WO2016120432A1

WO2016120432A1 - Compounds and methods for anticoagulation therapy

Info

Publication number: WO2016120432A1
Application number: PCT/EP2016/051892
Authority: WO
Inventors: Mikel ALLENDE RODRÍGUEZ; José HERMIDA SANTOS; Ramón MONTES DÍAZ; Julen Oyarzabal Santamarina
Original assignee: Fundación Para La Investigación Médica Aplicada
Priority date: 2015-01-30
Filing date: 2016-01-29
Publication date: 2016-08-04

Abstract

The invention relates to certain compounds that are inducers of Heat shock 70 kDa protein 1A/1B (HSPA1A/B) and their use for anticoagulation therapy; and to a method for anticoagulation therapy that comprises the administration of one of these inducer compounds. It has been here proved that induction of Heat shock 70 kDa protein 1A/1B by administration of one of these inducer compounds has antithrombotic effects without accelerating or altering bleeding time.

Description

COMPOUNDS AND METHODS FOR ANTICOAGULATION THERAPY

FIELD OF THE INVENTION

The present invention relates to compounds for use in anticoagulation therapy and methods of anticoagulation therapy which comprises the administration of one of these compounds to a subject that needs anticoagulation.

BACKGROUND ART

Thromboembolic disorders, such as venous thromboembolism (VTE), stroke- associated atrial fibrillation (AF), atherothrombotic stroke and acute coronary syndrome

(ACS) represent today a major health problem that has a great impact on patients' lives.

Several molecules are currently being used as anticoagulation therapies which are effective in treating and preventing thrombosis and thromboembolic events. Among them: coumarin derivatives (vitamin K antagonists, in particular Warfarin); heparin and heparin derivatives (e.g. low molecular weight Heparin, Fondaparinux and Idraparinux); and more recently, oral anticoagulants that are direct factor Xa inhibitors (e.g. rivaroxaban, apixaban and edoxaban, betrixaban, darexaban, letaxaban, and eribaxaban) or direct Thrombin inhibitors (e.g. the bivalent drugs hirudin, lepirudin, and bivalirudin; and the monovalent drugs argatroban and dabigatran).

However, the high frequency of major bleeding associated with anticoagulant drugs is a major concern that somehow limits their clinical use (Wysowski DK et al.

Arch Intern Med 2007; 167: 1414-1419). Several trials have demonstrated that new direct thrombin and coagulation factor X inhibitors are very similar to warfarin in preventing thrombosis. However, although intracranial hemorrhages are less frequent with these new anticoagulants, major bleeding complications are not insignificant (4.9% of AF patients during the trial period) (Dentali F. et al. Circulation 2012; 126:2381-

2391).

Thus, bleeding complications are still a major concern, particularly when considering that the duration of anticoagulant treatment in some patients is indefinite. This problem is even more dramatic in anticoagulated patients that are concomitantly treated with antiplatelet drugs (Steinberg BA et al. Circulation 2013; 128:721-728; Dans AL et al. Circulation 2013; 127:634-640). Due to the threat of these bleeding complications, a significant number of patients that must receive anticoagulant treatment are not treated (Sherwood MW et al. Circulation 2014; 129: 1850-1859).

Thus, development of new molecules that dissociate antithrombotic effect from bleeding is still necessary.

SUMMARY OF THE INVENTION

By conducting gene expression studies in patients that presented with permanent atrial fibrillation (AF), the inventors surprisingly realized that HSPA1B (one of the genes that encodes Heat shock 70 kDa protein 1 A/IB) plays a non-negligible protective effect against cardioembolic stroke in AF patients. Expression levels of HSPA1B gene in these patients were inversely associated with stroke: the risk of cardioembolic stroke decreased in parallel with the increase in HSPA1B expression after adjusting for CHAD index. Prompted by this first realization, they later corroborated in HSPA1A/B KO mice that the absence of Heat shock 70 kDa protein 1A/1B facilitates thrombus formation upon different thrombogenic challenges, although no obvious gross hemostatic alterations were displayed.

The inventors then hypothesized that a compound that directly or indirectly induces Heat shock 70 kDa protein 1 A/IB expression could hopefully prevent or reduce thrombus formation.

To test the hypothesis they assayed several compounds that: a) induce Heat shock factor 1 (HSF1) (compounds TRC051384 and BGP-15); b) inhibit HSP90 (compounds BIIB021, NVP-AUY922, and Geldanamycin); or c) inhibit histone deacetylase 6 (HDAC6) (tubastatin A, Rocillinostat, and recently developed HDAC6 inhibitor compounds 3-11 and 1-16). It was then confirmed and corroborated that these compounds did actually induce HSPA1B expression.

What it is more important, it was also probed that these compounds (TRC051384, tubastatin A and compounds 3-11 and 1-16), when administered to mice, had effective anticoagulant properties and did not produce longer bleeding times than the administration of the vehicle (control), while rivaroxaban treated animals displayed a prolonged bleeding time. This allows concluding that antithrombotic compounds that induce Heat shock 70 kDa protein 1A/1B do not induce bleeding. Therefore, in a first aspect the invention relates to a compound that induces the expression of Heat shock 70 kDa protein 1A/1B for use in anticoagulation therapy; and to the use of a compound that induces the expression of Heat shock 70 kDa protein 1 AJ IB in the preparation of a medicament for use in anticoagulation therapy.

In second aspect, the invention relates to a method for anticoagulation therapy in a subject that comprises administering to the subject a therapeutically effective amount of a compound that induces expression of Heat shock 70 kDa protein 1A/1B and one or more pharmaceutically or veterinary acceptable excipients or carriers, in a subject in need thereof, including a human.

In another aspect, the invention relates to a compound selected from the group consisting of compounds of formulae (3-18) to (3-30) (which are listed in Table 1); and in particular compounds of formulae 3-21, 3-22, 3-23, and 3-26; or a pharmaceutically or veterinary acceptable salt thereof, or any stereoisomer either of the compound of formula (3-18)-(3-30) or of any of its pharmaceutically or veterinary acceptable salts.

In another aspect, the invention relates to a pharmaceutical or veterinary composition which comprises a therapeutically effective amount of a compound selected from the group consisting of compounds of formulae (3-18) to (3-30), or a pharmaceutically or veterinary acceptable salt thereof, or any stereoisomer either of the compound of formula (3-18)-(3-30) or of any of its pharmaceutically or veterinary acceptable salts, together with one or more pharmaceutically or veterinary acceptable excipients or carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Effect of HSPA1A/B deficiency on thrombus formation and bleeding. (A) The time to carotid artery occlusion was determined in HSPA1A/B KO mice and their wild-type (WT) counterparts after Rose Bengal/laser light exposure. (B) The same end point was used after exposing HSPA1A/B KO and WT mice to ferric chloride. In (A) and (B), the median times to occlusion for each group are represented by horizontal bars. (C) The time to death caused by collagen/epinephrine-induced pulmonary thromboembolism was monitored for 30 minutes in HSPA1A/B KO (n=l l) and WT (n=9) mice. The Kaplan-Meier survival curves are represented for each group. (D) HSPA1A/B KO mice were subjected to a tail bleeding time assay, and the results compared with those obtained in WT mice. The median value of the tail bleeding time is indicated for each group. The P values for each comparison are also indicated.

Figure 2. Induction of HSPA1B expression in vivo. (A) TRC051384 (compound 7-01) increased HSPA1B expression in aortic tissue after i.p. administration of 9 mg / Kg of TRC051384 3 and 1 hours before sacrifice. The relative quantitation (RQ) (median and interquartile range), relative to vehicle treated mice is shown. The P value for the comparison is also indicated. (B) Aortic tissues from mice treated with TRC051384 were lysed and analyzed by western blot to confirm the increase in intracellular Heat shock 70 kDa protein lA/lB (Hsp70) levels. A representative experiment is shown.

Figure 3. Effect of TRC051384 on thrombus formation. Mice were administered 9 mg / Kg of TRC051384 (compound 7-01) 3 and 1 hours before the start of the thrombosis experiments. Subsequently, the time to carotid artery occlusion was determined and compared with their non-treated counterparts after (A) carotid artery exposure to Rose Bengal / laser light and (B) carotid artery exposure to ferric chloride. The median times to occlusion for each group are represented by horizontal bars. (C) The time to death caused by collagen/epinephrine-induced pulmonary thromboembolism was also monitored for 30 min in TRC051384 (n=l l) and vehicle- treated (n=13) mice. The Kaplan-Meier survival curves are represented for each group. (D) HSPA1A/B KO mice were also administered TRC051384 as above, and subsequently, together with a group of WT counterparts, carotid artery exposed to ferric chloride to induce thrombotic vessel occlusion. The median times to occlusion for each group are represented by horizontal bars. The P values for each comparison are also indicated.

Figure 4. Effect of BGP-15 on thrombus formation. Mice were administered

20 mg / Kg of BGP-15 (compound 7-02) once a day for 5 days before the start of the thrombosis experiment. The time to carotid artery occlusion was compared with their non-treated counterparts after carotid artery exposure to Rose Bengal / laser light. The median times to occlusion for each group are represented by horizontal bars. The P value for the comparison is also indicated.

Figure 5. Effect of tubastatin A on thrombus formation. Mice were administered 20 mg / Kg of tubastatin A (compound 6-04) 8 hours before the start of the thrombosis experiments. The time to carotid artery occlusion was compared with their non-treated counterparts after (A) carotid artery exposure to Rose Bengal / laser light, and (B) carotid artery exposure to ferric chloride. The median times to occlusion for each group are represented by horizontal bars. (C) The time to death caused by collagen / epinephrine-induced pulmonary thromboembolism was also monitored for 30 min in tubastatin A (n=9) and vehicle-treated (n=10) mice. The Kaplan-Meier survival curves are represented for each group. The P values for each comparison are also indicated.

Figure 6. Effect of compound 3-11 on thrombus formation. Mice were administered 20 mg / Kg of the molecule 1 hour before starting the thrombosis experiments. The time to carotid artery occlusion was compared with their non-treated counterparts after (A) carotid artery exposure to Rose Bengal / laser light, and (B) carotid artery exposure to ferric chloride. The median times to occlusion for each group are represented by horizontal bars. (C) The time to death caused by collagen / epinephrine-induced pulmonary thromboembolism was also monitored for 30 min in mice treated with compound 3-11 (n=10) and vehicle-treated (n=10) mice. The Kaplan- Meier survival curves are represented for each group. The P values for each comparison are also indicated.

Figure 7. Effect of compound 1-16 on thrombus formation. Mice were administered 20 mg / Kg of the molecule 3 hours before starting the thrombosis experiment. The time to carotid artery occlusion was compared with their non-treated counterparts after carotid artery exposure to Rose Bengal / laser light. The median times to occlusion for each group are represented by horizontal bars. The P value for the comparison is also indicated.

Figure 8. Effect of HSPA1B inducer compounds on the tail bleeding time. Mice treated with TRC051384 (7-01) (A), tubastatin A (6-04) (B), compound 3-11 (C) or compound 1-16 (D) according to the dose pattern followed in the previously performed thrombosis experiments, were subjected to the tail bleeding time assay, and the results compared with those obtained in vehicle-treated mice. The median value of the tail bleeding time is indicated for each group. The P values for each comparison are also indicated.

Figure 9. Effect of rivaroxaban treatment on tail bleeding time. Mice were treated with 3 mg / Kg rivaroxaban or vehicle 1 hour before the tail bleeding time assay. The median value of the tail bleeding time is indicated for each group. The P values for each comparison are also indicated.

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.

According to the present invention, the term "carbocyclic" ring system refers to a known ring system wherein all the ring members contain carbon atoms. The term "heterocyclic" ring system refers to a known ring system wherein one or more of the ring members, preferably 1, 2, 3, or 4 ring members, are selected from NH, N, O, and S, where chemically possible. The remaining ring members of the heterocyclic ring are independently selected from C, CH, CH₂, O, N, NH, and S. Unless otherwise specified, the "heterocyclic" ring system may be attached to the rest of the molecule through a C or a N atom of the ring system. Both the carbocyclic and heterocyclic rings can be saturated or partially unsaturated, and may be unsubstituted or substituted as described herein, being the substituents placed on any available position.

The term "polycyclic" ring refers to a ring system which is formed by two, three or four rings which can be fused, bridged-fused, spiro-fused or can contain different types of fusion. For the purposes of the present invention, in "fused" rings the fusion occurs through one bond which is common to two adjoining rings; in "bridged-fused" rings the fusion occurs through a sequence of atoms (bridgehead) which is common to two rings; and in "spiro-fused" rings, the fusion occurs through only one atom (spiro atom), preferably a carbon atom, which is common to two adjoining rings (including bridged rings).

