[1,2]Oxazolo[5,4-e]isoindoles as promising tubulin polymerization inhibitors

Accepted Manuscript [1,2]Oxazolo[5,4-e]isoindoles as promising tubulin polymerization inhibitors Virginia Spanò, Marzia Pennati, Barbara Parrino, Anna Carbone, Alessandra Montalbano, Alessia Lopergolo, Valentina Zuco, Denis Cominetti, Patrizia Diana, Girolamo Cirrincione, Nadia Zaffaroni, Paola Barraja PII:

S0223-5234(16)30739-5

DOI:

10.1016/j.ejmech.2016.09.013

Reference:

EJMECH 8879

To appear in:

European Journal of Medicinal Chemistry

Received Date: 20 July 2016 Revised Date:

31 August 2016

Accepted Date: 3 September 2016

Please cite this article as: V. Spanò, M. Pennati, B. Parrino, A. Carbone, A. Montalbano, A. Lopergolo, V. Zuco, D. Cominetti, P. Diana, G. Cirrincione, N. Zaffaroni, P. Barraja, [1,2]Oxazolo[5,4-e]isoindoles as promising tubulin polymerization inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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N OMe MeO

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Tumor Volume (mm3)

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Days after cells injection

ACCEPTED MANUSCRIPT [1,2]OXAZOLO[5,4-e]ISOINDOLES AS PROMISING TUBULIN POLYMERIZATION INHIBITORS Virginia Spanò1*, Marzia Pennati2*, Barbara Parrino1, Anna Carbone1, Alessandra Montalbano1,

Nadia Zaffaroni2, Paola Barraja1§°

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Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF),

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Università di Palermo, 90123 Palermo, Italy; 2

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Alessia Lopergolo2, Valentina Zuco2, Denis Cominetti2, Patrizia Diana1, Girolamo Cirrincione1,

Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine,

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Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milano, Italy.

*

°

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These authors contributed equally to this work

Corresponding author: Paola Barraja, Dipartimento di Scienze e Tecnologie Biologiche

Chimiche e Farmaceutiche (STEBICEF), Università di Palermo, via Archirafi 32, 90123 Palermo,

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Italy; e-mail: [email protected]

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Abstract A series of [1,2]Oxazolo[5,4-e]isoindoles has been synthesized through a versatile and high yielding sequence. All the new structures showed in the 1HNMR spectra, the typical signal in the

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8.34-8.47 ppm attributable to the H-3 of the [1,2]oxazole moiety. Among all derivatives, methoxy benzyl substituents at positions 3 and 4 or/and 5 were very effective in reducing the growth of different tumor cell lines, including diffuse malignant peritoneal mesothelioma (DMPM), an

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uncommon and rapidly malignancy poorly responsive to available therapeutic options. The most active compound 6j was found to impair tubulin polymerization, cause cell cycle arrest at G2/M

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phase and induce apoptosis in DMPM cells, making it as a new lead for the discovery of new potent

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antimitotic drugs.

Keywords: [1,2]oxazolo[5,4-e]isoindoles; α-hydroxyalkyl ketones; Antitubulin agents; Diffuse malignant peritoneal mesothelioma

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1. Introduction Despite the remarkable progress made in recent years, the treatment of cancer still remains the most challenging task for the mankind. In the last decades, both natural and synthetic products have

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appreciably contributed to the development of a large number of anticancer drugs [1-5]. Among these, tubulin-binding molecules represent an important class of antineoplastic agents, with broad activity in both solid and hematologic malignancies [6-8]. Such chemotherapeutic agents block cell

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division at the metaphase/anaphase junction of mitosis by affecting microtubule dynamics and interfering with the function of the mitotic spindle, thus leading to cell death [6-8]. The therapeutic

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potential of anti-microtubule agents (including taxanes, vinca alkaloids, macrolides and peptides) has been extensively exploited in clinical practice [7,8]. However, limitations in the use of these molecules are often related to their short half-life and the frequent incidence of tumor cell resistance (i.e., cellular efflux of the compound, ineffective interaction with the target, alterations in

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microtubules dynamics, and deficient activation of programmed cell death), as well as to the development of severe toxicity [7,9]. Recently, Combretastatin A4 (CA4, Chart 1), a natural product isolated from the bark of the South African bush willow Combretum caffrum, has been

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considered a promising lead compound for the design and synthesis of novel microtubule targeting agents able to overcome the above mentioned limitations [8,10,11]. The IC50 of CA4 against tubulin

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polymerization was found to range from 0.53 to 3.0 µM [12]. Besides its potent cytotoxicity against a broad spectrum of human cancer lines, CA4 shows high potency against MDR positive cancer cell lines. However, CA4 does not show in vivo efficacy, in part, due to its poor pharmacokinetics [13], and for this reason considerable effort has been addressed to the improvement of this issue. One example is the water soluble disodium phosphate of CA4 [14]. Moreover, cis double bond in CA4 has been replaced by several heterocycles, such as imidazole, oxazole, pyrazole, tetrazole or a thiazole [15-19]. In particular, an example of CA4 analogues demonstrating oral in vivo antitumor activity against solid tumor was achieved by incorporation of an N-methyl group into the central

ACCEPTED MANUSCRIPT imidazole ring, and much improved pharmacokinetic profiles. The in vivo studies confirmed the remarkable oral efficacy at the maximum tolerated doses (MTD) (30 mg/kg/day), with strong reduction in mean tumor mass in the animals studied [18]. Naphtylcombretastin and its analogues incorporating the isoxazole moiety displayed potent cytotoxic effect and inhibition of tubulin

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polymerization (Chart 1) [20]. In particular, 5-(naphthalen-2-yl)-4-(TMP)-1,2-oxazole (X = O, Y = CH) and 4-(naphthalen-2-yl)-5-(TMPl)-1,2-oxazole (X = CH, Y = O) (Chart 1) showed the same inhibitory potency of naphtylcombretastin and in the same order of magnitude of CA4. Another

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significative example is the indole-based oxazoline, A-289099, which was the S-isomer among two enantiomers, demonstrating as a potent and orally active antimitotic agent against various cancer

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cell lines including those that express the MDR phenotype [21].

A-289099 decreased the

proliferation of a variety of cells with EC50 values ranging from 5.1 to 12.8 nM, and studies on the mechanism of action indicated that A-289099 was able to depolymerize microtubules in human HCT-15 colon carcinoma cells at a concentration of 44 nM. In competition-binding assays, A-

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298099 competed with [3H]colchicine for binding to tubulin (Ki = 0.65 µM). Molecular modeling studies indicated that the structure of A289099 was superimposable to that of combretastatin A4, having the trimethoxylphenyl moiety as a common pharmacophore and the 2,5-disubstituted

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oxazoline mimicking the cis-double bond, with a binding constant at the colchicine site comparable to colchicine at 0.63 µM but 3-fold higher than that of combretastatin A4. The pharmacokinetic

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properties of A-289099 in four different species (mouse, rat, dog and monkey) indicated good oral bioavailabilities ranging from 6.5 to 18.6%. Moreover, in vivo studies against M5076 murine ovarian sarcoma in mice gave remarkable results, comparable to other antimitotic drugs such as E7010, a potent an orally active sulfonamide antitumor agent [22]. In fact, when administrated at the maximum tolerated dose (100 mg/kg/day), A-289099 achieved 206% increase in life span and an impressive 28 day delay to 1 g tumor volume in mice with M5076 murine ovarian sarcoma, copared to 27 of E7010. Chart 1. Structures of tubulin polymerization inhibitors.

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MeO

MeO

MeO

MeO OMe

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OR OMe

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CA4 CA4P

Naphtylcombretastatin

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Y N X

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N

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OMe

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R=H R = PO3Na2

TMP = Trimethoxyphenyl 5-(naphthalen-2-yl)-4-(TMP)-1,2-oxazole (X = O, Y = CH) 4-(naphthalen-2-yl)-5-(TMPl)-1,2-oxazole (X = CH, Y = O)

A-289099

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Since many years, we have been involved in studies dealing with nitrogen heterocycles which are widely represented in natural products and potentially active drugs [23-29]. Among them, several indole derivatives have been described as potent tubulin polymerization inhibitors, thus

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highlighting the indole scaffold as valuable pharmacophore [30-33].. However, only few examples

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have been reported in the literature about isoindole derivatives belonging to this class of anticancer agent [28,34, 35].

Isoxazoles or [1,2]oxazoles represent the core structure of many drug candidates, thus representing an attractive scaffold in medicinal chemistry continuously facing the challenge of designing new and leading drugs [36]. In particular, compounds containing the isoxazole moiety have been reported as pharmaceutically interesting agents because of their biological activities (i.e., antitumor, antiinflammatory, antidepressant, and antiviral) [37-43], and some of them were used for the treatment of pathologies, such as cystic fibrosis [44], neurological disorders [45] and tuberculosis [46].

ACCEPTED MANUSCRIPT Based on the evidence that small molecule therapeutic drugs are generally highly specific, exhibit best efficacy and mostly contain heterocyclic scaffolds, we have devoted our efforts to develop small molecules with antitumor properties [47-53]. Among these, we have studied the synthesis and biological properties of tricyclic systems of type 1-3 (Figure 1) based on the

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isoindole scaffold [35,54-58]. In addition, due to the promising antitumor properties of [1,2]oxazole derivatives, we have already investigated two classes of pyrazole- and pyrrole-fused systems incorporating the [1,2]oxazole unit of type 4 [58] and 5 [59]. In particular, two derivatives

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belonging to the latter class of compounds showed potent antiproliferative activity against the NCI human tumor cell lines panel at nanomolar concentrations. Starting from these promising results

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and in the search for novel promising antimitotic agents, here we report the synthesis and in vitro antitumor activity of [1,2]oxazolo[5,4-e]isoindoles 6 as positional isomers of compounds 5.

Results and Discussion

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2.1 Chemistry

Thus far, only two reports on the title ring system, have been reported describing the same synthetic approach to the tricyclic system with limited yields and poor possibility of obtaining

obtain

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derivatives with a wide substitution pattern [60,61]. The synthetic strategy optimized by us to [1,2]oxazolo[5,4-e]isoindoles

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outlined

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Scheme

1.

We

started

from

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tetrahydroisoindoles 7a-c, previously reported by us [35,54], which were ideal building blocks for our purpose considering that the α position to the carbonyl is suitable for the introduction of a second electrophilic centre, thus allowing cyclization with dinucleofiles.

