5-aminolevulinic acid esters as photosensitive agents in photochemotherapy

FIELD: organic chemistry, medicine.

SUBSTANCE: invention relates to compounds designated for applying in photochemotherapy or diagnosis and indicated compounds represent 5-aminolevulinic acid aryl-substituted esters, their derivatives or pharmaceutically acceptable salts. In particular, invention provides preparing compounds of the general formula (I): R

22
N-CH2COCH2CH2CO-OR1 wherein R1 represents aryl-substituted C1-alkyl group, preferably C1-alkyl group substituted with non-heteroaromatic aryl wherein indicated group aryl is substituted group, and especially preferable this radical is substituted with one or more alkyl groups (for examples, (C1-C2)-alkyl), alkoxy- (for example, methoxy-) groups, fluorine, chlorine atoms, nitro- or trifluoromethyl groups; R2 being each of that can be similar or different represents hydrogen atom or alkoxycarbonyloxy-; indicated alkyl group is broken optionally with one or more groups: -O-, -NR3-, -S- or -PR3- wherein R3 represents hydrogen atom or (C1-C6)-alkyl group, and their salts for applying in diagnosis and photochemotherapy of injures and disorders of internal and external surfaces of body, and products and sets for realization of this invention also.

EFFECT: valuable medicinal properties of compounds.

18 cl, 17 dwg, 2 tbl, 3 ex

 

The present invention relates to new derivatives of 5-aminolevulinic acid (ALA) and, in particular, ALA esters and their use as photosensitive agents in photochemotherapy or diagnosis.

Photochemotherapy or photodynamic therapy (PDT)as it is known, is a method of treatment of various disorders, or disorders of the skin or other epithelial organs, or mucous membranes, in particular cancer or precancerous lesions, as well as some non-malignant lesions, such as skin lesions such as psoriasis. Photochemotherapy includes applying a photosensitive (fotohimioterapiei) agents to be processed area of the body, with consequent impacts photoactivating light to activate photosensitive agents, and turning them into cytotoxic form, thereby killing the affected cells or slowing their proliferative ability.

A known number of photosensitive agents, including, mainly, psoralens, porphyrins, chlorins and phthalocyanines. Such drugs become toxic under the action of light.

Photosensitive drugs may exert their actions through a variety of mechanisms, either directly or indirectly. For example, some photosensitizers be directly toxic when the asset is AI light, while others operate by generating toxic particles, such as oxidizing agents, such as atomic oxygen or other free-radical derivatives of oxygen, which is extremely destructive to cellular material and biomolecules such as lipids, proteins and nucleic acids. Psoralens are an example of directly acting photosensitizers; the action of light, they form adducts and cross-links between the two strands of DNA, thereby inhibiting DNA synthesis. Unfortunately, the risk of this treatment is that it can be mutagenic and carcinogenic side effects.

This drawback can be avoided by choosing an alternative photosensitizers indirect mode of action. For example, porphyrins, which act indirectly through the generation of toxic oxygen particles do not have mutagenic side effects and are the most preferred candidates for photochemotherapy. Porphyrins are naturally occurring precursors in the synthesis of heme. In particular, heme get when iron (Fe3+) included in fetoprotein IX (Pp) under the action of the enzyme ferrochelatase. PP is extremely active photosensitizer, whereas heme does not have a photosensitizing effect.

Recently one such drug on the again of the porphyrin Photofrin (approved as a photosensitizer for the treatment of certain types of cancer. The main disadvantage is that the agent should be introduced parenterally, usually intravenously, and it causes photosensitization of the skin, which may persist for several weeks after intravenous injection. Photofrin consists of a large number of porphyrin oligomers and not easily penetrates the skin for local use. Similar problems exist for other photosensitizers based on porphyrin, such as the so-called “hematoporphyrin derivatives” (Hpd), which also indicated the use in photochemotherapy of cancer (see, for example, S. Dougherty, J. Natl. Cancer Ins., 1974, 52: 1333; Kelly and Snell, J. Urol., 1976, 115: 150). Hpd is a mixture of complexes obtained by treatment of hematoporphyrin acetic and sulphuric acids, after which the acetylated product is dissolved with addition of alkali.

To overcome these problems investigated predecessors PP on their photochemiluminescence activity. In particular, investigated the predecessor PP 5-aminolevulinic acid (ALA) as photochemotherapy agent for some types of skin cancer. ALA, which is obtained from succinyl of coenzyme a (succinyl SOA) and glycine at the first stage of the synthesis of heme, capable to a limited extent, to penetrate the skin and lead the localized formation of PP; because the action of ferrochelatase (enzyme metallation) is limiting the speed stage in the synthesis of heme, an excess of ALA leads to the accumulation of PP photosensitive agent. Thus, local application of ALA on skin tumor and after a few hours of exposure to light on tumors can provide useful photochemiluminescence effect (see, for example, WO 91/01727). Because the skin covering Basilone and squamous cell carcinoma, easier permeable for ALA than healthy skin, and since the concentration of ferrochelatase in skin tumors is low, found that topical application of ALA leads to a selective increase in the production of PP in tumors.

However, photochemotherapy with the use of ALA is not always entirely satisfactory. ALA is not able to penetrate all of the tumor and other tissues with efficiency, sufficient to treat a wide variety of tumors or other conditions, as well as ALA has a tendency to instability in pharmaceutical preparations. These problems are largely overcome with the use of ALA esters and unsubstituted linear Akilov that exhibit increased selectivity relative to the affected tissue, non-systemic localization of the introduced agents, improved absorption and production RRH and decreased pain perception in the introduction (see WO 96/28412).

Database Xfir, writings and 3060978, 5347132, 5499790, 5620924, 5633390, 5991317 and 6517740 (Beilstein); Cosmo Sogo Kenkyusho KK, Patent Abstracts of Japan, Vol.16, No. 156 (C-0930), 16.4.1992; EP-A-316179 (Tokuyama Soda KK); GB-A-2058077 (Hudson and others) and DE-A-2411382 (Boehringer Sohn Ingelheim) describe the alkyl ester derivatives of 5-aminolevulinic acid, their derivatives, and salts and methods for their preparation.

However, these compounds still have some limitations for use as pharmaceuticals in PDT, for example, relatively low efficiency, and therefore, there is a need for alternative fotohimioterapiei agents, particularly fotohimioterapiei agents that demonstrate the best performance in comparison with agents known in this field.

The present invention is directed to satisfying this need and, in particular, achieved the result is the provision of fotohimioterapiei agents who are the best of pharmaceuticals, for example, have a stronger photochemiluminescence action than those in the prototype.

Presently discovered that the class of ester derivatives of ALA, essentially comprising branched alkalemia ALA esters and substituted benzyl esters of ALA, suitable for use in photochemotherapy. In particular, we discovered that some of these connections provide unexpectedly more BC the favorable PDT properties in comparison with known compounds.

Thus, on the one hand, this invention provides a compound for use in photochemotherapy or diagnosis, and the specified connection is branched alkilany ether or substituted alkilany ester of 5-aminolevulinic acid, its derivative or salt.

In another aspect, the invention provides a compound for use in photochemotherapy or diagnosis, and the specified connection has the General formula I:

(where R1represents C1alkyl group substituted by one or more aryl groups); and,

R2independently represents a hydrogen atom or optionally substituted alkyl group), or

its pharmaceutically acceptable salt.

Preferably, the invention provides compounds of formula I:

(where R1is substituted by aryl (C1alkyl group, preferably C1alkyl group, a substituted vegetariantimes.com-aryl, where this aryl group is substituted, particularly preferably substituted by one or more alkyl (for example, C1-2alkyl), alkoxy (e.g. methoxy) groups, fluorine atoms, chlorine, nitro or triptoreline groups;

R2, each of which can be Odie is akovali or different, represents a hydrogen atom or optionally substituted alkyl group, preferably the group R1;

where these substituents selected from hydroxy-, alkoxy-, acyloxy, alkoxycarbonyl-, amino-, aryl, nitro, oxoprop, fluorine and groups-SR3, -NR

3
2
and PR
3
2
,

and each alkyl group optionally interrupted by one or more groups-O-, -NR3-, -S - or-PR3-; and

R3represents a hydrogen atom or a C1-6alkyl group)

and their salts for use in photochemotherapy or diagnosis.

Used herein, the term “alkyl”, unless otherwise indicated, includes any long or short, cyclic, linear or branched, aliphatic, saturated or unsaturated hydrocarbon group. Unsaturated alkyl groups may be mono - or polyunsaturated and include as alkeneamine and alkyline group. If not stated otherwise, these groups can contain up to 40 atoms. However, the preferred alkyl groups containing up to 10, preferably up to 8, more preferably up to 6 and especially preferably up to 4 carbon atoms.

Substituted alkyl group, R1may be mono - or polyamideimide. Thus, suitable groups R1include, for example, alkoxyalkyl, hydroxyalkoxy, polyhydroxyethyl, hydroxypolycarboxylic, oxaalkyl, polyoxyethyl and the like.

Used in this description, the term “acyl” includes both carboxylate and carbonate groups, thus acrosiphonia alkyl groups include, for example, alkylcarboxylic. In such groups any alkylene parts preferably have the number of carbon atoms as defined above for alkyl groups.

Preferred substituted alkyl groups of R1include groups bearing one or more oxoprop, the preferred linear C4-12alkyl (for example, C8-10alkyl) groups, substituted one, two or three (preferably two or three) exography. Examples of such groups include a 3.6-dioxa-1-octyl and 3,6,9-trioxa-1-decyl.

Particularly preferred substituted alkyl groups of R1that may be present in the compounds of formula I include With1-6alkyl, preferably1-4alkyl, particularly preferably1or4alkyl (e.g. methyl), substituted (preferably terminal-substituted) aryl group. Preferred aryl groups include phenyl, di is Anil and a 5-7 membered, for example, 5-or 6-membered heteroaromatic group, especially phenyl, and such groups can themselves be optionally substituted, for example, one or more (e.g. one or two)1-6alkyl groups (preferably1-4alkyl groups, for example, stands), alkoxygroup (for example, methoxy), nitro groups, fluorine atoms, chlorine or triptoreline groups. Suitable heteroaromatic groups include groups containing at least one heteroatom selected from oxygen, sulfur and nitrogen. Preferred heteroaromatic group is pyridine.

Typical examples of substituted alkyl groups, R1include arylalkyl, alkoxymethyl, alkoxyethyl and alkoxyaryl or acyloxymethyl, acyloxyacyl and acyloxymethyl, such as pivaloyloxymethyl.

