Enantioselective synthesis of a phenylalanine library containing alkyl groups on the aromatic moiety: Confirmation of stereostructure by X-ray analysis

Article abstract of DOI:10.1248/cpb.54.873

Six phenylalanine analogues containing 2′-methyl-, 2′,6′-dimethyl-, 2′-ethyl-6′-methyl-, 2′-isopropyl-6′-methyl-, 2′,4′,6′-trimethyl-, and 3′,5′-dimethyl-L-phenylalanine were synthesized enantioselectively through asymmetric hydrogenation of acetamidoacrylate derivatives. Enzymatic digestion and X-ray analysis supported the L-configuration of the phenylalanine derivatives obtained.

Full text of DOI:10.1248/cpb.54.873

June 2006
Chem. Pharm. Bull. 54(6) 873—877 (2006)
873
Enantioselective Synthesis of a Phenylalanine Library Containing Alkyl
Groups on the Aromatic Moiety: Confirmation of Stereostructure by
X-Ray Analysis
Tingyou LI,a Yuko TSUDA,a Katsuhiko MINOURA,b Yasuko IN,b Toshimasa ISHIDA,b
Lawrence H. LAZARUS,c and Yoshio OKADA*,a
a The Graduate School of Food and Medicinal Sciences and Faculty of Pharmaceutical Sciences, Kobe Gakuin University;
Nishi-ku, Kobe 651–2180, Japan: b Osaka University of Pharmaceutical Sciences; Takatsuki, Osaka 569–1094, Japan: and
c Medicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health
Sciences; Research Triangle Park, NC 27709, U.S.A.
Received February 16, 2006; accepted March 7, 2006; published online March 9, 2006
Six phenylalanine analogues containing 2ꢀ-methyl-, 2ꢀ,6ꢀ-dimethyl-, 2ꢀ-ethyl-6ꢀ-methyl-, 2ꢀ-isopropyl-6ꢀ-
methyl-, 2ꢀ,4ꢀ,6ꢀ-trimethyl-, and 3ꢀ,5ꢀ-dimethyl-L-phenylalanine were synthesized enantioselectively through
asymmetric hydrogenation of acetamidoacrylate derivatives. Enzymatic digestion and X-ray analysis supported
the L-configuration of the phenylalanine derivatives obtained.
Key words phenylalanine analogue; asymmetrical hydrogenation; L-configuration; enzymatic digestion; X-ray analysis
Introduction of unnatural amino acid into biologically ac- methyl- (Emp), 2ꢀ-isopropyl-6ꢀ-methyl- (Imp), 2ꢀ,4ꢀ,6ꢀ-
tive peptides is one of the most powerful approaches to de- trimethyl- (Tmp), and 3ꢀ,5ꢀ-dimethyl-L-phenylalnine
velopment of peptides and peptidomimetics with unique (3,5Dmp). Until now, 2ꢀ-methyl-L-phenylalanine was synthe-
properties. It permits an examination of the topographical re- sized through optical resolution with enzymes13); 2ꢀ,6ꢀ-di-
quirements for peptide bioactivities,1—3) improved enzymatic methyl-L-phenylalanine was prepared by separation of Ac-
stability,4) constrained spatial conformation,5) and the elicita- D,L-Dmp-Arg-OMe in preparative HPLC followed by acid
tion of novel biological properties.6,7)
hydrolysis8) or asymmetric alkylation of glycine equiva-
In opioid peptides for example, the introduction of unnatu- lent14—17); 3ꢀ,5ꢀ-dimethyl-L-phenylalanine was synthesized
ral amino acids, such as 2ꢀ,6ꢀ-dimethyl-L-tyrosine (Dmt) as through asymmetric alkylation of glycine equivalent without
an analogue of tyrosine and 1,2,3,4-tetrahydroisoquinoline-3- physicochemical data16); and 2ꢀ,4ꢀ,6ꢀ-trimethyl-D,L-phenyl-
carboxylic acid (Tic) as a conformationally constrained alanine was obtained by alkylation of diethyl acetamidoma-
phenylalanine analogue, play critical roles in the develop- lonate with the corresponding alkyl halide, however the
ment of unique ligands for the investigation of structure–ac- racemic mix failed to be optically resolved by enzymatic
tivity relationships of d- and m-opioid ligands.1—3) Similarly, treatment.13,18) Although several methods have been devel-
opioid studies indicated that 2ꢀ,6ꢀ-dimethyl-L-phenylalanine oped for preparing optically pure amino acids, the asymmet-
(Dmp) is an effective surrogate of phenylalanine in ric catalytic hydrogenation is still a very convenient method
enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH),8) dermorphin (H- due to the commercially availability of chiral catalysts and
Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), deltorphin II (H-Tyr- simple starting materials. In our report, the phenylalanine
D-Ala-Phe-Glu-Val-Val-Gly-NH2),9) YrFB (H-Tyr-D-Arg- ana-logues were synthesized by the method described by
Phe-bAla-NH2),10) and endomorphin-2 (H-Tyr-Pro-Phe-Phe- Dygos et al. for synthesis of Dmt through [Rh(1,5-
NH2).11) More interestingly, substitution of Tyr1 by Dmp in COD)(R,R-DIPAMP)]BF4 mediated asymmetric catalytic hy-
deltorphin II and enkephalin, yielded analogues which were drogenation of acetamidoacrylate (Chart 1).19) During the
nearly as effective as the parent peptides.12) In order to study synthesis, Z-configuration of acetamidoacrylate after Heck
the structure–activity relationships of this unnatural amino reaction and L-configuration of the final phenylalanine ana-
acid, phenylalanine analogues bearing different substituent(s) logues were confirmed by X-ray analysis.
on the aromatic ring were required. These analogues consist
According to the Chart 1, L-phenylalanine analogues were
of 2ꢀ-methyl- (Mmp), 2ꢀ,6ꢀ-dimethyl- (Dmp), 2ꢀ-ethyl-6ꢀ- prepared. The starting materials, 2-iodotoluene (1a; Sigma-
Reagents and conditions: a) methyl acetamidoacrylate, Pd(OAc)2, tri-o-tolylphosphine, Et3N, CH3CN, reflux 10 h; b) H2 (60 psig), [Rh(1,5-COD)(R,R-DIPAMP)]BF4, 60 °C,
12 h; c) 12 mol/l HCl conducted under reflux conditions for 6 h.
