Synthesis and antimalarial activity of new 4-aminoquinolines active against drug resistant strains

Article abstract of DOI:10.1039/c6ra14016e

In the present study we have synthesized a new class of 4-aminoquinoline derivatives via replacement of the diethylamine functionality of chloroquine (CQ) with acyclic and/or cyclic amine groups containing basic tertiary terminal nitrogen and bioevaluated them for antimalarial activity against Plasmodium falciparum in vitro (CQ-sensitive strain-3D7 & CQ-resistant strain-K1) and Plasmodium yoelii in vivo (N-67 strain). Among the series, thirteen compounds showed superior antimalarial activity against K1 strain as compared to CQ. In addition, all these analogs showed 100% suppression of parasitaemia on day 4 in the in vivo mouse model against N-67 strain when administered orally. Further, biophysical studies suggest that these compounds act on the heme polymerization target.

Full text of DOI:10.1039/c6ra14016e

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Page 1 of 39
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Synthesis and Antimalarial Activity of New 4-Aminoquinolines Active
Against Drug Resistant Strains
Srinivasarao Kondaparlaa, Awakash Sonib, Ashan Manhasb, Kumkum Srivastavab, Sunil K.
Purib, and S. B. Kattia*
aMedicinal & Process Chemistry Division, CSIR-Central Drug Research Institute Sector-10, Jankipuram
Extension, Sitapur Road, Lucknow -226031.
bParasitology Division, CSIR-Central Drug Research Institute, Sector-10, Jankipuram Extension, Sitapur Road,
Lucknow-226031.
*Corresponding author: To whom correspondence should be addressed.
Dr. S. B. Katti
Medicinal & Process Chemistry Division,
CSIR-Central Drug Research Institute,
Jankipuram Extension, Lucknow-226 031, India;
E-mail: setu_katti@yahoo.com
Tel: +91-522-2772454;
Fax: +91-522-2771941.
Abstract:
In the present study we have synthesized a new class of 4-aminoquinoline derivatives via
replacement of diethylamine functionality of chloroquine (CQ) with acyclic and/or cyclic
amine groups containing basic tertiary terminal nitrogen and bioevaluated for antimalarial
activity against Plasmodium falciparum in vitro (CQ-sensitive strain-3D7 & CQ-resistant
strain-K1) and Plasmodium yoelii in vivo (N-67 strain). Among the series, thirteen
compounds showed superior antimalarial activity against K1 strain as compared to CQ. In
addition, all these analogs showed 100% suppression of parasitaemia on day 4 in the in vivo
mouse model against N-67 strain when administered orally. Further, biophysical studies
suggest that these compounds act on heme polymerization target.
Keywords: 4-aminoquinoline; antimalarial; heam binding

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1. Introduction
Despite several advances in the drug development over a few decades, malaria still
remains a serious health problem in the world because of its high mortality and morbidity
burden, as well as its influence on the socio-economic development of the countries where it
is endemic1. Malaria is caused by parasitic protozoan of the genus Plasmodium. Among the
five species of Plasmodium (P. falciparum, vivax, ovale, malariae and knowlesi) known to
infect human’s P. falciparum, is the most virulent and is responsible for 300-500 million
clinical cases each year, and 3 million deaths annually2. Over the last 60 years the use of
quinine has declined owing to the development of synthetic 4-aminoquinolines such as
chloroquine (CQ)3 (Figure 1). Further, CQ and other aminoquinoline derivatives (Figure 1)
exert activity against all Plasmodial species in the erythrocytic stage and also they are
believed to function by inhibiting the formation of crystalline hemozoin in the P. falciparum
digestive vacuole (DV)4. The immense success of CQ and other aminoquinoline derivatives
is due to their ability to interfere with the heme polymerization process and the ability to
accumulate in biochemically relevant concentrations at the site of drug action5. However,
their clinical efficacy for treatment of malaria has been seriously eroded in recent years, due
to emergence and wide-spread of drug resistant parasites. Therefore, there is an urgent need
for the development of alternative drugs with unique mode of action that can circumvent the
problem of P. falciparum resistance. The CQ scaffold still could be a front line
pharmacophore for chemical modification despite emergence of resistance6.
Based on this premise a number of research groups have developed a number of 4-
aminoquinoline derivatives either by shortening (up to 3 carbons) or by lengthening (up to 10
carbon) the side chain length, and chemoselective modifications at diethylamino functionality
of CQ7. Among all CQ analogues, those having shorter chain length consistently show good
inhibition against CQ-resistant strain of P. falciparum in the in vitro studies. This observation
holds good only for quinoline derivatives that contain a diethyl substituent on the terminal
nitrogen8. However, shortening the chain length had no effect on the dealkylation of the distal
amino group when administered in vivo, which is the major metabolic pathway of CQ9.
However, substitution of bulkier groups at the distal amino side chain led to the
compounds that showed better in vivo activity and decrease in the cross resistance with CQ,
presumably by circumventing metabolic dealkylation10. However, Guy et al. synthesized a
side chain modified 4-aminoquinolines (Figure 1A) by incorporating bulkier substituent
including aromatic and heterocyclic ring systems at the lateral part of CQ. Consequently,

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increased efficacy has been observed against both 3D7 and W2 strains of P. falciparum11.
Furthermore, to overcome the problem of metabolic instability, Chibale et al. synthesized a
series of 4-aminoquinolines (Figure 1B) by incorporating bulkier substituent such as aromatic
and tetrazole rings while varying the length of the alkyl side chain and obtained good anti-
plasmodial activity in both in vitro (3D7 & K1) and in vivo (P. berghei) models12.
Inspired by these encouraging results and in continuation of our efforts to develop
effective antimalarial agents, earlier we have reported the design, synthesis and antimalarial
activity of some side chain modified 4-aminoquinolines13. Subsequently, we have synthesized
a series of 4-aminoquinolines with a variety of heterocyclic ring substituted thiourea
functionality in the side chain (Figure 2A). These analogues showed good activity against
CQ-resistant strain (Dd2) of P. falciparum in vitro14. Furthermore, we have demonstrated that
modification of 4-aminoquinoline lateral side chain with thiazolidine-4-one ring substitution
(Figure 2B) has led to compounds with improved antimalarial activity, and more importantly
some of these compounds were found to be more effective than CQ15. Further, we introduced
biologically privileged thiazolidine heterocyclic ring system (Figure 2C) at lateral part of the
side chain in order to enhance the lipophilic nature this could help the molecule to show
better antimalarial activity against resistant strains of parasite. These analogs exhibited
promising activity against CQ-sensitive strain of P. falciparum NF-54 in vitro and CQ-
resistant strain N-67 of P. yoelii in vivo16. Recently we reported a new series of side chain
modified 4-aminoquinolines constructed by amide bond, led to the compounds with
promising antiplasmodial activity against 3D7 & K1 strains of P. falciparum (Figure 2D).
Taking a note on above statement, earlier we have reported the synthesis of chiral
chloroquine and its analogues obtained by modifying amine side chain derived from various
amino acids (Figure 2E). These chloroquine analogues showed promising activity against P.
falciparum (3D7 & K1) in vitro and P. yoelii (N-67 strain) in vivo17. Based on the above
observations herein we have rationally designed the target compounds (Figure 2F) with
special emphasis on circumventing metabolic N-dealkylation by incorporating bulkier
substituents such as piperidinyl, substituted piperidinyl, pyrrolidinyl, morpholinyl and alkyl
amines in the side chain. The results are described in the present communication.
2. Results and Discussion
2.1 Chemistry
The strategy for the synthesis of the target compounds (10e-10m, 11e-11m and 12e-12j) is
outlined in scheme 1. The reaction of amino acids viz 4, 5 and 6 with Boc anhydride in the
presence of sodium hydroxide in dioxane at 0°C for 2-3 h with continuous stirring afforded

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4a, 5a and 6a in excellent yields. The intermediates obtained were used for the next reaction
without further purification. Subsequently, the Boc protected amino acids (4a, 5a and 6a)
were subjected to esterification with methyl iodide in the presence of potassium carbonate in
DMF at 0°C to afford 4b, 5b and 6b in good yields. Subsequently, methyl esters were
converted to the corresponding alcohols (4c, 5c, and 6c) by NaBH4 mediated reduction in
THF at 65°C18. The obtained alcohols were subjected to mesylation using 3 equiv. of mesyl
chloride in THF at 0°C to give 4d, 5d and 6d in excellent yields, followed by nucleophilic
substitution with corresponding amines in CH3CN afforded 4e-4m, 5e-5m and 6e-6j. Then
Boc group was finally deprotected using 15% HCl/dioxane to obtain the corresponding amine
hydrochloride salts (7e-7m), (8e-8m) and (9e-9j), which were finally treated with 4,7-
dichloroquinoline in phenol at 160°C to obtain the target compounds in good yields.
2.2 In vitro antiplsmodial activity
The final compounds synthesized (10e-10m, 11e-11m, and 12e-12j) in the present
study were evaluated for antiplasmodial activity against CQ-sensitive (3D7) & CQ-resistant
(K1) strains of P. falciparum in vitro according to the procedure reported by Kondaparla et
al. and the results are shown in Table 119. Some of the potent molecules, which have shown
better activity than CQ against K1 strain, were evaluated for in vivo activity against the
innately chloroquine resistant P. yoelii (N-67 strain) in Albino mice of Swiss strain (Table 2).
The in vitro activity data (IC50) showed that these derivatives, having bulky cyclic substituent
in the side chain as well as diversity in the amino acid, showed very good antiplasmodial
activity. Suggesting that modification at the side chain nitrogen atom is very well tolerated
for antimalarial activity.
Among the 24 compounds tested, four compounds showed IC50 range between 7-41
nM, three compounds had IC50 ranging between 74-80 nM, three compounds exhibited IC50
ranging between 112-945 nM, and IC50 ranging between 1,134-2,783 nM was observed in six
compounds. The remaining 8 compounds have shown IC50 values above 5,000 nM, when
screened against chloroquine sensitive (3D7) strain of P. falciparum. Further, out of these 24
compounds, 13 compounds were found to be more active (with IC50 between 21-183 nM)
against the chloroquine resistant (K1) strain as compared to CQ (IC50 = 255 nM). Two
compounds (10e and 11e) showed IC50 values of 395 and 395 nM which were comparable to
CQ. The differences in the IC50 values can be attributed to factors such as ring size, diversity
in the amino acid side chain, position of methyl group on the piperidyl ring and type of
dialkyl amine in the side chain.

