Synthesis and chemical reactivity of thiophenoxyphenylalanine bioisosteres, suitable synthons for the design of HIV protease inhibitors

Article abstract of DOI:10.1039/a907483j

Phenylalanine in which the methylene group is replaced by a sulfur atom could be a useful bioisostere for the design of HIV-protease inhibitors. Due to the chemical instability of hemiaminal intermediates, these bioisosteres have to be prepared from α-hyd

Full text of DOI:10.1039/a907483j

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Synthesis and chemical reactivity of thiophenoxyphenylalanine
bioisosteres, suitable synthons for the design of HIV protease
inhibitors
1
G. Priem, L. Rocheblave, C. De Michelis, J. Courcambeck and J. L. Kraus*
Laboratoire de Chimie Biomoléculaire, INSERM U322, Faculté des Sciences de Luminy,
case 901, Université de la Méditerranée, 70 route Léon Lachamp 13288 Marseille cedex 9,
France. Tel.: (33) 4 91 82 91 44; Fax: (33) 4 91 82 94 16; E-mail: kraus@luminy.univ-mrs.fr
Received (in Cambridge, UK) 15th September 1999, Accepted 17th January 2000
Phenylalanine in which the methylene group is replaced by a sulfur atom could be a useful bioisostere for the
design of HIV-protease inhibitors. Due to the chemical instability of hemiaminal intermediates, these bioisosteres
have to be prepared from α-hydroxyglycine following specific synthetic routes. In this paper, we report the synthesis
of sulfenylated phenylalanine bioisosteres 2 and 3, which represent two original building-blocks for peptide solid
phase synthesis.
Introduction
Results and discussion
HIV aspartylprotease represents an alternative target for a
therapeutic intervention. One of the interests of our research is
to find a new series of compounds which can inhibit the activity
of the HIV protease.
Several synthetic peptides, for example Ile-Arg-Lys-Ile-Leu-
Phe-Leu-Asp-Gly-Ile, were found to be substrates of the
recombinant HIV protease,1–3 and were cleaved specifically
between the Leu and Phe residues. Based on this observation,
we have attempted the synthesis of new modified thiophenoxy-
peptides containing a thiophenoxyphenylalanine bioisostere
moiety.
α-Hydroxyglycine derivatives 1 (Scheme 1), which are often
used as key synthons in the design of various protease inhibi-
tors,6 have been extensively studied.7,8 We therefore think that
α-hydroxyglycine could be a suitable synthon for the synthesis
of the various phenylalanine bioisosteres 2 and 3.
N-Carbonyl-α-thiophenoxyglycine derivatives are known to
be chemically stable; in contrast, when the amide function is
hydrolyzed (chemically or enzymatically), the resulting unstable
phenylthioaminal intermediate decomposed directly into thio-
phenol and aldehyde products9 as shown in Scheme 2. It is
We have previously reported through molecular model
studies4,5 that the bioisosteric replacement of a methylene
group by a sulfur atom in a benzyl group of various enzymatic
substrates does not induce drastic changes in the conform-
ational energies.
The synthesis and the anti-HIV properties of the two original
key-synthons 2 and 3 which are sulfenylated phenylalanine bio-
isosteres (Scheme 1) are described in this paper. The chemical
Scheme 2 Hydrolysis of N-carbonyl α-thiophenoxyglycine derivatives.
foreseeable that liberation of thiophenol and glyoxylic acid
derivatives may cause a dysfunction of the cellular mechanism
which will induce the death of the infected cell. One could take
advantage of this specific reactivity of N-carbonyl-α-thiophenoxy-
glycine compounds for the design of novel HIV-protease inhibi-
tors. Therefore the syntheses of bioisosteres 2 and 3 are of
major interest since they could be used as phenylalanine
peptidomimetic synthons suitable for modified peptide solid
phase synthesis.
Usually, the most straightforward synthesis of N-(1-hydroxy-
alkyl)amides involves the addition of primary or secondary
amides to aldehydes or ketones. This reaction is an equilibrium
process which usually disfavours the adduct except for special
cases. Indeed, reactive aldehydes like formaldehyde, chloral
Scheme 1 Structure of phenylalanine bioisosteres 2 and 3.
reactivity of these intermediates led us also to report their
chemical stabilities under various conditions.
DOI: 10.1039/a907483j
J. Chem. Soc., Perkin Trans. 1, 2000, 819–824
This journal is © The Royal Society of Chemistry 2000
819

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(trichloroacetaldehyde), glyoxylic acid and its derivatives react
with amides to give fairly stable N-(1-hydroxyalkyl)amides7,8
(Scheme 3).
In fact, we found that when glyoxylic acid 5 was condensed
with amide substrates 4 (BocLeuNH2 or BocPheNH2), isolation
of the corresponding carboxylic hemiaminals 6a and 6b was
not possible. Only highly hydrophobic amides like [bis(1-
naphthyl)methyl]acetamide led to pure hemiaminal 6c in 80%
yield6 (Scheme 3).
