Synthesis, antiproliferative and pro-apoptotic effects of nitrostyrenes and related compounds in Burkitt’s lymphoma

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Medicinal Chemistry, 2018, 14, 181-199

RESEARCH ARTICLE

ISSN: 1573-4064

eISSN: 1875-6638

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Synthesis, Antiproliferative and Pro-Apoptotic Effects of Nitrostyrenes

and Related Compounds in Burkitt’s Lymphoma

Andrew J. Byrne¹, Sandra A. Bright², Darren Fayne², James P. McKeown¹, Thomas McCabe³,

Brendan Twamley³, Clive Williams²and Mary J. Meegan^1,*

¹School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, Trinity College Dublin,

2

152-160 Pearse St, Dublin 2, Ireland; School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute,

3

152-160 Pearse St, Dublin 2, Trinity College Dublin, Ireland; School of Chemistry, Trinity Biomedical Sciences

Institute, Trinity College Dublin, Dublin, Ireland

Abstract: Background: Cancers of the lymphatic cells (lymphomas) account for approximately

12% of malignant diseases worldwide. The nitrostyrene scaffold is identified as a lead target

structure for the development of particularly effective compounds targeting Burkitt’s lymphoma

(BL).

Objectives: The aims of the curent study were to synthesise a panel of nitrostyrene compounds and

to evaluate their activity in Burkitt’s lymphoma (BL).

Methods: A panel of structurally varied compounds were designed and synthesised using Henry

Knoevenagel condensation reactions. Single crystal X-Ray analysis confirmed the E configuration

for six examples of these novel structures. A number of nitrostyrene-related compounds were also

investigated including 1,3-bis(aryl)-2-nitropropenes together with heterocyclic scaffolds contain-

ing the nitrovinyl pharmacophore such as 3-nitro-2-phenyl-2H-chromenes. The antiproliferative

activities of the compounds were evaluated using the BL cell lines EBV^-MUTU-1 and EBV⁺DG-

75 (chemoresistant) to establish preliminary structure-activity relationships.

A R T I C L E H I S T O R Y

Received: January 12, 2017

Revised: August 03, 2017

Accepted: September 21, 2017

DOI:

10.2174/1573406413666171002123907

Results: Lead compounds with optimized nitrostyrene scaffolds and 3-nitro-2-phenyl-2H-

chromene structures were successfully established with typical IC₅₀values of 0.45 ꢀM and 0.47

ꢀM in MUTU-1 cells and 1.41 ꢀM and 1.92 ꢀM, respectively, in DG-75 cells. The mechanism of

cell death was identified as apoptotic and the lead compound was found to elicit comparable apop-

totic effects to Taxol in Burkitt’s lymphoma cell lines MUTU-1 and DG-75.

Conclusion: This class of pharmaceutically active compounds with potential for the treatment of

Burkitt’s lymphoma suggest a potential role for nitrostyrene based agents in chemotherapy.

Keywords: Anticancer agent, B cell, Burkitt’s lymphoma, DG-75, MUTU-1, 3-Nitro-2-pheny-2H-chromene, nitrostyrene.

1. INTRODUCTION

opment for the treatment of Burkitt’s lymphoma (BL). The

current therapies for BL include cyclophosphamide/hy-

droxydaunorubicin/oncovin/prednisone (CHOP) or CHOP +

rituximab (R-CHOP). There are significant issues associated

with these drugs including the development of resistance and

there is an unmet need for alternative drug therapies for these

diseases.

Cancers of the lymphatic cells (lymphomas) account for

approximately 12% of malignant diseases worldwide [1].

Burkitt’s lymphoma (BL) is a non-Hodgkin’s lymphoma

(NHL) which manifests as tumours composed of small non-

cleaved B-cell lymphocytes [2]. Although many advances

have been made in treatment and management, chemo-

therapy-resistance requires development of new strategies

and drugs for treatment. We have now identified potential

candidate compounds which are suitable for further devel-

The serotonin transporter SERT is expressed in a number

of B cell malignancies including multiple myeloma, diffuse

large B cell lymphoma and Burkitt’s lymphoma and SERT

targetting ligands such as fluoxetine have been shown to

induce apoptosis in various cancer cell lines including

Burkitt’s lymphoma [3, 4]. This effect was also demon-

strated for the NET targetting antidepressant maprotiline

(Fig. 1). There has been much interest in the potential

*Address correspondence to this author at the School of Pharmacy and

Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, 152 - 160

Pearse St, Dublin 2, Trinity College Dublin, Dublin, Ireland;

E-mail: mmeegan@tcd.ie.

1875-6638/18 $58.00+.00

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Byrne et al.

2. MATERIAL AND METHODS

2.1. Chemistry

development of amphetamine derivatives as anticancer

agents [5, 6]. SERT has been associated with the cytotoxicity

observed for amphetamine analogues fenfluramine and

MDMA(3,4-methylenedioxymethamphetamine [7] (Fig. 1).

However, although these compounds act as non-selective

SERT ligands, the pro-apoptotoic action of these compounds

appears to be independent of SERT. Alternative molecular

targets for these compounds may be present in the BL cell

resulting in the antiproliferative effects observed. Structural

derivatives of illicit amphetamines such as MDMA

(‘Ecstasy’) [5, 6] and 4-MTA have been shown to

demonstrate anticancer effects in vitro and in BL cell lines

[8]. We have previously reported the synthesis and SERT

inhibitory activity of number of structurally related 1,3-

bis(aryl)-2-propanamine and 1,3-bis(aryl)-2-nitro-1-propene

compounds such as 85, some of which were originally

identified as synthetic impurities in amphetamines and we

identified their antiprolifertive activity against Burkitt’s

lymphoma [9]. Simple nitrostyrene compounds have been

reported to demonstrate antimicrobial [10], anti-tubulin and

related pro-apoptotic effects in tumour cells [10-15] together

with inhibition of protein tyrosine kinase [16-18] and protein

tyrosine phosphatases [19]. Trans-ꢀ-nitrostyrene has been

reported as a potent inhibitor of protein phosphatases PTB1

and PP2A and to display associated pro-apoptotic effects in

HT-29 and HCT116 colon cancer cells [20], while Hsieh et

al. reported the anti-platelet activity of ꢀ-nitrostyrenes [21].

Uncorrected melting points were measured on a Gallenk-

amp apparatus. Infra-red (IR) spectra were recorded on a

Perkin Elmer FT-IR Paragon 1000 spectrometer. ¹H and ¹³

C

nuclear magnetic resonance spectra (NMR) were recorded at

27 ^oC on a Brucker DPX 400 spectrometer (400.13 MHz, ¹H;

100.61 MHz, ¹³C) in either CDCl₃(internal standard

1

tetramethylsilane (TMS)) or CD₃OD or DMSO-d₆. For H-

NMR assignments, chemical shifts are reported: shift value

(number of protons, description of absorption, coupling con-

stant(s) where applicable). Electrospray ionisation mass

spectrometry (ESI-MS) was performed on a liquid chroma-

tography time-of-flight mass spectrometer (Micromass LCT,

Waters Ltd., Manchester, UK). Flash column chromatogra-

phy was performed on Merck Kieselgel 60 (particle size

0.040-0.063 mm). Microwave experiments were carried out

using the Discover CEM microwave synthesizer.

Compounds 1-57, 59-61, 63-65, 67-78, 81, 83, 84, 88, 90-91,

95, 98, 99, 102, 104-106, 120-132, 134-136, 138-140, 142-

148, 150-154, 156-162, 164-167, 169-180, 182, 184, 187,

189-193 were prepared as described in published procedures

(see Supporting Information). Details of the synthesis and

characterization of all novel compounds are also contained in

the Supporting Information and representative example are

provided below.

In this study, we evaluate the potential activity of an

extensive series of nitrostyrenes and related compounds as

antiproliferative agents specifically against BL. The

objective of the present project was to use the nitrostyrene

scaffold which we identified in the 1,3-bis(aryl)-2-nitro-1-

propene compound 85, as a starting point to synthesise a

number of possible pro-apoptotic agents and to evaluate their

in vitro antiprolifertive activity in BL cell lines. This will

facilitate the identification of potent and selective

nitrostyrenes which may be useful in the design of pro-

apoptotic anticancer agents.

2.2. General Procedure 1: Preparation of (E)-(2-

nitrovinyl)benzene (1-34, 71-74), (E)-(2-nitroprop-1-en-

1-yl)benzenes (35-59, 75-78) and (E)-(2-nitrobut-1-en-1-

yl)benzenes (60-70, 79-82)

To a solution of the appropriate benzaldehyde (3.6 mmol)

in glacial acetic acid (4 mL) was added the nitroalkane (7.2

mmol) followed by cyclohexylamine (3.6 mmol, 0.4 mL).

The reaction mixture was heated under microwave irradia-

o

tion at 120 C for 30 min. After cooling, water (10 mL) was

added to the reaction and it was allowed to stand. The pre-

cipitated nitroethene was isolated by filtration. The filtrate

was further diluted with water (20 mL) and extracted with

dichloromethane (3 x 10 mL). The organic phases were

combined and washed with saturated aqueous NaHCO₃(3 x

10 mL). The solution was dried over anhydrous Na₂SO₄,

filtered and all solvent removed in vacuo, to give an oil

which was purified by flash column chromatography over

silica gel (eluent: dichloromethane/hexane) and recrystallised

from ethanol.

H

N

O

CH₃

O

CF₃

HN

Fluoxetine

MDMA

Cl

NO₂

2.2.1. (E)-4-Methoxy-2-(2-nitroprop-1-en-1-yl)phenol (58)

o

Yield: 38 mg (18%), Mp: 110-114 C, yellow crystals.

IRꢁ_max: 3476 (OH), 3070, 2935 (C-H), 1659, 1585 (C=C),

Cl

1,3-Bis(aryl)-2-nitro-1-propene (85)

NH

1

1510, 1361 (NO₂), 1122 (C-N) cm^-1. H NMR (400 MHz,

CH₃

Maprotiline

CDCl₃) ꢀ 2.40 (s, 3 H, CH₃), 3.94 (s, 3 H, OCH₃), 6.06 (s, 1

H, H3), 6.92 (s, 2 H, 2 x ArH), 8.28 (s, 1 H, H1’). ¹³C NMR

(101 MHz, CDCl₃) ppm 14.3 (CH₃), 56.2 (OCH₃), 111.8

(CH), 118.8, 119.7 (CH), 121.6 (CH), 128.8 (C1’), 145.0

(C1), 146.6 (C2’), 147.8 (C4). HRMS: calculated for

C₁₀H₁₀NO₄[M⁺+H] 208.0610: found 208.0617.

Fig. (1). Chemical structures of the anti-depressants Fluoxetine,

Maprotiline together with methylenedioxymethamphetamine

(MDMA) and 1,3-bis(aryl)-2-nitro-1-propene 85.

Synthesis, Antiproliferative and Pro-Apoptotic Effects

Medicinal Chemistry, 2018, Vol. 14, No. 2 183

2.3. General Procedure 2: Preparation of Nitroethanes

(83, 84, 89)

matography over silica gel (eluent: hexane-ethyl acetate,

8:1).

2.5.1. (E)-3-(4-Hydroxyphenyl)-2-nitro-1-phenylprop-2-en-

1-one (92)

To a vigorously stirred mixture of the appropriate

nitroethene (3.68 mmol), silica gel (9.30 g) and propan-2-ol

(100 mL) in dichloromethane (350 mL) at room temperature

there was added NaBH₄(15.5 mmol, 0.59 g). The mixture

was stirred until the solution turned colourless (20 min). Ex-

cess NaBH₄was quenched with the addition of dilute HCl

(50 mL). The mixture was filtered and the silica gel was

washed with dichloromethane (100 mL). All organic phases

were combined and washed with brine (3 x 50 mL) and wa-

ter (3 x 50 mL). The organic phases were then dried over

anhydrous Mg₂SO₄and the solvent removed in vacuo, to

give an oil which was then purified by flash chromatography

over silica gel (eluent: hexane-ethyl acetate, 10:1) to afford

the product.

