Synthesis and biological evaluation of novel heterocyclic ionone-like derivatives as anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2006.04.007

Five- and six-membered heterocyclic ionone-like derivatives 4-6 have been synthesised in one step and with good yield from the key intermediate 3a and appropriate bifunctional reagents. Four were active as inhibitors of the respiratory burst of human neut

Full text of DOI:10.1016/j.bmc.2006.04.007

Bioorganic & Medicinal Chemistry 14 (2006) 5152–5160
Synthesis and biological evaluation of novel heterocyclic ionone-like
derivatives as anti-inflammatory agents
Alessandro Balbi,a,* Maria Anzaldi,a Mauro Mazzei,a Mariangela Miele,a
Maria Bertolotto,b Luciano Ottonellob and Franco Dallegrib
aDipartimento di Scienze Farmaceutiche, Viale Benedetto XV, 3 Genova, Italy
bDipartimento di Medicina Interna—Clinica di Medicina Interna 1, Viale Benedetto XV, 6 Genova, Italy
Received 31 January 2006; revised 29 March 2006; accepted 4 April 2006
Available online 8 May 2006
Abstract—Five- and six-membered heterocyclic ionone-like derivatives 4–6 have been synthesised in one step and with good yield
from the key intermediate 3a and appropriate bifunctional reagents. Four were active as inhibitors of the respiratory burst of human
neutrophils without affecting cell viability. The two most active compounds (5a,d) tested in neutrophil migration assays, were also
found to be potent inhibitors of neutrophil chemotactic responsiveness. These two molecules could be considered as lead compounds
of new drugs which can be an effective tool to treat psoriasis and related neutrophilic dermatoses.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
compounds have been studied and patented as anti-in-
flammatory agents.8–11 These so-called third generation
of retinoids are often associated with reduced toxicity,
while biological activity is maintained or even enhanced.
Therefore, to design selective retinoids devoid of danger-
ous side effects with high biological activity is not too far
from becoming reality.
Retinoic acid and its derivates have been used extensive-
ly in dermatological applications such as acne, psoriasis
and keratinization disorders, and studied in many other
anti-inflammatory applications.1,2
Unfortunately, their action has also been associated
with a high incidence of undesirable side effects: skin
and mucous membrane toxicity, hyperlipidemia, skeletal
side effects, visual disturbance and teratogenicity.3–7
Consequently, in an effort to both increase efficacy and
reduce toxicity, a great number of retinoids have been
synthesised. The original structure of vitamin A has
been modified in the cyclohexenyl ring, the polyene side
chain and the polar terminal group. Major transforma-
tions have led to compounds which barely resemble the
original retinoic acid. The wide spectrum of effects
which they produce suggests that they can interact with
a diverse range of receptors. Among these, adapalene
and tazarotene have been introduced on the market
for topical use and ‘short heteroretinoids’ are currently
studied as potent inducers of differentiation and apopto-
sis. Moreover, even if to a minor extent, retinoid-like
Although almost all retinoid-like compounds have a car-
boxylic function, there are few examples in which this
function is replaced by amides12 or amines.13,14 In many
cases, the authors’ statements regarding these com-
pounds are very optimistic. For example, some say that
amides maintain a noticeable activity both in vitro and
in vivo and are much less toxic and teratogenic than ret-
inoid acid itself. Nonetheless, these examples remain iso-
lated in the vast panorama of retinoid and retinoic-like
derivates.
We have already reported that heterocyclic ionone-like
derivatives, which can be linked to the so-called ‘short
heteroretinoids’, possess antimicrobial activity.15 We
would now like to demonstrate that this class of com-
pounds is endowed with anti-inflammatory and histo-
protective properties potentially useful in controlling
neutrophilic inflammatory responses.16–18
Keywords: Isoxazole; Pyrazole; Pyrimidine ionone-like derivatives;
Antiinflammatory activity; Neutrophil superoxide; Neutrophil
chemotaxis.
Since major pathologies targeted by retinoic acid and
retinoid-like compounds are represented by inflam-
matory dermatoses, such as acne and psoriasis, the
*
Corresponding author. Tel.: +39 0103538368; fax: +39
0103538358; e-mail: balbi@unige.it
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.007

A. Balbi et al. / Bioorg. Med. Chem. 14 (2006) 5152–5160
5153
evaluation of the new compounds potential anti-in-
flammatory activity was tested using purified human
neutrophils. Neutrophils play a crucial role in these
diseases and are indeed recruited in inflamed skin by
locally generated chemoattractants. In turn, extrava-
sated neutrophils undergo activation with a produc-
tion of oxidants and release of enzymes, favouring
both the amplification of the inflammatory reaction
and the development of tissue injury.16,19 In the pres-
ent study, the potential anti-inflammatory and histo-
protective properties of our short heterocyclic
ionone-like derivates were carried out by testing the
ability of these cells to migrate and to undergo activa-
tion of the respiratory burst as measured by determin-
ing superoxide production (O2ꢀ).
2. Chemistry
While studying the Vilsmeier reaction (VR) on a- and b-
ionones 1, we outlined both the formation of the classi-
cal b-chlorovinylaldehydes 2 and the unexpected prod-
ucts 3, which is unprecedented in a Vilsmeier reaction
(Scheme 1).
Somewhat surprisingly, the chlorine of compounds 2
had difficulty in reacting and consequently all attempts
to cyclise those derivatives failed. We thus opted to
examine a different approach, turning our attention to
compound 3.
Scheme 2. Synthesis of heterocyclic ionone-like derivatives.
We have already shown that enamines comparable to
compound 3 exhibit a propensity to amine exchange,
in particular when an electron-withdrawing group is
present in b-position.20 Moreover, cyclisation can occur
and become the only product when a bifunctional re-
agent is used. Compound 3 have shown similar behav-
iour. In fact, the reaction of 3 and a number of
bifunctional reagents allowed for the formation of a
series of heterocycles ionone-like derivatives. We spec-
ulate that this reaction, which involves both the formyl
and the dialkylamino groups, could be considered a
general reaction in which the primary amine of the
bifunctional reagent couples with the carbonyl group
giving the corresponding imine, and secondly, via a
concerted pathway, the other part of the reagent dis-
places the dialkylamino group which acts as an out-
standing leaving group (Scheme 2). Nonetheless,
particular care has to be taken in performing this reac-
tion. In fact, since the reaction is very sensitive both to
time and temperature, each reagent has its own story
(see Section 5).
