4-Aminoquinoline derivatives as novel Mycobacterium tuberculosis GyrB inhibitors: Structural optimization, synthesis and biological evaluation

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Graphical Abstract

4

-Amino Quinoline Derivatives as Novel Mycobacterium

tuberculosis GyrB Inhibitors: Structural Optimization,

Synthesis and Biological Evaluation

a†

a

Brahmam Medapi , Priyanka Suryadevara , Janupally Renuka , Jonnalagadda Padma Sridevi , Perumal Yogeeswari

a

and Dharmarajan Sriram *

Compound 53 was identified to be potent GyrB inhibitor with 0.86 µM and an MIC of 3.3 µM.

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SHORT COMMUNICATION

4

-Aminoquinoline Derivatives as Novel Mycobacterium tuberculosis GyrB

Inhibitors: Structural Optimization, Synthesis and Biological Evaluation

a†

a

Brahmam Medapi , Priyanka Suryadevara , Janupally Renuka , Jonnalagadda Padma Sridevi ,

a

Perumal Yogeeswari and Dharmarajan Sriram *

a

Department of Pharmacy, Birla Institute of Technology &Science Pilani Hyderabad Campus,

Hyderabad 500078, India.

Abstract

Mycobacterial DNA gyrase B subunit has been identified to be one of the potentially

underexploited drug targets in the field of antitubercular drug discovery. In the present study, we

employed structural optimization of the reported GyrB inhibitor resulting in synthesis of a series

of 46 novel quinoline derivatives. The compounds were evaluated for their in vitro

Mycobacterium smegmatis GyrB inhibitory ability and M. tuberculosis DNA supercoiling

inhibitory activity. The antitubercular activity of these compounds was tested over Mtb H37Rv

strain and their safety profile was checked against mouse macrophage RAW 264.7 cell line.

Among all, three compounds (23, 28, and 53) emerged to be active displaying IC values below

5

0

1

µM against Msm GyrB and were found to be non-cytotoxic at 50 µM concentration.

Compound 53 was identified to be potent GyrB inhibitor with 0.86±0.16 µM and an MIC

minimum inhibitory concentration) of 3.3 µM. The binding affinity of this compound towards

(

GyrB protein was analyzed by differential scanning fluorimetry which resulted in a positive shift

o

of 3.3 C in melting temperature (T ) when compared to the native protein thereby reacertaining

m

the stabilization effect of the compound over protein.

Keywords: Mycobacterium tuberculosis, DNA gyrase B, DNA supercoil assay, cytotoxicity,

differential scanning fluorimetry.

*

Corresponding Author

Prof. D. Sriram

Email: dsriram@hyderabad.bits-pilani.ac.in, drdsriram@yahoo.com

†

These authors have contributed equally.

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1

. Introduction

Tuberculosis (TB), caused by Mycobacterium tuberculosis, continues to be one of the deadliest

infectious diseases with mortality rate of around 1.5 million lives in 2013 as reported by World

Health Organization (WHO) [1]. Though several effective anti-tubercular drugs such as

isoniazid, rifampicin, ethambutol, fluoroquinolones, exist in the market, emergence of new Mtb

strains as like multi drug resistant (MDR), extensively drug resistant (XDR) and the latest total

drug resistant (TDR) TB indicate the urgency in the development of newer anti-tubercular agents

with newer combination therapy so as to be effective against these strains [2,3,4]. Unlike the

conventional anti-tubercular drugs in the market, there is a need to discover newer anti-

tubercular agents which can target a novel pathway thereby destroying the viability of the

pathogen.

DNA gyrase (topoisomerase type II) of Mtb can be an attractive target in this prospect due to the

uniqueness of the Mtb genome which codes for only two types of topoisomerases (type I and II)

unlike other pathogens. DNA gyrase, a crucial enzyme, causes negative supercoiling of DNA

which relieves strain during the DNA unwinding [5]. Functional DNA gyrase usually exists as a

2 2

heterotetramer (A B ) with two A subunits and two B subunits [6]. Fluoroquinolones which

target gyrase A subunit have been facing a major hurdle of their resistance developed by Mtb

which makes gyrase B subunit a druggable target for discovery of potent anti-tubercular agents.

DNA gyrase B subunit is involved in the process of ATP hydrolysis which in turn provides

energy to gyrase A subunit for maintaining the DNA topological state [7]. Novobiocin and

coumermycin are the reported Mtb GyrB inhibitors [7].

Several compounds have been reported as DNA GyrB inhibitors which include benzimidazole

ureas [8], pyrazinamides [9] and triazolopyridine ureas [10]. In the present study, structural

optimization of the reported lead, aminopyrazinamide derivative from AstraZeneca, was carried

out substituting at suitable positions and were synthetically derivatized to a series of 46

compounds. These compounds were subjected to in vitro M. smegmatis GyrB inhibition and Mtb

DNA supercoiling assay so as to study their inhibitory profile. Structure activity relationship of

the compounds was build up relating their GyrB activity and in silico docking pattern. Further,

the compounds were tested over in vitro drug sensitive Mtb cultures using microplate alamar

blue assay (MABA) and their cytotoxicity studies were carried out using macrophage cell line

from mouse.

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2

. Results and Discussion

In the present study, the reported aminopyrazinamide derivative from AstraZeneca [9] was

considered for further molecular optimization process which can be targeted against Mtb GyrB

enzyme. The interaction profile of the compound at the protein active site was analyzed using

molecular docking studies. The compound, 6-(3,4-dimethylphenyl)-3-[[4-[3-(4-methylpiperazin-

1

-yl)propoxy]phenyl]amino] pyrazine-2-carboxamide, was subjected to molecular docking at the

ATPase site of M. smegmatis GyrB using Glide v5.8 [11]. The interaction pattern of the

compound, shown in figure 1 [12], revealed the presence of two hydrogen bonds, one between

the free amino group of compound and Asp79 and the second one between the nitrogen atom of

piperazine and Arg82. The orientation of the compound at the active site revealed the presence of

a highly hydrophobic pocket where the 3,4-dimethyl phenyl moiety was found to be stabilized by

non-polar interactions with residues Val99, Met100, Val128, Leu135 and Leu171. Also residues

such as Ile84 and Pro85 were found to be involved in hydrophobic interactions with the

compound.

This compound was further considered for molecular reengineering by retaining the crucial

groups for GyrB activity and substituting particular sites with various suitable moieties. Figure 2

demonstrates the substitutions made over the aminopyrazinamide compound ending up with a

molecular lead. The 6-(3,4-dimethyl phenyl) pyrazine group which imparts for the hydrophobic

interactions of the compound was replaced by quinoline moiety so as to observe the effect of

fused ring system over the activity. The carboxamide group over pyrazine in the

aminopyrazinamide compound, which was reported to be crucial for activity, was also modified

by substituting amine group with ethoxy, hydrazine and hydroxyl moieties. The secondary amine

linking the pyrazine and phenyl moiety was retained. The oxygen connecting the butyl chain

with phenyl ring was retained and also substituted by amine group. The final modification was

done at the N-methyl group of piperazine by substituting with oxygen (O) and N-ethyl group.

The titled compounds were synthesized by following multi-step synthesis protocol. Initially, we

synthesized the amine derivatives (4a-b and 7a-b) starting with simple 1,4-phenylene diamine

and 4-aminophenol which were selectively alkylated with 1,3-dibromo propane using K CO in

2

3

o

DMF heating at 110 C for 3 h. This was followed by reacting them with morpholine and N-ethyl

piperazine under similar conditions, subsequent de-protection of BOC to get the amine

derivatives (4a-b and 7a-b). The reaction sequences were showed in Scheme 1. These amine

3

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derivatives were alkylated with different substituted 4-chloro quinoline analogues (11a-d) using

para-tolulene sulfonic acid (PTSA) as catalyst in methanol in a Biotage microwave vial by

o

irradiating at 120 C for 45 min to get substituted ethyl phenyl aminoquinoline-3-carboxylates

(12-27)[13]. These were further converted to their corresponding hydrazide (28-43) and acid

derivatives (44-57). The substituted 4-chloro quinoline analogues (11a-d) were prepared by a 3

step synthesis process according to the reported procedure[14,15] starting with 8a-d were

converted to diethyl phenyl amino methylidene propanedioate derivatives (9a-d) by condensing

with diethyl ethoxy methylene malonate. These (9a-d) were cyclized intramolecularly to 4-

o

hydroxy quinoline 3-carboxylate derivatives (10a-d) by heating them at 75 C for 12 h in

polyphosphoric acid and phosphorous oxy chloride (catalytic). In this method, the yield (89-

9

2%) was improved much better than the reported procedure using Dowtherm and subsequent

refluxing in phosphorous oxy chloride yielded the 4-chloroquinoline 3-carboxylate derivatives

11a-d). The reaction sequences are shown in Scheme 2.

(

Mycobacterium smegmatis (Msm) DNA GyrB was used for performing the gyrase ATPase

assay. The surrogate Msm GyrB protein was used as Mtb was found to be slow growing

organism[9]. Moreover, the sequence similarity between the GyrB protein domains of both the

organisms was found to be 87%, with most of the catalytic site being conserved which proves a

higher degree of homology in their ATP binding pocket[16]. The Msm GyrB gene was cloned

into a prokaryotic expression vector pQE2 and was expressed in BL21 (DE3) pLysS competent

cells. The expressed protein was further induced with IPTG (Isopropyl-β-D-

thiogalactopyranoside) for increased production of the desired protein and the desired GyrB

protein was subsequently purified by Ni-NTA column. The purified protein was confirmed by

running 10% SDS- PAGE (sodium dodecyl sulphate – polyacrylamide gel electrophoresis). Msm

DNA GyrB enzyme follows greater than first-order kinetics [17]. However, at a constant enzyme

concentration of 15 ꢀM a hyperbolic dependence of rate on substrate (ATP) concentration was

observed as the GyrB ATPase activity of Msm does not follow Michaelis–Menten kinetics. The

app

apparent K_mand V_m_a_xdetermined experimentally were 300 ꢀM and 2.1 ꢀmol/s respectively

as illustrated in figure 3.

Ideally, novobiocin was used as a standard inhibitor in these assays. Furthermore, according to

scientists from AstraZeneca, a tight correlation of <3–5 fold variation in IC₅₀values were

acceptable between the ATPase activity of Msm and the supercoiling activity of Mtb [9]; this

4

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had given us enough confidence to use Msm as a surrogate enzyme for DNA GyrB ATPase

activity. Moreover, to eliminate the possibilities of aggregation of the protein and also auto-

fluorescence artifacts, non-specific inhibition detergents were added in all our biological

experiments [18]. The series of 46 compounds synthesized were biologically evaluated for their

in vitro Msm GyrB ATPase assay using the protocol described in the experimental section. The

compounds showed an IC50 values ranging between 0.86 to 72.48 µM and are given in table 1.

Compound 53 was found to be most activewith an IC of 0.86 ± 0.42 µM against Msm GyrB

5

0

and its dose response curve was plotted using GraphPad Prism software (GraphPad Software

Inc., La Jolla, CA) by taking log (inhibitor concentration) on the X-axis and its response

(%inhibition) on the Y-axis as shown in figure 4, while the standard novobiocin showed an IC₅₀

of 180 ± 3.9 nM. Further, compound 53 selectively inhibited mycobacterial DNA GyrB and was

found inactive against other bacterial GyrB from Staphylococcus aureus and Escherichia coli

(

Inspiralis) even at 100 µM concentrations confirming its specificity towards mycobacterial

species.

Subsequently, the compounds inhibiting the ATPase activity of GyrB protein should also inhibit

the supercoiling activity of the gyrase holoenzyme as the GyrA and GyrB domains constitute the

DNA gyrase. The supercoiling assay was performed in our laboratory using the DNA

supercoiling assay kit (Inspiralis Pvt. limited, Norwich) as per the protocol described in the

experimental section given in supporting information. All the reactions were carried on Mtb

DNA gyrase enzyme using compounds dose dependently starting from 100 µM, 50 µM, 25 µM,

1

2.5 µM, 6.25 µM, 3.125 µM, 1.56 µM and 0.75 µM. The most active compound 53 showed an

IC of 0.63±0.19 µM as illustrated in table 1 and the agarose gel picture depicting the inhibitory

5

0

activity of compound 53 over DNA at various concentrations is shown in figure 5. Compounds

3, 28, 42 and 57 also exhibited sub-micromolar GyrB inhibitory concentrations highlighting this

2

series of 46 compounds as efficient inhibitors of DNA GyrB protein. To keep a check on the

assay conditions, novobiocin was considered as a standard compound in this assay too. IC50 for

all the compounds was calculated based on relative quantification using Image lab software, Bio-

Rad, compared to the control in the assay. These compounds were also screened for in vitro

antimycobacterial activity against Mtb H37Rv strain using microplate alamar blue assay

(MABA) [19]. The MICs for the compounds are given in table 1.

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The synthesized 46 compounds were subjected to molecular docking studies in order to analyze

their binding interactions and active site orientation so as to build a structure activity relationship

(SAR) for the series with respect to their GyrB activity. Among the series, compound 53 was

found to be exhibiting the most promising GyrB inhibitory activity with an IC50 of 0.86 µM. This

compound also correlated well in terms of its supercoiling activity with 0.63 µM IC and Mtb

5

0

MIC of 3.3 µM. The compound’s activity was well supported by its binding mode at the ATPase

site of Msm GyrB where it was found to be involved in two hydrogen bond interactions with

Arg82 and a cation-π interaction with Arg141 as shown in figure 6. The compound was oriented

such that the quinoline moiety was directed towards the solvent accessible surface area with the

carboxylic group over it involving in the hydrogen bonding. The N-ethyl piperazine was set in to

the GyrB hydrophobic pocket where it was found to be stabilized by hydrophobic interactions

with residues Val49, Ile84, Pro85, Val99, Met100, Val128, Leu135 and Ile171.