In contrast, when various esters of glyoxylic acid (7a:R3 =
ethyl; 7b:R3 = allyl) were condensed with BocLeuNH2 4a, the
corresponding hemiaminal esters 8a and 8b were isolated in
relatively good yields,10 50 and 60% respectively, (Scheme 4).
Our goal was to use intermediates 8a and 8b for the synthesis
of α-sulfenylated carboxylic acid 2 (R1 = Boc()Leu-; R2 =
-OH) or amide 3 (R1 = Boc()Phe-; R2 = -NH-CH2Ph) (Scheme
1) and we have investigated several routes. The most direct route
attempted with esters 8a (R3 = Et) and 8b (R3 = allyl) involved
initial O-acylation of the hydroxy group using acetic anhydride,
followed by the substitution of the O-acetoxy group of com-
pounds 9a and 9b by thiophenol, leading to the corresponding
α-thiophenoxy esters 10a and 10b (Scheme 4). Unfortunately,
classical hydrolysis of the ester function (1 M sodium hydroxide
solution)11 led to the decomposition products: thiophenol,
glyoxylic acid 5, and the starting amide 4a, according to the
mechanism shown in Scheme 4. This result prompted us to
study the hydrolysis stability of hemiaminal esters 8a and 8b.
Three different hydrolysis sites of cleavage 1, 2 and 3 of
intermediates 8a and 8b are indicated on Scheme 4. The sites of
cleavage depend on the following structural parameters: the
presence or not of a protecting group on the free hydroxy
function, and the nature of the terminal carbonyl function
(ester or amide). Direct basic hydrolysis of esters 8a and 8b gave
Scheme 3 Synthesis of N-(1-hydroxyalkyl)amides.
Scheme 4 Synthesis of phenylalanine bioisostere 2. a, Et3N, CH2Cl2, rt, 72 h, 50–60%; b, t-Bu(Ph)2SiCl, imidazole, DMF, 62%; c, 1 M NaOH,
CH3OH, quantitative; d, PhCH2NH2, BOP, Et3N, CH2Cl2, 50%; e, (nBu)4NϩFϪ, THF, 66%; f, Ac2O, Pyr, CH2Cl2; g, PhSH, (iPr)2NEt, DMF, rt, 72 h,
58%; h, potassium 2-ethylhexanoate, Pd(PPh3)4, PPh3, EtOAc, rt, 16 h, 70%.
820
J. Chem. Soc., Perkin Trans. 1, 2000, 819–824

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Scheme 5 Synthesis of diastereoisomers 3a and 3b. a, 1 M NaOH, CH3OH, quantitative; b, PhCH2NH2, BOP, Et3N, CH2Cl2, 96%; c, Pb(OAc)4,
molecular sieves 4 Å, EtOAc reflux, 1 h, quantitative; d, PhSLi, THF, 0 ЊC, 1 h, 61%.
Table 1 Anti-HIV activities of synthetic [-Leu-Phe-(S)-] peptides and
the initial amide 4a, glyoxylic acid 5, and the corresponding
alcohol (type 2 cleavage). In contrast, basic hydrolysis11 of
protected ester 11 (OH protected by a tert-butyldiphenylsilyl
group12) led to the expected corresponding acid 12 (type 3
cleavage).
O-Silyl deprotection of N-benzylamide 13 with tetrabutyl-
ammonium fluoride,12 and treatment of the resulting hydroxy-
derivative 14 with acetic anhydride did not lead to the expected
O-acetoxy analogue, but to the initial amide 4a and glyoxylic
amide 15 (type 2 cleavage).
mixture of compounds 3a and 3b
TIb
(CC50
EC50)
/µM
c
/
a
EC50
Compounds
/µM
Ile-Arg-Lys-Ile-Leu-Phe-(S)d-Leu-Asp-Gly-NH2
5
50
10
10
Ile-Arg-Lys-Ile-Leu-Phe-(S)d-Leu-Asp-Gly-Ile-OH 10
Boc-Phe-(S)d-NH-CH2Ph (mixture of 3a and 3b)
5
These results convinced us to use alternative routes to access
the desired synthons 2 and 3.
Hydrogenation of α-thiophenoxy allyl ester 10b using
a EC50: concentration required to inhibit 50% syncitia formation. b TI:
therapeutic index. c CC50: concentration required to cause death of 50%
of uninfected MT4 cells. d Phe-(S) represents thiophenoxyphenylalanine
bioisostere residue: –NH–CH(SPh)–CO–.
tetrakis(triphenylphosphine)palladium(0) [Pd(PPh3)4] in the
13
presence of PPh3 led to the desired α-thiophenoxy acid 2 in
acceptable yield (65%) (Scheme 4). α-Thiophenoxyamide bio-
isosteres 3a and 3b were obtained using the following synthetic
route (Scheme 5). Boc-protected ()-phenylalanyl-()-serine
ethyl ester 16 after saponification11 was condensed with benzyl-
amine in the presence of BOP (benzotriazol-1-yloxytris(dimeth-
ylamino)phosphonium hexafluorophosphate) reagent (96%
yield).14 The resulting amide 17 was refluxed with lead tetra-
acetate15 in ethyl acetate and led quantitatively to the corres-
ponding 1:1 ratio of α-acetoxybenzylamide diastereoisomers
18. Racemisation occurred during the nucleophilic addition
of acetic acid onto the two diastereofaces of the imine inter-
mediate. The asymmetric carbon of the phenylalanine residue
appears too far removed to affect the diastereoselectivity during
this process. Nucleophilic substitution of the O-acetoxy group
by lithium thiophenoxide led to the corresponding diastereo-
isomers 3a and 3b in acceptable yield (61%). The ratio of
diastereoisomers 3a and 3b was calculated from the integration
reported in specific cases of antiprotease inhibitors.18,19 Investi-
gations into the determination of the enzymatic mechanism of
action of such HIV inhibitors are currently in progress.
