Synthesis and bioevaluation of novel 4-aminoquinoline-tetrazole derivatives as potent antimalarial agents

Article abstract of DOI:10.1016/j.ejmech.2013.05.023

A series of novel tetrazole derivatives of 4-aminoquinoline were synthesized and screened for their antimalarial activities against both chloroquine-senstive (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum as well as for cytotoxicity

Full text of DOI:10.1016/j.ejmech.2013.05.023

Accepted Manuscript
Synthesis and bioevaluation of novel 4-aminoquinoline-tetrazole derivatives as potent
antimalarial agents
Shashi Pandey, Pooja Agarwal, Kumkum Srivastava, S. RajaKumar, Sunil K. Puri,
Pravesh Verma, J.K. Saxena, Abhisheak Sharma, Jawahar Lal, Prem. M.S. Chauhan
PII:
S0223-5234(13)00332-2
10.1016/j.ejmech.2013.05.023
EJMECH 6201
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 February 2013
Revised Date: 30 April 2013
Accepted Date: 19 May 2013
Please cite this article as: S. Pandey, P. Agarwal, K. Srivastava, S. RajaKumar, S.K. Puri, P.
Verma, J.K. Saxena, A. Sharma, J. Lal, P.M.S. Chauhan, Synthesis and bioevaluation of novel 4-
aminoquinoline-tetrazole derivatives as potent antimalarial agents, European Journal of Medicinal
Chemistry (2013), doi: 10.1016/j.ejmech.2013.05.023.
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ACCEPTED MANUSCRIPT
Graphical abstract:
Synthesis and bioevaluation of novel 4-aminoquinoline-tetrazole derivatives as
potent antimalarial agents
a
b
b
b
b
Shashi Pandey , Pooja Agarwal , Kumkum Srivastava , S. RajaKumar , Sunil K. Puri , Pravesh
c
c
d
d
a,
Verma , J. K. Saxena , Abhisheak Sharma , Jawahar Lal and Prem. M. S. Chauhan *
a
Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, India,
b
Parasitology Division, CSIR-Central Drug Research Institute, Lucknow 226001, India,
c
Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, India,
d
Pharmacokinetics and Metabolism Division CSIR-Central Drug Research Institute, Lucknow 226001, India
Cl
Br
R1
N
NH
N
N
N
NH
NH
N
N
N
N
N
N
N
N
HN
N
R2
HN
N
HN
N
Cl
Cl
Cl
4-aminoquinoline tetrazoles
Compound 20
Compound 23
Designed Prototype
(
(
3D7) IC : 10.66 nM
(3D7) IC50: 11.78 nM
(K1) IC50: 233.7 nM
50
K1) IC : 142.9 nM
5
0
Best Potent compounds of series
Series of novel 4-aminoquinoline-tetrazole derivatives were synthesized. Most of compounds
exhibited potent activity against resistant strain (K1). Compounds 20 and 23 also showed in vivo
suppression of 99.99% parasitaemia on day 4.

ACCEPTED MANUSCRIPT
Synthesis and bioevaluation of novel 4-aminoquinoline-tetrazole derivatives as
potent antimalarial agents
a
b
b
b
b
Shashi Pandey , Pooja Agarwal , Kumkum Srivastava , S. RajaKumar , Sunil K. Puri , Pravesh
c
c
d
d
a,
Verma , J. K. Saxena , Abhisheak Sharma , Jawahar Lal and Prem. M. S. Chauhan *
a
Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, India,
b
Parasitology Division, CSIR-Central Drug Research Institute, Lucknow 226001, India,
c
Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, India,
d
Pharmacokinetics and Metabolism Division CSIR-Central Drug Research Institute, Lucknow 226001, India
Abstract: A series of novel tetrazole derivatives of 4-aminoquinoline were synthesized and
screened for their antimalarial activities against both chloroquine-senstive (3D7) and
chloroquine-resistant (K1) strains of Plasmodium falciparum as well as for cytotoxicity against
VERO cell lines. Most of the synthesized compounds exhibited potent antimalarial activity as
compared to chloroquine against K1-strain. Compounds with significant in vitro antimalarial
activity were then evaluated for their in vivo efficacy in Swiss mice against P. yoelii following
both intraperitoneal (ip) and oral administration, wherein compounds 20 and 23 each showed in
vivo suppression of 99.99% parasitaemia on day 4.
Keywords: 4-Aminoquinoline, Chloroquine, Tetrazole, Antimalarial, Cytotoxicity.
*
Corresponding author. Tel.: +91 522 2612411; fax: +91 522 2623405
e-mail addresses: prem_chauhan_2000@yahoo.com; premsc58@hotmail.com

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1
. Introduction
Parasitic diseases are a global problem, affecting 30% of the world’s population. Among
parasitic diseases, Malaria is one of the most devastating infectious disease claiming more lives
than any other parasitic infections. There were at least 216 million cases of acute malaria
reported in 2010 and about 655, 000 people died from malaria, 86% of which are children under
5
years of age [1]. It is most common in tropical and subtropical areas and 90% of all cases are
found in sub-Saharan Africa. These facts are attributable almost exclusively to Plasmodium
falciparum, one of the five Plasmodium species responsible for malaria in humans.
Since the development of quinine, drugs with quinoline scaffold have immense significance as
antimalarial chemotherapeutic agents. Among the quinoline derivatives, 4-aminoquinolines
(Figure 1) especially Chloroquine (CQ, 1) have been extensively utilized for the treatment of
malaria [2]. However, the widespread resistance of P. falciparum to chloroquine and other
existing antimalarial drugs including the recent artemisinin class of antimalarial has hampered
the efforts to combat this deadly disease [3-6]. So, there is an urgent need for the development of
new antimalarial agents active against drug-resistant malaria strains. However, despite the
development of resistance, CQ is still attractive pharmacophore for chemical modification,
owing its excellent clinical efficacy, limited host toxicity, ease to use, simple cost-effective
synthesis [7]. Furthermore, literature survey on 4-aminoquinoline antimalarials clearly suggested
that antimalarial activity, particularly, inhibition of β-hematin formation and accumulation of the
drug at the target site, resides in 4-aminoquinoline core [8]. In the past two decades, a number of
new 4-aminoquinoline analogues with enhanced activity against CQR strains were developed by
synthetic modifications of the CQ side chain [9-12]. Consequently, researches regarding the
exploration of novel 4-aminoquinoline analogues that are equally active against chloroquine

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sensitive (CQ-S) and chloroquine resistant (CQ-R) strains has received much attention during
recent years.
4
-aminoquinoline hybridization is another quite attractive strategy which has been recently
introduced in medicinal chemistry and drug discovery process, in order to circumvent the
problem of antimalarial drug resistance [13-17]. Moreover, in recent years, incorporation of
other functionalities in the side chain of 4-aminoquinoline emerged as promising strategies to
construct the molecules with enhanced activity against drug-resistant P. falciparum and also with
improved metabolic stability.
Recently, Campiani et al. synthesized Clotrimazole (CLT) analogues [18] and subsequently
prepared hybrid of the 4-aminoquinoline with the clotrimazole-like pharmacophore [19] in which
imidazole ring of CLT behaves as a Fe(III) axial ligand and inhibit β-hematin formation and
reported promising in vitro (against both sensitive and resistant strain) and in vivo antimalarial
activities. On the other hand, tetrazole based compounds have received attention due to their
wide range of biological activities [20-24]. Moreover, various tetrazole-based compounds have
also shown good coordination properties and are able to form stable complexes with several
metal ions inhibitors [25]. Recently, Roman et al. had exploited the ability of the tetrazole ring to
coordinate with the iron centre of heme in the design of heme oxygenase inhibitors [26].
Inspired by these encouraging results, we envisaged that incorporation of tetrazole moiety (with
ability to coordinate with the heme) in the side chain of 4-aminoquinoline pharmacophore would
lead to develop the new antimalarial agents active against chloroquine resistant strains (CQ-R) of
P. falciparum. On the basis of the above fact and in the continuation of our ongoing programme
to develop new potent antimalarial agents [27-30] we herein, report the efficient synthesis of new
4
-aminoquinoline analogues containing tetrazole moiety (Figure 2) and there evaluation as
potentially active compounds against Plasmodium malaria parasite.