The compounds in the present series can be divided into three groups for convenience according

to their R

1

substitutions – (i) compounds 12-27, (ii) compounds 28-43 and (iii) compounds 44-

5

7. Of the compounds 12-27 with ethoxy substitution at R position, compound 23 was found to

1

be the top active one with GyrB inhibitory activity of 0.97 µM and Mtb MIC of 2.94 µM. The

interaction profile of the compound revealed the presence of polar contact between Arg82 and

nitrogen of piperazine ring as shown in figure 7(a). Unlike compound 53, compound 23 was

oriented such that the quinoline group was embedded into the hydrophobic pocket and involved

in non-polar interactions with Ile84, Pro85, Val99, Met100, Val128, and Leu135. The trifluoro

methyl group over quinoline added up the hydrophobicity of the moiety making it highly stable

at the pocket. Compounds 12-19 with morpholine ring in place of piperazine were found to be

inactive with an exception of compound 19 which was found with IC of 6.62 µM. This loss in

5

0

activity might be attributed for the absence of N-ethyl substitution at Y-position for hydrogen

bonding. The importance of -NC H can be understood from the interaction profiles of

2

5

compounds 23 and 15 given in figure S1 in supporting informtion. The compounds 20, 21 and

2 were found to be lesser active when compared to compound 23 which may be explained by

2

the presence of trifluoro methyl group at R substitution. It was observed that with an increase in

hydrophobic component at R position, as seen in compounds 20, 21, 22 and 23, the activity was

found to be improved starting from >50 µM to 0.97 µM. Replacement of oxygen at X-position in

compounds 20, 21 and 22 by nitrogen resulted in compounds 24, 25 and 26 with improved GyrB

6

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activity which can be explained by the participation of nitrogen in hydrogen bonding with

residues Glu48 or Glu56 which enhanced the inhibitory activity by ~2 folds.

Considering compounds 28-43 with hydrazine at R position, compound 28 was found to be the

1

best active one with GyrB IC50 of 0.97 µM and corresponding Mtb MIC of 2.4 µM. The

compound was found interacting by hydrogen bonding with residues Asn52, Val77 and Asp79 as

shown in figure 7(b). Interesting point is that the hydrazine group was found to be involved in

two hydrogen bonds of three orienting the compound in such a way that quinoline bound in to

the hydrophobic pocket. The compound was also involved in non-polar interactions with Ile84,

Pro85, Val99, Met100 and Val128. The difference in GyrB activity of compounds 12 and 28 by

almost 23 folds can be described by the presence of hydrazine in compound 28, as shown in

figure S2 in supporting information. On substitution of methoxy (compound 29), fluorine

(compound 30) and trifluoro methyl (compound 31) at R position of compound 28, activity was

found to be continuously decreased. This may be attributed to the steric bulk of the substitutions

which made the compound orient such that hydrazine was moved away from the interacting

residues thereby making them inactive. The substitution of nitrogen in place of oxygen at X-

position resulted in complete loss of GyrB inhibitory activity as observed in compounds 32-35.

The GyrB activity was found to be improved up on the presence of N-ethyl substitution at Y-

position making the compounds active by several folds when compared to their morpholine

counterparts. This might be reasoned for the addition of N-ethyl moiety over the compounds in

which it was found participating in strong hydrophobic interactions. The hydrazine group facing

the solvent was found to be involved in polar contacts with residues such as Asp55 (as in

compounds 36, 37, 38, 41), Asn52 (compounds 38, 40, 41 and 42).

The final set of compounds includes 44-57 with hydroxyl group with an IC of 7.89 µM. The

5

0

GyrB inhibitory activity and DNA supercoiling activity was found to be improved among the

compounds 52-57 with N-ethyl substitution. The difference in the activity of the compounds set

4

4-51 and 52-57 can be probably explained in terms of their hydroxyl and N-ethyl substitution.

In this set of 44-57 compounds, the presence of hydroxyl group at the R position reversed the

1

pose of the compounds making them orient such that the hydroxyl group facing the solvent due

to its high polar nature. In compounds 44-51, the morpholine group was oriented in the pocket

which was found with less non-polar interactions. Conversely, in compounds 52-57 the presence

of N-ethyl piperazine ring in the active site pocket was found to be highly stabilized and

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interacting with residues Val49, Ile84, Pro85, Val99, Met100, Val128 and Leu135. On the

whole, quinoline-3-carboxylic acid (-OH at R ) and quinoline-3-carbohydrazide (-NHNH at R )

1

2

1

derivatives with ethyl piperazine (-NC H at Y) moiety were found to be desirable substitutions

2

5

resulting in compounds with efficient GyrB inhibitory potential.

Additionally, the eukaryotic cell safety profile of the synthesized compounds was analyzed by

testing against mouse leukemic monocyte macrophage cell line RAW 264.7 cells at 50 µM

concentration using MTT assay[20]. All the tested compounds demonstrated a good safety

profile with very low inhibitory potential against the eukaryotic cell line. The assays were

performed in triplicates, twice to record the precise inhibitions [21].The inhibitions were within a

range of 0.07 to 44.56% at 50 µM inhibitor concentration. Further, the most active compound 53

showed 0.66% inhibition at 50 µM concentration illustrating its safety profile in the eukaryotes.

The cytotoxicity profiles of all the synthesized compounds are given in table 1.

Furthermore, to re-ascertain the binding affinity of the DNA GyrB protein with the ligand and to

estimate the thermal stabilityof the protein-ligand complex, differential scanning fluorimetry

experiments were performed. The DNA GyrB DSF experiments were performed as described in

the protocol in experimental section. Initially, purified concentrated protein of 2 mg/mL

o

concentration was subjected to sequential (0.6 C/min) increasing temperatures starting from an

o

optimized 25 C.

o

Subsequently, the native protein and the protein-ligand complex were heated from 25 C to 95 C

o

in steps of 0.1 C increment in the presence of 50 X SYPRO orange dye. As the dye has more

affinity towards the hydrophobic residues, the fluorescence increases with the increase in the

exposure and binding of the dye to the exposed hydrophobic residues of the protein[22]. Thus, it

can be concluded that the affinity of the protein-ligand complex is proportional to temperature

shift between the melting temperature (T ) of the native protein and the protein-ligand complex.

m

In short, greater the shift in the temperature towards right, higher is the stability of the complex.

Major shift was observed by taking the GyrB protein in complex with compound 53 as the shift

o

in T was found to be 3.3 C. The native protein T was observed at 45 C (green) whereas the

m

o

protein-ligand complex (red) showed T

m

of 48.3 C as depicted in figure 8.

8

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3

. Conclusions

Concluding the study, in the present work we report a new class of N-phenyl quinoline amines as

mycobacterial GyrB inhibitors with three compounds exhibiting activity below 1 µM. The in

vitro GyrB activity of the compounds was well correlated to their structures with respect to their

in silico binding pattern thereby establishing a valid structure activity relationship (SAR) for the

compounds. In the present scenario where there is an immediate necessity of newer potential

antitubercular drugs, these inhibitors, offering synthetic feasibility and least cytotoxicity, can be

handful for futher optimization to a drug candidate.

4

. Experimental Section

.1. General: All commercially available chemicals and solvents were used without further

purification. Melting points of the synthesized compounds were determined by Buchi B-540

open capillary instrument and wereuncorrected. The homogeneity of the compounds was

monitored by TLC (Thin layer chromatography) on silica gel 40 F254 coated on aluminium

1

13

4

plates, visualized by UV or iodine chamber and KMnO treatment. All H and C NMR spectra

were recorded on a Bruker AM-300 (300.12 MHz, 75.12 MHz) NMR spectrometer and

BrukerBioSpin Corp, Germany respectively. Molecular weights of the synthesized compounds

were checked by SHIMADZU LCMS-2020 series in ESI mode. Chemical shifts are reported in

ppm (δ) with reference to the internal standard TMS. The signals were designated as follows: s,

singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet. Elemental analyses were

carried out on ElementarVario MICRO CUBE CHN Analyser.

4

.1.1. Synthesis of tert-butyl (4-aminophenyl)carbamate (1): To a stirred solution of p-

phenylenediamine (10.8 g, 100 mmol ) and triethylamine (Et N) (21.8 g, 216 mmol) in N,N-

3

dimethyl formamide (DMF) (150 mL) Boc-anhydride (28.3 g, 130 mmol) was added slowly at 0

ºC allowed to stir for 3 h at room temperature (rt). The reaction was monitored by TLC, after

completion DMF was removed by high vacuum pump and diluted with ethylacetate (300 mL)

and the organic layer was washed with brine (150 mL). The organic layer was evaporated under

reduce pressure to get crude brown solid which was further purified by flash column

chromatography by 230-400 mesh silica gel using ethylacetate:hexane as eluent to afford brown

+

1

solid (15 g, 75%); mp: 112-114°C. ESI-MS was found at m/z 209.43 [M+H] . H NMR (300

MHz, CDCl , TMS):δ = 9.68 (s, 1H), 7.56 (d, J = 7.6 Hz, 2H), 6.79 (d, J = 7.8 Hz, 2H),5.89 (s,

3

2

H) 1.36 (s, 9H).

9

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4

.1.2. General synthetic procedure for the preparation of intermediates 2 and 6; General

procedure A: To a stirred solution of compound 1 or 5 (65.0 mmol), poatassium carbonate

K CO ) (97.5 mmol) and 1,3-dibromopropane (78.0 mmol) in DMF (100 ml) was heated to 110

(

2

3

ºC for 3h. After completion of the reaction (monitored by TLC), DMF was removed by under

vacuo. To the crude reaction mixture crushed ice was added and diluted with ethylacetate (150

mL). The organic layer was washed with brine (100 mL), separated and dried over anhydrous

sodium sulphate (anhy. Na SO ) and evaporated under reduced pressure to get crude product

2

4

which was further purified by flash column chromatography by 230-400 silica gel using

ethylacetate:hexane as eluent to afford corresponding bromo derivative as a solid.

4

.1.2.1. Tert-butyl (4-((3-bromopropyl) amino) phenyl)carbamate (2): The compound was

prepared according to the general procedure A using compound 1 (13.53g, 65mmol), 1,3-

dibromopropane (15.74 g, 78 mmol) as an off-white solid (12.8g, 60%);m.p: 113-115°C; ESI-

+

1

MS was found at m/z 330.43 [M+H] . H NMR (300 MHz, CDCl , TMS):δ = 9.98 (s, 1H), 7.53

3

(

d, J = 7.4 Hz, 2H), 6.72 (d, J = 8.1Hz, 2H), 5.68 (s, 1H), 3.56 (t, J = 7.0 Hz, 2H), 3.31 (t, J = 6.9

Hz, 2H), 2.01 (m, 2H), 1.39 (s, 9H).

.1.2.2. Tert-butyl (4-(3-bromopropoxy)phenyl)carbamate(6): The compound was prepared

4

according to the general procedure A using compound 5 (13.58 g, 65 mmol), 1,3-

dibromopropane (15.76 g, 78 mmol) as pale brown solid (10.4 g, 70%);m.p: 100-102°C; ESI-MS

+

1

was found at m/z 331.36 [M+H] . H NMR (300 MHz, CDCl

3

, TMS):δ = 9.76 (s, 1H), 6.78(d, J

=

7.8 Hz, 2H), 6.68 (d, J = 7.6 Hz, 2H),4.08 (t, J = 6.8 Hz, 2H), 3.58 (t, J = 7.0 Hz, 2H), 1.98 (m,

2

H), 1.29 (s, 9H).

4

.1.3. General synthetic procedure for the preparation of intermediate 3 and 7; General

procedure B: To a stirred solution of intermediate 3 (20 mmol) or 6 (20 mmol,), potassium

carbonate (30 mmol) (K CO ) and 1.1 equivalent of morpholine/1-ethylpiperazine in DMF was

2

3

heated to 110 ºC for 3 h. TLC analysis indicated that the reaction was complete. DMF was

removed by high vacuum pump. To the crude reaction mixture crushed ice was added and

diluted with ethylacetate (150 mL). The organic layer was washed with brine (100 mL),

separated, dried over anhydrous Na SO and evaporated to get crude product which was further

2

4

purified by flash column chromatography by 230-400 silica gel using ethyl acetate:hexane as

eluent to afford desired compounds (3a-b, 7a-b), followed by Boc de-protection of 3a-3b

1

&intermediateof step f with trifluoro acetic acid (TFA) 10 equiv (~10 mL) in 15 mL of DCM at

1

0

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0

ºC to room temperature for 4 h gives the corresponding amine (4a-b& 7a-7b ) derivatives in

good yields.

.1.3.1. Tert-butyl (4-((3-morpholinopropyl)amino)phenyl)carbamate (3a): The compound was

prepared according to the general procedure Busing compound 2 (6.58 g, 20 mmol), morpholine

4

(1.9 g, 22 mmol) as an off-white solid (4.75 g, 71%);m.p: 66-68 °C; ESI-MS was found at m/z

+

1

3

6

4

3

36.48 [M+H] . H NMR (300 MHz, CDCl , TMS):δ = 9.98 (s, 1H), 7.57 (d, J = 7.1 Hz, 2H),

.73 (d, J = 7.6 Hz, 2H), 5.49 (s, 1H), 3.40 (t,J = 6.8 Hz,4H), 2.41 (m, 2H),2.36 (t, J = 6.4 Hz,

H), 2.34 (s, 6H), 1.67 (m, 2H), 1.32 (s, 9H).

.1.3.2. Tert-butyl (4-((3-(4-ethylpiperazin-1-yl)propyl)amino)phenyl) carbamate (3b): The

compound was prepared according to the general procedure B using compound 2 (6.58 g, 20

mmol), 1-ethylpiperazine (2.5 g, 22 mmol) as a pale brown solid (5g, 70%);mp: 97-98°C; ESI-

+

1

MS was found at m/z 363.56 [M+H] . H NMR (300 MHz, CDCl , TMS):δ = 10.02 (s, 1H), 7.60

3

(d, J = 7.3 Hz, 2H), 6.68 (d, J = 7.7 Hz, 2H), 5.38 (s, 1H), 3.38 (t, J = 7.0 Hz, 2H), 2.40 (m, 2H),

2

.37 (t , J = 6.8 Hz, 4H), 2.31 (s, 6H), 1.69 (m, 2H), 1.21 (s, 9H), 1.02 (t, J = 6.8 Hz, 3H).

