The invention relates to acryloyl substituted pyrrole derivati- ves, to a process for their preparation and to pharmaceutical compositions containing them.

The pyrrole derivatives of the invention may be regarded as derivatives of Distaπycin A which is a known compound having the following formula

Literature referring to distamycin A includes, for examples Nature 203, 1064 (1964).

The invention provides acryloyl substituted pyrrole derivatives of the following formula (I)

wherein n is an integer of 1 to 5 ; eeaacchh ooff RR aanndd RR„_ ,, wwhhiicchh mmaayy bbee tthhie same or different , is 1 2 hydrogen , halogen , -CN , -NO. , C -C alkyl , or a group

CH, ,- ^> Ol-'

R3 is hydrogen, halogen, -CN, or -NO ; each R is, independently, hydrogen or C -C alkyl;

A is a bond, a group -NH— ά —C- , or a group -NH-Het-CO-,

0 wherein Het is a saturated or unsaturated pentatomic or hexa¬ tomic heteromonocyclic ring; and or

- (CH„ ) -N( Λ in which m is 1 , 2 or 3 and each R is , indepen-

2 m \ ^{s R} 5 dently, a C -C alkyl group.

The invention includes also the pharmaceutically acceptable salts of the compounds of formula (I) as well as the possible isomers covered by the formula (I), both separately and in mixture. In the above reported formula (I) n is preferably 3, 4 or 5; A is preferably a bond or a group or a group

-NH-He t-CO- ;

B is preferably a group wherein m is preferably 1 and each R_ is methyl . b

When A is a group -NH-Het-CO- wherein Het represents a heteromonocyclic ring as defined above, this is, preferably, an unsaturated pentatomic or hexatomic heteromonocyclic ring containing at least one, preferably one or two, heteroatom

chosen from 0, S and N. Examples of said heteromonocyclics are thiophene, thiazole, pyridine, isoxazole, furane, triazole and imidazole.

When R^ and R are the same, they are,preferably,hydrogen.

When R ₁ and R are different, R is, preferably, hydrogen and R ₂ is,preferably,a halogen; the halogen is, preferably, chlorine or bromine.

When R is.halogen, it is, preferably, chlorine or bromine.

Preferably each group R , independently, is C -C alkyl, in particular methyl and, most preferably, all groups R are methyl.

As already said, the invention includes also the pharmaceutical¬ ly acceptable salts of the compounds of formula (I). These salts are the salts with pharmaceutically acceptable acids, either inorganic acidisuch as, e.g., hydrochloric, hydro- bromic, nitric and sulfuric, or organic acids such as, e.g., citric , tartaric, maleic, fumaric, methanesulfonic and ethane- sulfonic.

A preferred class of compounds under this invention is represented by the compounds of formula (I) wherein n is

A is

B is ;

R and R. are hydrogen;

R is chlorine or bromine and R. is methyl,

3 ^A especially in the form of salts with hydrochloric acid.

Specific examples of preferred compounds under this invention, especially in the form of salts with hydrochloric acid, are the following:

N-deformyl-N-Φ*-chloroacryloyl) Distamycin A; β-(N-methyl-4-(N-methyl-4-(N-methyl-4-(N-methyl-4- ^-chloro-acrylamido) pyrrole-2-carboxamido) pyrrole-2-carbox- amido) pyrrole-2-carboxamido) pyrrole-2-carboxamido) propion- amidine;

N-deformyl-N-ft-bromoacryloyl) Distamycin A; -(N-methyl-4-(N-methyl-4-(N-methyl-4-(N-methyl-4-(o-bromoacr yl- a ido) pyrrole-2-carboxamido) pyrrole-2-carboxamido) pyrrole-2- carboxa ido) pyrrole-2-carboxamido) propionamidine; ^• * « -(N-methyl-4-(N-methyl-4_ (N-methyl-4-(N-methyl-4-(N-π ^* ethyl-4- (-K-bromoacrylamido ) pyrrole-2-carboxamido ) pyrrole-2-carboxamido ) pyrrole-2-carboxam o ) pyrrole-2-carboxamido) pyrrole-2-carbox- a ido ) propionamidine ;

N-deformyl-N-(4-ιj3(-bromoacrylamido) benzoyD-Distamycin A; 3- (N-methyl-4-(N-methyl-4- (N-methyl-4-(N-methyl-4-& -chloro- acryla ido ) pyrrole-2-carboxamido) pyrrole-2-carboxamido) pyrrole- 2-carboxamido) pyrrole-2-carboxamido) propyl-dimethylamine .

