Synthesis, evaluation, and CoMFA study of fluoroquinophenoxazine derivatives as bacterial topoisomerase IA inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.09.053

New antibacterial agents with novel target and mechanism of action are urgently needed to combat problematic bacterial infections and mounting antibiotic resistances. Topoisomerase IA represents an attractive and underexplored antibacterial target, as such, there is a growing interest in developing selective and potent topoisomerase I inhibitors for antibacterial therapy. Based on our initial biological screening, fluoroquinophenoxazine 1 was discovered as a low micromolar inhibitor against E.?coli topoisomerase IA. In the literature, fluoroquinophenoxazine analogs have been investigated as antibacterial and anticancer agents, however, their topoisomerase I inhibition was relatively underexplored and there is little structure-activity relationship (SAR) available. The good topoisomerase I inhibitory activity of 1 and the lack of SAR prompted us to design and synthesize a series of fluoroquinophenoxazine analogs to systematically evaluate the SAR and to probe the structural elements of the fluoroquinophenoxazine core toward topoisomerase I enzyme target recognition. In this study, a series of fluoroquinophenoxazine analogs was designed, synthesized, and evaluated as topoisomerase I inhibitors and antibacterial agents. Target-based assays revealed that the fluoroquinophenoxazine derivatives with 9-NH₂and/or 6-substituted amine functionalities generally exhibited good to excellent inhibitory activities against topoisomerase I with IC₅₀s ranging from 0.24 to 3.9?μM. Notably, 11a bearing the 6-methylpiperazinyl and 9-amino motifs was identified as one of the most potent topoisomerase I inhibitors (IC₅₀?=?0.48?μM), and showed broad spectrum antibacterial activity (MICs?=?0.78–7.6?μM) against all the bacteria strains tested. Compound 11g with the 6-bipiperidinyl lipophilic side chain exhibited the most potent antituberculosis activity (MIC?=?2.5?μM, SI?=?9.8). In addition, CoMFA analysis was performed to investigate the 3D-QSAR of this class of fluoroquinophenoxazine derivatives. The constructed CoMFA model produced reasonable statistics (q²?=?0.688 and r²?=?0.806). The predictive power of the developed model was obtained using a test set of 7 compounds, giving a predictive correlation coefficient r²_predof 0.767. Collectively, these promising data demonstrated that fluoroquinophenoxazine derivatives have the potential to be developed as a new chemotype of potent topoisomerase IA inhibitors with antibacterial therapeutic potential.

Full text of DOI:10.1016/j.ejmech.2016.09.053

Accepted Manuscript
Synthesis, evaluation, and CoMFA study of fluoroquinophenoxazine derivatives as
bacterial topoisomerase IA inhibitors
Xufen Yu, Mingming Zhang, Arasu Annamalai, Priyanka Bansod, Gagandeep Narula,
Yuk-Ching Tse-Dinh, Dianqing Sun
PII:
S0223-5234(16)30789-9
10.1016/j.ejmech.2016.09.053
EJMECH 8919
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 May 2016
Revised Date: 16 September 2016
Accepted Date: 17 September 2016
Please cite this article as: X. Yu, M. Zhang, A. Annamalai, P. Bansod, G. Narula, Y.-C. Tse-Dinh, D.
Sun, Synthesis, evaluation, and CoMFA study of fluoroquinophenoxazine derivatives as bacterial
topoisomerase IA inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.09.053.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis, evaluation, and CoMFA study of fluoroquinophenoxazine derivatives as bacterial topoisomerase IA inhibitors
Xufen Yu a,1, Mingming Zhang a,1, Arasu Annamalai b, Priyanka Bansod b, Gagandeep Narula b, Yuk-Ching Tse-Dinh b,c
Dianqing Sun a,*
,

ACCEPTED MANUSCRIPT
Synthesis, evaluation, and CoMFA study of fluoroquinophenoxazine derivatives as bacterial topoisomerase IA inhibitors
Xufen Yu a,1, Mingming Zhang a,1, Arasu Annamalai b, Priyanka Bansod b, Gagandeep Narula b, Yuk-Ching Tse-Dinh b,c
Dianqing Sun a,*
,
a Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawai’i at Hilo, 34
Rainbow Drive, Hilo, HI 96720, USA.
b Department of Chemistry & Biochemistry, Florida International University, Miami, FL 33199, USA
c Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, USA
* Corresponding author.
Dr. Dianqing Sun
Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawai’i at Hilo, 34
Rainbow Drive, Hilo, HI 96720, USA.
Tel: +1-(808)-933-2960
Fax: +1-(808)-933-2974
E-mail address: dianqing@hawaii.edu (D. Sun).
1 These two authors contributed equally to this paper.
1

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Abstract: New antibacterial agents with novel target and mechanism of action are urgently needed to combat problematic
bacterial infections and mounting antibiotic resistances. Topoisomerase IA represents an attractive and underexplored
antibacterial target, as such, there is a growing interest in developing selective and potent topoisomerase I inhibitors for
antibacterial therapy. Based on our initial biological screening, fluoroquinophenoxazine 1 was discovered as a low micromolar
inhibitor against E. coli topoisomerase IA. In the literature, fluoroquinophenoxazine analogs have been investigated as
antibacterial and anticancer agents, however, their topoisomerase I inhibition was relatively underexplored and there is little
structure-activity relationship (SAR) available. The good topoisomerase I inhibitory activity of 1 and the lack of SAR prompted
us to design and synthesize a series of fluoroquinophenoxazine analogs to systematically evaluate the SAR and to probe the
structural elements of the fluoroquinophenoxazine core toward topoisomerase I enzyme target recognition. In this study, a series
of fluoroquinophenoxazine analogs was designed, synthesized, and evaluated as topoisomerase I inhibitors and antibacterial
agents. Target-based assays revealed that the fluoroquinophenoxazine derivatives with 9-NH2 and/or 6-substituted amine
functionalities generally exhibited good to excellent inhibitory activities against topoisomerase I with IC50s ranging from 0.24-
3.9 µM. Notably, 11a bearing the 6-methylpiperazinyl and 9-amino motifs was identified as one of the most potent
topoisomerase I inhibitors (IC50 = 0.48 µM), and showed broad spectrum antibacterial activity (MICs = 0.78-7.6 µM) against all
the bacteria strains tested. Compound 11g with the 6-bipiperidinyl lipophilic side chain exhibited the most potent
antituberculosis activity (MIC = 2.5 µM, SI = 9.8). In addition, CoMFA analysis was performed to investigate the 3D-QSAR of
this class of fluoroquinophenoxazine derivatives. The constructed CoMFA model produced reasonable statistics (q2 = 0.688 and
r2 = 0.806). The predictive power of the developed model was obtained using a test set of 7 compounds, giving a predictive
correlation coefficient r2pred of 0.767. Collectively, these promising data demonstrated that fluoroquinophenoxazine derivatives
have the potential to be developed as a new chemotype of potent topoisomerase IA inhibitors with antibacterial therapeutic
potential.
Keywords: Antibacterial; CoMFA analysis; 3D-QSAR; fluoroquinophenoxazine; topoisomerase IA; SAR
Abbreviations:
SAR, structure-activity relationship; HTS, high-throughput screening; QSAR, quantitative structure-activity relationship;
CoMFA, comparative molecular field analysis; MIC, minimum inhibitory concentration; SI, selectivity index; 3D-QSAR, three
dimensional QSAR; PA, predicted activity; TLC, thin-layer chromatography; HPLC, high performance liquid chromatography;
MABA, microplate Alamar blue assay; PLS, partial least square; LOO, leave one out.
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1. Introduction
DNA topoisomerases maintain the helical and superhelical structure of DNA and are essential enzymes required for cellular
processes and functions including DNA replication, transcription, and repair [1, 2]. Therefore, poison inhibitors of
topoisomerase enzymes can lead to the accumulation of the intermediate topoisomerase-DNA cleavage complex and
subsequently result in bacterial or cancer cell death [2, 3]. Clinically, topoisomerase enzymes represent attractive and successful
targets for anticancer and antibacterial chemotherapy [4, 5]. In contrast, topoisomerase I is underexplored as antibacterial target,
thus potent and selective topoisomerase I inhibitors can serve as a promising class of chemotherapeutic agents toward the
treatment of problematic bacterial infections [6, 7].
In our ongoing efforts to discover new topoisomerase I inhibitors as antibacterial agents and to probe the topoisomerase I
recognition [8, 9], we identified during high-throughput screening (HTS) assay development a compound NSC648059 (1, Fig. 1)
with low micromolar inhibitory activity (IC50 = 0.8-2.0 µM) against Escherichia coli topoisomerase I. Structurally, 1 is a
fluoroquinophenzoxazine derivative with a unique planar tetracyclic ring system, belonging to a member of an extended
chemical class of fluoroquinolone antibiotics. In the clinic, fluoroquinolones including norfloxacin and ciprofloxacin (Fig. 1)
represent some of the most successful antibiotic classes, whose mechanisms of action are to inhibit bacterial DNA gyrase and
topoisomerase IV as well as relaxation of supercoiled DNA and thus to promote breakage of double-stranded DNA [10].
