Design, synthesis and biological evaluation of new tricyclic spiroisoxazoline derivatives as selective COX-2 inhibitors and study of their COX-2 binding modes via docking studies

Article abstract of DOI:10.1007/s00044-016-1534-x

A new series of 3′-(4-substitutedphenyl)-4′-(4-(methylsulfonyl)phenyl) spiroisoxazoline derivatives containing naphthalenone and chromanonespiro-bridge were synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. A synthetic reaction based on the 1,3-dipolar cycloaddition mechanism was used for the regiospecific formation of various spiroisoxazolines. One of the analogs, i.e., compound 7h, as the representative of the series was recrystallized and characterized structurally by single-crystal X-ray diffraction method. Moreover, the 3D structures of the synthesized compounds were docked into the COX-2 binding site to determine their most probable binding modes once the drug-receptor complexes are formed.

Full text of DOI:10.1007/s00044-016-1534-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1534-x
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of new tricyclic
spiroisoxazoline derivatives as selective COX-2 inhibitors
and study of their COX-2 binding modes via docking studies
Hoda Abolhasani1,2 Siavoush Dastmalchi2 Maryam Hamzeh-Mivehroud2
•
•
•
Bahram Daraei3 Afshin Zarghi4
•
Received: 29 October 2015 / Accepted: 10 February 2016
Ó Springer Science+Business Media New York 2016
Abstract A new series of 30-(4-substitutedphenyl)-40-(4-
(methylsulfonyl)phenyl) spiroisoxazoline derivatives con-
taining naphthalenone and chromanonespiro-bridge were
synthesized for evaluation as selective cyclooxygenase-2
(COX-2) inhibitors. A synthetic reaction based on the 1,3-
dipolar cycloaddition mechanism was used for the
regiospecific formation of various spiroisoxazolines. One
of the analogs, i.e., compound 7h, as the representative of
the series was recrystallized and characterized structurally
by single-crystal X-ray diffraction method. Moreover, the
3D structures of the synthesized compounds were docked
into the COX-2 binding site to determine their most
probable binding modes once the drug-receptor complexes
are formed.
Introduction
Cyclooxygenase (COX) (prostaglandin synthase), the key
enzyme of inflammatory process, exists at least in two
isoforms (COX-1 and COX-2). COX-2 is induced in
response to proinflammatory conditions, while COX-1 is
constitutive and responsible for the maintenance of physi-
ological homeostasis. This finding led to the theory that
inhibition of COX-1 causes the side effects of non-selec-
tive COX inhibitors such as NSAIDs like gastric ulcera-
tion, bleeding, and renal dysfunction, whereas inhibition of
COX-2 is responsible for their therapeutic effects (McA-
dam et al., 1999; Vane et al., 1998; WANG et al., 2005). In
normal condition, prostacyclin and thromboxane balance
each other’s opposing effects. Accordingly, introducing the
selective COX-2 inhibitor may alter the balance danger-
ously toward thromboxane, raising blood pressure, car-
diotoxicity and certainly promoting heart attack in some
cases (Hinz et al., 2006). This could describe the cardio-
vascular side effects caused by rofecoxib (Prasit et al.,
1999) and subsequently valdecoxib (Talley et al., 2000)
which were withdrawn from the market (Dogne’ et al.,
2005). Thus, there is a need to explore and evaluate
alternative selective COX-2 inhibitors with a mild thera-
peutic effect on COX-1, which could theoretically reduce
cardiovascular side effects due to their antiplatelet and anti-
thrombotic activities, and raise the safety profiles (Black
2004). Additionally, COX-2 inhibitors have emerged in
recent years as candidates for drugs due to their anti-cancer
activity (Koki and Masferrer 2002; Rizzo 2011; Ye et al.,
2004). Afterward COX-2 appears to be expressed at high
levels in many different types of cancer cells, but not in
bordering normal tissue, it appears plausible that the model
developed for colon cancer can be expanded to other
organs (Marnett and DuBois 2002). COX-2 is an enzyme
Keywords Spiroisoxazoline Á 1,3-Dipolar cycloaddition Á
Molecular modeling Á Docking
& Afshin Zarghi
azarghi@yahoo.com
1
Department of Pharmacology, Faculty of Medicine, Qom
University of Medical Sciences, Qom, Iran
2
Department of Medicinal Chemistry, School of Pharmacy,
Tabriz University of Medical Sciences, Tabriz, Iran
3
Department of Toxicology, Faculty of Medical Sciences,
Tarbiat Modares University, Tehran, Iran
4
Department of Medicinal Chemistry, School of Pharmacy,
Shahid Beheshti University of Medical Sciences, Tehran, Iran
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Med Chem Res
that is upregulated in colorectal cancer adenomas and
carcinoma (Eberhart et al., 1994). It has been shown that
PGE2 concentration is increased in colon cancer where
COX-2 overexpression (Chell et al., 2006; Yang et al.,
1998) leads to increased angiogenic and metastatic poten-
tial of tumor cells (Li et al., 2002). These findings are
supported by anticancer effect of selective COX-2 inhi-
bitor, celecoxib (Koki and Masferrer 2002; Ye et al.,
2004). Another well-documented cancer therapeutic target
is spiroisoxazoline compounds (1, 2 and 3, Fig. 1) that are
earning a lot of importance because of their extraordinary
biological properties such as anticancer activities (Al
Houari et al., 2008; Kang et al., 2000; Najim et al., 2010)
(CSID:13611429). Furthermore, Bennani and co-workers
have recently investigated the anti-breast cancer activity of
some spiroisoxazoline derivatives (Bennani et al., 2004;
Howe and Shelton 1990; Smietana et al., 1999). On the
other hand, the majority of selective COX-2 inhibitors
belong to diarylheterocycles that contain vicinal diaryl
substitution attached to a central ring system mainly mono
or bicyclic ring (van Ryn et al., 2000; Talley et al., 2000;
Zarghi et al., 2007a, b; Zarghi et al., 2009; Zebardast et al.,
2009) (Fig. 1). Extensive structure–activity relationship
(SAR) studies for this class of COX-2 inhibitors have
shown that a SO2NH2, or a SO2Me substituent at the para
position of one of the phenyl rings often provides optimum
selective COX-2 inhibitory potency (Palomer et al., 2002;
Talley et al., 2000; Zarghi et al., 2007a, b). In the current
work, we report the design and synthesis of a new series of
30-(4-substituted
phenyl)-40-(4-(methylsulfonyl)phenyl)
F3C
F3C
N
O
O
N
N
N
CH3
Br
SO2NH2
SO2NH2
SO2CH3
Celecoxib
SC-558
Rofecoxib
O
O
O
O
N
O
N
SO2CH3
1
2
O
X
N
O
R
SO2CH3
Designed
Fig. 1 Chemical structures of some known selective COX-2 inhibitors, spiro lead compounds (1, 2) and our designed scaffold
123

Med Chem Res
spiroisoxazoline derivatives containing naphthalenone and
chromanonespiro-bridge were synthesized for evaluation as
selective COX-2 inhibitors. The design strategy was to
combine pharmacophoric elements from COX-2 inhibitors
with added extra anticancer and anti-inflammatory prop-
erties as the result of bioisosteric replacement of the central
tricyclic bridge with spiroisoxazoline motif. Correspond-
ingly, it is desirable to develop safe, potent, profit selective
COX-2 inhibitors that show not only anti-inflammatory but
also anticancer effects.