Preferred specified branched alkyl groups in formula I are With4-8preferably5-8linear alkyl groups which are branched at the substitution of one or more optionally substituted C1-6alkyl (for example, C1-2alkyl) groups, preferably forming, so6-9alkyl groups. In those cases where R1represents a branched alkyl group, it preferably is unsubstituted. For example, R1can depict ablate unsubstituted branched C 5-10alkyl group, preferably5-8alkyl, for example With5or6alkyl.

If R1represents a substituted alkyl group, particularly preferred are aryl (e.g. phenyl) or alkoxygroup, which may themselves be substituted.

Thus, in a particularly preferred embodiment, R1is:

optionally substituted branched C5-30alkyl group, preferably6-9alkyl group, including linear C4-29alkyl group, preferably4-8more preferably5-8(for example, C5or6) alkyl group branched at the substitution of one or more alkyl groups, preferably With1or2where the specified location of the substitution is preferably in C2or higher atom, or

aryl - or alkoxy-substituted alkyl group, preferably1or2alkyl group,

where this aryl group is preferably substituted, particularly preferably substituted by one or more alkyl (for example, C1-2alkyl), alkoxy (e.g. methoxy) groups, fluorine atoms, chlorine, nitro or triptoreline groups, and

this alkoxygroup is preferably substituted by one or more alkoxygroup and, which, in turn, can be overridden.

Another aspect of the present invention provides new compounds. Thus, the present invention provides compounds of formula I:

(where R1represents optionally substituted branched C5-30alkyl group, preferably6-9alkyl group, including linear C4-9alkyl group, preferably4-8more preferably5-8(for example, C5or6) alkyl group branched at the substitution of one or more1-6alkyl groups, preferably With1or2where the specified location of the substitution is preferably in C2or higher carbon atom; or

alkyl group, a substituted vegetariantimes.com by aryl, preferably1or2alkyl group, where this aryl group is preferably substituted, particularly preferably substituted by one or more alkyl (for example, C1-2alkyl), alkoxy (e.g. methoxy) groups, fluorine atoms, chlorine, nitro or triptoreline groups; or

alkoxy-substituted alkyl group, preferably1or2alkyl group, where this alkyl group is substituted meth what syruppy or alkoxygroup, replaced by alkoxygroup, which can also be substituted,

R2, each of which may be the same or different, represents a hydrogen atom or optionally substituted alkyl group (for example, the group R1); and

where these substituents selected from hydroxy-, alkoxy-, acyloxy, alkoxycarbonyl-, amino-, aryl, nitro, oxoprop, fluorine and groups-SR3, -NR

3
2
and PR
3
2
and

the specified alkyl group optionally interrupted by one or more groups-O-, -NR3-, -S - or-PR3-; and

R3represents a hydrogen atom or a C1-6alkyl group) and their pharmaceutically acceptable salts.

Preferably, both R2was the hydrogen atoms and, thus, in the preferred embodiment, this invention provides substituted benzyl esters of ALA and optionally substituted branched C6-9alkalemia esters of ALA, which can be used in the methods of the present invention.

Thus, the compounds of this invention or compounds for use in the methods of this invention include 2-methylp nilowy ester of ALA, 4-methylpentanoic the ALA ester, 1-ethylbutylamine the ALA ester, 3,3-dimethyl-1-butyl ALA ester, benzyl ALA ester, para-methylbenzylamine the ALA ester, para-nitrobenzyloxy the ALA ester, para-[trifluoromethyl]benzyl ALA ester, para-tormentingly the ALA ester, 4-chlorbenzoyl the ALA ester, 3-methylbenzylamine the ALA ester and 2-methylbenzylamine ether ALA.

The compounds of this invention or compounds for use in this invention may be obtained using standard methods and techniques well known in the field to obtain derivatives of multifunctional compounds, and especially the esterification. As you know, this etherification of the compounds may include the introduction of the protect and unprotect appropriate groups, so only the necessary groups remain active and take part in the reaction under the conditions of esterification. Thus, during the esterification can be protected, for example, the substituents of substituted alkanols used to obtain esters. Similarly, a group NR

2
2
on the connection, making this group in the compounds of formula I, can be protected during the reaction and then remove the protection. Such techniques introduction/removal protection is well known in this field to obtain the derivatives and, in particular, complex e is IRow well known amino acids, see, for example, Mcomie “Protective Groups in Organic Chemistry”, Plenum, 1973 and T.W.Green in “Protective Groups in Organic Chemistry”, Wiley-Interscience, 1981.

Thus, another aspect of the present invention provides a method for producing compounds of this invention or compounds for use in this invention, including the production of ester on carboxypropyl 5-aminolevulinic acid.

As you can see, this invention provides a method for producing compounds of this invention or compounds for use in this invention, including the interaction of 5-aminolevulinic acid or its amenable esterifying derivative with alkanols or afrobrazil derived.

More specifically, this aspect of the invention provides a method for producing compounds of formula I, where the method includes at least one of the following stages:

(a) interaction of the compounds of formula II

(where X denotes a leaving group, such as hydroxyl group, halogen atom or alkoxygroup, or MOR denotes a group of the acid anhydride, and R2the same as defined above) with the compound of the formula III

(where R1the same as defined above); and

(b) the conversion of compounds of formula I, its pharmaceutically acceptable salt.

Stage (is) the reaction can be conveniently carried out in a solvent or mixture of solvents, such as water, acetone, diethyl ether, methylformamide, tetrahydrofuran, etc. at temperatures up to the boiling point of the mixture, preferably at ambient temperature.

Conditions of esterification reactions dependent on the use of alcohol, and these terms can be chosen in such a way as to obtain the maximum yield of ester. Since the esterification reaction is reversible equilibrium reactions, interaction terms can be chosen in such a way as to obtain the maximum yield of ester product. Such conditions can be obtained by choosing a solvent that is capable of removing the water generated during a typical esterification reaction, through the formation of azeotrope with water. Examples of such solvents are aromatic hydrocarbons or other, capable of forming azeotrope with water, such as some chlorinated hydrocarbons, such as chloroform. To obtain a lower ester of 5-ALA can shift the equilibrium of the reaction towards formation of ester, using a large excess of alcohol. Other esters can shift the equilibrium towards ester product using a large excess of acid.

The esterification reaction is well known in this field, for example, as described Saul Patai “The chemistry of The carboxylic acids and esters” (Ch.11, p.505, Interscience 1969) and Houban Weyl (ethoden der Orgnische Chemie, Band E5, “Carbonsauren und carbonsauren-derivate”, p.504, Georg Thieme Verlag, 1985). Receipt of amino acid derivatives described in Band XI/2 of the same series (Houben Weyl, Methods der Organische Chemie, Band XI/2, “Stickstoffverbindungen”, p.269, Georg Thieme Verlag, 1958).

Stage (a) the reaction is conveniently carried out in the presence of a catalyst, for example an inorganic or organic acid, or an agent that binds acid, such as the ground.

Used as starting compounds, the compounds known from the literature and in many cases commercially available or can be obtained using essentially known methods. For example, ALA available from Sigma or Photocure ASA, Oslo, Norway.

As mentioned above, the compounds of this invention or compounds for use in accordance with this invention may take the form of pharmaceutically acceptable salts. Such salts are preferably kislotoupornye salts of physiologically acceptable organic or inorganic acids. Suitable acids include, for example, hydrochloric, Hydrobromic, sulfuric, phosphoric, acetic, lactic, citric, tartaric, succinic, maleic, fumaric and ascorbic acid. Hydrophobic salts can also conveniently be obtained, for example, deposition. Suitable salts include, for example, acetate, bromide, chloride, citrate, hydrochloride, maleate, mesilate, nitrate, phosphate, sulfate, tartrate, Olea is, stearate, tosylate, calcium salts, meglumine, potassium and sodium. Methods of obtaining salts are well-known.

As mentioned above, the compounds of this invention and the compounds for use according to this invention and their salts have valuable pharmacological properties, namely properties of photosensitivity, which make them useful as fotohimioterapiei agents.

Another aspect of the present invention provides a pharmaceutical composition comprising the compound described above, or its pharmaceutically acceptable salt, at least one pharmaceutical carrier or excipient.

Another aspect provides a pharmaceutical composition, described above, for use as a drug, for example, in photochemotherapy or diagnosis.

Another aspect provides the use of compounds described above, or its pharmaceutically acceptable salt with the purpose of obtaining a therapeutic agent for treating disorders or disorders of the internal or external surfaces of the body which are responsive to photochemotherapy.

Disorders and disorders that can be treated according to the present invention, include any malignant, pre-malignant and non-malignant disorders or disorders that Rea is irout to photochemotherapy, for example tumors or other lesions, skin lesions such as psoriasis or senile keratosis and acne, abrasions on the skin and other diseases or infections, such as bacterial, viral or fungal infections, such as herpes virus infection. The invention is particularly suitable for the treatment of diseases, disorders or disorders, when the formation of discrete lesions that can directly be applied composition (the term “lesion” is used herein in a broad sense and includes the tumor and the like).

Internal and external surfaces of the body, which can be treated according to this invention, include the skin and all other epithelial and serosal surfaces, including, for example, the mucous membranes, the lining of organs such as the respiratory, gastrointestinal and urogenital tract, and glands with ducts that open onto such surfaces (e.g., liver, hair follicles with sebaceous glands, mammary glands, salivary glands, and seminal vesicles). In addition to the skin such surfaces include, for example, the lining of the vagina, endometrium and urately. Such surfaces may also include a cavity formed in the body after excision of diseased or cancerous tissue, for example, cavities in the brain after excision of these tumors, such as gliomas.

Therefore clicks the zoom, examples of surfaces include: (i) the skin and conjunctiva; (ii) the lining of the mouth, pharynx, esophagus, stomach, intestines and intestinal processes, rectum and anal canal; (iii) the lining of the nasal passages, nasal sinuses, nasopharynx, trachea, bronchi and bronchioles; (iv) the lining of the ureters, bladder, urethra; (v) the lining of the vagina, cervix and uterus; (vi) the parietal and visceral pleura; (vii) the lining of peritoneal and pelvic cavities and surfaces of organs inside these cavities; (viii) Dura and membranes of the brain; (ix) any tumors in solid tissues, which can be made available for photoactivating light, for example, during surgery or through the optical fiber is introduced through a needle.

The compositions of this invention can be in the usual way with one or more physiologically acceptable carriers or excipients well known in this field techniques. In a preferred aspect of the present invention, when it is prescribed, the compounds or compositions of the present invention is sterilized, for example by γ-irradiation treatment in an autoclave or heat sterilized before or after adding the substrate or excipients, if present, to ensure the sterility of products.

Comp the positions can be entered locally, oral or systemically. Compositions for local injection are preferred and include gels, creams, ointments, sprays, lotions, lubricants, pencils, Soaps, powders, pessaries, aerosols, drops, solutions, and any other well-known pharmaceutical forms.

