Antiplasmodial activity of new 4-aminoquinoline derivatives against chloroquine resistant strain

Article abstract of DOI:10.1016/j.bmc.2014.05.024

Emergence and spread of multidrug resistant strains of Plasmodium falciparum has severely limited the antimalarial chemotherapeutic options. In order to overcome the obstacle, a set of new side-chain modified 4-aminoquinolines were synthesized and screened against chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of P. falciparum. The key feature of the designed molecules is the use of methylpiperazine linked α, β³- and γ-amino acids to generate novel side chain modified 4-aminoquinoline analogues. Among the evaluated compounds, 20c and 30 were found more potent than CQ against K1 and displayed a four-fold and a three-fold higher activity respectively, with a good selectivity index (SI = 5846 and 11,350). All synthesized compounds had resistance index between 1.06 and >14.13 as against 47.2 for chloroquine. Biophysical studies suggested that this series of compounds act on heme polymerization target.

Full text of DOI:10.1016/j.bmc.2014.05.024

Accepted Manuscript
Antiplasmodial activity of new 4-aminoquinoline derivatives against chloro-
quine resistant strain
Manish Sinha, Vasanth R. Dola, Pooja Agarwal, Kumkum Srivastava, Wahajul
Haq, Sunil K. Puri, Seturam B. Katti
PII:
S0968-0896(14)00373-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.05.024
BMC 11585
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 April 2014
12 May 2014
12 May 2014
Please cite this article as: Sinha, M., Dola, V.R., Agarwal, P., Srivastava, K., Haq, W., Puri, S.K., Katti, S.B.,
Antiplasmodial activity of new 4-aminoquinoline derivatives against chloroquine resistant strain, Bioorganic &
Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.05.024
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Antiplasmodial activity of new 4-aminoquinoline derivatives against
chloroquine resistant strain.
Manish Sinhaa, Vasanth R. Dolaa, Pooja Agarwalb, Kumkum Srivastavab, Wahajul Haqa,
Sunil K. Purib and Seturam B. Kattia,*
aMedicinal and Process Chemistry Division, CSIR- Central Drug Research Institute,
Lucknow-226031, India.
bParasitology Division, CSIR-Central Drug Research Institute, Lucknow-226031, India.
Abstract
Emergence and spread of multidrug resistant strains of P. falciparum has severely limited the
antimalarial chemotherapeutic options. In order to overcome the obstacle, a set of new side-
chain modified 4-aminoquinolines were synthesized and screened against chloroquine-
sensitive (3D7) and chloroquine-resistant (K1) strains of P. falciparum. The key feature of
the designed molecules is the use of methylpiperazine linked α, β3-and γ-amino acids to
generate novel side chain modified 4-aminoquinoline analogues. Among the evaluated
compounds, 20c and 30 were found more potent than CQ against K1 and displayed a four-
fold and a three-fold higher activity respectively, with a good selectivity index (SI = 5846 &
11350). All synthesized compounds had resistance index between 1.06 and >14.13 as against
47.2 for chloroquine. Biophysical studies suggested that this series of compounds act on
heme polymerization target.
Keywords: 4-aminoquinoline; Chloroquine; Malaria; Resistant strains.
Corresponding author: Tel: +91-522-2772454, Fax; +91-522-2771941
Email address: setu_katti@yahoo.com, sb_katti@cdri.res.in (S. B. Katti)
1

1. Introduction
Malaria is a tropical disease caused by Apicomplexan parasite Plasmodium falciparum
severely affecting human well being. It is estimated that about 1 million deaths occur
annually, mostly children under five years of age.1-2 High morbidity is associated with the
failure of the established chemotherapies due to emergence of drug resistant P. falciparum
against most of the antimalarial drugs.3 Although, chloroquine (CQ) and other related 4-
aminoquinolines (Fig.1) have played important role during the past five decades drug
resistance to CQ has been a major problem in malaria eradication programme. So, the search
for novel, affordable, and structurally diverse drugs has become an urgent need to eradicate
this parasitic disease. Early biochemical studies carried out to understand the mechanism of
CQ resistance have led to two seminal findings namely, a mutation in the P. falciparum CQ-
resistance transporter (PfCRT) gene resulting in rapid efflux of CQ from the intracellular loci.
Secondly and more importantly study by Ridley et.al.4 has established that drug resistance is
compound specific. This has fuelled research activity towards modifying CQ side chain so as
to obtain molecules active against CQ-resistant parasite.
Several modifications have been done in the side chain of chloroquine and studied in detail. It
has been found that shortening of the chain length or incorporation of intramolecular
hydrogen-bonding motif in the side chain significantly increases the antimalrial activity.4-6
While replacement of 4-position nitrogen atom of the 7-chloro-4-aminoquinoline by oxygen
and sulphur significantly decreases the basicity of the quinonyl nitrogen which leads to
reduced antimalarial activity.7 Earlier in our research group we have also explored some
specific modifications by introducing, guanidine, tetramethylguandine, and thiazolidin-4-one
and substituted thiourea moieties at the side chain. Some of these compounds exhibited
superior in vitro activity and significant suppression of parasitemia in the in vivo assay as
2

compared to chloroquine. Based on the limited SAR-studies it was inferred that basicity of
the quinoline ring nitrogen (pKa1) and side chain nitrogen (pKa2) played an important role
for the antimalarial activity.8 Inspired by these observations, in this study we have
synthesized a set of compounds with specific modifications to CQ that are predicated to be
relevant for activity against chloroquine resistant parasites. The diversity in the synthesized
compounds has been generated through variations in the side chain of CQ. Initially we have
selected alanine and synthesized various amides (Scheme 1). Compound having N-
methylpiperzine as a pendant group showed improved activity against CQ-R strain. We
further preferred variety of α-amino acids by keeping the pendant group with N-
methylpiperzine. We have found that some of them exhibited promising antimalarial activity
than CQ in the case of CQ-R strain. Additionally we have also investigated the effect of chain
length variation between 2 to 4 carbon atoms via homologation of α-amino acids to get
corresponding β3 and γ- amino acids. The results are described in the present communication.
2. Chemistry
4,7- Dichloroquinoline (1), was fused with an excess of alanine (2) in the presence of phenol
at 140 °C to afford the compound (6).9 The obtained Compound (6) was coupled with
different amines using DCC, HOBt protocol taking DMF as solvent at 0 °C to afford (6a-
6f).10 A similar approach was utilized for some selected α-amino acids viz. glycine (3),
phenylalnine(4), methionine (5) directly fused to 4,7- Dichloroquinoline to get 7-9 in good
yields followed by coupling with N-methylpiperzine to afford 7a, 8a, and 9a as shown in
Scheme 1. We have observed poor yields in the case of remaining α-amino acids. Therefore,
in order to get the desired final products 14a-14j we have opted for different synthetic
strategy. The route is delineated in Scheme-2. Synthesis of compounds 20a-20c involve prior
preparation of β-amino acids followed by their conversion to piperazine amides and after
3

deprotection they were finally fused with 4,7-Dichloroquinoline as shown in Scheme 3.
Synthesis of compound 30 involves derivatization of γ-amino acid and its fusion with 4,7-
Dichloroquinoline. The synthetic steps involved are shown in Scheme 4. The compounds
synthesized in Scheme 1, Scheme 2, Scheme 3 and Scheme 4, essentially comprise of three
fragments namely, quinoline, amino acid, and the terminal alkyl amide fragment.
3. Results and Discussion
3.1 Antimalarial in vitro evaluation
The antimalarial activity of the targeted compounds 6a-6f were screened against the 3D7
(CQ-S) and K1 (CQ-R) strains of P. falciparum in vitro and the results are presented in Table
1. Amide 6a was found to exhibit comparable antimalarial activity in the case of resistant
strains. Other amides 6b-6f were noticeably less active against 3D7 strain, no detectable
antimalarial activity against K1 strain at the highest concentration tested (IC50 > 500 nM). It
was inferred from the preliminary screening data that amide 6a containing N-methylpiperzine
as a side chain showed comparable activity with reference to CQ against resistant strain. This
motivated us to synthesize various analogues of 4-aminoquinolines by using different α-
amino acids with methylpiperazine as pendent group. In this study we have also investigated
the effect of the chain length variation by homologation of selected α-amino acids to get the
corresponding β3 and γ-amino acids. All compounds exhibited activity in 11.51 nM to > 500
nM range against 3D7 strain and 56.36 nM to > 1378 nM range against K1 strain.
Compounds 7a and 14f exhibited 1.5 to 1.8 folds activity, whereas 8a and 9a displayed 2 fold
activities against resistant strain. We also examined the effect of hetroaromatic groups, bulky
alkyl groups and polar groups. However, these compounds showed no significant
improvements in the in vitro antimalarial activity. Another important finding is that
antimalarial activity does not improve by adding more basic groups 14i and 14j. Increase in
the chain length showed positive effects on the antimalarial activity against both 3D7 and K1
4

strain of P. falciparum. Compounds derived from β3-isoleucine (16b) showed better activity
in the case of K1 strain (IC50= 168 nM). The β3-phenylalanine derivative (16c) was found to
be 4.6 folds active in the case of K1 strain (IC50= 55.29 nM). Further increase of side chain
length of 16c by one carbon generated the γ-phenylalanine derived 4-aminoquinoline (30)
and its antimalarial activity against K1 strain was also very good (IC50 71.16 nM) nearly 3.6
folds active than chloroquine. Although both β3- and γ-phenylalanine derived 4-
aminoquinolines exhibited better activity but β3-compound (16c) was found to be the most
active in the series. It may be appropriate to reveal here that resistance factor which is
calculated as a ratio of IC50 in CQ-R vs CQ-S strains has been used as an index to assess
chances of parasite developing resistance to a particular class of compounds. Accordingly, it
is believed that smaller the resistance factor, the less likely is the chance of developing
resistance to that class of compounds. Interestingly, all the compounds in this series showed
resistance factors 1.06 and >14.13 as against 47.2 for chloroquine.
3.2 In vitro cytotoxicity.
The cytotoxicity of target compounds was determined by making use of standard protocol,
MTT assay against VERO cell line (Table-1). Our target compounds showed selectivity
index (SI) ranging from 392 to 9772 respectively. Some compounds namely, 6f, 9a, 14g, and
14h exhibited moderate activity against sensitive (3D7) strain with good selectivity indices
5834, 3821, 6472, and 4432 respectively. Compounds 20a-20c and 30 have shown promising
antimalarial activity against (3D7) as well as (K1) strain and showed better selectivity indices
6045, 5846, 7613, and 11,350 respectively. Thus these compounds demonstrated the
promising safe activity profile.
3.3 In vitro inhibition of β-hematin polymerization
5