determination of the in vitro ability (Zarghi et al., 2007a, b)
of the title compounds to inhibit the COX-1 and COX-2
isozymes showed that all compounds (7a–h) were selective
inhibitors of the COX-2 isozyme with IC50 values in the
0.09–0.20 lM range, and COX-2 selectivity indexes in the
63.2–116.2 range. Our results demonstrated that the COX
inhibition was affected by the type of substituent at the
para position of C-30 phenyl ring. Accordingly, compounds
having smaller groups (7a, 7b and 7g) were more potent
and selective COX-2 inhibitors compared with other ana-
logs which had larger ones. Based upon that, compound 7h
having chlorine group showed the lowest potency and
selectivity this may be explained by steric parameter for
interaction with COX-2 active binding site. These results
also indicated that the spiroisoxazoline derivatives con-
taining chromanone showed better both selectivity and
potency for COX-2 inhibitory activity compared with
naphthalenone analogs. This may be explained by the
ability of hydrogen binding ability of oxygen atom in
chromanones for better interaction with the COX-2 active
site. Therefore, the introduction of suitable substituents at
the para position of C-30 phenyl ring combined with
chromanone moiety improved the selectivity and potency
for COX-2 inhibitory activity. These data showed that the
size of substituent attached to para position of C-30 phenyl
ring and type of atom at position 1 of bicyclic ring can
influence both selectivity and potency for COX-2 inhibi-
tory activity. Our results indicated that 40-(4-(methylsul-
fonyl)phenyl)30-phenyl-40H-spiro [chroman-3,50-isoxazol]-
4-one 7b showed the highest COX-2 selectivity
(SI = 116.2) among the synthesized compounds which
may be due to better interaction with the COX-2 active site.
Result and discussion
Crystal structure of compound
Among these compounds (7a–h), the crystal structure of
compound (7h) was determined by single-crystal X-ray
diffraction method. The crystal data of compound (7h) are
presented in Table 1, and the corresponding perspective
view is shown in Fig. 2.
Molecular modeling
Docking simulation was performed to predict interaction of
compounds (7a–h) with COX-2 binding site (PDB code:
1CX2). All docking calculations were performed with the
GOLD program. Docking of compound 7b into the COX-2
binding site (Fig. 3) shows four hydrogen bonds His90 (angle
˚
O–H–N = 173.98°, distance = 2.10 A) and Arg513 (angle
˚
O–H–N = 157.79°, distance = 2.02 A), Arg120 (angle O–
˚
H–N = 120.18°, distance = 2.20 A) and Tyr355 (angle O–
˚
H–O = 143.29°, distance = 2.44A).AsshowninFig. 3, two
extra hydrogen bounds in compound 7b by interaction of
oxygen atom of chromanone with Arg120 and Tyr355 are
generated. Furthermore the para-substituted phenyl ring of
docked compound was embedded in a hydrophobic pocket
created by amino acids Tyr385, Trp387 and Phe518. In
addition, docking shows the other hydrophobic pocket sur-
rounding tricyclic spiroisoxazoline by the side chains of
residues Leu531, Leu359 and Val349. The mentioned resi-
dues were also found to be involved in the binding of SC-558
with COX-2 binding site. Figure 4 shows different three-di-
mensional superimposition of the SC-558 and synthesized
compounds while docked into the COX-2 binding site and
reveals that all compounds were well incorporated in the
selective COX-2 active pocket. The docking scores of syn-
thesized compounds and SC-558 are mentioned in Table 2.
Experimental
Materials
All reagents purchased from the Aldrich (USA) or Merck
(Germany) Chemical Company and were used without
further purifications.
General
Melting points (mp) were determined using a Thomas
Hoover capillary apparatus (Philadelphia, USA). Infrared
spectra were acquired on a Perkin-Elmer 1420 ratio
recording spectrometer. A Bruker FT-500 MHz instrument
1
(Bruker Biosciences, USA) was used to acquire HNMR
Biological activity
spectra, chloroform-D and DMSO-d6 used as solvents.
