Antiproliferative activity of diarylnaphthylpyrrolidine derivative via dual target inhibition

Journal Pre-proof

Amit Kumar Verma, Kaneez Fatima, Rajesh Kumar Dudi, Misbah Tabassum, Hina

Iqbal, Yogesh Kumar, Suaib Luqman, D.M. Mondhe, Debabrata Chanda, Feroz Khan,

Karuna Shanker, Arvind S. Negi

PII:

S0223-5234(19)31138-9

DOI:

https://doi.org/10.1016/j.ejmech.2019.111986

EJMECH 111986

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 15 October 2019

Revised Date: 5 December 2019

Accepted Date: 17 December 2019

Please cite this article as: A.K. Verma, K. Fatima, R.K. Dudi, M. Tabassum, H. Iqbal, Y. Kumar,

S. Luqman, D.M. Mondhe, D. Chanda, F. Khan, K. Shanker, A.S. Negi, Antiproliferative activity of

diarylnaphthylpyrrolidine derivative via dual target inhibition, European Journal of Medicinal Chemistry

(2020), doi: https://doi.org/10.1016/j.ejmech.2019.111986.

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Antiproliferative activity of diarylnaphthylpyrrolidine

derivative against via dual target inhibition

Amit Kumar Verma, Kaneez Fatima, Rajesh Kumar Dudi,

Misbah Tabassum, Hina Iqbal, Yogesh Kumar, Suaib Luqman,

D.M. Mondhe, Debabrata Chanda, Feroz Khan, Karuna Shanker,

Arvind S. Negi^*

Cell cycle

MCF-7: S- phase arrest,

MDA-MB-231: G2/M phase arrest

Antiproliferative activity

Breast cancer

MCF-7: IC₅₀=4.33µM;

MDA-MB-231: IC₅₀=5.21µM

Apoptosis induction

OCH₃

H₃CO

OCH-CH₂-N

Compound 50(R/S)

Dual target

-Antitubulin: IC₅₀=5.65µM;

-DNA-Topo-II inhibition:

25.9% inhibition at 17.3µM

Safety/Toxicity (in-vivo)

Well tolerable up to 300mg/kg dose.

Diarynaphthylpyrrolidine: A dual action antiproliferative agent against breast cancer.

Antiproliferative activity of diarylnaphthylpyrrolidine derivative via dual

target inhibition

Amit Kumar Verma,^aKaneez Fatima,^a,bRajesh Kumar Dudi,^cMisbah Tabassum,^cHina

Iqbal,^aYogesh Kumar,^a,bSuaib Luqman,^a,bD.M. Mondhe,^b,cDebabrata Chanda,^a,bFeroz

Khan,^a,bKaruna Shanker,^a,bArvind S. Negi^a,b*

^aCSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O. CIMAP, Kukrail Picnic Spot

Road, Lucknow-226 015, Uttar Pradesh, India.

^bAcademy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, Uttar Pradesh, India.

^cCSIR-Indian Institute of Integrative Medicine (CSIR-IIIM), Canal Road, Jammu-180001, India.

Abstract

Breast cancer is the second leading cause of deaths in women globally. Present communication

deals with design and synthesis of a few diarylnaphthyls as possible anti-breast cancer agents.

Among the thirteen representatives with significant antiproliferative activity compounds 23 and

50 were quite efficacious against human breast cancer cells. Compound 50 induced apoptosis

in both MCF-7 and MDA-MB-231 cells and exerted S phase and G2/M phase arrest

respectively via distinct mechanistic pathways. It showed moderate microtubule

destabilization. Further, it exhibited DNA topoisomerase-II inhibition effect in MCF-7 cells. It

was well tolerable and found safe up to 300 mg/kg dose in Swiss albino mice. The dual action

antiproliferative effect of compound 50 is quite interesting and warrants for future

development.

Keywords: breast cancer, apoptosis, microtubule destabilization, topoisomerase, molecular

docking studies, acute oral toxicity

1. Introduction

Breast cancer is the most prevalent among all cancer types in women around the world [1]. It is

the second leading cause of cancer deaths in women. Early stage diagnosis is very important

for the patient to control and eradicate the disease. Mammography is quite successful

technique though unaffordable to common person. However, mammography is significantly

associated in drastic reduction of breast cancer mortality [2,3]. Nevertheless, due to

heterogeneity of the disease, the effective treatment is still a challenge. There are several

successful clinical anticancer drugs with varied cancer targets which are used alone or in

combinations to treat breast cancer [4]. Some of the important drugs are tamoxifen

(Antiestrogen), anostrozole and letrozole (Aromatase inhibitors), doxorubicin (Topo-II

inhibitor), 5-fluorouracil (Antimetabolite), cisplatin (Alkylating agent), fulvestrant

......................................................................................................................................................................

*Corresponding authors: E mail: arvindcimap@rediffmail.com (a.s. negi ), Tel: +91-522- 2342676 x 583;

Fax: +91-522-2342666; )

1

(Estrogen receptor down-regulator, SERD), paclitaxel and docetaxel (Antitubulins),

abemaciclib (Cyclin dependent kinase inhibitor, CDKI), and olaparib (PARP inhibitor) etc.

[5, 6]. In advanced stage the breast cancer, cells may metastasise to spread to distant organs

such as lungs, bones, and brain [7, 8]. The treatment of advanced stage breast cancer is very

difficult and restricted due to chemotherapy resistance [9]. Although several effective drugs

have been developed, yet development of safe, effective and affordable anti-breast cancer drug

is still a challenge to scientific fraternity around the globe.

RO

O

HO

O

OH

N

OH

O

HN

O

NC

CN

O

CN

HO

H

O

N

OCH₃O

OH

O

HO

O

N

CN

OH

NH₂

O

N

Paclitaxel (Taxol), R=Ph;

Letrozole (Femara)

Tamoxifen (Nolvadex)

Anastrozole (Arimidex)

H

Doxorubicin (Adriamycin)

Docetaxel (Taxotere), R=t-Butoxy.

O

N

OH

F

O

N

F

N

O

Cl

N

NH₃

N

H

N

F

NH

Pt

N

O

HO

NH₃

Cl

N

H

O

N

F

O

S

F

Abemaciclib (Verzenio)

Olaparib (Lynparza)

Cisplatin (Platinol)

Fulvestrant (Falsodex)

5-Fluorouracil (Adrucil)

F

Figure 1: Some of the clinical drugs for breast cancer

Breast cancer is multifactorial in nature. Hence, it exhibits numerous targets due to

genetic, epigenetic and transcriptional factors [4]. Among various targets, tubulin is

considered an important target for anticancer drug development. α/β-Tubulin dimer

polymerises to microtubules and then microtubules depolymerise to tubulin [10, 11]. This

conversion is always in a dynamic equilibrium. Any disturbance exerted on this equilibrium

leads to cell cycle arrest and induces apoptosis [12]. Microtubule targeting agents are one of

the most reliable anticancer drugs with clinical efficacy. Paclitaxel, docetaxel (microtubule

stabilizers) and vincristine, vinblatine (microtubule destabilizers) are some of the clinical

drugs of this class [13, 14].

In the present study, we synthesized some naphthylstibenes and diarylnaphthylmethanes

as possible antibreast cancer agents. We designed these pharmacophores (I & II) based on

structure activity relationship of combretastatin A4 (CA4), a potent antitubulin agent [15-

17]. According to SAR of CA4, two aryl rings must be separated by a linker group

preferably in cis orientation, and one of the aryl rings must possess a 3,4,5-trimethoxy

system to induce antitubulin effect [14,18]. This 3,4,5-trimethoxyphenyl fragment is also

present is some of the naturally occurring antitubulin agents i.e. podophyllotoxin and

colchicine. Further, we planned to have some aminoalkyl chains at one of the aryl rings to

induce antiestrogenic effect. In hormone dependent breast cancer, there is a high level of

estrogenicity in tumour cells. Based on this hypothesis, we synthesized thirty four diverse

compounds as per prototypes-I & II compounds exhibited significant anticancer activity.

Compound 50, was the best representative of the series very close to our hypothesis. It was

extensively evaluated for cancer biology and safety aspects.

2

OH

O

H₃CO

O

H

N C CH₃

H₃CO

OH

O

H₃CO

OCH₃

O

OCH₃

H₃CO

OCH₃

3,4,5-Trimethoxyphenyl unit

Colchicine

Combretastatin A4

Podophyllotoxin

OCH₃

H₃CO

OCH₃

H₃CO

R₁

H

R₁

R₂

X

R₂

OR

OCH-CH-N

Prototype-III

Prototype-II

Compound 50

Prototype-I

17-22: (X=C & R1/R2=H/Ph);

23-28: (X=N, R1-R2=O-alkylester/acid)

32-41: R=alkyl/aryl)

9-16: R1=OH, R2=alkyl/aryl;

29: R1=OH, R2=H;

30, 31, 42-51: R1=H, R2=aryl

Figure 2: Some of natural microtubule destabilizers, trimethoxyphenyl fragment and

pharmacophores-I, II, & III.

2. Results

2.1. Chemistry

The synthetic strategy was as depicted in scheme 1. Pyrogallol (1) was fully methylated with

dimethyl sulphate in presence of anhydrous potassium carbonate to give 1,2,3-

trimethylpyrogallol (2). Trimethoxybenzene 2, underwent Friedel-Craft acylation on heating

at 80^oC with 1-naphthoic acid in presence of polyphosphoric acid to yield an inseparable

mixture of products i.e. trimethoxynaphthophenone (3a, 67%) and 3,4,-dimethoxy-2-

hydroxynapnthophenone (3b, 14%). This mixture was as such again methylated with methyl

iodide in presence of sodium hydride to get exclusively 3a. Naphthophenone 3a was

diversified towards the synthesis of four different pharmacophores I, II, II and IV. The yield

of the product 3a was very poor in three steps. Alternatively, acid catalysed methylation of

1-naphthoic acid was done to get the corresponding methyl ester 5, which was reduced with

lithium borohydride in dry THF to afford 1-naphthyl alcohol (6). Alcohol 6 was treated with

phosphorus tribromide to get corresponding 1-bromomethylnaphthalene (7). Bromo

derivative 7 underwent palladium chloride catalysed Suzuki coupling with 2,3,4-

trimethoxyphenylboronic acid in presence of anhydrous potassium carbonate to afford 2,3,4-

trimethoxybenzylnaphthalene (8). Compound 8 was oxidised with Collins reagent to get

naphthophenone 3a in very good yield.

Prototype-I (Scheme 1D): Naphthophenone 3a underwent Grignard reaction with various

Grignard reagents in dry diethylether to get various naphthyl-methyl alcohols (9-16), which

on dehydration with MeOH-HCl afforded the desired olefinic products (17-22, Series-I) in

excellent yields.

Prototype-II (Scheme 1E) Further, naphthophenone 3a was treated with hydroxylamine

in presence of DMAP to get corresponding oxime 23. Various fatty acid esters were hooked

3

up at hydroxyl group of the oxime 23 to afford oxime esters (24-27, Series-II) and

hydrolysed product 28 was obtained on hydrolysis of ester 25.

Prototype-III (Scheme 1F): Naphthophenone 3a was treated with ethyl magnesium

bromide (EtMgBr, diethyl ether) in dry THF to get a Grignard product at carbonyl carbon.

But, a reduced product i.e. 2,3,4-trimethoxyphenyl, 1'-naphthyl methanol (29) was achieved

in excellent yield. Alcohol 29 on treatment with conc. hydrochloric acid in dry benzene

underwent Friedel-Craft arylation with phenol to get a mixture of para and ortho substituted

products 30 and 31 as diarylnaphthalenes.

Prototype-IV (Scheme 1G): Aryl-naphthylmethanol 29 was treated with diverse alkyl

halides (MeI/EtBr/n-PrBr/Allyl bromide), aryl halides (BnBr/p-Me-BnBr/3,5-diMeO-

BnBr/p-COOMe-BnBr/p-CH₂OHBnBr) to get corresponding ethers (32-41, Series-III) at

alcoholic hydroxyl.

Further, diarylnaphthalenes 30 and 31 were modified by utilizing the o/p-phenolic

hydroxyl group (Scheme 1H & 1I). Compound 30 was condensed with various aminoalkyl

chains to get aliphatic amino chains at p-phenolic group (42-46, Series-IV). A fatty acid

ester chain was attached to phenolic group to get ethyl acetate chain at phenolic group (47).

Ester 49 was reduced with lithium borohydride in dry THF to get corresponding alcohol 48

in good yield. Ester 49 was hydrolysed with 6% alkali to get free carboxylic acid derivative

49 in excellent yield. Similarly, o-phenolic group of compound 31 was hooked up with two

amino alkyl chains to get corresponding o-aminoalkyl ethers 50 and 51 in excellent yields

(Series-IV). All the intermediates and final products were confirmed by spectroscopy.

Scheme 1

OCH₃

H₃CO

OCH₃

H₃CO

OH

OCH₃

O

OH

O

(A)

OH

ii

i

+

OCH₃

2

3a

1: Pyrogallol

3b

B(OH)₂

OCH₃

CH₂OH

CH₂Br

COOH

iii

COOCH₃

OCH₃

(B)

v

iv

vi

7

4

6

5

OCH₃

OCH3

H₃CO

OCH₃

OCH3

H₃CO

vii

O

8

3a

i) Me₂SO₄, K₂CO₃, dry acetone, reflux, 3h, 85%; ii) PPA, 1-Naphthoic acid, 80^oC, 3h, 3a (67%), 3b

(14%); iii) MeOH, Conc. H₂SO₄, 65^oC, 2h, 91%; iv) LiBH₄, dry THF, 4h, 60^oC, 86%; v) PBr₃, dry THF,

0-10^oC, 2h, 74%; vi) PdCl₂.(PPh₃)₂, K₂CO₃, DMF:H₂O(3:2), 80^oC, 4h, 51%; vii) Collins reagent (CrO₃

in DCM), pyridine, 0-10^oC (1h) then RT (28-30^oC), 9h, 56%.