Chart 1. Synthetic Procedure for Phenylalanine Analogues
∗ To whom correspondence should be addressed. e-mail: okada@pharm.kobegakuin.ac.jp
© 2006 Pharmaceutical Society of Japan

874
Vol. 54, No. 6
Aldrich Co., St. Louis, U.S.A.), 2,6-dimethylbromobenzene precipitated out from the homogeneous solution under reflux
(1b; Wako Pure Chemical Industries, Ltd., Osaka, Japan), conditions (60 min). The dehydroamino acids were then
and 3,5-dimethyliodobenzene (1f; Tokyo Kasei Kogyo Co., reduced with [Rh(1,5-COD)(R,R-DIPAMP)]BF4 mediated
Ltd., Tokyo, Japan) are commercially available. 2-Ethyl-6- asymmetric catalytic hydrogenation to give the protected
methyliodobenzene (1c), 2-isopropyl-6-methyliodobenzene amino acids 3a—f. The protecting groups of 3a—f were re-
(1d), and 2,4,6-trimethyliodobenzene (1e) were prepared moved by concentrated HCl to yield the final amino acid hy-
from 2-ethyl-6-methylaniline, 2-isopropyl-6-methylaniline, drochloride salts 4a—f. Racemic phenylalanine analogues
and 2,4,6-trimethylaniline, respectively, by diazotization of were also prepared through the reduction of dehydroamino
the aniline followed by replacement with iodide. [Rh(1,5- acids 2a—f with Pd/C catalyzed hydrogenation followed by
COD)(R,R-DIPAMP)]BF4 was purchased from Strem Chem- deprotection with concentrated HCl. The D and L configura-
icals (U.S.A.).
tions of the phenylalanine analogues were determined by di-
The Heck reaction was employed to produce (Z)-dehy- gestion of the racemic phenylalanine analogues using L-
droamino acid derivatives (2a—f).19) In this reaction, more amino acid oxidase according to a method described by Toth
than 95% Z-isomer over the corresponding E-isomer was et al.22) After digestion of each phenylalanine analogue, the
constructed through a complex between substrate and cata- isomer which exhibited longer retention time in a Chiralpak
lyst. Addition of hexane to the condensed reaction mixture WH column disappeared, and the one with shorter retention
precipitated the desired pure Z-isomer. In order to establish time remained intact. This result demonstrated that the iso-
the stereochemistry about the olefinic bond of 2a—f, the pro- mer with the longer retention time was the L-configuration,
ton coupled 13C-NMR spectra were examined. The magni- and the other was the D-antipode. The retention times and the
tude of the long range coupling constants (JCH) between the enantiomeric excesses of 3a—f determined by Chiralcel OD-
vinyl proton and the carbonyl carbon on the acrylic ester H column, as well as the retention times and the enan-
moiety of 2a—f ranging from 3.8—4.3 Hz20) are consistent tiomeric excesses of 4a—f determined by Chiralpak WH col-
with the desired Z-configuration.19) The Z-configuration of 2c umn are summarized in Tables 1 and 2, respectively; the L-
was also confirmed by X-ray analysis (Fig. 1). The Heck re- form of 4c was also confirmed by X-ray analysis. As shown
action of 1a and 1c—f gave dehydroamino acids 2a and 2c— in Tables 1 and 2, [Rh(1,5-COD)(R,R-DIPAMP)] is an excel-
f, respectively, with over 60% yields. It was reported that aryl lent catalyst for preparing 4b (Dmp), 4c (Emp), 4d (Imp),
bromide is a good substrate in the Heck reaction,21) but in and 4e (Tmp), which bear alkyl groups at the 2ꢀ and 6ꢀ posi-
this experimental protocol, the Heck reaction with 2,6-di- tions, while it is moderately effective for 4a (Mmp), with a
methylbromobenzene (1b) only gave 2b in low yield (38%), single alkyl group at the 2ꢀ or 6ꢀ position, and poor for the
probably due to the rapid deactivation of the catalyst which synthesis of 4f (3,5Dmp), with its absence of alkyl groups
at the 2ꢀ and 6ꢀ positions. Studies indicated that the re
face of (Z)-a-acylaminocinnamate interacts with [Rh(1,5-
COD)(R,R-DIPAMP)]BF4 producing a L-configuration prod-
Table 1. Retention Times and the Enantiomric Excesses of Compounds
3a—fa,b)
Yield
(%)
tR (D-isomer, tR (L-isomer,
ee
(%)
Compd.
min)
min)
3a Mmp
3b Dmp
3c Emp
3d Imp
3e Tmp
3f 3,5Dmp
97.5
94.0
94.6
93.7
95.7
87.8
10.96
9.97
10.49
8.74
12.86
12.19
11.27
9.49
11.39
ND
89.1
100
100
98.3
100
ND
9.95
NDc)
a) Enantiomeric excesses were determined with Chiralcel OD-H column
(4.6 mmꢁ250 mm), and the products were eluted with hexane : i-PrOHꢂ4 : 1 contain-
ing 0.1% TFA at a flow rate of 0.5 ml/min. b) The main peaks which have longer re-
tention time are tentatively assigned as L-isomers. c) DL-3f cannot be separated by
this condition.
Fig. 1. X-Ray Structure of Molecule 2c
The nitrogen and oxygen atoms are shown with blue and red colors, respectively.
