Syntheses of tetrahydroisoquinoline derivatives that inhibit NO production in activated BV-2 microglial cells

Article abstract of DOI:10.1016/j.ejmech.2007.09.009

Seventeen tetrahydroisoquinoline derivatives were designed, synthesized and evaluated for inhibition of NO production in lipopolysaccharide-stimulated BV-2 microglial cells. Compounds 5a, 9c and 11a potently attenuated NO production by >60%, and 5a and 11

Full text of DOI:10.1016/j.ejmech.2007.09.009

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European Journal of Medicinal Chemistry 43 (2008) 1160e1170
http://www.elsevier.com/locate/ejmech
Original article
Syntheses of tetrahydroisoquinoline derivatives that inhibit
NO production in activated BV-2 microglial cells
b,**
Jai Woong Seo a, Ekaruth Srisook a,1, Hyo Jin Son b, Onyou Hwang
,
Young-Nam Cha c, Dae Yoon Chi a,
*
a Department of Chemistry, Inha University, 253 Yonghyundong Namgu, Inchon 402-751, Republic of Korea
b Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736, Republic of Korea
c Department of Pharmacology and Toxicology, College of Medicine, Inha University, Inchon 402-751, Republic of Korea
Received 2 April 2007; received in revised form 31 July 2007; accepted 6 September 2007
Available online 22 September 2007
Abstract
Seventeen tetrahydroisoquinoline derivatives were designed, synthesized and evaluated for inhibition of NO production in lipopolysaccha-
ride-stimulated BV-2 microglial cells. Compounds 5a, 9c and 11a potently attenuated NO production by >60%, and 5a and 11a inhibited BH4
production by >48% at 100 mM. In particular, N-ethylcarbonyl-7-hydroxy-6-methoxy-1,2,3,4-tetrahydroisoquinoline (11a) reduced NO produc-
tion by 64% and tetrahydrobiopterin (BH4) production by 49%. Introducing longer alkyl component at C1 or N2 position led to attenuation of
the inhibitory effect. It is possible that 11a inhibits NO production by blocking BH4-dependent dimerization of newly synthesized iNOS mono-
mers.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Tetrahydroisoquinoline; Nitric oxide; Tetrahydrobiopterin; Nitric oxide synthase
1. Introduction
that the production of NO is controlled differently: eNOS
and nNOS are constitutively expressed and activated in
a Ca2þ-dependent manner, whereas iNOS gene expression is
minimal in unstimulated state and is dramatically induced in
response to stimulation [9]. Overproduction of NO by iNOS
has been implicated in pathogenesis of various diseases such
as stroke [10], Alzheimer’s disease [11], Parkinson’s disease
[12], atherosclerosis [13,14], and septic shock [15].
Nitric oxide (NO), a radical and yet a reactive gaseous
metabolite, is produced from L-arginine by the enzyme nitric
oxide synthase (NOS) [1]. Since NO became known as the
endothelium-derived relaxing factor [2], its biological roles
in cardiovascular, immune and neuronal systems have been
extensively investigated and identified [3e5].
The discovery of the subtypes of NOS, namely, the endo-
thelial (eNOS, type-3) [6], neuronal (nNOS, type-1) [7], and
inducible (iNOS, type-2) forms [8], triggered studies on their
physiological functions and structures [9]. It has been found
Formation of active iNOS enzyme requires a complex intra-
cellular process: homodimerization of newly synthesized NOS
monomers and binding of FMN, FAD and NADPH to the re-
ductase domain and heme and tetrahydrobiopterin (BH4) to
the oxygenase domain [16]. All these cofactors are essential
for effective electron transfer and generation of NO. Among
them, BH4 plays an important role in the dimerization and cat-
alytic activity of NOS [17,18]. Thus, inhibition of dimeriza-
tion even after the number of monomers has been increased
during iNOS induction would prevent NO overproduction
and attenuate NO-related damages.
* Corresponding author. Tel.: þ82 32 860 7686; fax: þ82 32 867 5604.
** Corresponding author. Tel.: þ82 2 3010 4279; fax: þ82 2 3010 4248.
E-mail addresses: oyhwang@amc.seoul.kr (O. Hwang), dychi@inha.ac.kr
(D.Y. Chi).
1
Present address: Department of Chemistry, Faculty of Science, Burapha
University, Chonburi 20131, Thailand.
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.09.009

J.W. Seo et al. / European Journal of Medicinal Chemistry 43 (2008) 1160e1170
1161
MeO
HO
N-Acetyl-3-O-methyldopamine (NAMDA, 1) was reported
to inhibit production of NO and BH4 in BV-2 microglial cells
that had been stimulated with lipopolysaccharide (LPS) but the
requirement of a large dose was disadvantageous for further ap-
plication [19,20]. Our initial approach aimed to find a com-
pound that is effective at lower concentrations. In this
connection, we previously synthesized NAMDA analogues
by using three modification methods and identified potential
lead compounds [21]. Among them, we have selected N-
acetyl-7-hydroxy-6-methoxy-1,2,3,4-tetrahydroisoquinoline (2)
because the chemistry of tetrahydroisoquinoline is well known
and C1 and/or N2 positions can easily be functionalized
(Fig. 1). We have synthesized 17 derivatives of 2 and evaluated
their activity against NO production in LPS-activated BV-2 cells.
In addition, to elucidate whether the reduction of NO production
is correlated with decreased BH4 generation, BH4 level has also
been measured in some cases. Herein, we report the syntheses
and structureeactivity relationships (SAR) of the newly synthe-
sized tetrahydroisoquinoline derivatives.
MeO
HO
a
NH2
NH
R
3
4a, R = Me (55%)
4b, R = Ph (74%)
b
MeO
HO
N
R
O
5a, R = Me (83%)
5b, R = Ph (89%)
Scheme 1. (a) Synthesis of 4a: acetaldehyde, 1 M HCl, 100 ꢀC, 24 h; synthesis
of 4b: (i) benzaldehyde, MgSO4, TEA, MeOH, reflux, 3 h; (ii) TFA, 80 ꢀC,
100 min; (b) Ac2O, Et3N, CH2Cl2, rt, 1 h.
tert-butyloxycarbonyl anhydride, benzylation of phenol with
benzyl bromide, and deprotection of tert-butyloxycarbonyl
group. Acylations with various acyl chlorides, such as pro-
pionyl, butyryl, isobutyryl, a-phenylacetyl, 4-methylbutyryl,
cyclopropanecarbonyl, and cyclobutanecarbonyl, afforded
amide derivatives 7aeg in high yields. These acylated com-
pounds 7aeg were treated with phosphorus oxychloride to ob-
tain cyclized dihydroisoquinoline and subsequent reductions by
sodium cyanoborohydride yielded 7-benzyloxy tetrahydroiso-
quinoline derivatives. Palladium catalyzed debenzylation gave
various C1 alkyl substituted tetrahydroisoquinoline derivatives
8aeg as hydrochloride salts. These compounds were reacted
with acetic anhydride to produce target compounds 9aeg. All
C1 substituted tetrahydroisoquinoline derivatives 5a, b, 9aeg
were isolated as racemic mixtures.
We also synthesized N2-acyl derivatives 11aee and their re-
duced forms of carbonyl groups 12aec. 7-Hydroxy-6-methoxy-
1,2,3,4-tetrahydroisoquinoline (10) was prepared according to
the known procedure [21]. Various N2-acyl tetrahydroisoquino-
lines 11aee were readily obtained in the presence of triethyl-
amine with commercially available acyl chloride or anhydride
in dichloromethane. The syntheses of N2-alkyl derivatives
12aec were accomplished from amide 11aec by reduction
with lithium aluminium hydride.
2. Chemistry
The synthesis of tetrahydroisoquinoline derivatives began
with the well-defined PicteteSpengler reaction between alde-
hyde and phenylethylamine to introduce alkyl component at
C1 position [22]. Cyclization with 3 and acetaldehyde under
acidic media readily afforded 1-methyl compound 4a but this
method failed to generate 1-phenyl analogue 4b because of
the solubility of benzaldehye in aqueous solvent. An alternative
method using reaction under methanol to generate imine first
and subsequent addition of trifluoroacetic acid (TFA) for ring
formation yielded 4b (Scheme 1). Acetylation of 4a and 4b
with acetic anhydride produced 5a and 5b, respectively. Our
endeavors to obtain various C1 alkyl derivatives with other al-
dehydes such as phenylacetaldehyde and phenylethylacetalde-
hyde were not fruitful because of the formation of pyridinium
salt as a side reaction.
Further syntheses of tetrahydroisoquinoline derivatives were
performed using the BischlereNapieralski reaction. Compound
6, an intermediate for branching to various alkyl amides 7aeg,
was obtained successively via protection of primary amine with
MeO
HO
MeO
HO
3. Results and discussion
HN
N
O
O
We synthesized 17 tetrahydroisoquinoline derivatives that
have a variable alkyl group at C1 position and/or an acyl group
at N2 position. Their effects on NO production in activated mi-
croglia were evaluated. For those compounds that led to de-
creased NO production, we also evaluated their cytotoxicity
in order to ascertain that the apparent NO decrease was not
due to cell death. Generally, the compounds did not show the
increase of cytotoxicity (Table 1) and these values were similar
to our previously reported results [21]. To compare the NO in-
hibition activity of the newly synthesized derivatives with those
of the previous study, we selected tetrahydroisoquinoline 2 as
1, NAMDA
2
MeO
N
R2
HO
R1
Tetrahydroisoquinoline derivatives
Fig. 1. NAMDA derivatives showing NO inhibitory effect.

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J.W. Seo et al. / European Journal of Medicinal Chemistry 43 (2008) 1160e1170
Table 1
Effects on the production of NO and cell survivala
NO inhibitory activities. As shown in Fig. 2, the amounts of
BH4 produced in the LPS-activated microglia decreased in
a manner dependent on the drugs’ concentration. Thus, it is
possible that these drugs inhibit BH4 production and therefore
lead to interruption of iNOS dimerization.
