WATER-SOLUBLE CORONENE DERIVATIVES ACTIVE AS INHIBITORS

OF HUMAN TELOMERASE BY INDUCTION OF G-QUADRUPLEX

STRUCTURES AND THEIR USE AS ANTICANCER AGENTS

The present invention concerns water-soluble coronene derivatives active as inhibitors of human telomerase by induction of G-quadruplex structures and their use as anticancer agents. More specifically, the invention concerns a new family of polycyclic aromatic compounds having coronene skeleton with hydrophilic chains bonded to the aromatic core, that can induce the formation of G-quadruplex structures in the telomeric DNA and thus act as inhibitors of the enzyme telomerase, thereby inhibiting the proliferation of cancer.

As is known, the enzyme telomerase is an interesting target for research in the pharmaceutical field since its involvement has been ascertained in cell senescence, apoptosis and immortalisation processes - the latter very often linked to carcinogenesis.

It is known that telomeres, nucleoprotein structures situated at the ends of linear chromosomes of eukaryotic organisms, have the function of defending the cell genetic material because they avoid chromosome degrada- tion and assure the complete genome replication, thereby preventing the loss of genetic information. Moreover, they prevent chromosome ends from being recognised as sites of DNA damage thereby impeding improper fusion and recombinant processes.

Telomeric DNA is mostly present as a DNA duplex, but it has a certain number of excess bases at the 3 ¹ end compared to the complementary strand, such to give rise to a single stranded portion. The telomere nucleotide sequence is composed of a repeated unit of 5-8 basic couples characterised by the massive presence of guanine (as much as 50% of the total bases). It is highly preserved at the evolutionary stage in all eukaryotic organisms; in man, and more generally in mammals, this sequence is 5'-TTAGGG-3' and appears repeated with its complement for 5-15 kbp, forming the duplex portion, while the single stranded portion is made up of 130-210 bases.

Telomeric DNA and its related proteins have an extremely important function of safeguarding chromosome ends from cell activity that jeopardises their integrity, protecting them from degradation and fusion with other DNA segments. Another important function performed by telomeres is that of preventing the loss of genetic material during replication. It is, in fact, known that DNA polymerase is incapable of copying the last bases of the 3 ¹ end of each strand, so that there is a progressive shortening of the chromosome ends at each mitotic division, of about 25-200 bp. This shortening is at the level of the te- lomeric DNA which, not being codifying, is sacrificed without suffering any loss of genetic material, thereby avoiding the onset of critical conditions of survival in the daughter cells.

It has been shown experimentally that, exceptionally, some cells can escape cell death (apoptosis) and become immortal, stabilising the length of their telomeres. This almost always happens through telomerase activation.

Telomerase is a ribonucleoproteic enzyme responsible for the addition of telomeric repetitions at the 3' end of chromosomes. It is composed of two subunits: the first (Telomerase RNA or TR) contains an RNA strand ending with a complementary sequence of single stranded telomeric DNA enabling it to bond to the telomere end and to act as a template; the second (TEIomerase Reverse Transcriptase or TERT) is the catalytic subunit, a reverse transcriptase responsible for the addition of nucleotides at the telomeric end starting from the template provided by TR.

Telomerase has recently become an important research target owing to its role in normal processes of senescence and cell death, and in pathological processes of cell immortalisation, often linked to cancer forms. Actually, the TRAP (Telomeric Repeat Amplification Protocol) assay has revealed the presence of telomerase activity in 90% of malignant tumours, while there is no trace of it in healthy somatic cells or in benign tumours (Incles, CM.; Schultes, CM.; Neidle, S. "Telomerase inhibitors in cancer therapy: current status and future directions" Curr. Opin. Investig. Drugs 2003, 4, 675). This has led to hypothesising that, in order for them to proliferate beyond the limits of normal

cell senescence, cancer cells must trigger telomerase or other mechanisms enabling the maintenance of telomere length. Telomerase activation, compensating for the shortening of the telomeres, is supposed to be able to prolong the life of a cell indefinitely, making it virtually immortal, where this is required for physiological functions (germinal and stem cells), or in pathological processes such as cancer.

This model has led to interest in telomerase, both for its possible use as a diagnostic tool and as a possible pharmaceutical target for creating new anti-tumourals that are less damaging for healthy cells. Actually, molecules that can inhibit this enzyme turn out to have the capacity to prevent cancer growth in culture mediums.

To confirm that an inhibitor can act specifically on mechanisms corre- latable with telomerase activity, it must meet a series of precise characteristics: 1. It must reduce telomerase activity without initially having effects on cell growth.

2. Its administration must lead to a progressive shortening of the telomeres at each cell cycle.

3. Prolonged treatment of a cell culture with the inhibitor must lead to the death of cancer cells or to a halt in their growth.

4. The time necessary to observe the decrease in cell proliferation must be proportional to the initial length of the telomeres.

5. Chemically correlated molecules that do not inhibit telomerase must not cause a decrease in cell proliferation or telomere shortening. In the search for telomerase inhibitors there are many strategies that can be followed because there are many targets one can work on, and namely the RNA of the TR subunit, the TERT catalytic subunit, the primer anchorage site, holoenzyme assembly or the factors involved in the association between the enzyme and the telomeres. One of the strategies followed was the one envisaging the sequestration of the primer necessary for reverse transcriptase of the enzyme: the single stranded telomehc DNA. Indeed, it is a question of acting on the telom-

- A -

erase substrate, the single stranded telomeric DNA, to induce it to take on a conformation such to be no longer recognised and ligated by the enzyme.

To this end, molecules were used that can induce and stabilise G- quadruplex structures on this DNA sequence. These particular quadruple helix structures are taken on by DNA sequences rich in guanine, such as the telomeric ones are. These structures ate characterised by a cyclic base unit (G- tetrad) generated by the association of four guanines bound to one another by means of Hoogsteen hydrogen bonds, that is non-conventional couplings in which each guanine is bound to its two neighbours by means of two hydrogen bonds having a slightly distorted geometry with respect to those of Watson and Crick (Neidle, S.; Parkinson, G.N. "The structure of telomeric DNA" Curr. Opin. Struct. Biol. 2003, 13, 275). The general scheme of a guanine tetrad (G- tetrad), compared to a classic duplex coupling according to Watson and Crick, is shown in Figure 1 of the attached drawings. Under the thermodynamic boost of hydrophobic forces and of Van der

Waals forces, the guanine tetrads can stack onto one another to create quadruple helix structures (as shown schematically in the attached Figure 2) known as G-quadruplex. The phenomenon is facilitated by the presence of monovalent cations (in particular, Na ⁺ and K ⁺ ) which, by placing themselves between the two layers of tetrads, go to coordinate the eight carbonyl oxygens stabilising the stack. The guanine stacking in the G-quadruplex produces four grooves which, depending on the shape of the tetraplex, can all be of the same size or different.

The research thus turned to possible G-quadruplex structure inducer agents that could induce and stabilise these structures on the telomeric DNA sequence in order to deprive the reverse transcriptase of its own substrate, preventing it from bonding to it and thus from catalysing the telomeric DNA synthesis, thus inhibiting telomerase activity.

Many compounds having such an activity have been identified. These molecules may have very different structural elements, but molecular modelling studies in all cases enabled the identification of two elements essential for an effective interaction with G-quadruplex: a planar aromatic structure favour-

ing stacking interactions with guanine tetrads and the presence of positive charges for a necessary electrostatic interaction with the negative charges of the phosphate groups present in the grooves. The positive charge is generally placed on side chains interacting with the DNA grooves. Most of the active compounds identified are thus characterised by the presence of large aromatic areas and by positively charged side chains of varying lengths.

A rather broad class of G-quadruplex inducers consists of porphyrins (international patent application published with No. WO 98/33503 in the name of the Board of Regents, The University of Texas System; Shi, D. F.; Wheel- house, R.T.; Sun, D.; Hurley, L.H. "Quadruplex-interactive agents as telom- erase inhibitors: synthesis of porphyrins and structure-activity relationship for the inhibition of telomerase" J. Med. Chem. 2001, 44, 4509-4523), of which many variants have been synthesised, depending on the substituents added on the macrocycle. A second series of compounds that turned out to be active consists of anthraquinone derivatives (1 ,4- 1 ,5- 2,6- or 2,7-disubstituted) (Sun, D.; Thompson, B.; Cathers, B.E.; et al. "Inhibition of human telomerase by a G- quadruplex-interactive compound" J. Med. Chem. 1997, 40, 2113-2116; Huang, H. S. et al. "Human telomerase inhibition and cytotoxicity of regioisom- eric disubstituted amidoanthraquinones and amino anthraquinones" Bioor- ganic & Medicinal Chemistry 2005, 13, 1435-1444), fluorenones (2,7 disubstituted) (Perry, P.J. et al. "2,7-disubstituites amidofluorenone derivatives as inhibitors of human telomerase" J. Med. Chem. 1999, 42, 2679-2684) and acridines (3,6 disubstituted) (Harrison, R.J.; Gowan, S. M.; Kelland, L.R.; Neidle, S. "Human telomerase inhibition by substituted acridine derivatives" Bioorganic & Medicinal Chemistry Letters 1999, 9, 2463-2468).

Also indoloquindoline derivatives have been found to be active as telomerase inhibitors, causing senescence and a ceasing of cell proliferation in human cancer cell cultures (Zhou, J-M. Zhu XF, Lu YJ, Deng R, Huang ZS, Mei YP, Wang Y, Huang WL, Liu ZC, Gu LQ, Zeng YX. "Senescence and telomere shortening induced by novel potent G-quadruplex interactive agents, quindoline derivatives, in human cancer cell lines" Oncogene 2006, 25, 503-

11).

