Synthesis of novel naphthoquinone aliphatic amides and esters and their anticancer evaluation

Article abstract of DOI:10.1016/j.ejmech.2012.12.006

Fourteen new naphthoquinone aliphatic amides and seventeen naphthoquinone aliphatic esters were synthesized in nine to ten steps from 1-hydroxy-2- naphthoic acid with 9-25% overall yield for the amides, and 16-21% overall yield for the esters. The key step of the amide synthesis is a coupling reaction between amine and various aliphatic acids using 4-(4,6-dimethoxy-1,3,5-triazin- 2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling agent while for the ester synthesis, DCC/DMAP or CDI was used as the coupling reagent between aliphatic acids and naphthoquinone alcohol. Both naphthoquinone amides and esters were evaluated for their anticancer activity against KB cells. It was found that naphthoquinone aliphatic amides showed stronger anticancer activity than those of the esters when the chains are longer than 7-carbon atoms. The optimum chain of amides is expected to be 16-carbon atoms. In addition, naphthoquinone aliphatic esters with α-methyl on the ester moiety possessed much stronger anticancer activity than the straight chains. Decatenation assay revealed that naphthoquinone amide with 16-carbon atoms chain at 15 μM and 20 μM can completely inhibit hTopoIIα activity while at 10 μM the enzyme activity was moderately inhibited. Molecular docking result also showed the same trend as the cytotoxicity and decatenation assay.

Full text of DOI:10.1016/j.ejmech.2012.12.006

European Journal of Medicinal Chemistry 60 (2013) 271e284
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel naphthoquinone aliphatic amides and esters and
their anticancer evaluation
a
a
a
b
Boonsong Kongkathip , Sunisa Akkarasamiyo , Komkrit Hasitapan , Pichamon Sittikul ,
b
a,
*
Nonlawat Boonyalai , Ngampong Kongkathip
Natural Products and Organic Synthesis Research Unit (NPOS), Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science,
Kasetsart University, 50 Phahon Yothin Rd, Chatuchak, Bangkok 10900, Thailand
a
b
Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Phahon Yothin Rd, Chatuchak, Bangkok 10900, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 September 2012
Received in revised form
Fourteen new naphthoquinone aliphatic amides and seventeen naphthoquinone aliphatic esters were
synthesized in nine to ten steps from 1-hydroxy-2-naphthoic acid with 9e25% overall yield for the
amides, and 16e21% overall yield for the esters. The key step of the amide synthesis is a coupling
reaction between amine and various aliphatic acids using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride (DMTMM) as a coupling agent while for the ester synthesis, DCC/DMAP
or CDI was used as the coupling reagent between aliphatic acids and naphthoquinone alcohol. Both
naphthoquinone amides and esters were evaluated for their anticancer activity against KB cells. It was
found that naphthoquinone aliphatic amides showed stronger anticancer activity than those of the esters
when the chains are longer than 7-carbon atoms. The optimum chain of amides is expected to be
16 November 2012
Accepted 5 December 2012
Available online 12 December 2012
Keywords:
Anticancer activity
Cancer
Naphthoquinone aliphatic amide
Naphthoquinone aliphatic ester
Topoisomerase II
16-carbon atoms. In addition, naphthoquinone aliphatic esters with
possessed much stronger anticancer activity than the straight chains. Decatenation assay revealed that
naphthoquinone amide with 16-carbon atoms chain at 15 M and 20 M can completely inhibit hTopoII
activity while at 10 M the enzyme activity was moderately inhibited. Molecular docking result also
showed the same trend as the cytotoxicity and decatenation assay.
Ó 2012 Elsevier Masson SAS. All rights reserved.
a-methyl on the ester moiety
m
m
a
m
0 0
2 ,2 -substituents [9]. This was the reason why novel naph-
1
. Introduction
0
0
thoquinone aromatic amides with 2 ,2 -dimethyl groups were
synthesized and evaluated for anticancer activity [10]. Although it
was revealed that naphthoquinone aromatic amides exhibited less
Rhinacanthins (1,4-naphthoquinone) and Rhinacanthone
(
1,2-naphthoquinone) isolated from Rhinacanthus nasutus (Thai
0
0
name: Thong Pan Chang) (Acanthaceae) showed biological activi-
ties [1e5], especially anticancer activity. Studies on apoptosis
mechanism of rhinacanthins and rhinacanthone were investigated
anticancer activity than those of aromatic esters with 2 ,2 -
dimethyl substituents [8], it is still worthwhile to further synthe-
size novel naphthoquinone aliphatic amides, as well as to evaluate
their anticancer activity in comparison with those of the aromatic
amides and the aliphatic esters, and to compare them with those of
the naphthoquinone ester and amide series.
[5]. Because of very interesting anticancer activity of rhinacanthins
and rhinacanthone, we have synthesized rhinacanthins [6], rhina-
canthone, 1,2-pyrano, 1,4-pyrano and furanonaphthoquinones [7],
naphthoquinone aromatic and aliphatic esters [6,8] and naph-
thoquinone 2 -cyclopentyl and 2 -cyclohexyl esters [9]. Most of
them exhibited anticancer activity especially naphthoquinone
aromatic esters, whereas some aliphatic esters showed very good
antimalarial activity with lesser or no toxicity against Vero cells [8].
Therefore this paper describes the synthesis of fourteen novel
naphthoquinone aliphatic amides and seventeen naphthoquinone
aliphatic esters starting from commercially available 1-hydroxy-2-
naphthoic acid in nine to ten steps. All thirty-one synthetic
aliphatic amides and esters were tested for cytotoxicity against KB
cells and normal Vero cells in comparison with the aromatic ones.
According to our previous work on naphthoquinone esters and
amides [6,10], the compound exhibited the TopoII inhibition
0
0
0
0
In addition, it was found that 2 ,2 -dimethyl substituents on
the propyl chain showed more potent activity than those of other
activity in corresponding with
and related naphthoquinones [11]. Therefore, in this paper, the
decatenation assay for human Topoisomerase II and molecular
b-lapachone (1,2-naphthoquinone)
*
Corresponding author. Tel.: þ66 25625555x2235; fax: þ66 25793955.
E-mail address: fscinpk@ku.ac.th (N. Kongkathip).
a
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.006

272
B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
docking analysis of some selected naphthoquinone compounds
were also carried out.
2.2. Biological activity
Fourteen novel synthetic naphthoquinone aliphatic amides and
seventeen naphthoquinone aliphatic esters were evaluated for
their cytotoxicity against human cancer cell lines KB (oral cavity
cancer) by the REsazurin Microplate Assay (REMA) [12] and normal
Vero cell lines by green fluorescent protein (GFP)-based assay [13].
The results are shown in Tables 1 and 2.
2
. Results and discussion
2.1. Synthesis
A series of naphthoquinone aliphatic amides was synthesized
and investigated for their anticancer activity. Methylation of 1-
hydroxy-2-naphthoic acid (1) followed by reduction, bromination,
alkylation and then reduction provided amine 6 [10]. Coupling
reactions between 6 and various aliphatic acids 7aen were done
using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride (DMTMM) in methanol to give various amides 8aen with
good to excellent yields (70e99% yield). Then demethylation using
boron tribromide gave naphthol amides 9aen with various acyl
groupsinmoderateto quantitativeyield. Thetarget naphthoquinone
aliphatic amides 11aen (Scheme 1), were obtained by oxidation of
Tables 1 and 2 demonstrate anticancer activity against KB cells
of naphthoquinone aliphatic amides and esters, respectively. The
results showed that chain lengths of amide and ester moieties
affected the anticancer activities. It can be seen that when the
chain lengths are between 3- to 7-carbon atoms, the ester series
(18aee, IC50 ¼ 1.76e41.01
amide series (11aee, IC50 ¼ 28.21e101.86
of amide 11d and ester 18d which showed similar results (IC50
values of 40.23 and 41.01 M, respectively). With respect to the
m
M) inhibited KB cells stronger than the
mM). With the exception
m
aliphatic amides, in contrast to those with shorter chain lengths,
the longer chain aliphatic amides with more than 7-carbon atoms
showed more potent anticancer activities. For example, amide 11f
9
aen with Fremy’s salt followed by hydroxylation with tert-butyl
hydroperoxide (TBHP). It was found that the hydroxylation step of
naphthoquinone aliphatic amides gave lower yields than those of
aromatic amides [10]. This may be accounted for by the easier
hydrolysis of the aliphatic amide group than the aromatic amide
group (less electrophilicity) under basic condition. In comparison to
the aliphatic amides, seventeen naphthoquinone aliphatic esters
were also synthesized by the same method as used in our previous
report [8] (Scheme 2) and their anticancer activity was also
determined.
(IC50 ¼ 24.64
activity than ester 18f (IC50 ¼ 38.29
carbon atoms) and 11n (18-carbon atoms) exhibited very strong
activity with IC50 values of 5.12 and 6.35 M, respectively, whereas
m
M) with an 8-carbon atoms showed stronger
mM) and amides 11m (16-
m
esters 18m (16-carbon atoms) and 18n (18-carbon atoms) showed
much lesser anticancer activities. However, it is interesting to note
that these two esters (18m and 18n) showed very good antima-
larial activity, which has been described in our previous paper [8].
Scheme 1. Synthesis of naphthoquinone aliphatic amides 11aen.

B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
273
Scheme 2. Synthesis of naphthoquinone aliphatic esters 18aen.
Regarding to the results, amides having 16- and 18-carbon atoms
2.3. Human Topoisomerase IIa inhibitory activity
chains could be the optimal chain length for the anticancer
activity.
The decatenation assay was used to determine the inhibitory
activity of novel naphthoquinone aromatic amides and esters
Interestingly, the esters containing
inhibited cancer cells stronger than those of the ones without
-methyl group whereas no difference of the amide series was
detected. In the same manner, most of naturally occurring naph-
thoquinone aliphatic esters containing -methyl isolated from R.
a-methyl on the chain
versus hTopoII
decatenation of long-chain, catenated DNA molecules into free
relaxed and supercoiled forms [14]. Fig. 2 shows hTopoII inhibi-
tory activity against four selected compounds (11c, 11m, 18c and
18m) at final concentration of 50 M. It can be clearly seen that
compound 11m, which is a naphthoquinone aliphatic amide with
16-carbon atoms side chain long, exhibited strong hTopoII inhib-
a. Enzyme hTopoIIa catalyzes the ATP-dependent
a
a
a
nasutus such as rhinacanthin-C, G, H, and K (Fig. 1) showed potent
anticancer activities [3]. Therefore, it would be very useful for
m
further comparison with and without
aliphatic chain of the ester series.
a
-methyl group on the
a
itory activity while compound 18m, a naphthoquinone aliphatic
Table 2 shows that naphthoquinone aliphatic esters with
methyl on the chain (18a, 18b, 18c and 18g, IC50 ¼ 14.38, 4.65, 1.76,
6.78 M, respectively) exhibited more potent cytotoxicity than the
naphthoquinone esters without -methyl group given with the
same chain lengths (18as, 18bs, 18cs and 18f, IC50 ¼ 84.40, 42.98,
7.79, 38.29 M, respectively).
According to our findings, it is evident that naphthoquinone
a
-
ester with 16-carbon atoms side chain long, can moderately inhibit
the hTopoIIa activity. This result correlated well with the cytotox-
icity test in that aliphatic amides with long carbon side chains
showed better cytotoxicity compared to the aliphatic esters. In
contrast to the naphthoquinone aliphatic ester or amide containing
a long side chain, those with shorter side chains, such as
1
m
a
2
m
compounds 11c and 18c, displayed low hTopoIIa inhibitory activity.
aliphatic amides showed stronger anticancer activity than the
aromatic amides [10] and aliphatic esters. In order to gain further
understanding on structureeactivity relationship (SAR), some
naphthoquinone aliphatic amides and esters (i.e. 11c, 11m, 18c
and 18m) were selected for the evaluation of human Topoisom-
However, by comparing between the naphthoquinone aliphatic
ester and amide with short side chains, the naphthoquinone
aliphatic ester can inhibit hTopoIIa activity better than the naph-
thoquinone aliphatic amide. Therefore, the trend of hTopoII
a
inhibition activity is altered when the aliphatic side chain grows.
Taken together, it can be concluded that with the short side chain,
the naphthoquinone aliphatic ester is a better hTopoIIa inhibitor
erase II
analysis.
a (hTopoIIa) inhibition and for molecular docking

