DOI: 10.1002/slct.

201800108 Full Papers

1
2 z Medicinal Chemistry & Drug Discovery
3
4
5
Synthesis, Antibacterial Activity and Molecular Docking of
6 Substituted Naphthyridines as Potential DNA Gyrase
7
8 Inhibitors
9
10 Farghaly A. Omar,*[a] Mariam Abelrasoul,[b] Mahmoud M. Sheha,[b] Hoda Y. Hassan,[b] and
11 Yasser Musa. Ibrahiem[c]
12
13
14 A series of naphthyridine-3-thiosemicarbazide 7,8(a–e) and the addition, the aryl substituted thioemicarbazides 7 c,d,e showed
15 corresponding cyclized analogs, naphthyridine-3-(1,3,4-oxadia- inhibitory effect of the growth of M. smegmatis at MIC ~ 5.4-
16 zoles) 9,10(a–e) were synthesized through modification of the 7.1 mM. Molecular docking to DNA-gyrase cleavage complex of
17 COOH in nalidixic acid (NA) and its 6-bromo analogue, as new S. aureus, Mycobac. (mTB) and Top. IV of K. pneumonia revealed
18 chemical entities (NCE) with enhanced antimicrobial potential. similar binding poses to the co-crystallized quinolone ligands
19 The compounds were screened for antibacterial activity against and indicate good correlation of the binding energy (DG) with
20 Gram positive (G + ve) strains (S. aureus, B. cereus); Gram the observed MIC values of the active compounds. Conse-
21 negative (G-ve) (E. coli, K. pneumonia, P. aeruginosa) and quently, DNA-gyrase assay was proposed and executed. Most
22 Mycobac. smegmatis. Compounds 7 b,c and 9 b,d displayed the prominent DNA-gyrase inhibition showed by the naphthyridin-
23 highest activity against S. aureus (minimal inhibitory concen- yl-3-thiosemicarbazides, 7 c and 8 e (IC50: 1.73 and 4.46 mg/mL
24 tration; MIC ~ 6–7 mM), whereas B. cereus was found to be respectively); and the oxadiazoles 9 b and 10 d (IC50: 3.36 and
25 more susceptible to the brominated oxadiazoles 10 b,d,e (MIC ~ 3.89 mg/mL respectively). Assessment of drug-likeness charac-
26 5.5-5.9 mM). Moreover, 10 b,c,d exhibit similar MIC values teristics illustrates that the synthesized compounds showed
27 against K. pneumonia and M. smegmatis. This demonstrates agreement to Lipinsiki’s and Veper’s parameters. The study
28 that bromination of the naphtyridone skeleton results in could offer an exceptional framework that may lead to the
29 broader spectrum and enhanced antibacterial profile. In discovery of new potent antimicrobial agents.
30
31 Introduction
further exacerbated by extensively drug-resistant (XDR) and
32
The widespread emergence of multidrug resistant (MDR) strains totally drug-resistant (TDR) forms.[5]
33
of microbial infections to clinically available drugs puts further In this context, chemical manipulation of proven leads or
34
momentum to the urgent need for discovery of new and drug candidate is a well established strategy affording new
35
effective antimicrobial agents with novel mechanisms of action. chemical entities (NCE) with improved therapeutic character-
36
WHO report (2015) on global surveillance of antimicrobial istics. Recent reports revealed approval of a brominated diary-
37
resistance stated that the evidence from around the world lquinoline derivative, Bedaquiline, for treatment of drug-
38
indicates an overall decline in the total stock of antibiotic sensitive and drug-resistant Mycobacterium forms. It has a
39
effectiveness.[1] Infections caused by S. aureus, E. coli, P. vulgaris, bactericidal effect against dormant tubercle bacilli through
40
P. aeruginosa, S.typhi and S. paratyphi become resistant to the inhibition of mycobacterial ATP synthase subunit C, thus
41
current antibacterials and are the most prevalent of fatal exhibiting a new mechanism of action.[6,7]
42
infectious diseases.[2–4] In case of M. tuberculosis, the problem is Compounds containing quinoline or its isosteric naphthyr-
43
idine scaffold represent important therapeutic classes that
44
exhibit a wide-spectrum of biological actions, including anti-
45
bacterial,[8] antiviral,[9] anticancer,[10] and antifungal activities.[11]
46 [a] Prof. Dr. F. A. Omar
Dept. Pharmaceutical Chemistry, Faculty of Pharmacy, October 6 Uni- The antibacterial quinolones has been enormously modified to
47
versity, www.o6u.edu.eg (Six October City, Giza/Egypt; Postal Code: 12585) enhance their antibacterial spectrum and attenuate the
48 E-mail: [email protected] developed bacterial resistance.[12] Nalidixic acid (NA), the
49 [email protected]
prototype of the quinolone antibacterials, has been considered
50 Homepage: https://www.researchgate.net/profile/Farghaly_Omar
[b] M. Abelrasoul, Prof. Dr. M. M. Sheha, Dr. H. Y. Hassan as outdated drug due to limited activity against Gram negative
51
Dept. of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, As- (G-ve) bacteria that infect urinary tract. Nevertheless, nalidixic
52 siut 71526, Egypt. acid is ineffective against Grampositive (G + ve) bacteria
53 [c] Dr. Y. M. Ibrahiem
including Staphylococcal, Streptococcal, and Pseudomonal spe-
54 Depart. of Microbiology, National Org. for Drug Control and Research
(NODCAR), Giza, Egypt. cies.[13] Trials involving modifications of the carboxylic function-
55
Supporting information for this article is available on the WWW under ality of nalidixic acid to the corresponding substituted
56
https://doi.org/10.1002/slct.201800108 hydrazides[14,15] or substituted 5-membered heterocycles lead to
57