Ointments, gels and creams can be prepared, for example, aqueous or oily base with the addition of suitable thickeners and/or gelling agents. Lotions can be prepared in water or oil-based, and they usually contain one or more emulsifiers, dispersing agents, suspendida agents, thickeners or dyes. Powders can be prepared using any suitable powder base. Drops and solutions can be prepared in an aqueous or nonaqueous basis, comprising one or more dispersing agents, agents that promote solubility, or suspendida agents. Aerosol sprays are usually produced in the form of packages under pressure using a suitable propellant.

Alternative compositions can be obtained in a form adapted for oral or parenteral administration, for example by intradermal, subcutaneous, intraperitoneal or intravenous injection. Alternative pharmaceutical forms, thus, include a simple pill or tablet and the shell, capsules, suspensions and solutions containing the active ingredient optionally together with one or more conventional inert carriers and/or diluents, for example, corn starch, lactose, sucrose, microcrystalline cellulose, magnesium stearate, polyvinylpyrrolidone, citric acid, tartaric acid, water, mixture of water/ethanol, water/glycerol, water/sorbitol, water/polyethylene glycol, propylene glycol, stearyl alcohol, carboxymethylcellulose or fatty substances such as hard fat or suitable mixtures.

The composition can additionally include lubricating and wetting agents, emulsifiers, suspendresume agents, preservatives, sweetening agents, flavoring agents, amplifiers adsorption, for example agents for penetration through the surface, which are listed below, and the like. The compositions of this invention can be designed to provide rapid, prolonged or delayed release of the active ingredient after administration to the patient, using techniques well known in the field. You can also apply agents that contribute to the dissolution and/or stabilizers, such as cyclodextrins (CD) α, β, γ and cyclodextrin HB-β. The composition can be in the form of any suitable dosage form, for example in the form of emulsions or liposomes, NRENs, microspheres, nanoparticles or the like. The compound of this invention may be absorbed on or associated with these forms.

Concentration is ia the above-described compounds in the compositions depends on the nature of the connection, composition, method of administration, subject to the treatment condition and the patient, and it can be varied or adjusted in accordance with the selection. However, it is usually preferred concentration range from 0.01 to 50%, for example from 0.05 to 20%, such as 1-10% (wt./mass.). Found that for therapeutic applications is appropriate concentration range from 0.1 to 50%, for example from 0.2 to 30% (wt./mass.). If they are highly lipophilic derivatives, it is possible to use smaller doses, for example, in the concentration range from 0.01 to 10%, for example from 0.02 to 1% (wt./mass.).

Local introduction in inaccessible places can make known in this area by, for example, using a catheter or other suitable delivery systems of drugs.

After application to the surface of the treated region is irradiated with light to achieve fotohimioterapiei effect. Period of time after application, during which the irradiation of light, depends on the nature of the composition that is subjected to the treatment condition and form of administration. Usually it can be from 0.5 to 48 hours, for example from 1 to 10 hours.

Usually applied irradiation dose of from 40 to 200 j/cm2for example , 100 j/cm2.

The wavelength of light used for exposure can be chosen in such a way as to achieve more effective photochemiluminescence the effect. Usually, when photochemotherapy is used porphyrins, their irradiate light at the maximum absorption of the porphyrin. Thus, for example, in the case of the prototype ALA in photochemotherapy of skin cancer used wavelength in the range 350-640 nm, preferably 610-635 nm. However, choosing a wide range of wavelengths for irradiation extending beyond the maximum absorption of the porphyrin can be enhanced photosensitizing effect. Not wanting to contact theory, believe that this is due to the fact that when PP and other porphyrins are exposed to light with wavelengths within its absorption spectrum, it breaks the various photoproduct, including, in particular, photoprotection (PPP). PPP represents chlorine and produces significant photosensitizing effect; its absorption spectrum extends to longer wavelengths, higher wavelengths, which absorbs the PP, namely almost to 700 nm (RR almost does not absorb light above 650 nm). Thus, under normal photochemotherapy used wavelengths do not initiate PPP and, therefore, cannot benefit from its additional photosensitizing effect. Found that the irradiation of light with a wavelength in the range of 500-700 nm is particularly effective. It is especially important to include wavelengths of 630 and 690 nm.

Another aspect of this invention, therefore about the time, provides a way fotohimioterapiei disorders or disorders of the internal or external surfaces of the body, including introduction to subject to the influence of surface composition, which is defined above, and the impact on the mentioned surface light, preferably light with a wavelength in the range of 300-800 nm, for example 500-700 nm.

Methods of exposure to different areas of the body, for example, a lamp or laser is well known in the art (see, for example, Van den Bergh, Chemistry in Britain, May 1986, p.430-439). For inaccessible areas, the irradiation can be performed using optical fibers.

The compounds of this invention or compounds for use in this invention can be prepared and/or used with other photosensitizing agents, such as ALA or Photofrin®or other active ingredients that may increase photochemiluminescence effect. For example, it is useful to include chelating agents to enhance the accumulation of PP; the chelation of iron chelating agents prevents its inclusion in PP with the formation of heme under the action of the enzyme ferrochelatase that leads to the creation of the RR. Thus, the enhanced photosensitizing effect.

In this respect, particularly suitable for use aminopolycarboxylate chelating agents, including any gelantinous the ligands, described in the literature for detoxification of metals or for chelation of paramagnetic metal ions in contrasting agents for obtaining a magnetic resonance image. In particular, it can be noted ethylenediaminetetraacetic acid (EDTA), cyclohexanedimethanol acid (CDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-N,N’,N’,N’’-tetraoxo acid (DOTA) and their well-known derivatives/analogues. The preferred EDTA. For the implementation of the chelating effect of iron can also be used desferrioxamine and the other siderophore, for example, in combination with aminopolycarboxylate chelating agents such as EDTA.

Typically, the chelating agent can be applied at a concentration of from 0.05 to 20%, for example from 0.1 to 10% (wt./mass.).

In addition, we discovered that the agents promoting penetration through the surface, especially diallylsulfide, such as dimethylsulfoxide (DMSO), can have a beneficial effect in enhancing fotohimioterapiei effect. This is described in detail in WO 95/07077.

Agents promoting penetration through the surface, can be any described in the pharmaceutical literature agents, such as chelat forming agents (such as EDTA), surface-active agents (for example sodium dodecyl sulphate), poverhnosti-active what gentami, salts of bile acids (for example, deoxycholate sodium) and fatty acids (e.g. oleic acid). Examples of suitable agents promoting penetration through the surface, include NRE-101 (Hisamitsu), DMSO and other diallylsulfide, in particular n-decylmethacrylate (NDMS), dimethylacetamide, dimethylformamide (DMF), dimethylacetamide, glycols, various derivatives of pyrrolidone (Woodford and others, J. Toxicol. Cut. & Ocular Toxicology, 1986, 5: 167-177) and Azone® (Stoughton and others, Drug Dvp. Ind. Pharm. 1983, 9: 725-744) and mixtures thereof.

However, the most preferred is DMSO due to its antihistamine and anti-inflammatory activity and stimulating effect on the activity of the enzyme ALA synthase and ALA-dehydrogenase (enzymes, which, respectively, form and condense ALA in porphobilinogen), thereby strengthening the formation of the active form PP.

Agents, penetrating through the surface, you can usually applied at a concentration in the range from 0.2 to 50% (wt./mass.), for example, about 10% (wt./mass.).

To increase the effectiveness of PDT compositions of the present invention or used according to this invention can also be prepared and/or used with other agents. In addition, for example, when processing tumors to achieve additional lesions of the vascular system of the tumor together with the compositions of this invention when PT can be applied inhibitors of angiogenesis (antiangiogenic drugs), which are detected as being useful in the treatment of tumors (O'reilly and others, Nature Medicine, 2, p.689-692, 1996; Yamamoto and others, Anticancer Research, 14, p.1-4, 1994; Brooks and others, J. Clin. Invest., 96, p.1815-1822, 1995). Inhibitors of angiogenesis that can be applied include TNP-470 (AGM-1470, a synthetic analogue of the product of fungal secretion, known as fumagillin, Takeda Chemical Industries Ltd., Osaka, Japan), angiostatin (Surgical Research Lab. at Children's Hospital Medical Center of Harvard Medical School) and antagonists of integrin αvβ3(for example, monoclonal antibody relatively integrin αvβ3, The Scripps Research Institute, LaJolla, CA).

Alternative or in addition, to improve PDT according to the present invention can be applied immunotherapy agents (e.g. antibodies or effectors, such as the factors of the activation of macrophages) or chemotherapeutic agents. The introduction of such additional agents should be carried out with the method, concentration of the drug in accordance with known methods for the introduction of such agents. Such additional agents can be entered before, after or simultaneously with PDT, depending on their function. For example, inhibitors of angiogenesis can add 5-10 days after PDT to prevent re-growth of the tumor.

Other anticancer agents can be applied similarly in combination with a composition of this invention, either as part of the drug is, or as a separate treatment simultaneously, separately or sequentially.

Also found that glucose promotes PDT for local or systemic use. Although not wishing to contact theory, it is clear that the introduction of glucose results in a decrease in pH, which increases the hydrophobic properties of protoporphyrin, such as ALA, so they can more easily penetrate into cells. If it is assumed the local introduction of the composition, for example a cream, can typically contain from 0.01 to 10% glucose (wt./mass.).

In accordance with the condition to be treated, and the nature of the composition of the compounds used in this invention, can be entered together with other optional agents, for example, in the form of a single composition, or sequentially, or separately. Indeed, in many cases, particularly useful photochemiluminescence effect can be obtained by using as a separate stage of pre-processing agent for promoting penetration through the surface, before the application of the compounds used in this invention.

In addition, in some situations, it may be useful pre-processing agent for promoting penetration through the surface with the subsequent introduction of fotohimioterapiei agent in combination with an agent that promotes penetration through the surface. If the seat reservation processing agent used, promote penetration through the surface, then it can be used at high concentrations, for example up to 100% (wt./mass.). When conducting this preliminary processing, photochemotherapy agent, you can enter in a few hours after pre-treatment, for example through 5-60 min after pre-processing.

Thus, another aspect of this invention provides a product comprising the above-described compound or its pharmaceutically acceptable salt together with at least one agent for promoting penetration through the surface, and optionally one or more chelating agents, as a combined preparation for simultaneous, separate or sequential use in the treatment of disorders or disorders of the internal or external surfaces of the body that are sensitive to photochemotherapy.

This alternative aspect of the present invention also provides a kit for use in treatment of disorders or disorders of the internal or external surfaces of the body, including:

a) a first container containing described here above compound or its pharmaceutically acceptable salt;

b) a second container containing at least one agent that promotes penetration through the surface, and long is Ino

c) one or more chelating agents, included in said first container or in a third container.

