WO2006092822A2

WO2006092822A2 - 4-fluorophenyl-derivatives of 1-{[1,5 -bis (4-chloro phenyl) -2-methyl-1h-pyrrol-3-yl]methyl} -4- methylpi pεrazine, synthesis process and uses thereof

Info

Publication number: WO2006092822A2
Application number: PCT/IT2006/000131
Authority: WO
Inventors: Mariangela Biava; Fabrizio Manetti; Delia Deidda; Raffaello Pompei; Maurizio Botta; Giulio Cesare Porretta
Original assignee: Università Degli Studi Di Siena
Priority date: 2005-03-04
Filing date: 2006-03-03
Publication date: 2006-09-08
Also published as: EP2172451A2; WO2006092822A3; US7910581B2; US8012970B2; EP2172451A3; ITRM20050094A1; US20110144109A1; EP1866283A2; US20090281094A1

Abstract

The present invention concerns pyrrole compounds, derivatives of 1-{[1,5-bis(4- chlorophenyl)-2-methyl-lH-pyrrol-3-yl]methyl}-4-methylpiperazine (BM212). The invention concerns the use of the described compounds as antitubercular agents having high activity and low toxicity and process to obtain intermediates and final compounds.

Description

Derivatives of 1-{[1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl]methyl}-4- methylpiperazine, synthesis process and uses thereof

FIELD OF THE INVENTION The present invention concerns novel pyrrole compounds, derivatives of l-{[l,5-bis(4- chlorophenyl)-2-methyl-1H-pyrrol-3 -yljmethyl} -4-methylρiperazine (BM212). The invention concerns the use of the described compounds as antitubercular agents and a process to obtain intermediates and final compounds. The compounds of the invention are found to be more active and much less toxic than previously known compounds.

STATE OF THE ART

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) responsible for a high death-rate in both industrialized and developing countries. According to a recent report compiled by the World Health Organisation (WHO), the total number of new cases of TB in 2004 has risen to 9 million worldwide (Duncan et al. 2004). This is particularly alarming considering that these cases represent only 32% of the actual incidence.

The recent recrudescence of TB, due in particular to the increased incidence of the M. avium complex (MAC) infection in HIV-infected individuals, has prompted a vigorous search for new drugs for the treatment of the disease. In fact, the progressive immunological deterioration associated with AIDS is often accompanied by opportunistic infections causing TB (M tuberculosis), a non-TB (M avium) mycobacterial disease and mycotic infections caused by Candida albicans and Cryptococcus neoformans. Treatment of these infections, along with other opportunistic infections which cause the majority of all AIDS-related deaths, is often complicated by patient intolerance to the drugs employed or pathogen resistance to conventional drug therapy.

Drugs currently used to treat TB are Isoniazid (INH), Rifampicin (RIF), Pyrazinamide (PZA), Ethambutol (EMB), Streptomycin (SM), Cycloserin, para-aminosalicylic acid (PAS). Moreover, for most of them, the mechanism of action is known. Indeed, INH and EMB inhibit mycolic acid biosynthesis, a fundamental component of the mycobacterial cell wall, acting at different steps of its synthesis. RIF inhibits the mRNA synthesis by binding to the β subunits of the DNA-dependent bacterial RNA polymerase. SM inhibits bacterial protein synthesis by interfering with molecular structures of the ribosomal 30S subunit. Cycloserin inhibits alanine racemase, which converts L-alanine to D-alanine, thus preventing its incorporation into the pentapeptide peptidoglycan of the bacterial cell wall. Finally, PAS is an antagonist of folates synthesis. Recent studies demonstrated that nearly 19% of TB isolates in a hospital were resistant to INH and RIF, the two most common antitubercular agents. Ih general, resistance to ESfH and SM is more common than resistance to RIF, EMB and PZA.

For an empiric treatment of all MTB infections, even if drug resistance is not suspected, the four-drug regimen of INH, RIF, PZA, and EMB (or SM) until susceptibility results, becomes available. Duration of therapy should be at least one year. However, very often this kind of therapy causes patient intolerance. For this reason, the search for new drugs is deemed necessary and the strategy followed has been to test known antibacterial drugs as antimycobacterial compounds. As a consequence, fluoroquinolones, oxazolidinones, β- lactams and macrolides are the newer drugs introduced in the therapy of antimycobacterial infections. Unfortunately, though these drugs revealed to be active, all of them rapidly develop resistance upon a prolonged treatment. Thus, they must always be used in conjunction with at least another antitubercular drug to which mycobacteria are susceptible.

Fluoroquinolones demonstrated in vitro and in vivo activity against MTB and they are also able to penetrate human macrophages in which mycobacteria live in their latent state. As an example, Levofloxacin is characterized by very favorable pharmacokinetic properties. However, new fluoroquinolones have been studied such as Sitafloxacin, Gatifloxacin and Moxifloxacin, all of them being more active than those already employed in therapy, m any case, quinolones are useful in association with other drugs because they can induce resistance. Moreover, many efforts have been made to broaden the activity of oxazolidinones (i.e., antibacterial effects) to mycobacteria.

Among β-Lactams, Amoxicillin-Clavulanate (amoxicillin-clavulanic acid association) is used as additive therapy for multidrug resistant (MDR)-TB, demonstrating a favorable patient response. Macrolides as Claritromycin, Azithromycin and, more recently, Rifapentine revealed less active in vitro against MTB than fluoroquinolones. In general, they are used in combination with at least another drug in order to prevent resistance. In this context, the search for new effective compounds endowed with a different mode of action seemed a possible solution to the above-mentioned intolerance and drug-resistance problems. Moreover, since in immunocompromized patients, tubercular pathology is very often accompanied by mycotic infections caused by Candida albicans, Candida sp. and Cryptococcus neoformans, this concomitance has suggested to search for new substances able to act both as antifungals and antimycobacterials.