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2
. Results and Discussion
2
.1. Chemistry
The targeted compounds were synthesized via two-step simple and efficient synthetic protocol.
The detailed synthetic route for the synthesis of intermediate (7-10) and target compounds (11-
3
2) is outlined in scheme 1. Reaction of commercially available 4, 7-dichloroquinoline with
corresponding diaminoalkanes/piperazine/p-phenylenediamine via ipso nucleophilic substitution
reaction resulted in the formation of compounds (7-10). These intermediate compounds (7-10)
were then utilized as amine input in isocyanide based multicomponent reaction. The target
compounds (11-32) were efficiently prepared by treating commercially available aldehyde with
amine (7-10) corresponding isocyanide and TMSN in methanol at room temperature. All
3
1
13
compounds were characterized using H NMR, C NMR, HRMS and IR spectroscopy.
.2. Biological assay
2
All the synthesized target compounds (11-32) were screened for their in vitro antimalarial
activity against CQ sensitive 3D7 strain and CQ-resistant K1 strain of P. falciparum (Table-1)
using a standardized inexpensive assay based on Malaria SYBR Green I nucleic acid staining
dye based fluorescence (MSF) assay. The IC values were calculated from experiments carried
5
0
out in triplicate. The cytotoxicity of all the synthesized molecules was determined against VERO
cell line using MTT assay. The in vivo drug responses of selected compounds were evaluated in
swiss mice infected with resistant strain N-67 of P. yoelii which is intrinsically resistant to CQ.
2
.2.1 In vitro antimalarial activity
Initially, all the synthesized 4-aminoquinoline-tetrazole derivatives (11-32) were evaluated for
their in vitro antimalarial efficacy against CQ-sensitive (3D7) and CQ-resistant (K1) strains of P.
falciparum. All tested compounds exhibited moderate activity against the CQ-S (3D7) strain,

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with IC value ranging from 10.66 to 216 nM. Among these 22 compounds, 3 compounds (20,
5
0
2
1 and 23) showed promising activity 10.66, 11.01, 11.78 nM respectively, 11 compounds
displayed moderate activity with the IC50 values ranging from 23.89 nM to 81.40 nM.
Furthermore, 13 compounds were found to be more active (with IC values between 73.70 and
5
0
2
33.70 nM) than CQ (IC = 254 nM), when screened against CQ-R (K1) strain of P. falciparum.
50
Two compounds 11 and 17 showed IC values 277.80 and 383.50 nM which was comparable to
5
0
CQ. Moreover, some of our synthesized compounds were also showing low resistance index
Table-1).
(
The structure-activity relationship studies on these compounds suggested that the activity of
these compounds were greatly influenced by type of linker, substitutions on aromatic ring as well
as substitution in the tetrazole ring. The activity data suggested that the type of linker had a
remarkable influence on the activity of 4-aminoquinoline tetrazole derivatives. According to our
results, compounds with ethyelene diamine linker (11, 12) exhibited mild inhibition against 3D7
strain in comparison to standard drug. However, the activity decreases dramatically on increasing
the chain length to 3 carbon (13-15). Interestingly, substitution of the flexible aliphatic linker
with rigid aromatic linker p-phenylene diamine resulted in manifold increased activity against
both the strains (3D7, K1). This increased activity may be due to increase in lipophilicity of
phenylene linker. Moreover, among the synthesized series, compound 25, 27 and 30 with
phenylenediamine linker were found to be most active against resistant strain (K1) and were
3
.44-3.38 fold more active than chloroquine. These SAR data clearly suggested that aromatic
linker have crucial role in activity of the compounds and analogues with aromatic linker
probably endowed with new leads that overcome drug resistance.
Another modification performed in the series was substitution of tert butyl group in tetrazole ring
with cyclohexyl ring. The cyclohexyl substituted analogues 28-30 showed manifold decrease in

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potency than analogues having tert butyl 18, 20 and 23 against 3D7 strain. Nevertheless, against
K1 strain, 4-chloro 28 and 4-bromo 29 analogue with cyclohexyl substituted tetrazole ring
showed increased activity while 3, 4 dimethoxy 30 resulted in decreased activity as compared to
respective analogue with tert butyl substituted tetrazole ring.
Furthermore, the activity of compounds also depends on the substitution on phenyl ring
however there is no obvious trend of activity with respect to substituted group and strains of
parasite. Compounds with unsubstituted phenyl ring 24 and 4-ethyl substituted group 26 were
showing mild activity IC50 59.64, 51.35 nM respectively against 3D7 strain but none of
compounds were active against resistant strain K1, while 4-methyl substituted analogue 25 was
found to be inactive against 3D7 strain but found to be most potent in series against resistant
strain with IC value 75.39 nM. Interestingly, despite the type of linker naphthalene substituted
5
0
derivatives were found to be active against K1 strain wherein compound 27 showed promising
activity with IC 77.79 nM and also showed better activity in 3D7 strain IC 28.00 nM. In case
5
0
50
of methoxy substituted compounds, 4-methoxy, 3, 4-dimethoxy and 3, 4, 5 trimethoxy
derivatives were showing good activity in sensitive strain (3D7) and showed mild activity in
resistant strain except 3, 4 dimethoxy compounds which exhibited promising activity against
CQ-R strain with IC 76.16 nM. Introduction of halogen substituent have immense influence on
5
0
activity. Compounds substituted with 4-chloro 20 and 4- bromo 23 on phenyl ring were found to
be most active against 3D7 strain and have very promising activity against K1 strain while 4-
flouro 22 substituted analogue showed mild activity against 3D7 and found to be inactive against
K1. Additionally analogue 20 and 23 were also found to be in vivo active.
Additionally, ferrocene containing heterocyclic compounds gaining interest because of their
wide applicability in organic, organometallic synthesis as well as in medicinal chemistry. [31-34]

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More precisely, the successful development of Ferroquine, Trioxaferroquines and ferrocenyl
derivative of Ciprofloxacin as antimalarial drugs having strong antimalarial activity against
resistant strain of plasmodium as compared to parent drug inspired us to synthesize ferrocene
containing analogues 31 and 32. However, disappointingly in our case these analogues were not
found to be active.
2
.2.2 In vitro cytotoxicity
The cytotoxicity of all the synthesized molecules 11-32 was determined against VERO cell line
using MTT assay (Table 1). 17 molecules showed high selectivity index (SI) ranging between
1
82.96 and 7076.29, rest of molecules showed selectivity index <100 but not less than 49.73.
Compounds 20 , 21 and 23 which were most potent against 3D7 also showed good selectivity
index 4616.14, 7076.29 and 2332.43 respectively (Table-1 ). Compound 30 which was most
potent against resistant strain K1, also showed good selectivity index of 1191.58. In general,
most of the compounds of the series exhibited reasonably promising activity against K1 strain,
less cytotoxic effect with fairly high selectivity index, and consequently these 4-aminoquinoline
tetrazole derivatives are good candidate for further lead optimization.
2
.2.3 β-Hematin inhibitory activity
To find out the mode of action of the synthesized molecules, the β-hematin inhibitory activity of
all the molecules was carried out (Table 2). All of the screened compounds inhibited β-hematin
formation in a concentration-dependent manner. All the tested compound showed IC values
5
0
between 3.81 µg/mL and 6.33 µg/mL. Among these, compound 29 showed better activity with
IC values = 3.81 µg/mL than CQ (3.95 µg/mL).
5
0
2
.2.4 In vivo antimalarial activity

