WO2019122151A1

WO2019122151A1 - Photoisomerizable derivatives of dihydrofolate reductase inhibitors

Info

Publication number: WO2019122151A1
Application number: PCT/EP2018/086233
Authority: WO
Inventors: Pablo Ignacio Gorostiza Langa; Concepció SOLER PRAT; Carlo MATERA; Nuria CAMARERO PALAO; Michela LIBERGOLI; Alexandre GOMILA JUANEDA
Original assignee: Fundació Institut De Bioenginyeria De Catalunya; Universitat De Barcelona; Consorcio Centro de Investigación Biomédica en Red, M.P.; Institució Catalana De Recerca I Estudis Avançats
Priority date: 2017-12-22
Filing date: 2018-12-20
Publication date: 2019-06-27

Abstract

The present invention relates a compound of formula (I) in Z-isomer form (Z-(l)) or a compound of formula (I) in E-isomer form (E-(l)) wherein: R2 is a radical of formula (II) and of formula (III); X1, X2, X3, X4 and X5 are CR3 and N; Y1 is NH, O and S; each R1, each R3 and R7 are H, halogen, -OH, -O-(C1-C8)alkyl, -N((C1-C8)alkyl)3, -NH((C1-C8)alkyl)2, and -NH2(C1-C8)alkyl; R4, R5 and R6 are H and (C1-C8)alkyl; - - - line indicates the position of R2 radical by which R2 is attached to the adjacent N; and the asterisk indicates a stereogenic carbon atom being in R-isomer, S-isomer and a mixture of R-isomer and S-isomer. It also relates to the compound Z-(l) for use as a medicament; and the compound E-(l) for use in the treatment of a disease or condition mediated by the dihydrofolate reductase, wherein the treatment comprises exposing a compound E-(I) to light irradiation to obtain the compound Z-(l).

Description

Photoisomerizable derivatives of dihydrofolate reductase inhibitors

This application claims the benefit of European Patent Application 17382894.8 filed on December 22th, 2017. The present invention relates to the field of the dihydrofolate reductase (DHFR) inhibitors. In particular, it relates to photoisomerizable derivatives of a folate antimetabolite such as methotrexate and pemetrexed; and to compositions containing them. It also relates to processes for their preparation and their uses in therapy.

Background Art

The dihydrofolate reductase is an enzyme which reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as the electron donor. Tetrahydrofolic acid is used for the de novo synthesis of purines, thymidylic acid, and certain amino acids. Because tetrahydrofolic acid is the active form of folate in humans, inhibition of dihydrofolate reductase can cause functional folate deficiency. However, as folate is needed by rapidly dividing cells to make thymine, this effect may be therapeutic.

Particularly, these inhibitors can be used as antiproliferative agent due to its inhibitory effect of cell divisions, such as for example in cancer chemotherapy because they can prevent cancer cells from dividing, and also in the treatment of psoriasis, rheumatoid arthritis, inflammatory diseases of the digestive tract because they can also prevent normal cells from dividing.

Particularly, methotrexate is the non-proprietary name of the compound (2S)-2-[(4-{[(2,4-diaminopteridin-6- yl)methyl](methyl)amino}benzoyl)amino]pentanedioic acid whose chemical structure is the following:

Methotrexate is an antimetabolite of the antifolate type which competitively inhibits dihydrofolate reductase (DHFR). Methotrexate was originally developed and continues to be used for chemotherapy. It is effective for the treatment of a number of cancers, including: breast, head and neck, leukemia, lymphoma, lung, osteosarcoma, bladder, and trophoblastic neoplasms. Furthermore, in low doses methotrexate is also used as a disease-modifying treatment for some autoimmune diseases, including rheumatoid arthritis, juvenile dermatomyositis, psoriasis, psoriatic arthritis, lupus, sarcoidosis, Crohn's disease eczema and many forms of vasculitis.

Besides, pemetrexed is the non-proprietary name of the compound (2S)-2-{ [4- [2-(2-a m i no-4-oxo- 1 ,7- dihydropyrrolo[2,3-c/]pyrimidin-5-yl)ethyl]benzoyl]amino}pentanedioic acid whose chemical structure is the following:

Pemetrexed is also an antimetabolite of the antifolate type which works by inhibiting three enzymes used in purine and pyrimidine synthesis, among others the dihydrofolate reductase (DHFR). Thus, pemetrexed prevents the formation of DNA and RNA, which are required for the growth and survival of both normal cells and cancer cells. Pemetrexed is indicated for the treatment of malignant pleural mesothelioma and for a metastatic non-small cell lung cancer (NSCLC).

However, serious adverse effects have been reported for these active ingredients. On one hand, the most common adverse effects of methotrexate include: hepatotoxicity (liver damage), ulcerative stomatitis, leukopenia and thus predisposition to infection, nausea, abdominal pain, fatigue, fever, dizziness, acute pneumonitis, rarely pulmonary fibrosis, and kidney failure. Furthermore, Methotrexate is teratogenic and it also may increase the risk of certain cancers for instance lung cancer and melanoma. Central nervous reactions to methotrexate have been also reported, which include myelopathies and leucoencephalopathies, as well as neurological damage and memory loss.

On the other hand, pemetrexed can suppress the function of the bone marrow which manifests itself as neutropenia, thrombocytopenia, and anemia (or pancytopenia). Serious renal events, including acute kidney failure, have been reported in connection with the use of pemetrexed. During clinical trials cardiovascular events, including myocardial infarction, and cerebrovascular events have been also reported.

Therefore, from what is known in the art it is derived that there is still the need of providing an inhibitor of the dihydrofolate reductase in which the side effects are reduced.

Summary of Invention

Inventors have provided a new class of azoaryl derivatives of the known inhibitors of the dihydrofolate reductase methotrexate and pemetrexed with high activity and low side effects. In particular, the compounds of formula (I) of the present invention are derivatives of the corresponding reference active ingredients wherein the main change is that a single bond has been replaced by the azo-photoisomerizable moiety -N=N-.

It is unexpected that, despite the changes in the structure of methotrexate and pemetrexed, in particular the replacement of the bridge (-CH2-N(CH3)-) between the two aromatic groups with a photoisomerizable moiety (-N=N-), together with other further changes in the structure of the reference active ingredient such as for examples the removal of one or more heteroatoms of the heterocyclic ring, the biological effect over the inhibition of the dihydrofolate reductase (DHFR) is not adversely affected. For instance, as reported by Pittolo S. et al. (Pittolo S et al.“An allosteric modulator to control endogenous G protein-coupled receptors with light”. Nature Chemical Biology, 2014, vol. 10, pp. 813-815), the replacement of one of the two amide bridge with a photoisomerizable moiety in the structure of VU0415374, a positive allosteric modulator of the metabotropic glutamate receptor 4, gave rise to a compound (Alloswitch-1) which is totally devoid of activity on the same receptor subtype.

Advantageously, the presence of this azo moiety allows having a regioisomeric geometry Z/E that changes from E-isomer to Z-isomer upon exposure to light at a specific wavelength. In fact, the photoisomerization induces a switch between the inactive and active configuration. The Z-isomer of the compound of formula (I) shows a significantly higher capacity to inhibit the dihydrofolate reductase (DHFR) compared to the E-isomer of the compound of formula (I). Therefore, the active azo-derivative compounds of formula (I) of the present invention are specially advantageous because they maintain the same desirable biological effect as do the reference active ingredient, but reducing the side effects due to the regulation of the pharmacological effect by light exposure. This control is reversible even in time and location. Thus, a first aspect of the present invention relates to a compound of formula (I) in Z-isomer form

or alternatively a compound of formula (I) in E-isomer form

or a pharmaceutically acceptable salt thereof, wherein: is selected from the group consisting of a radical of formula (II)

and a radical of formula

Xi, X2, X3, X4 and X5 are independently selected from the group consisting of CR3 and N; Y1 is selected from the group consisting of NH, 0 and S; each R-i, each R3 and R7 are independently selected from the group consisting of H, halogen, -OH, -0-(Ci-Ce)alkyi, -N((Ci-Ce)alkyl)3, -NH((Ci-Ce)alkyl)2, and -NH2(Ci-Ce)alkyl; R4, R5 and R6 are indepentently selected from the group consisting of H and (Ci-Ce)alkyl;— line indicates the position of R2 radical by which R2 is attached to the adjacent N; and the asterisk indicates a stereogenic carbon atom being in a form selected from R-isomer, S-isomer and a mixture of R-isomer and S-isomer.

The second aspect of the invention relates to a composition comprising a therapeutically effective amount of a compound of formula (I) in Z-isomer as defined in the first aspect of the invention; or alternatively an amount of a compound of formula (I) in E-isomer form as defined in the first aspect of the invention such that after light irradiation gives rise to a therapeutically effective amount of a compound of formula (I) as Z-isomer, together with one or more pharmaceutically acceptable excipients or carriers. The third aspect of the invention relates to a compound of formula (I) in Z-isomer form as defined in the first aspect of the invention for use as a medicament.

The fourth aspect of the invention relates to a compound of formula (I) in Z-isomer as defined in the first aspect of the invention for use in the treatment of a disease or condition mediated by the dihydrofolate reductase.

The fifth aspect of the invention relates to a compound of formula (I) in E-isomer form as defined in the first aspect of the invention for use in the treatment of a disease or condition mediated by the dihydrofolate reductase, wherein the treatment comprises: (a) first administering an amount of a compound of formula (I) in E-isomer form as defined in the first aspect of the invention such that after light irradiation gives rise to a therapeutically effective amount of a compound of formula (I) in Z-isomer; and then (b) light irradiating the treatment area.