The authors of the present invention have already synthesized antitubercular compounds with general structure 1 (Deidda et al., 1998, Biava et al., 1999, Biava et al., 1999b).

In particular, among them, the compound having the formula shown below, BM212, was identified as the most active, showing a potent and selective antifungal and antimycobacterial activity (Biava et al., 2003).

More recently, other compounds have been synthesized by the authors, having the general structure 2 (Biava et al., 2004, Biava et al., 2005).

These compounds showed an antitubercular activity against both MTB and other atypical mycobacteria. Patent application WO04/026828 describes pyrrole derivatives having antimycobacterial activity against clinically relevant strains of MTB. The compounds are assayed in vitro on MTB 27294 and on sensitive and resistant clinical isolates of this species. However, patent application WO04/026828 does not report data concerning maximum 50% non-toxic dose (MNTD₅₀), neither the protection index of compounds derived from N-methylpiperazine. It is important to note that the in vitro activity (MIC), the cytotoxicity (MNTD) and, consequently, the protection index (PI) of a compound should be all reported and evaluated at the same time to affirm that such a compound could be an efficient antitubercular agent. In fact, very low MIC values (i.e., high inhibitory properties) associated with high cytotoxicity lead a compound to be discarded. On the contrary, slightly higher MIC values (i.e., lower inhibitory properties) associated with no cytotoxicity (high values of MNTD) are optimal properties that a compounds should have to be classified as a putative antitubercular agent. Moreover, on the basis of the author's previous work, it is also important to note that, in general, compounds with a N-methylpiperazine moiety inserted in a pyrrole scaffold usually showed higher toxicity, with respect for example to the thiomorpholino moiety, even if MIC values of N-methylpiperazine derivatives were better than the corresponding thiomorpholino analogues.

The instant invention refers to compounds that are partially comprised in the general formula of WO04/026828. However, they are not there exemplified nor their activity is demonstrated or even suggested. The authors of the invention assayed active compounds on different species of mycobacteria, including species that are responsible of tubercular diseases in HIV positive patients, namely M. avium. As a matter of fact, WO04/026828 is silent on the activity on extracellular M. avium. As to the activity of compounds on intracellular M. avium, WO04/026828 reports some activity. Moreover, the authors of the instant invention are able to present data on MNTD₅₀ and protection index, which allow to determine the effectiveness of the compounds as antitubercular agents. In fact, only upon the achievement of all of data (inhibitory activity and cytotoxicity) it is possible to select compounds that are effectively usable as antimycobacteria, including relevant species of atypical mycobacteria such as M. avium. Best compounds are characterized by both a high activity and a low cytotoxicity. In addition, the compounds of the present invention display a high activity against intramacrophagic M. tuberculosis. These results indicate that such compounds are active in an early stage of the disease, where latent tuberculosis occurs before the development of active tuberculosis. Therefore the inactivation of mycobacteria in latent phase is crucial to reduce the percentage of progression towards active tuberculosis. Such percentage is particularly high in immunocompromised patients, for instance HIV positive subjects. WO04/026828 does not report any activity on intramacrophagic M. tuberculosis.

SUMMARY OF THE INVENTION

The invention aims at providing antimycobacterial compounds endowed with a high activity toward Mycobacteria and a low cytotoxicity, i.e. with a very good Protection Index (PI, defined as the ratio between cytotoxic concentration and inhibitory concentration). This is particularly important in view of the fact that antimycobacterial drugs are very often administered to immunocompromised patients for whom drug toxicity is the effective cause of death. The described derivatives possess excellent antimycobacterial activity, and they are

1. more active than existing drugs

2. less toxic than existing drugs

3. very active against dormient mycobacteria

4. very active against resistant mycobacteria

It is therefore an object of the present invention a compound having the general formula I or II,

in which: R represents a morpholinyl, thiomorpholinyl, N-methylpiperazinyl, JV- isopropylpiperazinyl, JV-acetylpiperazinyl, piperidyl or imidazolyl group; R¹ is either absent or represents a hydrogen or an σ-methylphenyl, m-methylphenyl, p- methylphenyl, o-etliylphenyl, m-ethylphenyl, j?-ethylphenyl, o-propylphenyl, m- propylphenyl, p-propylphenyl, o-isopropylphenyl, m-isopropylphenyl, p-isopropylphenyl, o-methoxyphenyl, 7n-methoxyphenyl, ^-methoxyphenyl, o-trifluoromethylphenyl, m- trifluoromethylphenyl, p- trifluoromethylphenyl, o-chlorophenyl, rø-chlorophenyl, p- chlorophenyl, ø,o-dichlorophenyl, m,m-dichlorophenyl, o,^-dichlorophenyl, o- fluorophenyl, rø-fluorophenyl, ^-fluorophenyl, o,o-difluorophenyl, w,m-difluorophenyl, ø,j9-difluorophenyl, 1-naphthyl; R² is H, methyl, ethyl, isopropyl, benzyl, o-chlorobenzyl, m-chlorobenzyl, p- chlorobenzyl, o-fluorobenzyl, m-fluorobenzyl, p- fluorobenzyl, o-methylbenzyl, m-methylbenzyl, p- methylbenzyl, o-trifluorobenzyl, m-trifluorobenzyl, p-trifluorobenzyl, o-methoxybenzyl, m-methoxybenzyl, j»-methoxybenzyl; wherein if the compound has the general formula I, X = N and Y = N or CH; wherein if the compound has the general formula II, X = O, S or N and Y = N or CH (for compounds with general formula II, wherein X = O or S, R¹ can not exist); wherein compound with the general formula I, in which X = N, Y = CH, R = N- methylpiperazinyl or thiomorpholinyl, R¹ = j9-chlorophenyl or ^-fluorophenyl and R² = CH₃ is not included in the present invention; wherein compound with the general formula II, in which X = N, Y = CH, R = N- methylpiperazinyl or thiomorpholinyl, R¹ = p- chlorophenyl or ^-fluorophenyl and R² = CH₃ is not included in the present invention; wherein compound with the general formula II, wherein X = O, Y = CH, R = imidazolyl, R¹ is not present and R² = CH₃ is not included in the present invention. In a preferred embodiment form, the compound has the general formula I, in which X = N; Y = CH; R = thiomorpholinyl or N-methylpiperazinyl; R² = methyl; R¹ = o-chlorophenyl, o-fluorophenyl,_jp-methyphenyl, cψ-dichlorophenyl, ojj-difluorophenyl, 1-naphthyl. Preferably, compounds bear the residues R e R¹ shown in Table 1. Table 1.

