USOO7524836B2

(12) United States Patent (10) Patent No.: US 7,524,836 B2 Del Soldato (45) Date of Patent: Apr. 28, 2009

(54) NITROOXYDERIVATIVESTEROIDAL JP 58-045724 3, 1983 COMPOUNDS JP 5-39.261 2, 1992 JP 4-273892 9, 1992 (75) Inventor: Piero Del Soldato, Milan (IT) WO 98.15568 4f1998 OTHER PUBLICATIONS (73) Assignee: Nicox S.A., Paris (FR) English language abstract of JP 58-045724. (*) Notice: Subject to any disclaimer, the term of this English language abstract of JP4-273892. patent is extended or adjusted under 35 English language abstract of DE 19634793. U.S.C. 154(b) by 173 days. K. B. Schwarz, “Oxidative Sress During Viral Infection: A Review”. Free Radical Biology & Medicine, vol. 21, No. 5, 1996, pp. 641-649. Goodman & Gillman, “The Pharmacological Basis of Therapeutics'. (21) Appl. No.: 11/643,887 9" Edition, 1996, McGraw-Hill Health preffsions Division, pp. 1-1. 1459–1465 and 1474. (22) Filed: Dec. 22, 2006 Martindale, The Extraphamacopeia,30" Edition, 1993, pp. 712-723. O O Nenseter et al., “Paracetamol inhibits Copper Ion-Induced, AZO Com (65) Prior Publication Data pound-Initialed, and Mononuclear Cell-Mediated Oxidative Modifi US 2007/O142342 A1 June 21, 2007 cation of LDL', Arterioscierosis, Thombosis, and Vascular Biology, s vol. 15, No. 9, Sep. 1995, pp. 1338-1344. O O Baylis et al. “Chronic Blockade of Nitric Oxide Synthesis in the Rat Related U.S. Application Data Produces Syntemic Hypertension and Glomemerular Damage'. J. (62) Division of application No. 1 1/028, 198, filed on Jan. 4, Clin. Investigation, vol.90, 1992, pp. 279-281. 2005, now Pat. No. 7,186,708, which is a division of International Search Report, Nov. 21, 2000. application No. 09/926,327, filed on Oct. 15, 2001, E.ederqvist Ny'She ... Biochen. E. OfSE7O. 1053, 1994 now Pat. No. 6,909,007. Bonn et al., Cardiovasc. Drug Rev. 16(3), 195-211, 1998. (51) Int. Cl McCance & Huetherm "Pathophysiology: The Biologic Basic for we Disease in Adults and Children.” Third Edition, Mosby, 1998, pp. A6 IK3I/56 (2006.01) 48-54, 71-77 and 1025. C07J 5/00 (2006.01) (52) U.S. Cl...... 514/182, 514/169,552/557 Primary Examiner Barbara P Badio (58) Field of Classification Search ...... 552/557 (74) Attorney, Agent, or Firm-Arent Fox, L.L.P. 514/169, 182 See application file for complete search history. (57) ABSTRACT (56) References Cited Steroidal compounds or their salts having general formulas (I) and (II) wherein: S is an integer equal to 1 or 2, preferably U.S. PATENT DOCUMENTS s=2; b0=0 or 1: A R , wherein R is the steroidal drug 3,702.335 A 11, 1972 Lafill radical, C and C are two bivalent radicals. The precursors of 3,963,707 A 6/1976 Högberg et al. the radicals B and B are such as to meet the pharmacological 4,913,852 A 4, 1990 Millioni et al. tests reported in the description. 4.956,384. A 9/1990 Chiesi et al. A-(B) to C N (O) (I) 5,318,987. A 6/1994 Weithmann et al. EO s 5,508,275 A 4, 1996 Weithmann et al. 5,707,984. A 1/1998 Tjoeng et al. 5,837,698 A 11/1998 Tjoeng et al. (II) A-C1-B FOREIGN PATENT DOCUMENTS o DE 19634.793 3, 1998 EP O 549 318 A2 6, 1993 EP O 562 497 A1 9, 1993 EP O 578 494 A1 1, 1994 7 Claims, No Drawings US 7,524,836 B2 1. 2 NITROOXYDERVATIVE STEROIDAL This happenss for example with . COMPOUNDS Drug research is directed to find new molecules having an improved therapeutic index (efficacy/toxicity ratio) or a lower This is a divisional of U.S. patent application Ser. No. risk/benefit ratio, also for pathological conditions as those 11/028,198, filed Jan. 4, 2005, now U.S. Pat. No. 7,186,708, above mentioned, wherein the therapeutic index of a great issued Mar. 6, 2007, which is a divisional of U.S. patent number of drugs results lowered. In fact in the above men application Ser. No. 09/926,327, filed Oct. 15, 2001, now tioned conditions of oxidative stress and/or endothelial dys U.S. Pat. No. 6,909,007, issued Jun. 21, 2005, which claims functions, many drugs show a lower activity and/or higher priority benefit to MI99A0000751, filed Apr. 13, 1999. The toxicity. disclosures of all applications is hereby incorporated by ref 10 It is well known that steroids represent a first choice phar erence in their entirety. macological intervention in the therapy of inflammatory dis The present invention relates to novel steroidal compounds eases. This class of drugs, among which can be mentioned for for systemic use and non systemic use, and their composi example , , , predniso tions, to be used in the conditions of oxidative stress and/or lone, , desoxycorticosterone, metilpredniso endothelial dysfuntions. Specifically it relates to compounds 15 lone, , , , dexam with a steroidal structure having antiinflammatory, immun ethasone, , acetonide, odepressive and angiostatic activity (the so called antiinflam beclomethasone, acetoxypregnelone, etc., elicits remarkable matory steroids), or gastrointestinal activity. pharmaco-toxicological effects on different organs, and for The compounds according to the present invention result this reason both their clinical use and its interruption cause a therapeutically useful in the treatment of morbid conditions series of side effects, some of which very serious. See for wherein the steroidal products are generally used with greater example Goodman & Gilman, “The pharmaceutical Basis of benefit, in terms both of a better tolerability and/or efficacy. Therapeutics' 9" ed., pages 1459-1465, 1996. By oxidative stress it is meant the generation of free radi Among said toxic effects can be mentioned those affecting cals on radicalic compounds, which causes injury both of the the bone tissue leading to an altered cellular metabolism and cell and of that of the Surrounding tissue (Pathophysiology: 25 an high osteoporosis incidence; those affecting the cardiovas the biological basis for disease in adults and children, cular system, generating an hypertensive response; those McCance & Huether 1998 pages 48-54). affecting the gastrointestinal apparatus giving gastric dam By endothelial dysfunctions are meant those relating to the ageS. Vasal endotheliun. The damage of the Vasal endothelium is See for example Martindale “The extrapharmacopoeia, known as one of those important events that can bring about 30 30" ed., pages 712-723, 1993. a series of pathological processes affecting various organs To the class of steroidal drugs belong also biliary acids, that and body apparatuses, as described hereinafter (Pathophysi have been used in the therapy of hepatic disorders and in ology: The biological basis for disease in adults and children, biliary colics. Ursodesoxycholic acid is also used in some McCance & Huether 1998 page 1025). hepatic dysfunctions (hepatic cirrhosis of biliary origin, etc.). As known, the oxidative stress and/or the endothelial dys 35 Their tolerability is strongly worsened in the presence of functions are associated to various pathologies as reported gastrointestinal complications (chronic hepatic damage, pep hereinafter. The oxidative stress can also be caused by toxic tic ulcer, intestinal inflammation, etc.). Also in the case of ity of a great variety of drugs, which significantly affects their biliary acids the oxidative stress remarkably affects drug per performances. formance: both the efficacy and the tolerability of chenode Said pathological events are of a chronic, debilitating char 40 oxycholic,and ursodesoxycholic acids are significantly acter and are very ofthen typical of the elderly. As already reduced. In particular the unwanted effects on liver are found said, in said pathological conditions the drugs used show a exalted. Among the steroidal compounds can be mentioned remarkably worsened performance. also estrogens for the treatment of dislipidaemias, hormonal Examples of pathological situations caused by the oxida troubles, female apparatus tumours treatment can be men tive stress and/or by the endothelial dysfunctions, or present 45 tioned. Also said steroids show side effects as above men in elderly, are the following: tioned, in particular at the hepatic level. For the cardiovascular system: myocardial and vascular According to the above mentioned prior artit seems almost ischaemia in general, hypertension, stroke, arterioscle impossible to separate therapeutic activity from side effects, rosis, etc. see Goodman et al, above mentioned, at p. 1474. For the connective tissue: rheumatoid arthritis and Con 50 The steroidal compounds are completely different from the nected inflammatory diseases, etc. antiinflammatory non steroidal compounds from the chemi For the pulmonary system: asthma and connected inflam cal, pharmacological and biochemical point of view, since the matory diseases, etc. pharmaco-toxicological mechanism of action of For the gastrointestinal system: ulcerative and non ulcer antiinflammatory products is based on the inhibition of one or ative dyspepsias, intestinal inflammatory diseases, etc. 55 more of the cyclooxygenases (COX), while steroids do not For the central nervous system: Alzheimer disease, etc. influence COX and have more complex pharmaco-toxico For the urogenital system: impotence, incontinence. logical mechanisms of action not yet fully cleared. For the cutaneous system: eczema, neurodermatitis, acne. Indeed it is well known that these two groups of drugs are The infective diseases in general (ref: Schwarz-KB, Brady classified in different classes in the pharmacopoeias. “Oxidative stress during viral infection: A review Free 60 The need was felt to have available steroids showing an radical Biol. Med. 21/5, 641-649 1996). improved therapeutic performance, i.e. endowed both of a Further the ageing process can be considered as a true lower toxicity and/or higher efficacy, so that they could be pathologic condition (ref. Pathophysiology: the biological administered to patients in morbid conditions of oxidative basis for disease in adults and children, pages 71-77). stress and/or endothelial dysfunctions, without showing the The known drugs when administered to patients having 65 drawbacks of the drugs of the prior art. pathologies associated to oxidative stress and/or endothelial It has been now Surprisingly and unexpectedly found that dysfunctions, show a lower efficacy and/or higher toxicity. the aforementioned technical problems shown in the admin US 7,524,836 B2 3 istration of steroidal drugs to patients affected by oxidative stress and/or endothelial dysfunctions, or to the elderly in general, are solved by a new class of drugs as described (CH)-O- hereinafter. An object of the invention are steroidal compounds or their 5 salts having the following general formulas (I) and (II): rSa. A-(B) to C N (O) (I) CCOHS-X (CH)- wherein: S is an integer equal to 1 or 2, preferably S-2; 10 wherein n3 and n3" have the above mentioned meaning A-R-T-, wherein R is the steroidal drug radical as defined hereunder, B-T-X-T- wherein 15 T and T are equal or different; (CH-CH-CH-O)- TB-(CO) when the reactive function in the precursor ste ONO. roid is —OH: TX when the reactive function in the -(CH-CH-CH-O)- precursor is —COOH: X=O, S, NR, R is H or a linear or branched alkyl ONO having from 1 to 5 carbon atoms, or a free Valence; T (CO), or (X), wherein tX and tXX have the value of wherein inf is an integer from 1 to 6 preferably from 1 to 0 or 1; with the proviso that tX=1 when tXX=0, tx=0 when tXX=1: X is as above defined; 4. X is a bivalent bridging bond as defined hereunder; 25 C is the bivalent radical-T-Y wherein - (CH-CH-O)- -(CH-CH-O)- T=(CO) when tx=0, T =X when tXX=0, X being as above defined; Rif Rif Y is: 30 wherein R, H, CH, and inf is an integer from 1 to 6; (III) preferably from 1 to 4: r us preferably Y= Yo-R'O— wherein R' is as above defined; o in-y glas -O- 35 preferably R' is a C-C alkylene; RTIX RTIDX (II) wherein: nIX is an integer between 0 and 3, preferably 1: 40 N(O), nIIX is an integer between 1 and 3, preferably 1: wherein: Rzza, Rzza, Rzna, Rzzy, equal to or different from each C = -Tcl-Y-Tcm other are H or a linear or branched C-C alkyl; preferably Rzza, Rzza, Rzza, Rzza, are H. Y is a saturated, unsaturated or aromatic heterocyclic 45 ring containing at least one nitrogenatom, preferably wherein T and T are equal or different, one or two nitrogen atoms, said ring having 5 or 6 T(CO) when the reactive function of the precursor ste atOmS. roid is —OH, TX when the reactive function of the or Y is Yo, selected from the following: precursor steroidis—COOH, X being as above defined; an alkylenoxy group RO wherein R is linear or 50 T (CO), or (X), whereint and t have the 0 or 1 branched when possible C-C preferably having value; with the-proviso that tI=1 when tII=0; t|=0 when from 1 to 6 carbon atoms, most preferably 2-4 carbon tII=1; X is as above defined; atoms, or a cycloalkylene having from 5 to 7 carbon atoms, in the cycloalkylenic ring one or more carbon Y" is as Y above defined, but with three free valences atoms can be substituted with heteroatoms, the ring 55 instead of two, preferably it is selected from the follow can have side chains of R' type, R' being as above ing: defined; or -RO-, 60 21 CC20 -(CH)-O- N -(CH2) group wherein R' is linear or branched preferably having 65 from 1 to 6 carbon atoms, most preferably 2-4, or a wherein n3 is an integer from 0 to 3 and n3' is an integer saturated, optionally Substituted, ring having from 5 from 1 to 3: to 7 carbon atoms; or US 7,524,836 B2 5 6 T(CO) when tI=0, TX when tII=0, X being as above defined; 21 X, bivalent radical, is such that the corresponding pre --(CH2)-O- cursor of B: -T-X-T- meets test 4 or test 5, pre Nx cursor in which the T and T free Valences are each - (CH) on saturated with OZ, with Zor with-Z'-N-Z', Z and Z' being equal or different and have the Z values as above defined, depending on whether T and/or TCO or wherein n3 is an integer from 0 to 3 and n3' is an integer X, in connection with the values oft, t', tX and tXX; from 1 to 3: 10 the C precursor when b0=0 is of-T-Y H type wherein the T free valence is saturated with OZ, Z, or with (CH)-O- -ZI-N-ZII, 21 15 -(CH), ls S Z and Z' being as above defined and is such as to COOH (CH)- meet test 5; X monovalent radical, such that the corresponding wherein n3 and n3' have the above mentioned meaning: precursor of B-T-X meets test 4 or test 5, pre cursor wherein the T free Valence is saturated with OZ or With Z or with