Coupling constant (J) values are estimated in hertz (Hz)
and spin multiples are given as s (singlet), d (double), t
(triplet), q (quartet), m (multiplet), and br (broad). The
The result of COX-1/COX-2 inhibition is summarized in
Table 3. SAR data (IC50 values) obtained by the
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Med Chem Res
absolute ethanol and NaOH 10 % solution produced the
corresponding oximes (6a–e). Methylsulfonylbenzylidene
chalcones (4a–b) were obtained by aldol condensation
reaction, where 4-(methylthio)benzaldehyde (2) were reac-
ted with 3,4-dihydronaphthalen-1(2H)-one(1a) or chroman-
4-one (1b) in the presence of sodium hydroxide and metha-
nol to obtain methylthiobenzylidene and chalcones (3a–
b) following by oxidation by OxonÒ. Subsequently the
methylsulfonylbenzylidene chalcones (4a–b) were reacted
with oximes (6a–e) in biphasic medium of aqueous sodium
hypochlorite 5 % and chloroform at cold temperature (-10
to 0 °C) to produce the corresponding spiroisoxazolines (7a–
h) using 1,3-dipolar cycloaddition reaction. The procedures
and the conditions for the synthesis of 30-(4-substituted
Table 1 Crystal data and structure refinement for compound 7h
Compound
7h
Empirical formula
Formula weight
Temperature (K)
C25H20ClNO4S
465.95
120(2)
˚
Wavelength (A)
0.71073
Triclinic
P - 1
Crystal system
Space group
˚
a (A)
8.1577(16)
10.200(2)
14.947(3)
97.83(3)
90.88(3)
106.41(3)
1180.0(5)
2
˚
b (A)
˚
c (A)
a (°)
b (°)
c (°)
phenyl)-40-(4-(methyl
sulfonyl)phenyl)spiroisoxazoline
3
˚
derivatives containing naphthalenone and chromanone (7a–
h) and their structures are shown in Scheme 1. Some of the
basic physicochemical properties as well as the production
yields of the synthesized compounds (7a–h) are shown in
Table 4. Figure 2 shows the crystal structure determined by
single-crystal X-ray diffraction method for one of com-
pounds as the representative of the series (i.e., 7h). Also the
structures of the synthesized compounds have been verified
using IR, MS and NMR (1H and 13C) spectroscopic methods.
Volume (A )
Z
D
calcd/(Mg/m-3
Absorption coefficient
(mm-1
Crystal size (mm)
)
1.401
0.289
)
0.35 9 0.30 9 0.25
h range for data collection (°) 2.34–29.13
F(0 0 0)
520
Limiting indices
11 B h B 8, -13 B k B 13,
-20 B l B 20
Reflections collected/unique 13224/6315 [R(int) = 0.0853]
Completeness to h = 29.13° 99.5 %
General Procedure for Preparation of Chalcones
(3a, 3b)
Absorption correction
Max. and min. transmission
Refinement method
Numerical
One of the (1a) 3, 4 dihydronaphthalen-1(2H)-one (0.146 g,
1 mmol) or (1b) chroman-4-one (0.148 g, 1 mmol) and
4-methylthiobenzaldehyde (2) (0.152 g, 1 mmol) were dis-
solved in the methanol (10 mL), a single NaOH pellet
(100 mg, 2.5 mmol) was then added to this solution, and the
reaction mixture was stirred at room temperature. In most
cases, a cream to dark yellow solid was formed within a few
minutes, but the reaction was allowed to proceed for 3 h. The
solid product 3a or 3b was collected on a filter, washed with
cold methanol, and the product was recrystallized from
methanol. (Yields: 65–72 %). The physical and spectral data
for 3a and 3b are listed below.
0.9312 and 0.9056
Full-matrix least-squares on F2
6315/1/313
R1 = 0.0620, wR2 = 0.1554
R1 = 0.0859, wR2 = 0.1690
1.008
Data/restraints/parameters
Final R indices [I [ 2r(I)]
R indices (all data)
Goodness-of-fit on F2
Largest diff. peak and hole (e 0.545 and -0.594
-3
˚
A
)
mass spectral measurements were performed on a 6410
Agilent LCMS triple quadruple mass spectrometer (LCMS)
with an electrospray ionization (ESI) interface.
(E)-2-(4-(Methylthio)benzylidene)-3,4-dihydronaphthalen-
1(2H)-one (3a) Yield: 72 %; yellow crystalline powder;
mp: 191 °C; IR (KBr): m (cm-1) 1590 (C=O); LC–MS (ESI)
m/z: 281.1 (M ? 1)), 303.1 (M ? 23); 1HNMR (CDCl3): d
ppm 2.94 (s, 3H, SMe), 2.99 (t, 2H, CH2), 3.17 (t, 2H, CH2),
7.29–7.30 (d, 1H, phenyl H5, J = 7.7 Hz), 7.32 (d, 2H,
4-methylthiophenyl H3 & H5, J = 8.3 Hz), 7.39–7.41 (t, 1H,
phenyl H7), 7.42–7.44 (d, 2H, 4-methylthiophenyl H2 & H6,
J = 8.3 Hz), 7.53 (t, 1H, phenyl H6), 7.87 (s, 1H, CH), 8.16
(d, 1H, phenyl H8, J = 7.0 Hz).
Chemistry
In the present work, the synthesis of 30-(4-substituted phe-
nyl)-40-(4-(methylsulfonyl)phenyl) spiroisoxazoline deriva-
tives containing naphthalenone and chromanone (7a–
h) from (E)-2-(4-(methylsulfonyl)benzylidene)-3,4-dihy-
dronaphthalen-1(2H)-one (4a) or (E)-3-(4-(methyl sul-
fonyl)benzylidene)chroman-4-one (4b) and (E)-4-substi-
tuted-benzaldehyde oxime (oximes) (6a–e) employing 1,3-
dipolar cycloaddition reaction was carried out. Reaction of
aldehydes (5a–e) with hydroxylamine hydrochloride in
(E)-3-(4-(Methylthio)benzylidene)chroman-4-one (3b)
Yield: 65 %; yellow crystalline powder; mp: 137–138 °C;
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Med Chem Res
the residue was extracted with chloroform, washed with 10 %
aqueous sodium bicarbonate and dried with anhydrous
sodium sulfate and then the solvent evaporated. In most cases,
off-white to pale yellow solid was formed. Yield: (70–94 %).
