reports, and in continuation to our work in the synthesis
of novel biologically active compounds [32-38], we designed a green synthesis of a
series of some novel bis-coumarin analogs aiming to introduce new DNA gyrase and
topo IV inhibitors as antibacterial agents starting from the key intermediate N,N-
bis(cyanoacetyl)hydrazines.
4

Fig. 2. Aminocoumarin inhibitors
5

2. Results and discussion
2.1. Chemistry
The reaction of 2-cyanoacetohydrazide (1) with 3-(3,5-dimethyl-1H-pyrazol-
1-yl)-3-oxopropanenitrile (2) in glacial acetic acid leads to the formation of N,N-
bis(cyano acetyl)hydrazine 3 (Scheme 1). The structural assignment of compound 3
was performed based on elemental analyses and spectral data (see experimental).
Scheme 1
Knoevenagel condensation of compound 3 with various aldehyde derivatives
4a-k via grinding in the presence of sodium hydroxide as a basic catalyst at room
temperature afforded the respective arylmethylene derivatives 5a-k (Scheme 2). The
structures of the desired products were confirmed by elemental analyses and spectral
1
data (IR, H NMR and MS). For example, the IR spectrum of 5b showed an
absorption bands at 3432, 2210 and 1649 cm-1 corresponding to NH, CN and CO
group. The 1H NMR spectrum of 5b revealed two singlet signals at δ = 10.64 and 7.94
ppm attributable to NH and vinylic protons, respectively. Mass spectrum exhibited a
molecular ion peak at m/z 402 (M+) which is consistent with the molecular formula
C22H18N4O4. Elemental analyses together with mass spectrum is in agreement to the
proposed structures (Scheme 2).
6

Fig. 3. Appearance of reaction media at different times for compound 5a
Scheme 2
Grinding compound 3 with 2-benzylidenemalononitrile (6) in the presence of
sodium hydroxide at room temperature provided compound 5a not compound 7 as
proved by mp., m.mp. and spectral data (Scheme 3).
7

Scheme 3
When compound 3 was grand with o-hydroxybenzaldehydes 8a,b in sodium
hydroxide, products 10a,b were isolated (Scheme 4). The IR spectrum of 10a
exhibited absorption band at 3433 cm-1 due to the NH function group and another
1
absorption band at 1669 cm-1 corresponding to CO group. Its H NMR spectrum
presented D2O exchangeable signals at 10.25 and 10.68 ppm owing to NH protons,
along with the signals for aryl protons in expected region. The latter compound was
refluxed in dioxane with few drops of concentrated hydrochloric acid to yield the
respective bis-coumarin derivatives 11a,b. The structures of the products were
established on the basis of elemental analysis and spectral data (Scheme 4).
Scheme 4
8

Condensation of compound 3 with different azosalicylaldehydes 12a-e under
the same reaction conditions furnished the isolated products 14a-e. Refluxing these
products in dioxane and few drops of conc. HCl afforded products 15a-e (Scheme 5).
The structure of the isolated products was supported by elemental analysis and
1
spectral data (IR, H NMR and MS). For example, the IR spectrum of the isolated
product 15e showed absorption bands at 3432 and 1665 cm-1 due to NH and CO
1
groups, respectively. The H NMR spectrum of this product revealed a singlet signal
at δ = 10.46 ppm attributable to NH, besides the aryl protons. Mass spectrum showed
a molecular ion peaks at m/z 620 (M+) which is consistent with the assigned structure
15e.
Fig. 4. Appearance of reaction media at different times for compound 15a
9

Scheme 5
It is interesting to synthesize azo dyes by grind stone method. Thus, a mixture
of equimolar amounts of compound 3, aromatic amines 16a-e and sodium nitrite in
presence of solid p-toluenesulfonic acid (P-TSA) was grand to afford colored
products 17a-e, (Scheme 6). The structure of the products 17a-e was proved by
1
13
elemental analysis and spectral data (IR, H NMR, C NMR and MS). For example,
the IR spectrum of the isolated product 17e showed absorption bands at 3433, 3215,
1
2216 and 1670 cm-1 corresponding to NH, CN and CO groups, respectively. The H
NMR spectrum of the same product revealed two singlet signals at δ = 10.23 and
11.28 ppm due to NH protons beside, the aryl proton signals in the expected regions.
Mass spectrum showed a molecular ion peaks at m/z 410 (M+) which is consistent
13
with the molecular formula C18H12F2N8O2. The C NMR spectrum of 17e exhibited
the following characteristic signals 105.7, 110.9, 115.1, 115.9, 128.0, 138.6, and
160.7 ppm.
10