25 (CH-CH-CH-O)- -Z-N-Z, ONO wherein one hydrogen atom on one of the carbon atoms Z" ' being equal or different and having the Z is substituted by a free valence: 30 values as above defined depending on whether TCO or X, in connection with the tI and tI values: A=R , has the following stricture: -(CH-CH-CH-O)-

ONO 35 wherein inf is an integer from 1 to 6 preferably from 1 to 4; wherein one hydrogen atom on one of the carbon atoms is substituted by a free valence; 40

-(CH-CH-O)- Rif 45 wherein in substitution of the hydrogens of the CH groups or wherein one hydrogen atom on one of the carbon atoms of the two hydrogens of the CH groups mentioned in the is substituted by a free valence: general formula, the following Substituents can be present: in position 1-2: there may be a double bond; - (CH-CH-O)- so in position 2-3: there may be the following Substituent: Rf

wherein R, H, CH, and nf is an integer from 1 to 6; 55 preferably from 1 to 4, wherein one hydrogenatom on one of the carbon atoms is substituted by a free Valences; preferably Y= R'O— wherein R is a linear or branched C-C, the oxygen which in Y is covalently 60 linked to the N(O) group is at the end of the free bond indicated in C formula: in position 2: there may be Cl, Br; orY =Y as defined in (I) but with three free valences in position 3: there may be CO, —O CH, CH, C1, OH: instead of 2: in position 3-4: there may be a double bond; 65 in position 4-5: there may be a double bond; B1-Tatr-X2, in position 5-6: there may be a double bond; wherein X2 is a monovalent radical, in position 5-10: there may be a double bond; US 7,524,836 B2 7 8 in position 6: there may be C1, F, CH, —CHO: radical production by DPPH is calculated as a percent in position 7: there may be C1, OH: age by means of the following formula: in position 9: there may be C1, F: in position 11: there may be OH, CO, C1, CH: in position 16: there may be CH, OH, =CH: wherein. As and A are respectively the absorbance values of in position 17: there may be OH, CH, OCO(O)(CH), the solution containing the test compound+DPPH and that of CH, C=CH or the solution containing only DPPH; the acceptance criterium of the compounds according to this test is the following: test O 4 is met by B or B precursor compounds if the inhibition 10 percentage as above defined is higher than or equal to 50%; wherein test 5 is the following: it is an analytical determi or ( ) nation carried out by adding aliquots of 10 M methanol wherein ua is an integer equal to 0 or 1, Va is an integer from solutions of the precursor of B or B or of C=-T-Y H. 15 having the free Valence saturated as above indicated, to a 0 to 4: solution formed by admixing a 2 mM solution of desoxyri in position 16-17: there may be the following groups: bose in water with 100 mM of phosphate buffer and 1 mM of the salt Fe' (NH) (SO); after having thermostatted the Solution at 37°C. for one hour, aliquots of aqueous solutions CH3 CH3 of trichloroacetic acid 2.8% and of thiobarbituric acid 0.5 M are added, in the order, heating is effected at 100° C. for 15 7 rr-I6 O 7 Nu.=1 minutes and the absorbance of the tested solutions is then read I6 at 532 nmi; the inhibition induced by the precursor of B or B CH3 or C-T-Y H with respect to radical production by Fe' is calculated as a percentage by means of the following formula: 7 ho 25 I6 O wherein. As and A are respectively the absorbance values of the Solution containing the tested compound and the iron salt Rand R', equal to or different from each other, can be hydro 30 and that of the solution containing only the iron salt, the gen or linear or branched alkyls from 1 to 4 carbon atoms, compound meets test 5 when the inhibition percentage as preferably R=R=CH: above defined of the precursor of B or B or C=-T-Y H. having the free Valence Saturated as above indicated, is higher whereint, t1 and t2 are integers equal to or different from each than or equal to 50%; provided that in the compounds of other, equal to 0 or 1, with the proviso that when t=0 t2=1 35 formula (I) are excluded the drugs with A=R-, wherein R is as and when t=1 t2=0, and that t and t1, or t2 and t1, cannot above defined, when bo () and C=-T-Yo - wherein the free contemporaneously be equal to 0 when A does not contain valence ofY is saturated as indicated above, s=1 or 2. —OH groups; Preferably the B or B precursor compound (precursor of the bivalent bridging group L is selected from: the X or X radical in formulas (I) and (II) respectively) (CRRs),(O)2(CRRs), (CO), cons,(CO), n, 40 which meets test 4, is selected from the following classes of (CRRs), compounds: whereinna, n'a, and n'a, equal to or different from each other, Aminoacids, selected from the following: L-carnosine are integers from 0 to 6, preferably 1-3; nb, n'b, n"b and (formula CI), anserine (CII), selenocysteine (CIII), sele n"b, equal to or different from each other, are integers nomethionine (CIV), penicillamine (CV), N-acetylpeni equal to 0 or 1; R Rs equal to or different from each other, 45 cillamine (CVI), cysteine (CVII), N-acetylcysteine are selected from H. linear or branched alkyl from 1 to 5 (CVIII), glutathione (CIX) or its esters, preferably ethyl carbon atoms, preferably from 1 to 3: or isopropyl ester: Xois Xas above defined, but R, is a linear or branched alkyl from 1 to 10 carbon atoms, or equal to X' wherein X is 50 (CI) equal to OH, CH, Cl, N(-CH CH). SCHF, SH, or O K OH M HN NH2 55 N r O (CII) wherein test 4 is the following: it is an analytical determi OH nation carried out by adding portions of methanol solu tions of the precursor of B or B at a 10 M concentra 60 H 1N1y tion, to a methanol solution of DPPH (2,2-diphenyl-1- O NYo NH2 picryl hydrazyl-free radical); after having maintained the solution at room temperature away from light for 30 N minutes, it is read the absorbance at the wave length of ls 517 nm of the test Solution and of a solution containing 65 V only DPPH in the same amount as in the test solution; CH and then the inhibition induced by the precursor towards US 7,524,836 B2 9 10