The mode of action of this series of 4-aminoquinoline derivatives (6a-6f, 7a, 8a, 9a, 14a-14j,
20a-20c, 30) was investigated by the reported method.11-13 and the results are shown in
(Table-1). All the synthesized compounds form complex with hematin and the range of Log
K was found to be 5.32-7.34. Among, all the compounds reported in the present study
compounds 16c and 30 have shown very strong binding to hematin. This result is concurrent
with the previous results from the reported literature evidences.14-17 The data suggest that the
principle interaction may be hydrophobic as well as electrostatic between the 4-
aminoquinoline ring and the porphyrin ring system that plays a role in hematin binding. All
the synthesized 4-aminoquinoline derivatives (6a-6f, 7a, 8a, 9a, 14a-14j, 20a-20c, 30)
inhibited the β-hematin formation in a concentration dependent manner (Table-1). Although
most of the synthesized compounds were good inhibitors of β-hematin formation, some of
them have shown moderate antimalarial activity against CQ-S and CQ-R strains of P.
falciparum. The most potent inhibitors were compounds 20c and 30 with an IC50 of 0.17 mM,
and 0.19 mM in the hemozoin inhibition assay. It may be inferred from the above data that
these compounds bind to hematin monomer or hematin µ-oxo dimer and inhibit the β-
hematin formation by blocking the growing face of crystal by a capping effect.14
4. Conclusion
In summary, we have prepared a series of new 4-aminoquinoline derivatives by varying the
substituents at the pendant group and modifying the trunk of the side chain with different
amino acids. Among all, N-methylpiperazine moiety as a pendant amino group showing
promising in vitro antimalarial activity against the chloroquine resistant strains of P.
falciparum. The most striking feature of this study is consistent antiplasmodial activity of
these derivative against both CQ-S and CQ-R strains of parasite. In some cases, the activity
against CQ-R strain is superior as compared to their activity against CQ-S strain of parasite.
The biophysical studies have suggested that this class of compounds form association
6

complex with hematin and thereby, inhibit the hemozoin formation. The N-methylpiperazine
amide functionality on 4-aminoquinoline-pendant amino group provided a promising lead
entry for designing the heme polymerization targeted antimalarials. The present 4-
aminoquinolines provide new opportunity for the development of potent antimalarials to
overcome the emerging problem of drug resistance.
5. Experimental Protocols
5.1 General
Meting points (mp) were determined on a complab melting point apparatus and are
uncorrected. The 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded in
CDCl3, CD3OD, and DMSO-d6, used as solvents on DPX-300 Bruker FT-NMR spectrometer.
Chemical shifts are reported in parts per million δ (ppm) with the residual protons of the
solvent as reference. 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, Agilent 6520 Q-Tof (ESI
HRMS) spectrometers respectively. Analytical thin-layer chromatography (TLC) was carried
out on Merck’s precoated silica-gel plates 60 F254 and spots were visualized by irradiation
with UV light (254 nm). Iodine was used as developing agent or by spraying with
Dragendorff’s reagent. Column chromatographic purification was performed over silica gel
(230-400 mesh) using a gradient solvent system (n-hexane/ ethyl acetate or chloroform/
methanol as the eluent unless otherwise specified). All chemicals and reagents were obtained
from Aldrich (USA), Lancaster (UK) or Spectrochem Pvt. Ltd (India) and were used without
further purification.
7

5.1.1 General procedure for the synthesis of 6-9:
4,7-Dichloroquinoline (10 mmol) and corresponding amino acid (alanine, glycine,
phenylalanine, methionine; 20 mmol) were heated in phenol for one hour at 140 °C. After
completion of the reaction as monitored by TLC to the reaction mixture was added a solution
of 10% KI 30 mL and diethyl ether 30 mL. The aqueous layer was then washed with ether
layer (3x30 mL). The combined ether layer was extracted with 10% KI (30 mL) solution. The
pH of the combined aqueous phase was adjusted to 7. The aqueous solution was let
uncovered for slow evaporation at room temperature. After 24h, the precipitated solid was
filtered and dried under vacuum.
5.1.2 (S)-2-(7-Chloroquinolin-4-ylamino)propanoic acid (6): white semi-solid; yield 1.25
g, (55%); 1H NMR (300 MHz, CDCl3) δ (ppm): 1.66 (br s, 3H, CHCH3), 4.25 (br s, 1H,
NHCH), 6.66 (br s, 1H, Ar-H quinoline), 7.53 (d, J = 9.5 Hz, 1H, Ar-H quinoline), 7.74 (d, J
= 6.3 Hz,1H, Ar-H quinoline), 8.30 (br s, 1H, Ar-H quinoline), 8.44 (br s, 1H, Ar-H
quinoline); ESI-MS: m/z 251 (M+H)+.
5.1.3 2-(7-Chloroquinolin-4-ylamino)-acetic acid (7): white semi-solid; yield 1.35 g,
(57%); 1H NMR (300 MHz, CDCl3) δ (ppm) : 3.84 (s, 2H, NHCH2), 6.15 (d, J = 5.6 Hz, 1H,
Ar-H quinoline), 7.24 (dd, J = 9.0, 2.1 Hz, 1H, Ar-H quinoline), 7.64 (d, J = 9.03 Hz,1H, Ar-
H quinoline), 8.19 (d, J = 5.6 Hz, 1H, Ar-H quinoline), 8.47 (br s, 1H, Ar-H quinoline).
5.1.4 (S)-2-(7-Chloroquinolin-4-ylamino)-3-phenylpropanoic acid (8): gummy residue;
yield 1.25 g, (55%); 1H NMR (300 MHz, DMSO-d6,) δ (ppm) : 3.55-3.61 (m, 2H, CH2Ph),
6.66 (d, J = 6.1 Hz, 1H, Ar-H quinoline), 7.18-7.33 (m, 5H, 5 Ar-H benzene), 7.72 (d, J =
3.8 Hz, 1H, Ar-H quinoline), 7.85 (d, J = 1.7 Hz, 1H, Ar-H quinoline), 8.26 (d, J = 5.5 Hz,
1H, Ar-H quinoline), 8.41 (d, J = 8.0 Hz, 1H, Ar-H quinoline). ESI-MS: m/z 327 (M+H+)
8

5.1.5 (S)-2-(7-Chloroquinolin-4-ylamino)-4-(methylthio)butanoic acid (9): gummy
residue; yield 1.3g, (57%); 1H NMR (300 MHz, CDCl3) δ (ppm): 2.03-2.11 (m, 2H, CHCH2),
2.14 (s, 3H, SCH3), 2.59-2.79 (m, 2H, CHCH2CH2), 4.70-4.74 (m, 1H, NHCH), 6.88 (d, J =
7.0 Hz, 1H, Ar-H quinoline), 7.88 (s, 1H, Ar-H quinoline), 8.41 (d, J = 6.9 Hz, 1H, Ar-H
quinoline), 8.51 (d, J = 9.0 Hz, 1H, Ar-H quinoline). ESI-MS: m/z 311 (M+H+).
6. General procedure for the synthesis of 6a-6f, 7a, 8a, 9a:
To a solution of (6-9, 2 mmol) in dimethylformamide (DMF, 10 mL) at 0 °C, 1-
hydroxybenzotriazole (HOBt, 2.2 mmol) was added. The reaction mixture was stirred for 2
minutes and the corresponding amine (3 mmol in 1.0 mL DMF) was added to it followed by
the addition of dicyclohexylcarbodimide (DCC, 2.2 mmol in 1.0 mL DMF). The reaction
mixture was stirred for next 4 hrs. After completion of reaction the dicyclohexylurea (DCU)
was filtered and the filtrate was evaporated under reduced pressure. The oily residue was
dissolved in chloroform, washed with 5% aqueous sodium bicarbonate followed by washing
with water and finally with brine. The organic layer was dried over anhydrous Na2SO4 and
evaporated under reduced pressure. The crude product was purified over silica gel column to
get the pure compound.
6.1 (S)-2-(7-Chloroquinolin-4-ylamino)-1-(4-methylpiperazin-1-yl)-propan-1-one (6a):
Yellow residue; yield: 410 mg, (62 %). 1H NMR (300 MHz, CDCl3) δ(ppm): 1.49 (d, J =
6.6, 3H, CHCH3), 2.35 (s, 3H, NCH3), 2.44-2.52 (m, 4H, N(CH2CH2)2), 3.59-3.62 (m, 2H,
CONCH2), 3.7-3.77 (m, 2H, CONCH2), 4.53-4.58 (m, 1H, NHCH), 6.30 (d, J = 5.4 Hz, 1H,
Ar-H quinoline), 6.51 (br s, 1H, NH), 7.40 (dd, J = 9.0, 2.1 Hz, 1H, Ar-H quinoline), 7.80 (d,
J = 8.97 Hz, 1H, Ar-H quinoline), 7.95 (d, J = 2.0 Hz, 1H, Ar-H quinoline), 8.51 (d, J = 5.3,
1H, Ar-H quinoline); 13C NMR (75 MHz ,CDCl3,) δ (ppm): 17.8, 42.3, 45.3, 45.9, 47.8, 54.5,
9

