New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of Mitotic Progression, Enhanced Stimulation of Natural Killer Cell Cytotoxic Activity, and Repression of Hedgehog-Dependent Cancer

Article abstract of DOI:10.1021/acs.jmedchem.5b00310

We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethoxyphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4-7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20-50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC₅₀ values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer.

Full text of DOI:10.1021/acs.jmedchem.5b00310

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Article
New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of
Mitotic Progression, Enhanced Stimulation of Natural Killer Cell
Cytotoxic Activity and Repression of Hedgehog-dependent Cancer
Giuseppe La Regina, Ruoli Bai, Antonio Coluccia, Valeria Famiglini, Sveva Pelliccia, Sara Passacantilli,
carmela mazzoccoli, Vitalba Ruggieri, Annalisa Verrico, Andrea Miele, Ludovica Monti, Marianna
Nalli, Romina Alfonsi, Lucia Di Marcotullio, Alberto Gulino, Biancamaria Ricci, Alessandra Soriani,
Angela Santoni, Michele Caraglia, Stefania Porto, Eleonora Da Pozzo, Claudia Martini, Andrea
Brancale, Luciana Marinelli, Ettore Novellino, Stefania Vultaggio, Mario Varasi, Ciro Mercurio,
Chiara Bigogno, Giulio Massimo Dondio, Ernest Hamel, Patrizia Lavia, and Romano Silvestri
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00310 • Publication Date (Web): 01 Jul 2015
Downloaded from http://pubs.acs.org on July 3, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Novellino, Ettore; University of Naples Federico II, Pharmaceutical and
Toxicological Chemistry
Vultaggio, Stefania; European Institute of Oncology,
Varasi, Mario; IEO_European Institute of Oncology, Expt. Therapy-Drug
Discovery Unit
Mercurio, Ciro; Genextra Group, DAC SRL,
Bigogno, Chiara; APHAD S.r.l.,
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Dondio, Giulio; Aphad S.r.l., Managment
Hamel, Ernest; Frederick National Laboratory for Cancer Research, National
Cancer Institute, National Institute of Health
Lavia, Patrizia; Institute of Molecular Biology and Pathology, CNR Consiglio
Nazionale delle Ricerche, c/o Sapienza Università di Roma,
Silvestri, Romano; Sapienza Università di Roma, Chimica e Tecnologie del
Farmaco
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New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of Mitotic Progression,
Enhanced Stimulation of Natural Killer Cell Cytotoxic Activity and Repression of
Hedgehog-dependent Cancer
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Giuseppe La Regina, Ruoli Bai, Antonio Coluccia, Valeria Famiglini, Sveva Pelliccia, Sara
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≠
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§
Passacantilli, Carmela Mazzoccoli, Vitalba Ruggieri, Annalisa Verrico, Andrea Miele, Ludovica
†
†
,
∂
Monti, Marianna Nalli, Romina Alfonsi,° Lucia Di Marcotullio,° Alberto Gulino,° Biancamaria
,
§
¢
¢
Ricci,° Alessandra Soriani,° Angela Santoni,° Michele Caraglia, Stefania Porto, Eleonora Da
œ
œ
∞
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Pozzo, Claudia Martini, Andrea Brancale, Luciana Marinelli, Ettore Novellino, Stefania
◊
◊
△
f
f
‡
Vultaggio, Mario Varasi, Ciro Mercurio, Chiara Bigogno, Giulio Dondio, Ernest Hamel, Patrizia
§
†,
Lavia, and Romano Silvestri *ꢀ
†
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco,
Sapienza Università 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
●
Dipartimento di Farmacia, Università di Napoli Federico II, Via Domenico Montesano 49, I-80131
Napoli, Italy
≠
Laboratorio di Ricerca Pre-Clinica e Traslazionale, IRCCS, Centro di Riferimento Oncologico della
Basilicata, Via Padre Pio 1, I-85028 Rionero in Vulture, Italy
§
Institute of Molecular Biology and Pathology, CNR Consiglio Nazionale delle Ricerche, c/o Sapienza
Università di Roma, Via degli Apuli 4, I-00185 Roma, Italy
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°
Department of Molecular Medicine, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza
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Università di Roma, Viale Regina Elena 291, I-00161 Roma, Italy
∂Center for Life NanoScience@Sapienza, Istituto Italiano di Tecnologia, Roma, Italy
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Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Via
S.M. Costantinopoli 16, I-80138 Naples, Italy
œ
Department of Pharmacy, University of Pisa, Via Bonanno Pisano 6, I-56126 Pisa, Italy
∞
Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII
Avenue, Cardiff, CF10 3NB, United Kingdom
◊
European Institute of Oncology, Via Adamello 16, I-20139 Milano, Italy
△
Genextra Group, DAC SRL, Via Adamello 16, I-20139 Milano, Italy
f
APHAD Srl, Via della Resistenza 65, I-20090 Buccinasco, Italy
ꢀ
In memory of Prof. Alberto Gulino,° an outstanding scientist and a dear colleague
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ABSTRACT
We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-
trimethoxyphenyl moiety with a sulfur, ketone or methylene bridging group at position 3 of the indole
and with halogen or methoxy substituent(s) at positions 4-7. Compounds 33 and 44 strongly inhibited
the growth of the P-glycoprotein-overexpressing multidrug resistant cell lines NCI/ADR-RES and
Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20-50 nM, 33 and 44
arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic
progression. Cell cycle arrest was followed by cell death. Indoles 33, 44 and 81 showed strong
inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50’s
of 19, 72 and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization
and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of
Hedgehog-dependent cancer.
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INTRODUCTION
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Microtubules (MTs) are cylindrical structures mainly composed of α,β-tubulin heterodimers. MT
assembly is a highly dynamic process because of continuous transitions between polymerization and
depolymerization. MTs are involved in a number of essential cellular functions, such as maintenance of
cell shape, cell motility, intracellular transport and cell division. Interfering with the MT dynamic
equilibrium, by either inhibiting tubulin polymerization or blocking MT disassembly, prevents proper
MT function and ultimately leads to cell death. Interfering with these cellular processes has resulted in
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a productive strategy for the development of efficient anticancer agents.
5,6
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Colchicine (1),
combretastatin A-4 (CSA4, 2) (Chart 1) and the Catharanthus alkaloids
vincristine (VCR) and vinblastine (VBL) inhibit MT assembly by preventing tubulin polymerization,
and this leads cell to death. In contrast, taxoids and epothilones bind at a luminal site on the β-
8
,9
10
subunit following entry into the MT through pores in its wall that are shaped by various tubulin
subunits on the MT surface. Some evidence indicates a transient binding of MT stabilizing agents at a
specific pore site. Paclitaxel (PTX) stimulates MT polymerization and stabilization at high
concentrations, whereas lower concentrations of PTX inhibit MT dynamics with little effect on the
1
1
proportion of tubulin in polymer.
12
The development of MT-targeting drugs with different mechanisms of action has achieved
substantial progress. However, drug resistance, toxicity and unwanted side effects still remain unsolved
problems. Therefore, the quest for new MT inhibitors as components of improved anticancer treatments
13
remains mandatory.
Arylthioindole (ATI) derivatives are potent inhibitors of tubulin polymerization that bind to the
colchicine site on β-tubulin. A number of ATIs proved to be more potent than 1, 2, VBL, and PTX and
1
4,15
thus are potential new anticancer agents.
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Chart 1. General Structures of Compounds 1−45.
MeO
MeO
MeO
A
B
NHCOMe
MeO
MeO
C
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MeO
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OH
O
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OMe
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OMe
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OMe
OMe
OMe
OMe
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R
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OMe
COOMe
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H
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4:(R(=(OMe,(5:(R(=(Cl
OMe
OMe
OMe
OMe
potent
activity
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X
C
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OMe
D
OMe
7
N
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drug(design
of(new(IASs
H
A
H
metabolic
stability
6'45
ATI derivatives bearing an aromatic ring at position 2 of the indole (A region) inhibit tubulin
polymerization and cancer cell growth. These compounds hamper mitotic progression, thus causing
1
4,15
cells to undergo apoptosis (i.e., ATI 3: tubulin assembly IC50 = 3.3 µM; MCF-7 IC50 = 52 nM)
(
Chart 1). In contrast, chemical modification of positions 4-7 of the indole (D region) were not
exhaustively explored, although a few ATI derivatives bearing an ethoxycarbonyl functionality at
position 2 and a halogen atom or a methoxy group at position 5 proved to be potent tubulin assembly
1
6
inhibitors (i.e., ATI 4: tubulin assembly IC50 = 2.0 µM; MCF-7 IC50 = 13 nM). It should also be noted
that major differences in structure-activity relationships (SAR) were found in comparing ATIs with the
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3
-aroylindoles reported by Hsieh and co-workers. These compounds are characterized by the
presence of a methoxy group at position 6 of the indole.