-continued -continued (CIII) COOH (DII) HSe

(CIV) NH2 10 H3 C Ns COOH (DIII) (CV) 15

HS OH

(DIV) (CVI)

OH (DV) 25 N COOH NHCOCH

(CVII)

30 orOH HS OH (DVI) > COOH

(CVIII) 35 orOH HS OH (DVII) COOH > NHCOCH 40 (CIX) HO SH r (DVIII) COOH N HO--- N NullsOH 45 H s H O MeO hydroxyacids, selected from the following: gallic acid (for 50 OH mula DI), ferulic acid (DII), gentisic acid (DIII), citric (DIX) acid (DIV), caffeic acid (DV), hydrocaffeic acid (DVI), HO, CCOH p-coumaric acid (DVII), Vanillic acid (DVIII), chloro genic acid (DIX), kynurenic acid (DX), Syringic acid O 55 (DXI): HO x N O OH (DI) ÖH HO

HO OH 65 2 OH CN COOH US 7,524,836 B2 11 12

-continued -continued CCOH (DXI) (EV) HO O 5

On O O OH MeO OMe OH (EVI) OH 10 CHOH Aromatic and heterocyclic mono- and polyalcohols, HC-OH Selected from the following: nordihydroguaiaretic acid O O (EI), quercetin (EII), catechin (EIII), kaempferol (EIV), sulphurethyne (EV), ascorbic acid (EVI), isoascorbic is o acid (EVII), hydroquinone (EVIII), gossypol (EIX), reductic acid (EX), methoxyhydroquinone (EXI), OH OH EWII hydroxyhydroquinone (EXII), propyl gallate (EXIII), CH2OH (EVII) saccharose (EXIV), vitamin E (EXV), vitamin A (EXVI), 8-quinolol (EXVII), 3-tert-butyl-4-hydroxya- HO-CH O nisole (EXVIII), 3-hydroxyflavone (EXIX),3,5-tert-bu- O tyl-p-hydroxytoluene (EXX), p-tert-butyl phenol (EXXI), timolol (EXXII),xibornol (EXXIII),3,5-di-ter- o butyl-4-hydroxybenzyl-thioglycolate (EXXIV), 4'-hy droxybutyranilide (EXXV), guaiacol (EXXVI), tocol 25 OH OH (EXXVII), isoeugenol (EXXVIII), eugenol (EXXIX), OH (EVIII) piperonyl alcohol (EXXX), allopurinol (EXXXI), cony feryl alcohol (EXXXII), 4-hydroxyphenetyl alcohol (EXXXIII), p-coumaric alcohol (EXXXIV), curcumin (EXXXV): HO EDX (EI) 30 (EDX) OH CH HO OH 35

HO O CH3 (EII) OH

OH 40 HO O O O (EX) OH 45

OH O (EIII) OH

OH 50 (EXI) OMe HO O w OH

55 OH

OH O (EIV) OH OH (EXII) O H 60 HO O OH

OH 65 OH O OH

US 7,524,836 B2 15 16

-continued -continued (EXXIX) (MIV) 2CH2 O

HN ls NH HO or OH OMe (EXXX) 10 O Compounds containing at least a free acid function, \, selected from the following: 3,3'-thiodipropionic acid (NI), fumaric acid (NII), dihydroxymaleic acid (NIII), thioctic acid (NIV), edetic acid (NV), bilirubin (NVI), 15 3.4-methylendioxycinnamic acid (NVII), piperonylic acid (NVIII): CHOH (EXXXII) N OH (NI) Hooc1N1 S N1)-cooli (NII) HO HOOC H OMe (EXXXIII) 25 OH H COOH (NIII) HOOC COOH

HO HO OH (EXXXIV) 30

(NIV)

HO rOH (NV) (EXXXV) 35 O

MeO OMe

O N 21 O 40 HO OH (NVI) aromatic and heterocyclic amines, selected from the fol lowing: N, N'-diphenyl-p-phenylenediamine (MI), ethoxyquin (MII), thionine (MIII), hydroxyurea (MIV): 45

(MI) COO 50 (MII) H 55 N CH3 COOH COOH CH3 p-\ (NVII) He1 No 21 O 60 CH (MIII) N n 21 2 65 HN S NH2 CI COOH US 7,524,836 B2 17 18 yvitamin D2 (QX) the compound derived from 1 O.25 -continued dihydroxyvitamin D2 (QXI), 2-mercaptoimidazol (NVIII) (QXII)

(QI) OH

COOH 10 r Nals SH The above mentioned substances precursors of B or B are (QII) prepared according to the known methods in the prior art, SH described, for example, in “The Merck Index, 12a Ed. (1996), Ho-1N1 herein incorporated by reference. When available, the corre 15 (QIII) sponding isomers and optical isomers can be used. OMe Preferably the precursor compound of B or of B (precur OH O O sor of the X or X radical in formulas (I) and (II) respec tively) which meets test 5, is selected from the following CH O Q OH compounds: OH Arminoacids: aspartic acid (PI), histidine (PII), 5-hydrox ytryptophan (PIII), 4-thiazolidincarboxylic acid (PIV), OH OH II 2-oxo-4-thiazolidincarboxylic acid (PV) OH (QIV) 25

(PI)

30 (PII)

35

(QV) (PIII) 40

45

(PIV) 50

(QVI) COOH (PV) 55

COOH 60 mono and polyalcohols or thiols: 2-thiouracil (QI), 2-mer captoethanol (QII), esperidine (QIII), secalciferol (QIV), 1-C-OH vitamin D2 (QV), flocalcitriol (QVI), 22-oxacalcitriol (QVII), the vitamin D3 derivative 65 esterified with the vitamin A radical (QVIII), the for mula (QIX) compound, 24.28-methylene-1C.-hydrox US 7,524,836 B2 19 20

-continued -continued (QVII) (QXI)

10

15 Ho (QVIII) Succinic acid (RI) 2O

(RI) O

OH 25 HO

The precursor compounds of B or B of the above men 30 tioned groups P, Q and Rare prepared according to the known methods in the prior art and described for example in “The Merck Index', 12" Ed. (1996), herein incorporated by refer (QIX) CCC.