A preferred compound is N-(p-fluorophenyl)-2-methyl-3-thiomorpholinomethyl-5-(p- methylphenyl)pyrrole (6e).

Alternatively, the compound of the invention has the formula II, preferably with X = N; Y = CH; R = thiomorpholinyl or N-methylpiperazinyl; R² = methyl; R¹ = one of the residues R¹ present in compound 7a-l and displayed in Table 2.

Table 2.

A preferred compound is N-(o-fluorophenyl)-2-methyl-3-thiomorpholinomethyl-5-(p- fluorophenyl)pyrrole, 7c.

The compounds of the invention are suitable for therapeutic use, in particular for the preparation of antitubercular pharmaceutical compositions, particularly active at the early stage of the disease (latent phase), preferably in association with at least one compound having an antitubercular activity.

The invention also refers to the synthesis methods for the preparation of the compounds having the general formula I or II and to the synthetic intermediates of such methods.

The invention will now be described by means of non limiting examples.

Structure and synthesis of compounds 6a-l.

The procedure for the synthesis of compounds having structure 6a-l is the following:

Preparation of compounds having formula 8:

wherein R¹ is o-chlorophenyl, o-fluorophenyl, p- methylρhenyl, o,p-dichlorophenyl, o,p- difluorophenyl, 1-naphthyl. a) Methyl vinyl ketone (0.016 mol) was reacted with the appropriate aryl aldehyde (0.016 mol) having the following formula 9:

RλCHO (9) wherein R¹ is o-chlorophenyl, o-fluorophenyl, j?-methylphenyl, ø,p- dichlorophenyl, o,p- difluorophenyl, 1-naphthyl; in the presence of 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide (0.0032 mol) and triethylamine (0.011 mol); b) the mixture was stirred at 75 °C under a nitrogen atmosphere for 5 h or 23 h, depending on the particular substrate; c) after cooling, treat the mixture with aqueous HCl until pH 2. d) keep the mixture under stirring for 30 min; e) extract the mixture with ethyl acetate and neutralize the aqueous phase with a NaHCO₃ solution; f) purify the product by column chromatography on aluminium oxide (Brockmann grade II-III), eluting with benzene.

Table 3 reports physico-chemical data of compounds 8. Table 3.

2) Preparation of compounds having formula 10:

wherein

R¹ is o-chlorophenyl, o-fiuorophenyl, jp-methylphenyl, oj?-dichlorophenyl, o,p- difluorophenyl, 1-naphthyl a) the appropriate compound 8 was reacted with an equimolar amount ofp-F-aniline; b) the mixture was heated at 100 °C for 3 h; c) the obtained products were purified by column chromatography on aluminium oxide (Brockmann grade H-III), eluting with cyclohexane. Table 4 reports physico-chemical data of compounds 10. Table 4.

3) Preparation of the compounds having general formula 6a-l:

wherein

R is morpholinyl, thiomorpholinyl, N-methylpiperazinyl, N-isopropylpiperazinyl, N- acetylpiperazinyl, piperidyl or imidazolyl; R¹ is o-chlorophenyl, o-fluorophenyl, ^-methylphenyl, o^-dichlorophenyl, o,p- difluorophenyl, 1-naphthyl a) allow to react 0.6 mol of a suitable amine (morpholine, thiomorpholine, N- methylpiperazine, N-acetylpiperazine, JV-isopropylpiperazine, piperidine, imidazole) with 0.6 mol of 36.5% (w/w) aqueous formaldehyde using 5 mL of glacial acetic acid as solvent; b) using a dropping funnel, add the appropriate compound 10 (0.6 mol), dissolved in 1:2 acetic acid/acetonitrile mixture, dropwise to the Mannich adduct; c) stir the mixture for 12 h at 25 °C; d) neutralize the mixture with 30 mL of 20% (w/v) aqueous NaOH; e) extract the solution with ethyl acetate and wash the organic phase with water to neutrality; f) purify the so obtained product by column chromatography on aluminium oxide (Brockmann grade II-III), eluting the derivatives containing the N-methylpiperazine moiety with chloroform and those containing the thiomorpholine moiety with benzene. Table 5 reports physico-chemical data of synthesized compounds 6a-l. Table 5.

Melting points were determined with a Fisher- Jones apparatus and are uncorrected. Elemental analyses are within ± 0.4% of theoretical values. As an example, the preparation of compound 6e starting from 8c and 10c is reported:

1) Preparation of compound 8c a) Methyl vinyl ketone (1.16 g, 0.016 mol) and p-tolualdehyde (2 g, 0.016 mol) were reacted in the presence of 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide (0,837 g, 0.0032 mol) and triethylamine (1.14 g, 0.011 mol). b) Stir the reaction mixture at 75 °C under a nitrogen atmosphere for 24 h. c) Cool the mixture to room temperature, add ice and 30 mL of concentrated HCl to the mixture until pH 2. d) Stir for 30 min/ e) Extract with ethyl acetate and neutralize the combined organic fractions with an aqueous solution OfNaHCO₃. f) Dry the organic fraction on anhydrous sodium sulphate for 3 h. g) Purify the product by column chromatography on aluminium oxide (Brockmann grade II-III), eluting with a 3:1 cyclohexane/ethyl acetate mixture (41% yield).

