Title of the invention

"New nuclear transcription factors regulators"

*****

Field of the invention

This invention relates to the field of compounds able to regulate nuclear transcription factors

(NTFs) . NTFs activation is involved in a variety of human diseases such as atherosclerosis, asthma, arthritis, cancer, diabetes, AIDS, inflammatory bowel diseases and stroke. The development of NTF

regulators should reduce side effects associated with drugs such as, for example, NSAIDs, glucocorticoids etc. Background of the invention

Many NTFs have been described in the literature. A non exclusive list of factors that are involved in the inflammatory process include nuclear factor kappa B (NF-kB) , activator protein 1 (AP-I) , specificity

protein 1 (SpI) , peroxisome proliferator-activated

receptors (PPARs) , Nr2/small Maf and other members

of the nuclear receptor superfamily (K. Kataoka et

al. in J. Biol. Chem. 2001, 276 (36) , pag. 34074-81 and Y. Lavrovsky et al . in Exp. Gerontol. 2000, 35, pag. 521-32) .

Among these NTFs, NF-kB is one of the most studied and will be described as non-limitative example. It

was discovered by David Baltimore's group in 1986,

ubiquitously expressed that represents a group of

five mammalian, structurally related, proteins also called Rel/NF-kB proteins (R. Sen and D. Baltimore in

Cell 1986, 47, pag. 921-928) . NF-kB plays a central role in regulating expression of a large number of genes that are critical for the regulation of

apoptosis, viral replication, tumorigenesis, inflammation etc. Cytokines that are stimulated NF- kB, such as IL-lβ and TNF-α, can also directly activate the NF-kB pathway, thus establishing loops

that can amplify the inflammatory response and increase the duration of chronic inflammation. NF-kB also stimulates the expression of enzymes whose products contribute to the pathogenesis of inflammatory process, including the inducible form of

nitric oxide synthase (iNOS) , which generates nitric

oxide (NO) , and the inducible cyclooxygenase (COX2) ,

which generates prostanoids (Y. Yamamoto and R.

Gaynor in J. Clin. Invest. 2001, 107, pag. 135-142) . The activation of NF-kB is thought to be part of a

stress response as it is activated by various factors

that include growth factor, cytokines, lymphokynes,

viral proteins, ischemia/reperfusion, UV,

pharmacological agents, and oxidative. Generally,

inflammation has been implicated in a very large

number of disorders either as a cause of primary

disease process or at least as consequence leading to

mild/severe complications. Therefore, there is a need in the art for more effective and safer drugs for inflammation-associated disorders in the cardiovascular system (for example myocardial and

vascular ischemia in general, hypertension systemic

and regional, stroke, atherosclerosis, etc), connective tissue (for example arthritis and connected inflammatory diseases, etc.) , pulmonary ssstem (for example asthma, COPD, etc.), gastrointestinal system (for example ulcerative and non-ulcerative diseases, intestinal inflammatory diseases, liver cirrhosis, etc) , urogenital system

(for example impotence, incontinence, etc.) , central

nervous system ( Alzheimer disease, Parkinson's

disease and neurogenerative diseases in general) ,

cutaneous system (eczema, neurodermatitis, acne,

etc.) , infective disease (of bacteria, viruses,

parasites origin) and for antineoplastic therapy in different organs (colon, lung, prostate, ovaries,

uterus, breast, tongue, liver, bone, etc.) , in

prevention and/or treatment as single therapy or

cotherapy with other chemiotherapic agents or

radiotherapic interventions.

Tissue damage by infection, trauma, toxins,

abnormally low or high temperatures and other

reasons, usually leads to formation of increased

amounts of pathogenetic mediators, such as prostaglandins, cytokines, leukotrienes, interleukins, oxy- , thiyl- and nitrogen-free radicals, interacting each other. A serious,

outstanding medical need is the development of NTF specific inhibitors that afford an efficacious treatment while minimizing unwanted effects. The research has been focused in the past to develop effective therapeutic agents able to counteract the deleterious effects of such mediators, but results

have been so far either unsatisfactory or only partially satisfactory. In fact it has been described

(see for example the review by Tak PP and Firestein

GS JCI, 107, 7-11,2001) that some compounds, including

corticosteroids, sulfasalazine, 5-aminosalicylic

acid, aspirin, NO-releasing aspirin, tepoxalin,

leflunomide, curcumin, antioxidants, proteasome

inhibitors are able to inhibit NF-kB only at relatively high concentrations. Thus this appears to

be the cause for the preclusion of the utilization of

NF-kB inhibitors in therapy. An alternative and/or

complementary approach is to develop agents able to

potentiate the natural defences. In this class of

agents a variety of moieties have been described,

including again in a non-limitative and exclusive way: heme oxygenase-1, y-glutamylcysteine synthetase, ILlO, Mn-SOD, Catalase, DT-diaphorase, glutathione peroxidase, thioredoxin system, NADPH-oxidase, NAG-I,

p53.

