S. X. Liu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2207–2211
2209
discovery of new NOS inhibitors, and structural complexity and
diversity provide options for clinical research, which supports the
theoretical basis for new clinical candidates with significant
activity.
vinyl acetate with compound 3 in the presence of Pd(OAc)2 and
KOH.14 (1R,2R,3S,4S)-Divinyl-2,4-bis(4-hydroxyphenyl)cyclobutane-
1,3-dicarboxylate (compound 7) was obtained by eliminating the
TBDMS-protecting groups of compound 6 with Bu4NF. Finally,
compound 8 was produced by acetylating compound 7 with acetic
anhydride in pyridine. The stereochemistry of compound 8 was
confirmed by X-ray analysis, indicating that compound 2 under-
The present study investigates the correlation between the
structures of 4,40-dihydroxy-
a
-truxillic acid derivatives and their
inhibition of NO release and reports the discovery of a new lead
compound for NOS inhibitors. 4,40-Dihydroxy-
-truxillic acid and
a
went topochemically controlled a-dimerisation to form the corre-
its derivatives, 5-1a–5-35a, were synthesised and evaluated in
LPS-induced RAW 264.7 macrophages in vitro. Cytotoxicity was
also evaluated using a CCK-8 assay.
sponding head-to-tail adduct 3. Experimental details and data for
this procedure are included in the Supplementary data.
The inhibition of NO production in LPS-induced RAW 264.7
A convenient method for the synthesis of 4,40-dihydroxy-
a-trux-
macrophages was tested at 25 lM following a previously described
illic acid derivatives is shown in Scheme 1. p-Coumaric acid (1) was
prepared according to a previously reported method:11 nucleophilic
substitution of 4-hydroxybenzaldehyde with malonic acid.
4-Hydroxybenzaldehyde was stirred at room temperature overnight
with b-alanine, malonic acid and 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU) in anhydrous alcohol to give compound 1. Compound 1
reacted with tert-butyldimethylsilyl (TBDMS) triflate in the
presence of triethylamine in CH2Cl2, and 1 N HCl was added into
the mixture after stirring for 12 h to yield (E)-3-(4-((tert-butyldi-
methylsilyl)-oxy)phenyl)acrylic acid (2). (1R,2R,3S,4S)-2,4-bis(4
((tert-butyldimethylsilyl)oxy)phenyl) cyclobutane-1,3-dicarbox-
ylic acid (3) was synthesised by photodimerising compound 2 with
a 400-W high-pressure mercury lamp in hexane.6 Compound 4 was
obtained by coupling hydrochloride (EDCI) and N,N-dimethylpyri-
din-4-amine (DMAP) in CH2Cl2. Finally, compound 5 was achieved
by eliminating the compound 3 with alcohols in the presence
of N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine
TBDMS protecting groups of compound 4 with tetrabutyl-ammo-
nium fluoride (Bu4NF). In particular, compound 5-15a was
synthesised by condensation of compound 3 with trans-4-(trans-
4-propylcyclohexyl)cyclohexanol under Mitsunobu reaction condi-
tions,12 followed by removal of the TBDMS-protecting groups with
Bu4NF. Experimental details and data for this procedure are pro-
vided in the Supplementary data.
procedure.15 Aminoguanidine was used as the positive control.16–
18
The corresponding effects on cell viability were determined
using a CCK-8 assay as previously reported19 (see Supplementary
data). The results are shown in Figure 2. Most of the tested com-
pounds exhibited no cytotoxic effect on cells up to a concentration
of 25 lM, except for compounds 5-3a, 5-4a, 5-5a, 5-12a, 5-24a,
5-26a, 5-30a and 5-31a. The relative cell viabilities were 62.6%,
58%, 67.8%, 37%, 78%, 32.6%, 40% and 56%, respectively.
Compound 5-1a displayed a weak effect on NO production.
Compared with compound 5-1a, nearly all derivatives were en-
hanced, except for compounds 5-9a, 5-14a and 5-15a, which
showed no activity.
Compounds 5-2a, 5-3a, 5-4a, 5-5a, and 5-6a have cyclopro-
pylmethyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl
groups in the R position, respectively. Among these five com-
pounds, 5-3a, 5-4a and 5-5a showed significant effects against
NO production, with inhibition rates of 55%, 62.1% and 76%, respec-
tively. Compound 5-2a (33.9%)20 displayed a moderate effect on
NO production, while 5-6a (16%) exhibited weak activity. The re-
sults indicated that a cyclohexyl R inhibited NO release best and
that a cycloheptyl R led to the lowest activity.
Compounds 5-7a and 5-8a were tetrahydrofurfuryl and tetrahy-
dro-4-pyranyl in R, respectively. Both showed weak activity
against NO production, with 10.2% and 18% inhibition, respectively.
On the basis of the results for compounds 5-4a, 5-5a, 5-7a and
5-8a, substitution with oxygen was likely responsible for the
decrease in inhibition of NO production.
Among analogues 5-5a, 5-9a, 5-10a, 5-11a, 5-14a, 5-15a and
5-33a, these results indicate that when the cyclohexane ring posi-
tion was modified, activity was reduced. Interestingly, the corre-
sponding cytotoxicities also decreased. Compared with
compound 5-5a (76%), the activity of compound 5-10a (72%) was
weaker, but the cytotoxicity of 5-10a was lower than that of
5-5a. Notably, when large substituents such as tert-butyl and
cyclohexyl were introduced into the cyclohexane ring, the activity
was lost (see compounds 5-9a, 5-14a and 5-15a).
Compounds 5-12a and 5-13a were 1-cyclohexylmethoxy and
2-cyclohexylethoxy in R, respectively. 5-12a (46.9%) displayed a
moderate effect on NO production, but 5-13a (19.1%) exhibited
poor activity. Among compounds 5-5a, 5-12a and 5-13a, the re-
sults indicated that extending the carbon chains, which connected
the cyclohexyl and hydroxyl groups, the effects on NO production
were decreased. Therefore, extension of the carbon chains resulted
in decreased inhibition of NO production in RAW 264.7 macro-
phage cell lines.
Among compounds 5-17a–5-22a, inhibition rates were 22%,
23.6%, 45.8%, 21.9%, 11.9% and 15.7%, respectively. The results
indicated that with increased numbers of oxygen atoms in the
substituted position, activity was reduced. When the number
of oxygen atoms in the R position was equal, the linear carbon
chains were lengthened, which led to increased inhibition of
NO production. However, when the oxygen atoms and the length
of the linear carbon chains increased, there was less inhibition of
NO production.
To explore the stereochemistry of compound 5, (1R,2R,3S,4S)-
divinyl-2,4-bis(4-acetoxyphenyl)cyclobutane-1,3-dicarboxylate
(compound 8) was synthesised according to Scheme 2, and its sin-
gle-crystal X-ray structure13 is provided in Figure 1. (1R,2R,3S,4S)-
Divinyl-2,4-bis(4-((tert-butyldimethyl-silyl)oxy)phenyl)cyclobutane-
1,3-dicarboxylate (6) was synthesised by transesterification of
TBDMSO
TBDMSO
O
C
COOH
ⅰ
O
O
C
O
HOOC
OTBDMS
OTBDMS
3
6
ⅱ
AcO
O
HO
O
O
C
O
C
ⅲ
O
O
C
O
C
O
OAc
OH
7
8
Scheme 2. Synthesis of compound 8. Reagents and conditions: (i) vinyl acetate,
Pd(OAc)2, KOH, 7 h, 80%; (ii) tetrabutylammonium fluoride, AcOH, THF, 90%; (iii)
acetic anhydride, pyridine, 95%.