Scheme 6
2.2. Biology
2.2.1. Antibacterial evaluation
Some selected newly synthesized compounds 3, 5a, 5b, 5c, 5d, 5e, 5h, 5i, 5j, 5k, 14a,
14b, 14c, 14d, 14e, 15a, 15b, 15c, 15d, 15e, 17c, 17d and 17e were tested in vitro for
their antibacterial activity against Staphylococcus aureus and Streptococcus mutans
(Gram positive bacteria), Escherichia coli, Pseudomonas aeruginosa and Klebsiella
pneumonia (Gram negative bacteria) using nutrient agar medium. Ampicillin and
gentamicin were used as standard drugs for Gram positive and Gram negative,
respectively. The antimicrobial activity of the newly synthesized compounds was
determined using agar well diffusion method [39], and the diameters (mm) of
inhibition zones are shown in Table 1.
From the results presented in Table 1, it was found that most of the tested
compounds exhibited toxicity against S. aureus, S. mutans and E. coli higher than P.
aeruginosa and K. pneumonia. Compound 5j is the most toxic compound against S.
aureus with activity index 171% followed by compound 15b with activity index
136%. In addition, compounds 15a and 15e with activity index 128 and 120%
respectively compared to standard drug ampicillin. Moreover, compounds 15a, 15b
and 15e exhibited toxicity against fungi (C. albicans) with activity index 104, 74 and
60%, respectively compared to standard drug nystatin. Compound 15a is the most
toxic against E. coli with activity index 111% compared to gentamicin activity. Also,
toxicity of compound 5j is better than 15b against E. coli, K. pneumonia and P.
aeruginosa with activity index 98, 94 and 57% respectively. The remaining tested
compounds showed activity between moderate and high compared to standard drug.
11

Table 1. Antibacterial activity of some tested compounds 3, 5a, 5b, 5d, 5e, 5h, 5i,
5j, 5k, 14b,14c, 14d, 14e, 15a, 15b, 15c, 15d, 15e, 17c, 17d and 17e against gram
positive, gram negative bacteria and fungi.
Diameter inhibition zone in mm (% activity index)
Gram negative bacteria
Gram positive bacteria
Pseudomonas Staphylococcus Streptococcus
aeruginosa aureus mutans
Fungi
Candida
albicans
Escherichia
coli
Klebsiella
pneumonia
Compounds
no.
(ATCC:3008) (ATCC:4415) (ATCC:27853) (ATCC:6538) (ATCC:25175) (ATCC:10231)
NA
11.7 ± 0.5
(46%)
19.9 ± 0.6
(79%)
19.7 ± 0.6
(78%)
NA
NA
NA
NA
NA
NA
13.7 ± 0.6
(65%)
NA
12.7 ± 0.5
(43%)
14.9 ± 0.5
(51%)
14.7 ± 0.5
(50%)
14.2 ± 0.5
(48%)
NA
NA
NA
NA
3
14.8 ± 0.5
(54%)
13.7 ± 0.5
(50%)
5a
5b
5c
5d
5e
5h
5i
NA
NA
NA
15.8 ± 0.5
(75%)
NA
14.6 ± 0.5
(54%)
NA
18.3 ± 0.6
(73%)
NA
NA
10.6 ± 0.5
(50%)
NA
13.3 ± 0.6
(63%)
12 ± 0.5
(57%)
15 ± 0.5
(71%)
12.5 ± 1.0
(43%)
19.8 ± 0.5
(68%)
25.1 ± 0.5
(86%)
13.8 ± 0.5
(51%)
17.9 ± 0.5
(66%)
NA
NA
14 ± 0.5
(48%)
NA
NA
NA
26.6 ± 0.6
(98%)
24.6 ± 0.6
(91%)
23.6 ± 0.6
(94%)
24.3 ± 0.6
(97%)
16.6 ± 0.5
(57%)
NA
36.3 ± 0.6
(171%)
NA
16.6 ± 0.6
(57%)
11.6 ± 0.6
(55%)
NA
5j
NA
5k
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
14a
14b
14c
14d
NA
NA
NA
NA
NA
NA
15.3 ± 0.5
(72%)
NA
10.6 ± 0.5
(36%)
NA
11.6 ± 0.5
(55%)
NA
NA
NA
NA
NA
14e
15a
30 ± 0.5
(111%)
NA
16 ± 0.5
(64%)
15.6 ± 0.5
(62%)
27 ± 0.5
(128%)
28.6 ± 0.6
(136%)
16.6 ± 0.6
(79%)
20.6 ± 0.6
(98%)
25.3 ± 0.6
(120%)
NA
37 ± 0.5
(127%)
20.3 ± 0.6
(70%)
13.3 ± 0.5
(45%)
14.6 ± 0.5
(50%)
15.3 ± 0.5
(52%)
22 ± 0.6
(104%)
15.6 ± 0.5
(74%)
10.6 ± 0.5
(50%)
NA
NA
NA
NA
NA
15b
15c
15d
15e
17c
NA
NA
NA
NA
NA
NA
NA
12.6 ± 0.5
(50%)
10.7 ± 0.5
(42%)
12.6 ± 0.5
(60%)
NA
NA
12

