Selective BH3-reduction of amide carbonyl groups of lithium salts of N- t-butoxycarbonyl (S)-O-benzyl tyrosyl (S)-proline and N, N'-ethylene-bridged dipeptides

Article abstract of DOI:10.1055/s-1998-2192

The selective reduction of the amide carbonyl group of the lithium salt of N-t-butoxycarbonyl (S)-O-benzyltyrosyl (S)-proline was carried out with 2 tool equivalents of BH₃ in tetrahydrofuran at r.t., providing a selectively reduced dipeptide i

Full text of DOI:10.1055/s-1998-2192

SYNTHESIS
November 1998
1623
Selective BH3-Reduction of Amide Carbonyl Groups of Lithium Salts of
N-t-Butoxycarbonyl (S)-O-Benzyl Tyrosyl (S)-Proline and N, N'-Ethylene-Bridged
Dipeptides
Kenichi Adachi, Eiji Tsuru, Eri Banjyo, Matsumi Doe, Kozo Shibata, Tetsushi Yamashita*
Department of Chemistry, Faculty of Science, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi-ku, Osaka 558, Japan.
Fax +81(6)6052522, E-mail: yamatetu@sci.osaka-cu.ac.jp
Received 5 February 1998; revised 20 April 1998
Abstract: The selective reduction of the amide carbonyl group of
HCl-DOX) for 24 h at r.t. in the presence of thioanisole as
a scavenger. Unexpectedly, it was found that the major
product (85 % yield) was a cyclic compound (piperazin-
2-one derivative 3), but not 2HCl-2 obtained by the re-
moval of the Boc-group of Boc-2. This fact suggests that
2HCl-2 was converted to 3 via the acid-catalyzed cyclisa-
tion as shown in our previous papers.4,5 The structure of 3,
recrystallized from benzene, was ascertained by IR, MS,
1H and 13C NMR measurements.
the lithium salt of N-t-butoxycarbonyl (S)-O-benzyltyrosyl (S)-
proline was carried out with 2 mol equivalents of BH3 in tetrahy-
drofuran at r.t., providing a selectively reduced dipeptide in 40 %
yield. Also, N-t-butoxycarbonyl N,N'-ethylene-bridged-dipep-
tides (piperazin-2-one derivatives) constructed from (S)-O-
benzyltyrosine, -methionine, -phenylalanine and -leucine were
similarly transformed into their corresponding piperazine deriva-
tives in 50–70 % yields according to the reductive method
described above.
Key words: piperazine and piperazin-2-one derivatives, lithium
Also, a β−casomorphin analog3 4 was obtained by the
coupling of Boc-2 with (S)-phenylalanine methyl ester
salts of dipeptides, selective BH3 reduction
Previously, Almquist et al.1 converted the amide carbonyl (DCC/HOBT method), followed by the removal4 of the
group of the Phe residue of the parent peptide (N-benzoyl- Boc- and O-benzyl groups in CF3COOH containing thio-
Phe-Gly-Pro-OH is an inhibitor of angiotensin converting anisole and successive hydrolysis in aq NaOH.
