BRPI0204489B1 - "rhodamine derivative, pharmaceutical composition, intermediate, and process for the synthesis of new rhodamine derivatives". - Google Patents

Abstract

"rhodamine derivatives, compound, intermediates, processes for the synthesis of rhodamine derivatives, use of a compound, methods for photodynamic therapy of patients, in vitro purification of human bone marrow, treatment of leukemia, immune disorder and bacterial infections and disease prevention, photoactivable pharmaceutical composition and bactericidal solution and drug ". novel compounds of formula (i) wherein: one of r1, r2, r3, r4 and (r10) n represents a halogen atom and each of the remaining r1, r2, r3, r4 and each of the remaining group r10 is independently selected from the group consisting of hydrogen, halogen atoms, an amino group, acylamino, dialkylamino, cycloalkylamino, azacycloalkyl, alkylcycloalkylamino, aroylamino, diarylamino, arylalkylamino, aralkylamino, arylalkylamino, arylalkyloxy, arylalkylamino, arylalkylamino aralkylthio, carboxyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, cyano, hydroxysulfonyl, amylsulfonyl, arylalkylsulfonyl, arylalkyl, arylalkyl, alkyl, arylalkyl, alkyl, arylalkyl, aryloxycarbonyl, alkyl, arylalkyl, alkyl, arylalkyl substituted: m = 0-1; n = 1-4, a is nothing, o or nh; R9 represents an alkylene group; z is h, amino, dialkylamino or trialkylamino salt; x- is an anion; 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 R7 or R8, or R4, in combination with R7 or R8, represents an alkylene alone or in combination with a pharmaceutically acceptable carrier. These compounds, which are useful as intermediates and as bactericides, as an antiviral agent and in the treatment of immune disorders.

Description

FIELD OF THE INVENTION The invention relates to novel rhodamine derivatives, which are useful for their pharmaceutical and non-pharmaceutical properties.

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

They are useful alone or in combination with a pharmaceutically acceptable carrier in the treatment and / or prevention of immune disorders.

In addition, those derivatives are useful as intermediates in the synthesis of other new rhodamine derivatives and also in the novel synthesis of known rhodamine derivatives.

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

BACKGROUND OF THE INVENTION Photodynamic therapy has been used as a method for the eradication of autologous graft neoplastic cells for cancer treatments. This method is based on the use of photosensitizing dyes, which, when activated with light of a particular wavelength, produce toxic O2 'radicals, ultimately resulting in cell death. Photochemical treatments have also been used for pathogenic inactivation, such as in the “decontamination” of blood and blood products. The danger of pathogenic transmission through whole blood transfusion, platelet concentrates, plasma and / or red blood cells is still a major medical concern. Although there has been impressive progress in preventing and maintaining blood safety regarding the presence of microorganisms, blood components continue to carry a risk of pathogen transfusion. In addition, the presence of viruses in the blood components is also of great concern, especially regarding the presence of Hepatitis C virus 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 human T-cell lymphotrophic virus type 1 (HTLV-1), hepatitis B (HBV) and cytomegalovirus. Photodynamic compounds such as pseuralenes, porphyrins, riboflavins and methylene blue dimethyl have been used in the treatment of blood product pathogens. These compounds require radiation by an ultraviolet A lamp (UVA) to be activated, thus leading to a possible mutagenic effect on the remaining cells present in the treated samples (Corash, L., Inactivation of infectious pathogens in labile blood components: meeting the challenge, Transfiis Clin Biol, 2001, 8, 138-145Lin, L., Londe, H., Janda, MJ, Hanson, CV and Corash, L., Photochemical inactivation of pathogenic bacteria in human platelet concentrates, Bloos, 1994, 83, 9, 2698 -2706; Lin, L, Londe, H., Hanson, CV, Wiesehahn, G., Isaacs, S., Cimino, G. and Corash, L., Photochemical inactivation of human immunodeficiency virus in platelet concentrates, Blood , 1993, 82, 1, 292-297; Lin, L., Eiesehahn, GP, Morei, PA 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, DN, Wiesehahn, GP, Alfonso, R., Behrman, B., Cimino, GD , 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 UVA exposure to blood components, these techniques are not entirely satisfactory. However, there was a need for new light-sensitive derivatives that do not require UVA exposure of blood components and that may also be a safer and more acceptable replacement for UVA-treated blood components.

Immunological disorders are uncontrolled cell proliferations, which 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 normal cell immunoreactivity induce breakdown in these normal cells and tissues. Such immunological disorders are usually divided into alloimmune conditions and autoimmune conditions. Alloimmune disorders occur mainly in the context of allogeneic transplantation (bone marrow and other organs; kidneys, heart, liver, lungs, etc.). In the bone marrow transplant setting, donor immune cells present in the hematopoietic stem cell graft react towards normal host tissues, causing graft-versus-host disease (GVHD). GVHD induces damage mainly to the liver, skin, colon, lung, eyes and mouth. Autoimmune disorders consist of numerous arthritic conditions, such as rheumatoid arthritis, sclerodema, and lupus erythematosus; endocrine conditions such as diabetes mellitus; neurological conditions such as multiple sclerosis and myasthenia gravis; haematological disorders such as autoimmune hemolytic anemia etc. The immune reaction, in both alloimmune and autoimmune disorders, progresses to generate dysfunction and organ damage.

Despite major advances in treatment, immunological complications remain the leading cause of allogeneic transplant failure, either in hematopoietic stem cell transplantation (GVHD) or solid organ transplantation (graft rejection). In addition, autoimmune disorders represent the leading cause of both morbidity and mortality. The prevention and treatment of these immune disorders has been based primarily on the use of immunosuppressive agents, monoclonal antibody-based therapies, radiation therapy and, more recently, molecular inhibitors. 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 transplants (bone marrow, kidneys, liver, heart and lungs) and for most immune disorders (rheumatoid arthritis, connective tissue diseases, multiple sclerosis etc.) the resolution of immune dysfunction and healing has not been achieved. Therefore, the development of new avenues for the prevention and treatment of patients with immune disorders is critically necessary, particularly for those patients who are at high risk or whose disease has progressed and are refractory to standard immunosuppressive therapy. Allogeneic stem cell transplantation (AlloSCT) has been employed to treat numerous malignant and nonmalignant 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 to replace the host hematopoietic system. AlloSCT has been shown to induce increased response rates compared to standard therapeutic options. An important problem that needs to be highlighted when employing AloSCT concerns the risk of reinfusing immune cells, which will subsequently recognize the patient's cells as foreign and cause GVHD. A variety of techniques have been developed that can deplete up to 105 cells of the marrow or peripheral blood. These techniques, including immunological and pharmacological purging, are not entirely satisfactory. A major consideration when purging stem cell grafts is to preserve non-host reactive T cells so that they can exert anti-infectious and anti-leukemic activity when grafting. The potential of photodynamic therapy, in combination with photosensitizing molecules, capable of destroying immunologically reactive cells while sparing normal nonreactive host 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 recurrent leukemic cells, has been largely unexplored. To achieve T cell eradication, several pathways have been proposed, including: 1) in vitro exposure of the graft to monoclonal antibodies and immunotoxins against T cell surface antigens (anti-CD3, anti-CD6, anti-CD8 etc.); 2) in vitro selection by soy agglutinin and rosette of red blood cells of sheep; 3) positive selection of CD34-I- 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 targeting monoclonal antibodies or immunotoxins to the interleukin 2 receptor or OX-40 antigen (Cavazzana-Calvo M. et al. (1990) Transplantation, 50: 1-7; Tittle TV and others (1997) Blood 89: 4652-58; Harris DT and others (1999) Bone Marrow Transplantation 23: 137-44).

However, most of these methods are not specifically targeted to the alloreactive T cell subset and are associated with numerous problems, including disease recurrence, graft rejection, secondary 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 to cancers of various origins and, more recently, to the eradication of viruses and pathogens (Raab O. (1990) Infusoria Z. Biol. 39: 524).

Early 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 VH (1903) Muench Med Wochneshr, 47: 2042; Hansman W. (1911) Biochem. Z., 30: 276). In the 1940s and 1960s, a variety of tumor types underwent photodynamic therapy, both in vitro and in vivo (Kessel, David (1990) Photodynamic Therapy of Neoplastic Disease, Vol. I, Π, CRC Press. David Kessel, Ed ISBN 08-8493-5816-7 (v. 1), ISBN 0-8493-5817-5 (v. 2)). Dougherty and colleagues and others in the 1970s and 1980s systematically explored the potential of oncological application of photodynamic therapy (Dougherty TJ (1974) J. Natl. Cancer Inst., 51: 1333-1336; Dougherty TJ et al. (1975) J Natl Cancer Inst., 55: 115-121; Dougherty TJ et al. (1978) Cancer Res., 38: 2628-2635; Dougherty TJ (1984) Urol Suppl., 23: 61; Dougherty TJ (1987) Photochem. Photobiol., 45: 874 - 889).

Treatment of immunoreactive cells with photodynamic therapy There is currently a lack of agents that allow selective destruction of immunoreactive cells while leaving the suppressed residual cell population intact. Preferred absorption of photosensitive dye and cytotoxicity of photodynamic therapy against leukemia (Jamieson CH et al. (1990) Leuk Res., 14: 209-219) and lymphoid cells (Greinix HT et al., Blood (1998) 92: 3098- 3104; are reviewed in Zic JA and others Therapeutic Apheresis (1999) 3: 50-62) have been previously demonstrated.

It would be highly desirable to provide photosensitizers which have at least one of the following characteristics: i) preferential localization and absorption by immunoreactive cells; ii) in the application of appropriate light intensities, kill those cells that have accumulated and retained the photosensitive agents; iii) spare the normal hemopoietic stem cell compartment from the destructive effects of activated photosensitizers; and iv) potential use of photosensitizers for hematopoietic stem cell purging of immunoreactive cells in preparation for allogeneic or autologous stem cell transplantation. v) potential use of photosensitizers for ex vivo elimination of reactive immune cells in patients with immunological disorders.

Rhodamine Dyes Rhodamine 123 (2- (6-amino-3-imino-3H-xanthene-9-yl) benzoic acid methyl ester hydrochloride), a pyrophilic lipophilic cationic dye that can disrupt cellular homeostasis and be cytostatic or cytotoxic at high concentration exposure and / or photodynamic therapy, albeit with very poor quantum production (Darzynkiewicz Z., Carter S. (1988) Cancer Res., 48: 1295 - 1299). It has been used in vitro as a fluorescent spot specific for living mitochondria. It is absorbed and is preferably retained by many tumor cell types, impairing their proliferation and survival by altering the membrane and mitochondrial function (Oseroff AR (1992) In Photodynamic therapy (Henderson BW, Dougherty TJ, eds)). , pp. 79-91). In vivo, rhodamine 123 chemotherapy may prolong the survival of cancerous mice, but despite initial attempts to use rhodamine 123 in the treatment of tomors, the systemic toxicity of rhodamine 123 may limit usefulness (Bemal, SD, et al. (1983). ) Science, 222: 169; Powers, SK et al. (1987) J. Neurosur., 67: 889). United States Patent No. 4,612,007, issued September 16, 1986, to Richard L. Edelson, describes a method for thoroughly treating human blood to reduce the lymphocyte population functioning in the blood system of a Human individual. Blood taken from the individual is passed through a field of ultraviolet radiation in the presence of a dissolved photoactive agent capable of forming photoducts with lymphocytic DNA. This method has the following disadvantages and shortcomings. The procedure described is based on the use of known commercially available photoactive chemical agents to thoroughly treat 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, not eradicates, the target cell population. In addition, the wavelength range of UV radiation used in the process proposed by Richard L. Edelson could be harmful to normal cells. The International Application, published January 7, 1993, under International Publication No. WO 93/00005, describes a method for inactivating pathogens in a body fluid while minimizing adverse effects caused by photosensitive agents. This method essentially consists of treating the cells in the presence of a photoactive agent under conditions that effect pathogen destruction and preventing the treated cells from contacting the additional extracellular protein for an extended period of time. This method refers to the eradication of infectious agents from the collected blood and its components prior to storage or transfusion.

It would be highly desirable to provide new rhodamine derivatives for the treatment of immunoreactive cells that overcome these disadvantages while having no systemic toxicity to the patient.

Halogenated rhodamine salts are dyes that have the ability to penetrate cells and are usually located in mitochondria. They have been used in conjunction with photoactivation to kill certain cell types, that is, cancer cells in leukemia, and activated T-cells in autoimmune diseases. The generally accepted mechanism for the effect of killing cells is the production of singlet oxygen, which is the reactive intermediate in disrupting biological life-sustaining processes in the cell. The role of rhodamine dye in the production of singlet oxygen is that of a photosensitizer, that is, of a molecule that absorbs the incident light energy and transfers it to normal state oxygen, thereby raising 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 increased by the presence of heavy atoms, such as halogens, in the dye aromatic chromophore.

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

However, it would be highly desirable to be provided with a series of novel halogenated rhodamine dyes containing a variety of substituents at different positions of the molecule, thereby making new selective dyes available for specific target cells.