The ideal approach is to develop compounds able to both counteract aggressive and pathogenetic

factors and potentiate defensive substances. This can be obtained by combining chemically a drug known to counteract one or more of the above mentioned pathogenetic mediators with other moieties able to regulate the activation of NTF.

The new compounds object of the present

invention, that are able to inhibit the formation of

a pathogenetic mediator, such as inducible NOS at non-toxic concentrations, and to stimulate the

formation of a defensive agent, such as heme

oxygenase-1, have the following chemical structure:

D-X-Y-S (I)

wherein D is a drug, X is zero or a chemical

functional group, such as C=O, COOR, CONH, R being straight or branched Ci-C ₄ -alkyl,

Y is zero or a bifuntional linker, such as HO-

(CH ₂ ) n-0H, HOOC- (CHa) _n -COOH, and S is a moiety capable

per se or in combination with the drug to modulate

the NTFs, with the proviso that when both X and Y are zero, the drug (D) and the moiety (S) are linked by an acid-base bond

D ^+/ \S ^'/+ wherein D is an acid (+) or basic (-) drug and S is a

basic (-) or acid (+) compound capable to modulate directly and/or indirectly the NTF that is a salt of an acid (drug ^" ) with a base (NFs ⁺ modulator) or a salt of a base (drug ⁺ ) with an acid

(NFs- modulator) . General

For the screening approach it was decided to test

the compounds object of the present invention and fulfilling the structural general criteria previously

described for heme oxygenase-1 (OH-I) and inducible

nitric oxide synthase (iNOS) . The enzyme tests were performed as described by Vicente AM et al .

(Br.J.Pharmacol . 2001,133,920-6) . The formation of

both these two enzymes was associated with the

activation of transcription factors, and NF-kB

particularly. The following reference compounds were

used: indomethacin as example of unselective

cyclooxygenase inhibitor, dexamethasone, as example ^■

of steroidal glucorticoid agent, N-acetylcysteine as

example of anti-oxidant agent, NS-398 as example of

COX-2 inhibitor, zileuton, as example of 5- lipooxygenase inhibitor,L-N-methylarginine (L-NAME), as example of NOS inhibitor.

In brief, Raw 264.7 murine macrophage cell line

were maintained in a proper culture medium supplemented with 10% fetal bovine serum, 2 mM L- glutamine and penicillin/streptomycin. Suspensions of zymosan A from Saccharomyces cerevisiae in saline were used. After incubation with zymosan and/or test compounds for 18 hrs, cells supernatants were

collected to measure protein expression, by western blot analysis. After incubation, macrophages were lysed in 100 microliters of buffer (1% Triton X-

100,1% deoxicholic acid, 20 mM NaCl and 25 mM Tris,pH

7.4) and then centrifuged at 4 ⁰ C for 5 min at 10,000 x g. The protein content was determined by the t

Bradford method using bovine serum albumin as

standard. Cell lysate (40 micrograms of protein) was

mixed with Laemmli sample buffer under reducing

conditions. Samples were sized-separated in 12.5%

SDS-PAGE and transferred into polyvinylidene

membranes, which were blocked in phosphate buffer

saline (0.02 M,pH 7.0)-Tween 20 (0.1%) containing 3%

fat-free dry milk. Membranes were incubated with

specific antibodies: polyclonal antibody against iNOS

(1/1000,Cayman Chemical) and anti-OH-1 monoclonal antibody (1/2000, Stressgen) . Blots were washed and incubated with peroxidase-conjugated goat IgG

(1/20,000) . The immunoreactive bands were visualized by an enhanced chemiluminescence system and band

intensity quantitated using computer-assisted densitometry.

Cell viability was assessed by the mitochondrial-

dependent reduction of 3- (4, 5-dimethyltiazol-2-yl) - 2.5 diphenyltetrazolium bromide (MTT) to formazan. After appropriate stimulation times, cells were incubated with MTT (200 micrograms/ml) for 60 min. The medium was then removed and cells were solubilized in dimethylsulphoxide to quantitate

formazan at 550 nm.

Results

The results on OH-I and iNOS were shown in table I . Only the test compound ATANPE was able to

stimulate OH-I production and inhibit iNOS formation.