The compounds of formula ( I ) are prepared by a process comprising

A) reacting a compound of formula (II ⁾

wherein n , R and B are as defined above _; or a salt thereof , with a compound of formula (ill)

wherein R , R , R and A are as defined above and X is <_- hydroxy or a leaving group, so obtaining a compound of formula (I) or a salt thereof; or

B) reacting a compound of formula (IV)

wherein Z is N-/ -CO-, or H N-Het-CO-, and n, R , B and Het are as defined above, or a salt thereof, with a compound of formula (V)

wherein R ___., R__, R___) and X are as defined above, so obtaining a compound of formula (I) wherein

A is a group -, or a salt thereof.

The leaving group X in the compounds (III) and (V) may be, for example, halogen, chlorine in particular, or another displaceable group such as, for instance, 2,4,5- trichlorophenoxy, 2,4-dinitrophenoxy, succinimido-N-oxy, imidazolyl, pivaloyloxy, [(-0-C-C(CH ₃ )3]

-O-Et) or isopropyloxy formate

A resulting compound of formula (I) may be converted into a pharmaceutically acceptable salt if desired.

The reaction between a compound of formula (II) and a compound of formula (III) wherein X is -OH is preferably carried out at a molar ratio of (II): (III) of from 1:1 to 1:2 in an organic solvent such as, e.g., dimethylsulphoxide, hexamethylphosphotriamide, dimethylacetamide or, preferably, dimethylformamide, or their aqueous mixtures, in the presence of an organic base such as, e.g., triethyla ine or diisopropyl ethyla ine or an inorganic base such as,e.g.,sα_i". ^* m bicarbonate and of a condensing agent such as, e.g., N-ethyl-N'-O-dimethylaminopropyl) carbodiimide or, preferably, N,N ^, -dicyclohexylcarbodiimide. The reaction temperature may vary from about -10°C to about 50°C and the reaction time from about 1 to about 12 hours.

The reaction between a compound of formula (II) and a compound of formula (III), wherein X is a leaving group, e.g., 2,4,5-trichlorophenoxy or succinimido-N-oxy or imidazolyl, is usually carried out at a molar ratio of (II): (III) of from 1:1 to 1:2 in an organic solvent such as, e.g. dimethylformamide or pyridine, in the presence of an organic base, e.g. diisopropylethylamine, at a temperature from about 0°C to about 25°C and for about two hours to about ten hours. Similar reaction conditions may be followed when X in the compound (III) is a halogen atom.

The reaction between a compound of formula (IV) and a compound of formula (V) may be carried out in analogous conditions as those reported hereabove for the reaction between a compounds of formula (II) and a compound of formula (III) having the corresponding meanings of X.

The compounds of formula (I) prepared according to the above described procedures may be purified by conventional methods such as, e.g., silica gel or alumina column chromatography, and/or by recrystallization from organic solvents such as, e.g. lower aliphatic alcohols or dimethylformamide.

The compounds of formula (II) are known compounds or may be prepared by known methods from known compounds: see, for instance, Arca one et al. Gazzetta Chim. Ital. _97, 1097 (1967). The compounds of formula (IV) may be prepared following methods well known in the organic chemistry. In particular, for example, the compounds of formula (IV) wherein Z is a group

NH-— \—-CO- may be prepared by reacting a compound of for¬ mula (II) with p-nitrobenzoyl chloride and reducing the obtained nitro compound by known methods.

The compounds of formula (III) are known compounds or may be prepared by standard methods, for example as described in J.C.S. 1947 - 1032 and JACS 2, 3495 (1940). For instance, compounds of formula (III) wherein A is -NH-<^^>-CO- may be prepared by reaction of p-aminobenzoic acid with the activated acrylic acid derivative in a conventional way.