An object of the present invention is to produce new photosensitizers having the following characteristics: i) preferential localization and absorption by immunoreactive cells; ii) the application of appropriate light intensities, killing those cells that have accumulated and retained photosensitizing agents; iii) spare the normal hemopoietic stem cell compartment from the destructive effects of activated photosensitizers; iv) potential use of photosensitizers for hematopoietic stem cell purging of immunoreactive cells in preparation for allogeneic or autologous stem cell transplantation; and v) Potential use of photosensitizers for ex vivo elimination of reactive immune cells in patients with immunological disorders.

Therefore, in accordance with the present invention, there are provided a number of novel rhodamine derivatives, either alone or in combination with a pharmaceutically acceptable carrier; whereby photoactivation of said derivatives induces cell killing, while inactivated derivatives of general structure represented by formula (I) and their salts are substantially non-toxic to cells.

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

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

In accordance with the present invention, there is provided a method for treating immune disorder in a patient comprising the steps of: a) harvesting said patient's hematopoietic cells; b) ex vivo treatment of the hematopoietic cells of step a) by photodynamic therapy, employing a therapeutic amount of a photoactivatable derivative or composition of claim 1 under irradiation of a suitable wavelength; and c) performing graft infusion or autograft transplantation using the hematopoietic cells of step b. The immune disorder may be selected from the group consisting of conditions in which the donor auto cells or cells react against host tissues or foreign targets, such as such as graft-versus-host disease, graft rejection, autoimmune disorders, and T-cell mediated immuno-disorders.

Hematopoietic cells can be selected from the group consisting of bone marrow, peripheral blood and cord blood mononuclear cells.

For purposes of the present invention, the following terms are defined below. The term "immunoreactive disorders" is intended to mean any reaction and / or alloimmune or autoimmune disorders.

According to other aspects of the present invention, these rhodamine compounds, which are prepared by observing the general strategy of known and readily available halogenating rhodamine dyes, thereby generating a first and varied series of intermediates, which may themselves be used. as potential photosensitizers or the use of these halogenated rhodamines as intermediates in the synthesis of a second series of rhodamine dyes, whereby one or more halogen (s) substituted one of the groups of structure (I). In the event that all halogens are replaced by new groups, a subsequent halogenation step is added to the sequence to obtain the desired compound of structure I (see Figures 1 to 5). Testing of these compounds on various cell types has surprisingly revealed that some of the candidate molecules are non-toxic, more efficient and more selective than known halogenated rhodamine dyes.

SUMMARY OF THE INVENTION The present invention relates to rhodamine derivatives of formula (I) wherein: one of R 1, R 2, R 3, R 4 and R [0 represents a halogen atom and each of the remaining R 1, R 2 R 3 and R 4 and each of the remaining R 10 group is independently selected from the group consisting of hydrogen and halogen atoms, amino, acylamino, dialkylamino, cycloalkylamino, azacycloalkyl, alkylcycloalkylamino, aroylamino, diarylamino, arylalkylamino, arylalkylamino, aralkylamino, arylalkylamino alkoxy, aryloxy, aralkyloxy, mercapto, alkylthio, arylthio, aralkylthio, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, cyano, hydroxysulfonyl, arylalkylalkylaminoalkylaminoalkylaminoalkyl , aryl, aralkyl, vinyl, alkynyl and the corresponding substituted groups. - m = 0 - 1; -n = 1-4; - A is nothing, O or NH; - R 9 represents an alkylene group; Z is H, amino, dialkylamino or trialkylamino salt; - X 'is an anion; and - R5, Re, R7 and R8 are independently H or C1 -C6 alkyl or R1 in combination with R5 or Re, or R2 in combination with R5 or Re, or R3 in combination with R7 or R8 or R4 in Combination with R 7 or R 8 represents an alkylene alone or in combination with a pharmaceutically acceptable carrier. The invention also relates to intermediates of formula (II) to (VII) and those of formula (Γ) as defined in 1 to 5, which are useful, among other things, in the synthesis of rhodamine derivatives of formula (I). . The invention further relates to novel processes for the synthesis of novel rhodamine derivatives of formula (I), wherein the various groups R 1 to R 1, A, X, Y, Y 'and Z emen are as previously defined, without exclusion. of the compounds listed in the condition at the end of the previous definition. These processes being defined by the schemes shown in Figures 1 to 5 and 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 Gra- bacteria. As well as treating diseases generated by enveloped viruses or non-enveloped viruses.

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

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is the general synthesis of substituted halogenated rhodamine derivatives 4 and 2.7. Fig. 2 is the general synthesis of substituted halogenated rhodamine derivatives 2 and 4,5. Fig. 3 is the general synthesis of substituted halogenated rhodamine derivatives 4 and 2.7. Fig. 4 is the general synthesis of substituted halogenated rhodamine derivatives 2 and 4,5. Fig. 5 is the general synthesis of substituted halogenated rhodamine derivatives 2 and 4,5. Fig. 6 is the bacteriostatic activity of rhodamine derivatives in relation to Escherichia coli. The bacterial strain Escherichia coli was treated with 50 µM rhodamine derivatives without extrusion time. Determined effects are expressed as log decreases in 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-VIII-92: 6.2 LOGS; TH9402: 7 logs. LB is growth without compounds. Fig. 7 is bacteriostatic activity of rhodamine derivatives relative to P. aeruginosa; The P. aeruginosa bacterial strain was treated with the 50 µM rhodamine derivatives without extrusion time. Determined effects are expressed as log decreases in bacterial growth: TH9402: 2 logs. LB is growth without compounds. Fig. 8 is bacteriostatic activity of rhodamine derivatives relative to S. typhimurium; The S. typhimurium bacterial strain was treated with 50 µM rhodamine derivatives without extrusion time. Determined effects are expressed as log decreases in bacterial growth: 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 relative to E. coli; The bacterial strain E. coli was treated with the 50 µM rhodamine derivatives without extrusion time. Determined effects are expressed as log decreases in 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-VIII-92: 6.2 logs; TH9402: 7 logs. LB is growth without compounds. Fig. 10 is bacteriostatic activity of rhodamine derivatives relative to P. aeruginosa; The P. aeruginosa bacterial strain was treated with the 50 µM rhodamine derivatives without extrusion time. Determined effects are expressed as log decreases in bacterial growth: TH9402: 2 logs. LB is growth without compounds. Fig. 11 is bacteriostatic activity of rhodamine derivatives relative to S. typhimurium; The S. typhimurium bacterial strain was treated with 50 µM rhodamine derivatives without extrusion time. Determined effects are expressed as log decreases in bacterial growth: XA-X-44: 5 logs; HA-X-164: 0.3 log; TH9402: 6.7 logs. LB is growth without compounds. Fig. 12 is bacteriostatic activity of rhodamine derivatives with respect to E. coli; The bacterial strain E. coli was treated with the 50 µM rhodamine derivatives without extrusion time. Determined effects are expressed as log decreases in bacterial growth: HZ-X-40: 0.6 log; XA-X-44: eradication; HA-X-164: 0.25 log; HA-X-171: 3.7 logs; HA-VIII-92: 6.2 logs; TH9402: 7 logs. LB is growth without compounds. Fig. 13 is bacteriostatic activity of rhodamine derivatives relative to P. aeruginosa; The P. aeruginosa bacterial strain was treated with the 50 µM rhodamine derivatives without extrusion time. Determined effects are expressed as log decreases in bacterial growth: TH9402: 2 logs. LB is growth without compounds.

Fig. 14 is bacteriostatic activity of rhodamine derivatives relative to S. typhimurium; The S. typhimurium bacterial strain was treated with 50 µM rhodamine derivatives without extrusion time. Determined effects are expressed as log decreases in bacterial growth: XA-X-44: 5 logs; HA-X-164: 0.3 log; TH9402: 6.7 logs. LB is growth without compounds.

Fig. 15 is antiviral activity of rhodamine derivatives tested on cytomegalovirus; log decreases in infectivity and viral proliferation in FS cells. Compounds were added at 50 µM, without extrusion time. Log decreases in infectivity and viral proliferation in FS cells; Compounds were added at 50 mM with no extrusion time and no light activation. Figure 16 is staphilococcus epidermitis; TH9402 inhibits S. epidermitis bacterial growth at 50 µM without extrusion time. Figure 17 is staphilococcus epidermitis; HA-X-40 exhibits a bacteriostatic effect on S. epidermitis growth with a 2 log decrease in bacterial growth at 50 µM, without extrusion time. Figure 18 is staphilococcus epidermitis; HA-X-40 eradicates S. epidermitis bacterial growth at 50 µM with 90 minutes extrusion time. Figure 19 is staphilococcus epidermitis; HA-X-44 eradicates S. epidermitis bacterial growth at 50 µM, without extrusion time. Figure 20 is staphilococcus epidermitis; HA-X-149 exhibits a bacteriostatic effect on S. epidermitis growth with a 4.5 log decrease in bacterial growth at 50 µM without extrusion time. Figure 21 is staphilococcus epidermitis; HA-X-164 exhibits a bacteriostatic effect on S. epidermitis growth with a 3 log decrease in bacterial growth at 50 µM with no extrusion time. Figure 22 is staphilococcus epidermitis; HA-X-171 exhibits a bacteriostatic effect on S. epidermitis growth with a 6.5 log decrease in bacterial growth at 10 µM, without extrusion time. Figure 23 is staphilococcus epidermitis; HA-VIII-92 exhibits a bacteriostatic effect on S. epidermitis growth with a 4 log decrease in bacterial growth at 10 µM with no extrusion time. Figure 24 is antiviral activity of rhodamine derivatives tested on cytomegalovirus; log decreases in infectivity and viral proliferation in MRC-5 cells. Compounds were added at 50 µM without extrusion time.

The following references mean: HA-X-164: 2,7-dibromorodamine B (8) methyl ester acetate salt HA-X-149: 2,7-dibromorodamine B (8) HA hexyl ester acetate salt -X-171: 4,49-dibromorhodamine 6G (11) HA-X-40: 2 '- (6-dimethylamino-3-dimethylimino-3H-xanthen-9-yl) 4', 5'-acid methyl ester hydrochloride dichloro-benzoic (10) HA-X-44: 4,5-dibromorodamine ethyl ester 110 2- (2-methoxy ethoxy) (13) HA-VIII-92: rhodamine bromopropyl ester B 3 (14) TH 9402: 4,5-dibromorodamine methyl ester 123.