And, the sixth aspect of the invention relates to a compound of formula (I) in E-isomer form as defined in the first aspect of the invention for use in the treatment of a disease or condition mediated by the dihydrofolate reductase, wherein the treatment comprises: (a) first exposing an amount of a compound of formula (I) in E- isomer form to light irradiation to obtain the compound of formula (I) in Z-isomer form; and then (b) administering a therapeutically effective amount of a compound of formula (I) in Z-isomer form obtained in step (a).

Brief Description of Drawings

Fig. 1 shows the in vitro inhibition of DHFR at three different concentrations (1 nM, 10 nM and 100 nM) of the compound IA’ in the E isomeric states in darkness (cf. black columns) versus the Z isomeric enriched form after ultraviolet (UV) light irradiation (cf. checkered columns). The spectrum expresses concentration of compound IA’ versus DHFR activity expressed in percentage (%). Data are means of at least three independent experiments in triplicate or quadriplicate ± SEM (standard error of the mean). Results were analyzed by two-way analysis of variance (ANOVA) with Bonferroni's multiple comparisons test for statistical significance (GraphPad Prism 6; adjusted p-value (^*) < 0.05).

Fig. 2 shows the results of viability assays in a HeLa cell line at different concentrations of compound IA’ in the E isomeric state in darkness (cf. solid line) versus Z-isomer enriched form after UV light irradiation (cf. pointed line). Results obtained with the reference compound methotrexate (MTX) are also shown in background (cf. crossed line). Data are means of at least three independent experiments in triplicate or quadriplicate ± SEM. Results were analyzed by two-way ANOVA with Bonferroni's multiple comparisons test for statistical significance (GraphPad Prism 6; adjusted p-value (^****) < 0.0001).

Fig. 3 sections (a) and (b) shows the results of the in vivo toxicity test on zebrafish (Danio rerio) embryos. Sections (a) and (b) show the anatomical profiles at 72 hours post fertilization (hpf) of the zebrafish treated with vehicle (dimethyl sulfoxide, DMSO) (cf. column A), methotrexate (MTX, 200 mM) (cf. column B) and compound IA’ (200 pM) in the Z-isomer enriched form after UV light irradiation (cf.column C) and in the E isomeric state in darkness (cf. column D). Figure 3 shows the percentage of zebrafish treated versus the treatment group, superposing abnormal larvae (cf. checkered part of the column) to viable embryos (cf. striped part of the columns) and viability to total number of fertilised embryos (cf. white part of the columns). Section (c) shows the illustrative pictures from individual larvae of every treatment at 72 hpf. Section (d) shows the individual larvae at 72 hpf after treatment with methotrexate (MTX, 200 pM). Black arrows point out observable aberrant developmental traits. Section (c2) shows the individual larvae at 72 hpf after treatment with vehicle (DMSO). Section (c3) shows the individual larvae at 72 hpf after treatment with compound IA’ (200 mM) in the Z-isomer enriched form after UV light irradiation. And, section (c4) shows the individual larvae at 72 hpf after treatment with compound IA’ (200 mM) in the E isomeric state in darkness.

Fig. 4 shows the mortality at 96 hours post fertilization (hpf) of the zebrafish treated with vehicle (dimethyl sulfoxide, DMSO) (cf. column A), methotrexate (MTX, 200 mM) (cf. column B) and compound IA’ (200 mM) in the Z-isomer enriched form after UV light irradiation (cf.column C) and in the E isomeric state in darkness (cf. column D). Figure 4 shows the percentage of dead zebrafish (cf. checkered part of the column) superposed to viability of embryos (cf. striped part of the columns) and total number of fertilised embryos (cf. white part of the columns) at 96 hpf.

Detailed description of the invention

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

For the purposes of the invention, any ranges given include both the lower and the upper end-points of the range. Ranges given, such as temperatures, times, and the like, should be considered approximate, unless specifically stated.

The terms“E-Z” configuration refers to the absolute stereochemistry of double bonds having two, three or four substituents following the IUPAC convention. In particular, the term“Z-isomer” refers to a double bond wherein the two groups of higher priority are on the same side of the double bond; and the term“E-isomer” refers to a double bond wherein the two groups of higher priority are on opposite sides of the double bond.

The term“ratio E/Z” refers to the ratio between the E-isomer and the Z-isomer obtained in the preparation process or after light irradiation. The measurement of the ratio between the E-isomer and the Z-isomer of the compounds of the present invention is performed by determination of the content at the photostationary state in methanol-d4 by ¹FI-NMR analysis.

In the compound of formula (I) of the present invention the asterisk indicates a stereogenic carbon atom being in a form selected from R-isomer, S-isomer and a mixture of R-isomer and S-isomer. The terms "stereogenic carbon atom", "chiral carbon atom" or "asymmetric carbon atom" have the same meaning, and are used interchangeably. A chiral carbon atom refers to a carbon atom to which four different atoms or groups of atoms are attached.

In the formula of the compounds of the present invention, the use of bold and dashed lines denotes particular configuration of groups that follows the IUPAC convention. A bond indicated by a broken line indicates that the group in question is below the general plane of the molecule as drawn (the "alpha" configuration), and a bond indicated by a bold line indicates that the group at the position in question is above the general plane of the molecule as drawn (the "beta" configuration). Besides, the use of the line denotes particular configuration of groups that follows the IUPAC convention, and indicates an indefinite bond wherein the group in question is part below the general plane of the molecule as drawn (the "alpha" configuration), and part above the general plane of the molecule as drawn (the "beta" configuration).

The term "stereoselectively enriched" refers to an enantioselectively and diastereoselectively enriched compound.

The term "enantioselectively enriched" refers to a chiral non-racemic compound, that is, a compound which has more of one enantiomer than another enantiomer. The degree of enrichment of one enantiomer is measured by the enantiomeric excess (ee).

The "enantiomeric excess" or "ee" is a measure of the excess of one enantiomer over a racemic mixture of a chiral compound, which is commonly expressed as a percentage. Enantiomeric excess is defined as the absolute difference between the mole fraction of each enantiomer [ee = F(+)- F(-)]. If the moles of each enantiomer are known, the percentage of the enantiomeric excess can be determined by the following formula: ee = ((R-S)/(R+S)) x 100, where R and S are the respective molar fractions of enantiomers in the mixture such that R+S=1.

The term "enantiomerically pure" compound refers to an optically active compound with an enantiomeric excess (ee) of at least 98%.

The term“halogen” refers to F, Cl, Br, and I.

The term alkyl refers to a saturated straight, or branched hydrocarbon chain which contains the number of carbon atoms specified in the description or claims. Examples include, among others, the group methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl.

The term“light” refers to the use of one or more light sources of any type that emits light with a defined wavelength, duration, intensity, and exposure pattern that can easily be applied by technologies of light sources (including lamps, light-emitting diodes (LEDs), organic LEDs (OLEDs), lasers, and monochromators) which may be coupled with methods for light focussing and delivery (including endoscopes and fibre optic cables, optical table setups, microscopy methods including confocal and spinning disk microscopy) in a manner which is well-defined in time and space. The light could be applied either continuously or in pulses. The application of light is preferably localised. The light can be applied to a cuvette, subcellular region, cell, tissue, tumour zone, organism or other region of interest by technologies of light sources as mentioned above.

As mentioned above, the first aspect of the present invention refers to a compound of formula (I) in Z-isomer form

Z-(l)

wherein:

R2 is selected from the group consisting of a radical of formula (II)

Rfi

and a radical of formula

Xi, X2, X3, X4 and X5 are independently selected from the group consisting of CR3 and N; Y1 is selected from the group consisting of NH, 0 and S; each R-i, each R3 and R7 are indepentently selected from the group consisting of H, halogen, -OH, -0-(Ci-Ce)alkyi, -N((Ci-Ce)alkyl)3, -NH((Ci-Ce)alkyl)2, and -NH2(Ci-Ce)alkyl; R4, R5 and R6 are indepentently selected from the group consisting of H and (Ci-Ce)alkyl;— line indicates the position of R2 radical by which R2 is attached to the adjacent N; and the asterisk indicates a stereogenic carbon atom being in a form selected from R-isomer, S-isomer and a mixture of R-isomer and S-isomer. Therefore, the compound of formula (I) in Z-isomer form of the present invention is selected from the group consisting of:

As mentioned above, the first aspect of the present invention also refers to a compound of formula (I) in E- isomer form

wherein:

is selected from the group consisting of a radical of formula (II)

and a radical of formula

Xi, X₂, X₃, X₄ and X₅ are independently selected from the group consisting of CR₃ and N; Y₁ is selected from the group consisting of NH, 0 and S; each R-i, each R₃ and R₇ are indepentently selected from the group consisting of H, halogen, -OH, -0-(Ci-Ce)alkyi, -N((Ci-Ce)alkyl)3, -NH((Ci-Ce)alkyl)2, and -NH2(Ci-Ce)alkyl; R4, Rs and R₆ are indepentently selected from the group consisting of H and (Ci-Ce)alkyl;— line indicates the position of R2 radical by which R2 is attached to the adjacent N; and the asterisk indicates a stereogenic carbon atom being in a form selected from R-isomer, S-isomer and a mixture of R-isomer and S-isomer.

