Novel tetrahydroisoquinoline derivatives with inhibitory activities against acyl-CoA: Cholesterol acyltransferase and lipid peroxidation

Article abstract of DOI:10.1248/cpb.58.1066

To find a novel acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor with anti-lipid peroxidative activity, a series of tetrahydroisoquinoline derivatives were synthesized and evaluated. A compound with a N-(4-hydroxy-2,3,5-trimethylphenyl)carbamoyl moi

Full text of DOI:10.1248/cpb.58.1066

1066
Regular Article
Chem. Pharm. Bull. 58(8) 1066—1076 (2010)
Vol. 58, No. 8
Novel Tetrahydroisoquinoline Derivatives with Inhibitory Activities
against Acyl-CoA: Cholesterol Acyltransferase and Lipid Peroxidation
Masaru OHTA,a,b Kenji TAKAHASHI,a Masayasu KASAI,a Yoshimichi SHOJI,a Kazuyoshi KUNISHIRO,a
a
a
b
,a
*
Tomohiro MIIKE, Mamoru KANDA, Chisato MUKAI, and Hiroaki SHIRAHASE
a Research Laboratories, Kyoto Pharmaceutical Industries, Ltd.; 38 Nishinokyo Tsukinowa-cho, Nakagyo-ku, Kyoto
604–8444, Japan: and b Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology,
Kanazawa University; Kakuma-machi, Kanazawa, Ishikawa 920–1192, Japan.
Received April 8, 2010; accepted May 19, 2010
To find a novel acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor with anti-lipid peroxidative activity,
a series of tetrahydroisoquinoline derivatives were synthesized and evaluated. A compound with a N-(4-hydroxy-
2,3,5-trimethylphenyl)carbamoyl moiety at the 3-position and an octanoyl moiety at the 2-position (7) was
demonstrated to show anti-foam cell formation activity stronger than and anti-lipid peroxidative activity compa-
rable to those of Pactimibe, while it was hardly absorbed orally. To increase its bioavailability, the acyl chain at
the 2-position was shortened and various polar or basic moieties were introduced at the 7-position of 7. Among
the synthesized derivatives, (S)-7-dimethylamino-N-(4-hydroxy-2,3,5-trimethylphenyl)-2-isobutyryl-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide hydrochloride (21) showed about 16-fold stronger anti-foam cell forma-
tion activity, 3-fold stronger hepatic ACAT inhibitory activity, similar anti-low density lipoprotein (LDL) oxida-
tive activity and 2-fold more potent protective activity against macrophage cell death by oxidative stress in com-
parison with Pactimibe. Compound 21 was efficiently absorbed after oral administration at 10 mg/kg in rats and
dogs and its Cmax values were higher than its IC50 values for in vitro activities. In conclusion, a tetrahydroiso-
quinoline structure is a useful scaffold for designing a phenolic anti-oxidative ACAT inhibitor, and compound 21
is expected to effectively prevent atherosclerosis.
Key words acyl-CoA: cholesterol acyltransferase; tetrahydroisoquinoline; foam cell formation; lipid peroxidation; oxidative
stress-induced cell death; Pactimibe
A great number of acyl-CoA: cholesterol acyltransferase ing dimethylamino moiety, such as NTE-122, also show po-
(ACAT) inhibitors have been reported as potential hypolipi- tent anti-oxidative activities11); however, the pharmacological
demic drugs and anti-atherosclerotic drugs, since ACAT effects of the aromatic amine-derived anti-oxidative activities
plays an important role in intestinal absorption and hepatic have not been elucidated in atherosclerosis. On the other
secretion of cholesterol and accumulation of cholesterol in hand, a number of phenolic compounds have been reported
macrophages in atherosclerotic plaque1—4); however, none of to have anti-oxidative activities. a-Tocopherol and Probucol
the reported inhibitors have been successfully developed. have been reported to show anti-atherosclerotic effects, prob-
Most of them were highly lipophilic, since ACAT inhibitory ably due to their preventive activities against LDL oxida-
activity is dependent on the inhibitor’s lipophilicity,5,6) and tion.12,13)
were thus demonstrated to have low bioavailability. We pre-
During the course of studies to find a new ACAT inhibitor
viously synthesized a new indoline-based ACAT inhibitor, with structural and biological properties different from
Pactimibe, with a carboxymethyl group at the 5-position, Pactimibe, a tetrahydroisoquinoline derivative with a N-(4-
which showed moderate ACAT inhibitory activities; however, hydroxy-2,3,5-trimethylphenyl)carbamoyl moiety at the 3-
it was highly water soluble, and showed good oral absorp- position and an octanoyl moiety at the 2-position (7) was
tion.7) Pactimibe also has potent anti-oxidative activities, found to have ACAT inhibitory and anti-oxidative activities
which were expected to exert synergetic anti-atherosclerotic comparable to those of Pactimibe, but to show poor bioavail-
effects with ACAT inhibitory activities, since oxidized low ability. To increase the bioavailability of 7, a series of
density lipoprotein (LDL) is taken up by macrophages and tetrahydroisoquinoline derivatives with polar or basic moi-
then cholesterol is esterified by ACAT to be accumulated in eties at the 7-position were synthesized and evaluated.
foam cells in atherosclerosis. Pactimibe decreased athero-
Chemistry The synthetic routes of various 2-acyl-N-
sclerotic areas in apo-E knockout mice and stabilized aortic aryl-1,2,3,4-tetrahydroisoquinoline-3-carboxamides are
plaques in Watanabe heritable hyperlipidemic rabbits shown in Chart 1—3. In Chart 1, the synthetic routes of N-
(WHHL rabbits)8,9); however, a clinical study using an in- aryl-2-octanoyl-1,2,3,4-tetrahydroisoquinoline-3-carboxam-
travascular-ultrasonography catheter did not show the retar- ides with no substitution at the 7-position (7, 8, 10) are out-
dation of plaques at a dose of 100 mg in patients with coro- lined. The starting material 1, which was easily prepared
nary artery disease (CAD).10) Pactimibe may have not fully from L-phenylalanine,14) was acylated with octanoyl chloride
inhibited plaque ACAT activity, and/or its ACAT-inhibitory to give 2. The methyl ester of 2 was hydrolyzed with NaOH
and anti-oxidative activities may have not exerted synergetic to provide carboxylic acid (3), which was condensed with
anti-atherosclerotic effects in humans. The anti-oxidative ac- three anilines, 4-amino-2,3,6-trimethylphenyl acetate (4),15)
tivity of Pactimibe is due to its indoline structure, of which 2,6-diisopropylaniline, and 2,6-diisopropyl-4-nitroaniline (5),
the tertiary amine at the 1-position chemically resembles that to give 6, 8, 9, respectively. Compound 6 was hydrolyzed
of aromatic dialkylamine. Indeed, ACAT inhibitors contain- with LiOH to give 7. The nitro moiety of compound 9 was
∗ To whom correspondence should be addressed. e-mail: shirahase@kyoto-pharm.co.jp
© 2010 Pharmaceutical Society of Japan

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Reagents: (a) n-octanoyl chloride, Et3N, CH2Cl2; (b) NaOH aq., MeOH; (c) 4, POCl3, pyridine, CH2Cl2; (d) 2,6-diisopropylaniline, EDC·HCl, CH2Cl2; (e) 5, POCl3,
pyridine, CH2Cl2; (f) LiOH aq., MeOH; (g) H2, Pd–C, MeOH; (h) HCl in i-PrOH, MeOH.
Chart 1. Synthesis of N-Aryl-2-octanoyl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Derivatives with No Substitution at the 7-Position
Reagents: (a) CbzCl, MgO, acetone; (b) Fe, HCl aq., MeOH; (c) Boc2O, CHCl3 or THF; (d) LiOH aq., MeOH–THF; (e) 4, EDC·HCl, CH2Cl2; (f) H2, Pd–C, MeOH;
(g) acyl chloride, Et3N, CH2Cl2; (h) HCl in i-PrOH, MeOH; (i) 33: H2, Pd–C, formalin, HCl aq., MeOH; 35: 1,4-dibromobutane, K2CO3, DMF; 39: Ac2O, Et3N, CH2Cl2;
(j) HCl in i-PrOH, HCO2H; (k) 38: (ClCH2CH2)2O, K2CO3, KI, NMP; (l) 28: MsCl, pyridine, CHCl3.
Chart 2. Synthesis of 2-Acyl-N-(4-hydroxy-2,3,5-trimethylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Derivatives with an Amino, Dimethy-
lamino, Acetylamino, Methanesulfonylamino, Pyrrolidinyl, and Morpholinyl Moiety at the 7-Position
reduced by hydrogenation, and the product was converted to Starting material 29, which was obtained from nitration of
a HCl salt (10).
1,16) was protected with Cbz group, and the nitro moiety was
In Chart 2, the synthetic routes of 2-acyl-N-(4-hydroxy- reduced selectively with iron, protected with Boc2O, and then
2,3,5-trimethylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-car- hydrolyzed to give 30. After condensation with the aniline 4,
boxamide derivatives with an amino, dimethylamino, ace- the Cbz group was removed to give 31. Compound 31 was
tylamino, methanesulfonylamino, pyrrolidinyl, and mor- acylated with eight acyl chlorides, and deprotected of the
pholinyl moiety at the 7-position (11—28) are outlined. acetyl moiety and the Boc group to afford 11—18. Sepa-

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Vol. 58, No. 8
rately, compound 29 was protected with Boc2O to give 32, prepared from L-tyrosine in accordance with a reported
and the nitro moiety of 32 was converted to dimethylamino method,17) was converted to 48 with the triflate moiety at the
moiety by reductive alkylation to afford compound 33, which 7-position by the usual methods. Compound 48 was reacted
was converted to 34 by hydrolyzation, condensation with the with 1-benzylpiperazine by palladium-catalyzed amination to
aniline 4 using 1-ethyl-3-(3-dimethylaminopropyl)carbodi- give 49,18) and the Boc group at the 2-position was changed
imide (EDC)·HCl, and removal of the Boc group. Com- to the hexanoyl moiety to afford 50. Compound 50 was hy-
pound 34 was acylated with five acyl chlorides, and the prod- drolyzed, and condensed with the aniline 4 to give 51. The
ucts were hydrolyzed and treated with hydrochloride to give acetyl moiety of 51 was removed, and then the benzyl protec-
19—23. Likewise, the nitro moiety of 32 was reduced, and tion group of piperazinyl moiety was removed by hydro-
the generated amino moiety was cyclized to 1-pyrrolidinyl genolyzation with Pd(OH)2–C to afford 41. To synthesize
moiety to give 35, and then compound 35 was converted to 42, compound 47 was protected with benzyl bromide at the
24 and 25 via 36 as described in the synthesis of 19—23. To 7-position by temporary protection with Boc group at the 2-
synthesize of 26, the Boc group of 32 was changed to hexa- position, to give 52. Compound 52 was acylated with hexa-
noyl moiety, and the nitro moiety was reduced to give 37, noyl chloride, and deiodination was achieved by hydro-
and then the amino moiety of 37 was cyclized with bis(2- genolyzation with Pd–C to give 53 without debenzylation.
chloroethyl) ether to give 38 having a morpholinyl moiety. Compound 53 was converted to 42 via 54 in a similar way to
Compound 38 was converted to 26 in a similar manner to the the conversion of 50 to 41. Separately, to synthesize 57—59,
synthesis of 19—23. Separately, compound 32 was reduced, the triflate moiety of 48 was replaced with a nitrile moiety by
and acetylated to give 39. Compound 39 was treated with hy- application of the palladium-catalyzed coupling reaction,19)
drochloride, and then acylated with hexanoyl chloride to af- and the nitrile was hydrogenated in the presence of ammonia
ford 40. Compound 40 was converted to 27 as described in and Pd–C, and then the generated aminomethyl moiety was
the synthesis of 19—23. Compound 28 was obtained directly protected with CbzCl to give 55. Conversion of 55 to 56 was
from 13 by mesylation with pyridine as a base.
accomplished as mentioned in the synthesis of 19—23. The
In Chart 3, the synthetic routes of 2-acyl-N-(4-hydroxy- Cbz group of 56 was removed by hydrogenolyzation, and the
2,3,5-trimethylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-car- aminomethyl moiety of the product was alkylated by reduc-
boxamide derivatives with a hydroxyl, piperazinyl, and di- tive alkylation to give 57 and 58, and cyclized with 1,4-di-
alkylaminomethyl moiety at the 7-position (41—46) are out- bromobutane to give 59. The synthesis of 62 with an asym-
lined. To synthesize 41, starting compound 47, which was metric tertiary amino moiety was difficult by this method;
Reagents: (a) Boc2O, Et3N, CHCl3; (b) BnBr, K2CO3, acetone; (c) HCl in i-PrOH, HCO2H; (d) acyl chloride, Et3N, CH2Cl2; (e) H2, Et3N, Pd–C, MeOH; (f) LiOH aq,
MeOH–THF; (g) 4, EDC·HCl, CH2Cl2; (h) H2, Pd(OH)2–C, MeOH; (i) H2, Pd–C, MeOH; (j) Tf2O, 2,6-lutidine, CH2Cl2; (k) 1-benzylpiperazine, Pd(OAc)2, BINAP,
Cs2CO3, 1,4-dioxane; (l) KCN, Pd(OAc)2, BINAP, NMP; (m) H2, Pd(OH)2–C, NH4OH, MeOH; (n) CbzCl, MgO, AcOEt; (o) 57: formalin, NaBH3CN, MeOH; 58: ac-
etaldehyde, NaBH3CN, MeOH; 59: 1,4-dibromobutane, K2CO3, DMF; (p) HCO2H, Raney nickel; (q) tert-butylamine borane, AcOEt; (r) MsCl, Et3N, CH2Cl2; (s)
PrNH(Me), THF; (t) 43, 44, 46: HCl in i-PrOH, MeOH.
