Solid-phase synthesis of HTLV-1 protease inhibitors containing hydroxyethylamine dipeptide isostere

Article abstract of DOI:10.1021/jo030063a

An efficient method has been developed for the first solid-phase synthesis of HTLV-1 protease inhibitors that contain hydroxyethylamine isostere as a transition-state mimetic. The synthetic procedure was designed to allow the evaluation of stereostructure

Full text of DOI:10.1021/jo030063a

Solid -P h a se Syn th esis of HTLV-1 P r otea se In h ibitor s Con ta in in g
Hyd r oxyeth yla m in e Dip ep tid e Isoster e
Kenichi Akaji,* Kenta Teruya, and Saburo Aimoto
Institute for Protein Research, Osaka University, Osaka 565-0871, J apan
akaji@protein.osaka-u.ac.jp
Received February 18, 2003
An efficient method has been developed for the first solid-phase synthesis of HTLV-1 protease
inhibitors that contain hydroxyethylamine isostere as a transition-state mimetic. The synthetic
procedure was designed to allow the evaluation of stereostructure-activity relationships at the
scissile site. All the possible configurations at the hydroxy- and side chain-bearing asymmetric
centers of the isostere were constructed by an ester-derived asymmetric aldol reaction. Each inhibitor
containing the isostere backbone was synthesized on solid support by using the newly developed
succinate ester linker. The configuration at the hydroxy- and side chain-bearing asymmetric center
showed remarkable effects on the inhibitory activity; the Ki value changed with approximately 2
orders of magnitude. The described approach enables an efficient preparation of the inhibitors
containing secondary alcohol as a transition-state mimetic.
In tr od u ction
suggested that the substrate binding site of HTLV-1
protease is different from the substrate binding site of
pepsin and HIV-1 protease.4b To address these difficul-
ties, we have reported a total chemical synthesis of [L40I,
C90A, C109A]-HTLV-1 protease, an auto-digestion re-
sistant mutant of the protease, and evaluated its kinetic
properties regarding enzymatic activity.5 In this paper,
we report the first solid-phase syntheses of HTLV-1
protease inhibitors containing the transition-state isos-
tere mimetic and evaluation of the stereostructure-
activity relationship of the inhibitors.6 By using a newly
developed succinic acid linker, each inhibitor containing
the isostere mimetic was efficiently synthesized on solid
support.
Human T-cell leukemia virus type-1 (HTLV-1) isolated
in the early 1980s was the first exogeneous retrovirus
shown to be associated with adult T-cell leukemia and a
number of other chronic diseases.1 The genome of HTLV-1
is approximately 9 kb in length and contains several open
reading frames that encode gag, pol, env, and regulatory
proteins. As in other retroviruses including HIV, HTLV-1
proteins are initially translated as large precursor polypro-
teins that undergo proteolytic processing by the viral
protease during virion assembly and maturation.2 The
protease is an aspartic protease that is encoded in a
separate reading frame overlapping the gag/ pol coding
sequence of the virus genome. The protease itself is auto-
processed from a precursor protein and thus the function
of the mature 125-amino acid-long HTLV-1 protease is
crucial for virus replication. Although several studies
have reported expression of the stabilized HTLV-1 pro-
tease in Escherichia coli as well as its kinetic character-
ization, the biochemical properties of the HTLV-1 pro-
tease have not been well-described because of the
difficulties in obtaining sizable amounts of the pro-
tease.3 To date, no structural information is available for
the HTLV-1 protease. Thus, no effective HTLV-1 protease
inhibitor has been reported; this contrasts with the
numerous potent inhibitors of HIV protease reported. The
inhibitors for HIV protease are unable to block the
processing of HTLV-1 derived precursor proteins.4 It is
Resu lts a n d Discu ssion
A key structural element in many of the aspartic
protease inhibitors is a hydroxy or hydroxy-like structure
that binds to the catalytic aspartates in the enzyme
activation site.7 Among numerous transition-state ana-
logues based on this principle, we selected a hydroxy-
ethylamine isostere, one of the most common transition-
state isosteres utilized in peptide-like aspartic protease
inhibitors.8 The absolute configuration of the hydroxy-
(4) (a) Daenke, S.; Schramm, H. J .; Bangham, C. R. M. J . Gen. Virol.
1994, 75, 2233. (b) Ding, Y. S.; Rich, D. H.; Ikeda, R. A. Biochemistry
1998, 37, 17514. (c) Tozser, J .; Zahuczky, G.; Bagossi, P.; Louis, J . M.;
Copeland, T. D.; Oroszlan, S.; Harrison, R. W.; Weker, I. T. Eur. J .
Biochem. 2000, 267, 6287.
* Address correspondence to this author.
(5) Teruya, K.; Kawakami, T.; Akaji, K.; Aimoto, S. Tetrahedron Lett.
2002, 43, 1487.
(1) Hinuma, Y.; Nagata, K.; Hanaoka, M.; Nakai, M.; Matsumoto,
T.; Kinoshita, K.; Shirakawa, S.; Miyoshi, I. Proc. Natl. Acad. Sci.
U.S.A. 1981, 78, 5476.
(2) (a) Nam, S. H.; Hatanaka, M. Biochem. Biophys. Res. Commun.
1986, 139, 129. (b) Nam, S. H.; Kidokoro, M.; Shida, H.; Hatanaka, M.
J . Viol. 1988, 62, 3718.
(3) (a) Ding, Y. S.; Owen, S. M.; Lal, R. B.; Ikeda, R. A. J . Virol.
1998, 72, 3383. (b) Louis, J . M.; Oroszlan, S.; Tozser, J . J . Biol. Chem.
1999, 274, 6660.
(6) A part of this work was preliminarily presented at the 39th
J apanese Peptide Symposium in Kobe, October 16-18, 2002.
(7) Babine, R. E.; Bender, S. L. Chem. Rev. 1997, 97, 1359.
(8) (a) Szelke, M.; J ones, D. M.; Hallett, A. Eurpean Patent Ap-
plication EP45665, 1982; Chem. Abstr. 1982, 97, 39405p. (b) Holladay,
M. W.; Rich, D. H. Tetrahedron Lett. 1983, 24, 4401. (c) Rich, D. H.;
Green, J .; Toth, M. V.; Marshall, G. R.; Kent, S. B. H. J . Med. Chem.
1990, 33, 1285.
10.1021/jo030063a CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/16/2003
J . Org. Chem. 2003, 68, 4755-4763
4755

Akaji et al.
SCHEME 1
bearing asymmetric center of the isostere would have a
pronounced effect on the inhibitory potency as in the case
of HIV protease inhibitors.9 In this study regarding the
HTLV-1 protease inhibitors, we attempted to evaluate
the effects of the amino acid derived substituent in
combination with the hydroxy group. Thus, a synthetic
scheme, in which the absolute configuration at the
hydroxy- and side chain-bearing asymmetric centers of
the scissile site can be selectively constructed, was
adopted for the construction of the isostere backbone.
Ster eoselective Con str u ction of th e Isoster e Back-
bon e. Scheme 1 shows our synthetic route for the
hydroxyethylamine isostere. Aminoalkyl epoxide is se-
lected as a key intermediate for the construction of the
isostere backbone. In most of the previous syntheses of
the aminoalkyl epoxide or related compounds, N-protect-
ed L-amino acid derivatives were employed as the start-
ing materials.10 These syntheses, however, have limita-
tions in stereochemical control at the hydroxy-bearing
centers as well as in the varieties of side-chain substit-
uents. To avoid these limitations, several stereoselective
syntheses of the aminoalkyl epoxide have been reported,
in which the hydroxy group and the side-chain substitu-
ent were introduced successively.11 In our synthetic
scheme, we selected stereochemical control of both groups
by a single step asymmetric aldol reaction, in which a
chiral ester-derived titanium enolate was employed.12
The enolate was reported to react with monodentate or
bidentate aldehydes to provide anti- or syn-aldol product,
respectively; thus each product would be selectively
prepared by using the same chiral template, cis-1-
arylsulfonamido-2-indanol derivative.
F IGURE 1. The preparation of the syn-type aminoalkyl
epoxide 5. Reagents and conditions: (a) -78 °C, 2 h; (b) 30%
H2O2, LiOH, 23 °C, 12 h; (c) DPPA/Et3N, 23 °C, 24 h; (d) KOH/
EtOH-H2O, reflux, 8 h; (e) di-tert-butyl dicarbonate, DIEA,
23 °C, 24 h; (f) H2/Pd-C, 23 °C, 12 h; (g) Ph3P/DEAD in CHCl3,
reflux, 6 h.
1). (1S,2R)-1-Arylsulfonamido-2-indanyl 4-methylvaler-
ate 1 was converted to titanium enolate and then reacted
with (benzyloxy)acetaldehyde according to the published
procedure to maintain the aldol product as a single
diastereomer. The chiral auxiliary of 2 was removed by
hydrolysis and the crude product, without further puri-
fication, was subjected to Curtius rearrangement. Treat-
ment of the mixture with diphenylphosphoryl azide and
triethylamine gave the oxazolidinone 3 in 61% yield. The
syn configuration at the asymmetric carbons of 3 was
confirmed by homonuclear decoupling experiments. The
coupling constant of the vicinal protons at the asym-
metric carbons (J A-B ) 7.9 Hz) was consistent with that
of the literature value of syn stereochemistry.14 The
oxazolidinone ring of 3 was then opened by hydrolysis
with aqueous KOH. Without further purification, the
amino group of the product was protected with a Boc
group by reaction with di-tert-butyl dicarbonate in DMF
to yield 4. After purification, the benzyl group of 4 was
removed by catalytic hydrogenation and the resulting diol
product was converted to the desired aminoalkyl epoxide
by Mitsunobu reaction. Exposure of the diol with triph-
enylphosphine and diethylazodicarboxylate gave 5 at 54%
yield after chromatography. The enantiomer of 5 was
similarly prepared starting from (1R,2S)-1-arylsulfona-
mido-2-indanyl ester-derived enolate.
The syn-type aminoalkyl epoxide 5 was prepared
according to the route developed by Ghosh et al.13 (Figure
(9) (a) Travins, J . M.; Bursavich, M. G.; Veber, D. F.; Rich, D. H.