4

(C)

OCH₃

H₃CO

H

N

O

R

17-22: Series-I

24-28: Series-II

OCH₃

H₃CO

O

3a

OCH₃

H₃CO

OCH₃

OR₁

O

R

R₂

32-41: Series-IV

42-51: Series-III

R₁=H/alkyl/aryl;

R₂=H/OH

(D)

O

OH

CH-R

ix

viii

R

3a

17-22

9-16

9: R=CH₃

;

17: R=H;

10: R=Isopropyl ;

11: R=allyl ;

18: R= -Ph ;

19: R= -C₆H₄-p-(CH₃) ;

12: R= -CH(CH₃)-COOEt ;

13: R= -CH₂-CH=CH-Ph;

14: R= -CH₂-Ph ;

20: R= -C₆H₃-3', 5'-(OCH₃)₂;

21 (E): R=-C₆H₄-p-F ;

22 (Z): R=-C₆H₄-p-F ;

15: R= -CH₂-C₆H₄-p-(CH₃) ;

16: R= -CH₂-C₆H₄-p-F

viii) RMgX, diethylether, THF, RT (26-30^oC), 30-60min, 68-90%; ix) MeOH, Conc. HCl, reflux, 2h,

72-90%;

(E)

OCH₃

H₃CO

OCH₃

H₃CO

OCH₃

H₃CO

N

OH

O

24: R=-CO-CH₃;

N

OR

25: R= -CH₂-COOC₂H₅;

26: R= -CH₂-CH₂-COOC₂H₅;

27: R=CH₂-CH=CH-COOC₂H₅;

28: R= -CH₂-COOH

xi

x

xii

3a

23

24-28

x) NH₂OH.HCl, EtOH, DMAP, reflux, 18h, 87%; xi) For 24: Ac₂O, DMAP, dry CHCl₃, RT, 2h, 86%,

for 25-27: ethylbromoester, dry THF, NaH, 65^oC, 2h, 86-92%; xii) 6% KOH in MeOH, reflux, 2h, 66%.

(F)

OCH₃

H₃CO

OH

O

OH

xiii

xiv

OH

+

31

30

3a

29

5

xiii) EtMgBr, diethyl ether, THF, RT (1h), reflux (3h), 86%; xiv) Phenol, dry benzene, Conc. HCl, 5-

6h, 30: 48%, 31: 26%.

(G)

32: R=CH₃;

OCH₃

OCH3

OCH₃

OCH3

H₃CO

33 R=-CH₂-CH₃;

H₃CO

34: R= -CH₂-CH₂-CH₃;

35: R= -CH₂-CH=CH₂;

36: R=-CH₂-CH₂-N(CH₃)₂;

37: R=CH₂-Ph ;

OR

H

OH

H

xv

38: R=CH₂-C₆H₄-p-CH₃;

39: R=CH₂-C₆H₃-3',5'-(OCH₃)₂;

40: R=CH₂-C₆H₄-p-COOCH₃;

41: R=CH₂-C₆H₄-p-CH₂-OH;

32-41

xvi

29

xv) NaH, dry THF, alkyl halide/aryl halide/benzylbromide, reflux, 1-2h, 63-86%; xvi) LiBH₄, THF,

reflux, 83%.

OCH₃

42: R= -CH_2-CH₂-N-(CH₃)₂;

H₃CO

(H)

OR

43: R= -CH_2-CH₂-N-(CH₂-CH₃)₂;

44: R= -CH(CH₃)CH₂-N-(CH₃₎₂

45: R= -CH_2-CH₂-N-(CH₂)₄;

46: R= -CH_2-CH₂-N-(CH₂)₅;

;

xvii

OCH₃

H₃CO

OH

42-46

OCH₃

H₃CO

OCH₂-CH₂OH

OCH₃

H₃CO

OCH₂-COOC₂H₅

xix

30

xviii

xx

48

47

OCH₃

H₃CO

OCH₂-COOH

49

xvii) NaH, dry THF, aminoalkylchain (HCl), 65^oC, 74-82% ; xviii) Ethylbromoacetate, dry THF, NaH,

65^oC, 85%; xix) LiBH₄, dry THF, 50^oC, 3h, 85%; xx) 6% KOH in MeOH-H₂O (9:1), 50^oC, 72%;

(I)

OCH₃

H₃CO

OCH₃

H₃CO

OCH₃

xxi

OX

OH

50 (R & S):X=-CH₂-CH₂-N-(CH₂)₄;

31

51:X=-CH₂-CH₂-N-(CH₂)₅

;

xxi) NaH, aminoalkyl chain (HCl), dry THF, reflux, 50: 87%, 51: 81%.

2.2. Purity profile of compound 50

Chromatographic conditions were optimised to get optimum separation. The peaks integration

performed at λ_max(210 nm). The column eluent was monitored in PDA range (190-400 nm) for

6

co-elution of possible impurities. The peak area and the retention time of compound 50 and

other unknown peaks were accounted to define the purity. Under optimised chromatographic

conditions, compound 50 was eluted at 4.295 min., while other impurities appeared at 3.331

min without any interference. As a reference methodology, the functions for peak purity

analysis in the chromatographic data processing software (Empower®, 3 version 7.40, Waters,

USA) were applied. The purity of compound 50 was 97.9%.

2.3. Biological evaluation

2.3.1. Cytotoxicity evaluation by Sulphorhodamine assay

All the synthesized compounds of prototype-I, II, III, and IV (17-48) were evaluated for

antiproliferative activity against a panel of seven human cancer cell lines i.e. MCF-7 (hormone

dependent breast cancer), MDA-MB-231 (breast cancer, triple negative), HCT-116 (Colon

cancer), A549 (lung cancer), and THP-1 (acute leukemia) by employing sulphorhodamine

assay.

Among the four diverse series of compounds i.e. series I-IV, compounds of series I (17-22)

and IV (42-51) exhibited significant antiproliferative activity against both MCF-7 and MDA-

MB-231 breast cancer cell lines. While, series II compounds (24-28) showed efficacy only

against MDA-MB-231 cells. Further, series III compounds were not much effective and only

three compounds (36-38) exhibited significant activity against MCF-7 cell lines. It will be

worth to mention that there was a distinct selective activity in series II compounds against

MDA-MB-231 and series-III compounds against MCF-7 cell lines. Compound 18 of series I

exhibited best cytotoxicity against MCF-7 while compound 23 from series II was the best

against MDA-MB-231 cell line. Compound 50 possessing potent cytotoxicity against both type

of breast cancer cell lines, was selected for detailed pharmacology.

In series IV (42-51), only the compounds with aminoalkyl group were active (42-46 &

50, 51). Compounds 47-49 without this group were found inactive. Among the active

compounds, compounds bearing amino alkyl chain at ortho position (50 & 51) were more

effective as compared to para substitution (42-46). The five member amino ring compound 50

was more effective as compared to six member amino ring compound 51.

7

Table 1: In-vitro cytotoxicity and tubulin polymerisation inhibition of synthesized compounds

S.

no.

Compd. No.

Cytotoxicity IC₅₀(µM)*

Tubulin

polymerisation

inhibition

IC₅₀(µM)

ND

MCF-7

MDA-

HCT-116

A549

THP-1

MB-231

1

2

---

10.66

4.00

---

11

12

ND

3

4

5

6

7

8

9

10.19

10.63

---

7.56

11.69

---

17.72

10.53

---

12.76

4.78

---

5.44

4.66

8.31

16.16

---

2.50

17.00

16.36

15.15

---

5.88

4.98

3.36

10.03

---

4.98

15.97

---

17.60

---

18.71

5.94

19.81

19.63

---

18.64

13.13

17.23

6.72

19.63

14.01

17.23

16.39

13.58

9.71

17.90

13.51

15.73

17.50

11.99

10.21

11 .47

11.36

15.78

15.00

---

19.42

12.38

---

5.86

12.54

---

9.08

18.85

---

9.45

---

7.95

13.88

15.01

5.40

16.58

16.64

---

12.57

19.13

11.91

19.55

ND

6.02

ND

9.03

ND

5.58

ND

5.65

ND

13

14

15

16

18

19

21

22

23

24

25

26

27

28

30

31

35

36

37

38

39

40

42

43

44

45

46

48

49

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

---

17.54

16.63

---

11.14

14.07

10.71

---

17.35

17.74

---

14.29

15.60

4.34

14.39

15.16

---

15.16

15.49

13.08

15.27

15.44

---

3.30

8.34

8.41

10.83

11.94

---

5.02

2.03

4.63

45.0

---

16.44

5.85

9.86

10.08

7.34

0.63

50

51

4.33

12.42

11.55

10.86

3.89

5.21

7.18

---

5.73

5.12

Tamoxifen

Podophyllotoxin

Doxorubicin

0.72

ND

* IC₅₀>20µM was considered as inactive, --- means inactive

2.3.2. Soft agar colony formation assay

The cytotoxicity of compound 50 was further authenticated by Soft agar colony formation in

MCF-7 cells, a well-established surrogate assay for tumorigenesis. This experiment estimates

the anchorage-independent growth ability of cells which is considered as gold standard assay

for cellular transformation in-vitro [19]. It gives an accurate quantitative measurement of

antiproliferative activity [20].

Compound 50 inhibited growth formation of MCF-7 cells in concentration dependent

manner. At 6.25 µg/mL concentration 29.2% cells were killed, while at 50 µg/mL

concentration, 87% MCF-7 cells were killed. It exhibited moderate cytotoxicity (IC₅₀=11.23

µg/mL). However, this effect was less than the standard drug tamoxifen (IC₅₀=1.31µg/mL).

8

The experiment confirmed the antiproliferative activity of compound 50 against MCF-7

cells.

Table 2: Effect of compound 50 on Colony Formation of MCF-7 cells

Condition

Concentration

(µg/mL)

Avg. % live cells

MCF-7

(% dead cells)

MCF-7

IC₅₀(µg/mL)

Control

Compound 50

-------

6.25

12.5

25

100

-----

------

70.77

47.20

20.64

12.98

70.50

42.20

5.69

29.23±3.39

52.80±1.22

79.36±2.28

87.02±1.50

29.50±2.18

57.80±1.03

94.31±0.43

11.23

50

Tamoxifen

1.07

5.39

26.95

1.31

No. of cells =12369±836; **p<0.01 (Dunnett test);

2.3.3. Cell cycle analysis

Cell division cycle is an ordered process having series of events which ultimately yields to

DNA duplication. Compound 50, exhibited S phase arrest at its half IC₅₀(2.17 µM) and IC₅₀

(4.33 µM) and induced apoptosis in MCF-7 cells. However, at higher concentration (double

IC₅₀) S phase arrest was less, but apoptosis was enhanced significantly.

Interestingly, in cell cycle analysis in MDA-MB-231 cells, compound 50, exerted

mainly G2/M phase arrest at all the three concentrations (Half IC₅₀, IC₅₀and double IC₅₀).

However, the induction of apoptosis was less up to IC₅₀concentration and it was significantly

high at double IC₅₀. There was slight effect at G0/G1 phase.

Compound 50 behaved distinctly with both the cell lines in cell division cycle, which

indicates independent mechanism in these cell lines for inducing cytotoxicity.

Table 3: Cell cycle analysis of compound 50 in MCF-7 and MDA-MB-231 cells

Compd.

Conc.

(µM)

Average %Population of

MCF-7 Cells

Conc.

(µM)

Average %Population of

MDA-MB-231 Cells

Apoptosis

G0/G1

S

G2/M

Apoptosi

s

G0/G1

S

G2/M

Control

Compd.

50

---

2.17

(half IC₅₀)

4.33

( IC₅₀)

8.66

2.1

2.2

74.5

74.6

6.9

10.9

15.2

12.1

---

2.60

(half IC₅₀)

5.21

( IC₅₀)

10.42

2.6±0.28

2.7±0.21

77.7±0.28 11.35±0.07 8.35±0.07

80.2±0.14

79.1±0.14

6.75±0.21 10.35±0.21

7.9±0.14 10.2±0.14

4.7

67.4

66.5

13.9

9.2

13.4

12.8

2.8±0.14

11.4

16.7±0.2

8

56.9±0.00 11.35±0.35 15.05±0.07

(2xIC₅₀)

9

(A) Control

Compd. half IC₅₀

Compd. IC₅₀

Compd. 2xIC₅₀

(B) Control

Compd. half IC₅₀

Compd. IC₅₀

Compd. 2xIC₅₀

Figure 3: Effect of compound 50 on cell division cycle ; (A) in MCF-7 cells; (B) in MDA-MB-231 cells

2.3.4. Apoptosis Vs Necrosis induction by 50 by Annexin V-FITC assay

Compound 50 substantially induced late apoptosis in MCF-7 cells at its half IC₅₀(2.17 µM)

and IC₅₀(4.33 µM). There was no early apoptosis in the treated cells. However, it also

induced necrosis at its IC₅₀. However, induction of necrosis was much less than the

apoptosis in MCF-7 cells.

Control

Compd. 50 (Half IC₅₀)

Compd. 50 (IC₅₀)

Compd. 50 (2xIC₅₀)

Figure 4A: Induction of late apoptosis and necrosis by compound 50 in MCF-7 cells by Annexin V-FITC assay

Surprisingly, compound 50 behaved differently with MDA-MB-231. It induced both

early and late apoptosis significantly. However, effect on late apoptosis was much higher

than the early apoptosis. There was some increase in necrosis also. But the induction of

apoptosis was much higher than necrosis. Nevertheless, induction of necrosis by compound

50 was much less than the control drug doxorubicin.

The induction of apoptosis effectively in both MCF-7 and MDA-MB-231 cells, indicate that

the cytotoxic effect of compound 50 is via a systematic mechanism.

10

Control

Compd. 50 (Half IC₅₀) Compd. 50 (IC₅₀) Compd. 50 (2xIC₅₀) Doxorubicin (1µg)

Figure 4B: Induction of late apoptosis and necrosis by compound 50 in MDA-MB-231 cells by Annexin V-FITC

assay

2.3.5. Molecular docking studies

Molecular docking studies of both the enantiomers of compound 50 i.e. 50R and 50S for the

interaction with β-tubulin showed good affinity and comparable binding energy of compound

50R (-8.0 Kcal/mol), 50S (-8.8 Kcal/mol) with standard destabilizers colchicine (-8.5

Kcal/mol) and podophyllotoxin (-7.2 Kcal/mol) (Table 4). Docked view and residual amino

acids showed that there were seven residual amino acids (LEU B:248, LEU B:255, ASN

B:258, MET B:259, ALA B:316, LYS B:352, and ALA B:354) common to all three within 4Ǻ.

It clearly indicates that all the four ligands occupied the same binding pocket of β-tubulin

(Figure 5).

Table 4. Interaction studies of compounds 50R and 50S, colchicine and podophyllotoxin with β-tubulin (PDB ID:

402B)

S.

No.

1.

Compounds

Docking Energy

Interacting Amino Acids with in region of 4Å

Compound 50R

-8

VAL B: 238, CYS B:241, LEU B: 242, LEU B:248, ASN B: 249,

ALA B:250, ASP B: 251, LEU B:252, LYS B: 254, LEU B:255,

ASN B:258, MET B:259, THR B:314, VAL B:315, ALA

B:316,ALA B: 317,ILE B:318, LYS B:352,THR B:353, ALA

B:354,

2.