NA
NA
12.5 ± 0.5
(50%)
11.3 ± 0.5
(45%)
25±0.5
(100%)
-
NA
NA
NA
NA
-
NA
NA
-
NA
17d
17e
NA
27±0.5
(100%)
-
29±0.5
(100%)
-
-
-
Gentamicin
Ampicillin
Nystatin
21±0.1
(100%)
-
29±0.5
(100%)
-
-
-
-
21±0.5
- Zone of inhibition is expressed in the form of mean± standard deviation (mm).
- NA: No activity
- Well diameter (6mm).
- 100 µ l was tested.
2.2.2. Structure activity relationships (SAR)
It was found that the parent compound 3 has a moderate antibacterial activity against
K. pneumonia, S. aureus and S. mutans with activity index 46, 65 and 43%,
respectively. Introducing phenyl groups in the parent compound 3 as in compound 5a,
13

resulted in an increase in the antibacterial activity towards both K. pneumonia and S.
mutans with activity index 79 and 51%, respectively. The introduction of one electron
donating group in each phenyl ring as in compound 5b, the antibacterial activity
decreased slightly towards E. coli (activity index 50%). On the other hand, the
presence of two electron donating groups in each phenyl group like compound 5d
resulted in a moderate activity to C. albicans (activity index 50%).
Introducing one or two electron withdrawing groups in each phenyl group such as 5c
or 5e, the activity against E. coli and K. pneumonia disappeared. But, they have
activity to S. aureus and S. mutans with activity index 75 and 48% for 5c and 63 and
43% for 5e, respectively.
In compounds 5h and 5i containing the hetero-moiety thiophene and furan
respectively, effected an increase of the antibacterial activity than the starting
compound 3. Increased antibacterial activity was observed on using substituted furan
as in 5j and 5k. Thus, compound 5j have a high activity towards all types of bacteria
and fungi especially, S. aureus and E. coli (activity index 171 and 98%, respectively).
Whereas, 5k was active only to E. coli and K. pneumonia (activity index 91 and 97%,
respectively).
Another category of synthesized compounds (imino coumarins) 14a-e showed no
activity against all types of the studied bacteria except for 14d which exhibited
activity to S. aureus, S. mutans and C. albicans. On the other hand, oxo-coumarins
15a-e showed much better antibacterial activity than the imino-coumarins 14a-e.
Compound 15a which contains unsubstituted azo phenyl group in the coumarin
moiety have a strong activity to all types of bacteria and fungi except P. aeruginosa.
Compound 15b (methyl derivative) have activity index 62, 136, 70 and 74% with
respect to K. pneumonia, S. aureus, S. mutans and C. albicans, respectively. Finally,
azo derivatives 17c, 17d and 17e have moderate activity against K. pneumonia only,
while being inactive towards the other studied bacteria.
14