The physical and spectral data for 4a and 4b are listed below.
(E)-2-(4-(methylsulfonyl) benzylidene)-3, 4-dihydronaphthalen-
1(2H)-one (4a)) Yield: 70 %; yellow crystalline powder;
mp: 200–202 °C; IR (KBr): m (cm-1) 1592 (C=O); 1301,
1157 (SO2); LC–MS (ESI) m/z: 313.1 (M ? 1); 335.1
(M ? 23).1HNMR (CDCl3): d ppm 3.01–3.04 (t, 2H, CH2),
3.13 (s, 1H, SO2Me), 3.13–3.15 (m, 2H, CH2), 7.31 (d,
J = 7.7 Hz, 1H, phenyl H5), 7.45 (t, 1H, phenyl H7), 7.58 (t,
1H, phenyl H6), 7.64 (d, J = 8.2 Hz, 2H, 4-methyl sul-
fonylphenyl H2 & H6), 7.88 (s, 1H, CH), 8.03 (d, J = 8.2 Hz,
2H, 4-methylsulfonylphenyl H3 & H5), 8.18 (d, J = 6.7 Hz,
1H, phenyl H8).
Fig. 2 Crystal structure diagrams of compound 7h
(E)-3-(4-(methylsulfonyl)benzylidene) chroman-4-one (4b)
Yield: 94 %; white crystalline powder; mp: 180–182 °C; IR
(KBr): m (cm-1) 1594 (C=O), 1306, 1141 (SO2); LC–MS
(ESI) m/z: 315.1 (M ? 1); 337.1 (M ? 23); 1HNMR
(CDCl3): d ppm 3.14 (s, 3H, SO2Me), 5.33 (s, 2H, CH2), 7.03
(d, J = 8.3 Hz, 1H, phenyl H8), 7.13 (t, 1H, phenyl H6), 7.53
(d, J = 7.5 Hz, 2H, 4-methylsulfonylphenyl H2 & H6),
7.57–7.58 (t, 1H, phenyl H7), 7.91 (s, 1H, CH), 8.06–8.08 (m,
3H, 4-methylsulfonylphenyl H3 & H5, phenyl H5).
IR (KBr): m (cm-1) 1600 (C=O); LC–MS (ESI) m/z: 283.1
(M ? 1); HNMR (CDCl3): d ppm 2.57 (s, 3H, SO2Me),
5.40 (s, 2H, CH2), 7.01 (d, 1H, phenyl H8, J = 8.2 Hz), 7.11
(t, 1H, phenyl H6), 7.29 (d, 4-methyl thiophenyl H3 & H5,
J = 8.4 Hz), 7.33 (d, 2H, 4-methylthiophenyl H2 & H6,
J = 8.4 Hz), 7.52 (t, 1H, phenyl H7), 7.65 (s, 1H, CH), 8.06
(d, 1H, phenyl H5, J = 7.1 Hz).
1
General Procedure for Preparation
of Methylsulfonyl chalcones (4a, 4b)
General Procedure for Preparation of p-substituted-
benzaldoximes (6a–e)
One gram of 3a or 3b was dissolved in 20 ml of THF and 5 g
Oxone in THF/water (30 ml) was added. The mixture was
stirred at room temperature for 2 h. After evaporation of THF,
One gram of compounds 5a–e was dissolved in 20 ml of
95 % ethanol. After the mixture was heated to 60 °C,
Fig. 3 3D representations of the binding of compound 7b (colored
according to the atom type) in the active site of murine COX-2 (COX-
2 structure with PDB code 1CX2 was used for docking). a The yellow
dotted lines show the hydrogen bonds in protein–ligand complex.
b The protein is represented by colored surface and shows superim-
position of SC-558 (hot pink and stick) and 7b (cyan and stick) with
COX-2 by the essential amino acid residues at the active site. All
pictures were prepared with PyMOL (Color figure online)
123

Med Chem Res
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Med Chem Res
bFig. 4 Different three-dimensional superimpose representations of the
SC-558(hot pink and stick) and synthesized compounds (7a, 7b, 7c, 7d,
7f, 7g and 7h) (cyan and stick) with COX-2 using PyMOL program with
the essential amino acid residues at the active site (Color figure online)
(E)-4-fluorobenzaldehyde oxime) 4-fluorobenzaldoxime
(6b) Yield: 69 %; white crystalline powder; mp:
70–72 °C; IR (KBr): m (cm-1) 1681 (C=N), 3084 (O–H),
859 (N–O); LC–MS (ESI) m/z: 140 (M ? 1).
(E)-4-chlorobenzaldehyde oxime) 4-chlorobenzaldoxime
(6c) Yield: 82 %; white crystalline powder; mp:
100–113 °C; IR (KBr): m (cm-1) 1588 (C=N), 3308 (O–H),
972 (N–O); LC–MS (ESI) m/z: 156 (M ? 1).
NaOH aq 10 % (10 ml) and NH2OHÁHCl (4 g) were
added. The mixture was refluxed for 2 h at 70 °C. Then the
reaction was terminated, and the majority of solvent was
evaporated under reduced pressure. Cool distilled water
and 2 ml HCl 6 M was added into the reaction mixture.
The solid product 6b–e was collected on a filter, washed
with cold water, and the product was recrystallized from
ethanol. But compound 6a was extracted with diethyl ether.
The organic layer dried over Na2SO4 and evaporated to
obtain the oily product.
(E)-4-methylbenzaldehyde oxime(4-methyl benzaldoxime)
(6d) Yield: 62 %; cream crystalline powder; mp:
70–74 °C;IR (KBr): m (cm-1) 1598 (C=N), 3273 (O–H), 954
(N–O); LC–MS (ESI) m/z: 136.1 (M ? 1).
(E)-4-methoxybenzaldehyde oxime) 4-methoxy benzal-
doxime) (6e) Yield: 65 %; cream crystalline powder; mp:
55–57 °C; IR (KBr): m (cm-1) 1682 (C=N), 3228 (O–H),
1170 (C–O), 950 (N–O); LC–MS (ESI) m/z: 152.3 (M ? 1).
