Preparation and anti-inflammatory activities of diarylheptanoid and diarylheptylamine analogs

Article abstract of DOI:10.1016/j.bmc.2005.06.058

Seven diarylheptylamine (12a-g) and four diarylheptanoid analogs (3-5, 9), structurally related to the natural anti-inflammatory agent oregonin (1), have been prepared from curcumin (2) for evaluation of their activity against the expression of iNOS and COX-2. Diarylheptylamine 12b and diarylheptanoid analogs can inhibit iNOS and COX-2 responses of LPS, although less potently than 1. These compounds, however, possess stronger potency than 1 against COX-2-derived PGE₂ formation, of which hexahydrocurcumin (4) is the most potent one with an IC₅₀ value of 0.7 μM.

Full text of DOI:10.1016/j.bmc.2005.06.058

Bioorganic & Medicinal Chemistry 13 (2005) 6175–6181
Preparation and anti-inflammatory activities of diarylheptanoid
and diarylheptylamine analogs
Su-Lin Lee,a Wei-Jan Huang,a Wan Wan Lin,b Shoei-Sheng Leea,*
and Chung-Hsiung Chena,*
aSchool of Pharmacy, College of Medicine, National Taiwan University, Taipei 100, Taiwan, ROC
bDepartment of Pharmacology, College of Medicine, National Taiwan University, Taipei 100, Taiwan, ROC
Received 28 April 2005; revised 14 June 2005; accepted 15 June 2005
Available online 9 August 2005
Abstract—Seven diarylheptylamine (12a–g) and four diarylheptanoid analogs (3–5, 9), structurally related to the natural anti-
inflammatory agent oregonin (1), have been prepared from curcumin (2) for evaluation of their activity against the expression of
iNOS and COX-2. Diarylheptylamine 12b and diarylheptanoid analogs can inhibit iNOS and COX-2 responses of LPS, although
less potently than 1. These compounds, however, possess stronger potency than 1 against COX-2-derived PGE2 formation, of which
hexahydrocurcumin (4) is the most potent one with an IC50 value of 0.7 lM.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
(1) could inhibit stimulation of murine macrophages
by LPS, iNOS mRNA expression, and NO synthesis.7
These studies indicate oregonin (1) to be a lead drug
against inflammation. Recent studies in our laboratory
have found that Alnus formosana also contains oregonin
(1).8 Curcumin (2), a naturally abundant diarylhepta-
noid containing two a,b-conjugated carbonyl functions,
also possesses strong anti-inflammatory activity.9–11
Various structures were derived from modification of
these two conjugated systems.12,13 Being interested in
exploration of anti-inflammatory agents, we used 2 as
starting material and applied the isosterism theory, i.e.,
converting the 5-hydroxy or 3-carbonyl function in the
aglycon of 1 into 3-alkylamino groups, which will enable
the preparation of organic salts and might help to im-
prove the pharmacokinetic profile. Based on this, four
diarylheptanoid analogs and a series of new diarylhep-
tylamine analogs were prepared. Using LPS-stimulated
macrophages for induction of iNOS and COX-2 as a
model system, the anti-inflammatory activity of the
prepared compounds was evaluated. In the following
section, the outcome of these efforts is described.
Chronic inflammation leads to destruction of normal
tissue integrity. Production of inflammatory mediators
through up-regulation of several inducible gene prod-
ucts, such as inducible nitric oxide (iNOS) and cyclo-
oxygenase-2 (COX-2), contributes to inflammatory
responses and tissue damage.1–3 Studies have indicated
the role of iNOS-derived NO and COX-2-derived
PGE2 in the amplification of inflammatory response.
Based on these recent findings, a lot of effort has been
bestowed on the development of anti-inflammatory
agents with regard to interference with the transcription-
al induction or inhibition of the enzymatic activities of
COX-2 and iNOS.4 On exposure to lipopolysaccharide
(LPS), macrophages undergo co-induction of iNOS
and COX-2 gene expression, leading to the formation
of two multifunctional inflammatory mediators, nitric
oxide (NO) and PGE2.5 Recently, oregonin (1), a
diarylheptanoid glycoside containing 3-carbonyl and
5-xylosyloxy groups, has been reported to have anti-in-
flammatory activity. It can inhibit COX-2 protein
expression in immortalized human mammary epithelial
MCF-10A cells.6 In addition, the presence of oregonin
2. Results and discussions
Keywords: Diarylheptanoid; Curcumin; Diarylheptylamine; Prepara-
tion; Anti-inflammatory activity.
