The present invention relates to novel analogs of 1,2,4-triazolophthalazines and a process for preparing analogs of 1,2,4-triazolophthalazines, in particular analogs having medicinal properties.

Depending on their structure, analogs of 1,2,4-triazoles fused to 6-membered carbocyclic and heterocyclic rings exhibit different biological activity. The compounds based on l,2,4-triazolo[3,4-a]phthalazine are primarily known as antagonists of GABA receptors (mainly a3 and a5 subunits) and as the ligands having a high affinity for the receptors of calcium channel gates. They are characterized by, among others, anticonvulsant activity. In the review of literature derivatives of l,2,4-triazolo[4,3- 6]pyridazine are also indicated as the agonists of GABA receptors. Furthermore, these compounds are characterized by anticonvulsant and antiviral activity, as well as cytostatic properties. In contrary, derivatives of l,2,4-triazoIo[4,3-«]pyrimidine act as antagonists of adenosine receptors. Many of them exhibit also antitumour activity. This type of activity is assigned to sulfonamide, hydrazine and pyrazole derivatives. Other derivatives have anti-inflammatory, analgesic and antibacterial properties.

l,2,4-triazolo[3,4-Gc]phthalazines are typically prepared by the reaction of 1- chlorophthalazine with suitable hydrazides. Reactions of this type take place both in solutions and using solid carriers, which is known from several publications. Another method comprises a cyclization reaction of hydrazino-4-phthalazines with carboxylic acids, as described in the publication Aly A. A. Gad El-Karim I. A., Phosphorus Sulfur, 2005, 180, 1997-201 1. Synthesis of S-triazolo[3,4-ot]phthalazine and Related Polynuclear Heterocyclic Systems.

None of the described synthetic methods includes the preparation of fused triazole derivatives with 2,4-dihydroxyphenyl substituent at position 3 of the fused system or a modified form thereof.

The present invention relates to new analogs of 1,2,4-triazolophthalazines of the general formula I:

General formula I

with different type of substitution of 2,4-dihydroxyphenyl system, wherein: R,= H, HO-, Me-, MeO-

R ₂ = H, Me-, Et-, Pr-, iPr-, C1-, HO-, MeO-, EtO-

R ₃ = H, HO-, Me-, MeO-.

Because of their antiproliferative properties, among numerous possible analogs of 1,2,4-triazolophthalazines, the most important compounds include, among others:

• 3-(2,4-dihydroxyphenyl)-l,2,4-triazolo[3,4-ct]phthalazine,

• 3 -(3 -methyl-2,4-dihydroxyphenyl)- 1 ,2,4-triazolo [3 ,4-a]phthalazine,

• 3-(5-chloro-2,4-dihydroxyphenyl)-l,2,4-triazolo[3,4- ]phthalazine,

• 3-(5-ethyl-2,4-dihydroxyphenyl)-l,2,4-triazolo[3,4-ot]phthal azine,

• 3-(2,3,4-trihydroxyphenyl)-l,2,4-triazolo[3,4-a]phthalazine,

• 3-(5-methyl-2,4-dihydroxyphenyl)-l,2,4-triazolo[3,4-a]phthal azine.

A process for preparing compounds of a group of 3-aryl-substituted 1,2,4- triazoles is based on a reaction of heteroarylhydrazines with a number of modified thioaryloylating agents E. The consecutive processes running "on pot" are a terminal thiohydrazide transformation, equilibrium regiospecific anular rearrangement and thione/thiol isomerization, conditioning endocyclization by eliminating H ₂ S.

Linear thiohydrazide moieties of intermediary compounds substituted to heterocyclic systems can be treated as ambident acids (or anions), which release readily leaving SH ^" ions deprotonating nitrogen atoms. At appropriate basicity and stereoaccessibility of nitrogen atoms of the heterocyclic system, the rate and yield of the reaction also determines the appropriate substitution of thiocarbonyl system of electrophilic reagents.

The fundamental of the process for preparation of analogs of 1 ,2,4- triazolophthalazines of the general formula I, wherein substrates are introduced into the solution, and then the product is isolated consists in that 1-hydrazinophthalazine hydrochloride is reacted in monohydric alcohol with the addition of pyridine with the substance of the general formula II:

General formula II

wherein: R = H, Me, Et, Pr, iPr, CI, OH, MeO, Et, and subsequently the resultant mixture is separated into filtrate I and precipitate I, then filtrate I is concentrated, while the resultant precipitate II is crystallized from a monohydric alcohol.