The compounds of the invention show cytostatic properties towards tumor cells so that they can be useful as antineoplastic agents, e.g. to inhibit the growth of various tumors, such as, for in¬ stance, carcinomas, e.g. mammary carcinoma, lung carcinoma, bladder carcinoma, colon carcinoma, ovary and endome rial tumors. Other neoplasias in which the compounds of the invention could find application are, for instance, sarcomas, e.g.soft tissue and bone sarcomas, and the he atological malignancies such as, e.g., leukemias.

The cytotoxicity of the compounds of the invention was tested, for instance, on urine L1210 leukemia cells with the following procedure. Cells were derived from in vivo tumors and established in cell culture. Cells were used until the tenth passage. Cytotoxicity was determined by counting surviving cells ^' after 4 hours treatment and 48 hours growth in drug-free medium. The percentage of cell growth in the treated cultures was compared with that of controls. ID values (doses inhibiting 509έ of the cellular growth in respect to controls) were calculated on dose- response curves.

Thus, for example, for the compound of the invention β-(N-methyl- -4-(N-methy1-4-(N-methy1-4-(N-me hy1-4-(eL-bromoacryla ido)pyrrole- -2-carboxami o)pyrrole-2-carboxamido)pyrrole-2-carboxami o)pyrro- le-2-carboxamido)propionamidine, hydrochloride, an ID- value of 0.003 j/ml was found in the above test.

The compounds of the invention can be administered by the usual routes, for example, parenterally, e.g. by intravenous injec¬ tion or infusion, intramuscularly, subcutaneously, topically or orally.

The dosage depends on the age, weight and conditions of the patient and on the administration route. For example, a suitable dosage for administration to adult humans may range f om about o.05 to about 100 mg pro dose 1-4 times a day.

The pharmaceutical compositions of the invention contain a compound of formula (I ) as the active substance, in association with one or more pharmaceutically acceptable excipients.

The pharmaceutical compositions of the invention are usually prepared following conventional methods and are administered in a pharmaceutically suitable form. For instance, solutions for intravenous injection or infusion may contain as carrier, for example, sterile water or. preferably,they may be in the form of sterile aqueous isotonic saline solutions.

Suspensions or solutions for intramuscular injections may contain, ogether with the active compound, pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride. In the forms for topical application, e.g. creams, lotions or pastes for use in dermatological treatment, the active

ingredient aay be aixed with conventional oleaginous or •■ulsifying βxcipients.

The solid oral foraa, e.g. tablets and capsules, aay contain, together with the active coapound, diluents, e.g., lactoaa, dextrose, aaccharoae, cellulose, corn atarch and potato atarch; lubricants, e.g. silica, talc, stearic acid, aagneaiua or calciua atearate, and/or polyethylene glycols; binding agents, e.g. atarchea, arabic guas, gelatin, aethyleelluloβe, carboxy- ■ethyl cellulose, polyvinylpyrrolidone; disaggregating agents, e.g. a atarch, alginic acid, alginates, aodiua atarch glycolate; effervescing aixtureβ; dyestuffs; sweeteners; wetting agenta, for instance, _* lecithin, polys_orbates, laurylaulphates; and, in general, non-toxic and pharaacologically inactive substances used in pharmaceutical foraulations. Said pharmaceutical prepara¬ tions aay be manufactured in a known Banner, for exaaple by aeans of aixing, granulating, tabletting, sugar-coating, or fila-coating processes. furthermore, according to the invention there la provided a method of treating viral infections and tuaora in a patient in need of it, cόapriβing adainistering to the βaid patient a com¬ position of the invention.

The following examples illustrate but do not limit the inven¬ tion.

The abbreviations DMF and THF stand, respectively, for dimethyl¬ formamide and tetrahydrofuran.

Example 1

To a solution of e rbromoacrylic acid (226 mg) in dry DMF (5 ml), N,N'-dicyclohexylcarbodiimide (228 mg) was added and the resulting suspension was stirred at room temperature for 20 minutes. The mixture was added to a solution of N-deformyl Distamycin A dihydrochloride (526 mg) in DMF (10 ml) and sodium bicarbonate (84 mg) .

