Synthesis and in silico biological activity evaluation of new N-substituted pyrazolo-oxazin-2-one systems

Article abstract of DOI:10.1016/j.bmc.2007.12.033

Cyclisation of pyrazolo-β-enaminones 3 readily obtained from 4-aceto acetyl pyrazol 2 with triphosgene led to the formation of N-substituted pyrazolo-1,3-oxazin-2-ones 4 in good yields. Estimation of pharmacotherapeutic potential, possible molecule mechan

Full text of DOI:10.1016/j.bmc.2007.12.033

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3059–3066
Synthesis and in silico biological activity evaluation of new
N-substituted pyrazolo-oxazin-2-one systems
Nora Benaamane,a Bellara Nedjar-Kolli,a,* Yamina Bentarzi,a Lamouri Hammal,a
Athina Geronikaki,b,* Phaedra Eleftherioub and Alexey Laguninc
a
`
Faculty of Chemistry, University of Sciences and Technology ‘‘Houari Boume´diene’’, Algiers, Algeria
bSchool of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
cInstitute of Biomedical Chemistry RAMS, Pogodinskaya Str., 10, Moscow 119832, Russia
Received 4 June 2007; revised 13 December 2007; accepted 18 December 2007
Available online 23 December 2007
Abstract—Cyclisation of pyrazolo-b-enaminones 3 readily obtained from 4-aceto acetyl pyrazol 2 with triphosgene led to the for-
mation of N-substituted pyrazolo-1,3-oxazin-2-ones 4 in good yields. Estimation of pharmacotherapeutic potential, possible mol-
ecule mechanisms of action, toxic/side effects and interaction with drug-metabolizing enzymes were made for synthesised
compounds on the basis of prediction of activity spectra for substances (PASS) prediction results and their analysis by PharmaEx-
pert software. COX inhibition predicted by PASS was confirmed by experimental evaluation.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Our research group has been working for a long time on
the development of new anti-inflammatory and antimi-
crobial agents. New thiazolidinone derivatives with
antimicrobial activities have emerged as a result of this
work.15 Moreover, new thiazole Shiff bases, thiazole
amides and thiazolidinones16–18 have been found with
good anti-inflammatory properties. In addition, we have
successfully attempted to develop new nootropic
agents.19 More recently, our attempts have been focused
on finding new anti-inflammatory compounds with com-
bined activities. As inflammation is often a consequence
of microbial infection, the production of agents with
combined anti-microbial and anti-inflammatory action
would be beneficial for the treatment of many diseases.
Moreover, inflammation is thought to be involved in
Alzheimer’s disease and other degenerative disorders
of CNS. The production of nootropics with combined
anti-inflammatory activity would probably result in
agents with improved properties.
Oxazinones constitute an important class of heterocy-
cles, which has attracted much synthetic interest due
to their wide range of biological activities. Considerable
attention has been focused on these compounds since
the discovery of Efavirenz, a trifluoromethyl-1,3-oxa-
zin-2-one which is a non-nucleoside reverse transcriptase
inhibitor and a selective anti-HIV drug.1 Several ben-
zoxazinones exhibit diverse pharmacological properties,
such as antagonism to progesterone receptor,2 antitu-
mour,3 antiviral,4 antithrombotic,5 antimycobacterial,6
anti-inflammatory,7 antidiabetic and hypolipidaemic8
effects. Further to these applications, they have also
been reported as inhibitors of human leucocyte elastase9
and serotonin reuptake.10 Oxazinones have also been
utilized as useful synthetic precursors for the prepara-
tion of some organic compounds.11–13 On the other
hand, it is well known that pyrazole derivatives are asso-
ciated with various pharmaceutical activities.14
As members of the oxazinones’ family are known to ex-
hibit a variety of properties including anti-inflammatory
and antimicrobial action, we designed and synthesised a
new series of oxazinone derivatives hoping to result in
novel multifunctional molecules. The in silico biological
activity evaluation of the compounds and the in vitro
evaluation of the cyclooxygenase inhibitory action of
Keywords: Enaminones; Triphosgene; Pyrazol; 1,3-Oxazine-2-ones;
SAR; PASS; PharmaExpert; COX.
*
Corresponding authors. Tel.: +30 23 10997616; fax: +30 23
10997612; e-mail: geronik@pharm.auth.gr
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.033

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N. Benaamane et al. / Bioorg. Med. Chem. 16 (2008) 3059–3066
the compounds as a first estimation of their anti-inflam-
matory potential are presented in this paper.
ones bearing a pyrazole side chain in the 6 position,
using as starting materials dehydroacetic acid which al-
lowed access to different pyranobenzodiazepines.33
Novel pharmacological actions can be found for N-
substituted pyrazolo-oxazin-2-ones on the basis of com-
puter-aided drug discovery approach with computer
program prediction of activity spectra for substances
(PASS).20–23 It is based on a robust analysis of struc-
ture–activity relationships21 in a heterogeneous training
set currently including about sixty thousand of biologi-
cally active compounds from different chemical series
with about 4500 types of biological activity.
