rivatives of Acetic and Butanoic Acid

Send Orders for Reprints to [email protected] Letters in Drug Design & Discovery, 2014, 11, 000-000

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Synthesis and Analgesic Activity of 3,7-dimethylpurine-2,6-dion-1-yl Derivatives of Acetic and Butanoic Acid Magorzata Zygmunta,*, Pawe mudzkib, Gra yna Cho-Rzepab, Jacek Sapaa and Maciej Pawowskib a

Department of Pharmacological Screening, Chair of Pharmacodynamics, Jagiellonian University Medical College, 9 Medyczna, 30-688 Kraków, Poland b

Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna, 30-688 Kraków, Poland Abstract: Hydrazones are a group of compounds possessing diversified biological activity, anti-inflammatory and analgesic activities. There are also known xanthine derivatives possessing such activity. The aim of our study was to investigate if introduction of hydrazone moiety to 3,7-dimethylpurine-2,6-dion-1-yl acetic and butanoic acid derivatives would enhance the analgesic activity. The designed series of compounds were synthesized in a multi-step procedure. Their pharmacological activity was investigated in the writhing syndrome test. Based on the results the structure-activity relationship was discussed. From the synthesized group of twenty compounds, nineteen were tested in vivo. The analgesic activity of most compounds, except for compound 4, was higher than for acetylsalicylic acid in the writhing syndrome test. Our study showed that the introduction of hydrazone moiety generally enhances analgesic activity of xanthine derivatives, compared to derivatives with free carboxylic group, ester, benzylamide and hydrazide moieties. The presence of hydroxyl moiety or substituent with high electron density does not seem to be necessary for the activity of hydrazone derivatives.

Keywords: Analgesic activity, Hydrazones, Theobromine, Xanthines INTRODUCTION Non-steroidal anti-inflammatory drugs (NSAIDs) are very often used in the treatment of pain and inflammation. The application of non-steroidal anti-inflammatory drugs NSAIDs is limited due to variety of side effects. Additionally, leukotrienes, particularly LTB4 which are the products of 5-LO (5-lipoxygenase) together with decrease in prostaglandin formation are incriminated in the acute ulceration induced by NSAID’s. Accordingly, compounds that obtain dual inhibition of COX and 5-LO enzymes can show safer profile of activity and enhanced efficacy in the battle of pain in inflammatory diseases. Therefore the research on preparing new analgesic agents is important [1-4]. Some reports suggest that the hydrazone moiety is a pharmacophore group for the inhibition of COX and 5-LO. According to some reports about the analgesic activity of hydrazide and hydrazine derivatives a new series of these compounds were synthesized in order to obtain new structures with analgesic activity [ 4-6]. Hydrazones are a group of compounds possessing several different biological activities, such as: anticonvulsant, antidepressant, antimalarial, antimicrobial, antimycobacterial and other activities [6-10]. Interestingly, some hydrazones have been also reported as potent anti-inflammatory and/or analgesic agents with comparable or even greater potency *Address correspondence to this author at the Department of Pharmacological Screening, Chair of Pharmacodynamics, Jagiellonian University Medical College, 9 Medyczna, 30-688 Kraków, Poland; Tel: +48 12 62-05-547; Fax: +48 12 62-05-530; E-mail: [email protected]

1570-1808/14 $58.00+.00

than currently used NSAIDs (non-steroid anti-inflammatory drugs), such as salicylic acid and its derivatives (aspirin, salicylamide), ibuprofen or indomethacin [4-6]. Barja-Fidalgo has a reported series of N-phenylpirazole arylhydrazone derivatives possessing high anti-inflammatory activity [5]. Their experiments suggested, that for this series of compounds not only the presence of electronegative substituent in aryl moiety, but also its position were crucial. Compound I (Fig. 1) with 4-hydroxy-3-methoxyphenyl substituent showed the highest activity. Authors suggested that the mechanism of its activity was based on inhibition of 5lipooxygenase (5-LO). Other series of potential 5-LO inhibitors, possessing 3,5-di-tert-butyl-4-hydroxyphenyl moiety, was investigated by Cuadro [4]. In case of these compounds hydrazine part also contained heterocyclic ring, such as thiazole, pyridine, pyrazine or pirymidines, with hydrophobic substituent. Researchers have noticed, that hydrophobic substituent at heterocyclic moiety was not necessary for antiinflammatory activity of this series of compounds, but might strongly influence it. Compound II (Fig. 1) with 4,6dibutylpirymidin-2-yl moiety showed the highest inhibiting activity (IC50 = 0.084 μM). Bispo Júnior has reported two compounds, H2LASSBio-466 and H2LASSBio-1064 (Fig. 1), with high anti-inflammatory and analgesic activity, comparable to indomethacin [6]. These compounds possessed ohydroxyphenyl moiety in their structure. Salgın-Göken has found highly active 2-[2-(5-methybenzoxazolin-2-one-3yl)acetyl]benzylidenenhydrazine derivatives (IV and V, Fig. 2) with anti-inflammatory and analgesic activity comparable to aspirin or morphine, respectively [12].

