Zinic, Mladen (Rudjer Boskovic Institute, Bijenicka cesta 54, Zagreb, 10002, HR)
Starcevic, Kristina (University of Zagreb, Faculty of chemical engineering and technology Marulicev trg 19, 1000 Zagreb, HR)
Karaminski-zamola, Grace (University of Zagreb, Faculty of chemical engineering and technology Marulicev trg 19, 1000 Zagreb, HR)
PIANTANIDA, Ivo (Rudjer Boskovic Institute, Bijenicka cesta 54, Zagreb, 10002, HR)
Zinic, Mladen (Rudjer Boskovic Institute, Bijenicka cesta 54, Zagreb, 10002, HR)
Starcevic, Kristina (University of Zagreb, Faculty of chemical engineering and technology Marulicev trg 19, 1000 Zagreb, HR)
Karaminski-zamola, Grace (University of Zagreb, Faculty of chemical engineering and technology Marulicev trg 19, 1000 Zagreb, HR)
| 1. | Chemical reaction of the photoinduced dehydrocyclization of the 1,2arenyl ethenes (prodrug) into tri or polycylic condensed aromatic or heteroaromatic products (drug) characterised by property that reactants (prodrugs) do not interact with DNA and RNA, while products (drags) form strong, stable interactions with DNA and RNA. |
| 2. | Chemical reaction corresponding to the one in Claim 1 characterised by feature that reactants (prodrugs) form much weaker complexes with DNA and RNA if compared to the corresponding products (drugs). |
| 3. | Chemical reaction corresponding to the one in Claim 1 characterised by significantly more pronounced antitumour activity of products (drags), if compared to activity of reactants (prodrugs), as a result of any direct or indirect cause. |
| 4. | Chemical reaction corresponding to the one in Claim 1 characterised by general formula of product F F Either when aromatic rings I and III are identical five or six membered aromatic or heteroaromatic moieties, or in the case I and III are not identical five or six membered aromatic or heteroaromatic moieties. |
| 5. | Chemical reaction corresponding to the one in Claim 1 characterised by general formula of product F substituted at any position of condensed aromatic moiety and by any number or combination of the following substituents: halogen (F, Cl, Br, I) nalkyl chain C1C5, alkyl amines, aklylhidroxy substituents, alkyl and/or aryl carboxylic acid, derivatives of carboxylic acid or tiocarboxylic acid like for example ester, amide, hidrazide, hydroxamate, C1C5 ether or tioether substituents, or C1C5 keto or aldehide substituents, aromatic substituents. |
| 6. | The procedure of tumour treatment characterised by introduction of a reactant (prodrug) in a form of suspension, solution, gel or any other pharmaceuticaly acceptable way directly on the targeted tumour tissue, followed by illumination with a goal of photoinduced conversion of the prodrug into the product (drug) according to the chemical reaction corresponding to the one in Claim 1. |
| 7. | The procedure of tumour treatment characterised by introduction of a reactant (prodrug) in a form of suspension, solution, gel or any other pharmaceutically acceptable way in the patient's body with the aim of accumulation of the reactant (prodrug) in the tumour tissue, followed by illumination with a goal of photoinduced conversion of the prodrug into the product (drug) according to the chemical reaction corresponding to the one in Claim 1. |
| 8. | Procedure of monitoring by spectroscopic methods (UV/vis , fluorescence) of any parameter of the chemical reaction corresponding to the one in Claim 1 characterised by the aim of following conversion of the reactant (prodrug) into product (drug) aimint to be used directly of indirectly in antitumour therapy. |
| 9. | Procedure of monitoring by spectroscopic methods (UV/vis , fluorescence) of any parameter characteristic for product (drug) obtained by chemical reaction corresponding to the one in Claim 1, characterised by the aim to follow migration of the drug and its metabolites within treated tumour tissue, and in general to follow migration through the patient's body and drug excretion. |
Field of the invention;