2) Preparation of compound 10c a) Compound 8c (1.29 g, 0.007 mol) was reacted at 100°C for 3 h withp-F-aniline (0.75 g, 0.007 mol) andj>toluenesulphonic acid (0.08 g). b) Purify the obtained product by column chromatography on aluminium oxide (Broclαnann grade U-UT), eluting with cyclohexane (75% yield). 3) Preparation of compound 6e a) Thiomorpholine (0.53 g, 0.0051 mol) and formaldehyde (0.153 g, 0.0051 mol, 36.5% w/w aqueous solution) were reacted using 5.5 mL of acetic acid as the solvent. b) Through a dropping funnel, add the obtained Mannich adduct dropwise to a solution containing compound 10c (1.35 g, 0.0051 mol) in glacial acetic acid (11.8 mL) and acetonitrile (23.5 mL). c) Stir the mixture for 12 h at 25 °C. d) Add 30 mL of 20% w/v aqueous NaOH to neutrality. e) Extract the mixture with ethyl acetate and wash the extracts with water to neutrality. f) Dry the organic phase on anhydrous sodium sulphate for 2 h. g) Purify the product by column chromatography on aluminium oxide (Brockmann, grade Il-πi), eluting with benzene (yield 19.4 %).

NMR data for compounds 6a-l

6a: ¹H NMR (CDCl₃) δ: 2.07 (s, 3H, CH₃), 2.70 (m, thiomorpholine 4H), 2.80 (m, thiomorpholine 4H), 3.49 (s, 2H, CH₂), 6.28 (s, 1H, H-4), 6.92-7.36 (m, 8H, aromatic protons). 6b: ¹H NMR (CDCl₃) δ: 2.08(s, 3H, N-CH₃), 2.30 (s, 3H, CH₃), 2.49 (m, JV- methylpiperazine 8H), 3.51 (s, 2H, CH₂), 6.31 (s, 1H, H-4), 6.28-7.27 (m, 8H, aromatic protons).

6c: ¹H NMR (CDCl₃) δ: 2.08 (s, 3H, CH₃), 2.71 (m, thiomorpholine 4H), 2.78 (m, thiomorpholine 4H), 3.48 (s, 2H, CH₂), 6.34 (s, 1H, H-4), 6.87-7.36 (m, 8H, aromatic protons).

6d: ¹H NMR (CDCl₃) δ: 2.08(s, 3H, N-CH₃), 2.30 (s, 3H, CH₃), 2.49 (m, N- methylpiperazine 8H), 3.51 (s, 2H, CH₂), 6.36 (s, 1H, H-4), 6.94-7.26 (m, 8H, aromatic protons).

6e: ¹H NMR (CDCl₃) δ: 2.05 (s, 3H, CH₃), 2.38 (s, 3H, CH₃), 2.71 (m, thiomorpholine 4H), 2.78 (m, thiomorpholine 4H), 3.46 (s, 2H, CH₂), 6.27 (s, 1H, H-4), 6.79-7.36 (m, 8H, aromatic protons). 6f: ¹H NMR (CDCl₃) δ: 2.05(s, 3H, N-CH₃), 2.30 (s, 3H, CH₃), 2.37 (s, 3H, CH₃), 2.38 (m,

N-methylpiperazine 8H), 3.48(s, 2H, CH₂), 6.30 (s, 1H, H-4), 6.79-7.30 (m, 8H, aromatic protons).

6g: ¹H NMR (CDCl₃) δ: 2.08 (s, 3H, CH₃), 2.71 (m, thiomorpholine 4H), 2.77 (m, thiomorpholine 4H), 3.48 (s, 2H, CH₂), 6.28 (s, 1H, H-4), 6.96-7.36 (m, 7H, aromatic protons).

6h: ¹H NMR (CDCl₃) δ: 2.09(s, 3H, N-CH₃), 2.30 (s, 3H, CH₃), 2.54 (m, N- methylpiperazine 8H), 3.50 (s, 2H, CH₂), 6.31 (s, 1H, H-4), 6.98-7.29 (m, 7H, aromatic protons). 6i: ¹H ΝMR (CDCl₃) δ: 2.06 (s, 3H, CH₃), 2.71 (m, thiomorpholine 4H), 2.78 (m, thiomorpholine 4H), 3.46 (s, 2H, CH₂), 6.29 (s, 1H, H-4), 6.66-7.36 (m, 7H, aromatic protons).

6j: ¹H ΝMR (CDCl₃) δ: 2.07(s, 3H, N-CH₃), 2.33 (s, 3H, CH₃), 2.49 (m, N- methylpiperazine 8H), 3.49 (s, 2H, CH₂), 6.31 (s, 1H, H-4), 6.65-7.27 (m, 7H, aromatic protons).

6k: ¹H ΝMR (CDCl₃) δ: 2.14 (s, 3H, CH₃), 2.74 (m, thiomorpholine 4H), 2.85 (m, thiomorpholine 4H), 3.56 (s, 2H, CH₂), 6.32 (s, 1H, H-4), 6.83-8.03 (m, HH, aromatic protons).

61: ¹H ΝMR (CDCl₃) δ: 2.14(s, 3H, N-CH₃), 2.32 (s, 3H, CH₃), 2.47 (m, N- methylpiperazine 8H), 3.60 (s, 2H, CH₂), 6.34 (s, 1H, H-4), 6.80-8.01 (m, HH, aromatic protons).

The ΝMR spectra were recorded with a Brucker 400 (MHz) spectrometer employing deuterochloroform (CDCI₃) as the solvent. Tetramethylsilane (TMS) was used as an internal standard.

Structure and synthesis of compounds 7a-l.

The following synthetic scheme was adopted for the synthesis of compounds having structure 7a-l.