Yield: 2.01 g (30%). IR

ꢁ

(KBr): 3390 (OH), 3066

1

max

(CH), 1672 (C=O), 1600 (C=C), 1516, 1307 (NO₂) cm^-1. H

NMR (400 MHz, CDCl₃) ꢀ 6.58 (br.s., 1 H, -OH), 6.77 (d, J

= 7 Hz, 2 H, 2 x ArH), 7.32 (d, J = 7 Hz, 2 H, 2 x ArH),

7.50-7.59 (m, 2 H, H3’’ & H5’’), 7.64-7.66 (m, 1 H, H4’’),

7.99 (d, J = 7.5 Hz, 2 H, 2 x ArH), 8.31 (s, 1 H, H3). ¹³C

NMR (101 MHz, CDCl₃) ppm 116.2 (CH), 120.7 (C1’),

128.8 (CH), 128.9 (CH), 133.4 (CH), 134.6 (CH), 137.4

(C3), 141.8 (C2), 159.6 (C4’), 188.8 (C=O). HRMS: calcu-

lated for C₁₅H₁₀NO₄[M⁺-H] 268.0610: found 268.0626.

2.6. General Procedure 5: Reaction of Amines and Thiols

with Nitrostyrenes (95-100)

2.3.1. 4,4'-(2-Nitropropane-1,3-diyl)bis(chlorobenzene)

(89)

To a mixture of the required nitrostyrene (1 mmol) in wa-

ter (4 mL) was added the appropriate thiol or amine (1.1

mmol). The reaction was stirred at room temperature over-

night. The aqueous reaction mixture was extracted with di-

chloromethane. The organic layer was washed with water,

brine and dried over anhydrous sodium sulphate. Solvent

was removed in vacuo. No further purification was required.

Yield: 309 mg (90%), colourless oil. IR ꢁ_max: 3089, 2981

1

(CH), 1620 (C=C), 1533, 1326 (NO₂) cm^-1. H NMR (400

MHz, CDCl₃) ꢀ 3.06 (dd, 2 H, J = 14.67, 5.87 Hz, 2 x H_a),

3.28 (dd, 2 H, J = 14.67, 5.87 Hz, 2 x H_b), 4.80-4.91 (m, 1 H,

CH), 7.09 (d, 4 H, J = 7.8 Hz, 4 x ArH), 7.33 (d, 4 H, J = 7.8

Hz, 4 x ArH). ¹³C NMR (101 MHz, CDCl₃) ppm 38.9 (2 x

CH₂), 90.6 (CH), 129.12 (CH), 130.15 (CH), 133.5, 133.6.

HRMS: calculated for C₁₅H₁₃Cl₃NO₂[M⁺+Cl] 344.0012:

found 344.0022.

2.6.1. (1-(5-Bromo-2-methoxyphenyl)-2-nitroethyl)(ethyl)

sulfane (97)

Yield: 286 mg (90%). IR

ꢁ

3011, 2966 (C-H), 1639

1

max

(C=C), 1550, 1374 (NO₂), 1132 (C-N) cm^-1. H NMR (400

MHz, CDCl₃) ꢀ 1.24 (t, J = 7.48 Hz, 3 H, CH₃), 2.55 (q, J =

7.46 Hz, 2 H, CH₂), 3.86 (s, 3 H, OCH₃), 4.65 - 4.85 (m, 3 H,

CH & CH₂), 6.79 (d, J = 8.71 Hz, 1 H, H3), 7.38 (d, J = 8.71

Hz, 1 H, H4), 7.46 (d, J = 2.49 Hz, 1 H, H6). ¹³C NMR (101

MHz, CDCl₃) ppm 14.5 (CH₃), 26.6 (CH₂S), 41.0 (C1’), 56.0

(OCH₃), 78.3 (C2’), 112.9 (C3), 113.1 (C5), 128.1 (C1),

131.2 (C4), 132.1 (C6), 156.0 (C2). HRMS: calculated for

C₁₁H₁₄NO₃BrS [M⁺] 318.9878: found 318.9878.

2.4. General Procedure 3: Preparation of (E)-1,3-

bis(aryl)-2-nitro-1-propenes (85-88)

A mixture of the appropriate 1-aryl-1-nitroethane (15.0

mmol), dimethylamine hydrochloride (30.0 mmol), potas-

sium fluoride (2.25 mmol) and the appropriate aldehyde

(15.0 mmol) in toluene (20 mL) was heated at reflux using a

Dean-Stark trap for 24 h. The reaction mixture was then di-

luted with toluene (100 mL) and washed with 10% aq. HCl

(3 x 30 mL). The organic phase was dried over anhydrous

Mg₂SO₄and solvent removed in vacuo. The product was

purified by flash chromatography over silica gel (eluent:

hexane-ethyl acetate, 10:1).

2.7. General Procedure 6: Preparation of Ethyl (E)-2-

hydroxy-3-nitro-4-phenylbut-3-enoates (101-104)

To a stirred solution of DMAP (50 mg, 40 mol %), the

required nitroalkene (1 mmol) in acetonitrile (1 mL) was

added ethyl glyoxylate (4 mmol). The reaction was stirred at

room temperature and monitored by TLC. After completion

the reaction was diluted with water and extracted with ethyl

acetate. The organic layer was extracted with water, brine

and dried over anhydrous sodium sulphate. The organic

phase was concentrated in vacuo. The product was purified

by column chromatography (eluent hexane-ethyl acetate,

2:1).

2.4.1. (E)-1-Chloro-4-(2-nitro-3-phenylprop-1-en-1-yl)ben-

zene (86)

Yield: 1.33 g (30%). IR

ꢁ

3111 (CH), 1674 (C=C),

max

1553, 1368 (NO₂) cm^-1. H NMR (400 MHz, CDCl₃) ꢀ 4.25

(s, 2 H, H3), 7.20 (d, 2 H, J = 7.6 Hz, H3), 7.32-7.42 (m, 6

H, 6 x ArH), 8.27 (s, 1 H, H1). ¹³C NMR (101 MHz, CDCl₃)

ppm 32.9 (C3’), 127.1, 127.52, 129.0, 130.4, 134.2, 135.8,

136.7 (C4), 149.1 (C2’). HRMS: calculated for

C₁₅H₁₂ClNO₂Na [M⁺+Na] 296.0454: found 296.0463.

1

2.7.1. Ethyl (E)-4-(5-bromo-2-methoxyphenyl)-2-hydroxy-

2.5. General Procedure 4: Preparation of (E)-2-nitro-1,3-

3-nitrobut-3-enoate (101)

diphenylprop-2-en-1-ones (90-94)

o

Yield: 208 mg (58%), Mp 123-125 C. IR ꢁ 3326 (O-

max

A mixture of nitroacetophenone (4.00 g, 25 mmol), the

appropriate benzaldehyde (40 mmol), ꢁ-alanine (0.188 g)

and glacial acetic acid (4 mL) in benzene (40 mL) was re-

fluxed for 3 h using a Dean-Stark trap. The reaction mixture

was washed with water (3 x 5 mL) and the benzene was re-

moved in vacuo. The product was purified using flash chro-

H), 3026, 2980 (C-H), 1730 (C=O), 1638, 1591 (C=C),

1528, 1325 (NO₂), 1190 (C-N) cm^-1. H NMR (400 MHz,

1

CDCl₃) ꢀ 1.31 (t, J = 7.05 Hz, 3 H, CH₃), 3.90 (s, 3 H,

OCH₃), 4.33 (dq, J = 1.87, 7.12 Hz, 2H, OCH₂), 5.18 (s, 1 H,

CH), 6.86 (d, J = 9.12 Hz, 1 H, H3), 7.57 (dd, J = 8.71, 2.49

Hz, 1 H, H4), 7.67 (d, J = 2.49 Hz, 1 H, H6), 8.40 (s, 1 H,

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Medicinal Chemistry, 2018, Vol. 14, No. 2

Byrne et al.

H4’). ¹³C NMR (101 MHz, CDCl₃) ppm 14.0 (CH₃), 56.0

(OCH₃), 63.1 (OCH₂), 65.9 (C2’), 112.6 (C3), 112.9 (C5),

121.9 (C1), 132.5 (C4), 134.1 (C6), 135.3 (C4’), 148.0

(C3’), 157.5 (C2), 170.4 (C=O). HRMS: calculated for

C₁₃H₁₄BrNO₆[M⁺] 359.0005: found 359.0005.

2.10.1. (5-Bromo-2-methoxybenzylidene)hydrazine (133)

o

Yield: 50 mg (22%), Mp 238-243 C, yellow solid. IR

ꢁ

_max3662 (N-H), 3004, 2988 (C-H), 1618, 1566 (C=C), 1128

(CN stretch) cm^-1. ¹H NMR (400 MHz, DMSO-d₆) ꢀ 3.89 (s,

3 H, OCH₃), 6.84 (d, J = 9.12 Hz, 1 H, H3), 7.51 (dd, J =

8.71, 2.49 Hz, 1 H, H4), 8.23 (d, J = 2.49 Hz, 1 H, H6), 8.98

(s, 1 H, H1’). ¹³C NMR (101 MHz, CDCl₃) ppm 55.9

(OCH₃), 113.0 (CH), 113.4, 129.8 (CH), 134.9 (CH), 156.6

(C1’), 158.1. HRMS: calculated for C₈H₉BrN₂O [M⁺]

227.9898: found 227.9894.

2.8. General Procedure 7: Preparation of 2-alkyl-2-nitro-

3-phenyloxiranes (107-111)

To

a

stirred solution of the required (E)-(2-

nitrovinyl)benzene (0.5 mmol), 1 8-diazabicyclo [5.4.0] un-

dec-7-ene 4 ꢁL (0.023 mmol) and hexane (1.5 mL) was

added tert-butyl hydroperoxide (100 ꢁL, 0.023 mmol). The

reaction was stirred at room temperature for 10 min. The

reaction was quenched with deionised water (10 mL) and

extracted into diethyl ether. The organic layer was washed

with water, brine and dried over sodium sulphate. Solvent

was removed in vacuo to afford pure product.

2.11. General Procedure 10: Preparation of 3-nitro-2-

phenyl-2H-chromenes (135-142)

To a mixture of salicylaldehyde (6 mmol) and ꢁ-

nitrostyrene (1 mmol) was added DABCO (1 mmol, 112 mg)

at room temperature. The reaction mixture was stirred at

60^oC for 1.5 h. The reaction was extracted with DCM (20

mL) and washed with 10 % HCl (2 x 5 mL). The organic

extracts were washed with H₂O and brine, then dried over

Na₂SO₄, and solvent removed in vacuo. The residue was

purified by flash column chromatography over silica gel

(eluent: hexane-ethyl acetate, 8:1) to afford the products

which were recrystallised from methanol.

2.8.1. 3-(3-Chlorophenyl)-2-methyl-2-nitrooxirane (107)

Yield: 180 mg (85%). IR ꢁ 3068, 3003 (C-H), 1493,

max

1

1454 (C=C), 1557, 1350 (NO₂) cm^-1. H NMR (400 MHz,

CDCl₃) ꢀ 1.73 - 1.78 (m, 3 H, CH₃), 4.49 (s, 1 H, CH), 7.14 -

7.21 (m, 1 H, H6’), 7.25 - 7.29 (s, 1 H, H2’), 7.29 - 7.38 (m,

2 H, H4’). ¹³C NMR (101 MHz, CDCl₃) ppm 12.4 (CH₃),

61.7 (CH), 88.5 (C2), 124.6 (CH), 126.5 (CH), 129.7 (CH),

130.1 (CH), 133.0, 135.0. HRMS: calculated for C₉H₇ClNO₃

[M⁺-H] 212.0114: found 212.0118.