Therefore, the compound of formula (I) in Z-isomer form of the present invention is selected from the group consisting of:

E-(IA)-R ,

E-(IB)-RS . The compounds of formula (I) of the present invention can be in form of pharmaceutically acceptable salt thereof. The term“pharmaceutically acceptable salts” used herein encompasses either an acid addition salt with an amine group present in the compound of formula (I), or a basic addition salt with the carboxylic acid present in the compound of formula (l).There is no limitation regarding the salts, except that if used for therapeutic purposes, they must be pharmaceutically acceptable. The“acid addition salt” of the compound of formula (I) as used herein refers to any salt formed by the addition of a non-toxic acid, including non-toxic organic or inorganic acids, to the compound of formula (I). Illustrative inorganic acids which form suitable salts include, without limitation, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid. Illustrative organic acids which form suitable salts include, without limitation, formic acid, acetic acid, propionic acid, oxalic acid, succinic acid, maleic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, p-chlorobenzenesulfonic acid, p- bromobenzenesulfonic acid, phthalic acid, isophthalic acid or benzoic acid. The“basic addition salt” of the compound of formula (I) as used herein means any salt formed by the addition of a non-toxic base, including non-toxic organic or inorganic base to the compound of formula (I). Illustrative inorganic bases which form suitable salts include without limitation lithium, sodium, potassium, calcium, magnesium, aluminium, zinc or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine. The preparation of pharmaceutically acceptable salts of the compounds of formula (I) can be carried out by methods known in the art. For instance, they can be prepared from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate pharmaceutically acceptable base or acid in water or in an organic solvent or in a mixture of them.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is enantiomerically enriched compound. In a preferred embodiment, the compound of formula (I) of the present invention has an enantiomeric enrichment (ee) equal or greater than 95%; more preferably equal or greater than 98%. In a still more preferred embodiment, the compound of formula (I) of the present invention has an ee. equal or greater than 99%.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein each Ri is H.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein is a radical of formula (II) and R-i, R3, R4 and R5, and X1-X5 are as defined in the present invention, thereby the compound of formula (I) is selected from the group consisting of

E-(IA)-RS.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein is a radical of formula (II), and and R₅ are H. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (II), each R1 is H; and R₄ and R₅ are H.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (II), and X-i, X₃, X₄ and X₅ are N; X₂ is CR₃; and each R₃ is as defined in the present invention; preferably each R₃ is selected from the group consisting of H and OH. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (II); X-i, X₃, X₄ and X₅ are N; X₂ is CR₃; each R₃ is selected from the group consisting of H and OH; and R-i, R₄ and R₅ are H. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (II); R-i, R₄ and R₅ are H; X-i, X₃, X₄ and X₅ are N; X₂ is CR₃; and each R₃ is H. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (II); R-i, R₄ and R₅ are H; X-i, X₃, X₄ and X₅ are N; X₂ is CR₃; and each R₃ is OH.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula

(II); Ci, X₂ and X₃ are CR₃; X₄ and X₅ are N; and R₃ is as defined in the present invention; preferably each R₃ is selected from the group consisting of H and OH. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (II); X-i, X₂ and X₃ are CR₃; X₄ and X₅ are N; each R₃ is selected from the group consisting of H and OH, and R-i, R₄ and R₅ are H. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (II); R-i, R₄ and R₅ are H; X₁, X₂ and X₃ are CR₃; X₄ and X₅ are N; and each R₃ is H (cf. Example 1).

In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (II); R₁, R₄ and R₅ are H; X₁, X₂ and X₃ are CR₃; X₄ and X₅ are N; and each R₃ is OH.

(III).

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein is a radical of formula (III) selected from the group consisting of (IMA)

and (NIB)

wherein: Y₂ is selected from the group consisting of NH, 0 and S; Y₃ is selected from the group consisting of NH and C; Ce and X₇ are independently selected from the group consisting of CR3 and N; Re is selected from the group consisting of H, SH, -N((Ci-Ce)alkyl)3, -NH((Ci-Ce)alkyl)2, and -NH2(Ci-Ce)alkyl; and R3, R6, R? and Yi are as defined in the present invention.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R2 is a radical of formula (IMA) and R-i, R₃, R₆ and R₇; C_Q; and Y1-Y2 are as defined in the present invention, thereby the compound of formula (I) is selected from the group consisting of

Z-(IB-1)-R , Z-(IB-1)-S , Rfi

E-(IB-1)-RS.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein is a radical of formula (IMA); and each f¾ and R7 are H. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein wherein R2 is a radical of formula (MIA); R1 is H; and each R6 and R7 are H.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein is a radical of formula (IMA); C_d is N; and Yi and Y₂ are NH.

In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R2 is a radical of formula (MIA); R1 is H; each R6 and R7 are H; C_d is N; and Y1 and Y2 are NH. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein is a radical of formula (NIB) and R-i, R₃, R₆ and R₇; X₇; and Y₁-Y₃ are as defined in the present invention, thereby the compound of formula (I) is selected from the group consisting of

E-(IB-2)-RS. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein is a radical of formula (NIB); and each f¾ and R₇ are H. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein wherein

is a radical of formula (NIB); R₁ is H; and each R₆ and R₇ are H.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is selected from the group consisting of SH and NH₂.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is selected from the group consisting of SH and NH₂ and Y₁ is selected from the group consisting of S, 0 and NH. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is SH and Y₁ is selected from the group consisting of S, O and NH. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is NH₂ and Y₁ is selected from the group consisting of S, 0 and NH.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is selected from the group consisting of SH and NH₂ and Y₃ is C.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is selected from the group consisting of SH and NH₂; Y₃ is C; and Y₁ is selected from the group consisting of S, 0 and NH. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is SH; Y₃ is C; and Y₁ is selected from the group consisting of S, 0 and NH. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is NH₂; Y₃ is C; and Y₁ is selected from the group consisting of S, 0 and NH.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is selected from the group consisting of SH and NH₂; and X₇ is N. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein is a radical of formula (NIB) and Re is selected from the group consisting of SH and Nhb; X₇ is N; and Y₁ is selected from the group consisting of S, 0 and NH.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is selected from the group consisting of SH and NH₂; X₇ is N; and Y₃ is C.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is selected from the group consisting of SH and NH₂; X₇ is N; Y₁ is selected from the group consisting of S, 0 and NH; and Y₃ is C.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (IIIB)and Re is selected from the group consisting of SH and NH₂; Y₁ is selected from the group consisting of S, 0 and NH; Y₃ is C; X₇ is CR₃; and R₃ is H.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is selected from the group consisting of SH and NH₂; Y₁ is selected from the group consisting of S, 0 and NH; Y₃ is C; X₇ is CR₃; R₃ is H; and each R₆ and R₇ are H.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is selected from the group consisting of SH and NH₂; Y₁ is selected from the group consisting of S, 0 and NH; Y₃ is C; X₇ is CR₃; R₃ is H; R₁ is H; and each R₆ and R₇ are H.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is H. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is H and Y₁ is selected from the group consisting of S, O and NH. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is H; and Y₃ is N. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is H, Y₁ is selected from the group consisting of S, O and NH; and Y₃ is N.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein is a radical of formula (NIB) and Re is H; and X₇ is N. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R₂ is a radical of formula (NIB) and Re is H; X₇ is N; and Y₃ is N. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R2 is a radical of formula (NIB) and Re is H; X₇ is N; Y₃ is N; and Y1 is selected from the group consisting of S, 0 and NH.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R2 is a radical of formula (NIB) and Re is H; Y₁ is selected from the group consisting of S, 0 and NH; Y₃ is N; and X₇ is N.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R2 is a radical of formula (NIB) and Re is H; Y₁ is selected from the group consisting of S, 0 and NH; Y₃ is N; X₇ is N; and each R₆ and R₇ are H. In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) of the present invention is one wherein R2 is a radical of formula (NIB) and Re is H; Y₁ is selected from the group consisting of S, O and NH; Y₃ is N; X₇ is N; R₁ is H; and each R₆ and R₇ are H.

The compounds of the present invention may be prepared by adapting known conventional techniques for forming azobenzenes and/or their azoheteroaryl analogues, along with such conventional techniques for modifying substituents on the thus-formed azo compounds or their precursors as may be found in the appropriate literature of azo compound chemistry or adapted from the state of the art, particularly from known reactions in e.g. aromatic or heterocyclic chemistry, or in the chemistry of prodrugs and protective groups (cf. E. Merino,“Synthesis of azobenzenes: the coloured pieces of molecular materials”, Chem. Soc. Rev. 2011 , vol. 40, pp. 3835-3853).

It is also part of the invention a process for the preparation of a compound of formula (I) in E-isomer form, which comprises reacting a compound of formula (IV)

with a compound selected from the group consisting of (V)

;and (VI)

wherein R₁-R₇, CI-C_Q, and Y₁-Y₂ are as defined in the present invention; and

Re and Rg are indepentendly selected from the group consisting of H and hydroxy protecting group.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the process for the preparation of a compound of formula (I) in E-isomer form at least one of Re and Rg is an hydroxy protecting group, and then the process further comprising an addicional step for the removal of the hydroxy protecting group. The introduction and/or removal of the hydroxy protective groups is carried out by standard methods well-known in the art (cf. Wuts PGM and Greene TW.“Greene's Protective Groups in Organic Synthesis”. Chapter 2, Protection for the Hydroxyl Group, Including 1,2- and 1 ,3-Diols,

John Wiley & Sons, Inc., 4^th edition, Hoboken, NJ, USA, 2006, pp. 16-299). Representative hydroxy protective groups include those where the hydroxy group is either acylated or alkylated such as benzyl, and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.

The appropriate reaction conditions such as for example temperature and time, as well as the amount of the reagents and the selection of solvents, can readily be determined by those skilled in the art according to the type of compound being prepared.

It is also part of the invention a process for the preparation of a compound of formula (I) in Z-isomer form. This process comprises exposing the compound of formula (I) in E-isomer form to light irradiation. Particularly, the process for the preparation of the compound of formula (I) in Z-isomer form from the E-isomer form comprises the irradiation of the E-isomer at a light having a wavelength comprised from 300 nm to 1000 nm. The term “light irradiation” refers to the use of one or more light sources of any type that emits light with a wavelength between about 300 nm and 1000 nm. The light irradiation occurs by the use of an emitter or light-generating device such as for example a lamp, for instance a flash lamp or a light output device. The light irradiation can be performed in a continuous or pulsed wave light irradiation between 300 nm and 1000 nm. Typically, light irradiation can be performed mantaining the fluence of the light during the entire treatment, or alternatively modifying the fluence from a relative low fluence to a high fluence. In general, a light application may include one or more exposures of a treatment area of one or more emitter light outputs.

