Diaryl-dithiolanes and -isothiazoles: COX-1/COX-2 and 5-LOX-inhibitory, {radical dot}OH scavenging and anti-adhesive activities

Article abstract of DOI:10.1016/j.bmc.2008.11.074

Three series of non-steroidal anti-inflammatory drugs (NSAIDs) inhibiting the cyclooxygenase/5-lipoxygenase (COX/5-LOX) pathways as such as formation of hydroxyl radicals and adhesion were prepared: 4,5-diaryl isothiazoles, 4,5-diaryl 3H-1,2-dithiole-3-thiones and 4,5-diaryl 3H-1,2-dithiole-3-ones. The aim of the present study was to develop substances which can intervene into the inflammatory processes via different mechanisms of action as multiple target non-steroidal anti-inflammatory drugs (MTNSAIDs) with increased anti-inflammatory potential. The current lead 11a was evaluated in COX-1/2, 5-LOX and ^{{radical dot}}OH scavenging in vitro assays and in a static adhesion assay where it proved to inhibit adhesion. Moreover, 11a treatment attenuated expression of macrophage adhesion molecule-1 (Mac-1) on extravasated polymorphonuclear leukocytes (PMNs) which indicates that the activation was reduced. The assays used are predictive for the in vivo efficacy of test compounds as shown for 11a in a peritonitis model of acute inflammation in mice. Thus, the novel 5-LOX/COX and ^{{radical dot}}OH inhibitor 11a possesses anti-inflammatory activity that, in addition to COX/5-LOX inhibition, implicates effects on leukocyte-endothelial interactions.

Full text of DOI:10.1016/j.bmc.2008.11.074

Bioorganic & Medicinal Chemistry 17 (2009) 558–568
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Diaryl-dithiolanes and -isothiazoles: COX-1/COX-2 and 5-LOX-inhibitory,
ÅOH scavenging and anti-adhesive activities
b
b
a
Michael Scholz a, , Holger K. Ulbrich a, , , Oliver Soehnlein , Lennart Lindbom , Andreas Mattern ,
*
Gerd Dannhardt a
a Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Johannes Gutenberg-University, Staudingerweg 5, DE-55128 Mainz, Germany
b Karolinska Institute, Department of Physiology and Pharmacology, von Eulers Väg 8, SE-171 77 Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of non-steroidal anti-inflammatory drugs (NSAIDs) inhibiting the cyclooxygenase/5-lipoxy-
genase (COX/5-LOX) pathways as such as formation of hydroxyl radicals and adhesion were prepared:
4,5-diaryl isothiazoles, 4,5-diaryl 3H-1,2-dithiole-3-thiones and 4,5-diaryl 3H-1,2-dithiole-3-ones. The
aim of the present study was to develop substances which can intervene into the inflammatory processes
via different mechanisms of action as multiple target non-steroidal anti-inflammatory drugs (MTNSAIDs)
with increased anti-inflammatory potential. The current lead 11a was evaluated in COX-1/2, 5-LOX and
ÅOH scavenging in vitro assays and in a static adhesion assay where it proved to inhibit adhesion. More-
over, 11a treatment attenuated expression of macrophage adhesion molecule-1 (Mac-1) on extravasated
polymorphonuclear leukocytes (PMNs) which indicates that the activation was reduced. The assays used
are predictive for the in vivo efficacy of test compounds as shown for 11a in a peritonitis model of acute
Received 20 August 2008
Revised 21 November 2008
Accepted 28 November 2008
Available online 6 December 2008
Keywords:
COX-1/2
5-LOX
Dual inhibitors
Hydroxyl radical scavenging
Anti-adhesive activities
Leukocyte–endothelial interactions
Å
inflammation in mice. Thus, the novel 5-LOX/COX and OH inhibitor 11a possesses anti-inflammatory
activity that, in addition to COX/5-LOX inhibition, implicates effects on leukocyte–endothelial
interactions.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
1999. Consequently the American Gastroenterological Association
(AGA) differentiated the therapy for patients with higher gastro-
NSAIDS are basically used for the treatment of pain, inflamma-
tion and fever. The main mechanism of action of these drugs is the
inhibition of the cyclooxygenase enzymes (COX-1 and COX-2),1,2
which are catalyzing the biotransformation of arachidonic acid to
the prostaglandins.
intestinal (GI) risks or higher cardiovascular (CV) risk in three
cases.5
Recently the target switched to a combined 5-LOX/COX-inhibi-
tion, interfering both pathways, the production of prostaglandins
and the biosynthesis of leukotrienes (LTs).4,6
Gastrointestinal haemorrhage, ulceration and kidney failure are
the major side effects associated with all currently available NSA-
IDS (e.g., celecoxib and diclofenac in Scheme 1).3 These unwanted
side activities are caused by the interference of the physiological
balance of prostaglandins. To overcome this several approaches
were undertaken. One attempt haunts the strategy of selective
inhibition of COX-2. Nevertheless, clinical studies have suggested
that selective COX-2 inhibitors could cause typical COX-mediated
side effects such as gastrointestinal injury, increased systemic
blood pressure and hypersensitivity.4 Due to safety concerns of
an increased risk of cardiovascular events (including heart attack
and stroke) in patients on VioxxTM a voluntary withdrawal of Vio-
xxTM (rofecoxib) was announced on the worldwide market since
This strategy i.g. led to tepoxalin which is a new non-steroidal
anti-inflammatory drug and up to now approved for veterinary
use only in the United States and the European Union under the
brand name ZubrinÒ for the relief of pain associated with musculo-
skeletal disorders. Tepoxalin inhibits in a dual mode of action both
the cyclooxygenase and 5-lipoxigenase pathway of the arachidonic
acid metabolism. In addition, Tepoxalin inhibits migration of neu-
trophilic granulocytes in the inflammatory tissue.7
Another example represents licofelone (formerly known as
ML3000), is an anti-inflammatory drug that inhibits the COX and
5-lipoxygenase pathway and is currently undergoing phase III tri-
als for osteoarthritis.8,9 The effectiveness of licofelone has been
demonstrated in animal models as well as in studies on humans,
and is attributed mainly to the efficient suppression of PGE2
formation.9
* Corresponding author. Tel.: +49 6131 39 25729; fax: +49 6131 39 23062.
E-mail address: ulbrich@uni-mainz.de (H.K. Ulbrich).
Both authors contributed equally.
Interestingly, in contrast to NSAIDs and selective COX-2 inhibi-
tors, licofelone shows an improved gastrointestinal and potentially
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.074

M. Scholz et al. / Bioorg. Med. Chem. 17 (2009) 558–568
559
H2NO2S
Cl
CH2COOH
CH2COOH
N
N
NH
CF3
Cl
Cl
Licofelone
Celecoxib
Diclofenac
OH
OH
O
HO
HO
OH
S
S
NDGA
Thiocticacid
Scheme 1. Chemical structures of some established NSAIDS and used standard compounds.
cardiovascular safety.10–12 This effect of licofelone might be attrib-
utable to the accompanied suppression of leukotrienes,8 which sig-
nificantly contribute to gastric epithelial injury as well as to
atherogenesis.13,14
Reactive oxygen species (ROS) especially the hydroxyl radicals
are very important in the inflammatory pathways. ROS like hydro-
gen peroxide, superoxide anion and HOCl can be easily converted
into the most reactive hydroxyl radical.15,16 These hydroxyl radi-
cals can cause a loss of the membrane lipid functions and induce
apoptosis. Another important inflammatory mechanism is the
enge hydroxyl radicals. The next step was the modification of the
dithiol thione core to achieve more potent leads.
In summary, we describe 4,5-diaryl isothiazoles, 4,5-diaryl
dithiol-3-thiones and 4,5-diaryl dithiol-3-ones as COX/5-LOX-
inhibitors which possess anti-adhesive activities.