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The compounds 10f and 12f having methyl substitution at 3rd position on piperidyl
ring exhibited mild inhibition against 3D7 strain and superior potency against K1 strain, as
compared to CQ. Whereas compound 11f was found to be inactive against 3D7 strain but
found to be second most potent molecule in the series against K1 strain with IC50 value 46
nM. On the other hand 4-methyl piperidyl analogue (10g, IC50 =141 nM) showed good
activity against K1 strain than corresponding phenylalanine (11g, IC50 = 938 nM) and alanine
(12g, IC50 = >5,000 nM) derivatives. Among these analogues compound 11g (IC50 = 7 nM)
exhibited very akin antiplasmodial activity against 3D7 strain as compared to CQ. It may be
appropriate to mention here that in the case of leucine (10e, 10f and 10g) improved activity
was observed against K1 strain when the methyl group was change to 2nd, 3rd and 4th position
on piperidyl ring.
Further, in the case of an unsubstituted piperidyl compound 10h (IC50 = 58 nM,
leucine analogue), the activity increased significantly against the K1 strain when compared to
its methyl substituted compounds. Whereas compound 11h (IC50 = 93 nM, phenylalanine
analogue) with similar moiety was found to be more active against K1 strain than its 2-
methyl piperidyl (11e, IC50 = 395 nM) and 4-methyl piperidyl (11g, IC50 = 938 nM)
analogues but it showed slightly less potency than 3-methyl piperidyl compound (11f, IC50=
46 nM) against K1 strain. This activity difference may be due to variation in the hydrophobic
nature of the amino acid side chain. Moreover, in the case of leucine slight reduction in the
antiplasmodial activity was observed in both strains (3D7 & K1) of P. falciparum when the
size of the ring reduced to pyrrolidino (compound 10i, IC50 = 1,845 & 86 nM). On the other
hand pyrrolidino analogue 11j (IC50 = 112 & 78 nM) of phenylalanine was found to be
slightly more active in both strains (3D7 & K1) of P. falciparum as compared to compound
11h (IC50 = 578 & 93 nM). Whereas coming to alanine pyrrolidino analogue 12i (IC50=164
nM) showed very good activity against K1 strain than piperidyl derivative 12h (IC50 = >5,000
nM). It is important to note that compounds (10i, 11j and 12i) exhibited 2.9 fold, 3.2 fold and
1.5 fold superior activities against K1 strain as compared to CQ. Further, introduction of
morpholino in the side chain led the compounds 10j and (IC50 = 2,783 & 613 nM) 11i (IC50 =
1,134 & 1,610 nM) with many fold decrease in the activity in both 3D7 & K1 strains of P.
falciparum. This difference in the activity may be due to diversity in their lipophilic nature.
Further, with a view to modify the lipopholic nature of the side chain, another
modification was performed in the series i.e. the replacement of cyclic amine with acyclic
amines (dimethyl, diethyl and di-n-propylamine) at the side chain of 4-aminoquinoline to

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furnish compounds 10k-10m, 11k-11m and 12j respectively. However, in the case of leucine
compounds 10k and 10l were found to be less active against 3D7 strain of P. falciparum,
when compared to its cyclic derivatives. Nevertheless, some of the compounds (10k, 10l, 11k
and 11l) exhibited much better activity (with IC50 values 99 nM, 89 nM, 183 nM and 118
nM) against K1 strain as compared to CQ. Furthermore, the activity of the compounds (10k,
10l and 10m) against K1 strain is directly proportional to the branching of the alkyl amine
side chain. The present results suggest that these 4-aminoquinolines appear to be promising
candidates for further lead optimization to obtain compounds active against CQ-resistant
parasites.
2.3 In vitro cytotoxicity
The cytotoxicity of all the synthesized molecules was determined against VERO cell line
using MTT assay (Table 1). Most of the compounds in the series showed high selectivity
indices (SI) values. Out of the 24 compounds, 17 compounds showed high selectivity indices
(SI) ranging between 65 and 4,803, rest of molecules showed selectivity index <50.
Compounds 10h, 10i, 10k, 10l and 11f which were most potent against K1 strain also showed
good selectivity index 1,421, 1,033, 396, 1,227 and 1,591 respectively (Table 1). Further,
compound 12f which was most potent in the series against resistant strain K1, also showed
good selectivity index of 4,803 In general, most of the compounds of the series exhibited
greater potency than CQ against K1 strain, less cytotoxic effect with fairly high selectivity
index, and therefore these 4-aminoquinoline derivatives appear to be good candidate for
further lead optimization.
2.4 In vitro inhibition of β-hematin polymerization
The mode of action of these 4-aminoquinoline derivatives (10e-10m, 11e-11m, and 12e-12j)
was investigated by the reported method and the results are shown in (Table 1). The results of
heme binding assay showed that all the synthesized compounds form complex with hematin
and the range of LogK was found to be 4.19-7.26. Among all the compounds reported,
compounds 11f and 12f have shown good binding to hematin. This result is concurrent with
the previous results reported in the literature. 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. The binding data suggest that the
mechanism of action is similar to chloroquine class of molecules and there is no linear
correlation between the heme binding and the in vivo activity because of PK/ADME
considerations that influence the in vivo activity. Therefore in the present study heme binding
data is used only to reiterate the mechanism of action.

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All the synthesized 4-aminoquinoline derivatives (10e-10m, 11e-11m, and 12e-12j) inhibited
the β-hematin formation in a concentration dependent manner (Table 1). The IC50 obtained
was in the range of 0.25–1.24 µM and the most active compound 12f in the series against K1
strain exhibited IC50 (0.25 0.02 µM).
2.5 In vivo antimalarial activity
Selected compounds that were significantly active in vitro (10f, 10g, 10h, 10i, 10k,
10l, 11f, 11g, 11h, 11j, 11k, 11l, 12f and 12i) were evaluated for in vivo activity against
innately chloroquine resistant P. yoelii (N-67 strain) in Albino mice of Swiss strain. Initially,
the in vivo activity of selected compounds were determined through oral route at the dose of
100 mg/kg administered once daily for four consecutive days and monitored for parasitaemia
reduction, and survival of mice until day 28 post-infection (Table 2).
Compound 10l, 11f, 11k, 11l and 12i showed 100% parasitaemia suppression on day 4 at a
dose of 100 mg/kg, but none of the mice survived up to day 28. Further, compounds 10g and
11j displayed 100% parasitaemia suppression on day 4 with 80% survival rate up to day 28 of
treatment at a dose of 100 mg/kg. Among these compound 10g cured 4 mice out of 5 whereas
compound 11j showed 60% curative rate only. Further, compounds 11g, 11h and 12f
administered at two different doses 100 and 50 mg/kg. At a dose of 100 mg/kg all three
compounds showed 100% parasitaemia suppression on day 4 with 100% survival and
curative rates as well. While, at lower dose 50 mg/kg compound 11h displayed 100 %
parasitaemia suppression on day 4 with 80% survival rate up to day 28 and four mice out of
five were cured. Whereas, compound 12f exhibited 100% parasitaemia suppression on day 4,
but none of the mice were cured. However, compound 11g showed 100% parasitaemia
suppression on day 4 with 100% curative rate at a dose of 50 mg/kg, but at lower dose 25
mg/kg one mouse out of five was cured with 100% parasitaemia suppression on day 4.
Furthermore, most active compounds (10h and 10i) against K1 strain, tested initially at a dose
of 100 mg/kg, showed 100% survival as well as curative rates up to day 28. Further, these
two compounds were administered at lower doses such as 50, 25 and 12.5 mg/kg. They
displayed 100% parasitaemia suppression on day 4 with all mice were cured up to day 28 at
doses of 50 and 25 mg/kg. Whereas, at a dose of 12.5 mg/kg the compound 10h exhibited
100% parasitaemia suppression on day 4 with 80% survival rate up to day 28 and 2 mice
were cured out of 5. Whereas, compound 10i at 12.5 mg/kg showed 100% parasitaemia on
day 4 but none of the mice survived up to day 28 of the treatment.

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3. Conclusion
In summary, a novel series of side chain modified 4-aminoquinoline derivatives were
prepared in an attempt to search for more potent molecule that can be effective against both
CQ sensitive and resistant strains of P. falciparum. The in vitro antimalarial result of these
compounds indicate that in comparison to reference drug CQ, 13 compounds (10f-10i, 10k-
10l, 11f, 11h, 11j, 11k, 11l, 12f and 12i) showed superior activity against K1 strain of P.
falciparum. Further, the in vivo results on selected compounds revealed that these compounds
also show good antimalarial activity in the mouse model. The biochemical studies confirm
that the mechanism of action is similar to that of CQ, as most of the compounds form an
association complex with hematin and thereby inhibit hemozoin formation. In summary
results discussed in the manuscript suggest that the presence of bulky cyclic substituent in the
side chain and less hindred amino acids at 4th position of quinoline exhibit potent activity
against K1 strain and require further lead optimization.
4. Experimental Procedures
4.1 General Information
Melting points (mp) were taken in open capillaries on Complab melting point apparatus and
1
are uncorrected. The H NMR (300, 400 MHz) and 13C NMR (100, 125 MHz) spectra were
recorded in CDCl3 solvent on DPX-300, and 400 Bruker FT-NMR spectrometers. All
chemical shifts (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 QTOF (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 (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 Sigma Aldrich (St. Louis, MO, USA), Lancaster
(Port of Heysham Industrial Park, Lancashire LA3 2XY, UK) and Spectrochem (Anand
Bhavan, Princess Street, Mumbai, India) and were used without further purification.

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4.2
General Procedure for the Synthesis of 4a, 5a and 6a
To a suspension of amino acid (leucine, phenylalanine and alanine) (1.0 equiv.) in dioxane-
water mixture (1:1, 100 mL) at room temperature was added 2N NaOH with stirring. The
reaction mixture was then cooled to 0°C and stirred for 15 min. Finally, (Boc)2O (1.1 equiv.)
in dioxane (10 mL) was added and the reaction mixture was allowed to stir at 0 °C for 45
min. The ice bath was then removed and the temperature of the reaction mixture was allowed
to rise to the room temperature. On completion of the reaction as monitored by the TLC
reaction mixture was concentrated under reduced pressure. The aqueous layer was acidified
with citric acid (pH 2–3) and extracted with EtOAc. The combined organic layer was washed
with brine, dried over Na2SO4 and concentrated under reduced pressure. The yields were
quantitative. These intermediates were used for the next step without further purification.
4.3.
General Procedure for the Synthesis of 4b, 5b and 6b
To a suspension of 4a, 5a and 6a (1.0 equiv.) in DMF (40 mL) at 0ºC, after 15 min K2CO3
(2.5 equiv.) was added. Then after 15 min, Methyl iodide (1.5 equiv.) was added drop wise
and the reaction mixture stirred (open air) from 0ºC to room temperature over 6-8 h. The
reaction mixture was diluted with EtOAc (300 mL) and the organic layer was washed twice
with 5% sodium bicarbonate and finally with brine. The organic layer was dried over
Na2SO4, and concentrated to a gummy substance to get 4b, 5b and 6b. The yields were
quantitative. This was used for the next step without further purification.
4.4
General Procedure for the Synthesis of 4c, 5c and 6c
Methanol (0.8 mL) was added drop wise over a period of 20 min to a mixture of N- protected
amino ester (1 mmol) and NaBH4 (2 mmol) in THF at 65 °C. The mixture was stirred for 1h,
and then water was added to the reaction mixture. Organic solvent was concentrated under
reduced pressure and brine was added and the mixture was extracted with EtoAc. The organic
layer was dried over Na2SO4 and concentrated under reduced pressure. The residue obtained
was purified on the silica gel column chromatography using hexane: EtoAc as eluant.
4.4.1 (S)-Tert-butyl 1-hydroxy-4-methylpentan-2-ylcarbamate (4c)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (300
MHz, CDCl3): δ 0.93 (d, J = 6.5 Hz, 3H, CH2CHCH3CH3), 1.05 (d, J = 6.5 Hz, 3H,
CH2CHCH3CH3), 1.39 (s, 9H, C(CH3)3), 1.61-1.55 (m, 2H, CH2CH(CH3)2), 1.75-1.70 (m,
1H, CH(CH3)2), 3.39-3.42 (dd, J = 5.4, 6.9 Hz, 2H, CH2OH), 4.43 (brs, 1H, NHCH); 13C
NMR (100 MHz, CDCl3): δ 23.4, 25.4, 28.3, 42.6, 55.3, 61.4, 78.5, 155.4; ESI-MS: (m/z)
218.2 (M+H+); HRMS calcd for [C11H24NO3+H]+ 218.1751, found 218.1757.

