6-(hydroxyphenyl)imidazo[2,1-b]thiazoles as potential antiinflammatory agents: Effects on human neutrophil functions

Article abstract of DOI:10.1135/cccc20000267

Synthesis of 6-(hydroxyphenyl)imidazo[2,1-b]thiazoles related to levamisole from the corresponding methoxy derivatives is reported. These compounds were tested on in vitro neutrophil activation, namely locomotion induced by chemoattractants, superoxide ge

Full text of DOI:10.1135/cccc20000267

6-(Hydroxyphenyl)imidazo[2,1-b]thiazoles
267
6-(HYDROXYPHENYL)IMIDAZO[2,1-b]THIAZOLES
AS POTENTIAL ANTIINFLAMMATORY AGENTS:
EFFECTS ON HUMAN NEUTROPHIL FUNCTIONS
a3
Aldo ANDREANIa1,*, Mirella RAMBALDIa2, Alberto LEONI
,
Alessandra LOCATELLIa4, Rita MORIGI , Serena TRANIELLO
,
a
b1
b
b
b2
Alessio CARIANI , Olivia RIZZUTI and Susanna SPISANI
a
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna,
Italy; e-mail: aldoandr@alma.unibo.it, mirella@alma.unibo.it, 3 aleoni@alma.unibo.it,
1
2
4
aleloc@alma.unibo.it
b
Department of Biochemistry and Molecular Biology, University of Ferrara, Via L. Borsari 46,
1
2
44100 Ferrara, Italy; e-mail: trs@unife.it, sps@unife.it
Received February 23, 1999
Accepted Decem ber 18, 1999
Syn th esis of 6-(h ydroxyph en yl)im idazo[2,1-b]th iazoles related to levam isole from th e corre-
spon din g m eth oxy derivatives is reported. Th ese com poun ds were tested on in vitro
n eutroph il activation , n am ely locom otion in duced by ch em oattractan ts, superoxide gen era-
tion an d lysozym e degran ulation triggered by agon ists. Th ese fun ction s were evaluated after
cell pulse with differen t con cen tration s of th e drugs. Several derivatives sh owed sign ifican t
in h ibitory effects, in som e cases m ore poten t th an th e paren t com poun d.
Key w ord s: Ph en ols; An tiin flam m atory agen ts; Im idazo[2,1-b]th iazoles; Levam isole; NSAIDs;
Neutroph il fun ction s.
Th e h ydroxyph en yl group of salicylic acid h as perh aps in spired th e design
of som e of th e subsequen t an tiin flam m atory/an tirh eum atic/an ti-
aggregatory agen ts con tain in g th is group as well as th e decision to test
structures kn own for differen t ph arm acological activities1–9. We just m en -
tion th e paper by Dewh irst10, wh o described structure–activity relation sh ips
in a series of 63 ph en ols en dowed with prostaglan din cyclooxygen ase in -
h ibitory activity. On e of th e m ost active com poun ds was 2-(2-h ydroxy-
ph en yl)ben zoth iazole (1). On th e basis of th ese con sideration s an d takin g
HO
N
N
S
S
N
1
2
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

268
Andreani et al.:
in to accoun t th at im idazoth iazoles related to levam isole 2 h ave been re-
ported as an tiin flam m atory agen ts11–15 an d th at levam isole itself is an
an tirh eum atic drug16–22 actin g on n eutroph ils23, we plan n ed th e syn th esis
of h ydroxyph en ylim idazo[2,1-b]th iazoles in order to test th eir effects on
h um an n eutroph il fun ction s.
It h as been ascertain ed th at n on steroidal an tiin flam m atory drugs
(NSAIDs) sh ow in vitro effects on n eutroph il fun ction s, con siderin g th e role
of th ese cells in th e in flam m atory process24. In fact, n eutroph ils are th e
prin cipal cell type of th e cellular in filtrate an d presen t an im portan t de-
fen se com pon en t of th e im m un e system in volved in th e in gestion an d deg-
radation of m icroorgan ism s. On th e oth er h an d, n eutroph ils are con sidered
h arm ful as m ediators of tissue dam age25 sin ce th e activation of th ese cells
leads to gen eration of reactive species an d extracellular release of gran ule
con stituen ts wh ich participate in th e propagation an d m ain ten an ce of
acute an d ch ron ic in flam m ation . Suppression of n eutroph il fun ction s m ay
con trol th e in flam m atory respon se an d h as been im plicated in th e m ech a-
n ism s of action of som e NSAIDs sin ce in h ibition of con stitutive an d in duc-
ible cyclooxygen ases does n ot seem to com pletely justify th eir an ti-
in flam m atory effects (for a review see ref.26). Som e NSAIDs in h ibit
n eutroph il activation by in flam m atory stim uli, such as C5a, LTB4, or
form yl–m eth ion in e–leucin e–ph en ylalan in e (FMLP), by in terferin g with
early even ts of sign al tran sduction 27–30. In dom eth acin , salicylic acid an d
acetylsalicylic acid were foun d to in h ibit superoxide gen eration by h um an
n eutroph ils exposed to ph orbol 12-m yristate 13-acetate (PMA) in
a
wh ole-cell system 31 wh ile predn isolon e in h ibited elastase release from n eu-
troph ils exposed to FMLP. Moreover, NSAIDs, such as acetylsalicylic acid,
in dom eth acin , ph en ylbutazon e, ibuprofen an d n aproxen , h ave lon g been
kn own to in h ibit platelet aggregation by actin g on th e cyclooxygen ase en -
zym e32,33. Th e tren d of m easurin g th e effects on n eutroph il fun ction s for
th e evaluation of n ew poten tial an tiin flam m atory agen ts is supported by a
recen t paper on 2-substituted 3-ch loro-1,4-n aph th oquin on e derivatives34.
Th e presen t paper is con n ected to our previous com m un ication 35 report-
in g syn th esis of rin g-open ed in dom eth acin an alogs in h ibitin g biological
fun ction s related to h um an n eutroph ils at n an om olar con cen tration s. Th e
n ew 6-(h ydroxyph en yl)im idazo[2,1-b]th iazoles described h ere (4a–4g, 6, 7,
9, 10, Sch em e 1) were tested for in vitro n eutroph il activation in duced by
differen t stim uli wh ich act eith er th rough specific cell-surface receptors
(e.g., FMLP) or by postreceptor sign alin g (e.g., PMA). Ch em otaxis, super-
oxide an ion gen eration an d lysozym e degran ulation were evaluated after
cell pulse with differen t con cen tration s of th e drugs.
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