.1.3.3. N1-(3-Morpholinopropyl)benzene-1,4-diamine (4a): The compound was prepared

4

according to the general procedure B using compound 3a (4.0 g, 12 mmol), TFA (13.64 g, 119

+

1

mmol) as pale brown liquid (2.52 g, 90%); ESI-MS was found at m/z 236.50 [M+H] . H NMR

300 MHz, CDCl , TMS):δ = 6.74 (d, J = 7.6 Hz, 2H), 6.56 (d, J = 7.2 Hz, 2H), 6.02-5.49 (bs,

(

3

H), 3.58 (t, J = 7.0 Hz, 4H), 3.31 (t, J = 6.8 Hz, 2H), 2.29 (s, 6H), 1.62 (m, 2H).

4

.1.3.4. N1-(3-(4-Ethylpiperazin-1-yl)propyl)benzene-1,4-diamine (4b): The compound was

prepared according to the general procedure B using compound 3b (4.35 g, 12 mmol), TFA

+

1

(

13.68 g, 119 mmol) as pale brown liquid; Yield ; ESI-MS was found at m/z 263.55 [M+H] . H

NMR (300 MHz, CDCl , TMS):δ = 6.57 (d, J = 7.7 Hz, 2H), 6.36 (d, J = 7.4 Hz, 2H), 5.56 (bs,

3

1

4

H), 3.35 (t, J = 7.0 Hz, 2H), 2.42 (m, 2H), 2.39 (t, J = 6.8 Hz, 4H), 2.28 (s, 6H), 1.26 (m, 2H),

.04 (t, J = 7.0 Hz, 3H).

.1.3.5. 4-(3-Morpholinopropoxy)aniline (7a): The compound was prepared according to the

general procedure B using compound 6 (3.22 g, 14 mmol), morpholine (1.34 g, 15.4 mmol)

+

1

aspale brown liquid (2.8 g, 85%); ESI-MS was found at m/z 237.20 [M+H] . H NMR (300 MHz,

CDCl , TMS):δ = 6.76 (d, J = 7.1 Hz, 2H), 6.62 (d, J = 7.5 Hz, 2H), 6.02 (bs, 2H), 4.03 (t, J=

.8 Hz, 2H), 3.64 (t, J = 7.0 Hz, 4H), 2.30 (s, 6H), 1.71 (m, 2H).

3

6

1

ACCEPTED MANUSCRIPT

4

.1.3.6. 4-(3-(4-Ethylpiperazin-1-yl)propoxy)aniline (7b): The compound was prepared

according to the general procedure B using compound 6 (3.22 g, 14 mmol), 1-ethylpiperazine

(1.75 g, 15.4 mmol) as pale brown liquid (3.2g, 87%); ESI-MS was found at m/z 264.73

+

1

[

M+H] . H NMR (300 MHz, CDCl

3

, TMS):δ = 6.74 (d, J = 7.6 Hz, 2H), 6.65 (d, J = 7.4 Hz,

2

4

H), 6.03 (bs, 2H), 4.03 (t, J = 7.0 Hz, 2H), 2.41 (q,J = 7.2 Hz, 2H), 2.34 (t, J = 6.8 Hz, 4H),

.29 (s, 6H), 1.77 (m, 2H), 1.05 (t,J = 7.0 Hz,3H).

.1.4. General procedure for synthesis of diethyl 2-((phenyl/4-methoxy/4-fluoro/4-

trifluoromethylphenylamino)methylene)malonate; General procedure C (9a-d): To a stirred

solution of substituted aniline 8 (50 mmol), diethyl 2-(ethoxymethylene)malonate(55 mmol) in

ethanol (EtOH) (250 mL) was refluxed for 3 h. TLC analysis indicated that the reaction was

complete. After cooling the reaction mixture solid was separated. The separated solid was

filtered and washed with 3% ethyl acetate:hexane for further purification to afford desired

compound as an off-white solid in good yield (89-92%).

4

.1.4.1. Diethyl 2-((phenylamino)methylene)malonate (9a): The compound was prepared

according to the general procedure C using compound 8a (4.65 g, 50 mmol), diethyl 2-

(ethoxymethylene)malonate (11.87 g, 55 mmol) as an off-white solid (11.83 g, 90%);m.p: 94-96

+

1

°

C; ESI-MS was found at m/z 264.23 [M+H] . H NMR (300 MHz, CDCl , TMS):δ = 8.46 (s,

3

1

H), 7.32-6.84 (m, 5H), 6.62 (s, 1H), 4.35 (q,J = 7.0 Hz, 4H), 1.32 (t,J = 6.8 Hz, 6H).

4

.1.4.2. Diethyl 2-(((4-methoxyphenyl)amino)methylene)malonate (9b): The compound was

prepared according to the general procedure C using compound 8b (6.15 g, 50 mmol), diethyl 2-

ethoxymethylene)malonate (11.86 g, 55 mmol) as an off-white solid (13.1 g, 89%);m.p: 40-42°

(

+

1

C; ESI-MS was found at m/z 294.42 [M+H] . H NMR (300 MHz, CDCl , TMS):δ = 8.43 (s,

3

1

3

4

H), 7.46 (d, J = 6.3Hz, 2H), 6.81 (d,J= 6.5Hz, 2H), 6.68 (s, 1H),4.37 (q,J = 7.0 Hz, 4H), 3.81 (s,

H), 1.36 (t,J = 6.8 Hz, 6H).

.1.4.3. Diethyl 2-(((4-fluorophenyl)amino)methylene)malonate (9c): The compound was

prepared according to the general procedure C using compound 8c (5.55 g, 50 mmol), diethyl 2-

(ethoxymethylene)malonate (11.87 g, 55 mmol) as an off white solid (12.68 g, 91%) ;m.p: 74-

+

1

7

1

6°C; ESI-MS was found at m/z 282.31 [M+H] . H NMR (300 MHz, CDCl , TMS):δ = 8.47 (s,

3

H), 7.11 (d, J = 7.2 Hz, 2H), 6.43 (d, J = 7.1Hz, 2H), 6.72 (s, 1H), 4.36 (q,J = 7.0 Hz,4H), 1.35

(t,J = 7.0 Hz, 6H).

1

2

ACCEPTED MANUSCRIPT

4

.1.4.4. Diethyl 2-(((4-(trifluoromethyl)phenyl)amino)methylene)malonate (9d): The

compound was prepared according to the general procedure C using compound 8d (8.0 g, 50

mmol), diethyl 2-(ethoxymethylene)malonate (11.87 g, 55 mmol) as an off white solid (14.8 g,

+

1

9

1%);m.p: 95-97°C; ESI-MS was found at m/z 332.32 [M+H] . H NMR (300 MHz, CDCl

3

,

TMS):δ = 8.45 (s, 1H), 7.46 (d, J = 6.8Hz, 2H), 6.46 (d, J = 6.7Hz, 2H), 6.70 (s, 1H), 4.37 (q,J =

7

4

.0 Hz, 4H), 1.33 (t,J = 6.8 Hz, 6H).

.1.5. General procedure for synthesis of ethyl 6-unsubtituted/methoxy/fluoro/trifluoromethyl-

-hydroxyquinoline-3-carboxylate (10a-d): Polyphosphoric acid (PPA) (2 equiv by weight) was

added to corresponding intermediate 1 (40 mmol) to thisphosphorousoxychloride (POCl

3

)

(10mmol) was added. The reaction mixture was heated to 75 ºC for 8 h, monitored by TLC, after

completion of the reaction. The reaction mixture was quenched slowly with 10% sodium

H

hydroxide solution by keeping it in an ice bath and the P was adjusted to a pH~7, the solid

separated was filtered, dried and washed with diethyl ether (3*20 mL) to afford desired

compound in good yield.

4

.1.5.1. Ethyl 4-hydroxyquinoline-3-carboxylate (10a): Pale yellow solid (5.64 g, 65%);m.p:

+

1

2

76-278 °C; ESI-MS was found at m/z 218.32 [M+H] . H NMR (300 MHz, DMSO-d6, TMS):δ

=

10.72 (brs, 1H), 8.91 (s, 1H), 8.46 -7.78 (m, 4H), 4.29 (q,J = 7.1 Hz, 2H), 1.29 (t, J = 7.1Hz,

3

4

6

H).

.1.5.2. Ethyl 4-hydroxy-6-methoxyquinoline-3-carboxylate (10b): yellow solid (6.51 g,

+

1

6%);m.p: 274-276°C; ESI-MS was found at m/z 248.32 [M+H] . H NMR (300 MHz, DMSO-

d6, TMS):δ = 10.67 (brs, 1H), 8.82 (s, 1H), 8.14 (d, J = 9.0Hz, 1H), 7.73 (d, J = 7.2 Hz, 1H),

7

4

7

.14 (s, 1H), 4.27 (q, J = 7.2 Hz, 2H), 1.29 (t,J = 7.0 Hz, 3H).

.1.5.3. Ethyl 6-fluoro-4-hydroxyquinoline-3-carboxylate (10c): Pale yellow solid (6.58 g,

+

1

0%);m.p: 288-290°C; ESI-MS was found at m/z 236.22 [M+H] . H NMR (300 MHz, DMSO-

d6, TMS):δ = 10.45 (brs, 1H), 8.86 (s, 1H), 8.16 (d, J = 7.7 Hz, 1H), 7.81 (s, 1H), 7.34 (d, J =

7

.4 Hz, 1H), 4.23 (q, J = 6.8 Hz, 2H), 1.29 (t, J = 6.8 Hz, 3H).

4

.1.5.4. Ethyl 4-hydroxy-6-(trifluoromethyl)quinoline-3-carboxylate (10d): Pale brown solid

+

1

(

7.75 g, 68%); m.p: 290-292° C; ESI-MS was found at m/z 286.34 [M+H] . H NMR (300 MHz,

DMSO-d6, TMS):δ = 10.52 (brs, 1H), 8.91 (s, 1H), 8.61 (d, J = 7.8 Hz, 1H), 8.37 (s, 1H), 8.11

d, J = 7.6 Hz, 1H), 4.27 (q, J = 7.0 Hz, 2H), 1.28 (t, J = 6.8 Hz, 3H).

(

1

3

ACCEPTED MANUSCRIPT

4

.1.6. General procedure for synthesis of ethyl 6-unsubtituted/methoxy/fluoro/trifluoromethyl-

-chloroquinoline-3-carboxylate (11a-d): To the corresponding intermediate 10 (25 mmol),

POCl (125 mmol) was added slowly and refluxed for 3 h at 105 ºC. TLC analysis indicated that

3

the reaction was completed. Excess POCl was removed under reduced pressure and the crude

reaction mass was quenched with crushed ice then neutralized with saturated

sodiumbicarbonatesolution (100 mL) and extracted with ethylacetate (3*100 mL). The organic

layer was dried over anhy.Na SO , filtered and evaporated under reduced pressure to get

2

4

corresponding desired chloro intermediate.

4

.1.6.1. Ethyl 4-chloroquinoline-3-carboxylate (11a): Yellow solid (4.74 g, 81%);m.p: 43-45

+

1

°

C; ESI-MS was found at m/z 236.67 [M+H] . H NMR (300 MHz, CDCl , TMS):δ = 8.92 (s,

3

1

4

8

H), 8.46-7.78 (m, 4H), 4.28 (q, J = 6.8 Hz, 2H), 1.27 (t, J = 7.3Hz, 3H).

.1.6.2. Ethyl 4-chloro-6-methoxyquinoline-3-carboxylate (11b): Pale yellow solid (5.44 g,

+

1

2%); m.p: 79-81° C; ESI-MS was found at m/z 266.68 [M+H] . H NMR (300 MHz, CDCl ,

3

TMS):δ = 8.84 (s, 1H), 8.13 (d, J = 9.1Hz, 1H), 7.72 (d, J = 7.2 Hz, 1H), 7.15 (s, 1H), 4.26 (q, J

7.0 Hz, 2H), 1.28 (t,J = 6.8 Hz, 3H).

.1.6.3. Ethyl 4-chloro-6-fluoroquinoline-3-carboxylate (11c): Yellow solid (5.64 g, 89%);m.p:

=

4

+

1

6

2-64°C; ESI-MS was found at m/z 254.60 [M+H] . H NMR (300 MHz, CDCl , TMS):δ = 8.87

3

(s, 1H), 8.17 (d, J = 7.5 Hz, 1H), 7.83 (s, 1H), 7.36 (d, J = 7.6 Hz, 1H), 4.22 (q, J = 7.0 Hz, 2H),

1

.29 (t, J = 7.0 Hz, 3H).

4

.1.6.4. Ethyl 4-chloro-6-(trifluoromethyl)quinoline-3-carboxylate (11d): Pale yellow solid (6.5

+

1

g, 86%);m.p: 90-92 °C; ESI-MS was found at m/z 304.64 [M+H] . H NMR (300 MHz, CDCl

TMS):δ = 8.95 (s, 1H), 8.62 (d, J = 7.7 Hz, 1H), 8.38 (s, 1H), 8.13 (d, J = 7.8 Hz, 1H), 4.28 (q, J

7.1 Hz, 2H), 1.29 (t, J = 7.0 Hz, 3H).

.1.7. General procedure for the preparation of titled phenyl-amino-quinoline ethyl ester

3

,

=

4

derivatives (12-27): The alkylation of amine intermediates (4a-b &7a-b) was carried in a

Biotage microwave vial. Compound 11 (4.5 mmol) (1 equiv), 4 or 7 (1.2 equiv) and para-

tolulenesufonic acid (PTSA) 5-6 mg catalytic amount in 15 ml methanol and then subjected to

microwave irradiation at 120 ºC for 45 min. The reaction mixture was concentrated under vacuo.

The residue was dissolved in water to this saturated NaHCO

alkaline, and extracted thrice with 5% MeOH:DCM solution. Combined organic layers were

dried over anhydrous Na SO and evaporated under vacuo, purification of the resultant product

3

solution was added to make

2

4

1

4

ACCEPTED MANUSCRIPT

in silica gel by flash column chromatography using 5-10 % MeOH:DCM as eluent to get final

ethyl ester derivatives.