DETAILED DESCRIPTION OF THE INVENTION

A first object of the present invention is the novel rhodamine derivatives of formula (I) wherein: one of R 1, R 2, R 3, R 4 and R 10 represents a halogen atom and each of the remaining R 1, R 2, R 3 and R 4 and each of the remaining R 10 group are independently selected from the group consisting of hydrogen and halogen atoms, amino, acylamino, dialkylamino, cycloalkylamino, azacycloalkyl, alkylcycloalkylamino, aroylamino, diarylamino, arylalkylamino, aralkylaminoalkylamino, arylalkylamino, arylalkylamino aryloxy, aralkyloxy, mercapto, alkylthio, arylthio, aralkylthio, carboxyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkylcarbamoyl, cyano, hydroxysulfonyl, amylsulfonyl, aryloxyalkyl, aryloxyalkyl, aryloxyalkyl, amylsulfonyl , aralkyl, vinyl, alchemy and the corresponding substituted groups - m = 0 - 1 ; - η = 1 - 4; - A is nothing, O or NH; - R 9 represents an alkylene group; Z is H, amino, dialkylamino or trialkylamino salt; - X 'is an anion; and - R5, R5, R7 and R5 are independently H or C1 -C6 alkyl or R1 in combination with R5 or R1 or R2 in combination with R5 or R1 or R3 in combination with R7 or R6 or R4. , in combination with R 7 or R 8, represents an alkylene, alone or in association with a pharmaceutically acceptable carrier. provided that the following specific compounds: - (4,5- (2- (4,5-Dibromo-6-amino-3-imino-3H-xanten-9-yl) benzoic acid) methyl ester hydrochloride) dibromorhodamine 123, also called TH9402; - (4,5-Dibromorodamine-2- (4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl) benzoic acid ethyl ester hydrochloride) 123; - (4,5-dibromorodamine-2- (4,5-dibromo-6-amino-3-imino-3H-xanthen-9-yl) benzoic acid) hydrochloride); - (2- (4,5-Dibromo-6-amino-3-imino-3H-xanthen-9-yl) benzoic acid n-butyl ester hydrochloride) of 4,5-dibromorodamine n-butyl ester 110; and - (2- (6-Ethylamino-3-ethylimino-3H-xanten-9-yl) -benzoic acid n-butyl ester hydrochloride) of rhodamine B-n-butyl ester 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 containing one or more substituents, which may be the same or different and which are selected from the group consisting of 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 straight alkyl chain, preferred alkyl groups including "lower alkyl" groups, which are those alkyl groups having from about 1 to about 6 carbons, exemplary alkyl groups being methyl, ethyl, isopropyl, hexyl, cyclohexylmethyl, methyl or ethyl which are most preferred; "Cycloalkyl" means a non-aromatic ring, preferably composed of 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 optionally be substituted with an aryl group substituent, cyclopentyl and cyclohexyl groups are preferred. "Alkenyl" means an alkyl group containing a carbon-carbon double bond and preferably having from 2 to 5 carbon atoms in the straight chain, exemplary groups include allyl vinyl; "Alkynyl" means an alkyl group containing a triple carbon-carbon bond and preferably having from 2 to 5 carbon atoms in the straight chain, exemplary groups include ethinyl, propargyl; - "aryl" means an aromatic carbocyclic radical or a substituted carbocyclic radical preferably containing from 6 to 10 carbon atoms, such as phenyl or naphthyl or phenyl or naphthyl substituted by at least one of the substituents selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamino, carbamoyl, alkylcarbylamoyl, alkylcarbamoyl, alkylamino NYY ', where Y and Y' are independently hydrogen, alkyl, aryl or aralkyl; - "aralkyl" means a radical in which an aryl group is substituted by an H-alkyl atom, exemplary aralkyl group is benzyl; - "acyl" means an alkyl-CO- group, wherein the alkyl group is as described above, preferred acyl has 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, wherein an aryl group is as previously described and preferably contains from 6 to 10 ring carbon atoms; exemplary groups include benzoyl and 1- and 2-naphthoyl; "Alkoxy" means an alkyl-O- group, where the alkyl group is as described above, exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy and heptoxy; "Aryloxy" means an aryl-O- group, wherein the aryl group is as described above, exemplary aryloxy groups include phenoxy and naphthoxy; "Alkylthio" means an S-alkyl group, wherein the alkyl group is as previously described, exemplary alkylthio groups include methylthio, ethylthio, i-propylthio and heptylthio; "Arylthio" means an aryl-S group, wherein the aryl group is as previously described, exemplary arylthio groups include phenylthio, naphthylthio; "Aralkyloxy" means an aralkyl-O group, wherein the aralkyl group is as previously described, exemplary aralkyloxy group is benzyloxy; "Aralkylthio" means an aralkyl-S group, wherein 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 described above, exemplary alkylamino includes 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 naphthoxycarbonyl; "Aralkoxycarbonyl" means an aralkyl-O-CO group, wherein aralkyl is as previously defined, aralkoxycarbonyl group is benzyloxycarbonyl; Carbamoyl is a group H2N-CO; - "alkylcarbamoyl" is a Υ'grupo-CO- group, wherein one of Y and Y 'is hydrogen and the other of Y and Y' is alkyl as defined above; - "dialkylcarbamoyl" is a group Y'YN-CO-, wherein both Y and Y 'are alkyl as defined above; - "acylamino" is an acyl-NH group, where acyl is as defined above; - "aroylamino" is an aroyl-NH group, wherein aroyl is as defined above; "Alkylene" means a straight or branched bivalent hydrocarbon chain group preferably having from 2 to 8 carbon atoms and the alkylene group may be interrupted by one or more substituted nitrogen atoms, wherein the nitrogen atom substituent is alkyl or oxygen or sulfur atoms, and is presently more preferred than the alkylene group, has from 2 to 3 carbon atoms, exemplary alkylene groups include ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), -CH2NMe-CH2 ”, 0-CH2-0 or -0-CH2CH2-0-; "Halo" preferably means fluoro, chloro, bromo or iodo; "Azacycloalkyl" preferably means a 4- to 9-membered saturated carbon ring, wherein one of the methylene groups is substituted by nitrogen; "Cycloalkylamine" means a group -NYY 'wherein one of Y and Y' is hydrogen and the other Y and Y 'is cycloalkyl as defined above; "Alkylcycloalkylamino" means a group -NYY ', wherein one of Y and Y' is alkyl as defined above and the other Y and Y 'is cycloalkyl as defined above; "Diarylamino" means a group -NYY 'wherein both Y and Y' are aryl groups as previously described; "Aralkylamino" means a group -NYY 'wherein one of Y and Y' is hydrogen and the other Y and Y 'is aralkyl as defined above; "Arylalkylamino" means a group -NYY ', wherein one of Y and Y' is alkyl as defined above and the other Ye Y 'is aryl as defined above; - "alkylaryalkylamino" means a group -NYY 'wherein one of Y and Y' is alkyl as defined above and the other Y and Y 'is aralkyl as defined above; "Arylalkylamino" means a group -NYY ', wherein one of Y and Y' is aryl as defined above and the other YeY 'is aralkyl as defined above; - "mercapto" is a group -SH or SR, where R may be any of the groups R 1 to R 10 above defined, the mercaptoaryl and mercaptoalkyl groups are preferred; - "hydroxysulfonyl" is a -SO 3 H; - "amidosulfonyl" is a -SO 2 NH 2; "Dialkyl aminosulfonyl" means a group -S02NYY ', wherein both YeY' are alkyl groups as previously described; "Arylalkylamido sulfonyl" means a group -SO 2 NYY wherein one of Y and Y 'is aryl as defined above and the other YeY' is aralkyl as defined above; "Anion" means the deprotonated form of an organic or inorganic acid and the anion is preferably selected from chlorides, bromides, sulfates, nitrates, borates, phosphates, oxalates, tartrates, maleates, citrates, akcetates, ascorbates, succinates, benzenesulfonates, methanesulfonates cyclohexanesulfonates, toluenesulfonates, sulfamates, lactates, malonates, ethanesulfonates, cyclohexyl sulfamates and quinates. Where the rhodamine derivative contains one or more acid substituents, the covered compound comprises the inner 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 those rhodamine derivatives wherein at least 2 of the groups R 1, R 2, R 3, R 4 and R 2 represent a halogen atom, which is preferably a bromide atom.

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

The following specific rhodamine derivatives are of particular interest: - 2 '- (6-Dimethylamino-3-dimethylimino-3H-xanthen-9-yl) 4', 5'-dichlorobenzoic acid methyl ester hydrochloride; 4,5-dibromo-rhodamine ethyl ester 110 2- (2-methoxy ethoxy); 2,7-dibromorodamine hexyl ester acetate salt B; 2,7-dibromorodamine hexyl ester acetate salt B; - 4,5-dibromorodamine 6G; and - rhodamine B 3-bromopropyl ester.

A second object of the present invention is the intermediates represented by formulas (II) to (VII) and (Γ), the formulas being as defined in Figures 1 to 5, wherein the various groups are as previously defined without any rejection. Intermediates being as defined in Figures 1 to 5.

A third object of the present invention is new processes for the synthesis of novel rhodamine derivatives of formula (I), wherein the various groups R 1 to R 10, A, X, Y, Y 'and Z emen are as previously defined; without excluding the compounds listed in the condition at the end of the previous definition. These processes being defined by the schemes shown in Figures 1 to 5 and the corresponding parts of the description.

A fourth object of the present invention is the use of at least one rhodamine derivative as defined in the first object of the invention without excluding the compounds listed in the condition at the end of the definition of the rhodamine derivative of formula (I) alone or in combination with a carrier, to treat infections generated by Gram + and / or Gram- bacteria.

According to a preferred embodiment of the present invention, rhodamine derivatives are used to treat infections generated by Staphylococcus epidermitis.

Of particular interest are: - the use of 4,5-dibromo rhodamine 110 2- (2-methoxy ethoxy) ethyl ester as a bacteriostatic agent Escherichia coli 0157: H7 and / or against Salmonella thyphimurium LT2; the use of the 2,7-dibromorhodamine B hexyl ester acetate salt as a bacteriostatic agent against Salmonella thyphimurium LT2; the use of 4,5-dibromorodamine 6G as a bacteriostatic agent against Escherichia coli 0157: H7; the use of rhodamine 3-bromopropyl ester B as a bacteriostatic agent against Escherichia coli 0157: H7; and - the use of 4,5-dibromorodamine methyl ester as a bacteriostatic agent against Escherichia coli 0157: H7, Salmonella thyphimurium LT2 and / or Pseudomonas aeruginosa.

Preferably for this therapeutic use the rhodamine derivative (s) is (are) combined with a carrier which is a pharmaceutically acceptable carrier and is preferably selected from the group consisting of 5% mannitol and / or DMSO.

Any carrier is possible, however, the acceptable carrier is preferably comprised of 5% mannitol in water or DMSO.

In the case of the HA-X-149, HA-X-149 hexyl ester acetate salt of HA-X-164, the carrier is preferably DMSO.

A fifth object of the present invention is a bactericidal composition for treating a Gram + and / or Gram- contaminated liquid, which composition comprises an effective amount of at least one rhodamine derivative as defined above, without excluding the compounds. listed in the condition at the end of claim 1, alone or in combination with a vehicle.

A sixth object of the present invention is a bactericidal solution for the treatment of a Gram + and / or Gram- contaminated site, a solution comprising an effective amount of at least one rhodamine derivative of formula (I) as above. defined, without any rejection, alone or in combination with a vehicle, to treat infections generated by Gram + and / or Gram- bacteria.

A seventh object of the present invention is a method for treating infections generated by Gram + and / or Gram- bacteria, which method comprises administering to a human or animal in need an effective amount of at least one rhodamine derivative of formula (I). as previously defined, without any rejection, alone or in combination with a vehicle.

According to a preferred embodiment of this method, the effective amount administered comprises between 0.5 and 200 mg per kilogram body weight per day.

An eighth version of the present invention is a medicament containing an effective amount of at least one rhodamine derivative of formula (I) as defined above, without excluding the compounds listed in the condition at the end of the definition alone or in combination with a vehicle for treating infections generated by Gram + and / or Gram- bacteria. A tenth object of the present invention is to use an effective amount of at least one rhodamine derivative or salt thereof as defined above, without any rejection, either alone or in combination with a vehicle in the treatment of diseases generated by enveloped viruses or non-enveloped viruses.

Preferably, the enveloped virus is one with double stranded DNA, more preferably one from the Herpes viridae family.

An eleventh object of the present invention is a medicament containing an effective amount of at least one rhodamine derivative or salt thereof, as above, without excluding the compounds listed at the end of the definition of the rhodamine derivatives of formula (I). alone or in combination with a vehicle to treat viral infections.

Most preferred version of the present invention is the use of: - 2 '- (6-Dimethylamino-3-dimethylimino-3H-xanten-9-yl) -4', 5'-dichloro benzoic acid methyl ester hydrochloride , 5-dibromo rhodamine; 4,5-dibromo-rhodamine 110 2- (2-methoxy ethoxy) ethyl as an antiviral agent against cytomegalovirus; 4,5-dibromorodamine methyl ester as an antiviral agent against cytomegalovirus; and 2,7-dibromorodamine B hexyl acetate acetate salt as an antiviral agent against Cytomegalovirus.

Another preferred embodiment of the invention is the 2,7-dibromorodamine B hexyl ester acetate salt as an antiviral agent against Cytomegalovirus.

Use of the 2,7-dibromorhodamine B hexyl ester acetate salt as an antiviral agent against Cytomegalovirus.

A twelfth object of the present invention is the use of the rhodamine derivatives of formula (I) as previously defined without any rejection in the treatment of immunological disorders.

According to a preferred embodiment, the use refers to increased production of high quantum yield and singlet oxygen generation in irradiation, while maintaining desirable differential retention of rhodamine between normal and cancer cells, said rhodamine derivatives of formula ( I) being as previously defined without rejection.

According to another embodiment, the use refers to photodynamic therapy of cancer patients by the destruction of human cancer cells, wherein appropriate intracellular levels of said derivatives are obtained and irradiation of an appropriate wavelength and intensity is applied.

According to a further preferred embodiment, the use of the invention relates to a method for photodynamic therapy of patients suffering from disseminated leukemia, multiple myelomas and lymphomas, comprising the steps of: a) harvesting said human bone marrow from the patient; b) purging the bone marrow from step a) by photodynamic therapy employing a therapeutic amount of a photoactivable derivative according to formula (I), without excluding the compounds listed in the condition at the end of the definition under irradiation of a length of suitable wave; and c) perform autologous stem cell transplantation using the purged bone marrow of step b).

Preferably, the purge of step b) further comprises intensive chemotherapy and total body irradiation (TBI) procedures.

Another preferred embodiment relates to a method for in vitro purging of human bone marrow containing solid tumor metastasis selected from the group consisting of breast, lung, prostate, pancreatic metastasis and disseminated colonic carcinomas, melanomas and sarcomas, in which excision or surgical thinning may be accomplished, which comprises the steps of: a) harvesting said patient bone marrow; b) purging the bone marrow of step a) by photodynamic therapy using a therapeutic amount of at least one photoactivable derivative of formula (I) as defined above without any rejection under irradiation of an appropriate wavelength; and c) perform autologous stem cell transplantation using the bone marrow of step b).

Preferably, the purge of step b) further comprises total chemotherapy and total body irradiation (TBI) procedures.

Another embodiment of this object of the invention is a method for photodynamic therapy of cancer patients, comprising administering to such patients a therapeutically acceptable intracellular level of at least one photoactivatable derivative of formula (I) as defined above, without rejection, and subjecting said patients irradiating a therapeutically effective wavelength.

Preferably, at least one photoactivatable derivative is administered by instillation, injection, diffusion of bloodstream into tumor sites directly accessible to light emission or tumor sites accessible to leiser bundles using rigid or flexible endoscopes.

More preferably, the leprosy accessible tumor site is selected from the group consisting of urinary bladder, oral cavity, esophagus, stomach, lower digestive tract, upper and lower respiratory tract.