Specifically, fluoroquinophenoxazines such as A-62176 and A-85226 (Fig. 1) have been reported as antibacterial [11, 12] and
anticancer [13-16] agents. For example, A-62176 exhibited good activity against several cancer cell lines with IC50 values
ranging from 0.87-4.34 µM [13]. However, their bacterial topoisomerase I inhibition was relatively underexplored and there is
little structure-activity relationship (SAR) of fluoroquinophenoxazine derivatives available in the literature [13]. Herein, on the
basis of this HTS hit 1, we describe the design, synthesis, and evaluation of a series of fluoroquinophenoxazine structural
analogs, probing their SAR toward bacterial topoisomerase I and other topoisomerases as well as their whole cell antibacterial
activity. Additionally, we built and developed a quantitative structure-activity relationship (QSAR) and comparative molecular
field analysis (CoMFA) model in an effort to guide further design and synthesis of this class of topoisomerase IA inhibitors.
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Fig. 1. Chemical structures of clinical fluoroquinolone antibiotics and fluoroquinophenzoxazine derivatives including our topo I
inhibitor 1.
2. Results and discussion
2.1. Chemistry
To validate the initial assay hit, we first resynthesized 9-amino-5, 6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-
carboxylic acid (1) and retested its biochemical activity against E. coli topoisomerase I. Resynthesized fluoroquinophenoxazine
hit 1 showed reproducible topoisomerase I inhibitory activity with an IC50 of 1.95 µM. The synthesis of 1 is shown in Scheme 1.
Briefly, commercially available 2,3-difluoro-6-nitrophenol was hydrogenated using Pd/C (20 mol %) as the catalyst to the
corresponding amino product 2, which can be used in next step without further purification [17]. Subsequent reaction of the
aniline derivative 2 with diethyl 2-(ethoxymethylene)malonate under ambient temperature gave 3 in 87% yield. For next
nucleophilic displacement cyclization, an improved protocol was developed for this intramolecular cyclization reaction under
microwave irradiation at 250 ºC instead of conventional heating [11]. Following simple filtration, 4 was obtained in 80% yield.
Subsequently, 4 reacted with 1-chloro-2,4-dinitrobenzene (5a) in DMF at 100 ºC to give the desired tetracyclic product 6a in 57%
yield. Upon treatment of 6a in acidic condition (AcOH/HCl) under reflux for 4 h, the free carboxylic acid derivative 7a was
.
obtained by simple filtration in 91% yield. Finally, hydrogenation of 7a under H2 (1.0 bar) using FeSO4 6H2O as the catalyst
failed to yield the amine product 1 after 14 h. However, when SnCl2 was used as catalyst and AcOH as the solvent, the nitro
group was smoothly reduced into the amino group under reflux for 3 h and the final product 1 was obtained in 86% yield.
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Scheme 1. Synthesis of 9-amino-5, 6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylic acid (1).
Under the optimized conditions, we next expanded the structural diversity of 1 to evaluate the effect of various substituents
on the quinophenoxazine skeleton and to explore their SAR. First, we used various substrates 5b-e in Scheme 2 in an effort to
generate a focused set of quinophenoxazine derivatives 7b-e. It is worthwhile noting that a dramatic difference was observed in
terms of the reactivity of the substrate 5 with different electronic and/or physiochemical properties. For example, 5b bearing an
electron-withdrawing nitrile group facilitated the completion of cyclization in 2 h and 6b was obtained in 59% yield. On the
other hand, 5c with the lipophilic trifluoromethyl group could finish the reaction by extending the reaction time, affording the
desired product 6c in a lower yield (10%). In addition, when the substrate 5d with an acetyl group was tried, 6d was obtained in
38% yield. However, for the substrate with the corresponding fluorine substitution, no reaction occurred even when the reaction
temperature was raised to 120 ºC. Furthermore, more harsh conditions were tried in an attempt to facilitate the reaction by
heating the reaction to 160 ºC in a sealed pressure tube or heating the reaction to 200 ºC under microwave irradiation, but with
not much success. In the case of 5e with additional nitro and chlorine substituents, the reaction proceeded smoothly and 6e was
obtained in 40% yield. Final ester hydrolysis was performed in acetic acid/hydrochloric acid under reflux, affording free
carboxylic acid products 7b-e in 36-95% yields.
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Scheme 2. Synthesis of fluoroquinophenoxazine derivatives 7b-e.
In the course of antibacterial evaluation, we observed compound 1 lost 2-4 fold whole cell antibacterial activity after 4 weeks
1
of storage, indicating that 1 may have stability and/or solubility issue. From our H NMR experiments, we also noted that 1
could be easily precipitated out in d6-DMSO solvent and no degradation-related evidence was observed following several weeks
of monitoring 1 in both d6-DMSO NMR and HPLC experiments (data not shown). Thus, to enhance the overall solubility
profile of this class of quinophenoxazine derivatives, we introduced a variety of solubilizing and polar groups into the
fluoroquinophenoxazine scaffold by displacing the 6-fluorine atom of 6a, 7a, or 1 with different amine functionalities such as
piperazine, 1-methylpiperazine, and morpholine (Scheme 3) [18]. Specifically, 6a reacted with 1-methylpiperazine in pyridine
at 110 ºC to give 9 in 53% yield. Accordingly, 7a reacted with 1-methylpiperazine and piperazine in pyridine under nitrogen
atmosphere at 90 ºC, and both reactions proceeded smoothly to afford 10a and 10b in 45% and 84% yields, respectively [12]. In
the cases of 1 and some other amine substrates, notably, reaction temperature appeared to play a critical role in this nucleophilic
displacement reaction. For example, when morpholine was used as a nucleophile, the reaction became complicated under 90 ºC,
which is suitable for 1-methylpiperazine and piperazine. In contrast, when temperature was reduced to 70 ºC, the reaction could
be completed in 16 h to produce 11d in 71% yield. Other functional amines were also subjected to this substitution reaction, and
most of the reactions could lead to the desired products 11c-k under 90 ºC in moderate yields except for 1-adamantylamine. We
found that the reaction of 1 with 1-adamantylamine could finish under reflux after 4 days in 29% yield, presumably due to steric
effect. In addition, to investigate the effect of the free amine functionality of 1 on topoisomerase inhibition and antibacterial
activity, we next tried to protect the free amino group with acetyl functionality. The N-acetyl derivative 12 was synthesized
from 1 and acetic anhydride in pyridine at 80-100 ºC and the solid product was collected by simple filtration in high yield (92%).
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Scheme 3. Synthesis of diverse amino-substituted fluoroquinophenoxazines 9-11 and acetyl-protected quinophenoxazine 12.
Finally, to evaluate the potential stereospecific effect at the 6 position of fluoroquinophenoxazine derivatives on biological
activity, we designed and synthesized several chiral fluoroquinophenoxazine amine derivatives from 6a or 1 and chiral amine
building blocks [12, 13]. The nucleophilic substitution reaction of 1 and (S)-3-(Boc-amino)pyrrolidine (13a) in pyridine was
completed in 20 h, affording 15a in 86% yield. Subsequent N-Boc deprotection of 15a produced 17a in 78% yield upon the
treatment with diluted hydrochloric acid. With regard to the reaction of 1 and the corresponding (R)-3-(Boc-amino)pyrrolidine
(13b), the substituted compound 15b could not be obtained by filtration upon the completion of reaction. Therefore, the crude
product 15b was used for the following deprotection reaction and the corresponding fluorophenoxazine derivative 17b (R) was
obtained in 48% yield over two steps. Accordingly, compound 16 was synthesized from 6a as the starting material in 32% yield
over two steps (Scheme 4).
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Scheme 4. Synthesis of fluoroquinophenoxazine chiral amine derivatives 16 and 17.