Chart 3. Synthesis of 2-Acyl-N-(4-hydroxy-2,3,5-trimethylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Derivatives with a Hydroxyl, Piper-
azinyl, and Dialkylaminomethyl Moiety at the 7-Position

August 2010
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therefore, an alternative route was utilized. Compound 47 and LDL oxidation, or both activities may not be effective in
was converted to 60, and the triflate moiety of 60 was re- human atherosclerotic plaque. To clarify these possibilities, it
placed with a nitrile moiety, and the nitrile was reduced to is important to find new bioavailable ACAT inhibitors with
aldehyde by Raney nickel.20) Selective reduction of the alde- structural and biological properties different from Pactimibe
hyde was accomplished with tert-butylamine borane com- and to experimentally and clinically investigate its effects on
plex,21) and the product was hydrolyzed to give 61. After atherosclerosis.
condensation of 61 with the aniline 4, the hydroxyl moiety
In the present study, a tetrahydroisoquinoline structure in-
was mesylated, and to the mesylate was attached N-methyl- stead of indoline structure was employed to design new
propylamine to give 62. Compounds 57—59, 62 were hy- ACAT inhibitors. To find the lead compound, 3 derivatives
drolyzed, and the products were converted to HCl salts (43, (7, 8, 10) were synthesized and their log D7.0, inhibitory ac-
44, 46) or obtained as a free form (45), respectively.
tivities against rabbit hepatic ACAT, foam cell formation in-
duced by acetyl LDL, and LDL peroxidation were deter-
mined (Table 1). Among them, compound 7 showed the most
Results and Discussion
Pactimibe exerted inhibitory effects on macrophage ACAT potent inhibitory activities against ACAT, foam cell forma-
activities and LDL oxidation, and was efficiently absorbed tion, and LDL peroxidation; however, all these compounds
orally, being expected to show potent anti-atherosclerotic ef- were highly lipophilic and were not detected in plasma after
fects by the synergism of both activities; however, Pactimibe oral administration at 10 mg/kg in rats. Therefore, to de-
failed to show a plaque-reducing effect in CAD patients.10) crease lipophilicity and increase bioavailability, the acyl
Pactimibe may have not fully inhibited plaque ACAT activity chain at the 2-position was shortened and a polar or basic
Table 1. Chemical Structures, Molecular Weights, log D7.0, and Inhibitory Activities against Liver ACAT, Foam Cell Formation, and LDL Oxidation of
Tetrahydroisoquinoline Derivatives
Liver ACATb)
(vs. Pac)
AFCFc)
(vs. Pac)
Anti-Oxd)
(vs. Pac)
Compound
Pactimibe
R
M.W.a)
416.6
log D7.0
3.26
1.0
10
1.0
6.3
1.9
1.0
0.74
7
8
436.6
462.7
3.35
4.84
2.2
Ͻ0.05
10
477.7
4.13
0.96
2.4
Ͻ0.05
a) Molecular weight of free form. b) Ratio of inhibitory activities against normocholesterolemic rabbit liver ACAT activity (IC50 of Pactimibe/IC50 of test compound), du-
plicate assay. c) Ratio of inhibitory activities against foam cell formation in THP-1 cells (IC50 of Pactimibe/IC50 of test compound), duplicate assay. d) Ratio of inhibitory ac-
tivities against LDL peroxidation (IC50 of Pactimibe/IC50 of test compound), duplicate assay.
Table 2. Chemical Structures, Molecular Weights, log D7.0, and Inhibitory Activities against Foam Cell Formation of Tetrahydroisoquinoline Derivatives
AFCFb)
(vs. Pac)
Compound
R1
R2
M.W.a)
log D7.0
Pactimibe
416.6
395.5
3.26
0.79
1.0
1.3
11
12
13
14
15
16
17
18
409.5
423.6
423.6
423.6
409.5
423.6
419.5
1.19
1.59
1.51
1.46
1.10
1.38
1.18
5.0
7.0
7.0
2.9
1.5
6.5
5.2
19
423.6
1.99
9.2
29
21
22
23
451.6
423.6
437.6
451.6
2.80
1.93
2.35
2.67
40
16
113
597
a) Molecular weight of free form. b) Ratio of inhibitory activities against foam cell formation in THP-1 cells (IC50 of Pactimibe/IC50 of test compound), duplicate assay.

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Vol. 58, No. 8
moiety was introduced at the 7-position in compound 7.
at 10 mg/kg (per os (p.o.)) in rats was 0.15, 0.91, 0.55 and
In Table 2, compounds with a primary amino group at the 0.23 mg/ml, respectively. The Cmax of 19 with butyryl was
7-position and various acyl groups (11—18) had lower lower than that of 21 with isobutyryl, suggesting the lower
log D7.0 than 1.6, and still had 1.3 to 7.0-fold more potent metabolic stability of a straight alkyl chain than a branched
anti-foam cell formation activities than Pactimibe. The activ- chain. In 21, 22 and 23, Cmax decreased dependently on
ities increased in a log D7.0-dependent manner, and tended to log D7.0.
be higher with straight alkyl chains than branched alkyl and
Finally, various basic and amide moieties were introduced
straight alkenyl chains. They showed poor oral absorption; a at the 7-position (Table 3). Compounds with pyrrolidinyl and
primary amino moiety may have been easily metabolized. morpholinyl groups (24—26) showed relatively high log D7.0
The most potent compound 13 with activity 7-fold higher (2.31—3.94) and markedly more potent activity (16—37-
than Pactimibe and comparable to 7 showed low plasma con- fold) than Pactimibe. Compounds with piperazinyl, acetyl-
centration after oral administration at 10 mg/kg in rats (Cmax
:
amino, methanesulfonylamino and hydroxyl groups (41, 27,
0.33 mg/ml). Compounds with a dimethylamino group at the 28, 42) showed lower log D7.0 (1.36—1.79) and higher activ-
7-position (19—23) had lower log D7.0 (1.93—2.80) and ity (2.7—9.8-fold). Compounds with a dialkylaminomethyl
markedly stronger anti-foam cell formation activity (9.2— group (43—46) showed low log D7.0 and activity. Compound
597-fold) than Pactimibe. Interestingly, unlike derivatives 43 with a dimethylaminomethyl group was markedly reduced
with a primary amino group, activity was higher at branched compared to compound 22 with a dimethylamino group.
alkyl chains than straight alkyl chains (19 vs. 21 and 20 vs. Compound 45 with N-methylpropylaminomethyl group
23), and was extremely higher than the corresponding deriva- showed relatively high activities, but was hardly absorbed
tives with a primary amino group. Further study is needed to orally in rats.
clarify the interaction of derivatives with amino and dimethyl-
In atherosclerosis, anti-oxidants are expected to inhibit
amino groups, and ACAT protein. The Cmax of 19, 21, 22, 23 LDL oxidation, resulting in reduction of the uptake of LDL
Table 3. Chemical Structures, Molecular Weights, log D7.0, and Inhibitory Activities against Foam Cell Formation of Tetrahydroisoquinoline Derivatives
AFCFb)
(vs. Pac)
Compound
R1
R2
M.W.a)
log D7.0
Pactimibe
416.6
463.6
3.26
3.22
1.0
29
24
25
26
41
27
477.6
493.6
492.7
465.6
3.94
2.31
1.36
1.74
16
37
6.9
2.7
28
42
501.6
424.5
1.61
1.79
5.7
9.8
43
44
45
46
451.6
479.7
479.7
477.6
0.42
1.23
1.21
1.00
0.56
1.1
5.2
0.45
a) Molecular weight of free form. b) Ratio of inhibitory activities against foam cell formation in THP-1 cells (IC50 of Pactimibe/IC50 of test compound), duplicate assay.
Table 4. Log D7.0, Inhibitory Activities against Liver ACAT, Foam Cell Formation, LDL Peroxidation, and Oxidative Stress-Induced Cell Death, and
Plasma Concentrations of Compound 21 and Pactimibe
e)
Cmax
Compound
log D7.0
Liver ACATa)
AFCFb)
Anti-Oxc)
THP-1 deathd)
Rat
Beagle
21
Pactimibe
1.93
3.26
0.113Ϯ0.004
0.312Ϯ0.027
0.15Ϯ0.02
2.45Ϯ0.25
4.8
3.8
1.5Ϯ0.1
3.1Ϯ0.4
0.91Ϯ0.13
0.15Ϯ0.02
1.63Ϯ0.19
6.98Ϯ0.35
a) IC50 (mM) against normocholesterolemic rabbit liver ACAT activity, meanϮS.E. (nϭ3). b) IC50 (mM) against foam cell formation in THP-1 cells, meanϮS.E. (nϭ3). c)
IC50 (mM) against LDL peroxidation. d) IC50 (mM) against oxidative stress-induced cell death, meanϮS.E. (nϭ6). e) Maximal plasma concentration (mg/ml) after oral adminis-
tration at 10 mg/kg, meanϮS.E. (nϭ3).

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1.88 mmol) in CH2Cl2 (7 ml) in an ice bath were added pyridine (0.46 ml,
5.7 mmol) and POCl3 (0.28 ml, 3.0 mmol), and the mixture was stirred for
15 min. AcOEt was added, and the mixture was washed with 10% citric acid
solution, saturated NaHCO3 solution and brine, dried over Na2SO4, and
evaporated under reduced pressure. The residue was purified by column
chromatography to give 2,3,6-trimethyl-4-{[(S)-2-octanoyl-1,2,3,4-tetrahydro-
isoquinoline-3-carbonyl]amino}phenyl acetate (6) as a powder (580 mg). To
a solution of 6 (550 mg, 1.15 mmol) in MeOH (11 ml) was added 1.0 M
LiOH aqueous solution (3.5 ml, 3.5 mmol), and the mixture was stirred at
room temperature for 1 h. After addition of 6 M hydrochloric acid (0.6 ml),
the mixture was concentrated under reduced pressure. The residue was ex-
tracted with CH2Cl2, and the organic layer was washed with brine, dried over
Na2SO4, and evaporated under reduced pressure. The residue was rinsed
with i-Pr2O, and the precipitate was collected by filtration to give 7 as a solid
(360 mg, 47% yield). mp 157—160 °C. IR (Nujol) cmϪ1: 1647. MS m/z: 437
(MϩHϩ). 1H-NMR (CDCl3) d: 0.87 (3H, br-t), 0.9—2.2 (13H, m), 2.04
(6H, s), 2.51 (2H, br-t), 2.8—3.4 (1H, m), 3.51 (1H, dd, Jϭ15.4, 4.1 Hz),
by macrophages and foam cell formation, and preventing ox-
idative stress-induced foam cell death, resulting in the reduc-
tion of cell debris, extracellular cholesterol deposition and
inflammatory response. Probucol and KY-455, an indoline-
based ACAT inhibitor, have been reported to attenuate the
oxidative stress-induced apoptosis of THP-1 derived macro-
phages.22,23) Compound 21 and Pactimibe concentration-
dependently decreased macrophage cell death induced by
co-incubation with LDL and Cu2ϩ (Table 4). The effect of
compound 21 was 2-fold stronger than Pactimibe, whereas
both compounds showed similar anti-LDL oxidative activi-
ties. The Cmax of compound 21 was higher in rats and lower
in dogs than Pactimibe, and exceeded the IC50 values for
foam cell formation, LDL oxidation and cell death, anticipat-
ing that 21 would exert these effects in vivo. Phenolic antiox- 4.3—5.4 (3H, m), 6.46 (1H, br-s), 6.95 (1H, s), 7.0—7.4 (5H, m), 7.86 (1H,
br-s). Anal. Calcd for C27H36N2O3: C, 74.28; H, 8.31; N, 6.42. Found: C,
74.11; H, 8.39; N, 6.39.
Procedure for the Synthesis of 8 and 10 (S)-N-(2,6-Diisopropyl-
phenyl)-2-octanoyl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (8): To a
idants such as Probucol are also known to have anti-inflam-
matory effects via inhibition of adhesion molecule expres-
sion.24) Future studies will reveal whether phenolic ACAT in-
hibitors have such anti-inflammatory effects beneficial for
atherosclerosis therapy.
solution of 3 (913 mg, 3.01 mmol) in CH2Cl2 (9 ml) were added 2,6-di-
isopropylphenylamine (0.57 ml, 3.0 mmol) and EDC·HCl (692 mg,
3.61 mmol), and the mixture was stirred at room temperature for 2 h. The
mixture was washed with 10% citric acid solution and brine, dried over
Na2SO4, and evaporated under reduced pressure. The residue was purified by
column chromatography. The residue was recrystallized from benzene to 8
as a crystalline solid (335 mg, 24% yield). mp 146—148 °C. IR (Nujol)
cmϪ1: 1655. MS m/z: 463 (MϩHϩ). 1H-NMR (CDCl3) d: 0.70—1.18 (15H,
m), 1.18—1.92 (10H, m), 2.30—2.80 (4H, m), 2.88—3.78 (2H, m), 4.49—
5.08 (2H, m), 5.40 (1H, br-t), 6.92—7.60 (8H, m). Anal. Calcd for
C30H42N2O2: C, 77.88; H, 9.15; N, 6.05. Found: C, 77.78; H, 9.28; N, 5.95.