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Letters in Drug Design & Discovery, 2014, Vol. 11, No. 10

Zygmunt et al.

NO2 N

N

N

N H

N

N

N NH

O OH

HO

I

II

O N

R1 H2LASSBio-466 R1=Cl, R2=H H2LASSBio-1064 R1=H, R2=Cl

N H

OH

R2

Fig. (1). Chemical formulas of compounds I, II and H2LASSBio-466, H2LASSBio-1064 [4-6].

O H N

N

O N

O

O O

N

N

IV R=Cl V R=CH3

O

O

N

N

R

VI

Fig. (2). Chemical formulas of compounds IV, V and VI [12, 13].

From methylxanthine derivatives ethyl 4-(7-methyl-2,6dioxo-3-propyl-2,3,6,7-tetrahydro-1H-purin-1-yl)butanoate (VI, Fig. 2) exhibited high anti-inflammatory activity by inhibiting synthesis of TNF (IC50 = 5 μM) [13]. Cottam demonstrated that replacement of 3-propyl substituent with methyl moiety did not change significantly inhibition of synthesis of TNF (IC50 = 6 μM), but influenced inhibiting activity of phosphodiesterase type IV, whereas changing butanoic acid with acetic acid strongly reduced this activity (IC50 = 200 μM). Free acids showed even lower activity (IC50 > 200 μM). The aim of our study was synthesis and biological evaluation of compounds possessing both above mentioned elements, that is hydrazone moiety and xanthine core. The synthesized 2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro1H-purin-1-yl)acetic acid and 4-(3,7-dimethyl-2,6-dioxo2,3,6,7-tetrahydro-1H-purin-1-yl)butanoic acid derivatives were submitted for biological evaluation in order to determine their analgesic activity. Since the pyrimidino-2,4-dione moiety of these compounds resembles oxazolin-2-one moiety of earlier mentioned compounds IV and V, we wanted to examine if introduction of hydrazone moiety enhances activity of these acids. Moreover for comparison we wanted to examine the activity of ethyl esters and hydrazide derivatives of both acids, as well as free acids themselves. In order to establish whether the hydrazone moiety is necessary for high

analgesic activity we also replaced it with N-benzylamide moiety. In order to determine the influence of position of hydroxy substituent we have synthesized series of hydrazones with o- and p-hydroxyphenyl moieties. We also synthesized a series of derivatives with o- and p-nitrophenyl moieties, to determine if replacement of hydroxy group by other substituent with high electron density will affect the activity of these compounds. We have also synthesized and evaluated the biological activity of phenyl derivatives, to determine if presence of substituent with high electron density is necessary for analgesic activity of these compounds. MATERIALS AND METHODS Chemistry Structures of the investigated compounds and their syntheses are presented in (Schemes 1 and 2). Melting points (mp) were determined with a Büchi Melting Point B-545 apparatus and are uncorrected. 1

H-NMR spectra were taken with a Varian Mercury-VX (300MHz) spectrometer in DMSO-d6 solutions, using signal of solvent’s residual 1H atoms as internal standard ( = 2.48 ppm). Chemical shifts were expressed in (ppm) and the coupling constants J in Hertz (Hz). Values of for both isomers E and Z are identical if not described differently. Num-

Analgesic 3,7-dimethylpurine-2,6-dion-1-yl Derivatives

N

O


3

N

HN

N O a

O

O

N

N

N

O

b

O H2N

N

n O

O

N

N

N

N H

c

O

R1

N

N

n

N N

N H

R2

O

1 n=1 2 n=3

O

N O

11 n=3, R1=H, R2=H 12 n=3, R1=H, R2=CH3 13 n=3, R1=o-OH, R2=H 14 n=3, R1=p-OH, R2=H 15 n=3, R1=o-NO2, R2=H 16 n=3, R1=p-NO2, R2=H

