Inhibitory effects of 2-substituted-1-naphthol derivatives on cyclooxygenase I and II

Article abstract of DOI:10.1016/j.bmc.2004.12.054

Naphthol derivatives, 2-(3′-hydroxypropyl)-naphthalen-1-ol (2), 2-(3′-hydroxy-2′-methylpropyl)-naphthalen-1-ol (3) and 2-(3′-hydroxy-2′,2′-dimethylpropyl)-naphthalen-1-ol (7) were synthesized and already reported by our group. Therefore in this paper we d

Full text of DOI:10.1016/j.bmc.2004.12.054

Bioorganic & Medicinal Chemistry 13 (2005) 2167–2175
Inhibitory effects of 2-substituted-1-naphthol derivatives
on cyclooxygenase I and II
Boonsong Kongkathip,a Chak Sangma,a,* Kanyawim Kirtikara,b Suwaporn Luangkamin,c
Komkrit Hasitapan,a Nipa Jongkon,a Supa Hannongbuaa and Ngampong Kongkathipa,*
aNatural Products and Organic Synthesis Research Unit (NPOS), Department of Chemistry, Faculty of Science,
Kasetsart University, Bangkok 10900, Thailand
bNational Center for Genetic Engineering and Biotechnology, Bangkok 10400, Thailand
cDepartment of Chemistry, Faculty of Science, Chiangmai University, Chiangmai 50200, Thailand
Received 24 September 2004; revised 28 December 2004; accepted 29 December 2004
Available online 19 January 2005
Abstract—Naphthol derivatives, 2-(30-hydroxypropyl)-naphthalen-1-ol (2), 2-(30-hydroxy-20-methylpropyl)-naphthalen-1-ol (3) and
2-(30-hydroxy-20,20-dimethylpropyl)-naphthalen-1-ol (7) were synthesized and already reported by our group. Therefore in this
paper we described further synthesis of their ether derivatives, 3-(1-methoxy-naphthalen-2-yl)-propan-1-ol (4), 3-(1-methoxy-naph-
thalen-2-yl)-2methyl-propan-1-ol (5), 3-(1-methoxy-naphthalen-2-yl)-2,2-dimethyl-propan-1-ol (8), 2-(3-methoxy-propyl)-naphtha-
len-1-ol (10) and 2-(3-methoxy-2,2-dimethyl-propyl)-naphthalen-1-ol (13). Compounds 4, 5 and 8 were prepared by methylation
of compounds 2, 3 and 7, respectively while compounds 10 and 13 were prepared in good yield from naphthols 2 and 7, respectively.
When tested for inhibitory activity, five compounds (2, 3, 7, 10 and 13) showed preferential inhibition of COX-2 over COX-1, while
compounds 4, 5 and 8 lacked inhibitory effect on either the COX-1 or COX-2 isozyme. The structure–activity relationships of these
naphthols analyzed by docking experiments, indicated that the presence of hydroxyl group at C-1 position on the naphthalene
nucleus enhanced the anti-inflammatory activity towards COX-2 via hydrogen bonding to the COX-2 Val 523 side chain. When this
hydroxyl group was replaced by methoxy group, there was no inhibition. C-20 Dimethyl substituents on the propyl chain also
increased the inhibitory activity. All active compounds have the C-1 hydroxyl group aligned so as to form hydrogen bond with
Val 523. The results provide a model for the binding of the naphthol derivatives to COX-2 and facilitate the design of more potent
or selective analogs prior to synthesis.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
and thus which reduce harmful side effects, is desirable.
Understanding the relationship of chemical structure
and enzyme activity is crucial to selecting inhibitory
compounds.
Non steroidal anti-inflammatory drugs (NSAIDs) such
as aspirin and naproxen are a group of drugs that inhibit
prostaglandin production via inhibition of the cyclooxy-
genase (COX) enzyme.1,2 There are two isoforms of
COX that is the constitutively induced COX-1, which
is associated with maintaining homeostasis, and the
inducible COX-2, which is normally not induced but
plays an important role during inflammation.3–5 Conse-
quently, a newer generation of NSAIDs, which selec-
tively inhibit COX-2 without affecting COX-1 activity,
As part of our studies of the syntheses of antitumour,
1,2-naphthoquinone,6 1,4-naphthoquinone,6 rhinacan-
thin and naphthoquinone ester derivatives,7 the synthe-
sis of naphthol derivatives was undertaken. The effects
of 1-naphthol itself on enzymatic arachidonic acid
oxidation have been previously reported by several
workers.8 Batt and co-workers described studies of
naphthalen-1-ol, bearing carbon-linked substituents at
the C-2 position, which are very potent inhibitors of 5-
lipoxygenase (the enzyme catalyzes the first step in the
oxidation of arachidonic acid to leukotrienes) and also
inhibit cyclooxygenase. An example of compounds from
this series is DuP 654 (2-benzyl-naphthalen-1-ol).8
Therefore, our synthesized naphthols were evaluated
Keywords: Naphthol; Anti-inflammatory; Activity; Molecular model-
ing; Cyclooxygenase I; Cyclooxygenase II; Synthesis.