In the first embodiment the resultant mixture is separated into filtrate I and precipitate I, then filtrate I is concentrated and filtrated, while the resultant precipitate II is combined with precipitate III obtained after adding water to filtrate II, and then the combined precipitates are subjected to crystallization from a monohydric alcohol.

In the second embodiment, when the improvement of yield is needed, the resultant mixture is separated into filtrate I and precipitate I, then filtrate I is concentrated and filtrated and the resultant precipitate II is combined with precipitate I, and then the combined precipitates are subjected to crystallization from a monohydric alcohol.

In contrary, in the third embodiment, the resultant mixture is separated into filtrate I and precipitate I, then filtrate I is concentrated and filtrated and the resultant precipitate II is subjected to crystallization from a monohydric alcohol. Filtrate I is preferably concentrated to dryness, then a monohydric alcohol is added, the mixture is stored for a specified time and then precipitate II is filtrated.

Preferably, the reaction is conducted at the reflux temperature of the mixture of the reagents in a monohydric alcohol. A monohydric alcohol is methanol or ethanol.

Depending on the location and type of R substituent, substance E can be in the form of:

STB: bis[(2,4-dihydroxyphenyl)methanethione] sulfoxide

S3MTB: bis[(2,4-dihydroxy-3-methylphenyl)methanethione] sulfoxide

S 5 MTB : bi s [(2 ,4-dihydroxy- 5 -methylphenyl)methanothione] sulfoxide

SETB: bis[(5-ethyl-2,4-dihydroxyphenyl)methanothione] sulfoxide

SC1TB: bis[(5-chloro-2,4-dihydroxyphenyl)methanothione] sulfoxide

S3TTB: bis[(2,3,4-trihydroxyphenyl)methanothione] sulfoxide

Mechanism of the reaction of thioaryloyl hydrazinephthalazine cyclocondensation to l,2,4-triazolo[3,4-oc]phthalazine system is presented in the Scheme.

Scheme

Transitional isomerizing linear forms may be considered a part of a rigid, sufficiently coplanar system and they have no possibility of free rotation across a multiple bond, which facilitates the processes of endocyclization. Isomerization rearrangements cause changes in the electronic structure, dipole moment and the shape of the equipotential surface. In the case of multicentric basicity of nucleophilic reagents, the condition is a suitable location of NHN¾ moiety, allowing for a directional protonation/deprotonation of nitrogen atoms and stabilization by forming geometrically favored rings.

In search for the correct directions of phenolic substituent modification, the ring was provided with the groups of a variable electron characteristic (polarity), taking into consideration a greater opportunity of approximating the molecule to hydrogen binding sites or location in hydrophobic spaces, given the function of benzenediol (benzenetriol) in interaction with molecular targets. This direction of -R changes includes also or above all the impact of substitution on the course of subsequent intermediary reactions, as well the properties of the final product.

Within these series also corresponding 2,3,4- and 2,4,6-trihydroxylphenyl analogs were obtained. It was interesting, because these directions of substitution affect both the nature of electronic and steric interactions in every area of hydrogen bonds and changes in particle energy.

The invention is further illustrated by the following examples.

Example 1

3-(2, 4-dihydroxyphenyl)-l , 2, 4-triazolo[3, 4-a Jphthalazine of Formula III.

rmula III

0.0013 moles of 1 -hydrazinophthalazine hydrochloride and 0.0013 moles of STB were transferred into 6.5 ml of methanol with the addition of pyridine (0.65 ml) and heated at reflux temperature (3 h). Hot reaction mixture was filtered through a Buchner funnel. Precipitate I and filtrate I were obtained. Filtrate I was concentrated, filtered and the resultant precipitate II was combined with precipitate III isolated from filtrate II obtained after addition of 6 ml of water. Subsequently, the combined precipitates were recrystallized from methanol (4 ml). The compound of Formula III was obtained.

Yield 86%, melting point 294-295°C. For the formula: Ci ₅ Hi ₀ N ₄ O ₂ , M=278.27 calculated C, 64.74; H, 3.62; N, 20.13; obtained: C, 64.81 ; H, 3.61 ; N, 20.19.