Additionally, those compounds have the potential to reduce the
level of reactive oxygen species such as hydroxyl radicals which
are well known for supporting inflammation processes in several
diseases and therefore could offer new opportunities for the treat-
ment of Parkinson’s disease,25 Alzheimer’s disease26 and rheuma-
toid arthritis.27
inactivation of IkappaB (I
a degradation of the I B and nuclear factor-kappaB (NF-
plex and to a nuclear translocation of NF- B. By virtue of its capa-
bility to modulate gene expression NF- can activate as
transcription factor the synthesis of cytokines like IL-1, IL-6, Il-8
and tumor necrosis factor-alpha (TNF- , the synthesis of inducible
jB) by hydroxyl radicals which leads to
j
jB) com-
j
2. Chemistry
j
B
We focused our synthesis on three different diaryl heterocyclic
ring systems. To receive the isothiazoles 2a–h (Scheme 3) different
a
nitric oxide synthase (iNOS) and adhesion molecules like E-selectin
a
-chloro formyl stilbene derivatives 1a–h were converted with
and vascular cell adhesion molecule-1 (VCAM-1).15–20
ammonium thiocyanate21,22 to the corresponding ring heterocy-
Polymorphonuclear leukocyte recruitment is fully accom-
plished through further homotypic and heterotypic cell–cell adhe-
sive interactions, sustained by adhesion receptors, including the
b2-integrin Mac-1 (CD11b/CD18). This molecule is in part constitu-
tively expressed on the surface of polymorphonuclear leukocytes
and is also stored in secretory granules. Upon activation by inflam-
matory stimuli, rapid translocation of Mac-1 from intracellular
pools to the cell surface occurs (upregulation) and, in parallel, con-
formational changes take place within the molecule to allow com-
petent binding to its counter receptors on polymorphonuclear
leukocytes (homotypic cell adhesion), as well as on platelets and
on the vascular cell surface (heterotypic cell adhesion).
cles. The synthesis of the a-chloro formyl stilbenes was described
previously.21,28,29 The isothiazole derivatives 2a–h were obtained
in yields between 30% and 80%. In an ether cleaving reaction 2a re-
acted with borontribromide to obtain 2i.30
For the dithiol-thiones different methods were applied. Proce-
dure 1 (Scheme 4) started with a Friedel Crafts reaction of the acid
chlorides 3a or 3b and thioanisole 4a to give the ketones 5a or
5b.31 The acid chloride 3a was prepared by heating the analogue
carboxylic acid in thionylchloride.32 The ketone 5c was synthesized
by heating a mixture of the carboxylic acid 3c, polyphosphoric acid
and thioanisole 4a. In the next step the ketones 5a–c were methyl-
ated with methyl iodide to afford the
a-methylketones 6a–c
The rationale for the synthesis of diaryl-dithiolanes and -iso-
thiazoles is that the iselenazoles previously showed high efficacies
in inhibiting COX-1, COX-2 and 5-LOX.21 Consequently, the aim of
this study was to develop compounds which maintain these activ-
ities and additionally possess significant hydroxyl radical scaveng-
ing potencies. Therefore the isoselenazol heterocycle was
exchanged by an isothiazole moiety unfortunately with no
improvement with regard to hydroxyl radical scavenging activities
(Scheme 2).
From there a new approach was needed. It is supposed that
compounds like anetholtrithion22 or oltipraz23,24 (Scheme 2) exhi-
bit potent hydroxyl radical scavenging activities. The dithiol thione
heterocycle of these compounds is a key structure for the radical
scavenging efficacy. This led us to the idea to introduce this hetero-
cycle system in our compound with potent COX-1, COX-2 and 5-
LOX inhibitory activity. As a consequence 11a was identified as
COX/5-LOX-inhibitor which additionally has the potential to scav-
(Scheme 4). It was important to oxidize the thioether derivatives
quantitatively with Oxon to get the methylsulfonyl derivatives
6d–f. For the ring closure reaction (Scheme 4) 6d–f reacted with
sulfur and Lawesson’s reagent to furnish the dithiol-thiones
11a–c.33
A Grignard reaction34 (Scheme 5) of the chloromethylbenzenes
7a–c and the ketones 8a–c provided the tertiary alcohols 9a–d in
good yields. The ketone 8a was generated by a Friedel Crafts reac-
tion of thioanisole 4a and acetyl chloride. The dehydration of the
tertiary alcohols 9a–d with phosphoric acid gave rise to the meth-
ylstilbenes 10a–d. The methylstilbene 10e was synthesized using
anisole 4b and chloroacetone in concentrated sulfuric acid. The thi-
oether intermediates 10c and 10d were oxidized with Oxon to the
methylsulfonyl derivatives 10f and 10g. The ring closure (Scheme
5) of 10d–g using sulfur on a graphite bath (220 °C) led to
dithiole-thiones 11d–h.35 Compound 11i was obtained by cleavage
of 11e with boron tribromide. The dithiol-thiones 11b or 11d–g

560
M. Scholz et al. / Bioorg. Med. Chem. 17 (2009) 558–568
R1
development through
F
variation of heterocycle
N
S
N
Se
R2
H3CO2S
2a - 2i
COX-1: ~ 20 µmol
COX-2: 0.6 µmol
chemistry: success
efficacy: fair success
but no improvement
5-LOX: 8 µmol
·OH scavenging: ~ 200 µmol
O
N
N
S
S
S
S
S
S
Oltipraz
Anetholtrithion
COX-1: > 50 µmol
COX-2: > 50 µmol
5-LOX: > 50 µmol
·OH scavenging: 4.5 µmol
COX-1: 2.1 µmol
COX-2: > 50 µmol
5-LOX: ~ 50 µmol
·OH scavenging: 7.5 µmol
H2NO2S
combination of key structures
of partially efficient compounds
N
N
CF3
R1
Celecoxib
COX-1: 8.0 µmol
COX-2: 0.8 µmol
S
·OH scavenging: ~ 500 µmol
11a - 11i
chemistry: success
efficacy: major success
S
S
consequential
development
R2
R1
O
12b, 12d - 12g
S
chemistry: success
efficacy: fair success
but no improvement
S
R2
Scheme 2. Rationale for the synthesis of 2a–i, 11a–i, 12b and 12d–g.
were synthesized using potassium permanganate to yield the
dithiole-ones 12b and 12d–g (Scheme 6).
pounds were also tested for their ability to inhibit leukocyte endo-
thelium interactions (Table 2). One compound was selected for
in vivo experiments in a mouse peritonitis model.
In general many substances inhibited the COX-1 enzyme inde-
pendently from the heterocyclic core systems. This phenomenon
was observed for the symmetrically substituted dimethoxyphenyl
(R1 and R2) isothiazole 2a and the dithiol-3-thione derivative 11f
3. Results and discussion
The 4,5-diaryl isothiazoles (2a–i), the 4,5-diaryl 3H-1,2-
dithiole-3-thiones (11a–i) and the 4,5-diaryl 3H-1,2-dithiole-3-
ones (12b–g) were initially evaluated in vitro to determine their
COX-1/2 and 5-LOX inhibitory activities. Standard compounds
were evaluated in parallel. The results are summarized in Table
1. The acquired structure activity relationship (SAR) data show a
broad range of inhibitory activities in all test systems (IC50 COX-
(IC50 COX-1: 0.003 vs IC50 COX-1: 0.03
12f (IC50: 0.0003 M). Substitution of the 4-position of the aryl
moiety with R1 = CH3 and R2 = Cl provided also potent inhibitors
of COX-1: 2c (IC50 COX-1: 0.04 M), 11d (IC50 COX-1: 2 M) and
12d (IC50 COX-1: 0.2 M).
Interestingly, in the series of the asymmetrically substituted
compounds such as 2c (R1 = CH3, R2 = Cl; IC50 COX-1: 0.04
M),
11e (R1 = Cl, R2 = CH3; IC50 COX-1: 0.006 M) and 12e (R1 = Cl,
lM), and analogously for
l
l
l
l
1: 0.0003
ꢀ10 M range, IC50 5-LOX: 3
scavenging: IC50: 5
M (11g) to ꢀ100
l
M (12f) to ꢀ10
l
M range, IC50 COX-2: 0.8
l
M (2a) to
Å
l
l
lM (11b) to ꢀ10
l
M range, OH
l
l
lM range). Some com-

M. Scholz et al. / Bioorg. Med. Chem. 17 (2009) 558–568
561
R1
R2 = OCH3
R2 = OCH3
R2 = Cl
1a, 2a R1 = OCH3
1b, 2b
R1
R1 = CH3
1c, 2c R1 = CH3
1d, 2d
(i)
R2 = CH3
R2 = Cl
R1 = Cl
1e, 2e R1 = Cl
1f, 2f
1g, 2g R1 = Cl
1h, 2h
N
O
S
Cl
R2 = Cl
R1 = OCH3
R2
R2 = OCH3
R2 = SO2CH3
R2
R1 = F
2a-2h
1a-1h
O
HO
(ii)
N
N
S
S
O
HO
2a
2i
Scheme 3. Synthesis of the isothiazoles: method i: 1 and ammonium thiocyanate in acetone reflux, 4 h, method ii: 2a and borontribromide in dichloromethane, ꢁ50 °C?rt.