*
Corresponding authors. Tel.: +662 9428900x211; fax: +662 5793955
(N.K.); tel.: +662 9428900x433; fax: +662 5793955 (C.S.); e-mail
addresses: fscinpk@ku.ac.th; fscicsm@ku.ac.th
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.054

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B. Kongkathip et al. / Bioorg. Med. Chem. 13 (2005) 2167–2175
OR1
methoxy group on the naphthalene ring and the pres-
ence of the hydroxyl group or methoxy group on the
C-30 position.
CH3
O
OH
OH
OR4
R2 R3
MeO
Type I naphthol
R1 = H ; R2, R3, R4 = H or Me
DuP 654
Naproxen
2. Results and discussion
2.1. Chemistry
Figure 1. Structures of DuP 654, naproxen and naphthol.
for anti-inflammatory activity and it was found that our
type I naphthol (Fig. 1) showed selective inhibition of
COX-2 over COX-1. So molecular docking procedure
of the tested naphthols was applied in order to establish
relationships between different modes of binding and the
activities of the compounds. We expect that the results
could provide a model for binding of naphthol deriva-
tives to COX-2 and assist the design of more potent or
selective analogs prior to synthesis.
We have previously reported6,7 the synthesis of naph-
thols 2 and 3 starting from naphthalen-1-ol (1) in three
steps6 as well as the synthesis of naphthol 7 starting
from 1-hydroxy-2-naphthoic acid (6) in six steps.7
In this experiment, methylation of compounds 2 and 3
gave the methyl naphthyl ethers 4 and 5, respectively
in quantitative yield. Compounds 3 and 5 were obtained
as racemic mixtures. Refluxing of naphthol 7 with
methyl iodide in the presence of potassium carbonate
gave methyl naphthyl ether 8 in 98% yield (Scheme 1).
Here, we report our synthesized anti-inflammatory naph-
thol derivatives, which are 2-(30-hydroxypropyl)-naph-
thalen-1-ol (2), 2-(30-hydroxy-20-methylpropyl)-naphthalen-
1-ol (3), 2-(30-hydroxy-20,20-dimethylpropyl)-naphtha-
len-1-ol (7), 2-(30-methoxypropyl)-naphthalen-1-ol (10)
and 2-(30-methoxy-20,20-dimethylpropyl)-naphthalen-1-
ol (13). These five naphthols can be prepared in several
successive steps with significantly high yield in each
step.6,7 To demonstrate whether the compounds and
their methyl naphthyl ether derivatives preferentially in-
hibit COX-2 isozyme over COX-1, we challenged mur-
ine COX-1 null and COX-2 null cell lines with these
compounds and measured the reduction in the amount
of prostaglandin E2(PGE2), which was used as an indi-
cator of COX-2 and COX-1 inhibition. Their inhibitory
effects on COX-1 and COX-2 were compared with a
commercially available NSAID, naproxen, which has a
naphthalene nucleus as a backbone. The biological
structure–activity relationships (SARs) are discussed
with respect to four features of the naphthol molecule:
the nature of the C-20 substituents, substitution on the
naphthalene ring, the presence of the C-1 hydroxyl or
Naphthol 10 was prepared from naphthol 2 by methyl-
ation at both hydroxyl groups and then selective
demethylation of the resulting methoxy-naphthalene 9
by using boron tribromide (Scheme 2). Naphthol 13
was synthesized from naphthol 7 in high yield. Selective
protection of the naphthol function as benzyl naphthyl
ether 11 followed by methylation of the primary hydr-
oxy group afforded compound 12 in 94% yield. Deben-
zylation of the benzyl ether 12 provided naphthol 13
in 87% yield (Scheme 3).
2.2. Bioactivity
To test whether these compounds exhibited anti-inflam-
matory activities, we used the COX-1 deficient and
COX-2 deficient murine fibroblast cell lines to investi-
gate the relative potencies and selectivity of these com-
pounds.9 The reduction of PGE2 produced in these
two cell lines corresponds to the inhibition of COX-2
and COX-1 enzymes.10 DMSO (0.1%) and aspirin were
OH
OH
OMe
3 steps
Methylation
OH
OH
R
R
2
3
R=H
R=Me
4
5
R=H (quanti)
R=Me (quanti)
1
OMe
OH
OH
CO2H
Methylation
6 steps
OH
OH
8
(98%)
OH
7
6
Scheme 1. Synthesis of naphthols 2, 3, 7 and naphthyl ethers 4, 5, 8.
OH
OMe
a
OH
b
OMe
OMe
2
9 (93%)
10 (45%)
Scheme 2. Reagents and conditions: (a) MeI, NaH, reflux, 3 h; (b) BBr3, CH2Cl2, 0 ꢁC, 1 h.

B. Kongkathip et al. / Bioorg. Med. Chem. 13 (2005) 2167–2175
2169
OH
OBn
used as control for 100% COX activities and as positive
control, respectively. Compounds 2 and 3 showed simi-
lar potency against COX-2 with IC50 values in the range
of 4.2–4.6 lM while compound 7 was more potent
against COX-2 with IC50 of 1.7 lM (Table 1). All these
compounds inhibited COX-2 in a dose dependent man-
ner (data not shown) and preferentially inhibited COX-2
over COX-1 as demonstrated by the lower COX-2 IC50
values. Their methyl ether derivatives, compounds 4, 5
and 8, showed no significant inhibition of either COX-
1 or COX-2. Naphthalen-1-ol (1) showed approximately
twofold selective inhibition of COX-2 over COX-1.