Starting from curcumin (2), various kinds of reductive
state products 3–5 have been described previously in
the literature.14 Further improvement with different
*
Corresponding authors. Tel.: +886 2 23123456; fax: +886 2 23211127
(S.-S. Lee); e-mail: shoeilee@ha.mc.ntu.edu.tw
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.058

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S.-L. Lee et al. / Bioorg. Med. Chem. 13 (2005) 6175–6181
conditions of Pt/C-catalyzed hydrogenations was
attempted (Scheme 1, Table 1), providing the opti-
mum yield of hexahydrocurcumin (4) (30%) from 2
and octahydrocurcumin (5) (91.8%) under higher pres-
sure and longer reaction time. Although 4 and 5 could
also be obtained from 3 by NaBH4 reduction, the
yields were low. Structure 4, possessing an asymmetri-
cal skeleton, has more potential for further structural
modification. Thus, direct reductive amination of 4
with propylamine yielded 6, though the yields (16%)
were not satisfactory. This could be due to intramolec-
ular hydrogen bonding between 3-carbonyl and 5-hy-
droxy in 4, reducing the possibility of Schiff base
formation.
yielded 8 (92.1%), a 1,7-diarylheptan-5-ol-3-one. Since
the Boc- and secondary alcoholic groups are labile un-
der stronger acidic conditions, we attempted to develop
facile reaction conditions to achieve deprotection, dehy-
dration, and hydrogenation in sequence to give 9. The
conditions for such a purpose were found to be de-
Boc- and dehydration under high concentrations of
TFA for a longer time (26.5 h), followed by catalytic
hydrogenation, which yielded 9 (51.5%), a key interme-
diate for the preparation of 1,7-diaryl-3-heptylamines. 9
was O,O-dibenzylated by BnCl/K2CO3 via a rapid
microwave reaction to give dibenzylmonoketone 10
(99%). Reductive amination of 10 with six primary
amines afforded the respective N-alkyl dibenzyldiaryl-
heptylamines, 11a–f, in yields of 43.3–67.5%, which
upon catalytic hydrogenation yielded the corresponding
N-alkyl diarylheptylamines, 12a–f, in yields of 61.8–
99.0%. Compound 12g, possessing a cyclopropylamine
moiety, which is labile to catalytic hydrogenation, was
prepared directly from 9 through reductive amination
(56.4% yield).
A synthetic scheme to improve the yield of diarylheptyl-
amine analogs was developed as shown in Scheme 2, in
which the 5-OH group was removed, followed by the
protection of two phenolic groups. The reaction of cur-
cumin (2) with Boc anhydride yielded 7. Catalytic
hydrogenation of 7 under neutral conditions (EtOAc)
Scheme 1. Preparation of compounds 3–6 from curcumin (2). Reagents and conditions: (a) Pt/C, H2, MeOH, rt*; (b) NaBH4 MeOH, rt, 6 h, 4 (7%),
5 (19%); (c) H+, propylamine, NaBH3CN, MeOH, rt, 4 days, 16%. *Reaction conditions and yields see Table 1.

S.-L. Lee et al. / Bioorg. Med. Chem. 13 (2005) 6175–6181
6177
Table 1. Hydrogenation conditions for curcumin (2) and yields of 3–5
of NO production; (3) inhibition of biosynthesis of
COX-2 downstream product PGE2; and (4) cytotoxicity.
Reaction condition
Product (yields)
H2 (1 atm), Pt/C, MeOH, rt, 1 h
H2 (200 psi), Pt/C, MeOH, rt, 16 h
H2 (200 psi), Pt/C, MeOH, rt, 27 h
3 (71.6%)
4 (30.2%), 5 (66.8%)
5 (91.8%)
MTT assay as an index of cell viability indicated that at
concentrations up to 100 lM, oregonin (1) and com-
pounds 3–5, 9, and 12b had no significant effect on cell
viability. However, a significant reduction of MTT val-
ues, cell toxicity, was observed for the other compounds
at 100 lM. The cytotoxic IC50 values are given in Table
2. In the measurement of NO production by LPS, we
found that, compared to oregonin (1), all these com-
pounds showed less potency (Table 2). As NO reduction
of 100 lM compounds 12a, 12c, 12d, 12e, 12f, and 12g
3. Bioactivity
Compounds 1, 3–5, 9, and 12a–g were tested in LPS
(1 lg/mL)-stimulated RAW264.7 macrophages pertain-
ing to: (1) inhibition of iNOS expression; (2) inhibition
Scheme 2. Preparation of diarylheptylamine 12a–g from curcumin (2). Reagents and conditions: (a) (Boc)2O, NaOH, MeOH, rt, 3 h; (b) Pd/C, H2
200 psi, EtOAc, rt, 60 h; (c) TFA-CH2Cl2 (2–3), rt, 26.5 h; (d) Pd/C, H2, MeOH, rt, 1 h; (e) BnCl, K2CO3, DMF, microwave, 20 min; (f) H+/NH2-R,
NaBH3CN, MeOH–CH2Cl2, rt; (g) Pd/C, H2, MeOH, rt, 1 h; (h) (i) H+, NH2–CH(CH2)2, toluene, n, 2 h; (ii) NaBH3CN, toluene, 40 ꢁC, 18 h *c-Pr,
cyclopropyl.