5 n=1, R1=H, R2=H 6 n=1, R1=H, R2=CH3 7 n=1, R1=o-OH, R2=H 8 n=1, R1=p-OH, R2=H 9 n=1, R1=o-NO2, R2=H 10 n=1, R1=p-NO2, R2=H

3 n=1 4 n=3

N

Scheme (1). Reagents and conditions: (a) ethyl 2-chloroacetate or ethyl 4-chlorobutyrate, K2CO3, TEBA, acetone, reflux; (b) hydrazine hydrate, ethanol, reflux; (c) appropriate aldehyde or ketone, conc. HCl, methanol, rt. O

N

N

HN

N O a

O O

O

N N

N N

n

O

b HO

O

N

N

N

N

n

O 1 n=1 2 n=3

c

O N H

O

N N

N N

n

O

O

17 n=1 18 n=3

19 n=1 20 n=3

Scheme (2). Reagents and conditions: (a) ethyl 2-chloroacetate or ethyl 4-chlorobutyrate, K2CO3, TEBA, acetone, reflux; (b) KOH, acetonewater (1:2, v/v), reflux, conc. HCl; (c) benzylamine, CDI, DMF, rt.

ber of 1H protons per peak was calculated for each isomer separately. E/Z isomers ratio was calculated using signals for protons of methyl group at N3 purine nitrogen atom or proton at C8 purine carbon atom. LC/MS analyses were performed on Waters Acquity TQD apparatus with e DAD detector. For mass spectrometry ESI+ (electrospray positive) ionization mode was used. UV spectra were taken in 200 – 700 nm range. For establishing the purity of compound UV chromatograms were used. All the investigated compounds have purity over 95%. All the compounds were routinely checked by TLC using Merck Kieselgel 60 F254 sheets with the following eluents: A: methylene chloride/methanol = 95:5 (v/v), B: methylene

chloride/methanol = 92.5:7.5 (v/v), C: methylene chloride/methanol = 90:10 (v/v). Spots were detected by UV absorption. Elemental analyses were taken with Elementar Vario EL III apparatus. All analyses were within ±0.4% of the theoretical values. A General Procedure for Preparing Ethyl -(3,7dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)alkylcarboxlates (1, 2) Mixture of 1 eq. (0.1 mol) of 3,7-dimethyl-1H-purine2,6(3H,7H)-dione, 1.5 eq. of appropriate ethyl chloroalkylcarboxylate, 2 eq. of K2CO3 and 0.1 eq. TEBA in

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Zygmunt et al.

acetone (100 ml) was refluxed for 20-40 h. The mixture was evaporated under reduced pressure and the solid residue was washed with water, than it was filtered off and purified by crystallization from ethanol.

amounts of HCl (2 drops of concentrate acid) in room temperature for 2 days. Afterwards water was added to the reaction mixture and the precipitate was filtered off. Crude product was purified by column chromatography.

Ethyl 2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)acetate (1) [13]

N'-benzylidene-2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)-acetohydrazide (5) [14]

From 3,7-dimethyl-1H-purine-2,6(3H,7H)-dione in 83% yield: mp 165.6 – 167.4 °C (lit. 162 – 165 °C) , Rf = 0.42 (A), 0.51 (B), 0.61 (C). LC/MS: purity 95.1%, m/z calc. 267.11, found 267.14. Analysis for C11H14N4O4 (266.25): C, H, N.

From 3 in 96% yield: mp 271.9 – 273.7 °C, Rf = 0.29 (A), 0.33 (B), 0.43 (C). LC/MS: purity 97.8%, m/z calc. 341.14, found 341.19. Analysis for C16H16N6O3 (340.34): C, H, N.

Ethyl 4-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)butanoate (2) [13] From 3,7-dimethyl-1H-purine-2,6(3H,7H)-dione in 63% yield: mp 80.8 – 82.2 °C (lit. 73 – 75 °C), Rf = 0.37 (A), 0.47 (B), 0.59 (C). LC/MS: purity 95.6%, m/z calc. 295.14, found 295.16. Analysis for C13H18N4O4 (294.31): C, H, N. A General Procedure for Preparing -(3,7-dimethyl-2,6dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)alkylohydrazides (3, 4) Mixture of 1 eq. (10 mmol) of appropriate ethyl -(3,7dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)alkylcarboxlate (1, 2) and 5 eq. of hydrazine hydrate in dry ethanol (30 ml) was refluxed for 10 h. Afterwards the reaction mixture was cooled and a precipitate of product was filtered off and washed with small amount of water. Hydrazides were crystallized from methanol.