enzyme) to the methylene one, through several steps con-
taining the BH3 reduction of both amide and carboxyl
groups and the successive CrO3 oxidation of an alcoholic
hydoxyl group to a carboxyl group in order to enhance the
biological activity. Also, Roeske et al.2 attempted selec-
tive BH3 reduction of the amide carbonyl groups of N-pro-
tected dipeptide esters. In this work, we reduced, selec-
tively, the tertiary (t) amide carbonyl group of N-t-
butoxycarbonyl-(S)-O-benzyltyrosyl-(S)-proline (Boc-1)
obtained by the coupling of Boc-(S)-O-benzyltyrosine
with (S)-proline methyl ester by dicyclohexylcarbodiim-
ide (DCC)/1-hydroxybenzotriazole (HOBT) method, suc-
cessively by hydolysis. This selectively reduced dipeptide
can be used as a unit in the first and second positions of
β−casomorphin analogs3 known as exogenous opioid pep-
tides isolated from casein hydrolysates. When the methyl,
ethyl, isopropyl, isobutyl and t-butyl esters of Boc-1 were
reduced, respectively, with 2–3 mol equivalents of BH3 in
anhydrous tetrahydrofuran according to the procedure
similar to that of Roeske et al.2, the yields of a selectively
reduced dipeptide (Boc-2) were below 5 % yields. More-
over, the separation of Boc-2 from its byproducts by chro-
matography was not easy. On the other hand, we found
that the lithium salt of Boc-1 (Boc-1-OLi) is readily solu-
ble not only in water, but also in common organic solvents
such as THF, CHCl3, CH2Cl2, benzene. Therefore, the se-
lective reduction of the amide carbonyl group of Boc-1-
OLi was carried out with 2 mol equivalents of BH3 in an-
hydrous THF at r.t. for 24 h. Silica gel chromatography of
the resulting products was able to easily separate a desired
product (Boc-2) as an oily material in 40 % yield from
various byproducts. In order to obtain the analytical,
physical and spectroscopic data of Boc-2 more exactly, it
was treated with 4 M hydrochloric acid–dioxane (4 M
Scheme 1

SYNTHESIS
Papers
1624
Scheme 1 shows a synthetic route to 3 and 4 using Boc-1- from commercial α-amino acids only in two steps4,5 via
OLi as a starting material. On the other hand, we attempt- acid-catalyzed cyclization. These macrocycles included
ed the selective BH3 reduction of the secondary amide car- several metal ions8,10 and differentiated various enanti-
bonyl groups of N-t-butoxycarbonyl-(S)-tyrosylglycine or omers,9,10 effectively. Therefore, the piperazine deriva-
-(S)-tyrosyl-(R)-alanine lithium salts, which are the units tives (basic amino acids) obtained in this work can be used
in the first and second positions of opioid peptides as the units of new types of macrocycles whose function-
(enkephalin4 and dermorphin6).However,the lithium salts alities are expected to be superior to those of previous8-10
of these dipeptides are hardly soluble in THF, diethylene macrocycles.
glycol dimethyl ether (diglyme), etc., and only easily sol-
uble in water. The heterogeneous reduction of these lithi-
um salts with 2, 3, 10 and 15 mol equivalents of BH3 in
anhydrous THF or diglyme proceeded very slowly (over a
week), and afforded the desired products only in below
3% yields, accompanying various byproducts containing
amino alcohols. These amino alcohols6 are obtained by
the reduction of amide and carboxylic acid groups of the
parent dipeptides. Recently, we inserted a chiral pipera-
zine derivative7 obtained by selective BH3 reduction of the
tertiary amide carbonyl group of the lithium salt of a pip-
erazin-2-one derivative7 (N-benzyloxycarbonyl N, N'-eth-
ylene-bridged alanyl-phenylalanine) and the parent piper-
azin-2-one derivative7 as the units in the second and third
positions of shorter dermorphin analogs (tetrapeptides).
As a result, it was found in the guinea pig ileum and the
mouse vas deferens assays of these analogs that the re-
placement of the piperazin-2-one ring with the piperazine
ring influenced the activities of these analogs. The piper-
azine derivative7 with both amino and carboxyl groups
can be inserted in every position in the main chains of
peptides. Especially, this selective BH3 method is consid-
ered to be useful for the preparation of the other pipera-