2.2. Biological testing
All the synthesized target molecules were tested for the ability to inhibit the relaxation activity of E. coli topoisomerase I in
target-based assay, as well as against a panel of bacterial strains including the wild-type E. coli MG1655 K12 strain, E. coli
strain BAS3023 with imp mutation conferring membrane permeability to small molecules [19, 20], the wild-type Gram-positive
B. subtilis (ATCC 6633) strain, and M. tuberculosis (H37Rv). The results are summarized in Table 1.
2.2.1. E. coli topoisomerase I inhibition
Biochemical evaluation for inhibition of the relaxation activity of E. coli topoisomerase I revealed that the majority of our
synthesized compounds possessed good activity against E. coli topoisomerase I. On the basis of these topoisomerase I inhibition
data (Table 1), an illuminating SAR has been obtained and is summarized in Fig. 2. i). The 9 position substituent plays a very
important role in topoisomerase I inhibitory activity. Among the 5,6-difluoroquinophenoxazine derivatives, the hit compound 1
with the electron-donating 9-NH2 group showed the most potent activity (IC50 = 1.95 µM) against E. coli topoisomerase I. Both
free carboxylic acid 7c and its ethyl ester derivative 6c with the 9-CF3 functionality were inactive against topoisomerase I when
tested at 125 µM. In addition, compared to 1 (9-NH2, 1.95 µM), all the other 5,6-difluoro derivatives with 9-substituted
electron-withdrawing groups (7a with 9-NO2, 15.6 µM; 7b with 9-CN, 31.25 µM; 7d with 9-Ac, 15.6 µM; 7e with 9-NO2 and
10-Cl, 31.25 µM ) were 8-16 fold less active with IC50 values ranging from 15.6-31.25 µM. ii). In general, the basic amine
functionality at the 6 position significantly enhanced topoisomerase I inhibitory activity. For example, the 6-substituted amine
derivatives 11a with 6-methylpiperazinyl, 11b with piperazinyl, 11d with morpholino, 11g with bipiperidinyl, 11h with
morpholinoethyl, as well as the 6-substituted aminopyrrolidinyl derivatives 16, 17a, and 17b demonstrated the most potent
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topoisomerase I inhibitory activity with IC50 values of 0.24-0.97 µM; and within this group, 11d and 11h with the morpholino
group had an IC50 value of 0.97 µM. In contrast, all the other 6-substituted amine derivatives with a more lipophilic side chain,
including 11c with methylpiperidinyl, 11e with phenethyl, 11f with adamantanyl, 11i with cyclohexyl, 11j with cyclopentyl,
11k with n-hexyl, and 15a with t-Boc-aminopyrrolidinyl, showed weaker topoisomerase I inhibition with IC50 values ranging
from 3.9 to 15.6 µM. Notably, both 6-substituted aminopyrrolidinyl S- and R- stereoisomers 17a and 17b exhibited the same
topoisomerase I inhibitory activity (IC50 = 0.48-0.97 µM), suggesting the stereochemistry at the 6 position is not required for
topoisomerase I inhibition. iii) Esterification of the carboxylic acid group had little effect on the E. coli topoisomerase I
inhibitory activity by comparing 6a and 7a (IC50 = 15.6 µM), 6b and 7b (IC50 = 31.25 µM), as well as 6c and 7c (IC50 >125
µM), indicating the ethyl ester functionality is tolerated for topoisomerase I enzyme inhibition. Representative inhibition results
of 11a and 11b against E. coli topoisomerase relaxation activity are shown in Fig. 3.
Fig. 2. SAR toward E. coli topoisomerase I inhibition.
2.2.2. Selectivity and specificity against other DNA topoisomerase enzymes
In addition, to determine the selectivity and specificity profiles of this class of fluoroquinophenoxazine derivatives, selected
compounds were also investigated for the ability to inhibit other DNA topoisomerases including E. coli DNA gyrase as well as
human topoisomerase I and IIα enzymes. Overall, these compounds were more selective toward E. coli topoisomerase I than
other enzymes tested. Given that this series of compounds has close structural similarity to quinolone antibiotic class, as such,
inhibition against E. coli gyrase can be also observed. Specifically, most of the compounds showed 4-64 fold selectivity toward
topoisomerase I over DNA gyrase except that the moderately active compound 7e with the 9-NO2 and 10-chloro substituents
showed less specificity with 2 fold selectivity toward topoisomerase I. With respect to human topoisomerases I and IIα
inhibition, these compounds also showed inhibitory activity against both human topoisomerase I (IC50 = 3.9-31.25 µM) and
topoisomerase IIα (IC50 = 0.97-250 µM), with approximate 4-32 fold selectivity. Taken together, compounds 11a (IC50 = 0.48
µM) and 11b (IC50 = 0.24 µM) (Fig. 3) bearing both 9-NH2 and 6-piperazinyl motifs exhibited the most potent topoisomerase I
inhibitory activity with the more favorable selectivity profile (8-64 fold) toward E. coli topoisomerase I against all the other
enzymes tested.
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2.2.3. Cell-based antibacterial activity
In addition to target-based topoisomerase enzyme inhibition, whole cell antibacterial activities of the synthesized compounds
were also assessed against a panel of bacterial strains. The results are also shown in Table 1. From these data, the majority of
these fluoroquinophenoxazine derivatives exhibited good to excellent antibacterial activity against the membrane permeable E.
coli strain BAS3023 and Gram-positive B. subtilis strain, and were inactive against the wide type E. coli strain. Additionally,
the antibacterial activity of most fluoroquinophenoxazine derivatives (e.g., 1, 7a, 7c, 9, 10a, 11a-d, 11f-i, 15a, 16, and 17a-b)
generally correlated with E. coli topoisomerase I inhibitory activity, suggesting that the antibacterial basis of these compounds
may be in part due to the inhibition of topoisomerase I. The only type IA topoisomerase present in M. tuberculosis has recently
been validated as an antitubercular target [21]. The topoisomerase I activity has been shown to be essential for viability and
infection in a murine model of tuberculosis [21, 22]. To further determine the antituberculosis profile for this chemical class of
fluoroquinophenoxazine derivatives, twelve compounds were selected and evaluated against M. tuberculosis. Among them, 11g
with the 6-bipiperidinyl lipophilic side chain and 11d with the 6-morpholino heterocyclic ring system showed the most potent
antituberculosis activity with minimum inhibitory concentration (MIC) values of 2.5 and 3.5 µM, respectively. In addition,
compared to 11d, its corresponding 6-piperazinyl structural analogs 11a with tertiary amine and 11b with secondary amine
functionality was about 2- and 8-fold less active with the MIC values of 7.6 and 29.5 µM, respectively. In contrast, both 6-
substituted aminopyrrolidinyl derivatives 17a (S) and 17b (R) with primary amine functionality were not active (MIC > 63.1
µM) against M. tuberculosis. These data strongly suggest that the decreased or lost whole cell antituberculosis activity of these
compounds are most likely due to their decreased lipophilicity and subsequent cell membrane penetration. Unfortunately,
cytotoxicity evaluation of our tested compounds against healthy normal Vero cells showed that they generally had narrow
selectivity index, with 11g (SI = 9.8) being the most promising compound. It is also worthwhile noting that, one of the most
potent topoisomerase I inhibitors, 11a (IC50 = 0.48 µM) bearing the 6-methylpiperazinyl and 9-amino motifs, showed broad
spectrum antibacterial activity against all the test bacteria strains with MICs ranging from 0.78 to 7.6 µM (SI = 3.8-37).
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Table 1. E. coli topoisomerase I inhibition and whole cell antibacterial activities (µM) of fluoroquinophenoxazine derivativesa
Topoisomerase inhibitory activity (IC50, µM)
Whole cell based antibacterial activity (MIC, µM)
Compd
E. coli
topo I
(type IA)
1.95
E. coli
Human
topo I
(type IB)
Human
topo IIα
(type IIA)
>500
E. coli
E. coli
(MG1655)
WT
B. subtilis
(ATCC 6633)
WT
M.
Vero cell SIb
IC50
DNA gyrase
(type IIA)
>125
Imp4213
(BAS3023)
0.78-1.56
tuberculosis
(H37Rv)
11.2
31.3
>200
6.25
75.7
6.8
1
15.6
31.25
>125
15.6
31.25
>125
15.6
31.25
1.95
1.95
3.9
0.48
0.24
3.9
0.97
3.9
3.9-7.8
0.48
0.97
3.9
3.9
15.6
7.8
50
>200
>200
>200
200
>200
>200
>200
50
>200
100
>200
6.25
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
25
200
6a
6b
6c
7a
7b
7c
7d
7e
>200
>200
0.78
200
>200
>200
0.39
25
1.56
>200
0.78
0.39
3.12
0.19-0.39
50
3.12
0.39-0.78
0.78
25
>200
12.5
>200
25
>200
3.12
50
0.78
>200
0.78
25
0.78
0.19
25-50
12.5
1.56
1.56
12.5
12.5
>200
>200
12.5
0.78
6.25
12.5
62.5
31.25
31.25
125
29
19
>127
95
>4.4
5.0
9
62.5-125
250-500
10a
10b
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
12
15.6-31.25
7.8-15.6
15.6
7.8
3.9-7.8
1.95-3.9
7.6
29.5
29
>126
3.8
>4.3
7.8
15.6
15.6
3.5
24.7
7.1
38.4
2.5
21.6
30.7
24.4
43.0
0.8
9.8
2.0
15.6
15.6-31.25
3.9
7.8-15.6
1.95-3.9
3.9-7.8
25
>200
>200
12.5
1.56
1.56
6.25
3.9
15a
16
17a
17b
0.48
0.48-0.97
0.48-0.97
3.9
3.9
3.9
7.8
3.9
3.9
1.95-3.9
0.97-1.95
3.9
>50
>63.1
>63.1
>50
>63.1
>63.1
a Blank cells indicate Not Determined.
b Selectivity index = cytotoxic IC50 against Vero cells/MIC against M. tuberculosis.
Fig. 3. Inhibition of E. coli topoisomerase relaxation activity by representative 11a and 11b. A) E. coli topoisomerase I
inhibition assays with supercoiled plasmid DNA. B) E. coli DNA gyrase inhibition assays with relaxed plasmid DNA. C:
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DMSO control; Nor: Norfloxacin (125 µM) control; S: Supercoiled plasmid DNA; N: Nicked DNA; FR: Fully relaxed DNA;
PR: Partially relaxed DNA.