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4.4.2 (S)-Tert-butyl 1-hydroxy-3-phenylpropan-2-ylcarbamate (5c)
The compound was obtained as a gummy substance in quantitative yield; 1H NMR (300MHz,
CDCl3) : δ 1.42 (s, 9H, C(CH3)3), 2.84 (d, J = 7.0 Hz, 2H, CH2C6H5), 3.49-3.70 (m, 2H,
CH2OH), 3.87 (brs, 1H, NHCH), 4.69 (t, J = 7.2 Hz, 1H, CH2OH), 7.20-7.30 (m, 5H,
CH2C6H5); 13C NMR (100MHz, CDCl3) : δ 28.3, 36.6, 58.6, 65.4, 78.4, 125.4, 127.7, 129.3,
137.7, 155.3; ESI-MS: (m/z) 252.3 (M+H+); HRMS calcd for [C14H22NO3+H]+ 252.1594,
found 252.1597.
4.4.3 (S)-Tert-butyl 1-hydroxypropan-2-ylcarbamate (6c)
The compound was obtained as a gummy substance in quantitative yield; 1H NMR (300MHz,
CDCl3): δ 1.26 (d, J = 6.4 Hz, 3H, CHCH3), 1.46 (s, 9H, C(CH3)3), 3.30 (d, J = 4.8 Hz, 2H,
CH2OH), 3.72 (m, 1H, CHCH3); 13C NMR (100MHz, CDCl3): δ 17.5, 28.5, 52.4, 65.4, 78.5,
155.4; ESI-MS: (m/z) 176.2 (M+H+); HRMS calcd for [C8H18NO3+H]+ 176.1281, found
176.1286.
4.5
General Procedure for the Synthesis of 4d, 5d and 6d
To a suspension of 4c, 5c and 6c (1.0 equiv.) in anhydrous THF under nitrogen atmosphere
was added triethylamine (3.0 equiv.). The mixture was cooled to 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 1h. After dilution with saturated NaHCO3 solution, the
aqueous layer was extracted with DCM. The combined organic extracts dried over Na2SO4
and concentrated under reduced pressure to afford 4d, 5d and 6d. These intermediates were
used for the next step without further purification.
4.6 General Procedure for the Synthesis of 4e-4m, 5e-5m and 6e-6j
To a suspension of 4d, 5d and 6d (1.0 equiv.) in acetonitrile under nitrogen atmosphere was
added triethylamine (2.0 equiv.) and amine (4.0 equiv.). The reaction mixture was stirred for
40 h at room temperature. After completion of the reaction (as monitored by TLC), the
solvent was concentrated under reduced pressure and the residue dissolved in DCM, the
organic layer was washed with 10% citric acid, finally the aqueous layer was basified with
NaHCO3 and extracted with DCM. The combine organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue obtained was purified on the silica gel (60-
120 mesh) column chromatography using hexane: EtoAc as eluant.
4.6.1 Tert-butyl (2S)-4-methyl-1-(2-methylpiperidin-1-yl)pentan-2-ylcarbamate (4e)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.94 (d, J = 6.3 Hz, 3H, CH2CHCH3CH3), 1.07 (d, J = 6.4 Hz, 3H,
CH2CHCH3CH3), 1.17 (d, J = 6.3 Hz, 3H, N(CH2)4CHCH3), 1.29 (s, 9H, C(CH3)3), 1.34-1.46

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(m, 6H, NCH2(CH2)3CHCH3), 1.56-1.78 (m, 5H, CH2NCH2(CH2)3CHCH3), 2.37-2.52 (m,
1H, CH2CH(CH3)2), 2.74-2.90 (m, 2H, NHCHCH2N), 3.57-3.64 (m, 1H, NHCH); 13C NMR
(100 MHz, CDCl3): δ 18.3, 22.2, 22.7, 23.6, 25.1, 26.3, 28.3, 34.3, 42.3, 54.4, 57.5, 63.2,
68.5, 78.5, 155.3; ESI-MS: (m/z) 299.2 (M+H+); HRMS calcd for [C17H35N2O2+H]+
299.2693, found 299.2696.
4.6.2 Tert-butyl (2S)-4-methyl-1-(3-methylpiperidin-1-yl)pentan-2-ylcarbamate (4f)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.92 (d, J = 6.5 Hz, 3H, CH2CHCH3CH3), 1.09 (d, J = 6.3 Hz, 3H,
CH2CHCH3CH3), 1.17 (d, J = 6.4 Hz, 3H, N(CH2)4CHCH3), 1.31 (s, 9H, C(CH3)3), 1.38-1.48
(m, 6H, NCH2(CH2)3CHCH3), 1.63-1.76 (m, 5H, CH2NCH2(CH2)3CHCH3), 2.36-2.54 (m,
1H, CH2CH(CH3)2), 2.74-2.89 (m, 2H, CH2CH(CH3)2), 3.61-3.64 (m, 1H, NHCH); 13C NMR
(100 MHz, CDCl3): δ 18.4, 22.3, 22.6, 23.6, 25.3, 27.3, 28.3, 34.2, 42.3, 53.4, 57.6, 63.7,
68.3, 78.2, 155.2; ESI-MS: (m/z) 299.2 (M+H+); HRMS calcd for [C17H35N2O2+H]+
299.2693, found 299.2699.
4.6.3 (S)-Tert-butyl 4-methyl-1-(4-methylpiperidin-1-yl)pentan-2-ylcarbamate (4g)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.87 (d, J = 6.4 Hz, 3H, CH2CHCH3CH3), 0.96 (d, J = 6.4 Hz, 3H,
CH2CHCH3CH3), 1.06 (d, J = 6.3 Hz, 3H, N(CH2)4CHCH3), 1.22-1.30 (m, 4H,
N(CH2)2(CH2)2CHCH3), 1.38 (s, 9H, C(CH3)3), 1.46-1.53 (m, 1H, N(CH2)2(CH2)2CHCH3),
1.63-1.84 (m, 4H, N(CH2)2(CH2)2CHCH3), 1.92-1.99 (m, 1H, CHC(H)(H)CH(CH3)2), 2.18-
2.26 (m, 1H, CHC(H)(H)CH(CH3)2), 2.58 (d, J = 6.6 Hz, 2H, NHCHCH2N), 2.68 (d, J = 11.4
Hz, 1H, CHC(H)(H)N), 2.89 (d, J = 11.3 Hz, 1H, CHC(H)(H)N), 3.64-3.71 (m, 1H,
NHCHCH2N); 13C NMR (100 MHz, CDCl3): δ 19.4, 23.3, 23.8, 24.3, 28.3, 33.2, 34.7, 42.3,
48.4, 51.6, 63.7, 78.2, 155.2; ESI-MS: (m/z) 299.3 (M+H+); HRMS calcd for
[C17H35N2O2+H]+ 299.2693, found 299.2692.
4.6.4 (S)-Tert-butyl 4-methyl-1-(piperidin-1-yl)pentan-2-ylcarbamate (4h)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.95 (d, J = 6.6 Hz, 3H, CH2CHCH3CH3), 1.06 (d, J = 6.4 Hz, 3H,
CH2CHCH3CH3), 1.33 (s, 9H, (CH3)3), 1.41-1.59 (m, 6H, piperidine), 1.68-1.74 (m, 2H,
CH2CHCH3CH3), 1.77-1.84 (m, 1H, CH2CHCH3CH3), 2.43-2.46 (m, 4H, piperidine), 2.58-
13
2.63 (m, 2H, NHCHCH2N), 3.66-3.73 (m, 1H, NHCHCH2N); C NMR (100 MHz, CDCl3):
δ 23.2, 23.6, 24.3, 24.7, 25.9, 28.4, 42.4, 52.2, 58.3, 62.8, 79.4, 155.4; ESI-MS: (m/z) 285.3
(M+H+); HRMS calcd for [C16H33N2O2+H]+ 285.2537, found 285.2539.

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4.6.5 (S)-Tert-butyl 4-methyl-1-(pyrrolidin-1-yl)pentan-2-ylcarbamate (4i)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.93 (d, J = 6.4 Hz, 3H, CH2CHCH3CH3), 1.04 (d, J = 6.4 Hz, 3H,
CH2CHCH3CH3), 1.52-1.59 (m, 1H, CH2CHCH3CH3), 1.64-1.69 (m, 2H, CH2CHCH3CH3),
1.73-1.82 (m, 4H, pyrrolidine), 2.57-2.61 (m, 4H, pyrrolidine), 2.69-2.74 (q, J = 12.1 Hz, 1H,
NHCHC(H)(H)N), 2.77-2.83 (q, J = 12.3 Hz, 1H, NHCHC(H)(H)N), 3.66-3.77 (m, 1H,
NHCHCH2N); 13C NMR (100 MHz, CDCl3): δ 23.3, 23.5, 24.6, 25.2, 28.3, 43.4, 50.4, 56.3,
62.7, 78.6, 155.4; ESI-MS: (m/z) 271.3 (M+H+); HRMS calcd for [C15H31N2O2+H]+
271.2380, found 271.2383.
4.6.6 (S)-Tert-butyl 4-methyl-1-morpholinopentan-2-ylcarbamate (4j)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.92 (d, J = 6.2 Hz, 3H, CH2CHCH3CH3), 1.08 (d, J = 6.6 Hz, 3H,
CH2CHCH3CH3), 1.29 (s, 9H, C(CH3)3), 1.39-1.47 (m, 2H, CH2CHCH3CH3), 1.71-1.80 (m,
1H, CH2CHCH3CH3), 2.46 (t, J = 4.7 Hz, 4H, morpholine), 2.62 (d, J = 6.4 Hz, 2H,
NHCHCH2N), 3.69 (t, J = 4.5 Hz, 4H, morpholine), 3.90-4.12 (m, 1H, NHCHCH2N); 13C
NMR (100 MHz, CDCl3): δ 23.6, 24.8, 28.8, 43.5, 51.2, 56.6, 64.5, 66.6, 78.4, 155.4; ESI-
MS: (m/z) 287.2 (M+H+); HRMS calcd for [C15H31N2O3+H]+ 287.2329, found 287.2331.
4.6.7 (S)-Tert-butyl 1-(diethylamino)-4-methylpentan-2-ylcarbamate (4k)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.96 (d, J = 6.4 Hz, 3H, CH2CHCH3CH3), 1.02 (t, J = 7.1 Hz, 6H,
N(CH2)2(CH3)2), 1.08 (d, J = 6.3 Hz, 3H, CH2CHCH3CH3), 1.38 (s, 9H, C(CH3)3), 1.44-1.52
(m, 1H, CH2CHCH3CH3), 1.67-1.72 (m, 2H, CH2CHCH3CH3), 1.74-1.85 (m, 2H,
NHCHCH2N), 2.48-2.61 (m, 4H, N(CH2)2(CH3)2), 2.69-2.73 (dd, J = 5.8, 13.0 Hz, 1H,
NHCHCH2N); 13C NMR (100 MHz, CDCl3): δ 14.3, 23.4, 24.8, 28.6, 42.4, 48.8, 51.2, 63.5,
78.8, 155.3; ESI-MS: (m/z) 273.2 (M+H+); HRMS calcd for [C15H33N2O2+H]+ 273.2537,
found 273.2539.
4.6.8 (S)-Tert-butyl 1-(dipropylamino)-4-methylpentan-2-ylcarbamate (4l)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.86 (t, J = 7.4 Hz, 6H, N(CH2)2(CH2)2(CH3)2), 0.93 (d, J = 6.4 Hz, 3H,
CH2CHCH3CH3), 1.06 (d, J = 6.4 Hz, 3H, CH2CHCH3CH3), 1.33 (s, 9H, C(CH3)3), 1.41-1.54
(m, 4H, N(CH2)2(CH2)2(CH3)2), 1.59-1.77 (m, 2H, CH2CHCH3CH3), 2.03-2.08 (m, 1H,
CH2CHCH3CH3), 2.34-2.45 (m, 4H, N(CH2)2(CH2)2(CH3)2), 2.56-2.69 (m, 2H,
NHCHCH2N), 3.53-3.59 (m, 1H, NHCH); 13C NMR (100 MHz, CDCl3): δ 11.4, 11.8, 23.6,