(S)-N-(4-Amino-2,6-diisopropylphenyl)-2-octanoyl-1,2,3,4-tetrahydroiso-
In conclusion, the present study demonstrated that a
tetrahydroisoquinoline structure with a phenol moiety is use-
ful for the design of bioavailable anti-oxidative ACAT in-
hibitors, and that 21 is expected to show anti-atherosclerotic
effects by ACAT inhibitory and phenolic anti-oxidative activ-
ities.
Experimental
General Procedures Chemicals were obtained from commercial quinoline-3-carboxamide Hydrochloride (10): To a solution of 3 (600 mg,
sources and used without purification. Reactions were monitored by TLC on
1.98 mmol) and compound 5 (396 mg, 1.80 mmol) in CH2Cl2 (6 ml) were
Merck precoated silica gel 60 F254 (0.25 mm) plates. Column chromatogra- added pyridine (0.48 ml, 5.9 mmol) and POCl3 (0.24 ml, 2.6 mmol) in an ice
phy was performed on silica gel (Daisogel No. 1001W; Daiso, Osaka, bath, and the mixture was stirred for 3 h. The mixture was washed with 10%
Japan). Melting points were measured on melting point apparatus (MP- citric acid solution, saturated NaHCO3 solution and brine, dried over
500P; Yanaco, Kyoto, Japan) and are uncorrected. IR spectra were obtained Na2SO4, and evaporated under reduced pressure. The residue was purified by
with an infrared spectrometer (FT-IR 8200PC; Shimadzu, Kyoto, Japan). 1H- column chromatography to give (S)-N-(2,6-diisopropyl-4-nitrophenyl)-2-oc-
NMR spectra were recorded on a nuclear magnetic resonance spectrometer tanoyl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (9) (812 mg). A solu-
at 90 MHz (R-1900; Hitachi, Tokyo, Japan) or 400 MHz (JNM-AL400; tion of 9 (795 mg, 1.57 mmol) in MeOH (10 ml) was hydrogenated at
JEOL, Tokyo, Japan) using tetramethylsilane as an internal standard. MS 0.3 MPa in the presence of 10% Pd–C (160 mg) at 40 °C for 2.5 h. After fil-
spectra were obtained on a QTRAP LC/MS/MS system (API2000; Applied
Biosystems, Foster, CA, U.S.A.).
Procedure for the Synthesis of 7 4-Amino-2,3,6-trimethylphenyl Ac-
tration, the filtrate was evaporated under reduced pressure. The residue was
purified by column chromatography. To a solution of the residue in MeOH
(2.6 ml) was added 10 M HCl in i-PrOH (0.13 ml, 1.3 mmol) in an ice bath.
etate (4): 2,3,6-Trimethylphenol was nitrated as described in a previous arti- After evaporation under reduced pressure, Et2O was added, and the precipi-
cle.15) The nitrated product was acetylated with AcCl and Et3N, and hydro- tate formed was collected by filtration to give 10 as a solid (453 mg, 56%
genated in the presence of Pd–C in a standard manner to give 4 as a crys-
yield). mp 187—197 °C. IR (Nujol) cmϪ1: 1636. MS m/z: 478 (MϩHϩ). 1H-
talline solid (78% yield). 1H-NMR (CDCl3) d: 2.04 (3H, s), 2.18 (6H, s), NMR (DMSO-d6) d: 0.35—1.80 (25H, m), 2.10—4.00 (6H, m), 4.38—5.32
2.30 (3H, s), 3.20—3.60 (2H, br), 6.41 (1H, s).
(3H, m), 7.03 (2H, s), 7.10—7.50 (4H, m), 8.91 (1H, s), 9.41 (1H, s), 8.60—
11.50 (2H, br). Anal. Calcd for C30H43N3O2·HCl·1.2H2O: C, 67.25; H,
8.73; N, 7.84. Found: C, 67.24; H, 8.67; N, 7.91.
Methyl (S)-2-Octanoyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (2):
To a solution of 1 (1.00 g, 5.23 mmol) in CH2Cl2 (10 ml) were added Et3N
(1.09 ml, 7.82 mmol) and n-octanoyl chloride (0.98 ml, 5.7 mmol), and the
Procedure for the Synthesis of 19—23 Compound 21 was synthesized
mixture was stirred at room temperature for 3 h. The mixture was washed from 29 via 32, 33, 34 as follows. Compounds 19, 20, 22, and 23 were syn-
with 10% citric acid solution and brine, dried over Na2SO4, and evaporated thesized in a similar manner to the synthesis of 21.
under reduced pressure. The residue was purified by column chromatogra-
phy to give 2 as an oil (1.32 g, 80% yield). H-NMR (CDCl3) d: 0.88 (3H, 3-carboxylate (32): To a solution of 29 (1.64 g, 6.94 mmol) in CHCl3 (30 ml)
br-t), 1.10—1.90 (10H, m), 2.21—2.60 (2H, m), 3.08—3.40 (2H, m), 3.61 was added Boc2O (1.89 g, 8.66 mmol), and the mixture was stirred at room
Methyl (S)-2-tert-Butoxycarbonyl-7-nitro-1,2,3,4-tetrahydroisoquinoline-
1
(3H, s), 4.50—4.98 (2H, m), 5.48 (1H, t, Jϭ5.4 Hz), 7.02—7.35 (4H, m).
(S)-2-Octanoyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid (3): To a was purified by column chromatography to give 32 as a crystalline solid
temperature for 14 h. After evaporation under reduced pressure, the residue
1
solution of 2 (1.00 g, 3.15 mmol) in MeOH (10 ml) was added 5.0 M NaOH
aqueous solution (1.26 ml, 6.3 mmol) at room temperature, and the mixture
(2.43 g, 100% yield). H-NMR (CDCl3) d: 1.51 (9H, s), 3.2—3.4 (2H, m),
3.64 (3H, s), 4.55 (1H, d, Jϭ17.1 Hz), 4.85 (1H, d, Jϭ17.1 Hz), 5.0—5.4
was stirred at 50 °C for 1 h. After evaporation under reduced pressure, water (1H, m), 7.2—7.4 (1H, m), 7.9—8.2 (2H, m).
and 6 M hydrochloric acid (1.1 ml) were added, and the mixture was ex-
Methyl (S)-2-tert-Butoxycarbonyl-7-dimethylamino-1,2,3,4-tetrahydroiso-
tracted with AcOEt. The organic layer was washed with brine, dried over quinoline-3-carboxylate (33): A solution of 32 (3.57 g, 10.6 mmol) in MeOH
Na2SO4, and evaporated under reduced pressure to give 3 as an oil (947 mg,
99% yield). 1H-NMR (CDCl3) d: 0.88 (3H, br-t), 1.10—1.85 (10H, m),
2.20—2.60 (2H, m), 2.90—3.50 (2H, m), 4.30—5.05 (2H, m), 5.40 (1H, t,
Jϭ5.2 Hz), 6.98—7.42 (4H, m), 8.80 (1H, br-s).
(50 ml) was hydrogenated at 0.3 MPa in the presence of 10% Pd–C (0.25 g)
at room temperature for 1.5 h. To the mixture were added formalin 2.4 ml
(32 mmol) and 2.0 M hydrochloric acid (1.05 ml, 2.1 mmol), and the mixture
was hydrogenated at 0.3 MPa for 3 h. After filtration, the filtrate was evapo-
(S)-N-(4-Hydroxy-2,3,5-trimethylphenyl)-2-octanoyl-1,2,3,4-tetrahy- rated under reduced pressure. A solution of the residue in AcOEt was
droisoquinoline-3-carboxamide (7): To a solution of 4-amino-2,3,6-tri- washed with saturated NaHCO3 solution and brine, dried over Na2SO4, and
methylphenyl acetate (4) (665 mg, 2.19 mmol) and compound 3 (364 mg,
evaporated under reduced pressure. The residue was recrystallized from

1072
Vol. 58, No. 8
AcOEt–hexane to give 33 as a crystalline solid (2.33 g, 66% yield). 1H-
(2H, br), 4.5—5.3 (3H, m), 6.60 (1H, s), 7.2—7.7 (3H, m), 9.18 (1H, br-s).
NMR (CDCl3) d: 1.45, 1.52 (total 9H, s, s), 2.8—3.3 (2H, m), 2.91 (6H, s), Anal. Calcd for C26H35N3O3·HCl·3H2O: C, 59.13; H, 8.02; N, 7.96. Found:
3.63 (3H, s), 4.2—5.2 (3H, m), 6.4—6.7 (2H, m), 7.00 (1H, d, Jϭ8.4 Hz).
4-{[(S)-7-Dimethylamino-1,2,3,4-tetrahydroisoquinoline-3-car-
bonyl]amino}-2,3,6-trimethylphenyl Acetate (34): To a solution of 33
(1.79 g, 5.35 mmol) in tetrahydrofuran (THF)–MeOH (3 : 1, 30 ml) in an ice
bath was added 1.0 M LiOH aqueous solution (11 ml, 11 mmol), and the mix-
C, 59.47; H, 7.83; N, 8.07.
(S)-7-Dimethylamino-2-(2,2-dimethylbutyryl)-N-(4-hydroxy-2,3,5-
trimethylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochlo-
ride (23): Compound 23 was synthesized from 34 using 2,2-dimethylbutyryl
chloride instead of isobutyryl chloride. A solid. mp 150—156 °C. IR (Nujol)
ture was stirred at room temperature for 2 h. After neutralization with 5% cmϪ1: 1614. MS m/z: 452 (MϩHϩ). 1H-NMR (DMSO-d6) d: 0.80 (3H, br-t),
citric acid solution, the mixture was concentrated under reduced pressure, 1.25 (6H, s), 1.6—2.0 (2H, m), 1.81 (3H, s), 2.06 (6H, s), 2.9—3.6 (2H, m),
and the residue was extracted with AcOEt. The organic layer was washed 3.05 (6H, s), 4.4—6.4 (2H, br), 4.62 (1H, d, Jϭ16.3 Hz), 4.99 (1H, d,
with brine, dried over Na2SO4, and evaporated under reduced pressure. To a Jϭ16.3 Hz), 5.11 (1H, br-t), 6.61 (1H, s), 7.2—7.7 (3H, m), 9.19 (1H, br-s).
solution of the residue in CH2Cl2 (50 ml) in an ice bath were added 4 (1.03 g,
5.33 mmol) and EDC·HCl (1.23 g, 6.42 mmol), and the mixture was stirred
for 30 min. After evaporation under reduced pressure, 5% citric acid solution
Anal. Calcd for C27H37N3O3·HCl·2.8H2O: C, 60.22; H, 8.16; N, 7.80.
Found: C, 60.22; H, 8.00; N, 7.94.
Procedure for the Synthesis of 11—18 Compound 11 was synthesized
was added to the residue, and the mixture was extracted with AcOEt. The via 30, 31 as follows. Compounds 12—18 were synthesized in a similar
organic layer was washed with brine, dried over Na2SO4, and evaporated manner to the synthesis of 11.
under reduced pressure. The residue was purified by column chromatogra-
Methyl (S)-2-Benzyloxycarbonyl-7-nitro-1,2,3,4-tetrahydroisoquinoline-3-
phy to give 4-{[(S)-2-tert-butoxycarbonyl-7-dimethylamino-1,2,3,4-tetrahy- carboxylate (65): To a suspension of 29 (8.00 g, 33.9 mmol) in acetone
droisoquinoline-3-carbonyl]amino}-2,3,6-trimethylphenyl acetate as a crys- (80 ml) in an ice bath were added MgO (2.05 g, 50.9 mmol) and CbzCl
talline solid (2.42 g, 91% yield). To a solution of the product (2.38 g,
4.80 mmol) in HCO2H (7 ml) in an ice bath was added 10 M HCl in i-PrOH
(1.5 ml, 15 mmol), and the mixture was stirred for 30 min. After neutraliza-
(5.3 ml, 37 mmol), and the mixture was stirred at room temperature for 16 h.
After filtration, the filtrate was evaporated under reduced pressure. Hexane
(20 ml) was added to the oily residue, and the mixture was stirred for 30 min.
tion with NaHCO3, the mixture was extracted with CH2Cl2. The organic The supernatant was removed by decantation, and the residue was dried
layer was washed with brine, dried over Na2SO4, and evaporated under re- under reduced pressure to give 65 as an oil (9.30 g, 74% yield). 1H-NMR
duced pressure. Et2O (50 ml) was added, and the precipitate formed was col- (CDCl3) d: 3.28 (2H, br-d), 3.57, 3.63 (total 3H, s, s), 4.5—5.4 (3H, m),
1
lected by filtration to give 34 as a crystalline solid (1.66 g, 87% yield). H-
NMR (CDCl3) d: 1.71 (3H, s), 1.9—2.1 (1H, br), 2.12 (6H, s), 2.32 (3H, s),
5.24 (2H, s), 7.2—7.6 (6H, m), 7.9—8.2 (2H, m).