Another preferred embodiment of this object of the invention is a method for treating leukemias in patients, comprising the steps of: a) purging the bone marrow cancer clones of said patients; b) subjecting said purged clones of step a) to photodynamic treatment, employing a therapeutic amount of at least one of the photoactivable derivatives of formula (I) as defined above, without the rejection present at the end of the definition, under irradiation of a suitable wavelength for selective destruction of leukemic cells without affecting normal patient cells; and c) administering said treated clones from step b) to the patients; thus not causing systemic toxicity to the patients.

A fourteenth object of the present invention is a photoactivatable pharmaceutical composition for the selective destruction and / or inactivation of immunologically reactive cells without affecting normal cells and without causing systemic toxicity to the patient, said composition comprising at least one derivative of photoactivatable rhodamine of formula (I) as defined above, without excluding the compounds listed in the condition at the end of the definition, and their photoactivatable derivatives; in association with a pharmaceutically acceptable carrier; whereby photoactivation of said derivatives induces cell killing while inactivated derivatives are substantially non-toxic to cells.

A fifteenth object of the present invention is the use of the photoactivatable derivatives of claim 1 for the photodynamic treatment for selective destruction and / or inactivation of immunologically reactive cells without affecting normal cells and without causing systemic toxicity to the patient in particular. that appropriate intracellular levels of said derivatives are achieved and irradiation of a suitable wavelength and intensity is applied.

A preferred embodiment is a method of preventing graft-versus-host disease associated with allogeneic stem cell transplantation in a patient, which comprises the steps of: a) activating a donor's lymphocytes by mixing donor cells with host cells long enough and long enough for an immune reaction to occur; b) substantially eliminating the activated lymphocytes of step a) with photodynamic therapy, employing a therapeutic amount of a photoactivatable composition of claim 24 under irradiation of a suitable wavelength; and c) perform allogeneic stem cell transplantation using the treated mixture from step b).

Another preferred embodiment is a method for treating immune disorder in a patient, comprising the steps of: a) harvesting the hematopoietic cells of said patient; b) ex vivo treatment of the hematopoietic cells of step a) by photodynamic therapy, employing a therapeutic amount of a photoactivatable composition of claim 24 under irradiation of a suitable wavelength; and c) performing graft infusion or autograft transplantation using the hematopoietic cells treated in step b).

Preferably, the immune disorder is selected from the group consisting of conditions in which donor auto-cells or cells react against host tissues or foreign targets, such as graft-versus-host disease, graft rejection, autoimmune disorders, and cell-mediated immunoallergies. -T.

More preferably, hematopoietic cells are selected from the group consisting of bone marrow, peripheral blood and cord blood mononuclear cells.

The compounds of structure I exhibit increased properties such as: labeling dyes for deoxynucleotides, dideoxynucleotides and polynucleotides; new dyes suitable for etching fluids for the inkjet process; new dyes for fiberglass and paper; new dyes for the eradication of infectious biological contaminants in body tissues; new dyes applicable in photographic processes; new dyes applicable in cancer chemotherapy; novel dyes applicable as herpes simplex virus thymidine kinase inhibitors and in the treatment and / or prophylaxis of herpes simplex virus infections; novel dyes for use as polymeric optical amplifiers and leysers; new dyes applicable in cell biology; new dyes applicable in doping silicon materials to provide solid dyestuffs; new pigments applicable for paints, inks and plastics; new organic reagents in solvent extraction of metal ions; new dyes applicable to the formation of new products in conjunction with other dyes; new dyes for the manufacture of CD-ROM optical memory discs; novel dyes applicable for fluorophore labeling of peptides; new dyes applicable in flow cytometry analysis; new dyes applicable as stains for the detection of Mycobacterium tuberculosis by fluorescence microscopy; new dyes applicable for fluorescent mapping of binding sites for substrates, ligands and inhibitors; new dyes for studying transport across the blood-brain barrier; new dyes for studying biofilm disinfection; new dyes applicable as fluorescent probes in cell biology; new dyes for use as water tracing; new dyes for visualization of peptide receptors by image intensified fluorescence microscopy; new dyes for the formation of metal chelates in analytical chemistry; new fluorescent dyes applicable in diagnostic therapy. Chemical Synthesis These compounds are prepared following the general halogenation strategy of known and readily available rhodamine dyes, thereby generating a first and varied series of intermediates, which may themselves serve as potential photosensitizers, or use of these halogenated rhodamines as intermediates. in the synthesis of a second series of rhodamine dyes, whereby one or more halogens replaced one of the groups of structure I. In the event that all halogens are replaced by the new groups, a subsequent halogenation step is added to the sequence for the desired compound of structure I is obtained (see illustrative schemes 1 and 2).

Due to the specific retention of rhodamine 123 dye class by abnormal malignant cells and the concomitant lack of their accumulation by normal hematopoietic stem cells, these results provide evidence for the potential use of these three new dyes for in vivo or in vitro photodynamic therapy.

According to the present invention, the use of such aforementioned dyes is provided, together with tumor specific antibodies, or poisonous substances, or liposomes or lipoproteins, or fluorochrome adducts.

In addition, the photosensitizers to be described have the potential to act synergistically in conjunction with other photoactive substances.

Moreover, the negative selection procedure provided by the use of photodynamic treatment does not preclude the use of other means to enrich hematopoietic stem cells, such as positive selection with anti-CD34 monoclonal antibodies.

Other clinical applications In addition to the use of photosensitizers in the context of in vitro bone marrow purging for leukemia and metastatic cancers, the molecules can also be used in vivo for tumor sites directly accessible to exposure to a light source and at appropriate local concentrations. of the drugs to be described. The molecules of the invention may also be used in photodynamic therapy of a patient suffering from multiple myelomas or disseminated lymphomas. Metastatic cancers, for which therapy of this invention is appropriate, include metastasis of breast, lung, prostate, pancreatic and colonic carcinomas, melanomas, and disseminated sarcomas. The photoactivatable derivatives of the present invention may be administered by instillation, injection, blood stream diffusion into tumor sites directly accessible to light emission from light-beam accessible tumor sites using rigid or flexible endoscopes.

DESCRIPTION OF PREFERRED VERSIONS

As a matter of illustration only, methods of treating immune disorders involving the rhodamine derivatives according to the invention are hereinafter illustrated.

METHOD / TREATMENT OF LEUKEMIA 1. Diagnostic Procedures Diagnosis of chronic myelogenous leukemia (CML) will be established using one or more of the following procedures in blood or bone marrow cells: a) Conventional cytogenetic studies, with identification of pH 1 metaphases + housing t (9:22); b) fluorescent in situ hybridization for detecting bcr / abl rearrangement; and c) Southern blot analysis for the detection of a rearranged ber fragment or PCR-RT for the detection of chimeric abl / ber messenger RNA. 2. Bone marrow harvesting After diagnosis, bone marrow (BM) or peripheral blood (PB) derived hemopoietic stem cells will be harvested using procedures previously described for cancer therapy autologous bone marrow transplantation (reviewed by Herzig GP (1981) Prog Hematol., 12: 1). Hemopoietic stem cells collected for autograft will be treated immediately ex vivo as described below. 3. In vitro Leukemia Purge Ex vivo treatment consists of short term incubation or BM or PB stem cells with one or several of the selected photoactive compounds. Incubation duration, cell concentration and drug molarity are to be determined for each patient using an aliquot of the harvested cell population. Excess dye will be removed by cell washes with sterile dye free medium supplemented with 2% autologous serum. The cells are then exposed to radiant energy of sufficient intensity to perform photodynamic purge of the leukemic cells. The efficacy of the photodynamic purge procedure is verified on an aliquot of the treated cell population before cryopreservation and / or reinfusion in the patient is performed. Until reinfusion in the patient, cells are cryopreserved in 10% dimethyl sulfoxide (DMSO) - 90% autologous serum medium at -196 ° C in the vapor phase of liquid nitrogen. 4. Systemic treatment of patients Following harvesting of stem cells, the patient will be treated with conventional regimens until the autograft is clinically indicated or immediately subjected to dose-intensive chemotherapy and full body irradiation where indicated. 5. Autologous stem cell transplantation Following appropriate treatment of the patient by high-dose chemotherapy and irradiation and at the appropriate clinical time, cryopreserved peripheral marrow or peripheral blood stem cells will be rapidly thawed and diluted in medium containing 25 IU DNase ml '. 1, -7 to minimize lump formation. A minimum of 2 X 10 / kg nucleated cells with 85% to 95% viability, as measured by Trypan ™ blue exclusion (trypan blue), will be resumed for the patient.

METHOD JJDO TREATMENT OF MALIGNITIES 1. Diagnostic procedures The diagnosis of malignancies will be established using conventional histopathological examination of the primary tumor. Detection of marrow involvement by neoplastic cells will be achieved by direct histological examination and ancillary procedures where indicated (ie, immunoperoxidase, immunohistochemistry, tumor markers, and hybridization studies). 2. Bone marrow harvesting After diagnosis, hemopoietic stem cells Bone marrow (BM) or peripheral blood (PB) derivatives will be collected using procedures previously described for autologous bone marrow transplantation in cancer therapy (reviewed by Herzig GP, (1981) Prog. Hematol., 12: 1). Hemopoietic stem cells collected for autograft will be treated immediately ex vivo as described below. 3. In vitro purging of leukemia Ex vivo treatment will consist of short term incubation of BM or PB stem cells with one or several of the selected photoactive compounds. Incubation duration, cell concentration and drug molarity will be determined for each patient using an aliquot of the harvested cell population. Excess dye will be removed by cell washes in sterile dye free medium supplemented with 2% autologous serum. The cells will then be exposed to radiant energy of sufficient intensity to perform the photodynamic purge of the leukemic cells. Whenever a sensitive molecular marker is available, an aliquot of the treated cell population will be tested for residual neoplastic cells before cryopreservation and / or reinfusion in the patient is attempted. The cells will be cryopreserved in 10% dimethyl sulfoxide (DMSO) - 90% autologous serum medium at 196 ° C in the vapor phase of liquid nitrogen. 4. Systemic treatment of patients Following stem cell harvesting, the patient will be treated with conventional regimens until the autograft is clinically indicated, or immediately subjected to dose-intensive chemotherapy and full body irradiation where indicated. 5. Autologous stem cell transplantation Following chemotherapy and high-dose irradiation, cryopreserved marrow or peripheral blood stem cells will be rapidly thawed and diluted in medium containing 25 IU DNase ΜΓ1 to minimize lump formation. A minimum of 2 x 10 / kg nucleated cells with 85% to 95% viability as measured by Trypan ™ blue exclusion will be resumed to the patient.

Graft-Versus-Host Disease Method III in the Stem Cell Transplant Context

ALLOGENIC 1. Diagnosis and identification of immunological differences between donor and recipient and graft-versus-host disease. Allogeneic stem cell transplantation is performed for numerous neoplastic and non-neoplastic conditions. Hematological malignancies consist of leukemia, lymphoma, multiple myeloma, myelodysplastic syndromes etc .; and non-hematologic malignancies: aplastic anemia, congenital disorders, severe immunodeficiency syndromes, rheumatoid arthritis, scleroderma, lupus erythematosus, multiple sclerosis, and other immune disorders. Graft-versus-host disease is a complication of allogeneic stem cell transplantation, where donor cells react against host cells, damaging target tissues (usually the skin, liver, intestines, lungs, lacrimal or salivary glands, etc.). . The diagnosis is based on several clinical and laboratory parameters, which are extensively reviewed in Graft-vs.-Host Disease, Ferrara JLM, Deeg HJ, Burakoff SJ eds, Marcei Dekker, New York, 1997. GVHD develops against the present antigens. in recipient cells but not in donor cells. Immunological differences between donor and recipient could be present at the level of the most histocompatible antigens, the least histocompatible antigens or the tumor associated antigens. The disparity will be established using one or more of the following procedures in blood or bone marrow cells: a) HLA typing: conventional or molecular serological typing to identify donor and recipient disparities in antigens of the largest class I and II histocompatibility complex ; and b) Mixed lymphocyte culture to identify differences in class II antigens; and c) Lower histocompatibility antigens: Although some cytotoxic T cell lines are available and can be used to identify lower histocompatibility antigens, these tests are currently only available for research purposes. 2. Collection of progenitor cells After diagnosis, hemopoietic stem cells derived from bone marrow (BM) or peripheral blood (PB) or donor cord blood will be harvested using procedures previously described for allogeneic progenitor cell transplantation (reviewed in Bone Marrow Transplantation, Forman SJ, Blume KG, Thomas ED es, Blackwell Scientific Publications, Cambridge MA, USA, 1994). Donor hemopoietic stem cells collected for allograft will be immediately incubated with host or other irradiated (25Gy) mononuclear cells. Host cells, mixed with donor cells, are incubated in sterile dye-free medium supplemented with 20% autologous serum and interleukin-2 for 2 days. This procedure elicits donor cell alloreactivity toward the host and the cell graft subsequently undergoes ex vivo photodynamic treatment as described below. 3. Selective in vitro purging of immunoreactive cells Ex vivo treatment will consist of short term incubation of previously activated BM or PB stem cells with one or more of the selected photoactive compounds. Incubation duration, cell concentration and drug molarity will be determined for each patient using an aliquot of the harvested cell population. Excess dye will be removed by cell washes with sterile dye free medium supplemented with 2% autologous serum. The cells will then be exposed to radiant energy of sufficient intensity to perform photodynamic purging of leukemia cells. The efficacy of the photodynamic purge procedure will be verified on an aliquot of the treated cell population before cryopreservation and / or reinfusion in the patient is performed. Until reinfusion in the patient, cells will be cryopreserved in 10% dimethylsulfoxide (DMSO) - 90% autologous serum medium, at -196 ° C in the vapor phase of liquid nitrogen. 4. Systemic treatment of patients Following stem cell harvesting, the patient will undergo chemotherapy and / or dose-intensive irradiation when indicated. 5. Allogeneic stem cell transplantation Following appropriate treatment of the patient by chemotherapy and / or high-dose irradiation and at the appropriate clinical time, cryopreserved marrow or peripheral blood or cord blood stem cells will be rapidly thawed and taken up again. the patient.