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S.-L. Lee et al. / Bioorg. Med. Chem. 13 (2005) 6175–6181
Table 2. IC50 values (lM) of oregonin (1) and diarylheptanoid
compounds on cytotoxicity and inhibition of NO and PGE2
production
LPS (1 ug/ml)
DMSO oregonin (1)
3
4
5
9
Compound
Inhibition Inhibition of LPS Inhibition of LPS
(1 lg/ml)-induced (1 lg/ml)-induced
of cell
viability
NO production
PGE2 production
120
100
80
60
40
20
0
Oregonin (1) >100
18
77
6.1
4.5
0.7
2.3
1.4
ND
1.0
7.4
16
3
>100
>100
>100
>100
95
4
100
42
5
9
72
61
12a
12b
12c
12d
12e
12f
12g
>100
73
73
100
68
b
62
61
78
69
6.6
ND
ND
c
a
a
67
58
86
a
ND, not determined.
DMSO oregonin(1)
3
4
5
9
was accompanied by displaying cell toxicity, NO re-
sponse had conceivably resulted from a toxic event. In
Figures 1 and 2, reduction of LPS-stimulated iNOS pro-
moter activity and protein expression by nontoxic com-
pounds, respectively, at 30 and 100 lM are given, which
implies that iNOS inhibition takes place primarily oc-
curs at the transcriptional level. In contrast to moderate
inhibition of NO, these compounds showed significant
activities inhibiting the biosynthesis of PGE2 (Table 2).
It should be pointed out that compounds 4 and 5 existed
as mixtures of enantiomeric and diastereomeric isomers,
respectively, which still await further resolution, and all
comparisons were made to a presumed active isomer in
the mixture.
Figure 2. Inhibitory effects of diarylheptanoid compounds (100 lM)
on LPS (1 lg/ml)-stimulated iNOS protein expression. Immunoblot-
ting of iNOS was performed from cell lysates (A) and immunoreac-
tivity quantified by densitometer is presented as percentages of control
LPS response without treatment with diarylheptanoid compounds.
P value: a < 0.005, b < 0.05, and c < 0.00005.
4. Experimental
4.1. General experimental conditions
The physical data of the prepared compounds were
obtained from the following instruments: Melting
points: Fisher–Johns melting apparatus (uncorrected);
IR (KBr): JASCO FT/IR-410 spectrometer; UV
(MeOH): Hitachi U-2000 spectrophotometer; NMR:
Brucker DPX-200 and Avance-400 in deuterated sol-
vent, using the residual solvent peak as internal stan-
dard (CDCl3: dH 7.24 and dC 77.0; methanol-d4:
dH 3.30, dC 49.0); Mass: Finnigan Mat TSQ-7000
Mass spectrometer (ESIMS), VG 70-250S GC/MS
(HFABMS). Chromatographic system: 230–400 mesh
silica gel for column.
From the scant information of these bioassays, it is still
too early for us to deduce meaningful SAR of this series
of compounds. However, most of them exhibit marked
inhibition of COX-2-derived PGE2 formation and have
the potential to develop as anti-inflammatory agents.
120
100
4.2. 1,7-Bis(4-hydroxy-3-methoxyphenyl)-heptane-3,5-
dione (tetrahydrocurcumin, 3)
c
b
80
60
40
20
0
a
b
To a solution of curcumin (2) (1.00 g, 2.7 mmol) in
MeOH (100 mL) was added 10% Pt/C (100 mg). After
degassing, the mixture was hydrogenated (H2, 1 atm)
at rt for 1 h, filtered though a Celite pad, and concen-
trated. The residue (1.05 g) was purified via a silica gel
column (40 g, CHCl3) to give 3 (724.3 mg, 71.6%) as a
white powder.
d
b
Compound 3: mp 95–97 ꢁC; Rf 0.53 (1% MeOH–CHCl3);
1
DMSO oregonin(1)
3
4
5
9
2b
key H NMR (CDCl3, 400 MHz) d 2.85 (4H, t, J = 7.4,
8.2 Hz, H-1, 7), 2.76 (2H, m, H-4), 2.53 (4H, t, J = 7.4,
8.2 Hz, H-2, 6); key 13C NMR (CDCl3) d 203.4 (s,
C-3, 5).
Figure 1. Inhibitory effect of diarylheptanoid compounds (30 lm) on
LPS-induced iNOS promoter activity. P value: a < 0.005, b < 0.05,
c < 0.01, and d < 0.001.