Since preliminary studies carried out in our laboratory
showed that the derivatives coming from a-ionones were
more easily obtainable and the activities were perfectly
comparable, we focused our attention on them.
Refining and improving our method, we then resynthes-
ised compounds 4, 5a,e,i and 6a,b, bettering the yield,
and prepared new derivatives 5b–d and 5f–h.
3. Biological results
3.1. Effect on neutrophil viability
In order to test the effects of the new compound on the
cell survival, neutrophil viability was evaluated in the
presence of different doses of the compounds by the ethi-
dium bromide–fluorescein diacetate test. As a control,
the cells were exposed to higher dose of vehicle EtOH
alone.
The data (Table 1) represent means of two independent
experiments. Only the compounds 5e is cytotoxic.
3.2. Effect on neutrophil superoxide production
Ten compounds were tested for their effect on neutro-
phil’s superoxide production triggered by 10 ng/mL
TNF-a on fibrinogen-coated wells. Human neutrophils
Scheme 1. Vilsmeier reaction on a- and b-ionones.

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A. Balbi et al. / Bioorg. Med. Chem. 14 (2006) 5152–5160
ꢀ
Table 1. Effect of different doses (100 lM ! 0.1 lM) of heterocyclic
it O2 production in a dose-dependent manner. Table 2
reports the IC50 for each of these compounds.
ionone-like derivatives and EtOH (vehicle) on cell viability
Compound
100 lM
10 lM
1 lM
0.1 lM
3.3. Effect on neutrophil chemotaxis
EtOH
4
98
—
—
—
99
97
95
95
100
—
94
94
94
92
96
—
98
100
94
99
100
—
98
98
92
94
99
100
100
99
Compounds 5a and 5d, which became more effective on
superoxide assay, were studied for their activity on neu-
trophil chemotaxis using fMLP as chemoattractant.
trans-Retinoic acid, which possesses anti-inflammatory
activity in several experimental models,21 was also tested
as reference. Neutrophils were incubated in the upper
compartment of the chemotaxis chamber in the absence
or in the presence of different doses of each compound.
Their locomotory response to 10 nM fMLP in the lower
compartment was tested after 45 min of incubation.
5a
5b
5c
98
—
—
97
5d
5e
—
100
—
<70
95
5f
99
99
5g
5h
6a
6b
95
93
94
94
93
96
95
Results are expressed as percentage of viable cells.
As shown in Figure 2 the chemotactic response of neu-
trophils was inhibited by compounds 5a and 5d in a
dose-dependent manner. Data are expressed as net
migration obtained subtracting spontaneous migration.
(5 · 104) exposed to 10 ng/mL TNF-a were found to
produce an average 3.5 0.5 (mean 1 SD, n = 3) nmol
of O2 as detected by superoxide dismutase-inhibitable
reduction of ferrocytochrome c. Under the same exper-
ꢀ
imental conditions, resting neutrophils generated
ꢀ
Tableꢀ2. IC50 (means 1SD, n = 3) of 5a,d,g and 6a on the production
0.4 0.2 (mean 1 SD, n = 3) nmol of O2
.
of O2 on human neutrophils
Compound 5a
6a
5d
5g
As shown in Figure 1, among the compounds examined
only four compounds (5a,d,g and 6a) were able to inhib-
IC50 (lM) 3.01 2.0 13.3 7.4 2.85 0.01 24.5 0.4
Figure 1. Effect of different doses (abscissa) of heterocyclic ionone-like derivatives on the superoxide anion production (O2ꢀ) in neutrophils adherent
to a biologic substrate (fibrinogen) triggered with TNF-a (10 ng/mL). Resting values are not reported in figure.

A. Balbi et al. / Bioorg. Med. Chem. 14 (2006) 5152–5160
5155
Figure 2. Dose-dependent inhibition of fMLP-induced neutrophil migration by different doses (abscissa) of 5a and 5d compared with trans-retinoic
acid. Results are expressed as net migration (lm).
Figure 3. Effect on neutrophil chemotaxis to fMLP in the presence of derivatives 5a (10 nM) and 5d (10 nM) and trans-retinoic Acid 10 nM. Tests
were carried out using neutrophils from different healthy volunteers. Statistical analysis: one-tailed P value is <0.01 for compound 5d and < 0.001 for
compound 5a. (Mann–Whitney test) Differences were accepted as significant when P < 0.05.
The dose of compounds 5a and 5d able to induce 50%
inhibition (IC50) was 0.46 and 0.9 nM.
drugs now available in clinical practice generally display
inhibitory activities at micromolar concentrations, as far
as neutrophil functional responses are concerned.22,23 In
addition, it also seems of interest that other related com-
pounds such as trans-retinoid acid and the retinoic-like
compound Adapalene are, respectively, inactive or ac-
tive at concentrations three-order higher21 than those
herein found in our heterocyclic ionone-like derivatives.
Therefore, these compounds appear to have features
consistent with the possibility of interfering with the
development of tissue injury during neutrophilic inflam-
matory reactions. For instance, it is known that an aro-
matic retinoid, Etretinate, is both active in skin diseases
such as acne and psoriasis and in inhibiting in vivo neu-
trophil migration.24,25 Similarly, and more importantly,
our compounds might reduce not only the recruitment
of neutrophils in skin during psoriasis or related neutro-
philic dermatoses but might also directly reduce neutro-
phil ability to cause skin damage. Present compounds
are indeed capable of inhibiting neutrophil respiratory
burst, known to be instrumental in the development of
oxidative tissue injury.26 Moreover, it is noteworthy that
this class of compounds also has direct anti-bacterial
activity.15 Taking into account that in certain inflamma-
tory dermatoses, such as acne, the intervention of micro-
organisms is a crucial pathogenetic event, coupled with
the consequent involvement of neutrophils, the present
In contrast, retinoic acid had no inhibitory effect on neu-
trophil chemotaxis in same conditions.
Moreover, the effects of compounds 5a and 5d (10 nM)
on chemotaxis were confirmed by using neutrophils
from different donors. As shown in Figure 3, the chemo-
tactic response of neutrophils versus fMLP was inhibit-
ed by equimolar concentrations (10 nM) of derivatives
5a and 5d, whereas trans-retinoic acid was ineffective.