Method IV Treatment of Graft-Versus-Host Disease and Autoimmune Diseases 1. Diagnostic Procedures Diagnosis of graft-versus-host disease or immunoreactive disorders shall be established using standard clinical, biochemical and / or histopathological examination of the appropriate blood or tissues. . Diagnostic and predictive aspects of GVHD are reviewed in Graft-vs.-Host Disease, Ferrara JLM, Deeg HJ, Burakoff SJ eds, Marcei Dekker, New York, 1997. 2. Peripheral Blood Cell Harvesting After GVHD diagnosis, autoimmune disorder or immunoreactive, peripheral blood mononuclear cells (PB) will be harvested using previously described or similar leukopheresis procedures (reviewed in Bone Marrow Transplantation, Forman SJ, Blume KG, Thomas ED eds, Blackwell Scientific Publications, Cambridge MA, USA, 1994). Patient peripheral blood mononuclear cells collected will be treated immediately ex vivo as described below.

3. In vitro Elimination of GVHD Medianto Cells Ex vivo treatment will consist of short term incubation of PB stem cells with one or several of the selected photoactive compounds. Incubation duration, cell concentration and drug molarity will be determined for each patient using an aliquot of the harvested cell population. Excess dye will be removed by cell washes in sterile dye free medium supplemented with 2% autologous serum. The cells will then be exposed to radiant energy of sufficient intensity to perform photodynamic purge of the activated cells, which mediate GVHD. 4. Administration of Photodynamically Treated Cells in Patients Leukoferesate cells, which are photodynamically treated, will be reinfused into the patient. This pathway will enable the elimination of a large number of circulating activated lymphocytes and other cells involved in GVHD. In addition, cells spared by photodynamic treatment are inactivated and their reinfuction in the patient may help restore normal immune balance.

METHOD KDE TREATMENT OF IMMUNOLOGICAL DISORDERS 1. Diagnostic Procedures Diagnosis of autoimmune disorders will be established using conventional clinical, biochemical and / or histopathological examination of the appropriate sange or tissues. Severe autoimmune diseases are receptive to autologous transplantation (reviewed in Sullivan KM et al., Am. Soc. Hematol., Educ. Book Program, 1998: 198-214). 2. Harvesting Hematopoietic Stem Cells After diagnosis, mononuclear bone marrow (BM), peripheral blood (PB) or cord blood (CB) cells will be harvested using the procedures previously described for autologous marrow transplantation in therapy. of cancer (reviewed in Bone Marrow Transplanation, Forman SJ, Blume KG, Thomas ED eds., Blackwell Scientific Publications, Cambridge MA, USA, 1994). Patient hemopoietic stem cells collected for autograft will be treated immediately ex vivo as described below. 3. In vitro elimination of cells mediating autoimmune disorders Ex vivo treatment will consist of short term incubation of BM or PB stem cells with one or more of the selected photoactive compounds. Incubation duration, cell concentration and drug molarity will be determined for each patient using an aliquot of the harvested cell population. Excess dye will be removed by cell washes in sterile dye free medium supplemented with 2% autologous serum. The cells will then be exposed to radiant energy of sufficient intensity to perform photodynamic purge of the immunoreactive cells that mediate the immune disorder. 4. Administration of Photodynamically Treated Cells in Patients Hematopoietic stem cells, which are photodynamically treated, will be stored (frozen or cultured). This pathway will enable the elimination of a large number of activated lymphocytes and other cells involved in the immune disorder. In addition, cells spared by photodynamic treatment are inactivated and their reinfusion may help restore normal immune balance. Following stem cell harvesting, the patient will be treated with conventional regimens until the autograft is clinically indicated or immediately subjected to intensive dose chemotherapy and full body irradiation, where indicated. 5. Autologous stem cell transplantation Following high-dose chemotherapy and cryopreserved irradiation, the marrow or peripheral blood stem cells will be rapidly thawed and infused into the patient. The preparation of those rhodamine derivatives of formula (I), as defined above, without the condition, will be more readily understood by reference to the following examples, which are given for illustrative purposes. I Synthesis of 2,7-dibromorodamine methyl ester acetate salt B (4) 1-1 Preparation of Rhodamine B methyl ether (1) Methanol ------> ~ HClgaz 1 To a stirred mixture of 1.63 g (3.40 mmol) of Rhodamine B and 100 ml of methanol, hydrochloric acid was bubbled through the solution for 45 min and the reaction mixture was refluxed overnight. Methanol was evaporated under reduced pressure and the dark red residue was then purified by scintillation chromatography, using a mixture of methanol and dichloromethane (1: 9) as eluent, to afford the desired product as a charged red viscous residue (1.0%). 54 g.).

Rf: 0.52 (MeOH: CH2Cl2 1.5: 8.5) Yield: 92% Ms (FAB): Calculated for C29H3303N2: (M-C1) +: 457.2491 Found (M-C1) +: 457, 2494 UV (MeOH): 555 nm 1-2 Preparation of dihydrorodamine methyl ester B (2) NaBH4 ---------- ► CH2 Cl2: H2 O 1 1 Rhodamine B methyl ester 1.73 g (3, 50 mmol) was dissolved in 250 mL of dichromethane and 100 mL of water. Excess NaBH4 (solid) was added portionwise with vigorous stirring for 30 min until the initial dark red color was discharged. The pale orange organic layer was separated and the aqueous phase extracted twice with dichloromethane. The combined organic layers were dried over Na 2 SO 4, filtered and evaporated under reduced pressure and the residue purified by scintillation chromatography using ethyl acetate as the eluent solvent. Fractions containing the product were combined and the solvent evaporated to afford product 2 as a pink oil (1.50 g). RF: 0.84 (AcOEt) Yield: 93.7% 1-3 Dihydrorhodamine B methyl ester housing (2) Propylene oxide ------------ * - Br2, MeOH 2 3 Em In a 250 ml round bottom flask we introduced dihydrorodamine methyl ester B (2) - 1.34 g (2.91 mmol) and 112 ml spectral grade methanol. The mixture was stirred at room temperature until all ester was dissolved. Propylene oxide 2 eq. (409 µL, 5.85 mmol) was added, followed by dropwise addition of bromine 2 eq. (300 µl, 5.85 mmol). Stirring was continued at room temperature for 1 h 30 min. The volatile solvent was evaporated under reduced pressure and the red oily residue was purified by scintillation chromatography using ethyl acetate and hexanes (0.5: 9.5) as eluent to afford the desired compound 3 as a solid. white (570 mg) Rf: 0.41 (AcOEt: kHexanes 0.5: 9.5) Yield: 31.6% Nmr: (CD 3 OD) δ 7.86 (dd, J = 1.44 and 7.8 Hz 1H); 7.44 (m, 1H); 7.32 (m, 1H); 7.16 ((s, 2H); 7.10 (dd, J = 1.45 and 7.8 Hz, 1H); 6.93 (s, 2H); 6.17 (s, 1H); 94 (s, 3H); 3.09 (q, J = 7.09 Hz, 8H); 1.04 (t, J = 7.09 Hz, 12H).

Ms (FAB): (MH) +615.1 1-4 Oxidation of 2,7-dibromodihydrodamine B methyl ester and formation of 2,7-dibromorodamine methyl ester acetate salt B (4) 2) Chloranil, CH 2 Cl 2) Purification AcOH, CH 2 Cl 2 In a stirred solution of 2,7-dibromodihydrorodamine B methyl ester (3) 400 mg (0.64 mmol) in 10 ml dichloromethane was added chloranil (1.2 eq. 0.77 mmol, 192 mg). The reaction mixture was stirred at room temperature overnight, then the reaction was stopped and the solvent was evaporated under reduced pressure to afford the purple residue. The oxidized compound obtained in the previous step was dissolved in 15 ml of dichloromethane and stirred for 5 min at room temperature followed by evaporation of the volatile solvent under reduced pressure to give a purple viscous residue. The residue was purified by scintillation chromatography using 10% methanol in dichloromethane as eluant to afford the desired compound 4 as a viscous purple solid (200 mg).

Rf: 0.29 (MeOH: CH 2 Cl 2: 9) Yield: 45.7% Nmr: (CD 3 OD) δ 8.48 (dd, J = 1.45 and 7.5 Hz, 1H); 7.95 (m, 2H); 7.52 (dd, J = 1.6 and 7.2 Hz, 1H); 7.45 (s, 2H); 7.38 (s, 2H); 3.79 (q, J = 8Hz, 8H); 3.71 (s, 3H); 1.99 (s, 3H); 1.37 (t, J = 7.02 Hz, 2H) Ms (FAB): Calculated for C29H3203N2Br2 (MH-Aco) +: 614.0779 Found: 614.0765 UV (MeOH): λ max 577 nm EXAMPLE II II Synthesis 2,7-dibromorhodamine hexyl ester acetate salt B (8) II-1 Preparation of Rhodamine B (5) 1-hexanol hexyl ester ------ HCl gas 5 In a stirred mixture of 2.39 Rhodamine g (4.98 mmol) B 120 ml of 1-hexanol, hydrochloric acid was bubbled through the solution for 45 min and the reaction mixture was refluxed overnight. 1-Hexanol was then distilled off under reduced pressure and the dark red residue was purified by scintillation chromatography using a mixture of methanol and dichloromethane (1: 9) as eluent. After evaporation of volatile solvents, a viscous red green residue (2.62 g) was obtained.

Rf: 0.45 (MeOH: CH2 Cl2.2: 8.8) Yield: 93.5% Ms (FAB): Calculated 034Η4303Ν2 (M-C1) +: 527.3273 Found: 527.3261 UV (MeOH): λmax 555 nm II-2 Preparation of dihydrorodamine hexyl ester B (6) NaBH 4 CHjClj · HjO 5 6 Rhodamine hexyl ester B (5) 940 mg (1.66 mmol) was dissolved in 200 ml of dichromethane and 150 ml of water. Excess NaBH4 (solid) was added portionwise with vigorous stirring for 30 min until the initial dark red color was discharged. The pale orange organic phase was separated and the aqueous phase extracted twice with dichloromethane. The combined organic layers were dried over Na 2 SO 4, filtered and evaporated under reduced pressure. The crude oil residue was purified by scintillation chromatography, using ethyl acetate as eluent, yielding 794 mg of 6 as a pink oil.

Rf: 0.92 (AcOEt) Yield: 90% II-3 Dihydrorhodamine hexyl ester block B (6) 7 Propylene oxide CH3 Br2, MeOH ^ 6 7 In a 100 ml round bottom flask we introduced dihydrorhodamine hexyl ester (6) 784 mg (1.48 mmol) and 25 mL of spectral grade methanol. The mixture was stirred at room temperature until all the ester had dissolved. Propylene oxide 2 eq. (208 µl, 2.96 mmol) was added, followed by dropwise addition of 2 eq. bromine (152 µl, 2.96 mmol). Stirring was continued at room temperature for 1 h 30 min. The volatile solvent was evaporated under reduced pressure and the red oily residue was subjected to purification by scintillation chromatography using ethyl acetate and hexanes (0.25: 9.75) as eluent to afford 207 mg of pure compound 7 as a foamy solid. white and 123 mg of crude product.

Rf: 0.61 (AcOEt: Hexanes 0.5: 9.5) Yield: 20.5% II-4 Oxidation of 2,7-dibromodihydrorodamine B hexyl ester and formation of 2.7-hexyl ester acetate salt dibromorhodamine B (81 1) Chloranil, CH 2 Cl 2 2) Purification of 3 AcOH, CH 2 Cl 2 2 8 To a stirred solution of 2,7-dibromo dihydro rhodamine B 207 mg (0.30 mmol) in 8 ml dichloromethane was added chloranil (1.2 eq., 0.36 mmol, 89 mg). The reaction mixture was stirred at room temperature overnight, then the reaction was stopped and the solvent was evaporated under reduced pressure to give a purple residue. The oxidized compound obtained in the previous step was dissolved in 8 ml of dichloromethane and acetic acid (0.8 ml) was added dropwise. The obtained translucent red solution was stirred for 5 min at room temperature, followed by evaporation of the volatile solvent under reduced pressure to afford a purple viscous residue, which is purified by scintillation chromatography using 10% methanol in dichloromethane as eluent to provide the solvent. desired compound 8 as a viscous purple solid (198 mg).