2.3. CoMFA analysis
To further understand the structural basis for topoisomerase I inhibitory activity of this set of fluoroquinophenoxazine
derivatives, we subsequently performed three dimensional QSAR (3D-QSAR) study using CoMFA analysis [23]. Because of
the relatively rigid core structural feature of this class of fluoroquinophenoxazine molecules, we directly applied core structure
based alignment to build reliable 3D-QSAR models. The CoMFA study was carried out using a total of 21 compounds (entries
1-21, Table 2). Statistical parameters of the CoMFA model showed a reasonable cross-validated correlation coefficient q2 of
0.688, indicating a good internal prediction of the model. The CoMFA model also exhibited a conventional correlation
coefficient r2 of 0.806. To evaluate the predictive ability of our developed model, a test set of 7 compounds (entries 22-28,
Table 2) which was not included in model generation was subsequently used. The predicative correlation coefficient r2
of
pred
0.767 indicates good external predicative ability of the CoMFA model. The experimental and predicted values as well as their
residuals from the training and test set molecules are listed in Table 2. The correlation between the predicted and experimental
values of all compounds was plotted and the resulting chart is shown in Fig. 4.
Table 2. Experimental (pIC50) and CoMFA predicted activity (PA) values and residuals for the training and test set compoundsa
c
Entry
1
Compd.
1
IC50 (
ꢀ
M)
pIC50
5.71
4.51
3.60
4.81
4.51
3.61
4.51
5.71
5.71
5.41
6.32
6.62
6.01
5.41
5.23
CoMFA PAb
5.05
∆
1.95
31.25
250
0.66
0.30
-0.40
0.18
0.24
-0.54
-0.03
-0.17
-0.34
-0.49
0.11
0.56
0.07
-0.42
-0.16
2
4.21
6b
3
4.00
6c
4
15.6
31.25
250
4.63
7a
5
4.27
7b
6
4.15
7c
7
31.25
1.95
1.95
3.9
4.54
7e
8
5.88
9
9
6.05
10a
10b
11a
11b
11d
11e
11f
10
11
12
13
14
15
5.90
0.48
0.24
0.97
3.9
6.21
6.06
5.94
5.83
5.85
5.39
13

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16
17
18
19
20
21
22
23
24
25
26
27
28
0.48
0.97
15.6
3.9
6.31
6.01
4.81
5.41
6.32
6.14
4.81
4.81
5.41
5.41
5.41
5.11
6.14
6.15
5.72
5.50
5.77
5.88
5.89
4.55
4.77
5.76
5.23
5.45
4.56
5.95
0.16
0.29
-0.69
-0.36
0.44
0.25
0.26
0.04
-0.35
0.18
-0.04
0.55
0.19
11g
11h
11k
15a
16
0.48
0.73
15.6
15.6
3.9
17b
6a
7d
11c
11i
11j
12
3.9
3.9
7.8
0.73
17a
a Entries 1-21 for training set; entries 22-28 for test set.
b Predicted activity.
c Residual of experimental and predicted activity values.
Fig. 4. The correlation chart of experimental versus predicted values for the training and test set compounds.
14

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The results of the CoMFA model were analyzed and visualized using the standard deviation coefficient (StDev*Coeff)
mapping option contoured by steric and electrostatic contributions. In order to probe the structure/activity correlation, the steric
and electrostatic contours were mapped onto their aligned chemical structures of these fluoroquinophenoxazine molecules to
identify the potential regions in which the molecules would favorably or unfavorably interact with the topoisomerase I enzyme.
The representative steric and electrostatic contour maps of the most active compound 11b and the least active 6c derived from
the CoMFA model are shown in Fig. 5. Briefly, the yellow areas in the steric contour maps indicate regions of steric hindrance
to activity, while the green areas indicating steric contribution to potency. From the electrostatic contour maps, the regions in
blue indicate positive electrostatic charge potential associated with increased activity, with the red regions show electronegative
groups with increased activity.
In Fig. 5A and Fig. 5B, one green contour was found near the piperazinyl moiety of compound 11b indicating that a
moderate steric substituent would be favored at the 6 position of the quinophenoxazine scaffold. This may offer a potential
explanation why the 6-substituted amino derivatives were generally more active than the 6-fluoro analogs. In addition, two
yellow contours were observed near the 9 position of inactive 6c, indicating that a steric bulkiness (e.g., NO2 in 6a and 7a, CF3
in 6c, and the acetyl group in 7d) would be disfavored for activity in this area.
The CoMFA electrostatic contour maps are displayed in Fig. 5C and Fig. 5D. A large blue contour was found around the 9
position of compounds 11b and 6c, indicating that the presence of electron rich functionalities and positively charged
environment at this position (e.g., NH2 vs. NO2, Ac, CN, and CF3) would be strongly favored for topoisomerase I inhibitory
activity. It was also observed that a big red contour region was present around the 2 position of fluoroquinophenoxazine
scaffold, suggesting that an electronegative group (e.g., COOH and COOR2) at this position may be required for activity.
Finally, two red contours were found at both sides of the fused heterocyclic skeleton of 11b and 6c, suggesting that electron
deficient functionalities would be favored in those regions.
15

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A)
B)
C)
D)
Fig. 5. Representative CoMFA steric and electrostatic contour maps. A). Steric contour maps with 6c; B). Steric contour maps
with 11b; C). Electrostatic contour maps with 6c; and D) Electrostatic contour maps with 11b.
3. Conclusions
On the basis of a HTS hit compound 1, a series of fluoroquinophenoxazine analogs were designed, synthesized, and
evaluated as topoisomerase IA inhibitors and antibacterial agents. Among the newly produced fluoroquinophenoxazine
derivatives, compounds with the 9-NH2 and/or 6-substituted amine functionalities exhibited good to excellent inhibitory
activities against E. coli topoisomerase IA with IC50 values ranging from 0.24 to 3.9 µM. An illuminating SAR against E. coli
topoisomerase I has also been obtained. For example, the 6-substituted amino motif significantly enhanced the topoisomerase I
inhibition and the 9-NH2 functionality was the most desirable compared to some other groups evaluated. Notably, 11a bearing
the 6-methylpiperazinyl and 9-amino motifs showed excellent topoisomerase I inhibition (IC50 = 0.48 µM) as well as broad
spectrum antibacterial activity against all the bacteria strains tested with MICs ranging from 0.78 to 7.6 µM (SI = 3.8-37). In
16

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addition, compound 11g with the 6-bipiperidinyl lipophilic side chain represents a promising antitubercular lead with the most
potent antituberculosis activity (MIC = 2.5 µM, SI = 9.8). Finally, CoMFA analysis was performed to investigate the 3D-QSAR.
The constructed CoMFA model produced reasonable statistics, with q2 = 0.688 and r2 = 0.806. The predictive power of the
developed model was obtained using a test set of 7 molecules, giving predictive correlation coefficient r2
of 0.767.
pred
Collectively, this work has generated valuable SAR and critical understanding for this chemotype class of
fluoroquinophenoxazine topoisomerase I inhibitors as antibacterial agents. Our developed CoMFA model can provide important
structural insights toward topoisomerase I recognition and guide future structure based design and synthesis of bacterial
topoisomerase I inhibitors.
4. Experimental
4.1. General methods for chemistry
All reagents and anhydrous solvents were purchased from Sigma-Aldrich and Fisher Scientific, and were used without
further purification. All reactions were monitored either by thin-layer chromatography (TLC) or by analytical high performance
liquid chromatography (HPLC) to detect the completion of reactions. TLC was performed using glass plates pre-coated with
silica gel (0.25 mm, 60-Å pore size, 230-400 mesh, Sorbent Technologies, GA) impregnated with a fluorescent indicator (254
nm). TLC plates were visualized by exposure to ultraviolet light (UV). Hydrogenation reactions were performed employing
domnick hunter NITROX UHP-60H hydrogen generator, USA. Microwave synthesis was performed using Biotage Initiator 8
Exp Microwave System. Compounds were purified by flash column chromatography on silica gel using a Biotage Isolera One
1
system and a Biotage SNAP cartridge. H and 13C NMR spectra were obtained on a Bruker Avance DRX-400 instrument with
chemical shifts (δ, ppm) determined using TMS as internal standard. Coupling constants (J) are in hertz (Hz). ESI mass spectra
in either positive or negative mode were provided by Varian 500-MS IT Mass Spectrometer. High-resolution mass spectra
(HRMS) were obtained on an Agilent 6530 Accurate Mass Q-TOF LC/MS. The purity of compounds was determined by
analytical HPLC using a Gemini, 3 ꢀm, C18, 110 Å column (50 mm × 4.6 mm, Phenomenex) and a flow rate of 1.0 mL/min.
Gradient conditions: solvent A (0.1% trifluoroacetic acid in water) and solvent B (acetonitrile): 0-2.00 min 100% A, 2.00-7.00
min 0-100% B (linear gradient), 7.00-8.00 min 100% B, UV detection at 254 and 220 nm.