4

.1.7.1. Ethyl 4-((4-(3-morpholinopropoxy)phenyl)amino) quinoline-3-carboxylate (12): Pale

1

yellow gammy (1.10 g, 60%); H NMR (300 MHz, DMSO-d

6

, TMS):δ = 10.21 (s, 1H), 9.31

(s,1H), 8.36 (d, J = 8.8 Hz, 1H), 7.99 (m, 3H),7.52 (d, J = 8.2 Hz, 2H), 6.97 (d,J = 8.4 Hz, 2H),

4

2

1

6

4

6

4

.32 (q, J = 7.0 Hz, 2H), 4.02 (t, J = 6.8 Hz, 2H), 3.67 (t, J = 7.0 Hz, 4H), 2.37 (s, 6H), 1.79 (m,

1

3

H), 1.28 (t, J = 7.0 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 168.5, 153.0, 152.2,

6

49.9, 141.3, 132.8(2C), 129.7, 127.4, 126.0, 121.7(2C), 120.9, 115.8(2C), 113.6, 73.4,

29 3 4

7.1(2C), 63.4(2C), 61.6, 59.1, 28.3, 14.6. ESI-MS m/z (Calcd. for C25H N O : 435.22); Found:

+

36.29 (M+H) . Anal calcd.for C H N O : C, 68.95; H, 6.71; N, 9.65; Found: C, 68.91; H,

2

5

29

3

4

.67; N, 9.62.

.1.7.2. Ethyl 6-methoxy-4-((4-(3-morpholinopropoxy)phenyl)amino) quinoline-3-carboxylate

1

(

13): Brown solid (1.2 g, 58%); m.p: 170-172°C; H NMR (300 MHz, DMSO-d , TMS): δ =

6

1

4

6

1

6

0.01 (s, 1H), 8.91 (s,1H), 8.31 (d, J = 8.6 Hz, 1H), 8.01-7.53 (m, 4H), 6.98 (d, J = 8.2 Hz, 2H),

.33 (q, J = 7.0 Hz, 2H), 4.03 (t, J = 6.8 Hz, 2H), 3.81 (s, 3H), 3.65 (t, J = 7.1 Hz, 4H), 2.41 (s,

1

3

6

H), 1.80 (m, 2H), 1.29 (t, J = 7.0 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 168.5,

57.3, 152.0, 150.2(2C), 136.9, 132.7, 131.4, 124.8, 122.3(3C), 115.9(2C), 114.2, 103.7, 73.5,

7.2(2C), 63.6(2C), 61.3, 59.0, 56.4, 28.5, 14.8. ESI-MS m/z (Calcd. for C H N O : 465.23);

26

31

3

5

-

Found: 464.05 (M-H) . Anal calcd.for C26

H, 6.74; N, 9.01.

31 3 5

H N O : C, 67.08; H, 6.71; N, 9.03; Found: C, 67.11;

4

.1.7.3. Ethyl 6-fluoro-4-((4-(3-morpholinopropoxy)phenyl)amino) quinoline-3-carboxylate

1

(

14): Off-white solid (1.16 g, 57%);m.p: 93-95°C; H NMR (300 MHz, DMSO-d , TMS):δ =

6

9

4

2

1

6

.89 (s, 1H), 9.01 (s,1H), 8.32 (d, J = 8.8 Hz, 1H), 7.96-7.49 (m, 4H), 6.98 (d, J = 8.1 Hz, 2H),

.33 (q, J = 7.1 Hz, 2H), 4.03 (t, J = 6.8 Hz, 2H), 3.59 (t, J = 6.9 Hz, 4H), 2.36 (s, 6H), 1.80 (m,

1

3

H), 1.26 (t, J = 7.0 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 168.3, 160.8, 153.0,

6

51.9, 149.7, 138.3, 133.0, 130.5, 124.2, 121.8(3C), 116.0(2C), 114.7, 104.5, 73.8, 67.5(2C),

3.2(2C), 61.4, 59.0, 28.6, 14.9. ESI-MS m/z (Calcd. for C H FN O : 453.21); Found: 452.21

2

5

28

3

4

-

(

M-H) . Anal calcd.for C H FN O : C, 66.21; H, 6.22; N, 9.27; Found: C, 66.19; H, 6.25; N,

25 28 3 4

9

4

.24.

.1.7.4. Ethyl 4-((4-(3-morpholinopropoxy)phenyl)amino)-6-(trifluoromethyl) quinoline-3-

1

carboxylate (15): Pale yellow solid (1.3 g, 58%);m.p: 98-100 °C; H NMR (300 MHz, DMSO-d

6

,

1

5

ACCEPTED MANUSCRIPT

TMS):δ = 10.20 (s, 1H), 8.87 (s,1H), 8.37 (d, J = 8.1 Hz, 1H), 7.96 (m, 2H), 7.51 (d, J = 8.4 Hz,

2

H), 6.98 (d, J = 8.2 Hz, 2H), 4.30 (q, J = 7.0 Hz, 2H), 4.04 (t, J = 6.8 Hz, 2H), 3.68 (t, J = 6.8

1

3

Hz, 4H), 2.37 (s, 6H), 1.80 (m, 2H), 1.29 (t, J = 6.4 Hz, 3H). C NMR (75 MHz, DMSO-d ,

6

TMS): δ = 168.5, 155.0, 154.3, 149.9, 142.5, 132.8, 131.6, 129.1, 127.9, 125.0, 121.8(2C),

1

20.5(2C), 116.2(2C), 115.4, 73.7, 67.3(2C), 63.5(2C), 61.8, 59.2, 28.5, 14.7. ESI-MS m/z

-

(Calcd. for C26

H

28

F

3

N

3

O

4

: 503.20); Found: 502.02 (M-H) . Anal calcd.for C26

H

28

F

3

N

3

O

4

: C,

6

4

2.02; H, 5.61; N, 8.35; Found: C, 62.04; H, 5.58; N, 8.32.

.1.7.5. Ethyl 4-((4-((3-morpholinopropyl)amino)phenyl)amino)quinoline-3-carboxylate (16):

1

Pale yellow solid (1.15 g, 59%);m.p: 240-242°C; H NMR (300 MHz, DMSO-d

6

, TMS):δ = 9.99

(s, 1H), 8.89 (s,1H), 7.99-7.48 (m, 4H), 6.85 (d, J = 8.1 Hz, 2H), 6.73 (d, J = 8.2 Hz, 2H), 5.51

(brs, 1H), 4.30 (q, J = 7.4 Hz, 2H), 3.68 (t, J = 7.0 Hz, 4H),3.35 (t, J = 6.8 Hz, 2H), 2.39 (s, 6H),

1

3

1

6

.69 (m, 2H), 1.26 (t, J = 7.0 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 168.3, 153.1,

6

52.4, 140.9, 138.2, 134.9, 132.7, 129.5, 127.0, 126.2, 121.5, 119.0(2C), 118.8(2C), 113.6,

30 4 3

7.3(2C), 63.5(2C), 61.2, 52.7, 41.5, 27.8, 14.6. ESI-MS m/z (Calcd. for C25H N O : 434.23);

+

Found: 435.25 (M+H) . Anal calcd.for C H N O : C, 69.10; H, 6.96; N, 12.89; Found: C,

2

5

30

4

3

6

9.13; H, 6.94; N, 12.87.

4

.1.7.6. Ethyl 6-methoxy-4-((4-((3-morpholinopropyl)amino)phenyl)amino) quinoline-3-

1

carboxylate (17): Brown solid (1.2 g, 60%);m.p: 138-140°C; H NMR (300 MHz, DMSO-d ,

6

TMS):δ = 10.01 (s, 1H), 8.89 (s,1H), 8.30 (d, J = 8.8 Hz, 1H), 8.04-7.55 (m, 2H), 6.61 (d, J =

9

.1 Hz, 2H), 6.59 (d, J = 8.4 Hz, 2H), 5.53 (brs, 1H), 4.31 (q, J = 7.2 Hz, 2H), 3.66 (t, J = 7.0

Hz, 4H),3.82 (s, 3H),3.43 (t, J = 6.8 Hz, 2H), 2.41 (s, 6H), 1.81 (m, 2H), 1.26 (t, J = 7.4 Hz, 3H).

1

3

C NMR (75 MHz, DMSO-d , TMS): δ = 168.5, 157.3, 152.0, 150.2, 138.0, 136.9, 135.1,

6

1

4

31.3, 125.0, 122.2, 119.4(2C), 119.0(2C), 114.3, 103.9, 67.4(2C), 63.6(2C), 61.3, 56.0, 52.8,

+

1.6, 27.9, 14.5. ESI-MS m/z (Calcd. for C26

H

32

N

4

O

4

: 464.24); Found: 465.31 (M+H) . Anal

calcd.for C H N O : C, 67.22; H, 6.94; N, 12.06; Found: C, 67.24; H, 6.91; N, 12.03.

2

6

32

4

.1.7.7. Ethyl 6-fluoro-4-((4-((3-morpholinopropyl)amino)phenyl)amino) quinoline-3-

1

carboxylate (18): Pale green solid (1.2 g, 59%);m.p: 116-118°C; H NMR (300 MHz, DMSO-d ,

6

TMS):δ = 9.96 (s, 1H), 8.82 (s,1H), 7.76-6.98 (m, 3H), 6.63 (d, J = 8.4 Hz, 2H), 6.59 (d, J = 8.2

Hz, 2H), 5.52 (brs, 1H), 4.35 (q, J = 7.1 Hz, 2H), 3.57 (t, J = 6.8 Hz, 4H),3.42 (t, J = 7.1 Hz,

1

3

2

H), 2.35 (s, 6H), 1.81 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS):

6

δ = 168.3, 160.9, 152.7, 151.6, 138.3(2C), 134.9, 130.4, 124.2, 121.8, 119.3(2C), 119.0(2C),

1

6

ACCEPTED MANUSCRIPT

1

14.9, 104.7, 67.4(2C), 63.5(2C), 61.7, 52.8, 41.6, 27.4, 14.7. ESI-MS m/z (Calcd. for

-

C H FN O : 452.22); Found: 451.09 (M-H) . Anal calcd.for C H FN O : C, 66.35; H, 6.46;

25

29

4

3

25 29

4

3

N, 12.38; Found: C, 66.33; H, 6.48; N, 12.41.

4

3

.1.7.8. Ethyl 4-((4-((3-morpholinopropyl)amino)phenyl)amino)-6-(trifluoro methyl)quinoline-

1

-carboxylate (19): Brown solid (1.25 g, 56%);m.p: 101-103°C; H NMR (300 MHz, DMSO-d ,

6

TMS):δ = 10.01 (s, 1H), 8.83 (s,1H), 7.96-7.01 (m, 3H), 6.54 (d, J = 8.0 Hz, 2H), 6.49 (d, J =

8

2

.1 Hz, 2H), 5.51 (brs, 1H), 4.32 (q, J = 7.0 Hz, 2H), 3.64 (t, J = 7.2 Hz, 4H),3.52 (t, J = 6.7 Hz,

1

3

H), 2.40 (s, 6H), 1.81 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS):

6

δ = 168.5, 154.9, 154.3, 142.6, 138.1, 134.8, 131.6, 129.0, 127.8, 125.1, 120.5(2C), 119.4(2C),

1

18.9(2C), 115.2, 67.2(2C), 63.4(2C), 61.7, 52.6, 41.8, 27.5, 14.7. ESI-MS m/z (Calcd. for

+

C

26

H

29

F

3

N

4

O

3

: 502.22); Found: 503.21 (M+H) . Anal calcd.for C26

H F

29 3

N

4

O

3

: C, 62.14; H,

5

.82; N, 11.15; Found: C, 62.08; H, 5.85; N, 11.12.

4

.1.7.9. Ethyl 4-((4-(3-(4-ethylpiperazin-1-yl)propoxy)phenyl)amino)quinoline-3-carboxylate

1

(

20): Brown solid(1.2 g, 59%);m.p: 110-112°C; H NMR (300 MHz, DMSO-d

6

, TMS):δ = 9.82

(s, 1H), 8.87 (s, 1H), 8.43-7.73 (m, 4H), 7.48 (d, J = 8.2 Hz, 2H), 6.58 (d, J = 8.2 Hz, 2H), 4.23

(q, J = 7.0 Hz, 2H), 4.02 (m, 2H), 2.47-2.32 (m, 12H), 1.83 (m, 2H), 1.29 (t, J = 7.0 Hz, 3H),

1

3

1

5

.04 (t, J = 7.1 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 167.3, 152.5, 151.7,

6

50.1,139.2, 132.3(2C), 129.1, 126.5, 125.2, 122.3 (3C), 115.2(2C), 113.3, 73.5, 61.1, 58.1(3C),

34 4 3

7.5(2C), 49.8, 27.8, 14.3, 13.4. ESI-MS m/z (Calcd. for C27H N O : 462.58); Found: 463.34

+

(

M+H) . Anal calcd.for C H N O : C, 70.10; H, 7.41; N, 12.11; Found: C, 70.09; H, 7.43; N,

27

34

4

3

1

4

2.24.

.1.7.10. Ethyl4-((4-(3-(4-ethylpiperazin-1-yl)propoxy)phenyl)amino)-6-methoxyquinoline-3-

1

carboxylate (21): Brown gammy (1.32 g, 60%); H NMR (300 MHz, DMSO-d , TMS):δ = 9.92

6

(

s, 1H), 8.86 (s, 1H), 7.81-7.45 (m, 3H), 7.43 (d, J = 8.4 Hz, 2H), 6.57 (d, J = 8.1 Hz, 2H), 4.19

q, J = 7.0 Hz, 2H), 4.01 (t, J = 6.8 Hz, 2H), 3.54 (s, 3H), 2.48-2.31 (m, 12H), 1.82 (m, 2H), 1.31

1

3

q, J = 7.1 Hz, 3H), 1.03 (t, J = 6.7 Hz, 3H). C NMR (75 MHz, DMSO-d

6

, TMS): δ = 168.3,

57.5, 156.2, 150.4(2C), 136.9, 133.0, 131.3, 125.1, 122.4(3C), 115.9(2C), 114.3, 103.7, 73.8,

1.4, 59.1(3C), 58.5(2C), 56.3, 50.1, 28.4, 14.7, 13.9. ESI-MS m/z (Calcd. for C H N O :

1

6

4

2

8

36

4

+

36 4 4

92.27); Found: 493.52 (M+H) . Anal calcd.for C28H N O : C, 68.27; H, 7.37; N, 11.37;

Found: C, 68.25; H, 7.33; N, 11.35.