Several analogs of compound 3, with various functions (OH,
COOH, NH2) at the para position of the benzamide group
could be prepared using a similar route to that described in
Scheme 5.
Experimental
General
Nuclear magnetic resonance spectra were recorded with a
Bruker AC-250 (1H, 13C NMR); chemical shifts are expressed as
δ units (parts per million) downfield from TMS. Infrared
spectra were obtained using a Perkin-Elmer 1600 FT-IR
spectrophotometer, values are expressed in cmϪ1. Fast atom
bombardment mass spectral analysis was performed by Dr
Astier (Laboratoire de Mesures Physiques-RMN, USTL,
Montpellier, France) on a JEOL DX-100 using a caesium ion
source and glycerol–thioglycerol (1:1) (GT) or m-nitrobenzyl
alcohol (NOBA) as matrix. Mass calibration was performed
using caesium iodide. Microanalyses were within 0.4% of
the theoretical values. Microanalyses were performed by the
Service Central d’Analyses, CNRS, Vernaison-Lyon, France.
Preparative flash column chromatography was carried out on
silica gel (230–240 mesh, G60 Merck). All reagents were of
commercial quality (Aldrich Company) from freshly opened
containers.
1
of the different H NMR signals: two broad doublet peaks for
the NH benzylamide protons at 6.81 and 6.94 ppm and for
the NH protons (4.91 and 5.05 ppm); two doublet peaks for the
CH-SPh protons at 5.69 and 5.72 ppm; and two singlet peaks
for the N-Boc protons at 1.24 and 1.27 ppm.
In conclusion, versatile sulfenylated phenylalanine bio-
isosteres 2 and 3 can be used as original building blocks in the
solid phase synthesis for the design of various protease inhibi-
tors. Indeed compound 2 was found to be stable enough for
solid phase synthesis of various HIV inhibitor peptides.10 The
fusogenic effect of HIV-1 on MT4 cell line (syncitia formation)
was determined for the peptides shown in Table 1 as described
by Rey and co-workers.16,17 EC50 values (concentration required
to inhibit 50% syncitia formation) ranging from 5 to 10 µM
were found for these peptides (Table 1). The mixture of
diastereoisomers 3a and 3b showed potent anti-HIV activity
(EC50 = 5 µM). However, the separation of the two isomers has
not yet been achieved and it can only be suggested that one
isomer should be more active than the other, as already
Anti-HIV evaluation assay
The CEM cell line and the T Leukaemia virus type one (HTLV-
1) CD4-positive T cell line were cultured in RPMI–10% FCS
(fetal calf serum) and refed twice a week. The laboratory-
adapted strain HIVLAV clade B stock was prepared from the
supernatant of the infected CEM cell line and aliquots were
J. Chem. Soc., Perkin Trans. 1, 2000, 819–824
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kept frozen at Ϫ80 ЊC until use. Anti-HIV activity was moni-
tored as the efficiency of drug compounds to inhibit syncytia
formation after HIV infection of MT4 as already described.
Briefly, 3 × 105 MT4 cells were initially pre-incubated with 100
µL of various concentrations of drug compounds dissolved in
phosphate buffer saline solution for 1 h at 37 ЊC. Then 100 µL
of an appropriate virus dilution was added to the mixture and
incubated at 37 ЊC for 1 h. After three washes, the cells were
resuspended in the culture medium in the presence or absence
of drug compounds. Cultures were then grown for 7 days at
37 ЊC, under 5% CO2 atmosphere and refed at day 3 post-
infection with culture medium supplemented (or not) with drug
compounds. Each culture well was duplicated. The appearance
of syncytia was followed each day with an inverted optical
microscope. Typically, the virus dilution used in the assay
(multiplicity of infection of 0.1 TCID50/CELL) allowed syn-
cytia formation at day 5 post-infection. The inhibitory concen-
tration of drug compounds was expressed as the concentration
that caused 50% inhibition of syncytia formation (EC50) with-
out direct toxicity for the cells. Cytotoxicity concentration
(CC50) of the drug compounds was monitored by the growth of
non-infected cells using the trypan blue exclusion assay and
corresponded to the concentration required to cause 50% of
cell death.
mmol) and N,N-diisopropylethylamine (0.79 g; 6.10 mmol)
were added. The solution was stirred for 72 hours and then the
solvent was evaporated. The crude residue was dissolved in
water and the aqueous phase was extracted twice with ethyl
acetate. The organic layer was dried on MgSO4 and evaporated.
