Synthesis and evaluation of chirally defined side chain variants of 7-chloro-4-aminoquinoline to overcome drug resistance in malaria chemotherapy

Article abstract of DOI:10.1128/AAC.01152-16

A novel 4-aminoquinoline derivative [(S)-7-chloro-N-(4-methyl-1-(4-methyl-piperazin-1-yl)pentan-2-yl)-quinolin-4-amine triphosphate] exhibiting curative activity against chloroquine-resistant malaria parasites has been identified for preclinical development as a blood schizonticidal agent. The lead molecule selected after detailed structure-activity relationship (SAR) studies has good solid-state properties and promising activity against in vitro and in vivo experimental malaria models. The in vitro absorption, distribution, metabolism, and excretion (ADME) parameters indicate a favorable drug-like profile.

Full text of DOI:10.1128/AAC.01152-16

AAC Accepted Manuscript Posted Online 12 December 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.01152-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Synthesis and evaluation of chirally defined side chain variants
of 7-chloro-4-aminoquinoline to overcome drug resistance in
malaria chemotherapy.
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Vasantha Rao Dola , Awakash Soni , Pooja Agarwal , Hafsa Ahmad , Kanumuri Siva Rama
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Raju Mamunur Rashid , Muhammad Wahajuddin , Kumkum Srivastava , W.Haq ,
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A.K.Dwivedi , S.K.Puri , and S.B.Katti *
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Divisions of Medicinal and Process Chemistry , Parasitology , Pharmaceutics , and
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Pharmacokinetics and Metabolism , Central Drug Research Institute, Sector-10, Jankipuram
Ext. Sitapur Road, Lucknow-226031.
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Running Title: Side chain modified 4-aminoquinoline antimalarials
CORRESPONDING AUTHOR INFORMATION
Dr. S. B. Katti,
Medicinal & Process Chemistry Division,
Central Drug Research Institute,
Sitapur Road, Sector-10, Jankipuram Extension, Lucknow-226021
Phone: +91 9415106737
E-mail: sb_katti@cdri.res.inand setu_katti@yahoo.com.
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ABSTRACT
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A novel 4-aminoquinoline derivative ((S)-7-chloro-N-(4-methyl-1-(4-methylpiperazin-1-
yl)pentan-2-yl)-quinolin-4-amine triphosphate) exhibiting curative activity against
chloroquine resistant malaria parasite has been identified for preclinical development as a
blood schizonticidal agent. The lead molecule selected after detailed SAR studies, has good
solid state properties, and has promising activity against in vitro and in vivo experimental
malaria models. The in vitro ADME parameters have indicated favourable drug like profile.
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INTRODUCTION
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Malaria is a major infectious disease affecting mainly tropical and subtropical areas. Of the
five species of Plasmodia which are responsible for human malaria, P. falciparum causes the
most severe form of the disease (1). There were an estimated 207 million malaria cases in
2012 and an estimated 627 000 deaths of which 90% of malaria deaths occur in sub-Saharan
Africa, and 77% occur in children under five years of the age (2). For several decades
chloroquine (CQ), a 4-aminoquinoline, has been the frontline drug in malaria chemotherapy
because of its therapeutic efficacy, ease of use and low cost (3). However due to emergence
of resistant P. falciparum and P. vivax strains against commonly used drugs such as CQ,
amodiaquine and artemisinin (Figure 1) there is urgent need to develop new chemical entities
with a goal to overcome parasite resistance.
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Aminoquinolines such as chloroquine had been the most important antimalarials for
more than four decades in view of their efficacy against all species of human malaria and
their safety profile. Emergence of resistance to this class of compounds during 1980’s created
a genuine health crisis in the developing world. Studies on elucidation of mechanism of
resistance and general trend emerging from the SAR-studies revealed that chloroquine
resistance does not involve any change to the target of this class of drugs but involves
compound specific efflux mechanism (4). Based on this premise a number of research groups
have developed short chain analogues of 4-aminoquinoline, which are active against CQ-
resistant strains of P.falciparum. However, these derivatives undergo biotransformation
(dealkylation) significantly affecting lipid solubility of the drug and decreasing the biological
efficacy (5). In view of this background information it was surmised that a focussed library of
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small molecules based on 4-aminoquinoline scaffold with suitable functionalities would
result in molecules with improved antiplasmodial activity against CQ resistant parasite.
Therefore, modifications employing side chain refinement and conformational rigidity were
considered. The seminal finding of our study is that a new chemical entity having significant
activity against CQ resistant parasites has been identified and the results are discussed in the
present manuscript.
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Stocks et al. synthesized and evaluated a series of short chain CQ derivatives, by
replacement of diethyl amino function with more metabolically stable side chain (tert-butyl)
as well as heterocyclic ring (piperidyl, pyrrolidino, morpholino) modifications, that led to a
substantial increase in the antimalarial activity against CQ-resistant parasite strains (6).
Madrid et al. have replaced diethylamino functionality with one propyl group as constant and
replacing the other ethyl group with bulky or aromatic ring and the results indicated that
some of these analogs are active against multi drug resistant strains (7).Some annals suggest
that 4-aminoquinoline analogs of altered side chain such as N'-(7-Chloro-quinolin-4-yl)-N,N-
diethyl-propane-1,3- diamine show potential leads for the development of new drugs (8).
Ryckebusch et al. evaluated new series (1,4-bis(3-aminopropyl) piperazine derivatives)
against the chloroquine resistant strains of P. falciparum. Compounds displayed moderate to
good activity when quinoline and/or aryl moieties were attached to the above mentioned
linker. In this series, compounds containing piperazine moiety were found to be active
against CQ resistant strains of P. falciparum (9-11).
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Based on these annotations, earlier from this laboratory Solomon et al. explored different
modifications at the pendant nitrogen of the CQ lateral side chain that led to compounds with
improved activity, particularly against CQ-resistant strains (12). By taking into account above
facts, more recently from our laboratory Sinha et al.developed a generic methodology for the
synthesis of chiral chloroquine and its analogues. The key feature of this methodology is that
it enables the use of amino acids to generate 4-aminoquinolines with chirally defined
substituted side-chain and also to address the role of hydrophobic substitution at the chiral
center. It was inferred from the antiplasmodial activity data that analogues containing N-
methylpiperazine at the terminal part of side chain showed excellent in vitro activity with
reference to CQ against the resistant strain. These authors also examined the chain length
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variation by homologation of selected α-amino acids to get the corresponding β -and γ-amino
acids (13). Most of the derivatives displayed excellent in vitro antiplasmodial activity, and a
few compounds in the in vivo studies showed 100% parasitaemia suppression on day 4 (14).
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The objective of the present study is to synthesize a new series of 4-aminoquinoline
derivatives with reduced side chain amide bond with a view to increase in vivo stability, and
also, to synthesize compounds with different substitutions at the chiral centre. These
compounds have been evaluated for antiplasmodial activity against chloroquine sensitive
(CQ-S) and chloroquine resistant (CQ-R) strains and the results are reported in the following
paragraphs.
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MATERIALS AND METHODS
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Chemistry
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Melting points (mp) were taken in open capillaries on Complab melting point apparatus and
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are uncorrected. The H NMR (200, 300MHz) and C NMR (50, 75 MHz) spectra were
recorded in CDCl , on DPX-200 and 300 Bruker FT-NMR spectrometers. All chemical shifts
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(δ) are reported in parts per million (ppm) downfield from tetramethylsilane. The splitting
pattern abbreviations are as follows: s (singlet), d (doublet), dd (doublet of doublet), t
(triplet), q (quartet), br s (broad singlet) and m (multiplet). Coupling constants are given in
hertz. Mass Spectra (ESI-MS), high resolution mass spectra HRMS (ESI-HRMS) were
recorded on Jeol (Japan)/SX-102, and Agilent 6520 Q-TOF (ESI-HRMS) spectrometers
respectively. Analytical thin-layer chromatography (TLC) was carried out on Merck’s pre-
coated silica-gel plates 60 F254 and spots were visualized by irradiation with UV light (254
nm). Iodine was used as developing agent and/or by spraying with Dragendorff’s reagent.
Column chromatographic purification was performed over neutral alumina and silica gel
(silica gel 60-120, 100-200 and 230-400 mesh) using a gradient solvent system (n-
hexane/EtOAc, DCM/Hexane or chloroform/methanol as the eluent unless otherwise
specified). All chemicals and reagents were obtained from Aldrich (USA), Lancaster (UK)
and Spectrochem Pvt. Ltd (India) and were used without further purification. Analytes were
eluted using isocratic mobile phase of methanol and 0.05% TFA in water (60:40) and
detected at 254 nm. The purities of compounds submitted for biological evaluation were
>98% as determined by HPLC. Yields are not optimized.
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General Procedure for the Synthesis of 2a-j
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Amino acids namely glycine, phenylalanine, tryptophan and methionine (1.0 equiv.) were
dissolved in dioxane-water mixture (1:1) and the reaction mixture was stirred at room
temperature. Addition of 2N NaOH to this reaction mixture dissolves the reactant thereby
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affording a miscible solution. The reaction mixture was then cooled to 0°C and stirred for 15
min. Finally, (Boc) O (1.1 equiv.) was added and the reaction mixture was allowed to stir at 0
2
°C for 10 min. The ice bath was then removed and the temperature of the reaction was
allowed to rise to the room temperature. On completion of the reaction, reaction mixture was
concentrated under reduced pressure. The aqueous layer was acidified with citric acid (pH 2-
3) and extracted with EtOAc and the organic layer was washed with brine, dried over Na
2 4
SO
and concentrated under reduced pressure. The yields were quantitative.
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Amino acids namely alanine, valine, leucine, and isoleucine (1.0 equiv) in a 2N aqueous
NaOH solution (17 mL) was cooled in an ice bath to 0 °C. Under vigorous stirring benzyl
chloroformate (1.1 equiv) and a 2N aqueous NaOH solution were simultaneously added
within 2 min. The mixture was stirred for 20 min at room temperature and extracted with
diethyl ether. The aqueous layer was separated and acidified with conc. hydrochloric acid to a
pH of 2-3. The resulting emulsion was extracted with EtOAc. The organic phases were
combined, washed with brine, and dried with Na
2-(Tert-butoxycarbonylamino) acetic acid (2a)
The compound was obtained as a white solid in quantitative yield. m. p. 86-88°C; H NMR
2 4
SO . The yields were quantitative.
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134
135
(CDCl
3
, 300 MHz) : δ 1.46 (s, 9H, C (CH
3
)
3
, 3.96 (s, 2H, CH
2
COOH); ESI-MS: (m/z) 176
+
(M+H) .
(S)-2-(Benzyloxycarbonylamino) propanoic acid (2b)
136
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The compound was obtained as a white solid in quantitative yield. m. p. 46-48°C; H NMR
(300 MHz, CDCl
3
): δ 1.49 (d, J = 6.7 Hz, 3H, CH CH
3
3
), 4.44 (br s, 1H, CHCH ), 5.15(s, 2H,
+
CH ), 7.37 (m, 5H, C
2
C
6
H
5
6
H
5
); ESI-MS: (m/z) 224 (M+H) .
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(S)-2-(Benzyloxycarbonylamino)-3-methylbutanoic acid (2c)
The compound was obtained as a gummy substance in quantitative yield. H NMR (300
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MHz, CDCl
CHCH(CH
3
): δ 0.92-1.01 (m, 6H, CH(CH
3
)
2
, 2.31-2.22 (m, 1H, CH(CH
3
)
2
, 4.43 (brs, 1H,
+
3
)
2
), 5.12 (s, 2H, CH ), 7.35 (s, 5H, C
2
6
C H
5
6
H
5
); ESI-MS: (m/z) 252 (M+H) .
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(S)-2-(Benzyloxycarbonylamino)-4-methylpentanoic acid (2d)
The compound was obtained as a gummy substance in quantitative yield. H NMR (300 MHz,
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46
1
CDCl
3
) : δ 0.98 (d, J = 6.5 Hz, 6H, CH (CH
3
)
2
, 1.55-1.61 (m, 1H, CH(CH
3
)
2
, 1.70-1.75 (m,
5

