HK1089160B - 7-imino derivatives of camptothecin having antitumor activity - Google Patents

Description

7-imino derivatives of camptothecin having antitumor activity

The present invention relates to compounds having antitumor activity, in particular to novel camptothecin derivatives, processes for their preparation, their use as antitumor agents and pharmaceutical compositions containing these compounds as active ingredients.

Background

Camptothecin is an alkaloid which was first isolated by Wall et al (j.am. chem. soc.88, 3888-3890(1966)) from a plant camptotecaacacetina tree (belonging to the family lakefruit tree) of chinese origin.

The molecule has a pentacyclic structure of a lactone in the E ring, which is essential for cytotoxicity.

The drug has been shown to have a broad spectrum of anti-tumor activity, particularly against colon tumors, other solid tumors and leukemia, with initial clinical trials beginning in the early 70 s. For clinical experiments, the National Cancer Institute (NCI) prepared a water-soluble sodium salt (NSC100880) due to the low water solubility of camptothecin (hereinafter, CPT). Phase I and II clinical trials were not completed because the compound showed higher toxicity (hemorrhagic upper-nexitis, gastrointestinal toxicity such as nausea, vomiting, diarrhea, and bone marrow suppression, especially leukopenia and thrombocytopenia).

In any case, the sodium salt showed less activity than CPT, since the inactive form (ring opening) was quantitatively superior to the lactone active form (ring closing) at pH7.4, which was predominant at pH < 4.0.

Subsequently, many CPT analogues were synthesized with the aim of obtaining compounds with lower toxicity and higher water solubility. Two drugs are currently marketed, irinotecan (CPT-11) by Upjohn under the registered trademark Camptosar ®, and Topotecan (Topotecan) by Smith Kline & Beecham under the registered trademark Hymcamptamin ® or Thycantin ®. Other derivatives are in different stages of clinical phase II development, such as NSC-603071 (9-aminocamptothecin), 9-NC or 9-nitrocamptothecin (a prodrug that can be converted to 9-aminocamptothecin), GG-211(GI147211) and DX-8591f, the latter being water-soluble. All derivatives identified to date contain the 5-ring parent structure necessary for cytotoxicity. Modification of the first ring, for example in the case of the above-mentioned drugs, has been shown to increase the water solubility of the drugs and their greater tolerance.

Water-soluble irinotecan is approved for the treatment of a variety of solid tumors and ascites (colorectal, skin, gastric, breast, small and non-small cell lung, cervical and ovarian cancers, and non-hodgkin's lymphoma). Moreover, irinotecan is active in solid tumors resistant to topotecan, vincristine or milflange and renders MDR-1 cells weakly resistant to the drug. The active metabolite was identified as a 10-hydroxy derivative (SN-38) which is produced by the action of carboxylesterases. CPT-11 showed good activity using different routes of administration, e.g. intraperitoneal, intravenous or oral (Costin, d., potmethylhexyl, m.. Advances in pharmacol., 29B, 51-721994).

Cisplatin or etoposide were also administered with CPT-11, which showed a synergistic effect due to their ability to block DNA repair. In this case, however, leukopenia and diarrhea of grade 3 and 4 occurred (Sinha, B.K., (1995) topoisomerase inhibitors, Drugs, 49, 11-19, 1995).

Topotecan has significant oral bioavailability. Oral administration has proven to be a convenient method of achieving prolonged exposure to drugs without the necessity of using temporary catheters (Rothenberg, m.l., ans of Oncology, 8, 837-855, 1997). In addition, the water-soluble analogs of CPT exhibit activity against different types of tumors when administered by different routes of administration (e.g., intraperitoneal, intravenous, subcutaneous, or oral). More assured results were obtained with 5 days of intravenous instillation of topotecan hydrochloride in various tumor cases, such as small and non-small cell lung cancer, ovarian cancer, breast cancer, gastric cancer, liver cancer, prostate cancer, soft tissue sarcoma, head and neck cancer, esophageal cancer, drug-resistant colorectal cancer, glioblastoma multiforme, chronic and acute myelocytic leukemia, and the like. However, also in this case severe side effects such as neutropenia and thrombocytopenia occur, but gastrointestinal toxicity such as nausea, vomiting and diarrhea is milder.

It has been demonstrated that the major conversion and elimination pathways of this drug include lactone hydrolysis and urinary excretion: in fact, the lactone form is 50% hydrolyzed to ring opening 30 minutes after instillation. Topotecan crosses the blood brain barrier 10 minutes after instillation (30% is present in cerebrospinal fluid relative to plasma). In contrast, the amount of camptothecin that crosses the blood brain barrier is not significant, probably because of its binding to proteins.

The clinical development of 9-aminocamptothecin is influenced by its extremely low water solubility. Recently, a colloidal dispersion has been prepared which makes it possible to enter phase II clinical trials for this compound. Due to the short half-life of the drug, prolonged exposure times (72 hours-21 days) appear to be necessary in order to exhibit its anti-tumor activity (Dahut et al, 1994). Note the response in those patients with untreated colo-rectal cancer and breast cancer and drug resistant lymphoma. The activity shown against Pgp-positive tumors suggests a lack of cross-resistance to drug-resistant MDR-1 cells, and in addition, bone marrow and gastrointestinal toxicity was again observed.