Rf: 0.47 (MeOH: CH 2 Cl 2: 9) Yield: 86.9% Nmr: (CD 3 OD) δ 8.29 (dd, J = 1.5 and 7.6 Hz, 1H); 7.82 (m, 2H): 7.40 (dd, J = 1.6 and 7.2 Hz, 1H); 7.37 (s, 2H); 7.28 (s, 2H); 3.96 (t, J = 7.2 Hz, 2H); 3.71 (q, J = 7.05 Hz, 8H); 1.91 (s, 3H); 1.29 (t, J = 7.095 Hz, 12H); 1.08 (m, 4H); 0.79 (t, j = 7.04 Hz, 3H).

Ms (FAB): Calculated for C34H4203N2Br2 (MH-AcO) +: 684.1565 Found: 684.1565 UV (MeOH): λ max 582 nm EXAMPLE III III Synthesis of 4'5'-dichloro-benzoic acid methyl ester hydrochloride 2 '- (6-Dimethylamino-3-dimethylimino-3H-xanten-9-yl) III-1 Preparation of 2' - (6-dimethylamino-3-dimethylimino) 4,5'-dichloro-benzoic acid hydrochloride 3H-xanthen-9-yl) 1) ZnCl 2 165 ° C, 5 h 2) NaOH 2) HCl 9 A mixture of 3- (dimethylamino) phenol 3.00 g (21.8 mmol), 3.00 g (13.8 mmol) 4,5-dichlorophthalic anhydride and 1.72 g zinc chloride is heated in an oil bath at 165 - 170 ° C for 5 h with stirring. The fusion is cooled and powdered to provide a red solid. The solid is washed with hot water, triturated with 10% sodium hydroxide and diluted with water. The separating gum is collected, washed with more sodium hydroxide and water. The resulting dye base is then triturated with concentrated hydrochloric acid. Water was then added and the obtained red precipitate was collected and dried. The dye was then dissolved in methanol and precipitated with diethyl ether to afford 9 as a red solid (3.27 g).

Rf: 0.48 (2: 8 MeOH: CH 2 Cl 2) Yield: 48% Nmr: (CD 3 OD) δ 8.47 (s, 1H); 7.72 (s, 1H); 7.22 (d, J = 9.47 Hz, 2H); 7.11 (m, 2H); 7.01 (d, J = 2.4 Hz, 2H); 3.32 (s, 12H) Ms (FAB): Calculated for C24H2103N2Cl2 (M-CI) +: 455.0929 Found: 455.0938 UV (MeOH): 511 nm III-2 Preparation of acid 4 methyl ester hydrochloride 2 '- (6-Dimethylamino-3-dimethylimino-3H-xanten-9-yl)', 5'-dichloro-beizozoic acid (10) DCC, DMAP MeOH 9 10 In a 250 ml round-bottom flask fitted with With magnetic stirrer, 738 mg (1.50 mmol) of acid 9 and 40 mL of anhydrous dichloromethane and 10 mL of anhydrous DMF were added. The mixture was stirred under nitrogen until all acid was dissolved. An amount of 309 mg (1.50 mmol) of 1,3-dicyclohexylcarbodiimide (DCC) was then added, followed by 200 µl of methanol and 18 mg of 4-N, N-dimethylamino pyridine (DMAP). The mixture was stirred at room temperature overnight. The solvent was then distilled off under reduced pressure to give a red residue which was purified by scintillation chromatography using MeOH: CH 2 Cl 2 (1.2: 8.8) as eluent to afford 10 as a reddish brown solid (350 mg).

Rf: 0.52 (MeOH: CH 2 Cl 2 2: 8) Yield: 46% Nmr: (CD 3 OD) δ 8.50 (s, 1H); 7.80 (s, 1H); 7.18 (d, J = 9.2 Hz, 2H); 7.12 (m, 2H); 7.04 (d, J = 1.31 Hz, 2H); 3.80 (s, 3H); 3.35 (s, 12H) Ms (FAB): calculated for C25H2303N2C12 (M-C1) +: 469.1085 Found: 469.1078 UV (MeOH): λ max 555 nm EXAMPLE IV IV Preparation of 4,5-dibromorodamine 6G (11) Br, MeOH 11 To a quantity of 600 mg (1.25 mmol) of 6G rhodamine dissolved in 50 ml of methanol was added dropwise at room temperature, a solution of 128 pL (2 eq., 250 mmol ) of bromine. A precipitate formed 10 min after the addition of bromine. The mixture was stirred for 3 hours and the solvent was evaporated under reduced pressure to give a red solid. The crude product was recrystallized from methanol: diethyl ether (80 mL: 400 mL) to give the product as a reddish green solid (585 mg).

Rf: 0.26 (MeOH: CH 2 Cl 2: 9) Yield: 68.5% Nmr (CD 3 OD) δ 8.36 (dd, J = 1.13 and 7.44 Hz, 1H); 7.89 ((m, 2H); 7.47 (dd, J = 1.46 and 6.76 HZ, 1H); 6.98 (s, 2H); 4.07 (m, 6H); 29 (s, 6H); 1.28 (t, J = 7.04 Hz, 6H); 1.02 (t, J = 7.05 Hz, 3H) Ms (FAB): Calculated for C28H30O3N2Br2 (MH-Br ) +: 600.0623 Found: 600.0605 UV (MeOH): λmax 546 nm EXAMPLE VV Synthesis of 4,5-dibromorodamine 2- (2-methoxy ethoxy) ethyl ester 110 (13) V1 Preparation of Rhodamine 2- (2-methoxy ethoxide 110 (12) C d, HOBT DMF, CH 2 Cl 2 In rhodamine 110 1.00 g (2.72 mmol) was added an anhydrous DMF mixture and dichloromethane (60 mL: 10 mL) and the mixture was stirred until all dye was dissolved 1,3-dicyclohexylcarbodiimide (DCC) 562 mg (1 eq, 2.72 mmol) was added, followed by HOBT 368 mg (1 eq., 2.72 mmol), 2- (2-methoxy ethoxy) ethanol 518 µl (1.60 eq., 4.36 mmol) and 33 mg (0.27 mmol) of 4- dimethylamino pyridine (DMAP) The reaction was stirred at room temperature overnight and DMF was then distilled under reduced pressure to give a m red residue loaded. This residue was subjected to purification by scintillation chromatography using methanol: dichloromethane (2: 8) as eluent to give (530 mg) a red solid. Thin layer chromatography (TLC) showed the presence of another product as desired. The obtained solid was then dissolved in methanol (10 ml) and downlink was added until a precipitate was obtained. The product was collected and dried to give the desired compound 12 (220 mg) as a red solid.

Rf: 0.33 (MeOH: CH 2 Cl 2 2: 8) Yield: 18.4% Ms (FAB): Calculated for C 25 H 25 N 2 O 5 (M-C 1) +: 433, 1736 Found: 433.1777 V-2 4,5-dibromo rhodamine 2- (2-methoxy ethoxy) 110 Br2, MeOH 12 13 In a 100 ml round bottom flask equipped with a magnetic stirrer, 235 mg (0.50 mmol) of ethyl ester was added. of rhodamine 2- (2-methoxy ethoxy) 110 12 and 15 ml of spectral grade methanol. The mixture was stirred until all rhodamine dye was dissolved. An amount of 50 μΕ (2 eq., 1.00 mmol) of bromine was then added and the reaction was stirred at room temperature for 1 h 30 min. At the end of the reaction, 10 µl of cyclohexane was added and the mixture was stirred for another 10 min. The volatile solvent was evaporated under reduced pressure to give a red solid. This solid was chromatographed on silica gel MeOH: CH 2 Cl 2 (1.2: 8.8) as eluting solvent.

The pure fractions were combined and evaporated to give compound J3 (250 mg) as red solid.

Rf: 0.76 (2: 8 MeOH: CH 2 Cl 2) Yield: 74.3% Nmr (CD 3 OD) δ 8.38 (dd, J = 1.5 and 6.87 Hz, 1H); 7.88 (m, 2H); 7.47 (dd, J = 1.48 and 7.02 Hz, 1H); 7.15 (d, J = 9.22 Hz, 2H); 7.04 (d, J = 9.21 Hz, 2H); 4.15 ((m, 2H); 3.39 - 3.25 (m, 9H) Ms (FAB): Calculated for C25H2305N2Br2 (M-Br) +: 588.9973 Found: 588.9962 UV (MeOH): 502 nm EXAMPLE VI VI Preparation of Rhodamine B 3-bromopropyl ester (14) DCC, DMAP CH 2 Cl 2 In Rhodamine B 300 mg (0.62 mmol) was added 5 ml dichloromethane and the mixture was stirred until all the dye was removed. An amount of 1,3-dicyclohexylcarbodiimide (DCC) 142 mg (1 eq, 0.62 mmol) was added followed by 139 mg (10.0 mmol) of 3-bromopropanol and 8 mg (0.06 mmol) 4-dimethyl aminopyridine (DMAP) The reaction was stirred at room temperature overnight AN, N-dicyclohexyl urea was filtered and the solvent evaporated in vacuo to give a charged red residue, which was subjected to purification by scintillation chromatography using methanol: dichloromethane (1: 9) as eluent Fractions containing the desired compound were combined and the solvent evaporated under reduced pressure to give 14 as a red viscous solid. O (300 mg) Rf: 0.71 MeOH: CH 2 Cl 2 1.5: 8.5) Yield: 79.8% Nmr (CD 3 OD) δ 8.29 (m, 1H); 7.85 (m, 2H); 7.43 M + R2 (R3) m; 7.05 (m, 6H); 4.08 (m, 2H); 3.68 (q, J = 7.06 Hz, 8H); 3.21 (m, 2H); 1.81 (m, 1H); 2.29 (t, J = 7.08 Hz, 12H) Ms (FAB): Calculated for C31 H3603 N2 Br! (M-Cl) +: 563.1909 Found: 563.1921 UV (MeOH): λ max 545 nm EXAMPLE VII VII Synthesis of 2,7-Dibromo-4'-carboxytetramethylrosamine methyl ester acetate salt VII-1 Preparation of 4'-carboxydihydrotetramethylrosamine methyl NaBH4 CH2 Cl2: H2 O 'ester (17) 15 16 Ester F5 910 mg (2.08 mmol) was dissolved in 250 mL dichloromethane and 150 mL water. Excess NaBH4 (solid) was added portionwise with vigorous stirring for 30 min until almost all color was discharged. The pale orange organic layer was separated and the aqueous phase extracted twice with dichloromethane. The combined organic layers were dried over Na 2 SO 4, filtered and evaporated under reduced pressure. The crude oil residue was purified by scintillation chromatography using ethyl acetate as eluant, yielding 530 mg of white foamy solid.

Rf: 0.83 (AcOEt) Yield: 63% VII-2 Dihydro-4'-carboxytetramethylamine methyl ester Br2, MeOH Propylene oxide 16 17 In a round bottom flask of 100 lifting mechanism we introduced dihydro rhodamine B hexyl ester 530 mg (1.31 mmol) and 50 ml spectral grade methanol. The mixture was stirred at room temperature until all ester was dissolved. Propylene oxide 2 eq. (185 μΐ, 2.63 mmol) was added, followed by dropwise addition of bromine 2 eq. (135 µl, 2.63 mmol). Stirring was continued at room temperature for 1 h 30 min. The volatile solvent was evaporated under reduced pressure and the red oily residue was purified by scintillation chromatography using ethyl acetate and hexanes (1: 9) as eluent to afford a white foamy solid (391 mg) Rf: 0, 36 (AcOEt: Hexanes 1: 9) Yield: 53.5% Nmr (CD 3 OD) δ 7.96 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 8.31 Hz, 2H ); 7.22 (s, 2H); 6.94 (s, 2H); 3.87 (s, 3H); 2.77 (s, 12H) VII-3 _______ Oxidation ________ of _______ 2,7-dibromodihydro-4'-carboxmexitetramethyl rosamino methyl ester (17) and 2,7 dibrom-4'-carboxytetramethylrosamino methyl ether acetate salt formation (18) Chloranil, CH2C 2) AcOH

13B To a stirred solution of 2,7-dibromodihydrotetramethylrodamino methyl ester 390 mg (0.69 mmol) in 15 ml dichloromethane was added chloranyl (1.2 eq., 0.83 mmol, 205 mg). The reaction mixture was stirred at room temperature overnight, then the reaction was stopped and the solvent was evaporated under reduced pressure to give a purple residue. The oxidized compound obtained was dissolved in 15 ml of dichloromethane and acetic acid (0.8 ml) was added dropwise. The obtained translucent purple solution was stirred for 5 min at room temperature, followed by evaporation of the volatile under reduced pressure to give a purple viscous residue, which is purified by scintillation chromatography using 10% methanol in dichloromethane as eluent to give the desired compound 18A which is in equilibrium with compound 18B. 18A Rf: 0.34 (MeOH: CH2 Cl2: 9) 18Β Rf: 0.93 (MeOH: CH2 Cl2 1: 9) Yield: 30% Nmr (CD 3 OD) δ 7.97 (d, J = 8.28 Hz, 2H ); 7.45 (d, J = 8.33 Hz, 2H); 7.19 (s, 2H); 6.99 (s, 2H); 3.89 (s, 3H); 2.93 (s, 2.64H); 2.83 (s, 12H); 2.01 (s, 0.356H) MS (FAB): Calculated for C25H2403N2Br2 (MH-AcO) +: 558.0153 Found: 558.0169 EXAMPLE VIII