2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)-N'-(1-phenyl-ethylidene)acetohydrazide (6) From 3 in 85% yield: mp 281.1 – 282.7 °C, Rf = 0.31 (A), 0.37 (B), 0.47 (C), E/Z ratio < 0.1, 1H-NMR 2.26 (s, 3H, CH3C=N), 3.43 (s, 3H, N3-CH3), 3.88 (s, 3H, N7-CH3), 5.01 (s, 2H, N1-CH2), 7.35 – 7.46 (m, 3H, 2,4,6-Ph), 7.73- 7.84 (m, 2H, 3,5-Ph), 8.06 (s, 1H, C8-H), 10.89 (s, 1H, CONH). LC/MS: purity 97.3%, m/z calc. 355.15, found 355.22. Analysis for C17H18N6O3 (354.36): C, H, N. 2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)-N'-(2-hydroxy-benzylidene)acetohydrazide (7) [14] From 3 in 71% yield: mp 294.5 – 295.3 °C, Rf = 0.31 (A), 0.39 (B), 0.51 (C). LC/MS: purity 100%, m/z calc. 357.13, found 357.15. Analysis for C16H16N6O4 (356.34): C, H, N. 2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)-N'-(4-hydroxy-benzylidene)acetohydrazide (8) [14]

2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)acetohydrazide (3) [14]

From 3 in 92% yield: mp 299.1 – 301.1 °C, Rf = 0.10 (A), 0.22 (B), 0.31 (C). LC/MS: purity 100%, m/z calc. 357.13, found 357.15. Analysis for C16H16N6O4 (356.34) C, H, N.

From 1 in 96% yield: mp 379.4 – 381.1 °C, Rf = 0.06 (A), 0.12 (B), 0.20 (C). LC/MS: purity 100%, m/z calc. 253.10, found 253.11. Analysis for C9H12N6O3 (252.23): C, H, N.

2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)-N'-(2-nitro-benzylidene)acetohydrazide (9)

4-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)butanehydrazide (4) From 2 in 72% yield: mp 197.2-197.8 °C, Rf = 0.23 (A), 0.31 (B), 0.43 (C), 1H-NMR 1.74 – 1.88 (m, 4H, CH2CH2 CH2CO), 3.39 (s, 3H, N3-CH3), 3.86 (s, 3H, N7 CH3), 3.88 (t, 3J = 6.4 Hz, 2H, N1-CH2), 7.99 (s, 1H, C8-H), 9.87 (s, 1H, CONHNH2). LC/MS: purity 98.9%, m/z calc. 281.14, found 281.17. Analysis for C11H16N6O3 (280.28): C, H, N. A General Procedure for Preparing N'-arylidene-4-(3,7dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)alkylhydrazides (5 – 16) Mixture of equimolar amounts of -(3,7-dimethyl-2,6dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)alkylohydrazide 3 or 4 (1 mmol) with appropriate aldehyde (benzaldehyde, 2hydroxybenzaldehyde, 4-hydroxybenzaldehyde, 2-nitrobenzaldehyde or 4-nitrobenzaldehyde) or ketone (acetophenone) was stirred in methanol (10 ml) in presence of catalytic

From 3 in 81% yield: mp 309.2 – 311.1 °C, Rf = 0.26 (A), 0.36 (B), 0.41 (C), E/Z ratio = 0.29, 1H-NMR 3.43 (s, 3H, N3-CH3), 3.87 (s, 3H, N7-CH3), 4.59 (s, 2H, (E)-9, N(1)CH2), 4.96 (s, 2H, (Z)-9, N1-CH2), 7.65 (dt, 3J = 7.7 Hz, 4J = 1.5 Hz, 1H, 4-Ph), 7.77 (dt, 3J = 7.7 Hz, 4J = 1.3 Hz, 1H, 5Ph), 7.97 – 8.13 (m, 3H, 3,6-Ph, C8-H), 8.37 (s, 1H, (Z)-9, HC=N), 8.60 (s, 1H, (E)-9, HC=N), 11.92 (s, 1H, (Z)-9, CONH), 11.97 (s, 1H, (E)-9, CONH). LC/MS: purity 100%, m/z calc. 386.12, found 386.20. Analysis for C16H15N7O5 (385.33): C, H, N. 2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)-N'-(4-nitro-benzylidene)acetohydrazide (10) From 3 in 81% yield: mp 335.7 – 336.3 °C, Rf = 0.26 (A), 0.36 (B), 0.41 (C), E/Z ratio = 0.83, 1H-NMR 3.43 (s, 3H, N3-CH3), 3.87 (s, 3H, N7-CH3), 4.61 (s, 2H, (E)-9, N1-CH2), 5.02 (s, 2H, (Z)-9, N1-CH2), 7.97 (td, 3J = 9.0 Hz, 4J = 2.0 Hz, 2H, 2,6-Ph), 8.06 (s, 1H, (Z)-9, C8-H), 8.12 (s, 1H, (E)-9, C8-H), 8.21 – 8.32 (m, 3H, 3,5-Ph, HC=N), 11.95 (s, 1H, CONH). LC/MS: purity 100%, m/z calc. 386.12, found 386.20. Analysis for C16H15N7O5 (385.33): C, H, N.


