Novel 1H-pyrrolo[2,3-b]pyridine derivative nortopsentin analogues: Synthesis and antitumor activity in peritoneal mesothelioma experimental models

Article abstract of DOI:10.1021/jm400842x

In this study, we describe the synthesis of new nortopsentin analogues, 1H-pyrrolo[2,3-b]pyridine derivatives and their biological effects in experimental models of diffuse malignant peritoneal mesothelioma (DMPM), a rare and rapidly fatal disease, poorly responsive to conventional therapies. The three most active compounds, 1f (3-[2-(5-fluoro-1-methyl-1H-indol-3-yl)-1,3- thiazol-4-yl]-1H-pyrrolo[2,3-b]pyridine), 3f (3-[2-(1H-indol-3-yl)-1,3-thiazol- 4-yl]-1-methyl-1H-pyrrolo[2,3-b]pyridine), and 1l (3-[2-(5-fluoro-1-methyl-1H- indol-3-yl)-1,3-thiazol-4-yl]-1-methyl-1H-pyrrolo[2,3-b] pyridine), which were shown to act as cyclin-dependent kinase 1 inhibitors, consistently reduced DMPM cell proliferation and induced a caspase-dependent apoptotic response, with a concomitant reduction of the expression of the active Thr³⁴- phosphorylated form of the antiapoptotic protein survivin. Moreover, the combined treatment of DMPM cells with 3f derivative and paclitaxel produced a synergistic cytotoxic effect, which was paralleled by an enhanced apoptotic response. In the mouse model, i.p. administration of 1f, 3f, and 1l derivatives was effective, resulting in a significant tumor volume inhibition of DMPM xenografts (range, 58-75%) at well-tolerated doses, and two complete responses were observed in each treatment group.

Full text of DOI:10.1021/jm400842x

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Article
Novel 1H-pyrrolo[2,3-b]pyridine derivatives nortopsentin analogues: synthesis
and antitumor activity in peritoneal mesothelioma experimental models
PATRIZIA DIANA, ANNA CARBONE, MARZIA PENNATI, BARBARA PARRINO, ALESSIA LOPERGOLO,
PAOLA BARRAJA, ALESSANDRA MONTALBANO, VIRGINIA SPANO', STEFANIA SBARRA, VALENTINA
DOLDI, MICHELANDREA DE CESARE, GIROLAMO CIRRINCIONE, and NADIA ZAFFARONI
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400842x • Publication Date (Web): 06 Aug 2013
Downloaded from http://pubs.acs.org on August 11, 2013
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Novel 1Hꢀpyrrolo[2,3ꢀb]pyridine derivatives nortopsentin analogues: synthesis and
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antitumor activity in peritoneal mesothelioma experimental models
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Anna Carbone,§,‡ Marzia Pennati,#,‡ Barbara Parrino,§ Alessia Lopergolo,# Paola Barraja,§
Alessandra Montalbano,§ Virginia Spanò,§ Stefania Sbarra,# Valentina Doldi,# Michelandrea De
Cesare,# Girolamo Cirrincione,§ Patrizia Diana*,§ and Nadia Zaffaroni*,#
‡ These authors contributed equally.
§ Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF),
Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
# Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto
Nazionale dei Tumori, Via Venezian 1, 20133 Milano, Italy
KEYWORDS: bisꢀindole alkaloids, CDK1, diffuse malignant peritoneal mesothelioma,
paclitaxel, survivin
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ABSTRACT
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In this study, we describe the synthesis of new nortopsentin analogues, 1Hꢀpyrrolo[2,3ꢀ
b]pyridine derivatives, and their biological effects in experimental models of diffuse malignant
peritoneal mesothelioma (DMPM), a rare and rapidly fatal disease, poorly responsive to
conventional therapies. The three most active compounds, 1f (3ꢀ[2ꢀ(5ꢀfluoroꢀ1ꢀmethylꢀ1Hꢀindolꢀ
3ꢀyl)ꢀ1,3ꢀthiazolꢀ4ꢀyl]ꢀ1Hꢀpyrrolo[2,3ꢀb]pyridine), 3f (3ꢀ[2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ1,3ꢀthiazolꢀ4ꢀyl]ꢀ1ꢀ
methylꢀ1Hꢀpyrrolo[2,3ꢀb]pyridine) and 1l (3ꢀ[2ꢀ(5ꢀfluoroꢀ1ꢀmethylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ1,3ꢀthiazolꢀ4ꢀ
yl]ꢀ1ꢀmethylꢀ1Hꢀpyrrolo[2,3ꢀb] pyridine), which were shown to act as cyclinꢀdependent kinase 1
inhibitors, consistently reduced DMPM cell proliferation and induced a caspaseꢀdependent
apoptotic response, with a concomitant reduction of the expression of the active Thr34ꢀ
phosphorylated form of the antiꢀapoptotic protein survivin. Moreover, the combined treatment of
DMPM cells with 3f derivative and paclitaxel produced a synergistic cytotoxic effect, which was
parallel by an enhanced apoptotic response. In the mouse model, i.p. administration of 1f, 3f, and
1l derivatives was effective, resulting in a significant tumor volume inhibition of DMPM
xenografts (range, 58%ꢀ75%) at wellꢀtolerated doses, and two complete responses were observed
in each treatment group.
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INTRODUCTION
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Diffuse malignant peritoneal mesothelioma (DMPM) is a rare tumor that develops from
mesothelial cells that line the peritoneal cavity and accounts for approximately 10ꢀ15% of all
malignant mesotheliomas.1,2 Therapeutic approaches proposed thus far for the treatment of
DMPM patients (i.e. palliative surgery, radiation therapy and systemic chemotherapy) have not
produced satisfactory results and the median patients survival commonly does not exceed 12
months.3 In the last decade, the introduction of an integrated therapeutic approach combining
aggressive cytoreductive surgery (CRS) with hyperthermic i.p. chemotherapy (HIPEC) has
significantly improved the median survival,4 although approximately 40–60% of patients
experienced recurrence.5 For patients not amenable to curative treatment, prognosis remains very
poor due to lack of valid alternative therapeutic options.6 In addition, little is known about the
mechanism responsible for the reduced responsiveness to the therapy of DMPM. These
considerations emphasize the need to develop novel therapeutic strategies to improve the
outcome of DMPM patients.
