Ring-opened analogs of indomethacin affecting human neutrophil functions

Article abstract of DOI:10.1135/cccc19990299

A series of ring-opened analogs of indomethacin was synthesized and tested in vitro (at concentrations ranging from 10^-9 to 10^-5 mol/1) on human neutrophil functions. Two compounds lacking the carboxylic group were subjected to the s

Full text of DOI:10.1135/cccc19990299

Ring-Opened Analogs of Indomethacin
299
RING-OPENED ANALOGS OF INDOMETHACIN AFFECTING HUMAN
NEUTROPHIL FUNCTIONS+
a3
Aldo ANDREANIa1,*, Alberto LEONIa2, Alessandra LOCATELLIa3, Rita MORIGI
,
a
b
c1
Mirella RAMBALDI , Jean-Claude GEHRET , Serena TRANIELLO ,
c2
c2
Alessio CARIANI and Susanna SPISANI
a
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna,
1
2
3
Italy; e-mail: aldoandr@alma.unibo.it, aleoni@alma.unibo.it, aleloc@alma.unibo.it
Novartis Crop Protection AG, P.O. Box, CH-4002 Basel, Switzerland;
e-mail: jc.gehret@cp.Novartis.com
b
c
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 July 27, 1998
Accepted Jan uary 8, 1999
Dedicated to the memory of Miroslav Protiva, professor at the University of Bologna in 1988.
A series of rin g-open ed an alogs of in dom eth acin was syn th esized an d tested in vitro (at con -
cen tration s ran gin g from 10–9 to 10–5 m ol/l) on h um an n eutroph il fun ction s. Two com -
poun ds lackin g th e carboxylic group were subjected to th e sam e tests an d on e of th ese
sh owed un expected activity. Am on g th e acidic derivatives, com poun d 12 bearin g th e sam e
substituen ts as in dom eth acin 10 (m eth oxy an d 4-ch loroben zoyl groups) was th e m ost ac-
tive: it sign ifican tly lowered n eutroph il respon ses in all five bioassays an d at th e th ree con -
cen tration s con sidered.
Key w ord s: Im idazo[2,1-b]th iazoles; Th iazoles; In dom eth acin ; In flam m ation ; Neutroph il
fun ction s; An tiin flam m atory agen ts.
Wh ile we were in volved1 in a project on th e syn th esis of an tiin flam m atory
agen ts derived from 6-ph en ylim idazo[2,1-b]th iazole-5-carboxylic acid (1)
(Sch em e 1), we decided to evaluate two routes for th e syn th esis of th is start-
in g m aterial: th e oxidation of th e correspon din g aldeh yde 2, accordin g to
th e m eth od we previously described2 an d th e h ydrolysis of th e correspon d-
in g ester 3, reported by Tedesch i3. Wh en we repeated th is syn th esis of 3
+ Presen ted at th e First Italian-Swiss Meeting on Medicinal Chemistry (Torino, Italy, September
23–26, 1997).
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

300
Andreani et al.:
from 2-am in oth iazole an d eth yl 2-ben zoyl-2-brom oacetate, we obtain ed
th e expected com poun d 3 an d a by-product wh ich , from th e first spectro-
scopic data, was con sidered to be 2-ben zoylim in oth iazole-3-acetate 4. Th e
com poun d was th en h ydrolyzed an d th e resultin g carboxylic acid 5 was
subjected to a series of agroch em ical tests. Th e level of h erbicidal activity
was sufficien t to con sider com poun d 5 as a lead for th e syn th esis of n ew
an alogs.
CH COOEt
2
COOEt
O
N
N
COOEt
N
N
+
S
+
NH
2
S
S
N
Br
O
3
4
CH COOH
2
CHO
COOH
N
N
N
N
S
S
N
S
N
O
2
1
5
SCHEME 1
Wh ile we were in volved in th is project an d were verifyin g th e structure
of com poun ds 4 an d 5 by th eir in depen den t syn th esis, a spectroscopic
dem on stration of th e structure of com poun d 4 an d a plausible m ech an ism
of its form ation was suggested by Abign en te et al.4. Th ese data are in agree-
m en t with th e results obtain ed from our in depen den t syn th esis wh ich , in
addition , was em ployed for th e preparation of a series of an alogs 6–9
(Sch em e 2) in order to evaluate th e effect of th e perform ed structural
ch an ges on th e h erbicidal activity. Th e ch an ges in cluded several substitu-
en ts R on th e ph en yl rin g in esters 8 an d acids 9, an d also correspon din g
N-m eth yl derivatives 7 were prepared.
All th e ch an ges were perform ed for th e paren t th iazole com poun d as well
as its 4,5-dih ydro derivative. However, th e data obtain ed from th e h erbi-
cidal screen in g did n ot disclose an y derivative m ore active th an 5. Sin ce th e
structure of com poun ds 9 is related to th at of in dom eth acin 10 with th e
five-m em bered rin g open ed, we th ough t to test a n um ber of th ese com -
poun ds as an tiin flam m atory agen ts. Th e activity of som e open ed an alogues
h as already been successfully explored, for exam ple, with th e syn th esis of
clam idoxic acid5 11. With th is in m in d, we used th e sam e m eth odology for
preparation of a m ore sim ilar an alog, com poun d 12. We subjected to bio-
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Ring-Opened Analogs of Indomethacin
301
logical tests all th e prepared acids an d, sin ce th e an tiin flam m atory activity
was n ot expected with in th is class of com poun ds, on ly two selected
N-m eth yl derivatives.