Org. Lett. 2001, 3, 2725. (b) Ghosh, A. K.; Thompson, W. J .; Lee, H.
Y.; Mckee, S. P.; Munson, P. M.; Duong, T. T.; Darke, P. L.; Zugay, J .
A.; Emini, E. A.; Schleif, W. A.; Huff, J . R.; Anderson, P. S. J . Med.
Chem. 1993, 36, 924.
(10) (a) Parkes, K. E. B.; Bushnell, D. J .; Crockett, P. H.; Dunsdon,
S. J .; Freeman, A. C.; Gunn, M. P.; Hopkins, R. A.; Lambert, R. W.;
Martin, J . A.; Merrett, J . H.; Redshaw, S.; Spurden, W. C.; Thomas,
G. J . J . Org. Chem. 1994, 59, 3656. (b) Albeck, A.; Persky, R.
Tetrahedron, 1994, 50, 6333. (c) Beaulieu, P. L.; Wernic, D. J . Org.
Chem. 1996, 61, 3635. (d) Branalt, J .; Kvarnstrom, I.; Classon, B.;
Samuelsson, B.; Nillroth, V.; Danielson, U. H.; Karlen, A.; Hallberg,
A. Tetrahedron Lett. 1997, 38, 3483. (e) Beaulieu, P. L.; Lavallee, P.;
Abraham, A.; Anderson, P. C.; Boucher, C.; Bousquet, Y.; Duceppe,
J .-S.; Gillard, J .; Gorys, V.; Grand-Maitre, C.; Grenier, L.; Guindon,
Y.; Guse, I.; Plamondon, L.; Soucy, F.; Valois, S.; Wernic, D.; Yoakim,
C. J . Org. Chem. 1997, 62, 3440.
For preparation of the anti-type aminoalkyl epoxide,
we first examined the synthesis of the anti-type oxazo-
lidinone 22 using the same bidentate aldehyde as above,
(11) (a) Kim, B. M.; Bae, S. J .; So, S. M.; Yoo, H. T.; Chang, S. K.;
Lee, J . H.; Kang, J . S. Org. Lett. 2001, 3, 2349. (b) Tamamura, H.;
Hori, T.; Otaka, A.; Fujii, N. J . Chem. Soc., Perkin Trans. 1 2002, 577.
(12) (a) Ghosh, A. K.; Onishi, M. J . Am. Chem. Soc. 1996, 118, 2527.
(b) Ghosh, A. K.; Fidanze, S.; Onishi, M.; Hussain, K. A. Tetrahedron
Lett. 1997, 41, 7171.
(14) Ghosh, A. K.; Mckee, S. P.; Thompson, W. J . Tetrahedron Lett.
1991, 32, 5729.
(13) Ghosh, A. K.; Fidanze, S. J . Org. Chem. 1998, 63, 6146.
4756 J . Org. Chem., Vol. 68, No. 12, 2003

Solid-Phase Synthesis of HTLV-1 Protease Inhibitor
which was converted to the desired aminoalkyl epoxide
by Mitsunobu reaction as described previously. The
enantiomer of 15 was similarly prepared starting from
(1R,2S)-1-arylsulfonamide-2-indanyl 4-methylvalerate.
Thus, the four stereoisomers of the aminoalkyl epoxide
were selectively prepared by using the asymmetric aldol
reaction.
Solid -P h a se Syn th esis of In h ibitor s. Although most
of the previous syntheses of protease inhibitors were
conducted with use of conventional solution-phase pro-
cedures, these methods were not suitable for library
preparation. In addition, in the syntheses of inhibitors
containing hydroxy-type isosters, formation of the byprod-
ucts derived from overreaction at the hydroxy group
yielded homobislactone or a half-ester dimer.15 To avoid
these limitations, several solid-phase approaches were
reported, in which a multistep conversion of the func-
tional groups would be necessary for the chain elongation
due to the acid-labile linkage to the solid support.16
A
F IGURE 2. Synthetic route for the anti-type aminoalkyl
epoxide 15. Reagents and conditions: (a) cinnamaldehyde/
nBu2BOTf, -78 °C, 1.5 h; (b) 30% H2O2, LiOH, 23 °C, 15 h; (c)
DPPA/Et3N in benzene, reflux, 10 h; (d) KOH/EtOH-H2O,
reflux, 15 h; (e) di-tert-butyl dicarbonate, DIEA, 23 °C, 4 h; (f)
O3, -78 °C, 30 min; (g) NaBH4, 23 °C, 1 h; (h) Ph3P/DEAD in
CHCl3, reflux, 6 h.
key feature of our solid-phase approach is the develop-
ment of an acid-stable linker, which can be incorporated
by simple reaction with succinic anhydride. The linkage
through the secondary alcohol of the isoster serves not
only to anchor the inhibitor to the solid support, but also
to protect the alcohol functionality. The linker can be
easily cleaved under an aqueous basic condition.
but containing a more sterically hindered TBDPS group
instead of the Bn group. The steric bulk could hinder the
effective chelation derived from the ether oxygen, which
could result in conversion of the stereoselectivity.12 The
aldol condensation of the tosyl-indanyl ester 6 with the
aldehyde precomplexed with TiCl4 gave a single product,
21, at 52% isolation yield leaving a small amount of the
starting ester. The product could be converted to the
desired anti-type oxazolidinone 22 according to the same
route as that employed for the syn-oxazolidinone 3.
However, the cyclization yield was poor (less than 10%)
probably due to the steric hindrance of the TBDPS group.
Thus, we decided to use a monodentate aldehyde for
preparation of the anti-type aldol product.
The synthetic scheme for HTLV-1 protease inhibitor
on solid support is shown in Figure 3. The aminoalkyl
epoxide 5 was reacted with proline amide derivative (H-
Pro-NHCH2C6H4I)17 at 70 °C to give the hydroxyethy-
lamine product 23 in 65% yield. A succinic acid linker
was introduced to the hydroxy group of 23 by reaction
with succinic anhydride in the presence of DMAP. The
resulting carbonyl group of the condensed product was
then used for the reaction with the amino group on solid
support to anchor the hydroxyethylamine derivative. As
the solid support, glycyl p-methylbenzhydrylamine poly-
styrene resin was employed, and the anchoring reaction
was carried out with DIPCDI-mediated reaction to yield
28. The Boc group of 28 was removed by treatment with
50% TFA/CH2Cl2. After neutralization with DIEA, Boc-
Ile-OH was condensed by reaction with DIPCDI and
HOBt. The same deprotection/condensation procedure
was employed for the successive introduction of Boc-Val-
OH and Boc-Pro-OH to afford the desired inhibitor resin
29. The product resin was then treated with HF at 4 °C
for 30 min to cleave the inhibitor precursor 30 from the
resin. The product showed a single major peak on an
analytical HPLC without any purification step (Figure
4a). In addition, the crude product had the mass number
expected for the precursor product. Finally, the precursor
product 30 was treated with aqueous AcONH4 at pH 8.0
to cleave the linker derivative.18 The progress of the
reaction could be easily monitored on an analytical
Figure 2 shows the synthetic route for the anti-type
aminoalkyl epoxide 15. Cinnamaldehyde was selected as
the monodentate aldehyde according to a previous re-
port.13 Reaction of the enolate of 1 with cinnamaldehyde
precomplexed with nBu2BOTf afforded the desired anti-
product as a major product (6:1 ratio) in 28% isolated
yield. Reaction with cinnamaldehyde precomplexed with
TiCl4, however, proceeded sluggishly and gave the un-
desired syn-product exclusively with 9% isolation yield.
Oxazolidinone 12 was prepared from the aldol product
11 and the anti stereochemistry of 12 was established
by the vicinal coupling constant (J A-B ) 6.3 Hz)14 as
described for the syn-type oxazolidinone 3. Basic hydroly-
sis of 12 and subsequent Boc-protection produced 13 in
90% yield without difficulties. Ozonolysis of the double
bond followed by reductive workup yielded the diol 14,
(15) Hayashi, Y.; Kinoshita, Y.; Hidaka, K.; Kiso, A.; Uchibori, H.;
Kimura, T.; Kiso, Y. J . Org. Chem. 2001, 66, 5537.
(16) (a) Lee, C. E.; Kick, E. K.; Ellman, J . A. J . Am. Chem. Soc.
1998, 120, 9735. (b) Zhaou, J .; Termin, A.: Wayland, M.; Taby, C. M.
Tetrahedron Lett. 1999, 40, 2729. (c) Chino, M.; Wakao, M.; Ellman,
J . A. Tetrahedron 2002, 58, 6305.
(17) Akaji, K.; Teruya, K.; Akaji, M.; Aimoto, S. Tetrahedron 2001,
57, 2293.
(18) Tamamura, H.; Ishihara, T.; Oyake, H.; Imai, M.; Otaka, A.;
Ibuka, T.; Arakaki, R.; Nakashima, H.; Murakami, T.; Waki, M.;
Matsumoto, A.; Yamamoto, N.; Fujii, N. J . Chem. Soc., Perkin Trans.
1 1998, 495.
J . Org. Chem, Vol. 68, No. 12, 2003 4757

Akaji et al.
F IGURE 4. Analytical HPLC spectra. Reagents and condi-
tions: (a) crude compound 30; (b) after 17 h treatment at pH
8.0, HPLC; YMC AM302 (reversed phase C18, 4.6 × 150 mm),
CH3CN in 0.1% aq TFA (25% to 70%/30 min), 0.9 mL/min flow.
TABLE 1. Ki Va lu es for 31-34a
F IGURE 3. Synthetic scheme for HTLV-1 protease inhibitor
on solid support. Reagents and conditions: (a) H-Pro-
NHCH2C6H4I in 2-propanol, 70 °C, 4 h; (b) succinic anhydride/
DMAP, 23 °C, 12 h; (c) H-Gly-MBHA resin, DIPCDI/HOBt,
23 °C, 15 h; (d) Boc-based solid-phase synthesis; (e) HF, 4 °C,
50 min; (f) 0.4 M AcONH4, pH 8.0, 23 °C, 17 h.
HPLC. After 17 h of treatment, most of the precursor
converted to the desired product having a single major
peak on HPLC (Figure 4b) and a desired mass number.
