Halogenated rhodamine derivatives and applications thereof   

This application is a division of co-pending U.S. patent application Ser. No. 10/297,088, which is the US national stage of international application no. PCT/CA02/00438, filed Mar. 27, 2002 designating the United States of America, the entire disclosures of which are incorporated herein by reference. Priority is claimed based on Canadian patent application no. 2,342,675, and U.S. patent application Ser. No. 09/822,223, both filed Apr. 2, 2001.

FIELD OF THE INVENTION

The invention relates to new rhodamine derivatives that are useful for their pharmaceutical and non-pharmaceutical properties.

The rhodamine derivatives of the invention exhibit powerful bactericidal and antiviral activities.

They are also useful, alone or in association with a pharmaceutically acceptable carrier, in the treatment and/or in the prevention of immunologic disorders.

Moreover, these derivatives are useful as intermediates I the synthesis of further new rhodamine derivatives and also in new synthesis of already known rhodamine derivatives.

Finally, the present invention also relates to new processes for the preparation of rhodamine derivatives.

BACKGROUND OF THE INVENTION

Photodynamic therapy has been used as a method for the eradication of neoplastic cells from autologous grafts for cancer treatments. This method relies on the use of photosensitizing dyes, which when activated with light of a particular wavelength, produce toxic O2— radicals, ultimately leading to cell death. Photochemical treatments have also been used for pathogen inactivation, such as in “decontamination” of blood and blood-derived products. The danger of pathogen transmission through transfusion of whole blood, platelets concentrates, plasma and/or red blood cells still represent major concerns in medicine. Although there has been impressive progress in the prevention and maintenance of blood safety regarding the presence of microorganisms, blood components continue to carry risk of pathogen transfusion. Moreover, the presence of viruses in blood components is also of great concerns, mainly for the presence of Hepatitis C and human immunodeficiency virus (HIV), even though the risk of contamination is reduced to negligible levels. The presence of other viruses is also required and includes the human T-cell lymphotrophic virus type 1 (HTLV-1), Hepatitis B (HBV) and cytomegalovirus. Photodynamic compounds such as pseuralens, porphyrins, riboflavines and dimethyl of methylene bleue have been used in the treatment of pathogen in blood product. These compounds necessitate radiation by a ultra violet A lamp (UVA) to get activated, thus leading to possible mutagenic effect in the remaining cells present in the treated samples. (Corash, L., Inactivation of infectious pathogens in labile blood components: meeting the challenge, Transfus Clin Biol, 2001, 8, 138-145Lin, L., Londe, H., Janda, M. J., Hanson, C. V. and Corash, L., Photochemical inactivation of pathogenic bacteria in human platelet concentrates, Blood, 1994, 83, 9, 2698-2706; Lin, L, Londe, H., Hanson, C. V., Wiesehahn, G., Isaacs, S., Cimino, G. and Corash, L., Photochemical inactivation of cell-associated human immunodeficiency virus in platelet concentrates, Blood, 1993, 82, 1, 292-297; Lin, L., Eiesehahn, G. P., Morel, P. A. and Corash, L., Use of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates, Blood, 1989, 74, 1, 517-525; Lin, L., Cook, D. N., Wiesehahn, G. P., Alfonso, R., Behrman, B., Cimino, G. D., Corten, L., Damonte, P. B., Dikeman, R., Dupuis, K., Fang, Y. M., Hanson, C. V., Heasrt, J. E., Lin, C. Y., Londe, H., Metchette, K., Nerio, A. T., Pu, J. T., Reames, A. A., Rheinschmidt, M., Tessman, J., Isaacs, S. T., Wollowitz, S. and Corash, L., Photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light, Transfusion, 1997, 37, 423-435). Because of the WA exposure to blood components, these techniques are not entirely satisfactory. There was therefore a need for new light sensitive derivatives that do not necessitate UVA exposure of blood components and that may also be a safer ad more acceptable replacement to UVA treated blood components.