Scheme 1 outlines our general strategy developed to ob-
tain the pyrazolo-oxazinones 4a–k. This procedure
starts with the reaction of the commercially available
dehydroacetic acid 1 with phenylhydrazine to give in
two steps the 1,3-dicarbonyl compound 2 according to
the literature procedure.34 Exposure of 2 to aliphatic
and aromatic primary amines led to pyrazolo-enami-
nones 3a–k using the method reported in the reference.35
Ring closure of compounds 3a–k with triphosgene in
dichloromethane in the presence of triethylamine pro-
vided the target compounds 4a–k in 70–85% yields (Ta-
ble 1). The reaction mechanism could be rationalized as
shown in Scheme 2.
Since only the structural formula of chemical compound
is necessary to obtain PASS predictions, this approach
can be used at the earliest stage of investigation. There
are many examples of successful use of PASS approach
for finding new pharmacological agents.24–29
All new structures of compounds 4a–k were satisfacto-
1
Thus, PASS application to pyrazolo-oxazin-2-ones was
done in order to identify prospective pharmacological
properties that could be confirmed by experimental
studies. The analysis of PASS prediction results includ-
ing estimation of potential pharmacotherapeutic effects,
mechanisms of action, interaction with the main drug-
metabolizing enzymes and side/toxic effects for new N-
substituted pyrazolo-oxazin-2-ones is presented.
rily confirmed by H and 13C NMR mass spectrometry
and elemental analysis. 13C NMR spectra display in par-
ticular signals at around 158–161 and 164–166 ppm
attributed, respectively, to the quaternary carbon of
junction and carbonyl of pyrazolic nucleus. The main
evidence for the formation of the oxazinone ring is the
appearance of the carbamate carbonyl in the range of
152–156 ppm and the lack of carbonyl signal around
1
182–185 ppm. H NMR spectrum indicates the absence
Consequently, the foregoing aspects have prompted us
to synthesise new heterocyclic derivatives combining
both pyrazol and 1,3-oxazinone in one molecular frame.
of O–H signal which was in agreement with the pro-
posed structures. The physico-chemical data of synthes-
ised compounds are given in Table 1.
A variety of synthetic procedures leading to compounds
with the oxazinone skeleton have been developed,30
including solid-phase synthesis.31
3. Biological activity spectra prediction
The analysis of biological activity spectra prediction for
the synthesised compounds made in this publication is a
good example of in silico study of chemical compounds
before their experimental investigations. Anyone can do
the same analysis using the free available web-site with
the internet version of PASS and PharmaExpert:
http://www.ibmc.msk.ru/PASS/.
Our research group reported earlier the synthesis of series
of benzimidazoles, benzimidazolones and 1,5-benzodi-
azepines based on the use of enaminones resulting from
2-pyrone.32 In continuation on these studies, we were
interested in pyrazolo-b-enaminones 3 as precursors for
the preparation of original pyrazolo-oxazinones.
A biological activity spectrum for a substance is a list of
biological activity types for which the probability to be
revealed (Pa) and the probability not to be revealed
(Pi) are calculated. Pa and Pi values are independent
and their values vary from 0 to 1. Biological activity
spectra were predicted for all 11 synthesised structures
with PASS 2005 version.20 The result of prediction is
valuable at planning of the experiment, but one should
2. Results and discussion
2.1. Synthesis
As an extension of our cyclisation strategy towards het-
erocyclic compounds, involving enaminone intermedi-
ates, we report in this work the synthesis of oxazin-2-
R
O
O
O
OH
O
O
CH3
NH
O
CH3
R
N
O
CH3
H3C
CH3
H3C
1) Ph-NH-NH2
2) CH3COOH
O) CO
(C
Cl
N
N
R-NH2
2
3
N
N
H3C
CH2Cl
2 / Et3N
N
N
H3C
O
HO
HO
Ph
Ph
2
3
4
1
O
Ph
g. R= 4-CH3C6H4
j. R= 4-ClC6H4
K. R= 4-BrC6H4
a. R= C2H5
d. R= 2-thienyl
e. R= C6H5-CH2
f. R= C6H5
h. R= 4-CH3OC6H4
b. R= n-C4H9
c. R= isobutyl
i. R= 4-CH3O2CC6H4
Scheme 1. Synthesis of N-substituted 1,3-oxazin-2-one 4a–k.