(iii)
with AlCl3
R2
or
(iv)
SCH3
Cl
+
O
O
R1
R1
with
polyphosphoric acid
4a
3a-3c
5a-5c
R2
R2
(v)
(vi)
O
O
R1
R1
6a-6c
6d-6f
R1
3a
3b
3c
R1 = CH3
S
R1 = H
R1 = F
R2 = SCH3
5a, 6a R1 = CH3
5b, 6b R1 = H
5c, 6c R1 = F
6d, 11a R1 = CH3
6e, 11b R1 = H
6f, 11c R1 = F
(vii)
S
S
R2 = SCH3
R2 = SCH3
R2 = SO2CH3
R2 = SO2CH3
R2 = SO2CH3
R2
11a-11c
Scheme 4. Synthesis of the 3H-1,2-dithiole-3-thiones (Part 1): method iii: 3a or 3b, with 4a, dichloromethane, AlCl3, 0 °C; method iv: 3c, 4a (thioanisole), polyphophoric acid,
90–120 °C, 5 min; method v: 5a, 5b or 5c NaH in DMF, methyl iodide, nitrogen atmosphere, rt; method vi: 6a–c in methanol/THF 1:1, Oxon, 0 °C?rt, over night, method vii:
6d–f, sulfur, Lawesson’s reagent in biphenyl, 210–220 °C.
R2 = CH3; IC50 COX-1: 0.02
was observed comparing the regioisomers 2d (R1 = Cl, R2 = CH3;
IC50 COX-1: 0.2 M)
M), 11d (R1 = CH3, R2 = Cl; IC50 COX-1: 2
l
M) a significant difference in potency
substituentes R1 and R2 at the phenyl rings. These findings are in
line with previous findings of Janusz et al. who investigated differ-
ent 5-substituted Dihydrodimethylbenzofurans as combined COX/
5-LOX inhibitors.36
No clear decisions can be drawn with regard to explain the
COX-2 inhibition activities. The introduction of a methylsulfonyl
group at both positions R1 or R2 resulted in a loss of COX-1 activity
(Table 1). This is in line with our previous findings.21,37 Three com-
pounds exhibited COX-2 inhibitory activities as potent as celecoxib
l
l
and 12d (R1 = CH3, R2 = Cl; IC50 COX-1: 0.2
lM).
With regard to the COX-1 inhibitory activities (R1 = CH3) and
(R2 = OCH3), (R1 = OCH3) and (R2 = Cl), and dichloro (R1 = R2) substi-
tution worked well for example, 2b (IC50 COX-1: 0.01
COX-1: 0.07 M), 2g (IC50 COX-1: 0.02 M) and, 2e (IC50 COX-1:
0.1 M), respectively.
Taken together the highest COX-1 inhibiting potency was
achieved by compounds bearing two methoxy groups (R1 = R2 2a
(IC50 COX-1: 0.003) and (12f) (IC50 COX-1: 0.0003 M). The data
lM), 2f (IC50
l
l
l
(IC50 COX-2: 1
lM): 2a (IC50 COX-2: 0.8
lM); 12b (IC50 COX-2:
1.1 M) and 12f (IC50 COX-2: 1
l
lM). However, only 12b is a meth-
l
ylsulfonyl (R2) derivative. But it is interesting to notice that the
combination of methylsulfonyl (R2) with fluorine (R1, 2h) or
methyl (R1, 11a) yielded further compounds with moderate COX-
discussed above demonstrate the weak impact of the heterocyclic
system towards COX-1 activity contrary to the influence of the

562
M. Scholz et al. / Bioorg. Med. Chem. 17 (2009) 558–568
R2
O
OH
R2
(ix)
+
R1
Cl
R1
8a-8c
7a-7c
9a-9d
9a-9d
R2
only 10a
and 10b
(x)
R1
10a-10d
R1
S
R2
(xii)
S
(vi)
(xi)
S
10c, 10d
R1
R2
10f-10g
11d - 11h
O
O
O
10e
R2 = Cl
anisole
7a
7b
7c
8a
8b
8c
R2 = CH3
R2 = H
R1 = SCH3
R1 = CH3
R1 = Cl
O
HO
S
S
S
(ii)
R2 = Cl
11d R1 = CH3
11e R1 = Cl
9a, 10a,
9b, 10b,
9c, 10c
9d, 10d
10e,
R2 = CH3
R2 = H
S
S
R1 = SCH3
S
R2 = CH3
R2 = OCH3
R2 = H
R1 = SCH3
O
11f R1 = OCH3
11g R1 = SO2CH3
11h R1 = SO2CH3
HO
11f
10f,
R2 = CH3
11i
10g,
Scheme 5. Synthesis of the 3H-1,2-dithiole-3-thiones (Part 2): method ii: 11f, borontribromide in dichloromethane, ꢁ50 °C?rt; method vi: 10c–d in methanol/THF 1:1,
Oxon, 0 °C?rt, over night; method ix: grignard: 7 and Mg in ether, addition of 8 stirred for 10 min, heated for 2 h; method x: 9a–d, H3PO4, reflux, 1 h; method xi: 4b (anisole),
acetone, H2SO4, 4–5 h rt; method xii: 10a–b or 10e–g, sulfur, 220 °C until the formation of hydrosulfite ended.
gard to the COX-2 inhibitory effect compared to a combination of
R1
R1
methylsulfonyl (R2) and fluorine (R1) (2h) (IC50 COX-2: 9.0
lM)
S
or methyl (R1) (11a) (IC50 COX-2: 9.0
lM). However, it is difficult
O
to draw conclusions about the impact of the isothiazole-, dithiol-
thione- or dithiolone-heterocyclic ring system on COX-2 activity
to generalize the results: In each series a candidate with a very
promising COX-2 inhibiting profile is present (2a, IC50 COX-2:
(xiii)
S
S
S
S
R2
R2
0.8
l
M; 11a, IC50 COX-2: 9.0
The 5-LOX inhibition values show a very interesting SAR. De-
spite it was not possible to meet the IC50-value of licofelone38
(IC50 5-LOX: 0.18 M) or NDGA (IC50 5-LOX: 1.0 M), we character-
ized three substances with a remarkable 5-LOX inhibiton: (11a,
IC50 5-LOX: 9 M; 11b, IC50 5-LOX: 3 M; 11i, IC50 5-LOX: 6 M).
lM; 12b, IC50 COX-2: 1.1 lM).
12b, 12d-12g
11b, 11d-11g
11b, 12b R1 = H
R2 = SO2CH3
R2 = Cl
11d, 12d R1 = CH3
11e, 12e R1 = Cl
l
l
R2 = CH3
R2 = OCH3
R2 = H
11f, 12f R1 = OCH3
11g, 12g R1 = SO2CH3
l
l
l
The substituents R1 and R2 at the isothiazole-, dithiolthione- or
dithiolone- systems showed no clear tendency to influence the 5-
LOX activity. The 5-LOX in vitro data for the different heterocyclic
systems illustrate that compounds containing a dithiolthione- or
an isothiazole-ring system seemed to be more active compared
to the heterocyclic dithiolone ring system. The replacement of
the dithiolthione sulfur of 11b (R1 = H, R2 = methylsulfonyl; IC50
Scheme 6. Synthesis of the 3H-1,2-dithiole-3-ones: method xiii: 11b, 11d–g,
acetone, potassium permanganate, 12 h, rt.