54.9, 98.7, 117.3, 121.5, 125.5, 128.5, 135.1, 147.7, 149.1, 151.6, 170.4; HRMS calculated
for [C17H21ClN4O + H+] 333.1477, found 333.1418; ESI-MS: m/z 333 (M+H)+.
6.1.1 (S)-2-(7-Chloroquinolin-4-ylamino)-N-(3-(diethylamino)-propyl)-propanamide
(6b): white solid; yield: 580 mg, (80 %), mp 150-152 °C); 1H NMR (300 MHz, CDCl3)
δ(ppm): 0.87 (t, J = 7.1 Hz, 6H, N(CH2)2(CH3)2), 1.57 (d, J = 6.8 Hz, 3H, CHCH3), 2.26-
2.48 (m, 8H, CH2CH2N(CH2)2), 3.24-3.49 (m, 2H, CONHCH2 ), 3.97-4.10 (m, 1H, NHCH),
5.72 (d, J = 4.9 Hz, 1H, NH), 6.31 (d, J = 5.3 Hz, 1H, Ar-H quinoline, 7.35 (dd, J = 9.0, 2.1
Hz, 1H, Ar-H quinoline), 7.74 (d, J = 9.0 Hz, 1H, Ar-H quinoline), 7.95 (d, J = 2.1, 1H, Ar-
H quinoline), 8.3 (br s, 1H, NH), 8.50 (d, 5.32 Hz, 1H, Ar-H quinoline); 13C NMR (75 MHz,
CDCl3) δ (ppm): 10.9, 18.2, 25.0, 39.5, 46.4, 46.5, 51.6, 58.2, 99.4, 117.3, 121.8, 125.5,
128.3, 135.1, 148.9, 151.8, 168.5; HRMS calculated for [C19H27ClN4O+ H+] 363.1946,
found 362.1899; ESI-MS: m/z 363 (M+H) +.
6.1.2 (S)-2-(7-Chloroquinolin-4-ylamino)-1-(4-phenylpiperazin-1-yl)-propan-1-one (6c):
brown gummy residue; yield 500 mg, (64 %). 1H NMR (300 MHz, CDCl3) δ (ppm): 1.54 (d,
J = 6.6, 3H, CHCH3) 3.24-3.29 (m, 4H, 2CH2CH2), 3.77 (br s, 2H, CONCH2), 3.89 (d, J =
9.5 Hz, 2H, CONCH2), 4.58-4.66 (m, H, NHCH), 6.34 (br s, 1H, NH), 6.48 (d, J = 6.3, 1H,
Ar-H quinoline), 6.96 (d, J = 7.47, 3H, Ar-H), 7.28-7.35 (m, 2H, Ar-H), 7.39 (dd, J = 9.0,
2.1 Hz, 1H, Ar-H quinoline), 7.82 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.97 (d, J = 2.0 Hz,
1H, Ar-H quinoline), 8.54 (d, J = 5.2 Hz, 1H, Ar-H quinoline); 13C NMR (75 MHz, CDCl3) δ
(ppm) : 17.9, 42.3, 45.4, 47.8, 49.5, 49.8, 98.7, 116.8, 121.0, 121.4, 125.5, 128.6, 129.3,
135.1, 147.7, 149.2, 150.6, 151.7, 170.6; HRMS calculated for [C22H23ClN4O+ H+] 395.1633,
found 395.1644; ESI-MS: m/z 395 (M+H) +.
6.1.3 (S)-2-(7-Chloroquinolin-4-ylamino)-N-(3-(dimethylamino)-propyl)-propanamide
(6d): Brown gummy residue; yield 475 mg, (71 %). 1H NMR (300 MHz, CDCl3) δ (ppm):
1.6 (d, J = 6.9 Hz, 3H, CHCH3 ), 1.9 (s, 6H, N(CH3)2, 2.22-2.32 (m, 4H, CH2CH2N), 3.4-
10

3.41 (m, 2H, NHCH2), 4.05-4.09 (m, 1H NHCH), 5.60 (d, J = 4.5 Hz, 1H, NH), 6.35 (d, J =
5.2 Hz, 1H, Ar-H quinoline), 7.39 (dd, J = 9.0, 2.1 Hz, 1H, Ar-H quinoline), 7.78 (d, J = 8.9
Hz, 1H, Ar-H quinoline) 7.97 (d, J = 2.0 Hz, 1H, Ar-H quinoline), 8.23 (br s, 1H, NH), 8.53
(d, J = 5.1, 1H, Ar-H quinoline); 13C NMR (75 MHz, CDCl3) δ (ppm) : 19.3, 24.8, 40.2,
45.0, 53.1, 59.0, 99.9, 120.9, 125.7, 128.9, 135.1, 148.3, 149.1, 152.0, 172.0 ; HRMS
calculated for [ C17H23ClN4O+H+] 335.1633, found 335.1593; ESI-MS: m/z 335 (M+H) +.
6.1.4 (S)-2-(7-Chloroquinolin-4-ylamino)-N-(2-(piperidin-1-yl)-ethyl)-propanamide (6e):
White gummy residue; yield: 420 mg, (62 %). 1H NMR (300 MHz, CDCl3) δ (ppm): 1.39 (br
s, 6H, cycNCH2(CH2)3CH2), 1.64 (d, J = 6.9 Hz, 3H, CHCH3) 2.18-2.51 (m, 6H,
CH2CH2N(CH2)2, 3.34-3.39 (m, 2H, CONHCH2), 4.11-4.20 (m, 1H, NHCH), 5.67 (d, J = 5.2
Hz, 1H, NH), 6.37 (d, J = 5.2 Hz, 1H, Ar-H quinoline), 7.21 (s, 1H, NH), 7.41 (dd, J = 9.0,
2.1 Hz, 1H, Ar-H quinoline), 7.82 (d, J = 8.9, 1H, Ar-H quinoline), 7.99 (d, J = 2.1 Hz, 1H,
Ar-H quinoline), 8.53 (d, J = 5.2 Hz, 1H, Ar-H quinoline ); 13C NMR (75 MHz, CDCl3) δ
(ppm): 19.0, 23.4, 24.7, 24.9, 33.9, 35.4, 49.1, 53.1, 54.0, 56.8, 99.8, 121.5, 125.6, 128.5,
135.2, 148.5, 151.7, 156.8, 172.7; HRMS calculated for [C19H25ClN4O+H+] 361.1790, found
361.1767; ESI-MS: m/z 361 (M+H) +.
6.1.5 (S)-2-(7-Chloroquinolin-4-ylamino)-N-(2-(diethylamino)-ethyl)-propanamide (6f):
white gummy residue ; yield: 467 mg, (67 %). 1H NMR (300 MHz, CDCl3) δ (ppm) : 0.92 (t,
J = 7.1 Hz, 6H, 2CH2CH3), 1.65 (d, J = 6.9 Hz, 3H, CHCH3), 2.50-2.55 (m, 4H, N(CH2)2,
2.57-2.65 (m, 2H, CH2CH2N), 3.32-3.47 (m, 2H, CONHCH2), 4.15-4.21 (m, 1H, NHCH),
5.81 (d, J = 4.1 Hz, 1H, NH), 6.37 (d, J = 5.37Hz, 1H, Ar-H quinoline), 7.40 (dd, J = 9.0,
2.1 Hz, 1H, Ar-H quinoline), 7.87 (d, J = 9.0 Hz, 1H, Ar-H quinoline), 7.97 (d, J = 2.0 Hz,
1H, Ar-H quinoline), 8.52 (d, J = 5.3 Hz, 1H, Ar-H quinoline); 13C NMR (75 MHz, CDCl3)
δ (ppm) : 19.1, 23.9, 25.3, 29.6, 35.8, 53.0, 54.0, 56.8, 99.8, 115.7, 117.2, 119.5, 121,3,
11

125.6, 128.5, 129.5, 135.1, 148.4, 149.0, 151.7, 172.3; HRMS calculated for
[C18H25ClN4O+H+] 349.1790, found 349.1721; ESI-MS: m/z 349 (M+H) +.
6.1.6 2-(7-Chloroquinolin-4-ylamino)-1-(4-methylpiperazin-1-yl)-ethanone (7a): brown
gummy residue; yield 520 mg, (75 %). 1H NMR (300 MHz, CDCl3) δ (ppm) : 2.22 (br s, 1H,
NH), 2.35 (s, 3H, NCH3), 2.46-2.51 (m, 4H, N(CH2CH2)2), 3.52 (t, J = 5.1 Hz, 2H,
CONCH2), 3.76 (t, J = 5.0 Hz, 2H, CONCH2), 3.98 (d, J = 3.5 Hz, 2H, NHCH2CO), 6.26 (d,
J = 5.3 Hz, 1H, Ar-H quinoline), 6.49 (s, 1H, NH), 7.38 (dd, J = 9.0, 2.1 Hz, 1H, Ar-H
quinoline), 7.83 ( d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.96 (d, J = 2.0 Hz, 1H, Ar-H
quinoline), 8.52 (d, J = 5.2 Hz, 1H, Ar-H quinoline); 13C NMR (75 MHz, CDCl3) δ (ppm) :
42.2, 43.7, 44.2, 46.0, 54.4, 54.7, 99.2, 117.0, 121.5, 125.7, 128.2, 135.3, 148.5, 148.6, 151.5,
165.8 ; HRMS calculated for C16H19ClN4O 319.1325, found 319.1291; ESI-MS: m/z 319
(M+H) +.
6.1.7 (S)-2-(7-Chloroquinolin-4-ylamino)-1-(4-methylpiperazin-1-yl)-3-phenylpropan-1-
one (8a): Yellow gummy residue; yield 357 mg,(65%); 1H NMR (300 MHz, CDCl3) δ
(ppm): 2.25 (s, 3H, NCH3), 2.34-2.37 (m, 4H, COcycN(CH2CH2)2NCH3), 3.00-3.25 (m , 4H,
COcycN(CH2CH2)2NCH3)), 3.56-3.74 (m, 2H, CHCH2Ph), 4.79-4.81 (m, 1H, NHCH), 6.33
(d, J = 5.3 Hz, 2H, Ar-H quinoline, NH), 7.19-7.731 (m, 5H, Ar-H), 7.37 (dd, J = 1.9, 8.9
Hz, 1H, Ar-H quinoline), 7.76 (d, J = 8.97 Hz, 1H, Ar-H quinoline), 7.99 (d, J = 1.8 Hz, 1H,
Ar-H quinoline), 8.50 (d, J = 5.3 Hz, 1H, Ar-H quinoline); 13C NMR (75 MHz, CDCl3) δ
(ppm): 41.5, 45.2, 45.8, 54.4, 54.7, 98.8, 117.3, 121.5, 125.5, 127.3, 128.4, 128.6, 129.3,
129.4, 135.0, 136.3, 148.4, 149.0, 151.6, 170.8; HRMS calculated for [C23H25ClN4O+H+]
409.1795, found 409.1762; ESI-MS: m/z 409 (M+H+).
6.1.8 (S)-2-(7-Chloroquinolin-4-ylamino)-1-(4-methylpiperazin-1-yl)-4-
(methylthio)butan-1-one (9a): Yellow residue; yield: 357 mg, (65%); 1H NMR (300 MHz,
CDCl3) δ (ppm) : 1.96-2.05 (m, 2H, CHCH2), 2.09 (s, 3H, SCH3), 2.31 (s, 1H, NCH3), 2.41-
12