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2H, CH
2
CH(CH
3
)
2
, 4.43 (brs, 1H, NCH), 5.14 (s, 2H, CH
2
C
6
H
5 2 6 5
), 7.37 (s, 5H, CH C H );
+
1
ESI-MS: (m/z) 266 (M+H) .
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(R)-2-(Benzyloxycarbonylamino)-4-methylpentanoic acid (2e)
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The compound was obtained as a gummy substance in quantitative yield. H NMR (300 MHz,
CDCl
3 3 2 3 2
) : δ 0.98 (d, J = 6.5 Hz, 6H, CH (CH ) , 1.55-1.61 (m, 1H, CH(CH ) , 1.70-1.75 (m,
2H, CH
2
CH(CH
3
)
2
, 4.43 (brs, 1H, NCH), 5.14 (s, 2H, CH
2
C
6
H
5
2 6 5
), 7.37 (s, 5H, CH C H );
+
ESI-MS: (m/z) 266 (M+H) .
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2-(Benzyloxycarbonylamino)-4-methylpentanoic acid (2f)
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The compound was obtained as a gummy substance in quantitative yield. H NMR (300 MHz,
CDCl
3
) : δ 0.98 (d, J = 6.5 Hz, 6H, CH (CH
3
)
2
, 1.55-1.61 (m, 1H, CH(CH
3
)
2
, 1.70-1.75 (m,
2H, CH
2
CH(CH
3
)
2
, 4.43 (brs, 1H, NCH), 5.14 (s, 2H, CH
2
C
6
H
5
), 7.37 (s, 5H, CH );
2 6 5
C H
+
ESI-MS: (m/z) 266 (M+H ).
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(2S,3S)-2-(Benzyloxycarbonylamino)-3-methylpentanoic acid (2g)
The compound was obtained as a gummy substance in quantitative yield. H-NMR (300 MHz,
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CDCl
1H, CHCH
6 5
5H, CH C H ); ESI-MS: (m/z) 266 (M+H) .
3
) : δ 0.93-1.08 (m, 6H, CHCH
3
CH
2
CH
3
), 1.15-1.45 (m, 2H, CH
2
CH
3
), 1.45-1.54 (m,
3
), 4.42 (brs,NHCH), 5.28(d, J = 8.3Hz, NHCH), 5.14 (s, 2H, CH C H ),7.37 (s,
2 6 5
+
2
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(S)-2-(Tert-butoxycarbonylamino)-4-(methylthio)butanoic acid (2h)
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The compound was obtained as a gummy substance in quantitative yield. H NMR (CDCl
3
,
300 MHz): δ 1.46 (s, 9H, C (CH
2.60 ( m, 2H, CH SCH
3
)
3
2 3
, δ 1.97-2.05 (m, 2H, CHCH ), 2.13 (s, 3H, SCH ), 2.51-
+
2
3
); ESI-MS: (m/z) 250.3 (M+H) .
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(S)-2-(Tert-butoxycarbonylamino)-3-phenylpropanoic acid (2i)
The compound was obtained as a white solid in quantitative yield. m.p 85-87°C; H NMR
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169
170
171
(CDCl
3
, 300 MHz):δ 1.41(s, 9H, C(CH
3
)
3
, 2.73-3.04 (m, 2H, CH
2
6
C H
5
), 4.07 (br s, 1H,
+
NCH), 7.17-7.27 (m,5H, C
6
H
5
); ESI-MS: (m/z) 266.5 (M+H) .
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(S)-2-(Tert-butoxycarbonylamino)-3-(1H-indol-3-yl)propanoic acid (2j)
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The compound was obtained as a white solid in quantitative yield. m.p 134-136 °C; H NMR
1
(CDCl
3
, 300 MHz):δ 1.42 (s, 9H, C(CH
3
)
3
, 3.21-3.31 (m, 2H, CH
2
-Ind), 4.64 (br s, 1H,
6

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NCH), 5.06 (br s, 1H, NHBoc), 7.02 (s, 1H, Ind-2H), 7.08 -7.21 (m, 2H, Ind-5, 6-H), 7.33 (d,
J = 7.5 Hz, 2H, Ind-4H), 7.59 (d, J =7.8 Hz, 2H, Ind-7H), 8.11(br s, 1H, Ind-NH).
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2.8.3. General Procedure for the Synthesis of 3a-j
°
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A solution of 2a-j (1.0 equiv.) in dry THF, was cooled to -15 C, and after 10 min, N-methyl
morpholine (NMM) (1.2 equiv.) and isobutylchloroformate (IBCF) (1.2 equiv.) were added
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with stirring. After 15 min, a solution of NaBH (2 equiv.) in water (10 mL) was added to this
reaction mixture. The reaction mixture was stirred for 1h. After completion of the reaction (as
monitored by TLC), water (100 mL) added to quench the reaction. Solvent was evaporated
under reduced pressure. The oily residue was taken in EtOAc, organic layer was washed with
3 2 4
5% NaHCO , and finally with brine. The organic layer was dried over anhydrous Na SO ,
evaporated under reduced pressure. The products obtained were purified by the silica gel
column chromatography, using a mixture of EtOAc and hexane as eluent to afford 3a-j.
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Tert-butyl 2-hydroxyethylcarbamate (3a)
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The compound was obtained as a gummy substance in quantitative yield. H NMR (300 MHz,
CDCl
3
): δ 1.46 (s, 9H, C (CH
3
)
3
), 3.30 (d, J = 4.8 Hz, 2H, CH
2
CH
2
OH), 3.72 (d, J = 3.5 Hz,
2H, CH
2
CH OH), 4.96 (s, 1H, CH
2
2
CH OH).
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(S)-Benzyl 1-hydroxypropan-2-ylcarbamate (3b)
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192
193
194
195
The compound was obtained as a white solid in quantitative yield. m.p 134-136°C; HNMR
(300MHz, CDCl
3.85 (m, 1H, CHCH
CH
3
): δ 1.17 (d, J= 6.7 Hz, 3H, CH(CH
3
), 3.53-3.68 (m, 2H, CHCH
2
OH), 3.80-
2
OH), 4.88 (brs, 1H, CHCH
2
OH), 5.10 (s, 2H, CH ),7.35 (s, 5H,
2 6 5
C H
+
2
C
6
H
5
); ESI-MS: (m/z) 209.9 (M+H) .
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(S)-Benzyl 1-hydroxy-3-methylbutan-2-ylcarbamate (3c)
The compound was obtained as a white solid in quantitative yield. m.p 114-116°C; IR (KBr)
197
198
199
200
201
-
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3360, 2975, 2881, 1688, 1523, 1456, 1226, 1057, 738 cm ; HNMR (300 MHz, CDCl
0.94-0.99 (2d, J = 6.7 Hz, 6H, CH(CH ), 1.84-1.89 (m, 1H, CH(CH , 3.52-3.66 (m, 2H,
CHCH OH), 3.68-3.75 (m, 1H, CHCH OH), 4.91 (brs, 1H,CHCH
CH ), 7.37 (m, 5H, C ); ESI-MS: (m/z) 260.2 (M+Na) .
3
): δ
3
)
2
3 2
)
2
2
2
OH), 5.13 (s, 2H,
+
2
C
6
H
5
6 5
H
202
(S)-Benzyl 1-hydroxy-4-methylpentan-2-ylcarbamate (3d)
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206
The compound was obtained as a gummy substance in quantitative yield; IR (neat) 3523,
-
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2925, 1713, 1517, 1466, 1251, 1106 cm ; H NMR (300 MHz, CDCl
6H, CH (CH , 1.61-1.55 (m, 2H, CH CH (CH , 1.75-1.70 (m, 1H, CH (CH
1H, NCH), 5.14 (s, 2H, CH ), 7.37 (s, 5H, CH
3
): δ 0.98 (d, J = 6.5 Hz,
3
)
2
2
3
)
2
3
)
2
, 4.43 (brs,
+
2
C
6
H
5
2
C
6
H
5
); ESI-MS: (m/z) 251.9 (M+Na) .
2
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(R)-Benzyl 1-hydroxy-4-methylpentan-2-ylcarbamate (3e)
The compound was obtained as a gummy substance in quantitative yield; IR (neat) 3523,
208
209
210
211
-
1 1
2925, 1713, 1517, 1466, 1251, 1106 cm ; H NMR (300 MHz, CDCl
6H, CH (CH , 1.61-1.55 (m, 2H, CH CH (CH , 1.75-1.70 (m, 1H, CH (CH
1H, NCH), 5.14 (s, 2H, CH ), 7.37 (s, 5H, CH
3
): δ 0.98 (d, J = 6.5 Hz,
3
)
2
2
3
)
2
3
)
2
, 4.43 (brs,
+
2
C
6
H
5
2
C
6
H
5
). ESI-MS: (m/z) 260.2 (M+Na) .
2
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Benzyl 1-hydroxy-4-methylpentan-2-ylcarbamate (3f)
The compound was obtained as a gummy substance in quantitative yield; IR (neat) 3523,
213
214
215
216
-
1 1
2925, 1713, 1517, 1466, 1251, 1106 cm ; H NMR (300 MHz, CDCl
6H, CH (CH , 1.61-1.55 (m, 2H, CH CH (CH , 1.75-1.70 (m, 1H, CH (CH
1H, NCH), 5.14 (s, 2H, CH ), 7.37 (s, 5H, CH
3
): δ 0.98 (d, J = 6.5 Hz,
3
)
2
2
3
)
2
3
)
2
, 4.43 (brs,
+
2
C
6
H
5
2
C
6
H
5
). ESI-MS: (m/z) 260.2 (M+Na) .
217
Benzyl (2S,3S)-1-hydroxy-3-methylpentan-2-ylcarbamate (3g)
1
218
219
220
221
The compound was obtained as a gummy substance in quantitative yield; H NMR (CDCl
300 MHz): δ 0.88-0.97 (m, 6H, CHCH CH CH ), 1.12-1.21 (m, 2H, CH CH ), 1.44-1.50 (m,
1H, CHCH ), 3.60-3.77 (m, 3H,CHCH OH), 4.94 (brs, CH
2H, CH ), 7.37 (s, 5H, CH
3
,
3
2
3
2
3
3
2
2
OH), 5.66 (brs, NHCH), 5.14 (s,
+
2
C
6
H
5
2
6
C H
5
); ESI-MS: (m/z) 252.0 (M+H) .
222
(S)-Tert-butyl 1-hydroxy-4-(methylthio)butan-2-ylcarbamate (3h)
1
223
224
225
226
The compound was obtained as a gummy substance in quantitative yield; H NMR (CDCl
300 MHz) :δ 1.46 (s, 9H, C (CH , δ 1.78-1.85 (m, 2H, CHCH CH SCH ), 2.13 (s, 3H,
SCH ), 3.61-3.78 ( m, 3H, NCHCH OH); ESI-MS: (m/z)
3
,
3
)
3
2
2
3
SCH
3
), 2.53-2.61 (m, 2H,CH
2
CH
2
3
2
+
236.4 (M+H) .
227
(S)-Tert-butyl 1-hydroxy-3-phenylpropan-2-ylcarbamate (3i)
228
229
230
231
The compound was obtained as a white solid in quantitative yield; m. p 95-97 °C; 3356,
1
2980, 2925, 1684, 1526, 1456, 1316, 1269, 1168, 1007, 885, 775, H NMR (300 MHz,
CDCl
3
) : δ 1.42 (s, 9H, C (CH
3
)
3
), 2.84 (d, J = 7.0 Hz, 2H ,CH
2
C
6
H
5
), 3.49-3.70 (m, 2H,
).
CH OH), 3.87 (brs, 1H, NCH), 4.69 (t, J = 7.2 Hz, 1H, CH
2
2
OH), 7.20-7.30 (m, 5H, C H
6 5
8

232
(S)-Tert-butyl 1-hydroxy-3-(1H-indol-3-yl)propan-2-ylcarbamate (3j)
1
233
234
235
236
237
The compound was obtained as a white solid in quantitative yield; m. p 118-120 °C; H NMR
(300 MHz ,CDCl ): δ 1.42 (s, 9H, C (CH , 2.99 (d, J = 6.72Hz,CH -Indole), 3.57-3.72 (m,
2H, CH OH), 3.98 (brs, 1H, NCH), 4.79 (brs, 1H, CH OH), 7.05 (s, 1H, Ind-2H), 7.10-7.22
3
3
)
3
2
2
2
(m, 2H, Ind-5, 6-H), 7.36 (d, J = 7.9 Hz, 2H, Ind-4H), 7.65 (d, J = 7.6 Hz, 2H, Ind-7H), 8.09
(br s, 1H, Ind-NH).
238
2.8.4. General Procedure for the Synthesis of 4a-j
239
240
241
242
243
244
245
246
To a suspension of 3a-j (1.0 equiv.) in anhydrous THF under nitrogen atmosphere was added
triethylamine (3.0 equiv.). The mixture was cooled to below 0°C. Methanesulfonyl chloride
(3.0 equiv.) was added slowly, keeping the temperature below 5°C, and the reaction mixture
was stirred in an ice bath for 45 min. After completion of the reaction as monitored by the
TLC the reaction mixture was diluted with saturated NaHCO
3
solution, the reaction mixture
SO and concentrated
was extracted with DCM. The combined organic extracts dried over Na
2
4
under reduced pressure to afford 4a-j. These intermediates were used without further
purification.
247
2-(Tert-butoxycarbonylamino)ethyl methanesulfonate (4a)
1
248
249
250
The compound was obtained as a yellow gummy substance in quantitative yield; H NMR
(CDCI
3
, 300 MHz) : δ 1.46 (s, 9H, C (CH
3
)
3
, 3.05 (s, 3H, SO
2
CH
3
), 3.49 (d, J = 5.4 Hz, 2H,
+
CH CH
2
2
OSO ), 4.30 (t, J = 9.9 Hz, 2H, CH
3
2
CH OSO
2
3
); ESI-MS: (m/z) 240 (M+H) .
251
(S)-2-(Benzyloxycarbonylamino) propyl methanesulfonate (4b)
1
252
253
254
255
The compound was obtained as a white solid in quantitative yield; m.p 84-86°C; HNMR
(300MHz, CDCl ) : δ 1.28 (d, J = 6.6 Hz, 3H, CH (CH ), 2.99 (s, 3H, SO CH ), 4.01-4.26
(m, 3H, CHCH OSO CH ), 4.89 (brs, 1H, NHCO), 5.12 (s, 2H, CH ), 7.37 (s, 5H,
CH ); ESI-MS: (m/z) 309 (M+Na) .
3
3
2
3
2
2
3
2 6 5
C H
+
2
6 5
C H
256
(S)-2-(benzyloxycarbonylamino)-3-methylbutyl methanesulfonate (4c)
1
257
258
259
The compound was obtained as a white solid in quantitative yield; m.p 62-64°C; H NMR
(300MHz, CDCl ) :δ 0.89-0.97 (2d, J = 6.6, Hz, 6H, CH(CH , 1.68-1.77 (m, 1H,
CH(CH , 2.96 (s, 3H, SO CH ), 3.57-3.68 (m, 1H, NCH), 4.28 (d, J = 4.1Hz,2H, SOCH
3
3 2
)
3
)
2
2
3
2
),
9