Yield: (60–92 %). The physical and spectral data for
6a–e are listed below.
(E)-benzaldehydeoxime(Benzaldoxime) (6a) Yield: 62 %;
yellow oily liquid; IR (neat): m (cm-1) 1720 (C=N), 3406
(O–H), 955 (N–O); LC–MS (ESI) m/z: 122.1 (M ? 1).
General Procedure for Preparation
of Spiroisoxazoline Compounds (7a-h)
Table 2 Docking scores for compounds 7a–7h
A mixture of a dipolarophile (4a or 4b; 1 mmol) and p-
substituted-benzaldoxime (6a–e; 2.5 mmol) was dissolved
in chloroform (15 ml) at cold temperature (-10 °C) that
placed in an ice-salt bath. To this mixture was added 2 ml
of sodium hypochlorite (5 %) that diluted in 5 ml water,
drop ways over 20 min. The temperature was controlled
and has not to excess (-5 °C) along addition of sodium
hypochlorite. After complete addition of NaOCl, the mix-
ture was stirred 1–2 h at room temperature. Compounds
were extracted by chloroform, washed several times with
water, and dried on anhydrous sodium sulfate. White solid
obtained after evaporation of the solvent and crystallization
in methanol.
Compound
R
X
Docking score
7a
H
CH2
O
31.84
33.71
32.82
32.80
33.94
30.60
32.78
15.09
7b
H
7c
CH3
CH3
OCH3
F
CH2
O
7d
7f
O
7g
CH2
CH2
–
7h
Cl
SC-558
–
Table 3 In vitro COX-1 and COX-2 enzyme inhibition assay data for
compounds 7a–7h
Yield: (32–56 %). The physical and spectral data for
7a–h are listed below.
Compound COX-1
ICa50 SD
(lM)
COX-2
ICa50 SD
(lM)
Selectivity indexb
(SI)
4-(4-(Methylsulfonyl)phenyl)-3-phenyl-30,40-dihydro-10H,4H-
spiro[isoxazole-5,20-naphthalen]-10-one (7a) Yield: 37 %;
white crystalline powder; mp: 130–132 °C; IR (KBr): m
(cm-1) 1686 (C=O), 1310, 1153 (SO2); LC–MS (ESI) m/z:
432.2 (M ? 1), 454.1 (M ? 23); 1HNMR (CDCl3,
500 MHz): d ppm 1.79–1.93 (m, 2H, CH2, naphtalenon
7a
7b
7c
7d
7e
7f
11.65
0.12
0.09
0.16
0.12
0.18
0.11
0.10
0.20
0.06
97.1
116.2
68.4
84.2
75.8
82.8
102.3
63.2
405
10.46
10.90
10.11
13.65
9.11
0
0
H4 ), 2.82–2.87 (m, 1H, CH2, naphtalenon H3 ), 3.36–3.41
0
(m, 1H, CH2, naphtalenon H3 ), 3.09 (s, 3H, SO2CH3), 5.67
(s, 1H, isoxazolin H4), 7.27–7.44 (m, 7H, phenyl H3 & H4
7g
7h
10.23
12.65
0
& H5, 4-methylsulfonylphenyl H2 & H6, naphtalenon H5 &
0
0
H7 ), 7.54–7.57 (t, 1H, naphtalenon H6 ), 7.62–7.64 (d,
Celecoxib 24.30
J = 8.5 Hz, 2H, phenyl H2 & H6), 7.95–7.97 (d,
a
J = 8.1 Hz, 2H, 4-methylsulfonylphenyl H3& H5),
8.11–8.13 (d, J = 7.7 Hz, 1H, naphtalenon H8 ); 13CNMR
Values are means of two determinations acquired using an ovine
COX-1/COX-2 assay kit and the deviation from the mean is\10 % of
the mean value
0
(CDCl3, 125 MHz): d ppm 24.40, 28.91 (CH2-CH2), 43.13
0
b
(SO2CH3), 54.36 (CH), 88.01 (Spiro C2 ,5), 123.96, 126.11,
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50
)
123

Med Chem Res
O
H
O
X
O
X
a
+
SCH3
SCH3
2
3a, X = CH2
3b, X = O
1a, X = CH2
1b, X = O
OH
N
b
O
H
O
c
+
R
R
X
SO2CH3
5a, R = H
6a, R = H
6b, R = F
6c, R = Cl
5b, R = F
4a, X = CH2
4b, X = O
5c, R = Cl
5d, R =CH3
5e, R= OCH3
6d, R =CH3
6e, R= OCH3
d
7a, X = CH2 , R = H
7b, X = O , R = H
O
X
N
O
7c, X = CH2 , R = CH3
7d, X = O , R = CH3
7e, X = CH2 , R = OCH3
7f, X = O , R = `OCH3
7g, X = CH2 , R = F
7h, X = CH2 , R = Cl
R
SO2CH3
Scheme 1 Synthesis of spiroheterocycles 7a–h. Reagents: a NaOH, CH3OH, stir, r.t; b Oxone, THF,H2O, stir, r.t.; c NH2OHCl, NaOH aq 10 %,
Ethanol, Reflux; d NaOCl 5 %, H2O, CHCl3, stir, -10 to 0 °C
0
(s, 1H, isoxazolin H4 ), 7.02–7.03 (d, 1H, chromanon H8),
126.13, 126.45, 127.10, 127.74, 127.88, 128.24, 128.54,,
129.22, 133.21, 139.29, 139.81, 140.19 (Aromatic C),
158.67 (C=N), 190.05 (C=O). Anal. Calcd for C25H21
NO4S: C, 69.59; H, 4.91; N, 3.25. Found: C, 69.71; H,
5.02; N, 3.01.
