Synthesis and Biological Evaluation of New 4,5-Disubstituted-Thiazolyl Amides, Derivatives of 4-Hydroxy-Piperidine or of 4-N-Methyl Piperazine

Abstract

4,5-disubstituted-thizolyl amides, derivatives of 4-hydroxy-piperidine and of 4-N-methyl piperazine, were synthesized and tested as anti-inflammatory agents. Log P values were theoretically calculated and experimentally determined. These compounds were tested for antioxidant activity, as hydroxyl radical scavengers and for their ability to in­teract with stable 1,1-diphenyl-2-picryl hydrazyl free radical (DPPH). The effect of the syn­thesized compounds on inflammation, using the carrageenin induced mice paw edema mo­del was studied. Both anti-inflammatory and antioxidant activities depended on some structural characteristics of the synthesized compounds.

Introduction

Various non-steroidal anti-inflammatory drugs (NSAIDs) are in widespread clinical use for the treatment of inflammation diseases. However, despite their great number, their mechanism of action as well as their precise therapeutic activities are still under investigation. Furthermore, almost all of them present a number of unwanted, often serious, side effects as consequence of interference with the arachi­do­nic cascade [1]. It is well known that free radicals play an important role in the inflammatory process [2]. Super­oxide anion radicals, hydrogen peroxide and hydroxyl radicals, produced by activation of phagocytes, are considered to be involved in inflammation and tissue destruction. Free radicals are also involved in the biosynthesis of prostaglandins, important mediators of inflammation [2]. Compounds with anti­oxidant properties are generally expected to protect against inflammation.

Several substituted thiazolyl derivatives [3,4] are reported to possess anti-inflammatory and/or antioxidant activities. This paper is an extension of previous work on the synthesis of thiazole derivatives with structures justifying anti-inflammatory activity.

Results and Discussion

The structures of the synthesized compounds are given in Figure 1 and the general method used to prepare the final compounds is shown in Figure 2.

Figure 1. Structures of synthesized compounds

Figure 1. Structures of synthesized compounds

Figure 2. Synthetic procedure

Figure 2. Synthetic procedure

Overall the reactions proceeded smoothly in good yields. The structure of the synthesized compounds and their physicochemical properties are shown in Table 1. The amides were identified both by elemen­tal analyses as well as by their spectroscopic analyses.

Table 1. Characterization data of the synthesized amides

N	R1	R2	Z	n	m.p. oC	ms	Mol. Structure b
1	H	H	OH	1	85-86	241	C12H14N3O2S
2	CH3	H	OH	1	89-92	254	C11H16N3O2S
3	Ph	H	OH	1	89.5-92	316	C16H18N3O2S
4	Ph	CH3(CH2)13	OH	1	79-80	512	C30H46N3O2S
5	Ph-OCH3	H	OH	1	115-117	346	C17H20N3O4S
6	CH2COOEt	H	OH	1	79-82c	326	C14H20N3O4S
7	H	H	OH	2	semisolid	255	C11H16N3O2S
8	CH3	H	OH	2	148-150	269	C12H19N3O2S
9	Ph	H	OH	2	227-8	331	C17H21N3O2S
10	Ph-OCH3	H	OH	2	193-5	360	C18H22N3O2S
11	Ph	CH3(CH2)13	OH	2	semisolid	526	C31H48N3O2S
12	CH2COOEt	H	OH	2	116-8	343	C15H23N3O4S
13a	H	H	CH3	1	225-228	240	C13H16N3OS
14a	Ph	H	CH3	1	semisolid	316	C17H20N3OS
15a	CH2COOEt	H	CH3	1	semisolid	326	C15H22N3O3S
16a	Ph-OCH3	H	CH3	2	248-50	382.5	C15H22N3O3S

Notes: ^a Piperazinyl – ring; ^b Elemental analyses for molecular formula; ^c m.p for hydrochloride salt