2 activities (both IC50 COX-2: 9.0
derivative 2e (IC50 COX-2: 4.0 M) provided also moderate COX-
2 activity. As already seen for the COX-1 activity the dimethoxy
substituents at R1 and R2 (2a and 12f) (IC50 COX-2: 0.8
M vs
IC50 COX-2: 1.0 M, respectively) proofed to be superior with re-
lM). Moreover, the dichloro
l
5-LOX: 3.0
LOX: > 50 M (13% inhibition at a concentration of 10
sulted almost in a loss of 5-LOX potency.
l
M) by oxygen of the dithiolone 12b (IC50 5-
l
l
lM)) re-
l

M. Scholz et al. / Bioorg. Med. Chem. 17 (2009) 558–568
563
Table 1
COX1/2, 5-LOX and OH scavenging activities of the 4,5-diaryl isothiazoles, 4,5-diaryl-3H-1,2-dithiole-3-thiones and 4,5-diaryl-3H-1,2-dithiole-3-ones
Å
R1
R1
X
N
S
S
S
R2
R2
2a-2i,
11a-11i, 12b,12d-12g
R1
R2
X
IC50
(l
M)
IC50
(lM)
Ratio
IC50
(
lM)
IC50
ÅOH
(lM)
a,*
a,*
a,*
b,**
Cpd
COX-1
COX-2
COX-1/COX-2
5-LOX
2a29
2b
2c
2d
2e
OCH3
CH3
CH3
Cl
Cl
OCH3
Cl
OCH3
OCH3
Cl
CH3
Cl
0.003
0.01
0.04
0.2
0.8
>50
>50
0.004
—
—
—
0.03
—
—
—
—
0.8
—
—
—
—
0.0015
—
—
—
7.3
—
—
ꢂ20
8.0
>500
>500
>500
>500
>500
500
>500
>500
500
ꢂ50
>50
0.1
4.0
2f
Cl
0.07
0.02
ꢂ50
1.0
ꢂ50
ꢂ20
ꢂ10
>50
9.0
2g
OCH3
SO2CH3
OH
SO2CH3
SO2CH3
SO2CH3
Cl
CH3
OCH3
H
CH3
OH
SO2CH3
Cl
CH3
>50
9.0
>50
9.0
>50
>50
>50
ꢂ50
ꢂ20
>50
>50
>50
1.1
2h49
2i
F
OH
CH3
H
F
CH3
Cl
11a
11b
11c
11d
11e
11f
11g
11h
11i
12b
12d
12e
12f
12g
S
S
S
S
S
S
S
S
7.0
9.0
7.0
3.0
>50
2.0
0.006
0.03
ꢂ20
0.6
ꢂ20
ꢂ20
>50
ꢂ10
>50
>50
6.0
>50
>50
>50
>50
>50
11.0
ꢂ100
ꢂ100
ꢂ200
5.0
OCH3
SO2CH3
SO2CH3
OH
H
CH3
Cl
OCH3
SO2CH3
9.0
S
ꢂ20
11.0
>500
>500
>500
>500
ꢂ500
O
O
O
O
O
8.0
0.2
0.02
0.0003
8.0
>50
>50
1.0
OCH3
H
0.0003
—
>50
Celecoxib
Diclofenac
Licofelone
Thioctic acid
NDGA
8.0
0.8
0.01
—
—
>50
10
0.3
—
—
—
—
ꢂ500
0.003
0.29
—
ꢂ50
>50
0.189
—
—
—
11.0
29
1.0
a
Values are means of at least two determinations.
Values are means of at least three determinations.
b
*
For clarity and better comparison the % inhibition values are represented by ranges of IC50-values (
tion = ꢂ20 mol, 25–34.9% inhibition = ꢂ50 mol, <24.9% inhibition = >50 mol.
For clarity and better comparison the % inhibition values are represented by ranges of IC50-values (
inhibition = ꢂ200 mol, 25–34.9% inhibition = ꢂ500 mol, <24.9% inhibition = >500 mol.
l
mol) as follows: 45–55% inhibition = ꢂ10
l
mol, 35–44.9% inhibi
l
l
l
**
l
mol) as follows: 45–55% inhibition = ꢂ100
lmol, 35–44.9%
l
l
l
Table 2
In vitro anti-adhesive activities the of 4,5-diaryl-3H-1,2-dithiole-3-thiones
Cpd
R1
R2
Static assaya,c %inhibition at 100
71 13%
lM
Mouse peritonitis model
11a
11b
11c
11h
11g
11i
Celecoxib
Licofelone
CH3
H
F
SO2CH3
SO2CH3
OH
—
—
SO2CH3
SO2CH3
SO2CH3
CH3
H
OH
49%*
—
—
R1
9
7%
S
85 7%
26 14%
38 16%
54 26%
85 13%b
56% at 30
—
S
—
S
—
—
—
l
M10
55%**
R2
11a-11i,
a
b
c
Values are the mean of SEM of at least six determinations.
Inhibition at a concentration of 40 M.
In this assay compounds at a concentration of 100
results for compounds which have been tested on different plates, the number of adherent MM6 cells on TNF-
Inhibition at a dose of 50 mg/kg.
l
l
M were evaluated for inhibition of adhesion of MM6 cells to BAEC and sLeX was chosen as reference. To compare the
a
-stimulated endothelial cells is set to 100%.43
*
**
Inhibition at a dose of 100 mg/kg, n.a.: not active (in adhesion assay, n.a. is defined as less than 10% inhibition at 100
lM).
However, the ÅOH inhibiting in vitro data acquired for the differ-
ent heterocyclic ring systems revealed a clear trend in the potency
of scavenging hydroxyl radicals. The dithiolthione heterocycles
(11a–i) exhibited very potent ÅOH scavenging activities (11a,
IC50: 9.0 M, 11b IC50: 7.0 M, 11c IC50: 11.0 M, 11g IC50
5.0 M, 11h IC50: 9.0 M, 11i IC50: 11.0 M). This is not very sur-
l
l
l
:
l
l
l

564
M. Scholz et al. / Bioorg. Med. Chem. 17 (2009) 558–568
prising since their structures are close related to thioctic acid (IC50
:
14
12
10
8
11.0 lM), a very potent and well known hydroxyl radical scaven-
48/80
ger in a variety of hydroxyl radical test assays.39 The dithiolthiones
derivatives bearing a methylsulfonyl moiety (11a–c, 11g–h) dis-
played ꢂ10-fold higher ÅOH scavenging activities compared to
compounds missing this structural feature (11d–f) (ÅOH scavenging
48/80 + 11a (50 mg/kg)
IC50 ꢂ 10.0
In contrast the corresponding dithiolones (12b, 12d–g) showed
no 12d–f (0% inhibition at a concentration of 100 M) or less hy-
droxyl radical scavenging effects (Table 1), for example, 12b
(R1 = H, R2 = SO2CH3; ÅOH scavenging IC50 > 500
M (22% inhibition
at a concentration of 100
M) and 12g (R1 = SO2CH3, R2 = H; (ÅOH
scavenging IC50 ꢂ 500 M (26% inhibition at a concentration of
100 M). The methylsulfonyl moiety seemed to be beneficial to
lM vs 100 lM) (Table 1).
l
6
l
l
4
l
l
support hydroxyl radical scavenging activities. Celecoxib (ÅOH
2
scavenging IC50 ꢂ 500
l
M (ÅOH inhibition at a concentration of
Å
100
l
M: 27%)) behaved as weak OH scavenging substance.
0
Among the isothiazoles only 2f exhibited a weak ÅOH inhibiting
activity (10% inhibition at a concentration of 100 M). It is interest-
Total cell count
PMN
Mono/Mac
l
Figure 1. Effect of 11a on leukocyte recruitment in a mouse peritonitis model.
Leukocyte extravasation into the peritoneal cavity was analyzed in mice after
intraperitoneal challenge with compound 48/80 for 4 h (25 lg/mouse, white bars).
One group of animals received 11a (50 mg/kg) intraperitoneally 1 h before injection
of compound 48/80 (black bars). Leukocytes harvested from the peritoneal cavity
were differentiated by FACS analysis. Values are mean SD of eight independent
experiments (*P < 0.05 vs control).
ing to notice that 2h (0% inhibition at a concentration of 100
l
M)
Å
did not show OH inhibiting activity at all even it bears a methyl-
sulfonyl group (R2).