The term "heteroaromatic" ring refers to a known aromatic ring system, wherein one or more of the ring members, preferably 1, 2, 3, or 4 ring members, are selected from NH, N, O, and S, where chemically possible. The remaining ring members of the heteroaromatic ring are independently selected from C, CH, O, N, NH, and S. The heteroaromatic ring may be unsubstituted or substituted as described herein, being the substituents placed on any available position.

The term "known ring system" refers to a ring system which is chemically feasible and is known in the art and so intends to exclude those ring systems that are not chemically possible.

For the purposes of the present invention, in all saturated or partially unsaturated rings, one or two members of the rings are optionally C(=0) and/or C(=NH) and/or C[=N(Ci-C₄)alkyl].

The term linear or branched, saturated or unsaturated (Ci-C_n)alkyl refers to a linear or branched hydrocarbon chain which contains from 1 to n carbon atoms. When the (Ci-C_n)alkyl is saturated it contains only single bonds. When the (Ci-C_n)alkyl is unsaturated it contains one or two double bonds and/or one or two triple bonds. The saturated or unsaturated (Ci-C_n)alkyl may be substituted or unsubstituted as described herein. Moreover, in any alkyl group one or two chain members selected from CH₂ or CH are optionally replaced by chain members independently selected from N, NR, O, C(=0), C(=0)NR, NRC(=0) and S; wherein R is as described herein. When it is not specified whether the term (Ci-C_n)alkyl is saturated or unsaturated, the term (Ci- C_n)alkyl has to be understood as a saturated linear or branched hydrocarbon chain which contains from 1 to n carbon atoms. The above definitions apply also for 0(Ci-C_n)alkyl.

A halogen substituent means fluoro, chloro, bromo or iodo.

In the embodiments of the invention referring to the compounds of formula (I) and formula (II), where the substitution or unsubstitution of a certain group is not specified, e.g. either by indicating a certain substitution for that group or by indicating that the group is unsubstituted, it has to be understood that the possible substitution of this group is the one as in the definition of the respective formula (I) or formula (II).

The expression "substituted with one or more" means that a group can be substituted with one or more, preferably with 1, 2, 3 or 4 substituents, provided that this group has enough positions susceptible of being substituted. Compounds that induce expression of Heat shock 70 kDa protein lA/lB

The "Heat shock 70 kDa protein lA/lB" is a protein of the HSP70 Heat-shock proteins family (Hsp70s), a class of molecular chaperones found in both prokaryotes and in several compartments of eukaryotic cells. In cooperation with other chaperones,

Hsp70s stabilize preexistent proteins against aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. These chaperones participate in all these processes through their ability to recognize nonnative conformations of other proteins. They bind extended peptide segments with a net hydrophobic character exposed by polypeptides during translation and membrane translocation, or following stress-induced damage.

In humans, the Heat shock 70 kDa protein 1A/1B is encoded by the genes with

HUGO Gene Nomenclature Committee 's approved symbol HSPAIA (HGNC ID:5232; which is also referred to as "heat shock 70kDa protein 1A", "HSPA1", and "HSP70-1"), and HSPAIB (HGNC ID:5233; which is also referred to as "heat shock 70kDa protein

IB", and "HSP70-2").

As used herein, a compound that induces the expression of Heat shock 70 kDa protein lA/lB is a compound that produces an increase of the amount of this protein within the cells and tissues; e.g. by enhancing the transcription of the genes encoding the protein; and/or by enhancing the translation from mRNA.

In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound that induces the expression of Heat shock 70 kDa protein lA/lB is a compound that increases the transcription of the genes HSPAIA, HSPAIB, or both. This way, the ability of a compound to induce expression of Heat shock 70 kDa protein 1 A/IB can be determined for example by performing conventional quantitative PCR techniques with primers specific for the gene of interest, HSPAIA and/or HSPAIB. HP AC 6 inhibitor compounds

In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound that induces the expression of Heat shock 70 kDa protein 1A/1B for use or to be administered according to the invention in anticoagulant therapy is an HDAC6 inhibitor compound.

The term "Histone deacetylase (HDAC)" as used herein refers to and comprises a group of enzymes that remove acetyl groups (0=C-CH3) from a ε-Ν-acetyl lysine amino acid on a histone, allowing the histones to wrap the DNA more tightly (EC Number 3.5.1.98). This is important because DNA is wrapped around histones, and DNA expression is regulated by acetylation and de-acetylation. HDACs are classified in four classes I to IV based on function and DNA sequence similarity. Class I includes isoforms HDAC1, HDAC2, HDAC3, and HDAC8; class II includes HDAC4, HDAC5, HDAC6, HDAC7, HDAC9 and HDAC10; Class IV includes HDAC11. Class II is grouped into two subclasses named Class Ila (HDAC4, HDAC5, HDAC7, and HDAC9) and class lib (HDAC6 and HDAC10). HDAC6 is a unique cytoplasmic deacetylase thanks to a Ser Glu-repeat domain (SE14), which acts as a cytoplasmic retention signal and mediates its stable anchorage in the cytoplasm, and targets tubulin, HSP90 and cortactin. Hence it can regulate cell adhesion, motility and chaperone function.

In humans, the Histone deacetylase 6 is identified with Uniprot accession number Q9UBN7 [http://www.uniprot.org/uniprot/Q9UBN7; Entry version 145 (29 Oct 2014), Sequence version 2 (02 Sep 2008)], and is encoded by the gene with HUGO Gene Nomenclature Committee's approved symbol HDAC6 (HGNC ID: 14064).

The term "inhibitor compound" as used herein refers to the capacity of a compound to inhibit partially or totally, directly or indirectly, a target molecule (in the present case HDAC6), by inhibiting its catalytic activity. The inhibition of activity can be total if the activity measured when inhibitor compound concentration is up to 10 μΜ is equal to or below than 10% compared to basal values. If the activity measured is higher than 10% and lower than 100%, more particularly higher than 10% and equal or lower than 90%, the activity is considered partially inhibited. Inhibitor compound also refers to a molecule which IC₅₀ vs target of interest (concentration to inhibit 50% its catalytic activity), in this case HDAC6, is lower than 10 μΜ.

Any HDAC6 inhibitor compound can be used or administered for anticoagulation therapy according to the invention. HDAC6 inhibitor compounds for use or to be administered in anticoagulation therapy according to the invention can be identified and selected, for example, by means of a specific biological activity test, as the assay described below (Example 6).

HDAC6 inhibitor compounds of Formula I

In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the HDAC6 inhibitor compound is a compound of formula (I),

wherein

Bi is a radical selected from the group consisting of formula (A"), formula (B"), formula (C"), and formula (D^M):

p, n and r are independently 0 or 1;

Ri and R₂ are independently selected from the group consisting of H; saturated or unsaturated (Ci-C₇)alkyl optionally substituted with one or more halogen atoms; and a 3- to 7-membered carbocyclic or heterocyclic monocyclic ring, which is saturated or partially unsaturated or aromatic, and which is optionally substituted with one or more substituents selected from halogen and (Ci-C3)alkyl;

Zi is a biradical selected from the group consisting of a formula (E), formula (F"), formula (G'), formula (Ff), formula (J'), and formula (K):

Z₂ is selected from the group consisting of -Z₅-; - Z₅-Cy⁴-; -Z₅-Cy⁴-Z₅-; and -Cy⁴-;

Z₃ and each Z5 are independently a biradical of a saturated or unsaturated (Ci-Ce)alkyl optionally substituted with one or more halogen atoms;

Z₄ is a biradical of a saturated or unsaturated (Ci-C₆)alkyl optionally substituted with one or more substituents selected from halogen, OH, and -0(Ci-C₃)alkyl optionally substituted with one or more halogen atoms; or alternatively Z₄ is -CRⁿR¹²-, wherein R¹¹ and R¹² taken together with the carbon they are attached to form C=0 or a 3- to 7- membered carbocyclic or heterocyclic monocyclic ring, which is saturated or partially unsaturated, and which is optionally substituted with one or more halogen atoms or (Ci-C₃)alkyl optionally substituted with one or more halogen atoms;

q and m are independently 0 or 1 ;

Cy¹, Cy³ and Cy⁴ are independently phenyl or a 3- to 7-membered carbocyclic or heterocyclic monocyclic ring, which is saturated or partially unsaturated or aromatic, and which is optionally substituted with one or more R₃ groups; or alternatively

Cy¹ is a 3- to 7-membered saturated or partially unsaturated heterocyclic monocyclic ring, which is fused, bridged-fused or spiro-fused to a 3- to 7-membered saturated or partially unsaturated carbocyclic or heterocyclic monocyclic ring, wherein Cy¹ is optionally substituted with one or more R₃ groups;

Cy² is a N-attached 3- to 7-membered saturated or partially unsaturated heterocyclic monocyclic ring, which is optionally fused, bridged-fused or spiro-fused to a 3- to 7- membered saturated or partially unsaturated carbocyclic or heterocyclic monocyclic ring, wherein Cy² is optionally substituted with one or more R₃ groups;

R₃ is selected from halogen; saturated or unsaturated (Ci-C₇)alkyl optionally substituted with one or more halogen atoms; saturated or unsaturated -0(Ci-C₇)alkyl optionally substituted with one or more halogen atoms; and a 3- to 7-membered carbocyclic or heterocyclic monocyclic ring, which is saturated or partially unsaturated or aromatic, and which is optionally substituted with one or more substituents selected from the group consisting of halogen and (Ci-Ce)alkyl optionally substituted with one or more halogen atoms;

R⁴ and R¹⁰ are independently H or (Ci-C₆)alkyl optionally substituted with one or more halogen atoms; and

wherein in any heterocyclic ring one or more of the ring members are selected from NH, N, O, and S;

wherein in all saturated or partially unsaturated rings one or two members of the rings are optionally C(=0) and/or C(=NH) and/or C[=N(Ci-C₄)alkyl],

wherein saturated alkyl refers to a linear or branched hydrocarbon chain which contains only single bonds; and unsaturated alkyl refers to a linear or branched hydrocarbon chain which contains one or two double bonds and/or one or two triple bonds;

wherein in any alkyl group one or two chain members selected from CH₂ or CH are optionally replaced by chain members independently selected from N, NR₄, O, C(=0),

C(=0)NR₄, NR₄C(=0), and S; and

with the condition that the moiety (Li) of the formula (A"), formula (B"), and formula (C"), and the moiety (L₂) of the formula (D")

(Li ) (L₂)

have a chain length comprised from 1 to 20 atoms.

The compounds of formula (I) of the invention are characterized in that they have a polycyclic ring system selected from the group consisting of formula (Α'), formula (Β'), formula (C), and formula (D'):

and a hydroxamic acid moiety. This polycyclic ring system comprises from 2 to 4 rings; being at least one ring an aromatic ring; and comprises at least 3 nitrogen atoms and 1 oxygen atom. The linker between the polycyclic ring system defined above and the hydroxamic acid moiet i.e. a structure of formula (Li) or (L₂),

(Li ) (L₂)

has a chain length comprised from 1 to 20 atoms and comprises a hydrocarbon chain, wherein one or more carbon atoms are optionally replaced by nitrogen, sulphur and/or oxygen atoms, which optionally contains one or more aromatic, heteroaromatic, carbocyclic and/or heterocyclic rings.

In a particular embodiment, the linker between the polycyclic ring system and the hydroxamic acid moiety is a structure of formula (Li') or (L₂')

(Li ') (L₂')

having a chain length comprised from 3 to 20 atoms. For the purposes of the invention, if there is more than one possibility for counting the chain length, the chain length corresponds to the highest number of atoms. As an example, in the compound 2-01 below the l

In another embodiment, the invention relates to a compound of formula (I),

wherein

p, n and r are independently 0 or 1;

Zi is a biradical selected from the group consisting of a formula (E), formula (F'), formula (G'), formula (Ff), formula (J') and formula (K):

Z₃, Z₄ and each Z5 are independently a biradical of a saturated or unsaturated (Ci-C₆)alkyl optionally substituted with one or more halogen atoms;

q and m are independently 0 or 1 ;

Cy¹, Cy³ and Cy⁴ are independently phenyl or a 3- to 7-membered carbocyclic or heterocyclic monocyclic ring, which is saturated or partially unsaturated or aromatic, and which is optionally substituted with one or more R₃ groups;

R₄ is H or (Ci-C₆)alkyl optionally substituted with one or more halogen atoms; and wherein in any heterocyclic ring one or more of the ring members are selected from NH, N, O, and S;

wherein in all saturated or partially unsaturated rings one or two members of the rings are optionally C(=0) and/or C(=NH) and/or C[=N(Ci-C₄)alkyl], wherein saturated alkyl refers to a linear or branched hydrocarbon chain which contains only single bonds; and unsaturated alkyl refers to a linear or branched hydrocarbon chain which contains one or two double bonds and/or one or two triple bonds;

wherein in any alkyl group one or two chain members selected from CH₂ or CH are optionally replaced by chain members independently selected from N, NR₄, O, C(=0), C(=0)NR4, NR4C(=0), and S; and

with the condition that the moiety (Li) of the formula (A"), formula (B"), and formula (C"), and the moiet (L₂) of the formula (D")

(Li ) (L₂)

have a chain length comprised from 1 to 20 atoms.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in a compound of formula (I), Bi is a radical selected from the group consisting of formula (A), formula (B), formula (C), and formula (D):

R₂ is H or saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms; Z_\ is a biradical selected from the group consisting of a formula (E), formula (F), formula (G), formula (H), formula (J), and formula (K):

and R₃ is selected from halogen, saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms; and saturated or unsaturated -0(Ci-C₄)alkyl optionally substituted with one or more halogen atoms. More particularly, Cy² is a N-attached 5- to 7-membered heterocyclic monocyclic ring, which is saturated or partially unsaturated, and which is optionally substituted with one or more R₃ groups.

