Synthesis, antimalarial activity, and target binding of dibenzazepine-tethered isoxazolines

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Koravangala, L. S. Gejjalagere, B. K. Yadaganahalli, A. C. Vinayaka, P. Bhat, P. K. S. Challanayakanahally, K.

S. Rangappa, C. D. Gowda and M. P. Sadashiva, RSC Adv., 2015, DOI: 10.1039/C5RA17926B.

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DOI: 10.1039/C5RA17926B

Synthesis, antimalarial activity, and target binding of dibenzazepine-tethered

isoxazolines

a

Koravangala S. Vinay Kumar,^aGejjalagere S. Lingaraju,^aYadaganahalli K. Bommegowda,

Ajjampura C. Vinayaka,^aPritesh Bhat,^bChallanayakanahally S. Pradeepa Kumara,^a

Kanchugarakoppal S. Rangappa,^*aD. Channe Gowda^*cand Maralinganadoddi P. Sadashiva^*a,c

^aDepartment of Chemistry, University of Mysore, Mysuru, Karnataka, India. Email. mpsadashiva@gmail.com, rangappaks@yahoo.com

^bManipal College of Pharmaceutical Sciences, Manipal University, Madhav Nagar, Manipal.

^cDepartment of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA. E-mail:

gowda@psu.edu.

Library of dibenzazepine tethered 3,5-disubstituted isoxazolines were synthesised via 1,3-dipolar cycloaddition reaction and further an additional

diversified group of dibenzazepine derivatives were accessed by Suzuki coupling of 6d with various organoboronic acids. All these compounds

were evaluated for their antimalarial activity against drug-sensitive Plasmodium falciparum 3D7 strain. Among the tested molecules, five

compounds (6j, 6k, 8c, 8k and 8l) that highly inhibited the parasite growth were further assessed for antimalarial activity using an additional

chloroquine-sensitive (D6) and two chloroquine-resistant (W2 and 7G8) P. falciparum strains. Molecular docking and dynamics simulation

studies were performed to understand the binding mode and binding strengths of the selected compounds with the enzyme.

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Synthesis, antimalarial activity, and target binding of dibenzazepine-

tethered isoxazolines

Received 00th January 20xx,

Accepted 00th January 20xx

Koravangala S. Vinay Kumar,^aGejjalagere S. Lingaraju,^aYadaganahalli K. Bommegowda,

a

Ajjampura C. Vinayaka,^aPritesh Bhat,^bChallanayakanahally S. Pradeepa Kumara,^a

DOI: 10.1039/x0xx00000x

Kanchugarakoppal S. Rangappa,^*aD. Channe Gowda^*cand Maralinganadoddi P.

Sadashiva^*a,c

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Malaria, a complex and deadly parasitic infectious disease, is a huge public health problem in many endemic countries

around the globe. The prevailing extensive resistance of malaria parasites to traditional drugs and emergence of resistance

to the currently used frontline artimisinin-based chemotherapy calls for the development of new drugs. Towards this

objective and since compounds containing dibenzazepine moiety are effective in treating both gametocyte and asexual

stage malaria parasites, including multi drug resistant parasites, a library of dibenzazepine tethered 3,5-disubstituted

isoxazolines were synthesised via 1,3-dipolar cycloaddition reaction. An additional diversified group of dibenzazepine

derivatives were accessed by Suzuki coupling of one the above dibenzazepine derivatives with various organoboronic

acids. All compounds were structurally characterized and were evaluated for their antimalarial activity. They exhibited

good to excellent inhibitory activity against the growth of drug-sensitive Plasmodium falciparum 3D7 strain with IC₅₀values

ranging from 0.2 to 7.7 µM. About 50% of the compounds were either minimally or not toxic to human cell lines. Five of

the compounds (6j, 6k, 8c, 8k and 8l) that highly inhibited the parasite growth were further assessed for antimalarial

activity using an additional chloroquine-sensitive (D6) and two chloroquine-resistant (W2 and 7G8) P. falciparum strains.

These compounds were effective against all four strains (3D7, D6, W2 and 7G8), exhibiting IC₅₀values of 0.1 to 1.75 µM.

The dibenzazepines were identified to target metalloamino-peptidase of parasites. Molecular docking and dynamics

simulation studies were performed to understand the binding mode and binding strengths of the selected compounds

with the enzyme. In agreement with their excellent antimalarial activity, the data suggested that the compounds can

strongly bind to the active site of the enzyme.

falciparum and P. vivax are responsible for almost all malarial

deaths. While P. falciparum is most prevalent in Africa, both P. vivax

Introduction

Malaria is

a deadly infectious disease in many tropical and

(majorly) and P. falciparum (to variable degrees) are prevalent in

Southeast Asia including Indian subcontinent, some parts of Africa,

and South America. Further, P. falciparum, the most virulent among

malaria parasites that can infect human, accounts for >90% of all

malarial deaths, whereas P. vivax is responsible for most of the

remainder of deaths.

subtropical countries of the world. Approximately 50% of the global

population is at risk of contracting the disease. According to a

recent WHO report, in 2013, ~200 million clinical cases of malaria

were diagnosed, and ~600,000 deaths have occurred worldwide,

most of which were in young children and in pregnant women.¹In

addition to the huge death toll it exerts, malaria vastly curtails

socioeconomic progress of people in the endemic areas, which are

mostly underdeveloped, by causing frequent debilitating clinical

conditions and thus, contributing to the loss of enormous

productive manpower. Although five species of Plasmodium family

of protozoan parasites can infect humans to cause malaria, P.

Traditionally chloroquine and other quinine derivatives have been

the most commonly used drugs to treat malaria. However, parasites

have developed widespread resistance against these drugs.²To

overcome this problem, artemisinin-based combination therapy

(ACT) has been developed as a frontline treatmnet.^3,4Although ACT

has been effective in treating malaria and is still being widely used,

resistance has emerged against this treatment in several Southeast

Asian countries.^5,6Hence, development of new, effective

antimalarial drugs is urgently needed. Towards this goal, our

research group has recently reported the potential utility of 1,2-

disubstituted-4-quinolones and abietane diterpenoids as new

antimalarial agents,^7,8and we have been continuing efforts to

identify new classes of antimalarial agents.

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The blood stage infection of Plasmodium parasites is responsible for usefulness of dibenzazepines as target-specific antimalarial agents

the clinical symptoms and severe illnesses of malaria. The initial and in view of exploring various new classes of compounds for

infection involves the entry of merozoites released from the antimalarial activity, we synthesized a library of dibenzazepines

matured liver stage parasites into erythrocytes, where they grow having substituted five-membered heterocyclic rings at the N-

into trophozoites, which upon maturation undergo schizogony to position of the azepine ring and evaluated their antimalarial

form merozoites. At the end of 48 h intraerythrocytic life cycle of activity.

parasites, the merozoites released from infected erythrocytes

invade new erythrocytes to start the next 48 h life cycle.^9,10During

their growth in erythrocytes, parasites depends on the host Chemistry

products for nutritional need. For example, amino acids formed by

Results and Discussion

At first, a series of 3,5-disubstituted isoxazolines (6a-6k) bearing

aryl, heteroaryl and alicyclic group at the C-3 position of isoxazoline

ring and having dibenzazepine moiety at the C-5 position were

synthesized³⁶through the reactions depicted in Scheme 1. The

synthon, dipolarophile 5-allyl-5H-dibenzazepine 2 was prepared by

N-allylation of 5H-dibenzo[b,f]azepine 1 using allyl bromide in

presence of tetra-n-butylammonium bromide (TBAB) as a phase

transfer catalyst, sodium hydroxide as a base in the biphasic

mixture of toluene-water in 1:1 ratio at 55 °C. The dipolar aryl nitrile

oxides (5a-5k) was generated in-situ in two-step processes by the

addition of hydroxylamine hydrochloride to aryl, heteroaryl or

alicyclic aldehydes (3a-3k) in the presence of sodium acetate in

methanol followed by the action of N-chlorosuccinimide (NCS) on

oximes formed (4a-4k) in the presence of triethyl amine base

(Scheme 1). Coupling of synthon 2 with aryl, heteroaryl or alicyclic

nitrile oxides in the presence of triethyl amine afforded

dibenzazepine tethered 3,5-disubstituted isoxazolines (6a-6k). The

the degradation of host cell haemoglobin by aminopeptidases are

the main source for parasite protein synthesis.^11,12P. falciparum M1

alanyl aminopeptidase (PfA-M1), a terminal zinc-metalloamino-

peptidase of hemoglobin digestion converts the cleaved

hemoglobin peptides into an amino acid pool, which is used by

parasites for its protein synthesis.^13-15Since hemoglobin-derived

amino acids are critical for parasite growth, PfA-M1 is considered as

a potential target for the development of antimalarial agents.^16-19

Compounds containing dibenzazepine 1 scaffold exhibit a wide

range of biological activity and hence, a variety of derivatives have

been synthesized and evaluated as antimicrobial and antifungal,²⁰

antioxidant,^21,22anticancer,²³antiepileptic,²⁴anticonvulasent,^25-27

and serotonin agents.²⁸Some examples of clinically important

dibenzazepine-based drugs are carbamazepine,^25,26,29opipramol,³⁰

and oxacarbazepine²⁴that used for treating epilepsy, anxiety, and

mood disorders.

A recent report describes dibenzazepine

derivatives as efficient antimalarial agents for both the gametocyte compounds were characterised by LC-MS, ¹H and ¹³C NMR, and

stage and the blood stage P. falciparum.³¹The dibenzazepine

elemental analyses (see the supplementary file)

moiety has also been used for the development of agents against

multi drug resistant Plasmodium parasites.³²Given the potential

i

N

H

90 %

N

1

2

iv

O

N

65-85 %

O

ii

N

iii

N

R₁

R₁CHO

R₁

5a-5k

OH

R₁

3a-3k

4a-4k

6a-6k

6j= p-(3-pyridyl)Ph

6k= 4-piperidyl

6g= p-(OH)Ph

6h= p-(OCOPh)Ph

6i= p-(CF₃)Ph

6d= p-(Br)Ph

6e= p, o-di-(Me)₂Ph

6f= p-(OMe)Ph

Where, R₁=

6a= p-(NO₂)Ph

6b= p-(F)Ph

6c= p-(Cl)Ph

Reagents and conditions: i) allyl bromide, TBAB, NaOH, toluene-water (1:1), 55 °C, 4h; ii) NH₂OH.HCl, CH₃COONa, CH₃OH, RT, 3h; iii) NCS,

Et₃N, CHCl₃, RT, 3h; iv) Et₃N, CHCl₃, 0 °C-RT, 6h.

Scheme 1 Synthesis of 3,5-disubstituted isoxazolines (6a-6k) from 5-allyl-5H-dibenzo[b,f]azepine 2.