(S)-2-Benzyloxycarbonyl-7-tert-butoxycarbonylamino-1,2,3,4-tetrahy-
2.6—3.5 (2H, m), 2.91 (6H, s), 3.5—4.0 (1H, m), 4.01 (2H, s), 6.47 (1H, d, droisoquinoline-3-carboxylic Acid (30): To a suspension of 65 (9.30 g,
Jϭ1.6 Hz), 6.62 (1H, dd, Jϭ8.5, 1.6 Hz), 7.08 (1H, d, Jϭ8.5 Hz), 7.67 (1H,
s), 9.23 (1H, s).
25.1 mmol) in 65% MeOH (260 ml) was added concentrated hydrochloric
acid (2.6 ml), and the mixture was heated to be refluxed. Under refluxing,
(S)-7-Dimethylamino-N-(4-hydroxy-2,3,5-trimethylphenyl)-2-isobutyryl- iron powder (5.61 g, 100 mmol) was added in portions over 10 min, and then
1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochloride (21): Com- the mixture was stirred for 1.5 h. Concentrated hydrochloric acid (2.6 ml)
pound 34 was acylated with isobutyryl chloride as described in the prepara- and iron powder (2.80 g, 50 mmol) were added, and the mixture was stirred
tion of 2. A crystalline solid. Yield 89%. To a solution of the product
(304 mg, 0.65 mmol) in THF–MeOH (3 : 1, 6 ml) in an ice bath was added
1.0 M LiOH aqueous solution (2.0 ml, 2.0 mmol), and the mixture was stirred
for 1 h. After allowing to cool, the mixture was neutralized with NaHCO3.
The mixture was filtrated, and the filtrate was concentrated under reduced
pressure. The residue was extracted with AcOEt, and the organic layer was
for 1 h at room temperature. After neutralization with 10% citric acid solu- washed with brine, dried over Na2SO4, and evaporated under reduced pres-
tion, the mixture was concentrated under reduced pressure, and the residue sure. To a solution of the residue in THF (40 ml) was added Boc2O (5.48 g,
was extracted with CHCl3. The organic layer was washed with brine, dried 25.1 mmol), and the mixture was stirred at 45 °C for 16 h. After evaporation
over Na2SO4, and evaporated under reduced pressure. To a suspension of the under reduced pressure, the residue was purified by column chromatography
residue (0.34 g) in MeOH (0.6 ml) in an ice bath was added 10 M HCl in i-
PrOH (0.10 ml, 1.0 mmol), and the mixture was stirred for 10 min. After ad-
dition of Et2O (20 ml), the mixture was stirred for 30 min, and the precipitate
formed was collected by filtration to give 21 as a solid (302 mg, 100%
yield). mp 161—164 °C. IR (Nujol) cmϪ1: 1622. MS m/z: 424 (MϩHϩ). 1H-
to give a Boc protected derivative. The product was hydrolyzed as described
in the preparation of 3 to give 30 as a crystalline solid (6.91 g, 65% yield).
1H-NMR (CDCl3) d: 1.50 (9H, s), 3.0—3.4 (2H, m), 4.3—5.3 (3H, m), 5.19
(2H, s), 5.4—6.6 (1H, br), 6.57 (1H, br-s), 6.8—7.6 (8H, m).
4-{[(S)-7-tert-Butoxycarbonylamino-1,2,3,4-tetrahydroisoquinoline-3-
NMR (DMSO-d6) d: 0.9—1.4 (6H, m), 1.71 (3H, s), 2.04 (6H, s), 2.65 (2H, carbonyl]amino}-2,3,6-trimethylphenyl Acetate (31): Compound 30 (6.91 g,
br-t), 2.8—3.5 (3H, m), 3.05 (6H, s), 4.0—6.0 (3H, m), 6.53 (1H, s), 7.2— 16.2 mmol) was condensed with compound 4 as described in the synthesis of
7.7 (3H, m), 9.01, 9.32 (total 1H, br-s, br-s). Anal. Calcd for 34, and then the Cbz group of the product was removed by hydrogenolyza-
C25H33N3O3·1.5HCl·2H2O: C, 58.39; H, 7.55; N, 8.17. Found: C, 58.77; H, tion in the presence of 10% Pd–C in MeOH to give 31. A crystalline solid.
1
7.25; N, 8.19.
Yield 58%. H-NMR (CDCl3) d: 1.51 (9H, s), 1.86 (1H, br-s), 2.07 (3H, s),
(S)-2-Butyryl-7-dimethylamino-N-(4-hydroxy-2,3,5-trimethylphenyl)- 2.12 (6H, s), 2.29 (3H, s), 2.85 (1H, dd, Jϭ16.0, 9.5 Hz), 3.27 (1H, dd,
1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochloride (19): Com- Jϭ16.0, 5.5 Hz), 3.70 (1H, dd, Jϭ9.5, 5.5 Hz), 4.01 (2H, s), 6.47 (1H, br-s),
pound 19 was synthesized from 34 using butyryl chloride instead of isobu-
tyryl chloride. A solid. mp 144—148 °C. IR (Nujol) cmϪ1: 1622. MS m/z:
424 (MϩHϩ). 1H-NMR (DMSO-d6) d: 0.86 (3H, br-t), 1.6—2.0 (5H, m),
6.9—7.2 (2H, m), 7.2—7.4 (1H, m), 7.64 (1H, s), 9.21 (1H, br-s).
(S)-7-Amino-2-butyryl-N-(4-hydroxy-2,3,5-trimethylphenyl)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide Hydrochloride (11): Compound 31
2.03 (3H, s), 2.05 (3H, s), 2.0—2.6 (2H, m), 2.9—3.6 (2H, m), 3.05 (6H, s), (400 mg, 0.856 mmol) was acylated with butyryl chloride, hydrolyzed, and
4.4—6.4 (5H, m), 6.50 (1H, s), 7.2—7.7 (3H, m), 8.96, 9.30 (total 1H, br-s, treated with HCl to give 11 as a solid (253 mg, 77% yield) in a manner simi-
br-s). Anal. Calcd for C25H33N3O3·HCl·2.5H2O: C, 59.45; H, 7.78; N, 8.32. lar to that described in the synthesis of 21. mp 166—175 °C. IR (Nujol)
Found: C, 59.71; H, 7.66; N, 8.45.
cmϪ1: 1622. MS m/z: 396 (MϩHϩ). 1H-NMR (DMSO-d6) d: 0.94 (3H, br-t),
(S)-7-Dimethylamino-2-hexanoyl-N-(4-hydroxy-2,3,5-trimethylphenyl)- 1.4—2.0 (2H, m), 1.67 (3H, br-s), 2.05 (6H, s), 2.1—2.7 (2H, m), 2.8—4.4
1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochloride (20): Com- (4H, br), 2.9—3.6 (2H, m), 4.4—5.3 (3H, m), 6.50 (1H, s), 7.0—7.5 (3H,
pound 20 was synthesized from 34 using hexanoyl chloride instead of isobu-
m), 9.00 (1H, br-s). Anal. Calcd for C23H29N3O3·HCl·2.5H2O: C, 57.91; H,
tyryl chloride. A solid. mp 133—139 °C. IR (Nujol) cmϪ1: 1651. MS m/z: 7.40; N, 8.81. Found: C, 57.79; H, 7.16; N, 8.71.
452 (MϩHϩ). 1H-NMR (DMSO-d6) d: 0.88 (3H, br-t), 1.0—1.8 (6H, m),
(S)-7-Amino-N-(4-hydroxy-2,3,5-trimethylphenyl)-2-pentanoyl-1,2,3,4-
1.66 (3H, s), 2.03 (6H, s), 2.2—2.7 (2H, m), 3.0—3.6 (2H, m), 3.05 (6H, s), tetrahydroisoquinoline-3-carboxamide Hydrochloride (12): Compound 12
4.0—6.0 (5H, m), 6.49 (1H, s), 7.2—7.7 (3H, m), 8.96, 9.33 (total 1H, br-s, was synthesized from 31 using pentanoyl chloride instead of butyryl chlo-
br-s). Anal. Calcd for C27H37N3O3·HCl·2.5H2O: C, 60.83; H, 8.13; N, 7.88. ride. A solid. mp 167—171 °C. IR (Nujol) cmϪ1: 1622. MS m/z: 410
1
Found: C, 60.56; H, 7.98; N, 7.87.
(MϩHϩ). H-NMR (DMSO-d6) d: 0.91 (3H, br-t), 1.2—1.8 (4H, m), 1.66,
(S)-7-Dimethylamino-2-(2,2-dimethylpropionyl)-N-(4-hydroxy-2,3,5- 1.71 (total 3H, s, s), 2.03 (3H, s), 2.05 (3H, s), 2.3—2.7 (2H, m), 2.9—3.6
trimethylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochlo- (2H, m), 4.4—5.3 (3H, m), 7.0—7.5 (4H, m), 8.99 (1H, br-s). Anal. Calcd
ride (22): Compound 22 was synthesized from 34 using 2,2-dimethylpropi- for C24H31N3O3·HCl·2H2O: C, 59.80; H, 7.53; N, 8.72. Found: C, 59.69; H,
onyl chloride instead of isobutyryl chloride. A solid. mp 162—166 °C. IR
7.47; N, 8.66.
1
(Nujol) cmϪ1: 1614. MS m/z: 438 (MϩHϩ). H-NMR (DMSO-d6) d: 1.29
(S)-7-Amino-2-hexanoyl-N-(4-hydroxy-2,3,5-trimethylphenyl)-1,2,3,4-
(9H, s), 1.79 (3H, s), 2.05 (6H, s), 2.9—3.4 (2H, m), 3.05 (6H, s), 4.0—6.0 tetrahydroisoquinoline-3-carboxamide Hydrochloride (13): Compound 13

August 2010
1073
1
464 (MϩHϩ). H-NMR (DMSO-d6) d: 1.04 (9H, s), 1.60—1.9.0 (4H, m),
2.04 (9H, s), 2.50 (2H, s), 2.90—3.60 (6H, m), 4.40—5.30 (3H, m), 6.54
(1H, s), 6.80—7.30 (4H, m), 8.95, 9.25 (total 1H, s, s). Anal. Calcd for
C28H37N3O3·HCl·2.5H2O: C, 61.69; H, 7.95; N, 7.71. Found: C, 61.73; H,
7.70; N, 7.76.
was synthesized from 31 using hexanoyl chloride instead of butyryl chlo-
ride. A solid. mp 198—204 °C. IR (Nujol) cmϪ1: 1653. MS m/z: 424
1
(MϩHϩ). H-NMR (DMSO-d6) d: 0.88 (3H, br-t), 1.0—1.8 (6H, m), 1.70
(3H, s), 2.03 (3H, s), 2.05 (3H, s), 2.2—2.7 (2H, m), 3.0—3.6 (2H, m),
4.4—5.3 (3H, m), 6.50 (1H, s), 7.0—7.5 (3H, m), 8.99, 9.34 (total 1H, br-s,
br-s).
(S)-2-(3,3-Dimethylbutyryl)-N-(4-hydroxy-2,3,5-trimethylphenyl)-7-
pyrrolidin-1-yl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochlo-
ride (25): Compound 25 was obtained from 36 using 3,3-dimethylbutyryl
chloride, as described in the synthesis of 24. A solid. Yield 54%. mp 144—
(S)-7-Amino-N-(4-hydroxy-2,3,5-trimethylphenyl)-2-(4-methylpen-
tanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochloride (14):
Compound 14 was synthesized from 31 using 4-methylpentanoyl chloride
instead of butyryl chloride. A solid. mp 165—175 °C. IR (Nujol) cmϪ1
:
148 °C. IR (Nujol) cmϪ1: 1622. MS m/z: 478 (MϩHϩ). H-NMR (DMSO-
1
1628. MS m/z: 424 (MϩHϩ). 1H-NMR (DMSO-d6) d: 0.91 (6H, d,
Jϭ5.0 Hz), 1.4—1.9 (3H, m), 1.66 (3H, s), 2.05 (6H, s), 2.1—2.6 (2H, m),
3.0—3.6 (2H, m), 4.4—5.3 (3H, m), 6.49 (1H, s), 7.1—7.6 (3H, m), 8.99,
9.36 (total 1H, br-s, br-s). Anal. Calcd for C25H33N3O3S·HCl·2.5H2O: C,
59.45; H, 7.78; N, 8.32. Found: C, 59.66; H, 7.60; N, 8.32.
d6) d: 0.9—1.4 (6H, m), 1.71 (3H, s), 2.04 (6H, s), 2.65 (2H, br-t), 2.8—3.5
(3H, m), 3.05 (6H, s), 4.0—6.0 (2H, br), 4.6—5.3 (3H, m), 6.53 (1H, s),
7.2—7.7 (3H, m), 9.01, 9.32 (total 1H, br-s, br-s). Anal. Calcd for
C29H39N3O3·HCl·2.5H2O: C, 62.29; H, 8.11; N, 7.52. Found: C, 62.08; H,
7.86; N, 7.45.