N
COCl
X
NH
N
O
S
X
NH
2
+
S
R
6
R
CH I
3
BrCH COOEt
2
BrCH COOH
2
CH
CH COOEt
2
CH COOH
2
3
N
N
N
X
X
X
N
N
N
O
O
O
S
S
S
KOH
R
R
R
7
8
9
X
R
6-9
CH=CH
CH=CH
CH=CH
CH=CH
H
a
b
c
d
e
f
Cl
CH
3
NO
2
CH -CH
2
H
2
2
2
2
CH -CH
2
Cl
CH -CH
2
CH
g
h
3
CH -CH
2
NO
2
SCHEME 2
Th e tests we decided to em ploy, evaluate th e effects th at a given com -
poun d in duces on h um an n eutroph il fun ction s: th e activity was com pared
to th at of in dom eth acin as th e referen ce drug. It is well kn own th at h um an
n eutroph ils play cen tral roles in both h ost defen ses again st m icroorgan ism s
an d tissue destruction associated with m an y in flam m atory processes. A
n um ber of h igh ly specialized fun ction s allow th ese cells to accom plish th is
com plicated task an d it seem s likely th at n on steroidal an tiin flam m atory
drugs (NSAIDs) m ay, at least in part, affect th e in flam m atory respon se
th rough th eir action on th ese cells, in depen den t of cyclooxygen ase in h ibi-
tion 6. It h as been dem on strated th at an tiin flam m atory agen ts in h ibits sev-
eral n eutroph il fun ction s in volved in th e developm en t of in flam m atory
respon se such as locom otion an d in tracellular killin g of in gested m icroor-
gan ism s th rough oxygen depen den t an d/or in depen den t m ech an ism s7. Th e
pattern of in h ibition varied from drug to drug an d was related to th e differ-
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

302
Andreani et al.:
en t stim ulatin g agen ts8. In th e presen t study, th e effect of com poun ds 7b,
7f, 9a–9h an d 12 was exam in ed on in vitro n eutroph il activation con sider-
in g several aspects of h um an n eutroph il an tim icrobial fun ction s, n am ely
m otility in duced by ch em oattractan ts, superoxide an ion (O −2 ) gen eration
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.
CH COOH
2
CH COOH
2
CH COOH
2
O
O
N
H C
3
H C
3
CH
3
N
O
O
N
S
NH
O
Cl
Cl
O
Cl
Cl
10
12
11
RESULTS AND DISCUSSION
Chemistry
For th e syn th esis of th e 2-(ben zoylam in o)th iazoles 6 (Sch em e 2), 2-am in o-
th iazole or its 4,5-dih ydro derivative was treated with th e appropriate
ben zoyl ch loride in th e presen ce of trieth ylam in e. Wh en th e reaction m ix-
ture was left at room tem perature for a few h ours, som e diben zoyl deriva-
tive was also obtain ed. However, wh en th e reaction was perform ed at lower
tem perature, th e desired m on osubstituted derivatives 6 were obtain ed as
th e on ly isolated products in good yields. All th ese com poun ds, with th e
exception of com poun d 6c, h ave already been reported in th e literature9–13
.
Com poun ds 6 treated with iodom eth an e in th e presen ce of potassium car-
bon ate in aceton e reacted as th eir im in o form s to provide correspon din g
N-m eth yl derivatives 7. An alogous treatm en t of sodium salts of com poun ds
6, gen erated in situ with sodium h ydride in DMF, with eth yl brom oacetate
afforded eth yl 2-(ben zoylim in o)th iazole-3-acetates 8. Carboxylic acids 9
an d 12 were prepared sim ilarly usin g directly brom oacetic acid in aqueous
eth an olic solution of sodium h ydroxide. Th ese carboxylic acids could also
be prepared by h ydrolysis of th e correspon din g esters 8 but th e yields are
lower.
Th e spectroscopic data of th e n ew com poun ds are in agreem en t with th e
assign ed structures. In th e IR spectra of ben zoyl derivatives 6 it is possible
to see th e stretch in g vibration s of th e carbon yl group at about 1 660 cm –1
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Ring-Opened Analogs of Indomethacin
303
in th e th iazole series an d at about 1 610 cm –1 in th e dih ydroth iazole series.
Th ese two series are quite differen t even in th e region of th e NH group: a
low in ten sity ban d is presen t in th iazoles (3 140 cm –1) wh ereas a m uch
stronger band characterizes the corresponding dihydro derivatives (3 200 cm –1).
1
Th e H NMR spectra of th iazole derivatives sh ow typical arom atic beh aviour
with two doublets in th e ran ge of 7–7.6 ppm , wh ereas th eir dih ydro
an alogs sh ow usual aliph atic pattern with two triplets in th e region of
3.2–4.2 ppm . Un der th e experim en tal con dition s em ployed, th e reson an ce
of th e carboxylic group (com poun ds 9a–9h an d 12) was usually un detect-
able.
Biological Activity
In th e first step of our study we verified if th e 2-(ben zoylim in o)th iazoles
an d in dom eth acin were ch em otaxin s for h um an n eutroph ils, secretagogue
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 (data n ot sh own ). In th e
secon d step we tested th e 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 in vitro
th e in fluen ce of th e tested com poun ds, in cludin g in dom eth acin (10) on
ran dom locom otion . All com poun ds sh owed sligh t effects: h owever th ese
were n ot statistically sign ifican t (p > 0.05) at an y of th e con cen tration s
tested (10–9, 10–7 an d 10–5 m ol/l). Con versely, wh en th e sam e com poun ds
were tested for th eir ability to m odulate directed m igration (Table I), com -
poun ds 9b an d 12 caused a 55% in h ibition (p < 0.01) on ch em otaxis
in duced by FMLP (N-form ylm ethionylleucylphenylalanine) at a concentration
as low as 10–9 m ol/l, com parable to th e effect of in dom eth acin at 10–5 m ol/l.
Com poun ds 9c, 9d an d 12 beh aved like th e referen ce drug at 10–5 m ol/l. A
sligh t but sign ifican t (p < 0.05) in h ibition of ch em otaxis was observed after
cell treatm ent with 7b, 7f, 9a and 9e. Other tested com pounds proved to be
inactive in this function. When chem otaxis was induced by casein (2 m g/m l),
com poun ds 9b, 9g, 9h an d 12 displayed 35–40% (p < 0.05) in h ibitory effect
at th e lowest con cen tration .