IR (KBr, cm ^"1 ): 3417 (OH), 1626 (C=N), 1475, 1334, 1251, 1 131, 977, 640; Ή NMR (500 MHz, CDCI3, S, ppm): 14.26 (s, 1H, C ₂ >-OH-N), 10.31 (s, 1H, C ₄ >-OH), 9.13 (s, 1H, C ₆ -H), 8.99 (m, 2H, C _AR -H), 8.24-8.09 (m, 3H, C _AR -H), 6.45 (s, 1H, J=2.2 Hz, C ₃ >- H), 6.38 (s, 1H, J=8.6 Hz and J=2.2 Hz, C ₅ -H); MS (EI, m/z): 279 (M+l, 100), 278 (M ⁺ , 87), 263 (6), 221 (10), 171 (59), 145 (49), 117 (17), 103 (9), 89 (13), 69 (5), 65 (6), 51 (7), 39 (9).

Example 2

3-(3-methyl-2, 4-dihydroxyphenyl)-l , 2, 4-triazolo[ 3, 4-a Jphthalazine of Formula IV.

Formula IV

0.0013 moles of 1 -hydrazinophthalazine hydrochloride and 0.0013 moles of S3MTB were transferred into 10.5 ml of methanol with the addition of pyridine (1 ml) and heated at reflux temperature (3 h). The reaction mixture was stored at room temperature (24 h), and then filtered through a Buchner funnel. Precipitate I and filtrate I were obtained. Filtrate I was concentrated, obtaining precipitate II, which was combined with precipitate I and subsequently the combined precipitates were recrystallized from methanol (6 ml). The compound of Formula IV was obtained.

Yield 80%, melting point 198-199°C. For the formula: Ci ₆ Hi ₂ N ₄ 0 ₂ , M=292.29 calculated C, 65.75; H, 4.14; N 19.17; obtained: C, 65.81 ; H, 4.12; N, 19.23.

IR (KBr, cm- ¹ ): 3213 (OH), 3030 (C _AR -H), 1621 (C-N), 1571 (C=C), 1501, 1481, 1379, 1316, 1252, 1197 (C-OH), 1 158, 1083, 1028, 999, 917, 901, 801 , 783, 763.

Ή NMR (500 MHz, CDC1 ₃ , S, ppm): 13.92 (s, 1H, C ₂ >-OH), 12.15 (s, 1H, C4-OH), 9.19 (s, 1H, C ₆ -H), 8.95 (m, 2H, CA _T -H), 8.48 (s, 1H, J=8.8 Hz, 1H, C ₆ <-H), 8.10 (m, 2H, CAR-H), 6.55 (d, J=8.8 Hz, 1H, C ₅ >-H), 2.11 (s, 3H, CH ₃ ); MS (EI, m/z): 294 (M+2, 96), 293 (M+l, 81), 292 (M ⁺ , 19), 265 (6), 237 (3), 209 (4), 171 (100), 146 (17), 145 (53), 131 (8), 1 17 (18), 103 (9), 89 (8), 77 (9), 63 (4), 51 (5), 36 (8).

Example 3 3-(5-methyl-2, 4-dihydroxyphenyl)-l , 2, 4-triazolo[3, 4-aJphthal zine of Formula V.

Formula V

0.0013 moles of 1-hydrazinophthalazine hydrochloride and 0.0013 moles of bis[(2,4- dihydroxy-5-methylphenyl)methanothione] sulfoxide (S5MTB) were transferred into 10.5 ml of ethanol with the addition of pyridine (1 ml) and heated at reflux temperature (3.5 h). The reaction mixture was stored at room temperature (24 h), and then filtered through a Biichner funnel. Precipitate I and filtrate were obtained. The filtrate was concentrated and precipitate I was combined with precipitate II isolated from the filtrate and the combined precipitates were recrystallized from methanol (6 ml). The compound of Formula V was obtained.

Yield 71%, melting point 242-243°C. For the formula: Ci ₆ H, ₂ N ₄ 0 ₂ , M=292.29 calculated C, 65.75; H, 4.14; N 19.17; obtained: C, 65.81 ; H, 4.1 1 ; N, 19.1 1.

IR (KBr, cm ^"1 ): 3213 (OH), 3030 (C _AR -H), 1621 (C-N), 1571 (C=C), 1501 , 1481 , 1379, 1316, 1252, 1 197 (C-OH), 1 158, 1083, 1028, 999, 917, 901 , 801 , 783, 763.

Ή NMR (500 MHz, DMSO-</ ₆ , δ, ppm): 10.86 (s, 1H, C ₂ -OH), 9.98 (s, 1H, C ₄ -OH), 9.19 (s, 1H, C ₆ -H), 8.54 (m, 2H, C _Ar -H), 8.27 (m, 2H, C _A R-H), 7.80 (s, 1H, C ₃ -H), 6.55 (s, 1H, C ₆ -H), 2.09 (s, 3H, CH ₃ ); MS (EI, m/z): 292 (M ⁺ , 100), 263 (9), 221 (5), 160 (4), 144 (5), 137 (4), 129 (3), 1 17 (4), 89 (4), 63 (6), 44 (6), 39 (3).