(S)-7-Amino-2-(3,3-dimethylbutyryl)-N-(4-hydroxy-2,3,5-tri-
methylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochloride
(15): Compound 15 was synthesized from 31 using 3,3-dimethylbutyryl
chloride instead of butyryl chloride. A solid. mp 165—170 °C. IR (Nujol)
Procedure for the Synthesis of 26 Methyl (S)-7-Amino-2-hexanoyl-
1,2,3,4-tetrahydroisoquinoline-3-carboxylate (37): Compound 29 (2.00 g,
8.47 mmol) was acylated with hexanoyl chloride as described in the prepara-
tion of 2. A solution of the obtained residue in MeOH (30 ml) was hydro-
genated at 0.3 MPa in the presence of 10% Pd–C (290 mg) at room tempera-
ture for 3.5 h. After filtration, the filtrate was evaporated under reduced pres-
sure, and the residue was purified by column chromatography to give 37 as
an oil (2.29 g, 89% yield). 1H-NMR (CDCl3) d: 0.91 (3H, br-t), 1.0—2.0
(6H, m), 2.2—2.6 (2H, m), 2.9—3.4 (2H, m), 3.4—4.0 (2H, br), 3.61 (3H,
s), 4.4—5.0 (2H, m), 5.43 (1H, br-t), 6.4—6.7 (2H, m), 6.93 (1H, d,
Jϭ7.9 Hz).
1
cmϪ1: 1622. MS m/z: 424 (MϩHϩ). H-NMR (DMSO-d6) d: 1.04 (9H, s),
1.74 (3H, s), 2.05 (6H, s), 2.4—2.6 (2H, m), 3.0—3.4 (2H, m), 4.4—5.3
(3H, m), 6.53 (1H, s), 7.0—7.4 (3H, m), 9.04 (1H, br-s). Anal. Calcd for
C25H33N3O3·1.4HCl·1.8H2O: C, 59.22; H, 7.55; N, 8.29. Found: C, 59.29;
H, 7.20; N, 8.42.
(S)-7-Amino-2-(2,2-dimethylpropionyl)-N-(4-hydroxy-2,3,5-tri-
methylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochloride
(16): Compound 16 was synthesized from 31 using 2,2-dimethylpropionyl
chloride instead of butyryl chloride. A solid. mp 171—185 °C. IR (Nujol)
Methyl (S)-2-Hexanoyl-7-(morpholin-4-yl)-1,2,3,4-tetrahydroisoquino-
line-3-carboxylate (38): A mixture of 37 (700 mg, 2.30 mmol), bis(2-
chloroethyl) ether (329 mg, 2.30 mmol), K2CO3 (636 mg, 4.60 mmol), KI
(191 mg, 1.15 mmol), and N-methyl-2-pyrrolidinone (3.5 ml) was heated at
100 °C for 5 h. After addition of water, the mixture was extracted with
AcOEt, and the organic layer was washed with brine, dried over Na2SO4,
and evaporated under reduced pressure. The residue was purified by column
1
cmϪ1: 1651. MS m/z: 410 (MϩHϩ). H-NMR (DMSO-d6) d: 1.28 (9H, s),
1.80 (3H, s), 2.05 (6H, s), 2.8—4.0 (3H, m), 4.57 (1H, d, Jϭ17.3 Hz), 4.95
(1H, d, Jϭ17.3 Hz), 5.06 (1H, br-t), 6.59 (1H, s), 7.0—7.4 (3H, m), 9.18
(1H, br-s). Anal. Calcd for C24H31N3O3·HCl·2.5H2O: C, 58.71; H, 7.60; N,
8.56. Found: C, 58.49; H, 7.54; N, 8.50.
1
(S)-7-Amino-2-(2,2-dimethylbutyryl)-N-(4-hydroxy-2,3,5-tri-
methylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochloride
(17): Compound 17 was synthesized from 31 using 2,2-dimethylbutyryl
chloride instead of butyryl chloride. A solid. IR (Nujol) cmϪ1:1655. MS m/z:
chromatography to give 38 as a viscous oil (626 mg, 73% yield). H-NMR
(CDCl3) d: 0.91 (3H, br-t), 1.1—1.8 (6H, m), 2.35 (2H, br-t), 2.8—3.5 (2H,
m), 3.11 (4H, br-t), 3.61 (3H, s), 3.85 (4H, br-t), 4.4—5.6 (3H, m), 6.6—6.9
(2H, m), 7.07 (1H, d, Jϭ8.3 Hz).
1
424 (MϩHϩ). H-NMR (DMSO-d6) d: 0.79 (3H, br-t), 1.24 (6H, s), 1.5—
(S)-2-Hexanoyl-N-(4-hydroxy-2,3,5-trimethylphenyl)-7-(morpholin-4-yl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochloride (26): Com-
pound 38 was hydrolyzed, condensed with compound 4, and then hydrolyzed
to give 26, as described in the synthesis of 21. A solid. Yield 87%. mp
133—137 °C. IR (Nujol) cmϪ1: 1634. MS m/z: 494 (MϩHϩ). 1H-NMR
(DMSO-d6) d: 0.88 (3H, br-t), 1.0—2.0 (6H, m), 1.65 (3H, s), 2.04 (6H, s),
2.2—2.6 (2H, m), 2.9—3.5 (6H, m), 3.7—4.2 (4H, m), 4.2—5.4 (5H, m),
6.51 (1H, s), 7.2—7.6 (3H, m), 8.94 (1H, br-s). Anal. Calcd for
C29H39N3O4·HCl·2H2O: C, 61.52; H, 7.83; N, 7.42. Found: C, 61.42; H,
7.84; N, 7.51.
Procedure for the Synthesis of 27 Methyl (S)-7-Acetylamino-2-tert-
butoxycarbonyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (39): A solu-
tion of 32 (1.00 g, 2.97 mmol) in MeOH (10 ml) was hydrogenated at
0.3 MPa in the presence of 10% Pd–C (100 mg) at room temperature for 2 h.
After filtration, the filtrate was evaporated under reduced pressure. To a solu-
tion of the residue in CH2Cl2 (10 ml) in an ice bath were added Et3N
(0.62 ml, 4.5 mmol) and Ac2O (0.25 ml, 3.5 mmol), and the mixture was
stirred for 15 min. The mixture was washed with 5% citric acid solution and
brine, dried over Na2SO4, and evaporated under reduced pressure. The solid
residue was washed with Et2O to give 39 as a crystalline solid (740 mg, 71%
yield). 1H-NMR (CDCl3) d: 1.50 (9H, s), 2.14 (3H, s), 2.9—3.4 (2H, m),
3.61 (3H, s), 4.3—5.2 (3H, m), 7.0—7.6 (4H, m).
Methyl (S)-7-Acetylamino-2-hexanoyl-1,2,3,4-tetrahydroisoquinoline-3-
carboxylate (40): Compound 39 was treated with HCl, and acylated with
hexanoyl chloride to give 40 in a manner similar to the preparation of 21. An
oil. Yield 68%. 1H-NMR (CDCl3) d: 0.91 (3H, br-t), 1.1—1.8 (6H, m), 2.15
(3H, s), 2.47 (2H, br-t), 2.8—3.5 (2H, m), 3.60 (3H, s), 4.3—5.0 (2H, m),
5.0—5.2 (0.2H, m), 5.50 (0.8H, dd, Jϭ5.6, 4.1 Hz), 6.8—7.2 (2H, m), 7.3—
7.8 (2H, m).
(S)-7-Acetylamino-2-hexanoyl-N-(4-hydroxy-2,3,5-trimethylphenyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (27): Compound 40 was con-
verted to 27 in a similar manner to the synthesis of 26. A crystalline solid.
Yield 65%. mp 147—151 °C. IR (Nujol) cmϪ1: 1651. MS m/z: 466
(MϩHϩ). 1H-NMR (CDCl3ϩDMSO-d6) d: 0.90 (3H, br-t), 1.1—1.8 (6H,
m), 1.85 (3H, s), 2.10 (9H, s), 2.51 (2H, br-t), 2.8—3.6 (3H, m), 4.3—5.5
(3H, m), 6.86 (1H, br-s), 6.5—8.1 (4H, m), 8.94 (1H, br-s). Anal. Calcd for
C27H35N3O4·H2O: C, 67.06; H, 7.71; N, 8.69. Found: C, 67.24; H, 7.61; N,
1.9 (2H, m), 1.83 (3H, s), 2.06 (6H, s), 3.0—3.6 (2H, m), 4.3—5.2 (3H, m),
6.60 (1H, s), 7.0—7.5 (3H, m), 9.17 (1H, br-s).
(S)-7-Amino-2-(2E,4E)-hexa-2,4-dienoyl-N-(4-hydroxy-2,3,5-tri-
methylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochloride
(18): Compound 18 was synthesized from 31 using trans-2,4-hexadienoyl
chloride instead of butyryl chloride. A solid. mp 224—230 °C (dec.). IR
1
(Nujol) cmϪ1: 1651. MS m/z: 420 (MϩHϩ). H-NMR (DMSO-d6) d: 1.70
(3H, s), 1.83 (3H, d, Jϭ4.6 Hz), 2.03 (3H, s), 2.14 (3H, s), 3.0—3.6 (2H, m),
4.5—5.4 (4H, m), 6.0—6.8 (3H, m), 6.52 (1H, s), 7.0—7.5 (4H, m), 9.0—
9.5 (1H, m). Anal. Calcd for C25H29N3O3·HCl·0.8H2O: C, 63.83; H, 6.77;
N, 8.93. Found: C, 63.70; H, 6.76; N, 8.91.
Procedure for the Synthesis of Compounds 24 and 25 Methyl (S)-2-
tert-Butoxycarbonyl-7-pyrrolidin-1-yl-1,2,3,4-tetrahydroisoquinoline-3-car-
boxylate (35): Compound 32 (2.92 g, 8.68 mmol) was hydrogenated as de-
scribed in the preparation of 33. A mixture of the residue (2.57 g), K2CO3
(2.90 g, 21.0 mmol) and 1,4-dibromobutane (1.2 ml, 10 mmol) in N,N-di-
methylformamide (DMF) (26 ml) was stirred at 60 °C for 18 h. After addi-
tion of water, the mixture was extracted with AcOEt, and the organic layer
was washed with water and brine, dried over Na2SO4, and evaporated under
reduced pressure. The residue was purified by column chromatography to
give 35 as a crystalline solid (2.25 g, 74% yield). 1H-NMR (CDCl3) d: 1.44,
1.51 (total 9H, s, s), 1.7—2.2 (4H, m), 2.9—3.5 (6H, m), 3.62 (3H, s), 4.2—
5.2 (3H, m), 6.2—6.5 (2H, m), 6.97 (1H, d, Jϭ8.4 Hz).
2,3,6-Trimethyl-4-{[(S)-7-pyrrolidin-1-yl-1,2,3,4-tetrahydroisoquinoline-
3-carbonyl]amino}phenyl Acetate (36): Compound 35 was hydrolyzed, and
condensed with compound 4, and then the Boc group was removed with HCl
as described in the preparation of 34 to afford 36. A crystalline solid. Yield
73%. 1H-NMR (CDCl3) d: 1.6—2.3 (4H, m), 2.07 (3H, s), 2.13 (6H, s), 2.32
(3H, s), 3.12 (1H, dd, Jϭ18.0, 10.0 Hz), 3.0—3.5 (6H, m), 3.68 (1H, dd,
Jϭ10.0, 5.5 Hz), 3.99 (2H, s), 6.29 (1H, br-s), 6.45 (1H, dd, Jϭ8.3, 2.3 Hz),
7.05 (1H, d, Jϭ8.3 Hz), 7.67 (1H, s), 9.26 (1H, s).
(S)-N-(4-Hydroxy-2,3,5-trimethylphenyl)-2-pentanoyl-7-pyrrolidin-1-yl-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochloride (24): Com-
pound 36 was acylated with pentanoyl chloride, hydrolyzed, and conver-
ted to a HCl salt in a similar manner to that described in the synthesis of 21.
A solid. Yield 73%. mp 141—145 °C. IR (Nujol) cmϪ1: 1647. MS m/z:

1074
8.64.
Vol. 58, No. 8
and the filtrate was evaporated under reduced pressure. The residue was pu-
Procedure for the Synthesis of 28 (S)-2-Hexanoyl-N-(4-hydroxy-2,3,5- rified by column chromatography. To a solution of the obtained residue in
trimethylphenyl)-7-methanesulfonylamino-1,2,3,4-tetrahydroisoquinoline-3- Et2O (30 ml) in an ice bath were added HCO2H (90 ml) and 8.8 M HCl in i-
carboxamide (28): To a solution of 13 (350 mg, 0.826 mmol) in CHCl3 PrOH (12.7 ml, 0.11 mol), and the mixture was stirred for 1.5 h. After addi-
(3.5 ml) in an ice bath were added pyridine (0.12 ml, 1.2 mmol) and MsCl tion of Et2O (360 ml), the precipitate formed was collected by filtration to
1
(0.06 ml, 0.78 mmol), and the mixture was stirred at room temperature for
1 h. After addition of 10% citric acid solution, the precipitate formed was
collected by filtration to give 28 as a crystalline solid (297 mg, 72% yield).
mp 168—173 °C. IR (Nujol) cmϪ1: 1649, 1634. MS m/z: 502 (MϩHϩ). 1H-
give 52 as a crystalline solid (18.8 g, 79% yield). H-NMR (DMSO-d6) d:
3.26 (2H, d, Jϭ6.5 Hz), 3.82 (3H, s), 4.13 (2H, s), 4.49 (1H, br-t), 4.93 (2H,
s), 7.3—8.0 (6H, m), 10.2—11.0 (2H, br).