2
60
61
2 6 5 6 5
4.92 (br s,1H, NH), 5.12 (s, 2H, CH C H ), 7.35 (s, 5H, C H ); ESI-MS: (m/z) 338.9
+
2
(M+Na) .
262
(S)-2-(Benzyloxycarbonylamino)-4-methylpentyl methanesulfonate (4d)
1
263
264
265
266
267
The compound was obtained as a yellow solid in quantitative yield; m.p 55-57°C; H NMR
(CDCl , 300 MHz): δ 0.95 (d, J = 3.9 Hz, 6H, CH (CH , 1.51-1.59 (m, 2H, CH CH
(CH , 1.60-1.70 (m, 1H, CH(CH , 2.96 (s, 3H, SO CH ), 3.91-3.99 (m, 1H,
NCHCH OSO ), 4.01-4.47 (m, 2H, CHCH
NHCO), 7.37 (s, 5H, CH
3
3
)
2
2
3
)
2
3
)
2
2
3
2
3
2
OSO
3
), 5.11 (s, 2H, CH
2
6
C H
5
), 5.44 (brs, 1H,
+
2
C
6
H
5
); ESI-MS: (m/z)352.1 (M+Na) .
268
(R)-2-(Benzyloxycarbonylamino)-4-methylpentyl methanesulfonate (4e)
1
269
270
271
272
273
The compound was obtained as a yellow solid in quantitative yield; m.p 55-57°C; H NMR
(CDCl
(CH
NCHCH
NHCO), 7.37 (s, 5H, CH
3
, 300 MHz) δ: 0.95 (d, J = 3.9 Hz, 6H, CH (CH
), 1.60-1.70 (m, 1H, CH(CH ), 2.96 (s, 3H, SO
OSO ), 4.01-4.47 (m, 2H, CHCH
3
)
2
, 1.51-1.59 (m, 2H, CH
2
CH
3
)
2
3
)
2
2
CH ), 3.91-3.99 (m, 1H,
3
2
3
2
OSO
3
), 5.11 (s, 2H, CH
2
6
C H
5
), 5.44 (brs, 1H,
+
2
C
6
H
5
); ESI-MS: (m/z)352.1 (M+Na) .
274
2-(Benzyloxycarbonylamino)-4-methylpentyl methanesulfonate (4f)
1
275
276
277
278
279
The compound was obtained as a yellow solid in quantitative yield; m.p 55-57°C; H NMR
(CDCl
(CH
NCHCH
NHCO), 7.37 (s, 5H, CH
3
, 300 MHz) : δ 0.95 (d, J = 3.9 Hz, 6H, CH (CH
), 1.60-1.70 (m, 1H, CH(CH ), 2.96 (s, 3H, SO
OSO ), 4.01-4.47 (m, 2H, CHCH
3
)
2
), 1.51-1.59 (m, 2H, CH
CH ), 3.91-3.99 (m, 1H,
), 5.44 (brs, 1H,
2
CH
3
)
2
3
)
2
2
3
2
3
2
OSO
3
2 6 5
), 5.11 (s, 2H, CH C H
+
2
C
6
H
5
); ESI-MS: (m/z)352.1 (M+Na) .
280
(2S, 3S)-2-(Benzyloxycarbonylamino)-3-methylpentyl methanesulfonate (4g)
1
281
282
283
284
285
The compound was obtained as a yellow solid in quantitative yield; m.p 55-57°C; H NMR
(CDCl , 300 MHz) : δ 0.87-0.95 (m, 6H, CHCH CH CH ), 1.07-1.19 (m, 2H, CH CH ),
1.31-1.50 (m, 1H, CHCH ), 2.90 (s, 3H, SO CH ), 3.65-3.73 (m, 1H, CHCH OSO ), 4.08-
4.46 (m, 2H, CHCH OSO ),5.24 (s, 2H, CH ), 5.39 (br s, 1H, NHCO),7.38 (s, 5H,
CH ).
3
3
2
3
2
3
3
2
3
2
3
2
3
2 6 5
C H
2
6 5
C H
286
(S)-2-(Tert-butoxycarbonylamino)-4-(methylthio)butyl methanesulfonate (4h)
1
2
87
88
The compound was obtained as a gummy substance in quantitative yield; H NMR (CDCl
3
,
2
3 3 2 2 3
300 MHz) :δ 1.43 (s, 9H, C (CH ) ), 1.81-2.01 (m, 2H, CHCH CH SCH ), 2.17 (s, 3H,
10

2
89
90
SCH
3
), 2.53-2.61 (m, 2H,CH
2
CH
2
SCH
3
), 3.67 (s, 3H, SO
2
3
CH ), 3.82-3.95 (m, 1H, NCH),
+
2
4.27 (d, J = 4.0Hz,2H, SOCH
2
); ESI-MS: (m/z) 313.5 (M+H) .
291
(S)-2-(Tert-butoxycarbonylamino)-3-phenylpropyl methanesulfonate (4i)
1
292
293
294
295
The compound was obtained as a yellow gummy substance in quantitative yield; H NMR
(300 MHz, CDCl ) : δ 1.43 (s, 9H, C (CH ), 2.84-2.97 (m, 2H, CH ), 3.03 (s, 3H,
SO CH ), 3.85 (brs, 1H, NCH), 4.12-4.27 (m, 2H, SOCH ), 4.71 (brs, NH), 7.22-7.33 (m,
5H, C ).
3
3
)
3
2 6 5
C H
2
3
2
6
H
5
296
(S)-2-(Tert-butoxycarbonylamino)-3-(1H-indol-3-yl)propyl methanesulfonate (4j)
1
297
298
299
300
301
The compound was obtained as a yellow gummy substance in quantitative yield; H NMR
(300 MHz, CDCl
3
): δ 1.43 (s, 9H, C(CH
3 3 2 3 2
) ), 2.98 (s, 3H, SO CH ), 2.72-2.86 (m, CH -
Indole), 3.01-3.13 (m, 2H, SOCH
2
), 4.28 (br s, 1H, NCH), 5.15(br s,1H,NHBoc), 7.05 (s, 1H,
Ind-2H), 7.10-7.22 (m, 2H, Ind-5, 6-H), 7.36 (d, J = 7.9 Hz, 2H, Ind-4H), 7.65 (d, J = 7.6
+
Hz, 2H, Ind-7H), 8.17 (br s, 1H, Ind-NH); ESI-MS: (m/z) 369.0 (M+H) .
302
2.8.5. General Procedure for the Synthesis of 5a-j
303
304
305
306
307
308
309
310
To a suspension of 4a-j (1.0 equiv.) in acetonitrile under nitrogen atmosphere was added
triethylamine (2.0 equiv.) and N-methyl piperazine (4.0 equiv.). The reaction mixture was
stirred for 40hrs at room temperature. After completion of the reaction (as monitored by
TLC), the solvent was evaporated under reduced pressure and the residue was dissolved in
DCM, the organic layer was washed with 10% citric acid, finally the aqueous layer was
basified with NaHCO
3
and extracted with DCM, and the organic layer was dried over
Na SO and concentrated under reduced pressure. The products obtained were used for the
2
4
next step without further purification.
311
Tert-butyl 2-(4-methylpiperazin-1-yl) ethylcarbamate (5a)
1
312
313
314
The compound was obtained as a gummy substance in 79% yield; H NMR (CDCl
MHz): 1.46 (s, 9H, C(CH ), 2.29 (s, 3H, NCH ), 2.45-2.48 (m, 10H,
CH cycN(CH CH NCH ), 3.23 (d, J = 4.8 Hz, 2H, CH CH cycN(CH CH NCH ).
3
, 300
δ
3
)
3
3
2
2
2
)
2
3
2
2
2
2
)
2
3
315
(S)-Benzyl 1-(4-methylpiperazin-1-yl) propan-2-ylcarbamate (5b)
1
3
16
17
The compound was obtained as a gummy substance in 89 % yield; H NMR (300 MHz,
CDCl ) : δ 1.19-1.21 (d, J = 6.3 Hz,3H, CH(CH )), 2.30 (s, 3H, NCH ), 2.35-2.58 (m, 10H,
3
3
3
3
11

3
18
19
CHCH
2
cycN(CH
2
CH
2
)
2
NCH
3
3 2 2 6 5
), 3.75-3.78 (m, 1H, CH CHCH ), 5.11 (s, 2H, CH C H ),7.32-
+
3
7.35 (m, 5H, CH
2
C
6
H
5
); ESI-MS: (m/z) 292.3 (M+H) .
320
321
322
323
324
(S)-benzyl3-methyl-1-(4-methylpiperazin-1-yl) butan-2-ylcarbamate (5c)
1
The compound was obtained as a gummy substance in 83% yield. H NMR (300 MHz,
CDCl
11H, CH(CH
CH ), 7.37 (s, 5H, C
3 3 2 3
) : δ 0.87-0.94 (2d, J = 6.8 Hz, 6H, CH(CH ) ), 2.27 (s, 3H, NCH ), 2.32-2.61 (m,
3
)CH
2
cycN(CH
2
CH
2
)
2
NCH
3
), 3.60-3.80 (m, 1H, NHCH), 5.13 (s, 2H,
+
2
C
6
H
5
6
H
5
); ESI-MS: (m/z)320.2 (M+H) .
325
326
327
328
329
330
(S)-Benzyl 4-methyl-1-(4-methylpiperazin-1-yl) pentan-2-ylcarbamate (5d)
1
The compound was obtained as a white solid in 89% yield. H NMR (300 MHz, CDCl
3
) : δ
0.91 (d, J = 6.5 Hz, 6H, CH(CH
3
)
2
), 1.35-1.28 (m, 2H, CH
2
CH(CH
CH
), 7.36-7.30 (m, 5H,
3
)
2
), 1.73-1.64 (m, 1H,
CH(CH ), 2.28 (s, 3H, NCH ), 2.59-2.30 (m, 10H, CH
3
)
2
3
2
cycN(CH
2
2
)
2
NCH ), 3.81-3.76
3
2 6 5
(m, 1H, NHCH), 4.70 (br s, 1H, NHCO), 5.11 (s, 2H, CH C H
+
CH
2
C
6
H
5
); ESI-MS: (m/z)334.2 (M+H) .
331
(R)-Benzyl 4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-ylcarbamate (5e)
1
332
333
334
335
336
The compound was obtained as a white solid in 89% yield. H NMR (300 MHz, CDCl
3
) : δ
0.91 (d, J = 6.5 Hz, 6H, CH(CH
3
)
2
), 1.35-1.28 (m, 2H, CH
2
CH(CH
CH
), 7.36-7.30 (m, 5H,
3
)
2
), 1.73-1.64 (m, 1H,
CH(CH ), 2.28 (s, 3H, NCH ), 2.59-2.30 (m, 10H, CH
3
)
2
3
2
cycN(CH
2
2
)
2
NCH ), 3.81-3.76
3
2 6 5
(m, 1H, NHCH), 4.70 (br s, 1H, NHCO), 5.11 (s, 2H, CH C H
+
CH
2
C
6
H
5
); ESI-MS: (m/z)334.2 (M+H) .
337
Benzyl 4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-ylcarbamate (5f)
1
338
339
340
341
342
The compound was obtained as a white solid in 89% yield. H NMR (300 MHz, CDCl
3
) : δ
0.91 (d, J = 6.5 Hz, 6H, CH(CH
3
)
2
), 1.35-1.28 (m, 2H, CH
2
CH(CH
CH
), 7.36-7.30 (m, 5H,
3
)
2
), 1.73-1.64 (m, 1H,
CH(CH ), 2.28 (s , 3H, NCH ), 2.59-2.30 (m, 10H, CH
3
)
2
3
2
cycN(CH
2
2
)
2
NCH ), 3.81-3.76
3
2 6 5
(m, 1H, NHCH), 4.70 (br s, 1H, NHCO), 5.11 (s, 2H, CH C H
+
CH
2
C
6
H
5
); ESI-MS: (m/z)334.2 (M+H) .
343
Benzyl (2S, 3S)-3-methyl-1-(4-methylpiperazin-1-yl) pentan-2-ylcarbamate (5g)
1
344
345
346
The compound was obtained as a gummy substance in 89% yield. H NMR (CDCl
3
, 300
MHz) : δ 0.87-1.05 (m, 6H, CHCH
3
CH
2
CH
3
), 1.08-1.18 (m, 2H, CH
2
CH
3
), 1.40-1.48 (m, 1H,
), 3.63-3.77 (m,
CHCH ), 2.27 (s, 3H, NCH ), 2.33-2.61 (m, 10H, CH
3
3
2
cycN(CH CH
2
2
)
2
NCH
3
12