ChemistrySelect 2018, 3, 2604 – 2612 2604  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Wiley VCH
1809 / 107990
Montag, 05.03.2018
[S. 2604/2612] 1
 Full Papers
remarkable antimicrobial activity against a wide range of tuted derivatives 7 a-8 a were prepared by refluxing the acid
1
bacterial strains.[16,17] Meanwhile, thiosemicarbazides and 1,3,4- hydrazides 5–6 with ammonium thiocyanate in 2 M HCl.[26]
2
oxadiazoles are known bioisosteric surrogates for carboxylic Meanwhile, the substituted congeners 7,8(b–e) were prepared
3
group in lead optimization procedures.[18] Both pharmacophoric by treating the hydrazides 5–6 with the appropriate arylisothio-
4
moieties have shown comprehensive antimicrobial and anti- cyanates in refluxing EtOH.[27] The naphthyridinyl-3-(2-amino-
5
mycobacterial activities.[19–22] 1,3,4-oxadiazoles) 9,10(a–e) were in turn prepared from the
6
The present investigation reports a new series of substi- corresponding thiosemicarbazides by cyclodesulfurization us-
7
tuted naphthyridine skeleton as hybrid entity with thiosemicar- ing mercuric acetate in acetic acid.[28]
8
bazide and/or 1,3,4-oxadiazole moieties. The modifications The structures of the synthesized compounds were con-
9
involve also introduction of bromosubstituent at C-6 of the firmed by spectral analyses involving IR, 1H-NMR, 13C-NMR
10
naphtyridine nucleus to delineate the effect of bromination on (Supporting information S:3 - 9) and MS (S:10 13) as well as
11
the antibacterial activity. The design concept is intended as to elemental analyses. A special feature in the IR spectra of the
12
maintain the pharmacophoric features established for inhib- thiosemicarbazides, characteristic for thiourea residue (NH-CS-
13
ition of bacterial DNA-gyrase cleavage complex as a wellknown NH), exhibited as strong absorption in the range of 1110–
14
mechanism for the antibacterial effect of quinolones and 1141 cm 1 attributable to C=S. The amidic carbonyl absorption
15
naphthyridones. Hybrid molecules have been reported as was observed as a broad band in the range of 1635–1680 cm 1.
16 1
approach for potentiation and/or attenuation of antibacterial H-NMR spectra of the unsubstituted thiosemicarbazides
17
activity towards resistant bacterial strains.[11,14,15] Moreover, 7 a, 8 a are characterized by three broad exchangeable singlets
18
halogention of lead compounds has been routinely adopted appeared at d = 7.5, 9.4 and 11.2 ppm, of the protons of CSNH2,
19
for improvement of the lipophilic and electronic characteristics NHCS and CONH respectively. The substituted counterparts
20
resulting in enhanced drug-target interactions. Unlike the 7,8(b–e) showed the presence of exchangeable singlets for the
21
known fluoroquinolones, shifting the 6-fluoro substituent by NH functions at d ~ 9.7; 10; and 12.2 ppm.
22
the corresponding 6-bromo might attain a clue to overcome In the oxadiazole series, the pattern of the three exchange-
23
bacterial resistance. This assumption resides on the fact that able singlets of the thiosemi-carbazide moiety has been
24
bromo- derivatives are rarely encountered within the molecular disappeared. Instead there is only one broad exchangeable
25
features of antibacterial agents. The synthesized compounds singlet, corresponds to NH2 or NH groups in compounds 9 a,
26
will be investigated for potential antimicrobial activity against 10 a appeared at d 7.1 or 7.4 ppm and at d ~ 10.6 - 12.5 ppm in
27
G + ve, G-ve; and Mycobacterial strains. Molecular docking to case of substituted amino analogues 9,10(b–e). In the 13C
28
selected bacterial DNAgyrase complexes will be adapted to spectra, the characteristic signals in the aliphatic region e. g.
29
assist understanding the possible binding modes and inter- CH3, one CH2, as well as CH are directly identified on basis of
30
actions between the compounds and their potential target. their characteristic chemical shifts. The C-atoms of the naph-
31
thyridine skeleton were assigned with reference to reported
32
data. Furthermore, DEPT experiments at 908 and 1358 were
33
Results and Discussion used for further verification of the respective C-atoms (Support-
34
ing information S: 4,6,8,9). Mass spectrum of compounds 9 d
35 Chemistry
and 10 d as representative for the non-brominated and 6-
36
The Synthesis of the intermediates and target compounds was bromo-naphthyridinyl series were carried out. The molecular
37
performed according to the reactions outlined in Scheme 1, ion peaks in each spectrum matched to the calculated values
38
using nalidixic acid (NA) 1 and its 6-bromo analog 2 as starting and additionally suggested fragmentation patterns have been
39
materials. The key intermediates, nalidixic acid methyl ester 3 proposed (Supporting information S:10-13).
40
and the corresponding 6-bromoester 4, were obtained accord-
41
ing to reported mild esterification method developed in our
42 Antimicrobial activity
laboratory.[23,24] 6-Bromonalidixic acid 2 has been obtained by
43
treatment of nalidixic acid methyl ester 3 with bromine in The primary screening test involved assessment of the
44
methanol followed by hydrolysis and acidification of the antibacterial effect of the synthesized compounds elicited as
45
resulting 6-bromonalidixic acid methyl ester 4. It is worth inhibition zones of the growth of the tested organisms. The
46
mentioning that direct bromination of nalidixic acid results in target compounds 710(a–e) were tested in vitro in comparison
47
decarboxylation and subsequent formation of 3-bromo or 3,6- with nalidixic acid for their antibacterial activity against selected
48
dibromo-naphtyridone derivatives.[25] The structure of 2 has Gram positive (G + ve) strains (S. aureus ATCC 25923, and B.
49
been confirmed on basis of 1H-NMR spectrum (Supporting cereus ATCC 11778); Gram negative (G–ve) (E. coli ATCC 25922,
50
information S1) characterized by the absence of pair of K. pneumonia ATCC 700603 and P. aeruginosa ATCC 27853) and
51
doublets corresponding to the naphthyridinyl H-5 & H-6 at d = Mycobac. smegmatis. Consequently, minimum inhibitory con-
52
8.5 & 7.5 ppm and the incidence of a singlet signal at d = centrations (MIC) were determined for compounds displaying
53
8.6 ppm due to H-5. The methyl esters 3–4 were converted to significant growth inhibition zones more than or equivalent to
54
the hydrazides 5–6 which were then used for synthesis of the that of nalidixic acid (NA) using the Broth micro dilution
55
targeted naphthy-ridinyl-3-thiosemicarbazides 7,8(a–e). Unlike, method.[29] The results summarized in (Table 1) indicate that
56
the aryl substituted thiosemicarbazide 7,8(b–e), the unsubsti- the thiosemicarbazides 7,8(a–e) display narrow antimicrobial
57