6-(Hydroxyphenyl)imidazo[2,1-b]thiazoles
269
RESULTS AND DISCUSSION
Chemistry
Th e h ydroxy com poun ds 4a–4g, 6, 7, 9 an d 10 were prepared from th e cor-
respon din g m eth oxy derivatives. Th e startin g m eth oxy derivatives h ave
been previously described, except 2-ch loro-6-(4-m eth oxyph en yl)im idazo-
[2,1-b]th iazole (3g) wh ich was prepared by th e sam e procedure36,37. Th e
first attem pts of eth er cleavage were perform ed on im idazo[2,1-b]-
th iazolin es 3a–3c by refluxin g with an h ydrous alum in um ch loride in
xylen e for 5 h (m eth od A) an d furn ish ed th e expected ph en ols 4a–4c. Th e
sam e reaction was successfully em ployed for th e syn th esis of com poun ds
4e an d 4g from th e respective m eth oxy derivatives 3, wh ereas, if th e eth er
5 was subjected to th e sam e treatm en t, a com plex m ixture was obtain ed.
Y
S
Y
S
N
N
AlCl3 or BBr3
X
X
O
N
OH
N
3a-3g
4a-4g
3,4
a
b
c
d
e
f
X-Y
position of OH
CH2-CH2
CH2-CH2
CH2-CH2
CH=CH
CH=CH
CH=CH
CCl=CH
4
3
2
4
3
2
4
g
HO
HO
N
AlCl3
(r.t.)
S
S
N
N
O
6
O
N
N
S
N
O
HBr or AlCl3
(reflux)
5
OH
7
O
O
HO
N
N
N
N
HBr
+
S
N
S
S
N
O
OH
OH
10
8
9
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

270
Andreani et al.:
Wh en th e reaction was perform ed at room tem perature, on ly on e m eth oxy
group was cleaved to give com poun d 6. Th e expected 2,5-dih ydroxyph en yl
derivative 7 was obtain ed after a sh ort reflux with alum in um ch loride or us-
in g azeotropic (48%, 8.8 M) h ydrobrom ic acid (m eth od B). Wh en attem pts
to dem eth ylate com poun d 8, bearin g a double bon d in th e 2,3 position , by
th is m eth od was don e, a m ixture dih ydroxy derivative 10 an d a partially
cleaved derivative 9 was obtain ed. On ly th e dih ydroxy derivative 10 was
obtain ed with th e use of boron tribrom ide (m eth od C). If com poun d 3f was
subjected to th e m eth ods A an d B, form ation of com poun d 4f was n ot ob-
served. We failed to prepare 4f also with iodotrim eth ylsilan e prepared in
situ from ch lorotrim eth ylsilan e an d sodium iodide accordin g to th e litera-
ture38. Com poun ds 4d , 4f were prepared from th e respective m eth oxy com -
poun ds 3d , 3f by m eth od C. Th e above m eth ods were n ot tested system -
atically for each com poun d an d th erefore th e yields reported in th e experi-
m en tal section could be im proved.
Th e spectroscopic data of com poun ds 4a–4g, 6, 7, 9 an d 10 are in agree-
m en t with th e assign ed structures. Th e h ydroxy groups of com poun ds
4a–4g, 6, 7, 9 an d 10 give a broad ban d in th e ran ge 2 500–3 300 cm –1. In
1H NMR spectra, im idazoth iazoles 4d –4g, 9 an d 10 sh ow a typical arom atic
pattern with two doublets at ≈7 ppm (H-2) an d ≈8 ppm (H-3) wh ereas th e
2,3-dih ydro an alogs 4a–4c, 6 an d 7 sh ow an aliph atic pattern with two trip-
lets at ≈3.9 an d 4.2 ppm . In order to establish th e structure of th e m on o-
h ydroxyph en yl derivatives 6 an d 9, som e NOE experim en ts were
perform ed. If th e h ydroxy group of com poun d 6 was irradiated (10.46 ppm ),
NOE was observed at th e H-5 of im idazoth iazole (7.80 ppm ) an d at th e H-3
of th e ph en yl rin g (6.75 ppm ); m oreover, if th e m eth oxy group (3.69 ppm )
was irradiated, NOE was observed with th e proton s adjacen t to position 5
of th e ph en yl rin g (6.64 ppm (H-4) an d 7.24 ppm (H-6)). Th e opposite situ-
ation was en coun tered wh en we repeated th e sam e experim en ts on com -
poun d 9: th e irradiation of th e h ydroxy group (8.96 ppm ) gave NOE on ly at
th e ph en yl rin g (6.63, 7.59 ppm ) an d th e irradiation of th e m eth oxy group
(3.84 ppm ) gave en h an cem en t at th e H-3 of th e ph en yl rin g (6.90 ppm )
an d at th e H-5 of im idazoth iazole (8.16 ppm ). Th ese data suggest th at th e
h ydroxy group is in position 2 in com poun d 6 an d in position 5 in com -
poun d 9.
Biological Activity
In th e first step of our study, we tested wh eth er com poun ds 4a–4g, 6, 7, 9,
10 an d levam isole were ch em otaxin s for h um an n eutroph ils, secretagogue
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