Table 2. Retention Times and Enantiomeric Excesses of the Analogues 4a—fa)
Before recrystallization
After recrystallization
Compd.
tR (D-isomer, min)
tR (L-isomer, min)
Yield (%)
ee (%)
Yield (%)b)
ee (%)
96.6
4a Mmp
4b Dmp
4c Emp
4d Imp
16.73
21.37
23.30
29.03
23.95
18.51
25.03
25.48
28.21
32.10
29.84
57.40
100
95.2
87.6
88.5
94.6
90.7
87.3
97.0
95.0
ꢃ99
ꢃ99
76.4
79.0
86.2
ꢃ99
4e Tmp
4f 3,5Dmp
35.7c)
90.0
a) Enantiomeric excesses were determined with Chiralpak WH column (4.6 mmꢁ250 mm), and the products were eluted with H2O : MeOHꢂ4 : 1 containing 1 mmol/l CuSO4
at 50 °C at a flow rate of 1.5 ml/min. b) Refers to the recovery of recrystallized product. c) Refers to the sum of two batches of crystals that have ee over 90.0%.

June 2006
875
2-Isopropyl-6-methyliodobenzene (1d) Title compound was prepared
from 2-isopropyl-6-methylaniline (14.9 g, 110 mmol) using the same method
described for the synthesis of 2-ethyl-6-methyliodobenzene (1c). Yield
7.90 g (27.6%), yellowish oil, Rf1ꢂ0.77. Anal. Calcd for C10H13I: C, 46.2; H,
uct, in contrast, when its si face interacts with the catalyst a
D-configurational product results.23) A molecular model of
the catalyst23) shows that the edge-exposed phenyl ring of the
catalyst prevents the substrate from closely approaching the
1
5.04; I, 48.8. Found: C, 46.1; H, 4.80; I, 49.0. H-NMR (CDCl3, 400 MHz)
metal with its si face, such that any group which increases d: 1.23 (6H, d, Jꢂ6.8 Hz), 2.49 (3H, s), 3.37 (1H, hept, Jꢂ6.8 Hz), 7.00—
7.20 (3H, m).
the steric hindrance in the phenyl ring of the catalyst or in the
phenyl ring in the substrate will increase enantiomeric selec-
tivity. From these results, we can deduce that the alkyl
2,4,6-Trimethyliodobenzene (1e) Title compound was prepared from
2,4,6-trimethylaniline (14.9 g, 110 mmol) using the same method described
for the synthesis of 2-ethyl-6-methyliodobenzene (1c). Yield 15.2 g (56.3%),
groups at the 2ꢀ and 6ꢀ positions (4b—e) are more effective
in improving enantioselectivity than only one group at the 2ꢀ
or 6ꢀ position (4a), or two groups at the 3ꢀ and 5ꢀ positions
(4f). Comparing with the ee data in the Tables 1 and 2, we
find that partial racemization occurred in 4a—c, and with
high probability in 4f, during the deprotection process by
using concentrated HCl, which was also observed in the dea-
cylation for other amino acids.24)
In summary, we synthesized a phenylalanine library with
various alkyl groups on the aromatic ring through enantiose-
lective catalytic hydrogenation reaction to yield high enan-
tiomeric purity. Following the Heck reaction, (Z)-dehy-
droamino acids were obtained and their stereostructures sup-
ported by NMR studies and X-ray analysis. L-Configuration
of the final phenylalanine derivatives was confirmed by enzy-
colorless oil, Rf1ꢂ0.77. Anal. Calcd for C9H11I: C, 43.9; H, 4.51; I, 51.6.
Found: C, 44.0; H, 4.40; I, 51.8. H-NMR (CDCl3, 400 MHz) d: 2.23 (3H,
s), 2.42 (6H, s), 6.87 (2H, s).
1
General Procedure for Synthesis of Methyl (Z)-2-Acetamido-3-
(alkylphenyl)-2-propenoate (2a—f)
A mixture of aryl halide (34.9
mmol), methyl-2-acetamidoacrylate (34.9 mmol), tri-o-tolylphosphine (1.85
mmol), triethylamine (69.9 mmol) and Pd(OAc)2 (0.65 mmol) in CH3CN
(50 ml) was subjected to reflux conditions for 10 h. The mixture was cooled
to room temperature and filtered. The solvent was removed in vacuo and the
residue was diluted with water (50 ml). The aqueous phase was extracted
with AcOEt (100 mlꢁ3). The combined organic phase was washed with sat-
urated NaCl solution (50 mlꢁ3), dried (Na2SO4) and treated with Florisil®
(10 g) overnight.
Methyl
(Z)-2-Acetamido-3-(2ꢀ-methylphenyl)-2-propenoate
(2a)
Starting from 2-methyliodobenzene (7.62 g, 34.9 mmol), after the reaction,
workup, and removal of Florisil® by filtration, the filtrate was concentrated
to about 60 ml. To this solution, hexane (80 ml) was added. The crystals ap-
peared were collected by filtration and dried in vacuo, yield 5.35 g. Another
matic digestion and X-ray analysis. The study on the struc- 770 mg of the product was obtained from the filtrate by flash chromatogra-
phy (SiO2, AcOEt). Total yield 6.12 g (75.0%), mp 129—130 °C, Rf2ꢂ0.44.
ture–activity relationship of these phenylalnine analogues by
incorporation of these unnatural amino acids to the third po-
sition of endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) is under
progress.
Anal. Calcd for C13H15NO3: C, 66.9; H, 6.48; N, 6.00. Found: C, 67.0; H,
6.51; N, 5.96. 1H-NMR (500 MHz, CDCl3) d: 2.03 (3H, s), 2.33 (3H, s),
3.86 (3H, s), 6.85 (1H, s), 7.13—7.40 (5H, m). 13C-NMR (125 MHz, CDCl3)
d: 19.9 (2ꢀ-CH3), 23.0 (2-NHCOCH3), 52.6 (1-OCH3), 125.7 (4ꢀ-C), 125.9
(2-C), 127.8 (6ꢀ-C), 128.9 (5ꢀ-C), 129.9 (3-C), 130.3 (3ꢀ-C), 132.9 (1ꢀ-C),
137.2 (2ꢀ-C), 165.5 (2-NHCO), 168.8 (1-CO).