2.48 (m, 4H, COcycN(CH2CH2)2NCH3), 2.58-2.63 (m, 2H, CHCH2CH2), 3.59-3.69 (m, 4H,
COcycN(CH2CH2)2NCH3), 4.81-4.82 (m, 1H, NHCH), 6.34 (br s, 1H, NH), 6.45 (d, J = 5.3
Hz, 1H, Ar-H quinoline), 7.30 (dd, J = 2.5, 9.0 Hz, 1H, Ar-H quinoline), 7.81 (d, J = 8.4 Hz,
1H, Ar-H quinoline), 7.97 (s, 1H, Ar-H quinoline), 8.54 (d, J = 5.2 Hz, 1H, Ar-H quinoline).
13C NMR (75 MHz, CDCl3) δ (ppm) : 15.8, 30.2, 32.1, 42.4, 45.4, 45.9, 50.7, 54.5, 55.0,
99.0, 117.4, 121.5, 125.5, 128.4, 128.5, 135.1, 149.1, 151.7, 151.8, 169.9. HRMS calculated
for [C19H25ClN4OS+H+] 393.1516 found 393.1514; ESI-MS: m/z 393 (M+H+).
7. General procedure for the synthesis of 12a-12j: Amino acids (10a-10j) were converted
to the corresponding Boc derivatives (11a-11j) in quantitative yields by using di-tert-
butylpyrocarbonate. 18 To a solution of (11a-11j; 2 mmol) in 10 mL THF, 1-
Hydroxybenzotriazole (HOBt; 2.2 mmol) was added. The reaction mixture was stirred for 2
minutes at 0 °C. The N-methylpiperazine (3 mmol) was added to above reaction mixture,
followed by addition of dicyclohexylcarbodimide (DCC; 2.2 mmol, 1.0 mL THF) at 0 °C.10
The reaction mixture was allowed to reach at room temperature and was stirred for next 4 hrs.
The dicyclohexylurea (DCU) was filtered and the filtrate was evaporated under reduced
pressure. The oily residue was dissolved in chloroform, organic layer was washed with 5%
aqueous sodium bicarbonate followed by washing with water and finally with brine. The
organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The
crude product was purified over silica gel column to afford compounds (12a-12j) in good
yields.
7.1. (S)-Tert-butyl 3-methyl-1-(4-methylpiperazin-1-yl)-1-oxobutan-2-ylcarbamate (12a):
Gummy residue; yield 559 mg, (77 %) 1H NMR (300 MHz, CDCl3) δ (ppm) : 0.80 (d, J = 6.6
Hz, 3H, CHCH(CH3)CH3), 0.87 (d, J = 6.6 Hz, 3H, CHCH(CH3)CH3), 1.36 (s, 9H, C(CH3)3),
1.80-1.89 (m, 1H, CHCH(CH3)CH3), 2.23 (s, 3H, NCH3), 2.31-2.33 (m, 4H,
13

COcycN(CH2CH2)2NCH3), 3.49-3.61 (m, 4H, COcycN(CH2CH2)2NCH3), 4.35-4.40 (m, 1H,
NHCHCH), 5.35 (d, J = 8.6 Hz, NH).
7.1.1 Tert-butyl (2S,3S)-3-methyl-1-(4-methylpiperazin-1-yl)-1-oxopentan-2-
ylcarbamate (12b): Gummy residue ; yield 579 mg, (79 %) 1H NMR (300 MHz, CDCl3) δ
(ppm) : 0.82-0.95 (m, 6H, CHCH(CH3)CH2CH3), 1.39 (s, 9H, C(CH3)3), 1.61-1.71 (m, 2H,
CHCH(CH3)CH2), 2.26 (s, 3H, NCH3), 2.35-2.36 (m, 4H, COcycN(CH2CH2)2NCH3), 2.58
(br s, 1H, NHCHCH), 3.54-3.65 (m, 4H, COcycN(CH2CH2)2NCH3), 4.40-4.45 (m, 1H,
NHCH)
7.1.2 (S)-Tert-butyl 3-Hydroxy-1-(4-methylpiperazin-1-yl)-1-oxopropan-2-ylcarbamate
(12c): Gummy residue; yield 413 mg, (72%) 1H NMR (300 MHz, CDCl3) δ (ppm) : 1.31 (s,
9H, C(CH3)3), 2.16 (s, 3H, NCH3), 2.28-2.30 (m, 4H, COcycN(CH2CH2)2NCH3), 3.50-3.59
(m, 4H, COcycN(CH2CH2)2NCH3), 3.76 (br s, 2H, NHCHCH2), 4.56-4.57 (s, 1H, NHCH),
5.83 (d, J = 8.3 Hz, 1H, NH).
7.1.3 Tert-butyl (2R,3S) 3-Hydroxy-1-(4-methylpiperazin-1-yl)-1-oxobutan-2-
ylcarbamate (12d): Gummy residue; yield 421 mg, (70%), 1H NMR (300 MHz, CDCl3) δ
(ppm) : 1.04 (d, J = 6.2 Hz, 3H, CHCH(OH)CH3), 1.34 (s, 9H, C(CH3)3), 2.18 (s, 3H,
NCH3), 2.22-2.38 (m, 4H, COcycN(CH2CH2)2NCH3), 3.47-3.59 (m, 4H,
COcycN(CH2CH2)2NCH3), 3.97-3.99 (m, 1H, CHCH(CH3)OH), 4.44 (d, J = 5.0 Hz, 1H,
NHCHCH(CH3)OH), 5.68 (d, J = 8.3 Hz, 1H, NH).
7.1.4 (S)-Tert-butyl-3-(1H-indol-3-yl)-1-(4-methylpiperazin-1-yl)-1-oxopropan-2-
ylcarbamate (12e): Gummy residue; yield: 557 mg, (72 %) 1H NMR (300 MHz, CDCl3)
δ(ppm) : 1.34 (s, 9H, C(CH3)3), 2.01-2.35 (m, 7H, cycN(CH2CH2)2NCH3), 3.04-3.10 (m, 2H,
CHCH2), 3.29-3.59 (m, 4H cycN(CH2CH2)2NCH3), 4.84-4.91 (m, 1H, NHCH), 7.05 (d, J =
14

2.0 Hz, 1H, Ar-H indole), 7.13-7.28 (m, 2H, Ar-H indole), 7.37-7.40 (m, 2H, Ar-H indole);
ESI-MS: m/z 387 (M+H+)
7.1.5 (S)-Tert-butyl4-methyl-1-(4-methylpiperazin-1-yl)-1-oxopentan-2-ylcarbamate
(12f): Gummy residue; yield 500 mg, (80 %). 1H NMR ( 300 MHz, CDCl3) δ (ppm): 0.91-
0.98 (m, 6H, CH(CH3)2), 1.43(s, 9H, C(CH3)3), 1.70-1.87 (m, 3H, CH2CH(CH3)2), 2.29 (s,
3H, NCH3), 2.36-2.41 (m, 4H, COcycN(CH2CH2)2NCH3), 2.58 (br s, 1H, NHCHCH), 3.58
(br s, 4H, COcycN(CH2CH2)2NCH3), 4.58 (br s, 1H, NHCH).
7.1.6 Tert-butyl 3-(4-methylpiperazin-1-yl)-3-oxopropylcarbamate (12g): Gummy
residue; yield 375 mg, (70%) 1H NMR (300 MHz, CDCl3) δ(ppm) : 1.37 (s, 9H, C(CH3)3),
2.25 (s, 6H, 2NCH3), 2.31-2.35 (m, 4H, COcycN(CH2CH2)2NCH3), 2.44-2.47 (m, 2H,
NHCH2CH2), 3.33-3.42 (m, 4H, COcycN(CH2CH2)2NCH3), 3.56-3.59 (m, 2H, NHCH2), 5.33
(s, 1H, NH).
7.1.7 Tert-butyl 4-(4-methylpiperazin-1-yl)-4-oxobutylcarbamate (12h): Gummy
residue,yield 407 mg, (71%), 1H NMR (300 MHz, CDCl3) δ(ppm): 1.37 (s, 9H, C(CH3)3),
1.71-1.80 (m, 2H, NHCH2CH2), 2.25 (s, 3H, NCH3), 2.27-2.36 (m, 6H,
CH2COcycN(CH2CH2)2NCH3), 3.06-3.13 (m, 2H, NHCH2), 3.40-3.58 (m, 4H,
CH2COcycN(CH2CH2)2NCH3), 4.99 (s, 1H, NH).
7.1.8 (S)-Tert-butyl 1,5-bis(4-methylpiperazin-1-yl)-1,5-dioxopentan-2-ylcarbamate
(12i): Gummy residue; yield : 600 mg, (73%) 1H NMR (300 MHz, CDCl3) δ (ppm) : 1.36 (s,
9H, C(CH3)3), 1.76-1.79 (m, 2H, CHCH2), 2.09-2.36 (m, 10H,
CH2COcyc2N(CH2CH2)2NCH3), 2.50 (s, 6H, 2NCH3), 3.33-3.51 (m, 8H,
COcyc2N(CH2CH2)2NCH3), 4.34 (br s, 1H, NHCH).
15