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24.7, 28.4, 42.6, 49.5, 58.4, 63.6, 79.6, 155.6; ESI-MS: (m/z) 301.2 (M+H+); HRMS calcd for
[C17H37N2O2+H]+ 301.2850, found 301.2854.
4.6.9 (S)-Tert-butyl 1-(dimethylamino)-4-methylpentan-2-ylcarbamate (4m)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.93 (d, J = 6.4 Hz, 3H, CH2CHCH3CH3), 1.07 (d, J = 6.3 Hz, 3H,
CH2CHCH3CH3), 1.34 (s, 9H, C(CH3)3), 1.49-1.57 (m, 1H, CH2CHCH3CH3), 1.65-1.72 (m,
2H, CH2CHCH3CH3), 2.27 (s, 6H, CH2N(CH3)2), 2.48-2.51 (dd, J = 5.6, 12.3 Hz, 1H,
NHCHC(H)(H)N), 2.54-2.59 (dd, J = 7.6, 12.3 Hz, 1H, NHCHC(H)(H)N), 3.64-3.70 (m, 1H,
NHCHCH2N); 13C NMR (100 MHz, CDCl3): δ 23.2, 23.7, 24.8, 28.6, 42.5, 46.2, 48.4, 63.6,
78.6, 155.6; ESI-MS: (m/z) 245.2 (M+H+); HRMS calcd for [C13H29N2O2+H]+ 245.2224,
found 245.2229.
4.6.10 Tert-butyl (2S)-1-(2-methylpiperidin-1-yl)-3-phenylpropan-2-ylcarbamate (5e)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.87 (d, J = 6.6 Hz, 3H, N (CH2)4CHCH3), 1.36 (s, 9H, C (CH3)3), 1.46-1.67
(m, 6H, NCH2CH2CH2CH2CHCH3), 2.36-3.17 (m, 7H, PhCH2 (CH) CH2NCH2
(CH2)3CHCH3), 3.83-3.90 (m, 1H, NHCH); 13C NMR (100 MHz, CDCl3): δ 14.4, 23.2, 26.2,
28.3, 33.2, 38.6, 55.3, 57.2, 61.4, 65.6, 79.1, 125.6, 128.3, 128.6, 129.6, 130.1, 135.4, 155.5;
ESI-MS: (m/z) 333.2 (M+H+); HRMS calcd for [C20H33N2O2+H]+ 333.2537, found 333.2539.
4.6.11 Tert-butyl (2S)-1-(3-methylpiperidin-1-yl)-3-phenylpropan-2-ylcarbamate (5f)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.84 (d, J = 6.2 Hz, 3H, N(CH2)3CHCH3), 1.36 (s, 9H, C(CH3)3), 1.76-2.29
(m, 7H, piperidine), 2.54-3.18 (m, 6H, PhCH2(CH)CH2NCH2CH), 3.82-3.86 (m, 1H,
NHCH); 13C NMR (100 MHz, CDCl3): δ 18.8, 23.8, 27.6, 28.4, 32.5, 39.1, 54.6, 57.7, 62.4,
65.5, 78.8, 125.4, 128.2, 128.4, 129.4, 130.3, 137.4, 155.4; ESI-MS: (m/z) 333.4 (M+H+);
HRMS calcd for [C20H33N2O2+H]+ 333.2537, found 333.2541.
4.6.12 (S)-Tert-butyl 1-(4-methylpiperidin-1-yl)-3-phenylpropan-2-ylcarbamate (5g)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.89 (d, J = 6.2 Hz, 3H, N(CH2)2(CH2)2CHCH3), 1.03-1.09 (m, 1H,
N(CH2)2(CH2)2CHCH3), 1.29 (s, 9H, C(CH3)3), 1.36-1.55 (m, 4H, N(CH2)2(CH2)2CHCH3),
1.86-1.91
NCH2C(H)(H)(CH2)2CHCH3), 2.44-2.48 (dd, J = 6.2, 12.4 Hz, 1H, NHCHC(H)(H)N), 2.56-
2.61 (dd, 8.4, 12.6 Hz, 1H, NHCHC(H)(H)N), 2.66-2.69 (m, 1H,
(m,
1H,
NCH2C(H)(H)(CH2)2CHCH3),
2.13-2.19
(m,
1H,
J
=
NC(H)(H)CH2(CH2)2CHCH3), 2.84-2.89 (m, 1H, NC(H)(H)CH2(CH2)2CHCH3), 2.95-2.99
(dd, J = 7.2, 13.7 Hz, 1H, NHCHC(H)(H)Ph), 3.15-3.19 (dd, J = 4.2, 13.6 Hz, 1H,

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NHCHC(H)(H)Ph), 3.86 (s, 1H, NHCHCH2Ph); 13C NMR (100 MHz, CDCl3): δ 21.4, 28.4,
32.1, 34.3, 34.6, 38.6, 47.6, 54.5, 61.8, 78.6, 125.3, 128.2, 128.6, 129.7, 130.5, 137.7, 155.1;
ESI-MS: (m/z) 333.3 (M+H+); HRMS calcd for [C20H33N2O2+H]+ 333.2537, found 333.2538.
4.6.13 (S)-Tert-butyl 1-phenyl-3-(piperidin-1-yl)propan-2-ylcarbamate (5h)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 1.38 (s, 9H, C(CH3)3), 1.47-1.58 (m, 6H, piperidine), 2.37-2.42 (m, 4H,
piperidine), 2.48-2.54 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N), 2.61-2.66 (q, J = 12.4 Hz, 1H,
NHCHC(H)(H)N), 2.89-2.94 (q, J = 13.7 Hz, 1H, NHCHC(H)(H)Ph), 3.14-3.18 (q, J = 13.7
Hz, 1H, NHCHC(H)(H)Ph), 3.86 (s, 1H, NHCH); 13C NMR (100 MHz, CDCl3): δ 24.3,
25.8, 28.3, 38.6, 51.8, 56.3, 61.8, 78.7, 125.7, 128.6, 128.1, 129.3, 130.4, 137.8, 155.4; ESI-
MS: (m/z) 319.3 (M+H+); HRMS calcd for [C19H31N2O2+H]+ 319.2380, found 319.2383.
4.6.14 (S)-Tert-butyl 1-morpholino-3-phenylpropan-2-ylcarbamate (5i)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 1.39 (s, 9H, C(CH3)3), 3.23-3.26 (m, 4H, morpholine), 3.37-3.42 (m, 1H,
NHCH), 3.66-3.72 (m, 4H, morpholine), 3.89 (m, 2H, CH2-morpholine), 3.94-3.99 (m, 2H,
CH2Ph); 13C NMR (100 MHz, CDCl3): δ 28.4, 38.7, 54.6, 56.2, 65.4, 79.2, 125.5, 128.4,
128.5, 129.6, 130.6, 137.4, 155.2; ESI-MS: (m/z) 321.4 (M+H+); HRMS calcd for
[C18H29N2O3+H]+ 321.2173, found 321.2177.
4.6.15 (S)-Tert-butyl 1-phenyl-3-(pyrrolidin-1-yl)propan-2-ylcarbamate (5j)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 1.39 (s, 9H, C(CH3)3), 1.74-1.78 (m, 4H, pyrrolidine), 2.49-2.52 (m, 4H,
pyrrolidine), 2.58-2.64 (q, J = 12.6 Hz, 1H, NHCHC(H)(H)N), 2.77-2.83 (q, J = 12.6 Hz, 1H,
NHCHC(H)(H)N), 2.95-3.01 (q, J = 13.6 Hz, 1H, NHCHC(H)(H)Ph), 3.10-3.16 (q, J = 13.6
Hz, 1H, NHCHC(H)(H)Ph), 3.88-3.92 (m, 1H, NHCHCH2N); 13C NMR (100 MHz, CDCl3):
δ 23.4, 28.6, 38.7, 53.8, 55.2, 61.1, 78.5, 125.2, 128.6, 128.6, 129.6, 130.4, 137.7, 155.2;
ESI-MS: (m/z) 305.2 (M+H+); HRMS calcd for [C18H29N2O2+H]+ 305.2224, found 305.2228.
4.6.16 (S)-Tert-butyl 1-(dimethylamino)-3-phenylpropan-2-ylcarbamate (5k)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 1.36 (s, 9H, C(CH3)3), 2.24 (s, 6H, CH2N(CH3)2), 2.39-2.44 (q, J = 12.2 Hz,
1H, CHC(H)(H)N(CH3)2), 2.53-2.59 (q, J = 12.2 Hz, 1H, CHC(H)(H)N(CH3)2), 2.91-2.96 (q,
J = 13.4 Hz, 1H, CHC(H)(H)Ph), 3.10-3.16 (q, J = 13.4 Hz, 1H, CHC(H)(H)Ph), 3.84-3.89
(m, 1H, NHCH); 13C NMR (100 MHz, CDCl3): δ 28.4, 38.1, 46.8, 51.2, 63.4, 78.4, 125.4,
128.5, 128.7, 129.3, 130.6, 137.4, 155.4; ESI-MS: (m/z) 279.3 (M+H+); HRMS calcd for
[C16H27N2O2+H]+ 279.2067, found 279.2069.

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4.6.17 (S)-Tert-butyl 1-(diethylamino)-3-phenylpropan-2-ylcarbamate (5l)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.93 (t, J = 7.2 Hz, 6H, N(CH2)2(CH3)2), 1.35 (s, 9H, C(CH3)3), 2.44-2.57
(m, 4H, N(CH2)2(CH3)2), 2.59 (d, J = 6.9 Hz, 2H, NHCHCH2N), 2.85-2.89 (dd, J = 7.1, 13.9
Hz, 1H, NHCHC(H)(H)Ph), 3.12-3.18 (dd, J = 4.3, 14.0 Hz, 1H, NHCHC(H)(H)Ph), 3.76-
3.84 (m, 1H, NHCH); 13C NMR (100 MHz, CDCl3): δ 12.1, 28.4, 38.6, 47.2, 55.4, 62.4,
78.4, 125.1, 128.3, 128.5, 129.6, 130.2, 137.6, 155.1; ESI-MS: (m/z) 307.3 (M+H+); HRMS
calcd for [C18H31N2O2+H]+ 307.2380, found 307.2384.
4.6.18 (S)-Tert-butyl 1-(dipropylamino)-3-phenylpropan-2-ylcarbamate (5m)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.82 (t, J = 7.4 Hz, 6H, N(CH2)2(CH2)2(CH3)2), 1.27 (s, 9H, C(CH3)3), 1.34-
1.45 (m, 4H, N(CH2)2(CH2)2(CH3)2), 2.33-2.45 (m, 4H, N(CH2)2(CH2)2(CH3)2), 2.61-2.65
(m, 2H, CH2N(CH2)2(CH2)2(CH3)2), 2.84-2.92 (m, 1H, CHCH(H)Ph), 3.11-3.17 (m, 1H,
CHC(H)HPh), 3.74-3.81 (m, 1H, NHCH); 13C NMR (100 MHz, CDCl3): δ 11.6, 20.6, 28.4,
38.6, 55.6, 58.4, 62.3, 78.5, 125.4, 128.7, 128.3, 129.5, 130.4, 137.7, 155.3; ESI-MS: (m/z)
335.3 (M+H+); HRMS calcd for [C20H35N2O2+H]+ 335.2693, found 335.2696.
4.6.19 Tert-butyl (2S)-1-(2-methylpiperidin-1-yl)propan-2-ylcarbamate (6e)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 1.04 (d, J = 6.5 Hz, 3H, N(CH2)4CHCH3), 1.15 (d, J = 6.2 Hz, 3H,
NHCHCH3), 1.34 (s, 9H, C(CH3)3), 1.43-1.76 (m, 6H, NCH2(CH2)3CHCH3), 2.32-2.48 (m,
4H, CH2NCH2(CH2)3CHCH3), 2.86-2.91 (m, 1H, CH2NCH2(CH2)3CHCH3), 3.68 (s, 1H,
NHCH); 13C NMR (100 MHz, CDCl3): δ 14.6, 19.4, 22.9, 26.1, 28.3, 33.6, 47.8, 56.3, 59.2,
64.7, 78.4, 155.3; ESI-MS: (m/z) 257.2 (M+H+); HRMS calcd for [C14H29N2O2+H]+
257.2224, found 257.2227.
4.6.20 Tert-butyl (2S)-1-(3-methylpiperidin-1-yl)propan-2-ylcarbamate (6f)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.86 (d, J = 6.3 Hz, 3H, piperidine), 1.29 (d, J = 6.2 Hz, 3H, NHCHCH3),
1.36 (s, 9H, C(CH3)3), 1.68-1.76 (m, 2H, piperidine), 1.83-1.91 (m, 3H, piperidine), 2.46-2.69
(m, 4H, piperidine), 2.77-2.83 (m, 2H, NHCHCH2N), 3.62-3.69 (m, 1H, NHCH); 13C NMR
(100 MHz, CDCl3): δ 18.6, 19.4, 24.3, 28.4, 29.2, 32.6, 47.6, 59.4, 64.4, 65.2, 78.6, 155.2;
ESI-MS: (m/z) 257.3 (M+H+); HRMS calcd for [C14H29N2O2+H]+ 257.2224, found 257.2225.
4.6.21 (S)-Tert-butyl 1-(4-methylpiperidin-1-yl)propan-2-ylcarbamate (6g)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.88 (d, J = 4.7 Hz, 6H, CH3CHCH2N(CH2)2(CH2)2CHCH3), 1.36 (s, 9H,