Immunologic disorders are uncontrolled cell proliferations that result from the production of immune cells recognizing normal cells and tissues as foreign. After a variable latency period during which they are clinically silent, cells with immunoreactivity towards normal cells induce damages in these normal cells and tissues. Such immunologic disorders are usually divided in alloimmune conditions and autoimmune conditions. Alloimmune disorders occur primarily in the context of allogeneic transplantation (bone marrow and other organs: kidney, heart, liver, lung, etc.). In the setting of bone marrow transplantation, donor immune cells present in the hematopoietic stem cell graft react towards host normal tissues, causing graft-versus-host disease (GVHD). The GVHD induces damage primarily to the liver, skin, colon, lung, eyes and mouth. Autoimmune disorders are comprised of a number of arthritic conditions, such as rhumatoid arthritis, scleroderma and lupus erythematosus; endocrine conditions, such as diabetes mellitus; neurologic conditions, such as multiple sclerosis and myasthenia gravis; hematological disorders, such as autoimmune hemolytic anemia, etc. The immune reaction, in both alloimmune and autoimmune disorders, progresses to generate organ dysfunction and damage.

Despite important advances in treatment, immunologic complications remain the primary cause of failure of allogeneic transplantations, whether in hematopoietic stem cell transplantation (GVHD) or in solid organ transplantation (graft rejection). In addition, autoimmune disorders represent a major cause of both morbidity and mortality. Prevention and treatment of these immune disorders has relied mainly on the use of immunosuppressive agents, monoclonal antibody-based therapies, radiation therapy, and more recently molecular inhibitor\'s. Significant improvement in outcome has occurred with the continued development of combined modalities, but for a small number of disorders and patients. However, for the most frequent types of transplantations (bone marrow, kidney, liver, heart and lung), and for most immune disorders (rheumatoid arthritis, connective tissue diseases, multiple sclerosis, etc.) resolution of the immunologic dysfunction and cure has not been achieved. Therefore, the development of new approaches for the prevention and treatment of patients with immunologic disorders is critically needed particularly for those patients who are at high risk or whose disease has progressed and are refractory to standard immunosuppressive therapy. Allogeneic stern cell transplantation (AlloSCT) has been employed for the treatment of a number of malignant and non-malignant conditions. Allogeneic stem cell transplantation is based on the administration of high-dose chemotherapy with or without total body irradiation to eliminate malignant cells, and host hematopoietic cells. Normal hematopoietic donor stem cells are then infused into the patient in order to replace the host hematopoietic system. AlloSCT has been shown to induce increased response rates when compared with standard therapeutic options. One important issue that needs to be stressed when using AlloSCT relates to the risk of reinfusing immune cells that will subsequently recognize patient cells as foreign and cause GVHD. A variety of techniques have been developed that can deplete up to 105 of T cells from the marrow or peripheral blood. These techniques, including immunologic and pharmacologic purging, are not entirely satisfactory. One major consideration when purging stem cell grafts is to preserve the non-host reactive T cells so that they can exert anti-infectious and anti-leukemia activity upon grafting. The potential of photodynamic therapy, in association with photosensitizing molecules capable of destroying immunologically reactive cells while sparing normal host-non-reactive immune cells, to purge hematopoietic cell grafts in preparation for AlloSCT or autologous stem cell transplantation (AutoSct), and after AlloSCT in the context of donor lymphocyte infusions to eliminate recurring leukemia cells has largely been unexplored. To achieve eradication of T cells, several approaches have been proposed including: 1) in vitro exposure of the graft to monoclonal antibodies and immunotoxins against antigens present on the surface of T cells (anti-CD3, anti-CD6, anti-CD8, etc.); 2) in vitro selection by soybean agglutinin and sheep red blood cell rosetting; 3) positive selection of CD34+ stem cells; and 4) in vivo therapy with combinations of anti-thymocyte globulin, or monoclonal antibodies. 5) In vitro exposure of recipient-reactive donor T cells by monoclonal antibodies or immunotoxins targeting the interleukin 2 receptor or OX-40 antigen (Cavazzana-Calvo M. et al. (1990) Transplantation, 50:1-7; Tittle T. V. et al (1997) Blood 89:4652-58; Harris D. T. et al. (1999) Bone Marrow Transplantation 23:137-44).

However, most of these methods are not specifically directed at the alloreactive T cell subset and associated with numerous problems, including disease recurrence, graft rejection, second malignancies and severe infections. In addition, the clinical relevance of several of these methods remains to be established.

There are many reports on the use of photodynamic therapy in the treatment of malignancies (Daniell M. D., Hill J. S. (1991) Aust. N. Z. J. Surg., 61: 340-348). The method has been applied for cancers of various origins and more recently for the eradication of viruses and pathogens (Raab O. (1990) Infusoria Z. Biol., 39: 524).