The vitamin D3 derivative with retinoic acid (QVIII) is 35 prepared as described in JP93039261 (ref.C.A. 119 117617); the formula (QIX) compound according to EP 562497; 24.28 methylene-1O-hydroxyvitamin D2 (QX) according to EP 57.8494; the derivative compound of dehydroxyvitamin D2 (QXI) according to EP 549,318. 40 The precursors of B or B which meet test 4, are preferred. The tests carried out to identify the precursors of B or B are in detail the following: Test 4 is a calorimetric test which affords to establish whether the orecursors of B or B inhibit the production of 45 radicals from DPPH (2,2-diphenyl-1-picryl-hydrazyl) (M. S. Nenseter et Al. Atheroscler. Thromb. 15, 1338-1344, 1995). 100 LM solutions in methanol of the tested substances are (QX) prepared, and an aliquot of each of said solutions is added to a DPPH solution in methanol 0.1 M. After having stored the 50 solutions at room temperature away from light for 30 min utes, their absorbances are read at the wave length of 517 nm, together with that of the corresponding DPPH solution at the same concentration. The absorbance decrease with respect to 55 that of the solution of DPPH at the same concentration of the test solutions is determined. The effectiveness of the tested compound in inhibiting formation of radicals by DPPH is expressed by the following formula:

wherein. As and A, are respectively the absorbance values of (QXII) the solution containing the test compound together with N DPPH and of the solution containing only DPPH; the com (> pounds precursor of B or B meet test 4 when the inhibition SH 65 percentage of radical production from DPPH, expressed as a H percentage according to the above equation, is higher than or equal co 50% at the indicated concentration (10 M). US 7,524,836 B2 21 22 If the precursors of B or B do not meet test 4, test 5 is carried out. -continued Test 5 is a calorimetric test wherein 0.1 ml aliquots of 10 H (Y8) M methanolic solutions of the tested products are added to -? - test tubes containing a solution formed by 0.2 ml of 2 mM 5 desoxyribose, 0.4 ml of phosphate buffer pH 7.4 100 mMand S. H 0.1 ml of 1 mM Fe'(NH) (SO) in 2 mM HC1. The test (Y9) tubes are then maintained at 37°C. for one hour. Then in each test tube 0.5 ml of a 2.8% solution in trichloroacetic acid 10 1SN water and 0.5 ml of an aqueous 0.1 M solution of thiobarbi turic acid are added, in the order. A reference blank is formed N4 by adding to a test tube containing only the above described (Y10) aqueous solution of reactants 0.1 ml of methanol. The test 1s tubes are closed and heated in an oil bath at 100° C. for 15 15 minutes. A pink coloration is developed the intensity of which NeN s is proportional to the quantity of desoxyribose undergone to N (Y11) radical oxidative degradation. The solutions are cooled at room temperature and their absorbances are read at 532 nm ?) against the blank. The inhibition induced by the precursor of 20 B or B or C=-T-Y H in comparison with the radical pro N4 duction by Fe" is determined by means of the following (Y12) formula: -- S (1-As? A)x100 25 N4 wherein. As and A are respectively the absorbance values of the solution containing the tested compound+the iron salt and 1. (Y13) that of the solution containing only the iron salt, the com pound meets test 5 when the inhibition percentage of radical N - production as above defined from the precursor of B or B or 30 H C=-T-Y H is higher than or equal to 50%. 1N (Y14) Y in formula (III) is preferably selected from the follow - I - ing: 35 N! H (Y15) (Y1) NC Y St.IfQ A\ Q2 N H H s 40 (Y2) N The most preferred of Y is Y12 (pyridyl) substituted in positions 2 and 6. The bonds can find also in asymumetric N H s position, for example Y12 (pyridyl) can be substituted also in (Y3) 45 position 2 and 3;Y 1 (pyrazol) may be 3,5-disubstituted. The compounds according to the present invention of for N mula (I) and (II) can be transformed into the corresponding H s salts. For example one way to form salts is the following: (Y4) when in the molecule one nitrogen atom Sufficiently basic to t( ) 50 be salified, in organic solvent such as for example acetoni N trile, tetrahydrofuran, is present, it is reacted with an equimo H s lecular amount of the corresponding organic or inorganic (Y5) acid. r 3. 55 Preferably in the formula of the invention compounds Yor N Y' of formula (III) is present. H s Examples of organic acids are: Oxalic, tartaric, maleic, (Y6) Succinic, citric acids. r AN 60 Examples of inorganic acids are: nitric, hydrochloric, Sul N1 phoric, phosphoric acids. H s In the steroid precursors preferably R"= CO CH-OH, (Y7) - N —CH(CH) CH-CH-COOH. N - Among the Steroid precursors those having the hydroxyl Q y 65 function in position 3 or in position 11, or having in R" an N hydroxyl or carboxylic function in terminal position, are pre ferred. US 7,524,836 B2 23 24 The steroid precursors of A which can be mentioned and Some syntnesis schemes for obtaining the compounds of which are preferred, are those listed hereinunder, obtainable the invention are reported hereinafter: according to the processes known in the art. As precursors and respective processes, those for example A) Synthesis of the Compounds of Formula (I). described in The Merck Index, ed. 12 of 1996, herein incor 5 1. Synthesis of the compound obtained by reaction between porated by reference, can be mentioned. The precursors (ac the C steroid and the compound precursor of B. cording to the Merck nomenclature) are the following, 1a. When the steroid contains a carboxylic function (general wherein H. H. R. R', R" have the meaning mentioned in the formula: R COOH) and the functional group of the pre compounds listed herein: , Hydrocortisone, cursor compound of B which binds itself to the carboxylic 10 function has the formula XZ, X being as above defined and Alclomethasone, Algestone, Beclomethasone, Betametha Z=H, the effected reactions depend on the nature of the sone, , , , Clocor second reactive group present in the precursor compound tolone, , Cortisone, , , of B. , Desoximethasone, , . , , Flucloronide, Flu 1a.1 When the second reactive group present in the precursor 15 compound of B is a carboxylic group, the synthesis general , , , Fluocino scheme expects the initial formation of the acyl halide of nide, Fluocortyn Butyl, , , the R COHal steroid (Hal=Cl, Br) and the subsequent Acetate, Acetate, Flupredniso reaction with the HX group of the precursor compound of lone, Flurandrenolide, Formocortal, , Halobeta B: sol Prorionate, Halomethasone, Acetate, (IA.1) , Etabonate, , RCOOH -----> RCOHal + H-X-X-COOH -----> , , Momethasone Furoate, Paramethasone, , , Prednisolone R-TR-X-COOH 25-Diethylaminoacetate, Prednisolone Sodium Phosphate, Prednisone, Prednival, , , Triamci 25 X, T being as above defined. nolone, Triamcinolone Acetonide, 21-Acetoxypregnenolone, When in the two reaction compounds other functional , , Propionate, groups COOH and/or HX are present, they must be , , Triamcinolone Hexacetonide, protected before the reaction according to the methods Ursodesoxycholic acid, Chenodeoxycholic acid, Mitatriene known in the prior art; for example as described in the diol, Moxestrol, Ethynylestradiol, Estradiol, Mestranol. publication by Th. W. Greene: “Protective groups in Unexpectedly the invention products of the formulas (I) 30 organic synthesis”. Harward University Press, 1980. and (II), in conditions of oxidative stress, have an improved The RCO Hal acylhalide is prepared according to the therapeutic index compared with the precursor steroids. For known methods in the prior art, for example by thionyl illustrative purposes the above mentioned tests are referred to or oxalyl chloride, p' or p halides in inert solvents the following compounds (see the tables attached to the under the reaction conditions. Such as for example tolu description): 35 ene, chloroform, DMF, etc. Specifically, when the HX group of the precursor com Test 4 (Test for the Precursor of B and B, Ref. Table III) pound of B is NH, or OH or SH, the steroid of formula N-acetylcysteine inhibits of 100% radical production from R—COOH is first converted into the corresponding acyl DPPH, therefore it meets test 4 and it can be used as precursor halide RCOHal, as above mentioned, and then reacted of B or B. 40 with the HXgroup of the precursor compound of B in the 4-thiazolidincarboxylic acid does not inhibit radical pro presence of an organic base. Such as triethylamine, pyri duction from DPPH, therefore it does not meet test 4: it can be dine, etc. using an inert Solvent in the reaction conditions used as precursor of B or B if it meets test 5. Such as toluene, tetrahydrofuran, etc. at a temperature in the range 0° C.-25°C. Test 5 (Test for the Precursor of Band B or of C=-T-Y-H, 45 Alternatively to the previous synthesis, the steroid of for Ref. Table IV) mula R—COOH can be treated with an agent activating 4-thiazolidincarboxylic acid meets test 5 since the inhibi the carboxyl group selected from N,N'-carbonyldiimi tion is of 100%. Therefore the compound can be used as dazol (CDI), N-hydroxybenzotriazol and dicyclohexyl precursor of B or B in formula (I). carbodiimide in solvent such as for example DMF, THF, 50 chloroform etc. at a temperature in the range -5°C.-50° The compounds of the invention can be used in the same C. and the obtained compound reacted in situ with the therapeutic indications of the precursor drug with the above reactive function of the precursor compound of B for mentioned advantages. obtaining the compound o. formula (IA.1). The compounds of formula (I) or (II) are prepared by 1a.2 When the precursor compound of B contains two func synthesis methods mentioned hereinunder. 55 tional groups XZ, equal to or different from each other, X The choice of the reactions for each method depends on the being as above defined and Z=H, the steroid having for reactive group present in the steroid molecule, in the precur mula R COOH is first treated with an agent activating the Sor compound of B or B, which can be, as above mentioned, carboxyl group, as above described in 1a. 1, and then with bivalent or monovalent, and in the precursor compound of C. the precursor compound of B, after having protected one of The reactions are carried out with well known methods in 60 the two reactive HX groups, for example with acetyl or the prior art, which allow to obtain bonds among the steroid, tert-butyloxycarbonyl, restoring the initial function at the the precursor compound of B or B and the precursor com synthesis end. The scheme is the following: pound of C as above defined. (IA.2) CDI, HX-X-X-G When the reactive function of the steroid (for example RCOOH ------se —COOH, -OH) is involved in a covalent bond, for example 65 of ester, amide, ether type, said function can be restored with R-TR-X-X-G -----> ----> R-TR-X-XH the well known methods in the prior art. US 7,524,836 B2 25 26 wherein X, TX areas above defined and G is a protective Alternatively, the halide Hal -X—COCl can be used, group of the HX function. wherein Hal is preferably bromine, which is let react 2. Nitroxyderivative synthesis. with the compound of formula (IA.2). 2a.1 When the compound obtained at the end of the previous 1b. When the reactive function of the steroidis—OH (general step 1a. has formula (IA.1), the acid can be converted into 5 formula: R-OH), the two functional groups present on the the corresponding sodic salt and one can then follow the precursor compound of B can be the following: known prior art methods for preparing the final compound, 1b. 1 A carboxylic group, which reacts with the steroid OH for example according to one of the following synthesis function, and a HX group, the latter reactive group of the Schemes: precursor compound of B being equal to or different from 10 the steroid functional group. The formula of the precursor compound of B is of the H X X COOH type, (1A.1b) A.) wherein X and X are as above defined. The H X— function of the precursor compound of B is R-TR-X-COONa + R-X-R protected according to the known prior art methods and 15 the carboxyl is reacted, as above mentioned, according R-TR-X-TE-T-X-R ------se to the following scheme: R-TR-X-TRI-T-Y-NO

