WO2004005298A1

WO2004005298A1 - Derivatives of rifabutine useful as antimicrobial agents

Info

Publication number: WO2004005298A1
Application number: PCT/PT2003/000009
Authority: WO
Inventors: Maria Medeiros; Maria Costa; Ricardo Figueiredo; Maria Rosa; Maria Curto; Lina Santos; Maria Cruz; Maria Gaspar; Sónia FEIO
Original assignee: Instituto Nacional De Engenharia E Tecnologia Industrial
Priority date: 2002-07-09
Filing date: 2003-07-09
Publication date: 2004-01-15
Also published as: PT102807A; AU2003245183A1

Abstract

This invention relates to new derivatives of rifabutine of general formula I, wherein R1 is hydrogen or COR2 in which R2 is independently selected from saturated or insaturated linear, branched or cyclic alkyl C1-C20; R3 is oxygen or hydroxyl; R4 is a nitrogen atom or N-oxide, and their acceptable salts and solvates, including hydrates. These derivatives are useful as anti-infectious agents, namely against Mycobacterium avium, and Mycobacterium tuberculosis. (I)

Description

FIELD OF THE INVENTION

DERIVATIVES OF RIFABUTINE USEFUL AS ANTIMICROBIAL AGENTS

BACKGROUND OF THE INVENTION

Rifabutin la is a known derivative of rifamycin S active against mycobateria including atypic organisms like Mycoba terium avium and Mycobacterium intracell ulare . It is also active against rifampicin resistant Mycoba cteri um tuberculosis isolates, and has comparable activity to rifampicin against Staphylococcus aureus, Haemophil us ducreyi e Chlamydia trachoma tis (cf . R.N. Brogden and A. Fitton, "Rifabutin A Review of its Antimicrobial Activity, Pharmacokinetic Properties and Therapeutic Efficacy", Drugs, 47(6): 983-1009, 1994).

Rifabutin is the generic name of the chemical compound 4-deoxo-3, - [2-spiro (N-isobutyl-4-piperidyl) - 2, 5-dihydro-lH-imidazo] -rifamicin S. Depending on the nomenclature system utilized it may also be identified as 6, 9-dihydro-5,17, 19, 21-tetrahydroxy-8, 9- [2-spiro- (N- isobutyl-4-piperidyl) -2, 5-dihydro-lH-imidazo] -23-metoxi- 2, 4, 12, 16, 18, 20, 22-heptamethyl-6-oxo-2, 7-epoxypentadeca- [1, 11, 13]-trienimine-2H-furo[2',3',7,8]nafto[l,2- d] imidazolo-2, 4 ' -piperidine-5, 10,26. (3H, 9H) -trione-16- acetate. Rifabutin is a compound of formula la:

la

Molecular Formula: C₄₆H_{62 4}0n Molecular Weight: 847.12

The preparation of Rifabutin la is reported in U.S. Patent No. 4.219.478 published on 26 August 1980. Rifabutin is a red-violet coloured amorphous powder freely soluble in chloroform and methanol, slightly soluble in water, melting point 148-156°C (with decomposition) . Rifabutin la is positioned as a especially relevant antibiotic in therapy. In 1994 Rifabutin la was the only studied antimycobacterial agent which was effective in placebo controlled prophylactic clinical tests. It is commercially known as Mycobutin^R (Upjohn & Pharmacia) and it is prescribed daily in 300 mg dosage units for prophylaxis of infections caused by disseminated Mycobacterium avium complex (MAC) in AIDS patients ( see : PCT/US93/00335 equivalent to WO 93/13774. 1993-07-21. BANKS PHILLIP L.,(US); WOLGEMUTH RICHARD L.,(US); WYNNE BEVERLEY A., Applicant ERBAMONT INC (US)) and also more recently as prophylactic agent in HIV and tuberculosis infected patients (Narita, M.; Stambaugh, J. ; Hollender, S.; Jones, D. ; Pitchenik, E.; Ashkin, D. , Clin . Infect . Dis . , 30(5), 779-783. 2000). Rifabutin la is not effective for the treatment of influenza or common colds, although it may be useful in the establishment of a prophylactic regimen for multiple opportunist pathogens

(see: Benson, A.; Williams, L.; Cohn, L.; Becker, S . ; Hojczyk, P.; Nevin, T . ; Korvick, A.; Heifets, L. ; Child, C; Lederman, M.; Reichman, C; Powderly, G.; Notario, F.; Wynne, A.; Hafner, R. , J. Infect . Dis . , 181(4), 1289- 1297, 2000; and, Cirioni, 0.; Giacometti, A.; Barchiesi, F.; Fortuna, M.; Scalise, ^' G. , J. Antimicrob . Chemother. , 44(5), 653-659, 1999) and against Mycobacteri um tuberculosis (MTB) isolates and multiresistances (MDR) (see, e.g., Chien, H.-P.; Yu, M.-C; Ong, T.-F.; Lin, T.- P.; Luh, K.-T., J. Formosan Med. Assoc , 99(5), 408-411, 2000) . Rifabutin la was also recently reported for the treatment of Chron's disease (see: US Patent No. 6297015. published 2 October 2001. by Shafran I. (US) untitled Chron 's disease diagnostic and trea tmen t methods and composi tions) and for treatment of patients where the eradication of infections caused by Helicobacter pylori had not occurred after triple reference therapies based on inhibitors of the proton pump (see: Patent WO9702039 untitled Anti-jbacterial synergistic composi tions comprising rifabutin, Date of publication: 1997-01-23. Inventor (s): ROSSI R. ; JABES D. ; CASTELLANI P. and applied by PHARMACIA & UPJOHN SPA (IT)).

Once approved as a medicine for a specific use, any pharmaceutical candidate may present secondary undesirable effects and, in the case of Rifabutin la, uveite has been associated (an inflammatory condition of the eye with possible unpaired vision) since the first reported studies on the tolerance to dose-limit (e.g.: Siegal, F.; Eilbott, D.; Burger, H.; Gehan, K. ; Davidson, B., et al . , Dose-limiting toxicity of rifabutin in AIDS- related complex: syndrome of arthralgia/arthritis, AIDS, 4:433-441, 1990; Shafran, S.; Deschenes, J. ; Miller, M. ; Phillips, P., Toma, E., Uveitis and pseudojaundice during a regimen of clarithromycin, rifabutin, and ethambutol. Correspondence. New England Journal of Medicine, 330:438- 439, 1994) .

The authors of the present invention became interested in the exceptional biological profile of Rifabutin la and considered an important challenge the optimization of its therapeutic activity. Accordingly, a first step was trying to understand the structure- activity relationships of Rifabutin la and related molecules through a few studies of conformational analysis using nuclear magnetic resonance spectroscopy techniques which were published in speciality journals (See: Santos, L.; Fant F. ; Medeiros, M. A.; Borremans, F. A. M.; Costa, M. C. and Curto, M. J. M., "Structural Characterization by NMR of Rifabutinol, A Derivative of Rifabutin," Magn . Reson . Chem . , 38: 937-945, 2000; Santos, L.; Medeiros, M. A.; Santos, S.; Costa, M. C; Tavares, R. and Curto, M. J. M. , "NMR STUDIES of Some Rifamycins," Journal of Molecular Structure, 563-564, 61- 78, 2001) . As a result of the above preliminary studies, active derivatives of Rifabutin were synthesized for the first time possessing high antibacterial activity, in particular against Mycobacterium avi um . Such compounds offered new advantageous perspectives for the therapy of diseases where Rifabutin la has been currently indicated and administered.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to new compounds derived from Rifabutin la with relevant antibiotic activity. Such derivatives are selected from a group consisting of compounds of general formula I

wherein: R¹ is hydrogen or -COR², where R² is independently selected from straight, branched or cyclic Cι-C₂₀ alkyl, saturated or unsaturated chain;

R³ is oxygen or hydroxyl;

R⁴ is a nitrogen atom or an N-oxide group, and their pharmaceutically acceptable salts and solvates, including hydrates .