In a more particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in a compound of formula (I), n is 0 and Cy¹ is selected from the group consisting of phenyl, 5- to 6- membered heteroaromatic ring, 3- to 6-membered carbocyclic ring, and 3- to 7- membered heterocyclic ring, wherein Cy¹ is optionally substituted with one or more R₃ groups. In a more particular embodiment, n is 0 and Cy¹ is phenyl optionally substituted with R₃, more particularly phenyl substituted with -0(Ci-C₄)alkyl or Cy¹ is pyrrolidine optionally substituted with R₃, more particularly pyrrolidine substituted with -(Ci-C₄)alkyl.

In another more particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in a compound of formula (I), n is 1 ; Z₃ is -CH(R₅)-, wherein R₅ is H or (Ci-C4)alkyl optionally substituted with one or more halogen atoms; and Cy¹ is a 3- to 6-membered carbocyclic ring or 3- to 7-membered heterocyclic ring, wherein Cy¹ is optionally substituted with one or more R3 groups. In a more particular embodiment, n is 1 ; Z₃ is -CH(R₅)-; R₅ is H or methyl and Cy¹ is piperidine or azetidine. In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (I), Ri is selected from the group consisting of (Ci-Cv)alkyl optionally substituted with one or more halogen atoms, 3- to 6-membered carbocyclic ring optionally substituted with one or more substituents selected from halogen and (Ci-C₃)alkyl, and 3- to 7-membered heterocyclic ring optionally substituted with one or more substituents selected from halogen and (Ci-C₃)alkyl. In a more particular embodiment, Ri is propyl, cyclopentane or tetrahydropyran.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (I), R₂ is H or optionally substituted (Ci-C4)alkyl. In a more particular embodiment, R₂ is H or methyl.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in a compound of formula (I), Cy² is optionally substituted saturated 5- to 7-membered heterocyclic ring. More particularly, Cy² is piperidine or piperazine.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in a compound of formula (I), Cy² is a N-attached 3- to 7-membered heterocyclic monocyclic ring, which is spiro-fused to a 3- to 7-membered carbocyclic or heterocyclic monocyclic ring, wherein Cy² is optionally substituted with one or more R₃ groups.

In another particular embodiment, in a compound of formula (I), optionally in combination with one or more features of the various embodiments described above or below, Cy³ is selected from the group consisting of phenyl, 5- to 6-membered heteroaromatic ring, 5- to 6-membered carbocyclic ring, 3- to 7-membered carbocyclic ring, 4- to 6-membered heterocyclic ring, and 5- to 7-membered heterocyclic ring, wherein Cy³ is optionally substituted with one or more R₃ groups. More particularly, Cy³ is phenyl, azetidine, piperidine, piperazine, pyrimidine, thiophene, furan, pyridine, cyclobutane, cyclopentane, cyclohexane, cyclohexene, or cycloheptane. In another particular embodiment, in a compound of formula (I), optionally in combination with one or more features of the various embodiments described above or below, Cy⁴ is selected from the group consisting of phenyl, and 5- to 6-membered heteroaromatic ring, wherein Cy⁴ is optionally substituted with one or more R₃ groups. More particularly, Cy⁴ is phenyl, pyridine or pyrimidine.

In another particular embodiment, in a compound of formula (I), optionally in combination with one or more features of the various embodiments described above or below, Zi is a biradical of formula (H), wherein m is 0 and q is 0 (Zi is absent).

In another particular embodiment, in a compound of formula (I), optionally in combination with one or more features of the various embodiments described above or below, Zi is selected from the group consisting of:

In another particular embodiment, in a compound of formula (I), optionally in combination with one or more features of the various embodiments described above or below, r is 1 and Z₂ is selected from the group consisting of:

In another particular embodiment, in a compound of formula (I), optionally in combination with one or more features of the various embodiments described above or below, m is 0.

In another particular embodiment, in a compound of formula (I), optionally in combination with one or more features of the various embodiments described above or below, m is 1, and Z₄ is -(CH₂)_r, wherein t is 1-3.

In another particular embodiment, in a compound of formula (I), optionally in combination with one or more features of the various embodiments described above or below, Z₅ is selected from the group consisting of: -(CH₂)_r, wherein t is 1-3, and - CH=CH-.

In another particular embodiment, in a compound of formula (I), optionally in combination with one or more features of the various embodiments described above or below, r is 1 and the group -Zi-Z₂- is selected from the group consisting of:

In another particular embodiment, in a compound of formula (I), optionally in combination with one or more features of the various embodiments described above or below, r is 1 and the group

is selected from the group consisting of:

wherein Bi' is selected from the group consisting of formula (Α'), formula (Β'), and formula (C)

(Α') (Β') (C)

wherein Z₃, n, Cy¹, r, Z_{l s} Z₂, Ri and R₂ are as previously defined.

The particular embodiments mentioned above for compounds of formula (I) are also particular embodiments of the compounds of formula (Γ).

In another embodiment, the compound of formula (Γ) is a compound of formula

(IA) or formula (IB):

wherein r, Z_{l s} Z₂, Ri and R₂ are as previously defined; and R₃' is selected from H; halogen; saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms; saturated or unsaturated -0(Ci-C₄)alkyl optionally substituted with one or more halogen atoms; and a 3- to 7-membered carbocyclic or heterocyclic monocyclic ring, which is saturated or partially unsaturated or aromatic, and which is optionally substituted with one or more substituents selected from the group consisting of halogen and (Ci-C₆)alkyl optionally substituted with one or more halogen atoms.

In a particular embodiment, in the compounds of formula (IA) or formula (IB),

R₃' is selected from H; halogen; saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms; and saturated or unsaturated -0(Ci-C₄)alkyl optionally substituted with one or more halogen atoms. The particular embodiments mentioned above for compounds of formula (I) are also particular embodiments of the compounds of formula (IA) and of formula (IB).

In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (IA) or formula (IB), Ri is optionally substituted (Ci-Cv)alkyl, more particularly, propyl.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (IA) or formula (IB), R₂ is optionally substituted (Ci-C₄)alkyl, more particularly, methyl.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (IA) or formula (IB), R₃' is optionally substituted -0(Ci-C₄)alkyl, more particularly, ethoxy, even more particularly ethoxy placed at the ortho position with respect to the carbon attached to the bicyclic ring.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (IA) or formula (IB), Z_\ is a biradical selected from the group consisting of formula (E), formula (F) and formula (G) as defined above.

In another embodiment, the invention relates to a compound of formula (I), which is a compound of formula (IC):

(IC)

wherein Ri-R₂, r, Zi-Z₃, and n are as previously defined, Cy¹ is 4- to 6-membered heterocyclic ring, and R₃' is selected from H; saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms; and 3- to 7-membered carbocyclic or heterocyclic monocyclic ring, which is saturated or partially unsaturated or aromatic, and which is optionally substituted with one or more substituents selected from the group consisting of halogen and (Ci-Ce)alkyl optionally substituted with one or more halogen atoms.

In a particular embodiment, in the compounds of formula (IC), R₃' is selected from H, and saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms.

The particular embodiments mentioned above for compounds of formula (I) are also particular embodiments of the compounds of formula (IC).

In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (IC), n is O.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (IC), n is 1 , and Z₃ is -CH(R₅)-, wherein R₅ is H or (Ci-C₄)alkyl optionally substituted with one or more halogen atoms, more particularly, R5 is H or methyl.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (IC), Cy¹ is pyrrolidine, piperidine, piperazine or azetidine.

In a particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (IC), Ri is saturated 3- to 7-membered carbocyclic or heterocyclic ring, more particularly, tetrahydropyran or cyclopentane.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (IC), R₂ is H.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (IC), and R₃' is H or (Ci-C₆)alkyl optionally substituted with one or more halogen atoms, more particularly, H or methyl.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the compound of formula (IC), Z_\ is a biradical selected from the group consisting of formula (G) and formula (H) as defined above. In another embodiment, the compound of formula (I) is a compound of formula

(ID) wherein Z₂, Z₃, n and Cy¹ are as previously defined, and R₃' is selected from H; halogen; saturated or unsaturated (Ci-C4)alkyl optionally substituted with one or more halogen atoms; and a 3- to 7-membered carbocyclic or heterocyclic monocyclic ring, which is saturated or partially unsaturated or aromatic, and which is optionally substituted with one or more substituents selected from the group consisting of halogen and (Ci-C₆)alkyl optionally substituted with one or more halogen atoms.

In a particular embodiment, in the compounds of formula (ID), R₃' is selected from H, halogen, and saturated or unsaturated (Ci-C4)alkyl optionally substituted with one or more halogen atoms.

The particular embodiments mentioned above for compounds of formula (I) are also particular embodiments of the compounds of formula (ID).

In a particular embodiment, in a compound of formula (ID), n is 0 and Cy¹ is selected from the group consisting of phenyl, 5- to 6-membered heteroaromatic ring, 5- to 6-membered carbocyclic ring, and 5- to 7-membered heterocyclic ring, wherein Cy¹ is optionally substituted with one or more R₃ groups. In a more particular embodiment, n is 0 and Cy¹ is optionally substituted phenyl, more particularly, unsubstituted phenyl.

In another particular embodiment, in a compound of formula (ID), n is 1; Z₃ is -CH(R₅)-, wherein R₅ is H or (Ci-C4)alkyl optionally substituted with one or more halogen atoms; and Cy¹ is 3- to 6-membered carbocyclic ring or 3- to 7-membered heterocyclic ring wherein Cy¹ is optionally substituted with one or more R₃ groups. In a more particular embodiment, n is 1 , Z₃ is -CH(R₅)-, R₅ is H, and Cy¹ is piperidine.

In another embodiment, in a compound of formula (ID), -Z₂- is selected from the group consisting of:

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), wherein Bi is a radical of formula (A").

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), wherein Bi is a radical of formula (B").

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), wherein Bi is a radical of formula (C").

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), wherein Ri is selected from the group consisting of saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms, 5- to 6-membered saturated carbocyclic ring optionally substituted with one or more substituents selected from halogen and (Ci-C₃)alkyl, and 5- to 6-membered saturated heterocyclic ring optionally substituted with one or more substituents selected from halogen and (Ci-C₃)alkyl.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), wherein R₂ is H or saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), wherein Z_\ is a biradical selected from the group consisting of a formula (E), formula (G'), and formula (Η'), more particularly, wherein q is 1 , and even more particularl wherein Z_\ is a biradical of formula (G) or formula (H):

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), wherein r is 0, or alternatively r is 1 and Z₂ is -Cy⁴-; more particularly, wherein r is 0.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), wherein Bi is a radical of formula (D").

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), wherein p is 1 ; more particularly, wherein

Cy¹ is selected from the group consisting of: phenyl, 5- to 6-membered heteroaromatic ring, and 4- to 6-membered saturated heterocyclic ring, wherein Cy¹ is optionally substituted with one or more R₃ groups.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), which is a compound of formula (IA):

CIA }

wherein R₃' is H or R₃. In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), which is a compound of formula (IB):

wherein R₃' is H or R₃.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), in particular a compound of formula (IA) or a compound of formula (IB), wherein R₃' is selected from H, halogen, saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms; and saturated or unsaturated -0(Ci-C₄)alkyl optionally substituted with one or more halogen atoms.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), which is a compound of formula (IC):

{IC}

wherein R₃' is H or R₃.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), which is a compound of formula (IC ) or a compound of formula (IC^IV):

wherein R₅ is selected from the group consisting of: H, halogen, and (Ci-C4)alkyl optionally substituted with one or more halogen atoms.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), in particular a compound of formula (IC), a compound of formula (IC¹¹¹) or a compound of formula (IC^IV), wherein R₃' is selected from H and saturated or unsaturated (Ci-C4)alkyl optionally substituted with one or more halogen atoms.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), which is a compound of formula (ID):

(ID) wherein R₃' is H or R₃.