2
74
B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
Table 1 (continued)
Table 1
Cytotoxicity of the synthetic naphthoquinone aliphatic amides 11aen against KB
cells and normal Vero cells.
Compounds
IC50
(m
M)a
M)a
IC50 (m
Anticancer
Cytotoxicity to
primary cells
(Vero cells)
Compounds
(KB cells)
Anticancer
Cytotoxicity to
primary cells
(KB cells)
(Vero cells)
1
2
2.82
5.10
13.86
9
4.63
Non-cytotoxic
Non-cytotoxic
48.09
1
1k
1
1a
9.81
5.60
1
01.86
1
1l
1
1b
5
6
.12
4
4
7.08
0.23
11m
1
1c
.35b
5.76b
23.94
1
1n
46.58
1
1d
Doxorubicinc
0.96
KB, human epidermoid carcinoma.
a
Data are typical values from six replicate experiments.
Partially soluble in 100% DMSO.
Used as a reference.
2
2
8.21
4.64
24.82
18.42
b
c
1
1e
Table 2
Cytotoxicity of the synthetic naphthoquinone aliphatic esters 18aen against KB cells
and normal Vero cells.
Compounds
IC50
(m
M)a
1
1f
Anticancer
Cytotoxicity to
primary cells
(KB cells)
(Vero cells)
2
1
3.73
9.65
15.54
16.67
1
8
4.38
4.40
123.10
60.06
1
1g
1
8a
1
1h
1
8as
1
1
8.50
7.63
15.04
14.92
4.65
0.87
1
1i
1j
18b
42.98
73.16
1
18bs

B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
Table 2 (continued )
275
Table 2 (continued )
Compounds
IC50
(
m
M)a
Compounds
IC50 (m
M)a
Anticancer
Cytotoxicity to
primary cells
Anticancer
(KB cells)
Cytotoxicity to
primary cells
(Vero cells)
(KB cells)
(Vero cells)
1
.76
0.12
7.84
5
2
7
1.36
7.07
58.43
4.41
1
8c
1
8l
2
7.79
1
8m
1
8cs
6.66
0.96
15.51
23.94
4
1
3
1
1.01
4.42
8.29
6.78
169.07
99.34
1
8n
1
8d
Doxorubicinb
KB, human epidermoid carcinoma.
a
Data are typical values from six replicate experiments.
Used as a reference.
b
1
8e
than the naphthoquinone aliphatic amide. However, when the side
chain is extended, the naphthoquinone aliphatic amide becomes
a potent hTopoII
To gain better understanding in structureeactivity relationship,
compound 11m was further evaluated for hTopoII inhibitory
activity at lower concentrations (5, 10, 15 and 20 M) as shown in
Fig. 3. The compound can completely inhibit hTopoII activity at
final concentrations of 15 M and 20 M while moderately
reducing hTopoII activity at final concentration of 10 M.
However, the compound did not show any hTopoII inhibitory
activity at final concentration of 5 M compared to doxorubicin at
a inhibitor.
>258.75
a
m
1
8f
a
m
m
a
m
a
0.45
m
the same concentration. In comparison with our previous naph-
thoquinone aromatic amides [10], it can be clearly seen that
1
8g
naphthoquinone aliphatic amide 11m is a better hTopoIIa inhibitor
than naphthoquinone aromatic amides. Therefore, molecular
docking of some selected naphthoquinone aliphatic amides and
esters was performed in order to obtain an insight into the binding
3
5
5
3
8.70
5.48
4.83
5.70
149.81
202.64
>233.34
248.54
of the compounds to DNA binding domain of hTopoII
a.
1
8h
2.4. Molecular docking analysis
According to our previous work [6,10], naphthoquinone
aromatic amides and esters were found to bind at the DNA binding
domain of hTopoII . The different spatial arrangement between
a
1
8i
naphthoquinone aromatic amides containing phenyl and naphthyl
groups was observed while for naphthoquinone aromatic esters the
quinone ring was found lying on the right-hand side and the propyl
chain pointing down. With difference in structural rigidity between
naphthoquinone aromatic amides/esters and the aliphatic ones,
molecular docking analysis of hTopoII
a with selected naph-
1
8j
thoquinone aromatic amides and esters was then carried out.
According to Fig. 4, in compounds 11c and 18c, the carbonyl
groups of the amide and ester form hydrogen bonds with R804
located near the active site Y805. The distance between hydrogen
bond donor and acceptor for compound 11c is slightly longer than
compound 18c, indicating a weaker hydrogen bonding interaction.
The amino acid R804 is conserved among TopoII family and the
1
8k

276
B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
Fig. 1. Structures of rhinacanthin-C, -G,-H, and -K.
R804A mutation decreased decatenation and DNA cleavage activity
about 5 and 10 folds, respectively [15]. Unlike compounds 11c and
of amides,16- and 18-carbon atoms, displayed very good anticancer
activity. In comparison with the aromatic amides, the aliphatic
amides showed stronger activity. However, among these, naph-
thoquinone aromatic esters [6] showed the strongest activity. For
18c, compounds 11m and 18m bound to hTopoIIa with slightly
different orientation. Compound 11m forms hydrogen bond with
K798 while no hydrogen bonding is observed in compound 18m
the ester series,
a pronounced effect on anticancer activity than those without
methyl group. The decatenation assay revealed that naph-
thoquinone amide 11m can inhibit hTopoII activity at 10 M,
a-methyl substituent of the aliphatic chain had
which may relate to the weaker hTopoII
a
inhibition compared to
a-
compound 11m. Residues K798 and R804 are solvent-accessible
positively-charged residues located at a loop near DNA recogni-
a
m
tion helix
a
4 of the HTH motif [16]. Collectively, our docking result
inhib-
which was much better than previously reported naphthoquinone
aromatic amides. Therefore, further development of this compound
correlated well with that of cytotoxicity assay and hTopoII
a
itory activity in that naphthoquinone aliphatic amide with longer
chain is a better inhibitor than the ester while with the shorter
chain length, the naphthoquinone aliphatic ester is a more potent
inhibitor than the amide.
can lead to a potent hTopoIIa inhibitor.
4. Experimental section
4.1. Chemistry
3
. Conclusion
The starting material, 1-hydroxy-2-naphthoic acid, was
Fourteen new naphthoquinone amides and seventeen naph-
purchased from FLUKA. All reagents were obtained from FLUKA,
MERCK, ACROSS and ALDRICH. Solvents were dried by distillation
from the appropriate drying reagents immediately prior to use.
Tetrahydrofuran and ether were distilled from sodium and benzo-
phenone under nitrogen atmosphere. Dichloromethane was
distilled from calcium hydride under nitrogen. Dimethylformamide
thoquinone aliphatic esters were synthesized and evaluated for
anticancer activity. Naphthoquinone aliphatic amides were
synthesized from 1-hydroxy-2-naphthoic acid in 9 steps whereas
naphthoquinone aliphatic esters were synthesized as described in
the previous report [8]. Naphthoquinone aliphatic amides with the
shorter length of aliphatic chain (3- to 7-carbon atoms) demon-
strated less anticancer activity than those of aliphatic ones with the
longer chains than 7-carbon atoms. Naphthoquinone aliphatic
amides with the chain length, longer than 7-carbon atoms showed
stronger anticancer activity than aliphatic esters containing linear
chains of the same number of carbons. The optimum aliphatic chain
ꢀ
was distilled under reduced pressure and stored over Type 3 A
molecular sieves. Moisture- and air-sensitive reactions were carried
out under an atmosphere of nitrogen. Analytical thin-layer chro-
matography (TLC) was conducted using Merck TLC aluminum sheet
(silica gel 60 F254). Compounds were visualized by ultraviolet light
and/or by heating the plate after dipping in a 1% solution of vanillin
Fig. 2. Topoisomerase II
concentration of 50
M. Lanes K: kDNA only, lane D: decatenated kDNA only, lane T: kDNA þ hTopoII
and 18m, respectively, lane I: kDNA þ hTopoII þ 50 M doxorubicin and lane L: linearized kDNA marker. Catenated kDNA does not migrate out of the loading wells.
a
inhibitory activity of naphthoquinone aliphatic amides (11c, 11m) and naphthoquinone aliphatic esters (18c, 18m). The compounds were examined in final
m
a
, lanes 11c, 18c, 11m and 18m: kDNA þ hTopoII þ compounds 11c, 18c, 11m
a
a
m

B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
277
Fig. 3. Topoisomerase II
a
inhibitory activity of compound 11m at various concentrations. The compound was examined in final concentration of 5, 10, 15 and 20
m
M, respectively, as
designated. Lanes K: kDNA only, lane D: decatenated kDNA only, lane T: kDNA þ hTopoII
a
, Lane 5, 10, 15, and 20: kDNA þ hTopoII
a
þ compounds 11m at 5, 10, 15 and 20
mM,
respectively, lane I: kDNA þ hTopoII þ 5 M doxorubicin and lane L: linearized kDNA marker. Catenated kDNA does not migrate out of the loading wells.
a
m
in 0.1 M sulfuric acid in ethanol. Flash column chromatography was
performed on silica gel (230e400 mesh, Merck 9385). Infrared (IR)
spectra were recorded on a PerkineElmer 2000 Fourier transform
infrared spectrophotometer. Proton and carbon nuclear magnetic
resonance (NMR) spectra were obtained using a Bruker AVANCE
UNITY
300 MHz spectrometer and VARIAN
INOVA 400 MHz spec-
trometer. Amine 6 and naphthoquinone aliphatic esters 18aen
were prepared as described in previous reports [8,10].
Fig. 4. H-bond interaction observed in DNA-binding domain of hTopoIIa. The amino acids of hTopoIIa are depicted in stick representation and colored by the atom type (carbon,
blue slate; oxygen, red; hydrogen, white and nitrogen, blue). The dotted red lines represent the hydrogen bonding interaction. Compounds 11c, 18c, 11m and 18m are shown in stick
and colored by the atom type (carbon, salmon, deep purple, orange, magenta respectively; oxygen, red; hydrogen, white and nitrogen, blue). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

