Toward highly potent cancer agents by modulating the C-2 group of the arylthioindole class of tubulin polymerization inhibitors

Article

pubs.acs.org/jmc

Toward Highly Potent Cancer Agents by Modulating the C‑2 Group

of the Arylthioindole Class of Tubulin Polymerization Inhibitors

†

‡

§

†

Giuseppe La Regina, Ruoli Bai, Whilelmina Maria Rensen, Erica Di Cesare, Antonio Coluccia,

†

∥

†

●

Francesco Piscitelli, Valeria Famiglini, Alessia Reggio, Marianna Nalli, Sveva Pelliccia,

∥

⊥

Eleonora Da Pozzo, Barbara Costa, Ilaria Granata, Amalia Porta, Bruno Maresca,

×

×,§

#

Alessandra Soriani, Maria Luisa Iannitto, Angela Santoni, Junjie Li, Marlein Miranda Cona,

#

∞

○

◆

Feng Chen, Yicheng Ni, Andrea Brancale, Giulio Dondio, Stefania Vultaggio, Mario Varasi,

Ciro Mercurio, Claudia Martini, Ernest Hamel, Patrizia Lavia, Ettore Novellino,

and Romano Silvestri*

△

∥

‡

§

●

,

†

Dipartimento di Chimica e Tecnologie del Farmaco, Istituto PasteurFondazione Cenci Bolognetti, Sapienza Universita

̀

di Roma,

Piazzale Aldo Moro 5, I-00185 Roma, Italy

‡

Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick

National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702,

United States

§

Institute of Molecular Biology and Pathology (IBPM), CNR National Research Council of Italy, c/o Sapienza Universita

Via degli Apuli 4, I-00185 Roma, Italy

̀

di Roma,

∥

Department of Psychiatry, Neurobiology, Pharmacology, and Biotechnology, University of Pisa, Via Bonanno Pisano 6, I-56126 Pisa, Italy

̂

⊥

̀

Dipartimento di Scienze Farmaceutiche, Sezione Biomedica, Universita di Salerno, Via Ponte don Melillo, I-84084 Fisciano, Salerno, Italy

#

Theragnostic Laboratory, Department of Imaging and Pathology, Faculty of Medicine, Biomedical Sciences Group, KU Leuven,

Herestraat 49, B-3000 Leuven, Belgium

∞

Welsh School of Pharmacy, Cardiﬀ University, King Edward VII Avenue, Cardiﬀ, CF10 3NB, U.K.

^●Dipartimento di Chimica Farmaceutica e Tossicologica, Universita

̀

di Napoli Federico II, Via Domenico Montesano 49, I-80131,

Napoli, Italy

×

Dipartimento di Medicina Sperimentale e Patologia, Sapienza Universita

̀

di Roma, Viale Regina Elena 324, I-00161 Roma, Italy

○

NiKem Research Srl, Via Zambeletti 25, I-20021 Baranzate, Milano, Italy

^◆European Institute of Oncology, Via Adamello 16, I-20139 Milano, Italy

^△Genextra Group, DAC SRL, Via Adamello 16, I-20139 Milano, Italy

*

S Supporting Information

ABSTRACT: New arylthioindole derivatives having diﬀerent

cyclic substituents at position 2 of the indole were synthesized

as anticancer agents. Several compounds inhibited tubulin

polymerization at submicromolar concentration and inhibited

cell growth at low nanomolar concentrations. Compounds 18

and 57 were superior to the previously synthesized 5. Com-

pound 18 was exceptionally potent as an inhibitor of cell

growth: it showed IC₅₀= 1.0 nM in MCF-7 cells, and it was

uniformly active in the whole panel of cancer cells and superior

to colchicine and combretastatin A-4. Compounds 18, 20, 55,

and 57 were notably more potent than vinorelbine, vinblastine,

and paclitaxel in the NCI/ADR-RES and Messa/Dx5 cell lines,

which overexpress P-glycoprotein. Compounds 18 and 57

showed initial vascular disrupting eﬀects in a tumor model of liver rhabdomyosarcomas at 15 mg/kg intravenous dosage.

Derivative 18 showed water solubility and higher metabolic stability than 5 in human liver microsomes.

INTRODUCTION

■

Microtubules (MTs) are involved in many essential cellular

functions, e.g., the maintenance of cell shape, cell motility,

Received: September 11, 2012

©

XXXX American Chemical Society

A

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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a

Chart 1. General Structure of ATI Derivatives 7−78 and Reference Compounds 1−6

a

R = azolyl, azinyl, phenyl, or substituted phenyl, naphthyl, biphenyl, benzofused heterocyclyl, alkyl, cycloalkyl; R = H; X = S, CO, CHOH, or

1

2

CH (Tables 1 and 2).

2

intracellular transport, and cell division. Cellular MTs undergo

continuous polymerization and depolymerization transitions.

Interference with this dynamic equilibrium, by either inhibiting

tubulin polymerization or blocking MT disassembly, prevents

proper MT function and ultimately leads to cell death. Because of

their crucial role in the formation of the mitotic spindle during

cell division, MTs are a highly attractive target for the develo-

proﬁle in the mouse characterized by low systemic clearance

and excellent oral bioavailability. On the basis of these observa-

tions, the current study was designed to explore further chemical

modiﬁcations of the ﬁve-membered heterocycle nucleus at

position 2 of the indole. We describe the synthesis and biological

evaluation of new ATI derivatives 7−78 with diﬀerent biososteric

rings at position 2, with a sulfur bridge between the ring systems

or with other bioisosteric groups at position 3 of the indole

(Table 1).

1

−5

pment of new eﬀective anticancer agents.

6

,7

Natural products such as colchicine (1), combretastatin A-4

8

(

CSA4, 2) (Chart 1), and the Catharanthus alkaloids vincristine

and vinblastine (VBL) inhibit MT assembly by preventing tubulin

polymerization. On the other hand, taxoids and epothilones

CHEMISTRY

■

9

,10

ATI derivatives 17, 18, 20, 27, 33, 34, 38, 39, 41, 43, 45−47, 53,

5, 57, 73, 75, and 77 were synthesized by microwave

target a lumenal site on the β-subunit. These drugs enter the

5

11

lumen through a binding site located at a pore on the MT

surface formed by diﬀerent tubulin heterodimers. At high con-

centrations, paclitaxel (PTX) stimulates MT polymerization and

stabilizes microtubules, whereas at lower concentrations it

inhibits MT dynamics with little eﬀect on the proportion of

reaction of an appropriate indole 79−87 with bis(3,4,5-tri-

methoxyphenyl)disulﬁde in the presence of sodium hydride in a

closed vessel at 110 °C while irradiating at 150 W for 2 min

Scheme 1). Compounds 14, 16, 37, 48, 59, 62, 72 were

obtained by heating at 60 °C in DMF the corresponding indoles

8−104 with bis(3,4,5-trimethoxyphenyl)disulﬁde in the

(

12

tubulin in polymer. Despite substantial interest in the develop-

9

13

ment of new MT-targeting drugs, several problems related to

drug resistance and secondary toxicity remain unresolved.

presence of sodium hydride. Acids 105 and 106 were similarly

transformed into the corresponding sulﬁdes 107 and 108,

respectively. Acid 107 was then converted into ATI 28 by

treatment with bromoethylamine hydrobromide in the presence

of benzotriazol-1-yloxytris(dimethylamino)phosphonium hexa-

ﬂuorophosphate (BOP reagent) and triethylamine. Iodo

derivative 108 was transformed into ATI 66 or 67 by heating

at 110 °C with an appropriate boronic acid pinacol ester in the

presence of 1,1′-bis(diphenylphosphino)ferrocene]dichloro-

palladium(II) complex with dichloromethane (Pd(dppf)-

Cl ·CH Cl ) and potassium phosphate in anhydrous DMF.

14

Therefore, new and improved tubulin inhibitors are needed.

Arylthioindoles (ATIs) are a class of potent inhibitors of

tubulin polymerization and cancer cell growth developed by our

1

5−19

research group.

The ATIs inhibit tubulin polymerization by

binding to the colchicine site, inhibiting the binding of

3

15

[

H]colchicine to tubulin.

19

In our previous work, we obtained new ATIs by replacing the

2

-alkoxycarbonyl group with a bioisosteric ﬁve-membered

heterocycle nucleus. The new ATIs (i.e., 3−6) inhibited tubulin

polymerization, inhibited the growth of a panel of human

transformed cell lines, and showed higher metabolic stability

than the reference ester (see example in Chart 1).

2

Organostannane reactions of 108 with 2-(triethylstannyl)-1H-

indole-1-carboxylic acid in the presence of bis(triphenylpho-

sphine)palladium(II) dichloride (PdCl (PPh ) ) in boiling

2

3 2

These new ATIs showed signiﬁcant therapeutic potential, as

they induced mitotic arrest and apoptosis similarly to 2 and VBL

and triggered caspase-3 expression in a signiﬁcant fraction of cells

in both p53-positive and p53-negative cell lines. Importantly,

some ATIs were more eﬀective than VRB, VBL, and PTX as

growth inhibitors of the P-glycoprotein-overexpressing cell line

NCI/ADR-RES. These compounds had a pharmacokinetic

ethanol for 48 h or 2-(tributylstannyl)oxazole, tris(dibenzyli-

deneacetone)dipalladium(0) (Pd (dba) ), and tricyclohexyl-

2

3

phosphine P(Cy) in anhydrous DMF at 150 °C and 150 W

3

20

aﬀorded 68 or 31, respectively. The synthesis of 69 was

performed by heating 108 at 100 °C in aqueous 1,4-dioxane in

the presence of 2-benzofuranylboronic acid, palladium(II)

acetate, and potassium phosphate. ATI 70 was synthesized by

B

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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a

Scheme 1

a

21

Reagents and reaction conditions: (a) (i) indoles 79−97 (R = imidazol-2-yl (79), imidazol-1-yl (80), 1,2,4-triazol-1-yl (81), thiazol-2-yl (82),

1

4,5-dihydro-1H-imidazol-2-yl (83), phenyl (84), 4-chlorophenyl (85), 2-ﬂuorophenyl (86), 3-ﬂuorophenyl (87), 4-ﬂuorophenyl (88), 2-methoxyphenyl

(89), 3-methoxyphenyl (90), 4-methoxyphenyl (91), pyridin-2-yl (92), pyridin-3-yl (93), pyridin-4-yl (94), cyclobutyl (95), cyclopentyl (96),

cyclohexyl (97)), NaH, anhydrous DMF, 25 °C, 10 min; (ii) closed vessel, 110 °C, 150 W, 2 min, yield 3−77%; (b) (i) indoles 98−104

2

(

R = pyrazol-4-yl (98), pyrazol-3-yl (99), 4-tolyl (100), 3-isopropoxy-4-methoxyphenyl (101), naphth-1-yl (102), naphth-2-yl (103), 1,1′-

1

2

3

biphenyl-4-yl (104), COOH (105), I (106) ), NaH, anhydrous DMF, 0 °C, 15 min, Ar stream; (ii) 60 °C, 12 h, Ar stream, 35−72%; (c) Et N,

3

BOP reagent, anhydrous DMF, 25 °C, 2 h, 30%; (d) 6-quinolineboronic acid pinacol ester (for 66) or 1-Boc-indole-5-boronic acid pinacol ester (for

24

6

7), Pd(dppf)Cl ·CH Cl , K PO , anhydrous DMF, Ar stream, 110 °C, 12 h, yield 26−70%; (e) 2-(triethylstannyl)-1H-indole-1-carboxylic acid

PdCl (PPh ) , ethanol, reﬂux, 78 °C, 48 h, Ar stream, yield 20%; (f) 2-(tributylstannyl)oxazole, Pd (dba) , P(Cy) , anhydrous DMF, closed vessel,

2

3

4

2

3

2

3

1

50 °C, 150 W, 10 min, yield 35%; (g) 2-benzofuranylboronic acid, Pd(OCOMe) , K PO , 1,4-dioxane/H O, 100 °C, 24 h, Ar stream, yield 88%;

2 3 4 2

(

h) benzo[b]thien-2-ylboronic acid, Pd(PPh ) , Na CO , THF, 60 °C, 24 h, Ar stream, yield 10%; (i) benzo[b]thien-3-ylboronic acid, Pd (dba) ,

3

4

2

3

2

3

SPhos, K PO , n-BuOH, Ar stream, 100 °C, 12 h, yield 36%.

3

4

reaction of 108 with benzo[b]thien-2-ylboronic acid in the pre-

solution in acetonitrile/methanol at 50 °C for 1 h aﬀorded the

corresponding methylene compounds 12, 22, 24, 26, 36, 52, 61,

and 65. Sodium hydroxide hydrolysis of 9, 11 (obtained by

sodium borohydride reduction of 9), or 12 in boiling methanol

provided ketone 8, 10, or 23, respectively.

sence of tetrakis(triphenylphosphine)palladium(0) (Pd(PPh ) )

3

4

and potassium carbonate in tetrahydrofuran at 60 °C. Com-

pound 71 was obtained from 108 and benzo[b]thien-3-ylboronic

acid in the presence of Pd (dba) , 2-dicyclohexylphosphino-

2

3

2

′,6′-dimethoxybiphenyl (SPhos), and potassium phosphate in

2-(1H-Pyrazol-4-yl)-1H-indole (98) was synthesized by

microwave reaction of 106 with 1-Boc-pyrazole-4-boronic acid

pinacol ester in the presence of PdCl (PPh ) , 1 M Na CO , and

n-butanol at 100 °C.

Ketones 7, 15, 19, 54, 56, 58, and 60 were prepared by

reaction of indoles 80, 92−94, 98, 102, and 109 with methyl-

magnesium bromide in the presence of anhydrous zinc chloride

at 25 °C and subsequent treatment with 3,4,5-trimethoxybenzoyl

chloride and tin(IV) chloride (Scheme 2). Ketones 9, 21, 23, 25,

2

3 2

2

3

DMF in a closed vessel at 160 °C while irradiating at 250 W for

10 min (Scheme 3). Solvent-free reaction at 150 °C of 106 with

1,2,4-triazole furnished 2-(1H-1,2,4-triazol-1-yl)-1H-indole

(81). 2-(1H-Indol-2-yl)oxazole (114) was obtained as 31 by

microwave reaction of 106 with 2-(tributylstannyl)oxazole in the

presence of Pd (dba) and P(Cy) in anhydrous DMF at 150 °C

3

2, 35, 40, 42, 44, 51, 63, 74, 76, and 78 were obtained by

reaction of the appropriate indoles 86−88, 91, 94−96, 103, and

10−114 with 3,4,5-trimethoxybenzoyl chloride in the presence

of anhydrous aluminum chloride. Reduction of ketones 9, 21, 23,

5, 35, 51, 60, and 63 with borane−tetrahydrofuran complex

2

3

1

and 150 W. 2-(4,5-Dihydro-1H-imidazol-2-yl)-1H-indole (83)

26

was prepared by treatment of 1H-indole-2-carbaldehyde (115)

2

with ethylenediamine and subsequent iodine oxidation in the

C

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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a

Scheme 2

a

21

Reagents and reaction conditions: (a) (i) indoles 80, 92−94, 98, 102, 109 (R = imidazol-1-yl (80), pyridin-2-yl (92), pyridin-3-yl (93), pyridin-

1

2

5

4

5

-yl (94), pyrazol-4-yl (98), naphth-1-yl (102), pyrrol-2-yl (109), MeMgBr, anhydrous ZnCl , anhydrous CH Cl , 25 °C, 1 h, Ar stream; (ii) 3,4,-

2 2 2

-trimethoxybenzoyl chloride, 25 °C, 1 h, Ar stream; (iii) SnCl , 25 °C, 12 h, Ar stream, yield 3−67%; (b) indoles 86−88, 91, 94−96, 103, 110−114

4

(

R = phenyl (84), 2-ﬂuorophenyl (86), 3-ﬂuorophenyl (87), 4-ﬂuorophenyl (88), 4-methoxyphenyl (91), cyclobutyl (94), cyclopentyl (95),

1

19 19 19 19

cyclohexyl (96), naphth-2-yl (103), 1-(phenylsulfonyl)-1H-pyrrol-3-yl (110), furan-2-yl (111), furan-3-yl (112), thiophen-3-yl (113), oxazol-

2

-yl (114)), 3,4,5-trimethoxybenzoyl chloride, anhydrous AlCl , 1,2-dichloroethane, closed vessel, 110 °C, 150 W, 2 min, yield 5−95%; (c): BH /

3

THF, MeCN/MeOH, 50 °C, 1 h, Ar stream, yield 4−99%; (d) 2 M NaOH, MeOH, reﬂux, 3 h, yield 20−98%; (e) NaBH , THF/H O, 80 °C, 2 h,

yield 44−97%.

4

2

presence of potassium carbonate. The 1-phenylethanones were

converted into 2-bromo-1-phenylethanones 116−121 with

bromine in dichloromethane and then transformed into the

corresponding 2-phenylindoles 86−90, 101, and 102 by heating

at 170 °C with aniline in N,N-dimethylaniline. Microwave

reaction of 2-bromo-1-(thiazol-2-yl)ethanone (116) with

aniline in a closed vessel at 150 °C and 100 W for 1 min

aﬀorded 88. o-Toluidides 123−127 were transformed into the

corresponding indoles 100, 104, and 95−97 with butyllithium in

anhydrous tetrahydrofuran, while the mixture was warmed from

and methylene derivatives as potential bioisosteres of previously

19

reported ATIs. As tubulin polymerization inhibitors, ketones 7

and 8 were less potent than their sulfur counterparts 3 and 4,

respectively, as well as less active than the similar compounds 5

and 6. Similar results were were also obtained with ketones 21,

23, and 25 in comparison with the corresponding arylthioin-

2

7

19

doles. Reduction of the ketone 8 to alcohol 10 resulted in an

equipotent inhibitor of tubulin polymerization. Replacement of

the sulfur bridging atom with a methylene group invariably

caused a drop, sometimes dramatic, in inhibitory activity (cf. 4

with 13, 34 with 36, 28 with 30, 47 with 52, and 62 with 65).

Replacement of the pyrrol-3-yl group of 4 with a pyrazol-4-yl

moiety provided ATI compound 14 (IC = 0.92 μM), which was

−

40 to 25 °C. Phenylhydrazono derivatives 128−133 underwent

Fischer cyclization in polyphosphoric acid at 120 °C to give

indoles 85, 91−94, and 103.

50

more potent than the parent compound as an inhibitor of tubulin

polymerization. This observation prompted the synthesis of new

ATIs bearing a pyrazol-3-yl (16), imidazol-2-yl (17), imidazol-1-

yl (18), 1,2,4-triazol-1-yl (20), thiazol-2-yl (27), or oxazol-2-yl

RESULTS AND DISCUSSION

■

7

Inhibition of Tubulin Polymerization. The eﬀects of ATIs

−78 on tubulin polymerization in vitro are shown in Table 1.

18,19

(

31) moiety at position 2 of the indole. With the exception of 17

On the basis of our previous results,

we synthesized these com-

and 31, these compounds potently inhibited tubulin polymer-

ⁱ^z^a^tⁱ^oⁿ^wⁱ^t^h^I^C50 ranging from 0.96 μM (27) to 1.3 μM (18).

Again, the ketones were less potent than the parent sulfur

compounds (compare 14 with 15, and 18 with 19).

Introduction of a methyl, methoxy, chlorine, or ﬂuorine moiety

at the para or meta position of a phenyl ring at indole position 2

also provided potent tubulin polymerization inhibitors (i.e., 37,

38, 41, 43, and 47), compared with the unsubstituted 34.

Interestingly, ATI 49, bearing the 3-hydroxy-4-methoxyphenyl

pounds to obtain structure−activity relationship (SAR) infor-

mation regarding position 2 of the indole moiety and the sulfur

bridging group. Several new ATIs (7, 8, 10, 15, 16, 18, 20, 21, 23,

3

8, 39, 41, 43, 49, 53, 55, 68−70, and 73) inhibited tubulin poly-

merization with IC of 1.0−2.0 μM, and six compounds (14, 27,

5

0

3

7, 47, 57, and 62) yielded IC ≤ 1.0 μM, compared with 1.0 μM

50

obtained for CSA4 (2) and 3.2 μM for colchicine (1).

For SAR studies regarding the sulfur bridging group, we

synthesized 3-aroylindoles and several corresponding alcohol

D

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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a

Scheme 3

a

R = 2-F-Ph (86), 3-F-Ph (87), 4-F-Ph (88), 2-MeO-Ph (89), 3-MeO-Ph (90), 3-i-PrO-4-MeO-Ph (101), 1-naphthyl (102); R = cyclobutyl (95),

2

3

cyclopentyl (96), cyclohexyl (97), 4-Me-Ph (100), 1,1′-biphenyl-4-yl (104); R = 4-Cl-Ph (85), 4-MeO-Ph (91), pyridin-2-yl (92), pyridin-3-yl (93)

4

pyridin-4-yl (94), 2-naphthyl (103). Reagents and reaction conditions: (a) PdCl (PPh ) , 1 M Na CO , DMF, closed vessel, 160 °C, 250 W, 10 min,

2

3

2

3

yield 26%; (b) 150 °C, 2 h, yield 42%; (c) Pd (dba) , P(Cy) , anhydrous DMF, closed vessel, 150 °C, 150 W, 10 min, yield 26%; (d) (i) t-BuOH,

2

3

Ar stream, 30 min; (ii) I , K CO , 70 °C, 3 h, yield 43%; (e) (diacetoxyiodo)benzene, K CO , DMSO, 25 °C, 12 h; (f) anhydrous DMF, closed vessel,

2

3

2

3

1

2

50 °C, 100 W, 1 min, PowerMAX, yield 52%; (g) Br , anhydrous CH Cl , 25 °C, 2 h, yield 21−90%; (h) R = 2-F (116), 3-F (117), 4-F (118),

2

28 28

-MeO (119), 3-MeO (120), 3-i-PrO-4-MeO (121), 1-naphthyl (122), N,N-dimethylaniline, 170 °C, 15 min, yield 12−42%; (i) R = 4-tolyl

3

(

123), 1,1′-biphenyl-4-yl (124), cyclobutyl (125), cyclopentyl (126), cyclohexyl (127), anhydrous THF, 25 °C, 12 h, yield 40−90%; (j) (i) n-BuLi,

anhydrous THF, −40 °C, 1 h, Ar stream; (ii) 0 °C, 1 h; (iii) 25 °C, 12 h, yield 22−59%; (k) R = 4-Cl-Ph (128), 4-MeO-Ph (129), pyridin-2-yl

4

29

(130), pyridin-3-yl (131), pyridin-4-yl (132), 2-naphthyl (133), CH COONa, EtOH, open vessel, 250 W, cooling-while-heating, 80 °C,

3

5

min, yield 92−95%; (l) polyphosphoric acid, 120 °C, 1 h, yield 37−80%.

substitution pattern (the same as 2) at indole position 2, inhib-

ited tubulin assembly with IC = 1.1 μM, compared with 1.0 μM

the binding reaction by at least 70%. Among them, 18 (92%), 55

(88%), and 57 (91%) were the strongest inhibitors of this

binding reaction, compared with 2 (98% inhibition).