In another particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a compound of formula (I), in particular a compound of formula (ID), wherein R₃' is selected from H, halogen, and saturated or unsaturated (Ci-C4)alkyl optionally substituted with one or more halogen atoms.

In another embodiment of the invention, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (I) is selected from the group of compounds listed in Table 1.

HDAC inhibitor compounds of formula (I) herein referred to as particular embodiments of the invention have been disclosed in the international patent application published as WO2014/131855A1, together with the methods for their preparation as well as their corresponding biochemical data vs different HDAC isoforms (IC50 values); for clarity, compounds numbering is kept identical to the numbering in this international patent application. Some additional HDAC inhibitor compounds of formula (I) (3-18 to 3-30), not disclosed in the above cited patent, have been also included in the Table 1 ; in these cases their corresponding synthetic routes, described in WO2014/131855A1, are explicitly mentioned.

Table 1. HDAC6 inhibitor compounds according to Formula (I) for use in anticoagulant therapy.

1-01 1-02 1-03

-33 1-34 1-35

-51 1-53 1-54

-70 1-71 1-72

* trans isomers

1-83 1-84 1-85

-07 3-08 3-09

3-19 (Route 3c) 3-20 (Route 3b) 3-21 (Route 3d)

-01 4-02 4-03

4-04 4-05

* Regarding these compounds, an aleatory absolute configuration of the cis and trans isomers is shown. In the examples it is clearly indicated which of the isomers is concerned in relative terms by differentiating unambiguously between cis and trans isomers by their physical and/or spectroscopic properties.

HDAC6 inhibitor compounds of Formula II

In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the HDAC6 inhibitor compound is a compound of formula (II),

(II)

wherein

q and s are independently 0 or 1 ;

Re and R₇ are independently selected from the group consisting of H; saturated or unsaturated (Ci-C₆)alkyl optionally substituted with one or more halogen atoms; and 3- to 6-membered carbocyclic or heterocyclic monocyclic ring containing from 1 to 3 ring members selected from NH, N, O, and S, which is saturated or partially unsaturated or aromatic, and which is optionally substituted with one or more Rs groups;

Z₆ is selected from the group consisting of-Z₈-; - Zs-Cy⁶-; -Zs-Cy⁶-Zs-; and -Cy⁷-; Z₇ and Z are independently a biradical of a saturated or unsaturated (Ci-Ce)alkyl optionally substituted with one or more halogen atoms;

Cy⁵ and Cy⁶ are independently phenyl or a 4- to 6-membered carbocyclic or heterocyclic monocyclic ring; which is saturated or partially unsaturated or aromatic; and which is optionally substituted with one or more Rs groups;

Rs is selected from halogen, saturated or unsaturated (Ci-C4)alkyl optionally substituted with one or more halogen atoms; and saturated or unsaturated -0(Ci-C4)alkyl optionally substituted with one or more halogen atoms;

wherein in all saturated or partially unsaturated rings one or two members of the rings are optionally C(=0) and/or C(=NH) and/or C[=N(Ci-C₄)alkyl]; and

wherein in any alkyl group one or two chain members selected from C¾ or CH are optionally replaced by chain members independently selected from N, NR₉, O, C(=0), C(=0)NR₉, NR₉C(=0) and S; and R₉ is H or (Ci-C₆)alkyl optionally substituted with one or more halogen atoms;

with the condition that the moiety (L₃) of the formula (II)

(L₃)

has a chain length comprised from 1 to 20 atoms.

In a particular embodiment, in a compound of formula (II), q is 0, s is 1 , and Cy⁵ is 3- to 6-membered carbocyclic ring or 3- to 6-membered heterocyclic ring, wherein Cy⁵ is optionally substituted with one or more Rs groups. In a more particular embodiment, q is 0, r is 1 and Cy⁵ is piperidine.

In another embodiment, in a compound of formula (II), q is 0 and s is 0.

In another embodiment, in a compound of formula (II), -Z₆- is selected from the group consisting of:

In another embodiment of the invention, optionally in combination with one or more features of the various embodiments described above or below, the compound of formula (II) is selected from the group of compounds listed in Table 2.

HDAC inhibitor compounds of Formula (II) herein referred to as particular embodiments of the invention have all been disclosed in the international patent application WO2014/131855A1, together with the methods for their preparation.

Table 2. HDAC6 inhibitor compounds according to Formula (II) for use in anticoagulant therapy.

-02 5-03 5-04

5-05

Compounds of Formula (I) and Formula (II) herein described are dual inhibitors of HDACs (in particular HDAC6) and phosphodiesterases (PDEs). Therefore, in an embodiment of the invention, optionally in combination with one or more features of the various embodiments described above or below, the HDAC6 inhibitor compound for use in anticoagulation therapy is a dual inhibitor of HDAC6 and PDEs. Other HDAC6 inhibitor compounds

In one embodiment of the invention, optionally in combination with one or more features of the various embodiments described above or below, the HDAC6 inhibitor compound for use in anticoagulation therapy is selected from the group of compounds listed in Table 3. According to the criteria described above, these compounds are all selective HDAC6 inhibitor compounds.

Table 3. Other HDAC6 inhibitor compounds for use in anticoagulant therapy.

6-01 6-02 Tubacin CAS: 1033390-84-9

CAS: 1350555-93-9 N-[4-[ 1 -[(4-fluorophenyl)methyl]triazol- N-[4-[(2i?,4i?,65)-4-[(4,5-diphenyloxazol-2- 4-y ljphenyl] - 8 -(hydroxy amino)- 8 -oxo - yl)sulfanylmethyl] -6- [4- octanamide

(hydroxymethyl)phenyl]- 1 ,3-dioxan-2- J. Med. Chem. 2008, 5L 3437-3448 yl]phenyl]-8-(hydroxyamino)-8-oxo- octanamide

PNAS, 2003, 100, 4389-4394

6-03 6-04

CAS: 1045792-66-2 Tubastatin A

tert-butyl N- [4- [3 - [ [7-(hydroxy amino)-7- CAS: 1252003-15-8

oxo-heptyl]carbamoyl]isoxazol-5- 4- [(2-methyl-3 ,4-dihydro- lH-pyrido [4,3 - yl]phenyl] carbamate b]indol-5- J. Med. Chem. 2008, 51, 4370-4373 yl)methyl]benzenecarbohydroxamic acid

JACS, 2010, 132, 10842-10846 J. Med. Chem. 2012, 55, 639-651

6-05 6-06 Nexturastat A CAS: 1259296-46-2

CAS: 1403783-31-2 2-( 1 -methylpyrrole-2-carbonyl)-3 ,4- 1 -butyl- 1 -[[4- dihydro- lH-isoquinoline-6- (hydroxycarbamoyl)phenyl]methyl] -3 - carbohydroxamic acid

phenyl-urea WO2010151318A1

WO2013134467A1 J. Med. Chem. 2013, 56, 7201-7211

6-07 6-08

CAS: 1338562-19-8 CAS: 1361380-97-3 2-( 1 -methylpyrrole-2-carbonyl)-3 ,4-dihydro- 6-(3-phenyl-lH-indole-2-carbonyl)-7,8- lH-pyrrolo[ 1 ,2-a]pyrazine-7- dihydro-5H- 1 ,6-naphthyridine-3- carbohydroxamic acid carbohydroxamic acid US20110251184A1 WO2012027564A1

6-09 6-10

CAS: 1355588-28-1 CAS: 1332871-00-7 1 - [7-(hydroxycarbamoyl)tetralin-2-yl] -3 - l-cyclopentyl-3-[l-[5- phenyl-urea (hydroxycarbamoyl)-2- US20120015943A1 thienyl] ethyljurea

WO2011106627A1

6-11 6-12

CAS: 1332891-37-8 CAS: 1259286-34-4 N-[(li?)-l-[4- 6-(2,2-dimethylpropanoyl)-5,7-dihydro- (hydroxycarbamoyl)phenyl] ethyl] adamantan 4H-thieno[2,3-c]pyridine-2- e-l-carboxamide carbohydroxamic acid

WO2011106632A1 WO2010151317A1

6-13 6-14

CAS: 928029-57-6 CAS: 422294-56-2

4- [ [(2R)-2 - [(4-hydroxyphenyl)methyl] -3 - cyclohexene- 1 -carbohydroxamic acid oxo-2,4-dihydroquinoxalin- 1 - J. Med. Chem. 2013, 56, 1772-1776 yl]methyl]benzenecarbohydroxamic acid WO2014100438A1

US20070155730A1

6-15 6-16

ACY-775 CAS: 1450591-25-9

CAS: 1375466-18-4 (E)-3-[5- 2-[[l-(3- [ [benzyl(propyl)amino]methyl] -2- fluorophenyl)cyclohexyl]amino]pyrimidine- furyl]prop-2-enehydroxamic acid

5 -carbohydroxamic acid Bioorg. Med. Chem. 2013, 21, 5339- US20120121502A1 5354

WO2014059306A1

6-17 6-18

CAS: 1609171-95-0 CAS: 1450618-63-9 (E)-3-(3-pyrrolo[2,3-£]pyridin- 1 - 4-[[2-ethyl-4-oxo-3-(2- ylsulfonylphenyl)prop-2-enehydroxamic phenylethyl)quinazo lin-7- acid yl]methyl]benzenecarbohydroxamic

J. Med. Chem. 2014, 57, 4009-4022 acid

US20130267542A1

6-19 6-20

CAS: 908860-17-3 CAS: 1260097-47-9 tert-butyl N- [( 1 S)- 1 -(cyclopentylcarbamoy 1)- tert-butyl N-[(\S)-\- 6-sulfanyl-hexyl] carbamate (cyclopentylcarbamoyl)-7- J. Med. Chem. 2006, 49, 4809-4812 (hydroxyamino)-7-oxo- J. Med. Chem. 2007, 50, 5425-5438 heptyljcarbamate

Bioorg. Med. Chem. Lett. 2010, 20,

7067-7070

6-21 6-22

CAS: 960130-78-3 CAS: 1257657-14-9

N-(8-quinolyl)-6- (S)-2-(6,18-Dioxo- (sulfamoylamino)hexanamide 5,6,7,9,10,11,12,13,18,19- Bioorg. Med. Chem. Lett. 2009, 19, 336-340 decahydrobenzo[5 ,6] [ 1 ,4,7]- oxadiazacyclotetradecino[ 10,9-b]indol- 7-yl)-N-hydroxyacetamide J. Med. Chem. 2010, 53, 8387-8399 WO2011047926A1

6-23 6-24

Rocillinostat; ACY-1215 HPOB

CAS: 1316214-52-4 CAS: 1429651-50-2 N-[7-(hydroxyamino)-7-oxo-heptyl]-2-(N- 2- [4-(hydroxycarbamoyl)phenyl] -N-(2- phenylanilino)pyrimidine-5-carboxamide hydroxyethyl)-N-phenyl-acetamide Blood, 2012, 119, 2579-2589 WO2013052110A1

6-25 6-26

CAS: 1350650-31-5 Tubathian A

2-[4-[[8-(hydroxyamino)-8-oxo- CAS: 1431080-97-5 octanoyl]amino]phenyl]-N,N'-bis(8- 4-[(2,2-dioxo-3,4-dihydro-lH- quino lyl)propanediamide thiopyrano [4 , 3 -b] indo 1-5 - WO2011146855A1 yl)methyl]benzenecarbohydroxamic acid

WO2014147178A1

6-27 6-28

CAS: 1432508-58-1 CAS: 1316215-08-3 4-[[6-fluoro-2,2-dimethyl-3-[(2- N- [7-(hydroxyamino)-7-oxo-heptyl] -4- methylimidazol- 1 -yl)methyl]-4-oxo- 1 ,3- [methoxy-phenyl-(2- dihydrocarbazo 1-9- pyridyl)methyl]benzamide yl]methyl]benzenecarbohydroxamic acid WO2013013113A2

WO2013062344A1

6-29 6-30

CAS: 1417535-37-5 CAS: 1416246-37-1 (E)-N-[4-[2-(hydroxycarbamoyl)-6,7- 4-[(5-methyl-2-oxo-3H-benzimidazol- 1 - dihydro-4H-thiazo lo [5 ,4-c]pyridine-5 - yl)methyl]benzenecarbohydroxamic carbonyljphenyl] -3 -phenyl-prop-2-enamide acid

CN102838625A WO2012178208A2

6-31 6-32

CAS: 1402160-24-0 CAS: 1383793-05-2 3 -(3 -furyl)-N- [6-(hydroxyamino)-6-oxo- tert-butyl 4-[6-(hydroxycarbamoyl)-3,4- hexyl]-5-(4-hydroxyphenyl)-lH-pyrrole-2- dihydro-lH-isoquinoline-2-carbonyl]-4- carboxamide methyl-piperidine- 1 -carboxylate WO2012136722A1 WO2012088015A2

6-33 6-34

CAS: 1372887-20-1 CAS: 1350750-34-3 3 - [(3 -chlorophenyl)methyl] -4-oxo- N- [3 -(hydroxycarbamoyl)-6- quinazo line-6-carbohydroxamic acid quino ly 1] eye lopropanecarboxamide

US20120094997A1 WO201 1 146591A1

6-35 6-36

CAS: 1355369-39-9 CAS: 1187303-88-3 N- [3 - [3 -(hydroxycarbamoyl)tetralin-6- 2-[2-(2-pyridyl)acetyl]-3,4-dihydro-lH- yl]phenyl]-2,2-dimethyl-propanamide isoquino line-6-carbohydroxamic acid

US20120015942A1 WO2009112550A1

In one embodiment of the invention, optionally in combination with one or more features of the various embodiments described above or below, the HDAC6 inhibitor compound is a derivative and/or analog compound of a compound listed in Table 3 which also has HDAC6 inhibitor activity. In a particular embodiment this derivative or analog compound is an HDAC6 inhibitor compound according to any of the structural formulae provided in the reference documents provided in Table 3.