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In recent years, a considerable effort has been made to identify and develop new molecules based
on natural compound scaffolds as possible novel cancer therapeutic agents.7ꢀ16 In particular, bisꢀ
indole alkaloids represent one of the most important class of pharmaceutically interesting
compounds due to their potent biological activities such as antiinflammatory, antimicrobial,
antiviral and antitumor.17ꢀ20 Such a class of compounds is characterized by two indole units
connected, through their 3 position, by a spacer whose structure can vary from carbocycles or
heteocycles differently sized to linear chains. Asterriquinone, whose symmetrical structure
involves a quinone moiety, was isolated from Aspergillus fungi and showed in vivo activity
against Ehrlich carcinoma, ascites hepatoma AH13 and mouse P388 leukemia (Chart 1).21
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Dragmacidins, isolated from a large number of deep water sponges including Dragmacidon,
Halicortex, Spongosorites, Hexadella and the tunicate Didemnum candidum, present different
spacers and different related activities. Dragmacidin and dragmacidins AꢀC, bearing the
saturated six membered heterocyclic link piperazine, showed modest cytotoxic activity.22
Conversely, dragmacidin D, which has a pyrazinone moiety as central core, exhibited several
biological properties such as inhibition of serineꢀthreonine protein phosphatases, antiviral,
antimicrobial and anticancer activities.23 Coscinamides, isolated from deep marine sponge
Coscinoderma sp. bearing a linear chain as a spacer, showed HIV inhibitory activity.24
Nortopsentins AꢀC represent the unique group of bisꢀindolyl alkaloids bearing a five membered
ring as spacer, imidazole. They were isolated from Spongosorites ruetzleri, and showed in vitro
cytotoxicity against P388 cells (IC50, 4.5ꢀ20.7 ꢁM). Substitution of the indole nitrogen with a
methyl group led to derivatives which showed a significant improvement in cytotoxicity against
P388 cells relative to that of the parent compounds (IC50, 0.8ꢀ2.1 ꢁM).25ꢀ27 The interesting
biological activities, of the bisꢀindolyl marine alkaloids attracted the attention of many
researchers who spent much effort in the discovery of new biologically active compounds
analogues of these important lead compounds. In this context, dragmacidin analogues bearing as
central heterocyclic moiety azine and diazine systems such as pyridine, pyrimidine, pyrazine and
pyrazinone were synthesized. Such analogues showed strong inhibitory activity against a wide
range of human tumor cell lines.28ꢀ31 Many reports describe the synthesis and the evaluation of
the antiproliferative activity of analogues of nortopsentins in which the imidazole ring of the
natural compound was replaced by many other five membered heterocycles such as bisꢀindolylꢀ
thiophenes,32 pyrazoles,33 furans,34 isoxazoles,34 pyrroles35 and 1,2,4ꢀthiadiazoles.36 Many of
these analogues showed remarkable antiproliferative activity in cancer cells, often reaching IC50
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values at subꢀmicromolar level. In addition, the structural manipulation of the natural
nortopsentins ꢀthat involved, beside the heterocyclic spacer, one or both indole unitsꢀ led to the
synthesis of 3ꢀ[(2ꢀindolyl)ꢀ5ꢀphenyl]pyridines and phenylthiazolylꢀ7ꢀazaindoles. Both these
series of compounds showed antiproliferative activity against a wide range of human tumor cell
lines at micromolar to subꢀmicromolar concentrations and were able to inhibit the activity of the
cyclinꢀdependent kinase 1 (CDK1) with IC50 values less than 1 ꢁM.37,38
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Among nortopsentin analogues bearing fiveꢀmembered heterocycles as spacer were also reported
bis indolylꢀthiazoles.28.39 Both papers dealt with the same series of eleven compounds describing
the synthesis and, as biological studies, only the antiproliferative tests with IC50 values in the
micromolar range.
In this paper we report the synthesis of a large number of substituted 3[2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ1,3ꢀ
thiazolꢀ4ꢀyl]ꢀ1Hꢀpyrrolo[2,3ꢀb]pyridines of type 1ꢀ3, nortopsentin analogues in which the spacer
is constituted by the thiazole ring and one of the indole units is replaced by a 7ꢀazaindole moiety
(CHART 1). Actually, our compounds are similar to the thiazole analogues with the exception of
the 7ꢀazaꢀsubstitution in one of the indole moieties. However, considering the already mentioned
good affinity for CDK1 shown by phenylthiazolylꢀ7ꢀazaindoles,38 we thought that the title
compounds might increase the interaction with kinases, their putative target.
The cytotoxic activity of all analogues was initially screened in a large panel of human cancer
cell lines. In addition, we extensively investigated the effects induced by the three most active
compounds, 1f, 3f and 1l, which were shown to act as CDK1 inhibitors, in DMPM experimental
models and demonstrated their ability to significantly impair tumor cell proliferation and growth
into athymic nude mice.
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CHART 1. Bis-Indolyl Alkaloids and analogues
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H
N
R1
N
Br
R3
N
O
O
HO
N
N
R
Br
N
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OH
R2
H
Dragmacidin
R=H; R1=Me; R2=Br; R3=OH
Dragmacidin A R=R2=R3=H; R1=Me
Dragmacidin B R=R1=Me; R2=R3=H
Dragmacidin C R=R1=R2=R3=H
Asterriquinone
H2N
N
+
N
H
N
H
H
N
O
H
Br
N
R
O
N
H
N
H
R1
N
H
O
_
N
CF3CO2
HO
H
Coscinamide A R=Br; R1=H
Coscinamide B R=R1=H
Coscinamide C R=Br; R1=OH
Dragmacidin D
R
R3
Y Z
X
R
S
N
N
N
R1
R2
N
N
N
Nortopsentin A X=N; Y=CH; Z=NH; R1=R2=H; R=R3=Br
Nortopsentin B X=N; Y=CH; Z=NH; R=R1=R2=H; R3=Br
Nortopsentin C X=N; Y=CH; Z=NH; R1=R2=R3=H; R=Br
Thiazoles X=N; Y=CH; Z=S; R1 or R2=H,Me; R or R3=H,OMe,Br
Thiophenes X=S; Y=Z=CH; R=R3=H,Me,OMe,Cl,Br; R1=R2=H,Me,SO2Ph
Pyrazoles X=CH; Y=N; Z=NH,NMe; R=R3=H,Me,OMe,Cl,Br;
R1=R2=Me
Furans X=O; Y=Z=CH; R=R3=H,Me,OMe; R1=R2=Me
[1,2]Oxazoles X=CH; Y=N; Z=O; R=R3=H,Me,OMe,Cl,Br;
R1=R2=Me
R1
R2
1ꢀ3
Pyrroles X=NH; Y=Z=CH; R1=R2=Me; R=R3= H,Me,OMe,Cl,Br
1,2,4ꢀThiadiazoles X=Z=N; Y=S; Ror R3=H,OMe,NO2,Br;
R1or R2=H
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RESULTS AND DISCUSSION
CHEMISTRY
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3ꢀ[2ꢀ(1HꢀIndolꢀ3ꢀyl)ꢀ1,3ꢀthiazolꢀ4ꢀyl]ꢀ1Hꢀpyrrolo[2,3ꢀb]pyridines 1aꢀn, 2aꢀj and 3aꢀj (Table
1) were conveniently prepared by Hantzsch reaction (Scheme 1) between thioamides 10f, 11aꢀf
and 12aꢀe and αꢀbromoacetyl compounds 14a,b.
