Original article

Synthesis and biological evaluation of newthiazolyl/benzothiazolyl-amides, derivatives of 4-phenyl-piperazine

Christina Papadopoulou, Athina Geronikaki *, Dimitra Hadjipavlou-Litina

Department of Pharmaceutical Chemistry, School of Pharmacy University Campus, Aristotle University, Thessaloniki 54124, Greece

Received 11 June 2005; accepted 22 June 2005

Available online 22 July 2005

Abstract

A series of thiazolyl-N-phenyl piperazines has been synthesised and tested for anti-inflammatory activity. Their RM values were deter-mined as an expression of their lipophilicity. Theoretical calculation of their lipophilicity, as clog P and logPsk also performed. The effect ofthe synthesised compounds on inflammation, using the carrageenin induced mouse paw oedema model was studied. In general, the studiedcompounds were found to be potent anti-inflammatory agents (44–74.1%). Anti-inflammatory activity was influenced by some structuralcharacteristics of the synthesised compounds. An attempt was made to correlate their biological activity with some physicochemical param-eters using a quantitative structure–activity relationship approach (QSAR).© 2005 Elsevier SAS. All rights reserved.

Keywords: Thiazole derivatives; Antiinflammatories; Lipophilicity

1. Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs) havebeen used to treat various ailments for over 100 years. As aclass, these drugs possess anti-inflammatory, anti-allergy,analgesic and antipyretic activity and are widely used to treatchronic inflammatory states such as arthritis, psoriasis andasthma. These agents exhibit an inhibitory action on thecyclooxygenase (COX) that catalyses the biosynthesis of pros-taglandins and thromboxane from arachidonic acid. For manyyears it was believed that PGs were formed via the activity ofa single enzyme, COX. In recent years, two COX enzymes,COX-1 and COX-2, have been discovered and it is agreed,that inhibition of COX-2 is more selective for anti-inflammatory effect [1–3]. All NSAIDs are approximatelyequivalent in terms of anti-inflammatory efficacy but alsocause untoward side effects (e.g. in gastrointestinal), in a sig-nificant fraction of treated patients and this frequently limitstherapy.

Most NSAIDs are weak acids with pKa values ranging from3 to 5 [4] and their side effects locally connected to their acidiccharacter. Thus, there is an increased interest in the develop-ment of effective non-acidic anti-inflammatory agents. BasicNSAIDs are gaining interest because of their improved bio-logical properties.

Thiazolyl ring derivatives are known to possess anti-inflammatory as well as antipyretic activities [5,6]. Meloxi-cam, for example is a new NSAID with a thiazolyl group. Anumber of thiazolyl-aminoketones was found to be stronganti-inflammatory agents [7]. These results, along with ear-lier studies, which showed that thiazolyl-amides exhibit sig-nificant anti-inflammatory activity [8,9], prompted us to inves-tigate other thiazolyl-amides.

NSAIDs have a broad spectrum of effects and it has beensuggested that the variations in both efficacy and their toler-ability are partly due to differences in their physicochemicalproperties which determine their distribution in the body andtheir ability to pass through and to enter the interior of mem-branes [10,11]. Thus, partition coefficients (log P values) asRM values are performed also and compared with the corre-sponding theoretically calculated values of n-octanol–water.

* Corresponding author. Tel.: +30 2310 997 616; fax: +30 2310 997 612.E-mail address: geronik@pharm.auth.gr (A. Geronikaki).

Il Farmaco 60 (2005) 969–973

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0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.doi:10.1016/j.farmac.2005.06.014

2. Experimental procedure

2.1. Materials

Melting points were obtained with a MELTEMP II capil-lary apparatus (LAB. Devices Holliston, MA, USA) and arereported without correction. Infrared spectra (Nujol mulls)were recorded on a spectrophotometer. UV–Vis were deter-mined on a Perkin–Elmer 554 UV–Vis spectrophotometer.Proton NMR spectra were obtained on a BruckerAW 80 appa-ratus at 80 MHz and are reported in ppm downfield from tet-ramethylsilane (TMS). Analyses indicated by the symbols ofelements were within 0.4% of theoretical values. Mass spec-tra (MS) were determined on a VG-250 instrument (VG Labs.Tritech, UK) with the ionisation energy maintained at 70 eV.The reactions were monitored by TLC on silica gel 60 F254

(Merck). All the chemicals used were of analytical grade andcommercially available by Merck (KgaA 64271 Darmstaadt,Germany). Carrageenin K-type was commercially available.