N. Benaamane et al. / Bioorg. Med. Chem. 16 (2008) 3059–3066
3061
Table 1. Physicochemical data of N-substituted 1,3-oxazin-2-ones 4a–
k compounds
lue is chosen as a threshold, therefore all compounds
with Pa > Pi are suggested to be active. Another crite-
rion for selection is the compounds’ novelty. If Pa value
is high, sometimes one may find close analogues of
known biologically active substances among the tested
compounds. For example, if Pa > 0.7 the chance to find
the activity in experiment is high, but in some cases the
compound may occur to be the close analogue of known
pharmaceutical agents. If 0.5 < Pa < 0.7 the chance to
find the activity in experiment is less, but the compound
is not so similar to known pharmaceutical agents. If
Pa < 0.5 the chance to find the activity in experiment
is even more less, but if it will be confirmed the com-
pound might occur to be a New Chemical Entity.
Compound
R
Yields (%)
Mp (°C)
4a
4b
4c
CH3CH2–
CH3CH2CH2–
(CH3)2CHCH2
78
76
74
213
217
210
S
4d
4e
4f
71
72
82
85
84
70
72
72
236
224
228
220
214
216
234
230
H2C
H2C
PharmaExpert36 was used for statistical and ‘activity–
activity’ relationship analysis of PASS prediction results
for the set of studied compounds. We calculated a num-
ber of activities predicted with different Pa values for the
studied compounds (Fig. 1).
CH3
4g
4h
4i
H3CO
H3COOC
Cl
The figure shows that synthesised compounds may re-
veal greater number of biological activities (327 at
Pa > 0.3; 98 at Pa > 0.5; 30 at Pa > 0.7). Keeping in
mind how PASS results should be interpreted, we se-
4j
350
327
Br
4k
300
250
200
150
take into account some additional factors: particular
interest to some kinds of activity, desirable novelty of
a substance, available facilities for experimental testing,
etc. Actually, each choice is always the compromise be-
tween the desirable novelty of studied substance and risk
to obtain the negative result in testing. The more is Pa
value, the less is the probability of false positives in
the set of compounds selected for biological testing.
For example, if one selects for testing only compounds
for which a particular activity is predicted with
Pa P 0.9, the expected probability to find inactive com-
pounds in the selected set is very low, but about 90% of
active compounds are missed. If only compounds with
Pa P 0.8 are chosen, the probability to find inactive
compounds is also low, but about 80% of active com-
pounds are missed etc. By default, in PASS Pa = Pi va-
98
100
50
0
30
Pa>0.3
Pa>0.5
Pa>0.7
Figure 1. PASS prediction results for the studied compounds.
Cl
Cl
Cl
O
O
Cl CO
Cl
3
O
R
OH
O
O
O
O
O
R
Cl
R
Cl
N
N
OH
O
CH
N
N
CH
N
O
3
3
CH
N
(Cl3CO)2CO
3
1/ NH NHPh
CH
2
3
H
C
H
C
3
3
H C
3
2/ CH3COOH
H C
H
3
N
N
N
O
3
O
1
Ph
Ph
H3C
Ph
NEt3
H
C
3
O
N
O
N
N
O
N
O
O
Cl
Cl
+
N
Et3NHCl + COCl2
N
Ph
R
Ph
Cl
R
O
O
H3C
H
C
3
4
Scheme 2. Mechanism of synthesis.

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N. Benaamane et al. / Bioorg. Med. Chem. 16 (2008) 3059–3066
Table 3. Pa values for predicted biological activities
lected predicted activities for studied compounds with
Pa value more than 0.5. The result of selection obtained
with computer program PharmaExpert is given in Table
2. Here, Rank reflects the position of predicted biologi-
cal activity in the activity list arranged in descending or-
der of biological activity occurrence for the analysed set
of compounds; N is the number of compounds for which
a particular biological activity is predicted.