In conclusion, we have designed and synthesized 17 tetra-
hydroisoquinoline derivatives and found a potent compound
11a that inhibits NO production by 64% in LPS-stimulated
BV-2 cells at 100 mM. BH4 production was also reduced by
49%. It appears that a small alkyl group at C1 position or
a small acyl group at N2 position yields compounds with
potentially stronger inhibitory effects on BH4 and NO produc-
tion. Compound 11a might serve as a useful candidate with
which inflammation-related damages can be controlled.
MeO
N
R2
HO
R1
Comp.
R1
R2
Nitriteb
LDHc
2
5a
H
CH3
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
46
40
50
84
39
113
51
96
60
69
36
74
82
90
64
82
111
63
107
104
e
9a
CH2CH3
CH2CH2CH3
9b
9c
e
e
CH(CH3)2
CH2CH(CH3)2
9e
e
5b
9d
9f
Ph
CH2Ph
104
80
e
Cyclopropyl
Cyclobutyl
4. Experimental protocols
9g
e
11a
11b
11d
11e
11c
12a
12b
12c
a
H
H
H
H
H
H
H
H
COCH2CH3
COCH2CH2CH3
COCH(CH3)2
104
91
90
84
82
86
93
87
4.1. Chemistry
COCH2CH(CH3)2
Cyclohexanecarbonyl
CH2CH2CH3
4.1.1. General information
Reagents were purchased from Aldrich Company and anhy-
drous solvents were purchased form Aldrich or Burdick &
Jackson Company. Reaction progress was followed by thin
layer chromatography using silica gel glass plates containing
F-254 indicator (Merck). Visualization of organic compounds
on TLC was monitored by UV light or phosphomolybdic acid
indicator. HPLC solvents were purchased from Burdick &
Jackson Company Analysis of mass trace with standard com-
CH2CH2CH2CH3
Cyclohexylmethyl
All values were obtained from triplicate experiments and averaged.
Amount of nitrite in the presence of each drug (100 mM) in LPS-induced
b
BV-2 cells, presented as percentage of LPS-activated BV-2.
c
Cytotoxicity of each drug was assessed by lactate dehydrogenase (LDH)
activity released into the medium, presented as percentage of LPS-activated
BV-2.
pounds was performed with Gilson HPLC system. 1H and 13
C
NMR spectra were btained on a Varian Gemini-200 or -400
and chemical shifts are reported in d unit (ppm) relative to tet-
ramethylsilane. Coupling constants are reported in hertz. Melt-
ing points were checked using OptiMelt apparatus (Stanford
research systems) and are uncorrected. Low-resolution elec-
tron impact (EI, 70 eV) spectra were obtained on a Varian
1200L single quadruple GCeMS system with CP-3800GC
a standard compound. As shown in Table 1, this compound at
100 mM inhibited NO production to 46% of that in the LPS-
alone control. Among the newly synthesized compounds, 5a,
9c and 11a showed a stronger inhibitory effect than 2.
In the series of non-aromatic C1 alkyl substituents 5a, 9a,
b, c, and e, the inhibitory activity was decreased with increas-
ing carbon length at C1 position in general. In the cases of C1
phenyl compound 5b and benzyl compound 9d, NO produc-
tion was inhibited by 49% and 4%, respectively. Although
cyclopropyl compound 9f and cyclobutyl compound 9g have
quite a large alkyl group, their inhibitory activity was more
effective compared to 9b and 9e that have a flexible alkyl
carbon. It is possible that the rigidity of the alkyl group can
facilitate binding.
100
90
11c
11a
5a
2
80
70
60
50
40
The compounds with acyl derivatives on N2, such as pro-
pionyl 11a, butyryl 11b, isobutyryl 11d, and isopentanoyl
11e, reduced NO production to 36%, 74%, 82%, and 90% of
control, respectively. This tendency was also very similar to
that observed with the C1 alkyl substituents mentioned above.
The N2-alkyl substituents 12a, b, and c were less effective.
However, cyclohexylmethyl compound 12c and cyclohexane-
carbonyl compound 11c were quite effective (63% and 64%,
respectively), and the reason for this is unknown.
0
25
50
75
100
conc. of compound (µM)
Fig. 2. Amount of BH4 produced in LPS-activated BV-2 cells in the presence of
various concentrations of drugs. Data points: 2 (50 mM e 79%, 100 mM e 55%),
5a (10 mM e 99%, 30 mM e 59%, 60 mM e 59%, 100 mM e 52%), 11a (10 mM
e 86%, 60 mM e 72%, 100 mM e 51%), 11c (30 mM e 95%, 60 mM e 71%,
100 mM e 63%). The data are presented as percentage of LPS-alone control.
It is possible that the inhibitory effect of these compounds
on NO production might be due to their effect on BH4. We
therefore evaluated the degrees of BH4 production in the pres-
ence of 2, 5a, 11a, and 11c, which have exhibited the highest

J.W. Seo et al. / European Journal of Medicinal Chemistry 43 (2008) 1160e1170
1163
and ESI low-resolution mass spectra were obtained on a Varian
1200L quadruple LC/MS system. Elemental analyses were
performed on Flash EA1112 (Thermo Electron Corporation)
by the Center for Collaborative Instruments at Inha University.
106 mg), and Et3N (2.0 mmol, 202 mg) in anhydrous MeOH
was refluxed for 3 h. The mixture was filtered through celite
and the solution was evaporated under reduced pressure. The
residual solid was refluxed in trifluoroacetic acid (TFA) for
1 h and then TFA was evaporated. After neutralization by
NaHCO3 solution, organics were extracted by CH2Cl2. The or-
ganic layer was dried over Na2SO4 and flash column chroma-
tography (5:95, MeOH:CH2Cl2) gave 4b (188 mg, 74%) as
a white foam: m.p. 176e178 ꢀC; 1H NMR (400 MHz,
DMSO-d6) d 8.57 (br s, 1H),7.22e7.32 (m, 5H), 6.63 (s,
1H), 6.04 (s, 1H), 3.72 (s, 3H), 3.01e3.10 (m, 1H), 2.76e
2.89 (m, 2H), 2.55e2.65 (m, 1H); 13C NMR (100 MHz,
DMSO-d6) d 146.0, 145.6, 144.1, 130.7, 128.9, 128.0, 126.8,
125.7, 114.4, 112.2, 60.8, 55.5, 42.0, 28.8; MS (CI) 284,
256 (Mþ þ 1, 100), 178. Registry: 72105-97-6. Compound
4b (57 mg, 0.223 mmol) dissolved in chloroform (5 mL) was
reacted with acetic anhydride (23 mg, 0.223 mmol) at room
temperature for 1 h. After neutralization by NaHCO3 solution,
organics were extracted by CH2Cl2. Compound 5b (59 mg,
89%) was recrystallized from CH2Cl2 and hexane as a white
solid: m.p. 168e169 ꢀC; 1H NMR (400 MHz, CDCl3)
d 7.32e7.18 (m, 5H), 6.86e6.58 (m, several conformer peaks
of C5eH and C8eH, 3H), 5.84e5.30 (m, several conformer
peaks of OeH, 1H), 4.33e4.27 (m, one conformer of C1eH),
3.89 (s, 3H) 3.72e3.66 (m, one conformer of C1eH), 3.46e
3.38 (m, one conformer of C3eH), 3.27e3.21 (m, one con-
former of C3eH), 2.96e2.82 (m, one conformer of C4eH),
2.76e2.60 (m, one conformer of C4eH), 2.74 (s, one con-
former of COCH3), 2.15 (s, one conformer of COCH3); 13C
NMR (100 MHz, CDCl3) d 169.7, 168.9, 145.8, 144.2,
143.9, 142.4, 141.4, 128.6, 129.5, 128.1, 127.8, 127.7,
127.5, 127.2, 126.8, 125.7, 114.5, 113.8, 110.8, 110.4, 60.2,
55.9, 54.4, 40.4, 37.6, 28.6, 27.4, 22.1, 21.7; MS (EI) 297
(Mþ), 254, 239, 220, 178 (100), 163. HRMS (EI) m/z calcd
for C18H19NO3 [M]þ 297.1365; found 297.1367.
4.1.2. Procedures for the preparation of 5a and 5b (Scheme 1)
4.1.2.1. 2-Acetyl-7-hydroxy-6-methoxy-1-methyl-1,2,3,4-tetra-
hydroisoquinoline (5a). To a solution of 3 (1.96 mmol,
400 mg) in 1 M HCl solution (10 mL) under pressure tube, ac-
etaldehyde (15.7 mmol, 692 mg) was added. After the tube was
surely capped, the reaction was heated at 100 ꢀC for 24 h. The
solution was neutralized by saturated NaHCO3 solution and
evaporated under vacuum. The powder was dissolved with
methanol and precipitates were removed. After the solution
was concentrated, flash column chromatography (1% MeOH/
EtOAc) was performed. Compound 4a was obtained as a hydro-
chloride salt from MeOH and 1.0 M hydrogen chloride in di-
ethyl ether (off-white powder, 250 mg, 55% yield): m.p.
175e177 ꢀC dec. (lit. 174e175 ꢀC [23]); 1H NMR
(400 MHz, DMSO-d6) d 9.93 (br s, 1H), 9.40 (br s, 1H), 9.06
(s, 1H), 6.72 (s, 1H), 6.67 (s, 1H), 4.32e4.30 (m, 1H), 3.47
(s, 3H), 3.28e3.36 (m, 1H), 3.21e3.22 (m, 1H), 2.29e2.00
(m, 1H), 1.52 (d, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6) d 147.1, 145.4, 126.0, 122.1, 112.7, 111.9, 55.6,
49.7, 38.6, 24.6, 19.1; MS (CI) 194 (Mþ þ 1, 100), 178, 164.
Registry: 98321-33-6. Compound 4a (0.44 mmol, 100 mg)
was poured in CH2Cl2 (5 mL) and then Et3N (1.0 mmol) and
acetic anhydride (0.44 mmol, 45 mg) were slowly added at
room temperature. After 1 h, solvent was evaporated under
vacuum. The residual solution was neutralized by saturated
NaHCO3 solution and extracted with CH2Cl2. The organic
layer was dried over Na2SO4 and solvent was evaporated under
reduced pressure. Compound 5a (91 mg, 83%) was obtained as
a white solid by crystallization in CH2Cl2 and hexane: m.p.