Preparation of 4,5-dibromo Rhodamine B lactone (191 AcOH: H2> --------> ~ Br2 12 Rhodamine B 500 mg (1.04 mmol) was dissolved in 25 ml acetic acid and 25 ml 107 pL bromine (2 eq., 2.08 mmol) was then added dropwise and the reaction mixture was then evaporated under reduced pressure and the residue obtained was redissolved in dichloromethane and 10% aqueous sodium bicarbonate solution. The organic layer was separated and washed twice with water, dried over Na2 SO4, filtered and evaporated to give a pink oil.The residue was chromatographed on silica gel using methanol: dichloromethane (0.2: 9.8) as eluent to give 544 mg of white foamy solid Rf: 0.88 (MeOH: CH 2 Cl 2: 9) Yield: 86.8% Nmr (CD 3 OD) δ 7.89 (dd, J = 1.45 and 7.8 Hz, 7.62 (m, 2H); 7.14 (dd, J = 1.6 and 7.2 Hz, 1H); 6.81 (d, J = 9.2 Hz, 2H); 58 (d, J = 9.2 Hz, 2H); 3.02 (q, J = 7.05 Hz, 8H); 0.93 (t, J = 7.04 Hz, 12H) Ms (FAB): Calculated for C28H2903N2Br2 (MH) +: 599.0545 Found: 5 99.0527 EXAMPLE IX

Preparation of 2,7-dibromo Rhodamine B lactone (20) j 1) Chloranil, CH-> C 12 ---------- 1 2) HCl (IN), dioxane 3 20 To a stirred solution of 2, 7-dibromodihydrorodamine B methyl ester (3) 46 mg (0.10 mmol) in 4 mL dichloromethane was added chloranil (1.2 eq. 0.12 mmol, 30 mg). The reaction mixture was stirred at room temperature overnight, then the reaction was stopped and the solvent was evaporated under reduced pressure to give a purple residue. The oxidized compound obtained in the previous step was dissolved in 4 ml of dioxane and HCl (1 M) (5 ml) was added dropwise and the resulting solution was heated in a water bath to give a translucent red solution. After evaporation to dryness under reduced pressure we obtained a purple viscous residue. The residue was purified by scintillation chromatography using ethyl acetate: hexanes (1.5: 8.5) as eluent to afford the desired compound 4 as a white foamy solid (35 mg).

Rf: 0.34 (AcOEt: hexanes 1.5: 8.5) Yield: 80% Nmr: (CD 3 OD) δ 7.92 (dd, J = 1.45 and 7.5 Hz, 1H), 7.63 (m, 4H); 7.18 (dd, J = 1.6 and 7.2 Hz, 1H); 7.02 ((m, 2H)).

Ms (FAB): Calculated for C28H2903N2Br2 (MH) +: 599.0545 Found: 599.0570 EXAMPLES OF USES OF RODAMINE DERIVATIVES ACCORDING TO THE INVENTION AS INTERMEDIARIES

EXAMPLE XX Synthesis of 4-bromo-5-phenyl methyl ester chloride Rhodamine B (22) X1 Preparation of 4-bromo-5-phenyl Rhodamine B lactone 21} PhB (OH) 1 Et 3 N; Pd (OAc) 2; P (Ar) 3 19 21 A mixture of 10 mmol dibromolactone 19 ± 10 mmol phenylboronic acid, 4.2 mL (30 mmol) Et 3 N, 0.067 g (0.3 mmol) Pd (OAc) 2 and 0, 19 g (0.62 mmol) tri-o-tolylphosphine catalyst or 0.16 g (0.62 mmol) PPh3 catalyst in 40 ml DMF is heated under a nitrogen atmosphere at 100 ° C for 2- 3 hours. The solvent is then distilled off under reduced pressure and the residue partitioned between CH 2 Cl 2 and 10% aqueous NH 3. The organic extracts are then dried (MgSO4) and concentrated under reduced pressure. Purification by silica gel scintillation chromatography gives pure monobromolactone 21. (See WJ Thompson and J. Gaudino, J. Org. Chem. 1984, 49, 5237-5243: N. Miyaura, T. Yanagi and A. Suzuki, Synthetic Communications, 1981, 11 (7), 513-519).

X-2 Preparation of 4-Bromo-5-phenyl methyl ester chloride Rhodamine B

MeOH / HQ

Reflux 21 Methanolysis and concomitant oxidation of monobromolactone 21 are performed by first stirring a mixture of 3-4 mmoles of the compound in 100 ml methanol, while bubbling into a thin stream of anhydrous HCl gas for a period of 5 min. then heating the mixture at reflux overnight. Methanol is then evaporated under reduced pressure and the dark red residue purified by scintillation chromatography to provide the desired dark red product 22.

EXAMPLE XI XI Synthesis of 2,7-dibromo-4,5-dimethyl methyl ester bromide Rhodamine B (241 XI-1 Preparation of 4,5-dimethyl Rhodamine B lactone (23) PhCH2Pd (PPl%) 2Br Me „Sn; HMPA To a 7.0 mmol solution of dibromolactone 19 in hexamethylphosphoramide (HMPA) is added 0.05 mmol of benzylbromobis (triphenylphosphine) palladium (II) catalyst and 16.0 mmol of tetramethyl tin. Stirring under air in a sealed tube until blackening occurs, the solution is then cooled to room temperature and 5 ml of water are added, the mixture is extracted with dichloromethane and the organic solution dried over MgSO4. the crude product, which is purified by silica gel scintillation chromatography to give pure lactone 23. (See D. Milstein and JK Stille, J. Am. Chem. Soc. 1979, 101 (17), 4992-4998). Preparation of 2,7-dibromo-4,5-dimethyl methyl ester bromide Rhodamine B (24) Br2 / MeOH ......... ■ .......- »» 22 2a A solution of 1.25 mmol of 4,5-dimethyl Rhodamine B lactone 23 in 50 ml of methanol was treated at room temperature. for 2.5 mmol bromine. A precipitate formed after the addition of bromine and the mixture was stirred for 3 hours. The solvent was evaporated under reduced pressure to give a red solid which was recrystallized from methanol: diethyl ether to give the desired dibromomethyl ester 24.

EXAMPLE XII XII Synthesis of 2-Bromo-7-ethynyl Rhodamine B methyl ester bromide (261. XII-I Preparation of 2-Bromo-7-ethynyl Rhodamine B lactone (25) HC = CSi (CH3) 3 (ΡΡ% Ρ <3 (ΟΑ <02, Εί3Ν 20 25 A mixture of 53 mmol dibromolactone 20 and 53 mmol ethinyltrimethylsilane, 300 mg triphenylphosphine and 150 mg palladium (II) acetate is prepared in 100 ml anhydrous triethylamine dehydrated at 30-40 ° C. The mixture is then heated under argon at 90-100 ° C for 22 h.The mixture is cooled and filtered to give the desired crude 25-trimethylsilyl derivative. Potassium carbonate treatment at 25 ° C. for 16 h, followed by neutralization, gives lactone 25 after purification by scintillation chromatography (See WB Austin, N. Bilow, WJ Kelleghan, and KSY Lau, J. Org. Chem. 1981, 46, 2280 - 2286; S. Takahashi , Y. Kuroyama, K. Sonogashira, N. Hagihara, Synthesis, 1980, 627-630) XII-2 Preparation of 2-Bromo-7-ethynyl Rhodamine B methyl ether chloride ( 26) MeOH / HCl Reflow 25 26 A stirred solution of 3.5 mmol 2-bromo-7-ethynyl Rhodamine B lactone 25 in 100 ml methanol is treated with a thin stream of bubbled HCl gas for 45 min. The reaction mixture is then heated at reflux overnight and the methanol evaporated under reduced pressure. The dark red residue is purified by scintillation chromatography using a mixture of methanol and dichloromethane to provide the desired ester 26.

EXAMPLE XIII XIII Synthesis of 4,5-dibromo-2,7-di-n-butyl rhodamine B methyl ester bromide (28). XIII- 1 Preparation of 2,7-di-n-butyl Rhodamine B lactone 27L

A solution of 7.0 mmol of dibromolactone 20 in 4 ml hexamethylphosphoramide (HMPA) is treated with 0.05 mmol benzylbromobis (triphenylphospho) palladium (II). and 16.0 mmol of the tetra-n-butyltin compound.The solution is then heated to 65Â ° C with stirring in air in a sealed tube until blackening occurs.The solution is then cooled to room temperature and 5 ml of water are added. The mixture is extracted with dichloromethane and the latter is evaporated in vacuo to give crude product, which is purified by scintillation chromatography to yield pure lactone 27 (see D. Milstein and JK Stile, J. Amer. Chem. Soc. 1979, 101 (17), 49924998).

XIII-2 Preparation of 4,5-dibromo-2,7-di-n-butyl methyl ester bromide Rhodamine B

Br2 / MeOH Reflux 27 28 A solution of 1.25 mmol Rhodamine B lactone 27 in 50 ml methanol is treated at room temperature with 2.5 mmol bromine. A precipitate is formed briefly after addition of bromine and the mixture is stirred for 3 hours. The solvent is then evaporated under reduced pressure to give a red solid which is recrystallized from methanol: diethyl ether to give the desired dibromomethyl ester bromide 28.

DETERMINATION OF BACTERIOSIDIC ACTIVITY AND / OR

BACTERIOSTATIC OF RODAMINE DERIVATIVES

Experimental Design The following experimental procedures were used to determine antibacterial activity.

Bacteriostasis: Escherichia coli: (0157) a The protocol used for bacteriosid inactivation was performed as described in Brasseur and colleagues, with some modifications for the evaluation of bacteriosid potential (Brasseur et al. 2000). Compounds TH9402, HA-X-44, HA-X-164, HA-X-171 and HA-VIII-92 showed antibacterial activity against E. coli using the following experimental procedure.

Bacteria were grown overnight in Lime Medium (LB), 100 μΐ («1 x 10 bacteria) from each bacterial suspension were added to 4 ml LB medium, a small aliquot of the bacterial suspension was taken for bacterial titration before expressed as CFU / mL (colony forming units per mL). To the bacterial suspension were added rhodamine derivatives, each derivative being tested in duplicate at a concentration of 50 μΜ. Each bacterial rhodamine derivative mixture was incubated at 37 ° C for 40 minutes. The bacteria-rhodamine suspensions were then treated and exposed to a wavelength light of 514 nm for 180 minutes for a total output energy of 30 Joules / cm. Following the treatment time, the bacteria-rhodamine suspensions were centrifuged at 3000g, resuspended in 4 ml and serial dilutions were made for each duplicate. 10 µl of the diluted bacterial suspensions were plated, the plates incubated overnight at 37 ° C. The bacteriostatic effect is expressed by the number of CFU / ml.

Pseudomonas aeruginosa: The protocol used for bacteriosid inactivation was performed as described in Brasseur et al. with some modifications for the evaluation of bacteriosidic potential (Brasseur et al. 2000). Compound TH9402 showed antibacterial activity against P. aeruginosa using the following experimental procedure.

Bacteria were grown overnight in Lime Medium (LB), 100 μΐ (1 x 107 bacteria) from each bacterial suspension were added to 4 ml LB medium, a small aliquot of the bacterial suspension was taken for bacterial titration before treatment expressed as CFU / mL. Rhodamine derivatives were added to the bacterial suspension, each derivative being tested in duplicate at a concentration of 50 μΜ. Each bacterial rhodamine derivative mixture was incubated at 37 ° C for 40 minutes. The bacteria-rhodamine suspensions were then treated and exposed to a wavelength light of 514 nm for 180 minutes for a total output energy of 30 Joules / cm2. Following the treatment time, the bacteria-rhodamine suspensions were centrifuged at 3000g, resuspended in 4 ml and serial dilutions were made for each duplicate. 10 µl of the diluted bacterial suspensions were plated, the plates incubated overnight at 37 ° C. The bacteriostatic effect is expressed by the number of CFU / mL.

Salmonella typhimurium The protocol used for bacteriosid inactivation was performed as described in Brasseur and colleagues, with few modifications for the evaluation of bacteriosid potential (Brasseur et al. 2000). The compounds TH9402, HA-X-44 and HA-X-164 showed antibacterial activity against Salmonella thyphimurium using the following experimental procedure.

Bacteria were grown overnight in Lime Medium (LB), 100 μΐ (1 x 107 bacteria) from each bacterial suspension were added to 4 ml LB medium, a small aliquot of the bacterial suspension was taken for bacterial titration before treatment expressed as CFU / mL. Rhodamine derivatives were added to the bacterial suspension, each derivative being tested in duplicate at a concentration of 50 μΜ. Each bacterial rhodamine derivative mixture was incubated at 37 ° C for 40 minutes. The bacteria-rhodamine suspensions were then treated and exposed to a wavelength light of 514 nm for 180 minutes for a total output energy of 30 Joules / cm. Following the treatment time, the bacteria-rhodamine suspensions were centrifuged at 3000g, resuspended in 4 ml and serial dilutions were made for each duplicate. 10 µl of the diluted bacterial suspensions were plated, the plates incubated overnight at 37 ° C. The bacteriostatic effect is expressed by the number of CFU / mL.

