Locking the two ends of tetrapeptidic HTLV-I protease inhibitors inside the enzyme

Article abstract of DOI:10.1016/j.bmc.2008.05.052

Adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy are only some of the more common end results of an infection with a human T-cell leukemia virus type 1 (HTLV-I). Expanding from our previous reports, we synthesized all different permutations of tetrapeptidic HTLV-I protease inhibitors using at least eight P₃-cap and five P₁^′-cap moieties. The inhibitors exhibited over 97% inhibition against HIV-1 protease and a wide range of inhibitory activity against HTLV-I protease.

Full text of DOI:10.1016/j.bmc.2008.05.052

Bioorganic & Medicinal Chemistry 16 (2008) 6880–6890
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Locking the two ends of tetrapeptidic HTLV-I protease inhibitors
inside the enzyme
Meihui Zhang a,b, Jeffrey-Tri Nguyen a, Henri-Obadja Kumada a, Tooru Kimura a, Maosheng Cheng b,
Yoshio Hayashi a,c, Yoshiaki Kiso a,
*
a Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science and 21st Century COE Program, Kyoto Pharmaceutical University, Yamashina-ku,
Kyoto 607-8412, Japan
b Key Laboratory of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China
c Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy are only some of
the more common end results of an infection with a human T-cell leukemia virus type 1 (HTLV-I).
Expanding from our previous reports, we synthesized all different permutations of tetrapeptidic HTLV-
I protease inhibitors using at least eight P3-cap and five P01-cap moieties. The inhibitors exhibited over
97% inhibition against HIV-1 protease and a wide range of inhibitory activity against HTLV-I protease.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 14 April 2008
Revised 22 May 2008
Accepted 23 May 2008
Available online 28 May 2008
Keywords:
HTLV-I
ATL
HIV-1
Aspartic protease inhibitor
1. Introduction
blood transfusion or sharing of contaminated needles. Despite sig-
nificant advances in understanding the pathogenesis of HTLV-I-
Identified in the laboratories of R.C. Gallo in the 1980s from pa-
tients with adult T-cell leukemia (ATL), the human T-cell leukemia
virus type 1 (HTLV-I) was the first human retrovirus to be clearly
linked with ATL, HTLV-I-associated myelopathy/tropical spastic
paraparesis (HAM/TSP), HTLV-I uveitis (HAU), HTLV-I-associated
arthropathy (HAAP) and a number of other chronic diseases.1–3
HTLV-I infection is currently considered intractable, because
20–30 million individuals worldwide are viral carriers, of whom
roughly 2–6% will succumb to the disease after a long latent period
of 20–50 years.4,5 HTLV-I infection is endemic in southwestern Ja-
pan, Central Africa, Caribbean Islands, South America and other
isolated populations in the world.6 In southwestern Japan, where
the virus is most prevalent in the world, antibodies to HTLV-I are
found in 6–37% of healthy adults.7 Estimations showed that 1.2
million Japanese people are infected with HTLV-I, and more than
700 cases of ATL are being diagnosed each year in Japan. HTLV-I
is not transmitted by casual contact because the three main routes
of viral transmission require close contact with infected T-lympho-
cytes.8–10 These routes include vertical transmission from mother
to child through breast-feeding; horizontal transmission by sexual
intercourse; and exposure to contaminated blood, either through
associated diseases and the wide experience with the virus over
the past twenty years, ATL patients generally have a very poor
prognosis and the disease remains difficult to treat.
HTLV-I is classified as a member of the family Retroviridae,
which also includes the human immunodeficiency virus (HIV).11
The virally encoded homodimeric aspartic protease, which is com-
posed of two identical chain-subunits with each chain containing
125 amino acid residues which is roughly 25% larger than the
HIV protease, mediates crucial proteolytic processing of viral pro-
tein precursors at the late stage of viral replication of the virus
and thus represents a virus-specific target for HTLV-I infection.12,13
The three main major proteins, Gag, Pol and Env, are encoded with-
in the retroviral genome.12,14 Upon viral maturation, the retroviral
protease cleaves the Gag protein into matrix (MA), capsid (CA) and
nucleocapsid (NC) proteins, while proteolytic cleavage of the Pol
precursor yields reverse transcriptase/ribonuclease H (RT–RH)
and integrase (IN). The third polyprotein, Env, contains two envel-
op proteins, surface glycoprotein (SU) and transmembrane protein
(TM), that are associated with the host cell-derived virus mem-
brane. Although HTLV-I protease shares 45% sequence identity
with HIV-1 protease at the substrate binding region and various
HIV-1 protease inhibitors are presently being used successfully in
therapies against AIDS, analogous HIV-1 protease inhibitors in
the treatment of HTLV-I infections do not cure ATL and HTLV-I
* Corresponding author. Tel.: +81 75 595 4635; fax: +81 75 591 9900.
E-mail address: kiso@mb.kyoto-phu.ac.jp (Y. Kiso).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.052

M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 6880–6890
6881
infections because of large differences in the specificity of the two
enzymes.15–17 HTLV-I protease continues to be one of the primary
targets of ATL drug discovery due to its central role in processing of
viral polypeptide precursors. The role of HTLV-I protease inhibitors
is just beginning to be investigated.