ChemistrySelect 2018, 3, 2604 – 2612 2605  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Wiley VCH
1809 / 107990
Montag, 05.03.2018
[S. 2605/2612] 1
 Full Papers

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
Scheme 1. Synthesis of the target compounds
54
55
56
57

ChemistrySelect 2018, 3, 2604 – 2612 2606  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Wiley VCH
1809 / 107990
Montag, 05.03.2018
[S. 2606/2612] 1
 Full Papers

1 Table 1. The antimicrobial MIC* (mM) values of thiosemicarbazide 7,8(a-e)

and oxadiazolyl naphthyridones 9,10(a–e):
2
3 Cpd. # S. aureus B. cereus E. coli K. pneum. P. aerug. M. smeg.
4 7a 16.4 - - - - -
5 7b 6.6 - - - 19.7 -
6 7c 6 - - - 18 6
7d 12.6 - - - 18.9 6.3
7
7e 12.2 - 6.1 - 18.2 6.1
8 8a 13 6.5 6.5 - 19.5 -
9 8b 10.8 10.8 - 10.8 16.3 -
10 8c 10 10 5 10 15 10
8d 10.5 10.5 - 10.5 15.8 10.5
11
8e 10.2 10.2 - 10.2 10.2 5.1
12 9a 18.4 - - - - 9.2
13 9b 7.2 7.2 - 7.2 21.6 -
14 9c 13 6.5 6.5 6.5 19.5 6.5
9d 6.9 - - - 20.7 6.9
15 Figure 2. Graphical representation of MIC-values of the naphthyridone-3-
9e 13.2 - - - 19.8 - oxadiazoles 9,10(a-e).
16 10a 14.2 - - 7.1 21.4 7.1
17 10b 11.7 5.9 5.9 5.9 17.6 -
18 10c 10.8 - 5.4 - 16.3 5.4
10d 11.4 5.7 5.7 5.7 17 5.7
19
10e 11 5.5 5.5 11 16.5 11 potency and selectivity towards B. cereus as evidenced by the
20 NA 21.5 10.8 4.3 10.8 21.5 10.8 thiosemicarbazide derivative 8 a and oxadiazoles 10(b,d,e); and
21
* MIC values (mean  SEM of n = 3. Values that are significantly higher than K. pneumonia by compounds 10(a,b,d).
22
nalidixic acid not considered. MIC values of the most active compounds are The observed MIC-values ( ~ 5.5 – 7.2 mM) are 1.5 – 2 times
23 highlighted.
less than that of the reference drug. In case of M. smegmatus
24
the MIC-values of the brominated thiosemicarbazide derivatives
25
e.g 8 e or oxadiazoles e. g. 10(a,c,d) are not significantly
26
spectrum (Figure 1) as compared to their cyclised counterparts different than the non-brominated analogues. The observed
27
(figure 2) of the oxadiazoles 9,10(a – e). It is evident that enhancement of MIC-values due to introduction of 6-bromo
28
substituent is likely due increased lipophilicity of the com-
29
pounds. This has been established through assessment of
30
molecular characteristics of the synthesized compounds,
31
(Table 4). The logP-values of the 6-bromo-3-thiosemicarbazides
32
8 a-e (miLogP = 1.13 – 3.25) and 6-bromo-3-oxadiazoles 10 a-e
33
(miLogP = 1.84 – 5.03) are approximately double that of the
34
non-brominated analogues 7 a-e (miLogP = 0.01 – 2.12) and 9 a-
35
e (miLogP = 0.72 3.90).
36
37
38 Docking study
39
Several X-ray crystal structures of bacterial gyrase illustrate that
40
it is a hetero-tetrameric (GyrA2GyrB2) enzyme. Only some of
41
them were DNA-gyrase cleavage complexes cocrystallised with
42
quinolones.[30–33] The crystal structures of DNA-gyrase cleavage
43
complex of S. aureus (PDB entry: 5cdq), Mycobac. mTB (PDB
44
Figure 1. Graphical representation of MIC-values of the naphthyridone-3- entry: 5btd) and the cleavage complex of topoisomerase Top. IV
45
thiosemicarbazides 7,8(a-e). of K. pneumonia (PDB entry: 5eix) co-crystalized with fluoroqui-
46
nolones, (Supporting information S:14), have been downloaded
47
from protein data bank and used for the present docking study.
48
Analysis of the cocrystallized S. aureus DNA-gyrase cleavage
49
compounds loaded by aryl substituent linked to the thiosemi- complex with moxifloxacin revealed binding between GyrA: Ser
50
carbazide 7,8(b – e) or the oxadiazole moieties 9,10(b – e) 84 and Glu 88 and the two C=O groups of quinolone nucleus
51
showed selective antibacterial activity against S. aureus (MIC = and COOH via water–Mg2 + bridge.[32]
52
6.0 - 13.0 mM), B. cereus and M. smegmatis (MIC = 5 – 6 mM). It is In addition there are hydrophobic interactions with adenine
53
worth mentioning that such bacterial strains are resistant to and guanine DNA bases and H-bonds with Arg 122, Glu 477 or
54
the reference drug. This might allocate the significance of the Asp 437. Similarly, levofloxacin binds ParC, subunit A of Topo IV
55
lipophilic characteristics for the antibacterial activity within this of K. pneumonia through Mg2 +; in addition to p–p stacking
56
series. Bromination of the naphthyridone nucleus enhances the interaction between DNA bases (adenine and guanine) as
57