Reactive oxygen species (ROS) produced by cytokines, growth
factors, and vasoactive agents contribute to the intracellular signal-
ling cascades associated with inflammatory responses.40,41 ROS in-
duce NF-
the kinase enzymes in the NF-
B is a major transcription factor involved in the transcriptional
regulation of cell adhesion molecules we studied the effect of some
potent ÅOH scavenging compounds (11a–c and 11h–i) on adhesion
of leukocytes on inflamed endothelium. In this assay compounds at
j
B activation by modifying the activity of one or more of
j
B activation cascades.42 Since NF-
lease of prostaglandins and leukotrienes which is related to a rapid
influx of PMN. As seen in Figure 1, pre-treatment with 11a (50 mg/
kg) reduced the total cell count in the peritoneal cavity, the major-
ity of which were PMNs (ꢃ49%) (n = 8, P < 0.02). Earlier mouse
peritonitis model studies revealed that treatment with the COX/
5-LOX inhibitor licofelone (100 mg/kg) reduced the total cell count
in the peritoneum, the majority of which were PMNs, from
24 ꢄ 106 cells in cytokine-treated mice to 13 ꢄ 106 cells (ꢂ55%)
(n = 4, P < 0.05).14 Only few monocytes and lymphocytes extrava-
sated in response to compound 48/80 or cytokine stimulation. This
indicates that our in vitro assays predicted anti-inflammatory effi-
cacy in vivo of the test compound. In order to investigate if the
anti-adhesive effect of 11a seen in vitro was mediated by changes
in expression of adhesion molecules we assessed the effect of 11a
on cell adnesion molecule (CAM) expression on protein level. Per-
itoneal PMNs from the compound 48/80-treated group responded
with strong expression of CD 11b (Mac-1) (n = 8, P < 0.008) (Fig. 2),
which was attenuated by 11a (50 mg/kg) by 40%, but not other
NSAIDs (data not shown), respectively. This indicates that the
activation of extravasated PMN was reduced. This effect may
j
a concentration of 100 lM were evaluated for inhibition of adhe-
sion of MM6 cells to BAEC.43 To compare the results for compounds
which have been tested on different plates, the number of adherent
MM6 cells on TNF-
(Table 2).
a-stimulated endothelial cells was set to 100%
Interestingly, we found that the dithiolthiones 11a (R1 = CH3,
R2 = SO2CH3) and 11c (R1 = F, R2 = SO2CH3) gave equivalent good
inhibitory activities in the cell based static assay (71% vs 85%) (Ta-
ble 2). It is worth to mention that the unsymmetrical substituted
regioisomer 11h (R1 = SO2CH3, R2 = CH3) was less active (26%).
The introduction of a hydroxyl group at the position R1 and R2 in
case of 11i resulted in an increase of inhibition of adhesion com-
pared to 11h (26% vs 54%). This is surprising since 11h and 11i be-
haved both as very potent hydroxyl radical scavenger (IC50: 9 vs
11 lM) in our hydroxyl radical test assays. In addition it is note-
worthy that introduction of hydrogen (11b) instead of a methyl
(11a) at position R1 led almost to a complete loss of ÅOH scavenging
activities (9% vs 71%).
Taken together we succeeded to prepare novel compounds able
to inhibit 4 targets. The overall best hit is the dithiolthione 11a
12
10
8
(COX-1 IC50: 7
l
M, COX-2 IC50: 9
l
M, 5-LOX IC50 ꢂ 10
lM, IC50
ÅOH: 9
lM) with a balanced inhibition of all 4 targets. Furthermore
11a inhibits the adhesion of leukocytes in vitro.
6
4. In vivo evaluations
4
Based on its overall biological profile, 11a was chosen for fur-
ther investigations. The effect of 11a on leukocyte recruitment
in vivo was tested in a mouse peritonitis model. Leukocyte extrav-
asation was induced by i.p. compound 48/8044 challenge. Treat-
ment of mice with compound 48/80 and the test substance did
not lead to any obvious signs of discomfort. The white blood count
and the relative amount of peripheral PMN and mononuclear cells
were not different in both groups (data not shown). Compound 48/
80 induces a strong activation of mast cells which involves the re-
2
0
48/80
48/80 + 11a
Figure 2. Effect of 11a on expression of the membrane receptor CD11b (Mac-1) on
neutrophils recruited to the peritoneum in mice exposed to compound 48/80.
Peritonitis was induced as described for Figure 1. Expression of CD11b was
measured by FACS analysis. Data are expressed as isotype-corrected MFI. Values are
mean SD of eight independent experiments (*P < 0.05 vs control).

M. Scholz et al. / Bioorg. Med. Chem. 17 (2009) 558–568
565
contribute to the markedly reduced leukocyte accumulation found
after 11a treatment in the mouse peritonitis model in vivo.
To exclude the possibility that inhibition of CD 11b expression
seen with 11a might have resulted from a cytotoxic effect of this
compound on leukocytes we investigated viability by trypan blue
exclusion. After 6 h, trypan blue exclusion was 96 4% and
94 2%, indicating that loss in cell viability does not account for
changes in CAM expression on EC.
value, to 192
cro-centrifuge tube. 2
solution (5 mM/l) and 2
l
L demineralized water (Millipore) in a 1.5 mL mi-
L DEPMPO solution (20 mM/l), 2 L FeSO4
L hydrogen peroxide solution (2.5 mM/
l
l
l
l) were put on different points on the wall of the tube. The reaction
was started by vortexing the tube. After 10 s a sample was gath-
ered with a 100 l
L ringcap from HIRSCHMANNÒ Laborgeräte and
closed with wax. After exact 90 s the EPR experiment was started.
Instrumental parameters were as follows: Receiver Gain
3.99 ꢄ 105, Modulation Amplitude 3.0 G, Time Constant 0.64 ms,
Conversion Time 81.92 ms, Attenuation 4 ms. The spectra were
analyzed with the WinEPRÒ (Version 1.0) software. The raw data
were integrated, the background noise was subtracted and the
heights of the second peaks were analyzed. Each concentration
was tested 3 times. Thioctic acid in concentrations of 10, 1 and
0.1 mM/l was used as standard substance in every test series. For
an amount of 30 spectra totalling 6 zero values as control were
analyzed. The EPR Spectra were acquired on a Bruker EMX EPR
spectrometer.
Melting points were determined with a Büchi apparatus accord-
ing to Dr. Tottoli and are uncorrected. 1H NMR spectra (300 MHz)
were recorded on a Bruker AC 300 spectrometer. Combustion anal-
yses were performed with a Carlo Erba Strumentiazione 1106
analyzer.
Column chromatography was performed with Merck Silica Gel
60 (0.063–0.200 mm). The progress of the reactions was monitored
by thinlayer chromatography (TLC) performed with Merck Silica
Gel 60 F-245 plates.
All reagents and solvents were obtained from commercial
sources and used as received. Reagents were purchased from Sig-
ma-Aldrich Chemie Steinheim, Germany, or Acros, Nidderau,
Germany.21
5. Conclusion
Three series of different substituted diaryl heterocycles were
synthesized. The 4,5-diaryl-dithiol-3-thione derivative 11a re-
ported here represents a candidate which is able to inhibit differ-
ent pathways involved for example, inflammation: (i) 11a
inhibits three arachidonic acid utilizing pathways: COX-1, COX-2
and 5-LOX; (ii) 11a reduces ROS via hydroxyl radical scavenging
activities; (iii) 11a inhibits the expression of Mac-1 and adhesion
and infiltration of leukocytes in vitro and in vivo.
Recapitulatory, we showed that this moderate overall in vitro
potency was translated in an additive in vivo activity. Up to date
we do not have an explanation for this phenomenon. One specula-
tion is that the intervention in the inflammatory cascade at differ-
ent signal transduction pathways might be beneficial for the good
in vivo activity. However, the precise mechanism of action remains
to be elucidated. Especially the effect of 11a on the proinflamma-
tory transcription factor nuclear factor-
jB (NF-jB) is interesting
in this regard because NF- B activation represents an essential
j
pathway for the induction of numerous proinflammatory genes
in the vascular wall, including cell CAMs and cytokines.