thiazolidine-4-carboxylic acid (Boc-Dmt-OH) using benzotriazol-
1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) in the presence of triethylamine (TEA), followed by Boc-
deprotection with 4 N HCl in dioxane to obtain general compound
5. General compound 6 was prepared by coupling general com-
pound 5 and Boc-Apns-OH with BOP. The same deprotection/con-
densation procedure was repeated for the successive introduction
Our research centres around HTLV-I protease inhibitors that
contain an allophenylnorstatine [(2S,3S)-3-amino-2-hydroxy-4-
phenylbutyric acid, Apns] residue as a hydroxymethylcarbonyl iso-
stere derived from a natural scissile amino acid sequence ‘Leu-Pro’
(1),18 which is the HTLV-I protease MA and CA cleavage site region
based on the concept of substrate-transition state mimicry, and
from which we derived potent peptidic HTLV-I protease inhibitors
KNI-10161 (2, IC50 = 159 nM),19 as shown in Figure 1. Believing that
smaller peptidic inhibitors with few hydrophilic moieties would
more readily penetrate intact cells, we began our natural amino
acid optimization studies and arrived at inhibitor KNI-10166 (3,
IC50 = 88 nM).20 Considering that the efficacy of peptides as in vivo
agents is severely compromising by their susceptibility to proteo-
lytic degradation and difficulty in penetrating intact cells, we ex-
plored non-natural amino acid residues and further reduce the
size of the inhibitor in inhibitors KNI-10247 (4, IC50 = 144 nM).21
After further optimization of compound 4 for inhibitory activity
against HTLV-I protease, we reported a series of potent HTLV-I pro-
tease inhibitors that possessed an isobutylamine P01-cap moiety and
various hydrophobic substituents at the P3-cap position.22
Although almost all of the inhibitors were highly potent with IC50
values ranging from 79 to 107 nM, the results from the structure–
activity relationship study remained ambiguous due to the narrow
range of inhibitory activity against HTLV-I protease. In the work de-
a
a
of N -tert-butyloxycarbonyl
L
-tert-leucine (Boc-Tle-OH) and N -
tert-butyloxycarbonyl -(+)-
L
a-phenylglycine (Boc-Phg-OH) to af-
ford compounds 8aa–8af, that were coupled one more time with
a respective carboxylic acid to afford compounds 8ba–8id.
3. Results and discussion
HTLV-I protease inhibitory activity assay was performed using
HTLV-I protease L40I mutant as previously reported.21,22 The L40I
mutation was necessary to prevent autolysis, thereby improving
the stability of the enzyme.23 In a preliminary report, we described
the HTLV-I protease inhibition of tetrapeptidic inhibitors having
the general structures of Ac-Phg-Tle-Apns-Dmt-R where the
a
-carbon of the P01-cap moiety was unbranched (8ba,bb,bc) (Table
1).21 In the current work, we studied the effect of branching at the
a
-carbon of the P01-cap moiety and lengthening the P01-cap moiety.
Among inhibitors 8ba, 8bb and 8bc, an isobutylamino P01-cap moi-
ety seemed to enhanced HTLV-I protease inhibitory activity, and
consequently, we reported very potent HTLV-I protease inhibitors
possessing an isobutylamino P0 -cap moiety and various P3-cap
1
moieties (8ab,bb,cb,db,eb,fb,gb,hb).22 However, within the isobu-
tylamino P01-cap moiety series, the inhibition range was fairly nar-
row (IC50 = 83–107 and 332 nM), thereby suggesting that our
structure–activity relationship observations could be fairly specu-
lative. To clarify our deduced structure–activity relationships
trend, in the current study, we varied the P01-cap moiety along with
the P3-cap moiety, and statistically validated the observed trends
(Eq. 1). Among the 44 inhibitors that are shown in Table 1, 34 com-
pounds have not been previously described.
scribed herein, using at least eight P3-cap and five P0 -cap hydropho-
1
bic moieties, we applied combinatorics to the synthesis of all
possible permutations to compounds that possessed a similar
P3 ꢀ P0 sequence as inhibitor 4. As a result of varying both the P3-
1
cap and P01-cap moieties in our attempts to ‘lock’ the two ends
and trap the inhibitor within the enzyme, we developed potent tet-
rapeptidic HTLV-I protease inhibitors and obtained a statistically
reliable quantitative structure–activity relationship (QSAR) equa-
tion to support the observed structure–activity trends.
Quantitative structure–activity relationship equation correlat-
ing HTLV-I protease inhibition and structural features with enzyme
inhibition, IC50 (nM).
2. Chemistry
ꢀ logðIC50Þ ¼ 0:520ðP3-cap moietyÞ þ 0:269ðP0 -cap moietyÞ ð1Þ
1
ꢀ 2:665
Compounds 8aa–8id were prepared by standard solution phase
n ¼ 42; r2 ¼ 0:87; F ¼ 131; p ¼ 0:001
where P3-cap moiety : H ¼ 0:000; Ac ¼ 0:761; EtCO ¼ 0:817;
peptide coupling chemistry (Scheme 1). A respective alkylamine
a
was coupled with N -tert-butyloxycarbonyl (R)-5,5-dimethyl-1,3-
nPrCO ¼ 1:000; cPrCO ¼ 0:986; iPrCO ¼ 0:977; tBuCO ¼ 0:846;
iBuCO ¼ 0:975; ranging from 0 to 1:
P0 -cap moiety : CH2cPr ¼ 0:000; iBu ¼ 0:734; tPe ¼ 1:000;
1
i
t
ðRÞ ꢀ CHðMeÞ Pr ¼ 0:454; ðRÞ ꢀ CHðMeÞ Bu ¼ 0:923; iPe ¼ 0:484;
ranging from 0 to 1:
Equation 1. Quantitative structure–activity relationship equa-
tion correlating HTLV-I protease inhibition and structural features
with enzyme inhibition, IC50 (nM).
We derived a QSAR equation (Eq. 1) to correlate chemical struc-
ture and HTLV-I protease inhibition. At first glance, it was apparent
that compound 8dd did not fit the HTLV-I inhibition trend due to
its very low inhibitory activity, and the compound was conse-
quently excluded as an outlier (Fig. 2). The scores given for the
P3-cap and P0 -cap moieties were awarded on averaged normalized
1
percentile based on IC50 values. In simpler words, groups of com-
pounds, where only one portion of the structure was changed
(either the P3-cap or P0 -cap moiety), were ranked as normalized
1
percentiles from 0 to 1, and based on their IC50 values. To compare
between the groups, the average of the respective normalized per-
Figure 1. From an HTLV-I protease substrate (1), potent HTLV-I protease inhibitors,
KNI-10161 (2), KNI-10166 (3) and KNI-10247 (4), were developed.

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M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 6880–6890
Scheme 1. Synthesis of compounds 8aa–8id. Reagents and conditions: (a) Boc-Dmt-OH, BOP, TEA, DMF, rt, 15 h; (b) 4 N HCl/dioxane, rt, 1 h; (c) Boc-Apns-OH, BOP, TEA,
DMF, rt, 15 h; (d) Boc-Tle-OH, BOP, TEA, DMF, rt, 15 h; (e) Boc-Phg-OH, BOP, TEA, DMF, rt, 15 h; (f) R2OH, BOP, TEA, DMF, rt, 15 h.