3.

4.

Compound 50S

Colchicine

-8.8

-8.5

-7.2

CYS B:241, GLN B:247, LEU B:248, ALA B:250, ASP B:251,

LYS B:254, LEU B:255, ASN B:258, MET B:259, THR B:314,

VAL B:315, ALA B:316, ALA B:317, ILE B:318, ASN B:349,

ASN B:350 , LYS B:352, ALA B:354

VAL B:238, CYS B:241, LEU B:242, LEU B:248, ALA B:250,

ASP B:251, LYS B:254, LEU B:255, ASN B:258, MET B:259,

THR B:314, VAL B:315, ALA B:316, ALA B:317, ILE B:318,

ASN B:350, LYS B:352, ALA B:354, ILE B:378,

Podophyllotoxin

CYS B:241, LEU B:242, LEU B:248, ALA B:250, ASP B:251,

LYS B:254, LEU B:255, ASN B:258, MET B:259, ALA B:316,

ILE B:318, LYS B:352, THR B:353, ALA B:354

11

Figure 5. The molecular docking pose showing the binding of compounds represented in different colours as

mentioned (colchicine (red), Compd. 50R (blue),Compd. 50S (green) and Podophyllotoxin (pink)) with the

Tubulin-Colchicine complex PDB ID: 4O2B. The 2D representation showing the interacting residues within the

docking region of 4Å.

2.3.6. Tubulin polymerisation inhibition activity

The effect of potent compounds 14, 18, 23, and 50 was determined on polymerisation of

tubulin protein. All these compounds exhibited moderate to weak antitubulin effect

(IC₅₀=5.58-9.44 µM) by inhibiting polymerisation of tubulin to microtubules (Table 1).

However, the standard inhibitor, podophyllotoxin had potential antitubulin effect (IC₅₀=0.78

µM).

2.3.7. DNA-Topoisomerase-II inhibition activity

In DNA topoisomerase inhibition activity in MCF-7 cells, compound 50 showed

concentration dependent effect on enzyme inhibition (Table 5). The maximum inhibition of

25.89% was observed at 17.32 µM concentration (4*IC₅₀). The standard inhibitor etoposide

caused 47.66% inhibition at 10.76 µM (4*IC₅₀) while podophyllotoxin, a known poor

inhibitor of Topo-II exhibited 26.86% inhibition at 50µM concentration.

Table 5: DNA-Topoisomerase-II inhibition activity of compound 50

S. No.

1.

Compound

Compound 50

Podophyllotoxin^#

Concentration

(µM)

% Inhibition of Topo-II

4.33

( IC₅₀)

8.66

( 2*IC₅₀)

17.32

(4*IC₅₀)

120

0.22

13.68

25.89

26.86

2.

3.

4.

12

5.

Etoposide

10.76 (4*IC₅₀)

47.66

^#Podophyllotoxin is strong antitubulin but weak topo-II inhibitor

2.3.8. Safety Studies

Compound 50 was further evaluated for safety studies by acute oral toxicity at three different

oral doses i.e. 5 mg/kg, 50 mg/kg, and 300mg/kg in Swiss-albino mice. There were no

observational changes, morbidity and mortality throughout the experimental period up to the

dose level of 300 mg /kg body weight. Blood and serum samples upon analysis showed non-

significant changes in all the parameters studied like haematological (total haemoglobin level,

differential leucocyte count), lipid (serum total cholesterol, triglycerides), Kidney function

(creatinine level), Liver function (SGOT, SGPT, and ALKP) activity (Table 6 and Figure 7).

Animals on gross pathological study showed no changes in any of the organs studied including

their absolute and relative weights (Figure 6). Therefore, the experiment showed that

compound 50 is well tolerated by the Swiss albino mice up to the dose level of 300 mg/kg body

weight as a single acute oral dose. However, the compound should be evaluated for sub-acute

and chronic experiments to look for any adverse effect on repeated exposure to the compound

50 and changes in biochemical parameters for its future development [21].

Table 6: Acute oral toxicity of compound 50 at 5, 50 and 300 mg/kg in Swiss albino mice. (Mean±SE; n=6; *,

P<0.05 compared to control).

Dose of compd. 50 at mg/kg body weight as a single oral dose

Parameters

Control

31.62±1.64

12.05±0.88

10.56±1.22

17.06±1.33

195.80±12.06

22.12±1.91

21.81±2.10

0.56±0.02

5 mg/kg

30.58±1.67

11.35±0.97

13.06±1.58

11.11±1.09

198.57±28.63

27.32±2.95

21.18±1.21

0.65±0.04

50 mg/kg

30.44±0.94

9.96±0.88

9.84±0.89

13.46±1.59

182.40±21.32

25.42±2.56

19.12±1.96

0.57±0.05

0.62±0.07

2.52±0.15

300 mg/kg

31.03±0.97

10.13±0.44

11.64±1.67

7.19±1.14*

137.70±11.23

25.61±3.95

22.34±1.33

0.54±0.04

Body weight (gm)

Haematological

Haemoglobin (gm/dL)

RBC (million/mm³)

WBC(thousands/mm³)

ALP (U/L)

Profile

Liver Function Test

SGOT (U/L)

SGPT (U/L)

Albumin (g/dL)

0.62±0.05

2.83±0.09

0.66±0.08

2.27±0.08

1.01±0.08^*

2.87±0.16

Total Bilirubin (mg/dL)

Serum Protein (mg/ml)

Creatinine (mg/dL)

Kidney Function Test

Lipid Profile

0.27±0.02

78.34±2.99

103.62±7.45

0.22±0.05

77.24±6.56

111.17±5.02

0.34±0.02

94.42±9.56

100.11±12.23

0.20±0.05

106.09±8.86

120.54±6.33

Triglycerides (mg/dL)

Cholesterol (mg/dL)

13

Figure 6. Effect of compound 50 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg on absolute and

relative organ weight in Swiss albino mice.

Figure 7. Effect of compd. 50 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg body weight on differential

leucocytes counts in Swiss albino mice.

3. Discussion

Breast cancer is a complex and heterogeneous disease. On an average 65-70% of breast

cancer cases are hormone dependent, 15-20% cases are hormone independent and 10-15% are

metastatic breast cancer. Among the various treatment options chemotherapy is one of the

most effective strategy to tackle the disease. However, at advanced stages metastatic breast

cancer is difficult to treat which is one of the greatest challenges encountered by the

clinicians. Drug resistance is another problem which is frequently faced due to prolonged

treatments.

During Grignard reaction on naphthophenone derivative 3a, no adduct was formed rather

ketone was reduced to alcohol 29. It was the case only when there was a β-hydrogen

available in Grignard reagent (RMgX). In case of MeMgX and BnMgBr only Grignard

product was obtained. However, EtMgBr and PrMgBr yielded exclusively reduced products.

In 3a, carbonyl group is flanked with phenyl and naphthyl ring and presence of β-hydrogen

14

in Grignard reagent resulted in reduction of ketone to alcohol and no Grignard adduct was

formed. We also tried some more naphthophenones and benzophenones to give similar

results.

Compound 50 was designed as microtubule destabilizer applying Fragment Based Drug

Discovery (FBDD) approach [22]. In FBDD, a fragment is identified that has quality

interactions with the target protein. When this fragment is incorporated at an appropriate

position in a pharmacophore, it may induce desired biological response [23]. We

incorporated a trimethoxyphenyl fragment in our all the three pharmacophores (Figure 2)

which are supposed to have quality interactions with β-tubulin and induces antitubulin effect

[14, 24]. Further, triarylmethanes show affinity for estrogen receptor (ER). It is well

established that ER-α is over-expressed in hormone dependent breast cancers [25-27]. Due

to higher level of estrogenicity in breast tumour cells, there is excessive proliferative.

Introduction of an aminoalkyl chain at an appropriate position of estrogen receptor ligands,

exhibits estrogen antagonistic activity, which might suppress the proliferation of MCF-7

cells [28-30]. Therefore, compound 50 possessed a pyrrolidinoethyl chain at phenolic

hydroxyl.

Compound 50 exhibited potential cytotoxicity against both MCF-7 and MDA-MB-231

cells. Interestingly, it had distinct action on these breast cancer cell lines. In cell cycle

analysis, it induced apoptosis in both the cell lines. However, in MCF-7 it induced S phase

arrest while, in MDA-MB-231 cells it caused G2/M phase arrest. During the S-phase the

entire DNA content of the nucleus is replicated completely and precisely [31]. Induction of

cell cycle arrest in MCF-7 cells at S phase indicates the inhibition of any of the cell cycle

regulator or enzyme involved in DNA duplication at S phase. Compound 50 exhibited DNA-

Topoisomerase-II inhibition activity which is actively involved in S phase of cell cycle

process. Topoisomerase-II alters DNA topology and plays roles in the replication,

transcription and segregation [32]. Inhibition of this enzyme by several anticancer drugs like

etoposide and teniposide etc. lead to Topo-II mediated chromosome DNA breakage and

cancer cell death.

Contrarily, in MDA-MB-231 cells, G2/M phase was arrested which might be due to

antitubulin effect of compound 50. Microtubule dynamics plays a crucial role in mitosis

process during the G2/M phase of cell division cycle. Modulation of tubulin-microtubules

dynamics is one of the most effective targets for cancer chemotherapeutics [14, 10].

Molecular docking studies showed that both the enantiomers i.e. compounds 50R, 50S,

podophyllotoxin, and colchicine occupied the same binding pocket at β-tubulin. There were

seven residual amino acids common to these three ligands. Both the enantiomers differed

slightly for their binding energies with β-tubulin. Isomer 50S had better affinity as compared to

50R. However, both the isomers possessed ALA β:316 and ILE β:318 amino acids, which are

considered to be crucial for inducing antitubulin effect by the 3,4,5-trimethoxyphenyl fragment

[14].

Compound 50 was safe and well tolerated by Swiss albino mice up to 300 mg/kg dose in

acute oral toxicity. However, there were some non-significant changes in some of the

parameters like increase in bilirubin and cholesterol levels and lowering in total WBC count in

15

the group of animals treated with the test drug at 300 mg/kg. It is quite possible that a potent

cytotoxic compound given through oral route might have such effects. However, sub-acute and

chronic experiments with the compound 50 need to be carried out to look for any adverse effect

on prolonged exposure of it. Notably, safety is an essential aspect in the development of a drug

candidate for a particular pharmacological activity [33]. Nowadays, it is denoted with a unique

terminology, ‘Pharmacovigilance’ [34]. Acute oral and sub-acute oral toxicity are most

frequent experiments used to assess the preliminary toxicity of the compounds [35].

4. Conclusion

In summary, we synthesized several compounds based on three designed pharmacophores.

Eight analogues exhibited potential antiproliferative activity against breast cancer cell lines.

Compound 50 exhibited potential anticancer activity against both the cell lines. It exerted

cell cycle arrest in cancer cells and induced apoptosis. In mechanistic studies, it was found

to be potent DNA topoisomerase-II inhibitor and moderate tubulin polymerisation inhibitor.

Compound 50 was non-toxic and safe up to 300mg/kg dose in experimental mice.

Compound 50, a triarylmethane is a potent anti-breast cancer agent. Its further optimization

is underway for better efficacy.

5. Experimental

5.2. Chemical synthesis

5.2.1. Synthesis of 2,3,4-trimethoxyphenyl-naphthlen-1-yl-methanone (3a)

1-Napthoic acid (2.06g, 12 mmol) and 1,2,3-trimethoxybenzene (1.68g, 10mmol) were

mixed well with polyphosphoric acid (6g). The reaction mixture was heated at 80^oC for 3h.

Reaction mixture was cooled to room temperature and poured into crushed ice, extracted

with ethyl acetate (60mLx3). The organic layer was washed with water (30mLx2), dried

over anhydrous sodium sulphate and dried in-vacuo. The residue obtained was purified

through column chromatography over silica gel eluting with hexane-ethyl acetate to get

compound 3a as amorphous solid. Further elution yield a mono demethylated product of 3a

i.e. 3,4-dimethoxy-2-hydroxynaphthophenone (3b) as minor side product.

1

3a:Yield=67%. White crystalline Mp= 75-77^oC; H NMR (CDCl₃, 500 MHz): δ3.49 (s, 3H,

OCH₃), 3.83 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 6.71-6.73 (d, 1H, CH, aromatic, J=9.0 Hz),

7.35-7.36 (d, 1H, CH, aromatic, J=9.0 Hz), 7.42-7.56 (m, 4H, 4xCH, aromatic), 7.88-7.95 (m,

2H, 2xCH, aromatic), 8.36-8.37 (dd, 1H, CH, aromatic, J=2.5, 6.5 Hz); ¹³C NMR (CDCl₃,

125MHz): δ56.15, 60.86, 61.39, 106.80, 124.32, 125.68, 126.30, 126.39, 127.37, 127.71,

128.09, 128.36, 130.68, 131.52, 133.73, 138.20, 142.24, 153.84, 157.22, 196.86; HRMS

(MeOH): m/z [M+H]⁺calcd for C₂₀H₁₉O₄, 323.1283, found 323.1283.

2-Hydroxy-3,4-dimethoxyphenyl-naphthlen-1-yl-methanone (3b): Yield=14%. Yellowish

viscous,¹H NMR (CDCl₃, 500 MHz): δ3.89 (s, 3H, OCH₃), 3.98 (s, 3H, OCH₃), 6.32-6.34 (d,

1H, CH, aromatic, J=9.5 Hz), 7.05-7.07 (d, 1H, CH, aromatic, J=9.0 Hz), 7.47-7.54 (m, 4H,

4xCH, aromatic), 7.89-7.92 (m, 2H, 2xCH, aromatic), 7.97-7.99 (d, 1H, CH, aromatic, J=8.0

Hz), 12.66 (s, 1H, OH_,exchangeable); ¹³C NMR (CDCl₃, 125MHz): δ56.17, 60.80, 102.98,

116.04, 125.39, 126.16, 126.56, 127.19, 128.42, 128.63, 130.40, 130.57, 130.65, 133.58,

16

135.84, 136.59, 157.93, 159.08, 202.37; Electrospray mass for C₁₉H₁₆O₄(MeOH)- 309

[M+H]⁺, 331 [M+Na]⁺; 347 [M+K]⁺, 307 [M-H]^-.