Further improvements on the potency of this compound could
lead to novel and safe anti-inflammatory agents with potential
therapeutic utilities. These drugs represent a novel class of multi-
ple target non-steroidal anti-inflammatory drugs (MTNSAIDs).
6.3. Static assay
The adhesion assay was performed as described previously.43
Briefly, BAEC monolayer 7 days post seeding; passage 1-3, were
grown in 24 well plates. Prior to assay, the BAEC were examined
microscopically to confirm the integrity and uniformity of the
monolayer. After the culture medium was removed and the endo-
thelial cell monolayer was washed with DPBS (2 ꢄ 1 mL/well at
37 °C) the cells were treated with cell culture medium containing
6. Experimental
The docking experiments (see SI-file),21 COX-1, 5-LOX,45 COX-
2,46 Hydroxyl radical scavenging, inhibition assays21 and the adhe-
sion assay42,47 were performed as described previously. For clarity
a brief summary of the procedures are given below.
20 ng/mL recombinant tumor necrosis factor alpha (TNF-a) for 4
or 16 h and the respective test substance dissolved in DMSO or
vehicle for 20 min (in other settings for 4 or 16 h) at 37 °C/5%
CO2. The BAEC were washed with DPBS (2 ꢄ 1 mL/well at 37 °C)
6.1. Enzyme assays
COX-1 inhibition was determined by using platelets isolated
from bovine blood, incubated with the test substance, and stimu-
lated by the Ca ionophor A23187. The inhibition of COX-1 was
assessed by quantitative HPLC determination of the formation of
12-hydroxyheptadecatrienoic acid.45
5-LOX inhibition was investigated by using bovine polymorpho-
nuclear leucocytes, incubated with the test substance, and stimu-
lated by the Ca ionophor A23187. The inhibition of 5-LOX was
assessed by quantitative HPLC determination of the formation of
leukotriene B4.45
before 900 lL of culture medium and 100 lL of PMN suspension
(1 ꢄ 106 cells/mL) per well were added and incubated for 30 min
at 37 °C/5% CO2. Non-adherent PMNs were removed by washing
with DPBS (3 ꢄ 1 mL/well at 37 °C). Culture medium (1 mL/well
at 37 °C) was added prior to microscopic examination. (In inhibi-
tion experiments, various concentrations of drugs were used
(1 ꢄ 10ꢁ4–1 ꢄ 10ꢁ7 mol/L)). After incubating with cytokines or
drugs the cell viability of BAEC was confirmed by trypan blue
exclusion and additional microscopic examination. IC50 values
were calculated using the program GRAFIT, Erithacus Software
Ltd., UK.42,47
COX-2 inhibition was determined by using aliquots of human
blood samples, containing sodium heparin. The samples were incu-
bated in absence and presence of LPS and the test substances as
described.48
6.4. Compound 48/80-induced Mouse peritonitis
Female Balb/c mice were obtained from B&K, Stockholm, Swe-
den. Animals of 20–25 g were fed normal chow and water ad
libitum. Peritonitis was induced by a single injection of com-
6.2. EPR spin trapping
Test assay for hydroxyl radical scavenging: DEPMPO was pur-
chased from Calbiochem (San Diego, USA). The test solution was
pound 48/80 (Sigma) at 25 lg/mL in 1 mL sterile PBS. The test
compound was injected intraperitoneally at a concentration of
50 mg/kg 1 h before challenge with compound 48/80, control
animals were injected with vehicle only. Four hours after injec-
prepared by adding 2
lL of a solution of the test substance in ace-
tonitrile (concentration 10, 1 or 0.1 mM/l) or acetonitrile, as zero

566
M. Scholz et al. / Bioorg. Med. Chem. 17 (2009) 558–568
tion of compound 48/80, peritoneal cells were harvested by
injecting 4 mL cold HBSS containing 5 mM EDTA. Cells were
spun down, counted after staining with turck solution and the
proportion of PMN and monocytes/macrophages was assessed
by FACS analysis using cell specific antibodies. PMN were stained
with rat anti-Gr1 PE/Cy5 (Biolegend), monocytes/macrophages
were stained with rat anti-F4/80 FITC (Serotec). After 30 min of
incubation, cells were washed twice, resuspended in PBS and
analyzed in FACSort (Becton Dickinson). Gating for leukocytes
was performed in forward and side scatter, and the relative
amount of PMN and monocytes/macrophages was estimated by
the number of Gr1+/F4/80ꢁ (PMN) or F4/80+/Gr1ꢁ and F4/80+/
Gr1lo (both monocytes). The activation of emigrated PMN was
assessed by staining with rat anti-mouse CD11b PE antibody
(Serotec). All experiments were approved by the local ethical
committee for animal experimentation (N149/06).
220 °C until the formation of hydrosulfite ended. After addition
of ethyl acetate (25 mL) the mixture was refluxed for 30 min, fil-
tered over CelliteÒ, washed and concentrated under reduced pres-
sure. By column chromatography purification (SiO2, ethyl acetate/
petroleum ether 1:9) the dithiole-3-thiones 11d–h (9–19%) were
obtained as red solids.
6.5.5. Method xiii
To a solution of the dithiole-3-thione 11b or 11d–g (1 mmol) in
acetone a solution of potassium permanganate (5 mmol) in ace-
tone was added slowly. The violet mixture was stirred for 12 h at
rt until it was decolourized. The mixture was filtered, dried over
anhydrous Na2SO4, concentrated under reduced pressure, and
recrystallized with ethyl acetate to obtain of the dithiole-3-ones
12b or 12d–g (29–83%).
6.6. Compounds
6.5. Chemistry
Compounds 1a–h and 2a were described previously.21,28,29
Compounds 5a–c, 6a–f, 8a, 9a–d and 10a–g are described in the
Supplementary data.
Compounds were analyzed for C, H, N, S and elemental analyses
were within 0.4% for elements unless indicated otherwise.
Methods iii–vi and methods viii–xi are described in the Supple-
mentary data.
6.6.1. 4,5-Bis(4-methoxyphenyl)isothiazole (2a)29
Method i; Reactant: 1a; yield: 60%; mp: 81 °C; 1H NMR (DMSO)
8.61 (s, 1H), 7.245 (d, 8.69 Hz, 4H, A00A000 and B00B000), 6.95 (m, 4H,
AA0 and BB0), 3.76 (s, 3H), 3.75 (s, 3H). FD: m/z (rel. Int.) = 297.6
(100%). Anal. Calcd for C17H15NO2S: C, 68.66; H, 5.08; N, 4.71; S,
10.78. Found: C, 69.01; H, 5.00; N, 4.64; S, 10.62.
6.5.1. Method i
A mixture of the 3-chloro-acrylaldehyde 1a–h (0.1 mol) and
ammonium thiocyanate (22.8 g, 0.3 mol) in acetone (200 mL) was
refluxed for 4 h (CAUTION: HCN formation!). After cooling to rt
the mixture was poured in a saturated solution of aqueous sodium
hydrogen carbonate (300 mL) and extracted with diethyl ether
(3 ꢄ 200 mL). The combined organic layers were dried over anhy-
drous Na2SO4, concentrated under vacuum and the residue purified
by SiO2, ethyl acetate/petroleum ether 3:7 to yield the isothiazoles
2a–h (30–80% yield) as colourless solids.
6.6.2. 5-(4-Methoxyphenyl)-4-p-tolylisothiazole (2b)
Method i; Reactant 1b; yield: 40%; mp: 94 °C; 1H NMR (DMSO
8.62 (s, 1H), 7.24 (d, 8.65 Hz, 2H, BB0), 7.18 (m, 4H), 6.96 (d,
8.73 Hz, 2H, AA0), 3.76 (s, 3H), 2.30 (s, 3H); FD: m/z (rel.
Int.) = 281.2 (100%). Anal. Calcd for C17H15NOS: C, 72.57; H, 5.37;
N, 4.98; S, 11.40. Found: C, 72.65; H, 5.32; N, 4.77; S, 10.97.
6.5.2. Method ii
The anisol derivative 2a or 11e (4.8 mmol) was dissolved in
50 mL anhydrous dichloromethane. The reaction mixture was
cooled to ꢁ50 °C and stirred vigorously in a nitrogen atmosphere.