All the compounds did not affect cell integrity and

viability under the experimental conditions above

described and at the concentrations used.

Table I

Effect of some compounds on zymosan-induced OH-I and iNOS protein expression. Band intensity was

calculated as percentage with respect to zymosan.

It has been found that the compounds fulfilling these

criteria surprisingly showed not only an efficacy greatly increased, with additive or synergistic

implementation, but also a safety profile

dramatically improved.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that

it is possible to link regulators of NTF to a pharmacologically active compound helpful for

treating disorders in the cardiovascular, connective

tissue, pulmonary, gastrointestinal, respiratory,

urogenital, nervous, or cutaneous systems or tumoral

and infective diseases. The resulting compounds have good bioavailability, increased activity and/or

safety.

The parent drugs (i.e. the drugs in which the modification with NF-regulating moiety can be applied) in the present invention can be selected within a wide class of compounds, such as NSAIDs

(i.e. unselective cyclo-oxygenase (COX) inhibitors, - ketoprofen, flurbiprofen, suprofen, indobufen,

etodolac, indomethacin, naproxen etc.) , analgesics (i.e. paracetamol, capsaicin), steroids (chenodeoxycholic acid, flunisolide) , antibiotics (bacilysin) , bronchodilators (albuterol) ,

expectorants and mucolitic agents (ambroxol) , anti-

asthma, antiallergic and anti-histaminic drugs

(epinastine) , ACE-inhibitors (captopril) , /3-blockers

(atenolol) , drugs for vascular disorders

(brovincamine) , antidiabetics (glibomuride) ,

antitumorals (ancitabine, mopidamol) , antiulcer

(arbaprostil) , antihyperlipidemics (fluvastatine) ,

antibacterials (amoxicilline) , antivirals

(amantadine) , cGMP phosphodiesterase inhibitors

(sildenafil, verdenafil) , drugs against dementia

(mofegiline) and the bone reabsorption (alendronic

acid) . Also compounds that are nitric oxide donors

are useful in the present invention.

When the compounds include at least one asymmetric carbon atom, the products can be used in racemic mixture or in form of single enantiomer.

In the present invention the parent compound is considered in its original form or in a proper modification to allow the chemical manipulation with NF-regulating moieties.

Also parent drugs able to release nitric oxide

exogenously or endogenously are useful for the present invention.

NF-regulating moieties include α-lipoic acid, α- tocoferol, butylated hydroxyanisole (BHA) , caffeic

acid derivatives, catechol derivatives, cinnamic

acid, curcumin, epigallocatechin-3-gallate,

ergothioneine, N- (p-aminobenzoyl) glutamic acid, glutathione, hematein, magnolol, nordihydroguaiaretic

acid (NDGA) , phenolic antioxidants, pyrrolidine

dithiocarbamate (PDTC) , quercetin, resveratrol,

Vitamin C, vitamin E, salicylic acid

derivatives (such as cresotic acid isomers and 4-

hydroxyisophthalic acid) .

Other substances releasing or stimulating the

release of hydrogen sulfide and NF-regulating that

can be linked to drugs are N-acetyl-penicillamine,

S-allyl-cysteine, bucillamine, carbocysteine, cysteamine, cystathionine, homocysteine, mecysteine,

methionine, pantetheine, penicillamine, penicillamine disulfide, thioacetic acid, thiodiglycolic acid, thioglycolic acid, thiolactic acid, 2-thiolhistidine,

thiomalic acid, thiosalicylic acid, tiopronin. Other substances releasing and/or stimulating the release of hydrogen sulfide and NF-regulating that can be linked to drugs are 5- (p-hydroxyphenyl) -3H- l,2-dithiol-3-thione, 1,3 dithiol-2-thione-5-

carboxylic acid, 3-thioxo-3H-l,2-dithiole-5- carboxylic acid, 3-thioxo-3H-l, 2-dithiole-4 carboxylic acid.

More groups NF-regulating by releasing H ₂ S also

in combination with groups that release nitric oxide

or carbon oxide or that release nitric oxide or

carbon oxide per se are part of the present

invention.

The substances can be linked via different

linking groups such as esters, amides, imides,

sulfonamides, azo groups, carbamates, carbonates,

anhydrides, acetals, thioacetals, etc.

Bifunctional linkers known to the expert in the

field (such as ethyl, propyl, or butyl diols; di- amines; hydroxy amines, etc..) can be optionally

present when they are necessary to link the drug to

the NF-regulating moieties.