Th e m odulation of th e selected com poun ds an d in dom eth acin on
FMLP-in duced respiratory burst is sh own in Table I. On ly com poun ds 7f,
9b, 9f an d 12 were able to in duce a statistically sign ifican t (p < 0.05) in h ibi-
tion on free radical production : th e greatest activity was at 10–5 m ol/l for
com poun ds 9b, 12 an d th e referen ce drug an d at 10–9 m ol/l for com poun ds
7f an d 9f. Th e oth er an alogs gave n o effect. Moreover, n o effect was seen
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

304
Andreani et al.:
TABLE I
Activity of com poun ds 7b, 7f, 9a–9h an d 12 on h um an n eutroph il fun ction s (in %) in com -
parison with in dom eth acin 10 (on ly th e sign ifican t values are reporteda)
Con cen -
tration
m ol/l ch em otaxis ch em otaxis
FMLP-
in duced
Casein -
in duced
FMLP-
FMLP-in duced PMA-in duced
Com -
poun d
triggered O−2
gen eration
lysozym e
secretion
lysozym e
secretion
10
7b
7f
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
55
48
41
77
68
63
78
58
56
55
57
55
46
46
41
85
65
39
62
62
51
84
68
67
85
67
66
78
78
64
78
76
65
65
64
59
73
65
64
67
67
53
71
79
77
75
69
69
9a
9b
9c
9d
9e
9f
75
68
68
64
67
71
86
80
70
80
80
70
70
75
72
69
68
70
72
72
71
63
64
69
64
64
59
82
72
62
73
70
55
75
83
84
9g
9h
12
10–9
10–7
10–5
10–9
10–7
10–5
10–9
10–7
10–5
58
70
76
58
59
63
65
65
62
77
76
75
78
78
75
71
70
69
71
70
68
68
70
69
51
55
54
45
44
41
89
82
78
a
Each value is th e average ± SEM from 8–10 separate experim en ts carried out in triplicate
an d SEM are aroun d 10%. Statistical sign ifican ce is reported in th e text.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Ring-Opened Analogs of Indomethacin
305
for all tested com poun ds wh en respiratory burst was triggered by PMA
(ph orbol 12-m yristate 13-acetate). Th ese data are in agreem en t with previ-
ous reports8,15,16 wh ere th e in h ibitory action of NSAIDs was predom in an tly
on respon ses m ediated th rough th e FMLP receptor, probably by disruptin g
m olecular in teraction s of key com pon en ts of th e tran sduction m ach in ery
with in th e plasm a m em bran e17
.
Wh en th e derivatives were tested for th eir ability to in fluen ce FMLP- or
PMA-depen den t lysozym e release, about 35% dose in depen den t-in h ibition
(very close to th at of in dom eth acin ) was seen for several derivatives (Table I).
Com poun d 12 sh owed a h igh in h ibition (p < 0.01) at th e lowest con cen tra-
tion wh en th e agon ist was PMA.
Th e aim of th is study was th e search for n ew com poun ds wh ich could be
able to act at low con cen tration s, with th e capability to depress n eutroph il
respon ses to in flam m atory stim uli th us reducin g un wan ted tissue dam age.
Th e un expected broad activity of com poun d 7f m ay be con sidered as a
h elpful suggestion for our n ext projects. As far as th e carboxylic acids are
con cern ed, th e bioch em ical beh aviour of com poun d 9b is n oteworth y sin ce
a sign ifican t depression of n eutroph il ch em otaxis an d O −2 production were
foun d. Com poun d 12, structurally th e closest to in dom eth acin , was th e
m ost active derivative. It was capable to in h ibit all aspects of n eutroph il
fun ction s reported h ere: locom otion , release of oxygen radical an d
lysozym e secreted by activated n eutroph ils wh ich m ay be im portan t causes
of tissue dam age in a n um ber of in flam m atory con dition s18. It is in terestin g
to observe th at both 9b an d 12 were able to depress directed m igration to-
wards th e two ch em otaxin s tested, at a con cen tration as low as 10–9 m ol/l.
EXPERIMENTAL
Ch em istry
Meltin g poin ts are un corrected. IR spectra were taken on a Perkin –Elm er 683 spectro-
ph otom eter (waven um bers in cm –1). 1H NMR spectra were recorded on a Varian Gem in i in -
strum en t (300 MHz) in DMSO-d6 usin g TMS as th e in tern al stan dard. Ch em ical sh ifts are
given in ppm (δ-scale), couplin g con stan ts (J) in Hz. Th in layer ch rom atograph y was don e
on pre-coated TLC plates Bakerflex (silica gel IBF-2) usin g m ixtures of petroleum eth er an d
aceton e as eluen ts.
2-(Ben zoylam in o)th iazoles 6a–6h an d 2-(4-Ch loroben zoylam in o)-5-m eth oxyth iazole.
Gen eral Procedure
Trieth ylam in e (3 g, 30 m m ol) was added to a solution of 30 m m ol of 2-am in oth iazole (prep-
aration of com poun ds 6a–6d ) or 2-am in o-4,5-dih ydroth iazole (preparation of com poun ds
6e–6h ) or 2-am in o-5-m eth oxyth iazole14 (preparation of 2-(4-ch loroben zoylam in o)-
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

306
Andreani et al.:
5-m eth oxyth iazole) in tetrah ydrofuran (70 m l) an d th e m ixture was cooled to –5 °C. A solution
of th e appropriate ben zoyl ch loride (28 m m ol) in tetrah ydrofuran (20 m l) was th en added
dropwise wh ile th e tem perature of th e m ixture was kept between –5 an d 0 °C. Th e form ed
precipitate of trieth ylam in e h ydroch loride was filtered off, th e filtrate was evaporated un der
reduced pressure an d th e crude product was recrystallized from eth an ol to provide com -
poun ds 6a–6h .