6-(Hydroxyphenyl)imidazo[2,1-b]thiazoles
271
agen ts or were able per se to trigger superoxide an ion production . Non e of
th e m olecules was able to m ediate th ese activities. In th e secon d step, we
tested a possible in fluen ce of th e com poun ds on n eutroph il fun ction s.
In order to study th e in itial even t of in flam m ation , we an alyzed th e in -
fluen ce of th e prepared com poun ds an d levam isole on ran dom locom otion
in vitro. All com poun ds sh owed sligh t effects: h owever, th ese were n ot sta-
tistically sign ifican t (p > 0.05) at th e con cen tration s tested (10–7 an d 10–5
M). Con versely, wh en th e sam e com poun ds were tested for th eir ability to
m odulate directed m igration in duced by casein (Table I), a sign ifican t in h i-
bition of ch em otaxis was observed in th e presen ce of com poun ds 4c, 7, 9
an d levam isole. Th is in h ibition was con cen tration -depen den t but was
greater with levam isole at 10–5 M (p < 0.01) th an with th e h ydroxyph en yl
derivatives. On th e oth er h an d, com poun d 4c was able to in h ibit about
40% even at 10–7 M. Th e ch em otactic m otility in respon se to FMLP an d in
th e presen ce of th e tested com poun ds sh owed th at com poun ds 7 an d 10 at
th e h igh er con cen tration h ad th e stron gest in h ibitin g effect followed by 4c,
4d , 4g wh ich sh owed a tren d sim ilar to th at foun d for levam isole (Table I).
Th e oth er com poun ds sh own were weaker in h ibitors (p < 0.05) at th e sam e
con cen tration . It is to be poin ted out th at com poun ds 4c, 4d , 7, 9 an d 10,
at th e lower con cen tration displayed beh avior an alogous to th at of
levam isole with about 40% (p < 0.05) in h ibitory effect. Th ese data suggest
th at com poun ds 4c, 7, 9 an d levam isole probably act at th e sam e level,
sin ce th ey caused sim ilar respon ses n ot depen din g on th e ch em oattractan ts
used to activate n eutroph il ch em otaxis.
Com poun ds 4a–4g, 6, 7, 10 an d levam isole were n ot able to affect PMA-
or FMLP-in duced respiratory burst (n ot sh own ) at th e two con cen tration s
tested. Com poun d 9 was th e on ly on e sh owin g a m oderate in h ibitory effect
(35% at 10–5 M, FMLP, p = 0.05).
Testin g th e im idazoth iazoles for th eir ability to in fluen ce PMA- an d
FMLP-depen den t lysozym e release, an in h ibitory effect was seen for several
derivatives (Table I). Th e in h ibition of th e lysozym e release in duced by
PMA decreased sign ifican tly (p < 0.01) wh en th e com poun ds added were 4a
an d 7 with out a dose-effect relation sh ip. Levam isole was un able to reduce
degran ulation . If th e agon ist was FMLP, com poun d 4c, followed by 4b an d
9, were th e m ost poten t in h ibitors (p < 0.01) even at th e lower dose tested,
a beh avior very close to th at of levam isole. Th e oth er com poun ds sh own
were less effective (p < 0.05); h owever, with th e exception of com poun d 10,
all th e oth er derivatives sh owed a h igh er in h ibitory effect at 10–7 M.
It is eviden t th at th e tested 6-(h ydroxyph en yl)im idazo[2,1-b]th iazoles
4a–4g, 6, 7, 9 and 10 are not toxic (measurement of cell viability, see Experim en -
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

272
Andreani et al.:
TABLE I
Activity of com poun ds 4a–4g, 6, 7, 9 an d 10 on h um an n eutroph il fun ction s in com parison
with levam isole 2 (on ly th e sign ifican t values are reported)
Com -
Con cen tration FMLP-in duced Casein -in duced PMA-in duced FMLP-in duced
poun d
m ol/l
ch em otaxis
%
ch em otaxis
%
lysozym e
secretion , %
lysozym e
secretion , %
2
4a
4b
4c
4d
4e
4f
4g
6
10–7
10–5
10–7
10–5
10–7
10–5
10–7
10–5
10–7
10–5
10–7
10–5
10–7
10–5
10–7
10–5
10–7
10–5
10–7
10–5
10–7
10–5
10–7
10–5
60
50
75
65
69
70
61
48
61
53
79
75
78
80
83
56
80
67
62
30
57
67
60
34
80
44
105
102
59
60
60
74
81
65
76
50
53
72
90
59
56
54
87
73
74
70
67
67
60
54
65
64
75
77
70
79
67
83
62
71
87
73
7
76
73
77
70
9
10
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