175e177 ꢀC; 1H NMR (400 MHz, CDCl3) d 6.96 (s, one con-
former of C5eH), 6.66 (s, one conformer of C5eH), 6.59 (s,
one conformer of C8eH), 6.57 (s, one conformer of C8eH),
5.83 (s, one conformer of OeH), 5.78 (s, one conformer of
OeH), 5.53 (q, J ¼ 6.6 Hz, one conformer of C1eH), 4.83
(q, J ¼ 6.6 Hz, one conformer of C1eH), 4.70e4.65 (m, one
conformer of C3eH), 3.82e3.76 (m, one conformer of
C3eH), 3.52e3.44 (m, one conformer of C3eH), 3.02e2.95
(m, one conformer of C3eH), 2.90e2.79 (m, 1H), 2.75e2.70
(m, one conformer of C4eH), 2.69e2.60 (m, one conformer
of C4eH), 2.18 (s, one conformer of C1eCH3), 2.15 (s, one
conformer of C1eCH3), 1.49 (d, J ¼ 6.4 Hz, one conformer
of COCH3), 1.40 (d, J ¼ 6.8 Hz, one conformer of COCH3);
13C NMR (100 MHz, CDCl3) d 168.8, 168.7, 145.5, 145.3,
144.3, 144.1, 131.3, 130.0, 125.6, 124.4, 112.8, 112.3, 110.8,
110.4, 55.9, 52.3, 47.9, 40.4, 34.8, 29.0, 28.2, 22.5, 21.9,
21.5, 21.4; MS (EI) 235 (Mþ, 100), 220. HRMS (EI) m/z calcd
for C13H17NO3 [M]þ 235.1208; found 235.1209.
4.1.3. Procedures for the preparation of 9aeg
4.1.3.1. N-[2-(4-Hydroxy-3-O-methylphenyl)ethyl]amine hy-
drochloride (6). To a solution of 3 (1.3 g, 6.4 mmol) in chloro-
form (20 mL) was added tert-butyloxycarbonyl anhydride
(1.67 g, 7.6 mmol) and Et3N (1.93 g, 19.5 mmol) (Scheme 2).
After 24 h, the reaction was quenched by NH4Cl solution. Or-
ganics were extracted by CH2Cl2 and washed by additional
H2O (100 mL ꢁ 2). Column chromatography (1:1, EtOAc:hex-
ane) gave a white solid (1.32 g, 59%): m.p. 117e119 ꢀC (lit.
115 ꢀC [26]). Registry: 23699-77-6. It was dissolved in acetone
(20 mL). After addition of K2CO3 and benzylbromide, the re-
action mixture was refluxed for 12 h. Solvent was evaporated
under reduced pressure and water was poured. Organics were
extracted by ethyl acetate and dried over sodium sulfate. Col-
umn chromatography (3:7, EtOAc:hexane) gave white solid
(1.57 g, 95%): m.p. 74e75 ꢀC (lit. 68e70 ꢀC [26]). Registry:
23428-81-1. The white solid (16.8 mmol, 6.0 g) in CH2Cl2
(20 mL) was treated with trifluoroacetic acid (20 mL) slowly
under N2 at 0 ꢀC. The reaction was stirred for 40 min. After
the reaction, the reaction mixture was slowly poured to
4.1.2.2. 2-Acetyl-7-hydroxy-6-methoxy-1-phenyl-1,2,3,4-tetra-
hydroisoquinoline (5b). A mixture of 3 (1.0 mmol, 204 mg),
MgSO4 (2.49 mmol, 300 mg), benzaldehyde (1.0 mmol,

1164
J.W. Seo et al. / European Journal of Medicinal Chemistry 43 (2008) 1160e1170
MeO
MeO
BnO
91 (100), 65, 57, 30. HRMS (EI) m/z calcd for C19H23NO3
[M]þ 313.1678; found 313.1675.
d
a,b,c
3
NH2
HN
R
BnO
O
4.1.3.3. N-[2-(4-Benzyloxy-3-methoxyphenyl)ethyl]butyramide
(7b). Same procedure as for 7a was performed with 6
(3.5 mmol, 0.9 g), butyryl chloride (4.2 mmol, 0.45 g), and
Et3N (10.5 mmol, 1.06 g) to obtain 7b (1.1 g, 94%) as a white
foam: m.p. 100e101 ꢀC; 1H NMR (200 MHz, CDCl3)
d 7.46e7.26 (m, 5H), 6.81 (d, J ¼ 8.0 Hz, 1H), 6.73 (d,
J ¼ 1.8 Hz, 1H), 6.64 (dd, J ¼ 8.0, 1.8 Hz, 1H), 5.56 (s, 1H),
5.12 (s, 2H), 3.86 (s, 3H), 3.47 (q, J ¼ 6.6 Hz, 2H), 2.74 (t,
J ¼ 7.0 Hz, 2H), 2.09 (t, J ¼ 7.6 Hz, 2H), 1.61 (sext,
J ¼ 7.4 Hz, 2H), 0.91 (t, J ¼ 7.3 Hz, 3H); 13C NMR (50 MHz,
CDCl3) d 172.9, 149.7, 146.7, 137.2, 132.0, 128.4, 127.7,
127.2, 120.5, 114.2, 112.4, 71.1, 55.9, 40.5, 38.6, 35.3, 19.1,
13.7; MS (EI) 327 (Mþ), 240, 149, 137, 91 (100), 43. HRMS
(EI) m/z calcd for C20H25NO3 [M]þ 327.1834; found 327.1831.
6, (59%)
7a~7g
MeO
HO
MeO
HO
h
e,f,g
N
NH
R
O
R
9a~9g
8a~8g
7a (90%), 8a (18%), 9a (58%), R = CH2CH3
7b (94%), 8b (77%), 9b (45%), R = CH2CH2CH3
7c (96%), 8c (70%), 9c (67%), R = CH(CH3)2
7d (63%), 8d (63%), 9d (94%), R = CH2Ph
7e (93%), 8e (67%), 9e (73%), R = CH2CH(CH3)2
7f (88%), 8f (58%), 9f (72%), R = cyclopropyl
7g (79%), 8g (81%), 9g (80%), R = cyclobutyl
Scheme 2. (a) (Boc)2O, Et3N, CHCl3, rt, 24 h; (b) benzyl bromide, K2CO3, ac-
etone, reflux, 12 h; (c) TFA, CH2Cl2, 0 ꢀC, 40 min; (d) acyl chloride, TEA,
CH2Cl2, rt, 30 mine1 h; (e) POCl3, CH3CN, reflux, 2e5 h; (f) NaBH4,
0 ꢀCert, 24 h; (g) Pd/C, H2, HCl, MeOH, rt, 12 h; (h) Ac2O, CH2Cl2,
0 ꢀCert, 2 h.
4.1.3.4.
N-[2-(4-Benzyloxy-3-methoxyphenyl)ethyl]isobutyr-
amide (7c). Same procedure as for 7a was performed with 6
(3.50 mmol, 0.9 g), isobutyryl chloride (4.2 mmol, 0.45 g),
and Et3N (11 mmol, 1.1 g) to obtain 7c (1.1 g, 96%) as a white
solid: m.p. 110e112 ꢀC; 1H NMR (200 MHz, CDCl3) d 7.46e
7.26 (m, 5H), 6.82 (d, J ¼ 8.2 Hz, 1H), 6.73 (d, J ¼ 1.8 Hz, 1H),
6.64 (dd, J ¼ 8.0, 1.4 Hz, 1H), 5.56 (br s, 1H), 5.13 (s, 2H), 3.87
(s, 3H), 3.47 (q, J ¼ 6.6 Hz, 2H), 2.74 (t, J ¼ 6.9 Hz, 2H), 2.28
(quin, J ¼ 7.1 Hz, 1H), 1.11 (d, J ¼ 7.0 Hz, 2H); 13C NMR
(50 MHz, CDCl3) d 176.8, 149.7, 146.7, 137.1, 132.1, 128.4,
127.7, 127.2, 120.6, 114.2, 112.4, 71.1, 55.9, 40.4, 35.5, 35.2,
19.5; MS (EI) 327 (Mþ), 240, 149, 137, 91 (100), 43. HRMS
(EI) m/z calcd for C20H25NO3 [M]þ 327.1834; found 327.1836.
saturated sodium bicarbonate solution containing ice. Excess
of diethyl ether was poured and extracted. Solvent was evapo-
rated and the mixture was dissolved in chloroform, and washed
with saturated NaHCO3. Solvent was evaporated. The mixture
was dissolved in diethyl ether. The mixture was solidified as
HCl salt by pouring 1 M HCl ether (20 mL) and stirring. Filtra-
tion gave 6 as a white solid (1.32 g, 64%): m.p. 170e172 ꢀC
1
(lit. 173e174 ꢀC [27]); H NMR (200 MHz, CDCl3) d 8.24
(br s, 2H), 7.28e7.45 (m, 5H), 6.96 (d, J ¼ 8.0 Hz, 1H), 6.90
(d, J ¼ 1.8 Hz, 1H), 6.73 (dd, J ¼ 8.0, 1.8 Hz, 1H), 5.04 (s,
1H), 3.77 (s, 3H), 2.96e3.03 (m, 2H), 2.80e2.88 (m, 2H);
13C NMR (50 MHz, CDCl3) d 149.3, 146.6, 137.2, 130.3,
128.2, 127.6, 127.5, 120.5, 114.1, 113.0, 70.1, 55.6, 32.4;
MS (CI) 258 (Mþ), 241 (100), 228, 91. Registry: 1860-57-7.