If separate pre-processing stage is used, the agent that promotes penetration through the surface, then it can be used at high concentrations, for example up to 100% (wt./mass.).

It is clear that the method of treatment using the above-described compounds inevitably includes fluorescence in the field of disorders or disorders to be treated. Though the intensity of this fluorescence can be used to destroy diseased cells, the localization of fluorescence can be used to visually determine the size, extent and location of this disorder or disturbance.

The disorder or violation identified in this way or confirmed in this study place, you can then handle alternative therapeutic methods, such as surgically or chemically, or therapeutic method of the present invention, while continuing to generate fluorescence, or additionally, applying the compounds of this invention in place. It is clear that the diagnostic methods may require to render smaller levels of fluorescence than used in therapeutic treatment. Thus, suitable concentrations usually the range from about 0.2 to 30%, for example 1-5% (wt./masses). Places, methods and models of application of the above from the point of view of therapeutic applications, but is applicable also described for diagnostic purposes.

The compounds of this invention or compounds for use in this invention can also be used for in vitro diagnostic methods for the study of cells contained in the body fluids. Higher fluorescence corresponding to the unhealthy tissue, can be a useful indicator of disorder or disturbance. This method is highly sensitive and can be used for easy detection of disorders or disorders, for example, carcinoma of the bladder or lungs, through the study of epithelial cells in samples of urine or sputum, respectively. Other useful body fluid that can be used for diagnosis, in addition to urine and sputum specimens include blood, semen, tears, cerebrospinal fluid, etc. Can also be evaluated samples or preparations of tissue, for example tissue taken at biopsy, or samples of bone marrow. Thus, the present invention extends to the use of compounds of this invention or their salts for the diagnosis of the above methods in photochemotherapy and products and kits for carrying out the specified diagnostic.

The another aspect of the present invention relates to a method of in vitro diagnosis of disorders or disorders by studying a sample of fluid or tissue of the patient, moreover, the method includes at least the following stages:

mixing the specified fluid or tissue of the body with the above-described connection;

irradiation of this mixture of light;

determining the level of fluorescence

comparing the level of fluorescence with the control levels.

The invention will be described in more detail in the following non-restrictive examples with reference to the drawings, where:

the figure 1 shows the formation of cellular porphyrin induced by ALA (filled circles), 1-methylpentylamino ether ALA (empty circles), para-isopropylbenzylamine the ALA ester (filled inverted triangles) and pair-methylbenzylamine ether ALA (empty inverted triangles);

the figure 2 shows the formation of cellular porphyrin induced by ALA (filled circles), benzyl ALA ester (filled triangles) and 2-phenethyl ether ALA (empty triangles), the bars indicate the standard deviation;

the figure 3 shows the formation of cellular porphyrin induced by ALA (filled circles), hexyl ether ALA (empty circles), cyclohexylamin the ALA ester (filled inverted triangles) and 4-methylpentylamino ether ALA (empty inverted triangles), lines indicate standard error;

the figure 4 shows the formation of cellular porphyrin in ucirvine ALA (filled circles), steam-[trifluoromethyl]benzyl ALA ester (empty circles), para-[tert-butyl]benzyl ALA ester (filled inverted triangles) and pair-nitrobenzyl ether ALA (empty inverted triangles);

the figure 5 shows the fluorescence of the skin after local application of hexyl ALA ester (filled squares), 1-methylpentylamino of ALA ester (filled triangles), 1-ethylbutanol of ALA ester (filled inverted triangles) and 2-methylpentylamino of ALA ester (filled circles), the bars indicate standard error;

the figure 6 shows the fluorescence of the skin after local application of hexyl ALA ester (empty squares), cyclohexylamino of ALA ester (filled triangles), 2-phenethyl ester ALA (shaded squares) and 4-methylpentylamino of ALA ester (filled circles), the bars indicate standard error;

the figure 7 shows the fluorescence of the skin after local application of hexyl ALA ester (empty squares) and pair-methylbenzylamino ether ALA (shaded triangles), the bars indicate standard error;

the figure 8 shows the fluorescence of the skin after local application of hexyl ALA ester (filled squares), para-(isopropyl)benzyl ALA ester (filled triangles) and 4-phenylbutyramide ether ALA (shaded inverted triangles), lines indicate the conventional error;

the figure 9 shows the fluorescence of the skin after local application of hexyl ALA ester (filled squares), performancelevel of ALA ester (filled triangles) and pair-nitrobenzylamine of ALA ester (filled circles), the bars indicate standard error;

the figure 10 shows the fluorescence of the skin after local application of hexyl ALA ester (filled squares), para-(tert-butyl)benzyl ALA ester (shaded triangles) and para-[trifluoromethyl]benzyl ALA ester (filled circles), the bars indicate standard error;

the figure 11 shows the fluorescence of the skin after local application of hexyl ALA ester (filled squares) and benzyl ALA ester (filled circles), the bars indicate standard error;

the figure 12 shows the fluorescence of the skin after local application of 1% of hexyl ALA ester (filled squares) and 3% 3,3-dimethyl-1-butyl ALA ester (filled triangles), the bars indicate standard error;

the figure 13 shows the fluorescence of the skin after local application of 1% hexyl ester of ALA (empty squares), 10% 2-forbeslife ether ALA (shaded triangles), 10% 2,3,4,5,6-pentafluorobenzyl ether ALA (shaded diamonds) and 10% 4-chlorobenzylamino of ALA ester (filled circles), the bars indicate standard error;

the figure 14 shows luorescence skin after local application of hexyl ALA ester (empty triangles), 2-methoxyethanol of ALA ester (filled squares), 3-nitrobenzyl ether ALA (shaded diamonds) and 3,4-[dichloro]benzyl ALA ester (empty circles), the bars indicate standard error;

the figure 15 shows the fluorescence of the skin after local application of hexyl ALA ester (filled squares), 3,6-dioxa-1-oktilovom of ALA ester (filled triangles), 3-fermentelos ether ALA (empty diamonds) and 3,6,9-trioxa-1-delovogo ether ALA (shaded circles), the bars indicate standard error;

the figure 16 shows the fluorescence of the skin after local application of hexyl ALA ester (filled squares), 3-pyridinylmethyl of ALA ester (filled triangles), 4-diphenylmethylene of ALA ester (filled diamonds) and 4-methoxybenzylamine of ALA ester (filled circles), the bars indicate standard error;

the figure 17 shows the fluorescence of the skin after local application of 1% hexyl ester of ALA (empty squares), 3% 2-methylbenzylamino of ALA ester (filled triangles), 3% benzyl-5-[(1-acetylacetone)carbonyl]new ALA ester (filled diamonds) and 3% of 3-methylbenzylamino ether ALA (empty circles), the bars indicate the standard error.

Example 1: production of esters of 5-amino-4-oxopentanenitrile

General method I

Thionyl chloride (1.0 ml) is added drop by drop to mix alcohol (6.0 ml ili-6 g), cooled to 0°C (bath temperature), and then add one portion hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The mixture was stirred at 70 to 90°C for 1-4 hours to obtain a transparent solution. The reaction mixture control TLC [silica gel 60 on aluminum foil, elution with a mixture of acetone-Meon (3:2)]. The mixture is cooled to room temperature and added dropwise with stirring diethyl ether (50 ml). Filtration gives the crude ester; excess alcohol can be regenerated from the filtrate.

The crude ester purified flash chromatography on a column of 150 to 200 × 25 mm silica gel 60, elwira acetonitrile (250 ml) and 5-10% Meon in acetonitrile (500-1000 ml). The fractions containing the product is evaporated, the residue is washed with ether and dried at 30-40°and 0.2 mm Hg

This method are the following esters:

Ethyl 5-amino-4-oxopentanenitrile

From ethanol (6.0 ml) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 70°C. the Reaction is completed within 2 hours. Yield 1.0 g (85%).

1H NMR (200 MHz, DMSO-d6) δ: 1,19 (3H, t, J=7.2 Hz), of 2.54 (2H, t, J=6.3 Hz), and 2.83 (2H, t, J=6.6 Hz), of 3.96 (2H, CL), of 4.05 (2H, q, J=7.0 Hz), charged 8.52 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 14,0, 27,1, 34,2, 46,4, 60,0, 171,9, 202,4.

1-Methylpentyl 5-amino-4-oxopentanenitrile [compound 1]

2-hexanol (0 ml) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 70°C. the Reaction is finished after about 4 hours. Yield 1.0 g (66%).

1H NMR (200 MHz, DMSO-d6) δ: of 0.87 (3H, t, J=6.5 Hz)and 1.15 (3H, d, J=6.2 Hz), 1,25 (4H, m)and 1.51 (2H, m), of 2.51 (2H, t, J=6.5 Hz), of 2.81 (2H, t, J=6.6 Hz), 3.95 to (2N, c), 4,78 (1H, m, J=6.5 Hz), of 8.47 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 13,6, 19,4, 21,6, 26,6, 27,0, 33,8, 34,4, 44,9, 69,6, 169,5, 200,1.

3-Hexyl 5-amino-4-oxopentanenitrile [compound 12]

3-hexanol (6,0 ml) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 100°C. the Reaction is finished after about 2 days. Yield 0.87 g (58%) of yellow oil.

1H NMR (200 MHz, DMSO-d6) δ: or 0.83 (3H, t, J=7.4 Hz), 0,86 (3H, t, J=7.2 Hz), 1.27mm (2H, m, J=7,7 Hz)to 1.48 (4H, m, J=7.5 Hz), of 2.54 (2H, t, J=6.3 Hz), and 2.83 (2H, t, J=6.0 Hz), 3.95 to (2N, c), to 4.73 (1H, m, J=5,9 Hz), 8,55 (3H, SHS).

13With NMR (50 MHz, DMSO-d6) δ: 9,3, 13,6, 17,8, 26,2, 26,8, 33,9, 34,8, 45,9, 73,7, 169,8, 200,1.

2-Methyl-1-pentyl 5-amino-4-oxopentanenitrile [compound 13]

From 2-methyl-1-pentanol (6,0 ml) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 70°C. the Reaction is finished after about 3.5 hours. The output of 1.43 g (95%).

1H NMR (200 MHz, DMSO-d6) δ: 0,85-0,90 (6N, m), 1.1 to 1.4 (4H, m), 1,74 (1H, m), of 2.56 (2H, t, J=6,7 Hz), 2,84 (2H, t, J=6.6 Hz), 3.75 to 3,9 (2H, m), of 3.95 (2H, CL), charged 8.52 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 13,9, 16,4, 19,1, 26,7, 31,3, 33,9, 34,5, 45,9, 67,9, 170,0, 200,1.