2.11.1. 4-(3-Nitro-2H-chromen-2-yl)phenol (137)

o

Yield: 122 mg (45%), Mp 193-195 C. IR

ꢁ

(KBr):

max

3477 (OH), 3071 (CH), 1648, 1604 (C=C), 1509, 1319

(NO₂) cm^-1. H NMR (400 MHz, DMSO-d₆) ꢀ 6.52 (s, 1 H,

1

H2), 6.62 - 6.80 (m, 2 H, H3’ & H5’), 6.84 (d, J =8.03 Hz, 1

H, H8), 7.03 (td, J = 7.53, 1.00 Hz, 1 H, H6), 7.14 - 7.26 (m,

2 H, H2’, H6’), 7.26 - 7.43 (m, 1 H, H7), 7.60 (dd, J= 7.53,

1.51 Hz, 1 H, H5), 8.38 (s, 1 H, H4), 9.69 (s, 1 H, OH). ¹³C

NMR (101 MHz, DMSO-d₆) ppm 73.3 (C2), 115.5, 116.8

(C8, C6), 118.2 (C4a), 122.4 (C6), 127.1 (C1’), 128.6, 129.8

(C4), 131.1 (C5), 134.4 (C7), 140.8 (C3), 152.8 (C8a), 158.5

(C4’). HRMS: calculated for C₁₅H₁₁NO₄[M⁺] 269.0688:

found 269.0682.

2.9. General Procedure 8: Preparation of 5-alkyl-4-

phenyl-1H-1,2,3-triazoles (112-119)

To a stirred solution of the (E)-(2-nitrovinyl)benzene (1

mmol) in DMSO (2 mL) was added sodium azide (2 mmol).

The reaction was stirred at 90^oC for 90 min. After allowing

the reaction to cool to room temperature deionised water (10

mL) was added and the aqueous mix was extracted into di-

ethyl ether. The organic layer was washed with water, brine

and dried over sodium sulphate. Solvent was removed in

vacuo to afford crude product. The product was purified by

flash column chromatography over silica gel (dichloro-

methane – methanol gradient).

2.11.2. 3-Nitro-2-phenyl-2H-chromen-7-ol (141)

To 7-methoxy-3-nitro-2-phenyl-2H-chromene (80 mg,

2.83 mmol) was added pyridine hydrochloride (3.2 g, 28.3

mmol). The mixture was heated to 170 ^oC for 6 h. The reac-

tion was allowed to come to room temperature and water (50

mL) was added and the mixture was extracted with di-

chloromethane. The organic phases were combined and

washed with water, brine and dried over anhydrous sodium

sulphate. The solvent was removed in vacuo. The crude

product was purified by column chromatography on silica

2.9.1. 4-(3-Chlorophenyl)-5-methyl-1H-1,2,3-triazole (112)

Yield: 119 mg (62%). Mp. 166-169^oC. IR

ꢁ

3089,

max

2981 (C-H), 1594, 1464 (C=C), 1231 (C-N) cm^-1. H NMR

(400 MHz, DMSO-d₆) ꢀ 2.45 (s, 3 H, CH₃), 7.40 (d, J = 8.29

Hz, 1 H, H4’), 7.48 (t, J = 7.88 Hz, 1 H, H5’), 7.66 (d, J =

7.88 Hz, 1 H, H6’), 7.73 (s, 1 H, H2’). ¹³C NMR (101 MHz,

DMSO-d₆) ppm 10.4 (CH₃), 125.0 (CH), 126.0 (CH), 127.3

(CH), 130.7 (CH), 133.5 (C5), 135.3, 141.3, 145.2. HRMS:

calculated for C₉H₇ClN₃[M⁺-H] 192.0329: found 192.0335.

1

gel (eluent: dichloromethane) and recrystallised from ethanol

o

as orange crystals, 29 mg (10 %), Mp 153-156 C. IR ꢁ

max

(KBr): 3321 (OH), 3055, 2971 (CH), 1605, 1572 (C=C),

1

1542, 1323 (NO₂) cm^-1. H NMR (400 MHz, DMSO-d₆)

2.10. General Procedure 9: Preparation of Benzylide-

nehydrazines (130-134)

ꢀ6.35 (d, J = 1.66 Hz, 1 H, H8), 6.49 (dd, J = 8.29, 2.07 Hz,

1 H, H6), 6.57 (s, 1 H, H2), 7.21 (d, J = 8.29 Hz, 1 H, H5),

7.30 - 7.40 (m, 5 H, 5 x ArH), 8.05 (s, 1 H, H4). ¹³C NMR

(101 MHz, CDCl₃) ppm 74.5 (C2), 104.3 (CH), 110.0 (CH),

110.6 (CH), 111.4, 127.0 (CH), 128.8 (CH), 129.4 (CH),

129.7 (CH), 132.1 (C4), 136.9, 138.5 (C3), 155.6 (C8a),

161.3 (C7). HRMS: calculated for C₁₅H₁₀NO₄[M⁺-H]

286.0610: found 286.0607.

To a solution of hydrazine hydrate (1 mL) in ethanol (1

mL) was added the appropriate benzaldehyde (1 mmol). The

reaction mixture was refluxed for three hours and

subsequently cooled to room temperature. The precipitate

was isolated by filtration and recrystallized with ethanol. No

further purification was required.

Synthesis, Antiproliferative and Pro-Apoptotic Effects

Medicinal Chemistry, 2018, Vol. 14, No. 2 185

precipitate was washed with acetic acid (20 mL, 10%) and

water (20 mL). The product was then recrystallised from

methanol.

2.12. General Procedure 11: Preparation of 3-nitro-2-

phenyl-1,2-dihydroquinolines (148, 149)

To a mixture of 2-aminobenzaldehyde (1.2 mmol) and

the appropriate (E)-(2-nitrovinyl)benzene (1 mmol) (1 mmol,

0.149 g) in benzene (5 mL) was added DABCO (0.5 mmol,

56 mg) at room temperature. The reaction mixture was re-

2.14.1. 1-(4-Chloro-2-hydroxyphenyl)-3-(4-chlorophenyl)-

3-hydroxyprop-2-en-1-one (163)

o

Yield: 0.489 g (20%), Mp 154-157 C. IR

ꢁ

(film):

o

3078 (C-H), 1688 (C=C), 1594 (C=O), 1488 (C=C^m)^a,^x780 (C-

fluxed at 100 C for 10 h. The reaction was cooled and ex-

tracted with DCM (20 mL) and washed with 10 % HCl (2 x

5 mL). The organic extracts were washed with water and

brine, then dried over anhydrous Na₂SO₄, and solvent re-

moved in vacuo. The residue was purified by flash column

chromatography over silica gel (eluent: dichloromethane-

hexane, 3:1) and recrystallised from ethanol.

1

Cl). H NMR (400 MHz, DMSO-d₆) ꢀ6.75 (s, 1 H, =CH),

6.92 (dd, 1 H, ArH, H5), 7.29 (s, 1 H, ArH, H3), 7.49 (d, 2

H, J = 8 Hz, 2 x ArH, H3’ & H5’), 7.71 (d, 1 H, J = 8 Hz,

ArH, H6), 7.89 (d, 2 H, J = 8.5 Hz, 2 x ArH, H2’ & H6’),

12.23 (s, 1 H, phenol OH), 15.42 (s, 1 H, enol OH). ¹³C

NMR (101 MHz, CDCl₃) ppm 92.2 (=CH), 118.9, 119.8,

128.2, 128.4, 128.9, 129.2, 129.5, 131.7, 131.8, 138.9

(C4’/C4), 141.8 (C4’/C4), 163.2 (C2), 176.4 (C=C(OH)),

194.8 (CO). HRMS: calculated for C₁₅H₉Cl₂O₃[M⁺-H]

306.9929: found 306.9931.

2.12.1. 4-(3-Nitro-1,2-dihydroquinolin-2-yl)phenol (149)

o

117 mg (40%), Mp 207-209 C, purple crystals, IR ꢁ

(KBr): 3356 (OH), 2917 (CH), 1603 (C=C), 1492, 13^m3^a5^x

1

(NO₂) cm^-1. H NMR (400 MHz, DMSO-d₆) ꢀ5.84 (s, 1 H,

H2), 6.53-6.58 (m, 2 H, 2 x ArH), 6.69 (d, J = 8.6 Hz, 2 H, 2

x ArH), 7.10-7.14 (m, 3 H, 3 x ArH), 7.31 (d, J = 7.5 Hz, 1

H, 1 x ArH), 8.15 (s, 1 H, H4), 9.48 (s, 1 H, OH). ¹³C NMR

(101 MHz, DMSO-d₆) ppm 53.5 (C2), 113.3, 114.1, 115.3,

116.8, 127.3, 131.5, 131.9, 133.7, 134.2, 139.5 (C3), 145.7

(C8a), 157.4 (C4’). HRMS: calculated for C₁₅H₁₂N₂O₃Na

[M⁺+Na] 291.0746: found 291.0736.

2.15. General Procedure 14: Preparation of 3-

nitroflavones (179-188)

To the appropriately substituted flavone (1.58 mmol) was

added of ammonium nitrate (5 mmol, 400 mg) followed by

dry dichloromethane (25 mL) and dry acetonitrile (25 mL).

Trifluoroacetic acid (8.49 mmol (1.2 mL) was then added

with cooling in an ice bath. The reaction mixture was heated

to 40 ^oC and allowed to react for 1.5 hours. The mixture was

neutralised with water (100 mL), extracted with dichloro-

methane(2 x 20 mL). The solvent was removed in vacuo and

the 3-nitroflavone was recrystallised from methanol.

2.13. General Procedure 12: Preparation of o-benzo-

yloxyacetophenones (152-160)

The appropriately substituted o-hydroxyacetophenone

(11.7 mmol, 2 g) was added to a solution of 4-chlorobenzoyl

chloride (16.4 mmol, 2.866 g) and pyridine (4 mL) and the

mixture was stirred for 30 min. The reaction mixture was

poured onto 100 mL of 1 M hydrochloric acid and 50 g of

crushed ice. The product was then filtered and washed with

water (20 mL) and ice-cold methanol (20 mL). The product

was then recrystallised from methanol.

2.15.1. 7-Chloro-2-(4-chlorophenyl)-3-nitrochromen-4-one

(181)

o

Yield: 109 mg (33%), Mp 197-199 C. IR

ꢁ

(KBr):

3068 (C-H), 1660 (C=O), 1613 (C=C), 1599 (N^m-O^ax), 1426

(C=C), 794 (C-Cl) cm^-1. H NMR (400 MHz, DMSO-d₆)

1

ꢀ7.51-7.58 (m, 3 H, 3 x ArH, H3’ H5’ & H6), 7.66-7.71 (m,

3 H, 3 x ArH, H2’, H6’ & H8), 8.27 (d, 1 H, J = 8 Hz, ArH,

H5). ¹³C NMR (101 MHz, CDCl₃) ppm 118.1 (C7), 121.4,

125.9, 127.1, 127.4, 128.7 (C3’ & C5’), 129.4 (C2’ & C6’),

139.4, 141.2 (C3), 154.9 (C8a), 158.9 (C2), 167.5 (C4).

HRMS: calculated for C₁₅H₇Cl₃NO₄[M⁺+Cl] 369.9441:

found 369.9428. 3-Nitro-2-(3,4,5-trimethoxy-2-nitrophenyl)

chromen-4-one (185) was obtained as a minor product , 36

mg (7%). IR ꢁ_max(film): 2947 (C-H), 1671 (C=O), 1532 (N-

2.13.1. 4-Chlorobenzoic Acid 2-acetyl-5-chlorophenyl Ester

(155)

Yield: 2.93 g (76%), Mp 93-95 ^oC, colourless crystals. IR

ꢁ

max

(film): 3095 (CH), 1741 (C=O), 1595 (C=C), 1261 (C-

1

O), 752 (C-Cl) cm^-1. H NMR (400 MHz, DMSO-d₆) ꢀ 2.54

(s, 3 H, CH₃), 7.29 (s, 1 H, ArH, H3), 7.38(d, 1 H, J = 8 Hz,

ArH, H5), 7.53 (d, 2 H, J = 8.5 Hz, 2 x ArH, H3’, H5’), 7.84

(d, 1 H, J = 8.5 Hz, ArH, H6), 8.14 (d, 2 H, J = 8 Hz, 2 x

ArH, H2’, H6’). ¹³C NMR (101 MHz, CDCl₃) ppm 29.1

(CH₃), 124.1, 126.2, 126.9, 128.4, 128.7, 128.8, 131.0,

131.3, 138.7 (C4’), 140.2 (C4), 149.4 (C2), 163.6(CO-O),

195.7(CO). HRMS: calculated for C₁₅H₁₀Cl₂O₃Na [M⁺+Na]

330.9905: found 330.9904.