The suspension was stirred at room temperature for 4 hours; after filtration, the solvent was evaporated in vacuum to dryness, The residue was chro atographed on silica gel with methylene- chloride:methanol 80:20 as eluant, affording N-deformyl-N- (t-A-bromoacryloyl) Distamycin A, hydrochloride (310 mg), U.V. λmax (EtOH 95') (£): 242 (23772), 312 (33961) n ;

FD -M.S.: m/z 586, M ⁺ +l; 568, M -NH ; 505, M -HBr;

N.M.R. (DMSC-d.):fe2.62(_H,t); 3.45 (2H,m); 3.81 (3H,s); 3.85 (6H,s); 6

6.20 (IH.d); 6.70 (lH,d); 6.9-7.3 (6H,m); 8.18 (lH.t); 8.6 (2H, bs);

8.96 (2H, bs); 9.88 (lH.s); 9.93 (lH.s); 10.29 (lH,s).

By analogous procedure the following compounds were obtained:

N-Deformyl-N-(^-chloroacryloyl) Distamycin A, hydrochloride,

U.V. Amax (EtOH 95%) (£) : 242 (23080), 310 (32972) nm ;

FD-MS: m/z: 542, M ⁺ +l; 505, M ⁺ -HC1; 524, M ⁺ -NH ;

N.M.R. (DMSO-d : _.2.65 (2H,t); 3.50 (2H,m); 3.80 (3H,s); 3.83 (3H,s); o

3.84 (3H,s); 5.98 (IH.d); 6.40 (lH,d); 6.90-7.30 (6H,m); 8.20 (lH.t); 8.75 (2H,bs); 9.04 (2H,bs); 9.89 (lH,s); 9.95 (1H, s); 10.32 (lH,s).

β-(N-methyl-4-(N-methyl-4-(N-methyl-4-(N-methyl-4-(v< -chloroacryl- a ido) pyrrole-2-carboxamido) pyrrole-2-carboxamido) pyrrole-2- carboxa ido) pyrrole-2-carboxamido) propionamidine, hydrochloride.

U.V. λmax (EtOH) 95°) ( ): 244 (30.055), 314 (46098) nm;

FAB-MS: m/z: 664, M ⁺ +l; 602, M ⁺ -CH -CC1- ;

N.M.R. (DMSO-d ):&2.62 (2H,t); 3.2-4.00 (14H,m); 5.99 (IH.d); 6.39 (IH.d); b

6.90-7.30 (8H,m); 8.20 (lH.t); 8.80 (2H,bs); 9.00 (2H,bs);

9.90 (2H,s); 9.93 (lH,s); 10.30 (lH,s). β- ( N-me thy 1-4- ( N-me thy l-4-( N-msthyl-4- ( N-π ^* et_hyl-4-.(_\-b-_roπoacrylamido) pyττOle-2-c_r_oxa_mido) pyrrole-2-carboxamido) pyrτole-2-car_ox___mido)pyr-role-

2-car_oxamido) propionamidine, hydrochloride _/

U.V. max (EtOH 95") (£): 242 (29876), 314 (45224) nm;

FAB-MS: m/z: 708, M ⁺ l; 628, M ⁺ -Br;

N.M.R. (DMS0-d _c ): .-2.63 (2H,t); 3.50 (2H,t); 3.80 (3H,s); 3.84 (3H,s); b

3.85 (6H,s); 6.19 (lH,d); 6.69 (IH.d); 6.90-7.25 (8H,m); 8.12 (lH,t); 8.63 (2H,bs); 8.89 (2H, bs); 9.80 (lH.s); 9.83 (lH.s); 9.86 (lH.s); 10.30 (lH.s).

3-(N-methyl-4-(N-methyl-4-(N-methyl-4-(N-methyl-4-(c, -chloro- acrylamido) pyrrole-2-carboxamido) pyrrole-2-carboxamido) pyrrole- 2-carboxamido) pyrrole-2-carboxamido ⁾ propyl-dimethylamine,

U.V. λmax (EtOH 95 ^e ): 239 (29707), 313 (43738);

FAB-MS : m z; 679, M+l; 589, M ⁺ -CH ₂ =CC1-C0-;

N.M.R. (DMSO-d.): l.66 (2H,m); 2.17 (6H,s); 2.25 (2H,t); 3.20 (2H,m);

3.80 (3H,s); 3.83 (9H,s); 5.99 (IH.d); 6.37 (IH.d);

6.75-7.30 (8H,m); 8.03 (lH,t); 9.83 (lH.s); 9.90 (lH.s);

9.92 (lH.s); 10.23 (lH,s).