2-(Benzoylamino)thiazole (6a). Yield 85%, m .p. 150–152 °C (ref.9, m .p. 150–151 °C).
2-(4-Chlorobenzoylamino)thiazole (6b). Yield 82%, m .p. 211–214 °C (ref.10, m .p. 212.5–213 °C).
2-(4-Methylbenzoylamino)thiazole (6c). Yield 80%, m .p. 211–214 °C. For C11H10N2OS
(218.3) calculated: 60.53% C, 4.62% H, 12.83% N; foun d: 60.57% C, 4.93% H, 13.05% N. IR:
1 300, 1 540, 1 610, 1 660. 1H NMR: 2.38 s, 3 H (CH3); 7.27 d, 1 H, J = 4.5 (H-4 or H-5); 7.34 d,
2 H, J = 9 (arom . H); 7.55 d, 1 H, J = 4.5 (H-4 or H-5); 7.99 d, 2 H, J = 9 (arom . H); 12.55 s,
1 H (NH).
2-(4-Nitrobenzoylamino)thiazole (6d). Yield 81%, m .p. 292–296 °C (ref.11, m .p. 297–298 °C).
2-(Benzoylamino)-4,5-dihydrothiazole (6e). Yield 84%, m .p. 168–170 °C (ref.12, m .p. 168 °C).
2-(4-Chlorobenzoylamino)-4,5-dihydrothiazole (6f). Yield 82%, m .p. 223–225 °C (ref.13, m .p.
220–221 °C).
2-(4-Methylbenzoylamino)-4,5-dihydrothiazole (6g). Yield 82%, m .p. 224–226 °C (ref.13, m .p.
225 °C).
2-(4-Nitrobenzoylamino)-4,5-dihydrothiazole (6h ). Yield 80%, m .p. 275–279 °C (ref.13, m .p.
261–262 °C).
2-(4-Chlorobenzoylamino)-5-methoxythiazole. Yield 81%, m .p. 203–207 °C. For
C
11H9ClN2O2S (268.7) calculated: 49.17% C, 3.38% H, 10.42% N; foun d: 48.96% C, 3.49% H,
10.24% N. IR: 750, 1 310, 1 590, 1 655. 1H NMR: 3.88 s, 3 H (CH3O); 6.94 s, 1 H (H-4); 7.60 d,
2 H, J = 8.5 (arom . H); 8.06 d, 2 H, J = 8.5 (arom . H); 12.45 s, 1 H (NH).
2-(Ben zoylim in o)-3-m eth yl-2,3-dih ydroth iazoles 7a–7h . Gen eral Procedure
A m ixture of appropriate com poun d 6 (5 m m ol), an h ydrous potassium carbon ate (1.4 g,
10 m m ol), iodom eth an e (7 g, 50 m m ol) an d aceton e (100 m l) was refluxed for 12 h . Th e
residue after evaporation of th e reaction m ixture was triturated with water, th e in soluble
portion was filtered off an d recrystallized from eth an ol to give com poun ds 7a–7h .
2-(Benzoylimino)-3-methyl-2,3-dihydrothiazole (7a ). Yield 90%, m .p. 112–116 °C. For
C11H10N2OS (218.3) calculated: 60.53% C, 4.62% H, 12.83% N; foun d: 60.81% C, 4.44% H,
12.59% N. IR: 710, 1 480, 1 545, 1 590. 1H NMR: 3.81 s, 3 H (CH3N); 7.04 d, 1 H, J = 4 (H-4
or H-5); 7.50 m , 3 H (arom . H); 7.55 d, 1 H, J = 4 (H-4 or H-5); 8.23 m , 2 H (arom . H).
2-(4-Chlorobenzoylimino)-3-methyl-2,3-dihydrothiazole (7b). Yield 86%, m .p. 165–168 °C.
For C11H9ClN2OS (252.7) calculated: 52.28% C, 3.59% H, 11.08% N; foun d: 51.97% C,
3.68% H, 10.88% N. IR: 970, 1 490, 1 550, 1 595. 1H NMR: 3.82 s, 3 H (CH3N); 7.08 d, 1 H,
J = 4.5 (H-4 or H-5); 7.53 d, 2 H, J = 8.5 (arom . H); 7.58 d, 1 H, J = 4.5 (H-4 or H-5); 8.22 d,
2 H, J = 8.5 (arom . H).
3-Methyl-2-(4-methylbenzoylimino)-2,3-dihydrothiazole (7c). Yield 87%, m .p. 143–147 °C.
For C12H12N2OS (232.3) calculated: 62.04% C, 5.21% H, 12.06% N; foun d: 61.86% C, 4.92% H,
11.92% N. IR: 760, 1 170, 1 550, 1 590. 1H NMR: 2.36 s, 3 H (CH3); 3.79 s, 3 H (CH3N); 7.01 d,
1 H, J = 4.5 (H-4 or H-5); 7.27 d, 2 H, J = 8.5 (arom . H); 7.53 d, 1 H, J = 4.5 (H-4 or H-5);
8.10 d, 2 H, J = 8.5 (arom . H).
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Ring-Opened Analogs of Indomethacin
307
3-Methyl-2-(4-nitrobenzoylimino)-2,3-dihydrothiazole (7d ). Yield 86%, m .p. 231–233 °C. For
C11H9N3O3S (263.3) calculated: 50.18% C, 3.45% H, 15.96% N; foun d: 50.24% C, 3.67% H,
16.03% N. IR: 845, 1 335, 1 490, 1 575. 1H NMR: 3.86 s, 3 H (CH3N); 7.16 d, 1 H, J = 4.5
(H-4 or H-5); 7.65 d, 1 H, J = 4.5 (H-4 or H-5); 8.32 d, 2 H, J = 9 (arom . H); 8.43 d, 2 H, J = 9
(arom . H).