Lurtotecan is the most water soluble analog, with in vitro activity comparable to topotecan. Two schemes are adopted: one is instillation for 30 minutes per day, 5 days every 3 weeks, and the other is instillation once every 3 weeks, 72 hours each time. Responses were observed in patients with cervical, ovarian, breast, liver tumors. Also in this case, hematological toxicity was detected.

The molecule is as follows:

9-Nitrocetothecin is an oral prodrug that converts to 9-aminocamptothecin rapidly after administration. A response was observed in those patients with pancreatic, ovarian and breast cancer.

Although a major proportion of tumor cells are highly sensitive to topoisomerase I inhibitors due to the high enzyme levels, there are also some tumor lines that develop resistance. This is due to other mechanisms than overexpression of MDR1 and MRP (protein associated with multiple drug resistance) genes and their products, P (Pgp) glycoprotein and MRP protein, for which topotecan or CPT-11 is not a good substrate (Kawato, Y. et al, J.Pharm.Pharmaco., 45, 444-.

Indeed, it has been observed that some resistant tumor cells contain mutated forms of topoisomerase I, thus impairing the formation of the topoisomerase I-DNA complex, or that some cells lack the carboxylesterase activity necessary for the conversion of CPT-11 to its active metabolite SN-38, thus conferring resistance to this drug (Rothenberg, 1997, ibid).

Among the drugs employed in the treatment of tumors, the interest in topoisomerase I inhibitors is mainly due to the following considerations: a) efficacy against tumors that are naturally resistant to conventional drugs including topoisomerase I I, b) the level of topoisomerase I enzyme remains elevated throughout the entire phase of the cycle; c) many tumors express high levels of target enzymes; d) lack of recognition of proteins (Pgp or MRP) involved in multiple drug resistance phenomena, and lack of detoxification enzyme-mediated mechanisms associated with glutathione-dependent systems (glutathione peroxidase and glutathione S-transferase) (Gerrits CJH et al, Brit. J. cancer, 76, 952-962).

When considering the potential clinical advantages of topoisomerase I inhibitors, including broad spectrum of anti-tumor activity and less susceptibility to drug resistance, the goal of this study was to identify topoisomerase I inhibitors that have lower toxicity than the drugs on the market or that are still in the clinical trial phase. Factors that determine the relative potency of camptothecin analogs include: a) intrinsic activity of topoisomerase I inhibition; b) mean life of the drug; c) interaction with plasma proteins; d) the ratio between the active (lactone) and inactive (carboxylate) forms of the cycle; e) drug sensitivity relative to cellular efflux mediated by glycoprotein P or MRP; f) stability of binding to topoisomerase I (Rothenberg, 1997, ibid).

Among the major adverse effects of irinotecan and other camptothecin derivatives, myelosuppression and gastrointestinal toxicity (e.g., diarrhea and vomiting) have been observed. Diarrhea can occur early or late and can be a dose limiting factor. Many antineoplastic drugs can cause emesis and late-stage diarrhea, while early-stage diarrhea, which occurs during or immediately after instillation, is almost specific for irinotecan and some camptothecin derivatives.

Toxic reactions occur mainly in the intestinal tract.

In order to alleviate diarrhea, CPT-11 was combined in several clinical trials with jamesonide (a synthetic opioid, mu opioid intestinal receptor agonist (Abigerges, 1994; Abigerges, 1995) and enkephalinase inhibitor (acetyltolfentra) or ondansetron (a 5-HT3 receptor antagonist) or diphenidamine (an H)₁Receptor antagonist).

To date, the problems associated with the use of camptothecin derivatives as antitumor agents can be summarized as follows:

camptothecin (CPT) and many of its active derivatives have a low water solubility;

subsequent derivatives have severe side effects at gastrointestinal and bone marrow levels;

some tumor lines develop resistance to topoisomerase I inhibitors;

drugs with better therapeutic indices are constantly being sought.

Patent application WO97/31003 discloses camptothecin derivatives substituted in the 7, 9 and 10 positions. The following substitutions are provided at the 7 position: -CN, -CH (CN) -R₄、-CH＝C(CN)-R₄、-CH₂-CH＝C(CN)-R₄、-C(＝NOH)-NH₂、-CH＝C(NO₂)-R₄、-CH(CN)-R₅、-CH(CH₂NO₂)-R₅5-tetrazolyl, 2- (4, 5-dihydrooxazolyl), 1, 2, 4-oxadiazolidin-3-yl-5-one, wherein R₄Hydrogen, straight or branched chain alkyl of 1 to 6 carbon atoms, nitrile, carboxyalkoxy. In theseOf the possible compounds, WO97/31003 discloses only carrying the groups-CN and-CH ═ C (CN) in position 7₂And camptothecin derivatives unsubstituted in the 9 and 10 positions.

Of these compounds, the best one has proven to be the 7-nitrile (R)₄-CN), also referred to herein as CPT83, which has cytotoxic activity against non-small cell lung tumors (non-SCLC, H-460). This tumor line is inherently resistant to cytotoxic therapy and, despite overexpression of the target enzyme, responds only to a moderate extent to topoisomerase I inhibitors. CPT83 is more active than topotecan as reference compound, and in general it provides better pharmacological profile, even in terms of tolerability and better therapeutic index.