4. Discussion and conclusion
The present data show that among eleven heterocyclic
ionone-like derivatives tested, four are active as inhibi-
tors of the respiratory burst of human neutrophils with-
out affecting cell viability. Moreover, the two most
active compounds, that is, 5a and 5d, were tested in neu-
trophil migration assays and were found to be potent
inhibitors of neutrophil chemotactic responsiveness as
well. In particular, inhibition of neutrophil chemotaxis
could be detected at very low concentrations (i.e.,
0.1 nM) of the compounds. This appears to be a relevant
finding in as much as nonsteroidal anti-inflammatory

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A. Balbi et al. / Bioorg. Med. Chem. 14 (2006) 5152–5160
findings are in agreement with the concept of retinoic-
like compounds as a new strategy to simultaneously tar-
get cutaneus infection and inflammation with one single
treatment.
for 2) with a diluted solution of sodium hydroxide and
allowed to stand overnight at room temperature. The
resulting water–oil mixture led to an oil which solidified
on standing, furnishing 3a [3-dimethylamino-5-(2,6,6-
trimethyl-2-cyclohexen-1-yl)-2,4-pentadienal] as an
essentially pure pale yellow powder (40% yield); mp
147 ꢁC from n-hexane.27
From a structural–activity point of view it can be pointed
out that the pyrazole ring and its substituents in position-
1 play a fundamental role in determining and increasing
the activity. In fact, the substitution of one nitrogen of
the pyrazole with oxygen, which led to the isoxazole
derivative 4, resulted in a significant loss of activity.
The introduction of a substituent in position-1 led to a
general decrease of activity. Only the 2-chlorophenyl
and the aminotriazole substituents were able to maintain
and even increase the activity. The positive effect of the
amino group seems confirmed by the good activity of
6a which is the only nonpyrazole ring having significant
activity. The high activity of 5d (IC50 2.85 lM), which
sums in his structure the pyrazole and the amino group,
which is in turn linked to the triazole ring, substantiates
the promising characteristics of these groups which
deserve further and more profound studies.
5.3. Synthesis of 4, 5a–i and 6a–b
5.3.1. 5-(2,6,6-Trimethyl-2-cyclohexen-1-yl)ethenyl-isox-
azole (4). To a solution of 3a (0.4 g, 1.6 mmol) in
10 mL of ethanol, 0.5 g (7.0 mmol) of hydroxylamine
hydrochloride dissolved in 3 mL of water and 2 mL
of a 10% water solution of sodium hydroxide was add-
ed portionwise. The mixture was allowed to stand at
room temperature for 3 days. The resulting mixture
was added with 10 mL of water and extracted with
chloroform. The combined chloroform extracts were
washed with brine, dried and evaporated under re-
duced pressure. The residue was purified by chroma-
tography on silica gel (toluene/ethyl acetate 1:1),
giving 4 in 90% yield as a thick oil.
In conclusion, our synthetic method proved to be versa-
tile enough to obtain ionone-like derivatives in which
the heteroaromatic ring and their substituents can affect
both the strength and the character of the biological
activity, eventually addressing the activity towards dif-
ferent targets. In the present case, the two most active
compounds 5a and 5d are good candidates for prototype
drugs in psoriasis and related neutrophilic dermatoses.
IR (film): m 3100, 2900, 1640, 1570, 1449, 1370, 915,
1
870 cmꢀ1; H NMR (200 MHz): d 0.82 (3H, s, CH3),
0.91 (3H, s, CH3), 1.22 (1H, m, H-5), 1.42 (1H, m, H-
5), 1.56 (3H, s, CH3), 2.01 (2H, m, H-4), 2.23 (1H, d,
H-1 J = 9.50 Hz), 5.46 (1H, m, H-3), 5.85 (1H, dd,
ethene, J = 9.50; 15.23 Hz), 6.28 (1H, d, ethene,
J = 15.23 Hz), 6.32 (1H, d, CH), 7.45 (1H, d, CH); 13C
NMR (50 MHz): d 23.3 (CH3), 24.5 (CH2), 27.2
(CH3), 28.4 (CH3), 31.6 (CH2), 32.9 (C), 55.2 (CH),
93.1 (CH), 121.1 (CH), 122.3 (CH), 133.2 (CH), 133.2
(C), 142.4 (CH), 158.01 (C). Anal. Calcd for
C14H19NO: C, 77.38; H, 8.81; N, 6.45; O, 7.36. Found:
C, 76.99; H, 8.90; N, 6.22.
5. Experimental
5.1. General
Melting points were determined with Fisher–Johns
apparatus and are uncorrected. The IR spectra were
recorded in film or in potassium bromide disks on a Per-
kin-Elmer 398 spectrometer. The 1H and 13C NMR
spectra were recorded a Varian Gemini 200 (200 MHz,
1H; 50 MHz, 13C) spectrometers in deuteriochloroform
solutions with tetramethylsilane as the internal standard
(d = 0). The purity of all compounds was checked by
thin-layer chromatography on silica gel 60-F-254 pre-
coated plates and the spots were located in UV light
or by vanillin in sulfuric acid. Elemental analyses were
performed on a Carlo Erba 1106 Elemental Analyser
in the Microanalysis Laboratory in our Department.
5.3.2. 5-(2,6,6-Trimethyl-2-cyclohexen-1-yl)ethenyl-1H-
pyrazole (5a). Hydrazine hydrate (1 mL, 20 mmol) was
added in a single portion to a solution of 3a (0.25 g,
1 mmol) in 10 mL of ethanol and the mixture was stirred
at room temperature for 30 min and was allowed to
stand at ꢀ10 ꢁC for 2 days. After evaporation to dry-
ness, the residue was purified by chromatography on
silica gel (toluene/ethyl acetate 1:1) giving 5a in 89%
yield as thick oil.
IR (film): m 3200, 2915, 1650, 1560, 1500, 1380,
1
1360 cmꢀ1; H NMR (200 MHz): d 0.86 (3H, s, CH3),
0.91 (3H, s, CH3), 1.22 (1H, m, H-5), 1.45 (1H, m, H-
5), 1.62 (3H, s, CH3), 2.05 (2H, m, H-4), 2.28 (1H, d,
H-1, J = 9.80 Hz), 5.48 (1H, m, H-3), 6.05 (1H, dd, eth-
ene, J = 15.68 Hz and J = 9.80 Hz), 6.32 (1H, d, CH),
6.38 (1H, d, ethene, J = 15.68 Hz), 7.51 (1H, d, CH),
14.4 (1H, s, NH); 13C NMR (50 MHz): d 23.5, 23.6,
27.4, 28.3, 32.0, 33.0, 55.3, 102.6, 121.2, 121.9, 134.1,
134.4, 135.1, 146.7. Anal. Calcd for C14H20N2: C,
77.73; H, 9.32; N, 12.95. Found: C, 77.44; H, 9.11; N,
13.02.