Also salts that directly or indirectly are capable to inhibits NF-kB, such as, for example, salts of arginine, agmatine, aminoguanidine, penicillamine, lipoic acid, caffeic acid, 1,3 dithiol-2-thione-5-carboxylic acid, thiosalicylic

acid, cinnamic acid etc. are part of the present invention. The compounds of the present invention can be administered in the form of any pharmaceutical

formulation, the nature of which will depend upon the route of administration and the nature of the disease

to be treated. These pharmaceutical compositions can be prepared by conventional methods, using

compatible, pharmaceutically acceptable excipients or

vehicles. Examples of such compositions include capsules, tablets, syrups, powders and granulates for

the preparation of extemporaneous solutions,

injectable preparations, rectal, nasal, ocular,

vaginal etc. A preferred route of administration is

the oral route .

The following non-limitative examples further

describe and enable an ordinary skilled in the art to make and use the invention.

EXAMPLE 1. Synthesis of 5- (p-hydroxyphenyl) -3H-1,2- dithiol-3-thione.

To 280 mmol of sulfur 40 mmol of anethole were added. After heating at 200 ⁰ C for 6 hours, 2.5 g of anethole dithiolethione were obtained. The product, washed with ether, was crystallized by ethyl acetate: melting point HO-IIl ⁰ C. Then 1.5 g of anethole dithiolethione were mixed with 7.5 g of pyridine HCl and the mixture was heated for 25 minutes at 215 ⁰ C. After cooling, IN HCl in excess was added and the precipitate was filtered, washed and crystallized

from ethanol . The obtained compound melted at 191-

192 ⁰ C. EXAMPLE 2. Synthesis of valproic acid ester with 5-

(p-hydroxyphenyl) -3H-I ₇ 2-dithiol-3-thione.

The ester of valproic acid with 5- (p- hydroxyphenyl) -3H-1, 2-dithiol-3-thione was prepared

via the acyl chloride of valproic acid. The acyl

chloride of valproic acid (3.0 mmol) and the compound

prepared in the example 1 (1.59 mmol) were refluxed

in THF anhydrous for 6 hours under nitrogen

atmosphere. After removal of THF, the mixture was

chromatographed on silica gel eluting with

dichloromethane. The compound was crystallized from ethyl ether and showed a melting point of 69.5-

70.5 ⁰ C. Anal, calcd. C ₁₇ H ₂₀ O ₂ S ₃ C% 57.92; H% 5.72; S% 27.29; found C% 58.05; H% 5.69; S% 27.34. EXAMPLE 3. Synthesis of ketoprofen ester with 5-(p- hydroxyphenyl) -3H-1,2-dithiol-3-thione.

The ketoprofen ester was prepared via its acyl chloride and 5- (p-hydroxyphenyl) -3H-1, 2-dithiol-3- thione. 2.3 mmol of ketoprofen were suspended in 7 ml of anhydrous THF with 0.92 ml of thionyl chloride,

heating at 67 ⁰ C for 6 hours. After evaporation of THF and thionyl chloride, the ketoprofen acyl chloride was dissolved in anhydrous THF, and a solution of 5-

(p-hydroxyphenyl) -3H-1, 2-dithiol-3-thione (1.77

mmol.) , pyridine (0.2 ml) in THF anhydrous (1 ml) was

dropped therein, and the reaction was kept under

stirring overnight at room temperature. After evaporation of THF, the product thus obtained was

chromatographed on silica gel eluting with

dichloromethane. The purified product had a gummy

glue-like appearance and its crystallization was

obtained adding few drops of ether to the

refrigerated compound. The orange crystals showed a

melting point of 111.5 - 112.5°C. Anal. calcd.

C ₂₅ Hi ₈ O ₃ S ₃ C% 64.91; H% 3.92; S% 20.79; found C%

64.83; H% 4.02; S% 20.49.

EXAMPLE 4. Synthesis of ketorolac ester with 5- (p- hydroxyphenyl) -3H-1,2-dithiol-3-thione.

The ketorolac ester was prepared employing dicyclohexylcarbodiimide (DCC) as coupling agent in

) presence of 4-pyrrolidinylpyridine. The solvent used

was methylene chloride purified on basic Al ₂ O ₃ to remove traces of ethanol . In a 50 ml flask, 1.17 mmol of ketorolac and 2.15 ml of methylene chloride were charged as well as 0.014 mol of 4- pyrrolidinylpyridine, 1.17 mmol of 5- (p-

hydroxyphenyl) -3H-1, 2-dithiol-3-thione and 1.47 mmol of DCC (25% excess) in 1.5 ml of methylene chloride. The mixture was stirred for 30 minutes at room temperature. At the end of the reaction, after

filtration the solution was extracted with 10% acetic

acid, then with 0. IN NaOH and finally with brine.