5.2.2. Alternative synthesis of compound 3a:

In a cool mixture of dry pyridine and dichloromethane (2:1, 15mL), chromium trioxide

(300mg, 3 mmol) was stirred. To this compound 8 (200mg, 0.61 mmol) was added and

reaction mixture was stirred for an hour with cooling after that at RT for 8h. On completion

1mL methanol was added and stirred for 10 min to decompose excess of CrO₃. Reaction

mixture was evaporated and residue was taken in ethyl acetate (20mL), washed with water

(20mL), organic layer dried over anhydrous sodium sulphate and evaporated in vacuo.

Residue thus obtained was purified through silica gel column and eluted with ethyl acetate-

hexane to get desired product 3a at 12% EA-hexane as amorphous solid.

5.2.3. Synthesis of 2,3,4-trimethoxyphenyl-1-naphthylmethane (8)

To a stirred solution of compound 7 (241 mg, 1.4 mmol) in DMF-H₂O (3:2, 10mL), bis

triphenylphosphine palladium chloride (35 mg, 0.05 mmol) and potassium carbonate (345

mg, 2.5 mmol) were added. To this reaction mixture 2,3,4-trimethoxyphenylboronic acid (

212 mg, 1mmol) was added and the reaction mixture was refluxed for 4h. Reaction mixture

was diluted with water (10mL), extracted with ethyl acetate (10mLx3), and washed with

water. Organic layer was dried over anhydrous sodium sulphate, evaporated in-vacuo to get

a residue which was charged over silica gel column and eluted with ethyl acetate-hexane to

get the desired product 8.

1

8: Yield= 51%; Mp=106-108^oC, white amorphous solid; H NMR (CDCl₃, 300 MHz): δ3.86-

3.95(s, 9H, 3xOCH₃), 4.42 (s, 2H, CH₂), 6.52-6.55 (d, 1H, CH, aromatic, J=8.7Hz), 6.61-6.64

(d, 1H, CH, aromatic, J=8.4Hz), 7.25-7.17 (d, 1H, CH, aromatic, J=6.6Hz), 7.37-7.51 (m, 3H,

3xCH, aromatic), 7.75-7.78 (d, 1H, CH, aromatic, J=8.1Hz), 7.87-7.90 (dd, 1H, CH, aromatic,

13

J=7.5, 3.3 Hz), 8.06-8.09 (m, 1H, CH, aromatic); C NMR (CDCl₃, 75MHz): δ32.70, 56.38,

61.23, 61.29, 107.72, 124.65, 125.60, 126.30, 127.31, 127.33, 128.29, 129.04, 132.65, 134.30,

137.41, 142.76, 152.17, 152.67; Electrospray mass for C₂₀H₂₀O₃(MeOH)- 309[M+H]⁺,

331[M+Na]⁺; 347 [M+K]⁺.

5.2.4. General procedure for the syntheses of compounds 9-16 and 29 by Grignard reaction:

Synthesis of 1-Naphthalen-1-yl-1-(2,3,4-trimethoxyphenyl)-ethanol (9): Activated and dry

magnesium turning (150g, 6.3 mmol) and methyl iodide (0.4mL, 912mg, 6.42 mmol) were

stirred in dry diethyl ether (15mL). A small crystal of iodine was added to initial the

reaction. On disappearance of magnesium, compound 3a (200mg, 0.63 mmol), dissolved in

2mL THF) was added dropwise and stirred at RT for 30 min. Solvents were evaporated and

residue was diluted with water (10mL), and extracted with ethyl acetate (3x15mL). Organic

layer was dried over anhydrous sodium sulphate and evaporated to dryness under vacuum to

get a residue. Residue was charged on a silica gel column and eluted with hexane-ethyl

acetate to get desired product at 3% ethyl acetate-hexane as viscous oil.

1

9: Yield=87%; Yellowish viscous; H NMR (CDCl₃, 500 MHz): δ2.08 (s, 3H, CH₃), 3.09 (s, 3H,

OCH₃), 3.79 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 4.41 (s, 1H, exchangeable_,OH), 6.67-6.69 (d, 1H, CH,

aromatic, J=8.5 Hz), 7.18-7.19 (d, 1H, CH, aromatic, J=9.0 Hz), 7.28-7.44 (m, 3H, 3xCH, aromatic),

7.64-7.65 (d, 1H, CH, aromatic, J= 7.0 Hz), 7.74-7.76 (d, 1H, CH, aromatic, J= 8.0 Hz), 7.08-7.82 (d,

17

1H, CH, aromatic, J= 8.0 Hz), 8.36-8.38 (d, 1H, CH, aromatic, J= 8.5 Hz); ¹³C NMR (CDCl₃,

125MHz): δ29.52, 55.93, 60.03, 60.51, 106.53, 121.13, 123.70, 124.84, 124.92, 125.08, 126.83, 128.28,

128.77, 130.66, 134.24, 134.70, 142.73, 144.36, 151.52, 153.23; Electrospray mass (MeOH)-

361[M+Na]⁺, 377[M+K]⁺.

2-Methyl-1-naphthalen-1-yl-1-(2,3,4-trimethoxyphenyl)-propan-1-ol (10): Yield=78%; Yellowish

viscous; ¹H NMR (CDCl₃, 500 MHz): δ0.75-0.76 (d, 3H, CH_3,J= 7.0 Hz), 1.17-1.18 (d, 3H, CH_3,J= 6.5

Hz), 3.04 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.85 (s, 1H, CH), 3.88 (s, 3H, OCH₃), 5.08 (s, 1H,

exchangeable, OH), 6.72-6.74 (d, 1H, CH, aromatic, J=9.0 Hz), 7.28-7.44 (m, 4H, 4xCH, aromatic),

7.69-7.70 (d, 1H, CH, aromatic, J=9.0 Hz), 7.76-7.77 (d, 1H, CH, aromatic, J=7.5 Hz), 7.80-7.81 (d,

13

1H, CH, aromatic, J=8.0 Hz), 7.87 (s, br, 1H, CH, aromatic); C NMR (CDCl₃, 125MHz): δ17.43,

18.19, 35.09, 55.93, 60.31, 60.53, 80.48, 106.25, 121.57, 124.07, 124.80, 125.42, 125.81, 127.13,

127.42, 128.10, 132.03, 132.43, 133.00, 142.77, 146.17, 152.82; Electrospray mass (MeOH)- 389

[M+Na]⁺, 365 [M-H]^-.

1-Naphthalen-1-yl-1-(2,3,4-trimethoxyphenyl)-but-3-en-1-ol (11): Yield=73%; Yellowish gum; ¹H

NMR (CDCl₃, 500 MHz): δ3.00 (s, 3H, OCH_3,J= 7.0 Hz), 3.29-3.31 (d, 2H, CH_2,J= 6.0 Hz, olefinic),

3.77 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 4.24 (s, 1H, exchangeable, OH), 5.01-5.11 (m, 2H, CH_2,

olefinic), 5.78-5.84 (m, 1H, CH, olefinic), 6.68-6.69 (d, 1H, CH, aromatic, J=8.5 Hz), 7.28-7.29 (t, 2H,

2xCH, aromatic, J=3.5 Hz), 7.34-7.37 (m, 1H, CH, aromatic), 7.43-7.46 (d, 1H, CH, aromatic, J=7.5

Hz), 7.67-7.69 (d, 1H, CH, aromatic, J=7.0 Hz), 7.74-7.76 (d, 1H, CH, aromatic, J=8.5 Hz), 7.79-7.80

(d, 1H, CH, aromatic, J=7.0 Hz), 8.36-8.38 (d, 1H, CH, aromatic, J=9.0 Hz); ¹³C NMR (CDCl₃,

125MHz): δ45.23, 55.86, 59.91, 60.46, 78.00, 106.36, 117.88, 121.67, 124.58, 124.66, 124.84, 125.03,

126.74, 128.34, 128.75, 130.87, 132.92, 134.42, 134.70, 142.50, 151.59, 153.06; Electrospray mass

(MeOH)- 365 [M+H]⁺, 387 [M+Na]⁺, 403 [M+K]⁺.

Ethyl-3-Hydroxy-3-naphthalen-1-yl-3-(2,3,4-trimethoxyphenyl)-2-methyl-propionate (12): Yield= 68%;

1

Yellowish viscous; H NMR (CDCl₃, 500 MHz): δ1.15-1.17 (d, 3H, CH_3,J=7.0Hz), 1.29-1.32 (t, 3H,

CH_3,J=7.5Hz), 2.65 (s, 3H, OCH₃), 3.71 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 4.01-4.03 (q, 1H, CH of

CH_2,J=7.0 Hz,14.0Hz), 4.14-4.18 (m, 1H, CH), 4.25-4.27 (m, 1H, CH of CH₂), 5.44 (s, 1H,

exchangeable, OH), 6.77-6.79 (d, 1H, CH, aromatic, J=9.0Hz), 7.25-7.33 (m, 2H, 2xCH, aromatic),

7.40-7.43 (t, 1H, CH, aromatic, J=8.0Hz), 7.69-7.74 (m, 3H, 3xCH, aromatic), 7.90-7.92 (d, 1H, CH,

aromatic, J=8.5Hz), 8.61-8.63 (d, 1H, CH, aromatic, J=8.5Hz); ¹³C NMR (CDCl₃, 125MHz): δ13.96,

14.17, 44.80, 55.81, 59.27, 60.44, 60.88, 78.60, 106.24, 122.83, 124.07, 124.11, 124.85, 128.02, 128.41,

128.48, 129.73, 131.89, 134.86, 141.64, 142.64, 150.24, 152.82, 178.91; Electrospray mass (MeOH)-

447 [M+Na]⁺.

1-Naphthalen-1-yl-1-(2,3,4-trimethoxyphenyl)-4-phenyl-but-3-en-1-ol (13): Yield=74%; Yellowish

viscous.¹H NMR (CD₃OD, 500 MHz): δ2.61 (s, br, 3H, OCH₃), 3.67 (s, 6H, 2xOCH₃), 5.10-5.20 (dd,

2H, CH_2,J= 10.5 Hz, 12.0 Hz ), 6.36-6.37 (d, 1H, CH, olefinic, J=9.0 Hz), 6.65-6.68 (m, 1H, CH,

aromatic), 6.98-7.22 (m, 8H, 8xCH, aromatic), 7.25-7.28 (t, 1H, CH, aromatic, J= 8.0 Hz), 7.48-7.51 (t,

1H, CH, aromatic, J= 8.0 Hz), 7.74-7.77 (m, 2H, 2xCH, aromatic), 8.10 (bs, 1H, CH, aromatic), 8.54-

8.55 (d, 1H, CH, olefinic, J=8.5 Hz); ¹³C NMR (CD₃OD, 125MHz): δ56.42, 60.74, 79.58, 107.54,

117.69, 124.31, 125.40, 125.62, 125.77, 126.78, 126.89, 127.19, 127.50, 128.08, 128.29, 128.51,

128.65, 129.23, 129.42, 129.68, 131.34, 133.64, 136.53, 142.15, 142.38, 143.44, 144.84, 153.43,

153.95; Electrospray mass (MeOH)- 463[M+Na]⁺.

1-Naphthalen-1-yl-1-(2,3,4-trimethoxyphenyl)-2-phenylethanol (14): Yield=90%; Yellowish viscous;

¹H NMR (CDCl₃, 500 MHz): δ2.89 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.82 (s, 2H, CH_2,benzylic),

3.86 (s, 3H, OCH₃), 4.42 (s, 1H, exchangeable, OH), 6.63-6.65 (d, 1H, CH, aromatic, J=9Hz), 6.80-6.81

(d, 1H, CH, aromatic, J=7Hz), 7.10-7.14 (m, 4H, CH, aromatic), 7.28-7.43 (m, 4H, 4xCH, aromatic),

18

7.41-7.43 (d, 1H, CH, aromatic, J=8Hz), 7.67-7.69 (d, 1H, CH, aromatic, J=7.5Hz), 7.76-7.77 (d, 1H,

CH, aromatic, J=8.5Hz), 8.20-8.22 (d, 1H, CH, aromatic, J=8.5Hz); ¹³C NMR (CDCl₃, 125MHz):

δ55.89, 59.78, 60.46, 78.43, 106.62, 121.70, 124.47, 124.59, 124.77, 125.05, 126.17, 126.56, 127.45,

128.37, 128.77, 130.93, 131.14, 132.56, 134.64, 136.87, 142.37, 142.53, 151.62, 152.87; Electrospray

mass (MeOH)- 437 [M+Na]⁺, 453 [M+K]⁺.

1-Naphthalen-1-yl-1-(2,3,4-trimethoxyphenyl)-2-(4-tolyl)-ethanol (15): Yield=84%; Yellowish viscous;

¹H NMR (CDCl₃, 500 MHz): δ2.25 (s, 3H, CH₃), 2.86 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 3.77 (s, 2H,

CH_2,benzylic), 3.84 (s, 3H, OCH₃), 4.32 (s, 1H, exchangeable, OH), 6.61-6.66 (m, 3H, 3xCH,

aromatic), 6.89-6.91 (d, 2H, 2xCH, aromatic, J=8Hz), 7.12-7.13 (d, 1H, CH, aromatic, J=9Hz), 7.21-

7.24 (m, 1H, CH, aromatic), 7.32-7.42 (m, 2H, 2xCH, aromatic), 7.66-7.68 (d, 1H, CH, aromatic,

J=7.5Hz), 7.74-7.75 (d, 1H, CH, aromatic, J=8Hz), 7.78-7.80 (d, 1H, CH, aromatic, J=8Hz), 8.19-8.21

13

(d, 1H, CH, aromatic, J=9Hz); C NMR (CDCl₃, 125MHz): δ21.03, 46.71, 55.88, 59.75, 60.46, 78.27,

106.53, 121.75, 124.54, 124.62, 124.74, 125.02, 126.60, 128.15, 128.22, 128.31, 128.74, 130.96,

132.64, 133.56, 134.62, 135.64, 142.38, 142.51, 151.61, 152.83; Electrospray mass (MeOH)- 451

[M+Na]⁺, 467 [M+K]⁺.