5.2. Synthesis of key intermediate (3a)
Phosphorus oxychloride (50.0 mmol, 4.57 mL) was add-
ed dropwise, for 15 min at 0 ꢁC, to 3.87 mL of N,N-di-
methylformamide in a two-necked flask protected from
atmospheric moisture and efficiently stirred with a mag-
netic bar. A solution of a-ionone (25.0 mmol) in 3 mL of
dimethylformamide was added dropwise into the above
Vilsmeier reagent cooled at ꢀ20 ꢁC. The reaction mix-
ture was allowed to stir while the temperature rose to
0 ꢁC in 45 min, then poured onto crushed ice. The
aqueous layer was separated, alkalinized (neutralized
5.3.3. 2-{5-[2-(2,6,6-Trimethyl-2-cyclohexen-1-yl)ethen-
yl]-1H-pyrazol-1-yl}-ethanol (5b). A solution of 2-

A. Balbi et al. / Bioorg. Med. Chem. 14 (2006) 5152–5160
5157
hydrazinoethanol (0.14 mL, 2 mmol) in 1 mL of ethanol
was added in a single portion to a solution of 3a (0.5 g,
2 mmol) in 10 mL of ethanol. The mixture was allowed
to stir at room temperature until the starting product
had almost disappeared (control by TLC, 15 days).
After evaporation to dryness, the residue was chromato-
graphed on silica gel eluting first with toluene and sec-
ondly with toluene/ethyl acetate 1:1. The second eluate
gave 5b in 60% yield as a thick oil.
and crystallized from cyclohexane: yield 65%; mp
138–139 ꢁC.
IR (KBr) m 3300, 2930, 2830, 1650, 1600, 1490, 1450,
1
1360 cmꢀ1; H NMR: d 0.85 (3H, s, CH3), 0.92 (3H,
s, CH3), 1.22 (1H, m, H-5), 1.48 (1H, m, H-5), 1.59
(3H, s, CH3), 2.04 (2H, m, H-4), 2.30 (1H, d, H-1),
5.38 (2H, s, NH2, deuterium oxide exchangeable),
5.48 (1H, m, H-3), 6.20 (1H, dd, ethene, J = 8.60 Hz,
J = 15.05 Hz), 6.45 (1H, d, ethene, J = 15.05 Hz), 6.54
(1H, d, CH), 7.75 (1H, d, CH), 11.5 (1H, br s, SH,
deuterium oxide exchangeable); 13C NMR (50 MHz)
d 23.38 (CH3), 23.51 (CH2), 27.32 (CH3), 28.25
(CH3), 31.89 (C), 33.06 (CH2), 55.32 (CH), 105.00
(CH), 112.35 (C), 117.10 (CH), 122.60 (CH), 133.23
(C), 135.70 (C), 139.64 (CH), 143.88 (CH), 145.14
(C). Anal. Calcd for C16H22N6S: C, 58.15; H, 6.71;
N, 25.43; S, 9.70. Found: C, 58.16; H, 6.84; N, 25.15;
S, 9.45.
1
IR (film) m 3350, 2900, 1630, 1450, 1380, 1360 cmꢀ1; H
NMR: d 0.87 (3H, s, CH3), 0.93 (3H, s, CH3), 1.22 (1H,
m, H-5), 1.48 (1H, m, H-5), 1.61 (3H, s, CH3), 2.03 (2H,
m, H-4), 2.25 (1H, d, H-1, J = 8.98 Hz), 2.60 (1H, s, OH,
deuterium oxide exchangeable), 3.98 (2H, m, CH2), 4.18
(2H, m, CH2), 5.48 (1H, m, H-3), 6.04 (1H, dd, ethene,
J = 8.98 Hz, J = 15.05 Hz), 6.30 (1H, d, CH), 6.35 (1H,
d, ethene, J = 15.05 Hz), 7.33 (1H, d, CH); 13C NMR
(50 MHz) d 23.40 (CH3), 23.47 (CH2), 27.40 (CH3),
28.20 (CH3), 31.95 (CH2), 32.94 (C), 54.06 (CH2),
55.13 (CH), 62.29 (CH2), 102.54 (CH), 121.68 (CH),
122.36 (CH), 133.78 (CH), 134.40 (C), 139.30 (CH),
151.92 (C). Anal. Calcd for C16H24N2O: C, 73.81; H,
9.29; N, 10.76, O, 6,14. Found: C, 74.11; H, 9.51; N,
10.43.
5.3.6. 1-Phenyl-5-(2,6,6-trimethyl-2-cyclohex-1-yl)ethen-
yl-1H-pyrazole (5e). Phenylhydrazine hydrochloride
[0.5 g (3.2 mmol) in 5 mL of water and 0.8 g of sodium
acetate trihydrate] was added to a stirred solution of
3a (0.4 g,1.6 mmol) in 10 mL of ethanol. After 15 min
stirring at reflux, the reaction mixture was allowed to
stand at room temperature for 1 day. The mixture was
poured into ice-water and the resulting oil was extracted
with chloroform. The combined extracts were washed
with brine, dried and concentrated. The red oil was puri-
fied by chromatography on silica gel (toluene/ethyl
acetate 1:1): thick oil; yield 85%.
5.3.4.
N,N-Dimethyl-5-[(2,6,6-trimethyl-2-cyclohex-1-
yl)ethenyl]-1H-pyrazole-1-carbothioamide (5c). A mix-
ture of 3a (0.5 g, 2 mmol) and thiosemicarbazide
(0.21 g, 2,3 mmol) in 10 mL of ethanol was allowed to
stir for 20 days at room temperature (reaction moni-
tored on TLC). After evaporation to dryness, the resi-
due was chromatographed on silica gel eluting first
with toluene and second with toluene/ethyl acetate 1:1.
The crude product obtained from the second eluate
was purified by silica gel chromatography (toluene/ethyl
acetate 1:1): red oil, yield 51%.