CPT83 can be prepared by a synthetic route involving oxidation of 7-hydroxymethylcamptothecin to camptothecin 7-aldehyde, followed by conversion of the latter to an oxime and finally to a nitrile.

Starting compounds and intermediates are disclosed in Sawada et al, Chem, Pharm, Bull, 39(10)2574 (1991). This paper refers to a family of patents with priority in 1981, such as the european patent application EP0056692 disclosed in 1982. In these publications, the compound camptothecin 7-aldehyde and its oxime compounds are disclosed. These derivatives are utilized in order to provide compounds with antitumor activity with low toxicity starting from 7-hydroxymethylcamptothecin. In the paper published in chem. pharm. bull., 39, (10)2574, (1991), the authors demonstrated that 7-alkyl and 7-acyloxymethyl derivatives, which were not foreseen in the above patent application, are more active compounds for the murine leukemia L1210 line than camptothecin, whereas always less activity than camptothecin, such as hydrazone and oxime-CH (═ NOH), can be observed in compounds with highly polar 7-substitution.

EP1044977 discloses camptothecin derivatives bearing oximes (O-substituted in position 7). The formula also includes camptothecin derivatives having an enamine group at the 7-position. In this reference, the main teaching relates to the antitumor activity of oxime derivatives, while imines are given in only a few examples of synthetic preparations, but no pharmacological data are provided. Subsequent work by the inventors of the above-mentioned patent has focused on oxime derivatives, in particular tert-butoxyoxime derivatives, which are known under the name Gimatecan and are now in clinical trials. Imines were considered to be only an alternative to oximes, but initial pharmacological assays prevented further development of this class of compounds.

Summary of The Invention

It has now surprisingly been found that camptothecin bearing an aromatic enamine group in the 7-position has anti-tumour activity. The compound has better therapeutic index.

Accordingly, one subject of the present invention is the compounds of formula (I), their N₁Oxides, their single isomers, in particular-C (R)₅)＝N-R₄Cis and trans isomers of the radicals, their possible enantiomers, diastereomers and related mixtures, their pharmaceutically acceptable salts and active metabolites

Wherein: r₁is-C (R)₅)＝N-R₄Group, wherein R₄Is phenyl, optionally substituted with one or more groups selected from: halogen, hydroxy, keto, C₁-C₈Alkyl radical, C₁-C₈Alkoxy, phenyl, cyano, nitro, -NR₆R₇Wherein R is₆And R₇Are hydrogen, (C), the same or different from each other₁-C₈) A linear or branched alkyl group; -S-S- (2-aminophenyl), -S-S- (4-aminophenyl), -SCH₃and-CH₂ON＝C(CH₃₎₂；

R₅Is hydrogen, C₁-C₈Straight or branched chainChain alkyl radical, C₁-C₈Straight-chain or branched alkenyl, C₃-C₁₀Cycloalkyl group, (C)₃-C₁₀) Cycloalkyl- (C)₁-C₈) Straight or branched alkyl, C₆-C₁₄Aryl group, (C)₆-C₁₄) Aryl radical- (C)₁-C₈) A linear or branched alkyl group;

R₂and R₃Hydrogen, hydroxy, C, equal to or different from each other₁-C₈Straight or branched alkoxy.

The invention comprises the use of a compound of formula (I) as described above as an active ingredient of a medicament, in particular a medicament for the treatment of tumors. Another aspect of the invention is also the use of a compound of formula (I) as an active ingredient of a medicament for the treatment of viral infections. Another object of the present invention is also the use of the compounds of formula (I) as active ingredients of medicaments having activity against plasmodium falciparum.

The invention encompasses pharmaceutical compositions comprising a compound of formula (I) as an active ingredient in combination with pharmaceutically acceptable carriers and excipients.

The invention also includes processes for the preparation of compounds of formula (I) and related key intermediates.

Detailed Description

Within the scope of the invention as C₁-C₈Examples of linear or branched alkyl radicals are methyl, ethyl, propyl, butyl, pentyl, octyl and their possible isomers such as isopropyl, isobutyl, tert-butyl.

Halogen means fluorine, chlorine, bromine, iodine.

Examples of pharmaceutically acceptable salts are salts with pharmaceutically acceptable acids, inorganic and organic acids, such as hydrochloric acid, sulfuric acid, acetic acid in the case of basic nitrogen atoms, or pharmaceutically acceptable bases, inorganic and organic bases, such as alkali and alkaline earth metal hydroxides, ammonium hydroxides, amines and heterocyclic bases in the case of acidic groups, such as carboxyl groups.

A first group of preferred compounds are those wherein R4 is phenyl substituted with at least one residue selected from: methyl, tert-butyl, methoxy, hydroxy, chlorine, iodine, nitro, -S-S- (2-aminophenyl), -S-S- (4-aminophenyl), -SCH₃and-CH₂ON＝C(CH₃)₂. In particular, phenyl is most preferably substituted in the ortho position.