I) Preparation of compounds having formula 11:

a) allow to react methyl vinyl ketone (0.016 mol), j?-F-benzaldehyde (0.016 mol), 3-ethyl- 5-(2-hydroxyethyl)-4-methylthiazolium bromide (0.0032 mol) and triethylamine (0.011 mol); b) stir the mixture at 75 °C under a nitrogen atmosphere for 5 h or for 23 h, depending on the substrate; c) cool the mixture and treat it with aqueous HCl until pH 2; d) stir the mixture for 30 min; e) extract the mixture with ethyl acetate and neutralize the extracts washing with aqueous NaHCO₃; f) purify the product by column chromatography on aluminium oxide (Brockmann, grade H-III),, using benzene as eluant. 2) Preparation of compounds having formula 12:

12

wherein R¹ is o-chlorophenyl, o-fiuorophenyl, ju-methylphenyl, oj>dichlorophenyl, o,p- difluorophenyl, 1-naphthyl a) make compound 11 to react with an equimolar amount of the suitable aromatic amine having the general formula 13;

R¹ -NH₂ (13)

wherein

R¹ is o-chlorophenyl, o-fluorophenyl, p-methylphenyl, o,p-dichlorophenyl, o,p- difluorophenyl, 1-naphthyl. b) allow the mixture to react at 100 °C for 3 h; c) purify the obtained products by column chromatography on aluminium oxide, eluting with cyclohexane.

Table 6 reports physico-chemical data for compounds 12. Table 6.

3) Preparation of compounds having general formula 7a-l:

wherein

R is morpholinyl, thiomorpholinyl, N-methylpiperazinyl, N-isopropylpiperazinyl, N- acetylpiperazinyl, piperidyl or imidazolyl; R¹ is o-chlorophenyl, o-fluorophenyl, p-methylρhenyl, o,p-dichlorophenyl, o,p- difluorophenyl, 1-naphthyl. a) react 0.6 mol of a suitable base (morpholine, thiomorpholine, N~methylpiperazine, N- acetylpiperazine, N-isopropylpiperazine, piperidine, imidazole) with 0.6 mol of 36.5% w/w aqueous formaldehyde employing 5 mL of glacial acetic acid as solvent; b) through a dropping funnel, add the formed Mannich adduct dropwise to a solution of the appropriate compound 12 (0.6 mol) in a 1:2 acetic acid/acetonitrile mixture; c) stir the mixture for 12 h at 25 °C; d) neutralize the mixture with 30 mL of 20% w/v aqueous NaOH; e) extract the solution with ethyl acetate and wash the organic phase with water to neutrality; f) purify the obtained product by column chromatography on aluminium oxide (Brockmann, grade II-III), eluting the derivatives containing the N-methylpiperazine moiety with chloroform and the derivatives containing the thiomorpholine one with benzene. In Table 7, physico-chemical data for some of the compounds 7a-l are reported. Table 7.

Melting points were determined with a Fisher- Jones apparatus and are uncorrected. AU synthesized derivatives have been subjected to elemental analysis. Elemental analyses are within ± 0.4% of theoretical values.

Hereafter, the preparation of compound 7c starting from 12b is described.

Preparation of compound 12b a) Compound 11 (1.25 g, 0.006 mol) was reacted with o-F-aniline (0.71 g, 0.006 mol) and p-toluenesulphonic acid (0.08 g); b) Allow the reaction to proceed at 100 °C for 5 h; c) Purify the obtained product by column chromatography on aluminium oxide (activity grade II-III, according to Brockmann), eluting with cyclohexane (yield 82%). Preparation of compound 7c a) Thiomorpholine (0.53 g, 0.0051 mol) was reacted with formaldehyde (0.153 g, 0.0051 mol, 36.5% w/w aqueous solution) using 5.5 mL of acetic acid as solvent. b) Through a dropping funnel, add the Mannich adduct dropwise to a solution containing compound 12b (1.4 g, 0.0053 mol) in glacial acetic acid (12.2 mL) and acetonitrile' (24.4 mL). c) Stir the reaction for 12 h at 25 °C. d) Neutralize with 30 mL of a 20% w/v aqueous solution of NaOH. e) Extract the mixture with ethyl acetate and wash the organic extracts with water to neutrality. f) Dry the organic extracts on anhydrous sodium sulphate for 2 h. g) Purify the product by column chromatography on silica gel eluting with ethyl acetate (yield 23%).

NMR data for compounds 7a-l

7a: ¹H NMR (CDCl₃) δ: 1.97 (s, 3H, CH₃), 2.72 (m, thiomorpholine 4H), 2.78 (m, thiomorpholine 4H), 3.48 (s, 2H, CH₂), 6.31 (s, 1H, H-4), 6.79-7.48 (m, 8H, aromatic protons).

7b: ¹H NMR (CDCl₃) δ: 1.94(s, 3H, N-CH₃), 2.30 (s, 3H, CH₃), 2.33 (m, N- methylpiperazine 8H), 3.50 (s, 2H, CH₂), 6.34 (s, 1H, H-4), 6.36-7.48 (m, 8H, aromatic protons).

7c: ¹H ΝMR (CDCl₃) δ: 2.03 (s, 3H, CH₃), 2.71 (m, thiomorpholine 4H), 2.77 (m, thiomorpholine 4H), 3.47 (s, 2H, CH₂), 6.29 (s, 1H, H-4), 6.80-733 (m, 8H, aromatic protons).

7d: ¹H ΝMR (CDCl₃) δ: 2.04(s, 3H, N-CH₃), 2.35 (s, 3H, CH₃), 2.55 (m, N- methylpiperazine 8H), 3.55 (s, 2H, CH₂), 6.34 (s, 1H, H-4), 6.83-7.34 (m, 8H, aromatic protons). 7e: ¹H NMR (CDCl₃) δ: 2.05 (s, 3H, CH₃), 2.25 (s, 3H, CH₃), 2.72 (m, thiomorpholine

4H), 2.80 (m, thiomorpholine 4H), 3.48 (s, 2H, CH₂), 6.29 (s, 1H, H-4), 6.92-7.12 (m, 8H, aromatic protons).