Table 1
Structures and IC50 values (nM) of HTLV-I protease inhibitors 8aa–8id
R2
R1
CH2cPr
iBu
tPe
(R)-CH(Me)iPr
(R)-CH(Me)tBu
iPe
H
8aa, KNI-10679,
928 (462)
8ba, KNI-10592,
146 (186)
8ab, KNI-10673,
332 (293)
8bb, KNI-10516,
107 (118)
8ac, KNI-10680,
180 (249)
8bc, KNI-10600,
113 (100)
8ad, KNI-10681,
354 (349)
8bd, KNI-10586,
136 (140)
8ae, KNI-10683,
156 (261)
8be, KNI-10627,
106 (105)
8af, KNI-10684,
372 (342)
NA
Ac
EtCO
8ca, KNI-10589,
145 (174)
8da, KNI-10591,
113 (140)
8ea, KNI-10670,
142 (142)
8fa, KNI-10613,
134 (144)
8ga, KNI-10669,
187 (168)
8ha, KNI-10668,
132 (144)
8cb, KNI-10571,
105 (110)
8db, KNI-10570,
104 (89)
8eb, KNI-10575,
97 (90)
8fb, KNI-10574,
94 (91)
8gb, KNI-10572,
83 (107)
8hb, KNI-10573,
102 (91)
8cc, KNI-10599,
98 (94)
8dc, KNI-10635,
74 (75)
8ec, KNI-10649,
86 (76)
8fc, KNI-10636,
78 (77)
8gc, KNI-10631,
97 (90)
8hc, KNI-10648,
82 (77)
8cd, KNI-10577,
129 (131)
8dd, KNI-10587,
659 (105)
8ed, KNI-10601,
90 (107)
8fd, KNI-10688,
122 (108)
8gd, KNI-10602,
133 (127)
8hd, KNI-10667,
107 (109)
8id, KNI-10603,
87 (105)
8ce, KNI-10578,
103 (98)
8de, KNI-10588,
94 (79)
8ee, KNI-10628,
85 (80)
8fe, KNI-10590,
84 (81)
8ge, KNI-10630,
97 (95)
8he, KNI-10629,
84 (81)
NA
nPrCO
8df, KNI-10671,
95 (103)
NA
cPrCO
iPrCO
NA
NA
tBuCO
iBuCO
8hf, KNI-10672,
107 (107)
NA
CH2@CH(CH2)2CO
NA
NA
NA
NA
The data are listed as the compound’s referred name in bold, followed by its proprietary name, its experimental HTLV-I protease L40I mutant inhibition (nM, the largest
standard-error-of-the-mean is 1.8 nM), and its predicted HTLV-I protease inhibition (nM) in parentheses as calculated from Eq. 1.
NA (not available): The compound was not synthesized.
Of the 44 compounds (8aa–8id), the HTLV-I and HIV-1 protease inhibitory activities of 10 compounds (8ab,ba,bb,bc,cb,db,eb,fb,gb,hb) have been previously reported.21,22
centiles represented the score of the moiety being examined. To
corroborate the scoring method, a well fitted, normalized, multiple
collinear, QSAR equation (Eq. 1) was derived that associated HTLV-
I protease inhibitory activity with the structures of the P3-cap and
P01-cap moieties. The equation is well fitted (r2 = 0.87) and statisti-
cally significant (p < 0.001). Moreover, the equation was reliable
because it consisted of only two parameters. A form of K-fold
cross-validation, namely leave-one-out cross-validation, was per-
formed using a single observation from the original sample as
the validation data and the remaining observations as the training
data, and the process was repeated such that each observation in
the sample was used once as the validation data. In other words,
many QSAR equations, in which one of the compounds was ex-
cluded, were derived to predict the HTLV-I protease inhibition of
the excluded compound. Variations in the equations determined
the validity of the overall equation, Eq. 1. The overall equation
was valid, because the root-mean-square-deviations of the coeffi-
cients and intercept were relatively small (0.520 0.014,
0.269 0.008, ꢀ2.665 0.015), and the coefficient of determina-
tion did not greatly vary during cross-validation (r2 = 0.85–0.90).
Cross-validation also confirmed the inhibitory activity of com-
pound 8dd as an outlier datum. We were unable to elucidate a rea-
son for the lower HTLV-I protease inhibitory activity exhibited by
Figure 2. HTLV-I protease inhibition correlations between observed and predicted
ꢀlog(IC50) values, based on Eq. 1, ranging from the least (8aa) to most (8dc) potent
compound from 44 compounds, with compound 8dd as an outlier.

M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 6880–6890
6883
compound 8dd. To account for the outlier, we synthesized com-
pound 8id as an analogue of compound 8dd. We predicted that
inhibitor 8id’s IC50 value would be approximately 105 nM, which
was the expected value for inhibitor 8dd. Indeed, the experimental
IC50 value of 87 nM was similar to the predicted value. Inhibitor
8id was not used in deriving the QSAR equation due to the lack
of comparable compounds.
contribution to the equation was reversed, in that the P01-cap moiety
contributed twice as much as the alkyl group on the P3-cap moiety.
The reason for such a reversal of equation contribution was eluci-
dated from computer-assisted docking experiments that suggested
that the P3-cap moiety resided near the edge of the active site (Fig.