3
47
48
2 6 5 2 6 5
1H, NHCH), 4.90 (br s, 1H, NHCH) 5.13 (s, 2H, CH C H ), 7.32-7.38 (m, 5H, CH C H );
+
3
ESI-MS: (m/z) 334.2 (M+H) .
349
350
351
352
353
(S)-tert-butyl 1-(4-methylpiperazin-1-yl)-4-(methylthio) butan-2-ylcarbamate (5h) The
1
compound was obtained as a off-white solid in 85% yield. H NMR (300 MHz, CDCl
3
) : δ
), 2.39-2.67 (m, 12H,
), 3.81-3.88 (m, 1H, NHCH); ESI-MS: (m/z) 318.0
1.97-2.05 (m, 2H, CHCH
2
), 2.13 (s, 3H, SCH
CH NCH
3 3
), 2.25 (s, 3H, NCH
CH
2
SCH
3
CHCH
2
cycN(CH
2
2
)
2
3
+
(M+H) .
354
(S)-Tert-butyl 1-(4-methylpiperazin-1-yl)-3-phenylpropan-2-ylcarbamate (5i)
1
355
356
357
358
The compound was obtained as a off-white solid in 86 % yield. H NMR (300 MHz, CDCl
3
) :
δ 1.42 (s, 9H, C(CH
2.85 (d, J = 5.8 Hz, 2H, CH
(m, 5H, C
3 3 3 2 2 2 2
) ), 2.28 (s, 3H, NCH ), 2.30-2.61 (m, 10H, CH cycN(CH CH ) NCH3),
2
C
6
H
5
), 3.91-4.21 (m, 1H, NCH), 4.59 (br s, 1H, NH), 7.26-7.30
+
6
H
5
); ESI-MS: (m/z) 334.2 (M+H) .
359
(S)-Tert-butyl 1-(1H-indol-3-yl)-3-(4-methylpiperazin-1-yl) propan-2-ylcarbamate (5j)
1
360
361
362
363
364
The compound was obtained as a off-white solid in 86 % yield. H NMR (300 MHz ,CDCl
3
):
δ
1.43 (s, 9H, (CH
C
3 3 3
) ), 2.27 (s, 3H, NCH ), 2.29-2.44 (m, 12H, Ind-
CH
2
CH cycN(CH CH ) , 4.02 (br s, 1H, NCH), 4.66 (br s, 1H, NHBoc), 7.02 (s, 1H, Ind-
2
2
2 2
2H), 7.08-7.20 (m, 2H, Ind-5, 6-H), 7.37 (d, J = 7.8 Hz, 2H, Ind-4H), 7.66 (d, J = 7.3 Hz,
+
2H, Ind-7H), 8.09 (brs, 1H, NH-Ind); ESI-MS: (m/z) 373.2 (M+H) .
365
2.8.6. General Procedure for the Synthesis of 6a-j
366
367
368
369
370
To a suspension of 5a, 5h, 5i and 5j in methanol (1.0 equiv. in 10.0 ml) were added 20%
HCl/dioxane and stirred for 1 h at room temperature. After completion of the reaction, the
solvent was evaporated under reduced pressure. The product was purified by trituration with
diethyl ether. The hydrochloride salt was basified with the triethylamine and the products
obtained were used for the next step without further purification.
371
372
373
374
375
To a solution of 5b, 5c, 5d, 5e, 5f, and 5g, in MeOH (1.0 equiv. in 10.0.ml) 10 % (w/w)
Pd/C was added, and flushed two times with hydrogen gas, and the reaction mixture was
agitated at room temperature for 2 h under hydrogen gas, at 30 Psi. After complete
deblocking of the protecting group (as monitored by the TLC) the reaction mixture was
filtered through Celite and concentrated under reduced pressure to afford the product as a
13

3
76
77
gummy residue. The products obtained were used for the next step without further
purification.
3
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
2-(4-methylpiperazin-1-yl)-ethanamine (6a)
The compound was obtained as a gummy substance in quantitative yield.
(S)-1-(4-methylpiperazin-1-yl) propan-2-amine (6b)
The compound was obtained as a gummy substance in quantitative yield.
(S)-3-methyl-1-(4-methylpiperazin-1-yl)butan-2-amine (6c)
The compound was obtained as a gummy substance in quantitative yield.
(S)-4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-amine (6d)
The compound was obtained as a gummy substance in quantitative yield.
(R)-4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-amine (6e)
The compound was obtained as a gummy substance in quantitative yield.
4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-amine (6f)
The compound was obtained as a gummy substance in quantitative yield.
(2S,3S)-3-methyl-1-(4-methylpiperazin-1-yl)pentan-2-amine (6g)
The compound was obtained as a gummy substance in quantitative yield.
(S)-1-(4-methylpiperazin-1-yl)-4-(methylthio) butan-2-amine (6h)
The compound was obtained as a gummy substance in quantitative yield.
(S)-1-(4-methylpiperazin-1-yl)-3-phenylpropan-2-amine (6i)
The compound was obtained as a gummy substance in quantitative yield.
(S)-1-(1H-indol-3-yl)-3-(4-methylpiperazin-1-yl) propan-2-amine (6j)
The compound was obtained as a gummy substance in quantitative yield.
398
14

399
2.8.7. General Procedure for the Synthesis of 7a-r
400
401
402
403
404
405
The free amines 6a-j were (2.0 equiv.) heated with 4,7-dichloroquinoline (1.0 equiv.) in
presence of phenol. After completion of the reaction (as monitored by the TLC) the reaction
mixture was dissolved in chloroform. The organic layer was washed with 10% aqueous
2 4
NaOH solution and finally with brine. The organic layer was dried over Na SO , and the
solvent was concentrated under reduced pressure. The crude products obtained were purified
by column chromatography by using methanol-chloroform-triethylamine as eluent.
406
7-Chloro-N-(2-(4-methylpiperazin-1-yl) ethyl) quinolin-4-amine (7a)
1
407
408
409
410
411
412
413
414
The compound was obtained as a white solid in 78% yield; m.p 104-106 °C; H NMR (300
MHz, CDCl
3
) : δ 2.34 (s, 3H, NCH
3
), 2.52-2.60 (m, 8H, CH
2
cycN(CH
2
CH
2
)
2
NCH
CH
3
2
), 2.79-
NCH ),
2.83 (t, J = 6.2 Hz, CH
2
cycN(CH CH
2
2
)
2
NCH ), 3.34 (s, 2H, CH
3
2
CH cycN(CH
2
2
2
)
3
6.07 (br s, NH), 6.39 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.39-7.43 (dd, J = 2.1, 8.9 Hz, 1H,
Ar-H quinoline), 7.70 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.98 (d, J = 2.0 Hz, 1H, Ar-H
1
3
3
quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar-H quinoline); CNMR (CDCl , 50 MHz) :δ 45.9,
52.5, 55.3, 99.2, 117.2, 121.1, 125.4, 128.4, 135.0, 148.6, 150.0, 151.6; HRMS calculated for
+
[C20
H
27ClN
4
+H] 305.1528, found 305.1521; ESI-MS: (m/z) 305.1 (M+H) .
415
416
417
418
419
420
421
422
423
424
425
N-(2-(4-methylpiperazin-1-yl)ethyl)-7-(trifluoromethyl)quinolin-4-amine (7b)
1
The compound was obtained as a white solid in 78% yield; m.p 72-74°C; H NMR (300
MHz, CDCl
CH cycN(CH
Hz, 2H, CH CH
3
) : δ 2.34 (s, 3H, NCH
NCH ), 2.83 (t, J = 5.8 Hz, 2H, CH
cycN(CH CH NCH ), 6.09 (brs, NH), 6.48 (d, J = 6.8 Hz, 1H, Ar-H
3
), 2.52 (brs, 4H, CH
2
cycN(CH
2
)
2
), 2.61 (brs, 4H,
2
2
)
2
3
2
cycN(CH
2
CH NCH
2
)
2
3
), 3.35 (d, J = 4.6
2
2
2
2
)
2
3
quinoline), 7.64 (d, J = 8.5 Hz, 1H, Ar-H quinoline), 7.88 (d, J = 8.5 Hz, 1H, Ar-H
1
quinoline), 8.29 (s, 1H, Ar-H quinoline), 8.63 (d, J = 5.0 Hz, 1H, Ar-H quinoline); H NMR
(50 MHz, CDCl
3
) : δ 38.9, 45.9, 52.5, 55.1, 100.1, 120.6, 121.0, 126.7, 127.4, 130.4, 131.1,
+
147.5, 149.6, 152.2; HRMS calculated for [C20
H
21
F
3
N
4
+H] 339.1791, found 339.1788; ESI-
+
MS: (m/z) 339.1 (M+H) .
(S)-7-Chloro-N-(1-(4-methylpiperazin-1-yl) propan-2-yl) quinolin-4-amine (7c)
1
426
427
428
429
The compound was obtained as a white solid in 70% yield; m.p 96-98°C; H NMR (300 MHz,
CDCl
CHCH
6.44 (d, J = 5.2 Hz, 1H, Ar-H quinoline), 7.37-7.40- (dd, J = 1.9, 8.9 Hz,1H, Ar-H
3
) : δ 1.31 (d, J = 6.0 Hz, 3H, CH (CH
3
), 2.26 (s, 3H, NCH
3
), 2.42-2.67 (m, 10H,
2
cycN (CH CH NCH ), 3.66-3.74 (m, 1H, CHCH
2
2
)
2
3
3
CH ), 5.99 (br s, 1H, NHCH),
2
15

430
431
432
433
quinoline), 7.72 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.96 (d, J = 1.8 Hz, 1H, Ar-H
1
3
quinoline), 8.52 (d, J = 5.2 Hz, 1H, Ar-H quinoline); C NMR (CDCl
3
, 50 MHz) : δ 18.8,
45.9, 50.0, 55.1, 62.7, 99.6, 117.9, 121.2, 125.3, 128.6, 134.8, 149.1, 149.8, 151.9; HRMS
+
+
4
calculated for [C17H23ClN +H] 319.1684, found 319.1694; ESI-MS: (m/z)319.2 (M+H) .
434
(S)-N-(1-(4-methylpiperazin-1-yl)propan-2-yl)-7-(trifluoromethyl)quinolin-4-amine (7d)
1
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
The compound was obtained as a white solid in 66% yield; m.p 88-90°C; H NMR (300
MHz, CDCl
3
) : δ 1.33 (d, J = 6 Hz, 3H, CH (CH
3
)), 2.26 (s, 3H, NCH
3
), 2.42-2.75 (m, 10H,
CHCH cycN(CH
2
2
CH NCH ), 3.67-3.79 (m, 1H, CHCH
2
)
2
3
3
CH ), 6.08 (brs, 1H, NHCH), 6.53
2
(d, J = 5.3 Hz, 1H ,Ar-H quinoline), 7.61 (d, J = 8.6 Hz, 1H, Ar-H quinoline), 7.89 (d, J =
8.5 Hz, 1H, Ar-H quinoline), 8.27 (s, 1H, Ar-H quinoline), 8.61 (d, J = 5.1 Hz, 1H, Ar-H
1
3
quinoline); C NMR (CDCl
3
, 75 MHz) : δ 18.7, 45.1, 45.9, 52.9, 55.1, 62.7, 100.6, 120.1,
121.0, 121.2, 122.2, 125.8, 127.6, 130.5, 131.0, 147.7, 149.6, 152.2; HRMS calculated for
+
+
23 3 4
[C18H F N +H] 353.1948; found 353.1946; ESI-MS: (m/z) 353.2 (M+H) .
(S)-7-Chloro-N-(3-methyl-1-(4-methylpiperazin-1-yl) butan-2-yl) quinolin-4- amine (7e)
1
The compound was obtained as a white solid in 78% yield; m.p 95-97°C; H NMR (300
MHz, CDCl
3
) : δ 0.96-1.06 (2d, J = 6.8 Hz, 6H, CH (CH
3 2 3
) ), 2.24 (s, 3H, NCH ), 2.31-2.70
(m, 11H, CH(CH
3
)CH cycN(CH CH NCH ), 3.53-3.59 (m, 1H, NHCH) 5.99 (br s, 1H,
2
2
2
)
2
3
NHCH), 6.43 (d, J = 5.3 Hz, 1H, Ar-H quinoline), 7.38-7.42 (dd, J = 2.0, 8.9 Hz, 1H, Ar-H
quinoline), 7.76 (d, J = 8.8 Hz, 1H, Ar-H quinoline), 7.97 (d, J = 1.9 Hz, 1H, Ar-H
1
3
3
quinoline), 8.52 (d, J = 5.3 Hz, 1H, Ar-H quinoline); C NMR (CDCl , 50 MHz) : δ 17.3,
18.3, 29.6, 45.9 , 53.1, 54.3, 55.1 , 56.7, 99.4, 117.8, 121.1, 125.2, 128.7, 134.85, 150.2,
+
151.9; HRMS calculated for [C19
H
27ClN
4
+H] 347.1997, found 347.2003; ESI-MS: (m/z)
+
347.2 (M+H) .
(S)-N-(3-methyl-1-(4-methylpiperazin-1-yl) butan-2-yl)-7-(trifluoromethyl) quinolin-4-
amine (7f)
1
The compound was obtained as a white -off solid in 78% yield; m.p 87-89°C; H NMR (300
MHz, CDCl
3
): δ 0.97-1.07 (2d, J = 6.8 Hz, 6H, CH (CH
3
)
2
), 2.25-2.29 (m, 1H, CH(CH
3 2
) ),
2.34 (s, 3H, NCH
3
), 2.56-2.82 (m, 10H, CHCH cycN(CH
2
2
CH NCH ), 3.54-3.64 (m, 1H,
2
)
2
3
NHCH), 6.0 (br s, 1H, NHCH), 6.54 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.64 (d, J = 8.3 Hz,
1H, Ar-H quinoline), 8.05 (d, J = 8.7 Hz, 1H, Ar-H quinoline), 8.29 (s, 1H, Ar-H quinoline),
1
3
8.60 (d, J = 5.5 Hz, 1H, Ar-H quinoline); C NMR (CDCl
3
, 50 MHz) : δ 17.3, 18.4, 28.4,
29.2, 29.6, 45.8, 53.0, 54.4, 55.1, 56.7, 100.0, 120.1, 120.9, 121.1, 122.6, 125.3, 127.6, 130.7,
16