Methyl (S)-7-Benzyloxy-2-hexanoyl-1,2,3,4-tetrahydroisoquinoline-3-car-
NMR (DMSO-d6) d: 0.70—1.10 (3H, br-t), 1.10—1.80 (6H, m), 2.03 (9H, boxylate (53): Compound 52 (4.00 g, 6.83 mmol) was acylated with hexa-
s), 2.10—2.70 (2H, m), 2.70—3.50 (2H, m), 2.94 (3H, s), 4.40—5.20 (3H, noyl chloride, as described in the preparation of 2. A solution of the residue
m), 6.45 (1H, s), 6.80—7.30 (3H, m), 7.60—8.20 (1H, br), 8.88, 9.15 (total in MeOH (40 ml) was hydrogenated at 0.3 MPa in the presence of 10%
1H, s, s), 9.62 (1H, s). Anal. Calcd for C26H35N3O5S·0.5H2O: C, 61.15; H, Pd–C (200 mg) at 25 °C for 2 h. After filtration, the filtrate was evaporated
7.11; N, 8.23. Found: C, 61.21; H, 6.91; N, 8.07.
Procedure for the Synthesis of 41 Methyl (S)-2-tert-Butoxycarbonyl- phy to give 53 as an oil (1.93 g, 71% yield). 1H-NMR (CDCl3) d: 0.90 (3H,
7-trifluoromethanesulfonyloxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate br-t), 1.1—1.9 (6H, m), 2.2—2.7 (2H, m), 2.8—3.4 (2H, m), 3.66 (3H, s),
under reduced pressure. The residue was purified by column chromatogra-
(48): Compound 47 was protected with Boc2O at the 2-position, and hy- 4.3—4.9 (2H, m), 4.8—5.0 (0.2H, m), 5.03 (2H, s), 5.39 (0.8H, dd, Jϭ5.7,
drogenolyzed to give methyl (S)-2-tert-butoxycarbonyl-7-hydroxy-1,2,3,4- 4.4 Hz), 6.4—7.0 (2H, m), 7.11 (1H, d, Jϭ7.8 Hz), 7.2—7.6 (5H, m).
tetrahydroisoquinoline-3-carboxylate (63) as described in the synthesis of
32 and 53. To a solution of 63 (5.00 g, 16.3 mmol) in CH2Cl2 (50 ml) in an
ice bath were added 2,6-lutidine (2.7 ml, 23 mmol) and Tf2O (5.53 g,
4-{[(S)-7-Benzyloxy-2-hexanoyl-1,2,3,4-tetrahydroisoquinoline-3-car-
bonyl]amino}-2,3,6-trimethylphenyl Acetate (54): Compound 53 was hy-
drolyzed, and condensed with compound 4 in a manner similar to the syn-
19.6 mmol) slowly, and then the mixture was stirred for 30 min. After evapo- thesis of 21 to give 54. A crystalline solid. Yield 61%. 1H-NMR (CDCl3) d:
ration under reduced pressure, 1.0 M hydrochloride acid (25 ml) was added,
0.90 (3H, br-t), 1.1—2.0 (6H, m), 1.58 (3H, s), 2.04 (6H, s), 2.2—2.6 (2H,
and the mixture was extracted with Et2O. The organic layer was washed with m), 2.30 (3H, s), 2.7—3.6 (2H, m), 4.4—5.4 (3H, m), 5.04 (2H, s), 6.7—7.0
saturated NaHCO3 solution and brine, dried over Na2SO4, and then evapo- (2H, m), 7.0—7.6 (7H, m), 8.13 (1H, br-s).
rated under reduced pressure. The residue was purified by column chro-
matography to give 48 as an oil (6.92 g, 97% yield). H-NMR (CDCl3) d:
1.51 (9H, s), 3.0—3.3 (2H, m), 3.64 (3H, s), 4.3—5.3 (3H, m), 6.9—7.3 from 54 in a manner similar to the synthesis of 41. A crystalline solid. Yield
(S)-2-Hexanoyl-7-hydroxy-N-(4-hydroxy-2,3,5-trimethylphenyl)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide (42): Compound 42 was obtained
1
(3H, m).
63%. mp 213—217 °C. IR (Nujol) cmϪ1: 3600—3100, 1636. MS m/z: 425
Methyl (S)-7-(4-Benzylpiperazin-1-yl)-2-hexanoyl-1,2,3,4-tetrahydroiso- (MϩHϩ). H NMR (DMSO-d6) d: 0.88 (3H, br-t), 1.1—2.0 (6H, m), 1.68
1
quinoline-3-carboxylate (50): Under a nitrogen atmosphere, a suspension of
(3H, s), 2.03 (6H, s), 2.2—2.6 (2H, m), 2.7—3.6 (2H, m), 4.3—5.0 (2H, m),
Pd(OAc)2 (85 mg, 0.32 mmol) and rac-BINAP (262 mg, 0.42 mmol) in dehy- 5.01 (1H, br-t), 6.4—6.8 (3H, m), 6.97 (1H, d, Jϭ7.9 Hz), 7.92, 8.84, 9.11,
drated 1,4-dioxane (1 ml) was stirred for 30 min, and a solution of 48 9.19 (total 3H, br-s, br-s, br-s, br-s). Anal. Calcd for C25H32N2O4·0.3H2O: C,
(1.42 g, 3.23 mmol) and 1-benzylpiperazine (684 mg, 3.88 mmol) in 1,4-
dioxane (4 ml) and Cs2CO3 (1.47 g, 4.51 mmol) were added. The mixture
69.84; H, 7.64; N, 6.52. Found: C, 69.75; H, 7.55; N, 6.49.
Procedure for the Synthesis of 43, 44, and 46 Compound 43 was syn-
was aerated with nitrogen for 10 min, and stirred at 80 °C for 5 h. After al- thesized from 48 via 55, 56 as follows. Compounds 44 and 46 were synthe-
lowing to cool, AcOEt was added to the mixture. After filtration, the filtrate sized from 56 in a similar manner to the synthesis of 43.
was evaporated under reduced pressure, and the residue was purified by col-
umn chromatography to give 49 as a viscous oil (745 mg, 50% yield). The
Methyl (S)-2-tert-Butoxycarbonyl-7-cyano-1,2,3,4-tetrahydroisoquino-
line-3-carboxylate (64): After suspension of Pd(OAc)2 (153 mg,
a
Boc group of 49 was changed to hexanoyl moiety, as described in the syn- 0.579 mmol) and rac-BINAP (467 mg, 0.750 mmol) in dehydrated N-methyl-
1
thesis of 38 to give 50. An oil. Yield 83%. H-NMR (CDCl3) d: 0.91 (3H, 2-pyrrolidinone (NMP) (3 ml) was stirred at room temperature for 1 h under
br-t), 1.1—2.0 (6H, m), 2.2—2.8 (6H, m), 3.0—3.4 (6H, m), 3.56 (2H, s), a nitrogen atmosphere, KCN (892 mg, 13.7 mmol) and a solution of 48
3.60 (3H, s), 4.3—5.6 (3H, m), 6.6—6.9 (2H, m), 7.03 (1H, d, Jϭ8.1 Hz),
7.2—7.5 (5H, m).
4-{[(S)-7-(4-Benzylpiperazin-1-yl)-2-hexanoyl-1,2,3,4-tetrahydroiso-
(3.00 g, 6.83 mmol) in NMP (6 ml) were added, and the mixture was stirred
at 80 °C for 10 h. After allowing to cool, water (60 ml) and Et2O (20 ml)
were added, and the mixture was stirred for 10 min. After filtration, the fil-
quinoline-3-carbonyl]amino}-2,3,6-trimethylphenyl Acetate (51): Com- trate was extracted with Et2O. The organic layer was washed with brine,
pound 50 was hydrolyzed, and condensed with compound 4 in a manner dried over Na2SO4, and evaporated under reduced pressure. After the residue
similar to the synthesis of 21 to give 51. A crystalline solid. Yield 74%. 1H- had been purified by column chromatography, the residue was washed with
NMR (CDCl3) d: 0.90 (3H, br-t), 1.0—2.0 (6H, m), 1.98 (3H, s), 2.04 (6H, hexane, and the precipitate was collected by filtration to give 64 as a crys-
s), 2.2—2.8 (6H, m), 2.30 (3H, s), 2.9—3.5 (6H, m), 3.57 (2H, s), 4.3—5.4 talline solid (1.65 g, 76% yield). IR (neat) cmϪ1: 2230. 1H-NMR (CDCl3) d:
(3H, m), 6.6—7.0 (2H, m), 7.0—7.6 (7H, m), 8.09 (1H, br-s). 1.50 (9H, s), 3.0—3.4 (2H, m), 3.63 (3H, s), 4.3—5.3 (3H, m), 7.25 (1H, d,
(S)-2-Hexanoyl-N-(4-hydroxy-2,3,5-trimethylphenyl)-7-(piperazin-1-yl)- Jϭ8.1 Hz), 7.3—7.5 (2H, m).
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (41): Compound 51 was
7-Benzyloxycarbonylaminomethyl-2-tert-butoxycarbnoyl-2-(2,2-di-
deacetylated by usual method. Yield 76%. The obtained deacetylated com- methylpropionyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (55):
A
pound (570 mg, 0.98 mmol) in MeOH (20 ml) was hydrogenolyzed at mixture of 64 (2.19 g, 6.92 mmol), 28% aqueous ammonia (8.4 g, 0.14 mol),
0.4 MPa in the presence of 20% Pd(OH)2–C (110 mg) at 40 °C for 9 h. After and MeOH (22 ml) was hydrogenated at 0.4 MPa in the presence of 20%
filtration, the filtrate was evaporated under reduced pressure. The residue Pd(OH)2–C (0.22 g) at 35 °C for 15 h. After filtration, the filtrate was evapo-
was purified by column chromatography to give 41 as a crystalline solid
rated under reduced pressure. A solution of the residue in CHCl3 was dried
over Na2SO4, and evaporated under reduced pressure. The residue was puri-
fied by column chromatography to give an aminomethyl derivative as an oil
(283 mg, 59% yield). mp 155—174 °C. IR (Nujol) cmϪ1: 1622. MS m/z: 493
1
(MϩHϩ). H-NMR (DMSO-d6) d: 0.87 (3H, br-t), 1.0—1.8 (6H, m), 1.69
(3H, s), 2.04 (6H, s), 2.1—4.0 (14H, m), 4.4—5.2 (3H, m), 6.51 (1H, s), (712 mg, 32% yield). To a solution of the product (900 mg, 2.81 mmol) in
6.6—7.2 (3H, m), 9.13 (1H, br-s). Anal. Calcd for C29H40N4O3·2H2O: C, AcOEt (9 ml) were added MgO (340 mg, 8.43 mmol) and CbzCl (0.60 ml,
65.88; H, 8.39; N, 10.60. Found: C, 66.13; H, 8.13; N, 10.48.
4.2 mmol), and the mixture was stirred at room temperature for 24 h. After
Procedure for the Synthesis of 42 Methyl (S)-7-Benzyloxy-6,8-diiodo- filtration, the filtrate was evaporated under reduced pressure. The residue
1,2,3,4-tetrahydroisoquinoline-3-carboxylate Hydrochloride (52): To a sus- was purified by column chromatography to give 55 as an oil (882 mg, 69%
pension of compound 47 (20.0 g, 40.4 mmol) in CHCl3 (150 ml) in an ice
bath were added Et3N (12.4 ml, 89.0 mmol) and a solution of Boc2O (9.69 g,
44.4 mmol) in CHCl3 (50 ml), and the mixture was stirred at room tempera-
ture for 16 h. The mixture was washed with 1.0 M hydrochloric acid and
yield). 1H-NMR (CDCl3) d: 1.47, 1.51 (total 9H, s, s), 3.0—3.3 (2H, m),
3.61 (3H, s), 4.33 (2H, d, Jϭ5.8 Hz), 4.4—5.4 (3H, m), 5.14 (2H, s), 7.0—
7.2 (4H, m), 7.2—7.5 (5H, m).
4-{[(S)-7-Benzyloxycarbonylaminomethyl-2-(2,2-dimethylpropionyl)-
brine, dried over Na2SO4, and evaporated under reduced pressure. The 1,2,3,4-tetrahydroisoquinoline-3-carbonyl]amino}-2,3,6-trimethylphenyl
residue was purified by column chromatography. To a solution of the ob- Acetate (56): Compound 55 was hydrolyzed, condensed with compound 4,
tained residue in acetone (200 ml) were added benzyl bromide (5.4 ml, treated with acid to remove the Boc group, and acylated with 2,2-dimethyl-
45 mmol) and K2CO3 (7.90 g, 57.2 mmol), and the mixture was stirred at
60 °C for 2.5 h. After addition of AcOEt (100 ml), the mixture was filtered, solid. Yield 63%. H-NMR (CDCl3) d: 1.37 (9H, s), 1.98 (3H, br-s), 2.03
propionyl chloride in a similar manner to the preparation of 21. A crystalline
1

August 2010
1075
with 2,2-dimethylpropionyl chloride, and hydrogenolyzed as described in the
(3H, s), 2.06 (3H, s), 2.31 (3H, s), 3.05 (1H, dd, Jϭ16.2, 6.4 Hz), 3.49 (1H,
dd, Jϭ16.2, 4.6 Hz), 4.35 (2H, br-t), 4.50 (1H, d, Jϭ16.1 Hz), 4.96 (1H, d,
Jϭ16.1 Hz), 5.00—5.10 (1H, br), 5.13 (2H, s), 5.32 (1H, dd, Jϭ6.4, 4.6 Hz),
7.07 (1H, s), 7.16 (1H, d, Jϭ7.8 Hz), 7.21 (1H, d, Jϭ7.8 Hz), 7.30—7.40
(5H, m), 7.44 (1H, s), 7.96 (1H, br-s).
preparation of 48 to give 66. The obtained crude crystal was recrystallized
1
from AcOEt–hexane. A crystalline solid. Yield 78%. H-NMR (CDCl3) d:
1.34 (9H, s), 3.00—3.25 (2H, m), 3.65 (3H, s), 4.56 (1H, br-d), 4.93 (1H, d,
Jϭ16.4 Hz), 5.05—5.25 (1H, m), 5.50—6.50 (1H, br), 6.60—6.80 (2H, m),
7.00 (1H, d, Jϭ8.6 Hz).