3B), whereas the P01-cap moiety was deeper within the active site
(Fig. 3A). This implied that location-wise the hydrophobic region
of the P3-cap moiety was a lesser determinant of activity than that
In our most recent work, we suggested that the amide oxygen
from the P3-cap moiety was involved in a hydrogen bond interaction
with Leu57A from the flap of the HTLV-I protease (Fig. 3C).22 Indeed,
the inhibitory activity data from the current work confirmed such an
observation. Because of this important hydrogen bond interaction,
the P3-cap moiety contributed two-times more to the regression
equationthantheP0 -capmoiety(P3-capmoiety, 66%;P0 -capmoiety,
of the P0 -cap moiety. From the scores derived for the P3-cap moiety
1
in Eq. 1, a statistically supported structure–activity trend suggested
that lengthening alkyl group increased inhibitory potency against
HTLV-I protease. Although not included in the derivation of Eq. 1,
the potent HTLV-I protease inhibition profile of inhibitor 8id also
supported the observed alkyl-lengthening trend of the P3-cap moi-
ety. This observation agreed with our previously suggested trend
thatwasderivedfromtheinhibitoryprofilesof analogues containing
an isobutylamino P01-cap moiety.22 In the same former study, we also
1
1
34%). Interestingly, in an equation derived from a data set in which
compounds lacking a P3-cap moiety (8aa–8af) were excluded, the
suggested methyl branching at either the P3-cap moiety’s a- or b-
carbon increased inhibitory activity against HTLV-I protease. How-
ever, in contrast, the scores from the current and statistically rele-
vant study suggested that increasing volumetric bulk at either the
P3-cap moiety’s a- or b-carbon reduced HTLV-I protease inhibitory
activity. We believe that the current study describes a more accurate
trend. According to the overall scores, considering that lengthening
the P3-cap moiety enhanced HTLV-I protease inhibition whereas
branching at the a- or b-carbon would diminish inhibitory activity,
a flexible and non-bulky P3-cap moiety would be more adequately
accommodated by the protease.
According to the scores derived for the P01-cap moiety, increas-
ing volumetric bulk at the P01-cap moiety’s b-carbon enhanced
HTLV-I protease inhibitory activity (Eq. 1). This observation agreed
with our previous suggested trend that was based on the inhibitory
activity values of a limited number of compounds.21 Interestingly,
the scores implied that methyl branching at the P01-cap moiety’s
(R)-a-carbon had an undesired effect on enzyme inhibition. More-
over, the scores also suggested that lengthening the P01-cap moiety
also decreased inhibitory activity.
We were not compelled to complete the isopentyl P01-cap
groups of compounds (e.g., inhibitors 8af, 8df and 8hf). Synthesiz-
ing compounds 8bf, 8cf, 8ef, 8ff and 8gf would only confirm that
lengthening the P01-cap moiety reduced inhibitory activity against
HTLV-I proteases, and we were more interested in designing more
inhibitory active compounds. Moreover, we determined an ideal
‘fit’ within the enzyme for the P01-cap moiety’s
a- and b-carbon.
The P01-cap moiety’s
c-carbon would have less influence on binding
than the a- and b-carbon, because the c-carbon was relatively fur-
ther away from the important catalytic S1—S0 region. Note that we
1
previously suggested that the hydrophobic region of the P3-cap
moiety had lesser influence on inhibitory activity than that of the
P0 -cap moiety, because the P3-cap moiety was more distant from
1
the S1—S0 region than the P01-cap moiety.
1
Inhibitor 8dc was predicted and observed as our more potent
inhibitor described in the current study. To visualize and elucidate
the correlations between inhibitory activity and chemical struc-
ture, we generated a computer-assisted docking model of inhibitor
8dc in complex with an HTLV-I protease L40I mutant (Fig. 3). From
the model, we observed various possible hydrogen bond interac-
tions throughout the backbone of the inhibitor and the dimeric
HTLV-I protease’s Asp32A, Asp32B, Gly34A, Asp36A, Leu57A,
Ala59A and Ala59B (Fig. 3C). A hydrogen bond network between
Ala59A and Ala59B in the hairpin regions of HTLV-I protease’s flaps
and the inhibitor’s P01 and P2 amide oxygen was mediated by a
water molecule in a similar fashion as that of HIV-1 and Plasmep-
sin protease inhibitors. Also in common, another hydrogen bond
network was also formed between the oxygens of the inhibitor’s
P1 hydroxymethylcarbonyl group and HTLV-I protease’s catalytic
Figure 3. Computer model of KNI-10635 (8dc) in the active site of HTLV-I protease.
Dotted lines represent possible hydrogen bond interactions. (A) The P01-cap moiety
is located deeper within the active site than (B) the P3 moiety which resides near
the exposed surface of the HTLV-I protease. (C) The surface of some HTLV-I protease
heavy atoms is hidden to clearly depict various hydrogen bond interactions.

6884
M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 6880–6890
Asp32A and Asp32B, and thereby forming a transition-state mimic.
Indeed, the P3-cap moiety was accommodated near the edge of the
active site region (Fig. 3B), whereas the location of the P01-cap moi-
ety was deeper within the active site (Fig. 3A). Hence, the P01-cap
moiety would have a greater location-wise influence than the P3-
cap moiety on the inhibitor’s activity profile. It should however
be noted that throughout the current study, we assumed that the
5.1.2. General method A: amide bond formation
To a solution of substituted amine (1.0 mmol) in N,N-dimethyl
formamide (DMF, 5 mL) were added Boc-Xaa-OH or carboxylic
acid (1.1 mmol), BOP (1.1 mmol) and Et3N (adjusted to pH 8),
and stirred for 15 h at rt. After removal of the solution in vacuo,
the residue was dissolved in EtOAc (20 mL), washed with 10% cit-
ric acid aq (3ꢁ 15mL), 10% NaHCO3 aq (3ꢁ 15 mL) and brine (1ꢁ
15 mL), dried over anhydrous Na2SO4, and concentrated in vacuo
to give the crude product which was used directly to the next step
without purification, or purified by preparative HPLC in the case
of the target compounds. The purified target compounds were
immediately lyophilized to afford their respective white amor-
phous powders.
P3-cap and P0 -cap moieties did not have any influence on each
1
other. In reality, especially for the case of these small tetrapeptidic
compounds, a slight structural change at one portion of the mole-
cule would also affect the average orientation of other regions in
the same molecule as well as regions of the HTLV-I protease.