278
B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
4
4
.2. Synthetic procedures
13C NMR (100 MHz, CDCl
C]O), 155.0 (Ar), 135.1 (ArH), 133.3 (ArH), 132.8 (Ar), 129.3 (Ar),
127.1 (ArH), 126.3 (ArH), 121.6 (Ar), 46.8 (CH ), 38.2 (C), 36.1 (CH),
31.9 (CH
3
):
d
¼ 186.0 (C]O), 180.9 (C]O), 177.2
(
.2.1. General procedure for amide coupling
To a mixture of amine 6 (1 mmol) and carboxylic acids 7aen
2
2
), 26.2 (2ꢁ CH
3
), 19.8 (2ꢁ CH ); IR (KBr) ¼ 3291 (NH),
3
(
1.1 mmol) in methanol (10 mL) was added 4-(4,6-dimethoxy-
,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)
1.1 mmol). The reaction mixture was stirred at room temperature
2968, 2930 (CH), 1668, 1641, 1624 (C]O), 1595, 1577 (C]C), 1272
þ
1
(
(CeN), 1216 (CeO); HRMS (ESI ): m/z [M þ Na] calcd for
19 4
C H23NNaO : 352.1519, found: 352.1517.
for 30 min. The solvent was removed under reduced pressure, and
then the residue was extracted with ethyl acetate. The combined
organic layers were dried over anhydrous Na SO , filtered and
2 4
concentrated under reduced pressure. The crude product was
purified by flash column chromatography to give N-[3-(1-methoxy-
4.2.4.2. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
2,2-dimethylpropyl]-2-methylbutanamide (11b). Flash column chro-
matography, eluting with 2:1 hexane:ethyl acetate afforded the
product 11b (11%) as a yellow amorphous solid, which was recrys-
ꢀ
1
2
-naphthyl)-2,2-dimethylpropyl]amide (8aen). All spectroscopic
tallized from hexane to give yellow crystals, mp 120e121 C;
NMR (400 MHz, CDCl ):
¼ 8.10 (dd, J ¼ 7.6, 1.4 Hz, 1H, ArH), 8.07
dd, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.75 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.68
(td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 6.95 (t, J ¼ 6.7 Hz, 1H, NH), 2.99e2.87
(m, 2H, CH N), 2.52 (s, 2H, CH Ar), 2.32e2.22 (m, 1H, CH),1.80e1.68
(m, 1H, CH ), 1.56e1.43, (m, 1H, CH ),
), 1.21 (d, J ¼ 6.9 Hz, 3H, CH
0.95e0.94 (overlapping, 9H, 3ꢁ CH ):
¼ 186.0 (C]O), 180.9 (C]O), 176.9 (C]O), 155.1 (Ar), 135.1 (ArH),
133.2 (ArH), 132.7 (Ar), 129.3 (Ar), 127.1 (ArH), 126.3 (ArH), 121.5
(Ar), 47.0 (CH ), 43.6 (CH), 38.1 (C), 31.8 (CH ), 27.3 (CH
), 26.2 (2ꢁ
CH ), 17.6 (CH ), 12.0 (CH
H
data of compounds 8aen are shown in the Supplementary data.
3
d
(
4.2.2. General procedure for demethylation reaction of N-[3-(1-
methoxy-2-naphthyl)-2,2-dimethylpropyl] amide (8aen)
2
2
To a solution of N-[3-(1-methoxy-2-naphthyl)-2,2-dimethylpropyl]
2
2
3
ꢀ
13
amide (8aen) (1 mmol) in dry CH
BBr
Cl
2 2
(50 mL) at 0 C was slowly added
3 3
); C NMR (100 MHz, CDCl
3
(2 mmol). The reaction mixturewaswarmed toroomtemperature
d
and stirred under nitrogen atmosphere for 3 h. Water was added. The
organic phase was separated and the aqueous phase was extracted
2
2
2
with CH
Na SO ,filtered and concentrated under reduced pressure to give N-[3-
1-hydroxy-2-naphthyl)-2,2-dimethylpropyl] amide (9aen). The
crude product was used in the next step without further purification.
2 2
Cl . The combined organic layers were dried over anhydrous
3
3
3
); IR (KBr) ¼ 3294 (NH), 2967, 2920, 2873
2
4
(CH), 1668, 1641, 1623 (C]O), 1596, 1566 (C]C), 1272 (CeN), 1215
þ
(
(CeO); HRMS (ESI ): m/z [M þ Na] calcd for C20
4
H25NNaO :
366.1676, found: 366.1692.
4
.2.3. General procedure for Fremy’s salt oxidation reaction of N-[3-
4.2.4.3. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
2,2-dimethylpropyl]-2-methyl pentanamide (11c). Flash column
chromatography, eluting with 2:1 hexane:ethyl acetate afforded
the product 11c (21%) as a yellow amorphous solid, which was
(
1-hydroxy-2-naphthyl)-2,2-dimethylpropyl] amide (9aen)
To a solution of N-[3-(1-hydroxy-2-naphthyl)-2,2-dimethylpropyl]
amide (9aen) (1 mmol) inMeOH(10 mL)andDMF(30 mL) was added
NaOAc (1 M, 10 mL) followed by Fremy’s salt (6 mmol) in water
ꢀ
recrystallized from hexane to give yellow crystals, mp 145e147 C;
1
(
20 mL). After stirring at room temperature for 12 h, the reaction
mixturewasextractedwithdiethylether.Thecombinedorganiclayers
were dried over anhydrous Na SO , filtered and concentrated under
H NMR (400 MHz, CDCl
3
):
d
¼ 8.06 (ddd, J ¼ 7.6, 1.4, 0.4 Hz, 1H,
ArH), 8.03 (ddd, J ¼ 7.6, 1.4, 0.4 Hz, 1H, ArH), 7.71 (td, J ¼ 7.6, 1.4 Hz,
1H, ArH), 7.64 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 6.84 (t, J ¼ 6.7 Hz, 1H,
2
4
reduced pressure. The crude product was purified by flash column
chromatography (silica gel, ethyl acetate:hexane,1:2)to give the N-[3-
NH), 2.93e2.82 (m, 2H, CH
2
N), 2.48 (s, 2H, CH
2
Ar), 2.33e2.25 (m,
), 1.16
), 0.87 (t, J ¼ 7.3 Hz, 3H,
¼ 186.0 (C]O), 180.9 (C]O),
176.8 (C]O), 155.1 (Ar), 135.0 (ArH), 133.2 (ArH), 132.8 (Ar), 129.4
Ar), 127.1 (ArH), 126.3 (ArH), 121.6 (Ar), 46.9 (CH ), 41.8 (CH), 38.1
(C), 36.5 (CH ), 31.8 (CH ), 20.7 (CH ), 18.0 (CH ), 14.0
), 26.1 (2ꢁ CH
(CH
1H, CH), 1.70e1.63 (m, 1H, CH
(d, J ¼ 6.9 Hz, 3H, CH ), 0.90 (s, 6H, 2ꢁ CH
); C NMR (100 MHz, CDCl ):
2
), 1.39e3.28 (m, 1H þ 2H, CH
2
(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-dimethylpropyl] amide
3
3
13
(10aen). All spectroscopic dataofcompounds10aenare showninthe
CH
3
3
d
Supplementary data.
(
2
4.2.4. GeneralprocedureforhydroxylationreactionofN-[3-(1,4-dioxo-
2
2
3
2
3
1,4-dihydronaphthalen-2-yl)-2,2-dimethylpropyl] amide (10aen)
3
); IR (KBr) ¼ 3295 (NH), 2963, 2930, 2871 (CH), 1669, 1642,
To a solution of N-[3-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
,2-dimethylpropyl] amide (10aen) (1 mmol) in THF (13 mL), was
1623 (C]O), 1596, 1566 (C]C), 1271 (CeN), 1215 (CeO); HRMS
þ
2
(ESI ): m/z [M þ Na] calcd for C21
4
H27NNaO : 380.1832, found:
added TBHP (12 mmol) followed by triton B (3 mmol) until the
solution turned red. The reaction mixture was stirred at room
temperature for 20 min and then 10% HCl was added until the
solution turned yellow. The mixture was extracted with ethyl
acetate. The combined organic layers were dried over anhydrous
Na SO , filtered and concentrated under reduced pressure. The
2 4
crude product was purified by flash column chromatography (silica
gel, ethyl acetate:hexane, 1:2) to give N-[3-(3-hydroxy-1,4-dioxo-
380.1836.
4.2.4.4. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
2,2-dimethylpropyl] hexanamide (11d). Flash column chromatog-
raphy, eluting with 2:1 hexane:ethyl acetate afforded the product
11d (22%) as a yellow amorphous solid, which was recrystallized
ꢀ
1
from hexane to give yellow crystals, mp 79e80 C; H NMR
(400 MHz, CDCl ):
3
d
¼ 8.07 (dd, J ¼ 7.6, 1.4 Hz, 1H, ArH), 8.04 (dd,
1,4-dihydronaphthalen-2-yl)-2,2-dimethylpropyl] amide (11aen).
J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.72 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.65 (td,
J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.53 (s, 1H, OH), 6.79 (t, J ¼ 6.8 Hz, 1H, NH),
4
2
.2.4.1. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
,2-dimethylpropyl]-2-methylpropanamide (11a). Flash column
2.87 (d, J ¼ 6.8 Hz, 2H, CH
2
N), 2.48 (s, 2H, CH
2
Ar), 2.24 (t, J ¼ 7.5 Hz,
2H, CH
CH
), 0.91 (s, 6H, 2ꢁ CH
(100 MHz, CDCl ):
2
CO), 1.66 (quintet, J ¼ 7.5 Hz, 2H, CH
2
), 1.35e1.27 (m, 4H, 2ꢁ
1
3
chromatography, eluting with 2:1 hexane:ethyl acetate afforded
the product 11a (9%) as a yellow amorphous solid, which was
2
3
), 0.86 (t, J ¼ 6.5 Hz, 3H, CH
3
); C NMR
3
d
¼ 186.0 (C]O),180.9 (C]O),173.4 (C]O),155.0
ꢀ
recrystallized from hexane to give yellow crystals, mp 127e129 C;
(Ar), 135.1 (ArH), 133.2 (ArH), 132.8 (Ar), 129.3 (Ar), 127.1 (ArH),
126.3 (ArH), 121.6 (Ar), 47.1 (CH ), 38.1 (C), 37.2 (CH ), 31.9 (CH ),
31.6 (CH ), 25.6 (CH ), 22.4 (CH ), 14.0 (CH ); IR
1
H NMR (400 MHz, CDCl
.08 (dd, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.76 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH),
.69 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 6.92 (t, J ¼ 6.7 Hz, 1H, NH), 2.91 (d,
J ¼ 6.7 Hz, 2H, CH N), 2.53 (s, 2H, CH Ar), 2.50 (heptet, J ¼ 6.9 Hz,
H, CH), 1.24 (d, J ¼ 6.9 Hz, 6H, 2ꢁ CH ), 0.95 (s, 6H, 2ꢁ CH );
3
):
d
¼ 8.11 (dd, J ¼ 7.6, 1.4 Hz, 1H, ArH),
2
2
2
8
7
2
), 26.2 (2ꢁ CH
3
2
2
3
(KBr) ¼ 3357 (NH), 2960, 2924, 2855 (CH), 1673, 1660, 1632 (C]O),
þ
2
2
1594, 1578, 1550 (C]C), 1273 (CeN), 1216 (CeO); HRMS (ESI ): m/z
1
3
3
[M þ Na] calcd for C21
4
H27NNaO : 380.1832 found 380.1850.