2.2.3. Minimum inhibitory concentration (MIC)
MIC was carried out for compounds 5c, 5j, 15a, 15b, 15c, 15d and 15e with high
antibacterial activity against S. aureus, S. mutans, E. coli and K. pneumonia. The
results presented in Table 2 indicated that compound 5j with high toxicity against S.
aureus has the best minimum inhibitory concentration (MIC) against s. aureus (7.8
μg/ml), compared to the other tested compounds which is also better than MIC of the
standard drug ampicillin (MIC = 62.5 μg/ml). Also, the MIC of compounds 15b and
15e with high toxicity against s. aureus is equal to that of the standard ampicillin
(MIC = 62.5 μg/ml). The MIC of compound 15a with the highest toxicity against S.
mutans is 31.25 μg/ml and it is better than MIC of the standard drug ampicillin (MIC
= 62.5 μg/ml). Regarding results in Table 3, it showed that both compounds 5j and 5k
have MIC values against K. pneumonia which are equal to the standard gentamicin
(MIC = 62.5 μg/ml). Also, 5j is the only compound which has MIC value with respect
to P. aeruginosa (MIC = 250 μg/ml). Moreover, compound 15a has MIC value
against E. coli which is equal to that of gentamicin (MIC = 31.25 μg/ml).
Table 2. Minimum inhibitory concentration of some tested compounds 5c, 5j, 15a, 15b,
15c, 15d and 15e on Gram positive bacteria.
Minimum inhibitory concentration (MIC) in μg/ml
Gram positive bacteria
Compound
no.
Staphylococcus
aureus
Streptococcus
mutans
(ATCC:6538)
(ATCC:25175)
5c
5j
125
7.8
-
250
31.25
250
-
15a
125
62.5
125
250
62.5
62.5
15b
15c
15d
-
15e
250
62.5
Ampicillin
- Stock concentration 1 mg/ml
- Concentrations unit of MIC are represented as μg/ml
15

Table 3. Minimum inhibitory concentration of some tested compounds 5d, 5j, 5K, 15a
and 15b on Gram negative bacteria.
Minimum inhibitory concentration (MIC) in μg/ml
Gram negative bacteria
Compound
no.
Escherichia
coli
(ATCC:9637)
Klebsiella
pneumonia
(ATCC:10031)
125
Pseudomonas
aeruginosa
(ATCC:27853)
-
5d
250
5j
5k
125
500
31.25
-
62.5
62.5
250
500
62.5
250
-
-
15a
15b
-
Gentamicin
31.25
125
- Stock concentration 1 mg/ml
- Concentrations unit of MIC are represented as μg/ml
2.2.4. Enzyme inhibition of DNA gyrase and topoisomerase IV
DNA gyrase and topoisomerase IV inhibition activity was investigated for the most
potent cytotoxic active compounds 5j, 5k, 15a, 15b and 15e using methotrexate as a
reference drug. The results deduced from Table 4 showed that all tested compounds
exhibited potent inhibitory effect towards DNA gyrase and topoisomerase IV.
Compound 15a can strongly inhibit the two enzymes (IC50 = 27.30 and 25.52 μM
respectively), compared to methotrexate. This means that the tested compounds may
induce their inhibitory effect by binding to the enzymes active site causing inhibition
of their activity which affected bacterial cell growth. This may be due to the similarity
of structure 15a to coumermycin A1 (Fig.2).
The data listed in Table 5 showed that methotrexate have the highest inhibitory effect
on DNA gyrase and topo IV (92 and 94.7%, respectively). Among the tested
compounds, it was found that compound 15a have inhibitory effect on DNA gyrase
and topoisomerase IV higher than the other tested compounds. Compound 15a
effected inhibition percent 88.9 and 93.7 followed by 5k 86.4 and 88.3 of DNA
gyrase and topoisomerase IV, respectively.
16

Table 4.IC50 (μM) of the tested compounds 5j, 5k, 15a, 15b, 15e and methotrexate
on DNA gyrase and topoisomerase IV.
Compound no.
IC50 (μM) on
IC50 (μM) on
topoisomerase
IV
DNA gyrase
5j
33.40±1.56
30.34±1.82
27.30±1.66
30.96±1.44
51.10±2.39
29.01±1.27
44.92±2.95
33.98±2.08
25.52±1.39
34.45±1.86
46.15±3.11
23.55±1.71
5k
15a
15b
15e
Methotrexate
IC50 (μM)
IC50 (μM)
60
50
40
30
20
10
0
50
40
30
20
10
0
Fig. 5. IC50 (μM) on DNA gyrase
Fig. 6. IC50 (μM) on topoisomerase IV
17