The crude residue was purified by flash column chromato-
graphy on silica gel (solvent: toluene–methanol 9:1) to give
compound 10b (1.28 g, 58%).
HMRS calcd for C22H32N2O5S: 436.5747. Found: 436.5741.
νmax (film/cmϪ1) 1788, 1655, 1592, 750, 698. δH (250 MHz;
CDCl3) 7.40–7.30 (m, 5H, CHarom), 6.30 (m, 1H, CH᎐CH ),
᎐
2
5.80 (m, 1H, CH᎐CH ), 5.20–5.05 (m, 1H, CH᎐CH ), 4.60–4.50
᎐
᎐
2
2
(m, 2H, CH-SPh ϩ O-CH2-CH), 4.20 (m, 1H, NH-CH-CH2),
1.60–1.40 (m, 3H, CH2-CH(CH3)2), 1.40 (s, 9H, (CH3)3CO),
0.90 (2d, 6H, (CH3)2CH). δC (62.8 MHz; CDCl3) 172.3 (CO),
154.2 (CO), 136.5 (CH᎐CH ), 135.4 (Cquat.arom), 130.4, 129.5,
᎐
2
128.6 (CHarom), 112.8 (CH᎐CH ), 71.6 (CH -CH᎐CH ), 68.4
᎐
᎐
2
2
2
(CH-SPh), 40.2 (CH2CH(CH3)2), 28.4 ((CH3)3CO), 20.3
((CH3)2CH).
N-tert-Butyloxycarbonyl-L-leucyl-DL-ꢀ-thiophenoxyglycine (2)
To a solution of compound 10b (1.07 g; 2.45 mmol) in dry ethyl
acetate (10 cm3) a 0.5 M potassium 2-ethylhexanoate solution
in ethyl acetate (0.63 cm3; 0.3 mmol) was added. The solution
was stirred at room temperature for 6 hours, then Pd(PPh3)4
(113.0 mg; 0.09 mmol) and PPh3 (25.0 mg; 0.04 mmol) were
successively added. The mixture was stirred for 16 hours at
room temperature. The solvent was removed and the desired
compound 2 (0.67 g, 70%) was obtained after purification by
flash column chromatography on silica gel (solvent: dichloro-
methane–methanol 85:15).
HMRS calcd for C19H28N2O5S: 396.5094. Found: 396.4088
(Found: C, 57.7; H, 7.4; N, 7.2. Requires C, 57.5; H, 7.1; N,
7.0%). νmax (film/cmϪ1) 2312, 1718, 1654, 752, 702, 658. δH (250
MHz; CDCl3) 8.00 (br s, 1H, NH), 7.40–7.10 (m, 5H, CHarom),
4.50–4.20 (m, 2H, CH-SPh ϩ NH-CH-CH2), 4.10 (br s, 1H,
NH), 1.60–1.40 (m, 3H, CH2-CH(CH3)2), 1.40 (s, 9H, (CH3)3-
CO), 0.90 (2d, 6H, (CH3)2CH). δC (62.8 MHz; CDCl3) 180.1
(COOH), 156.3 (CO), 136.0 (Cquat.arom), 130.4, 129.5, 128.6
(CHarom), 70.6 (Cquat.Boc), 68.5 (CH-SPh), 55.0 (CH-CH2(CH3)2),
40.3 (CH2CH(CH3)2), 28.5 ((CH3)3CO), 20.4 ((CH3)2CH).
FABMS m/z 395 (MH)Ϫ.
N-tert-Butyloxycarbonyl-L-leucyl-DL-ꢀ-hydroxyglycine allyl
ester (8b)
To a solution of allyl glyoxalate (2.0 g; 16.0 mmol) in dry
dichloromethane (40 cm3) N-tert-butyloxycarbonyl--leucine
(2.8 g; 12.0 mmol) and triethylamine (4.0 cm3; 29.0 mmol)
were added. The solution was stirred at 40 ЊC for 72 hours and
then dichloromethane was removed. The crude residue was
purified by flash column chromatography on silica gel (solvent:
dichloromethane–methanol 98:2) to give compound 8b (1.81 g,
44%).
HMRS calcd for C16H28N2O6: 344.4111. Found: 344.4120.
νmax (film/cmϪ1) 3310, 1790, 1653, 1596. δH (250 MHz; CDCl3)
8.10 (br s, 1H, NH), 6.00–5.80 (m, 2H, CH᎐CH ϩ NH-CH-
᎐
2
OH), 5.40–5.20 (m, 2H, CH ᎐CH), 4.60 (m, 3H, NH-CH-
᎐
2
CH2 ϩ O-CH2-CH), 4.20 (br s, 1H, OH), 1.80–1.50 (m,
3H, CH2-CH(CH3)2), 1.40 (s, 9H, (CH3)3CO), 0.80 (2d, 6H,
(CH3)2CH). δC (62.8 MHz; CDCl3) 175.4 (CO), 154.3 (CO),
135.5 (CH᎐CH ), 110.3 (CH᎐CH ), 80.1 (CH-OH), 70.3 (CH -
᎐
᎐
2
2
2
N-tert-Butyloxycarbonyl-L-phenylalanyl-DL-ꢀ-(tert-butyl-
diphenylsilyloxy)glycine ethyl ester (11)
CH᎐CH ), 39.6 (CH2CH(CH3)2), 28.5 ((CH3)3CO), 20.3
᎐
2
((CH3)2CH).