50

for 2. Introduction of a pyridin-2-yl (53, IC = 1.5 μM), pyridin-

5

0

3

-yl (55, IC = 1.3 μM), or pyridin-4-yl (57, IC = 0.95 μM)

Cell Growth Inhibition. ATIs 7, 8, 10, 14−16, 18, 20, 21,

23, 25, 34, 39, 40, 47, 55, and 57 inhibited the growth of human

MCF-7 nonmetastatic breast cancer epithelial cells with IC ≤

50

group resulted in potent inhibitors of tubulin assembly. The

-naphthyl derivative 62 inhibited potently the tubulin polymer-

2

5

0

ization (IC₅₀= 1.0 μM), but the 1-naphthyl derivative 59 was

inactive. Compounds 68−70, bearing a benzofused heterocycl-2-yl

substituent at position 2 of the indole, inhibited tubulin assembly

with IC in the range of 1.5−1.8 μM.

Molecular modeling studies were consistent with the

conclusion that the binding modes of these new ATIs in the

colchicine site on tubulin were similar to those of previously

60 nM (Table 1). Five compounds (7, 18, 20, 55, and 57) had

IC ≤ 16 nM, with 18 (IC = 1.0 nM) being the most potent.

5

0

50

As an inhibitor of MCF-7 cell growth, 18 was 5 and 13 times

more potent than 1 and 2, respectively. Moreover, this ATI

was 18−39 times more eﬀective than the reference ATI

compounds 3−6.

Many of the new agents were potent inhibitors of tubulin

polymerization; however, compounds bearing the heterocycle

nucleus at the indole position 2 were more eﬀective as inhibitors

of the growth of MCF-7 cells. ATI 49, having the same substitution

pattern as 2, was only a relatively weak inhibitor of MCF-7 cell

50

1

9

reported ATIs (Supporting Information).

The new ATIs were also examined for potential inhibition of

3

the binding of [ H]colchicine to tubulin (Table 1). Compounds

7

, 8, 10, 14, 16, 18, 20, 21, 23, 27, 39, 41, 43, 55, and 57 inhibited

E

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

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Table 1. Inhibition of Tubulin Polymerization, Growth of MCF-7 Human Breast Carcinoma Cells, and Colchicine Binding by

Compounds 7−78

F

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

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Table 1. continued

G

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

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Table 1. continued

H

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Table 1. continued

a

b

Inhibition of tubulin polymerization. Tubulin was 10 μM during polymerization. Inhibition of growth of MCF-7 human breast carcinoma cells.

c

3

d

Inhibition of [ H]colchicine binding. Tubulin was at 1 μM. Both [ H]colchicine and inhibitor were at 5 μM. Compounds that inhibited tubulin

e

19

assembly with IC₅₀≤ 5 μM were tested in the cellular and colchicine binding assays. Lit. Plots of inhibition of tubulin polymerization and MCF-7

cell growth by compounds 7−78 are shown in Supporting Information.

growth (IC = 140 μM). ATI derivatives 18 (IC = 1.0 nM) and

Compounds 7, 8, 10, 14, 15, 18, 20, 23, 25, 34, 47, 55, and 57

were evaluated for growth inhibition of HeLa, HT29 (human

colon adenocarcinoma), A549 (human lung carcinoma), HCT

116, and HCT 15 (human colon carcinoma cell lines) in

comparison with 1, 2, vinblastine (VBL), and paclitaxel (PTX)

(Table 2). In terms of average IC₅₀for the ﬁve cell lines, the

control compounds ranged from 17 to 40 nM (ignoring the high

value obtained with 2 in the A549 cells). The average value for

50

2

0 (IC₅₀= 11 nM), bearing an imidazol-1-yl or 1,2,4-triazol-1-yl

nucleus, respectively, at indole position 2, were powerful

inhibitors of the MCF-7 cell line. In contrast to 18, compounds

1

4 (IC = 35 nM), 16 (IC = 50 nM), 17 (IC = 270 nM), 27

50 50 50

(

IC₅₀= 80 nM), and 32 (IC₅₀= 270 nM), each having two

heteroatoms in the 2-heterocyclyl moiety, were less active

inhibitors of the growth of this cell line.

I

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Table 2. Growth Inhibition of the HeLa, HT29, A549, HCT 116, and HCT 15 Cell Lines by Compounds 7, 8, 10, 14, 15, 18, 20, 23,

a

2

5, 34, 47, 55, and 57

IC₅₀± SD (nM)

b

compd

HeLa

HT-29

A549

HCT 116

HCT 15

average

7

8

1

2

3

4

5

1

2

94 ± 4

182 ± 1

80 ± 16

88 ± 3

94 ± 30

282 ± 5

30 ± 4

90 ± 25

184 ± 70

282 ± 6

105 ± 10

188 ± 10

45 ± 6

50 ± 6

68 ± 4

94 ± 4

80 ± 6

112 ± 20

20 ± 7

75 ± 5

82 ± 30

90 ± 5

137 ± 3

285 ± 2

28 ± 1

75 ± 2

20 ± 8

>10000

20 ± 2

7 ± 2

89 ± 37

62 ± 5

177 ± 41

67 ± 2

154 ± 34

16 ± 6

68 ± 2

90 ± 43

533 ± 95

186 ± 1

593 ± 18

183 ± 5

59 ± 5

20 ± 2

5 ± 0.4

3 ± 2

38 ± 22

52 ± 2

75 ± 14

62 ± 0.4

175 ± 2

11 ± 3

91

120 ± 10

134 ± 60

188 ± 20

97 ± 3

76

0

4

5

8

0

3

5

4

7

5

7

114

98

164

21

30 ± 7

180 ± 15

154 ± 80

350 ± 9

288 ± 80

482 ± 60

48 ± 4

47 ± 1

28 ± 9

20 ± 5

10 ± 0.6

5 ± 1

50 ± 0.9

53 ± 4

93

113

275

175

388

78

119 ± 18

160 ± 24

394 ± 91

84 ± 20

47 ± 13

86 ± 10

3 ± 9

78 ± 32

18 ± 4

130 ± 12

30 ± 0.8

61

34

>2032

17

VBL

PTX

24 ± 10

90 ± 17

8 ± 1.5

4 ± 0.4

23

a

b

Inhibition of growth of the indicated cell lines. Average activity of each compound was established for comparative purposes.

1

8, 21 nM, was in the same range, and six other compounds (7, 8,

4, 20, 55, and 57) had average IC values less than 100 nM. All

compound solvent, was used as a control. Representative images

(DNA distribution by ﬂow cytometry and wide-ﬁeld microscopic

images) of unﬁxed cultures taken after 24 h of treatment are

shown in Figure 1A. Compound 57 at 100 nM induced a

signiﬁcant increase in the proportion of cells in the G2/M phase

and, presumably, in mitosis, based on their rounded morphology

in the microscopic image. After treatment with 10 nM 18, a

signiﬁcant proportion of the cells had assumed the rounded

shape characteristic of mitotic arrest, and at 50 nM virtually the

entire population was rounded and arrested in G2/M, based on

DNA content.

5

0

these compounds had IC vlues of 35 nM or less in the screening

assay in the MCF7 cells.

MDR Cell Lines. Compounds 18, 20, 55, and 57 were com-

pared with vinorelbine (VRB), VBL, PTX 1 and 2 in the ovarian

carcinoma cell lines OVCAR-8 and its cognate P-glycoprotein

5

0

(

Pgp) overexpressing line NCI/ADR-RES and with the human

uterine sarcoma cell line Messa and its cognate line MDR line

Messa/Dx5 (Table 3). Except for 2, the standard agents were

Table 3. Growth Inhibition of the OVCAR-8 and NCI/ADR-

RES and Messa and Messa/Dx Cell Lines by Compounds 18,

To conﬁrm the initial microscopic observations, treated

cultures were incubated with propidium iodide and subjected

to quantitative ﬂow cytometric analysis of the cell cycle phase

distribution. Typical cell cycle proﬁles of PI-stained cultures after

2

0, 55, and 57 and Reference Compounds VRB, VBL, PTX, 1,

a

and 2

2

4 h of treatment are shown in Figure 1A, and average values

IC₅₀± SD (nM)

calculated from three to ﬁve independent assays per compound

are shown in Figure 1B. Both compounds 18 and 57 arrested cell

cycle progression in the G2/M phases (4C DNA content) when

used at 100 nM. Plots of the concentration of the tested ATI

against the fraction of G2/M-arrested cells in treated cultures

b

compd

OVCAR-8

NCI/ADR-RES

Messa

Messa/Dx5

18

20

55

57

1

2

16 ± 6

28 ± 10

17 ± 6

7.0 ± 3

20 ± 10

15 ± 7

23 ± 10

14 ± 7

7 ± 0.3

45 ± 4

60 ± 7

49 ± 2

11 ± 6

2.7 ± 2

13 ± 3

39 ± 6

71 ± 2

53 ± 14

329 ± 166

2.6 ± 1

(

Figure 1B) indicated that 18 was a potent inhibitor of cell cycle

c

nd

progression already at 20 nM and induced a signiﬁcant

proportion of cells (46%) to arrest with a 4C DNA content,

compared with 10.5% G2/M cells in control cultures. At higher

concentrations, 18 progressively increased cell cycle arrest: at 50

nM, over 60% cells in treated cultures were in G2/M phase

1.3 ± 0.6

300 ± 0

15 ± 7

1.3 ± 0.6

5000 ± 1000

200 ± 0

c

VRB

VBL

PTX

nd

3 ± 2

4 ± 1

144 ± 61

5.0 ± 2

3300 ± 1000

1764 ± 477

a

b

Inhibition of growth of the indicated cell lines. Growth inhibition

(

Figure 1B), similar to the values obtained with both VBL and

data of compounds 7, 8, 10, 14, 15, 23, 25, 34, and 47 are shown in

c

CSA-4 (2). The accumulation of cells with a replicated genome

demonstrated that 18, like the control drugs, prevented or

impaired mitotic cell division.

Compound 57 had somewhat milder eﬀects on cell cycle

progression (Figure 1B), compared with 18. Only about 48% of

cells accumulated in the G2/M region with 57 at 50 nM; lower

doses were virtually ineﬀective, with the proportion of G2/M

cells essentially identical to the values observed in untretated

controls. Only when the concentration was raised to 100 nM was

the majority (65%) of cells arrested in G2/M.

Supporting Information No data.

relatively inactive in both MDR lines. The four ATIs 18, 20, 55,

and 57 closely ressembled 2 in having full activity in both MDR

lines.

Eﬀects on Cell Cycle Progression. The most potent

compound 18, as well as 57, was tested in dose−response

experiments in comparison with 2 and VBL for eﬀects on cell

cycle distribution. HeLa cell cultures were treated with increasing

concentrations of each compound, and 0.1% DMSO, the

J

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Figure 1. (A) Analysis of HeLa cell cultures treated with 18 or 57 for 24 h by wide-ﬁeld microscopy (left panels) and ﬂow cytometry (right panels). The

left panels in each group show representative ﬁelds from unﬁxed cultures (10× objective): cells arrested in mitosis detach from the culture dish surface,

leaving ample empty spaces, and are recognizable from their rounded-up refractive morphology. Cycling cultures are adherent with only rare rounded

mitotic cells. Flow cytometry proﬁles of the cultures are shown on the right. The histrograms represent the ditribution of cells according to their DNA

content calculated from the emitted PI ﬂuorescence. Fluorescence intensity values are plotted on the x axis and cell numbers are plotted on the y axis.

The 2C peak (in gray) identiﬁes G1 cells. The 4C peak (in blue) identiﬁes G2/M cells. Cells with intermediate DNA content are in S phase. (B) Mean

frequency and SD of the frequency of cells with a 4C content (G2 + M) under the indicated conditions. From three to ﬁve assays were carried out for

every treatment, and data from 20 000 cells per assay were acquired.

Inhibition of Microtubule Assembly and Induction of

Mitotic Arrest. We analyzed cell cultures in dose−response

experiments using ﬂuorescence microscopy to gain information

on the eﬀects of 18 and 57 on cellular MTs. After treatment with

increasing concentrations of 18 or 57 for 24 h, we stained treated

cells for α-tubulin, cyclin B1, and DAPI in order to discriminate

between arrest in G2 interphase (i.e., interphase cells expressing

cyclin B1, Figure 2A, left panel) or in prometaphase (Figure 2A,

central panel). This is relevant because the molecular features of

mitotic cell death are distinct, at least in part, from the classical

K

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This arrest was not absolutely stringent, since 16% of cells in

the culture were aberrant, multinucleated, postmitotic species.

1

00 nM 57 arrested almost 65% of cells stably in prometaphase

with completely absent MTs and condensed chromosomes.

Virtually no mitotic slippage was observed with this concen-

tration. At 200 nM, the mitotic index decreased while the fraction

of G2 interphases correspondingly increased, indicating such a

strong eﬀect on interphase MTs that cells were prevented from

31

entering mitosis altogether. Indeed, a similar decrease in mitotic

arrest, with a corresponding increase in G2 cells, is observed at

high concentrations of 2 or VBL.

Compound 18 showed the highest eﬀectiveness, in terms of

inducing mitotic arrest at 50 nM in HeLa cell cultures (Figure 2B,

top panel). At this concentration, 18 induced eﬀective mitotic

arrest in prometaphase with totally absent MTs and no

signiﬁcant mitotic slippage. ATI 18 blocked mitotic progression

even at lower concentration (20 nM), yet the arrest was not fully

sustained with this concentration, and some cells slipped through

mitosis with a defective mitotic apparatus, generating multi-

nucleated cells (18% of all cells). At 100 nM, 18 prevented M

entry, suggesting an excessively strong eﬀect on interphase MTs.

Together, these data indicate that 18 is a strong inhibtior of MT

polymerization and induced eﬀective mitotic arrest at 50 nM,

similar to 2 or VBL, whereas 57 induced a durable mitotic block

when used at 100 nM.

Induction of Mitotic Cell Death. We examined whether the

new ATIs 18 and 57, in addition to arresting mitosis, also

triggered cell death. HeLa cell cultures were treated for 24 h as

described above, and they were then incubated with annexin V in

the absence of permeabilization in order to detect apoptotic cells

with a damaged plasma membrane. They were then analyzed by

ﬂow cytometry (Figure 3A). Signiﬁcant cell death was detected in

Figure 2. (A) Examples of immunoﬂuorescenctly stained cells. In all

panels, the DNA is blue (stained with DAPI), α-tubulin is green, and

cyclin B1 is red, as indicated. Left: A G2 interphase cell identiﬁed by

cyclin B1 expression. Middle: A cell arrested in prometaphase with

depolymerized microtubules (shown with and without merging with

cyclin B1 staining). Right: A multinucleated cell derived from mitotic

slippage. (B) Distribution of cells in dose−response experiments with

1

8 and 57. The histograms represent the mean frequency, and bars

represent the SD of interphases (I), mitoses (M), and multinculeated

cells (MN) from three independent experiments.

30

apoptotic pathway induced in interphase. In some experiments,

we also stained lamin B1 to visualize the integrity of the nuclear

membrane and assess the stringency of mitotic arrest induced by

ATI compounds. Indeed, cells with a defective mitotic

apparatus do not necessarily arrest stably in prometaphase,

but depending on the extent of MT damage, they may ﬁnally

exit mitosis (mitotic slippage) with abnormal chromosome

segregation and/or failed cell division giving rise to multi-

nucleated cells (Figure 2A, right panel).

In dose−response experiments (Figure 2 B, bottom panel), we

found that 57 at 10 and 20 nM did not aﬀect mitotic progression

(

Figure 3. (A) Frequency of apoptotic cells in cultures treated with the

indicated concentrations of 18 or 57. The histograms represent mean

frequencies, and bars represent the SDs of annexin V reactive cells from

three independent experiments. (B) Biparametric analysis of cell death

in dose−response experiments with 18. Early apoptotic, late apotptotic,

and necrotic cells are distinguished by their diﬀerential reactivity to

annexin V but not PI, to both annexin V and PI, and to PI but not

annexin V, respectively. Results from three independent experiments are

shown for each concentration. No condition induced signiﬁcant necrosis

compared with the baseline level seen in DMSO-treated controls (data

from 20 000 cells acquired per sample).

only 10% of all cells displayed mitotic properites, similar to the

baseline frequency observed in control cells treated with

DMSO). Although these concentrations induced many

abnormal mitotic spindles (aberrantly shaped, multipolar, and/

or with sparse and rare MTs), cells were distributed in all mitotic

stages, indicating that these abnormalities did not evoke a

stringent spindle checkpoint, and cells progressed to divide.

At 50 nM, 57 induced some mitotic arrest: 47% of all cells

were arrested in prometaphase with reduced numbers of MTs.

L

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cultures exposed to 18 (50 and 100 nM) or 57 (100 nM)

concentrations at which the cell populations displayed signiﬁcant

mitotic arrest (Figures 1 and 2). Biparametric analysis of annexin

V reactivity versus genomic DNA content revealed that necrosis

was negligible (Figure 3B). These data therefore show that both

18 and 57 induce mitotic cell death.

Loss of Mitochondrial Potential. We evaluated the loss of

mitochondrial potential (ΔΨ ), a hallmark for early induction of

m

32

cellular apoptosis, to elucidate further whether compounds 18

and 57 would induce apoptosis or necrotic death. The loss of

ΔΨ was indicated by a decrease in red 5,5′,6,6′-tetrachloro-

m

1

,1′3,3′-tetraethylbenzimidazoylcarbocyanine iodide (JC-1) ag-

gregates, accompanied by a concomitant accumulation in green

JC-1 monomers. The collapse in ΔΨ , as measured as reduction

m

in the red/green ﬂuorescence ratio, was assessed after cell

treatment with 18 or 57. As shown in Figure 4, after a 24 h

Figure 5. Eﬀects of treatment with 100 nM 57 or 18 and 400 mM

H O on ROS formation in U87MG cells after exposures of 1, 5, 15,

2

and 60 min. The results are expressed as % of mean ﬂuorescence

intensity relative to the control and normalized for cell number. Data

were the mean of two independent experiments performed in

triplicate ((∗) p < 0.05, (∗∗) p < 0.01, and (∗∗∗) p < 0.001, one-way

ANOVA, Bonferroni’s corrected t test for post hoc pairwise

comparisons).

In Vivo Vascular Disrupting Eﬀects. In preclinical

development of new anticancer drugs such as vascular disrupting

agents (VDAs), in vivo validation proved to be an indispensible

step in the bench-to-bedside chain, where animal models with

tumor growth in visceral organs could better simulate clinical

scenarios in comparison with the mostly applied subcutaneous

Figure 4. Eﬀects of a 24 h treatment with 100 nM 57 or 18 or 30 μM

CCCP on mitochondrial transmembrane potential as assessed by JC-1

staining. Graphs indicate the ΔΨ dissipation expressed as red/green (R/

G) ﬂuorescence ratio. Each value has been normalized versus the R/G

ratio of the vehicle treated control to which an arbitrary value of 100%

has been assigned. Data were the mean of two independent experiments

performed in triplicate ((∗∗) p < 0.01 and (∗∗∗) p < 0.001, one-way

ANOVA, Bonferroni’s corrected t test for post hoc pairwise

comparisons).

34

implants. Magnetic resonance imaging (MRI) has greatly

facilitated in vivo noninvasive assessment of tumoricidal eﬀects

35

on deeply seeded lesions in the animal models. Consequently,

we were able to observe that at the same intravenous dosage of 15

mg/kg, both compounds 18 and 57 exerted initial vascular

disrupting eﬀects in the applied tumor model of liver

rhabdomyosarcomas. The observed tumor vascular shutdown

eﬀect seems stronger and lasts longer with 18 (B′ and C′)

compared to 57 (B and C). The reason for such diﬀerent eﬀects

will be further elucidated (Figure 6).

treatment, 57 was able to induce a more signiﬁcant reduction in

ΔΨ than was 18. The standard compound carbonyl cyanide

m

m-chlorophenylhydrazone (CCCP) caused about 50% potential

dissipation at 30 μM.

ROS Generation. Mitochondria are an important intra-

Metabolic Stability. Compounds 18, 20, 55, and 57 were

examined in a microsomal stability assay in comparison with

7-ethoxycoumarin and propranolol as control compounds, using

both human and mouse liver microsomes, to estimate compound

stability to phase I oxidative metabolism (Table 4). Compound

18 showed the highest metabolic stability with 48.6% and 10.3%

remaining after 30 min in human and mouse liver microsomes,

respectively. Compounds 20, 55, and 57 showed medium

metabolic stability with human liver microsomes and low

metabolic stability with mouse liver microsomes (relative

stabilities are deﬁned in a Table 4 footnote).

Aqueous Solubility. The solubility in aqueous pH 7.4 buﬀer

of compounds 18, 20, 55, and 57 was measured in a high

throughput screening solubility assay. The solubility of

compound 18 was 64.5 μM, while under the same conditions

compounds 20 (4 μM), 55 (20.5 μM), and 57 (8 μM) showed

lower solubility (Table 4). The higher solubility of compound 18

could explain its greater activity as a cell growth inhibitor (see

Tables 1 and 2), although these compounds were comparable

inhibitors of tubulin assembly in the biochemical assays.

33

cellular source of reactive oxygen species (ROS). We measured

the ability of compounds 18 and 57 to generate ROS in U87MG

cells, using hydrogen peroxide speciﬁc probe 6-carboxy-2′,

7

′-dichlorodihydroﬂuorescein diacetate (DCFH2-DA).

The IC values of compounds 18 and 57 in U87MG (human

50

glioblastoma) cell growth/survival after a 24 h treatment were

determined. Compounds 18 and 57 showed dose-dependent

inhibitory eﬀects on cell survival, with compound 18 having IC₅₀

=

85 ± 8 nM and 57 having IC₅₀= 103 ± 9 nM. Compound-

treated cells showed an increase in the percentage of cell death

compared with control cells, reaching statistical signiﬁcance

at100 nM.