4-Methyl-1-pentyl 5-amino-4-oxopent atherogenic [compound 8]

From 4-methyl-1-pentanol (6,0 ml) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 70°C. the Reaction is completed within 2 hours. The output of 1.32 g (87%).

1H NMR (200 MHz, DMSO-d6) δ: 0,87 (6N, d, J=6.6 Hz), 1,15-1,25 (2H, m), 1,45-of 1.65 (3H, m)to 2.55 (2H, t, J=6.5 Hz), and 2.83 (2H, t, J=6.4 Hz), of 3.96 (2H, CL), to 3.99 (2H, t, J=6.8 Hz), 8,53 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 22,0, 22,2, 25,6, 26,7, 26,8, 34,0, 45,9, 63,5, 169,9, 200,1.

3,3-Dimethyl-1-butyl 5-amino-4-oxopentanenitrile [compound 16]

From 3,3-dimethyl-1-butanol (5.0 g, 49 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 70°C. the Reaction is finished after 4 hours. The output of 0.91 g (60%), TPL 146-148°C.

1H NMR (200 MHz, DMSO-d6) δ: 0,91 (N,) and 1.51 (2H, t, J=7,6 Hz), 2,53 (2H, t, J=6.2 Hz), and 2.83 (2H, t, J=6.2 Hz), of 3.96 (2H, CL), 4,07 (2H, t, J=7.0 Hz), 8,54 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,0, 29,3, 29,34, 34,3, 46,4, 61,5, 171,9, 202,4.

Cyclohexyl 5-amino-4-oxopentanenitrile [compound 7]

From cyclohexanol (6,0 ml) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 80°C. thionyl chloride added at room temperature. The reaction is finished after 3 hours. Yield 1.48 g (99%).

1H NMR (200 MHz, DMSO-d6) δ: 1,1-1,5 (6N, m), of 1.5-1.9 (4H, m), 2,52 (2H, t, J=6.3 Hz), 2,82 (2H, t, J=6.3 Hz), 3,95 (2H, CL), with 4.64 (1H, m), charged 8.52 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 22,8, 24,5, 27,1, 30,6, 33,9, 459, 71,2, 169,3, 200,1.

2-Phenylethyl 5-amino-4-oxopentanenitrile [compound 5]

2-phenylethanol (5.0 g, 41 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 70°C. the Reaction is finished after 3 hours. The output of 1.43 g (88%).

1H NMR (200 MHz, DMSO-d6) δ: of 2.50 (2H, t, J=6.4 Hz), 2,82 (2H, t, J=6.4 Hz), 2,89 (2H, t, J=7.0 Hz), 3,95 (2H, CL), to 4.23 (2H, t, J=7,0 Hz), and 7.1 to 7.4 (5H, m).

13With NMR (50 MHz, DMSO-d6) δ: 24,6, 28,0, 35,2, 47,4, 65,5, 127,2, 129,2, 129,7, 172,8, 203,3.

4-Phenyl-1-butyl 5-amino-4-oxopentanenitrile [compound 14]

From 4-phenyl-1-butanol (5.0 g, 33 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 70°C. the Reaction is terminated after 29 hours. Yield 1.22 g (68%).

1H NMR (200 MHz, DMSO-d6) δ: 1,60 (4H, CL), 2,5-2,6 (4H, m), 2,84 (2H, t, J=6.4 Hz), of 3.97 (2H, CL), 4,0 (2H, m), 7,15-to 7.35 (5H, m), 8,56 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,0, 27,2, 27,6, 34,3, 34,6, 46,4, 63,8, 125,6, 128,2, 141,8, 171,9, 202,4.

2-Methoxyethyl 5-amino-4-oxopentanenitrile [compound 20]

2-methoxyethanol (5.0 g, 66 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 70°C. the Reaction is finished after 1 hour. The output of 1.25 g (93%) of a yellowish oil.

1H NMR (200 MHz, DMSO-d6) δ: to 2.57 (2H, t, J=6.2 Hz), and 2.83 (2H, t, J=6.2 Hz), with 3.27 (3H, s), of 3.54 (5H, CL), of 3.95 (2H, CL), of 4.12 (2H, CL), charged 8.52 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 3,3, 46,5, 58,0, 63,2, 69,6, 172,0, 202,5.

3,6-Dioxa-1-octyl 5-amino-4-oxopentanenitrile [compound 23]

Of monoethylene ether of diethylene glycol (5.0 g, 37 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 70°C. the Reaction is finished after 4 hours. The output of 0.90 g (53%) of light tan solid.

1H NMR (200 MHz, DMSO-d6) δ: of 1.10 (3H, t, J=7.0 Hz), 2,58 (2H, t, J=6.0 Hz), 3,35-the 3.65 (8H, m), of 3.96 (2H, CL), of 4.13 (2H, t, J=4, 2 Hz), charged 8.52 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 15,0, 27,1, 34,3, 46,5, 63,4, 65,5, 68,1, 69,1, 69,8, 172,0, 202,5.

3,6,9-Trioxa-1-decyl 5-amino-4-oxopentanenitrile [compound 25]

Of monoethylene ether of triethylene glycol (5.0 g, 30 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) at 70°C. the Reaction is completed within 20 hours. Yield 1.19 g (63%) of a brownish oil.

1H NMR (200 MHz, DMSO-d6) δ: of 2.58 (2H, t, J=6.2 Hz), 2,84 (2H, t, J=6,4 Hz)at 3.25 (3H, s), 3,4-of 3.65 (10H, m), of 3.96 (2H, CL), of 4.13 (2H, m), 8,49 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 34,3, 46,5, 58,0, 63,4, 68,1, 69,5, 69,6, 69,7, 71,2, 171,9, 202,5.

A General method II

The mixture of alcohol (5.0 g) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol) was heated to 90-100°With (up to 135°if you want to melt alcohol) and add 12M hydrochloric acid (0.1 ml). The heat continue to obtain a clear rest the RA (5-6 days). Treated as in the previous method.

This method are the following esters:

Benzyl 5-amino-4-oxopentanenitrile [compound 4]

Of benzyl alcohol (50 ml) and hydrochloride 5-amino-4-oxopentanoic acid (10.0 g, 60 mmol). After 23 h at 90°With distilled off excess benzyl alcohol at 90°C (bath temperature) and 0.33 mm Hg Output of 8.1 g (53%) of reddish-brown powder.

1H NMR (200 MHz, DMSO-d6) δ: 2,63 (2H, t, J=6.5 Hz), 2,87 (2H, t, J=6.5 Hz), 3,98 (2H, CL), 5,11 (2N, c), 7.3 to 7.4 (5H, CL), charged 8.52 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 26,7, 33,8, 45,9, 64,8, 126,3, 126,4, 126,8, 134,4, 169,8, 200,1.

4-Nitrobenzyl 5-amino-4-oxopentanenitrile [compound 11]

4-nitrobenzyl alcohol (5.0 g, 33 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is terminated after 15 min at 135°C. the Yield of 0.95 g (52%).

1H NMR (200 MHz, DMSO-d6) δ: of 2.72 (2H, t, J=5.8 Hz), only 2.91 (2H, t, J=6.2 Hz), was 4.02 (2H, CL), 5,28 (2N, c), to 7.67 (2H, d, J=8.0 Hz), of 8.25 (2H, d, J=9.1 Hz), 8,58 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 34,3, 46,5, 64,4, 123,5, 128,3, 143,9, 146,9, 171,8, 202,5.

4-Terbisil 5-amino-4-oxopentanenitrile [compound 15]

4-fermentelos alcohol (5.0 g, 40 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is finished after 3 hours at 90°C. the Yield of 1.02 g (62%).

1N the Mrs (200 MHz, DMSO-d6) δ: 2,63 (2H, t, J=6.4 Hz), is 2.88 (2H, t, J=6.6 Hz), 3,99 (2H, CL), 5,10 (2N, c), 7,22 (2H, t, J=8,8 Hz), was 7.45 (2H, d, J=8.0 Hz), to 8.57 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 34,3, 46,5, 64,9, 115,0, 115,4, 130,1, 130,2, 132,2, 132,3, 159,3, 164,2, 171,8, 202,4.

4-Methylbenzyl 5-amino-4-oxopentanenitrile [3]

4-methylbenzylamino alcohol (5.0 g, 41 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is finished after 2 days. Yield 0.84 g (52%).

1H NMR (200 MHz, DMSO-d6) δ: 2,30 (3H, s), 2,60 (2H, t, J=6.4 Hz), of 2.86 (2H, t, J=6.2 Hz), of 3.96 (2H, CL), of 5.05 (2H, s), 7,1-7,3 (4H, m) 8,55 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 21,7, 28,1, 35,2, 47,4, 66,4, 128,9, 129,8, 133,9, 138,2, 172,8, 203,4.

4-Isopropylbenzyl 5-amino-4-oxopentanenitrile [compound 2]

4-isopropylbenzyl alcohol (5.0 ml) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is finished after 2 days. Yield 1.0 g (56%).

1H NMR (200 MHz, DMSO-d6) δ: 1,19 (6N, d, J=6.6 Hz), 2,61 (2H, t, J=6.4 Hz), 2,84 (1H, m), is 2.88 (2H, t, J=6,8gts), 3,98 (2H, CL), is 5.06 (2H, s), 7,2-7,4 (4H, m), 8,56 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 23,7, 27,1, 33,1, 34,3, 46,4, 65,5, 126,2, 128,0, 133,3, 148,2, 171,8, 202,6.

4-tert-Butylbenzyl 5-amino-4-oxopentanenitrile [compound 10]

From 4-tert-butylbenzyl alcohol (5.0 g, 30 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is finished the via 2 days. Yield 0.84 g (52%).

1H NMR (200 MHz, DMSO-d6) δ: 1,27 (N, C), 2,61 (2H, t, J=6.4 Hz), 2,87 (2H, t, J=6.6 Hz), 3,98 (2H, CL), is 5.06 (2H, s), 7,29 (2H, d, J=8,4 Hz), 7,39 (2H, d, J=8,4 Hz), 8,55 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 31,0, 34,16, 34,26, 46,4, 65,4, 125,0, 127,7, 133,0, 150,4, 171,8, 202,4.

4-(Trifluoromethyl)benzyl 5-amino-4-oxopentanenitrile [compound 9]

From 4-(trifluoromethyl)benzyl alcohol (4.9 g, 28 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is completed within 2 hours. Yield 1.24 g (64%).

1H NMR (200 MHz, DMSO-d6) δ: 2,69 (2H, t, J=6.2 Hz), of 2.92 (2H, t, J=6.4 Hz), 4,00 (2H, CL), 5,23 (2N, c), a 7.62 (2H, d, J=8,2 Hz), of 7.75 (2H, d, J=8,2 Hz), 8,58 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 34,4, 46,5, 64,7, 121,4, 125,2, 125,3, 125,4, 126,9, 128,2, 140,9, 171,8, 202,5.