4.1.3.5. N-[2-(4-Benzyloxy-3-methoxyphenyl)ethyl]-2-phenyl-
acetamide (7d). Same procedure as for 7a was performed with
6 (3.4 mmol, 1.0 g), Et3N (10 mmol, 1.0 g) and phenylacetyl
chloride (3.8 mmol, 0.52 g) to obtain 7d (0.8 g, 63%) as a white
1
solid: H NMR (200 MHz, CDCl3) d 7.71e7.47 (m, 10H), 6.65
(d, J ¼ 8.2 Hz, 1H), 6.62 (d, J ¼ 1.8 Hz, 1H), 6.44 (dd, J ¼ 8.2,
2.2 Hz, 1H), 5.39 (br s, 1H), 5.12 (s, 2H), 3.82 (s, 3H), 3.51 (s,
2H), 3.42 (q, J ¼ 6.4 Hz, 2H), 2.65 (t, J ¼ 6.7 Hz, 2H); 13C
NMR (50 MHz, CDCl3) d 170.8, 149.6, 146.7, 137.2, 134.7,
131.7, 129.4, 128.9, 128.5, 127.8, 127.2, 127.1, 120.5, 114.1,
112.2, 71.0, 55.9, 43.8, 40.6, 35.0; MS (EI) 375 (Mþ), 240,
149, 137, 91 (100), 65. Registry: 4876-01-1.
4.1.3.2.
N-[2-(4-Benzyloxy-3-methoxyphenyl)ethyl]propion-
amide (7a). To a solution of 6 (3.5 mmol, 0.9 g) in CH2Cl2
were added Et3N (11 mmol, 1.1 g) and propionyl chloride
(4.6 mmol, 0.42 g) dropwise at 0 ꢀC. Ice bath was removed
and stirred for 30e60 min. After removal of solvent by evapo-
ration, H2O was added and then extracted with ethyl acetate.
The organic layer was washed with brine and water, and dried
over Na2SO4. Solvent was evaporated and the product was pu-
rified by flash column chromatography (3:2:5, EtOAc:CH2Cl2:-
hexane). Compound 7a (0.98 g, 90%) was obtained as a white
4.1.3.6. N-[2-(4-Benzyloxy-3-methoxyphenyl)ethyl]-3-methyl-
butyramide (7e). Same procedure as for 7a was performed
with 6 (3.5 mmol, 0.9 g), isovaleryl chloride (4.2 mmol,
0.51 g), and Et3N (11 mmol, 1.1 g) to obtain 7e (1.1 g, 93%)
1
1
foam: m.p. 88e89 ꢀC; H NMR (200 MHz, CDCl3) d 7.26e
as a white solid: m.p. 114e115 ꢀC (lit. 114e115 ꢀC [28]); H
7.46 (m, 5H), 6.81 (d, J ¼ 8.0 Hz, 1H), 6.73 (d, J ¼ 2.2 Hz,
1H), 6.64 (dd, J ¼ 8.2, 1.9 Hz, 1H), 5.65 (br s, 1H), 5.12 (s,
2H), 3.86 (s, 3H), 3.46 (q, J ¼ 6.6 Hz, 2H), 2.73 (t,
J ¼ 7.0 Hz, 2H), 2.14 (q, J ¼ 7.5 Hz, 2H), 1.11 (t, J ¼ 7.7 Hz,
3H); 13C NMR (50 MHz, CDCl3) d 173.7, 149.6, 146.7,
137.1, 132.1, 128.4, 127.7, 127.2, 120.5, 114.2, 112.4, 71.0,
55.8, 40.5, 35.2, 29.6, 9.8; MS (EI) 313 (Mþ), 240, 149, 137,
NMR (200 MHz, CDCl3) d 7.46e7.25 (m, 5H), 6.82 (d,
J ¼ 8.0 Hz, 1H), 6.73 (d, J ¼ 1.8 Hz, 1H), 6.63 (dd, J ¼ 8.2,
2.0 Hz, 1H), 5.51 (br s, 1H), 5.13 (s, 2H), 3.87 (s, 3H), 3.49
(q, J ¼ 6.7 Hz, 2H), 2.74 (t, J ¼ 6.9 Hz, 2H), 2.15e1.95 (m,
3H), 0.90 (d, J ¼ 6.6 Hz, 6H); 13C NMR (50 MHz, CDCl3)
d 172.4, 149.7, 146.7, 137.1, 132.0, 128.5, 127.7, 127.2,
120.5, 114.2, 112.4, 71.1, 55.9, 46.1, 40.4, 35.3, 22.4; MS

J.W. Seo et al. / European Journal of Medicinal Chemistry 43 (2008) 1160e1170
1165
(EI) 341 (Mþ), 240, 149, 137, 91 (100), 65, 57, 30. Registry:
95290-23-6.
1H NMR (400 MHz, DMSO-d6) d 9.69 (br s, 1H), 9.07 (br s,
1H), 9.02 (s, 1H), 6.73 (s, 1H), 6.66 (s, 1H), 4.23 (br s, 1H),
3.74 (s, 3H), 3.16 (br s, 1H), 3.01e2.93 (m, 1H), 2.86e2.80
(m, 1H), 1.97e1.87 (m, 2H), 1.01 (t, J ¼ 3.7 Hz, 3H); 13C
NMR (100 MHz, DMSO-d6) d 147.1, 145.2, 124.5, 122.5,
113.0, 112.0, 55.6, 55.0, 54.6, 26.2, 24.5, 9.8; LC MS 208.2
(Mþ þ 1, 100). Registry: 19886-92-1.
4.1.3.7.
N-[2-(4-Benzyloxy-3-methoxyphenyl)ethyl]-2-cyclo-
propylacetamide (7f). Same procedure as for 7a was per-
formed with 6 (3.9 mmol, 1.0 g), cyclopropanecarbonyl
chloride (5.5 mmol, 0.59 g), and Et3N (12 mmol, 1.2 g) to ob-
1
tain 7f (1.12 g, 88%)as a white solid: m.p. 115e116 ꢀC; H
NMR (200 MHz, CDCl3) d 7.45e7.26 (m, 5H), 6.82 (d,
J ¼ 8.0 Hz, 1H), 6.74 (d, J ¼ 1.8 Hz, 1H), 6.58 (dd, J ¼ 8.0,
1.8 Hz, 1H), 5.77 (br s, 1H), 5.13 (s, 2H), 3.87 (s, 3H), 3.48
(q, J ¼ 6.6 Hz, 2H), 2.74 (t, J ¼ 7.0 Hz, 2H), 1.85e1.19 (m,
1H), 0.95 (quin, J ¼ 3.8 Hz, 2H), 0.74e0.64 (m, 2H); 13C
NMR (50 MHz, CDCl3) d 173.4, 149.6, 146.7, 137.2, 132.1,
128.4, 127.7, 127.2, 120.5, 114.2, 112.4, 71.1, 55.9, 40.8,
35.3, 14.6, 7.0; MS (EI) 325 (Mþ), 240, 149, 137, 91 (100),
69, 41. HRMS (EI) m/z calcd for C20H23NO3 [M]þ
325.1678; found 325.1678.
4.1.3.10. 7-Hydroxy-6-methoxy-1-propyl-1,2,3,4-tetrahydro-
isoquinoline hydrochloride (8b). Same procedure as for 8a
was followed with 7b (3.0 mmol, 1.0 g), POCl3 (9.0 mmol,
1.38 g), NaBH4 (24 mmol, 0.91 g), and 10% palladium char-
coal (0.1 g) to obtain 8b (0.59 g, 77%) as a white solid: m.p.
1
209e211 ꢀC; H NMR (400 MHz, DMSO-d6) d 9.80e9.20
(br s, 1H), 9.04 (br s, 1H), 6.72 (s, 1H), 6.66 (s, 1H), 4.26
(t, J ¼ 3.2 Hz, 1H), 3.74 (s, 3H), 3.36e3.30 (m, 1H), 3.00e
2.93 (m, 1H), 2.85e2.78 (m, 1H), 1.88e1.81 (m, 2H),
1.52e1.42 (m, 2H), 0.92 (t, J ¼ 7.2 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6) d 147.1, 145.2, 124.9, 122.5, 123.0,
112.0, 55.6, 53.5, 35.6, 24.5, 18.2, 13.7; LC MS (ESI) 222.2
(Mþ þ 1, 100). HRMS (FAB) m/z calcd for C13H20NO2
[MH]þ 222.1494; found 222.1495.
4.1.3.8. N-[2-(4-Benzyloxy-3-methoxyphenyl)ethyl]-2-cyclobu-
tylacetamide (7g). Same procedure as for 7a was performed
with 6 (3.5 mmol, 0.9 g), cyclobutanecarbonyl chloride
(4.6 mmol, 0.54 g), and Et3N (10.5 mmol, 1.06 g) to obtain
7g (0.74 g, 79%) as a white solid: m.p. 104e105 ꢀC; 1H
NMR (200 MHz, CDCl3) d 7.46e7.25 (m, 5H), 6.81 (d,
J ¼ 8.0 Hz, 1H), 6.73 (d, J ¼ 1.8 Hz, 1H), 6.64 (dd, J ¼ 8.2,
2.2 Hz, 1H), 5.49 (br s, 1H), 5.13 (s, 2H), 3.87 (s, 3H), 3.47
(q, J ¼ 6.6 Hz, 2H), 2.92 (quin, J ¼ 8.5 Hz, 1H), 2.73 (t,
J ¼ 7.0 Hz, 2H), 2.37e1.98 (m, 4H), 1.94e1.78 (m, 2H);
13C NMR (50 MHz, CDCl3) d 174.9, 149.7, 146.7, 137.2,
132.1, 128.4, 127.7, 127.2, 120.6, 114.2, 112.4, 71.1, 55.9,
40.4, 39.9, 35.2, 25.3, 18.1; MS (EI) 339 (Mþ), 240, 149,
137, 91 (100), 55. HRMS (EI) m/z calcd for C22H27NO3
[M]þ 339.1834; found 339.1838.
4.1.3.11. 7-Hydroxy-1-isopropyl-6-methoxy-1,2,3,4-tetrahydr-
oisoquinoline hydrochloride (8c). Same procedure as for 8a
was followed with 7c (3.0 mmol, 0.98 g), POCl3 (9.0 mmol,
1.38 g), NaBH4 (24 mmol, 0.91 g), 10% palladium charcoal
(0.1 g) to obtain 8c (536 mg, 70%) as a white solid: m.p.