Table 1. Characterization data of the synthesized amides

N	R1	R2	Z	n	m.p. oC	ms	Mol. Structure b
1	H	H	OH	1	85-86	241	C12H14N3O2S
2	CH3	H	OH	1	89-92	254	C11H16N3O2S
3	Ph	H	OH	1	89.5-92	316	C16H18N3O2S
4	Ph	CH3(CH2)13	OH	1	79-80	512	C30H46N3O2S
5	Ph-OCH3	H	OH	1	115-117	346	C17H20N3O4S
6	CH2COOEt	H	OH	1	79-82c	326	C14H20N3O4S
7	H	H	OH	2	semisolid	255	C11H16N3O2S
8	CH3	H	OH	2	148-150	269	C12H19N3O2S
9	Ph	H	OH	2	227-8	331	C17H21N3O2S
10	Ph-OCH3	H	OH	2	193-5	360	C18H22N3O2S
11	Ph	CH3(CH2)13	OH	2	semisolid	526	C31H48N3O2S
12	CH2COOEt	H	OH	2	116-8	343	C15H23N3O4S
13a	H	H	CH3	1	225-228	240	C13H16N3OS
14a	Ph	H	CH3	1	semisolid	316	C17H20N3OS
15a	CH2COOEt	H	CH3	1	semisolid	326	C15H22N3O3S
16a	Ph-OCH3	H	CH3	2	248-50	382.5	C15H22N3O3S

Notes: ^a Piperazinyl – ring; ^b Elemental analyses for molecular formula; ^c m.p for hydrochloride salt

Physicochemical parameters, e.g. log P have been calculated theoretically, as an expression of li­po­phi­licity (Table 2). The in vivo antiinflammatory activity of the synthesized compound was determined using carrageenin induced mice paw edema (26.1-64.3 %) (see Table 2). The compounds have also been screened for their in vitro reducing activity towards the free stable radical DPPH (15.6-26.6 %) and for their hydroxyl free radical scavenging activity (44.6-100 %) (Table 3). For the 4-OH-piperidinyl ami­des the inhibition on the soybean LOX has been performed (77.5-100 %). The preliminary results re­ve­al that the tested compounds exhibit in generally good biological activity. The results are discussed in terms of structural characteristics. The presence of a R₁ = phenyl group seems to be crucial for high ac­ti­vity in vivo. The length of the chain also plays an important role. The derivatives with n = 1 are stronger inhibitors. The nature of the alicyclic amine is an important feature, since the 4-N-methyl pi­pe­razinyl derivatives possess higher in vivo results.

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

N	RM	logPsk	Clog P	CPE%
1	-0.494± 0.03	-4.287	-0.34	52.3
2	nt	-1.878	0.159	59
3	-0.578± 0.031	-0.434	1.758	62.6
4	0.911± 0.037	6.322	6.322	44.2
5	-0.490± 0.031	-1.062	1.777	45
6	nt	-2.473	-0.133	33.4
7	-0.504± 0.021	-1.751	-0.143	26.1
8	-0.534± 0.037	-0.365	0.212	27.5
9	-0.589± 0.039	1.081	-1.0955	47.5
10	-0.504± 0.024	0.453	1.974	57
11	0.894± 0.033	6.5	8.684	32.2
12	nt	-0.958	0.014	59.6
13	nt	-3.747	1.102	61
14	nt	-1.969	3.2	64.3
15	nt	-2.161	1.309	47.9
16	nt	0.97	3.219	38.5

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

N	RM	logPsk	Clog P	CPE%
1	-0.494± 0.03	-4.287	-0.34	52.3
2	nt	-1.878	0.159	59
3	-0.578± 0.031	-0.434	1.758	62.6
4	0.911± 0.037	6.322	6.322	44.2
5	-0.490± 0.031	-1.062	1.777	45
6	nt	-2.473	-0.133	33.4
7	-0.504± 0.021	-1.751	-0.143	26.1
8	-0.534± 0.037	-0.365	0.212	27.5
9	-0.589± 0.039	1.081	-1.0955	47.5
10	-0.504± 0.024	0.453	1.974	57
11	0.894± 0.033	6.5	8.684	32.2
12	nt	-0.958	0.014	59.6
13	nt	-3.747	1.102	61
14	nt	-1.969	3.2	64.3
15	nt	-2.161	1.309	47.9
16	nt	0.97	3.219	38.5

Table 3. Biological testing results. In vitro effects of the examined and reference drugs (in % at 0.1 mmol/L and 0.2 mmol/L) on reducing ability (DPPH), on hydroxyl radical scavenging activity (^. OH) and on soybean lipoxygenase activity (LOX).