4.1.1. 6-Amino-2,3-difluorophenol (2)
2,3-Difluoro-6-nitrophenol (700 mg, 4 mmol) was dissolved in ethanol (5 mL) and palladium on activated carbon (Pd/C) (84.8
mg, 20%) was added. The reaction was stirred at room temperature under H2 atmosphere (1.0 bar). After 7 h, all starting
17

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material was consumed and Pd/C was filtered through Celite. The solvent was evaporated under reduced pressure to afford 6-
1
amino-2,3-difluorophenol (550.5 mg, 95% yield). H NMR (400 MHz, CDCl3):
δ (ppm) 6.59 (dt, J = 9.9, 8.6 Hz, 1H), 6.47-
6.31 (m, 1H), 4.27 (br s, 2H). ESI-HRMS: calc. for C6H6F2NO [M + H]+: 146.0412, found: 146.0418.
4.1.2. Diethyl 2-(((3,4-difluoro-2-hydroxyphenyl)amino)methylene)malonate (3)
6-Amino-2,3-difluorophenol (2) (550 mg, 3.8 mmol) was dissolved in ethanol (15 mL) and diethyl 2-
(ethoxymethylene)malonate (819 mg, 3.8 mmol) was added. The reaction was stirred at room temperature until there was no
starting material left. Ethanol was removed and the residue was purified by flash column chromatography on silica gel (EtOAc /
hexane = 1 / 3 to 3 / 1) to give product 3 as a brown solid (1.04 g, 87%). 1H NMR (400 MHz, d6-DMSO):
δ (ppm) 10.96 (d, J =
14.0 Hz, 1H), 8.45 (d, J = 13.9 Hz, 1H), 7.31-7.22 (m, 1H), 6.98-6.86 (m, 1H), 4.20 (q, J = 7.1 Hz, 2H), 4.12 (q, J = 7.1 Hz, 2H),
1.26 (t, J = 7.1 Hz, 3H), 1.24 (t, J = 7.1 Hz, 3H). ESI-MS: calc. for C14H14F2NO5 [M - H]-: 314.3, found: 314.3.
4.1.3. Ethyl 6,7-difluoro-8-hydroxy-4-oxo-1,4-dihydroquinoline-3-carboxylate (4)
Compound 3 (445 mg, 1.4 mmol) was added in a microwave sealed tube. Diphenyl ether (2.5 mL) was added and the tube was
sealed with cap. The reaction was then set up at 250 ºC in a Biotage Microwave Initiator instrument for 30 min. Hexane was
added and the solid was filtered and washed with hexane. The product was then dried and obtained in 80% yield (299.4 mg),
1
which was used for next step without further purification. H NMR (400 MHz, d6-DMSO):
δ (ppm) 8.36 (s, 1H), 7.45 (dd, J =
10.9, 7.9 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H). ESI-MS: calc. for C12H8F2NO4 [M - H]-: 268.2, found:
268.2.
4.1.4. Ethyl 5,6-difluoro-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate (6a
)
Compound 4 (269 mg, 1 mmol) and 1-chloro-2,4-dinitrobenzene (202 mg, 1 mmol) were dissolved in DMF (2 mL). NaHCO3
(252 mg, 3 mmol) was added and the reaction was stirred at 100 ºC until no starting materials were detected by HPLC. The
solid base was removed by filtration through Celite. The filtrate was concentrated and the residue was purified through Biotage
reverse phase C18 cartridge to give 220 mg of 6a as yellow solid (yield: 57%). 1H NMR (400 MHz, d6-DMSO):
1H), 8.15 (d, J = 9.6 Hz, 1H), 8.05-8.01 (m, 2H), 7.60-7.54 (m, 1H), 4.27 (q, J = 7.2 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H); 13C NMR
δ (ppm) 9.01 (s,
(100 MHz, d6-DMSO): δ (ppm) 170.4, 163.7, 145.8, 142.7, 138.1, 133.2, 129.4, 124.6, 122.2, 120.8, 117.0, 113.0, 105.4, 105.2,
60.8, 14.2; ESI-MS: calc. for C18H10F2N2O6Na [M + Na]+: 411.3, found: 411.2. ESI-HRMS: calc. for C18H11F2N2O6 [M + H]+:
389.0580, found: 389.0582. HPLC purity: 100% (254 nm), tR: 6.92 min; 100% (220 nm), tR: 6.92 min.
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Compounds 6b-e were prepared according to the experimental procedure above for the preparation of 6a.
4.1.5. Ethyl 9-cyano-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate (6b
Yellow solid. Yield: 59%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 9.04 (s, 1H), 8.11 (d, J = 8.8 Hz, 1H), 7.87 (d, J = 1.6 Hz,
1H), 7.72 (dd, J = 1.6 and 8.8 Hz, 1H), 7.63-7.59 (m, 1H), 4.28 (q, J = 7.2 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H); ESI-MS: calc. for
C19H10F2N2O4Na [M + Na]+: 391.3, found: 391.2. ESI-HRMS: calc. for C19H11F2N2O4 [M + H]+: 369.0681, found: 369.0684.
HPLC purity: 100% (254 nm), tR: 6.73 min; 100% (220 nm), tR: 6.73 min.
4.1.6. Ethyl 5,6-difluoro-3-oxo-9-(trifluoromethyl)-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate (6c
Yellow solid. Yield: 10%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 9.05 (s, 1H), 8.12 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 1.6 Hz,
1H), 7.63-7.58 (m, 2H), 4.27 (q, J = 7.2 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H); ESI-MS: calc. for C19H10F5NO4Na [M + Na]+: 434.3,
found: 434.1. ESI-HRMS: calc. for C19H11F5NO4 [M + H]+: 412.0603, found: 412.0603. HPLC purity: 100% (254 nm), tR: 7.25
min; 100% (220 nm), tR: 7.25 min.
4.1.7. Ethyl 9-acetyl-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate (6d
)
1
Dark yellow solid. Yield: 38%. H NMR (400 MHz, d6-DMSO):
δ (ppm) 8.97 (s, 1H), 8.06-7.93 (m, 1H), 7.73 (d, J = 8.4 Hz,
1H), 7.69 (s, 1H), 7.55 (dd, J = 10.2, 8.0 Hz, 1H), 4.27 (q, J = 7.1 Hz, 2H), 2.58 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H). ESI-MS: calc.
for C20H13F2NNaO5 [M + Na]+: 408.3, found: 408.1. HPLC purity: 100% (254 nm), tR: 6.62 min; 100% (220 nm), tR: 6.62 min.
4.1.8. Ethyl 10-chloro-5,6-difluoro-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate (6e
Dark yellow solid. Yield: 40%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 9.13 (s, 1H), 8.48 (s, 1H), 8.12 (s, 1H), 7.69-7.60 (m,
1H), 4.30 (q, J = 7.0 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). ESI-MS: calc. for C18H9ClF2N2NaO6 [M + Na]+: 445.0, found: 445.1.
HPLC purity: 98.5% (254 nm), tR: 6.92 min; 96.9% (220 nm), tR: 6.92 min.
4.1.9. 5,6-Difluoro-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (7a
)
Compound 6a (180 mg, 0.46 mmol) was dissolved in AcOH (15 mL). Hydrochloric acid (1 N, 2 mL) was added and the
reaction was stirred under reflux for 2 h. Upon completion, water was added and yellow solid was precipitated. The solid was
1
collected by filtration and washed with water, then dried and afforded product 7a (150 mg) as yellow solid in 91% yield. H
19

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NMR (400 MHz, d6-DMSO):
δ
(ppm) 9.36 (s, 1H), 8.42 (d, J = 9.2 Hz, 1H), 8.17 (d, J = 2.8 Hz, 1H), 8.09 (dd, J = 2.4 and 9.2
Hz, 1H), 7.86-7.82 (m, 1H); ESI-MS: calc. for C16H7F2N2O6 [M + H]+: 361.2, found: 361.3. ESI-HRMS: calc. for C16H7F2N2O6
[M + H]+: 361.0267, found: 361.0268. HPLC purity: 100% (254 nm), tR: 6.79 min; 100% (220 nm), tR: 6.79 min.
Compounds 7b-e were prepared according to the experimental procedure above for the preparation of 7a.
4.1.10. 9-Cyano-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (7b
Yellow solid. Yield: 36%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 9.34 (s, 1H), 8.35 (d, J = 8.8 Hz, 1H), 7.98 (d, J = 1.6 Hz,
1H), 7.82 (d, J = 2.4 Hz, 1H), 7.78-7.75 (m, 2H); ESI-MS: calc. for C17H7F2N2O4 [M + H]+: 341.2, found: 341.1. ESI-HRMS:
calc. for C17H7F2N2O4 [M + H]+: 341.0368, found: 341.0378. HPLC purity: 95.1% (254 nm), tR: 6.37 min; 97.7% (220 nm), tR:
6.37 min.