2-Terbisil 5-amino-4-oxopentanenitrile [compound 17]

2 fermentelos alcohol (5.7 g, 45 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is terminated after 27 hours at 100°C. the Yield 0.64 g (44%). TPL 91-94°C (decomposition).

1H NMR (200 MHz, DMSO-d6) δ: 2,63 (2H, t, J=6.0 Hz), is 2.88 (2H, t, J=6.0 Hz), 3,98 (2H, CL), 5,16 (2N, c)of 7.2 and 7.6 (4H, m), 8,56 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,0, 34,3, 46,4, 59,7, 59,8, 115,1, 115,5, 122,7, 123,0, 124,5, 130,4, 130,6, 130,7, 130,8, 157,8, 162,7, 171,8, 202,4.

3-Terbisil 5-amino-4-oxopentanenitrile [compound 24]

3-verbenia the CSOs alcohol (5,1 g, 40 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is finished after 20 hours at 100°C. the Yield 1.04 g (72%). TPL 115-119°C.

1H NMR (200 MHz, DMSO-d6) δ: to 2.67 (2H, t, J=6.2 Hz), 2,90 (2H, t, J=6.2 Hz), 4,00 (2H, d, J=5.0 Hz), 5,14 (2H, s), 7,1-7,3 (3H, m), between 7.4 to 7.5 (1H, m), 8,54 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 34,3, 46,5, 64,7, 114,1, 114,5, 114,9, 123,1, 123,6, 130,4, 130,5, 138,8, 139,0, 159,6, 164,5, 171,8, 202,5.

2,3,4,5,6-Pentafluorobenzyl 5-amino-4-oxopentanenitrile [compound 18]

From 2,3,4,5,6-pentafluorobenzyl alcohol (5.1 g, 26 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is finished after 6 days at 100°C. the Yield 0.25 g (13%). TPL 146-148°C.

1H NMR (200 MHz, DMSO-d6) δ: 2,59 (2H, t, J=6.4 Hz), 2,85 (2H, t, J=6.4 Hz), 3,95 (2H, CL), 5,22 (2N, c), 8,51 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 26,8, 34,2, 46,5, 53,2, 109,9, 123,2, 134,5, 139,5, 142,7, 147,7, 171,6, 202,4.

4-Chlorbenzyl 5-amino-4-oxopentanenitrile [compound 19]

4-chlorobenzylamino alcohol (5.0 g, 35 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is finished after 24 hours at 100°C. the Yield 0.56 g (32%). TPL 127-129°C.

1H NMR (200 MHz, DMSO-d6) δ: to 2.65 (2H, t, J=5.8 Hz), 2,89 (2H, t, J=6.0 Hz), 3,99 (2H, CL), 5,11 (2N, c), 7,44 (4H, s), 8,56 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 34,3, 46,5, 64,7, 128,4, 129,7, 132,6, 135,1, 171,8, 202,.

3,4-Dichlorobenzyl 5-amino-4-oxopentanenitrile [compound 22]

From 3,4-amyl-metacresol (5.0 g, 28 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is finished after 45 hours at 100°C. the Yield 1.12 g (57%).

1H NMR (200 MHz, DMSO-d6) δ: to 2.67 (2H, t, J=6.4 Hz), 2,90 (2H, t, J=6.4 Hz), 4,00 (2H, CL), 5,12 (2N, c), 7,2-7,5 (1H, m), and 7.5 and 7.6 (2H, m), and 8.50 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 34,3, 46,7, 64,1, 129,8, 130,7, 137,3, 173,7, 202,8.

3-Nitrobenzyl 5-amino-4-oxopentanenitrile [compound 21]

3 nitrobenzyl alcohol (5.0 g, 33 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is finished after 20 hours at 100°C. the Yield of 1.00 g (55%).

1H NMR (200 MHz, DMSO-d6) δ: of 2.68 (2H, CL), 2,90 (2H, CL), 3,98 (2H, CL), 5,26 (2N, c), and 7.6 to 7.9 (2H, m), 8,1-8,3 (2H, m), of 8.47 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 34,3, 46,5, 64,4, 122,3, 122,8, 130,1, 134,3, 138,4, 147,7, 171,8, 202,5.

2-methylbenzyl 5-amino-4-oxopentanenitrile [compound 29]

2 methylbenzylamino alcohol (5.0 g, 41 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is finished after 4 days at 100°C. Output 0,72 g (44%). TPL 107-109°C.

1H NMR (200 MHz, DMSO-d6) δ: to 2.29 (3H, c), 2,62 (2H, t, J=6.4 Hz), of 2.86 (2H, t, J=6.4 Hz), of 3.96 (2H, CL), 5,10 (2N, c), 7,2-7,4 (4H, m), 8,54 (3H, CL).

13With NMR (50 MHz, DMSO-d6 ) δ: 18,4, 27,0, 34,3, 46,4, 64,1, 123,1, 125,8, 128,2, 128,8, 130,0, 133,8, 136,5, 171,8, 202,5.

3-Methylbenzyl 5-amino-4-oxopentanenitrile [compound 31]

3 methylbenzylamino alcohol (5.0 g, 40 mmol) and hydrochloride 5-amino-4-oxopentanoic acid (1.0 g, 6.0 mmol). The reaction is finished after 2 days at 80°C. the Yield of 1.11 g (68%). TPL 96-98°C.

1H NMR (200 MHz, DMSO-d6) δ: 2,31 (3H, c), 2,62 (2H, t, J=6.2 Hz), 2,87 (2H, t, J=6.4 Hz), of 3.97 (2H, CL), is 5.06 (2H, c), and 7.1 to 7.4 (4H, m), 8,55 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 20,9, 27,1, 34,3, 46,4, 65,6, 124,9, 128,3, 128,4, 128,5, 135,9, 137,5, 171,9, 202,5.

A General method III

Benzyl alcohol (5.0 mmol) is added drop by drop to a mixture of N,N’-diisopropylcarbodiimide (0,63 g, 5.0 mmol) and copper chloride (I) (1 mg), cooled to 0°C (bath temperature). After 1 hour at 0°the mixture is stirred for about 24 hours at room temperature. The dark green mixture was diluted with pentane (3 ml) and filtered through a layer of Celite. The residue is washed with a small amount of pentane and the combined filtrates evaporated. The residue is dissolved in dry tetrahydrofuran and added dropwise N-tert-BOC-5-amino-4-oxopentanoic acid (0.43 mmol) in dry dichloromethane (10 ml). After 5 days at room temperature the mixture is filtered and the residue washed with a small amount of diethyl ether. The residue is dissolved in ethyl acetate (20 ml) and treated with a solution of hydrogen chloride in etelaat is those (2 ml). The precipitated hydrochloride appears after 4 hours up to 3 days and washed as described below.

N-tert-BOC-5-amino-4-oxopentanoic acid

The triethylamine (13,9 ml, 10.1 g, 0.10 mmol) is added drop by drop to a stirred mixture of the hydrochloride of 5-amino-4-oxopentanoic acid (10.0 g, 59,7 mmol) and di-tert-BUTYLCARBAMATE (21.8 g, 0.10 mol) in dry N,N-dimethylformamide (25 ml). The reaction mixture is stirred for 6 hours at 50°and over night at room temperature. The solvent is evaporated at 35-40°C (bath temperature) and 7-1 mm Hg, the Residue is acidified chilled on ice for 10% citric acid (50 ml) and immediately extracted with ethyl acetate (6×15 ml). The combined extracts washed with water (2×5 ml) and saturated NaCl (1×5 ml). After drying (Na2SO4in over night in the refrigerator, the mixture is filtered and evaporated. Red oil is purified flash chromatography on a column of 50×60 mm silica gel, elwira a mixture of ethyl acetate-hexane (2:1), collecting 50 ml fractions, receiving 12.2 g (88%) of a viscous yellow oil which partially hardens when stored in the refrigerator.

1H NMR (200 MHz, DMSO-d6) δ: 1,39 (N, c), 2,42 (2H, t, J=6.4 Hz), 2,63 (2H, t, J=6.4 Hz), of 3.78 (2H, d, J=5.8 Hz),? 7.04 baby mortality (1H, t, J=5.6 Hz), 12,1 (1H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,4, 28,1, 38,6, 49,5, 78,1, 155,7, 173,6, 206,1.

4-Methoxybenzyl 5-amino-4-oxopentanenitrile [with the unity 28]

Obtained from 4-methoxybenzylthio alcohol (1.0 g, 7.2 mmol), N,N’-diisopropylcarbodiimide (0,91 g, 7.2 mmol), CuCl (9 mg) and N-tert-BOC-5-amino-4-oxopentanoic acid (1.7 g, 7.2 mmol). Flash chromatography on a column of 175×25 mm silica gel 60 with elution by acetonitrile (200 ml), 5% methanol in acetonitrile (500 ml) and 10% methanol in acetonitrile (750 ml) and collecting 50 ml fractions gives 0,24 mg (11%) of product. So pl. 110-112°C.

1H NMR (200 MHz, DMSO-d6) δ: of 2.58 (2H, t, J=6.4 Hz), and 2.83 (2H, t, J=6,4 Hz in), 3.75 (3H, c), of 3.94 (2H, CL), 5,02 (2N, c), 6,93 (2H, d, J=8,4 Hz), 7,31 (2H, d, J=8,4 Hz), 8,48 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 34,2, 46,4, 55,0, 65,4, 113,7, 127,9, 129,8, 159,1, 171,8, 202,5.

3-Pyridinylmethyl 5-amino-4-oxopentanenitrile [compound 26]

3-pyridinemethanol (0.55 g, 5.0 mmol) and 0.43 m solution of N-tert-BOC-5-amino-4-oxopentanoic acid in dichloromethane (10 ml, 4.3 mmol). Evaporation of the solvent from the filtrate gives 1.0 g of residue which is dissolved in ethyl acetate. A solution of hydrogen chloride in ethyl acetate (2 ml) is added to the yellow solution. After 4 hours at room temperature, the residue is filtered off and dried over silica gel at 30°and 0.01 mm Hg, getting to 0.63 g (68%) of a hygroscopic solid.

1H NMR (200 MHz, DMSO-d6) δ: 2,69 (2H, t, J=6.4 Hz), only 2.91 (2H, t, J=6.4 Hz), 3,9-4,0 (2H, m), 5,35 (2N, c), 8,0-8,2 (1H, m), 8,54 (3H, CL), 8,5-8,7 (1H, m), 8,9-9,0 (2H, m).