This invention is based on the photo-induced dehydrocyclization of the 1,2-arenyl ethenes of low or no biological activity into poly-condensed aromatic compounds of high biological activity due to the DNA/RNA intercalation. Here we show that this conversion can be done in aqueous solutions, thus allowing implementation of presented method as an alternative to the photodynamic anticancer therapy (PDT). Also, this invention shows application of highly sensitive spectroscopic methods in the monitoring of progress of photo-induced anticancer therapy. By international classification this invention can be correlated to the C07D307/00, A61K31/343, A61K49/00P4F, A61K31/34. State of the art:
The photodynamic anticancer therapy (PDT) is one of the most promising fields of non¬ invasive antitumour therapies. It is based on the introduction of the small, non-toxic organic molecules into the patient's body. The drug localizes preferentially in the tumour within the irradiation volume. The patient's tissues in the zone of macroscopic tumour is then irradiated with a beam of red laser light. The biochemical mechanism of cell damage in PDT is believed to be mediated largely by singlet oxygen. Singlet oxygen is produced by transfer of energy from the light-excited small molecule to an oxygen molecule. The resultant singlet oxygen is highly reactive chemically and is believed to react with and disable DNA and cell membranes. The vascular cells of the irradiated tumour and some of the tumour cells are rendered incapable of further mitotic activity or may be killed outright if the light penetrates the tissue sufficiently. l'2'3 Acceptable antitumour activity was achieved by application of many small organic molecules as for example phtalocianine derivatives (as described in the patent US2003170178 and there cited other sources), porphyrin derivatives (as described in the patent US2003032799, DE10127544, US 6069140 and there cited other sources), 1,3,4,6- tetrahydroxy-helianthrone derivatives (as described in the patent US US6229048, and there cited other sources), conjugates of already known photoactive species with other biologically active substances (for example porphyrin-protein conjugate, patent US2002137901). All afore mentioned patents do not solve adequately issues as low selectivity of drug action between tumour and normal tissue, undesirable reactions of formed radicals, complicated monitoring of the progress of the therapy. In addition, antitumour action of PDT is stopped soon after switching off the light source. One of the most important drawbacks of PDT is the transparency of treated tissue since even in the most efficient systems (light wavelength λ>650 nm) light penetrates only few cm through the tissue.
1 W. M. Sharman, C. M. Allen and J. E. van Lier, DDT 4, (11) 507-517 (1999). Photodynamic therapeutics: basic principles and clinical applications; G. Stochel, A. Wanat, E. Kulis, Z. Stasicka; Coordination Chemistry Reviews 171 (1998) 203-220, Saito, L; Takayama, M.; Sugiyama, H.; Nakamura, T. In DNA and RNA Cleavers and Chemotherapy of Cancer and Viral Diseases; Menunier, B., Ed.; Kluwer: Netherlands, 1996; pp. 163-176. 2 R. Bonnet, Chem. Soc. Rev. 24 (1995) 19. 3 Henderson, B. W.; Dougherty, T. J. Photochem. Photobiol. 1992, 55, 145. This invention has shown that a number of 1,2-arenyl ethenes do not intercalate or show any
measurable interaction with DNA/RNA because of unfavourable structural characteristics.
Nevertheless, most of these compounds can be efficiently converted by photo-induced
dehydrocyclization4 into condensed tricyclic aromatics, which strongly interact with
DNA/RNA by intercalation5 (Scheme 1).
This novel approach offers an attractive answer to the some of afore mentioned drawbacks of
PDT. The photo-induced dehydrocyclization is an irreversible chemical reaction and
condensed aromatic products are in most cases highly stable, though antitumour action is
localised to the lighted area but time of action is not dependent on the permanent impact of
the light. Common strong fluorescence of the condensed aromatic products allows easy
spectroscopic monitoring of the progress of the active substance in the treated tissue.