S.-L. Lee et al. / Bioorg. Med. Chem. 13 (2005) 6175–6181
6179
4.3. 1,7-Bis(4-hydroxy-3-methoxyphenyl)-5-hydroxy-3-hep-
tanone (Hexahydrocurcumin, 4) and 1,7-Bis(4-hydroxy-3-
methoxyphenyl)-3,5-heptanediol (octahydrocurcumin, 5)
Compound 7. mp 139.5–142 ꢁC; Rf 0.4 (50% hexane–
1
CHCl3); H NMR (CDCl3, 200 MHz) d 7.59 (2H, d,
J = 15.8 Hz, H-1, 7), 7.12 (4H, br s, H-50,500,60,600), 7.10
(2H, br s, H-20,200), 6.53 (2H, d, J = 15.8, H-2,6), 5.84
(1H, s, H-4), 3.88 (6H, s, 30,300-OCH3), 1.54 (18H, s,
Boc-CH3); ESIMS m/z (rel. int. %) [M+Na]+ 591.2
(100), [M+H]+ 569.1 (37).
The mixture of 2 (1.64 g, 4.4 mmol), MeOH (175 mL),
and Pt/C (10%, 163 mg) in a stainless steel autoclave
was hydrogenated (H2, 200 psi) at rt for 16 h. The resi-
due obtained (1.65 g) after a similar workup procedure
as above was chomatographed over a silica gel column
(50 g, CHCl3) to give 5 (1.12 g, 66.8 %) and 4 (502 mg,
30.2%). While prolonging the reaction time to 27 h
yielded 5 exclusively (91.8%).
4.6. O,O-Di-tbutyloxycarbonyl-hexahydrocurcumin (8)
To a stainless steel autoclave were added 7 (3.20 g,
5.6 mmol), EtOAc (150 mL), and 10% Pd/C (600 mg)
in sequence. After degassing, the mixture was hydroge-
nated (H2, 200 psi) at rt for 60 h. The usual workup pro-
cedure gave a crude product (3.39 g), which was purified
via a silica gel column (100 g, CHCl3) to yield 8 as a
colorless solid (2.98 g, 92.1%).
Compound 4. Greenish crystal, mp 80–82 ꢁC; Rf 0.42
(3% MeOH–CHCl3); key 1H NMR (CDCl3,
400 MHz) d 4.02 (1H, m, H-5), 3.84 and 3.83 (6H,
s, 30,300-OMe), 2.84–2.50 (8H, m, H-1, 2, 4, 7), 1.78–
1.58 (2H, m, H-6).
Compound 8. Rf 0.34 (1% MeOH–CHCl3); key 1H NMR
(CDCl3, 400 MHz) d 4.02 (1H, m, H-5), 1.52 (18H, s,
Boc-CH3); key 13C NMR (CDCl3) d 209.6 (s, C-3),
83.1 and 83.0 (s, Boc-OCMe3), 66.5 (d, C-5); ESIMS
m/z [M+Na]+ 597.3.
Compound 5. Yellowish viscous liquid, Rf 0.36 (5%
MeOH–CHCl3); key 1H NMR (CDCl3, 400 MHz) d
3.84 (2H, m, H-3, 5), 3.79 (6H, s, 30-,300-OMe); key 13C
NMR (CDCl3) d 72.4 (d, C-3, 5), 55.9 (q, 30, 300-OMe),
42.9 (t, C-4), 40.0 (t, C-2, 6), 31.4 (t, C-1,7).
4.7. 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3-heptanone (9)
4.4. 1,7-Bis(4-hydroxy-3-methoxyphenyl) 5-(propylamino)-
3-heptanol (6)
The mixture of 8 (2.39 g, 4.1 mmol), CH2Cl2 (15 mL),
and TFA (10 mL) was stirred under N2 at rt for 14 h
and then concentrated to give an oily residue, which
was partitioned between water (100 mL) and CH2Cl2
(100 mL · 3). The combined CH2Cl2 layers were con-
centrated to give a viscous residue (1.65 g), which with-
out further purification was catalytically hydrogenated
(MeOH, 40 mL; 10% Pd/C, 146 mg; H2, 1 atm; 1 h).
After the usual workup procedure, the crude residue
(1.39 g) was separated over a silica gel column (40 g,
25% hexane–CHCl3) to give 4 (532 mg, 34.4%) and 9
(761 mg, two steps 51.5%).
The mixture of 4 (100 mg, 0.3 mmol), MeOH (15 mL),
12 N HCl (5 drops), and propylamine (115 mg,
1.9 mmol) was stirred at rt for 1 h. Then, NaBH3CN
˚
(82 mg, 1.3 mmol) and molecular sieve (4 A) were added
and the reaction mixture was stirred for 4 days. After the
removal of molecular sieve by filtration, the filtrate was
concentrated and the residue suspended in 1 N HCl
(50 mL) was extracted with CH2Cl2 (50 mL) to remove
unreacted 4. The aqueous layer after neutralization with
ammonia water was partitioned with CH2Cl2
(50 mL · 3). The combined CH2Cl2 layers were dried
over Na2SO4 and concentrated to give a crude product
(42 mg), which was purified via a silica gel column
(5 g, 3–10% MeOH in CHCl3) to give 6 (18 mg, 16%)
as a yellowish solid.