Table 5. Inhibition percentage of the tested compounds 5j, 5k, 15a, 15b, 15e
and methotrexate on DNA gyrase and topoisomerase IV.
Code
conc
µM
log
conc
%inhibition %inhibition of
of DNA
gyrase
topoisomerase
IV
100
50
25
2
92.00836
70.08258
45.49292
19.87869
94.73674
71.61601
45.29972
35.90105
Methotrexate
1.69897
1.39794
1.09691
12.5
5j
100
50
25
2
85.35236
60.45163
43.03395
15.37059
85.52604
63.9091
32.95246
10.71261
1.69897
1.39794
1.09691
12.5
5k
100
50
25
2
86.47569
62.09094
48.1568
88.34565
65.60086
35.5251
1.69897
1.39794
1.09691
12.5
25.00154
16.16384
15a
15b
15e
100
50
25
2
88.93465
68.53469
44.66446
29.83598
93.79688
82.14252
54.51042
17.66763
1.69897
1.39794
1.09691
12.5
100
50
25
2
80.7381
67.82853
36.68163
25.41136
81.59155
60.84304
38.22249
22.3101
1.69897
1.39794
1.09691
12.5
100
50
25
2
71.51698
48.36171
23.97697
14.75585
85.90199
51.69082
13.34424
6.577191
1.69897
1.39794
1.09691
12.5
2.2.5. Agarose gel electrophoresis.
Investigation the influence of compounds 5j, 5k, 15a, 15b, 15e and methotrexate at
different concentrations of 100, 50, 25, 12.5 and 7 μM on the enzymes using the
supercoiling assay for DNA gyrase and the decantation assay for topo IV (Fig 7 and
8). The first lane, labeled (-) contains a control assay without enzymes while the
second lane, labeled (+) contains enzyme assays (DNA gyrase or topoisomerase IV)
which indicates supercoiled DNA in case of DNA gyrase and decanted DNA in case
of topoisomerase IV [6,40]. At higher drug concentrations, inhibition of the enzyme is
18

very high like the negative control and the enzymes activity increase by decreasing
the drug concentration. Compounds 15a and 5k have high ability to inhibit the
enzymes activity which reflected on the bands pattern of the gel, while 15e is the
lowest compound.
Fig. 7. Electrophoretic analysis of bacterial DNA gyrase assays with the
Inhibitory effect of 5j, 5k, 15a,15b, 15e.
Fig. 8. Electrophoretic analysis of bacterial topoisomerse IV assays with the
Inhibitory effect of 5j, 5k, 15a,15b, 15e.
2.2.6. Molecular modeling and docking study
Docking simulation for the newly synthesized compounds 5j, 5k, 15a, 15b, 15e and
methotrexate was carried out using MOE 2009.10 software. The crystal structure of
the selected protein was downloaded from a protein data bank (http://www. PDB.org)
with pdb code = 6ENH (Fig 9). Refinement of the crude pdb structure was performed,
and then saved as a moe file to be used for docking simulation. The 2D structures of
19

the selected compounds 5j, 5k, 15a, 15b, 15e and methotrexate were converted into
their 3D forms using Chem 3D ultra 12.0 software and then the energy was minimized
and saved as mol. file. Docking simulation was performed as in the MOE 2009.10
manual; all these compounds were docked into the same active site of the ligand and
the binding affinities were calculated. The results of the docking simulation together
with the measured affinities and distances are shown in Table 6.
According to the docking simulation results, compounds 5j, 5k, 15a, 15b, 15e and
methotrexate have binding energies of -8.6143, -10.2332, -7.1273, -8.1949, -9.1186
and -13.4633 kcal/mole, respectively. Docking of methotrexate with the active site of
the selected protein showed three hydrogen bonds, two are formed between oxygen
and Arg 123 with bond length (1.95 Å) and CO with Arg 123 (2.09 Å). The third one
between NH2 and Asp 70 (1.97 Å) (Fig. 10). The mode of interactions for compounds
15a and 5k which appeared to have the highest potency against DNA gyrase and
topoisomerase IV are illustrated. Thus compound 15a showed four interactions three
of them are hydrogen bond interactions and one arene-cation interaction. All
hydrogen bonds are between CO linked to Arg 133 and Arg 73 (1.89, 2.32 and 2 Ǻ)
respectively, and the arene-cation interaction is between phenyl ring and Arg 73 (Fig.
13). Also, compound 5k exhibited three hydrogen bond between CN and Arg 73, Gly
74 and Thr 164 (2.49, 2.07 and 2.53 Å, respectively) (Fig. 12). Moreover, the
molecular docking of compound 15b with active site of enzyme presented three
interactions two of them for arene-cation and one for hydrogen bond between CO
linked to Asn 43 (2.08 Å) (Fig. 14). Compound 5j have a moderate activity towards
the enzymes which showed two interactions, a hydrogen bonding between CO and
Arg 73 (2.26 Å) and arene-cation interaction between furan ring and Arg 73 (Fig. 11).
Molecular docking of compound 15e which has the lowest cytotoxic activity showed
only one hydrogen bonding interaction between CO and Arg 73 (2.11 Å) (Fig. 15).
20