1

O), 1464 (C=C), 1375 (C-O) cm^-1. H NMR (400 MHz,

DMSO-d₆) ꢀ3.95 (s, 3 H, OCH₃), 4.02 (s, 3 H, OCH₃), 4.10

(s, 3 H, OCH₃) 6.92 (s, 1 H, ArH, H6’), 7.49 (d, 1 H, J = 8.5

Hz, ArH, H8), 7.53-7.56 (m, 1 H, ArH, H6), 7.78-7.84 (m, 1

H, ArH, H7), 8.31(d, 1 H, J = 7.5 Hz, ArH, H5). ¹³C NMR

(101 MHz, CDCl₃) ppm 55.9 (2 x OCH₃on C3’ & C5’), 60.6

(OCH₃on C4’), 106.9, 117.5, 117.9 (C7), 123.1, 126.1,

126.4, 135.1, 145.6 (C3), 147.9, 154.8 (C8_a), 155.3, 156.7

(C2), 167.9 (CO). HRMS: calculated for C₁₈H₁₄N₂O₉Na

[M⁺+Na] 425.0597: found 425.0583.

2.14. General Procedure 13: Preparation of 3-hydroxy-3-

phenylprop-2-en-1-ones (161-169)

The appropriately substituted o-benzoyloxyacetophenone

(15.8 mmol) was dissolved in dry pyridine (15 mL) in a

o

2.16. X-Ray Crystallography

pressure flask and heated to 50 C. Potassium hydroxide (25

o

mmol, 1.42 g), (powdered and preheated at 100 C), was

Data for samples 35, 54, 68, 94 and 118 were collected

on a Bruker APEX DUO using Mo Kꢀ radiation (ꢁ =

0.71073 Å) using a Mitegen cryoloop and at 100(2) K (Ox-

ford Cobra Cryosystem). Bruker APEX [22] software was

o

then added and the mixture was heated at 50 C for 20 min.

The reaction mixture was then cooled and acidified with

acetic acid (21 mL, 10%). The mixture was then filtered and

186

Medicinal Chemistry, 2018, Vol. 14, No. 2

Byrne et al.

used to collect and reduce data, determine the space group,

solve and refine the structure. Absorption corrections were

applied using SADABS 2014 [23]. Data for sample 85 were

collected on a Rigaku Saturn 724 at 90(2) K (X-Stream) and

CrystalClear [24, 25] was used for cell refinement and data

reduction and absorption corrections. Bruker APEX software

was used to determine the space group, solve and refine the

structure. All non-hydrogen atoms were refined anisotro-

pically. Hydrogen atoms were assigned to calculated

positions using a riding model with appropriately fixed

isotropic thermal parameters.

desired drug concentration for a predetermined time frame

and incubated as required. Alamar blue (20 ꢁL) was then

added to each well and incubated at 37 °C in the dark for 4 h.

Plates were then read on a fluorescence plate reader (Spec-

traMax Gemini, Molecular Devices) with excitement and

emission wavelengths of 544 nm and 590 nm respectively.

Experiments were performed in triplicate. A sample contain-

ing only reagent and medium (without any cells) was used as

a blank. Vehicle samples were set as 100% viability from

which any decrease in viability was calculated. Taxol (10

ꢀM) was used as an internal standard and resulted in 90%

cytotoxicity in each of the cell lines.

^aR₁= ꢀ||F_o| ꢀ |F_c||/ ꢀ |F_o|, wR₂= [ꢀw(F_o²ꢀ F_c²)²/ꢀw(F_o) ]

.

2 2 1/2

2.18.4. Flow Cytometry

2.17. Molecular Modelling

BL cells (750,000) were treated with the appropriate

concentration of compound and incubated for the designated

time. Samples were harvested by centrifugation and the

pellets were rinsed with 0.5 mL of Ca²⁺Annexin V binding

buffer (0.1 M HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl₂)

each and pelleted again by centrifugation. Supernatants were

decanted and the pellets were resuspended in 50 ꢁL FITC

Annexin V (diluted 1:33 in Ca²⁺Annexin V binding buffer),

the samples were then placed on ice and incubated for 10

min in the dark. After the incubation period 0.5 mL of Ca²⁺

Annexin V binding buffer was added and samples were

pelleted by centrifugation. The supernatant was decanted and

the pellet was resuspended in 500 ꢁL of PI solution (0.5 ꢁg/

mL). Samples were analysed within 1 h using CyAn ADP

flow cytometer (Beckman Coulter) counting 10,000 cells and

analysed using the Flowjo software package. Taxol was used

as a positive control for cell death.

Using MOE (Molecular Operating Environment)

2015.V10 [26], the structures of interest (Compound 66: (E)-

4-bromo-1-methoxy-2-(2-nitrobut-1-en-1-yl)benzene, Com-

pound 85: (E)-4,4'-(2-nitroprop-1-ene-1,3-diyl)bis(chloro-

benzene) and Compound 145: 7-methoxy-3-nitro-2-phenyl-

2H-chromene) were flexibly aligned with one another for

structural comparison. The molecular structures were

processed using the MMFF94s force field, commonly used

for small molecule modelling. The chirality of the chiral

centre of compound 145 was defined as either (R) or (S) for

separate runs of 100, 200 and 500 iterations respectively

(See Supporting Information). Default parameters were

utilised for other settings. Both proposed alignments featured

as the top ranked alignment of generated poses, ranked in

order of ascending F score (measure of configuration

similarity).

2.18. Biochemistry

2.18.1. Materials

3. RESULTS AND DISCUSSION

3.1. Chemistry

MUTU-I (c179) and DG-75 BL cell lines were gifts from

Dr. Dermot Walls (School of Biotechnology, Dublin City

University, Ireland). Alamar blue was obtained from

BioSource, Belgium and Fetal Bovine Serum(FBS) was

sourced from Invitrogen, U.K. RPMI 1640 medium, DMEM,

HEPES, sodium pyruvate, Gentamycin (G418) and glutami-

ne was sourced from Biosciences, Ireland. Cell culture con-

sumables were from Greiner Bio-One Ltd., U.K. while all

other reagents used were obtained from Sigma-Aldrich, Ire-

land.

Following this, based on the initial positive results with

1,3-bis(aryl)-2-nitro-1-propene compound 85 in BL cell

lines, we now report the synthesis of a diverse library of

novel nitrostyrenes, 1,3-bis(aryl)-2-nitro-1-propenes and

related heterocyclic analogues (n=193); all of which contain

the classic nitrostyrene scaffold structure, and evaluate their

potential activity as antiproliferative agents against BL. The

objective of the present research was to use the nitrostyrene

scaffold structure as a starting point to synthesise a number

of possible pro-apoptotic agents and to evaluate their in vitro

anti-proliferative activity in BL cell lines. This will not only

facilitate the synthesis and development of potent and

selective nitrostyrenes which may be useful in the design of

proapoptotic anticancer agents.

2.18.2. Cell Culture

MUTU-I and chemoresistant DG-75 cell lines were

grown in RPMI 1640 (Glutamax) medium containing phenol

red and supplemented with 10% (v/v) foetal bovine serum

(FBS), L-glutamine (2 mM) and 50 ꢁg/mL penicil-

lin/streptomycin. The MUTU-I cell line also required the

additional supplements of alpha-thioglycerol (5 mM in

phosphate buffered saline (PBS) with 20 ꢂM bathocuprione

disulfonic acid), sodium pyruvate (100 mM) and HEPES

(1 mM). All cells were grown in a humidified environment

maintained at 95% O₂and 5% CO₂and passaged at least

twice weekly depending on their levels of confluency.

The structures of the nitrostyrenes and related com-

pounds to be investigated in the present study are arranged in

six distinct groups as follows (Types I -VI): (see Schemes 1-

5 and Supplementary Information).

Type I. Structures based on nitrostyrenes with aryl

substitution (1-34).

2.18.3. Alamar Blue Viability Assay

Type II. Structures based on methylnitrostyrenes with

aryl substitution (35-59).

BL cells were seeded in 200 ꢁL medium in a 96-well

plate (1-5x10⁴cells per well). Cells were treated with the

Synthesis, Antiproliferative and Pro-Apoptotic Effects

R₂

Medicinal Chemistry, 2018, Vol. 14, No. 2 187

substituents were observed to be unfavourable. When using

aliphatic nitro compounds with increased chain length (when

compared to nitromethane), the yield of nitrostyrene ob-

tained was significantly reduced. The pKa values of the rele-

vant aliphatic nitro compounds nitromethane (9.63),

nitroethane (9.99) and nitropropane (9.77) did not demon-

strate a correlation between increased chain length, reduction

in acidic strength of nitroalkane and reduced reactivity of

these nitroalkanes in the Henry-Knoevenagel condensation

reaction.

R₂

R₃

NO₂

R₃

R₄

CHO

R₆

(a)

R₁

R₄

R₆

R₅

1-34 R₁= H,

35-59 R₁= CH₃

60-70 R₁= CH₂CH₃

NO₂

R₅

CHO

Nitrostyrenes like many alkene systems can exist as

either cis (Z) or trans (E) isomers. However, in these reac-

tions, the trans(E) configuration is sterically favoured and

the structure was confirmed by analysis of the reaction prod-

R₁

(a)

R₂

R₃

X

R₂

R₃

X

71,72 R₁=H

75,76 R₁=CH₃

1

ucts by H NMR spectroscopy, and by X-Ray crystallogra-

phy of compounds (E)-4-bromo-1-methoxy-2-(2-nitroprop-

1-en-1-yl)benzene (54), (E)-1-chloro-4-(2-nitrobut-1-en-1-

yl)ben-zene (60), and (E)-3-(2-nitrobut-1-en-1-yl)phenol

(68), (Figs. 2A, 2B and 2C respectively). The corresponding

Z isomer was not detected or isolated in these products.

79,80 R₁=CH₂CH₃

R₂

R₁

R₂

(a)

NO₂

R₃

X

CHO

R₃

X

73,74 R₁=H

77,78 R₁=CH₃

71,82 R₁=CH₂CH₃

Fig. (2A) represents the asymmetric unit for compound

54, with both independent molecules shown. The two inde-

pendent molecules in the asymmetric unit have small varia-

tions in the angles between the methyl/NO₂groups on the

side chain with an overall non-hydrogen overlay RMS of

0.074. Angstroms indicating that both molecules are essen-

tially identical. However the orientation of the two inde-

pendent molecules in the structure leads to differences in the

hydrogen bonding synthons which affect the packing in the

solid state. The crystal and experimental data for compounds

54, 60 and 68 are shown in Table 2. The bond distances and

torsional angles for the aromatic ring and the nitropropene

and nitrobutene substituents are in close agreement with the

expected values and reported values for ꢀ-methyl-ꢀ-

nitrostyrenes [30, 31]. The nitrostyrene C=C bond was ob-

served at 1.3387(16)Å for compound 60(C(7)-C(8)), at

1.331(3)Å for compound 54((C6)-C(5)) and at 1.3398(19)Å

for compound 68(C(7)-C(8)). These values are similar to the

value of 1.332(2) Å reported for the (E)-1-(3,4-methylene-

dioxy-6-fluorophenyl)-2-nitropropene [30]. The torsional

angle of 31.0(3)°, C(6)-(C5)-(C9)-(C10) was observed for

the ꢀ-methyl-ꢀ-nitrostyrene compound 54, ꢀ-ethyl-ꢀ-nitro-

styrene compounds 60 (C(3)-(C4)-(C7)-(C8) =20.59(18)°

and 68 C(2)-(C3)-(C7)-(C8) = 23.0(2)°, resulting in the nitro

and alkyl substituents on the alkene lying out of the plane of

the aromatic ring.

Scheme 1. Preparation of nitrostyrenes 1-82: Scheme reagents and

conditions: (a) Nitroalkane (CH₃NO_2,CH₃CH₂NO₂, CH₃CH₂CH₂

NO₂), CH₃CO₂H, cyclohexylamine, microwave, 120 °C, 25 min.