Compound 9. Colorless liquid, Rf 0.60 (2% MeOH–
CHCl3); UV kmax (log e) 228.4 (4.11), 281.2 (3.82) nm;
1H NMR (CDCl3, 400 MHz)
d
6.80 (2H, d,
J = 7.9 Hz, H-50,500), 6.66–6.61 (4H, m, H-20,200,60,600),
5.47 (1H, s) and 5.45 (1H, s) (OH), 3.85 and 3.83 (6H,
s, 30,300-OMe), 2.80 (2H, t, J = 7.4 Hz, H-1), 2.66 (2H,
t, J = 7.6 Hz, H-2), 2.51 (2H, t, J = 7.3 Hz, H-7), 2.37
(2H, t, J = 6.7 Hz, H-4), 1.58–1.53 (4H, m, H-6, 5);
13C NMR (CDCl3) d 210.4 (s, C-3), 146.3 (s, C-30,300),
143.8 and 143.5 (s, C-40,400), 133.0 and 134.1 (s, C-
10,100), 120.7 and 120.8 (d, C-60, 600), 110.9, 111.0 and
114.1, 114.3 (each d) (C-20,200,50,500), 55.8 (q, 30,300-
OMe), 44.6 (t, C-2), 42.8 (t, C-4), 35.3 (t, C-7), 31.2 (t,
C-6), 29.5 (t, C-1), 23.3 (t, C-5); HRFABMS m/z
358.1779 (Calcd for C21H26O5, 358.1780).
Compound 6. Rf 0.10 (3% MeOH–CHCl3); UV kmax (log
1
e) 227.2 (3.92), 280.2 (3.59) nm; key H NMR (CDCl3,
200 MHz) d 3.83 (1H, m, H-3), 2.85–2.45 (7H, m, H-5,
1, 7, NCH2C2H5), 1.73–1.23 (8H, m, H-2, 4, 6,
NCH2CH2CH3), 0.89 (3H, t, J = 7.2 Hz, NC2H4CH3);
key 13C NMR (CDCl3) d 72.0 (d, C-3), 58.5 (d, C-5),
46.8 (t, NCH2C2H5), 40.3 (t, C-4), 38.5 (t, C-2),
35.1 (t, C-6), 31.7 (t, C-7), 31.4 (t, C-1), 22.4
(t, NCH2CH2CH3), 11.5 (q, NC2H4CH3); ESIMS:
m/z [M+H]+ 418.2.
4.5. O,O-Di-tbutyloxycarbonylcurcumin (7)
4.8. 1,7-Bis(4-benzyloxy-3-methoxyphenyl)-3-heptanone
(10)
The mixture of
2
(5.00 g, 13.6 mmol), MeOH
(200 mL), NaOH (1.60 g, 40.7 mmol), and (Boc)2O
(11.8 g, 54.3 mmol) was stirred under N2 at rt for
3 h and then filtered. The residue was washed with
water and dried to give 7 (6.84 g, 88.7%) as a yellow
powder.
The mixture of 9 (1.07 g, 3.0 mmol), DMF (15 mL),
BnCl (3.16 g, 24.9 mmol), and K2CO3 (1.22 g, 8.9 mmol)
was reacted for 15 min in a microwave digester (Prolabo
Maxidigest MX350) with 60% power. After cooling, the
reaction mixture was concentrated and the residue

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S.-L. Lee et al. / Bioorg. Med. Chem. 13 (2005) 6175–6181
dissolved in CH2Cl2 (80 mL) was washed with water
(80 mL · 3). The CH2Cl2 layer was dried over Na2SO4
and concentrated to give a solid residue (1.68 g), which
upon treatment with acetone gave pure 10 (1.59 g, 99
%) as a white powder: Rf 0.60 (0.5% MeOH–CHCl3);
NCH2C2H5), 23.5 (t, NCH2CH2CH3), 11.9 (q,
NC2H4CH3).
Compound 11e. Rf 0.31 (CHCl3, satd with NH3(aq)); key
1H NMR (CDCl3, 200 MHz) d 2.53 (6H, m, H-1, 3, 7
and NH), 2.37 (2H, d, J = 6.7 Hz, NCH2 Pr), 0.87
i
key 1H NMR (CDCl3)
d 7.43–7.24 (10H, m,
OCH2C6H5), 5.09 (4H, s, OCH2C6H5).
(6H, d, J = 6.6 Hz, NCH2CH(CH3)2); key 13C NMR
i
(CDCl3) d 57.1 (d, C-3), 54.4 (t, NCH2 Pr), 28.1 (d,
NCH2CH(CH3)2), 20.7 (q, NCH2CH(CH3)2).