According to the observed loss of mitochondrial potential,

both compounds 18 and 57 were able to increase ROS levels.

The eﬀect was not detectable immediately, but it reached

statistical signiﬁcance after a 1 h treatment (ﬂuorescence

intensity with respect to control: 18, 216%; 57, 254%; both

greater than the value of 208% obtained with the usual standard,

hydrogen peroxide (Figure 5)).

M

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Figure 6. Contrast enhanced MRI was used in rats with bifocal liver growths of rhabdomyosarcoma (smaller and larger arrows denote smaller and larger

lesions, respectively). At baseline prior to treatment, the liver tumors appear as homogeneous hyperintense spheroidal nodules, suggesting their

hypervascularity (A, A′). At 1 h after intravenous injection of either 57 (B) or 18 (B′), the tumors were partially enhanced because of their early vascular

disrupting eﬀects, which appeared stronger with 18 (B′) than 57 (B). At 24 h after treatment, the tumors treated with 57 were enhanced completely (C),

suggesting only transient early vascular disruption eﬀect and the absence of any consequent tumor necrosis. However, tumors treated with 18 showed

rim (arrowheads) and partial central enhancement (C′), suggesting the presence of both tumor necrosis and viable residues.

Table 4. Metabolic Stability with Human and Mouse Liver

Microsomes and Aqueous Solubility of Compounds 18, 20,

permeability in both A → B (P_a_p_p= 132.5 nm/s) and B → A

(P_a_p_p= 133.2 nm/s) directions. Compounds 18, 55, and 57

showed medium−high permeability (Table 5). None of the

compounds tested showed eﬄux phenomena (i.e., ratio of

P (B→A) to P (A→B) of <2).

a

5

5, and 57

b

%

remaining at 30 min

app

human liver

microsomes

mouse liver

microsomes

CYP450 Inhibition. To assess further the safety of

compounds 18, 20, 55, and 57, we carried out a CYP inhibition

assay. CYP inhibition can cause drug−drug interactions by

increasing concentrations to other drugs to toxic levels. The

inhibition of the enzymatic activity of human cloned P450

isoforms CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4

was measured using speciﬁc substrates for each isoform that

produced a ﬂuorescent metabolite upon CYP metabolism.

Compounds 18, 20, 55, and 57 at 1 μM were weak inhibitors

of the isoforms CYP1A2 and CYP2D6. For these isoforms,

inhibition was <50%, ranging from <5% (20 in CYP1A2 and

CYP2D6 isoforms) to 43% (57 in CYP2D6 isoform). The

isoform CYP3A4 was inhibited by 20, 55, and 77 by 51%, 76%,

and 57%, respectively; CYP2C19 and CYP2C9 were inhibited to

a greater extent by 18, 20, 55, and 57.

compd

solubility (μM) ^,

bd

1

2

5

7

8

0

5

7

48.6 ± 1.9

12.0 ± 1.1

19.3 ± 0.9

17.7 ± 0.5

6.6 ± 0.2

10.3 ± 0.4

0.6 ± 0.1

2.8 ± 0.3

7.9 ± 0.2

0.07 ± 0.02

20.6 ± 0.5

64.5 ± 0.7

4.0 ± 0.1

20.5 ± 0.7

5.0 ± 0.1

c

-ethoxycoumarin

c

propranolol

54.1 ± 0.4

a

b

Metabolic stability: >50, good; 10−50, medium; <10, low. Results

are expressed as mean ± SD, n = 2. The standard compounds

c

7

-ethoxycoumarin and propranolol showed metabolic stability in

36 d

agreement with the literature and internal validation data.

pH 7.4,

high throughput screening solubility assay.

Caco-2 Cell Permeability. The intestinal permeability of

compounds 18, 20, 55, and 57 was evaluated in the human Caco-

2

model in comparison with caﬀeine (high permeability) and

CONCLUSION

cimetidine (low permeability, Pgp substrate). The apparent

permeability (P ) of 18, 20, 55, or 57 from the A (apical) to B

■

We synthesized 71 new arylthioindole/aroylindole compounds

as potential anticancer agents having diﬀerent (hetero)cyclic

substituents at indole position 2. Several new ATIs inhibited

tubulin polymerization with IC ranging from <1.0 to 2.0 μM,

app

(

basolateral) side together with B to A was measured in order to

predict the absorption from the lumen of the gut and potential

eﬄux phenomena. Compound 20 showed the highest

50

Table 5. Caco-2 Cell Permeability and CYP450 Isoform Inhibition of Compounds 18, 20, 55, and 57

a

P_a_p_p(nm/s)

CYP450 isoform (% inhibition at 1 μM)

compd

P_A_→_B

P_B_→_A

CYP1A2

CYP2C19

CYP2C9

CYP2D6

CYP3A4

18

20

55

57

55.3 ± 11.1

132.5 ± 19.7

53.1 ± 6.0

44.5 ± 4.0

60.0 ± 5.6

133.2 ± 4.2

65.8 ± 2.5

54.2 ± 5.3

22.9 ± 0.03

<5

82.5 ± 0.01

83.9 ± 0.21

84.9 ± 0.70

100.2 ± 0.09

95.9 ± 0.05

93.1 ± 0.18

101.8 ± 1.10

100.4 ± 1.36

10.6 ± 0.16

<5

94.5 ± 0.55

51.4 ± 0.18

76.4 ± 1.42

58.0 ± 1.26

23.4 ± 0.31

14.5 ± 1.66

6.6 ± 0.64

43.1 ± 3.21

a

P_a_p_p(nm/s): >50, high; 10−50, medium; <10, low; caﬀeine P_A₋_Breference control, 206 ± 35 nm/s; cimetidine P_A₋_Band P_B₋_Areference control,

.1 ± 0.1 and 21.3 ± 3.4 nm/s, respectively.

1

N

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and six compounds yielded submicromolar IC . These com-

pounds inhibited the growth of human MCF-7 cells, and ﬁve of

Developed plates were visualized by a Spectroline ENF 260C/FE UV

apparatus. Organic solutions were dried over anhydrous Na SO .

Evaporation of the solvents was carried out on a Buchi rotavapor R-210

equipped with a Buchi V-850 vacuum controller and a Buchi V-700 or V-

5

0

2

4

them inhibited the growth of these cells with low nanomolar IC .

50

ATI 18 strongly inhibited MCF-7 cell growth (IC = 1.0 nM) and

50

7

10 vacuum pump. All reagents and solvents are commercially available

was uniformly active in a larger panel of cancer cells. Compound

and were used as purchased, without further puriﬁcation. Elemental

analyses of the compounds were found within ±0.4% of the theoretical

values. The purity of tested compounds was >95%.

1

8 was superior to reference compounds 1 and 2 and ATIs

19

3−6. ATIs 18, 20, 55, and 57 were more potent than 1, 2, VRB,

2

1

VBL, and PTX in two MDR cell lines, the Pgp overexpressing

NCI-ADR-RES line and the Messa/Dx5 line. Compounds 18

and 57 arrested cell cycle progression at the G2/M phase.

Compound 18 was a potent inhibitor of cell cycle progression at

2

-(1H-Imidazol-1-yl)-1H-indole (80), 2-(1H-pyrazol-3-yl)-1H-in-

22 23

dole (99), 2-iodo-1H-indole (106), 2-(1H-pyrrol-2-yl)-1H-indole

(109), 2-(1-(phenylsulfonyl)-1H-pyrrol-3-yl)-1H-indole (110),

2-(furan-2-yl)-1H-indole (111), 2-(furan-3-yl)-1H-indole (112),

2

5

19

1

9

2

6

1

H-indole-2-carbaldehyde (115), 2-(1-(2-phenylhydrazono)ethyl)-

29 29

20 nM, strongly inhibited MT polymerization, and induced

pyridine (130), 3-(1-(2-phenylhydrazono)ethyl)pyridine (131),

eﬀective mitotic arrest and cell death, while 57 showed somewhat

weaker eﬀects. Compounds 18 and 57 elicited dose- and time-

dependent inhibition of U87MG cell growth and induced

mitochondrial membrane potential collapse, a hallmark of early

apoptosis. The compounds also evoked signiﬁcant ROS

generation in GBM cells. At 15 mg/kg intravenous dosage,

both compounds 18 and 57 exerted initial vascular disrupting

eﬀects in a tumor model of liver rhabdomyosarcomas. In a

microsomal stability assay, compound 18 showed the highest

metabolic stability with both human and mouse liver microsomes

of all the ATIs examined. The higher solubility in aqueous pH 7.4

buﬀer of compound 18 (64.5 μM) could explain its greater

ⁱⁿ^hⁱ^bⁱ^t^o^r^y^e^ﬀ^e^c^t^s^oⁿ^c^e^l^l^g^r^o^w^t^h⁽^M^C^F^-⁷^c^e^l^l^s^,^I^C⁵0 = 1.0 nM)

among compounds with comparable inhibitory eﬀects on tubulin

assembly. In conclusion, we succeeded in improving the ATI

class of cancer agents through the synthesis of new compounds.

Compounds 18 and 57 proved to be superior to 5, the reference

29

4

2

-(1-(2-phenylhydrazono)ethyl)pyridine (132), and 1-(1-(naphthalen-

-yl)ethylidene)-2-phenylhydrazine (133) were synthesized accord-

29

ing to the indicated literature. 2-Phenyl-1H-indole (84), 1H-indole-2-

carboxylic acid (105), 2-bromo-1-(2-methoxyphenyl)ethanone (119),

and 2-bromo-1-(3-methoxyphenyl)ethanone (120) were purchased

from Sigma-Aldrich.

Arylthio-1H-indoles. General Procedure for the Preparation

of Compounds 17, 18, 20, 27, 33, 34, 38, 39, 41, 43, 45−47, 53,

5

5, 57, 73, 75, and 77. Example: 2-(1H-Imidazol-2-yl)-3-((3,4,5-

trimethoxyphenyl)thio)-1H-indole (17). 2-(1H-Imidazol-2-yl)-1H-

indole (79) (0.044 g, 0.00024 mol) was added to a suspension of NaH

(0.014 g, 0.00036 mol, 60% dispersion in mineral oil) in anhydrous

DMF (2 mL). After 10 min, bis(3,4,5-trimethoxyphenyl)disulﬁde (0.1 g,

0.000 264 mol) was added, and the reaction mixture was placed into the

microwave cavity (closed vessel mode, P_m_a_x= 250 psi). A starting

microwave irradiation of 150 W was used, the temperature being ramped

from 25 to 110 °C while stirring. Once 110 °C was reached, taking about

1

min, the reaction mixture was held at this temperature for 2 min. The

19

reaction mixture was quenched on crushed ice and extracted with ethyl

acetate. The organic layer was washed with brine, dried, and ﬁltered.

Removal of the solvent gave a residue that was puriﬁed by column

chromatography (silica gel, ethyl acetate/n-hexane = 7:3 as eluent) to

ATI of the previous series, in the whole cancer cell panel,

including two MDR cell lines. Derivative 18 showed higher

metabolic stability than 5 in human liver microsomes (48.6%

remaining), showed the highest water solubility, and displayed

medium-high Caco-2 cell permeability. The present results

highlight the therapeutic potential of the ATI class as anticancer

agents and prompt their further development.

1

furnish 17 (0.02 g, 22%), mp 135−140 °C (from ethanol). H NMR

(

7

CDCl ): δ 3.62 (s, 6H), 3.76 (s, 3H), 6.38 (s, 2H), 7.14−7.26 (m, 4H),

3

.36−7.38 (m, 1H), 7.63 (d, J = 7.9 Hz, 1H), 11.01 (broad s, disappeared

on treatment with D O, 1H), 11.58 ppm (broad s, disappeared on

treatment with D O, 1H). IR: ν 2923, 3345 cm . Anal. (C H N O S

2

−1

2

20 19

3

(

381.45)) C, H, N, S.

EXPERIMENTAL SECTION

■

2-(1H-Imidazol-1-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-in-

21

Microwave-assisted reactions were performed on a CEM Discover SP

single mode reactor, controlling the instrument settings with PC-running

CEM Synergy 1.49 software. Closed vessel experiments were carried out

in capped microwave-dedicated vials (10 mL) with cylindrical stirring

bar (length 8 mm, diameter 3 mm). Open vessel experiments were

carried out in 100 mL round-bottom ﬂasks equipped with a Dimroth

reﬂux condenser and a cylindrical stirring bar (length 20 mm, diameter 6 mm).

Stirring, temperature, irradiation power, maximum pressure (P_m_a_x),

PowerMAX (simultaneous cooling-while-heating), ActiVent (simultaneous

venting-while-heating), and ramp and hold times were set as indicated.

Temperature of the reaction was monitored by a built-in infrared sensor.

After completion of the reaction, the mixture was cooled to 25 °C via air-jet

cooling. Melting points (mp) were determined on a Stuart Scientiﬁc SMP1

apparatus and are uncorrected. Infrared spectra (IR) were run on a Perkin-

Elmer SpectrumOne FT-ATR spectrophotometer. Band position and

dole (18). 18 was synthesized similarly to 17, starting from 80. Yield

1

77%, mp 193−197 °C (from ethanol). H NMR (DMSO-d ): δ 3.57 (s,

6

6H), 3.58 (s, 3H), 6.31 (s, 2H), 7.17−7.21 (m, 2H), 7.26−7.30 (m, 1H),

7.49−7.55 (m, 2H), 7.68−7.69 (m, 1H), 8.14−7.15 (m, 1H), 12.23 ppm

13

(broad s, disappeared on treatment with D O, 1H). C NMR (DMSO-d ):

2

6

δ 56.33, 60.48, 91.97, 103.95, 112.62, 119.17, 120.49, 121.66, 123.68,

129.27, 129.77, 132.91, 133.81,136.2, 136.23, 137.78, 153.79 ppm. IR: ν

2930 cm . Anal. (C H N O S (381.45)) C, H, N, S.

−

1

20

19

3

2-(1H-1,2,4-Triazol-1-yl)-3-((3,4,5-trimethoxyphenyl)thio)-

1H-indole (20). 20 was synthesized similarly to 17, starting from 81.

1

Yield 34%, mp 140−145 °C (from ethanol). H NMR (DMSO-d ): δ

6

3.58 (s, 9H), 6.37 (s, 2H), 7.21 (t, J = 7.7 Hz, 1H), 7.31 (t, J = 8.0 Hz,

1H), 7.52−7.58 (m, 2H), 8.41 (s, 1H), 9.12 (s, 1H), 12.80 ppm (broad s,

1

3

disappeared on treatment with D O, 1H). C NMR (DMSO-d ): δ

2

6

56.32, 60.47, 93.24, 104.45, 113.01, 119.50, 121.78, 124.14, 128.78,

132.15, 134.11, 135.32, 136.38, 145.67, 153.04, 153.75 ppm. IR: ν 3354 cm .

Anal. (C H N O S (382.44)) C, H, N, S.

−1

absorption ranges are given in cm . Proton nuclear magnetic resonance

1

(

H NMR) spectra were recorded on a Bruker 400 MHz FT

19

18

4

3

spectrometer in the indicated solvent and corresponding ﬁd ﬁles pro-

cessed by MestreLab Research S.L. MestreReNova 6.2.1-769 software.

Chemical shifts are expressed in δ units (ppm) from tetramethylsilane.

Column chromatography was performed on columns packed with

alumina from Merck (70−230 mesh) or silica gel from Macherey-Nagel

2-(3-((3,4,5-Trimethoxyphenyl)thio)-1H-indol-2-yl)thiazole

(27). 27 was synthesized similarly to 17, starting from 82. Yield 10%, mp

1

160−165 °C (from ethanol). H NMR (CDCl ): δ 3.65 (s, 6H), 3.77

3

(s, 3H), 6.41 (s, 2H), 7.19−7.23 (m, 1H), 7.31−7.35 (m, 1H), 7.43−

7.45 (m, 2H), 7.72 (dd, J = 0.8 and 7.9 Hz, 1H), 7.90 (d, J = 3.2 Hz, 1H),

(

70−230 mesh). Aluminum oxide thin layer chromatography (TLC)

9.87 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν 3339

2

−

1

cards from Fluka (aluminum oxide precoated aluminum cards with

ﬂuorescent indicator visualizable at 254 nm) and silica gel TLC cards

from Macherey-Nagel (silica gel precoated aluminum cards with

ﬂuorescent indicator visualizable at 254 nm) were used for TLC.

cm . Anal. (C H N O S (379.47)) C, H, N, S.

20 18 2 3 2

2-(4,5-Dihydro-1H-imidazol-2-yl)-3-((3,4,5-trimethoxyphenyl)-

thio)-1H-indole (33). 33 was synthesized similarly to 17, starting from

1

83. Yield 8%, mp 150−155 °C (from ethanol). H NMR (CDCl ): δ

3

O

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

3

1

.64 (s, 6H), 3.76 (s, 3H), 3.92 (m, 4H), 6.33 (s, 2H), 7.15 (t, J = 7.1 Hz,

H), 7.32 (t, J = 7.0 Hz, 1H), 7.49 (d, J = 8.3 Hz, 1H), 7.61 (d, J = 8.1 Hz,

188−191 °C (from ethanol). H NMR (CDCl ): δ 3.65 (s, 6H), 3.76

3

(s, 3H), 6.33 (s, 2H), 7.22 (m, 1H), 7.31 (m, 1H), 7.37−7.41 (m 1H),

7.48 (d, J = 8.1 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 8.16−8.17 (m, 1H),

8.62 (m, 1H), 9.02 (m, 1H), 9.26 ppm (broad s, disappeared on

treatment with D O, 1H). C NMR (DMSO-d ): δ 56.28, 60.48, 98.8,

103.33, 112.63, 119.31, 121.21, 123.66, 124.12, 127.75, 130.72, 133.85,

135.99, 136.82, 139.7, 149.19, 149.68, 153.77 ppm. IR: ν 2923, 3674

cm . Anal. (C H N O S (392.47)) C, H, N.

2-(Pyridin-4-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole

(57). 57 was synthesized similarly to 17, starting from 94. Yield 60% as a

H), 8.31 (broad s, disappeared on treatment with D O, 1H), 12.10

2

ppm (broad s, disappeared on treatment with D O, 1H). IR: ν 2852,

2

−1

13

921 cm . Anal. (C H N O S (383.46)) C, H, N, S.

20

21

3

2

6

2-Phenyl-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (34). 34

was synthesized similarly to 17, starting from 84. Yield 3%, mp 158−160

1

−1

°

C (from ethanol). H NMR (CDCl ): δ 3.64 (s, 6H), 3.78 (s, 3H), 6.37

3

22 20

2

3

(

s, 2H), 7.18−7.22 (m, 1H), 7.27−7.30 (m, 2H), 7.41−7.50 (m, 3H),

7

.69 (d, J = 7.9 Hz, 1H), 7.80−7.82 (m, 2H), 8.56 ppm (broad s,

−1

1

disappeared on treatment with D O, 1H). IR: ν 3329 cm . Anal.

yellow solid, mp 215−218 °C (from ethanol). H NMR (DMSO-d ): δ

2

6

(

C H NO S (391.48)) C, H, N, S.

3.53 (s, 6H), 3.57 (s, 3H), 6.32 (s, 2H), 7.14−7.18 (m, 1H), 7.27−7.31

(m, 1H), 7.53−7.57 (m, 2H), 7.92 (dd, J = 1.7 and 4.5 Hz, 2H), 8.71 (dd,

J = 1.6 and 4.5 Hz, 2H), 12.36 ppm (broad s, disappeared on treatment

23

21

3

2

-(4-Chlorophenyl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-in-

dole (38). 38 was synthesized similarly to 17, starting from 85. Yield

1

13

1

6

7

1%, mp 190−195 °C (from ethanol). H NMR (CDCl ): δ 3.64 (s,

3

with D O, 1H). C NMR (DMSO-d ): δ 56.27, 60.47, 100.49, 103.60,

2

6

H), 3.78 (s, 3H), 6.34 (s, 2H), 7.19−7.23 (m, 1H), 7.29−7.31 (m, 1H),

.43−7.46 (m, 3H), 7.68−7.70 (m, 1H), 7.72−7.75 (m, 2H), 8.59 ppm

1

12.82, 119.63, 121.38, 122.5, 124.25, 130.89, 133.39, 135.99, 136.92,

38.76, 139.02, 150.52, 153.8 ppm. IR: ν 3320 cm⁻. Anal.

1

−1

(

broad s, disappeared on treatment with D O, 1H). IR: ν 3335 cm .

2

(C H N O S (392.47)) C, H, N.

2-Cyclobutyl-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole

(73). 73 was synthesized similarly to 17, starting from 95. Yield 56%, mp

22 20 2 3

Anal. (C H ClNO S (425.93)) C, H, N, Cl, S.

23

20

3

2

-(2-Fluorophenyl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-in-

dole (39). 39 was synthesized similarly to 17, starting from 86. Yield

1

49−153 °C (from ethanol). H NMR (DMSO-d ): δ 1.85−1.92

6

1

3

9% mp 175−180 °C (from toluene). H NMR (DMSO-d ): δ 3.54

6

(m, 1H), 1.98−2.10 (m, 1H), 2.23−2.28 (m, 2H), 2.28−2.30 (m, 2H),

(

s, 6H), 3.56 (s, 3H), 6.26 (s, 2H), 7.13 (t, J = 7.6 Hz, 1H), 7.24 (t, J =

2

7

.57 (s, 9H), 3.94−4.03 (m, 1H), 6.27 (s, 2H), 7.01−7.05 (m, 1H),

.11−7.15 (m, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H),

7

1

.8 Hz, 1H), 7.33−7.42 (m, 2H), 7.49−7.54 (m, 3H), 7.65 (t, J = 7.5 Hz,

H), 12.07 ppm (broad s, disappeared on treatment with D O, 1H). IR:

2

11.74 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν

2

−1

ν 3342 cm . Anal. (C H FNO S (409.47)) C, H, N, F, S.

−1

23

20

3

3361 cm . Anal. (C H NO S (369.48)) C, H, N, S.

