(19)
(11)EP 2 029 545 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
17.12.2014 Bulletin 2014/51

(21)Application number: 07825822.5

(22)Date of filing:  15.06.2007
(51)Int. Cl.: 
C07D 221/14  (2006.01)
(86)International application number:
PCT/IB2007/052311
(87)International publication number:
WO 2008/007262 (17.01.2008 Gazette  2008/03)

(54)

Bisnaphthalimidopropyl derivatives with anti-parasite and anti-cancer activity

Bisnaphthalimidopropylderivate mit Wirkung gegen Parasiten und Krebs

Dérivés de bisnaphtalimidopropyle à activité anticancéreuse et antiparasitaire


(84)Designated Contracting States:
AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

(30)Priority: 19.06.2006 PT 10350306

(43)Date of publication of application:
04.03.2009 Bulletin 2009/10

(73)Proprietor: Universidade Do Porto
4050 345 Porto (PT)

(72)Inventors:
  • CORDEIRO DA SILVA, Anabela
    P-4250-325 Porto (PT)
  • PINTO DA COSTA TAVARES, Joana Alexandra
    P-3720-201 Oliveira De Azeméis (PT)
  • KONG THOO LIN, Paul
    Kingswells Aberdeen AB15 8EF (GB)

(74)Representative: Ferreira, Maria Silvina 
Clarke, Modet & Co. Rua Castilho, 50-9°
1269-163 Lisboa
1269-163 Lisboa (PT)


(56)References cited: : 
EP-A- 0 281 902
WO-A-95/29895
WO-A-91/18884
DE-A1- 4 232 739
  
  • TAVARES J ET AL: "Differential effects of polyamine derivative compounds against Leishmania infantum promastigotes and axenic amastigotes" INTERNATIONAL JOURNAL OF PARASITOLOGY, PERGAMON PRESS, GB, vol. 35, no. 6, 1 May 2005 (2005-05-01), pages 637-646, XP004867333 ISSN: 0020-7519
  • DANCE A M ET AL: "Synthesis and biological activities of bisnaphthalimido polyamines derivatives: cytotoxicity, DNA binding, DNA damage and drug localization in breast cancer MCF 7 cells" BIOCHEMICAL PHARMACOLOGY, PERGAMON, OXFORD, GB, vol. 69, no. 1, 1 January 2005 (2005-01-01), pages 19-27, XP004664589 ISSN: 0006-2952 cited in the application
  • LIN P K T ET AL: "The synthesis and in vitro cytotoxic studies of novel bis-naphthalimidopropyl polyamine derivatives" BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, OXFORD, GB, vol. 10, no. 14, 17 July 2000 (2000-07-17), pages 1609-1612, XP004209715 ISSN: 0960-894X cited in the application
  • P. KONG THOO LIN ET AL: "The biological activities of new polyamine derivatives as potential therapeutic agents" BIOCHEMICAL SOCIETY TRANSACTIONS, vol. 31, no. 2, 2003, pages 407-410, XP002482636 cited in the application
  • J. TAVARES ET AL.: "Bisnaphthalimidopropyl Derivatives as Inhibitors of Leishmania SIR2 Related Protein 1", CHEMMEDCHEM, vol. 5, no. 1, January 2010 (2010-01), pages 140-147, DOI: 10.1002/cmdc.200900367
  • BARRON G A ET AL: "Synthesis, cytotoxicity and DNA-binding of novel bisnaphthalimidopropyl derivatives in breast cancer MDA-MB-231 cells", EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY, EDITIONS SCIENTIFIQUE ELSEVIER, PARIS, FR, vol. 45, no. 4, 1 April 2010 (2010-04-01), pages 1430-1437, XP026932313, ISSN: 0223-5234, DOI: 10.1016/J.EJMECH.2009.12.047 [retrieved on 2010-03-02]
 
Remarks:
The file contains technical information submitted after the application was filed and not included in this specification
 
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

Technical domain of the invention



[0001] The new bisnaphthalimidopropyl derivatives, preparation process thereof, pharmaceutical composition comprising them and their use in cancer and parasitic diseases, namely leishmaniasis, trypanosomiasis and malaria, with pharmaceutical industry application.

[0002] The present invention referred to a preparation process of the new bisnaphthalimidopropyl derivative compounds and pharmaceutical compositions comprising them. These compounds have a role in the growth inhibition of the parasite protozoa Leishmania infantum and cytotoxic properties on cancer cells.

Previous state of art



[0003] Naphthalimido derivatives exhibit considerable potential as cytotoxic agents for cancer chemotherapy (Brana et al., 2001). We previously reported the synthesis and biological activities of a novel series of bisnaphthalimidopropyl polyamines compounds (Kong et al., 2000). Subsequent work revealed the presence of the bisnaphthalimidopropyl functionality to be essential for optimum biological activity since the presence of an oxygen atom in the alpha-position of the naphthalimido ring tends to reduce activity (Pavlov et al., 2001).