+
4
62
63
27 3 4
131.0, 147.8, 150.0, 152.2; HRMS calculated for [C20H F N +H] 381.2161, found
+
4
381.2278. ESI-MS: (m/z) 381.2 (M+H) .
464
(S)-7-Chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)quinolin-4-amine (7g)
1
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
The compound was obtained as a white solid in 72% yield; m. p.103-105 °C H NMR (300
MHz, CDCl
1.53 (m, 2H, CH
(m, 10H, CHCH
3
): δ0.96 (d, J = 6.0 Hz, 3H, CH(CH
CH(CH ), 1.67-1.83 (m, 1H, CH(CH
cycN(CH CH NCH ), 3.68 (d, J = 5.7 Hz,1H, NCH), 5.71 (d, J = 4.1 Hz,
3
), 1.06 (d, J = 6.2 Hz, 3H, CH(CH
3
), 1.44-
2
3
)
2
3
)
2
), 2.27 (s, 3H, NCH ), 2.63-2.41
3
2
2
2
)
2
3
1H, NH), 6.44 (d, J = 5.3 Hz, Ar-H quinoline), 7.37-7.41 (dd, J = 2.0, 8.8 Hz, 1H, Ar-H
quinoline), 7.51 (d, J = 8.9 Hz,1H,Ar-H quinoline), 7.97 (dd, J = 2.0 Hz,1H,Ar-H quinoline),
1
3
8.53 (d, J = 5.3 Hz, 1H,Ar-H quinoline); C NMR (CDCl
3
, 50 MHz) : δ 22.5, 23.3, 42.6,
45.9, 53.3, 55.2, 61.2, 99.2, 117.7, 121.1, 125.3, 128.7, 134.8, 149.2, 149.8, 151.9; HRMS
+
+
4
calculated for [C20H29ClN +H] 361.2154 found 361.2151; ESI-MS: (m/z) 361.2 (M+H) .
(R)-7-Chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)quinolin-4-amine (7h)
1
The compound was obtained as a white solid in 72% yield; m. p. 103-105°C; H NMR (300
MHz, CDCl
1.44-1.51 (m, 2H, CH
2.61 (m, 10H ,CHCH cycN(CH
3
): δ 0.96 (d, J = 6.15 Hz, 3H, CH(CH
CH(CH ), 1.66-1.75 (m, 1H, CH(CH
CH NCH ), 3.66 (d, J = 5.8 Hz, 1H, NCH), 5.70 (d, J = 4.1
3
), 1.06 (d, J = 6.18 Hz, 3H, CH(CH
3
)),
2
3
)
2
3
)
2
), 2.25 (s, 3H, NCH ), 2.40-
3
2
2
2
)
2
3
Hz, 1H, NH), 6.43 (d, J = 5.3Hz, Ar-Hquinoline),7.36-7.39 (dd, J = 1.9, 8.9 Hz, 1H, Ar-H
quinoline), 7.69 (d, J = 8.9 Hz, 1H,Ar-H quinoline), 7.95 (dd, J = 1.8 Hz, 1H, Ar-H
1
3
3
quinoline), 8.52 (d, J = 5.2 Hz, 1H, Ar-H quinoline); CNMR (CDCl , 50 MHz) : δ 22.4,
23.3, 24.9, 42.6, 45.9, 53.3, 55.2, 61.2, 99.2, 117.7, 121.1, 125.2, 128.7, 134.7, 149.2, 149.7,
+
151.9;HRMS calculated for [C20
H
29ClN
4
+H] 361.2154 found 361.2175; ESI-MS: (m/z)
+
361.2 (M+H) .
7-Chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)quinolin-4-amine (7i)
1
The compound was obtained as a white solid in 72% yield; m.p.103-105 °C H NMR (300
MHz, CDCl
1.53 (m, 2H, CH
(m, 10H, CHCH
3
): δ0.96 (d, J = 6.0 Hz, 3H, CH(CH
3
)), 1.06 (d, J = 6.2 Hz, 3H, CH(CH
), 1. 67-1.83. (m, 1H, CH(CH ), 2.27 (s, 3H, NCH ), 2.63-2.41
CH NCH ), 3.68 (d, J = 5.7 Hz,1H, NCH), 5.71 (d, J = 4.1 Hz,
3
)), 1.44-
2
CH(CH
3
)
2
3
)
2
3
2
cycN(CH
2
2
)
2
3
1H, NH), 6.44 (d, J = 5.3 Hz, Ar-H quinoline), 7.37-7.41 (dd, J = 2.0, 8.8 Hz, 1H, Ar-H
quinoline), 7.51 (d, J = 8.9 Hz,1H,Ar-H quinoline), 7.97 (dd, J = 2.0 Hz,1H,Ar-H quinoline),
1
3
8.53 (d, J = 5.3 Hz, 1H,Ar-H quinoline); C NMR (CDCl
3
, 50 MHz) : δ 22.5, 23.3, 42.6,
45.9, 53.3, 55.2, 61.2, 99.2, 117.7, 121.1, 125.3, 128.7, 134.8, 149.2, 149.8, 151.9; HRMS
+
+
4
calculated for [C20H29ClN +H] 361.2154 found 361.2151; ESI-MS: (m/z) 361.2 (M+H) .
17

495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
(S)-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-7-(trifluoromethyl)quinolin-4-
amine (7j)
1
The compound was obtained as a white solid in 72% yield; m. p. 96-98°C; H NMR (300
MHz, CDCl
1.50-1.52 (m, 2H, CH
2.65 (m, 10H ,CHCH
3
) : δ 0.97 (d, J = 6.2 Hz, 3H, CH(CH
3
)), 1.07 (d, J = 6.1 Hz, 3H, CH(CH
), 1.69-1.74 (m, 1H, CH (CH ), 2.27 (s, 3H, NCH ), 2.43-
CH NCH ), 3.70 (d, J = 5.3 Hz, 1H, NHCH), 5.81 (br s,
3
)),
2
CH (CH
3
)
2
3
)
2
3
2
cycN(CH
2
2
)
2
3
1H, NH), 6.53 (d, J = 5.19 Hz, Ar-H quinoline), 7.36-7.39 (dd, J = 1.9, 8.9 Hz, 1H, Ar-H
quinoline), 7.89 (d, J = 8.4 Hz, 1H, Ar-H quinoline), 8.28 (s, 1H, Ar-H quinoline), 8.63 (d, J
1
3
= 5.1 Hz, 1H, Ar-H quinoline); C NMR (50 MHz, CDCl
3
) : δ 22.4, 23.3, 24.9, 42.4, 45.9,
53.3, 55.2, 61.2, 100.2, 120.0, 120.9, 127.6, 130.4, 131.1, 147.8, 149.5, 152.2; HRMS
+
+
29 3 4
calculated for [C21H F N +H] 395.2417, found 395.2410. ESI-MS: (m/z) 395.2 (M+H) .
7-Chloro-N-(2S,3S)-3-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)quinolin-4-amine
(7k)
1
The compound obtained as a white solid in 66% yield; m. p 101-103°C; H NMR (300 MHz,
CDCl
(m, 2H, CH
2.42 (m, 10H, CHCH
3
) : δ 0.92 (d, J = 6.7 Hz, 3H, CHCH
CH CHCH ), 1.50-1.71 (m, 1H, CH
cycN(CH CH NCH ), 3.68-3.63 (m,1H, NHCH), 5.92 (br s,1H,
3
), 1.02 (t, J = 7.3 Hz, 3H, CH
2
CH
3
), 1.39-1.48
3
2
3
3
CH CHCH ), 2.26 (s, 3H, NCH
2
3
3
), 2.74-
2
2
2
)
2
3
NHCH), 6.45 (d, J = 5.6 Hz, 1H, Ar-H quinoline), 7.42-7.45 (t, J = 6.9 1H, Ar-H quinoline),
7.88 (d, J = 1.7 Hz, 1H, Ar-H quinoline), 8.03 (d,. J = 1.7 Hz, 1H, Ar-H quinoline), 8.50 (d.
1
3
J = 5.4 Hz, 1H, Ar-H quinoline); C NMR (CDCl
3
, 50 MHz) : δ 12.3, 14.0, 25.9, 35.8, 45.7,
55.0, 56.0, 99.2, 117.7, 121.5, 125.4, 127.9, 135.2, 148.3, 150.5, 151.1; HRMS calculated for
+
+
[C20
H
29ClN
4
+H] 361.2154 found 361.2172; ESI-MS: (m/z) 361.2 (M+H) .
3S)-3-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-7
quinolin-4-amine (7l)
N-(2S,
(trifluoromethyl)
1
The compound was obtained as a pale yellow solid in 60% yield; m.p 97-99 °C; H NMR
(300MHz, CDCl
1.33-1.44 (m, 2H, CH
2.50-2.80 (m, 10H, CHCH
3
): δ 0.95 (d, J = 6.7 Hz, 3H, CHCH
CH CHCH ), 1.51-1.60 (m, 1H, CH
cycN(CH CH NCH
3
), 1.05 (t, J = 7.2 Hz, 3H, CH
2
CH
3
),
),
3
2
3
3
CH CHCH ), 2.31 (s, 3H, NCH
2
3
3
2
2
2
)
2
3
), 3.70 (d, J = 4.5 Hz, 1H, NHCH), 5.94 (br
s, 1H, NHCH), 6.52 (d, J = 5.2 Hz, 1H, Ar-H quinoline), 7.64 (d, J = 7.9 Hz, 1H, Ar-H
quinoline), 7.99 (dd, J = 8.2 Hz, 1H, Ar-H quinoline), 8.29 (s,1H, Ar-H quinoline), 8.62 (d. J
1
3
3
= 5.3 Hz, 1H, Ar-H quinoline); C NMR (50 MHz, CDCl ) : 12.3, 13.9, 25.9, 35.6, 45.7,
53.0, 55.0, 56.0, 100.3, 120.1, 121.1, 122.6, 125.3, 129.3, 130.7, 131.0, 147.6, 149. 9, 152.0;
+
HRMS calculated for [C21
H
29
F
3
N
4
+H] 395.2417, found 395.2410. ESI-MS: (m/z) 395.2
+
(M+H) .
18