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Figureꢀ1.ꢀProposedꢀbindingꢀmodesꢀofꢀ1ꢀ(cyan),ꢀ3ꢀ(gray)ꢀandꢀ44ꢀ(orange).ꢀTubulinꢀisꢀrepresentedꢀasꢀaꢀcartoonꢀforꢀtheꢀ
α-ꢀ(red)ꢀandꢀβ-ꢀ(green)ꢀsubunits.ꢀResiduesꢀformingꢀinteractionsꢀwithꢀtheꢀATIꢀDꢀregionꢀareꢀdepictedꢀinꢀwhite.
Docking studies of compounds 1 and 3 into the colchicine site of tubulin showed a good
superimposition of the trimethoxyphenyl (TMP) moieties and of the tropolone ring C with the phenyl
14,15
core of the indole
(Figure 1). These findings suggested that appropriate substituents at positions 4-7
of the indole could resemble the methoxy and carbonyl groups at positions 10 and 9, respectively, of
1
8
the tropolone ring. Docking simulations with PLANTS, using our methodology described
19
previously, revealed binding poses consistent with the previous ATI series: (i) the TMP group formed
polar interactions with βCys241, βMet259 and βLeu255; (ii) the indole NH established an H-bond
with αThr179; (iii) the phenyl ring at position 2 of the indole set up hydrophobic interactions with the
βLys254 and βLeu248 side chains. In comparison with 3, the chlorine atom(s) of 10, 18, 28, 37 and 44
fitted into a new hydrophobic pocket formed by the βLys353, βAsn258, βMet259 and αVal181 residue
side chains (Figure 1S, Supporting Information). The 7-fluoro atom of 40, the most potent tubulin
polymerization inhibitor within the series, behaved as a H-bond acceptor with αVal181 (Figure 2S,
Supporting Information). The methoxy group of 33 mimicked the corresponding group at position 10
of 1, resembling its interaction with the εN of βLys353.
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Schemeꢀ1.ꢀSynthesisꢀofꢀCompoundsꢀ6-45.ꢀ
OMe
OMe
R
a
S
R
OMe
N
H
N
0
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H
4
6+62
6
2
3
,#8,#11,#13,#15,#18,
1,#23,#25,#28,#31,#33,
5,#37,#40,#42,#44
b
OMe
OMe
OMe
O
OMe
OMe
R
c#or#d
R
OMe
N
N
H
H
7,ꢀ9,ꢀ12,ꢀ14,ꢀ16,ꢀ19,ꢀ22,
24,ꢀ27,ꢀ29,ꢀ32,ꢀ34,ꢀ36,
38,ꢀ41,ꢀ43,ꢀ45.
10,#17,ꢀ20,
25,#30,#39
6
4
5
5
5
,ꢀ7,ꢀ46,ꢀRꢀ=ꢀ4-Br;ꢀ8-10,ꢀ47,ꢀRꢀ=ꢀ4-Clꢀ;ꢀ11,ꢀ12,ꢀ48,ꢀRꢀ=ꢀ4-F;ꢀ13,ꢀ14,ꢀ
9,ꢀRꢀ=ꢀ4-OMe;ꢀ15-17,ꢀ50,ꢀRꢀ=ꢀ5-Br;ꢀ18-20,ꢀ51,ꢀRꢀ=ꢀ5-Cl;ꢀ21,ꢀ22,ꢀ
ꢀ
2,ꢀRꢀ=ꢀ5-F;ꢀ23-25,ꢀ53, Rꢀ=ꢀ5-OMe;ꢀ26,ꢀ27,ꢀ54,ꢀRꢀ=ꢀ6-Br;ꢀ28-30,ꢀ
5,ꢀRꢀ=ꢀ6-Cl;ꢀ31,ꢀ32,ꢀ56,ꢀRꢀ=ꢀ6-F;ꢀ33,ꢀ34,ꢀ57,ꢀRꢀ=ꢀ6-OMe;ꢀ35,ꢀ36,ꢀ
8,ꢀRꢀ=ꢀ7-Br;ꢀ37-39,ꢀ59,ꢀRꢀ=ꢀ7-Cl;ꢀ40,ꢀ41,ꢀ60,ꢀRꢀ=ꢀ7-F;ꢀ42,ꢀ43,ꢀ61,ꢀ
Rꢀ=ꢀ7-OMe;ꢀ44,ꢀ45,ꢀ62,ꢀRꢀ=ꢀ6,7-Cl .ꢀ
2
ꢀ
aReagentsꢀ andꢀ reactionꢀ conditions:ꢀ (a)ꢀ bis-(3,4,5-
trimethoxyphenyl)disulfide,ꢀ anhydrousꢀ DMF,ꢀ closedꢀ vessel,ꢀ
30ꢀ°C,ꢀ120ꢀW,ꢀ2ꢀmin,ꢀventing-while-heating,ꢀ20-77%;ꢀ(b)ꢀ3,4,5-
trimethoxybenzoylꢀ chloride,ꢀ anhydrousꢀ AlCl ,ꢀ 1,2-
dichloroethane,ꢀclosedꢀvessel,ꢀ110ꢀ°C,ꢀ150ꢀW,ꢀ2ꢀmin,ꢀ20-78%;ꢀ
c)ꢀ(10,ꢀ 20,ꢀ25,ꢀ30ꢀandꢀ39)ꢀboraneꢀtetrahydrofuranꢀcomplex,ꢀ
acetonitrile/methanol,ꢀ 50ꢀ °C,ꢀ 1ꢀ h,ꢀ 10-41%;ꢀ (d)ꢀ (17)ꢀ
triethylsilane,ꢀ trifluoroaceticꢀ acid,ꢀ 1,2-dichloroethane,ꢀ 250ꢀ °C,ꢀ
50ꢀW,ꢀ20ꢀmin,ꢀ15%.ꢀ
1
3
(
2
These observations prompted us to undertake SAR investigations at positions 4-7 of the indole
(
Chart 1 and Table 1). We planned the synthesis of 2-phenyl-1H-indole derivatives, keeping the 2-
phenyl group fixed at position 2, because of the better metabolic profile compared to indole derivatives
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bearing an ester function at this position.
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CHEMISTRY
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-Phenyl-3-((3,4,5-trimethoxyphenyl)thio)-1H-indoles 6, 8, 11, 13, 15, 18, 21, 23, 25, 28, 31, 33, 35,
3
7, 40, 42 and 44 were prepared by reacting 2-phenylindoles 46-62 with bis-(3,4,5-
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16
trimethoxyphenyl)disulfide in the presence of sodium hydride at 130 °C (120 W) for 2 min according
2
0
to our previously reported venting-while-heating microwave-assisted procedure (Scheme 1).
Microwave-assisted Friedel-Crafts reaction of the indoles 46-62 with 3,4,5-trimethoxybenzoyl chloride
in the presence of AlCl
methanones 7, 9, 12, 14, 16, 19, 22, 24, 27, 29, 32, 34, 36, 38, 41, 43 and 45. Methylene derivatives
0, 17, 20, 25, 30 and 39 were prepared by reducing the appropriate ketones with borane
3
in 1,2-dichloroethane at 110 °C (150 W) for 2 min furnished the corresponding
2
1
1
2
2
tetrahydrofuran complex in acetonitrile/methanol at 50 °C for 1 h (10, 20, 25, 30 and 39) or with
2
3
triethylsilane/trifluoroacetic acid in 1,2-dichloroethane at 250 °C (250 W) for 20 min (17).
Reaction of the appropriate 1-methyl-2-nitrobenzene with benzaldehyde in the presence of sodium
ethoxide in anhydrous dimethyl sulfoxide at 25 °C for 12 h furnished the alcohols 63-65. After
oxidation of 63-65 to ketones 66-68 with pyridium chlorochromate in anhydrous dichloromethane at
2
5 °C for 1.5 h, the nitro group was reduced to amino with tin(II) chloride dihydrate/1 N HCl in acetic
acid at reflux temperature for 12 h; the intermediate aminoketone underwent intramolecular cyclization
2
4
to give the corresponding 2-phenylindoles 46, 54 and 56 (Scheme 2a). 2-Phenylindoles 47-49, 55, 57
and 61 were prepared by reaction of the N-(2-tolyl)benzamides 69-74 with tert-butyllithium in
2
5
anhydrous tetrahydrofuran at -40 °C. Amides 69-74 were obtained by treatment of an appropriate
ortho-toluidine with benzoyl chloride in the presence of triethylamine in anhydrous tetrahydrofuran at
reflux temperature for 2 h (Scheme 2b). Polyphosphoric acid-mediated cyclization at 110 °C for 1 h of
phenylhydrazones 75-80, prepared from the appropriate phenylhydrazine hydrochloride and
26
acetophenone in the presence of sodium acetate in ethanol at 80 °C (250 W) for 5 min, gave the
2
7
corresponding 2-phenylindoles 50-52, 58, 60 and 62 (Scheme 2c).