Scheme 1. Synthesis of [1,2]oxazolo[5,4-e]isoindoles 6a-r

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R2 NH

O

O

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a

NR

R1

b

HO

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c

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3

R2 7

NR 4

9a-r

8a-r

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7a-c

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5

6

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6a-r (Table 1)

a R= Me, R1= R2= H; b R= Bn, R1= R2= H; c R= 4-MeBn, R1= R2= H; d R= 4-OMeBn, R1= R2= H; e R= 3-OMeBn, R1= R2= H; f R= 2-OMeBn, R1= R2= H; g R= 2,3(OMe)2Bn, R1= R2= H; h R= 2,5-(OMe)2Bn, R1= R2= H; i R= 3,4-(OMe)2Bn, R1= R2= H; j R= 3,5-(OMe)2Bn, R1= R2= H; k R= 3,4,5-(OMe)3Bn, R1= R2= H; l R= Ph, R1= R2= H; m R= 4-OMePh, R1= R2= H; n R= R2= Me, R1= COOEt; o R= Bn, R1= COOEt, R2= Me; p R= 4-OMeBn, R1= COOEt, R2= Me; q R= R2= Me, R1= H; r R= Bn, R1= H, R2= Me

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a R1= R2= H b R1= COOEt, R2= Me c R1 = H, R2= Me

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Reagents and conditions: (a) for 8a-d,n-r,[35,54] for 8e-k NaH, DMF, 0 °C to rt, 1 h, then benzylchlorides at 0 °C to rt, 1-24 h, 60-79%; for 8l,[24,43] 8m K2CO3, N-methylpyrrolidone, N2,

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rt, 1 h, then Cu(I)Br, rt, 1 h, then 4-OMePhI, reflux, 3 h, 60%; (b) t-BuOK, toluene, N2, 0 °C to rt, 3 h, then HCOOEt, 0 °C to rt, 24 h, for 9a-d,l,n-r,[35,54] 9e-k,m 63–97%; (c) NH2OH•HCl, ethanol, reflux, 50 min, 56–78%.

Functionalization of the nitrogen atom was achieved by nucleophilic reactions with the proper

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alkyl or aralkyl halides in dimethylformamide to give N-substituted ketones 8a-d,n-r [35,54] and 8e-k (60-79%). Instead, the N-phenyl derivatives 8l [35,54] and 8m (60%) were obtained by a modified Ulmann cross-coupling reaction in the presence of potassium carbonate as the base,

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copper(I)bromide and iodobenzene [35] or iodoanisole. Subsequent formylation with ethyl formate in the presence of potassium t-buthoxyde gave the corresponding α-hydroxymethylidene ketones

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9a-d,l,n-r [35,54] and 9e-k,m (63-97%), which were in turn subjected to annelation of the [1,2]oxazole moiety using hydroxylamine hydrochloride as dinucleofile in refluxing ethanol yielding the corresponding [1,2]oxazole[5,4-e]isoindoles 6a-r in 56–78% yields (Table 1).

2.2 Biology 2.2.1 [1,2]oxazole[5,4-e]isoindoles impair tumor cell growth The cytotoxic activity of new [1,2]oxazole[5,4-e]isoindole derivatives was examined in a panel of human tumor cell lines of different histological origin, including melanoma (JR8 and M14),

ACCEPTED MANUSCRIPT breast cancer (MCF-7 and MDA-MB-231), castration-resistant prostate carcinoma (PC3 and DU145) and diffuse malignant peritoneal mesothelioma (DMPM; STO and MP8). Cells were exposed to increasing concentrations (0.01 to 100 µM) of each derivative for 72 h, and the effect on cell proliferation was determined by MTS assay. Results indicated that only [1,2]oxazolo[5,4-

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e]isoindoles unsubstituted at positions 6 and 8 (R1= R2= H) of the pyrrole moiety were active at micromolar/submicromolar level in all tested human cell lines with the exception of 6c and 6g. Moreover, within this series two compounds (6d and 6h) did not reduce the growth of DMPM cells

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(Table 2).

Although a structure-activity relationship of general application is not easy, some observations

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can be done. The presence of a benzyl or substituted benzyl groups at the pyrrole nitrogen is crucial in conferring good activity to [1,2]oxazolo derivatives (Table 2). By contrast, the presence of a methyl (6a), phenyl (6l) or 4-methoxyphenyl group (6m) significantly reduced their activity. Moreover, among the N-benzyl substituted derivatives, the presence of a methoxy substitution

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generally seems to be relevant. In fact, the most potent compounds are 6e, 6i, 6j and 6k, bearing a 3-methoxy, 3,4-dimethoxy, 3,5-dimethoxy and 3,4,5-trimethoxy benzyl substitution, respectively. The presence of a methoxy group in the position 2 (6f, 6g and 6h) or its removal (6b) significantly

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decreased the activity, whereas the replacement of the 4-methoxy substituent with a 4-methyl in the benzyl moiety (6c) led to the inactivation of the compound.

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Interestingly, the most active compound 6j showed a remarkable antiproliferative activity against DMPM, a highly chemoresistant tumor type [62,63], without affecting the growth of both normal human lung fibroblast (WI38) and adult human breast (MCF10A) cell lines (IC50 >100 µM). The antitumor activity of 6j was also evaluated on STO cells following subcutaneous xenotransplantation into athymic nude mice. As shown in Figure 2B, the intraperitoneal administration of the compound resulted in a tumor growth delay, with a maximum tumor volume inhibition of 51%. Moreover, the compound was well tolerated without any appreciable sign of toxicity.

ACCEPTED MANUSCRIPT 2.2.2 6j derivative inhibits tubulin polymerization, promotes cell cycle arrest and induces apoptosis Since previous evidence indicated that derivatives structurally related to our [1,2]oxazolo[5,4e]isoindoles interfere with tubulin polymerization [64], we investigated whether 6j affected tubulin dynamics in DMPM models. Western blot results revealed that 6j markedly reduced the

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polymerized compared to soluble fraction of tubulin in both STO and MP8 cells, with a mechanism superimposable to that observed following exposure to vinorelbine but opposite to that induced by taxol (Figure 3).

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The exposure of DMPM cells to 6j also resulted in a marked alteration of the distribution of the cells in the different phases of the cell cycle (Figure 4A), as assessed by flow cytometric analysis.

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Specifically, the treatment of STO and MP8 cells with 6j (at specific IC50 and IC80 of each cell line) induced a time- and dose-dependent accumulation of cells in the G2/M phase, which was paralleled by a marked reduction in the percentage of cells in the G1 phase (Figure 4A). Such an effect was comparable to that observed in the same cell lines following exposure to taxol and vinorelbine

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(Figure 4A). Consistently, fluorescence microscopy analysis of DMPM cells revealed the presence of 35-70% mitotic cells within the overall cell population following 72-h exposure of STO and MP8 cells to equitoxic (IC50) concentrations of 6j, vinorelbine and taxol (Figure 4B). In addition, at the

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molecular level, treatment of STO and MP8 cells with 6j resulted in a significant dose- and timedependent increase in caspase-3 catalytic activity, determined in vitro by the hydrolysis of the

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specific fluorogenic substrate (Figure 5). Based on the evidence that a cell cycle arrest at G2/M phase could be also due to a reduced activity of the cyclin-dependent kinase 1 (CDK1) [65], we investigated the ability of compound 6j to interfere with the enzyme’s in vitro catalytic activity. To this purpose, increasing amounts of 6j were incubated with purified recombinant CDK1/cyclin B complex and its potential kinase inhibitory effect was measured using a fluorimetric assay. Results revealed that the compound failed to modify the activity of the kinase at doses up to 100 µM, whereas Purvalanol A -a well-

ACCEPTED MANUSCRIPT known CDK1 inhibitor [66] used as a positive control- showed activity toward CDK1 at a very low concentration (IC50=0.61±0.03 µM).

3. Conclusions

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In this study, we have reported the synthesis of a new series of [1,2]oxazolo[5,4-e]isoindoles through a convenient, versatile and high yielding sequence by annelation of the [1,2]oxazole ring into the isoindole moiety. The analysis of the structure-activity relationship indicated that the

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existence of a benzyl (or substituted benzyl) groups at the pyrrole nitrogen is crucial in conferring good activity to the compounds. In particular, the presence of N-methoxy groups at positions 3, 4

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and/or 5 allowed to obtain the greatest response. Indeed, in vitro studies indicated that these derivatives significantly impaired the growth of human cancer cells lines of different histological origin, including experimental models of DMPM, a lethal disease lacking of effective therapeutic approaches. Notably, these derivatives did not appreciably interfere with the growth of human cells,

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thus suggesting a preferential activity against malignant cells.

As far as the mechanisms underlying the antiproliferative effect of 6j were concerned, we demonstrated that the compound impaired microtubule assembly during mitosis, in a vinca alkaloid-

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like manner, and consequently induced a dose- and time-dependent cell cycle arrest at the G2/M compartment, which was paralleled by an induction of apoptosis.

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Overall, our results indicated this derivative as a new lead for the discovery of new potent antimitotic drugs, and suggest [1,2]oxazolo[5,4-e]isoindoles as a possible new therapeutic approach for the treatment of DMPM.

4. Experimental Section 4.1. Chemistry 4.1.1. General Methods

ACCEPTED MANUSCRIPT All melting points were taken on a Büchi melting point M-560 apparatus. IR spectra were determined in bromoform with Shimadzu FT/IR 8400S spectrophotometer. 1H and

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spectra were measured at 200 and 50.0 MHz, respectively, in DMSO-d6 or CDCl3 solution using a Bruker Avance II series 200 MHz spectrometer. Column chromatography was performed with

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Merck silica gel (230−400 mesh ASTM) or a 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. The purity of all the tested compounds was

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>95%, determined by HPLC (Agilent 1100 series).

Compounds 7a-c, 8a-d,l,n-r and 9a-d,l,n-r were prepared according to procedures earlier reported

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by us.[24,43] Representative spectra of compounds 6e, 6i, 6j, 6k, 6m were included as supplementary material.