(S)-7-Dimethylaminomethyl-2-(2,2-dimethylpropionyl)-N-(4-hydroxy-
2,3,5-trimethylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hy-
drochloride (43): A solution of 56 (315 mg, 0.525 mmol) in MeOH (6 ml)
was hydrogenolyzed at 0.3 MPa in the presence of 10% Pd–C (30 mg) at
25 °C for 4 h. After filtration, the filtrate was evaporated under reduced pres-
sure. To a solution of the residue in MeOH (2 ml) were added formalin
(172 mg, 2.1 mmol) and NaBH3CN (74 mg, 1.1 mmol), and the mixture was
stirred at room temperature for 1 h. After evaporation under reduced pres-
sure, saturated NaHCO3 solution was added, and the mixture was extracted
with AcOEt. The organic layer was washed with brine, dried over Na2SO4,
and evaporated under reduced pressure to give 57. To a solution of the
residue in THF–MeOH (3 : 1, 5 ml) in an ice bath was added 1.0 M LiOH
aqueous solution (1.6 ml, 1.6 mmol), and the mixture was stirred for 1.5 h.
After addition of 2.0 M hydrochloric acid (0.8 ml), the mixture was concen-
trated under reduced pressure. Saturated NaHCO3 solution was added, and
the mixture was extracted with AcOEt. The organic layer was washed with
saturated NaHCO3 solution and brine, dried over Na2SO4, and evaporated
under reduced pressure. The residue was purified by column chromatogra-
phy. To a solution of the residue in MeOH (0.5 ml) in an ice bath was added
8.7 M HCl in i-PrOH (0.05 ml, 0.4 mmol), and the mixture was stirred for
5 min. Et2O (60 ml) was added, and stirred for 30 min. The precipitate
formed was collected by filtration to give 43 as a solid (128 mg, 50% yield).
IR (Nujol) cmϪ1: 1668. MS m/z: 452 (MϩHϩ). 1H-NMR (DMSO-d6) d:
1.29 (9H, s), 1.77 (3H, br-s), 2.04 (3H, s), 2.06 (3H, s), 2.66 (3H, s), 2.67
(3H, s), 3.15—3.40 (2H, m), 4.21 (2H, br-d), 4.60 (1H, d, Jϭ16.7 Hz), 4.91
(1H, d, Jϭ16.7 Hz), 4.95—5.20 (1H, m), 6.56 (1H, br-s), 7.31 (1H, d,
Jϭ7.6 Hz), 7.39 (1H, d, Jϭ7.6 Hz), 7.44 (1H, s), 8.00 (1H, br-s), 9.21 (1H,
br-s), 10.53 (1H, br-s).
Methyl (S)-2-(2,2-Dimethylpropionyl)-7-trifluoromethanesulfonyloxy-
1,2,3,4-tetrahydroisoquinoline-3-carboxylate (60): Compound 66 was tri-
flated as described in the preparation of 48 to give 60. A crystalline solid.
Yield 90%. 1H-NMR (CDCl3) d: 1.35 (9H, s), 3.17 (1H, dd, Jϭ16.2,
6.1 Hz), 3.28 (1H, br-dd), 3.67 (3H, s), 4.61 (1H, br-d), 5.04 (1H, d,
Jϭ16.8 Hz), 5.31 (1H, dd, Jϭ6.1, 4.4 Hz), 7.06 (1H, d, Jϭ2.4 Hz), 7.12 (1H,
dd, Jϭ8.4, 2.4 Hz), 7.25 (1H, d, Jϭ8.4 Hz).
Methyl (S)-7-Cyano-2-(2,2-dimethylpropionyl)-1,2,3,4-tetrahydroiso-
quinoline-3-carboxylate (67): Compound 60 was reacted with KCN as de-
scribed in the preparation of 64 to give 67. A crystalline solid. Yield 80%.
IR (Nujol) cmϪ1: 2235. 1H-NMR (CDCl3) d: 1.35 (9H, s), 3.21 (1H, dd,
Jϭ16.6, 6.2 Hz), 3.31 (1H, br-dd), 3.67 (3H, s), 4.61 (1H, br-d), 5.04 (1H, d,
Jϭ16.8 Hz), 5.33 (1H, dd, Jϭ6.2, 4.4 Hz), 7.29 (1H, d, Jϭ7.9 Hz), 7.44 (1H,
s), 7.49 (1H, d, Jϭ7.9 Hz).
Methyl (S)-2-(2,2-Dimethylpropionyl)-7-formyl-1,2,3,4-tetrahydroiso-
quinoline-3-carboxylate (68): To a solution of 67 (34.1 g, 114 mmol) in 80%
formic acid (340 ml) heated at 90 °C was added Raney nickel (34 g) in por-
tions, and the mixture was refluxed for 20 min. The mixture was poured into
ice-water (3.4 l), and stirred for 1 h at room temperature. After collecting the
precipitate by filtration, a solution of the residue in AcOEt was washed with
water and brine, dried over Na2SO4, and evaporated under reduced pressure.
The residue was recrystallized from AcOEt–hexane to give 68 as a crys-
talline solid (18.1 g, 53% yield). 1H-NMR (CDCl3) d: 1.36 (9H, s), 3.23
(1H, dd, Jϭ16.4, 6.2 Hz), 3.64 (1H, br-dd), 3.66 (3H, s), 4.68 (1H, br-d),
5.09 (1H, d, Jϭ16.4 Hz), 5.31 (1H, dd, Jϭ6.2, 4.9 Hz), 7.35 (1H, d,
Jϭ7.7 Hz), 7.67 (1H, s), 7.72 (1H, d, Jϭ7.7 Hz), 9.97 (1H, s).
(S)-2-(2,2-Dimethylpropionyl)-7-hydroxymethyl-1,2,3,4-tetrahydroiso-
quinoline-3-carboxylic Acid (61): To a solution of 68 (23.0 g, 75.8 mmol) in
AcOEt (230 ml) in an ice bath was added tert-butylamine borane (7.25 g,
83.4 mmol), and the mixture was stirred at room temperature for 1.5 h. 2.0 M
hydrochloric acid was added, and the mixture was stirred for 1 h. The AcOEt
layer was washed with 1.0 M hydrochloric acid and brine, dried over Na2SO4,
and evaporated under reduced pressure. The residue was hydrolyzed as de-
scribed in the preparation of 34 to give 61 as a crystalline solid (18.2 g, 82%
yield). 1H-NMR (DMSO-d6) d: 1.25 (9H, s), 3.00—3.20 (2H, m), 4.35—
4.60 (3H, m), 4.80—5.20 (2H, m), 4.90 (1H, br-d), 7.10—7.30 (3H, m),
12.30—13.10 (1H, br).
7-Diethylaminomethyl-2-(2,2-dimethylpropionyl)-N-(4-hydroxy-2,3,5-
trimethylphenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hydrochlo-
ride (44): Compound 44 was obtained from 56 via 58 in a similar manner to
the synthesis of 43 using acetaldehyde instead of formalin. A solid. Yield
1
49%. mp 157—165 °C. IR (Nujol) cmϪ1:1674. MS m/z: 480 (MϩHϩ). H-
NMR (DMSO-d6) d: 1.24 (6H, t, Jϭ16.2 Hz), 1.29 (9H, s), 1.75 (3H, br-s),
2.04 (3H, s), 2.06 (3H, s), 2.90—3.20 (4H, m), 3.20—3.50 (2H, m), 4.23
(2H, br-s), 4.60 (1H, d, Jϭ16.1 Hz), 4.92 (1H, d, Jϭ16.1 Hz), 4.95—5.20
(1H, m), 6.57 (1H, br-s), 7.25—7.55 (3H, m), 8.00 (1H, br-s), 9.20 (1H, br-
s), 10.40—10.70 (1H, br). Anal. Calcd for C29H41N3O3·HCl·1.7H2O: C,
63.71; H, 8.37; N, 7.69. Found: C, 63.73; H, 8.30; N, 7.63.
(S)-3-(4-Acetoxy-2,3,5-trimethylphenylcarbamoyl)-2-(2,2-dimethylpro-
pionyl)-1,2,3,4-tetrahydroisoquinolin-7-ylmethyl Methanesulfonate (69):
Compound 61 was condensed with compound 4 as described in the prepara-
tion of 21. Yield 98%. To a solution of the amide derivative (1.50 g,
3.21 mmol) and Et3N (0.58 ml, 4.2 mmol) in CH2Cl2 (15 ml) in an ice bath
was added MsCl (0.30 ml, 3.9 mmol), and the mixture was stirred for 3 h.
The mixture was washed with saturated NaHCO3 solution, 5% citric acid so-
lution and brine, dried over Na2SO4, and evaporated under reduced pressure
4-{[(S)-2-(2,2-Dimethylpropionyl)-7-(pyrrolidin-1-ylmethyl)-1,2,3,4-
tetrahydroisoquinoline-3-carbonyl]amino}-2,3,6-trimethylphenyl Acetate
(59): Compound 56 (4.02 g, 8.71 mmol) was hydrogenolyzed. A mixture of
the residue, DMF (40 ml), 1,4-dibromobutane (1.14 ml, 9.55 mmol), and
K2CO3 (3.01 g, 21.8 mmol) was heated at 55 °C for 3 h. After allowing to
cool, 0.26 M hydrochloric acid (200 ml) was added, and NaHCO3 was added
until pHϾ8. The mixture was extracted with AcOEt, and the organic layer
was washed with brine, dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by column chromatography to give 59 as
a crystalline solid (1.50 g, 33% yield). 1H-NMR (CDCl3) d: 1.37 (9H, s),
1.70—1.85 (4H, m), 1.98 (3H, br-s), 2.03 (3H, s), 2.07 (3H, s), 2.31 (3H, s),
2.40—2.60 (4H, m), 3.06 (1H, dd, Jϭ16.0, 6.7 Hz), 3.50 (1H, dd, Jϭ16.0,
5.0 Hz), 3.58 (1H, d, Jϭ12.9 Hz), 3.62 (1H, d, Jϭ12.9 Hz), 4.53 (1H, d,
Jϭ15.6 Hz), 4.96 (1H, d, Jϭ15.6 Hz), 5.29 (1H, dd, Jϭ6.7, 5.0 Hz), 7.10—
7.25 (3H, m), 7.41 (1H, s), 7.93 (1H, br-s).
(S)-2-(2,2-Dimethylpropionyl)-N-(4-hydroxy-2,3,5-trimethylphenyl)-7-
(pyrrolidin-1-ylmethyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide Hy-
drochloride (46): Compound 59 was converted to 46 as described in the syn-
thesis of 43. A solid. Yield 89%. mp 169—173 °C. IR (Nujol) cmϪ1: 1674.
MS m/z: 478 (MϩHϩ). 1H-NMR (DMSO-d6) d: 1.29 (9H, s), 1.70—1.95
(4H, m), 2.00 (3H, br-s), 2.04 (3H, s), 2.06 (3H, s), 2.95—3.10 (2H, m),
3.15—3.45 (4H, m), 4.28 (2H, br-s), 4.60 (1H, d, Jϭ16.7 Hz), 4.91 (1H, d,
Jϭ16.7 Hz), 4.95—5.20 (1H, m), 6.57 (1H, br-s), 7.30 (1H, d, Jϭ7.1 Hz),
7.35—7.55 (2H, m), 7.99 (1H, br-s), 9.18 (1H, br-s), 10.20—11.00 (1H, br).
Anal. Calcd for C29H39N3O3·HCl·1.6H2O: C, 64.15; H, 8.02; N, 7.74.
Found: C, 64.01; H, 7.72; N, 7.69.