Moreover, various natural substances have been recognised as being effective G-quadruplex inducers and thus as valid telomerase inhibitors, including alkaloids, such as berbehne (Kuo, L.C.; Chou, CC; Yung, B.Y.-M. "Berberine complexes with DNA in the berberine-induced apoptosis in human leukemic HL-60 cells" Cancer Letters 1995, 93, 193-200, Jantova, S. et al. "Effect of berberine on proliferation, cell cycle and apoptosis in HeLa and L1210 cells", J. Pharm. Pharmacol. 2003, 55, 1143-1149) and its synthetic derivatives (Franceschin M., Rossetti L., D'Ambrosio A., Schirripa S., Bianco A., Ortaggi G., Savino M., Schultes C ₁ Neidle S. "Natural and synthetic G- quadruplex interactive berberine derivatives" Bioorg. Med. Chem. Lett. 2006, 16, 1707-1711), and telomestatin, a substance produced by Streptomyces Annulatus, having a macrocyclic structure composed of seven oxazole rings, two of which are methyl-substituted, and one being a tiazoline ring (Shin-Ya, K. et al. "Telomestatin, a novel telomerase inhibitor from Streptomyces annulatus" J. Am. Chem. Soc. 2001 , 123, 1262-1263, Kim, M.Y.; Vankayalapati, H.; Shin-ya, K.; Wierzba, K.; Hurley, L.H. "Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular G-quadruplex" J. Am. Chem. Soc. 2001 , 124, 2098-2099). The capacity of perylene derivatives (in particular, N,N'-bis[2-(1- piperidine)-ethyl]-3,4,9,10-perylenetetracarboxyl diimide) to inhibit telomerase and to bond to G-quadruplex structures has been known since 1998 (Fedoroff, O.Y.; Salazar, M.; Han, H.; Chemeris, V.V.; Kerwin, S.M.; Hurley, L.H. "NMR- based model of telomerase-inhibiting compound bound to a G-quadruplex DNA" Biochemistry 1998, 37, 13367-12374). European patent EP 1 053 237 in the name of the Board of Regents, The University of Texas System, of the same research group, describes a family of perylene derivatives that are not only able to bond to the G-quadruplex structure, but also to induce their formation, and proposes their use for the production of pharmaceutical drugs with antiproliferative activity.

Subsequent studies have also concerned the synthesis of perylene derivatives having different side chains both as regards pK _a and the distance

of the positive charge from the aromatic core (Rossetti L., Franceschin M., Bianco A., Ortaggi G., Savino M. "Perylene diimides with different side chains are selective in inducing different G-quadruplex DNA structures and in inhibiting telomerase" Bioorg. Med. Chem. Lett. 2002, 12, 2527-2533; Rossetti L., Franceschin M., Schirripa S., Bianco A., Ortaggi G., Savino M. "Selective interactions of perylene derivatives having different side chains with inter- and intramolecular G-quadruplex structures. A correlation with telomerase inhibition" Bioorg. Med. Chem. Lett. 2005, 15, 413-420; Franceschin M., Alvino A., Ortaggi G., Bianco A. "New hydrosoluble perylene and coronene derivatives" Tetrahedron Lett. 2004, 45, 9015-9020). These showed that the chemical and structural characteristics of the side chains strongly influence both the affinity for the quadruplex DNA and the capacity to induce its formation and even the specific structure formed.

With reference to the research lines summarised above, it must be noted that many telomerase inhibitors that are effective in vitro and widely studied in the literature did not arrive at a clinical study for their ineffectiveness on cell cultures, probably due to uptake problems. On the basis of this prior art, an aspect of the present invention is thus to provide new telomerase inhibitors that can act by means of a G-quadruplex structure induction mecha- nism in order to effectively and selectively prevent cancer growth, and that can be validly applicable for the production of pharmaceuticals with anticancer activity.

In the frame of the studies that lead to the present invention, there have been considered some derivatives of coronene, a polycyclic aromatic molecule composed of seven ortho-fused benzene rings, and - with the help of molecular modelling studies - a structure was researched in which the interactions with the macromolecular biological target (G-quadruplex) would be optimised.

It was thus found that a specific family of coronene derivatives having six or seven fused benzene rings and in which the central aromatic area is further enlarged by two dicarboximide rings in a symmetrical position (thereby taking the number of condensed hexa-atomic rings to eight or nine) and is

provided with hydrophilic side chains bound to the highly hydrophobic core of coronene, shows a quite considerable efficiency in inducing the G-quadruplex structure and in inhibiting telomerase. Actually, the enlarged central aromatic area is particularly suitable for interacting via stacking with the terminal tetrad of G-quadruplex, while the positively charged side chains bond to the grooves by means of electrostatic interactions with the negatively charged DNA phosphates.

Moreover, with respect to the hitherto known G-quadruplex ligands, the broad aromatic area of the coronene suggests a potential selectivity for the target structures (G-quadruplex) with respect to the conventional DNA duplex, which presents an area of interaction of the bases that is definitely less broad. This element is certainly significant for a valid pharmacological use of these compounds.

The family of derivatives proposed includes water-soluble coronene derivatives that turn out to be new per se, or new in relation to their pharmacological activity as potential anticancer agents.

Hence, the present invention specifically provides coronene derivatives of the general formula (I)

wherein:

R2, R2' and R4 are null or have the same meanings as R1 , R3 and R4' below, V and J represent, independently from one another, H, Cl, Br, I, or C, N, O, NH or NR - R having the same meaning as R1 , R3 and R4' below, - or has one of the structures defined under A and B below,

the dotted line between V and J is null, or represents a bond or a double bond only when V and J are C or N, each one of the dotted lines between V and R2' and between J and R2 represents a bond when R2 and R2' are not null, T represents C or N,

R1 , R3 and R4' represent, each one independently of the other, a group of general formula:

-[L ₁ -X] _n -L ₂ -Y wherein: n = O ₁ 1 , 2 or 3

X = NH, O, S ₁ NMe or NCOMe

- wherein Me represents a methyl group, -CH ₃

Li and L ₂ , independently of one another, represent a connection group of formula:

wherein: m = 0, 1 , 2, 3 or 4

R5 = H, OH, Me, OMe,

Y is chosen from among H, NH ₂ , OH, OCOMe, NHCOMe, NMe ₂ , N ⁺ Me ₃ , or has one of the following structures:

A) pyrrolidine/piperidine/morpholine/piperazine structure

wherein: s = 0, 1 or 2 q = 0, 1 or 2

Z = CH ₂ , O, NH, NMe, NEt, N ⁺ Me ₂ , NCOMe R6 = H, Me

B) pyridine/pyrrole structure

wherein: o = 0 or 1 p = 1 , 2 or 3

R7 = H ₁ Me or null W = CH ₁ O ₁ S ₁ N ₁ NH with the proviso that at least two from among R1 , R2, R2', R3, R4 and R4 ¹ are different from null and from H. A specific compound according to the invention which is represented by the general formula (I) above is the coronene derivative of formula (Vl):

the chemical name of which is N,N'-bis[2-(1-piperidino)-ethyl]-5-(1-piperidinyl)- 12-[2-(1-piperidino)-ethyl]- benzoperylene-2,3,8,9-tetracarboxyl-diimide.

According to some specific embodiments of the present invention, the coronene derivative has the following general formula (I ¹ )

wherein R1 , R2, R2', R3, R4 and R4' represent, each one independently of the other, a group of general formula: -[L ₁ -X] _n -L ₂ -Y wherein n, X ₁ Li, L ₂ and Y have the same meanings as specified above.

Preferably, in the general formulae (I) or (I'), R1 and R3 are different from H and equal to each other, while according to another variant of the invention, either R2 or R2' and either R4 or R4 ¹ are different from H and equal to one another.

According to some preferred embodiments of the present invention, in the general formula (I ¹ ) R1 , R3, R2 and R4 are different from hydrogen, and preferably they may be selected, equal in couples, from the group consisting of:

2-(1-piperidine)-ethyl

2-(4-methyl-1 -piperazine)-ethyl

,N, 3-(dimethylamine)-propyl

Some coronene derivatives studied as specific embodiments of the present invention have one of the following structural formulas:

The present invention further specifically provides the following, as new compounds: the derivatives of formula (II), N,N'-bis[2-(1-piperidine)- ethyll-δ.i i-bis^-^-methyl-i-piperazineJ-ethyH-coronene^.S.δ.θ-tetrac ar- boxyl-diimide or CORON2; of formula (III), N,N'-bis[3-(dimethylamine)-propyl]- 5,11 -bis[2-(4-methyl-1 -piperazineJ-ethylJ-coronene-Z.S.δ.θ-tetracarboxyl- diimide or CORON3; and formula (IV), N,N'-bis[3-(dimethylamine)-propyl]- 5,11-bis[2-(1-piperidine)-ethyl]-coronene-2,3 _l 8,9-tetracarboxyl-diimide or

CORON4. The coronone derivatives, object of the invention, can be prepared starting from 3,4,9, 10-perylenetetracarboxyl dianhydride, indicated as (1) in the following reaction schemes, where the synthesis is exemplified relatively to the preparation of the compounds of formula (II), (III) and (IV) (CORON2,

CORON3 and CORON4). The general scheme of the synthesis is as follows. a) Preparation of 1 ,7-dibromoperylene-3,4,9,10-tetracarboxyl dianhydride

(2) through bromuration catalysed by I ₂ in positions 1 and 7 of 3,4,9,10- perylenetetracarboxyl dianhydride (1). This reaction enables obtaining two functionalised positions on the aromatic skeleton of perylene that will then be exploited in the next synthetic steps. b) Formation of diimides through the reaction between a primary amine having a suitably functionalised chain and bromurated perylene anhydride (2). Two compounds are thus prepared: by reacting with 1-(2-

aminoethyl)piperidine N,N ^l -bis[2-(1-piperidine)-ethyl]-1 ,7-dibromoperyl- ene-3,4,9,10-tetracarboxyl diimide (3) is obtained, while using 3- dimethylamine-1 -propylamine N,N'-bis[3-(dimethylamine)-propyl]-1 ,7- dibromoperylene-3,4,9,10-tetracarboxyl diimide - compound (4) is ob- tained. c) Preparation of suitable end alkynes having, in position ω, 1-piperidine or 1-(4-methyl)-piperazine. The starting compound, 3-butynyl-1-ole (5), a commercially available product, is treated with methanesulfonyl chloride. The 3-butynyl-methanesulfonate (6) thus obtained is made to react with piperidine or with N-methyl-piperazine in a nucleophilic substitution reaction, by exploiting the excellent qualities of methanesulfonyl as the leaving group. Hence, in the first case, 1-(3-butynyl)-piperidine (7) is obtained and, in the second, 1-(3-butynyl)-2-methyl-piperazine (8) is obtained. d) Addition of the triple bond on the aromatic core, in the bromurated positions, by Sonogashira coupling, catalysed by Pd(PPh ₃ ^ and CuI. This step yields three products: compounds (9), (10) and (11), but in the same reaction environment there occurs, to a lesser degree, also the cyclisation reaction described in the next step. e) Coronene closure via a base-catalysed electrocyclic reaction. It is necessary to use a strong, but not nucleophilic, base in order to avoid saponification of the imidic function. This yields the final products: (12) CORON2 (N,N'-bis[2-(1-piperidine)-ethyl]-5,11-bis[2-(4-methyl-1-pip er- azine)-ethyl]-coronene-2,3,8,9-tetracarboxyldiimide), (13) CORON3 (N,N'-bis[3-(dimethylamine)-propyl]-5,11-bis[2-(4-methyl-1-p iperazine)- ethyl]-coronene-2,3,8,9-tetracarboxyldiimide) and (14) CORON4 (N ₁ N'- bis[3-(dimethylamine)-propyl]-5,11-bis[2-(1-piperidine)-ethy l]-coronene- 2,3,8,9-tetracarboxyldiimide).