21

23

3

2

-(3-Fluorophenyl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-in-

2

-Cyclopentyl-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole

dole (41). 41 was synthesized similarly to 17, starting from 87. Yield

(

75). 75 was synthesized similarly to 17, starting from 96. Yield 56%, mp

1

6

3

7

5%, mp 180−185 °C (from toluene). H NMR (DMSO-d ): δ 3.59 (s,

1

6

178−183 °C (from ethanol). H NMR (DMSO-d ): δ 1.66−1.68 (m,

6

H), 3.64 (s, 6H), 6.79 (s, 2H), 7.07−7.35 (m, 5H), 7.45−7.48 (m, 1H),

.52 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 12.27 (broad s,

2

H), 1.17−1.85 (m, 4H), 1.95−1.98 (m, 2H), 3.52−3.54 (m, 1H), 3.56

(

s, 6H), 3.57 (s, 3H), 6.28 (s, 2H), 7.0−7.04 (m, 1H), 7.09−7.13

−1

disappeared on treatment with D O, 1H). IR: ν 1571, 3187 cm . Anal.

2

m, 1H), 7.36−7.41 (m, 2H), 11.51 ppm (broad s, disappeared on treat-

(

C H FNO (405.42)) C, H, N, F, S.

−1

24

20

4

ment with D O, 1H). IR: ν 3331 cm . Anal. (C H NO S (383.50)) C,

2

22 25

3

2

-(4-Fluorophenyl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-in-

H, N, S.

-Cyclohexyl-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole

77). 77 was synthesized similarly to 17, starting from 97. Yield 48%, mp

dole (43). 43 was synthesized similarly to 17, starting from 88. Yield

2

1

3

7%, mp 170−175 °C (from toluene). H NMR (DMSO-d ): δ 3.53

6

(

s, 6H), 3.57 (s, 3H), 6.30 (s, 2H), 7.12 (t, J = 7.0 Hz, 1H), 7.23 (t, J =

.4 Hz, 1H), 7.38 (t, J = 8.9 Hz, 2H), 7.49 (t, J = 7.2 Hz, 2H), 7.90−7.93

m, 2H), 12.09 (broad s, disappeared on treatment with D O, 1H). IR:

1

2

14−219 °C (from ethanol). H NMR (DMSO-d ): δ 1.24−1.42

6

7

(

m, 3H), 1.66−1.83 (m, 7H), 3.09−3.17 (m, 1H), 3.56 (s, 6H), 3.57

2

s, 3H), 6.28 (s, 2H), 7.0−7.02 (m, 1H), 7.09−7.13 (m, 1H), 7.35−7.41

−1

ν 3318 cm . Anal. (C H FNO S (409.47)) C, H, N, F, S.

23

20

3

m, 2H), 11.52 ppm (broad s, disappeared on treatment with D O, 1H).

2

-(2-Methoxyphenyl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-

−1

IR: ν 3327 cm . Anal. (C H NO S (397.53)) C, H, N, S.

23

27

3

indole (45). 45 was synthesized similarly to 17, starting from 89. Yield

1

General Procedure for the Preparation of Compounds 14, 16,

7, 48, 59, 62, and 72. Example: 2-(1H-Pyrazol-4-yl)-3-((3,4,5-

5

3

%, mp 160−165 °C (from ethanol). H NMR (CDCl ): δ 3.64 (s, 6H),

3

.77 (s, 3H), 3.92 (s, 3H), 6.35 (s, 2H), 7.04−7.08 (m, 2H), 7.15−7.19

trimethoxyphenyl)thio)-1H-indole (14). 2-(1H-Pyrazol-4-yl)-1H-

indole (98) (0.08 g, 0.000 44 mol) was added to an ice-cooled sus-

pension of NaH (0.058 g, 0.001 45 mol, 60% dispersion in mineral oil)

in anhydrous DMF (3 mL) under an Ar stream. After 15 min, bis(3,4,5-

trimethoxyphenyl)disulﬁde (0.21 g, 0.000 53 mol) was added, and the

reaction mixture was heated at 60 °C for 12 h under an Ar stream. After

cooling, the reaction mixture was quenched on crushed ice and extracted

with ethyl acetate. The organic layer was washed with brine, dried, and

ﬁltered. Removal of the solvent gave a residue that was puriﬁed by

column chromatography (silica gel, ethyl acetate/n-hexane = 7:3) to

(

m, 1H), 7.25−7.29 (m, 1H), 7.36−7.40 (m, 1H), 7.45−7.47 (m, 1H),

.67−7.69 (m, 1H), 7.97−7.99 (m, 1H), 9.40 ppm (broad s, disappeared

7

−1

on treatment with D O, 1H). IR: ν 3338 cm . Anal. (C H NO S

2

24 23

4

(

421.51)) C, H, N, S.

-(3-Methoxyphenyl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-

indole (46). 46 was synthesized similarly to 17, starting from 90. Yield

2

1

4

3

%, mp 192−194 °C (from ethanol). H NMR (CDCl ): δ 3.63 (s, 6H),

3

.75 (s, 3H), 3.76 (s, 3H), 6.36 (s, 2H), 6.93−6.96 (m, 1H), 7.18−7.21

(

m, 1H), 7.25−7.40 (m, 4H), 7.44−7.46 (m, 1H), 7.69−7.71 (m, 1H),

.57 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν 3333

8

2

1

−1

furnish 14 (0.1 g, 59%), mp 178−182 °C (from ethanol). H NMR

cm . Anal. (C H NO S (421.51)) C, H, N, S.

24

23

4

(

DMSO-d ): δ 3.53 (s, 6H), 3.57 (s, 3H), 6.33 (s, 2H), 7.06−7.10

6

2

-(4-Methoxyphenyl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-

m, 1H), 7.15−7.19 (m, 1H), 7.44−7.46 (m, 2H), 8.23 (m, 2H), 11.88

indole (47). 47 was synthesized similarly to 17, starting from 91. Yield

1

(broad s, disappeared on treatment with D O, 1H), 13.17 ppm (broad s,

8

3

%, mp 165−170 °C (from ethanol). H NMR (CDCl ): δ 3.63 (s, 6H),

2

3

−1

disappeared on treatment with D O, 1H). IR: ν 3158, 3291 cm . Anal.

C H N O S (381.45)) C, H, N, S.

.78 (s, 3H), 3.85 (s, 3H), 6.37 (s, 2H), 6.97−6.99 (m, 2H), 7.17−7.23

2

(

m, 2H), 7.41−7.43 (m, 1H), 7.66−7.74 (m, 3H), 8.68 ppm (broad s,

20 19 3 3

−1

2-(1H-Pyrazol-3-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-in-

22

disappeared on treatment with D O, 1H). IR: ν 3324 cm . Anal.

2

dole (16). 16 was synthesized similarly to 14, starting from 99. Yield

(

C H NO S (421.51)) C, H, N, S.

24 23 4

1

3

5%, mp 194−197 °C (from ethanol). H NMR (DMSO-d ): δ 3.51

2

-(Pyridin-2-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole

6

(

s, 6H), 3.55 (s, 3H), 6.35 (s, 2H), 6.97−6.98 (m, 1H), 7.07 (t, J =

(

53). 53 was synthesized similarly to 17, starting from 92. Yield 26% as

1

7

1

.6 Hz, 1H), 7.17 (t, J = 7.9 Hz, 1H), 7.46−7.49 (m, 2H), 7.83 (s, 1H),

an oil. H NMR (CDCl ): δ 3.63 (s, 6H), 3.76 (s, 3H), 6.39 (s, 2H), 7.18

3

1.97 (broad s, disappeared on treatment with D O, 1H), 13.23 ppm

(

7

(

t, J = 7.1 Hz, 1H), 7.24−7.27 (m, 2H), 7.45 (d, J = 8.1 Hz, 1H), 7.70−

.77 (m, 2H), 8.63−8.64 (m, 1H), 8.73 (d, J = 8.1 Hz, 1H), 10.23 ppm

broad s, disappeared on treatment with D O, 1H). IR: ν 2828, 2933,

2

(broad s, disappeared on treatment with D O, 1H). IR: ν 2936, 3348

cm . Anal. (C20H N O S (381.45)) C, H, N, S.

19 3 3

2-(p-Tolyl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (37).

37 was synthesized similarly to 14, starting from 100. Yield 72%, mp

2

−

1

2

−1

3

003, 3056 cm . Anal. (C H N O S (392.47)) C, H, N, S.

22

20

2

3

2

-(Pyridin-3-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole

1

(55). 55 was synthesized similarly to 17, starting from 93. Yield 20%, mp

168−171 °C (from ethanol). H NMR (DMSO-d ): δ 2.36 (s, 3H), 3.52

6

P

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(

1

3

s, 6H), 3.56 (s, 3H), 6.29 (s, 2H), 7.09−7.13 (m, 1H), 7.19−7.23

m, 1H), 7.32−7.34 (m, 2H), 7.47−7.51 (m, 2H), 7.78−7.80 (m, 2H),

6-(3-((3,4,5-Trimethoxyphenyl)thio)-1H-indol-2-yl)quinoline

(66). A mixture of 108 (0.1 g, 0.000 23 mol), 6-quinolineboronic acid

pinacol ester (0.076 g, 0.0003 mol), and potassium phosphate (0.14 g,

0.000 68 mol) in anhydrous DMF (10 mL) was degassed for 30 min.

2.02 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν

2

−1

344 cm . Anal. (C H NO S (405.51)) C, H, N, S.

24

23

3

2-(3-Isopropoxy-4-methoxyphenyl)-3-((3,4,5-trimethoxyphenyl)-

Pd(dppf)Cl .CH Cl (0.0054 g, 0.000 006 6 mol, complex with

2 2 2

thio)-1H-indole (48). 48 was synthesized similarly to 14, starting from

1

δ 1.29 (d, J = 6.1 Hz, 6H), 3.65 (s, 6H), 3.77 (s, 3H), 3.90 (s, 3H), 4.35

dichloromethane (1:1), Pd 13%) was added under an Ar stream, and

the reaction mixture was heated at 110 °C for 12 h. After cooling, the

reaction mixture was diluted with water and extracted with ethyl acetate.

The organic layer was washed with brine, dried, and ﬁltered. Removal of

the solvent gave a residue that was puriﬁed by column chromatography

1

01. Yield 53%, mp 110−113 °C (from ethanol). H NMR (CDCl ):

3

(

m, 1H), 6.36 (s, 2H), 6.97 (d, J = 8.4 Hz, 1H), 7.19−7.29 (m, 3H), 7.41

m, 1H), 7.44−7.46 (m, 1H), 7.70−7.72 (m, 1H), 8.56 ppm (broad s,

−1

disappeared on treatment with D O, 1H). IR: ν 3337 cm . Anal.

(silica gel, ethyl acetate/n-hexane = 8:2 as eluent) to furnish 66 as a

2

1

(

C H NO S (479.59)) C, H, N, S.

slurry (0.07 g, 70%). H NMR (DMSO-d

): δ 3.52 (s, 6H), 3.55 (s, 3H),

6

27

29

5

2

-(Naphthalen-1-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-in-

6.35 (s, 2H), 7.14−7.16 (m, 1H), 7.25−7.28 (m, 1H), 7.54−7.61

(m, 3H), 8.15 (d, J = 8.8 Hz, 1H), 8.32 (dd, J = 2.0 and 8.8 Hz, 1H), 8.42

(d, J = 7.3 Hz, 1H), 8.47 (d, J = 1.8 Hz, 1H), 8.95 (dd, J = 1.7 and 4.2 Hz,

dole (59). 59 was synthesized similarly to 14, starting from 102. Yield

4

(

7

1

4%, mp 227−230 °C (from ethanol). H NMR (CDCl ): δ 3.56

3

s, 6H), 3.71 (s, 3H), 6.30 (s, 2H), 7.22−7.26 (m, 1H), 7.29−7.33

1H), 12.29 ppm (broad s, disappeared on treatment with D O, 1H). IR:

2

−1

m, 1H), 7.39−7.55 (m, 4H), 7.61 (dd, J = 1.2 and 7.1 Hz, 1H), 7.74−

ν 2922, 3305 cm . Anal. (C H N O S (442.53)) C, H, N, S.

26

22

2

3

.76 (m, 1H), 7.82−7.84 (m, 1H), 7.90−7.95 (m, 2H), 8.59 ppm (broad

3-((3,4,5-Trimethoxyphenyl)thio)-1H,1′H-2,5′-bisindole (67).

−1

s, disappeared on treatment with D O, 1H). IR: ν 3229 cm . Anal.

67 was synthesized similarly to 66, starting from 108 and 1-Boc-indole-

2

1

(

C H NO S (441.54)) C, H, N, S.

5-boronic acid pinacol ester. Yield 26% as a slurry. H NMR (CDCl ): δ

27

23

3

2

-(Naphthalen-2-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-in-

3.64 (s, 6H), 3.79 (s, 3H), 6.41 (s, 2H), 6.61−6.62 (m, 1H), 7.21−7.28

dole (62). 62 was synthesized similarly to 14, starting from 103. Yield

4

(

8

(m, 3H), 7.45−7.47 (m, 2H), 7.64−7.71 (m, 2H), 8.05−8.06 (m, 1H),

1

4%, mp 157−160 °C (from ethanol). H NMR (CDCl ): δ 3.65

8.32 (broad s, disappeared on treatment with D O, 1H), 8.65 ppm

3

2

s, 6H), 3.79 (s, 3H), 6.42 (s, 2H), 7.22−7.34 (m, 2H), 7.50−7.56

(broad s, disappeared on treatment with D O, 1H). IR: ν 2963, 2918,

2

−

1

m, 3H), 7.74−7.75 (m, 1H), 7.88−7.90 (m, 2H), 7.93−8.00 (m, 2H),

3350 cm . Anal. (C H N O S (430.52)) C, H, N, S.

25

22

2

3

.25 (m, 1H), 8.71 ppm (broad s, disappeared on treatment with D O,

3-((3,4,5-Trimethoxyphenyl)thio)-1H,1′H-2,2′-bisindole (68).

2

−1

1

H). IR: ν 3318 cm . Anal. (C H NO S (441.54)) C, H, N, S.

A mixture of 108 (0.15 g, 0.000 34 mol), 2-(triethylstannyl)-1H-indole-

27

23

3

2

4

2

-((1,1′-Biphenyl)-4-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-

1-carboxylic acid (0.25 g, 0.000 683 mol), and ethanol (5 mL) was

degassed for 30 min. PdCl (PPh ) (0.021 g, 0.000 031 mol) was added

indole (72). 72 was synthesized similarly to 14, starting from 104. Yield

2

3 2

1

5

(

2%, mp 210−215 °C (from ethanol). H NMR (DMSO-d ): δ 3.54

6

under an Ar stream, and the reaction mixture was heated at 78 °C for 48

h. After cooling, the mixture was ﬁltered through a pad of Celite, then

diluted with water and extracted with ethyl acetate. The organic layer

was washed with brine, dried, and ﬁltered. Removal of the solvent gave a

residue that was puriﬁed by column chromatography (silica gel, ethyl

s, 6H), 3.57 (s, 3H), 6.33 (s, 2H), 7.11−7.15 (m, 1H), 7.23−7.25

m, 1H), 7.38−7.42 (m, 1H), 7.48−7.54 (m, 4H), 7.75−7.77 (m, 2H),

7

.84−7.86 (m, 2H), 8.01−8.03 (m, 2H), 12.14 ppm (broad s, dis-

−1

appeared on treatment with D O, 1H). IR: ν 3329 cm . Anal.

2

(

C H NO S (467.58)) C, H, N, S.

29 25 3

acetate/n-hexane = 1:2 as eluent) to furnish 68 (0.03 g, 20%), mp 220−

1

2-(3-((3,4,5-Trimethoxyphenyl)thio)-1H-indol-2-yl)-4,5-dihy-

2

25 °C (from ethanol). H NMR (DMSO-d ): δ 3.52 (s, 6H), 3.55

6

droxazole (28). 2-Bromoethylamine hydrobromide (0.63 g, 0.0031

mol) was added to a solution of 3-((3,4,5-trimethoxyphenyl)thio)-1H-

indole-2-carboxylic acid (107) (1.0 g, 0.0028 mol), triethylamine (0.85

g, 1.2 mL, 0.0084 mol), and BOP reagent (1.24 g, 0.0028 mol) in

anhydrous DMF (15 mL). The reaction mixture was stirred at 25 °C for

(

7

s, 3H), 6.39 (s, 2H), 7.02−7.06 (m, 1H), 7.13−7.18 (m, 3H), 7.22−

.26 (m, 1H), 7.49−7.55 (m, 3H), 7.59−7.61 (m, 1H), 11.37 (broad s,

disappeared on treatment with D O, 1H), 12.02 ppm (broad s,

disappeared on treatment with D O, 1H). IR: ν 3326, 3388 cm . Anal.

2

−1

2

(

C H N O S (430.52)) C, H, N, S.

25 22 2 3

2

h and then diluted with water and extracted with ethyl acetate. The

organic layer was washed with brine, dried, and ﬁltered. Removal of the

solvent gave a residue that was puriﬁed by column chromatography

silica gel, ethyl acetate/n-hexane = 7:3 as eluent) to furnish 28 (0.31 g,

0%), mp 190−195 °C (from ethanol). H NMR (DMSO-d ): δ 3.55

s, 6H), 3.56 (s, 3H), 4.00 (t, J = 9.7 Hz, 2H), 4.43 (t, J = 9.5 Hz, 2H),

.42 (s, 2H), 7.07−7.11 (m, 1H), 7.23−7.27 (m, 1H), 7.43−7.49 (m,

H), 12.21 ppm (broad s, disappeared on treatment with D O, 1H). IR:

ν 3198 cm . Anal. (C H N O S (384.45)) C, H, N, S.

2-(Benzofuran-2-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-in-

dole (69). A mixture of 108 (0.1 g, 0.000 23 mol), 2-benzofuranylbor-

onic acid (0.29 g, 0.0018 mol), and potassium carbonate (0.28 g,

0.02 mol) in 1,4-dioxane (12 mL) containing water (2 mL) was degassed

(

3

(

6

1

6

for 30 min. Pd(OCOMe) (0.052 g, 0.002 34 mol) was added under an

2

Ar stream, and the reaction mixture was heated at 100 °C for 24 h. After

cooling, the mixture was diluted with water and extracted with ethyl

acetate. The organic layer was washed with brine, dried, and ﬁltered.

Removal of the solvent gave a residue that was puriﬁed by column

chromatography (silica gel, ethyl acetate/n-hexane = 1:2 as eluent) to

2

−1

20

2

4

2

-(3-((3,4,5-Trimethoxyphenyl)thio)-1H-indol-2-yl)oxazole

31). A mixture of 2-iodo-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole

108) (0.1 g, 0.000 23 mol), 2-(tributylstannyl)oxazole (0.25 g, 0.1 mL,

.000 69 mol), P(Cy) (0.0064 g, 0.000 023 mol) in anhydrous DMF

1

(

0

(

furnish 69 (0.11 g, 88%), mp 190−195 °C (from ethanol). H NMR

(

DMSO-d ): δ 3.54 (s, 6H), 3.56 (s, 3H), 6.42 (s, 2H), 7.14−7.18

6

3

(m, 1H), 7.27−7.33 (m, 2H), 7.37−7.42 (m, 1H), 7.54−7.57 (m, 2H),

2 mL) was degassed for 10 min. Pd (dba) (0.01 g, 0.000 011 mol) was

2 3

7.64−7.67 (m, 2H), 7.75−7.76 (m, 1H), 12.49 ppm (broad s, disappeared

−1

added, and the reaction mixture was placed into the microwave cavity

^c^l^o^s^e^d^v^e^s^s^e^l^m^o^d^e^,^Pmax = 250 psi). A starting microwave irradiation of

50 W was used, the temperature being ramped from 25 to 150 °C while

on treatment with D O, 1H). IR: ν 3338 cm . Anal. (C H NO S

2

25 21

4

(

1

(431.50)) C, H, N, S.

-(Benzo[b]thiophen-2-yl)-3-((3,4,5-trimethoxyphenyl)thio)-

2

stirring. Once 150 °C was reached, taking about 2 min, the reaction

mixture was held at this temperature for 10 min. The reaction was

quenched on a mixture of a saturated aqueous solution of potassium

carbonate (20 mL) and ethyl acetate while stirring for 30 min.

The organic layer was separated and washed with brine, dried, and

ﬁltered. Removal of the solvent gave a residue that was puriﬁed by

column chromatography (silica gel, ethyl acetate/n-hexane = 1:3 as

1H-indole (70). A mixture of 108 (0.1 g, 0.000 23 mol), benzo[b]thien-

2-ylboronic acid (0.04 g, 0.000 225 mol), and 1 M potassium carbonate

(0.6 mL) in THF (5 mL) was degassed for 30 min. Pd(PPh ) (0.01 g,

0.009 mmol) was added under an Ar stream, and the reaction mixture

was heated at 60 °C for 24 h. After cooling, the mixture was diluted with

water and extracted with ethyl acetate. The organic layer was washed

with brine, dried, and ﬁltered. Removal of the solvent gave a residue that

was puriﬁed by column chromatography (silica gel, dichloromethane/

3

4

1

eluent) to furnish 31 (0.03, 35%), mp 80−83 °C (from ethanol). H

NMR (DMSO-d ): δ 3.56 (s, 6H), 3.57 (s, 3H), 6.38 (s, 2H), 7.06−7.10

ethyl acetate = 99:1 as eluent) to furnish 70 (0.01 g, 10%), mp 220−225 °C

6

1

(

1

3

m, 1H), 7.16−7.20 (m, 1H), 7.40−7.52 (m, 3H), 7.76−7.77 (m, 1H),

(from ethanol). H NMR (CDCl ): δ 3.66 (s, 6H), 3.78 (s, 3H), 6.44 (s,

3

1.66 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν

2H), 7.21−7.23 (m, 1H), 7.28−7.39 (m, 3H), 7.46 (d, J = 7.6 Hz, 1H),

7.70 (d, J = 7.6 Hz, 1H), 7.80−7.86 (m, 3H), 8.83 ppm (broad s,

2

−1

340 cm . Anal. (C H N O S (382.43)) C, H, N, S.

20

18

2

4

Q

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

−

1

disappeared on treatment with D O, 1H). IR: ν 3303 cm . Anal.

(2-(1H-Pyrazol-4-yl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

2

(

C H NO S (447.57)) C, H, N, S.

methanone (15). 15 was synthesized similarly to 7, starting from 98.