B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
279
4
2
.2.4.5. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
,2-dimethylpropyl] heptanamide (11e). Flash column chromatog-
J ¼ 7.6, 1.1 Hz, 1H, ArH), 7.76 (td, J ¼ 7.6, 1.3 Hz, 1H, ArH), 7.69 (td,
J ¼ 7.6, 1.3 Hz, 1H, ArH), 6.90 (t, J ¼ 6.3 Hz, 1H, NH), 2.93 (d,
raphy, eluting with 2:1 hexane:ethyl acetate afforded the product
1e (10%) as a yellow amorphous solid, which was recrystallized
J ¼ 6.3 Hz, 2H, CH
2
N), 2.54 (s, 2H, CH
2
Ar), 2.30 (t, J ¼ 7.7 Hz, 2H,
1
CH
2
CO), 1.71 (quintet, J ¼ 7.7 Hz, 2H, CH
), 0.96 (s, 6H, 2ꢁ CH ), 0.86 (t, J ¼ 7.0 Hz, 3H, CH
):
¼ 186.0 (C]O),180.9 (C]O),173.4 (C]O),155.1
(Ar), 135.0 (ArH), 133.2 (ArH), 132.8 (Ar), 129.4 (Ar), 127.1 (ArH),
126.3 (ArH), 121.6 (Ar), 47.1 (CH ), 38.0 (C), 37.3 (CH
), 31.8 (2ꢁ
CH ), 29.4 (CH ), 29.3 (CH ), 29.2 (CH ), 25.9 (CH ),
), 26.2 (2ꢁ CH
22.6 (CH ), 14.0 (CH
2
), 1.40e1.24 (m, 10H, 5ꢁ
ꢀ
1
13
from hexane to give yellow crystals, mp 85e86 C; H NMR
400 MHz, CDCl ):
CH
2
3
3
); C NMR
(
3
d
¼ 8.16 (dd, J ¼ 7.6, 1.4 Hz, 1H, ArH), 8.13 (dd,
(100 MHz CDCl
3
d
J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.81 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.74 (td,
J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.63 (s, 1H, OH), 6.87 (t, J ¼ 6.8 Hz, 1H, NH),
2
2
2
2
CH
.96 (d, J ¼ 6.8 Hz, 2H, CH
H, CH
), 0.99 (s, 6H, 2ꢁ CH
100 MHz, CDCl ):
2
N), 2.57 (s, 2H, CH
2
Ar), 2.32 (t, J ¼ 7.8 Hz,
2
2
2
2
3
2
2
CO), 1.74 (quintet, J ¼ 7.8 Hz, 2H, CH
2
), 1.47e1.31 (m, 6H, 3ꢁ
2
3
); IR (KBr) ¼ 3357 (NH), 2959, 2923, 2853 (CH),
1
3
2
3
), 0.92 (t, J ¼ 6.8 Hz, 3H, CH
3
); C NMR
1665, 1633 (C]O),1594,1578,1550 (C]C),1273 (CeN),1215 (CeO);
þ
(
(
1
3
(
3
d
¼ 186.0 (C]O),180.9 (C]O),173.3 (C]O),155.0
HRMS (ESI ): m/z [M þ Na] calcd for C24
4
H33NNaO : 422.2302,
Ar), 135.1 (ArH), 133.2 (ArH), 132.8 (Ar), 129.3 (Ar), 127.1 (ArH),
26.3 (ArH), 121.6 (Ar), 47.1 (CH ), 38.1 (C), 37.3 (CH ), 31.9 (CH ),
1.6 (CH ), 29.4 (CH ), 25.9 (CH ), 22.5 (CH ), 14.0
CH
found: 422.2320.
2
2
2
2
2
), 26.2 (2ꢁ CH
3
2
2
4.2.4.9. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
2,2-dimethylpropyl]decanamide (11i). Flash column chromatog-
raphy, eluting with 2:1 hexane:ethyl acetate afforded the product
11i (17%) as a yellow amorphous solid, which was recrystallized
3
); IR (KBr) ¼ 3358 (NH), 2928, 2870 (CH), 1673, 1632 (C]O),
þ
1594, 1578, 1549 (C]C), 1274 (CeN), 1216 (CeO); HRMS (ESI ): m/z
[M þ Na] calcd for C22
4
H29NNaO : 394.1989, found: 394.2008.
ꢀ
1
from hexane to give yellow crystals, mp 107e108 C; H NMR
(300 MHz, CDCl ):
4
2
.2.4.6. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
,2-dimethylpropyl] octanamide (11f). Flash column chromatog-
3
d
¼ 8.16 (dd, J ¼ 7.6, 1.1 Hz, 1H, ArH), 8.13 (dd,
J ¼ 7.6, 1.1 Hz, 1H, ArH), 7.81 (td, J ¼ 7.6, 1.6 Hz, 1H, ArH), 7.74 (td,
raphy, eluting with 2:1 hexane:ethyl acetate afforded the product
1f (13%) as a yellow amorphous solid, which was recrystallized
J ¼ 7.6, 1.6 Hz, 1H, ArH), 6.88 (t, J ¼ 6.8 Hz, 1H, NH), 2.96 (d,
1
J ¼ 6.8 Hz, 2H, CH
2
N), 2.57 (s, 2H, CH
2
Ar), 2.32 (t, J ¼ 7.8 Hz, 2H,
ꢀ
1
from hexane to give yellow crystals, mp 113e114 C; H NMR
400 MHz, CDCl ):
CH
2
CO), 1.74 (quintet, J ¼ 7.8 Hz, 2H, CH
), 0.99 (s, 6H, 2ꢁ CH ), 0.89 (t, J ¼ 7.1 Hz, 3H, CH
):
¼ 186.0 (C]O), 180.9 (C]O), 173.5 (C]O),
155.1 (Ar), 135.0 (ArH), 133.2 (ArH), 132.8 (Ar), 129.4 (Ar), 127.1
(ArH), 126.3 (ArH), 121.5 (Ar), 47.0 (CH ), 38.0 (C), 37.2 (CH ), 31.8
(CH ), 29.5 (CH ), 29.4 (CH ), 29.3 (CH ), 26.2 (2ꢁ
), 29.6 (2ꢁ CH
CH ), 25.9 (CH ), 22.6 (CH ), 14.1 (CH
); IR (KBr) ¼ 3358 (NH), 2921,
2853 (CH), 1664, 1631 (C]O), 1594, 1578, 1551 (C]C), 1273 (CeN),
2
), 1.44e1.24 (m, 12H, 6ꢁ
13
(
3
d
¼ 8.09 (dd, J ¼ 7.6, 1.4 Hz, 1H, ArH), 8.06 (dd,
CH
2
3
3
); C NMR
J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.74 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.67 (td,
J ¼ 7.6, 1.4 Hz, 1H, ArH), 6.85 (t, J ¼ 6.3 Hz, 1H, NH), 2.92 (d,
(100 MHz, CDCl
3
d
J ¼ 6.3 Hz, 2H, CH
2
N), 2.53 (s, 2H, CH
2
Ar), 2.27 (t, J ¼ 7.7 Hz, 2H,
2
2
CH
CH
2
CO), 1.70 (quintet, J ¼ 7.7 Hz, 2H, CH
), 0.95 (s, 6H, 2ꢁ CH ), 0.86 (t, J ¼ 6.9 Hz, 3H, CH
):
¼ 186.0 (C]O), 180.9 (C]O), 173.4 (C]O),
55.1 (Ar), 135.1 (ArH), 133.2 (ArH), 132.8 (Ar), 129.4 (Ar), 127.1
ArH), 126.3 (ArH), 121.6 (Ar), 47.1 (CH ), 38.0 (C), 37.3 (CH ), 31.9
CH ), 31.7 (CH ), 29.3 (CH ), 29.0 (CH ), 25.9 (CH ),
), 26.2 (2ꢁ CH
2.6 (CH ), 14.0 (CH
); IR (KBr) ¼ 3355 (NH), 2957, 2925, 2853 (CH),
2
), 1.40e1.21 (m, 8H, 4ꢁ
2
2
2
2
2
1
3
2
3
3
); C NMR
3
2
2
3
(
100 MHz, CDCl
3
d
þ
þ
1
(
(
1216 (CeO); HRMS (ESI ): m/z [M þ H] calcd for C25
4
H36NO :
2
2
414.2644, found 414.2628.
2
2
2
2
3
2
2
2
3
4.2.4.10. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
2,2-dimethylpropyl]undecanamide (11j). Flash column chromatog-
raphy, eluting with 2:1 hexane:ethyl acetate afforded the product
11j (14%) as a yellow amorphous solid, which was recrystallized
1671, 1632 (C]O), 1594, 1578, 1551 (C]C), 1274 (CeN), 1216 (CeO);
þ
HRMS (ESI ): m/z [M þ Na] calcd for C23
4
H31NNaO : 408.2145,
found: 408.2152.
ꢀ
1
from hexane to give yellow crystals, mp 93e94 C; H NMR
(400 MHz, CDCl ):
4
2
.2.4.7. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
,2-dimethylpropyl]-2-methyl octanamide (11g). Flash column
3
d
¼ 8.10 (dd, J ¼ 7.6, 1.1 Hz, 1H, ArH), 8.07 (dd,
J ¼ 7.6, 1.1 Hz, 1H, ArH), 7.75 (td, J ¼ 7.6, 1.1 Hz, 1H, ArH), 7.68 (td,
chromatography, eluting with 2:1 hexane:ethyl acetate afforded
the product 11g (19%) as a yellow amorphous solid, which was
recrystallized from hexane to give yellow crystals, mp 102e104 C;
J ¼ 7.6, 1.1 Hz, 1H, ArH), 6.89 (t, J ¼ 6.3 Hz, 1H, NH), 2.92 (d,
J ¼ 6.3 Hz, 2H, CH
2
N), 2.53 (s, 2H, CH
2
Ar), 2.29 (t, J ¼ 7.7 Hz, 2H,
ꢀ
CH
2
CO), 1.69 (quintet, J ¼ 7.7 Hz, 2H, CH
), 0.95 (s, 6H, 2ꢁ CH ), 0.84 (t, J ¼ 6.3 Hz, 3H, CH
):
¼ 186.0 (C]O), 180.9 (C]O), 173.5 (C]O),
155.1 (Ar), 135.0 (ArH), 133.2 (ArH), 132.8 (Ar), 129.4 (Ar), 127.1
(ArH), 126.3 (ArH), 121.5 (Ar), 47.0 (CH ), 38.0 (C), 37.2 (CH ), 31.8
(2ꢁ CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (2ꢁ CH ), 29.3 (CH ), 26.2 (2ꢁ
CH ), 25.9 (CH ), 22.6 (CH ), 14.1 (CH
2
), 1.40e1.19 (m, 14H, 7ꢁ
1
13
H NMR (400 MHz, CDCl
.09 (dd, J ¼ 7.8, 1.4 Hz, 1H, ArH), 7.77 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH),
.70 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.58 (s, 1H, OH), 6.86 (t, J ¼ 6.6 Hz,
N), 2.54 (s, 2H, CH Ar),
.39e2.30 (m, 1H, CH), 1.79e1.67 (m, 1H, CH ), 1.37e1.22 (m,
H þ 8H, CH ),1.21 (d, J ¼ 6.4 Hz, 3H, CH ), 0.96 (s, 6H, 2ꢁ CH ), 0.86
); C NMR (100 MHz, CDCl ):
¼ 186.0 (C]O),
80.9 (C]O),178.8 (C]O),155.0 (Ar),135.1 (ArH),133.3 (ArH),132.9
Ar), 129.3 (Ar), 127.2 (ArH), 126.3 (ArH), 121.6 (Ar), 47.0 (CH ), 42.1
CH), 38.2 (C), 34.5 (CH ), 31.9 (CH ), 31.8 (CH ), 29.3 (CH ), 27.6
CH ), 26.3 (CH ), 26.2 (CH ), 22.6 (CH ), 18.1 (CH ), 14.1 (CH ); IR
3
):
d
¼ 8.12 (dd, J ¼ 7.8, 1.4 Hz, 1H, ArH),
CH
2
3
3
); C NMR
8
7
1
2
1
(100 MHz, CDCl
3
d
H, NH), 2.94 (dd, J ¼ 6.6, 4.5 Hz, 2H, CH
2
2
2
2
2
2
2
2
2
2
2
3
3
3
2
2
3
); IR (KBr) ¼ 3361 (NH), 2921,
13
(
1
t, J ¼ 7.3 Hz, 3H, CH
3
3
d
2853 (CH), 1665, 1632 (C]O), 1594, 1578, 1550 (C]C), 1273 (CeN),
þ
1215 (CeO); HRMS (ESI ): m/z [M þ Na] calcd for C26
4
H37NNaO :
(
(
(
(
2
450.2615, found: 450.2618.
2
2
2
2
2
3
3
2
3
3
4.2.4.11. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
2,2-dimethylpropyl]dodecanamide (11k). Flash column chromatog-
raphy, eluting with 2:1 hexane:ethyl acetate afforded the product
11k (14%) as a yellow amorphous solid, which was recrystallized
KBr) ¼ 3295 (NH), 2965, 2922, 2855 (CH), 1668, 1642, 1623 (C]O),
þ
1
597, 1560 (C]C), 1271 (CeN), 1214 (CeO); HRMS (ESI ): m/z
[M þ Na] calcd for C24
4
H33NNaO : 422.2302, found 422.2321.
ꢀ
1
from hexane to give yellow crystals, mp 97e98 C; H NMR
(400 MHz, CDCl ):
4
2
.2.4.8. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
,2-dimethylpropyl]nonanamide (11h). Flash column chromatog-
3
d
¼ 8.09 (d, J ¼ 7.6 Hz, 1H, ArH), 8.06 (d,
J ¼ 7.6 Hz, 1H, ArH), 7.73 (td, J ¼ 7.6, 1.1 Hz, 1H, ArH), 7.67 (td, J ¼ 7.6,
1.1 Hz, 1H, ArH), 6.89 (t, J ¼ 6.6 Hz, 1H, NH), 2.92 (d, J ¼ 6.6 Hz, 2H,
raphy, eluting with 2:1 hexane:ethyl acetate afforded the product
1h (25%) as a yellow amorphous solid, which was recrystallized
1
CH
2
N), 2.52 (s, 2H, CH
2
Ar), 2.28 (t, J ¼ 7.4 Hz, 2H, CH
2
CO), 1.68
), 0.94 (s,
3
); C NMR (100 MHz,
ꢀ
1
from hexane to give yellow crystals, mp 111e113 C; H NMR
400 MHz, CDCl ):
¼ 8.11 (dd, J ¼ 7.6, 1.1 Hz, 1H, ArH), 8.08 (dd,
(quintet, J ¼ 7.4 Hz, 2H, CH
2
), 1.40e1.15 (m, 16H, 8ꢁ CH
2
13
(
3
d
6H, 2ꢁ CH ), 0.83 (t, J ¼ 6.7 Hz, 3H, CH
3