4.1.2 General procedure for the synthesis of 2-substituted-2,5,6,7-tetrahydro-4H-isoindol-4-ones (8).

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To a solution of 7a (1.35 g, 10 mmol) in anhydrous DMF (15 mL) NaH (0.24 g, 10 mmol) was added at 0 °C and the reaction was stirred for 1 h at rt. The suitable benzyl chloride (15 mmol) was added at 0 °C and the reaction mixture was stirred at rt up to completeness (TLC). Then it was

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poured into crushed ice. The aqueous solution was extracted with dichloromethane (3 x 50 mL), and the organic phase was dried over Na2SO4. After evaporation of the solvent at reduced pressure, the

eluent.

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crude residue was purified by chromatography column using dichloromethane/ethyl acetate as

4.1.2.1. 2-(3-Methoxybenzyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (8e). This compound was obtained from reaction of 7a with 3-methoxybenzylchloride after 1 h and 30 min. White solid; yield 69%; Rf=0.41 (CH2Cl2/EtOAc, 9:1); m.p.:120–121 °C; IR cm-1: 1649 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 1.83–1.96 (m, 2H, CH2), 2.29 (t, 2H, J = 6.0 Hz, CH2), 2.57 (t, 2H, J = 6.0 Hz, CH2), 3.73 (s, 3H, CH3), 5.06 (s, 2H, CH2), 6.67 (s, 1H, H-1), 6.82–6.88 (m, 3H, Ar), 7.26 (t, 1H, J = 8.0 Hz, Ar), 7.43 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 21.1 (t), 24.8 (t), 38.8 (t),

ACCEPTED MANUSCRIPT 52.6 (t), 55.0 (q), 112.9 (d), 113.6 (d), 117.3 (d), 119.8 (d), 121.3 (s), 122.1 (d), 126.4 (s), 129.7 (d), 139.3 (s), 159.4 (s), 193.6 (s). Anal calcd for C16H17NO2 (255.31): C 75.27, H 6.71, N 5.49. Found: C 75.43, H 6.90, N 5.15. 4.1.2.2 2-(2-Methoxybenzyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (8f). This compound was

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obtained from reaction of 7a with 2-methoxybenzylchloride after 1 h and 30 min. Yellow oil; yield 69%; Rf=0.44 (CH2Cl2/EtOAc, 9:1); IR cm-1: 1658 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 1.83–1.96 (m, 2H, CH2), 2.29 (t, 2H, J = 6.1 Hz, CH2), 2.57 (t, 2H, J = 6.1 Hz, CH2), 3.83 (s, 3H,

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CH3), 5.06 (s, 2H, CH2), 6.63 (s, 1H, H-1), 6.91 (t, 1H, J = 7.3 Hz, Ar), 7.01–7.08 (m, 2H, Ar), 7.24–7.43 (m, 2H, H-3 and Ar); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 21.6 (t), 25.3 (t), 39.3 (t),

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48.2 (t), 55.9 (q), 111.4 (d), 118.0 (d), 121.0 (d), 121.6 (s), 122.6 (d), 125.9 (s), 126.6 (s), 129.5 (d), 130.0 (d), 157.1 (s), 194.0 (s). Anal calcd for C16H17NO2 (255.31): C 75.27, H 6.71, N 5.49. Found: C 75.40, H 6.82, N 5.36.

4.1.2.3 2-(2,3-Dimethoxybenzyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (8g). This compound was

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obtained from reaction of 7a with 2,3-dimethoxybenzylchloride after 24 h. Yellow oil; yield 79%; Rf=0.34 (CH2Cl2/EtOAc, 9:1); IR cm-1: 1653 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 1.83– 1.95 (m, 2H, CH2), 2.29 (t, 2H, J = 6.0 Hz, CH2), 2.57 (t, 2H, J = 6.0 Hz, CH2), 3.72 (s, 3H, CH3),

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3.80 (s, 3H, CH3), 5.08 (s, 2H, CH2), 6.62 (s, 1H, H-1), 6.69–6.77 (m, 1H, Ar), 7.02–7.05 (m, 2H,

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Ar), 7.34 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 21.6 (t), 25.3 (t), 39.3 (t), 48.2 (t), 56.2 (q), 60.6 (q), 113.3 (d), 117.9 (d), 121.3 (d), 121.5 (s), 122.6 (d), 124.6 (d), 126.7 (s), 131.3 (s), 146.7 (s), 152.8 (s), 194.0 (s). Anal calcd for C17H19NO3 (285.34): C 71.56, H 6.71, N 4.91. Found: C 71.75, H 6.60, N 5.21.

4.1.2.4 2-(2,5-Dimethoxybenzyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (8h). This compound was obtained from reaction of 7a with 2,5-dimethoxybenzylchloride after 24 h. Yellow oil; yield 64%; Rf=0.38 (CH2Cl2/EtOAc, 9:1); IR cm-1: 1651 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 1.83– 1.96 (m, 2H, CH2), 2.29 (t, 2H, J = 6.1 Hz, CH2), 2.57 (t, 2H, J = 6.1 Hz, CH2), 3.67 (s, 3H, CH3), 3.77 (s, 3H, CH3), 5.01 (s, 2H, CH2), 6.64 (s, 1H, H-1), 6.70 (d, 1H, J = 2.9 Hz, Ar), 6.83–6.98 (m,

ACCEPTED MANUSCRIPT 2H, Ar), 7.33 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 21.6 (t), 25.2 (t), 39.3 (t), 48.3 (t), 55.9 (q), 56.4 (q), 112.7 (d), 114.0 (d), 116.4 (d), 118.2 (d), 121.4 (s), 122.7 (d), 126.7 (s), 127.0 (s), 151.4 (s), 153.8 (s), 194.5 (s). Anal calcd for C17H19NO3 (285.34): C 71.56, H 6.71, N 4.91. Found: C 71.30, H 6.60, N 5.13.

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4.1.2.5 2-(3,4-Dimethoxybenzyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (8i). This compound was obtained from reaction of 7a with 3,4-dimethoxybenzylchloride after 1 h. Light brown solid; yield 60%; Rf=0.31 (CH2Cl2/EtOAc, 9:1); m.p.: 102–103 °C; IR cm-1: 1651 (CO); 1H NMR (DMSO-d6,

SC

200 MHz, ppm): δ 1.82–1.95 (m, 2H, CH2), 2.28 (t, 2H, J = 6.1 Hz, CH2), 2.56 (t, 2H, J = 6.1 Hz, CH2), 3.72 (s, 3H, CH3), 3.74 (s, 3H, CH3), 4.99 (s, 2H, CH2), 6.67 (s, 1H, H-1), 6.81–7.01 (m, 3H,

M AN U

Ar), 7.41 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 21.1 (t), 24.8 (t), 38.8 (t), 52.5 (t), 55.4 (q), 55.5 (q), 111.8 (d), 112.0 (d), 117.1 (d), 120.3 (d), 121.1 (s), 121.8 (d), 126.3 (s), 130.0 (s), 148.4 (s), 148.7 (s), 193.5 (s). Anal calcd for C17H19NO3 (285.34): C 71.56, H 6.71, N 4.91. Found: C 71.69, H 6.58, N 5.15.

TE D

4.1.2.6 2-(3,5-Dimethoxybenzyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (8j). This compound was obtained from reaction of 7a with 3,5-dimethoxybenzylchloride after 1 h. White solid; yield 63%; Rf=0.34 (CH2Cl2/EtOAc, 9:1); m.p.: 90–91 °C; IR cm-1: 1649 (CO); 1H NMR (DMSO-d6, 200

EP

MHz, ppm): δ 1.84–1.96 (m, 2H, CH2), 2.29 (t, 2H, J = 6.1 Hz, CH2), 2.58 (t, 2H, J = 6.1 Hz, CH2),

AC C

3.72 (s, 6H, 2 x CH3), 5.01 (s, 2H, CH2), 6.44 (s, 1H, H-1), 6.45–6.47 (m, 2H, H-2’ and H-6’), 6.68 (s, 1H, H-4’), 7.43 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 21.1 (t), 24.8 (t), 38.9 (t), 52.6 (t), 55.2 (2 x q), 99.1 (d), 105.9 (2 x d), 117.4 (d), 121.2 (s), 122.1 (d), 126.3 (s), 139.9 (s), 160.6 (2 x s), 193.6 (s). Anal calcd for C17H19NO3 (285.34): C 71.56, H 6.71, N 4.91. Found: C 71.67, H 6.55, N 5.09. 4.1.2.7 2-(3,4,5-Trimethoxybenzyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (8k). This compound was obtained from reaction of 7a with 3,4,5-trimethoxybenzylchloride after 24 h. White solid; yield 60%; Rf=0.28 (CH2Cl2/EtOAc, 9:1); m.p.: 101–102 °C; IR cm-1: 1657 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 1.83–1.95 (m, 2H, CH2), 2.29 (t, 2H, J = 6.4 Hz, CH2), 2.57 (t, 2H, J = 6.4 Hz,

ACCEPTED MANUSCRIPT CH2), 3.62 (s, 3H, CH3), 3.75 (s, 6H, 2 xC H3), 4.98 (s, 2H, CH2), 6.73 (s, 3H, H-2’, H-6’ and H-1), 7.45 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 21.1 (t), 24.8 (t), 38.8 (t), 52.9 (t), 55.9 (2 x q), 59.9 (q), 105.7 (2 x d), 117.2 (d), 121.1 (d), 121.9 (s), 126.3 (s), 133.1 (s), 137.0 (s), 152.9 (2 x s), 193.6 (s). Anal calcd for C18H21NO4 (315.36): C 68.55, H 6.71, N 4.44. Found C 68.36, H 6.87,

RI PT

N 4.32.