Naproxen, an NSAID based on a naphthalene structure,
was reported to slightly selectively inhibit COX-1 over
OH
OH
a
7
11 (95%)
b
OH
OBn
c
OMe
OMe
13 (87%)
12 (94%)
Scheme 3. Reagents and conditions: (a) BnCl, K2CO3, acetone, reflux,
5 h; (b) NaH, MeI, THF, reflux, 5 h; (c) TMSCl, KI, CH3CN, rt, 1 h.
Table 1. Structure of naphthol compounds, the experimental activity (IC50 values) of compounds tested as inhibitors of COX-1 and COX-2 and their
cytotoxicity against Vero cells
Compound
Structure
IC50 of COX-1
(lM)a
IC50 of COX-2
(lM)a
Ratio of COX-1/COX-2
IC50 of Vero cells
(lM)
OH
Naphthalen-1-ol (1)
0.97
93.9
0.40
4.2
2.4
215.0
OH
OH
2
22.4
19.1
2
>247.2
OH
OH
3
87.8
3.4
4.6
1.7
171.1
>217.1
—
OH
7
OH
OMe
OMe
OMe
OH
4
Inactive
Inactive
Inactive
15.7
Inactive
Inactive
Inactive
0.25
—
OH
5
—
—
OH
8
—
—
OH
10
13
62.8
>10.0
>231.2
>204.6
OMe
OMe
OH
>40.9
4.1
Br
O
S
O
SC-558
17.7
0.0093
—
—
NH2
N
N
F3C
(continued on next page)

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B. Kongkathip et al. / Bioorg. Med. Chem. 13 (2005) 2167–2175
Table 1 (continued)
Compound
Structure
IC50 of COX-1
(lM)a
IC50 of COX-2
(lM)a
Ratio of COX-1/COX-2
IC50 of Vero cells
(lM)
H3C
Flurbiprofen
Naproxen
2.56
27.8
0.29
43.4
8.8
0.6
—
HOOC
F
OH
>217.1
O
MeO
HOOC
O COCH3
Aspirin
77.7
321.9
0.2
—
a Values are averaged from two to four experiments.
COX-2 and inhibit COX activities in the gastric muco-
sa.11,12 In our test system, naproxen also preferentially
inhibited COX-1 over COX-2 to a slight degree (Fig.
7). Interestingly, both compounds 10 and 13 exhibited
at least 10-fold selective inhibition of COX-2 over
COX-1, suggesting an important role of the C-30meth-
oxy group. While COX-2 is a target of inhibition by
NSAIDs, the harmful side effects such as gastric damage
by currently used NSAIDs are associated with COX-1
inhibition.12–14 When tested for cytotoxicity employing
the colorimet- ric method modified from an anti-cancer
screening method15 with Vero cell line, compound 3
showed toxic effects to Vero cells similar to naphtha-
len-1-ol (1), while compounds 2, 7, 10, 13 and naproxen
are not toxic to Vero cells at concentrations as high as in
the range of 217–247 lM. From our results of COX-1/
COX-2 IC50 ratios and cytotoxicity IC50 values, it is
conceivable that compounds 2, 7 and 10 might confer
less damaging side effects than naproxen. In all of our
experiments, aspirin was employed as a nonselective
COX-2 inhibitor with IC50 values for COX-1 and
COX-2 of 77.7 and 321.9 lM, respectively.
Figure 2. The COX-1 and COX-2 active sites are shown superimposed
(COX-1, blue; COX-2, violet). Two inhibitors are seen: flurbiprofen
(magenta), a nonselective inhibitor, and SC-558 (yellow), a COX-2-
selective inhibitor. Note how the COX-2-selective inhibitor projects
rightward into a side pocket that is not exploited by the nonselective
inhibitor.
(compounds 2, 3 and 7) and COX-2 activity. We have
also done docking experiment of the two stereoisomers
of compound 3 and we have found that both isomers
can occupy the binding site in the same orientation
(Fig. 5).
2.3. Molecular docking
From the results of the inhibitory testing (Table 1), it
was found that the presence of the hydroxyl group at
the C-1 position of the naphthalene ring plays an impor-
tant role in conferring inhibitory activity whereas the
presence of the methoxy group gave no inhibition. Fur-
thermore, the substituent at C-20 of the propyl chain has
some inhibitory effect against the COX-1 and COX-2.
These results were verified by docking experiments as
shown in Figure 4.