^β -(N-methyl-4-(N-methyl-4-(N-methyl-4-(N-methyl-4-(N-me thyl-4- (e* -bromoacrylamido) pyrrole-2-carboxamido) pyrrole-2-carbox- a ido) pyrrole-2-carboxamido) pyrrole-2-carboxamido)pyrrole-2- -.carboxamido)propionamidine, hydrochloride, U.V. Λmax (EtOH 95") (_.): 240 (34947), 312 (53018); N.M.R. (DMSO-dg): _>2.61 (2H,t); 3.48 (2H,m); 3.82 (ISH.bs); 6.21 (IH.d);

, 6.80 (IH.d); 6.9-7.3 (lCH.m); 8.19 (lH.t); 8.73 (2H,bs); 8.93 (2H, bs); 9.90 (4H,bs); 10.28 (lH.bs).

Example 2

To a solution of (E)-β-(p-methoxybenzoyl)-β-bromo-acrylic acid (428 mg), prepared according to J.O. C.2_6 755 (1961), in dry D. M.F, (ml 10), cooled to 0 ^e C, N,N'-dicyclohexylcarbodiimide (288 mg) was added and the resulting solution was stirred at 0°C for 20 minutes.

N-Deformyl-Distamycin A-dihydrochloride (526 mg) was added, and the mixture was stirred for 30 minutes at 0°C, and then for 4 hours at room temperature. After filtration, the solvent was evaporated in vacuum to dryness, and the residue was chromato- graphed on silica gel with methylene chloride: methanol 80:20 as eluant affording N-deformyl-N-(E)-β-(p-methoxybenzoyl)β-bromo- acryloyl)-distamycin A, hydrochloride (265 mg)

U.V.Λmax (EtOH 95 ^e ) (_): 225 (33007), 304 (32704) nm

FAB-J-3: m/z 720, M ⁺ +l; 511,389,267

N.M.R. (_-MS0-d :&2.62 (2H,t); 3.48 (2H,m); 3.71 (3H,s); 3.74 (3H,s); o

3.79 (3H,s); 3.81 (3H,s); 6.68 (lH,s); 6.75-7.20 (6H,m); 6.88 (2H,m); 7.25 (2H,m); 8.19 (lH.t); 8.54 (2H,bs); 8.93 (2H,bs); 9.88 (lH.s); 9.90 (lH.s); 10.30 (lH.s).

By analogous procedure the following compound was obtained:

N-Deformyl-N-(4-(« ^* < -bromoacrylamido) benzoyD-Distamycin A, hydrochloride,

U.V. λmax (EtOH 95°) (£): 241 (31387), 311 (48156) nm;

N.M.R. (DMSO-d : 2.62 (2H,t); 3.45 (2H,m); 3.81 (3H,s); 3.85 (3H,s); b

3.86 (3H,s); 6.34 (IH.d); 6.84 (IH.d); 6.9-7.4 (6H,m); 7.7-8.1 (4H,m); 8.20 (lH,t); 8.72 (2H,bs); 9.00 (2H,bs); 9.90 (lH.s); 9.96 (lH,s); 10.30 (IH.s); 10.56 (lH.s).

Example 3

To a solution of acrylic acid (245 mg) in dry THF(iθml), cooled to -10 ^β C, triethylamine (0.47 ml) was added, and then pivaloyl chloride (0.41 ml).

The resulting suspension was stirred at -10°C for 20 minutes, then the whole was added to a cooled solution of N-deformyl

Distamycin A dihydrochloride (526 mg) in DMF (10 ml) and NaHCO

(84 mg).

The mixture was stirred for 30* at 0 ^β C, and then for 4 hours at room temperature.

The solvent was evaporated in vacuum to dryness, andthe residue was chromatographed on silica gel with methylene chloride methanol 80:20 as eluant, affording N-deformyl-N-acryloyl-

Distamycin A, hydrochloride (290 mg)^

U.V. max (EtOH 95°) (£): 242 (23229), 308 (34164)

FD-MS: m/z 508, M ⁺ +l; 490, M ⁺ -«H ; 437.