N'-benzylidene-4-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)-butanehydrazide (11) From 4 in 59% yield: mp 204.9 – 206.1 °C, Rf = 0.31 (A), 0.39 (B), 0.51 (C), E/Z ratio = 0.57, 1H-NMR 1.78 – 1.93 (m, 2H, CH2CH2CH2 CO), 2.14 – 2.28 (m, 2H, CH2CH2 CH2CO), 3.38 (s, 3H, (Z)-11, N3-CH3), 3.40 (s, 3H, (E)-11, N3-CH3), 3.83 (s, 3H, (Z)-11, N7-CH3), 3.86 (s, 3H, (E)-11, N7-CH3), 3.87 – 4.00 (m, 2H, N1-CH2), 7.32 – 7.46 (m, 3H, (E)-11, 4-Ph, (Z)-11, 2,4,6-Ph), 7.47 – 7.54 (m, 2H, (E)-11, 2,6-Ph), 7.54 – 7.61 (m, 2H, (Z)-11, 3,5-Ph), 7.63 (dd, 3J = 7.6 Hz, 4J = 2.2 Hz, 2H, (E)-11, 3,5-Ph), 7.93 (s, 1H, (E)-11, C8-H), 7.98 (s, 1H, (Z)-11, C8-H), 8.11 (s, 1H, (Z)-11, HC=N), 8.70 (s, 1H, (E)-11, HC=N), 11.19 (s, 1H, (Z)-9, CONH), 11.30 (s, 1H, (E)-11, CONH). LC/MS: purity 96.5%, m/z calc. 369.17, found 369.18. Analysis for C18H20N6O3 (368.39): C, H, N. 4-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)-N'-(1-phenyl-ethylidene)butanehydrazide (12) From 4 in 50% yield: mp 210.1 – 212.1 °C, Rf = 0.29 (A), 0.40 (B), 0.43 (C), E/Z ratio = 0.54, 1H-NMR 1.88 (quin, 3J = 7.2 Hz, 2H, CH2CH2CH2CO), 2.19 (s, 3H, (Z)-12, CH3C=N), 2.23 (s, 3H, (E)-12, CH3C=N), 2.32 (t, 3J = 7.4 Hz, 2H, (E)-12, CH2CH2CH2CO), 2.66 (t, 3J = 7.6 Hz, 2H, (Z)-12, CH2CH2 CH2CO), 3.38 (s, 3H, (Z)-12, N3-CH3), 3.40 (s, 3H, (E)-12, N3-CH3), 3.83 (s, 3H, (Z)-12, N7-CH3), 3.86 (s, 3H, (E)-12, N7-CH3), 3.89 – 4.01 (m, 2H, N1-CH2), 7.28 – 7.43 (m, 3H, 2,4,6-Ph), 7.62 – 7.80 (m, 2H, 2,6-Ph), 7.97 (s, 1H, (Z)-12, C8-H), 7.99 (s, 1H, (E)-12, C8-H), 10.27 (s, 1H, (E)-12, CONH), 10.38 (s, 1H, (Z)-12, CONH). LC/MS: purity 99.2%, m/z calc. 383.18, found 383.20. Analysis for C19H22N6O3 (382.43): C, H, N. 4-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)-N'-(2-hydroxy-benzylidene)butanehydrazide (13) From 4 in 64% yield: mp 221.3 – 222.7 °C, Rf = 0.26 (A), 0.36 (B), 0.43 (C), E/Z ratio = 1.69, 1H-NMR 1.86 (quin, 3J = 7.3 Hz, 2H, CH2 CH2CH2CO), 2.23 (t, 3J = 7.7 Hz, 2H, (E)13, CH2CH2CH2 CO), 2.57 (t, 3J = 7.6 Hz, 2H, (Z)-13, CH2CH2 CH2CO), 3.38 (s, 3H, (Z)-13, N3-CH3), 3.49 (s, 3H, (E)-13, N3-CH3), 3.84 (s, 3H, (Z)-13, N7-CH3), 3.86 (s, 3H, (E)-13, N7-CH3), 3.88 – 3.98 (m, 2H, N1-CH2), 6.74 – 6.93 (m, 2H, 3,5-Ph), 7.15 – 7.30 (m, 1H, 6-Ph), 7.41 – 7.55 (m, 1H, 4Ph), 7.97 (s, 1H, C8-H), 8.20 (s, 1H, (Z)-13, HC=N), 8.29 (s, 1H, (E)-13, HC=N), 10.06 (s, 1H, (Z)-13, CONH), 11.13 (s, 1H, (E)-13, CONH), 11.17 (s, 1H, (Z)-13, OH), 11.54 (s, 1H, (E)-13, OH). LC/MS: purity 100%, m/z calc. 385.16, found 385.20. Analysis for C18H20N6O4 (384.39): C, H, N. 4-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)-N'-(4-hydroxy-benzylidene)butanehydrazide (14) From 4 in 54% yield: mp 249.5 – 251.4 °C, Rf = 0.12 (A), 0.24 (B), 0.31 (C), E/Z ratio = 0.74, 1H-NMR 1.85 (quin, 3J = 7.2 Hz, 2H, CH2 CH2CH2CO), 2.17 (t, 3J = 7.2 Hz, 2H, (E)14, CH2CH2CH2 CO), 2.57 (t, 3J = 7.7 Hz, 2H, (Z)-14, CH2CH2 CH2CO), 3.39 (s, 3H, (Z)-14, N3-CH3), 3.40 (s, 3H,