4.1.3 Synthesis of 2-(4-methoxyphenyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (8m).

To a solution of 7a (20 mmol) in N-methyl-2-pyrrolidone (40 mL) anhydrous K2CO3 (2.76 g, 20

SC

mmol) was added under nitrogen atmosphere and the reaction mixture was stirred at rt for 1 h. Then Cu(I)Br (5.74 g, 40 mmol) was added and the reaction mixture was stirred at rt for 1 h and finally 4-

M AN U

iodoanisole (16.38 g, 70 mmol) was added. The reaction mixture was heated under reflux for 3 h. After cooling, HCl (5%, 20 mL) was added and the mixture was stirred for 1 h, then ethyl acetate (20 mL) was added and the mixture was stirred for further 30 min. Then the resulting solution was filtered through celite and washed with ethyl acetate (20 mL). The organic layer was stirred for 1 h

TE D

with ice and water, separated, dried over Na2SO4 and the solvent evaporated at reduced pressure. The crude product was purified by chromatography column using dichloromethane/ethyl acetate (9:1) as eluent. Brown solid; yield 60%; Rf=0.50 (CH2Cl2/EtOAc, 9:1); m.p.: 88–89 °C; IR cm-1:

EP

1655 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 1.91–2.04 (m, 2H, CH2), 2.38 (t, 2H, J = 6.4

AC C

Hz, CH2), 2.68 (t, 2H, J = 6.4 Hz, CH2), 3.79 (s, 3H, CH3), 7.03 (d, 2H, J = 9.0 Hz, H-3’ and H-5’), 7.14 (d, 1H, J = 2.2 Hz, H-1), 7.57 (d, 2H, J = 9.0 Hz, H-2’ and H-6’), 7.77 (d, 1H, J = 2.2 Hz, H3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 21.1 (t), 24.6 (t), 38.9 (t), 55.4 (q), 114.7 (2 x d), 116.1 (d), 119.6 (d), 121.7 (2 x d), 122.7 (s), 127.4 (s), 132.6 (s), 157.8 (s), 193.9 (s). Anal calcd for C15H15NO2 (241.29): C 74.67, H 6.27, N 5.81. Found: C 74.49, H 6.16, N 5.99. 4.1.4 General procedure for the synthesis of 5-(hydroxymethylidene)-2-substituted-2,5,6,7tetrahydro-4H-isoindol-4-ones (9). To a suspension of t-BuOK (3.7 g, 36 mmol) in anhydrous toluene (30 mL), a solution of 8 (12 mmol) in the same solvent (40 mL) was added dropwise under N2 at 0 °C. After 3 h stirring at rt the

ACCEPTED MANUSCRIPT reaction was cooled at 0 °C and a solution of ethyl formate (2.90 mL, 36 mmol) in anhydrous toluene (20 mL) was added and the mixture was kept stirring at rt for 24 h, then the solvent was removed at reduced pressure. The residue was dissolved in water and the solution was washed with diethyl ether. The acqueous solution was then acidified with HCl 6 M and in case of a precipitate

RI PT

formed it was filtered and dried, otherwise the solution was extracted with dichloromethane (3 x 30 mL). The organic phase was dried over Na2SO4 and the solvent evaporated at reduced pressure. The crude product was purified by chromatography column using dichloromethane as eluent.

SC

4.1.4.1 5-(Hydroxymethylidene)-2-(3-methoxybenzyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (9e). This compound was obtained from reaction of 8e. Yellow oil; yield 87%; Rf=0.36 (CH2Cl2); IR cm: 2935 (OH), 1633 (CO); 1H NMR (CDCl3, 200 MHz, ppm): δ 2.47 (t, 2H, J = 6.9 Hz, CH2), 2.63

M AN U

1

(t, 2H, J = 6.9 Hz, CH2), 3.77 (s, 3H, CH3), 4.98 (s, 2H, CH2), 6.42 (s, 1H, H-1), 6.69–6.87 (m, 3H, Ar), 7.22 (s, 1H, H-3), 7.26–7.30 (m, 1H, Ar), 7.48 (s, 1H, CH), 14.35 (bs, 1H, OH);

13

C NMR

(CDCl3, 50 MHz, ppm): δ 21.1 (t), 25.8 (t), 54.0 (t), 55.3 (q), 109.4 (s), 113.3 (d), 113.4 (d), 117.4

TE D

(d), 119.7 (d), 121.0 (s), 122.7 (d), 125.9 (s), 130.0 (d), 137.9 (s), 160.0 (s), 165.5 (d), 186.8 (s). Anal calcd for C17H17NO3 (283.32): C 72.07, H 6.05, N 4.94. Found: C 72.38, H 6.34, N 4.82. 4.1.4.2 5-(Hydroxymethylidene)-2-(2-methoxybenzyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (9f).

: 2937 (OH), 1633 (CO); 1H NMR (CDCl3, 200 MHz, ppm): δ 2.47 (t, 2H, J = 6.7 Hz, CH2), 2.63

AC C

1

EP

This compound was obtained from reaction of 8f. Yellow oil; yield 70%; Rf=0.40 (CH2Cl2); IR cm-

(t, 2H, J = 6.7 Hz, CH2), 3.84 (s, 3H, CH3), 5.02 (s, 2H, CH2), 6.45 (s, 1H, H-1), 6.87–7.03 (m, 3H, Ar), 7.25–7.34 (m, 2H, H-3 and Ar), 7.46 (s, 1H, CH), 14.39 (bs, 1H, OH);

13

C NMR (CDCl3, 50

MHz, ppm): δ 21.1 (t), 25.9 (t), 49.0 (t), 55.4 (q), 109.4 (s), 110.5 (d), 117.5 (d), 120.6 (s), 120.7 (d), 122.9 (d), 124.8 (s), 125.4 (s), 129.2 (d), 129.7 (d), 157.0 (s), 165.0 (d), 187.0 (s). Anal calcd for C17H17NO3 (283.32): C 72.07, H 6.05, N 4.94. Found: C 72.32, H 6.26, N 4.87. 4.1.4.3

2-(2,3-Dimethoxybenzyl)-5-(hydroxymethylidene)-2,5,6,7-tetrahydro-4H-isoindol-4-one

(9g). This compound was obtained from reaction of 8g. Yellow oil; yield 65%; Rf=0.28 (CH2Cl2); IR cm-1: 2935 (OH), 1633 (CO); 1H NMR (CDCl3, 200 MHz, ppm): δ 2.47 (t, 2H, J = 6.6 Hz, CH2),

ACCEPTED MANUSCRIPT 2.62 (t, 2H, J = 6.6 Hz, CH2), 3.75 (s, 3H, CH3), 3.87 (s, 3H, CH3), 5.04 (s, 2H, CH2), 6.46 (s, 1H, H-1), 6.67–6.72 (m, 1H, Ar), 6.88–7.07 (m, 2H, Ar), 7.30 (s, 1H, H-3), 7.47 (s, 1H, CH), 14.39 (bs, 1H, OH);

13

C NMR (CDCl3, 50 MHz, ppm): δ 21.1 (t), 25.9 (t), 49.0 (t), 55.8 (q), 60.6 (q), 109.4

(s), 112.7 (d), 117.4 (d), 120.7 (s), 121.1 (d), 122.7 (d), 124.3 (d), 125.6 (s), 130.1 (s), 146.9 (s),

RI PT

152.8 (s), 165.2 (d), 186.9 (s). Anal calcd for C18H19NO4 (313.35): C 68.99, H 6.11, N 4.47. Found: C 68.67, H 6.33, N 4.35. 4.1.4.4

2-(2,5-Dimethoxybenzyl)-5-(hydroxymethylidene)-2,5,6,7-tetrahydro-4H-isoindol-4-one

SC

(9h). This compound was obtained from reaction of 8h. Yellow oil; yield 72%; Rf=0.38 (CH2Cl2); IR cm-1: 2933 (OH), 1633 (CO); 1H NMR (CDCl3, 200 MHz, ppm): δ 2.47 (t, 2H, J = 6.7 Hz, CH2),

M AN U

2.63 (t, 2H, J = 6.7 Hz, CH2), 3.73 (s, 3H, CH3), 3.80 (s, 3H, CH3), 5.00 (s, 2H, CH2), 6.45 (s, 1H, H-1), 6.59 (s, 1H, Ar), 6.81 (d, 2H, J = 1.6 Hz, Ar), 7.30 (s, 1H, H-3), 7.48 (s, 1H, CH), 14.37 (bs, 1H, OH);

13

C NMR (CDCl3, 50 MHz, ppm): δ 21.1 (t), 25.9 (t), 49.0 (t), 55.7 (q), 55.9 (q), 109.4

(s), 111.5 (d), 113.5 (d), 115.7 (d), 117.5 (d), 120.7 (s), 122.9 (d), 125.5 (s), 125.9 (s), 151.2 (s),

C 68.72, H 6.34, N 4.19.

TE D

153.6 (s), 165.1 (d), 187.0 (s). Anal calcd for C18H19NO4 (313.35): C 68.99, H 6.11, N 4.47. Found:

4.1.4.5 2-(3,4-Dimethoxybenzyl)-5-(hydroxymethylidene)-2,5,6,7-tetrahydro-4H-isoindol-4-one (9i).

: 2933 (OH), 1633 (CO); 1H NMR (CDCl3, 200 MHz, ppm): δ 2.44 (t, 2H, J = 7.4 Hz, CH2), 2.62

AC C

1

EP

This compound was obtained from reaction of 8i. Yellow oil; yield 71%; Rf=0.24 (CH2Cl2); IR cm-

(t, 2H, J = 7.4 Hz, CH2), 3.84 (s, 3H, CH3), 3.87 (s, 3H, CH3), 4.96 (s, 2H, CH2), 6.42 (s, 1H, H-1), 6.69–6.86 (m, 3H, Ar), 7.28 (s, 1H, H-3), 7.48 (s, 1H, CH), 14.36 (bs, 1H, OH); 13C NMR (CDCl3, 50 MHz, ppm): δ 21.1 (t), 25.9 (t), 53.9 (t), 55.8 (q), 55.9 (q), 109.4 (s), 110.8 (d), 111.2 (d), 117.2 (d), 120.3 (d), 120.9 (s), 122.4 (d), 125.9 (s), 128.6 (s), 149.1 (s), 149.3 (s), 165.3 (d), 186.9 (s). Anal calcd for C18H19NO4 (313.35): C 68.99, H 6.11, N 4.47. Found: C 68.71, H 6.40, N 4.12. 4.1.4.6 2-(3,5-Dimethoxybenzyl)-5-(hydroxymethylidene)-2,5,6,7-tetrahydro-4H-isoindol-4-one (9j). This compound was obtained from reaction of 8j. Yellow oil; yield 63%; Rf=0.25 (CH2Cl2); IR cm1

: 2935 (OH), 1637 (CO); 1H NMR (CDCl3, 200 MHz, ppm): δ 2.49 (t, 2H, J = 6.6 Hz, CH2), 2.64

ACCEPTED MANUSCRIPT (t, 2H, J = 6.6 Hz, CH2), 3.76 (s, 6H, 2 x CH3), 4.94 (s, 2H, CH2), 6.31 (s, 2H, Ar), 6.38–6.42 (m, 2H, H-1 and Ar), 7.27 (s, 1H, H-3), 7.49 (s, 1H, CH), 14.36 (bs, 1H, OH);

13

C NMR (CDCl3, 50

MHz, ppm): δ 21.1 (t), 25.8 (t), 54.1 (t), 55.4 (2 x q), 99.7 (d), 105.7 (2 x d), 109.4 (s), 117.4 (d), 121.0 (s), 122.7 (d), 125.9 (s), 138.6 (s), 161.3 (2 x s), 165.5 (d), 186.8 (s). Anal calcd for

4.1.4.7

RI PT

C18H19NO4 (313.35): C 68.99, H 6.11, N 4.47. Found: C 68.77, H 6.34, N 4.15.