2.2. Synthesis

2.2.1. Synthesis of precursors2-Aminothiazole, 2-amino-4-methylthiazole, 2-amino-4-

phenylthiazole and 2-amino-4-(4-methoxyphenyl) thiazolewere synthesised as described previously [12].

General synthetic procedure for the synthesis of the2-chloroacetamido- and 3-chloropropionylamido thiazolesand benzothiazoles [12].

To a solution of 2-aminothiazole/aminobenzothiazole orsuitable 4-substituted aminothiazole/benzaminothiazole(0.02 mol) in dry benzene a cooled solution of chloroacetylor 3-chloropropionyl chloride (0.033 mol) in dry benzene(7.5 ml) was added dropwise. The reaction mixture wasrefluxed in a water bath at 80 °C for 3 h. Benzene and excess3-chloropropionyl chloride were removed by distillation. Theresidue was washed with aqueous sodium bicarbonate (5%w/v) followed by cold water. The crude product was driedand crystallised from ethanol.

General method of the synthesis of the 2-(N-substitutedaminoacetamido)/3-(N-substituted aminopropioamido) thia-zoles [12].

A mixture of 2-chloroacetamido or 3-chloropropionamidothiazole/benzothiazole (0.006 mol), amine (phenyl pipera-zine 0.7 mol), absolute ethanol (15 ml) and anhydrous sodiumcarbonate (1.48 g) was heated under reflux in a water bath for12 h. The excess of amine and ethanol was removed by dis-tillation and the residue was treated with 5% sodium bicar-bonate solution to remove acid impurities, filtered, washedwith water and dried. It was crystallised from ethanol (95%)to give white crystals.

2-(4-Phenylpiperazin-1-yl)-N-(thiazol-2-yl)acetamide (1).1H-NMR: (CDCl3, DMSO) d: 2.7–2.9 (m, 4H,

a-piperazine), 3.15–3.5 (m, 6H, b-piperazine, –CH2–), 6.75–7.1 (m, 5H phenyl), 7.1 (d, H, thiazole C5–H), 7.6 (d, H,C4–thiazole) .MS: M+ 302(10%).

N-(4-Methylthiazol-2-yl)-2-(4-phenylpiperazin-1-yl)ace-tamide (2).

1H-NMR: (CDCl3, DMSO) d: 2.4 (s, CH3), 2.85–2.98 (m4H, a-piperazine), 3.18–3.33 (m, 6H, b-piperazine, –CH2–),6.82–7.22 (m, 5H phenyl, H–C5 thiazole) , 8.96 (s, NH). MS:M+ 317(22%).

2-(4-Phenylpiperazin-1-yl)-N-(phenylthiazol-2-yl)acetamide (3).

1H-NMR: (CDCl3, DMSO) d: 2.59–3.0 (m, 4H,a-piperazine), 3.25–3.45 (m, 6H, b-piperazine, –CH2–), 6.59–7.08 (m, 5H–Ph–N), 7.22–7.48 (m, 6H, Ph, thiazole, C5–H).MS: M+ 378(93%).

2-(4-Phenylpiperazin-1-yl)-N-(methoxyphenylthiazol-2-yl)acetamide (4).

1H-NMR: (CDCl3, DMSO) d: 2.6–3 (m, 4H, a-piperazine),3.15–3.6 (m, 6H b-piperazine, CH2), 3.75 (s, 3H, CH3O–Ph), 6.75–7.1 (m, 5H–Ph–N), 7.1–7.9 (m, 5H thiazole C5–H,CH3O–Ph). MS: M+ 408(73%).

3-(4-Phenylpiperazin-1-yl)-N-(thiazol-2-yl)propionamide (5).

1H-NMR: (CDCl3, DMSO) d: 2.28–2.64 (m, 4H,a-piperazine), 3.45 (m, 8H, 2CH2–, b-piperazine), 6.59–7.08(m, 5H–Ph–N), 6.56 (d, H, thiazole C5–H) 7.53 (d, H, C4–thia-zole). MS: M+ 317(8%).

3-(4-Phenylpiperazin-1-yl)-N-(phenylthiazol-2-yl)propio-namide (6).