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Scheme 1. Synthesis of substituted 3[2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ1,3ꢀthiazolꢀ4ꢀyl]ꢀ1Hꢀpyrrolo[2,3ꢀ
b]pyridines 1aꢀn, 2aꢀj and 3aꢀj.
S
O
R
R
NH2
NH2
R
iii
iv
N
N
N
R1
R1
R1
10f
R1 = H
7f
R1 = H
4aꢀf R1 = H
5aꢀf R1 = Me
6aꢀe R1 = Boc
i
11aꢀf R1 = Me
12aꢀe R1 = Boc
8aꢀf R1 = Me
9aꢀe R1 = Boc
ii
R
S
vii
N
a R = H, b R = Me, c R = OMe, d R = Cl, e R = Br, f R = F
N
N
N
R1
R2
1aꢀn
2aꢀj
3aꢀj
viii
O
Br
vi
N
N
N
N
R2
R2
13a R2 = H
13b R2 = Me
14a R2 = H
14b R3 = Me
v
Reagents and conditions: for compounds 4bꢀf: (i) (a) tꢀBuOK, toluene, TDAꢀ1, rt, 1ꢀ24 h; (b)
MeI, rt, 0.5ꢀ1 h, 96ꢀ98%; (ii) (Boc)2O, TEA, THF, reflux, 24ꢀ48 h, 90ꢀ100%; (iii) (a) CSI,
MeCN, 0°Cꢀrt, then rt 0.5ꢀ2 h or reflux 15 min; (b) 10% KOH, aq. acetone, 40ꢀ70%; (iv)
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Lawesson’s reagent, toluene or benzene, reflux, 0.5ꢀ24 h, 90ꢀ98%; (v) (a) tꢀBuOK, benzene,
TDAꢀ1, rt, 3 h; (b) CH3I, rt, 1 h, 96%; (vi) AlCl3, CH2Cl2, BrCOCH2Br, reflux, 40 min, 80% for
14a and 92% for 14b; (vii) EtOH, reflux, 30 min, 53ꢀ95%; (viii) (a) TFA, CH2Cl2, reflux, 24 h¸
(b) aqueous NaHCO3, 54ꢀ99%.
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The key intermediates of type 10f, 11aꢀf and 12aꢀe were synthesized from the corresponding
carboxamides 7f, 8aꢀf and 9aꢀe easily prepared (40ꢀ70%) by reaction of the corresponding
indoles 4f, 5a (commercially availables), 5bꢀf and 6aꢀe with chlorosulfonyl isocyanate (CSI) in
acetonitrile followed by alkaline hydrolysis of the chlorosulfonyl group. The treatment of amides
7ꢀ9 with Lawesson’s reagent under reflux in toluene or benzene gave the desired thioamides 10ꢀ
12 in excellent yields (90ꢀ98%). αꢀBromoacetyl derivatives 14a,b were obtained from the
corresponding indoles 13a,b as previously reported.39 The reaction of thioamides of type 10f,
11aꢀf and 12aꢀe with αꢀbromoacetyl compounds 14a,b in ethanol under reflux gave the desired
3ꢀ[2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ1,3ꢀthiazolꢀ4ꢀyl]ꢀ1Hꢀpyrrolo[2,3ꢀb]pyridines 1aꢀn and 2aꢀj in good to
excellent yields (53ꢀ95%). The subsequent deprotection of Nꢀtertꢀbutylcarboxylate derivatives
2aꢀj using trifluoroacetic acid in dichloromethane under reflux afforded, after neutralization, the
corresponding compounds 3aꢀj in good to excellent yields (54ꢀ99%) (Table 1).
BIOLOGY
1HꢀPyrrolo[2,3ꢀb]pyridine Derivatives Significantly Impair Tumor Cell Growth both in
Vitro and in Vivo. All the synthesized nortopsentin analogues 1aꢀn, 2aꢀj and 3aꢀj were
submitted to the National Cancer Institute (Bethesda MD) and preꢀscreened, at one dose
concentration (10ꢀ5 M), in a panel of 60 cell lines of different tumor types (data not shown).
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Compounds 1a, 1d, 1f, 1g, 1lꢀn, 2f, 3bꢀf were further selected for full evaluation at five
concentration levels (10ꢀ4 – 10ꢀ8 M). The growth inhibition activity of compounds was defined in
terms of pGI50 value (which represents the ꢀlog of the molar concentration of the compound that
inhibits 50% net cell growth, GI50). Data reported in the Supporting Table 1 revealed that all the
nortopsentin analogues assayed were cytotoxic, and that 9 out of 13 compounds showed GI50
values against the total number of cell lines investigated in the micromolar to subꢀmicromolar
range. In spite of the large number of nortopsentin analogues synthesized, the evaluation of the
pGI50 values did not allow good SAR. However, it was possible to obtain some interesting
indications. The most active compound was 3f, bearing a methyl substituent at the pyrrole
nitrogen of the pyrrolo[2,3ꢀb]pyridine moiety (azaindole portion). The introduction of a methyl
group at the indole moiety maintained the good activity although lower (compare 3f and 1g)
whereas the further introduction of a fluorine in position 5 of the indole moiety had no
significant influence on the biological activity of 1g (compare 3f, 1g and 1l); conversely the
change of the fluorine with chlorine in the same position brought about a decrease of the activity
(compare 1f and 1d). Replacement of one of the two methyl groups of 1l with a hydrogen
provoked only a very slight decrease of the activity (compare 1l, 1f and 1n). On the contrary,
removal of both the methyl groups led to the decrease of the activity of almost one magnitude
order (compare 1f and 1m). The other components of the bisꢀdemethyl series, 3bꢀ3e, resulted the
less active compounds.
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The five most active compounds 1f, 3f, 1g, 1l and 1n, which did not show particular selectivity
against any of the tumor subꢀpanels, were further investigated in two additional cell lines, STO
and MesoII, derived from human DMPM, a tumor type not included in the NCI panel. Seventyꢀ
twoꢀhours exposure to increasing concentrations of each compound resulted in a doseꢀdependent
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inhibition of cell proliferation in both cellular models (Figure 1). As indicated by the drug
concentrations required to inhibit growth by 50% (IC50 values which ranged from 0.33 to 0.61
ꢁM), compounds 1f, 3f, 1g, 1l and 1n exhibited a comparable activity in STO cells (Table 2). By
contrast, a variable growth inhibitory effect was induced by the different compounds in MesoII
cells (IC50 values ranging from 4.11 to 25.12 ꢁM) (Table 2). In addition, while compounds 1f,
3f, and 1l did not interfere with the proliferation of normal cells, compounds 1g and 1n displayed
an important inhibitory effect on the growth of normal cells (Table 2).