(scheme follows)

follows

The specific examples of synthesis of the three exemplified coronene derivatives are reported in detail in the experimental section presented further on.

According to another different aspect thereof, the present invention also concerns the use of a coronene derivative of general formula (I), as de-

fined above, for the production of a pharmaceutical preparation. More specifically, the preparation is a pharmaceutical drug with anticancer activity, or a medicament having telomerase inhibiting activity, useful for the treatment of proliferative pathologies. On the basis of some specific embodiments of the present invention, the use as anticancer agent is proposed particularly for the compounds of formula (I) wherein R1 and R3 are different from H and the same as one another, and either R2 or R2' and either R4 and R4' are different from H and the same as one another. Among these, the specific compounds already men- tioned, which correspond to formulas (V), (II), (III) and (IV), were extensively assayed and turned out to be efficient telomerase inhibitors, showing promising growth inhibiting properties on cancer cell cultures. In particular, the preferred compounds according to this invention correspond to one of the following names: • (N,N'-bis[2-(1-piperidine)-ethyl]-5,11-bis [2-(1-piperidine)-ethyl]-coron- ene-2,3,8,9-tetracarboxyl diimide) (CORON);

• (N,N'-bis[2-(1-piperidine)-ethyl]-5,11-bis[2-(4-methyl-1-pip erazine)-eth- yl]-coronene-2,3,8,9-tetracarboxyl diimide) (CORON2);

• (N,N'-bis[3-(dimethylamine)-propyl]-5,11-bis[2-(4-methyl-1-p iperazine)- ethyl]-coronene-2,3,8,9-tetracarboxyl diimide) (CORON3);

• (N,N'-bis[3-(dimethylamine)-propyl]-5,11-bis[2-(1-piperidine )-ethyl]- coronene-2,3,8,9-tetracarboxyl diimide) (CORON4).

According to a further aspect thereof, the present invention also provides a pharmaceutical composition for the treatment of cancer including, as active ingredient, at least one coronene derivative of general formula (I) ₁ together with one or more coadjuvants and/or pharmaceutically acceptable vehicles. Preferably, the said coronene derivative is a compound of general formula (II), (III) or (IV).

The specific characteristics of the present invention, as well as its advantages both in terms of synthesis of the proposed derivatives and in terms of their biological and pharmacological activity, will be all the more evident with reference to the detailed description presented merely for exemplifi-

cation purposes below, along with the results of the experimentations carried out on it. Some experimental results are also illustrated in the attached drawings, wherein:

Figure 1 shows the general scheme of a guanine tetrad, in compari- son with a duplex DNA coupling according to Watson and Crick;

Figure 2 shows a general scheme of a guanine tetrad and the way that several tetrads stack in order to yield a G-quadruplex structure.

Figure 3 shows the induction of G-quadruplex structures by electrophoresis on polyacrylamide gel (PAGE), carried out on the four coronene derivatives called CORON ₁ CORON2, CORON3 and CORON4: panels A) and B) show the relative radiographics, while panels a) and b) show the corresponding quantitative analyses; and

Figure 4 shows human telomerase inhibition in a cell-free system studied by means of Telomerase Repeat Protocol Assay (TRAP) on the same four coronene derivatives as in Figure 3, of which the relative radiographics are reported.

As regards the synthesis, as already noted, all the coronene derivatives described below were obtained starting from the perylene intermediate of formula (1), 3,4,9, 10-perilenetetracarboxyl dianhydride, which in turn is com- mercially available, for example, from Sigma-Aldrich.

All the commercially available reagents and anhydrous solvents used in the preparations were purchased from Sigma-Aldrich. The chromatographic techniques used were thin-layer chromatography (TLC) and column chromatography. As regards the former, 60 F ₂₅₄ silica gel slides from the Merck com- pany were used. The eluent mixtures used were: 9 CHCI ₃ : 1 CH ₃ OH with the addition of ammonia up to saturation, for the Sonogashira reaction products and those of the subsequent reaction, and 9 CHCb: 1 CH ₃ OH for the purification of 1-(3-butynyl)-2-methyl-piperazine. The column chromatography used Silica gel 60 (0.063-0.200 mm) from the Merck company. The NMR spectra were carried out with Varian Gemini 200 and Varian

Mercury 300 devices. Several deuterated solvents were used: sulphuric acid was chosen for dibromurated anhydride, which is insoluble in the other sol-

vents; chloroform, thanks to its ease of use and capacity to dissolve most of the organic compounds worked with, was widely used for diagnostic purposes in order to identify (by ¹ H NMR) the products isolated during the many chromatographic separations and to identify the desired ones, and, where possible, to characterise the molecules synthesised; trifluoroacetic acid was rarely used and only in order to have a greater quantity of spectroscopic data; water was used for studies on the aggregation of the synthesised coronene derivatives.

The elemental analysis was carried out with an EA1110 CHNS-O element analyser from CE Instruments: this device provides percentages in weight of nitrogen, carbon, hydrogen and sulphur, obtained by burning a few milligrams of the compound.

The mass spectra were obtained by means of a Micromass Q-TOF MICRO spectrometer.

EXAMPLE 1

1,7-dibromoperylene-3,4,9,10-tetracarboxyl dianhydride (2)

(2) The 3,4,9, 10-perylenetetracarboxyl dianhydride (1) is dissolved in

96% H ₂ SO ₄ and left under stirring for 2 hours at room temperature. Then, iodine (30-40 mmoles per mole of anhydride) is added and the composition is heated to 8O ⁰ C. When the purple vapours of iodine are given off, the bromide is added drop by drop (molar ratio with the anhydride 2-2.5:1) and the temperature is increased to about 100°C. After 4-6 hours, the reaction is extinguished by adding ice until cooling. The resulting precipitate is then filtered on Buchner filter and washed first in a 5% solution of sodium metabisulfite to eliminate the residual bromine and then several times in water. The precipitate

is then dried first in an oven and then on a thimble for drying from solid form. A red solid is thus obtained, having a sufficient degree of purity to be used as such in the subsequent preparations.

One of the preparations is reported below: 5 g 3,4,9, 10-perylenetetracarboxyl dianhydride (1)

1.65 cc Br ₂ 113 mg I ₂ 100 cc 96% H ₂ SO ₄

6 g of 1 ,7-dibromoperylene-3,4,9,10-tetracarboxyl dianhydride (2) was obtained. The yield was 87%.

Note. Besides the 1 ,7 dibromurated isomer, the reaction also yields a small percentage of 1 ,6 disubstituted product, as can be seen from a careful examination of the ¹ H NMR spectra (see further on, for the complete characterisation). There is no way of separating the two isomers, neither here nor in the next steps. In the characterisations of the products obtained in the next synthesis steps, reference will exclusively be made - when not indicated otherwise - to the 1 ,7 isomer.

Empirical formula: C ₂₄ H ₆ Br ₂ Oe Molecular weight: 550 Solubility: in concentrated H ₂ SO ₄ Elemental analysis:

Theoretical data Experimental data

% C = 52.36 % C = 51.61

% H = 1.09 % H = 1.08

¹ H NMR spectrum, 300 MHz: solvent D ₂ SO ₄ The spectrum is composed of three signals present in the aromatic area: δ 10.71(d 2H J=8 Hz), 10.04(s 2H), 9.82(d 2H J=8 Hz). Some lower signals are also often present, with chemical shifts very close to the ones described above: in all likelihood, these signals are to be attributed to the small percentage of 1 ,6 isomer that is formed together with the main product. EXAMPLE 2

N,N'-bis[2-(1-piperidine)-ethyl]-1,7-dibromoperylene-3,4, 9,10- tetracarboxyl diimide (3)

The 1 ,7-dibromoperylene-3,4,9,10-tetracarboxyl dianhydride (2) is dissolved in anhydrous dioxane and anhydrous N,N-dimethylacetamide. Then 1-(2-aminoethyl)piperidine is added in a molar ratio of 2.2:1 with the substrate. In order to avoid any side reactions to the bromine atoms, owing to the presence of atmospheric oxygen, the reaction environment is saturated with argon. The composition is heated under stirring for 6 hours at 120 ⁰ C.

The reaction is worked by adding water, checking that the pH is not acidic (in which case soda must be added). The resulting precipitate is then filtered with a Buchner filter and washed extensively in water. Finally, it is first dried in an oven and then on thimble. The red solid obtained is sufficiently pure to be used as such in the subsequent synthetic steps.

The quantity of reagents and solvents used in one of the preparations was as follows:

6 g 1 ,7-dibromoperylene-3,4,9, 10-tetracarboxyl dianhydride (2)

3,6 cc 1-(2-aminoethyl)piperidine

60 cc anhydrous dioxane

60 cc anhydrous DMA 6 g of the product was obtained, with a 71 % yield.