2-(4-Fluorophenyl)-1-naphthalen-1-yl-1-(2,3,4-trimethoxyhenyl)-ethanol (16): Yield=77%; Yellowish

1

viscous; H NMR (CDCl₃, 500 MHz): δ2.86 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 3.78 (s, 2H, CH_2,

benzylic), 3.85 (s, 3H, OCH₃), 4.31 (s, 1H, exchangeable, OH), 6.63-6.65 (d, 1H, CH, aromatic,

J=9Hz), 6.69-6.98 (m, 4H, 4xCH, aromatic), 7.15-7.25 (m, 2H, 2xCH, aromatic), 7.33-7.39 (m, 2H,

2xCH, aromatic), 7.59-7.61 (d, 1H, CH, aromatic, J=7.5Hz), 7.73-7.74 (d, 1H, CH, aromatic, J=8Hz),

7.79-7.80 (d, 1H, CH, aromatic, J=8Hz), 8.14-8.16 (d, 1H, CH, aromatic, J=9Hz); ¹³C NMR (CDCl₃,

125MHz): δ46.04, 55.90, 59.79, 60.46, 78.17, 106.65, 114.02, 114.19, 121.47, 124.52, 124.63, 124.80,

125.14, 126.26, 130.54, 130.74, 132.35, 132.41, 132.49, 132.66, 134.57, 142.18, 142.61, 151.60,

152.99, 160.68, 162.62 Electrospray mass (MeOH)- 455 [M+Na]⁺, 471 [M+K]⁺.

Naphthalen-1-yl-(2,3,4-trimethoxy-phenyl)-methanol (29): Yield=86%; Mp=108-110^oC, White

amorphous solid. ¹H NMR (CDCl₃, 500 MHz): δ3.80 (s, 3H, OCH₃), 3.90-3.92 (s, 6H, 2xOCH₃), 6.51-

6.53 (d, 1H, CH, aromatic, J=9.0Hz), 6.70-6.71 (d, 1H, CH, aromatic, J=8.50Hz), 6.79 (s, 1H, CH),

7.42-7.51 (m, 3H, 3xCH, aromatic), 7.67-7.68 (d, 1H, CH, aromatic, J=7.0 Hz), 7.79-7.81 (d, 1H, CH,

aromatic, J=8.5 Hz), 7.85-7.87 (dd, 1H, CH, aromatic, J=2.5Hz, 6.5Hz), 7.99-8.01 (dd, 1H, CH,

aromatic, J=2.5Hz, 6.0Hz); ¹³C NMR (CDCl₃, 125MHz): δ55.90, 60.77, 61.24, 68.35, 107.07, 122.63,

123.96, 124.11, 125.29, 125.46, 125.98, 128.09, 128.63, 129.17, 130.78, 133.74, 138.55, 142.01,

151.50, 153.48; Electrospray mass (MeOH)- 347 [M+Na]⁺; 363 [M+K]⁺.

5.2.5. General procedure for the synthesis of compound 21-22:

Synthesis of (Z/E)1-[2-(4-Fluorophenyl)-1-(2,3,4-trimethoxyphenyl)-vinyl]-naphthalene (21 & 22):

Alcohol 16 (150mg, 0.36 mmol) was taken in methanol (5mL). To this stirred solution 2-3

drops Conc. hydrochloric acid was added and reaction mixture was refluxed for 2h. Solvent

was evaporated and water (10mL) was added to it. It was extracted with ethyl acetate

(10mLx3), washed with water, dried over anhydrous sodium sulphate and evaporated in

vacuo. The crude mass was charged on a silica gel column and carefully eluted with ethyl

acetate-hexane to get compound 21 and 22 as pure isomers.

(Z)1-[2-(4-Fluorophenyl)-1-(2,3,4-trimethoxyphenyl)-vinyl]-naphthalene

(21): Yield=46%; Yellowish

1

viscous; H NMR (CDCl₃, 500 MHz): δ 3.43 (s, 3H, OCH₃), 3.97 (s, 3H, OCH₃), 3.99 (s, 3H, OCH₃),

6.74-6.80 (m, 3H, 3xCH, aromatic), 6.93-6.95 (m, 2H, 2xCH, aromatic), 7.12-7.13 (d, 1H, CH,

aromatic, J=8.5Hz), 7.40 (s, 1H, CH, olefinic, Cis-diphenyl), 7.42-7.48 (m, 2H, 2xCH, aromatic), 7.53-

7.58 (m, 2H, 2xCH, aromatic), 7.93-7.95 (d, 1H, CH, aromatic, J=8.5Hz), 7.96-7.98 (d, 1H, CH,

19

aromatic, J=8.5Hz), 8.08-8.10 (d, 1H, CH, aromatic, J=8.5Hz); ¹³C NMR (CDCl₃, 125MHz): δ 55.95,

60.03, 60.68, 106.93, 114.67, 114.84, 124.71, 125.63, 125.70, 125.92, 126.18, 127.62, 128.18, 130.48,

130.54, 131.31, 131.37, 133.52, 133.86, 137.41, 137.42, 138.90, 142.66, 151.98, 153.35, 160.37-

162.33 (d, J_C,F=245Hz); Electrospray mass (MeOH): 415[M+H]⁺.

(E)1-[2-(4-Fluorophenyl)-1-(2,3,4-trimethoxyphenyl)-vinyl]-naphthalene (22): Yield=26%; Yellowish

1

viscous; H NMR (CDCl₃, 500 MHz): δ 3.21 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃),

6.59-6.60 (d, 1H, CH, aromatic, J=8.5Hz), 6.74 (s, 1H, CH, olefinic, Trans diphenyl), 6.86-6.93 (m, 3H,

3xCH, aromatic), 7.09-7.11 (m, 2H, 2xCH, aromatic), 7.32-7.46 (m, 4H, 4xCH, aromatic), 7.74-7.76

(dd, 1H, CH, aromatic, J=8Hz & 1.5Hz), 7.84-7.86 (dd, 1H, CH, aromatic, J=8Hz & 2Hz), 8.39-8.41(d,

1H, CH, aromatic, J=8Hz); ¹³C NMR (CDCl₃, 125MHz): δ 55.91, 59.97, 60.74, 107.23, 114.88, 115.05,

125.03, 125.49, 125.55, 125.76, 126.21, 126.30, 127.39, 128.07, 128.26, 130.61, 130.67, 131.76,

133.69, 134.13, 137.24, 142.33, 142.81, 151.85, 153.58, 160.59-162.55 (d, J_C,F=245Hz); Electrospray

mass (MeOH): 415 [M+H]⁺, Negative mode: 413 [M-H]^-.

5.2.6. Synthesis of Naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methanone oxime (23):

Naphthophenone 3a (200mg, 0.62 mmol) was taken in ethanol (8mL). To this stirred solution

DMAP (18mg, 0.15mmol) and hydroxylamine hydrochloride (207 mg, 3 mmol) were added and

reaction mixture was refluxed for 18h. Solvent was evaporated and water (10mL) was added to it. It

was extracted with ethyl acetate (15mLx3), washed with water and dried over anhydrous sodium

sulphate. Organic solvent was dried in vacuo and residue was charged on a silica gel column and

eluted with chloroform-acetone to get oxime derivative 23 as amorphous solid after recrystallization

with chloroform-hexane (1:3).

1

23: Yield=87%; Mp=175-178 ^oC, White solid; H NMR (CDCl₃, 500 MHz): δ3.13 (s, 3H, OCH₃), 3.75

(s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 6.58-6.60 (d, 1H, CH, aromatic, J=9Hz), 7.16-7.17 (d, 1H, CH,

aromatic, J=9Hz), 7.29-7.30 (d, 1H, CH, aromatic, J=1Hz), 7.30-7.53 (m, 3H, 3xCH, aromatic), 7.86-

7.98 (m, 3H, 3xCH, aromatic), 9.47 (s, 1H, exchangeable, N-OH); ¹³C NMR (CDCl₃, 125MHz): δ

55.99, 60.18, 60.69, 106.98, 124.27, 124.92, 124.98, 125.48, 125.99, 126.21, 126.95, 128.21, 128.96,

130.18, 133.29, 133.79, 142.60, 152.71, 154.97, 155.97; Electrospray mass (MeOH): 338 [M+H]⁺, 360

[M+Na]⁺; HRMS (MeOH): m/z [M+H]⁺calcd for C₂₀H₂₀NO₄, 338.1392, found 338.1389.

5.2.7. Synthesis of naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methanone oxime acetate (24):

Oxime 23 was acetylated using acetic anhydride, DMAP in dry chloroform at RT as per reported

method.

1

24: Yield=86%. Mp=104-105 ^oC, White crystalline solid; H NMR (CDCl₃, 500 MHz): δ 1.79 (s, 3H,

CH₃), 3.05 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 6.69-6.71 (d, 1H, CH, aromatic,

J=9Hz), 7.21-7.22 (d, 1H, CH, aromatic, J=7Hz), 7.38-7.51 (m, 4H, 4xCH, aromatic), 7.82-7.86 (m, 3H,

13

3xCH, aromatic); C NMR (CDCl₃, 125MHz): δ19.46, 56.07, 60.28, 60.67, 106.97, 123.09, 124.62,

125.08, 125.58, 126.09, 126.13, 126.56, 128.25, 129.46, 129.82, 133.10, 133.50, 142.60, 153.15,

156.10, 163.74, 168.58; Electrospray mass (MeOH)- 380 [M+H]⁺, 402 [M+Na]⁺, 418 [M+K]⁺; HRMS

(MeOH): m/z [M+H]⁺calcd for C₂₂H₂₂NO₅, 380.1497, found 380.1539.

5.2.8. General procedure for the synthesis of compounds 25-27 and 47:

Synthesis of Ethyl-naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyleneaminooxy]-acetate (25):

Ketoxime 23 (100mg, 0.30 mmol) was stirred in dry THF (10mL). To this sodium hydride (60mg,

2.5mmol) and ethylbromoacetate (100mg, 0.6mmol) were added and reaction mixture was refluxed

for 2h. On completion, reaction mixture was acidified with dil. HCl (5%, 5mL), extracted with ethyl

acetate (10mLx3) and washed with water (15mLx2). The organic layer was dried over anhydrous

sodium sulphate and evaporated in vacuo. The crude mass thus obtained was purified through

20

column chromatography over silica gel using chloroform-hexane to get ester 25 at 80% chloroform-

hexane as amorphous solid.

25: Yield=92%; Mp= 84-85 ^oC, White crystalline Solid; ¹H NMR (CDCl₃, 500 MHz): δ1.28-1.31 (t, 3H,

CH_3,J= 7.0 Hz), 3.12 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 4.20-4.24 (q, 2H, CH_2,J=

7.0 Hz, 14Hz ), 4.62 (s, 2H, CH₂), 6.66-6.68 (d, 1H, CH, aromatic, J=8.5 Hz), 7.24-7.53 (m, 5H, 5xCH,

aromatic), 7.83-7.86 (m, 2H, 2xCH, aromatic), 8.08-8.09 (d, 1H, CH, aromatic, J= 8.0 Hz,). ¹³C NMR

(CDCl₃, 125MHz): δ 14.24, 56.04, 60.30, 60.68, 60.79, 107.06, 124.35, 124.83, 124.96, 125.40, 125.93,

126.08, 127.19, 128.02, 130.13, 133.21, 133.92, 142.72, 152.78, 155.14, 156.64, 170.05; Electrospray

mass (MeOH)- 424 [M+H]⁺, 446 [M+Na]⁺, 418 [M+K]⁺; HRMS (MeOH): m/z [M+H]⁺calcd for

C₂₄H₂₆NO₆, 424.1760, found 424.1757.

Ethyl-3-[Naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyleneaminooxy]-propionate (26): Yield=87%;

1

Yellowish viscous; H NMR (CDCl₃, 500 MHz): δ1.12-1.52 (t, 3H, CH₃, J=7.5Hz), 2.65-2.67 (t, 2H,

CH₂, J=6.5Hz), 3.08 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 4.01-4.02 (t, 2H, CH₂,

J=7Hz), 4.36-4.39 (q, 2H, CH₂, J=7Hz), 6.67-6.69 (d, 1H, CH, aromatic, J=8.5Hz), 7.20-7.24 (m, 2H,

2xCH, aromatic), 7.40-7.48 (m, 3H, 3xCH, aromatic), 7.84-7.89 (m, 3H, 3xCH, aromatic); ¹³C NMR

(CDCl₃, 125MHz): δ14.08, 34.86, 56.04, 60.19, 60.38, 60.64, 69.72, 107.02, 124.74, 124.80, 125.18,

125.78, 125.97, 126.90, 128.05, 128.71, 130.09, 133.17, 134.36, 142.68, 152.73, 155.02, 155.64,

171.30; Electrospray mass for C₂₅H₂₇NO₆, (MeOH) 460 [M+Na]⁺, 476 [M+K]⁺.

Ethyl 4-[naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyleneaminooxy]-but-2-enoate (27): Yield=86%;

Yellowish viscous; ¹H NMR (CDCl₃, 500 MHz): δ1.27-1.29 (t, 3H, CH_3,J= 7.0Hz), 3.12 (s, 3H, OCH₃),

3.77 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 4.15-4.20 (q, 2H, CH_2,J= 7.17 Hz), 4.75-4.77 (dd, 2H, CH_2,J=

2.0Hz, 5.0Hz), 5.82-5.85 (dd, 1H, CH, olefinic, J=14.0Hz, 16.0Hz), 6.67-6.68 (d, 1H, CH, aromatic,

J=8.5Hz), 6.96-6.99 (d, 1H, CH, olefinic, J=16.0 Hz), 7.22-7.85 (m, 8H, 8xCH, aromatic); ¹³C NMR

(CDCl₃, 125MHz): δ14.19, 55.94, 60.23, 60.27, 60.60, 72.74, 107.05, 121.93, 124.33, 124.79, 124.81,

125.09, 125.88, 126.10, 126.72, 128.11, 128.80, 130.00, 133.16, 134.14, 142.71, 143.92, 152.69,

155.08, 156.24, 166.13; Electrospray mass (MeOH)- 450 [M+H]⁺, 472 [M+Na]⁺, 488 [M+K]⁺; HRMS

(MeOH): m/z [M+H]⁺, calcd for C₂₆H₂₈NO₆, 450.1916, found 450.1911.