Experimental
Melting points were determined on a Yanagimoto micromelting point ap-
paratus and are uncorrected. TLC was performed on precoated plates of sil-
ica gel F254 (Merk, Darmstadt, Germany). Rf1, Rf2, Rf3, and Rf4 values refer
to hexane, AcOEt : hexane (2 : 1), n-BnOH : AcOH : Py : H2O (4 : 1 : 1 : 2),
Methyl (Z)-2-Acetamido-3-(2ꢀ,6ꢀ-dimethylphenyl)-2-propenoate (2b)
Starting from 2,6-dimethylbromobenzene (15.5 g, 84.0 mmol), after the re-
action, workup, and removal of Florisil® by filtration, the filtrate was con-
centrated to about 60 ml. To this solution, hexane (80 ml) was added. The
and n-BnOH : AcOH : H2O (4 : 1 : 5, upper layer), respectively. [a]D values crystals appeared were collected by filtration and dried in vacuo, yield
were measured with a DIP-1000 automatic polarimeter (Japan Spectro- 5.08 g. Another 1.50 g of the product was obtained from the filtrate by flash
scopic Co.) and are given in 10ꢄ1 deg cm2 gꢄ1. Analytical RP-HPLC was chromatography (SiO2, AcOEt : hexaneꢂ1 : 1). Total yield 6.58 g (38.0%),
performed with a Waters Delta 600 with a COSMOSIL C18 column
(4.6 mmꢁ250 mm). The solvents for analytical HPLC are: A, 0.05% TFA in N, 5.66. Found: C, 68.3; H, 6.94; N, 5.61. H-NMR (500 MHz, CDCl3) d:
water; B, 0.05% TFA in CH3CN. The column was eluted at a flow rate of 1.92 (3H, s), 2.21 (6H, s), 3.88 (3H, s), 6.62 (1H, br), 7.05 (1H, s), 7.07 (1H,
1 ml/min with a linear gradient: 90% A to 10% A in 30 min; the retention
time was reported as tR (min). The enantiomeric ratio was determined by 22.8 (2-NHCOCH3), 52.5 (1-OCH3), 127.5 (3ꢀ,5ꢀ-C), 128.0 (4ꢀ-C), 128.3 (2-
mp 131—132 °C, Rf2ꢂ0.52. Anal. Calcd for C14H17NO3: C, 68.0; H, 6.93;
1
s), 7.12—7.18 (2H, m). 13C-NMR (125 MHz, CDCl3) d: 20.1 (2ꢀ,6ꢀ-CH3),
using
a
Chiralcel OD-H column (Daisel Chemical Industries, Ltd.,
C), 128.5 (3-C), 132.2 (1ꢀ-C), 136.0 (2ꢀ,6ꢀ-C), 164.8 (1-CO), 168.3 (2-
NHCO).
Methyl (Z)-2-Acetamido-3-(2ꢀ-ethyl-6ꢀ-methylphenyl)-2-propenoate
(2c) Starting from 2-ethyl-6-methyliodobenzene (8.60 g, 34.9 mmol), after
4.6 mmꢁ250 mm) or a Chiralpak WH column (Daisel Chemical Industries,
Ltd., 4.6 mmꢁ250 mm). H- (400 MHz) and 13C- (100 MHz) NMR spectra
1
except 2a—f were recorded on a Bruker DPX-400 spectrometer at 25 °C.
For 2a—f, H- (500 MHz) and 13C- (125 MHz) NMR spectra were recorded the reaction, workup, and removal of Florisil® by filtration, the filtrate was
on a Varian unity INOVA500 spectrometer at 25 °C. Chemical shift values concentrated to about 50 ml. To this solution, hexane (50 ml) was added. The
are expressed as ppm downfield from tetramethylsilane. Elemental analyses crystals were collected by filtration and dried in vacuo, yield 6.27 g. Filtrate
were performed at the Laboratory for Organic Elemental Microanalysis, was concentrated to about 20 ml, and hexane (20 ml) was added, and crystals
1
Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
2-Ethyl-6-methyliodobenzene (1c) To suspension of 2-ethyl-6- 148 °C, Rf2ꢂ0.56. Anal. Calcd for C15H19NO3: C, 68.9; H, 7.33; N, 5.36.
methylaniline (14.9 g, 110 mmol), in conc. HCl (50 ml) and 30 g of ice, a so-
(500 mg) were collected by filtration. Total yield 6.77 g (74.1%), mp 147—
a
1
Found: C, 68.8; H, 7.34; N, 5.41. H-NMR (500 MHz, CDCl3) d: 1.15 (3H,
lution of NaNO2 (7.95 g, 115 mmol) in H2O (35 ml) was added dropwise t, Jꢂ7.6 Hz), 1.95 (3H, s), 2.20 (3H, s), 2.53 (2H, q, Jꢂ7.6 Hz), 3.88 (3H,
over 30 min at 0 to 5 °C. The solution was stirred for another 30 min at the
same temperature. Then, a solution of KI (24.9 g, 150 mmol) in H2O (35 ml)
s), 6.49 (1H, s), 7.00—7.20 (4H, m). 13C-NMR (125 MHz, CDCl3) d: 14.9
(2ꢀ-CH2CH3), 20.1 (6ꢀ-CH3), 22.8 (2-NHCOCH3), 26.5 (2ꢀ-CH2CH3), 52.6
was added dropwise over 20 min at 0 to 5 °C. The reaction mixture was al- (1-OCH3), 125.7 (3ꢀ-C), 127.7 (5ꢀ-C), 127.8 (3-C), 128.3 (4ꢀ-C), 128.8 (2-
lowed to warm to room temperature during which time an exothermic reac- C), 131.4 (1ꢀ-C), 136.0 (6ꢀ-C), 142.2 (2ꢀ-C), 164.8 (1-CO), 168.5 (2-
tion occurred with gas evolution. The resulting red colored solution was
stirred for 18 h. The mixture was extracted with ethyl acetate (AcOEt,
NHCO).