The product was purified by preparative HPLC to
produce the inhibitor 31 with 46% yield calculated from
the starting MBHA-resin. Three other diastereomers
were also prepared according to the same route starting
from the corresponding aminoalkyl epoxide synthesized
above.
In h ibitor y Activity. The activity of each inhibitor
was examined by using the auto-digestion resistant
protease mutant prepared by chemical synthesis.5 Syn-
thetic dodecapeptide, which contains the cleavage site
corresponding to the C-terminal portion of the protease
itself, was employed as the substrate. Cleavage of the
substrate by the mutant enzyme was monitored on an
analytical HPLC. Inhibitory activity of each inhibitor was
evaluated by using the corresponding Ki value deter-
mined by the least-squares method in a Dixon plot.19 For
a
R1 ) H-Pro-Val-Ile, R2 ) CH2C6H4I.
comparison, pepstatin was used as the standard inhibitor
for aspartic protease.
As shown in Table 1, all four diastereomers prepared
above inhibited the enzyme reaction more strongly than
pepstatin. Among them, the inhibitor 31, which has the
hydroxyethylamine isoster composed of the R-configura-
tion for the hydroxy group and the S-configuration for
the scissile-site side chain, was most effective. The
inhibitor 31 showed a Ki value of 38 nM, a 250-fold
increase in potency over pepstatin. As expected, the
(19) Dixon, M. Biochem. J . 1953, 55, 170.
4758 J . Org. Chem., Vol. 68, No. 12, 2003

Solid-Phase Synthesis of HTLV-1 Protease Inhibitor
6.32 g (65%) of 6 as a white solid: mp 118-119 °C, [R]25D +73.8
configuration at the hydroxy-bearing asymmetric center
showed remarkable effects on the inhibitory activity. The
Ki value decreased by about 2 orders of magnitude by
the configuration change of the hydroxy group (compound
31 vs 33). The decrease by the change at the side chain-
bearing center was less than 1 order of magnitude
(compound 31 vs 32). Details of the structure-activity
relationship are the subject of ongoing investigation, and
will be published in the future.
1
(c 0.5, CHCl3); H NMR (400 MHz, CDCl3) δ 0.84 (d, J ) 6.5
Hz, 3H), 0.85 (d, J ) 6.5 Hz, 3H), 1.37-1.41 (m, 2H), 1.43-
1.53 (m, 1H), 2.13 (dd, J ) 7.4, 7.4 Hz, 2H), 2.45 (s, 3H), 2.88
(d, J ) 17.3 Hz, 1H), 3.08 (dd, J ) 17.3, 5.0 Hz, 1H), 4.99 (dd,
J ) 10.3, 5.0 Hz, 1H), 5.12 (dd, J ) 5.0, 5.0 Hz, 1H), 5.18 (d,
J ) 10.3 Hz, 1H), 7.19-7.23 (m, 4H), 7.32 (d, J ) 8.2 Hz, 2H),
7.81 (d, J ) 8.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 21.55,
22.14, 27.55, 32.06, 33.35, 37.47, 59.49, 74.56, 124.25, 124.96,
126.91, 127.38, 128.61, 129.86, 137.81, 138.61, 139.68, 143.81,
172.82. Anal. Calcd for C22H27O4NS: C, 65.70; H, 6.72; N, 3.48.
Found: C, 65.62; H, 6.77; N, 3.48; HRFAB MS, m/z 402.1745
for [M + H]+ (calcd for C22H28O4NS 402.1739).
Con clu sion
(1S,2R)-cis-N-(2,3-Dih yd r o-2-[((2S,3S)-3-h yd r oxy-2-
(isobu tyl)-4-(p h en ylm eth oxy)-1-oxobu toxy)in d en -1-yl]-4-
m eth ylben zen esu lfon a m id e (2). To a solution of 1 (2.00 g,
5.0 mmol) in CH2Cl2 (15 mL) was added a 1 M solution of TiCl4
(6.00 mL) in CH2Cl2 at 4 °C. The mixture was stirred at 23 °C
for 15 min. To this solution was added DIEA (3.30 mL, 19
mmol) and the mixture was stirred for 2 h at 23 °C. The
resulting solution was added to the aldehyde solution prepared
at -78 °C by the addition of the 1 M solution of TiCl4 (10.5
mL in CH2Cl2) to a stirred solution of (benzyloxy)acetaldehyde
(1.57 g, 10 mmol) in CH2Cl2 (10 mL) followed by 15 min of
stirring. The mixture was stirred at -78 °C for 2 h and then
quenched by the addition of aqueous ammonium chloride. The
organic layer was washed with H2O, dried over MgSO4, and
rotary evaporated. The crude product was purified by silica
gel column chromatography with CHCl3 followed by flash
chromatography with hexane:AcOEt 3:1 to yield 2.61 g (95%)
We have achieved the first solid-phase synthesis of
HTLV-1 protease inhibitor containing a hydroxyethyl-
amine isostere backbone. The key features of the present
synthesis are the stereoselective construction of the
isostere by the ester-derived asymmetric aldol reaction
and the development of a new succinic acid linker, which
enables an efficient preparation of the inhibitors on solid
support. Using the synthetic inhibitors prepared stereo-
selectively, we have succeeded in evaluating the stereo-
structure-activity relationship of HTLV-1 protease in-
hibitors. Configuration of the hydroxy group at the
isostere backbone influenced the activity by 2 orders of
magnitude, while the influence of the side chain at the
scissile bond was less than 1 order of magnitude. The
present synthetic approach should provide an efficient
procedure to prepare the hydroxy-containing inhibitors
on solid support. Application of this method in the
construction of an HTLV-1 protease inhibitor library is
currently in progress.
of 2 as an oil: [R]25 -20.4 (c 1.1, CHCl3); 1H NMR (400 MHz,
D
CDCl3) δ 0.78 (d, J ) 6.6 Hz, 3H), 0.83 (d, J ) 6.6 Hz, 3H),
1.17 (ddd, J ) 13.5, 9.4, 3.9 Hz, 1H), 1.34-1.41 (m, 1H), 1.55
(ddd, J ) 13.5, 11.2, 4.5 Hz, 1H), 2.44 (s, 3H), 2.57-2.62 (m,
1H), 2.73 (d, J ) 4.0 Hz, 1H), 2.86 (d, J ) 17.2 Hz, 1H), 3.06
(dd, J ) 17.2, 4.6 Hz, 1H), 3.33 (dd, J ) 9.6, 7.3 Hz, 1H), 3.42
(dd, J ) 9.6, 3.8 Hz, 1H), 3.93-3.98 (m, 1H), 4.39 (d, J ) 11.8
Hz, 1H), 4.43 (d, J ) 11.8 Hz, 1H), 4.92 (dd, J ) 9.9, 4.8 Hz,
1H), 5.32 (dd, J ) 4.8, 4.6 Hz, 1H), 5.82 (d, J ) 9.9 Hz, 1H),
7.13-7.36 (m, 1H), 7.81 (d, J ) 8.2 Hz, 2H); 13C NMR (100
MHz, CDCl3) δ 21.56, 21.61, 23.30, 26.33, 35.32, 37.19, 46.93,
59.67, 71.14, 71.53, 73.38, 75.83, 124.30, 124.77, 127.07,
127.40, 127.63, 127.83, 127.89, 128.00, 128.45, 128.47, 128.60,
129.79, 137.51, 137.91, 138.40, 140.01, 143.60, 173.04; HRFAB
MS, m/z 552.2413 for [M + H]+ (calcd for C31H38O6NS
552.2420).
Exp er im en ta l Section
Gen er a l. Solvents were reagent grade and dried prior to
use. Fmoc-Gly-OH, Boc amino acid derivatives, and p-meth-
ylbenzhydrylamine resin were obtained from Peptide Institute,
Inc. (Osaka, J apan) and were used without further purifica-
tion. Melting points were uncorrected. The 1H and 13C NMR
spectra were recorded on a 400-MHz spectrometer with TMS
as an internal standard. HPLC was carried out on a reversed-
phase column, which was eluted with CH3CN in 0.1% aqueous
TFA and detected at OD 220 nm.
The (1R,2S) derivative 7 was similarly prepared from 6 (1.00
g, 2.5 mmol) to give 1.29 g (94%) of 7 as an oil: [R]25D +19.0 (c
0.7, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.78 (d, J ) 6.5 Hz,
3H), 0.83 (d, J ) 6.5 Hz, 3H), 1.18 (ddd, J ) 13.5, 9.4, 3.9 Hz,
1H), 1.34-1.41 (m, 1H), 1.55 (ddd, J ) 13.5, 11.2, 4.4 Hz, 1H),
2.44 (s, 3H), 2.56-2.61 (m, 1H), 2.69 (d, J ) 4.0 Hz, 1H), 2.86
(d, J ) 17.2 Hz, 1H), 3.06 (dd, J ) 17.2, 4.6 Hz, 1H), 3.33 (dd,
J ) 9.6, 7.3 Hz, 1H), 3.42 (dd, J ) 9.6, 3.9 Hz, 1H), 3.92-3.97
(m, 1H), 4.39 (d, J ) 11.8 Hz, 1H), 4.43 (d, J ) 11.8 Hz, 1H),
4.92 (dd, J ) 9.9, 4.8 Hz, 1H), 5.33 (dd, J ) 4.8, 4.6 Hz, 1H),
5.78 (d, J ) 9.9 Hz, 1H), 7.13-7.37 (m, 1H), 7.81 (d, J ) 8.3
Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 21.54, 21.61, 23.29,
26.34, 35.35, 37.19, 46.95, 59.67, 71.15, 71.54, 73.38, 75.83,
124.31, 124.76, 127.07, 127.39, 127.62, 127.82, 127.87, 128.02,
128.20, 128.46, 128.59, 129.78, 137.53, 137.95, 138.41, 140.02,
143.58, 173.04; HRFAB MS, m/z 552.2426 for [M + H]+ (calcd
for C31H38O6NS 552.2420).