Dibenzazepine derivatives carrying substituents on the Pd(dppf)Cl₂.CH₂Cl₂catalyst³⁷using Cs₂CO₃as base in dioxane-water

dibenzazepine ring were found to exhibit effective biological and biphasic solvent under nitrogen atmosphere (Scheme 2). The

pharmacological properties with minimal side effects.^27,32products were purified by column chromatography on silica gel (60-

Therefore, these class of compounds have been of special interest 120 mesh) using mixtures of ethyl acetate and hexane in suitable

for exploiting as drug candidates. Considering this, an additional ratios and were characterised by LC-MS, ¹H and ¹³C NMR, and

series of compounds (8a-8l) were prepared by introducing various elemental analyses (see the supplementary file). Additionally,

substituents on the para-position of the C-3 phenyl moiety of compound 6d was characterized by single crystal XRD studies and

isoxazoline ring in 6d. This was accomplished via Suzuki coupling of the crystallographic data have been deposited at the CCDC No

6d with various organoboronic acids (7a-7l) in the presence of 1049585³⁸(Fig. S1, see the supplementary file).

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N

OH

O

i

O

N

R₂

B

N

70-90 %

8 (a-l)

6d

7 (a-l)

Br

R₂

8i= p-(CH₂NHPh)Ph

8j= 2-thiophene

8k= 4-pyridyl

8e= p-(CHO)Ph

8f= p-(OMe)Ph

8g= p-(OCF₃)Ph

8h= p-(CN)Ph

8a= p-(Cl)Ph

Where, R₂=

8b= p-(OH)Ph

8c= p-(OH)Ph

8d= p-(CH₂OH)Ph

8l= 5-(6-Chloro-1,3-dihydro-indol-2-one)

Reagents and conditions: i) Pd(dppf)Cl₂.CH₂Cl₂, Cs₂CO₃, dioxane/water (6:1), 100 °C, 12 h.

Scheme 2 Suzuki coupling of 5-[3-(4-bromo-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-5H-dibenzo[b,f]azepine 6d with various boronic

acids.

Biology

Structure-activity relationship

All the synthesized compounds (6a-6k and 8a-8l) were initially

examined for antimalarial activity against chloroquine-sensitive P.

falciparum 3D7 strain. The parasite growth inhibition was assessed

at concentrations ranging from 0.1 to 100 µM by a SYBR Green

assay.^39,40All compounds showed good to excellent antimalarial

activity with IC₅₀values of 0.2 to 7.7 µM (Table 1). The cytotoxicity

of the compounds was examined using human kidney embryonic

(HEK 293) cells and human lung tumor epithelial cell (A549) by the

MTS assay. Many of the compounds exhibited either minimal or no

cytotoxicity (IC₅₀>40 µM), and others displayed low cytotoxicity (20

In efforts to understand the structure-activity relationship, first the

activity of dibenzazepine -series derivatives having substituents at

the para-position of the phenyl moiety at the C-3 position of

isoxazoline ring was compared. Regardless of whether the

substituents are electron-withdrawing groups such as -NO₂(6a), -F

6

(

6b), -Cl (6c), -Br (6d) and -CF₃(6i) or electron-releasing groups such

as -CH₃6f) and -OH (6g) groups, the compounds showed good to

(

excellent parasite growth inhibitory activity. Interestingly, nitrogen-

containing hetero-alicyclic substituent piperidyl (6k) or hetero-

aromatic substituent pyridyl (6j) in place of phenyl moiety exhibited

much higher inhibitory activity than the above compounds.

However, compounds containing hydrophobic methyl substituents

to 40 µM) (Tables 1). The compounds, 6j, 6k, 8c, 8k, and 8l that

showed inhibitory activity less than 2 µM were further assessed for

their ability to inhibit an additional chloroquine-sensitive parasite

(D6) strain and two chloroquine-resistant (W2 and 7G8) strains at

concentrations ranging from 0.10 µM to 1.75 µM. Each of the five

compounds inhibited both chloroquine-sensitive and chloroquine-

resistant parasites to similar extents (Table 2). Further, we tested

(

6e) or ester substituent (6h) were relatively less active. This could

be due to their expected lower solubility compared to other

compounds. In 8a-8l series, in general, compounds having hydroxy-

or methoxy-phenyl substituents

excellent inhibitory activity. As is the case of

8k and 8l, bearing pyridyl or 2-indolone substituents, exhibited

more potent activity. The remaining compounds, 8a 8e 8g 8i, and

(

8b

,

8c

,

8d

,

and 8f) showed

6

-series, compounds

6j, 6k, 8c, 8k and 8l for lysis of red blood cells at concentration

ranging from 2.5 µM to 40 µM. The compounds were not lytic at or

below 20 µM, suggesting that the antimalarial activity of

dibenzazepines is not due to the lysis of red blood cells.

,

8j, were relatively less active than others and there was no clear

distinct structure-activity relationship was evident.

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Table 1

Antimalarial activity of 3,5-disubstituted isoxazolines

Target identification

(6a-6k and 8a-8l) against drug-sensitive P. falciparum 3D7 strain

and cytotoxicity against A549 and HEK 293 cells.

PASS Online structure-activity predictive program was used to

identify the target molecule involved in blocking of malaria parasite

growth by dibenzazepine derivatives.^33,34This program predicts

many types of biological activity, including pharmacological effects,

interaction with metabolic enzymes, transporters, and mechanisms

of action by comparing the structures of test compounds with the

data base. The prediction is based on statistical probability data

that was developed based on the structure-activity relationships of

a collection of structurally similar molecules. Searching for the

probable targets suggested that PfA-M1 aminopeptidase is the

most likely target candidate. Hence, binding mode of the

dibenzazepine derivatives with PfA-M1 was analyzed by

computational modeling studies.

Parasite growth inhibition IC₅₀(µM ± SD)

Product

R₁

3D7 Strain ^a

A549 cells ^b

HEK293

cells^b

6a

6b

6c

NO₂

F

2.65 ± 0.95

2.11 ± 0.74

1.69 ± 0.28

>100

18.40 ± 2.61

18.60 ± 2.30

21.60 ± 1.51

16.60 ± 3.14

Cl

Br

6d

6e

6f

1.42 ± 0.96

3.35 ± 0.95

2.27 ± 0.88

26.80 ± 4.51

>100

31.60 ± 4.53

>100

H₃C

Table 2

Activity of selective dibenzazepine derivatives against

CH₃

drug-sensitive and drug-resistant P. falciparum.

CH₃

O

43.20 ± 3.66

24.10 ± 3.16

40.16 ± 6.23

23.60 ± 4.36

Parasite growth inhibition (IC₅₀, µM ± SEM)

Compound

3D7

D6

W2

7G8

6g

OH

6j

0.58 ± 0.18

0.19 ± 0.06

1.75 ± 0.84

0.99 ± 0.21

1.15 ± 0.67

0.13 ± 0.06

0.04 ± 0.02

0.40 ± 0.08

0.16 ± 0.04

1.66 ± 0.16

1.66 ± 0.04

1.06 ± 0.13

0.12 ± 0.08

0.03 ± 0.06

0.36 ± 0.09

0.12 ± 0.09

1.22 ± 0.09

1.10 ± 0.12

0.92 ± 0.09

0.32 ± 0.09

0.16 ± 0.07

0.42 ± 0.06

0.10 ± 0.06

1.30 ± 0.12

1.20 ± 0.02

0.93 ± 0.15

0.28 ± 0.03

0.11 ± 0.03

O

6k

4.17 ± 1.20

42.10 ± 5.03

43.40 ± 5.20

O

Ph

6h

6i

8c

CF₃

2.23 ± 0.85

0.58 ± 0.18

0.19 ± 0.06

23.40 ± 4.41

28.40 ± 3.17

45.60 ± 5.62

26.60 ± 3.22

22.10 ± 3.22

44.80 ± 6.11

8k

N

8l

6j

6k

8a

8b

Chloroquine

Mefloquine

NH

Cl

7.70 ± 1.46

2.69 ± 1.49

1.75 ± 0.84

2.83 ± 1.06

5.67 ± 1.63

2.70 ± 0.94

4.79 ± 0.93

2.63 ± 1.22

5.20 ± 1.10

3.14 ± 0.93

0.995 ± 0.21

1.15 ± 0.67

20.10 ± 3.94

36.70 ± 4.68

41.60 ± 3.13

33.60 ± 4.43

21.20 ± 3.82

56.20 ± 6.22

18.60 ± 4.20

39.50 ± 4.63

46.40 ± 6.63

42.40 ± 4.93

>100

36.40 ± 5.27

42.30 ± 5.09

31.90 ± 6.73

41.20 ± 3.50

31.20 ± 3.61

>100

Molecular docking analysis

OH

The aim of the molecular docking study was to elucidate how

dibenzazepine derivatives bind to the target PfA-M1, which cleaves

hemoglobin-derived peptides. The docking results provided

appropriate information about the binding affinity, binding energy

and orientation of ligand-enzyme interactions to inhibit the

function of PfA-M1. The molecular docking was carried out for all

substituted dibenzazepine tethered 3,5-disubstituted isoxazoline

derivatives with PfA-M1 and the interaction of ligands with protein

was analyzed (Table S1, Supplementary file). The docking protocol

was validated using the reported structure of PfA-M1 bound to its

ligand. The root mean square (RMS) deviation between the actual

and the predicted pose was 0.8 Å, which was well within the

acceptable limit of 2.0 Å.³⁹

OH

8c

8d

8e

8f

OH

O

CH₃

O

OCF₃

N

8g

8h

8i

29.10 ± 5.36

44.30 ± 5.03

41.30 ± 5.15

58.40 ± 4.66

>100

The compound 6d showed docking score of -6.68 kcal/mol. It

exhibited combination of both electrostatic and hydrophobic

interactions resulting in strong binding affinity. The oxygen and

nitrogen atom of isoxazoline ring formed co-ordination bond with

the Zn⁺²ion with the bond length 2.2 Å and 2.5 Å, respectively; the

dibenzazepine ring formed π−π parallel staking interactions with

His496; the terminal aromatic ring formed π−π staking interactions

with Tyr575; the bromine formed week halogen interactions with

backbone carbonyl of Glu319; the residues Val493, Val523, Tyr580,

Tyr575, Val459, Met462 formed hydrophobic interaction with

ligand (Fig. 1, Table 3).

HN

S

8j

8k

8l

N

O

56.6 ± 6.72

50.10 ± 4.70

N

Cl

H

^aThe fluorescence intensity was measured in a microliter plate reader.

^bThe absorbance was measured in a microliter plate spectrophotometer.