The presence of hydroxyl group at C-1 on the naphtha-
lene nucleus seemed to enhance COX-2 selectivity and
inhibition, whereas the methoxy group (compounds 4,
5 and 8) at the same position reduced inhibitory activity
as shown in Figure 4. Investigation on the COX-2
docked conformations revealed that all active com-
pounds have the C-1 hydroxyl group aligned so as to
form hydrogen bond with Val 523 (Figs. 4 and 8). Un-
like the COX-2 selective inhibitory mechanism of SC-
558,16 the selectivity of this series was not contributed
by the group bound to the hydrophilic pocket on the
other side of Val 523. Also in the experiments, it was
found that C-20 dimethyl substituents increased inhibi-
tory activity (compound 7 compared with 2) in support
to the finding of the other researchers.8
Docking outputs showed that compounds having naph-
thalene moieties have the ability to occupy the hydro-
phobic cavity (Fig. 2) formed by residues Phe 381, Tyr
385, Tyr 387, Phe 513 and Ser 530 in both COX-1 and
COX-2 even in the case of naphthalen-1-ol as shown
in Figure 6. This implied that affinity-binding to the
hydrophobic pocket alone would not confer enzyme
inhibition and selectivity. The data from enzyme assay
experiments suggested that there was a relationship be-
tween the types of C-20 substituents on the propyl chain
Almost all active compounds showed very low inhibi-
tion against COX-1. Comparison between the bound

B. Kongkathip et al. / Bioorg. Med. Chem. 13 (2005) 2167–2175
2171
Figure 3. Conformational comparison of SC-558 from the crystal
structure (grey) and that from AutoDock result (yellow).
Figure 5. Superimposition of the docked conformation of the R-
configuration (yellow) and the S-configuration (sky blue) of compound
3, indicating similar binding orientation of both isomers in the COX-2
binding site.
Figure 6. Possible orientations of naphthalen-1-ol in the binding site.
(Left: COX-2, Right: COX-1).
Figure 7. Comparison of the bound conformation of naproxen in the
binding site. (Left: COX-2, Right: COX-1).
Figure 4. Comparison of the orientations of bound naphthol (inhibitor
as obtained from AutoDock) in the binding site of COX-2. Val 523 is
colored in blue. The hydrogen-bonding interactions are shown as blue
lines. All tested compounds are shown in yellow: compounds 2, 3, 7, 4,
5 and 8. The orientation of the naphthol group of active compounds
(2, 3 and 7) is upward to form hydrogen bonding while the methyl
naphthyl ether group of inactive compounds (4, 5 and 8) is oriented
downward.
conformation of compound 2 in the binding site of
COX-2 and COX-1 (Fig. 8) revealed that residue Ile
523 of COX-1 could not provide hydrogen bonding con-
dition as good as that provided by Val 523 of COX-2.
Table 1 showed that compound 10 has the same potent
inhibitory effect as Flurbiprofen against COX-2 but
compound 10 is much more selective for COX-2 (7
times) than Flurbiprofen. Compound 10 is much less
active than SC-558.17 The hydrophilic and hydrophobic
moieties of compound 10 and Flurbiprofen are almost
of the same configuration whereas compound SC-558,16
which has bezenesulfonamide group as hydrophilic
moiety and bromobenzene group as the hydrophobic
portion has distinctly different configuration than
compound 10.
Figure 8. Comparison of the bound conformation of naphthol 2 in the
binding site. (Left: COX-2, Right: COX-1).
3. Conclusions
The synthesized naphthols 2, 3, 7, 10 and 13 showed
inhibitory activity against cyclooxygenase. Most espe-
cially, compounds 2, 3 and 10 showed significant activity
with preferential COX-2 selectivity, while their methyl
naphthyl ether derivatives (4, 5 and 8) did not inhibit
both COX-1 and COX-2 indicating the negative effect
of an additional methyl group. Furthermore, all active

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B. Kongkathip et al. / Bioorg. Med. Chem. 13 (2005) 2167–2175
compounds showed no cytotoxic effect against Vero cell
lines.
washed with acetone. The filtrate was concentrated in
vacuo, then diethyl ether was added and washed with
water, dried over anhydrous sodium sulfate, filtered
and concentrated in vacuo. The residue was purified
by flash column chromatography.
Molecular modeling experiments indicated no signifi-
cant COX-1 inhibitory effect as a result of having some
polarity (hydroxyl group) at the C-1 position of the
naphthalene nucleus. Contrarily, the hydroxyl groups
enhanced inhibition of COX-2 by hydrogen bonding
to the COX-2 Val 523 side chain. Interestingly, naphthol
10 showed similar inhibitory effect as Flurbiprofen with
both compounds occupying the binding site in the same
orientation. However, COX-2 inhibition of our naph-
thols was lower than that of SC-558 since the com-
pounds had no moiety comparable to the hydrophilic
binding structure of SC-558 bound to the hydrophilic
pocket of COX-2.
4.1.1. 3-(1-Methoxy-naphthalen-2-yl)-propan-1-ol (4).
Yield: quantitative; colourless oil; FTIR (neat, cmꢀ1):
1
3380 (OH), 1572 (C@C), 1085 (C–O), 1056 (C–O); H
NMR (CDCl3, 400 MHz) d 1.87 (m, 2H, CH2), 2.20
(br s, 1H, OH), 2.87 (t, 2H, J = 7.2 Hz, CH2), 3.53 (t,
2H, J = 6.0 Hz, OCH2), 3.89 (s, 3H, OCH3), 7.25 (d,
1H, J = 8.4 Hz, ArH), 7.43 (m, 2H, ArH), 7.53 (d, 1H,
J = 8.4 Hz, ArH), 7.76 (d, 1H, J = 7.7 Hz, ArH), 8.02
(d, 1H, J = 8.2 Hz, ArH); 13C NMR (CDCl3,
100 MHz) d 26.1, 33.9, 62.0, 62.8, 122.5, 125.1, 126.2,
126.7, 128.6, 128.9, 128.4, 130.2, 134.5, 154.0; EIMS
(m/z, %): 216 (M+, 100), 171 (79), 156 (40), 128 (60);
Anal. Calcd for C14H16O2: C, 77.75; H, 7.46. Found:
C, 77.47; H, 7.64.