zine derivatives containing α-amino acid residues such as
tyrosine and methionine with phenol and thioether groups,
which are oxidized easily and relatively unstable. There-
fore, we tried to reduce, selectively, the amide carbonyl
groups of the lithium salts of N-t-butoxycarbonyl N,N'-eth-
ylene-bridged (S)-O-benzyltyrosyl-(S)-O-benzyltyrosine
(Boc-5-OLi)5,7, -(S)-methionyl-(S)-methionine5 (Boc-6-
OLi), -(S)-phenylalanyl-(S)-phenylalanine4,7 (Boc-7-OLi)
and -(S)-leucyl-(S)-leucine4,5,8–10 (Boc-8-OLi) homoge-
neously with 3 mol equivalents of BH3 for 5 h in anhy-
drous THF at r.t. These piperazine derivatives thus ob-
tained were chromatographically separated out from vari-
ous byproducts, providing the purified Boc-5-sRed, -6-
sRed, -7-sRed and -8-sRed in 50, 70, 62 and 63 % yields,
respectively. Moreover, these oily materials were con-
verted, through the removal of the Boc-group in 4 M-HCl/
DOX, into the corresponding dihydrochloride salts
(2HCl•5-sRed, 6-sRed, 7-sRed and 8-sRed). The struc-
tures of 2HCl•5-sRed, 6-sRed, 7-sRed and 8-sRed recrys-
tallized from CH3CN/MeOH, isopropanol, MeOH/ether
and isopropanol, respectively, were established by IR, MS
Scheme 2
and 1H NMR measurements. Scheme 2 shows a route for
obtaining the dihydrochloride salts described above. Pre-
viously, the N,N'-ethylene-bridged dipeptides used as
starting materials for the piperazine derivatives described
here were utilized as the units of many kinds of macrocy-
clic pseudo-peptides8–10 because these dipeptides can be
obtained conveniently and economically in a large scale
All samples were measured at r.t. using JASCO IRA-1(IR spectra),
JEOL D-300 (MS spectra), JASCO DIP-370 (optical rotations),
JEOL GX400, Varian Unity-500 spectrometers (1H and 13C NMR
spectra). Anhydrous THF (99.9 %) used as the solvent for BH3 reduc-
tion, α-amino acids, their derivatives and reagents for synthesis of

SYNTHESIS
November 1998
1625
•
peptides were purchased from Aldrich Chemical Co. Inc., Peptide In-
stitute Inc., and Kokusan Chemical Works Ltd.
Anal. Calcd. for C23H31N3O4Cl2 9/4 H2O: C, 52.63; H, 6.81; N, 8.00.
Found: C, 52.65; H, 6.77; N, 8.01.
Selective BH3 Reduction of the Lithium Salt of N-t-Butoxycarbo-
nyl (S)-O-Benzyltyrosyl (S)-Proline (Boc-1-OLi):
N-t-Butoxycarbonyl (S)-O-benzyltyrosyl (S)-proline methyl ester
(Boc-1: 4.82 g, 10.0 mmol) obtained from commercial N-t-butoxycar-
bonyl (S)-O-benzyltyrosine and (S)-proline methyl ester hydrochlo-
ride by the usual solution method was treated with lithium hydroxide
Preparation of Piperazine Derivative Salts:
Boc-5-OLi was obtained by the reaction of t-butoxycarbonyl N,N'-
ethylene-bridged (S)-tyrosyl-(S)-tyrosine ethyl ester7 with 2 mol
equivalents of benzyl bromide in THF in the presence of 2 mol equiv-
alents of NaH, sucessively by hydrolysis with aq LiOH. mp 110–
113°C. [α]D= +12.4 (c = 0.5, DMF).
•
monohydrate (LiOH H2O: 0.42 g, 10.0 mmol) in H2O (50 mL). The
MS: m/z = 650.
solution was evaporated to dryness under reduced pressure, and was
washed thoroughly with anhyd Et2O, providing a lithium salt (Boc-1-
OLi: 4.26 g, 9.0 mmol) as a powder in 90 % yield. Boc-1-OLi: mp
136–140°C, [α]D= –24 (c = 0.9, MeOH).
•
Anal. Calcd. for C39H41LiN2O7 3.5 H2O: C, 65.09; H, 6.72; N, 3.89.
Found: C, 65.08; H, 6.47; N, 3.90.
Boc-5-OLi was similarly reduced by the method for Boc-1-OLi on the
same scale except the use of 3 mol equivalents of BH3. Crude Boc-5-
sRed thus obtained was purified by silica gel column chromatography
[CHCl3–MeOH (30 : 1)].
MS: m/z = 474.
•
Anal. Calcd. for C26H31LiN2O6 3.3 H2O: C, 58.48; H, 7.10; N, 5.24.
Found: C, 58.37; H, 6.90; N, 5.19.
After the treatment of Boc-5-sRed with 4M HCl-DOX in the presence
NaBH4 (0.24 g, 6.32 mmol) was suspended in anhyd THF (20 mL) at
0°C, and MeI (0.90 g, 6.32 mmol) was added. After 2 h, the THF
solution (20 mL) of Boc-1-OLi (1.50 g, 3.16 mmol) was dropped into
the BH3-containing solution with stirring, and then the reaction was
continued at r.t. for 24 h. At the end of the reaction, the solution of
•
of thioanisole, the resulting powder (2HCl 5-sRed) was recrystallized
from CH3CN/MeOH. mp 220 – 222°C, [α]D= –9.4 (c = 0.5, MeOH).