5-(Hydroxymethylidene)-2-(3,4,5-trimethoxybenzyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one

(9k). This compound was obtained from reaction of 8k. Brown solid; yield 64%; Rf=0.29 (CH2Cl2);

SC

m.p.: 105–106 °C; IR cm-1: 2931 (OH), 1631 (CO); 1H NMR (CDCl3, 200 MHz, ppm): δ 2.49 (t, 2H, J = 7.4 Hz, CH2), 2.65 (t, 2H, J = 7.4 Hz, CH2), 3.83 (s, 6H, 2 x CH3), 3.84 (s, 3H, CH3), 4.96

M AN U

(s, 2H, CH2), 6.40 (s, 2H, H-2’ and H-6’), 6.43–6.45 (m, 1H, H-1), 7.27–7.29 (m, 1H, H-3), 7.47– 7.50 (s, 1H, CH), 14.35 (s, 1H, OH); 13C NMR (CDCl3, 50 MHz, ppm): δ 21.1 (t), 25.9 (t), 54.3 (t), 56.2 (2 x q), 60.9 (q), 104.7 (2 x d), 109.4 (s), 117.3 (d), 121.0 (s), 122.5 (d), 125.9 (s), 131.7 (s), 137.8 (s), 153.6 (2 x s), 165.3 (d), 187.0 (s). Anal calcd for C19H21NO5 (343.37): C 66.46, H 6.16, N

TE D

4.08. Found: C 66.30, H 5.95, N 4.20.

4.1.4.8 5-(hydroxymethylidene)-2-(4-methoxyphenyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (9m). This compound was obtained from reaction of 8m. Light brown solid; yield 97%; Rf=0.38

EP

(CH2Cl2); m.p.: 126–127 °C; IR cm-1: 2928 (OH), 1635 (CO); 1H NMR (CDCl3, 200 MHz, ppm): δ

AC C

2.54 (t, 2H, J = 6.0 Hz, CH2), 2.73 (t, 2H, J = 6.0 Hz, CH2), 3.84 (s, 3H, CH3), 6.75–6.73 (m, 1H, H-1), 6.96 (d, 2H, J = 9.0 Hz, H-3’ and H-5’), 7.31 (d, 2H, J = 9.0 Hz, H-2’ and H-6’), 7.54–7.59 (m, 2H, H-3 and CH), 14.40 (bs, 1H, OH);

13

C NMR (CDCl3, 50 MHz, ppm): δ 21.0 (t), 25.7 (t),

55.6 (q), 109.4 (s), 114.8 (2 x d), 116.4 (d), 120.6 (d), 122.1 (s), 122.4 (2 x d), 126.4 (s), 133.3 (s), 158.5 (s), 166.4 (d), 186.5 (s). Anal calcd for C16H15NO3 (269.30): C 71.36, H 5.61, N 5.20. Found: C 71.49, H 5.72, N 5.07. 4.1.5 General procedure for the Synthesis of [1,2]oxazolo[5,4-e]isoindoles (6). To a solution of the suitable hydroxymethylideneketones 9 (5.0 mmol) in ethanol (15 mL) hydroxylamine hydrochloride (0.38 g, 5.5 mmol) was added and the reaction mixture was heated

ACCEPTED MANUSCRIPT under reflux for 50 min. After cooling, the solvent was evaporated at reduced pressure. The crude product was purified by chromatography column using dichloromethane as eluent. 4.1.5.1 7-Methyl-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (6a). This compound was obtained from reaction of 9a. Yellow oil; yield 64%; Rf=0.19 (CH2Cl2); 1H NMR (DMSO-d6, 200 MHz,

RI PT

ppm): δ 2.60–2.68 (m, 4H, 2 x CH2), 3.62 (s, 3H, CH3), 6.64 (s, 1H, H-6), 7.08 (s, 1H, H-8), 8.36 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 20.0 (t), 20.5 (t), 36.3 (q), 108.6 (s), 110.3 (s),

5.79, N 16.08. Found: C 69.16, H 5.43, N 16.21.

SC

116.8 (d), 119.7 (d), 120.4 (s), 149.5 (d), 163.1 (s). Anal calcd for C10H10N2O (174.20): C 68.95, H

4.1.5.2 7-Benzyl-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (6b). This compound was obtained

M AN U

from reaction of 9b. White solid; yield 64%; Rf=0.36 (CH2Cl2); m.p.: 88–89 °C; 1H NMR (DMSOd6, 200 MHz, ppm): δ 2.55–2.80 (m, 4H, 2 x CH2), 5.10 (s, 2H, CH2), 6.76 (s, 1H, H-6), 7.26–7.39 (m, 6H, H-8 and Ar), 8.37 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 19.4 (t), 20.0 (t), 52.4 (t), 108.3 (s), 110.0 (s), 115.7 (d), 118.4 (d), 120.2 (s), 127.4 (2 x d), 127.5 (d), 128.6 (2 x d),

TE D

138.4 (s), 149.0 (d), 162.5 (s). Anal calcd for C16H14N2O (250.30): C 76.78, H 5.64, N 11.19. Found: C 76.52, H 5.35, N 11.50.

4.1.5.3 7-(4-Methylbenzyl)-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (6c). This compound was

EP

obtained from reaction of 9c. White solid; yield72%; Rf=0.38 (CH2Cl2); m.p.: 141–142 °C; 1H

AC C

NMR (DMSO-d6, 200 MHz, ppm): δ 2.27 (s, 3H, CH3), 2.63–2.66 (m, 4H, 2 x CH2), 5.03 (s, 2H, CH2), 6.73 (s, 1H, H-6), 7.08 (d, 2H, J = 8.6 Hz, H-3’ and H-5’), 7.20 (d, 2H, J = 8.6 Hz, H-2’ and H-6’), 7.22 (s, 1H, H-8), 8.36 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 19.4 (t), 20.1 (t), 20.6 (q), 52.2 (t), 108.3 (s), 110.0 (s), 115.6 (d), 118.3 (d), 120.1 (s), 127.5 (2 x d), 129.1 (2 x d), 135.3 (s), 136.8 (s), 149.0 (d), 162.5 (s). Anal calcd for C17H16N2O (264.32): C 77.25, H 6.10, N 10.60. Found: C 77.52, H 6.40, N 10.38. 4.1.5.4 7-(4-Methoxybenzyl)-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (6d). This compound was obtained from reaction of 9d. White solid; yield 60%; Rf=0.39 (CH2Cl2); m.p.: 102–103 °C; 1H

ACCEPTED MANUSCRIPT (DMSO-d6, 200 MHz, ppm): δ 2.50–2.64 (m, 4H, 2 x CH2), 3.73 (s, 3H, CH3), 5.00 (s, 2H, CH2), 6.73 (s, 1H, H-6), 6.91 (d, 2H, J = 8.5 Hz, H-3’ and H-5’), 7.21 (s, 1H, H-8), 7.25 (d, 2H, J = 8.5 Hz, H-2’ and H-6’), 8.36 (s, 1H, H-3);

C NMR (DMSO-d6, 50 MHz, ppm): δ 19.4 (t), 20.1 (t),

13

51.9 (t), 55.0 (q), 108.3 (s), 110.0 (s), 113.9 (2 x d), 115.4 (d), 118.2 (d), 120.1 (s), 129.0 (2 x d),

RI PT

130.3 (s), 149.0 (d), 158.7 (s), 162.5 (s). Anal calcd for C17H16N2O2 (280.32): C 72.84, H 5.75, N 9.99. Found: C 72.57, H 5.93, N 9.69.

4.1.5.5 7-(3-Methoxybenzyl)-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (6e). This compound

SC

was obtained from reaction of 9e. White solid; yield 78%; Rf=0.46 (CH2Cl2); m.p.: 83–84 °C; 1H (DMSO-d6, 200 MHz, ppm): δ 2.64–2.67 (m, 4H, 2 x CH2), 3.73 (s, 3H, CH3), 5.05 (s, 2H, CH2),

M AN U

6.77 (s, 1H, H-6), 6.81–6.87 (m, 3H, Ar), 7.22–7.30 (m, 2H, H-8 and Ar), 8.37 (s, 1H, H-3);

13

C

NMR (DMSO-d6, 50 MHz, ppm): δ 19.4 (t), 20.0 (t), 52.3 (t), 55.0 (q), 108.3 (s), 110.0 (s), 112.7 (d), 113.3 (d), 115.7 (d), 118.4 (d), 119.6 (d), 120.1 (s), 129.7 (d), 139.9 (s), 149.0 (d), 159.4 (s), 162.5 (s). Anal calcd for C17H16N2O2 (280.32): C 72.84, H 5.75, N 9.99. Found: C 72.69, H 5.87, N

TE D

9.71.

4.1.5.6 7-(2-Methoxybenzyl)-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (6f). This compound was obtained from reaction of 9f. White solid; yield 75%; Rf=0.39 (CH2Cl2); m.p.: 126–127 °C; 1H

EP

NMR (DMSO-d6, 200 MHz, ppm): δ 2.64–2.67 (m, 4H, 2 x CH2), 3.84 (s, 3H, CH3), 5.05 (s, 2H,

AC C

CH2), 6.73 (s, 1H, H-6), 6.87–7.05 (m, 3H, Ar), 7.19 (s, 1H, H-8), 7.25–7.34 (m, 1H, Ar), 8.37 (s, 1H, H-3);

13

C NMR (DMSO-d6, 50 MHz, ppm): δ 19.4 (t), 20.1 (t), 47.5 (t), 55.4 (q), 108.3 (s),

109.8 (s), 110.8 (d), 115.8 (d), 118.6 (d), 119.9 (s), 120.4 (d), 126.1 (s), 128.6 (d), 129.2 (d), 149.1 (d), 156.4 (s), 162.5 (s). Anal calcd for C17H16N2O2 (280.32): C 72.84, H 5.75, N 9.99. Found: C 73.02, H 5.47, N 10.08. 4.1.5.7

7-(2,3-Dimethoxybenzyl)-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole

(6g).