[0004] Majority research traditionally focused on the modification of the naphthalimido rings to enhance anticancer activities through increased DNA binding and cleavage. For example, acenaphthalimide was introduced into the naphthalimide chromophore to increase the solubility of the bisnaphthalimide compounds (Patten et al., 1992). Furan heterocycles were added to the naphthalimide chromophore and those compounds exhibited strong DNA binding properties with toxicity to CEM leukaemia cells to be in the nanomolar concentration (Brana et al., 1995). Pyrazine heterocycles have also recently been fused to naphthalimides and those pyrazino-naphthalimides exhibited in vitro toxicity with IC50 values ranging from 0.002 to 7.8 µM after 72 hour treatment in cancer HT 29, HeLa, and PC 3 cells (Bailly et al., 2003).

[0005] However, in our laboratory we have developed bisnaphthalimidopropyl fragments linked to natural polyamines such as putrescine, spermidine and spermine. The spermidine and spermine derivatives exhibited enhanced aqueous solubility while maintaining good biological activity (Carrasco et al., 2003 and Kong et al., 2003). In MCF 7 breast cancer cells, compounds were observed within the cell nuclei after 6 and 12 hour drug exposure, with transport being potentially energy dependent (Dance et al., 2005). Within MCF7 cells, the bisnaphthalimidopropyl compounds inflicted significant quantitative DNA damage. We also found for the first time that bisnaphthalimido propyl derivatives exert significant antiproliferative effects on the life cycle of Leishmania infantum, the causative agent of visceral leismaniasis and these drugs also induced the death of promastigotes by apoptosis (Tavares et al., 2005).

[0006] In the leishmaniasis the first line chemotherapy is restricted to the use of pentavalent antimony derivatives (Murry et al., 2001). As a consequence of the long course of the therapy, the adverse reactions appear and the resistances induce the search of new drug more efficient. The efficacy of the treatment be also comprised in immunodeficiency situation, namely in the co infection Leismania/HIV.

[0007] The natural polyamine, putresceine, spermidine, and spermine are present in most of the eukaryotic cells and have an important role on proliferation and cellular differentiation (Muller et al., 2001). In the case of trypanosomatids the polyamines have an additional role in the endogenous oxi-redox equilibrium by the trypanothione compound [N1, N8-bis (glutathione) spermidine]. These molecules and enzymatic reactions have been considered as a good drug targets (Fairlamb and Cerami, 1992; Barrete et al., 1999). Interfere with the regulatory function of the polyamines have been a strategy to search for new compounds with anti-cancer and anti-parasitic activity. Polyamine synthesis inhibitors as a DFMO (alpha-difluormetylornitine) showed to be active against different parasite stages of Plasmodium sp. This parasite has a bifunctional enzyme with ornithine and S-adenosylmethionine decarboxylase activity, that became attractive as a drug target since it is absent in the host cells (Müller et al., 2001).

[0008] Interfere with regulatory function of polyamines became a strategy to search the efficacy compounds with anti-cancer and anti-parasitic activity.

[0009] In this invention we report the synthesis of analogues bisnaphthalimidopropyl di-and tri-amines: BNIPDaoct, BNIPDanon, BNIPDadec and BNIPDadod, based on our leading compound BNIPPut (I) (spermidine derivative) with modification of the central chain. The modification consists of different alkyl length of the central chain with 2 or 3 nitrogen atoms, thus modulating the number of positive charges in the molecules. We also discuss the in vitro cytotoxic properties of these newly synthesised compounds in colon cancer cells (CaCo-2) and parasites (Leishmania infantum, promastigotes). The modifications include the power, specificity, oral biodistribution, penetration into target tissue and action duration, concerning the new compounds from the general formulation A.

wherein:

the alkyl central chain has modifications in the length and in the introduction of nitrogen atoms.



[0010] The compounds showed in formulation A are:

5.

        Bisnaphthalimidopropyldiaminooctane - BNIPDaoct     (V)

6.

        Bisnaphthalimidopropyldiaminononane - BNIPDanon     (VI)

7.

        Bisnaphthalimidopropyldiaminodecane - BNIPDadec     (VII)

8.

        Bisnaphthalimidopropyldiaminododecane - BNIPDadod     (VIII)



[0011] Another aspect of the invention concern the preparation process, pharmaceutical formulation made in combination of one compound of formula V, VI, VII, VIII with a vehicle or safety pharmaceutical excipient.

Description of the invention



[0012] The synthesis of the protected compounds in this invention was based on methods previously described by our group (Kong et al., 2003). The bisnaphthalimido compounds with linker chain containing 2 nitrogens were previously synthesised by simply reacting the corresponding alkyltetraamine with 1,8-naphthalic anhydride (Brana et al., 1995). In order to introduce more heteroatoms in the linker chain, N-alkylation reaction was chosen according to a Kong modified method (Kong et al., 1998). The common intermediate for the synthesis of different compounds was toluenesulfonyloxypropylnaphthalimide. This was prepared by first reacting 1,8-naphthalic anhydride with aminopropanol to give N-(3-hydroxypropyl)naphthalimide which upon reaction with tosyl chloride gave toluenesulfonyloxypropylnaphthalimide, with 60% yield. To obtain the bisnaphthimide the polyamine used depends on the compound to synthesise and were first protected with 2, 4, 5- trimethylsulphonyl chloride (Mts-Cl) in pyridine followed by their N-alkylation with toluenesulfonyloxypropylnaph- thalimide produced the bisnaphthalimide derivative protected. The deprotection was obtained with hydrobromic acid/glacial acetic acid in dichloromethane to give the respective derivative as their hydrobromide salts.