(1B.1) wherein TX, TT are as above defined, R is selected H-X-X-COOH ------G-X-X-COOH + from Cl, Br. Y is as above defined, X is theY radical free R-XH ------R-TR-X-X-G ----> from the oxygen atom, R is Cl, Br, Iodine, OH. If R-OH the compound of formula (1A.1b) is subjected R-TR-X-X-H to halogenation, for example with PBral PCls, SOCl, PPh+I, and then reacted with AgNO in organic sol vent such as acetonitrile, tetrahydrofuran. If R is Cl, Br, 25 At the end of the reaction the HX function of the precursor Iodine, the compound of formula (1 A.1b) is directly compound of B is restored. reacted with AgNO, as above mentioned. 1b.2 When the precursor compound of B contains two car boxylic groups, it is treated with an equimolaramount of an agent activating the carboxyl group under the conditions B.) 30 previously described in 1a.1, and then reacted with the reactive OH function of the steroid molecule. Possible R-T-X-COONa + Hal-Y-NO - - - - - other reactive functions of HX type present in the two R-TR-X-TR-T-Y-NO compounds must be carefully protected as previously men (1A.1c) tioned. Lastly a compound of formula R-T-X. —COOH C.) 35 (1B.2) is obtained. R-T-X-COC + R-X-R -----> 2b. Nitroxyderivative synthesis. R-TR-X-TR-T-X-R 2b. 1 To obtain the final nitroxyderivative starting from the AgNO3 compound of formula R-T-X. X—H (1B.1), obtained R-TR-X-TR-T-X-R ------r at the end of the synthesis described in 1b.1, the (1B.1) R-TR-X-TR-T-Y-NO 40 compound is reacted with an halogenacid of formula Hal X COOH, which has been treated as previously described in paragraph 1 a.1, or with the corresponding wherein R-OH or NHR, R, R and the other symbols halogenacid chloride, the resulting compound is dissolved being as above defined. in organic solvent, for example acetonitrile or tetrahydro When X is a linear Calkyl, the corresponding acid R-T- 45 furan and reacted with silver nitrate. X COOH is reacted with triphenylphosphine in the 2b.2 To obtain the final nitroxyderivative starting from the presence of an halogenating agent such as CBr or compound of formula R-T-X COOH (1B.2), obtained N-bromosuccinimide in tetrahydrofuran obtaining the at the end of the synthesis described in 1b.2, the acid is compound (1A.1C) wherein R.Br. transformed into the corresponding sodic salt, it is reacted 2a.2 When the compound obtained at the end of the previous 50 with a R X-R compound, Previously defined in the step 1a. has formula (IA.2), the corresponding nitroy reaction A. Scheme of paragraph 2a.1, obtaining according derivative is obtained by treating an halogen-carboxylic to the same process therein mentioned the final nitroxy acid of formula Hal-X—COOH, X being as above derivative. Alternatively, when X is a linear Calkyl, the defined, first with an agent activating the carboxyl group as acid (1B.2) is reacted with triphenyl-phosphine in the pres ence of an halogenating agent Such as CBr or N-bromo described in 1A.1, and then with the compound of formula 55 (IA.2), obtaining an halogen derivative, which is isolated Succinimide in tetrahydrofuran and the resulting com and then dissolved in organic solvent, (ref. paragraph 2a.1), pound dissolved in organic solvent for example and treated with silver nitrate. The global reaction scheme acetonitrile, tetrahydrofuran, is reacted with silver nitrate. is the following: 2b.3 Alternatively to the synthesis process according to 1b. 1 1) CDI, 2) R-TR-X-XH and 2b.1, it is possible to react in a first step the HX Hal-X-COOH ...... - 60 function of the precursor compound of B HX X COOH with the acyl chloride of an halogenacid of formula Hal-X, CO Cl, wherein Hal is preferably Br, and sub R-TR-X-TR-T-X-Hal sequently the carboxylic function of the so obtained com R-TR-X-TR-T-Y-NO pound, with the steroid of formula R OH. In the third and 65 last step the Hal group is substituted with —ONO accord ing to the process described in 2b. 1. The reaction scheme is wherein T, X, T, T, Y are as above defined. the following: US 7,524,836 B2 28

-continued (2B.3) R-TCI-X-TR-X. HX-X-COOH + Hal-X-COC ------ONO Hal-X-To-TR-X-COOH 5 Hai-X-To-Tai-x,-COOH ---- 1b. When the steroid reactive function is a OH group and the Hal-X-To-TR-X-TR-R precursor compound of B contains a reactive group of general formula XH. HX wherein X is as above defined, Hal-X-To-TR-X-TR-R 10 being equal to or different from OH, the synthesis is carried out starting from an halogendiacid compound of formula HOOC-X"-COOH wherein T, T, TX, X, Y are as above defined. Hal In the previous scheme the nitration can alternatively be 15 carried out on the acid compound of formula (2B.3). In the above mentioned processes the steroid reaction with X' being as above defined, said compound being prepared the precursor of B for the compounds of formula (I) is not from the corresponding hydroxy-diacid as described for carried out whenbo O, and in the reaction with the precursor the halogenation of the compound (1A.1b) in 2a.1. The compound of C the steroid with its reactive function is halogendiacid compound is treated with an equimolar directly used. amount of an agent activating the carboxyl group, under the conditions previously described in 1a. 1., and then it B) Synthesis of Compounds of Formula (II). is reacted with the reactive function of the steroid mol 1a. When the steroid reactive function is a carboxylic group ecule. In the Subsequent step the second carboxylic func and the precursor compound of B contains only one func 25 tion is treated with an activating agent, as previously tional reactive group of formula XH. X being as above made for the first, and reacted with the precursor com defined, the steroid is initially converted into the corre pound of B according to the following scheme: sponding acyl-halide (RCOCl), or treated with an agent activating the carboxyl group as described in 1a. 1, and then HOOC-X"-COOH reacted with the HX function of an halogen-acid com pound, said function being equal to or different from that 30 Hal present on the precursor compound of B, said halogen HOOC-X-Ti-R -----> acid having the formula: Hal (IIA.1) CDI, HX-X HX-X"-COOH HOOC-X-Ti-R ------se 35 Hal Hal X, TRI-TCII-X-TI-R wherein X" is Yas above defined without the oxygenatom Hal through which the NO. group is linked, X and Hal are as above defined. 40 The halogenatom is then substituted with the ONO group The compound (IIA. 1) can be obtained with the known as above mentioned. method of the prior art. 3. Synthesis of the nitroso (s=1) derivatives of formula (I). For example when X=NH, it can be obtained from the 3a.1 The compound of formula (1 A.1b) wherein R-OH is corresponding hydroxy-aminoacid, by protecting the reacted with sodium nitrite in a solventformed of a mixture aminic group by the corresponding tert-butyl-oxycarbo 45 nyl derivative and transforming the hydroxyl function of water with tetrahydrofuran in the presence of hydrochlo into halogen group as described for the compound halo ric acid. The reaction is widely illustrated in the prior art. genation (1A.1b) in 2a.1. The general scheme is the following: The free carboxylic function of the compound resulting R-TR-X-TR-T-X-OH + NaNO - - - - - from the reaction with the steroid molecule is reacted 50 with the function present in the molecule of the precur A-B-C-NO sor of B, as previously illustrated in 1a. 1 for the reaction between the steroid of formula R COOH and the pre cursor compound of B. In the final step the halogenatom 3a.2 If the compound obtained at the end of step A has (Hal) present on the radical X is substituted with an 55 formula (IA.2) the corresponding nitroso derivative is ONO group by adding AgNOs to an organic Solution of obtained by treating an hydroxyacid of formula the compound. The reaction scheme is the following, HO—X COOH, X being as above defined, first with an exemplified starting from the RCOCl halide: agent activating the carboxyl group, as described in 1a. 1, (IIA.2) R-COCl + HX-X"-COOH ----> then with 1A.2 and the resulting product with sodium 60 nitrite as described in 3a.1. Hal 3b. 1. To obtain the nitroso derivative starting from the com R-T-X"-COOH + HX-X -----> pound of formula R T X. XH (1B.1) obtained at the end of the synthesis described in 1b. 1, the compound Hal (1B.1) is reacted with an hydroxyacid as described in 3a.2. R-Tcl-X-To-TR-X. + AgNO3 - - - - -ss 65 3b.2 To obtain the nitroso derivative from the compound of Hal formula R-T-X, COOH (1B.2) obtained at the end of the synthesis described in 1b.2, the acid is transformed into US 7,524,836 B2 29 30 the sodic salt and reacted with a compound Hal-X—OH, HOOC-X"-COOH as previously described, and the obtained alcchol is treated as described in 3a.1. OH 4) Synthesis of the nitroso derivatives of formula (II) 4a.1 When the steroid reactive function is a carboxylic group 5 X' being as above defined, said hydroxydiacid compound (general formula R COOH) and the precursor compound is treated with an equimolar amount of an agent activat of B contains only one functional reactive group of for ing C the carboxyl group, under the conditions previ mula XH. X being as above defined, R-COOH is initially ously described in 1a. 1., and then it is reacted with the converted into the corrsponding acyl-halide or treated with steroid reactive function. In the Subsequent step the sec an agent activating the carboxyl group as described in 1a. 1, 10 ond carboxylic function is treated with an activating and then reacted with the HX function of an hydroxy-acid agent, as previously made for the first one, and reacted compound, said function being equal to or different from with the precursor compound of B according to the that present on the precursor compound of B, said following scheme: hydroxy-acid having the formula: HOOC-X"-COOH (4A.1) 15 HX-X"-COOH OH e OH HOOC-X-To-R OH wherein X" is Y'as above defined without the oxygenatom HOOC-X-T-R through which the —NO group is linked, X is as above defined. OH The free carboxylic function of the compound resulting X-TR-Tcl-X'-To-R from the reaction with the steroid molecule is reacted with the function present in the molecule of the precur 25 Sor compound of B, as previously illustrated in 1a. 1 for the reaction between the R COOH acid and the pre The obtained compound is reacted as described in 3a.1. cursor compound of B. in the final step the alcohol is The compounds object of the present invention are formu transformed into the nitroso-derivative as described in lated in the corresponding pharmaceutical compositions for 3a. 1. 30 parenteral, oral and topic use according to the well known The reaction scheme is the following, exemplified starting methods in the art, together with the usual excipients; see for from the RCOCl acid halide: example the Volume “Remington’s Pharmaceutical Sciences 15a Ed. R-COCl + HX-X"-COOH ----> The amount on molar basis of the active principle in these OH 35 formulations is the same, or lower, in comparison with that R-Tcl-X"-COOH (4A.2) + HX-X -----> used of the corresponding precursor drug. The daily administrable doses are those of precursor drugs, OH or in the case lower. The daily doses can be found in the R-Tcl-X-To-TR-X + NaNO -----> publications of the field, such as for example in “Physicians OH Desk reference’. R-Tcl-X-TR-X 40 The following examples have the purpose to illustrate the ONO invention and are not to be considered as limitative of the SaC. 4b. When the reactive steroid function is a OH group and the EXAMPLE 1. precursor compound of B contains a reactive group of general formula XH, HX in which X is as above defined Preparation of 3-4-(3C.5 B.7B)-3,7-dihydroxycolan being equal to or different from OH, the synthesis is carried 24-oiloxy-3-methoxyphenyl-2-propenoic acid 4-ni out starting from an hydroxydiacid compound of formula troxybutyl ester