7f: ¹H NMR (CDCl₃) δ: 2.05(s, 3H, N-CH₃), 2.30 (s, 3H, CH₃), 2.37 (s, 3H, CH₃), 2.50 (m,

7g: ¹H NMR (CDCl₃) δ: 1.97 (s, 3H, CH₃), 2.70 (m, thiomorpholine 4H), 2.76 (m, thiomorpholine 4H), 3.47 (s, 2H, CH₂), 6.30 (s, 1H, H-4), 6.86-7.49 (m, 7H, aromatic protons).

7h: ¹H NMR (CDCl₃) δ: 1.97(s, 3H, N-CH₃), 2.27 (s, 3H, CH₃), 2.48 (m, N- methylpiperazine 8H), 3.43 (s, 2H, CH₂), 6.32 (s, 1H, H-4), 6.82-7.48 (m, 7H, aromatic protons). 7i: ¹H NMR (CDCl₃) δ: 2.04 (s, 3H, CH₃), 2.71 (m, thiomorpholine 4H), 2.77 (m, thiomorpholine 4H), 3.46 (s, 2H, CH₂), 6.29 (s, 1H, H-4), 6.84-7.26 (m, 7H, aromatic protons).

7j: ¹H NMR (CDCl₃) δ: 2.05(s, 3H, N-CH₃), 2.29 (s, 3H, CH₃), 2.50 (m, N- methylpiperazine 8H), 3.50 (s, 2H, CH₂), 6.32 (s, 1H, H-4), 6.79-7.37 (m, 7H, aromatic protons).

7k: ¹H NMR (CDCl₃) δ: 1.87 (s, 3H, CH₃), 2.76 (m, thiomorpholine 4H), 2.83 (m, thiomorpholine 4H), 3.54 (s, 2H, CH₂), 6.41 (s, 1H, H-4), 6.64-7.89 (m, HH, aromatic protons). 71: ¹H NMR (CDCl₃) δ: 1.90 (s, 3H, N-CH₃), 2.32 (s, 3H, CH₃), 2.53 (m, JV- methylpiperazine 8H), 3.56 (s, 2H, CH₂), 6.44 (s, 1H, H-4), 6.63-7.90 (m, HH, aromatic protons ).

The NMR spectra were recorded with a Brucker 400 (MHz) spectrometer employing deuterochloroform (CDCl₃) as solvent. Tetramethylsilane (TMS) was used as an internal standard.

Microbiological activity

Compounds 6a-l and 7a-l were tested in DMSO. a) Antimycobacterial activity Compounds 6a-l and 7a-l were preliminarily assayed against two freshly isolated clinical strains, M. fortuitum CAlO and M. tuberculosis B814, according to the dilution method in agar (Hawkins et al., 1991)

Growth media were Mueller-Hinton (Difco) containing 10% of OADC (oleic acid, albumine and dextrose complex) for M.fortuitum and Middlebrook 7Hl 1 agar (Difco) with 10% of OADC (albumine and dextrose complex) for M. tuberculosis. Substances were tested at the single dose of 100 μg/mL. The active compounds were then assayed for inhibitory activity against a variety of mycobacterium strains in Middlebrok 7H9 broth using the NCCLS procedure. The mycobacterium species used for biological tests were M. tuberculosis 103471 and, among atypical mycobacteria, M. smegmatis 103599, M. marinum 6423 and M. avium 103317 (from the Institute Pasteur collection).

In all cases, minimum inhibitory concentrations (MICs in μg/mL) for each compound were determined. The MIC was defined as the lowest concentration of drug that yielded an absence of visual turbidity. Stock solutions of substances were prepared by dissolving a known weight of the compound in DMSO. The stock solutions were sterilized by passage throught a 0.2 μm Nylon membrane filter. Serial 2-fold dilutions of the compounds with water were prepared. The tubes were incubated at 37 °C for 3-21 days. A control tube without any compound was included in each experiment. BM212, Isoniazid (INH), streptomycin and rifampin were used as reference compounds.

Inhibitory activity of 6e and 7c on intramacrophagic mycobacteria. The bactericidal activity of such compounds on intracellular mycobacteria was studied on U937 cells (INC-FLOW), a human hystiocytic cell line. Cells were differentiated into macrophages with 20 ng/mL of phorbol myristate acetate (PMA, Sigma) and grown in RPMI 1640 medium with 10% fetal calf serum.

Inhibitory activity of 6e and 7c on multidrug-resistant mycobacteria A panel of twelve mycobacteria resistant to currently available antitubercular drugs, were used. Compounds 6e and 7c were tested on multiresistant M. tuberculosis strains in Middlebrook 7119 broth enriched with 10% ADC (Difco) using the macrodilution broth method. b) Cytotoxic activity assays

The cytotoxicity was evaluated on Vero cell monolayers (ICN-Flow). They were inoculated in 6-well plates each containing 9x10⁴ cells and incubated in DMEM with 5% FCS for 24 h at 37 °C in a 5% CO₂ incubator. After 24 h of culture, the medium was changed and a new medium containing decreasing doses of the substances under study was added.

After 5 days, the cells were trypsinized and counted in a Neubauer chamber under a light microscope. All the tests were done in triplicate. The maximum 50% non-toxic dose (MNTD₅₀) was defined as the drug concentration that decreased cell multiplication less than 50% with respect to the control. c) Protection Index

Protection Index (PI), is the MNTD₅₀/MIC ratio.