The structure–activity relationships of the naphthols
analyzed by docking experiments, indicated that the
C-1 hydroxyl group and C-20 dimethyl substituents were
the groups that enhanced the inhibition as shown in
Table 1. The study showed that all active compounds
formed hydrogen bonding with residue Val 523 of the
enzyme of COX-2. In COX-1, no tested compound
clearly gained the benefits of having hydrogen bond with
Ile 523.
4.1.2. 3-(1-Methoxy-naphthalen-2-yl)-2methyl-propan-1-
ol (5). Yield: quantitative; colourless oil; FTIR (neat,
cmꢀ1): 3400 (OH), 1462 (C@C), 1258 (C–O), 1035
(C–O); 1H NMR (CDCl3, 400 MHz) d 0.93 (d, 3H,
J = 6.8 Hz, CH3), 1.94 (m, 1H, CH), 2.32 (br s, 1H,
OH), 2.65 and 2.81 (2 · m, 2 · 1H, CH2), 3.30 (m, 2H,
OCH2), 3.85 (s, 3H, OCH3), 7.20 (d, 1H, J = 8.4 Hz,
ArH), 7.40 (m, 2H, ArH), 7.49 (d, 1H, J = 8.4 Hz,
ArH), 7.73 (d, 1H, J = 7.8 Hz, ArH), 7.98 (d, 1H,
J = 8.3 Hz, ArH); 13C NMR (CDCl3, 100 MHz) d 17.7
(CH3), 33.5 (CH2), 37.5 (CH), 62.8 (OCH3), 66.9
(OCH2), 122.6, 124.9, 126.2, 126.6, 128.6, 129.6 (CH
arom), 128.3, 129.1, 134.5, 154.1 (C arom); EIMS
(m/z, %): 230 (M+, 50), 171 (100), 156 (44), 128 (69);
Anal. Calcd for C15H18O2: C, 78.23; H, 7.88. Found:
C, 78.32; H, 7.92.
The results of this study provide a model for the binding
of naphthol derivatives to COX-2. We are able to estab-
lish the relationships between two types of substituents
(monomethyl and dimethyl) and the mode of interaction
to the amino acid residue Val 523 of COX-2, which have
not been reported before. This finding should be very
useful to design more potent COX-2 selective analogs.
The synthesis of other naphthol derivatives and their
anti-inflammatory evaluation as well as molecular dock-
ing by our group are being currently investigated.
4.2. 3-(1-Methoxy-naphthalen-2-yl)-2,2-dimethyl-propan-
1-ol (8)
4. Experimental
Prepared by the general method for the methylation of
naphthol. Yield: 98%; colourless oil; FTIR (neat,
cmꢀ1): 3437 (OH), 1466 (C@C), 1080 (C–O), 1045 (C–
O); 1H NMR (CDCl3, 400 MHz) d 0.94 (s, 6H,
2 · CH3), 2.71 (s, 2H, CH2), 2.99 (s, 2H, OCH2), 3.11
(br s, 1H, OH), 3.91 (s, 3H, OCH3), 7.22 (d, 1H,
J = 8.4 Hz, ArH), 7.46 (m, 2H, ArH), 7.53 (d, 1H,
J = 8.4 Hz, ArH), 7.79 (m, 1H, ArH), 8.02 (m, 1H,
ArH); 13C NMR (CDCl3, 100 MHz) d 25.8 (2 · CH3),
38.2 (C), 38.7 (CH2), 62.8 (OCH3), 70.0 (OCH2),
122.6, 124.4, 126.3, 126.7, 128.6, 131.1 (CH arom),
127.8, 128.0, 134.7, 154.3 (C arom); EIMS (m/z, %):
244 (M+, 52), 171 (100), 156 (29), 128(47); Anal. Calcd
for C16H20O2: C, 78.65; H, 8.25. Found: C, 78.72, H,
8.09.
General remarks. Melting points were determined on a
Fisher John apparatus and are uncorrected. The IR
spectra were recorded on an FTIR Perkin–Elmer system
2000. Mass spectral data were obtained on the GCMS-
QP-5050A. Nuclear magnetic resonance spectra were re-
corded at 400 MHz on a Brucker Advance DPX-400.
Chemical shifts are given in parts per million (d) down-
field from tetramethylsilane (TMS) as internal standard.
Coupling constants are given in Hertz (Hz). The follow-
ing abbreviations are used: s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, br s = broad sin-
glet. Column chromatography was performed with flash
silica gel (Merck 9385).