1
194e196 ꢀC; H NMR (400 MHz, DMSO-d6) d 9.81 (br s,
1H), 9.04 (br s, 1H), 8.66 (br s, 1H), 6.69 (s, 1H), 4.26 (br t,
J ¼ 3.4 Hz, 1H), 3.75 (s, 3H), 3.37 (br s, 1H), 3.10e3.08 (m,
2H), 2.78e2.73 (m, 1H), 2.37e2.29 (m, 1H), 1.08 (d,
J ¼ 7.2 Hz, 3H), 0.84 (d, J ¼ 7.2 Hz, 3H); 13C NMR
(100 MHz, DMSO-d6) d 146.9, 145.3, 123.7, 123.4, 113.0,
111.9, 58.9, 55.5, 43.3, 30.8, 24.6, 19.2, 16.2; LC MS (ESI)
222.2 (Mþ þ 1, 100). HRMS (FAB) m/z calcd for
C13H20NO2 [MH]þ 222.1494; found 222.1489.
4.1.3.9. 1-Ethyl-7-hydroxy-6-methoxy-1,2,3,4-tetrahydroisoqu-
inoline hydrochloride (8a). POCl3 (9.0 mmol, 1.38 g) was
added to a solution of 7a (3.0 mmol, 0.9 g) in anhydrous ace-
tonitrile under N2. The reaction mixture was refluxed for 2 h
and then cooled to room temperature. Solvent was evaporated
and vacuumed. The crude mixture was dissolved in anhydrous
methanol and NaBH4 (15 mmol, 0.59 mg) was slowly added
under ice bath under nitrogen atmosphere. Ice bath was re-
moved and the reaction was stirred for 24 h. After reaction,
the mixture was quenched by 0.1 M NaOH solution and filtered
with silica gel and Na2SO4 fitted drying tube. Saturated sodium
bicarbonate solution was poured and then extracted with
CH2Cl2 two times. The organic layer was dried over Na2SO4
and evaporated in vacuo. The crude, a slightly yellow solid,
was dissolved in methanol and 10% palladium charcoal
(0.1 g) was added. Hydrogenation was performed with H2 bal-
loon with vigorous stirring for 12 h. After reaction, the mixture
was filtered with celite, and solvent was evaporated. Flash col-
umn chromatography (3e5% MeOH/CH2Cl2) was performed
and the product was crystallized as hydrochloride salt of 8a
(128 mg, 18%) from 1.0 M hydrogen chloride in diethyl ether
as a white powder: m.p. 150e152 ꢀC (lit. 218e220 ꢀC [29]);
4.1.3.12. 1-Benzyl-7-hydroxy-6-methoxy-1,2,3,4-tetrahydroiso-
quinoline (8d). Same procedure as for 8a was followed with
7d (1.6 mmol, 0.6 g), POCl3 (13 mmol, 2.0 g), NaBH4
(15 mmol, 0.59 mg), and 10% palladium charcoal (0.1 g) to
obtain 8d (0.1 g, 74%) as a white solid: m.p. 153e156 ꢀC
1
(lit. 115e119 ꢀC w/HCl [30]); H NMR (400 MHz, CDCl3)
d 7.22e7.36 (m, 5H), 6.62 (s, 1H), 6.57 (s, 1H), 4.04e4.10
(m, 1H), 3.82 (s, 3H), 3.13e3.22 (m, 2H), 2.70e2.90 (m,
3H), 2.62e2.70 (m, 1H); 13C NMR (400 MHz, CDCl3)
d 145.1, 143.6, 139.1, 131.2, 129.3, 128.6, 126.6, 126.4,
112.0, 111.1, 56.8, 55.9, 42.4, 40.9, 29.5; MS (CI) 270
(Mþ þ 1), 178 (100). Registry: 57256-36-7.
4.1.3.13. 7-Hydroxy-1-isobutyl-6-methoxy-1,2,3,4-tetrahydro-
isoquinoline hydrochloride (8e). Same procedure as for 8a
was followed with 7e (3.1 mmol, 1.1 g), POCl3 (9.4 mmol,
1.44 g), NaBH4 (25 mmol, 0.95 mg), and 10% palladium char-
coal (0.1 g) to obtain 8e (567 mg, 67%) as a white solid: m.p.
1
231e233 ꢀC dec.; H NMR (400 MHz, DMSO-d6) d 9.75 (br

1166
J.W. Seo et al. / European Journal of Medicinal Chemistry 43 (2008) 1160e1170
s, 1H), 9.36 (br s, 1H), 9.06 (s, 1H), 6.72 (s, 1H), 6.64
(s, 1H), 4.27 (br d, J ¼ 3.6 Hz, 1H), 3.74 (s, 3H), 3.37e3.11
(m, 1H), 3.20e3.08 (m, 1H), 3.02e2.90 (m, 1H), 2.90e2.78
(m, 1H), 2.00e1.89 (m, 1H), 1.88e1.77 (m, 1H), 1.65e
1.55 (m, 1H), 0.98 (d, J ¼ 6.4 Hz, 3H), 0.95 (d, J ¼ 6.8 Hz,
3H); 13C NMR (100 MHz, DMSO-d6) d 147.0, 145.2, 125.4,
122.5, 113.0, 112.0, 55.6, 51.8, 43.3, 38.7, 24.4, 23.8, 23.0.
21.6; LC MS (ESI) 236.2 (Mþ þ 1, 100). HRMS (FAB) m/z
calcd for C14H22NO2 [MH]þ 236.1651; found 236.1650.
one conformer of C1eH), 3.859 (s, one conformer of
OCH3), 3.850 (s, one conformer of OCH3), 3.78e3.73 (m,
one conformer of C3eH), 3.55e3.42 (m, one conformer of
C3eH), 3.06e2.97 (m, one conformer of C3eH), 2.94e2.79
(m, one conformer of C4eH, 1H), 2.79e2.70 (m, one con-
former of C4eH), 2.65e2.57 (m, one conformer of C4eH),
2.17 (s, one conformer of eNCOCH3), 2.16 (s, one conformer
of eNCOCH3), 1.88e1.70 (m, 2H), 0.96 (td, J ¼ 29.2, 7.2 Hz,
3H). Anal. calculated (C14H19NO3) C, 67.45; H, 7.68; N, 5.62;
found C, 67.32; H, 7.84; N, 5.67.
4.1.3.14. 1-Cyclopropyl-7-hydroxy-6-methoxy-1,2,3,4-tetrahy-
droisoquinoline hydrochloride (8f). Same procedure as for
8a was followed with 7f (3.1 mmol, 1.0 g), POCl3
(9.1 mmol, 1.40 g), NaBH4 (25 mmol, 0.93 g), and 10% palla-
dium charcoal (0.1 g) to obtain 8f (458 mg, 58%) as a white
4.1.3.17. 2-Acetyl-7-hydroxy-6-methoxy-1-propyl-1,2,3,4-tetra-
hydroisoquinoline (9b). Same procedure as for 9a was fol-
lowed with 8b (0.78 mmol, 0.20 g), acetic anhydride
(0.78 mmol, 79 mg) and Et3N (2.3 mmol, 0.24 g) and it gave
9b (92 mg, 45%) as a white solid: m.p. 121e122 ꢀC; 1H
NMR (400 MHz, CDCl3) d 6.69 (s, one conformer of C6eH
or C8eH), 6.64 (s, one conformer of C6eH or C8eH), 6.58
(s, one conformer of C6eH or C8eH), 6.56 (s, one conformer
of C6eH or C8eH), 5.54e5.48 (m, one conformer of C1eH),
4.70e4.56 (m, one conformer of C1eH and C3eH), 3.854 (s,
one conformer of OCH3), 3.845 (s, one conformer of OCH3),
3.79e3.71 (m, one conformer of C3eH), 3.57e3.47 (m, one
conformer of C3eH), 3.10e3.00 (m, one conformer of C3e
H), 2.95e2.58 (m, one conformer of C4eH, 2H), 2.164 (s,
one conformer of eNCOCH3), 2.157 (s, one conformer of
eNCOCH3), 1.88e1.60 (m, 2H), 1.50e1.24 (m, 2H), 1.12e
0.84 (m, 3H); 13C NMR (100 MHz, CDCl3) d 169.4, 145.5,
144.1, 143.8, 130.8, 129.9, 125.5, 124.4, 113.3, 112.5, 110.9,
110.4, 57.1, 55.9, 51.9, 40.7, 29.4, 38.7, 35.4, 28.7, 27.6,
21.8, 21.7, 19.9, 19.6, 14.0. Anal. calculated (C15H21NO3) C,
68.42; H, 8.04; N, 5.32; found C, 68.48; H, 8.04; N, 5.30.
1
solid: m.p. 210e213 ꢀC dec.; H NMR (400 MHz, DMSO-
d6) d 9.64 (br s, 2H), 9.09 (s, 1H), 6.97 (s, 1H), 6.73 (s,
1H), 3.74 (s, 3H), 3.55 (d, J ¼ 9.6 Hz, 1H), 3.41e3.36 (m,
1H), 3.14e2.97 (m, 2H), 2.86e2.77 (m, 1H), 1.18e1.09
(m, 1H), 0.88e0.81 (m, 1H), 0.81e0.72 (m, 1H), 0.72e0.64
(m, 1H), 0.60e0.52 (m, 1H); 13C NMR (100 MHz, DMSO-
d6) d 147.3, 145.2, 125.1, 122.4, 113.1, 111.9, 58.9, 55.6,
39.8, 24.5, 14.6, 5.8, 2.8; LC MS (ESI) 220.2 (Mþ þ 1,
100), 203.2. HRMS (FAB) m/z calcd for C13H18NO2 [MH]þ
220.1338; found 220.1336.