This

compound was obtained from reaction of 9g. White solid; yield 65%; Rf=0.29 (CH2Cl2); m.p.: 131– 132 °C; 1H NMR (DMSO-d6, 200 MHz, ppm): δ 2.60–2.70 (m, 4H, 2 x CH2), 3.73 (s, 3H, CH3),

ACCEPTED MANUSCRIPT 3.80 (s, 3H, CH3), 5.07 (s, 2H, CH2), 6.67–6.74 (m, 2H, H-6 and Ar), 6.99–7.08 (m, 2H, Ar), 7.19 (s, 1H, H-8), 8.37 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 19.4 (t), 20.0 (t), 47.5 (t), 55.7 (q), 60.1 (q), 108.3 (s), 109.8 (s), 112.6 (d), 115.8 (d), 118.5 (d), 119.9 (s), 120.5 (d), 124.1 (d), 131.5 (s), 146.1 (s), 149.1 (d), 152.3 (s), 162.5 (s). Anal calcd for C18H18N2O3 (310.35): C 69.66, H

4.1.5.8

RI PT

5.85, N 9.03. Found: C 69.50, H 5.99, N 9.15.

7-(2,5-Dimethoxybenzyl)-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole

(6h).

This

compound was obtained from reaction of 9h. White solid; yield 75%; Rf=0.35 (CH2Cl2); m.p.: 119–

SC

120 °C; 1H NMR (DMSO-d6, 200 MHz, ppm): δ 2.60–2.69 (m, 4H, 2 x CH2), 3.66 (s, 3H, CH3), 3.79 (s, 3H, CH3), 5.01 (s, 2H, CH2), 6.65 (s, 1H, H-6), 6.72–6.75 (m, 1H, Ar), 6.81–6.87 (m, 1H, 13

M AN U

Ar), 6.93–6.97 (m, 1H, Ar), 7.19 (s, 1H, H-8), 8.37 (s, 1H, H-3);

C NMR (DMSO-d6, 50 MHz,

ppm): δ 19.4 (t), 20.1 (t), 47.5 (t), 55.3 (q), 55.8 (q), 108.3 (s), 109.8 (s), 111.7 (d), 112.8 (d), 115.4 (d), 115.8 (d), 118.5 (d), 119.9 (s), 127.2 (s), 149.1 (d), 150.6 (s), 153.1 (s), 162.5 (s). Anal calcd for C18H18N2O3 (310.35): C 69.66, H 5.85, N 9.03. Found: C 69.55, H 5.98, N 9.21.

TE D

4.1.5.9 7-(3,4-Dimethoxybenzyl)-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (6i). This compound was obtained from reaction of 9i. Yellow oil; yield 65%; Rf=0.23 (CH2Cl2); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 2.59–2.66 (m, 4H, 2 x CH2), 3.72 (s, 3H, CH3), 3.74 (s, 3H, CH3), 4.99 (s, 2H,

EP

CH2), 6.76 (s, 1H, H-6), 6.80–6.94 (m, 2H, H-5’ and H-6’), 7.01 (s, 1H, H-2’), 7.25 (s, 1H, H-8),

AC C

8.37 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 19.4 (t), 20.1 (t), 52.2 (t), 55.4 (q), 55.5 (q), 108.2 (s), 109.8 (s), 111.7 (d), 111.8 (d), 115.5 (d), 118.2 (d), 120.0 (d), 120.1 (s), 130.6 (s), 148.3 (s), 148.6 (s), 149.0 (d), 162.6 (s). Anal calcd for C18H18N2O3 (310.35): C 69.66, H 5.85, N 9.03. Found: C 69.74, H 5.61, N 8.84. 4.1.5.10

7-(3,5-Dimethoxybenzyl)-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole

(6j).

This

compound was obtained from reaction of 9j. Yellow oil; yield 74%; Rf=0.28 (CH2Cl2); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 2.64–2.67 (m, 4H, 2 x CH2), 3.72 (s, 6H, 2 x CH3), 5.00 (s, 2H, CH2), 6.42–6.46 (m, 3H, Ar), 6.77 (s, 1H, H-6), 7.26 (s, 1H, H-8), 8.37 (s, 1H, H-3);

13

C NMR

ACCEPTED MANUSCRIPT (DMSO-d6, 50 MHz, ppm): δ 19.4 (t), 20.0 (t), 52.4 (t), 55.2 (2 x q), 98.9 (d), 105.7 (2 x d), 108.3 (s), 109.9 (s), 115.8 (d), 118.5 (d), 120.1 (s), 140.6 (s), 149.1 (d), 160.6 (2 x s), 162.5 (s). Anal calcd for C18H18N2O3 (310.35): C 69.66, H 5.85, N 9.03. Found: C 69.80, H 5.55, N 8.79. 4.1.5.11 7-(3,4,5-Trimethoxybenzyl)-5,7-dihydro-4H-[1,2]oxazolo[5,4-e] isoindole (6k). This

RI PT

compound was obtained from reaction of 9k. White solid; yield 61%; Rf=0.13 (CH2Cl2); m.p.: 77– 78 °C; 1H NMR (DMSO-d6, 200 MHz, ppm): δ 2.65 (s, 4H, 2 x CH2), 3.63 (s, 3H, CH3), 3.76 (s, 6H, 2 x CH3), 4.98 (s, 2H, CH2), 6.71 (s, 2H, H-2’ and H-6’), 6.81 (s, 1H, H-6), 7.29 (s, 1H, H-8), 13

C NMR (DMSO-d6, 50 MHz, ppm): δ 19.4 (t), 20.1 (t), 52.7 (t), 55.9 (2 x q),

SC

8.37 (s, 1H, H-3);

59.9 (q), 105.4 (2 x d), 108.3 (s), 109.9 (s), 115.6 (d), 118.3 (d), 120.0 (s), 133.8 (s), 136.9 (s),

Found: C 66.92, H 5.84, N 8.39.

M AN U

149.1 (d), 152.9 (2 x s), 162.5 (s). Anal calcd for C19H20N2O4 (340.37): C 67.05, H 5.92, N 8.23.

4.1.5.12 7-Phenyl-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (6l). This compound was obtained from reaction of 9l. White solid; yield 58%; Rf=0.48 (CH2Cl2); m.p.: 123–124 °C; 1H NMR

1H, H-8), 8.47 (s, 1H, H-3);

TE D

(DMSO-d6, 200 MHz, ppm): δ 2.69–2.82 (m, 4H, 2 x CH2), 7.24–7.67 (m, 6H, H-6 and Ar), 7.80 (s, 13

C NMR (DMSO-d6, 50 MHz, ppm): δ 19.8 (t), 20.5 (t), 110.3 (s),

EP

112.7 (s), 113.7 (d), 117.0 (d), 119.8 (2 x d), 122.7 (s), 126.1 (d), 130.2 (2 x d), 139.9 (s), 149.8 (d), 162.2 (s). Anal calcd for C15H12N2O (236.27): C 76.25, H 5.12, N 11.86. Found: C 76.03, H 5.39, N

AC C

11.58.

4.1.5.13 7-(4-Methoxyphenyl)-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (6m). This compound was obtained from reaction of 9m. White solid; yield 62%; Rf=0.33 (CH2Cl2); m.p.: 134–135 °C; 1H NMR (DMSO-d6, 200 MHz, ppm): δ 2.68–2.81 (m, 4H, 2 x CH2), 3.79 (s, 3H, CH3), 7.03 (d, 2H, J = 9.0 Hz, H-3’ and H-5’), 7.25 (s, 1H, H-6), 7.55 (d, 2H, J = 9.0 Hz, H-2’ and H-6’), 7.67 (s, 1H, H8), 8.45 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 19.3 (t), 20.0 (t), 55.4 (q), 109.4 (s), 111.8 (s), 113.4 (d), 114.7 (2 x d), 116.8 (d), 120.9 (2 x d), 121.7 (s), 133.0 (s), 149.2 (d), 157.2 (s),

ACCEPTED MANUSCRIPT 161.9 (s). Anal calcd for C16H14N2O2 (266.29): C 72.16, H 5.30, N 10.52. Found: C 72.32, H 5.19, N 10.38. 4.1.5.14 Ethyl 7,8-dimethyl-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindol-6-carboxylate (6n). This compound was obtained from reaction of 9n. White solid; yield 72%; Rf=0.35 (CH2Cl2); m.p.: 84–

RI PT

85 °C; IR cm-1: 1689 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 1.30 (t, 3H, J = 7.1 Hz, CH3), 2.46 (s, 3H, CH3), 2.69 (t, 2H, J = 8.1 Hz, CH2), 3.01 (t, 2H, J = 8.1 Hz, CH2), 3.76 (s, 3H, CH3), 4.23 (q, 2H, J = 7.1 Hz, CH2), 8.42 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 11.2 (q),

SC

13.5 (q), 18.6 (t), 21.6 (t), 32.5 (q), 59.6 (t), 108.3 (s), 109.0 (s), 118.5 (s), 128.3 (s), 131.4 (s),

Found: C 64.93, H 6.47, N 10.60.

M AN U

148.8 (d), 160.8 (s), 162.0 (s). Anal calcd for C14H16N2O3 (260.29): C 64.60, H 6.20, N 10.76.