Staphilococcus epidermitis The protocol used for bacteriosid inactivation was performed as described in Brasseur and colleagues, with few modifications for the evaluation of bacteriosid potential (Brasseur et al. 2000). Compounds TH9402, HAX-40, HA-X-44, HA-X-149, and HA-X-164 showed antibacterial activity against S. epidermitis using the following experimental procedure.

Bacteria were grown overnight in Lime Medium (LB), 100 μΐ (1 x 107 bacteria) from each bacterial suspension were added to 4 ml LB medium, a small aliquot of the bacterial suspension was taken for bacterial titration before treatment expressed as CFU / mL. Rhodamine derivatives were added to the bacterial suspension, each derivative being tested in duplicate at a concentration of 50 μΜ. Each rhodamine-bacteria derivative mixture was incubated at 37 ° C for 40 minutes. Bacteria-rhodamine suspensions were then treated and exposed to a wavelength light of 514 nm for 180 minutes for a total output energy of 30 Joules / cm. Following the treatment time, the bacteria-rhodamine suspensions were centrifuged at 3000g, resuspended in 4 ml and serial dilutions were made for each duplicate. 10 µl of the diluted bacterial suspensions were plated, the plates incubated overnight at 37 ° C. The bacteriostatic effect is expressed by the number of CFU / mL.

Staphilococcus epidermitis The protocol used for bacteriosid inactivation was performed as described in Brasseur and colleagues, with few modifications for the evaluation of bacteriosid potential (Brasseur et al. 2000). The compounds HA-X-171 and HA-VIII-92 showed antibacterial activity against S. epidermitis using the following experimental procedure, except that a concentration of 10 μΜ was used in the experimental procedure.

Bacteria were cultured overnight in Lubria Syrup (LB) medium, 100 μΐ («1 x 10 bacteria) from each bacterial suspension were added to 4 ml LB medium, a small aliquot of the bacterial suspension was removed for bacterial titration. before treatment expressed as CFU / mL. To the bacterial suspension were added rhodamine derivatives, each derivative being tested in duplicate at a concentration of 10 μΜ. Each rhodamine-bacteria derivative mixture was incubated at 37 ° C for 40 minutes. The bacteria-rhodamine suspensions were then treated and exposed to a wavelength light of 514 nm for 180 minutes for a total output energy of 30 Joules / cm2. Following the treatment time, the bacteria-rhodamine suspensions were centrifuged at 3000g, resuspended in 4 ml and serial dilutions were made for each duplicate. 10 µl of the diluted bacterial suspensions were plated, the plates incubated overnight at 37 ° C. The bacteriostatic effect is expressed by the number of CFU / mL.

Staphilococcus epidermitis The protocol used for bacteriosid inactivation was performed as described in Brasseur and colleagues, with few modifications for the evaluation of bacteriosid potential (Brasseur et al. 2000). HAX-40 showed antibacterial activity against S. epidermitis using the following experimental procedure, except that an extraction time of 90 minutes was performed prior to radiation treatment. S. epidermitis was grown overnight in Calda n Lubria (LB) medium, 100 μΐ («1 x 10 bacteria) from each bacterial suspension was added to 4 ml LB medium. A small aliquot of the bacterial suspension was taken for bacterial titration prior to treatment. To the bacterial suspension were added the rhodamine derivatives, the derivative being tested in duplicate at a concentration of 50 μΜ. Each rhodamine-bacteria derivative mixture was incubated at 37 ° C for 40 minutes. Bacteria-rhodamine suspensions were then centrifuged at 3000 g for 10 minutes, resuspended in 4 ml LB media and incubated for 90 minutes at 37 ° C to allow extraction of the derivatives. The bacteria-rhodamine suspensions were then treated and exposed to a wavelength light of 514 nm for 180 minutes for a total output energy of 30 Joules / cm. Following the treatment time, serial dilutions were made for each duplicate and 10 µl of the diluted bacterial suspension was plated, plates incubated overnight at 37 ° C. The bacteriostatic effect is expressed by the number of colony forming units / mL. DETERMINATION OF ANTIVIRAL ACTIVITY OF THE FORMULA RODAMINE DERIVATIVES (I) Antiviral Assay References Lin L., Inactivation of cytomegalovirus in platelet concentrates using Helinx ™ technology, Seminar in Hematology, 2001, 38, 4, Supp. # 11, 27-33;

Brasseur, N., Menard, I., Forget, A., El Jastimi, R., Hamel, R., Molflno. N.A. and E van Lier, J., Eradication of Multiple Myeloma and Breast Cancer Cells by Th9402-mediated Photodynamic Therapy: Implication of Clinical Ex vivo Purging of Autologous Stem Cell Transplant, Photochemistry and Photobiology, 2000, 72, 6, 780-878;

Lin.B., Londe, H., Janda, J.M., Hanson, C.V. and Corash, L., Photochemical Inactivation of Pathogenic, Bacteria in Human Platelet Concentrates, Blood, 1994, 83, 9, 2698-2706;

Lin, BL, Londe, H., Hanson, CV, Wioesehahn, G., Isaacs, S., Cimino, G. and Corash, L., Photochemical Inactivation of Cell-Associated Human Immunodeficiency Virus in Platelets Concentrates, Blood, 1993, 82, 1.292-297;

Li, B.L., Viesehahn, G., P., Morei, 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.

Purpose: The antiviral assay was performed as described in Lin, L (2001). Human diploid and foreskin fibroblast (FS) cells were used in this assay. The antiviral activity of rhodamine derivatives was tested and the results showed that all compounds HA-X-40, HA-X-149, HA-X-164, HA-X-171 and HA-VIII-92 followed by PDT treatment had antiviral activity against Cytomegalovirus. Method: FS cells were cultured to confluence in cartridge vials. At the time of infection, 2.5 - 3.5 x 105 cells were being cultured on each overleaf. CMV stock solution (ADI69) containing 1 mL virus was rapidly thawed, seeded and diluted following 100-fold dilutions in MEM (Earle's salt) supplemented with L-glutamine and 2% FBS, total volume 30 ml. The titration of the vms was determined at 10 em2 (104 TCID50) plaque forming units (pfu) in 0.2 ml. Therefore, 1 ml used in the PDT experiments represents 1.4 x 105 pfu. A Μ O.I. 0.4 - 0.5 CMV was used throughout this experiment.

Plates not containing rhodamine derivatives were light-treated in parallel to un-light-treated plates. The concentration used throughout the assay for rhodamine derivatives was maintained at 50 μΜ. Following the addition of the derivatives to the viral stock solution, the plates were placed into the Theraluz L6.30 device and illuminated for 180 minutes with 210 rpm agitation. Power output was measured to be 30 Joules / cm2. The non-PDT plate was placed inside a 37 ° C incubator for the same amount of time. Following this treatment time, dilutions are made and inoculated with the FS cells under centrifugation (2000 g, 60 minutes). Following centrifugation, cells are incubated 60 minutes at 37 ° C, 5% CO 2, then washed with culture media and incubated for 18-24ha37 ° C 5% CO 2. The inoculation volume of each dilution was 0.2 ml, the dilutions made were 10 -3 to 10.5 in duplicate.

The cells were fixed, removed from the flasks and stained with FITC labeled (fluorescein isothiocyanate) Mab with early CMV antigen immediately. CMV viral particles were counted. A fluorescent (kidney-shaped) virus particle represents a plaque forming unit.

Here, it is the protocol for the two other compounds TH9402 and XA-X-44 that inhibits cytomegalovirus infectivity without PDT treatment, as well as a new version of the table to be added in the patent.

Purpose: The antiviral assay was performed as described in Lin, L (2001). Human diploid fibroblast foreskin (FS) cells were used in this assay. The antiviral activity of rhodamine derivatives were tested and the results showed that the compounds, TH9402 and HA-X-44 do not require PDT treatment to possess antiviral activity against cytomegalovirus. Method: FS cells were cultured at confluence in cartridge vials. At the time of infection, 2.5-3.5 x 105 cells were being cultured on each overlever. CMV stock solution (ADI69) containing 1 ml virus was rapidly thawed, seeded and diluted following 100-fold dilutions in MEM (Earle's salt) supplemented with L-glutamine and 2% FBS, total volume 30 ml. . Virus titer was determined in ΙΟ "2 (104 TCID50) plaque forming units (pfu) in 0.2 ml. Therefore, 1 ml used in the PDT experiment represents 1.4 x 105 pfu. An MOI of 0.4 - 0.5 CMV was used throughout this experiment.

Plates not containing rhodamine derivatives were treated with light in parallel with untreated plates. The concentration used throughout the assay for rhodamine derivatives was maintained at 50 μΜ. Following addition of the derivatives to the viral stock solution, dilutions were made and inoculated with FS cells under centrifugation (2000 g, 60 minutes). Following centrifugation, cells are incubated 60 minutes at 37 ° C, 5% CO 2, then washed with culture media and incubated for 18 - 24 h at 37 ° C at 5% CO 2. The inoculation volume of each dilution was 0.2 ml, the dilutions made were from 10'3 to 10'5 in duplicate.

The cells were fixed, removed from the flasks and stained with FITC (fluorescein isothiocyanate) labeled Mab with early CMV antigen immediately.

CMV viral particles were counted. A fluorescent (kidney-shaped) virus particle represents a plaque forming unit.

Although the invention has been described with respect to its specific versions, it should be understood that it is capable of further modification and this application is intended to cover any variations, uses, or adaptations of the invention, generally following the principles including deviations from the present disclosure which fall within the known or customary practice of the art to which the invention belongs and how it may be applied to the essential aspects set forth herein and as follows within the scope of the appended claims.

Claims (6)

1. Rhodamine derivative, characterized in that it is selected from the group consisting of: 2 '- (6-dimethylamino-3-dimethylimino-3H-xanten-9-yl) 4', 5'-dichloric acid methyl ester hydrochloride benzoic; 4,5-dibromorodamine ethyl ester hydrobromide 2- (2-methoxy ethoxy); 2,7-dibromorodamine H hexyl ester acetate salt; 2,7-dibromorodamine methyl ester acetate salt B; 4,5-dibromorodamine 6G hydrobromide; Rhodamine 3-bromopropyl; 2,7-dibromo-4'-carboxytetramethylrosamine methyl ester acetate salt; 4-bromo-5-phenyl methyl ester chloride Rhodamine B; 2,7-dibromo-4,5-dimethyl methyl ester bromide Rhodamine B; 2-bromo-7-ethynyl methyl ester bromide Rhodamine B; and 4,5-dibromo-2,7-di-n-butyl rhodamine B methyl ester bromide. 2. Rhodamine derivative, characterized in that it is for use in the treatment of infections generated by Gram positive and / or Gram negative bacteria, wherein said rhodamine derivative is selected from the group consisting of: acid methyl ester hydrochloride 2, - (6-dimethylamino-3-dimethylimino-3H-xanten-9-yl) 4 ', 5'-dichloro-benzoic acid; 4,5-dibromorodamine ethyl ester hydrobromide 2- (2-methoxy ethoxy); 2,7-dibromorodamine H hexyl ester acetate salt; 2,7-dibromorodamine methyl ester acetate salt B; 4,5-dibromorodamine 6G hydrobromide; Rhodamine 3-bromopropyl; 2,7-dibromo-4'-carboxytetramethylrosamine methyl ester acetate salt; 4-bromo-5-phenyl methyl ester chloride Rhodamine B; 2,7-dibromo-4,5-dimethyl methyl ester bromide Rhodamine B; 2-bromo-7-ethynyl methyl ester bromide Rhodamine B; and 4,5-dibromo-2,7-di-n-butyl rhodamine B methyl ester bromide. Pharmaceutical composition, characterized in that it comprises a rhodamine derivative as defined in claim 1 and a pharmaceutically acceptable carrier. 4. Pharmaceutical composition for use in the treatment of infections caused by Gram positive and / or Gram negative bacteria, said composition characterized in that it comprises a rhodamine derivative selected from the group consisting of: 2 '- (6 - 6) methyl ester hydrochloride dimethylamino-3-dimethylimino-3H-xanthen-9-yl) 4 ', 5'-dichloro-benzoic acid; 4,5-dibromorodamine ethyl ester hydrobromide 2- (2-methoxy ethoxy); 2,7-dibromorodamine H hexyl ester acetate salt; 2,7-dibromorodamine methyl ester acetate salt B; 4,5-dibromorodamine 6G hydrobromide; Rhodamine 3-bromopropyl; 2,7-dibromo-4'-carboxytetramethylrosamine methyl ester acetate salt; 4-bromo-5-phenyl methyl ester chloride Rhodamine B; 2,7-dibromo-4,5-dimethyl methyl ester bromide Rhodamine B; 2-bromo-7-ethynyl methyl ester bromide Rhodamine B; and 4,5-dibromo-2,7-di-n-butyl methyl ester bromide RhodamineB. 5. Intermediate, characterized in that it is selected from the group consisting of 4,5-dibromo Rhodamine B lactone and 2,7-dibromo Rhodamine B lactone. Process for the synthesis of new rhodamine derivatives as defined in claim 1 or 2, characterized in that it comprises at least one reaction step of any of the following reaction schemes: wherein R, Ar and Y are such as to enable the preparation of the above-mentioned derivatives. .