Tözsér and co-workers reported narrower inhibition profiles
from peptidic substrates for HTLV-I protease than HIV-1 prote-
ase.17 In a similar pattern, we observed that HTLV-I protease ex-
presses higher specificity than its HIV-1 protease cousin for the
case inhibitors, in that all synthesized inhibitors exhibited almost
complete HIV-1 protease inhibition at 50 nM (P98%). Indeed, the
current work supported our past observations that, in general,
HTLV-I protease inhibitors also exhibit extremely potent HIV-1
protease inhibition; however, potent HIV-1 protease inhibitors
may not potently inhibit HTLV-I protease.20–22 Hence, HTLV-I pro-
tease inhibitors might also provide a different design approach for
novel HIV-1 protease inhibitors.
5.1.3. General method B: Boc-deprotection
4 N HCl in 1,4-dioxane (5 mL) was added to the afforded crude
Boc-protected product and stirred for 1 h at rt. The solution was
evaporated under reduced pressure and used directly to the next
step without purification. A small portion of the product was puri-
fied by preparative HPLC. The desired fractions were collected and
immediately lyophilized to afford a white amorphous powder,
compound 5, 6, or 7.
5.1.3.1. (R)-N-Cyclopropylmethyl-5,5-dimethyl-1,3-thiazolidine-
4-carboxamide (H-Dmt-NHCH2cPr, 5a). Compound 5a was
prepared from Boc-Dmt-OH and aminomethylcyclopropane by
general methods A and B. Yield: 98%; 1H NMR (CDCl3) d = 0.20 (d,
2H), 0.50 (d, 2H), 0.90–1.02 (m, 1H), 1.43 (s, 3H), 1.65 (s, 3H),
3.09–3.24 (m, 2H), 4.33 (s, 1H), 4.45–4.60 (m, 2H), 6.22 (br s,
1H), 7.48–7.60 (m, 1H); MS (LC) m/z = 214.89 [M+H]+; Analytical
HPLC: tR = 15.06 min (system A).
4. Conclusions
The current study described the design, synthesis and HTLV-I and
HIV-1 protease inhibitory activity of 34 novel small tetrapeptidic
inhibitors containing allophenylnorstatine as a substrate transi-
tion-state mimicking moiety. HTLV-I protease inhibitory activity
was statistically correlated to the inhibitor’s respective P3- and P0 -
1
cap moieties. The IC50 value of the most potent inhibitor described
in this study, KNI-10635 (8dc), was determined to be
73.8 0.6 nM, and quantitative structure–activity relationship
equations along with computer-assisted docking models were gen-
erated to explain the compound’s highly potent inhibitory activity
against HTLV-I protease.
5.1.3.2. (R)-N-Cyclopropylmethyl-3-[(2S,3S)-3-amino-2-hydroxy-
4-phenyl]butanoyl-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
(H-Apns-Dmt-NHCH2cPr, 6a). Compound 6a was prepared from
Boc-Apns-OH and compound by general methods A and B. Yield:
36%; 1H NMR (CDCl3) d = 0.15 (d, 2H), 0.45 (d, 2H), 0.80–0.90 (m,
1H), 1.39–1.56 (m, 2ꢁ 3H), 2.7–3.1 (br s, 1H), 2.88–2.95 (m, 2H),
3.17–3.26 (m, 2H), 3.75–3.83 (br s, 1H), 4.21–4.39 (m, 1H+2H),
4.82 (d, 1H), 7.08–7.20 (m, 2H), 7.21–7.32 (m, 5H), 7.8–8.2 (br s,
1H); MS (TOF) m/z = 391.84 [M+H]+, 414.007 [M+Na]+; Analytical
HPLC: tR = 20.44 min (system A).
5. Experimental
5.1. Chemistry
5.1.1. General methods
5.1.3.3. (R)-N-Cyclopropylmethyl-3-{(2S,3S)-3-[(2S)-2-amino-3,3-
dimethyl]butanoylamino-2-hydroxy-4-phenyl}butanoyl-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide (H-Tle-Apns-Dmt-NHCH2
cPr, 7a). Compound 7a was prepared from Boc-Tle-OH and com-
pound 6a by general methods A and B. Yield: 53%; 1H NMR (CDCl3)
d = 0.10–0.18 (m, 2H), 0.40–0.48 (m, 2H), 0.77–0.93 (m, 1H+3ꢁ
3H), 1.41–1.58 (m, 2ꢁ 3H), 1.7–2.5 (br s, 1H), 2.80–3.20 (m, 2ꢁ
2H), 3.51–3.64 (m, 1H), 4.29–4.40 (m, 1H), 4.48–4.84 (m,
1H+2H), 5.00–5.12 (m, 1H), 6.7–6.8 (m, 1H), 7.10–7.26 (m, 5H),
7.6–7.8 (br s, 1H), NH2 not observed; MS (TOF) m/z = 504.425
[M+H]+; Analytical HPLC: tR = 21.67 min (system A).
The detailed preparation of compounds 8ab,bb,cb,d-
b,eb,fb,gb,hb has already been reported.22 Reagents and solvents
were purchased from commercial suppliers and used without fur-
ther purification. 1H NMR spectra were measured on a JEOL JNM-
AL300 FT (300 MHz) spectrometer with tetramethylsilane (TMS)
as an internal standard at ambient temperature. Data are reported
as follows: chemical shift (ppm), multiplicity (s = singlet, d = dou-
blet, t = triplet, q = quartet, dd = double doublet, bs = broad singlet,
m = multiplet) and number of protons. LC–MS was recorded on a
Finnigan SSQ-7000 spectrometer. MALDI-TOF MS was performed
on a Voyager-DE RP spectrometer and all target compounds were
less than 1 m/z from calculated values. Preparative HPLC was ob-
tained from a C18 reverse phase column (20 ꢁ 250 mm; YMC Pack
ODS SH-343-5AM). Assay HPLC was performed on a C18 reverse
phase column (3.0 ꢁ 75 mm; YMC Pack ODS AS-3E7). Analytical
5.1.3.4. (R)-N-Cyclopropylmethyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-2-
amino-2-phenyl]acetylamino-3,3-dimethyl}butanoylamino-2-
hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (H-Phg-Tle-Apns-Dmt-NHCH2cPr, 8aa). Compound
8aa was prepared from Boc-Phg-OH and compound 7a by general
methods A and B. Yield: 41%; 1H NMR (CDCl3) d = 0.14–0.20 (m,
2H), 0.40–0.48 (m, 2H), 0.64–0.73 (m, 3ꢁ 3H), 0.82–0.98 (m, 1H),
1.40–1.60 (m, 2ꢁ 3H), 1.6–2.0 (br s, 1H), 2.60–3.32 (m, 2ꢁ 2H),
3.83–4.35 (m, 2ꢁ 1H), 4.54–4.89 (m, 2ꢁ 1H+2H), 5.10–5.22 (m,
1H), 6.30–6.42 (m, 1H), 6.70–6.80 (m, 1H), 6.90–6.98 (m, 1H),
7.05–7.26 (m, 5H), 7.27–7.61 (m, 5H), NH2 not observed; MS
HPLC was performed using
a C18 reverse phase column
(4.6 ꢁ 150 mm; YMC Pack ODS AM-302) with a binary solvent sys-
tem: linear gradient of CH3CN in 0.1% aqueous trifluoroacetic acid
(TFA) at a flow rate of 0.9 mL/min, detected at UV 230 nm. The purity
of the desired compounds and intermediates was confirmed by ana-
lytical HPLC and was greater than 99% in two different systems (gra-
dient: system A, 0–100%/40 min; system B, 30–90%/40 min).