A solution of borontribromide (9.6 mmol) in 20 mL dichlorometh-
ane was added using a septum and the mixture was stirred over
night slowly warming up to rt. The mixture was poured into ice
water; The organic layer was separated and extracted with a di-
luted solution of NaOH (3 ꢄ 50 mL). The aqueous layers were acid-
ified by dropwise addition of aqueous hydrochloric acid (1.0 M)
and extracted with diethyl ether (3 ꢄ 100 mL). The organic layers
were dried over anhydrous Na2SO4 and concentrated under re-
duced pressure to obtain the phenol derivative 2i or 11i (15–25%
yield).
6.6.3. 5-(4-Chlorophenyl)-4-p-tolylisothiazole (2c)
Method i; Reactant: 1c; yield: 76%; mp: 79 °C; 1H NMR (DMSO)
8.69 (s, 1H), 7.49 (d, 8.51 Hz, 2H, BB0), 7.33 (d, 8.51 Hz, 2H, AA0),
7.19 (m, 4H), 2.3 (s, 3H); FD: m/z (rel. Int.) = 285.4 (100%).
6.6.4. 4-(4-Chlorophenyl)-5-p-tolylisothiazole (2d)
Method i; Reactant: 1d; yield: 59%; mp: 108 °C; 1H NMR
(DMSO) 8.7 (s, 1H), 7.45 (d, 8.42 Hz, 2H, B00B000), 7.34 (d, 8.29 Hz,
2H, BB0), 7.24 (m, 4H, AA0and A00A000), 2.31(s, 3H); FD: m/z (rel.
Int.) = 285.4 (100%).
6.6.5. 4,5-Bis(4-chlorophenyl)isothiazole (2e)
Method i; Reactant: 1e; yield: 57%; mp: 94 °C; 1H NMR (DMSO)
8.73 (s, 1H), 7.5 (d, 8.51 Hz, 2H, B00B000), 7.46 (d, 8.54 Hz, 2H, A00A000),
7.34 (d, 8.44 Hz, 4H, BB0 and AA0); FD: m/z (rel. Int.) = 305.4 (100%).
6.5.3. Method vii
To a mixture of ketone 6d, 6e or 6f (13.87 mmol), sulfur (4.44 g,
138.75 mmol) and Lawesson’s reagent (5.6 g, 13.87 mmol) biphe-
nyl were added and heated on a graphite bath (210–220 °C). After
completion of hydrosulfite formation, the mixture was cooled to
50 °C and ethyl acetate (100 mL) was added. The reaction mixture
was refluxed for 1 h, stirred at rt over night, filtered over CeliteÒ
and concentrated under reduced pressure. By column chromatog-
raphy purification (SiO2, ethyl acetate/petroleum ether 5:95) and
recrystallization with methanol the 5-(4-methylsulfonyl)phenyl)-
4-phenyl-3H-1,2-dithiole-3-thiones 11a, 11b or 11c (5–9% yield)
were obtained as dark red crystals.
6.6.6. 5-(4-Chlorophenyl)-4-(4-methoxyphenyl)isothiazole (2f)
Method i; Reactant: 1f; yield: 66%; 1H NMR (DMSO) 8.67 (s, 1H),
7.49 (d, 8.54 Hz, 2H, B00B000), 7.33 (d, 8.54 Hz, 2H, A00A000), 7.24 (d,
8.77 Hz, 2H, BB0), 6.94 (d, 8.78 Hz, 2H, AA0); FD: m/z (rel.
Int.) = 301.5 (100%). Anal. Calcd for C16H12ClNOS: C, 63.68; H,
4.01; N, 4.64; S, 10.62. Found: C, 63.73; H, 4.08; N, 4.44; S, 10.41.
6.6.7. 4-(4-Chlorophenyl)-5-(4-methoxyphenyl)isothiazole (2g)
Method i; Reactant: 1g; yield: 70%; mp: 99 °C; 1H NMR (DMSO)
8.67 (s, 1H), 7.44 (d, 8.46 Hz, 2H, B00B000), 7.34 (d, 8.51 Hz, 2H, A00A000),
7.24 (d, 8.68 Hz, 2H, BB0), 6.98 (d, 8.71 Hz, 2H, AA0), 3.76 (s, 3H);
FD: m/z (rel. Int.) = 301.5 (100%). Anal. Calcd for C16H12ClNOS: C,
63.68; H, 4.01; N, 4.64; S, 10.62. Found: C, 63.68; H, 4.11; N,
4.55; S, 10.58.
6.5.4. Method xii
A
mixture of (E)-1,2-diphenylprop-1-ene 10a–b or 10e–g
(1 mmol) and sulfur (4 mmol) was heated on a graphit bath to

M. Scholz et al. / Bioorg. Med. Chem. 17 (2009) 558–568
567
6.6.8. 4-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)isothia-
zole (2h)49
EI: m/z (rel. Int.) = 364.5. Anal. Calcd for C16H12O2S4: C, 52.72; H,
3.32; N, 0; S, 35.18. Found: C, 52.65; H, 3.48; N, 0; S, 34.92.
Method i; reactant 1h; yield: 48%; mp: 104 °C; 1H NMR
(DMSO 8.77 (s, 1H), 7.96 (d, 8.23 Hz, 2H, BB0), 7.58 (d, 8.26 Hz,
2H, AA0), 7.39 (m, 2H), 7.25 (m, 2H), 3.26 (s, 3H); FD:
m/z = 333.1 (M+).
6.6.17. 4-(4-(Methylsulfonyl)phenyl)-5-p-tolyl-3H-1,2-dithiole-
3-thione (11h)
Method xii; Reactant: 10g; yield: 9%; 1H NMR (DMSO) 7.88 (d,
8.1 Hz, 2H, BB0), 7.4 (d, 8.4 Hz, 2H, AA0), 7.19 (s, 4H), 3.23 (s, 3H),
2.27 (s, 3H);); 13C NMR: (DMSO) 21.2 (Ar-CH3); 43.6 (SO2CH3);
127.1 (2CH); 129.1 (2CH); 129.7 (Cq); 130.1 (2CH); 132.3 (2CH);
140.0 (Cq); 140.6 (Cq); 141.6 (Cq); 144.0 (Cq); 173.0 (Cq); 214.3
(C@S); EI: m/z (rel. Int.) = 378.5. Anal. Calcd for C17H14O2S4: C,
53.94; H, 3.73; N, 0; S, 33.88. Found: C, 53.71; H, 3.60; N, 0; S,
33.68.
6.6.9. 4-(5-(4-Hydroxyphenyl)isothiazol-4-yl)phenol (2i)
Method ii; Reactant: 2a; yield: 25%; mp: 227 °C; 1H NMR
(DMSO) 9.86 (s, 1H), 9.59 (s, 1H), 7.13 (d, 7.94 Hz, 4H, BB0 and
AA0), 6.76 (m, 4H), 8.55 (s, 1H); FD: m/z (rel. Int.) = 269.1 (100%).
6.6.10. 5-(4-(Methylsulfonyl)phenyl)-4-p-tolyl-3H-1,2-dithiole-
3-thione (11a)
Method vii; Reactant: 6d; yield: 5%; mp: 243 °C; 1H NMR
(DMSO) 7.91 (d, 8.4 Hz, 2H, B00B000), 7.69 (d, 8.6 Hz, 2H, A00A000),
7.13 (d, 7.9 Hz, 2H, BB0), 7.01 (d, 7.9 Hz, 2H, AA0), 3.23 (s, 3H),
2.26 (s, 3H); 13C NMR: (DMSO) 21.5 (Ar-CH3); 44.3 (SO2CH3);
128.0 (2CH); 129.7 (2CH); 130.0 (2CH); 130.2 (Cq); 130.6 (2CH);
138.7 (Cq); 139.0 (Cq); 142.1 (Cq); 146.8 (Cq); 166.4 (Cq); 215.1
(C@S); EI: m/z = 378.7 (M+). Anal. Calcd for C17H14O2S4: C, 53.94;
H, 3.73; N, 0; S, 33.88. Found: C, 53.62; H, 3.61; N, 0; S, 33.58.