7.1.9 (S)-Tert-butyl 1,4-bis(4-methylpiperazin-1-yl)-1,4-dioxobutan-2-ylcarbamate (12j):
Gummy residue,yield, 595 mg. (75%) 1H NMR (300 MHz, CDCl3) δ (ppm): 1.42 (s, 9H,
C(CH3)3), 2.28 (s, 6H, 2NCH3), 2.30-2.34 (m, 8H, COcyc2N(CH2CH2)2NCH3), 2.57-2.64 (m,
1H, CHCHH), 2.86-2.94 (m, 1H, CHCHH), 3.36-3.70 (m, 8H, COcyc2N(CH2CH2)2NCH3),
5.01-5.03 (m, 1H, NHCH), 5.42 (d, J = 8.64 Hz, NH).
8. General procedure for the synthesis of 14a-14j: Boc protection of (12a-12j) was
removed using 15% HCl/Dioxane at 0 °C.19 Compounds 13a-13j obtained in quantitative
yields as corresponding hydrochloride salts. Hydrochloride salts were converted to free base
by triethylamine and fused to the 4,7-Dichloroquinoline in the presence of phenol to obtain
compounds 14a-14j.9
8.1 (S)-2-(7-Chloroquinolin-4-ylamino)-3-methyl-1-(4-methylpiperazin-1-yl)butan-1-one
(14a): Gummy residue; yield 164 mg, 45.4%).1H NMR (300 MHz, CDCl3) δ (ppm) : 1.04-
1.12 (m, 6H, CHCH(CH3)2, 2.35 (s, 3H, NCH3), 2.44-2.51 (m, 5H, CHCH(CH3)2,
cycN(CH2CH2)2NCH3), 3.71-3.73 (m, 4H, cycN(CH2CH2)2NCH3)), 4.54-4.58 (m, 1H,
NHCH), 6.50 (d, J = 6.1 Hz, 1 H, Ar-H quinoline), 7.43 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H
quinoline), 7.88-7.91 (m, 1H, Ar-H quinoline), 8.06 (d, J = 1.9 Hz, 1H, Ar-H quinoline), 8.49
(d, J = 4.4 Hz, 1H, Ar-H quinoline). 13C NMR (75 MHz, CDCl3) δ (ppm) :18.4, 29.6, 41.5,
45.6, 45.8, 54.4, 54.9, 55.8, 98.6, 117.0, 121.9, 125.5, 127.1, 135.5, 147.7, 150.4, 169.6.
HRMS calculated for [C19H25ClN4O+H+] 361.1795, found 361.1791; ESI-MS: m/z 361
(M+H+).
8.1.1 (2S,3S)-2-(7-Chloroquinolin-4-ylamino)-3-methyl-1-(4-methylpiperazin-1-
yl)pentan-1-one (14b): Gummy residue; yield 182 mg, 48 %), 1H NMR (300 MHz, CDCl3)
δ (ppm) : 0.88-1.00 (m, 6H, CHCH3, CH2CH3), 1.48-1.78 (m, 3H, NHCHCHCH2), 2.36 (s,
1H, NCH3), 2.44-2.55 (m, 4H, cycN(CH2CH2)2NCH3), 3.62-3-78 (m, 4H,
16

cycN(CH2CH2)2NCH3), 4.92-4.97 (m, 1H, NHCH), 6.36 (s, 1H, NH), 6.55 (d, J = 6.2 Hz,
1H, Ar-H quinoline), 7.46-7.50 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.88 (d, J = 9.1
Hz, 1H, Ar-H quinoline), 8.20 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.43 (d, J = 5.4 Hz, 1H,
Ar-H quinoline); 13C NMR (75 MHz; CDCl3+DMSO-d6) δ (ppm) : 16.2, 20.6, 30.0, 41.9,
42.3, 46.8, 50.7, 60.0, 104.3, 122.6, 128.4, 129.4, 132.5, 139.1, 155.0, 156.5, 166.0, 175.1.
HRMS calculated for [C20H27ClN4O+H+] 375.1952, found 375.1946; ESI-MS: m/z 375.2
(M+H+).
8.1.2 (S)-2-(7-Chloroquinolin-4-ylamino)-3-hydroxy-1-(4-methylpiperazin-1-
yl)propan-1-one (14c): Gummy residue; yield 178 mg, 51%); 1H NMR (300 MHz, DMSO-
d6,) δ (ppm) : 2.20 (s, 3H, NCH3), 2.31-2.51 (m, 4H, cycN(CH2CH2)2NCH3), 3.33-3.43 (m,
4H, cycN(CH2CH2)2NCH3), 3.65-3.79 (m, 2H, CHCH2), 4.75-4.78 (m, 1H, NHCH), 5.15
(br s, 1H, NH), 6.44 (d, J = 6.1 Hz, 1H, Ar-H quinoline ), 7.17 (d, J = 7.2 Hz, 1H, Ar-H
quinoline), 7.47 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.81 (d, J = 2.0 Hz, 1H, Ar-H
quinoline), 8.39 (d, J = 4.4 Hz, 1H, Ar-H quinoline); 13C NMR (75 MHz, DMSO-d6) δ:
40.2, 45.3, 54.2, 54.8, 61.1, 98.4, 117.3, 122.1, 125.3, 127.4, 135.1, 148.2, 150.2, 151.0,
169.6. HRMS calculated for [C17H22ClN4O2+H+] 349.1431, found 349.1355; ESI-MS: m/z
349 (M+H+).
8.1.3 (2S,3S)-2-(7-Chloroquinolin-4-ylamino)-3-hydroxy-1-(4-methylpiperazin-1-
yl)butan-1-one (14d): Gummy residue; yield 168 mg, 46 %); 1H NMR (300 MHz, CDCl3) δ
(ppm) : 1.31 (d, J = 6.36 Hz, CHCH(OH)CH3), 2.36 (s, NCH3), 2.46-2.52 (m, 4H,
cycN(CH2CH2)2NCH3), 3.51-3.78 (m, 4H, cycN(CH2CH2)2NCH3)), 4.26-4.29 (m, NCHCH,
1H), 4.61 (m, 1H, NCH), 5.15 (br s, 1H, NH), 6.28 (d, J = 6.1 Hz, 1 H, Ar-H quinoline), 7.40
(dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.82 (d, J = 2.0 Hz, 1H, Ar-H quinoline), 7.97 (d,
J = 1.9 Hz, 1H, Ar-H quinoline), 8.52 (d, J = 4.4 Hz, 1H, Ar-H quinoline). 13C NMR (75
17

MHz, CDCl3) δ (ppm): 19.3, 42.4, 45.8, 54.3, 55.2, 57.9, 67.5, 99.5, 117.1, 122.1, 125.6,
127.3, 135.5, 147.9, 149.6, 150.8, 168.7; HRMS calculated for [C18H23ClN4O2+ H+]
363.1588, found 363.1591; ESI-MS: m/z 363.1 (M+H+).
8.1.4 (S)-2-(7-Chloroquinolin-4-ylamino)-3-(1H-indol-3-yl)-1-(4-methylpiperazin-1-
yl)propan-1-one (14e): Gummy residue; yield 197 mg, (44%); 1H NMR (300 MHz, CDCl3)
δ (ppm): 2.01-2.35 (m, 7H, cycN(CH2CH2)2NCH3), 3.04-3.10 (m, 2H, CHCH2), 3.29-3.59
(m, 4H cycN(CH2CH2)2NCH3), 4.84-4.91 (m, 1H, NHCH), 6.33-6.41( m, 2H, NH, Ar-H
quinoline), 7.05 (d, J = 2.0 Hz, 1H, Ar-H indole), 7.13-7.28 (m, 2H, Ar-H indole), 7.37-7.40
(m, 2H, Ar-H indole), 7.57 (d, J = 7.7 Hz, 1H, Ar-H quinoline), 7.77 (d, J = 8.9 Hz, 1H, Ar-
H quinoline), 7.98 (d, J = 1.9 Hz, 1H, Ar-H quinoline), 8.34 (s, 1H, NH), 8.46 (d, J = 5.4 Hz,
1H, Ar-H quinoline); 13C NMR (75 MHz, DMSO-d6) δ(ppm) :27.4, 44.2, 44.8, 53.2, 53.6,
57.0, 99.5, 109.2, 111.4, 117.1, 118.1, 118.4, 120.9, 124.3, 126.4, 127.3, 134.1, 135.9, 149.8,
150.9, 158.9, 169.8; HRMS calculated for [C25H26ClN5O+ H+] 448.1904, found 448.1898;
ESI-MS: m/z 448 (M+H+).
8.1.5. (S)-2-(7-Chloroquinolin-4-ylamino)-4-methyl-1-(4-methylpiperazin-1-yl)pentan-1-
one (14f): Gummy residue; yield:170 mg, 45 %). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.99-
1.02 (m, 6H, CH2CH(CH3)2), 1.71 (m, 1H, CH2CH), 1.87 (m, 2H, NHCHCH2), 2.33 (s, 1H,
NCH3), 2.43 (m, 4H, COcycN(CH2CH2)2NCH3), 3.56 (m, 4H, COcycN(CH2CH2)2NCH3),
4.58 (m, 1H, NHCH), 6.32 (d, J = 5.5 Hz, 1H, Ar-H quinoline), 7.36 (dd, J = 2.1, 8.9 Hz,
1H, Ar-H quinoline), 7.82 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.96 (d, J = 2.1 Hz, 1H, Ar-H
quinoline), 8.54(d, J = 5.4 Hz, 1H, Ar-H quinoline); 13C NMR (75 MHz, CDCl3) δ (ppm):
22.3, 23.4, 24.7, 41.5, 42.3, 45.3, 45.9, 54.6, 55.0, 98.7, 117.2, 121.6, 125.5, 127.7, 135.4,
148.2, 149.6, 151.0, 170.6; HRMS calculated for [C20H27ClN4O+H+] 375.1952, found
375.1946; ESI-MS: m/z 375.2 (M+H+).
18