In an embodiment, the light irradiation is performed with a continuous wave illumination at a wavelength comprised from 300 nm to 1000 nm for an appropriate period of time to converse a compound of formula (I) in E-isomer into a compound of formula (I) in Z-isomer form. In an embodiment, the light irradiation is performed with a continuous wave illumination at a wavelength comprised from 300 nm to 800 nm for an appropriate period of time to converse a compound of formula (I) in E-isomer into a compound of formula (I) in Z-isomer form. In a particular embodiment, the light irradation is performed with continuous wave illumination for an appropriate period of time to converse at least 50% of the E-isomer. This process allows obtaining the compound of formula (I) in Z-isomer form in a“ratio E/Z”from 1 :1 to 1 :3.

In an embodiment, the light irradiation is performed with a pulsed wave illumination at a wavelength comprised from 300 nm to 1000 nm for an appropriate period of time to converse a compound of formula (I) in E-isomer into a compound of formula (I) in Z-isomer form. In an embodiment, the light irradiation is performed with a pulsed wave illumination at a wavelength comprised from 700 nm to 1000 nm for an appropriate period of time to converse a compound of formula (I) in E-isomer into a compound of formula (I) in Z-isomer form. In a particular embodiment, the light irradiation is performed with pulsed wave illumination (i.e., multiphoton excitation) for an appropriate period of time to converse at least 50% of the E-isomer. This process allows obtaining the compound of formula (I) in Z-isomer form in a“ratio E/Z” from 1 :1 to 1 :3.

The second aspect of the invention relates to a composition comprising a therapeutically effective amount of a compound of formula (I) in Z-isomer as defined in the first aspect of the invention together with one or more pharmaceutically acceptable excipients or carriers; or alternatively a composition comprising an amount of a compound of formula (I) in E-isomer form as defined in the first aspect of the invention such that after light irradiation gives rise to a therapeutically effective amount of a compound of formula (I) in Z-isomer of the present invention, together with one or more pharmaceutically acceptable excipients or carriers.

The expression "therapeutically effective amount" as used herein, refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed. The particular dose of compound administered according to this invention will be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations. In an embodiment, the composition is one wherein the therapeutically effective amount of a compound of formula (I) in Z-isomer is comprised from 2.5 mg to 500 mg, typically comprised from 10 mg to 100 mg together with one or more pharmaceutically acceptable excipients or carriers.

The expression“amount of a compound of formula (I) in E-isomer form such that after light irradiation gives rise to a therapeutically effective amount of a compound of formula (I) in Z-isomer” as used herein, refers to the amount of the compound of formula (I) in E-isomer that, after light irradiation gives rise to a therapeutically effective amount of a compound of formula (I) in Z-isomer sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed. The particular amount of the compound of formula (I) in E-isomer form according to this invention will be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.

In an embodiment, optionally in combination with one or more features of the various embodiments described above or below, the composition is one wherein the amount of a compound of formula (I) in E-isomer form is such that after light irradiation gives rise to a therapeutically effective amount of a compound of formula (I) in Z-isomer comprised from 2.5 mg to 500 mg, typically comprised from 10 mg to 100 mg.

The expression "pharmaceutically acceptable excipients or carriers" refers to pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.

The compositions of the invention can be formulated in several forms that include, but are not limited to, oral, topical, transdermal and parenteral compositions.

The oral compositions defined above comprise appropriate excipients or carriers for oral administration including, but not limited to, binder, lubricant, surfactant and diluent. The oral compositions can be formulated in several forms that include, but are not limited to solutions, tablets, capsules, granules, suspensions, dispersions, powders, lozenges, concentrates, drops, elixirs, emulsions, pastilles and pellets.

The topical or transdermal compositions defined above comprise appropriate excipients or carriers for topical administration including, but not limited to, a hydrating agent, an emollient, an emulsifier, a thickener, a humectant, a pH-regulating agent, an antioxidant, a preservative agent, a vehicle, or their mixtures. The excipients or carriers used have affinity for the skin, are well tolerated, stable, and are used in an amount adequate to provide the desired consistency, and ease application. The topical compositions of the invention can be formulated in several forms that include, but are not limited to, solutions, aerosols and non-aerosol sprays, creams, powders, mousses, lotions, gels, sticks, ointments, pastes, and emulsions.

The parenteral compositions defined above are suitable for their injection, infusion, or implantation into the body. The parenteral compositions defined above should be sterile, and pyrogen-free, and they can be in form of liquid such as solutions, emulsions, or suspensions, or in solid form packaged in either single-dose or multidose containers suitably diluted before use. Parenteral compositions can comprise appropriate excipients or carriers for parenteral administration including, but not limited to, solvents, suspending agents, buffering agents, substances to make the preparation isotonic with blood, stabilizers, or antimicrobial preservatives.

The addition of excipients should be kept to a minimum. When excipients are used, they should not adversely affect the stability, bioavailability, safety, or efficacy of the polymers and/or the active agents, or cause toxicity or undue local irritation. There should not be any incompatibility between any of the components of the dosage form.

Additionally, the compositions of the present invention may contain other ingredients, such as fragrances, colorants, and other components known in the state of the art.

The above mentioned composition can be prepared according to methods well known in the state of the art. The appropriate excipients and/or carriers, and their amounts, can readily be determined by those skilled in the art according to the type of formulation being prepared.

As mentioned above, the third aspect of the invention relates to a compound of formula (I) in Z-isomer form as defined in the first aspect of the invention for use as a medicament. As it is shown in the examples, the compounds of formula (I) in Z-isomer form of the present invention allow inhibiting the dihydrofolate reductase (DHFR) similarly to the reference active ingredient, meanwhile the compounds of formula (I) in E-isomer form have no or lower capacity to inhibit the DHFR. Therefore, the compounds of formula (I) of the present invention can reduce the side effects of the reference active ingredient without compromising their pharmacological effect.

All the embodiments disclosed above for the compound of formula (I) in Z-isomer of the invention of the first aspect of the invention also apply for its use as a medicament of the third aspect of the invention.

Furthermore, the fourth aspect of the invention relates to the compound of formula (I) in Z-isomer as defined in the present invention for use in the treatment of a disease or condition mediated by the dihydrofolate reductase. The terms "dihydrofolate reductase- mediated disease or disorder" or“disease or condition mediated by the dihydrofolate reductase” have the same meaning, and are used interchangeably. They refer to a disease or disorder that is characterized by an abnormal cell proliferation. A dihydrofolate reductase- mediated disorder may be completely or partially mediated by modulating dihydrofolate reductase activity. In particular, a dihydrofolate reductase- mediated disease or disorder is one in which inhibiting dihydrofolate reductase activity results in some effect on the underlying disease or disorder, particularly this inhibition results in some improvement in at least some of the patients being treated. The term "inhibiting dihydrofolate reductase activity" or "inhibition of dihydrofolate reductase activity" refers to altering the function of dihydrofolate reductase by administering a dihydrofolate reductase inhibitor. In an embodiment, the compound of formula (I) in Z-isomer as defined in the present invention for use in the treatment of a disease or condition mediated by the dihydrofolate reductase. In an embodiment, the compound of formula (I) in Z-isomer as defined in the present invention for use in the treatment of a disease or condition mediated by the dihydrofolate reductase, wherein the disease or condition is a hyperproliferative disease or condition. In an embodiment, the compound of formula (I) in Z-isomer as defined in the present invention for use in the treatment of a disease or condition mediated by the dihydrofolate reductase, wherein the disease or condition is a hyperproliferative disease or condition selected from cancer, psoriasis and rheumatoid arthritis. In an embodiment, the compound of formula (I) in Z-isomer as defined in the present invention for use in the treatment of a disease or condition mediated by the dihydrofolate reductase selected from the group consisting of breast cancer, skin cancer, head cancer, neck cancer, and lung cancer, psoriasis and rheumatoid arthritis.

All the embodiments disclosed above for the compound of formula (I) in Z-isomer of the invention of the first aspect of the invention also apply for its use in the treatment of a disease or condition mediated by the dihydrofolate reductase of the fourth aspect of the invention.

As mentioned above, the fifth aspect of the invention relates to a compound of formula (I) in E-isomer form as defined in the first aspect of the invention for use in the treatment of a disease or condition mediated by the dihydrofolate reductase as defined above, wherein the treatment comprises: (a) first administering an amount of a compound of formula (I) in E-isomer form as defined in the present invention such that after light irradiation gives rise to a therapeutically effective amount of a compound of formula (I) in Z-isomer; and then (b) light irradiating the treatment area.

This process allows firstly administering the inactive compound of formula (I) in E-isomer form by any appropriate route of administration and then carrying out the conversion into the active compound (that is the Z-isomer) by light exposure. Thus, the active compound of formula (I) (i.e. in the Z-isomer form) only inhibits the DHFR in the target treatment area reducing the side effects associated with the inhibition of the DHFR in the areas not exposed to light.

In an embodiment, the compound of formula (I) in E-isomer form for use in the treatment of a disease or condition mediated by the DHFR as defined above, wherein in step (b) of the treatment the light is a light having a wavelength comprised from 300 nm to 1000 nm. All mentioned above for the process of the compound of formula (I) in Z-isomeric state from the E-isomer state with light irradation also applies in the compound of formula (I) in E-isomer form for use in the treatment of a disease or condition mediated by the dihydrofolate reductase as defined above. It is advantegeous because the range of wavelength used in step (b) as defined above includes the range of wavelength used in the light-coadjuvated treatment of some hyperproliferative disease such as for example psoriasis (cf. Hamblin MR et al. Flandbook of Photomedicine, Chapter 19,“PUVA Therapy”, pp. 197-204, Chapter 22,“Recent Advances in Developing Improved Agents for Photodynamic Therapy”, pp. 227-266, Chapter 40,“Photodynamic Therapy in Dermatology”, pp. 465-474, Taylor & Francis. X ed. Boca Raton, 2013). It means that a unique light treatment can be used for preparing in situ at the target region the Z-isomer of the compounds of formula (I) of the present invention for carrying out the treatment of the disease or condition mediated by the dihydrofolate reductase, while keeping the less active E-isomer at other regions.