The initial experiments on the use of photodynamic therapy for cancer treatment using various naturally occurring or synthetically produced photoactivable substances were published early this century (Jesionek A., Tappeiner V. H. (1903) Muench Med Wochneshr, 47: 2042; Hausman W. (1911) Biochem. Z., 30: 276). In the 40\'s and 60\'s, a variety of tumor types were subjected to photodynamic therapy both in vitro and in vivo (Kessel, David (1990) Photodynamic Therapy of neoplastic disease, Vol. I, II, CRC Press. David Kessel, Ed. ISBN 0-8493-5816-7 (v. 1), ISBN 0-8493-5817-5 (v. 2)). Dougherty et al. and others, in the 70\'s and 80\'s, systematically explored the potential of oncologic application of photodynamic therapy (Dougherty T. J. (1974) J. Natl Cancer Inst., 51: 1333-1336; Dougherty T. J. et al. (1975) J. Natl Cancer Inst., 55: 115-121; Dougherty T. J. et al. (1978) Cancer Res., 38: 2628-2635; Dougherty T. J. (1984) Urol. Suppl., 23: 61; Dougherty T. J. (1987) Photochem. Photobiol., 45: 874-889).

Treatment of Immunoreactive Cells with Photodynamic Therapy

There is currently a lack of agents which allow selective destruction of immunoreactive cells while leaving intact the normal but suppressed residual cellular population. Preferential uptake of photosensitive dye and cytotoxicity of photodynamic therapy against leukemia (Jamieson C. H. at al. (1990) Leuk. Res., 14: 209-219) and lymphoid cells (Greinix H. T., et al. Blood (1998) 92:3098-3104; are reviewed in Zic J. A. et al. Therapeutic Apheresis (1999) 3:50-62) have been previously demonstrated.

It would be highly desirable to be provided with photosensitizers which possess at least one of the following characteristics: i) preferential, localization and uptake by the immunoreactive cells; ii) upon application of appropriate light intensities, killing those cells which have accumulated and retained the photosensiting agents; iii) sparing of the normal hemopoietic stem cell compartment from the destructive effects of activated photo sensitizers; and iv) potential utilization of photosensitizers for hematopoietic stem cell purging of immunoreactive cells in preparation for allogeneic or autologous stem cell transplantation. v) Potential utilization of photosensitizers for ex vivo elimination of reactive immune cells in patients with immunological disorders.

Rhodamine 123 (2-(6-amino-3-imino-3H-xanthen-9-yl)benzoic acid methyl ester hydrochloride), a lipophilic cationic dye of the pyrylium class which can disrupt cellular homeostasis and be cytostatic or cytotoxic upon high concentration exposure and/or photodynamic therapy, although with a very poor quantum yield (Darzynkiewicz Z., Carter S. (1988) Cancer Res., 48: 1295-1299). It has been used in vitro as a specific fluorescent stain for living mitochondria. It is taken up and is preferentially retained by many tumor cell types, impairing their proliferation and survival by altering membrane and mitochondrial function (Oseroff A. R. (1992) In Photodynamic therapy (Henderson B. W., Dougherty T. J., eds) New York: Marcel Dekker, pp. 79-91). In vivo, chemotherapy with rhodamine 123 can prolong the survival of cancerous mice, but, despite initial attempts to utilize rhodamine 123 in the treatment of tumors, the systemic toxicity of rhodamine 123 may limit the usefulness (Bernal, S. D., at al. (1983) Science, 222: 169; Powers, S. K. et al. (1987) J. Neurosur., 67: 889).

U.S. Pat. No. 4,612,007 issued on Sep. 16, 1986 in the name of Richard L. Edelson, discloses a method for externally treating human blood, with the objective of reducing the functioning lymphocyte population in the blood system of a human subject. The blood, withdrawn from the subject, is passed through an ultraviolet radiation field in the presence of a dissolved photoactive agent capable of forming photoadducts with lymphocytic-DNA. This method presents the following disadvantages and deficiencies. The procedure described is based on the utilization of known commercially available photoactive chemical agents for externally treating patient\'s blood, leaving the bone marrow and potential resident leukemic clones intact in the process. According to Richard L. Edelson, the method only reduces, does not eradicate, the target cell population. Moreover, the wavelength range of UV radiation used in the process proposed by Richard L. Edelson could be damageable to the normal cells.