Such compounds are obtained as a powder having a colour ranging from orange-reddish to red-violet, they are soluble in polar organic solvents and water insoluble. New compounds provided by this invention may be obtained by reaction of Rifabutin la above with well known mild oxidative reagents, for example Oxone^® (DuPont) , which is a stable and convenient source of potassium monoperoxosulfate (caroate) , and may be used for oxidation purposes in a great variety of conditions known to one skilled in the art. Thus, aqueous solutions of Oxone may be useful to carry out oxidations either in homogeneous conditions, or in biphasic systems with an immiscible co-solvent and a phase transfer catalyst. In a similar manner to other currently used oxidation procedures, there is an in si tu formation and reaction of a secondary reactant in the particular reaction conditions, because in reactions involving aqueous Oxone- ketone systems, dioxirane production occurs as an active intermediate (See, e.g.: Burke S. D. e Danheiser R. L.; "Handbook of reagents for organic synthesis - oxidizing and reducing agents," John Wiley & Sons, 2000; Yang, D. ; Yip, Y. C. and Wong, M. K. , J. Org. Chem . , 64. 1635-1639, 1999) . Owing to the presence of available electron pairs, the heteroatoms are good acceptors of oxygen, the latter through the side presenting these unpaired electron pairs, the dimethyldioxirane behaving as an electrophilic oxidant able to selectively oxidize the tertiary amine function of Rifabutin la (See: Boyd, D. R. ; Coulter, P. B.; McGuckin, M. R.; Sharma, N. D.; Jennings, W. B. and Wilson, V. E., J. Chem . Soc . Perkin Trans . I, pp. 301- 306, 1990) and analogous molecules prepared in the field of present invention, producing further preferred compounds of this invention, in particular N-oxides of general Formula II

II

wherein:

R¹ is hydrogen or -COR , where R is independently selected from hydrogen or straight, branched or cyclic Cι~ C₂rj alkyl, saturated or unsaturated chain;

R^J is oxygen or hydroxyl; and their pharmaceutically acceptable salts and solvates, including hydrates. Another embodiment of the present invention is the preparation of more preferred N-acylated derivatives of Rifabutin la with relevant antibiotic activity, using conventional techniques for selective acylation of the tertiary amine known to those skilled in the art thereby producing compounds of formula III

12'

III

wherein:

R¹ is hydrogen or -COR², where R² is independently selected from straight, branched or cyclic Cι-C₂₀ alkyl, saturated or unsaturated chain;

R³ is oxygen or hydroxyl;

R⁴ is a nitrogen atom or an N-oxide group, and their pharmaceutically acceptable salts and solvates, including hydrates . EXPERIMENTAL

All the reagents and the solvents used were analytical pure products. The reactions were followed by thin layer chromatography (t.l.c.) on Merck 60 F254 (0.2 mm), silica gel pre-prepared plates. The isolation of the products was performed by column chromatography using silica gel Merck 60 (230-400 mesh, 0.04-0.063 mm), at normal atmospheric pressure or flash with nitrogen pressure, or also by preparative thin layer chromatography on silica gel Merck 60 F254 (0.5 mm) plates. In the case of 25-hydroxy-Rifabutin-N-oxide lie, column chromatography purification took place with RP-18 silica of Fluka (>400 mesh, 0.015-0.035 mm).

The mass spectra were obtained in a Brucker Daltonics Esquire 3000 spectrometer, with an ESI source/interface detecting negative ions; the concentration of the analysed solutions was: 1 μg of

^'substrate/ mL of 1:1 solution of 0.1% acetic acid/acetonitrile. "M" is the molecular ion, "Ch" is the chromophore (molecule core without the ansa chain) and a, b, and c are ions (cf. Studies of Vekey, K. ; Edwards, D. M. F. and Zerilli L. F. ; Journal of Chroma tography, 474, 317-327, 1989).

With the exception of the Rifabutin-N-oxide Ila and 25-hydroxy-Rifabutin-N-oxide lie, nuclear magnetic resonance spectra were registered in a General Electric GE-NMR spectrometer, with a proton frequency of 300.65 MHz and a carbon-13 frequency of 75.6 MHz, all the other spectra were registere in a Brucker spectrometer with a proton frequency of 400.13 MHz and a carbon-13 frequency of 100.62 MHz. With the exception of Rifabutinol N-oxide lib that was dissolved in deuterated methanol, all the samples were dissolved in 99% deuterated chloroform, with tetramethylsilane as internal standard. The obtained results are presented in the following order: solvent, chemical shift (δ in ppm) ; relative intensity (as number of protons, H) ; multiplicity (s-singlet, bs-broad singlet, d-duplet, dd-double duplet, bd-broad duplet, t- triplet, q-quartet, m-multiplet) ; coupling constant J in Hz; and location in the molecule structure.

The infrared spectra were obtained in a Perkin-Elmer FT-IR 1725X spectrometer, as film. The dichloromethane used was previously purified (cf. Perrin D.D., Armarego L.F., "Purification of laboratory chemicals", 2nd Edition, Oxford, Pergamon Press, 1980) . The exact mass was determined by mass spectrometry operating with a laser ionisation-desorption source, at 70 eV and close to 200°C, with negative charged ions detection.

Example 1. Synthesis of Rifabutinol-N-oxide lib 1.1. Synthesis of Rifabutinol lb

To Rifabutin la (300.0 mg; 0.354 mmol) previously dissolved in THF (9.2 mL) in a round-bottomed flask, was added Pt0₂ (10.7 mg; 0.047 mmol) and sodium borohydrate (278.1 mg; 7.35 mmol) dissolved in ethanol (6.5 mL) dropwise. The reaction mixture was maintained under stirring at room temperature, and the reaction was followed by t.l.c. using a mixture of petroleum ether/diethyl acetate/methanol (55:35:10) as eluant, till completion (ca. 4 h 05 min). The reaction mixture was filtered through Celite, and the Celite washed with diethyl ether until the absence of colour was noted. A few mL of distilled water were added and the resulting solution transferred to a separatory funnel. A few extractions of the aqueous layer with diethyl ether were made until the ether layer was discoloured, and collected in a Erlenmeyer, dried over NaS0₄, filtered to produce a crude red coloured solid product dried in vacuum (282.9 mg; η=94.2%), and further purified by Flash Column Chromatography, using mixtures of increasing polarity: Petroleum ether/ethyl acetate/methanol (62.5:35:2.5) and Petroleum ether/ethyl acetate/methanol (60:35:5), to give Rifabutinol lb (143.1 mg; η=47.6%). Rifabutinol lb :