A second group of particularly preferred compounds includes:

7- (2-methylphenyl) iminomethylcamptothecin (ST2212)

7- (2-chlorophenyl) iminomethylcamptothecin (ST2228)

7- (2, 6-dimethylphenyl) iminomethylcamptothecin (ST2317)

7- (2-iodophenyl) iminomethylcamptothecin (ST2316)

7- (2-methoxyphenyl) iminomethylcamptothecin (ST2343)

7- (4-methylphenyl) iminomethylcamptothecin (ST2478)

7- (2-hydroxyphenyl) iminomethylcamptothecin (ST2389)

7- (4-chlorophenyl) iminomethylcamptothecin (ST2412)

7- (4-methoxyphenyl) iminomethylcamptothecin (ST2477)

7- [ (4-isopropylidene-amino-oxymethyl) phenyl ] iminomethylcamptothecin (ST2460)

7- (2-Tert-butylphenyl) iminomethylcamptothecin (ST2388)

7-Phenyliminomethylcamptothecin (ST1546)

7- (4-Nitrophenyl) Iminomethylcamptothecin (ST1561)

7-2- (2-Aminophenyldithio) phenyliminomethylcamptothecin (ST1737)

7-4- (4-Aminophenyldithio) phenyliminomethylcamptothecin (ST2034)

7-4- (4-Aminothiophenyl) phenyliminomethylcamptothecin (ST2069)

7- (2-methylthiophenyl) iminomethylcamptothecin (ST2138)

7- (4-tert-butylphenyl iminomethyl) -camptothecin (ST2619)

7- (4-methylthiophenyliminomethyl) -camptothecin (ST2667)

7- (4-Hydroxyphenyliminomethyl) -camptothecin (ST 2616).

The compounds of formula (I) can be prepared in different ways depending on the nature of the R4 group attached to the nitrogen of the 7-iminomethyl group.

R₄The compound of formula (I) as defined above may be prepared from camptothecin 7-aldehyde (formula Ia, R)₅Hydrogen) or 7-ketocamptothecin (formula Ia, R)₅Other than hydrogen) to start the production,

wherein R is₁Is a group-C (R)₅) Is not O, but R₅As defined for formula (I), R₂And R₃As defined in formula (I). Reacting a compound of formula (Ia) with R₄As defined above of formula (IIb) R₄-NH₂To give a compound of the formula (I), wherein R₁Is a group-C (R)₅)＝N-R₄，R₄As defined in formula I. This reaction can be carried out by conventional methods known to those skilled in the art, usually to form an imine. Preferably the molar ratio between the 7-aldehyde or 7-ketocamptothecin and the amine is from 1: 3 to 3: 1. Salts of the amines of interest may also be used. The reaction is carried out in the presence of a base, for example an inorganic base such as potassium carbonate, or an organic base such as triethylThe reaction is carried out in the presence of an amine or diazabicyclononene using a polar solvent, preferably methanol or ethanol, and is carried out at a temperature between room temperature and the boiling point of the solvent, optionally in the presence of a dehydrating agent such as sodium or magnesium sulfate, molecular sieves. If desired, the reaction may also be carried out in the presence of a catalyst, for example a Lewis acid (as disclosed by Moretti and Torre, Synthesis, 1970, 141; or Kobayashi et al, Synlett, 1977, 115).

Camptothecin 7-aldehyde and camptothecin 7-oxime are disclosed in patent application EP0056692 and said Sawada et al, chem. pharm. bull, 39, (10)2574 (1991).

The N1-oxides of the compounds of formula (I) are prepared according to known heteroaromatic nitroxide processes, preferably by oxidation with acetic acid or trifluoroacetic acid and hydrogen peroxide, or by reaction with organic peroxy acids (a. albini and s. pietra, Heterocyclic N-oxides, CRC, 1991).

With respect to R present in the different reactants of formula II₄These reactants are commercially available or can be prepared according to methods known in the literature, to which experts in the field can turn and use their own judgment capabilities to complete the preparation.

The pharmaceutically acceptable salts are obtained by conventional methods found in the literature and need not be further disclosed.

The compounds disclosed in the present invention exhibit antiproliferative activity and are therefore useful for their therapeutic activity, and they possess physical and chemical properties that make them suitable for use in formulating pharmaceutical compositions.

The pharmaceutical compositions comprise at least one compound of formula (I) in an amount such as to produce a significant therapeutic effect, in particular an anti-tumour effect. The compositions comprised in the present invention are conventional and can be obtained using common methods in the pharmaceutical industry. The compositions may take solid or liquid forms suitable for oral, parenteral, intravenous administration, depending on the route of administration desired. According to the invention, the composition comprises, together with the active ingredient, at least one pharmaceutically acceptable carrier or excipient. Formulation aids such as co-solvents, dispersants, suspending agents, emulsifiers are very useful.

The compounds of formula (I) may also be used in combination with other active ingredients, for example other antineoplastic agents, either in separate or single dosage forms.

The compounds of the invention are useful as medicaments with anti-tumor activity, for example in the treatment of lung tumors, such as non-small cell lung tumors, colon-rectal tumors, prostate tumors, gliomas and the like.

The cytotoxicity of the compounds of the invention was analyzed in the human tumor cell system using the antiproliferative activity assay as an evaluation method of the cytotoxic potential.

The cell line used was a lung non-small cell carcinoma that was of a non-small cell tissue type, designated NCIH 460.