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Compounds with significant activity in vitro (16, 18, 20, 21, 23, 25, 27, 29 and 30) were selected
for in vivo activity in Swiss mice against with CQR N-67 strain of P. yoelli. Initially the in vivo
activity of selected molecules was determined through intraperitoneal route at the dose of 50 mg/
kg administered once daily for four consecutive days and monitored for parasitemia, and survival
of mice until day 28 postinfection (Table 3). The compounds 20 and 23 exhibited 99.99 %
parasitaemia suppression on day four, while compounds 16, 21, 27 and 30 suppressed
parasitaemia by 73.91%, 71.49%, 57.03%, 69.56% respectively. Furthermore, these data clearly
showed that halogen substituted phenyl derivatives have better in vivo activity profile.
Compound 20, one of the potent compound of the series, showed 99.99% parasitaemia
suppression on day 4 and 60% survival on day 28 of treatment at a dose of 50 mg/kg × 4 days,
while compound 23, showed 99.99% parasite suppression on day 4 and 80% survival on day 28
of treatment. Furthermore, compound 20, cured 60% of treated mice while compound 23, cured
4
0% of treated mice. Compound 20 and 23, with promising result by intraperitoneal route were
further screened at 100 mg/ kg by oral route. Both compounds exhibited promising results with
9
9.99% parasite suppression on day 4, and 60% survival on day 28 of treatment.
.2.5 Preliminary Pharmacokinetic Studies
2
The compelling in vitro and in vivo antimalarial activity exhibited by compound 20 and
3 encouraged us to perform in vivo oral pharmacokinetic study of compound 20 and 23.
2
Pharmacokinetic studies revealed that the animals tolerated the treatment as no peculiarities in
the animals behavior were observed. Calculations of the pharmacokinetic data were based on the
mean serum concentrations and were performed by the use of noncompartmental approaches
using WinNonlin program, version 5.1 (Scientific Consulting Inc.). 20 and 23 were rapidly
absorbed and slowly eliminated (Figure 3 and Table 4). Both compounds showed multiple peaks
phenomenon which may be due to the enterohepatic recirculation, delayed gastric emptying, and

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variability of absorption in different regions of gastrointestinal tract [35]. For both compound 20
and 23, the volume of distribution (V , 28.6 L and 63.38 L, respectively) is greater than the total
d
blood volume (13.5 ml; Davies and Morris, 1993), [36] indicating high extra-vascular
distribution of the compound. Moreover, the systemic clearance of both 20 and 23 was higher
than the hepatic blood flow of the rat (Davies and Morris, 1993) which indicates an extra-hepatic
elimination. On comparison with reported pharmacokinetic profile of CQ, [37] compound 23
showed higher terminal half-life than CQ. Both compounds 20 and 23 have higher volume of
distribution which indicates their extensive distribution in the body than that of CQ. A 1.9-fold
lower clearance, 1.9-fold higher AUC and 3.3-fold higher MRT of 23 than that of 20 suggest that
2
3 could be a better candidate drug than 20.
3
. Conclusion
In summary, we have synthesized new tetrazole derivatives of 4-aminoquinoline by utilizing
highly efficient TMSN -Ugi multicomponent reaction. The majority of these synthesized
3
molecules exhibited promising in vitro antimalarial activity especially against CQ-R strain (K1).
Most of the compounds have shown the fairly high selectivity index. In present series,
Compound 20 and 23 showed promising in vitro activity against both CQ-S as well as CQ-R
strain of P. falciparum and also excellent in vivo antimalarial activity against P.yoelli. Taken as a
whole, these studies highlights the potential of these new 4-aminoquinolne-tetrazole derivatives
especially compound 20 and 23 for developing novel antimalarials that address CQ resistance as
well as able to reduce parasitaemia after oral administration.
4
. Experimental Methods
4
.1. General information

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Commercially available reagents and solvents were used without further purification. Thin-layer
chromatography (TLC) was carried out with silica gel plates (silica gel 60 F254), that were
visualized by exposure to ultraviolet light. IR spectra were recorded on a FTIR
-
spectrophotometer Shimadzu 8201 PC and are reported in terms of frequency of absorption (cm
1
1
13
)
. H NMR and C NMR spectra were recorded on Bruker Supercon Magnet Avance DRX-400,
3
00 or DPX 200 FT spectrometers using TMS as an internal reference and the samples were
dissolved in suitable deuterated solvents (Chemical shifts (δ) are given in ppm relative to TMS
and coupling constants (J) in Hz). HR-DART MS were recorded on JEOL, JMS T100LC Accu
TOF. Column chromatography purifications were performed in flash using 60-120 or 100-200
mesh silica gel. The melting points were recorded on an electrically heated melting point
apparatus and are uncorrected.
4
.2 General procedure for the synthesis of compounds 7 and 8
A mixture of 4, 7-dichloroquinoline (1 equiv.) and 1, 2-diaminoethane/ 1, 3-diaminopropane (5
equiv.) were heated for 6-8 h with continued stirring to drive the reaction to completion. After
completion of the reaction the excess solvent was removed under vacuum. The reaction mixture
was then poured into ice cold water and precipitate was filtered and washed with ethyl acetate to
furnish compound 7 and 8 in quantitative yields.
4
.3 General procedure for the synthesis of compounds 9 and 10
A solution of 4, 7-dichloroquinoline (1 equiv.) and p-phenylenediamine/ piperazine (2.0 equiv.)
in absolute ethanol was refluxed in presence of catalytic amount of p-TSA for 3-5 h. During the
refluxing, precipitation of product occurred. The precipitate was collected through filtration,
washed with ethanol and dried under vacuum to get the desired 9 and 10 in excellent yields.
4
.4. General procedure for the synthesis of compounds 11–32

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To a stirred solution of amine 7-10 (1mmol) in methanol (6 mL) were added successively
commercially available benzaldehyde (1.1 mmol), isocyanide (1 mmol) and TMSN (1 mmol).
3
The mixture was stirred at room temperature for 24 h. After completion of the reaction, solvent
was evaporated at reduced pressure and purified through column chromatography (eluent:
CHCl / MeOH) using 60-120 or 100-200 mesh silica gel to afford the desired products (11-32).
3
1
2
4
.4.1.
N -((1-tert-butyl-1H-tetrazol-5-yl)(4-chlorophenyl)methyl)-N -(7-chloroquinolin-4-
yl)ethane-1,2-diamine (11)
-1
1
White semi solid; Yield: 76 %; IR (cm , KBr): 3269, 2954, 1581, 1434, 1234; H NMR (300
MHz, CDCl ): δ = 8.48 (d, 1H, J = 5.1 Hz), 8.04 (d, 1H, J = 8.8 Hz), 7.94 (s, 1H), 7.44 (d, 1H,
3
J = 8.9 Hz), 7.34 (d, 2H, J = 7.9 Hz), 7.21 (d, 2H, J = 7.9 Hz), 6.37 (br s, 1H), 6.32 (d, 1H, J =
1
3
5
.1 Hz), 5.37( s, 1H), 3.34 (br s, 2H), 3.31-2.89 (m, 2H), 2.74 (br s, 1H), 1.60( s, 9H); C NMR
(
75 MHz, CDCl ) δ = 155.3, 151.4, 150.2, 148.5, 136.8, 134.9, 134.7, 129.3, 127.9, 125.4,
3
1
22.2, 117.3, 98.8, 61.8, 58.3, 45.8, 42.5, 29.9; HRMS (ESI TOF (+)) calcd for [C H Cl N +
23 25 2 7
+
H ] 470.1621 found 470.1622.
1
2
4
.4.2.
N -((1-tert-butyl-1H-tetrazol-5-yl)(naphthalen-1-yl)methyl)-N -(7-chloroquinolin-4-yl)
ethane-1,2-diamine (12)
-1
1
White solid; Yield: 70 %; mp 115-116 ºC; IR (cm , KBr): 3370, 2977, 1587, 1235; H NMR
(
300 MHz, CDCl ): δ = 8.49 (d, 1H, J = 5.3 Hz), 8.23 (d, 1H, J = 8.0 Hz), 7.97-7.92 (m, 2H),
3
7
1
3
1
.86 (d, 1H, J = 8.2 Hz), 7.79 (d, 1H, J = 8.9 Hz), 7.63-7.53 (m, 2H), 7.35-7.27 (m, 2H), 6.80 (d,
H, J = 7.0 Hz), 6.38 (br s, 1H), 6.33 (d, 1H, J = 5.3 Hz), 6.18 (s, 1H), 3.49-3.38 (m, 2H), 3.29-
1
3
.13( m, 2H), 1.51 (s, 9H); C NMR (75 MHz, CDCl ) δ = 155.4, 151.7, 150.0, 148.8, 134.8,
3
34.4, 134.2, 130.6, 129.7, 129.5, 128.2, 127.5, 126.2, 125.4, 125.1, 122.1, 122.0, 117.3, 98.9,