4.1.11. 5,6-Difluoro-3-oxo-9-(trifluoromethyl)-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (7c
)
1
Yellow solid. Yield: 95%. H NMR (400 MHz, d6-DMSO):
δ (ppm) 9.35 (s, 1H), 8.37 (d, J = 8.4 Hz, 1H), 7.84-7.79 (m, 2H),
7.65-7.62 (dd, J = 1.2 and 8.8 Hz, 1H); ESI-MS: calc. for C17H7F5NO4 [M + H]+: 384.2, found: 384.1. ESI-HRMS: calc. for
C17H7F5NO4 [M + H]+: 384.0290, found: 384.0293. HPLC purity: 100% (254 nm), tR: 7.15 min; 100% (220 nm), tR: 7.15 min.
4.1.12. 9-Acetyl-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (7d
Yellow solid. Yield: 62%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 9.36 (s, 1H), 8.30 (d, J = 11.2 Hz, 1H), 7.85-7.80 (m, 3H),
2.63 (s, 3H); ESI-MS: calc. for C18H10F2NO5 [M + H]+: 358.3, found: 358.2. ESI-HRMS: calc. for C18H10F2NO5 [M + H]+:
358.0522, found: 358.0521. HPLC purity: 97.3% (254 nm), tR: 6.70 min; 97.2% (220 nm), tR: 6.70 min.
4.1.13. 10-Chloro-5,6-difluoro-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (7e
)
1
Brown solid. Yield: 39%. H NMR (400 MHz, d6-DMSO):
δ (ppm) 9.43 (s, 1H), 8.74 (s, 1H), 8.18 (s, 1H), 7.83-7.79 (m, 1H);
ESI-MS: calc. for C16H6ClF2N2O6 [M + H]+: 394.7, found: 394.9. ESI-HRMS: calc. for C16H6F2N2ClO6 [M + H]+: 394.9877,
found: 394.9879. HPLC purity: 95.4% (254 nm), tR: 6.90 min; 97.0% (220 nm), tR: 6.90 min.
4.1.14. 9-Amino-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (1)
Compound 7a (150 mg, 0.42 mmol) was added to a mixture of AcOH / HCl (1 / 1). SnCl2 (236 mg, 1.25 mmol) was added and
the reaction was stirred under reflux for 2 h. No starting material was observed in the reaction and then water was added. The
20

ACCEPTED MANUSCRIPT
large amount of solid precipitated and was collected by filtration. Product 1 was obtained in 86% yield (236 mg) as yellow solid.
1H NMR (400 MHz, d6-DMSO):
δ (ppm) 9.08 (s, 1H), 7.79 (d, J = 9.2 Hz, 1H), 7.76-7.73 (m, 1H), 6.48 (dd, J = 2.4 and 9.2 Hz,
1H), 6.43 (d, J = 2.4 Hz, 1H); ESI-MS: calc. for C16H9F2N2O4 [M + H]+: 331.3, found: 331.1. ESI-HRMS: calc. for
C16H9F2N2O4 [M + H]+: 331.0525, found: 331.0526. HPLC purity: 100% (254 nm), tR: 6.47 min; 100% (220 nm), tR: 6.47 min.
4.1.15. Ethyl-5-fluoro-6-(4-methylpiperazin-1-yl)-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate (9)
Compound 6a (159 mg, 0.4 mmol) was dissolved in pyridine (1 mL) and 1-methylpiperazine (120 mg, 1.2 mmol) was added.
The reaction was heated to 110 ºC until no starting material was observed. Pyridine was removed under reduced pressure and
the residue was purified by flash column chromatography on silica gel (MeOH / CH2Cl2 = 1 / 19), affording product 9 (100.1
mg) in 53% yield as orange solid. 1H NMR (400 MHz, d6-DMSO):
δ (ppm) 8.87 (d, J = 1.6 Hz, 1H), 8.04-7.97 (m, 2H), 7.87 (t,
J = 1.2 Hz, 1H), 7.32 (dd, J = 1.2 and 8.0 Hz, 2H), 4.25 (q, J = 7.2 Hz, 2H), 3.34 (s, 4H), 3.27 (s, 4H), 2.27 (s, 3H), 1.32 (t, J =
7.2 Hz, 3H); ESI-MS: calc. for C23H22FN4O6 [M + H]+: 469.4, found: 469.3. HPLC purity: 100% (254 nm), tR: 5.48 min; 100%
(220 nm), tR: 5.48 min.
4.1.16. 5-Fluoro-6-(4-methylpiperazin-1-yl)-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (10a
)
Compound 7a (108 mg, 0.3 mmol) was dissolved in pyridine (4 mL), and then the reaction was heated to 90 ºC. 1-
Methylpiperazine (100 µL, 0.9 mmol) was added and the reaction was stirred under nitrogen atmosphere until there was no
starting material. Upon completion, pyridine was removed under reduced pressure and the residue was dissolved in ethanol (10
mL) and heated under reflux for additional 30 min. The solid was filtered and washed with water, then dried to obtain product
1
10a (60 mg) in 45 % yield as yellow solid. H NMR (400 MHz, d6-DMSO):
δ (ppm) 9.18 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H),
8.02-7.99 (m, 2H), 7.53 (d, J = 12.6 Hz, 1H), 3.30 (br s, overlapping with H2O peak, 4H), 2.57 (br s, 4H), 2.32 (s, 3H); ESI-MS:
calc. for C21H18FN4O6 [M + H]+: 441.4, found: 441.4. ESI-MS: calc. for C21H18FN4O6 [M + H]+: 441.1205, found: 441.1216.
HPLC purity: 100% (254 nm), tR: 5.37 min; 100% (220 nm), tR: 5.37 min.
Compounds 10b and 11a-k were prepared following the similar procedure for the preparation of 10a.
4.1.17. 5-Fluoro-9-nitro-3-oxo-6-(piperazin-1-yl)-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (10b
Yellow solid. Yield: 84%. 1H NMR (400 MHz, d6-DMSO):
1H), 6.43-6.36 (m, 2H), 3.22 (s, 4H), 2.87 (s, 4H); ESI-MS: calc. for C20H16FN4O6 [M + H]+: 427.4, found: 427.2. ESI-HRMS:
)
δ
(ppm) 8.94 (s, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 12.0 Hz,
21

ACCEPTED MANUSCRIPT
calc. for C20H16FN4O6 [M + H]+: 427.1048, found: 427.1041. HPLC purity: 100% (254 nm), tR: 5.36 min; 100% (220 nm), tR:
5.36 min.
4.1.18. 9-Amino-5-fluoro-6-(4-methylpiperazin-1-yl)-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (11a
Yellow solid. Yield: 48%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 8.94 (s, 1H), 7.65 (d, J = 9.2 Hz, 1H), 7.45 (d, J = 12.0 Hz,
1H), 6.43-6.36 (m, 2H), 5.77 (s, 2H), 3.31 (s, 4H), 2.57 (s, 4H), 2.32 (s, 3H); ESI-MS: calc. for C21H20FN4O4 [M + H]+: 411.4,
found: 411.2. ESI-MS: calc. for C21H20FN4O4 [M + H]+: 411.1463, found: 411.1466. HPLC purity: 100% (254 nm), tR: 5.22
min; 100% (220 nm), tR: 5.22 min.
4.1.19. 9-Amino-5-fluoro-3-oxo-6-(piperazin-1-yl)-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (11b
Yellow solid. Yield: 97%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 8.97 (s, 1H), 7.68 (d, J = 9.2 Hz, 1H), 7.48 (d, J = 12.4 Hz,
1H), 6.44-6.38 (m, 2H), 5.77 (s, 2H), 3.22 (s, 4H), 2.86 (s, 4H); ESI-MS: calc. for C20H18FN4O4 [M + H]+: 397.4, found: 397.2.
ESI-HRMS: calc. for C20H18FN4O4 [M + H]+: 397.1307, found: 397.1303. HPLC purity: 100% (254 nm), tR: 5.20 min; 100%
(220 nm), tR: 5.20 min.
4.1.20. 9-Amino-5-fluoro-6-(4-methylpiperidin-1-yl)-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (11c
Yellow solid. Yield: 33%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 8.92 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 11.6 Hz,
1H), 6.42-6.36 (m, 2H), 5.75 (s, 2H), 3.13 (t, J = 10.8 Hz, 2H), 1.71 (d, J = 11.2 Hz, 2H), 1.57 (s, 1H), 1.32-1.26 (m, 2H), 0.98
(s, 3H); 13C NMR (100 MHz, d6-DMSO):
δ
(ppm) 175.4, 166.4, 150.7, 144.6, 138.5, 136.0, 131.7, 125.3, 119.6, 119.5, 117.6,
112.2, 111.1, 107.0, 104.0, 101.2, 51.1, 35.1, 30.6, 22.5; ESI-MS: calc. for C22H21FN3O4 [M + H]+: 410.4, found: 410.2. ESI-
HRMS: calc. for C22H21FN3O4 [M + H]+: 410.1511, found: 410.1510. HPLC purity: 100% (254 nm), tR: 6.38 min; 100% (220
nm), tR: 6.38 min.