1H-NMR: (CDCl3, DMSO) d: 2.28–2.64 ((m, 4H,a-piperazine), 3.45 (m, 8H, 2CH2–, b-piperazine), 6.59–7.08(m, 5H–Ph–N), 6.6–7.48 (m, 5H, Ph, thiazole C5–H).

MS: M+ 392(13%).3-(4-Phenylpiperazin-1-yl)-N-(methoxyphenylthiazol-2-

yl)propionamide (7).1H-NMR: (CDCl3, DMSO) d: 2.6–2.68 (m, 4H,

b-piperazine), 3.15–3.6 (m, 6H a-piperazine, CH2), 3.77 (s,3H, CH3O–Ph), 6.75–7.1 (m, 5H–Ph–N), 7.1–7.9 (m, 5H thia-zole C5–H, CH3O–Ph). MS: M+ 422(1%).

N-(benzo[d]-thiazol-2-yl)-2-(4-phenylpiperazin-1-yl)acetamide (8).

1H-NMR: (CDCl3, DMSO) d: 2.7–3 (m, 4H, a-piperazine),3.2–3.55 (m, 6H, b-piperazine, CH2), 6.75–7.95 (m, 8H, aro-matic). MS: M+ 352(24%).

N-(6-fluorobenzo[d]-thiazol-2-yl)-2-(4-phenylpiperazin-1-yl)acetamide (9).

1H-NMR: (CDCl3, DMSO) d: 2.7–3 (m, 4H, a-piperazine),3.2–3.5 (m, 6H, b-piperazine, –CH2–), 6.8–7.9 (m, 8H, aro-matic). MS: M+ 370(18%).

N-(6-ethoxybenzo[d]-thiazol-2-yl)-2-(4-phenylpiperazin-1-yl acetamide (10).

1H-NMR: (CDCl3, DMSO) d: 1.33 (t, 3H, CH3–CH2O),3.98 (q, 2 h OCH2–CH3).

2.59–3.25 (m, 4H, a-piperazine),3.45 (m, 6H, b-piperazine,–CH2–), 6.59–7.08 (m, 6H, –Ph–N, thiazole C5–H), 8.12 (d,H C4–thiazole).

N-(benzo[d]-thiazol-2-yl)-3-(4-phenylpiperazin-1-yl) pro-pionamide (11).

1H-NMR: (CDCl3, DMSO) d: 2.28–2.64 (m, 4H,a-piperazine), 3.45 (m, 8H, b-piperazine, 2CH2–), 6.59–7.08

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(m, 5H,Ph–N), 7.55 (q, 2H, C5–C6 benzothiazole), 8.12 (d,H, C7–benzothiazole), 8.23 (d, H, C4–benzothiazole).

2.2.2. Physicochemical studies [8]Reversed phase TLC (RPTLC) was performed on silica

gel plates impregnated with 5% (v/v) liquid paraffin in lightpetroleum ether [8]. Mobile phase: methanol/water mixture(70:30, v/v) containing 2% aqueous ammonia (27%). Theplates were developed in closed chromatography tanks satu-rated with the mobile phase at 24 °C. Spots were detectedunder UV light or by iodine vapours. RM values were deter-mined from the corresponding Rf values (from 10 individualmeasurements) using the equation RM = log [(1/Rf) – 1]. Forresults see Table 2.

Theoretical calculations of lipophilicity as clog P andSuzuki–Kudo’s method [13] were performed (Table 2). Theprogram CLOG P [14], has been designed to calculate thelipophilicity of a molecule using the method of additivity.

2.2.3. Inhibition of carrageenin induced paw oedema [4]Oedema was induced in the right hind paw of AKR or A

mice (20–30 g, 2–3 months old) by the intradermal injectionof 0.05 ml 2% carrageenin in water. Both sexes were used.Females pregnant were excluded. The animals were housedunder standard conditions and received a diet of commercialfood pellets and water ad libitum during the maintenance,but they were entirely fasted during the experiment period.Our studies were in accordance with recognised guidelineson animal experimentation (Guidelines for the care and useof laboratory animals published by the Greek Government160/1991, based on EU regulations 86/609).