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C(CH3)3), 1.68-1.72 (m, 1H, N(CH2)2(CH2)2CHCH3), 1.94-2.06 (m, 4H,
N(CH2)2(CH2)2CHCH3), 2.23-2.26 (m, 1H, NCH(H)CH2(CH2)2CHCH3), 2.40-2.46 (m, 1H,
NC(H)HCH2(CH2)2CHCH3), 2.53-2.58 (m, 1H, N(CH2)2CH(H)CH2CHCH3), 2.66-2.72 (m,
1H, N(CH2)2C(H)HCH2CHCH3), 2.77-2.82 (m, 1H, NHCHCH(H)N), 2.91-2.99 (m, 1H,
NHCHC(H)HN), 3.72-3.78 (m, 1H, NHCH); 13C NMR (100 MHz, CDCl3): δ 18.5, 21.4,
28.4, 32.3, 34.5, 45.5, 49.5, 64.3, 78.8, 155.5; ESI-MS: (m/z) 257.2 (M+H+); HRMS calcd for
[C14H29N2O2+H]+ 257.2224, found 257.2229.
4.6.22 (S)-Tert-butyl 1-(piperidin-1-yl)propan-2-ylcarbamate (6h)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 1.29 (d, J = 6.3 Hz, 3H, NHCHCH3), 1.37 (s, 9H, C(CH3)3), 1.44-1.64 (m,
6H, piperidine), 2.39 (s, 2H, NHCHCH2N), 2.46-2.60 (m, 4H, piperidine), 3.62-3.73 (m, 1H,
NHCHCH3); 13C NMR (100 MHz, CDCl3): δ 18.1, 24.6, 25.7, 28.3, 46.8, 56.4, 64.3, 78.6,
155.3; ESI-MS: (m/z) 244.3 (M+H+); HRMS calcd for [C13H27N2O2+H]+ 243.2067, found
243.2070.
4.6.23 (S)-Tert-butyl 1-(pyrrolidin-1-yl)propan-2-ylcarbamate (6i)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 1.34 (d, J = 6.4 Hz, 3H, NHCHCH3), 1.39 (s, 9H, C(CH3)3), 1.68-1.75 (m,
4H, pyrrolidine), 1.86 (s, 2H, NHCHCH2N), 2.52-2.57 (m, 4H, pyrrolidine), 2.83-2.86 (m,
1H, NHCH); 13C NMR (100 MHz, CDCl3): δ 18.4, 23.4, 28.4, 48.8, 55.2, 63.5, 78.5, 155.3;
ESI-MS: (m/z) 229.3 (M+H+); HRMS calcd for [C12H25N2O2+H]+ 229.1911, found 229.1915.
4.6.24 (S)-Tert-butyl 1-(dipropylamino)propan-2-ylcarbamate (6j)
1
The compound was obtained as a gummy substance in quantitative yield; H NMR (400
MHz, CDCl3): δ 0.85 (t, J = 7.3 Hz, 6H, N(CH2)2(CH2)2(CH3)2), 1.27 (d, J = 6.2 Hz, 3H,
NHCHCH3), 1.37 (s, 9H, C(CH3)3), 1.41-1.49 (m, 4H, N(CH2)2(CH2)2(CH3)2), 2.34-2.45 (m,
4H, N(CH2)2(CH2)2(CH3)2), 2.63 (d, J = 7.4 Hz, 2H, NHCHCH2N), 3.56-3.64 (m, 1H,
NHCH); 13C NMR (100 MHz, CDCl3): δ 11.5, 17.8, 21.2, 28.3, 46.7, 58.6, 62.3, 78.3, 155.4;
ESI-MS: (m/z) 259.3 (M+H+); HRMS calcd for [C14H31N2O2+H]+ 259.2380, found 259.2385.
4.7
General Procedure for the Synthesis of 7e-7m, 8e-8m and 9e-9j
To a suspension of 4e-4m, 5e-5m and 6e-6j (1.0 equiv.) dissolved in 15% HCl/dioxane and
stirred for 1h at room temperature. After completion of the reaction, the solvent was
concentrated under reduced pressure. The product was purified by trituration with diethyl
ether and finally the hydrochloride salt basified with triethylamine. These intermediates were
used for the next step without further purification.

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4.8 General procedure for the synthesis of 10e-10m, 11e-11m and 12e-12g, 12h20 12i21
and 12j
The free amines 7e-7m, 8e-8m and 9e-9j (2.0 equiv.) were added to 4,7-dichloroquinoline
(1.0 equiv.) and heated the reaction mixture in phenol. After completion of the reaction as
monitored by the TLC, chloroform (100 mL) was added to the reaction mixture. The organic
layer was washed with 10% NaOH solution and finally with brine. The organic layer was
dried over anhydrous Na2SO4, concentrated under reduced pressure and the product obtained
was purified by column chromatography by using methanol-chloroform-triethylamine as
eluent.
4.8.1
7-Chloro-N-((2S)-4-methyl-1-(2-methylpiperidin-1-yl)pentan-2-yl)quinolin-4-
amine (10e)
1
The compound was obtained as a gummy substance in 79% yield; H NMR (400 MHz,
CDCl3): δ 0.95 (d, J = 6.4 Hz, 3H, CH2CHCH3CH3), 1.05 (d, J = 6.2 Hz, 3H,
CH2CHCH3CH3), 1.12 (d, J = 6.2 Hz, 3H, N(CH2)4CHCH3), 1.25-1.45 (m, 6H,
NCH2(CH2)3CHCH3), 1.58-1.77 (m, 5H, CH2NCH2(CH2)3CHCH3), 2.36-2.51 (m, 1H,
CH2CH(CH3)2), 2.72-2.89 (m, 2H, CH2CH(CH3)2), 3.59-3.67 (m, 1H, NHCH), 6.42 (d, J =
5.4 Hz, 1H, Ar-H quinoline), 7.35-7.38 (dd, J = 2.2, 8.8 Hz, 1H, Ar-H quinoline), 7.70 (d, J =
8.8 Hz, 1H, Ar-H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.52 (d, J = 5.3 Hz,
1H, Ar-H quinoline); 13C NMR (100 MHz, CDCl3): δ 18.6, 22.3, 22.8, 23.6, 25.0, 26.1, 34.5,
42.5, 55.6, 56.5, 56.8, 57.5, 99.4, 121.2, 125.2, 128.8, 134.7, 149.3, 149.7, 150.0, 152.0; ESI-
MS: (m/z): 360.2 (M+H)+; HRMS calcd for [C21H31ClN3+H]+ 360.2201, found 360.2203.
4.8.2
7-Chloro-N-((2S)-4-methyl-1-(3-methylpiperidin-1-yl)pentan-2-yl)quinolin-4-
amine (10f)
1
The compound was obtained as a gummy substance in 76% yield; H NMR (400 MHz,
CDCl3): δ 0.93 (d, J = 6.5 Hz, 3H, CH2CHCH3CH3), 1.12 (d, J = 6.4 Hz, 3H,
CH2CHCH3CH3), 1.19 (d, J = 6.2 Hz, 3H, piperidine), 1.26-1.48 (m, 6H, piperidine), 1.60-
1.78 (m, 5H, piperidine), 2.35-2.53 (m, 1H, CH2CH(CH3)2), 2.72-2.89 (m, 2H,
CH2CH(CH3)2), 3.60-3.67 (m, 1H, NHCH), 6.45 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.35-
7.38 (dd, J = 2.2, 8.8 Hz, 1H, Ar-H quinoline), 7.73 (d, J = 8.8 Hz, 1H, Ar-H quinoline), 7.93
(d, J = 2.2 Hz, 1H, Ar-H quinoline), 8.54 (d, J = 5.3 Hz, 1H, Ar-H quinoline); 13C NMR (100
MHz, CDCl3): δ 18.6, 22.5, 22.9, 23.6, 25.5, 26.1, 34.8, 42.5, 55.7, 56.6, 56.8, 57.8, 99.4,
117.2, 125.2, 127.8, 133.7, 149.3, 149.9, 150.0, 152.2; ESI-MS: (m/z): 360.1 (M+H)+; HRMS
calcd for [C21H31ClN3+H]+ 360.2201, found 360.2198.

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4.8.3 (S)-7-Chloro-N-(4-methyl-1-(4-methylpiperidin-1-yl)pentan-2-yl)quinolin-4-amine
(10g)
1
The compound was obtained as a gummy substance in 83% yield; H NMR (400 MHz,
CDCl3): δ 0.89 (d, J = 6.3 Hz, 3H, CH2CHCH3CH3), 0.93 (d, J = 6.4 Hz, 3H,
CH2CHCH3CH3), 1.03 (d, J = 6.3 Hz, 3H, N(CH2)4CHCH3), 1.25-1.33 (m, 4H,
N(CH2)2(CH2)2CHCH3), 1.43-1.52 (m, 1H, N(CH2)2(CH2)2CHCH3), 1.62-1.76 (m, 4H,
N(CH2)2(CH2)2CHCH3), 1.92-1.93 (m, 1H, CHC(H)(H)CH(CH3)2), 2.15-2.21 (m, 1H,
CHC(H)(H)CH(CH3)2), 2.56 (d, J = 6.8 Hz, 2H, NHCHCH2N), 2.69 (d, J = 11.7 Hz, 1H,
NHCHC(H)(H)N), 2.87 (d, J = 11.2 Hz, 1H, NHCHC(H)(H)N), 3.64-3.66 (m, 1H, NHCH),
6.41 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.35-7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline),
7.72 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.94 (d, J = 2.0 Hz, 1H, Ar-H quinoline), 8.50 (d, J
13
= 5.3 Hz, 1H, Ar-H quinoline); C NMR (100 MHz, CDCl3): δ 21.7, 22.4, 23.4, 24.9, 30.6,
34.4, 34.6, 42.6, 48.3, 53.3, 55.2, 61.6, 99.2, 117.9, 121.2, 125.2, 128.7, 134.7, 149.3, 149.8,
152.0; ESI-MS: (m/z): 360.2 (M+H)+; HRMS calcd for [C21H31ClN3+H]+ 360.2201, found
360.2207.
4.8.4 (S)-7-Chloro-N-(4-methyl-1-(piperidin-1-yl)pentan-2-yl)quinolin-4-amine (10h)
1
The compound was obtained as a white solid in 85% yield; m.p 104-106°C; H NMR (400
MHz, CDCl3): δ 0.94 (d, J = 6.4 Hz, 3H, CH2CHCH3CH3), 1.04 (d, J = 6.3 Hz, 3H,
CH2CHCH3CH3), 1.40-1.60 (m, 6H, piperidine), 1.67-1.71 (m, 2H, CH2CHCH3CH3), 1.75-
1.82 (m, 1H, CH2CHCH3CH3), 2.41-2.44 (m, 4H, piperidine), 2.56-2.58 (m, 2H,
NHCHCH2N), 3.62-3.69 (m, 1H, NHCHCH2N), 6.42 (d, J = 5.4 Hz, 1H, Ar-H quinoline),
7.35-7.38 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.74 (d, J = 8.9 Hz, 1H, Ar-H quinoline),
7.94 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.51 (d, J = 5.3 Hz, 1H, Ar-H quinoline); 13C NMR
(100 MHz, CDCl3): δ 22.4, 23.4, 24.2, 24.9, 26.1, 42.6, 48.1, 54.8, 61.9, 99.2, 117.9, 121.2,
125.2, 128.7, 134.7, 149.2, 149.9, 151.9; ESI-MS: (m/z): 346.1 (M+H)+; HRMS calcd for
[C20H29ClN3+H]+346.2045, found 346.2037.
4.8.5 (S)-7-Chloro-N-(4-methyl-1-(pyrrolidin-1-yl)pentan-2-yl)quinolin-4-amine (10i)
1
The compound was obtained as a white solid in 80% yield; m.p 100-102°C; H NMR (400
MHz, CDCl3): δ 0.94 (d, J = 6.5 Hz, 3H, CH2CHCH3CH3), 1.03 (d, J = 6.5 Hz, 3H,
CH2CHCH3CH3), 1.50-1.57 (m, 1H, CH2CHCH3CH3), 1.63-1.68 (m, 2H, CH2CHCH3CH3),
1.69-1.83 (m, 4H, pyrrolidine), 2.55-2.59 (m, 4H, pyrrolidine), 2.67-2.72 (q, J = 12.4 Hz, 1H,
NHCHC(H)(H)N), 2.75-2.80 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N), 3.65-3.73 (m, 1H,
NHCH), 6.43 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.34-7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H
quinoline), 7.71 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar-H