IR (nujol): ν = 1735 cm–1.
MS: m/z = 536.
•
Anal. Calcd. for C34H38N2O4Cl2 3/2H2O: C, 64.15; H, 6.49; N, 4.40.
•
LiOH H2O (0.42 g, 10.0 mmol) in H2O (10 mL) was added slowly to
Found: C, 64.03; H, 6.29; N, 4.65.
the reaction solution in order to decompose excess BH3. After remov-
al of the solvent, CHCl3 (100 mL) was added to the residue obtained,
and the insoluble materials were removed by filtration. The CHCl3
solution was evaporated to dryness, and the resulting residue was sub-
jected to silica gel column chromatography [CHCl3 –MeOH (4 : 1)],
yielding Boc-2 (0.59 g, 1.30 mmol) in 40 % yield as a syrupy oil.
Furthermore, Boc-2 (0.49 g, 1.10 mmol) was treated in 4 M HCl/
DOX (40 mL) at r.t. in the presence of thioanisole (0.14 g,
1.10 mmol). After 24 h, the solution was evaporated to dryness and
the residue formed was washed with anhyd Et2O. The residue was
dissolved in CHCl3 (50 mL), and the solution was washed with aq
NaHCO3, successively with H2O, and dried (Na2SO4). After removal
of the solvent, a solid (3: 0.31 g, 0.93 mmol) obtained in 85 % yield
was recrystallized from benzene.
1H NMR (CD3OD, TMS): δ = 2.84, 2.94 (m, 1H each, iHA, iHB),
2.97 (m, 1H, H1a), 3.04 (m, 2H, oHA = oHB), 3.25 (m, 1H, H4a),
3.28 (m, 1H, H5a), 3.30 (m, 1H, H1e), 3.38 (m, 1H, H5e), 3.45 (m,
1H, H4e), 3.48 (m, 1H, H2), 3.92 (t, 1H, 7.3 Hz, H7), 5.04, 5.08 (s,
2H, and s, 2H, benzyl -CH2), 6.88 (d, 2H, 8.7 Hz, Phenol-o-CH), 6.99
(d, 2H, 8.6 Hz, Phenol-o-CH), 7.05 (d, 2H, 8.7 Hz, Phenol-m-CH),
7.11 (d, 2H, 8.6 Hz, Phenol-m-CH), 7.27–7.41 (m, 10H, aromatic).
The analytical, physical and spectroscopic data of Boc-6-OLi
•
•
•
(2HCl 6-sRed), Boc-7-OLi (2HCl 7-sRed) and Boc-8-OLi (2HCl 8-
sRed) obtained from N,N'-ethylene-bridged-(S)-methionyl-(S)-
methionine5,-(S)-phenylalanyl-(S)-phenylalanine4,6 and -(S)-leucyl-
(S)-leucine5,8–10 according to the procedure similar to that for Boc-5-
•
OLi (2HCl 5-sRed) are shown as follows.
Boc-6-OLi: mp 123–127°C, [α]D= +34 (c = 0.5, MeOH).
MS: m/z = 406.
A piperazin-2-one derivative (3): mp 167–170˚C, [α]D= +4.8 (c = 0.9,
MeOH).
•
Anal. Calcd. for C17H29LiN2O5S2 3/2 H2O: C, 46.46; H, 7.34; N,
IR (nujol): ν = 1665 cm–1.
6.37. Found: C, 46.38; H, 7.33; N, 6.17.
•
MS: m/z = 336.
2HCl 6-sRed: mp 145–147°C, [α]D= –16.4 (c = 0.5, MeOH).
Anal. Calcd. for C21H24N2O2: C,74.97; H, 7.19; N, 8.32. Found: C,
75.00; H, 7.13; N, 8.19.
IR (nujol): ν = 1730 cm–1.
MS: m/z = 292.