1
to give 69 as a crystalline solid (1.77 g, 100%). H-NMR (CDCl3) d: 1.38
(9H, br-s), 2.02 (3H, br-s), 2.04 (3H, s), 2.07 (3H, s), 2.32 (3H, s), 2.92 (3H,
s), 3.08 (1H, dd, Jϭ16.4, 6.8 Hz), 3.54 (1H, dd, Jϭ16.4, 4.5 Hz), 4.52 (1H,
d, Jϭ16.0 Hz), 5.02 (1H, d, Jϭ16.0 Hz), 5.29 (2H, s), 5.37 (1H, dd, Jϭ6.8,
4.5 Hz), 7.18 (1H, s), 7.20—7.30 (2H, m), 7.45 (1H, br-s), 8.03 (1H, br-s).
4-{[(S)-2-(2,2-Dimethylpropionyl)-7-[(methylpropylamino)methyl]-
1,2,3,4-tetrahydroisoquinoline-3-carbonyl]amino}-2,3,6-trimethylphenyl
Acetate (62): To a solution of 69 (909 mg, 1.87 mmol) in THF (4.5 ml) was
added N-methylpropylamine (0.51 ml, 5.1 mmol), and the mixture was
stirred at room temperature for 19 h. After evaporation under reduced pres-
sure, 1.0 M hydrochloric acid was added, and washed with Et2O. After neu-
tralization with NaHCO3, the mixture was extracted with AcOEt. The or-
ganic layer was washed with brine, dried over Na2SO4, and evaporated under
1
reduced pressure to give 62 as a crystalline solid (683 mg, 70% yield). H-
NMR (CDCl3) d: 0.89 (3H, t, Jϭ7.4 Hz), 1.37 (9H, br-s), 1.52 (2H, sextet,
Jϭ7.4 Hz), 1.98 (3H, br-s), 2.03 (3H, s), 2.07 (3H, s), 2.18 (3H, s), 2.25—
2.35 (2H, m), 2.31 (3H, s), 3.06 (1H, dd, Jϭ16.0, 6.8 Hz), 3.45 (2H, s), 3.51
(1H, dd, Jϭ16.0, 4.9 Hz), 4.52 (1H, d, Jϭ15.9 Hz), 4.96 (1H, d, Jϭ15.9 Hz),
5.56 (1H, dd, Jϭ6.8, 4.9 Hz), 7.13 (1H, s), 7.15—7.25 (2H, m), 7.41 (1H,
br-s), 7.94 (1H, br-s).
(S)-2-(2,2-Dimethylpropionyl)-N-(4-hydroxy-2,3,5-trimethylphenyl)-7-
[(methylpropylamino)methyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
(45): Compound 62 was hydrolyzed as described in the synthesis of 43 to
Procedure for the Synthesis of 45 Compound 45 was obtained from 47
via 60, 61, and 62.
Methyl (S)-2-(2,2-Dimethylpropionyl)-7-hydroxy-1,2,3,4-tetrahydroiso-
quinoline-3-carboxylate (66): Compound 47 was acylated at the 2-position

1076
Vol. 58, No. 8
give 45. A crystalline solid. Yield 32%. mp 165—170 °C. IR (Nujol) cmϪ1
;
cubated at 4ϫ104 cells/well in a humidified atmosphere of 95% air, 5% CO2
at 37 °C for 24 h. Differentiated cells were incubated with LDL (400 mg/ml),
CuSO4 (5 mM) and test compounds in serum-free RPMI-1640 medium for
24 h, and cell death was observed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyltetrazolium bromide (MTT) assay (DOJINDO LABORATORIES,
Kumamoto, Japan). LDL was prepared from the serum of male KHC rabbits
(12 months old; Japan Laboratory Animals, Inc.).
Plasma Levels after Oral Administration: Test compounds were sus-
pended in 5% arabic gum and administered orally at 10 mg/kg to male
Sprague-Dawley (SD) rats (6—7 weeks old; Japan SLC), and beagles (9—
14 months; NARC Co., Yamatake, Japan). Blood samples were drawn from
the jugular vein using a heparinized syringe at 0.25, 0.5, 1, 2, 3, 5, 8, and
24 h after administration. Blood was centrifuged at 3000 rpm for 10 min at
room temperature. The concentrations of test compounds in the plasma were
determined using HPLC.
1680. MS m/z: 480 (MϩHϩ). H-NMR (CDCl3) d: 0.88 (3H, t, Jϭ7.5 Hz),
1.38 (9H, br-s), 1.52 (2H, sextet, Jϭ7.5 Hz), 1.90 (3H, br-s), 2.09, 2.10,
2.12, 2.13 (total 6H, s, s, s, s), 2.18 (3H, s), 2.31 (2H, t, Jϭ7.5 Hz), 3.06
(1H, dd, Jϭ15.8, 6.8 Hz), 3.45 (2H, s), 3.51 (1H, dd, Jϭ15.8, 5.1 Hz), 4.56
(1H, d, Jϭ15.9 Hz), 4.95 (1H, d, Jϭ15.9 Hz), 4.70—5.00 (1H, br), 5.35 (1H,
dd, Jϭ6.8, 5.1 Hz), 6.95—7.05 (1H, br), 7.13 (1H, s), 7.15—7.25 (2H, m),
7.69 (1H, br-s). Anal. Calcd for C29H41N3O3: C, 72.62; H, 8.62; N, 8.70.
Found: C, 72.27; H, 8.63; N, 8.70.
1
Partition Coefficient at pH 7.0 Log D7.0 values (logarithm of oc-
tanol–water partition coefficients at pH 7.0) were determined by HPLC
methods.25) Acetanilide, benzonitrile, benzene, bromobenzene, biphenyl and
hexachlorobenzene, the log D7.0 values of which are known, were used as
reference substances. Test compounds and reference substances were dis-
solved in acetonitrile containing 1% dimethylsulfoxide (DMSO) at
10 mg/ml, and then 10 ml of the solution was injected into the HPLC system.
The HPLC equipment consisted of a pump (PU-980; JASCO, Tokyo, Japan),
a UV detector (UV-970; JASCO), an autoinjector (AS-950; JASCO), and a
Cosmosil 5C18-AR-II column (5 mm, 4.6 mmϫ150 mm; Nacalai Tesque,
Kyoto, Japan). Phosphate buffer (pH 7.0)–MeOH (8 : 2) was used as the elu-
ent. The capacity factors of test substances and reference substances were
calculated from their retention time. The log D7.0 values of test compounds
were calculated using these capacity factors and the reported log D7.0 values
of reference substances.
References
1) Sliskovic D. R., White A. D., Trends Pharmacol. Sci., 12, 194—199
(1991).
2) Roth B. D., Drug Discov. Today, 3, 19—25 (1998).
3) Matsuda K., Med. Res. Rev., 14, 271—305 (1994).
4) Chang C., Dong R., Miyazaki A., Sakashita N., Zhang Y., Liu J., Guo
M., Li B. L., Chang T. Y., Acta Biochim. Biophys. Sin., 38, 151—156
(2006).
Biological Evaluations of Compounds All experiments were con-
ducted according to the guidelines for animal experiments of our institute
and the Guidelines for Animal Experimentation approved by the Japanese
Association of Laboratory Animal Science and the Japanese Pharmacologi-
cal Society.
Effect on in Vitro LDL Peroxidation: The effects of test compounds on
LDL oxidation were determined according to the method reported previ-
ously.6) Briefly, the LDL fraction was isolated from the plasma of male KHC
rabbits and incubated with CuSO4 (5 mM) at 37 °C for 1 h in the presence of
vehicle or test compounds. Oxidized LDL was determined as MDA. IC50
values were calculated using data from duplicate assay tubes at each concen-
tration of test compounds.
5) Homan R., Hamelehle K. L., J. Pharm. Sci., 90, 1859—1867 (2001).
6) Takahashi K., Kunishiro K., Kasai M., Miike T., Kurahashi K., Shira-
hase H., Arzneim.-Forsch., 58, 666—672 (2008).
7) Takahashi K., Kasai M., Ohta M., Shoji Y., Kunishiro K., Kanda M.,
Kurahashi K., Shirahase H., J. Med. Chem., 51, 4823—4833 (2008).
8) Terasaka N., Miyazaki A., Kasanuki N., Ito K., Ubukata N., Koieyama
T., Kitayama K., Tanimoto T., Maeda N., Inaba T., Atherosclerosis,
190, 239—247 (2007).
9) Kitayama K., Koga T., Maeda N., Inaba T., Fujioka T., J. Pharmacol.,
539, 81—88 (2006).
10) Nissen S. E., Tuzcu E. M., Brewer H. B., Sipahi I., Nicholls S. J., Ganz
P., Schoenhagen P., Waters D. D., Pepine C. J., Crowe T. D., Davidson
M. H., Deanfield J. E., Wisniewski L. M., Hanyok J. J., Kassalow L.
M., N. Engl. J. Med., 354, 1253—1263 (2006).
11) Kawasaki T., Azuma Y., Ikemoto K., Mikami H., Yamada T.,
Nobuhara Y., “Abstracts of Papers,” The 69th Annual Meeting of The
Japanese Pharmacological Society, Nagasaki, Japan, 1996, p. 249.
12) Cyrus T., Yao Y., Rokach J., Tang L. X., Pratico D., Circulation, 107,
521—523 (2003).
13) Kita T., Nagano Y., Yokode M., Ishii K., Kume N., Narumiya S.,
Kawai C., Am. J. Cardiol., 62, 13B—19B (1988).
14) Whaley W. M., Govindachari T. R., Organic Reactions, 6, 151—190
(1951).
In Vitro ACAT Activity: Male Japanese white rabbits (2.5 kg, Japan SLC)
were anesthetized with sodium pentobarbital (30 mg/kg, intravenously (i.v.)),
exsanguinated from the common carotid artery, and then the liver was iso-
lated. Microsomes were prepared according to the method of Field and
Mathur.26) Briefly, each sample was homogenized in a buffered sucrose solu-
tion (250 mM sucrose, 5 mM K2HPO4/KH2PO4, 1 mM ethylenediaminete-
traacetic acid (EDTA), and 1 mM dithioerythritol, pH 7.4) using a Teflon-
glass homogenizer. The homogenate was centrifuged at 12000ϫg for 15 min
at 4 °C. The resulting supernatant was centrifuged at 105000ϫg for 30 min
at 4 °C. The microsomal fraction was used as the ACAT preparation. ACAT
activity was determined according to the method described by Heider et
al.27) The microsomes were incubated in 154 mM phosphate buffer (pH 7.4)
containing bovine serum albumin. Test compounds were applied and pre-
incubated at 37 °C for 5 min, then 30 nmol of [1-14C]oleoyl-CoA
(PerkinElmer, Waltham, MA, U.S.A.) was added. The reaction mixture was
incubated at 37 °C for 20 min and EC was extracted with CHCl3/MeOH
(2 : 1) and separated by thin-layer chromatography. The EC produced in mi-
crosomes treated with vehicle and test compounds was determined. IC50 val-
ues were calculated using data from duplicate assay tubes at the concentra-
tions of test compounds in each experiment.
Esterified Cholesterol (EC) Accumulation in THP-1 Cell-Derived
Macrophages: The effects of the test compounds on EC accumulation in
THP-1 cells were determined during differentiation and foam cell formation.
In order to cause them to differentiate into macrophages and to form foam
cells, THP-1 cells were suspended in RPMI-1640 medium containing fetal
bovine serum (FBS, 10%) and phorbol 12-myristate 13-acetate (PMA,
200 nM) with acetyl LDL (400 mg protein/ml), and then they were incubated
at 4ϫ105 cells/well in a humidified atmosphere of 95% air, 5% CO2 at 37 °C
for 3 d in the presence or absence of the test compounds. Acetyl LDL was
prepared from the serum of male KHC rabbits (6—8 months old; Japan Lab-
oratory Animals, Inc., Tokyo, Japan). Cellular cholesterol was extracted by
hexane/i-PrOH (3 : 2) and determined by enzymatic methods. EC content
was calculated by subtracting the amount of free cholesterol from the total
amount of cholesterol. Cellular protein was measured by Lowry’s method.
Oxidative Stress-Induced Cell Death: THP-1 cells were suspended in
RPMI-1640 medium containing FBS (10%) and PMA (200 nM), and then in-
15) Cross G. G., Fischer A., Henderson N. H., Smyth T. A., Can. J. Chem.,
62, 1446—1451 (1984).
16) MacDonald J., Matz J., Shakespeare W., Astra Aktiebolag, AMIDINE
AND ISOTHIOUREA DERIVATIVES, COMPOSITIONS CON-
TAINING THEM AND THEIR USE AS INHIBITORS OF NITRIC
OXIDE SYNTHASE. U.S. Patent US005929085, Jul. 27, 1999.
17) Verschueren K., Toth G., Tourwé D., Lebl M., Van Binst G., Hruby V.,
Synthesis, 5, 458—460 (1992).
18) Wolfe J. P., Buchwald L. S., J. Org. Chem., 62, 1264—1267 (1997).
19) Takagi K., Sakakibara Y., Chem. Lett., 18, 1957—1958 (1989).
20) van Es T., Staskun B., Org. Synth., 51, 20—23 (1971).
21) Andrews G. C., Crawford T. C., Tetrahedron. Lett., 21, 693—696
(1980).
22) Su B., Yang Y. B., Tuo Q. H., Zhu B. Y., Lei X. Y., Yin W., Liao D. F.,
Cardiovasc. Drugs Ther., 21, 37—45 (2007).
23) Miike T., Shirahase H., Jino H., Kunishiro K., Kanda M., Kurahashi
K., Life Sci., 82, 79—84 (2008).
24) Fruebis J., Gonzalez V., Silvestre M., Palinski W., Arterioscler.
Thromb. Vasc. Biol., 17, 1289—1302 (1997).
25) Masumoto K., Takeyasu A., Oizumi K., Kobayashi T., Yakugaku
Zasshi, 115, 213—220 (1995).
26) Field F. J., Mathur S. N., J. Lipid Res., 24, 409—417 (1983).
27) Heider J. G., Pickens C. E., Kelly L., J. Lipid Res., 24, 1127—1134
(1983).