^-N.M.R. (CDCI₃) δ(ppm) : 0.04 (3H, d, J₃4/26= 7.2Hz, Me- 34); 0.61 (3H, d, J_33/24= 6.8Hz, Me-33) ; 0.85 (3H, d, J₃ι_/20= 7.0Hz, Me-31); 0.93 (6H, d, Jιr/ιo= Ji2'/ιo= 6.4Hz, Me-11' e Me-12') ; 1.04 (3H, d, J_{32 22}= 7.0Hz, Me-32); 1.52 (1H, ddd, J_26/25=9.9Hz, J_26/27=2.5Hz, J_26/3₄=7.3. H-26) ; 1.67- 1.85 (4H, m, H-24/H-22/H-10' ) ; 1.94 (3H, s, Me-13) ; 2.00 (4H, m, CH₂-4'/CH₂-8' ) ; 2.06 (6H, s, Me-30 e Me-36) ; 2.22 (3H, s, Me-14) ; 2.23 (2H, d, J₉-_/ιo' = 6.8Hz, CH₂-9' ) ; 2.38 (1H, m, H-20) ; 2.69-2.79 (4H, m, CH₂-5'e CH₂-7'); 3.03 (1H, m, H-23); 3.06 (3H, s, Me-37); 3.48 (1H, m, H-27); 3.72 (1H, s, 21-OH); 3.79 (1H, bd, J₂ι_/20= 9.9Hz, H-21) ; 4.08 (1H, d, J_OH/₂₃= 4.5Hz, 23-OH) ; 4.95 (1H, dd, J_{28 27}= 4.2Hz/ J_28/29= 12.3Hz, H-28); 5.05 (1H, d, J_25/26= 10.1Hz, H- 25); 5.55 (1H, s, H-ll) ; 5.98 (1H, dd, J_29/28= 12.3Hz, J_{29 27}= 1.2Hz H-29) ; 6.15 (1H, dd, Jι_9/ι₈= 15.7Hz, Jι_{9 20}= 6.8Hz, H-19); 6.25 (1H, bd, Jι_7/ι₈= 10.8Hz, H-17); 6.43 (1H, dd, Ji_8/ι₇= 10.9Hz, Jι_8/ι₉= 15.8Hz, H-18) ; 6.80 (1H, s, 11-OH) ; 8.12 (1H, bs, NH' ) ; 8.33 (1H, s, NH) ; 13.70 (1H, s, 8-OH)

¹³C-N.M.R. (CDC1₃) δ(ppm) : 8.2 (C-14); 8.7 (C-33) ; 8.8 (C- 34); 11.2 (C-32) ; 17.7 (C-31); 20.5 (C-30) ; 20.8 (C- ll'/C-12') ; 20.9 (C-36) ; 25.1 (C-13) ; 25.8 (C-10' ) ; 33.3 (C-22) ; 36.3 (C-4')*; 36.4 (C-8')*; 37.2 (C-24); 37.4 (C- 20) ; 39.0 (C-26); 51.5 (C-5'/C-7'); 57.5 (C-37) ; 66.4 (C- 9'); 73.2 (C-21); 74.2 (C-25) ; 76.6 (C-ll) ; 77.2 (C-23) ; 77.5 (C-27); 92.9 (C-2'); 105.8 (C-3) ; 108.3 (C-9) ; 111.5 (C-28) ; 111.8 (C-12); 113.1 (C-7) ; 120.0 (C-5) ; 120.7 (C- 10); 124.0 (C-18) ; 130.3 (C-16) ; 133.7 (C-17); 140.8 (C- 2); 141.7 (C-19); 142.4 (C-29) ; 159.0 (C-4); 159.9 (C-6) ; 163.2 (C-8); 168.6 (C-15) ; 172.7 (C-35) ; 182.1 (C-l). * - interchangeable values

I.V. _fu_m (NaCl) ,v_mέx(cm^_1) : 3252 (N-H) ; 2964. 2821 (C-H) 1714 (C₃₅=0) ;1674 (Cι₅=0) ; 1645 (C₄=N) ; 1616. 1574 (C=C) 1505 δ(N-H);1417 (C₈-0) e δ(O-H); 1384 δ(C-H); 1344 (C-N) 1283 δ(O-H); 1262 (C₃₅-0-C₂₅) ; 1149 (C-N); 1063 (C-O) e (C₃₅-0-C₂₅) ; 974 γ(C-H); 769 (chromophore ' s vibration).

Mass/ESI (m/z) : 847 [M-H] ^•"; 755 [ (M-H) -CH₃C00H-CH₃0H] ^■"; 729 [M- iC₄H₉-N(CH₃)=CH₂)-H₂0] ^■" ; 545 [(M-H)-c] -^~;423[Ch- H] ^•"; 323[Ch-(iC₄H₉-N(CH₃)=CH₂) ] ^•"

Exact Mass (m/z)- C₄₆H_{64 4}0ιι (M)^" obtained: 848.45769/ (M) ^" caic = 848.45771.

1.2. Synthesis of Rif abutinol-N-oxide lib

To a round-bottomed flask with Rifabutinol lb (181.1 mg; 0.213 mmol) in dichloromethane (9.1 mL) , was added benzyl triethylammonium chloride (74.0 mg; 0.325 mmol), sodium hydrogenocarbonate (12.1 mg; 0.144 mmol), Oxone

(263.6 mg; 0.429 mmol) in acetone (1.8 mL; 24.5 mmol), distilled water (9.2 mL) and a Metrohm pH=7 buffer (12.7 mL) , stirring at room temperature. The reaction was followed by t.l.c, using petroleum ether/ethyl acetate/methanol (55:35:10) as eluant; 1 h 15 min after the beginning of the reaction it was progressively added

2.25 eq. more of Oxone (593.4 mg; 0.965 mmol) and buffer

(2.5 mL) to adjust the medium pH. The reaction was followed by t.l.c. and it was complete after 26 h 30 min. The flask content was transferred to a separatory funnel, and extracted with dichloromethane (2x25 mL) ; the combined organic layers were collected in a Erlenmeyer, transferred to a new separatory funnel, washed with distilled water and the resulting organic layers were collected and dried over anhydrous sodium sulfate. The resulting organic layers were filtered, the solvent evaporated at reduced pressure, and a crude red coloured solid produced was dried in vacuum (152.1 mg; η=82.4%) and further purified by Flash Column Chromatography, using mixtures of increasing polarity: Petroleum ether/ethyl acetate/methanol (65:25:10), Petroleum ether/ethyl acetate/methanol (55:35:10), Petroleum ether/ethyl acetate/methanol (50:35:15), Petroleum ether/ethyl acetate/methanol (45:30:25), ethyl acetate/methanol (40:60), ethyl acetate/methanol (30:70), ethyl acetate/methanol (20:80) and ethyl acetate/methanol

(10:90), to give Rifabutinol-N-oxide lib (94.2 mg; η=51.0%) . Rifabutinol-N-oxide lib:

^-N.M.R. (MeOD) δ(ppm) : -0.04 (3H, d, J_34/26= 6.6Hz, Me- 34) ; 0.56 (3H, d, J_{33 24}=6.6Hz, Me-33) ; 0.84 (3H, d, J₃ι_/20= 6.6Hz, Me-31) ; 0.95 (3H, d,J_{32 22}= 6.8Hz, Me-32) ; 1.13 (6H, d, Jιi'_/ιo= Ji_2'/ιo= 6.6Hz, Me-11' e Me-12' ) ; 1.21-1.28 (IH, m, H-26) ; 1.62 (IH, m, H-24) ; 1.72 (IH, m, H-22) ; 1.85 (3H, s, Me-13) ; 1.94 (3H, s, Me-36) ; 2.03 (3H, s, Me-30) ; 2.08 (3H, s, Me-14) ; 2.32 (IH, m, H-20) 2.40 (IH, m, H-

10') ; 2.98 (3H, s, Me-37) ; 3.04 (1. m, H-23) ; 3.32 (2H, m, CH₂-9' ) ; 3.38 (IH, m, H-27) 1.3-3.8 (m, CH₂-7'/ CH₂-5' /

CH₂-4'/ CH₂-8'); 3.85 (IH, bd, J₂ι_/20= 9.6Hz, H-21) ; 4.89

(IH, m, H-28) ; 5.18 (IH, d, J_25/26= 10.0Hz, H-25) ; 5.59 (IH, s, H-ll) ; 5.93 (IH, d, J_29/28= 12.4Hz, H-29) ; 6.02 (IH, dd, Ji₉ i₈= 15.6Hz, Jι_{9 20}= 6.8Hz, H-19) ; 6.27 (IH, bd, Ji7_/ιβ= 10.4Hz, H-17); 6.59 (IH, dd, Jι_8/ι₇= 11.0Hz, Jι_8/ιg= 15.6Hz, H-18) ; 1³C-N.M.R. (MeOD) δ(ppm) : 8.4 (C-14); 9.5 (C-33) ; 9.7 (C- 34) ; 11.3 (C-32) ; 18.0 (C-31); 20.8 (C-30) ; 21.0 (C-36) ; 23.5 (C-ll'/C-12' ) ; 24.6 (C-10' ) ; 25.3 (C-13); 31.7 (C- 4' /C-8') ; 34.3 (C-22); 39.0 (C-20/C-24); 40.7 (C-26) ; 57.5 (C-37) ; 63.7 (C-5' ) ; 64.0 (C-7' ) ; 74.6 (C-21) ; 74.9 (C-25) ; 77.6 (C-ll); 78.1 (C-23) ; 78.2 (C-27); 79.5 (C- 9' ); 91.5 (C-2' ) ; 105.4 (C-3) ; 110.2 (C-9) ; 113.0 (C-12) 114.2 (C-7) ; 114.8 (C-28) ; 121.3 (C-5); 121.9 (C-10) 127.1 (C-18); 132.0 (C-16) ; 134.6 (C-17) ; 140.9 (C-19) 143.4 (C-29) ; 145.1 (C-2); 160.8 (C-4) ; 161.9 (C-6) ; 164.1 (C-8) ; 169.3 (C-15) ; 172.8 (C-35) ; 184.9(C-1) . I.V. _n, (NaCl) v_mέx(cm^_1): 3418 (N-H) ; 3252 (O-H) ; 2964.

2938. 2880 (C-H) ; 1730 (C_u=0) ; 1673 (Cι₅=0) ; 1645 (C₄=N) ;

1633 (Cι=0) ; 1616. 1605. 1582 (C=C) ; 1520 δ(N-H); 1418

(C₈-0) e δ(O-H); 1374 δ(C-H); 1285 δ(O-H); 1256 (C₃₅-0-C₂₅) ;

1149 (C-N); 1078 (N-O); 1062 (C-O) e (C₃₅-0-C₂₅) ; 974 γ(C-

H) ; 768 (chromophore's vibration) .

Mass/ESI (m/z): 864 [M] ^•"; 848 [M-O] '^"; 820 [ (M-H) -C₃H₈] ^•";

735 [ (M-H) -iC₄H₉N(CH₃)=CH₂] ^•"; 703 [ (Ch-H) - (iC₄H₉N (CH₃) =CH₂) -

CH₃OH]-^~; 543 [ (M-3H) -O-c] ^•"; 422 [ (Ch-2H) -O] ^•";

Exact Mass (m/z ) C₄₆H₆₄N₄Oι₂ (M) ^" obtained : 848 . 45779 (M-O) ^" .

Example 2 . Synthesis of 25-hydroxy-Rif abutin N-oxide l ie

2 . 1 . Synthesis of 25-hydroxy-Rif abutin Ic

To a solution of Rifabutin la (300.1 mg; 0.355 mmol) in methanol (15 mL) in a round-bottomed flask, a solution of NaOH (0.184M in MeOH/H₂0 1:1) was added followed by an aqueous solution of ZnCl₂ (48.7 mg; 0.357 mmol). The extraction mixture was stirred at room temperature and followed by t.l.c, and ^ eq. more of ZnCl₂ (24.4 mg; 0.179 mmol) was added and NaOH (27.2 mg) until complete conversion of starting material. After completion of the reaction (ca. 6 h 30 min) the mixture was extracted with ethyl acetate (5x25 mL) and the organic layers washed with water (30 mL) . Drying agent (MgS0₄) was added, the solution filtered, evaporated in a rotary evaporator and dried in vacuum, to give a chromatographic homogeneous 25-hydroxy-Rifabutin Ic, as a purple coloured solid (284.4 mg; η=99.8%) .

25-hydroxy-Rifabutin Ic:

^-N.M.R. (CDC1₃) δ(ppm) : -0.12 (3H, d, J_34/26= 6.8Hz, Me- 34) ; 0.56 (3H, d, J₃3_/24= 6.8Hz, Me-33) ; 0.84 (3H, d, J₃ι ₂o= 6.8Hz, Me-31) ; 0.95 (6H, d, Jιr_/ιo= Ji2'_/ιo= 6.8Hz, Me-11' e Me-12'); 1.02 (IH, m, H-24); 1.09 (3H, d, J_32/22= 6.8Hz, Me-32); 1.74 (3H, s, Me-13) ; 1.7-1.8 (IH, m, H-22) ; 1.8- 1.9 (5H, m, H-10'/H-26/CH₂-8'*) ; 2.05 (3H, bs, Me-30) ; 2.0-2.1 (2H, m, CH₂-4')*; 2.30 (3H, s, Me-14); 2.32 (2H, m, CH₂-9' ) ; 2.43 (IH, m, J_20/3ι=6.8Hz, H-20) ; 2.67 (2H, m, CH₂-5')*; 2.96-2.99 (4H, m, 21-OH/23-OH/CH₂-7' *) ; 3.18 (3H, s, Me-37); 3.32 (IH, bd, J_25/26= 10.4Hz, H-25) ; 3.40 (IH, dd, J_{27 26}=3.8Hz/ J_27/28=10.1Hz, H-27) ; 3.55 (IH, t, J₂₃/_23OH=8.3HZ, H-23) ; 3.72 (IH, d, J₂ι_/20= 9.7Hz, H-21) ; 4.20 (IH, s, 25-OH) ; 5.20 (IH, dd, J_{28 27}= 10.3Hz/J_{28 29}= 12.6Hz, H-28); 5.91 (IH, dd, Jι_9/ι₈= 14.9Hz, Jι_9/20= 6.0Hz, H-19) ; 6.24-6.32 (2H, m, H-18/H-17) ; 6.36 (IH, bd, J_{29 28}= 12.8Hz, H-29); 8.29 (IH, s, NH) ; 9.71 (IH, s, NH' ) ; 14.60 (IH, s, 8-OH) . 1³C-R.M.C. (CDCI3) δ(ppm) : 7.7 (C-14) ; 8.3 (C-33) ; 10.8 (C- 32) ; 12.1 (C-34); 17.1 (C-31) ; 20.0 (C-30); 20.8 (C- ll'/C-12'); 22.8 (C-13) ; 25.8 (C-10'); 32.7 (C-22) ; 35.2 (C-4')*; 36,0 (C-8')*; 37.7 (C-26) ; 38.8 (C-24); 39.4 (C- 20); 51.5 (C-5' /C-7') ; 56.0 (C-37) ; 66.3 (C-9' ) ; 70.8 (C- 25); 71.5 (C-21) ; 76.3 (C-23) ; 85.4 (C-27); 94.7 (C-2' ) ; 104.2 (C-3) ; 108.0 (C-12) ; 109.4 (C-10) ; 111.4 (C-9) ; 114.6 (C-7/C-28); 123.2 (C-18) ; 124.8 (C-5) ; 132.4 (C- 16); 132.8 (C-17); 140.9 (C-19) ; 141.5 (C-2) ; 147.4 (C-29) ; 154.9 (C-4); 168.1 (C-8); 168.2 (C-15) ; 170.9 (C- 6) ; 180.3(C-1) ; 192.0 (C-ll) . * - interchangeable values