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The antitumor activity of 1f, 3f, and 1l derivatives was then evaluated on STO cells
xenotransplanted in athymic nude mice. As shown in Figure 2, the treatment with the different
compounds resulted in marked tumor growth inhibition. Specifically, at the end of the
experiment, a statistically significant tumor volume inhibition (TVI) compared to control (73%,
75% and 58%, for 1f, 3f, and 1l derivatives, respectively) was observed (Table 3). In addition,
two complete responses were observed in each treatment group (Table 3). Moreover, the
compounds were well tolerated without any appreciable sign of toxicity (Table 3).
1HꢀPyrrolo[2,3ꢀb]Pyridine Derivatives Activity is Mediated by the Inhibition of CDK1
Activity. Since different nortopsentin analogues (i.e. 3ꢀ[(2ꢀindolyl)ꢀ5ꢀphenyl]pyridines and
phenylꢀthiazolylꢀ7ꢀazaindoles) exerted the cellular effects mainly through the inhibition of kinase
activity,38,39 we verified whether compounds 1f, 3f, and 1l could inhibit the in vitro catalytic
activity of several protein kinases (i.e. CDK1, CDK5, EGFR, FGFR1, RET, MET, KIT, JAK2,
PKCA, PKCB, CHK1, MAPK12, SGK, PKA, GSK3α and GSK3β). Nortopsentin derivatives 1f,
3f, and 1l markedly inhibited CDK1 activity, with IC50 values (0.89±0.07, 0.75±0.03 and
0.86±0.04 ꢁM, respectively) (Table 4) comparable to those reported for two wellꢀknown CDK1
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inhibitors, roscovitine and purvalanol A.39 In addition, our compounds were able to inhibit
GSK3β but only at very high concentrations (> 30 ꢁM) (Table 4), whereas they failed to
appreciably modify the activity of other protein kinases (IC50 values > 50 ꢁM) (data not shown).
To confirm the ability of the three selected derivatives to inhibit CDK1 activity in living cells,
we assessed the phosphorylation status of histone H1, a specific substrate for CDK1, in STO
cells after exposure to 1f, 3f, and 1l. A significant and timeꢀdependent reduction of kinase
activity was observed for all the compounds (Figure 3).
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Derivatives 1f, 3f, and 1l Impair CellꢀCycle Phase Distribution and Induce Apoptosis by
Inhibiting Survivin Activation. Since CDK1/Cyclin B complex is required for G2 to M
transition,40 we assessed the consequence of nortopsentin derivative treatment on cell cycle
progression. Flow cytometry profiles of nuclear DNA content revealed that the treatment of STO
and MesoII cells with 1f, 3f, and 1l (at specific IC50 of each cell line) was able to induce an
accumulation of cells in the G2/M phase (33.3ꢀ45.6% and 41.9ꢀ54.3% of the overall cell
population, after 48 and 72 h of treatment, respectively), which was parallel by a marked
reduction in the percentage of cells in G1 phase (Figure 4A). Moreover, the percentage of cells
accumulated in the subꢀG1 phase significantly increased, with the maximum peak after 72 h of
treatment (27.6±1.1%, 25.8±1.2% and 26.5±1.1% of the overall cell population after treatment
with 1f, 3f, and 1l in the STO cell line, and 28.6±1.1%, 19.9±1.2% and 27.9±1.1% in the MesoII
cell line, respectively) (Figure 4A), suggesting a drugꢀinduced apoptotic response.
Consistently, drugꢀtreated STO and MesoII cells showed a significant increase in the number of
cells with an apoptotic morphology (in terms of chromatin condensation and DNA
fragmentation) (Figure 4B). Specifically, the presence of spontaneous apoptosis was detected
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only in a small fraction of control cells (0.5±0.1% and 1.2±0.4% in STO and MesoII cell lines,
respectively) but the percentage of apoptotic cells significantly (p<0.01) increased in a timeꢀ
dependent fashion after treatment with the three derivatives (Figure 4B). In addition, a marked
and significant (p<0.02) increase in caspaseꢀ9 and caspaseꢀ3 catalytic activity (determined in
vitro by the hydrolysis of specific fluorogenic substrates) was observed after treatment with
derivatives 1f, 3f, and 1l. In particular, in STO cells exposed for 72h to derivative 1l (IC80) the
catalytic activity of caspaseꢀ9 and caspaseꢀ3 was 12ꢀ and 10ꢀfold higher, respectively, than that
observed in control samples (Figure 4C). Similarly, an 11.9ꢀ and 7.8ꢀfold increase in caspaseꢀ9
and caspaseꢀ3 catalytic activity, respectively, was also observed in MesoII cells (Figure 4C).
We previously reported that one of the main events associated with DMPM chemoꢀresistance is
the dysregulation of apoptotic pathways, mainly due to the overꢀexpression of members of the
inhibitors of apoptosis protein (IAP) family, such as survivin,41 which has essential roles in cell
division and apoptosis, and is selectively overexpressed in most human tumors.42 Based on these
findings, we investigated whether one possible mechanism underlying the apoptotic response of
DMPM cells after 1Hꢀpyrrolo[2,3ꢀb]pyridine derivatives exposure was the interference whit
survivin activation. In fact, it has been showed that to exert its functions, survivin requires to be
phosphorylated on Thr34 residue by CDK1/Cyclin B1 complex.42,43 Previous studies carried out
by us and other groups also demonstrated that loss of phosphorylation on Thr34, accomplished
through the use of survivin dominant negative mutants or CDK1 inhibitors, destabilizes survivin
favoring accelerated clearance and promotes the induction of apoptosis.42ꢀ45 Consistently, results
from immunoblotting experiments showed that nortopsentin derivativesꢀinduced apoptosis in
DMPM cells occurred as a consequence of a significant and timeꢀdependent reduction of the
levels of the active, Thr34ꢀphosphorylated form of survivin (Figure 5).