2-(Benzoylimino)-3-methylthiazoline (7e). Yield 85%, m .p. 101–105 °C. For C11H12N2OS
(220.3) calculated: 59.97% C, 5.49% H, 12.72% N; foun d: 60.07% C, 5.49% H, 12.72% N. IR:
710, 950, 1 550, 1 605. 1H NMR: 3.17 t, 2 H, J = 7.5 (H-4 or H-5); 3.21 s, 3 H (CH3N); 3.72 t,
2 H, J = 7.5 (H-4 or H-5); 7.50 m , 3 H (arom . H); 8.14 m , 2 H (arom . H).
2-(4-Chlorobenzoylimino)-3-methylthiazoline (7f). Yield 85%, m .p. 161–165 °C. For
C
11H11ClN2OS (254.7) calculated: 51.87% C, 4.35% H, 11.00% N; foun d: 52.02% C, 4.58% H,
10.87% N. IR: 950, 1 085, 1 545, 1 610. 1H NMR: 3.19 t, 2 H, J = 7.5 (H-4 or H-5); 3.22 s, 3 H
(CH3N); 3.74 t, 2 H, J = 7.5 (H-4 or H-5); 7.52 d, 2 H, J = 8.5 (arom . H); 8.14 d, 2 H, J = 8.5
(arom . H).
3-Methyl-2-(4-methylbenzoylimino)thiazoline (7g). Yield 86%, m .p. 176–179 °C. For
C
12H14N2OS (234.3) calculated: 61.51% C, 6.02% H, 11.95% N; foun d: 61.61% C, 5.86% H,
12.08% N. IR: 1 165, 1 260, 1 550, 1 600. 1H NMR: 2.35 s, 3 H (CH3); 3.16 t, 2 H, J = 8 (H-4
or H-5); 3.20 s, 3 H (CH3N); 3.70 t, 2 H, J = 8 (H-4 or H-5); 7.25 d, 2 H, J = 8 (arom . H); 8.03 d,
2 H, J = 8 (arom . H).
3-Methyl-2-(4-nitrobenzoylimino)thiazoline (7h ). Yield 88%, m .p. 222–226 °C. For
C
11H11N3O3S (265.3) calculated: 49.80% C, 4.18% H, 15.84% N; foun d: 50.03% C, 4.22% H,
16.00% N. IR: 1 520, 1 560, 1 585, 1 615. 1H NMR: 3.24 t, 2 H, J = 8 (H-4 or H-5); 3.27 s, 3 H
(CH3N); 3.78 t, 2 H, J = 8 (H-4 or H-5); 8.29 d, 2 H, J = 9 (arom . H); 8.35 d, 2 H, J = 9 (arom . H).
Eth yl 2-(Ben zoylim in o)-2,3-dih ydroth iazole-3-acetates 8a–8h . Gen eral Procedure
A solution of appropriate com poun d 6 (4 m m ol) in N,N-dim eth ylform am ide (30 m l) was
treated portion wise with sodium h ydride (0.3 g, 7 m m ol, 50% dispersion in m in eral oil). Af-
ter 15 m in of stirrin g at 25–30 °C, th e m ixture was cooled to 5 °C an d treated with eth yl
brom oacetate (1.7 g, 10 m m ol) an d th e m ixture was stirred at 90 °C for 4 h . Th en th e m ix-
ture was evaporated un der reduced pressure, triturated with water an d th e in soluble portion
was filtered off an d crystallized from eth an ol (com poun d 8a from petroleum eth er) to give
com poun ds 8a–8h .
Ethyl 2-(benzoylimino)-2,3-dihydrothiazole-3-acetate (8a). Yield 90%, m .p. 99–102 °C. (ref.4,
m .p. 100–102 °C).
Ethyl 2-(4-chlorobenzoylimino)-2,3-dihydrothiazole-3-acetate (8b). Yield 86%, m .p. 110–112 °C.
For C14H13ClN2O3S (324.8) calculated: 51.77% C, 4.03% H, 8.62% N; foun d: 51.58% C,
3.95% H, 8.62% N. IR: 1 010, 1 220, 1 595, 1 740. 1H NMR: 1.20 t, 3 H, J = 7 (CH3CH2); 4.18 q,
2 H, J = 7 (CH3CH2); 5.12 s, 2 H (CH2N); 7.09 d, 1 H, J = 4.5 (H-4 or H-5); 7.53 d, 2 H, J = 8
(arom . H); 7.59 d, 1 H, J = 4.5 (H-4 or H-5); 8.13 d, 2 H, J = 8 (arom . H).
Ethyl 2-(4-methylbenzoylimino)-2,3-dihydrothiazole-3-acetate (8c). Yield 85%, m .p. 106–107 °C.
For C15H16N2O3S (304.4) calculated: 59.19% C, 5.30% H, 9.20% N; foun d: 58.97% C, 5.59% H,
9.24% N. IR: 1 020, 1 210, 1 605, 1 740. 1H NMR: 1.23 t, 3 H, J = 7 (CH3CH2); 2.37 s, 3 H
(CH3); 4.20 q, 2 H, J = 7 (CH3CH2); 5.11 s, 2 H (CH2N); 7.06 d, 1 H, J = 4.5 (H-4 or H-5);
7.28 d, 2 H, J = 8 (arom . H); 7.57 d, 1 H, J = 4.5 (H-4 or H-5); 8.05 d, 2 H, J = 8 (arom . H).
Ethyl 2-(4-nitrobenzoylimino)-2,3-dihydrothiazole-3-acetate (8d ). Yield 80%, m .p. 161–165 °C.