N	DPPH (0.1 mmol/L) 20min	DPPH (0.1 mmol/L) 1hr	DPPH (0.2 mol/L) 1 hr	. OH	LOX
1	25.4	24.6	23.5	97.2	no
2	13.5	17.3	18.1	100	100
3	nt	nt	nt	75.3	100
4	15.6	29.8	17.5	78.4	100
5	no	27.6	no	44.6	100
6	nt	nt	nt	no	100
7	nt	nt	nt	no	100
8	nt	nt	nt	nt	nt
9	23.7	25.1	36	no	100
10	26.7	32	35.3	95.9	100
11	17.8	17.7	16.1	40	100
12	13.3	14	20.6	87	77.5
ASA	80.5	–	–	–	–
NDGA	94	–	–	–	91

Table 3. Biological testing results. In vitro effects of the examined and reference drugs (in % at 0.1 mmol/L and 0.2 mmol/L) on reducing ability (DPPH), on hydroxyl radical scavenging activity (^. OH) and on soybean lipoxygenase activity (LOX).

N	DPPH (0.1 mmol/L) 20min	DPPH (0.1 mmol/L) 1hr	DPPH (0.2 mol/L) 1 hr	. OH	LOX
1	25.4	24.6	23.5	97.2	no
2	13.5	17.3	18.1	100	100
3	nt	nt	nt	75.3	100
4	15.6	29.8	17.5	78.4	100
5	no	27.6	no	44.6	100
6	nt	nt	nt	no	100
7	nt	nt	nt	no	100
8	nt	nt	nt	nt	nt
9	23.7	25.1	36	no	100
10	26.7	32	35.3	95.9	100
11	17.8	17.7	16.1	40	100
12	13.3	14	20.6	87	77.5
ASA	80.5	–	–	–	–
NDGA	94	–	–	–	91

Conclusions

We have presented a facile route for the formation of new 4,5-disubstituted thiazolyl amides, possessing anti-inflammatory activity. These compounds also present mild antioxidant activity. They highly compete with DMSO for hydroxyl radical and strongly inhibit LOX. A phenyl group in R_1, the length of the chain and a value of n = 1 are all significant structural features for the activity observed.

Experimental

General

Melting points were obtained with a MELTEMP II capillary apparatus (LAB. Devices Holliston, MA, USA) and are reported without correction. Infrared spectra (Nujol mulls) were recorded on a Perkin Elmer 597 spectrophotometer. UV-Vis were determined on a Perkin Elmer 554 UV-Vis spec­tro­photometer. Proton NMR spectra were obtained on a Brucker AW 80 apparatus at 80 MHz and are re­ported in ppm downfield from tetramethylsilane (TMS). Analyses indicated by the symbols of ele­ments were within 0.4 % of theoretical values. Mass spectra (MS) were determined on a VG-250 in­stru­ment (VG Labs. Tritech, England) with the ionization energy maintained at 70 eV. The reactions we­re monitored by TLC on silica gel 60 F₂₅₄ (Merck). All reagents were obtained from commercial sources. 2-Aminothiazole, 2-amino-4-methylthiazole, 2-amino-4-phenylthiazole and 2-amino-4-(4-metho­xy­­phenyl)thiazole were synthesized as described previously [5].

General synthetic procedure for the synthesis of the 2-chloroacetamido- and 3-chloropropionylamido thiazoles [5].

To a solution of 2-aminothiazole or suitable 4-substituted aminothiazole (0.02 mol) in dry benzene a cooled solution of chloroacetyl or 3-chloropropionyl chloride (0.033 mol) in dry benzene (7.5 mL) was added dropwise. The reaction mixture was refluxed in a water bath at 80 ^oC for 3 h. Benzene and ex­cess 3-chloropropionyl chloride were removed by distillation. The residue was washed with aqueous sodium bicarbonate (5 % w/v) followed by cold water. The crude product was dried and crystallized from ethanol.

General synthetic procedure for the synthesis of the 2-(N-substituted aminoacetamido)/3-(N-substituted aminopropioamido) thiazoles [5].

A mixture of 2-chloroacetamido or 3-chloropropionamido thiazole (0.006mol), amine (4-OH-piperidine or N-methyl-piperazine, 0.7 mol), absolute ethanol (15 mL) and anhydrous sodium car­bo­na­te (1.48 g) was heated under reflux in a water bath for 12 h. The excess of amine and ethanol was re­mo­ved by distillation and the residue was treated with 5 % sodium bicarbonate solution to remove acid im­purities, filtered, washed with water and dried. It was crystallized from ethanol (95 %) to give white crystals.

Preparation of the hydrochlorides of 2-(N-substituted- aminoacetamido) / 3-(N-substituted-amino-propionamido) thiazoles.

A solution of the base in anhydrous ethanol was saturated with dry hydrogen chloride gas. The salt was filtered off, washed with dry ether and recrystallized from absolute ethanol to give white crystals.

The structure of all compounds was identified both by elemental analyses as well as by spectroscopic analysis (IR, ¹H-NMR, MS).