Ethyl-2-[4-(naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyl)]-phenoxy]-ethanoate (47): Yield=84%;

1

Yellowish viscous; H NMR (CDCl₃, 500 MHz): δ1.29-1.30 (t, 3H, CH₃, J=3.5Hz), 3.47(s, 3H, OCH₃),

3.80 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 4.24-4.28 (q, 2H, OCH₂, J=7.17Hz), 4.58 (s, 2H, 2H, OCH₂),

6.43-6.44 (d, 3H, 2xCH, aromatic, J=8.5Hz), 6.50-6.51 (d, 2H, 2xCH, aromatic, J=8.5Hz), 6.50 (s, 1H,

1H, CH), 6.81-6.83 (d, 2H, 2xCH, aromatic, J=9.0Hz), 6.94-6.96 (d, 1H, CH, aromatic, J=7.0Hz), 7.03-

7.05 (d, 2H, 2xCH, aromatic, J=8.50Hz), 7.33-7.44 (m, 3H, 3xCH, aromatic), 7.71-7.73 (d, 1H, CH,

aromatic, J=8.0Hz), 7.82-7.84 (d, 1H, CH, aromatic, J=7.5Hz), 7.98-7.99 (d, 1H, CH, aromatic,

J=8.0Hz); ¹³C NMR (CDCl₃, 125MHz): δ14.16, 45.38, 55.84, 60.64, 60.73, 61.32, 65.57, 106.66,

114.51, 124.34, 124.45, 125.08, 125.41, 126.03, 127.03, 127.13, 128.58, 130.50, 130.62, 131.84,

133.94, 137.13, 140.41, 142.40, 151.40, 152.40, 156.33, 169.03; ESI-MS (MeOH) for C₃₀H₃₀O₆: 487

[M+H]⁺, 509 [M+Na]⁺, 525 [M+K]⁺.

5.2.9. General procedure for the synthesis of compounds 28 and 49:

Synthesis of [Naphthalen-1-yl-(2,3,4-trimethoxy-phenyl)-methyleneaminooxy]-acetic acid (28):

Ester 25 (100mg, 0.24 mmol) was taken in 6% KOH in MeOH:Water (9:1) (5mL). It was refluxed

for 2h. Reaction mixture was acidified with dil. HCl (5%, 5mL), extracted with ethyl acetate

(10mLx3), washed with water (15mLx2) and dried over anhydrous sodium sulphate. The organic

layer was evaporated under vacuum and residue was purified through chromatography eluting with

chloroform-acetone to get acid 28 at 5% acetone-chloroform and recrystallized with

chloroform:pentane (1:3) as amorphous solid.

21

28: Yield=66%; Mp= 103-104^oC, White solid; ¹H NMR (CDCl₃, 500 MHz): δ2.96 (s, 3H, OCH₃), 3.60

(s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 4.47 (s, 2H, CH₂), 6.64-6.66 (d, 1H, CH, aromatic, J=8.5Hz), 7.10-

7.12 (d, 1H, CH, aromatic, J=8.5Hz), 7.15-7.16 (d, 1H, CH, aromatic, J=7.0Hz), 7.31-7.37(m, 3H,

3xCH, aromatic), 7.73-7.77 (m, 2H, 2xCH, aromatic), 7.90-7.92 (d, 1H, CH, aromatic, J=8.5Hz); ¹³C

NMR (CDCl₃, 125MHz): δ59.15, 63.47, 63.57, 74.09, 111.05, 128.16, 128.39, 128.82, 129.19, 129.44,

129.47, 130.88, 131.54, 132.36, 134.10, 137.32, 137.94, 146.43, 156.32, 159.12, 160.36; Electrospray

mass (MeOH): Positive mode: 396 [M+H]⁺, 418 [M+Na]⁺, negative mode: 394 [M-H]^-; HRMS

(MeOH): m/z [M+H]⁺, calcd for C₂₂H₂₂NO₆, 396.1447, found 396.1441.

2-[4-(naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyl)]-phenoxy]-ethanoic acid (49): Yield=72%;

1

Yellowish viscous; H NMR (CDCl₃, 500 MHz): δ3.48 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.88 (s, 3H,

OCH₃), 4.62 (s, 2H, OCH₂), 6.43-6.51 (m, 3H, 3xCH, aromatic), 6.50-6.51 (d, 2H, 2xCH, aromatic,

J=4.5Hz), 6.82-6.84 (d, 1H, CH, aromatic, J= 8.5 Hz), 6.94-6.95 (d, 1H, CH, aromatic, J=7.0 Hz), 7.04-

7.06 (d, 1H, CH, aromatic, J=8.5 Hz), 7.32-7.44 (m, 3H, 3xCH, aromatic), 7.71-7.73 (d, 1H, CH,

aromatic, J=8.0 Hz), 7.83-7.84 (d, 1H, CH, aromatic, J=7.5 Hz), 7.97-7.99 (d, 1H, 1xCH, aromatic,

J=8.0 Hz); ¹³C NMR (CDCl₃, 125MHz): δ45.36, 55.85, 60.68, 60.75, 65.05, 106.71, 114.51, 124.30,

124.47, 125.10, 125.45, 126.07, 127.02, 127.18, 128.61, 130.41, 130.73, 131.81, 133.95, 137.47,

140.31, 142.36, 151.35, 152.40, 155.94, 173.28; Electrospray mass for C₂₈H₂₇O₆(MeOH):

481[M+Na]⁺; Negative mode: 457[M-H]^-.

5.2.10. Synthesis of 4-[Naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyl]-phenol (30) and 2-

[Naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyl]-phenol (31):

Alcohol 29 (200mg, 0.62 mmol) was taken in a solution of dry benzene (12mL) and Conc. HCl

(0.2mL). To this stirred reaction mixture phenol (71mg, 0.75 mmol) was added and further stirred at

RT for 6h. Water (20 mL) was added to it, extracted with ethyl acetate (20mLx3), and washed with

water. Organic layer was dried over anhydrous sodium sulphate and dried in vacuo. The residue thus

obtained was purified through column chromatography over silica gel eluting with ethyl acetate-

hexane to get phenolic derivatives 30 and 31 at 6% and 8% ethyl acetate-hexane respectively as

amorphous solids.

1

30: Yield=48%; Mp=168-170^oC, White crystalline solid; H NMR (CDCl₃, 500 MHz): δ 3.48 (s, 3H,

OCH₃), 3.80 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 5.17 (s, 1H, OH_,exchangeable), 6.44-6.52 (m, 3H,

3xCH, aromatic), 6.73-6.74 (d, 2H, 2xCH, aromatic, J=8.5Hz), 6.95 (s, 1H, CH), 6.97-6.99 (d, 1H, CH,

aromatic, J=8.50Hz), 7.33-7.44 (m, 3H, 3xCH, aromatic), 7.71-7.73 (d, 1H, CH, aromatic, J=9.0Hz),

7.82-7.84 (d, 1H, CH, aromatic, J=7.5Hz), 7.98-8.00 (d, 1H, CH, aromatic, J=8.0Hz); ¹³C NMR

(CDCl₃, 125MHz): δ 45.38, 55.85, 60.70, 60.77, 106.71, 115.20, 120.65, 124.37, 124.51, 125.09,

125.42, 126.03, 127.00, 127.11, 128.58, 129.65, 130.69, 130.72, 131.85, 133.94, 135.97, 140.54,

142.35, 151.35, 152.32, 154.04; Electrospray mass for C₂₆H₂₄O₄(MeOH)- 423[M+Na]⁺; 439[M+K]⁺.

1

31: Yield=26%; Mp=119-120^oC, White amorphous solid; H NMR (CDCl₃, 500 MHz): δ 3.53 (s, 3H,

OCH₃), 3.81 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 5.21 (s, 1H, OH_,exchangeable), 6.55 (s, 2H, 2.xCH,

aromatic), 6.73 (s, 1H, CH, aromatic), 6.77-6.84 (m, 3H, 3xCH, aromatic), 7.04-7.06 (d, 1H, CH,

aromatic, J=7.5Hz), 7.11-7.14 (m, 1H, CH, aromatic), 7.35-7.45 (m, 3H, 3xCH, aromatic), 7.75-7.77 (d,

1H, CH, aromatic, J=8.0Hz), 7.84-7.86 (d, 1H, CH, aromatic, J=8.0Hz), 7.99-8.00 (d, 1H, CH,

aromatic, J=3.5Hz); ¹³C NMR (CDCl₃, 125MHz): δ 40.12, 55.86, 60.74, 60.80, 106.93, 115.96, 120.61,

124.30, 124.45, 125.18, 125.57, 126.22, 126.58, 127.53, 127.80, 128.85, 129.63, 130.08, 130.48,

131.87, 134.09, 138.90, 142.52, 151.54, 152.65, 153.36; Electrospray mass (MeOH)- 423[M+Na]⁺;

439[M+K]⁺, 399[M-H]^-; HRMS (MeOH): m/z [M+H]⁺, calcd for C₂₆H₂₅O₄, 401.1752, found 401.1743.

5.2.11. General procedure for the synthesis of compounds 32-40:

Synthesis of Methoxy-1-[naphthalen-1-yl- 1-(2,3,4-trimethoxyphenyl)-methane (32):

22

Alcohol 29 (100mg, 0.31 mmol) was stirred in dry THF (10mL) and sodium hydride (40mg, 1.67

mmol) was added to it. After 10 min methyl iodide (0.1mL, 228mg, 1.61 mmol) was added and

reaction mixture was refluxed for an hour. It was acidified with dil. HCl (5%, 5mL), extracted with

ethyl acetate (10mLx3), and washed with water (10mLx2). Organic layer was dried over anhydrous

sodium sulphate and evaporated in rotavapour. The residue was purified through a filter column

eluting with ethyl acetate-hexane to get compound 32 as amorphous solid.

1

32: Yield=77%; yellowish viscous; H NMR (CDCl₃, 500 MHz): δ3.50 (s, 3H, OCH₃), 3.80 (s, 3H,

OCH₃), 3.88 (s, 3H, OCH₃), 3.90 (s, 3H, OCH₃), 6.34 (s, 1H, CH), 6.56-6.57(d, 1H, CH, aromatic,

J=9.0Hz), 6.85-6.87 (d, 1H, CH, aromatic, J=8.5Hz), 7.43-7.52 (m, 3H, 3xCH, aromatic), 7.64-7.66 (d,

1H, CH, aromatic, J=7.0 Hz), 7.78-7.80 (d, 1H, CH, aromatic, J=13.5Hz), 7.83-7.86 (m, 1H, CH,

13

aromatic), 8.02-8.04 (m, 1H, CH, aromatic); C NMR (CDCl₃, 125MHz): δ55.92, 57.50, 60.78, 61.17,

76.79, 107.27, 122.84, 123.76, 124.30, 125.31, 125.44, 125.99, 127.42, 127.99, 128.66, 131.33, 133.87,

136.96, 142.05, 151.85, 153.35; Electrospray mass (MeOH)- 361[M+Na]⁺; 377[M+K]⁺.

1

Ethoxy-1-[naphthalen-1-yl-1-(2,3,4-trimethoxyphenyl)-methane (33):Yield=76%; Yellow viscous; H

NMR (CDCl₃, 500 MHz): δ 1.31-1.33 (t, 3H, CH₃, J=7.0Hz), 3.66-3.71 (q, 2H, O-CH₂, J=7.0, 14.0Hz),

3.80 (s, 3H, OCH₃), 3.90 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 6.47 (s, 1H, CH), 6.55-6.57 (d, 1H, CH,

aromatic, J=9.0Hz), 6.86-6.87 (d, 1H, CH, aromatic, J=9.0Hz), 7.43-7.52 (m, 3H, 3xCH, aromatic),

7.67-7.69 (d, 1H, CH, aromatic, J=7.0Hz), 7.79-7.80 (d, 1H, CH, aromatic, J=8.5Hz), 7.83-7.86 (m, 1H,

CH, aromatic), 8.04-8.06 (m, 1H, CH, aromatic); ¹³C NMR (CDCl₃, 125MHz): δ 15.60, 55.92, 60.79,

61.16, 65.18, 74.47, 107.30, 122.98, 123.83, 124.42, 125.34, 125.42, 125.94, 127.86, 127.94, 128.65,

131.37, 133.89, 137.43, 142.09, 151.82, 153.30; Electrospray mass (MeOH)- 375[M+Na]⁺; 391[M+K]⁺.

Propoxy-1-[naphthalen-1-yl-1-(2,3,4-trimethoxyphenyl)-methane (34):Yield=81%; Yellowish viscous;

¹H NMR (CDCl₃, 500 MHz): δ0.95-0.98 (t, 3H, CH₃, J=7.5Hz), 1.70-1.74 (m, 2H, CH₂), 3.56-3.58 (t,

2H, OCH₂, J=6.5Hz), 3.81 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 3.90 (s, 3H, OCH₃), 6.44 (s, 1H, CH),

6.57-6.58 (d, 1H, CH, aromatic, J=8.5Hz), 6.89-6.91(d, 1H, CH, aromatic, J=8.5Hz), 7.43-7.51 (m, 3H,

3xCH, aromatic), 7.65-7.67 (d, 1H, CH, aromatic, J=7.0Hz), 7.78-7.79 (d, 1H, CH, aromatic, J=8.0Hz),

7.84-7.86 (m, 1H, CH, aromatic), 8.06-8.08 (d, 1H, CH, aromatic, J=8.5Hz); ¹³C NMR (CDCl₃,

125MHz): δ 10.87, 23.33, 55.91, 60.76, 61.11, 71.61, 74.58, 107.29, 122.99, 123.92, 124.54, 125.30,

125.39, 125.89, 127.86, 127.90, 128.62, 131.42, 133.88, 137.63, 142.06, 151.81, 153.24; Electrospray

mass (MeOH)- 389[M+Na]⁺; 405[M+K]⁺.

Allyloxy-1-[naphthalen-1-yl-1-(2,3,4-trimethoxyphenyl)-methane (35): Yield=86%; Yellowish viscous;

¹H NMR (CDCl₃, 500 MHz): δ3.80 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 3.90(s, 3H, OCH₃), 4.13-4.16

(m, 2H, CH₂), 5.19-5.21 (dd, 1H, CH of =CH₂, J=1.5, 10.5Hz), 5.29-5.33 (dd, 1H, CH of =CH₂, J=1.5,

17.5Hz), 6.05-6.06 (m, 1H, CH, olefinic), 6.31 (s, 1H, CH), 6.53 (s, 1H, CH), 6.56-6.58 (d, 1H, CH,

aromatic, J=8.5Hz), 6.90-6.92 (d, 1H, CH, aromatic, J=9.0Hz), 7.44-7.52 (m, 3H, 3xCH, aromatic),

7.70-7.71 (d, 1H, CH, aromatic, J=7.0Hz), 7.79-7.80 (d, 1H, CH, aromatic, J=8.5Hz), 7.84-7.86 (m, 1H,

CH, aromatic, J=7.0Hz), 8.06-8.08 (m, 1H, CH, aromatic, J=7.0Hz); ¹³C NMR (CDCl₃, 125MHz):

δ55.92, 60.75, 61.13, 70.57, 73.92, 107.26, 117.03, 123.05, 123.82, 124.62, 125.34, 125.42, 125.95,

127.58, 127.99, 128.65, 131.33, 133.87, 134.98, 137.20, 142.05, 151.79, 153.34; Electrospray mass for

C₂₃H₂₄O₄(MeOH)- 387[M+Na]⁺; 403[M+K]⁺.