M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 6880–6890
6885
(TOF) m/z = 638.654 [M+H]+; Analytical HPLC: tR = 23.14 min (sys-
tem A), 9.60 min (system B).
pound 8aa by general method A. Yield: 13%; 1H NMR (CDCl3)
d = 0.16–0.24 (m, 2H), 0.42–0.51 (m, 2H), 0.75–0.80 (m, 3ꢁ 3H),
0.85–0.97 (m, 1H), 1.10–1.22 (m, 2ꢁ 3H), 1.47–1.63 (m, 2ꢁ 3H),
1.8–2.2 (br s, 1H), 2.43–2.53 (m, 1H), 2.64–2.90 (m, 2H), 2.91–
3.25 (m, 2H), 3.97–4.96 (m, 4ꢁ 1H+2H), 5.40–5.50 (m, 1H), 6.19
(d, 1H), 6.25 (d, 1H), 6.70–6.80 (m, 2ꢁ 1H), 6.90–7.27 (m, 5H),
7.28–7.42 (m, 5H); MS (TOF) m/z = 730.829 [M+Na]+, 746.932
[M+K]+; Analytical HPLC: tR = 29.79 min (system A), 21.19 min
(system B).
5.1.3.5. (R)-N-Cyclopropylmethyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-2-
acetylamino-2-phenyl]acetylamino-3,3-dimethyl}butanoylamino-
2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazolidine-
4-carboxamide(Ac-Phg-Tle-Apns-Dmt-NHCH2cPr, 8ba). Compound
8ba was prepared from acetic acid and compound 8aa by general
method A. Yield: 14%; 1H NMR (CDCl3) d = 0.15–0.23 (m, 2H),
0.42–0.54 (m, 2H), 0.76–0.80 (m, 3ꢁ 3H), 0.88–0.98 (m, 1H),
1.47–1.59 (m, 2ꢁ 3H, partly covered by H2O), 2.04–2.06 (m, 3H),
2.64–2.83 (m, 2H), 2.90–3.25 (m, 2H), 3.94–4.96 (m, 4ꢁ 1H+2H),
5.45 (t, 1H), 6.00 (d, 1H), 6.06 (d, 1H), 6.70 (d, 1H), 6.78 (d, 1H),
6.90–7.26 (m, 5H), 7.27–7.39 (m, 5H), OH not observed; MS
(TOF) m/z = 680.806 [M+H]+, 702.814 [M+Na]+, 718.843 [M+K]+;
Analytical HPLC: tR = 27.27 min (system A), 16.91 min (system B).
5.1.3.10. (R)-N-Cyclopropylmethyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-2-
pivaloylamino-2-phenyl]acetylamino-3,3-dimethyl}butanoyl-
amino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazo-
lidine-4-carboxamide
(tBuCO-Phg-Tle-Apns-Dmt-NHCH2cPr,
8ga). Compound 8ga was prepared from pivalic acid and com-
pound 8aa by general method A. Yield: 11%; 1H NMR (CDCl3)
d = 0.10–0.24 (m, 2H), 0.35–0.52 (m, 2H), 0.79 (s, 3ꢁ 3H), 0.90–
1.02 (m, 1H), 1.23 (s, 3ꢁ 3H), 1.47–1.60 (m, 2ꢁ 3H), 2.4–2.8 (br
s, 1H), 2.64–2.85 (m, 2H), 2.88–3.25 (m, 2H), 3.98–5.04 (m, 4ꢁ
1H+2H), 5.43 (d, 1H), 6.20 (d, 1H), 6.28 (d, 1H), 6.78 (t, 1H), 6.87
(d, 1H), 6.92–7.25 (m, 5H), 7.26–7.37 (m, 5H); MS (TOF) m/
z = 744.498 [M+Na]+, 760.702 [M+K]+; Analytical HPLC:
tR = 30.94 min (system A), 23.64 min (system B).
5.1.3.6. (R)-N-Cyclopropylmethyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-2-
propionylamino-2-phenyl]acetylamino-3,3-dimethyl}butanoyl-
amino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazol-
idine-4-carboxamide (EtCO-Phg-Tle-Apns-Dmt-NHCH2cPr,
8ca). Compound 8ca was prepared from propionic acid and com-
pound 8aa by general method A. Yield: 16%; 1H NMR (CDCl3)
d = 0.14 (d, 1H), 0.19 (d, 1H), 0.42 (d, 1H), 0.48 (d, 1H), 0.75–0.95
(m, 1H+3ꢁ 3H), 1.16 (t, 3H), 1.40–1.60 (m, 2ꢁ 3H), 2.30 (q, 2H),
2.6–3.2 (br s, 1H), 2.61–3.20 (m, 2ꢁ 2H), 3.95–4.99 (m, 4ꢁ
1H+2H), 5.51 (t, 1H), 6.15–6.25 (m, 1H), 6.30–6.45 (m, 1H), 6.70–
6.80 (m, 1H), 6.81–6.89 (br s, 1H), 6.90–7.25 (m, 5H), 7.26–7.37
(m, 5H); MS (TOF) m/z = 695.001 [M+H]+, 716.968 [M+Na]+,
732.969 [M+K]+; Analytical HPLC: tR = 28.55 min (system A),
18.83 min (system B).