Empirical formula: C ₃₈ H ₃₄ Br ₂ N ₄ O ₄ Molecular weight: 770

Solubility: soluble in chloroform and in trifluoroacetic acid

¹ H NMR 300 MHz spectrum: Solvent CDCI ₃ .

Three signals are present in the aromatic area: δ 9.44 (2H ₁ d, J=8 Hz) ₁ 8.89 (2H, s), 8.67 (2H, d, J=8 Hz). The aliphatic signals are: methylene in α at the imide group: δ 4.41 (4H ₁ 1, J=7 Hz). Methylene in β at the imidic group: δ 2.8 (4H ₁ unresolved). Methylene protons (diastereotopical) in α in nitrogen, on the piperidine ring: δ 2.7 (8H, unresolved, partly overlapping the previous signal). Protons in β and y in nitrogen, on the piperidine ring: δ 1.67 (8H ₁ unresolved), 1.50 (4H ₁ unresolved, partly overlapping the previous signal).

¹³ C NMR spectrum: Solvent CDCI ₃ . APT also carried out. Two non-equivalent carbonyl signals at 162.14 and 161.64 ppm were obtained. Ten signals belonging to the aromatic carbons, between 137.31 and 120.18 ppm, of which three tertiary (identified with APT) and seven quaternary; the three CH drop at 137.31 , 129.29 and 127.78 ppm. Five aliphatic signals, thus assigned: δ 55.74 (CH ₂ in α in amine nitrogen, on an ethylenic bridge), 54.24 (CH ₂ in α at the amine group, on a piperidine ring), 37.41 (CH ₂ in β in amine nitrogen, on an ethylene bridge), 25.48 (CH ₂ in β in aminic nitrogen on a piperidine ring), 23.81 (CH ₂ in y in amine nitrogen, on piperidine ring).

Mass spectrometry: m/z: 769.1005 [(M+H) ⁺ ] (calculated C ₃₈ H ₃₅ N ₄ O ₄ Br ₂ : 769.1025).

EXAMPLE 3

N,N'-bis[3-(dimethylamine)-propyl]-1 ,7-dibromoperylene-3,4,9,10- tetracarboxyl diimide (4)

The reaction takes place in the same conditions as the previous one above. The 1 ,7-dibromoperylene-3,4,9,10-tetracarboxyl dianhydride (2) is dissolved in anhydrous dioxane and anhydrous N,N-dimethylacetamide. Then 3-dimethylamine-1 -propylamine is added in a molar ratio of 2.2:1 with the substrate and, after creating an argon atmosphere, the composition is taken to reflux for 6 hours.

The product is made to precipitate by adding water and (if the reaction environment is acidic) a moderate quantity of soda. The precipitate is filtered using a Buchner filter and washed abundantly in water. It is finally dried, first in an oven and then on thimble.

One of the preparations is reported below: 4 g 1 ,7-dibromoperylene-3,4,9.10-tetracarboxyl dianhydride (2) 2 cc 3-dimethylamine-1 -propylamine

40 cc anhydrous dioxane 40 cc anhydrous DMA

Hence, 4.4 g of the product was obtained, with a yield of 83.6%. Empirical formula: C ₃₄ H ₃₀ Br ₂ N ₄ O ₄ Molecular weight: 718 Solubility: soluble in chloroform

Elemental analysis:

Theoretical data Experimental data

% C = 56.7 % C = 57.4

% H = 4.2 % H = 4.5

% N = 7.8 % N = 8.0

¹ H NMR 300MHz spectrum: solvent CDCI ₃

The aromatic area presents the usual three characteristic signals of disubstituded perylene: δ 9.44 (2H, d, J=8 Hz); 8.88 (2H, s); 8.66 (2H, d, J=8 Hz).

The aliphatic area shows the signals of the imidide side chains: δ 4.25

(4H, t, J=7 Hz) corresponding to methylene in α at the imide group; δ 2.44

(4H, t, J=7 Hz) of methylene in α at the amine group; δ 2.26 (12H ₁ s) ascrib- able to the methyls ligated to nitrogen; δ 1.92 (4H, t, J=7 Hz) methylene in β at the imide group.

¹³ C NMR spectrum: Solvent CDCI ₃ .

The expected signals are present: δ 162.64 (C=O), 162.14 (C=O), 137.80 (ar.), 132.64 (ar.), 132.51 (ar.), 129.78 (ar.), 128.99 (ar.), 128.29 (ar), 123.07 (ar.), 122.62 (ar.), 120.67 (ar.), 126.74 (ar.), 57.23, 45.34, 39.17, 26.01. Mass spectrometry: m/z: 717.0752 [(M+H) ⁺ ] (calculated C ₃₄ H ₃₁ Br ₂ N ₄ O ₄ : 717.0712).

EXAMPLE 4 3-butynyl-methanesulfonate (6)

CH ₃ SO ₂ CI (C)

OSO ₂ CH ₃

Et ₃ N / CH ₂ CI _{2 (6)} 2h r.t.

3-butyn-1-ol (5) (previously dissolved in anhydrous dichloromethane) and anhydrous triethylamine (present in a molar excess of 50% with respect to alcohol) are both placed under stirring. The methanesulfonyl chloride (in a molar ratio of 1.1 :1 with 3-butyn-1-ol), diluted with anhydrous dichloromethane, is slowly added by means of a drip-funnel. These additions should be carried out in an ice bath because the esterification reaction is very exothermic and tends to give off volatile and irritant substances (HCI and mesyl chloride itself). After the liquids have been added, the composition is left to

react for 2 hours under stirring at room temperature. During the reaction we observe the appearance of a whitish precipitate due to the formation of a salt between the triethylamine and the hydrochloric acid that is released following the reaction of the acidic chloride with alcohol. The product is transferred to a separator funnel and washed first in water to eliminate the salts and then in 0.5 M HCI - using litmus paper to check that the washing water is acidic - to eliminate the excess triethylamine, and then a saturated sodium bicarbonate solution, all the while checking the pH, to eliminate the HCI and, finally, with a saturated NaCI solution. It is then dried with anhydrous Na ₂ SO ₄ and most of the solvents are eliminated by reduced pressure evaporation (rotavapor). An oleous liquid is obtained, which is then purified of the last traces of solvents by bubbling nitrogen in the solution.

One of the preparations is reported below: 4 cc 3-butyn-1-ol (5)

4,3 cc methanesulfonyl chloride

11 cc anhydrous Et ₃ N

80 cc anhydrous CH ₂ CI ₂

7.15 g of the product were obtained, with a 94% yield. Empirical formula: C ₅ H ₈ O ₃ S Molecular weight: 148

¹ H NMR 200MHz spectrum: solvent CDCI ₃

The signals present are: δ 4.269 (2H ₁ t, J=7 Hz); 3.029 (3H,s); 2.627 (2H, td, Ji=3 Hz, J ₂ =7 Hz); 2.069 (1 H, t, J=3 Hz).

EXAMPLE 5 1-(3-butynyl)piperidine (7)

Piperidine and absolute ethanol are placed under stirring and taken to

reflux. 3-butynyl-methanesulfonate (6), previously dissolved in a small quantity of ethanol, is slowly added by means of a drip-funnel. The piperidine must be at a molar ratio of 2:1 with the alkyne. It is left to react for 15-20 hours.

The reaction mixture is then transferred to a separator funnel, and dichloromethane is added. The composition is washed repeatedly in water to eliminate the excess piperidine. In checking the pH of the wash water, it was found that it did not become neutral, probably because the alkyne itself is slightly soluble also in water.

The mixture is then dried with anhydrous Na ₂ SO ₄ and most of the solvents is eliminated by reduced pressure evaporation (Rotavapor). An oleous liquid is obtained, which is then purified of the last traces of solvents by bubbling nitrogen in the solution.

One of the preparations is reported below: 4.5 g 3-butynyl-methanesulfonate (6) 6 cc piperidine

40 cc absolute ethanol

2.4g of the product was obtained, with a 58% yield. Empirical formula: C ₉ Hi ₅ N Molecular weight: 137

¹ H NMR 200 MHz spectrum: solvent CDCI ₃ The signals present in the spectrum are the following: δ 2.49 (2H ₁ m),

2.35 (6H, m), 1.90 (1 H, t, J=3 Hz), 1.53 (4H, m), 1.37 (2H, m). 1 ³ C NMR spectrum: solvent CDCI ₃ . δ 82.50 (C alkyne), 68.30 (CH alkyne), 57.29 (CH ₂ on chain, in β to nitrogen), 53.65 (CH ₂ on chain, in α to nitrogen), 25.36 (CH ₂ on ring, in α to nitrogen), 23.74 (CH ₂ on ring, in β to nitrogen), 16.10 (CH ₂ on ring, in Y to nitrogen).

EXAMPLE 6 1 -(3-butynyl)-4-methy l-piperazine (8)

Absolute ethanol and N-methyl-piperazine are placed under stirring and then taken to reflux. 3-butynyl-methenesulfonate (6) is then added slowly by means of a drip-funnel. The amine must be at a molar ratio of 2:1 with the alkyne. The mixture is left to react for 15-20 hours.

Since the product is more soluble in water with respect to 1-(3- butynyl)-piperidine, it is not possible to purify it as was done with the latter, if not with very low yields. As a result, the product was transferred to a separator funnel and, after adding dichloromethane, it was washed slowly in a saturated

NaCI solution. After adding anhydrous Na ₂ SO ₄ to the organic phase, the latter was reduced in volume by reduced pressure evaporation on rotavapor and the product was then purified by column chromatography by using silica packed with just chloroform. A 9:1 mixture of chloroform and methanol was used as eluent.

One of the preparations is reported below:

3 g 3-butynyl-methanesulfonate (6)

4.5 cc N-methyl-piperazine

20 cc ethanol 2.8 g of the product was obtained, with a 91 % yield.

Empirical formula: Cg Hi ₆ N ₂ Molecular weight: 152

¹ H NMR 200 MHz spectrum: solvent CDCI ₃

It is easy to see the peak concerning the acetylenic proton δ 1.97 (1 H, t, J=3 Hz) and the one of the methyl bound to nitrogen δ 2.28 (3H, s). The remaining protons give rise to an enlarged unresolved signal between 2.3 and 2.6 ppm.