4.1.3.15. 1-Cyclobutyl-7-hydroxy-6-methoxy-1,2,3,4-tetrahy-
droisoquinoline hydrochloride (8g). Same procedure as for
8a was followed with 7g (3.0 mmol, 1.0 g), POCl3
(8.8 mmol, 1.36 g), NaBH4 (24 mmol, 0.89 g), and 10% palla-
dium charcoal (0.1 g) to obtain 8g (645 mg, 81%) as a white
1
solid: m.p. 269e271 ꢀC dec.; H NMR (400 MHz, DMSO-
d6) d 9.90e9.00 (br s, 2H), 9.07 (s, 1H), 6.73 (s, 1H), 6.62
(s, 1H), 4.20 (d, J ¼ 9.2 Hz, one conformer of C1eH), 4.14
(br d, J ¼ 4.8 Hz, one conformer of C1eH), 3.73 (s, 3H),
3.31e3.25 (m, 1H), 3.16 (d, J ¼ 4.0 Hz, 1H), 3.13e3.07 (m,
1H), 2.98e2.91 (m, 1H), 2.85e2.76 (m, 1H), 2.78e2.67
(m, 1H), 2.17e2.02 (m, 3H), 2.02e1.92 (m, 1H), 1.89e1.70
(m, 2H); 13C NMR (100 MHz, DMSO-d6) d 147.1, 145.1,
124.1, 122.4, 112.9, 112.1, 57.5, 55.5, 48.6, 38.3, 26.8, 25.2,
24.5, 17.6; LC MS (ESI) 234.2 (Mþ þ 1, 100). HRMS (FAB)
m/z calcd for C14H20NO2 [MH]þ 234.1494; found 234.1497.
4.1.3.18. 2-Acetyl-7-hydroxy-1-isopropyl-6-methoxy-1,2,3,4-
tetrahydroisoquinoline (9c). Same procedure as for 9a was fol-
lowed with 8c (0.78 mmol, 0.20 g), acetic anhydride
(0.78 mmol, 79 mg) and Et3N (3.9 mmol, 0.39 g) and it gave
9c (141 mg, 67%) as a white solid: m.p. 136e137 ꢀC; 1H
NMR (400 MHz, CDCl3) d 6.73 (s, one conformer of C6eH
or C8eH), 6.67 (s, one conformer of C6eH or C8eH), 6.61
(s, one conformer of C6eH or C8eH), 6.60 (s, one conformer
of C6eH or C8eH), 5.18 (d, J ¼ 8.8 Hz, one conformer of
C1eH), 4.54e4.44 (m, one conformer of C3eH), 4.16 (d,
J ¼ 8.2 Hz, one conformer of C1eH), 3.857 (s, one conformer
of OCH3), 3.850 (s, one conformer of OCH3), 3.74e3.62 (m,
one conformer of C3eH), 3.26e3.16 (m, one conformer of
C3eH), 2.95e2.72 (m, one conformer of C4eH, 2H), 2.15
(s, 3H), 2.08e1.90 (m, 1H), 1.14e0.92 (m, 6H); 13C NMR
(100 MHz, CDCl3) d 169.9, 169.8, 145.7, 145.5, 143.5,
143.1, 130.0, 128.9, 125.6, 124.9, 114.6, 113.8, 111.0, 110.4,
63.6, 57.7, 55.9, 42.0, 36.5, 33.7, 33.4, 28.0, 26.8, 22.0,
21.98, 20.5, 20.2, 20.0, 19.8. Anal. calculated (C15H21NO3)
C, 68.42; H, 8.04; N, 5.32; found C, 68.17; H, 8.28; N, 5.35.
4.1.3.16. 2-Acetyl-1-ethyl-7-hydroxy-6-methoxy-1,2,3,4-tetra-
hydroisoquinoline (9a). To a solution of 8a (0.36 mmol,
85 mg) in CH2Cl2 (10 mL) were added acetic anhydride
(0.35 mmol, 36 mg) and Et3N (1.8 mmol, 0.18 g) at room tem-
perature. After stirring for 2 h, solvent was evaporated at re-
duced pressure. Flash column chromatography (1:1,
EtOAc:hexane) was performed and recrystallization from
CH2Cl2 and hexane gave 9a (51 mg, 58%) as a white solid:
m.p. 131e132 ꢀC; 1H NMR (400 MHz, CDCl3) d 6.70 (s,
one conformer of C6eH or C8eH), 6.65 (s, one conformer
of C6eH or C8eH), 6.58 (s, one conformer of C6eH or
C8eH), 6.56 (s, one conformer of C6eH or C8eH), 5.46e
5.40 (dd, J ¼ 8.8, 6.0 Hz, one conformer of C1eH), 4.66e
4.59 (m, one conformer of C3eH), 4.59e4.53 (t, J ¼ 7.2 Hz,
4.1.3.19. 2-Acetyl-1-benzyl-7-hydroxy-6-methoxy-1,2,3,4-tetra-
hydroisoquinoline (9d). Same procedure as for 9a was fol-
lowed with 8d (0.26 mmol, 69 mg) and acetic anhydride

J.W. Seo et al. / European Journal of Medicinal Chemistry 43 (2008) 1160e1170
1167
(0.28 mmol, 29 mg) and it gave 9d (75 mg, 94%) as a white
1
(m, one conformer of C4eH, 2H), 2.17 (s, one conformer of
eNCOCH3), 2.12 (s, one conformer of eNCOCH3), 1.30e
1.10 (m, 1H), 0.77e0.48 (m, 3H), 0.44e0.34 (m, 6H); 13C
NMR (100 MHz, CDCl3) d 169.0, 145.5, 144.0, 143.7, 129.9,
128.5, 125.7, 124.5, 114.6, 113.3, 112.7, 110.8, 110.4, 60.4,
55.9, 55.5, 41.2, 36.1, 28.8, 27.7, 21.7, 21.5, 18.0, 17.8, 5.31,
5.25, 2.9, 2.5. Anal. calculated (C15H19NO3) C, 68.94; H,
7.33; N, 5.36; found C, 68.94; H, 7.47; N, 5.35.
foam: m.p. 66e70 ꢀC; H NMR (400 MHz, CDCl3) d 7.40e
6.95 (m, 5H), 6.72 (s, one conformer of C6eH or C8eH),
6.58 (s, one conformer of C6eH or C8eH), 6.50 (s, one con-
former of C6eH or C8eH), 6.48 (s, one conformer of C6eH
or C8eH), 4.62e4.40 (m, one conformer of C1eH), 3.85 (s,
one conformer of OCH3), 3.82 (s, one conformer of OCH3),
3.15e2.95 (m, one conformer of C3eH and one conformer of
C4eH), 2.90e2.80 (m, one conformer of benzyl-H), 2.55e
2.52 (m, one conformer of benzyl-H), 2.55e2.45 (m, one con-
former of benzyl-H), 2.06 (s, one conformer of eNCOCH3),
1.40 (s, one conformer of eNCOCH3); 13C NMR (100 MHz,
CDCl3) d 169.8, 169.3, 145.7, 145.4, 144.0, 143.9, 138.0,
129.7, 129.4, 129.2, 128.9, 128.7, 128.0, 127.0, 126.4, 126.0,
125.2, 113.4, 112.6, 110.9, 110.2, 59.3, 55.9, 55.8, 53.6, 42.9,
42.0, 41.7, 34.9, 28.6, 28.0, 22.0, 20.8; MS (CI) 312 (Mþ þ 1,
100), 220, 178. HRMS (FAB) m/z calcd for C19H21NO3
[MH]þ 312.1600; found 312.1597.
4.1.3.22. 2-Acetyl-1-cyclobutyl-7-hydroxy-6-methoxy-1,2,3,4-
tetrahydroisoquinoline (9g). Same procedure as for 9a was
followed with 8g (0.88 mmol, 0.24 g), acetic anhydride
(0.88 mmol, 89 mg) and Et3N (4.4 mmol, 0.44 g) and it gave
1
9g (195 mg, 80%) as a white solid: m.p. 123e124 ꢀC; H
NMR (400 MHz, CDCl3) d 6.70 (s, one conformer of C6eH
or C8eH), 6.65 (s, one conformer of C6eH or C8eH), 6.58
(s, one conformer of C6eH or C8eH), 6.56 (s, one conformer
of C6eH or C8eH), 5.46 (d, J ¼ 9.6 Hz, one conformer of
C1eH), 4.68e4.60 (m, one conformer of C3eH), 4.50 (d,
J ¼ 9.2 Hz, one conformer of C1eH), 3.85 (s, one conformer
of OCH3), 3.84 (s, one conformer of OCH3), 3.77e3.69 (m,
one conformer of C3eH), 3.58e3.44 (m, one conformer of
C3eH), 3.04e2.95 (m, one conformer of C3eH), 2.92e2.68
(m, one conformer of C4eH, 2H), 2.66e2.55 (m, 1H), 2.21
(s, one conformer of eNCOCH3), 2.15 (s, one conformer of
eNCOCH3), 2.10e1.90 (m, 3H), 1.89e1.68 (m, 3H); 13C
NMR (100 MHz, CDCl3) d 169.4, 169.3, 145.6, 145.4,
143.8, 143.5, 129.5, 128.4, 125.2, 124.1, 113.0, 112.2,
111.1, 110.6, 61.5, 56.0, 55.9, 41.4, 41.1, 41.0, 35.7, 28.6,
27.8, 27.6, 27.4, 26.1, 25.5, 21.9, 21.7, 17.6. Anal. calculated
(C16H21NO3) C, 69.79; H, 7.69; N, 5.09; found C, 70.01; H,
7.81; N, 5.06.