ChemistrySelect 2018, 3, 2604 – 2612 2607  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Wiley VCH
1809 / 107990
Montag, 05.03.2018
[S. 2607/2612] 1
 Full Papers

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18 Figure 3. Ligand interaction diagram(A) and 3D representation (B) of (7b) into S. aureus DNA-gyrase cleavage complex
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37 Figure 4. Ligand interaction diagram (A) and 3D-representaion of (10d) into K. pneumoniae topoisomerase
38
39
40
second important interaction.[33] Likewise, the crystal structure Chemical Computing Group, Canada) to predict their binding
41
of Mycobac. DNA-gyrase complex co-crystallized with gatiflox- modes to bacterial DNA-gyrase cleavage complex active site.
42
acin shows binding of the keto-acid moiety of gatifloxacin The studies have successfully identified binding poses for all
43
molecule to GyrA: Asp 94 through a hydrated Mg2 + . Other test molecules, with comparable docking scores, indicating that
44
significant interactions include vdWs interactions of the the proposed compounds could potentially bind to the active
45
quinolone ring with two adenine bases of DNA and that of the site with analogous strengths. The ligand interaction diagrams
46
cyclopropyl group with one thymine base.[32] and the corresponding 3D representations of the binding
47
In the present investigation the proposed docking algo- modes of selected active compounds are illustrated in
48
rithm was initially validated by self-docking of the co-crystal- Figures 3–5
49
lized ligands to each of the aforementioned targets. Whereby, The following binding features are observed regarding the
50
the ligands were removed from the complex and then docked docked compounds:
51
back into the binding site. Heavy-atom root mean square 1. The studied compounds showed similar binding modes as
52
deviation (RMSD) values between top-ranked poses and the displayed in case of the co-crystallized ligands. As evident in
53
experimental crystal structure ranged from ~ 0.52 – 0.9 Å. Figure (3) the thiosemicarbazide derivative 7 b coordinate
54
Subsequently, a docking procedures have been achieved for through C=O oxygen of naphthyridone ring and C=S sulfur
55
the tested compounds 7,8(a–e) and 9,10(a–e) using Molecular of the side chain with Mg2 +, which in turn linked via a water
56
Operating Environment software (MOE-dock tool, ver. 2014.0901, bridge to Ser 84 and Glu 88. In case of the oxadiazole
57

ChemistrySelect 2018, 3, 2604 – 2612 2608  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Wiley VCH
1809 / 107990
Montag, 05.03.2018
[S. 2608/2612] 1
 Full Papers