¹³ C NMR spectrum: solvent CDCI ₃

The signals relative to acetylene carbons are easy assignable: δ

82.68 (C), 69.13 (CH). The other five signals present fall at δ: 56.97, 54.99, 52.82, 46.01 , 16.77.

EXAMPLE 7 Addition of the triple bond by Sonogashira coupling

The substrate (N,N'-bis[2-(1-piperidine)-ethyl]-1 ,7-dibromoperylene- 3,4,9, 10-tetracarboxyl diimide (3) or N,N'-bis[3-(dimethylamine)-propyl]-1 ,7- dibromoperylene-3,4,9,10-tetracarboxyl diimide) (4) is disolved in an equal volume of anhydrous THF and anhydrous trietilamine. CuI and Pd(PPh ₃ ) ₄ (at a molar ratio of 10% with respect to the substrate) are then added as catalysts. The reaction environment is saturated with argon and the mixture is heated under stirring until reflux. The alkyne (1-(3-butynyl)-4-methyl-piperazine (8) or 1-(3-butynyl)-piperidine) (7), at a molar ratio of 4:1 with the substrate, is slowly added by means of a drip-funnel. The latter is left to reflux and under stirring

for about 20 hours. The reaction is then worked by adding HCI 1 :3 and transferring it to the separator funnel. Chloroform and water are then added and the latter is basified with soda to prevent the product from dissolving in an aqueous phase as a hydrochloride. The separation of the two phases may be diffi- cult owing to the formation of emulsions. The organic phase is washed repeatedly in water until the wash water has a neutral pH. It is then dried with anhydrous Na ₂ SO ₄ and the solvent is evaporated at a reduced pressure. The raw product is then purified by column chromatography carried out with silica packed in choloform. The eluent used is initially only chloroform, which is then gradually enriched with methanol, starting at 5%, then, 10%, 20% and finally 30%. The product generally comes out with the eluent at 30%, but sometimes fractions containing it were collected with an eluent at 20%. A first indication on the presence of the compound in a fraction may be obtained by looking at the colour, which becomes a very bright amber-yellow. An example of the preparation is reported below for each of the three different products obtained through this reaction.

N,N'-bis[2-(1 -piperidine)-ethy l]-1 ,7-bis[3-(4-methy 1-1 -piperidine)-butynyl]- perylene-3,4,9,10-tetracarboxyl diimide (9):

2.7 g N,N'-bis[2-(1-piperidine)-ethyl]-1 ,7-dibromoperylene- 3,4,9, 10-tetracarboxyl diimide (3)

2 g 1-(3-butynyl)-4-methyl-piperazine

386 mg Pd(PPh ₃ J ₄ 65 mg CuI 70 cc anhydrous THF 70 cc anhydrous Et ₃ N

1 g of raw product was obtained.

N,N'-bis[3-(dimethylamine)-propyl]-1 ,7-bis[3-(4-methyl-1-piperidine)- butynyl]-perylene-3,4,9,10-tetracarboxyl diimide (10):

2.2 g N,N'-bis[3-(dimethylamine)-propyl]-1 ,7-dibromoperylene- 3,4,9,10-tetracarboxyl diimide (4)

1.9 g 1 -(3-butynyl)-4-methyl-piperazine 353 mg Pd(PPh ₃ ) ₄

58 mg CuI

60 cc anhydrous THF

60cc anhydrous Et ₃ N

650 mg of the raw product was obtained. N,N'-bis[3-(dimethylamine)-propyl]-1 ,7-bis[3-(4-methyl-1 -piperidine)- butynyl]-perylene-3,4,9,10-tetracarboxyl diimide (11):

1.5 g N,N'-bis[3-(dimethylamine)-propyl]-1 ,7-dibromoperylene-

3,4,9, 10-tetracarboxyl diimide (4) 1.14 g 1 -(3-butynyl)-piperidine 240 mg Pd(PPh ₃ ) ₄

39 mg CuI 30 cc anhydrous THF 30 cc anhydrous Et3N

140 mg of the raw product was obtained. These intermediates were not characterised because they are difficult to isolate with any sufficient degree of purity. The column fractions considered good were used as such in the subsequent cyclisation reaction. The aspect of the ¹ H NMR spectrum was adopted as a selection criterion, by selecting the fractions which did not show any impurities and whose spectrum, however complicated, was decipherable. In all likelihood, these were mixtures in which the product of the Sonogashira cross-coupling is accompanied by an already partly or totally cyclisised derivative. In view of their structure, it is highly likely that these compounds tend to cyclise spontaneously when heated in the presence of bases. EXAMPLE 8

Coronene closure: base-catalysed electrocyclic reaction

The substrate (9), (10) or (11) is dissolved in toluene and then 1 ,8- diazabicycle[5.4.0]undec-7-ene (DBU), 0.6 ml per mmol of reagent, is added, whci is a strong, but not nucleophilic, base whose function is that of catalysing the ring closure reaction without any saponification of the imide function. The reaction environment is saturated with argon by bubbling and insufflating from bladder and the mixture is heated to reflux for 20 hours under vigorous stirring. The product is then transferred to a separator funnel. Chloroform is added and the organic phase is washed several times in water until the wash water has a neutral pH, and then with a NaCI saturated solution. The organic phase is then dried on anhydrous Na ₂ SO ₄ and the solvents are removed by reduced pressure evaporation. Finally, the raw product is purified if necessary by column chromatography, by using silica packed in chloroform and chloroform as an eluent, subsequently enriched in methanol, firstly at 2%, then 5%, 10%, 20% and finally 30% methanol. The products can be eluted already with

the solvent at 10%, but sometimes they may not drop until 30% is used. The fractions of the clean product obtained from the column are taken to dry state by reduced pressure evaporation.

To obtain a compound that is soluble in water, the product must be transformed into a hydrochloride. To this end, it is dissolved in the minimal quantity of 1M HCI. The obtained solution is filtered on filter paper to eliminate any insoluble impurities. After adding some drops of concentrated HCI, to assure the complete salification of all the base sites, the filtrate is heated in a bain-marie (50-60 ⁰ C) and the product is precipitated as a hydrochloride by adding acetone, a little at a time, which is a solvent miscible with water but unable to dissolve the product itself. The volume of acetone necessary to obtain precipitation is about three times the starting volume of acidic aqueous solution. The precipitate that forms is left to digest for one night. It is then centrifuged, eliminating the supernatant, and washed several times in acetone to which has been added a small amount of water that is sufficient to slightly solubilise the product, giving the solution a pale yellow colour. The product is finally dried in an oven and then on thimble.

This reaction yielded three new molecules which are reported below, together with the quantity of reagents and solvents used in a preparation thereof.

CORON2 (N,N'-bis[2-(1 -piperidine)-ethyl]-5,11 -bis[2-(4-methyl-1 -piper- azine)-ethyl]-coronene-2,3,8,9-tetracarboxyl diimide) (12)

1 g substrate (fractions selected in the previous synthetic step)

0.34 cc DBU 100 cc toluene After column chromatography, 230 mg of the product was obtained, with a 7.2% yield. The yield was calculated starting from the reagents of the previous synthetic step.

Of the product obtained, 170 mg was transformed into hydrochloride, obtaining 137 mg with a yield of 65%. Empirical formula: C ₅₆ H ₆₄ N ₈ O ₄ (base form)

C ₅₆ H( _M N ₈ O-TeHCI (hydrochloride) Molecular weight: 913 (base form)

1125 (hydrochloride)

Solubility: the base form turns out to be very soluble in chloroform and in organic solvents generally, while the hydrochloride is soluble in water, DMSO and in polar solvents such as methanol.

Elemental analysis: (performed on hydrochloride) Theoretical data Experimental data

% C = 59.7 % C = 54.3 % H = 6.2 % H = 7.3

% N = 9.9 % N = 8.4

The differences between the theoretical and experimental data are

due to the humidity in the sample, as demonstrated by the constant ratio between the percentage of nitrogen and carbon.

¹ H NMR 300MHz spectrum: solvent CDCI ₃ δ 9.38 (s ^* ), 9.33 (s, 2H, aromatic H), 9.13 (s, 2H, aromatic H), 9.08 (s ^* ), 8.36 (s, 2H, aromatic H), 4.46 (t, J = 7Hz, 4H, N _imid e-CH ₂ ), 3.73 (t, J=7

Hz, 4H, Car-CH ₂ ), 3.0-2.6 (broad, 32H, N-CH ₂ ), 2.41 (s, 6H, N-CH ₃ ) 1.72 (br,

8H, CH ₂ piperidine), 1.54 (br, 4H ₁ CH _2p iperidine) PPTI.

^* aromatic singlets due to the lesser amount of isomer: 1/6 of the total. 1 ³ C NMR spectrum: solvent CDCI ₃ δ 163.93, 163.76 (C=O). Twelve aromatic signals: δ 138.58, 128.98,

128.21 , 127.91 , 127.60, 124.98, 121.57, 121.08. 120.61 , 120.34 (two overlapping) and 118.90. Ten aliphatic signals: δ 59.31 , 56.52, 55.18, 54.90, 53.32, 46.05, 38.11 , 30.96, 26.06 and 24.41. Mass spectrometry: m/z: 913.5139 [(IvRH) ⁺ ] (calculated C ₅₆ H ₆₅ N ₈ O ₄ : 913.5129).

CORON3 (N,N'-bis[3-(dimethy lamine)-propyl]-5,11 -bis[2-(4-methyl-1 - piperazine)-ethyl]-coronene-2,3,8,9-tetracarboxyl diimide (13)

650 mg substrate (fractions selected in the previous synthetic step) 0.24 cc DBU

50 cc toluene

After column chromatography, 260 mg of the product was obtained,

with a 9.5% yield. 200mg was transformed into hydrochloride, obtaining 180mg, with a 72% yield.