In an embodiment, the compound of formula (I) in E-isomer form for use in the treatment of a disease or condition mediated by the dihydrofolate reductase as defined above, wherein in step (b) the light irradiation is the unique light treatment.

In an embodiment, the compound of formula (I) in E-isomer form for use in the treatment of a disease or condition mediated by the dihydrofolate reductase as defined above, wherein in step (b) the light irradiating is combined with one or more additional light treatments. In an embodiment, the compound of formula (I) in E- isomer form for use in the treatment of a disease or condition mediated by the dihydrofolate reductase as defined above, wherein in step (b) the light irradiating is combined with one or more additional light treatments (cf. Hamblin MR et al. Handbook of Photomedicine, Chapter 19,“PUVA Therapy”, pp. 197-204, Chapter 22, “Recent Advances in Developing Improved Agents for Photodynamic Therapy”, pp. 227-266, Chapter 40, “Photodynamic Therapy in Dermatology”, pp. 465-474, Taylor & Francis. X ed. Boca Raton, 2013). Illustrative additional light treatments appropriate for the present invention include, without limitation, psoralen photochemotherapy (PUVA) or the photodynamic therapy (PDT). It is advantegeous because the compound for use of the present invention might then be used synergistically/complementarily in some of the light treatments known in the state of the art or simply exploit the same kind of devices commonly used and clinically approved for other light therapy treatments.

All the embodiments disclosed above for the compound of formula (I) in Z-isomer of the invention of the first aspect of the invention, as well as the dihydrofolate reductase- mediated disease or disorder of the third aspect of the invention, also apply for its use in the treatment of a disease or condition mediated by the dihydrofolate reductase of the fifth aspect of the invention.

As mentioned above, the sixth aspect of the invention relates to a compound of formula (I) in E-isomer form as defined in the first aspect of the invention for use in the treatment of a disease or condition mediated by the dihydrofolate reductase, wherein the treatment comprises: (a) first exposing an amount of a compound of formula (I) in E-isomer form to light irradiation to obtain the compound of formula (I) in Z-isomer form; and then (b) administering a therapeutically effective amount of a compound of formula (I) in Z-isomer form obtained in step (a).

This process allows firstly converting the inactive compound of formula (I) in E-isomer form into the active active compound of formula (I) in Z-isomer by light irradiation; and then administering by any appropriate route of administration a therapeutically effective amount of the compound of formula (I) in Z-isomer thus obtained. Therefore, the active compound of formula (I) (i.e. in the Z-isomer form) only inhibits the DHFR in the target treatment area reducing the side effects associated with the inhibition of the DHFR in the non-target treatment area, because the diffusion of the compound of formula (I) in Z-isomer form to other areas not exposed to light allows its thermal relaxation to the inactive E-isomer form.

All the embodiments disclosed above for the compound of formula (I) in E-isomer of the invention of the first aspect of the invention, as well as the dihydrofolate reductase-mediated disease or disorder of the third aspect of the invention, and the conditions of the treatment such as light an therapeutically effective amount of the active compound also apply for its use in the treatment of a disease or condition mediated by the dihydrofolate reductase of the sixth aspect of the invention.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word“comprise” encompasses the case of“consisting of. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Reference signs related to drawings and placed in parentheses in a claim, are solely for attempting to increase the intelligibility of the claim, and shall not be construed as limiting the scope of the claim. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

Examples

CHEMICAL SYNTHESIS

Abbreviations

DCM: dichloromethane

DMF: dimethyl formamide

DMSO: dimethylsulfoxide

Pd-C: palladium on activated charcoal

Rf. retention factor (TLC)

R{. retention time (HPLC)

Materials and methods

All the chemicals and solvents are from commercial suppliers and used without purification. Reactions were monitored by thin layer chromatography (TLC: EMD/Millipore, silica gel 60 on aluminium support, layer thickness: 200 pm, particle size: 10-12 pm) by visualisation under 254 and/or 365 nm lamp. Flash column chromatography: Panreac Silica Gel 60, 40-63 pm RE. Nuclear magnetic resonance spectrometry (NMR): Varian-Mercury 400 MHz. Chemical shifts (6) are reported in parts per million (ppm) against the reference compound tetramethylsilane using the signal of the residual non-deuterated solvent (Chloroform-cf d = 7.26 ppm (¹H), d = 77.16 ppm (¹³C); Dimethyl sulfoxide-c/e d = 2.50 ppm (¹H), d = 39.52 ppm (¹³C); Methanol-c/4 d = 3.31 ppm (¹H), d = 49.00 ppm (¹³C). High-performance Liquid Chromatography (HPLC) apparatus: Waters Alliance 2695 separation module coupled to Waters 2996 photodiode detector (PDA) with MassLynx 4.1 software for data acquisition; SunFire C18 Column (100A, 5 mhi, 4.6 mm X 150 mm); injection volume: 5 pL; mobile phase: water w/0.1% formic acid (solvent A) and acetonitrile w/0.1% formic acid (solvent B); elution method: flow 1 mL/min, gradient 0.0-1.0 min, 5% B; 1.0-7.0 min, 5-100% B; 7.0-8.0min, 100% B; 8.0-10.0 min, 100-5% B; runtime 10 min. Mass spectroscopy (MS) apparatus: Waters ACQUITY QDa detector (single quad mass detector) equipped with an electrospray ionization (ESI) interface. Spectra have been scanned between 200 and 800 Da with values every 0.1 seconds and peaks are given as mass/charge ( mlz ) ratio.

The compounds of formula IA of the present invention have been prepared as shown in the following reaction schemes and description thereof. EXAMPLE 1. Preparation of (S,E)-2-(4-((2,4-diaminoquinazolin-6-yl)diazenyl)benzamido)pentanedioic acid (compound IA’ in E-isomer form).

The preparation of the compound IA’ in E-isomer form is summarized below in Scheme 1 :

(E)-IA· Scheme 1. (a) 50 psi H₂, 10% Pd-C, DMF, CH₃COOH, 5 h; (b) Oxone®, DCM, H₂0, 3 h; (c) CH3COOH, 16 h; (d) EtOH, 1 M NaOH, 1 h, from 0 °C to room temperature.

A. Preparation of quinazoline-2, 4, 6-triamine (2):

6-nitroquinazoline-2, 4-diamine (1.50 g, 7.31 mmol), 10% Pd-C (150 mg), DMF (20 mL) and acetic acid (2 mL) were charged into a Parr apparatus and hydrogenated under pressure (50 psi) for 5 hours, during which the reaction mixture turned from an orange to a yellow-greenish color. The catalyst was then filtered off through Celite^® and the filtrate was concentrated under reduced pressure. Ethyl acetate (100 mL) was added to the concentrated residue and the resulting suspension was stirred for 30 minutes. The yellow-greenish solid was filtered, washed with fresh ethyl acetate (3x10 mL) and dried under vacuum to afford 1.25 g of quinazoline- 2, 4, 6-triamine ((2); 98% yield).

¹H NMR (400 MHz, DMSO-de) d 7.99 - 6.98 (m, 3H).

¹³C NMR (101 MHz, DMSO-de) d 162.50, 153.36, 145.81 , 130.00, 124.04, 117.41 , 110.12, 105.16.

B. Preparation of (S)-diethyl 2-(4-nitrosobenzamido)pentanedioate (3):

To a solution of (S)-diethyl 2-(4-aminobenzamido)pentanedioate (4.00 g, 12.41 mmol) in DCM (70 mL) was added a solution of Oxone^® (7.63 g, 24.82 mmol) in water (210 mL) and the resulting mixture was stirred under nitrogen at room temperature for 3 h, during which the reaction mixture turned from pale yellow to green (TLC in cyclohexane/ethyl acetate = 6:4). After separation of the layers, the aqueous phase was extracted with DCM (2x150 mL). The combined organic layers were washed with 1 M HCI, saturated sodium bicarbonate solution, water and brine, dried over magnesium sulfate and evaporated to dryness to afford 3.80 g of (S)-diethyl 2-(4-nitrosobenzamido)pentanedioate as a dark green solid which was immediately used in the following step without further purification ((3); 91% yield). /¾ = 0.46 (TLC in cyclohexane/ethyl acetate = 6:4).

C. Preparation of (S,E)-diethyl 2-(4-((2,4-diaminoquinazolin-6-yl)diazenyl)benzamido)pentanedioate (4):

A solution of quinazoline-2, 4, 6-triamine (2) obtained in step A (0.50 g, 2.85 mmol) and (S)-diethyl 2-(4- nitrosobenzamido)pentanedioate (3) obtained in step B (1.44 g, 4.28 mmol) in acetic acid (100 mL) was stirred under nitrogen at room temperature for 16 h (TLC in DCM/MeOH = 8:2, desired final compound identified as a yellow spot). The dark red mixture was then filtered to remove unreacted quinazoline, evaporated under vacuum to dryness and purified via column chromatography (mobile phase DCM/MeOH with a gradient from 95:5 to 8:2) to afford 0.77 g of (S,£)-diethyl 2-(4-((2,4-diaminoquinazolin-6- yl)diazenyl)benzamido)pentanedioate as an orange solid ((4); 55% yield). R_f = 0.46 (TLC in DCM/MeOH = 8:2).