2-[(Naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methoxy]-N,N-dimethyethanamine (36): Yield= 67%;

1

Brown-viscous; H NMR (CDCl₃, 500 MHz): δ2.31 (s, 6H, N(CH₃)₂), 2.68-2.70 (t, 2H, N-CH_2,

J=5.5Hz), 3.71-3.73 (t, 2H, OCH₂, J=5.0Hz), 3.82 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 3.90 (s, 3H,

OCH₃), 6.43 (s, 1H, CH), 6.54-6.56 (d, 1H, CH, aromatic, J=8.5Hz), 6.87-6.89(d, 1H, CH, aromatic,

J=9.0Hz), 7.43-7.49 (m, 3H, 3xCH, aromatic), 7.64-7.65 (d, 1H, CH, aromatic, J=7.0Hz), 7.76-7.78 (d,

1H, CH, aromatic, J=8.0Hz), 7.83-7.84 (d, 1H, CH, aromatic, J=6.0Hz), 8.04-8.05 (d, 1H, CH,

23

aromatic, J=5.0Hz). ¹³C NMR (CDCl₃, 125MHz): δ45.62, 55.91, 58.73, 60.72, 61.06, 67.36, 74.97,

107.24, 122.99, 123.79, 124.56, 125.29, 125.72, 125.94, 126.37, 127.61, 127.99, 128.63, 131.26,

133.83, 137.23, 141.95, 151.74, 153.32; Electrospray mass (MeOH)- 396[M+H]⁺.

1-Benzyloxy-1-[naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methane (37): Yield=83%; Brown-viscous;

¹H NMR (CDCl₃, 500 MHz): δ3.76 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 4.63-4.70 (

q, 2H, OCH₂, benzylic, J=12.2Hz), 6.59-6.60 (d, 2H, 2xCH, J=7.0Hz), 7.00-7.01(d, 1H, CH, aromatic,

J=9.0Hz), 7.29-7.53 (m, 8H, 8xCH, aromatic), 7.74-7.75 (d, 1H, CH, aromatic, J=7.0Hz), 7.80-7.81 (d,

1H, CH, aromatic, J=8.5Hz), 7.85-7.87 (m, 1H, CH, aromatic), 8.04-8.06 (m, 1H, CH, aromatic); ¹³C

NMR (CDCl₃, 125MHz): δ 55.93, 60.75, 61.02, 71.42, 73.99, 107.29, 123.11, 123.89, 124.80, 125.34,

125.45, 125.96, 127.47, 127.58, 128.06, 128.09, 128.33, 128.64, 131.41, 133.89, 137.27, 138.44,

142.02, 151.81, 153.33; Electrospray mass (MeOH)- 437[M+Na]⁺; 453[M+K]⁺.

1-(4-methylbenzyloxy)-1-[naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methane (38):Yield=83%; Yellow

viscous; ¹H NMR (CDCl₃, 500 MHz): δ2.35 (s, 3H, CH₃), 3.75 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 3.88

(s, 3H, OCH₃), 4.57-4.65 (m, 2H, OCH₂, benzylic, J=12.5Hz), 6.57-6.59 (m, 2H, 2xCH, aromatic & one

CH), 6.98-7.00 (d, 1H, CH, aromatic, J=10Hz), 7.14-7.18 (m, 2H, 2xCH, aromatic), 7.26-7.28 (m, 2H,

2xCH, aromatic), 7.42-7.52 (m, 3H, 3xCH, aromatic), 7.71-7.73 (d, 1H, CH, aromatic, J=7.0Hz), 7.79-

7.80 (d, 1H, CH, aromatic, J=8.0Hz), 7.84-7.86 (m, 1H, CH, aromatic), 8.02-8.04 (m, 1H, CH,

aromatic). ¹³C NMR (CDCl₃, 125MHz): δ21.17, 55.92, 60.73, 61.01, 71.27, 73.83, 107.27, 123.12,

123.92, 124.82, 125.32, 125.41, 125.91, 127.11, 127.55, 128.00, 128.19, 128.61, 128.98, 129.24,

131.43, 133.88, 135.38, 137.24, 137.34, 142.02, 151.81, 153.28; Electrospray mass (MeOH)-

451[M+Na]⁺; 467[M+K]⁺.

1-(3,5-dimethoxybenzyloxy)-1-[naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methane (39): Yield=81%;

1

Yellow viscous; H NMR (CDCl₃, 500 MHz): δ3.74(s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.82 (s, 3H,

OCH₃), 3.89 (s, 6H, 2xOCH₃), 4.58-4.64 (q, 2H, OCH₂, benzylic J=10.8Hz), 6.40-6.41 (m, 1H, CH),

6.55-6.61(m, 4H, 4xCH, aromatic), 7.02-7.04 (d, 1H, CH, aromatic, J=9.0Hz), 7.44-7.52 (m, 3H, 3xCH,

aromatic), 7.71-7.73 (d, 1H, CH, aromatic, J=7.0Hz), 7.79-7.81 (d, 1H, CH, aromatic, J=8.5Hz), 7.85-

7.86 (m, 1H, CH, aromatic), 8.06-8.08 (m, 1H, CH, aromatic); ¹³C NMR (CDCl₃, 125MHz): δ55.30,

55.94, 60.73, 60.99, 71.38, 74.06, 99.75, 105.82, 107.27, 123.08, 123.94, 124.92, 125.34, 125.45,

125.94, 127.38, 128.09, 128.65, 131.40, 133.90, 137.28, 140.83, 142.03, 151.80, 153.34, 160.83;

Electrospray mass (MeOH)- 497[M+Na]⁺, 513[M+K]⁺.

Methyl-4-[naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methoxymethyl]-benzoate

(40):

Yield=63%;

1

yellow viscous; H NMR (CDCl₃, 500 MHz): δ3.77 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 3.88(s, 3H,

OCH₃), 3.91 (s, 3H, OCH₃), 4.67-4.74 (q, 2H, OCH₂, benzylic, J=11.7Hz), 6.58 (s, 1H, CH, benzylic),

6.58-6.60 (d, 1H, CH, aromatic, J=9.0Hz), 6.97-6.99 (d, 1H, CH, aromatic, J=8.5Hz), 7.44-7.53 (m, 5H,

5xCH, aromatic), 7.72-7.74 (d, 1H, CH, aromatic, J=7.0Hz), 7.80-7.82 (d, 1H, CH, aromatic, J=8.5Hz),

7.85-7.87 (m, 1H, CH, aromatic), 8.00-8.08 (m, 1H, CH, aromatic). ¹³C NMR (CDCl₃, 125MHz):

δ52.10, 55.92, 60.76, 61.05, 70.75, 74.41, 107.22, 123.03, 123.74, 124.73, 125.36, 125.53, 126.06,

127.11, 127.50, 128.71, 129.26, 129.65, 130.67, 131.29, 133.87, 136.95, 141.95, 143.78, 151.78,

153.43, 167.04; Electrospray mass (MeOH)- 495[M+Na]⁺, 511[M+K]⁺.

5.2.12. General procedure for the synthesis of compounds 41 and 48:

Synthesis of 4-[Naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methoxymethyl]-phenyl-methanol (41):

To a cold stirred solution of ester 40 (50 mg, 0.11 mmol) in dry THF (8mL), lithium borohydride

(20mg, 0.95 mmol) was added in portions and further stirred at 50^oC for an hour. Reaction mixture

was acidified with dil. HCl (5%, 5mL) and extracted with ethyl acetate (10mLx3), washed with

water (10mLx2) and dried over anhydrous sodium sulphate. On evaporation of organic layer a

24

residue was obtained which on recrystallaised from chloroform:pentane (1:3) afforded alcohol 41 as

viscous liquid.

41: Yield= 83%; viscous gum; ¹H NMR (CDCl₃, 500 MHz): δ3.72 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃),

3.80 (s, 3H, OCH₃), 3.90 (s, 3H, OCH₃), 4.63-4.69 (m, 4H, 2xCH₂, 2xbenzylic, J=10.8Hz), 6.58 (s, 1H,

CH benzylic), 6.58-6.59 (d, 1H, CH, aromatic, J=6Hz), 6.99-7.01(d, 1H, CH, aromatic, J= 9.0Hz), 7.31-

7.53 (m, 7H, 7xCH, aromatic), 7.73-7.75 (d, 1H, CH, aromatic, J=7.0Hz), 7.80-7.82 (d, 1H, CH,

aromatic, J=8.0Hz), 7.86-7.87 (d, 1H, CH, aromatic, J=5.0Hz), 8.05-8.06 (m, 1H, CH, aromatic); ¹³C

NMR (CDCl₃, 125MHz): δ55.93, 60.79, 61.08, 65.06, 71.17, 74.07, 107.27, 123.13, 123.88, 124.82,

125.38, 125.51, 126.02, 127.01, 127.39, 128.13, 128.22, 128.29, 128.39, 128.69, 131.39, 133.90,

137.20, 137.79, 140.33, 141.99, 151.81, 153.34; Electrospray mass (MeOH)- 467[M+Na]⁺, 483[M+K]⁺.

2-[4-(Naphthalen-1-yl-(2,3,4-trimethoxy-phenyl)-methyl)-phenoxy]-ethanol

(48): Yield= 85%;

Yellowish viscous; ¹H NMR (CDCl₃, 500 MHz): δ3.49 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.89 (s, 3H,

OCH₃), 3.94-3.94 (bs, 2H, OCH₂), 4.04-4.05 (bs, 2H, OCH₂), 6.44-6.45 (d, 2H, 2xCH, aromatic,

J=8.5Hz), 6.51-6.52 (bs, 1H, CH), 6.83-6.84 (d, 2H, 2xCH, aromatic, J=8.5Hz), 6.95-6.96 (d, 1H, CH,

aromatic, J=7.0Hz), 7.04-7.05 (d, 2H, 2xCH, aromatic, J=8.0Hz), 7.33-7.44 (m, 3H, 3xCH, aromatic),

7.72-7.73 (d, 1H, CH, aromatic, J= 8.5Hz), 7.83-7.84 (d, 1H, CH, aromatic, J=7.5Hz), 7.99-8.01 (d, 1H,

CH, aromatic, J=8.0Hz); ¹³C NMR (CDCl₃, 125MHz): δ45.39, 55.85, 60.69, 60.75, 61.52, 69.10,

106.66, 114.32, 124.36, 124.45, 125.10, 125.43, 126.04, 127.00, 127.12, 128.60, 130.59, 130.62,

131.86, 133.95, 136.49, 140.52, 142.39, 151.40, 152.38, 157.04; Electrospray mass (MeOH)-

467[M+Na]⁺; 483[M+K]⁺; HRMS (MeOH): m/z [M+H]⁺, calcd for C₂₈H₂₉O₅, 445.2015, found

445.1935.

5.2.13. Synthesis of compounds 42-46:

Synthesis

of

2-[4-(naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyl)]-phenoxy]-N,N-Dimethyl-

ethnamine (42):

Phenol 30 (50mg, 0.13 mmol) was taken in dry THF (10mL) and sodium hydride (20mg, 0.84

mmol). To this stirred reaction mixture, 2-chloro-N,N-dimethylethanamine hydrochloride (56 mg,

0.39 mmol) was added and refluxed for 2h. Water was added to it and reaction mixture was

extracted with ethyl acetate (10mLx3), washed with water and dried in-vacuo to get a residue which

was charged on a silica gel column, eluted with chloroform-methanol to get desired product 42 at

2% methanol-chloroform as gummy viscous oil.

1

42: Yield=77%; Brown viscous; H NMR (CDCl₃, 500 MHz): δ2.95 (s, br, 6H, N(CH₃)₂), 3.48 (s, br,

5H, OCH₃, N-CH₂), 3.78 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 4.45 (s, br, 2H, O-CH₂), 6.41(s, 1H, CH),

6.47 (s, br, 2H, 2xCH, aromatic), 6.81-7.03 (m, br, 5H, 5xCH, aromatic), 7.33-7.39 (m, br, 3H, 3xCH,

13

aromatic), 7.70-7.96 (m, br, 3H, 3xCH, aromatic); C NMR (CDCl₃, 125MHz): δ 44.01, 45.32, 55.86,

56.75, 60.73, 62.92, 106.74, 114.40, 124.24, 124.43, 125.11, 125.45, 126.07, 126.96, 127.17, 128.62,

130.23, 130.77, 131.75, 133.92, 137.44, 140.30, 142.35, 151.30, 152.41, 155.65; Electrospray mass

(MeOH)- 472 [M+H]⁺; 494[M+Na]⁺; HRMS (MeOH): m/z [M+H]⁺, calcd for C₃₀H₃₄NO₄, 472.2487,

found 472.2481.

2-[4-(naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyl)]-phenoxy]-N,N-Diethyl-ethnamine

1

(43): Yield=81%; Brown viscous; H NMR (CDCl₃, 500 MHz): δ1.23-1.26 (t, 6H, 2xCH₃, J=7.0 Hz),

2.96-3.01 (q, 2H, N-CH₂, J=7.0Hz), 3.19-3.21 (t, 2H, N-CH₂, J=5.0Hz), 3.48 (s, 3H, OCH₃), 3.86 (s,

3H, OCH₃), 3.87 (s, 3H, OCH₃), 4.24-4.26 (t, 2H, OCH₂, J=5.0Hz), 6.42-6.44 (d, 1H, CH, aromatic,

J=8.5Hz), 6.49 (s, 1H, CH benzylic), 6.50-6.51 (d, 1H, CH, aromatic, J=9.0Hz), 6.78-6.80 (d, 2H,

2xCH, aromatic, J=8.5Hz), 6.93-6.94 (d, 1H, CH, aromatic, J=7.0Hz), 7.02-7.04 (d, 2H, 2xCH,

aromatic, J=8.5Hz), 7.32-7.43 (m, 3H, 3xCH, aromatic), 7.71-7.72 (d, 1H, CH, aromatic, J=8.5Hz),

7.82-7.83 (d, 1H, CH, aromatic, J=7.5Hz), 7.97-7.98 (d, 1H, CH, aromatic, J=8.0Hz); ¹³C NMR

25

(CDCl₃, 125MHz): δ9.70, 45.36, 47.03, 50.74, 55.84, 60.68, 60.74, 64.10, 106.68, 114.24, 124.32,

124.44, 125.09, 125.41, 126.99, 127.13, 128.59, 130.47, 130.67, 131.82, 133.94, 136.80, 140.44,

142.38, 151.37, 152.39, 156.35.Electrospray mass (MeOH)- 500[M+H]⁺; HRMS (MeOH): m/z

[M+H]⁺calcd for C₃₂H₃₈NO₄, 500.2800, found 500.2790.