4.9. N-Alkyl-1,7-bis(4-benzyloxy-3-methoxyphenyl)-3-hep-
tylamines (11a: N-Bn; 11b: N-Me; 11c: N-Et; 11d: N-nPr;
11e: N-iBu; 11f: N-nBu)
Compound 11f. Rf 0.33 (CHCl3, satd with NH3(aq)
,
develop twice); key 1H NMR (CDCl3, 400 MHz) d
2.65 (4H, m, H-1 and H-7), 2.59 (1H, m, H-3), 2.53
(2H, t, J = 7.7 Hz, NCH2C3H7), 1.59 (6H, m, H-2, 6,
NCH2CH2C2H5), 0.87 (3H, t, J = 7.4 Hz, NC3H6CH3);
key 13C NMR (CDCl3) d 57.2 (d, C-3), 45.8 (t,
NCH2C3H7), 20.4 (t, NC2H4CH2CH3), 13.7 (q,
NC3H6CH3).
The mixture of 10 (436 mg, 0.8 mmol), MeOH–CH2Cl2
(5:4, 18 mL), benzylamine (893 mg, 8.3 mmol, 10.3
equiv), and 12 N HCl (3 drops) was stirred at rt under
N2 for 30 min and then NaBH3CN (524 mg, 8.3 mmol,
10.3 equiv) was added. The reaction took place for
89 h. The reaction mixture was concentrated and the res-
idue was partitioned between CH2Cl2 (200 mL) and
water (50 mL · 3). The CH2Cl2 layer was dried over
Na2SO4 and concentrated to give a residue (1.10 g),
which was purified via two silica gel columns (20 g,
50–100% CHCl3–hexane, satd with NH4OH; 10 g 0–
3% MeOH–CHCl3) to give compound 11a as colorless
oil (211 mg, 43.3%).
4.10. 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3-heptylamine
(12a)
To a stainless steel autoclave were added 11a (129 mg
0.2 mmol), MeOH (10 mL), and 10% Pd/C (29 mg) in
sequence. After degassing, the mixture was hydrogenat-
ed (H2, 200 psi) for 17 h. The usual workup procedure
gave the product 12a (48 mg, 61.8%) as a greenish
liquid.
Under similar reaction conditions and workup proce-
dures for the preparation of 11a, compounds 11b
(253 mg, 61.8%), 11c (288 mg, 67.5%), 11d (327 mg,
64.1%), 11e (233 mg, 65.9%), and 11f (197 mg, 62.9%)
were produced from five batches of 10 (398 mg,
0.7 mmol for 11b; 406 mg, 0.8 mmol for 11c; 473 mg,
0.9 mmol for 11d; 320 mg, 0.6 mmol for 11e; 283 mg,
0.5 mmol for 11f) by reacting with methylamine solution
(25%, 922 mg, 7.4 mmol, 10 equiv for 11b), ethylamine
(491 mg, 7.6 mmol, 10 equiv for 11c), propylamine
(591 mg, 11.0 mmol, 12 equiv for 11d), isobutylamine
(382 mg, 5.2 mmol, 9 equiv for 11e), and butylamine
(412 mg, 5.6 mmol, 11 equiv for 11f), respectively.
Compound 12a. Rf 0.22 (3% MeOH–CHCl3, satd with
NH3(aq)); IR mmax: 3445, 2929, 1519 cmꢀ1; UV kmax
(log e) 226.6 (3.87), 280.6 (3.53) nm; 1H NMR (CD3OD,
200 MHz) d 6.47 (2H, m, H-50,500), 6.70 (2H, m, H-20,200),
6.62 (2H, m, H-60,600), 3.80 (6H, s, 30,300-OCH3), 2.58–
2.54 (5H, m, H-1, 3, 7), 1.64–1.61, 1.35–1.13 (8H, m,
H-2, 4, 5, 6); HRFABMS m/z [M+H]+ 360.2173 (Calcd
for C21H30O4N, 360.2175).
4.11. N-Alkyl-1,7-bis-(4-hydroxy-3-methoxyphenyl)-3-hep-
tylamines (12b: N-Me; 12c: N-Et; 12d: N-nPr; 12e: N-iBu;
12f: N-nBu)
Compound 11a. Rf 0.33 (CHCl3, satd with NH3(aq)); key
1H NMR (CDCl3, 400 MHz) d 7.44–7.29 (15H, m, 2·
OCH2C6H5, NCH2C6H5), 3.74 (2H, s, NCH2C6H5);
key 13C NMR (CDCl3) d 56.2 (d, C-3), 53.0 (t,
NCH2C6H5).
To a solution of 11b (137 mg 0.3 mmol) in MeOH
(10 mL) was added 10% Pd/C (12 mg). After degassing,
the mixture was hydrogenated (H2, 1 atm) for 1 h. After
the usual workup procedure, the product 12b (91 mg,
99%) was obtained as a brownish liquid. Compound
12b tartrate was prepared by evaporating the methanolic
solution of 12b and L-(+)-tartaric acid (1 equiv).