The compound 6j exhibited docking score of -8.06 kcal/mol; the

oxygen and nitrogen atom of isoxazoline ring of 6j formed co-

ordination bond with the Zn⁺²ion with the bond length 2.3 Å and

2.6 Å, respectively; the nitrogen atom of isoxazoline ring formed

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week H-bond with hydroxyl of Tyr580; the dibenzazepine ring co-ordination bond with the Zn⁺²ion with the bond length 2.3 Å and

formed π−π interactions with His496; the terminal pyridine ring 2.8 Å, respectively; the dibenzazepine ring formed π−π interactions

formed edge to face type of π−π interactions with Tyr575; the with His496; the terminal piperidine ring formed strong ionic

residues Val493, V523, Tyr580, Tyr575, Val459, Met462, Met1034, interaction (like salt bridge) with Glu572 and the protonated

Ala320 formed hydrophobic interaction with ligand (Table 3).

nitrogen of the piperidine ring formed π-cation interaction with

Tyr575 resulting in high gain in binding affinity; the residues Val493,

Tyr580, Tyr575, Val459, Met462, Met1034, Ala320 formed

hydrophobic interaction with ligand (Fig. 1, Table 3).

The compound 6k exhibited potent in vitro antimalarial activity. The

docking pose of 6k with protein showed strong combination of

electrostatic and hydrophobic interactions with active site amino

acid residues of PfA-M1, the observed docking score was -7.90

kcal/mol. The oxygen and nitrogen atom of isoxazoline ring formed

Fig. 1 Docking pose for compounds 6d

are shown in blue colour dotted lines and

,

6k

, 8k and 8l with PfA-M1. The H-bonds are shown in pink colour dotted lines, π−π interactions

π-cation interactions are shown in green colour dotted lines.

To improve the mode of interaction and binding ability with the The compound 8l showed a docking score of -7.30 kcal/mol. The

target, the structural modification on compound 6d was carried out oxygen and nitrogen atom of isoxazoline ring formed co-ordination

and obtained several additional compounds (8

series) and tested for bond with the Zn⁺²ion with the bond length 2.2 Å and 2.3 Å

antimalarial activity. Of these, two compounds, 8k and 8l, that respectively; the dibenzazepine ring formed π−π interactions with

showed excellent activity were analysed for binding to PfA-M1. The His496; the phenyl ring formed π−π staking interactions with

docking score for compound 8k was -7.80 kcal/mol. The oxygen and Tyr575, the terminal carbonyl of 2-indolone formed H-bond with

nitrogen atom of isoxazoline ring formed co-ordination bond with Asn458 and formed π−π staking interactions with Tyr575; the

the Zn⁺²ion with the bond length 2.1 Å and 2.5 Å, respectively; the residues Val493, Val523, Tyr580, Tyr575, Val459, Met462, Met1034,

nitrogen atom of isoxazoline ring formed weak H-bond with Ala320 formed hydrophobic interaction with ligand (Fig. 1, Table 3).

hydroxyl group of Tyr580; the dibenzazepine ring formed π−π The ligand displayed combination of electrostatic and hydrophobic

interactions with His496; the terminal pyridine ring and the interactions, but the binding pose bumped on Van der Waals radii

penultimate phenyl ring formed edge to face π-π interaction with of the protein atoms, which could be the reason for observed

Tyr575; the residues V523, Val493, Tyr580, Tyr575, Val459, Met462, decreased activity of 8l compared to 8k (Fig. 1, Table 3).

Met1034, Ala320 formed hydrophobic interaction with ligand (Fig.

1, Table 3).

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Table 3

Docking score and interaction residues for the binding of dibenzazepine derivatives with PfA-M1.

Docking score

(kcal/mol)

Metal

coordinate bond

π−π stacking or π−cat H-bond forming

Compound

Hydrophobic interaction residues

residues

ion interaction

6d

6j

-6.68

-8.06

-7.90

-7.80

-7.30

With Zn⁺²

His496, Tyr575

-

Val493, Val523, Tyr580, Tyr575,

Val459, Met462

Val493, V523, Tyr580, Tyr575, Val459,

Met462, Met1034, Ala320

Val493, Tyr580, Tyr575, Val459,

Met462, Met1034, Ala320

His496, Tyr575

Tyr580

Glu572

Tyr580

Asn458

6k

8k

8l

Val493, Tyr580, Tyr575, Val459,

Met462, Met1034, Ala320

Val493, Val523, Tyr580, Tyr575,

Val459, Met462, Met1034, Ala320

Molecular dynamics simulation

To confirm the stability of binding mode predicted by Glide docking

of the compounds to PfA-M1 and to monitor the structural changes

in the form of conformations and ligand-protein interactions,

molecular dynamics (MD) simulation was performed. The docked

complex of the most potent compound 6k was considered for the

MD simulation. The complex was adequately soaked into simulation

box consisted of 24,244 water molecules. The system was simulated

for 50 ps for equilibration. The final simulation run was for a total of

10 ns, during which 1000 structures enumerated were saved in the

trajectory. To understand the stability of the complex during MD

simulation, the protein backbone frames were aligned to the

backbone of initial frame and then the RMS deviation was

calculated with respect to the initial frame. The RMS deviations

between the original structure and the structure enumerated

during MD simulation were plotted (Fig. 2). The protein backbone

RMS deviation recorded during simulation shows large deviation for

the initial 500 ps due to the initial protein structural stabilization;

after about 500 ps the system showed a steady state dynamics. The

backbone structural deviations observed in protein for the latter

phase of 2 to 10 ns was in the range of 1.28 Å to 1.69 Å compared

to that of original structure. From 2 ns to till the end of simulation,

the total RMS deviation of protein was within a range of 0.41 Å. This

clearly suggests that 10 ns of simulation are sufficient for stabilizing

this complex. To further confirm the structure stability of the

protein, the RMS deviations between the side chain conformations

of the amino acid residues was measured and found minimal RMS

deviations of 0.43 Å after 2 ns (Fig. 2).

Fig. 2 The RSM deviations between the original structure and the

structure enumerated during MD simulation; the backbone

fluctuations are shown in blue, ligand fluctuations are shown in

green colour and the protein side chain fluctuations are shown in

red colour.

Further the RMS deviation of the ligand as shown in the Fig. 2,

drastically reduced to 0.52 Å after 2 ns simulation. This strongly

demonstrates that the ligand is well stabilized in binding site of the

protein. Various inter-molecular interactions, including H-bond,

hydrophobic, ionic, and electrostatic interactions were formed

between ligand and protein during the MD simulation, making the

ligand well stabilized in the binding pocket. The Fig. 3 shows the

summary of the total interactions observed during the MD

simulation. The stacked bar charts are normalized over the course

of the trajectory. Values over 1.0 are possible as some amino acid

residues could make multiple contacts of same subtype with the

ligand. All the ligand-protein interactions found in the docking study

were retained throughout the MD simulation. Fig. 4 shows a

schematic diagram of the ligand interacting with the amino acid

residues of protein structure evolved during MD simulation.

Residues with the ligand interactions that occurred more than 25%

of the simulation time in the trajectory are shown. The total

number of specific contacts the protein made with the ligand over

the course of the trajectory is shown in Fig. 5. From the structure

evolved from the MD simulation, it is evident that ionic interaction

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with Glu572, co-ordination bond formation with Zn⁺², the π-cation

interaction with Tyr575 and hydrophobic interaction with Tyr580 of

ligand is contributing strongly for the binding affinity; the π-cation

interaction with Arg489 and π−π interactions with His496 were also

observed. The water mediated bridged interaction between the

amino acid residues Glu319 and Glu463 further stabilizing the

ligand in the pocket.

Fig. 5 A timeline representation of the interactions and contacts

(H-bonds, and hydrophobic and ionic interactions, and water

bridges). The top panel shows the total number of specific contacts

the protein makes with ligand over the course of trajectory. The

bottom panel shows the residues that interact with the ligand in

each trajectory frame. The residues making more than one contact

are shown in darker colour shade.

Fig. 3 Interaction between PfA-M1 and 6k evolved during MD Experimental Section

simulation.

Materials and methods: The starting chemicals used here were

purchased from commercial sources. The solvents were of

analytical grade and were used without further purification.

Reactions were monitored by thin-layer chromatography (TLC)

using pre-coated sheets of silica gel 60 (Merck 60F₂₅₄, 0.25 mm

thickness) and visualization under UV light. The specific molecule

purified by preparative HPLC, waters SPD-4245, USA. The melting

points were determined on SELACO melting point apparatus and

are uncorrected. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz)

spectra were obtained using Agilent and Bruker NMR spectrometer,

respectively. Chemical shifts (δ) are given in parts per million (ppm)

using the residue solvent (CDCl₃and DMSO-d₆) peaks as reference

relative to TMS. Coupling constant (J) values are given in Hz.

Electrospray ionization (ESI) mass spectral analysis was performed

using Waters-Synapt G2 mass spectrometer. The C, H, and N

analysis were performed using CE-400 CHN analyser. Infrared

spectra were recorded on Shimadzu FT-IR model 8300

spectrophotometer.

Experimental procedure for the synthesis of 5-allyl-5H-

dibenzo[b,f]azepine (2)

To 5H-dibenzazepine (1, 25.8 mmol) in a mixture of toluene and

water in the ratio of 1:1 was added sodium hydroxide (51.7 mmol)

followed by TBAB (2.5 mmol) at room temperature. After 15

minutes, allyl bromide was added drop wise (38.8 mmol) at room

temperature. The reaction mixture was heated at 55 °C for 4 h.

After completion of the reaction as monitored by TLC, the reaction

mixture was diluted with water (100 mL) and the aqueous layer was

extracted with ethyl acetate (3 x 50 mL). The combined ethyl

acetate layer was washed with 0.1 N hydrochloric acid (2 x 50 mL),

followed by brine solution (2 x 50 mL). The organic layer was dried

Fig. 4 Schematic diagram of ligand interaction with the amino acid over anhydrous sodium sulphate, filtered, and concentrated under

residues of protein evolved during MD simulation. Interactions that

occur more than 25% of the simulation time are shown.

reduced pressure. The crude compound 2, was purified by column

chromatography over silica gel (60-120 mesh) using hexane:ethyl

acetate mixture in 9.5:0.5 ratios as eluent. The crude compound,

allyl-5H-dibenzo[b,f]azepine (2), was crystallized in ethyl acetate

and hexane mixture to obtain yellow crystals.