Methyl (Z)-2-Acetamido-3-(2ꢀ-isopropyl-6ꢀ-methylphenyl)-2-prope-
100 mlꢁ3). The combined extracts were washed with 15% sodium thiosul- noate (2d) Starting from 2-isopropyl-6-methyliodobenzene (7.80 g,
fate solution (80 mlꢁ2), brine solution and dried over MgSO4. After removal 30.0 mmol), after the reaction, workup, and removal of Florisil® by filtration,
of MgSO4, the solvent was removed under vacuum. The colored residue was the filtrate was concentrated to about 30 ml. To this solution, hexane (70 ml)
purified by silica gel flash chromatography to give pure 2-ethyl-6- was added. The crystals appeared were collected by filtration and dried in
methyliodobenzene (1c). Yield 14.0 g (51.8%), colorless oil, Rf1ꢂ0.77. vacuo. Yield 5.60 g (67.9%), mp 149—150 °C, Rf2ꢂ0.62. Anal. Calcd for
Anal. Calcd for C9H11I: C, 43.9; H, 4.51; I, 51.6. Found: C, 44.1; H, 4.43; I, C16H21NO3: C, 69.8; H, 7.69; N, 5.09. Found: C, 69.8; H, 7.64; N, 5.19. 1H-
1
51.5. H-NMR (CDCl3, 400 MHz) d: 1.23 (3H, t, Jꢂ7.5 Hz), 2.49 (3H, s),
2.81 (2H, q, Jꢂ7.5 Hz), 7.05—7.10 (2H, m), 7.15 (1H, t, Jꢂ7.4 Hz).
NMR (500 MHz, CDCl3) d: 1.16 (6H, d, Jꢂ6.8 Hz), 1.95 (3H, s), 2.20 (3H,
s), 2.95 (1H, hept, Jꢂ6.8 Hz), 3.88 (3H, s), 6.44 (1H, br), 7.05—7.26 (4H,

876
Vol. 54, No. 6
m). 13C-NMR (125 MHz, CDCl3) d: 20.2 (6ꢀ-CH3), 22.8 (2-NHCOCH3), 2.81 g (87.8%), mp 72—74 °C, [a]D25 ꢅ15.7° (cꢂ0.54, MeOH), Rf2ꢂ0.36.
23.7 (2ꢀ-CH-(CH3)2), 30.3 (2ꢀ-CH-(CH3)2), 52.6 (1-OCH3), 122.8 (3ꢀ-C),
127.6 (5ꢀ-C), 127.8 (3-C), 128.5 (4ꢀ-C), 129.0 (2-C), 130.6 (1ꢀ-C), 135.8
(6ꢀ-C), 146.9 (2ꢀ-C), 164.7 (1-C), 168.3 (2-NHCO).
Anal. Calcd for C14H19NO3: C, 67.5; H, 7.68; N, 5.62. Found: C, 67.3; H,
7.69; N, 5.54. 1H-NMR (400 MHz, CDCl3) d: 1.99 (3H, s), 2.27 (6H, s),
3.00 (1H, dd, Jꢂ13.8, 5.9 Hz), 3.06 (1H, dd, Jꢂ13.8, 5.9 Hz), 3.73 (s, 3H),
Methyl (Z)-2-Acetamido-3-(2ꢀ,4ꢀ,6ꢀ-trimethylphenyl)-2-propenoate 4.84 (1H, q, Jꢂ7.8 Hz), 5.87 (1H, d, Jꢂ7.2 Hz), 6.70 (2H, s), 6.88 (1H, s).
(2e) Starting from 2,4,6-trimethyliodobenzene (8.60 g, 34.9 mmol), after 2ꢀ-Methyl-L-phenylalanine Hydrochloride (4a) N-Acetyl-2-methyl-L-
the reaction, workup, and removal of Florisil® by filtration, the filtrate was phenylalanine methyl ester (3a, 4.87 g, 20.7 mmol) in concentrated HCl
concentrated to about 60 ml. To this solution, hexane (50 ml) was added. The (27.6 ml, 331 mmol) was subjected to reflux conditions for 6 h. After the
crystals were collected by filtration and dried in vacuo, yield 4.59 g. Another temperature of the solution was automatically decreased to room tempera-
1.90 g of the product was obtained from the filtrate by flash chromatography ture, the solution became a solid mass. The solid was dried directly in vacuo,
(SiO2, AcOEt : hexaneꢂ2 : 1). Total yield 6.45 g (70.6%), mp 165—166 °C, yield 4.84 g (100%): ee 87.3%. The optically crude sample (4.00 g) was dis-
Rf2ꢂ0.57. Anal. Calcd for C15H19NO3: C, 68.9; H, 7.33; N, 5.36. Found: C, solved in MeOH (10 ml), Et2O (35 ml) was added to precipitate the product.
69.0; H, 7.26; N, 5.35. 1H-NMR (500 MHz, CDCl3) d: 1.95 (3H, s), 2.17 After 1 h, the crystals were collected by filtration and dried in vacuo. Yield
(6H, s), 2.28 (3H, s), 3.87 (3H, s), 6.57 (1H, s), 6.89 (2H, s), 7.13 (1H, br).
13C-NMR (125 MHz, CDCl3) d: 20.0 (2ꢀ,6ꢀ-CH3), 20.9 (4ꢀ-CH3), 22.8 (2-
NHCOCH3), 52.5 (1-OCH3), 128.2 (2-C), 128.4 (3ꢀ,5ꢀ-C), 128.6 (3-C),
129.2 (1ꢀ-C), 135.9 (2ꢀ,6ꢀ-C), 137.7 (4ꢀ-C), 164.9 (1-C), 168.2 (2-NHCO).
3.16 g (79.0%), mp 226—227 °C, [a]D25ꢄ3.63° (cꢂ1.0, H2O), ee 96.6%,
Rf3ꢂ0.54, Rf4ꢂ0.29, tRꢂ12.45 min. Anal. Calcd for C10H13NO2·HCl: C,
55.7; H, 6.54; N, 6.49. Found: C, 55.5; H, 6.40; N, 6.45. 1H-NMR
(400 MHz, DMSO-d6) d: 2.30 (3H, s), 3.03—3.20 (2H, m), 3.99 (1H, br),
Methyl (Z)-2-Acetamido-3-(3ꢀ,5ꢀ-dimethylphenyl)-2-propenoate (2f) 7.10—7.23 (4H, m), 8.57 (3H, br), 13.73 (1H, br).