4.1. General method for the methylation of naphthol (4
and 5)
4.3. 1-Methoxy-2-(3-methoxy-propyl)-naphthalene (9)
A
mixture of alcohol (0.5 mmol), methyl iodide
To a stirred suspension of sodium hydride (0.14 g,
6 mmol) in dry THF (5 mL), a solution of naphthol 2
(0.2 g, 1 mmol) in dry THF (5 mL) was added dropwise.
After refluxing for 3 h, the reaction mixture was cooled
(2.0 mmol) and potassium carbonate (1 mmol) in ace-
tone (5 mL) was refluxed for 4 h. Then the reaction mix-
ture was cooled to room temperature, filtered and

B. Kongkathip et al. / Bioorg. Med. Chem. 13 (2005) 2167–2175
2173
to room temperature and quenched with water, then
extracted with diethyl ether (3 · 50 mL). The combined
organic phase was washed with water, dried over
anhydrous sodium sulfate, filtered and concentrated in
vacuo. The residue was purified by flash column chro-
matography eluting with 49:1 v/v hexane–ethyl acetate
to afford the methyl ether product (93%) as colourless
oil. FTIR (neat, cmꢀ1): 2931 (CH@C), 1450 (C@C),
(s, 2H, OCH2), 7.34 (d, 1H, J = 8.4 Hz, ArH), 7.43–
7.62 (m, 7H, ArH), 7.65 (d, 1H, J = 8.4 Hz, ArH),
7.91 (m, 1H, ArH), 8.18 (m, 1H, ArH); 13C NMR
(CDCl3, 100 MHz) d 25.7 (2 · C), 38.2, 39.0, 69.9,
77.5, 122.6, 124.6, 126.4, 126.8, 128.7, 128.8 (2 · C),
129.3, 129.5 (2 · C), 131.1, 128.2, 128.4, 134.7, 137.0,
153.0; EIMS (m/z, %): 320 (M+, 4), 212 (5), 157 (16),
128 (11), 91 (100). HRMS calcd for C22H24O2 [M+H]
321.1849, found 321.1851.
1
1116 (C–O); H NMR (CDCl3, 400 MHz) d 2.00 (m,
2H, CH2), 2.92 (t, 2H, J = 7.7 Hz, CH2), 3.40 (s, 3H,
OCH3), 3.48 (t, 2H, J = 6.4 Hz, OCH2), 3.97 (s, 3H,
OCH3), 7.37 (d, 1H, J = 8.4 Hz, ArH), 7.47 (m, 1H,
ArH), 7.53 (m, 1H, ArH), 7.61 (d, 1H, J = 8.4 Hz,
ArH), 7.85 (m, 1H, ArH), 8.12 (m, 1H, ArH); 13C
NMR (CDCl3, 100 MHz) d 26.9, 31.3, 59.2, 62.6, 72.9,
122.6, 124.6, 126.0, 126.5, 128.6, 128.9, 128.7, 130.8,
134.4, 154.0; EIMS (m/z, %): 230 (M+, 71), 171 (30),
141 (30), 57 (100). HRMS calcd for C15H18O2 [M+Na]
253.1204, found 253.1201.
4.6. 1-Benzyloxy-2-(3-methoxy-2,2-dimethyl-propyl)-
naphthalene (12)
To a stirred suspension of sodium hydride (0.12 g,
5 mmol) in dry THF (10 mL), a solution of compound
11 (0.26 g, 0.8 mmol) in dry THF (5 mL) was added
dropwise. Then methyl iodide (0.3 mL, 5 mmol) was
added dropwise to the reaction mixture. After refluxing
for 5 h, the reaction mixture was cooled to room temper-
ature and quenched with water, then extracted with
diethyl ether (3 · 30 mL). The combined organic layer
was washed with brine and water, dried over anhydrous
sodium sulfate, filtered and concentrated in vacuo.
The residue was purified by flash column chromatogra-
phy eluting with 9:1 v/v hexane–dichloromethane to af-
ford the product 12 (94%) as colourless oil. FTIR (neat,
cmꢀ1): 1457 (C@C), 1183 (C–O), 1109 (C–O); 1H NMR
(CDCl3, 400 MHz) d 1.00 (s, 6H, 2 · CH3), 2.85 (s, 2H,
CH2), 3.11(s, 2H, OCH2), 3.41 (s, 3H, OCH3), 5.05 (s,
2H, OCH2), 7.38 (d, 1H, J = 8.4 Hz, ArH), 7.45 (m,
1H, ArH), 7.51 (m, 4H, ArH), 7.64 (m, 3H, ArH),
7.90 (m, 1H, ArH), 8.17(m, 1H, ArH); 13C NMR
(CDCl3, 100 MHz) d 25.5 (2 · C), 37.7, 39.3, 59.7,
76.4, 82.1, 122.9, 123.9, 126.1, 126.4, 128.2 (2 ·
C), 128.5 (2 · C), 129.2 (2 · C), 131.2, 128.8 (2 · C),
134.6, 138.4, 153.8; EIMS (m/z, %): 334 (M+, 4), 212
(22), 157 (20), 128 (13), 91 (100). HRMS calcd for
C23H26O2 [M+H] 335.2006, found 335.2006.