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18 Figure 5. Ligand interaction diagram (A) and 3D-representaion (B) of (10c) into Mycobac. DNA-gyrase cleavage complex
19
20
21 Table 2. Interaction energies (Kcal/mol) and MIC (mM) of the tested
derivatives (Figures 4 & 5), the coordination initiated by compounds with DNA-gyrase/ topoisomerase-IV cleavage complexes
22
one of the nitrogen atoms of the oxadiazole ring and/or
23 S. aureus K. pneumon. mTB
naphthyridone C=O oxygen. Cpd. # DG MIC DG MIC DG MIC
24
2. Additionally, p-p interactions of adenine and thymidine
25 7a 5.20 16.4 -5.68 16.4 2.54 16.4
DNA bases and naphthyridone, oxadiazole and/or the
26 7b -24.7 6.6 -18.7 13.1 -12.3 13
aromatic moieties of C-3 substituents were observed. 7c -26.1 6 -14.1 12 -26.3 6
27
3. H-bonds were perceived between DNA bases and NH or C= 7d -15.4 12.6 -16.6 12.6 -23.8 6.3
28
O of the thiosemi-carbazide moiety. 7e -12.8 12.2 -18.3 12.2 -26.7 6.1
29 8a -15.5 13 -16.2 13 -17.0 13.1
Docking simulations based on London DG, expressed as E-
30 8b -14.5 10.8 -15.9 10.8 8.82 16.3
refine values, as a scoring function were performed to estimate 8c -18.2 10 -14.5 10 -17.1 10
31
the free energy of interactions of the studied thiosemicarba- 8d -13.0 10.5 -16.1 10.5 -17.6 10.5
32
zides 7,8(a–e) and oxadiazoles 9,10(a–e) from a given pose 8e -14.5 10.2 -17.6 10.2 -24.6 5.1
33 9a 4.32 18.4 -1.61 18.4 -19.0 9.2
with the respective DNA-gyrase cleavage complexes. This
34 9b -23.3 7.2 -26.6 7.2 6.57 14.4
parameter considered as more representative, since it involves 9c -14.2 13 -26.9 6.5 -25.8 6.5
35
energy minimization procedure for both the ligand and target. 9d -23.3 6.9 -22.3 13.8 -27.2 6.9
36
Consequently, it illustrates the least energy interaction value of 9e -13.0 13.2 -19.7 13.2 -12.5 13.2
37 10a -5.02 14.2 -22.6 7.1 -22.7 7.1
the selected pose. (Table 2) summarizes the docking scores
38 10b -14.5 11.7 -25.2 5.9 -12.7 11.7
and the respective MIC-values of the tested compounds. 10c -16.1 10.8 -20.93 10.8 -25.42 5.4
39
The results indicate that the most active compounds 10d -10.41 11.4 -25.63 5.7 -24.77 5.7
40
against S. aureus (7 b,c; 9 b and 9 d) with MIC values 6.6, 6.0, 7.2 10e -13.80 11 -23.53 11 -13.61 11
41 NA 7.86 21.5 -18.12 10.8 -14.11 10.8
and 6.9 mM, showed the lowest DG values (E-refine): 24.68,
42
26.09, 23.34 and 23.30 Kcal/mol respectively. It is obvious
43
that the reference compound, nalidixic acid, which is practically
44
inactive against S. aureus exhibits very low binding with the mTB DNA–gyrase cleavage complex as evident in compounds:
45
DNA-gyrase cleavage complex (E-refine = 7.86). Similar pattern 7 c,d,e; 8 e; 9 c,d; and 10 a,c,d.
46
has been logged in case of topoisomerase (Top. IV) DNA-gyrase
47
cleavage complex of K. pneumonia. The most active com-
48 Inhibition of DNA-gyrase supercoiling activity
pounds are the substituted amino oxadiazoles 9 b,c and
49
10 a,b,d (MIC values: 7.2, 6.5 and 7.1, 5.9, and 5.7) displayed the The observed binding characteristics of the synthesized com-
50
lowest binding energy values corresponding to: 26.57, pounds with DNA Gyrases in molecular docking inspire the
51
26.89, 22.62, 25.63, and 25.53. The results illustrate also assessment of their potential inhibitory activity against the
52
that the active site of Top. IV of K. pneumonia can embrace enzyme. Consequently, DNA-gyrase assay has been proposed
53
bulky substituents of the 6-bromo derivatives 10 a,b,d. Remark- and executed. Representative compounds of the synthesized
54
able correlation is also observed between the in vitro antimyco- series, specifically those having the lowest MIC-values and
55
bacterial activity and binding free energy (DG) with Mycobac. docking scores, were used for assessment of the IC50.
56
57

ChemistrySelect 2018, 3, 2604 – 2612 2609  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Wiley VCH
1809 / 107990
Montag, 05.03.2018
[S. 2609/2612] 1
 Full Papers
E. Coli DNA-gyrase microplate assay kit (Inspiralis) has been significance of an electron withdrawing group on the aryl
1
used for the anticipated assay according to the optimized substituent for enzyme inhibition.
2
protocol by the manufacturer (Supporting information S:15). In case of the cyclized analogues i. e. naphthyridinyl-3-
3
This assay is based upon the fact that negatively-supercoiled oxadiazoles, 9 b and 10 d are the most active compounds with
4
plasmids form intermolecular triplex DNA more readily than do almost equal IC50 values of 3.36 and 3.89 mg/ml respectively.