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Derivative 3f Increases Paclitaxel Cytotoxicity in DMPM Cells. Since we previously found
that paclitaxel is able to induce survivin phosphorylation on Thr34,46 we investigated whether the
most active derivative (3f) could sensitize DMPM cells to this anticancer agent. To this purpose,
STO and MesoII cells were exposed to the taxane for 24 h, followed by a 72 h exposure to
derivative 3f. At all concentrations, 3f was very effective in cooperating with paclitaxel to inhibit
DMPM cell survival (Figure 6A). In fact, when cells were treated with the drugs in combination,
an inhibition of the cell proliferation greater than that expected by simple additivity of the effects
of the two agents was consistently observed (Figure 6A). Specifically, the synergistic interaction
progressively increased by increasing drug concentrations, as indicated by combination index
(CI) values, which progressively lowered from 0.81 to 0.42 and from 0.94 to 0.57 in STO and
MesoII cells, respectively (Figure 6A). Moreover, the caspaseꢀ3 catalytic activity was
consistently and significantly (p<0.001) higher in cells treated with the combination than in those
exposed to each single agent (Figure 6B).
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CONCLUSIONS
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In this study, we reported the synthesis, the main action mechanism and the preclinical antitumor
activity of novel analogues of the marine alkaloid nortopsentin, 1Hꢀpyrrolo[2,3ꢀb]pyridine
derivatives. Newly synthesized nortopsentin derivatives were active against a broad spectrum of
human cancer cells lines. However, among the compounds that exhibited the greatest
antiproliferative activity in the whole NCI cell line panel, only compounds 1f, 3f and 1l did not
appreciably interfere with the proliferation of human normal fibroblasts, suggesting their
preferential activity against cancer cells. It is worthy of note that these compounds significantly
impaired the growth of two experimental models of DMPM, a rapidly lethal human malignancy,
generally refractory to currently available therapeutic options.2 Since the cellular and molecular
bases responsible for the treatmentꢀresistance of DMPM are still poorly understood, the
identification of new therapeutic targets and strategies to improve the outcome for DMPM
patients are urgently needed.
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As far as the mechanisms underlying the antiproliferative effect of 1f, 3f and 1l derivatives were
concerned, we showed that the compounds potently inhibited CDK1 activity, and consequently
induced a marked timeꢀdependent cell cycle arrest at G2/M compartment, which was paralleled
by an increase in the apoptotic rate, with a concomitant reduction of the phosphorylated form of
the antiꢀapoptotic protein survivin. Moreover, addition of compound 3f to paclitaxelꢀtreated cells
resulted in a synergistic cytotoxic effect, as a consequence of an increased apoptotic response.
Most importantly, our results documented a significant antiꢀtumor activity of the novel
nortopsentin derivatives at wellꢀtolerated doses in a DMPM experimental model.
In conclusion, the preclinical evidence that selected nortopsentin derivatives can be active as
single agents in a DMPM in vivo model, together with the cellular study results showing that
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they impair cell cycle progression by inhibiting CDK1 activity and restore the apoptotic response
by reducing the active form of the antiꢀapoptotic protein survivin, provides a solid rationale for
the clinical development of these agents in this malignancy.
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EXPERIMENTAL SECTION
CHEMISTRY
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General Methods
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All melting point were taken on a BüchiꢀTottoly capillary apparatus and are uncorrected. IR
spectra were determined in bromoform with a Jasco FT/IR 5300 spectrophotometer. 1H and 13C
NMR spectra were measured at 200 and 50.0 MHz, respectively, in DMSOꢀd6 or CDCl3 solution,
using a Bruker Avance II series 200 MHz spectrometer. Compounds 1f, 1l, 1m, 1n were
characterized only by 1H NMR spectra, for their poor solubility the 13C spectra were not
performed. Column chromatography was performed with Merk silica gel 230ꢀ400 mesh ASTM
or with Büchi Sepacor chromatography module (prepacked cartridge system). Elemental
analyses (C, H, N) were within ± 0.4% of theoretical values and were performed with a VARIO
EL III elemental analyzer. Purity of all the tested compounds was greater than 95%, determined
by HPLC (Agilent 1100 Series).
General procedure for the synthesis of 5ꢀsubstitutedꢀ1ꢀmethylꢀ1Hꢀindoles (5bꢀf). To a cold
solution of indoles 4bꢀf (5 mmol) in anhydrous toluene (50 mL), potassium tꢀbutoxide (0.76 g,
6.8 mmol) and tris[2ꢀ(2ꢀmethoxyethoxy)ethyl]amine (TDAꢀ1) (1 or 2 drops) were added. The
reaction mixture was stirred at room temperature for 1ꢀ24 h and then methyl iodide (0.31 mL, 5
mmol) was added. TLC analysis (dichloromethane/petroleum ether 9/1) revealed that
methylation was complete after 0.5ꢀ1 h. The solvent was evaporated under reduced pressure. The
residue, treated with water, was filtered off and air dried or extracted with dichloromethane (3 x
15 mL), dried (Na2SO4) and evaporated to afford the pure methyl derivatives 5bꢀe35 and 5f (see
supporting information).
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General procedure for the synthesis of 5ꢀsubstitutedꢀtertꢀbutyl 1Hꢀindoleꢀ1ꢀcarboxylates
(6aꢀe). Diꢀtertꢀbutyl dicarbonate (8.73 g, 40 mmol) and triethylamine (3.3 mL, 23.7 mmol) were
added to a solution of appropriate indoles 4aꢀe (20 mmol) in tetrahydrofuran (10 mL). The
reaction mixture was heated under reflux for 24ꢀ48 h. After cooling the solvent was evaporated
under reduced pressure and the residue (derivatives 6a and 6c) was purified by column
chromatography using dichloromethane or dichloromethane /petroleum ether (9/1) as eluent or
recrystallized from ethanol (derivatives 6b, 6d, 6e). For compounds 6a, cꢀe see supporting
information. Derivatives 6b, 6c and 6e were also reported with a different method.48
tertꢀButyl 5ꢀmethylꢀ1Hꢀindoleꢀ1ꢀcarboxylate (6b). Conditions: 48 h at reflux. Oil; yield: 100%;
IR (cmꢀ1) 1732; 1H NMR (200 MHz, DMSOꢀd6) δ: 1.62 (s, 9H, 3xCH3), 2.39 (s, 3H, CH3), 6.62
(d, 1H, J = 3.7 Hz, Hꢀ3), 7.13 (dd, 1H, J = 8.5, 1.5 Hz, Hꢀ6), 7.38 (d, 1H, J = 1.5 Hz, Hꢀ4), 7.63
(d, 1H, J = 3.7 Hz, Hꢀ2), 7.99 (d, 1H, J = 8.5 Hz, Hꢀ7); 13C NMR (50 MHz, DMSOꢀd6) δ: 20.8
(q), 27.5 (3xq), 83.3 (s), 107.1 (d), 114.3 (d), 120.8 (d), 125.4 (d), 125.9 (d), 130.4 (s), 131.5 (s),
132.8 (s), 149.0 (s). Anal. Calcd for C14H17NO2: C, 72.70; H, 7.41; N, 6.06. Found: C, 72.40; H,
7.08; N, 6.30.