Compound
Focal predicted activity
4a
4b
4c
4d
4e
4f
(À)-(4S)-Limonene synthase inhibitor (0.965)
Nootropic (0.865)
Nootropic (0.780)
Amyotrophic lateral sclerosis treatment (0.799)
Nootropic (0.849)
Analgesic, non-opioid (0.843)
Analgesic, non-opioid (0.829)
Analgesic, non-opioid (0.804)
Analgesic, non-opioid (0.771)
Analgesic, non-opioid (0.828)
Prolyl aminopeptidase inhibitor (0.879)
4g
4h
4i
As one may see from Table 2, the most frequently pre-
dicted types of biological activity are: amyotrophic lat-
eral sclerosis treatment, analgesic, analgesic non-
opioid, antiepileptic, anti-inflammatory, anxiolytic, cog-
nition disorders treatment, corticotropin releasing factor
antagonist, CYP2A1 substrate, CYP2A2 substrate, neu-
roprotector, nootropic, psychotropic, transplant rejec-
tion treatment, anticonvulsant, antipyretic, etc. All
types of activity that are predicted for more than 2/3
of all compounds from the set can be considered as ‘typ-
ical’, all others—as ‘minor’.37 Since the purpose of this
study was finding of new anti-inflammatory compounds,
as well as COX inhibitors, one may conclude that anti-
inflammatory (fifth rank) activity is typical. Such activ-
ities as antiasthmatic (rank 24) and others with higher
rank are minor ones.
4j
4k
effects with Pa > 0.5 (Table 4). It may help to plan an
experiment to find mechanism of predicted effects.
For example, ‘amyotrophic lateral sclerosis treatment’
activity was predicted with high probability for all com-
pounds. At the same time the mechanism of amyotro-
phic lateral sclerosis treatment was predicted only for
several compounds. Compounds 4a–c were predicted
as nerve growth factor agonists and compounds 4a–c,
4e–g, 4i, 4j were predicted as neurotrophic factors.
Analgestic and anti-inflammatory activity were pre-
dicted for all compounds with Pa > 0.5. Besides, all
compounds were predicted as cyclooxygenase-1 inhibi-
tors with Pa > 0.3. Five of them were predicted to be
cyclooxygenase-1 inhibitors with Pa > 0.5 (4f–h, 4j, 4k).
We also selected the types of activity that were predicted
for a particular compound with the highest probability
(focal activities). They are presented in Table 3.
Using PharmaExpert we analysed probable mechanisms
of action for predicted ‘typical’ pharmacotherapeutical
We also predicted the toxic effects of the compounds.
Only compound 4a has high probability to have many
toxic/side effects with Pa > 0.5 (convulsant, eye/skin irri-
tation, teratogenic and carcinogenic effect, acute toxic-
ity, cardiotoxicity, hematotoxicity, embryotoxicity,
arrhythmogenic effect, torsades de pointes). No other
Table 2. Analysis of biological activity predictions (Pa > 0.5) with
Pharma Expert
Rank
N
Type of activity
1
2
11
11
11
11
11
11
11
11
11
11
11
11
11
11
10
9
Amyotrophic lateral sclerosis treatment
Analgesic
3
Analgesic, non-opioid
Antiepileptic
4
Table 4. ‘Mechanism-effect’ analysis of the prediction results with
Pa > 0.5 for synthesised compounds
5
Anti-inflammatory
Anxiolytic
6
Effect
Mechanism of action
7
Cognition disorders treatment
Corticotropin releasing factor antagonist
CYP2A1 substrate
CYP2A2 substrate
Neuroprotector
8
Amyotrophic lateral Nerve growth factor agonist: 4a–ca
9
sclerosis treatment
Nootropic
Anti-inflammatory
Neurotrophic factor: 4a–c, 4e–g, 4i, 4j
Nerve growth factor agonist: 4a–c
Cyclooxygenase 1 inhibitor: 4f–h, 4j, 4k
Cyclooxygenase inhibitor: 4f–h, 4j
Prostaglandin antagonist: 4j
Tumour necrosis factor alpha antagonist:
4a–c, 4e–j
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
. . .
70
. . .
98
Nootropic
Psychotropic
Transplant rejection treatment
Anticonvulsant
Antipyretic
Analgesic
Cyclooxygenase 1 inhibitor: 4f–h, 4j, 4k
Cyclooxygenase inhibitor: 4f–h, 4j
Prostaglandin antagonist: 4j
Prostaglandin E1 antagonist: 4d
Tumour necrosis factor alpha antagonist:
4a–c, 4e–j
9
Mediator release inhibitor
Tumour necrosis factor alpha antagonist
Antithrombocytopenic
Neurotrophic factor
Antiischaemic
9
8
8
6
5
Cyclooxygenase 1 inhibitor
Cyclooxygenase inhibitor
Antiasthmatic
Neuroprotector
Nerve growth factor agonist: 4a–c
Nerve growth factor agonist: 4a–c
Neurotrophic factor: 4a–c, 4e–g, 4i, 4j
Nootropic: 4a–k
4
3
. . .
1
. . .
Immunomodulator
. . .
Urologic disorders treatment
Anxiolytic
Corticotropin releasing factor antagonist:
4a–k
. . .
1
a ID of compounds.