Empirical formula: C ₅₂ H _6O N ₈ O ₄ (base form)

C ₅₂ H _6O N ₈ O ₄ -BHCI (hydrochloride) Molecular weight: 861 (base form)

1080 (hydrochloride)

Solubility: the basic form turns out to be very soluble in chloroform and in organic solvents generally, while the hydrochloride is soluble in water, DMSO and in polar solvents such as methanol. Element analysis:

Theoretical data Experimental data

% C = 57.8 % C = 52.9

% H = 6.1 % H = 6.8

% N = 10.37 % N = 9.4 The differences between the theoretical and experimental data are due to the humidity in the sample.

¹ H NMR 300MHz spectrum: solvent CDCI ₃ δ 9.01 (s ^* ), 8.89 (s, 2H ₁ aromatic H), 8.73 (s, 2H, aromatic H), 8.59 (s*), 8.02 (s, 2H, aromatic H) ₁ 8.00 (s*), 4.37 (br, 4H, N _im i _de -CH ₂ ), 3.48 (br, 4H, C _a r-CH ₂ ), 2.9-2.1 (br) ppm.

^* aromatic singlets due to the lesser amount of isomer: 1/6 of the total. 1 ³ C NMR spectrum: solvent CDCI ₃

The two carbonyl carbons are visible at δ 163.42 and 163.33, twelve aromatic carbons: δ 138.25, 128.35, 127.61, 127.28, 127.03, 124.28, 120.68, 120.39, 120.07, 119.78, 119.50, 118.04 and nine aliphatic carbons: δ 58.93, 57.31 , 55.22, 53.37, 46.07, 45.35, 39.34, 30.55, 26.10. Mass spectrometry: m/z: 861.4794 [(M+H) ⁺ ] (calculated C ₅₂ H ₆₁ N ₈ O ₄ : 861.4816). CORON4 (N,N'-bis[3-(dimethylamine)-propyl]-5,11-bis[2-(1-piperidine )- ethyl]-coronene-2,3,8,9-tetracarboxyl diimide (14)

140 mg substrate (fractions selected in the previous synthetic step) 0.06 cc DBU 30 cc toluene 170 mg of the raw product was obtained (9.8% yield). Since it already presented a considerable degree of purity, it did not undergo purification by column chromatography, but was used as such to be turned into hydrochloride. Starting from 125 mg of base form, 78 mg of hydrochloride was obtained, with a 53% yield. Empirical formula: C ₅₂ H ₅₈ N ₆ O ₄ (base form)

C ₅₂ H ₅₈ N ₆ O ₄ ^HCI (hydrochloride) Molecular weight: 831 (base form)

977 (hydrochloride)

Solubility: the basic form turns out to be very soluble in chloroform and in organic solvents generally, while the hydrochloride is soluble in water, DMSO and in polar solvents such as methanol. Elemental analysis: Theoretical data Experimental data

% C = 63.9 % C = 58.3 % H = 6.3 % H = 7.0

% N = 9.2 % N = 7.7

The differences between the theoretical and experimental data are due to the humidity in the sample.

¹ H NMR 200MHz spectrum: solvent CDCI ₃

δ 9.36 (s ^* ) 9.30 (s, 2H, aromatic H) ₁ 9.08 (s, 2H, aromatic H), 9.00 (s ^* ), 8.32 (S ₁ 2H, aromatic H), 8.30 (s ^* ), 4.50 (br, 4H, N _im i _d e-CH ₂ ), 3.72 (br, 4H, Car-CH ₂ ), 2.95 (br, 4H ₁ N-CH ₂ ), 2.8-2.5 (br, 12H, N-CH ₂ ), 2.37 (s, 12H ₁ CH ₃ - N) ₁ 1.78 (br, 12H ₁ CHa-CHz-Naminic), 1-58 (br, 4H, CH _2pipe πdine) ppm. * aromatic singlets due to the lesser amount of isomer: 1/6 of the total.

¹³ C NMR spectrum: solvent CDCI ₃

Two peaks are present relative to the carbonyl carbons: δ 162.47, 162.38; twelve aromatic carbons: δ 137.59, 127.35 126.73, 126.42, 126.18, 123.48, 119.95, 119.51 , 119.11, 118.87, 118.75, 117.31; and nine aliphatic carbons: δ 58.94, 56.26, 53.60, 44.28, 38.25, 29.57, 28.45, 25.02, 23.31.

Mass spectrometry: m/z: 831.4552 [(M+H) ⁺ ] (calculated C ₅₂ H ₅₉ N ₆ O ₄ : 831.4598).

Study of the biological activity of the coronene derivatives a) PAGE (PolyAcrylamide Gel Electrophoresis) assay

The capacity of the coronene derivatives to induce the formation of inter- and intramolecular G-quadruplex structures on telomeric DNA sequences was studied by means of polyacrylamide gel electrophoresis.

The oligonucleotides used were 2HTR (5'- AATCCGTCGAGCAGAGTTAGGGTTAGGGTTAG-S') and TSG4 (5'- GGGATTGGGATTGGGATTGGGATT-3'): the first is composed of a TS sequence (a usual primer for telomerase) followed by two human telomeric repetitions and can only form intermolecular, dimeric or tetrameric G-quadruplex structures; the second, instead, is composed of four telomeric repetitions and preferentially forms intramolecular structures.

Oligonucleotide solutions are prepared at a concentration of 12 μM, to which is added the ("hot) marked oligonucleotide in a concentration of about 10 nM (and thus negligible with respect to the unmarked oligonucleotide), corresponding to a radioactivity of about 1 -10 ⁴ cpm. The solutions are buffered with 10 IΎIM MES at a pH of 6.5 in the presence of KCI (5 mM in the case of TSG4 and 50 mM in the case of 2HTR). The samples are heated at 95°C for 10 min and then cooled in ice for another 10 min, in order to break down any

kind of preformed structure. The coronene derivatives are then added (except to the reference solution) in order to have solutions of increasing drug concentrations for each derivative, and a final volume of 30 μl. As a further reference, a sample containing PIPER was also prepared. The solutions are left for 2 hours at 30°C under stirring (350 rpm).

Subsequently, a suitable volume of each sample is subjected to poly- acrylamide gel electrophoresis at 15%, with run buffer TBE/KCI 0.5X (KCI 20 mM in the case of 2HTR and 5 mM in the case of TSG4). Electrophoresis continues for about 12 hours at 100 V and at room temperature. At the end of the 12 hours, the dried gel is subjected to autoradiography and the intensity of the bands are quantified by means of the "Instant Imager" (Packard), which enables an evaluation of the radioactivity present in the different bands.

Considering the data obtained in previous studies carried out in similar experimental conditions and the electrophoretic mobility obtained with the dimeric G-quadruplex structures induced by potassium ions, the bands corresponding to the single stranded (ss) DNA and to the dimeric (D) and mono- meric (M) G-quadruplex structures were identified, as shown in Figures 3 A) and 3 B).

In order to compare the activity of the coronene derivatives with that of perylene derivatives (which was the object of a study in the prior art), a 20 or 40 μM solution of the perylene derivative called PIPER (N,N'-bis[2-(1- pipehdine)-ethyl]-3,4,9,10-perylenetetracarboxyl diimide) was used as reference.

To make the analysis of the results clearer, Figures 3 C) and 3 D) graphically show the percentage of single stranded DNA as a function of the drug concentration. This type of reprocessing was preferred to the evaluation of G-quadruplex formed because the bands, especially as regards the experiment performed with TSG4, appear to be widespread, suggesting the existence of different types of complexes. Given the great tendency towards aggregation seen for these compounds even at very low concentrations, it is reasonable to suppose that they do not interact with oligonucleotides only as monomers, but also in aggregate form, changing the weight and probably

even the structure of the resulting complexes. Still not being possible to further explore this aspect and to provide a characterisation of these structures, we limited ourselves to studying the disappearance of free DNA, this being the biologically relevant datum in order to study the activity of the derivatives of the present invention as telomerase inhibitors, which has single stranded DNA as substrate.

As regards the activity of coronene derivatives, these turned out to be inducers of G-quadruplex selective with respect to the intramolecular structure. In the experiments carried out with 2HTR, actually, these molecules revealed a modest activity. Clearly better results were obtained with TSG4: with this oligonucleotide, actually, the coronene derivatives at a concentration of even 20 μM cause the virtually complete disappearance of free DNA. b) TRAP (Telomeric Repeat Amplification Protocol) assay

The TRAP assay, initially developed as a diagnostic assay to identify cancer cells, is useful in order to characterise the capacity that small organic molecules have of inhibiting telomerase.

To carry out the assay, samples containing the oligonucleotide substrate of telomerase (TSG4) are prepared at a final concentration of 0.5 μM, and the four nucleotides (dATP, dCTP, dGTP, dTTP) at a final concentration of 50 μM. The reaction takes place in a buffer solution (TRAP buffer) composed of 20 mM Tris HCI at a pH of 8.3, 68 rtiM KCI, 1.5 mM MgCI ₂ , 1 mM EGTA and 0.05% Tween20 ^® (a surfactant). The solutions thus prepared are heated in thermoblock at 95°C for 10 min and then cooled in ice for 10 min. 5 μl of the coronene derivative (or DMSO, in the case of zero) to be tested is added to the solutions, from the stocks of increasing concentrations, and the samples are placed in a thermomixer for two hours. After which time the HeLa cell extract at a final concentration of 62.5 μM in proteins is added to the solutions (The concentration of the cell extract solution used in the TRAP assay refers to the total protein content and is assessed by means of a standard, obtained by reading the adsorbance on the spectrophotometer at suitable wavelengths, of standard samples of known protein content). The final volume

of the samples was 50 μl. The samples were heated at 30 ⁰ C and the enzymatic reaction continued for 30 min at 30 ⁰ C in a thermomixer.

At the same time, a solution not containing the cell extract was also prepared. The reference was placed in the thermomixer along with the other samples at 30 ⁰ C for half an hour.

At the end of the reaction, all the solutions were purified via phenol- chloroform extraction and the nucleic acids were precipitated with ethanol. The solutions were left at -20 ⁰ C for at least 12 hours.

The precipitates were recovered and dissolved in water. To these solutions were added the necessary reagents for PCR (Polimerase Chain

Reaction) amplification in order to obtain a final volume of 50 μl:

• Forward primer (TSG4) for amplifying the multimers synthesised from telomerase: radioactive oligonucleotide in a final concentration of 0.001 g/l.