25

21

3 2

1

2

-(Benzo[b]thiophen-3-yl)-3-((3,4,5-trimethoxyphenyl)thio)-

H-indole (71). A mixture of 108 (0.1 g, 0.000 23 mol), SPhos (0.0075

g, 0.0182 mmol), Pd (dba) (0.0042 g, 0.004 54 mmol), and potassium

Yield 21%, mp 112−117 °C (from ethanol). H NMR (DMSO-d

6

): δ

1

3.65 (s, 6H), 3.71 (s, 3H), 6.95 (s, 2H), 7.06−7.10 (m, 1H), 7.16−7.20

(m, 1H), 7.44−7.51 (m, 2H), 7.86 (m, 2H), 12.02 (broad s, disappeared

2

3

carbonate (0.0964 g, 0.000 454 mol) was degassed for 30 min. A

solution of benzo[b]thien-3-ylboronic acid (0.061 g, 0.000 34 mol) in

n-BuOH (2 mL) was added under an Ar stream, and the reaction mixture

was heated at 100 °C for 12 h. After cooling, the mixture was diluted with

water, made acidic with 1 M HCl (pH ≈ 3−4), and extracted with ethyl

acetate. The organic layer was washed with brine, dried, and ﬁltered.

Removal of the solvent gave a residue that was puriﬁed by column

chromatography (silica gel, ethyl acetate/n-hexane = 1:2 as eluent) to

on treatment with D O, 1H), 13.04 ppm (broad s, disappeared on

2

−1

treatment with D O, 1H). IR: ν 1634, 2962, 3243 cm . Anal.

2

(C H N O (377.39)) C, H, N.

21

19

3

4

( 2 - ( 1 H - I m i d a z o l - 1 - y l ) - 1 H - i n d o l - 3 - y l ) ( 3 , 4 , 5 -

trimethoxyphenyl)methanone (19). 19 was synthesized similarly to

2

1

7 starting from 80. Yield 26%, mp 210−215 °C (from ethanol). H

NMR (DMSO-d

): δ 3.66 (s, 3H), 3.69 (s, 6H), 6.79 (s, 2H), 6.88−6.89

6

(m, 1H), 7.24−7.32 (m, 2H), 7.39−7.40 (m, 1H), 7.50−7.52 (m, 1H),

1

furnish 71 (0.037 g, 36%), mp 170−174 °C (from ethanol). H NMR

7.79−7.80 (m, 1H), 7.97−7.99 (m, 1H), 12.66 ppm (broad s,

−

1

(

DMSO-d ): δ 3.48 (s, 6H), 3.53 (s, 3H), 6.25 (s, 2H), 7.16−7.18 (m,

disappeared on treatment with D

O, 1H). IR: ν 1618, 3354 cm .

2

6

1

7

H), 7.24−7.28 (m, 1H), 7.45−7.47 (m, 2H), 7.53−7.56 (m, 2H),

.87−7.89 (m, 1H), 8.18 (s, 1H), 8.09−8.12 (m, 1H), 12.17 ppm (broad

Anal. (C21 (377.39)) C, H, N.

H N O

19 3 4

(2-(Pyridin-2-yl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

−

1

s, disappeared on treatment with D O, 1H). IR: ν 3237 cm . Anal.

methanone (54). 54 was synthesized similarly to 7, starting from 92.

2

1

(

C H NO S (447.57)) C, H, N, S.

Yield 67%, mp 65−70 °C (from ethanol). H NMR (DMSO-d

6

): δ 3.61

25

21

3 2

2

-Methoxy-5-(3-((3,4,5-trimethoxyphenyl)thio)-1H-indol-2-

(s, 6H), 3.64 (s, 3H), 6.89 (s, 2H), 7.19−7.21 (m, 1H), 7.26−7.28 (m,

2H), 7.39−7.41 (m, 1H), 7.56−7.58 (m, 1H), 7.63−7.65 (m, 1H),

7.79−7.81 (m, 1H), 8.55−8.57 (m, 1H), 12.36 ppm (broad s,

yl)phenol (49). Anhydrous aluminum chloride (0.14 g, 0.001 mol) was

added to a solution of 48 (0.17 g, 0.000 35 mol) in anhydrous

dichloromethane (10 mL). The reaction mixture was stirred at 25 °C for

−1

disappeared on treatment with D O, 1H). IR: ν 1625, 3241 cm .

2

1.5 h. After dilution with a saturated aqueous solution of ammonium

Anal. (C H N O (388.42)) C, H, N.

23

20

2

4

chloride, the mixture was extracted with chloroform. The organic layer

was washed with brine, dried, and ﬁltered. Removal of the solvent gave a

residue that was puriﬁed by column chromatography (silica gel, ethyl

(2-(Pyridin-3-yl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

methanone (56). 56 was synthesized similarly to 7, starting from 93.

1

Yield 45%, mp 130−135 °C (from ethanol). H NMR (DMSO-d

): δ

6

acetate/n-hexane = 1:1 as eluent) to furnish 49 (0.05 g, 35%), mp 110−

3.60 (s, 3H), 3.61 (s, 6H), 6.82 (s, 2H), 7.21−7.31 (m, 3H), 7.53−7.55

(m, 1H), 7.75−7.78 (m, 1H), 7.96 (d, J = 7.7 Hz, 1H), 8.44 (dd, J = 1.6

and 4.8 Hz, 1H), 8.56−8.57 (m, 1H), 12.38 ppm (broad s, disappeared

1

3

7

13 °C (from ethanol). H NMR (CDCl ): δ 3.65 (s, 6H), 3.77 (s, 3H),

3

.95 (s, 3H), 5.69 (s, 1H), 6.36 (s, 2H), 6.94 (d, J = 8.3 Hz, 1H), 7.16−

.20 (m, 1H), 7.24−7.28 (m, 1H), 7.34 (dd, J = 2.2 and 8.3 Hz, 1H),

.38 (d, J = 2.2 Hz, 1H), 7.42−7.44 (m, 1H), 7.66−7.68 (m, 1H), 8.49

−

1

on treatment with D

(388.42)) C, H, N.

O, 1H). IR: ν 1597, 3094 cm . Anal.

2

(C23H N O

20 2 4

ppm (broad s, disappeared on treatment with D O, 1H). IR: ν 2933,

(2-(Pyridin-4-yl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

2

−1

methanone (58). 58 was synthesized similarly to 7, starting from 94.

3

329 cm . Anal. (C H NO S (437.51)) C, H, N, S.

24 23 5

1

Yield 10%, mp 50−55 °C (from ethanol). H NMR (DMSO-d ): δ 3.62

2

-(3,4-Dimethoxyphenyl)-3-((3,4,5-trimethoxyphenyl)thio)-

6

1

H-indole (50). Iodomethane (0.033 g, 0.01 mL, 0.000 23 mol) was

(s, 3H), 3.63 (s, 6H), 6.86 (s, 2H), 7.21−7.25 (m, 1H), 7.29−7.33 (m,

1H), 7.35−7.37 (m, 2H), 7.55−7.57 (m, 1H), 7.87−7.90 (m, 1H),

8.46−8.47 (m, 2H), 12.43 ppm (broad s, disappeared on treatment with

added to a mixture of 49 (0.1 g, 0.000 23 mol) and anhydrous potassium

carbonate (0.032 g, 0.000 23 mol) in anhydrous DMF (5 mL). The

reaction mixture was stirred at 25 °C for 2 h, then diluted with water and

extracted with ethyl acetate. The organic layer was washed with brine,

dried, and ﬁltered. Removal of the solvent gave a residue that was

puriﬁed by column chromatography (silica gel, ethyl acetate/n-hexane =

−1

D

O, 1H). IR: ν 1602, 3304 cm . Anal. (C23

H N O (388.42)) C,

20 2 4

2

H, N.

( 2 - ( N a p h t h a l e n - 1 - y l ) - 1 H - i n d o l - 3 - y l ) ( 3 , 4 , 5 -

trimethoxyphenyl)methanone (60). 60 was synthesized similarly to

1

:1 as eluent) to furnish 50 (0.02 g, 20%), mp 117−118 °C (from

7, starting from 102. Yield 50%, mp 177−179 °C (from ethanol). H

1

ethanol). H NMR (CDCl ): δ 3.65 (s, 6H), 3.77 (s, 3H), 3.78 (s, 3H),

NMR (CDCl ): δ 3.40 (s, 6H), 3.64 (s, 3H), 6.61 (s, 2H), 7.26−7.30

3

7

1

.94 (s, 3H), 6.38 (s, 2H), 6.97 (d, J = 8.3 Hz, 1H), 7.19−7.23 (m, 1H),

(m, 1H), 7.34−7.36 (m, 1H), 7.37−7.41 (m, 2H), 7.43−7.53 (m, 3H),

7.73 (d, J = 8.2 Hz, 1H), 7.78−7.83 (m, 1H), 7.94−8.00 (m, 1H), 8.31−

.26−7.31 (m, 2H), 7.40 (d, J = 2.1 Hz, 1H), 7.45−7.47 (m, 1H), 7.70−

.72 (m, 1H), 8.57 ppm (broad s, disappeared on treatment with D O,

8.36 (m, 1H), 8.81 ppm (broad s, disappeared on treatment with D O,

2

−1

H). IR: ν 3344 cm . Anal. (C H NO S (451.53)) C, H, N, S.

1H). IR: ν 1605, 3158 cm . Anal. (C28H23NO (437.49)) C, H, N.

4

25

5

Aroyl-1H-indoles. General Procedure for the Preparation of

General Procedure for the Preparation of Compounds 9, 21,

23, 25, 32, 35, 40, 42, 44, 51, 63, 74, 76, and 78. Example: (2-(1-

(Phenylsulfonyl)-1H-pyrrol-3-yl)-1H-indol-3-yl)(3,4,5-

trimethoxyphenyl)methanone (9). A mixture of 2-(1-(phenyl-

Compounds 7, 15, 19, 54, 56, 58, and 60. Example: (2-(1H-

Pyrrol-2-yl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)methanone

(

7). Methylmagnesium bromide (1.4 mL, 0.0041 mol, 3.0 M in Et O)

2

1

9

was added dropwise over 5 min into a mixture of 2-(1H-pyrrol-2-yl)-1H-

sulfonyl)-1H-pyrrol-3-yl)-1H-indole (110) (0.26 g, 0.000 82 mol),

3,4,5-trimethoxybenzoyl chloride (0.23 g, 0.000 98 mol), and anhydrous

aluminum chloride (0.13 g, 0.000 98 mol) in anhydrous 1,2-dichloro-

ethane (2 mL) was placed into the microwave cavity (closed vessel

mode, Pmax = 250 psi). A starting microwave irradiation of 150 W was

used, the temperature being ramped from 25 to 110 °C while stirring.

Once 110 °C was reached, taking about 1 min, the reaction mixture was

held at this temperature for 2 min. The reaction mixture was quenched

on 1 M HCl/crushed ice and extracted with chloroform. The organic

layer was washed with brine, dried, and ﬁltered. Removal of the solvent

gave a residue that was puriﬁed by column chromatography (silica gel,

25

indole (109) (0.5 g, 0.003 mol) and anhydrous ZnCl (0.82 g, 0.006

2

mol) in anhydrous dichloromethane (20 mL) at 25 °C under an Ar

stream. After 1 h, a solution of 3,4,5-trimethoxybenzoyl chloride (0.76 g,

.0033 mol) in the same solvent (16 mL) was added dropwise over 5

0

min at 25 °C under an Ar stream. After an additional 1 h, SnCl (3 mL,

4

0

.003 mol, 1.0 M in dichloromethane) was added dropwise, and the

reaction mixture was stirred at 25 °C for 12 h under an Ar stream. The

reaction was quenched on crushed ice and extracted with dichloro-

methane The organic layer was washed with brine, dried, and ﬁltered.

Removal of the solvent gave a residue that was puriﬁed by column

chromatography (silica gel, ethyl acetate/n-hexane = 1:1 as eluent) to

ethyl acetate/n-hexane = 1:1 as eluent) to furnish 9 (0.27 g, 65%), mp

1

furnish 7 (0.03 g, 3%), mp 59−62 °C (from toluene). H NMR

124−126 °C (from ethanol). H NMR (DMSO-d ): δ 3.57 (s, 6H), 3.67

6

(

6

1

(

DMSO-d ): δ 3.70 (s, 6H), 3.75 (s, 3H), 6.24−6.26 (m, 1H), 6.84−

(s, 3H), 6.44 (s, 1H), 6.92 (s, 2H), 7.07 (t, J = 7.4 Hz, 1H), 7.18 (t, J = 7.4

Hz, 1H), 7.35 (s, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H),

7.66 (t, J = 7.8 Hz, 2H), 7.76−7.78 (m, 2H), 7.96 (d, J = 7.4 Hz, 2H),

6

.85 (m, 1H), 6.96−7.07 (m, 5H), 7.14−7.18 (m, 1H), 7.43−7.45 (m, 1H),

1.94 (broad s, disappeared on treatment with D O, 1H), 12.15 ppm

2

broad s, disappeared on treatment with D O, 1H). IR: ν 1623,

12.04 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν

2

−1

3

344 cm . Anal. (C H N O (376.41)) C, H, N.

1603, 3303 cm . Anal. (C H N O S (516.56)) C, H, N, S.

22

20

2

4

28 24

2

6

R

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(

2-(Furan-2-yl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

(2-Cyclobutyl-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

methanone (21). 21 was synthesized similarly to 9, starting from

methanone (74). 74 was synthesized similarly to 9, starting from 94.

19

1

3

7

11. Yield 5%, mp 115−117 °C (from ethanol). H NMR (CDCl ): δ

Yield 75%, mp 60−63 °C (from ethanol). H NMR (DMSO-d ): δ

3

6

.80 (s, 6H), 3.95 (s, 3H), 6.50−6.51 (m, 1H), 7.10−7.15 (m, 3H),

.22−7.26 (m, 2H), 7.41−7.46 (m, 2H), 7.52−7.53 (m, 1H), 9.11 ppm

1.84−1.94 (m, 2H), 2.14−2.22 (m, 2H), 2.29−2.39 (m, 2H), 3.76

(s, 6H), 3.77 (s, 3H), 3.79−3.81 (m, 1H), 6.94 (s, 2H), 7.02−7.06

(m, 1H), 7.12−7.16 (m, 1H), 7.40−7.42 (m, 1H), 7.44−7.47 (m, 1H),

−1

(

broad s, disappeared on treatment with D O, 1H). IR: ν 1643, 3358 cm .

2

Anal. (C H NO (377.99)) C, H, N.

11.95 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν

22

19

5

2

−1

(

2-(Furan-3-yl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

1600, 3217 cm . Anal. (C H NO (365.42)) C, H, N.

22 23 4

methanone (23). 23 was synthesized similarly to 9, starting from

(2-Cyclopentyl-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

19

1

12. Yield 32%, mp 115−117 °C (from ethanol). H NMR (CDCl ):

methanone (76). 76 was synthesized similarly to 9, starting from 95.

3

1

δ 3.78 (s, 6H), 3.93 (s, 3H), 6.60−6.61 (m, 1H), 7.13 (s, 2H), 7.15−7.19

m, 1H), 7.25−7.27 (m, 1H), 7.43−7.46 (m, 2H), 7.58−7.60 (m, 1H),

.05−8.06 (m, 1H), 8.70 ppm (broad s, disappeared on treatment with

Yield 68%, mp 110−115 °C (from ethanol). H NMR (DMSO-d

6

): δ

(

8

1.57−1.62 (m, 2H), 1.75−1.85 (m, 4H), 1.95−1.99 (m, 2H), 3.37−3.45

(m, 1H), 3.76 (s, 6H), 3.78 (s, 3H), 6.97 (s, 2H), 7.0−7.04 (m, 1H),

7.10−7.14 (m, 1H), 7.29−7.31 (m, 1H), 7.41−7.43 (m, 1H), 11.77 ppm

−1

D O, 1H). IR: ν 1598, 3345 cm . Anal. (C H NO (377.99)) C, H, N.

2

(

22 19

5

2-(Thiophen-3-yl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

(broad s, disappeared on treatment with D O, 1H). IR: ν 1603, 3261

2

−

1

methanone (25). 25 was synthesized similarly to 9, starting from

cm . Anal. (C H NO (379.45)) C, H, N.

23

25

4

19

1

13. Yield 25%, mp 118−120 °C (from ethanol). H NMR (CDCl ):

(2-Cyclohexyl-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

3

δ 3.74 (s, 6H), 3.87 (s, 3H), 7.04 (s, 2H), 7.11−7.12 (m, 1H), 7.23−7.28

m, 2H), 7.29−7.33 (m, 1H), 7.44−7.47 (m, 1H), 7.50−7.51 (m, 1H),

.91−7.93 (m, 1H), 8.60 ppm (broad s, disappeared on treatment with

methanone (78). 78 was synthesized similarly to 9, starting from 96.

1

(

7

Yield 68%, mp 140−145 °C (from ethanol). H NMR (DMSO-d

6

): δ

1.08−1.26 (m, 3H), 1.59−1.70 (m, 3H), 1.77−1.83 (m, 4H), 2.89−2.95

(m, 1H), 3.75 (s, 6H), 3.76 (s, 3H), 6.95 (s, 2H), 7.01−7.05 (m, 1H),

7.10−7.14 (m, 1H), 7.38−7.42 (m, 2H), 11.80 ppm (broad s,

−1

D O, 1H). IR: ν1606, 3337 cm . Anal. (C H NO S (393.46)) C, H, N, S.

2

22 19

4

(

2-(Oxazol-2-yl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

−1

methanone (32). 32 was synthesized similarly to 9, starting from 114.

disappeared on treatment with D O, 1H). IR: ν 1602, 3285 cm .

2

1

Yield 28%, mp 208−210 °C (from ethanol). H NMR (CDCl ): δ 3.79

Anal. (C H NO (393.48)) C, H, N.

3

24 27

4

(

7

s, 6H), 3.94 (s, 3H), 7.20−7.26 (m, 4H), 7.35−7.39 (m, 1H), 7.49−

((2-(1H-Pyrrol-3-yl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

methanone (8). A mixture of 9 (0.1 g, 0.000 19 mol) and 2 M NaOH

(0.6 mL) in methanol (2 mL) was heated at reﬂux for 3 h. After cooling,

the reaction mixture was made acidic with 2 M HCl (pH ≈ 3−4) and

extracted with ethyl acetate. The organic layer was washed with brine,

dried, and ﬁltered. Removal of the solvent gave a residue that was

puriﬁed by column chromatography (silica gel, ethyl acetate/n-hexane =

.56 (m, 2H), 7.73−7.75 (m, 1H), 9.69 ppm (broad s, disappeared on

−1

treatment with D O, 1H). IR: ν 1608, 3137 cm . Anal. (C H N O

2

21 18

2

5

(

378.38)) C, H, N.

(

methanone (35). 35 was synthesized similarly to 9, starting from 84.

2-Phenyl-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

1

Yield 95%, mp 207−212 °C (from ethanol). H NMR (DMSO-d ): δ

6

3

(

.59 (s, 3H), 3.60 (s, 6H), 6.82 (s, 2H), 7.18−7.28 (m, 5H), 7.36−7.38

1:1 as eluent) to furnish 8 (0.7 g, 98%), mp 114−115 °C (from ethanol).

1

m, 2H), 7.50−7.53 (m, 1H), 7.92−7.94 (m, 1H), 12.20 ppm (broad s,

H NMR (CDCl ): δ 3.77 (s, 6H), 3.90 (s, 3H), 6.42−6.44 (m, 1H),

3

−1

disappeared on treatment with D O, 1H). IR: ν 1590, 3213 cm . Anal.

6.77−6.79 (m, 1H), 7.10 (s, 2H), 7.14−7.25 (m, 3H), 7.40−7.42 (m,

1H), 7.67−7.71 (m, 1H), 8.38 (broad s, disappeared on treatment with

D O, 1H), 8.53 ppm (broad s, disappeared on treatment with D O, 1H).

2

(

C H NO (387.43)) C, H, N.

24 21 4

(

2-(2-Fluorophenyl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

2

−

1

methanone (40). 40 was synthesized similarly to 9, starting from 86.

IR: ν 1614, 3241, 3333 cm . Anal. (C H N O (376.41)) C, H, N.

22 20 2 4

1

Yield 65%, mp 180−185 °C (from toluene). H NMR (DMSO-d ): δ

(2-(4,5-Dihydrooxazol-2-yl)-1H-indol-3-yl)(3,4,5-trimethoxy-

phenyl)methanone (29). 29 was synthesized similarly to 28, starting

6

3

(

.59 (s, 3H), 3.64 (s, 6H), 6.79 (s, 2H), 7.07−7.35 (m, 5H), 7.45−7.48

m, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 12.27 ppm

from 3-(3,4,5-trimethoxybenzoyl)-1H-indole-2-carboxylic acid. Yield

1

broad s, disappeared on treatment with D O, 1H). IR: ν 1591, 3187

47%, mp 220−225 °C (from ethanol). H NMR (CDCl ): δ 3.84 (s,

2

3

−

1

cm . Anal. (C H FNO (405.42)) C, H, N, F.

6H), 3.94 (s, 3H), 3.99 (m, 2H), 4.19 (m, 2H), 7.19 (s, 2H), 7.21−7.23

(m, 1H), 7.34−7.37 (m, 1H), 7.45−7.47 (m, 1H), 7.70−7.72 (m, 1H),

10.24 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν

24

20

4

(

2-(3-Fluorophenyl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

methanone (42). 42 was synthesized similarly to 9, starting from 87.

2

1

−1

Yield 52%, mp 190−195 °C (from toluene). H NMR (DMSO-d ): δ

.60 (s, 3H), 3.62 (s, 6H), 6.78 (s, 2H), 7.11 (t, J = 8.8 Hz, 2H), 7.20 (t,

J = 7.6 Hz, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.39−7.42 (m, 2H), 7.51 (d, J =

.9 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H), 12.21 ppm (broad s, disappeared

on treatment with D O, 1H). IR: ν 1594, 3221 cm . Anal. (C H FNO

405.42)) C, H, F, N.

3182 cm . Anal. (C H N O (380.39)) C, H, N.

6

21 20

2

5

3

3-(3,4,5-Trimethoxybenzoyl)-1H-indole-2-carboxylic Acid. A

mixture of methyl 3-(3,4,5-trimethoxybenzoyl)-1H-indole-2-carboxy-

1

8

7

late (0.42 g, 0.000 65 mol) and 3 N NaOH (2 mL) was placed into the

microwave cavity (closed vessel mode, P_m_a_x= 250 psi). A starting

microwave irradiation of 150 W was used, the temperature being ramped

from 25 to 110 °C, while stirring. Once 110 °C was reached, taking

about 1 min, the reaction mixture was held at this temperature for 2 min,

then made acidic with 3 N HCl (pH ≈ 3−4) and extracted with ethyl

acetate. The organic layer was washed with brine, dried, ﬁltered, and

−1

2

24 20

4

(

2-(4-Fluorophenyl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

methanone (44). 44 was synthesized similarly to 9, starting from 88.