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ꢀ
Schemeꢀ2.ꢀSynthesisꢀofꢀIntermediatesꢀ46-62.aꢀ
a)
R
Me
a
b
R
0
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0
OH
NO2
NO2
6
3#65
c
46,ꢀ54,(56
R
O
NO2
66#68
b)
R
Me
Me
O
e
d
R
47#49,ꢀ55,
57,ꢀ61
N
H
NH2
6
9#74
c)
R
R
f
N
Me g
5
0#52,ꢀ58,
60,ꢀ62
N
H
NHNH2
.
HCl
75#80
ꢀ
63,ꢀ66,ꢀRꢀ=ꢀ2-Br;ꢀ64,ꢀ67,ꢀRꢀ=ꢀ4-Br;ꢀ65,ꢀ68,ꢀRꢀ=ꢀ4-F;ꢀ69,ꢀRꢀ=ꢀ3-Cl;ꢀ
70,ꢀRꢀ=ꢀ3-F;ꢀ71,ꢀRꢀ=ꢀ3-OMe;ꢀ72,ꢀRꢀ=ꢀ5-Cl;ꢀ73,ꢀRꢀ=ꢀ5-OMe;ꢀ74,ꢀRꢀ=ꢀ
2-OMe;ꢀ75,ꢀRꢀ=ꢀ4-Br;ꢀ76,ꢀRꢀ=ꢀ4-Cl;ꢀ77,ꢀRꢀ=ꢀ4-F;ꢀ78,ꢀRꢀ=ꢀ2-Br;ꢀ79,ꢀ
2
Rꢀ=ꢀ2-F;ꢀ80,ꢀRꢀ=ꢀ5,6-Cl .ꢀ
ꢀ
a
Reagentsꢀ andꢀ reactionꢀ conditions:ꢀ (a)ꢀ benzaldehyde,ꢀ sodiumꢀ
ethoxide,ꢀanhydrousꢀDMSO,ꢀ25ꢀ°C,ꢀ12ꢀh,ꢀ40-44%;ꢀ(b)ꢀpyridiumꢀ
chlorochromate,ꢀanhydrousꢀdichloromethane,ꢀ25ꢀ°C,ꢀ1.5ꢀh,ꢀ16-
4
0%;ꢀ(c)ꢀtin(II)ꢀchlorideꢀdihydrate/1ꢀNꢀHCl,ꢀaceticꢀacid,ꢀrefluxꢀ
temperature,ꢀ12ꢀh,ꢀ19-24%;ꢀ(d)ꢀbenzoylꢀchloride,ꢀtriethylamine,ꢀ
anhydrousꢀTHF,ꢀrefluxꢀtemperature,ꢀ2ꢀh,ꢀ49-88%;ꢀ (e)ꢀ (i)ꢀ tert-
butyllithium,ꢀ anhydrousꢀ THF,ꢀ 40ꢀ °C,ꢀ Arꢀ stream;ꢀ (ii)ꢀ 1ꢀ h,ꢀ 0ꢀ °C;ꢀ
iii)ꢀ 25ꢀ °C,ꢀ 12ꢀ h,ꢀ 18-52%;ꢀ (f)ꢀ acetophenone,ꢀ sodiumꢀ acetate,ꢀ
ethanol,ꢀopenꢀvessel,ꢀ80ꢀ°C,ꢀ250ꢀW,ꢀ5ꢀmin,ꢀcooling-while-heating,ꢀ
(
2
5-95%;ꢀ(g)ꢀpolyphosphoricꢀacid,ꢀ110ꢀ°C,ꢀ1ꢀh,ꢀ25-82%.ꢀ
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RESULTS AND DISCUSSION
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6
Inhibition of Tubulin Polymerization, the Binding of Colchicine to Tubulin and MCF-7 Breast
Cancer Cell Growth. We synthesized compounds 6-45, including ATI derivatives and some
corresponding ketone and methylene compounds, to obtain SAR information on the substituent(s)
introduced at positions 4-7 of the indole nucleus. The activities of compounds 6-45 as inhibitors of
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tubulin polymerization in vitro, the growth of MCF-7 human breast cancer cells and the binding of
3
[
H]colchicine to tubulin are shown in Table 1. The majority of these new derivatives (23 compounds:
8, 11, 12, 15, 18, 21, 26-30, 33-38 and 40-45) inhibited tubulin polymerization with IC50 values in the
1
.0−2.0 µM range, as compared with colchicine (1) (IC50 = 3.2 µM) and CSA4 (2) (IC50 = 1.0 µM).
Among the group of compounds 6-14, bearing the substituent at position 4 of the indole, the 4-
chloro derivative 8 inhibited tubulin assembly with an IC50 of 1.6 µM and was 2.2-fold more active
than corresponding ketone 9 (IC50 = 3.6 µM). The thio/keto 4-fluoro derivatives 11 (IC50 = 1.7 µM) and
1
2 (IC50 = 1.6 µM) were almost equipotent as tubulin assembly inhibitors and showed the greatest
inhibition of MCF-7 cell growth (11, IC50 = 80 nM, and 12, IC50 = 65 nM). Neither the bromine atom
nor the methoxy group were found among the most active compounds with position 4 substituents in
both the thio and ketone series. In terms of effects on MCF-7 cell growth, there is no evident major
difference between the thio and keto series with substituents at position 4, although the data are limited.
Compounds 15-25 were characterized by substituents at position 5 of the indole nucleus. As tubulin
polymerization inhibitors, the arylthioindoles 15 (IC50 = 1.3 µM), 18 (IC50 = 1.5 µM), 21 (IC50 = 1.5
µM) and 23 (IC50 = 2.1 µM) were all significantly more potent than the corresponding ketones 16, 19,
2
2 and 24 and slightly more active than the methylene compounds 17, 20 and 25. The tubulin
inhibitory activity of these compounds seemed to be weakly affected by the nature of the substituent. In
contrast to the corresponding ester derivatives, all these compounds were weak inhibitors of MCF-7
cell growth with IC50’s ≥100 nM (compare 4 (IC50 = 13 nM) with 23, and 5 (IC50 = 42 nM) with 18).
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Further, within the limitations of the data, there are no major differences in effects on MCF-7 cell
growth dependent on the bridging group (thio, ketone or methylene).
Compounds 26-34, bearing the substituent at position 6, inhibited tubulin polymerization with IC50’s
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<
2.5 µM, with the exception of the 6-fluoro derivative 32 (IC50 = 10 µM). As inhibitors of MCF-7 cell
growth, ketones were less potent than the corresponding thio derivatives. Among the thio derivatives,
the 6-bromo- (26, IC50 = 9.0 nM) and 6-methoxy- (33, IC50 = 1.3 nM) derivatives were the most potent
cell growth inhibitors. Compounds 35-43, with the substituent at position 7 of the indole, were all
potent inhibitors of tubulin assembly, with IC50’s < 2.5 µM. With the exception of 39 and 43, these
compounds potently inhibited the growth of the MCF-7 cells, with compounds 36 (IC50 = 4 nM) and 38
(IC50 = 9 nM) being the most active. In contrast to the findings with the 6-substituted indoles, in the 7-
substituted series, the bromo and chloro ketones tended to be more potent than the corresponding thio
counterparts as inhibitors of MCF-7 cell growth (compare 26, 27 with 35, 36; and 28, 29 with 37, 38).
Finally, we tried to join the features of 28, 29 with those of 37, 38 to further enhance activity. This
effort yielded the 6,7-dichloroindole derivatives 44 and 45, both of which indeed were highly potent
inhibitors of tubulin polymerization (IC50 = 1.2 and 1.5 µM, respectively) and MCF-7 cell growth (IC50
= 7 and 15 nM, respectively). SAR summary of tubulin polymerization inhibition (TPI) and inhibition
of MCF-7 cell growth of ATI derivatives 6-45 is depicted in Figure 2.
The results obtained from the docking simulations described above, provided us with a general
binding mode that was able to justify the biological activity of the compounds. However, there were
some exceptions that could not be fully rationalized (7, 13, 14 and 16) and we did not see any
correlation between docking scores and experimental data. This has prompted us to further investigate
the binding mode of ATIs with another set of docking simulations using a more recent tubulin structure
29
(
pdb code 4O2A), crystallized at higher resolution than the structure used previously (2.50Å vs 3.58Å
2
9
respectively). Furthermore, we have performed the docking calculations with two other software that
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each rely on a different search algorithm: Glide and Autodock.