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+
6
4
1.8, 55.5, 46.5, 42.3, 29.7; HRMS (ESI TOF (+)) calcd for [C H ClN + H ] 486.2167 found
27 28 7
86.2167.
1
3
4
.4.3
N -((1-tert-butyl-1H-tetrazol-5-yl)(4-chlorophenyl)methyl)-N -(7-chloroquinolin-4-
yl)propane-1,3-diamine (13)
-1
1
White solid; Yield: 72 %; mp 130-132 ºC; IR (cm , KBr): 3432, 2988, 1636, 1455, 1217; H
NMR (300 MHz, CDCl ): δ = 8.41 (d, 1H, J = 5.2 Hz), 7.88 (d, 1H, J = 1.7 Hz), 7.37-7.27 (m,
3
6
2
1
4
H), 7.06 (dd,1H, J = 1.8 Hz, J = 8.8 Hz), 6.29 (d, 1H, J = 5.5 Hz), 5.32 (s, 1H), 3.44 (br s, 2H),
1
3
.92-2.78 (m, 3H), 1.94 (br s, 2H), 1.51 (s, 9H); C NMR (75 MHz, CDCl ) δ = 154.9, 150.7,
3
50.6, 147.7, 136.6, 135.1, 134.8, 129.5, 129.4, 127.2, 125.2, 122.0, 116.9, 98.1, 61.6, 58.6, 47.5,
+
3.4, 29.9, 27.3; HRMS (ESI TOF (+)) calcd for [C H Cl N + H ] 484.1778 found 484.1782.
24
27
2
7
1
3
4
.4.4
N -((1-tert-butyl-1H-tetrazol-5-yl)(naphthalen-1-yl)methyl)-N -(7-chloroquinolin-4-
yl)propane-1,3-diamine (14)
-1
1
Off white solid Yield: 75 %; mp 68-70 ºC; IR (cm , KBr): 3269, 2830, 1580, 1371; H NMR
(
300 MHz, DMSO-d ): δ = 8.37-8.31 (m, 2H), 8.06 (d, 1H, J = 8.9 Hz), 7.97 (d, 1H, J = 8.2 Hz),
6
7
.91 (d, 1H, J = 8.1 Hz), 7.77 (s, 1H), 7.54-7.51( m, 2H), 7.45-7.39 (m, 2H), 7.29 (d, 1H, J = 9.8
Hz), 6.99 (d, 1H, J = 7.1 Hz), 6.43 (d, 1H, J = 5.3 Hz), 6.19 (s, 1H), 3.34 (br s, 2H), 2.84-2.79
1
3
(
m, 2H), 1.86 (br s, 2H), 1.51 (s, 9H); C NMR (75 MHz, DMSO-d ) δ = 155.1, 151.2, 149.7,
6
1
1
48.3, 134.8, 133.1, 132.8, 130.3, 128.2, 128.1, 126.7, 126.2, 125.4, 124.7, 124.4, 123.4, 123.4,
23.0, 116.8, 98.1, 60.9, 54.1, 45.4, 28.6, 27.7 ; HRMS (ESI) calcd for HRMS (ESI TOF (+))
+
calcd for [C H ClN + H ] 500.2324 found 500.2326.
28
30
7
1
3
4
.4.5. N -((1-tert-butyl-1H-tetrazol-5-yl)(3,4-dimethoxyphenyl)methyl)-N -(7-chloroquinolin-4-
yl)propane-1,3-diamine (15)

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-1
1
White solid; Yield: 68%; mp 146-147 ºC; IR (cm , KBr): 3356, 2973, 1584, 1216; H NMR
(
300 MHz, DMSO-d ): δ = 8.43 (d, 1H, J = 5.8 Hz), 8.18 (d, 1H, J = 9.0 Hz), 7.8 (s, 1H), 7.46
6
(d, 1H, J = 8.9 Hz), 7.16 (s, 1H), 6.88 (s, 2H), 6.58 (d, 1H, J = 5.9 Hz), 5.42 (s, 1H), 3.71 (s, 3H),
1
3
3
.68 (s, 3H), 3.39-3.35 (m, 2H), 2.62-2.58 (m, 2H), 1.86-1.83 (m, 2H), 1.62 (s, 9H);
C NMR
(
75 MHz, CDCl ) δ = 156.0, 152.4, 148.8, 148.6, 147.9, 144.4, 135.4, 131.1, 125.2, 124.7,
3
1
23.8, 120.7, 116.5, 112.2, 111.5, 98.6, 61.4, 57.1, 55.6, 55.5, 45.2, 41.6, 29.4, 27.5; HRMS (ESI
+
TOF (+)) calcd for [C H ClN O + H ] 510.2379 found 510.2379.
26
32
7
2
4
.4.6.
4-(4-((1-tert-butyl-1H-tetrazol-5-yl)(4-chlorophenyl)methyl)piperazin-1-yl)-7-
chloroquinoline (16)
-1
1
Off white solid Yield: 78 %; mp 200-210 ºC; IR (cm , KBr): 3421, 3018, 1575, 1493, 1215; H
NMR (300 MHz, CDCl ): δ = 8.67 (d, 1H, J = 4.9 Hz), 8.05 (s, 1H), 7.89 (d, 1H, J = 8.9 Hz),
3
7
2
1
.39-7.37 (m, 5H), 6.79 (d, 1H, J = 5.0 Hz), 5.44 (s, 1H), 3.23 (s, 4H), 3.08-3.05 (m, 2H), 2.74-
1
3
.71 (s, 2H), 1.72 (s, 9H); C NMR (75 MHz, CDCl ) δ = 156.6, 153.4, 151.8, 150.0, 134.8,
3
34.7, 133.4, 130.9, 128.9, 128.7, 126.0, 125.1, 121.7, 108.9, 64.1, 61.5, 52.2, 49.8, 30.2;
+
HRMS (ESI TOF (+)) calcd for [C H Cl N + H ] 496.1778 found 496.1782.
25
27
2
7
1
4
4
.4.7.
N -((1-tert-butyl-1H-tetrazol-5-yl)(4-methoxyphenyl)methyl)-N -(7-chloroquinolin-4-yl)
benzene-1,4-diamine (17)
1
1
Yellow solid; Yield: 78 %; mp 106-107 ºC; IR (cm- , KBr): 3269, 2954, 1581, 1434,1234; H
NMR (300 MHz, CDCl3): δ = 8.35 (d, 1H, J = 5.3 Hz), 7.92 (s, 1H), 7.79 (d, 1H, J = 8.8 Hz),
7
6
.34 (d, 2H, J = 8.7 Hz), 7.19-7.17 (m, 3H), 7.04 (d, 2H, J = 8.3 Hz), 6.83 (d, 2H, J = 8.5 Hz),
.64 (d, 2H, J = 8.4 Hz,), 6.54 (d, 1H, J = 5.3 Hz), 6.03 (d, 1H, J = 9.2 Hz), 4.84 (d, 1H, J = 9.2
1
3
Hz), 3.73 (s, 3H), 1.62 (s, 9H); C NMR (50 MHz, CDCl ) δ = 159.8, 155.3, 151.7, 149.3,
3
1
43.9, 135.0, 130.4, 129.7, 129.1, 128.5, 126.2, 125.5, 121.6, 117.5, 115.1, 114.5, 101.3, 61.8,