280
B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
CDCl
35.0 (ArH), 133.2 (ArH), 132.7 (Ar), 129.4 (Ar), 127.0 (ArH), 126.2
ArH),121.6 (Ar), 47.1 (CH ), 38.0 (C), 37.2 (CH ), 31.8 (CH
), 29.6 (2ꢁ
CH ), 29.5 (CH ), 29.3 (CH ), 26.2 (2ꢁ CH ), 25.9
), 29.4 (3ꢁ CH
CH ), 22.6 (CH ), 14.1 (CH
); IR (KBr) ¼ 3355 (NH), 2917, 2853 (CH),
673, 1662, 1633 (C]O), 1594, 1578, 1551 (C]C), 1273 (CeN), 1216
3
):
d
¼ 186.0 (C]O), 180.9 (C]O), 173.5 (C]O), 155.3 (Ar),
(KBr) ¼ 3317 (NH), 2918, 2849 (CH), 1671, 1645 (C]O), 1594, 1579,
þ
1
(
1554 (C]C), 1274 (CeN), 1214 (CeO); HRMS (ESI ): m/z [M þ Na]
2
2
2
4
calcd for C33H51NNaO : 548.3710, found: 548.3700.
2
2
2
2
3
(
1
2
2
3
4.2.5. Compounds 12e17 were prepared as described in the
previous report [6,7]
þ
(
CeO); HRMS (ESI ): m/z [M þ Na] calcd for C27
H
39NNaO
4
:
Their spectral data are shown in Supplementary data.
4
64.2771, found: 464.2775.
4
.2.6. General procedure for coupling of naphthoquinone alcohol 17
with fatty acids to naphthoquinone esters 18aen [8]
To solution of carboxylic acid (1.3 mmol) and 4-
4
2
.2.4.12. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
,2-dimethylpropyl]tetradecanamide (11l). Flash column chroma-
a
tography, eluting with 2:1 hexane:ethyl acetate afforded the
dimethylaminopyridine (DMAP) (0.3 mmol) in dry dichloro-
methane (10 mL) was added naphthoquinone alcohol 17 (1 mmol)
in dry dichloromethane (10 mL). The reaction mixture was stirred
product 11l (12%) as a yellow amorphous solid, which was recrys-
ꢀ
1
tallized from hexane to give yellow crystals, mp 94e95 C; H NMR
(
(
7
400 MHz, CDCl
ddd, J ¼ 7.6, 1.4, 0.4 Hz, 1H, ArH), 7.77 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH),
.71 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 6.86 (t, J ¼ 6.5 Hz, 1H, NH), 2.94 (d,
N), 2.55 (s, 2H, CH
CO), 1.71 (quintet, J ¼ 7.5 Hz, 2H, CH
), 0.96 (s, 6H, 2ꢁ CH ), 0.87 (t, J ¼ 6.5 Hz, 3H, CH
):
¼ 186.0 (C]O), 180.9 (C]O), 173.4 (C]O),
55.0 (Ar), 135.1 (ArH), 133.2 (ArH), 132.8 (Ar), 129.4 (Ar), 127.1
ArH), 126.3 (ArH), 121.6 (Ar), 47.1 (CH ), 38.1 (C), 37.3 (CH ), 31.9
) 29.5 (CH ), 29.3
), 29.4 (3ꢁ CH
), 25.9 (CH ), 22.7 (CH ), 14.1 (CH ); IR
3
):
d
¼ 8.13 (ddd, J ¼ 7.6, 1.4, 0.4 Hz, 1H, ArH), 8.10
at room temperature for 5 min when a solution of 1,3-
dicyclohexylcarbodiimide (DCC) (1.3 mmol) in dry dichloro-
methane (15 mL) was added. Stirring was continued overnight at
room temperature. The precipitate of dicyclohexylurea was filtered
off and the filtrate washed with saturated ammonium chloride
solution then water. The organic layer was dried over anhydrous
Na SO , filtered and concentrated under reduced pressure. The
2 4
crude product was purified by flash column chromatography (silica
gel) to give the target product, naphthoquinone aliphatic esters.
J ¼ 6.5 Hz, 2H, CH
2
2
Ar), 2.30 (t, J ¼ 7.5 Hz, 2H,
), 1.43e1.17 (m, 20H, 10ꢁ
CH
CH
(
2
2
1
3
2
3
3
); C NMR
100 MHz, CDCl
3
d
1
(
(
(
(
2
2
CH
CH
2
2
), 29.7 (2ꢁ CH
2
), 29.6 (2ꢁ CH
2
2
2
), 26.4 (2ꢁ CH
3
2
2
3
4.2.6.1. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dimethylpropyl-2-methyl propanoate (18a). Flash column chroma-
tography, eluting with 30:1 hexane:ethyl acetate afforded the
product 18a (86%) as a yellow gum, which was recrystallized from
KBr) ¼ 3354 (NH), 2918, 2851 (CH), 1673, 1661, 1631 (C]O), 1594,
þ
1
578, 1552 (C]C), 1273 (CeN), 1216 (CeO); HRMS (ESI ): m/z
[M þ Na] calcd for C29
4
H43NNaO : 492.3084, found: 492.3072.
ꢀ
hexaneedichloromethane to give yellow crystals, mp 70.5e71.5 C;
1
4
2
.2.4.13. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
,2-dimethylpropyl]hexadecanamide (11m). Flash column chroma-
H NMR (400 MHz, CDCl
3
):
d
¼ 8.13 (dd, J ¼ 7.5,1.2 Hz,1H, ArH), 8.10
(dd, J ¼ 7.5, 1.2 Hz, 1H, ArH), 7.77 (td, J ¼ 7.5, 1.2 Hz, 1H, ArH), 7.70
(td, J ¼ 7.5, 1.2 Hz, 1H, ArH), 7.44 (s, 1H, OH), 3.85 (s, 2H, OCH ), 2.69
(s, 2H, CH
Ar), 2.54 (heptet, J ¼ 7.0 Hz,1H, CH),1.17 (d, J ¼ 7.0 Hz, 6H,
2ꢁ CH ), 0.99 (s, 6H, 2ꢁ CH
(C]O), 181.2 (C]O), 177.0 (C]O), 154.3 (Ar), 134.9 (ArH), 132.9
(ArH, Ar), 129.3 (Ar), 127.0 (ArH), 126.1 (ArH), 121.6 (Ar), 72.4 (CH ),
37.0 (C), 34.2 (CH), 31.8 (CH ); IR
tography, eluting with 2:1 hexane:ethyl acetate afforded the
product 11m (19%) as a yellow amorphous solid, which was
2
2
ꢀ
1
13
recrystallized from hexane to give yellow crystals, mp 94e95 C; H
NMR (400 MHz, CDCl ):
¼ 8.11 (ddd, J ¼ 7.6, 1.3, 0.4 Hz, 1H, ArH),
.03 (ddd, J ¼ 7.6, 1.3, 0.4 Hz, 1H, ArH), 7.76 (td, J ¼ 7.6, 1.4 Hz, 1H,
3
3
); C NMR (100 MHz, CDCl
3
):
d
¼ 184.8
3
d
8
2
ArH), 7.69 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 6.89 (t, J ¼ 6.4 Hz, 1H, NH),
2
), 25.0 (2ꢁ CH
3
), 19.0 (2ꢁ CH
3
2
2
1
(
1
(
(
.93 (d, J ¼ 6.4 Hz, 2H, CH
H, CH
CO), 1.71 (quintet, J ¼ 7.7 Hz, 2H, CH
2ꢁ CH ), 0.96 (s, 6H, 2ꢁ CH ), 0.86 (t, J ¼ 6.7 Hz, 3H, CH
100 MHz, CDCl ):
¼ 186.0 (C]O), 180.9 (C]O), 173.4 (C]O),
55.1 (Ar), 135.0 (ArH), 133.2 (ArH), 132.8 (Ar), 129.4 (Ar), 127.1
ArH), 126.3 (ArH), 121.5 (Ar), 47.1 (CH ), 38.0 (C), 37.3 (CH ), 31.9
2ꢁ CH ), 29.7 (3ꢁ CH ), 29.6 (3ꢁ CH ), 29.5 (CH ), 29.4 (2ꢁ CH ),
9.3 (CH ), 25.9 (CH ), 22.6 (CH ), 14.0 (CH ); IR
2
N), 2.54 (s, 2H, CH
2
Ar), 2.30 (t, J ¼ 7.7 Hz,
(KBr) ¼ 3362 (OH), 2969, 2932, 2873 (CH), 1729, 1666, 1644 (C]O),
1594, 1466, 1371, 1275 (C]C), 1216, 1151 (CeO); MS (EI), m/z (%
2
2
), 1.42e1.21 (m, 24H,
1
3
þ
2
3
3
); C NMR
relative intensity): 331 ([M þ H] , 21), 243 (74), 187 (100), 159 (45),
3
d
145 (33); Anal. Calcd for C19
6.31.
22 5
H O : C 69.07, H 6.71. Found: C 69.26, H
2
2
2
2
2
2
2
4.2.6.2. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dimethylpropyl propanoate (18as). Flash column chromatography,
eluting with 49:1 hexane:ethyl acetate afforded the product 18as
(90%) as a yellow gum, which was recrystallized from hexanee
2
2
), 26.2 (2ꢁ CH
3
2
2
3
(
1
KBr) ¼ 3355 (NH), 2918, 2850 (CH), 1673, 1662, 1631 (C]O), 1594,
þ
578, 1551 (C]C), 1273 (CeN), 1216 (CeO); HRMS (ESI ): m/z
ꢀ
1
[M þ Na] calcd for C31
H
47NNaO
4
: 520.3397, found: 520.3393.
dichloromethane to give yellow crystals, mp 89e90 C; H NMR
400 MHz, CDCl ):
¼ 8.13 (dd, J ¼ 7.6, 1.2 Hz, 1H, ArH), 8.10 (dd,
J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.77 (td, J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.70 (td,
J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.43 (s, 1H, OH), 3.86 (s, 2H, OCH ), 2.69 (s,
2H, CH ), 1.13 (t, J ¼ 7.6 Hz, 3H, CH ),
Ar), 2.31 (q, J ¼ 7.6 Hz, 2H, CH
0.91 (s, 6H, 2ꢁ CH ); C NMR (100 MHz, CDCl ): ¼ 184.8 (C]O),
181.2 (C]O), 174.5 (C]O), 154.2 (Ar), 135.0 (ArH), 132.9 (ArH, Ar),
129.3 (Ar), 127.0 (ArH), 126.1 (ArH), 121.7 (Ar), 72.5 (CH ), 36.8 (C),
31.9 (CH ), 27.6 (CH ), 9.2 (CH
); IR (KBr) ¼ 3323
(
3
d
4
2
.2.4.14. N-[3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-
,2-dimethylpropyl]octadecanamide (11n). Flash column chroma-
2
tography, eluting with 2:1 hexane:ethyl acetate afforded the
product 11n (25%) as a yellow amorphous solid, which was
2
2
3
13
3
3
d
ꢀ
1
recrystallized from hexane to give yellow crystals, mp 95e96 C; H
NMR (400 MHz, CDCl ):
¼ 8.06 (ddd, J ¼ 7.6, 1.4, 0.4 Hz, 1H, ArH),
.04 (ddd, J ¼ 7.6, 1.4, 0.4 Hz, 1H, ArH), 7.71 (td, J ¼ 7.6, 1.4 Hz, 1H,
3
d
2
8
2
2
), 25.0 (2ꢁ CH
3
3
ArH), 7.65 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 6.84 (t, J ¼ 6.7 Hz, 1H, NH),
(OH), 2985, 2953, 2925 (CH), 1708, 1668, 1646 (C]O), 1592, 1460,
1368, 1271 (C]C), 1213, 1022 (CeO); MS (EI), m/z (% relative
2
2
1
(
.88 (d, J ¼ 6.7 Hz, 2H, CH
H, CH
CO), 1.66 (quintet, J ¼ 7.7 Hz, 2H, CH
), 0.91 (s, 6H, 2ꢁ CH ), 0.81 (t, J ¼ 6.7 Hz, 3H, CH
100 MHz, CDCl ):
¼ 186.0 (C]O), 180.9 (C]O), 173.5 (C]O),
55.1 (Ar), 135.1 (ArH), 133.2 (ArH), 132.8 (Ar), 129.4 (Ar), 127.1
ArH), 126.3 (ArH), 121.5 (Ar), 47.1 (CH ), 38.1 (C), 37.3 (CH ), 31.9
2ꢁ CH ), 29.7 (8ꢁ CH ), 29.6 (CH ), 29.5 (CH ), 29.4 (CH ), 29.3
CH ), 26.0 (CH ), 22.7 (CH ), 14.1 (CH ); IR
), 26.2 (2ꢁ CH
2
N), 2.49 (s, 2H, CH
2
Ar), 2.25 (t, J ¼ 7.7 Hz,
þ
2
2
), 1.37e1.11 (m, 28H,
intensity): 316 (M , 5), 257 (5), 244 (11), 243 (100), 187 (2); Anal.
1
3
4ꢁ CH
2
3
3
); C NMR
20 5
Calcd for C18H O : C 68.34, H 6.37. Found: C 68.46, H 6.49.
3
d
1
4.2.6.3. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dimethylpropyl-2-methyl butanoate (18b). Flash column chroma-
tography, eluting with 25:1 hexane:ethyl acetate afforded the
product 18b (81%) as a yellow gum, which was recrystallized from
(
(
(
2
2
2
2
2
2
2
2
3
2
2
3