7.14–7.17 (t, 1H, chromanon H6), 7.33–7.47 (m, 5H, phenyl
H3 & H4 & H5 & 4-methylsulfonylphenyl H2& H6),
7.58–7.62 (m, 3H, phenyl H2 & H6 &chromanon H7),
7.99–8.00 (d, J = 8 Hz, 2H, 4-methylsulfonylphenyl H 3&
H5), 8.01–8.03 (d, J = 7.5 Hz, 1H, chromanonH5);13CNMR
40-(4-(Methylsulfonyl)phenyl)30-phenyl-40H-spiro[chroman-
3,50-isoxazol]-4-one (7b) Yield: 35 %;white crystalline
powder; mp: 168–170 °C; IR (KBr): m (cm-1) 1709 (C=O),
1331, 1171 (SO2); LC–MS (ESI) m/z: 433.8 (M ? 1);
1HNMR (CDCl3, 500 MHz): d ppm 3.12 (s, 3H, SO2CH3),
4.01–4.03 (d, J = 12.7 Hz, 1H, CH2, chromanon H2),
4.23–4.26 (d, J = 12.7 Hz, 1H, CH2, chromanon H2), 5.53
(CDCl3, 125 MHz): d ppm 43.55 (SO2CH3), 54.66 (CH),
0
60.9 (CH2), 89.88 (Spiro C2 ,5), 115.56, 126.31, 126.53,
126.88, 127.60, 127.84, 128.08, 128.34, 128.66, 130.12,
135.44, 142.11, 143.21, 146.24 (Aromatic C), 160.17 (C=N),
191.10 (C=O). Anal. Calcd for C24H19NO5S: C, 66.50; H,
4.42; N, 3.23. Found: C, 66.71; H, 4.63; N, 3.39.
123

Med Chem Res
Table 4 Structure and characteristics of the synthesized compounds 7a–7h
O
N
O
R
X
SO2CH3
Compound
X
R
Empirical formula
C25H21NO4S
MW
mp (°C)
Yield (%)
Color
7a
7b
7c
7d
7e
7f
CH2
O
H
431
433
445
447
461
463
449
465
130–132
168–170
140–142
138–140
150–152
140–141
122–123
171–173
37
35
41
38
50
48
32
54
White
White
White
White
White
White
White
White
H
C24H19NO5S
CH2
O
CH3
CH3
OCH3
OCH3
F
C26H23NO4S
C25H21NO5S
C26H23NO5S
C25H21NO6S
C25H20FNO4S
C25H20ClNO4S
CH2
O
7g
7h
CH2
CH2
Cl
4-(4-(Methylsulfonyl)phenyl)-3-p-tolyl-30,40-dihydro-10H,4H-
spiro[isoxazole-5,20-naphthalen]-10-one (7c) Yield: 41 %;
white crystalline powder; mp: 140–142 °C; IR (KBr): m
(cm-1) 1697 (C=O), 1320, 1162 (SO2); LC–MS (ESI) m/z:
445.8 (M ? 1); 1HNMR (CDCl3, 500 MHz): d ppm
J = 12.7 Hz, 1H, CH2, chromanon H2), 4.22–4.25 (d,
J = 12.7 Hz, 1H, CH2, chromanon H2), 5.51 (s, 1H, isox-
0
azolin H4 ), 7.01–7.02 (d, 1H, chromanon H8), 7.13–7.16
(m, 3H, chromanon H6& 4-methylphenyl H3 & H5),
7.42–7.49 (m, 4H, 4-methylphenyl H2 & H6 & 4-methyl-
sulfonylphenyl H2& H6), 7.57–7.61 (t, 1H, chromanon H7),
7.97–7.99 (d, J = 8.2 Hz, 2H, 4-methylsulfonylphenyl
H3& H5), 8.01–8.03 (d, J = 7.9 Hz, 1H, chromanon H5);
0
1.78–1.92 (m, 2H, CH2, naphtalenon H4 ), 2.35 (s, 3H,
CH3, 4-methylphenyl), 2.81–2.86 (m, 1H, CH2, naphtal-
0
0
enon H3 ), 3.35–3.42 (m, 1H, CH2, naphtalenon H3 ), 3.09
(s, 3H, SO2CH3), 5.65 (s, 1H, isoxazolin H4), 7.13–7.15 (d,
J = 8.1 Hz, 2H, 4-methylphenyl H3 & H5), 7.26–7.43 (m,
13CNMR (CDCl3, 125 MHz): d ppm 21.11 (CH3), 44.05
0
(SO2CH3), 54.46 (CH), 60.2 (CH2), 88.24 (Spiro C2 ,5),
0
4H, 4-methylsulfonylphenyl H2& H6, naphtalenon H5
&
115.26, 126.41, 126.33, 126.77, 127.55, 127.91, 128.33,
128.48, 129.44, 129.92, 135.14, 141.33, 142.31, 145.51
(Aromatic C), 159.10 (C=N), 190.25 (C=O). Anal. Calcd.
for C25H21NO5S: C, 67.10; H, 4.73; N, 3.13. Found: C,
67.26; H, 4.51; N, 3.15.
0
H7 ), 7.51–7.53 (d, J = 8.2 Hz, 2H, 4-methylphenyl H2 &
0
H6), 7.54–7.57 (t, 1H, naphtalenon H6 ), 7.94–7.96 (d,
J = 8.2 Hz, 2H, 4-methylsulfonylphenyl H3
& H5),
8.11–8.13 (d, J = 8 Hz, 1H, naphtalenon H8 ); 13CNMR
0
(CDCl3, 125 MHz): d ppm 20.51 (CH3), 24.38, 28.85
3-(4-Methoxyphenyl)-4-(4-(methylsulfonyl)phenyl)-30,40-dihy-
(CH2-CH2), 43.33 (SO2CH3), 54.16 (CH), 87.81 (Spiro
dro-10H,4H-spiro[isoxazole-5,20-naphthalen]-10-one
(7e)
0
C2 ,5), 124.06, 126.13, 126.47, 127.17, 127.55, 127.85,
128.32, 128.47, 128.66, 129.19, 133.16, 139.78, 140.22,
142.33 (Aromatic C), 158.49 (C=N), 189.97 (C=O). Anal.