IR (film): m 3100, 2920, 1670, 1600, 1500, 1450,
1
1390 cmꢀ1; H NMR (200 MHz): d 0.88 (3H, s, CH3),
0.92 (3H, s, CH3), 1.20 (1H, m, H-5), 1.45 (1H, m, H-
5), 1.60 (3H, s, CH3), 2.03 (2H, m, H-4), 2.25 (1H, d,
H-1, J = 8.98 Hz), 5.45 (1H, m, H-3), 6.07 (1H, dd,
ethene, J = 8.98; 15.76 Hz), 6.20 (1H, d, ethene,
J = 15.76 Hz), 6.47 (1H, d, CH), 7.47 (5H, m, ArH)
7.61 (1H, d, CH); 13C NMR (50 MHz) d 23.44 (CH3),
23.53 (CH2), 27.39 (CH3), 28.23 (CH3), 32.00 (CH2),
33.11 (C), 55.35 (CH), 104.37 (CH), 119.44 (CH),
122.17 (CH), 125.72 (CH), 125.72 (CH), 128.17 (CH),
129.50 (CH), 129.50 (CH), 133.69 (C), 136.62 (CH),
140.17 (C), 140.56 (CH), 141.71 (C). Anal. C20H24N2:
C, 82.15; H, 8.27; N, 9.58. Found: C, 81.86; H, 8.01;
N, 9.82.
IR (film) m 2900, 2830, 1520, 1450, 1390, 1370, 1280,
1
1100 cmꢀ1; H NMR: d 0.86 (3H, s, CH3), 0.92 (3H, s,
CH3), 1.22 (1H, m, H-5), 1.42 (1H, m, H-5), 1.61 (3H,
s, CH3), 2.05 (2H, m, H-4), 2.20 (1H, d, CH), 3.01
(3H, s, CH3), 3.54 (3H, s, CH3), 5.48 (1H, m, H-3),
6.12 (1H, dd, ethene), 6.37 (1H, d, CH), 6.50 (1H, d, eth-
ene), 7.52 (1H, d, CH); 13C NMR (50 MHz) d 23.46
(CH3), 23.54 (CH2), 27.38 (CH3), 28.21 (CH3), 32.00
(CH2), 33.11 (C), 42.96 (CH3), 44.38 (CH3), 55.30
(CH), 104.55 (CH), 118.86 (CH), 122.18 (CH), 133.75
(C), 136.99 (CH), 140.84 (CH), 142.87 (C), 180.37 (C).
Anal. Calcd for C17H25N3S: C, 67.28; H, 8.30; N,
13.85; S, 10.57. Found: C, 67.58; H, 8.22; N, 13.57; S,
10.28.
5.3.7. 1-(4-Bromophenyl)-5-(2,6,6-trimethyl-2-cyclohex-
1-yl)ethenyl-1H-pyrazole (5f). 4-Bromophenylhydrazine
hydrochloride (0.45 g, 2 mmol) in 3 mL of water fol-
lowed by 1 mL of NaOH 10% w/v was added to a
stirring solution of 3a (0.5 g, 2 mmol) in 10 mL etha-
nol. The reaction was monitored with TLC. After
24 h of stirring at room temperature, ice-water was
added to the reaction and extracted with chloroform.
The combined extracts were washed with brine, dried
and evaporated under reduced pressure. The residue
was purified by silica gel chromatography eluting first
with toluene and second with toluene/ethyl acetate
9:1. The second eluate gave 5f as a red oil, yield
68%.
5.3.5. 4-Amino-5-{5-[2-(2,6,6-trimethyl-2-cyclohexen-1-
yl)ethenyl]pyrazol-1-yl}-4H-[1,2,4]triazole-3-thiol (5d).
To a stirring solution of 3a (0.5 g, 2 mmol) in
10 mL of ethylene glycol, 0.29 g (2 mmol) of Purp-
aldꢂ and 2 N NaOH (3 mL) were added portionwise.
The solution changed colour from green to red. After
24 h of stirring at room temperature, the reaction
was treated dropwise with a dilute solution of HCl
until the solution tested was neutral. The resulting
yellow solid was filtered, washed with water, dried

5158
A. Balbi et al. / Bioorg. Med. Chem. 14 (2006) 5152–5160
IR (film): m 3100, 2920, 2860, 1890, 1670, 1600, 1590,
1490, 1380, 1360 cmꢀ1 1H NMR (200 MHz): d 0.87
C20H23N2Cl: C, 73.49; H, 7.09; Cl, 10.85; N, 8.57.
Found: C, 73.40; H, 7.19; Cl, 10.74; N, 8.65.
;
(3H, s, CH3), 0.92 (3H, s, CH3), 1.25 (1H, m, H-5),
1.44 (1H, m, H-5), 1.59 (3H, s, CH3), 2.05 (2H, m, H-
4), 2.20 (1H, d, H-1, J = 8.60 Hz), 5.48 (1H, m, H-3),
6.12 (2H, m, ethene), 6.45 (1H, d, CH), 7.38 (2H, d,
ArH) 7.58 (2H, d, ArH), 7.59 (1H, d, CH); 13C NMR
(50 MHz) d 23.44 (CH3), 23.52 (CH2), 27.36 (CH3),
28.26 (CH3), 31.94 (CH2), 33.14 (C), 55.35 (CH),
104.80 (CH), 119.06 (CH), 121.83 (C), 122.35 (CH),
127.08 (2CH), 132.64 (2CH), 133.50 (C), 137.24 (CH),
139.84 (C), 140.94 (CH), 141.78 (C). Anal. C20H23N2Br:
C, 64.69, H, 6.24; Br, 21.52; N, 7.54. Found: C, 64.82,
H, 6.35; Br, 21.23; N, 7.68.
5.3.10. 1-(2,4-Nitrophenyl)-5-(2,6,6-trimethyl-2-cyclohex-
1-yl)ethenyl-1H-pyrazole (5i). A solution of 0.25 g
(1 mmol) of 3a and 2,4-dinitrophenylhydrazine [0.2 g
(1 mmol) in ethanol/H2SO4] in 10 mL of ethanol was al-
lowed to stand at ꢀ10 ꢁC and 2 h at room temperature.
The precipitated solid was collected by filtration and
washed with ethanol, yielding 5i as already pure yellow
crystals (90% yield, mp 116–117 ꢁC from ethanol).