6-(Hydroxyphenyl)imidazo[2,1-b]thiazoles
273
tal) an d possess differen t spectra of biological activities regardin g th e pa-
ram eters related to n eutroph il fun ction s. Levam isole displayed good levels
of in h ibition in both ch em otaxis stim ulated by eith er FMLP or casein (in a
dose-depen den t m an n er) an d in degran ulation in duced by FMLP. Th e pres-
en t derivatives, with th e exception of 9 wh ich gave about 35% in h ibition ,
did n ot affect n eutroph il-gen erated superoxide. Neverth eless, alm ost all th e
com poun ds in h ibited th e degran ulation in duced by eith er th e receptor ago-
n ist FMLP or th e postreceptor sign allin g PMA (with som e differen ces in po-
ten cy depen din g on th e stim ulus used) as well as ch em otaxis activated by
FMLP. Com poun ds 7 an d 9 appeared to be en dowed with a wide ran ge of
activity. Th ey m ain tain th e in h ibitory power of levam isole, bein g in som e
cases m ore active; for exam ple both com poun ds in PMA-in duced
degran ulation an d com poun d 7 also in FMLP-in duced ch em otaxis. Com -
poun d 4c is as poten t as levam isole in FMLP-triggered degran ulation an d
in duced a 40% in h ibition at th e lower con cen tration in casein -activated
ch em otaxis.
In con clusion , som e derivatives display in terestin g properties as in h ibi-
tors of h um an n eutroph il fun ction s an d, for th is reason , m ay be con sidered
as poten tial an tiin flam m atory agen ts. Th ey could block recruitm en t of n eu-
troph ils in to in flam m atory lesion s an d con trol n eutroph il degran ulation ,
sh owin g a protective effect again st th e direct in jury to th e target tissue de-
rived from en zym e release.
EXPERIMENTAL
Ch em istry
Meltin g poin ts are un corrected. IR spectra were taken on
a
Perkin –Elm er 683
spectroph otom eter (waven um bers in cm –1). 1H NMR spectra were recorded on a Varian
Gem in i (300 MHz) m ach in e, an d were referen ced to solven t sign als (dim eth yl sulfoxide-d6).
Ch em ical sh ifts are given in ppm (δ-scale), couplin g con stan ts (J) in Hz. Th in layer ch rom a-
tograph y was don e on pre-coated TLC plates Bakerflex (silica gel IB2-F) usin g m ixtures of pe-
troleum eth er an d aceton e as eluen ts. Silica gel 60 (Merck) was used for colum n
ch rom atograph y.
2-Ch loro-6-(4-m eth oxyph en yl)im idazo[2,1-b]th iazole (3g)
2-Am in o-5-ch loroth iazole (6.7 g, 50 m m ol) was dissolved in aceton e (100 m l) an d treated
with 2-brom o-1-(4-m eth oxyph en yl)eth an on e (11.4 g, 50 m m ol). Th e reaction m ixture was
refluxed for 2 h an d th e resultin g precipitate was collected by filtration . Th is in term ediate
2-(2-im in o-5-ch loroth iazol-3-yl)-1-(4-m eth oxyph en yl)eth an on e was treated, with out furth er
purification , with 2 M solution of h ydrobrom ic acid (400 m l) an d refluxed for 1 h . Th e solu-
tion th us obtain ed was basified with 5 M solution of am m on ium h ydroxide an d th e result-
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