529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
(S)-7-Chloro-N-(1-(4-methylpiperazin-1-yl)-4-(methylthio)butan-2-yl)quinolin-4-amine
(7m)
1
The compound was obtained as a off-white solid in 65% yield; m.p 92-94°C; H NMR (300
MHz, CDCl
3
): δ 1.97-2.05 (m, 2H, CHCH
2
), 2.12 (s, 3H, SCH
3 3
), 2.25 (s, 3H, NCH ), 2.37-
2.66 (m, 12H, CH
2
SCH CHCH cycN(CH CH
3
2
2
2
)
2
NCH ), 3.80-3.87 (m, 1H, NHCH), 5.77 (d,
3
J = 1H, 3.78 Hz, NH), 6.53 (d, J = 4.05 Hz, 1H, Ar-H quinoline), 7.76 (d , J = 6.9 Hz, 1H,
Ar-H quinoline), 7.71 (d, J = 6.7 Hz, 1H, Ar-H quinoline), 7.97 (d, J = 1.17 Hz, 1H, Ar-H
1
3
3
quinoline), 8.53 (d, J = 4.0 Hz, 1H, Ar-H quinoline); C NMR (50 MHz, CDCl ): δ 15.8,
30.2, 32.1, 42.4, 45.4, 45.9, 50.7, 54.5, 55.0, 59.8. 99.0, 117.4, 121.5, 125.5, 128.4, 128.5,
+
135.1, 149.1, 151.7, 151.8; HRMS calculated for [C19
H
27ClN
4
S+H] 379.1718, found
+
379.1716; ESI-MS: (m/z) 379.2 (M+H) .
(S)-N-(1-(4-methylpiperazin-1-yl)-4-(methylthio)butan-2-yl)-7-
(trifluoromethyl)quinolin-4-amine (7n)
The compound was obtained as a white solid in 65% yield; m.p 89-91°C; H NMR (300 MHz,
CDCl ): δ 1.97-2.09 (m, 2H, CHCH ), 2.13 (s, 3H, SCH ), 2.27 (s, 3H, NCH ), 2.44-2.73 (m,
12H, CH SCH CHCH cycN(CH CH NCH ), 3.57-3.62 (m, 1H, NHCH), 5.90 (d, J = 3.7
3
2
3
3
2
3
2
2
2
)
2
3
Hz, 1H, NH), 6.63 (d, J = 4.0 Hz, 1H, Ar-H quinoline), 7.60-7.63 (dd, J = 1.2, 6.5 Hz, 1H,
Ar-H quinoline), 7.91 (d, J = 6.5 Hz, 1H, Ar-H quinoline), 7.97 (s, 1H, Ar-H quinoline), 8.62
1
3
3
(d, J = 4.02 Hz, 1H, Ar-H quinoline); C NMR (50 MHz, CDCl ): δ15.9, 30.7, 32.1, 42.4,
45.4, 45.9, 51.7, 54.5, 55.4, 59.8. 99.0, 117.4, 121.5, 125.5, 128.4, 128.5, 135.1, 149.1, 151.7,
+
151.8; HRMS calculated for [C20
H
27
F
3
N
4
S+H] 413.1981, found 413.1993; ESI-MS: (m/z)
+
413.2 (M+H) .
(S)-7-Chloro-N-(1-(4-methylpiperazin-1-yl)-3-phenylpropan-2-yl)quinolin-4-amine (7o)
1
The compound was obtained as a yellowish white solid in 63% yield; m.p 98-100 °C H
NMR (300 MHz, CDCl
CH cycN(CH CH NCH ), 2.92-3.13 (m, CH
4.4Hz, 1H, NHCH), 6.50 (d, J = 5.3 Hz, 1H, Ar-H quinoline), 7.16-7.29 (m, 5H, CH
3
)
:
δ
2.24 (s, 3H, NCH
), 3.83-3.90 (m, 1H, NCH), 5.72 (d, J =
),
3
), 2.40-2.79 (m, 10H,
2
2
2
)
2
3
2 6 5
C H
2
6 5
C H
7.36-7.40 (dd, J = 2.0, 8.9 Hz, 1H, Ar-H quinoline), 7.66 (d, J = 8.9 Hz, 1H, Ar-H
quinoline), 7.97 (d, J = 2.0 Hz, 1H, Ar-H quinoline), 8.54 (d, J = 5.1 Hz, 1H, Ar-H
1
3
3
quinoline); CNMR (CDCl , 50 MHz) :δ 38.1, 45.8, 55.1, 59.8, 99.4, 117.7, 121.7, 125.5,
126.7, 128.5, 129.6, 135.0, 137.0, 138.3, 148.9, 149.6, 151.7; HRMS calculated for
+
[C23
H
27ClN
4
+H] 394.1997 found 395.1995; ESI-MS: (m/z) 395.4
(S)-N-(1-(4-methylpiperazin-1-yl)-3-phenylpropan-2-yl)-7-(trifluoromethyl) quinolin-4-
amine (7p)
19

1
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
The compound was obtained as a yellowish white solid in 65% yield; m.p 91-93°C; H NMR
(300MHz, CDCl ): δ2.24 (s, 3H, NCH ), 2.40-2.66 (m, 10H, CH cycN(CH CH NCH ),
2.92-3.14 (m, CH ), 3.92 (s, 1H, NCH), 5.83 (brs, 1H, NHCH), 6.58 (d, J = 5.1 Hz, 1H,
Ar-H quinoline), 7.19-7.30 (m, 5H, CH ), 7.60 (d, J = 8.3 Hz, 1H, Ar-H quinoline), 7.84
(d, J = 8.4 Hz, 1H, Ar-H quinoline), 8.28 (s, 1H, Ar-H quinoline), 8.63 (d, J = 5.2 Hz, 1H,
3
3
2
2
2
)
2
3
2
6 5
C H
2
6 5
C H
1
3
Ar-H quinoline); C NMR (CDCl
3
, 50 MHz) :δ 38.0, 45.9, 50.3, 52.9, 55.1, 59.8, 100.4,
120.2, 121.03, 126.7, 127.6, 128.5, 129.6, 130.5, 131.2, 136.9, 147.8, 149.2, 152.2;HRMS
+
+
27 3 4
calculated for [C24H F N +H] 429.2261, found 429.2272; ESI-MS: (m/z) 429.2 (M+H) .
(S)-N-(1-(1H-indol-3-yl)-3-(4-methylpiperazin-1-yl)propan-2-yl)-7-chloroquinolin-4-
amine (7q)
1
The compound was obtained as a pale yellow solid in 60% yield; m.p 102-104 °C; H NMR
(300 MHz, CDCl
3.16-3.30 (m, CH
3
): δ 2.29 (s, 3H, NCH
3 2 2 2 2 3
), 2.35-2.69 (m, 10H, CH cycN(CH CH ) NCH ),
2
-Indole), 4.05 (d, J = 4.7 Hz, 1H, NHCH), 5.75 (br s, 1H, NHCH), 6.56 (d,
J = 5.3 Hz, 1H, Ar-H quinoline), 7.00 (d, J = 2.0 Hz, 1H, Ind-2H), 7.09-7.23 (m, 2H, Ind-5,
6-H), 7.33-7.36 (m, 3H, Ind-4H and Ar-H quinoline), 7.61 (d, J = 2.9 Hz, 2H, Ind-7H), 8.00
(d, J = 1.9 Hz, 1H, Ar-H quinoline), 8.21 (d, J = 9.3 Hz, 1H, Ar-H quinoline),8.54 (d, J =
1
3
3
5.4Hz, 1H, Ar-H quinoline); C NMR (CDCl , 50MHz) : δ 29.6, 45.9, 55.1, 60.0, 99.4,
110.7, 111.3, 117.6, 118.8, 119.6, 121.3, 122.1, 123.1, 125.3, 128.0, 128.5, 134.9, 136.2,
+
149.1, 149.6, 151.8; HRMS calculated for [C25
H28ClN
5
+H] 434.9763, found 434. 9743; ESI-
+
MS: (m/z) 434.3 (M+H) .
(S)-N-(1-(1H-indol-3-yl)-3-(4-methylpiperazin-1-yl)propan-2-yl)-7-
(trifluoromethyl)quinolin-4-amine (7r)
1
The compound was obtained as a pale yellow solid in 60% yield; m.p 96-98 °C; H NMR
(300 MHz, CDCl ) : δ 2.26 (s, 3H, NCH ), 2.41-2.65 (m, 10H, CH cycN(CH CH NCH ),
3.20-3.25 (m, 2H, CH -Indole), 4.04 (d, J = 4.0 Hz, 1H, NHCH), 5.79 (d, J = 3.7 Hz,
3
3
2
2
2
)
2
3
2
NHCH), 6.63 (d, J = 5.3 Hz, 1H, Ar-H quinoline), 7.09 (d, J = 2.0 Hz, 1H, Ind-2H), 7.10-
7.23 (m, 2H, Ind-5, 6-H), 7.54 (d, J = 1.9 Hz,1H, Ar-H quinoline), 7.60 (d, J = 6.1 Hz, Ind-
4H ), 7.73 (d, J = 2.9 Hz, 1H, Ind-7H), 8.18 (d, J = 6.4 Hz, 1H, Ar-H quinoline), 8.27 (s, 1H,
1
3
3
Ar-H quinoline),8.61 (d, J = 5.3Hz, 1H, Ar-H quinoline); C NMR (50 MHz, CDCl ): δ
29.6, 45.7, 45.8, 49.8, 53.0, 54.9, 55.1, 60.0, 61.1, 110.4, 110.6, 111.3, 118.7, 119.6, 121.0,
121.1, 122.1,122.6, 123.5, 125.3, 127.4, 127.9, 130.7, 131.0, 136.2, 147.7, 149.4, 152.2;
+
HRMS calculated for [C26
H
28
F
3
N
5
+H] 468.2370, found 434. 2368; ESI-MS: (m/z) 468.1
+
(M+H) .
20

597
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599
600
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602
603
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605
606
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608
609
610
611
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615
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623
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629
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2.8.8. General Procedure for the synthesis of 8a-b
Starting from glycine and α-phenylalanine, compounds 8a-b were prepared by the procedure
described for (2b).
2-(benzyloxycarbonylamino)acetic acid (8a)
1
The compound was obtained as a white solid in quantitative yield; m.p 119-120°C; H-NMR
(300 MHz, CDCl
3
): δ 3.67 (s, 2H, CH
2
), 5.07 (s, 2H, CH
2
), 7.35 (s, 5H, OCH
2
C
6
H
5
); ESI-
+
MS: (m/z) 210 (M+H) .
(S)-2-(benzyloxycarbonylamino)-3-phenylpropanoic acid (8b)
1
The compound was obtained as a white solid in quantitative yield; m.p 100-102°C; H NMR
(300 MHz, CDCl
NHCH), 5.09 (s, 2H, OCH
2.8.9. General procedure for the synthesis of 9a-b
3
): δ 2.80 (d, J = 7.0 Hz, 2H, CH
2
Ph), 4.50 (m, 1H, NHCH), 4.95 (s, 1H,
2
Ph), 7.13-7.28 (m, 10H, ArH).
To a stirred solution of N-Cbz protected amino acids 8a-b (1.0 equiv.) in dry THF at - 15°C
under nitrogen atmosphere were added successively isobutylchloroformate (IBCF) (1.1
equiv.), and N-methylmorpholine (NMM) (1.1 equiv.).The mixture was stirred for 15 min,
2
and then treated drop wise with an ethereal solution of excess CH N2. The yellow solution
was allowed to warm to room temperature and stirring was continued until there was no N-
protected amino acid remaining (TLC control). The reaction mixture was concentrated under
reduced pressure, and the residue was taken up in EtOAc. The organic phase was washed
successively with aqueous NaHCO
3
solution and brine. The organic layer was dried over
Na SO and concentrated under reduced pressure. The products were purified by the silica gel
2
4
column chromatography (EtOAc/hexane) to obtain the diazoketones 9a-b.
Benzyl 3-diazo-2-oxopropylcarbamate (9a)
1
The compound was obtained as a white off solid in 80% yield; m.p 68-69°C; H NMR (300
MHz, CDCl ) δ 3.97 (d, J = 4.8 Hz, NHCH ), 5.12 (s, 2H, CH Ph), 5.37 (br s, 1H, CHN ),
5.54 (brs, 1H, NH), 7.35 (5 H, m, C ).
(S)-benzyl 4-diazo-3-oxo-1-phenylbutan-2-ylcarbamate (9b)
The compound was obtained as a yellow solid in 80% yield; m.p 80-81 C; H NMR (300
MHz, CDCl ): δ 3.03 (d, 2H, J = 6.8 Hz, CH Ph); 4.45-4.49 (m, 1H, CHNH), 5.21 (s, 2H,
OCH Ph), 5.31 (s, 1H, CHN ), 5.35 (1H, br, NH), 7.18-7.42 (m, 10H, ArH).
3
2
2
2
6
H
5
°
1
3
2
2
2
2.8.10. General procedure for the synthesis of 10a-b
To a solution of the diazo ketone 9a-b (1.0 equiv.) in MeOH at -25 °C under N
2
with the
N (2.9
exclusion of light was treated with a solution of silver benzoate (0.11 equiv.) in Et
3
21