5
relaxed plasmids under some conditions. The assay overcomes This advocate that bromination of the naphtyridine nucleus is
6
some of the problems of gel-based assays, which are time not a critical modification for inhibition activity of the
7
consuming and are therefore inherently low-throughput. In this synthesized compounds against E. coli DNA gyrase. These
8
assay the substrate is relaxed pNO1, a modified form of pBR322 results matched well with the observed binding scores of the
9
which contains a ‘triplex-forming sequence’. The assay can be two compounds (E-refine: -26.57 and 25.53 respectively).
10
used to determine the activity of compounds as inhibitors of
11
DNA-gyrase either as an initial screen or in the determination of
12 Molecular properties and drug-likeness
IC50 values.
13
The IC50 values of the selected compounds are listed in Molecular properties are a complex balance of various struc-
14
(Table 3) and represented in (Figure 6). The results indicate that tural features which determines whether a particular molecule
15
is similar to the known drugs or not. It generally means
16
“molecules which contain functional groups and/or have
17 Table 3. IC50 (mg/ml) against E. Coli DNA-gyrase physical properties consistent with most of the known drugs”.
18
Cpd. # IC50 (mg/ml) Hydrophobicity, molecular size, flexibility, and presence of
19
7a 10.29
various pharmacophoric features are the main physical proper-
20
8a 51.02 ties that influence the behavior of molecules in a living
21
7c 1.73 organism. Good bioavailability can be achieved with an
22 7e 7.66 appropriate balance between solubility and partitioning prop-
23 8e 4.64
9b 3.36
erties. Thus, the compliance of the newly synthesized com-
24
9c 9.69 pounds to the Lipinski’s rule of five was evaluated.[34] In
25
10b 40.5 addition, topological polar surface area (TPSA) and number of
26 10d 3.89 rotatable bonds (nrotb) have been linked to drug bioavail-
27 NA 1.74
ability.[35]
28
Molecular properties (TPSA, nrotb, miLogP, OH–NH inter-
29
action, O–N interaction, molecular weight, and number of
30
violations from Lipinski’s rule) of the newly synthesized
31
compounds were calculated using molinspiration software and
32
compared to the values of the standard drug, nalidixic acid
33
(Table 4). Topological polar surface area (TPSA) and lipophilicity
34
(logP) values are two important properties for the prediction of
35
oral bioavailability of drug molecules.[36–39] TPSA is calculated
36
based on the methodology published by Ertl et al[39] as the
37
surface areas that are occupied by oxygen and nitrogen atoms
38
and by hydrogen atoms attached to them. Thus, it is closely
39
related to the hydrogen bonding potential of a compound.
40
Moreover, it is considered as a very good descriptor character-
41
izing drug absorption, including intestinal absorption, bioavail-
42
ability, and blood–brain barrier penetration. Molecules with
43 Figure 6. Inhibitory activity (IC50 mg/ml) of selected compounds against E. coli
TPSA values around 140 Å2 or more are expected to exhibit
44 DNA gyrase
poor intestinal absorption.[35] The results shown in Table (4)
45
indicate that all the synthesized compounds have TPSA values
46
< 140 Å2; thus, they are expected to have good intestinal
47
within the naphthyridinyl-3-thiosemicarbazide series the pres- absorption.
48
ence of aryl substituent on thiosemicarbazide moiety is crucial Additionally, the total number of rotatable bonds in drug
49
as previously evidenced by the MIC-value. Compounds 7a and molecules represents an important molecular characteristic.
50
8a, the non-substituted thiosemicarbazides, showed much Compounds with more than 10 rotatable bonds may have
51
higher IC50values (10.29 and 51.02 mg/ml respectively). Com- problems with bioavailability.[35] All compounds under inves-
52
pound 7c showed the lowest IC50 value (1.73 mg/ml), which is tigation (Table 4) have two to eight rotatable bonds and they
53
similar to that of nalidixic acid. This compound is a 4- might not have problems with bioavailability. The lipophilic
54
chlorophenyl substituted thiosemicarbazide derivative. characteristics expressed as (LogP) value have been calculated
55
Its analogue 7 e with 4-methoxyphenyl substituent showed adopting the methodology developed by Molinspiration
56
relatively moderate IC50 value (7.66 mg/ml) illustrating the (MiLogP), which depends on summation of fragment-based
57