Table 6. The protein-ligand interactions of some tested compounds and
methotrexate (standard drug) with 6ENH active site pocket.
Compounds
Binding
energy (S)
Kcal/mol
Groups of
the ligand
involved in
the
Amino acids
involved in
the
Type of the bond and length of
Hydrogen bonds
interaction
Ǻ
interaction
-13.4633
O-
Arg 123
Arg 123
Asp 70
Arg 73
Arg 73
Arg 73
Gly 74
Thr 164
Arg 133
Arg 133
Arg 73
Arg 73
Basic side chain acceptor1.95 Ǻ
Basic side chain acceptor 2.09 Ǻ
Acidic side chain donor 1.97 Ǻ
Basic side chain acceptor 2.26 Ǻ
Basic (Arene-cation interaction)
Basic side chain acceptor 2.49 Ǻ
Polar backbone acceptor 2.07 Ǻ
Polar side chain acceptor 2.53 Ǻ
Basic side chain acceptor 1.89 Ǻ
Basic side chain acceptor 2.32 Ǻ
Basic side chain acceptor 2 Ǻ
Basic (Arene-cation interaction)
Polar side chain acceptor 2.08 Ǻ
Basic (Arene-cation interaction)
Methotrexate
C=O
NH2
-8.6143
C=O
5j
Furan ring
CN
-10.2332
5k
CN
CN
-7.1273
C=O
15a
C=O
C=O
Phenyl ring
C=O
-8.1949
-9.1186
Asn 43
Arg 133
Arg 73
Arg 73
15b
15e
Phenyl ring
Phenyl ring
C=O
Basic (Arene-cation interaction)
Basic side chain acceptor 2.11 Ǻ
21

Fig. 9. Structure of 6ENH.
2D structure
3D structure
Fig. 10. Binding pattern of methotrexate (2D and 3D) structure with the active site of
DNA gyrase and topoisomerase IV enzymes (PDB code = 6ENH).
22

2D structure
3D structure
Fig. 11. Binding pattern of compound 5j (2D and 3D) structure with the active site of
DNA gyrase and topoisomerase IV enzymes (PDB code = 6ENH).
2D structure
3D structure
Fig. 12. Binding pattern of compound 5k (2D and 3D) structure with the active site of
DNA gyrase and topoisomerase IV enzymes (PDB code = 6ENH).
23

2D structure
3D structure
Fig. 13. Binding pattern of compound 15a (2D and 3D) structure with the active site
of DNA gyrase and topoisomerase IV enzymes (PDB code = 6ENH).
24

2D structure
3D structure
Fig. 14. Binding pattern of compound 15b (2D and 3D) structure with the active site
of DNA gyrase and topoisomerase IV enzymes (PDB code = 6ENH).
25

2D structure
3D structure
Fig. 15. Binding pattern of compound 15e (2D and 3D) structure with the active site
of DNA gyrase and topoisomerase IV enzymes (PDB code = 6ENH).
26