4.1.21. 9-Amino-5-fluoro-6-morpholino-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (11d
Yellow solid. Yield: 71%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 8.93 (s, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 12.0 Hz,
1H), 6.43-6.37 (m, 2H), 5.75 (s, 2H), 3.69 (s, 4H), 3.28 (s, 4H); ESI-MS: calc. for C20H17FN3O5 [M + H]+: 398.4, found: 398.2.
ESI-HRMS: calc. for C20H17FN3O5 [M + H]+: 398.1147, found: 398.1146. HPLC purity: 100% (254 nm), tR: 6.38 min; 100%
(220 nm), tR: 6.38 min.
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4.1.22. 9-Amino-5-fluoro-3-oxo-6-(phenethylamino)-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (11e
)
Yellow solid. Yield: 55%. 1H NMR (400 MHz, d6-DMSO):
δ (ppm) 8.90 (s, 1H), 7.61 (d, J = 9.2 Hz, 1H), 7.45 (d, J = 13.2 Hz,
1H), 7.29-7.18 (m, 5H), 6.43-6.37 (m, 2H), 6.15 (br s, 1H), 5.76 (br s, 2H), 3.68-3.61 (m, 2H), 2.89-2.85 (m, 2H); ESI-MS: calc.
for C24H18FN3O4 [M + H]+: 432.4, found: 432.1. ESI-HRMS: calc. for C24H19FN3O4 [M + H]+: 432.1354, found: 432.1362.
HPLC purity: 100% (254 nm), tR: 7.07 min; 100% (220 nm), tR: 7.07 min.
4.1.23. 6-(((3s,5s,7s)-Adamantan-1-yl)amino)-9-amino-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (11f
Brown solid. Yield: 29%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 9.00 (s, 1H), 7.71 (d, J = 9.1 Hz, 1H), 7.54 (d, J = 10.7 Hz,
1H), 6.50-6.38 (m, 2H), 5.81 (s, 2H), 4.34 (s, 1H), 2.09-2.04 (m, 3H), 1.85 (s, 6H), 1.65-1.55 (m, 6H). ESI-MS: calc. for
C26H23FN3O4 [M - H]+: 460.2, found: 460.0. HPLC purity: 85.2% (254 nm), tR: 7.41 min; 91.1% (220 nm), tR: 7.41 min.
4.1.24. 6-([1,4'-Bipiperidin]-1'-yl)-9-amino-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (11g
Yellow solid. Yield: 64%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 8.98 (s, 1H), 7.68 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 9.6 Hz,
1H), 6.45-6.41 (m, 2H), 5.77 (br s, 2H), 3.20-3.15 (m, 1H), 1.84-1.82 (m, 3H), 1.64-1.41 (m, 14H), 1.06-1.04 (m, 1H); ESI-MS:
calc. for C26H28FN4O4 [M + H]+: 479.5, found: 479.3. ESI-HRMS: calc. for C26H28FN4O4 [M + H]+: 479.2089, found: 479.2093.
HPLC purity: 100% (254 nm), tR: 5.53 min; 100% (220 nm), tR: 5.53 min.
4.1.25. 9-Amino-5-fluoro-6-((2-morpholinoethyl)amino)-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (11h
)
1
Yellow solid. Yield: 74%. H NMR (400 MHz, d6-DMSO):
δ (ppm) 8.90 (s, 1H), 8.55 (s, 1H), 7.46 (d, J = 6.0 Hz, 1H), 7.39-
7.34 (m, 2H), 6.42-6.37 (m, 2H), 5.95 (s, 1H), 5.78 (br s, 2H), 2.57 (s, 2H), 2.44 (br s, 6H); ESI-MS: calc. for C22H22FN4O5 [M
+ H]+: 441.4, found: 441.3. ESI-MS: calc. for C22H22FN4O5 [M + H]+: 441.1569, found: 441.1579. HPLC purity: 97.2% (254
nm), tR: 5.22 min; 99.3% (220 nm), tR: 5.22 min.
4.1.26. 9-Amino-6-(cyclohexylamino)-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (11i
Brown solid. Yield: 74%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 8.86 (s, 1H), 7.58-7.56 (m, 1H), 7.43-7.40 (m, 1H), 6.40 (s,
1H), 5.72 (s, 2H), 5.40-5.38 (m, 1H), 1.92 (s, 3H), 1.73-1.67 (m, 3H), 1.31-1.22 (m, 6H); ESI-MS: calc. for C22H21FN3O4 [M +
H]+: 410.4, found: 410.3. ESI-HRMS: calc. for C22H21FN3O4 [M + H]+: 410.1511, found: 410.1494. HPLC purity: 100% (254
nm), tR: 7.27 min; 100% (220 nm), tR: 7.27 min.
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4.1.27. 9-Amino-6-(cyclopentylamino)-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (11j
)
Black solid. Yield: 63%. 1H NMR (400 MHz, d6-DMSO):
δ (ppm) 8.91 (s, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 12.4 Hz,
1H), 6.46-6.41 (m, 2H), 5.73 (s, 2H), 5.53 (d, J = 2.4 Hz, 1H), 4.30 (s, 1H), 1.93 (br s, 4H), 1.73 (br s, 4H); ESI-MS: calc. for
C21H19FN3O4 [M + H]+: 396.4, found: 396.1. ESI-HRMS: calc. for C21H19FN3O4 [M + H]+: 396.1354, found: 396.1358. HPLC
purity: 96.7% (254 nm), tR: 7.07 min; 96.0% (220 nm), tR: 7.06 min.
4.1.28. 9-Amino-5-fluoro-6-(hexylamino)-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (11k
Yellow solid. Yield: 49%. 1H NMR (400 MHz, d6-DMSO):
)
δ
(ppm) 8.88 (s, 1H), 7.64 (s, 1H), 7.44 (s, 1H), 6.56-6.36 (m, 2H),
5.88-5.50 (m, 2H), 1.57 (s, 2H), 1.27 (s, 7H), 0.84 (s, 4H); ESI-MS: calc. for C22H23FN3O4 [M + H]+: 412.4, found: 412.2. ESI-
HRMS: calc. for C22H23FN3O4 [M + H]+: 412.1667, found: 412.1672. HPLC purity: 100% (254 nm), tR: 7.47 min; 100% (220
nm), tR: 7.47 min.
4.1.29. 9-acetamido-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (12
)
To the solution of 1 (33 mg, 0.1 mmol) and pyridine (1 mL), acetic anhydride (12.2 mg, 0.12 mmol) was added. The reaction
was stirred at 80 ºC for 3 h, then at 100 ºC until no starting material was detected by HPLC. The solid was filtered and dried to
give 34.1 mg of 12 in 92% yield. Yellow solid. 1H NMR (400 MHz, d6-DMSO):
δ (ppm) 10.31 (s, 1H), 9.20 (s, 1H), 8.05 (d, J
= 9.2 Hz, 1H), 7.78 (t, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.29 (d, J = 7.6 Hz, 1H), 2.06 (s, 3H); ESI-MS: calc. for C18H11F2N2O5 [M
+ H]+: 373.3, found: 373.2. ESI-MS: calc. for C18H11F2N2O5 [M + H]+: 373.0631, found: 373.0638. HPLC purity: 98.6% (254
nm), tR: 6.42 min; 99.1% (220 nm), tR: 6.42 min.
Compounds 14 and 15a-b were prepared at 70 ºC following the similar procedure for the preparation of 10a
4.1.30.
(S)-9-amino-6-(3-((tert-butoxycarbonyl)amino)pyrrolidin-1-yl)-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-
carboxylic acid (15a
)
Yellow solid. Yield: 86%. 1H NMR (400 MHz, d6-DMSO):
δ
(ppm) 8.77 (s, 1H), 7.49 (d, J = 9.6 Hz, 1H), 7.31 (d, J = 14.0 Hz,
1H), 7.17 (d, J = 5.6 Hz, 1H), 6.38 (dd, J = 2.4 and 9.2 Hz, 2H), 6.29 (d, J = 2.0 Hz, 1H), 5.67 (s, 2H), 3.82-3.80 (m, 5H), 2.05-
2.04 (m, 1H), 1.83 (br s, 1H), 1.38 (s, 9H); ESI-MS: calc. for C25H26FN4O6 [M + H]+: 497.5, found: 497.3. ESI-HRMS: calc. for
C25H26FN4O6 [M + H]+: 497.1831, found: 497.1831. HPLC purity: 100% (254 nm), tR: 6.94 min; 100% (220 nm), tR: 6.94 min.