1H NMR (CDCl3, TMS): δ = 1.76, 1.91 (m, 1H each, Pro-γ-CH2),
1.93, 2.15 (m, 1H each, Pro-β-CH2), 2.74, 2.95 (m, 1H and ddd, 1H,
3.4, 7.6, 10.5 Hz, Pro-δ-CH2), 3.21 (dd, 1H, 8.0, 9.4 Hz, Pro-α-CH),
2.63, 2.74 (dd, 1H, 6.6, 11.2 Hz and dd, 1H, 3.8, 11.2 Hz, piperazin-
2-one CH2), 3.71 (dddd, 1H, 3.8, 5.8, 6.6, 9.0 Hz, Tyr-α-CH ), 2.65,
2.82 (dd, 1H, 9.0, 13.6 Hz and dd, 1H, 5.8, 13.6, Hz,Tyr-β-CH2), 5.05
(s, 2H, benzyl-CH2), 6.94 (d, 2H, 8.7 Hz, Phenol-o-CH), 7.09 (d, 2H,
8.7 Hz, Phenol-m-CH), 7.33– 7.43 (m, 5H, aromatic).
13C NMR (CDCl3, TMS): δ = 21.7 (Pro-γ-C), 26.7 (Pro-β-C), 54.7
(Pro-δ-C), 64.0 (Pro-α-C), 52.7 (piperazin-2-one methylene),
40.2(Tyr-β-C), 53.6 (Tyr-α-C), 70.0 (benzyl-C), 115.3 (Phenol-o-C),
128.7 (Phenol-p-C), 130.2 (Phenol-m-C), 157.8 (Phenol quartery C),
127.4 (benzyl o-C), 128.0 ( benzyl p-C), 128.6 ( benzyl m-C), 136.9
(benzyl quartery C), 172.2 (amide C=O).
Anal. Calcd. for C12H26N2O2S2Cl2: C, 39.44; H, 7.17; N, 7.67.
Found: C, 39.14; H, 7.23; N, 7.56.
1H NMR (D2O, DSS): δ = 2.00 (m, 2H, iHA = iHB), 2.08, 2.09, (s,
3H each , SCH3), 2.15 (m, 2H, oHA = oHB), 2.56, 2.64 (br, 4H,
SCH2), 3.16 (br, 1H, H1a), 3.28 (br, 1H, H5a), 3.42 (m, 1H, H4a),
3.65 (br, 1H, H1e), 3.66 (br, 1H, H5e), 3.67 (br, 1H, H4e), 3.74 (br,
1H, H2), 3.85 (br, 1H, H7).
Furthermore, the 13C NMR data of 2HCl 6-sRed is added as follows
•
because of its broad 1H NMR signals.
13C NMR (D2O, DSS): δ = 14.0, 14.3 (SCH3), 26.9, 28.9 (Met-β-C),
28.3, 29.3 (Met-γ-C), 41.5 (C4), 46.2 (C5), 50.9 (C1), 52.9 (C2), 67.9
(C7), 171.8 (carboxylic acid C=O).
Boc-7-OLi: mp 130–133°C, [α]D= –27 (c = 0.5, MeOH).
•
MS: m/z = 437. Anal. Calcd. for C25H29LiN2O5 2/5H2O: C, 66.48; H,
6.65; N, 6.20. Found: C, 66.59; H, 6.75; N, 6.20.
•
Preparation of â-Casomorphin Analog (4):
2HCl 7-sRed: mp 218 – 221°C, [α]D= –24 (c = 0.5, MeOH).
The coupling of Boc-2 (0.45 g, 1.00 mmol) with (S)-phenylalanine
methyl ester hydrochloride (0.22 g, 1.00 mmol) by the DCC/HOBT
method and the successive deprotection of the blocking groups pro-
vided crude 4 as a solid. Furthermore, 4 was purified by silica gel
column chromatography [CHCl3–MeOH (4:1)], and transformed to
its dihydrochloride (0.15 g, 0.30 mmol). mp 126 – 128°C, [α]D=
–34.3 (c = 0.8, MeOH).
IR (nujol): ν = 1736 cm–1.
MS: m/z = 324.
Anal. Calcd for C20H26N2O2Cl2: C, 60.49; H, 6.59; N, 7.05. Found:
C, 60.55; H, 6.75 ; N,7.21.