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General procedure for the synthesis of 5ꢀsubstitutedꢀ1ꢀmethylꢀ1Hꢀindoleꢀ3ꢀcarboxamides,
5ꢀsubstitutedꢀtertꢀbutyl 3ꢀcarbamoylꢀ1Hꢀindoleꢀ1ꢀcarboxylates and 5ꢀfluoroꢀ1Hꢀindoleꢀ3ꢀ
carboxamide (7f, 8aꢀf and 9aꢀe). To a solution of the appropriate indoles 4f (0.80 g, 5.9 mmol),
5bꢀf or 6aꢀe (16 mmol) in anhydrous acetonitrile (20 mL) were added dropwise at 0°C
chlorosulfonyl isocyanate (CSI) (0.8 mL, 9.1 mmol for compound 4f or 1.4 mL, 16 mmol for
compounds 5bꢀf and 6aꢀe). The reaction mixture was warmed to room temperature and stirred
for 0.5ꢀ2 h (for compounds 5bꢀf and 6aꢀe) or at reflux for 15 min (for compound 4f). A solution
of acetone (16 mL) and water (2 mL) was added and the solution was basified using 10%
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aqueous solution of potassium hydroxide. The mixture was extracted with ethyl acetate (3 x 20
mL), dried (Na2SO4) and the solvent evaporated under reduced pressure. The residue was
purified by column chromatography using ethyl acetate (for compounds 7f and 8aꢀf) or
dichloromethane /ethyl acetate (1/1) (for compounds 9aꢀe) as eluent. For compounds 7f, 8aꢀe
and 9a,cꢀe see supporting information. For compound 8f analytical and spectroscopic data are
previously reported.49
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tertꢀButyl 3ꢀcarbamoylꢀ5ꢀmethylꢀ1Hꢀindoleꢀ1ꢀcarboxylate (9b). Conditions: 2 h after CSI
addition. White solid; yield: 60%; mp: 171°C; IR (cmꢀ1) 3336, 3294, 1720, 1664; 1H NMR (200
MHz, DMSOꢀd6) δ: 1.65 (s, 9H, 3xCH3), 2.41 (s, 3H, CH3), 7.18 (d, 1H, J = 8.7 Hz, Hꢀ6), 7.20
(s, 1H, NH), 7.86 (s, 1H, NH), 7.95 (d, 1H, J = 8.7 Hz, Hꢀ7), 8.01 (s, 1H, Hꢀ4), 8.42 (s, 1H, Hꢀ
2); 13C NMR (50 MHz, DMSOꢀd6) δ: 21.0 (3xq), 27.6 (q), 84.5 (s), 114.2 (d), 114.7 (s), 121.7
(d), 126.0 (d), 128.4 (d), 128.5 (s), 132.3 (s), 133.0 (s), 148.8 (s), 165.1 (s). Anal. Calcd for
C15H18N2O3: C, 65.68; H, 6.61; N, 10.21. Found: C, 65.61; H, 6.97; N, 9.93.
General procedure for the synthesis of 5ꢀsubstitutedꢀ1ꢀmethylꢀ1Hꢀindoleꢀ3ꢀ
carbothioamides, 5ꢀsubstitutedꢀtertꢀbutyl 3ꢀcarbamothioyl ꢀ1Hꢀindoleꢀ1ꢀcarboxylates and
5ꢀfluoroꢀ1Hꢀindoleꢀ3ꢀcarbothioamide (10f, 11aꢀf and 12aꢀe). Lawesson’s reagent (0.38 g, 0.96
mmol for compound 7f or 0.80 g, 2 mmol for compounds 8aꢀf and 9aꢀe) was added to a
solution of appropriate derivatives 7f (0.17 g, 0.95 mmol) or 8aꢀf and 9aꢀe (4 mmol) in
anhydrous toluene or benzene (20 mL). The mixture was heated at reflux under nitrogen
atmosphere for 0.5ꢀ24 h. After cooling the solvent was evaporated under reduced pressure and
the residue was purified by column chromatography using ethyl acetate (for compound 10f),
dichloromethane /ethyl acetate (98/2) (for compounds 11aꢀf) or dichloromethane (for compounds
12aꢀe) as eluent. For compounds 10f, 11aꢀf and 12a,cꢀe see supporting information.
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tertꢀButyl 3ꢀcarbamothioylꢀ5ꢀmethylꢀ1Hꢀindoleꢀ1ꢀcarboxylate (12b). Conditions: benzene as
solvent, 1 h at reflux. Orange solid; yield: 98%; mp: 130°C; IR (cmꢀ1) 3479, 3365, 1737, 1597;
1H NMR (200 MHz, DMSOꢀd6) δ:1.64 (s, 9H, 3xCH3), 2.42 (s, 3H, CH3), 7.20 (d, 1H, J = 8.7
Hz, Hꢀ6), 7.97 (d, 1H, J = 8.7 Hz, Hꢀ7), 8.27 (s, 1H, Hꢀ2), 8.39 (s, 1H, Hꢀ4), 9.31 (s, 1H, SH),
9.52 (s,1H, NH); 13C NMR (50 MHz, DMSOꢀd6) δ: 21.2 (q), 27.6 (3xq), 84.7 (s), 114.3 (d),
120.2 (s), 122.2 (d), 126.1 (d), 126.3 (d), 127.9 (s), 132.3 (s), 133.4 (s), 148.7 (s), 192.9 (s). Anal.
Calcd for C15H18N2O2S: C, 62.04; H, 6.25; N, 9.65. Found: C, 61.80; H, 6.63; N, 9.73.
General procedure for the synthesis of derivatives (1aꢀn and 2aꢀj). A suspension of the
appropriate derivatives 10ꢀ12 (5 mmol) and 14 (5 mmol) in anhydrous ethanol (20 mL) was
heated under reflux for 30 min. The precipitate, obtained after cooling, was filtered off, dried and
crystallized with ethanol to afford derivatives 1aꢀn and 2aꢀj. For compounds 1aꢀn and 2a,cꢀj see
supporting information.