Type III. Structures based on ethylnitrostyrenes with

aryl substitution (60-70).

Type IV Structures based on nitrostyrenes with hetero-

cycle substitution (71-82).

Type V. Structures based on modified nitrostyrenes

(83-119), acrylonitriles (120-129) and hydrazones (130-134).

Type VI. Structures based on heterocycles containing

the nitrostyrene pharmacophore (135-151, 179-190, 193).

The synthetic routes used in the preparation of the ni-

trostyrenes, types I-IV, are illustrated in Scheme 1 and Table

1, and are based on modifications of the previously described

procedures [9, 27]. In the present work, libraries of structur-

ally diverse (E)-(2-nitrovinyl)benzenes (1-34), (E)-(2-nitro-

prop-1-en-1-yl)benzenes (35-59), (E)-3/2-(2-nitrovinyl)hete-

rocycles (71-82) and (E)-(2-nitrobut-1-en-1-yl)benzenes (60-

70) were prepared the microwave assisted Henry-

Knoevenagel condensation reaction [28, 29] of nitromethane,

nitroethane and nitropropane with the appropriately substi-

tuted aryl aldehydes catalysed by cyclohexylamine. Aryl

substitution patterns achieved included halogen, alkyl,

methoxy, hydroxy, benzyloxy and phenoxy, including previ-

ously unreported compounds 58, 62, 66, 79-82. Related het-

erocyclic (E)-(2-nitrovinyl) compounds (71-82) were synthe-

sised utilising the Henry-Knoevenagel condensation of the

required furan or thiophene aldehydes and the appropriate

nitroalkane (Scheme 1).

A number of structural modifications of the nitrostyrene

structures (83-111) were also prepared together with struc-

turally related acrylonitriles (120-129) and hydrazones (130-

134) for evaluation (Type V compounds). The synthesis of a

series of structurally varied (E)-1,3-bis(aryl)-2-nitro-1-

propenes 85-88 was achieved using a three step synthetic

route, (Scheme 2) [9]. The nitrostyrenes 1 and 21 were re-

duced under mild conditions with sodium borohydride to the

subsequent nitroalkanes 83 and 84. A second Henry-

Knoevenagel condensation afforded the required products

85-88. X-ray crystallography of (E)-4,4'-(2-nitroprop-1-ene-

1,3-diyl)bis(chlorobenzene) (85) confirmed the trans con-

figuration of the nitrovinyl group after the second Henry-

Knoevenagel condensation reaction, (Fig. 3A). The nitrosty-

On the analysis of the yields obtained for the nitrostyrene

products 1-70, (see Supplementary Information, Tables S1-

S4), it was observed that electron withdrawing groups such

as the halogens at the para position on the aryl ring, seemed

to favour the Henry-Knoevenagel reaction, whereas phenolic

188

Medicinal Chemistry, 2018, Vol. 14, No. 2

Byrne et al.

Table 1. (E)-(2-Nitrovinyl)benzenes 1-34 (Type I), (E)-(2-Nitroprop-1-en-1-yl)benzenes 35-59 (Type II), (E)-(2-Nitrobut-1-en-1-

yl)benzenes 60-70 (Type III), (E)-(2-Nitrovinyl) substituted furans and thiophenes 71-82 (Type IV). (R=H unless otherwise

specified).

Yield

(%)

Yield

(%)

Yield

(%)

Compound

R₂-R₅

Compound

R₂-R₅

Compound

R₂-R₅

1

2

3

R₄=Cl

R₄=OH

83

40

75

29

30

31

R₄=Br

69

58

29

57

58

59

R₂=Cl

56

18

39

R₄=OCH₂CH₃

R₂=OH, R₃=OCH₃

R₂=OH, R₅=OCH₃

R₄=OCH₂CH₃

R₃R₄=C₄H₄

4

5

6

R₄=F

78

79

76

32

33

34

R₃=Br, R₄=OCH₃

R₃=OH, R₄=OCH₃

R₃=NO₂

39

62

33

60

61

62

R₄=Cl

R₄=OH

20

7

R₄=CH=CHNO₂

R₂R₃=C₄H₄

R₃R₄=C₄H₄

30

7

8

R₂R₃=R₅R₆=C₄H₄

R₃=OCH₂C₆H₅

99

82

86

70

62

35

36

37

38

39

R₄=Cl

R₄=OH

64

8

63

64

65

66

67

R₄=F

R₄=CH=CH(CH₂CH₃)NO₂

R₂R₃=C₄H₄

40

35

62

94

9

R₂=OCH₃, R₅=Br

R₄=OCH₃

R₃R₄=C₄H₄

R₄=F

60

50

60

10

11

R₂=OCH₃, R₅=Br

R₂=Cl

R₄=OCH_3,R₃=OCH₂C₆H₅

R₄=CH=CH(CH₃)NO₂

12

13

R₂=R₄=R₅=OCH₃

R₂=OH, R₅=OCH₃

48

58

40

41

R₂R₃=C₄H₄

55

73

68

69

R₃=OH

R₃=Cl

13

23

R₂R₃=R₅R₆=C₄H₄

14

15

R₃=Cl

73

78

42

43

R₃=OCH₂C₆H₅

60

35

70

71

R₂=OH, R₅=OCH₃

X=O

9

R₄=CH₃

R₃=OCH₂C₆H₅, R₄=OCH₃

75

16

17

18

R₂=R₄=OCH₃

R₂=R₅=OCH₃

70

80

60

44

45

46

R₂=R₄=R₅=OCH₃

R₄=CH₃

25

40

18

72

73

74

X=S

74

89

50

R₃=R₄=R₅=OCH₃

R₄=Br

R₃=CH_3,X=S

19

20

21

22

23

R₂=OCH₂CH₃

R₄=OC₆H₅

67

70

80

70

40

47

48

49

50

51

R₂=R₄=OCH₃

R₂=R₅=OCH₃

R₃=R₄=R₅=OCH₃

R₂=OCH₂CH₃

R₄=OC₆H₅

35

80

50

40

72

75

76

77

78

79

R₁=CH₃, X=O

R₁=CH₃, X=S

70

62

65

55

30

R₁-R₅=H

R₁=CH₃, X=S

R₃R₄=OCH₂O

R₃=R₄=OCH₃

R₁= R₃= CH_3,X=S

R₁=CH₂CH₃, X=O

24

25

R₄=OCH₂C₆H₅

R₂=R₄=Cl

40

52

53

R₄=OCH₂C₆H₅

R₁-R₅=H

50

68

80

81

R₁=CH₂CH₃, X=S

48

58

R₁=CH₂CH₃, R₃= CH_3,

X=S

26

R₃=OH

50

54

R₂=OCH₃, R₅=Br

85

82

56

27

28

R₂=OH

R₂=Cl

30

84

55

56

R₃=Cl

61

37

-

R₃=OH

rene C=C bond (C(4)-C(12)), was observed at 1.338(7)Å for

compound 85; a torsional angle of 38.6(8)° for C(4)-(C12)-

(C13)-(C14) was determined, indicating that the benzyl and

nitro-substituents are out of the plane of the aromatic ring.

Reduction of 85 with sodium borohydride afforded 89.

The structurally related (E)-2-nitro-1,3-diphenylprop-2-

en-1-ones (90-94) with phenylprop-2-en-1-one substitution

at C-2 of the nitrovinyl unit, were obtained using methodol-

ogy reported by Sagitullina et al., [32] the Henry-

Knoevenagel condensation of nitroacetophenone with vari-

Synthesis, Antiproliferative and Pro-Apoptotic Effects

Medicinal Chemistry, 2018, Vol. 14, No. 2 189

A

B

C

Fig. (2). Molecular structure of (A) compound 54 (both independent molecules shown), (B) compound 60 and (C) compound 68 with atomic

displacement parameters shown at 50% probability. Hydrogen atoms omitted for clarity.

A

B

C

Fig. (3). Molecular structure of (A) compound 85, (B) compound 94 and (C) compound 118 with atomic displacement parameters shown at

50% probability. Hydrogen atoms omitted for clarity.

190

Medicinal Chemistry, 2018, Vol. 14, No. 2

Byrne et al.

Table 2. X-ray crystal data and refinement parameters for compounds 54, 68, 85, 94 and 118.

Compound

54

60

68

85

94

118

Empirical formula

C₁₀H₁₀BrNO₃

272.10

Monoclinic

P2₁/c

C₁₀H₁₀ClNO₂

211.64

Monoclinic

P2₁/c

C₁₀H₁₁NO₃

193.20

Monoclinic

P2₁/n

C₁₅H₁₁Cl₂NO₂

308.15

Monoclinic

P2₁/c

C₁₆H₁₂BrNO₄

362.18

C₁₁H₁₂BrN₃O

282.15

Orthorhombic

Pna2₁

fw

Crystal System

SG

Triclinic

Pꢃ

a (Å)

7.7190(3)

18.8942(7)

14.4098(5)

90

13.1822(4)

5.1986(2)

15.1731(4)

90

4.9914(2)

12.9543(4)

14.1875(5)

90

7.8204(16)

10.598(2)

16.791(3)

90

7.2329(3)

9.8229(4)

10.7899(4)

88.8374(14)

78.3347(14)

76.4676(14)

729.63(5)

100(2)

8.1530(2)

15.0687(5)

9.4971(3)

90

b (Å)

c (Å)

ꢀ (°)

ꢁ (°)

93.1571(12)

90

113.5340(12)

90)

91.0844(12)

90

96.69(3)

90

ꢂ (°)

90

V (Å3)

T (K)

2098.40(13)

100(2)

953.31(5)

100(2)

4

917.20(6)

100(2)

4

1382.2(5)

90(2)

1166.77(6)

100(2)

4

Z

8

4

2

ꢃ (Mg/cm³)

1.723

1.475

1.399

1.481

1.649

1.606

ꢄ (mm^ꢀ1

)

3.903

0.371

0.104

0.469

2.835

3.506

Total reflns

Indep. reflns

R(int)

67821

51382

33675

10469

31389

33128

4412

2832

2109

2375

3599

4423

0.0346

0.998

0.0388

1.009

0.0353

1.070

0.0649

1.068

0.0195

0.0198

1.038

S

1.040

R₁^a[I>2ꢁ(I)]

wR₂^#[I>2ꢁ(I)]

0.0218

0.0515

0.0295

0.0666

0.0381

0.0946

0.0764

0.2396

0.0199

0.0156

0.0373

0.0477

CCDC deposition

number

1497901

1497900

1497902

1497903

1497904

1497905

(75%) and 106 (62%) respectively [35]. Epoxidation of the

2-(nitrovinyl)benzenes 55, 35, 46, 69 and 85 with tert-butyl

hydrogen peroxide [36] afforded the novel 2-alkyl-2-nitro-3-

phenyloxiranes 107-111 in good yield (85-92%), while reac-

tion of the 2-(nitrovinyl)benzenes 55, 46, 54, 48, 35, 69, 66,

104 with sodium azide in DMSO [37] gave the novel 5-

alkyl-4-phenyl-1H-1,2,3-triazoles 112-119 respectively

(Scheme 2). X-ray crystallography confirmed the structure

of 4-(5-bromo-2-methoxyphenyl)-5-ethyl-1H-1,2,3-triazole

(118), Fig. (3C). The bond lengths and angles are within the

normal ranges with the triazole C=C bond at C(10)-C(14)

observed at 1.375(2)Å, N(11)-N(12) at 1.319(2)Å, and

N(12)-N(13) at 1.337(2)Å [38]. The triazole ring C(10)-

N(11)-N(12)-N(13)-C(14) makes a dihedral angle of 79.02°

with the phenyl ring C(2)-C(3)-C(4)-C(5)-C(6)-C(7). The X-

Ray crystal data and refinement parameters for compounds

35, 54, 68, 85, 94 and 118 are displayed in Table 1.

ous benzaldehydes catalysed by ꢄ-alanine, Scheme 2. The

molecular structure of 3-(5-bromo-2-methoxyphenyl)-2-

nitro-1-phenylprop-2-en-1-one (94) is displayed in Fig. (3B)

which confirms the E configuration for the nitrovinyl unit,

with nitrostyrene C=C bond at C(10)-C(11) observed at

1.3332(18)Å and the torsional angle (C5)-C(6)-C(10)-C(11)

of -30.8(2)°.