Compound 11b. Rf 0.51 (3% MeOH–CHCl3, satd with
1
NH3(aq)); key H NMR (CDCl3, 200 MHz) d 2.54 (4H,
t, J = 7.7 Hz, H-1 and H-7), 2.43 (1H, m, H-3), 2.37
(3H, s, NMe); key 13C NMR (CDCl3) d 58.6 (d, C-3),
33.5 (q, N-Me).
Under similar reaction conditions and workup proce-
dures for the preparation of 12b, compounds 12c
(140 mg, 99%), 12d (139 mg, 98%), 12e (144 mg, 99%),
and 12f (103 mg, 99%) were produced from 11c
(209 mg, 0.4 mmol), 11d (201 mg, 0.4 mmol), 11e
(207 mg, 0.4 mmol), and 11f (150 mg, 0.3 mmol),
respectively.
Compound 11c. Rf 0.40 (1% MeOH–CHCl3, satd with
1
NH3(aq)); key H NMR (CDCl3, 400 MHz) d 2.62–2.52
(7H, m, H-1, 3, 7, NCH2CH3), 1.07 (3H, t, J = 7.1 Hz,
NCH2CH3); key 13C NMR (CDCl3) d 57.0 (d, C-3),
41.2 (t, NCH2CH3), 15.6 (q, NCH2CH3).
Compound 11d. Rf 0.36 (3% MeOH–CHCl3); key 1H
NMR (CDCl3, 200 MHz) d 2.56–2.47 (7H, m, H-1, 3,
7, NCH2C2H5), 1.51–1.36 (6H, m, H-4, 5,
NCH2CH2CH3), 0.89 (3H, t, J = 7.3 Hz, NC2H4CH3);
key 13C NMR (CDCl3) d 57.0 (d, C-3), 48.9 (t,
Common physical data for 12b–f. UV kmax (log e) 228 (ca.
1
3.9), 281 (ca. 3.5) nm; H NMR d (CDCl3) 6.79 (2H, d,
J = 7.7 Hz, H-50,500), 6.64 (4H, d, J = 8.1 Hz, H-
20,200,60,600), 3.84 and 3.83 (6H, s, 30,300-OMe); 13C
NMR d (CDCl3) 146.5 and 146.4 (s, C-30,300), 143.7

S.-L. Lee et al. / Bioorg. Med. Chem. 13 (2005) 6175–6181
6181
and 143.6 (s, C-40,400), 134.5 and 134.3 (s, C-10,100), 120.8
and 120.7 (d, C-60,600), 114.3 and 111.0 (d, C-20,200,50,500),
55.8 (q, 30,300-OMe).
(CD3OD, 400 MHz) d 2.60 (1H, m, H-3), 2.04 (1H, m,
NCH(CH2)2), 0.43 (2H, m) and 0.31 (2H, m)
(NCH(CH2)2); key 13C NMR (CD3OD) d 58.7 (d, C-
3), 29.5 (d, NCH(CH2)2), 6.5 (t, NCH(CH2)2);
HRFABMS m/z [M+H]+ 400.2488 (Calcd for
C24H34O4N, 400.2488).
Compound 12b tartrate. Rf 0.38 (7% MeOH–CHCl3,
satd with NH3(aq)); IR mmax 3123, 1519, 1683 cmꢀ1; key
1H NMR (CD3OD, 400 MHz) d 2.64 (3H, s, NCH3);
key 13C NMR (CD3OD) d 60.0 (d, C-3), 30.8 (q,
NCH3); HRFABMS m/z [M+H]+ 374.2334 (Calcd for
C22H32O4N, 374.2331).
4.13. Biological assay
The samples tested were dissolved in DMSO and the fi-
nal concentration of DMSO for each assay was 0.1%.
Measurements of nitrite production, as an assay of
NO release, and PGE2 production were carried out, as
we have previously described.15 Immunoblotting was
performed to assess the protein level of iNOS. Promoter
activity of the iNOS gene as reflected by the reporter
gene assay was performed, as we have previously de-
scribed.15 Cells were co-transfected with iNOS promot-
er–luciferase reporter and b-galactosidase expression
vector. After compoundsÕ treatment, luciferase activity
was determined and normalized to transfection efficien-
cy, as indicated by the expressed b-galactosidase
activity.
Compound 12c. Rf 0.18 (3% MeOH–CHCl3, satd with
NH3(aq)); IR mmax 3318, 1558, 1134 cmꢀ1; key 1H
NMR (CDCl3) d 2.61 (2H, q, J = 7.1 Hz, NCH2CH3),
1.07 (3H, t, J = 7.1 Hz, NCH2CH3); key 13C NMR
(CDCl3) d 57.0 (d, C-3), 41.2 (t, NCH2CH3), 15.5 (q,
NCH2CH3); HRFABMS m/z [M+H]+ 388.2486 (Calcd
for C23H34O4N, 388.2488).