N. Benaamane et al. / Bioorg. Med. Chem. 16 (2008) 3059–3066
3063
compound was predicted to have toxic/side effects at
Pa > 0.5. It means that toxic/side effects of compound
4a should be scrutinized in further investigation.
Compound 4i was not tested. Thus, PASS program
in collaboration with PharmaExpert software success-
fully predicted COX-1 inhibition for the studied
compounds.
The PASS prediction results allow us to study the possi-
ble interactions with drug-metabolizing enzymes. It ap-
peared that all synthesised compounds may be
CYP2A1 and CYP2A2 substrates with high probability.
‘CYP3A1 substrate’ activity was predicted for com-
pounds 4a–c. Compound 4h was predicted to be
CYP2C9 inhibitor.
In conclusion, we have developed a useful and efficient
procedure for the preparation of new N-substituted pyr-
azolo-1,3-oxazin-2-ones as potent pharmaceutical
agents. Furthermore, we have been able to use for the
first time suitable pyrazolo-enaminones in cycloconden-
sation reaction with triphosgene. Studies directed to-
wards the synthesis of thiazinones using this method
are currently being pursued.
Thus, our study showed that prediction of biological
activity spectra for synthesised compounds by PASS
and its analysis made by PharmaExpert may estimate
pharmacotherapeutic potential, possible molecular
mechanisms of action, toxic/side effects and interaction
with drug-metabolizing enzymes. These results may be
used in further experimental studies.
5. Experimental
All NMR spectra were recorded on a Brucker AC 2 300
spectrometer at 300 MHz (1H) or 75 MHz (13C), chem-
ical shifts are expressed in d (ppm) relative to TMS as
internal standard. Mass data were acquired on a Ner-
mag R10-10C mass spectrometer. Melting points were
determined on a Buchi 512 Apparatus and was uncor-
rected. Microanalysis was performed at the INP-ENSI-
ACET in Toulouse (France).
4. Evaluation of cyclooxygenase inhibitory activity
As half of the compounds were predicted to inhibit
cyclooxygenase-1 (COX-1) isoenzyme (Pa > 0.5), thus
exhibiting anti-inflammatory and analgestic activity,
we tested the inhibitory action of eight of the synthesised
compounds (Table 5). Seven of the tested compounds
exhibited COX-1 inhibitory activity when they were
added at the reaction mixture at a concentration of
100 lM. Compound 4a had no inhibitory effect when
it was added at this concentration. However, it exhibited
low inhibition (19.9%) at a final concentration of
200 lM. Compared to naproxen (inhibition: 39.3%),
two of the tested compounds, 4f and 4g, showed higher
inhibitory activity at the same experimental conditions.
As gathered by the results, phenyl- (4f) and p-CH3-phe-
nyl groups are the substituents that mostly favor inhib-
itory action (inhibition: 61.8%, IC50 = 79.4 lM and
54.6%, IC50 = 91.2 lM, respectively). Ethyl- (4a) and
n-propyl- (4b) substitution diminished inhibitory activity
to 0% and 16%, respectively. p-OCH3 (4h), p-Cl (4j) and
p-Br (4k) substitution of the phenyl-ring lowered inhibi-
tion to 22.6%, 21.9% and 15%, as well. Phenyl-ring con-
nection via a methylen-bridge (4e) also does not favor
inhibition (inhibition: 18%). The four compounds exhib-
iting the best inhibitory action were among the five com-
pounds predicted to be COX-1 inhibitors with Pa > 0.5.
5.1. General procedure for the preparation of compounds
4
To a cooled (0 °C) solution of the appropriate enami-
none 3 (5 mmol) and triethylamine (10 mmol) in 40 ml
of dichloromethane, triphosgene (1.66 mmol) was slowly
added dropwise under magnetic stirring. After being
gradually warmed to room temperature, the resulting
mixture was stirred for 48 h. Water was added, followed
by extraction with dichloromethane. The organic layer
was dried over sodium sulfate and evaporated to dryness
to give the desired compounds 4a–k as a white solid.
5.1.1. 3-Ethyl-4-methyl-6-(3-methyl-5-oxo-1-phenyl-1,5-
dihydro-1H-4-pyrazolyliden)-3,6-dihydro-2H-1,3-oxazin-
2-one (4a). Yield: 78%; mp 213 °C; 1H NMR (300 MHz,
CDCl3 + CF3CO2H) d (ppm): 1.41 (t, 3H, J = 7.3 Hz),
2.57 (s, 3H), 2.65 (s, 3H), 3.56 (q, 2H), 5.30 (s, 1H),
7.28–7.97 (m, 5H); 13C NMR (75 MHz, CDCl3 +
CF3CO2H) d (ppm): 14.0, 14.4, 18.3, 39.2, 96.5, 100.1,
120.8, 126.8, 129.3, 137.2, 145.1, 147.3, 154.0, 158.5,
165.0. MS (70 eV) m/z 311 (M+, 26), 294 (2), 283 (1),
138 (20). Anal. Calcd for C17H17N3O3: C, 71.30; H,
5.46; N, 10.85. Found: C, 71.20; H, 5.39; N, 10.79.