8.1.6. 3-(7-Chloroquinolin-4-ylamino)-1-(4-methylpiperazin-1-yl)propan-1-one (14g):
Gummy residue; yield 164, (45%). 1H NMR (300 MHz, CDCl3) δ (ppm): 2.28 (s, 3H,
NCH3), 2.34-2.38 (m, 4H, cycN(CH2CH2)2NCH3), 2.69-2.72 (m, 2H, HNCH2CH2CO),
3.43-3.48 (m, 2H, HNCH2CH2CO), 3.63-3.66 (m, 4H, 2 COcycN(CH2CH2)2NCH3), 6.32 (s,
1H, NH), 6.40 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.31 (dd, J = 8.9, 2.1, Hz, 1H, Ar-H
quinoline), 7.72 (d, J = 9 Hz, 1H, Ar-H quinoline), 7.91 (d, J = 2.1 Hz, 1H, Ar-H
quinoline), 8.47 (d, J = 5.4 Hz, 1H, Ar-H quinoline); 13C NMR (75 MHz, CDCl3) δ (ppm):
31.2, 38.7, 41.5, 45.2, 45.8, 54.4, 54.7, 98.4, 117.3, 122.1, 125.3, 127.4, 135.1, 148.2, 150.2,
151.0, 169.6; HRMS calculated for [C17H22ClN4O+H]+ 333.1482, found 333.1471; ESI-
MS: m/z 333.2 (M+H+).
8.1.7. 4-(7-Chloroquinolin-4-ylamino)-1-(4-methylpiperazin-1-yl)butan-1-one (14h):
1
Gummy residue; yield:188 mg, (54%). H NMR (300 MHz, CDCl3) δ (ppm) : 1.84 (br s,
2H, CH2CH2), 2.22-2.64 (m, 9H, CH2CH2COcycN(CH2CH2)2NCH3), 3.36-3.51 (m, 4H,
COcycN(CH2CH2)2NCH3), 3.71 (br s, 2H, NHCH2), 6.34 (d, J = 5.2 Hz, 1H, Ar-H
quinoline), 7.38 (d, J = 7.4 Hz, 1H, Ar-H quinoline), 7.58 (s, 1H, NH), 7.92 (d, J = 9.0 Hz,
1H, Ar-H quinoline), 8.03 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.45 (br s, 1H, Ar-H
quinoline); 13C NMR (75 MHz, CDCl3) δ (ppm): 25.2, 33.0, 43.7, 45.3, 45.8, 45.8, 54.4,
55.1, 98.1, 117.1, 123.1, 125.4, 126.1, 135.6, 146.8, 149.7, 151.4, 171.6; HRMS
calculated for [C18H23ClN4O+H]+ 346.1633, found 346.1639 ; ESI-MS: m/z 347 (M+H+).
8.1.8. (S)-2-(7-Chloroquinolin-4-ylamino)-1,5-bis(4-methylpiperazin-1-yl)pentane-1,5-
dione (14i): Gummy residue; yield 171 mg, (36%). 1H NMR (300 MHz, CDCl3) δ (ppm):
1.95 (br s, 4H, CHCH2CH2), 2.29 (s, 3H, NCH3), 2.34 (s, 3H, NCH3), 2.39-2.52 (m, 8H, 2
cycN(CH2CH2)2NCH3), 3.30-3.90 (m, 8H, 2 cycN(CH2CH2)2NCH3), 4.88-4.90 (m, 1H,
NHCH), 6.35 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 6.68 (s, 1 H, NH), 7.41 (dd, J = 2.1, 8.9
19

Hz, 1H, Ar-H quinoline), 7.88 (d, J = 9 Hz, 1H, Ar-H quinoline), 8.00 (d, J = 2.1 Hz, 1H,
Ar-H quinoline), 8.46 (d, J = 5.3 Hz, 1H, Ar-H quinoline); HRMS calculated for
[C24H34ClN6O2+ H+] 473.2432, found 473.2424; ESI-MS: m/z 473 (M+H+).
8.1.9. (S)-2-(7-Chloroquinolin-4-ylamino)-1,4-bis(4-methylpiperazin-1-yl)butane-1,4-
dione (14j): Brown gummy residue; yield 206 mg, (44%). 1H NMR (300 MHz, CDCl3) δ
(ppm): 2.20 (s, 3H, NCH3), 2.30 (s, 3H, NCH3), 2.35-2.44 (m, 8H,
COcycN(CH2CH2)2NCH3), 2.84-2.87 (m, 2H, CHCH2), 3.42-3.71 (m, 8H,
COcycN(CH2CH2)2NCH3), 5.16-5.21 (m, 1H, NHCH), 6.20 (d, J = 6.1 Hz, 1H, NH), 6.58
(d, J = 4.1 Hz, 1 H, Ar-H quinoline), 7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.76
(d, J = 6.7 Hz, 1H, Ar-H quinoline), 7.96 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.53 (d, J =
5.6 Hz, 1H, Ar-H quinoline); HRMS calculated for [C23H31ClN6O2+H]+ 459.2275, found
459.2286; ESI-MS: m/z 459.1 (M+H+).
9. General procedure for the synthesis of 16a-16c: Boc-protected amino acids of valine,
phenylalanine and isoleucine were converted to the corresponding diazoketones .20
9.1 Boc-L-valyl-diazoketone (16a): Gummy residue; yield: 1.62 gm, (45%); 1H NMR
(300 MHz, CDCl3) δ (ppm): 0.92 (d, J = 4.3 Hz, 6H, CH(CH3)2, 1.43 (s, 9H, C(CH3)3),
1.95-2.10 (m, 1H, CHCH(CH3) 2), 3.50-3.95 (m, 1H, NHCH), 5.13 (s, 1H, COCHN2).
9.1.1. Boc-L-phenylalanyl-diazoketone (16b): Gummy residue; yield: 2.9 gm, (68%); 1H
NMR (300 MHz, CDCl3) δ (ppm) : 1.42 (s, 9H, C(CH3)3), 3.00-3.22 (m, 2H, CHCH2Ph),
4.38 (br s, 1H, NHCH), 5.12 (s, 1H, COCHN2), 5.23 (br, NH), 7.16-7.28 (m, 3H, Ar-H),
7.29-7.36 (m, 2H, Ar-H).
20

9.1.2. Boc-L-isoleucyl-diazoketone (16c): Gummy residue; yield: 1.79 gm, (47%); 1H NMR
(300 MHz, CDCl3) δ (ppm): 0.85-1.38 (m, 8H, CHCH(CH3)CH2CH3), 1.43 (s, 9H, C(CH3)3),
1.46-1.58 (m, 1H, CHCH(CH3)CH2CH3), 4.10-4.25 (m, 1H, NHCH), 5.13 (s, 1H, COCHN2).
10. General procedure for the synthesis of 17a-17c: Purified diazoketone derivatives were
then stirred in dioxane/water (5:1) at 70 °C in the presence of silver benzoate to get
corresponding β-amino acids (17a-17c) via wolf rearrangement.21
10.1. Boc-L-β3h-valine (17a): Gummy residue; yield: 1.01 gm, (88%); 1H NMR (300 MHz,
CDCl3) δ (ppm): 0.90 (d, J = 4.4 Hz, 6H, CH(CH3)2, 1.43 (s, 9H, C (CH3)3), 1.82-1.98 (m,
1H, NHCH(CH2)CH(CH3)2), 2.45-2.71(m, 2H, NHCH(CH2)CH(CH3)2), 3.45 (s, 1H, NHCH).
10.1.1. Boc-L-β3h-phenylalanine (17b): Gummy residue; yield: 1.14 gm, (82%); 1H NMR
(300 MHz, CDCl3) δ (ppm): 1.42 (s, 9H, C(CH3)3), 2.50-2.65 (m, 2H, NHCH(CH2)CH2),
2.82-3.12 (m, 2H, NHCH(CH2)CH2 Ph)), 4.11-4.23 (m, 1H, NHCH), 5.17 (br, NH), 7.18-
7.26 (m, 3H, Ar-H), 7.29-7.36 (m, 2H, Ar-H).
10.1.2. Boc-L-β3h-isoleucine (17c): Gummy residue; yield: 1.11 gm, (91%); 1H NMR (300
MHz, CDCl3) δ (ppm): 0.90-1.20 (m, 6H, CH(CH3)CH2CH3), 1.43 (s, 9H, C (CH3)3), 1.48-
1.60 (m, 2H, CH(CH3)CH2CH3), 1.93-2.05 (m, 1H, NHCH(CH)CH2), 2.40-2.73 (m, 2H, ,
NHCH(CH)CH2), 3.48 (br s, 1H, NHCH).
11. General procedure for the synthesis of 18a-18c: The compounds obtained 17a-17c
were coupled with methylpiperazine using DCC, HOBt protocol in dry THF.10
11.1. (S)-Tert-butyl 4-methyl-1-(4-methylpiperazin-1-yl)-1-oxopentan-3-ylcarbamate
(18a): Gummy residue; yield 500 mg, (80 %) 1H NMR (300 MHz, CDCl3) δ (ppm): 0.80 (d,
J = 6.6 Hz, 3H, CHCH(CH3)CH3), 0.87 (d, J = 6.6 Hz, 3H, CHCH(CH3)CH3), 1.36 (s, 9H,
21