5

(E)-14, N3-CH3), 3.84 (s, 3H, (Z)-14, N7-CH3), 3.86 (s, 3H, (E)-14, N7-CH3), 3.87 – 3.98 (m, 2H, N1-CH2), 6.72 – 6.91 (m, 2H, 2,6-Ph), 7.35 – 7.50 (m, 2H, 3,5-Ph), 7.82 (s, 1H, (Z)-14, HC=N), 7.98 (s, 1H, C8-H), 7.99 (s, 1H, (E)-14, HC=N), 10.97 (s, 1H, (Z)-13, CONH), 11.08 (s, 1H, (E)-13, CONH). LC/MS: purity 95.4%, m/z calc. 385.16, found 385.27. Analysis for C18H20N6O4 (384.39): C, H, N. 4-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)-N'-(2-nitro-benzylidene)butanehydrazide (15) From 4 in 69% yield: mp 264.8 – 265.5 °C, Rf = 0.26 (A), 0.36 (B), 0.41 (C), E/Z ratio = 0.66, 1H-NMR 1.72 – 1.93 (m, 2H, CH2CH2CH2 CO), 2.24 (t, 3J = 7.8 Hz, 2H, (E)-15, CH2CH2 CH2CO), 2.62 (t, 3J = 7.4 Hz, 2H, (Z)-15, CH2CH2 CH2CO), 3.38 (s, 3H, (Z)-15, N3-CH3), 3.40 (s, 3H, (E)-15, N3-CH3), 3.83 (s, 3H, (Z)-15, N7-CH3), 3.86 (s, 3H, (E)-15, N7-CH3), 3.88 – 3.98 (m, 2H, N1-CH2), 7.56 – 7.81 (m, 2H, 4,5-Ph), 7.92 – 8.07 (m, 3H, C8-H, 3,6-Ph), 8.30 (s, 1H, (Z)-15, HC=N), 8.52 (s, 1H, (E)-15, HC=N), 11.48 (s, 1H, (Z)-15, CONH), 11.64 (s, 1H, (E)-15, CONH). LC/MS: purity 96.8%, m/z calc. 414.15, found 414.25. Analysis for C18H19N7O5 (413.39): C, H, N. 4-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)-N'-(4-nitro-benzylidene)butanehydrazide (16) From 4 in 73% yield: mp 274.1 – 276.0 °C, Rf = 0.26 (A), 0.36 (B), 0.41 (C), E/Z ratio = 0.89, 1H-NMR 1.75 – 1.95 (m, 2H, CH2CH2CH2 CO), 2.25 (t, 3J = 7.7 Hz, 2H, (E)-16, CH2CH2 CH2CO), 2.67 (t, 3J = 7.4 Hz, 2H, (Z)-16, CH2CH2 CH2CO), 3.38 (s, 3H, (Z)-16, N3-CH3), 3.39 (s, 3H, (E)-16, N3-CH3), 3.82 (s, 3H, (Z)-16, N7-CH3), 3.86 (s, 3H, (E)-16, N7-CH3), 3.89 – 3.99 (m, 2H, N1-CH2), 7.79 – 8.00 (m, 2H, 2,6-Ph), 8.03 (s, 1H, C8-H), 8.18 – 8.30 (m, 3H, HC=N, 3,5-Ph), 11.51 (s, 1H, (Z)-16, CONH), 11.61 (s, 1H, (E)-16, CONH). LC/MS: purity 99.4%, m/z calc. 414.15, found 414.18. Analysis for C18H19N7O5 (413.39): C, H, N. A General Procedure for Preparing -(3,7-dimethyl-2,6dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)alkylcarboxlic Acids (17, 18) Mixture of 1 eq. (10 mmol) of appropriate ethyl -(3,7dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)alkylcarboxlate (1, 2) and 2 eq. of KOH in acetone/water mixture (50 ml, 1:2, v/v) was refluxed for 30 h. Afterwards the reaction mixture was evaporated under reduced pressure and the residue was dissolved in a small amount of water (ca. 10 ml). Solution was acidified with conc. HCl to pH ca. 3, cooled and a precipitate of product was filtered off. Products were crystallized from water. 2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)acetic acid (17) [15] From 1 in 66% yield: mp 262.9 – 264.6 °C, Rf = 0.06 (A), 0.06 (B), 0.06 (C). LC/MS: purity 100%, m/z calc. 239.08, found 239.09. Analysis for C9H10N4O4 (238.20): C, H, N.