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quinoline), 8.51 (d, J = 5.4 Hz, 1H, Ar-H quinoline); 13C NMR (100 MHz, CDCl3): δ 22.5,
23.1, 23.6, 25.0, 42.5, 49.9, 54.5, 59.2, 99.1, 117.7, 121.3, 125.2, 128.6, 134.8, 149.2, 149.7,
151.9; ESI-MS: (m/z): 332.1 (M+H)+; HRMS calcd for [C19H27ClN3+H]+332.1888, found
332.1877.
4.8.6 (S)-7-Chloro-N-(4-methyl-1-morpholinopentan-2-yl)quinolin-4-amine (10j)
1
The compound was obtained as a off-white solid in 79% yield; m.p 97-99°C; H NMR (400
MHz, CDCl3): δ 0.93 (d, J = 6.0 Hz, 3H, CH2CHCH3CH3), 1.04 (d, J = 6.4 Hz, 3H,
CH2CHCH3CH3), 1.37-1.44 (m, 2H, CH2CHCH3CH3), 1.74-1.83 (m, 1H, CH2CHCH3CH3),
2.48 (t, J = 4.3 Hz, 4H, morpholine), 2.59 (d, J = 6.7 Hz, 2H, NHCHCH2N), 3.66 (t, J = 4.1
Hz, 4H, morpholine), 3.89-4.01 (m, 1H, NHCH), 6.43 (d, J = 5.4 Hz, 1H, Ar-H quinoline),
7.37-7.39 (dd, J = 2.0, 8.8 Hz, 1H, Ar-H quinoline), 7.69 (d, J = 8.9 Hz, 1H, Ar-H quinoline),
7.97 (d, J = 1.8 Hz, 1H, Ar-H quinoline), 8.51 (d, J = 5.3 Hz, 1H, Ar-H quinoline); 13C NMR
(100 MHz, CDCl3): δ 23.2, 25.1, 42.6, 44.4, 48.1, 50.9, 53.9, 61.9, 67.0, 70.6, 99.2, 117.6,
120.9, 125.4, 128.7, 134.9, 149.1, 149.6, 151.8; ESI-MS: (m/z): 348.2 (M+H)+; HRMS calcd
for [C19H27ClN3O+H]+348.1837, found 348.1811.
4.8.7 (S)-N2-(7-Chloroquinolin-4-yl)-N1,N1-diethyl-4-methylpentane-1,2-diamine (10k)
1
The compound was obtained as a gummy substance in 71% yield; H NMR (400 MHz,
CDCl3): δ 0.94 (d, J = 6.4 Hz, 3H, CH2CHCH3CH3), 1.00 (t, J = 7.0 Hz, 6H,
N(CH2)2(CH3)2), 1.04 (d, J = 6.4 Hz, 3H, CH2CHCH3CH3), 1.43-1.50 (m, 1H,
CH2CHCH3CH3), 1.65-1.69 (m, 2H, CH2CHCH3CH3), 1.72-1.83 (m, 2H, NHCHCH2N),
2.47-2.62 (m, 4H, N(CH2)2(CH3)2), 2.67-2.72 (dd, J = 5.6, 13.0 Hz, 1H, NHCHCH2N), 6.42
(d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.34-7.37 (dd, J = 2.2, 8.9 Hz, 1H, Ar-H quinoline), 7.68
(d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.51 (d, J = 5.3
Hz, 1H, Ar-H quinoline); 13C NMR (100 MHz, CDCl3): δ 11.9, 22.5, 23.3, 25.0, 42.4, 47.2,
48.8, 56.6, 99.3, 117.9, 121.2, 125.2, 128.7, 134.7, 149.2, 149.8, 151.9; ESI-MS: (m/z): 334.1
(M+H)+; HRMS calcd for [C19H29ClN3+H]+ 334.2045, found 334.2049.
4.8.8 (S)-N2-(7-Chloroquinolin-4-yl)-4-methyl-N1,N1-dipropylpentane-1,2-diamine (10l)
1
The compound was obtained as a gummy substance in 70% yield; H NMR (400 MHz,
CDCl3): δ 0.83 (t, J = 7.3 Hz, 6H, N(CH2)2(CH2)2(CH3)2), 0.94 (d, J = 6.4 Hz, 3H,
CH2CHCH3CH3), 1.04 (d, J = 6.4 Hz, 3H, CH2CHCH3CH3), 1.39-1.51 (m, 4H,
N(CH2)2(CH2)2(CH3)2), 1.59-1.77 (m, 2H, CH2CHCH3CH3), 2.02-2.04 (m, 1H,
CH2CHCH3CH3), 2.35-2.45 (m, 4H, N(CH2)2(CH2)2(CH3)2), 2.61-2.67 (m, 2H,
NHCHCH2N), 3.58-3.59 (m, 1H, NHCHCH2N), 6.42 (d, J = 5.4 Hz, 1H, Ar-H quinoline),
7.33-7.36 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.67 (d, J = 8.9 Hz, 1H, Ar-H quinoline),

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7.94 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.52 (d, J = 5.3 Hz, 1H, Ar-H quinoline); 13C NMR
(100 MHz, CDCl3): δ 11.1, 11.8, 20.3, 22.0, 22.5, 23.4, 25.0, 42.3, 48.8, 49.5, 56.2, 58.0,
99.3, 117.9, 121.2, 125.1, 128.7, 134.7, 149.2, 149.8, 152.0; ESI-MS: (m/z): 362.1 (M+H)+;
HRMS calcd for [C21H33ClN3+H]+ 362.2358, found 362.2354.
4.8.9 (S)-N2-(7-Chloroquinolin-4-yl)-N1,N1,4-trimethylpentane-1,2-diamine (10m)
1
The compound was obtained as a gummy substance in 76% yield; H NMR (400 MHz,
CDCl3): δ 0.94 (d, J = 6.5 Hz, 3H, CH2CHCH3CH3), 1.04 (d, J = 6.5 Hz, 3H,
CH2CHCH3CH3), 1.47-1.54 (m, 1H, CH2CHCH3CH3), 1.63-1.70 (m, 2H, CH2CHCH3CH3),
2.25 (s, 6H, CH2N(CH3)2), 2.45-2.49 (dd, J = 5.8, 12.4 Hz, 1H, NHCHC(H)(H)N), 2.52-2.57
(dd, J = 7.8, 12.4 Hz, 1H, NHCHC(H)(H)N), 3.59-3.68 (m, 1H, NHCHC(H)(H)N), 6.43 (d, J
= 5.4 Hz, 1H, Ar-H quinoline), 7.34-7.37 (dd, J = 2.2, 8.9 Hz, 1H, Ar-H quinoline), 7.70 (d, J
= 8.9 Hz, 1H, Ar-H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.52 (d, J = 5.3 Hz,
1H, Ar-H quinoline); 13C NMR (100 MHz, CDCl3): δ 22.5, 23.2, 25.0, 42.3, 45.8, 48.8, 62.8,
99.1, 117.7, 121.3, 125.2, 128.6, 134.8, 149.2, 149.7, 151.9; ESI-MS: (m/z): 306.1 (M+H)+;
HRMS calcd for [C17H25ClN3+H]+ 306.1732, found 306.1723.
4.8.10
7-Chloro-N-((2S)-1-(2-methylpiperidin-1-yl)-3-phenylpropan-2-yl)quinolin-4-
amine (11e)
1
The compound was obtained as a off-white solid in 82% yield; m.p 112-114°C; H NMR
(400 MHz, CDCl3): δ 0.89 (d, J = 6.8 Hz, 3H, NCHCH3), 1.47-1.69 (m, 6H,
NCH2CH2CH2CH2CHCH3), 2.34-3.18 (m, 7H, PhCH2CHCH2NCH2(CH2)3CHCH3), 3.82-
3.87 (m, 1H, NHCHCH2Ph), 6.48 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.20-7.24 (m, 3H, Ar-
H Ph), 7.27-7.29 (m, 2H, Ar-H Ph), 7.35-7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.67
(d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.97 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.53 (d, J = 5.4
Hz, 1H, Ar-H quinoline); 13C NMR (100 MHz, CDCl3): δ 14.1, 22.6, 26.0, 31.9, 38.7, 50.4,
51.5, 55.7, 56.4, 99.5, 114.0, 121.1, 121.3, 125.3, 125.4, 126.6, 128.5, 128.7, 129.4, 134.8,
149.2, 149.5, 149.8, 151.8; ESI-MS: (m/z): 394.6 (M+H)+; HRMS calcd for [C24H29ClN3+H]+
394.2045, found 394.2043.
4.8.11
7-Chloro-N-((2S)-1-(3-methylpiperidin-1-yl)-3-phenylpropan-2-yl)quinolin-4-
amine (11f)
1
The compound was obtained as a off-white solid in 84% yield; m.p 112-114°C; H NMR
(400 MHz, CDCl3): δ 0.86 (d, J = 6.4 Hz, 3H, piperidine), 1.78-2.47 (m, 7H, piperidine),
2.53-3.16 (m, 6H, CH2NCH2CHCH2Ph), 3.84-3.88 (m, 1H, NHCH), 6.51 (d, J = 5.4 Hz, 1H,
Ar-H quinoline), 7.18-7.20 (m, 2H, Ar-H Ph), 7.27-7.31 (m, 3H, Ar-H Ph), 7.35-7.38 (dd, J =
2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.67 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.96 (d, J = 2.1