ACCEPTED MANUSCRIPT
+
5
5
5.3, 54,3, 30.0 ; HRMS (ESI TOF (+)) calcd for [C H ClN O + H ] 514.2117 found
28 28 7
14.2117.
1
4
4
.4.8. N -((1-tert-butyl-1H-tetrazol-5-yl)(3,4-dimethoxyphenyl)methyl)-N -(7-chloroquinolin-4-
yl)benzene-1,4-diamine (18)
-1
1
Yellow solid; Yield: 80 %; mp 80-81 ºC; IR (cm , KBr): 3412, 3018, 1576, 1422, 1251; H
NMR (300 MHz, CDCl ): δ = 8.46 (d, 1H, J = 5.3 Hz), 7.99 (d, 1H, J = 1.83 Hz), 7.82 (d, 1H, J
3
=
8.9 Hz), 7.42 (dd,1H, J = 1.9 Hz, J = 8.9 Hz), 7.11 (d, 2H, J = 8.5 Hz), 6.93 (s, 1H) 6.85-6.82
(m, 2H), 6.73 (d, 2H, J = 8.6 Hz), 6.62 (d, 1H, J = 5.3 Hz,), 6.53 (s, 1H), 6.09 (d, 1H, J = 9.3
1
3
Hz), 4.92 (d, 1H, J = 9.2 Hz), 3.87 (s, 3H), 3.85 (s, 3H), 1.71 (s, 9H); C NMR (75 MHz, CDCl
3
)
δ = 155.1, 151.3, 149.6, 149.4, 149.3, 149.0 143.9, 135.0, 130.3, 130.1, 128.1, 126.1, 125.5,
1
21.6, 120.3, 117.4, 115.1, 111.1, 110.7, 101.2, 61.8, 55.9, 55.8, 54.7, 29.9 HRMS (ESI TOF
+
(
+)) calcd for [C H ClN O + H ] 544.2222 found 544.2222.
2
9
30
7
2
1
4
4
4
.4.9. N -((1-tert-butyl-1H-tetrazol-5-yl)(3,4,5-trimethoxyphenyl)methyl)-N -(7-chloroquinolin-
-yl)benzene-1,4-diamine (19)
-1
Pale Yellow solid Yield: 74 %; mp 168-170 ºC; IR (cm , KBr): 3428, 3017, 1578, 1373,1234,
1
1
7
2
3
1
6
5
105, 848; H NMR (300 MHz, CDCl ): δ = 8.45 (d, 1H, J = 5.3 Hz), 7.98 (d, 1H, J = 2.0 Hz),
3
.84 (d, 1H, J = 8.9 Hz), 7.41 (dd, 1H, J = 2.1 Hz, J = 8.9 Hz), 7.11 (d, 2H, J = 8.6 Hz), 6.62 (d,
H, J = 5.3 Hz), 6.55 (s, 2H), 6.06 (d, 1H, J = 9.3 Hz), 4.91 (d, 1H, J = 9.3 Hz), 3.84 (s, 3H),
1
3
.80 (s, 6H) 1.73 (s, 9H); C NMR (75 MHz, CDCl ) δ = 154.9, 153.7, 151.7, 149.4, 149.1,
3
43.9, 138.3, 135.0, 133.2, 130.6, 128.6, 126.1, 125.5, 121.3, 117.5, 115.2, 105.0, 101.3, 61.8,
+
0.8, 56.2, 55.1, 30.0 HRMS (ESI TOF (+)) calcd for [C H ClN O + H ] 574.2328 found
3
0
32
7
3
74.2328.

ACCEPTED MANUSCRIPT
1
4
4
.4.10.
N -((1-tert-butyl-1H-tetrazol-5-yl)(4-chlorophenyl)methyl)-N -(7-chloroquinolin-4-yl)
benzene-1,4-diamine (20)
-1
Yellow solid; Yield: 84 %; mp 128-130 ºC; IR (cm , KBr): 3435, 3019, 2927, 1576, 1374,1215;
1
H NMR (300 MHz, CDCl ): δ = 8.44 (d, 1H, J = 5.2 Hz), 7.98 (d, 1H, J = 1.6 Hz), 7.84 (d, 1H,
3
J = 8.9 Hz), 7.41-7.30 (m, 5H), 7.12 (d, 2H, J = 8.5 Hz), 6.72 (d, 2H, J = 8.5 Hz), 6.61 (d, 1H, J
1
3
=
5.3 Hz), 6.13 (d, 1H, J = 8.6 Hz), 4.93 (d, 1H, J = 8.6 Hz), 2.50 (br s, 1H), 1.62 (s, 9H); C
NMR (75 MHz, CDCl ) δ = 154.7, 151.3, 149.3, 149.0, 143.6, 136.3, 135.2, 134.8, 130.6, 129.4,
3
1
29.1, 128.3, 126.2, 125.7, 121.4, 117.4, 115.2, 101.3, 61.9, 54.1, 30.1, HRMS (ESI TOF (+))
+
calcd for [C H Cl N + H ] 518.1621 found 518.1622.
27
25
2
7
1
4
4
.4.11. N -((1-tert-butyl-1H-tetrazol-5-yl)(3,4-dichlorophenyl)methyl)-N -(7-chloroquinolin-4-
yl)benzene-1,4-diamine (21)
-1
1
Yellow solid; Yield: 82 %; mp 200-201 ºC; IR (cm , KBr): 3412, 3018, 1610, 1515,1215; H
NMR (300 MHz, DMSO-d ): δ = 10.82 (br s, 1H), 8.74 (d, 1H, J = 9.1 Hz), 8.43 (d, 1H, J = 6.9
6
Hz), 8.09 (s, 1H), 7.86 (s, 1H), 7.83 (d, 1H, J = 9.1 Hz), 7.70 (d, 1H, J = 8.3 Hz), 7.57 (d, 1H, J =
8
.2 Hz), 7.35 (d, 1H, J = 8.6 Hz), 7.20 (d, 2H, J = 8.1 Hz), 6.89 (d, 2H, J = 8.2 Hz), 6.63 (d, 1H,
13
J = 6.9 Hz), 6.37 (d, 1H, J = 8.7 Hz), 1.76(s, 9H) ; C NMR (75 MHz, DMSO-d6 ) δ = 154.9,
1
1
54.2, 145.4, 143.2, 140.0, 139.2, 138.1, 130.9, 130.5, 130.4, 130.2, 128.4, 127.1, 126.6, 126.5,
25.8, 119.3, 115.6, 113.9, 100.03 62.4, 50.7, 29.2; HRMS (ESI TOF (+)) calcd for
+
[
C H Cl N + H ] 552.1232 found 552.1231.
2
7
24
3
7
1
4
4
.4.12.
N -((1-tert-butyl-1H-tetrazol-5-yl)(4-fluorophenyl)methyl)-N -(7-chloroquinolin-4-yl)
benzene-1,4-diamine (22)