4.4. 2-(3-Methoxy-propyl)-naphthalen-1-ol (10)
To a stirred solution of the methyl ether 9 (0.2 g,
0.9 mmol) in dry dichloromethane (20 mL) at 0 ꢁC,
boron tribromide (0.3 mL, 3.5 mmol) was added drop-
wise and the solution was stirred for 1 h at the same tem-
perature. Then water was added and extracted with
dichloromethane (3 · 50 mL). The organic phase was
washed with water, dried over anhydrous sodium sul-
fate, filtered and concentrated in vacuo. The residue
was purified by flash column chromatography eluting
with 49:1 v/v hexane–ethyl acetate to afford the desired
product 10 (45%) as a brown oil. FTIR (neat, cmꢀ1):
1
3290 (OH), 1662 (C@C), 1265 (C–O), 1105 (C–O); H
NMR (CDCl3, 400 MHz) d 2.00 (m, 2H, CH2), 2.92 (t,
2H, J = 6.4 Hz, CH2), 3.39 (t, 2H, J = 6.4 Hz, OCH2),
3.49 (s, 3H, OCH3), 7.22 (d, 1H, J = 8.3 Hz, ArH),
7.40 (d, 1H, J = 8.3 Hz, ArH), 7.48 (m, 2H, ArH),
7.79 (m, 1H, ArH), 7.94 (s, 1H, OH), 8.33 (m, 1H,
ArH); 13C NMR (CDCl3, 100 MHz) d 26.0, 29.9, 59.1,
70.4, 120.5, 123.0, 125.6, 126.2, 127.9, 129.3, 120.0,
125.9, 134.2, 151.1; EIMS (m/z, %): 216 (M+, 63), 184
(100), 156 (59), 128 (50). HRMS calcd for C14H16O2
[M+Na] 239.1048, found 239.1046.
4.7. 2-(3-Methoxy-2,2-dimethyl-propyl)-naphthalen-1-ol
(13)
To a mixture of compound 12 (0.12 g, 0.37 mmol),
potassium iodide (0.18 g, 1.1 mmol) in acetonitrile
(6 mL), trimethylsilylchloride (0.14 mL, 1.1 mmol) was
added dropwise and the reaction mixture was stirred
at room temperature for 1 h. After that water was added
to the reaction mixture and extracted with diethyl ether
(3 · 30 mL). The combined organic layer was washed
with saturated sodium hydrogen carbonate and water,
dried over anhydrous sodium sulfate, filtered and con-
centrated in vacuo. The residue was purified by flash col-
umn chromatography eluting with 19:1 v/v hexane–
dichloromethane to afford the desired product 13
(87%) as a colourless solid. Mp 67–68 ꢁC. FTIR (KBr,
cmꢀ1): 3229 (OH), 1569 (C@C), 1290 (C–O), 1083
(C–O); 1H NMR (CDCl3, 400 MHz) d 1.10 (s, 6H,
2 · CH3), 2.78 (s, 2H, CH2), 3.00 (s, 2H, OCH2), 3.50
(s, 3H, OCH3), 7.18 (d, 1H, J = 8.3 Hz, ArH), 7.35 (d,
1H, J = 8.3 Hz, ArH), 7.48 (m, 2H, ArH), 7.79 (m,
1H, ArH), 8.36 (m, 1H, ArH), 8.55 (s, 1H, OH); 13C
NMR (CDCl3, 100 MHz) d 26.3 (2 · C), 36.8, 39.5,
59.8, 79.9, 119.3, 123.2, 125.4, 126.2, 127.8, 131.4,
4.5. 3-(1-Benzyloxy-naphthalen-2-yl)-2,2-dimethyl-pro-
pan-1-ol (11)
A mixture of naphthol 2 (0.5 g, 2 mmol), potassium car-
bonate (0.6 g, 4 mmol) and benzyl chloride (0.5 mL,
4 mmol) in acetone (10 mL) was stirred under refluxing
for 5 h. After that the reaction mixture was cooled to
room temperature. Water was added to the reaction
mixture and then extracted with diethyl ether
(3 · 50 mL). The combined organic phase was washed
with water, dried over anhydrous sodium sulfate, filtered
and concentrated in vacuo. The residue was purified by
flash column chromatography eluting with 23:2 v/v hex-
ane–ethyl acetate to afford the product 11 (95%) as col-
ourless oil. FTIR (neat, cmꢀ1): 3447 (OH), 1467 (C@C),
1077 (C–O), 1044 (C–O); 1H NMR (CDCl3, 400 MHz) d
1.00 (s, 6H, 2 · CH3), 2.78 (s, 2H, CH2), 3.05 (d, 2H,
J = 7.0 Hz, OCH2), 3.41 (t, 1H, J = 7.0 Hz, OH), 5.08

2174
B. Kongkathip et al. / Bioorg. Med. Chem. 13 (2005) 2167–2175
4.10. Molecular modeling
4.10.1. Molecular docking. Crystal structures (1eqh and
1c · 2) from Brookhaven Protein Data Bank (http://
www.rcsb.org/pdb/) provided enzyme structures and
binding site information for COX-2 complexed with
SC-55818 and COX-1 bound to flurbiprofen19,20 as refer-
ence structures. Initial structures of eight naphthol com-
pounds (compounds 2–13 in Table 1) were generated by
molecular modeling software Sybyl 6.8.21 The geome-
tries of these compounds were subsequently optimized
using the semi-empirical parameters AM1.