631
632
633
634
635
636
637
638
639
640
641
642
643
equiv.).The reaction mixture was allowed to warm to room temperature within 3.0 h in the
dark and then concentrated under reduced pressure. The oily residue dissolved in EtOAc and
2 4
washed with brine solution. The organic layer was dried over Na SO and concentrated under
reduced pressure. The residue was purified by the silica gel column chromatography.
Methyl 3-(benzyloxycarbonylamino) propanoate (10a)
°
1
The compound was obtained as a yellow solid in 75% yield; m.p 80-81 C; H NMR (300
MHz, CDCl ): δ2.41-2.51 (m, 2H, CH COOCH ), 3.43-3.51 (m, 2H, NHCH ), 3.65 (s, 3H,
OCH ), 5.09 (s, 2H, CH Ph), 5.30 (1H, br, NH), 7.35 (m, 5H, ArH).
(S)-Methyl 3-(benzyloxycarbonylamino)-4-phenylbutanoate (10b)
The compound was obtained as a gummy substance in 75% yield; H NMR (300 MHz,
CDCl ): δ 2.49-2.53 (m, 2H, CH COOCH ), 2.80-2.99 (m, 2H, CH Ph), 3.67 (s, 3H, OCH ),
Ph), 5.29 (d, J = 7.3 Hz, NH), 7.15-7.35 (m,
3
2
3
2
3
2
1
3
2
3
2
3
4.17-4.24 (m, 1H, NHCH), 5.07 (s, 2H, OCH
2
+
10H, ArH); ESI-MS: (m/z) 328.3 (M+H) .
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
2.8.11. General procedure for the synthesis of 11a-b
MeOH was added drop wise over a period of 20 min to a mixture of ester of N- protected
amino acid (10a-b, 1.0 equiv.) and NaBH (2 equiv.) in THF at 50-55 °C. The mixture was
4
stirred for 10-25 min, and then water was added to the reaction mixture. Organic solvent was
concentrated under reduced pressure. The residue was taken in EtOAc and extracted with
2 4
brine. The organic layer was dried over Na SO and concentrated under reduced pressure.
The residue was purified by the silica gel column chromatography using hexane:EtOAc as
eluent.
Benzyl 3-hydroxypropylcarbamate (11a)
1
The compound was obtained as a gummy substance in 75% yield; H NMR (300 MHz,
CDCl
3H, CH
(S)-benzyl 4-hydroxy-1-phenylbutan-2-ylcarbamate (11b)
3
): δ 1.47-1.59 (m, 2H, CH
2
CH
2
CH
2
OH),2.87-3.31 (m,CH
2
CH
2
CH
2
OH), 3.47-3.61 (m,
2
CH CH OH), 3.94 (brs, 1H, NHCH), 5.09 (s, 2H, O-CH
2
2
2
Ph), 7.35 (s, 5H, Ar-H).
1
The compound was obtained as a gummy substance in 80% yield. H NMR (300MHz,
CDCl
3
): δ
1.80-1.85 (m, 2H, CH
2
CH
2
OH),2.82-2.87 (m,CH
2
C
6
H
5
), 3.65 (s, 1H,
CH
2
2
CH
2
OH),3.94 (br s, 1H, NHCH), 4.72-4.75 (m, 2H, CH CH OH), 5.07 (s, 2H, O-
2 2
+
CH
Ph), 7.15-7.35 (m, 10H, Ar-H); ESI-MS: (m/z) 300 (M+Na) .
2.8.12. General procedure for the synthesis of 12a-b
Compounds 12a-b were prepared by similar method described for 4a.
3-(Benzyloxycarbonylamino)propyl methanesulfonate (12a)
22

1
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667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
The compound was obtained as a gummy substance in quantitative yield. H NMR (300MHz,
CDCl ): δ 1.65-1.79 (m, 2H, CH CH CH OSO CH ), 2.87-3.07 (m,CH CH CH OSO CH ),
3.15 (s, 3H, OSO CH ), 3.91(br s, 1H, NHCH), 4.07 (t, 4.7
Hz,2H,CH CH CH OSO CH ), 5.07 (s, 2H, O-CH Ph), 7.35 (s, 5H, Ar-H).
3
2
2
2
2
3
2
2
2
2
3
2
3
J
=
2
2
2
2
3
2
(S)-3-(Benzyloxycarbonylamino)-4-phenylbutyl methanesulfonate (12b)
1
The compound was obtained as a gummy substance in quantitative yield. H NMR (300MHz,
CDCl
OSO
CH Ph), 7.15-7.35 (m, 10H, Ar-H); ESI-MS: (m/z) 400 (M+Na) .
2.8.13. General procedure for the synthesis of 13a-b
3
): δ 1.69-1.80 (m, 2H, CH
2
CH
2
OSO
2
CH
3
),2.88-2.98 (m,CH
2
C
6
H
5
), 3.15 (s, 3H,
2
CH ), 3.96 (br s, 1H, NHCH), 4.26 (t, J = 4.5 Hz, CH
3
2
CH OSO
+
2
2
CH ), 5.08 (s, 2H, O-
3
2
Compounds 12a-b were prepared by similar method described for 5a
Benzyl 3-(4-methylpiperazin-1-yl)propylcarbamate (13a)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (300MHz,
CDCl
3
): δ 1.95 (s, 2H, CH
CH cycN(CH CH
2
CH
2
cycN(CH
2 2 2 3 3
CH ) NCH ), 2.41 (s, 3H, NCH ), 2.61-2.67 (m,
10H, CH
2
2
2
2
)
2
NCH
3
), 3.35-3.42 (m, 2H, NHCH )
2 .
(S)-Benzyl 4-(4-methylpiperazin-1-yl)-1-phenylbutan-2-ylcarbamate (13b)
1
The compound was obtained as a gummy substance in 73% yield. H NMR (300 MHz,
CDCl
3
): δ1.67-1.79 (m, 2H, CH
2
CH
2
cycN(CH
2 2 2 3 3
CH ) NCH ), 2.26 (s, 3H, NCH ), 2.32-2.49
(m, 10H, CH
2
CH cycN (CH CH
2
2
2
)
2
NCH
3
), 2.76-3.09 (m,CH
2
C
6
H
5
), 3.94 (br s, 1H, NHCH),
+
6.0 (brs, 1H, NHCH), 7.15-7.35 (m, 10H, Ar-H); ESI-MS: (m/z) 382.3 (M+H) .
2.8.14. General procedure for the synthesis of 14a-b
Compounds 15a-c were prepared by similar method described for 6a
3-(4-methylpiperazin-1-yl) propan-1-amine (14a)
The compound was obtained as a gummy substance and used without further purification.
(S)-4-(4-methylpiperazin-1-yl)-1-phenylbutan-2-amine (14b)
The compound was obtained as a gummy substance and used without further purification.
2.8.15. General procedure for the synthesis of 15a-c
The final compounds 15a-c were prepared by similar method described for 7a-r.
7-Chloro-N-(3-(4-methylpiperazin-1-yl)propyl)quinolin-4-amine (15a)
°
1
The compound obtained as off-white solid in 75 % yield. m.p 108-110 C; H NMR (300
MHz, CDCl ) : δ 1.97 (s, 2H, CH CH cycN(CH CH NCH ), 2.41 (s, 3H, NCH ), 2.62-2.68
(m, 10H, CH CH cycN(CH CH NCH ), 3.37-3.43 (m, 2H, NHCH 6.34 (d, J = 5.4 Hz,
3
2
2
2
2
)
2
3
3
2
2
2
2
)
2
3
2 ,
)
1H, Ar-H quinoline), 7.33-7.37 (dd, J = 2.0, 8.8 Hz, 1H, Ar-H quinoline), 7.91 (d, J = 8.9
Hz, 1H, Ar-H quinoline), 7.97 (d, J = 1.9 Hz 1H, Ar-H quinoline), 8.51 (d, J = 5.2 Hz, 1H,
23

1
3
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
Ar-H quinoline); C NMR (50 MHz, CDCl
3
) : δ 23.4, 44.3, 46.2, 53.5, 55.2, 58.6,
98.4,117.4, 122.4, 124.6, 128.3, 134.7, 148.8, 150.6, 151.8; HRMS calculated for
+
+
[C17
H
23ClN
4
+H] 319.1684, found 319.1683; ESI-MS: (m/z) 319.3 (M+H) .
N-(3-(4-methylpiperazin-1-yl) propyl)-7-(trifluoromethyl)quinolin-4-amine (15b)
°
1
The compound obtained as off-white solid in 70 % yield. m.p 76-78 C; H NMR (300 MHz,
CDCl ): δ 1.95-2.00 (m, 2H, CH CH cycN(CH CH NCH ), 2.41 (s, 3H, NCH ), 2.63-2.68
(m, 10H, CH CH cycN(CH CH NCH ), 3.38-3.42 (m, 2H, NHCH 6.41 (d, J = 4.05 Hz,
3
2
2
2
2
)
2
3
3
2
2
2
2
)
2
3
2 ,
)
1H, Ar-H quinoline), 7.53-7.56 (dd, J = 1.2, 6.5 Hz, 1H, Ar-Hquinoline), 8.07 (d, J = 6.5 Hz,
1H, Ar-H quinoline), 8.25 (s, 1H, Ar-H quinoline), 8.59 (d, J = 4.0 Hz, 1H, Ar-H quinoline);
1
3
C NMR (75 MHz, CDCl
3
): δ 23.3, 44.4, 46.3, 53.5, 55.2, 58.6, 99.4, 119.3, 119.3, 120.7,
122.2, 125.9, 127.4, 127.5, 130.4, 130.9, 131.3, 147.6, 150.4, 152.4; HRMS calculated for
+
+
23 3 4
[C18H F N +H] 353.1948, found 353.1951; ESI-MS: (m/z) 353.2 (M+H) .
(S)-7-Chloro-N-(4-(4-methylpiperazin-1-yl)-1-phenylbutan-2-yl)quinolin-4-amine (15c)
1
The compound was obtained as a gummy substance in 45 % yield. H NMR (300 MHz,
CDCl
(m, 10H, CH
6.40 (d, J = 4.2 Hz, 1H, Ar-H quinoline), 7.11-7.27 (m, 5H, CH
1H, Ar-H quinoline), 7.88 (d, J = 8.7 Hz, 1H, Ar-H quinoline), 7.95 (d, J = 6.5 Hz, 1H, Ar-H
3
) : δ 1.67-1.89 (m, 2H, CH
2
CH
2
cycN (CH
2
CH
2
)
2
NCH
3
), 2.28 (s, 3H, NCH
), 3.91 (br s, 1H, NHCH),
), 7.76 (d, J = 6.9 Hz,
3
), 2.36-2.47
2
CH cycN (CH CH
2
2
2
)
2
NCH
3
), 2.76-3.09 (m,CH
2 6 5
C H
2
6 5
C H
1
3
quinoline), 8.35 (d, J = 4.0 Hz, 1H, Ar-H quinoline); C NMR (CDCl
3
, 50 MHz): δ 37.9,
40.8, 45.0, 51.7, 55.1, 58.9, 97.6, 117.7, 122.0, 124.6, 125.9, 127.6, 128.6, 128.5, 128.2,
4
134.6, 138.2, 146.4, 149.0, 149.6; HRMS calculated for [C17H23ClN +H] 409.2154, found
+
409.2151; ESI-MS: (m/z) 409.3 (M+H) .
720
721
722
723
724
725
726
727
728
729
730
Biological methods
In vitro antimalarial assay
The compounds were evaluated for antimalarial activity against 3D7 (CQ-sensitive) and K1
(CQ-resistant) strains of P. falciparum using Malaria SYBR Green I nucleic acid staining dye
based fluorescence (MSF) assay as mentioned by Singh et al (22).The stock (5 mg/mL)
solution was prepared in DMSO and test dilutions were prepared in culture medium (RPMI-
1640-FBS). The final concentration of DMSO in Plasmodium cultures was< 1%.
Chloroquine diphosphate was used as a reference drug.
Test technique: 50mL of culture medium was dispensed in 96 well plate followed by
addition of 50mL of highest concentration of test compounds (in duplicate wells) in row B.
Subsequent two-fold serial dilutions were prepared and finally 50mL of 1.0% parasitized cell
24

731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
suspension containing 0.8% parasitaemia was added to each well except 4 wells in row‘A’
received non parasitized erythrocyte suspension. The plates were incubated at 37 °C in CO
incubator in an atmosphere of 5% CO and air mixture and 72 h later 100 μL of lysis buffer
2
2
containing 2x concentration of SYBR Green-I (in Nitrogen) was added to each well and
incubated for 1 h at 37 °C. The plates were examined at 485±20 nm of excitation and 530±20
nm of emission for relative fluorescence units (RFUs) per well using the fluorescence plate
reader (FLX800, BIOTEK).
Statistical analysis: Data was transferred into a graphic programme (EXCEL) and IC50
values were obtained by Logit regression analysis of dose response curves using
preprogrammed Excel spreadsheet.
In vitro assay for evaluation of cytotoxic activity
Cytotoxicity of the compounds was carried out using VERO cell line (C1008; Monkey
kidney fibroblast) following the method as mentioned in M. Sinha et al (26).The cells were
incubated with compound-dilutions for 72 h and MTT was used as reagent for detection of
cytotoxicity. 50% cytotoxic concentration (CC50) was determined using nonlinear regression
analysis of dose response curves using pre-programmed Excel spreadsheet. Selectivity Index
(SI) was calculated as SI = CC50/IC50
In vivo antimalarial assay
The in vivo drug response was evaluated in Swiss mice infected with P. yoelii (N-67 strain)
which is innately resistant to CQ (23).In addition selected compounds were also tested on
6
multidrug resistance P. yoelii and P. vinckei. The mice (22±2 g) were inoculated with 1x10
parasitized RBC on day 0 and treatment was administered to a group of five mice from day 0-
6, once daily. The aqueous suspensions of compounds were prepared with 0.5%v/v Tween
80. The efficacy of test compounds was evaluated at 100 mg/kg/day and the required daily
dose was administered in 0.5mL volume via oral route. Parasitemia levels were recorded
from thin blood smears at regular intervals of four days throughout the period of experiment.
The mean value determined for a group of five mice was used to calculate the percent
suppression of parasitemia with respect to the untreated control group. Mice treated with CQ
served as reference controls. Whereas, in the case of arteether, the drug was dissolved in
neutralized ground nut oil and administered via intramuscular route.
761
762
763
Determination of hematin-4-aminoquinoline derivatives association constant
Hematin and amino quinoline association constants for the compounds synthesized in the
present study were determined by spectrophotometric titration procedure in aqueous DMSO
25