3. Conclusion
Some novel bis(cyanoacetyl)hydrazines and bis-coumarin derivatives were
synthesized and evaluated in vitro against bacterial cell lines. Results revealed that the
bis-coumarin derivative 15a is the most potent inhibitor of DNA gyrase and
topoisomerase IV (essential for bacterial cell growth), which can be attributed to its
structural similarity to 6ENH native ligand.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were determined on an Electrothermal (9100) apparatus and are
uncorrected. The IR spectra were recorded as KBr pellets on a Perkin Elmer 1430
1
spectrophotometer. H NMR and 13C NMR spectra were recorded in deuterated
dimethylsulfoxide at 300 and 75 MHz, respectively, on a Varian Gemini NMR
spectrometer using tetramethylsilane as internal reference and the results are expected
as δ value. Mass spectra were taken on a Shimadzu GCMS-QP 1000 Ex mass
spectrometer at 70 eV. Elemental analyses were carried out at the Microanalyses
Center of Cairo University, Giza-Egypt. Antibacterial activity and MIC carried out in
laboratory of Biochemistry at Faculty of Science-Cairo University-Egypt. Enzyme
inhibition tests were carried out in Science Way laboratory in Cairo. Physical and
spectral data of all newly synthesized compounds are found in supplementary
information 1.
4.1.2. 2-Cyano-N'-(2-cyanoacetyl)acetohydrazide (3).
A mixture of 2-cyanoacetohydrazide (1) (0.01mol) and 3-(3,5-dimethyl-1H-pyrazol-
1-yl)-3-oxopropanenitrile (2) (0.01mol) was heated in glacial acetic acid in water
o
bath at 40 C for one hour, then allowed to cool. The solid collected by filtration and
recrystallized from an ethanol-dioxane mixture.
o
White crystals; yield 90%; mp. 195 C (lit., 200-202 oC) [41]; IR (KBr) 3203, 3063
1
(NH), 2263 (CN), 1617 (CO) cm-1; H NMR (DMSO-d6)  = 3.75 (s, 4H, 2CH2),
10.38 (s, 2H, 2NH). Anal. Calcd. C6H6N4O2 (166.14): C, 43.38; H, 3.64; N, 33.72.
Found: C, 43.58; H, 3.46; N, 33.94 %.
27

4.1.3. Synthesis of compounds 5a-k and 10a,b.
A mixture of 2-cyano-N'-(2-cyanoacetyl)acetohydrazide (3) (0.01mol) and the
appropriate aromatic aldehyde 4a-k and / or 8a,b (0.01mol) was ground in presence of
sodium hydroxide (0.01mol). The ice cold water was poured into the mixture, then the
solid was filtered off and washed well with water, then recrystallized from N,N-
dimethylformamide.
4.1.3. 1. 2-Cyano-N'-(2-cyano-3-phenylacryloyl)-3-phenylacrylohydrazide (5a).
Yellow crystals; yield 60%; mp. 285 oC; IR (KBr) 3431, 3296 (NH), 2212 (CN), 1639
1
(CO) cm-1; H NMR (DMSO-d6)  = 7.29-7.53 (m, 10H, Ar), 7.94 (s, 2H, 2CH),
10.62 (s, 2H, 2NH); m/z = 342 (M+, 2.99%), 239 (3.35%), 186 (4.61%), 165 (29.4%),
120 (13.3%), 110 (12.6%), 103 (22.2%), 91 (54.4%), 83 (26.4%), 60 (47.6%), 57
(94.4%), 55 (100%), 51 (55.5%). Anal. Calcd. C20H14N4O2 (342.11): C, 70.17; H,
4.12; N, 16.37. Found: C, 70.35; H, 4.30; N, 16.15 %.
4.1.3.2. 2-Cyano-N'-(2-cyano-3-(2-methoxyphenyl)acryloyl)-3-(2-methoxyphenyl)
acrylohydrazide (5b).
Orange crystals; yield 58%; mp. 250 oC; IR (KBr) 3432 (NH), 2210 (CN), 1649 (CO)
1
cm-1; H NMR (DMSO-d6)  = 3.90 (s, 6H, 2OCH3), 7.08-8.02 (m, 8H, Ar), 8.38 (s,
2H, 2CH), 10.52 (s, 2H, 2NH); 13C NMR (DMSO-d6)  = 56.2, 112.2, 113.1, 118.3,
121.9, 128.4, 133.4, 136.9, 158.4; m/z = 402 (M+, 1.01%), 375 (1.47%), 285 (2.85%),
215 (12.6%), 186 (20.3%), 160 (11.9%), 121 (100%), 111 (10.8%), 99 (10.3%), 89
(16.4%), 77 (20.0%), 69 (13.0%). Anal. Calcd. C22H18N4O4 (402.13): C, 65.66; H,
4.51; N, 13.92. Found: C, 65.49; H, 4.71; N, 14.15 %.
4.1.3.3.
2-Cyano-N'-(2-cyano-3-(2,5-dimethoxyphenyl)acryloyl)-3-(2,5-
dimethoxyphenyl)acrylohydrazide (5c).
Orange crystals; yield 69%; mp. 235 oC; IR (KBr) 3428, 3288 (NH), 2209 (CN), 1642
(CO) cm-1; 1H NMR (DMSO-d6)  = 3.75 (s, 6H, 2OCH3), 3.83 (s, 6H, 2OCH3), 6.76-
7.64 (m, 6H, Ar), 8.32 (s, 2H, 2CH), 10.42 (s, 2H, 2NH). Anal. Calcd. C24H22N4O6
(462.15): C, 62.33; H, 4.80; N, 12.12. Found: C, 62.52; H, 4.62; N, 12.36 %.
28