After removal of the solvent, the product was chromatographed on silica gel with dichoromethane and

crystallized by ether: melting point 146-147°C. Anal,

calcd. C ₂₄ Hi ₇ NO ₃ S ₃ C% 62.18; H% 3.69; N% 3.02; S%

20.75; found C% 61.85; H% 3.60; N% 2.83; S% 21.10.

EXAMPLE 5. Synthesis of acetyl thiosalicylic acid 4-

(nitroxymethyl)phenyl ester(ATANPE) .

To a solution of thiosalicylic acid (39.9 mmol) in KOH (5.6 g in 56 ml of water) , 48.9 mmol of acetic

anhydride were added under stirring for 1 hour on a ice bath. Adding 4N HCl a white precipitate was

obtained. The solid was filtered and dried.

To a suspension of 5 mmol of the solid in 5 ml of chloroform, 10.3 mmol of oxalyl chloride were added. The mixture was heated at 86 ⁰ C and at the end of the reaction the chloroform and oxalyl chloride were evaporated under reduced pressure.

To the acyl chloride solubilized in chloroform, 4.17 mmol of p-hydroxybenzaldehyde and 5 mmol of triethylamine were added. The temperature of the reaction was maintained at 0 ⁰ C. The acetyl thiosalicylic p-hydroxybenzaldheide ester was

chromatographed on silica gel eluting with

cycloexane/ethylacetate (8/2) . After crystallization

with ether, the compound showed a melting point of

93-94 ⁰ C. The ester (1.32 mmol) was transformed in

the corresponding p-hydroxybenzyl alcohol with sodium

triacetoxy borohydride (5.3 mmoles) in dichloroethane heating under reflux.

The last step was the nitration of the alcohol

previously prepared. The mixture for the nitration was constituted by 0.5 ml of fuming HNO ₃ , 1.25 ml of

acetic anhydride and 10.75 ml of methylene choride.

This mixture (2.7 ml) was added to the alcohol derivative (2.7 mmol in 5 ml of dichloromethane) and the reaction was maintained at 0 ⁰ C for 1 hour. After

dilution with dichloromethane, the solution was

extracted with iced water. The organic phase, dried on anhydrous sodium sulphate, was evaporated. The oily ^' compound, after purification on silica column eluting with dichloromethane and crystallization by ether, had a melting point of 65-66 ⁰ C.

EXAMPLE 6 - Pharmacological

The NF-kB-CAT reporter gene cell line (Lenardo MJ

et al.,Cell 58,227-9,1989) and. L29/Ji6 cells (Fratelli M et al, Antioxidants & Redox Signalling 5,229-235,2003) were prepared as previously described. Human TNF alfa (100 ng/ml) was used. The

chloramphenicol-acetyltransferase (CAT) activity was

measured by adding 40 microliters of Tris-HCl buffer

(250 mM, ph 7.5) and D-threo- [dichloroacetyl] 1,2 ¹⁴ C

chloramphenicol (10 microliters, 8.3 x 10 ^"2 mCi/sample) The reaction was started by the addition

of n-butyryl coenzyme A. After 2 h at 37 ⁰ C, the

reaction was stopped by extraction with hexane/xylene

(2:1) . The upper organic phase containing the

reaction product was then recovered, and

radioactivity was counted on a beta-counter. The

activity on NF-kB was expressed as % of CAT formation.

Table II

Effect on NF-kB activity by test compound (100 μM) , as assessed by a reporter gene synthesis: % of CAT synthesis

EXAMPLE 7. Inhibition of the expression of NF-kB regulated cytokine IL-6 and IL-8 in HeLa cells by compound described in example 5.

The cells were stimulated with 100 μmol of compound described in example 6 with TNF (200 E/ml)

or with PMA (50ng/ml)and the concentration of IL-6 or

IL-8 was measured in the cell supernatant at various

interval using ELISA (British Biotechnology Products

Ltd.) . The expression values are listed in the following table III.

TABLE III

IL-6 (pg/tnl) IL-8 (pg/ml)

15 120' 15' 120

TNF —— 945 7640 61

TNF+EX 5 -- 945 1738 6

PMA -- 870 11813 445 PMA+EX 5 -- 920 3138 12

The values show that after 120 minutes the compound described in example 5 is able to inhibit the production of IL-6 by 87% or 74% (TNF

stimulation) and the production of IL-8 by 90% or 97% (PMA stimulation) .