Starting from 3,5-dimethyliodobenzene (5.00 g, 21.6 mmol), after the reac-
tion, workup, and removal of Florisil® by filtration, the filtrate was concen-
trated to about 50 ml. To this solution, hexane (50 ml) was added. The crys-
2ꢀ,6ꢀ-Dimethyl-L-phenylalanine Hydrochloride (4b) N-Acetyl-2,6-di-
methyl-L-phenylalanine methyl ester (3b, 5.50 g, 22.0 mmol) in concentrated
HCl (30 ml, 360 mmol) was subjected to reflux conditions for 6 h. After
tals appeared were collected by filtration and dried in vacuo, yield 2.96 g. cooling to room temperature, the solvent was removed in vacuo to afford 4b
Another 370 mg of the product was obtained from the filtrate by flash chro-
matography (SiO2, EtOAc : hexaneꢂ2 : 1). Total yield 3.30 g (62.1%), mp
as a white solid. Yield 5.19 g (95.2%), mp 251—253 °C, [a]D25 ꢅ59.7°
(cꢂ0.5, MeOH), ee 97.0%, Rf3ꢂ0.45, Rf4ꢂ0.29, tRꢂ12.78 min. Anal. Calcd
139—139 °C, Rf2ꢂ0.45. Anal. Calcd for C14H17NO3: C, 68.0; H, 6.93; N, for C11H15NO21·HCl·0.5H2O: C, 55.4; H, 7.18; N, 5.87. Found: C, 55.2; H,
5.66. Found: C, 67.9; H, 6.94; N, 5.68. 1H-NMR (500 MHz, CDCl3) d: 2.13 7.14; N, 5.84. H-NMR (400 MHz, CD3OD) d: 2.36 (6H, s), 3.17 (1H, dd,
(3H, s), 2.31 (6H, s), 3.84 (3H, s), 6.88 (1H, s), 6.96 (1H, s), 7.08 (2H, s), Jꢂ14.4, 7.8 Hz), 3.39 (1H, dd, Jꢂ14.4, 8.2 Hz), 4.07 (1H, t, Jꢂ8.0 Hz),
7.31 (1H, s). 13C-NMR (125 MHz, CDCl3) d: 21.2 (2-NHCOCH3), 23.2 7.00—7.10 (3H, m).
(3ꢀ,5ꢀ-CH3), 52.5 (1-OCH3), 124..13, 133.44 (2-C, 1ꢀ-C), 127.4 (2ꢀ,6ꢀ-C),
131.2 (4ꢀ-C), 132.8 (3-C), 138.0 (3ꢀ,5ꢀ-C), 165.8 (1-CO), 168.9 (2-NHCO).
2ꢀ-Ethyl-6ꢀ-methyl-L-phenylalanine Hydrochloride (4c) N-Acetyl-2-
methyl-6-ethyl-L-phenylalanine methyl ester (3c, 5.60 g, 21.3 mmol) in con-
General Procedure for Synthesis of N-Acetylalkylphenylalanine centrated HCl (28 ml, 340 mmol) was subjected to reflux conditions for 6 h.
Methyl Ester (3a—f) An N2-purged reaction vessel was charged with a The temperature of the solution was automatically decreased to room tem-
slurry of [Rh(1,5-COD)(R,R-DIPAMP)]BF4 (0.46 mmol), methyl (Z)-2-ac- perature, and the crystals were collected by filtration and dried in vacuo,
etamido-3-(alkylphenyl)-2-propenoate (25.7 mmol) and AcOEt (60 ml). The yield 4.88 g (87.6%), ee 95.0%. The optically crude product (4.70 g) was re-
vessel was repeatedly aspirated to boiling of the AcOEt and pressurized to crystallized from 95 ml of hot hydrochloric acid solution (5 mol/l) to give
10 psig with H2 (five times). The last reaction mixture was pressurized to 60 optically pure 4c 4.05 g (86.2%), mp 233—235 °C, [a]D25 ꢅ62.5° (cꢂ1.0,
psig with H2 and stirred vigorously at 60 °C for 12 h. The mixture was
H2O), ee ꢃ99%, Rf3ꢂ0.57, Rf4ꢂ0.35, tRꢂ12.89 min. Anal. Calcd for
cooled to room temperature, vented with N2, filtered, and the filtrate was C12H17NO2·HCl·0.1H2O: C, 58.7; H, 7.47; N, 5.70. Found: C, 58.6; H, 7.40;
treated with Florisil® (12 g) overnight. The mixture was filtered, and the fil- N, 5.70. 1H-NMR (400 MHz, DMSO-d6) d: 1.12 (3H, t, Jꢂ7.5 Hz), 2.29 (3H
trate was concentrated, and the product was precipitated with hexane.
N-Acetyl-2ꢀ-methyl-L-phenylalanine Methyl Ester (3a) Yield 5.85 g
(97.5%), oil, [a]D25 ꢄ3.06° (cꢂ0.53, MeOH), Rf2ꢂ0.32. Anal. Calcd for
C13H17NO3: C, 66.4; H, 7.28; N, 5.95. Found: C, 66.5; H, 7.31; N, 6.24. 1H-
s,), 2.55—2.68 (2H, m), 3.12—3.25 (2H, m), 3.79 (1H, dd, Jꢂ9.9, 6.2 Hz),
6.95—7.03 (2H, m), 7.06—7.11 (1H, m), 8.74 (3H, br), 13.49 (1H, br).
2-Isopropyl-6-methyl-L-phenylalanine Hydrochloride (4d) N-Acetyl-
2-isopropyl-6-methyl-L-phenylalanine methyl ester (3d, 4.41 g, 15.9 mmol)
NMR (400 MHz, CDCl3) d: 1.96 (3H, s), 2.24 (3H, s), 3.04 (1H, dd, in concentrated HCl (21 ml, 252 mmol) was subjected to reflux conditions
Jꢂ14.1, 6.9 Hz), 3.15 (1H, dd, Jꢂ14.1, 6.8 Hz), 3.68 (3H, s), 4.86 (1H, q, for 6 h. The temperature of the solution was automatically decreased to room
Jꢂ8.0 Hz), 5.91 (1H, d, Jꢂ7.3 Hz), 6.97—7.04 (1H, m), 7.10—7.20 (3H, temperature, and the crystals were collected by filtration and dried in vacuo.
m).