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tertꢀButyl
5ꢀmethylꢀ3ꢀ[4ꢀ(1Hꢀpyrrolo[2,3ꢀb]pyridinꢀ3ꢀyl)ꢀ1,3ꢀthiazolꢀ2ꢀyl]ꢀ1Hꢀindoleꢀ1ꢀ
carboxylate (2b). Yellow solid; yield: 60%, mp; 199ꢀ201°C; IR (cmꢀ1) 3303, 1788; 1H NMR
(200 MHz, DMSOꢀd6) δ: 1.68 (s, 9H, 3xCH3), 2.51 (s, 3H, CH3), 7.31 (dd, 1H, J = 8.8, 1.4 Hz,
Hꢀ6’), 7.41 (dd, 1H, J = 7.9, 5.0 Hz, Hꢀ5’’), 7.99 (s, 1H, Hꢀ2’), 8.06 (d, 1H, J = 8.8 Hz, Hꢀ7’),
8.24ꢀ8.30 (m, 3H, Hꢀ2’’, Hꢀ4’ and Hꢀ5), 8.43 (dd, 1H, J = 5.0, 1.3 Hz, Hꢀ6’’), 8.85 (d, 1H, J = 7.9
13
Hz, Hꢀ4’’), 12.40 (s, 1H, NH); C NMR (50 MHz, DMSOꢀd6) δ: 21.1 (q), 27.6 (3xq), 84.7 (s),
108.4 (s), 109.7 (s), 110.6 (d), 112.1 (d), 114.5 (d), 116.5 (d), 120.9 (d), 124.3 (s), 125.5 (d),
127.0 (d), 127.3 (d), 130.0 (s), 133.2 (s), 135.0 (s), 139.1 (d), 144.1 (s), 148.8 (s), 149.4 (s),
160.2 (s). Anal. Calcd for C24H22N4O2S: C, 66.96; H, 5.15; N, 13.01. Found: C, 66.67; H, 5.41;
N, 13.28.
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General procedure for the synthesis of derivatives (3aꢀj). To a suspension of appropriate
derivative 2aꢀj (0.78 mmol) in dichloromethane (10 mL) trifluoacetic acid (1.1 mL) was added.
The reaction was heated at reflux for 24 h. The mixture was neutralized with saturated aqueous
sodium hydrogen carbonate solution. The solvent was dried (Na2SO4), evaporated under reduced
pressure and the residue recrystallized with ethanol to afford derivatives 3aꢀj. For compounds
3a,cꢀj see supporting information.
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3ꢀ[2ꢀ(5ꢀMethylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ1,3ꢀthiazolꢀ4ꢀyl]ꢀ1Hꢀpyrrolo[2,3ꢀb]pyridine (3b). Green solid;
yield: 95%; mp: 243ꢀ244°C; IR (cmꢀ1) 3303, 3337; 1H NMR (200 MHz, DMSOꢀd6) δ: 2.51 (s,
3H, CH3), 7.10 (d, 1H, J = 8.4 Hz, Hꢀ6’), 7.44 (d, 1H, J = 8.4 Hz, Hꢀ7’), 7.58 (dd, 1H, J = 7.6,
4.6 Hz, Hꢀ5’’), 7.92 (s, 1H, Hꢀ2’), 8.07 (s, 1H, Hꢀ2’’), 8.23ꢀ8.36 (m, 2H, Hꢀ4’ and Hꢀ5) 8.55 (d,
1H, J = 4.6 Hz, Hꢀ6’’), 9.16 (d, 1H, J = 7.6 Hz, Hꢀ4’’), 11.8 (s, 1H, NH), 12.90 (s, 1H, NH); 13C
NMR (50 MHz, DMSOꢀd6) δ: 21.6 (q), 108.7 (d), 109.6 (s), 111.1 (s), 112.1 (d), 116.2 (d), 119.7
(d), 121.0 (s), 124.1 (d), 124.5 (s), 126.8 (d), 127.2 (d), 129.7 (s), 134.9 (d), 135.0 (s), 137.4 (d),
142.1 (s), 147.5 (s), 163.0 (s). Anal. Calcd for C19H14N4S: C, 69.07; H, 4.27; N, 16.96. Found: C,
68.79; H, 4.56; N, 16.69.
BIOLOGY
Drugs. Paclitaxel was purchased from Santa Cruz Biotechnology. The nortopsentin derivatives
were prepared as described above. To obtain a 50 ꢁM stock solution, each compound was
completely dissolved in 1% dimethylsulfoxide (DMSO), stored at ꢀ20°C protected from light,
and then diluted in complete culture medium immediately before use at the appropriate
concentration.
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Cell Culture. Human DMPM cell lines (STO and MesoII) were established from surgical
specimens of patients admitted to Fondazione IRCCS Istituto Nazionale dei Tumori of Milan as
previously described.43 Their origin was authenticated through microsatellite analysis by the
AmpFISTR Identifiler PCR amplification kit (Applied Biosystem, PN4322288). The normal
human lung fibroblast cell line (WI38) was obtained from the American Type Culture Collection
(ATCC). Cells were tested for the absence of Mycoplasma fortnightly and maintained in the
logarithmic growth phase as a monolayer in appropriate culture media supplemented with 10%
heatꢀinactivated fetal bovine serum in a humidified incubator at 37°C with a supply of 5%
CO2/95% air atmosphere.
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Cell Proliferation Assay. After harvesting in the logarithmic growth phase, 4500 cells /50 ꢂl
were plated in 96ꢀwell flatꢀbottomed microtiter plates (EuroClone) for 24 h and then treated with
increasing concentrations of derivatives 1f, 3f, and 1l (0.1–100 ꢁM) for 72 h. Control cells
received vehicle alone (DMSO). In the combination experiments, STO and MesoII cells were
treated with paclitaxel for 24h and then exposed to 3f for additional 72h. All studies were
performed in eight replicates and repeated at least three times independently. At the end of drug
exposure, the antiproliferative potential was determined with the CellTiter 96® AQueous One
Solution Cell Proliferation Assay (MTS, purchased from Promega) according to the
manufacturer’s protocols. Optical density was read at 490 nm on a microplate reader and the
results were expressed as a percentage, relative to DMSOꢀtreated cells. Doseꢀresponse curves
were created and IC50 and IC80 values (i.e., concentrations able to inhibit cell growth by 50% and
80%, respectively) were determined graphically from the curve for each compound.
The method described by Chou&Talalay47 was used to determine the nature of the interaction
between paclitaxel and 3f. Drugs were combined at a constant ratio of paclitaxel and 3f
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concentrations (1:100 and 1:333 in STO and MESOII, respectively). The type of interaction was
assessed in terms of combination index (CI). CI values of <1 or >1 indicated synergy or
antagonism, respectively, whereas a CI value of 1 indicates additivity.