¹H NMR (400 MHz, Methanol-^) d 8.67 (d, J = 2.1 Hz, 1H), 8.23 (dd, J = 9.0, 2.1 Hz, 1 H), 8.07 - 7.95 (m, 4H), 7.43 (d, J = 9.0 Hz, 1 H), 4.67 (dd, J = 9.4, 5.2 Hz, 1 H), 4.23 (q, J = 7.1 Hz, 2H), 4.13 (p, J = 7.2 Hz, 2H), 2.53 (t, J = 7.2 Hz, 2H), 2.32 (m, 1 H), 2.14 (m, 1H), 1.30 (t, J = 7.1 Hz, 3H), 1.24 (t, J = 7.1 Hz, 3H).

¹³C NMR (101 MHz, DMSO-de) d 172.17, 171.66, 165.99, 163.19, 163.17, 153.67, 146.33, 135.25, 128.87, 126.76, 125.06, 122.03, 121.87, 109.85, 108.84, 60.63, 59.95, 52.15, 30.19, 25.69, 14.08.

D. Preparation of (S,£)-2-(4-((2,4-diaminoquinazolin-6-yl)diazenyl)benzamido)pentanedioic acid (compound (I A’) in E- isomer form)

In the dark, to a solution of (S,£)-diethyl 2-(4-((2,4-diaminoquinazolin-6-yl)diazenyl)benzamido)pentanedioate (4) obtained in step C (700 mg, 1.42 mmol) in ethanol (50 mL) at 0 °C was added 1 M NaOH (50 mL) and the resulting mixture was stirred at room temperature for 1 h (TLC in DCM/MeOH = 8:2 to monitor the disappearance of the starting material). The reaction mixture was concentrated to remove the ethanol and diluted with 10 mL additional water. 2 M HCI was added under stirring at 0 °C to adjust the pH to 4 causing the precipitation of an orange solid. The mixture was allowed to settle under refrigeration for about 10 min, and then the orange solid was collected on paper by suction filtration, scraped into a round-bottom flask, triturated with diethyl ether (3 x 30 mL) and finally dried under vacuum to afford 587 mg of (S,E)-2-(4-((2,4- diaminoquinazolin-6-yl)diazenyl)benzamido)pentanedioic acid as an orange solid ((IA’) in E-isomer form; 95% yield).

¹H NMR (400 MHz, Methanol-^) d 8.80 (d, J = 2.0 Hz, 1H), 8.39 (dd, J = 8.9, 2.1 Hz, 1 H), 8.10 - 7.96 (m, 4H), 7.62 - 7.55 (m, 1 H), 4.68 (dd, J = 9.4, 4.9 Hz, 1 H), 2.52 (t, J = 7.5 Hz, 2H), 2.35 (m, 1 H), 2.14 (m, 1 H).

¹³C NMR (101 MHz, Methanol-^) d 176.59, 174.95, 169.50, 165.62, 156.77, 155.43, 150.39, 142.76, 137.77, 129.83, 129.17, 123.95, 123.21 , 119.13, 111.31 , 53.94, 31.57, 27.62.

R_t (HPLC-PDA) = 4.48 min.

MS calculated for C2oH2oN₇05⁺ [M+H]: 438, found: 438.

EXAMPLE 2. Preparation of (S,Z)-2-(4-((2,4-diaminoquinazolin-6-yl)diazenyl)benzamido)pentanedioic acid (compound (IA’) in Z-isomer form).

The preparation of the compound IA’ in Z-isomer form is summarized below in Scheme 2:

(E)-IA· (Z)-IA·

Scheme 2. Light (375 nm), MeOH, 15 min.

A solution of (S,£)-2-(4-((2,4-diaminoquinazolin-6-yl)diazenyl)benzamido)pentanedioic acid (compound IA’ in E-isomer prepared in Example 1) in methanol (volume < 3.5 mL, concentration < 3 mM) was placed into a standard quartz cuvette (pathlength 10 mm, size 45 mm ^c 12.5 mm ^c 12.5 mm) in a dark room and irradiated at a distance of 10 mm or less with a LED light source (measured power > 2 mW, wavelength range 370-380 nm, maximum wavelength 375 nm, focus size 150 mm x 80 mm) for 15 min. The solution was then transferred into a suitable flask and the solvent was removed by evaporation or freeze drying to afford an orange mixture of (S,E)-2-(4-((2,4-diaminoquinazolin-6-yl)diazenyl)benzamido)pentanedioic acid and (S,Z)-2- (4-((2,4-diaminoquinazolin-6-yl)diazenyl)benzamido)pentanedioic acid in a ratio of 1 :3.

¹H NMR (400 MHz, Methanol-^) d 7.86 (d, J = 2.1 Hz, 1 H), 7.81 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 8.8 Hz, 1 H), 7.19 (dd, J = 8.8, 2.0 Hz, 1 H), 6.99 (d, J = 8.5 Hz, 2H), 4.59 (dd, J = 9.4, 5.0 Hz, 1 H), 2.46 (t, J = 7.4 Hz, 2H), 2.31 - 2.24 (m, 1 H), 2.11 - 1.99 (m, 1 H).

R_t (HPLC-PDA) = 4.29 min. EXAMPLE 3. Preparation of (S,E)-2-(4-((2,4-diaminopteridin-6-yl)diazenyl)benzamido)pentanedioic acid (compound (IA”) in E- isomer form).

The preparation of the compound (IA”) in E-isomer form is summarized in Scheme 3:

(E)- IA"

Scheme 3. (a) CH₃COOH; (b) aqueous NaOH, EtOH. A. Preparation of the compound of formula 6

Alternative A: the preparation of compound 6 is summarized in Scheme 4:

5 6 Scheme 4. (a) guanidine, Na, MeOH; (b) Zn, HCI, DCM or H , Pd-C, MeOH or Na S, MeOH.

The compound of formula (6) is prepared in two steps following the synthetic pathway depicted in Scheme 4. In the first step (a), intermediate (5) is obtained by reacting freshly prepared methanolic sodium methoxide or ethanolic sodium ethoxide with a guanidine salt in a standard reactor or flask followed by addition of 3-amino- 6-nitropyrazine-2-carbonitrile and stirring the corresponding mixture at room temperature or upon heating until completion of the reaction. In the second step (b), intermediate (5) is converted into compound (6) by:

(i) reduction of the nitro group with zinc in the presence of dichloromethane in a standard reactor or flask at a temperature between -30 °C and 100 °C. This step can be also performed using iron, tin or tin (II) chloride as a metal or metal salts instead of Zn; and using ethyl acetate, ethanol, methanol, isopropyl alcohol, tetrahydrofuran, N,/\/-dimethylformamide or acetic acid as a solvent instead of dichloromethane. Further, step (b) can be optionally carried out in the presence of an acid, for instance HCI, H₂SO₄ and acetic acid.

(ii) reduction of the nitro group in a hydrogen atmosphere with a pressure between 1 and 5 bar in the presence of a palladium catalysts in the presence of methanol in a standard hydrogenation reactor at a temperature between 0 °C and 50 °C. This step can be also performed using a catalyst comprising rhodium, platinum, copper, vanadium or iron instead of palladium; and using ethanol, isopropyl alcohol, tetrahydrofuran, N,/\/-dimethylformamide, water or 1 ,4-dioxane as a solvent instead of methanol.

(iii) reduction of the nitro group with sodium sulfide in the presence of methanol in a standard reactor at a temperature between 0 °C and 150 °C. This step can be also performed using sodium hydrogensulfide as a sulfide salt instead of sodium sulfide; and using ethanol, isopropyl alcohol, tetrahydrofuran, N,N- dimethylformamide, water or 1 ,4-dioxane as a solvent instead of methanol. This step can be optionally prepared by reduction with sulfide hydrates. Alternative B: the preparation of compound 6 is summarized in Scheme 5:

Scheme 5. (a) NH₃, MeOH.

The compound of formula (6) is prepared in one step as depicted in Scheme 5 starting from commercially available 6-chloropteridine-2, 4-diamine (Enamine, EN300-319515) with ammonia or ammonium hydroxide in methanol in a standard reactor or flask between 0 °C and 120 °C. This reaction can be also carried out using ethanol, isopropyl alcohol, tetrahydrofuran, N,/\/-dimethylformamide, water or 1 ,4-dioxane as a solvent instead of methanol.

B. Preparation of the compound (IA”) in E-isomer form

The compound (6) prepared in previous section and the compound (3) prepared in Example 1 section B react in presence of acetic acid in a standard reactor or flask at room temperature or upon heating (Mills reaction), to afford the intermediate compound (7). In a second step (b), compound (7) is hydrolized in aqueous solution of NaOH in a standard reactor or flask at a temperature between 0 °C and 110 °C, to afford final compound (S,E)-2-(4-((2,4-diaminopteridin-6-yl)diazenyl)benzamido)pentanedioic acid [(E)-(IA”)]. Step b can be also carried out using KOH or LiOH instead of NaOH. This step can be optionally carried out in the presence of an additional solvent such as methanol, ethanol, isopropyl alcohol, tetrahydrofuran and 1,4-dioxane.

EXAMPLE 4. Preparation of (S,Z)-2-(4-((2,4-diaminopteridin-6-yl)diazenyl)benzamido)pentanedioic acid (compound (IA”) in Z-isomer form):

The preparation of the compound compound (IA”) in Z-isomer form is summarized in Scheme 6:

(E)- IA" (Z)-IA"

Scheme 6. Light (315-400 nm), MeOH, 15 min.

Compound (IA”) in Z-isomer (in a diastereomeric mixture with the corresponding E-isomer) is prepared in one step by irradiating for 15 min a solution of (S,E)-2-(4-((2,4-diaminopteridin-6- yl)diazenyl)benzamido)pentanedioic acid in methanol in a standard quartz cuvette in a dark room equipped with a light source emitting light at a wavelength comprised between 315 nm and 400 nm.

EXAMPLE 5. Preparation of (S,E)-2-(4-((2-amino-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-c(]pyrimidin-5- yl)diazenyl)benzamido)pentanedioic acid (compound (IB’) in E- isomer form).