[0013] CaCo-2 cells (ECACC, 86010202) were obtained from the European Collection of Cell Cultures. All reagents were purchased from Aldrich, Fluka and Lancaster and were used without purification. TLC was performed on Kieselgel plates (Merck) 60 F254 in chloroform: methanol (97:3 or 99:1). Column Chromatography was done with silica gel 60, 230-400 meshes using chloroform and methanol as eluent. FAB-mass spectra were obtained on a VG Analytical AutoSpec (25Kv) spectrometer; EC/CI spectra were performed on a Micromass Quatro II (low resolution) or a VG Analytical ZAB-E instrument (accurate mass). 1H and 13C NMR spectra were recorded on a JEOL JNM-EX90 FT NMR spectrometer.

[0014] BNIPSpd and BNIPPut were synthesised according to our methods previously reported (Kong et al.., 2003 and Tavares et al., 2005).

Cytotoxicity studies



[0015] Cytotoxicity was evaluated for CaCo-2 colon carcinoma using the MTT assay with protocols appropriate for the individual test system.11, 13 CaCo-2 cells were maintained in Earle's Minimum Essential Medium (Sigma), supplemented with 10% fetal calf serum (Biosera), 2mM L-glutamine (Sigma), 1% non-essential amino acids (Sigma), 100 IU mL-1 penicillin and 100 mg mL-1 streptomycin (Sigma). Exponentially growing cells were plated at 2 x 104 cells cm-2 into 96-well plates and incubated for 24 h before the addition of drugs. Stock solutions of compounds were initially dissolved in 20% DMSO and further diluted with fresh complete medium.

[0016] After 24 and 48 h of incubation at 37 °C, the medium was removed and 200 µl of MTT reagent (1 mg/mL) in serum free medium was added to each well. The plates were incubated at 37 °C for 4 h. At the end of the incubation period, the medium was removed and pure DMSO (200 µl) was added to each well. The metabolized MTT product dissolved in DMSO was quantified by reading the absorbance at 560 nm on a micro plate reader (Dynex Technologies, USA). IC50 values are defined, as the drug concentrations required to reduce the absorbance by 50% of the control values. The IC50 values were calculated from the equation of the logarithmic line determined by fitting the best line (Microsoft Excel) to the curve formed from the data. The IC50 value was obtained from the equation for y=50 (50% value).

[0017] Leishmania infantum (clone MHOM/MA671TMA-P263) promastigotes transfected with reporter gene that encode to the luciferase enzyme (Roy et al., 2000) were grown at 27 °C in RPMI medium (Gibco) supplemented with 10% of heat inactivated fetal bovine serum (FBS- Gibco), 2mM L-glutamine (Gibco), 20mM Hepes (Gibco), 100U/ml penicillin (Gibco) and 100 µ/ml streptomycin (Gibco). The parasites (106/ml) in the logarithmic phase (2 days of culture) were incubated with a serial range of concentrations of each drug for 3 days at 27°C and the growth of parasites was determined by using the luciferase activity using luciferin as subtract.

[0018] The axenic amastigote of Leishmania infantum (clone MHOM/MA671TMA-P263) transfected with reporter gene that encode to the luciferase enzyme (Roy et al., 2000) were grown at 37°C with 5% CO2 in a cell-free medium called MAA (medium for axenically grown amastigote). The medium MAA/20, consisted of modified medium 199 (Hanks' balanced salts) supplemented with 0.5% soya trypto-casein, 15mM D-glucose, 5mM L-glutamine, 4mM NaHCO3, 0.023mM bovine hemin, 25mM HEPES final pH, 6.5 and 20% inactivated fetal calf serum. The parasites were incubated with different concentrations during 3 days at 37°C with 5% CO2. The growth of the parasites was done by measuring the luciferase activity using luciferin as a substract. The intracellular amastigotes of L. infantum were cultured in a macrophage differentiated human leukemia monocyte cell line (THP-1 cells). The THP-1 cells were differentiated during 2 days with 20ng/ml of PMA in RPMI - 1640 medium supplemented with 10% FCS, 2mM glutamine, 100 IU of penicillin/ml and 100 m g/ml of streptomycin. The non differentiated cells were washed with prewarmed medium and the adherent cells infected with luciferase-expressing axenic amastigotes at a parasite/macrophage ratio of 3:1 for 4 h at 37°C with 5% CO2. Noninternalized parasites were removed and serial dilutions of each drug were made in the RPMI medium supplemented with 10%FCS. After 3 days of drug exposure, wells containing adherent differentiated THP-1 cells were washed and luciferase activity was determined.