1

Yield 64%, mp 210−215 °C (from toluene). H NMR (DMSO-d ):

δ 3.60 (s, 3H), 3.62 (s, 6H), 6.78 (s, 2H), 7.10 (t, J = 8.9 Hz, 2H), 7.20

t, J = 7.3 Hz, 1H), 7.27 (t, J = 7.1 Hz, 2H), 7.38−7.42 (m, 2H), 7.51 (d,

J = 8.2 Hz, 1H), 12.22 ppm (broad s, disappeared on treatment with D O,

6

(

evaporated to give 3-(3,4,5-trimethoxybenzoyl)-1H-indole-2-carboxylic

1

2

acid (0.23 g, 98%). H NMR (CDCl ): δ 3.84 (s, 6H), 1.09 (s, 3H), 7.11

3

−1

1

H). IR: ν 1592, 3218 cm . Anal. (C H FNO (405.42)) C, H, F, N.

24 20 4

(s, 2H), 7.16−7.20 (m, 1H), 7.23−7.25 (m, 1H), 7.39−7.43 (m, 1H),

(

2-(4-Methoxyphenyl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

7

1

.67 (m, 1H), 10.88 (broad s, disappeared on treatment with D O, 1H),

2

methanone (51). 51 was synthesized similarly to 9, starting from 91.

2.95 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν

2

1

Yield 9%, mp 170−175 °C (from ethanol). H NMR (CDCl ): δ 3.72

−1

3

1696, 2837, 2933, 3265 cm . Anal. (C H NO (355.34)) C, H, N.

19 17 6

(

7

(

s, 6H), 3.78 (s, 3H), 3.84 (s, 3H), 6.76−6.79 (m, 2H), 7.00 (s, 2H),

.27−7.34 (m, 4H), 7.46−7.48 (m, 1H), 8.03−8.05 (m, 1H), 8.70 ppm

broad s, disappeared on treatment with D O, 1H). IR: ν 1614, 3144

Arylmethyl-1H-indoles. General Procedure for the Prepara-

tion of Compounds 12, 22, 24, 26, 36, 52, 61, and 65. Example:

2-(1-(Phenylsulfonyl)-1H-pyrrol-3-yl)-3-(3,4,5-trimethoxyben-

zyl)-1H-indole (12). Borane−tetrahydrofuran complex, 1 M solution

in THF (0.85 mL, 0.000 85 mol), was added to an ice-cooled solution of 9

(0.1 g, 0.019 mol) in acetonitrile (1.2 mL) containing methanol (0.017 mL)

under an Ar stream. The reaction mixture was heated at 50 °C for 1 h. After

cooling, the reaction mixture was quenched on crushed ice and extracted

with ethyl acetate. The organic layer was washed with brine, dried, and

ﬁltered. Removal of the solvent gave a residue that was puriﬁed by

column chromatography (silica gel, ethyl acetate/n-hexane = 1:1 as

2

−

1

cm . Anal. (C H NO (417.45)) C, H, N.

25

23

5

(

2-(Naphthalen-2-yl)-1H-indol-3-yl)(3,4,5-trimethoxyphenyl)-

methanone (63). 63 was synthesized similarly to 9, starting from 103.

1

Yield 33%, mp 182−186 °C (from ethanol). H NMR (CDCl ): δ 3.56

3

(

s, 3H), 3.63 (s, 6H), 6.98 (s, 2H), 7.29−7.37 (m, 2H), 7.43 (dd, J = 1.8

and 8.5 Hz, 1H), 7.47−7.52 (m, 3H), 7.68−7.78 (m, 3H), 7.87 (m, 1H),

8

.10−8.11 (m, 1H), 8.80 ppm (broad s, disappeared on treatment with

−1

D O, 1H). IR: ν 1609, 3170 cm . Anal. (C H NO (437.49)) C, H, N.

2

28 23

4

S

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1

eluent) to furnish 12 (0.08 g, 84%), mp 176−180 °C (from ethanol). H

(from ethanol). H NMR (CDCl ): δ 3.77 (s, 6H), 3.80 (s, 3H), 4.09 (t,

3

NMR (DMSO-d ): δ 3.55 (s, 6H), 3.59 (s, 3H), 4.15 (s, 2H), 6.46

J = 9.5 Hz, 2H), 4.44−4.52 (m, 4H), 6.55 (s, 2H), 7.07−7.11 (m, 1H),

6

(

7

s, 2H), 6.81−6.82 (m, 1H), 6.97−6.99 (m, 1H), 7.06−7.08 (m, 1H),

7.25−7.29 (m, 1H), 7.36 (dd, J = 0.75 and 8.3 Hz, 1H), 7.56 (d, J = 8.2

.32 (d, J = 8.0 Hz, 1H), 7.48−7.50 (m, 2H), 7.62−7.66 (m, 3H), 7.75−

.76 (m, 1H), 7.94−7.96 (m, 2H), 11.16 ppm (broad s, disappeared on

Hz, 1H), 9.10 ppm (broad s, disappeared on treatment with D O, 1H).

2

−1

IR: ν 3257 cm . Anal. (C H N O (366.41)) C, H, N.

21

22

2

4

−1

treatment with D O, 1H). IR: ν 3293 cm . Anal. (C H N O S

3-(3,4,5-Trimethoxybenzyl)-1H-indole-2-carboxylic Acid. The

2

28 26

2

5

(

502.58)) C, H, N, S.

compound was obtained from methyl 3-(3,4,5-trimethoxybenzyl)-1H-

1

8

1

2-(Furan-2-yl)-3-(3,4,5-trimethoxybenzyl)-1H-indole (22). 22

indole-2-carboxylate. Yield 96%, mp 240−245 °C (from ethanol). H

was synthesized similarly to 12, starting from 21. Yield 9%, mp 78−80

NMR (DMSO-d ): δ 3.56 (s, 3H), 3.66 (s, 6H), 4.37 (s, 2H), 6.65

6

1

°

C (from ethanol). H NMR (CDCl ): δ 3.76 (s, 6H), 3.82 (s, 3H), 4.30

(s, 2H), 6.99−7.03 (m, 1H), 7.18−7.23 (m, 1H), 7.38 (d, J = 8.0 Hz, 1H),

7.67 (d, J = 8.0 Hz, 1H), 11.48 (broad s, disappeared on treatment with

D O, 1H), 13.05 ppm (broad s, disappeared on treatment with D O,

3

(

s, 2H), 6.51−6.55 (m, 4H), 7.09−7.13 (m, 1H), 7.21−7.24 (m, 1H),

7

.40−7.43 (m, 1H), 7.52−7.54 (m, 2H), 8.49 ppm (broad s, disappeared

2

−1

on treatment with D O, 1H). IR: ν 3347 cm . Anal. (C H NO

1H). IR: ν 1663, 2840, 3354 cm . Anal. (C H NO (341.36)) C, H, N.

2

22 21

4

19 19 5

(

363.41)) C, H, N.

Arylmethanol-1H-indoles. (2-(1H-Pyrrol-3-yl)-1H-indol-3-yl)-

(3,4,5-trimethoxyphenyl)methanol (10). 10 was synthesized

2-(Furan-3-yl)-3-(3,4,5-trimethoxybenzyl)-1H-indole (24). 24

was synthesized similarly to 12, starting from 23. Yield 13%, mp 102−

similarly to 8, starting from 11. Yield 98%, mp 189−191 °C (from

1

4

7

05 °C (from ethanol). H NMR (CDCl ): δ 3.73 (s, 6H), 3.80 (s, 3H),

.18 (s, 2H), 6.44 (s, 2H), 6.65−6.66 (m, 1H), 7.07−7.11 (m, 1H),

.17−7.19 (m, 1H), 7.37−7.39 (m, 1H), 7.47−7.49 (m, 1H), 7.52−7.53

ethanol). H NMR (CDCl

3

): δ 3.57 (s, 6H), 3.86 (s, 3H), 5.13 (m, 1H),

3

6.00 (s, disappeared on treatment with D O, 1H), 6.38−6.39 (m, 2H),

2

6.54 (s, 1H), 6.62−6.64 (m, 1H), 6.75−6.78 (m, 3H), 6.85−6.88 (m,

1H), 7.00−7.03 (m, 1H), 8.01 (broad s, disappeared on treatment with

(

m, 1H), 7.63−7.64 (m, 1H), 8.07 ppm (broad s, disappeared on treat-

−1

ment with D O, 1H). IR: ν 3320 cm . Anal. (C H NO (363.41)) C,

D

2

O, 1H), 8.16 ppm (broad s, disappeared on treatment with D

2

O, 1H).

IR: ν 2852, 2922, 3382 cm . Anal. (C22H N O (378.42)) C, H, N.

22 2 4

2

22 21

4

−

1

H, N.

-(Thiophen-3-yl)-3-(3,4,5-trimethoxybenzyl)-1H-indole

26). 26 was synthesized similarly to 12, starting from 25. Yield 61%, mp

2

General Procedure for the Preparation of Compounds 11 and

64. Example: (2-(1-(Phenylsulfonyl)-1H-pyrrol-3-yl)-1H-indol-3-

yl)(3,4,5-trimethoxyphenyl)methanol (11). To a solution of 9 (0.5

g, 0.0013 mol) in THF (2.6 mL) containing 0.08 mL of water was

carefully added sodium borohydride (0.049 g, 0.0013 mol). The reaction

mixture was heated at 80 °C for 2 h. After cooling, the mixture was

diluted with water and extracted with ethyl acetate. The organic layer

was washed with brine, dried, and ﬁltered. Removal of the solvent gave a

residue that was puriﬁed by column chromatography (silica gel, acetone/

(

1

65−168 °C (from ethanol). H NMR (CDCl ): δ 3.74 (s, 6H), 3.83

3

(

7

s, 3H), 4.25 (s, 2H), 6.48 (s, 2H), 7.10−7.14 (m, 1H), 7.21−7.25 (m, 1H),

.34−7.35 (m, 1H), 7.40−7.46 (m, 3H), 7.51−7.53 (m, 1H), 8.22 ppm

−

1

(

broad s, disappeared on treatment with D O, 1H). IR: ν 3353 cm .

2

Anal. (C H NO S (379.47)) C, H, N, S.

22

21

3

2-Phenyl-3-(3,4,5-trimethoxybenzyl)-1H-indole (36). 36 was

synthesized similarly to 12, starting from 35. Yield 26%, mp 187−192 °C

1

n-hexane = 1:1 as eluent) to furnish 11 (0.54 g, 97%), mp 90−94 °C

(

7

from ethanol). H NMR (DMSO-d ): δ 3.59 (s, 3H), 3.60 (s, 6H), 4.17

6

1

(

5

from ethanol). H NMR (DMSO-d ): 3.58 (s, 6H), 3.60 (s, 3H), 5.71−

s, 2H), 6.46 (s, 2H), 6.97−7.01 (m, 1H), 7.09−7.13 (m, 1H), 7.37−

6

.73 (m, 1H), 6.05 (s, disappeared on treatment with D O, 1H), 6.63 (s,

.41 (m, 2H), 7.47−7.53 (m, 3H), 7.64−7.66 (m, 2H), 11.28 ppm

2

−1

2H), 6.83−6.86 (m, 2H), 7.01 (t, J = 6.6 Hz, 1H), 7.27 (d, J = 8.4 Hz,

(

broad s, disappeared on treatment with D O, 1H). IR: ν 3351 cm .

2

1

7

H), 7.36 (d, J = 7.4 Hz, 1H), 7.47−7.50 (m, 1H), 7.63−7.67 (m, 2H),

.74−7.77 (m, 2H), 7.98 (d, J = 7.1 Hz, 2H), 11.14 ppm (broad s,

Anal. (C H NO (373.44)) C, H, N.

24

23

3

2

-(4-Methoxyphenyl)-3-(3,4,5-trimethoxybenzyl)-1H-indole

−1

disappeared on treatment with D O, 1H). IR: ν 2938, 3341 cm . Anal.

(

52). 52 was synthesized similarly to 12, starting from 51. Yield 4% as an

2

1

oil. H NMR (CDCl ): δ 3.73 (s, 6H), 3.82 (s, 3H), 3.86 (s, 3H), 4.19

(C28H N O S (518.58)) C, H, N, S.

26 2 6

3

(

2 - ( N a p h t h a l e n - 2 - y l ) - 1 H - i n d o l - 3 - y l ) ( 3 , 4 , 5 -

(

7

s, 2H), 6.46 (s, 2H), 6.98−7.00 (m, 2H), 7.07−7.11 (m, 1H), 7.18−

trimethoxyphenyl)methanol (64). 64 was synthesized similarly to

.22 (m, 1H), 7.39−7.41 (m, 1H), 7.46−7.49 (m, 3H), 8.09 ppm (broad

1

−

1

11, starting from 63. Yield 44%, mp 145−150 °C (from ethanol). H

NMR (CDCl ): δ 2.29 (d, J = 4.2 Hz, 1H), 3.76 (s, 6H), 3.84 (s, 3H),

s, disappeared on treatment with D O, 1H). IR: ν 3356 cm . Anal.

2

(

C H NO (403.47)) C, H, N.

3

25

4

6

.29 (d, J = 4.2 Hz, 1H), 6.79 (s, 2H), 7.07−7.11 (m, 1H), 7.22−7.26

2

-(Naphthalen-1-yl)-3-(3,4,5-trimethoxybenzyl)-1H-indole

(

m, 1H), 7.43−7.46 (m, 1H), 7.55−7.60 (m, 3H), 7.72 (dd, J = 1.8 and

(61). 61 was synthesized similarly to 12, starting from 60. Yield 99%, mp

1

8

3

.5 Hz, 1H), 7.87−7.91 (m, 2H), 7.96 (d, J = 8.4 Hz, 1H), 8.06 (m, 1H),

1

3

7

42−147 °C (from ethanol). H NMR (CDCl ): δ 3.60 (s, 6H), 3.74 (s,

H), 4.03 (s, 2H), 6.26 (s, 2H), 7.18−7.20 (m, 1H), 7.25−7.29 (m, 1H),

.43−7.47 (m, 2H), 7.52−7.58 (m, 3H), 7.63−7.65 (m, 1H), 7.84−7.86

3

.35 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν

2

−1

286, 3295, 3293 cm . Anal. (C H NO (439.50)) C, H, N.

28 25

4

Synthetic Intermediates. 2-(1H-Imidazol-2-yl)-1H-indole (79).

Diacetoxyiodo)benzene (0.52 g, 0.0016 mol) was added to a mixture of

-(4,5-dihydro-1H-imidazol-2-yl)-1H-indole (83) (0.27 g, 0.0015 mol)

(

m, 1H), 7.94−7.96 (m, 2H), 8.19 ppm (broad s, disappeared on treat-

−1

(

2

ment with D O, 1H). IR: ν 3317 cm . Anal. (C H NO (423.50)) C,

2

28 25

3

H, N.

-(Naphthalen-2-yl)-3-(3,4,5-trimethoxybenzyl)-1H-indole

65). 65 was synthesized similarly to 12, starting from 63. Yield 73%, mp

and potassium carbonate (0.22 g, 0.0016 mol) in DMSO (15 mL). The

reaction mixture was stirred at 25 °C for 12 h under an Ar stream, then

diluted with a saturated aqueous solution of sodium hydrogen carbonate

and extracted with ethyl acetate. The organic layer was washed with

brine, dried, and ﬁltered. Removal of the solvent gave a residue that was

puriﬁed by column chromatography (silica gel, ethyl acetate/n-hexane =

2

(

1

3

7

1

38−140 °C (from ethanol). H NMR (CDCl ): δ 3.73 (s, 6H), 3.83 (s,

3

H), 4.29 (s, 2H), 6.52 (s, 2H), 7.13−7.17 (m, 1H), 7.24−7.28 (m, 1H),

.46 (d, J = 8.1 Hz, 1H), 7.51−7.58 (m, 3H), 7.70 (dd, J = 1.8 and 8.5 Hz,

H), 7.82−7.94 (m, 3H), 7.99 (m, 1H), 8.30 ppm (broad s, disappeared

−1

on treatment with D O, 1H). IR: ν 3359 cm . Anal. (C H NO

1:1 as eluent) to furnish 79 (0.08 g, 29%), mp 130−133 °C (from

2

28 25

3

1

(423.50)) C, H, N.

ethanol). H NMR (DMSO-d

6

): δ 7.12 (t, J = 7.5 Hz, 1H), 7.20 (t, J =

2

-(1H-Pyrrol-3-yl)-3-(3,4,5-trimethoxybenzyl)-1H-indole (13).

7.4 Hz, 1H), 7.23−7.25 (m, 3H), 7.32 (d, J = 7.8 Hz, 1H), 7.41 (d, J = 7.8

Hz, 1H), 11.90 (broad s, disappeared on treatment with D O, 1H),

12.19 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν

1

(

7

1

3 was synthesized similarly to 8, from 12. Yield 20%, mp 195−197 °C

from ethanol). H NMR (DMSO-d ): δ 3.59 (s, 3H), 3.61 (s, 6H), 4.14

2

1

6

2

−1

s, 2H), 6.48−6.49 (m, 1H), 6.53 (s, 2H), 6.86−6.92 (m, 2H), 6.97−

2920, 3060, 3120, 3380 cm .

2-(1H-1,2,4-Triazol-1-yl)-1H-indole (81). A mixture of 2-iodo-

.01 (m, 1H), 7.12−7.13 (m, 1H), 7.29 (d, J = 7.8 Hz, 1H), 7.37 (d, J =

2

3

.7 Hz, 1H), 10.88 (broad s, disappeared on treatment with D O, 1H),

1H-indole (106) (0.5 g, 0.0021 mol) and triazole (0.14 g, 0.021 mol)

was heated at 150 °C for 2 h. After cooling, the mixture was puriﬁed by

2

1.03 ppm (broad s, disappeared on treatment with D O, 1H). IR:

ν 3242 cm . Anal. (C H N O (362.42)) C, H, N.

2

−1

column chromatography (silica gel, ethyl acetate as eluent) to give 81

22

2

3

1

2

-(3-(3,4,5-Trimethoxybenzyl)-1H-indol-2-yl)-4,5-dihydroxa-

(0.16 g, 42%) as a powder. H NMR (DMSO-d

): δ 6.76 (s, 1H), 7.08

6

zole (30). 30 was synthesized similarly to 28, starting from 3-(3,4,5-tri-

methoxybenzyl)-1H-indole-2-carboxylic acid. Yield 80%, mp 160−165 °C

(t, J = 7.2 Hz, 1H), 7.17 (t, J = 7.1 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.58

(d, J = 7.9 Hz, 1H), 8.32 (s, 1H), 9.22 (broad s, disappeared on

T

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

treatment with D O, 1H), 12.19 ppm (broad s, disappeared on

11.48 ppm (broad s, disappeared on treatment with D2O, 1H). IR: ν

2

−1

43

treatment with D O, 1H). IR: ν 3112 cm .

3412 cm . Lit. mp 97−98.5 °C.

2

-(1H-Indol-2-yl)thiazole (82). A mixture 2-bromo-1-(thiazol-2-

yl)ethanone (122) (0.5 g, 0.0024 mol) and aniline (0.67 g, 0.6 mL,

.0072 mol) in anhydrous DMF (2 mL) was placed into the microwave

2-(3-Fluorophenyl)-1H-indole (87). 87 was synthesized similarly

to 86, starting from 2-bromo-1-(3-ﬂuorophenyl)ethanone (117). Yield

4

0

42%, mp 142−144 °C (from toluene). Lit. mp 144 °C.

cavity (closed vessel mode, P_m_a_x= 250 psi). A starting microwave

irradiation of 100 W was used, the temperature being ramped from 25 to

2-(4-Fluorophenyl)-1H-indole (88). 88 was synthesized similarly

to 86, starting from 2-bromo-1-(4-ﬂuorophenyl)ethanone (118). Yield

4

150 °C, while stirring. Once 150 °C was reached, taking about 1 min, the

42%, mp 182−187 °C (from toluene). Lit. mp 185 °C.

reaction mixture was held at this temperature for 1 min, while cooling,

then diluted with water and extracted with ethyl acetate. The organic

layer was washed with brine, dried, and ﬁltered. Removal of the solvent

gave a residue that was puriﬁed by column chromatography (silica gel,

ethyl acetate/n-hexane = 2:7 as eluent) to furnish 82 (0.25 g, 52%), mp

2-(2-Methoxyphenyl)-1H-indole (89). 89 was synthesized

2

8

similarly to 86, starting from 2-bromo-1-(2-methoxyphenyl)ethanone

45

(119). Yield 41%, mp 75−80 °C (from toluene). Lit. mp 78.5−81.0 °C.

2-(3-Methoxyphenyl)-1H-indole (90). 90 was synthesized

28

similarly to 86, starting from 2-bromo-1-(3-methoxyphenyl)ethanone

37

1

12−115 °C (from ethanol).

-(4,5-Dihydro-1H-imidazol-2-yl)-1H-indole (83). Ethylenedi-

amine (0.23 g, 0.25 mL, 0.0038 mol) was added to a solution of 1H-

(120). Yield 40%, mp 138−141 °C (from toluene). Lit. mp 139.4−

2

140.7 °C.

2-(3-Isopropoxy-4-methoxyphenyl)-1H-indole (101). 101 was

synthesized similarly to 86, starting from 2-bromo-1-(4-ﬂuorophenyl)-

26

indole-2-carbaldehyde (115) (0.5 g, 0.0034 mol) in tert-butanol

34 mL). The reaction mixture was stirred at 25 °C for 30 min under an

Ar stream. Iodine (1.08 g, 0.00425 mol) and potassium carbonate (1.41 g,

1

(

ethanone (121). Yield 12%, mp 137−141 °C (from ethanol). H NMR

(CDCl ): δ 1.42 (s, 3H), 1.43 (s, 3H), 3.90 (s, 3H), 4.64 (m, 1H), 6.72

3

0

3

.01 mol) were added, and the reaction mixture was heated at 70 °C for

(s, 1H), 6.95 (d, J = 8.2 Hz, 1H), 7.10−7.27 (m, 5H), 7.39 (d, J = 8.0 Hz,

1H), 7.62 (d, J = 8.0 Hz, 1H), 8.3 ppm (broad s, disappeared on

h. After cooling, the mixture was diluted with a saturated aqueous

−1

solution of sodium sulﬁte and extracted with chloroform. The organic

layer was washed with brine, dried, and ﬁltered. Removal of the solvent

gave a residue that was puriﬁed by column chromatography (silica gel,

chloroform/ethanol = 8:2 as eluent) to furnish 83 (0.27 g, 43%). H

NMR (D MSO-d ): δ 3.70 (s, 4H), 7.13 (t, J = 7.6 Hz, 1H), 7.25 (t, J =

treatment with D O, 1H). IR: ν 3366 cm .