C(CH3)3), 1.80-1.89 (m, 1H, CHCH(CH3)CH3), 2.23 (s, 3H, NCH3), 2.31-2.33 (m, 4H,
COcycN(CH2CH2)2NCH3), 2.45-2.71 (m, 2H, NHCH(CH2)CH(CH3)2), 3.49-3.61 (m, 4H,
COcycN(CH2CH2)2NCH3), 4.35-4.40 (m, 1H, NHCHCH), 5.35 (d, J = 8.6 Hz, NH).
11.1.1. (S)-Tert-butyl 4-(4-methylpiperazin-1-yl)-4-oxo-1-phenylbutan-2-ylcarbamate
(18b): Gummy residue; yield 575 mg, (80%). 1H NMR (300 MHz, CDCl3) δ (ppm): 1.42 (s,
9H, C(CH3)3), 2.31-2.33 (m, 4H, COcycN(CH2CH2)2NCH3), 2.52-2.66 (m, 2H,
NHCH(CH2)CH2), 2.83-3.14 (m, 2H, NHCH(CH2)CH2 Ph), 3.51-3.64 (m, 4H,
COcycN(CH2CH2)2NCH3), 4.14-4.23 (m, 1H, NHCH), 5.13 (br, NH), 7.16-7.27 (m, 3H, Ar-
H), 7.28-7.35 (m, 2H, Ar-H).
11.1.2. Tert-butyl (3S)-4-methyl-1-(4-methylpiperazin-1-yl)-1-oxohexan-3-ylcarbamate
(18c): Gummy residue; yield 525 mg, (84%) 1H NMR (300 MHz, CDCl3) δ (ppm): 0.82-0.95
(m, 6H, CHCH(CH3)CH2CH3), 1.39 (s, 9H, C(CH3)3), 1.61-1.71 (m, 2H, CHCH(CH3)CH2
CH3), 1.93-2.05 (m, 1H, NHCH(CH)CH2), 2.26 (s, 3H, NCH3), 2.34-2.76 (m, 6H, 2.40-2.73
NHCH(CH)CH2COcycN(CH2CH2)2NCH3), 3.54-3.65 (m, 4H, COcycN(CH2CH2)2NCH3),
4.40-4.45 (m, 1H, NHCH)
12. General procedure for the synthesis of 19a-19c:
Boc protection of 18a-18c was removed using 15% HCl/Dioxane at 0 °C.19 Compounds 19a-
19c obtained in quantitative yields as corresponding hydrochloride salt. Hydrochloride salts
were converted to free base by triethylamine and used for the next step without further
purification.
12.1 General procedure for the synthesis of 20a-20c: The compounds 19a-19c obtained
were fused with the 4,7-Dichloroquinoline in the presence of phenol resulting in compounds
20a -20c.9
22

12.1.1 (S)-3-(7-Chloroquinolin-4-ylamino)-4-methyl-1-(4-methylpiperazin-1-yl)pentan-
1-one (20a): Gummy residue; yield 168 mg, (45 %). 1H NMR (300 MHz, CDCl3) δ (ppm):
1.01-1.06 (m, 6H, CH(CH3)2), 2.10 (br s, 1H, CHCH(CH3)2), 2.25 (s, 3H, NCH3), 2.32-2.37
(m, 4H, cycN(CH2CH2)2NCH3), 2.59-2.84 (m, 2H, NHCHCH2), 3.49-3.63 (m, 4H,
COcycN(CH2CH2)2NCH3), 3.84 (br s, 1H, NHCH), 6.47 (d, J = 5.4 Hz, 1H, Ar-H quinoline),
6.58 (d, J = 7.8 Hz, 1H, NH), 7.36 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline),7.76 (d, J = 8.9
Hz, Ar-H quinoline), 7.96 (d, J = 2.1 Hz, 1H, A Ar-H quinoline ), 8.47 (d, J = 5.4, 1H, Ar-H
quinoline); 13C NMR (75 MHz, CDCl3) δ (ppm): 19.4, 31.9, 33.3, 41.5, 45.6, 45.8, 54.4,
54.9, 55.9, 98.8, 117.0, 122.1, 125.5, 126.9, 135.6, 147.7, 150.2, 150.5, 169.6; HRMS
calculated for [C20H27ClN4O+H+] 375.1952, found 375.1946; ESI-MS: m/z 375.2 (M+H+).
12.1.2. (3R,4S)-3-(7-Chloroquinolin-4-ylamino)-4-methyl-1-(4-methylpiperazin-1-
yl)hexan-1-one (20b): Yellow gummy residue; yield 160 mg, ( 41%). 1H NMR (300 MHz,
CDCl3) δ (ppm) : 0.94-1.00 (m, 6H, CH(CH3)CH2CH3), 1.58-1.66 (m, 2H,
CH(CH3)CH2CH3), 1.93-2.39 (m, 8H, NHCHCH, COcycN(CH2CH2)2NCH3), 2.59-2.89 (m,
2H, NHCHCH2), 3.52-3.63 (m, 4H, COcycN(CH2CH2)2NCH3), 3.95 (br s, 1H), 6.47 (d, J =
5.49, 1H, Ar-H quinoline), 6.97 (br s,1H, NH), 7.35 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H
quinoline), 7.82 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.92 (d, J = 2.1 Hz, 1H, Ar-H
quinoline), 8.44 (d, J = 5.4 Hz, 1H, Ar-H quinoline): 13C NMR (75 MHz, CDCl3+DMSO-d6)
δ (ppm) : 16.2, 20.6, 30.0, 33.2, 41.9, 42.3, 46.8, 50.7, 60.0, 104.3, 122.6, 128.4, 129.4,
132.5, 139.1, 155.0, 156.5, 166.0, 175.1; HRMS calculated for [C21H30ClN4O+H+] 389.2108,
found 389.2139; ESI-MS: m/z 389.2 (M+H+).
12.1.3 (S)-3-(7-Chloroquinolin-4-ylamino)-1-(4-methylpiperazin-1-yl)-4-phenylbutan-1-
one (20c): Brown gummy residue; yield 190 mg, 45 %. 1H NMR (300 MHz, CDCl3) δ (ppm):
2.29 (s, 3H, NCH3), 2.30-2.64 (m, 4H, COcycN(CH2CH2)2NCH3), 2.93-3.34 (m, 4H,
23

NHCHCH2CO, CHCH2Ph), 3.63-3.70 (m, 4H, COcycN(CH2CH2)2NCH3), 4.2 (br s, 1H),
6.44 (d, J = 5.49 Hz, 1H, Ar-H quinoline), 6.99 (d, J = 7.86 Hz, 2H, Ar-H quinoline), 7.18
(m, 4 H, 3Ar-H, 1Ar-H quinoline ),7.74 (d, J = 8.9, 1H, Ar-H quinoline), 7.92 (s, 1H, Ar-H
quinoline), 8.46 (d, J = 5.4, 1H, Ar-H quinoline). 13C NMR (75 MHz, CDCl3): 33.2, 39.2,
41.5, 45.3, 45.4, 51.4, 54.9, 54.8, 98.5, 117.3, 121.9, 125.4, 126.6, 126.8, 127.8, 128.5, 128.7,
129.1, 135.5, 137.8, 148.5, 148.2, 151.1, 169.5; HRMS calculated for [C24H28ClN4O+H+]
423.1952, found 423.1947; ESI-MS: m/z 423.2 (M+H+).
13. General procedure for the synthesis of (30): The L- phenylalanine was converted to its
methyl ester (22) by drop wise addition of thionyl chloride to the methanolic solution of 21 at
0 °C.22 After completion of the reaction solvent was evaporated and the compound was
crystallized by dry ether. The hydrochloride salt obtained was basified by triethylamine and
dissolved in dry dichloromethane. Di-tert-butylpyrocarbonate was added to the solution at 0
°C and reaction was stirred for next three hours. This afforded the Boc protected methyl ester
(23) in quantitative yields. A carefully controlled reduction of 23 by DIBAL-H at -78 °C
afforded 24 (the N protected amino aldehyde) and the aldehyde was directly used for the
Wittig reaction without any purification.23 The reaction of aldehyde with the stabilized ylide
in THF at 0 °C led to α, β-unsaturated esters (25). Crude product was purified by the silica
gel column chromatography. Compound 25 obtained in 70% yield after 2 steps. Catalytic
hydrogenation of the double bond.23 led to Boc-protected ethyl ester of γ-amino acid (26) in
quantitative yield. Compound 26 was hydrolyzed in a mixture of THF and water (9:1 ratio)
by lithium hydroxide produced the Boc protected γ-amino acid (27). Compound 22 was
reacted at -15 °C in dry THF with N-methylpiperazine using isobutylchloroformate (IBCF) in
the presence of N-methylmorpholine (NMM) to afford compound 28 in 90% yield.
Deprotection of 28 by 15% HCl/Dioxane at 0 °C gave compound 29 in quantitative yield.19
24