In one embodiment of the invention, optionally in combination with one or more features of the various embodiments described above or below, the HDAC6 inhibitor compound is a selective HDAC6 inhibitor compound.

The term "selective inhibitor compound" as used herein refers to a compound that is able to inhibit a particular isoform (HDAC6 in the present case) of an enzyme target family over other isoform(s) from the same enzyme target family (in the present case HDAC class I; in particular: HDACl and HDAC2) with at least 1 log unit difference in inhibitory potency (IC50). In addition, its corresponding IC50 for HDAC6 is lower than 500nM.

In one embodiment of the invention, optionally in combination with one or more features of the various embodiments described above or below, selective HDAC6 inhibitor compound is selected from the group consisting of compounds 1-15, 1-16, 1-17, 1-40, 1-55, 1-68, 1-71, 1-76, 2-11, 3-09, 3-11, 3-21, 3-22, 3-23, 3-26, and compounds 6-01 to 6-36. Other compounds that induce expression of Heat shock 70 kDa protein 1A/1B

In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the compound that induces the expression of Heat shock 70 kDa protein 1 AJ IB for use or to be administered according to the invention in anticoagulant therapy is selected from the group of compounds listed in Table 4. These compounds induce the activity of HSF1.

Table 4. Other compounds that induce the expression of Heat shock 70 kDa protein 1A/1B for use in anticoagulant therapy.

7-01 7-02

TRC051384 BGP 15

CAS: 867164-40-7 CAS: 6661 1-38-9

Urea, N-[2-(4-morpholinyl)ethyl]-N'-[4-[3-[6-(4- 3-Pyridinecarboximidamide, N-[2- morpholinyl)-2-pyridinyl] - 1 -oxo-2-propen- 1 -yl] hydroxy-3-( 1 -piperidinyl)propoxy] - phenyl]-

7-03 7-04 BIIB021 NVP-AUY922

CAS: 848695-25-0 CAS: 747412-49-3 6-chloro-9-((4-methoxy-3,5- 5-(2,4-dihydroxy-5-isopropylphenyl)-N- dimethylpyridin-2-yl)methyl)-9H-purin-2- ethyl-4-(4- amine (morpholinomethyl)phenyl)isoxazo le-3 - carboxamide

7-05

Geldanamycin

CAS: 30562-34-6

In all embodiments of the invention referring to the compounds of formula (I), formula (II) and compounds listed in Tables 1, 2, 3, and 4, and their derivatives and/or analogs, the pharmaceutically or veterinary acceptable salts thereof and the stereoisomers or mixtures thereof, either of any of the compounds or of any of their pharmaceutically or veterinary acceptable salts, are always contemplated even if they are not specifically mentioned. There is no limitation on the type of salt that can be used, provided that these are pharmaceutically or veterinary acceptable when they are used for therapeutic purposes. The term "pharmaceutically or veterinary acceptable salts", embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases.

The preparation of pharmaceutically or veterinary acceptable salts of the compounds that induce the expression of Heat shock 70 kDa protein 1A/1B mentioned above 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 or veterinary acceptable base or acid in water or in an organic solvent or in a mixture of them. The compounds that induce the expression of Heat shock 70 kDa protein 1A/1B and their salts may differ in some physical properties but they are equivalent for the purposes of the present invention.

Some of the compounds that induce the expression of Heat shock 70 kDa protein

1A/1B may be in crystalline form either as free solvation compounds or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art. In general, the solvated forms with pharmaceutically or veterinary acceptable solvents such as water, ethanol and the like are equivalent to the unsolvated forms for the purposes of the invention.

Some of the compounds can have chiral centres that can give rise to various stereoisomers. As used herein, the term "stereoisomer" refers to all isomers of individual compounds that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers (enantiomers), mixtures of mirror image isomers (racemates, racemic mixtures), geometric (cis/trans or syn/anti or E/Z) isomers, and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereoisomers).

Diastereoisomers and enantiomers can be separated by conventional techniques such as chromatography or fractional crystallization. Optical isomers can be resolved by conventional techniques of optical resolution to give optically pure isomers. This resolution can be carried out on any chiral synthetic intermediate or on compounds of formula (1-01) to (1-86), formula (2-01) to (2-13), formula (3-01) to (3-30), formula (4-01) to (4-05), formula (5-02) to (5-05), formula (6-01) to (6-36) and formula (7-01) to (7-02). Optically pure isomers can also be individually obtained using enantiospecific synthesis.

Anticoagulation therapy

According to the different aspects of the invention, the compound that induces expression of Heat shock 70 kDa protein 1A/1B is used and/or administered to a subject as anticoagulation therapy.

As used herein the term "subject" refers to an animal, in particular a mammal, and preferably a human subject.

As used herein "Thrombosis" refers to the formation and development of a thrombus or blood clot in the blood vessel, reducing the flow of blood through the circulatory system.

As used herein "Thromboembolism" refers to the obstruction of a blood vessel

(embolism) by a blood clot that breaks free (embolus) and begins to travel in the bloodstream around the body. This embolus can cause a blockage in a blood vessel that may affect a part of the body distanced from the actual site of origin; in contrast to a thrombus, which causes a blockage at the site of origin.

As used herein the terms "Anticoagulation therapy" and "Antithrombotic therapy" indistinctively and interchangeably refer to a drug therapy administered to a subject to prevent or reduce the formation of thrombi and/or emboli that can later cause vascular occlusions.

As used herein the term anticoagulation therapy and antithrombotic therapy indistinctively refers to a drug therapy administered to a subject for the treatment and prevention of thrombosis and/or thromboembolism, i.e. to prevent or reduce the formation of thrombi and/or emboli causing thrombotic and/or thromboembolic occlusion of blood vessels (thrombotic or thromboembolic events).

According to the invention, anticoagulation therapy can be administered to treat a subject that suffers, or have recently suffered, an acute thrombotic or thromboembolic event, in order to avoid the enlargement of the occlusion and/or to prevent or diminish the risk of a re-occlusion (to prevent recurrence). Anticoagulation therapy can also be administered to a subject as a prophylactic therapy to prevent or diminish the risk of a later thrombotic or thromboembolic event. This can be indicated for example for subject's groups that are at a higher risk of suffering a thrombosis and/or thromboembolism (e.g. stroke prophylaxis in subjects presented with atrial fibrillation).

In one embodiment of the invention, optionally in combination with any of the embodiments above or below, the compound that induces the expression of Heat shock 70 kDa protein 1A/1B is used or administered as anticoagulation therapy to a subject that presented with atrial fibrillation, arterial thrombosis, acute coronary syndrome associated with coronary thrombosis, metallic prosthetic cardiac valves, stroke, systemic embolism, venous thrombosis, deep venous thrombosis, venous thromboembolism, and/or pulmonary embolism.

In a particular embodiment, optionally in combination with any of the embodiments above or below, the compound that induces the expression of Heat shock 70 kDa protein 1A/1B is used or administered for stroke prophylaxis in a subject presented with atrial fibrillation.

In another embodiment, optionally in combination with any of the embodiments above or below, the compound that induces the expression of Heat shock 70 kDa protein 1A/1B is used or administered as anticoagulation therapy to a subject receiving antiplatelet drug therapy. In a more particular embodiment, optionally in combination with any of the embodiments above or below, the compound that induces the expression of Heat shock 70 kDa protein 1A/1B is used or administered as anticoagulation therapy to a subject receiving as antiplatelet drug therapy a compound selected from the group consisting of Aspirin, Triflusal (Disgren), Clopidogrel (Plavix), Prasugrel (Effient), Ticagrelor (Brilinta), Ticlopidine (Ticlid), Cilostazol (Pletal), Vorapaxar (Zontivity), Eptifibatide (Integrilin), Tirofiban (Aggrastat), Dipyridamole (Persantine), and Terutroban.

The compound that induces the expression of Heat shock 70 kDa protein 1A/1B does not increase the bleeding risk and thus can be used particularly in those clinical situations in which the bleeding risk is high such as during antiplatelet therapy, during bleeding complication induced by anticoagulant or antiplatelet agent or in clinical situations in which anticoagulant or antiplatelet agent must be stopped. In one embodiment of the invention, optionally in combination with any of the embodiments above or below, the compound that induces expression of Heat shock 70 kDa protein lA/lB as defined above is the active pharmaceutical or veterinary ingredient of a pharmaceutical or veterinary composition, which comprises effective amounts of the compound that induces expression of Heat shock 70 kDa protein 1A/1B, together with one or more pharmaceutically or veterinary acceptable excipients or carriers.

The expression "effective amount" as used herein, refers to the amounts of the compound that induces expression of Heat shock 70 kDa protein lA/lB that, when administered to the subject, is sufficient to prevent or significantly reduce the development of thrombosis and/or thromboembolism, to prevent or significantly reduce the risk of developing a thrombotic and/or thromboembolic event, and/or its recurrence. The specific doses of the compound to obtain a therapeutic and/or prophylactic benefit may vary depending on the particular circumstances of the individual subject including, among others, the size, weight, age and sex of the subject, the nature and stage of the disease, the aggressiveness of the disease, and the route of administration. For example, in one embodiment a dose of from about 0.01 to about 300 mg of compound / kg of body weight may be use; in a more particular embodiment a dose comprised within the range of 1 to 40 mg / Kg may also be used.

The expression "pharmaceutically or veterinary acceptable excipients or carriers" refers to pharmaceutically or veterinary acceptable materials, compositions or vehicles. Each component must be pharmaceutically or veterinary acceptable in the sense of being compatible with the other ingredients of the pharmaceutical or veterinary 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 election of the pharmaceutical or veterinary formulation will depend upon the nature of the active compound and its route of administration. Any route of administration may be used. In one embodiment of the invention, optionally in combination with any of the embodiments above or below, the pharmaceutical or veterinary composition is administered orally, topically or parenterally. For example, the pharmaceutical or veterinary composition may be formulated for oral administration and may contain one or more physiologically compatible carriers or excipients, in solid or liquid form. These preparations may contain conventional ingredients such as binding agents, fillers, lubricants, and acceptable wetting agents.

The pharmaceutical or veterinary composition may be formulated for parenteral administration in combination with conventional injectable liquid carriers, such as water or suitable alcohols. Conventional pharmaceutical or veterinary excipients for injection, such as stabilizing agents, solubilizing agents, and buffers, may be included in such compositions. These pharmaceutical or veterinary compositions may be injected subcutaneously, intramuscularly, intraperitoneally, or intravenously.

The pharmaceutical or veterinary composition may be formulated for topical administration. Formulations include creams, lotions, gels, powders, solutions and patches wherein the compound is dispersed or dissolved in suitable excipients. The topical compositions of the invention may be administered by means of a carrier material, which can be a solid support. Thus, it also forms part of the invention a topical composition comprising a carrier material, which can be a solid support. Illustrative, non-limiting examples of solid supports include intelligent textiles, dressings, coatings, sponges, band-aids, sanitary pads, compresses, plasters, etc. The manufacture of such compositions can be obtained by conventional methods, for example, by mixing the combinations of the invention and the material carrier.

The pharmaceutical or veterinary compositions may be in any form, including, among others, tablets, pellets, capsules, aqueous or oily solutions, suspensions, emulsions, or dry powdered forms suitable for reconstitution with water or other suitable liquid medium before use, for immediate or retarded release.

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.

Throughout the description and claims the word "comprise" and variations of thereof, 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 are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

EXAMPLES

Synthesis of the compounds of formula (I) (3-18) to (3-30)

The compounds of formula (I) 3-18 to 3-30 were synthesized as disclosed in the PCT application WO2014/131855 using the synthetic route indicated in Table 1 of this invention.