(4S,5S)-4-Isobu tyl-5-[(p h en ylm eth yloxy)m eth yl]oxa zo-
lid in -2-on e (3). To a stirred solution of 2 (2.61 g, 4.7 mmol)
in THF:H2O (3:1, 20 mL) were added 30% H2O2 (4.5 mL) and
LiOH‚H2O (840 mg, 20 mmol), and the mixture was stirred at
23 °C for 12 h. Aqueous Na2SO3 (1.5 M) and saturated aqueous
NaHCO3 were added to the mixture, and the organic solvent
was removed in vacuo. The resulting solution was acidified
with AcOH, and then extracted with CHCl3. The organic layer
was washed with H2O, dried over MgSO4, and rotary evapo-
(1S,2R)-cis-N-[2,3-Dih ydr o-2-(4-m eth yl-1-oxopen tyloxy)-
in d en -1-yl]-4-m eth ylben zen esu lfon a m id e (1). To a stirred
solution of (1S,2R)-cis-N-tosyl-1-aminoindan-2-ol (7.76 g, 26
mmol) in CHCl3 (100 mL) were added 4-methylvaleric acid
(3.90 mL, 31 mmol), (dimethylamino)pyridine (DMAP, 940 mg,
7.7 mmol), diisopropylethylamine (DIEA, 4.50 mL, 26 mmol),
and diisopropylcarbodiimide (DIPCDI, 6.10 mL, 0.45 mmol).
The mixture was stirred at 25 °C for 24 h and then filtered.
The filtrate was washed with H2O and dried over anhydrous
MgSO4. The solvent was removed and the resulting residue
was recrystallized from ether to yield 6.50 g (63%) of 1 as a
o
white solid: mp 118-119.5 C, [R]25 -77.6 (c 0.5, CHCl3); 1H
D
NMR (400 MHz, CDCl3) δ 0.84 (d, J ) 6.5 Hz, 3H), 0.85 (d, J
) 6.5 Hz, 3H), 1.36-1.42 (m, 2H), 1.44-1.52 (m, 1H), 2.14 (dd,
J ) 7.2, 7.2 Hz, 2H), 2.45 (s, 3H), 2.89 (d, J ) 17.3 Hz, 1H),
3.09 (dd, J ) 17.3, 5.0 Hz, 1H), 4.98 (dd, J ) 10.2, 5.2 Hz,
1H), 5.14 (dd, J ) 5.2, 5.0 Hz, 1H), 5.14 (d, J ) 10.2 Hz, 1H),
7.16-7.29 (m, 4H), 7.31 (d, J ) 8.2 Hz, 2H), 7.81 (d, J ) 8.2
Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 21.53, 22.13, 27.56,
32.08, 33.39, 37.47, 59.51, 74.58, 124.27, 124.96, 126.93,
127.39, 128.62, 129.85, 137.88, 138.63, 139.71, 143.80, 172.81.
Anal. Calcd for C22H27O4NS: C, 65.70; H, 6.72; N, 3.48.
Found: C, 65.59; H, 6.77; N, 3.56; HRFAB MS, m/z 402.1733
for [M + H]+ (calcd for C22H28O4NS 402.1739).
The (1R,2S) derivative 6 was similarly prepared from
(1R,2S)-cis-N-tosyl-1-aminoindan-2-ol (7.38 g, 24 mmol) to give
J . Org. Chem, Vol. 68, No. 12, 2003 4759

Akaji et al.
1
rated. The residue was dissolved in CH2Cl2 (10 mL). To this
solution were added Et3N (2.79 mL, 20 mmol) and diphe-
nylphosphoryl azide (4.30 mL, 20 mmol). The mixture was
stirred at 23 °C for 24 h, and the solvent was removed in vacuo.
The product was purified by silica gel column chromatography
with CHCl3 followed by flash chromatography with hexane:
5 as an oil: [R]25 -25.4 (c 1.0, CHCl3); H NMR (400 MHz,
D
CDCl3) δ 0.91 (d, J ) 6.6 Hz, 3H), 0.93 (d, J ) 6.6 Hz, 3H),
1.36-1.41 (m, 2H), 1.44 (s, 9H), 1.71-1.78 (m, 1H), 2.75 (br d,
J ) 3.8 Hz, 2H), 2.83-2.85 (m, 1H), 3.51 (br s, 1H), 4.41 (br s,
1H); 13C NMR (100 MHz, CDCl3) δ 21.79, 23.29, 24.44, 28.33,
40.82, 46.09, 50.30, 54.50, 79.42, 155.41; HRFAB MS, m/z
230.1750 for [M + H]+ (calcd for C12H24O3N 230.1756).
AcOEt 3:1 to yield 0.760 g (61%) of 3 as an oil: [R]25 -13.7 (c
D
0.8, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.87 (d, J ) 6.6 Hz,
3H), 0.92 (d, J ) 6.6 Hz, 3H), 1.23 (ddd, J ) 13.4, 10.0, 3.4
Hz, 1H), 1.50 (ddd, J ) 13.4, 11.0, 4.3 Hz, 1H), 1.58-1.66 (m,
1H), 3.67 (d, J ) 5.8 Hz, 2H), 3.93-3.99 (m, 1H), 4.53 (d, J )
12.0 Hz, 1H), 4.59 (d, J ) 12.0 Hz, 1H), 4.71-4.76 (m, 1H),
7.06 (br s, 1H), 7.29-7.37 (m, 5H); 13C NMR (100 MHz, CDCl3)
δ 20.83, 23.73, 24.61, 38.05, 52.53, 67.37, 73.48, 77.78, 127.69,
127.75, 128.33, 137.36, 159.72; HRFAB MS, m/z 264.1606 for
[M + H]+ (calcd for C15H22O3N 264.1600).
The (2R,1′R)-oxirane 10 was similarly prepared starting
from 9 (0.640 g, 1.9 mmol) to yield 0.240 g (55%) of 10 as an
1
oil: [R]25 +25.1 (c 0.7, CHCl3); H NMR (400 MHz, CDCl3) δ
D
0.91 (d, J ) 6.6 Hz, 3H), 0.94 (d, J ) 6.6 Hz, 3H), 1.35-1.41
(m, 2H), 1.44 (s, 9H), 1.71-1.78 (m, 1H), 2.75 (br d, J ) 3.8
Hz, 2H), 2.83-2.85 (m, 1H), 3.51 (br s, 1H), 4.40 (br s, 1H);
13C NMR (100 MHz, CDCl3) δ 21.83, 23.33, 24.49, 28.37, 40.87,
46.14, 50.35, 54.54, 79.48, 155.44; HRFAB MS, m/z 230.1763
for [M + H]+ (calcd for C12H24O3N 230.1756).
The (4R,5R) derivative 8 was similarly prepared from 7 (1.25
(1S,2R)-N-[2,3-Dih ydr o-2-((2S,3S)-(E)-3-h ydr oxy-5-ph en -
yl-2-(isob u t yl)-1-oxop en t -4-en oxy)in d en -1-yl]-4-m et h yl-
ben zen esu lfon a m id e (11). To a stirred solution of 1 (400 mg,
1.0 mmol) in CH2Cl2 (8 mL) was added a 1 M solution of TiCl4
in CH2Cl2 (1.20 mL) at 4 °C, and the mixture was stirred at
23 °C for 15 min. To this solution was added DIEA (660 µL,
3.8 mmol). The mixture was stirred for 90 min at 23 °C, and
then cooled to -78 °C. The aldehyde solution, prepared at -78
g, 2.3 mmol) to give 0.360 g (60%) of 8 as an oil: [R]25 +13.1
D
1
(c 0.6, CHCl3); H NMR (400 MHz, CDCl3) δ 0.89 (d, J ) 6.6
Hz, 3H), 0.94 (d, J ) 6.6 Hz, 3H), 1.29 (ddd, J ) 13.3, 9.7, 3.4
Hz, 1H), 1.50 (ddd, J ) 13.3, 10.7, 4.4 Hz, 1H), 1.56-1.622
(m, 1H), 3.68 (d, J ) 5.8 Hz, 2H), 3.94-3.99 (m, 1H), 4.54 (d,
J ) 12.0 Hz, 1H), 4.59 (d, J ) 12.0 Hz, 1H), 4.72-4.77 (m,
1H), 6.07 (br s, 1H), 7.28-7.37 (m, 5H); 13C NMR (100 MHz,
CDCl3) δ 21.05, 23.72, 24.95, 38.14, 52.66, 67.40, 73.66, 77.85,
127.82, 127.90, 128.46, 137.45, 159.16; HRFAB MS, m/z
264.1595 for [M + H]+ (calcd for C15H22O3N 264.1600).
n
°C by the addition of a 1 M solution of Bu2BOTf (3.5 mL in
CH2Cl2) to a stirred solution of cinnamaldehyde (440 µL, 3.5
mmol) in CH2Cl2 (8 mL) followed by 30 min of stirring, was
added to the above solution at -78 °C. The mixture was stirred
at -78 °C for 90 min. MeOH (3 mL), pH 7 buffer (2 mL), and
30% H2O2 (1 mL) were added successively, and the mixture
was allowed to warm to 23 °C. CHCl3 (10 mL) was added and
the organic layer was washed with 5% aqueous NaHCO3 and
H2O, dried over MgSO4, and rotary evaporated. The crude
product was purified by silica gel column chromatography with
CHCl3 followed by flash chromatography with hexane:AcOEt
(2S,3S)-3-[N-(ter t-Bu tyloxyca r bon yl)a m in o]-5-m eth yl-
1-(p h en ylm eth yl)h exa n -2-ol (4). To a stirred solution of 3
(0.750 g, 2.9 mmol) in EtOH (10 mL) was added 85% KOH
(0.750 g, 11 mmol) in H2O (4 mL), and the mixture was heated
at reflux for 8 h. The solution was neutralized with 1 N HCl
and the solvent was removed to dryness. The residue was
suspended in DMF (5 mL), and di-tert-butyl dicarbonate (1.24
g, 5.7 mmol) and DIEA (400 µL, 2.3 mmol) were added to the
suspension. The mixture was stirred at 23 °C for 24 h, and
the solvent was removed in vacuo. The product was extracted
with CHCl3 and the organic layer was washed with H2O, dried
over MgSO4, and rotary evaporated. The crude product was
purified by silica gel column chromatography with CHCl3
followed by flash chromatography with hexane:AcOEt 3:1 to
6:1 to yield 150 mg (28%) of 11 (and 25 mg of a syn-
1
diastereomer) as an oil: [R]25 -15.5 (c 0.8, CHCl3); H NMR
D
(400 MHz, CDCl3) δ 0.83 (d, J ) 6.4 Hz, 3H), 0.87 (d, J ) 6.4
Hz, 3H), 1.14-1.21 (m, 1H), 1.45-1.53 (m, 2H), 2.42 (s, 3H),
2.60-2.66 (m, 1H), 2.82 (d, J ) 17.1 Hz, 1H), 2.83 (d, J ) 4.7
Hz, 1H), 3.08 (dd, J ) 17.1, 4.8 Hz, 1H), 4.20-4.25 (m, 1H),
4.83 (dd, J ) 9.4, 5.0 Hz, 1H), 5.42 (dd, J ) 5.0, 4.8 Hz, 1H),
6.05 (dd, J ) 15.8, 7.3 Hz, 1H), 6.28 (d, J ) 9.4 Hz, 1H), 6.54
(d, J ) 15.8 Hz, 1H), 7.05-7.07 (m, 1H), 7.22-7.31 (m, 9H),
7.38-7.40 (m, 1H), 7.81 (d, J ) 8.3 Hz, 2H); 13C NMR (100
MHz, CDCl3) δ 21.54, 21.66, 23.30, 26.18, 37.33, 38.09, 50.63,
59.68, 74.50, 74.91, 124.55, 124.76, 126.65, 127.23, 127.34,
128.10, 128.32, 128.62, 128.86, 129.70, 133.29, 135.98, 137.63,
138.37, 140.22, 143.46, 172.56; HRFAB MS, m/z 556.2141 for
[M + Na]+ (calcd for C31H35O5NSNa 556.2134).