To a solution of compound 8a (200 mg; 0.55 mmol) in dry N,N-
dimethylformamide (15 cm3), imidazole (260 mg; 3.82 mmol)
and tert-butyldiphenylsilyl chloride (1.05 g; 3.82 mmol) were
added. The mixture was stirred overnight, then N,N-dimethyl-
formamide was removed and the crude residue was dissolved in
ethyl acetate. The organic layer was washed twice with 5% citric
acid solution, water, dried on MgSO4 and evaporated. The
crude residue was purified by flash column chromatography on
silica gel (solvent: n-hexane–ethyl acetate 4:1) to afford 11 as a
pale yellow oil (0.20 g, 62%).
HMRS calcd for C34H44N2O6Si: 604.8255. Found: 604.8310.
νmax (film/cmϪ1) 1701, 1592, 909, 737, 668. δH (250 MHz;
CDCl3) 7.56–7.50 (m, 5H, CHarom-Si), 7.28–7.19 (m, 5H,
CHarom-CH2), 7.19–6.99 (m, 5H, CHarom-Si), 5.56 (2d, 1H,
CH-OSi, J = 8.42 Hz), 4.73 (br s, 1H, NH), 4.55 (br s, 1H,
NH), 4.12 (m, 1H, CH-CH2Ph), 3.97–3.76 (2q, 2H, O-CH2-
CH3, J = 7.41 Hz), 2.92–2.69 (m, 2H, Ph-CH2-CH), 1.20 (2s,
9H (CH3)3C-O), 0.95 (2t, 3H, O-CH2-CH3, J = 7.20 Hz),
0.90 (2s, 9H (CH3)3C-Si). δC (62.8 MHz; CDCl3) 170.9
(CO), 168.7 (CO), 158.2 (CO), 136.7, 136.2, 136.1, 135.5, 135.4,
135.3, 132.9, 132.7, 132.3, 130.6, 130.3, 129.6, 128.9, 127.8,
127.1 (CHarom), 73.1 (NH-CH-OSi), 61.9 (O-CH2-CH3), 38.1
(CH2-Ph), 28.2 ((CH3)3C-O), 27.0 ((CH3)3C-Si), 14.3 (O-CH2-
CH3).
N-tert-Butyloxycarbonyl-L-leucyl-DL-ꢀ-acetoxyglycine allyl
ester (9b)
Compound 8b (1.70 g; 5.10 mmol) was dissolved in dry pyridine
(10 cm3) and cooled to 0 ЊC, before acetic anhydride (20 cm3;
0.21 mol) was added. The mixture was stirred at room temper-
ature for 72 hours. After evaporation of solvent, ethyl acetate
was added. The organic layer was washed with 5% sodium
hydrogen carbonate solution and brine, dried on MgSO4 and
evaporated to afford compound 9b (2.0 g, quantitative) which
was used without further purification.
HMRS calcd for C18H30N2O7: 386.4490. Found: 386.4485.
νmax (film/cmϪ1) 1788, 1774, 1654, 1588. δH (250 MHz; CDCl3)
6.35 (m, 1H, CH᎐CH ), 5.80 (m, 1H, CH᎐CH ), 5.20–5.00 (m,
᎐
᎐
2
2
1H, CH᎐CH ), 4.60 (m, 2H, CH-OCOCH ϩ O-CH -CH),
᎐
2
3
2
4.20–4.00 (m, 1H, NH-CH-CH2), 2.5 (s, 3H, CH3CO), 1.80–
1.30 (m, 3H, CH2-CH(CH3)2), 1.20 (s, 9H, (CH3)3CO), 0.80 (2d,
6H, (CH3)2CH).
N-tert-Butyloxycarbonyl-L-leucyl-DL-ꢀ-thiophenoxyglycine allyl
ester (10b)
To a solution of compound 9b (1.91 g; 5.10 mmol) dissolved in
dry N,N-dimethylformamide (10 cm3), thiophenol (0.67 g; 6.10
822
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N-tert-Butyloxycarbonyl-L-phenylalanyl-L-serine ethyl ester (16)
N-tert-Butyloxycarbonyl-L-phenylalanyl-DL-ꢀ-(tert-butyl-
diphenylsilyloxy)glycine (12)
To a solution of N-tert-butyloxycarbonyl--phenylalanine (2.34
g; 8.84 mmol) in dry dichloromethane (70 cm3) -serine ethyl
ester hydrochloride (1.50 g; 8.84 mmol), BOP reagent (4.69 g;
10.60 mmol) and triethylamine (3.10 cm3; 22.0 mmol) were
added. The mixture was stirred overnight at room temperature.
The solvent was removed and the crude residue was dissolved in
ethyl acetate. After four washes with 5% sodium hydrogen carb-
onate solution, brine, 5% citric acid solution and brine, succes-
sively, the organic layer was dried on MgSO4 and evaporated.