Table 5. Experimental data and prediction results for the studied
compounds
5.1.2. 4-Methyl-6-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-
1H-4-pyrazolyliden)-3-propyl-3,6-dihydro-2H-1,3-oxa-
zin-2-one (4b). Yield: 76%; mp 217 °C; 1H NMR
Compound
COX-1a inhibition %
IC50 (lM)
Pa
4a
4b
0.0
16.0
18.0
61.8
54.6
22.6
21.9
15.0
39.3
—
—
—
—
(300 MHz, CDCl3 + CF3CO2H)
d
1.08 (t, 3H,
4e
—
—
J = 7.3 Hz), 1.79 (sex, 2H, J = 7.3 Hz), 2.57 (s, 3H),
2.64 (s, 3H), 3.47 (m, 2H), 5.31 (s, 1H), 7.28–7.87 (m,
5H); 13C NMR (75 MHz, CDCl3 + CF3CO2H) d 11.5,
14.0, 18.4, 22.5, 46.1, 97.6, 100.5, 120.9, 126.8, 129.3,
137.2, 145.1, 147.3, 155.0, 159.0, 165.2. MS (70 eV) m/z
325 (M+, 17), 310 (2), 283 (4), 152 (22). Anal. Calcd
for C18H19N3O3: C, 71.80; H, 5.77; N, 10.47. Found:
C, 71.77; H, 5.70; N, 10.40.
4f
4g
79.4
91.2
269
251
—
0.546
0.541
0.586
0.563
0.550
0.852
4h
4j
4k
Naproxen
a Values are means of three determinations and deviation from the
mean is <10% of the mean value.

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N. Benaamane et al. / Bioorg. Med. Chem. 16 (2008) 3059–3066
5.1.3. 3-Isobutyl-4-methyl-6-(3-methyl-5-oxo-1-phenyl-
1,5-dihydro-1H-4-pyrazolyliden)-3,6-dihydro-2H-1,3-
5.1.8. 3-(4-Methoxyphenyl)-4-methyl-6-(3-methyl-5-oxo-
1-phenyl-1,5-dihydro-1H-4-pyrazolyliden)-3,6-dihydro-
1
1
oxazin-2-one (4c). Yield: 74%; mp 210 °C; H NMR
2H-1,3-oxazin-2-one (4h). Yield: 84%; mp 214 °C; H
(300 MHz, CDCl3 + CF3CO2H)
d
1.08 (d, 6H,
NMR (300 MHz, CDCl3 + CF3CO2H) d 1.96 (s, 3H),
2.50 (s, 3H), 3.87 (s, 3H), 5.31 (s, 1H), 6.95–7.98 (m,
J = 6.9 Hz), 2.04 (sex, 1H, J = 6.9 Hz), 2.56 (s, 3H),
2.64 (s, 3H), 3.33 (d, 2H, J = 6.9 Hz), 5.32 (s, 1H),
7.28–7.87 (m, 5H); 13C NMR (75 MHz, CDCl3 +
CF3CO2H) d 14.0, 18.5, 20.2, 28.6, 51.9, 97.2, 100.0,
120.8, 126.8, 129.3, 137.2, 145.2, 147.4, 155.5, 159.5,
165.0. MS (70 eV) m/z 339 (M+, 31), 324 (7), 283
(35), 166 (6). Anal. Calcd for C19H21N3O3: C,
72.27; H, 6.06; N, 10.10. Found: C, 72.20; H, 6.00; N,
10.01.
9H); 13C NMR (75 MHz, CDCl3 + CF3CO2H)
d
17.20, 21.20, 55.6, 97.7, 102.7, 115.3, 118.9, 124.4,
127.4, 138.7, 128.9.7, 138.8, 146.2, 147.5, 154.0 160.1,
160.5, 164.7. MS (70 eV) m/z 389 (M+, 63), 313 (1),
344 (6), 200 (10). Anal. Calcd for C22H19N3O4: C,
67.86; H, 4.92; N, 10.79. Found: C, 67.76; H, 4.80; N,
10.71.