N

OMe US 7,524,836 B2 31 32 wherein the precursor steroid is ursodesoxycholic acid of di-methylacetamide (25 ml), 3-(4-hydroxy-3-methoxyphe formula (XL), the precursor of B is ferulic acid of formula nyl)-2-propenoic acid 4-bromobutyl ester (2.73 g, 8.28 (DII): mmoles) is added under stirring. To the solution cooled at 0° C., kept under stirring, N,N'-dicyclohexylcarbodiimide (2 g, (XL) 9.7 mmoles) and 4-dimethylamino pyridine (100 mg, 0.81 COOH mmoles) are added. After 1 hour the mixture is heated to room temperature, after 24 hours the precipitate is filtered, the Solvent is evaporated at reduced pressure. The residue is 10 treated with ethyl acetate (150 ml) and washed with water (3x100 ml). After the organic phase is anhydrified with sodium sulphate the solvent is evaporated. The obtained crude product is purified by chromatography on silica gel Ho column eluting with n-hexane/ethyl acetate 1/9. 2.5g of 3-4- 15 (3C,53.7(3)-3,7-dihydroxycolan-24-oiloxy-3-methoxyphe (DII) nyl-2-propenoic acid 4-bromobutyl ester are obtained. c) Synthesis of the 3-4-(3C., 53.7(3)-3,7-dihydroxy collan-24-oiloxy-3-methoxyphenyl-2-propenoic HO acid 4-nitroxybutyl ester To a solution of 3-4-(3C.5 B.7(3)-3,7-dehydroxycolan-24 oiloxy-3-methoxyphenyl-I-2-propenoic acid 4--bromobutyl 25 ester (2.3 g, 3.27 mmoles) in acetonritrile (20 ml) and tetrahy a) Synthesis of the drofuran (5 ml) silver nitrate (0.84g, 4.94 mmoles) is added 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid urder stirring and the mixture is heated to 80°C. under mag 4-bromobutyl ester netic stirring for 6 hours. When the reaction is over the pre cipitate is filtered and the solvent is evaporated. The obtained To a solution of 3-(4-hydroxy-3-methoxyphenyl)-2-pro 30 crude product is purified by chromatography on silica gel penoic acid (10 g, 51.5 mmoles) in THF (400 ml) triph column eluting with methylene chloride/ethylacetate 3/7. 1.5 enylphosphine (2.7g, 10.3 mmoles) and carbon tetrabromide g of 3-4-(3C.5 B.7f8)-3,7-dehydroxycolan-24-oiloxy-3- (34.16 g. 10.3 mmoles) are added and the solution is left at methoxyphenyl-2-propencic acid 4-nitroxybutyl ester are room temperature, under magnetic stirring, for 48 hours. The 35 obtained. Total yield 32%. solid is filtered and then evaporated at reduced pressure. The obtained crude product is purified by chromatogrphy on silica gel eluting with n-hexane/ethyl acetate 7/3.9 g of 3-(4-hy droxy-3-methoxyphenyl)-2-propenoic acid 4-bromobutyl Elementary analysis ester are obtained. M.p.-86-89° C. 40 Calculated C 66.55% H 8.08% N 2.04% Found C 66.59% H 8.14% N 1.99% b) Synthesis of the 3-4-(3C.5 B.7(3)-3,7-dihydroxy collan-24-oiloxy-3-methoxyphenyl-2-propenoic acid 4-bromobutyl ester EXAMPLE 2 45 Preparation of 3-4-(3C.5 B.7Cl)-3,7-dihydroxycolan To a solution of (3C.5 B.7 B)-3,7-dehydroxycolan-24-oic 24-oiloxy-3-methoxyphenyl-2-propenoic acid 4-ni acid (2.9 g, 7.38 mmoles) dissolved in chloroform (25 ml) and troxybutyl ester

S

OMe US 7,524,836 B2 33 34 wherein the precursor steroid is chenodeoxycholic acid of formula (XLI) and the B precursor is ferulic acid of formula -continued (DII) COOH (CVIII)

H3Cr, COOH (XLI) 5 r NHCOCH

a) Synthesis of 10 N-acetyl-S-(4-bromobutyroyl)cysteine A solution of 4-bromobutyric acid (5.1 g, 30.6 mmoles) and 1,1'-carbonyldiimidazol (5.61 g, 34.6 mmoles)) in chlo H roform (50 ml) is left at room temperature under stirring for 1 (DII) 15 hour. To the reaction mixture N-acetyl cysteine (5 g, 30.6 N-COOH mmoles) dissolved in N,N-dimethylformamide (5 ml) and sodium ethylate (50 mg) is added under stirring. After 24 hours the solution is washed with HCl 1% and brine, the HO organic phase is anhydrified with sodium Sulphate and evapo 20 rated at reduced pressure. The obtained crude product is puri -O fied by chromatography on silica gel column, eluting with H3C ethyl acetate/chloroform 7/3. N-acetyl-S-(4-bromobutyroyl) cysteineis obtained. The compound is prepared following the procedure 25 b) Synthesis of (11B)-11,17-Dihydroxy-21 N-acetyl reported in Example 1. Total yield 28%. S-(4-bromobutyroyl)cysteinyloxy)-pregn-1,4-diene 3.20-dione To a solution of N-acetyl-S-(4-bromobutyroyl)cysteine Elementary analysis (2.7g, 8.64 mmoles) and (11f)-11, 17,21-trihydroxypregn-1, 4-diene-3,20-dione (3.2g, 8.86 mmoles) in tetrahydrofuran