274
Andreani et al.:
in g free base was crystallized from eth an ol to give pure 3g (7.9 g, 60%); m .p. 204–208 °C.
For C12H9ClN2OS (264.7) calculated: 54.44% C, 3.43% H, 10.58% N; foun d: 54.72% C,
3.19% H, 10.71% N. IR: 995, 1 030, 1 250, 1 545. 1H NMR: 3.77 s, 3 H (CH3O); 6.97 d, 2 H,
J = 9 (arom . H); 7.76 d, 2 H, J = 9 (arom . H); 8.12 s, 1 H (H-3); 8.28 s, 1 H (H-5).
Dem eth ylation with An h ydrous Alum in um Ch loride (Meth od A) – Gen eral Procedure
A m ixture of th e startin g m eth oxy derivative (8 m m ol) an d xylen e (100 m l) was treated with
an h ydrous alum in um ch loride (2.7 g, 20 m m ol). Th e reaction m ixture was refluxed for 5 h
an d evaporated un der reduced pressure. Water was added to th e residue an d th e m ixture
was basified with 2 M solution of am m on ium h ydroxide. Th e resultin g precipitate was col-
lected by filtration an d crystallized from eth an ol to give yields of 55–60%. Com poun d 6
could be obtain ed after 48 h at room tem perature an d com poun d 7 after 40 m in reflux.
6-(4-Hydroxyphenyl)imidazo[2,1-b]thiazoline (4a ). Yield 58%, m .p. 280–282 °C. For
C
11H10N2OS (218.3) calculated: 60.53% C, 4.62% H, 12.83% N; foun d: 60.65% C, 4.88% H,
13.04% N. IR: 820, 1 270, 1 490, 1 545. 1H NMR: 3.86 t, 2 H, J = 7 (H-2); 4.16 t, 2 H, J = 7
(H-3); 6.72 d, 2 H, J = 8 (arom . H); 7.48 d, 2 H, J = 8 (arom . H); 7.49 s, 1 H (H-5); 9.35 s, 1 H
(OH).
6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazoline (4b ). Yield 56%, m .p. 237–239 °C. For
C
11H10N2OS (218.3) calculated: 60.53% C, 4.62% H, 12.83% N; foun d: 60.35% C, 4.33% H,
12.75% N. IR: 965, 1 220, 1 475, 1 575. 1H NMR: 3.88 t, 2 H, J = 7 (H-2); 4.19 t, 2 H, J = 7
(H-3); 6.58 m , 1 H (arom . H); 7.09 m , 3 H (arom . H); 7.63 s, 1 H (H-5); 9.32 s, 1 H (OH).
6-(2-Hydroxyphenyl)imidazo[2,1-b]thiazoline (4c). Yield 60%, m .p. 165–169 °C. For
C
11H10N2OS (218.3) calculated: 60.53% C, 4.62% H, 12.83% N; foun d: 60.84% C, 4.58% H,
12.64% N. IR: 750, 830, 1 250, 1 580. 1H NMR: 3.80 t, 2 H, J = 7 (H-2); 4.15 t, 2 H, J = 7
(H-3); 6.90 m , 3 H (arom . H); 7.62 m , 1 H (arom . H); 7.72 s, 1 H (H-5); 11.13 s, 1 H (OH).
6-(3-Hydroxyphenyl)imidazo[2,1-b]thiazole (4e). Yield 59%, m .p. 207–211 °C. For
C
11H8N2OS (216.3) calculated: 61.09% C, 3.73% H, 12.95% N; foun d: 61.37% C, 3.94% H,
12.66% N. IR: 1 155, 1 175, 1 215, 1 590. 1H NMR: 6.66 m , 1 H (arom . H); 7.20 m , 3 H
(arom . H); 7.25 d, 1 H, J = 4.4 (H-2); 7.91 d, 1 H, J = 4.4 (H-3); 8.14 s, 1 H (H-5); 9.43 s, 1 H
(OH).
2-Chloro-6-(4-hydroxyphenyl)imidazo[2,1-b]thiazole (4g). Yield 60%, m .p. 185–189 °C. For
C
11H7ClN2OS (250.7) calculated: 52.70% C, 2.81% H, 11.17% N; foun d: 52.91% C, 2.63% H,
10.94% N. IR: 1 240, 1 260, 1 550, 1 610. 1H NMR: 6.78 d, 2 H, J = 8 (arom . H); 7.63 d, 2 H,
J = 8 (arom . H); 8.03 s, 1 H (H-3); 8.25 s, 1 H (H-5); 9.49 s, 1 H (OH).
6-(2-Hydroxy-5-methoxyphenyl)imidazo[2,1-b]thiazoline (6).Yield 58%, m .p. 227–230 °C. For
C
12H12N2O2S (248.3) calculated: 58.05% C, 4.87% H, 11.28% N; foun d: 57.86% C, 5.06% H,
11.49% N. IR: 1 155, 1 200, 1 265, 1 500. 1H NMR: 3.69 s, 3 H (CH3O); 3.93 t, 2 H, J = 7
(H-2); 4.25 t, 2 H, J = 7 (H-3); 6.64 dd, 1 H, J = 3, J = 8 (arom . H-4); 6.75 d, 1 H, J = 8 (arom .
H-3); 7.24 d, 1 H, J = 3 (arom . H-6); 7.80 s, 1 H (H-5); 10.46 s, 1 H (OH).
6-(2,5-Dihydroxyphenyl)imidazo[2,1-b]thiazoline (7). Yield 60%, m .p. 188–190 °C. For
C
11H10N2O2S (234.3) calculated: 56.40% C, 4.30% H, 11.96% N; foun d: 56.69% C, 4.12% H,
12.12% N. IR: 965, 1 195, 1 335, 1 480. 1H NMR: 3.94 t, 2 H, J = 7 (H-2); 4.26 t, 2 H, J = 7
(H-3); 6.45 dd, 1 H, J = 3, J = 8 (arom . H-4); 6.62 d, 1 H, J = 8 (arom . H-3); 7.07 d, 1 H, J = 3
(arom . H-6); 7.71 s, 1 H (H-5); 8.72 s, 1 H (OH); 10.29 s, 1 H (OH).
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