yield 0.610 g (63%) of 4 as an oil: [R]25 -29.0 (c 0.8, CHCl3);
D
1H NMR (400 MHz, CDCl3) δ 0.91 (t, J ) 6.8 Hz, 6H), 1.25-
1.37 (m, 2H), 1.43 (s, 9H), 1.65-1.67 (m, 1H), 2.85 (br s, 1H),
3.49-3.58 (m, 2H), 3.77 (br s, 2H), 4.55 (s, 2H), 4.72 (br d, J
) 7.0 Hz, 1H), 7.29-7.37 (m, 5H); 13C NMR (100 MHz, CDCl3)
δ 21.70, 23.55, 24.70, 28.34, 39.73, 51.55, 71.81, 73.04, 73.54,
79.30, 127.70, 127.78, 128.44, 137.86, 156.25; HRFAB MS, m/z
338.2327 for [M + H]+ (calcd for C19H32O4N 338.2331).
The (2R,3R) derivative 9 was similarly prepared starting
from 8 (0.750 g, 2.9 mmol) to give 0.650 g (68%) of 9 as an oil:
The (1R,2S) derivative 16 was similarly prepared starting
from 6 (1.26 g, 3.1 mmol) to give 0.430 g (26%) of 16 as an oil:
[R]25 +26.2 (c 0.6, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.91
D
(t, J ) 6.8 Hz, 6H), 1.25-1.37 (m, 2H), 1.43 (s, 9H), 1.63-1.67
(m, 1H), 2.83 (br s, 1H), 3.49-3.58 (m, 2H), 3.77 (br s, 2H),
4.55 (s, 2H), 4.71 (br d, J ) 6.5 Hz, 1H), 7.29-7.37 (m, 5H);
13C NMR (100 MHz, CDCl3) δ 21.71, 23.55, 24.71, 28.35, 39.74,
51.57, 71.81, 73.05, 73.55, 79.32, 127.71, 127.79, 128.45,
137.86, 156.26; HRFAB MS, m/z 338.2328 for [M + H]+ (calcd
for C19H32O4N 338.2331).
[R]25 +14.9 (c 0.6, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.83
D
(d, J ) 6.4 Hz, 3H), 0.88 (d, J ) 6.4 Hz, 3H), 1.18-1.22 (m,
1H), 1.46-1.54 (m, 2H), 2.43 (s, 3H), 2.61-2.66 (m, 1H), 2.82
(d, J ) 17.2 Hz, 1H), 2.78 (d, J ) 4.5 Hz, 1H), 3.05 (dd, J )
17.2, 4.8 Hz, 1H), 4.21-4.26 (m, 1H), 4.84 (dd, J ) 9.4, 5.0
Hz, 1H), 5.43 (dd, J ) 5.0, 4.8 Hz, 1H), 6.06 (dd, J ) 15.8 Hz,
7.3 Hz, 1H), 6.24 (d, J ) 9.4 Hz, 1H), 6.56 (d, J ) 15.8 Hz,
1H), 7.06-7.07 (m, 1H), 7.21-7.32 (m, 9H), 7.38-7.40 (m, 1H),
7.81 (d, J ) 8.3 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 21.54,
21.68, 23.30, 26.20, 37.34, 38.13, 50.64, 59.70, 74.51, 74.94,
124.55, 124.77, 126.66, 127.24, 127.35, 128.12, 128.34, 128.63,
128.89, 129.72, 133.31, 135.99, 137.66, 138.38, 140.23, 143.48,
172.57; HRFAB MS, m/z 556.2128 for [M + Na]+ (calcd for
2(S)-[1′-(S)-N-(ter t-Bu tyloxyca r bon yl)a m in o-3′-m eth yl-
2-bu tyl]oxir a n e (5). To a stirred solution of 4 (0.600 g, 1.8
mmol) in MeOH (10 mL) was added a catalytic amount of 10%
Pd-C, and the mixture was stirred at 23 °C for 12 h under H2
atmosphere. Pd-C was removed by filtration, and the filtrate
was rotary evaporated. The residue was dissolved in CHCl3
(5 mL), and Ph3P (1.40 g, 5.3 mmol) and diethyl azodicarboxy-
late (1.20 mL, 7.5 mmol) were added to the solution. The
mixture was heated at reflux for 6 h. After cooling, the reaction
mixture was washed with H2O, dried over MgSO4, and rotary
evaporated. The crude product was purified by silica gel
column chromatography with CHCl3 followed by flash chro-
matography with hexane:AcOEt 3:1 to yield 0.220 g (54%) of
C
31H35O5NSNa 556.2134).
(4S ,5S )-4-Isob u t yl-5-[(E )-p h e n ylvin yl]oxa zolid in -2-
on e (12). To a stirred solution of 11 (0.740 g, 1.4 mmol) in
THF:H2O (3:1, 20 mL) were added 30% H2O2 (1.6 mL) and
LiOH‚H2O (0.290 g, 6.9 mmol), and the mixture was stirred
at 23 °C for 15 h. Aqueous Na2SO3 (1.5 M) and saturated
aqueous NaHCO3 were added to the mixture and the organic
4760 J . Org. Chem., Vol. 68, No. 12, 2003

Solid-Phase Synthesis of HTLV-1 Protease Inhibitor
solvent was removed in vacuo. The resulting solution was
acidified with AcOH and then extracted with CHCl3. The
organic layer was washed with H2O, dried over MgSO4, and
rotary evaporated. The residue was dissolved in benzene (10
mL). To this solution were added Et3N (0.580 mL, 4.1 mmol)
and diphenylphosphoryl azide (0.900 mL, 4.1 mmol). The
mixture was stirred at reflux for 10 h, and the solvent was
removed in vacuo. The product was purified by silica gel
column chromatography with CHCl3 followed by flash chro-
H2O, dried, and rotary evaporated. The product was purified
by silica gel column chromatography with CHCl3:MeOH 20:1
to yield 71 mg (76%) of 14 as an oil: [R]25D -26.3 (c 0.5, CHCl3);
1H NMR (400 MHz, CDCl3) δ 0.93 (d, J ) 6.4 Hz, 3H), 0.94 (d,
J ) 6.4 Hz, 3H), 1.32 (ddd, J ) 13.8, 8.5, 5.0 Hz, 1H), 1.45 (s,
9H), 1.53 (ddd, J ) 13.8, 9.9, 5.5 Hz, 1H), 1.64-1.74 (m, 1H),
2.27 (br d, J ) 5.0 Hz, 1H), 3.28 (br t, J ) 6.5 Hz, 1H), 3.45-
3.51 (m, 1H), 3.54-3.60 (m, 1H), 3.64-3.66 (m, 1H), 3.77-
3.82 (m, 1H), 4.62 (br d, J ) 9.0 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 21.97, 23.07, 24.82, 28.31, 40.89, 49.21, 63.64, 73.69,
80.02, 157.23; HRFAB MS, m/z 248.1858 for [M + H]+ (calcd
for C12H26O4N 248.1862).
matography with hexane:AcOEt 3:1 to yield 0.225 g (66%) of
1
12 as an oil: [R]25 -59.4 (c 0.7, CHCl3); H NMR (400 MHz,
D
CDCl3) δ 0.94 (d, J ) 6.6 Hz, 3H), 0.96 (d, J ) 6.6 Hz, 3H),
1.48 (ddd, J ) 13.5, 8.3, 5.0 Hz, 1H), 1.57 (ddd, J ) 13.5, 8.5,
6.0 Hz, 1H), 1.65-1.74 (m, 1H), 3.67-3.72 (m, 1H), 4.69-4.73
(m, 1H), 5.96 (br s, 1H), 6.22 (dd, J ) 15.8, 7.5 Hz, 1H), 6.72
(d, J ) 15.8 Hz, 1H), 7.30-7.42 (m, 5H); 13C NMR (100 MHz,
CDCl3) δ 21.97, 23.01, 25.14, 43.64, 56.90, 83.64, 124.60,
126.82, 128.59, 128.71, 134.53, 135.45, 158.99; HRFAB MS,
m/z 246.1499 for [M + H]+ (calcd for C15H20O2N 246.1494).