I.V. _fu_n, (NaCl) v_mέx(cm^_1) : 3493 (O-H) ; 3360 (N-H) ; 2960. 2824 (C-H) ; 1728 (Cu=0) ; 1674 (Cι₅=0) ; 1646 (C₄=N) ; 1602. 1562 (C=C); 1515 δ(N-H); 1455. 1422 (C₈-0) e δ(O-H); 1372 δ(C-H) ; 1342 (C-N) ; 1294 δ(O-H) ; 1157 (C-N); 1071 (C-O); 981 γ(C-H); 766 (chromophore ' s vibration) .

Mass/ESI (m/z) : 804 [M] ^•" ; 771 [(M-H) - CH₃OH] ^•" ; 753 [(M-H) - CH₃OH - H₂0]-^"; 571 [(M-H) - b] '^"; 543 [(M-H) - c]-^"; 421 [Ch-H] ^■"; 322 [Ch - (iC H₉-N (CH₃) =CH₂) ] '^" Exact Mass (m/z) C ₄H60N₄O₁0 (M-H)^" obtained: 803. 2332/ (M- H)^" _caι = 803.423670.

2.2. Synthesis of 25-hydroxy-Rifabutin-N-oxide lie

25-hydroxy-Rifabutin Ic 25-hydroxy-Rifabutin N-oxide lie M=804 , 98g/mol M=820, 98g/mol

In a round-bottomed flask containing 25-hydroxy- Rifabutin Ic (150.0 mg; 0.186 mmol) in dichloromethane (7.9 mL) the following reactants were added with stirring at room temperature: benzyl triethylammonium chloride (70.0 mg; 0.307 mmol); NaHC0₃ (13.5 mg; 0.161 mmol), distilled water (7.9 mL) , pH=7 buffer (10 mL) and Oxone (179.1 mg; 0.291 mmol). The Oxone was slowly added to the reactional mixture during a period ca. 3 h 15 min of the reaction.

The reaction was followed by t.l.c. (petroleum ether/ethyl acetate/methanol - 45:35:20); after ca. 19 h of reaction, more Oxone was added (282.0 mg; 0.458 mmol), as well as pH=7 buffer in order to maintain the pH ca . 7. The reaction was complete after ca. 28 h 30 min. The aqueous and organic layers were separated and the aqueous layer extracted with dichloromethane. The collected organic layers were washed with water, dried over anhydrous sodium sulfate, filtered and the solvent evaporated at reduced pressure. A crude product was produced after drying in vacuum (102.5 mg; η=67.0%), which was further purified by Flash column chromatography, using reverse phase silica with the petroleum ether/methanol (2:98) eluant, to give 25- hydroxy-Rifabutin-N-oxide lie (29.3 mg; η=19.1%).

25-hydroxy-Rifabutin-N-oxide lie:

^-N.M.R. (CDC1₃) δ(ppm) : -0.16 (3H, d, J_34/26= 6.8Hz, Me-

34) ; 0.53 (3H, d, J_33/24= 6.8Hz, Me-33) ; 0.82 (3H, d, J₃ι_/2o= 6.8Hz, Me-31); 1.16 (6H, d, Jιr_/ιo= Jι_2'/ιo= 6.8Hz,

Me-11' e Me-12' ) ; 1.04 (3H, d, J_32/22= 6.8Hz, Me-32);

1.16 (IH, m, H-24) ; 1.24 (IH, m, H-10' ) ; 1.64 (IH, m, H-

26) ; 1.73 (3H, s, Me-13) ; 1.7-1.8 (IH, m, H-22); 2.06

(3H, bs, Me-30) ; 2.28 (3H, s, Me-14); 2.36 (IH, m, H- 20) ; 3.16 (3H, s, Me-37); 3.32 (2H, m, CH₂-9' ) ; 3.3-3.6 (3H, m, H-23/H-25/H-27) ; 3.73 (IH, d, J₂ι_/20= 9.3Hz, H-21) ; 4.20 (IH, s, 25-OH) ; 5.11 (IH, dd, J_{28 27}= 8.7Hz/ J_{28 29}= 12.4Hz, H-28) ; 5.91 (IH, dd, Jι_9/ι₈= 14.6Hz, Jι_{9 20}= 6.2Hz, H-19); 6.22 (IH, bd, J_{29 28}= 12.7Hz, H-29) ; 6.29-6.36 (2H, m, H-18/H-17) .

¹³C-N.M.R. (CDC1₃) δ(ppm) : 7.7 (C-14) ; 8.4 (C-33) ; 11.0 (C- 32); 11.2 (C-34); 17.4 (C-31) ; 20.2 (C-30); 22.7 (C-13) ; 23.4 (C-ll'/C-12') ; 23.6 (C-10'); 32.8 (C-22) ; 38.6 (C- 24) ; 39.1 (C-20); 56.5 (C-37); 62.8 (C-5' /C-7' ) ; 70.4 (C- 25) ; 72.0 (C-21) ; 77.3 (C-23) ; 79.2 (C-9') ; 91.6 (C-2') ; 105.1 (C-3) ; 107.8 (C-12) ; 109.2 (C-10); 111.6 (C-9); 114.7 (C-7/C-28) ; 123.9 (C-18) ; 124.8 (C-5) ; 132.2 (C- 16) ; 132.7 (C-17) ; 140.5 (C-19) ; 156.1 (C-4); 168.1 (C- 8) ; 168.3 (C-15) ; 171.2 (C-6) ; 181.8 (C-l) ; 192.6 (C-ll) . I.V. _fii_m (NaCl) v_mέx(cm^"1) : 3402 (O-H) ; 2965. 2934 (C-H) ; 1724 (Cu=0); 1672 (C₁₅=0) ; 1602. 1568 (C=C) ; 1521 δ(N-H) ; 1422 (C₈-0) e δ(O-H) ; 1374 δ(C-H); 1328 (C-N) ; 1295 δ(0- H) ; 1160 (C-N) ; 1083 (N-O); 1059 (C-O); 978 γ(C-H) ; 770 (chromophore' s vibration)

Mass/ESI (m/z) : 819 [M-H]-^" ; 803 [ (M-H) -O] '^"; 323 [Ch- iC₄H₉N(CH₃)=CH₂] ^•"] .