Synthesis of 1-substituted-2-nitroethylbenzenes (95-100)

was achieved the aqueous Michael addition of thiols and

aniline to selected nitrostyrenes [33], Scheme 2. The ethyl

(E)-2-hydroxy-3-nitro-4-phenylbut-3-enoates(101-104) were

prepared using a 4-dimethylaminopyridine (DMAP) cata-

lyzed Baylis-Hillman reaction of the corresponding (E)-2-

nitrovinylbenzene and ethyl glyoxylate [34]. Oxidation of

the (E)-1,4-dimethoxy-2-(2-nitrovinyl)benzenes 17 and 48

with cerium ammonium nitrate afforded the diones 105

Synthesis, Antiproliferative and Pro-Apoptotic Effects

Medicinal Chemistry, 2018, Vol. 14, No. 2 191

R₁

NO₂

R₂

R₃

NO₂

(a)

(b)

R₁

Cl

O

R₁

(c)

83 R₁= Cl

83 R₁= H

R₂

85 R₁= R₂=Cl

89

R₄

Cl

86 R₁= Cl, R₂=H

87 R₁= H, R₂= Cl

88 R₁= R₂= H

90 R₁=R₂=R₃=R₄=H

(a)

91 R₁=R₂=R₃=R₄=H, R₃=Cl

92 R₁=R₂=R₃=R₄=H, R₃=OH

93 R₁=R₂=C₄H₄, R₃=R₄=H

94 R₁=OCH₃, R₂=R₃=H, R₄=Br

R₁

R₄

R₁

R₂

R₃

NO₂

R₂

R₃

NO₂

(d)

O

(f)

R₁

R₂

NO₂

O

R

HO

R₃

O

R₄

101 R₁= OCH₃, R₂=R₃=H, R₄= Br

102 R₁= R₂= R₄= H, R₃=Cl

103 R₁= R₂=R₄=H, R₃=OH

104 R₁= R₃=R₄=H, R₂=Cl

107 R₁=Cl, R₂=H, R₃= CH₃

108 R₁=H, R₂=Cl, R₃= CH₃

109 R₁=H, R₂=Br, R₃= CH₃

110 R₁=Cl, R₂=H, R₃= CH₂CH₃

111 R₁=H, R₂=Cl, R₃=CH₂(C₆H₄Cl)

(g)

(h)

N

(e)

R₁

N

O

R₄

NH

R₁

X

R₂

R₃

NO₂

R₅

R

R₂

R₄

O

105 R = H106 R = CH₃

R₃

112 R₁=R₃=R₄=H, R₂=Cl, R₅=CH₃

113 R₁=R₂=C₄=H, R₃=Br, R₄=CH₃

95 R₁=R₂=R₃=H, R₄=CH₂CH₃, X=S

96 R₁=OCH₃, R₂=H, R₃=Br, R₄=CH₃, X=S

97 R₁=OCH₃, R₂=H, R₃=Br, R₄=C₆H₅, X=S

98 R₁=R₂=R₃=H, R₄=CH₆CH₅, X=NH

99 R₁=R₃=H, R₂=Cl, R₄=C₆H₅, X=NH

100 R₁=R₃=H, R₂=Br, R₄=C₆H₅, X=NH

114 R₁=OCH₃, R₂=R₃=H, R₄=Br, R₅=CH₃

115 R₁=R₄=OCG₃, R₂=R₃=H, R₅=CH₃

116 R₁=R₂=R₄=H, R₃=Cl, R₅=CH₃

117 R₁=R₃=R₄=H, R₂=Cl, R₅=CH₂CH₃

118 R₁=OCH₃, R₂=R₃=H, R₄=Br, R₅=CH₂CH₃

119 R₁=R₃=R₄=H, R₂=Cl, R₅=C(OH)CO₂CH₂CH₃

Scheme 2. Chemistry of nitrostyrenes (Type V) Scheme reagents and conditions: (a) NaBH₄, silica gel, CH₃CH(OH)CH₃, CH₂Cl₂, RT; (b)

R₂C₆H₄CHO, HN(CH₃)₂.HCl, KF, toluene, 24 h, reflux; (c) C₆H₅COCH₂NO₂, ꢀ-Alanine, C₆H₆, CH₃CO₂H, reflux; (d) (CH₃)₃C-OOH, DBU,

RT 20 min; (e) R₄-X-H, X=S, NH, H₂O, RT, 20 h; (f) DMAP, CH₃CN, RT, 2-7 days; (g) NaN₃, DMSO, 90ꢀC, 1 h; (h) (NH₄)₂Ce(NO₃)₆,

CH₃CN, H₂O, 10 min, RT.

R₁

R₄

R₁

R₄

(b)

R₂

R₃

CN

(a)

R₂

R₃

CHO

R₂

R₃

NH₂

N

R₅

R₄

130 R₁=R₂=R₃=R₄=H

120 R₁=R₂=R₄=H, R₃=Cl, R₅=CO₂CH₃

131 R₁=R₃=R₄=H, R₂=Cl

132 R₁=R₂=R₄=H, R₃=Cl

121 R₁=OCH₃, R₄=Br, R-R₃=H, R₅=CO₂CH₃

122 R₁=R₃=R₄=H, R₂=Cl, R₅=CO₂CH₃

123 R₁=R₂=R₄=H, R₃=Cl, R₅=CO₂CH₂CH₃

124 R₁=OCH₃=R₄=Br, R₂=R₃=H, R₅=CO₂CH₂CH₃

125 R₁=R₃=R₄=H,R₂=Cl, R₅=CO₂CH₂CH₃

126 R₁=R₂=R₄=H, R₃=Cl, R₅=CN

133 R₁=OCH₃, R₂=R₃=H, R₄=Br

134 R₁=R₂=R₄=H, R₃=Br

127 R₁=R₂=R₄=H, R₃=Br, R₅=CN

128 R₁=OCH₃=R₄=Br, R₂=R₃=H, R₅=CN

129 R₁=R₃=R₄=H, R₂=Cl, R₅=CN

Scheme 3. Acrylonitriles and hydrazones (Type V) Scheme reagents and conditions: (a) R₅CH₂CN, piperidine, 0.5-1 h, reflux for compounds

126-129; 80ꢀC. 30 min microwave for compounds 120-125; (b) NH₂NH₂, ethanol, 3 h.

192

Medicinal Chemistry, 2018, Vol. 14, No. 2

Byrne et al.

R₅

R₄

NO₂

R₅

R₄

CHO

XH

(a)

X

R₂

R₃

135-149

142 X=O, R₁R₂=C₄H₂

143 X=O, R₃=OCH₃, R₂=Cl

144 X=O, R₃=OCH₃

145 X=O, R₄=OCH₃

146 X=O, R₅=Cl

R₂

R₁

R₃

R₁

135 X=O

136 X=O, R₂=Cl

137 X=O, R₂=OH

138 X=O, R₂=OCH₂C₆H₅

139 X=O, R₁R₂=OCH₂O

140 X=O, R₁=R₂=OCH₃

141 X=O, R₄=OH

147 X=O, R₃=Cl

148 X=NH

149 X=NH, R₂=OH

Scheme 4. Preparation of 3-nitro-2-phenyl-2H-chromenes and 2-nitro-1,2-dihydroquinolines 135-149 (Type VI). Scheme reagents and condi-

tions: (a) 1,4-diazabicyclo[2.2.2]octane (DABCO), 60 ^oC, 1.5 h R=H unless otherwise specified.

O

A

O

OH

O

NO₂

R₄

R₃

CH₃

(b)

(c)

(a)

R₄

R₃

R₄

R₃

R₅

O

R₅

O

R₅

OH R₁

R₅

O

R₄

R₂

R₁

O

R₁

R₂

R₁

R₃

R₂

179-188

161-169

170-178

152-160

170 R₁=R₂=R₃=R₄=R₅=H=

171 R₃=Cl

179 R₁=R₂=R₃=R₄=R₅=H

180 R₃=Cl

152 R₁=R₂=R₃=R₄=R₅=H

153 R₃=Cl

161 R₁=R₂=R₃=R₄=R₅=H

162 R₁₃=Cl

172 R₅=R₃=Cl

181 R₅=R₃=Cl

154 R₅=R₃=Cl

163 R₅=R₃=Cl

173 R₁=NO₂, R₃=OCH₃

174 R₂=R₃=R₄=OCH₃

175 R₁=NO₂, R₂=R₃=R₄=OCH₃

176 R₅=R₃=OCH₃

177 R₂=Cl

182 R₂=NO₂, R₃=OCH₃

184 R₂=R₃=R₄=OCH₃

155 R₂=NO₂, R₃=OCH₃

156 R₂=R₃=R₄=OCH₃

164 R₂=NO₂, R₃=OCH₃

165 R₂=R₃=R₄=OCH₃

185 R₁=NO₂, R₂=R₃=R₄=OCH₃

186 R₅=R₃=OCH₃

187 R₂=Cl

157 R₁=NO₂=R₂=R₃=OCH₃

158 R₅=R₃=OCH₃

597 R₂=Cl

166 R₁=NO₂, R₂=R₃=R₄=OCH₃

167 R₁=R₃=OCH₃

168 R₂=Cl

178 R₅=OCH₃

187 R₅=OCH₃

160 R₅=OCH₃

169 R₅=OCH₃

O

N

NO₂

S

B

NO₂

O₂N

NO₂

N

H

N

H

N

H

O

150

193

151

189

190

Scheme 5. (A) 3-Nitro-2-phenylchromene-4-one library (Type VI). Scheme reagents and conditions: (a) pyridine, KOH, 50°C, 20 min; (b)

H₂SO₄, CH₃CO₂H, 1 h, reflux; (c) NH₄NO₃, (CF₃CO)₂O, CH₂Cl₂, CH₃CN, 1.5 h, 0-40°C. (B) Nitro-substituted heterocycles 150, 151, 189,

190 and 193.

steps to give the 3-nitro-2-phenyl-2H-chromenes (135-147)

and 3-nitro-1,2-dihydroquinolines (148-149), Scheme 4

[39, 40]. The demethylation of 7-methoxy-3-nitro-2-phenyl-

2H-chromene (145) was achieved by heating with pyridine

hydrochloride neat to give 3-nitro-2-phenyl-2H-chromen-7-

ol (141).

A series of 2-substituted-3-phenylacrylonitriles (120-

129) was prepared by Knoevenagel condensation of the

required benzaldehyde with activated cyanomethylene com-

pounds (Scheme 3). The related benzylidenehydrazones 130-

134 were also prepared for biochemical investigation.