B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
281
ꢀ
); 13
C
3
hexaneedichloromethane to give yellow crystals, mp 70.5e71.5 C;
(m, 2H, CH
NMR (100 MHz, CDCl
154.2 (Ar),135.0(ArH),133.0 (ArH),132.9 (Ar),129.3 (Ar),127.0(ArH),
126.1 (ArH), 121.7 (Ar), 72.5 (CH ), 36.8 (C), 34.1 (CH ), 32.0 (CH ),
27.0 (CH ), 22.3 (CH ), 13.7 (CH
); IR (KBr) ¼ 3373
2
), 0.99 (s, 6H, 2ꢁ CH
3
), 0.88 (t, J ¼ 7.3 Hz, 3H, CH
1
H NMR (400 MHz, CDCl
3
):
d
¼ 8.13 (ddd, J ¼ 7.5, 1.2, 0.6 Hz, 1H,
3
):
d
¼ 184.8 (C]O),181.2 (C]O),173.8 (C]O),
ArH), 8.10 (ddd, J ¼ 7.5, 1.2, 0.6 Hz, 1H, ArH), 7.77 (td, J ¼ 7.5, 1.2 Hz,
1
H, ArH), 7.70 (td, J ¼ 7.5, 1.2 Hz, 1H, ArH), 7.45 (s, 1H, OH), 3.87 (s,
H, OCH ), 2.69 (s, 2H, CH
), 1.52e1.38 (m, 1H, CH
2
2
2
2
2
2
Ar), 2.35 (hextet, J ¼ 7.0 Hz, 1H, CH),
2
), 25.0 (2ꢁ CH
3
2
3
1
3
(
.73e1.60 (m, 1H, CH
H, CH
), 0.99 (s, 6H, 2ꢁ CH
100 MHz, CDCl ):
2
2
), 1.14 (d, J ¼ 7.0 Hz,
(OH), 2957, 2925, 2865 (CH), 1739, 1666, 1647 (C]O), 1596, 1461,
1374, 1275 (C]C), 1217, 1157 (CeO); MS (EI), m/z (% relative inten-
13
3
3
), 0.89 (t, J ¼ 7.2 Hz, 3H, CH
3
); C NMR
þ
3
d
¼ 185.5 (C]O), 181.9 (C]O), 177.3 (C]O),
sity): 344 (M , 5), 243 (100), 225 (9),187 (13),159 (2); Anal. Calcd for
1
54.9 (Ar), 135.6 (ArH), 133.6 (ArH, Ar), 130.0 (Ar), 127.7 (ArH), 126.7
ArH), 122.3 (Ar), 73.1 (CH ), 41.9 (CH), 37.6 (C), 32.6 (CH ), 27.3
CH ), 17.2 (CH ), 12.2 (CH ); IR (KBr) ¼ 3371 (OH),
), 25.6 (2ꢁ CH
967, 2932, 2873 (CH), 1731, 1667, 1650 (C]O), 1594, 1461, 1368,
274 (C]C), 1217, 1049 (CeO); MS (EI), m/z (% relative intensity):
20 24 5
C H O : C 69.75, H 7.02. Found: C 69.94, H 7.36.
(
(
2
2
2
3
3
3
4.2.6.7. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dimethylpropyl hexanoate (18d). Flash column chromatography,
eluting with 30:1 hexane:ethyl acetate afforded the product 18d
(74%) as a yellow gum, which was recrystallized from hexanee
2
1
3
þ
44 (M , 10), 243 (100), 225 (43), 187 (97), 159 (71); Anal. Calcd for
: C 69.75, H 7.02. Found: C 96.64, H 7.20.
ꢀ
1
C
H O
20 24 5
dichloromethane to give yellow crystals, mp 54.5e55.5 C;
NMR (400 MHz, CDCl ):
¼ 8.12 (dd, J ¼ 7.6, 1.4 Hz, 1H, ArH), 8.08
(dd, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.77 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.69
),
H
3
d
4
.2.6.4. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dimethylpropyl butanoate (18bs). Flash column chromatography,
eluting with 14:1 hexane:ethyl acetate afforded the product 18bs
(td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.62 (br s, 1H, OH), 3.87 (s, 2H, OCH
2
2.69 (s, 2H, CH
2H, CH
), 1.31e1.25 (m, 4H, 2ꢁ CH
J ¼ 6.7 Hz, 3H, CH
2
Ar), 2.27 (t, 2H, J ¼ 7.5 Hz, CH
2
), 1.51 (quintet, J ¼ 7.5,
(
87%) as a yellow gum, which was recrystallized from hexanee
2
2
), 1.00 (s, 6H, 2ꢁ CH
3
), 0.89 (t,
¼ 185.5 (C]O),
ꢀ
1
13
dichloromethane to give yellow crystals, mp 66e67 C; H NMR
400 MHz, CDCl ):
¼ 8.16e8.04 (m, 1H, ArH), 8.04e8.01 (m, 1H,
ArH), 7.69 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.62 (td, J ¼ 7.6, 1.4 Hz, 1H,
ArH), 7.37 (s, 1H, OH), 3.79 (s, 2H, OCH ), 2.61 (s, 2H, CH Ar), 2.18 (t,
J ¼ 7.4 Hz, 2H, CH ), 1.59e1.47 (m, 2H, CH ), 0.92 (s, 6H, 2ꢁ CH ),
); C NMR (100 MHz, CDCl ):
¼ 184.8
C]O), 181.2 (C]O), 173.7 (C]O), 154.2 (Ar), 135.0 (ArH), 132.9
ArH, Ar), 129.3 (Ar), 127.0 (ArH), 126.1 (ArH), 121.7 (Ar), 72.5 (CH ),
6.8 (C), 36.3 (CH ), 31.9 (CH ),18.4 (CH ),13.7 (CH );
3
); C NMR (100 MHz, CDCl
3
):
d
(
3
d
181.8 (C]O), 174.6 (C]O), 154.9 (Ar), 135.6 (ArH), 133.5 (ArH, Ar),
130.0 (C), 127.6 (ArH), 126.7 (ArH), 122.4 (Ar), 73.2 (CH ), 37.5 (C),
35.0 (CH ), 32.6 (CH ), 31.9 (CH ), 25.3 (CH ), 22.9
), 25.7 (2ꢁ CH
(CH ), 14.5 (CH
2
2
2
2
2
2
3
2
2
2
3
2
3
); IR (KBr) ¼ 3366 (OH), 2940, 2865 (CH), 1738, 1644
13
0
.85 (t, J ¼ 7.4 Hz, 3H, CH
3
3
d
(C]O), 1372, 1272 (C]C), 1212, 1155 (CeO); MS (EI), m/z (% relative
þ
(
(
intensity): 358 (M , 2), 243 (100), 225 (12), 187 (16), 158 (2), 149
2
(18); Anal. Calcd for C21
7.57.
26 5
H O : C 70.37, H 7.31. Found: C 70.49, H
3
2
2
), 25.0 (2ꢁ CH
3
2
3
IR (KBr) ¼ 3378 (OH), 2968, 2932, 2873 (CH), 1701, 1667, 1646 (C]
O), 1593, 1459, 1367, 1271 (C]C), 1214, 1015 (CeO); MS (EI), m/z (%
relative intensity): 330 (M , 11), 243 (9), 229 (64), 211 (96), 187
4.2.6.8. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dimethylpropyl heptanoate (18e). Flash column chromatography,
eluting with 30:1 hexane:ethyl acetate afforded the product 18e
(83%) as a yellow gum, which was recrystallized from hexanee
þ
(
48), 159 (28), 149 (100); Anal. Calcd for C19
22 5
H O : C 69.07, H 6.71.
Found: C 69.47, H 6.69.
ꢀ
1
dichloromethane to give yellow crystals, mp 57.5e58.5 C;
NMR (400 MHz, CDCl ):
¼ 8.13 (dd, 1H, J ¼ 7.6, 1.1 Hz, ArH), 8.10
(dd,1H, J ¼ 7.6,1.1 Hz, ArH), 7.78 (td, J ¼ 7.6,1.1 Hz,1H, ArH), 7.70 (td,
J ¼ 7.6, 1.1 Hz, 1H, ArH), 7.58 (br s, 1H, OH), 3.87 (s, 2H, OCH ), 2.69
(s, 2H, CH ), 1.60 (quintet, J ¼ 7.4 Hz,
Ar), 2.28 (t, 2H, J ¼ 7.4 Hz, CH
2H, CH ), 1.00 (s, 6H, 2ꢁ CH ), 0.88 (t,
), 1.35e1.21 (m, 6H, 3ꢁ CH
J ¼ 6.9 Hz, 3H, CH ); C NMR (100 MHz, CDCl ): ¼ 185.5 (C]O),
181.8 (C]O), 174.6 (C]O), 154.9 (Ar), 135.6 (ArH), 133.5 (ArH, Ar),
130.0 (C), 127.6 (ArH), 126.7 (ArH), 122.4 (Ar), 73.2 (CH ), 37.5 (C),
35.0 (CH ), 32.6 (CH ), 32.0 (CH ), 29.5 (CH ), 25.6
), 25.7 (2ꢁ CH
(CH ), 23.1 (CH ), 14.6 (CH
H
4
.2.6.5. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
3
d
dimethylpropyl-2-methyl pentanoate (18c). Flash column chroma-
tography, eluting with 30:1 hexane:ethyl acetate afforded the
product 18c (77%) as a yellow gum, which was recrystallized from
2
2
2
ꢀ
1
hexaneedichloromethane to give yellow crystals, mp 57e58 C; H
NMR (400 MHz, CDCl ):
dd, J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.77 (td, J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.70
td, J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.45 (br s, 1H, OH), 3.86 (s, 2H, OCH ),
2
2
3
13
3
d
¼ 8.14 (dd, J ¼ 7.6, 1.2 Hz, 1H, ArH), 8.10
3
3
d
(
(
2
2
2
1
0
.69 (s, 2H, CH
H, CH
.99 (s, 6H, 2ꢁ CH
):
2
Ar), 2.43 (hextet, J ¼ 7.2 Hz, 1H, CH), 1.68e1.59 (m,
2
2
2
2
3
2
), 1.24e1.42 (m, 1H þ 2H, CH
2
), 1.14 (d, J ¼ 7.2 Hz, 3H, CH
3
),
2
2
3
); IR (KBr) ¼ 3370 (OH), 2928, 2858 (CH),
13
3
), 0.88 (t, J ¼ 7.2 Hz, 3H, CH
3
); C NMR (100 MHz,
1739, 1644 (C]O), 1374, 1276 (C]C), 1212, 1156 (CeO); MS (EI), m/z
þ
CDCl
3
d
¼ 185.5 (C]O),181.9 (C]O),177.5 (C]O),154.9 (Ar),135.6
(% relative intensity): 372 (M , 9), 242 (26), 227 (11), 188 (28), 159
(
(
(
(
ArH), 133.6 (ArH, Ar), 130.0 (Ar), 127.7 (ArH), 126.7 (ArH), 122.3
Ar), 73.1 (CH ), 40.1 (CH), 37.6 (C), 36.5 (CH ), 32.6 (CH ), 25.7
CH ), 25.6 (CH ), 21.0 (CH ), 17.6 (CH ), 14.6 (CH
); IR (KBr) ¼ 3381
OH), 2959, 2932, 2863 (CH), 1730, 1660, 1650 (C]O), 1594, 1456,
(12), 113 (100); Anal. Calcd for C22
70.68, H 7.85.
28 5
H O : C 70.94, H 7.58. Found: C
2
2
2
3
3
2
3
3
4.2.6.9. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dimethylpropyl octanoate (18f). Flash column chromatography,
eluting with 20:1 hexane:ethyl acetate afforded the product 18f
(96%) as a yellow gum, which was recrystallized from hexanee
1
370, 1275 (C]C), 1218, 1048 (CeO); MS (EI), m/z (% relative
þ
intensity): 357 ([M ꢂ H] , 29), 243 (100), 225 (46), 187 (99), 159
(
26 5
69); Anal. Calcd for C21H O : C, 70.37; H, 7.31. Found: C, 70.18, H,
ꢀ
1
7.42.
dichloromethane to give yellow crystals, mp 56e57 C; H NMR
400 MHz, CDCl ):
¼ 8.12 (dd, J ¼ 7.6, 1.4 Hz, 1H, ArH), 8.09 (dd,
J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.76 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.69 (td,
J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.44 (s, 1H, OH), 3.85 (s, 2H, OCH ), 2.68 (s,
2H, CH ), 1.63e1.55 (m, 2H, CH ), 1.27
Ar), 2.26 (t, J ¼ 7.6 Hz, 2H, CH
(br s, 8H, 4ꢁ CH ), 0.99 (s, 6H, 2ꢁ CH ), 0.87 (t, J ¼ 6.9 Hz, 3H, CH );
C NMR (100 MHz, CDCl ):
¼ 184.8 (C]O), 181.2 (C]O), 173.9
(C]O), 154.2 (Ar), 134.9 (ArH), 132.9 (Ar), 132.9 (ArH), 129.3 (Ar),
127.0 (ArH), 126.1 (ArH), 121.7 (Ar), 72.5 (CH ), 36.8 (C), 34.4 (CH ),
), 25.0 (2ꢁ CH ), 24.9
); IR (KBr) ¼ 3366 (OH), 2924, 2865 (CH),
(
3
d
4
.2.6.6. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dimethylpropyl pentanoate (18cs). Flash column chromatography,
eluting with 24:1 hexane:ethyl acetate afforded the product 18cs
2
2
2
2
(
45%) as a yellow gum, which was recrystallized from hexanee
2
3
3
ꢀ
1
13
dichloromethane to give yellow crystals, mp 51e52 C; H NMR
400 MHz, CDCl ):
¼ 8.13 (dd, J ¼ 7.6, 1.4 Hz, 1H, ArH), 8.09 (dd,
J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.69 (td, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.67 (td,
J ¼ 7.6,1.4 Hz,1H, ArH), 7.44 (s,1H, OH), 3.85 (s, 2H, OCH ), 2.68 (s, 2H,
CH ),1.65e1.58 (m, 2H, CH ),1.38e1.25
Ar), 2.27 (t, J ¼ 7.6 Hz, 2H, CH
3
d
(
3
d
2
2
2
32.0 (CH
2
), 31.6 (CH
2
), 29.1 (CH
2
), 28.9 (CH
2
3
2
2
2
(CH ), 22.6 (CH
2
),14.0 (CH
2 3