2-[4-(naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyl)]-phenoxy]-N,N-Diethyl-2-methylpropanamine

1

(44):Yield=82%; Yellowish viscous; H NMR (CDCl₃, 500 MHz): δ2.37 (s, 3H, CH₃), 2.48 (s, 6H,

N(CH₃)₂), 3.16-3.19 (q, 1H, O-CH, 5.5 Hz), 3.47 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.88 (s, 3H,

OCH₃), 3.96-3.97 (t, 1H, CH of –N-CH₂, J=5.0 Hz), 4.04-4.05 (t, 1H, CH of –N-CH₂, J=4.0 Hz), 6.43-

6.51 (m, 3H, 2xCH, aromatic & 1H, CH), 6.81-6.82 (d, 2H, 2xCH, aromatic, J=8.5Hz), 6.94-7.04 (m,

3H, 3xCH, aromatic), 7.32-7.43 (m, 3H, 3xCH, aromatic), 7.71-7.72 (d, 1H, CH, aromatic, J=8.0 Hz),

7.82-7.83 (d, 1H, CH, aromatic, J=7.5 Hz), 7.98-8.00 (d, 1H, CH, aromatic, J= 8.5 Hz); ¹³C NMR

(CDCl₃, 125MHz): δ41.03, 45.37, 45.75, 55.84, 58.77, 60.68, 60.73, 68.89, 106.68, 114.32, 115.76,

124.34, 124.39, 124.45, 125.09, 125.41, 125.99, 126.02, 126.99, 127.11, 128.59, 130.59, 131.87,

133.95, 136.47, 140.53, 142.40, 151.39, 152.34, 152.37, 156.85; Electrospray mass (MeOH)-

486[M+H]⁺; HRMS (MeOH): m/z [M+H]⁺, calcd for C₃₁H₃₆NO₄, 486.2644, found 486.2641.

2-[4-(naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyl)]-phenoxy]-pyrrolidine

(45): Yield=81%;

Yellowish viscous; ¹H NMR (CDCl₃, 500 MHz): δ2.07-2.10 (bd, 4H, 2xCH₂, J=12.0 Hz), 3.23-3.46

(m, 4H, N(CH₂)₂), 3.83 (bs, 2H, N-CH₂), 3.46 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃),

4.43-4.55 (t, 2H, O-CH₂, J= 6.5 Hz), 6.39-6.48 (m, 2H, 2xCH, aromatic), 6.50 (s, 1H, CH benzylic),

6.78-6.79 (d, 2H, 2xCH, aromatic, J= 8.5 Hz), 6.89-6.91 (t, 1H, CH, aromatic, J= 6.0 Hz), 7.00-7.02 (d,

2H, 2xCH, aromatic, J= 8.5 Hz), 7.30-7.40 (m, 3H, 3xCH, aromatic), 7.69-7.70 (d, 1H, CH, aromatic,

J=8.5 Hz), 7.80-7.81 (d, 1H, CH, aromatic, J=7.5 Hz), 7.94-7.96 (d, 1H, CH, aromatic, J=8.0 Hz); ¹³C

NMR (CDCl₃, 125MHz): δ23.24, 45.33, 53.76, 54.38, 55.85, 60.70, 60.72, 63.52, 106.75, 114.36,

124.26, 124.45, 125.09, 125.41, 126.04, 126.98, 127.14, 128.59, 130.28, 130.75, 131.78, 133.93,

137.37, 140.33, 142.37, 151.32, 152.41, 155.75; Electrospray mass (MeOH)- 498 [M+H]⁺; HRMS

(MeOH): m/z [M+H]⁺, calcd for C₃₂H₃₆NO₄, 498.2644, found 498.2638.

2-[4-(naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyl)]-phenoxy]-piperazine

(46):

Yield=74%;

1

Yellowish viscous; H NMR (CDCl₃, 500 MHz): δ0.85-0.88 (t, 2H, CH₂, J=7.0Hz), 1.24-1.41 (m, 4H,

2xCH₂), 2.01-2.09 (m, 4H, N(CH₂)₂, 3.32 (br s, 2H, N-CH₂), 3.63 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃),

3.87 (s, 3H, OCH₃), 4.46 (br s, 2H, OCH₂), 6.41-6.48 (m, 2H, 2xCH, aromatic), 6.51 (s, 1H, CH

benzylic), 6.78-6.80 (d, 2H, 2xCH, aromatic, J= 9.0Hz), 6.91-6.93 (d, 1H,CH, aromatic, J= 7.0Hz),

7.02-7.04 (d, 2H, 2xCH, aromatic, J= 8.5 Hz), 7.32-7.42 (m, 3H, 3xCH, aromatic), 7.71-7.72 (d, 1H,

CH, aromatic, J=8.0 Hz), 7.82-7.83 (d, 1H, CH, aromatic, J=8.0 Hz), 7.96-7.97 (d, 1H, CH, aromatic,

J=8.5 Hz); ¹³C NMR (CDCl₃, 125MHz): δ22.07, 22.97, 45.34, 53.97, 54.04, 54.20, 56.45, 60.70, 60.75,

63.00, 106.69, 114.31, 124.28, 124.44, 125.09, 125.43, 126.06, 126.99, 127.17, 128.61, 130.31, 130.76,

131.79, 133.94, 137.32, 140.33, 142.37, 151.35, 152.41, 155.74; Electrospray mass (MeOH)-

512[M+H]⁺; HRMS (MeOH): m/z [M+H]⁺, calcd for C₃₃H₃₈NO₄, 512.2800, found 512.2790.

5.2.14. General procedure for the synthesis of compounds 50 and 51:

Synthesis of [2-(2-[Naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyl]-phenoxy]-ethyl-1-pyrrolidine

(50):

To a stirred solution of phenol 31 (50mg, 0.13 mmol) in dry THF (10mL), sodium hydride (Pre-

washed, 20mg, 0.84 mmol) was added. After 10 min, 2-pyrrolidinoethyl chloride hydrochloride (88

mg, 0.39 mmol) was added to it and reaction mixture was refluxed for 2h. Water was added to it and

extracted with ethyl acetate (10mL x3) and washed with water (20mL). Organic layer was dried over

anhydrous sodium sulphate and evaporated to dryness under vacuum. The residue was charged on a

26

silica gel column and eluted with chloroform-methanol to get desired product 50 at 5%

methanol:chloroform as gummy viscous liquid.

1

50: Yield=87%; Yellowish viscous; Specific rotation [α] (EtOH), (20^oC)=+40^o; H NMR (CDCl₃, 500

MHz): δ1.23-1.26 (t, 4H, 2xCH_2,J=6.5Hz), 2.60 (br s, 4H, N(CH₂)₂, 3.07 (s, br, 2H, N-CH₂), 3.44 (s,

3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 4.32 (s, br, 2H, OCH₂), 6.38-6.44 (m, 2H, 2xCH,

aromatic), 6.70 (s, 1H, CH), 6.79-6.91 (m, 4H, 4xCH, aromatic), 7.19-7.40 (m, 4H, 4xCH, aromatic),

7.67-7.69 (d, 1H, CH, aromatic, J=8.5Hz), 7.80-7.81 (d, 1H, CH, aromatic, J=7.5Hz), 7.90-7.92 (d, 1H,

CH, aromatic, J=7.0Hz); ¹³C NMR (CDCl₃, 125MHz): δ22.79, 39.74, 53.63, 54.05, 55.79, 60.79, 63.98,

106.79, 111.34, 121.23, 124.01, 125.51, 126.20, 126.43, 126.56, 127.05, 127.98, 128.69, 129.24,

130.46, 131.79, 132.03, 133.89, 140.04, 142.34, 151.12, 152.51, 154.94; Electrospray mass (MeOH)-

498[M+H]⁺; 520[M+Na]⁺; HRMS (MeOH): m/z [M+H]⁺, calcd for C₃₂H₃₆NO₄, 498.2644, found

498.2641.

[2-(2-[Naphthalen-1-yl-(2,3,4-trimethoxyphenyl)-methyl]-phenoxy]-ethyl-1-piperrdine(51):

Yield=81%; Yellowish viscous; ¹H NMR (CDCl₃, 500 MHz): δ1.26 (s, br, 4H, 2xCH₂), 1.45 (br s, 4H,

N(CH₂)₂), 2.30 (br s, 2H, CH₂), 2.80 (br s, 2H, N-CH₂), 3.47 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.87

(s, 3H, OCH₃), 4.18-4.24 (m, 2H, OCH₂), 6.40-6.47 (m, 2H, 2xCH, aromatic), 6.76 (s, 1H, CH), 6.80-

6.93 (m, 4H, 4xCH, aromatic), 7.18-7.32 (m, 2H, 2xCH, aromatic), 7.39-7.41 (m, 2H, 2xCH, aromatic),

7.67-7.69 (d, 1H, CH, aromatic, J=8.0Hz), 7.80-7.82 (d, 1H, CH, aromatic, J=7.0Hz), 7.96-7.97 (d, 1H,

CH, aromatic, J=7.5Hz); ¹³C NMR (CDCl₃, 125MHz): δ22.43, 23.73, 24.99, 39.53, 53.65, 54.64, 55.80,

56.99, 60.70, 60.75, 64.19, 106.67, 111.30, 114.57, 120.87, 124.11, 124.42, 125.32, 126.09, 126.47,

126.94, 127.79, 128.61, 129.39, 130.36, 131.91, 132.31, 133.94, 140.24, 142.37, 151.32, 152.42,

155.21; Electrospray mass (MeOH)- 512[M+H]⁺; 534[M+Na]⁺; HRMS (MeOH): m/z [M+H]⁺, calcd

for C₃₃H₃₈NO₄, 512.2800, found 512.2783.

5.3. Purity profile of compound 50 by UPLC

A reverse phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the

determination of purity of compound 50 in the presence of its possible process impurities and storage

degradation products. Liquid chromatographic system was consisting ACQUITY- UPLC H-Class Bio

System (Waters, USA) equipped with a PDA detector.205nm). The UPLC column was C-18 (BEH

130Å, 1.7×50mm, 1.7µm, Waters, Milford, USA). The binary mobile phases were A (water with0.1%

formic acid) and B (acetonitrile). The column was thermostated at 30±0.1°C. A gradient elution

composition of mobile phase was selected 10 % to 90% B in 5 min, and held for next 10 min. A

constant flow rate was maintained at 0.3 mL/min.The injection volume was 3.0 µL. In absence of

reference with defined potency, the purity of the compound was calculated by peak area normalization

method.

5.4. Biological evaluation

All the biological evaluations were done as per standard protocols. Detailed descriptions are in

Supplementary information (S1 to S9).

5.4.1. Cytotoxicity by Sulphorhodamine assay

As per Akindele et al. [36].

5.4.2. Soft agar colony assay

Soft agar colony formation assay was performed as per the reported method Kakuguchi et al. [37].

Colonies of MCF-7 cells were formed and treated with compound 50 at various concentrations 6.25-

50 µg/mL. Tamoxifen was used at various concentrations of 1-27 µg/mL as positive control.

5.4.3. Cell cycle analysis of MCF-7 cells

27

The effect of compound 50 on cell division cycle of MCF-7 cells as well as MDA-MB-231 cells

different concentrations (Half IC₅₀, IC₅₀, and double IC₅₀) was assessed by flow cytometry with PI-

stained cellular DNA as per Riccardi et al. [38].

5.4.5. Annexin V-FITC staining assay

To assess the apoptosis Vs necrosis induction by compound 50, we followed Annexin V-FITC

apoptosis assay by Flow cytometry as per Looi et al. [39]. Different concentrations of compound 50

were used i.e. half IC₅₀, IC₅₀, and double IC_50.The assay was performed against both MCF-7 and MDA-

MB-231 cells separately.

5.4.6. Molecular docking studies

Performed by AutoDockVina [40] to know the binding conformation of ligands in active site of proteins

structures. The ligands bound protein 3D crystallographic structure of Tubulin-Colchicine complex

PDB ID: 402B to know the binding of enantiomers 50R and 50S along with colchicine and

podophyllotoxin as control drugs.

5.4.7. Tubulin polymerisation assay

Tubulin Polymerization assay was performed using ‘assay kit’ from Cytoskeleton, USA, as per

Manufacturer’s reported protocol [41, 42]. Podophyllotoxin (PDT) was used as standard inhibitor and

paclitaxel as standard stabilizer of tubulin polymerase and DMSO as negative control. The IC₅₀value

was determined from dose-dependent analysis and is defined as the concentration that inhibits the rate

of polymerization by 50%.

5.4.8. Topoisomerase-II inhibition assay

Topoisomerase-II inhibition assay was performed on ELISA kit from Cloud-Clone Corp., USA

(Catalog no. SEA792Hu) as per reported method [43]. Compound 50 was used at multiple IC₅₀

concentrations. Etoposide and podophyllotoxin were used as positive controls.

5.4.9. Acute oral toxicity

Safety assessment was done as per reported method [44]. Compound 50 was given as single acute

dose at 5 mg/kg, 50mg/kg, and 300mg/kg oral doses. The study and number of animals used were

approved via CIMAP/IAEC/2016-19/32 dated 09-02-2017 by the Institutional Animal Ethics

Committee (IAEC) of CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India.

5.4.10. Statistical analysis

All data have been expressed as mean ± standard deviation (SD) were calculated using MS-Excel.

Statistical analysis of differences was carried out by ANOVA followed by Tukey’s multiple

comparison test. Comparisons are made relative to the untreated controls. Differences with a p

value<0.05 were considered significant.

Acknowledgement

The study was financially supported from CSIR CIMAP under MLP-08. Mr. Amir Kumar Verma

acknowledges to UGC and Ms. Kaneez Fatima acknowledges to CSIR for their Senior Research

Fellowships. Biological Central Facility and Chemical Central Facility of CSIR-CIMAP are duly

acknowledged for Sophisticated Instruments support.

References

[1] B. Park, H.S. Lee, J.W. Lee, S. Park, Association of white blood cell count with breast cancer

burden varies according to menopausal status, body mass index, and hormone receptor status: a

case-control study, Sci. Rep. 9 (2019) 5762-.

[2] The Independent UK Panel on Breast Cancer Screening, Lancet 380 (2012) 1778–1786.

28

Article Doi

Antiproliferative activity of diarylnaphthylpyrrolidine derivative via dual target inhibition

DOI: 10.1016/j.ejmech.2019.111986

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Full text of DOI:10.1016/j.ejmech.2019.111986

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