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5-Allyl-5

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Journal Name

H

-dibenzo[b,f]azepine (2)

(dd, J_1-2= 16.8 Hz, J_1-1= 10.4 Hz, 1H, N-CH), 3.83-3.52 (m, 2H, C₄-2H

isoxazoline), 4.35 (dd, J_1-2= 12.8 Hz, J_1-1= 4.0 Hz, 1H, N-CH), 4.87-

4.94 (m, 1H, C₅-H isoxazoline), 6.73 (d, J = 2.4 Hz, 2H, ArH), 7.10-

6.98 (m, 7H, ArH), 7.18-7.23 (m, 2H, ArH), 7.27-7.32 (m, 1H, ArH),

7.56-7.60 (m, 2H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 38.3, 54.2,

78.7, 115.9, 116.3, 121.3, 121.4, 124.0, 126.4, 126.5, 129.1, 129.2,

129.4, 129.5, 132.3, 132.5, 133.9, 150.4, 150.7, 155.9, 162.1, 164.6;

Yield 95%; yellow crystal; mp 40-42 °C; FT-IR (cm^-1): 3015, 1623,

1515, 1475, 1262; H NMR (400 MHz, CDCl₃): δ 4.40 (d, J = 5.6 Hz,

1

2H, -C=CH₂), 5.10 (dd, J_1-2= 10.4 Hz, J_1-3= 1.2 Hz, 1H, N-CH), 5.30

(dd, J_1-2= 17.2 Hz, J_1-3= 1.2 Hz, 1H, N-CH), 5.74-5.84 (m, 1H, -C=C-H),

6.75 (s, 2H, ArH), 6.98 (t, J = 6.26 Hz, 4H, ArH), 7.06 (dd, J_1-2= 8 Hz,

J_1-3= 1.6 Hz, 2H, ArH), 7.24 (td, J_1-2= 8 Hz, J_1-3= 1.2 Hz, 2H, ArH); ¹³

C

MS: m/z

= 370.4 (calculated), m/z =

371.5 [M+H]⁺(found);

NMR (100 MHz, CDCl₃): δ 53.54, 117.5, 120.5, 123.3, 128.6, 129.1,

132.2, 133.7, 135.1, 150.7; MS (ESI): m/z = 234.1 [M+H]⁺; elemental

composition calculated for C₁₇H₁₅N: C, 87.52; H, 6.48; N, 6.00;

Found: C, 87.56; H, 6.55; N, 6.04.

elemental composition calculated for: C₂₄H₁₉FN₂O: C, 77.82; H, 5.17;

F, 5.13; N, 7.56; O, 4.32; found: C, 77.84; H, 5.21; N, 7.58.

5-[3-(4-Chloro-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-5H-

dibenzo[b,f]azepine (6c)

General procedure for the synthesis of isoxazolines

Yield 82%; Yellow solid; mp 155-157 °C; FT-IR (cm^-1): 2925, 1593,

1494, 1485, 1456, 1434, 1355, 1238, 1179; ¹H NMR (400 MHz,

CDCl₃): δ 3.18 (dd, J_1-2= 16.8 Hz, J_1-1= 10.4 Hz, 1H, C₄-H isoxazoline),

3.40 (dd, J_1-2= 16.8 Hz, J_1-1= 6.0 Hz, 1H, C₄-H isoxazoline), 3.51 (dd,

Preparation of aldoxime (4a-4k)

To a solution of aldehyde (7.1 mmol) in methanol was added

hydroxyl amine hydrochloride (7.8 mmol) followed by sodium

acetate (7.8 mmol). The mixture was stirred at room temperature.

After completion of the reaction as monitored by TLC, the reaction

was quenched by adding crushed ice and the white precipitate

formed was isolated by filtration, washed with hexane, and dried.

J_1-2= 12.8 Hz, J_1-1= 9.2 Hz, 1H, N-CH), 4.33 (dd, J_1-2= 12.8 Hz, J_1-1

=

4.4 Hz, 1H, N-CH), 4.87-4.95 (m, 1H, C₅-H isoxazoline), 6.73 (q, J =

11.2 Hz, 2H, ArH), 6.98- 7.05 (m, 4H, ArH), 7.09 (d, J = 7.6 Hz, 1H,

ArH), 7.18-7.23 (m, 2H, ArH), 7.27-7.33 (m, 3H, ArH), 7.51-7.54 (m,

2H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 38.1, 54.2, 79.0, 121.3,

124.1, 128.6, 128.8, 129.2, 129.5, 132.2, 132.5, 133.9, 134.9, 156.0;

Synthesis of isoxazoline derivatives (6a-6k)

MS: m/z

= 386.8 (calculated), m/z =

387.5 [M+H]⁺(found);

For chlorination of aldoximes (4a-4k), their solutions of aldoximes

(4.1 mmol) in chloroform at room temperature was added NCS (4.5

mmol) and stirring was continued for 3 h, reaction progress was

monitored by TLC. The reaction mixture was cooled to 0 °C and

slowly added triethyl amine (10.25 mmol). After 10 minutes, the

elemental composition calculated for: C₂₄H₁₉ClN₂O: C, 74.51; H,

4.95; Cl, 9.16; N, 7.24; O, 4.14; found: C, 74.53; H, 4.99; N, 7.25.

5-[3-(4-Bromo-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-5H-

dibenzo[b,f]azepine (6d)

Yield 83%; brown solid; mp 138-140 °C; FT-IR (cm^-1): 2923, 1590,

1486, 1457, 1398, 1347, 1235, 1158, 1114; ¹H NMR (400 MHz,

CDCl₃): δ 3.17 (dd, J_1-2= 17.2 Hz, J_1-1= 10.8 Hz, 1H, C₄-H isoxazoline),

3.39 (dd, J_1-2= 16.8 Hz, J_1-1= 6.0 Hz, 1H, N-CH), 3.50 (dd, J_1-2= 12.8

solution of 5-allyl-5H-dibenzazepine

2 in DMF (4.1 mmol) was

°

added dropwise at 0 C. The stirring was continued for 6-8 h at 0 °C

to room temperature. The reaction mixture was diluted with

chloroform (2 x 25 mL), the combined organic layer was washed

with 0.1 N hydrochloric acid (2 x 20 mL), followed by brine solution

(2 x 20 mL). Then, the organic layer was dried over anhydrous

sodium sulphate, filtered and concentrated under reduced

pressure. The crude products (6a-6k), were purified by column

chromatography over silica gel (60-120 mesh) using hexane:ethyl

acetate mixture in 8:2 ratios as eluent. The compounds (6a-6k)

were crystallized in ethyl acetate and hexane mixture.

Hz, J_1-1= 9.2 Hz, 1H, C₄-H isoxazoline), 4.31 (dd, J_1-2= 12.8 Hz, J_1-1

=

4.4 Hz, 1H, N-CH), 4.89-4.91 (m, 1H, C₅-H isoxazoline), 6.72 (d, J =

3.2 Hz, 2H, ArH), 7.04 (q, J = 15.6 Hz, 3H, ArH), 7.09 (d, J = 6.4 Hz,

1H, ArH), 7.18 (d, J = 8.0 Hz, 1H, ArH), 7.22-7.30 (m, 3H, ArH), 7.43-

7.49 (m, 4H, ArH); ¹³C NMR (100 MHz, CDCl₃): δ 38.2, 53.6, 78.8,

120.7, 120.9, 123.9, 124.0, 124.2, 128.1, 128.5, 129.0, 129.1, 129.2,

129.4, 131.7, 131.8, 132.2, 133.5, 134.2, 148.4, 151.4, 155.6; MS:

m/z = 431.3 (calculated), m/z = 431.9 [M+H]⁺(found); elemental

composition calculated for: C₂₄H₁₉BrN₂O: C, 66.83; H, 4.44; Br,

18.53; N, 6.49; O, 3.71; found: C, 66.86; H, 4.49; N, 6.71.

5-[3-(4-Nitro-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-5H-

dibenzo[b,f]azepine (6a)

5-[3-(2,4-Dimethyl-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-5H-

dibenzo[b,f]azepine (6e)

Yield 72%; yellow crystal; mp 166-168 °C; FT-IR (cm^-1): 2993, 1727,

1519, 1484, 1269, 1169, 909, 764, 704; ¹H NMR (400 MHz, CDCl₃): δ

3.21 (dd, J_1-2= 16.8 Hz, J_1-1= 10.8 Hz, 1H, C₄-H isoxazoline), 3.46 (dd,

Yield 78%; yellow gummy solid; FT-IR (cm^-1): 2935, 1590, 1484,

1481, 1453, 1424, 1359, 1231, 1199; ¹H NMR (400 MHz, CDCl₃): δ

2.23 (S, 3H, CH₃), 2.37 (S, 3H, CH₃), 3.18 (dd, J_1-2= 16.8 Hz, J_1-1= 10.4

Hz, 1H, C₄-H isoxazoline), 3.36-3.42 (m, 2H, N-CH and C₄-H

isoxazoline), 4.25 (dd, J_1-2= 12.8 Hz, J_1-1= 4.4 Hz, 1H, N-CH), 4.75 (t,

J = 4.8 Hz, 1H, C₅-H isoxazoline), 6.65 (S, 2H, ArH), 6.90 -7.02 (m, 7H,

ArH), 7.08-7.22 (m, 4H, ArH); ¹³C NMR (100 MHz, CDCl₃): δ 21.1,

22.9, 41.1, 53.5, 77.0, 120.7, 120.9, 123.8, 124.0, 125.7, 126.3,

128.9, 129.02, 129.09, 129.1, 129.4, 131.9, 132.1, 132.3, 133.5,

J_1-2= 17.2 Hz, J_1-1= 6.4 Hz, 1H, N-CH), 3.58 (dd, J_1-2= 12.8 Hz, J_1-1

=

8.8 Hz, 1H, C₄-H isoxazoline), 4.32 (dd, J_1-2= 13.2 Hz, J_1-1= 4.4 Hz,

1H, N-CH), 4.94 -5.02 (m, 1H, C₅-H isoxazoline), 6.70 (q, J = 11.6 Hz,

2H, ArH), 6.98-7.04 (m, 4H, ArH), 7.09 (d, J = 6.4 Hz, 1H, ArH), 7.18

(d, J = 8.0 Hz, 1H, ArH), 7.23-7.31 (m, 2H, ArH), 7.73 (d, J = 8.8 Hz,

2H, ArH), 8.20 (d, J = 8.8 Hz, 2H, ArH); ¹³C NMR (100 MHz, CDCl₃): δ

37.7, 53.5, 79.7, 120.7, 120.9, 123.8, 124.0, 124.1, 127.3, 129.0,

129.2, 129.4, 131.8, 132.1, 133.5, 134.2, 135.7, 148.3, 148.4, 151.1,

154.9; MS: m/z = 397.4 (calculated), m/z = 398.8 [M+H]⁺(found);

elemental composition calculated for C₂₄H₁₉N₃O₃: C, 72.53; H, 4.82;

N, 10.57; O, 12.08; found: C, 72.56; H, 4.89; N, 10.59.

134.2, 137.8, 139.2, 148.5, 151.7, 157.3; MS: m/z

= 380.48

(calculated), m/z = 381.3 [M+H]⁺(found); elemental composition

calculated for C₂₆H₂₄N₂O: C, 82.07; H, 6.36; N, 7.36; O, 4.21; Found:

C, 82.09; H, 6.40; N, 4.23.