ACCEPTED MANUSCRIPT
-1
1
Yellow solid; Yield: 65 %; mp 110-112 ºC; IR (cm , KBr): 3258, 2989, 1574, 1515,1230; H
NMR (300 MHz, CDCl ): δ = 8.45 (d, 1H, J = 5.4 Hz), 7.99 (d, 1H, J = 1.7 Hz), 7.83 (d, 1H, J =
3
9
.0 Hz), 7.42-7.33 (m, 3H), 7.12-7.05 (m, 4H), 6.73 (d, 2H, J = 8.6 Hz), 6.62 (d, 1H, J = 5.4
1
3
Hz), 6.14 (d, 1H, J = 8.7 Hz), 4.91 (d, 1H, J = 9.6 Hz), 2.11 (br s, 1H), 1.72 (s, 9H); C NMR
(
75 MHz, DMSO-d ) δ = 158.8, 155.4, 151.5, 149.6, 149.1, 144.4, 133.7, 130.7, 129.3, 129.0,
6
1
27.2, 125.7, 124.4, 124.2, 117.5, 113.9, 113.7, 100.3, 61.8, 55.0, 51.7, 29.3; HRMS (ESI TOF
+
(
+)) calcd for [C H ClFN + H ] 502.1917 found 502.1918.
2
7
25
7
1
4
4
.4.13.
N -((4-bromophenyl)(1-tert-butyl-1H-tetrazol-5-yl)methyl)-N -(7-chloroquinolin-4-
yl)benzene-1,4-diamine (23)
-
1
1
Yellow solid; Yield: 84 %; mp : >240ºC; IR (cm , KBr): 3436, 3019, 1571, 1516; H NMR (300
MHz, DMSO-d ) = 10.88 (s, 1H), 8.76 (d, 1H, J = 8.9 Hz), 8.43 (d, 1H, J = 6.9 Hz), 8.11 (s, 1H),
6
7
.84 (d, 1H, J = 9.1 Hz),7.61-7.48 (m, 4H),7.25(d, 1H, J = 8.3 Hz), 7.18 (d, 2H, J = 7.9 Hz), 6.68
1
3
(
d, 2H, J = 7.9 Hz), 6.62 (d, 1H, J = 6.8 Hz), 6.32 (d, 1H, J = 8.1 Hz), 1.74 (s, 9H); C NMR (75
MHz, DMSO-d )) δ = 154.5, 154.1, 145.2, 142.3, 138.5, 137.7, 137.6, 130.7, 129.8, 126.5,
6
1
26.0, 125.7, 125.6, 120.6, 118.5, 115.1, 113.3, 99.4, 61.7, 50.7, 28.7; HRMS (ESI TOF (+))
+
calcd for [C H BrClN + H ] 562.1116 found 562.1116.
27
25
7
1
4
4
1
.4.14.
N -((1-tert-butyl-1H-tetrazol-5-yl)(phenyl)methyl)-N -(7-chloroquinolin-4-yl)benzene-
,4-diamine (24)
1
1
Yellow solid; Yield: 72 %; mp 112-114 ºC; IR (cm- , KBr): 3433, 3020, 1575, 1517, 1216; H
NMR (300 MHz, CDCl ): δ = 8.44 (d, 1H, J = 5.3 Hz), 7.98 (d, 1H, J = 1.8 Hz), 7.84 (d, 1H, J =
3
8
5
.9 Hz), 7.39-7.36 (m, 6H), 7.10 (d, 2H, J = 8.5 Hz), 6.72 (d, 2H, J = 8.6 Hz), 6.61 (d, 1H, J =
1
3
.3 Hz), 6.15 (d, 1H, J = 9.3 Hz), 4.95 (d, 1H, J = 9.3 Hz), 1.70 (s, 9H); C NMR (75 MHz,

ACCEPTED MANUSCRIPT
CDCl ) δ = 155.0, 151.6, 149.3, 149.2, 143.8, 137.7, 134.9, 130.4, 129.1, 128.8, 128.4, 127.7,
3
1
26.1, 125.5, 121.5, 117.5, 115.0, 101.3, 61.8, 54.8, 30.0; HRMS (ESI TOF (+)) calcd for
+
[
C H ClN + H ] 484.2011 found 484.2012.
2
7
26
7
1
4
4
1
.4.15.
N -((1-tert-butyl-1H-tetrazol-5-yl)(p-tolyl)methyl)-N -(7-chloroquinolin-4-yl)benzene-
,4-diamine (25)
-1
1
Yellow solid; Yield: 70 %; mp 128-130 ºC; IR (cm , KBr): 3431, 2921, 1577, 1248; H NMR
(
300 MHz,CDCl ) δ = 8.45 (d, 1H, J = 4.9 Hz), 7.98 (s, 1H), 7.82 (d, 1H, J = 9.1 Hz), 7.41 (d,
3
1
H, J = 8.6 Hz), 7.22 (d, 4H, J = 5.7 Hz), 7.10 (d, 2H, J = 7.9 Hz), 6.71 (d, 2H, J = 7.8 Hz), 6.62
(d, 1H, J = 4.9 Hz), 6.56 (s, 1H), 6.11 (d, 1H, J = 9.1 Hz), 4.91 (d, 1H, J = 9.5 Hz), 2.34 (s, 3H),
1
3
1
1
.69 (s, 9H); C NMR (75 MHz, CDCl ) δ = 155.2, 151.6, 149.3, 143.9, 138.7, 134.9, 134.8,
3
30.3, 129.8, 128.4, 127.6, 126.1, 125.4, 121.5, 117.5, 115.0, 101.2, 61.8, 54.6, 29.9, 21.0 ;
+
HRMS (ESI TOF (+)) calcd for [C H ClN + H ] 498.2167 found 498.2174.
28
28
7
1
4
4
.4.16.
N -((1-tert-butyl-1H-tetrazol-5-yl)(4-ethylphenyl)methyl)-N -(7-chloroquinolin-4-
yl)benzene-1,4-diamine (26)
-1
1
Yellow solid; Yield: 74 %; mp 136-138ºC; IR (cm , KBr): ): 3455, 2956, 1564, 1248; H NMR
(
300 MHz, DMSO-d6): δ = 8.42 (d, 1H, J = 5.3 Hz), 8.01 (s, 1H), 7.90 (d, 1H, J = 8.9 Hz), 7.42
d, 1H, J = 7.4 Hz), 7.28-7.21 (m, 5H), 7.14 (d, 2H, J = 8.3 Hz), 6.73 (d, 2H, J = 8.3 Hz), 6.62
d, 1H, J = 5.3 Hz), 6.14 (d, 1H, J = 8.3 Hz), 4.94 (d, 1H, J = 8.0 Hz), 2.70 (q, 2H, J = 7.5 Hz),
(
(
1
3
1
1
5
5
.71(s, 9H), 1.27-1.21 (m, 3H); C NMR (50 MHz, CDCl ) δ = 155.2, 150.9, 149.6, 148.6,
3
45.1, 144.1, 135.4, 134.9, 130.2, 128.7, 127.8, 126.1, 125.7, 121.7, 117.3, 115.1, 101.2, 61.8,
+
4.6, 30.1, 28.4, 15.3;HRMS (ESI TOF (+)) calcd for [C H ClN + H ] 512.2324 found
29
30
7
12.2327.