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Hz, 1H, Ar-H quinoline), 8.54 (d, J = 5.3 Hz, 1H, Ar-H quinoline); 13C NMR (100 MHz,
CDCl3): δ 19.5, 25.4, 25.5, 32.7, 38.3, 54.9, 60.5, 60.9, 62.7, 99.6, 117.9, 121.2, 125.3, 126.6,
128.5, 128.8, 129.5, 134.8, 137.3, 149.3, 149.6, 152.0; ESI-MS: (m/z): 394.2 (M+H)+; HRMS
calcd for [C24H29ClN3+H]+ 394.2045, found 394.2046.
4.8.12
(S)-7-Chloro-N-(1-(4-methylpiperidin-1-yl)-3-phenylpropan-2-yl)quinolin-4-
amine (11g)
1
The compound was obtained as a off-white solid in 81% yield; m.p 111-113°C; H NMR
(400 MHz, CDCl3): δ 0.88 (d, J = 6.2 Hz, 3H, N(CH2)2(CH2)2CHCH3), 1.01-1.04 (m, 1H,
N(CH2)2(CH2)2CHCH3), 1.22-1.52 (m, 4H, N(CH2)2(CH2)2CHCH3), 1.86-1.87 (m, 1H,
NCH2C(H)(H)(CH2)2CHCH3), 2.11-2.16 (m, 1H, NCH2C(H)(H)(CH2)2CHCH3), 2.43-2.47
(dd, J = 6.0, 12.6 Hz, 1H, NHCHC(H)(H)N), 2.54-2.59 (dd, J = 8.6, 12.6 Hz, 1H,
NHCHC(H)(H)N), 2.65-2.68 (m, 1H, NC(H)(H)CH2(CH2)2CHCH3), 2.83-2.87 (m, 1H,
NC(H)(H)CH2(CH2)2CHCH3), 2.88-2.93 (dd, J = 7.1, 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.10-
3.15 (dd, J = 4.1, 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.87 (s, 1H, NHCHCH2Ph), 6.50 (d, J =
5.4 Hz, 1H, Ar-H quinoline), 7.18-7.20 (m, 2H, Ar-H Ph), 7.23-7.24 (m, 1H, Ar-H Ph), 7.27-
7.31 (m, 2H, Ar-H Ph), 7.36-7.38 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.67 (d, J = 8.9
Hz, 1H, Ar-H quinoline), 7.96 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.53 (d, J = 5.3 Hz, 1H,
Ar-H quinoline); 13C NMR (100 MHz, CDCl3): δ 21.7, 30.7, 34.4, 34.6, 38.3, 50.4, 52.8,
54.9, 60.3, 99.5, 117.9, 121.2, 125.3, 126.6, 128.5, 128.8, 129.6, 134.8, 137.3, 149.3, 149.6,
152.0; ESI-MS: (m/z): 394.1 (M+H)+; HRMS calcd for [C24H29ClN3+H]+ 394.2045, found
394.2033.
4.8.13 (S)-7-Chloro-N-(1-phenyl-3-(piperidin-1-yl)propan-2-yl)quinolin-4-amine (11h)
1
The compound was obtained as a white solid in 79% yield; m.p 114-116°C; H NMR (400
MHz, CDCl3): δ 1.48-1.57 (m, 6H, piperidine), 2.35-2.40 (m, 4H, piperidine), 2.44-2.48 (q, J
= 12.6 Hz, 1H, NHCHC(H)(H)N), 2.55-2.60 (q, J = 12.6 Hz, 1H, NHCHC(H)(H)N), 2.88-
2.93 (q, J = 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.11-3.16 (q, J = 13.8 Hz, 1H,
NHCHC(H)(H)Ph), 3.88 (s, 1H, NHCHCH2N), 6.50 (d, J = 5.4 Hz, 1H, Ar-H quinoline),
7.18-7.20 (m, 2H, Ar-H Ph), 7.22-7.24 (m, 1H, Ar-H Ph), 7.27-7.31 (m, 2H, Ar-H Ph), 7.36-
7.38 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.70 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.97
(d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar-H quinoline); 13C NMR (100
MHz, CDCl3): δ 24.1, 26.0, 31.6, 38.3, 50.3, 54.4, 60.5, 99.5, 115.9, 117.8, 121.3, 123.4,
123.9, 125.5, 126.7, 128.5, 129.6, 135.0, 137.2, 149.0, 149.8, 151.7; ESI-MS: (m/z): 380.2
(M+H)+; HRMS calcd for [C22H27ClN3+H]+ 380.1888, found 380.1893.

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4.8.14 (S)-7-Chloro-N-(1-morpholino-3-phenylpropan-2-yl)quinolin-4-amine (11i)
1
The compound was obtained as a off-white solid in 75% yield; m.p 117-119°C; H NMR
(400 MHz, CDCl3): δ 3.20-3.24 (m, 4H, morpholine), 3.38-3.41 (m, 1H, NHCH), 3.64-3.71
(m, 4H, morpholine), 3.88 (m, 2H, NHCHCH2N), 3.96-3.99 (m, 2H, NHCHCH2Ph), 6.52 (d,
J = 5.4 Hz, 1H, Ar-H quinoline), 7.16-7.23 (m, 2H, Ar-H Ph ), 7.26-7.32(m, 3H, Ar-H Ph ),
7.38-7.40 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.65 (d, J = 8.9 Hz, 1H, Ar-H quinoline),
8.01 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.54 (d, J = 5.4 Hz, 1H, Ar-H quinoline); 13C NMR
(100 MHz, CDCl3): δ 48.5, 52.5, 53.5, 60.4, 66.6, 66.8, 66.9, 99.3, 115.5, 119.9, 121.0,
125.9, 126.9, 127.8, 128.6, 129.5, 135.6, 136.7, 149.9, 150.3, 151.1, 156.6; ESI-MS: (m/z):
382.2 (M+H)+; HRMS calcd for [C22H25ClN3+H]+ 382.1681, found 382.1686.
4.8.15 (S)-7-Chloro-N-(1-phenyl-3-(pyrrolidin-1-yl)propan-2-yl)quinolin-4-amine (11j)
1
The compound was obtained as a white solid in 75% yield; m.p 111-113°C; H NMR (400
MHz, CDCl3): δ 1.73-1.76 (m, 4H, pyrrolidine), 2.53-2.56 (m, 4H, pyrrolidine), 2.57-2.62 (q,
J = 12.4 Hz, 1H, NHCHC(H)(H)N), 2.75-2.81 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N), 2.92-
2.97 (q, J = 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.09-3.13 (q, J = 13.8 Hz, 1H,
NHCHC(H)(H)Ph), 3.86-3.90 (m, 1H, NHCH), 6.50 (d, J = 5.4 Hz, 1H, Ar-H quinoline),
7.18-7.20 (m, 2H, Ar-H Ph), 7.21-7.24 (m, 1H, Ar-H Ph), 7.27-7.31 (m, 2H, Ar-H Ph), 7.34-
7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.66 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.96
(d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar-H quinoline); 13C NMR (100
MHz, CDCl3): δ 23.6, 38.2, 52.2, 54.1, 57.9, 99.4, 117.8, 121.3, 125.3, 126.6, 128.5, 128.7,
129.5, 134.9, 137.3, 149.2, 149.4, 151.9; ESI-MS: (m/z): 366.1 (M+H)+; HRMS calcd for
[C22H25ClN3+H]+ 366.1732, found 366.1703.
4.8.16
(11k)
(S)-N2-(7-Chloroquinolin-4-yl)-N1,N1-dimethyl-3-phenylpropane-1,2-diamine
1
The compound was obtained as a off-white solid in 72% yield; m.p 109-111°C; H NMR
(400 MHz, CDCl3): δ 2.22 (s, 6H, CH2N(CH3)2), 2.36-2.40 (q, J = 12.4 Hz, 1H,
NHCHC(H)(H)N(CH3)2), 2.51-2.57 (q, J = 12.4 Hz, 1H, NHCHC(H)(H)N(CH3)2), 2.90-2.95
(q, J = 13.8 Hz, 1H, NHCHC(H)(H)Ph), 3.08-3.13 (q, J = 13.8 Hz, 1H, NHCHC(H)(H)Ph),
3.83-3.90 (m, 1H, NHCHC(H)(H)Ph), 6.51 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.17-7.19
(m, 2H, Ar-H Ph), 7.22-7.25 (m, 1H, Ar-H Ph), 7.28-7.31 (m, 2H, Ar-H Ph), 7.34-7.37 (dd, J
= 2.2, 8.9 Hz, 1H, Ar-H quinoline), 7.65 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.96 (d, J = 2.1
Hz, 1H, Ar-H quinoline), 8.54 (d, J = 5.3 Hz, 1H, Ar-H quinoline); 13C NMR (100 MHz,
CDCl3): δ 37.9, 45.5, 50.9, 61.5, 99.5, 117.8, 121.3, 125.3, 126.7, 128.5, 128.8, 129.6, 134.9,

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137.2, 149.3, 149.4, 152.0; ESI-MS: (m/z): 340.6 (M+H)+; HRMS calcd for
[C20H23ClN3+H]+ 340.1575, found 340.1580.
4.8.17 (S)-N2-(7-Chloroquinolin-4-yl)-N1,N1-diethyl-3-phenylpropane-1,2-diamine (11l)
1
The compound was obtained as a gummy substance in 76% yield; H NMR (400 MHz,
CDCl3): δ 0.96 (t, J = 7.1 Hz, 6H, N(CH2)2(CH3)2), 2.43-2.58 (m, 4H, N(CH2)2(CH3)2), 2.62
(d, J = 7.1 Hz, 2H, NHCHCH2N), 2.86-2.92 (dd, J = 7.2, 13.9 Hz, 1H, NHCHC(H)(H)Ph),
3.10-3.15 (dd, J = 4.4, 14.0 Hz, 1H, NHCHC(H)(H)Ph), 3.76-3.84 (m, 1H, NHCH), 6.50 (d, J
= 5.4 Hz, 1H, Ar-H quinoline), 7.19-7.31 (m, 5H, Ar-H Ph), 7.35-7.37 (dd, J = 2.1, 8.9 Hz,
1H, Ar-H quinoline), 7.65 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.96 (d, J = 2.0 Hz, 1H, Ar-H
quinoline), 8.53 (d, J = 5.3 Hz, 1H, Ar-H quinoline); 13C NMR (100 MHz, CDCl3): δ 11.8,
38.5, 46.8, 51.1, 55.7, 99.6, 118.0, 121.2, 125.3, 126.6, 128.5, 128.8, 129.4, 134.8, 137.5,
149.3, 149.6, 152.0; ESI-MS: (m/z): 368.6 (M+H)+; HRMS calcd for [C22H27ClN3+H]+
368.1888, found 368.1895.
4.8.18
(11m)
(S)-N2-(7-Chloroquinolin-4-yl)-3-phenyl-N1,N1-dipropylpropane-1,2-diamine
1
The compound was obtained as a gummy substance in 72% yield; H NMR (400 MHz,
CDCl3): δ 0.80 (t, J = 7.3 Hz, 6H, N(CH2)2(CH2)2(CH3)2), 1.31-1.43 (m, 4H, N
(CH2)2(CH2)2(CH3)2), 2.31-2.42 (m, 4H, N (CH2)2(CH2)2(CH3)2), 2.60-2.64 (m, 2H,
NHCHCH2N), 2.85-2.91 (m, 1H, NHCHCH(H)Ph), 3.10-3.15 (m, 1H, NHCHC(H)HPh),
3.75-3.82 (m, 1H, NHCH), 6.50 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.19-7.25 (m, 3H, Ar-H
Ph), 7.27-7.31 (m, 2H, Ar-H Ph), 7.34-7.37 (dd, J = 2.2, 8.9 Hz, 1H, Ar-H quinoline), 7.64
(d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.96 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.53 (d, J = 5.3
Hz, 1H, Ar-H quinoline); 13C NMR (100 MHz, CDCl3): δ 11.8, 20.1, 38.5, 51.2, 55.9, 57.1,
99.6, 118.0, 121.2, 125.3, 126.6, 128.5, 128.7, 129.4, 134.8, 137.6, 149.2, 149.7, 151.9; ESI-
MS: (m/z): 396.6 (M+H)+; HRMS calcd for [C21H31ClN3+H]+ 396.2201, found 396.2202.
4.8.19 7-Chloro-N-((2S)-1-(2-methylpiperidin-1-yl)propan-2-yl)quinolin-4-amine (12e)
1
The compound was obtained as a gummy substance in 78% yield; H NMR (400 MHz,
CDCl3): δ 1.06 (d, J = 6.5 Hz, 3H, N (CH2)4CHCH3), 1.18 (d, J = 6.2 Hz, 3H, NHCHCH3),
1.41-1.73 (m, 6H, NCH2(CH2)3CHCH3), 2.35-2.50 (m, 4H, CH2NCH2(CH2)3CHCH3), 2.84-
2.89 (m, 1H, CH2NCH2(CH2)3CHCH3), 3.66 (s, 1H, NHCH), 6.44 (d, J = 5.3 Hz, 1H, Ar-H
quinoline), 7.37-7.39 (dd, J = 2.2, 8.9 Hz, 1H, Ar-H quinoline), 7.76 (d, J = 9.1 Hz, 1H, Ar-H
quinoline), 7.97 (d, J = 2.0 Hz, 1H, Ar-H quinoline), 8.51 (d, J = 5.3 Hz, 1H, Ar-H
quinoline); 13C NMR (100 MHz, CDCl3): δ 19.1, 21.6, 25.5, 32.9, 44.7, 46.0, 56.4, 57.9,