1

7

ACCEPTED MANUSCRIPT

4

.1.7.11. Ethyl 4-((4-(3-(4-ethylpiperazin-1-yl)propoxy)phenyl)amino)-6-fluoroquinoline-3-

1

carboxylate (22): Pale yellow solid (1.27 g, 59%);m.p: 99-101°C; H NMR (300 MHz, DMSO-

d₆, TMS):δ = 9.94 (s, 1H), 8.87 (s, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.63

(dd, J = 8.1 Hz,2.7 Hz, 1H), 6.92 (d, J = 8.6 Hz, 2H), 6.58 (d, J = 8.0 Hz, 2H), 4.28 (q, J = 6.8

Hz, 2H), 3.98 (t, J = 7.0 Hz, 2H), 2.49-2.32 (m, 12H), 1.83 (m, 2H), 1.32 (t, J = 6.8 Hz, 3H),

1

3

1

5

6

.03 (t, J = 7.2 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 168.6, 160.5, 152.7, 151.9,

50.2, 138.0, 132.8, 130.5, 124.2, 121.9(3C), 115.7(2C), 114.8, 104.5, 73.7, 61.4, 59.4(3C),

8.6(2C), 50.2, 28.2, 14.5, 13.8. ESI-MS m/z (Calcd. for C H FN O : 480.25); Found: 479.16

2

7

33

4

3

-

(

4 3

M-H) . Anal calcd.for C27H33FN O : C, 67.48; H, 6.92; N, 11.66; Found: C, 67.51; H, 6.64; N,

1

1.67.

4

.1.7.12.

Ethyl4-((4-(3-(4-ethylpiperazin-1-yl)propoxy)phenyl)amino)-6-

1

(

trifluoromethyl)quinoline-3-carboxylate (23): Pale yellow gammy (1.3 g, 54%);m.p: 240°C; H

NMR (300 MHz, DMSO-d , TMS):δ = 10.01 (s, 1H), 8.89 (s, 1H), 8.47 (d, J = 2.6 Hz, 1H), 8.29

6

(dd, J = 8.4 Hz, 2.4 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 8.6 Hz, 2H), 6.59 (d, J = 8.2

Hz, 2H), 4.16 (q, J = 7.0 Hz, 2H), 3.99 (t, J = 7.1 Hz, 2H), 2.47-2.35 (m, 12H), 1.82 (m, 2H),

1

3

1

6

.31 (t, J = 6.8 Hz, 3H), 1.03 (t, J = 7.0 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ =

68.5, 154.8(2C), 150.1, 142.6, 132.9, 131.6, 129.1, 127.8, 125.0, 121.8(2C), 120.9(2C),

15.7(3C), 73.5, 70.3, 59.0(3C), 58.5(2C), 50.2, 28.4, 14.6, 13.7. ESI-MS m/z (Calcd. for

-

C

28

H

33

F

3

N

4

O

3

: 530.25); Found: 529.11 (M-H) . Anal calcd.for C28

H

33

F

3

N

4

O

3

: C, 63.38; H, 6.27;

N, 10.56; Found: C, 63.35; H, 6.24; N, 10.58.

4

.1.7.13. Ethyl 4-((4-((3-(4-ethylpiperazin-1-yl)propyl)amino)phenyl)amino) quinoline-3-

1

carboxylate (24): Pale yellow solid (1.2 g, 57%);m.p: 90-92°C; H NMR (300 MHz, DMSO-d ,

6

TMS):δ = 9.97 (s, 1H), 8.81 (s, 1H), 8.39-7.71 (m, 4H), 6.57 (d, J = 8.0 Hz, 2H), 6.45 (d, J = 8.2

Hz, 2H), 5.54 (brs, 1H), 4.19 (q, J = 7.0 Hz, 2H), 3.41 (t, J = 6.8 Hz, 2H), 2.48-2.32 (m, 12H),

1

3

1

.82 (m, 2H), 1.36 (t, J = 7.0 Hz, 3H), 1.02 (t, J = 6.8 Hz, 3H). C NMR (75 MHz, DMSO-d ,

6

TMS): δ = 168.3, 153.5, 152.4, 141.0, 138.2, 134.9, 132.6, 129.7, 127.3, 126.1, 121.4,

1

19.3(2C), 119.0(2C), 113.8, 61.5, 58.9(2C), 38.1(2C), 52.4, 50.2, 41.7, 27.5, 14.8, 13.9. ESI-

+

MS m/z (Calcd. for C H N O : 461.28); Found: 462.41 (M+H) . Anal calcd.for C H N O :

2

7

35

5

2

27 35

5

2

C, 70.25; H, 7.64; N, 15.17; Found: C, 70.27; H, 7.61; N, 15.19.

4

.1.7.14.

Ethyl4-((4-((3-(4-ethylpiperazin-1-yl)propyl)amino)phenyl)amino)-6-

1

methoxyquinoline-3-carboxylate (25): Yellow gummy (1.2 g, 55%); H NMR (300 MHz,

1

8

ACCEPTED MANUSCRIPT

DMSO-d

6

, TMS):δ = 9.97 (s, 1H), 8.87 (s, 1H), 7.78 (d, J = 8.6 Hz, 1H), 7.65 (d, J = 2.2 Hz,

1

H), 7.39 (dd, J = 8.2 Hz, 2.4 Hz, 1H), 6.60 (d, J = 8.2 Hz, 2H), 6.54 (d, J = 8.1 Hz, 2H), 5.52

(brs, 1H), 4.21 (q, J = 7.0 Hz, 2H), 3.58 (s, 3H), 3.42 (t, J = 6.8 Hz, 2H), 2.47-2.30 (m, 12H),

1

3

1

6

.83 (m, 2H), 1.35 (t, J = 7.0 Hz, 3H), 1.02 (t, J = 6.7 Hz, 3H). C NMR (75 MHz, DMSO-d ,

TMS): δ = 168.5, 157.3, 152.0, 150.2, 138.4, 136.9, 135.1, 131.2, 124.9, 122.2, 119.3(2C),

1

18.9(2C), 114.4, 103.7, 61.5, 59.1(2C), 58.4(2C), 56.3, 52.5, 50.2, 41.7, 27.9, 14.5, 13.8. ESI-

-

MS m/z (Calcd. for C H N O : 491.29); Found: 490.18 (M-H) . Anal calcd.for C H N O : C,

2

8

37

5

3

28 37

5

3

6

8.41; H, 7.59; N, 14.25; Found: C, 68.43; H, 7.61; N, 14.27.

4

.1.7.15.

Ethyl

4-((4-((3-(4-ethylpiperazin-1-yl)propyl)amino)phenyl)amino)-6-

1

fluoroquinoline-3-carboxylate (26): Pale yellow solid (1.27 g, 59%);m.p: 106-108 °C; H NMR

(

300 MHz, DMSO-d

.2 Hz, 1H), 7.49 (dd, J = 8.6 Hz, 2.4 Hz, 1H), 6.91 (d, J = 8.1 Hz, 2H), 6.59 (d, J = 8.2 Hz, 2H),

.51(brs, 1H), 4.20 (q, J = 7.0 Hz, 2H), 3.54 (t, J = 6.7 Hz, 2H), 2.47-2.31 (m, 12H), 1.84 (m,

6

, TMS):δ = 9.97 (s, 1H), 8.88 (s, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.60 (d, J =

2

5

2

1

3

6

H), 1.33 (t, J = 7.1 Hz, 3H), 1.01 (t, J = 6.7 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ =

68.3, 160.7, 152.5, 151.8, 138.3(2C), 134.9, 130.4, 124.2, 121.9, 119.1(2C), 118.8(2C), 115.0,

04.7, 61.4, 59.2(2C), 58.6(2C), 52.4, 50.2, 41.7, 27.9, 14.5, 13.7. ESI-MS m/z (Calcd. for

+

C H FN O : 479.27); Found: 480.11 (M+H) . Anal calcd.for C H FN O : C, 67.62; H, 7.15;

27

34

5

2

27 34

5

2

N, 14.60; Found: C, 67.65; H, 7.13; N, 14.57.

4

.1.7.16.

Ethyl

4-((4-((3-(4-ethylpiperazin-1-yl)propyl)amino)phenyl)amino)-6-(trifluoro

1

methyl)quinoline-3-carboxylate (27): Yellow solid (1.28 g, 54%);m.p: 98-100°C; H NMR (300

6

MHz, DMSO-d , TMS):δ = 9.99 (s, 1H), 8.87 (s, 1H), 8.51 (d, J = 2.2 Hz, 1H), 8.29 (dd, J = 8.6

Hz, 2.4 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 6.56 (d, J = 8.4 Hz, 2H), 6.48 (d, J = 8.1 Hz, 2H), 5.51

(brs, 1H), 4.18 (q, J = 7.1 Hz, 2H), 3.51 (t, J = 6.7 Hz, 2H), 2.48-2.34 (m, 12H), 1.83 (m, 2H),

1

3

1

6

.36 (t, J = 7.0 Hz, 3H), 1.03 (t, J = 6.9 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ =

68.6, 155.0, 154.5, 142.7, 138.2, 134.9, 131.8, 129.0, 127.9, 125.2, 120.6, 119.8, 118.9(2C),

18.7(2C), 115.4, 61.6, 59.1(2C), 58.5(2C), 52.7, 50.3, 41.7, 27.5, 14.9, 13.8. ESI-MS m/z

+

(

Calcd. for C H F N O : 529.27); Found: 530.29 (M+H) . Anal calcd.for C H F N O : C,

2

8

34

3

5

2

28 34

3

5

2

6

4

3.50; H, 6.47; N, 13.22; Found: C, 63.48; H, 6.44; N, 13.26.

.1.8. General procedure for the preparation of titled phenyl-amino-quinolineacidhydrazide

derivatives (28-43): The corresponding ethyl ester derivatives (0.5 mmol) (12-27) were

converted to acid hydrazides by refluxing them with hydrazine hydrate (1.5 mmol) in ethanol for

1

9

ACCEPTED MANUSCRIPT

6

h to get corresponding acid hydrazide derivative which were further purified by

recrystallization from ethanol.

4

.1.8.1. 4-((4-(3-Morpholinopropoxy)phenyl)amino)quinoline-3-carbohydrazide (28): Yellow

1

solid (0.126 g, 60%);m.p: 289-291°C; H NMR (300 MHz, DMSO-d

6

, TMS):δ = 10.03 (s, 1H),

9

.29 (s, 1H), 8.82 (s, 1H), 8.30 (d, J = 8.8 Hz, 1H), 8.01 (m, 3H), 7.52 (d,J = 8.2 Hz, 2H), 6.95

(d, J = 8.5 Hz, 2H), 4.01 (t, J = 7.1 Hz, 2H), 3.68 (t, J = 6.8 Hz, 4H), 2.46 (s, 6H), 2.21 (brs,

1

3

2

1

5

H),1.98 (m, 2H). C NMR (75 MHz, DMSO-d , TMS): δ = 165.5, 152.2, 150.0, 146.9, 138.1,

6

33.0, 132.6, 129.4, 127.1, 126.3, 123.9, 121.7(2C), 115.9(2C), 113.4, 73.6, 67.4(2C), 63.7(2C),

+

9.1, 28.4. ESI-MS m/z (Calcd. for C23

H

27

N

5

O

3

: 421.21); Found: 422.19 (M+H) . Anal calcd.for

C H N O : C, 65.54; H, 6.46; N, 16.62; Found: C, 65.51; H, 6.48; N, 16.64.

23

27

5

3

4

.1.8.2.

6-Methoxy-4-((4-(3-morpholinopropoxy)phenyl)amino)quinoline-3-carbohydrazide

1

(

29): Yellow solid (0.13 g, 58%);m.p: 298-300°C; H NMR (300 MHz, DMSO-d , TMS):δ =

6

1

0.02 (s, 1H), 8.93 (s,1H), 8.84 (s, 1H), 8.12 (d, J = 8.6 Hz, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.54

d, J = 8.4 Hz, 2H), 7.39 (d, J = 2.3 Hz, 1H), 7.28 (dd, J = 8.6 Hz, 2.4 Hz, 1H), 6.96 (d, J = 8.4

Hz, 2H), 4.04 (t, J = 7.1 Hz, 2H), 3.82 (s, 3H), 3.64 (t, J = 6.8 Hz, 4H), 2.31 (s, 6H), 2.19 (brs,

(

1

3

2

6

H), 1.81 (m, 2H). C NMR (75 MHz, DMSO-d , TMS): δ = 163.3, 157.5, 151.2, 150.0, 144.5,

1

33.7, 132.9, 131.1, 124.8(2C), 122.0(2C), 115.7(2C), 113.9, 103.6, 73.8, 67.3(2C), 63.5(2C),

+

5

9.1, 56.3, 28.5. ESI-MS m/z (Calcd. for C H N O : 451.22); Found: 452.45 (M+H) . Anal

2

4

29

5

4

29 5 4

calcd.for C24H N O : C, 63.84; H, 6.47; N, 15.51; Found: C, 63.87; H, 6.45; N, 15.56.

4

.1.8.3.