Yield 3.88 g (88.5%), mp 192—194 °C, [a]D25 ꢅ56.7° (cꢂ0.94, H2O), ee
N-Acetyl-2ꢀ,6ꢀ-dimethyl-L-phenylalanine Methyl Ester (3b) Yield
ꢃ99%, Rf3ꢂ0.58, Rf4ꢂ0.39, tRꢂ14.00 min. Anal. Calcd for C13H19NO2·
5.62 g (94.0%), mp 127—128 °C, [a]D25 ꢅ4.09° (cꢂ0.55, MeOH), Rf2ꢂ0.43. HCl·H2O: C, 56.6; H, 8.04; N, 5.08. Found: C, 56.4; H, 7.89; N, 5.08. H-
1
Anal. Calcd for C14H19NO3: C, 67.5; H, 7.68; N, 5.62. Found: C, 67.2; H, NMR (400 MHz, DMSO-d6) d: 1.15 (6H, t, Jꢂ6.9 Hz), 2.29 (3H, s), 3.10—
7.54; N, 5.63. 1H-NMR (400 MHz, CDCl3) d: 1.96 (3H, s), 2.34 (6H, s),
3.08 (1H, dd, Jꢂ14.0, 8.2 Hz), 3.13 (1H, dd, Jꢂ14.0, 7.7 Hz), 3.61 (3H, s),
4.82 (1H, q, Jꢂ8.0 Hz), 6.05 (1H, d, Jꢂ7.4 Hz), 6.90—7.10 (3H, m).
N-Acetyl-2ꢀ-ethyl-6ꢀ-methyl-L-phenylalanine Methyl Ester (3c) Yield
3.27 (3H, m), 3.70—3.80 (1H, m), 6.94—7.00 (1H, m), 7.09—7.15 (2H, m),
8.76 (3H, br), 13.42 (1H, br).
2ꢀ,4ꢀ,6ꢀ-Trimethyl-L-phenylalanine Hydrochloride (4e) N-Acetyl-
2,4,6-trimethyl-L-phenylalanine methyl ester (3e, 4.66 g, 17.7 mmol) in con-
5.68 g (94.6%), mp 94—95 °C, [a]D25 ꢅ8.37° (cꢂ0.57, MeOH), Rf2ꢂ0.44. centrated HCl (23.6 ml, 283 mmol) was heated and subjected to reflux condi-
Anal. Calcd for C15H21NO3: C, 68.4; H, 8.04; N, 5.32. Found: C, 68.2; H, tions. After 30 min, the solution turned into a gel, at which time HCl
1
8.07; N, 5.17. H-NMR (400 MHz, CDCl3) d: 1.20 (3H, t, Jꢂ7.6 Hz), 1.96
(3H, s), 2.35 (3H, s), 2.67 (2H, q, Jꢂ7.5 Hz), 3.08 (1H, dd, Jꢂ14.0, 7.6 Hz),
3.15 (1H, dd, Jꢂ14.0, 7.6 Hz), 3.60 (3H, s), 4.79 (1H, q, Jꢂ7.9 Hz), 6.02
(1H, d, Jꢂ7.2 Hz), 6.98—7.12 (3H, m).
(6 mol/l, 50 ml) was added, and reflux conditions were continued for total
6 h. The temperature of the solution was automatically decreased to room
temperature, and the resulting crystals were collected by filtration and dried
in vacuo. Yield 4.38 g (94.6%), mp 270—275 °C (dec.), [a]D25 ꢅ64.6°
(cꢂ0.50, MeOH), ee ꢃ99%, Rf3ꢂ0.56, Rf4ꢂ0.35, tRꢂ13.24 min. Anal.
Calcd for C12H17NO2·HCl·0.5H2O: C, 57.0; H, 7.58; N, 5.54. Found: C,
N-Acetyl-2ꢀ-isopropyl-6ꢀ-methyl-L-phenylalanine Methyl Ester (3d)
Yield 5.00 g (93.7%), mp 97—98 °C, [a]D25 ꢅ3.96° (cꢂ0.55, MeOH),
Rf2ꢂ0.46. Anal. Calcd for C16H23NO3: C, 69.3; H, 8.36; N, 5.05. Found: C, 57.0; H, 7.52; N, 5.57. 1H-NMR (400 MHz, DMSO-d6) d: 2.19 (3H, s), 2.24
68.9; H, 8.32; N, 4.97. 1H-NMR (400 MHz, CDCl3) d: 1.22 (3H, d, (6H, s), 3.06—3.17 (2H, m), 3.79 (1H, t, Jꢂ8.0 Hz), 6.81 (2H, s), 8.68 (3H,
Jꢂ7.0 Hz), 1.24 (3H, d, Jꢂ6.8 Hz), 1.96 (3H, s), 2.34 (3H, s), 3.07—3.28 br), 13.45 (1H, br).
(3H, m), 3.61 (3H, s), 4.77 (1H, q, Jꢂ8.0 Hz), 5.98 (1H, d, Jꢂ7.4 Hz),
6.93—7.00 (1H, m), 7.12—7.14 (2H, m).