For in vivo studies, dissolution was performed in 10% DMSO in double distilled water in the event dissolution in saline was not possible, and oral administration was performed in a volume of 10 ml/kg.

Antitumor activity

Atimicnu/nu Swiss mice (Charles River, Calco, Italia) aged 10-12 weeks were used. Animals were kept in the laminar flow chamber according to the United Federal coordination Commission Cancer Research (United kingdomCo-addressing Committee Cancer Research) guidelines. The protocol was approved by the animal Experimental ethics Committee of Istituto Nazionale per lo study la Curadei Tumori.

Approximately 2X 2mm are derived from mice which are inoculated subcutaneously with 10⁶Individual cell NCIH 460/mouse) were implanted bilaterally subcutaneously, 5 mice per group.

When the tumor is accessible, the animal is treated with the compound.

One weekThe width, minimum diameter (1), length and maximum diameter (L) of the tumor were measured twice using a Vernier caliper in mm. According to equation 1²X L/2 tumor volume (mm)³). The efficacy of the molecules was assessed as the percentage TVI of the treated versus the control group based on the TVI% ═ 100- (T/C × 100), where T is the mean tumor volume of the treated group and C is the value of the control group. The compound is considered active when TVI%. gtoreq.50.

Further advantages of these molecules can be identified in a wide effective dose range, which indicates an increased therapeutic index and a higher operability of the therapeutic application, especially if a longer administration time is foreseen, above all injectable formulations, with variable regimens and dosages.

An important drawback of conventional camptothecins is the reversibility of their binding in ternary complexes (drug-DNA-enzyme). This reversibility affects the efficacy of the drug, as it does not allow the conversion of single stranded DNA cleavage to double stranded DNA cleavage during DNA synthesis.

The advantages offered by the compounds of the present invention with respect to the prior art are clearly that of overcoming the limitation of the reversibility of the ternary complex.

In preclinical studies, the compounds of the invention showed cytotoxic activity against a variety of tumor cell lines.

This broad spectrum of anti-cancer activity was demonstrated in mice transplanted with human tumor xenografts, including NSCLC (H460, a549), prostate cancer (JCA-1), glioblastoma (GBM/7), gastric cancer (MKN28), osteosarcoma (U2OS), ovarian cancer (a2780/Dx, a2780/DDP) and colon cancer (HT29, CoBA) as well as murine lung cancer (M109) and leukemia model (L1210).

Preclinical data indicate that the compounds of the invention are active anticancer agents against human cancers, in particular against non-small cell lung cancer (NSCLC), glioblastoma and prostate cancer.

The antitumor activity of exemplary compounds of the invention is shown in Table 1 below

TABLE 1

Compound IC₅₀(mg/ml)

ST 17370.0295 + -0.07 (Water soluble)

ST2034 0.235±0.02

ST2069 0.14±0.04

ST2138 0.033±0.004

ST2228 0.036±0.01

ST2316 0.062±0.03

ST2317 0.075±0.02

ST1561(CPT160) 0.14±0.03

ST2412 0.042±0.0012

ST2212 0.235±0.06

ST2388 0.033±0.004

ST2389 0.015±0.008

ST2343 0.028±0.012

ST2477 0.08±0.002

The in vivo activity of compound ST1737 is shown in table 2 below.

TABLE 2

Effect of orally administered Q4dx4 on ST1737 in athymic nude mice with MKN-28 human gastric carcinoma growing thereunder

Medicine	Dosage (mg/kg)	TVI％	BWL％	Tox
					ST1737	4712	695668	4	0/40/40/4

The mean tumor doubling time of control mice was 7.1. + -. 1.3 and 5.4. + -. 1.4 days (exp.656 and 664, respectively).

¹Tumor volume inhibition% of treated versus control tumors at 11-12 days post-final treatment.

²Weight loss due to drug treatment.

³Mice were sacrificed/treated.

The high cytotoxic potency of the compounds of the invention represented here as examples by a preferred compound ST1737 is also reflected by a strong antitumor activity. With a panel of tumor xenografts characterized by significant response to topotecan (i.e., TVI > 80%), the spectrum of antitumor activity of the compounds of the invention against a significant number of human tumor models was greatly improved. In particular, impressive antitumor efficacy was found in the treatment of many tumor models, with very high regression obtained in a large number of treated animals. Further, the compounds of the invention are capable of inducing high amounts of CR in tumors characterized by an MDR-phenotype. This observation is very important, indicating that the compounds of the invention are not substrates for P-glycoprotein.

Other therapeutic advantages of the compounds of the invention are related to the following factors: a) improvement in therapeutic index, b) efficacy of the drug over a larger dose range, c) evidence of efficacy with widely differing treatment regimens, which makes the compounds of the invention less dependent on treatment regimen than topotecan.

The following examples further illustrate the invention.

Example 1

General procedure: to 5ml of anhydrous CH containing 4 Å MS₂Cl₂Yb (OTf) of (1)₃(16mg, 0.03mmol) in 20ml CH₂Cl₂To a solution of 7-formylcamptothecin (100mg, 0.26mmol) in (A) was added 0.5ml of CH₂Cl₂Amine (0.26 mmol). The resulting mixture was stirred at room temperature until the reaction was complete. After filtration through a sieve, 20ml of water are added and the two phases are separated. The aqueous layer was extracted rapidly three times with dichloromethane. The combined organic phases were dried and evaporated and the product was purified by flash chromatography on silica gel.