The HPLC measurement was performed using Gilson 281 from 233 pump (binary), an autosampler, and a UV detector. The fractions were detected by LC-MS. The MS detector was configured with an electrospray ionization source. The source temperature was maintained at 300-350 °C. The HPLC method used was method 2 as defined in PCT application WO2014/131855.

Example 1. HSPA1B gene expression in a cohort of atrial fibrillation patients

1.1 Recruited patients and methodology

200 consecutive patients with permanent atrial fibrillation (AF) were recruited from University Hospital of Salamanca, Spain. Patients with any of the following criteria were excluded: cancer in progress, leukocytosis (more than 7,000 cells/mL), leukopenia (less than 3,500 cells/mL), history of venous thromboembolism, acute coronary syndrome, infection, autoimmune disease or surgery in the last three months. Renal failure (creatinine value more than double of normal value), oral contraceptive use, hormonal therapy, and corticoid consumption were also excluding criteria. 101 patients had suffered a first episode of cardioembolic stroke while 99 did not. AF was diagnosed by electrocardiography and cardioembolic stroke was diagnosed clinically and confirmed by imaging techniques (magnetic resonance imaging or X-ray computed tomography). Blood samples were collected at least three months after the stroke episode. All patients were under anticoagulant treatment with anti-vitamin K drugs. Clinical characteristic of the patients are shown in Table 4. Table 4. Clinical characteristics of the AF patients

Non-stroke Stroke P (n=99) (n=101)

Age (years) 74.61±8.72 76.02±7.40 0.214

Gender (female) 43 53 0.201

CHAD

Hypertension, n (%) 1.44±0.82 1.65±0.89 0.088

Congestive heart failure, n (%) 65 (65.7) 75 (74.3) 0.184

Diabetes, n (%) 6 (6.1) 9 (8.9) 0.444

Leukocytes (xlO³, cells/mL) 6.44±1.58 6.84±1.78 0.093

Neutrophils (%) 59.03±19.08 59.78±19.36 0.121

Monocytes (%) 8.04±2.38 7.15±2.10 0.319

Lymphocytes (%) 27.42±8.49 26.80±9.82 0.442

Values for age, CHAD, leukocytes, neutrophils, monocytes and lymphocytes are the mean±SD. CHAD is the CHADS2 index (Congestive heart failure, Hypertension, Age, Diabetes, previous Stroke) after subtracting the previous stroke punctuation. Neutrophils, monocytes and lymphocytes are expressed as the percentage of the total number of leukocytes.

1.2 Determination ofHSPA!B gene expression

1.2.1 RNA isolation

Whole blood (2.5 mL) was collected from each subject into a PAXgene tube

(BD, Franklin Lakes, USA). PAXgene tubes were kept for 2 hours at room temperature and then frozen at -80 °C until their use. Total RNA was isolated according to the manufacturer^'s protocol using the PAXgene blood RNA kit (Pre-AnalytiX, Feldbachstrasse, Switzerland).

1.2.2 cDNA synthesis

One μg of each RNA sample was reverse-transcribed using RNase H-MMLV reverse transcriptase (Supercript II, Invitrogen, Illkirch, France) and random primers (Invitrogen). 1.2.3 Quantitative real time PCR (qRT-PCR)

A mixture of 10 of 2x TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA), 2 μΐ, (20 ng) of cDNA, 1 μΐ, of Taqman Gene Expression Assay for HSPA1B (HsOl 04050 l sH) or 1 μΕ of Taqman Gene Expression Assay for endogenous control [DECR1, 2,4-dienoyl CoA reductase 1 (Hs00154728_ml)] (Applied Biosystems) and 7 μΐ, of water was prepared. This mix was submitted to a preheating at 60 °C for 20 seconds (sec), followed by 40 cycles which consisted of shuttle heating at 95 °C for 1 sec and subsequent annealing at 60 °C for 20 sec, utilizing an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). The relative quantity of HSPA1B mRNA was normalized to the relative quantity of DECR1. All samples were run in triplicate. Samples exhibiting cycle threshold (Ct) differences among triplicates higher than 0.5 cycles were discarded.

1.2.4 Statistical analyses

A non-conditional logistic regression model was used to evaluate the risk of stroke associated with HSPA1B expression levels. Goodness of fit was tested with the Hosmer-Lemeshow goodness-of-fit statistic. The main independent variable was the level of HSPA1B categorized into quartiles according to the distribution in the non- stroke group. Univariate and multivariate, adjusting for traditional risk factors for cardioembolic stroke, analyses were performed. To assess the p value for linear trend, the quartile specific median was assigned to each quartile and the resulting variable was treated as quantitative. Product-terms were introduced in the non-conditional logistic models to analyze interaction (effect modification).

1.3 Results and conclusions

HSPA1B expression levels were inversely associated with stroke. Taking the lowest HSPA1B quartile as the reference, the risk of cardioembolic stroke decreased in parallel with the increase in HSPA1B expression after adjusting for CHAD index (CHAD stands for Congestive heart failure, Hypertension, Age and Diabetes), sex and leukocyte counts, (P for trend<0.001) (Table 2). Accordingly, maximum protection against stroke was shown for patients in the highest HSPA1B quartile: odds ratio (OR) [95% confidence interval (CI)] = 0.21 (0.09-0.51) (Table 5). Finally, we observed no significant multiplicative interaction between HSPAIB expression and age, gender or components of the CHAD index.

These results suggest that HSPAIB plays a non-negligible protective effect against cardioembolic stroke in AF patients.

Table 5. Risk of stroke according to HSPAIB expression

Qi Q2 Q3 Q4

Range (AU) 0.07-0.89 0.90-1.35 1.36-2.13 2.14-5.15

Cases/Controls 48/25 21/24 20/25 12/25 P

OR ¹ 1 (ref.) 0.46 0.42 0.25 0.001

(95% CI) (0.21-0.97) (0.20-0.89) (0.11-0.58)

OR ¹ 1 (ref.) 0.43 0.38 0.21 <0.001

(95% CI) (0.19-0.94) (0.17-0.85) (0.09-0.51)

Q, quartile. 1, non-adjusted model; 2, CHAD, sex and leukocyte counts-adjusted model (leukocyte counts were categorized into tertiles for this purpose). The P value for lineal trend is indicated. AU, arbitrary units.

Example 2. Thrombus formation in HSPA1A/B knockout mice

2.1 Background

HSPAIB codes for the Heat shock 70 kDa protein lA/lB (Hsp70) which is a chaperone aimed to protect the cell against a variety of insults. A cardioembolic stroke takes place when a thrombus formed in the left atrium travels to the brain. Therefore, the next aim was to investigate the role of Heat shock 70 kDa protein lA/lB in thrombus formation.

Heat shock 70 kDa protein lA/lB is produced not only by HSPAIB but also by the HSPA1A gene. For this reason, mice simultaneously knocked-out (KO) for both HSPA1A and HSPAIB (HSPA1A/B KO) were chosen to study the influence of Hsp70 in thrombosis. To assess this issue, three different thrombosis models were used. 2.2 Material and methods

2.2.1 HSPAIA/B KO mice

HSPAIA/B KO mice (B6;129S7-Hspala/Hspalb^tmlDix/Mmcd) were purchased from the Mutant Mouse Regional Resource Centers (University of California, Davis, CA, USA). The strain used to generate the HSPAIA/B KO mice was used as a control and was obtained from Harlan Laboratories (Harlan Interfauna Iberica S.A., Barcelona, Spain). 7-8 weeks female mice were used for the assay.

2.2.2 Thrombosis experimental models

2.2.2.1. Thrombus formation in carotid artery after administration of Rose Bengal and exposure to laser light.

A mixture of 100 mg/kg ketamine (Merial, Lyon, France) and 10 mg/kg xylazine (Bayer HealthCare, Kiel, Germany) was administered intraperitoneally (i.p.) to each mouse at the beginning of the experiment. The left common carotid artery was fitted with a Doppler flow probe (Model 0.5 VB; Transonic System, Ithaca, NY, USA). Rose Bengal (Sigma-Aldrich, St Louis, MO, USA) was intravenously infused (100 mg per kg of body weight), and the carotid artery was immediately exposed to 1.5-mW 540-nm laser (Melles Griot Inc, Carlsbad, CA, USA). Flow was monitored for 30 minutes (min) after the thrombus had formed, which was assumed to occur when the blood flow was totally interrupted or for 120 min in the cases that no occlusive thrombi were formed.

2.2.2.2. Thrombus formation in carotid artery exposed to ferric chloride

In this model, ferric chloride was used as the thrombogenic trigger. A 2x2 mm piece of Whatman paper soaked in 15% ferric chloride was applied to the left carotid artery. After 5 min, the Whatman paper was removed and the vessel was washed with saline. Flow through the carotid artery was monitored for 30 min after the thrombus formation or for 40 min in the cases that no occlusive thrombi were formed.

2.2.2.3. Intravenous administration of collagen and epinephrine

Mice were anesthetized and thromboembolism was induced by an intravenous injection of a mixture of 0.8 mg/kg of collagen (Roche, Mannheim, Germany) and 60 μg/kg of epinephrine (Sigma-Aldrich). Animals were followed for 30 min and mortality was registered. 2.2.3 Coagulation test

Blood from mice was extracted by cardiac puncture, mixed with sodium citrate, and centrifugate 3,000 rpm for 10 min at 4°C to obtain blood plasma. Prothrombin times (PT) and activated partial thrombloplastin times (APTT) were determined using an automated blood coagulation system BSC Coagulation System (Siemens, Berlin, Germany).

2.2.4 Tail bleeding time test

Mice were anesthetized and the distal 3-mm segment of the tail was removed with a scalpel. Bleeding was monitored by absorbing the bead of the blood with a filter paper at 15 sec intervals without touching the wound. Bleeding was stopped manually if it continued for more than 30 min.

2.2.5 Statistical analyses

To assess the differences in the occlusion and bleeding times between wild-type and KO mice the Mann- Whitney U test was used. The statistical significance of the Kaplan-Meier curves was calculated by the Log-Rank test.

2.3 Results and conclusions

First, the time lasted until an occlusive thrombus formed in the carotid artery of Rose Bengal-administered and laser light-exposed mice was significantly shorter in animals lacking Heat shock 70 kDa protein 1A/1B (Figure 1A). Carotid artery exposure to ferric chloride was also used as an alternative thrombogenic stimulus. The thrombogenic role of Heat shock 70 kDa protein 1 A/IB deficiency was confirmed since the vessel occlusion time was again significantly shorter in the HSPA1A/B KO animals (Figure IB). A methodologically different approach to thrombosis was finally used: the intravenous administration of collagen and epinephrine would cause the mouse to die within few minutes due to the massive pulmonary thromboembolism. Of note, Heat shock 70 kDa protein lA lB deficiency accelerated mice death (Figure 1C). Taking all these evidences together, it is concluded that the absence of Heat shock 70 kDa protein 1 A/IB facilitates thrombus formation upon thrombogenic challenge.

In order to understand why HSPA1A/B KO mice were prone to thrombosis their hemostatic system was explored. Surprisingly, plasma from wild type animals (n=5) and the ones obtained from HSPA1A/B KO animals (n= 4) displayed similar PT [11.3 sec (10.1-12.7) vs. 10.7 sec (9.6-11.1) respectively] and APTT [39.6 sec (38.1-59.7) vs. 44.4 sec (37.3-48.3) respectively], and similar values between both groups were also obtained in the tail bleeding time test (Figure ID). Thus, HSPAIA/B KO animals are prone to thrombosis, but they do not display an obvious gross hemostatic alteration.

Example 3. Effect of compounds of the invention on in vitro HSPA1B gene expression

3.1 Background

Heat shock factor 1 (HSF1) is the main transcription factor involved in the induction of Heat shock 70 kDa protein 1A/1B expression. Compounds that increase the HSF1 activity increase the Heat shock 70 kDa protein lA/lB expression levels like TRC051384 (compound of formula 7-01) or BGP-15 (compound 7-02), among many others. There is also a group of compounds that inhibits histone deacetylase 6 (HDAC6). These compounds can keep heat shock protein 90 (Hsp90) acetylated. Unlike the deacetylated form, acetylated Hsp90 cannot bind to HSF1, which is then able to trimerize and induce HSPAIA/B transcription and thus Heat shock 70 kDa protein 1A/1B expression. The experiments described in this section were conducted to determine the effect of several compounds on the expression of HSPA1B gene in cultured cells.