ACCEPTED MANUSCRIPT
1
4
4
.4.17. N -((1-tert-butyl-1H-tetrazol-5-yl)(naphthalen-1-yl)methyl)-N -(7-chloroquinolin-4-yl)
benzene-1,4-diamine (27)
-1
1
Yellow solid; Yield: 74 %; mp 140-142 ºC; IR (cm , KBr): 3358, 2933, 1573, 1375; H NMR
(
300 MHz, CDCl ): δ = 8.49 (d, 1H, J = 5.3 Hz), 8.14 (d, 1H, J = 8.2 Hz), 8.00 (d, 1H, J = 1.7
3
Hz), 7.97 (d, 1H, J = 7.9 Hz), 7.91 (d, 1H, J = 8.1 Hz), 7.84 (d, 1H, J = 9.0 Hz), 7.67-7.57 (m,
2
6
H), 7.43-7.36 (m, 2H), 7.14 (d, 2H, J = 8.5 Hz), 6.92-6.86 (m, 2H), 6.77 (d, 2H, J = 8.5 Hz),
13
.68 (d, 1H, J = 5.3 Hz), 6.54 (s, 1H), 4.79 (d, 1H, J = 9.7 Hz), 1.59 (s, 9H); C NMR (50 MHz,
DMSO-d6) δ = 154.9, 151.6, 149.5, 149.2, 144.4, 133.8, 133.7, 133.5, 130.6, 129.1, 128.7,
1
27.4, 126.8, 125.9, 125.3, 124.4, 124.1, 122.9, 117.6, 113.6, 100.4, 62.3, 49.7, 29.9; HRMS
+
(
ESI TOF (+)) calcd for [C H ClN + H ] 534.2167 found 534.2167.
31
28
7
1
4
4
.4.18.
N -(7-chloroquinolin-4-yl)-N -((1-cyclohexyl-1H-tetrazol-5-yl)(3,4-dimethoxyphenyl)
methyl)benzene-1,4-diamine (28)
-1
1
Yellow solid; Yield: 76 %; mp 138-140 ºC; IR (cm , KBr): 3432, 2933, 1634, 1516, 1254; H
NMR (300 MHz, CDCl ): δ = 8.43 (d, 1H, J = 5.3 Hz), 7.97 (d, 1H, J = 1.8 Hz), 7.85 (d, 1H J =
3
8
.9 Hz), 7.40 (dd, 1H, J = 1.8 Hz, J = 8.9 Hz), 7.10 (d, 2H, J = 8.5 Hz), 6.97-6.84 (m, 3H), 6.73
d, 2H, J = 8.6 Hz), 6.59 (d, 1H, J = 5.3 Hz), 5.82 (d, 1H, J = 5.9 Hz,), 5.18 (d, 1H, J = 5.9 Hz),
.30-4.23 (m, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 2.09-1.95 (m, 5H),1.83-1.72 (m, 3H), 1.37-1.29
(
4
1
3
(
m, 3H); C NMR (100 MHz, CDCl +DMSO-d ) δ = 154.2, 151.1, 149.5, 149.3, 149.0 143.7,
3 6
1
5
5
34.5, 130.1, 129.6, 127.7, 125.9, 124.9, 122.4, 119.4, 117.4, 114.4, 110.8, 109.9, 100.6, 57.9,
+
5.7, 55.6, 53.2, 32.0, 25.0, 24.9, 24.3; HRMS (ESI TOF (+)) calcd for [C H ClN O + H ]
3
1
32
7
2
70.2379 found 570.2372.
1
4
4
.4.19.
N -((4-chlorophenyl)(1-cyclohexyl-1H-tetrazol-5-yl)methyl)-N -(7-chloroquinolin-4-
yl)benzene-1,4-diamine (29)

ACCEPTED MANUSCRIPT
-1
Pale yellow solid; Yield: 76 %; mp 206 -208 ºC; IR (cm , KBr): 3480, 2933, 1574, 1517,1247;
1
H NMR (300 MHz, CDCl ): δ = 8.45 (d, 1H, J = 7.2 Hz), 7.98 (s, 1H), 7.84 (d, 1H, J = 10.1
3
Hz), 7.37 (s, 5H), 7.11 (d, 1H, J = 9.2 Hz), 6.72-6.67 (m, 3H), 6.60 (d, 1H, J = 5.7 Hz), 5.87 (d,
1
2
1
2
H, J = 7.5 Hz), 5.18 (d, 1H, J = 6.8 Hz ), 4.27-4.20 (m, 1H), 2.07-1.75 (m, 8H), 1.45-1.28 (m,
1
3
H) ; C NMR (75MHz, DMSO- d ) δ = 154.7, 151.7, 149.5, 149.4, 144.2, 137.5, 133.6, 132.6,
6
29.6, 129.4, 128.5, 127.5, 125.8, 124.4, 124.1, 117.6, 114.0, 100.3, 56.9, 50.2, 32.5, 32.4, 24.7,
+
4.6, 24.4; HRMS (ESI TOF (+)) calcd for [C H Cl N + H ] 544.1778 found 544.1738.
29
27
2
7
1
4
4
.4.20
N -((4-bromophenyl)(1-cyclohexyl-1H-tetrazol-5-yl)methyl)-N -(7-chloroquinolin-4-
yl)benzene-1,4-diamine (30)
-1
1
Yellow solid; Yield: 80 %; mp 208-209 ºC; IR (cm , KBr): 3358, 2933, 1573, 1315; H NMR
(
300 MHz, CDCl ): δ = 8.96 (d, 1H, J = 5.1 Hz), 8.68 (d, 1H, J = 8.7 Hz), 8.49 (s, 1H), 8.37(br
3
s, 1H), 8.11 (d, 2H, J = 7.5 Hz), 7.94-7.90 (m, 3H), 7.69 (d, 2H, J = 8.2 Hz), 7.32 (d, 2H, J = 8.2
Hz), 7.17 (d, 1H, J = 5.6 Hz), 6.53 (d, 1H, J = 6.3 Hz), 6.29 (d, 1H, J = 7.1 Hz), 4.95-4.87 (m,
1
3
1
1
1
H), 2.31-2.16 (m, 4H), 1.86-1.82 (m, 6H); C NMR (75 MHz, DMSO-d ) δ = 154.6, 151.5,
6
49.6, 149.2, 144.2, 137.9, 133.7, 131.4, 129.9, 129.3, 127.3, 125.8, 124.4, 124.1, 121.2, 117.6,
14.0, 100.3, 56.9, 50.3, 32.5, 32.3, 24.7, 24.6, 24.4; HRMS (ESI TOF (+)) calcd for [C H
27
29
+
BrClN + H ] 583.1273 found 583.1271.
7
1
4
4
1
.4.21 N -((1-tert-butyl-1H-tetrazol-5-yl)(ferrocenyl)methyl)-N -(7-chloroquinolin-4-yl)benzene-
,4-diamine (31)
-1
Yellow solid; Yield: 86 %; mp 118 - 120 ºC; IR (cm , KBr): 3437, 3020, 1610, 1575,1216; 1H
NMR (300 MHz, CDCl ): δ = 8.47 (d, 1H, J = 5.2 Hz), 7.99 (s, 1H), 7.86 (d, 1H J = 8.9 Hz),
3
7
.41 (d, 1H, J = 8.3 Hz), 7.14 (d, 2H, J = 8.3 Hz), 6.76 (d, 2H, J = 8.3 Hz), 6.67 (br s, 1H), 6.63
(d, 1H, J = 5.2 Hz), 5.83 (d, 1H, J = 9.7 Hz), 5.06 (d, 1H, J = 9.7 Hz), 4.48 (s, 1H), 4.28 (s, 6H),

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1
3
4
.17 (s, 1H), 3.60 (s, 1H), 1.76 (s, 9H); C NMR (50 MHz, CDCl ) δ = 155.6, 151.5, 149.4,
3
1
49.1, 144.3, 135.2, 130.5, 128.3, 126.3, 125.6121.5, 117.5, 115.6, 101.3, 89.8, 69.1, 68.6, 68.1,
+
6
7.6, 67.2, 61.9, 49.6, 30.4; HRMS (ESI TOF (+)) calcd for [C H ClFeN + H ] 592.1673
3
1
30
7
found 592.1677.
1
2
4
.4.21 N -((1-tert-butyl-1H-tetrazol-5-yl)(phenyl)methyl)-N -(7-chloroquinolin-4-yl)ethane-1,2-
diamine (32)
-1
1
Yellow solid; Yield: 84 %; mp 120-121 ºC; IR (cm , KBr): 3435, 2928, 1636, 1583, 1334; H
NMR (300 MHz, CDCl ): δ = 8.54 (d, 1H, J = 5.1 Hz), 8.03 (d, 1H, J = 8.9 Hz), 7.96 (s, 1H),
3
7
1
.47 (d,1H, J = 8.5 Hz), 6.42 (d, 1H, J = 4.8 Hz), 6.11 (s, 1H), 5.06 (s, 1H), 4.52 (s, 1H), 4.24 (s,
1
3
H), 4.11 (s, 6H), 3.54 (s, 1H), 3.40-3.34 (m, 2H), 3.05-2.85 (m, 2H), 1.73 (s, 9H); C NMR (50
MHz, CDCl ) δ = 156.4, 151.9, 150.0, 148.9, 134.9, 128.5, 125.5, 121.6, 117.4, 99.1, 89.2, 68.9,
3
+
6
5
8.6, 68.2, 67.4, 67.1, 61.6, 52.8, 45.3, 42.5, 30.3; HRMS (ESI) calcd for [C H ClFeN + H ]
2
7
30
7
44.1673; found 544.1679.
.5 Bioevaluation methods:
.5.1. In vitro antimalarial assay
4
4
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 (2011) [38]. The stock
(
(
5mg/ml) solution was prepared in DMSO and test dilutions were prepared in culture medium
RPMI-1640-FBS). Chloroquine-diphosphate was used as reference drug.
(
4
.5.1.1 Test Technique -