N.M.R. (DMS0-d ₆ ): b 2.63 (2H,t); 3.50 (2H,m); 3.81 (3H,s); 3.85 (3H,s);

3.89 (3H,s); 5.66 (lH.dd); 6.19 (IH.d d); 6.46 (IH.d d);

6.90-7.30 (6H,m); 8.20 (lH.t); 8.60-9.20 (4H,b); 9.88

(lH,s); 9.90 (lH,s); 10.18 (lH.s).

By analogous procedure the following compounds were obtained:

N-deformyl-N-( (Z)-β-chloroacryloyl) Distamycin A, hydrochloride

U.V. Λmax (EtOH 95°) (£): 243 (23254), 311 (35288) nm ;

FD-MS: m/z 541,M ⁺ ; 505, M ⁺ -HC1; 478;

N.M.R. (DMS0-d _c ): £>2.64 (2H,t); 3.50 (2H,m); 3.80 (3H,s); o

3.86 (6H,s); 6.52 (IH.d); 6.89 (lH,d); 6.9-7.3 (6H,m); 8.22 (lH.t); 8.85 (4H,b t); 9.89 (lH.s); 9.93 (lH.s); 10.28 (lH.s). N-deformyl-N-( (E)-β-chloroacryloyl) Distamycin A, hydrochloride

U.V. Λmax (EtOH 95°) (£,) : 241 (24584), 312 (35517) nm;

FD-MS: m/z 505, M* ^" -HC1, 478.

N.M.R. (DMSC-<i _e ): 2.64 (2H,t); 3.50 (2H,m); 3.80 (3H,s); 3.86 (6H,s); o

6.70 (IH.d); 7.30 (IH.d); 6.9-7.3 (6H,m); 8.22 (lH.t); 8 ^' .85 (4H,bt); 9.89 (lH.s); 9.93 (lH,s); 10.5 (lH.s).

Example 4

To a stirred solution of N-deformyl Distamycin A dihydrochloride (2.5 g) in water (30 ml) and dioxane (40 ml), sodium bicarbonate (2 g) was added with caution. The mixture was cooled to 5'C and then a solution of £- nitrobenzoyl chloride (2.5 g) in dioxane (25 ml) was added in 1 hour, with vigorous stirring.

The reaction mixture was stirred for 1 hour,acidified with ■HC12N to pH 4, and then evaporated to dryness in vacuum. ^' The residue was treated with acetone (200 ml), stirred for 1 hour and filtered, to obtain N-deformyl-N-(p-nitrobenzoyl) Distamycin A hydrochloride (2.6 g).

Example 5

The compound N-deformyl-N-(p-nitrobenzoyl) Distamycin A hydro¬ chloride (2.6 g) was dissolved into a mixture of CH OH (150 ml) and 2N HC1(10 ml) and reduced over a Pd catalyst (10% on carbon) under H pressure (50 p.s.i.) for 4 hours.

The catalyst was filtered and the resulting solution was con¬ centrated in vacuum to dryness.

The residue was treated with ethanol (10 ml), stirred for 1 hour and filtered, to obtain N-deformyl-N-(p-a inobenzoyl) Distamycin A, dihydrochloride (2 g),

N.M.R. (H-EO-dg): & 2.62 (2H,t); 3.45 (2H,m); 3.81 (3H,s); 3.85 (3H,s);

3.86 (_H,s); 6.90-7.40 (6H,m); 7.10-7.70 (4H,m); 8.20 (LH.t); 8.52 Orl.bs); 8.72 (_H,b_); 9.00 (2H,__); 9.90 (lH.s); 9.96 (lH.s); 10.30 (lH.s).

Example 6

Intramuscular injection 20 mg/ml

An injectable pharmaceutical composition can be manufactured by dissolving 20 g of β-(N-methyl-4-(N-methyl-4-(N-methyl-4- -(N-methyl-4-(e -bromoacrylamido)pyrrole-2-carboxamido)pyrrole- -2-carboxamido)pyrrole-2-carboxamido)pyrrole-2-carboxamido)p ro¬ pionamidine, hydrochloride in water for injection (1000 ml) and sealing ampoules of 1-5 ml.