The salt was converted to free amine (29) and it was fused with 4,7-DCQ in phenol at 140 °C
afforded the final product (30).9
13.1. (S)-Methyl 2-(tert-butoxycarbonylamino)-3-phenylpropanoate (23): At 0 °C di-tert-
butylpyrocarbonate (7 mL, 30 mmol) was added drop wise under a nitrogen atmosphere to a
solution of L-phenylalanine methyl ester hydrochloride 22 (5 g, 23 mmol) and triethylamine
(5.5 mL, 56 mmol) in CH2Cl2, (120 mL). The resulting mixture was stirred at room
temperature for 5 h. After completion of the reaction organic layer was washed with 5% citric
acid (100 mL), then with 5% NaHCO3, (100 mL) and finally with H2O (50 mL). The organic
layer was dried over Na2SO4, and evaporated to give an oily residue (Yield: quantitative).
Compound was purified by column chromatography with 5% EtOAc/hexane. ESI-MS: m/z
280 (M+H+).
13.1.1. (S)-Tert-butyl 1-oxo-3-phenylpropan-2-ylcarbamate (24): Protected ester (7.4 g,
26.5 mmol) obtained above was dissolved in dry THF (300 mL) and cooled to -80 °C. To this
diisobutylaluminum hydride (52 mL, 1 M solution in cyclohexane, 0.052 mol) was added via
needle over a period of 45 minutes while maintaining the internal temperature below -69 °C.
The mixture was stirred for 3 hrs, and precooled (-75 °C) methanol (20 mL) was added.
During the addition, the reaction mixture was maintained below -69 °C. The mixture was
then allowed to warm to 5 °C, and 300 g of ice was added with heavy agitation, the mixture
was filtered through sintered funnel and extracted with chloroform. Organic layer was
washed with brine (100 mL), dried over MgSO4, and concentrated. The crude product thus
obtained stored at -20 °C for next step.
13.1.2. (S,E)-Ethyl 4-(tert-butoxycarbonylamino)-5-phenylpent-2-enoate (25): To a
solution of the above N-protected α-amino aldehyde in dry DCM (50 mL) at 0 °C EtOOCCH
= PPh3 (26.5 mmol) was added and the solution stirred for 2-3 h. After completion of the
25

reaction (as monitored by TLC), the reaction mixture was concentrated on a rotary evaporator
to give the crude product. This was purified by column chromatography using a mixture of
EtOAc and hexane as eluent. (Yield : 5.5g, 65%). 1H NMR (300 MHz, CDCl3) δ (ppm): (E-
isomer) 1.24-1.29 (t, J = 7.11 Hz, 3H, CH2CH3), 1.39 (s, 9H, C(CH3)3), 2.88-2.90 (m, 2H,
CHCH2Ph), 4.14-4.21 (m, 2H, COOCH2CH3), 4.66-4.69 (m, 2H, NHCH), 5.84 (d, J = 17.3
Hz, 1H, CH=CH), 6.88 (dd, J = 15.6 Hz, 4.89, 1H, CH-CH=CH), 7.18-7.32 (m, 5H, Ar-H).
13.1.3. (R)-Ethyl 4-(tert-butoxycarbonylamino)-5-phenylpentanoate (26): To a solution of
the olefin (5.5g, 16.5mmol) in MeOH 10 % (w/w) Pd/C (10%) was added. The apparatus
flushed 2 times with hydrogen gas, and the mixture was agitated at room temperature for 2 h
under hydrogen gas at 30 Psi. Filtration through Celite and concentration under reduced
pressure yielded the ethyl carboxylate which was used without further purification. (Yield 5.3
g, quantitative). 1H NMR (300 MHz, CDCl3) δ (ppm): 1.20 (t, J = 6.4 Hz, 3H, CH2CH3),
1.39 (s, 9H, C(CH3)3 ), 1.58-1.65 (m, 1H, CHCHH), 1.79-1.89 (m, 1H, CHCHH), 2.32-2.38
(m, 2H, CH2CH2), 2.69-2.80 (m, 2H, CH2Ph), 3.82 (br s 1H, NHCH), 4.06-4.03 (m, 2H,
COOCH2), 7.16-7.29 (m, 5H, Ar-H).
13.1.4. (R)-4-(Tert-butoxycarbonylamino)-5-phenylpentanoic acid (27): Ethyl ester (1g,
3.1 mmol) was dissolved in 20 mL of THF, and LiOH solution (0.51 g, 12 mmol) in water
was added to it. The mixture was stirred at room temperature for 30 h. After completion of
the reaction the reaction mixture was acidified with citric acid solution to (pH 3) and
extracted with chloroform 25 mL (2 x), the organic phases were washed with saturated NaCl
solution, dried over sodium sulfate and evaporated to afford oily residue.(Yield:
Quantitative). 1H NMR (300 MHz, DMSO-d6) δ (ppm): 1.32 (s, 9H, C(CH3)3), 1.54-1.46 (m,
1H, CHCHH), 1.70-1.62 (m, 1H, CHCHH), 2.20 (m, 2H, CHCH2CH2), 2.65 (m, 2H,
26

CHCH2Ph), 3.60 (m, 1H, NHCH), 6.72 (brs, NHCH, 1H), 7.29-7.16 (m, 5H), 11.99 (br s,
1H).
13.1.5. (R)-Tert-butyl 5-(4-methylpiperazin-1-yl)-5-oxo-1-phenylpentan-2-ylcarbamate
(28):
To a solution of 27 (586 mg, 2 mmol) in 10 mL THF with 1.0 mL of DMF, NMM (0.22 mL,
2 mmol) and IBCF (0.26 mL, 2 mmol) were added successively at -15 ºC under vigorous
stirring. The temperature was maintained at -15 ºC for 10 min. Subsequently N-
methylpiperazine (0.270 mL, 3 mmol) was added to the reaction mixture. The reaction was
stirred at -15 ºC for 15 min and then allowed to stir at room temperature for additional 2 hr.
Solvent was evaporated under reduced pressure. The oily residue was taken in EtOAc and
washed with 5 % aqueous sodium bicarbonate, water and finally with brine. The organic
layer was dried over anhydrous Na2SO4, evaporated under reduced pressure and purified by
column chromatography (1% methanol chloroform was used as eluent). This compound was
obtained as oil (yield 562 mg, 75%). 1H NMR (300 MHz, CDCl3) δ (ppm) : 1.31 (s, 9H,
C(CH3)3), 2.04-2.22 (m, 2H, CHCH2CH2), 2.24 (s, 3H, NCH3), 2.30-2.62 (m, 4H,
CHCH2CH2COcycN(CH2CH2)2NCH3), 2.83-2.90 (m, 2H, CHCH2Ph), 3.11-3.60 (m, 4H,
COcycN(CH2CH2)2NCH3), 3.82 (br s, 1H, NHCH), 7.22-7.35 (m, 5H, Ar-H).
13.1.6. (R)-4-Amino-1-(4-methylpiperazin-1-yl)-5-phenylpentan-1-one (29): Compound
28 was deprotected by 15 % HCl/Dioxane. After evaporation of solvent the hydrochloride
salt was crystallized by dry ether. The hydrochloride salt was converted to free base 29 by
triethylamine and used without further purification.
13.1.7. (R)-4-(7-Chloroquinolin-4-ylamino)-1-(4-methylpiperazin-1-yl)-5- phenylpentan-
1-one (30): To a solution of 29 (412.5 mg, 1.5 mmol) in phenol 4,7-Dichloroquinoline (198
mg, 1mmol) was added. The reaction mixture was stirred at 140 °C for 5 hours. After
27

completion of the reaction the reaction mixture was dissolved in chloroform and extracted
with sodium hydroxide solution and then with water and finally with brine. Organic layer was
dried over sodium sulfate and concentrated in vacuo. Compound was purified by column
chromatography using chloroform: methanol: triethylamine in 9.5:0.3:0.2 ratio. Compound
obtained as dark brown gummy residue. (Yield: 201 mg; 41%). 1H NMR (300 MHz, CDCl3)
δ: 2.03-2.22 (m, 2H, CHCH2CH2), 2.24 (s, 3H, NCH3), 2.30-2.62 (m, 4H,
CHCH2CH2COcycN(CH2CH2)2NCH3), 2.83-3.13 (m, 2H, CHCH2Ph), 3.37-3.62 (m, 4H,
COcycN(CH2CH2)2NCH3), 3.80 (br s, 1H, NHCH), 6.42 ( d, J = 8.9 Hz, 1H, Ar-H
quinoline), 7.21-7.34 (m, 5H, Ar-H), 7.41 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.92 (d,
J = 9.0 Hz, 1H, Ar-H quinoline), 8.02 (d, J = 1.9 Hz, 1H, Ar-H quinoline), 8.41 (d, J = 5.8
Hz, 1H, Ar-H quinoline); HRMS calculated for [C25H29ClN4O+H+] 437.2103, found
437.2098; ESI-MS: m/z 437 (M+H+).
14. Biological testing assay.
14.1 In vitro antimalarial assay.
The compounds were dissolved in DMSO to get 10 mM concentration. Two fold serial
dilutions of the test samples were made in 96 well plates and incubated with 1.0% parasitized
cell suspension containing 0.8% parasitemia (an asynchronous culture with more than 80%
ring stages). The plates were incubated at 37 °C in CO2 incubator in an atmosphere of 5%
CO2 and air mixture. Later (72 h) 100µl of lysis buffer containing 2 x concentration of SYBR
Green-1 (Invitrogen) was added to each well and incubated for 1 h at 37 ˚C. The plates were
examined at 485 20 nm of emission for relative fluorescence units (RFUs) per well using the
fluorescence plate reader (FLUO star, BMG lab technologies). Data was transferred into a
graphic programme (EXCEL) and IC50 values were obtained by Logit regression analysis of
dose response curve.24 Chloroquine (CQ) was used as the standard reference drug.
28

14.1.1. 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 Mosmann et al.25 The cells were incubated
with compound-dilutions for 72 h and MTT was used as reagent for detection of cytotoxicity,
50% cytotoxic concentration (CC50) wasdetermined using nonlinear regression analysis of
dose response curves using pre-programmed Excel spreadsheet. Selectivity Index (SI) was
calculated as
SI = CC50 / IC50
14.1.2. Determination of hematin - 4-aminoquinoline derivatives association constant
Association constant for hematin - 4-aminoquinoline derivatives complex formation were
determined by spectrophotometric titration procedure in aqueous DMSO at pH-7.5.26 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.
14.1.3. In vitro inhibition of β-Hematin polymerization:
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.27 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 GraphPad Prism
29