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58.5, 99.5, 115.6, 125.5, 128.0, 129.5, 135.1, 148.6, 150.1, 151.4; ESI-MS: (m/z): 318.1
(M+H)+; HRMS calcd for [C18H25ClN3+H]+ 318.1732, found 318.1738.
4.8.20 7-Chloro-N-((2S)-1-(3-methylpiperidin-1-yl)propan-2-yl)quinolin-4-amine (12f)
1
The compound was obtained as a gummy substance in 76% yield; H NMR (400 MHz,
CDCl3): δ 0.88 (d, J = 6.4 Hz, 3H, piperidine), 1.30 (d, J = 6.0 Hz, 3H, NHCHCH3), 1.65-
1.75 (m, 2H, piperidine), 1.81-1.90 (m, 3H, piperidine), 2.45-2.67 (m, 4H, piperidine), 2.75-
2.79 (m, 2H, NHCHCH2N), 3.60-3.67 (m, 1H, NHCH), 6.43 (d, J = 5.2 Hz, 1H, Ar-H
quinoline), 7.35-7.37 (dd, J = 1.3, 8.9 Hz, 1H, Ar-H quinoline), 7.72 (d, J = 8.9 Hz, 1H, Ar-H
quinoline), 7.94 (d, J = 1.8 Hz, 1H, Ar-H quinoline), 8.51 (d, J = 5.3 Hz, 1H, Ar-H
quinoline); 13C NMR (100 MHz, CDCl3): δ 18.9, 19.5, 25.4, 31.0, 32.7, 45.0, 60.5, 62.9,
63.3, 99.7, 118.1, 121.3, 125.2, 128.7, 134.7, 149.2, 149.9, 152.0; ESI-MS: (m/z): 318.6
(M+H)+; HRMS calcd for [C18H24ClN3+H]+ 318.1732, found 318.1745.
4.8.21 (S)-7-Chloro-N-(1-(4-methylpiperidin-1-yl)propan-2-yl)quinolin-4-amine (12g)
1
The compound was obtained as a gummy substance in 79% yield; H NMR (400 MHz,
CDCl3): δ 0.89 (d, J = 4.7 Hz, 6H, CH3CHCH2N(CH2)2(CH2)2CHCH3), 1.66-1.70 (m, 1H,
N(CH2)2(CH2)2CHCH3), 1.92-2.07 (m, 4H, N(CH2)2(CH2)2CHCH3), 2.21-2.27 (m, 1H,
NCH(H)CH2(CH2)2CHCH3), 2.38-2.42 (m, 1H, NC(H)HCH2(CH2)2CHCH3), 2.51-2.55 (m,
1H, N(CH2)2CH(H)CH2CHCH3), 2.65-2.71 (m, 1H, N(CH2)2C(H)HCH2CHCH3), 2.77-2.80
(m, 1H, NHCHCH(H)N), 2.90-2.94 (m, 1H, NHCHC(H)HN), 3.70-3.76 (m, 1H, NHCH),
6.45 (d, J = 5.6 Hz, 1H, Ar-H quinoline), 7.35-7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline),
7.76 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 8.01 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.50 (d, J
13
= 5.5 Hz, 1H, Ar-H quinoline); C NMR (100 MHz, CDCl3): δ 18.7, 21.7, 29.7, 34.0, 34.3,
45.0, 52.2, 55.2, 62.9, 99.4, 115.6, 119.8, 121.6, 125.7, 127.4, 129.5, 150.9, 156.8; ESI-MS:
(m/z): 318.1 (M+H)+; HRMS calcd for [C18H25ClN3+H]+ 318.1732, found 318.1729.
4.8.22 (S)-7-Chloro-N-(1-(piperidin-1-yl)propan-2-yl)quinolin-4-amine (12h)
1
The compound was obtained as a gummy substance in 78% yield; H NMR (400 MHz,
CDCl3): δ 1.30 (d, J = 6.1 Hz, 3H, NHCHCH3), 1.41-1.60 (m, 6H, piperidine), 2.36 (s, 2H,
NHCHCH2N), 2.42-2.58 (m, 4H, piperidine), 3.59-3.68 (m, 1H, NHCH), 6.43 (d, J = 5.3 Hz,
1H, Ar-H quinoline), 7.36-7.39 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.73 (d, J = 8.9 Hz,
1H, Ar-H quinoline), 7.95 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.52 (d, J = 5.3 Hz, 1H, Ar-H
quinoline); 13C NMR (100 MHz, CDCl3): δ 18.9, 24.2, 26.1, 45.0, 54.3, 63.6, 99.7, 118.1,
121.3, 125.3, 128.7, 134.7, 149.2, 150.0, 152.0; ESI-MS: (m/z): 304.1 (M+H)+; HRMS calcd
for [C17H23ClN3+H]+ 304.1575, found 304.1575.

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4.8.23 (S)-7-Chloro-N-(1-(pyrrolidin-1-yl)propan-2-yl)quinolin-4-amine (12i)
1
The compound was obtained as a gummy substance in 81% yield; H NMR (400 MHz,
CDCl3): δ 1.32 (d, J = 6.2 Hz, 3H, NHCHCH3), 1.72-1.79 (m, 4H, pyrrolidine), 1.88 (s, 2H,
NHCHCH2N), 2.54-2.58 (m, 4H, pyrrolidine), 2.81-2.87 (m, 1H, NHCHCH2N), 6.44 (d, J =
5.4 Hz, 1H, Ar-H quinoline), 7.34-7.37 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline), 7.72 (d, J =
8.9 Hz, 1H, Ar-H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.51 (d, J = 5.3 Hz,
1H, Ar-H quinoline); 13C NMR (100 MHz, CDCl3): δ 18.8, 23.6, 47.0, 54.1, 61.0, 99.6,
117.9, 121.4, 125.2, 128.6, 134.7, 149.2, 149.7, 151.9; ESI-MS: (m/z): 290.1 (M+H)+; HRMS
calcd for [C16H21ClN3+H]+ 290.1419, found 290.1430.
4.8.24 (S)-N2-(7-Chloroquinolin-4-yl)-N1,N1-dipropylpropane-1,2-diamine (12j)
1
The compound was obtained as a gummy substance in 83% yield; H NMR (400 MHz,
CDCl3): δ 0.83 (t, J = 7.3 Hz, 6H, N(CH2)2(CH2)2(CH3)2), 1.29 (d, J = 6.0 Hz, 3H,
NHCHCH3), 1.39-1.51 (m, 4H, N(CH2)2(CH2)2(CH3)2), 2.33-2.49 (m, 4H,
N(CH2)2(CH2)2(CH3)2), 2.61 (d, J = 7.3 Hz, 2H, NHCHCH2N), 3.54-3.60 (m, 1H, NHCH),
6.44 (d, J = 5.4 Hz, 1H, Ar-H quinoline), 7.34-7.36 (dd, J = 2.1, 8.9 Hz, 1H, Ar-H quinoline),
7.68 (d, J = 8.9 Hz, 1H, Ar-H quinoline), 7.94 (d, J = 2.1 Hz, 1H, Ar-H quinoline), 8.51 (d, J
13
= 5.3 Hz, 1H, Ar-H quinoline); C NMR (100 MHz, CDCl3): δ 11.8, 18.6, 20.2, 45.6, 55.9,
59.7, 99.8, 118.1, 121.3, 125.2, 128.6, 134.7, 149.1, 149.9, 151.9; ESI-MS: (m/z): 320.1
(M+H)+; HRMS calcd for [C18H27ClN3+H]+ 320.1888, found 320.1890.
4.9 Biological methods
4.9.1 In vitro antimalarial assay
The compounds were evaluated for antimalarial activity against 3D7 (CQ- sensitive) and K1
(CQ-resistant) strains of Plasmodium falciparum using Malaria SYBR Green I nucleic acid
staining dye based fluorescence (MSF) assay as mentioned by Singh et al.22 The stock (10
mM) 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 (SIGMA) was used as a reference drug. For assessment of
antimalarial activity 50µl of culture medium was dispensed in 96 well plate followed by
addition of 50µl of highest concentration (containing less than 0.5% of DMSO) of test
compounds (in duplicate wells) in row B. Subsequent two-fold serial dilutions were prepared
in culture medium and finally 50 µl of 2.0% parasitized cell suspension containing 0.8%
parasitaemia (Asynchronous culture containing more than 80% ring stages) was added to
each well except 4 wells in row ‘A’ received non parasitized erythrocyte suspension. The
plates were incubated at 37 °C in CO2 incubator in an atmosphere of 5% CO2 and air mixture

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and 72 hours later 100 µl of lysis buffer containing 2 x concentration of SYBR Green-I
(Invitrogen) was added to each well and incubated for one hour at 37°C23. The plates were
examined at 485 20nm of excitation and 530 20nm of emission for relative fluorescence
units (RFUs) per well using the fluorescence plate reader (FLX800, BIOTEK). Data were
transferred into a graphic programme (EXCEL) and IC50 values were obtained by Logit
regression analysis of dose response curves using pre-programmed Excel spreadsheet.
4.9.2 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 Mosmann24. 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 the
dose response curves using pre-programmed Excel spreadsheet. Selectivity Index (SI) was
calculated as SI = CC50 / IC50
4.9.3 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 CQ13, 25-26. The study received ethical approval from CSIR-
Central Drug Research Institute’s ‘Institutional Animal Care and Use Committee’ recognized
by ‘Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA)’, Government of India. The mice (22 2 g) were inoculated with 1x106 parasitized
RBC on day 0 and treatment was administered to a group of five mice from day 0-3, once
daily. The aqueous suspensions of compounds were prepared with a few drops of Tween 80.
The final concentration of Tween 80 was 0.5 vol%. The efficacy of test compounds was
evaluated at 100 mg/kg/day and the required daily dose was administered in 0.5 mL 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.
4.9.4 Determination of hematin 4-aminoquinoline derivatives association constant
Association constant for hematin and 4-aminoquinoline derivatives complex formation were
determined by spectrometric titration procedure in aqueous DMSO at pH-7.527. In this assay
condition, hematin is strictly in monomeric state and interpretation of the results is not
complicated by the need to consider hematin disaggregation process. Association constant

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calculated in this technique is a good reflection of the interaction that would occur in the
acidic food vacuole of the parasite and pH-7.5 improves the stability of hematin solutions and
quality of the data.
4.9.5 In vitro inhibition of β-hematin formation assay
The ability of the 4-aminoquinoline derivatives to inhibit β-hematin formation induced by 1-
oleoyl-rac-glycerol. Spectroscopic measurements were done using UV spectrophotometer
wave length 405 nm and at pH 528. The IC50 values obtained from the assay are expressed as
percent inhibition relative to β-hematin formation in a drug free control. The 50% inhibitory
concentration values for the compounds were obtained from the sigmoidal dose-response
curves using non-linear regression curve fitting analyses with GraphPad Prism v.3.00
software29.
Acknowledgment.
One of the authors (K.S.R) thanks the CSIR, New Delhi, for Senior Research Fellowship.
Authors thank the Director, CDRI, for the support, and the SAIF division for the spectral
data. The CDRI Communication No is 9354.
Associated Content.
1
Electronic supplementary information (ESI) available: H NMR, 13C NMR and HRMS data
of selected compounds.

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