6-Fluoro-4-((4-(3-morpholinopropoxy)phenyl)amino)quinoline-3-carbohydrazide

1

(

30): Brown solid (0.124 g, 57%);m.p: 290-292°C; H NMR (300 MHz, DMSO-d

6

, TMS):δ =

9

.90 (s, 1H), 9.02 (s, 1H), 8.87(s, 1H), 7.99 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.2 Hz, 2H),7.46 (d,

J = 2.4 Hz, 1H), 7.28 (dd, J = 8.6 Hz, 2.5 Hz, 1H), 6.86 (d, J = 8.3 Hz, 2H), 4.02 (t, J = 7.0 Hz,

1

3

2

H), 3.57 (t, J = 7.1 Hz, 4H), 2.34 (s, 6H), 2.20 (brs, 2H), 1.84 (m, 2H). C NMR (75 MHz,

DMSO-d , TMS): δ = 165.4, 160.6, 151.9, 150.0, 145.8, 135.2, 132.7, 130.1, 124.6, 123.9,

6

1

22.2(2C), 115.8(2C), 114.4, 104.5, 73.8, 67.5(2C), 63.7(2C), 59.1, 28.3. ESI-MS m/z (Calcd.

+

for C H FN O : 439.20); Found: 440.51 (M+H) . Anal calcd.for C H FN O : C, 62.86; H,

2

3

26

5

3

23 26

5

3

5

.96; N, 15.94; Found: C, 68.84; H, 5.93; N, 15.89.

4

.1.8.4.

4-((4-(3-Morpholinopropoxy)phenyl)amino)-6-(trifluoromethyl)

quinoline-3-

1

carbohydrazide (31): Yellow solid (0.143 g, 59%);m.p: 281-283°C; H NMR (300 MHz, DMSO-

6

d , TMS):δ = 10.09 (s, 1H), 8.99 (s,1H), 8.87 (s, 1H), 8.51 (d, J = 2.1 Hz, 1H), 8.19 (dd,J = 8.6

2

0

ACCEPTED MANUSCRIPT

Hz, 2.3 Hz, 1H), 8.14 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.5 Hz, 2H), 4.03

1

3

(

t, J = 7.0 Hz, 2H), 3.69 (t, J = 6.7 Hz, 4H), 2.33 (s, 6H), 2.03 (brs, 2H), 1.82 (m, 2H). C NMR

(

75 MHz, DMSO-d , TMS): δ = 165.3, 153.1, 149.9, 148.8, 139.2, 132.7, 131.4, 128.8, 127.6,

6

1

25.2, 122.9(3C), 120.7, 116.1(2C), 114.6, 73.8, 67.3(2C), 63.5(2C), 59.1, 28.4. ESI-MS m/z

+

(

Calcd. for C H F N O : 489.20); Found: 490.25 (M+H) . Anal calcd.for C H F N O : C,

2

4

26

3

5

3

24 26

3

5

3

5

4

8.89; H, 5.35; N, 14.31; Found: C, 58.91; H, 5.33; N, 14.28.

.1.8.5. 4-((4-((3-Morpholinopropyl)amino)phenyl)amino)quinoline-3-carbohydrazide (32):

1

Pale green solid (0.128 g, 61%);m.p: 310-312°C; H NMR (300 MHz, DMSO-d , TMS):δ =

6

1

8

0.01 (s, 1H), 8.90 (s, 1H), 8.88 (s, 1H), 8.20-7.46 (m, 4H), 6.86 (d, J = 8.4 Hz, 2H), 6.41 (d, J =

.6 Hz, 2H), 5.52 (brs, 1H), 3.65 (t, J = 6.8 Hz, 4H),3.36 (t, J = 7.0 Hz, 2H), 2.35 (s, 6H), 2.02

1

3

(

s, 2H), 1.78 (m, 2H). C NMR (75 MHz, DMSO-d

6

, TMS): δ = 165.3, 152.0, 146.7, 138.2(2C),

1

34.9, 132.5, 129.3, 127.0, 126.2, 123.9, 119.1(2C), 118.7(2C), 113.2, 67.5(2C), 63.3(2C), 52.9,

-

4

1.6, 27.8. ESI-MS m/z (Calcd. for C H N O : 420.23); Found: 419.05 (M-H) . Anal calcd.for

23 28 6 2

C

23

H

28

N

6

O

2

: C, 65.69; H, 6.71; N, 19.99; Found: C, 65.64; H, 6.73; N, 19.96.

4

.1.8.6.

6-Methoxy-4-((4-((3-morpholinopropyl)amino)phenyl)amino)quinoline-3-

1

carbohydrazide(33): Yellow solid (0.126 g, 56%);m.p: 291-293°C; H NMR (300 MHz, DMSO-

d₆, TMS):δ = 9.98 (s, 1H), 8.90 (s, 1H), 8.79 (s, 1H), 8.05 (d, J = 8.6 Hz, 1H), 7.52 (d, J = 2.4

Hz, 1H), 7.31 (dd, J = 8.2 Hz, 2.3 Hz, 1H), 6.61 (d, J = 7.8 Hz, 2H), 6.42 (d, J = 8.4 Hz, 2H),

5

1

5

.52 (brs, 1H), 3.83 (s, 3H), 3.65 (m, 4H), 3.44 (t, J = 7.0 Hz, 2H),2.36 (s, 6H), 2.04 (brs, 2H),

1

3

.85 (m, 2H). C NMR (75 MHz, DMSO-d , TMS): δ = 165.4, 157.1, 150.9, 144.3, 138.5,

6

34.8, 133.6, 130.9, 125.0(2C), 119.2(2C), 118.9(2C), 113.7, 103.5, 67.5(2C), 63.7(2C), 56.3,

+

2.8, 41.9, 27.6. ESI-MS m/z (Calcd. for C H N O : 450.24); Found: 451.26 (M+H) . Anal

2

4

30

6

3

calcd.for C H N O : C, 63.98; H, 6.71; N, 18.65; Found: C, 63.95; H, 6.73; N, 18.67.

2

4

30

6

3

4

.1.8.7.

6-Fluoro-4-((4-((3-morpholinopropyl)amino)phenyl)amino)quinoline-3-

1

carbohydrazide (34): Brown solid (0.127 g, 58%);m.p: 289-291°C; H NMR (300 MHz, DMSO-

d

6

, TMS):δ = 9.99 (s, 1H), 9.21 (s, 1H), 8.85 (s,1H), 7.78 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 2.4

Hz, 1H), 7.29 (dd, J = 8.6 Hz, 2.5 Hz, 1H), 6.58 (d, J = 8.2 Hz, 2H), 6.40 (d, J = 8.4 Hz, 2H),

.51 (brs, 1H), 3.58 (t, J = 7.0 Hz, 4H),3.44 (t, J = 6.8 Hz, 2H), 2.33 (s, 6H), 2.03 (s, 2H), 1.81

5

1

3

(

m, 2H). C NMR (75 MHz, DMSO-d

6

, TMS): δ = 165.3, 160.5, 151.8, 146.0, 138.2,

1

35.1(2C), 130.3, 124.9(2C), 119.1(2C), 118.8(2C), 114.2, 104.7, 67.3(2C), 63.6(2C), 52.9, 41.6,

2

1

ACCEPTED MANUSCRIPT

-

2

6 2

7.8. ESI-MS m/z (Calcd. for C23H27FN O : 438.22); Found: 437.11 (M-H) . Anal calcd.for

C H FN O : C, 63.00; H, 6.21; N, 19.17; Found: C, 63.04; H, 6.22; N, 19.15.

23

27

6

2

4

.1.8.8.

4-((4-((3-Morpholinopropyl)amino)phenyl)amino)-6-(trifluoromethyl)quinoline-3-

1

carbohydrazide (35): Brown solid (0.14 g, 57%);m.p: 302-304°C; H NMR (300 MHz, DMSO-

d₆, TMS):δ = 9.98 (s, 1H), 9.38 (s, 1H), 8.81 (s,1H), 8.28 (d, J = 2.4 Hz, 1H), 8.18 (dd, J = 8.8

Hz, 2.2 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 8.4 Hz, 2H), 6.38 (d, J = 8.2 Hz, 2H), 5.52

(brs, 1H), 3.66 (t, J = 7.0 Hz, 4H),3.54 (t, J = 6.7 Hz, 2H), 2.31 (s, 6H), 2.06 (s, 2H), 1.86 (m,

1

3

2

1

2

C

H). C NMR (75 MHz, DMSO-d , TMS): δ = 165.3, 153.1, 148.9, 139.2, 138.4, 134.8, 131.5,

6

28.9, 127.7, 125.0, 122.8, 120.6, 119.2(2C), 118.8(2C), 114.7, 67.3(2C), 63.5(2C), 52.7, 41.4,

-

7.6. ESI-MS m/z (Calcd. for C H F N O : 488.21); Found: 487.05 (M-H) . Anal calcd.for

2

4

27

3

6

2

24

H

27

F

3

N

6

O

2

: C, 59.01; H, 5.57; N, 17.20; Found: C, 59.05; H, 5.61; N, 17.15.

4

.1.8.9.

4-((4-(3-(4-Ethylpiperazin-1-yl)propoxy)phenyl)amino)quinoline-3-

1

carbohydrazide(36): Brown solid (0.134 g, 60%);m.p: 320-322°C; H NMR (300 MHz, DMSO-

d

2

1

5

4

7

4

6

, TMS):δ = 9.86 (s, 1H), 8.90 (s, 1H), 8.86 (s, 1H), 8.34-7.59 (m, 4H), 7.56 (d, J = 8.6 Hz,

H), 6.59 (d, J = 8.4 Hz, 2H), 4.03 (t, J = 7.0 Hz, 2H), 2.49-2.31 (m, 12H), 2.11 (s, 2H), 1.86 (m,

1

3

6

H), 1.05 (t, J = 6.8 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 165.2, 152.0, 149.9,

46.6, 137.8, 132.7(2C), 129.3, 127.1, 126.3, 123.8, 122.0(2C), 115.9(2C), 113.3, 73.7,

9.1(3C), 58.3(2C), 50.4, 28.3, 14.0. ESI-MS m/z (Calcd. for C H N O : 448.26); Found:

2

5

32

6

2

+

49.46 (M+H) . Anal calcd.for C25

H

32

N

6

O

2

: C, 66.94; H, 7.19; N, 18.74; Found: C, 66.97; H,

.21; N, 18.69.

.1.8.10.

4-((4-(3-(4-Ethylpiperazin-1-yl)propoxy)phenyl)amino)-6-methoxy

quinoline-3-

1

carbohydrazide (37): Yellow solid (0.14 g, 59%);m.p: 302-304°C; H NMR (300 MHz, DMSO-

d₆, TMS):δ = 9.97 (s, 1H), 8.97 (s, 1H), 8.74 (s, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.8

Hz, 2H), 7.43 (d, J = 2.4 Hz, 1H), 7.28 (dd, J = 8.4 Hz, 2.5 Hz, 1H), 6.78 (d, J = 8.2 Hz, 2H),

4

.04 (t, J = 7.0 Hz, 2H), 3.55 (s, 3H), 2.49-2.30 (m, 12H), 2.02 (s, 2H), 1.79 (m, 2H), 1.04 (t, J =

1

3

7

6

.1 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 165.3, 157.1, 150.9, 149.7, 144.5,

1

33.8(2C), 130.8, 125.1(2C), 121.9(2C), 115.7(2C), 113.4, 103.6, 73.4, 59.2(3C), 58.5(2C), 56.1,

-

5

0.3, 28.1, 13.9. ESI-MS m/z (Calcd. for C H N O : 478.27); Found: 477.04 (M-H) . Anal

2

6

34

6

3

34 6 3

calcd.for C26H N O : C, 65.25; H, 7.16; N, 17.56; Found: C, 66.28; H, 7.11; N, 17.52.

4

.1.8.11.

4-((4-(3-(4-Ethylpiperazin-1-yl)propoxy)phenyl)amino)-6-fluoro

quinoline-3-

1

carbohydrazide (38): Brown solid (0.128 g, 55%);m.p: 320-322°C; H NMR (300 MHz, DMSO-

2

ACCEPTED MANUSCRIPT

d

6

, TMS):δ = 9.96 (s, 1H), 9.34 (s, 1H), 8.89 (s, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.64 (d, J= 8.8

Hz, 2H), 7.39 (d, J = 2.5 Hz, 1H), 7.31 (dd, J = 8.4 Hz, 2.2 Hz, 1H),6.57 (d, J = 8.2 Hz, 2H),

.99 (t, J = 7.1 Hz, 2H), 2.48-2.30 (m, 12H), 2.05 (s, 2H), 1.86 (m, 2H), 1.03 (t, J = 6.8 Hz, 3H).

3

1

3

6

C NMR (75 MHz, DMSO-d , TMS): δ = 165.4, 160.6, 151.9, 150.1, 145.8, 135.0, 132.7,

1

30.2, 124.5(2C), 121.9(2C), 115.7(2C), 114.3, 104.5, 73.8, 59.5(3C), 58.3(2C), 50.2, 28.4, 13.9.

+

ESI-MS m/z (Calcd. for C25

H31FN

6

O

2

: 466.25); Found: 467.48 (M+H) . Anal calcd.for

C H FN O : C, 64.36; H, 6.70; N, 18.01; Found: C, 64.39; H, 6.72; N, 17.98.

25

31

6

2

4

3

.1.8.12. 4-((4-(3-(4-Ethylpiperazin-1-yl)propoxy)phenyl)amino)-6-(trifluoromethyl)quinoline-

1

-carbohydrazide (39): Yellow solid (0.154 g, 60%);m.p: 300-302°C; H NMR (300 MHz,

DMSO-d , TMS):δ = 9.99 (s, 1H), 9.48 (s, 1H), 8.90 (s, 1H), 8.38 (d, J = 2.5 Hz, 1H), 8.20 (dd,

6

J = 8.6 Hz, 2.4 HZ, 1H), 8.14 (d, J = 8.8 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H), 6.61 (d, J = 8.2 Hz,

2

H), 4.01 (t, J = 6.8 Hz, 2H), 2.49-2.33 (m, 12H), 2.10 (brs, 2H), 1.86 (m, 2H), 1.02 (t, J = 7.0

1

3

Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 165.4, 153.1, 150.0, 149.2, 139.4, 132.9,

6

1

31.5, 128.7, 127.6, 125.1, 122.8, 121.6(2C), 120.4, 115.8(2C), 114.6, 73.4, 59.0(3C), 58.5(2C),

+

5

0.2, 28.6, 14.0. ESI-MS m/z (Calcd. for C H F N O : 516.25); Found: 517.29 (M+H) . Anal

2

6

31

3

6

2

31 3 6 2

calcd.for C26H F N O : C, 60.45; H, 6.05; N, 16.27; Found: C, 60.47; H, 6.01; N, 16.29.