282
B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
1
(
(
735, 1666, 1645 (C]O), 1595, 1461, 1376, 1275 (C]C), 1218, 1156
CeO); MS (EI), m/z (% relative intensity): 385 ([M ꢂ H]þ, 45), 243
100); Anal. Calcd for C23 : C 71.48, H 7.82. Found: C 71.72, H
4.2.6.13. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dimethylpropyl undecanoate (18j). Flash column chromatography,
eluting with 30:1 hexane:ethyl acetate afforded the product 18j
(83%) as a yellow gum, which was recrystallized from hexanee
30 5
H O
7.71.
ꢀ
1
dichloromethane to give yellow crystals, mp 64.5e65.5 C;
NMR (400 MHz, CDCl ):
¼ 8.15 (dd, J ¼ 7.6, 1.1 Hz, 1H, ArH), 8.12
(dd, J ¼ 7.6,1.1 Hz,1H, ArH), 7.80 (td, J ¼ 7.6,1.1 Hz,1H, ArH), 7.73 (td,
J ¼ 7.6, 1.1 Hz, 1H, ArH), 7.46 (s, 1H, OH), 3.88 (s, 2H, OCH ), 2.71 (s,
2H, CH ), 1.70e1.57 (m, 2H, CH ),
Ar), 2.29 (t, J ¼ 7.6 Hz, 2H, CH
1.39e1.22 (m, 14H, 7ꢁ CH ), 1.01 (s, 6H, 2ꢁ CH ), 0.90 (t,
); C NMR (100 MHz, CDCl ):
¼ 184.8 (C]O),
181.2 (C]O), 173.9 (C]O), 154.2 (Ar), 135.0 (ArH), 133.0 (Ar), 132.9
H
4
.2.6.10. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
3
d
dimethylpropyl-2-methyl octanoate (18g). Flash column chroma-
tography, eluting with 25:1 hexane:ethyl acetate afforded the
product 18g (84%) as a yellow gum, which was recrystallized from
2
2
2
2
ꢀ
hexaneedichloromethane to give yellow crystals, mp 57.5e58.5 C;
2
3
1
13
H NMR (400 MHz, CDCl
3
):
d
¼ 8.15e8.11 (m, 1H, ArH), 8.11e8.05
J ¼ 6.8 Hz, 3H, CH
3
3
d
(
1
m, 1H, ArH), 7.76 (td, J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.69 (td, J ¼ 7.6,
.2 Hz,1H, ArH), 3.85 (s, 2H, OCH ), 2.68 (s, 2H, CH Ar), 2.40 (hextet,
J ¼ 7.0 Hz, 1H, CH ), 1.68e1.89 (m, 2H, CH ), 1.40e1.32 (m, 2H, CH ),
), 1.13 (d, J ¼ 7.0 Hz, 3H, CH ), 0.99 (s, 6H,
); C NMR (100 MHz, CDCl ):
¼ 185.5 (C]O), 181.9 (C]O), 177.5 (C]O), 154.9 (Ar), 135.6 (ArH),
33.6 (ArH, Ar), 130.0 (C), 127.7 (ArH), 126.7 (ArH), 122.3 (Ar), 73.1
2
2
(ArH), 129.3 (Ar), 127.0 (ArH), 126.1 (ArH), 121.7 (Ar), 72.5 (CH
2
2
),
),
3
2
2
36.8 (C), 34.4 (CH
29.3 (CH ), 29.2 (CH
(CH ), 14.1 (CH
2
), 32.0 (CH
2
), 31.9 (CH
2
), 29.5 (CH
2
), 29.5 (CH
1
2
d
.31e1.18 (m, 6H, 3ꢁ CH
2
3
2
2
), 29.2 (CH
2
), 25.1 (2ꢁ CH
3
2
), 25.0 (CH ), 22.7
13
ꢁ CH
3
), 0.86 (t, J ¼ 6.8 Hz, 3H, CH
3
3
2
3
); IR (KBr) ¼ 3365 (OH), 2917, 2850 (CH), 1738, 1645
(C]O), 1518, 1462, 1377, 1276 (C]C), 1212, 1157 (CeO); MS (EI), m/z
þ
1
(% relative intensity): 428 (M , 9), 243 (28), 241 (56), 227 (22), 188
(
(
CH
CH
2
), 40.4 (CH), 37.6 (C), 34.4 (CH
2
), 32.6 (CH
2
), 32.3 (CH
2
), 29.8
),
(82), 187 (50), 159 (56), 71 (100); Anal. Calcd for C26
H 8.47. Found: C 72.59, H 8.32.
36 5
H O : C 72.87,
2
), 27.8 (CH ), 25.7 (CH ), 25.6 (CH
2
3
3
), 23.2 (CH
2
), 17.7 (CH
3
1
4.7 (CH
3
); IR (KBr) ¼ 3389 (OH), 2959, 2930, 2856 (CH), 1722, 1650
(
(
(
C]O), 1461, 1373, 1268 (C]C), 1214, 1049 (CeO); MS (EI), m/z
4.2.6.14. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dimethylpropyl dodecanoate (18k). Flash column chromatography,
eluting with 30:1 hexane:ethyl acetate afforded the product 18k
(79%) as a yellow gum, which was recrystallized from hexanee
þ
% relative intensity): 400 ([M ꢂ H] , 29), 243 (100), 225 (46), 187
32 5
99), 159 (69); Anal. Calcd for C24H O : C, 71.97; H, 8.05. Found: C,
7
1.68, H, 8.41.
ꢀ
1
dichloromethane to give yellow crystals, mp 68e69 C; H NMR
(400 MHz, CDCl ):
¼ 8.16 (dd, J ¼ 7.6, 1.3 Hz, 1H, ArH), 8.13 (dd,
J ¼ 7.6, 1.3 Hz, 1H, ArH), 7.80 (td, J ¼ 7.6, 1.3 Hz, 1H, ArH), 7.73 (td,
J ¼ 7.6, 1.3 Hz, 1H, ArH), 7.46 (s, 1H, OH), 3.88 (s, 2H, OCH ), 2.71 (s,
2H, CH ), 1.69e1.59 (m, 2H, CH ),
Ar), 2.29 (t, J ¼ 7.6 Hz, 2H, CH
1.35e1.22 (m, 16H, 8ꢁ CH ), 1.02 (s, 6H, 2ꢁ CH ), 0.91 (t,
); C NMR (100 MHz, CDCl ):
¼ 185.5 (C]O),
181.8 (C]O), 174.5 (C]O), 154.8 (Ar), 135.6 (ArH), 133.6 (Ar), 133.5
(ArH), 130.0 (Ar), 127.6 (ArH), 126.7 (ArH), 122.4 (Ar), 73.2 (CH ),
4
.2.6.11. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
3
d
dimethylpropyl nonanoate (18h). Flash column chromatography,
eluting with 30:1 hexane:ethyl acetate afforded the product 18h
2
(
73%) as a yellow gum, which was recrystallized from hexanee
2
2
2
ꢀ
1
dichloromethane to give yellow crystals, mp 52.5e53.5 C;
NMR (400 MHz, CDCl ):
¼ 8.06 (d, J ¼ 7.6 Hz, 1H, ArH), 8.03 (d,
J ¼ 7.6 Hz,1H, ArH), 7.80 (t, J ¼ 7.6 Hz,1H, ArH), 7.73 (t, J ¼ 7.6 Hz,1H,
ArH), 7.47 (s, 1H, OH), 3.87 (s, 2H, OCH ), 2.71 (s, 2H, CH Ar), 2.29 (t,
J ¼ 7.5 Hz, 2H, CH ), 1.62e1.58 (m, 2H, CH ), 1.28e1.20 (m, 10H, 5ꢁ
CH ), 0.90 (t, J ¼ 6.7 Hz, 3H, CH
), 1.02 (s, 6H, 2ꢁ CH
100 MHz, CDCl ):
¼ 185.5 (C]O), 181.8 (C]O), 174.5 (C]O),
54.9 (Ar), 135.6 (ArH), 133.5 (ArH, Ar), 130.0 (Ar), 127.6 (ArH), 126.7
ArH), 122.4 (Ar), 73.2 (CH ), 37.5 (C), 35.0 (CH ), 32.6 (CH ), 32.4
CH ), 29.8 (CH ), 29.8 (CH ), 29.7 (CH ), 25.6 (CH ),
), 25.7 (2ꢁ CH
3.2 (CH ), 14.7 (CH
); IR (KBr) ¼ 3366 (OH), 2923, 2850 (CH), 1739,
644 (C]O), 1462, 1373, 1268 (C]C), 1212, 1155 (CeO); MS (EI), m/z
H
2
3
13
3
d
J ¼ 6.8 Hz, 3H, CH
3
3
d
2
2
2
2
2
37.5 (C), 35.0 (CH
2
), 32.6 (CH
), 29.9 (2ꢁ CH ), 29.8 (CH
), 14.7 (CH
2
), 32.5 (CH
2
), 30.2 (2ꢁ CH
2
), 30.1
), 23.3
13
2
3
3
); C NMR
(CH
(CH
2
2
2
), 25.7 (2ꢁ CH
3
), 25.6 (CH
2
(
1
(
(
2
3
d
2
3
); IR (KBr) ¼ 3365 (OH), 2920, 2850 (CH), 1735, 1645
(C]O), 1515, 1462, 1378, 1272 (C]C), 1210, 1153 (CeO); MS (EI), m/z
þ
2
2
2
(% relative intensity): 442 (M , 6), 243 (21), 242 (50), 229 (7), 227
2
2
2
2
3
2
(24), 188 (81), 187 (88), 159 (91), 71 (100); Anal. Calcd for C27
C 73.27, H 8.65. Found: C 73.40, H 8.64.
38 5
H O :
2
3
1
þ
(
(
% relative intensity): 400 (M , 4), 242 (28), 227 (18), 188 (58), 187
35), 159 (38), 141 (100); Anal. Calcd for C24 : C 71.97, H 8.05.
4.2.6.15. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dimethylpropyl tetradecanoate (18l). Flash column chromatog-
raphy, eluting with 25:1 hexane:ethyl acetate afforded the product
32 5
H O
Found: C 71.82, H 8.35.
1
8l (80%) as a yellow gum, which was recrystallized from hexanee
ꢀ
1
4
.2.6.12. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
dichloromethane to give yellow crystals, mp 79e80 C; H NMR
(400 MHz, CDCl ):
¼ 8.13 (dd, J ¼ 7.6, 1.2 Hz, 1H, ArH), 8.10 (dd,
J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.77 (td, J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.70 (td,
J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.44 (s, 1H, OH), 3.86 (s, 2H, OCH ), 2.68 (s,
2H, CH ), 1.63e1.57 (m, 2H, CH ),
Ar), 2.26 (t, 2H, J ¼ 7.6 Hz, CH
1.33e1.21 (m, 20H, 10ꢁ CH ), 0.99 (s, 6H, 2ꢁ CH ), 0.88 (t,
); C NMR (100 MHz, CDCl ):
¼ 185.5 (C]O),
181.9 (C]O), 174.5 (C]O), 154.8 (Ar), 135.6 (ArH), 133.6 (Ar), 133.5
(ArH), 130.0 (Ar), 127.7 (ArH), 126.7 (ArH), 122.4 (Ar), 73.2 (CH ),
37.5 (C), 35.0 (CH ), 32.6 (CH ), 32.5 (CH ), 30.2
), 30.3 (3ꢁ CH
(CH ), 30.1 (CH ), 30.0 (CH ), 29.9 (CH ), 29.8 (CH ),
), 25.7 (2ꢁ CH
25.6 (CH ), 23.3 (CH ), 14.7 (CH
dimethylpropyl decanoate (18i). Flash column chromatography,
eluting with 30:1 hexane:ethyl acetate afforded the product 18i
3
d
(
72%) as a yellow gum, which was recrystallized from hexanee
2
ꢀ
1
dichloromethane to give yellow crystals, mp 61e62 C; H NMR
400 MHz, CDCl ):
¼ 8.15 (d, J ¼ 7.6 Hz,1H, ArH), 8.12 (d, J ¼ 7.6 Hz,
H, ArH), 7.80 (td, J ¼ 7.6,1.2 Hz,1H, ArH), 7.73 (td, J ¼ 7.6,1.2 Hz,1H,
ArH), 7.46 (s,1H, OH), 3.88 (s, 2H, OCH ), 2.71 (s, 2H, CH Ar), 2.29 (t,
J ¼ 7.6 Hz, 2H, CH ), 1.69e1.59 (m, 2H, CH ), 1.32e1.25 (m, 12H, 6ꢁ
CH ), 0.91 (t, J ¼ 6.8 Hz, 3H, CH
), 1.02 (s, 6H, 2ꢁ CH
100 MHz, CDCl ):
¼ 185.5 (C]O), 181.8 (C]O), 174.5 (C]O),
54.8 (Ar), 135.6 (ArH), 133.6 (ArH, Ar), 130.0 (Ar), 127.6 (ArH), 126.7
ArH), 122.4 (Ar), 73.2 (CH ), 37.5 (C), 35.0 (CH ), 32.6 (CH ), 32.5
), 29.9 (2ꢁ CH ), 29.8 (CH ), 25.7 (2ꢁ CH ), 25.6
), 14.7 (CH
); IR (KBr) ¼ 3364 (OH), 2921, 2850 (CH),
2
2
2
(
1
3
d
2
3
13
J ¼ 6.8 Hz, 3H, CH
3
3
d
2
2
2
2
2
1
3
3
3
3
); C NMR
2
2
2
2
(
1
(
(
(
1
(
(
3
d
2
2
2
2
2
3
2
2
3
); IR (KBr) ¼ 3364 (OH), 2918, 2850
2
2
2
(CH), 1733, 1667, 1645 (C]O), 1595, 1472, 1376, 1276 (C]C), 1218,
1155 (CeO); MS (EI), m/z (% relative intensity): 470 (M , 62), 469
þ
CH
CH
2
2
), 30.0 (CH
), 23.3 (CH
2
2
2
3
2
3
(64), 243 (100), 187 (52), 159 (47); Anal. Calcd for C29
H 8.99. Found: C 74.06, H 8.69.
42 5
H O : C 74.01,
734, 1660, 1645 (C]O), 1376, 1277 (C]C), 1216, 1156 (CeO); MS
þ
EI), m/z (% relative intensity): 414 (M , 5), 243 (10), 242 (28), 227
43), 188 (61), 187 (64), 159 (65), 155 (79), 71 (100); Anal. Calcd for
4.2.6.16. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
C
H
25 34
O
5
: C 72.43, H 8.27. Found: C 72.21, H 8.16.
dimethylpropyl hexadecanoate (18m). Flash column chromatography,