Example 3. Synthesis of Rifabutin-N-oxide Ila

To a round-bottomed flask containing Rifabutin la (100.0 mg; 0.118 mmol) in dichloromethane (4 mL) , was added benzyl triethylammonium chloride (40.9 mg; 0.180 mmol), sodium hydrogenocarbonate (6.7 mg; 0.080 mmol); Oxone (145.5 mg; 0.237 mmol) in acetone (1.0 mL) , distilled water (5 mL) and a Metrohm pH=7 buffer (7.0 mL) , in permanent agitation and at room temperature. The reaction was followed by t.l.c, using Petroleum ether/ethyl acetate/methanol (55:35:10) as eluant; after ca . 17 h it was progressively added 1 eq. more of Oxone (146.0 mg; 0.237 mmol) and buffer (0.5 mL) to adjust the pH of the medium. The reaction was complete after ca. 26 h 30 min. The flask content was transferred to a separatory funnel, and 3 extractions with dichloromethane were followed by 2 washings with water. The combined organic layers were collected in an Erlenmeyer, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated at reduced pressure, and the crude red coloured solid obtained was dried in vacuum (152.1 mg; η=82.4%) and purified by Flash Column Chromatography, using as eluant: Petroleum ether/ethyl acetate/methanol (55:35:10), giving Rifabutin-N-oxide Ila (94.2 mg; η=51.0%) .

Rif abutin-N-oxide Ila:

^-N.M.R. (CDC1₃) δ(ppm) : -0.08 (3H, d, J_34/26= 7.0Hz, Me- 34); 0.61 (3H, d, J_33/24=6.9Hz, Me-33) ; 0.86 (3H, d, J₃ι ₂₀= 6.9Hz, Me-31); 1.03 (3H, d, J_32/22= 6.9Hz, Me-32); 1.1-1.7 (m, CH₂-4' /CH₂-8' ) 1.21 (6H, d, J_n-_/10= Jι₂'_/ιo= 6.6Hz, Me- 11' e Me-12') ; 1.42 (IH, m, H-24) ; 1.68-1.82 (2H, m, H- 22/H-26) ; 1.76 (3H, s, Me-13) ; 1.98 (3H, s, Me-36) ; 2.06 (3H, s, Me-30) ; 2.35 (3H, s, Me-14) ; 2.30-2.41 (IH, m,

H-20); 2.50 (IH, m, H-10' ) ; 3.01 (1. m, H-23) ; 3.09 (3H, s, Me-37); 3.34 (IH, m, H-27); 3.40 (IH, bs, 21-OH) ; 3.48 (2H, m, CH₂-9' ) ; 3.58 (IH, d, 23-OH) ; 3.69 (IH, bd, J₂ι_/20= 9.9Hz, H-21) ; 3.2-4.2 (m, CH₂-7'/ CH₂-5' ) ; 4.81 (IH, bd, J_25/26=9.9Hz, H-25) ; 5.12 (IH, dd, J_{28 27}= 7.2Hz /J_28/29= 12.3Hz, H-28) ; ) ; 6.00 (IH, dd, Jι₉ ι₈= 15.3Hz, Jι_9/20= 6.9Hz, H-19) ; 6.14 (IH, d, J_29/28= 12.6Hz, H-29) ; 6.29 (IH, bd, Ji7_/i8= 10.2Hz, H-17); 6.39 (IH, dd, Jι_8/ι₇= 10.2Hz, 15.3Hz, H-18); 8.43 (IH, s, NH) ; 8.74 (IH, s, NH' ) ; 14.54 (IH, bs, 8-OH) . 1³C-N.M.R. (MeOD) δ(ppm) : 7.6 (C-14) ; 8.8 (C-33) ; 10.7 (C- 34); 11.1 (C-32) ; 17.3 (C-31); 20.3 (C-30); 21.0 (C-36) ; 21.8 (C-13) ; 23.2 (C-ll' /C-12' ) ; 23.7 (C-10' ) ; 31.6 (C- 4') ; 31.9 (C-8'); 32.9 (C-22) ; 37.6 (C-24/C-26) ; 38.4 (C- 20) ; 56.9 (C-37); 62.8 (C-5' /C-7' ) ; 72.5 (C-21) ; 73.0 (C- 25) ; 76.9 (C-23) ; 78.4 (C-9' ) ; 80.5 (C-27); 91.3 (C-2') 105.4 (C-3); 107.3 (C-12); 108.8 (C-10); 111.8 (C-9) 114.8 (C-7) ; 115.4 (C-28) ; 124.2 (C-18) ; 124.7 (C-5) 131.0 (C-16) ; 133.2 (C-17) ; 140.8 (C-19) ; 141.8 (C-2) 144.0 (C-29) ; 156.2 (C-4); 168.1 (C-8) ; 168.3 (C-15) 171.6 (C-6); 172.1 (C-35) ; 181.9(C-1) .

I.V. film (NaCl) v_mόx(cm^_1) : 3423 (O-H) ; 2965. 2931 (C-H) ; 1725 (Cu=0) ; 1672 (C₁₅=0) ; 1602. 1562 (C=C) ; 1524 δ(N-H) ; 1422 (Cβ-O) e δ(O-H) ; 1376 δ(C-H) ; 1295 δ(O-H) ; 1248 (C₃₅- 0-C₂₅) ; 1161 (C-N); 1085 (N-O); 1062 (C-O) e (C₃₅-0-C₂₅) ; 976 γ(C-H) ; 770 (chromophore ' s vibration) .

Mass/ESI (m/z) : 861 [M-H]"^" ; 845 [(M-H)-O]"^" ; 785 [ (M-H) - O-CH3COOH] '^"; 753 [ (M-H) -O-CH₃COOH-CH₃OH] ^■"; 709 [(M-H)-O- CH₃COOH-CH₃OH-C₃H₈] ^•"; 651 [ (M-H) -0-CH₃COOH-CH₃OH-C₃H₈-

C₃H₈N]-^~; 571 [ (M-3H) -O-b) ] '^"; 543 [ (M-3H) - c] ^•"; 421 [Ch- H] ^•";

Exact Mass (m/z) - C₄₆H₆₂N₄θι₂ (M(-H)-O)^" obtained: 846.44133 ((M-H)-O)^" Example 4. Synthesis of N' -acetyl-Rifabutin Ilia

To a round-bottomed flask containing a solution of Rifabutin la (500 mg; 0.59 mmol) in tetrahydrofuran (15 mL) , triethylamine (248 μL; 1.9 mmol) was added dropwise, at 0°C, with stirring, followed by an acetyl chloride solution (129 μL; 1.9 mmol) in tetrahydrofuran (10 mL) , and the resulting mixture was left stirred till it reached room temperature. The reaction was complete 5 minutes later, the solvent removed under nitrogen flux, and the diluted mixture in ethyl acetate, filtered and evaporated to dryness in a rotary evaporator and at high vacuum, to originate N' -Acetyl-Rifabutin Ilia, as a chromatographic homogeneous orange-coloured solid in 95% yield.