International Application published on Jan. 7, 1993 under International publication number WO 93/00005, discloses a method for inactivating pathogens in a body fluid while minimizing the adverse effects caused by the photosensitive agents. This method essentially consists of treating the cells in the presence of a photoactive agent under conditions that effect the destruction of the pathogen, and of preventing the treated cells from contacting additional extracellular protein for a predetermined period of time. This method is concerned with the eradication of infectious agents from collected blood and its components, prior to storage or transfusion.

It would be highly desirable to be provided with new rhodamine derivatives for the treatment of immunereactive cells which overcomes these drawbacks while having no systemic toxicity for the patient.

Halogenated rhodamine salts are dyes that have the property of penetrating cells and generally localising at the mitochondria. They have been used in conjunction with photoactivation to kill certain types of cells, namely cancer cells in Leukemia, and activated T-cells in autoimmune diseases.

The generally accepted mechanism for the cell killing effect is the production of singlet oxygen which is the reactive intermediate in the disruption of the life-sustaining biological processes of the cell.

The role of the rhodamine dye in the production of singlet oxygen is that of a photosensitizer, i.e. that of a molecule which absorbs the incident light energy and transfers it to ground state oxygen, thereby elevating it to its singlet excited state which is the reactive intermediate.

It is further known that the efficiency of the energy transfer process is greatly enhanced by the presence of heavy atoms such as halogens on the aromatic chromophore of the dye.

One critical problem that has not been addressed however is the differential uptake of the photosensitizer by the target cells relative to the other, normal, cells. Indeed, it is known that uptake is generally a function of the molecular structure of the dye being absorbed and that this property varies with different cell types.

It would therefore be highly desirable to be provided with a series of new halogenated rhodamine dyes bearing a variety of substituents at different positions of the molecule thereby making available new selective dyes for specific target cells.

One aim of the present invention is to produce new photosensitizers endowed with the following characteristics: i) preferential localization and uptake by the immunoreactive cells; ii) upon application of appropriate light intensities, killing those cells which have accumulated and retained the photosensiting agents; iii) sparing of the normal hemopoietic stem cell compartment from the destructive effects of activated photosensitizers; iv) potential utilization of photosensitizers for hematopoietic stem cell purging of immunoreactive cells in preparation for allogeneic or autologous stem cell transplantation; and v) Potential utilization of photosensitizers for ex vivo elimination of reactive immune cells in patients with immunological disorders.

Therefore, in accordance with the present invention, there is provided a series of new rhodamine derivatives alone or in association with a pharmaceutically acceptable carrier; whereby photoactivation of said derivatives induces cell killing while unactivated derivatives of general structure represented by the formula (I), and salts thereof, are substantially non-toxic to cells.

In accordance with the present invention, there is also provided with the use of the photoactivable rhodamine derivatives according to the invention for the photodynamic treatment for the selective destruction and/or inactivation of immunologically reactive cells without affecting the normal cells and without causing systemic toxicity for the patient, wherein appropriate intracellular levels of said derivatives are achieved and irradiation of a suitable wavelength and intensity is applied.

In accordance with the present invention, there is also provided a method of prevention of graft-versus-host disease associated with allogeneic stem cell transplantation in a patient, which comprises the steps of: a) activating lymphocytes from donor by mixing donor cells with host cells for a time sufficient for a period of time sufficient for an immune reaction to occur; b) substantially eliminating the activated lymphocytes of step a) with photodynamic therapy using a therapeutic amount of a photoactivable derivative or composition of claim 1 under irradiation of a suitable wavelength; and c) performing allogenic stem cell transplantation using the treated mix of step b).

In accordance with the present invention, there is provided a method for the treatment of immunologic disorder in a patient, which comprises the steps of: a) harvesting said patient\'s hematopoietic cells; b) ex vivo treating of the hematopoietic cells of step a) by photodynamic therapy using a therapeutic amount of a photoactivable derivative or composition of claim 1 under irradiation of a suitable wavelength; and c) performing graft infusion or autograft transplantation using the treated hematopoietic cells of step b).

The immunologic disorder may be selected from the group consisting of conditions in which self cells or donor cells react against host tissues or foreign targets, such as garft-versus-host disease, graft rejection, autoimmune disorders and T-cell mediated immunoallergies.

The hematopoietic cells may be selected from the group consisting of bone marrow, peripheral blood, and cord blood mononuclear cells.