4

.1.8.13.

4-((4-((3-(4-Ethylpiperazin-1yl)propyl)amino)phenyl)amino)quinoline-3-

1

carbohydrazide (40): Yellow solid (0.127 g, 57%);m.p: 310-312°C; H NMR (300 MHz, DMSO-

d

6

, TMS):δ = 9.98 (s, 1H), 9.39 (s, 1H), 8.86 (s, 1H), 7.54 (m, 4H), 6.73 (d, J = 8.4 Hz, 2H), 6.58

(d, J = 8.2 Hz, 2H), 5.52 (brs, 1H), 3.42 (t, J = 7.0 Hz, 2H), 2.50-2.33 (m, 12H), 2.06 (brs, 2H),

1

3

1

5

6

.71 (m, 2H), 1.01 (t, J = 6.8 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 165.4, 152.1,

46.6, 138.3, 137.9, 134.7, 132.5, 129.3, 126.8, 125.9, 123.7, 119.1(2C), 118.8(2C), 113.4,

9.0(2C), 58.6(2C), 52.4, 50.2, 41.8, 27.5, 14.0. ESI-MS m/z (Calcd. for C H N O: 447.27);

2

5

33

7

+

33 7

Found: 448.15 (M+H) . Anal Calcd.for C25H N O: C, 67.09; H, 7.43; N, 21.91; Found: C,

6

7.12; H, 7.39; N, 21.93.

4

.1.8.14. 4-((4-((3-(4-Ethylpiperazin-1-yl)propyl)amino)phenyl)amino)-6-methoxyquinoline-3-

1

carbohydrazide (41): Yellow solid (0.13 g, 55%);m.p: 306-308°C; H NMR (300 MHz, DMSO-

d₆, TMS):δ = 10.01 (s, 1H), 9.16 (s, 1H), 8.89 (s, 1H), 7.81 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 2.4

Hz, 1H), 7.29 (dd, J = 8.4 Hz, 2.5 Hz, 1H), 6.56 (d, J = 8.2 Hz, 2H), 6.51 (d, J = 8.4 Hz, 2H),

5

2

.52 (brs, 1H), 3.58 (s, 3H), 3.46 (t, J = 6.8 Hz, 2H), 2.49-2.29 (m, 12H), 2.08 (s, 2H), 1.70 9m,

1

3

6

H), 1.01 (t, J = 7.0 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 165.4, 157.2, 150.9,

2

3

ACCEPTED MANUSCRIPT

1

5

44.5, 138.3, 135.0, 133.6, 130.9, 125.1(2C), 119.4(2C), 119.0(2C), 113.7, 103.5, 59.0(2C),

8.4(2C), 56.2, 52.5, 50.1, 41.8, 27.5, 13.9. ESI-MS m/z (Calcd. for C H N O : 477.29);

26

35

7

2

+

Found: 478.36 (M+H) . Anal calcd.for C H N O : C, 65.38; H, 7.39; N, 20.53; Found: C,

2

6

35

7

2

6

5.41; H, 7.37; N, 20.55.

4

.1.8.15. 4-((4-((3-(4-Ethylpiperazin-1-yl)propyl)amino)phenyl)amino)-6-fluoroquinoline-3-

1

carbohydrazide (42): Yellow solid (0.13 g, 56%);m.p: 298-300°C; H NMR (300 MHz, DMSO-

d₆, TMS):δ = 10.02 (s, 1H), 9.28 (s, 1H), 8.67 (s, 1H), 7.91 (d, J = 8.6 Hz, 1H), 7.42 (d, J = 2.5

Hz, 1H), 7.31(dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.61 (d, J = 8.2 Hz, 2H), 6.39 (d, J = 8.4 Hz, 2H),

5

.52(brs, 1H), 3.55 (t, J = 6.8 Hz, 2H), 2.50-2.30 (m, 12H), 2.09 (s, 2H), 1.71 (m, 2H), 1.02 (t, J

1

3

=

7.0 Hz, 3H). C NMR (75 MHz, DMSO-d , TMS): δ = 165.5, 160.6, 151.8, 146.0, 138.2,

6

1

4

35.1(2C), 130.3, 124.9(2C), 119.3(2C), 119.0(2C), 114.3, 104.9, 59.0(2C), 58.7(2C), 52.4, 50.2,

+

1.5, 27.8, 14.2.ESI-MS m/z (Calcd. for C H FN O: 465.27); Found: 466.42 (M+H) . Anal

2

5

32

7

calcd.for C H FN O: C, 64.50; H, 6.93; N, 21.06; Found: C, 64.53; H, 6.95; N, 21.01.

2

5

32

7

4

.1.8.16.

4-((4-((3-(4-Ethylpiperazin-1-yl)propyl)amino)phenyl)amino)-6-

(trifluoromethyl)quinoline-3-carbohydrazide (43): Yellow solid (0.138 g, 54%);m.p: 268-

1

2

70°C; H NMR (300 MHz, DMSO-d

6

, TMS):δ = 9.98 (s, 1H), 9.45 (s, 1H), 8.43 (s, 1H), 8.40

(d, J = 2.5 Hz, 1H), 8.21 (dd, J = 8.6 Hz, 2.5 Hz, 1H), 6.59 (d, J = 8.4 Hz, 2H), 6.44 (d, J = 8.2

Hz, 2H), 5.50 (brs, 1H), 3.52 (t, J = 6.8 Hz, 2H), 2.49-2.33 (m, 12H), 2.11 (s, 2H), 1.73 (m, 2H),

1

3

1

5

6

4

.01 (t, J = 7.0 Hz, 3H). C NMR (75 MHz, DMSO-d

6

, TMS): δ = 165.4, 153.2, 149.0, 139.5,

38.3, 134.9, 131.4, 128.7, 127.8, 125.3, 122.6, 120.8, 119.4(2C), 118.9(2C), 114.5, 59.1(2C),

32 3 7

8.4(2C), 52.6, 50.3, 41.6, 27.8, 14.0. ESI-MS m/z (Calcd. for C26H F N O: 515.26); Found:

+

16.31 (M+H) . Anal calcd.for C H F N O: C, 60.57; H, 6.26; N, 19.02; Found: C, 60.54; H,

2

6

32

3

7

.29; N, 19.04.

.1.9. General procedure for the preparation of titled phenyl-amino-quinoline acid derivatives

44-57): The corresponding ethyl ester derivatives (0.5 mmol) (12-27) were hydrolyzed by

(

lithium hydroxide monohydrate (1.25 mmol) in 6mL of 1:1:1 ratio of methanol; tetrahydrofuran;

distilled water as a solvent and acidified with 2N HCl by maintaingpH 5-6. The solid precipitated

was filtered, dried and diethyl ether, ethyl acetate washings were given for further purification to

get the corresponding acid derivatives in moderate yield.

4

.1.9.1. 4-((4-(3-Morpholinopropoxy)phenyl)amino)quinoline-3-carboxylic acid (44): Yellow

1

gammy (0.11 g, 55%); H NMR (300 MHz, DMSO-d

6

, TMS):δ = 11.51 (s, 1H), 9.86 (s, 1H),

2

4

ACCEPTED MANUSCRIPT

9

7

.34 (s,1H), 8.18 (dd, J = 8.6 Hz, J = 2.4 Hz, 1H), 7.83 (dd,J = 8.8 Hz, J = 2.5 Hz, 1H), 7.76-

.60 (m, 2H), 7.49 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.2 Hz, 2H), 4.03 (t, J = 7.0 Hz, 2H), 3.66

1

3

(

t,J = 6.8 Hz, 4H), 2.34 (s, 6H), 1.79 (m, 2H). C NMR (75 MHz, DMSO-d , TMS): δ = 170.0,

6

1

6

53.2, 152.4, 149.8, 140.7, 132.5(2C), 129.3, 127.0, 126.2, 121.9(3C), 115.8(2C), 114.3, 73.6,

7.4(2C), 63.6(2C), 59.2, 28.5. ESI-MS m/z (Calcd. for C H N O : 407.18); Found: 408.25

2

3

25

3

4

+

(

25 3 4

M+H) . Anal calcd.for C23H N O : C, 67.80; H, 6.18; N, 10.31; Found: C, 67.76; H, 6.21; N,

1

0.33.

4

.1.9.2. 6-Methoxy-4-((4-(3-morpholinopropoxy)phenyl)amino)quinoline-3-carboxylic acid

1

(

45): Pale yellow solid (0.11 g, 53%);m.p: 239-241°C; H NMR (300 MHz, DMSO-d

6

, TMS):δ =

1

1.65 (brs, 1H), 9.92 (s, 1H), 8.94 (s,1H), 7.84 (d, J = 8.6 Hz, 1H), 7.63 (d, J = 8.2 Hz, 2H), 7.43

(d, J = 2.5 Hz, 1H), 7.28 (dd, J = 8.4Hz, 2.5 Hz, 1H), 6.86 (d, J = 8.0 Hz, 2H), 4.01 (t, J = 7.1

13

Hz, 2H), 3.82 (s, 3H), 3.66 (t, J = 6.8 Hz, 4H), 2.36 (s, 6H), 1.80 (m, 2H). C NMR (75 MHz,

DMSO-d , TMS): δ = 169.9, 157.3, 151.8, 150.2(2C), 136.4, 132.7, 130.9, 125.0, 121.8(3C),

6

1

16.1(2C), 114.9, 103.5, 73.7, 67.4(2C), 63.5(2C), 59.2, 56.4, 28.6. ESI-MS m/z (Calcd. for

-

C H N O : 437.20); Found: 436.11 (M-H) . Anal calcd.for C H N O : C, 65.89; H, 6.22; N,

24

27

3

5

24 27

3

5

9

.60; Found: C, 65.87; H, 6.25; N, 9.61.

4

.1.9.3. 6-Fluoro-4-((4-(3-morpholinopropoxy)phenyl)amino)quinoline-3-carboxylic acid (46):

1

Off-white solid (0.11 g, 56%);m.p: 199-201°C; H NMR (300 MHz, DMSO-d , TMS):δ = 11.69

6

(brs, 1H), 9.93 (s, 1H), 8.92 (s,1H), 7.90 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.45 (d, J

=

2.4 Hz, 1H), 7.36 (dd, J = 8.6 Hz, 2.5 Hz, 1H), 6.98 (d, J = 8.1 Hz, 2H), 4.02 (t, J = 6.9 Hz,

1

3

2

H), 3.61 (t, J = 7.0 Hz, 4H), 2.35 (s, 6H), 1.81 (m, 2H). C NMR (75 MHz, DMSO-d

δ = 170.1, 160.8, 152.4, 151.6, 149.9, 137.7, 133.0, 130.4, 124.1, 121.9(3C), 116.0(2C), 115.3,

04.5, 73.7, 67.3(2C), 63.6(2C), 59.1, 28.4. ESI-MS m/z (Calcd. for C H FN O : 425.18);

6

, TMS):

1

2

3

24

3

4

+

3 4

Found: 426.35 (M+H) . Anal calcd.for C23H24FN O : C, 64.93; H, 5.69; N, 9.88; Found: C,

6

4.95; H, 5.66; N, 9.91.

4

.1.9.4.

4-((4-(3-Morpholinopropoxy)phenyl)amino)-6-(trifluoromethyl)

quinoline-3-

1

carboxylic acid (47): Pale green solid (0.144 g, 61%);m.p: 248-250°C; H NMR (300 MHz,

DMSO-d , TMS):δ = 11.72 (brs, 1H), 9.91 (s, 1H), 8.87 (s,1H), 8.31 (d, J = 2.5 Hz, 1H), 8.24

6

(dd, J = 8.8 Hz, 2.4 Hz, 1H), 8.18 (d, J = 8.6 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 6.96 (d, J = 8.0

1

3

Hz, 2H), 3.99 (t, J = 7.0 Hz, 2H), 3.60 (t, J = 6.8 Hz, 4H), 2.31 (s, 6H), 1.82 (m, 2H). C NMR

75 MHz, DMSO-d , TMS): δ = 169.9, 154.6(2C), 149.8, 142.2, 132.7, 131.5, 129.0, 127.8,

(

6

2

5

ACCEPTED MANUSCRIPT

1

25.3, 122.1(2C), 119.9(2C), 115.7(3C), 73.5, 67.2(2C), 63.6(2C), 59.1, 28.3. ESI-MS m/z

+

(

Calcd. for C H F N O : 475.17); Found: 476.38 (M+H) . Anal calcd.for C H F N O : C,

2

4

24

3

4

24 24

3

4

6

4

0.63; H, 5.09; N, 8.84; Found: C, 60.68; H, 5.06; N, 8.81.

.1.9.5. 4-((4-((3-Morpholinopropyl)amino)phenyl)amino)quinoline-3-carboxylic acid (48):

1

Brown solid (0.118 g, 58%);m.p: 187-189°C; H NMR (300 MHz, DMSO-d , TMS):δ = 11.67

6

(brs, 1H), 9.94 (s, 1H), 8.87 (s, 1H), 8.21 (d, J = 2.5 Hz, 1H), 7.92 (d, J = d, J = 8.4 Hz, 1H),

7

.81-7.61 (m, 2H), 6.71 (d, J = 8.2 Hz, 2H), 6.40 (d, J = 8.2 Hz, 2H), 5.51 (brs, 1H), 3.64 (t, J =

1

3

.0 Hz, 4H), 3.34 (t, J = 6.8 Hz, 2H), 2.30 (s, 6H), 1.69 (m, 2H). C NMR (75 MHz, DMSO-d ,

6

TMS): δ = 170.1, 153.3, 152.0, 140.9, 138.2, 134.8, 132.6, 129.3, 127.0, 125.9, 120.7,

1

19.2(2C), 118.6(2C), 114.2, 67.1(2C), 63.6(2C), 52.9, 41.6, 27.5. ESI-MS m/z (Calcd. for

+

C

23

H

26

N

4

O

3

: 406.20); Found: 407.19 (M+H) . Anal calcd.for C23

H

26

N

4

O

3

: C, 67.96; H, 6.45; N,

1

3.78; Found: C, 67.94; H, 6.47; N, 13.81.