Results


Chemistry



[0019] The synthetic strategy adopted to synthesise bisnaphthalimidopropyl derivatives BNIPDaoct, BNIPDanon, BNIPDadec and BNIPDadod, based on methods previously developed in our laboratory (Kong et al., 2000). Protection and activation of all the di-and tri- amines were carried with mesitylene chloride in pyridine at room temperature to give compounds 1-5 in high yield. N-alkylation of the latter compounds with O-tosylpropylnaphthalimide 6 with Ceasium carbonate in anhydrous DMF, afforded the fully protected Bisnaphthalimidopropyl derivatives which upon deprotection with hydrobromic acid/glacial acetic acid in CH2Cl2 gave BNIPDaoct, BNIPDanon, BNIPDadec and BNIPDadod in yield varying from 50-70%.


Biological Activities



[0020] The in vitro cytotoxicity of all the bisnaphthalimidopropyl derivatives described above were studied against colon cancer cell lines CaCo-2 and parasite Leishmania infantum. In the cancer cell line the IC50 values of each compound were determined after 24 and 48 hr drug exposure (Table 1). All compounds exerted IC50 values between 0.15 and 8.00 µM. In the same order of activity, the compound BNIPDadec showed a IC50 0.77 and 0.36 µM, after 24h and 48h of incubation, respectively.

[0021] The removal of a nitrogen atom from the linker chain does not appear to substantially affect the cytotoxic properties of these compounds. We previously reported that when the central alkyl group is a butyl chain, the compound (BNIPPut) is not soluble in most solvents and the aqueous solubility of bisnaphthalimidopropyl compounds is enhanced by introducing a heteroatom like nitrogen in the central chain (Kong et al., 2000). Here, by increasing the length of the alkyl central chain such as in BNIPDaoct, BNIPDanon and BNTPDadec, also helps aqueous solubility. We reason that with the longer alkyl chain, the two naphthalimido rings do not tend to stack on top of each other by T-T interactions between the aromatic rings and hence favour aqueous solubility. Among the latter compounds, BNIPDadec showed the highest cytotoxicity against CaCo-2 cells with IC50 values of 0.36 µM (48 hr) and 0.77 µM (24 hr).
Table 1: Cytotoxicity of polyamine analogues against CaCo-2 cancer cells
CompoundaIC50 (µM)
24h48h
BNIPDaoct 6,20 3,20
BNIPDanon 3,60 0,67
BNIPDadec 0,77 0,36
BNIPDadod 4,50 2,70
a Cytotoxicity determined by MTT assay. Data obtained after treating CaCo-2 cells with varying concentrations of analogues (0.01-40 µM) for 24 and 48 hours. Data are mean ± SD of 6 replicates. ND: not determined.


[0022] Figure 1. The growth curve of luciferase promastigote and axenic amastigote forms. In vitro effect of different bisnaphthalimidopropyl derivatives on parasite growth. The results are representative of 5 assays made independently. The promastigote and axenic amastigote were incubated with a serial range of drug concentrations 0.30 to 12.5 µM, at 27 °C and 3.125 to 100 µM at 37 °C in 5% CO2, respectively, during 3 days. The growth curves represented indicate the percentage growth related to the control for each concentration after luciferase activity determination. Each point represents a mean of 3 assays ± STD.

[0023] The treatment of different forms of the parasite Leishmania infantum, promastigote, axenic amastigote and intracellular amastigote, with the compounds, BNIPDaoct and BNIPDanon, in range of concentration 0.39 to 12.50 µM resulted in a dose dependent inhibition of parasite growth, except to the axenic amastigote incubated with BNIPDaoct, which didn't inhibited the parasite growth up to 50 µM concentration (Figure 1). We have observed that the parasite growth was completely blocked after 6.25 µM to the promastigote and 50 µM to the axenic amastigote, except to the BNIPDaoct.

[0024] In the case of promastigote, the IC50± SD determined were ≤0.39, 2.09±0.54 µM for BNIPDaoct, BNIPDanon, respectively. The most active compound for this parasite form was BNIPDaoct.

[0025] In case of axenic amastigote form the IC50± SD determined were >50.00, 17.42±0.97, µM, for the following compounds BNIPDaoct, BNIPDanon, respectively. The most active compound for this parasitic form was BNIPDanon.

[0026] In the case of intracellular amastigote form the IC50± SD determined were 2.43±0.19, 6.03±0.67, µM, for the following compounds, BNIPDaoct, BNIPDanon, respectively. The most active compound for this parasitic form was BNIPDaoct.

[0027] According to the results obtained all the compounds in study have anti-parasitic activity that give potential drugs to the leishmaniase treatment.
Table 2. Cytotoxicity of bisnaphthalimidopropyl derivative compounds in different forms of the parasite Leishmania infantum
IC50 (µM)
CompoundsaPromastigotesAmastigotes axenicAmastigote intracellular
BNIPDaoct ≤ 0,39 ≥ 50,00 2,43 ± 0,19
BNIPDanon 2,09 ± 0,54 17,42 ± 0,97 6,03 ± 0,67
BNIPDadec ND ND ND
BNIPDadod ND ND ND
Cytotoxicity determined by luciferase assay. The results were obtained after treatment of different parasite forms with a range of different drug concentrations 0.30 to 100 µM after 72h of incubation. The results were representative of medium ± SD at least 5 assays. ND: not determined.

[0028] In conclusion, the new bisnaphthalimidopropyl derivatives exhibit cytotoxicity that may be further developed as anti-tumour and/or anti-parasitic therapeutic agents.