IR (KBr): m 3100, 2900, 2860, 1610, 1530, 1450, 1390,
1
1350 cmꢀ1; H NMR (200 MHz): d 0.86 (3H, s, CH3),
0.91 (3H, s, CH3), 1.22 (1H, m, H-50), 1.42 (1H, m, H-
5), 1.58 (3H, s, CH3), 2.05 (2H, m, H-4), 2.20 (1H, d,
H-1, J = 8.6 Hz), 5.48 (1H, m, H-3), 6.12 (2H, m, eth-
ene), 6.54 (1H, d, CH), 7.68 (1H, d, CH), [7.75 (1H,
d), 8.56 (1H, dd), 8.85 (1H, d) ArH]; 13C NMR
(50 MHz): d 23.42 (CH3), 23.50 (CH2), 27.30 (CH3),
28.30 (CH3), 31.85 (CH2), 33.20 (C), 55.41 (CH),
105.94 (CH), 116.92 (CH), 121.61 (CH), 122.87 (CH),
127.74 (CH), 130.30 (CH), 133.00 (C), 138.11 (C),
139.84 (CH), 143.10 (C), 143.44 (CH), 146.02 (C),
147.00 (C). Anal. Calcd for C20H22N4O4: C, 62.82; H,
5.80; N, 14.65; O, 16.74. Found: C, 62.80; H, 5.71; N,
14.59.
5.3.8. 1-(2-Chlorophenyl)-5-(2,6,6-trimethyl-2-cyclohex-
1-yl)ethenyl-1H-pyrazole (5g). 2-Chlorophenylhydrazine
hydrochloride (0.36 g, 2 mmol) in 3 mL of water fol-
lowed by 1 mL of NaOH 10% w/v was added to a stir-
ring solution of 3a (0.5 g, 2 mmol) in 10 mL ethanol.
After 28 h of stirring at room temperature, the reaction,
which was monitored by TLC, was poured into ice-
water and extracted with chloroform. The combined ex-
tracts were washed with brine, dried and evaporated un-
der reduced pressure. The oil residue was purified by
silica gel chromatography eluting first with toluene
and second with toluene/ethyl acetate 9:1. The second
eluate gave 5g as a red oil, yield 70%.
5.3.11. 6-(2,6,6-Trimethyl-2-cyclohexen-1-yl)ethenyl-2-
aminopyrimidine (6a). A solution of 0.5 g (2.0 mmol) of
3a and guanidine carbonate [0.36 g (2.0 mmol) in 3 mL
of water] in 30 mL of ethanol was refluxed for 48 h.
The resulting oil was extracted with chloroform. The
combined extracts were dried (sodium sulfate) and evap-
orated to afford an oil which was purified by chromato-
graphy on silica gel (toluene/ethyl acetate 1:1), giving 6a
in 85% yield as a thick oil.
IR (film): m 2900, 2860, 1890, 1670, 1590, 1490, 1380,
1
1360 cmꢀ1; H NMR (200 MHz): d 0.78 (3H, s, CH3),
0.86 (3H, s, CH3), 1.25 (1H, m, H-5), 1.43 (1H, m, H-
5), 1.53 (3H, s, CH3), 2.01 (2H, m, H-4), 2.10 (1H, d,
H-1, J = 8.60 Hz), 5.48 (1H, m, H-3), 5.90 (1H, m, eth-
ene), 6.47 (1H, d, CH), 7.43 (3H, m, ArH), 7.56 (1H, m,
ArH), 7.65 (1H, d, CH,); 13C NMR (50 MHz) d 23.25
(CH3), 23.47 (CH2), 27.35 (CH3), 27.95 (CH3), 32.07
(CH2), 32.90 (C), 55.37 (CH), 103.18 (CH), 118.48
(CH), 122.01 (CH), 127.95 (CH), 130.45 (CH), 130.78
(CH), 131.11 (C), 131.28 (C), 133.71 (CH), 136.72
(CH), 137.10 (C), 140.95 (CH), 141.89 (C). Anal.
C20H23N2Cl: C, 73.49; H, 7.09; Cl, 10.85; N, 8.57.
Found: C, 73.42; H, 7.15; Cl, 10.66; N, 8.70.
IR (film):
m 3300, 3180, 2900, 2860,1680, 1560,
1
1450 cmꢀ1; H NMR (200 MHz): d 0.87 (3H, s, CH3),
0.93 (3H, s, CH3), 1.25 (1H, m, H-5), 1.45 (1H, m, H-
5), 1.59 (3H, s, CH3), 2.04 (2H, m, H-40), 2.30 (1H, d,
H-10 J = 8.98 Hz), 5.17 (2H, s, NH2), 5.48 (1H, m, H-
3), 6.20 (1H, d, ethene, J = 8.60 Hz), 6.58 (1H, d,
ArH), 6.65 (1H, dd, ethene, J = 15.05, 8.60 Hz), 8.19
(1H, d, ArH); 13C NMR (50 MHz,): d 23.4 (CH3),
23.6 (CH2), 27.4 (CH3), 28.4 (CH3), 31.8 (CH2), 33.1
(C), 55.1 (CH), 108.9 (CH), 122.5 (CH), 130.2 (CH),
133.3 (C), 141.6 (CH), 158.6 (CH),163.5 (C), 164.4 (C).
Anal. Calcd for C15H21N3: C, 74.03; H, 8.70; N, 17.27.
Found: C, 73.82; H, 8.61; N, 17.11.
5.3.9. 1-(4-Chlorophenyl)-5-(2,6,6-trimethyl-2-cyclohex-
1-yl)ethenyl-1H-pyrazole (5h). 4-Chlorophenylhydrazine
hydrochloride (0.36 g, 2 mmol) in 3 mL of water fol-
lowed by 1 mL of NaOH 10% w/v was added to a stir-
ring solution of 3a (0.5 g, 2 mmol) in 10 mL ethanol.
Following the above procedure the second eluate gave
5h as a red oil, yield 69%.
IR (film): m 2900, 2860, 1890, 1670, 1590, 1490, 1380,
5.3.12. 2-Methylthio-4-[(2,6,6-trimethyl-2-cyclohexen-1-
yl)ethenyl]-pyrimidine (6b). To the solution of 0.5 g
(2.0 mmol) of 3a in 10 mL of ethanol, 0.55 g (2.0 mmol)
of S-methylisothiourea hydrogen sulfate dissolved in
2 mL of sodium hydroxide 2 N and 10 mL of water
was added and the mixture refluxed for 48 h. The result-
ing oil was extracted with chloroform. The combined ex-
tracts were dried (sodium sulfate) and evaporated to
afford an oil which was purified by chromatography
on silica gel (toluene/ethyl acetate 1:1), giving 6b in 85
% yield as a thick yellow oil.