Mode of antitumour action of condensed aromatic products is well known since number of
already used anticancer drugs base their activity on the intercalation into DNA (amsacrine,
elipticine, daunomicine, adriamicine).6
4 J. March, in Advanced Organic Chemistry, J. Wiley and Sons, Fourth Edition, (1992), page 1120. 5 W. Saenger, Principles of Nucleic Acid Structure, Springer, New York, N.Y.,(1988). 6 G. Capranico, F. Zunino, Eur. J. Cancer., 28a(1992) 2055.; P. D'Arpa, L.F. Liu, Biochim. Biophys. Acta, 989(1989)163. Summary of the invention:
Basics of this invention is efficient application of reaction presented on the Scheme 1 in the conditions relevant to the antitumour therapy at which nonintercalative prodrug can be converted by photoinduced cyclisation into DNA intercalative product. Conversion should be possible within acceptable time interval and formed active product should be formed in high yield. In addition, active product should be highly stable thus minimizing the undesired side-reactions and diminishing of high local concentration of active substance. Advantage of common strong fluorescence of active substances formed can be exploited for monitoring their concentration in the treated tissue and migration within and out of addressed body region. Difference in the spectroscopic properties (UV/VTS spectra) of starting prodrug and active drug can be used for determination of conversion effectiveness. Object of the present invention is therefore in vivo photoinduced synthesis of tricyclic products of general formula F by chemical reaction presented on the Scheme 1.
Obtained products are characterised by structure containing:
The aromatic rings I and III are identical and present five- or six-membered aryl or heteroaryl or The aromatic rings I and III are not identical and present five- or six-membered aryl or heteroaryl a) The identical aromatic rings I and III substituted by same substituents at any position of aromatic ring but equivalent for both I and III, like for example halogen (F, Cl, Br, I) n-alkyl chain C1-C5, alkyl amines, aklylhidroxy substituents, alkyl and/or aryl carboxylic acid, derivatives of carboxylic acid or tiocarboxylic acid like for example ester, amide, hidrazide, hydroxamate, C1-C5 ether or tioether substituents, or C1-C5 keto- or aldehide substituents, aromatic substituents. and b) The not identical aromatic rings I and III substituted by the same substituents mentioned in a), and c) The identical aromatic rings I and III substituted by different substituents at any position of aromatic ring by the same substituents mentioned in a) Detailed description of the invention:
Invention is here demonstrated by following examples. Model reaction is presented by Scheme 2, also in more details described in the literature.7
EXAMPLE 1 Conversion of Methyl-E-3-(5-(N- isopropyl)αwi<iwo-2-furyl)-2-phenylacrylate (1) into Methyl-2-(N-isopropyl)amidinonaphtho[2,l-b]furan-5-carboxyla te (2) at high concentration conditions (c—10' mol dm 3) and isolation of larger quantities of product 2.
To the aqueous solution of 1 (0.134g, 0.384mmol in 250 ml), the air was bubbled through. The prepared solution was then irradiated using a high pressure mercury arc lamp for 2 h. The water was evaporated and recrystallization from ethanol-ether gave 2 as a white powder (0.04Og, 30.05%). Mp 221-223 0C, Elemental analysis (Found: C5 49.22; H 7.60; N 6.32. C18H19O3N2- 5H2O requires: C, 49.60; H, 6.66; N, 6.43%); δH(300 MHz; DMSO; Me4Si) 10.1 (IH, br s, NH), 9.90 (IH, br s NH), 9.41 (IH, br s, NH), 8.80 (IH, d, J 8.6, Ph), 8.79 (IH, s, Ph), 8.41-8.39 (2H, m, Ph), 7.81 (2H, dd, J 8.4 and J 1.3, Ph), 4.15 (IH, m, CH(CH3)2, 4.0 (3H, s, - COOCH3), 1.34 (6H, d, J 6.37, CH(CH3)2; δc(300 MHz; DMSO; Me4Si) 21.1 (2 x q), 45.1 (d), 52.8 (q), 112.5 (d), 115.3 (d), 123.9 (d), 125.9 (s), 126.6 (d), 127.3 (d), 127.4 (s), 127.5 (s), 128.3 (d), 128.4 (s), 144.6 (s), 150.7 (s), 151.1 (s) and 166.7 (s); υmax (KBr)/cm4 3010, 2930, 1705, 1650, 1610, 1435, 1420, 1325, 1260, 1190, 1115, 1080, 1010.