4.1.3.20. 2-Acetyl-7-hydroxy-1-isobutyl-6-methoxy-1,2,3,4-tet-
rahydroisoquinoline (9e). Same procedure as for 9a was fol-
lowed with 8e (0.73 mmol, 0.20 g), acetic anhydride
(0.73 mmol, 75 mg) and Et3N (3.7 mmol, 0.37 g) and it gave
9e (148 mg, 73%) as a white solid: m.p. 136e137 ꢀC; 1H
NMR (400 MHz, CDCl3) d 6.66 (s, one conformer of C6eH
or C8eH), 6.61 (s, one conformer of C6eH or C8eH), 6.58
(s, one conformer of C6eH or C8eH), 6.54 (s, one conformer
of C6eH or C8eH), 5.64e5.56 (m, one conformer of C1eH),
4.70e4.64 (m, one conformer of C1eH), 4.58e4.39 (m, one
conformer of C3eH), 3.86 (s, one conformer of OCH3),
3.84 (s, one conformer of OCH3), 3.80e3.71 (m, one con-
former of C3eH), 3.58e3.47 (m, one conformer of C3eH),
3.18e3.07 (m, one conformer of C3eH), 2.94e2.81 (m, one
conformer of C4eH, 1H), 2.76e2.69 (m, one conformer of
C4eH, 1H), 2.17 (s, one conformer of eNCOCH3), 2.157
(s, one conformer of eNCOCH3), 1.83e1.38 (m, 3H),
1.10e0.88 (m, 6H); 13C NMR (100 MHz, CDCl3) d 169.8,
169.5, 145.5, 145.2, 144.1, 143.8, 131.1, 129.9, 125.5,
124.4, 113.3, 112.5, 110.9, 110.5, 55.9, 55.6, 50.4, 46.5,
46.0, 40.3, 35.8, 28.6, 27.4, 25.0, 24.7, 23.4, 23.1, 22.7,
22.4, 21.6, 21.4. Anal. calculated (C16H23NO3) C, 69.29; H,
8.36; N, 5.05; found C, 69.26; H, 8.60; N, 5.02.
4.1.4. Procedures for the preparation of 11aee
4.1.4.1. 2-Ethylcarbonyl-7-hydroxy-6-methoxy-1,2,3,4-tetrahy-
droisoquinoline (11a). To a solution of 10 in CH2Cl2
(10 mL) were added propionic anhydride and Et3N and stirred
for 1 h at room temperature (Scheme 3). The reaction was
quenched by addition of water and the organic layer was ex-
tracted with additional CH2Cl2 (5 mL). After CH2Cl2 was
evaporated at reduced pressure, residual oil was dissolved in
methanol (10 mL) and K2CO3 was added. The reaction was re-
fluxed for 3 h. The solution was filtered and excess CH2Cl2
was poured. The organic layer was washed with 1.0 M HCl so-
lution (20 mL) and brine (10 mL). The organic solution was
dried over Na2SO4 and concentrated by evaporation. Flash col-
umn chromatography (1:1, EtOAc:hexane) gave 11a as a white
4.1.3.21. 2-Acetyl-1-cyclopropyl-7-hydroxy-6-methoxy-1,2,3,4-
tetrahydroisoquinoline (9f). Same procedure as for 9a was fol-
lowed with 8f (0.77 mmol, 0.20 g), acetic anhydride
(0.77 mmol, 78 mg) and Et3N (3.9 mmol, 0.39 g) and it gave
9f (145 mg, 72%) as a white solid: m.p. 175e176 ꢀC; 1H
NMR (400 MHz, CDCl3) d 6.83 (s, one conformer of C6eH
or C8eH), 6.74 (s, one conformer of C6eH or C8eH), 6.59
(s, one conformer of C6eH or C8eH), 6.56 (s, one conformer
of C6eH or C8eH), 4.95 (d, J ¼ 8.8 Hz, one conformer of
C1eH), 4.72e4.63 (m, one conformer of C3eH), 4.17 (d,
J ¼ 8.0 Hz, one conformer of C1eH), 3.858 (s, one conformer
of OCH3), 3.848 (s, one conformer of OCH3), 3.85e3.78 (m,
one conformer of C3eH), 3.72e3.63 (m, one conformer of
C3eH), 3.30e3.18 (m, one conformer of C3eH), 2.96e2.60
1
solid: m.p. 95e96 ꢀC; H NMR (200 MHz, CDCl3) d 6.71 (s,
one conformer of C5eH or C8eH), 6.66 (s, one conformer of
C5eH or C8eH), 6.62 (s, one conformer of C5eH or C8eH),
6.60 (s, one conformer of C5eH or C8eH), 6.10 (s, one con-
former of OeH), 5.96 (s, one conformer of OeH), 4.62 (s, one
conformer of C1eH), 4.50 (s, one conformer of C1eH), 3.86
(s, O CH3, 3H), 3.95e3.49 (m, C4eH, 2H), 2.85e2.70 (m,
C3eH, 2H), 2.46 (q, J ¼ 7.4 Hz, 2H), 1.15e1.05 (m, 3H);
13C NMR (50 MHz, CDCl3) d 172.8, 145.5, 144.6, 144.3,

1168
J.W. Seo et al. / European Journal of Medicinal Chemistry 43 (2008) 1160e1170
(m, 5H), 1.64e1.44 (m, 2H), 1.38e1.28 (m, 3H); 13C NMR
MeO
HO
MeO
HO
a, b
(100 MHz, CDCl3) d 174.9, 174.8, 145.4, 145.3, 144.4,
144.1, 126.3, 126.3, 125.2, 125.0, 112.4, 111.6, 110.9,
110.42, 56.0, 46.9, 44.0, 43.2, 41.1, 41.0, 40.0, 29.6, 29.4,
29.3, 28.1, 26.0, 25.9; MS (EI) 289 (Mþ, 100), 274, 178,
163, 150. HRMS (EI) m/z calcd for C17H23NO3 [M]þ
289.1678; found 289.1682.
NH
N
R
10
O
11a, R = CH2CH3 (31%)
c
11b, R = CH2CH2CH3 (75%)
11c, R = cyclohexyl (57%)
11d, R = CH(CH3)2 (71%)
11e, R = CH2CH(CH3)2 (60%)
MeO
HO
N
R
4.1.4.4. 7-Hydroxy-2-isopropylcarbonyl-6-methoxy-1,2,3,4-tet-
rahydroisoquinoline (11d). Same procedure as for 11a was
followed with 10 (1.0 mmol, 0.22 g), isobutyryl chloride
(2.0 mmol, 21 mL), Et3N (0.5 mL), and K2CO3 (0.16 g) and
it gave 11d (178 mg, 71%) as a white solid: m.p. 115e
117 ꢀC; 1H NMR (200 MHz, CDCl3) d 6.76 (s, one conformer
of C5eH or C8eH), 6.67 (s, one conformer of C5eH or
C8eH), 6.61 (s, one conformer of C5eH or C8eH), 6.20 (s,
one conformer of OeH), 6.00 (s, one conformer of OeH),
4.61 (s, one conformer of C1eH), 4.55 (s, one conformer of
C1eH), 3.86 (s, O CH3, 3H), 3.95e3.60 (m, C4eH, 2H),
3.00e2.65 (m, 3H), 1.45 (t, J ¼ 6.6 Hz, 6H); 13C NMR
(50 MHz, CDCl3) d 175.8, 145.5, 144.4, 126.3, 125.0, 112.6,
111.7, 111.0, 110.7, 56.0, 46.8, 44.0, 43.1, 40.1, 30.4, 29.4,
28.0, 19.3; MS (EI) 249 (Mþ, 100), 234, 206, 178, 163, 150.
HRMS (EI) m/z calcd for C14H19NO3 [M]þ 249.1365; found
249.1368.
12a, R = CH2CH3 (92%)
12b, R = CH2CH2CH3 (70%)
12c, R = cyclohexyl (90%)
Scheme 3. (a) Propionic anhydride, butyryl chloride, cyclohexanecarbonyl
chloride, isobutyryl chloride, or 3-methylbutyryl chloride, TEA, CH2Cl2, rt,
1 h; (b) K2CO3, MeOH, reflux, 2 h; (c) LAH, THF, reflux, 3e5 h.
126.12, 126.11, 112.5, 111.8, 111.0, 110.6, 56.0, 46.8, 43.8,
43.2, 39.8, 29.1, 28.1, 26.9, 26.7, 9.4; MS (EI) 235 (Mþ,
100), 220, 178, 163, 150, 135. HRMS (EI) m/z calcd for
C13H17NO3 [M]þ 235.1208; found 235.1208.
4.1.4.2. 7-Hydroxy-6-methoxy-2-propylcarbonyl-1,2,3,4-tetra-
hydroisoquinoline (11b). Same procedure as for 11a was fol-
lowed with 10 (1.0 mmol, 0.22 g), butyryl chloride
(1.0 mmol, 93 mg), Et3N (4.5 mmol, 0.45 g), and K2CO3
(0.1 g) and it gave 11b (187 mg, 75%) as a white solid: m.p.
115e116 ꢀC; 1H NMR (200 MHz, CDCl3) d 6.71 (s, one con-
former of C5eH or C8eH), 6.66 (s, one conformer of C5eH or
C8eH), 6.61 (s, one conformer of C5eH or C8eH), 6.60 (s,
one conformer of C5eH or C8eH), 6.21 (s, one conformer
of OeH), 6.03 (s, one conformer of OeH), 4.62 (s, one con-
former of C1eH), 4.50 (s, one conformer of C1eH), 3.85 (s,
O CH3, 3H), 3.95e3.60 (m, C4eH, 2H), 2.90e2.70 (m, C3e
H, 2H), 2.46 (t, J ¼ 7.4 Hz, 2H), 1.80e1.60 (m, 2H), 1.05e
0.90 (m, 3H); 13C NMR (50 MHz, CDCl3) d 172.1, 145.8,
144.7, 144.5, 126.2, 125.2, 112.7, 111.9, 111.2, 110.8, 56.1,
47.1, 43.9, 43.5, 39.9, 35.8, 35.7, 29.3, 28.2, 18.7, 14.1; MS
(EI) 249 (Mþ, 100), 220, 178, 163, 150. HRMS (EI) m/z calcd
for C14H19NO3 [M]þ 249.1365; found 249.1361.