1
1360 cmꢀ1; H NMR (200 MHz): d 0.88 (3H, s, CH3),
0.92 (3H, s, CH3), 1.25 (1H, m, H-5), 1.43 (1H, m, H-
5), 1.60 (3H, s, CH3), 2.01 (2H, m, H-4), 2.20 (1H, d,
H-1, J = 8.60 Hz), 5.48 (1H, m, H-3), 6.12 (2H, m, eth-
ene), 6.46 (1H, d, CH), 7.43 (4H, m, ArH), 7.59 (1H, d,
CH); 13C NMR (50 MHz) d 23.44 (CH3), 23.52 (CH2),
27.36 (CH3), 28.26 (CH3), 31.94 (CH2), 33.13 (C),
55.35 (CH), 104.74 (CH), 119.07 (CH), 122.34 (CH),
126.80 (2CH), 129.67 (2CH), 133.50 (C), 133.91 (C),
137.19 (CH), 138.20 (C), 140.88 (CH), 141.79 (C). Anal.

A. Balbi et al. / Bioorg. Med. Chem. 14 (2006) 5152–5160
5159
ꢀ
IR (film): m 3100, 2900, 2860,1680, 1560, 1450, 1375,
The amounts of O2 produced by neutrophils were
determined from OD550 of sample without SOD minus
the OD550 of matched samples with SOD, using an
extinction coefficient of 0.0095 · 106 Mꢀ1 calculated
according to Leslie.30
1
1200 cmꢀ1; H NMR (200 MHz): d 0.85 (3H, s, CH3),
0.92 (3H, s, CH3), 1.24 (1H, m, H-5), 1.44 (1H, m, H-
5), 1.59 (3H, s, CH3), 2.04 (2H, m, H-4), 2.27 (1H, d,
H-1, J = 8.98 Hz), 2.29 (3H, s, SCH3), 5.47 (1H, m,
H-3), 6.19 (1H, d, ethene, J = 8.65 Hz), 6.59 (1H, d,
ArH), 6.70 (1H, dd, ethene, J = 15.15, 8.65 Hz), 8.15
(1H, d, ArH); 13C NMR (50 MHz): d 18.95 (CH3S),
23.41 (CH3), 23.56 (CH2), 27.81 (CH3), 28.35 (CH3),
31.78 (CH2), 33.11 (C), 55.18 (CH), 108.82 (CH),
122.53 (CH), 130.17 (CH), 133.27 (C), 141.89 (CH),
158.34 (CH),164.408 (C), 164.55 (C). Anal. Calcd for
C16H22N2S: C, 70.03; H, 8.08; N, 10.21; S, 11.68.
Found: C, 69.09; H, 7.78; N, 10.00; S, 11.29
5.4.4. Neutrophil incubation and chemotaxis assays. Neu-
trophils were incubated in absence and presence of
appropriate doses of each compound or medium alone
for 15 min in air at 37 ꢁC temperature and then, washed
and resuspended in medium to test their chemotactic
activity. Neutrophil chemotaxis was assessed in a 48-
well micro chemotaxis chamber (from Neuro Probe
Inc., Gaithersburg, MD) using: a 3 lm pore size,
150 lm-thick cellulose ester filter.31
5.4. Biological assay
In the assay with cellulose filter, 10 nM N-formyl-L-
methionyl-L-leucyl-L-phenylalanine (fMLP) or control
solution medium was seeded into the lower wells, while
the upper wells were filled with 50 lL of untreated or
short time incubated neutrophil suspension (2· 106/
mL). The whole chamber was incubated at 37 ꢁC in air
with 5% CO2 for 45 min and, at the end, the filters were
removed, fixed in ethanol, stained with Harris haema-
toxylin, dehydrated, cleared with xylene and mounted
in Eukitt (Kindler, Freiburg, Germany). All treatments
were performed in duplicate wells and the distance
(micrometers) travelled by the leading front of cells
was measured at 500· magnification, reading five ran-
domly chosen fields for each filter. Data are expressed
in net migration that was determined by subtracting
neutrophils spontaneous migration to medium from
the distance travelled by neutrophils toward the ligand.
5.4.1. Neutrophils. Heparinized (heparin 10 U/mL) ve-
nous blood was obtained from healthy volunteers. Neu-
trophils were isolated by dextran sedimentation, and
subsequent centrifugation on a Ficoll–Hypaque density
gradient, as described.28
Contaminating erythrocytes were removed by hypotonic
lysis.28 Neutrophils resuspended in incubation medium
were >97% pure, as determined by morphologic analysis
of Giemsa-stained cytopreps.
5.4.2. Neutrophil membrane integrity assay. Neutrophil
viability measured as integrity of membrane was as-
sessed according to Dankberg and Persidsky.29 Briefly,
cells (4· 104/100 lL) were mixed with 50 lL staining
solution (2 lg/mL fluorescein diacetate, 4 lg/mL ethi-
dium bromide in HBSS) and incubated for 10 min at
room temperature. Thereafter, a drop of cell suspension
was placed on a slide, sealed with a coverslip and ana-
lysed under ultraviolet light in a dark field illumination.
Neutrophils with intact membrane (i.e., viable cells) ap-
peared as green fluorescent cells, whereas neutrophils
with damaged and ethidium bromide-permeable mem-
brane (i.e., necrotic cells) displayed a fluorescent red
nucleus.
Acknowledgment
Financial support of this research by the MIUR (pro-
gram 2002) is gratefully acknowledged.
References and notes
1. Camisa, C.; Eisenstat, B.; Ragaz, A.; Weissmann, G. J.
Am. Acad. Dermatol. 1982, 6, 620.
2. Orfanos, C. E.; Zouboulis, C. C.; Almond-Roesler, B.;
Geilen, C. C. Drugs 1997, 53, 358.
5.4.3. Superoxide anion assay. The production of super-
ꢀ
oxide anion O2 by neutrophils was measured as SOD-
inhibitable reduction of cytochrome c, using a micro-
plate reader (Titertek Twinreader Plus, Flow Lab, Ltd,
Irvine, Scotland).26 The assay was carried out in 96-well,
flat-bottomed, polystyrene plates (Primaria plates, Fal-
con, Becton Dickinson, Oxnard, CA, USA). The wells
were pre-treated with Fibrinogen by incubation in 5%
CO2 at 37 ꢁC for 2 h. Immediately after washing, incu-
bation medium containing 15 nmol cytochrome c was
added to each well and the temperature brought to
37 ꢁC in the microplate reader (usually within 15 min).