6-(Hydroxyphenyl)imidazo[2,1-b]thiazoles
275
Dem eth ylation with Hydrobrom ic Acid (Meth od B) – Gen eral Procedure
A m ixture of th e startin g m eth oxy derivative (2 m m ol) an d 20 m l of azeotropic aqueous
h ydrobrom ic acid (8.8 M) was refluxed for 18 h . Th e m ixture was diluted with water (40 m l)
an d treated with 15% (5 M) am m on ium h ydroxide un til pH 6 was reach ed. Th en th e m ix-
ture was extracted with m eth ylen e ch loride, dried an d evaporated un der reduced pressure.
Th e crude product th us obtain ed was purified by colum n ch rom atograph y (h exan e–
butan -2-on e, 70 : 30).
6-(2,5-Dihydroxyphenyl)imidazo[2,1-b]thiazoline (7). Yield 30%. Th e sam ple was in all aspects
iden tical with th e com poun d prepared by m eth od A.
6-(5-Hydroxy-2-methoxyphenyl)imidazo[2,1-b]thiazole (9). Yield 20%, m .p. 165–169 °C. For
C
12H10N2O2S (246.3) calculated: 58.52% C, 4.09% H, 11.37% N; foun d: 58.81% C, 3.86% H,
11.49% N. IR: 1 160, 1 195, 1 275, 1 495. 1H NMR: 3.84 s, 3 H (CH3O); 6.63 dd, 1 H, J = 3, J = 8
(arom . H-4); 6.90 d, 1 H, J = 8 (arom . H-3); 7.23 d, 1 H, J = 4.4 (H-2); 7.59 d, 1 H, J = 3
(arom . H-6); 7.92 d, 1 H, J = 4.4 (H-3); 8.16 s, 1 H (H-5); 8.96 s, 1 H (OH).
6-(2,5-Dihydroxyphenyl)imidazo[2,1-b]thiazole (10). Yield 20%, m .p. 197–200 °C. For
C
11H8N2O2S (232.3) calculated: 56.88% C, 3.47% H, 12.06% N; foun d: 57.04% C, 3.62% H,
11.88% N. IR: 1 260, 1 325, 1 485, 1 530. 1H NMR: 6.54 dd, 1 H, J = 3, J = 8 (arom . H-4);
6.71 d, 1 H, J = 8 (arom . H-3); 7.26 d, 1 H, J = 3 (arom . H-6); 7.28 d, 1 H, J = 4.4 (H-2);
7.96 d, 1 H, J = 4.4 (H-3); 8.22 s, 1 H (H-5); 8.77 s, 1 H (OH); 10.20 s, 1 H (OH).
Dem eth ylation with Boron Tribrom ide (Meth od C) – Gen eral Procedure
A solution of th e startin g m eth oxy derivative (3 m m ol) in m eth ylen e ch loride (40 m l) was
treated un der n itrogen at 0–5 °C with
ch loride (4 m l, 4 m m ol). Th e m ixture was stirred overn igh t at room tem perature, treated with
M solution of sodium h ydrogen carbon ate (pH 6), stirred for addition al 10 m in an d ex-
1 M solution of boron tribrom ide in m eth ylen e
1
tracted with m eth ylen e ch loride. After th e usual procedures, th e crude product was crystal-
lized from a m ixture of aceton e an d petroleum eth er.
6-(4-Hydroxyphenyl)imidazo[2,1-b]thiazole (4d ). Yield 42%, m .p. 242–245 °C. For
C
11H8N2OS (216.3) calculated: 61.09% C, 3.73% H, 12.95% N; foun d: 60.98% C, 4.02% H,
13.05% N. IR: 1 165, 1 280, 1 540, 1 605. 1H NMR: 6.79 d, 2 H, J = 8 (arom . H); 7.25 d, 1 H,
J = 4.4 (H-2), 7.63 d, 2 H, J = 8 (arom . H); 7.93 d, 1 H, J = 4.4 (H-3); 8.06 s, 1 H (H-5); 9.50 s,
1 H (OH).
6-(2-Hydroxyphenyl)imidazo[2,1-b]thiazole (4f). Yield 45%, m .p. 170–173 °C. For C11H8N2OS
(216.3) calculated: 61.09% C, 3.73% H, 12.95% N; foun d: 61.15% C, 3.73% H, 12.78% N. IR:
1 020, 1 240, 1 255, 1 570. 1H NMR: 6.88 m , 2 H (arom . H); 7.12 t, 1 H, J = 8 (arom . H);
7.31 d, 1 H, J = 4.4 (H-2); 7.87 d, 1 H, J = 8 (arom . H); 7.99 d, 1 H, J = 4.4 (H-3); 8.29 s, 1 H
(H-5); 10.97 s, 1 H (OH).
Biology
Dextran an d Ficoll–Paque were purch ased from Ph arm acia (Uppsala, Sweden ). Levam isole,
FMLP, PMA, cytoch alasin B, ferricytoch rom e C, bovin e serum album in , superoxide
dism utase an d Micrococcus lysodeicticus were obtain ed from Sigm a Ch em ical Co. (St. Louis
(MO), U.S.A.); filters for th e ch em otactic ch am ber were from Millipore (Rom a, Italy) an d ca-
sein “Ham m arsten ” was from Merck (Darm stadt, Germ an y). Sterile 96-well m icrotitre plates
with flat-bottom ed wells were from Falcon Microtest III, Becton –Dickin son Labware (Milan o,
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