Compound 12d. Rf 0.27 (3% MeOH/CHCl3, satd with
NH3(aq)); IR mmax 2934, 1698, 1508 cmꢀ1; key 1H
NMR (CDCl3, 400 MHz) d 2.56–2.49 (7H, m, H-1, 3,
7, NCH2C2H5), 1.49–1.43 (4H, m, H-4, 10), 0.89 (3H,
t, J = 7.4 Hz, NC2H4CH3); key 13C NMR (CDCl3) d
57.1 (d, C-3), 49.0 (t, NCH2C2H5), 23.5 (t,
NCH2CH2CH3), 11.9 (q, NC2H4CH3); HRFABMS
m/z [M+H]+ 402.2645 (Calcd for C24H36O4N, 402.2644).
Acknowledgment
This work was supported by the National Science Coun-
cil, Taiwan, ROC, under Grant NSC 92-2320-B-002-082.
Compound 12e. Rf 0.42 (3% MeOH–CHCl3, satd with
;
NH3(aq)); IR mmax 3324, 1518, 1732, 1698, 1263 cmꢀ1
1
key H NMR (CD3OD, 200 MHz) d 2.54 (5H, m, H-1,
3, 7), 2.38 (2H, d, J = 6.8 Hz, NCH2 Pr), 0.887 (3H, d,
i
References and notes
J = 6.6 Hz)
and
0.880
(3H,
d,
J = 6.6 Hz)
(NCH2CH(CH3)2); key 13CNMR (CD3OD) d 58.4 (d,
C-3), 55.5 (t, NCH2 Pr), 28.8 (d, NCH2CH(CH3)2),
i
1. Szabo, C.; Billiar, T. R. Shock 1999, 12, 1–9.
2. Taylor, E. L.; Megson, I. L.; Haslett, C.; Rossi, A. G. Cell
Death Diff. 2003, 10, 418–430.
21.0 (q, NCH2CH(CH3)2); HRFABMS m/z [M+H]+
416.2803 (Calcd for C25H38O4N, 416.2801).
3. Vila, L. Med. Res. Rev. 2004, 24, 399–424.
4. Flower, R. J. Nat. Rev. Drug Discov. 2003, 2, 179–191.
5. Swierkosz, T. A.; Mitchell, J. A.; Warner, T. D.; Botting,
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2000, 66, 551–553.
Compound 12f. Rf 0.13 (3% MeOH–CHCl3, satd with
1
NH3(aq)); IR mmax 3225, 1716, 1698, 1507 cmꢀ1; key H
NMR (CD3OD, 400 MHz) d 2.52 (7H, m, H-1, 3, 7,
NCH2C3H7), 1.67 (2H, m), 1.59 (2H, m), 1.47 (2H, m),
1.39 (2H, m), 1.30 (4H, m), 0.90 (3H, t, J = 7.2 Hz,
NC3H6CH3); key 13C NMR (CD3OD) d 58.1 (d, C-3),
47.6 (t, C-9), 21.6 (t, NC2H4CH2CH3), 14.4 (q,
NC3H6CH3); HRFABMS m/z [M+H]+ 416.2799 (Calcd
for C25H38O4N, 416.2801).
8. Chen, S. C. In Studies on the chemical constituents of Alnus
formosana, Master Thesis, School of Pharmacy, National
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9. Jovanovic, S. V.; Boone, C. W.; Steenken, S.; Trinoga, M.;
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4.12. N-Cyclopropyl-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-
heptylamine (12g)
The mixture of 9 (203.5 mg, 0.57 mmol), toluene (7 mL),
cyclopropylamine (269 mg, 4.7 mmol) and 12 N HCl (4
drops) was stirred at 100 ꢁC under N2 for 2 h and then
NaBH3CN (199 mg, 3.2 mmol) was added and stirred
at 40 ꢁC for additional 13 h. After a similar workup
as that for the preparation of 11a, the crude product
obtained (199 mg) was purified via a flash column
(silica gel, 10 g, 0–1% MeOH–CHCl3, saturated with
NH4OH) to give 12g as a bluish liquid (129 mg, 56%).
11. Zhang, F.; Altorki, N. K.; Mestre, J. R.; Subbaramaiah,
K.; Dannenberg, A. J. Carcinogenesis 1999, 20, 445–451.
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13. Hori, Y.; Miura, T.; Hirai, Y.; Fukumura, M.; Nemoto,
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14. Roughley, P. J.; Whiting, D. A. J. Chem. Soc., Perkin
Trans. 1 1973, 2379–2388.
Compound 12g. Rf 0.42 (7% MeOH–CHCl3); IR mmax
15. Ho, F. M.; Lai, C. C.; Huang, L. J.; Kuo, T. C.; Chao, C.
M.; Lin, W. W. Br. J. Pharmacol. 2004, 141, 1037–1047.
3322, 2938, 1601, 1517, 1269 cmꢀ1; key 1H NMR