For the purpose of the present invention the following terms are defined below.

The term “immunoreactive disorders” is intended to mean any alloimmune or autoimmune reaction and/or disorders:

In accordance with other aspects of the present invention, these rhodamine compounds which are prepared following the general strategy of halogenating known and readily available rhodamine dyes thereby generating a first and varied series of intermediates, which themselves can serve as potential photosensitizers or, use these halogenated rhodamines as intermediates in the synthesis of a second series of rhodamine dyes whereby one or more halogen has been substituted for one of the groups of structure (I). In the case where all of the halogens are replaced by new groups, a subsequent halogenation step is added to the sequence to obtain the desired compound of structure I (see FIGS. 1 to 5).

Testing of these compounds on various types of cells surprisingly revealed some of the candidate molecules to be non-toxic, more efficient and more selective than the known halogenated rhodamine dyes.

SUMMARY

The present invention relates to rhodamine derivatives of the formula (I)

wherein: one of R1, R2, R3, R4, and R10 represents an halogen atom and each of the remaining R1, R2, R3, R4, and each of the remaining R10 group is independently selected in the group constituted by hydrogen, halogen atoms, an amino, acylamino, diarylamino, cycloalkyl amino, azacycloalkyl, alkylcycloalkylamino, aroylamino, diarylamino, arylalkylamino, aralkyl amino, alkylaralkyl amino, arylaralkyl amino, hydroxy, alkoxy, aryloxy, aralkyloxy, mercapto, alkylthio, arylthio, aralkylthio, carboxyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, cyano, hydroxysulfonyl, amidosulfonyl, dialkylamidosulfonyl, arylalkylamidosulfonyl, formyl, acyl, aroyl, alkyl, alkylene, alkenyl, aryl, aralkyl, vinyl; alkynyl group and by the corresponding substituted groups; m=0-1; n=1-4; A is nil, O, or NH; R9 represents an alkylene group; Z is H, amino, dialkylamino, or trialkylamino salt; X− is an anion; and R5, R6, R7 and R8 are independently H or C1-C6 alkyl or R1 in combination with R5 or R6, or R2 in combination with R5 or R6, or R3 in combination with R7 or R8, or R4 in combination with R7 or R8 represents an alkylene, alone or in association with a pharmaceutically acceptable carrier.

The invention also relates to intermediates of the formula (II) to (VII) and to those of formula (I′) as defined in 1 to 5, which are useful inter alia in the synthesis of the rhodamine derivatives of formula (I).

The invention further relates to the new processes for the synthesis of new rhodamines derivatives of formula (I), wherein the various groups R1 to R10, A, X, Y, Y′ and Z, and m and n are as previously defined, without the exclusion of the compounds listed in the proviso at the end of the previous definition. This processes being defined by the schemes represented in FIGS. 1 to 5 and by the corresponding parts of the description.

The rhodamine derivatives of the invention are useful alone or in combination with a carrier, for treating infections generated by Gram+ and/or by Gram− bacteria. As well as in the treatment of diseases generated by enveloped viruses or by non-enveloped viruses.

Those compounds are also useful in the in-vivo and ex-vivo treatment of immunologic disorders.

FIG. 1 is the general synthesis of substituted 4 and 2,7 halogenated rhodamine derivatives.

FIG. 2 is the general synthesis of substituted 2 and 4,5 halogenated rhodamine derivatives.

FIG. 3 is the general synthesis of substituted 4- and 2,7-halogenated rhodamine derivatives.

FIG. 4 is the general synthesis of substituted 2- and 4,5-halogenated rhodamine derivatives.

FIG. 5 is the general synthesis of substituted 2- and 4,5-halogenated rhodamine derivatives.

FIG. 6 is the bacteriostatic activity of rhodamine derivatives against E coli; the bacterial strain E coli was treated with the rhodamine derivatives at 50 uM without extrusion time. The determined effects are expressed in log decrease of bacterial growth: HA-X-40: 0.6 log; XA-X-44: eradication; HA-X-164: 0.25 log; HA-X-171: 3.7 logs; HA-VM-92: 6.2 logs; TH9402: 7 logs. LB is growth without compounds.

FIG. 7 is bacteriostatic activity of rhodamine derivatives against P. aeruginosa; the bacterial strain P. aeruginosa was treated with the rhodamine derivatives at 50 μM without extrusion time. The determined effects are expressed in log decrease of bacterial growth; TH9402: 2 logs. LB is growth without compounds.