Compound 16 (3.09 g, 92%), mp 113–115 ЊC was purified by
flash column chromatography on silica gel (solvent: n-hexane–
ethyl acetate 2:3) as a white solid.
HMRS calcd for C19H28N2O6: 380.4448. Found: 380.4398.
νmax (film/cmϪ1) 3598, 1702, 1658, 908, 731, 658. δH (250 MHz;
CDCl3) 7.40 (br d, 1H, NH, J = 7.21 Hz), 7.19–7.14 (m, 5H,
CHarom), 5.57 (d, 1H, NH, J = 7.73 Hz), 4.55 (m, 1H, CH-
CH2OH ), 4.45 (m, 1H, CH-CH2Ph), 4.15–4.00 (q, 2H, O-CH2-
CH3, J = 10.65 Hz), 3.91 (d, 2H, CH2-OH, J = 5.70 Hz),
3.81 (br s, 1H, OH), 3.10–2.75 (m, 2H, Ph-CH2-CH), 1.26
(s, 9H, (CH3)3C-O), 1.13 (t, 3H, O-CH2-CH3, J = 7.13 Hz).
δC (62.8 MHz; CDCl3) 172.8 (CO), 170.6 (CO), 156.2 (CO),
137.0 (Cquat.arom), 129.6, 128.6, 126.9 (CHarom), 80.4 (Cquat.t-Bu),
62.7 (CH2-OH), 62.0 (O-CH2-CH3), 56.0 (Ph-CH2-CH), 50.5
(CH-CH2-OH), 38.7 (CH-CH2-Ph), 28.4 ((CH3)3C-O), 14.2
(CH3-CH2). FABMS m/z 381 (MH)ϩ.
To a solution of compound 11 (120 mg; 0.20 mmol) in metha-
nol (5 cm3) 1 M sodium hydroxide solution (0.5 cm3; 0.45
mmol) was slowly added. The mixture was stirred for one
hour then 1 M hydrochloric acid solution (0.20 cm3) was
added before methanol was evaporated. The residue was
cooled to 0 ЊC before slow addition of 1 M hydrochloric acid
solution (0.3 cm3). The mixture was extracted three times with
ethyl acetate. The organic layer was dried and evaporated
to give 12 (0.09 g, 85%) which was used without further
purification.
HMRS calcd for C32H40N2O6Si: 576.7714. Found: 576.7742.
νmax (film/cmϪ1) 2358, 1698, 1662, 913, 737, 650. δH (250 MHz;
CDCl3) 7.50–7.47 (m, 5H, CHarom-Si), 7.16–7.07 (m, 5H,
CHarom-CH2), 7.03–6.89 (m, 5H, CHarom-Si), 5.52 (2d, 1H, CH-
OSi, J = 9.42 Hz), 4.93 (br s, 1H, NH), 4.59 (br s, 1H, NH),
4.07 (m, 1H, CH-CH2Ph), 2.79–2.47 (m, 2H, Ph-CH2-CH), 1.16
(2s, 9H, (CH3)3C-O), 0.86 (2s, 9H, (CH3)3C-Si).
N-Benzyl-[N-tert-butyloxycarbonyl-L-phenylalanyl-DL-ꢀ-(tert-
butyldiphenylsilyloxy)]glycinamide (13)
Compound 12 (770 mg; 1.33 mmol) was dissolved in dry
dichloromethane (10 cm3) then benzylamine (0.15 cm3; 1.33
mmol), BOP reagent (710 mg; 1.60 mmol) and triethylamine
(0.46 cm3; 3.32 mmol) were added. The mixture was stirred
overnight before the solvent was evaporated. The crude residue
was dissolved in ethyl acetate and the organic layer was succes-
sively washed with 5% sodium hydrogen carbonate solution,
brine, dried on MgSO4 and evaporated. The crude residue was
purified by flash column chromatography on silica gel (solvent:
n-hexane–ethyl acetate 7:3) to give 13 (0.39 g, 45% ) as a yellow
oil.
HMRS calcd for C39H47N3O5Si: 665.9125. Found: 665.9128.
νmax (film/cmϪ1) 2359, 1683, 1558, 973, 737, 650. δH (250 MHz;
CDCl3) 7.42–6.92 (m, 20H, CHarom), 6.76 (d, 1H, NH, J = 8.09
Hz), 6.65 (d, 1H, NH, J = 8.43 Hz), 6.37 ϩ 6.29 (2br m, 2H,
2NH), 5.34 (2s, 1H, CH-OSi, J = 8.47 Hz), 4.54 (br m, 1H, NH),
4.54 ϩ 4.45 (2br s, 2H, NH), 4.20–4.08 (m, 2H, Ph-CH2-NH),
4.08–3.90 (m, 1H, CH-CH2Ph), 2.84–2.40 (m, 2H, Ph-CH2-
CH), 1.12 (2s, 9H, (CH3)3C-O), 0.80 (2s, 9H, (CH3)3C-Si).