Conclusion



[0029] The use of the compounds in formula V, VI, VII, VIII could be an advantage in the treatment of cancer and parasitic disease namely, treatment of leishmaniasis, trypanosomiasis and malaria.

[0030] For preparation of the pharmaceutical compositions with the compounds of formula V, VI, VII, VIII a inert pharmaceutical adjuvant are mixed with active compounds. The adjuvant used could be solid or liquid. The solid forms include powder, pill, grainy disperse and capsule. The solid adjuvant could be one or more substance that could be diluents, flavouring agents, sweeteners, solvents, lubricants, suspension agents, binding agents or disaggregating agents and could be a encapsulating agents.

[0031] The pharmaceutical preparation is preferentially presented on single dose form, the package contains discrete quantities of the preparation as cover pills, capsules, power in flask or ampoule and liposome formula.

[0032] The dose could range according to the needs of the animal or the patient, the severity of the disease and the compound to be used. The determinations of the dose for a particular situation regards to the people skilled in the art. For convenience, the total daily dose could be devised and distributed administration during the day.

Detailed description of the invention


General method for the synthesis of mesitylated diamine



[0033] Corresponding diamine or triamine was dissolved in anhydrous pyridine followed by the addition of mesitylene chloride (2.1 molar excess for diamine and 3.1 molar excess for triamine). The resulting solution was stirred at room temperature for 4 hours. Removal of the pyridine followed by the addition of cold water resulted in the formation of a precipitate. The latter was filtered off and washed thoroughly with water. The crude product was recrystallised from absolute ethanol.

[0034] N1,N8-Dimesityloctane 2- (70%), 13 C NMR (CDCl3) d: 20.82 (CH3, Mts), 22.85 (CH3, Mts), 26.34 (CH2), 28.70 (CH2), 29.41 (CH2), 41.05 (N-CH2), 47.58 (CH2), 133 (Aromatic carbons, Mts).

[0035] N1,N9-Dimesitylnonane 3- (36%), 13 C NMR (CDCl3) d : 20.82 (CH3, Mts), 22.85 (CH3, Mts), 26.34 (CH2), 28.70 (CH2), 29.41 (CH2), 41.05 (N-CH2), 47.58 (CH2), 133 (Aromatic carbons, Mts).

[0036] N1,N10-Dimesityldecane 4- (48%), 13 C NMR (CDCl3) d : 20.82 (CH3, Mts), 22.85 (CH3, Mts), 26.34 (CH2), 28.70 (CH2), 29.41 (CH2), 41.05 (N-CH2), 47.58 (CH2), 133 (Aromatic carbons, Mts).

Synthesis of O-tosylpropylnaphthalimide



[0037] Naphthalic anhydride (6.34g, 0.032 mol) was dissolved in DMF (50 mL) followed by the addition of aminopropanol 3 (2.45g, 0.032 mol) and DBU (4.87g, 0.032 mol). The solution was left stirring at 85 °C for 4 hr. The solvent DMF was removed under reduced pressure and the resulting residue was poured into cold water with stirring (200 mL) to form a precipitate. The latter was filtered using a buchner funnel and washed thoroughly with (i) water (ii) saturated bicarbonate solution. The yield of the reaction was found to be 95%. This compound, Naphthalimidopropanol was pure enough and was used in the next step with no further purification. NMR (CDCl3): d = 8.65-7.80 (m, 6H, aromatic protons), 4.39 (t, 2H, -N-CH2), 3.69 (t, 2H, CH2-O-), 3.20 (s, broad, 1 H, OH), 2.06 (p, 2H, CH2). 13C NMR (CDCl3): d = 161.70 (C=O), 135.70-122.90 (aromatic carbons), 74.90, 59.90, 30.90 (3xCH2).

[0038] Naphthalimidopropanol (5.10g, 20 mmol) was dissolved in anhydrous pyridine (80 mL). The solution was stirred for 15 mins at 4 °C. Tosyl Chloride (5.72g, 30 mmol) were added, in small portions, over 30 mins. The solution was left overnight at 4°C and was poured into ice water (200 mL) to form a solid on standing. The solid formed was filtered off and washed thoroughly with water. The crude product was recrystallised from either ethanol or ethylacetate to give O-tosylpropylnaphthalimide 6 (53%) . 1H NMR (CDCl3): d = 8.65-7.80 (m, 6H, aromatic protons), 4.45 (t, 2H, CH2), 4.35 (t, 2H, CH2), 2.50 (s, 3H, CH3), 2.25 (p, 2H, CH2). 13C NMR (CDCl3): d = 161.30 (C=O), 145.10-123.10 (aromatic carbons), 73.10, 67.90, 28.70 (3 x CH2), 22.10 (CH3). LRMS (FAB): Calcd. for C12H19NO6S 425.09, Found: 426 [MH]+.