With NMR (50 MHz, DMSO-d6) δ: 27,0, 34,3, 46,4, 61,9, 126,9, 140,8, 141,3, 144,4, 171,7, 202,5.

4-Diphenylmethyl 5-amino-4-oxopentanenitrile [compound 27]

4-diphenylethanol (5.0 g, 40 mmol) and a solution of N-tert-BOC-5-amino-4-oxopentanoic acid (10 ml, 4.3 mmol). The crude hydrochloride is purified flash chromatography on a column of 190×25 mm silica gel 60 with elution by acetonitrile (300 ml), 10% methanol in acetonitrile (1000 ml) and 20% methanol in acetonitrile (250 ml), collecting 50 ml fractions. Yield 0.39 g (16%). TPL 163-166°C.

1H NMR (200 MHz, DMSO-d6) δ: to 2.65 (2H, t, J=6.4 Hz), is 2.88 (2H, t, J=6.4 Hz), 3,98 (2H, CL), 5,16 (2N, c), a 7.2 to 7.5 (5H, m), of 7.6 to 7.8 (4H, m), charged 8.52 (3H, CL).

13With NMR (50 MHz, DMSO-d6) δ: 27,1, 34,3, 46,4, 65,3, 126,6, 126,7, 127,5, 128,5, 128,9, 135,2, 139,6, 139,8, 171,9, 202,5.

Benzyl 5-[[1-(atomic charges)ethoxy]carbonyl]amino-4-oxopentanoate [connection 30]

Pyridine (of 0.32 ml, 0.32 g, 4.0 mmol) is added drop by drop to a stirred mixture of benzyl 5-amino-4-oxopentanenitrile (0.52 g, 2.0 mmol) and 1-chloroethylphosphonic (0,29 g, 2.0 mmol) in dry tetrahydrofuran (10 ml). After stirring for 2 hours at room temperature the mixture is washed with water (1 x 2 ml) and saturated NaCl (1×2 ml) and dried (MgSO4). Filtration and evaporation give 0.65 g of amber oil. This oil is dissolved in glacial acetic acid (10 ml) and add a acetate mercury (II) (0.64 g, 2.0 mmol). the donkey stirring for 3 days at room temperature the excess acetic acid is evaporated at about 30° C (bath temperature) and 8-10 mm Hg, the Residue is stirred with diethyl ether (10 ml) and filtered. After washing the residue with ether (15 ml) and the combined filtrates are neutralized NaHCO3and washed with saturated NaCl solution (1×10 ml). After drying (MgSO4), filtration and evaporation the crude product was then purified flash chromatography on a column of 190×25 mm silica gel 60, elwira a mixture of ethyl acetate-hexane (2:1) (500 ml), collecting 25 ml fractions. The yield of 0.43 g (61%) of light yellow oil.

1H NMR (200 MHz, DMSO-d6) δ: of 1.41 (3H, d, J=5.6 Hz), from 2.00 (3H, s), to 2.57 (2H, t, J=6.0 Hz), 2,73 (2H, t, J=6.0 Hz), 3,90 (2H, d, J=6.0 Hz), to 5.08 (2H, s), of 6.68 (1H, q, J=5.4 Hz), was 7.36 (5H, s), 7,71 (1H, t, J=5.8 Hz).

13With NMR (50 MHz, DMSO-d6) δ: 19,6, 20,6, 27,3, 33,7, 49,5, 65,5, 88,8, 127,8, 127,9, 128,4, 136,1, 154,2, 168,6, 172,0, 205,1.

Example 2 - Determining formation of porphyrin in cell culture in vitro

Ways

The following compounds have been studied when compared with ALA:

1. 1-methylpentylamine ether ALA

2. pair-isopropylbenzylamine ether ALA

3. pair-methylbenzylamine ether ALA

4. benzyl ALA ester

5. 2-phenethyl ester ALA

6. hexyl ester of ALA

7. cyclohexyloxy ether ALA

8. 4-methylpentanoic ether ALA

9. steam-[trifluoromethyl]benzyl ALA ester

10. steam-[tert-butyl]benzyl ALA ester

11. pair-nitrobenzyloxy ether ALA

The compounds are dissolved in DMSO to a concentration of the emission 100 mm (basic solution). The desired concentration is obtained by dilution of the stock solution with saline phosphate buffer (PBS) or culture medium.

Cultivation of cells

The WiDr cells derived from primary adenocarcinoma of the rectosigmoid Department of the colon, subcultured in medium RPMI 1640 (Gibco)containing 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 1% glutamine. Cells are split 1:100 twice a week and maintained at 37°C and 5% CO2in a wet environment.

The machining conditions

5×105of WiDr cells in 2 ml of RPMI medium, described in the previous paragraph, add to each well of a 6-hole plastic tablet for cultivation of tissues (Nunc) and left for 48 hours at 37°C and 5% CO2in a humid environment for the proper adherence to the substrate. The cells are washed twice with RPMI medium 1640 containing no serum and then added to the wells with the appropriate dilutions of the compounds under study in 2 ml of fresh nutrient medium to the final concentrations of 0.001, 0.01, 0.1, and 1 mm in duplicate. Cells are incubated at 37°C for four hours.

Determination of porphyrins in cells

After the treatment, described in the paragraph “terms of processing, the cells are washed twice by PBS and transferred into a 1 M solution HCl450% Meon, scraping the cells from the substrate when p is power cell scraper Costarer. Cellular debris is removed by centrifugation. The porphyrin concentration in each sample determine fluorimetrically using spectrofluorimeter Perkin Elmer LS50B (excitation at 407 nm, emission measured at 606 nm using a filter that cuts off the passage of wavelengths (530 nm) of the emission). Calculate the fluorescence of each sample relative to the protein content in control cells, as measured by the method of Bradford.

Results

The results for the studied compounds 1-3, 4 and 5, 6-8 and 9-11 are shown on the drawings 1-4, respectively.

On figure 1 you can see that the steep curves dose-response found for the pair-methylbenzylamino and para-isopropylbenzyl esters. These esters are equally effective in the induction of cellular synthesis of porphyrin and about 200 times more effective than ALA. The curve for 1-methylpentylamino ether increases slowly with increasing concentration of the ether. 1-Methylpentylamine ether at low concentrations only slightly better than ALA in inducing the formation of porphyrin.

From figure 2 one can see that the benzyl and 2-phenethyl esters significantly more effective than ALA in the induction of cellular synthesis of porphyrin. Indeed, the formation of porphyrin-induced 0.001 mm benzyl ether, is the formation of porphyrin obtained at 0.6 mm ALA. Similarly, we can see that h is of the fluorescence after addition of 0.001 mm 2-phenethyl ester corresponds to fluorescence, caused 0.4 mm ALA.

The figure 3 shows that the hexyl and 4-methylpentanoic esters are equally effective. From the figure you can see that the ability of these esters to induce the formation of cell porphyrin about 100 times higher than that of ALA. Cyclohexyloxy ether at low concentrations only slightly more effective than ALA in inducing the formation of porphyrin. At high concentrations cyclohexyloxy ether less effective than ALA.

From figure 4 one can see that the pair-[trifluoromethyl]benzyl, para-[tert-butyl]benzyl and para-nitrobenzoyl esters is approximately 200 times better than ALA in inducing the formation of porphyrin.

The results can be summarized as follows:

Table 1
Connection (i.e. ester side chain) [no connection]Efficiency*
ALA1
1-methylpentyl [1]3
4-methylpentyl [8]100
2-phenylethyl [5]500
benzyl [4]100-600
hexyl [6]100
para-isopropylbenzyl [2]200
para-methylbenzyl [3]200
cyclohexyl [7]1/td>
para-nitrobenzyl [11]200
steam-[trifluoromethyl]benzyl [9]200
steam-[tert-butyl]benzyl [10]200
* the relative ability to induce the formation of porphyrin

Example 3 - the Formation of porphyrin after local application on the skin of rats

The ways. The following compounds were investigated when compared with ALA (including compounds 1-11, studied in example 2)

1. 1-methylpentylamine ether ALA

2. pair-isopropylbenzylamine ether ALA

3. pair-methylbenzylamine ether ALA

4. benzyl ALA ester

5. 2-phenethyl ester ALA

6. hexyl ester of ALA

7. cyclohexyloxy ether ALA

8. 4-methylpentanoic ether ALA

9. steam-[trifluoromethyl]benzyl ALA ester

10. steam-[tert-butyl]benzyl ALA ester

11. pair-nitrobenzyloxy ether ALA

12. 1-ethylbutylamine ether ALA

13. 2-methylpentylamine ether ALA

14. 4-privately ether ALA

15. pair-tormentingly ether ALA

16. 3,3’-dimethyl-1-butyl ether ALA

17. 2-tormentingly ether ALA

18. 2,3,4,5,6-pentafluorobenzoyl ether ALA

19. 4-chlorbenzoyl ether ALA

20. 2-methoxyethylamine ether ALA

21. 3-nitrobenzyloxy ether ALA

22. 3,4-[dichloro]benzyl ALA ester

23. 3,6-dioxa-1-oktilovom ether ALA

24. 3-tormentingly ether ALA

25. 3,6,9-trioxa-1-decroly ether LA

26. 3-pyridinylmethyl ether ALA

27. 4-diphenylmethylene ether ALA

28. 4-methoxybenzyloxy ether ALA

29. 2-methylbenzylamine ether ALA

30. benzyl 5-[(1-acetylacetone)carbonyl]aminolevulinat

31. 3-methylbenzylamine ether ALA

Ways

The compounds prepared in Unguentum Merck (based cream from Merck consisting of silicon dioxide, paraffin oil, vaseline, albumin (album), sitosterol, Polysorbate 40, glycerylmonostearate, Miglyol®812 (a mixture of vegetable fatty acids), polypropylenglycol and purified water) with the desired concentration. Concentrations expressed in % (mass./mass.) based on the hydrochloride. Drugs get the day before the experiment and stored in the refrigerator until the day of use. The study used female Nude naked mice Balb/weight 22 g, obtained from the Department of Laboratory Animals, The Norwegian Radium Hospital Montebello, Oslo, Norway).

Using three mice per group. Each mouse receives 0.05-0.1 g of the drug is applied locally on the left side of the body, evenly distributed and covered with a dressing (Opsite Flexigrid; Smith and Nephew Medical Ltd., Hull, England). Optic device for measuring point consists of a bundle of optical fibers attached to the an, which gives exciting light at 407 nm. Exciting light, which can penetrate 0.1-0.5 mm in tissue, spend over half of the fibers to the same mouse. The obtained emission spectrum of fluorescence (550-750 nm) collect and spend on the remaining fibers on the photomultiplier for the quantitative determination.

After application of the drug in the form of creams measured fluorescence spectrum of porphyrins in the skin fiber way through different intervals of time after application.