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5
0ꢀl of culture medium was dispensed in 96 well plate followed by addition of 50ꢀl of highest
concentration of test compounds (in duplicate wells) in row B. Subsequent two-fold serial
dilutions were prepared and finally 50 µl of 1.0% parasitized cell suspension containing 0.8%
parasitaemia was added to each well except 4 wells in row ‘A’ received non parasitized
erythrocyte suspension. The plates were incubated at 37 °C in CO incubator in an atmosphere of
2
5
% CO and air mixture and 72 hours later 100 µl of lysis buffer containing 2x concentration of
2
SYBR Green-I (In Nitrogen) was added to each well and incubated for one hour at 37 °C. 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)
4
.5.1.2 Statistical analysis- Data was 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
.5.2. 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 Sashidhara et al. (2012) [39]. The cells were
incubated with compound-dilutions for 72h and MTT was used as reagent for detection of
cytotoxicity. 50% cytotoxic concentration (CC ) was determined using nonlinear regression
50
analysis of dose response curves using pre-programmed Excel spreadsheet. Selectivity Index (SI)
was calculated as
SI = CC / IC
50
5
0
4
.5.3. In vitro assay for evaluation of β-hematin inhibition
Inhibition of in vitro β-hematin formation was analysed by using the method of Pandey et al
(1999) with some modifications [40]. Male swiss albino mice, weighing 15-20g were inoculated
5
with 1 x 10 P. yoelii infected RBCs. Blood of infected animal at 50% parasitemia was collected

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by cardiac puncture in 2.0% citrate buffer and centrifuged at 5000 rpm for 10 min at 4 °C. The
plasma was used in assay of β-hematin formation. The assay mixture contained 100 mM sodium
acetate buffer pH (5.1), 50 µL plasma, 100 µM hemin as the substrate and 1-20 µg
compound/drug in a total reaction volume of 1.0 mL. The control tubes contained all reagents
except compound. The reaction mixture in triplicate was incubated at 37 °C for 16 h in a rotary
shaker. The reaction was stopped by centrifugation at 10,000 rpm for 10 min at 30°C. The pellet
was suspended in 100 mM Tris-HCl buffer pH (7.4) containing 2.5% SDS. The pellet obtained
after centrifugation was washed thrice with distilled water (TDW) to remove free heme attached
to β-hematin. The pellet was solubilized in 50 µL of 2N NaOH and volume was made up to 1.0
mL with TDW. Absorbance was measured at 400 nm. The 50% inhibitory concentration (IC )
5
0
was determined using non-linear regression analysis of dose response curves.
.5.4 In vivo antimalarial assay
4
The in vivo drug response was evaluated in Swiss mice infected with P. yoelii (N-67 strain)
6
which is innately resistant to CQ [41]. The mice (22±2g) were inoculated with 1 ×10 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. Initially,
the efficacy of test compounds was evaluated at 50.0 mg/kg/day and required daily dose was
administered in 0.2 mL volume via intraperitoneal route. The efficacy of test compounds was
evaluated at 100 mg/kg/day and required daily dose was administered in 0.1 mL volume via oral
route. Parasitemia levels were recorded from thin blood smears between days 4 and 6. The mean
value determined for a group of 5 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.
4
.5.5 Pharmacokinetic Evaluation.

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The pharmacokinetic studies of compounds 20 and 23 were carried out in young and
healthy male Sprague-Dawley rats weighing 250 ± 25 g. The rats were obtained from
Laboratory Animal Division of the Institute and were housed in plastic cages under standard
laboratory conditions with a regular 12 h day-night cycle. Standard pelleted laboratory chow
(Goldmohar Laboratory Animal Feed, Lipton India Ltd, Chandigarh, India) and water were
allowed ad libitum. The rats were acclimatized to this environment for at least two days before
conducting the experiments. In all the studies mentioned below the dose was administered after
overnight fasting (12-16 h). The study was conducted in three rats per time point. In all
experiments, euthanasia and disposal of carcasses were carried out as per the guidelines of Local
Ethics Committee for animal experimentation.
Suspension formulation of the compounds were separately prepared by triturating the
compound with gum acacia and water in a pestle with mortar, and a single oral dose of 10 mg/Kg
was given to conscious rats by oral gavage in a volume of approximately 1 mL/250 g rat and the
time of dosing was recorded. Rodent food and water were provided 2 h after the dose. Blood
samples were collected at 0.5, 1, 1.5, 2, 4, 6, 8, 10, 18, and 24 h post dose. Two blood samples
were withdrawn from each animal. An initial 1.5 mL blood sample was drawn by cardiac
puncture under light anesthesia followed by a sample drawn from the inferior vena cava
(terminal sample), from the dosed rats using a 24G needle and a syringe in a clean and dry test
tube. The total volume of blood collected from each rat was not more than 3% of the total body
volume. The blood was allowed to clot, by keeping the tube on a slant, approximately for 45
min. Then it was centrifuged at 2000 rpm for 10 min and the serum was separated into clean and
neatly labeled tubes. All samples were stored at -20°C until analysis.
Chromatographic separations and quantification of the compounds was achieved by a
reverse phase HPLC method on a Discovery HS C-18 column (5 ꢀm, 100 x 4.6 mm id) preceded

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with a guard column (5 ꢀm, 20 x 4.0 mm, id) packed with the same material under isocratic
condition at a flow rate of 1 mL/min. The HPLC system used in this study consisted of a pump
(
LC-10AT VP with FCV-10AL VP), degasser (DGU-14A) and auto-injector (SIL-HTc, fixed
with a 100 ꢀl loop) (Shimadzu, Japan). Eluents were monitored at 253 nm for 20 and 257 nm for
3 with UV-Vis multiple wavelength detector (Shimadzu, Japan) and chromatograms were
2
integrated using Class-VP (version 6.12 SP5) software. The mobile phase composition was 0.01
M aqueous ammonium acetate : acetonitrile (35:65, v/v) and it was degassed by ultrasonication
for 15 min before use. The HPLC system was equilibrated for approximately 30 min before
commencement of analysis and chromatography was carried out at ambient temperature. The
lower limit of quantification for the analytical method was 10 ng/mL of test analyte in serum.
The mean and SEM of the serum concentrations of the candidate drug at each time point was
calculated using Microsoft Excel for Windows. All pharmacokinetic parameters were calculated
by noncompartmental models using WinNonlin program, version 5.1 (Scientific Consulting
Inc.).
Acknowledgments
SP is thankful to University Grant Commission, New Delhi, for financial support in the form of
SRF, SAIF-CDRI Lucknow for providing spectral and analytical data and Mrs. Rima Ray Sarkar
for technical support in β-Hematin inhibitory activity evaluation.
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Captions:
Table 1 In vitro antimalarial activity of compounds against 3D7 and K1 strains of P. falciparum
and their cytotoxicity against VERO cell line
Table 2 β -Hematin inhibitory activity of selected compounds.
Table 3 In vivo antimalarial activity of selected compounds against CQ resistant N-67 strain of
in swiss mice.
Table 4. . Table 4. Pharmacokinetic parameters of 20, 23 and CQ in male rats.
Figure 1. Structures of some 4-aminoquinoline derivatives with antimalarial activity.
Figure 2 Basis for the synthesis of tetrazole derivatives of 4-aminoquinoline .
Figure 3 Concentration-time profile of 20 and 23 after a single oral (10 mg/kg) dose in male
Sprague-Dawley rats. Bar represents SEM
Scheme 1 Synthesis of 4-aminoquinoline-tetrazole derivatives.