The following compounds were obtained. In some cases, antitumor activity (IC for H-460) was shown₅₀，μM)

7- (2-methylphenyl) iminomethylcamptothecin (ST2212)

IC₅₀(H-460，μM)：0.10

M.P.247-248℃dec.，¹H NMR(DMSO-d₆)δ：0.87(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，2.5(s，Ar-CH₃)，5.4(s，H₂-17)，5.60(s，H₂-5)，6.55(s，-OH)，7.25-7.50(m，4H Ar，H-14)，7.75(m，H-11)，7.95(m，H-10)，8.25(dd，H-12)，9.10(dd，H-9)，9.65(s，CH＝N)。

7- (2-chlorophenyl) iminomethylcamptothecin (ST2228)

IC₅₀(H-460，μM)：0.07

M.P.>240℃dec.，¹H NMR(DMSO-d₆)δ：0.83(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，5.45(s，H₂-17)，5.60(s，H₂-5)，6.50(s，-OH)，7.35-7.50(m，H-14；4H Arom.)，7.85(m，H-11)，7.95(m，H-10)，8.30(dd，H-12)，9.10(dd，H-9)，9.70(s，CH＝N)。

7- (2, 6-dimethylphenyl) iminomethylcamptothecin (ST2317)

IC₅₀(H-460，μM)：0.15

M.P.250℃dec.，¹H NMR(DMSO-d₆)δ：0.87(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，2.25(s，2Ar-CH₃)，5.4(s，H₂-17)，5.60(s，H₂-5)，6.55(s，-OH)，7.0-7.30(m，3H Ar)，7.40(s，H-14)，7.8(m，H-11)，7.9(m，H-10)，8.25(dd，H-12)，8.85(dd，H-9)，9.5(s，CH＝N)。

7- (2-iodophenyl) iminomethylcamptothecin (ST2316)

IC₅₀(H-460，μM)：0.06

M.P.240℃dec.，¹H NMR(DMSO-d₆)δ：0.87(t，J＝7Hz，H₃-18)，1.8-1.9(m，H₂-19)，5.45(s，H₂-17)，5.75(s，H₂-5)，6.55(s，-OH)，7.1-7.6(m，4H Ar，H-14)，7.8(m，H-11)，7.9(m，H-10)，8.30(dd，H-12)，9.10(dd，H-9)，9.65(s，CH＝N)。

7- (2-methoxyphenyl) iminomethylcamptothecin (ST2343)

IC₅₀(H-460，μM)：0.06

M.P.244-246℃dec.，¹H NMR(DMSO-d₆)δ：0.83(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，3.95(s，OCH₃)，5.45(s，H₂-17)，5.55(s，H₂-5)，6.45(s，-OH)，7.0-7.50(m，H-14；4H A r om.)，7.7(m，H-11)，7.85(m，H-10)，8.25(dd，H-12)，8.9(dd，H-9)，9.70(s，CH＝N)。

7- (4-methylphenyl) iminomethylcamptothecin (ST2478)

IC₅₀(H-460，μM)：0.18

M.P.159-160℃dec.，¹H NMR(DMSO-d₆)δ：0.83(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，2.35(s，Ar-CH₃)，5.37(s，H₂-17)，5.5(s，H₂-5)，6.45(s，-OH)，7.25-7.35(m，H-14；2H Arom.)，7.4-7.5(m，2Harom.)，7.7(m，H-11)，7.85(m，H-10)，8.16(dd，H-12)，8.9(dd，H-9)，9.55(s，CH)。

7- (2-hydroxyphenyl) iminomethylcamptothecin (ST2389)

IC₅₀(H-460，μM)：0.06

M.P.252-254℃dec.，¹H NMR(DMSO-d₆)δ：0.87(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，5.4(s，H₂-17)，5.60(s，H₂-5)，6.55(s，-OH)，6.90-7.5(m，4H A r，H-14)，7.85-8.0(m，H-11，H-10)，8.35(dd，H-12)，8.90(dd，H-9)，9.70(s，CH＝N)。

7- (4-chlorophenyl) iminomethylcamptothecin (ST2412)

IC₅₀(H-460，μM)：0.08

M.P.246-247℃dec.，¹H NMR(DMSO-d₆)δ：0.83(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，5.40(s，H₂-17)，5.55(s，H₂-5)，6.45(s，-OH)，7.35(s，H-14)，7.50-7.60(m，4H arom.)，7.85(m，H-11)，7.95(m，H-10)，8.25(dd，H-12)，8.95(dd，H-9)，9.55(s，CH＝N)。

7- (4-methoxyphenyl) iminomethylcamptothecin (ST2477)

IC₅₀(H-460，μM)：0.16

M.P.252-255℃dec.，¹H NMR(DMSO-d₆)δ：0.87(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，3.8(s，-OCH₃)，5.4(s，H₂-17)，5.45(s，H₂-5)，6.55(s，-OH)，7.05(d，2H Ar)，7.35(s，H-14)，7.60(d，2H Ar)，7.85(m，H-11)，7.9(m，H-10)，8.25(dd，H-12)，8.8(dd，H-9)，9.5(s，CH)。