6


4-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)butanoic Acid (18) [13] From 2 in 66% yield: mp 221.9 – 223.8 °C, Rf = 0.19 (A), 0.27 (B), 0.37 (C). LC/MS: purity 95.5%, m/z calc. 267.11, found 267.14. Analysis for C11H14N4O4 (266.25): C, H, N. A General Procedure for Preparing N-benzyl--(3,7dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1yl)alkylamides (19, 20) 1 eq. (2 mmol) of appropriate acid 17 or 18 was dissolved in a small amount of DMF (3 ml) and 1.5 eq. of CDI was added. Mixture was stirred in room temperature for 30 min, and afterwards 1.1 eq. of benzylamine was added. Reaction mixture was stirred in room temperature for 2 days. Afterwards 2-3 drops of water were added and mixture was evaporated under reduced pressure. Crude products were purified with column chromatography. N-benzyl-2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1Hpurin-1-yl)acetamide (19) [16] From 17 in 92% yield: mp 261.5 – 262.9 °C, Rf = 0.29 (A), 0.40 (B), 0.43 (C). LC/MS: purity 95.8%, m/z calc. 328.14, found 328.16. Analysis for C16H17N5O3 (327.34): C, H, N. N-benzyl-4-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1Hpurin-1-yl)butanamide (20) From 18 in 55% yield: mp 176.7 – 178.2 °C, Rf = 0.33 (A), 0.45 (B), 0.47 (C), 1H-NMR 1.79 (quin, 3J = 7.4 Hz, 2H, CH2 CH2CH2 CO), 2.25 (t, 3J = 7.6 Hz, 2H, CH2CH2 CH2CO), 3.31 (s, 3H, N3-CH3), 3.39 (s, 3H, N7 CH3), 3.87 (t, 3J = 6.9 Hz, 2H, N1-CH2), 4.21 (d, 3J = 5.9 Hz, 2H, CH2Ph), 7.14 – 7.33 (m, 5H, Ph), 7.98 (s, 1H, C8-H), 8.27 (t, 3J = 5.9 Hz, 1H, CONH). LC/MS: purity 100%, m/z calc. 356.17, found 356.22. Analysis for C18H21N5O3 (355.39): C, H, N. PHARMACOLOGY Animals The experiment was carried out on male Albino Swiss mice weighing 18-30 g. The animals were housed in constant temperature facilities exposed to 12:12 light-dark cycle and maintained on a standard pellet diet and tap water was given ad libitum. Control and experimental groups consisted of 6-8 animals each. The investigated compounds were administered intraperitoneally (ip) in a form of a suspension in 0.9% physiological saline. Control animals received the equivalent volume of physiological saline. All procedures were conducted according to guidelines of ICLAS (International Council on Laboratory Animals Science) and approved by The Local Ethic Committee on Animal Experimentation. Acetylsalicylic acid (ASA) was used as a reference drug. The data are expressed as mean ± SEM (standard error). The data were evaluated by one-way analysis of variance (ANOVA) followed by Duncan test. The difference of means was statistically significant if p< 0.05.