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The docking results obtained from these simulations were entirely consistent with the one obtained
using PLANTS. Indeed, we observed a virtually identical binding pose for the ATIs, regardless of the
tubulin structure or the algorithm used in the simulation (Figure 3S, Supporting Information). It should
be noted that we have not observed any correlation between the docking score and the experimental
data in any of the calculations (Table 1S, Supporting Information). This observation is not entirely
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surprising considering this correlation is rarely observed.
We analyzed the data from Table 1 comparing the microtuble assembly inhibitory concentrations
mM) with % inhibition of colchicine binding since these data provide an indirect measure of the
(
affinity of the compounds for the colchicine site (Figure 3). The inhibition of tubulin polymerization
was in good agreement with inhibition of colchicine binding. The compounds that inhibited tubulin
assembly with IC50 values in the 1.0-1.5 µM range, inhibited colchicine binding by 39-96% (mean
value of 79%); those that inhibited assembly with IC50’s in the 1.6-2.0 µM range, inhibited colchicine
binding by 48-82% (mean value of 70%); and assembly inhibitors with IC50’s in the 2.1-3.0 µM range,
inhibited colchicine binding by 33-74% (mean value of 53%) (CSA4: assembly IC50 = 1.0 µM,
colchicine binding inhibition = 98%). We analyzed the MCF-7 cell growth inhibition (nM) of ATIs 6-
4
5 with the corresponding inhibitory concentration (µM) on tubulin assembly (Panel A, Figure 4S,
3
Supporting Information) and with % inhibition on the binding of [ H]colchicine to tubulin (Panel B,
Figure 4S, Supporting Information). In these plots we observed good correlation of the antiproliferative
data with the biochemical data, particularly with the ligand binding data (inhibition of colchicine
binding).
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TPI00S0=0CO
MCFꢀ70R0=0F0good
TPI0S0>0CO0good
OMe
OMe
MCFꢀ70weak
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TPI0good0(except0F)
MCFꢀ700S0>0CO0potent
X
R
OMe
6
2
N
7
R0=06,7ꢀCl2
H
TPI0and0MCFꢀ70potent
R0=0halogen,0TPI0good
MCFꢀ70CO0>0S;0Br,0Cl0potent
Figureꢀ 2.ꢀ SARꢀ summaryꢀ ofꢀ tubulinꢀ polymerizationꢀ
inhibitionꢀ (TPI)ꢀ andꢀ inhibitionꢀ ofꢀ MCF-7ꢀ cellꢀ growthꢀ ofꢀ
ATIꢀderivativesꢀ6-45.
Figureꢀ3.ꢀCorrelationꢀbetweenꢀtubulinꢀassemblyꢀ(IC50ꢀvalues,ꢀμM)ꢀandꢀinhibitionꢀofꢀcolchicineꢀbindingꢀ
%ꢀvalues).ꢀDataꢀofꢀATIꢀderivativesꢀ 6-45ꢀareꢀshownꢀasꢀopenꢀcircles.ꢀBlackꢀcircleꢀrepresentsꢀCSA4ꢀasꢀ
(
referenceꢀcompound.ꢀ
ꢀ
ꢀ
ꢀ
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Tableꢀ 1.ꢀ Inhibitionꢀ ofꢀ Tubulinꢀ Polymerization,ꢀ Bindingꢀ ofꢀ Colchicineꢀ toꢀ Tubulinꢀ andꢀ Growthꢀ ofꢀ MCF-7ꢀ
HumanꢀBreastꢀCarcinomaꢀCellsꢀbyꢀCompoundsꢀ6-45ꢀandꢀReferencesꢀ1-5.ꢀ
4
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1
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2
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5
5
5
5
6
OMe
OMe
X
R
OMe
0
1
2
3
4
5
6
7
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9
0
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N
H
6-45
TubulinꢀAssemblyaꢀ
IC50ꢀ±ꢀSDꢀ
MCF-7b,cꢀ
IC50ꢀ±ꢀSDꢀ
(nM)ꢀ
Colchicineꢀ
Bindingdꢀ
(%ꢀ±ꢀSD)ꢀ
compdꢀ
Rꢀ
Xꢀ
(µM)ꢀ
6ꢀ
7ꢀ
4-Brꢀ
4-Brꢀ
4-Clꢀ
Sꢀ
2.4ꢀ±ꢀ0.1ꢀ
>20ꢀ(partialꢀactivity)eꢀ
1.6ꢀ±ꢀ0.07ꢀ
400ꢀ±ꢀ70ꢀ
430ꢀ±ꢀ100ꢀ
290ꢀ±ꢀ50ꢀ
260ꢀ±ꢀ50ꢀ
>5000ꢀ
33ꢀ±ꢀ2ꢀ
ndꢀ
C=Oꢀ
Sꢀ
8
9
ꢀ
ꢀ
ꢀ48ꢀ±ꢀ5ꢀ
38ꢀ±ꢀ3ꢀ
ndfꢀ
4-Clꢀ
C=Oꢀ
3.6ꢀ±ꢀ0.2ꢀ
1
1
1
1
1
1
1
1
1
1
2
2
0ꢀ
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
7ꢀ
8ꢀ
9ꢀ
0ꢀ
1ꢀ
4-Clꢀ
CH
Sꢀ
2
ꢀ
17ꢀ±ꢀ0.2ꢀ
4-Fꢀ
1.7ꢀ±ꢀ0.07ꢀ
80ꢀ±ꢀ30ꢀ
80ꢀ±ꢀ0.4ꢀ
63ꢀ±ꢀ1ꢀ
ndꢀ
4-Fꢀ
C=Oꢀ
Sꢀ
1.6ꢀ±ꢀ0.08ꢀ
65ꢀ±ꢀ7ꢀ
ꢀ
e
4-MeOꢀ
4-MeOꢀ
5-Brꢀ
5-Brꢀ
5-Brꢀ
5-Clꢀ
>20ꢀ(partialꢀactivity) ꢀ
>20ꢀ(noꢀactivity)gꢀ
1.3ꢀ±ꢀ0.1ꢀ