The (2S,3R) derivative 19 was similarly prepared starting
from 18 (130 mg, 0.41 mmol) to yield 97 mg (96%) of 19 as an
1
oil: [R]25 +30.4 (c 0.5, CHCl3); H NMR (400 MHz, CDCl3) δ
D
0.93 (d, J ) 6.3 Hz, 3H), 0.94 (d, J ) 6.3 Hz, 3H), 1.32 (ddd,
J ) 13.8, 8.7, 5.1 Hz, 1H), 1.45 (s, 9H), 1.53 (ddd, J ) 13.8,
9.8, 5.5 Hz, 1H), 1.63-1.72 (m, 1H), 2.18 (br d, J ) 5.1 Hz,
1H), 3.22 (br t, J ) 6.5 Hz, 1H), 3.47-3.51 (m, 1H), 3.54-3.61
(m, 1H), 3.64-3.66 (m, 1H), 3.77-3.82 (m, 1H), 4.59 (br d, J
) 8.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 21.97, 23.07,
24.82, 28.30, 40.88, 49.21, 63.65, 73.69, 80.03, 157.23; HRFAB
MS, m/z 248.1866 for [M + H]+ (calcd for C12H26O4N 248.1862).
The (4R,5R) derivative 17 was similarly prepared from 16
(0.420 g, 0.79 mmol) to yield 123 mg (64%) of 17 as an oil:
[R]25 +65.4 (c 0.6, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.93
D
(d, J ) 6.6 Hz, 3H), 0.96 (d, J ) 6.6 Hz, 3H), 1.48 (ddd, J )
13.5, 8.2, 5.0 Hz, 1H), 1.57 (ddd, J ) 13.5, 8.5, 6.0 Hz, 1H),
1.67-1.74 (m, 1H), 3.67-3.72 (m, 1H), 4.69-4.73 (m, 1H), 6.01
(br s, 1H), 6.22 (dd, J ) 15.8 , 7.5 Hz, 1H), 6.72 (d, J ) 15.8
Hz, 1H), 7.29-7.42 (m, 5H); 13C NMR (100 MHz, CDCl3) δ
21.98, 23.01, 25.14, 43.67, 56.92, 83.63, 124.64, 126.83, 128.56,
128.71, 134.52, 135.48, 159.02; HRFAB MS, m/z 246.1488 for
[M + H]+ (calcd for C15H20O2N 246.1494).
2(R)-[1′-(S)-N-(ter t-Bu tyloxyca r bon yl)a m in o-3′-m eth yl-
2-bu tyl]oxir a n e (15). To a stirred solution of 14 (60 mg, 0.24
mmol) in CHCl3 (5 mL) were added Ph3P (220 mg, 0.84 mmol)
and diethyl azodicarboxylate (160 µL, 1.0 mmol), and the
mixture was heated at reflux for 6 h. After cooling, the reaction
mixture was washed with H2O, dried, and rotary evaporated.
The crude product was purified by flash chromatography with
(3S,4S)-(E)-4-[N-(ter t-Bu tyloxycar bon yl)am in o]-6-m eth -
yl-1-p h en ylh ep t-1-en e-3-ol (13). To a stirred solution of 12
(220 mg, 0.90 mmol) in EtOH (5 mL) was added 85% KOH
(240 mg, 3.6 mmol) in H2O (5 mL), and the mixture was heated
at reflux for 15 h. The solution was neutralized with 1 N HCl
and the solvent was removed to dryness. The residue was
suspended in DMF (5 mL), and di-tert-butyl dicarbonate (390
mg, 1.8 mmol) and DIEA (160 µL, 0.90 mmol) were added to
the suspension. The mixture was stirred at 23 °C for 4 h, and
the solvent was removed in vacuo. The product was extracted
with CHCl3 and the organic layer was washed with H2O, dried,
and rotary evaporated. The crude product was purified by flash
chromatography with hexane:AcOEt 5:1 to yield 260 mg (91%)
of 13 as an oil: [R]25D -15.5 (c 0.6, CHCl3); 1H NMR (400 MHz,
CDCl3) δ 0.93 (s, 3H), 0.95 (s, 3H), 1.40 (s, 9H), 1.40-1.47 (m,
2H), 1.68-1.73 (m, 1H), 2.53 (br s, 1H), 3.75 (br s, 1H), 4.24
(br s, 1H), 4.64 (br d, J ) 8.4 Hz, 1H), 6.23 (dd, J ) 15.9, 6.4
Hz, 1H), 6.62 (d, J ) 15.9 Hz, 1H), 7.21-7.38 (m, 5H); 13C
NMR (100 MHz, CDCl3) δ 21.93, 23.34, 24.86, 28.31, 40.64,
53.35, 75.20, 79.44, 126.54, 127.64, 128.50, 129.52, 131.44,
136.68, 156.45; HRFAB MS, m/z 320.2221 for [M + H]+ (calcd
for C15H20O2N 320.2226).
hexane:AcOEt 6:1 to yield 35 mg (63%) of 15 as an oil: [R]25
D
-9.3 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.95 (s, 3H),
0.97 (s, 3H), 1.43 (s, 9H), 1.43-1.51 (m, 2H), 1.70-1.77 (m,
1H), 2.59 (br s, 1H), 2.73 (t, J ) 4.4 Hz, 1H), 2.98 (br s, 1H),
3.97 (br s, 1H), 4.29 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ
22.12, 23.04, 24.70, 28.32, 42.34, 44.39, 47.18, 53.94, 79.38,
155.65; HRFAB MS, m/z 230.1760 for [M + H]+ (calcd for
C
12H24O3N 230.1756).
The (2S,1′R)-oxirane 20 was similarly prepared starting
from 19 (90 mg, 0.36 mmol) to yield 52 mg (62%) of 20 as an
1
oil: [R]25 +10.5 (c 0.4, CHCl3); H NMR (400 MHz, CDCl3) δ
D
0.95 (s, 3H), 0.96 (s, 3H), 1.43 (s, 9H), 1.43-1.51 (m, 2H), 1.70-
1.77 (m, 1H), 2.59 (br s, 1H), 2.73 (t, J ) 4.3 Hz, 1H), 2.98 (br
s, 1H), 3.97 (br s, 1H), 4.29 (br s, 1H); 13C NMR (100 MHz,
CDCl3) δ 22.12, 23.04, 24.70, 28.32, 42.34, 44.38, 47.18, 53.94,
79.38, 155.65; HRFAB MS, m/z 230.1752 for [M + H]+ (calcd
for C12H24O3N 230.1756).
(1R ,2S )-cis-N -(2,3-Dih yd r o-2-[(2R ,3S)-3-h yd r oxy-2-
(isobu tyl)-4-(ter t-bu tyld ip h en ylsilyoxy)-1-oxobu toxy]in -
d en -1-yl)-4-m eth ylben zen esu lfon a m id e (21). To a stirred
solution of 6 (400 mg, 1.0 mmol) in CH2Cl2 (5 mL) was added
a 1 M solution of TiCl4 in CH2Cl2 (1.2 mL) at 4 °C. The mixture
was stirred at 23 °C for 15 min. To this solution was added
DIEA (660 µL, 3.8 mmol), and the mixture was stirred for 90
min at 23 °C. The resulting solution was added to the aldehyde
solution prepared at -78 °C by the addition of the 1 M TiCl4/
CH2Cl2 (3.9 mL) to a stirred solution of (tert-butyldiphenylsi-
lyoxy)acetaldehyde (1.05 g, 3.5 mmol) in CH2Cl2 (10 mL)
followed by 15 min of stirring. The mixture was stirred at -78
°C for 2 h and then quenched by the addition of aqueous
ammonium chloride. The organic layer was washed with H2O,
dried, and rotary evaporated. The crude product was purified
by silica gel column chromatography with CHCl3 followed by
flash chromatography with hexane:AcOEt 6:1 to yield 360 mg
The (3R,4R) derivative 18 was similarly prepared starting
from 17 (120 mg, 0.49 mmol) to give 140 mg (90%) of 18 as an
1
oil: [R]25 +18.8 (c 0.4, CHCl3); H NMR (400 MHz, CDCl3) δ
D
0.93 (s, 3H), 0.95 (s, 3H), 1.40 (s, 9H), 1.40-1.45 (m, 2H), 1.68-
1.74 (m, 1H), 2.40 (br s, 1H), 3.74 (br s, 1H), 4.24 (br s, 1H),
4.61 (br d, J ) 7.7 Hz, 1H), 6.23 (dd, J ) 15.9, 6.4 Hz, 1H),
6.62 (d, J ) 15.9 Hz, 1H), 7.21-7.39 (m, 5H); 13C NMR (100
MHz, CDCl3) δ 21.94, 23.35, 24.87, 28.32, 40.69, 53.38, 75.26,
79.46, 126.56, 127.66, 128.51, 129.53, 131.49, 136.68, 156.46;
HRFAB MS, m/z 320.2230 for [M + H]+ (calcd for C15H20O2N
320.2226).
(2R,3S)-3-[N-(ter t-Bu th yloxyca r bon yl)a m in o]-3-isobu -
tyl-2-h yd r oxyp r op a n -1-ol (14). To a stirred solution of 13
(120 mg, 0.39 mmol) in EtOH (5 mL) was bubbled a stream of
ozonized oxygen at -78 °C until the blue color persisted (30
min). The solution was flushed with N2 for 5 min, and then
NaBH4 (150 mg, 3.9 mmol) in EtOH (5 mL) was added. The
reaction mixture was stirred at 23 °C for 1 h, and acetone was
added. The solvent was removed in vacuo and the residue was
extracted with AcOEt. The organic layer was washed with
(52%) of 21 as a powder: mp 127-129 °C, [R]25 +17.0 (c 1.2,
D
CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.74 (d, J ) 6.5 Hz, 3H),
0.78 (d, J ) 6.5 Hz, 3H), 1.03 (s, 9H), 1.11 (ddd, J ) 13.5, 9.4,
3.8 Hz, 1H), 1.27-1.34 (m, 1H), 1.52 (ddd, J ) 13.5, 9.3, 4.4
Hz, 1H), 2.43 (s, 3H), 2.51-2.56 (m, 1H), 2.76 (d, J ) 17.0 Hz,
1H), 2.77 (d, J ) 3.1 Hz, 1H), 3.02 (dd, J ) 17.0, 4.7 Hz, 1H),
3.53 (d, J ) 6.2 Hz, 2H), 3.87-3.92 (m, 1H), 4.91 (dd, J ) 9.9,
5.0 Hz, 1H), 5.31 (dd, J ) 5.0, 4.7 Hz, 1H), 5.82 (d, J ) 9.9 Hz,
J . Org. Chem, Vol. 68, No. 12, 2003 4761

Akaji et al.