4.1.4.5. 7-Hydroxy-2-isobutylcarbonyl-6-methoxy-1,2,3,4-tet-
rahydroisoquinoline (11e). Same procedure as for 11a was
followed with 10 (1.0 mmol, 0.22 g), 3-methylbutyryl chloride
(2.0 mmol, 25 mL), Et3N (0.5 mL), and K2CO3 (0.16 g) and it
gave 11e (158 mg, 60%) as a white solid: m.p. 141e142 ꢀC;
1H NMR (200 MHz, CDCl3) d 6.71 (s, one conformer of
C5eH or C8eH), 6.66 (s, one conformer of C5eH or C8e
H), 6.61 (s, one conformer of C5eH or C8eH), 6.60 (s, one
conformer of C5eH or C8eH), 6.21 (s, one conformer of
OeH), 6.04 (s, one conformer of OeH), 4.63 (s, one con-
former of C1eH), 4.51 (s, one conformer of C1eH), 3.85
(s, O CH3, 3H), 3.95e3.60 (m, C4eH, 2H), 2.85e2.67 (m,
2H), 2.38e2.05 (m, 3H), 1.10e0.95 (m, 6H); 13C NMR
(50 MHz, CDCl3) d 171.4, 145.7, 145.5, 144.6, 144.3, 126.2,
126.1, 125.0, 112.5, 111.7, 111.0, 110.6, 55.9, 47.2, 43.8,
43.6, 42.5, 42.3, 39.8, 29.2, 28.1, 25.6, 22.7; MS (EI) 263
(Mþ), 220 (100), 206, 178, 163, 150. HRMS (EI) m/z calcd
for C15H21NO3 [M]þ 263.1521; found 263.1523.
4.1.4.3. 2-Cyclohexylcarbonyl-7-hydroxy-6-methoxy-1,2,3,4-
tetrahydroisoquinoline (11c). Same procedure as for 11a
was followed with 10 (2.0 mmol, 0.43 g), cyclohexanecar-
bonyl chloride (4.0 mmol, 0.59 mg), Et3N (0.9 mL), and
K2CO3 (0.32 g) and it gave 11c (302 mg, 57%) as a white
4.1.5. Procedures for the preparation of 12aec (Scheme 3)
1
solid: m.p. 160e161 ꢀC; H NMR (400 MHz, CDCl3) d 6.71
(s, one conformer of C5eH or C8eH), 6.68 (s, one conformer
of C5eH or C8eH), 6.61 (s, one conformer of C5eH or C8e
H), 6.59 (s, one conformer of C5eH or C8eH), 5.83 (s, one
conformer of OeH), 5.68 (s, one conformer of OeH), 4.61
(s, one conformer of C1eH), 4.54 (s, one conformer of C1e
H), 3.861 (s, one conformer of O CH3), 3.857 (s, one con-
former of O CH3), 3.79 (t, J ¼ 6.0 Hz, one conformer of
C4eH), 3.68 (t, J ¼ 6.0 Hz, one conformer of C4eH), 2.81
(t, J ¼ 5.6 Hz, one conformer of C3eH), 2.74 (t, J ¼ 5.6 Hz,
one conformer of C3eH), 2.59e2.49 (m, 1H), 1.90e1.64
4.1.5.1. 7-Hydroxy-6-methoxy-2-propyl-1,2,3,4-tetrahydroiso-
quinoline hydrochloride (12a). To
a solution of 11a
(0.68 mmol, 0.16 g) in freshly distilled THF (10 mL) was
added 1.0 M lithium aluminium hydride (0.6 mL) in THF so-
lution. The reaction mixture was refluxed for 4 h and cooled to
room temperature. The reaction was quenched by EtOAc,
1.0 M NaOH solution, and H2O and the solution was filtered.
Organics were extracted by CH2Cl2 (20 mL) and concentrated
under reduced pressure. Flash column chromatography (5%
MeOH/CH2Cl2) was performed and 12a (138 mg, 92%) was

J.W. Seo et al. / European Journal of Medicinal Chemistry 43 (2008) 1160e1170
1169
crystallized as a hydrochloride salt (off-white solid) from eth-
anol and 1.0 M hydrochloride diethyl ether: m.p. 203e205 ꢀC
dec.; H NMR (400 MHz, DMSO-d6) d 9.14 (br s, OH, 1H),
4.2.2. NO production
Accumulated nitrite, a stable oxidation metabolite of NO,
was measured by the Griess reaction [24]. Briefly, 200 mL al-
iquots of the culture medium were mixed with 100 mL of Gri-
ess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine
dihydrochloride, and 2.5% H3PO4) in a 96-well microtiter
plate, and the absorbance was read at 540 nm. The effect of
each compound on NO production was expressed as the per-
centage of NO produced by the LPS-treated control cells.
1
6.74 (s, 1H), 6.59 (s, 1H), 4.27 (d, J ¼ 12.6 Hz, 1H), 4.06
(dd, J ¼ 16.0, 8.0 Hz, 1H), 3.75 (s, 3H), 3.37 (br s, 1H),
3.25e3.00 (m, 4H), 2.95e2.90 (br m, 1H), 1.85e1.75 (m,
2H), 0.90 (t, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, DMSO-
d6) d 147.3, 145.4, 121.6, 120.1, 112.9, 111.8, 56.4, 55.6,
51.0, 48.7, 24.3, 16.8, 11.0; MS (EI) 221 (Mþ), 192 (100),
150. HRMS (EI) m/z calcd for C13H19NO2 [M]þ 221.1416;
found 221.1415.
4.2.3. BH4 production
BH4 produced was determined according to the method
reported previously [25]. To the 900 mL aliquot of culture
medium, 100 mL of 1 M phosphoric acid and 200 mL of acidic
iodine solution (0.5% I2 and 1.0% KI in 0.2 M trichloroacetic
acid) were added and incubated for 1 h in the dark. The oxida-
tion reaction was terminated by addition of 0.1 mL of 0.1%
ascorbic acid. The reaction mixture was then centrifuged for
15 min at 8000ꢁg and the supernatant was diluted with
distilled water. BH4 was separated isocratically with 5% meth-
anol as mobile phase using HPLC and detected by a fluores-
cence detector (Waters, Boston, MA, USA). BH4 standard
curve was prepared every time. The BH4 content was calcu-
lated using Waters 991 computerized integrator system as
nanograms of BH4 per milligram of cellular protein and ex-
pressed as percentage of LPS-treated control.
4.1.5.2. 2-Butyl-7-hydroxy-6-methoxy-1,2,3,4-tetrahydroiso-
quinoline hydrochloride (12b). Same procedure as for 12a
was performed with 11b (0.56 mmol, 0.14 g) and 1.0 M lith-
ium aluminium hydride (0.6 mL) and it gave 12b (92 mg,
70%) as a hydrochloride salt (white solid): m.p. 105e110 ꢀC
1
dec.; H NMR (200 MHz, DMSO-d6) d 6.77 (s, 1H), 6.60 (s,
1H), 4.34 (d, J ¼ 13.6 Hz, 1H), 4.06 (dd, J ¼ 15.2, 8.0 Hz,
1H), 3.75 (s, 3H), 3.37 (br s, 1H), 3.25e3.00 (m, 4H),
2.95e2.90 (br m, 1H), 1.85e1.75 (m, 2H), 1.50e1.25 (m,
2H), 0.93 (t, J ¼ 7.0 Hz, 3H); 13C NMR (50 MHz, DMSO-
d6) d 147.3, 145.4, 121.6, 120.1, 112.9, 111.8, 55.6, 54.6,
51.0, 48.7, 25.1, 24.3, 19.5, 13.5; MS (EI) 235 (Mþ), 192
(100), 150. HRMS (EI) m/z calcd for C14H21NO2 [M]þ
235.1572; found 235.1571.
4.2.4. Determination of cytotoxicity by LDH activity
4.1.5.3. 2-Cyclohexylmethyl-7-hydroxy-6-methoxy-1,2,3,4-tet-
rahydroisoquinoline hydrochloride (12c). Same procedure as
for 12a was performed with 11c (0.6 mmol, 0.18 g) and
1.0 M lithium aluminium hydride (0.4 mL) and it gave 12c
(149 mg, 90%) as a hydrochloride salt (off-white solid): m.p.
193e195 ꢀC; 1H NMR (200 MHz, DMSO-d6) d 6.76 (s,
1H), 6.61 (s, 1H), 4.34 (d, J ¼ 13.2 Hz, 1H), 4.11 (dd,
J ¼ 15.2, 7.2 Hz, 1H), 3.74 (s, 3H), 3.65e2.65 (m, 6H),
2.00e1.65 (m, 6H), 1.40e1.80 (m, 5H); 13C NMR (50 MHz,
DMSO-d6) d 147.3, 145.4, 121.5, 119.9, 113.1, 111.8, 60.6,
55.9, 55.6, 32.1, 30.7, 25.4, 25.0, 23.9, 18.5; MS (EI) 275
(Mþ), 192 (100), 150. HRMS (EI) m/z calcd for C17H25NO2
[M]þ 275.1885; found 275.1885.
Cytotoxicity of the newly synthesized compounds was as-
sessed by determining the activity of LDH released into the
culture medium. Aliquots (50 mL) of cell culture medium
were incubated at room temperature in the presence of
0.26 mM NADH, 2.87 mM sodium pyruvate, and 100 mM po-
tassium phosphate buffer (pH 7.4) in a total volume of 200 mL.
The rate of NADþ formation was monitored for 5 min at 2-s
intervals at 340 nm using a microplate spectrophotometer
(SPECTRA MAX 340 pc; Molecular Devices, Menlo Park,
CA). Cytotoxicity values were expressed as percentage of
LDH released by the tetrahydroisoquinoline derivatives com-
pared with that released from LPS-treated control cells and
thus, numbers approaching 100 indicate no toxicity.
Acknowledgements
4.2. Biology
This work was supported by the Brain Research Center of the
21st Century Frontier Research Program of the Ministry of Sci-
ence and Technology (M103KV010011-06K2201-01110), the
Korea Health 21 R&D Project (A05-0242-A20718-05N1-
00010A), and KOSEF grant funded by National Research and
Development Program of MOST, Korea (2006-03770).
4.2.1. Cell culture
BV-2 cells, a murine microglial cell line, were grown and
maintained in DMEM supplemented with 10% fetal calf se-
rum and penicillinestreptomycin at 37 ꢀC in a humidified in-
cubator under 5% CO2. For experiments, the cells were plated
on polystyrene tissue culture dishes at a density of 2 ꢁ 105
cells/well in 24-well culture plates. After 24 h, the cells
were changed into fresh medium and treated with bacterial
LPS (0.2 mg/mL). The tetrahydroisoquinoline derivatives (at
various concentrations dissolved in DMSO) were added at
the same time with LPS treatment. After 24 h, the medium
was taken to measure NO and BH4 and lactate dehydrogenase
(LDH) activity.
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