Spectral Data: ¹H-NMR (CDCl₃, DMSO) for representative compounds ( δ):

Compound 1: 1.94-2.16 (d, 4H, piperidyl C3-H,), 2.157 (s, 2H, COCH₂), 3.2 (s, 1H, piperidyl C4-H), 3.2-3.38 (d, 4H, piperidyl NCH₂), 6.4 (d, 1H, thiazolyl C5-H), 6.86 (d, 1H, thiazolyl C4-H). Compound 9: 1.71-1.89 (d, 4H, piperidyl C3-H), 2.49-2.53 (m, 8H, NCH₂, COCH₂CH₂ ), 7.42-7.45 (m, 3H, phenyl C3,4,5-H), 7.46-7.48 (m, 2H, phenyl C-2.6-H), 9.05 (b, 1H, NH). Compound 16: 2.78 (s, 3H, NCH₃), 2.78-3.53 (m, 8H, piperidyl), 3.35 (s, 2H, COCH₂), 3.71 (s, 3H, OCH₃), 6.96-6.99 (d, 2H, phenyl C2,6-H), 7.51 (s, 1H, thiazolyl C5),7.8-7.89 (d, 2H, phenyl C3,5-H); IR cm^-1: 3190-3230, 1680, 1620

Biological assays

1.

Physicochemical Studies [4]

Lipophilicity is an important factor affecting the distribution and fate of drug molecules. Increased lipophilicity is correlated with increased biological activity and more rapid metabolism. Theoretical calculations of lipophilicity as clog P and Suzuki-Kudo’s method [10] were performed (Table 2). The program CLOG P [11], has been designed to calculate the lipophilicity of a molecule using the additivity method. Reversed phase TLC (RPTLC) was performed on silica gel plates impregnated with 55 (v/v) liquid paraffin in light petroleum ether. Mobile phase: methanol/water mixture (70/30, v/v) containing 2 % aqueous ammonia (27 %). The plates were developed in closed chromatography tanks saturated with the mobile phase at 24 ^oC. Spots were detected under UV light or by iodine vapours. R_M values were determined from the corresponding R_f values (from ten individual measurements) using the equation R_M = log [( 1/R_f ) -1 ]. For results see Table 2.

2.

Inhibition of carrageenin induced paw oedema [4]

All tested compounds were suspended in water with few drops of Tween-80 and ground in a mortar be­fore use. Groups of 5-6 AKR mice weighing 20-30 g were used. A single dose of 0.2 mmol/Kg or in­domethacin (0.11 mmol/Kg) was administered intraperitoneally i.p., simultaneously to the admi­nis­tra­tion of the phlogistic agent : carrageenin (2%, 0.05 ml was injected intradermally i.d. into the right fo­ot pad, the left serving as control). Results are given in Table 2.

3.

Competition of the tested compounds with DMSO for hydroxyl radicals [6]

The hydroxyl radicals generated by the Fe³⁺/ascorbic acid system were detected by the deter­mi­nation of formaldehyde produced from the oxidation of DMSO. The reaction mixture contained EDTA (0.1mM), Fe³⁺(167 μM, as a 1:2 mixture with EDTA) and DMSO (33mM) in phosphate buffer (50 mM, pH 7.4) and the tested compounds (final concentration 1mM - final volume of the samples 1 mL). As­cor­bic acid (150 μL, 10 mM in phosphate buffer) was added at the end in order to initiate the reaction. The mixture was incubated at 37 ^oC for 30 min. The reaction was stopped by the addition of tri­chlo­ro­ace­tic acid (250 μL, 17.5 % w/v) and the formaldehyde formed was detected spectrophotometrically at 412 nm by the method of Nash [7].

Interaction of the synthesized compounds with DPPH [8]

To a solution of DPPH (0.1 mM) in absolute ethanol, an equal volume of the compounds dissolved in ethanol was added (0.1 mM). A control solution containing ethanol was also used. After 20 and 60 mins at room temperature, absorbance was recorded at 517 nm. For the results see Table 3.

4.

Soybean lipoxygenase inhibition [9]

The conversion of sodium linoleate to 13-hydroxy-peroxylinoleic acid at 234 nm, was recorded and compared with appropriate standard, see Table 3.

Each in vitro experiment was performed at least in triplicate and the standard deviation in ab­sor­ban­ce was less than ±10 %. Acetyl salicylic acid (ASA) and/or nor-dihydroguaeretic acid (NDGA) were used as standards.