Example 4

3-(5-ethyl-2, 4-dihydroxyphenyl)- 1 , 2, 4-triazolo[ 3, 4-a Jphthalazine of Formula VI.

Formula VI

0.0013 moles of 1-hydrazinophthalazine hydrochloride and 0.0013 moles of SEBT were transferred into 6.5 ml of methanol and heated at reflux temperature for 3 h. Hot reaction mixture was filtered through a Biichner funnel. Precipitate I and filtrate were obtained. The filtrate was concentrated and precipitate I was combined with precipitate II isolated from the filtrate, and the mixture of both precipitates was recrystallized from methanol (4 ml). The compound of Formula VI was obtained. Yield 78%, melting point 312-314°C. For the formula: C, ₇ H, ₄ N ₄ 0 ₂ , M=306.32 calculated C, 66.66; H, 4.61; N 18.29; obtained: C, 66.58; H, 4.62; N, 18.25.

IR (KBr, cm ^"1 ): 3425 (OH), 1630 (C=N), 1536 (C=C), 1506, 1411, 1266, 1 185 (C-OH), 1 149, 905, 759; Ή NMR (500 MHz, CDC1 ₃ , δ, ppm): 11.18 (s, IH, C ₂ -OH), 9.92 (s, IH, C4-OH), 9.17 (s, IH, C ₆ -H), 8.56 (d, J=7.9 Hz, IH, C ₁₀ -H), 8.24 (d, J=7.7 Hz, IH, C ₇ -H), 8.18 (s, IH, C ₆ -H), 8.09 (td, IH, C ₉ -H), 7.97 (td, IH, C ₈ -H), 6.54 (s, IH, C ₃ <-H), 2.56 (q, 2H, CH ₂ CH ₃ ), 1.17 (t, 3H, CH?CH ₃ ; MS (EI, m/z): 306 (M ⁰⁺ , 51), 291 (M ⁺ - CH ₃ °, 100), 279 (4), 212 (6), 196 (6), 162 (40), 146 (28), 144 (12), 130 (10), 1 17 (20), 103 (14), 89 (36), 77 (15), 75 (10), 71 (6), 69 (20), 63 (16), 52 (29), 51 (2), 44 (39), 40 (36), 39 (20), 38 (26). 36 (86).

Example 5

3-(5-chloro-2, 4-dihydroxyphenyl)-l, 2, 4-triazolo[3, 4-a Jphthalazine of Formula VII.

Formula VII

0.0076 moles of 1-hydrazinophthalazine hydrochloride and 0.0076 moles of bis[(5- chloro-2,4-dihydroxyphenyl)methanothione] sulfoxide (SCITB) were transferred into 38 ml of methanol with the addition of pyridine (3.8 ml) and heated at reflux temperature (3 h). Hot reaction mixture was filtered. Precipitate I and filtrate were obtained. The filtrate was concentrated to dryness, then 16 ml of methanol were added, and the mixture was stored for 24 h at room temperature. The isolated solid product (precipitate II) was filtered and recrystallized from methanol (25 ml). The compound of Formula VII was obtained.

Yield 77%, melting point 334-335°C. For the formula: Ci ₅ H ₉ ClN ₄ 0 ₂ , M=312.71 calculated C, 57.61 ; H, 2.90; N 17.92; obtained: C, 57.70; H, 2.92; N, 17.87.

IR (KBr, cm ^-1 ): 3398 (OH), 3234 (OH), 3041 (C _AR -H), 2984 (C-H), 1626 (C=N), 1598 (C=C), 1512 (C=C), 1444, 1375, 1308, 1273, 1242, 1217 (C-OH), 1 156, 1 124, 1093, 1045, 1024, 978, 920, 891, 857, 806, 761, 718. 1H NMR (500 MHz, DMSO-i ₆ , δ, ppm): 14.34 (s, IH, C ₂ -OH), 10.91 (s, IH, C4 -OH), 9.05 (s, IH, C ₆ -H), 8.96 (m, IH, C _Ar -H), 8.45 (s, IH, C ₆ -H), 8.37 (s, IH, C ₆ -H), 8.21 (m, I H, C _Ar -H), 8.14 (m, IH, C _Ar -H), 6.71 (s, IH, C ₃ <-H); MS (El, m/z): 312 (100), 278 (4), 249 (2), 221 (2), 144 (4), 1 15 (8), 88 (10), 63 (5), 39 (7).