5-[3-(4-Fluoro-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-5H-

5-[3-(4-Methoxy-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-5H-

dibenzo[b,f]azepine (6b)

dibenzo[b,f]azepine (6f)

Yield 76%; yellow solid; mp 98-100 °C; FT-IR (cm^-1): 2980, 1731,

1523, 1489, 1260, 1153, 901, 764; ¹H NMR (400 MHz, CDCl₃): δ 3.19

8 | RSC Adv., 2012, 00, 1-3

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Yield 75%; orange gummy solid; FT-IR (cm^-1): 2953, 1575, 1491, 5-[3-(4-Pyridin-3-yl-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-5

1466, 1376, 1339, 1237, 1160, 1101; ¹H NMR (400 MHz, CDCl₃): δ dibenzo[b,f]azepine (6j)

3.26 (dd, J_1-2= 16.4 Hz, J_1-1= 10.0 Hz, 1H, C₄-H isoxazoline), 3.44-

H-

Yield 76%; pale brown solid; mp 162-164 °C; FT-IR (cm^-1): 2922,

2853,1732, 1583, 1583, 1571, 1484, 1433, 1407, 1358, 1334, 1298,

3.49 (m, 2H, C₄-H isoxazoline and N-CH), 3.81 (s, 3H, OCH₃), 4.33

(dd, J_1-2= 12.8 Hz, J_1-1= 4.4 Hz, 1H, N-CH), 6.73 (s, 2H, ArH), 4.80-

4.85 (m, 1H, C₅-H isoxazoline), 6.97-7.03 (m, 7H, ArH), 7.09 (dd, J_1-2

= 7.6 Hz, J_1-3= 1.6 Hz, 1H, ArH), 7.15-7.23 (m, 3H, ArH), 7.26-7.32

(m, 1H, ArH); ¹³C NMR (100 MHz, CDCl₃): δ 38.3, 55.5, 55.6, 79.7,

114.5, 120.3, 120.6, 120.7, 122.0, 123.7, 129.0, 129.1, 130.5, 132.1,

133.5, 146.5, 150.1, 159.6; MS: m/z = 382.4 (calculated), m/z =

383.3 [M+H]⁺(found); elemental composition calculated for:

C₂₅H₂₂N₂O₂: C, 78.51; H, 5.80; N, 7.32; O, 8.37; found: C, 78.54; H,

5.88; N, 7.35.

1

1240, 1225, 1162; H NMR (400 MHz, CDCl₃): δ 3.26 (dd, J_1-2= 16.8

Hz, J_1-1= 10.4 Hz, 1H, N-CH), 3.45-3.57 (m, 2H, C₄-2H isoxazoline),

4.35 (dd, J_1-2= 12.8 Hz, J_1-1= 4.4 Hz, 1H, N-CH), 4.90-4.97 (m, 1H, C₅-

H isoxazoline), 6.73 (d, J = 2.8 Hz, 2H, ArH), 6.98-7.05 (m, 4H, ArH),

7.10 (d, J = 7.6 Hz, 1H, ArH), 7.22 (t, J = 8.4 Hz, 2H, ArH), 7.30 (t, J =

7.2 Hz, 1H, ArH), 7. 02-7.84 (m, 5H, ArH), 8.00 (d, J = 8.4 Hz, 2H,

ArH), 8.34 (d, J = 4.4 Hz, 1H, ArH); ¹³C NMR (100 MHz, CDCl₃): δ

38.1, 54.2, 78.2, 120.2, 120.3, 120.4, 123.8, 124.0, 127.8, 129.1,

129.2, 132.1, 133.5, 133.6, 141.4, 147.4, 149.6, 149.9; MS: m/z =

4-(5-Dibenzo[b,f]azepin-5-ylmethyl-4,5-dihydro-isoxazol-3-yl)-

429.5 (calculated), m/z =

430.0 [M+H]⁺(found); elemental

phenol (6g)

composition calculated for: C₂₉H₂₃N₃O: C, 81.09; H, 5.40; N, 9.78; O,

3.73; found: C, 81.10; H, 5.46; N, 9.79.

Yield 70%; buff white solid; mp 169-171 °C; FT-IR (cm^-1): 3210, 2924,

1598, 1521, 1485, 1459, 1437, 1360, 1276, 1188, 1128; ¹H NMR 5-(3-Piperazin-1-yl-4,5-dihydro-isoxazol-5-ylmethyl)-5

(400 MHz, CDCl₃): δ 3.23 (dd, J_1-2= 16.8 Hz, J_1-1= 10.8 Hz, 1H, C₄-H dibenzo[b,f]azepine (6k)

isoxazoline), 3.42-3.54 (m, 2H, C₄-H isoxazoline and N-CH), 4.35 (dd,

H-

Yield 67%; buff white solid; mp 126-128 °C; FT-IR (cm^-1): 3400, 2712,

1593, 1483, 1458, 1308, 1224, 1160; ¹H NMR (400 MHz, CDCl₃): δ

1.85-2.01 (m, 4H, 2CH₂piperidine), 2.5-2.52 (m, 1H, CH piperidine),

2.78 (dd, J_1-2= 17.2 Hz, J_1-1= 10.8 Hz, 1H, C₄-H isoxazoline), 2.90-

3.01 (m, 3H, CH₂piperidine and N-CH), 3.32 (S, 2H, CH₂piperidine),

3.41 (dd, J_1-2= 12.8 Hz, J_1-1= 9.2 Hz, 1H, C₄-H isoxazoline), 4.22 (dd,

J_1-2= 12.8 Hz, J_1-1= 4.0 Hz, 1H, N-CH), 4.67-4.73 (m, 1H, C₅-H

isoxazoline), 5.18 (br, 1H, NH), 6.72 (d, J = 2.0 Hz, 2H, ArH), 7.04 (t, J

= 6.4 Hz, 3H, ArH), 7.05-7.07 (m, 2H, ArH), 7.12 (d, J = 8.4 Hz, 1H,

ArH), 7.23-7.28 (m, 2H, ArH); ¹³C NMR (100 MHz, CDCl₃): δ 26.0,

J_1-2= 12.8 Hz, J_1-1= 4.4 Hz, 1H, N-CH), 4.89-4.96 (m, 1H, C₅-H

isoxazoline), 6.74 (d, J = 1.2 Hz, 2H, ArH), 6.99-7.06 (m, 4H, ArH),

7.10 (dd, J_1-2= 7.6 Hz, J_1-3= 1.6 Hz, 1H, ArH), 7.19-7.25 (m, 4H, ArH),

7.28-7.33 (m, 1H, ArH), 7.49-7.53 (m, 2H, ArH), 9.20 (s, 1H, ArH); ¹³C

NMR (100 MHz, CDCl₃): δ 38.2, 54.5, 77.2, 120.2, 120.3, 121.5,

123.8, 129.1, 129.2, 129.7, 132.1, 133.5, 134.2, 135.5, 147.0, 149.9;

MS: m/z = 368.43 (calculated), m/z = 369.0 [M+H]⁺(found);

elemental composition calculated for: C₂₄H₂₀N₂O₂: C, 78.24; H, 5.47;

N, 7.60; O, 8.69; found: C, 78.25; H, 5.49; N, 7.61.

Benzoic acid 4-(5-dibenzo[b,f]azepin-5-ylmethyl-4,5-dihydro- 29.6, 33.1, 38.8, 43.0, 53.4, 77.6, 120.7, 121.0, 123.9, 124.0, 129.0,

isoxazol-3-yl)-phenyl ester (6h)

Yield 80%; yellow solid; mp 140-142 °C; FT-IR (cm^-1): 2990, 1727,

129.1, 129.2, 129.4, 131.9, 132.1, 133.5, 134.2, 148.5, 151.3, 159.2;

MS: m/z = 359.464 (calculated), m/z = 360.189 [M+H]⁺(found);

elemental composition calculated for C₂₃H₂₅N₃O: C, 76.85; H, 7.01;

N, 11.69; O, 4.45; found: C, 76.88; H, 7.05; N, 11.70; O, 4.49.

1

1484, 1269, 1169, 909, 764, 704; H NMR (400 MHz, CDCl₃): δ 3.30-

3.43 (m, 2H, C₄-2H isoxazoline), 3.82 (dd, J_1-2= 13.6 Hz, J_1-1= 6.4 Hz,

1H, N-CH), 4.05 (dd, J_1-2= 13.6 Hz, J_1-1= 6.4 Hz, 1H, N-CH), 4.75-4.80

(m, 1H, C₅-H isoxazoline), 6.77 (d, J = 1.6 Hz, 2H, ArH), 7.03 (d, J =

5.6 Hz, 2H, ArH), 7.10-7.23 (m, 4H, ArH), 7.30-7.37 (m, 4H, ArH),

Experimental procedure for Suzuki coupling reaction

5-[3-(4-Bromo-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-5H-

7.59-7.69 (m, 4H, ArH), 7.76 (t, J = 7.2 Hz, 1H, ArH), 8.14 (d, J = 7.2 dibenzo[b,f]azepine 6d (1.1 mmol) and aryl boronic acid (7a-7l)

,

Hz, 2H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 38.3, 54.2, 78.8, (1.1 mmol) were dissolved in a mixture of 1,4-dioxane (6 mL), H₂O

121.3, 121.4, 122.8, 124.1, 127.7, 128.2, 129.1, 129.4, 129.5, 130.3, (1mL) in a seal tube under nitrogen atmosphere. To the solution

132.3, 132.5, 133.9, 134.6, 150.4, 150.7, 152.1, 156.1, 164; MS: m/z was added Pd(dppf)Cl₂.DCM (0.1 mmol) followed by Cs₂CO₃

= 472.5 (calculated), m/z = 473.6 [M+H]⁺(found); elemental (3.3mmol). The resulting mixture was heated under reflux at 100 °C

composition calculated for: C₃₁H₂₄N₂O₃: C, 78.79; H, 5.12; N, 5.93; for 12h. The reaction was monitored by TLC and the solution was

O, 10.16; found: C, 78.80; H, 5.17; N, 5.95.

filtered on celite. To the clear filtrate was added ethyl acetate (3 x

25 mL), washed sequentially with water (2 x 25 mL) and brine (2 x

25 mL), dried over anhydrous Na₂SO₄, filtered and distilled under

reduced pressure. The crude products (8a-8l) were purified by

column chromatography on silica gel (60-120 mesh) using

hexane:ethyl acetate mixture in 7:3 ratios as eluent.