RESULTS

The microbiological results relative to the tests against extracellular M. tuberculosis and atypical Mycobacteria are reported in Tables 8-13, as well as the PI, the cytotoxicity, and the activity against intracellular M. tuberculosis and multi drag resistant strains (MDR- TB). The inhibitory activity toward extracellular M. tuberculosis accounts for the ability of tested compounds to treat active tuberculosis. Differently, assays on intracellular M. tuberculosis assess the ability of tested compounds to inhibit mycobacteria during the latent phase of tuberculosis, before latent tuberculosis infection itself progresses to active disease.

Table 8. Cytotoxicity, antimycobacterial activity toward M. tuberculosis and protection index of compounds 6a-l.

Table 9. Antimycobacterial activity of compounds 6a-l toward atypical mycobacteria.

Table 10. Cytotoxicity, antimycobacterial activity toward M. tuberculosis and protection index of compounds 7a-l.

Table 11. Antimycobacterial activity of compounds 7a-l toward atypical mycobacteria.

Table 12. Activity of compounds 6e and 7c against intracellular M. tuberculosis.

Table 13. Activity toward multi drug resistant strains (MDR-TB).

The compounds can be usefully employed in medical care. For example, compounds 6e and 7c are characterized by a very interesting biological profile. In particular, their activity against M. tuberculosis 103471 (0.4 μg/mL for 6e and 0.5 μg/mL for 7c, Table 8 and Table

10, respectively) is comparable to that shown by isoniazid (0.25 μg/mL), streptomycin (0.50 μg/mL) and rifampin (0.30 μg/mL), as well as to that of the parent compound BM212 (0.70 μg/mL). In addition, compounds 6e and 7c have the advantage to be less toxic, and particularly 6e is endowed with a very good protection index (PI = 160), which is better than that found for isoniazid and streptomycin (PI = 128) and slightly lower than that found for rifampin (PI = 213). Moreover, compounds 6c, 6i, and 7g also showed good antimycobacterial activity (0.5, 0,.5, and 1 μg/mL, respectively).

In general, tested compounds showed activity toward atypical mycobacteria at concentrations higher than 8 μg/mL (Tables 9 and 11), suggesting a significant selectivity toward M. tuberculosis with respect to atypical mycobacteria. Significant exceptions were represented by activity toward M. avium, found to be in the range between 2 and 4 μg/mL for compounds 6f-h and 7g-h. Finally, 7d showed a 0.3 μg/mL activity toward M. smegmatis.

Compounds 6e and 7c, showing the best activity toward M. tuberculosis, were also tested against intracellular and resistant mycobacteria. Biological results reported in Table 12 showed that both of them exerted bactericidal activity on intracellular mycobacteria at 3 μg/mL concentration, comparable to that of rifampin. This result was very important because mycobacteria can reside for years inside lymphoid cells and macrophages (latent tuberculosis) and many traditional drugs were unable to get throw it. Moreover, combating latent tuberculosis infection is one of the major challenges mainly for reducing the high rate of progression to active disease in immuno-compromised individuals.

Finally, Table 13 showed that all of the tested strains were inhibited by compounds 6e and 7c at concentrations ranging from 0.5 to 2 μg/mL. The sole exception was represented by the 326/04 strain, sensitive to such compounds at concentrations higher than 32 μg/mL. The present experimental evidences make these compounds extremely interesting when compared to the compounds now used in therapy, which tend to be less active against drug-resistant mycobacteria. As a consequence, toward drug-resistant mycobacteria a multi-drug therapy is needed today. In this context, considering the reduced toxicity of the pyrrole derivatives reported here, they could be usefully employed, alone or in combination, for the therapy of tuberculosis.

BIBLIOGRAPHY

Duncan, K. et al. Curr. Opin. Microbiol. 7, 460-465, 2004 Deidda, D. et al, Antimicrob. Agents Chemother. 42, 3035-3037, 1998 Biava M., et al Bioorg. Med. Chem. Lett. 9, 2983-2988, 1999 Biava M., et al Med. Chem. Res. 9, 19-34, 1999b Biava M., et al Bioorg. Med. Chem. 11, 515-520, 2003 Biava M., et al Bioorg. Med. Chem. 12, 1453-1458, 2004 Biava M., et al Bioorg. Med. Chem. 13, 1221-1230, 2005

Hawkins, J. E.; Wallace Jr., R. J.; Brown, A.; 1991, Antibacterial susceptibility test: Mycobacteria: in A. Balows, W. J. Hausler Jr., K. L. Hermann, H. D. Isenberg, H. J.

Shadomy (eds.). Manual of Clinical Microbiology, 5 edn., American Society for Microbiology, Washington, D. C.

Claims

1. Compounds having general formula I or II:

wherein

R is morpholinyl, thiomorpholinyl, N-methylpiperazinyl, N-isopropylpiperazinyl, N- acetylpiperazinyl, piperidyl or imidazolyl;

R¹ is either absent or is hydrogen, o-methylphenyl, m-methylphenyl, p-methylphenyl, o- ethylphenyl, m-ethylphenyl, p- ethylphenyl, o-propylphenyl, m-propylphenyl, p- propylphenyl, o-isopropylphenyl, m-isopropylphenyl, p-isopropylphenyl, o- methoxyphenyl, m-methoxyphenyl, p-methoxyphenyl, o-trifluoromethylphenyl, m- trifluoromethylphenyl, p-trifluoromethylphenyl, o-chlorophenyl, m-chlorophenyl, p- chlorophenyl, o,o-dichlorophenyl, m,m-dichlorophenyl, o,p-dichlorophenyl, o- fluorophenyl, m-fiuorophenyl, p-fluorophenyl, o,o-difluorophenyl, m,m-difluorophenyl, o,p-difluorophenyl, 1-naphthyl;