7- [ (4-isopropylidene-amino-oxymethyl) phenyl ] iminomethylcamptothecin (ST2460)

IC₅₀(H-460，μM)：0.01

M.P.147℃dec.，¹H NMR(DMSO-d₆)δ：0.85(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19，C(CH₃)，₂)，5.05(s，CH₂-O)，5.40-5.55(m，H₂-17，H₂-5)，6.50(s，-OH)，7.35(s，H-14)，7.40-7.60(m，4H arom.)，7.75-7.85(m，H-11)，7.86-7.95(m，H-10)，8.25(dd，H-12)，8.95(dd，H-9)，9.60(s，CH＝N)。

7- (2-Tert-butylphenyl) iminomethylcamptothecin (ST2388)

IC₅₀(H-460，μM)：0.07

M.P.215℃dec.，¹H NMR(DMSO-d₆)δ：0.85(t，J＝7Hz，H₃-18)，1.45(s，9H，t Bu t)，1.7-1.9(m，H₂-19)，5.35-5.75(m，H₂-17，H₂-5)，6.50(s，-OH)，7.05-7.5(m，H-14；4H a r om.)，7.75-7.85(m，H-11)，7.88-7.95(m，H-10)，8.25(dd，H-12)，8.95(dd，H-9)，9.45(s，CH＝N)。

7-Phenyliminomethylcamptothecin (ST1546)

IC₅₀(H-460，μM)：0.13

¹H NMR(DMSO-d₆)δ：0.83(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，5.37(s，H₂-17)，5.5(s，H₂-5)，6.45(s，-OH)，7.25-7.35(m，H-14；H arom.)，7.4-7.5(m，4H arom)，7.7(m，H-11)，7.85(m，H-10)，8.16(dd，H-12)，8.9(dd，H-9)，9.55(s，CH＝N)。

7- (4-Nitrophenyl) Iminomethylcamptothecin (ST1561)

IC₅₀(H-460，μM)：0.28

M.P.260-265℃dec.¹H NMR(DMSO-d₆)δ：0.85(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，5.35(s，H₂-17)，5.48(s，H₂-5)，6.45(s，-OH)，7.3(s，H-14)，7.6-7.7(m，2Ar)，7.8(m，H-11)，7.9(m，H-10)，8.25(dd，H-12)，8.35-8.40(m，2Ar)，8.9(dd，H-9)，9.67(s，CH＝N)。

7-2- (2-Aminophenyldithio) phenyliminomethylcamptothecin (ST1737)

IC₅₀(H-460，μM)：0.017

¹H NMR(DMSO-d₆)δ：0.83(t，J＝7Hz，H₃-18)1.7-1.9(m，H₂-19)5.35-5.75(6H，m，H₂-5+H-17+NH₂)，6.40(1H，m，ArH)，6.5-6.6(2H，m，1ArH+OH)，6.90(1H，m，ArH)，7.25-7.45(4H，m，3ArH+H-14)，7.15-8.0(4H，m，4ArH)，8.25(1H，dd)，9.75(1H，s，CH＝N)。

7-4- (4-Aminophenyldithio) phenyliminomethylcamptothecin (ST2034)

IC₅₀(H-460，μM)：0.39

M.P.154-155℃dec.，¹H NMR(DMSO-d₆)δ：0.83(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，5.40(s，H₂-17)，5.55(s，H₂-5+NH₂)，6.50(s，-OH)，6.55(m，2H Ar)，7.25(m，2H Ar)，7.35(s，H-14)，7.60(m，4H Ar)，7.8(m，H-11)，7.9(m，H-10)，8.25(dd，H-12)，9.0(dd，H-9)，9.70(s，CH＝N)。

7-4- (4-Aminothiophenyl) phenyliminomethylcamptothecin (ST2069)

IC₅₀(H-460，μM)：0.24

M.P.187-188℃dec.，¹H NMR(DMSO-d₆)δ：0.87(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，5.4(s，H₂-17)，5.55(s，H₂-5+NH₂)，6.55(s，-OH)，6.65(m，2H Ar)，7.10-7.50(m，6H Ar+H-14)，7.8(m，H-11)，7.9(m，H-10)，8.30(dd，H-12)，9.0(dd，H-9)，9.5(s，CH＝N)。

7- (2-methylthiophenyl) iminomethylcamptothecin (ST2138)

IC₅₀(H-460，μM)：0.06

M.P.>250℃dec.，¹H NMR(DMSO-d₆)δ：0.83(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，2.50(s，SCH₃)，5.40(s，H₂-17)，5.70(s，H₂-5)，6.45(s，-OH)，7.25-7.35(m，H-14；3H arom.)，7.6(m，1H arom)，7.8(m，H-11)，7.95(m，H-10)，8.30(dd，H-12)，9.10(dd，H-9)，9.55(s，CH＝N)。

Example 2

To 7mL of anhydrous CH₂Cl₂To a suspension of 20S-camptothecin-7-aldehyde (1) (100mg, 0.26mmol) was added the appropriate amine (0.78mmol) and Yb (OTf)₃(16mg, 0.03 mmol). The resulting mixture was stirred at room temperature until the reaction was complete. After filtration through a sieve, the solvent was evaporated and the product was purified by flash chromatography on silica gel (Merck230-400 mesh).