2

2-(Naphthalen-1-yl)-1H-indole (102). 102 was synthesized

similarly to 86, starting from 2-bromo-1-(naphthalen-1-yl)ethanone

1

46

(122). Yield 28%, mp 95−97 °C (from petroleum ether). Lit. mp 97−

99 °C.

6

.9 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 8.68

General Procedure for the Preparation of Compounds 95−

97, 100, and 104. Example: 2-(p-Tolyl)-1H-indole (100). n-

(

broad s, disappeared on treatment with D O, 1H), 11.79 ppm (broad s,

2

−1

Butyllithium (4.2 mL, 0.0067 mol, 1.6 M in n-hexane) was added

dropwise to a −40 °C solution of 4-methyl-N-(o-tolyl)benzamide (123)

(0.5 g, 0.0022 mol) in anhydrous THF (18 mL). After being stirred at

−40 °C for 1 h, the reaction mixture was warmed at 0 °C for 1 h and then

kept at 25 °C for 12 h. The mixture was diluted with water and extracted

with ethyl acetate. The organic layer was washed with brine, dried, and

ﬁltered. Removal of the solvent gave a residue that was puriﬁed by

column chromatography (silica gel, ethyl acetate/n-hexane = 1:5 as

eluent) to furnish 100 (0.2 g, 43%), mp 217−219 °C (from ethanol).

disappeared on treatment with D O, 1H). IR: ν 3387, 2855 cm .

2

General Procedure for the Preparation of Compounds 85,

1−94, and 103. Example: 2-(4-Chlorophenyl)-1H-indole (85). 1-

9

(

1-(4-Chlorophenyl)ethylidene)-2-phenylhydrazine (128) (2.82 g,

0

1

.011 mol) was added in portions to polyphosphoric acid (28 g) at

20 °C. The reaction mixture was stirred at the same temperature for 1

h. After cooling, it was quenched on crushed ice and treated with 3 N

NaOH with stirring, then extracted with ethyl acetate. The organic layer

was washed with brine, dried, and ﬁltered. Removal of the solvent gave a

residue that was puriﬁed by column chromatography (silica gel, ethyl

acetate/n-hexane = 1:1 as eluent) to furnish 85 (2.1 g, 80%), mp 210−

4

5

Lit. mp 218.0−219.9 °C.

2-((1,1′-Biphenyl)-4-yl)-1H-indole (104). 104 was synthesized

similarly to 100, starting from N-(o-tolyl)-(1,1′-biphenyl)-4-carbox-

37

2

12 °C (from ethanol). Lit. mp 206.3−209.0 °C.

-(4-Methoxyphenyl)-1H-indole (91). 91 was synthesized

similarly to 85, starting from 1-(1-(4-methoxyphenyl)ethylidene)-

-phenylhydrazine (129). Yield 37%, mp 222−224 °C (from toluene).

1

2

amide (124). Yield 22%, mp 296−298 °C (from ethanol). H NMR

(DMSO-d ): δ 6.97−7.04 (m, 2H), 7.10−7.14 (t, J = 8.0 Hz, 1H), 7.37−

6

2

7.41 (m, 2H), 7.44−7.56 (m, 3H), 7.75−7.80 (m, 4H), 7.97 (d, J =

8.0 Hz, 2H), 11,61 ppm (broad s, disappeared on treatment with D O,

38

Lit. mp 224−227 °C.

2

−

1

47

2

-(Pyridin-2-yl)-1H-indole (92). 92 was synthesized similarly to

1H). IR: ν 3681 cm . Lit. mp 297−298 °C.

29

8

7

5, starting from 2-(1-(2-phenylhydrazono)ethyl)pyridine (130). Yield

2-Cyclobutyl-1H-indole (95). 95 was synthesized similarly to 100,

39

3%, mp 154−156 °C (from ethanol). Lit. mp 156.0−156.5 °C.

starting from N-(o-tolyl)cyclobutanecarboxamide (125). Yield 40%, mp

1

2-(Pyridin-3-yl)-1H-indole (93). 93 was synthesized similarly to

73−76 °C (from toluene). H NMR (DMSO-d

6

): δ 1.86−2.03 (m, 2H),

2

9

8

5, starting from 3-(1-(2-phenylhydrazono)ethyl)pyridine (131).

2.19−2.33 (m, 2H), 2.58−2.54 (m, 2H), 3.57−3.66 (m, 1H), 6.16−6.17

(m, 1H), 6.89−6.93 (m, 1H), 6.98−7.01 (m, 1H), 7.26−7.29 (m, 1H),

7.39−7.41 (m, 1H), 10.90 (broad s, disappeared on treatment with D

40

Yield 73%, mp 168−170 °C (from ethanol). Lit. mp 169−170 °C.

2

-(Pyridin-4-yl)-1H-indole (94). 94 was synthesized similarly to

2

O,

2

9

−1

8

5, starting from 4-(1-(2-phenylhydrazono)ethyl)pyridine (132).

1H). IR: ν 3334 cm .

41

Yield 68%, mp 237−239 °C (from ethanol). Lit. mp 238−239 °C.

-(Naphthalen-2-yl)-1H-indole (103). 103 was synthesized

similarly to 85, starting from 1-(1-(naphthalen-2-yl)ethylidene)-

2-Cyclopentyl-1H-indole (96). 96 was synthesized similarly to

2

100, starting from N-(o-tolyl)cyclopentanecarboxamide (126). Yield

4

8

40%, mp 80−82 °C (from toluene). Lit. mp 84−85 °C.

29

2

-phenylhydrazine (133). Yield 45%, mp 165−168 °C (petroleum

2-Cyclohexyl-1H-indole (97). 97 was synthesized similarly to 100,

starting from N-(o-tolyl)cyclohexanecarboxamide (127). Yield 59%, mp

42

ether). Lit. mp 163−165 °C.

4

9

General Procedure for the Preparation of Compounds 86−

101−103 °C (from toluene). Lit. mp 103−105 °C.

9

0, 101, and 102. Example: 2-(2-Fluorophenyl)-1H-indole (86).

2-(1H-Pyrazol-4-yl)-1H-indole (98). A mixture of 106 (0.1 g,

0.000 41 mol), 1-Boc-pyrazole-4-boronic acid pinacol ester (0.18 g,

0.000 615 mol), and 1 M potassium carbonate (4.1 mL) in DMF (2 mL)

was degassed for 30 min. PdCl (PPh ) (0.003 g, 0. 0041 mmol) was

A solution of bromo-1-(2-ﬂuorophenyl)ethanone (116) (0.9 g, 0.0041)

in N,N-dimethylaniline (1.8 mL) was added dropwise to aniline (1.26 g,

1

.2 mL, 0.0135 mol) in the same solvent (2.4 mL). The reaction mixture

2

3 2

was heated at 170 °C for 15 min. After cooling to 0 °C, the mixture was

made acidic with 1 M HCl (pH ≈ 3−4) and extracted with ethyl acetate.

The organic layer was washed with brine, dried, and ﬁltered. Removal of

the solvent gave a residue that was puriﬁed by column chromatography

added, and the reaction mixture was placed into the microwave cavity

(closed vessel mode, P_m_a_x= 250 psi). A starting microwave irradiation of

250 W was used, the temperature being ramped from 25 to 160 °C,

while stirring. Once 160 °C was reached, taking about 2 min, the

reaction mixture was held at this temperature for 10 min. The reaction

mixture was diluted with water, neutralized with 1 M HCl, and extracted

with ethyl acetate. The organic layer was washed with brine, dried, and

ﬁltered. Removal of the solvent gave a residue that was puriﬁed by

(

silica gel, acetone/n-hexane = 1:3 as eluent) to furnish 89 (0.3 g, 35%),

1

mp 102−104 °C (from toluene). H NMR (DMSO-d ): δ 6.92 (s, 1H),

7

6

.03 (t, J = 7.8 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 7.34−7.40 (m, 3H), 7.45

(

d, J = 8.0 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.93 (t, J = 7.6 Hz, 1H),

U

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

column chromatography (silica gel, diethyl ether/methanol/n-hexane =

The mixture was diluted with a saturated solution of sodium hydrogen

carbonate and extracted with ethyl acetate. The organic layer was

washed with brine, dried, and ﬁltered. Removal of the solvent gave a

residue that was triturated with diethyl ether to furnish 123 (8.05 g,

8

:1:2 as eluent) to furnish 79 (0.02 g, 26%), mp 220−225 °C (from

1

ethanol). H NMR (DMSO-d ): δ 6.55 (s, 1H), 6.97 (t, J = 7.6 Hz, 1H),

6

7

1

.00 (t, J = 7.1 Hz, 1H), 7.32 (d, J = 7.9 Hz, 1H), 7.44 (d, J = 7.7 Hz, 1H),

5

.90−8.22 (m, 2H), 11.24 (broad s, disappeared on treatment with D O,

76%), mp 118−120 °C (from ethanol). Lit. mp 121 °C.

2

H), 13.05 ppm (broad s, disappeared on treatment with D O, 1H). IR:

N-(o-Tolyl)(1,1′-biphenyl)-4-carboxamide (124). 124 was syn-

thesized similarly to 123, starting from (1,1′-biphenyl)-4-carbonyl

2

−1

ν 2851, 2921, 3111, 3400 cm .

-((3,4,5-Trimethoxyphenyl)thio)-1H-indole-2-carboxylic

Acid (107). 107 was synthesized similarly to 14, starting from 105. Yield

1

3

chloride. Yield 40%, mp 176−178 °C (from ethanol). H NMR

(

CDCl ): δ 2.39 (s, 3H), 7.14−7.18 (m, 1H), 7.27−7.33 (m, 2H), 7.42−

3

1

4

5%, mp 240−245 °C (from ethanol). H NMR (DMSO-d ): δ 3.33

6

7.44 (m, 1H), 7.45−7.54 (m, 2H), 7.65−7.68 (m, 2H), 7.65−7.77 (m,

3H), 7.98−8.02 (m, 3H). IR: ν 1637−3280 cm . Lit. mp 179.5−180 °C.

N-(o-Tolyl)cyclobutanecarboxamide (125). 125 was synthesized

−1

56

(

broad s, disappeared on treatment with D O, 1H), 3.56 (s, 6H), 3.57

2

(

7

s, 3H), 6.42 (s, 2H), 7.06−7.10 (m, 1H), 7.26−7.30 (m, 1H), 7.40−

.42 (m, 1H), 7.47−7.49 (m, 1H), 12.18 (broad s, disappeared on

similarly to 123, starting from cyclobutanecarbonyl chloride. Yield 90%,

treatment with D O, 1H), 13.10 ppm (broad s, disappeared on

treatment with D O, 1H). IR: ν 1738, 2574, 3329 cm .

1

2

mp 106−110 °C (from toluene). H NMR (CDCl ): δ 1.91−2.07 (m,

3

−1

2

2H), 2.23 (s, 3H), 2.26−2.44 (m, 4H), 3.16−3.24 (m, 1H), 6.86 (broad

2-Iodo-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (108). 108

s, disappeared on treatment with D O, 1H), 7.03−7.07 (m, 1H), 7.16−

2

was synthesized similarly to 14, starting from 106. Yield 93%, mp 135−

−1

7

.22 (m, 2H), 7.86 ppm (d, J = 7.8 Hz, 1H). IR: ν 1646, 3256 cm .

N-(o-Tolyl)cyclopentanecarboxamide (126). 126 was synthe-

1

6

(

40 °C (from ethanol). H NMR (CDCl ): δ 3.71 (s, 6H), 3.79 (s, 6H),

3

.68 (s, 2H), 7.13−7.22 (m, 2H), 7.37−7.39 (m, 1H), 7.61−7.63

m, 1H), 8.65 ppm (broad s, disappeared on treatment with D O, 1H).

IR: ν 3331 cm .

-(1H-Indol-2-yl)oxazole (114). 114 was synthesized similarly to

sized similarly to 123, starting from cyclopentanecarbonyl chloride.

1

2

Yield 86%, mp 125−129 °C (from toluene). H NMR (DMSO-d ): δ

6

−

1

.52−1.88 (m, 8H), 2.18 (s, 3H), 2.81−2.88 (m, 1H), 7.05−7.08 (m,

2

H), 7.13−7.16 (m, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 7.6 Hz,

50

3

7

8

1, using 106 and 2-(tributylstannyl)oxazole. Yield 26%, mp 33−

H), 9.19 ppm (broad s, disappeared on treatment with D O, 1H). IR: ν

1

2

5 °C (from toluene). H NMR (CDCl ): δ 7.16−7.20 (m, 2H), 7.27−

−1

3

648, 3268 cm .

.32 (m, 2H), 7.40−7.43 (m, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.77 (s, 1H),

N-(o-Tolyl)cyclohexanecarboxamide (127). 127 was synthe-

.65 (broad s, disappeared on treatment with D O, 1H), 10.05 ppm

2

sized similarly to 123, starting from cyclohexanecarbonyl chloride. Yield

−1

(

broad s, disappeared on treatment with D O, 1H). IR: ν 3271 cm .

1

2

8

1

7

5%, mp 150−155 °C (from toluene). H NMR (DMSO-d ): δ 1.18−

6

General Procedure for the Preparation of Compounds 116−

.47 (m, 5H), 1.64−1.84 (m, 5H), 2.17 (s, 3H), 2.37−2.42 (m, 1H),

.05−7.08 (m 1H), 7.12−7.16 (m, 1H), 7.19 (d, J = 7.4 Hz, 1H), 7.32

d, J = 7.1 Hz, 1H), 9.13 ppm (broad s, disappeared on treatment with

1

22. Example: 2-Bromo-1-(2-ﬂuorophenyl)ethanone (116). A

solution of bromine (3.47 g, 1.1 mL, 0.0217 mol) in anhydrous dichloro-

methane (7 mL) was dropped into a solution of 1-(2-ﬂuorophenyl)-

ethanone (3.0 g, 2.6 mL, 0.002 17 mol) in the same solvent (14 mL).

The reaction mixture was stirred at 25 °C for 2 h, then diluted with a

saturated aqueous solution of sodium hydrogen carbonate and extracted

with dichloromethane. The organic layer was washed with brine, dried,

and ﬁltered. Removal of the solvent gave a residue that was puriﬁed by

column chromatography (silica gel, dichloromethane/petroleum ether

(

−1

D O, 1H). IR: ν 1649, 3284 cm .

2

General Procedure for the Preparation of Compounds 128

and 129. Example: 1-(1-(4-Chlorophenyl)ethylidene)-2-phenyl-

hydrazine (128). A mixture of phenylhydrazine hydrochloride (2.2 g,

0

.015 mol), 4′-chloroacetophenone (1.054 g, 1.3 mL, 0.01 mol), and

sodium acetate (1.23 g, 0.015 mol) in ethanol (25 mL) was placed into

the microwave cavity (open vessel mode). Microwave irradiation of

5

1

=

7:3 as eluent) to furnish 116 as a slurry (0.9 g, 20%).

-Bromo-1-(3-ﬂuorophenyl)ethanone (117). 117 was synthe-

sized similarly to 116, starting from 1-(3-ﬂuorophenyl)ethanone. Yield

2

8

50 W was used, the temperature being ramped from 25 to 80 °C. Once

0 °C was reached, taking about 1 min, the reaction mixture was held at

2

this temperature for 5 min with stirring. The reaction mixture was

1

2

(

1% as a slurry. H NMR (DMSO-d ): δ 4.97 (s, 2H), 7.53−7.65

6

cooled to 0 °C, ﬁltered, washed with petroleum ether, and dried to give

−1

m, 2H), 7.79−7.81 (m, 1H), 7.85−7.87 ppm (m, 1H). IR: ν 1686 cm .

-Bromo-1-(4-ﬂuorophenyl)ethanone (118). 118 was synthe-

sized similarly to 116, starting from 1-(4-ﬂuorophenyl)ethanone. Yield

1

28 (3.38 g, 92%), mp 114−116 °C (from ethanol). H NMR (CDCl ):

3

2

δ 2.23 (s, 3H), 6.91 (broad s, disappeared on treatment with D O, 1H),

2

1

7.17−7.19 (m, 2H), 7.28−7.37 (m, 5H), 7.28−7.75 ppm (m, 2H). IR: ν

7

(

3%, mp 47−49 °C (from n-hexane). H NMR (DMSO-d ): δ 4.93

57

6

−1

3349 cm . Lit. mp 112−113 °C.

s, 2H), 7.37−7.49 (m, 2H), 8.03−8.12 ppm (m, 2H). IR: ν 1694 cm .

52

1-(1-(4-Methoxyphenyl)ethylidene)-2-phenylhydrazine

Lit. mp 48−49 °C.

-Bromo-1-(3-isopropoxy-4-methoxyphenyl)ethanone

121). A solution of 1-(3-hydroxy-4-methoxyphenyl)ethanone (0.25 g,

(

129). 129 was synthesized similarly to 128, starting from 4′-meth-

2

oxyacetophenone (166). Yield 95%, mp 102−104 °C (from ethanol).

(

1

H NMR (CDCl ): δ 2.23 (s, 3H), 3.85 (s, 3H), 6.87 (broad s, dis-

3

0

2

.0015 mol) in anhydrous DMF (5 mL) was added to a mixture of

-iodopropane (0.28 g, 0.2 mL, 0.001 65 mol) and potassium carbonate

appeared on treatment with D O, 1H), 6.90−6.94 (m, 2H), 7.17−7.19

2

−1

(

m, 2H), 7.28−7.31 (m, 2H), 7.75−7.77 ppm (m, 2H). IR: ν 3336 cm .

(

0.415 g, 0.003 mol) in the same solvent (5 mL). The mixture was

heated at 50 °C for 3 h. After cooling, the mixture was neutralized with

M HCl and extracted with ethyl acetate. The organic layer was washed

58

Lit. mp 97−98 °C.

Biology. Tubulin Assembly. The reaction mixtures contained 0.8

M monosodium glutamate (pH 6.6 with HCl in a 2 M stock solution),

1

with brine, dried, and ﬁltered. Removal of the solvent gave a residue that

was puriﬁed by column chromatography (silica gel, ethyl acetate/

n-hexane = 3:7 as eluent) to furnish 1-(3-isopropoxy-4-methoxyphenyl)-

1

0 μM tubulin, and varying concentrations of drug. Following a 15 min

preincubation at 30 °C, samples were chilled on ice, GTP to 0.4 mM was

added, and turbidity development was followed at 350 nm in a

temperature controlled recording spectrophotometer for 20 min at 30 °C.

Extent of reaction was measured. Full experimental details were previ-

5

3

ethanone (0.27 g, 86%), mp 53−56 °C (from toluene/n-hexane). Lit

mp 56 °C. As described above for 116, 1-(3-isopropoxy-4-

methoxyphenyl)ethanone was converted to 121, yield 65%, mp 75−

5

9

ously reported.

80 °C (from ethanol).

3

[

H]Colchicine Binding Assay. The reaction mixtures contained

.0 μM tubulin, 5.0 μM [ H]colchicine, and 5.0 μM inhibitor and were

Bromo-1-(naphthalen-1-yl)ethanone (122). The compound was

3

1

synthesized similarly to 116, starting from 1-(naphthalen-1-yl)-

5

4

incubated 10 min at 37 °C. Complete details were described previo-

ethanone. Yield 90%, mp 175−177 °C (petroleum ether). Lit. mp

6

0

usly.

1

77−179 °C.

Cell Cultures and Treatment. Cell lines were obtained from the

American Tissue Culture Collection (ATCC), unless speciﬁed

otherwise. Cells were grown in Dulbecco’s modiﬁed Eagle medium

General Procedure for the Preparation of Compounds 123−

1

27. Example: N-(o-Tolyl)-4-methylbenzamide (123). To an ice-

cooled solution of o-toluidine (5.0 g, 5 mL, 0.0047 mol) and triethyl-

amine in anhydrous THF (160 mL) was added dropwise p-tolyl chloride

(DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C

2

(

8.66 g, 7.4 mL, 0.056 mol). The mixture was stirred at 25 °C for 12 h.

with 5% CO . In all experiments 300 000 cells were plated in 9 cm

2

V

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

dishes and exposed to test compound dissolved in DMSO (0.1% ﬁnal

concentration) at the indicated concentrations.

HeLa, HT29, and A549 cell lines were grown at 37 °C in DMEM

containing 10 mM glucose supplemented with 10% FBS, 100 units/mL

each of penicillin and streptomycin, and 2 mM glutamine. At the onset

of each experiment, cells were placed in fresh medium and cultured in

the presence of test compounds from 0.01 to 25 μM.

The eﬀects of the treatments with 18 or 57 on U87MG cell survival/

32

growth were estimated by a colorimetric MTS conversion assay and

5

9

trypan blue exclusion assay. The MTS assay was used to determine

IC₅₀values on U87MG cell survival following 24 h of treatment.

U87MG cells were seeded in 96-well plates and incubated with

compound concentrations ranging from 0.1 nM to 20 μM (DMSO as

control). After 24 h, MTS reagent was added, and the absorbance at 590

nm was measured by a microplate reader (Wallac, Victor 2, 1420

multilabel counter, PerkinElmer). The percentage of proliferating cells

after compound exposure was calculated with respect to control cells

(100%). Sigmoid dose response curves were performed using GraphPad

Prism 4 software (GraphPad Software Inc., San Diego, CA).

231-MDA and A549 cells were cultured in DMEM supplemented

with 10% FBS for 24, 48, and 72 h in a 96-well tissue culture plate at 37 °C

and 5% CO in the absence or presence of diﬀerent drug concentrations.

2

HCT 116, HCT 15, Messa, and Messa/Dx5 were seeded into 96-well

3

plates (Corning Inc., Costar) at a density of 2 × 10 cells/well in a

volume of 50 μL of the appropriate tissue culture medium. Compounds

were added with diﬀerent concentrations for the indicated incubation

time at 37 °C with 5% CO₂.