4

.1.9.6. 6-Methoxy-4-((4-((3-morpholinopropyl)amino)phenyl)amino) quinoline-3-carboxylic

1

acid (49): Brown solid (0.128 g, 59%);m.p: 199-201°C; H NMR (300 MHz, DMSO-d

6

, TMS):δ

=

11.52 (brs, 1H), 9.93 (s, 1H), 8.88 (s, 1H), 8.34 (m, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.54 (d, J =

2

5

.5 Hz, 1H), 7.31 (d, J = 8.2 Hz, 2.4 Hz, 1H), 6.54 (d, J = 8.2 Hz, 2H), 6.40 (d, J = 8.0 Hz, 2H),

.51 (brs, 1H), 3.84 (s, 3H), 3.64 (t, J = 6.8 Hz, 4H),3.45 (t, J = 7.0 Hz, 2H), 2.36 (s, 6H), 1.82

1

3

(

m, 2H). C NMR (75 MHz, DMSO-d , TMS): δ = 170.2, 157.5, 151.8, 150.2, 138.3, 136.7,

6

1

34.9, 131.0, 125.2, 121.8, 119.0(2C), 118.7(2C), 114.9, 103.6, 67.3(2C), 63.4(2C), 56.0, 52.9,

+

4

1.7, 27.5. ESI-MS m/z (Calcd. for C H N O : 436.21); Found: 437.17 (M+H) . Anal calcd.for

2

4

28

4

C

24

H

28

N

4

O

4

: C, 66.04; H, 6.47; N, 12.84; Found: C, 66.07; H, 6.51; N, 12.81.

4

.1.9.7.

6-Fluoro-4-((4-((3-morpholinopropyl)amino)phenyl)amino)quinoline-3-carboxylic

1

acid (50): Red solid (0.118 g, 56%);m.p: 216-218°C; H NMR (300 MHz, DMSO-d , TMS):δ =

6

1

7

1

1.71 (brs, 1H), 9.97 (s, 1H), 8.82 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 2.5 Hz, 1H),

.34 (dd, J = 8.2 Hz, 2.5 Hz, 1H), 6.56 (d, J = 8.0 Hz, 2H), 6.40 (d, J = 8.2 Hz, 2H), 5.52 (brs,

1

3

H), 3.63 (t, J = 7.0 Hz, 4H), 3.45 (t, J = 6.8 Hz, 2H), 2.34 (s, 6H), 1.81 (m, 2H). C NMR (75

MHz, DMSO-d , TMS): δ = 169.9, 160.7, 152.5(2C), 138.3(2C), 134.9, 130.1, 124.3, 121.8,

6

1

19.5(2C), 119.1(2C), 115.4, 104.2, 67.5(2C), 63.4(2C), 52.9, 41.5, 27.7. ESI-MS m/z (Calcd.

+

for C23

H25FN

4

O

3

: 424.19); Found: 425.15 (M+H) . Anal calcd.for C23

H

25FN

4

O

3

: C, 65.08; H,

5

.94; N, 13.20; Found: C, 65.09; H, 5.91; N, 13.22.

2

6

ACCEPTED MANUSCRIPT

4

.1.9.8. 4-((4-((3-Morpholinopropyl)amino)phenyl)amino)-6-(trifluoromethyl) quinoline-3-

1

carboxylic acid (51): Brown solid (0.141 g, 60%);m.p: 223-224°C; H NMR (300 MHz, DMSO-

d₆, TMS):δ = 11.89 (brs, 1H), 9.98 (s, 1H), 8.80 (s, 1H), 8.29 (d, J = 2.5 Hz, 1H), 8.20-8.16 (m,

2

H), 6.57 (d, J = 8.2 Hz, 2H), 6.38 (d, J = 8.0 Hz, 2H), 5.53 (brs, 1H), 3.64 (t, J = 7.0 Hz, 4H),

1

3

.44 (t, J = 6.7 Hz, 2H), 2.33 (s, 6H), 1.82 (m, 2H). C NMR (75 MHz, DMSO-d , TMS): δ =

6

1

69.8, 155.2, 154.0, 142.5, 138.3, 134.9, 131.6, 128.8, 127.9, 125.1, 120.3(2C), 119.0(2C),

18.7(2C), 115.8, 67.5(2C), 63.6(2C), 52.7, 41.4, 27.6. ESI-MS m/z (Calcd. for C H F N O :

1

2

4

25

3

4

3

+

4

74.19); Found: 475.13(M+H) . Anal calcd.for C H F N O : C, 60.75; H, 5.31; N, 11.81;

24 25 3 4 3

Found: C, 60.71; H, 5.28; N, 11.76.

4

.1.9.9. 4-((4-(3-(4-Ethylpiperazin-1-yl)propoxy)phenyl)amino)quinoline-3-carboxylic acid

1

(

52): Brown gammy (0.127 g, 59%); H NMR (300 MHz, DMSO-d

.86 (s, 1H), 8.85 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.80-7.63 (m, 2H),

.59 (d, J = 8.0 Hz, 2H), 6.92 (d, J = 8.2 Hz, 2H), 4.01 (t, J = 7.0 Hz, 2H), 2.48-2.30 (m, 12H),

6

, TMS):δ = 11.79 (brs, 1H),

9

7

1

5

4

6

4

1

3

.83 (m, 2H), 1.05 (t, J = 6.8 Hz, 3H). C NMR (75 MHz, DMSO-d

6

, TMS): δ = 170.0, 152.9,

52.3, 150.1, 140.8, 132.9(2C), 128.9, 127.4, 126.2, 121.7(2C), 120.6, 115.8(2C), 114.3, 73.6,

30 4 3

9.2(3C), 58.6(2C), 50.2, 28.3, 14.1. ESI-MS m/z (Calcd. for C25H N O : 434.23); Found:

-

33.16 (M-H) . Anal calcd.for C H N O : C, 69.10; H, 6.96; N, 12.89; Found: C, 69.14; H,

2

5

30

4

3

.92; N, 12.91.

.1.9.10.

4-((4-(3-(4-Ethylpiperazin-1-yl)propoxy)phenyl)amino)-6-methoxyquinoline-3-

1

carboxylic acid (53): Pale green solid (0.131 g, 57%);m.p: 282-284°C; H NMR (300 MHz,

DMSO-d

d, J = 8.4 Hz, 2H), 7.52 (d, J = 2.5 Hz, 1H), 7.31 (dd, J = 8.1 Hz, 2.5 Hz, 1H), 6.94 (d, J = 8.4

Hz, 2H), 4.03 (t, J = 7.0 Hz, 2H), 3.55 (s, 3H), 2.47-2.30 (m, 12H), 1.82 (m, 2H), 1.04 (t, J = 6.8

6

, TMS):δ = 11.72 (brs, 1H), 9.96 (s, 1H), 8.87 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.62

(

1

3

Hz, 3H). C NMR (75 MHz, DMSO-d

6

, TMS): δ = 169.9, 157.3, 151.7, 150.2(2C), 136.4,

1

2

32.8, 131.1, 125.0, 121.9(3C), 115.7(2C), 114.9, 103.5, 73.7, 59.3(3C), 58.3(2C), 56.5, 50.1,

+

8.3, 14.1. ESI-MS m/z (Calcd. for C26

H

32

N

4

O

4

: 464.24); Found: 465.28 (M+H) . Anal calcd.for

C, 67.22; H, 6.94; N, 12.06; Found: C, 67.25; H, 6.91; N, 12.09.

4

.1.9.11.

4-((4-(3-(4-Ethylpiperazin-1-yl)propoxy)phenyl)amino)-6-fluoroquinoline-3-

1

carboxylic acid (54): Yellow gammy (0.137 g, 61%); H NMR (300 MHz, DMSO-d

6

, TMS):δ =

1

7

1.54 (brs, 1H), 9.95 (s, 1H), 8.86 (s, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H),

.46 (s, 1H), 7.31 (dd, J = 8.2 Hz, 2.4 Hz, 1H), 6.68 (d, J = 8.2 Hz, 2H), 3.99 (t, J = 7.0 Hz, 2H),

2

7

ACCEPTED MANUSCRIPT

1

3

2

.47-2.30 (m, 12H), 1.82 (m, 2H), 1.02 (t, J = 6.8 Hz, 3H). C NMR (75 MHz, DMSO-d

6

,

TMS): δ = 170.1, 160.7, 152.4, 151.5, 149.9, 137.6, 132.8, 130.3, 124.1, 122.0(3C), 115.9(3C),

1

4

04.5, 73.7, 59.2(3C), 58.4(2C), 50.1, 28.5, 14.0. ESI-MS m/z (Calcd. for C H FN O :

25 29 4 3

+

4 3

52.22); Found: 453.38 (M+H) . Anal calcd.for C25H29FN O : C, 66.35; H, 6.46; N, 12.38;

Found: C, 66.33; H, 6.42; N, 12.41.

4

3

.1.9.12. 4-((4-(3-(4-Ethylpiperazin-1-yl)propoxy)phenyl)amino)-6-(trifluoromethyl)quinoline-

1

-carboxylic acid (55): Yellow solid (0.14 g, 56%);m.p: 153-155°C; H NMR (300 MHz,

DMSO-d , TMS):δ = 11.56 (brs, 1H), 9.97 (s, 1H), 8.88 (s, 1H), 8.36 (s, 1H), 8.21 (dd, J = 8.0

6

Hz, 2.5 Hz, 1H), 8.13 (d, J = 8.2 Hz, 1H), 7.82 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 3.98

1

3

(

t, J = 7.0 Hz, 2H), 2.46-2.34 (m, 12H), 1.82 (m, 2H), 1.03 (t, J = 7.1 Hz, 3H). C NMR (75

MHz, DMSO-d , TMS): δ = 169.9, 154.7(2C), 150.1, 142.3, 132.9, 131.6, 129.2, 128.0, 125.2,

21.8(2C), 120.5(2C), 115.9(3C), 73.6, 59.1(3C), 58.3(2C), 50.0, 28.4, 14.1. ESI-MS m/z

6

1

+

(

Calcd. for C H F N O : 502.22); Found: 503.36 (M+H) . Anal calcd.for C H F N O : C,

26 29 3 4 3 26 29 3 4 3

6

4

2.14; H, 5.82; N, 11.15; Found: C, 62.11; H, 5.85; N, 11.12.

.1.9.13. 4-((4-((3-(4-Ethylpiperazin-1-yl)propyl)amino)phenyl)amino) quinoline-3-carboxylic

1

acid (56): Brown solid (0.127 g, 59%);m.p: 188-190°C; H NMR (300 MHz, DMSO-d

6

, TMS):δ

=

11.72 (brs, 1H), 9.96 (s, 1H), 8.86 (s, 1H), 8.13-7.61 (m, 4H), 6.58 (d, J = 8.2 Hz, 2H), 6.34 (d,

J = 8.0 Hz, 2H), 5.52 (brs, 1H), 3.51 (t, J = 7.0 Hz, 2H), 2.46-2.31 (m, 12H), 1.83 (m, 2H), 1.04

1

3

(

t, J = 6.8 Hz, 3H). C NMR (75 MHz, DMSO-d

6

, TMS): δ = 169.8, 153.0, 152.5, 140.9, 138.2,

1

5

34.8, 132.6, 129.3, 127.1, 126.2, 120.9, 119.4(2C), 118.9(2C), 114.3, 59.0(2C), 58.5(2C), 52.7,

-

31 5 2

0.2, 41.8, 27.6, 13.9. ESI-MS m/z (Calcd. for C25H N O : 433.25); Found: 432.09 (M-H) .

Anal calcd.for C H N O : C, 69.26; H, 7.21; N, 16.15; Found: C, 69.21; H, 7.23; N, 16.18.

2

5

31

5

2

4

.1.9.14. 4-((4-((3-(4-Ethylpiperazin-1-yl)propyl)amino)phenyl)amino)-6-methoxyquinoline-3-

1

carboxylic acid (57): Yellow gummy (0.138 g, 60%); H NMR (300 MHz, DMSO-d

6

, TMS):δ =

1

7

3

1.58 (brs, 1H), 9.98 (s, 1H), 8.89 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.51 (s, 1H), 7.34 (dd, J =

.8 Hz, 2.4 Hz, 1H), 6.57 (d, J = 8.2 Hz, 2H),6.46 (d, J = 8.0 Hz, 2H), 5.51 (s, 1H), 3.86 (s, 3H),

1

3

.36 (t, J = 7.0 Hz, 2H), 2.48-2.29 (m, 12H), 1.83 (m, 2H), 1.04 (t, J = 6.8 Hz, 3H). C NMR

(

75 MHz, DMSO-d , TMS): δ = 170.0, 157.3, 151.9, 150.2, 138.4, 136.7, 134.8, 131.0, 124.9,

6

1

22.1, 119.2(2C), 118.7(2C), 115.2, 103.4, 59.2(2C), 58.5(2C), 56.3, 52.4, 50.1, 41.9, 27.7, 14.1.

+

ESI-MS m/z (Calcd. For C H N O : 463.26); Found: 464.35 (M+H) . Anal calcd.for

2

6

33

5

3

26 33 5 3

C H N O : C, 67.36; H, 7.18; N, 15.11; Found: C, 67.32; H, 7.21; N, 15.07.

2

8

Article Doi

4-Aminoquinoline derivatives as novel Mycobacterium tuberculosis GyrB inhibitors: Structural optimization, synthesis and biological evaluation

DOI: 10.1016/j.ejmech.2015.06.032

Source and publish data:

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Article abstract of DOI:10.1016/j.ejmech.2015.06.032

Full text of DOI:10.1016/j.ejmech.2015.06.032

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