CalcdforC26H23NO4S: C, 70.09; H, 5.20; N, 3.14. Found:
C, 70.21; H, 5.43; N, 2.99.
Yield: 50 %; white crystalline powder; mp: 150–152 °C;
IR (KBr): m (cm-1) 1688 (C=O), 1304, 1148 (SO2); LC–
MS (ESI) m/z: 462.1 (M ? 1); 1HNMR (CDCl3,
500 MHz): d ppm 1.79–1.92 (m, 2H, CH2, naphtalenon
0
H4 ), 2.82–2.87 (m, 1H, CH2, naphtalenon H3 ), 3.36–3.42
0
40-(4-(Methylsulfonyl)phenyl)30-p-methylphenyl-40H-spiro
0
(m, 1H, CH2, naphtalenon H3 ), 3.1 (s, 3H, SO2CH3), 3.82
[chroman-3,50-isoxazol]-4-one (7d) Yield: 38 %;white
(s, 3H, OCH3, 4-methoxyphenyl), 5.63 (s, 1H, isoxazolin
H4), 6.85–6.88 (d, J = 8.8 Hz, 2H, 4-methoxylphenyl H3
& H5), 7.27–7.44 (m, 4H, 4-methylsulfonylphenyl H2& H6,
crystalline powder; mp: 138–140 °C; IR (KBr): m (cm-1
1677 (C=O), 1300, 1142 (SO2); LC–MS (ESI) m/z: 448.1
)
1
0
naphtalenon H5 & H7 ), 7.57–7.58 (d, J = 8.8 Hz, 2H,
0
(M ? 1); HNMR (CDCl3, 500 MHz): d ppm 2.36 (s, 3H,
CH3, 4-methylphenyl), 3.11 (s, 3H, SO2CH3), 4.00–4.02 (d,
4-methoxylphenyl H2 & H6), 7.54–7.57 (t, 1H, naphtalenon
123

Med Chem Res
0
H6 ), 7.96–7.97 (d, J = 8.0 Hz, 2H, 4-methylsul-
C25H20FNO4S: C, 66.80; H, 4.48; N, 3.12. Found: C,
66.61; H, 4.27; N, 3.25.
fonylphenyl H3 & H5), 8.12–8.13 (d, J = 7.8 Hz, 1H,
13
0
naphtalenon H8 ); CNMR (CDCl3, 125 MHz): d ppm
24.39, 28.85 (CH2-CH2), 43.32 (SO2CH3), 54.20 (CH),
4-(4-(Methylsulfonyl) phenyl)-3-p-chlorophenyl-30, 40-di-
hydro-10H, 4H-spiro [isoxazole-5, 20-naphthalen]-10-one
(7h) Yield: 54 %; white crystalline powder; mp:
171–173 °C; IR (KBr): m (cm-1) 1690 (C=O), 1315, 1160
(SO2); LC–MS (ESI) m/z: 465.8 (M ? 1); 1HNMR
(CDCl3, 500 MHz): dppm 1.80–1.93 (m, 2H, CH2, naph-
0
54.29 (OCH3), 87.67 (Spiro C2 ,5), 113.11, 119.37, 126.02,
126.96, 127.15, 127.63, 127.83, 129.20, 129.42, 133.12,
139.33, 140.29, 142.34, 158.15 (Aromatic C), 160.25
(C=N), 190.04 (C=O). Anal. Calcd. for C26H23NO5S: C,
67.66; H, 5.02; N, 3.03. Found: C, 67.90; H, 5.23; N, 3.21.
0
talenon H4 ), 2.83–2.88 (m, 1H, CH2, naphtalenon H3 ),
0
40-(4-(Methylsulfonyl)phenyl)30-p-methoxyphenyl-40H-spiro
0
3.36–3.43 (m, 1H, CH2, naphtalenon H3 ), 3.11 (s, 3H,
[chroman-3,50-isoxazol]-4-one (7f) Yield: 48 %; white
SO2CH3), 5.64 (s, 1H, isoxazolin H4), 7.28–7.29 (d,
crystalline powder; mp: 140–142 °C; IR (KBr): m (cm-1
1693 (C=O), 1308, 1150 (SO2); LC–MS (ESI) m/z: 463.7
)
J = 8.6 Hz, 2H, 4-chlorophenyl H3 &H5), 7.28–7.49 (m,
0
4H, 4-methylsulfonylphenyl H2& H6, naphtalenon H5 &
1
0
0
(M ? 1); HNMR (CDCl3, 500 MHz): d ppm 3.11 (s, 3H,
H7 ), 7.56–7.59 (t, 1H, naphtalenon H6 ), 7.56–7.58 (d,
J = 8.6 Hz, 2H, 4-chlorophenyl H2 & H6), 7.97–7.99 (d,
J = 8.1 Hz, 2H, 4-methylsulfonylphenyl H3& H5),
SO2CH3), 3.82 (s, 3H, OCH3, 4-methoxyphenyl),
4.00–4.02 (d, J = 12.7 Hz, 1H, chromanon H2), 4.22–4.24
(d, J = 12.7 Hz, 1H, chromanon H2), 5.48 (s, 1H, isoxa-
0
8.12–8.13 (d, J = 8 Hz, 1H, naphtalenon H8 ); 13CNMR
0
zolin H4 ), 6.84–6.86 (d, 2H, 4-methoxyphenyl H3 & H5),
(CDCl3, 100 MHz): d ppm 24.31, 28.78 (CH2–CH2), 43.32
0
7.01–7.02 (d, J = 8 Hz, 1H, chromanon H8), 7.13–7.16 (t,
1H, chromanon H6), 7.47–7.52 (d, 2H, J = 8 Hz,
(SO2CH3), 53.89 (CH), 88.27 (Spiro C2,5 ), 125.40, 126.11,
127.31, 127.70, 127.71, 127.86, 128.09, 129.13, 129.19,
133.32, 135.46, 139.50, 139.66, 142.30 (Aromatic C),
157.63 (C=N), 189.75 (C=O). Anal. Calcd. for C25H20
ClNO4S: C, 64.44; H, 4.33; N, 3.01. Found: C, 64.59; H,
4.45; N, 3.20.