B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
283
eluting with 25:1 hexane:ethyl acetate afforded the product 18m
77%) as a yellow gum, which was recrystallized from hexanee
with 10% heat-inactivated fetal bovine serum, 2 mM
L
-glutamine,
(
1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate and 0.8 mg/mL
ꢀ
1
ꢀ
dichloromethane to give yellow crystals, mp 76e77 C; H NMR
400 MHz, CDCl ):
¼ 8.13 (dd, J ¼ 7.6, 1.2 Hz, 1H, ArH), 8.10 (dd,
J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.77 (td, J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.70 (td,
J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.45 (s, 1H, OH), 3.86 (s, 2H, OCH ), 2.68 (s,
H, CH ), 1.65e1.48 (m, 2H, CH ), 1.25
), 0.99 (s, 6H, 2ꢁ CH ), 0.88 (t, J ¼ 6.8 Hz, 3H,
); C NMR (100 MHz, CDCl ):
¼ 185.5 (C]O), 181.9 (C]O),
geneticin, at 37 C in a humidified incubator with 5% CO
2
. The assay
4
(
3
d
was carried out by adding 45
mL to each well of 384-well plates containing 5
m
L of cell suspension at 3.3 ꢁ 10 cells/
m
L of test
2
compounds previously diluted in 0.5% DMSO, and then incubating
ꢀ
2
2
Ar), 2.26 (t, 2H, J ¼ 7.6 Hz, CH
2
2
for 4 days in 37 C incubator with 5% CO
2
. Fluorescence signals were
(
br s, 24H, 12ꢁ CH
2
3
measured by using SpectraMax M5 multi-detection microplate
reader (Molecular Devices, USA) in the bottom-reading mode with
excitation and emission wavelengths of 485 and 535 nm. Fluores-
cence signal at day 4 was subtracted with background fluorescence
at day 0. IC50 values were obtained as previously described in
Section 4.3.
1
3
CH
3
3
d
174.5 (C]O), 154.8 (Ar), 135.6 (ArH), 133.6 (Ar), 133.5 (ArH), 130.0
(
(
2
Ar), 127.6 (ArH), 126.7 (ArH), 122.4 (Ar), 73.2 (CH ), 37.5 (C), 35.0
2
CH
9.9 (CH
4.7 (CH
2
), 32.6 (CH
2
), 32.5 (CH
2
), 30.3 (6ꢁ CH
2
), 30.1 (CH
2
), 30.0 (CH
2
),
),
2
), 29.8 (CH
2
), 25.7 (2ꢁ CH
3
), 25.6 (CH ), 23.3 (CH
2
2
1
3
); IR (KBr) ¼ 3363 (OH), 2917, 2849 (CH), 1733, 1667, 1645
(
(
C]O), 1595, 1471, 1376, 1276 (C]C), 1218, 1154 (CeO); MS (EI), m/z
% relative intensity): 498 (M , 31), 497 (69), 243 (100); Anal. Calcd
: C 74.66, H 9.30. Found: C 74.36, H 9.45.
4.5. Topoisomerase assay
þ
for C31
H O
46 5
Human Topoisomerase II
Topoisomerase II assay kit (TopGEN Inc.). The reaction mixture
containing 60 ng of kinetoplast DNA (kDNA) and 2 units of hTopoII
a activity was determined using
4
.2.6.17. 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2,2-
a
dimethylpropyl octadecanoate (18n). Flash column chromatog-
raphy, eluting with 25:1 hexane:ethyl acetate afforded the product
was incubated with and without naphthoquinone aliphatic amides
and esters at 37 C for 1 h in complete assay buffer (50 mM Tris-Cl
ꢀ
1
8n (74%) as a yellow gum, which was recrystallized from hexanee
2
pH 8.0, 150 mM NaCl, 10 mM MgCl , 0.5 mM dithiothreitol, and
2 mM ATP). Doxorubicin was used as a positive control while
decatenated and linearized kDNA were used as markers. The
reaction in a final volume of 20 mL was stopped by adding stop
buffer/gel loading dye (1% Sarkosyl, 0.025% bromophenol blue and
ꢀ
1
dichloromethane to give yellow crystals, mp 82e83 C; H NMR
400 MHz, CDCl ):
¼ 8.13 (dd, J ¼ 7.6, 1.2 Hz, 1H, ArH), 8.10 (dd,
J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.77 (td, J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.70 (td,
J ¼ 7.6, 1.2 Hz, 1H, ArH), 7.44 (s, 1H, OH), 3.86 (s, 2H, OCH ), 2.68 (s,
H, CH ), 1.68e1.58 (m, 2H, CH ), 1.25
), 0.99 (s, 6H, 2ꢁ CH ), 0.88 (t, J ¼ 6.8 Hz, 3H,
); C NMR (100 MHz, CDCl ):
¼ 185.5 (C]O), 181.9 (C]O),
(
3
d
2
2
2
Ar), 2.26 (t, 2H, J ¼ 7.2 Hz, CH
2
2
5% glycerol). Reaction products were run on 1% agarose gel in 0.5ꢁ
(
CH
br s, 28H, 14ꢁ CH
2
3
TBE buffer with 0.5 mg/mL ethidium bromide included in the gel.
1
3
3
3
d
Electrophoresis was performed at 50 V for 1.30 h. After electro-
phoresis, the gel was destained with distilled water for 30 min and
photographed over a UV transilluminator using DNr Bio-Imaging
174.5 (C]O), 154.8 (Ar), 135.6 (ArH), 133.6 (Ar), 133.5 (ArH), 130.0
(
(
2
Ar), 127.6 (ArH), 126.7 (ArH), 122.4 (Ar), 73.2 (CH ), 37.5 (C), 35.0
2
CH
9.9 (CH
4.7 (CH
2
), 32.6 (CH
2
), 32.5 (CH
2
), 30.3 (8ꢁ CH
2
), 30.1 (CH
2
), 30.0 (CH
2
2
),
),
system. One unit of Topoisomerase II is defined as the amount of
ꢀ
2
), 29.8 (CH
2
), 25.7 (2ꢁ CH
3
), 25.6 (CH ), 23.3 (CH
2
enzyme that decatenates 0.2
m
g of kDNA in 30 min at 37 C.
1
3
); IR (KBr) ¼ 3363 (OH), 2917, 2849 (CH), 1733, 1667, 1645
(
C]O), 1595, 1471, 1376, 1276 (C]C), 1217, 1155 (CeO); MS (EI), m/z
4.6. Molecular docking studies
þ
(
% relative intensity): 526 (M , 90), 243 (100), 229 (6), 187 (9); Anal.
: C 75.25, H 9.57. Found: C 75.08, H 9.69.
Calcd for C33
H
50
O
5
Three-dimensional structure of human TopoII
modeled by SWISS-MODEL using human TopoII
a template (PDB: 3qx3A) [17]. Amino acid sequence identity
between hTopoII and TopoII was 74.1%. The quality of the
modeled hTopoII was evaluated using the program PROCHECK
a
(hTopoII
a
) was
b
(hTopoII
b
) as
4.3. Resazurin microplate assay (REMA) [12]
a
b
Novel naphthoquinone aliphatic amides and esters were sub-
jected to cytotoxic evaluation against KB human-cell lines
epidermoid carcinoma of oral cavity, ATCC CCL-17). Cells at a log-
a
[18]. The model possesses a good geometry with 87% of all residues
in the most favored region and 10.8% in the allowed region of the
Ramachandran plot. Naphthoquinone aliphatic amides (11c and
11m) and esters (18c and 18m) structures were built and optimized
at the calculation level hf/6-31g with Gaussian 03. Gasteiger
charges were added by using Auto Dock4.0 [19]. All hydrogen atoms
were added and water molecules were removed from the protein
structure. Lamarckian genetic algorithm (LGA) was used in this
study with default search parameters being used except for 100
docking runs. The grid size was set to 60 ꢁ 60 ꢁ 60 points with
(
4
arithmic growth phase were harvested and diluted to 7 ꢁ 10 cells/
mL for KB in fresh medium. Successively, 5 mL of a test sample
diluted in 0.5% DMSO and 45 mL of cell suspension were added into
3
incubator. After the incubation period (3 days for KB), 12.5 mL of
6
plates were then incubated at 37 C for 4 h. Fluorescence signals
were measured using SpectraMax M5 multi-detection microplate
reader (Molecular Devices, USA) at the excitation and emission
wavelengths of 530 nm and 590 nm, respectively. Does response
curves were plotted from 6 concentrations of 2-fold serially diluted
test compounds and the 50% inhibition concentration (IC50) was
determined by curve fitting using the SOFTMax Pro software
ꢀ
84-well plates. The plates were incubated at 37 C in 5% CO
2
2.5 mg/mL resazurin solution was added to each well, and the
ꢀ
ꢀ
a grid spacing of 0.375 A. The structures with relative lower binding
free energy and the most cluster members were chosen for the
optimum docking conformation. Three-dimensional structures
with the best-docked conformation were visualized and analyzed
by PyMOL v. 0.99 and Discovery Studio 2.5 (Accelrys, Inc., CA, USA).
(
Molecular Devices, USA). Doxorubicin and 0.5% DMSO were used
as a positive and a negative control, respectively.
Acknowledgments
4
.4. Green fluorescent protein (GFP)-based assay [13]
Financial supports from the Thailand Research Fund (TRF)
through the Royal Golden Jubilee (RGJ) Ph.D. program, the Center of
Excellence for Innovation in Chemistry (PERCH-CIC), Commission
on Higher Education, Ministry of Education, and Kasetsart Univer-
sity Research and Development Institute (KURDI) (v-t(d)38.51 and
v-t(d)49.53 for N.B.) are gratefully acknowledged. S.A. is a Ph.D.
The GFP-expressing Vero cell lines were generated in-house by
stably transfecting the African green monkey kidney cell lines
Vero, ATCC CCL-81), with pEGFP-N1 plasmid (Clontech). The cell
lines were maintained in minimal essential medium supplemented
(

284
B. Kongkathip et al. / European Journal of Medicinal Chemistry 60 (2013) 271e284
student under the Royal Golden Jubilee (RGJ) PhD program and
PERCH-CIC.
antitumor
activity
of
synthetic
1,2-naphthoquinones
and
1,4-
naphthoquinones, Bioorg. Med. Chem. 11 (2003) 3179e3191.
[
8] N. Kongkathip, N. Pradidphol, K. Hasitapan, R. Grigg, W.-C. Kao, C. Hunte,
N. Fisher, A.J. Warman, G.A. Biagini, P. Kongsaeree, P. Chuawong,
B. Kongkathip, Transforming rhinacanthin analogues from potent anticancer
agents into potent antimalarial agents, J. Med. Chem. 53 (2010) 1211e1221.
9] N. Kongkathip, N. Pradidphol, K. Hasitapan, B. Kongkathip, Synthesis and
anticancer evaluation of naphthoquinone esters with 2
cyclohexyl substituents, Biosci. Biotechnol. Biochem. 74 (2010) 1205e1214.
10] N. Pradidphol, N. Kongkathip, P. Sittikul, N. Boonyalai, B. Kongkathip, First
synthesis and anticancer activity of novel naphthoquinone aromatic amides,
Eur. J. Med. Chem. 49 (2012) 253e270.
11] K. Neder, L.J. Marton, L.F. Liu, B. Frydman, Reaction of beta-lapachone and
related naphthoquinones with 2-mercaptoethanol: a biomimetic model of
topoisomerase II poisoning by quinones, Cell. Mol. Biol. (Noisy-le-grand) 44
(1998) 465e474.
[12] J.O. Brien, I. Wilson, T. Orton, F. Pognan, Investigation of the alamar blue
(resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity,
Eur. J. Biochem. 267 (2000) 5421e5426.
[13] L. Hunt, M. Jordan, M. De Jesus, F.M. Wurm, GFP-expressing mammalian cells
for fast, sensitive, non-invasive cell growth assessment in a kinetic mode,
Biotechnol. Bioeng. 64 (1999) 201e205.
Appendix A. Supplementary data
[
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.12.006.
0 0
-cyclopentyl and 2 -
[
[
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