Calculated C 66.55%0. H 8.08%0. N 2.04%0. S.1cycloneXylcarbod11m1de R SR i Ka (1.9p g,ks; 9.2 mmoles)t ity, aed 4-d1m N. Found C 66.64% H 8.13% N 1.94% ethylaminopyridine (100 mg, 0.8 mmoles) are added. AMter 1 hour the mixture is heated to room temperature, after 24 hours the precipitate is filtered, the solvent is evaporated at reduced pressure. The residue is -treated with ethyl acetate EXAMPLE 3 (150 ml) and washed with water (3x100 ml). After having anhydrified the organic phase with sodium Sulphate the Sol Preparation of (11 B)-1 1,17-dihydroxy-21 (N-acetyl- vent is evaporated. The obtained crude product is purified by S-(4-nitroxybutyroyl)cysteinyloxy)-pregn-1,4-diene- 40 chromatography on silica gel column eluting with chloro 3,20-dione form/ethyl acetate 3/7. 0.94 g of (11B)-11,17-dehydroxy-21 N-acetyl-S-(4-bromobutyroyl)cysteinyloxy)-pregn-1,4-di O O ene-320-dione are obtained. -Yr-sco, 45 c) Synthesis of (11B)-11,17-Dihydroxy-21 N-acetyl OH S-(4-nitroxybutyroyl)cysteinyloxy)-pregn-1,4-diene NHCOCH 3.20-dione To a solution of (11B)-11,17-dehydroxy-21 (N-acetyl-S- so (4-bromobutyroyl)cysteinyloxy)-pregn-1,4-diene-3,20-di one (0.8 g. 1.28-moles) in acetonitrile (10 ml) and tetrahy drofuran (5 ml) silver nitrate (0.4g, 2.35 mmoli) is added under stirring and the mixture is heated to 80° C. under herein th id i dnisol f f 1 magnetic stirring for 20 hours. At the end of the reaction the wherein the precursor Stero1d 1s prednisolone of formula precipitate is filtered and the solvent is evaporated. The (XLII) and the precursor, of B is N-acetyl cysteine of formula 5S obtained crude product is purified by chromatography on (CVIII) silica gel column eluting with methylene chloride/ethylac

(XLII) etate 3/7. (11B)-11,17-dehydroxy-21 N-acetyl-S-(4-nitroxy butyroyl)cysteinyloxy)-pregn-1,4-diene-320-dione is obtained. Total yield 12%. 60

Elementary analysis Calculated C 56.59% H 6.33% N 4.40% S 5.04% 65 Found C 56.63% H 6.38% N 4.36% S 5.01% US 7,524,836 B2 35 36 EXAMPLE 4 wherein the precursor steroid is desamethasone of formula (X-LIV) and the precursor of B is N-acetyl cysteine of for Preparation of (11B)-11, 17-Dihydroxy-21N-acetyl mula (CVIII) S-(4-nitroxybutyroyl)cysteinyloxy)-pregn-4-ene-3, 20-dione

(XLIV)

O O O ls 10 O S (CH2)3ONO OH NHCOCH

O 15 (CVIII) COOH HS ~ NHCOCH wherein the precursor steroid is hydrocortisone of formula (XLIII) and the precursor of B is N-acetyl cysteine of formula The compound is prepared according to the procedure (CVIII) reported in Example 3. Total yield 17%.

(XLIII) 25 Elementary analysis Calculated C 55.68% H 6.18% N 4.19% S 4.79% Found C 55.72% H 6.22% N 4.15% S 4.75%

30 Pharmacological Tests

O EXAMPLE (CVIII) COOH 35 Acute Toxicity HS ~ Acute toxicity has been evaluated by administering to a NHCOCH group of 10 rats weighing 20 g a single dose of each of the tested compounds, by cannula, by os in an aqueous Suspen sion of carboxymethylcellulose 2% w/v. The compound is prepared according to the procedure The animals are kept under observation for 14 days. In no reported in Example 3. Total yield 15%. 40 animal of the group toxic symptoms appeared even after a 100 mg/kg dose administration. Example F1 Elementary analysis 45 Calculated C 56.37% H 6.78% N 4.39% S 5.02% Experimental in vivo model with N'-nitro-L-arginine-me Found C 56.39% H 6.81% N 4.31% S 4.93% thyl ester (L-NAME): effect of the precursor steroids and of the corresponding compounds according to the present inven tion on the endothelial dysfunction induced L-NAME. EXAMPLE 5 The experimental model adopted is according to J. Clin. 50 Investigation 90,278-281, 1992. Preparation of (11B, 16C.)-9-Fluoro-1 1,17-dihydroxy The endothelial dysfunction is evaluated by determining 21 N-acetyl-S-(4-nitroxybutyroyl)cysteinyloxy-16 the damage the hepatic damage (GPT increase), and the vas methylpregn-1,4-diene-320-dione cular endothelium or cardiovascular damage (blood hyper tension) induced by L-NAME admirnistration. 55 The animals (Long Evans rats, average weight 350-450 g) are divided in groups as herein below described. The group O O receiving L-NAME is treated for 4 weeks with said com

pound dissolved at the concentration of 400 mg/litre in drink ing water. The following groups (No. 10 animals for group) VOH or sco, 60 are constituted: CH3 NHCOCH, A) Control Groups: 1 group: treatment: only carrier (physiologic solution), 2 group: trealtmrent: carrier +L-NAME, 65 B) Groups Treated with the Drug: 3° group: treatment: carrier-drug, 4° group: treatment: carrier--drug-i-L-NAME. US 7,524,836 B2 37 38 The drugs screened in the test are hydrocortisone, desa The ccmpound to be used as precursor of B or B according methasone, prednisolone, chenodeoxycholic acid, ursodes to the present invention meets test 4 if it inhibits radical oxycholic acid and the corresponding derivatives according production From DPPH in a percent equal to or higher than to the present invention. 50%. In those groups of rats treated, respectively, with hydrocor- 5 In Table III are reported the results obtained in said test tisone, desamethasone, prednisolone and thereof correspond with the following compounds: N-acetylcysteine, cysteine, ing compounds according to the present invention, the blood ferulic acid, (L)-carnosine, gentisic acid, 4-thiazolidin car pressure is determined. boxylic acid and 2-oxc-4-thiazolidincarboxylic acid. In those groups of rats treated, respectively, with ursodes Table III shows the following: oxycholic acid and chenodeoxycholic acid and thereof cor- 10 N-acetylcysteine, cysteine, ferulic acid, (L)-carnosine, responding compounds according to the present invention, gentisic acid meet test 4 since they inhibit the production GPT is determined. of radicals induced by DPPH to an extent higher than Each drug is administered by intraperitoneal route once a 50%. Therefore they can be used as precursors of the B day for 4 weeks. compound in the synthesis of the compounds according At the end of the four weeks access to water is prevented 15 to the present invention. and after 24 hours the animals are sacrificed. 4-thiazolidin carboxylic acid and the 2-oxo-4-thiazolidin Four hours after the last administration the blood-pressure carboxylic acid do not meet test 4 since they do not is determined. inhibit radical production from DPPH. Therefore they Damage to the vascular endothelium is determined, as said 20 can be used as precursors of B or B if they meet test 5. by the cardiovascular effects induced by L-NAME (increase of the blood pressure). The hepatic damage is determined by Example F3 evaluation of the glutamic-pyruvic transaminase (GPT increase) after sacrifice. Test 5: Inhibition of the Radical Production from Fe' from Results are reported in Tables I and II. The % blood- 25 Compounds Used as Precursors of B, B, or C=-T-Y H. pressure and GPT values are referred to the corresponding 0.1 ml aliauo.s of 10 M methanolic solutions of 4-thia value found in the animals of the 1st control group. The Zolidin carboxylic acid and 2-oxo-4-thiazolidin carboxylic average value of the blood pressure in this group was of 105 acid are added to test tubes containing an aqueous solution mmHg. formed by mixing 0.2 ml of 2 mM desoxyribose, 0.4 ml of The results obtained show that the steroidal precursors buffer phosphate pH 7.4 100 mM and 0.1 ml of 1 mM Fe' cause hepatic damage (ursodesoxycholic acid and chenode (NH) (SO) in 2 mM HC1. The test tubes are then kept at a oxycholic acid) and arterial hypertension (hydrocortisone, temperature of 37° C. for one hour. Then in each test tube 0.5 desamethasone, prednisolone). GPT and blood pressure val ml of a 2.8% solution in trichloroacetic acid in water and 0.5 ues of the treated rats are higher compared both with the ml of an aqueous solution 0.1 Mthiobarbituric acid are added corresponding groups treated with drug in the absence of in the order. A reference blank is constituted by substituting L-NAME and with the controls treated with L-NAME. The the above 0.1 ml aliquots of the test compound methanolic products of the invention are instead better tolerated in com solutions with 0.1 ml of methanol. The test tubes are closed parison with the corresponding precursors, even in animals and heated in an oil bath at 100° C. for 15 minutes. A pink not pretreated with L-NAME. coloration develops the intensity of which is proportional to 40 the quantity of desoxyribose undergone to radical oxidative Example F2 degradation. The Solutions are cooled at room temperature and their absorbances at 532 nm are read against the blank. The inhibition induced by the precursor of B or B or Test 4: Inhibition of the Radical Production from DPPH of C=-T-Y H (wherein the free valence is saturated as above Some Substances Used to Prepare the Precursors of B or B1 45 defined) with respect to radical production from Fe' is deter The method is based on a colorimetric test in which DPPH mined as a percentage by means of the following formula: (2,2-diphenyl-1-picryl-hydrazyl) is used as the compound forming radicals (M. S. Nenseter et Al.., Atheroscler. Thromb. 15, 1338-1344, 1995). wherein. As and A, are respectively the absorbance values of Solutions in methanol of the tested substances at a final the solution containing the tested compound+the iron salt and concentration 100 uMare initially prepared. 0.1 ml of each of that of the solution containing only the iron salt. these solutions are added to aliquots of 1 ml of a methanol The results are reported in Table IV, which shows that both solution 0.1 M of DPPH and then the final volume is brought acids meet test 5 since they inhibit radical production from to 1.5 ml. After having stored the solutions at room tempera Fe' in a percentage higher than 50%. Therefore both 4-thia ture away from light for 30 minutes, the absorbance at the 55 Zolidin carboxylic acid and 2-oxo-4-thiazolidin carboxylic wave length of 517 mm is read. It is determined the absor acid can be used as precursors of B, B, or C=-T-Y H for bance decrease with respect to the absorbance of a solution obtaining compounds of the present invention. containing the same concentration of DPPH. The efficacy of the test compound to inhibit the production Example F4 of radicals, otherwise said antiradical activity, is expressed by 60 the following formula: Example F1 was repeated with three groups of rats (each group of ten animals), one control group not receiving L-NAME and two groups receiving L-NAME, and i.p. wherein. As and Aare, respectively, the absorbance values of 65 administered as it follows: the solution containing the test compound together+DPPH a. control group (not receiving L-NAME): the carrier (physi and of the solution containing only DPPH. ologic Solution), US 7,524,836 B2 39 40 b. 1st group receiving L-NAME (group b-comparative) administered at the same time with 25 mg/Kg (0.064 TABLE II mmoles/Kg) of dexamethasone--10.4 mg/Kg (0.064 mmoles/Kg) of N-acetylcysteine in the same above carrier, c. 2nd group receiving L-NAME (group c) administered with Study of hepatic damage, determined by GPT assay, of 42.5 mg/Kg (0.064 mmoles/Kg) of the dexamethasone chenodeoxycholic acid and ursodesoxycholic acid, and derivative according to the invention (ref.ex. 5) in the same of the corresponding derivatives according to the above carrier. In this experiment vascular tolerability, i.e. the rise in blood invention, in animals (rats) both not treated and treated pressure (vascular damage) was determined in the animals of 10 with L-NAME. The % variation of GPT with respect to groups b and c and expressed as percentages to that of the the controls not treated with L-NAME and treated with control group a, assumed to be 100%. the only carrier (physiological solution) The results are reported in Table V and show that the mixture administered to group b (comparative) induced in the animals an higher blood pressure increase than the compound animals of the invention (group c). 15 non treated with Animals treated L-NAME with L-NAME Example F5 Example F1 was repeated with three groups of rats (each dose dose group of ten animals), one control group not receiving mg/Kg mg/Kg L-NAME and two groups receiving L-NAME, and i.p. Compound i.p. GPT war. 90 i.p. GPT war. 90 administered as it follows a. control group (not receiving L-NAME): the carrier (physi carrier 1OO 230 ologic Solution), chenodeoxycholic 1OO 150 1OO 350 b. 1st group receiving L-NAME (group d-comparative) 25 administered at the same time with 100 mg/Kg (0.25 acid mmoles/Kg) of ursodesoxycholic acid--49.5 mg/Kg (0.25 chenodeoxycholic 1OO 105 1OO 130 mmoles/Kg) of ferulic acid in the same above carrier, acid der. c. 2nd group receiving L-NAME (group e) administered with Ex. 2 175 mg/Kg (0.25 mmoles/Kg) of the ursodesoxycholic 30 derivative according to the invention (ref. ex. 1) in the same ursodesoxycholic 1OO 130 1OO 276 above carrier. acid In this experiment hepatic tolerability, i.e. the rise in GPT ursodesoxycholic 1OO 103 1OO 123 (hepatic damage) was determined in the animals of groups d acid der. 35 and e and expressed as percentages to that of the control group Ex. 1 a, assumed to be 100%. The results are reported in Table VI and show that the mixture administered to group d (comparative), induced in TABLE III the animals an higher GPT increase than the compound of the invention (groupe). 40 Test 4: Screening of the effectiveness of some substances to inhibit radical production from DPPH. TABLE I % inhibition radical Study of vascular tolerability of hydrocortisone, production from Compound DPPH dexamethasone and prednisolone, and of the corresponding 45 derivatives according to the invention, in animals (rats) both not treated and treated with L-NAME. Vascular Solvent tolerability is indicated as % variation of the blood N-acetylcysteine pressure (hypertension) with respect to the controls not Cysteine treated with L-NAME and treated with the only carrier Ferulic acid physiological solution (L)-carnosine 50 Gentisic acid Animals 2-oxo-4-thiazolidin non treated with Animals treated carboxylic acid L-NAME with L-NAME 4-thiazolidin carboxylic acid dose Blood dose Blood mg/Kg pressure mg/Kg pressure 55 Compound i.p. variation% i.p. variation% TABLE IV carrier 100 40 hydrocortisone 10 115 5 60 Test 5: study on the effectiveness of the listed hydrocortisone 10 98 5 2O Substances to inhibit radical production induced der. Ex. 4 60 by Fe". dexamethasone 5 125 25 70 dexamethasone 5 103 25 25 % Radical der. Ex. 5 Compound Inhibition from Fe' prednisolone 10 119 15 65 prednisolone 10 102 15 10 White O der. Ex. 3 2-oxo-4-thiazolidin carboxylic acid 100 65 4-thiazolidin carboxylic acid 100 US 7,524,836 B2 41 42