5-[3-(4-Trifluoromethyl-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-

5H-dibenzo[b,f]azepine azepine (6i)

Yield 83%; yellow solid; mp 143-145 °C; FT-IR (cm^-1): 2940, 1593,

1485, 1457, 1457, 1410, 1323, 1229, 1163, 1127; ¹H NMR (400 MHz,

CDCl₃): δ 3.21 (dd, J_1-2= 17.2 Hz, J_1-1= 10.8 Hz, 1H, N-CH), 3.42-3.57

(m, 2H, C₄-2H isoxazoline), 4.33 (dd, J_1-2= 12.8 Hz, J_1-1= 4.4 Hz, 1H,

N-CH), 4.92-4.99 (m, 1H, C₅-H isoxazoline), 6.72 (q, J = 11.2 Hz, 2H,

5-[3-(4'-Chloro-biphenyl-4-yl)-4,5-dihydro-isoxazol-5-ylmethyl]-5H-

dibenzo[b,f]azepine (8a)

ArH), 6.90-7.05 (m, 4H, ArH), 7.10 (d, J = 6.4 Hz, 1H, ArH), 7.19 (d, J Yield 82%; yellow solid; mp 165–167 °C; FT-IR (cm^-1): 2986, 2873,

= 8.0 Hz, 1H, ArH), 7.23-7.32 (m, 2H, ArH), 7.61 (d, J = 8.4 Hz, 2H, 1762, 1576, 1573, 1561, 1474, 1433, 1360, 1278, 1230, 1152; ¹H

ArH), 7.70 (d, J = 8.4 Hz, 2H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ NMR (400 MHz, DMSO-d₆): δ 3.25 (dd, J_1-2= 17.2 Hz, J_1-1= 6.8 Hz,

37.9, 54.1, 79.4, 121.3, 121.4, 123.1, 124.1, 125.8, 126.03, 126.07, 1H, C₄-H isoxazoline), 3.37 (dd, J_1-2= 17.2 Hz, J_1-1= 10.8 Hz, 1H, C₄-H

126.1, 127.6, 129.3, 129.5, 130.0, 130.3, 130.6, 132.2, 132.5, 133.8, isoxazoline), 3.75 (dd, J_1-2= 14.0 Hz, J_1-1= 6.4 Hz, 1H, N-CH), 4.01

133.9, 150.4, 150.6, 156.0; MS: m/z = 420.4 (calculated), m/z = (dd, J_1-2= 19.6 Hz, J_1-1= 6.4 Hz, 1H, N-CH), 4.69-4.74 (m, 1H, C₅-H

421.5 [M+H]⁺(found); elemental composition calculated for: isoxazoline), 6.72 (d, J = 2.4 Hz, 2H, ArH), 6.97-7.27 (m, 8H, ArH),

C₂₅H₁₉F₃N₂O: C, 71.42; H, 4.56; F, 13.56; N, 6.66; O, 3.81; found: C, 7.41(d, J = 8.4 Hz, 2H, ArH), 7.61 (d, J = 8.0 Hz, 2H, ArH), 7.66-7.69

71.45; H, 4.59; N, 6.67.

(m, 4H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 38.2, 54.2, 78.7,

121.4, 124.0, 127.3, 127.5, 128.8, 129.2, 129.4, 129.5, 129.5, 132.2,

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133.2, 133.9, 138.4, 140.4, 150.3, 150.7, 156.4; MS: m/z = 462.97 4.76 (m, 1H, C₅-H isoxazoline), 6.73 (d, J = 2.8 Hz, 2H, ArH), 6.97-

(calculated), m/z = 463.4 [M+H]⁺(found); elemental composition 7.31 (m, 8H, ArH), 7.67 (t, J = 7.2 Hz, 3H, ArH), 7.79 (dd, J_1-2= 6.8 Hz,

calculated for C₃₀H₂₃ClN₂O: C, 77.83; H, 5.01; Cl, 7.66; N, 6.05; O, J_1-1= 1.6 Hz, 2H, ArH), 7.87-7.90 (m, 1H, ArH), 8.00-8.03 (m, 1H,

3.46; found: C, 77.85; H, 5.08; N, 6.06; O, 3.47.

ArH), 8.20 (t, J = 1.6 Hz, 1H, ArH), 10.06 ( s, 1H, CHO); ¹³C NMR (100

MHz, DMSO-d₆): δ 38.2, 54.2, 78.8, 121.4, 124.1, 127.5, 127.6,

128.3, 128.8, 129.5, 130.3, 132.2, 132.4, 132.9, 133.9, 137.3, 140.4,

140.5, 150.3, 150.6, 156.4, 193.6; MS: m/z = 456.53 (calculated),

m/z = 457.0 [M+H]⁺(found); elemental composition calculated for

C₃₁H₂₄N₂O₂: C, 81.56; H, 5.30; N, 6.14; O, 7.01; found: C, 81.58; H,

5.37; N, 6.16; O, 7.02.

4'-(5-Dibenzo[b,f]azepin-5-ylmethyl-4,5-dihydro-isoxazol-3-yl)-

biphenyl-4-ol (8b)

Yield 79%; yellow solid; mp 119–121 °C; FT-IR (cm^-1): 2985, 1585,

1

1482, 1306, 1200, 1047; H NMR (400 MHz, DMSO-d₆): δ 3.25 (dd,

J_1-2= 16.0 Hz, J_1-1= 5.6 Hz, 1H, C₄-H isoxazoline), 3.40 (d, J = 3.2 Hz,

1H, C₄-H isoxazoline), 3.75 (dd, J_1-2= 12.8 Hz, J_1-1= 5.6 Hz, 1H, N-

CH), 4.02 (dd, J_1-2= 13.6 Hz, J_1-1= 5.2 Hz, 1H, N-CH), 4.72-4.74 (m,

1H, C₅-H isoxazoline), 6.75 (s, 2H, ArH), 6.85 (d, J = 7.6 Hz, 2H, ArH),

5-[3-(4'-Methoxy-biphenyl-4-yl)-4,5-dihydro-isoxazol-5-ylmethyl]-

5H-dibenzo[b,f]azepine (8f)

7.00-7.36 (m, 8H, ArH), 7.51 (d, J = 7.6 Hz, 2H, ArH), 7.60 (d, J = 8.1 Yield 87%; pale yellow solid; mp 187–189 °C; FT-IR (cm^-1): 2926,

Hz, 4H, ArH), 9.63 (s, 1H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 2853,1777, 1551, 1529, 1424, 1369, 1258, 1222, 1172; ¹H NMR (400

38.3, 54.2, 78.5, 116.0, 116.2, 121.3, 124.0, 126.4, 127.4, 127.7, MHz, DMSO-d₆): δ 3.28 (dd, J_1-2= 16.8 Hz, J_1-1= 6.4 Hz, 1H, C₄-H

128.2, 129.4, 129.5, 130.2, 132.2, 132.4, 133.8, 141.9, 156.5, 158.0; isoxazoline), 3.40 (dd, J_1-2= 16.8 Hz, J_1-1= 10.4 Hz, 1H, C₄-H

MS: m/z = 444.52 (calculated), m/z = 445.1 [M+H]⁺(found); isoxazoline), 3.75-3.80 (m, 4H, N-CH and OCH₃), 4.05 (dd, J_1-2= 13.6

elemental composition calculated for C₃₀H₂₄N₂O₂: C, 81.06; H, 5.44; Hz, J_1-1= 6.0 Hz, 1H, N-CH), 4.72-4.77 (m, 1H, C₅-H isoxazoline), 6.77

N, 6.30; O, 7.20; found: C, 81.09; H, 5.50; N, 6.32; O, 7.21.

(d, J = 1.6 Hz, 2H, ArH), 7.03 (d, J = 8.8 Hz, 4H, ArH), 7.10-7.32 (m,

6H, ArH), 7.61-7.69 (m, 6H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ

38.2, 54.2, 55.6, 78.5, 114.9, 124.0, 126.7, 127.4, 128.1, 128.2,

129.5, 131.9, 132.2, 132.5, 133.9, 141.5, 156.5, 159.7; MS: m/z =

458.55 (calculated), m/z

composition calculated for C₃₁H₂₆N₂O₂: C, 81.20; H, 5.72; N, 6.11; O,

6.98; found: C, 81.23; H, 5.79; N, 6.13; O, 6.99.

4'-(5-Dibenzo[b,f]azepin-5-ylmethyl-4,5-dihydro-isoxazol-3-yl)-

biphenyl-3-ol (8c)

Yield 89%; pale brown solid; mp 108-110 °C; FT-IR (cm^-1): 3419,

2988, 2254, 1659, 1481, 1244, 1050, 1023; ¹H NMR (400 MHz,

DMSO-d₆): δ 3.23 (dd, J_1-2= 17.2 Hz, J_1-1= 6.4 Hz, 1H, C₄-H

isoxazoline), 3.36 (dd, J_1-2= 16.8 Hz, J_1-1= 10.4 Hz, 1H, C₄-H

isoxazoline), 3.73 (dd, J_1-2= 11.2 Hz, J_1-1= 6.4 Hz, 1H, N-CH), 4.00

(dd, J_1-2= 13.2 Hz, J_1-1= 6.0 Hz, 1H, N-CH), 4.68-4.72 (m, 1H, C₅-H

=

459.10 [M+H]⁺(found); elemental

5-[3-(4'-Trifluoromethoxy-biphenyl-4-yl)-4,5-dihydro-isoxazol-5-

ylmethyl]-5H-dibenzo[b,f]azepine (8g)

isoxazoline), 6.73 (d, J = 1.6 Hz, 2H, ArH), 6.80-6.83 (m, 2H, ArH), Yield 80%; yellow solid; mp 165–167 °C; FT-IR (cm^-1): 2902, 2260,

6.98-7.27 (m, 8H, ArH), 7.47-7.50 (m, 12H, ArH), 7.54-7.61 (m, 6H, 2128, 1662, 1075, 1021, 987; ¹H NMR (400 MHz, DMSO-d₆): δ 3.25

ArH), 9.32 (s, 1H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 38.3, 54.2, (dd, J_1-2= 17.2 Hz, J_1-1= 6.8 Hz, 1H, C₄-H isoxazoline), 3.37 (dd, J_1-2

=

78.5, 115.9, 116.2, 121.3, 124.0, 126.4, 127.4, 127.7, 128.1, 129.5, 17.2 Hz, J_1-1= 10.9 Hz, 1H, C₄-H isoxazoline), 3.75 (dd, J_1-2= 14.0 Hz,

130.3, 131.6, 132.2, 133.9, 141.9, 150.3, 150.7, 156.5, 156.6, 158.0; J_1-1= 6.4 Hz, 1H, N-CH), 4.01 (dd, J_1-2= 13.6 Hz, J_1-1= 6.4 Hz, 1H, N-

MS: m/z = 444.52 (calculated), m/z = 445.1 [M+H]⁺(found); CH), 4.70-4.75 (m, 1H, C₅-H isoxazoline), 6.73 (d, J = 2.4 Hz, 2H,

elemental composition calculated for C₃₀H₂₄N₂O₂: C, 81.06; H, 5.44; ArH), 6.96-7.29 (m, 8H, ArH), 7.41 (d, J = 8 Hz, 2H, ArH), 7.62 (d, J =

N, 6.30; O, 7.20; found: C, 81.09; H, 5.50; N, 6.32; O, 7.21.