Example 6

3-(2,3,4-trihydroxyphenyl)-l,2,4-triazolo[3,4-a]phthalazine of the Formula VIII.

Formula VIII

0.0013 moles of 1 -hydrazinophthalazine hydrochloride and 0.0013 moles S3TTB were transferred into 6.5 ml of methanol with the addition of pyridine (0.65 ml) and heated at reflux temperature for 3 h. Hot reaction mixture was filtered through a Buchner funnel. Precipitate I and filtrate I were obtained. Filtrate I was concentrated and filtered to give precipitate II, which was combined with precipitate III isolated from filtrate II obtained after the addition of 6 ml of water. The combined precipitates were recrystallized from methanol (4 ml). The compound of Formula VIII was obtained.

Yield 64%, melting point 220-222°C. For the formula: C ₁₅ H, ₀ N ₄ O ₃ , M=294.26 calculated C, 61.22; H, 3.43; N 19.04; obtained: C, 61.30; H, 3.45; N, 18.99.

IR (KBr, cm ^"1 ): 3236, 3132 (OH), 1615 (C=N), 1505 (C=C), 1493, 1456, 1423, 1388, 1340, 1266, 1214, 1 179 (C-OH), 1 137, 1109, 1042, 1017, 978, 955, 903, 876, 850, 786, 716. 'H NMR (500 MHz, CDC1 ₃ , 3, ppm): 14.17 (s, 1H, C ₂ '-OH~N), 11.96 (s, 1H, C ₂ - OH), 9.99 (s, 1H, C ₄ -OH), 9.44 (s, 1H, C ₃ -OH), 9.21 (s, 1H, C ₆ -H), 8.56 (m, 2H, C _AR - H), 8.15 (m, 2H, C _A R-H), 8.05 (d, 1H, C ₆ -H), 6.71 (d, J=8.7 Hz, 1H, C ₅ -H), MS (EI, m/z, %): 294 (M ⁺ , 100), 265 (5), 153 (13), 144 (8), 126 (35), 1 17 (4), 108 (10), 105 (5), 88 (4), 79 (12), 52 (18), 44 (22), 36 (8).

1 -hydrazinophthalazine hydrochloride used in the examples above was purchased according to Alfa Aesar catalog, Cat. No. B22995 or Sigma, Cat. No. HI 753.

Comparative example

The obtained compounds were examined for their antitumour activity. To this end tests of antiproliferative activity against such human cancer cells as: bladder cancer HCV 29T, lung cancer A549, breast cancer T47D and colon adenocarcinoma SW707 were performed. The results of the experiments in the form of ID ₅₀ (the dose inducing a 50% inhibition of proliferation of the tumour cell population), determined for each compound are summarized in Table 1. An accepted activity criterion for novel compounds in in vitro screening tests is the ID ₅₀ level not higher than 4 μg/ml [Geran RI et al. Cancer Chemotherapy Reports, 3, 2 (part3):59-61, 1972].

SRB Cytotoxicity Assay

The tests were performed using the SRB cytotoxicity assay, which measures the inhibition of target cell proliferation in 72-hour in vitro culture. [Skehan et al. J. Natl. Cancer Inst., 82: 1 107-1 1 12, 1990]. Stock solutions of the test compounds at a concentration of 1 mg/ml were prepared ex tempore for each experiment dissolving 1 mg of the preparation in 100 μΐ DMSO + 900 μΐ of culture medium. The solvent for further dilutions was culture medium. The compounds were tested at concentrations of 100, 10, 1, 0.1 μg/ml. Cisplatin at concentrations of 100, 10, 1, 0.1 μg/ml and DMSO at concentrations corresponding to its concentration in the samples of the compounds: 1, 0.1, 0.01 and 0.001% were applied as a control. In each experiment samples containing specified concentrations of the preparation were applied in triplicate. The experiments were performed in triplicate.

Cell lines

In the experiments cell lines from the cell line bank of the Institute of Immunology and Experimental Therapy of Polish Academy of Sciences in Wroclaw were used. The cells were cultures in RPMI + opti-MEM (1 :1) medium supplemented with 5% FBS, 2 mM of glutamine, 1 mM of sodium pyruvate and 0.8 mg/1 of insulin in the case of the T47D line. All media contained antibiotics: 100 mg/ml of streptomycin and lOO U/ml of penicillin. The cells were cultured in humidified atmosphere of 5% C0 ₂ at 37°C.

Table 1. Antiproliferative activity of the compounds