A series of 3-nitro-2-phenyl-2H-chromenes (135-147)

and 3-nitro-1,2-dihydroquinolines (148-149) containing the

(E)-nitrostyrene pharmacophore were synthesised from the

appropriate (E)-(2-nitrovinyl)benzenes(Type VI). The syn-

thetic strategy chosen involves a one-pot Baylis-Hillman

reaction of a (E)-(2-nitrovinyl)benzenes with appropriately

substituted salicylaldehydes or 2-amino-benzaldehydes cata-

lyzed by 1,4-diazabicyclo[2.2.2]octane (DABCO), which

takes place in tandem with the cyclisation and dehydration

The flavonoids have proven a versatile source of poten-

tial therapeutic agents to inhibit the growth of various types

of cancer cells in vivo and in vitro [41, 42]. In the present

study, the 3-nitro-2-phenylchromen-4-ones (179-188) were

prepared which contain the (2-nitrovinyl)benzene pharma-

cophore as a substructure, with a constrained (Z) stereochem-

istry for the C-3 group and the C-2 aryl ring at of the

chromene-4-one ring (Type VI, Scheme 5A). The 2-phenyl-

Synthesis, Antiproliferative and Pro-Apoptotic Effects

Medicinal Chemistry, 2018, Vol. 14, No. 2 193

1-chromen-4-ones (170-178) were obtained by the acid cata-

lysed intramolecular cyclisation of the 3-hydroxy-3-

phenylprop-2-en-1-ones (161-169), (prepared from the cor-

responding O-benzyloxyacetophenones (152-160) by treat-

ment with KOH and pyridine). These flavones are then sub-

sequently nitrated in a solution of ammonium nitrate and

trifluoroacetic anhydride (TFAA) [43] to afford the 3-nitro-

2-phenylchromen-4-ones, 179-188. Nitration side-products

2-(4-methoxy-3-nitrophenyl)-3-nitro-4H-chromen-4-one

(182) and 3-nitro-2-(3,4,5-trimethoxy-2-nitrophenyl)-4H-

chromen-4-one (185) were also isolated. The following het-

erocycles containing the nitrostyrene pharmacophore were

also prepared by reported procedures for inclusion in the

biochemical study: 3-nitro-2-phenylquinoline (150) [44], 3-

nitroquinolin-2(1H)-one (151) [45], 3-nitro-2,4-diphenyl-

2,5-dihydrobenzo[b][1,4]thiazepine (189) [46], 3-nitro-2-

phenyl-1H-indole (190) [47] and 4-nitro-3,5-diphenyl-

isoxazole (193) [48], (Scheme 5B).

methyl and ethyl generally leads to increased anti-

proliferative effects; this trend continued throughout the

screen with some exceptions particularly in the anthracene,

bis-nitrovinyl, furan and C-2 thiophene based nitrovinyl

compounds, (See Supplementary Information, Tables S1-

S3). Some examples of this trend include compounds (9, 54,

66), (1, 35, 60) and (2, 36, 61). Less active compounds in-

clude the 2-hydroxy-5-methoxyphenyl 70, while the

naphthyl 41 and 3-benzyloxyphenyl compound 42 were in-

active (>80% viability in both cell lines at 10 ꢁM).

The results for the preliminary in vitro antiproliferative

screen for some examples of the (E)-2-nitroprop-1-en-1-

yl)benzenes, compounds (50, 59, 44, 47, 48, 54, 58) (Type

II) are presented in Fig. (4B). Compound 54 exhibited the

most promising anti-proliferative activity and induced a cy-

totoxic effect at 1 ꢁM in both cell lines with only ~55% vi-

able cells remaining in the MUTU-I cell line and ~82% in

the DG-75 cell line. Replacing the C-2 methoxy substitution

observed in compound 45 with a C-2 hydroxy substituent

(58) reduced the anti-proliferative activity in the chemoresis-

tant DG-75 cell line at 10 ꢁM by ~20%.

3.2. Biochemical Evaluation

Burkitt’s lymphoma MUTU-I (positive for Epstein-Barr

virus) and chemoresistant DG-75 cell lines (negative for Ep-

stein-Barr virus) were treated with 10 ꢀM and 1 ꢁM of each

compound and assessed for viability via the Alamar Blue

assay. The full results of this screen are represented

in Tables S1-S6, Supplementary Information. Compound

85 ((E)-1-chloro-3-(3-(4-chlorophenyl)-2-nitroprop-1-en-1-

yl) benzene), the lead compound was included as a control in

the screen. For Type I compounds (1-34), which are nitrosty-

renes with aryl substitution, the majority of compounds were

found to be active, with the viability of both cell lines falling

to 50% or below when treated with 10 ꢁM of each com-

pound. Less active compounds (>50% viability in 1 cell line

at 10 ꢁM) include 3, 11, 13, 18, 20, 21 and 23. A number of

the more potent (E)-(2-nitrovinyl)benzenes possessed C-3

halogen or hydroxy and C-2 alkoxy substitutions and these

characteristics were found to be favourable for promoting

anti-proliferative activity e.g. compounds (9, 14, 17, 19, 26,

32) in both cell lines (See Supplementary Information, Table

S1).

The results for the preliminary in vitro antiproliferative

screen for some selected examples of the (E)-2-nitrobut1-en-

1-yl)benzenes (Type III) are presented in Fig. (4C) while

related naphthyl, furfuryl and thienyl compounds are dis-

played in Fig. (5A). Introduction of an ethyl substituent to

the C-4 chloro nitrostyrene 60 once again increased the anti-

proliferative effect in both the DG-75 (~80% 1 ꢁM) and

MUTU-I (~59% 1 ꢁM) cell lines when compared to the pre-

vious lead compound 85, (Fig. 4C). Compound 66 possessed

the most potent anti-proliferative activity with good activity

at 1 ꢁM in both cell lines (MUTU-I ~20%, DG-75 ~67%). In

a study of the position of chloro substituent it was observed

that C-3 and C-4 chloro substitutions (60, 69) were more

favourable for increased anti-proliferative activity in both

cell lines over the C-2 chloro analogue (67). Increased elec-

tronegativity provided by fluoro substitution (63) was shown

to decrease the anticancer effect in the MUTU-I cell line to

~26% viable cells remaining at 10 ꢁM, (Fig. 4C). By com-

paring the results obtained by the hydroxy compounds (68,

70, 61) it was concluded that C-3 hydroxyl substitution

(compound 68, 1 ꢁM ~64%) was more favourable for anti-

proliferative behaviour in the MUTU-I cell line over its C-4

analogue (61, 1 ꢁM ~80%), (Fig. 4C).

The naphthalene based (E)-(2-nitrobut-1-en-1-yl)ben-

zenes 62 and 65 exhibited significant anti-proliferative activ-

ity in both cell lines at the higher concentration with ~2-35%

viable cells remaining after treatment (Fig. 5A). The C-3

thiophene based analogue (80) induced a ~17% response at

10 ꢁM in the DG-75 cell line outperforming its furan based

analogue (79) and the C-2 thiophene analogues (81, 82)

which had >50% viable cells remaining, (Fig. 5A). For Type

IV compounds, nitrostyrenes with a heterocycle substitution

(71-82), compounds with the methyl and ethyl substituents

tended to be less active (see Supplementary Information,

Table S4).

The anti-proliferative effect of selected alkoxy and phe-

nolic substituted (E)-(2-nitrovinyl)benzenes (Type I) is dis-

played in Fig. (4A). Compounds 9 and 32 (both bearing meta

bromo and a para/ortho methoxy substituent) elicited potent

anticancer effects at both concentrations in the MUTU-I cell

lines. Compound 9 with a C-2 methoxy substituent proved

more active than its C-4 methoxy analogue compound 32.

Compound 9 was the most active in the chemoresistant DG-

75 cell line of this cohort also, with below 20% viable cells

remaining at 10 ꢁM and reducing the amount of viable cells

in the experiments to ~80% at 1 ꢁM. Compound 26 (C-3

hydroxy) was found to be the most active of the hydroxy

substituted compounds (e.g. 2, 13, 26, 27, 31) with <15% of

viable cells remaining in both lines at 10 ꢁM.

Similar results for Type II and III compounds, methyl

(35-59) and ethyl (60-70) nitrostyrenes with aryl substitution

were also found with the majority of these compounds sig-

nificantly reducing cell viability. Increasing the alkyl chain

length at the vinyl C-2 of the nitrostyrene pharmacophore to

The (E)-1,3-bis(aryl)-2-nitro-1-propenes 85-87, 1-

subsituted-(2-nitroethyl)benzenes 97, 99, 100, and ethyl (E)-

2-hydroxy-3-nitro-4-phenylbut-3-enoate 101-104 proved to

be the most active modified nitrostyrenes from the Type V

194

Medicinal Chemistry, 2018, Vol. 14, No. 2

Byrne et al.

A

MUTU-I Cell line (24 h)

DG-75 Cell line (48 h)

100

80

60

40

20

0

100

80

60

40

20

0

m)

M)

ꢀ

0

9 (1

2 (1

9 (1

2 (1

31 (1

85 (1

32 (1

33 (1

13 (1 9 (1

2 (1

27 (1

26 (1

85 (1

31 (1

32 (1

33 (1

13 (1 9 (1

2 (1

27 (1

26 (1

13 (10

85 (10

31 (10

32 (10

33 (10

27 (10

26 (10

85 (10

31 (10

32 (10

33 (10

13 (10

27 (10

26 (10

Compound # (Treatment Concentration ꢀM)

B

MUTU-I Cell line (24 h)

DG-75 Cell line (48 h)

100

80

60

40

20

0

100

80

60

40

20

0

M)

ꢀ

1

(

60 (1

68

85 (1

69 (1

67 (1

63 (1

61 (1

70 (1

66 (1

85 (1

60 (1

69 (1

67 (1

63 (1

61 (1

68 (1

70 (1 66 (1

66 (10

85 (10

60 (10

69 (10

67 (10

63 (10

61 (10

68 (10

70 (10

66 (10

85 (10

60 (10

69 (10

67 (10

63 (10

61 (10

68 (10

70 (10

Compound # (Treatment Concentration ꢀM)

C

MUTU-I Cell line (24 h)

DG-75 Cell line (48 h)

100

80

60

40

20

0

100

80

60

40

20

0

m)

M)

ꢀ

50 (1

59 (10

85 (1

59 (1

44 (1

47 (1

48 (1

54 (1

58 (1

85 (1

59 (1

44 (1

47 (1

48 (1

54 (1

58 (10

58 (1

47 (10

85 (10

50 (10

59 (10

44 (10

48 (10

54 (10

58 (10

85 (10

50 (10

44 (10

47 (10

48 (10

54 (10

Compound # (Treatment Concentration ꢀM)

Fig. (4). Preliminary screen for anti-proliferative activity of selected (E)-(2-nitrovinyl)benzenes (Type I) selected (A), (E)-(2-nitroprop-1-en-

1-yl)benzenes (Type II) (B) and (E)-(2-nitrobut-1-en-1-yl)benzenes (Type III) (C).

were identified as the most potent compounds evaluated con-

taining a constrained nitrostyrene pharmacophore together

with hydroxyl and alkoxy substituents on the aryl rings.

These compounds were found to exert anti-proliferative ef-

fects comparable to the previous lead compound 85, but less

active when compared to the nitrostyrenes with aryl substitu-

tions such as 66 (see Fig. 5C and Supporting Information).

Contrasting the heterocyclic structures evaluated, it was con-

cluded that compounds inducing a constrained trans (E) con-

figuration on (2-nitrovinyl)benzene (nitrostyrene) and 1,3-

bis(aryl)-2-nitro-1-propene scaffolds (135-148) were more

potent than compounds inducing a cis (Z) configuration (179

- 188, 189, 190, 193), 3-nitro-1,2-dihydroquinolines (148,

149) or nitro-substituted heterocycles (150, 151) (Table S6).

Compound 145 (C-7 methoxy chromene ring) exerted the

category, indicating the requirement for the preservation of

the nitrostyrene pharmacophore, (Fig. 5B). Reduction of the

nitrostyrenes (1, 10, 85) to their nitroalkane adducts (83, 84,

89) inactivated the pharmacophore while the modified cya-

noacrylates (120-129) and hydrazones (130-134) proved

largely inactive or minimally active (see Supporting Infor-

mation, Table S5).

The anticancer effects of flavonoids have been reported

including 3-nitro-2H-chromenes in A549 cancer cells [49],

3-nitroquinolines and 3-nitro-2-phenyl-2H-chromenes in

breast cancer cell lines MCF-7 and MDA-MB-468 [50, 51].