N' -acetyl-Rifabutin Ilia: ^-N.M.R. (CDC1₃) δ(ppm): -0.07 (3H, bs, Me-34); 0.59 (3H, d, J_33/24=6.0Hz, Me-33); 0.86 (3H, d, J₃ι_/20= 6.0Hz, Me-31); 0.99 (6H, d, J_UM2 IO'= 6.3Hz, Me-11' e Me-12' ) ; 1.03 (3H, d, J_32/22= 7.5 Hz, Me-32); 1.6 (IH, m, H-24); 1.6 (IH, m, H-26); 1.77 (3H, s, Me-13) ; 1.8 (IH, m, H-22) ; 1.91 (IH, , Jιi_',i₂Vio-= 6.4Hz, H-10' ) ; 1.7* (2H, m, CH₂-8') ; 1.7*

(2H, m, CH₂-4') ; 2.02 (3H, s, Me-36) ; 2.05 (3H, s, Me-30) ;

2.44 (3H, bs, Me-39); 2.4 (2H, m, CH₂-9') ; 2.4 (IH, m, H- 20); 2.44 (3H, s, Me-14); 2.9* (2H, m, CH₂-5') ; 2.9* (2H, m, CH₂-7^'); 2.99 (IH, m, H-23) ; 3.01 (IH, bs, H-21); 3.09

(3H, s, Me-37) ; 3.41 (IH, m, H-27); 3.77 (IH, bs, 23-OH)

3.87 (IH, d, 21-OH); 4.97 (IH, bd, J_{25 26}=10.2Hz, H-25)

5.19 (IH, bs, H-28); 6.07 (IH, bd, Jι_{9 20}= 6.3Hz, H-19) 6.09 (IH, m, H-29) ; 6.23 (IH, bd,

10.8Hz, H-17)

6.72 (IH, m, H-18); 8.21 (IH, s, NH) ; 13.89 (IH, s, 8-

OH) .

13 C-N.M.R. (CDC1₃) δ(ppm) : 7.36 (C-14); 8.44 (C-33) ; 9.44

(C-34) 10.78 (C-32); 17.39 (C-31) ; 20.45 (C-30); 20.71 ((CC--3366)) ; 20.87 (C-ll^'); 20.87 (C-12') ; 21.22 (C-13) ; 25.24 (C-39) 25.88 (C-10'); 32.97 (C-22) ; 35.34 (C-4') ; 35.34 (C-8') 37.30 (C-24); 38.16 (C-26) ; 38.23 (C-20); 51.13* (C-7') 51.29* (C-5') ; 57.20 (C-37) ; 65.92 (C-9') ; 72.90 (C-21) 73.81 (C-25); 77.11 (C-23) ; 79.1 (C-27) ; 96.38 ((CC--22'')); 106.86 (C-3) ; 108.93 (C-12); 110.97 (C-10)

113.34 (C-9); 113.77 (C-7) ; 116.73 (C-28) ; 125.67 (C-18)

126.41 (C-5); 129.5 (C-16) ; 134.95 (C-17) ; 140.77 (C-19)

140.77 (C-2); 142.37 (C-29) ; 153.92 (C-4) ; 166.58 (C-8)

166.66 (C-38); 167.61 (C-15) ; 172.08 (C-6) ; 177.22(C-35) 186.86 (C-l); 191.97 (C-ll) .

Mass/FAB (m/z) : 888 [M]⁺

Example 5. Synthesis of N' -acetyl-Rifabutinol Illb

To a round-bottomed flask containing a solution of Rifabutinol lb (300 mg; 0.35 mmol) in tetrahydrofuran (9 mL) , triethylamine (149 μL; 1.08 mmol) was added dropwise, at 0°C, with stirring, followed by an acetyl chloride solution (77.1 μL; 1.08 mmol) in tetrahydrofuran (6 mL) , and the resulting mixture was left stirred till it reached room temperature. The reaction was completed 5 minutes later, the solvent removed under nitrogen flux, and the diluted mixture in ethyl acetate, filtered and evaporated to dryness in a rotary evaporator and at high vacuum, to originate the chromatographic homogeneous N'- Acetyl-Rifabutinol Illb, in a 95% yield, identified by ¹ti- N.M.R., ¹³C-N.M.R. and Mass/FAB (m/z): 888 [M]⁺.

Biological activity screening tests

Microbiology Assay for the Evaluation of Mycobacterium avium 1581 Susceptibility to Rifabutin and Derivatives .

To the culture medium agar 7H10 enriched with OADC from Difco the molecules derived from Rifabutin la of this invention were added at the final concentrations 0.1. 0.2 and 0.4 μg/mL and also Rifabutin la was added at final concentrations 0.1 and 0.2 μg/mL. Plates containing Rifabutin la or its derivatives of this invention were inoculated with a strain of M. avium 1581 at dilutions 10⁵ and 10^"4. Plates without antibiotic were inoculated with a strain of M. avium at dilutions 10^"7' 10^"6, 10^"5 e 10^~4. Plates were then incubated 15 days in an oven at 37°C in the presence of 5% C0₂. By comparing with control plates (inoculated with the strain M. avium but in the absence of antibiotic) the concentration of the Rifabutin derivative which is related with the strain whose colonies number is smaller than 1% in relation to the colonies number in control plates. The results obtained while testing the molecules of this invention are summarized in Table 1. Rifabutin la produced an inhibition greater than 99% in the tested concentrations (0.15 and 0.2 μg/mL). Rifabutinol lb and 25-hydroxy-Rifabutin Ic showed inibitions from 86 to 91%, respectively, at a concentration 0.2 μg/mL. The N-oxide of Rifabutin Ila, and N-oxide of 25-hydroxy-rifabutin lie were inactives at the tested concentrations (0.2 and 0.4 μg/mL). N' -acetyl-rifabutinol Illb and N-oxide of Rifabutinol lib showed inhibitions between 44 and 54% at concentration 0.2 μg/mL and the derivative N'-acetyl- rifabutin Ilia presented an activity similar to Rifabutin la. Table 1- Results from assays with Myc . avium 1581

Evaluation of the activity against Gram-positive and Gram-negative bacteria MATERIALS AND METHODS

Compounds tested: N-oxide of Rifabutin Ila N-oxide of Rifabutinol lib Rifabutin la

N' -acetyl-Rifabutin Ilia N' -acetyl-rifabutinol Illb Rifampicin

Microorganisms

Staphylococcus aureus CCMI 335; Streptococcus faecalis CCMI 338. Escherichia coli CCMI 270. Pseudomonas aeruginosa CCMI 331. Salmonella enteri tidis CCMI 859.

Culture Media

S . aureus e S . faecium were incubated in brain and heart infusion (BHI) (Merck) . Escherichia coli , Pseudomonas aeruginosa and Salmonella enteri tidis were incubated in nutritive agar (Na) (Oxoid) .