3.2 Experimental test and expression measurement procedure

3.2.1 Cell culture conditions

EA.hy926 and C2C12 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were seeded in a 6-well plate (Corning, New York, NY, USA), at a density of 100,000 cells/well in 2 mL culture medium consisting of Dulbecco's Modified Eagle Medium (DMEM) (Gibco, NY, USA) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic (Gibco), and allowed to grow for 24 hours at 37 °C. Each compound was assayed at 1 and 10 μΜ. The plate was incubated at 37 °C in a C0₂ incubator for 3 and 24 hours in the case of EA.hy926 cells and for 3 hours in the case of C2C12 cells. Rocillinostat was purchased from Selleckhem (Munich, Germany). 3.2.2 RNA isolation and quantification

After incubation, cells were detached and RNA was isolated following TriReagent® (Sigma- Aldrich) recommended instructions.

3.2.3 cDNA synthesis and qRT-PCR

These procedures were performed as described in Example 1 using TaqMan

Gene Expression Assay Hs01040501_sH for HSPAIB gene and Hs03929097_gl for GAPDH gene, which was used as endogenous control.

3.2.4 Data analysis

Data analysis was performed by comparative Ct method. HSPAIB expression was normalized to the expression of GAPDH for each sample. The results for test compounds were expressed as fold induction of HSPAIB relative to vehicle-treated control, and are shown in Table 6.

3.3 Results and conclusions

As it is shown in Table 6, HSPAIB levels were increased over control after treatment with compounds of the invention at least in one experimental condition or cell line. Thus, it is concluded that the compounds of the invention have the ability to induce the expression of HSPAIB gene. Table 6. HSPAIB gene induction in different conditions

0, less than 2 fold induction; +, 2-3 fold induction; ++, 3.1-10 fold induction; +++ 10.1- 20, fold induction; ++++, >20.1 fold induction.

* Cells were pre-heated at 42 °C for 40 minutes and then incubated for 3 hours at 37 °C in the presence of the compound or vehicle.

Example 4. Evaluation of anticoagulant activity of compounds that induce

HSPAIB expression

4.1 Material and methods

4.1.1 Compound treatment

All compounds were i.p. administered at the following times before the start of the thrombosis models: the Heat shock 70 kDa protein lA/lB inducer compound TRC051384 (compound 7-01) (WuXi AppTec, Shanghai, China) (9.00 mg Kg^"1 in saline solution) or its vehicle, 1 and 3 hours; tubastatin A (compound 6-04) (Sigma- Aldrich) (20 mg Kg^"1 in 10% DMSO) or its vehicle, 8 hours; compound 3-11 (20 mg Kg^"1 in saline solution) or its vehicle, 1 hour; compound 1-16 (20 mg Kg-1 in 10% DMSO) or its vehicle, 3 hours; and BGP-15 (compound 7-02) (Sigma- Aldrich) (20 mg Kg^"1 in saline solution) or its vehicle were administered once a day for five days before the start of the thrombosis induction procedures.

4.1.2 Thrombosis models

To assess the anticoagulant activity of the compounds, they were evaluated in the three thrombosis models described above (see example 2). Eight weeks female CD1 mice obtained from Harlan Laboratories (Harlan Interfauna Iberica S.A.) were used for this purpose. TRC051384 was also assayed in HSPA1A/B KO female mice purchased from the Mutant Mouse Regional Resource Centers (University of California) using the model of thrombus induced by ferric chloride.

4.1.3 Measurement of HSPAIB gene expression in vascular tissue

The mice were sacrificed and perfused with 10 mL of PBS. Aortic samples were extracted, and then snap frozen and stored at -80 °C until RNA extraction with TriReagent® (Sigma- Aldrich). After retrotranscription, qRT-PCR was performed on an ABI PRISM 7900 detector (Applied Biosystems) using TaqMan Gene Expression Assays for murine HSPAIB, (Mm03038954_sl, Applied Biosystems). Mouse β-Actin (ACTB, Mm00607939_sl, Applied Biosystems) was used to normalize results. 4.1.4 Measurement of Heat shock 70 kDa protein 1A/1B in vascular tissue Aortic tissue was incubated with RIPA buffer (50 mM Tris; 150 mM NaCl;

0.1% SDS; 0.5% DOC; 1% Triton-X) and homogenized with Polytron Homogenizer (Kinematica, Eschbach, Germany) for 2 min. Tissue homogenate was maintained for 2 hours at 4 °C in shaking and, finally, centrifuged at 16,000 g at 4 °C to collect the supernatant. The proteins obtained from the cell lysate were quantified using a BCA assay (Pierce, Rockford, IL, USA). 10 μg of the proteins extract were denatured under reducing conditions and submitted to a western blot analysis. The primary antibodies were a rabbit anti- heat shock 70 kDa protein 1A/1B polyclonal antibody (pAb) (Thermo, Walthman, MA, USA) and a rabbit anti-P-actin pAb (R&D Systems, Minneapolis, MN, USA).

4.1.5 Statistical analyses

To assess the differences in the occlusion times between treated and non-treated mice the Mann- Whitney U test was used. The Kaplan-Meier curve significance was calculated by the Log-Rank test.

4.2 Results and conclusions

TRC051384 induced both the mRNA of HSPAIB and the heat shock 70 kDa protein 1A/1B levels in vessel tissue from treated mice (Figure 2). TRC051384 significantly delayed the time to thrombus formation or death in the three thrombosis models (Figure 3A-C). Interestingly, TRC051384 was unable to delay the time to thrombus formation in HSPAIA/B KO mice (Figure 3D), strongly suggesting that the overexpression of HSPAIB and, as a result, the induction of Heat shock 70 kDa protein 1 A/IB is directly involved in preventing thrombosis.

It was also studied the effect of Heat shock 70 kDa protein lA/lB inducer compound BGP-15 to further substantiate that HSPAIA/B induction can prevent thrombosis. This compound induced the expression of HSPAIB in the vessel tissue of mice after five days of i.p. treatment (4.24 fold-increase over vehicle-treated mice) and consistently delayed the onset of thrombosis (Figure 4).

On the other hand, inventors hypothesized that the use of HDAC6 inhibitors like tubastatin A could induce Hsp70 by avoiding Hsp90 deacetylation. Tubastatin A was able to induce HSPAIB expression in vivo in murine vascular vessel (5.34 fold-increase over vehicle-treated mice). Accordingly, a significant antithrombotic effect was also observed in the three murine thrombosis models (Figure 5).

To go deeper into the HDAC6 inhibitors as antithrombotic molecules, two other HDAC6 inhibitors, compounds 3-11 and 1-16, were assayed to assess their antithrombotic effect. Figures 6 and 7 show that these compounds also have an antithrombotic effect.

Thus, it is concluded that the compounds included in the invention have an anticoagulant activity. Example 5. Evaluation of the effect on bleeding time of the treatment with a compound able to induce HSPA1B expression

5.1 Background

In this example the mice bleeding risk associated to the treatment with antithrombotic Heat shock 70 kDa protein lA/lB inducer compounds was compared with the risk associated to the treatment with rivaroxaban, one of the anticoagulant drugs more widely used to prevent and treat thrombosis currently.

5.2 Material and methods

5.2.1 Compound treatment

Compound treatment pattern for TRC051384 (compound 7-01), tubastatin A

(compound 6-04), compound 3-11 and compound 1-16 have already been indicated in example 4. 3 mg/Kg rivaroxaban (Selleckchem) in DMSO or vehicle were intravenously administered 1 hour before starting the experiment.

5.2.2 Bleeding time

The tail bleeding time in CD1 female mice has been described in example 2.

5.2.3 Statistical analyses

To assess the differences in the bleeding time between treated and non-treated mice, the Mann- Whitney U test was used.

5.3 Results and conclusions

Of note, mice treated with each compound of the invention (TRC051384, tubastatin A, compound 3-11 and compound 1-16) did not show longer bleeding times than those animals treated with vehicle (Figure 8). On the contrary, mice treated with rivaroxaban displayed a prolonged bleeding time (Figure 9).

Thus, it is concluded that antithrombotic compounds that induce Heat shock 70 kDa protein 1A/1B do not induce bleeding.

Example 6. Enzymatic activity assays

The biochemical assay to measure HD AC 1 , HDAC2, HDAC3, and HDAC6 enzyme activities relies on the fluorescence signal produced by a specific labelled substrate (BPS Biosciences, Cat # 50037) after its deacetylation by HDACs. Fluorogenic substrate, containing a acetylated lysine side chain, can be deacetylated and then sensitized to subsequent treatment with the lysine developer (BPS Biosciences, Cat# 50030), which produced a fluorophore that can be measured with a fluorescence plate reader.

The enzymes were obtained from BPS Biosciences. The enzyme HDAC1 (GenBank Accession number No. NM_004964; Cat. # 50051) is full-length with C- terminal his tag and C-terminal Flag tag. The enzyme HDAC2 (GenBank Accession number No. NM_001527; Cat. # 50002) is full-length with C-terminal his tag. The enzyme HDAC3 (GenBank Accession number No. NM 003883; Cat. # 50003) is full- length with C-terminal his tag and human NCOR2, N-terminal GST tag. The enzyme HDAC6 (GenBank Accession number No. BC069243; Cat. # 50006) is full-length with N-terminal GST tag. The enzymes were expressed in a baculovirus infected Sf9 cell expression system.

Enzyme activity assay was carried out in a black 96-well plate in a final volume of 100 μί, as follows:

- 5 of vehicle or studied compound 10 x concentrated prepared in assay buffer

(BPS Biosciences, Cat # 50031). Final percentage of DMSO was 1%;

- 5 μΐ, of HDAC1 (4 μg/mL HDAC1) or 5 μΐ, of HDAC2 (15 μg/mL) or 5 μΐ, of HDAC3 (10 μg/mL) or 5 μΙ_, of HDAC6 (36 μg/mL) diluted in assay buffer. Final concentration was 0.4 μg/mL (HDAC1), 1.5 μg/mL (HDAC2) or 1 μg/mL (HDAC3) or 3.6 μg/mL (HDAC6); Start the reaction by adding 40 of reaction mixture containing 0.125 mg/mL BSA and 12.5 μΜ of fluorogenic HDACs substrate. Final concentrations of BSA and substrate were 0.1 mg/mL and 10 μΜ, respectively;

- After plate sealing, mixture was incubated for 30 minutes at 37 °C;

- Reaction was stopped by adding 50 μΐ, of lysine assay developer;

Plate was incubated 20 minutes at room temperature.

Fluorescence of each at 355 nm excitation and 460nm emission was determined using the plate reader Mithras (Berthold). Positive control was obtained in the presence of the vehicle of the compounds. Negative control was obtained in the absence of HDAC1 enzyme activity.

On the other hand, PDE9A enzyme activity assay was also conducted for some new compounds. The biochemical assay to measure PDE9A isoform b enzyme activities relies on the HTRF cGMP assay kit from CisBio (CisBio, Cat. # 62GM2PEB), which determines the amount of cGMP present in the reaction. The enzyme was obtained from BPS Biosciences (GenBank Accession number for PDE9A:

NM 001001567, Cat. # 60090) and it is full-length with N-terminal GST tag. It was expressed in a baculovirus infected Sf9 cell expression system.

Enzyme activity assay was carried out in a 384-well plate in a final volume of 20 μί, as follows:

2.5 μΐ, οΐ vehicle or studied compound 4 x concentrated prepared in assay buffer containing 50 mM Tris-HCl, 6 mM MgC12, pH 7.4 and additionally 0.03%

Tween-20. Final percentage of DMSO was 0.5%.

2.5 μΐ_^ PDE9A (0.2 μg/mL) diluted in assay buffer. Final concentration was 0.05 μg/mL (PDE9A.

5 μΐ_^ of substrate cGMP 4 x concentrated to reach a final concentration of cyclic nucleotide of 100 nM (PDE9A).

After plate sealing, mixture was incubated for 30 minutes at 37 oC.

Reaction was stopped by adding 5 μΐ, of labelled cGMP labelled with the dye D2 (cGMP-D2) and 5 μΕ of Mab anti-cGMP labelled with cryptate (cGMP- cryptate) as recommended by the assay kit of CisBio.

Plate was sealed and incubated 1 hour at room temperature. Fluorescence of each well was determined at 665nm excitation and 620nm emission using the plate reader EnVision (Perkin-Elmer). Results were calculated from the 665 nm/620 nm ratio. Positive control was obtained in the presence of the vehicle of the compounds. Negative control was obtained in the absence of cGMP and labelled cGMP-D2 cyclic nucleotide.

Table 7 shows the inhibition values for recombinant enzymes (IC₅₀).

Table 7. Inhibition values for recombinant enzymes (IC50) of some new compounds.

(+) IC50 > 10μΜ; (++) 1 μΜ < IC50 < 10 μΜ; (+++) 10 nM < IC50 < 1 μΜ; and (++++) IC50 < 10 nM .

As can be seen in the above table, tested compounds of the invention show a dual inhibition of PDE9 and HDAC.