TABLEV -continued (DI) Study of vascular tolerability in animals (rats) O OH treated with L-NAME and i.p. administered with a mixture of dexamethasone + N-acetylcysteine and with the derivative of dexamethasone of ex. 5 according to the invention. Vascular tolerability is indicated as % variation of the blood pressure (hypertension) with respect to the controls not treated with L-NAME and treated with the only carrier. HO1 OH, 10 dose Blood OH mg/Kg pressure (DIII) Compound i.p. variation% O controls 100 HO group b - comparative 25(A) + 10.4(B) 170 15 OH, dexamethasone (A) + N acetyl cysteine (B) group c 42.5 125 OH dexamethasone (DV) der. Ex. 5 N-COOH,

TABLE VI HO or OH Study of hepatic tolerability in animals (rats) (DVI) treated with L-NAME and i.p. administered with a 25 COOH, mixture of ursodesoxycholic acid + ferulic acid and with the derivative of ursodesoxycholic acid of ex. 1 according to the invention. Hepatic damage is indicated as % variation of GPT with respect to the controls not treated with L-NAME and treated with the only carrier. HO or OH dose 30 mg/Kg GPT (DVII) Compound i.p. variation% controls 100 r COOH, group d - comparative 100 (C) + 49.5 (D) 18O HO ursodesoxycholic 35 (DVIII) acid (C) + ferulic COOH, acid (D) groupe 175 123 ursodesoxycholic acid der, ex. 1 40 MeO The invention claimed is: 1. A steroidal compound or a salt thereof having the fol OH (DXI) lowing general formula: COOH A-B-T-Y NO2 (I) 45 wherein: A=R , wherein R is a steroidal drug radical as defined hereunder, B=-T-X-T - wherein MeO OMe T is (CO), 50 OH T is O: X is a bivalent bridging group Such that the following precursors of B, having the formula Z-T-X-T-Z T, is (CO); in which Z and Z are independently H or OH, is Y has the following meanings: 55 selected from the following compounds: a linear or branched C-Co alkylenoxy group or a cycloalkylene having from 5 to 7 carbon atoms, in the cycloalkylenic ring one or more carbon atoms can be (DII) Substituted by heteroatoms, the ring can have side chains of R' type, R' being a linear or branched C-C alkyl; or 60 OH, N HO --(CH)-O- 2 65 -(CH) CH US 7,524,836 B2 43 44 wherein n is an integer from 0 to 3 and n is an integer R" and R', equal or different from each other, are hydrogen from 1 to 3, or CH: -(CH-CH-O)-; - (CH-CH-O)- R" is C(O)CHO Rf Rif 2. The steroidal compound or salt thereof according to claim 1 wherein the steroidal drug radical is derived from one of the following: Budesonide, Hydrocortisone, Beclometha wherein R, H, CH, and nf is an integer from 1 to 6; Sone, Betamethasone, Chloroprednisone, , Cor R has the following formula: tisone, Corticosterone, Desonide, DeSoximethasone, Dexam ethasone, Diflucortolone, Flumethasone, Flunisolide, 10 , Flurandrenolide, Meprednisone, Methyl prednisolone, Paramethasone, Prednisolone, Prednisone, or Triamcinolone. 3. The steroidal compound or salt thereof according to 15 claims 1 or 2 wherein the precursor of B is

(DII) wherein in substitution of the hydrogenatom of the CH groups or of OH. the two hydrogenatoms of the CH groups mentioned in the general formula, the following Substituents can be HO present: in position 1-2: there may be a double bond; O in position 3: there may be C=O, OH: 25 YCH, in position 4-5: there may be a double bond; in position 6: there may be C1, F, CH: in position 7: there may be C1, OH: 4. The steroidal compound or salt thereof according to in position 9: there may be Cl, F: claim 1 wherein Y is a linear or branched (C-C)-alkylenoxy in position 11: there may be OH, C=O, C1, CH: 30 group. in position 16: there may be CH, OH: 5. A method for the treatment of oxidative stress and/or in position 17: there may be OH, CH: endothelian dysfunctions comprising administering the Ste in position 16-17: there may be the following groups: roidal compound or salt thereof according to claim 1. CH3 6. A method for the treatment of inflammation comprising 35 administering the steroidal compound or salt thereof accord ing to claim 1. 7 os-n-" 7 rfs.I6 O 7. A pharmaceutical formulation comprising the steroidal O compound or salt thereof according to claim 1 and a pharma I6 40 ceutical acceptable carrier.

k k k k k