4.1.5.15 Ethyl 7-benzyl-8-methyl-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindol-6-carboxylate (6o). This compound was obtained from reaction of 9o. White solid; yield 65%; Rf=0.45 (CH2Cl2); m.p.: 120–121 °C; IR cm-1: 1687 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 1.23 (t, 3H, J = 7.1 Hz,

TE D

CH3), 2.41 (s, 3H, CH3), 2.74 (t, 2H, J = 8.1 Hz, CH2), 3.09 (t, 2H, J = 8.1 Hz, CH2), 4.17 (q, 2H, J = 7.1 Hz, CH2), 5.62 (s, 2H, CH2), 6.97 (d, 2H, J = 6.6 Hz, H-2’ and H-6’), 7.20–7.37 (m, 3H, H-3’, H-4’ and H-5’), 8.46 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 11.7 (q), 14.6 (q), 19.1

EP

(t), 22.2 (t), 48.1 (t), 60.2 (t), 109.3 (s), 110.2 (s), 118.8 (s), 126.2 (2 x d), 127.4 (d), 129.1 (2 x d),

AC C

129.6 (s), 132.0 (s), 138.5 (s), 149.4 (d), 161.1 (s), 162.3 (s). Anal calcd for C20H20N2O3 (336.38): C 71.41, H 5.99, N 8.33. Found: C 71.75, H 6.23, N 8.12. 4.1.5.16

Ethyl

7-(4-methoxybenzyl)-8-methyl-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindol-6-

carboxylate (6p). This compound was obtained from reaction of 9p. White solid; yield 63%; Rf=0.35 (CH2Cl2); m.p.: 102–103 °C; IR cm-1: 1689 (CO); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 1.25 (t, 3H, J = 7.1 Hz, CH3), 2.42 (s, 3H, CH3), 2.73 (t, 2H, J = 7.8 Hz, CH2), 3.07 (t, 2H, J = 7.8 Hz, CH2), 3.71 (s, 3H, CH3), 4.19 (q, 2H, J = 7.1 Hz, CH2), 5.54 (s, 2H, CH2), 6.86 (d, 2H, J = 8.0 Hz, H-3’ and H-5’), 6.94 (d, 2H, J = 8.0 Hz, H-2’ and H-6’), 8.45 (s, 1H, H-3); 13C NMR (DMSOd6, 50 MHz, ppm): δ 11.3 (q), 14.1 (q), 18.6 (t), 21.7 (t), 47.0 (t), 55.0 (q), 59.7 (t), 108.7 (s), 109.7

ACCEPTED MANUSCRIPT (s), 114.0 (2 x d), 118.2 (s), 127.2 (2 x d), 129.1 (s), 129.8 (s), 131.5 (s), 148.9 (d), 158.2 (s), 160.7 (s), 161.8 (s). Anal calcd for C21H22N2O4 (366.41): C 68.84, H 6.05, N 7.65. Found: C 68.99, H 5.81, N 7.39. 4.1.5.17 7,8-Dimethyl-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (6q). This compound was

RI PT

obtained from reaction of 9q. Yellow oil; yield 58%; Rf=0.42 (CH2Cl2); 1H NMR (DMSO-d6, 200 MHz, ppm): δ 2.37 (s, 3H, CH3), 2.55–2.72 (m, 4H, 2 x CH2), 3.48 (s, 3H, CH3), 6.55 (s, 1H, H-6), 8.34 (s, 1H, H-3);

13

C NMR (DMSO-d6, 50 MHz, ppm): δ 11.2 (q), 20.1 (t), 20.7 (t), 33.58 (q),

SC

107.9 (s), 108.5 (s), 118.3 (s), 118.7 (d), 124.7 (s), 149.2 (d), 164.0 (s). Anal calcd for C11H12N2O (188.23): C 70.19, H 6.43, N 14.88. Found: C 70.07, H 6.64, N 14.54.

M AN U

4.1.5.18 7-Benzyl-8-methyl-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (6r). This compound was obtained from reaction of 9r. White solid; yield 56%; Rf=0.53 (CH2Cl2); m.p.: 98–99 °C; 1H NMR (DMSO-d6, 200 MHz, ppm): δ 2.33 (s, 3H, CH3), 2.60–2.82 (m, 4H, 2 x CH2), 5.09 (s, 2H, CH2), 6.71 (s, 1H, H-6), 7.12 (d, 2H, J = 6.8 Hz, H-2’ and H-6’), 7.26–7.38 (m, 3H, H-3’, H-4’ and H-5’),

TE D

8.36 (s, 1H, H-3); 13C NMR (DMSO-d6, 50 MHz, ppm): δ 11.4 (q), 20.0 (t), 20.7 (t), 49.8 (t), 108.3 (s), 109.1 (s), 118.6 (d), 118.9 (s), 124.4 (s), 127.2 (2 x d), 127.8 (d), 129.1 (2 x d), 138.7 (s), 149.3

AC C

6.33, N 10.28.

EP

(d), 163.8 (s). Anal calcd for C17H16N2O (264.32): C 77.25, H 6.10, N 10.60. Found: C 77.59, H

4.2. Biology

4.2.1. Drugs.

For in vitro studies, vinorelbine (purchased from Pierre Fabre Pharma), taxol (Paclitaxel; purchased from Santa Cruz Biotechnology), Purvalanol A (Tocris) and 6j were completely dissolved in 1% dimethylsulfoxide (DMSO), stored at -20°C, and then diluted in complete culture medium immediately before use at the appropriate concentration. For in vivo studies, 6j was dissolved in DMSO 10% and diluted in saline solution.

ACCEPTED MANUSCRIPT 4.2.2. Cell Culture and In Vivo Experiments. The Mycoplasma-free melanoma (JR8 and M14), breast cancer (MCF-7 and MDA-MB-231), castration-resistant prostate carcinoma (PC3 and DU145) and DMPM (STO and MP8), and the normal human lung fibroblast (WI38) and adult breast (MCF10A) cell lines were maintained in the

RI PT

logarithmic growth phase as a monolayer in the appropriate culture medium (Lonza): RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; JR8, M14, PC3 and DU145), DMEMF12 supplemented with 5% (MCF-7 and MDA-MB-231) or 10% (STO and MP8) FBS, or DMEM

with a supply of 5% CO2/95% air atmosphere.

SC

supplemented with 10% FBS (WI38), or in MEBM (MCF10A), in a humidified incubator at 37°C

M AN U

For in vivo experiments, female nude mice (6-8 weeks-old, purchased from Charles River) were maintained in laminar flow rooms keeping temperature and humidity constant. The mice had free access to food and water. Experimental protocols were approved by the Ethics Committee for Animal Experimentation of the Fondazione IRCCS Istituto Nazionale Tumori of Milan according to

TE D

institutional guidelines that are in compliance with national and international laws and policies. Exponentially growing STO cells were subcutaneously injected into the right flank mice (107 cells/flank). Eight mice for each experimental group were used. 6j was dissolved in a mixture of

EP

DMSO (10%) and saline solution (90%) and delivered i.p. every seven days for four weeks (q7dx4) starting from the tenth day after cell inoculum at a dose of 5 mg/kg. Tumor growth was followed by

AC C

weekly measurements of tumor diameters with a Vernier caliper and tumor volume (TV) was calculated according to the formula: TV (mm3) = d2 × D/2 where d and D are the shortest and the longest diameter, respectively. The efficacy of the drug treatment was assessed as tumor volume inhibition percentage (TVI %) in treated versus control mice, calculated as TVI% = 100 − (mean TV treated/mean TV control × 100). The toxicity of the drug treatment was determined as body weight loss and lethal toxicity. 4.2.3. Cell Proliferation Assay.

ACCEPTED MANUSCRIPT After harvesting in the logarithmic growth phase, 4500 cells/50 µL were plated in 96-well flatbottomed microtiter plates (EuroClone) for 24 h and then treated with increasing concentrations of 6j (0.01-100 µM) for 72 h. Control cells received vehicle alone (0.01% DMSO). Studies were performed in eight replicates and repeated at least three times independently. At the end of drug

RI PT

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 (POLARstar OPTIMA, BMG

SC

Labtech GmbH), and the results were expressed as a percentage, relative to DMSO-treated cells. Dose-response curves were created, and IC50 and IC80 values (i.e., concentrations able to inhibit cell

compound. 4.2.4. Tubulin polymerization assays.

M AN U

growth by 50% and 80%, respectively) were determined graphically from the curve for each

Cells were seeded in 6 wells plates (Eppendorf) and were exposed the next day to the 15r

TE D

derivative for 24 h at concentrations corresponding to the IC50 at 72 h. Samples were then processed for the tubulin polymerization assay [67,68]. To separate cytosolic and cytoskeletal-associated

EP

proteins, cells were rinsed twice in PIPES-EGTA-MgCl2 (PEM) buffer (85 mmol/L PIPES, pH 6.94; 10 mmol/L EGTA; 1 mmol/L MgCl2; 2 mol//L glycerol; 1 mmol/L phenylmethylsulfonyl

AC C

fluoride; 0.1 mmol/L leupeptin; 1 µmol/L pepstatin; 2 µg/mL aprotinin), lysed at room temperature for 10 min with PEM buffer supplemented with 0.1% v/v Triton X-100, and rinsed in PEM buffer. The Triton X-100-soluble fractions were then diluted 3:1 with 4 × SDS-PAGE sample buffer. The insoluble material that remained attached to the dish was scraped into SDS-PAGE sample buffer containing protease inhibitors. Proteins were separated by SDS-PAGE, and tubulin distribution was analyzed by immunoblotting using anti α-tubulin antibody (Sigma-Aldrich). 4.2.5. Cell cycle distribution analysis.

ACCEPTED MANUSCRIPT Both adherent and floating cells were fixed in 70% EtOH and incubated at 4°C for 30 min in staining solution containing 50 µg/mL of propidium iodide, 50 mg/mL of RNase, and 0.05% Nonidet-P40 in PBS. Samples were analyzed with BD Accuri c6 flow cytometer (Becton

software according to the Modfit model (Becton Dickinson). 4.2.6. Fluorescence microscopy analysis

RI PT

Dickinson). At least 30000 events were read, and histograms were analyzed using the CellQuest

Cells seeded on glass cover-slips were treated for 72 h with equitoxic (IC50) concentrations of

SC

6j, taxol and vinorelbine, and then fixed in 2% paraformaldehyde for 30 minutes and permeabilized

M AN U

with ice-cold methanol for 20 minutes at -20°C. After blocking with PBS containing 1% bovine serum albumin, cells were probed overnight with a mouse anti-MPM-2 (mitotic protein monoclonal #2; Upstate Biotechnology) at room temperature, followed by a 1-h incubation at room temperature with goat antimouse AlexaFluor594® dye secondary antibody (Life Technologies). Images were acquired using a Leika fluorescence microscope (Leica). The percentage of mitotic cells was

TE D

determined by scoring at least 300 cells for each sample run in duplicate. 4.2.7. Apoptosis analysis. The catalytic activity of caspase-3 was measured in the same cellular

EP

samples as the ability to cleave the specific substrate N-acetyl-Asp-Glu-Val-Asp-pNA (DEVDpNA) by means of the APOPCYTO/caspase-3 kit (MBL), according to manufacturer's instructions.