EXAMPLE 2 Conversion of Methyl-E-3-(5-(N- isopropyyαw2zWmo-2-furyl)-2-phenylacrylate (1) into Methyl-2-(N-isopropyl)amidinonaphtho[2,l-b]furan-5-carboxyla te (2) at low, biologically relevant concentrations (c=10~5 mol dm 3)
Air-saturated aqueous solutions of 1 (c=2.5 x 10 ~5 mol dm"3) were irradiated with a high- pressure Hg lamp (400 W) immersed into a Pyrex water-cooled coat (cut off light λ< 300
D K. Starcevic, G. Karminski-Zamola, I. Piantanida, M. Zinic, L. Suman and M. Kralj; ; J. Am. Chem. Soc, 127 (2005), 1074-1075. nm). The progress of changes in the UV/Vis spectra was continuously followed using the through-flow quartz cell system (total volume V=2 ml). The sharp maximum of 1 at λ = 327 nm changed into two peaks with maxima at λ = 321 nm and λ = 342 nm, agreeing well with the UV/Vis spectrum of 2 (Figure IA). Also, new maximum at λ = 258 nm (Figure IA) characteristic for product 2 appeeared. The reaction was completed in about 60 min, as it can be seen by following time dependent increase of the absorption maximum at λ = 258 nm (Figure IB) characteristic for product 2.
EXAMPLE 3 Study of interactions of compounds 1 and 2 with DNA i RNA
Interactions of 1 and 2 with double stranded (ds-) DNA and RNA were studied in biologically relevant buffered aqueous solutions by spectrophotometric titrations (UV/vis and fluorimetric) and the changes of the thermal denaturation points of ds- polynucleotides caused by added compound. Addition of DNA (c(ct-DNA)= 0 - 3.7 x 10"4mol dm"3 ) didn't yield any measurable change in the UV/vis and fluorescence spectra of 1. Also, addition of 1 to the ds-DNA even at excess of 1 over DNA intercalation binding sites didn't result in any change of the thermal denaturation point of DNA. Under same experimental conditions, strong spectroscopic (UV/vis and fluorimetric) changes observed for 2 upon addition of ds- DNA and RNA allowed calculation of binding constants and rations n^ound 2]/ [polynucleotide]: (for ct-DNA: logKs « 5, ratio n[bOUnd2]/[DNA]=0.22; ds- RNA: poliA-poliU - logKs « 4.2; ratio n=0.2; poliG- poliC - logKs « 4.4; n » 0.15). In addition, 2 stabilised double stranded helices of polynucleotides. All afore mentioned results point toward formation of stable intercalative complex of 2 with ds- DNA and RNA under biologically relevant conditions, while it's precursor (prodrug) 1 under same experimental conditions didn't show any measurable interactions.
Presented Examples 1-3 do not limit this invention only on the presented structures of model reaction (Scheme 2), but should serve as an experimental example of the main idea of this invention about implementation of the chemical reaction shown on the Scheme 1 for the photoinduced antitumour therapy. Possible application procedures of the invention in the antitumour therapy:
1. Aqueous solution of the prodrug (1,2-arenyl ethenes; c= 1- 100 x 10"5mol dm"3) can be applied directly on the targeted tumour. After incubation time neccesary for the penetration of the prodrug into tumour cells, targeted area is exposed to the strong light source of narrow bandwith wavelength (laser) corresponding to the absorbance maximum of the prodrug. The illumination time neccesary for the optimal conversion of the prodrug into the active drug (DNA intercalator) is determined previously by in vitro experiments. By monitoring the fluorescence parameters characteristic for the active substance (drug) in the treated tissue, efficiency of conversion and concentration of active drag can be estimated. In addition, monitoring the characteristic fluorescence parameters of a drug over longer perion of time can give an overview about migrration of the drug in treated tissue, in the body and drug degradation and exctretion form the body. 2. Aqueous solution of the prodrug (1,2-arenyl ethenes; c= 1- 100 x 10"5mol dm"3) can be directly injected into well defined solid tumour. After neccesary incubation time tumour tissue can be illuminated by wavelength corresponding to the absorbance maximum of the prodrug by introduced fiber optic. In the same manner, by application of fiber optic fluorescence parameters characteristic for the drug formed can be monitored, thus allowing application described afore in point 1.
Next Patent: DOORMAT SYSTEM TO BE ASSEMBLED BY ELEMENTS