General Methods Qualitative Microbiological Assays

Disc diffusion disc method was used according to the procedure described by Hong and Song, 2001 (see: Hong-Xi and Song, F. Lee, (2001) "Activity of plant flavonoids against antibiotic-resistant bacteria", Phytot er. Res . , 15. 39-43) as a diagnostic assay for antibacterial activity. Petri dishes were prepared with an adequate culture medium and containing the microorganism beeing tested at the concentration 10⁸ cfu mL^-1. The compounds were dissolved in dimethylsulfoxide (DMSO) at the concentration lmg.mL^"1 and applied in Oxoid discs at the ratio 20 μg/disc After evaporation of the solvent, the compounds of the invention were applied over each Petri dish medium prepared as indicated above and incubated at 37 °C, followed by determination of the diameters of the inhibition zones 24h later. Discs containing DMSO, without any compound of the invention added, were used as control, and did not show any inhibition of colonies. Rifampicin (Sigma) was used as Reference for the evaluation of the activity of the compounds to be tested against Gram-positive and Gram-negative bacteria.

Quantitative Microbiological Assays

The Minimum Inhibitory Concentration (MIC) of the active compounds provided by the present invention was determined through the medium dilution method as described by Muroi and Kubo, 1996 (see: Muroi, H and Kubo, I, "Antibacterial activity of anacardic acid and totarol, alone and in combination with methicillin, against methicillin-resistant Staphylococcus aureus" , J. Appl . Bacteriol . 80. 387-394 (1996)). After incubation during 24h, the bacteria growth was examined and the value for the MIC defined as the minimum concentration of the tested compound, from which there is a null result after 24h incubation at 37°C. The minimum inhibitory concentration of each compound derived from Rifabutin provided by the present invention was determined at least in duplicate assays.

Bacteriostatic and Bactericide Activities

From the samples used for the MIC determination, where there was not consequently any observed bacteria growth, Petri dishes were inoculated with the strains in a BHI agar medium, and they were incubated for 24h at 37°C; after this period the plates were observed. Those where bacteria growth was observed were considered as bacteriostatic and conversely those where the bacteria growth was absent corresponded to the bactericide activities . RESULTS

The results from the evaluation of the antibacterial activity for compounds derived from Rifabutin provided this invention and tested against S. aureus, S. faecalis, E. coli, P. aeruginosa e S. enteritidis using the diffusion disc method are presented in Table 2.

Tabela 2: Antibacterial Activity of Test Compounds determined by the Method of Diffusion with Discs

Compounds S. S. E. P. S.

(lmg/mL) aureus faecalis coli aeruginosa enteretidis

Ila 22 15 10 ± 8 lib 20 13 ± - ± la 26 18 15 7 14

Ila + la 22 16 8 8 10 lib + la 23 18 8 6 10

Ila + 21 17 8 10 11

Rifampicin lib + 20 16 6 ± 9

Rifampicin

He + 24 19 10 9 13

Rifampicin

Ilia 30 16 25 16 19

Illb 25 10 - - -

Rifampicin 25 20 7 7 12

±: reduction of microorganism growth The N-oxide of Rifabutin Ila inhibited the development of Gram-positive bacteria, and also the growth of Gram-negative bacteria, E. coli e S . enteri tidis . The N-oxide of Rifabutinol lib and the N'- Acetyl-Rifabutinol Illb did not promote any inhibition of the growth of Gram-negative bacteria. The results suggested that N-oxide of Rifabutinol lib is especially active against Gram-positive bacteria. This compound did not show activity against P. aeruginosa and only partially inhibited the growth of E. coli and S . enteri tidis . N ' -Acetyl-Rifabutin Ilia as well as Rifabutin la inhibited the growth of all microorganisms tested, presenting large activity spectra. None of the mixtures of the compounds tested showed any synergistic effect.

The compounds provided by the present invention which showed any antibacterial activity using the diffusion disc method were also tested by the liquid medium method using successive dilutions, for the determination of the minimum inhibitory concentration (MIC) .

The results on the MICs for Rifabutin and derivatives are presented in Table 3. Table 3 : Determination of MICs for Rifabutin la and some derivatives

MIC (μg . mL^-1 )

Compounds S. S. E. P. S. aureus faecalis coli aeruginosa enteretidis

Rifabutin

N-oxide Ila 0.1^* 5^* 5" n.t. 10^* Rifabutinol -

N-oxide lib 0.1* 2.5^* n.t. - n.t. Rifabutin la 0.003* 0.25^* 1^* 0.6* 1.0* N' -Acetyl- Rifabutin 0.006* 0.25^* 1^* 0.6^* 1.0^* Ilia

N'-Acetyl- rifabutinol 0.03* 1.0^* n.t. - n.t. Illb

Rifampicin <0.001 0.06^* 1^* 2.5* 1.0^* n.t.: not tested *: bactericide activity; ^* : bacteriostatic activity.

The N-oxide of Rifabutin Ila presented a weak activity against Gram-negative bacteria, with MIC values much higher (5 and 10 μg mL^-1) for E. coli e S . enteri tidis than Rifabutin la and N ' -Acetil-Rifabutin Ilia. At the same MIC, Rifabutin la is bactericide against S . enteri tidis while N' -Acetyl-Rifabutin Ilia and rifampicin are bacteriostatic compounds. Rifabutin la and N' -Acetyl-Rifabutin Ilia showed a more efficient inhibition of the growing of P. aeruginosa than rifampicin, presenting MIC values of 6 μg mL-¹. Rifampicin demonstrated to be more active against Gram-positive bacteria than Rifabutin la and N ' -Acetyl-Rifabutin Ilia. The bacteriostatic compounds are bactericides at superior concentrations.

Claims

1. A compound of general formula I,

33 32 31

wherein :

R³ is oxygen or hydroxyl;

R⁴ is a nitrogen atom or an N-oxide group, and their pharmaceutically acceptable salts and solvates, including hydrates.

2. A compound according to claim 1 wherein R¹ is an acetyl group and R⁴ is a nitrogen atom.

3. A compound according to claim 1 wherein R¹ is an acyl group R² is an hydrogen or optionally an alkyl or acyl group and R⁴ is N-oxide.

4. A process for preparing compounds according to claims 1-3 wherein R¹ is an acyl group introduced in Rifabutin la, by reaction of an acylating agent in the presence of basic catalyst.

5. A process for preparing a compound according to claims 1 and 2 wherein R¹ is an acyl group introduced in Rifabutin la, by reaction of acetyl chloride in the presence of triethylamine in the temperature range between 0 and 30°C.

6. A process for preparing a compound according to claims 1 to 3 wherein R⁴ is introduced in Rifabutin la by reaction with a proper oxidative reagent able to selectively form an N-oxide.

7. A process for preparing a compound according to claims 1 to 3 wherein the oxidative reagent is

Oxone in buffered reaction medium.

8. A pharmaceutical composition containing as active ingredient a compound according to claims 1, 2 or 3, in a mixture with a pharmaceutical acceptable carrier.

9. The use of compounds according to claims 1, 2 or 3 in the preparation of a pharmaceutical drug directed to the treatment of infectious related to the presence of microorganisms susceptible to any compound of this invention.

10. A pharmaceutical composition to the treatment of bacterial infection in a mammal, that comprises the adminsitration to the mammal of a therapeutical effective quantity of a compound of general formula I according to claim 1 and a pharmaceutical acceptable carrier.

11. A method for the treatment of a bacterial infection in a mammal that comprises the administration of a therapeutical effective quantity of any compound according to claim 1 to 3 to the refered animal.

12. The use of compounds according to claims 1, 2, or 3 for the preparation of a composition to be administered to an animal as growth promoter.

13. The use of compounds according to claims 1 , 2 or 3, in the human Acquired Immunodeficiency Syndrome prophylaxis.