FIG. 8 is bacteriostatic activity of rhodamine derivatives against S. typhimurium; the bacterial strain S. typhimurium was treated with the rhodamine derivatives at 50 μM without extrusion time. The determined effects are expressed in log decrease of bacterial browth: XA-X-44: 5 logs; HA-X-164; 0.3 log; TH9402: 6.7 logs. LB is growth without compounds.

FIG. 9 is bacteriostatic activity of rhodamine derivatives against P. aeruginosa; the bacterial strain P. aeruginosa was treated with the rhodamine derivatives at 50 μM without extrusion time. The determined effects are expressed in log decrease of bacterial growth: TH9402: 2 logs. LB is growth without compounds.

FIG. 10 is antiviral activity of rhodamine derivatives tested on cytomegalovirus; log decreases of viral infectivity and proliferation in FS cells. Compounds were added at 50 μM without extrusion time. Log decreases of viral infectivity and proliferation in FS cells; compounds were added at 50 μM without extrusion time and without light activation.

FIG. 11 is staphylococcus epidermitis; TH9402 inhibits bacterial growth of S. epidermitis at 50 μM without extrusion time.

FIG. 12 is staphylococcus epidermitis; HA-X-40 exhibits a bacteriostatic effect on the growth of S. epidermitis with a 2 logs decrease of bacterial growth at 50 μM without extrusion time.

FIG. 13 is staphylococcus epidermitis; HA-X-40 eradicates bacterial growth of S. epidermitis at 50 μM with 90 minutes extrusion time.

FIG. 14 is, staphylococcus epidermitis; XA-X-44 eradicates bacterial growth of S. epidermitis at 50 μM without extrusion time.

FIG. 15 is staphylococcus epidermitis; HA-X-149 exhibits a bacteriostatic effect on the growth of S. epidermitis with a 4.5 logs decrease of bacterial growth at 50 μM without extrusion time.

FIG. 16 is staphylococcus epidermitis; HA-X-164 exhibits a bacteriostatic effect on the growth of S. epidermitis with a 3 logs decrease of bacterial growth at 50 μM without extrusion time.

FIG. 17 is staphylococcus epidermitis; HA-X-171 exhibits a bacteriostatic effect on the growth of S. epidermitis with a 6.5 logs decrease of bacterial growth at 10 μM without extrusion time.

FIG. 18 is staphylococcus epidermitis; HA-VII-92 exhibits a bacteriostatic effect on the growth of S. epidermitis with a 4 logs decrease of bacterial growth at 10 μM without extrusion time.

The following references mean: HA-X-164: the acetate salt of 2,7-dibromorhodamine B methyl ester (4) HA-X-149: the acetate salt of 2,7-dibromorhodamine B hexyl ester (8) HA-X-171: 4,5-dibromorhodamine 6G (11) HA-X-40: 2′-(6-dimethylamino-3-dimethylimino-3H-xanthen-9-yl) 4′,5′-dichloro-benzoic acid methyl ester hydrochloride (10) HA-X-44: 4,5-dibromorhodamine 110 2-(2-methoxy ethoxy)ethyl esther (13) HA-VIII-92: rhodamine B 3-bromopropyl ester (14) TH 9402: 4,5-dibromorhodamine methyl ester 123.

DETAILED DESCRIPTION

A first object of the present invention is constituted by the new rhodamines derivatives of the formula (I)