ChemistrySelect 2018, 3, 2604 – 2612 2610  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Wiley VCH
1809 / 107990
Montag, 05.03.2018
[S. 2610/2612] 1
 Full Papers

Table 4. nonbrominated (7 b,c) and the 6-brominated thiosemicarbazide

1
Molecular characteristics* of 7,8(a-e) and 9,10(a-e): (8 c-e) as well as their corresponding oxadiazoles 9 b,d exhibit
2
code Mwt miLog HBA HBD TPSA #RT Drug Solubility
superior antibacterial activity (MIC = 6.0 - 10.5 mM). However, in
3
P score (mg/mL) case of B. cereus the presence of 6-bromo substituent seems to
4
be promising for their antibacterial action as manifested by
5 NA 232.23 0.81 5 1 72.20 2 0.82 68.5
7a 305.36 0.01 7 4 102.05 4 1.30 100.3
compounds 8 a; 10 b,d,e (MIC = 5 – 6 mM). The antibacterial
6
7b 381.45 1.45 7 3 88.05 6 1.03 5.27 activity of the tested compounds is not significantly deviating
7
7c 415.90 2.12 7 3 88.05 6 1.41 0.63 than the reference drug against the G –ve bacterial strains.
8 7d 395.48 1.89 7 3 88.05 6 0.99 1.92 Only in case of K. pneumonia some of the naphthyridinyl-3-
9 7e 411.48 1.50 8 3 97.28 7 0.90 3.12
8a 384.25 1.13 7 4 102.05 4 1.28 8.23
oxadiazole derivatives (9 b,c and 10 a,b,d) are twice as potent
10
8b 460.35 2.57 7 3 88.05 6 0.84 0.42 as the reference drug. As regards the antimycobacterial activity
11
8c 494.80 3.25 7 3 88.05 6 1.45 0.05 against M. smegmatis, it can be concluded that the achieved
12 8d 474.38 3.02 7 3 88.05 6 0.86 0.15 structural modifications result in two folds enhancement of the
13 8e 490.38 2.62 8 3 97.28 7 0.90 0.24
9a 271.28 0.72 7 2 99.84 2 0.69 18.67
MIC-value as illustrated by compounds 7 c,d,e; 8 e; 9 c,d; and
14
9b 347.37 3.23 7 1 85.85 4 0.74 0.92 10 c,d. Molecular docking and DNA gyrase inhibition assay
15
9c 381.82 3.90 7 1 85.85 4 1.08 0.11 illustrate that compound 7 c is the most active compound with
16 9d 361.40 3.67 7 1 85.85 4 0.65 0.33 IC50value = 1.73 mg/ml against E. coli DNA gyrase. Furthermore,
17 9e 377.40 3.28 8 1 95.08 5 0.59 0.54
10a 350.17 1.84 7 2 99.84 2 0.75 1.54
Molecular properties and drug-likeness data illustrate that the
18
10b 426.27 4.35 7 1 85.85 4 0.69 0.07 studied compounds fulfill lipinski’s rule requirements and
19
10c 460.71 5.03 7 1 85.85 4 1.20 0.01 possess good drug score values. These preliminary encouraging
20 10d 440.30 4.80 7 1 85.85 4 0.63 0.03 results of the in vitro antibacterial activity of the newly
21 10e 456.29 4.41 8 1 95.08 5 0.68 0.04
synthesized compounds could offer an exceptional framework
22
* Mwt: molecular weight, miLog P: lipophilicity parameter, HBD: # of that may lead to the discovery of new potent antimicrobial
23 hydrogen bond donor, HBA: # of hydrogen bond acceptor, TPSA:
agents.
24 topological polar surface area, # RT: # of rotatable bonds.
25
26 Supporting Information Summary
27
contributions and correction factors (http://www.molinspiration.- Experimental details from the synthetic procedures of the
28
com). It has been shown that for the compound to have a intermediates (1 6) the targeted naphthyridinyl-3-thiosemi-
29
reasonable probability of being well absorbed, miLogP value carbazides 7,8(a–e) and naphthyridinyl-3-(2-Amino-1,3,4-oxa-
30
must be in the range of 0.4 to 5.[35] On this basis, all the tested diazoles) 9,10(a–e) as will as their spectral data (involving 1H-;
31 13
compounds were found to have miLogP values within the C-NMR and MS) are presented in the supporting Information
32
acceptable criteria and are expected to have reasonable oral file. Moreover, this section includes the antibacterial screening
33
absorption. It is worth mentioning that all compounds have method against the selected G + ve, G-ve, Mycopbac. strains
34
one or zero violation of Lipinski’s rule; therefore, they are (Comprising: assessment of MIC values, IC50 against E. coli DNA-
35
expected not to have problems with bioavailability. gyrase), the 3D representation and docking scores of reference
36
The drug score combines drug-likeness, miLogP, solubility, compounds with DNA- gyrase.
37
molecular weight, and toxicity risks in one handy value that
38
may be used to judge the compound‘s overall potential to
39 Conflict of Interest
qualify for a drug.[36] A value of 0.5 or more makes the
40
compound a promising lead for future development of safe The authors declare no conflict of interest.
41
and efficient drugs.
42
The overall drug score values for the synthesized com- Keywords: Antimicrobial · DNA Gyrase · Drug-likeness ·
43
pounds were calculated using molsoft software (http://molsoft.- Molecular Docking Naphthyridone · Oxadiazole ·
44
com/ mprop.) and compared to that of the parent drug, Thiosemicarbazide
45
nalidixic acid (Table 4). Almost all the synthesized compounds
46
possess good drug score values above 0.5.
47 [1] H. Gelband, M. Miller-Petrie, S. Pant, A. White, S. Gandra, J. Levinson, D.
48 Barter, R. Laxminarayan, The state of world’s antibiotics, Center for Disease
Dynamics, Economics & Policy (CDDEP): Washington, D.C, USA, 2015, p. 8.
49 Conclusions [2] D. L. Paterson, Am. J. Med. 2006,119, 20–28.
50 [3] V. Talla, P. Veerareddy, J. Young Pharm. 2011, 3, 304–309.
A synthetic strategy for structural modification of nalidixic acid
51 [4] C. Kang, S. Kim, D. M. Kim, W. B. Park, K. Lee, H. Kim, M. Oh, E. Kim,
involving isosteric replacement of the COOH in 3-position and Epidemiol. 2014, 25, 860–867.
52
introduction of bromo substituent at C-6 was developed. [5] T. H. Dellit, R. C. Owens, J. E. Mc Gowan. J. Clin. Infect. Dis. 2007, 44, 159.
53 [6] A. H. Diacon, A. Pym, R. Rustomjee, M. Grobusch, R. Patientia, L. Page-
Results of antimicrobial screening showed that all the synthe-
54 Shipp, J. Allen, J. C. Palomino, T. De Marez, R. P. G. Van Heeswijk, N. Engl.
sized compounds showed selective antibacterial activity against
55 J. Med. 2009, 360, 2397–2405.
the tested G + ve bacterial strains that are known to be [7] N. Veziris, M. Ibrahim, N. Lounis, K. Andries, V. Jarlier, PLoS One 2011, 6,
56
resistant to nalidixic acid. In case of S. aureus both the 2–7.
57