Claims

1. A compound that induces expression of Heat shock 70 kDa protein lA/lB for use in anticoagulation therapy.

2. The compound for use according to claim 1, wherein the compound is selected from the group consisting of

a) a histone deacetylase 6 (HDAC6) inhibitor compound;

b)

BGP-15, TRC051384:

and

c) a pharmaceutically or veterinary acceptable salt of the compound, or any stereoisomer either of the compound or of any of its pharmaceutically or veterinary acceptable salts.

3. The compound for use according to claim 2, wherein the compound is an HDAC6 inhibitor selected from, a) a compound of formula (I):

(I)

wherein

p, n and r are independently 0 or 1;

Zi is a biradical selected from the group consisting of a formula (E), formula (F"), formula G'), formula (Ff), formula (J'), and formula (K):

Z₃ and each Z5 are independently a biradical of a saturated or unsaturated (Ci-Ce)alkyl optionally substituted with one or more halogen atoms; Z₄ is a biradical of a saturated or unsaturated (Ci-C₆)alkyl optionally substituted with one or more substituents selected from halogen, OH, and -0(Ci-C₃)alkyl optionally substituted with one or more halogen atoms; or alternatively Z₄ is -CRⁿR¹²-, wherein R¹¹ and R¹² taken together with the carbon they are attached to form C=0 or a 3- to 7- membered carbocyclic or heterocyclic monocyclic ring, which is saturated or partially unsaturated, and which is optionally substituted with one or more halogen atoms or (Ci-C₃)alkyl optionally substituted with one or more halogen atoms;

q and m are independently 0 or 1 ;

(Li ) (L₂

a chain length comprised from 1 to 20 atoms; b) a compound of formula (II):

(II)

wherein

q and s are independently 0 or 1 ;

Z₆ is selected from the group consisting of-Z₈-; - Z₈-Cy⁶-; -Z8-Cy⁶-Z₈-; and -Cy⁷-; Z₇ and Z are independently a biradical of a saturated or unsaturated (Ci-Ce)alkyl optionally substituted with one or more halogen atoms; Cy⁵ and Cy⁶ are independently phenyl or a 4- to 6-membered carbocyclic or heterocyclic monocyclic ring; which is saturated or partially unsaturated or aromatic; and which is optionally substituted with one or more Rs groups;

with the condition that the moiety (L₃) of the formula (II)

(L₃)

has a chain length comprised from 1 to 20 atoms; c) a compound selected from the group consisting of:

N-[4-[(2i?,4i?,65)-4-[(4,5-diphenyloxazol-2-yl)sulfanylmethyl]-6-[4- (hydroxymethyl)phenyl]- 1 ,3-dioxan-2-yl]phenyl]-8-(hydroxyamino)-8-oxo- octanamide (6-01);

N-[4-[l-[(4-fluorophenyl)methyl]triazol-4-yl]phenyl]-8-(hydroxyamino)-8-oxo- octanamide (6-02);

tert-butyl N- [4- [3 - [[7-(hydroxyamino)-7-oxo-heptyl]carbamoyl] isoxazo 1-5 - yljphenyl] carbamate (6-03);

4-[(2-methyl-3,4-dihydro-lH-pyrido[4,3-b]indol-5-yl)methyl]benzenecarbohydroxamic acid (6-04);

1 -butyl- 1 -[[4-(hydroxycarbamoyl)phenyl]methyl]-3-phenyl-urea (6-05); 2-( 1 -methylpyrrole-2-carbonyl)-3 ,4-dihydro- lH-isoquinoline-6- carbohydroxamic acid (6-06);

2-( 1 -methylpyrrole-2-carbonyl)-3 ,4-dihydro- lH-pyrrolo[ 1 ,2-a]pyrazine-7- carbohydroxamic acid (6-07);

6-(3-phenyl- lH-indole-2-carbonyl)-7,8-dihydro-5H- 1 ,6-naphthyridine-3- carbohydroxamic acid (6-08);

1 -[7-(hydroxycarbamoyl)tetralin-2-yl]-3-phenyl-urea (6-09);

1 -cyclopentyl-3-[ 1 -[5-(hydroxycarbamoyl)-2-thienyl]ethyl]urea (6-10);

N-[(IR)- 1 -[4-(hydroxycarbamoyl)phenyl]ethyl]adamantane- 1 -carboxamide (6- ii);

6-(2,2-dimethylpropanoyl)-5,7-dihydro-4H-thieno[2,3-c]pyridine-2- carbohydroxamic acid (6-12);

4- [ [(2R)-2- [(4-hydroxyphenyl)methyl] -3 -oxo-2,4-dihydroquinoxalin- 1 - yl]methyl]benzenecarbohydroxamic acid (6-13);

cyclohexene-l-carbohydroxamic acid (6-14);

2-[[ 1 -(3-fluorophenyl)cyclohexyl]amino]pyrimidine-5-carbohydroxamic acid (6

15) ;

(E)-3 - [5- [ [benzyl(propyl)amino]methyl] -2-furyl]prop-2-enehydroxamic acid (6-

16) ;

(E)-3-(3-pyrrolo[2,3-¾]pyridin-l-ylsulfonylphenyl)prop-2-enehydroxamic acid (6-17);

4-[[2-ethyl-4-oxo-3-(2-phenylethyl)quinazolin-7-yl]methyl]benzenecarbo- hydroxamic acid (6-18);

tert-butyl N- [( 1 S)- 1 -(cyclopentylcarbamoy l)-6-sulfanyl-hexyl] carbamate (6- 19); tert-butyl N- [( 1 S)- 1 -(cyclopentylcarbamoy l)-7-(hydro xyamino)-7-oxo- heptyljcarbamate (6-20);

N-(8-quinolyl)-6-(sulfamoylamino)hexanamide (6-21);

(S)-2-(6,18-Dioxo-5,6,7,9,10,l 1,12,13, 18,19-decahydrobenzo[5,6][l,4,7]- oxadiazacyclotetradecino[ 10,9-b]indol-7-yl)-N-hydroxyacetamide (6-22);

N- [7-(hydroxyamino)-7-oxo-heptyl] -2-(N-phenylanilino)pyrimidine-5 - carboxamide (6-23); 2- [4-(hydroxycarbamoyl)phenyl] -N-(2-hydroxyethyl)-N-phenyl-acetamide (6- 24);

2- [4-[[8-(hydroxyamino)-8-oxo-octanoyl]amino]phenyl]-N,N'-bis(8- quinolyl)propanediamide (6-25);

4-[(2,2-dioxo-3,4-dihydro-lH-thiopyrano[4,3-b]indol-5- yl)methyl]benzenecarbohydroxamic acid (6-26);

4-[[6-fluoro-2,2-dimethyl-3-[(2-methylimidazol- 1 -yl)methyl]-4-oxo- 1 ,3- dihydrocarbazol-9-yl]methyl]benzenecarbohydroxamic acid (6-27);

N- [7-(hydroxyamino)-7-oxo-heptyl] -4- [methoxy-phenyl-(2- pyridyl)methyl]benzamide (6-28);

(E)-N-[4-[2-(hydroxycarbamoyl)-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5- carbonyl]phenyl]-3-phenyl-prop-2-enamide (6-29);

4- [(5 -methyl-2-oxo-3H-benzimidazol- 1 -yl)methyl]benzenecarbohydroxamic acid (6-30);

3 - (3 -furyl)-N- [6-(hydroxyamino)-6-oxo-hexyl] -5 -(4-hydroxyphenyl)- 1H- pyrrole-2-carboxamide (6-31);

tert-butyl 4-[6-(hydroxycarbamoyl)-3,4-dihydro-lH-isoquinoline-2-carbonyl]-4- methyl-piperidine- 1 -carboxylate (6-32);

3-[(3-chlorophenyl)methyl]-4-oxo-quinazoline-6-carbohydroxamic acid (6-33);

N-[3-(hydroxycarbamoyl)-6-quinolyl]cyclopropanecarboxamide (6-34);

N- [3 - [3 -(hydroxycarbamoyl)tetralin-6-yl]phenyl] -2,2-dimethyl-propanamide (6-

35) ;

2-[2-(2-pyridyl)acetyl]-3,4-dihydro-lH-isoquinoline-6-carbohydroxamic acid (6-

36) ;

, and any derivative and/or analog compound thereof that has HDAC6 inhibitor activity; and d) a pharmaceutically or veterinary acceptable salt of any of the compounds included in a), b), and c), or any stereoisomer either of the compound or of any of its pharmaceutically or veterinary acceptable salts.

4. The compound for use according to claim 3, wherein the compound is a compound of formula (I), in which Bi is a radical selected from the group consisting of formula (A), formula (B), formula (C), and formula (D):

R₂ is H or saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms;

Zi is a biradical selected from the group consisting of a formula (E), formula (F), formula (G), formula (H), formula (J), and formula (K):

and R₃ is selected from halogen, saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms; and saturated or unsaturated -0(Ci-C₄)alkyl optionally substituted with one or more halogen atoms.

5. The compound for use according to any of claims 3 or 4, wherein the compound is a compound of formula (I), in which wherein Cy² is a N-attached 5- to 7-membered heterocyclic monocyclic ring.

6. The compound for use according to any of claims 3 - 5, wherein the compound is a compound of formula (I), in which n is 0 and Cy¹ is selected from the group consisting of optionally substituted phenyl, optionally substituted 5- to 6-membered heteroaromatic ring, optionally substituted 3- to 6-membered carbocyclic ring, and optionally substituted 3- to 7-membered heterocyclic ring.

7. The compound for use according to any of claims 3 - 5, wherein the compound is a compound of formula (I), in which n is 1 ; Z₃ is -CH(R₅)-, wherein R₅ is H or optionally substituted (Ci-C4)alkyl; and Cy¹ is a optionally substituted 3- to 6-membered carbocyclic ring or optionally substituted 3- to 7-membered heterocyclic ring.

8. The compound for use according to any of claims 3 - 7, wherein the compound is a compound of formula (I), in which Ri is selected from the group consisting of optionally substituted (Ci-C₇)alkyl, optionally substituted 3- to 6-membered carbocyclic ring, and optionally substituted 3- to 7-membered heterocyclic ring.

9. The compound for use according to any of claims 3 - 8, wherein the compound is a compound of formula (I), in which Z_\ is selected from the group consisting of:

10. The compound for use according to any of claims 3 - 9, wherein the compound is a compound of formula (I), in which r is 1 and Z₂ is selected from the group consisting of:

1 1. The compound for use according to any of claims 3 - 10, wherein the compound is a HDAC6 inhibitor compound of formula (Γ):

( )

in which Bi' is selected from the group consisting of formula (Α'), formula (Β'), and formula (C)

(Α') (Β') (C)

12. The compound for use according to claim 1 1 , wherein the compound is a HDAC6 inhibitory compound of formula (IA) or formula (IB):

in which R₃' is selected from H; halogen; saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms; and saturated or unsaturated -0(Ci-C₄)alkyl optionally substituted with one or more halogen atoms.

13. The compound for use according to claim 1 1 , wherein the compound is a HDAC6 inhibitor compound of formula (IC):

(IC)

in which Cy¹ is 4- to 6-membered heterocyclic ring, and R₃' is selected from H; saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms.

14. The compound for use according to any of claims 3 - 10, wherein the compound is a HDAC6 inhibitor compound of formula (ID):

(ID) in which R₃' is selected from H; halogen; and saturated or unsaturated (Ci-C₄)alkyl optionally substituted with one or more halogen atoms.

15. The compound for use according to any of claims 3 - 14, wherein the compound is a selective HDAC6 inhibitor compound.

16. The compound for use according to any of claims 1 - 15, in a subject presented with atrial fibrillation, arterial thrombosis, acute coronary syndrome associated with coronary thrombosis, metallic prosthetic cardiac valves, stroke, systemic embolism, venous thrombosis, deep venous thrombosis, venous thromboembolism, and/or pulmonary embolism.

17. The compound for use according to any of claims 1 - 15, for stroke prophylaxis in a subject presented with atrial fibrillation.

18. The compound for use according to any of claims 1 - 17, in a subject receiving antiplatelet drug therapy. 19. The compound for use according to claim 18, wherein the subject receives as antiplatelet drug therapy a compound selected from the group consisting of Aspirin, Triflusal, Clopidogrel, Prasugrel, Ticagrelor, Ticlopidine, Cilostazol, Vorapaxar, Eptifibatide, Tirofiban, Dipyridamole, and Terutroban.

H

3-30

or a pharmaceutically or veterinary acceptable salt thereof, or any stereoisomer either of the compound of formula (3-18)-(3-30) or of any of its pharmaceutically or veterinary acceptable salts.

21. A pharmaceutical or veterinary composition which comprises a therapeutically effective amount of a compound as defined in claim 20, together with one or more pharmaceutically or veterinary acceptable excipients or carriers.