δC (62.8 MHz; CDCl3) 171.3, 170.2 (CO), 158.4 (CO), 136.7,
136.2, 136.1, 135.5, 135.4, 135.3, 132.9, 132.7, 132.3, 130.6,
130.3, 129.6, 128.9, 127.8, 127.1 (CHarom), 73.5 (NH-CH-OSi),
47.5 (NH-CH2-Ph), 38.2 (CH-CH2-Ph), 28.3 ((CH3)3C-O), 27.1
((CH3)3C-Si).
N-Benzyl-(N-tert-butyloxycarbonyl-L-phenylalanyl)-L-serin-
amide (17)
Compound 16 (1.50 g; 3.94 mmol) was dissolved in methanol
(10 cm3), then 1 M sodium hydroxide solution (8.70 cm3; 8.67
mmol) was added slowly. After 3 hours, 1 M hydrochloric acid
solution (4 cm3) was added and methanol was removed, then
1 M hydrochloric acid solution (4.7 cm3) was added slowly at
0 ЊC. The aqueous layer was extracted twice with ethyl acetate,
and the organic layer was dried on MgSO4. The crude carb-
oxylic acid intermediate, obtained in a quantitative yield (3.05
g; 8.67 mmol), was dissolved in dry dichloromethane (30 cm3)
then benzylamine (0.94 cm3; 8.67 mmol), BOP reagent (4.60 g;
10.40 mmol) and triethylamine (3.01 cm3; 21.67 mmol) were
added. After one night’s stirring at room temperature, the
solvent was removed and the residue dissolved in ethyl acetate.
The organic layer was washed successively with 5% sodium
hydrogen carbonate solution, 5% citric acid solution and brine,
and then dried and evaporated. The crude residue was purified
by flash column chromatography on silica gel (solvent: ethyl
acetate) to afford 17 (1.66 g, 96%), mp 150–154 ЊC as a white
solid.
N-Benzyl-(N-tert-butyloxycarbonyl-L-phenylalanyl-DL-ꢀ-
HMRS calcd for C24H31N3O5: 441.5317. Found:
441.5220. νmax (film/cmϪ1) 3573, 1705, 1662, 1562, 910, 736, 660.
δH (250 MHz; CDCl3) 7.40 (br m, 1H, NH), 7.20–6.95 (m, 10H,
CHarom), 6.82 (d, 1H, NH, J = 7.78 Hz), 5.00 (br m, 1H, NH,
J = 7.21 Hz), 4.39 (m, 1H, CH-CH2OH), 4.31–4.25 (m, 3H,
CH-CH2Ph ϩ NH-CH2Ph), 4.16–3.97 (m, 2H, CH2-OH), 2.97
(m, 2H, Ph-CH2-CH), 1.26 (s, 9H, (CH3)3C-O). δC (62.8 MHz;
CDCl3) 172.8, 170.5 (CO), 137.1 (Cquat.arom), 129.6, 128.6, 128.4,
127.3, 127.2, 127.0 (CHarom), 80.3 (Cquat.t-Bu), 62.6 (CH2-OH),
56.0 (Ph-CH2-CH), 43.2 (Ph-CH2-NH), 38.5 (CH-CH2-Ph),
28.3 ((CH3)3C-O). FABMS m/z 442(MH)ϩ.
hydroxy)glycinamide (14)
To a solution of compound 17 (400 mg; 0.60 mmol) in dry
tetrahydrofuran (5 cm3) tetrabutylammonium fluoride (0.60
cm3; 0.62 mmol) was added. The mixture was stirred for 30
minutes, then tetrahydrofuran was evaporated and the crude
residue was dissolved in dichloromethane. The organic layer
was washed twice with water, dried on MgSO4 and evaporated.
Compound 14 (0.17 g, 66%), mp 135–137 ЊC was obtained after
purification by flash column chromatography on silica gel
(solvent: n-hexane–ethyl acetate 2:3) as a white solid.
HMRS calcd for C23H29N3O5: 427.5046. Found: 427.5120.
νmax (film/cmϪ1) 2360, 1694, 1592, 909, 737, 668. δH (250 MHz;
CDCl3) 7.25–7.12 (m, 10H, CHarom), 6.07 (br s, 1H, NH), 5.86
(br m, 2H, NH ϩ OH), 5.16 (br m, 1H, NH), 5.00 (2d, 1H,
CH-OH, J = 8.08 Hz), 4.31 (m, 2H, Ph-CH2-NH), 4.06 (m, 1H,
CH-CH2Ph), 2.96 (m, 2H, Ph-CH2-CH), 1.31 (s, 9H, (CH3)3-
C-O). δC (62.8 MHz; CDCl3) 136.8, 135.2, 129.5, 129.1, 128.9,
128.7, 128.3, 127.2 (CHarom), 78.6 (NH-CH-OH), 47.4 (NH-
CH2-Ph), 38.7 (CH-CH2-Ph), 28.5 ((CH3)3C-O). FABMS m/z
428 (MH)ϩ.