■ Reverse primer (CX _ex t) for amplifying the multimers synthesised from te- lomerase: final concentration of 0.006 g/l.

■ Reference oligonucleotide (IS) for the PCR: final concentration of 0.006 g/l.

■ Forward primer (UP) for amplifying the IS oligonucleotide: "cold" oligonucleotide at a final concentration of 0.002 g/l and radioactive oligonucleotide at a final concentration of 0.001 g/l. ■ Reverse primer (DOWN) for amplifying the IS oligonucleotide: final concentration of 0.006 g/l

■ Nucleotides (dATP, dCTP, dGTP, dTTP): final concentration of 50 μM. Taq DNA po//merase/HotMaster™: 2 U (HotMaster™ is a Taq polymerase inhibitor which reversibly blocks its activity below 55°C. The inhibitor pres- ence prevents the synthesis of oligonucleotides deriving from erroneous couplings that could result at low temperatures ("hot start" PCR)).

The solutions are buffered ("Hot Master™ Taq DNA polymerase buffer") at a pH of 8 with 25 mM Tris - HCI. The buffer also contains KCI (35 mM), MgCI ₂ (2.5 mM), EDTA (0.1 mM), dithiothreitol (DTT, 1 mM), glycerol (50%), Tween20 ^® (0.5%), Igepal ^® CA-620 (0.5%) and stabilisers, all being necessary components for Taq DNA polymerase activity during the PCR.

25 PCR cycles are carried out (denaturation at 94°C for 20 s, primer coupling at 50-70 ⁰ C for 10 s, primer extension at 60-70 ⁰ C for 20 or 30 s).

At the end of the PCR, 8 μl of the amplified samples underwent poly- acrylamide gel electrophoresis at 12% in run buffer TBE 1X for about 12 hours at 130 V. After the 12 hours, the gel is dried and subjected to autoradiography. If the enzyme has added telomeric repetitions to the starting oligonucleotide, then a series of bands is seen on the gel autoradiograph that are ascribable to the fragments which differ from one another for a telomeric repetition (six nucleotides). As shown in Figure 4, in the presence of a telomerase inhibitor, such as a molecule that can promote the formation of G-quadruplex structures, the synthesis of telomeric repetitions on the oligonucleotide is reduced or absent and the TRAP assay shows a decrease in the number and/or intensity of the electrophoretic bands owing to the various fragments in the solutions contain- ing the drug, compared to a solution not containing it. In each electrophoretic track there is a very strong band with a low electrophoretic mobility, indicated with the abbreviation "IS" (Internal Standard). This internal standard is composed of a single stranded oligonucleotide about 150 bases in length that is amplified by the PCR along with the fragments containing the telomeric repeti- tions, useful for finding any Taq polymerase inhibition by the chemical species present in the reaction environment.

It is interesting to note that the KCI concentration used in the TRAP assay is, per se, enough to induce the formation of G-quadruplex in the TSG4 oligonucleotide, used as a substrate of the enzyme. However, in the absence of any molecules capable of stabilising them, these structures are unstable and are thus unable to inhibit telomerase.

The efficiency of the coronene derivatives was evaluated by carrying out this assay on 10 μM solutions of the drug. As can be seen in the following Table 1 , which reports the percentage inhibition of the enzyme, even at this concentration all the coronene derivatives assayed proved to be able to inhibit telomerase.

TABLE 1 Quantitative analysis of telomerase inhibition by coronene derivatives

It is interesting to note how the reported assay shows a difference in activity between the various coronene derivatives. In particular, CORON turned out to be the least efficient in inhibiting telomerase. CORON2 and CORON4, characterised by two chains the same as those of CORON but also including two chains chosen from among the perylene derivatives that proved to be more efficient, demonstrated a greater activity than CORON, with concentrations being equal, but a similar activity with one another. CORON3, in which the chains that interacted better with the G-quadruplex grooves in the PIPER model were inserted on both axes, demonstrated the greatest capacity to inhibit telomerase.

Study of the anticancer activity of coronene derivatives Assays on cell lines - inhibition of cell growth and cytotoxicity

The effect of CORON (first member of the coronene derivative family) on cancer cell cultures was tested at the National Cancer Institute (Division of Cancer Treatment and Diagnosis) - National Institutes of Health (Bethesda, Maryland 20892), within the anticancer screening programme. The activity of the coronene derivative was assayed against the whole panel of tumour cell lines (Monks, A. et al. Feasibility of a High-Flux Anticancer Drug Screen Using a Diverse Panel of Cultured Human Tumor Cell Lines J. Natl. Cancer Inst, 1991 , 83, 757-766), comprising 60 different human tumour cell lines grouped into nine tumour subgroups: leukaemia, non-small cell pulmonary tumour,

colon tumour, tumour of the CNS, ovary carcinoma, renal tumour, prostate carcinoma and mammary carcinoma.

The procedure used may be summarised as follows. The tumour line cells were grown in a 1640 RPMI culture containing 5% of bovine foetal serum and 2 rtiM L-glutamine. In a typical screening experiment, the cells are inoculated on 96-well microtitration plates in 100 μl_, at densities ranging from 5,000 to 40,000 cells/well depending on individual cell line doubling time. After cell inoculation, the microtitration plates are incubated at 37°C in 5% CO ₂ , 95% air and 100% of relative humidity for 24 hours before adding the agents under trial.

After 24 hours, two plates of each cell line are fixed in situ with TCA in order to represent a measure of the cell population for each line at the moment of adding the agent (Tz). The agents tested are solubilised in dimethyl- sulfoxide at 400 times the final maximum concentration desired and stored frozen before use. At the time of adding the agent, a quantity of frozen concentrate is defrosted and diluted to double the maximum final concentration desired, with a complete medium containing 50 μg/ml of gentamycin. Additional serial four-fold, ten-fold or ^λ A log dilutions are made in order to give a total of five serial concentrations of agent plus the control. 100 μl aliquots of these different dilutions of agent are added to the appropriate microtitration wells already containing 100 μl of medium, yielding the required final concentrations of agent.

After addition, the plates are incubated for another 48 hours at 37°C, 5% CO ₂ , 95% air and 100% relative humidity. For the adhering cells, the as- say ends by adding cold TCA. The cells are fixed in situ by gently adding 50 μl of cold 50% TCA (p/v) (final concentration, 10% TCA) and incubated for 60 minutes at 4 ⁰ C. The supernatant is discarded and the plates are washed five times in running water and then dried in air.

A sulforhodamine B solution (SRB) (100 μl) at 0.4% (w/v) in 1% acetic acid is added to each well, and the plates are incubated for 10 min at room temperature. After staining, the unbound colorant is removed by washing five times in 1% acetic acid and the plates are dried in air. The bound colorant is

then solubilised with 10 mM of trizma base and the adsorbance is read on an automatic slide reader at a wavelength of 515 nm. For the cells in suspension, the methodology is the same except for the fact that the assay is ended by fixing the cells deposited at the bottom of the wells by gently adding 50 μl of 80% TCA (final concentration, 16% TCA). Using the seven measures of adsorbance [time zero, (Tz), control growth, (C), and the test growths in the presence of the drug in the five concentrations (Ti)], the percentage growth is calculated at each of the concentrations of agent, as follows:

[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz

[(Ti-Tz)ATz] x 100 for concentrations for which Ti<Tz.

Three response parameters for the dose are calculated for each agent being tested:

Gl ₅₀ (concentration inhibiting 50% of the net cell growth), calculated by [(Ti-Tz)/(C-Tz)] x 100 = 50, is the concentration causing a 50% reduction in the net protein increment (measured by staining with SRB) in the control cells during incubation with the agent.

TGI (concentration causing total growth inhibition) is calculated by Ti = Tz.

LC ₅₀ (concentration causing 50% of net cell death) is the concentra- tion of agent causing a 50% reduction in proteins measured at the end of treatment with the drug compared to the initial value, indicating a net cell loss following treatment. It is calculated by [(Ti-Tz)/Tz] x 100 = -50.

For each of these three parameters, the values are calculated if the level of activity is reached. However, if the level of activity is not reached or is exceeded, the value of the relative parameter is expressed as greater or lower than the maximum or minimum concentration tested.

The data obtained are summarised in Table 2 below.

TABLE 2

In-vitro inhibition of tumour cell lines by CORON ^a

Cell line pGUo ^b pLC ₅₀ ^c pTGI ^d

Leukaemia

CCRF-CEM 6.18 4.30 Nd ^e

HL-60 (TB) 6.93 5.71 6.19

K-562 7.55 6.07 6.74

MOLT-4 7.44 5.85 6.65

RPMI-8226 5.85 4.30 4.90

SR 6.87 4.30 6.06

Non-small cell lung carcinoma

EKVX 7.30 6.09 6.72

HOP-62 7.68 6.19 6.83

HOP-92 7.76 5.95 6.75

NCI-H226 6.67 4.51 5.84

NCI-H23 6.85 5.35 6.02

NCI-H322M 7.73 5.31 6.16

NCI-H460 7.30 6.03 6.68

NCI-H522 6.93 4.30 6.04

Colon tumour

COLO-205 6.30 Nd ^e 5.91

HCC-2998 6.62 5.50 5.98

HCT-116 7.23 Nd ^e 6.64

HCT-15 7.14 5.56 6.29

HT29 7.49 5.89 6.72

KM12 7.55 5.36 6.26

SW-620 7.17 5.64 6.27

Tumour of the CNS

SF-268 8.30 6.17 7.30

SF-295 6.93 4.30 6.05

SF-539 8.30 6.97 7.70

SNB-19 7.43 5.51 6.45

U251 7.25 5.88 6.51

Melanoma

LOX IMVI 7.19 6.40 6.79

MALME-3M 7.93 5.43 5.98

M14 6.57 5.64 6.02

Cell line pGUo ^b pLC ₅₀ ^c pTGI ^d

SK-MEL-2 5.94 4.34 5.39

SK-MEL-28 6.97 5.91 6.48

SK-MEL-5 6.19 5.60 5.89

UACC-62 6.25 5.59 5.92

Ovary carcinoma

IGROV1 7.50 4.30 5.83

OVCAR-3 6.83 4.30 5.25

OVCAR-4 7.28 6.15 6.75

OVCAR-5 6.54 5.34 5.91

SK-OV-3 6.52 5.39 5.89

Renal tumour

786-0 7.04 5.80 6.33

A498 6.69 5.62 6.12

ACHN 7.19 5.69 6.14

CAKI-1 7.09 4.30 6.51

SN12C 7.20 5.66 6.21

TK-10 7.03 5.32 6.18

UO-31 7.16 Nd ^e 6.17

Prostate carcinoma

PC-3 7.65 6.33 6.93

DU-145 7.39 6.52 6.92

Mammary tumour

MCF7 7.01 5.84 6.41

NCI/ADR-RES 7.22 5.57 6.14

MDA-MB-231/ATCC 7.03 5.51 6.13

HS 578T 6.81 4.30 6.02

MDA-MB-435 6.73 Nd ^e 5.97

T-47D 6.71 5.32 5.88 ^a Data obtained from the in-vitro disease-oriented human tumour cells screen of the NCI. ^b pGI ₅₀ is the -log of the molar concentration inhibiting 50% of net cell growth. ^c pLCso is the -log of the molar concentration yielding 50% of net cell death. ^d pTGI is the -log of the molar concentration yielding total growth inhibition. ^e Undetermined.