For C14H13N3O5S (335.3) calculated: 50.14% C, 3.91% H, 12.53% N; foun d: 49.99% C,
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4.06% H, 12.89% N. IR: 1 220, 1 520, 1 580, 1 745. 1H NMR: 1.23 t, 3 H, J = 7 (CH3CH2);
4.20 q, 2 H, J = 7 (CH3CH2); 5.19 s, 2 H (CH2N); 7.18 d, 1 H, J = 4.5 (H-4 or H-5); 7.65 d,
1 H, J = 4.5 (H-4 or H-5); 8.34 m , 4 H (arom . H).
Ethyl 2-(benzoylimino)thiazoline-3-acetate (8e). Yield 88%, m .p. 118–121 °C. For
C14H16N2O3S (292.4) calculated: 57.52% C, 5.52% H, 9.58% N; foun d: 57.58% C, 5.31% H,
9.83% N. IR: 1 200, 1 525, 1 615, 1 730. 1H NMR: 1.19 t, 3 H, J = 7 (CH3CH2); 3.25 t, 2 H, J = 8
(H-4 or H-5); 3.81 t, 2 H, J = 8 (H-4 or H-5); 4.14 q, 2 H, J = 7 (CH3CH2); 4.53 s, 2 H (CH2N);
7.45 t, 2 H, J = 7(arom . H); 7.54 t, 1 H, J = 7 (arom . H); 8.09 d, 2 H, J = 7 (arom . H).
Ethyl 2-(4-chlorobenzoylimino)thiazoline-3-acetate (8f). Yield 87%, m .p. 108–111 °C. For
C14H15ClN2O3S (326.8) calculated: 51.45% C, 4.63% H, 8.57% N; foun d: 51.72% C, 4.38% H,
8.65% N. IR: 1 275, 1 525, 1 615, 1 730. 1H NMR: 1.19 t, 3 H, J = 7 (CH3CH2); 3.27 t, 2 H, J = 8
(H-4 or H-5); 3.81 t, 2 H, J = 8 (H-4 or H-5); 4.14 q, 2 H, J = 7 (CH3CH2); 4.53 s, 2 H (CH2N);
7.52 d, 2 H, J = 8 (arom . H); 8.08 d, 2 H, J = 8 (arom . H).
Ethyl 2-(4-methylbenzoylimino)thiazoline-3-acetate (8g). Yield 82%, m .p. 125–128 °C. For
C
15H18N2O3S (306.4) calculated: 58.80% C, 5.92% H, 9.14% N; foun d: 59.11% C, 6.16% H,
9.29% N. IR: 1 275, 1 525, 1 615, 1 730. 1H NMR: 1.20 t, 3 H, J = 7 (CH3CH2); 2.35 s, 3 H
(CH3); 3.24 t, 2 H, J = 8 (H-4 or H-5); 3.80 t, 2 H, J = 8 (H-4 or H-5); 4.14 q, 2 H, J = 7
(CH3CH2); 4.51 s, 2 H (CH2N); 7.25 d, 2 H, J = 8 (arom . H); 7.98 d, 2 H, J = 8 (arom . H).
Ethyl 2-(4-nitrobenzoylimino)thiazoline-3-acetate (8h ). Yield 82%, m .p. 129–132 °C. For
C
14H15N3O5S (337.4) calculated: 49.84% C, 4.48% H, 12.46% N; foun d: 50.12% C, 4.56% H,
12.21% N. IR: 1 525, 1 590, 1 620, 1 725. 1H NMR: 1.20 t, 3 H, J = 7 (CH3CH2); 3.31 t, 2 H, J = 8
(H-4 or H-5); 3.85 t, 2 H, J = 8 (H-4 or H-5); 4.16 q, 2 H, J = 7 (CH3CH2); 4.58 s, 2 H (CH2N);
8.30 m , 4 H (arom . H).
2-(Ben zoylim in o)-2,3-dih ydroth iazole-3-acetic Acids 9a–9h an d 12. Gen eral Procedure
A solution of 2-(ben zoylam in o)th iazole 6a–6h or 2-(4-ch loroben zoylam in o)-5-m eth oxy-
th iazole (4 m m ol) in eth an ol (150 m l) was treated with brom oacetic acid (1 g, 7 m m ol) an d
a 2 M solution of sodium h ydroxide was added dropwise to reach pH 8. Th e m ixture was
th en refluxed for 16 h , evaporated un der reduced pressure, th e residue was treated with wa-
ter an d acidified with 2 M solution of h ydroch loric acid. Th e in soluble portion was filtered
off an d crystallized from eth an ol to give com poun ds 9a–9h an d 12.
2-(Benzoylimino)-2,3-dihydrothiazole-3-acetic acid (9a). Yield 70%, m .p. 225–227 °C. For
C
12H10N2O3S (262.3) calculated: 54.95% C, 3.84% H, 10.68% N; foun d: 55.27% C, 4.03% H,
10.79% N. IR: 710, 1 530, 1 590, 1 730. 1H NMR: 5.08 s, 2 H (CH2N); 7.05 d, 1H, J = 4.50
(H-4 or H-5); 7.50 m , 3 H (arom . H); 7.58 d, 1 H, J = 4.5 (H-4 or H-5); 8.20 m , 2 H (arom . H).
2-(4-Chlorobenzoylimino)-2,3-dihydrothiazole-3-acetic acid (9b). Yield 68%, m .p. 224–227 °C.
For C12H9ClN2O3S (296.7) calculated: 48.57% C, 3.06% H, 9.44% N; foun d: 48.29% C,
2.95% H, 9.68% N. IR: 760, 1 525, 1 580, 1 730. 1H NMR: 5.06 s, 2 H (CH2N); 7.07 d, 1 H,
J = 4.5 (H-4 or H-5); 7.54 d, 2 H, J = 8.5 (arom . H); 7.59 d, 1 H, J = 4.5 (H-4 or H-5); 8.16 d,
2 H, J = 8.5 (arom . H).
2-(4-Methylbenzoylimino)-2,3-dihydrothiazole-3-acetic acid (9c). Yield 67%, m .p. 242–247 °C.