1H), 7.03-7.05 (m, 1H), 7.18-7.45 (m, 11H), 7.58-7.62 (m,
4H), 7.80 (d, J ) 8.2 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ
19.14, 21.55, 21.56, 23.29, 26.36, 26.79, 34.62, 37.20, 46.51,
59.62, 65.38, 72.65, 75.63, 124.35, 124.71, 127.05, 127.34,
127.85, 128.40, 129.76, 129.90, 129.94, 132.77, 132.86, 135.49,
138.05, 138.33, 139.99, 143.53, 173.16; HRFAB MS, m/z
722.2945 for [M + Na]+ (calcd for C40H49O6NSSiNa 722.2948).
Three other diastereomers of 23 were similarly prepared
starting from 10, 15, and 20, respectively.
Boc-HEA[R-isobu tyl,S-h ydr oxy]-P r o-NH-CH2-C6H4I (24).
Yield 77%; [R]25 -52.4 (c 0.4, CHCl3); 1H NMR (400 MHz,
D
CDCl3) δ 090 (d, J ) 6.6 Hz, 3H), 094 (d, J ) 6.6 Hz, 3H),
1.10-1.17 (m, 1H), 1.30-1.37 (m, 1H), 1.43 (s, 9H), 1.59-1.68
(m, 1H), 1.76-1.83 (m, 2H), 1.87-1.95 (m, 1H), 2.18-2.25 (m,
1H), 2.27-2.35 (m, 1H), 2.44 (dd, J ) 12.6, 1.5 Hz, 1H), 2.65
(dd, J ) 12.6, 10.6 Hz, 1H), 3.19 (dd, J ) 9.7, 5.5 Hz, 1H),
3.23-3.27 (m, 1H), 3.37 (br s, 1H), 3.67 (br s, 1H), 3.74-3.76
(m, 1H), 4.27-4.33 (m, 1H), 4.39-4.44 (m, 1H), 4.55 (br d, J
) 7.0 Hz, 1H), 7.04 (t, J ) 7.8 Hz, 1H), 7.25 (d, J ) 7.8 Hz,
1H), 7.57 (d, J ) 7.8 Hz, 1H), 7.62 (br s, 1H), 8.20 (br s, 1H);
13C NMR (100 MHz, CDCl3) δ 21.62, 23.52, 24.20, 24.84, 28.33,
30.43, 38.74, 42.15, 52.74, 53.65, 58.26, 68.09, 73.04, 80.02,
94.41, 126.81, 130.26, 136.21, 136.55, 141.12, 174.69; HRFAB
MS, m/z 560.1977 for [M + H]+ (calcd for C24H39O4N3I
560.1985).
(4R ,5S )-4-Isob u t yl-5-[(t er t -b u t yld ip h e n ylsilyloxy)-
m eth yl]oxa zolid in -2-on e (22). To a stirred solution of 21
(350 mg, 0.50 mmol) in THF: H2O (3:1, 4 mL) were added 30%
H2O2 (450 µL) and LiOH‚H2O (84 mg, 2.0 mmol), and the
mixture was stirred at 23 °C for 90 min. Aqueous Na2SO3 (1.5
M) and saturated aqueous NaHCO3 were added to the mixture
and the organic solvent was removed in vacuo. The resulting
solution was acidified with AcOH and extracted with CHCl3.
The organic layer was washed with H2O, dried, and evapo-
rated. The residue was extracted with hexane. N-Tosyl-
aminoindanol was precipitated as a powder. The solvent of the
extract was removed in vacuo and the residue was dissolved
in CH2Cl2 (4 mL). To this solution were added Et3N (280 µL,
2.0 mmol) and diphenylphosphoryl azide (430 µL, 2.0 mmol).
The mixture was stirred at 23 °C for 48 h, and the solvent
was removed in vacuo. The product was purified by silica gel
column chromatography with CHCl3 followed by flash chro-
matography with hexane:AcOEt 3:1 to yield 12 mg (6%) of 22
Boc-HEA[S-isobu tyl,S-h ydr oxy]-P r o-NH-CH2-C6H4I (25).
1
Yield 75%; [R]25 -116.4 (c 0.3, CHCl3); H NMR (400 MHz,
D
CDCl3) δ 0.919 (d, J ) 6.6 Hz, 3H), 0.923 (d, J ) 6.6 Hz, 3H),
1.22-1.29 (m, 2H), 1.43 (s, 9H), 1.43-1.52 (ms, 1H), 1.58-
1.65 (m, 1H), 1.72-1.94 (m, 2H), 2.17-2.27 (m, 1H), 2.32-
2.38 (m, 1H), 2.50-2.54 (m, 1H), 2.67-2.73 (m, 1H), 3.19-
3.24 (m, 2H), 3.58 (br s, 1H), 3.64-3.67 (m, 1H), 4.34-4.44
(m, 2H), 4.63 (br d, J ) 9.0 Hz, 1H), 7.07 (t, J ) 7.8 Hz, 1H),
7.25 (d, J ) 7.8 Hz, 1H), 7.59 (d, J ) 7.8 Hz, 1H), 7.65 (br s,
1H), 7.89 (br s, 1H); 13C NMR (100 MHz, CDCl3) δ 22.08, 23.22,
24.24, 24.77, 28.38, 30.42, 41.68, 42.21, 51.10, 53.94, 59.62,
68.18, 71.87, 76.68, 79.50, 94.47, 126.85, 130.34, 136.31,
136.54, 141.08, 156.21, 174.54; HRFAB MS, m/z 560.1979 for
[M + H]+ (calcd for C24H39O4N3I 560.1985).
as an oil: [R]25 -15.3 (c 0.3, CHCl3); 1H NMR (400 MHz,
D
CDCl3) δ 0.89 (d, J ) 6.4 Hz, 3H), 0.93 (d, J ) 6.4 Hz, 3H),
1.06 (s, 9H), 1.36-1.43 (m, 1H), 1.54-1.63 (m, 1H), 3.85 (d, J
) 5.4 Hz, 2H), 3.97-4.02 (m, 1H), 4.60-4.65 (m, 1H), 5.65 (br
s, 1H), 7.37-7.47 (m, 6H), 7.65-7.68 (m, 4H); 13C NMR (100
MHz, CDCl3) δ 19.13, 21.16, 23.71, 25.15, 26.70, 38.14, 52.73,
61.37, 78.91, 127.83, 129.93, 132.65, 132.79, 135.55, 135.62,
159.08; HRFAB MS, m/z 412.2304 for [M + H]+ (calcd for
C24H34O3NSi 412.2308).
Boc-HEA[R-isobu tyl,R-h ydr oxy]-P r o-NH-CH2-C6H4I (26).
Yield 75%; [R]25 -50.7 (c 0.3, CHCl3); 1H NMR (400 MHz,
D
CDCl3) δ 0.91 (d, J ) 6.6 Hz, 3H), 0.92 (d, J ) 6.6 Hz, 3H),
1.13-1.19 (m, 1H), 1.42 (s, 9H), 1.38-1.45 (m, 1H), 1.55-1.62
(m, 1H), 1.70-1.80 (m, 2H), 1.95-2.01 (m, 1H), 2.12-2.23 (m,
1H), 2.48-2.54 (m, 1H), 2.58-2.67 (m, 2H), 3.12-3.18 (m, 2H),
3.39 (br s, 1H), 3.59 (br s, 1H), 4.30-4.35 (m, 1H), 4.41-4.46
(m, 1H), 4.54 (br d, J ) 9.4 Hz, 1H), 7.05 (t, J ) 7.8 Hz, 1H),
7.26 (d, J ) 7.8 Hz, 1H), 7.59 (d, J ) 7.8 Hz, 1H), 7.65 (br s,
1H), 7.83 (br t, J ) 5.9 Hz, 1H); 13C NMR (100 MHz, CDCl3)
δ 22.05, 23.29, 24.54, 24.76, 28.35, 30.91, 42.01, 42.25, 50.84,
56.20, 60.53, 68.08, 72.31, 79.40, 94.53, 126.97, 130.38, 136.39,
136.67, 141.42, 156.28, 175.06; HRFAB MS, m/z 560.1981 for
[M + H]+ (calcd for C24H39O4N3I 560.1985).
Solid -P h a se Syn th esis of In h ibitor s. (a ) Gen er a l P r o-
ced u r e for th e Dep r otection of th e NR-Boc Gr ou p . TFA
(50%) in CH2Cl2 was added to a Boc-containing resin and the
mixture was agitated at 23 °C for 3 min. After filtration, the
resin was re-treated with 50% TFA/CH2Cl2 for 15 min. The
resin was washed with CH2Cl2 and DMF, and then agitated
with 5% DIEA/DMF for 30 s (three times). The resulting resin
was washed with DMF, and was used for the next reaction.