The appropriate doses of each compound and the stim-
ulus (TNF-a 10 ng/mL) were added followed by cells
(5 · 104 neutrophils). This number of neutrophils per
well was on the linear portion of the dose–response
curve for this system. Experiments were carried out in
triplicate, in presence and absence of SOD. The reduc-
tion of cytochrome c was monitored (3 h) at intervals
(15 min) by reading the plate at 550 nm.
3. Boehm, M. F.; Zhang, L.; Zhi, L.; McClurg, M. R.;
Berger, E.; Wagoner, M.; Mais, D. E.; Suto, C. M.;
Davies, J. A.; Heyman, R. A.; Nadzan, A. M. J. Med.
Chem. 1995, 38, 3146.
4. Kamm, J. J.; Ashenfelter, K. O.; Ehmann, C. W. In Sporn,
M. B., Roberts, A. B., Goodman, D. S., Eds.; The
Retinoids; Academic Press: Orlando, FL, 1984; pp 288–326.
5. Amstrong, R. B.; Ashenfelter, K. O.; Eckhoff, C.; Levin,
A. A.; Shapio, S. S. In The Retinoid 2nd ed.; Sporn, M. B.,
Roberts, A. B., Goodman, D. S., Eds.; Raven Press: New
York, 1994; pp 545–572.
6. Standeven, A. M.; Beart, R. L.; Johnson, A. T.; Boehm,
M. F.; Escobar, M.; Heyman, R. A.; Chandraratna, R. A.
S. Fundam. Appl. Toxicol. 1996, 33, 264.
7. Soprano, D. R.; Soprano, K. J. Annu. Rev. Nutr. 1995, 15,
111.
8. (a) Zusi, F. C.; Reczek, P. R.; Ostrowski, J. U.S. Patent
6,008,251, 1999; . Chem. Abstr. 1998, 132, 35518.

5160
A. Balbi et al. / Bioorg. Med. Chem. 14 (2006) 5152–5160
9. Oh, Gi-Su.; Pae, Hyun-Ock.; Seo, Won-Gil.; Shin, Min-
18. Dallegri, F.; Ottonello, L.; Ballestrero, A.; Bogliolo, F.;
Ferrando, F.; Patrone, F. Gut 1990, 31, 184.
19. Dallegri, F.; Ottonello, L. Inflamm. Res. 1997, 46, 382.
20. Sottofattori, E.; Grandi, T.; Balbi, A. Tetrahedron Lett.
1995, 36, 1331.
21. Hensby, C. N.; Eustache, J.; Shroot, B.; Bouclier, M.;
Chatelus, A.; Luginbuhl, B. Agents Actions 1987, 21, 238.
22. Duberttret, L.; Lebreton, C.; Touraine, R. Br. J. Derma-
tol. 1982, 107, 681.
23. Hensby, C.; Cavey, M.; Bouclier, A.; D’algate, C.;
Eustache, J.; Shroot, B. Agents Actions 1990, 29, 1.
24. Shalita, A. J. Eur. Acad. Dermatol. Venereol. 2001, 15,
43.
25. Bershad, S. V. Mt. Sinai J. Med. 2001, 68, 279.
26. Ottonello, L.; Dapino, P.; Amelotti, M.; Barbera, P.;
Arduino, N.; Bertolotto, M.; Dallegri, F. Inflamm. Res.
1998, 47, 345.
27. Sottofattori, E.; Anzaldi, M.; Balbi, A. J. Heterocycl.
Chem. 1998, 35, 1377.
28. Ottonello, L.; Dapino, P.; Scirocco, M. C.; Balbi, A.;
Bevilacqua, M.; Dallegri, F. Clin. Sci. 1995, 88, 331.
29. Dankberg, F.; Persidsky, M. D. Cryobiology 1976, 13, 430.
30. Leslie, R. G. J. Immunol. Methods 1987, 103, 253.
31. Ottonello, L.; Gnerre, P.; Bertolotto, M.; Mancini, M.;
Dapino, P.; Russo, R.; Garibotto, G.; Barreca, T.;
Dallegri, F. J. Am. Soc. Nephrol. 2004, 15, 2366.
Kyo.; Kim, Il-Kwang.; Chai, Kyu-Yun.; Yun, Young-
Gab.; Kwon, Tae-Oh.; Lim, Joung-Sik.; Chung, Hun-
Taeg. Immunopharmacol. Immunotoxicol. 2001, 23, 335.
10. Datta, P. K.; Lianos, E. A. Kidney Int. 1999, 56, 486.
11. Kim, B. H.; Kang, K. S.; Lee, Y. S. Toxicol. Lett. 2004,
150, 191.
12. Manfredini, S.; Simoni, D.; Caminiti, G.; Vertuani, S.;
Invidiata, F.; Moscato, B.; Hatse, S.; De Clercq, E.;
Balzarini J. Med. Chem. Res. 1998, 8, 291.
13. Garattini, E.; Parrella, E.; Diomede, L.; Gianni, M.;
Kalac, Y.; Merlini, L.; Simoni, D.; Zanier, R.; Ferrara, F.
F.; Chiarucci, I.; Carminati, P.; Terao, M.; Pisano, C.
Blood 2004, 103, 194.
14. Simoni, D.; Grisolia, G.; Giannini, G.; Roberti, M.;
Rondanin, R.; Piccagli, L.; Baruchello, R.; Rossi, M.;
Romagnoli, R.; Invidiata, F. P.; Grimaudo, S.; Jung, M.
K.; Hamel, E.; Gebbia, N.; Crosta, O. L.; Abbadessa, O.
V.; Di Cristina, A.; Dusonchet, L.; Meli, M.; Tolomeo, M.
J. Med. Chem. 2005, 48, 723.
15. Anzaldi, M.; Sottofattori, E.; Rizzetto, R.; Granello Di
Casaleto, B.; Balbi, A. Eur. J. Med. Chem. 1999, 34, 837.
16. Dallegri, F.; Ottonello, L.; Dapino, P.; Sacchetti, C.
J. Rheumatol. 1992, 19, 419.
17. Dallegri, F.; Dapino, P.; Arduino, N.; Bertolotto, M.;
Ottonello, L. Antimicrob. Agents Chemother. 1999, 43, 2307.