For C13H12N2O3S (276.3) calculated: 56.51% C, 4.38% H, 10.14% N; foun d: 56.75% C,
4.50% H, 9.99% N. IR: 750, 1 200, 1 530, 1 720. 1H NMR: 2.37 s, 3 H (CH3); 5.04 s, 2 H
(CH2N); 7.03 d, 1 H, J = 4.5 (H-4 or H-5); 7.27 d, 2 H, J = 8 (arom . H); 7.56 d, 1 H, J = 4.5
(H-4 or H-5); 8.06 d, 2 H, J = 8 (arom . H).
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2-(4-Nitrobenzoylimino)-2,3-dihydrothiazole-3-acetic acid (9d ). Yield 65%, m .p. 218–222 °C.
For C12H9N3O5S (307.3) calculated: 46.90% C, 2.95% H, 13.67% N; foun d: 47.13% C, 3.03% H,
13.58% N. IR: 1 220, 1 510, 1 540, 1 730. 1H NMR: 5.11 s, 2 H (CH2N); 7.15 d, 1 H, J = 4.5
(H-4 or H-5); 7.63 d, 1 H, J = 4.5 (H-4 or H-5); 8.31 d, 2 H, J = 8 (arom . H); 8.37 d, 2 H, J = 8
(arom . H).
2-(Benzoylimino)thiazoline-3-acetic acid (9e). Yield 68%, m .p. 181–183 °C. For C12H12N2O3S
(264.3) calculated: 54.53% C, 4.58% H, 10.60% N; foun d: 54.61% C, 4.86% H, 10.72% N. IR:
1 175, 1 510, 1 595, 1 730. 1H NMR: 3.23 t, 2 H, J = 8 (H-4 or H-5); 3.80 t, 2 H, J = 8 (H-4 or
H-5); 4.46 s, 2 H (CH2N); 7.45 t, 2 H, J = 8 (arom . H); 7.53 t, 1 H, J = 8 (arom . H); 8.10 d,
2 H, J = 8 (arom . H).
2-(4-Chlorobenzoylimino)thiazoline-3-acetic acid (9f). Yield 65%, m .p. 187–191 °C. For
C
12H11ClN2O3S (298.7) calculated: 48.25% C, 3.71% H, 9.38% N; foun d: 47.98% C, 3.63% H,
9.57% N. IR: 1 520, 1 555, 1 590, 1 720. 1H NMR: 3.25 t, 2 H, J = 8 (H-4 or H-5); 3.81 t, 2 H,
J = 8 (H-4 or H-5); 4.47 s, 2 H (CH2N); 7.52 d, 2 H, J = 8 (arom . H); 8.10 d, 2 H, J = 8 (arom . H).
2-(4-Methylbenzoylimino)thiazoline-3-acetic acid (9g). Yield 66%, m .p. 199–201 °C. For
C13H14N2O3S (278.3) calculated: 56.10% C, 5.07% H, 10.06% N; foun d: 55.88% C, 5.22% H,
9.97% N. IR: 1 500, 1 520, 1 585, 1 725. 1H NMR: 2.36 s, 3 H (CH3); 3.23 t, 2 H, J = 8 (H-4 or
H-5); 3.79 t, 2 H, J = 8 (H-4 or H-5); 4.46 s, 2 H (CH2N); 7.26 d, 2 H, J = 8 (arom . H); 8.00 d,
2 H, J = 8 (arom . H).
2-(4-Nitrobenzoylimino)thiazoline-3-acetic acid (9h ). Yield 66%, m .p. 189–193 °C. For
C
12H11N3O5S (309.3) calculated: 46.60% C, 3.58% H, 13.58% N; foun d: 46.67% C, 3.75% H,
13.72% N. IR: 1 530, 1 590, 1 615, 1 720. 1H NMR: 3.29 t, 2 H, J = 8 (H-4 or H-5); 3.85 t,
2 H, J = 8 (H-4 or H-5); 4.51 s, 2 H (CH2N); 8.30 m , 4 H (arom . H).
2-(4-Chlorobenzoylimino)-5-methoxy-2,3-dihydrothiazole-3-acetic acid (12). Yield 65%, m .p.
216–220 °C. For C13H11ClN2O4S (326.8) calculated: 47.79% C, 3.39% H, 8.57% N; foun d:
48.01% C, 3.11% H, 8.63% N. IR: 1 180, 1 495, 1 605, 1 735. 1H NMR: 3.86 s, 3 H (CH3O);
4.96 s, 2 H (CH2N); 7.07 s, 1 H (H-4); 7.53 d, 2 H, J = 8.5 (arom . H); 8.12 d, 2 H, J = 8.5
(arom . H).
Biology
Dextran an d Ficoll–Paque were purch ased from Ph arm acia (Uppsala, Sweden ); FMLP, PMA,
cytoch alasin B, ferricytoch rom e C, bovin e serum album in , superoxide dism utase, Micrococcus
lysodeicticus an d NADH were 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 casein “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, Italy. FMLP stock solution
(10–2 m ol/l in DMSO) was diluted before use in Krebs–Rin ger-ph osph ate con tain in g 0.1%
glucose (KRPG), pH 7.4. PMA stock solution (1 m g/m l in DMSO) was diluted before use in
KRPG. Casein stock solution con tain in g 10 m g/m l in KRPG was diluted before use in KRPG
con tain in g 1 m g/m l bovin e serum album in (KRPG-A). Stock solution s (10–2 m ol/l in DMSO)
of th e tested com poun ds an d in dom eth acin were diluted before use in KRPG. Dim eth yl
sulfoxide did n ot in terfere with an y of th e biological assays perform ed.