Figure 9. A possible orientation of naphthol 13 in the binding site of
COX-2. The hydrogen-bonding interaction is shown as a blue line.
Binding conformations of naphthol derivatives with
COX-2 and COX-1 were analyzed by the program
AutoDock 3.0.522,23 using the Lamarckian genetic
algorithm (LGA)23 in conjunction with an empirical
force field to calculate ligand free energy of binding.
Kollman-all-atom24 charges were assigned to enzyme
117.9, 125.9, 134.3, 151.7; EIMS (m/z, %): 244 (M+, 35),
212 (35), 157 (100), 128 (78) 87 (46). HRMS calcd for
C16H20O2 [M+Na] 267.1361, found 267.1359 (Fig. 9).
4.8. Anti-COX (PGHS) assay
electrostatic contributions whereas Gasteiger-Huckel25
¨
4.8.1. Materials. All tissue culture components were pur-
chased from Gibco BRL (Gaithersburg, MD). Aspirin
and calcium ionophore A23187 were purchased from
charges were assigned to all ligands. All calcul-
ations were performed by representing the enzyme
affinity by a 90 · 90 · 90 grid box of 0.25 A grid
spacing.
˚
3
Sigma (St. Louis, MO). H-PGE2 was from NEN Life
Science (Boston, MA) and anti-PGE2 antibody was
from the Upstate Biotechnology (Upstate, NY) or
Sigma (St. Louis, MO).
The conditions applied throughout the docking simula-
tions were those, which reproduced the co-crystals of
flurbiprofen and SC-558 bound to COX-1 and COX-2
enzymes with root mean square deviation (RMSD)
4.8.2. Cell culture and treatment. Immortalized mouse
PGHS-1 (PGHS-1ꢀ/ꢀ) and PGHS-2 (PGHS-2ꢀ/ꢀ) null
cells were seeded at 1 · 105 cells/mL in complete Dub-
elccoÕs Modified Eagle Medium (DMEM) supplemented
with non essential amino acids (0.1 mM), glutamine
(292 mg/L), ascorbic acid (50 mg/L) and 10% FCS in
96-well (83 lL/well) flat-bottomed tissue culture plates.
The cells were incubated at 37 ꢁC in a humidified incu-
bator with 5% CO2 for 72 h. The cells were then washed
with DMEM medium without FCS and preincubated
for 30 min with 83 lL serum-free DMEM medium con-
taining vehicle or drug. Aspirin or the test compounds
were dissolved and serially diluted in ethanol or DMSO
before they were added to the medium. The final con-
centrations of ethanol and DMSO were 1% and 0.1%,
respectively. Following the preincubation period, the
medium was removed and the cells were immediately
treated with serum-free medium containing vehicle or
drug and 2 lM A23187 for 30 min. Medium samples
were then collected from the wells and analyzed for
PGE2 concentration by RIA as previously described.
The test compounds were first screened at 10ꢀ5 g/mL.
IC50 values were further determined for samples
(10ꢀ5 g/mL) that show inhibitory effect on PGE2 pro-
duction. Aspirin, which was found to have PGHS-1
and PGHS-2 IC50 values of 0.014 0.008 and
0.058 0.053 mg/mL, respectively was employed as a
nonselective COX-2 inhibitor.
˚
value of 0.7 A for both cases using flurbiprofen and
SC-558 at the binding sites in 1eqh and 1cx2 as refer-
ences (Fig. 3). The estimated free energy of binding
using these docking conditions for SC-558 binding to
COX-2 was ꢀ11.65 kcal/mol, which was slightly differ-
ent from the value from the literature (ꢀ11.35 kcal/
mol). Therefore, the AutoDock method and the param-
eter set could be extended to search the enzyme binding
conformations for other inhibitors accordingly.
Finally, the docked complexes of inhibitor–enzyme were
selected according to the criteria of interacting energy
combined with geometrical matching quality. All amino
˚
acid residues within a 5.0 A radius of the inhibitor atom
were considered and analyzed for their activity
contributions.
Acknowledgements
This work was supported by the Thailand Research
Fund (TRF), Kasetsart University Research and Devel-
opment Institute (KURDI), and the National Center for
Genetic Engineering and Biotechnology as well as Post-
graduate Education and Research program in Petro-
leum and Petrochemical Technology (MUA-ADB
funds). K.H. is a Ph.D. student under the Royal Golden
Jubilee program, the Thailand Research Fund (TRF).
Biodiversity Research and Training Program (BRT) is
gratefully acknowledged for supporting bioactivity
assays. Postgraduate on Education and Research in
Petroleum and Petrochemical Technology and LCAC
are gratefully acknowledged for software and comput-
ing facilities.
4.9. Cytotoxicity assay
The cytotoxicity assay against Vero cells (ATCC CCL-
81) was performed employing the colorimetric meth-
od.15 IC50 values of the standard compound, ellipticine
was found to be 0.4 0.1 lg/mL.

B. Kongkathip et al. / Bioorg. Med. Chem. 13 (2005) 2167–2175
2175
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