Of the flavonoid and chromene derivatives making up the

last type of compounds in this screen, Type VI, the nitro-2-

phenyl-2H-chromenes 135, 137, 139, 140, 144, 146 and 147

Synthesis, Antiproliferative and Pro-Apoptotic Effects

Medicinal Chemistry, 2018, Vol. 14, No. 2 195

MUTU-I Cell line (24 h)

DG-75 Cell line (48 h)

A

100

80

60

40

20

0

40

20

0

Compound # (Treatment Concentration mM)

MUTU-I Cell line (24 h)

DG-75 Cell line (48 h)

B

100

80

60

80

60

40

20

0

40

20

0

Compound # (Treatment Concentration mM)

MUTU-I Cell line (24 h)

DG-75 Cell line (48 h)

C

100

80

60

40

20

0

100

80

60

40

20

0

Compound # (Treatment Concentration mM)

Fig. (5). Preliminary screen for anti-proliferative activity of selected (E)-(2-nitrobut-1-en-1-yl)benzenes (Type IV) (A), ethyl (E)-2-hydroxy-

3-nitro-4-phenylbut-3-enoates (Type V) (B) and 3-nitro-2-phenyl-2H-chromenes (Type VI) (C).

greatest anti-proliferative effect in the DG-75 cell line at 1

ꢁM with 58% viable cells remaining. Compound 141 (the

phenol analogue of 145) was relatively inactive with >85%

viable cells were remaining with this analogue. A number of

other chromene ring substitutions (C-6 / C-8 chloro, C-8

methoxy) were also evaluated and found to possess good

anti-proliferative effects in both BL lines at 10 ꢁM (<10%)

(146, 147, 144, 143). The quinoline and quinolone analogues

(149, 150, 151) possessed little anti-proliferative effects at

both concentrations (>90% remaining). Compound 148 was

the most promising quinoline with activity at 10 ꢁM in both

cell lines (~45% viable cells remaining).

Pgp substrate status, blood-brain barrier partition, plasma

protein binding and human intestinal absorption properties

(see Supplementary Information, Tables S7 and S8). All

compounds selected for further analysis were found to sat-

isfy all the Lipinski rule of five criteria, with no particular

correlation observed between anti-proliferative activity and

lipophilicity. The previous lead compound 85, did not have

an optimal logP value (5.377). Compounds 7, 9, 17, 19 and

26 (Type I) were selected from the initial (E)-(2-nitrovinyl)

benzene library, compounds 35, 36, 54, 55, 57 and 59 (Type

II) were selected from the (E)-(2-nitroprop-1-en-1-yl)ben-

zene library. The (E)-(2-nitrobut-1-en-1-yl)benzene library

provided the active compounds 60, 61, 66, 67 and 69 (Type

III). All of these compounds contained aryl substitu-

tions(OH, OR, Br or Cl). The ester 101 (Type V) represented

the most active ethyl (E)-2-hydroxy-3-nitro-4-phenylbut-3-

enoates, and compounds 137 and 145 (Type VI) represented

the most active 3-nitro-2-phenyl-2H-chromene. IC₅₀values

From the initial BL screen of >190 compounds, 19 of the

more active compounds were selected for further detailed

biological analysis and determination of IC₅₀values for each

selected compound against the BL cell lines (Table 3), fol-

lowing analysis of their drug-like (Lipinski) properties, to-

gether with predictions of permeability, metabolic stability,

196

Medicinal Chemistry, 2018, Vol. 14, No. 2

Byrne et al.

Table 3. IC₅₀values in Burkitt’s lymphoma cell lines MUTU-1 and DG 75 of the most potent members of the nitrostyrene screen.

R= H unless otherwise stated.

IC₅₀Value

Name

General Structure

#

logP

R Group

MUTU-I

DG-75

(ꢀ M)

Paclitaxel

Taxol

7

7.380

4.763

3.228

2.474

2.856

2.163

3.501

2.553

3.618

3.501

3.246

3.891

2.943

4.009

3.891

0.30

2.57

0.88

0.79

0.75

5.65

1.30

2.18

0.77

0.88

0.82

0.98

1.18

1.68

0.45

2.18

1.22

1.32

2.08

3.97

3.74

3.20

8.43

2.75

5.57

1.66

2.25

2.75

3.30

3.11

3.35

1.41

2.05

2.36

(E)-(2-Nitrovinyl)benzene

R₂R₃= R₅R₆= C₄H₄

R₂= OCH_3,R₅= Br

R₂= R₅= OCH₃

R₂= OCH₂CH₃

R₃= OH

R₂

(Type I)

9

R₃

NO₂

17

19

26

35

36

54

55

57

59

60

61

66

67

69

R₄

R₆

R₅

(E)-(2-Nitroprop-1-en-1-yl)benzene

R₄= Cl

(Type II)

R₄= OH

R₂

R₅

R₃

R₄

NO₂

CH₃

R₅= Br, R₂= OCH₃

R₃= Cl

R₂= Cl

R₄= OCH₂CH₃

R₄= Cl

(E)-(2-Nitrobut-1-en-1-yl)benzene

R₂

R₅

(Type III)

R₄= OH

R₃

R₄

NO₂

CH₃

R₂= OCH₃, R₅= Br

R₂= Cl

R₃= Cl

NO₂

(E)-4,4'-(2-Nitroprop-1-ene-1,3-

diyl)bis (chlorobenzene)

85

5.377

2.523

-

2.97

2.29

6.39

2.66

Cl

R₆

Ethyl (E)-2-hydroxy-3-nitro-4-

phenylbut-3-enoate

Cl

(Type V)

R₁

R₄

R₂

NO₂

101

R₁= OCH₃, R₄= Br

O

CH₃

R₃

HO

O

NO₂

3-Nitro-2-phenyl-2H-chromenes

137

145

3.666

3.969

R₂= OH

0.87

0.47

2.54

1.92

(Type VI)

R₁

O

R₁= OCH₃

R₂

were also obtained for the previous lead nitropropene com-

pound 85 (2.82-6.03 ꢁM) in the BL cell lines and the che-

motherapeutic agent taxol (IC₅₀values 0.32-1.32 ꢁM) was

used as a control drug. Most of the nitrostyrene compounds

evaluated gave IC₅₀values <6 ꢁM in the BL cell lines, with

many <1 ꢁM (Table 3). The IC₅₀determination identified

compound 66 (Type III) as the most potent anti-proliferative

agent with an IC₅₀of 0.45 ꢁM in the MUTU-I and 1.26 ꢁM

in the DG-75. The 3-nitro-2-phenyl-2H-chromene compound

145 was also identified as potent with an IC₅₀of 0.47 ꢁM in

the MUTU-I and 1.92 ꢁM in the DG-75. Flexible alignment

of compounds 66 and 85 as examples of the most potent

compounds shows the common nitrostyrene pharmacophore

(see Figs. 1 and 2, Supplementary Information); a similar

result has been observed for flexible alignment of compound

66 and 145. The close correspondence between overlays of

these compounds highlights the presence of three main

shared molecular features: the (E)-configuration nitrovinyl

unit, an aromatic/aliphatic functionality adjacent to the nitro

group and the presence of an aromatic core. In addition,

compounds 66 and 145 possess a hydrogen bond acceptor in

the form of an ether oxygen atom.

Synthesis, Antiproliferative and Pro-Apoptotic Effects

Medicinal Chemistry, 2018, Vol. 14, No. 2 197

MUTU-I cell line (24 h)

DG-75 cell line (48 h)

100

80

60

40

20

0

100

80

60

40

20

0

e

l

c

M)

ꢀ

M)

ꢀ

hi

ve

hi

ve

0

5 (1

14

5 (1

14

85 (10

XOL (1

TA

XOL (1

TA

Compound # (Treatment Concentration)

Fig. (6). Nitrostyrene compounds 85 and 145 potently induce apopotosis in MUTU-I and DG-75 cell lines (Annexin V / PI FACS).

Nitrovinyl pharmacophore

R₁= Br, Cl, OH

NO₂

R₁

NO₂

R₄

O

R₂

O

R

R₃

R₂

Range of aryl/

heteroaryl rings tolerated

R₁

R₂= H < CH₃< C₂H₅

R₄= OCH₃

R₃= OCH₃, Cl

R₂= Cl, OCH₃, OH

R = CH₃< C₂H₅

R₁= OCH₃,

R₁R₂= OCH₂O, -CH=CH-CH=CH^-

Fig. (7). Optimal SAR for nitrostyrenes (1-82, Type I – IV) and related 2-phenyl-2H-chromenes (143-147, Type VI) in Burkitt’s lymphoma.

To examine the potential anti-proliferative effects of

these nitrostyrene type compounds, the ability of the most

potent compounds identified from the cell viability study to

induce apoptosis in the MUTU-1 and DG-75 cell lines was

investigated using Annexin V and propidium iodide staining,

initially using 10 ꢀM concentration. The chemotherapeutic

drug Taxol was used as a positive control. In the MUTU-I

cell line, the selected 3-nitro-2-phenyl-2H-chromene

compound 145 elicited a significant increase in apoptosis

(>80%) comparable to the effect induced by Taxol at the

same concentration, and slightly greater than that observed

for the initial lead 1,3-bis(aryl)-2-nitro-1-propene compound

85, (Fig. 6). The mode of cell death was identified as apop-

totic and 3-nitro-2-phenyl-2H-chromene compound 145 was

found to elicit comparable apoptotic effects to Taxol (>80%)

at both 10 ꢁM and 1 ꢁM concentrations. This antiprolifera-

tive effect was superior to that of our previous lead com-

pound 85 at 1 ꢁM (~40%) [9], possibly due to the difference

in the electronic effect of the methoxy group in 145 com-

pared to the chloro substituent in 85. In the DG-75 cell line,

the 1,3-bis(aryl)-2-nitro-1-propene compound 85 was found

to induce ~40% apoptosis at 10 ꢀM, while compound 145

produced a significant increase in apoptosis (>85%) which is

double that of compound 85. A similar increase in apoptosis

(>90% in MUTU-1 cells and >80% in DG-75 cells) was

observed on treatment with the nitrostyrene compound 66 at

10 ꢀM. The effect of these compounds was compared

favourablely with Taxol at this concentration (MUTU-I

>80%, DG-75 >60%). The identification of compounds

which can induce programmed cell death (PCD) or apoptosis

in cancer cells is potentially important in the development of

effective anticancer drugs. Collectively, these results suggest

the suitability of this cohort of selected nitrostyrene com-

pounds for further development.

Overall, of the large number of compounds analysed in

this screen, by far the most promising nitrostyrene structures

were those with aryl substitutions (Type I – III) and the 3-

nitro-2-phenyl-2H-chromenes(Type VI), indicating the sig-

nificant contribution of the nitrostyrene pharmacophore to

the activity of the compounds. This biochemical library also

allowed for the development of a structure-activity relation-

ship (SAR) and an optimised nitrosytrene analogue for anti-

proliferative activity. This optimized structure contains a

phenyl ring (C-5 halogen of hydroxyl, and a C-2 alkoxy sub-

stituent), a nitrovinyl unit and an extended ethyl chain at C-

2, or constrained in the 2-phenyl-2H-chromene structure as

illustrated in Fig. (7).

198

Medicinal Chemistry, 2018, Vol. 14, No. 2

Byrne et al.

Supplementary material is available on the publisher’s

web site along with the published article.

CONCLUSION

In this study, we have evaluated the potential activity of

an extensive series of nitrostyrenes and related compounds

as antiproliferative agents specifically useful against BL. A

comprehensive series of structurally varied nitrostyrene

based compounds (n=193) was synthesized and evaluated for

toxicity against two BL cell lines. The current therapies for

BL include cyclophosphamide/ hydroxydaunorubicin/on-

covin/prednisone (CHOP) or CHOP + rituximab (R-CHOP).

There are significant issues associated with these drugs in-

cluding the development of resistance and an unmet need is

emerging for alternative drug therapies for these diseases. In

the present study, the most active nitrostyrene compounds

evaluated potently reduce cellular viability in Burkitt’s lym-

phoma cells. The E-nitrovinyl pharmacophore when incorpo-

rated into a simple aryl scaffold structure demonstrated sig-

nificant antiproliferative activity in both BL cell lines

(MUTU-1 and the chemoresistant DG-75) with IC₅₀values

in the low micromolar range for the most active compounds.

The is the first report of the synthesis of a number of the

compounds, while for compounds whose synthesis has pre-

viously been reported, this is the first account of their poten-

tial for the treatment of BL. Optimized lead compounds (E)-

(2-nitrobut-1-en-1-yl)benzene 66 and 2-phenyl-2H-chromene

145 were identified and will form the basis of future devel-

opment for modified structures with activity in Burkitt’s

lymphoma.

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Article Doi

Synthesis, antiproliferative and pro-apoptotic effects of nitrostyrenes and related compounds in Burkitt’s lymphoma

DOI: 10.2174/1573406413666171002123907

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