R² is H, methyl, ethyl, isopropyl, benzyl, o-chlorobenzyl, m-chlorobenzyl, p-chlorobenzyl, o-fluorobenzyl, m-fluorobenzyl, p- fluorobenzyl, o-methylbenzyl, m-methylbenzyl, p- methylbenzyl, o-trifluorobenzyl, m-trifluorobenzyl, p-trifluorobenzyl, o-methoxybenzyl, m-methoxybenzyl, p-methoxybenzyl; if the compound has general formula 1, X = N and Y = N or CH; if the compound has general formula II, X = O, S or N and Y = N or CH (for compounds with general formula 7, wherein X = O or S, R¹ can not exist); compound with general formula I, wherein X = N, Y = CH, R = N-methylpiperazinyl or thiomorpholinyl, R¹ = p-chlorophenyl or ^-fluorophenyl and R² = CH₃ is not inclusive in the present invention; compound with general formula II, wherein X = N, Y = CH, R = N-methylpiperazinyl or thiomorpholinyl, R¹ = ^-chlorophenyl or ^-fluorophenyl and R² = CH₃ is not inclusive in the present invention; compound with general formula II, wherein X = O, Y = CH, R = imidazolyl, R¹ is not present and R² = CH₃ is not inclusive in the present invention.

2. A compound according to claim 1 wherein X = N; Y = CH; R = thiomorpholinyl or N- methylpiperazinyl; R² = methyl; R¹ = o-chlorophenyl, o-fluorophenyl, /j-methylphenyl, o,j!7-dichlorophenyl, oj?-difluorophenyl, 1-naphthyl.

3. A compound according to claim 2 being N-(4-fluorophenyl)-2-methyl-3- thiomorpholinomethyl-5-(4-methylphenyl)pyrrole.

4. A compound according to claim 2 being N-(2-fluorophenyl)-2-methyl-3- thiomorpholinomethyl-5-(4-fluorophenyl)pyrrole.

5. A compound for medical use according to claims from 1 to 4.

6. Use of the compound according to any of previous claims for the preparation of a medicine endowed with, antitubercular activity.

7. Use of the compound according to claim 6 wherein the antitubercular activity is exerted at the latent phase of the disease.

8. Use of the compound according to claim 6 or 7 in which the medicine includes at least another compound endowed with antitubercular activity.

9. Use of the compound according to claim 6 to 8, in which the compound is N-(4- fluorophenyl)-2-methyl-3-thiomoφholinomethyl-5-(4-methylphenyl)pyrrole.

10. Use of the compound according to claim 6 to 8, in which the compound is N-(2- fluorophenyl)-2-methyl-3-thiomorpholinomethyl-5-(4-fluorophenyl)pyrrole.

11. A pharmaceutical composition comprising the compound according to claims 1-5, and appropriate excipients and diluents.

12. The pharmaceutical composition according to claim 11 further comprising at least another compound endowed with antitubercular activity.

13. Process for the preparation of a compound having general formula I according to claim 1 involving the following steps: a) reaction of methyl vinyl ketone with the suitable aryl aldehyde having formula 9 R^CHO (9) wherein R¹ is o-chlorophenyl, o-fluorophenyl, p- methylphenyl, o,j9-dichlorophenyl, o,p- difluorophenyl, 1-naphthyl in the presence of 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide and triethylamine under conditions such as to obtain the appropriate intermediate 8;

wherein

R¹ is o-chlorophenyl, o-fluorophenyl, ^-methylphenyl, oj?-dichlorophenyl, o,p- difluorophenyl, 1-naphthyl b) extract and/or purify compound 8 as obtained under a); c) allow to react compound 8 with p-F-aniline under conditions suitable for obtaining the appropriate intermediate 10;

wherein

O

R¹ is o-chlorophenyl, o-fluorophenyl, ^-methylphenyl, o,j?-dichlorophenyl, o,p- difluorophenyl, 1-naphthyl d) purify compound 10 as obtained under c); e) allow the appropriate amine (morpholine, thiomorpholine, N-methyilpiperazine, N- acetylpiperazine, JV-isopropylpiperazine, piperidine, imidazole) to react with formaldehyde adding compound 10 under conditions suitable for obtaining the appropriate compound 6; f) extract and/or purify product 6 as obtained under e).

14. Intermediate having formula 8:

wherein

R , 1 is o-chlorophenyl, o-fluorophenyl, p-methylphenyl, o,p-dichlorophenyl, o,p~ difluorophenyl, 1-naρhthyl

15. Intermediate having formula 10:

wherein

R¹ is o-chlorophenyl, o-fluorophenyl, p-methylphenyl, o.p- dichlorophenyl, o,p- difluorophenyl, 1-naphthyl

16. Process for the preparation of a compound having general formula II according to claim 1 including the following reaction steps: a) allow methyl vinyl ketone to react with ^-F-benzaldehyde in the presence of 3-ethyl-5-

(2-hydroxyethyl)-4-methylthiazolium bromide and triethylamine under conditions suitable for obtaining compound 11 having the following formula:

b) extract and/or purify compound 11 as obtained under a); c) allow compound 11 to react with the appropriate aromatic amine having general formula

13:

R^NH₂ (13) wherein

R¹ is o-chlorophenyl, o-fluorophenyl, p-methylphenyl, oj?-dichlorophenyl, o,p- difluorophenyl, 1-naphthyl under conditions suitable for obtaining the appropriate compound 12;

wherein R¹ is o-chlorophenyl, o-fluorophenyl, p-methylphenyl, oj?-dichlorophenyl, o,p- difluorophenyl, 1-naphthyl d) purify compound 12 as obtained under c); e) allow the appropriate amine (morpholine, thiomorpholine, N-methyilpiperazine, N- acetylpiperazine, N-isopropylpiperazine, piperidine, imidazole) to react with formaldehyde adding a solution of compound 12 under conditions suitable for obtaining the appropriate compound 7; f) extract and/or purify product 7 as obtained under e).

17. Intermediate having formula 11:

18. Intermediate having formula 12:

wherein

R¹ is o-chlorophenyl, o-fluorophenyl, p-methylphenyl, o,p-dichlorophenyl, o,p- difluorophenyl, 1-naphthyl.