276
Andreani et al.:
Italy). NADH was from Fluka. A 10–2 M FMLP stock solution in dim eth yl sulfoxide (DMSO)
was diluted before use in Krebs–Rin ger-ph osph ate con tain in g 0.1% (w/v) D-glucose (KRPG),
pH 7.4. A PMA stock solution of 1 m g/m l in DMSO was diluted before use with KRPG. A
casein stock solution 10 m g/m l in KRPG was diluted before use with KRPG con tain in g 1 m g/m l
of bovin e serum album in (KRPG-A). Stock solution s (10–2 M) of th e tested com poun ds an d
levam isole in DMSO were diluted before use with KRPG. DMSO used did n ot in terfere with
an y of th e biological assays perform ed.
Cell Preparation
Cells were obtain ed from th e blood of h ealth y subjects an d n eutroph ils were purified em -
ployin g th e stan dard tech n iques of dextran sedim en tation , cen trifugation on Ficoll–Paque
an d h ypoton ic lysis of con tam in atin g red cells. Th e cells were wash ed twice, resuspen ded in
KRPG to obtain a fin al con cen tration of 50 · 106 cells/m l an d kept at room tem perature un -
til used. Th e percen tage of n eutroph ils was 98–100% pure an d >99% viable as determ in ed
by Trypan blue exclusion test.
Ran dom Locom otion
Ran dom locom otion was evaluated with a 48-well m icroch em otaxis ch am ber, by estim atin g
th e distan ce in m icrom eters wh ich th e leadin g fron t of th e cell m igrated, usin g th e m eth od
of Zigm on d an d Hirsch 39 after 90 m in in cubation at 37 °C. A 3-µm pore-size filter separated
upper an d lower com partm en ts. Th e actual con trol for ran dom locom otion was 40 ± 4 µm
SE from 15 separate experim en ts.
Ch em otaxis
Ch em otaxis was studied by addin g th e ch em oattractan t to th e lower com partm en t. Th e
ch em otactic factors used were FMLP (10–8 M in KRPG-A) an d casein (2 m g/m l in KRPG-A).
Th e actual con trol of ch em otaxis was 95 ± 4 µm or 83 ± 2 µm SE, from 15 separate experi-
m en ts, in duced by 10–8 M FMLP an d 2 m g/m l casein , respectively.
Superoxide An ion Production
Th e superoxide an ion was m easured by th e superoxide dism utase-in h ibitable reduction of
ferricytoch rom e40 m odified for m icroplate-based assays. Th e tests were carried out in a fin al
volum e of 200 µl con tain in g 4 · 105 n eutroph ils, 100 n m ol cytoch rom e C an d KRPG. At zero
tim e, th e stim ulan t was added an d th e plates were in cubated in a m icroplate reader (Ceres
900, Bio-TeK in strum en ts, INC) with th e com partm en t T set at 37 °C. Th e absorban ce was
recorded at wavelen gth s of 550 an d 468 n m . Differen ces in absorban ce at th e two wave-
len gth s were used to calculate th e O−2 produced in n m ol, usin g a m illim olar absorption coef-
ficien t for cytoch rom e C of 15.5. Th e stim ulan ts em ployed were PMA (100 n g/m l in KRPG)
an d FMLP (10–6 M in KRPG). Neutroph ils were prein cubated with cytoch alasin B 5 µg/m l for
5 m in prior to activation by FMLP.
En zym e Assay
Th e release of n eutroph il gran ule en zym es was evaluated by determ in in g lysozym e activ-
ity40, m odified for m icroplate-based assays. 3 · 106 cells were in cubated in m icroplates wells
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

6-(Hydroxyphenyl)imidazo[2,1-b]thiazoles
277
in th e presen ce of th e stim ulus for 15 m in at 37 °C. Th e plates were th en cen trifuged for
5 m in at 400 g an d th e lysozym e was quan tified n eph elom etrically by th e lysis rate of
cell-wall suspen sion of Micrococcus lysodeikticus. Th e reaction rate was m easured with
a
m icroplate reader at 465 n m . Th e en zym e was expressed as a n et percen tage of th e total en -
zym e con ten t released by 0.1% Triton X-100. Th e total en zym e activity was 85 ± 1 µg/1 · 107
cells/m in . Spon tan eous release was less th an 10%. Th e degran ulatin g agen ts used were PMA
(0.1 µg/m l) an d FMLP (10–6 M) in KRPG. Cells were prein cubated with cytoch alasin
B
(5 µg/m l) for 15 m in prior to activation by FMLP. Assays were curren tly don e in triplicate
for each experim en tal con dition . Cells were in cubated with 10–7 an d 10–5 M of th e com -
poun ds un der test for 10 m in before th e addition of stim ulus. Th e actual con trol of
lysozym e release was or 30 ± 2% or 45 ± 4%/3 · 106 cells/15 m in from 15 separate experi-
m en ts, in duced by 100 n g/m l PMA an d 10–6 M FMLP, respectively.
Measurem en t of Cell Viability
In order to assess possible cytotoxic effects of th e tested com poun ds, th e cytoplasm ic m arker
en zym e, lactate deh ydrogen ase (LDH), was determ in ed by m easurin g th e rate of oxidation of
NADH. Th e absorban ce ch an ge was followed at 340 n m (ref.41) (data n ot sh own ).
Treatm en t with th e Com poun ds un der Test
Wh en required, 10–7 an d 10–5 M solution s of com poun ds 4a–4g, 6, 7, 9, 10 an d levam isole
were added to n eutroph ils 10 m in before th e in cubation step for cell fun ction ality.
Statistical An alysis
Th e n on param etric Wilcoxon test was used in th e statistical evaluation of differen ces be-
tween groups.
This work was supported by a grant from the Italian National Research Council (CNR) and
MURST. We are grateful to “Banca del Sangue di Ferrara” for supplying fresh blood.
REFERENCES
1. Ozaki Y.: Chem. Pharm. Bull. 1992, 40, 954.
2. Bendele A. M., Benslay D. N., Hom J. T., Spaethe S. M., Ruterbories K. J., Lindstrom T. D.,
Lee S. J., Naismith R. W.: J. Pharmacol. Exp. Ther. 1992, 260, 300.
3. Mullican M. D., Wilson M. W., Connor D. T., Kostlan C. R., Schrier D. J., Dyer R. D.:
J. Med. Chem. 1993, 36, 1090.
4. Schrier D. J., Baragi V. M., Connor D. T., Dyer R. D., Jordan J. H., Imre K. M., Lesch M. E.,
Mullican M. D., Okonkwo G. C., Conroy M. C.: Prostaglandins 1994, 47, 17.
5. Unangst P. C., Connor D. T., Cetenko W. A., Sorenson R. J., Kostlan C. R., Sircar J. C.,
Wright C. D., Schrier D. J., Dyer R. D.: J. Med. Chem. 1994, 37, 322.
6. Fukumoto S., Terashita Z., Ashida Y., Terao S., Shirashi M.: Chem. Pharm. Bull. 1996, 44,
749.
7. Yamamoto S., Jiang H., Kato R.: Pharmacology 1994, 48, 273.
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