5.1.3.11. (R)-N-Cyclopropylmethyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-2-
(3-methylbutanoyl)amino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide (iBuCO-Phg-Tle-Apns-Dmt-
NHCH2cPr, 8ha). Compound 8ha was prepared from isovaleric
acid and compound 8aa by general method A. Yield: 17%; 1H
NMR (CDCl3) d = 0.14–0.24 (m, 2H), 0.40–0.52 (m, 2H), 0.75–0.80
(m, 3ꢁ 3H), 0.90–1.00 (m, 1H+2ꢁ 3H), 1.47–1.60 (m, 2ꢁ 3H),
1.8–2.2 (br s, 1H), 2.05–2.19 (m, 1H+2H), 2.58–2.93 (m, 2H),
2.94–3.29 (m, 2H), 3.97–4.99 (m, 4ꢁ 1H+2H), 5.43–5.51 (m, 1H),
6.09 (d, 1H), 6.16 (d, 1H), 6.70–6.80 (m, 2ꢁ 1H), 6.88–7.26 (m,
5H), 7.27–7.38 (m, 5H); MS (TOF) m/z = 745.088 [M+Na]+,
761.321 [M+K]+; Analytical HPLC: tR = 30.03 min (system A),
22.60 min (system B).
5.1.3.7. (R)-N-Cyclopropylmethyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-2-
butyrylamino-2-phenyl]acetylamino-3,3-dimethyl}butanoyla-
mino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazo-
lidine-4-carboxamide (nPrCO-Phg-Tle-Apns-Dmt-NHCH2cPr,
8da). Compound 8da prepared from n-butyric acid and com-
pound 8aa general method A. Yield: 15%; 1H NMR (CDCl3)
d = 0.15–0.24 (m, 2H), 0.41–0.54 (m, 2H), 0.75–0.82 (m, 3ꢁ
3H), 0.90–0.98 (m, 1H+3H), 1.47–1.73 (m, 2ꢁ 3H, partly cov-
ered by H2O), 2.24 (t, 2H), 2.55–2.85 (m, 2H), 2.94–3.29 (m,
2H), 3.94–4.98 (m, 4ꢁ 1H+2H), 5.40–5.49 (m, 1H), 5.90–6.05
(m, 1H), 6.00 (d, 1H), 6.09 (d, 1H), 6.65–6.75 (m, 1H), 6.91–
7.25 (m, 5H), 7.27–7.38 (m, 5H), OH not observed; MS (TOF)
m/z = 730.888 [M+Na]+, 746.916 [M+K]+; Analytical HPLC:
tR = 30.03 min (system A), 20.38 min (system B).
5.1.3.12. (R)-N-(2,2-Dimethyl)propyl-5,5-dimethyl-1,3-thiazolidine-
4-carboxamide (H-Dmt-NHtPe, 5c). Compound 5c was prepared
from 2,2-dimethylpropylamine and Boc-Dmt-OH by general meth-
ods A and B. Yield: 99%; 1H NMR (CDCl3) d = 0.90 (s, 3ꢁ 3H), 1.43 (s,
3H), 1.65 (s, 3H), 3.03–3.20 (m, 2H), 4.35 (s, 1H), 4.52 (s, 2H), 6.2–
6.5 (br s, 1H), 7.20–7.30 (m, 1H); MS (LC) m/z = 230.89 [M+H]+,
252.89 [M+Na]+; Analytical HPLC: tR = 17.84 min (system A).
5.1.3.8. (R)-N-Cyclopropylmethyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-2-
cyclopropanecarbonylamino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide (cPrCO-Phg-Tle-Apns-Dmt-
NHCH2cPr, 8ea). Compound 8ea was prepared from cyclopropane-
carboxylic acid and compound 8aa by general method A. Yield:
13%; 1H NMR (CDCl3) d = 0.11–0.21 (m, 2H), 0.40–0.51 (m, 2H),
0.74–1.05 (m, 1H+2ꢁ 2H+3ꢁ 3H), 1.45–1.60 (m, 1H+2ꢁ 3H), 1.7–
2.1 (br s, 1H), 2.60–2.90 (m, 2H), 2.91–3.22 (m, 2H), 3.60–4.99
(m, 4ꢁ 1H+2H), 5.41–5.50 (m, 1H), 6.02–6.10 (m, 1H), 6.11–6.20
(m, 1H), 6.26 (d, 1H), 6.75 (t, 1H), 6.85–7.26 (m, 5H), 7.27–7.49
(m, 5H); MS (TOF) m/z = 728.893 [M+Na]+, 744.72 [M+K]+; Analyt-
ical HPLC: tR = 29.02 min (system A), 20.24 min (system B).
5.1.3.13. (R)-N-(2,2-Dimethyl)propyl-3-[(2S,3S)-3-amino-2-
hydroxy-4-phenyl]butanoyl-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (H-Apns-Dmt-NHtPe, 6c). Compound 6c was pre-
pared from Boc-Apns-OH and compound 5c by general methods
A and B. Yield: 70%; 1H NMR (CDCl3) d = 0.85–0.93 (m, 3ꢁ 3H),
1.38–1.54 (m, 2ꢁ 3H), 1.9–2.3 (br s, 1H), 2.90–3.25 (m, 2ꢁ 2H),
3.77–3.86 (m, 1H), 4.25–4.87 (m, 1H+2H), 4.73–4.85 (m, 1H),
6.85–6.95 (m, 1H), 7.13–7.20 (m, 2H), 7.26–7.35 (m, 5H); MS
(TOF) m/z = 408.544 [M+H]+; Analytical HPLC: tR = 22.46 min (sys-
tem A).