The trypan blue exclusion assay was used to examine the eﬀects of

compound treatments on U87MG cell survival. U87MG cells were

seeded in 24-well plates and exposed to 1 nM to 10 μM compound

concentrations for 8, 24, and 48 h. After exposure, the number of viable

(white) and dead (blue) cells was counted using a microscope, and the

percentage of viable or dead cells was calculated with respect to control

cells (100%).

The human glioblastoma multiforme (GBM) cell line U87MG was

purchased from the National Institute for Cancer Research (ICLC) of

6

Genoa (Italy). The U87MG cells (1 × 10 ) were seeded onto T-75 ﬂasks

and cultured in RPMI medium supplemented with 10% FBS, 2 mM

L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 1%

nonessential amino acids at 37 °C in humidiﬁed atmosphere composed

of oxygen (95%) and carbon dioxide (5%). For U87MG cell treatments,

the cells were seeded at appropriate densities (5000 cells/well in 96-well

plates or 50 000 cells/well in 24-well plates) in complete medium. After

Immunoﬂuorescence (IF) and Microscopy. HeLa epithelial cells

were grown in Dulbecco’s modiﬁed Eagle medium (DMEM)

supplemented with 10% FBS at 37 °C with 5% CO . All compounds

2

were dissolved in DMSO (0.1% ﬁnal concentration) and used at the

indicated concentrations for 24 h.

HeLa cells were seeded at 3 × 10 cells in culture dishes containing

5

12 or 24 h the medium was replaced either with complete culture

medium containing DMSO (untreated control cells) or with complete

medium supplemented with the compound 18 or 57 dissolved in

DMSO (<1% v/v of medium).

sterile coverslips coated with poly-L-lysine. At the end of the treatment,

they were directly ﬁxed in methanol for 10 min at −20 °C and processed

for IF with either mouse anti-α-tubulin antibody (Sigma clone B5.1.2,

1:2000 dilution) followed by FITC-conjugated goat anti-mouse

secondary antibody (Jackson Immunoresearch Laboratories, 115-095-

068, 1:200) or with mouse anti-cyclin B1 antibody (Santa Cruz, GNS1,

1:50) followed by Texas Red conjugated horse anti-mouse (Vector, TI-

2000, 1:800) secondary antibody. Slides were counterstained with

0.05 μg/mL DAPI (Sigma) and mounted in Vectashield (Vector). Cells

were analyzed under an epiﬂuorescence Olympus AX70 microscope

with a CCD camera (Diagnostic Instrument, Spot RT slider model

2.3.0). Unﬁxed cell cultures were observed under an inverted NIKON

TE 300 microscope with a 10× objective, and images were acquired

using the ACT-1 software and a DMX1200 CCD (resolution 1280 ×

1024 pixels).

Cell Viability Assay. The methodology for the evaluation of the

growth of human MCF-7 breast carcinoma, OVCAR-8, and NCI/ADR-

RES cells, obtained from the National Cancer Institute drug screening

laboratory, was previously described except that cells were grown for 96

61

h for IC₅₀determinations.

Cell viability in Hela, HT-29, and A549 cells was determined using

the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT) colorimetric assay. The test is based on the ability of mitochondrial

dehydrogenase to convert, in viable cells, the yellow MTT reagent into a

soluble blue formazan dye. Cells were seeded into 96-well plates to a

3

density of 7 × 10 /100 μL well. After 24 h of growth to allow attachment

of cells to the wells, compounds were added at various concentrations

(

from 0.01 to 25 μM). After 48 h of growth and after removal of the

Flow Cytometric Analysis. Cell cycle distribution was analyzed

after incubation with PI (Sigma-Aldrich), whereas apoptosis was

analyzed with annexin V-FITC (Immunological Sciences, IK-11120)

staining alone or with annexin V-FITC in combination with PI. Cell

samples were analyzed in a Coulter Epics XL cytoﬂuorometer (Beckman

Coulter) equipped with EXPO 32 ADC software. Data from at least 10

000 cells per sample were acquired.

culture medium, 100 μL/well medium containing 1 mg/mL MTT was

added. Cell cultures were further incubated at 37 °C for 2 h in the dark.

The solution was then gently aspirated from each well, and the formazan

crystals within the cells were dissolved with 100 μL of DMSO. Optical

densities were read at 550 nm using a Multiskan Spectrum Thermo

Electron Corporation reader. Results were expressed as a percentage

relative to vehicle-treated control (0.5% DMSO was added to untreated

cells), and the IC₅₀values were determined by linear and polynomial

regression. Experiments were performed in triplicate.

Evaluation of Mitochondrial Potential. JC-1 (Invitrogen, T3168,

Carlsbad, CA) is a cationic dye that exhibits potential-dependent

accumulation in mitochondria indicated by a ﬂuorescence emission shift

from green (∼525 nm) to red (∼590 nm). Consequently, mitochondrial

depolarization is indicated by a decrease in the red/green ﬂuorescence

intensity ratio and can be quantiﬁed by using ﬂuorescence micro-

231-MDA and A549 cells were plated at diﬀerent cellular densities in

order to test the compounds on logarithmic phase cells. Test com-

pounds were added after cell adhesion. On the day of the assay, 10 μL of

MTT (5 mg/mL) was added to each well, and cells were incubated for 2

3

3b

scopy. To evaluate the mitochondrial depolarization induced by drug

treatment, U87MG cells were seeded in 96-well plates and stained for

10 min in medium containing JC-1 at a ﬁnal concentration of 50 μg/mL.

After removal of JC-1, fresh medium containing the drug at 100 nM

(approximately its IC₅₀value in U87MG cells) or 30 μM CCCP, a

standard mitochondrial potential dissipation drug, was added to the

cells. Immediately and 24 h after the treatment, pictures were taken

under an Axiovert ﬂuorescent microscope (Zeiss) using the ﬁlter set 10,

488010-0000 (Zeiss) (excitation 450−490 nm, emission 515−565 nm).

Pictures were then split in the RGB channels (red and green) and

h (37 °C, 5% CO ). When dark crystals appeared at the well bottom,

2

culture medium was discarded, and ethanol (100 μL) was added to each

well to solubilize the crystals, yielding a purple solution. Absorbance was

read with an enzyme-linked immunosorbent assay (ELISA) reader at

5

70 nm.

The antiproliferative eﬀect of the tubulin polymerization inhibitors

on cell proliferation was evaluated against HCT 116, HCT 15, Messa,

and Messa/Dx5 tumor cell lines using the CellTiter-Glo luminescent

cell viability assay (Promega, Madison, WI) according to the

manufacturer’s instructions. The cells, in exponential growth, were

incubated for 72 h at diﬀerent concentrations of the inhibitors. Then an

equivalent of the CellTiter-Glo reagent was added, the solution was

mixed for 2 min in order to induce cell lysis, and the luminescence was

recorded after a further 10 min. GI₅₀values were calculated using the

parameters of a nonlinear regression analysis program (GraphPad Prism

statistics software).

6

2

analyzed by using the program ImageJ. For each treatment, the red/

green ﬂuorescence ratio obtained at 24 h was normalized with respect to

red/green ﬂuorescence ratio obtained at 0 h.

ROS Production. The generation of ROS was assessed by the

ﬂuorogenic probe DCFH2-DA (Molecular Probes, Invitrogen) in

U87MG cells after compound exposure (DMSO, H O as control

2

standard). DCFH2-DA is a reduced and acetylated form of ﬂuorescein

W

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

6

3

used as an indicator for ROS in cells. This nonﬂuorescent molecule is

readily converted to a green-ﬂuorescent form (FDA) when the acetate

group is removed by intracellular esterases and an oxidation by ROS

occurs within the cell.

a reference standard. A ﬁxed concentration of verapamil was added in

every sample as an internal standard for LC−MS/MS. The percent of

test compound remaining after a 30 min incubation period was

calculated from the peak area relative to the area of the compound after

the 30 min incubation time.

Aqueous Solubility. The solubilities of compounds 18, 20, 55, and

57 were measured using a high throughput screening assay format. Samples

prepared at the target concentration of 200 μM were placed in a 96-well

ﬁlter plate and incubated at room temperature for 90 min. The plate was

then ﬁltered, and solutions were analyzed by LC/MS−UV. Final

concentrations are evaluated by comparing the area under the curve of

the MeOH stock solution with those of the test compound solutions.

Caco-2 Cell Permeability. Caco-2 cells (ECACC) were cultured in

DMEM, 10% FBS, 1% NEAA, 10 mM Hepes buﬀer, 50 U penicillin, and

50 ug/mL streptomycin and split at conﬂuence by trypsinization. For

transport studies, 200 000 cells/well were seeded on Millicell 24-well

U87MG cells were seeded in 96-wells plate, washed in PBS/10 mM

glucose, andloadedwith10μM DCFH2-DA for 30 min in the dark at 37 °C.

Afterward, the cells were washed with the same medium and incubated

with or without the compound at 100 nM. Fluorescence increase was

estimated in a plate reader at 485 nm (excitation) and 520 nm

(

emission) after 1, 5, 15, and 60 min (Wallac, Victor 2, 1420 multilabel

counter, PerkinElmer). The ﬂuorescence values were normalized

between samples for cell number content and assessed by a crystal

violet cell staining assay. Crystal violet is an intense stain that binds to

cell nuclei and gives an A₅₉₅reading that is proportional to cell number.

After washing with PBS/10 mM glucose, the cells were incubated with

crystal violet for 30 min at room temperature. After washing, a solution

of 1% SDS was added to each well, and the plates were shaken

mechanically for 1 h at room temperature. A₅₉₅was then determined on

a plate reader. The measured optical density readings indicated the

relative number of cells in each well, and this was used to normalize the

absorbance obtained with FDA.

Statistical Analyses. For data analysis and graphic presentations,

the nonlinear multipurpose curve-ﬁtting program GraphPad Prism

GraphPad) was used. All data are presented as the mean ± SEM.

Statistical analysis was performed by one-way analysis of variance

ANOVA) with Bonferroni’s corrected t test for posthoc pairwise

cell culture plates. After 24 h at 37 °C in a humidiﬁed, 5% CO

2

atmosphere, the medium was exchanged for enterocyte diﬀerentiation

medium with additives (Becton Dickinson), which allows Caco-2 cells

to establish within 3 days a diﬀerentiated enterocyte monolayer. TEER,

measured with a Millicell-ERS (Millipore, Corp), must be >1000 Ω.

Transport across the Caco-2 monolayer was determined by adding a

10 μM solution of compound in DMEM (1% ﬁnal concentration of

DMSO) to the side from which permeability was to be determined. The

A → B transport across Caco-2 monolayer cells was determined by

adding the compound to the apical side at pH 6.5. After 2 h, the

basolateral side solution at pH 7.4 and the apical and starting solutions

were analyzed by LC−MS/MS. In the B → A experiment, the

compound was added to the basolateral side and collected on the apical

side. Monolayer integrity was assessed with a lucifer yellow assay at the

end of each experiment. The apparent permeability (Papp) was calculated

with the following equation:

(

comparisons. P < 0.05 was considered statistically signiﬁcant.

In Vivo Vascular Disrupting Eﬀects. This study was approved by

the institutional ethical committee for the use and care of laboratory

animals. Six adult WAG/Rij rats (Iﬀa Credo, Brussels, Belgium) weighing

50 g received intrahepatic implantation of bifocal rhabdomyosarcoma

allografts, which mimics hypervascular human liver metastases as

2

34

previously described. Three rats bearing six liver tumors in total were

thus prepared for studying 18 and 57 separately. The growth of tumors

was monitored with MRI until their diameter reached about 10 mm for

therapy. MRI was carried out with a clinical 3.0 T whole-body MR

magnet (Trio; Siemens, Erlangen, Germany) with a maximum gradient

capability of 45 mT/m by using an eight-channel phased-array wrist coil

P

⁼^J^/^C0

app

where J is the ﬂux (dX/dt per A) and C is the donor concentration

μM) at t = 0; dX/dt is the change in mass (X, nmol) per time (t, s), and

A is theﬁlter surface area (cm ).

0

(

2

CYP450 Inhibition. Compounds 18, 20, 55, and 57 were evaluated

in a 96-well plate containing an appropriate buﬀer and a NADPH

regenerating system. Cytochrome P450 inhibition experiments were

carried out according to the manufacturer’s instructions (BD

Biosciences, Franklin Lakes, NJ, U.S.). The compounds were dissolved

in a 96-well plate at a 10 μM ﬁnal concentration in potassium phosphate

buﬀer (pH 7.4) containing an NADPH regenerating system. For all

enzyme/substrate pairs, the ﬁnal cofactor concentrations were 1.3 mM

(

In Vivo, Latham, NY). The rat was gas-anesthetized with 2% isoﬂurane

in a mixture of 20% oxygen and 80% room air, through a mask con-

nected by a tube to a Harvard Apparatus system (Holliston, MA) and

placed supine in a plastic holder. The penile vein of the rat was

cannulated for MRI contrast agent or VDA compound administration.

With intravenous bolus of gadoterate meglumine (Dotarem; Guerbet,

Brussels, Belgium) at 0.1 mmol/kg, contrast enhanced T weighted MRI

1

35

(

CE-T1WI) was performed at baseline at 1 and 24 h post-VDA

+

NADP , 3.3 mM glucose 6-phosphate, and 0.4 U/mL glucose

-phosphate dehydrogenase. The reaction was initiated by the addition

treatment to document intratumoral vascular disrupting event. After

baseline CE-T1WI, a VDA solution of 18 or 57 was intravenously

injected at a dose of 15 mg/kg for subsequent evaluation of the

therapeutic eﬀects.

LC−MS/MS Analytical Method. Samples from the metabolic

stability, solubility, and permeability assays were analyzed under the

following conditions: UPLC instrument (Waters) interfaced with a

Premiere XE triple quadrupole instrument (Waters). Eluents were the

6

of speciﬁc isoenzymes (Supersomes, Gentest) and substrates at 37 °C.

Furafylline (for CYP1A2, 100 mM), sulfaphenazole (for CYP2C9, 10 mM),

tranylcypromine (for CYP2C19, 500 μM), quinidine (for CYP2D6, 0.5

mM), and ketoconazole (for CYP3A4, 1.66 mM) were employed as

control inhibitors in one-third serial dilution. Incubations were carried

out for 15 min (0.5 pmol of CYP1A2, 5 mM 3-cyano-7-ethoxycoumarin),

0 min (0.5 pmol of CYP2C19, 25 mM 3-cyano-7-ethoxycoumarin, 1

pmol of CYP3A4, 50 mM 7-benzyloxy-4-(triﬂuoromethyl)coumarin;

.5 pmol of CYP2D6, 1.5 mM 3-[2-(N,N-diethyl-N-methylamino)-

ethyl]-7-methoxy-4-methylcoumarin), or 45 min (1 pmol of CYP2C9,

5 mM 7-methoxy-4-(triﬂuoromethyl)coumarin). The reaction was

3

following: phase A consisting of 95% H O, 5% acetonitrile + 0.1%

2

HCOOH; phase B consisting of 5% H O, 95% acetonitrile + 0.1%

2

1

HCOOH. Parameters were as follows: ﬂow rate, 0.6 mL/min; column,

Acquity BEH C18, 50 mm × 2.1 mm, 1.7 μm at 50 °C; injection volume,

7

5

μL. LC−MS/MS analyses were carried out using an ESI(+) interface

then quenched by adding 75 mL of a mixture containing 80% CH CN

3

in multiple reaction monitoring mode.

and 20% Tris base (0.5 m), and plates were read on a ﬂuorimeter at the

appropriate emission/excitation wavelengths. The percentage inhibition

was calculated relative to enzyme samples without inhibitors.

Metabolic Stability. Compounds 18, 20, 55, and 57 were dissolved

in DMSO in duplicate at a ﬁnal concentration of 1 μM and preincubated

for 10 min at 37 °C in potassium phosphate buﬀer, pH 7.4, 3 mM MgCl₂,

with rat liver microsomes (Xenotech) at a ﬁnal concentration of 0.5 mg/

mL. After the preincubation period, reactions were started by adding the

cofactors mixture (NADP, Glc6P, G6P-DH). Samples were taken at times

ASSOCIATED CONTENT

■

*

S

Supporting Information

0

and 30 min and added to acetonitrile to stop the reaction and centrifuged.

Molecular modeling results, binding modes, inhibition data, cell

death data, and elemental analysis results. This material is

available free of charge via the Internet at http://pubs.acs.org.

Supernatants were analyzed and quantiﬁed by LC−MS/MS. A control

sample without cofactors was always added in order to check the stability of

test compounds in the reaction mixtures. 7-Ethoxycoumarin was used as

X

dx.doi.org/10.1021/jm3013097 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

mechanism of their inhibition of the binding to colchicine to tubulin.

AUTHOR INFORMATION

■

*

Biochemistry 1989, 28, 6984−6991.

Corresponding Author

Phone: +39 06 4991 3800. Fax: +39 06 4991 3133. E-mail:

romano.silvestri@uniroma1.it.

(9) Nogales, E.; Whittaker, M.; Milligan, R. A.; Downing, K. H. High-

resolution model of the microtubule. Cell 1999, 96, 79−88.

(10) Nettles, J. H.; Li, H.; Cornett, B.; Krahn, J. M.; Snyder, J. P.;

Downing, K. H. The binding mode of epothilone A on alpha, beta-

tubulin by electron crystallography. Science 2004, 305, 866−869.

Notes

The authors declare no competing ﬁnancial interest.

(11) Buey, R. M.; Calvo, E.; Barasoain, I.; Pineda, O.; Edler, M. C.;

Matesanz, R.; Cerezo, G.; Vanderwal, C. D.; Day, B. W.; Sorensen, E. J.;

Lopez, J. A.; Andreu, J. M.; Hamel, E.; Diaz, J. F. Cyclostreptin binds

covalently to microtubule pores and lumenal taxoid binding sites. Nat.

Chem. Biol. 2007, 3, 117−125.

ACKNOWLEDGMENTS

■

This research was supported by PRIN 2008 (Grant

2

00879X9N9), Progetti di Ricerca di Universita,

di Roma (Grant 2011), and Bando Futuro in Ricerca

010 (Grant RBFR10ZJQT). The authors also thank FILAS

Finanziaria Laziale di Sviluppo, Grant 2010) for partial support.

̀

Sapienza

(

12) Beckers, T.; Mahboobi, S. Natural, semisynthetic and synthetic

microtubule inhibitors for cancer therapy. Drugs Future 2003, 28, 767−

85.

13) Sridhare, M.; Macapinlac, M. J.; Goel, S.; Verdier-Pinard, D.; Fojo,

Universita

̀

2

(

7

(

A.C. thanks Istituto PasteurFondazione Cenci Bolognetti for

his Borsa di Studio per Ricerche in Italia. A.R. thanks Bando

Futuro in Ricerca 2010 for her Borsa di Studio. We are grateful to

Alessio Bolognesi, Maria Giubettini, and Annalisa Verrico for

experimental work in cell cycle analysis. This work was supported

in part by funds from AIRC (Grant IG 10164) to P.L.

T.; Rothenberg, M.; Colevas, D. The clinical development of new

mitotic inhibitors that stabilize the microtubule. Anti-Cancer Drugs 2004,

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(

5, 553−555.

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2

(

ABBREVIATIONS USED

M. C.; Brancale, A.; Wilcox, E.; Hamel, E.; Artico, M.; Silvestri, R.

Arylthioindoles, potent inhibitors of tubulin polymerization. J. Med.

Chem. 2004, 47, 6120−6123.

■

MT, microtubule; ATI, arylthioindole; CSA4, combretastatin A-

; VBL, vinblastine; PTX, paclitaxel; VRB, vinorelbine; SAR,

^s^t^r^u^c^t^u^r^e⁻^a^c^tⁱ^vⁱ^t^y^r^e^l^a^tⁱ^oⁿ^s^hⁱ^p^;^Pmax, maximum pressure; BOP,

benzotriazol-1-yloxytris(dimethylamino)phosphonium hexa-

4

(16) De Martino, G.; Edler, M. C.; La Regina, G.; Coluccia, A.; Barbera,

M. C.; Barrow, D.; Nicholson, R. I.; Chiosis, G.; Brancale, A.; Hamel, E.;

Artico, M.; Silvestri, R. Arylthioindoles, potent inhibitors of tubulin

polymerization. 2. Structure activity relationships and molecular

modeling studies. J. Med. Chem. 2006, 49, 947−954.

ﬂuorophosphate; Pd (dba) , tris(dibenzylideneacetone)-

2

3

dipalladium(0); P(Cy), tricyclohexylphosphine; Pd(dppf)-

Cl ·CH Cl , 1,1′-bis(diphenylphosphino)ferrocene]-

(17) La Regina, G.; Edler, M. C.; Brancale, A.; Kandil, S.; Coluccia, A.;

Piscitelli, F.; Hamel, E.; De Martino, G.; Matesanz, R.; Díaz, J. F.;

Scovassi, A. I.; Prosperi, E.; Lavecchia, A.; Novellino, E.; Artico, M.;

Silvestri, R. New arythioindoles inhibitors of tubulin polymerization. 3.

Biological evaluation, SAR and molecular modeling studies. J. Med.

Chem. 2007, 50, 2865−2874.

2

dichloropalladium(II), complex with dichloromethane;

PdCl (PPh ) , bis(triphenylphosphine)palladium(II) dichlor-

2

3 2

ide; Pd(OAc) , palladium(II) acetate; Pd(PPh ) , tetrakis-

2

3 4

(

triphenylphosphine)palladium(0); SPhos, 2-dicyclohexylphos-

phino-2′,6′-dimethoxybiphenyl; MI, mitotic index; DAPI, 4′,6-

diamidino-2-phenylindole; Pgp, P-glycoprotein; FBS, fetal

bovine serum; CCCP, carbonyl cyanide m-chlorophenylhydra-

zone; DCFH2-DA, 6-carboxy-2′,7′-dichlorodihydroﬂuorescein

diacetate; ROS, reactive oxygen species; JC-1, 5,5′,6,6′-

tetrachloro-1,1′3,3′-tetraethylbenzimidazoylcarbocyanine io-

dide; FDA, green-ﬂuorescent form of 6-carboxy-2′,7′-dichlor-

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Toward highly potent cancer agents by modulating the C-2 group of the arylthioindole class of tubulin polymerization inhibitors

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