8.4 Hz, 2H, ArH), 7.70 (d, J = 8 Hz, 2H, ArH), 7.77 (d, J = 8.8 Hz, 2H,

ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 38.2, 54.1, 78.7, 119.2,

121.3, 121.3, 121.8, 121.9, 124.0, 127.5, 127.6, 129.0, 129.3, 129.4,

129.5, 132.2, 132.4, 133.9, 138.9, 140.3, 148.5, 150.3, 150.7, 156.4;

MS: m/z = 512.525 (calculated), m/z = 513.10 [M+H]⁺(found);

elemental composition calculated for C₃₁H₂₃F₃N₂O₂: C, 72.65; H,

4.52; F, 11.12; N, 5.47; O, 6.24; found: C, 72.67; H, 4.57; N, 5.48; O,

6.25.

[4'-(5-Dibenzo[b,f]azepin-5-ylmethyl-4,5-dihydro-isoxazol-3-yl)-

biphenyl-4-yl]-methanol (8d)

Yield 90%; yellow solid; mp 145-147 °C; FT-IR (cm^-1): 3445, 2250,

1

2135, 1672, 1055, 1001; H NMR (400 MHz, DMSO-d₆): δ 3.28 (dd,

J_1-2= 16.8 Hz, J_1-1= 6.4 Hz, 1H, C₄-H isoxazoline), 3.40 (dd, J_1-2= 16.8

Hz, J_1-1= 10.8 Hz, 1H, C₄-H isoxazoline), 3.75 (dd, J_1-2= 13.6 Hz, J_1-1

=

6.4Hz, 1H, N-CH), 4.01 (dd, J_1-2= 13.6 Hz, J_1-1= 6.4 Hz, 1H, N-CH),

4.54 (d, J = 5.6 Hz, 2H, OCH₂), 4.70-4.74 (m, 1H, C₅-H isoxazoline),

5.22 (t, J = 11.6 Hz, 1H, ArH), 6.73 (d, J = 2.4 Hz, 2H, ArH), 6.98-7.31

4'-(5-Dibenzo[b,f]azepin-5-ylmethyl-4,5-dihydro-isoxazol-3-yl)-

biphenyl-4-carbonitrile (8h)

(m, 9H, ArH), 7.39 (t, J = 8.4 Hz, 1H, ArH), 7.51 (d, J = 7.6 Hz, 1H, Yield 84%; pale yellow solid; mp 190–193 °C; FT-IR (cm^-1): 2971,

ArH), 7.62 (d, J = 8.4 Hz, 3H, ArH), 7.68 (d, J = 8.8 Hz, 2H, ArH); ¹³C 1483, 1457, 1226, 1052, 907; H NMR (400 MHz, DMSO-d₆): δ 3.29

1

NMR (100 MHz, DMSO-d₆): δ 38.2, 54.2, 63.2, 78.6, 121.3, 124.0, (dd, J_1-2= 16.8 Hz, J_1-1= 6.4 Hz, 1H, C₄-H isoxazoline), 3.40 (dd, J_1-2

=

125.0, 125.2, 125.4, 126.4, 127.3, 127.5, 128.8, 129.2, 129.4, 129.5, 12.0 Hz, J_1-1= 6.8 Hz, 1H, C₄-H isoxazoline), 3.78 (dd, J_1-2= 13.2 Hz,

132.2, 132.4, 139.4, 133.9, 142.0, 143.8, 150.3, 150.7, 156.4; MS: J_1-1= 6.0 Hz, 1H, N-CH), 4.04 (dd, J_1-2= 14.0 Hz, J_1-1= 6.4 Hz, 1H, N-

m/z = 458.55 (calculated), m/z = 459.1 [M+H]⁺(found); elemental CH), 4.73 (m, 1H, C₅-H isoxazoline), 6.72 (d, J = 2.8 Hz, 2H, ArH),

composition calculated for C₃₁H₂₆N₂O₂: C, 81.20; H, 5.72; N, 6.11; O, 6.99-7.28 (m, 8H, ArH), 7.64-7.76 (m, 2H, ArH), 7.76-7.85 (m, 2H,

6.98; found: C, 81.23; H, 5.79; N, 6.13; O, 6.99.

ArH), 7.85-7.91 (m, 4H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 38.1,

54.2, 78.9, 110.8, 119.2, 121.3, 124.0, 127.6, 127.8, 127.9, 129.5,

130.1, 132.2, 132.4, 133.3, 133.9, 139.8, 144.0, 150.3, 150.6, 156.3;

MS: m/z = 453.53 (calculated), m/z = 454.0 [M+H]⁺(found);

elemental composition calculated for C₃₁H₂₃N₃O: C, 82.10; H, 5.11;

N, 9.27; O, 3.53; Found: C, 82.13; H, 5.16; N, 9.29; O, 3.54.

4'-(5-Dibenzo[b,f]azepin-5-ylmethyl-4,5-dihydro-isoxazol-3-yl)-

biphenyl-4-carbaldehyde (8e)

Yield 78%; yellow solid; mp 119–121 °C; FT-IR (cm^-1): 2251, 2125,

1

1661, 1052, 1024, 1005, 821, 758, 624; H NMR (400 MHz, DMSO-

d₆): δ 3.27 (dd, J_1-2= 17.2 Hz, J_1-1= 4.4 Hz, 1H, C₄-H isoxazoline),

3.39-3.43 (m, 1H, C₄-H isoxazoline), 3.78 (dd, J_1-2= 13.2 Hz, J_1-1= 6.0

Hz, 1H, N-CH), 4.01 (dd, J_1-2= 13.6 Hz, J_1-1= 6.4 Hz, 1H, N-CH), 4.72-

[4'-(5-Dibenzo[b,f]azepin-5-ylmethyl-4,5-dihydro-isoxazol-3-yl)-

biphenyl-4-ylmethyl]-phenyl-amine (8i)

10 | RSC Adv., 2012, 00, 1-3

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Yield 77%; pale brown solid; mp 173–175 °C; FT-IR (cm^-1): 3444, 176.7; MS: m/z = 518.00 (calculated), m/z = 520.2 [M+2H]⁺(found);

1

2251, 1661, 1053, 1005; H NMR (400 MHz, DMSO-d₆): δ 3.26 (dd, elemental composition calculated for C₃₂H₂₄ClN₃O₂: C, 74.20; H,

J_1-2= 13.6 Hz, J_1-1= 6.8 Hz, 1H, C₄-H isoxazoline), 3.36 (dd, J_1-2= 14.8 4.67; Cl, 6.84; N, 8.11; O, 6.18; found: C, 74.23; H, 4.72; N, 8.13; O,

Hz, J_1-1= 8.4 Hz, 1H, C₄-H isoxazoline), 3.76 (dd, J_1-2= 14.0 Hz, J_1-1

=

6.19.

6.4 Hz, 1H, N-CH), 4.01 (dd, J_1-2= 14.0 Hz, J_1-1= 6.4 Hz, 1H, N-CH),

4.30 (d, J = 5.6 Hz, 2H, N-CH₂), 4.72 (m, 1H, C₅-H isoxazoline), 6.22 (t,

J = 6.0 Hz, 1H, ArH), 6.47 (t, J = 7.2 Hz, 1H, ArH), 6.58 (dd, J_1-2= 8.8

Hz, J_1-1= 0.8 Hz, 2H, ArH), 6.73 (d, J = 2.4 Hz, 2H, ArH), 6.99-7.02 (m,

4H, ArH), 7.06-7.26 (m, 6H, ArH), 7.33-7.40 (m, 2H, ArH), 7.51 (dd,

J_1-1= 7.6 Hz, J_1-2= 1.6 Hz, 1H, ArH), 7.61 (d, J = 8.8 Hz, 2H, ArH),

6.65-7.67 (m, 3H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 38.2, 46.9,

54.2, 78.6, 112.8, 116.2, 121.3, 124.0, 125.4, 125.9, 126.0, 127.3,

127.5, 128.8, 129.2, 129.4, 129.5, 132.2, 132.4, 133.9, 139.6, 141.6,

141.9, 149.0, 150.3, 150.7, 156.4; MS: m/z = 533.66 (calculated),

m/z = 534.8 [M+H]⁺(found); elemental composition calculated for

C₃₇H₃₁ClN₃O: C, 83.27; H, 5.86; N, 7.87; O, 3.00; found: C, 83.29; H,

5.90; N, 7.88; O, 3.01.

Assessment of antimalarial activity

P. falciparum parasites were cultured according to the method of

Trager and Jensen.⁴⁰Briefly, 3D7, D6, W2 and 7G8 P. falciparum

strains were cultured in RPMI 1640 medium (Gibco) supplemented

with 25 mM HEPES, 29 mM sodium bicarbonate, 0.005%

hypoxanthine, p-aminobenzoic acid (2 mg/liter), gentamycin sulfate

(50 mg/liter) and 5% AlbuMAX II (Invitrogen) using fresh O-positive

human red blood cells at 2% hematocrit. The cultures were

maintained at 37 °C under 5% O₂, 5% CO₂, and 90% N₂. The

antimalarial activity of isoxazoline compounds were determined by

a

fluorometric method using SYBR Green I.^41,42Briefly, stock

5-[3-(4-Thiophen-2-yl-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-

solutions of isoxazoline were prepared in DMSO (10 mM) and were

serially diluted with complete culture medium to give

concentrations ranging from 0.2 to 200 µM and the final

concentration of DMSO was less than 0.1%. 100 µL of each testing

solution was mixed with 100 µL of 0.2% parasitized red blood cells

in the ring stage at 2% hematocrit in complete medium and

dispensed into 96-well plates. Untreated- and DMSO (0.1%) vehicle-

treated parasites were used as controls. The plates were incubated

at 37 °C for 72 h and the experiment was performed in triplicate. To

each well, 100 µL of lysis buffer (20 mM Tris-HCl, pH 7.5, 5 mM

EDTA, 0.008% saponin, 0.08% Triton X-100) containing 0.2 µL/mL of

SYBR Green I (Life Technology) was added. The plates were

wrapped with aluminium foil and incubated in the dark at room

temperature for 1 h. The fluorescence intensity was measured using

multi-well plate fluorescence reader at an excitation and emission

wavelengths of 485 and 535 nm, respectively. The values were

expressed as relative fluorescence units. The IC₅₀values (the

effective concentrations that inhibit parasite growth by 50%) of

three independent experiments were plotted using nonlinear

regression (Sigmoidal dose response) equation (GraphPad Prism

version 4.01, GraphPad Software, La Jolla, CA).