5.1.9. 4-[4-Methyl-6-(3-methyl-5-oxo-1-phenyl-1,5-dihy-
dro-1H-4-pyrazolyliden)-2-oxo-3,6-dihydro-2H-1,3-oxa-
5.1.4. 4-Methyl-6-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-
1H-4-pyrazolyliden)-3-(2-thienylmethyl)-3,6-dihydro-2H-
1
zin-3-yl]phenylacetate (4i). Yield: 70%; mp 216 °C; H
1
1,3-oxazin-2-one (4d). Yield: 70%; mp 236 °C; H NMR
NMR (300 MHz, CDCl3 + CF3CO2H) d 1.91 (s, 3H),
2.50 (s, 3H), 3.98 (s, 3H), 5.30 (s, 1H), 7.14–8.24 (m,
(300 MHz, CDCl3 + CF3CO2H) d 2.59 (s, 3H), 2.70 (s,
3H), 5.01–5.08 (m, 2H), 5.32 (s, 1H), 6.93–7.54 (m,
9H); 13C NMR (75 MHz, CDCl3 + CF3CO2H)
d
8H); 13C NMR (75 MHz, CDCl3 + CF3CO2H)
d
17.20, 21.00, 52.60, 97.0, 103.2, 118.9, 124.5, 128.2,
128.7, 131.4, 131.9, 138.7, 139.2, 145.5, 147.4, 152.3
159.8, 164.5, 165.6. MS (70 eV) m/z 417 (M+, 6), 372
(2), 314 (1), 200 (2). Anal. Calcd for C23H19N3O5: C,
66.18; H, 4.59; N, 10.07. Found: C, 66.12; H, 4.49; N,
10.01.
13.74, 20.1, 44.8, 98.0, 104.0, 121.9, 127.4, 127.7,
129.9, 130.3, 138.9, 140.8, 145.4, 148.5, 156.0, 158.5,
166.0. MS (70 eV) m/z 379 (M+, 2), 335 (1), 268
(1), 241 (2). Anal. Calcd for C20H17N3O3S: C, 63.31;
H, 4.52; N, 11.07. Found: C, 63.27; H, 4.57; N,
11.01.
5.1.10. 3-(4-Chlorophenyl)-4-methyl-6-(3-methyl-5-oxo-
1-phenyl-1,5-dihydro-1H-4-pyrazolyliden)-3-phenyl-3,6-
dihydro-2H-1,3-oxazin-2-one (4j). Yield: 72%; mp
234 °C; 1H NMR (300 MHz, CDCl3 + CF3CO2H) d
2.23 (s, 3H), 2.73 (s, 3H), 5.31 (s, 1H), 7.14–7.86 (m,
9H); 13C NMR (75 MHz, CDCl3 + CF3CO2H) d
15.10, 22.3, 97.3, 100.04, 120.3, 122.1 123.6, 126.7,
128.5, 129.9, 132.1, 141.5, 146.2, 149.0, 156.0, 161.0,
165.0. MS (70 eV) m/z 393 (M+, 48), 348 (14), 314
(5), 199 (18). Anal. Calcd for C21H16N3O3Cl: C,
64.05; H, 4.09; N, 10.67. Found: C, 64.03; H, 4.12;
N, 10.70.
5.1.5. 3-Benzyl-4-methyl-6-(3-methyl-5-oxo-1-phenyl-1,5-
dihydro-1H-4-pyrazolyliden)-3,6-dihydro-2H-1,3-oxazin-
2-one (4e). Yield: 72%; mp 224 °C; 1H NMR (300 MHz,
CDCl3 + CF3CO2H) d 2.61 (s, 3H), 2.71 (s, 3H), 4.85–
4.93 (m, 2H), 5.31 (s, 1H), 7.27–7.73 (m, 10H); 13C
NMR (75 MHz, CDCl3 + CF3CO2H) d 13.80, 20.1,
44.7, 97.5.0, 105.0, 122.4, 128.3, 129.1, 129.2, 129.5,
130.0, 134.6, 144.2, 145.6, 149.3, 156.0, 158.4, 166.5.
MS (70 eV) m/z 373 (M+, 3), 329 (1), 267 (1), 241 (2).