N-Benzyl-(N-tert-butyloxycarbonyl-L-phenylalanyl-DL-ꢀ-
acetoxy)glycinamide (18)
Compound 17 (750 mg; 1.51 mmol) was dissolved in dry ethyl
acetate (30 cm3) then lead tetraacetate (2.00 g; 4.53 mmol) and
molecular sieves 4 Å (3 g) were added. The mixture was refluxed
under nitrogen for 2 hours. The precipitate was filtrated on
Celite and the organic layer was stirred with 10% citric acid
solution during 20 minutes. The organic layer was washed with
J. Chem. Soc., Perkin Trans. 1, 2000, 819–824
823

View Article Online
water and dried on MgSO4. The crude product 18 (0.67 g, 96%)
was used in the next step without purification.
CH), 44.0 (Ph-CH2-NH), 38.3 (Ph-CH2-CH), 28.2 ((CH3)3CO).
FABMS m/z 520 (MH)ϩ.
νmax (film/cmϪ1) 3405, 2245, 1690, 1497, 908, 731, 668. δH (250
MHz; CDCl3) 7.79 (d, 1H, NH, J = 8.53 Hz), 7.68 (d, 1H,
NH, J = 8.72 Hz), 7.20–6.93 (m, 10H, CHarom), 6.45 (2d, 1H,
NH-CH-OCOCH3, J = 8.88 Hz), 5.20 (br m, 1H, NH), 4.50–
4.40 (m, 1H, CH-CH2Ph), 4.31 (m, 2H, NH-CH2Ph), 3.08–2.93
(m, 2H, Ph-CH2-CH), 1.95 (2s, 3H, CH3-CO), 1.26 (2s, 9H,
(CH3)3C-O). δC (62.8 MHz; CDCl3) 173.1, 172.1 (CO), 165.6
(CO), 137.5, 137.0 (Cquat.arom), 129.6, 129.5, 129.3, 128.9, 128.8,
127.8, 127.3, 127.2 (CHarom), 73.3 (NH-CH-OCOCH3), 44.0
(Ph-CH2-NH), 28.4 ((CH3)3C-O), 21.0 (CH3-CO). FABMS
m/z 470 (MH)ϩ.
Acknowledgements
INSERM is gratefully acknowledged for financial support.
References
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N-Benzyl-(N-tert-butyloxycarbonyl-L-phenylalanyl-DL-ꢀ-
thiophenoxy)glycinamide (3a) and (3b)
Compound 18 (2.71 g; 5.77 mmol) dissolved in dry tetrahydro-
furan (20 cm3) was cooled to 0 ЊC. A solution of 1.0 M lithium
thiophenoxide in tetrahydrofuran (6.92 cm3; 6.92 mmol) was
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layer was washed with water, and then dried on MgSO4 and
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Compound 3a: HMRS calcd for C29H33N3O4S: 519.6680.
Found: 519.6695 (Found: C, 67.1; H, 6.6; N, 8.2. Requires C,
67.0; H, 6.4; N, 8.0%). νmax (film/cmϪ1) 3418, 2253, 1667, 1495,
908, 735, 660. δH (250 MHz; CDCl3) 1.24 (s, 9H, (CH3)3CO),
2.76–2.99 (m, 2H, PhCH2-CH), 4.02–4.38 (m, 3H, CH-
CH2PhϩNH-CH2Ph), 4.91 (br d, 1H, NH, J = 7.45 Hz), 5.68
(d, 1H, NH-CH-SPh, J = 8.21 Hz), 6.81 (br d, 1H, NH), 7.00–
7.28 (m, 15H, CHarom). δC (62.8 MHz; CDCl3) 171.1, 170.9,
167.0 (CO), 137.5, 136.5, 134.6 (Cquat.arom), 130.4, 129.4, 129.0,
128.9, 128.6, 128.5, 127.8, 127.5, 126.8 (CHarom), 57.7 (Ph-CH2-
CH), 44.0 (Ph-CH2-NH), 38.3 (Ph-CH2-CH), 28.2 ((CH3)3CO).
FABMS m/z 520 (MH)ϩ.
Compound 3b: HMRS calcd for C29H33N3O4S: 519.6680.
Found: 519.6695 (Found: C, 67.1; H, 6.6; N, 8.2. Requires C,
67.0; H, 6.4; N, 8.0%). νmax (film/cmϪ1) 3418, 2253, 1667, 1495,
908, 735, 660. δH (250 MHz; CDCl3) 1.27 (s, 9H, (CH3)3CO),
2.76–2.99 (m, 2H, PhCH2-CH), 4.02–4.38 (m, 3H, CH-
CH2PhϩNH-CH2Ph), 5.05 (br d, 1H, NH, J = 9.66 Hz), 5.72
(d, 1H, NH-CH-SPh, J = 8.25 Hz), 6.94 (br d, 1H, NH), 7.00–
7.28 (m, 15H, CHarom). δC (62.8 MHz; CDCl3) 171.1, 170.9,
167.0 (CO), 137.5, 136.5, 134.6 (Cquat.arom), 130.4, 129.4, 129.0,
128.9, 128.6, 128.5, 127.8, 127.5, 126.8 (CHarom), 57.7 (Ph-CH2-
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Paper a907483j
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J. Chem. Soc., Perkin Trans. 1, 2000, 819–824