1H NMR (D2O, DSS): δ = 3.01 (d, 2H , 7.1 Hz, iHA = iHB), 3.04 (dd,
1H, 11.2, 13.4 Hz, H1a), 3.14 (dd, 1H, 7.3, 14.2 Hz, oHA), 3.19 (dd,
1H, 7.8, 14.2 Hz, oHB), 3.25 (td, 1H, 3.0, 12.6 Hz, H5a), 3.32 (td, 1H,
3.1, 12.8 Hz, H4a), 3.47 (ddd, 1H, 2.2, 2.7, 12.9 Hz, H1e), 3.60 (m,
MS: m/z = 411.

SYNTHESIS
Papers
1626
1H, H4e), 3.61 (m, 1H, H5e), 3.70 (dtd, 1H, 3.1, 7.1, 11.0 Hz, H2),
3.95 (dd, 1H, 7.3, 7.8 Hz, H7), 7.24–7.45 (m, 10H, aromatic).
Boc-8-OLi: mp 153–157°C, [α]D= +9 (c = 0.5, MeOH).
MS: m/z = 370.
(1) Almquist, R. G.; Christie, P.H.; Chao, W-Ru,;Johnson, H.L.
J. Pharm. Sci. 1983, 72, 63.
(2) Roeske, R.W.; Weitl, F. L.; Prasad, K.U.; Thompson, R.M.
J. Org. Chem. 1976, 41, 1260.
(3) Yoshikawa, M.; Tani, F.; Yoshimura, T.; Chiba, H. Agric. Biol.
Chem. 1986, 50, 2419.
(4) Takenaka, H.; Miyake, H.; Kojima, Y.; Yasuda, M.; Gemba,
M.; Yamashita, T. J. Chem. Soc. Perkin Trans. 1. 1993, 933.
(5) Yamashita, T.; Takenaka, H.; Kojima, K. Amino Acids 1993, 4,
187.
(6) Yamashita, T.; Hatamoto, E.; Takenaka, H.; Kojima, Y.; Inoue,
Y.; Gemba, M.; Yasuda, M. Chem. Pharm. Bull. 1996, 44, 856.
(7) Yamashita, T.; Tsuru, E.; Banjyo, E.; Doe, M.; Shibata, K.; Ya-
suda, M.; Gemba, M. Chem. Pharm. Bull. 1997, 45, 1940.
(8) Kojima, Y.; Ikeda, Y.; Miyake, H.; Iwado, I.; Hirotsu, K.;
Shibata, K.; Yamashita, T.; Ohsuka, A.; Sugihara, A. Polymer
J. 1991, 23, 1359.
.
Anal. Calcd. for C19H33LiN2O5 4/5H2O: C, 58.39; H, 8.92; N, 7.17.
Found: C, 58.26; H, 8.98; N, 7.15.
2HCl.8-sRed: mp 243–245°C, [α]D= –13 (c = 0.5, MeOH).
IR (nujol): 1736 cm–1.
MS: m/z = 256.
Anal. Calcd for C14H30N2O2Cl2: C, 51.06; H, 9.18; N, 8.51. Found:
C, 51.22; H, 9.31 ; N, 8.31.
1H NMR (D2O, DSS): δ = 0.94 (d, 6H, 6.4 Hz, isobutyl CH3), 0.96,
0.98 (d, 3H each, 6.1 Hz, isobutyl CH3), 1.61 (m, 2H, iHA, iHB), 1.71
(m, 1H, oHA), 1.72 (m, 1H, isobutyl CH), 1.73 (m, 1H, isobutyl CH),
1.86 (m, 1H, oHB), 3.33 (br, 2H, H1a, H1e), 3.41 (td, 1H, 2.7, 13.2
Hz, H5a), 3.52 (1H, td, 2.6, 13.0 Hz, H4a), 3.73 (br , 1H, H2), 3.77
(m, 1H, H4e), 3.92 (m, 1H, H7), 3.93 (m, 1H, H5e).
(9) Miyake, H.; Kojima, Y.; Yamashita, T.; Ohsuka, A. Makromol.
Chem. 1993, 194, 1925.
We thank Mrs. Rika Yutani for the elemental analysis and the mass
spectral measurements.
(10) Miyake, H.; Yamashita, T.; Kojima, Y.; Tsukube, H. Tetrahe-
dron Lett. 1995, 36, 7669.