4.1.31. (S)-6-(3-aminopyrrolidin-1-yl)-5-fluoro-9-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (16
)
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Compound 16 was prepared following the same procedure for the synthesis of 7. Brown solid. Yield: 32% (two steps). 1H NMR
(400 MHz, d6-DMSO):
δ (ppm) 9.10 (s, 1H), 8.41 (s, 2H), 8.25 (d, J = 9.3 Hz, 1H), 8.09 (s, 1H), 8.01 (d, J = 7.2 Hz, 1H), 7.48
(d, J = 13.6 Hz, 1H), 4.07 – 3.74 (m, 5H), 2.28 (s, 1H), 2.08 (s, 1H). ESI-MS: calc. for C20H16FN4O6 [M + H]+: 427.4, found:
427.1. HPLC purity: 100% (254 nm), tR: 5.45 min; 100% (220 nm), tR: 5.46 min.
4.1.32. (S)-9-amino-6-(3-aminopyrrolidin-1-yl)-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (17a
)
Compound 15a (85 mg, 0.17 mmol) was dissolved in 10 mL of hydrochloric acid (1 N). The reaction was stirred under reflux
for 2.5 h. The solvent was removed and ethanol (10 mL) was added. The mixture was heated under reflux for 30 min. The solid
was collected by filtration and dried to yield 52 mg of 17a in 78% yield. Yellow solid. 1H NMR (400 MHz, d6-DMSO):
δ (ppm)
8.94 (s, 1H), 8.24 (s, 3H), 7.65 (d, J = 9.2 Hz, 1H), 7.50 (d, J = 13.6 Hz, 1H), 6.46-6.40 (m, 2H), 3.99-3.85 (m, 5H), 2.34-2.26
(m, 1H), 2.09-1.98 (m, 1H); ESI-MS: calc. for C20H18FN4O4 [M + H]+: 397.4, found: 397.1. ESI-MS: calc. for C20H18FN4O4 [M
+ H]+: 397.1307, found: 397.1298. HPLC purity: 100% (254 nm), tR: 5.20 min; 100% (220 nm), tR: 5.20 min.
4.1.33. (R)-9-amino-6-(3-aminopyrrolidin-1-yl)-5-fluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid (17b
)
Compound 17b was prepared using the same procedure for the synthesis of 17a. Brown solid. Yield: 48% (two steps). 1H NMR
(400 MHz, d6-DMSO):
δ (ppm) 8.88 (s, 1H), 8.31 (s, 3H), 7.59 (d, J = 9.2 Hz, 1H), 7.43 (d, J = 13.2 Hz, 1H), 6.42 (d, J = 9.2
Hz, 1H), 6.38 (s, 1H), 3.98-3.88 (m, 2H), 3.73-3.68 (m, 3H), 2.30-2.25 (m, 1H), 2.06-2.03 (m, 1H); ESI-MS: calc. for
C20H18FN4O4 [M + H]+: 397.4, found: 397.2. ESI-HRMS: calc. for C20H18FN4O4 [M + H]+: 397.1307, found: 397.1302. HPLC
purity: 100% (254 nm), tR: 5.24 min; 100% (220 nm), tR: 5.24 min.
4.2. Target-based biochemical assays
Topoisomerase enzymes
Recombinant E. coli topoisomerase I and gyrase expressed in E. coli were purified as described previously [24, 25]. Human
topoisomerase I and topoisomerase IIα were purchased from TopoGen (Buena vista, CO, USA).
4.2.1. E. coli topoisomerase I relaxation activity inhibition assay
The relaxation activity of E. coli topoisomerase I was assayed in a buffer containing 10 mM Tris-HCl, pH 8.0, 50 mM NaCl,
0.1 mg/mL gelatin, and 0.5 mM MgCl2. Half microliter from the appropriate stock solutions of compounds dissolved in the
solvent (DMSO) or the solvent alone (control) was mixed with 9.5 µL of the reaction buffer containing 10 ng of enzyme before
25

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the addition of 10 µL of reaction buffer containing 200 ng of supercoiled pBAD/Thio plasmid DNA purified by cesium chloride
gradient as substrate. Following incubation at 37 oC for 30 min, the reactions were terminated by the addition of 4
ꢀL of a stop
buffer (50% glycerol, 50 mM EDTA, and 0.5% (v/v) bromophenol blue), and analyzed by agarose gel electrophoresis. The gels
were stained in ethidium bromide and photographed under UV light.
4.2.2. DNA gyrase supercoiling inhibition assay
DNA gyrase supercoiling assays were carried out by mixing the compounds and the enzyme in a similar manner as above
(EcTopI relaxation inhibition assay) but in a gyrase assay buffer (35 mM Tris-HCl, 24 mM KCl, 4 mM MgCl2, 2 mM DTT,
1.75 mM ATP, 5 mM spermidine, 0.1 mg/mL BSA, 6.5 % glycerol at pH 7.5), followed by the addition of 300 ng of relaxed
covalently closed circular DNA (New England Biolabs, Ipswich, MA, USA) to a final reaction volume of 20 µL. The samples
were incubated at 37 °C for 30 minutes before being terminated by the addition of a buffer containing 5% SDS, 0.25%
bromophenol blue, and 25% glycerol. The reactions were then analyzed by agarose gel electrophoresis.
4.2.3. Human topoisomerase I relaxation inhibition assay
Human topoisomerase I relaxation assays were carried out with 0.5 U of enzyme in reaction buffer supplied by the
manufacturer. The enzyme was mixed with the indicated concentration of compound dissolved before 200 ng of supercoiled
pBAD/Thio plasmid DNA was added in the same buffer, for a final volume of 20 µL. Following incubation at 37 °C for 30
minutes, the reactions were terminated with a buffer containing 5% SDS, 0.25% bromophenol blue, and 25% glycerol, and
analyzed by agarose gel electrophoresis.
4.2.4. Human topoisomerase IIα decatenation inhibition assay
Human Topoisomerase IIα assays were carried out by adding the compounds to 185 ng of kinetoplast DNA (kDNA, from
TopoGen) in the buffer supplied by the manufacturer before the addition of 2 U of the enzyme. The samples were incubated for
15 minutes at 37 °C before the addition of 4 µL of a stop buffer containing 5% sarkosyl, 0.25% bromophenol blue, and 25%
glycerol. The reactions were then analyzed by electrophoresis in 1% agarose gels containing 0.5 µg/mL ethidium bromide
before being photographed under UV light.
4.3. Cell-based assays
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The minimum inhibitory concentrations (MIC) of the compounds were determined against E. coli and B. subtilis in cation-
adjusted Mueller-Hinton Broth according to standard microdilution protocol [26].
MICs of compounds against M. tuberculosis were determined by a modified microplate Alamar blue assay (MABA) [27].
Vero cell cytotoxicity assay was performed as previously described [27].
4.4. CoMFA modeling
4.4.1. Dataset
All the synthesized and tested fluoroquinophenoxazine derivatives were used for CoMFA study. The topoisomerase I
inhibitory activity (IC50, µM) from biochemical enzyme assay was converted to pIC50 values for correlation purpose (pIC50 = –
logIC50). The total compound set is divided into two subsets: a training set of 21 compounds for generating 3D-QSAR models
and a test set of 7 compounds for validating the quality of the model (Table 2). The compound selections of training and test
sets were done manually so that compounds ranging from weak, moderate, to strong topoisomerase I inhibitory activities were
present in both sets and were in approximately equal proportions.
4.4.2. Conformational model analysis and molecular alignment
In the 3D-QSAR studies, alignment rule and biological conformation selection are two important factors to construct reliable
models. For both training and test set molecules, conformational models representing their available conformational space were
calculated. All the molecules were subjected to produce a maximum of 255 conformations within 20 kcal/mol in energy from
global minimum. Due to the relatively rigid structural feature of these molecules, the core structure of quinophenoxazine was
used for the alignment.
4.4.3. CoMFA model generation
CoMFA was performed using the QSAR module of SYBYL-X [28]. The steric and electrostatic field energies were
calculated using the Lennard-Jones and the Coulomb potentials, respectively, with a 1/r distance-dependent dielectric constant
in all intersections of a regularly spaced (0.2 nm) grid. The electrostatic fields were computed using Gasteiger-Huckel charge
calculation methods. A sp3 hybridized carbon atom with a radius of 1.53 Å and a charge of +1.0 was used as a probe to calculate
the steric and electrostatic energies between the probe and the molecules using the Tripos force field. The standard parameters
implemented in SYBYL-X were used. The truncation for both steric and electrostatic energies was set to 30 kcal/mol.
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4.4.4. Partial least square (PLS) analysis
PLS methodology [29] was used for 3D-QSAR analysis. The cross-validation analysis [30, 31] was performed using the leave
one out (LOO) methods in which one compound is removed from the dataset and its activity is then predicted using the model
derived from the rest of the dataset. The cross validated r2 that resulted in the optimum number of components and the lowest
standard error of prediction were considered for further analysis. To speed up the analysis and reduce noise, a minimum filter
value of 2.00 kcal/mol was used. A final analysis was performed to calculate conventional r2 using the optimum number of
components obtained from the cross-validation analysis.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This project was supported in part by the National Institutes of Health grants (P20GM103466 and R15AI092315 to DS,
R01AI069313 to YT). MICs against M. tuberculosis and Vero cell cytotoxicity were provided by the service available at the
Institute for Tuberculosis Research at University of Illinois at Chicago. NSC648059 was provided by the NCI Developmental
Therapeutics Program.
Appendix A: Supplementary data
Supplementary data related to this article can be found at
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