3ꢀ,5ꢀ-Dimethyl-L-phenylalanine Hydrochloride (4f) N-Acetyl-3,5-di-
methyl-L-phenylanine methyl ester (3f, 2.71 g, 10.9 mmol) in concentrated
HCl (14.5 ml, 174 mmol) was heated under reflux conditions. After 1 h,
N-Acetyl-2ꢀ,4ꢀ,6ꢀ-trimethyl-L-phenylalanine Methyl Ester (3e) Yield
5.78 g (95.7%), mp 141—142 °C, [a]D25 ꢅ12.2° (cꢂ0.55, MeOH), Rf2ꢂ0.37. crystals precipitated, HCl (6 mol/l, 25 ml) was added, and the reflux condi-
Anal. Calcd for C15H21NO3: C, 68.4; H, 8.04; N, 5.32. Found: C, 68.0; H, tions were continued for total 6 h. The temperature of the solution was auto-
7.98; N, 5.20. 1H-NMR (400 MHz, CDCl3) d: 1.95 (3H, s), 2.24 (3H, s),
2.30 (6H, s), 3.00—3.12 (2H, m), 3.63 (3H, s), 4.78 (1H, q, Jꢂ7.9 Hz), 5.98
(1H, d, Jꢂ7.5 Hz), 6.83 (2H, s).
matically decreased to room temperature, and the resulting crystals were col-
lected by filtration and dried in vacuo, yield 2.44 g (90.7%), ee 76.4%. The
optically crude compound (2.38 g) was dissolved in MeOH (10 ml), the
product was precipitated with Et2O (10 ml), and the crystals formed were
N-Acetyl-3ꢀ,5ꢀ-dimethyl-L-phenylalanine Methyl Ester (3f) Yield

June 2006
877
collected by filtration and dried in vacuo, yield 740 mg, ee 90.0%. The fil-
trate was evaporated to dryness, and the residue was dissolved again in
MeOH (10 ml), and acetone (22 ml) was added. The solution was kept at
7 °C overnight. The crystals that appeared were collected by filtration and
dried in vacuo. Yield 110 mg, mp 212—214 °C, [a]D25 ꢄ13.5° (cꢂ0.48,
H2O), ee ꢃ99%, Rf3ꢂ0.57, Rf4ꢂ0.35, tRꢂ12.45 min. Anal. Calcd for
C11H15NO2·HCl·0.33H2O: C, 56.1; H, 7.13; N, 5.94. Found: C, 56.0; H,
6.97; N, 5.93. H-NMR (400 MHz, DMSO-d6) d: 2.24 (6H, s), 3.01—3.12
(2H, m), 4.08 (1H, t, Jꢂ6.2 Hz), 6.88 (2H, s), 6.90 (1H, s), 8.49 (3H, br),
13.73 (1H, br).
5) Okada Y., Fujita Y., Motoyama T., Tsuda Y., Yokoi T., Li T., Sasaki Y.,
Ambo A., Jinsmaa Y., Bryant S. D., Lazarus L. H., Bioorg. Med.
Chem., 11, 1983—1994 (2003).
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Med. Chem. Lett., 11, 327—329 (2001).
1
General Procedure for Enzymatic Digestion of Phenylalanine Ana-
logues Racemic phenylalanine analogues (0.3 mg) were dissolved in 1 ml
of Tris–HCl buffer (0.1 mol/l, pH 7.5), and 0.3 mg of L-amino acid oxidase
9) Ambo A., Murase H., Niizuma H., Ouchi H., Yamamoto Y., Sasaki Y.,
Bioorg. Med. Chem. Lett., 12, 879—881 (2002).
(Bothrops atrox crude dried venom, Sigma) was added. The solutions were 10) Ambo A., Niizuma H., Sasaki A., Kohara H., Sasaki Y., Bioorg. Med.
incubated for 24 h at 37 °C. After 24 h fresh enzyme was added and the incu- Chem. Lett., 13, 1269—1272 (2003).
bation continued for another 24 h. The digestion was stopped by addition of 11) Sasaki Y., Sasaki A., Niizuma H., Goto H., Ambo A., Bioorg. Med.
HCl (1 ml, 0.1 mol/l) and further denaturation of the enzyme by heating the Chem., 11, 675—678 (2003).
solution in boiling water for 3 min. The solutions were filtered, lyophilized, 12) Sasaki Y., Sasaki A., Ariizuma T., Igari Y., Sato K., Kohara H., Ni-
and the residue dissolved in water (300 ml) for chiral HPLC analysis. izuma H., Ambo A., Biol. Pharm. Bull., 27, 244—247 (2004).
X-Ray Structure Determination The X-ray diffraction data were col- 13) Porter J., Dykert J., Rivier J., Int. J. Peptide Protein Res., 30, 13—21
lected with a Bruker AXS SMART APEX CCD camera using graphite- (1987).
monochromated MoKa radiation (lꢂ0.71073 Å) at 120 K for 2c and 278 K 14) Soloshonok V. A., Tang X., Hruby V. J., Tetrahedron, 57, 6375—6382
for 4c. The crystal structures were solved by a direct method using the (2001).
SHELXS97 program.25) Atomic scattering factors were taken from Inter- 15) Ooi T., Kameda M., Maruoka K., J. Am. Chem. Soc., 125, 5139—5151
national Tables for X-Ray Crystallography.26) Positional parameters of
(2003).
non-H atoms were refined by a full matrix least squares method with 16) Ouchi H., Kumagai M., Sakurada S., Takahata H., Heterocycles, 64,
anisotropic thermal parameters using the SHELXL97 program.27) The struc-
tural data were deposited with the following designations: 2c: CCDC
288681, 4c: CCDC 288682. These can be obtained free of charge at
505—514 (2004).
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Acknowledgements This work was supported in part by a Grant-in-Aid 20) 2a, 2b, and 2e have JCHꢂ3.8 Hz, 2c and 2d have JCHꢂ4.3 Hz, JCH of 2f
for Japan Society for the Promotion of the Science (JSPS) Fellows cannot be determined.
(1503306) to T. L. and in part by the Intramural Research Program of the 21) Patel B. A., Ziegler C. B., Cortese N. A., Plevyak J. E., Zebovitz T. C.,
NIH, and NIEHS.
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References and Notes
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Salvadori S., Drug Disc Today, 3, 284—294 (1998).
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Biopolymers (Peptide Science), 71, 86—102 (2003).
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Y., Bryant S. D., Lazarus L. H., Okada Y., J. Med. Chem., 48, 586—
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Bull., 47, 1506—1509 (1999).
27) Sheldrick G. M., ‘SHELXL97. Program for the Refinement of Crystal
Structure,’ University of Gottingen, Germany, 1997.