1400ꢀ±ꢀ300ꢀ
>5000ꢀ
C=Oꢀ
Sꢀ
ndꢀ
320ꢀꢀ±ꢀ100ꢀ
530ꢀꢀ±ꢀ100ꢀ
700ꢀꢀ±ꢀ300ꢀ
280ꢀ±ꢀ70ꢀ
330ꢀ±ꢀ100ꢀ
310ꢀ±ꢀ10ꢀ
300ꢀ±ꢀ0ꢀ
39ꢀ±ꢀ3ꢀ
ndꢀ
C=Oꢀ
>40ꢀ
CH
Sꢀ
2
ꢀ
4.0ꢀ±ꢀ0.5ꢀ
31ꢀ±ꢀ2ꢀ
49ꢀ±ꢀ5ꢀ
ndꢀ
1.5ꢀ±ꢀ0.2ꢀ
5-Clꢀ
C=Oꢀ
>20ꢀ
5-Clꢀ
CH
Sꢀ
2
ꢀ
4.4ꢀ±ꢀ0.8ꢀ
38ꢀ±ꢀ3ꢀ
61ꢀ±ꢀ4ꢀ
ndꢀ
5-Fꢀ
1.5ꢀ±ꢀ0.1ꢀ
22ꢀ
23ꢀ
24ꢀ
25ꢀ
5-Fꢀ
C=Oꢀ
Sꢀ
13ꢀ±ꢀ0.4ꢀ
240ꢀ±ꢀ90ꢀ
200ꢀ±ꢀ80ꢀ
170ꢀ±ꢀ60ꢀ
100ꢀ±ꢀ0ꢀ
5-OMeꢀ
5-OMeꢀ
5-OMeꢀ
2.1ꢀ±ꢀ0.01ꢀ
8.3ꢀ±ꢀ1ꢀ
58ꢀ±ꢀ0.5ꢀ
ndꢀ
C=Oꢀ
CH
2
ꢀ
3.5ꢀ±ꢀ0.4ꢀ
41ꢀ±ꢀ0.9ꢀ
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6-Brꢀ
6-Clꢀ
Sꢀ
1.9ꢀ±ꢀ0.2ꢀ
9.0ꢀ±ꢀ2ꢀ
30ꢀ±ꢀ2ꢀ
87ꢀ±ꢀ0.6ꢀ
86ꢀ±ꢀ0.4ꢀ
ꢀ88ꢀ±ꢀ3ꢀ
71ꢀ±ꢀ5ꢀ
72ꢀ±ꢀ6ꢀ
74ꢀ±ꢀ2ꢀ
ndꢀ
C=Oꢀ
Sꢀ
1.3ꢀ±ꢀ0.2ꢀ
1.2ꢀ±ꢀ0.2ꢀ
1.4ꢀ±ꢀ0.06ꢀ
1.6ꢀ±ꢀ0.01ꢀ
2.4ꢀ±ꢀ0.2ꢀ
10ꢀ±ꢀ0.4ꢀ
1.1ꢀ±ꢀ0.1ꢀ
1.2ꢀ±ꢀ0.06ꢀ
1.6ꢀ±ꢀ0.2ꢀ
1.7ꢀ±ꢀ0.1ꢀ
1.3ꢀ±ꢀ0.2ꢀ
1.8ꢀ±ꢀ0.05ꢀ
2.4ꢀ±ꢀ0.2ꢀ
1.0ꢀ±ꢀ0.1ꢀ
1.7ꢀ±ꢀ0.1ꢀ
1.2ꢀ±ꢀ0.01ꢀ
1.8ꢀ±ꢀ0.1ꢀ
1.2ꢀ±ꢀ0.1ꢀ
1.5ꢀ±ꢀ0.03ꢀ
3.2ꢀ±ꢀ0.4ꢀ
1.0ꢀ±ꢀ0.1ꢀ
3.3ꢀ±ꢀ0.1ꢀ
2.0ꢀ±ꢀ0.2ꢀ
2.5ꢀ±ꢀ0.3ꢀ
20ꢀ±ꢀ10ꢀ
35ꢀ±ꢀ7ꢀ
6-Clꢀ
C=Oꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6-Clꢀ
CH
Sꢀ
2
ꢀ
55ꢀ±ꢀ20ꢀ
80ꢀ±ꢀ20ꢀ
600ꢀ±ꢀ0ꢀ
1.3ꢀ±ꢀ0.6ꢀ
30ꢀ±ꢀ10ꢀ
25ꢀ±ꢀ5ꢀ
6-Fꢀ
6-Fꢀ
C=Oꢀ
Sꢀ
6-MeOꢀ
6-MeOꢀ
7-Brꢀ
7-Brꢀ
7-Clꢀ
96ꢀ±ꢀ1ꢀ
95ꢀ±ꢀ0.4ꢀ
82ꢀ±ꢀ1ꢀ
72ꢀ±ꢀ0.3ꢀ
ꢀ82ꢀ±ꢀ0.3ꢀ
69ꢀ±ꢀ0.5ꢀ
50ꢀ±ꢀ4ꢀ
92ꢀ±ꢀ0.3ꢀ
67ꢀ±ꢀ3ꢀ
87ꢀ±ꢀ2ꢀ
62ꢀ±ꢀ3ꢀ
92ꢀ±ꢀ2ꢀ
93ꢀ±ꢀ0.7ꢀ
ndꢀ
C=Oꢀ
Sꢀ
C=Oꢀ
Sꢀ
4.0ꢀ±ꢀ0.5ꢀ
50ꢀ±ꢀ10ꢀ
9.0ꢀ±ꢀ1ꢀ
200ꢀ±ꢀ0ꢀ
20ꢀ±ꢀ9ꢀ
38ꢀ
39ꢀ
40ꢀ
41ꢀ
42ꢀ
7-Clꢀ
C=Oꢀ
7-Clꢀ
CH
Sꢀ
2
ꢀ
7-Fꢀ
7-Fꢀ
C=Oꢀ
Sꢀ
30ꢀ±ꢀ0ꢀ
7-OMeꢀ
7-OMeꢀ
19ꢀ±ꢀ10ꢀ
200ꢀ±ꢀ0ꢀ
7.0ꢀ±ꢀ3ꢀ
15ꢀ±ꢀ5ꢀ
4
4
4
3ꢀ
4ꢀ
5ꢀ
C=Oꢀ
Sꢀ
6,7-Cl
6,7-Cl
—ꢀ
2
2
ꢀ
ꢀ
C=Oꢀ
—ꢀ
1
ꢀ
ꢀ
5.0ꢀ±ꢀ1ꢀ
13ꢀ±ꢀ3ꢀ
2
—ꢀ
—ꢀ
98ꢀ±ꢀ0.6ꢀ
ndꢀ
3h
ꢀ
—ꢀ
—ꢀ
52ꢀ±ꢀ7ꢀ
4i
5i
ꢀ
—ꢀ
—ꢀ
13ꢀ±ꢀ3ꢀ
93ꢀ±ꢀ0.8ꢀ
76ꢀ±ꢀ0.2ꢀ
ꢀ
—ꢀ
—ꢀ
42ꢀ±ꢀ10ꢀ
a
b
Inhibitionꢀofꢀtubulinꢀpolymerization.ꢀTubulinꢀwasꢀatꢀ10ꢀμMꢀinꢀtheꢀassemblyꢀassay.ꢀ InhibitionꢀofꢀgrowthꢀofꢀMCF-7ꢀ
humanꢀbreastꢀcarcinomaꢀcells.ꢀ CompoundsꢀthatꢀinhibitedꢀtubulinꢀassemblyꢀwithꢀIC50ꢀ≤5ꢀμMꢀwereꢀtestedꢀinꢀtheꢀ
cellularꢀ andꢀ colchicineꢀ bindingꢀ assays.ꢀ Inhibitionꢀ ofꢀ [ H]colchicineꢀ binding.ꢀ Tubulinꢀ wasꢀ atꢀ 1ꢀ μM.ꢀ Bothꢀ
c
d
3
3
e
f
g
[
h
H]colchicineꢀandꢀinhibitorꢀwereꢀatꢀ5ꢀμM.ꢀ Partialꢀinhibitionꢀatꢀ20ꢀµM.ꢀ Noꢀdata.ꢀ Littleꢀorꢀnoꢀactivityꢀatꢀ20ꢀµM.ꢀ
15 16
. i
Lit. ꢀ Lit. ꢀ
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ꢀ
Tableꢀ2.ꢀGrowthꢀInhibitionꢀofꢀMDA-MB-468,ꢀMDA-MB-436,ꢀMDA-MB-231,ꢀA-549,ꢀMV4-11,ꢀNB4ꢀandꢀNCI-H1975ꢀꢀ
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CellꢀLinesꢀbyꢀCompoundsꢀ33ꢀandꢀ44ꢀandꢀReferenceꢀPTX.ꢀ
ꢀ
ꢀ
IC50ꢀ±ꢀSDꢀ(nM)ꢀ
MV4-11bꢀ
ꢀ
MDA-ꢀ
MB-468aꢀ
MDA-ꢀ
MB-436aꢀ
MDAꢀ
MB-23aꢀ
NB4bꢀ
ꢀ
NCI-ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compdꢀ
(AML_M9)ꢀꢀ (AML_M3)ꢀ
A-549a
H1975aꢀ
3
4
3ꢀ
4ꢀ
37ꢀ±ꢀ0.5ꢀ
33ꢀ±ꢀ0.3ꢀ
5ꢀ±ꢀ1ꢀ
62ꢀ±ꢀ1ꢀ
75ꢀ±ꢀ1.1ꢀ
8ꢀ±ꢀ1.5ꢀ
39ꢀ±ꢀ1.2ꢀ
47ꢀ±ꢀ0.1ꢀ
2.5ꢀ±ꢀ2.1ꢀ
10.5ꢀ±ꢀ0.7ꢀ
ndbꢀ
4ꢀ±ꢀ1ꢀ
10ꢀ±ꢀ4ꢀ
28ꢀ±ꢀ6
120ꢀ±ꢀ10
7ꢀ±ꢀ2
195ꢀ±ꢀ158ꢀ
305ꢀ±ꢀ122ꢀ
2.5ꢀ±ꢀ5ꢀ
PTXꢀꢀ
7ꢀ±ꢀ2ꢀ
2.3ꢀ±ꢀ0.3ꢀ
a
b
b
Incubationꢀtimeꢀwasꢀ72ꢀh.ꢀ Incubationꢀtimeꢀwasꢀ48ꢀh.ꢀ Noꢀdata.ꢀ
Cell Growth Inhibition. ATIs 33 and 44 were assayed as growth inhibitors of a panel of cancer cell
lines, including MDA-MB-468, MDA-MB-436, MDA-MB-231, A-549, MV4-11, NB4 and NCI-
H1975, using PTX as reference drug. As a growth inhibitor of MDA-MB-468, MDA-MB-436, and
MDA-MB-231 breast cancer cells, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay, ATI 33 at 72 h showed IC50 values of 37, 62 and 39 nM, respectively, with the
corresponding values for 44 being 33, 75 and 47 nM, respectively (Table 2). Compounds 33 and 44
induced a dose and time-dependent growth inhibition of each treated cell line (Figures 5S-10S,
Supporting Information). Compounds 33 and 44 inhibited the human acute myelocytic leukemia
(AML) cell lines MV4-11 and NB4, showing at 48 h IC50’s of 2.5 and 10.5, and 4 and 10 nM,
respectively. As growth inhibitors of the A-549 and NCI-H1975 human lung adenocarcinoma cancer
cells bearing the KRAS mutation and resistance to EGFR inhibitors, respectively, ATIs 33 and 44
yielded at 72 h IC50 values in the nanomolar concentration range.