o
Orange crystals; yield 79%; mp. >300 C; IR (KBr) 3433, 3303 (NH), 2211 (CN),
1642 (CO) cm-1; 1H NMR (DMSO-d6)  = 2.38 (s, 6H, 2CH3), 6.40-7.15 (m, 4H, Ar),
7.75 (s, 2H, 2CH), 10.44 (s, 2H, 2NH); 13C NMR (DMSO-d6)  = 14.2, 110.7, 115.8,
118.3, 129.0, 135.4, 148.6, 155.8, 164.3; m/z = 350 (M+, 0.3%), 328 (1.5%), 313
(1.2%), 270 (15.8%), 268 (21.8%), 139 (11.6%), 125 (20.1%), 111 (30.6%), 97
(46.1%), 83 (54.3%), 71 (59.2%), 57 (100%), 55 (66.5%). Anal. Calcd. C18H14N4O4
(350.10): C, 61.71; H, 4.03; N, 15.99. Found: C, 61.88; H, 4.20; N, 15.75 %.
4.1.3.12. 2-Imino-N'-(2-imino-2H-chromene-3-carbonyl)-2H-chromene-3-carbo-
hydrazide (10a).
o
Orange crystals; yield 62%; mp. >300 C; IR (KBr) 3433 (NH), 1669 (CO), 1611
1
(C=N) cm-1; H NMR (DMSO-d6)  = 6.72-7.57 (m, 8H, Ar), 9.00 (s, 2H, 2CH),
10.25 (s, 2H, 2NH), 10.68 (s, 2H, 2NH); m/z = 374 (M+, 4.88%), 357 (18.13%), 305
(49.21%), 268 (100%), 240 (58.10%), 223 (41.60%), 214 (40.58%), 186 (15.57%),
143 (72.91%), 118 (41.34%), 107 (40.06%), 88 (54.10%), 78 (52.40%), 63 (95.82%),
57 (35.58%), 51 (84.20%). Anal. Calcd. C20H14N4O4 (374.10): C, 64.17; H, 3.77; N,
14.97. Found: C, 64.34; H, 3.60; N, 14.73 %.
4.1.3.13. 6-Hydroxy-N'-(6-hydroxy-2-imino-2H-chromene-3-carbonyl)-2-imino-
2H-chromene-3-carbohydrazide (10b).
o
Red crystals; yield 71%; mp. >300 C; IR (KBr) 3430 (NH), 3399 (OH), 1662 (CO),
1580 (C=N) cm-1; 1H NMR (DMSO-d6)  = 6.70-7.60 (m, 6H, Ar), 9.12 (s, 2H, 2CH),
9.92 (s, 2H, 2OH), 10.30 (s, 2H, 2NH), 10.64 (s, 2H, 2NH). Anal. Calcd. C20H14N4O6
(406.09): C, 59.12; H, 3.47; N, 13.79. Found: C, 58.93; H, 3.27; N, 13.54 %.
4.1.4. Synthesis of compounds 11a,b.
Compounds 10a,b (0.01mol) were refluxed in dioxane containing few drops of conc.
HCl for three hours. The solid was collected by filtration, and recrystallized from
N,N-dimethylformamide.
4.1.4.1. 2-Oxo-N'-(2-oxo-2H-chromene-3-carbonyl)-2H-chromene-3-carbohydra-
zide (11a).
o
1
Orange crystals; yield 59%; mp. >300 C; IR (KBr) 3438 (NH), 1680 (CO) cm-1; H
NMR (DMSO-d6)  = 6.70-7.58 (m, 8H, Ar), 8.89 (s, 2H, 2CH), 10.68 (s, 2H, 2NH).
29