278
Andreani et al.:
8. Krause M., Rouleau A., Stark H., Luger P., Lipp R., Garbar M., Schwartz J. C., Schunack W.:
J. Med. Chem. 1995, 38, 4070.
9. Rouleau A., Garbar M., Ligneau X., Mantion C., Lavie P., Advenier C., Lecomte J. M.,
Krause M., Stark H., Schunack W., Schwartz J. C.: J. Pharmacol. Exp. Ther. 1997, 281,
1085.
10. Dewhirst F. E.: Prostaglandins 1980, 20, 209.
11. Paolini J. P., Lendvay L. J.: J. Med. Chem. 1969, 12, 1031.
12. Powers L. J., Fogt S. W., Ariyan Z. S., Rippin D. J., Heilman R. D., Matthews R. J.: J. Med.
Chem. 1981, 24, 604.
13. Andreani A., Bonazzi D., Rambaldi M., Fabbri G., Rainsford K. D.: Eur. J. Med. Chem.
1982, 17, 271.
14. Isomura Y., Ito N., Sakamoto S., Homma H., Abe T., Kubo K.: Chem. Pharm. Bull. 1983,
31, 3179.
15. Palagiano F., Arenare L., Luraschi E., de Caprariis P., Abignente E., D’Amico M., Filippelli W.,
Rossi F.: Eur. J. Med. Chem. 1995, 30, 901.
16. Hunneyball I. M.: Prog. Drug Res. 1980, 24, 101.
17. Veys E. M., Mielants H., Verbruggen G., Dhondt E., Goethals L., Cheroutre L.: J. Rheumatol.
1981, 8, 45.
18. Dayrens P., Ivanoff B., Cussac M., Fontanges R.: Arzneim. Forsch. 1983, 33, 372.
19. Wildfeuer A.: Arzneim. Forsch. 1983, 33, 780.
20. Huck F., de Medicis R., Lussier A., Dupuis G., Federlin P.: J. Rheumatol. 1984, 11, 605.
21. Veys E. M., Mielants H., Verbruggen G.: Clin. Exp. Rheumatol. 1987, 5, 111.
22. Nielsen O. H., Ahnfelt-Ronne I., Elmgreen J.: Agents Actions 1989, 26, 233.
23. Nielsen O. H., Ahnfelt-Ronne I., Elmgreen J.: Pharmacol. Toxicol. (Copenhagen) 1988, 62,
322.
24. Raghoebar M., Tiemessen H. L., van den Berg W. B., van Ginneken C. A.: Pharmacology
1989, 39, 350.
25. Berradia N., Marchand-Arvier M., Humbert J. C., Vigneron C.: J. Pharm. Pharmacol. 1988,
40, 806.
26. Kankaanranta H., Moilanen E., Vapaatalo H.: Naunyn–Schmiedeberg’s Arch. Pharmacol.
1994, 350, 685.
27. Crowell R. F., van Epps D. E.: Inflammation 1990, 14, 163.
28. Partsch G., Schwarzer C., Eberl R.: J. Rheumatol. 1990, 17, 583.
29. Kankaanranta H., Moilanen E., Vapaatalo H.: Inflammation 1991, 15, 137.
30. Gonzales-Alvaro I., Carmona L., Diaz-Gonzales F., Gonzales-Amaro R., Mollinedo F.,
Sanchez-Madrid F., Laffon A., Garcia-Vicuna R.: J. Rheumatol. 1996, 23, 723.
31. Umeki S.: Biochem. Pharmacol. 1990, 40, 559.
32. Adeyemi E. O., Chadwick V. S., Hodgson J. F.: J. Pharm. Pharmacol. 1990, 42, 487.
33. Petrusewicz J., Turowski M., Foks H., Pilarski B., Kaliszan R.: Life Sci. 1995, 56, 667.
34. Lien J. C., Huang L. J., Wang J. P., Teng C. M., Lee K. H., Kuo S. C.: Bioorg. Med. Chem.
1997, 5, 2111.
35. Andreani A., Leoni A., Locatelli A., Morigi R., Rambaldi M., Gehret J. C., Traniello S.,
Cariani A., Spisani S.: Collect. Czech. Chem. Commun. 1999, 64, 299.
36. Andreani A., Rambaldi M., Bonazzi D., Lelli G., Bossa R., Galatulas I.: Eur. J. Med. Chem.
1984, 19, 219.
37. Andreani A., Rambaldi M., Bonazzi D., Bossa R., Galatulas I.: Arch. Pharm. (Weinheim,
Ger.) 1985, 318, 1003.
Collect. Czech. Chem. Commun. (Vol. 65) (2000)

6-(Hydroxyphenyl)imidazo[2,1-b]thiazoles
279
38. Olah G. A., Narang S. C., Gupta B. G. B., Malhotra R.: J. Org. Chem. 1979, 44, 1247.
39. Zigmond S. H., Hirsch J. G.: J. Exp. Med. 1973, 137, 387.
40. Torrini I., Pagani Zecchini G., Paglialunga Paradisi M., Lucente G., Mastropietro G.,
Gavuzzo E., Mazza F., Pochetti G., Traniello S., Spisani S.: Biopolymers 1996, 39, 327.
41. Bergmeyer H. U. in: Methods of Enzymatic Analysis (H. U. Bergmeyer, Ed.), 3rd ed., Vol. 3,
p. 118. Verlag Chemie, Weinheim 1983.
Collect. Czech. Chem. Commun. (Vol. 65) (2000)