5H-dibenzo[b,f]azepine (8j)

Yield 87%; pale brown solid; mp 173–175 °C; FT-IR (cm^-1): 2958.

1736, 1592, 1483, 1458, 1434, 1360, 1336, 1298, 1246, 1161; ¹H

NMR (400 MHz, DMSO-d₆): δ 3.25 (dd, J_1-2= 17.2 Hz, J_1-1= 6.8 Hz,

1H, C₄-H isoxazoline), 3.37 (dd, J_1-2= 17.2 Hz, J_1-1= 10.8 Hz, 1H, C₄-H

isoxazoline), 3.76 (dd, J_1-2= 14.0 Hz, J_1-1= 6.4 Hz, 1H, N-CH), 3.97-

4.02 (m, 1H, N-CH), 4.69-4.73 (m, 1H, C₅-H isoxazoline), 6.73 (d, J =

2.8 Hz, 2H, ArH), 6.98-7.28 (m, 9H, ArH), 7.55-7.56 (m, 4H, ArH),

7.67 (d, J = 8.4 Hz, 2H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 38.1,

54.2, 78.7, 121.3, 124.0, 124.9, 125.9, 126.8, 127.6, 128.7, 129.1,

129.5, 132.2, 132.4, 133.8, 135.4, 142.8, 150.37, 150.70, 156.3; MS:

m/z = 434.55 (calculated) m/z = 435.2 [M+H]⁺(found); elemental

composition calculated for C₂₈H₂₂N₂OS: C, 77.39; H, 5.10; N, 6.45; O,

3.68; S, 7.38; found: C, 77.41; H, 5.17; N, 6.47; O, 3.69.

5-[3-(4-Pyridin-4-yl-phenyl)-4,5-dihydro-isoxazol-5-ylmethyl]-5H-

dibenzo[b,f]azepine (8k)

Yield 86%; yellow solid; mp 108-110 °C; FT-IR (cm^-1): 2269, 2246,

2177, 1622, 1085, 901; ¹H NMR (400 MHz, DMSO-d₆): δ 3.25 (dd, J_1-2

= 17.2 Hz, J_1-1= 6.8 Hz, 1H, C₄-H isoxazoline), 3.37 (dd, J_1-2= 17.2 Hz,

J_1-1= 10.9 Hz, 1H, C₄-H isoxazoline), 3.75 (dd, J_1-2= 14.0 Hz, J_1-1= 6.4

Hz, 1H, N-CH), 4.01 (dd, J_1-2= 13.6 Hz, J_1-1= 6.4 Hz, 1H, N-CH), 4.71-

4.76 (m, 1H, C₅-H isoxazoline), 6.73 (d, J = 2.4 Hz, 2H, ArH), 6.98-

7.27 (m, 8H, ArH), 7.68 (d, J = 8.0 Hz, 2H, ArH), 7.83 (d, J = 8.4 Hz,

2H, ArH), 8.64 (s, 2H, ArH); ¹³C NMR (100 MHz, DMSO-d₆): δ 38.1,

54.1, 78.9, 121.3, 124.0, 127.5, 127.6, 129.3, 129.5, 130.6, 132.2,

132.5, 133.9, 138.7, 146.3, 150.3, 150.7, 156.4; MS: m/z = 429.51

(calculated), m/z = 430.1 [M+H]⁺(found); elemental composition

calculated for C₂₉H₂₃N₃O: C, 81.09; H, 5.40; N, 9.78; O, 3.73; found:

C, 81.91; H, 5.45; N, 9.79; O, 3.74.

Cytotoxicity assay

The toxicity of 3,5-disubstituted isoxazoline derivaives against

human cells was assessed by the MTS assay^43,44using human

embryonic kidney cells (HEK 293 cell line) and human

adenocarcinoma epithelial cells (A549 cell line). Briefly, 5×10³

cells/well in 100 µL of DMEM medium supplemented with 10%

bovine fetal serum were plated in 96-well plates. After 24 h, the

stock solutions of 3,5-disubstituted isoxazoline in DMSO that were

serially diluted to obtain concentrations ranging from 0.5 µM to 200

µM in 100 µl complete medium was added to each well and

incubated at 37 °C for 72 h. The cells treated with DMSO (1%) alone

were used as vehicle controls. To each well added 20 µl of

MTS/PMS reagent (Promega, Madison, WI), incubated for 3 h at 37

°C and the absorbance measured at 490 nm. The assay was

performed in triplicate and the values from three independent

experiments were used to calculate the IC₅₀values (GraphPad Prism

version 4.01).

6-Chloro-5-[4-(5-dibenzo[b,f]azepin-5-ylmethyl-4,5-dihydro-

isoxazol-3-yl)-phenyl]-1,3-dihydro-indol-2-one (8l)

Yield 70%; yellow solid; mp 160–162 °C; FT-IR (cm^-1): 3441, 2988,

2251, 2125, 1661, 1052, 1024, 1005; H NMR (400 MHz, DMSO-d₆):

δ 3.29 (dd, J_1-2= 16.8 Hz, J_1-1= 6.4 Hz, 1H, C₄-H isoxazoline); 3.40

(dd, J_1-2= 12.0 Hz, J_1-1= 6.8 Hz, 1H, C₄-H isoxazoline), 3.45 (s, 2H,

CH₂), 3.78 (dd, J_1-2= 13.2 Hz, J_1-1= 6.0 Hz, 1H, N-CH), 4.04 (dd, J_1-2

14.0 Hz, J_1-1= 6.4 Hz, 1H, N-CH), 4.74-4.78 (m, 4H, C₅-H isoxazoline),

6.77 (d, J = 1.6 Hz, 2H, ArH), 6.94 (s, 1H, ArH), 7.02-7.32 (m, 9H,

ArH), 7.41-7.44 (m, 2H, ArH), 7.62-7.64 (m, 2H, ArH), 10.58 (s, 1H,

NH); ¹³C NMR (100 MHz, DMSO-d₆): δ 35.82, 38.25, 54.20, 78.6,

110.4, 121.3, 124.0, 126.0, 126.7, 127.3, 127.4, 128.7, 129.5, 130.1,

130.2, 132.1, 132.2, 132.4, 133.9, 140.9, 145.0, 150.3, 150.7, 156.5,

1

=

Molecular modelling methods

The software used for all computational calculations is Schrödinger

Software Suite 2015-2 with hardware 2x Intel Xeon 1.9 GHz E5-

J. Name., 2013, 00, 1-3 | 11

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DOI: 10.1039/C5RA17926B

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2420/ 6C/15MB Cache RAM 6 x 4Gb DDR-3 1333MHz ECC RDIMM docking studies revealed the mode of the dibenzazepine

4x 500 Gb Graphics Card NvidiaQuadro 600 machine.

derivatives binding to PfA-M1. The molecular dynamics

simulation confirmed the stability of the binding mode of the

ligand 6k predicted by docking studies. The co-ordination bond

formed between the isoxazoline moiety of the ligand and the

enzyme-bound Zn⁺²ion, π−π interaction between

dibenzazepine and His496, and π−cation interactions between

piperidine moiety and Tyr575, and hydrophobic interactions

with residues Val493, Val523, Tyr580, Tyr575, Val459, Met462,

Met1034, Ala320 stabilized the dibenzazepine binding to PfA-

M1. Our experimental and theoretical finding data show that,

several compounds possess excellent antimalarial activity with

minimal or no cytotoxicity. Thus, overall our results suggest

that the dibenzazepine tethered 3,5-disubstituted isoxazolines

are promising candidates for the development of antimalarial

drugs.

The docking studies of 3,5-disubstituted isoxazoline tethered

dibenzazepine derivatives with PfA-M1 were performed by using

Schrödinger Glide.⁴⁵The structures were drawn using 2D sketcher

of Schrödinger maestro 10.2. The ligands were optimised for the

docking using LigPrep^46,47where the structures were converted to

3D and energy optimised using OPLS2005. Ionisation states were

generated for all molecules at pH range 7.0 ± 2 using Epik module,

in LigPrep with all other default options. 3D structure of inhibitor

bound PfA-M1 was retrieved in form from protein data bank³⁵(PDB

ID: 4X2U). The downloaded protein structure was refined using

Protein preparation wizard where in missing hydrogen atoms,

amino acid side chains and missing residues were added and bond

order were set. The unnecessary hetero atoms and water molecules

were deleted except the Zn⁺²and co-crystallised inhibitor. The

structure was optimized to create H-bond network and finally

minimized to remove any steric clashes using OPLS2015 force

field.⁴⁸The fully optimized protein structure was used for the

receptor grid generation using Glide software.⁴⁹The grid was

generated by choosing the centroid of bound ligand as the centroid

of the grid. The prepared ligands were docked to protein grid using

extra precession (XP) mode. The protein-ligand complex was further

analysed and visualized using Maestro.

Acknowledgements

KSV would like to thank UGC-BSR for financial support and IOE,

University of Mysore, Karnataka for NMR, mass spectrometry

facility. MPS thank UGC-SAP-DRS-Phase III, New Delhi and

VGST-SMYSR, Bangalore, Karnataka for funding. ACV would

like to thank CSIR-SRF, New Delhi for financial support. All

authors acknowledge Department of Biochemistry and

Molecular Biology, Penn State University College of Medicine,

Hershey Medical Centre, Hershey, PA, for providing facility for

assessing antimalarial activity, and Schrödinger, for providing

evaluation licence.

Molecular dynamics simulation analysis

The molecule (6k) that exhibited excellent antimalarial activity and

low toxicity to human cells was assessed by molecular dynamics

analysis to determine the binding stability and the binding pattern.

The protein complex docked with the inhibitor was submitted for

10 ns simulation. The molecular dynamics simulation was run using

Desmond.⁵⁰The orthorhombic simulation box was prepared using

the system builder panel of Desmond with TIP3P explicit water

model so that the minimum distance between the protein surface

and the solvent surface is 10 Å. The counter ions were added to

neutralise the system and 0.15 M NaCl was used to provide the iso-

osmotic salt environment. The system was minimized with 2000

iterations and with convergence criteria of 1 kcal/mol/Å. The

minimised system was subjected to the 10 ns molecular dynamic

simulation using NPT ensemble at temperature of 300K and

atmospheric pressure (1.013 bars) with the default setting of

relaxation before simulation. The Nose-Hoover Chain thermostat

and Matrtyna-Tobias-Klein barostate was used to maintain the

temperature and pressure, respectively. The time step of 2 fs was

considered during the simulation and every 10 ps, the energy and

structure was recorded and saved in the trajectory. From the

trajectory generated, the simulation interaction diagram was

generated to analyse the results.

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Synthesis, antimalarial activity, and target binding of dibenzazepine-tethered isoxazolines

DOI: 10.1039/c5ra17926b

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