General N-alkylation Reaction (Step 1 in scheme 1)



[0039] Mesitylated polyamines (2-6) (0.651 mmol) were dissolved in anhydrous DMF (13.5 mL) followed by the addition of 7 (0.13 mmol) and cesium carbonate (1.06g). The solution was left at 85°C and completion of the reaction was monitored by thin layer chromatography. DMF was removed under vacuo and the residue was poured into cold water and the resulted precipitate filtered and washed thoroughly with water. After drying the crude was recrystallised from ethanol to give the fully protected pure product in high yield (75-85%).

General Deprotection Reaction (step 2 in scheme 1)



[0040] The fully protected polyamine derivatives (0.222 mmol) were dissolved in anhydrous dichloromethane (10 mL) followed by the addition of hydrobromic acid/ glacial acetic acid (1 mL). The solution was left stirring at room temperature for 24 h. The yellow precipitate formed, was filtered off and washed with dichloromethane, ethylacetate and ether.

[0041] Using the process described and related processes, currently used by the ones skilled in the art, using alkyl chain appropriated, were synthesized,
  • Bisnaphthalimidopropyldiaminooctane (BNIPDaoct) (85%), DMSO-d6, d: 24.43 (CH2), 25.30 (CH2), 25.66 (CH2), 28.07 (CH2), 44,72 (N-CH2), 46.60 (N-CH2), 121.99, 127.13, 130.62, 131.21, 134.31 (Aromatic Carbons), 163.61 (C=O). LRMS (FAB): Calcd. for C38H42N4O4 H 619.3279, Found: 619.3282 [M-H-2Br]+.
  • Bisnaphthalimidopropyldiaminononane (BNIPDanon) (85%), DMSO-d6, d: 24.88 (CH2), 25.84 (CH2), 26.16 (CH2), 28.76 (CH2), 45.29 (N-CH2), 47.29 (N-CH2), 121.94, 127.51, 131.12, 131.42, 134.76 (Naphthalimido aromatic Carbons), 164.00 (C=O). LRMS (TAB): Calcd. for C39H44N4O4 H 633.3435, Found: 633.3440 [M-H-2Br]+.
  • Bisnaphthalimidopropyldiaminodecane (BNIPDadec) (75%), DMSO-d6, d: 24.97 (CH2), 25.90 (CH2), 26.22 (CH2), 28.79 (CH2), 29.00 (CH2), 45.35 (N-CH2), 47.38 (N-CH2), 121.05, 127.66, 131.30, 131.51, 134.94 (Naphthalimido aromatic Carbons), 164.21 (C=O). LRMS (FAB): Calcd. for C40H46N4O4 H 647.4, Found: 647.4 [M-H-2Br]+.
  • Bisnaphthalimidopropyldiaminododecane (BNIPDadod). LRMS (FAB): Calcd. for C42H50N4O4 H 836.71, Found: 675.4 [M-2H-2Br]+.

Example 1: Synthesis of BNIPDaoct



[0042] The diamine was dissolved in anhydrous pyridine followed by the addition of mesitylene chloride (2.1 molar excess). The resulting solution was stirred at room temperature for 4 hours. Removal of the pyridine followed by the addition of cold water resulted in the formation of a precipitate. The latter was filtered off and washed thoroughly with water. The crude product was recrystallised from absolute ethanol.

[0043] N1,N8-Dimesityloctane 2- (70%), 13C NMR (CDCl3) d : 20.82 (CH3, Mts), 22.85 (CH3, Mts), 26.34 (CH2), 28.70 (CH2), 29.41 (CH2), 41.05 (N-CH2), 47.58 (CH2), 133 (Aromatic carbons, Mts).

[0044] The naphthalic anhydride (6.34g, 0.032 mol) was dissolved in DMF (50 mL) followed by the addition of aminopropanol 3 (2.45 g, 0.032 mol) and DBU (4.87g, 0.032 mol). The solution was left stirring at 85°C for 4 hr. The solvent DMF was removed under reduced pressure and the resulting residue was poured into cold water with stirring (200 mL) to form a precipitate. The latter was filtered using a buchner funnel and washed thoroughly with (i) water (ii) saturated bicarbonate solution. The yield of the reaction was found to be 95%. This compound, Naphthalimidopropanol was pure enough and was used in the next step with no further purification. NMR (CDCl3): d = 8.65-7.80 (m, 6H, aromatic protons), 4.39 (t, 2H, -N-CH2), 3.69 (t, 2H, CH2-O-), 3.20 (s, broad, 1 H, OH), 2.06 (p, 2H, CH2). 13C NMR (CDCl3): d = 161.70 (C=O), 135.70-122.90 (aromatic carbons), 74.90, 59.90, 30.90 (3xCH2).

[0045] Naphthalimidopropanol (5.10g, 20 mmol) was dissolved in anhydrous pyridine (80 rnL). The solution was stirred for 15 mins at 0 °C. Tosyl Chloride (5.72g, 30 mmol) were added, in small portions, over 30 mins. The solution was left overnight at 4°C and was poured into ice water (200 mL) to form a solid on standing. The solid formed was filtered off and washed thoroughly with water. The crude product was recrystallised from either ethanol or ethylacetate to give O-tosylpropylnaphthalimide 6 (53%). 1H NMR (CDCl3): d = 8.65-7.80 (m, 6H, aromatic protons), 4.45 (t, 2H, CH2), 4.35 (t, 2H, CH2), 2.50 (s, 3H, CH3), 2.25 (p, 2H, CH2). 13C NMR (CDCl3): d = 161.30 (C=O), 145.10-123.10 (aromatic carbons), 73.10, 67.90, 28.70 (3xCH2), 22.10 (CH3). LRMS (FAB): Calcd. for C12H19NO6S 425.09, Found: 426 [MH]+.