Zygmunt et al.

“Writhing Syndrome” Test In the writhing syndrome test mice were treated with 0.25 ml 0.02% phenylbenzoquinone (INC Pharmaceuticals, Inc, NY) solution 30 minutes after ip administration of the investigated compound or the vehicle. Afterwards the mice were placed individually into glass beakers and during 5 minutes were allowed to elapse. After that period of time a 10-minute observation was conducted for each animal – the number of characteristic writhes was counted. The analgesic effect of the tested substances was determined by a decrease in the number of writhes observed [17]. The ED50 values and their confidence limits were estimated by the method of Litchfield and Wilcoxon [18]. Rota-Rod Test For examining possible neurological deficits due to the test substances, which may interfere with the test results to give false positives such as muscle relaxant or impairment of motor coordination, Rota-Rod test was performed. Animals were placed on a 1-inch diameter knurled plastic rod rotating at 24 rpm. Non-toxic (normal) mice can remain on a rod rotating at this speed almost indefinitely. Neurological toxicity is defined as the failure of the animal to remain on the rod for 1 min and is expressed as the number of animals exhibiting toxicity/number of animals tested. Animals are considered toxic if they fail this test on three successive attempts [19]. CONCLUSION Hydrazones may exist in two isomeric forms, E and Z, depending on the configuration of substituents attached to the carbon atom of hydrazone moiety and thermal isomerization rate may be relatively high even in room temperature [20-22]. Analysis of 1H-NMR spectra of newly synthesized hydrazones 9 - 16 suggests, that the predominant form of these compounds was Z isomer (E/Z ratio < 0.90), except for the compound 13 for which E isomer seemed to be more preferable (E/Z ratio = 1.69). This may be explained by steric interactions between large phenyl substituent and free electron pair of nitrogen atom creating hydrazone moiety. In case of compound 13, the o-hydroxy substituent on the phenyl ring may interact with the nitrogen free electron pair and stabilize E isomer by creating hydrogen bond. Further analysis of 1H-NMR spectra showed lack of signals for protons of 4-hydroxy group of compound 14 and amino group of compound 4. This may be explained by rapid exchange of this protons with residual water in polar solvent such as DMSO. Nineteen of the synthesized compounds were tested in the writhing syndrome test. The in vivo activity of compound 14 was not determined due to its very low solubility in water. Results of the test are presented in (Tables 1-2). The writhing test consists of intraperitoneal injection of a chemical irritant followed by subsequent counting of “writhes” that is characteristic contractions of abdominal muscles accompanied by a hind limb extensor motion. This test detects peripheral analgesic activity; however, some



Table 1. Influence of the Investigated Compounds on the Pain Reaction in the Writhing Syndrome Test. Dose Comp.

Mean Number of Writhing ±SEM

control

–

31.4 ± 2.8

3

4

5

100

9.8 ± 7.0

50

17.3 ± 5.0

10

23.5 ± 9.5

5

32.0 ± 10.0

100

10 ± 2.3*

50

13.3 ± 5.5

10

25.3 ± 5.7

100

13.7 ± 5.5

50

14.7 ± 5.5

10

16.0 ± 2.0

100

11.0 ± 3.1*

50

24.3 ± 4.8

10

29.3 ± 3.7

100

13.0 ± 8.3

50

17.0 ± 6.3

10

18.3 ± 2.7

100

6.3 ± 2.7**

50

5.7 ± 1.2**

10

9.3 ± 4.7*

5

16.7 ± 5.1

10

11.0 ± 1.5*

5

10.3 ± 1.9*

1

20.7 ± 10.7*

100

1.0 ± 0.6**

50

5.0 ± 3.1**

25

6.0 ± 2.7**

10

13.3 ± 4.9*

1

20.7 ± 8.0

100

3.0 ± 2.5**

50

3.7 ± 1.9**

25

5.3 ± 1.9**

10

8.7 ± 5.2*

1

27.0 ± 8.0

45.8

6

7

8

9

–

*

1

2

ED50 [mg/kg]

[mg/kg]

8.7

14.6

83.8

45.7

8.5

2.4

8.6

8.2

7

8


Zygmunt et al.

Table 1. contd… Dose Comp.

Mean Number of Writhing ±SEM

[mg/kg]

[mg/kg]

10

ASA

*

**

p