(b) Hyd r oxyeth yla m in a ted P r o-a m id e, Boc-HEA[S-
isobu tyl,R-h yd r oxy]- P r o-NH-CH 2-C6H4I (23). To a stirred
solution of anisole (0.760 mL, 7.0 mmol) in TFA (10 mL) in an
ice bath was added Boc-Pro-NH-CH2-C6H4I (1.50 g, 3.5 mmol),
and the mixture was stirred at 4 °C for 60 min. The TFA was
removed by azeotroping with hexane (three times) and the
product was extracted with CHCl3. The organic layer was
washed with 5% NaHCO3 and H2O, dried over MgSO4, and
evaporated. The residue was triturated with hexane to yield
a powder (0.780 g). The product was dissolved in 2-propanol
(10 mL). To this solution was added the (2S,1′S)-aminoalky-
lepoxide 5 (220 mg, 1.0 mmol), and the mixture was stirred at
70 °C for 4 h. The solvent was removed in vacuo, and the
residue was extracted with CHCl3. The organic layer was
washed with 5% citric acid and H2O, dried over MgSO4, and
then rotary evaporated. The residue was triturated with
(c)
H -P r o-Va l-Ile-H E A[S-isob u t yl,R-h yd r oxy](Gly-
MBHA)-P r o-NH-CH2-C6H4I (29). To a stirred solution of 23
(180 mg, 0.32 mmol) in CHCl3 (3 mL) were added succinic
anhydride (42 mg, 0.42 mmol) and DMAP (12 mg, 98 µmol),
and the mixture was stirred at 23 °C for 12 h. The reaction
mixture was washed with 5% citric acid and H2O, dried over
MgSO4, and then evaporated. The product was purified by
flash chromatography with hexane:AcOEt 1:5 followed by
CHCl3:MeOH 5:1 as the eluant to yield 105 mg (48%) of the
desired carboxylic acid, Boc-HEA(COCH2CH2COOH)-Pro-NH-
CH2-C6H4I (27):1H NMR (400 MHz, CDCl3) δ 0.82 (d, J ) 6.6
Hz, 3H), 0.88 (d, J ) 6.6 Hz, 3H), 1.15-1.18 (m, 2H), 1.42 (s,
9H), 1.55-1.71 (m, 2H), 1.76 (br s, 2H), 1.85-1.90 (m, 1H),
2.10-2.18 (m, 1H), 2.51-2.55 (m, 1H), 2.62-2.86 (m, 6H),
3.15-3.16 (m, 1H), 3.30-3.32 (m, 1H), 3.91 (br s, 1H), 4.23-
4.28 (m, 2H), 4.41-4.53 (m, 1H), 4.56 (br d, J ) 7.1 Hz, 1H),
4.82-4.86 (m, 1H), 7.04 (t, J ) 7.7 Hz, 1H), 7.28 (d, J ) 7.7
Hz, 1H), 7.57 (d, J ) 7.7 Hz, 1H), 7.65 (br s, 2H), 8.45 (br s,
1H); 13C NMR (100 MHz, CDCl3) δ 21.35, 23.53, 24.47, 24.56,
28.31, 29.03, 29.32, 30.93, 39.43, 42.10, 49.37, 55.59, 56.60,
68.82, 75.47, 79.64, 94.34, 127.01, 130.23, 136.11, 136.59,
141.39, 155.69, 172.15, 175.32, 176.04; HRFAB MS, m/z
660.2143 for [M + H]+ (calcd for C28H43O7N3I 660.2146).
hexane to yield 350 mg (65%) of 23 as a powder: mp 144-146
1
°C; [R]25 -54.8 (c 1.2, CHCl3); H NMR (400 MHz, CDCl3) δ
D
0.87 (d, J ) 6.6 Hz, 3H), 0.90 (d, J ) 6.6 Hz, 3H), 1.14-1.30
(m, 2H), 1.44 (s, 9H), 1.60 (br s, 1H), 1.70-1.77 (m, 2H), 1.95-
2.00 (m, 1H), 2.13-2.23 (m, 1H), 2.46-2.53 (m, 1H), 2.55-
2.65 (m, 1H), 3.10-3.14 (m, 1H), 3.22-3.26 (m, 1H), 3.49 (br
s, 1H), 3.67 (br s, 1H), 4.36-4.39 (m, 2H), 4.48 (br d, J ) 7.7
Hz, 1H), 7.06 (t, J ) 7.7 Hz, 1H), 7.25 (d, J ) 7.7 Hz, 1H),
7.59 (d, J ) 7.7 Hz, 1H), 7.63 (br s, 1H), 8.00 (br s, 1H); 13C
NMR (100 MHz, CDCl3) δ 21.56, 23.52, 24.56, 24.77, 28.33,
30.97, 39.13, 42.21, 52.68, 56.30, 59.20, 67.84, 73.75, 79.91,
94.49, 126.84, 130.33, 136.29, 136.53, 141.18, 156.69, 175.26;
HRFAB MS, m/z 560.1990 for [M + H]+ (calcd for C24H39O4N3I
560.1985).
To 100 mg of p-methylbenzhydrylamine resin (0.63 mmol/g
resin) swelled in DMF (3 mL) were added. Fmoc-Gly-OH (49
mg, 0.16 mmol), N-hydroxybenzotriazole (HOBt, 25 mg, 0.16
4762 J . Org. Chem., Vol. 68, No. 12, 2003

Solid-Phase Synthesis of HTLV-1 Protease Inhibitor
mmol), DIEA (29 µL, 0.16 mmol), and DIPCDI (26 µL, 0.16
mmol) The mixture was agitated for 90 min at 23 °C. After
the resin was washed with DMF, piperidine (20%) in DMF
was added to the resin, and the mixture was agitated for 20
min at 23 °C. The NR-deprotected resin was filtered and
washed with DMF. To this resin were added the carboxylic
acid 27 (90 mg, 0.13 mmol, 2 equiv for the resin) prepared
above, HOBt (20 mg, 0.13 mmol), DIEA (29 µL, 0.16 mmol),
and DIPCDI (26 µL, 0.16 mmol). The resin mixture was
agitated for 15 h at 23 °C to yield the anchored resin 28. After
the resin was washed with DMF, the NR-Boc group was
removed by treatment with 50% TFA in CH2Cl2 according to
the general procedure described above. Boc-Ile-OH was then
introduced by a coupling reaction (90 min) with 0.16 mmol of
each Boc-Ile-OH, HOBt, DIEA, and DIPCDI in DMF (3 mL).
The Boc group of the resulting resin was removed with 50%
TFA/CH2Cl2 as above. Boc-Val-OH and Boc-Pro-OH were then
introduced by using the same coupling reaction followed by
the NR-deprotection reaction. The resin obtained was filtered,
washed with MeOH, and dried to yield 120 mg of the desired
resin 29.
(d ) H-P r o-Va l-Ile-HEA[S-isobu tyl,R-h yd r oxy]-P r o-NH-
CH2-C6H4I (31). An aliquot of the inhibitor resin 29 (50 mg)
was treated with an anhydrous HF (5 mL) in the presence of
anisole (0.10 mL) at 4 °C for 50 min. After evaporation of HF,
ether (10 mL) was added to the reaction cylinder. The resulting
precipitate was washed with ether (10 mL) and dissolved in
0.1% aqueous TFA. An analytical HPLC chromatogram of this
solution is shown in Figure 4a. The solution was freeze-dried
to yield 14.5 mg of the inhibitor precursor 30 as a white
amorphous powder: HPLC, 15.08 min [YMC AM302 (4.6 ×
150 mm), 0.9 mL/min, CH3CN (25% to 70%, 30 min)], HRFAB
MS, m/z 925.4056 for [M + H]+ (calcd for C41H66O8N8I
925.4048).
Three other inhibitors were prepared from the correspond-
ing Boc-HEA-Pro-NH-CH2-C6H4I, 24, 25, and 26, using the
same anchoring reaction followed by solid-phase synthesis.
H-P r o-Val-Ile-HEA[R-isobu tyl,S-h ydr oxy]-P r o-NH-CH2-
C6H4I (32). Yield, overall 7% (white amorphous powder);
HPLC, 16.96 min [YMC AM302 (4.6 × 150 mm), 0.9 mL/min,
CH3CN (25% to 70%, 30 min)], HRFAB MS, m/z 769.3535 for
[M + H]+ (calcd for C35H58O5N6I 769.3513).
H-P r o-Val-Ile-HEA[S-isobu tyl,S-h ydr oxy]-P r o-NH-CH2-
C6H4I (33). Yield, overall 9% (white amorphous powder);
HPLC, 13.24 min [YMC AM302 (4.6 × 150 mm), 0.9 mL/min,
CH3CN (25% to 70%, 30 min)], HRFAB MS, m/z 769.3506 for
[M + H]+ (calcd for C35H58O5N6I 769.3513).
H-P r o-Val-Ile-HEA[R-isobu tyl,R-h ydr oxy]-P r o-NH-CH2-
C6H4I (34). Yield, overall 12% (white amorphous powder);
HPLC, 17.16 min [YMC AM302 (4.6 × 150 mm), 0.9 mL/min,
CH3CN (25% to 70%, 30 min)], HRFAB MS, m/z 769.3518 for
[M + H]+ (calcd for C35H58O5N6I 769.3513).
Mea su r em en t of In h ibitor y Activity. Enzyme assays
were carried out with use of the synthetic HTLV-1 protease
mutant at an enzyme concentration of 5.6 nM. Hydrolyzate of
the substrate in a reaction solution (0.5 M sodium acetate
buffer, pH 5.6, containing 10% glycerol, 10 mM DTT and 4 M
NaCl) were analyzed on a Cosmosil 5C18-AR11 column (4.6
× 250 mm), employing a linear gradient of CH3CN (10-40%)
in aq 0.1% TFA over 30 min. Under the two different substrate
concentrations (30 and 15 µM), the initial velocities of the
enzymatic reaction were measured in the presence of different
amounts of the inhibitor. The incubation time was 12 min in
the absence of the inhibitor and 40 min at the highest inhibitor
concentration. Each experiment was repeated 3 times. The
inhibitory constant, Ki, was then calculated from the Dixon
plot. The Ki values obtained are summarized in Table 1.
The precursor product 30 was dissolved in 0.4 M AcONH4
(10 mL) and the pH of the solution was adjusted to 8.0. The
mixture was stirred at 23 °C for 17 h, and then the pH was
adjusted to 5.5 with 1 N AcOH. The solution was freeze-dried
and the crude product was purified by preparative HPLC
[YMC Pro C18 column (10 × 250 mm), CH3CN (25-70%/60
min) in 0.1% aq TFA, 2.5 mL/min] to yield 7.1 mg (overall 46%)
of inhibitor 31 as a white amorphous powder: HPLC, 15.98
min [YMC AM302 (4.6 × 150 mm), 0.9 mL/min, CH3CN (25%
to 70%, 30 min)], HRFAB MS, m/z 769.3520 for [M + H]+ (calcd
for C35H58O5N6I 769.3513).
Ack n ow led gm en t. We thank Professor Yoshiaki
Kiso for useful suggestions. We are grateful to Ms.
Kayoko Oda (Kyoto Pharmaceutical University) for
measuring the HRFAB MS spectra.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra for compound 1-27, and HPLC chromatograms for
compound 31-34. This material is available free of charge via
the Internet at http://pubs.acs.org.
J O030063A
J . Org. Chem, Vol. 68, No. 12, 2003 4763