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Tableꢀ3.ꢀInhibitionꢀofꢀGrowthꢀofꢀtheꢀOVCAR-8ꢀandꢀNCI/ADR-RES,ꢀandꢀMessaꢀandꢀ
Messa/DxꢀCellꢀLineꢀPairsꢀbyꢀCompoundsꢀ33ꢀandꢀ44,ꢀandꢀReferenceꢀCompoundsꢀ1,ꢀ
2
,ꢀVRB,ꢀVBLꢀandꢀPTX.aꢀ
ꢀ
ꢀ
ꢀ
IC50ꢀ±ꢀSDꢀꢀ (nM)ꢀ
NCI/ADR-RESꢀ
ꢀ
compdꢀ
OVCAR-8ꢀ
4.3ꢀ±ꢀ1ꢀ
14ꢀ±ꢀ2ꢀ
30ꢀ±ꢀ1ꢀ
2.8ꢀ±ꢀ1ꢀ
300ꢀ±ꢀ0ꢀ
15ꢀ±ꢀ7ꢀ
ꢀꢀMessabꢀ
20.7ꢀ±ꢀ1.7ꢀ
3.5ꢀ±ꢀ0.9ꢀ
11ꢀ±ꢀ6ꢀ
Messa/Dx5bꢀ
28.0ꢀ±ꢀ1.0ꢀ
4.5ꢀ±ꢀ1.9ꢀ
329ꢀ±ꢀ166ꢀ
2.6ꢀ±ꢀ1ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4
3ꢀ
4ꢀ
2.5ꢀ±ꢀ1ꢀ
10ꢀ±ꢀ4ꢀ
1
ꢀ
ꢀ
420ꢀ±ꢀ100ꢀ
1.8ꢀ±ꢀ1ꢀ
2
2.7ꢀ±ꢀ2ꢀ
ndcꢀ
VRBꢀ
VBLꢀ
PTXꢀ
5000ꢀ±ꢀ1000ꢀ
ndꢀ
200ꢀ±ꢀ0ꢀ
6000ꢀ±ꢀ500ꢀ
b
3ꢀ±ꢀ2ꢀ
4ꢀ±ꢀ1ꢀ
144ꢀ±ꢀ61ꢀ
3
.7ꢀ±ꢀ1ꢀ
1764ꢀ±ꢀ477ꢀ
a
c
Inhibitionꢀofꢀgrowthꢀofꢀtheꢀindicatedꢀcellꢀlines.ꢀ Incubationꢀtimeꢀwasꢀ72ꢀh.ꢀ Noꢀdata.ꢀ
Multidrug Resistant (MDR) Cell Lines. Compounds 33 and 44 were evaluated as inhibitors of the
ovarian carcinoma cell lines OVCAR-8 and its cognate P-glycoprotein (Pgp) overexpressing line
NCI/ADR-RES and of the human uterine sarcoma cell line Messa and its cognate MDR line
Messa/Dx5, using 1, 2, vinorelbine (VRB), VBL and PTX as reference compounds (Table 3). The
reference agents 1, VRB, VBL and PTX weakly inhibited these MDR cell lines. Compounds 33 and 44
and the reference 2 were potent inhibitors of the MDR cell lines. ATI 33 (IC50 = 2.5 ± 1 nM) was
comparable to 2 (IC50 = 1.8 ± 1 nM) as an inhibitor of the NCI/ADR-RES cell line, and 44 (IC50 = 4.5
±
1 nM) was comparable to 2 (IC50 = 2.6 ± 1 nM) in the Messa/Dx5 cells.
Arrest of Mitotic Progression and Concomitant Cell Death Induction. To assess whether the
growth suppressive effect of 33 and 44 reflected their antimitotic activity, we assessed their ability to
induce mitotic arrest. We previously found that treatment with 20 nM VBL effectively arrested the cell
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cycle of HeLa cells in mitosis; at lower concentrations VBL did not fully prevent mitotic progression,
such that cells assemble defective mitotic spindles, “slip” through the mitotic checkpoint and progress
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towards aberrant chromosome segregation. We examined HeLa cell cultures treated with 20, 50 and
0
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9
0
1
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1
00 nM 33 or 44 in DMSO, 20 nM VBL or DMSO vehicle. Treatments were carried out for 24 h,
allowing all cells to enter mitosis during the treatment. Cell cultures were then harvested, and their cell
cycle profile was analyzed by flow cytometry after incubation with propidium iodide (PI) to reveal
their genomic DNA content (linear scale). ATIs 33 and 44 at 20 nM induced >80% of HeLa cells to
arrest with a G2/M genomic content, similar to VBL; very few cells progressed past the G2/M phase
(
Figure 4A). Thus, both 33 and 44 effectively arrested mitotic progression and prevented mitotic
slippage, and hence the generation of hyperdiploid or polyploid cells. To assess whether G2/M-arrested
cells underwent cell death over time, PI-stained cell samples were analyzed by plotting their DNA
content on a logarithmic scale to resolve the sub-G1 region, in which hypodiploid cells represent the
terminal products of cell death. ATIs 33 and 44 at 20 nM induced both cell death and concomitant
G2/M phase arrest and were comparable to VBL; these effects were even more substantial at 50 nM
(
Figure 4B). The induction of cell death by 33 seemed to be superior to 44 at all tested concentrations.
Biparametric analysis after simultaneous incubation of non-permeabilized cells with annexin V, which
reacts with phosphatidylserine residues on the outer cell membrane during early apoptosis, and PI, to
which viable cells are not spontaneously permeable, discriminates early and late stages of apoptotic cell
death (Figure 4C) from necrotic cells, which are permeable to PI but do not react with annexin V. We
found that 33 and 44 induced cell death with apoptotic-like phenotypic features with comparable
effectiveness and early-to-late kinetics as observed with VBL.