[0046] The mesitylated polyamines (0.651 mmol) were dissolved in anhydrous DMF (13.5 mL) followed by the addition of 7x (0.13 mmol) and cesium carbonate (1.06g). The solution was left at 85°C and completion of the reaction was monitored by thin layer chromatography. DMF was removed under vacuo and the residue was poured into cold water and the resulted precipitate filtered and washed thoroughly with water. After drying the crude was recrystallised from ethanol to give the fully protected pure product in high yield (75-85%).

[0047] The fully protected polyamine derivatives (0.222 mmol) were dissolved in anhydrous dichloromethane (10 mL) followed by the addition of hydrobromic acid/ glacial acetic acid (1 mL). The solution was left stirring at room temperature for 24 h. The yellow precipitate formed, was filtered off and washed with dichloromethane, ethylacetate and ether.

[0048] By this way were synthesized the Bisnaphthalimidopropyldiaminooctane-(BNIPDaoct) (85%), DMSO-d6, d : 24.43 (CH2), 25.30 (CH2), 25.66 (CH2), 28.07 (CH2), 44.72 (N-CH2), 46.60 (N-CH2), 121.99, 127.13, 130.62, 131.21, 134.31 (Aromatic
Carbons), 163.61 (C=O). LRMS (FAB): Calcd. for C38H42N4O4H 619.3279, Found: 619.3282 [M-H-2Br]+.

Bibliography



[0049] 
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Claims

1. The compounds derived from formula A,

characterized in that the compounds are selected from:

1.

        Bisnaphthalimidopropyldiaminooctane - BNIPDaoct     (V)

2.

        Bisnaphthalimidopropyldiaminononane - BNIPDanon     (VI)

3.

        Bisnaphthalimidopropyldiaminodecane - BNIPDadec     (VII)

4.

        Bisnaphthalimidopropyldiaminododecane - BNIPDadod     (VIII)


 
2. Compounds, accordingly to claim 1 for use in the treatment and/or prevention of cancer.
 
3. Compounds, accordingly to claim 1 for use in the treatment and/or prevention of parasitic diseases, namely trypanosomiasis, leishmaniasis and malaria.
 
4. Compounds, accordingly to claim 1 for use in the treatment and/or prevention of infections conditions induced by the genus parasite Leishmania, including the species, L. infantum, L. donovani, L. major. L. tropica, L. mexicana, L. amazonensis and L. braziliensis, Trypanosoma including the species, T. cruzi, T. brucei, T. gambiense, and Pasmodium, including the species, P. falciparum, P. vivax, P. ovale.
 
5. Pharmaceutical compositions, characterized in that they comprise at least one compound of claim 1, in association with a vehicle or excipient pharmaceutically accepted.
 
6. Pharmaceutical compositions, according to claim 5, characterized in that they further comprise a combination of other kind of active substance, besides the compounds defined in claim 1.
 
7. Pharmaceutical compositions, according to claim 6 wherein the active substances are miltefosine, penthavalent antymonial derivates, amphotericyne B, pentamidine and derivates, melarsoprol, benznidazol, nifurtimox, ketoconazol, difluorometilornitine, cloroquine and their derivates and quinine.
 
8. Pharmaceutical compositions, according to claims 5 to 7, for use in the treatment and/or prevention of cancer.
 
9. Pharmaceutical compositions, according to claims 5 to 7 for use in the treatment and/or prevention of parasitic diseases, namely trypanosomiasis, leishmaniasis and malaria.
 
10. Pharmaceutical compositions, according to claims 5 to 6, for use in the treatment and/or prevention of parasitic conditions induced by the genus parasite Leishmania including the species, L. infantum, L. donovani, L. major. L. tropica, L. mexicana, L. amazonensis and L. braziliensis, Trypanosoma including the species, T. cruzi, T. brucei, T. gambiense, and Pasmodium, including the species, P. falciparum, P. vivax, P. ovale.
 


Ansprüche

1. Verbindungen aus Formel A:

dadurch gekennzeichnet, dass die Verbindungen aus:

1.

        Bisnaphthalimidopropyldiaminooktan - BNIPDaoct     (V)

2.

        Bisnaphthalimidopropyldiaminononan - BNIPDanon     (VI)

3.

        Bisnaphthalimidopropyldiaminodecan - BNIPDadec     (VII)

4.

        Bisnaphthalimidopropyldiaminododecan - BNIPDadod     (VIII)

ausgewählt sind.
 
2. Verbindungen nach Anspruch 1 zur Verwendung bei der Behandlung und/oder Prävention von Krebs.
 
3. Verbindungen nach Anspruch 1 zur Verwendung bei der Behandlung und / oder Prävention von parasitären Erkrankungen, insbesondere Trypanosomiasis, Leishmaniose und Malaria.
 