As it may be seen from the data of the table above, CORON showed an effective cytotoxic activity on all 53 human tumour cell lines, starting - in some cases - from concentrations of 0.1 μM or less. Actually, considering the first column of the table, it can be seen that the concentration at which 50% inhibition of cell growth is obtained lies, in the vast majority of cases, in an interval (on an inverse logarithmic scale) ranging between 6 and 8 - values corresponding to concentrations of 1 μM and 10 nM, respectively.

Moreover, it can be seen how the antiproliferation effect is clearly visible on all the tested cell lines, and thus it is possible to hypothesise a broad range of application of the possible pharmaceutical agent. On the other hand, it is important to note that the best results are achieved with brain tumours, in which this parameter fluctuates between a value of 0.1 μM and 5 nM. This, therefore, seems potentially to be the field of greatest interest of the present invention. The results obtained in the case of certain types of prostate tumour (PC-3), pulmonary carcinoma (HOP-62/92 and NCI-H322M) and melanoma (MALME-3M) are also significant, with a 50% effectiveness at a concentration of about 20 nM. These pathologies, thus, appear particularly promising as fields of application of the present invention.

Very interesting results have been achieved also with CORON2 and CORON4, as it is shown in the following Tables 3 and 4.

TABLE 3

In-vitro inhibition of tumour cell lines by CORON 2

Cell line pGlso* ¹ pLC ₅₀ ^c pTGI ^c

Leukemia

CCRF-CEM 6.68 4.00 5.91

HL-60 (TB) 6.50 5.21 5.88

K-562 6.72 5.45 6.19

MOLT-4 6.74 5.19 6.08

RPMI-8226 6.06 4.00 5.31

SR 7.37 Nd ^e 6.35

Non-small cell lung cancer

A549/ATCC 6.56 4.00 5.67

EKVX 6.32 4.00 5.30

HOP-62 7.34 Nd ^e 6.68

HOP-92 6.23 4.00 5.29

NCI-H226 6.56 Nd ^e 6.12

NCI-H23 Nd ^e 4.00 Nd ^e

NCI-H322M 6.38 4.00 Nd ^e

NCI-H460 6.80 5.48 6.35

NCI-H522 6.73 Nd ^e 6.37

Colon cancer

COLO-205 6.86 6.12 6.49

HCT-116 7.54 6.33 6.80

HCT-15 6.54 4.00 4.99

HT29 6.50 4.00 4.85

KM12 7.23 4.00 6.39

SW-620 6.87 4.00 6.12

CNS cancer

SF-268 7.08 5.92 6.51

SF-295 6.74 4.00 6.28

SF-539 6.68 Nd ^e 6.33

SNB-19 6.80 5.52 6.35

SNB-75 6.49 Nd ^e 5.97

U251 7.32 4.00 6.33

Cell line pGI ₅ o ^b pLC ₅₀ ^c pTGI ^d

Melanoma

LOX IMVI 6.55 4.00 5.83

MALME-3M 6.61 Nd ^e 5.96

M14 6.73 5.85 6.33

SK-MEL-2 6.72 Nd ^e Nd ^e

SK-MEL-28 6.75 Nd ^e 6.32

SK-MEL-5 6.71 Nd ^e 6.36

UACC-257 6.61 4.00 5.83

UACC-62 6.15 4.00 5.40

Ovarian cancer

IGROV1 6.61 4.00 6.12

OVCAR-3 6.61 4.57 6.02

OVCAR-4 6.07 4.00 4.00

OVCAR-5 6.30 4.00 Nd ^e

OVCAR-8 6.67 4.00 5.88

SK-OV-3 6.64 5.11 6.04

Renal cancer

786-0 7.84 Nd ^e 7.28

A498 6.84 6.17 6.51

ACHN 6.42 4.00 5.46

CAKI-1 6.67 4.00 6.26

RXF 393 6.96 6.12 6.54

SN12C 6.53 4.00 4.98

TK-10 6.00 4.00 5.01

UO-31 6.57 4.00 5.94

Prostate cancer

PC-3 6.46 4.00 4.00

DU-145 6.70 5.03 6.15

Breast cancer

MCF7 6.71 4.00 6.27

NCI/ADR-RES Nd ^e 4.00 4.00

MDA-MB-231/ATCC 6.73 4.18 6.02

HS 578T 6.62 4.00 6.08

CeII line pGUo ^b pLC ₅₀ ^c pTGI ^d

MDA-MB-435 6.55 Nd ^e 5.94

BT-549 6.12 4.00 5.31

T-47D 6.68 4.00 5.49 ^a Data obtained from the in-vitro disease-oriented human tumour cells screen of the NCI. ^b pGI ₅₀ is the -log of the molar concentration inhibiting 50% of net cell growth. ^c pLCso is the -log of the molar concentration yielding 50% of net cell death. ^d pTGI is the -log of the molar concentration yielding total growth inhibition. ^e Undetermined.

TABLE 4

In-vitro inhibition of tumour cell lines by CORQN 4 ^a

Cell line pGlso ^b pLC ₅₀ ^c pTGI ^d

Leukemia

CCRF-CEM 6.93 4.00 6.37

HL-60 (TB) 7.16 6.14 6.62

K-562 7.46 6.25 6.75

MOLT-4 7.55 6.19 6.77

RPMI-8226 6.87 4.00 6.33

SR 8.00 Nd ^e 7.19

Non-small cell lung cancer

A549/ATCC 7.32 4.00 6.37

EKVX 6.80 4.00 6.27

HOP-62 7.60 6.29 6.96

HOP-92 6.77 4.00 6.26

NCI-H226 6.95 Nd ^e 6.51

NCI-H23 7.15 4.00 6.29

NCI-H322M 6.79 4.00 6.11

NCI-H460 7.67 6.26 6.99

NCI-H522 7.57 Nd ^e 6.91

Colon cancer

COLO-205 7.68 Nd ^e 7.07

HCC-2998 6.63 5.43 6.07

HCT-116 8.00 6.58 7.41

Cell line pGI ₅ o ^b pLC ₅₀ ^c pTGI ^c

HCT-15 7.17 4.00 6.46 HT29 7.43 4.78 6.36 KM12 7.98 6.27 7.05 SW-620 8.00 6.02 6.95

CNS cancer

SF-268 8.00 6.71 7.62 SF-295 7.33 4.00 6.52 SF-539 7.37 Nd ^e 6.73 SNB-19 7.76 6.15 6.92 SNB-75 7.25 Nd ^e 6.68 U251 8.00 Nd ^e 6.87

Melanoma

LOX IMVI 6.97 4.00 6.32

MALME-3M 7.45 Nd ^e 6.76

M14 7.84 6.55 7.23

SK-MEL-2 7.45 Nd ^e 6.80

SK-MEL-28 7.65 Nd ^e 6.95

SK-MEL-5 7.34 6.38 6.76

UACC-257 6.86 Nd ^e 6.40

UACC-62 6.82 Nd ^e 6.49

Ovarian cancer

IGROV1 7.28 6.06 6.62

OVCAR-3 7.66 6.34 6.95

OVCAR-4 6.85 4.00 6.10

OVCAR-5 7.16 Nd ^e 6.36

OVCAR-8 7.87 Nd ^e 6.72

SK-OV-3 7.50 6.23 6.82

Renal cancer

786-0 8.00 6.99 7.62 A498 7.70 6.78 7.32 ACHN 7.31 Nd ^e 6.43 CAKI-1 7.37 4.03 6.60 RXF 393 6.89 6.07 6.48

Cell line pGlso ^b pLC ₅₀ ^c pTGI ^d

SN12C 7.35 Nd ^e 6.37

TK-10 6.65 4.00 6.08

UO-31 6.93 Nd ^e 6.49

Prostate cancer

DU-145 7.52 6.25 6.86

Breast cancer

MCF7 7.34 5.95 6.61

NCI/ADR-RES 6.74 4.00 5.95

MDA-MB-231/ATCC 7.68 6.05 6.83

HS 578T 7.31 4.00 6.47

MDA-MB-435 7.07 Nd ^e 6.66

BT-549 6.51 Nd ^e Nd ^e

T-47D 7.54 Nd ^e 6.73 ^a Data obtained from the in-vitro disease-oriented human tumour cells screen of the NCI. ^b pGI ₅ o is the -log of the molar concentration inhibiting 50% of net cell growth. ^c pLC ₅ o is the -log of the molar concentration yielding 50% of net cell death. ^d pTGI is the -log of the molar concentration yielding total growth inhibition. ^e Undetermined.

The present invention has been disclosed with reference to some specific embodiments thereof, but it is to be understood that variations or modifications can be brought by persons skilled in the art without departing from the scope of the appended claims.