Synthesis and anti-inflammatory activity of ethynylthiazoles

Article abstract of DOI:10.1007/s10593-006-0145-8

A series of acetylene derivatives of thiazole using the Sonogashira cross-coupling method was synthesized and evaluated in vivo for their anti-inflammatory activity. Four compounds exhibited good anti-inflammatory activity and two inhibited soybean lipoxy

Full text of DOI:10.1007/s10593-006-0145-8

Chemistry of Heterocyclic Compounds, Vol. 42, No. 5, 2006
SYNTHESIS AND ANTI-INFLAMMATORY
ACTIVITY OF ETHYNYLTHIAZOLES
A. Geronikaki1, S. Vasilevsky2, D. Hadjipavlou-Litina1, A. Lagunin, and B. V. Poroikov3
A series of acetylene derivatives of thiazole using the Sonogashira cross-coupling method was
synthesized and evaluated in vivo for their anti-inflammatory activity. Four compounds exhibited good
anti-inflammatory activity and two inhibited soybean lipoxygenase.
Keywords: ethynylthiazoles, lipoxygenase (LOX), anti-inflammatory.
Inflammation is a complex phenomenon, involving the interrelationship between humoral and cellular
reactions through a number of inflammatory mediators. A substantial body of evidence suggests the involvement
of the products of the arachidonic acid metabolic pathway as mediators of a variety of cellular functions [1].
Excessive production of arachidonic acid metabolites has been shown to be important in the pathophysiology of
inflammation and thrombosis [2]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are of huge therapeutic
benefit in the treatment of inflammatory diseases. Recently the synthesis and biological activity of substituted
functional ethynylarenes and -hetarenes have been reported as a rapidly developing field of bioorganic chemistry
[3]. It is also well established that the thiazolyl group is of great importance in biological systems [4-11]. Several
thiazolyl derivatives were found to possess anti-inflammatory activity [4, 12]. Recently some acetylene
derivatives of thiazole were reported as potent and highly selective metabotropic glutamate subtype 5 receptor
antagonists with anxiolytic activity [13].
Computer analysis of structure-activity relationships and molecular modelling are widely used in
developing new leads by pharmaceutical chemists [14]. The majority of currently available molecular modelling
methods are designed to study the ligand-receptor interaction for one particular biological target at a time, while
quantitative structure-activity relationship (QSAR) analysis is mostly applicable to the optimization of properties
of the lead compounds within the same chemical series. In contrast to these methods, computer software PASS
(Prediction of Activity Spectra for Substances) predicts simultaneously several hundred types of biological
activity for drug-like substances from different chemical classes on the basis of their structural formulas [15-17].
It estimates probabilities not only for the desirable pharmacological effect but also for molecular mechanisms of
action and different undesirable and side effects. Such analysis of heterogeneous sets increases considerably the
chance to discover new chemical entities (NCEs).
The latest version of PASS 1.917 (July, 2005) predicts about 2,000 kinds of biological activity with a
mean prediction accuracy of about 87%. The list of predictable biological activities includes main and side
pharmacological effects (e.g., antihypertensive, hepatoprotective, sedative, etc.), mechanisms of action
__________________________________________________________________________________________
1
Aristotles University of Thessaloniki, School of Pharmacy, Thessaloniki 54124, Greece; e-mail:
2
geronik@pharm.auth.gr. Institute of Chemical Kinetics and Combustion, Siberian Branch of the Russian
Academy of Sciences, 630090, Novosibirsk, Russian Federation; e-mail: vasilev@ns.kinetics.usc.ru.
3 V. N. Orekhnovich Institute of Biomedical Chemistry, RAMS, 119992 Moscow, Russian Federation;
e-mail: vvp@ibmh.msk.ru. Published in Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 769-774, May,
2006. Original article submitted December 23, 2005.
0009-3122/06/4205-0675©2006 Springer Science+Business Media, Inc.
675