6.6.18. 4,5-Bis(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (11i)
Method ii; Reactant: 11f; yield: 24%; mp: 249 °C; 1H NMR
(DMSO) 10.16 (s, 1H), 9.56 (s, 1H), 7.11(d, 8.6 Hz, 2H, AA0), 6.97–
6.62 (m, 6H); EI: m/z (rel. Int.) = 318.4.
6.6.19. 5-(4-(Methylsulfonyl)phenyl)-4-phenyl-3H-1,2-dithiol-
3-one (12b)
Method xiii; Reactant: 11b; yield: 28%; mp: 183 °C; 1H NMR
(DMSO) 7.93 (d, 7.9 Hz, 2H, BB0), 7.58 (d, 7.6 Hz, 2H, AA0), 7.31 (s,
3H), 7.14 (s, 2H), 3.24 (s, 3H); 13C NMR: (DMSO) 43.4 (SO2CH3);
127.9 (2CH); 128.7 (CH); 128.8 (2CH); 129.9 (2CH); 130.5 (2CH);
132.2 (Cq); 132.3 (Cq); 138.6 (Cq); 142.7 (Cq); 164.4 (Cq); 193.2
(C@O) EI: m/z (rel. Int.) = 348.4. Anal. Calcd for C16H12O3S3: C,
55.15; H, 3.47; N, 0; S, 27.60. Found: C, 54.98; H, 3.17; N, 0; S,
27.63.
6.6.11. 5-(4-(Methylsulfonyl)phenyl)-4-phenyl-3H-1,2-dithiole-
3-thione (11b)
Method vii; Reactant: 6e; yield 6%; mp: 193 °C; 1H NMR
(DMSO) 7.9 (d, 8.4 Hz, 2H), 7.58 (d, 8.4 Hz, 2H), 7.39–7.24 (m,
3H), 7.18–7.07 (m, 2H), 3.23 (s, 3H); 13C NMR: (DMSO) 43.3
(SO2CH3); 127.7 (2CH); 128.66 (2CH); 128.72 (CH); 130.3 (2CH);
131.1 (2CH); 134.0 (Cq); 137.8 (Cq); 142.6 (Cq); 146.8 (Cq);
169.3 (Cq); 215.0 (C@S); EI: m/z = 364.5 (M+). Anal. Calcd for
C16H12O2S4: C, 52.72; H, 3.32; N, 0; S, 35.18. Found: C, 52.60; H,
3.24; N, 0; S, 34.93.
6.6.20. 5-(4-Chlorophenyl)-4-p-tolyl-3H-1,2-dithiol-3-one (12d)
Method xiii, Reactant: 11d; yield: 83%; mp: 130 °C; 1H NMR
(DMSO) 7.47 (d, 8.53 Hz, 2H, BB0), 7.32 (d, 8.52 Hz, 2H, AA0), 7.12
(d, 8.02 Hz, 2H, B00B00), 6.99 (d, 7.96 Hz, 2H, A00A000), 2.26 (s, 2H);
FD: m/z (rel. Int.) = 318,5 (100%).
6.6.12. 4-(4-Fluorophenyl)-5-(4-(methylsulfonyl)phenyl)-3H-
1,2-dithiole-3-thione (11c)
Method vii; Reactant: 6f; yield: 5%; mp: 201 °C; 1H NMR (CDCl3)
8.01–7.92 (m, 2H), 7.7–7.62 (m, 2H), 7.25–7.17 (m, 2H), 7.04–6.94
(m, 2H), 3.24 (s, 3H); EI: m/z = 382.5 (M+). Anal. Calcd for
C16H11FO2S4: C, 50.24; H, 2.90; N, 0; S, 33.53. Found: C, 50.22; H,
3.06; N, 0; S, 33.46.
6.6.21. 4-(4-Chlorophenyl)-5-p-tolyl-3H-1,2-dithiol-3-one (12e)
Method xiii; Reactant: 11e; yield: 59%; mp: 110 °C; 1H NMR
(DMSO) 7.38 (d, 8.19 Hz, 2H, BB0), 7.2 (s, 4H), 7.13 (d, 8.26 Hz,
2H, AA0), 2.29 (s, 3H); FD: m/z (rel. Int.) = 318.5 (100%). Anal. Calcd
for C16H11ClOS2: C, 60.27; H, 3.48; N, 0; S, 20.11. Found: C, 60.27; H,
3.52; N, 0; S, 19.77.
6.6.13. 5-(4-Chlorophenyl)-4-p-tolyl-3H-1,2-dithiole-3-thione
(11d)
6.6.22. 4,5-Bis(4-methoxyphenyl)-3H-1,2-dithiol-3-one (12f)
Method xiii; Reactant: 11f; 1H NMR (CDCl3) 7.21 (d, 8.87 Hz, 2H,
BB0), 7.08 (d, 8.80 Hz, 2H, AA0), 6.87–6.77 (m, 4H), 3.80 (s, 3H), 3.79
(s, 3H).
Method xii; Reactant: 10a; yield: 16%; mp: 175 °C; 1H NMR
(DMSO) 7.45 (d, 8.5 Hz, 2H, BB0), 7.32 (d, 8.5 Hz, 2H, AA0), 7.12 (d,
8.0 Hz, 2H, B00B000), 6.97 (d, 8.0 Hz, 2H, A00A000), 2.265 (s, 3H); EI: m/
z = 334.9 (M+). Anal. Calcd for C16H11ClS3: C, 57.38; H, 3.31; N, 0;
S, 28.72. Found: C, 57.36; H, 3.34; N, 0; S, 28.81.
6.6.23. 4-(4-(Methylsulfonyl)phenyl)-5-phenyl-3H-1,2-dithiol-
3-one (12g)
6.6.14. 4-(4-Chlorophenyl)-5-p-tolyl-3H-1,2-dithiole-3-thione (11e)
Method xii; Reactant: 10b; yield: 19%; mp: 156 °C; 1H NMR
(DMSO) 7.39 (d, 8.35 Hz, 2H, BB0), 7.19 (s, 4H), 7.12 (d, 8.35 Hz,
2H, AA0), 2.27 (s, 3H); FD: m/z (rel. Int.) = 334.6 (100%).
Method xiii; Reactant: 11g; yield: 69%; mp: 176 °C; 1H NMR
(CDCl3) 7.86 (d, 8.6 Hz, 2H, AA0), 7.49–7.31 (m, 5H), 7.29–7.19
(m, 2H), 3.03 (s, 3H); EI: m/z (rel. Int.) = 348.4 (100%). Anal. Calcd
for C16H12O3S3: C, 55.15; H, 3.47; N, 0; S, 27.60. Found: C, 55.27;
H, 3.62; N, 0; S, 27.61.
6.6.15. 4,5-Bis(4-methoxyphenyl)-3H-1,2-dithiole-3-thione (11f)
Method xii; Reactant: 10e; yield: 15%; mp: 179 °C; 1H NMR
(CDCl3) 7.19 (d, 8.8 Hz, 2H, B00B000), 7.07 (d, 8.8 Hz, 2H, A00A000),
6.89 (d, 8.8 Hz, 2H, BB0), 6.81 (d, 9.1 Hz, 2H, AA0), 3.79 (s, 6H); EI:
m/z (rel. Int.) = 346.5. Anal. Calcd for C17H14O2S3: C, 58.93; H,
4.07; N, 0; S, 27.76. Found: C, 58.79; H, 4.07; N, 0; S, 27.73.
Acknowledgement
O.S. is recipient of a postdoctoral fellow grant from the Deut-
sche Forschungsgemeinschaft (SO 876/1-1).
Supplementary data
6.6.16. 4-(4-(Methylsulfonyl)phenyl)-5-phenyl-3H-1,2-dithiole-
3-thione (11g)
Method xii; Reactant: 10f; yield: 18%; mp: 191 °C; 1H NMR
(DMSO) 7.86 (d, 8.1 Hz, 2H, AA0), 7.5–7.24 (m, 7H), 3.22 (s, 3H);
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.11.074.

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M. Scholz et al. / Bioorg. Med. Chem. 17 (2009) 558–568
29. Scholz, M.; Mattern, A.; Ulbrich, H. K.; Kramb, J.-P.; Kiefer, W.; Dannhardt, G.
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