The following compounds were obtained.

7- (4-tert-butylphenyl iminomethyl) -camptothecin (ST2619)

The solution was stirred for 1.5 hours. Flash chromatography (eluent: CH)₂Cl₂MeOH 99: 1). Yellow powder. Yield 50%, m.p.250 ℃ dec.,¹H NMR(DMSO-d₆)δ：0.88(t，J＝7Hz，H₃-18)，1.30(s，t But)，1.75-1.95(m，H₂-19)，5.45(s，H₂-17)，5.55(s，H₂-5)，6.55(s，-OH)，7.35(s，H-14)，7.45-7.60(m，4H Ar)，7.80(m，H-11)，7.95(m，H-10)，8.25(dd，H-12)，8.95(dd，H-9)，9.7(s，CH＝N)。

IC₅₀(H-460，μM)：0.09

7- (4-methylthiophenyliminomethyl) -camptothecin (ST2667)

The solution was stirred for 22 hours. Flash chromatography (eluent: CH)₂Cl₂MeOH 98: 2). Yellow powder. Yield 36%, m.p.160 ℃ dec.,¹H NMR(DMSO-d₆)δ：0.87(t，J＝7Hz，H₃-18)，1.7-1.9(m，H₂-19)，2.55(s，-SCH₃)，5.45(s，H₂-17)，5.55(s，H₂-5)，6.50(s，-OH)，7.35(s，H-14)，7.40(d，2H Ar)，7.55(d，2H Ar)，7.80(m，H-11)，7.9(m，H-10)，8.20(dd，H-12)，8.95(dd，H-9)，9.7(s，CH＝N)。

IC₅₀(H-460，μM)：0.074

7- (4-Hydroxyphenyliminomethyl) -camptothecin (ST2616)

The solution was stirred for 3 hours. Flash chromatography (eluent: CH)₂Cl₂MeOH 96: 4). Yellow powder. Yield 79%, m.p.250 ℃ dec.,¹H NMR(DMSO-d₆)δ：0.90(t，J＝7Hz，H₃-18)，1.75-2.0(m，H₂-19)，5.4(s，H₂-17)，5.55(s，H₂-5)，6.50(s，-OH)，6.90(d，2H Ar)，7.35(s，H-14)，7.55(d，2H Ar)，7.80(m，H-11)，7.90(m，H-10)，8.25(dd，H-12)，9.0(dd，H-9)，9.70(s，CH＝N)。

IC₅₀(H-460，μM)：0.22

Claims (8)

1. Compounds of formula (I), their N₁-oxides, or pharmaceutically acceptable salts thereof

Formula (I)

Wherein: r₁is-C (R)₅)＝N-R₄Group, wherein R₄Is phenyl, substituted by one or more of-S-S- (2-aminophenyl), -S-S- (4-aminophenyl), -S- (4-aminophenyl)Base) or-SCH₃Substitution;

R₅is hydrogen, C₁-C₈Straight or branched alkyl, C₁-C₈Straight-chain or branched alkenyl and excluding the presence of only 1 carbon, C₃-C₁₀Cycloalkyl radical, C₃-C₁₀cycloalkyl-C₁-C₈Straight or branched alkyl, C₆-C₁₄Aryl or C₆-C₁₄aryl-C₁-C₈A linear or branched alkyl group;

R₂and R₃Hydrogen, hydroxy or C, equal to or different from each other₁-C₈Straight or branched alkoxy.

2. A compound according to claim 1, wherein the phenyl group is substituted in the ortho position.

3. A compound according to claim 1, selected from:

7-2- (2-aminophenyldithio) phenyliminomethylcamptothecin

7-4- (4-aminophenyldithio) phenyliminomethylcamptothecin

7-4- (4-aminophenylthio) phenyliminomethylcamptothecin

7- (2-methylthiophenyl) iminomethylcamptothecin,

their N₁-an oxide, or a pharmaceutically acceptable salt thereof.

4. A process for the preparation of a compound according to any one of claims 1 to 3, which process comprises reacting a compound of formula (Ia) with R of formula (IIa)₄-NH₂By reaction of a compound of

Wherein R is₁Is a group-C (R)₅) Is not O, but R₅As defined in claim 1 in formula (I), R₂And R₃As defined in formula (I) in claim 1; r₄As defined in claim 1, wherein the first and second groups are,

and optionally converting the resulting compounds of formula (I) into their N₁-oxides, or their pharmaceutically acceptable salts.

5. A process according to claim 4, wherein the molar ratio between the compound of formula (Ia) and the compound of formula (IIa) is from 1: 3 to 3: 1.

6. A pharmaceutical composition comprising a therapeutically effective amount of at least one compound of any one of claims 1-3 in admixture with pharmaceutically acceptable carriers and excipients.

7. Use of a compound according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of tumours.

8. Use according to claim 7, wherein the tumour is selected from the group consisting of non-small cell lung tumours, colon-rectal tumours, prostate tumours, gliomas, ovarian carcinomas, osteosarcomas and leukaemias.