All tested compounds were suspended in water with fewdrops of Tween-80 and ground in a mortar before use. Groupsof five to six AKR mice weighing 20–30 g were used. A doseof 0.2 mmol kg was chosen in order to be possible for us tocompare the new results with previous findings [9]. The com-pounds as well as indomethacin were administered intraperi-toneally i.p., simultaneously to the administration of the phlo-gistic agent: carrageenin (2%, 0.05 ml was injectedintradermal i.d. into the right foot pad, the left serving as con-trol). Results are given in Table 2.

3. Results and discussion

3.1. Chemistry

The general method employed is shown in Fig. 1. Thestructure of the derivatives and their physicochemical prop-erties are given in Tables 1a, b. The compounds of the titlewere prepared in two steps. Initial amino/substituted aminothiazoles and benzothiazoles were treated withchloracetyl/propionyl chloride in benzene to give the corre-sponding chloroacetamides/propionamides, which in the sec-ond step undergo condensation with phenylpiperazine in etha-nol to give the title compounds. Overall the reactions proceedsmoothly in good yields. The amides were identified both byelemental analyses as well as by their spectroscopic analy-ses.

The structure of all compounds was identified both byelemental analyses as well as by spectroscopic analysis (UV,IR, 1H-NMR, MS). The UV and the IR spectra were in agree-ment with the proposed structures: in the UV spectra charac-teristic absorptions ekmax = 14242 at kmax = 275.5 nm,ekmax = 25575 at kmax = 226.5 nm and ekmax 24818 at 210 nm(2-phenylpiperazin-N-benzothiazole acetamide). IR spectrashowed a sharp band in the region 1692 cm−1 (NHC = O), aswell as in region 1606 (aromatic). Also there is a week band

Fig. 1. The synthetic pathway.

Table 1aCharacterisation data of the synthesised thiazole amides

N R N Yield(%)

Rf m.p.(°C)

Ms Molecularstructure b

1 H 1 74.2 0.759 170–1 302 C15H18N4OS2 CH3 1 98.3 0.827 219–21 316 C16H20N4OS3 Ph 1 84.0 0.810 142–3 378 C21H22N4OS4 Ph–OCH3 1 95.1 0.879 170–

0.5408 C22H24N4O2S

5 H 2 73.6 0.824 135–6 316 C16H20N4OS6 Ph 2 60.3 0.760 154–5 392 C22H24N4OS7 Ph–OCH3 2 78.0 0.808 196–

0.5422 C23H26N4O2S

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at 3175 cm−1 corresponding to the protonated tertiary aminegroup (+N–H). Coordination of aromatic and side chain pro-tons was observed in the 1H-NMR spectra and the exact num-ber of protons was given by integration. In the MS spectraalmost all substances gave stable molecular ions. Character-istic fragments were formed by the loss of HCl (M+–HCl).

The lipophilicity of the synthesised compounds expressedas RM values ranging from −0.204 − 0.618, as well as calcu-lated theoretically log P values are given in Table 2. Poorcorrelation was obtained between RM and clog P values.

3.2. Biological studies

Basic anti-inflammatory agents are gaining interest becausethey possess better pharmacokinetic properties and cause lessgastric irritation compared to acidic agents. The in vivo anti-inflammatory effects of the synthesised amides were assessedby using the functional model of carrageenin induced mousepaw oedema the most frequently used model for anti-inflammatory activity (Table 2), as percentage of weightincrease at the right hind paw in comparison to the unin-jected left hind paw. We have previously reported thatthiazolyl-amides demonstrated good anti inflammatory as wellas local anaesthetic properties [9]. Thus, with modificationsperformed with the synthesis of more compounds presentedherein we further elucidated the relative significance of struc-tural features for activity.

The main chemical features of the synthesised amides canbe divided into four basic parts:

• (1) a substituted aromatic heterocyclic group (thiazolyl–/benzothiazolyl–);

• (2) a 4-phenyl-substituted piperazine;• (3) a central ligand consisting of a NHCO amide group;• (4) a methylenic linkage (CH2)n.

The nature of the moieties (1) and (4) seems to have amajor effect on the activity.