(5-hydroxytryptamine agonist, acetylcholinesterase inhibitor, adenosine uptake inhibitor, etc.), and specific
toxicities (mutageniсity, carcinogenicity, teratogenicity, etc.).
The current PASS training set includes 57978 substances. It is a complex knowledge base, containing
vocabularies of MNA (Multilevel Neighborhoods of Atoms), descriptors and activity names, the database for the
substance structures presented by MNA descriptors, their biological activity types, and data on the structure–
activity relationships.
PASS presents the biological activity spectra for each substance as a result of prediction. This is a list of
biological activity types for which the calculated probability to be present (Pa) is greater than the calculated
probability not to be present (Pi). Taking into account that some substances from the training set are formally
considered inactive, the estimated value of Pa is more reliable. However, even in this case there are some other
factors that significantly influence its absolute value: the number and diversity of substances revealing such
activity in the training set, recall ratio, etc. In general, the higher the Pa value, the higher is the probability for a
studied substance to be structurally similar to the known biologically active substances from the training set. The
result of prediction is valuable in planning the experiment, but one should take into account some additional
factors: a particular interest in some kinds of activity, desirable novelty of a substance, available facilities for
experimental testing, etc. Actually, each choice is always a compromise between the desirable novelty of the
studied substance and the risk to obtain a negative result in testing.
Taking into consideration the above mentioned and using PASS, we designed and synthesized
compounds in which the acetylenic triple bond as well as the thiazole ring were both involved in the same
molecule. Current investigation is targeted towards the development of a synthetic method for the preparation of
new ethynylthiazoles and the biological evaluation and correlation of their structure with biological activities.
HC
CR
N
N
PdCl2 (PPh3)2 , PPh3
CuI, Et3N
Br
S
C
CR
S
1
2–6
NaOH, 80oC
KOH, 20oC
R = Me3Si
R = Me2COH
, (CH O)
N
N
HN
O
2
n
C
CCH2N
O
S
S
7
C
CH
CuCl
8
2 R = Ph, 3 R = CH2OH, 4 R = Me2COH, 5 R = Me3Si, 6 R = α-Py
TABLE 1. Physicochemical Data and MS Spectra of Compounds 2-8
MS, m/z
Yield, %
Compound
Empirical formula
Rf*
Reaction time, h
2
3
4
5
6
7
8
C11H7NS
0.90
0.50
0.46
0.82
0.57
0.76
0.49
4
5
5
4
3
2
1
185
139
167
181
186
109
208
82
65
62
75
53
70
85
C6H5NOS
C8H9NOS
C8H11NSSi
C10H6N2S
C5H3NS
C10H12N2OS
_______
* Benzene–petroleum ether, 5:1 (compound 2), chloroform–ethylacetate, 3:1
(compounds 3-5, 7), chloroform–ethylacetate, 4:1 (compounds 6, 8).
676