ChemistrySelect 2018, 3, 2604 – 2612 2611  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Wiley VCH
1809 / 107990
Montag, 05.03.2018
[S. 2611/2612] 1
 Full Papers
[8] A. Naeem, S. Badshah, M. Muska, N. Ahmad, K. Khan, Molecules 2016, 21, [26] M. A. Metwally, S. Bondock, H. El-azap, E. M. Kandeel, J. Sulfur Chem.
1 268–287. 2011, 32, 489–519.
2 [9] S. Huang, J. Qing, S. Wang, H. Wang, Org. Biomol. Chem. 2014, 1, 2344– [27] G. L. Almajan, I. Saramet, C. Draghici, C. Enache, Rev. Chim. 2008, 59,
3 2348. 305–308.
[10] C. Manera, A. M. Malfitano, T. Parkkari, V. Lucchesi, F. Stefano, S. Bertini, [28] N. R. Rivera, J. Balsells, K. B. Hansen, Tetrahedron Lett. 2006, 47, 4889–
4
C. Laezza, A. Ligresti, J. R. Savinainen, E. Ciaglia, S. Pisanti, P. Gazzerro, V. 4891.
5 Di Marzo, P. Nieri, M. Macchia, M. Bifulco, Eur. J. Med. Chem. 2015, 97, [29] J. M. Andrews, J. Antimicrob. Chemother. 2001, 48(1), 5–16. doi:10.1093/
6 10–18. jac/48.suppl_1.5
7 [11] N. Aggarwal, R. Kumar, P. Dureja, J. M. Khurana, Eur. J. Med. Chem. 2011, [30] P. F. Chan, V. Srikannathasan, J. Huang, H. Cui, A. P. Fosberry, M. Gu, M. M.
46, 4089–4099. Hann, M. Hibbs, P. Homes, K. Ingraham, J. Pizzollo, C. Shen, A. J. Shillings,
8
[12] C. M. Oliphant, W. Casper, G. M. Green, K. Permanente, Clin. Pharmacol. C. E. Spitzfaden, R. Tanner, A. J. Theobald, R. A. Stavenger, B. D. Bax, M. N.
9 2002, 65, 455–464. Gwynn, Nat. Commun. 2015, 6, 10048.
10 [13] R. R. Somani, A. G. Agrawal, P. P. Kalantri, P. S. Gavarkar, E. D. Clercq, Int. J. [31] B. D. Bax, P. F. Chan, D. S. Eggleston, A. Fosberry, D. R. Gentry, F. Gorrec, I.
11 Drug Des. Discov. 2011, 2, 353–360. Giordano, M. Hann, A. Hennessy, M. Hibbs, J. Huang, E. Jones, J. Jones,
[14] G. Grover, S. G. Kini, Eur. J. Med. Chem. 2006, 41, 256–262. K. K. Brown, C. J. Lewis, E. W. May, M. R. Saunders, O. Singh, C. E.
12
[15] T. Aboul-Fadl, A. A. Radwan, H. A. Abdel-Aziz, M. Baseeruddin, M. I. Attia, Spitzfaden, C. Shen, A. Shillings, A. J. Theobald, A. Wohlkonig, N. D.
13 A. Kadi, Dig. J. Nanomater. Biostructures. 2012, 7, 329–338. Pearson, M. N. Gwynn, Natur. 2010, 466, 935–940.
14 [16] N. Aggarwal, R. Kumar, P. Dureja, J. M. Khurana, Chem. Biol. Drug Des. [32] T. R. Blower, B. H. Williamson, R. J. Kerns, J. M. Berger, Proc. Natl. Acad.
15 2012, 79, 384–397. Sci. 2016, 113, 1706–1713.
[17] Ł. Popiołek, M. Gawrońska-grzywacz, Int. Res. J. Pure Appl. Chem. 2015, 7, [33] D. A. Veselkov, I. Laponogov, X. S. Pan, J. Selvarajah, G. B. Skamrova, A.
16
191–202. Branstrom, J. Narasimhan, J. V. N. V. Prasad, L. M. Fisher, M. R. Sanderson,
17 [18] A. Kadi, N. El-Brollosy, O. Al-Deeb, E. Habib, T. Ibrahim, A. El-emam, Eur. J. Acta Crystallogr. Sect. D, Struct. Biol. 2016, 72, 488–96.
18 Med. Chem. 2007, 42, 235–343. [34] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug Deliv.
19 [19] P. Shetty, M. Raghavendra, B. M. Praveen, S. Cheruku, K. Manjunath, Mol. Rev. 2001, 46, 3–26.
Divers. 2016, 20, 391–398. [35] D. F. Veber, S. R. Johnson, H. Y. Cheng, B. R. Smith, K. W. Ward, K. D.
20
[20] A. Mazumder, M. Shaharyar, Med. Chem. Res. 2015, 24, 2514–2528. Kopple, J. Med. Chem. 2002, 45, 2615–2623.
21 [21] A. P. Liesen, T. M. De Aquino, C. S. Carvalho, V. T. Lima, J. M. De Araffljo, [36] A. Jarrahpour, J. Fathi, M. Mimouni, T. Ben Hadda, J. Sheikh, Z. H.
22 J. G. De Lima, A. R. De Faria, E. De Melo, A. J. Alves, E. W. Alves, A. Q. Chohan, A. Parvez, Med. Chem. Res. 2012, 21, 1984–1990.
23 Alves, A. Ges, Eur. J. Med. Chem. 2010, 45, 3685–3691. [37] A. Parvez, M. Jyotsna, M. H. Youssoufi, T. Ben Hadda, Phosphorus Sulfur
[22] O. U. Tan, K. Ozadali, P. Yogeeswari, D. Sriram, A. Balkan, Med. Chem. Res. Silicon Relat. Elem. 2010, 7, 1500–1510.
24
2012, 21, 2388–2394. [38] A. Parvez, J. Meshram, V. Tiwari, J. Sheikh, R. Dongre, M. H. Youssoufi, T.
25 [23] F. A. Omar, Alex. J. Pharm. Sci. 1994, 8, 65–69. Ben Hadda, Eur. J. Med. Chem. 2010, 45, 4370–4378.
26 [24] A. F. Youssef, F. A. Omar, H. A. El-sherief, G. A. Abuo-Rahma, Design, Bull. [39] P. Ertl, B. Rohde, P. Selzer, J. Med. Chem. 2000, 43, 3714–3717.
27 Pharm. Sci. Assiut University 1998, 21, 15–26.
[25] A. F. Youssef, F. A. Omar, H. A. El-Sherief, G. A. Abou-Rahma, 1st Pharm.
28 Submitted: January 11, 2018
Sci. Conf. (faculty of pharmacy-Assiut University- Assiut/Egypt) 1998, 20–
29 34. Accepted: February 20, 2018
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

ChemistrySelect 2018, 3, 2604 – 2612 2612  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Wiley VCH
1809 / 107990
Montag, 05.03.2018
[S. 2612/2612] 1