The preparation of the compound (IB’) in E-isomer form) is summarized in Scheme 7:

Scheme 7. (a) NH₃, MeOH; (b) CH₃C00H; (c) aqueous NaOH, EtOH.

In the first step (a), 2-amino-5-bromo-1 H-pyrrolo[2,3-d]pyrimidin-4(7H)-one or alternatively 2-amino-5-iodo-1 H- pyrrolo[2,3-d]pyrimidin-4(7H)-one, which are both commercially available (Oxchem, AX8271656 and AX8271657), is converted into the corresponding amino derivative (8) by reaction with ammonia or ammonium hydroxide in methanol in a standard reactor or flask at a temperature between 0 °C and 120 °C. Step (a) can be carried out using ethanol, isopropyl alcohol, tetrahydrofuran, N,/\/-dimethylformamide, water or 1 ,4-dioxane as a solvent instead of methanol..

In the second step (b), the compound (8) thus obtained and compound (3) obtained in Example 1 section B react in presence of acetic acid in a standard reactor or flask at room temperature or upon heating (Mills reaction), to afford the intermediate compound (9). Finally, in the third step (c), compound (9) thus obtained is hydrolized in aqueous solution of NaOH in a standard reactor or flask at a temperature between 0 °C and 110 °C, to afford final compound (S,E)-2-(4-((2- amino-4-oxo-4,7-dihydro-1H-pyrrolo[2,3-c(]pyrimidin-5-yl)diazenyl)benzamido)pentanedioic acid [(E)-(IB’)].

Step (c) can also be carried out with KOH or LiOH, instead of NaOH; and optionally in presence of an additional solvent such as methanol, ethanol, isopropyl alcohol, tetrahydrofuran or 1 ,4-dioxane. EXAMPLE 6. Preparation of (S,Z)-2-(4-((2-amino-4-oxo-4,7-dihydro-1 /--pyrrolo[2,3-c/]pyrimidin-5- yl)diazenyl)benzamido)pentanedioic acid (compound (IB’) in Z-isomer form).

The preparation of the compound (IB’) in Z-isomer form is summarized in Scheme 8:

Scheme 8. Light (315-400 nm), MeOH, 15 min.

Compound (IB’) in Z-isomer form is prepared in one step by irradiating a solution of (S,E)-2-(4-((2-amino-4- oxo-4, 7-dihydro-1H-pyrrolo[2,3-c(]pyrimidin-5-yl)diazenyl)benzamido)pentanedioic acid in methanol for 15 min in a standard quartz cuvette in a dark room equipped with a light source emitting light at a wavelength comprised between 315 nm and 400 nm.

EXAMPLE 7: Pharmacological Experiments Antifolate activity, cytotoxicity and safety of the compounds of the present invention, particularly of the compound IA’, were assessed by: 1) in vitro enzyme inhibition assays with purified dihydrofolate reductase, 2) in vitro viability assays on a HeLa cell line, and 3) in vivo toxicity assay on zebrafish (Danio rerio) larvae.

7A. Dihydrofolate reductase (DHFR) inhibition assays.

Materials and methods: DHFR activity was measured using the DHFR assay kit (CS0340, Sigma-Aldrich) as per the manufacturer's instructions (unless otherwise stated) using Nunc 96-Well MicroWell microplates (Thermo Scientific) and a BMG FLUOstar OPTIMA microplate reader measuring the decrease in absorbance at 340 nm every 15 sec over 2.75 min. The extent of enzyme inhibition was expressed as the percentage of remaining enzyme activity (slope) compared with the control (100%). Photoisomerization of the tested photochromic compound was achieved by illuminating a 300 nM stock solution of the specimen following the procedure described in Example 2. All experiments were performed in dim light. Data are means of at least three independent experiments performed in triplicate or quadriplicate ± SEM. Results were analyzed by two- way ANOVA with Bonferroni's multiple comparisons test for statistical significance. Results: Compound IA’ of the present invention was preliminarily tested in“in vitro” enzyme experiments to assess its capacity to inhibit DHFR activity in both states: the all-E isomer form in the dark versus the Z isomer-enriched form obtained after ultraviolet irradiation. A statistically significant difference in the inhibitory effect against DHFR activity was observed between the two forms at 10 nM and 100 nM, notably with a stronger inhibitory capacity of the Z-enriched form, which inhibited DHFR activity by more than 70% at both concentrations, against the about 21 % and about 34% respectively observed with the all-E isomer form (cf.

Fig. 1).

7B. Cytotoxicity assays.

Materials and methods: Cytotoxicity of the compounds of the present invention, particularly the compound IA’ was evaluated in“in vitro” by MTT assay for cell viability using Nunc 96-Well MicroWell microplates (Thermo Scientific) and a Thermo Scientific Multiskan FC microplate reader. MTT (thiazolyl blue tetrazolium bromide) was purchased from Sigma-Aldrich (M5655). Immediately before the treatment, to obtain the Z-enriched form of compound IA’, a stock solution of this compound was irradiated as described above in Example 2.

Methotrexate (A6770, Sigma-Aldrich) was also tested for comparison as the positive control. Data are means of at least three independent experiments performed in triplicate or quadriplicate ± SEM (unless otherwise stated). Results were analyzed by two-way ANOVA with Bonferroni's multiple comparisons test for statistical significance (GraphPad Prism 6). IC50 values were estimated by a nonlinear regression analysis

[log(inhibitor) versus response with variable slope (four parameters)] in GraphPad Prism 6. All experiments were performed in dim light.

HeLa (human cervix adenocarcinoma ) cell line : HeLa cells were cultured in DMEM (31885-023, Thermo Scientific) with 10% fetal bovine serum and 1% streptomycin/penicillin. Cells were dispensed in a sterile 96- well plate at a cell density of 5000 cells/well (160 pL/well) and maintained at 37 °C and 5% CO2 for 24 hours before treatments. For background wells, no cells were seeded. Then the medium was aspirated and replaced by 160 pL of fresh medium containing different concentrations of the inhibitors. Control wells received the same amount of the vehicle (0.1 mM NaOH) of the inhibitors. After 24 hours of incubation at 37 °C and 5% CO2, medium was carefully removed and each well was washed with 200 pL of phosphate buffered saline (PBS). Then 200 pi of MTT (0.5 mg/mL) in PBS were added in the dark to each well and microplates were incubated at 37 °C for 4 hours. The medium was cautiously aspirated and formazan crystals were dissolved in DMSO (200 pl/well). Microplates were covered with aluminum foil and gently shaken for 15 minutes (orbital shaking), then absorbance was measured at 570 nm. Cytotoxicity was expressed as a relative viability of cells compared to control wells.

Results: The global cytotoxic activity of compound IA’ was evaluated by cell viability assays in a HeLa cancer cell line. Cells were incubated with the all-E isomeric form or the Z-isomer enriched form at different concentrations followed by measurement of cell viability. Methotrexate was also tested in the same conditions as the positive control (cf. Fig. 2). Notably, compound IA’ in the Z-enriched form killed about 73% of the cells at 1 mM, whereas the all-Eform of compound IA’ gave only a poor decrease of cell viability (-12%). About 77% of cell growth inhibition was obtained at the maximum concentration tested (100 mM) for the Z-enriched form, against the about 22% given by the E isomer. We estimated an IC50 of about 6 nM for the Z-enriched form against an IC50 of about 34 pM for the pure E-isomer. More similarly to the Z-enriched mixture, the reference drug methotrexate killed about 60% of the cells at 1 pM and about 67% of the cells at the maximum concentration tested (100 pM).

In conclusion, the Z-enriched form of compound IA’ resulted significantly more effective in killing HeLa cancer cells than the E-isomer, i.e. the Z-isomer displayed a higher antiproliferative activity than the E-isomer. In view of the results of Fig.1 on DHFR activity assays, this effect can be reasonably ascribed to the disruption of the folate pathway, which plays an essential role in the synthesis of DNA and RNA, in the metabolism of amino acids, and ultimately in the process of cell division. Considering the fact that compound IA’ in Z-isomeric state has a relatively short half-life (about 218 minutes) of thermal relaxation to the E-isomer in the test conditions (incubation at 37 °C), the differences observed in terms of pharmacological activity are quite remarkable.

7C. In vivo toxicity test.

Materials and methods: The safety profile of the compounds of the present invention, particularly the compound IA’, was evaluated by observation of its effects on the development of zebrafish (Danio rerio) embryos. Danio rerio (Tupfel-Lon) fertilized eggs were placed in a 12-well plate (TPP tissue culture test plate, cat. 92024) with 1 mL of treatment solution per well [4 treatment groups: vehicle VEH (dimethylsulfoxide, DMSO), methotrexate MTX 200 pM, compound IA’ (E-isomer) 200 pM, and compound IA’ (Z-enriched form) 200 pM],