The Sonogashira Pd–Cu-catalyzed cross-coupling [18] of 2-bromothiazole with terminal acetylenes
(Scheme 1) was used to prepare the substituted functional ethynyl derivatives of thiazoles (Table 1). The yields
of the synthesized compounds are 62-85%.
1
Compounds 2-4 and 6-8 were characterized by IR, MS, and H NMR spectroscopic data (Tables 1
and 2).
The in vivo anti-inflammatory effects of the tested compounds were assessed by using the functional
model of carrageenin-induced mice paw edema and are presented in Table 3 as percentage of weight increase in
the right hind paw in comparison to the uninjected left hind paw. Carrageenin-induced edema is a nonspecific
inflammation resulting from a complex of diverse mediators [19]. All the investigated compounds protected
against carrageenin-induced paw edema. The protection ranged from 37 to 80%, while the reference drug
indomethacin induced 44% protection at an equivalent concentration. Compound 6 was the most potent followed
by compounds 4 and 2.
Lipophilicity is an important physicochemical parameter for the kinetics of biologically active
compounds. In our case lipophilicity seems to affect the biological activities of the tested compounds, with the
exception of compound 3, which, although it presents the lowest clog P value, is highly potent (73%).
Representative compounds 2 and 6 were tested against soybean lipoxygenase in vitro in order to delineate the
TABLE 2. 1H NMR Spectra of Synthesized Compounds
Compound
δ, ppm (J, Hz)
2
7.54-7.55 (1H, d, J = 2, H-4 thiazole); 7.23-7.24 (1H, d, J = 2, H-5 thiazole);
7.31-7.35 (5H, m, C6H5)
3
4
6
7.56-7.60 (1H, d, J = 6, H-4 thiazole); 7.25-7.28 (1H, d, J = 6, H-5 thiazole);
4.5 (2H, s, CH2); 2.4 (H, s, OH)
7.77-7.82 (1H , d, J = 10, H-4 thiazole); 7.50-7.57 (1H, d, J = 10, H-5 thiazole);
5.4 (1H, br, OH); 1.40-1.75 (6H, m, CH3)
8.65-8.68 (1H, d, J = 6, α-H pyridine); 7.89-7.93 (1H, d, J = 6, H-4 thiazole);
7.69-7.76 (1H, m, γ-H pyridine); 7.60-7.69 (1H, d, J = 7, β'-H pyridine);
7.42-7.47 (1H, d, J = 6, H-5 thiazole); 7.28-7.36 (1H, m, β-Η pyridine)
7
8
7.20-7.29 (1H, d, J = 8, H-4 thiazole); 7.15-719 (1H, d, J = 8, H-5 thiazole);
2.4 (1H, s, C≡CH)
7.78-7.82 (1H, d, J = 8, H-4 thiazole); 7.33-7.38 (1H, d, J = 8, H-5 thiazole);
3.40-3.82 (10H, m, morpholine and CH2)
TABLE 3. Calculated Lipophilicity and Biological Evaluation of
Compounds 2-4, 6-8*
Inhibition, %
LOX (0.1 mM)
Compound
Clog P
LOX (1 mM)
CPE
2
3
4
6
7
8
3.12
-0.70
0.01
1.63
0.76
0.64
67
73
58
80
37
38
98.7
Nt
100
Nt
Nt
Nt
80
Nt
100
Nt
Nt
Nt
_______
* CPE – carrageenin paw edema; LOX – lipoxygenase; Clog P – calculated
lipophilicity.
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TABLE 4. Prediction of Spectrum of Biological Activities of
2-Ethynylthiazoles
Compound
Predicted activities
Pa
2
4
6
5-Lipoxygenase inhibitor
5-Lipoxygenase inhibitor
5-Lipoxygenase inhibitor
0.561
0.820
0.661
role of the ring nature in the biological activity. Both compounds present significantly high inhibition. The
phenyl ring (compound 2) correlates with higher activity as compared with the inhibition induced by the
pyridine analogue 6. These results occur in parallel to the lipophilicity values.
For compounds 2 and 6 the values predicted for biological activity using the PASS program showed
satisfactory inhibitory activity against lipoxygenase and significant antagonistic activity against leukotrienes
(Table 4). The results concerning prediction of lipoxygenase inhibitory activity coincide with the experimental
findings.
Thus, ethynylthiazoles could be used for the further synthesis of new biologically active compounds.
EXPERIMENTAL
Melting points were obtained with a MELTEMP II capillary apparatus (LAB Devices Holliston, MA,
USA) without correction. Infrared spectra (nujol mulls) were carried out on a Perkin Elmer 597
1
spectrophotometer, UV-Vis Hitachi U-2001. H NMR spectra were obtained on a Brucker AW 200 (200 MHz)
apparatus and were reported downfield from TMS. Mass spectra were determined on a VG-250 instrument (VG
Labs Tritech, England) with 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.
Synthesis of 2-Ethynyl-1,3-thiazoles 2-6 (General Procedure). Compounds 2–6 were synthesized
according to the scheme by refluxing a mixture of 2-bromo-1,3-thiazole (0.03 mol) and the corresponding
terminal acetylene (0.035 mol) in the presence of PdCl2(PPh3)2 (120 mg), triphenylphosphine (60 mg) and CuI
(60 mg), triethylamine (0.04 mol), and benzene at 30-80°C for 3-5 h under argon with stirring.
Compound 3. Mp 119-121°C (filtration from column with Al2O3 and washing with benzene). IR
spectrum, ν, cm-1: 2235 (C≡C).
2-Methyl-4-(1,3-thiazol-2-yl)butyn-2-ol (4). Bis(triphenylphospine)palladium(II) chloride (160 mg),
copper(I) iodide (80 mg), and triphenylphosphine (80 mg) were added successively to 2-bromothiazole (9.85 g,
0.06 mol) and then 2-methylbut-3-yn-2-ol (5.5g, 0.066 mol) in benzene (100 ml) and Et3N (11 ml, 0.08 mol)
under nitrogen at room temperature. The mixture was stirred under nitrogen for 4 h at 80°C. After cooling, the
mixture was filtered off, and the filtrate was decolorized through silica gel (3×3 cm). Evaporation of the filtrate,
addition of benzene, and recrystallization from benzene gave 6.2 g (62%) of compound 4 as sandy crystals;
mp 119-120°C (petroleum ether). IR spectrum, ν, cm-1: 2175 (C≡C), 3340 (OH).
Compound 6. Mp 52-54°C (benzene–petroleum ether). IR spectrum, ν, cm-1: 2277 (C≡C).
Compound 7 was synthesized from 2-methyl-4-(1,3-thiazol-2-yl)-3-butyn-2-ol 4 (0.04 mol) by heating
with sodium hydroxide (0.035 mol) in dry benzene under argon for 2 h. After cooling, the reaction mixture was
filtered off through Al2O3 (3 × 3 cm) and solvent was removed to give a brown powder. The product was
purified by column chromatography using SiO2 (12 × 1.5 cm) and petroleum ether (50 ml) as eluent followed by
a mixture of petroleum ether–benzene, 1:1 (50 ml). TLC of the last fraction showed one spot.
As an alternative method for preparation of compound 7 we used the treatment of 2-(trimethyl-
silylethynyl)-1,3-thiazole (5) at 20°C in the presence of KOH in methanol during 1 h. Methanol (25 ml) and
678