Preparation of Hum an Neutroph ils
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
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

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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 at 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 usin g 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 19 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 casein (2 m g/m l in KRPG-A) an d FMLP (10–8 m ol/l in
KRPG-A). Cells were in cubated with 10–9, 10–7 an d 10–5 m ol/l of th e com poun ds for 10 m in
before th e ch em otactic assay. Th e actual con trol of ch em otaxis in duced by 10–6 M FMLP an d
2 m g/m l casein was 78 ± 3 µm an d 87.6 ± 2 µm , respectively (12 separate experim en ts don e
in triplicate).
Superoxide An ion Production
Th e O−2 was m easured by th e superoxide dism utase-in h ibitable reduction of ferricyto-
ch rom e20 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 of 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 to 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. Absorban ce was re-
corded 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 n m ol of O−2 produced, usin g
a m illim olar extin ction
coefficien 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 ol/l in KRPG). Neutroph ils were prein cubated with cytoch alasin B
(5 µg/m l) 5 m in prior th e activation by FMLP. Assays were don e in triplicate for each experi-
m en tal con dition . Cells were in cubated with 10–9, 10–7 an d 10–5 m ol/l of th e com poun ds for
10 m in before th e addition of stim ulus. Th e actual con trol of O−2 gen eration by 10–6 M FMLP
was 20 ± 2 n m ol/4 · 105 cells/5 m in (12 separate experim en ts don e in triplicate).
En zym e Assay
Release of n eutroph il gran ule en zym es was evaluated by determ in in g lysozym e activity20
,
m odified for m icroplate-based assays. Cells were in cubated with 10–9, 10–7 an d 10–5 m ol/l of
th e com poun ds 10 m in before th e addition of stim ulus. 3 · 106 cells were in cubated in
m icroplate wells 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
rate of lysis of cell wall suspen sion of Micrococcus lysodeikticus. Reaction rate was m easured
with a m icroplate reader at 465 n m . En zym e was expressed as a n et percen tage of total en zym e
con ten t released by 0.1% Triton X-100. Total en zym e activity was 85 ± 1 µg/1 · 107 cells/m in .
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

Ring-Opened Analogs of Indomethacin
311
Th e degran ulatin g agen ts used were PMA 0.1 µg/m l an d FMLP 10–6 m ol/l, both in KRPG.
Th e actual con trol of lysozym e release by 10–6 M FMLP an d 100 n g/m l PMA was 45 ± 4%
an d 52 ± 5% of release/3 · 106 cells/10 m in , respectively (12 separate experim en ts don e in
triplicate). Cells were prein cubated with cytoch alasin B (5 µg/m l) 15 m in prior th e activa-
tion by FMLP. Assays were don e in triplicate for each experim en tal con dition .
Treatm en t of th e Tested Com poun ds
Wh en requested, n eutroph ils were prein cubated for 10 m in at room tem perature with 10–9
,
10–7 an d 10–5 M (ben zoylim in o)th iazoles or in dom eth acin before th e in cubation step for cell
fun ction ality.
Measurem en t of En zym atic Activity
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.21).
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), MURST
and Associazione Italiana per la Ricerca sul Cancro (AIRC). We are grateful to Banca del Sangue di
Ferrara for supplying fresh blood and to Merck Sharp & Dohme (NJ, U.S.A.) for the gift of
indomethacin. We thank Prof. E. Abignente (University of Naples) for the helpful discussion on
2-(benzoylimino)thiazole-3-acetate.
REFERENCES
1. Andreani A., Rambaldi M., Bonazzi D., Fabbri G., Greci L., Galatulas I., Bossa R.: Arch.
Pharm. (Weinheim, Ger.) 1983, 316, 141.
2. Andreani A., Rambaldi M., Bonazzi D., Greci L.: Boll. Chim. Farm. 1980, 119, 647.
3. Tedeschi E. ( Plantex Ltd., Israel): Ger. Offen. 2,505,068; Chem. Abstr. 1975, 83, 206269.
4. Abignente E., De Caprariis P., Fattorusso E., Mayol L.: J. Heterocycl. Chem. 1989, 26,
1875.
5. Drain D. J., Daly M. J., Davy B., Horlington M., Howes J. G. B., Scruton J. M., Selvay R. A.:
J. Pharm. Pharmacol. 1970, 22, 684.
6. Abramson S., Korchak H., Ludewig R., Edelson E., Haines K., Levin R. I., Herman R.,
Rider L., Kimmel S., Weissmann G.: Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 7227.
7. Abramson S. B., Cherksey B., Gude D., Leszczynska-Piziak J., Philips M. R., Blau L.,
Weissmann G.: Inflammation (N. Y.) 1990, 14, 11.
8. Crowell R. E., Van Epps D. E.: Inflammation (N. Y.) 1990, 14, 163.
9. Coburn R. A., Glennon R. A.: J. Heterocycl. Chem. 1973, 10, 487.
10. Kaye I. A., Parris C. L.: J. Org. Chem. 1951, 16, 1761.
11. Schmelkes F. C., Rubin M.: J. Am. Chem. Soc. 1944, 66, 1631.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

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12. Douglass I. B., Dains F. B.: J. Am. Chem. Soc. 1934, 56, 719.
13. Viallet M.-P., Boucherle A., Cohen-Addad C.: Eur. J. Med. Chem. 1979, 14, 553.
14. Forlani L., Medici A., Todesco P. E.: Tetrahedron Lett. 1976, 201.
15. Abramson S. B., Leszczynska-Piziak J., Clancy R. M., Philips M., Weissmann G.: Biochem.
Pharmacol. 1994, 47, 563.
16. Kaplan H. B., Edelson H. S., Korchak H. M., Given W. P., Abramson S., Weissmann G.:
Biochem. Pharmacol. 1984, 33, 371.
17. Abramson S. B., Weissmann G.: Arthritis Rheum. 1989, 32, 1.
18. Crouch S. P. M., Fletcher J.: Infect. Immun. 1992, 60, 4504.
19. Zigmond S. H., Hirsch J. G.: J. Exp. Med. 1973, 137, 387.
20. 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.
21. 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. 64) (1999)