Immediately before the treatment, to obtain the Z-enriched form of compound IA’, a stock solution of this compound was irradiated as described above in Example 2. Methotrexate (A6770, Sigma-Aldrich) was tested for comparison. Treatment solutions were daily prepared from stocks and replaced every twenty-four hours during five consecutive days. Before solution replacement, every individual embryo was observed, analyzed and described in terms of developmental aspects, such as period and stages. Once larvae were developed, behavioral traits were also analyzed and described in addition to their anatomical development. For statistical purposes, acquired data were interpreted in terms of anatomical development and mortality. Total number of embryos refers to the number of fertilized embryos placed per treatment at zero hours post-fertilization (hpf). Viability is described as the number of fertilized embryos whose development has not been interrupted during the first twenty-four hours and therefore no decomposition occurred. Notwithstanding, viability does not describe any anatomical trait and includes any embryo that reached the pharyngula period according to Kimmel et al. (cf. Kimmel SR et al.“Stages of embryonic development of the zebrafish”. Developmental Dynamics, 1995, vol. 203(3), pp. 253-310).“Mortality” refers to the number of dead larvae at ninety-six hours, hence the number of deceased hatched larvae, regardless their anatomical development.“Abnormality” is referred as any number of aberrant anatomical deviations observed in a hatched individual in comparison to the development described by Kimmel et al. For our purposes, three specific aberrant anatomical traits have been observed: one affecting the pigmentation pattern of the larvae, another referring to the volume of the heart cavity, and a third trait which includes the bending or the turning of the larva tail. Results: At 72 hpf, hatched larvae should have reached the stage of protruding-mouth (cf. Kimmel SR et al.“Stages of embryonic development of the zebrafish”. Developmental Dynamics, 1995, vol. 203(3), pp. 253-310), with an embryo length of 3.5 mm. Anatomical traits would include iridiophores in the yolk and half of the eye, with yellow pigmentation on the head and in the dorsal part and black pigmented lateral and ventral stripes from the head to the tail, as well as observable heart beat and blood flow. Embryos treated with methotrexate presented a low viability at 24 hpf, with six out of seventeen total embryos viable. At 72 hpf, all six individuals presented three abnormalities: a deficient iridiophore ocular pigmentation, with complete black colored eyes and a deficient black body stripe patterns; an abnormal volume of the cardiac cavity; and tail angle deviations, with prominent bent or curved tails. Only one larva from the vehicle group (VEH treatment) presented malformation, a non-hatched individual with unformed tail. Viability in the vehicle group (VEH) and Z-IA’ groups was high (only two and one non-viable embryos, respectively) and perfect in the E-IA’ group (Fig. 3).

Overall, compound IA’ in the Z-enriched form showed the same toxicity as the reference compound methotrexate, producing the same kind of abnormalities to the animal development (72 hpf) together with a high rate of mortality (96 hpf), whereas the pure E-IA (all-E isomer) showed a general lower toxicity, in fact it did not produce any abnormality at 72 hpf and no mortality at 96 hpf (Fig. 4).

EXAMPLE 8. Preparation of (S,E)-2-(4-((4,6-diaminobenzo[b]thiophen-3-yl)diazenyl)benzamido)pentanedioic acid (compound (IB”) in E-isomer form).

The preparation of the compound (IB”) in E-isomer form) is summarized in Scheme 9:

Scheme 9. (a) CH3COOH; (b) aqueous NaOH, EtOH. In the first step (a), commercially available benzo[b]thiophene-3, 4, 6-triamine (Ambinter, Amb24611355) and compound (3) obtained in Example 1 section B react in presence of acetic acid in a standard reactor or flask at room temperature or upon heating (Mills reaction), to afford the intermediate compound (10). In the second step (b), compound (10) thus obtained is hydrolized in aqueous solution of NaOH in a standard reactor or flask at a temperature between 0 °C and 110 °C, to afford final compound (S,E)-2-(4-((4,6-diaminobenzo[b]thiophen-3- yl)diazenyl)benzamido)pentanedioic acid [(E)-(IB”)]. Step (b) can also be carried out with KOH or LiOH, instead of NaOH; and optionally in presence of an additional solvent such as methanol, ethanol, isopropyl alcohol, tetrahydrofuran or 1,4-dioxane.

EXAMPLE 9. Preparation of (S,Z)-2-(4-((4,6-diaminobenzo[b]thiophen-3-yl)diazenyl)benzamido)pentanedioic acid (compound (IB”) in Z-isomer form).

The preparation of the compound (IB”) in Z-isomer form is summarized in Scheme 10:

Scheme 10. Light (350-460 nm), MeOH, 15 min.

Compound (IB”) in Z-isomer form is prepared in one step by irradiating a solution of (S,E)-2-(4-((4,6- diaminobenzo[b]thiophen-3-yl)diazenyl)benzamido)pentanedioic acid in methanol for 15 min in a standard quartz cuvette in a dark room equipped with a light source emitting light at a wavelength comprised between 350 nm and 460 nm.

References Cited In The Application

1. Pittolo S et al.“An allosteric modulator to control endogenous G protein-coupled receptors with light”. Nature Chemical Biology, 2014, vol. 10, pp. 813-815.

2. Wuts PGM and Greene TW.“Greene's Protective Groups in Organic Synthesis”. Chapter 2, Protection for the Hydroxyl Group, Including 1 ,2- and 1 ,3-Diols, John Wiley & Sons, Inc., 4^th edition, Hoboken, NJ, USA,

2006, pp. 16-299.

3. Hamblin MR et al.“Handbook of Photomedicine”, Chapter 19,“PUVA Therapy”, Chapter 22,“Recent Advances in Developing Improved Agents for Photodynamic Therapy”, Chapter 40,“Photodynamic Therapy in Dermatology”, CRC Press, Boca Raton, FL, USA, 2013, pp. 197-204, pp. 227-266, pp. 465-474.

4. E. Merino,“Synthesis of azobenzenes: the coloured pieces of molecular materials”, Chem. Soc. Rev. 2011, vol. 40, pp. 3835-3853.

5. Kimmel SR et al.“Stages of embryonic development of the zebrafish”. Developmental Dynamics, 1995, vol. 203(3), pp. 253-310.

Claims

1. A compound of formula (I) in Z-isomer form

or alternatively a compound of formula (I) in E-isomer form

or a pharmaceutically acceptable salt thereof, wherein:

is selected from the group consisting of a radical of formula (II)

and a radical of formula (III)

Xi, X2, X3, X4_, and X5 are independently selected from the group consisting of CR3 and N;

Yi is selected from the group consisting of NH, 0 and S; each R-i, each R3 and R7 are indepentently selected from the group consisting of H, halogen, -OH, -0-(Cr C₈)alkyl, -N((Ci-C₈)alkyl)₃, -NH((Ci-C₈)alkyl)₂, and -NH₂(Ci-C₈)alkyl;

R₄, R₅ and R6 are indepentently selected from the group consisting of H and (Ci-Ce)alkyl;

— line indicates the position of R2 radical by which R2 is attached to the adjacent N; and the asterisk indicates a stereogenic carbon atom being in a form selected from R-isomer, S-isomer and a mixture of R-isomer and S-isomer.

2. The compound according to claim 1 , wherein each R1 is H.

3. The compound according to any of the claims 1-2 wherein R2 is a radical of formula (II).

4. The compound according to claim 3, wherein R₄ and R₅ are H; X-i, X₃, X₄ and X₅ are N; X₂ is CR₃; and each

R3 is selected from the group consisting of H and OH.

5. The compound according to any of the claims 3-4, wherein R₄ and R₅ are H; X-i, X₂ and X₃ are CR₃; X₄ and Xs are N; and each R3 is H.

6. The compound according to any of the claims 1-2, wherein R2 is a radical of formula (III).

7. The compound according to claim 6, wherein R2 is a radical of formula (III) selected from the group consisting of (IMA)

(111 A)

and (NIB)

wherein:

Y2 is selected from the group consisting of NH, 0 and S;

Y₃ is selected from the group consisting of NH and C;

Ce and X7 are independently selected from the group consisting of CR3 and N; Re is selected from the group consisting of H, SH, -N((Ci-Ce)alkyl)3, -NH((Ci-Ce)alkyl)2, and -NH2(Ci-Ce)alkyl; and

R₃, Re, R₇ and Y₁ are as defined in any of the claims 1-6.

8. The compound according to claim 7, wherein R₂ is a radical of formula (IMA) and each R₆ and R₇ are H; C_d is N; and Y₁ and Y₂ are NH.

9. The compound according to claim 7, wherein R₂ is a radical of formula (NIB) and Re is selected from the group consisting of SH and NH2; Y1 is selected from the group consisting of S, 0 and NH; Y3 is C; X7 is CR3; and R₃ is H.

10. The compound according to claim 7, wherein R2 is a radical of formula (NIB) and Re is H; Y1 is selected from the group consisting of S, 0 and NH; Y3 is N; and X7 is N.

11. A composition comprising a therapeutically effective amount of a compound of formula (I) in Z-isomer as defined in any of the claims 1-10; or alternatively an amount of a compound of formula (I) in E-isomer form as defined in any of the claims 1-10 such that after light irradiation gives rise to a therapeutically effective amount of a compound of formula (I) as Z-isomer, together with one or more pharmaceutically acceptable excipients or carriers.

12. The compound of formula (I) in Z-isomer form as defined in any of the claims 1-10 for use as a medicament.

13. The compound of formula (I) in Z-isomer as defined in any of the claims 1-10 for use in the treatment of a disease or condition mediated by the dihydrofolate reductase.

14. The compound of formula (I) in E-isomer form as defined in any of the claims 1-10 for use in the treatment of a disease or condition mediated by the dihydrofolate reductase, wherein the treatment comprises:

(a) first administering an amount of a compound of formula (I) in E-isomer form as defined in any of the claims 1-10 such that after light irradiation gives rise to a therapeutically effective amount of a compound of formula (I) in Z-isomer; and then

(b) light irradiating the treatment area.

15. The compound of formula (I) in E-isomer form as defined in any of the claims 1-10 for use in the treatment of a disease or condition mediated by the dihydrofolate reductase, wherein the treatment comprises:

(a) first exposing an amount of a compound of formula (I) in E-isomer form to light irradiation to obtain the compound of formula (I) in Z-isomer form; and then

(b) administering a therapeutically effective amount of a compound of formula (I) in Z-isomer form obtained in step (a).

16. The compound of formula (I) for use according to any of the claims 13-15, wherein the light is a light having a wavelength comprised from 300 nm to 1000 nm.

17. The compound of formula (I) for use according to any of the claims 13-16, wherein the disease or condition mediated by the dihydrofolate reductase is selected from the group consisting of breast cancer, skin cancer, head cancer, neck cancer, lung cancer, psoriasis and rheumatoid arthritis.