Anal. Calcd for C22H19N3O3: C, 70.67; H, 5.13; N,
11.25. Found: C, 70.72; H, 5.09; N, 11.21.
5.1.6. 4-Methyl-6-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-
1H-4-pyrazolyliden)-3-phenyl-3,6-dihydro-2H-1,3-oxa-
zin-2-one (4f). Yield: 80%; mp 228–229 °C; H NMR
(300 MHz, CDCl3 + CF3CO2H) d 2.05 (s, 3H), 2.56 (s,
3H), 5.31 (s, 1H), 7.23–7.77 (m, 10H); 13C NMR
(75 MHz, CDCl3 + CF3CO2H) d 16.70, 21.4, 98.1,
101.7, 121.5, 126.5 128.0, 129.0, 129.5, 129.8, 133.0,
137.9, 145.4, 149.1, 154.5, 159.2, 164.0. MS (70 eV) m/
z 359 (M+, 100), 314 (25), 313 (12), 200 (1). Anal. Calcd
for C21H17N3O3: C, 70.18; H, 4.77; N, 11.69. Found: C,
70.10; H, 4.72; N, 11.64.
5.1.11. 3-(4-Bromophenyl)-4-methyl-6-(3-methyl-5-oxo-1-
phenyl-1,5-dihydro-1H-4-pyrazolyliden)-3-phenyl-3,6-dihy-
dro-2H-1,3-oxazin-2-one (4k). Yield: 72%; mp 230 °C; 1H
NMR (300 MHz, CDCl3 + CF3CO2H) d 2.28 (s, 3H),
2.74 (s, 3H), 5.31 (s, 1H), 7.09–7.67 (m, 9H); 13C
NMR (75 MHz, CDCl3 + CF3CO2H) d 15.40, 22.9,
97.2, 100.1, 122.4, 123.2, 124.7, 126.4, 128.8, 129.8,
133.5, 135.3, 146.2, 149.0, 155.7, 161.2, 166.0. MS
(70 eV) m/z 439 (M+, 26), 394 (5), 314 (7), 198 (25).
Anal. Calcd for C21H16N3O3Br: C, 57.55; H, 3.68; N,
9.59. Found: C, 57.49; H, 3.59; N, 9.54.
1
5.1.7. 4-Methyl-6-(3-methyl-5-oxo-1-phenyl-1,5-dihydro-
1H-4-pyrazolyliden)-3-(4-methylphenyl)-3,6-dihydro-2H-
1,3-oxazin-2-one (4g). Yield: 85%; mp 220 °C; 1H
NMR (300 MHz, CDCl3 + CF3CO2H) d 2.09 (s, 3H),
2.54 (s, 3H), 2.61 (s, 3H), 5.39 (s, 1H), 7.14–7.99
(m, 9H); 13C NMR (75 MHz, CDCl3 + CF3CO2H) d
17.00, 21.12, 21.20, 97.8, 102.7, 120.1, 125.4, 127.4,
128.9, 130.3, 132.7, 138.0, 140.7, 145.8, 148.2, 155.6
158.2, 164.6. MS (70 eV) m/z 373 (M+, 58), 328 (14),
314 (2), 200 (15). Anal. Calcd for C22H19N3O3: C,
70.67; H, 5.13; N, 11.25. Found: C, 70.69; H, 5.09;
N, 11.19.
5.2. Biological activity spectra prediction
Prediction of activity spectra for substances (PASS) is
software for prediction of more than 2800 types of bio-
logical activity (PASS 2005 version) including pharma-
cotherapeutical effects, mechanisms of actions,
interactions with drug-metabolizing enzymes, side and
toxic effects on the basis of structural formula of chem-
ical compounds. Average accuracy of prediction in
leave-one-out cross-validation procedure (for ꢀ68,000
compounds and ꢀ2800 kinds of biological activity from
the PASS training set) was about 93%. PharmaExpert is

N. Benaamane et al. / Bioorg. Med. Chem. 16 (2008) 3059–3066
3065
Stemp, G.; Thewlis, K. M.; Thomas, D. R.; Thompson,
M.; Vong, A. K. K.; Watson, J. M. Bioorg. Med. Chem.
Lett. 2005, 15, 737.
software for analysis of PASS prediction results on the
basis of activity–activity relationships. Details of the
method used for biological activity prediction are pre-
sented at the web-site17 and described in the publica-
tions.21–24,28
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activity of the compounds was measured using ovine
COX-1 isoenzyme included in the ‘COX Inhibitor
Screening Assay’ kit provided by Cayman (Cayman
Chemical Co., Ann Arbor, MI, USA). The assay di-
rectly measures PGF2a produced by SnCl2 reduction of
COX-derived PGH2. The prostanoid production was
quantified via enzyme immunoassay using a broadly
specific antibody that binds to all the major prostaglan-
din compounds.38 Arachidonic acid was added at the
reaction mixture at a final concentration of 0.1 lM.
The compounds were added at a final concentration of
100 lM unless otherwise mentioned. IC50 values were
calculated for the most active compounds. Naproxen
used as a positive control was added to the reaction mix-
ture at the same concentration, 100 lM, as the tested
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