We tried to delineate the contribution of the 4-phenyl-substituted piperazine in the biological activity, since fromprevious results a 4-CH3–piperazinyl group leads to 72.1%inhibition of carrageenin paw oedema [9]. The insertion ofthe benzothiazolyl group does not increase the activity. Com-pounds 8–11 present higher lipophilicity values and may bethis is the reason for the decrease of activity. Comparing com-pounds 1–4, with our previous findings [9] it seems that thenew compounds inhibit more the carrageenin induced micepaw oedema at the same dose level (0.2 mmol kg). On thecontrary our new compounds 6 and 7 present lower efficacywith the exception of compound 5, which was found to be themost potent within the series. It contains the 4-phenyl-substituted piperazinyl group and a (CH2)2. The tested com-pounds did not present any side effects from the gastrointes-tinal route (results not shown). On the contrary indomethacin,the standard drug, was responsible for side effects in someanimals.

Regression analysis was performed to find out whether anycorrelation exists between the % in vivo anti-inflammatoryactivity (expressed as log % CPE, carrageenin induced paw

Table 1bCharacterisation data of the synthesised benzothiazole amides

N R N Yield (%) Rf m.p. (°C) Ms Molecular structure b

8 H 1 76.0 0.853 180–2 352 C19H20N4OS9 F 1 77.5 0.787 190–2 370 C19H19N4OSF10 OC2H5 1 67.0 0.795 191.5–2 380 C21H24N4OS11 H 2 58.6 0.811 205–0.5 366 C20H22N4OS

Elemental analysis for molecular formulas ( ± 0.40).

Table 2Lipophilicity values a) experimentally performed RM values; b) theoretically calculated lipophilicity values using: i) Suzuki–Kudo’s method ii) the clog Pprogram from Biobyte. In vivo carrageenin rat paw oedema % inhibition after 3.5 h (CPE %).

Compounds R N RM Clog P logPsk CPE a %1 H 1 0.618 ± 0.06 3.02 –1.429 512 CH3 1 Nt 3.52 0.980 493 C6H5 1 0.115 ± 0.011 5.11 –0.011 494 p-OmePh 1 0.094 ± 0.005 5.13 0.033 445 H 2 0.419 ± 0.019 3.21 0.086 746 C6H5 2 Nt 5.31 1.740 447 p-OmePh 2 –0.468 ± 0.028 5.33 1.548 458 H 1 –0.204 ± 0.020 4.61 0.017 449 F 1 Nt 4.75 0.265 4610 OC2H5 1 –0403 ± (0.024) 5.43 0.310 3611 H 2 Nt 4.81 1.532 44

Nt—non testeda Indomethacin as standard drug for comparison reasons (0.1 mmol/kg–1) 56%.

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oedema) of the examined amides and several physicochemi-cal parameters (lipophilicity, steric and electronic param-eters). The % inhibition of CPE of the tested compounds andtheir lipophilicity as clog P values, calculated using theC-QSAR program [14], were correlated (Eq. (1)) in a linearmodel with the presence of an indicator variable I1 whichtakes the value = 1 for the congeners where N = 1:

(1)log %� CPE � = −0.072� 0.035 �clog P − 0.085

� 0.068 �I1+2.052� 0.170 �

n = 9, r = 0.918, r2 = 0.842, q2 = 0.480, s = 0.039, F2,6 = 16, � = 0.01

Equation includes: 95% confidence limits for each term inparentheses, the correlation coefficient r, between observedvalues of the dependent and the values calculated from theequation, the s standard deviation, q2 square of cross-validated correlation coefficient (a measure of the quality ofmodel), is often computed in order to test the stability of modeland the F-values for the individual term. N, s, F are the num-bers of data points, standard deviation from regression, F-testof significance. In terms of r, r2 values, Eq. (1) is statisticallysignificant with a significant F-value. Both the intercept andthe slope are contributing to the equation in a significant man-ner. This indicates that the basic nucleus of thiazolyl/benzothiazolyl substituted groups and the amide group arecontributing to the anti-inflammatory activity to an appropri-ate extent. The negative sign with lipophilicity indicates thathydrophilicity (low lipophilicity) is significant for the anti-inflammatory activity of the examined compounds. The pres-ence of one CH2– group has a negative effect for the biologi-cal response whereas no role for the contribution of the secondCH2 group was found. Although no parameterisation for thevariously substituted R– has been done all of the log % CPEvalues are well predicted and all data fit the pattern of QSARwith the exception of compounds 3 and 11. No interrelation-ship between the used parameters was observed. No role forelectronic effects was found. The role of the steric effects for

substituents R was not well defined, since the numbers of thedata were low.

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