The intensity of fluorescence varies from experiment to experiment. Therefore, in each experiment consists of 1% (wt./mass.) hexyl 5-aminolevulinat HCl as an external control.

Results

In figures 5-7 shows the results analyzed for the following compounds: figure 5 - connection 1, 6, 12, and 13; figure 6 - connection 5-8; figure 7 - connections 3 and 6; figure 8 - connections 2, 6 and 14; figure 9 - connection 6, 11 and 15; figure 10 - connection 6, 9, and 10; figure 11 - connection 4 and 6; figure 12 - connection 6 and 16; figure 13 - connection 6 and 17-19; figure 14 - connection 6 and 20-22; figure 15 connection 6 and 23-25; figure 16 - connection 6 and 26-28; figure 17 connection 6 and 29-31. All connections used in 1% (wt./mass.) except for figures 12 and 17, where 3,3’-dimethyl-1-butyl ALA ester, 2-methylbenzylamine the ALA ester, benzyl-5-[(acetylacetone)carbonyl]new ester of ALA and 3-methylbenzylamine ester of ALA used at a concentration of 3% (wt./mass.), and figure 13, where 2-tormentingly the ALA ester, 2,3,4,5,6-pentafluorobenzoyl the ALA ester and 4-chlorbenzoyl EPE the ALA is applied at 10% (wt./mass.).

From figure 5 we can see that hexyl, 1-methylpentylamine, 1-ethylbutylamine and 2-methylpentylamine esters give all the formation of porphyrins. In addition, hexyl ester gives the greatest fluorescence, followed by 2-methylpentylamine and 1-ethylbutylamine esters. 1-Methylpentylamine ether gives an intermediate level of fluorescence in this study.

In figure 6 it is shown that the hexyl ester and 4-methylpentanoic ether to give high levels of fluorescence, whereas cyclohexylamine and 2-phenethyl esters give low levels of fluorescence.

The figure 7 shows that as hexyl, and the pair-methylbenzylamine ALA esters give the same high fluorescence.

From figure 8 it is obvious that the pair-isopropylbenzyl 5-aminolevulinat gives intermediate levels of fluorescence, whereas 4-phenylbutyl 5-aminolevulinat gives only low levels of fluorescence. In this regard, it is interesting that the analogue of the latter substances (2-phenylbutyl 5-aminolevulinat) also gives a relatively moderate levels of fluorescence (figure 6). This shows that if benzyl 5-aminolevulinat (figure 11) “extend” one or more methylene (-CH2-) groups between benzil and ALA, the result is a reduced ability to induce the formation of porphyrin.

The figure 9 shows that the pair-nitrobenzyloxy and hexyl esters both give high ur the attention of fluorescence. A little lower to get a pair of fermentelos ether.

The figure 10 shows that the high education porphyrins (which indicate high levels of fluorescence) to get hexyl and para-[trifluoromethyl]benzyl esters. For steam-[tert-butyl]benzyl ester receive an intermediate level of fluorescence. Perhaps this is due to the rather bulky ester group.

From figure 11 it is evident that for hexyl and benzyl esters receive high levels of fluorescence.

From figure 12 it is seen that for 1% hexyl ether and 3% of the drug 3,3’-dimethyl-1-tert-butyl ester receive the same levels of fluorescence. Thus, the two data broadcast about equally effective in inducing the formation of porphyrin on the skin.

From figure 13 we can see that 4-chlorbenzoyl ether gives high levels of fluorescence, whereas 2,3,4,5,6-pentafluorobenzoyl and 2-tormentingly esters give intermediate/high levels of fluorescence.

From figures 14 and 15 we can see that all the investigated esters of ALA, non-hexyl ether, to give the intermediate fluorescence of the skin after local application.

From figure 16 it is obvious that hexyl ether gives high levels of fluorescence, and 3-pyridinylmethyl and 4-methoxybenzyloxy esters give intermediate fluorescence. 4-Diphenylmethyl the th ether gives a relatively low fluorescence.

From figure 17 we can see that N-substituted benzyl ALA ester gives intermediate/high fluorescence, while others investigated three ether to give high levels of fluorescence.

The results can be summarized as follows:

Table 2
Connection (i.e. ester side chain) [no connection]The level of fluorescence
LowElapsed.High
1-methylpentyl [1]X
2-methylpentylamine [13]X
4-methylpentyl [8]X
1-ethylbutyl [12]X
hexyl [6]X
3,3-dimethyl-1-butyl [16]X
2-phenylethyl [5]X
4-phenylbutyl [14]X
benzyl [4]X
para-isopropylbenzyl [2]X
Para-methylbenzyl [3]X
para-nitrobenzyl [11]X
steam-[trifluoromethyl]benzyl [9]X
steam-[tert-butyl]benzyl [10]X
para-terbisil [15]X
Cyclohexyl [7]X
2-terbisil [17]X
3-terbisil [24]X
2,3,4,5,6-pentafluorobenzyl [18]X
4-chlorbenzyl [19]X
3,4-[dichloro]benzyl [22]X
3-nitrobenzyl [21]X
4-methoxybenzyl [28]X
3-pyridinylmethyl [26]&x0200A; X
4-diphenylmethyl [27]X
3-methylbenzyl [31]X
2-methylbenzyl [29]X
2-methoxyethyl [20]X
3,6-dioxa-1-octyl [23]X
3,6,9-trioxa-1-decyl [25]X

Benzyl 5-[(1-acetylacetone)carbonyl]aminolevulinat [30]X

1. Connection for use in photochemotherapy or diagnosis, and the specified connection has the formula I:

R

2
2
N-CH2COCH2-CH2CO-OR1(I)

where R1represents C1alkyl group substituted by one or more aryl groups;

R2independently represents a hydrogen atom or alkoxycarbonyl,

or its pharmaceutically acceptable salt.

2. Connection on p. 1, the de-mentioned aryl group substituted by one or more alkilani (for example, C1-2alkilani), alkoxy (e.g. methoxy), fluorine atoms, chlorine, nitro or triptoreline groups.

3. The compound according to claim 1 or 2 where the specified aryl group represents a phenyl, diphenyl or monocyclic 5-7-membered heteroaromatic group, preferably phenyl.

4. The compound according to claim 3 where the specified heteroaromatic group is pyridinyl.

5. The compound according to any one of claims 1 to 4, where each R2represents a hydrogen atom.

6. The compound of the formula I:

R

2
2
N-CH2COCH2-CH2CO-OR1(I)

where R1is substituted by aryl (C1alkyl group, preferably C1alkyl group, a substituted vegetariantimes.com-aryl, where this aryl group is substituted, particularly preferably substituted by one or more alkyl (for example, C1-2alkyl), alkoxy (e.g. methoxy) groups, fluorine atoms, chlorine, nitro or triptoreline groups;

R2, each of which may be the same or different, represents a hydrogen atom or alkoxycarbonyl;

the specified alkyl group optionally interrupted by one or more groups-O-, -NR3-, -S - or-PR3-

R3represents a hydrogen atom or a C1-6alkyl group,

and its pharmaceutically acceptable salt.

7. The connection according to claim 6, where the specified aryl group represents a phenyl, diphenyl or monocyclic 5-7-membered heteroaromatic group, preferably phenyl.

8. The connection according to claim 6 or 7, where in formula I, each R2represents a hydrogen atom.

9. The connection of claim 8, where the group R1specified aryl group is phenyl.

10. The connection according to claim 6, which is selected from parameterbinding ester of 5-aminolevulinic acid, paranitroaniline ester of 5-aminolevulinic acid, pair[trifluoromethyl]benzyl ester of 5-aminolevulinic acid, performancelevel ester of 5-aminolevulinic acid, 4-chlorobenzylamino ester of 5-aminolevulinic acid, 3-methylbenzylamino ester of 5-aminolevulinic acid and 2-methylbenzylamino ester of 5-aminolevulinic acid.

11. The compound of claim 10 or benzyl ester of 5-aminolevulinic acid for use in photochemotherapy or diagnosis.

12. Method of producing compounds of the formula I according to any one of p-10, and the method includes at least one of the following stages:

(a) interaction of the compounds of formula II

R

2
2
N-CH2COCH2-CH2CO-X (II)

where X denotes a leaving group, for example, hydroxyl group, halogen atom or alkoxygroup, or X denotes a group of the acid anhydride, and R2such as defined in claim 6

with the compound of the formula III

R1-OH, (III)

where R1such as defined in claim 6;

(b) the conversion of compounds of formula I, its pharmaceutically acceptable salt.

13. Pharmaceutical composition for use in photochemotherapy or diagnosis, comprising the compound according to any one of claims 1 to 11, or its pharmaceutically acceptable salt together with at least one pharmaceutically acceptable carrier or excipient.

14. The compound according to any one of claims 1 to 11, or its pharmaceutically acceptable salt, suitable for receiving therapeutic agent used in photochemotherapy, or diagnostic agent used in the diagnosis.

15. The connection 14, where the photochemotherapy or diagnosis of conduct disorders or disorders of the internal or external surfaces of the body that are sensitive to photochemotherapy.

16. The product comprising the compound according to any one of claims 1 to 11, or its pharmaceutically acceptable salt together with at least one agent for promoting penetration through the surface, and not necessarily od is them or more chelating agents, in the form of a combined preparation for simultaneous, separate or sequential use in the treatment of disorders or disorders of the internal or external surfaces of the body that are sensitive to photochemotherapy.

17. Set for use in photochemotherapy disorders or disorders of the internal or external surfaces of the body, including

a) a first container containing a compound according to any one of claims 1 to 11, or its pharmaceutically acceptable salt;

b) a second container containing at least one agent that promotes penetration through the surface, and is optional

c) one or more chelating agents, included in said first container, or in a third container.

18. The wayin vitrodiagnosis of disorders or disorders by studying a sample of fluid or tissue of the patient, and the method includes at least the following stages:

i) mixing the specified fluid or tissues with the compound according to any one of claims 1 to 11;

ii) irradiation of this mixture of light;

iii) determining the level of fluorescence

iv) comparing the level of fluorescence with the control levels.



 

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FIELD: medicine, pharmaceutical, cosmetic and food industry.

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EFFECT: improved producing method, valuable medicinal properties of preparation.

11 cl, 3 ex

FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

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22 cl, 19 dwg, 18 tbl, 117 ex

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10 cl, 7 tbl, 6 ex

FIELD: organic chemistry, chemical technology, medicine, biochemistry, pharmacy.

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EFFECT: valuable medicinal properties of compounds.

15 cl, 7 tbl, 56 ex

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33 cl, 3 tbl, 9 dwg, 23 ex

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EFFECT: improved preparing method, enhanced and valuable medicinal properties of compounds.

2 cl, 3 ex

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