4-methylsulfonylphenyl H2
&
H6), 7.52–7.54 (d,
J = 8.9 Hz, 2H, 4-methoxyphenyl H2 & H6), 7.57–7.61 (t,
1H, chromanon H7), 7.98–8.00 (d, J = 8 Hz, 2H,
4-methylsulfonylphenyl H3
&
H5), 8.01–8.03 (d,
J = 7.9 Hz, 1H, chromanon H5); 13CNMR (CDCl3,
125 MHz): d ppm 44.05 (SO2CH3), 54.39 (CH), 54.61
0
(CH3), 59.86.2 (CH2), 88.55 (Spiro C2 ,5), 114.87, 126.31,
126.55, 126.95, 127.75, 128.12, 128.43, 128.85, 129.33,
129.88, 135.44, 141.56, 142.51, 144.88 (Aromatic C),
158.92 (C=N), 191.05 (C=O). Anal. Calcd. for C25H21
NO6S: C, 64.78; H, 4.57; N, 3.02. Found: C, 64.99; H,
4.65; N, 3.22.
Crystal structure determination
Crystal structure determination of compound 7h was car-
ried out by single-crystal X-ray diffraction method. The
crystal data, data collection, and refinement parameter for
the compound 7h is listed in Table 1.
3-(4-Fluorophenyl)-4-(4-(methylsulfonyl)phenyl)-30,40-dihydro-
10H,4H-spiro[isoxazole-5,20-naphthalen]-10-one (7g) Yield:
32 %; white crystalline powder; mp: 122–124 °C; IR
(KBr): m (cm-1) 1685 (C=O), 1317, 1152 (SO2); LC–MS
Molecular modeling (docking) studies
3D structures of the synthesized compounds were gener-
ated using the Hyper Chem (version 7.0). The initial
structures were first minimized using molecular mechanics
MM ? force field (Allinger1977). Then, those structures
were fully optimized based on the semi-empirical quantum
mechanics AM1 method, available in HyperChem (Dewar
and Thiel 1977). The output structures were converted to
SYBYL Cartesian coordinate files (mol2 file format) using
Open Babel program (version 2.3.2) in order to be used as
an acceptable format in GOLD program (version 5.0) with
the aim of docking (Jones et al., 1995, Morris et al., 1998).
Flexible docking of all synthesized compounds was carried
out using GOLD program running under Linux OS. The
crystal structure of SC-558 (PDB code: 1CX2) was
obtained from Protein Data Bank at RCSB (http://www.
rcsb.org/pdb/home/home.do). The protein structure was
1
(ESI) m/z: 449.8 (M ? 1); HNMR (CDCl3, 500 MHz):
0
dppm 1.80–1.93 (m, 2H, CH2, naphtalenon H4 ), 2.83–2.88
0
(m, 1H, CH2, naphtalenon H3 ), 3.37–3.43 (m, 1H, CH2,
0
naphtalenon H3 ), 3.11 (s, 3H, SO2CH3), 5.64 (s, 1H,
isoxazolin H4), 7.03–7.09 (t, 2H, 4-fluorophenyl H3 &H5),
7.28–7.51 (m, 4H, 4-methylsulfonylphenyl H2& H6,
0
0
0
naphtalenon H5 & H7 ), 7.56–7.59 (t, 1H, naphtalenon H6 ),
7.62–7.65 (m, 2H, 4-fluorophenyl H2 & H6), 7.98–7.99 (d,
J = 8.1 Hz, 2H, 4-methylsulfonylphenyl H3& H5), 8.12-
8.14 (d, J = 6.9 Hz, 1H, naphtalenon H8 ); 13CNMR
0
(CDCl3, 100 MHz): d ppm 24.33, 28.79 (CH2–CH2), 43.29
0
(SO2CH3), 53.92 (CH), 88.34 (Spiro C2,5 ), 125.46, 126.05,
127.21, 127.62, 127.74, 127.84, 128.12, 129.21, 129.28,
133.22, 135.40, 139.47, 139.71, 142.36 (Aromatic C),
157.71 (C=N), 189.97 (C=O). Anal. Calcd. for
123

Med Chem Res
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prepared for docking using GOLD program. Docking was
performed by applying a point assigned based on the
important residues involved in the binding. All atoms
˚
within a 10A radius were selected, and flexible docking
was carried out. Chem PLP method of GOLD suite was
selected as the scoring function. The binding cavity was
determined based on the binding location of SC-558 co-
crystallized with cyclooxygenase-2, and then SC-558
molecule was removed and the synthesized compounds
were docked. The interactions between ligands and COX-2
have been visualized via the 3D representation using
PyMOL (v0.99) programs. The results from docking study
using GOLD program indicates specific interactions.
Superimposition of the Sc-558 and the synthesized com-
pounds once bound into the COX-2 binding site has been
illustrated in Fig. 4 in surface and cartoon representations.
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In vitro cyclooxygenase (COX) inhibition assays
The ability of the test compounds listed in Table 3 to
inhibit ovine COX-1 and COX-2 (IC50 value, lM) was
determined using chemiluminescent enzyme assays kit
(Cayman Chemical, Ann Arbor, MI, USA) according to
our previously reported method (Zarghi et al., 2007a, b).
Acknowledgments The authors would like to thank the Research
Deputy of Shahid Beheshti University of Medical Sciences and the
Research Office, Biotechnology Research Center and School of
Pharmacy of Tabriz University of Medical Sciences for providing
financial support of this project.
Najim N, Bathich Y, Zain MM, Hamzah AS, Shaameri Z (2010)
Evaluation of the bioactivity of novel spiroisoxazoline type-
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