wherein: one of R1, R2, R3, R4, and R10 represents an halogen atom and each of the remaining R1, R2, R3, R4, and each of the remaining R10 group is independently selected in the group constituted by hydrogen, halogen atoms, an amino, acylamino, dialkylamino, cycloalkylamino, azacycloalkyl, alkylcycloalkylamino, aroylamino, diarylamino, arylalkylamino, aralkylamino, alkylaralkylamino, arylaralkylamino, hydroxy, alkoxy, aryloxy, aralkyloxy, mercapto, alkylthio, arylthio, aralkylthio, carboxyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, cyano, hydroxysulfonyl, amidosulfonyl, dialkylamidosulfonyl, arylalkylamidosulfonyl, formyl, acyl, aroyl, alkyl, alkylene, alkenyl, aryl, aralkyl, vinyl, alkynyl group and by the corresponding substituted groups; m=0-1; n=1-4 A is nil, O, or NH; R9 represents an alkylene group; Z is H, amino, dialkylamino, or trialkylamino salt; X− is an anion; and R5, R6, R7 and R5 are independently H or C1-C6 alkyl or R1 in combination with R5 or R6, or R2 in combination with R5 or R6, or R3 in combination with R7 or R8, or R4 in combination with R7 or R8 represents an alkylene, alone or in association with a pharmaceutically acceptable carrier, with the proviso that the following specific compounds: 4,5-dibromorhodamine 123 (2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid methyl ester hydrochloride) also called TH9402; 4,5-dibromorhodamine 123 (2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid ethyl ester hydrochloride); 4,5-dibromorhodamine 123 (2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid octyl ester hydrochloride); 4,5-dibromorhodamine 110 n-butyl ester (2-(4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride); and rhodamine B n-butyl ester (2-(6-ethyl amino-3-ethyl imino-3H-xanthen-9-yl)-benzoic acid n-butyl ester hydrochloride) are excluded.