4. Verbindungen nach Anspruch 1 zur Verwendung bei der Behandlung und/oder Prävention von Infektionen die von Parasiten der Gattung Leishmania, einschließlich der Arten L. infantum, L. donovani, L. major. L. tropica, L. mexicana, L. amazonensis und L. brasiliensis, Trypanosoma, einschließlich der Arten T. cruzi, T. brucei und T. gambiense und Plasmodium, einschließlich der Arten, P. falciparum, P. vivax und P. ovale induziert sind.
 
5. Pharmazeutische Zusammensetzungen dadurch gekennzeichnet, dass sie mindestens eine Verbindung nach Anspruch 1, in Verbindung mit einem Träger oder pharmazeutisch annehmbaren Hilfsstoff umfassen.
 
6. Pharmazeutische Zusammensetzungen nach Anspruch 5 dadurch gekennzeichnet, dass sie ferner eine Kombination von anderen Arten von Wirkstoff neben den Verbindungen nach Anspruch 1 umfassen.
 
7. Pharmazeutische Zusammensetzungen nach Anspruch 6, wobei die Wirkstoffe Miltefosin, penthavalent antymonial Derivate, amphotericyne B, Pentamidin und Derivate, melarsoprol, benznidazol, Nifurtimox, ketoconazol, difluorometilornitine, cloroquine und deren Derivate und Chinin sind.
 
8. Pharmazeutische Zusammensetzungen nach Ansprüche 5 bis 7, zur Verwendung bei der Behandlung und / oder Prävention von Krebs.
 
9. Pharmazeutische Zusammensetzungen nach Ansprüche 5 bis 7 zur Verwendung bei der Behandlung und / oder Prävention von parasitären Erkrankungen, insbesondere Trypanosomiasis, Leishmaniose und Malaria.
 
10. Pharmazeutische Zusammensetzungen nach Ansprüche 5 bis 6, zur Verwendung bei der Behandlung und/oder Prävention parasitärer Erkrankungen die von Parasiten der Gattung Leishmania, einschließlich der Arten L. infantum, L. donovani, L. major. L. tropica, L. mexicana, L. amazonensis und L. brasiliensis, Trypanosoma, einschließlich der Arten T. cruzi, T. brucei und T. gambiense und Plasmodium, einschließlich der Arten, P. falciparum, P. vivax und P. ovale induziert sind.
 


Revendications

1. Les composés dérivés de la formule A

caractérisés en ce que les composés sont choisis parmi:

1.

        Bis-naphthalimide-propyl-diaminooctane - BNIPDaoct     (V)

2.

        Bis-naphtalimide-propyl-diaminononane - BNIDanon     (VI)

3.

        Bis-naphthalimide-propyl-diaminodécane - BNIPDadec     (VII)

4.

        Bis-naphthalimide-propyl-diaminododécane -BNIPDadod     (VIII).


 
2. Composés, selon la revendication 1, pour utilisation dans le traitement et/ou prévention du cancer.
 
3. Composés, selon la revendication 1 pour utilisation dans le traitement et/ou prévention des maladies parasitaires à savoir trypanosomiase, leishmaniose et malaria.
 
4. Composés, selon la revendication 1 pour utilisation dans le traitement et/ou prévention des infections induites par le genre parasite Leishmania, comprenant les espèces: L. infantum, L. donovani, L. major. L. tropica, L. mexicana, L. amazonensis et L. braziliensis; Trypanosoma comprenant les espèces: T. cruzi, T. brucei et T. gambiense ; Pasmodium, comprenant les espèces: P. falciparum, P. vivax et P. ovale.
 
5. Compositions pharmaceutiques, caractérisées en ce qu' elles comprennent au moins un composé selon la revendication 1, associé au véhicule ou à l'excipient pharmaceutiquement acceptable.
 
6. Compositions pharmaceutiques, selon la revendication 5, caractérisées en ce qu' elles comprennent de plus une combinaison d'un autre type de substance active, outre les composés définis à la revendication 1.
 
7. Compositions pharmaceutiques, selon la revendication 6, dont les substances actives sont le miltéfosine, les dérivés d'antimoine pentavalent, l' amphotéricine B, la pentamidine et ses dérivés, le mélarsoprol, la difluorométhylornithine le benznidazole, la chloroquine et ses dérivés et la quinine.
 
8. Compositions pharmaceutiques, selon les revendications 5 à 7, pour utilisation dans le traitement e/ou prévention du cancer.
 
9. Compositions pharmaceutiques, selon les revendications 5 à 7, pour utiliser dans le traitement e/ou prévention des maladies parasitaires, à savoir les trypanosomiases, la leishmaniose et la malaria.
 
10. Compositions pharmaceutiques, selon les revendications 5 à 6, pour utilisation dans le traitement et/ou prévention des infections parasitaires induites par le genre parasite Leishmania comprenant des espèces: L. infantum, L. donovani, L. major. L. tropica, L. mexicana, L. amazonensis et L. braziliensis: Trypanosoma comprenant les espèces: T. cruzi, T. brucei, T. gambiense; Pasmodium : comprenant les espèces: P. falciparum, P. vivax et P. ovale.
 




Drawing






REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description