1N KOH solution (20 ml) was added to compound 5 (5.2 g, 0.37 mol). The mixture was stirred at room
temperature for 1 h. MeOH was removed under vacuum, and the water layer was extracted by ether (3 times) and
dried over Na2SO4. IR spectrum, ν, cm-1: 2106 (C≡C), 3283 C≡CH).
Compound 8 was prepared from 2-(1-ethynyl)-1,3-thiazole 7 (0.25 mmol), CuCl (50 mg),
paraformaldehyde (0.02 g), and morpholine (0.05 mol) in dioxane by heating at 65°C for 4 h under argon. After
cooling, the mixture was filtered off through silica gel (2 × 2 cm). Compound 8 was purified by recrystallization;
mp 154-156°C (from Al2O3 column, eluent petroleum ether), IR spectrum, ν, cm-1: 2235 (C≡C).
BIOLOGICAL EVALUATION
Animals. AKR or A mice (20-30 g, groups of ten, 2-3 months old), both male and female were used.
Pregnant females were excluded. The animals, bred in our laboratory, were housed under standard conditions
and received a diet of commercial food pellets and water ad libitum. Four hours before carrageenin treatment,
food and water were withdrawn from all animals.
Inhibition of Carrageenin-induced Edema [12]. Edema was induced in the right hind paw of AKR or
A mice (20-30 g, 2-3 months old) by an intradermal injection of 0.05 ml 2% carrageenin in water. These studies
were in accordance with recognized guidelines on animal experimentation (Guidelines for the care and use of
laboratory animals published by the Greek Government 160/1991, based on EU regulations 86/609).
The tested compounds of 0.1 mmol/kg body weight were suspended in water with a few drops of Tween-
80 and ground in a mortar before use and were given intraperitoneally (ip) at the same time as carrageenin. The
animals were euthanized 3.5 h after carrageenin injection. The experiment was repeated twice for each
compound (two groups of 6 animals). The difference between the weight of the injected and uninjected paws
was calculated for each animal. The change in paw weight was compared with that in control animals (injected
with water) and expressed as percent inhibition of the edema (CPE % values, Table 3). Indomethacin in
0.1 mmol/kg was administered as a standard comparative drug (44% inhibition).
Inhibition of the Soybean Lipoxygenase [20]. The tested compounds dissolved in DMSO (final
concentration 0.1 and 1mM) were incubated at room temperature with sodium linoleate (0.1 mM) and 0.2 ml of
enzyme solution (1/3 × 104 w/v in saline). The conversion of sodium linoleate to 13-hydroperoxylinoleic acid was
recorded at 234 nm and compared with nordihydroguaretic acid ((NDGA) = 94.4% at 0.1 mM), being an appropriate
standard inhibitor. The results are summarized in Table 3.
This research is supported by a NATO Fellowship and grant CRDF 008-XI.
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