AC C

Briefly, cells were washed, pelleted, and lyzed according to the manufacturer's instructions. Total protein and the specific fluorogenic substrate N-acetyl-Asp-Glu-Val-Asp-pNA (DEVD-pNA) were mixed for 1 h at 37 °C and transferred to 96-well microtiter plates. The hydrolysis of the specific substrates was monitored by a spectrofluorometer (POLARstar OPTIMA) with 380-nm excitation and 460-nm emission filters. Results were expressed as relative fluorescence units (rfu). 4.2.8. In Vitro Kinase Assay. The effect of 6j on CDK1 activity was evaluated using the OmniaTM Recombinant Kit (Invitrogen) according to the manufacturer’s protocols. Briefly, increasing concentrations (0.1-100 µM) of 6j and Purvalanol A were mixed with recombinant CDK1/cyclin B

ACCEPTED MANUSCRIPT complex (Invitrogen) in 1× kinase buffer containing a peptide substrate, ATP, and dithiothreitol, and the kinase reaction was performed at 30°C for 30 min. Fluorescences were measured upon excitation at 360 nm and emission at 485 nm.

Acknowledgements

SC

test. Ps < 0.05 was considered statistically significant.

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4.2.9. Statistical Analysis. Statistical evaluation of data was done with the two-tailed Student’s t

This work was financially supported by Ministero dell’Istruzione dell’Università e della Ricerca

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EP

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(MIUR). DC is recipient of an AIRC fellowship (#16360).

ACCEPTED MANUSCRIPT

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ACCEPTED MANUSCRIPT [8]

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(1R,2R)1,2-dihydroxy-1-(2′,3′′-dihydroxy-4′-methoxyphenyl)-2-

(3′′,4′′,5′′trimehoxyphenyl)ethane, J. Nat. Prod. 63 (2000) 969–974. [13] K. Ohsumi, R. Nakagawa, Y. Fukuda, T. Hatanaka, Y. Morinaga, Y. Nihei, K. Ohishi, Y.

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ACCEPTED MANUSCRIPT

Tables Table 1.

Sbt

R

R1

R2

Yieldsa (%)

6a

9a

Me

H

H

64

6b

9b

Bn

H

H

64

6c

9c

4-MeBn

H

H

72

6d

9d

4-OMeBn

H

H

60

6e

9e

3-OMeBn

H

H

78

6f

9f

2-OMeBn

H

H

75

6g

9g

2,3-(OMe)2Bn

H

H

65

6h

9h

2,5-(OMe)2Bn

H

H

75

6i

9i

3,4-(OMe)2Bn

H

H

65

6j

9j

3,5-(OMe)2Bn

H

H

74

6k

9k

3,4,5-(OMe)3Bn

H

H

61

6l

9l

Ph

H

H

58

9m

4-OMePh

H

H

62

9n

Me

COOEt Me

72

9o

Bn

COOEt Me

65

9p

4-OMeBn

COOEt Me

63

6q

9q

Me

H

Me

58

6r

9r

Bn

H

Me

56

6n 6o

a

AC C

EP

6p

M AN U

TE D

6m

SC

[1,2]Oxazoles

RI PT

[1,2]Oxazolo[5,4-e]isoindoles 6a-r.

Figures represent the yield obtained at the final reaction step.

ACCEPTED MANUSCRIPT

Table 2.

IC50 (µM)[a] cpd

JR8

M14

MCF7

MDA-MB-231

PC3

RI PT

Cytotoxic activity of [1,2]Oxazolo[5,4-e]isoindoles 6a-r in human cell lines.

DU145

STO

MP8

6a

58.37±2.36 65.85±3.65 86.64±1.95

55.37±3.52

77.14±4.99 69.32±3.51 28.13±4.08 26.69±1.72

6b

5.14±1.19

6.88±1.17

11.26±3.21

19.64±2.44

13.47±3.54 15.19±2.32

6c

>100

>100

>100

>100

6d

9.15±1.81

12.45±3.20

7.89±1.56

15.56±2.25

18.63±3.09 15.21±3.65

6e

0.96±0.12

1.02±0.17

1.17±0.22

0.85±0.07

0.96±0.11

6f

16.21±3.48 20.47±2.89 19.37±3.65

14.38±2.56

>100

>100

>100

>100

n.a.

n.a.

0.69±0.22

>100

>100

>100

>100

n.a.

n.a.

>100

>100

>100

>100

1.37±0.24

0.46±0.06

0.51±0.07

>100

>100

18.57±4.08 17.54±2.99

0.79±0.14

1.52±0.34

>100

>100

>100

>100

n.a.

n.a.

>100

>100

>100

>100

>100

SC

0.79±0.13

>100

M AN U

6g

WI38 MCF10A

>100

>100

19.16±4.44 16.83±3.85 15.15±2.82

19.63±1.89

23.65±3.93 19.47±2.87

6i

0.85±0.11

0.96±0.09

1.03±0.04

0.88±0.02

0.79±0.12

0.87±0.04

0.64±0.16

0.51±0.11

>100

>100

6j

0.23±0.04

0.31±0.07

0.33±0.09

0.32±0.02

0.27±0.10

0.31±0.06

0.06±0.01

0.07±0.02

>100

>100

6k

0.63±0.05

0.89±0.08

0.92±0.03

0.75±0.04

0.87±0.03

0.99±0.01

0.62±0.04

0.73±0.03

>100

>100

6l

59.55±3.97 63.38±4.12 81.15±5.32

6m

61.55±4.90 58.46±2.62 79.64±2.56

EP

TE D

6h

70.45±2.08 76.44±3.33 29.47±3.11 25.33±1.96

n.a.

n.a.

57.49±3.29

79.87±4.17 81.13±2.77 25.46±3.22 21.19±2.13

n.a.

n.a.

AC C

68.44±3.64

6n

>100

>100

>100

>100

>100

>100

>100

>100

n.a.

n.a.

6o

>100

>100

>100

>100

>100

>100

>100

>100

n.a.

n.a.

6p

>100

>100

>100

>100

>100

>100

>100

>100

n.a.

n.a.

6q

>100

>100

>100

>100

>100

>100

>100

>100

n.a.

n.a.

6r

>100

>100

>100

>100

>100

>100

>100

>100

n.a.

n.a.

ACCEPTED MANUSCRIPT

[a]

Data are reported as IC50 values (concentration of drug required to inhibit growth by 50%) determined by MTS assay after 72 h of continuous

exposure of both human tumor (JR8, M14, MCF7, MDA-MB-231, PC3, DU145, STO and MP8) and normal (WI38 and MCF10A) cell lines to each

AC C

EP

TE D

M AN U

SC

RI PT

compound. Data represent mean values±SD of three independent experiments. n.a.: not assessed.

ACCEPTED MANUSCRIPT Figure Legends Figure 1. Structures of pyrano[2,3-e]isoindoles (1), pyrrolo[3,4-h]quinolines (2), pyrrolo[3,4h]quinazolines

(3),

[1,2]oxazolo[5,4-e]indazoles

(4),

[1,2]oxazolo[4,5-g]indoles

(5),

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[1,2]oxazolo[5,4-e]isoindoles (6). Figure 2. Effect of 6j derivative on DMPM growth. (A) Cytotoxic activity of 6j in DMPM cell lines. STO (●) and MesoII (■) cells were cultured for 72 h in the presence of increasing concentrations of the compound and the cytotoxic activity was assessed by MTS assay. Data are

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expressed as percentage values with respect to untreated cells (only DMSO) and represent the mean

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values±SD (standard deviation) of three independent experiments. (B) Antitumor activity of 6j on STO cells xenotransplanted on athymic nude mice. Drugs were administered ip at 5 mg/kg, q7dx4, starting 10 days after cell inoculum. Data are expressed as mean values±SE. Figure 3. Effect of 6j derivative on tubulin polymerization. Representative western blot showing

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the soluble (S) or polymerized (P) tubulin fraction in STO and MP8 cells after 24 h of exposure to 6j at the concentrations corresponding to the IC50 at 72 h. Vinorelbine (Vin: 2 and 13 nM for STO and MP8, respectively) and taxol (Tx: 1 and 18 nM for STO and MP8, respectively) were selected

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as reference drugs due to their opposite mechanism of action on tubulin polymerization.

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Figure 4. Effect of 6j derivative on cell cycle progression. (A) Flow cytometric analysis of the cell cycle distribution in STO and MP8 cells exposed for 48 and 72 h to 1% (v/v) DMSO (control cells; Ctr) and 6j (IC50 and IC80) or taxol (Tx) and vinorelbine (Vin) (IC50). Data are reported as the percentage of cells in G1, S , and G2/M phases and represent the mean values of three independent experiments; SDs were always within 5%. (B) Percentage of mitotic cells upon the exposure of STO and MP8 cells to equitoxic concentrations (IC50) of 6j, vinorelbine (Vin; 2 and 13 nM for STO and MP8, respectively) and taxol (Tx; 1 and 18 nM for STO and MP8, respectively). Data are expressed as mean values±SD of three independent experiments.

ACCEPTED MANUSCRIPT Figure 5. Effect of 6j derivative on apoptosis induction. The catalytic activity of caspase-3 was assessed by the in vitro hydrolysis of the fluorogenic substrates (DEVD-pNA) after exposure of STO and MP8 cells to 1% (v/v) DMSO (control cells; Ctr) or to 6j derivative. Data are expressed as

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mean values±SD of three independent experiments. ***P<0.001, **P<0.01, *P< 0.05.

Figure 1

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Figure 2

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ACCEPTED MANUSCRIPT HIGHLIGHTS • A new series of [1,2]oxazolo[5,4-e]isoindoles were prepared by a versatile sequence • Remarkable and selective antiproliferative activity against DMPM

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• 6j impairs tubulin polymerization in a vinca alkaloid-like manner • 6j causes cell cycle arrest at G2/M phase and apoptosis in DMPM cells

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• 6j determined tumor volume inhibition (51%) without any appreciable sign of toxicity