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In Vitro Kinase Assay. Drug effect on protein kinase activity was evaluated using the
OmniaTM® Recombinant Kit (Invitrogen) according to the manufacturer’s protocols. Briefly,
increasing concentrations of 1f, 3f, and 1l (0.05ꢀ50 ꢁM) were mixed to recombinant
CDK1/cyclin B, CDK5/p25, EGFR, FGFR1, RET, MET, KIT, JAK2, PKCA, PKCB, CHK1,
MAPK12, SGK, PKA, GSK3α and GSK3β proteins (Invitrogen) in 1X kinase buffer containing
a peptide substrate, ATP, and dithiothreitol, and the kinase reaction was performed at 30°C for
30min. Fluorescences were measured upon excitation at 360 nm and emission at 485 nm. IC50
values (drug concentration at which enzyme activity is reduced by 50%) were determined
graphically from the curve obtained for each compound.
CDK1 Activity Assay in Cells. Cellular lysates (0.5 mL) were combined with the monoclonal
antibody antiꢀCyclin B1 (Abcam Inc.) in the presence of 100 ꢁL of a mixture of AꢀSepharose
20% (v/v) protein (GE Healthcare) and lysis buffer (10mM TrisꢀHCl pH 7.4, 150mM NaCl, 1%
Triton Xꢀ100, 1mM PMSF, 5ꢁg/mL Aprotinin e 20ꢁg/mL Leupeptin). The samples were
immunoprecipitated for 4 h at 4˚C and then incubated with 3ꢁg of Histone H1 (Upstate
Biotechnology Inc.) for 20 min at 30°C in a specific buffer containing 20mM TrisꢀHCI, pH 7.5,
10mM MgCl2, 5pM cold ATP, and 10ꢁCi of [γ32P] ATP. The reaction was stopped adding an
equal volume of loading buffer with SDS and then analyzed by 12% polyacrylamide gel.
Resolved bands were quantified by densitometric analysis using the ImageꢀQuant software
(Molecular Dynamics).
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Cell Cycle Phase Distribution and Apoptosis Analyses. Both adherent and floating cells were
fixed in 70% EtOH and incubated at 4°C for 30 min in staining solution containing 50ꢁg/mL of
propidium iodide, 50mg/mL of RNase, and 0.05% NonidetꢀP40 in PBS. Samples were analyzed
with a FACSꢀCalibur cytofluorimeter (Becton Dickinson). At least 30000 events were read and
histograms were analyzed using the CellQuest software according to the Modfit model (Becton
Dickinson).
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For apoptosis analysis, an aliquot of propidiumꢀstained cells was scored for nuclear morphology
of apoptosis (chromatin condensation and DNA fragmentation) by fluorescence microscopy. The
percentage of cells with an apoptotic nuclear morphology was determined by scoring at least 500
cells in each sample. In the same cellular samples, the catalytic activity of caspaseꢀ9 and
caspaseꢀ3 was measured as the ability to cleave the specific substrates NꢀacetylꢀLeuꢀGluꢀHisꢀ
Aspꢀ pNA (LEHDꢀpNA) and NꢀacetylꢀAspꢀGluꢀValꢀAspꢀpNA (DEVDꢀpNA) by means of the
APOPCYTO/caspaseꢀ9 and APOPCYTO/caspaseꢀ3 kits (MBL), respectively, according to
manufacturer’s instructions. The hydrolysis of the specific substrates was monitored by
spectrofluorometry with 380ꢀnm excitation and 460ꢀnm emission filters. Results were expressed
as relative fluorescence units (rfu).
Immunoprecipitation and Western Blot Analysis. To assess the phosphorylation status of the
antiꢀapoptotic protein survivin, total protein extract (250ꢁg) was immunoprecipitated with the
antiꢀhuman survivin antibody (Abcam Inc.) for 16 h at 4°C with a mixture of AꢀSepharose 20%
(v/v) protein (GE Healthcare) and lysis buffer. After separation by 15% SDS gel electrophoresis,
samples were transferred to nitrocellulose. The filters were blocked in PBS with 5% skim milk
and incubated overnight with the primary antibody specific for Thr34ꢀphosphorylated survivin
(Abcam Inc.). The filters were then incubated with the secondary antiꢀrabbit peroxidaseꢀlinked
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antibody (GE Healthcare). Bound antibodies were detected using the SuperSignals West PICO
chemiluminescent substrate (Pierce Biotechnology Inc.). The results were quantified by
densitometric analysis using the ImageꢀQuant software (Molecular Dynamics).
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In Vivo Experiments. Female athymic Swiss nude mice (8ꢀ10 weeksꢀold, purchased from
Charles River) were maintained in laminar flow rooms keeping temperature and humidity
constant. Mice had free access to food and water. Experiments were approved by the Ethics
Committee for Animal Experimentation of the Fondazione IRCCS Istituto Nazionale Tumori of
Milan according to institutional guidelines that are in compliance with national and international
laws and policies. Exponentially growing STO cells were subcutaneously injected into mouse
right flank (107cells/flank). Eight mice for each group experimental group were used. All
compounds were dissolved in a mixture of DMSO (10%), Cremophor EL (5%) and saline
solution (85%) and delivered i.p. every day a week for three weeks (qdx4ꢀ5/wx3w) starting from
the day after cell inoculum. Tumor growth was followed by biweekly measurements of tumor
diameters with a Vernier caliper. Tumor volume (TV) was calculated according to the formula:
TV (mm3) = d2xD/2 where d and D are the shortest and the longest diameter, respectively. The
efficacy of the drug treatment was assessed as: i) Tumor volume inhibition percentage (TVI %)
in treated versus control mice, calculated as: TVI% = 100ꢀ(mean TV treated/mean TV control x
100), and ii) Complete regression (CR), i.e. disappearance of the tumor lasting until the end of
experiment. The toxicity of the drug treatment was determined as body weight loss and lethal
toxicity. Deaths occurring in treated mice before the death of the first control mouse were
ascribed to toxic effects.
Statistical Analysis. Statistical evaluation of data was done with twoꢀtailed Student’s t test. Ps <
0.05 were considered statistically significant.
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SUPPORTING INFORMATION
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Table 1 and spectroscopic data (IR, 1H, 13C NMR) and elemental analysis for compounds
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5f, 6a,cꢀe, 7f, 8aꢀe, 9a,cꢀe, 10f, 11aꢀf, 12a,cꢀe, 1aꢀn, 2a,cꢀj and 3a,cꢀj.
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CORRESPONDING AUTHORS
*Phone: +39ꢀ091ꢀ23896815. Eꢀmail: patrizia.diana@unipa.it
*Phone: +39ꢀ02ꢀ23903260. Eꢀmail: nadia.zaffaroni@istitutotumori.mi.it
ACKNOWLEDGMENT
This work was financially supported by Ministero dell’Istruzione dell’Università e della Ricerca
(MIUR).
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