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Figureꢀ 4.ꢀ A.ꢀ Cellꢀ cycleꢀ profilesꢀ ofꢀ HeLaꢀ cellꢀ culturesꢀ exposedꢀ toꢀ 33,ꢀ 44ꢀ orꢀ VBLꢀ forꢀ 24ꢀ hꢀ atꢀ theꢀ indicatedꢀ
concentrations;ꢀtheꢀ%ꢀofꢀcellsꢀwithꢀ2Cꢀ(G1ꢀphase,ꢀblackꢀbars),ꢀ4Cꢀ(G2/Mꢀphases,ꢀwhiteꢀbars),ꢀorꢀbetweenꢀ2Cꢀandꢀ4Cꢀ(Sꢀ
phase,ꢀ dashedꢀ bars)ꢀ genomicꢀ DNAꢀ contentꢀ isꢀ shown.ꢀ Meanꢀ valuesꢀ wereꢀ calculatedꢀ fromꢀ threeꢀ independentꢀ
experiments.ꢀ B.ꢀ Flowꢀ cytometryꢀ analysisꢀ ofꢀ PI-stainedꢀ cellsꢀ withꢀ sub-G1ꢀ DNAꢀ content,ꢀ representingꢀ terminalꢀ cellꢀ
death,ꢀ afterꢀ 24ꢀ h.ꢀ Meanꢀ valuesꢀ wereꢀ calculatedꢀ fromꢀ fourꢀ independentꢀ experiments.ꢀ C.ꢀ Distributionꢀ ofꢀ cellsꢀ
simultaneouslyꢀprocessedꢀforꢀannexinꢀVꢀreactivityꢀandꢀPIꢀincorporation,ꢀrepresentingꢀearlyꢀ(reactiveꢀtoꢀannexinꢀV,ꢀ
notꢀpermeableꢀtoꢀPI)ꢀandꢀlateꢀ(reactiveꢀtoꢀbothꢀannexinꢀVꢀandꢀPI)ꢀstagesꢀofꢀtheꢀcellꢀdeathꢀprocessꢀinꢀculturesꢀtreatedꢀ
forꢀ24ꢀh.ꢀMeanꢀvaluesꢀwereꢀcalculatedꢀfromꢀthreeꢀindependentꢀexperiments.ꢀ
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Figureꢀ 5.ꢀ Mitoticꢀ phenotypesꢀ inꢀ HeLaꢀ cellꢀ culturesꢀ exposedꢀ toꢀ ATIsꢀ 33ꢀ andꢀ 44ꢀ forꢀ 24ꢀ hꢀ andꢀ processedꢀ forꢀ IFꢀ toꢀ
tubulinꢀ(inꢀred).ꢀTheꢀbarꢀgraphꢀonꢀtheꢀleftꢀindicatesꢀtheꢀstatisticalꢀdistributionꢀofꢀtheꢀcytologicalꢀphenotypesꢀdepictedꢀ
underꢀtheꢀindicatedꢀconditions;ꢀtheꢀfrequencyꢀofꢀscoredꢀphenotypesꢀisꢀshownꢀasꢀ%ꢀofꢀallꢀcountedꢀmitoticꢀfiguresꢀ(330ꢀ
toꢀ 400ꢀ countedꢀ mitoticꢀ figuresꢀ perꢀ condition).ꢀ Greyꢀ barsꢀ representꢀ theꢀ frequencyꢀ ofꢀ mitoticꢀ cellsꢀ withꢀ severeꢀ
inhibitionꢀofꢀtubulinꢀpolymerization,ꢀwithꢀnoꢀrecognizableꢀMTsꢀandꢀunstructuredꢀtubulinꢀaggregatesꢀ(exemplifiedꢀinꢀ
theꢀtopꢀIFꢀpanel).ꢀPurpleꢀbarsꢀrepresentꢀtheꢀfrequencyꢀofꢀmitoticꢀcellsꢀwithꢀinhibitedꢀMTꢀpolymerization,ꢀyieldingꢀ
shortꢀ MTꢀ stretchesꢀ (middleꢀ panel).ꢀ Blackꢀ barsꢀ representꢀ theꢀ frequencyꢀ ofꢀ normalꢀ mitosesꢀ withꢀ polymerizedꢀ MTꢀ
arraysꢀformingꢀaꢀbipolarꢀspindleꢀ(bottomꢀpanel).ꢀ
ꢀ
ꢀ
ATIs 33 and 44 Strongly Inhibit Mitotic MT Assembly. To correlate cell cycle arrest and cell
death induction with inhibition of MT dynamics in HeLa cells, we employed immunofluorescence (IF)
to examine the effects of ATIs on tubulin and MTs. HeLa cells were treated with 20 or 50 nM 33 or 44
for 24 h, the same conditions that induced cell cycle arrest (Figure 4), then processed for tubulin IF and
examined under fluorescence microscopy. We found that at both concentrations 33 or 44 arrested cells
in mitosis with a prometaphase-like appearance. Consistent with the absence of hyperdiploid cells in
flow cytometry analysis, both ATIs at 20 nM or higher effectively prevented mitotic slippage. At 20
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nM, the ATIs inhibited MT polymerization and left tubulin foci and/or small asters of short MT
fragments (an example is shown in Figure 5A, middle panel). The proportion of mitotic cells displaying
this inhibited MT phenotype was very similar to that observed with VBL (Figure 5B). Raising the ATI
concentrations to 50 nM yielded a phenotype with no recognizable mitotic MT remnants, but only
tubulin aggregates that formed an unstructured meshwork throughout the mitotic cells (Figure 5A, top
panel). In summary, the newly synthesized ATIs strongly affect cellular MT polymerization, resulting
in effective inhibition of formation of the mitotic apparatus, particularly when used at 50 nM, and this
phenotype was associated with a durable mitotic arrest and concomitant induction of cell death.
Inhibition of PC-3, RD, and HepG2 Cancer Cell Growth. We determined the effects of ATIs 33
and 44 as growth inhibitors of the human prostate cancer PC-3, rhabdomyosarcoma RD and human
liver hepatocellular carcinoma HepG2 cell lines, with VBL and PTX as reference compounds (Table 4).
ATIs 33 and 44 strongly inhibited these cell lines at nanomolar concentrations and were superior to the
references VBL and PTX. Compound 33 was generally more effective than 44 as an inhibitor of the
growth of these three cell lines.
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PC-3, RD, and HEPG2 cell cultures were treated for 24 h with increasing concentrations of ATIs 33
and 44 and of PTX as reference drug at 500, 1000, and 2000 nM. After treatment, cells and vehicle
controls (0.1% DMSO) were incubated with PI to analyze their DNA content in flow cytometry assays.
Both ATIs arrested cell cycle progression in all three cell lines at low concentrations. ATIs 33 and 44
induced an accumulation in the G2/M phase in PC-3 and RD cells, as did PTX, whereas in HepG2 cells
these compounds caused a stronger effect on cell cycle progression as compared with the reference
drug (Figure 11S, Supporting Information).
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Tableꢀ4.ꢀGrowthꢀInhibitionꢀofꢀPC-3,ꢀRDꢀandꢀHepG2ꢀCellꢀLinesꢀ
byꢀCompoundsꢀ33ꢀandꢀ41ꢀandꢀReferencesꢀVBLꢀandꢀPTX.ꢀ
ꢀ
IC50ꢀ±ꢀSDꢀ(nM)aꢀ
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compdꢀ
PC3ꢀ
RDꢀ
HepG2ꢀ
ꢀꢀ0.1ꢀ±ꢀ0.02ꢀꢀꢀꢀ
62ꢀ±ꢀ2ꢀ
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3ꢀ
4ꢀ
0.3ꢀ±ꢀ0.06ꢀ
19ꢀ±ꢀ1ꢀ
0.2ꢀ±ꢀ0.04ꢀ
16ꢀ±ꢀ1.3ꢀ
53ꢀ±ꢀ2.5ꢀ
>10000ꢀ
VBLꢀ
PTXꢀꢀ
766ꢀ±ꢀ1000ꢀꢀ
4900ꢀ±ꢀ1.3ꢀ
81ꢀ±ꢀ2.4ꢀ
2600ꢀ±ꢀ1,5ꢀ
aIncubationꢀtimeꢀwasꢀ48ꢀh.ꢀ
ꢀ
After a 24 h exposure and subsequent incubation in drug-free medium for 24 h, the PC-3, RD, and
HepG2 cell lines treated with 33 or 44 showed a strong accumulation of cells in G2/M (Figure 6).
Moreover, 33 and 44 induced an irreversible cell cycle arrest at the concentrations of 500, 1000, and
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000 nM in the PC-3 and RD cells.
A correlation between cell cycle arrest and cell death was investigated by exposing the cells to a
500, 1000 or 2000 nM concentration of 33, 44 or PTX, followed by incubation with fluorescently
conjugated annexin V and PI (Figure 7). All the cell lines treated with 33 or 44 at 500 nM showed
higher rates of cell death than the same populations treated with 500 nM PTX. A dose-response trend in
cell death was observed in the RD cell line after a 48 h exposure with either 44 or 33 (Figure 7B) and in
the PC3 and HepG2 cell lines following treatment with PTX (Figure 7A and 7C).
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Figureꢀ6.ꢀꢀCellꢀcycleꢀanalysisꢀofꢀPC-3ꢀ(A),ꢀRDꢀ(B)ꢀandꢀHepG2ꢀ(C)ꢀcellsꢀtreatedꢀwithꢀ0.1%ꢀDMSOꢀorꢀ500,ꢀ1000ꢀorꢀ2000ꢀ
nMꢀ33,ꢀ44ꢀorꢀPTXꢀforꢀ24ꢀh,ꢀfollowedꢀbyꢀaꢀ24ꢀhꢀrecoveryꢀinꢀdrug-freeꢀmedium.ꢀHistogramsꢀrepresentꢀ%ꢀofꢀcellsꢀwithꢀ
G0/G1,ꢀSꢀandꢀG2/MꢀDNAꢀcontentꢀexpressedꢀasꢀmeanꢀvaluesꢀ±ꢀSDꢀofꢀthreeꢀindependentꢀexperiments.ꢀꢀ
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ꢀ
Figureꢀ7.ꢀCellꢀdeathꢀcytometricꢀanalysisꢀofꢀPC-3ꢀ(A),ꢀRDꢀ(B)ꢀandꢀHepG2ꢀ(C)ꢀcellsꢀtreatedꢀwithꢀ0.1%ꢀDMSOꢀorꢀ500,ꢀ1000ꢀ
orꢀ2000ꢀnMꢀ33,ꢀ44ꢀorꢀPTXꢀforꢀ48ꢀh.ꢀFlowꢀcytometricꢀprofilesꢀofꢀcellꢀpopulationsꢀfollowingꢀtreatmentꢀwithꢀDMSOꢀorꢀ
2000ꢀnMꢀ33ꢀorꢀ44ꢀareꢀatꢀtheꢀtopꢀofꢀeachꢀpanelꢀ(annexin-V-FITCꢀstainingꢀonꢀtheꢀxꢀaxisꢀandꢀPIꢀonꢀtheꢀyꢀaxis).ꢀHistogramsꢀ
representꢀ%ꢀofꢀcellsꢀinꢀearlyꢀapoptosisꢀ(annexinꢀV-FITCꢀstaining)ꢀandꢀlateꢀapoptosisꢀ(annexinꢀV-FITCꢀandꢀPIꢀstaining)ꢀ
expressedꢀasꢀmeanꢀvaluesꢀ±ꢀSDꢀcalculatedꢀfromꢀthreeꢀindependentꢀexperiments.ꢀꢀ
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Inhibition of T98G and U343MG Cancer Cell Growth. Malignant gliomas develop from gradual
accumulation of multiple genetic alterations, resulting in either activation of oncogenes or inactivation
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of tumor suppressor genes. Human glioblastoma multiforme T98G and U343MG cells show typical
hallmarks of glioblastoma multiforme tumors in patients. We evaluated the ability of compounds 33 and
44 to inhibit the growth of T98G and U343MG cancer cells, which show different genetic profiles for
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the expression of key cell survival proteins, such as p53, MDM2, EGFR, RB, cyclin D, and MMPs.