According to a preferred embodiment of this object of the invention “alkyl” means a straight or branched aliphatic hydrocarbon group and the corresponding substituted alkyl group bearing one or more substituents which may be the same or different and which are selected in the group constituted by halo, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, and cycloalkyl and “branched” means that a lower alkyl group such as methyl, ethyl or propyl is attached to a linear alkyl chain, preferred alkyl groups include the “lower alkyl” groups which are those alkyl groups having from about 1 to about 6 carbons., exemplary alkyl groups are methyl, ethyl, isopropyl, hexyl, cyclohexylmethyl, methyl or ethyl groups are more preferred; “cycloalkyl” means a non-aromatic ring preferably composed from 4 to 10 carbon atoms, and the cyclic alkyl may be partially unsaturated, preferred cyclic alkyl rings include cyclopentyl, cyclohexyl, cycloheptyl, the cycloalkyl group may be optionally substituted with an aryl group substituent, the cyclopentyl and the cyclohexyl groups are preferred; “alkenyl” means an alkyl group containing a carbon-carbon double bond and having preferably from 2 to 5 carbon atoms in the linear chain, exemplary groups include allyl vinyl; “alkynyl” means an alkyl group containing a carbon-carbon triple bond and having preferably from 2 to 5 carbon atoms in the linear chain; exemplary groups include ethynyl, propargyl; “aryl” means an aromatic carbocyclic radicalor asubstituted carbocyclic radical containing preferably from 6 to 10 carbon atoms, such as phenyl or naphtyl or phenyl or naphtyl substituted by at least one of the substituents selected in the group constituted by alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, alkylthio, arylthio, alkylene or -NYY′ where Y and Y′ are independently hydrogen, alkyl, aryl, or aralkyl; “aralkyl” means a radical in which an aryl group is substituted for an alkyl H atom, exemplary aralkyl group is benzyl; “acyl” means an alkyl-CO— group in which the alkyl group is as previously described, preferred acyl have an alkyl containing from 1 to 3 carbon atoms in the alkyl group, exemplary groups include acetyl, propanoyl, 2-methylpropanoyl, butanoyl or palmitoyl; “aroyl” means an aryl-CO— group in which the aryl group is as previously described and preferably contains from 6 to 10 carbon atoms in the ring, exemplary groups include benzoyl and 1- and 2-naphtoyl; “alkoxy” means an alkyl-O— group in which the alkyl group is as previously described, exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, and heptoxy; “aryloxy” means an aryl-O— group in which the aryl group is as previously described, exemplary aryloxy groups include phenoxy and naphthoxy; “alkylthio” means an alkyl-5-group in which the alkyl group is as previously described, exemplary alkylthio groups include methylthio, ethylthio, i-propylthio and heptylthio; “arylthio” means an aryl-5-group in which the aryl group is as previously described, exemplary arylthio groups include phenylthio, naphthylthio; “aralkyloxy” means an aralkyl-O— group in which the aralkyl group is as previously described, exemplary aralkyloxy group is benzyloxy; “aralkylthio” means an aralkyl-S— group in which the aralkyl group is as previously described, exemplary aralkylthio group is benzylthio; “dialkylamino” means an —NYY′ group wherein both Y and Y′ are alkyl groups as previously described, exemplary alkylamino include ethylamino, dimethylamino and diethylamino; “alkoxycarbonyl” means an alkyl-O—CO— group wherein the alkyl group is as previously described., exemplary alkoxycarbonyl groups include methoxy- and ethoxy-carbonyl; “aryloxycarbonyl” means an aryl-O—CO— group wherein the aryl group is as previously described, exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl; “aralkoxycarbonyl” means an aralkyl-O—CO— group wherein the aralkyl is as previously defined, exemplary aralkoxycarbonyl group is benzyloxycarbonyl; “carbamoyl” is an H2N—CO— group; “alkylcarbamoyl” is an Y′YN—CO— group wherein one of Y and Y′ is hydrogen and the other of Y and Y′ is alkyl as defined previously; “dialkylcarbamoyl” is an Y′YN—CO— group wherein both Y and Y′ are alkyl as defined previously; “acylamino” is an acyl-NH group wherein acyl is as defined previously; “aroylamino” is an aroyl-NH group wherein aroyl is as defined previously; “alkylene” means a straight or branched bivalent hydrocarbon chain group having preferably from 2 to 8 carbon atoms, and the alkylene group may be interrupted by, one or more substituted nitrogen atoms wherein the substituent of the nitrogen atom is alkyl or oxygen or sulfur atoms, and it is presently more preferred that the alkylene group has from 2 to 3 carbon atoms, exemplary alkylene groups include ethylene (—CH2CH2—), propylene (—CH2CH2CH2—), —CH2NMe-CH2—, O—CH2—O or —O—CH2CH2—O—; “halo” preferably means fluoro, chloro, bromo or iodo; “azacycloalkyl” preferably means a 4 to 9 membered saturated carbon ring where one of the methylene groups is replaced by nitrogen; “cycloalkylamine” means an —NYY′ group wherein one of the Y and Y′ is hydrogen and the other Y and Y′ is cycloalkyl as defined previously; “alkylcycloalkylamino” means an NYY′ group wherein one of the Y and Y′ is alkyl as defined previously and the other Y and Y′ is cycloalkyl as defined previously; “diarylamino” means an —NYY′ group wherein both Y and Y′ are aryl groups as previously described; “aralkylamino” means an —NYY′ group wherein one of the Y and Y′ is hydrogen and the other Y and Y′ is aralkyl as defined previously; “arylalkylamino” means an —NYY group wherein one of the Y and Y′ is alkyl as defined previously and the other Y and Y′ is aryl as defined previously; “alkylaralkylamino” means an —NYY′ group wherein one of the Y and Y′ is alkyl as defined previously and the other Y and Y′ is aralkyl as defined previously; “arylaralkylamino” means an —NYY′ group wherein one of the Y and Y′ is aryl as defined previously and the other Y and Y′ is aralkyl as defined previously; “mercapto” is a —SH or a SR group wherein R may be any of the above defined groups R1 to R10, the —SH, the mercaptoaryl and the mercaptoalkyl groups are preferred; “hydroxysulfonyl” is an —SO3H; “amidosulfonyl” is an —SO2NH2; “dialkylamidosulfonyl” means an —SO2NYY′ group wherein both Y and Y′ are alkyl groups as previously described; “arylaralkylamidosulfonyl” means an —SO2NYY′ group wherein one of the Y and Y′ is aryl as defined previously and the other Y and r is aralkyl as defined previously; and “anion” means the deprotonated form of an organic or inorganic acid and the anion is preferably selected from hydrochlorides, hydrobromides, sulfates, nitrates, borates, phosphates, oxalates, tartrates, maleates, citrates, acetates, ascorbates, succinates, benzenesulfonates, methanesulfonates, cyclohexanesulfonates, toluenesulfonates, sulfamates, lactates, malonates, ethanesulfonates, cyclohexylsulfamates, and quinates. In the case where the rhodamine derivative bears one or more acidsubstituents, the covered compound comprise the internal salt or any salt derived from neutralization by any of the following bases: sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, ammonia, ethylene diamine, lysine, diethanolamine, piperazine and the like.

A preferred embodiment of the invention is constituted by those rhodamine derivatives wherein at least 2 of the R1, R2, R3, R4, and R10 groups represent an halogen atom which is preferably a bromide atom.

More preferred are those rhodamine derivatives, wherein the halogen(s) atom is(are) on the 2-7, 4-5 or 4′-5′ position on the ring or is(are) at the end of the ester chain.

The following specific rhodamine derivatives are particularly interesting, the:

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