764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
at pH 7.5 (24). In this assay condition, hematin is strictly in monomeric state and
interpretation of results is not complicated by need to consider hematin disaggregation
process. Association constant calculated in this technique is a good reflection of the
interaction that would occur in the acidic food vacuole. The pH 7.5 improves the stability of
hematin solutions and quality of data.
In vitro Inhibition of β-hematin formation assay
The ability of the 4-aminoquinoline derivatives to inhibit β-Hematin polymerization was
induced by 1-oleoyl-rac-glycerol using UV spectrophotometer and measurements were
carried out at 405 nm (25).The triplicate values obtained from the assay are expressed as
percent inhibition relative to hemozoin formation in a drug free control. The 50% inhibitory
concentration (IC50) values for the compounds were obtained from the sigmoidal dose
response curves using non-linear regression curve fitting analyses with Graph Pad Prism 30
v.3.00 software (26). Each IC50 value is the result of at least three separate experiments
performed in duplicate.
Molecular Docking Method
779
Database Preparation
780
781
782
783
784
785
786
Compounds were used to perform docking study against heme. To do a comparison study, we
used chloroquine drug for docking. A database was prepared by using SYBYL-X 1.3 (Tripos
Inc, St. Louis, MO, USA) modeling package (27).All compounds were drawn through sketch
module in sybyl. Structures were minimized further by adding Gasteiger-Huckel charges
along with distance-dependent dielectric and the Powell conjugate gradient algorithms with a
convergence criterion of 0.001 kcal/mol. All structures were put into a database and finally
aligned with each other by way of 'Fit Atom' method.
787
Protomol-Based Docking Study
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The molecular docking studies of synthesized compounds were performed using the Surflex-
Dock module with standard protocols in SYBYL-X 1.3.We have extracted heme molecule
from a protein (XX) which was collected from a protein database bank(PDB ID). Hydrogen
atoms were added to heme structure to get a correct configuration. Charges were also added
to it using Gasteiger-Huckel charge and energy-minimizations were performed with standard
protocol using the Tripos force field with same Gasteiger-Huckel charges along with
distance-dependent dielectric and the Powell conjugate gradient algorithms with a
convergence criterion of 0.001 kcal/mol. After that, automatic protomol generation method
26

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was utilized to create a grid over heme. Molecular docking was performed by placing the
molecules including reference into the grid with reasonable scoring function to score the
ligands and protomol guided docking. Finally, the protomol-based method and empirically
derived scoring function (e.g. Total score, crash score, polar etc.) were used to calculate the
binding affinities.
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Experimental Procedures for the pharmacokinetic studies of 7g and 16 (phosphate salt
of 7g)
In vitro Pharmacokinetics of 7g
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Simulated gastric fluid (SGF) was prepared by dissolving 234 mg of NaCl and 37.2 mg of
KCl in 100 mL triple distilled water and pH was adjusted to 1.2 with concentrated HCl.
2 4
Simulated intestinal fluid (SIF) was prepared by dissolving 680 mg of KH PO and 90 mg of
NaOH in 100 mL triple distilled water and pH was adjusted to 6.8 with orthophosphoric acid.
SGF/SIF (2 mL) was taken in a test tube and pre-incubated in shaking water bath for 10-15
min at 37 ± 2°C. 5 µL of 7g stock solution (100µg/mL) was spiked in preincubated SGF/SIF
to produce a concentration of 250 ng/ml and immediately subjected to incubation. 50 µl of
the incubation mixture was sampled at 0, 15, 30, 60, 90, 120 min, diluted 5 times with
acetonitrile and analyzed by LC-MS/MS. Metabolic stability of 7g was performed in
duplicate using glass tubes. To each tube, 460 μL of phosphate buffer (0.1 M, pH 7.4) and
12.5 μL of microsomal protein (20.0 mg/mL) were added and incubated for at 37±0.2°C for 5
min. Then, 2.5 μL of test compound (1mM) was added. For positive control testosterone was
used as the test compound. Reaction was initiated by addition of 25μL of NADPH (24 mM)
and incubated for 0, 5, 10, 15, 30, 45 and 60 min. In negative control, NADPH was replaced
by 25 μL of phosphate buffer. Reaction was stopped by addition of 450 μL ice-cold
acetonitrile to 50 μL reaction mixture collected at predefined time intervals, followed by
centrifugation for 15 min at 10,000 rpm. 100µL of supernatant was directly analyzed by LC-
MS/MS. For the assessment of plasma stability the blank rat plasma (2 mL) in a test tube was
pre-incubated in shaking water bath for 10 min at 37 ± 2°C. 2 µl of 7g stock solution (100
µg/mL) was spiked to preincubated plasma to produce a concentration of 100 ng/mL and
immediately subjected to incubation. 50 µl of the plasma was sampled at 0, 5, 10, 15, 30, 60,
90 and 120 min. Plasma proteins were precipitated by addition of 400μL acetonitrile at
predefined time intervals, followed by centrifugation for 15 min at 10,000 rpm. 100µL of
supernatant was directly analyzed by LC-MS/MS.
27

828
In vivo Pharmacokinetic study:
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Invivo pharmacokinetic study was performed in male SpragueDawley rats (n=4). Intravenous
formulation of 7g was prepared by dissolving accurately weighed quantity of 7g (20 mg) in 2
mL of DMSO followed by addition of 400 µL of ethanol and 800 µL of glycerol and
vortexing for 2 min. Volume was then made up to 4 ml with TDW followed by vortexing for
2 min. For its oral formulation, accurately weighed quantity of 7g (30 mg) was transferred to
mortar and triturated with a pestle using 200 µL of Tween 20. Volume was then made up to 4
ml with 0.25% CMC Suspension. The intravenous and oral formulations of its phosphate salt
form (16) were prepared by dissolving in distilled water. Blood samples were collected from
the retroorbital plexus of rats under light ether anesthesia into microfuge tubes containing
heparin as an anti-coagulant at 0.083, 0.25, 0.5, 1, 2, 3, 5, 7, 9, 24, 30 and 48 hours post-
dosing for intravenous study, while 0.25, 0.5, 1, 2, 3, 5, 7, 9, 24, 30 and 48 hours post-dosing
for oral study. Plasma was harvested by centrifuging the blood at 13000 rpm for 10 min on
Sigma 1-15 K (Frankfurt, Germany) and stored frozen at -70 ± 10°C until bioanalysis. Each
plasma sample (100 μl) was processed using protein precipitation method using 200 μl
acetonitrile containing piracetam as internal standard (I.S.) as protein precipitant, and 10 µl of
the supernatant was injected for LC-MS/MS.
845
RESULTS AND DISCUSSION
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Chemistry. The target compounds envisaged in the present study having the general
structure shown in figure 2 were obtained by strategies using α-amino acids and β-amino
acids as shown in schemes depicted in figures 3 and 4.
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Synthesis of compounds 7a-r: Synthesis of 7a-r involves following steps starting from the
preparation of the Boc and/or Cbz protected α-amino acids. Compounds 1a-j were converted
to the corresponding Boc and/or Cbz derivatives 2a-j in quantitative yields (14, 15). The
Boc/Cbz protected amino acids were reduced to the corresponding alcohols by mixed
anhydride protocol (16). Boc/Cbz amino alcohols were subjected to mesylation to afford 4a-j
in good yields (16). The mesylated products were treated with N-methylpiperazine under
nitrogen atmosphere to get 5a-j (17). Boc deprotection was done by using 20% HCl/Dioxane
at room temperature in quantitative yields and Cbz group was removed by using 10% Pd/C
catalyst to afford free amines 6a-j. (14, 16) Compounds 6a-j thus obtained were fused to 4,7-
Dichloroquinoline or 4-chloro-7-(trifluoromethyl)quinoline in the presence of phenol to
obtain compounds 7a-r (18) (Figure 3).
28

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Synthesis of compounds 15a-c: Synthesis of 15a-c involves following steps starting from
the preparation of the N-Cbz protected amino acids (scheme-2). Amino acids 1a & 1i were
converted to the corresponding N-Cbz derivatives 8a-b in quantitative yields (19). N-Cbz
protected amino acids were converted into the corresponding β-amino esters by Arndt-Eistert
reaction. The reaction involves two steps in the first step Cbz-amino acids were converted
into their corresponding diazoketones 9a-b by mixed anhydride reaction and treating the
mixed anhydride intermediate with diazomethane in THF at -15°C. Diazoketones were
1
purified by the silica gel column chromatography and characterized by H NMR. Purified
diazoketone derivatives were converted into the corresponding β-amino esters via Wolf
rearrangement in the presence of silver benzoate to get 10a-c (20).The β-amino esters were
converted to β-amino alcohols 11a-b (21), subsequently alcohols were transformed to
mesylates 12a-b followed by replacing the mesyl group with N-methyl piperazine to obtain
compounds 13a-b (17). The N-Cbz protecting group was deprotected by using Pd/C to give
amines 14a-b (16) The amines so obtained were fused with the 4,7-Dichloroquinoline or 4-
chloro-7-(trifluoromethyl)quinoline in the presence of phenol resulting in compounds 15a-c
(18). The details of the synthetic steps and the reaction conditions are depicted in figure 4
(scheme 2).
877
Preparation of Phosphate salt of compound 7g (16)
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A cold solution of 400 mg of the compound 7g (> 99% pure) in methanol, was added to a
methanolic solution of phosphoric acid made from 85% phosphoric acid (3.0 equiv). After
complete conversion of free base as seen from TLC, isopropyl alcohol was added to the
reaction mixture and the phosphate salt separated as oil. The alcohol layer was decanted;
acetone was added to the oil and triturated /stirred until it solidified. The mixture was then
filtered and the phosphate salt thus obtained was washed with acetone and quickly placed in a
vacuum desiccator. Further, it was inferred by differential scanning calorimetry (DSC)
(figure 5), that no freebase (7g) was present in the phosphate salt (16). The yield was
quantitative. m. p 195-198°C.
887
In vitro antiplasmodial activity
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The synthesized compounds 7a-r and 15a-c were screened against the 3D7 (CQ-S) and K1
(CQ-R) strains of P. falciparum in vitro for antiplasmodial activity and the results are
presented in table 1. Most of the compounds displayed excellent antiplasmodial activity in the
nanomolar range. As expected reduction of the amide bond has led to a substantial increase in
29

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antiplasmodial activity against both the strains. Our data (table 1) suggest that compounds
derived from glycine, leucine and phenylalnine i.e 7a-b, 7g, 7j and 7o-p displayed similar
antiplasmodial activity when chloro substituent was replaced with a trifluoromethyl group
against both the strains. Whereas some compounds exhibited moderate to substantial
difference in the antiplasmodial activity.
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Among the 21 compounds tested (7a-r & 15a-c), fourteen compounds namely 7a-c, 7e, 7g-
k,7o-q, 15a & 15c exhibited potent antiplasmodial activity in the range of 3.27 to 25.1 nM
against CQ-S strain and IC50 9.79 to 167.4 nM range against CQ-R strain. Whereas, seven
compounds7d, 7f, 7l-n, 7r & 15b displayed antiplasmodial activity against CQ-S strain in the
range of IC50 81.22 to 723 nM and one compound 7r showed comparable activity against
CQ-R with IC50 259 nM, two compounds7d & 15b exhibited IC50 548 and 585 nM, four
compounds 7f & 7l-n demonstrated no detectable antiplasmodial activity against K1 strain at
the highest concentration tested (IC50>1000 nM). On the other hand, compounds having a
iso-butyl group (7g-j) and benzyl group (7o-p & 15c) at the chiral centre were found to be
the most active in this series. Moreover, the enantiomeric pairs of 7g, 7h and racemic
compound 7i did not show any difference in the activities against both 3D7 and K1 strains.
There was more than a 1.7-fold increase in the activity against CQ-S for compound-7a (3.27
nM) when compared to the CQ (5.46 nM). In fact, compounds namely 7g-7j, 7p, 15a and 15c
exhibited almost 20 to 28 fold increases in the activity against the CQ resistant strain with
IC50 values of 13.57, 11.16, 11.79, 12.13, 9.97 and 11.52 nM when compared to the
chloroquine. In this study we have also explored the effect of the chain length variation by
3
homologation of selected α-amino acids to get the corresponding β amino acids. Increase in
the chain length showed positive effects on the antiplasmodial activities against both 3D7 and
K1 strain of P. falciparum. Compounds derived from glycine (15a) (IC50 9.79 nM) enhanced
ten folds the activity when compared to the CQ in the case of resistant strain. Whereas, in the
case of phenylalanine (15c) (IC50 11.52 nM) the activity was increased four folds with respect
to the CQ.
919
In vitro cytotoxicity
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The cytotoxicity of all the synthesized molecules was determined against VERO cell line
using MTT assay (table 1). Our target compounds showed selectivity index ranging from
83.20 to 37,281. Compounds namely 7a, 7g, and 15c exhibited excellent selectivity indices
37, 281, 22,727 and 9,852 respectively. Whereas, compounds 7e, 7k, and 7p, displayed
30