Treatment of T98G and U343MG cells with increasing concentrations of 33 or 44 for 24, 48 or 72 h
significantly inhibited cell growth in a dose- and time-dependent manner (Figures 12S and 13S,
Supporting Information). The IC50 values were calculated taking into account the relative doubling time
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(
CDT).
after 48 h for the T98G cells and after 72 h for the U343MG cells. As a cell growth inhibitor,
compound 33 yielded IC50 values of 15.2 ± 1.6 nM in T98G cells, and 0.5 ± 0.05 nM in U343 cells; for
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4, IC50 values of 16.3 ± 1.5 nM nM in T98G cells and 0.6 ± 0.05 nM nM in U343 cells were obtained.
Expression of MICA and MICB ligands in HeLa Cells, Resulting in Enhanced Natural Killer
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(
NK) Cell Degranulation. In previous studies, treatment of HeLa and HepG2 tumor cell lines with
sodium butyrate, a potent repressor of histone deacetylases that causes spindle abnormalities and
mitotic arrest, resulted in up-regulation of the expression of NK cell receptor activating ligand MICA
and MICB at both the mRNA and protein levels and in enhanced susceptibility of both cell lines to NK
lysis. We evaluated the expression of NKG2D and DNAM-1 ligands in HeLa cells after treatment with
either ATI 33, 37 or 44, in particular whether the compounds could modulate their expression. We first
characterized HeLa cell growth inhibition by 33, 37 or 44, at a sub-lethal concentration after a 48 h
treatment (MTT assay). HeLa cells were more sensitive to 33 and 44 (IC50 value = 10 nM) than to 37
(
IC50 = 76 nM). After a 48 h treatment with 10 nM ATI, flow cytometric biparametric analysis of HeLa
cells by annexin V/PI staining showed only a weak increase of early apoptotic cells compared to
control cultures (Figure 14S, Supporting Information).
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Figureꢀ 8.ꢀATIsꢀ33,ꢀ37ꢀandꢀ44ꢀup-regulateꢀDNAM-1ꢀandꢀNKG2DꢀligandsꢀinꢀHeLaꢀcells.ꢀNKG2DꢀandꢀDNAM-1ꢀligandꢀ
surfaceꢀexpressionꢀwasꢀanalyzedꢀbyꢀflowꢀcytometryꢀafterꢀaꢀ48ꢀhꢀtreatmentꢀwithꢀtheꢀindicatedꢀATIꢀcompound.ꢀDataꢀ
areꢀrepresentativeꢀofꢀoneꢀoutꢀofꢀthreeꢀindependentꢀexperiments.ꢀ
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NK cell receptor activating ligand analysis by combined IF and flow cytometry revealed a different
modulation of NKG2D and DNAM-1 ligands in ATI-treated HeLa cells after a 48 h treatment with
sub-lethal doses. ATI 33, 37 or 44 behaved as strong enhancers of MICA, ULBP3 and PVR expression,
while treatment with the compounds had weaker effects on MICB, ULBP1 and ULBP2 ligand
expression (Figure 8) and no effect on the expression of the Nec-2 ligand (data not shown).
Interestingly, (1-(3-aminophenyl)-1H-pyrrol-3-yl)(3,4,5-trimethoxyphenyl)methanone, a potent tubulin
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polymerization inhibitor belonging to the ARAP class, was unable to induce the NKG2D and
DNAM-1 ligands (data not shown). The expression of ligand surface on treated HeLa cells was not
accompanied by a corresponding increase in mRNA levels, as indicated by real-time PCR data (Figure
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5S, Supporting Information).
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Figureꢀ 9.ꢀNKꢀcell-mediatedꢀkillingꢀofꢀHeLaꢀcellsꢀincreasedꢀafterꢀaꢀ48ꢀhꢀtreatmentꢀwithꢀATIꢀ 33ꢀorꢀ44.ꢀResultsꢀareꢀ
expressedꢀ asꢀ %ꢀ ofꢀ CD107a ꢀ cellsꢀ afterꢀ subtractionꢀ ofꢀ theꢀ %ꢀ ofꢀ theꢀ controlꢀ antibodyꢀ andꢀ representꢀ oneꢀ ofꢀ twoꢀ
+
independentꢀexperiments.ꢀTheꢀbasalꢀCD107aꢀexpressionꢀonꢀNKꢀcellsꢀwasꢀaboutꢀ2%.ꢀ
Based on these findings, we evaluated whether ATIs 33 and 44 would increase NK-cell
degranulation towards HeLa cells. The expression of the lysosomal marker CD107a, which correlates
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with NK cell cytotoxicity, was evaluated by IF and flow cytometry analysis by gating on NK cells
upon their interaction with treated or untreated HeLa cells, used as targets. The up-regulation of
NKG2D and DNAM-1 ligands was verified before the degranulation assay (data not shown). The
expression of CD107a on NK cells contacting treated-HeLa target cells indicated that those cells were
more susceptible to NK cell lysis (Figure 9).
Hedgehog Inhibiting Activity. Hedgehog (Hh) signaling pathway is deeply involved in
tumorigenesis, and inhibitors of the Hh pathway have shown great potential as cancer therapeutics. In
recent years, an ever growing interest in the Hh pathway has led to the development of a variety of
small molecules targeting key Hh components, i.e., Smoothened (Smo), Sonic hedgehog protein (Shh),
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and Gli1. Currently, some antagonists of Smo, the positive signaling transducer in Hh pathway, are
undergoing clinical trials.
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Figureꢀ 10.ꢀInhibitionꢀofꢀendogenousꢀHhꢀsignalingꢀinꢀShh-LꢀIIꢀcellsꢀbyꢀ33,ꢀ44ꢀandꢀ81.ꢀDose–responseꢀcurveꢀofꢀtheꢀ
indicatedꢀ compoundsꢀ inꢀ SAG-treatedꢀ cellsꢀ inꢀ comparisonꢀ withꢀ untreatedꢀ Shh-Lightꢀ IIꢀ cellsꢀ asꢀ aꢀ controlꢀ (CTR).ꢀ
Treatmentꢀ timeꢀ wasꢀ 48ꢀ h,ꢀ andꢀ normalizationꢀ wasꢀ againstꢀ Renillaꢀ luciferase.ꢀ Dataꢀ fromꢀ threeꢀ independentꢀ
experiments.ꢀErrorꢀbarsꢀindicateꢀSD.ꢀ*P,ꢀ0.05ꢀvsꢀCTR.
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We wished to investigate whether ATI compounds, like the structurally related ARAP derivatives,
could behave as inhibitors of Hh signaling. We selected three highly potent ATI derivatives, 33, 44 and
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-(1H-imidazol-1-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (81),
and their effects were
evaluated by a luciferase assay performed in NIH3T3 Shh-Light II (Shh-LII) cells. In these cells, in
which is stably incorporated an Hh-responsive (Gli-RE) reporter, the induction of the pathway occurs
following treatment with the SMO agonist SAG. ATIs 33, 44 and 81 showed a strong reduction of
luciferase activity in cells treated with SAG in a dose-dependent manner (Figure 10), yielding IC50’s of
19, 72 and 38 nM, respectively. The treatment did not decrease the control Renilla luciferase activity,
preventing any cytotoxicity mediated effects on the inhibition of Hh signaling. To address the ability of
these compounds to affect D283 medulloblastoma cell proliferation and survival, we performed a
trypan blue count assay. The in vitro treatment with ATI 33, 44 or 81 at 1 µM impaired cell growth and
the percentage of cell death, as shown in Figure 16S, Supporting Information.
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