5.1.3.14. (R)-N-(2,2-Dimethyl)propyl-3-{(2S,3S)-3-[(2S)-2-amino-3,
3-dimethyl]butanoylamino-2-hydroxy-4-phenyl}butanoyl-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide
(H-Tle-Apns-Dmt-
t
5.1.3.9. (R)-N-Cyclopropylmethyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-2-
isobutyrylamino-2-phenyl]acetylamino-3,3-dimethyl}butanoyl-
amino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazo-
lidine-4-carboxamide (iPrCO-Phg-Tle-Apns-Dmt-NHCH2cPr,
8fa). Compound 8fa was prepared from isobutyric acid and com-
NHCH2 Bu, 7c). Compound 7c was prepared from Boc-Tle-OH
and compound 6c by general methods A and B. Yield: 85%; 1H
NMR (CDCl3) d = 0.80–0.93 (m, 6ꢁ 3H), 1.43–1.52 (m, 2ꢁ 3H),
2.1–2.5 (br s, 1H), 2.80–3.20 (m, 2ꢁ 2H), 3.50–3.68 (m, 1H),
4.31–5.12 (m, 3ꢁ 1H+2H), 6.4–6.7 (br s, 1H), 7.06–7.29 (m, 5H),

6886
M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 6880–6890
7.8–8.0 (br s, 1H), NH2 not observed; MS (TOF) m/z = 521.376
[M+H]+. Analytical HPLC: tR = 23.10 min (system A).
6.32 (d, 1H), 6.55 (d, 1H), 6.69 (t, 1H), 6.91–7.24 (m, 5H),
7.26–7.38 (m, 5H). MS (TOF) m/z = 744.939 [M+Na]+, 760.936
[M+K]+. Analytical HPLC: tR = 31.76 min (system A), 23.95 min
(system B).
5.1.3.15. (R)-N-(2,2-Dimethyl)propyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-
2-amino-2-phenyl]acetylamino-3,3-dimethyl}butanoylamino-
2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazolidine-
4-carboxamide (H-Phg-Tle-Apns-Dmt-NHtPe, 8ac). Compound
8ac was prepared from Boc-Phg-OH and compound 8ac by general
methods A and B. Yield: 57%. 1H NMR (CDCl3) d = 0.72–0.82 (m, 3ꢁ
3H), 0.83–0.95 (m, 3ꢁ 3H), 1.44–1.60 (m, 2ꢁ 3H), 2.07 (s, 3H), 2.5–
2.9 (br s, 1H), 2.65–3.26 (m, 2ꢁ 2H), 3.97–4.99 (m, 4ꢁ 1H+2H),
5.53–5.60 (m, 1H), 6.15 (br s, 1H), 6.33 (br s, 1H), 6.46 (d, 1H),
6.68 (t, 1H), 6.90–7.24 (m, 5H), 7.28–7.38 (m, 5H). MS (TOF) m/
z = 696.772 [M+H]+, 718.777 [M+Na]+, 734.805 [M+K]+. Analytical
HPLC: tR = 29.62 min (system A), 20.98 min (system B).
5.1.3.20. (R)-N-(2,2-Dimethyl)propyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-
2-isobutyrylamino-2-phenyl]acetylamino-3,3-dimethyl}butanoyl-
amino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazo-
lidine-4-carboxamide (iPrCO-Phg-Tle-Apns-Dmt-NHtPe,
8fc). Compound 8fc was prepared from isobutyric acid and com-
pound 8ac by general method A. Yield: 62%. 1H NMR (CDCl3)
d = 0.74–0.96 (m, 8ꢁ 3H), 1.4–1.8 (br s, 1H), 1.45–1.60 (m, 2ꢁ
3H), 2.05–2.14 (m, 1H+2H), 2.61–2.80 (m, 2H), 2.83–3.29 (m,
2H), 3.93–4.99 (m, 4ꢁ 1H+2H), 5.45–5.50 (m, 1H), 6.01 (d, 1H),
6.09 (d, 1H), 6.68 (t, 1H), 6.74 (d, 1H), 6.85–7.26 (m, 5H),
7.27–7.40 (m, 5H). MS (TOF) m/z = 761.022 [M+Na]+, 777.014
[M+K]+. Analytical HPLC: tR = 33.07 min (system A), 25.92 min
(system B).
5.1.3.16. (R)-N-(2,2-Dimethyl)propyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-
2-acetylamino-2-phenyl]acetylamino-3,3-dimethyl}butanoyl-
amino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazo-
lidine-4-carboxamide
(Ac-Phg-Tle-Apns-Dmt-NHtPe,
5.1.3.21. (R)-N-(2,2-Dimethyl)propyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-
2-pivaloylamino-2-phenyl]acetylamino-3,3-dimethyl}butanoyl-
amino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazo-
lidine-4-carboxamide (tBuCO-Phg-Tle-Apns-Dmt-NHtPe,
8gc). Compound 8gc was prepared from pivalic acid and com-
pound 8ac by general method A. Yield: 59%. 1H NMR (CDCl3)
d = 0.75–0.98 (m, 6ꢁ 3H), 1.12–1.29 (m, 3ꢁ 3H), 1.47–1.60 (m,
2ꢁ 3H), 2.6–3.0 (br s, 1H), 2.60–3.29 (m, 2ꢁ 2H), 3.97–5.05 (m,
4ꢁ 1H+2H), 5.45 (d, 1H), 5.50 (d, 1H), 6.19 (d, 1H), 6.24 (d, 1H),
6.70 (t, 1H), 6.91–7.25 (m, 5H), 7.26–7.40 (m, 5H). MS (TOF) m/
z = 760.994 [M+Na]+, 776.945 [M+K]+. Analytical HPLC:
tR = 33.66 min (system A), 27.15 min (system B).
8bc). Compound 8bc was prepared from acetic acid and compound
8ac by general method A. Yield: 57%. 1H NMR (CDCl3) d = 0.72–0.82
(m, 3ꢁ 3H), 0.83–0.95 (m, 3ꢁ 3H), 1.44–1.60 (m, 2ꢁ 3H), 2.07 (s, 3H),
2.5–2.9 (br s, 1H), 2.65–3.26 (m, 2ꢁ 2H), 3.97–4.99 (m, 4ꢁ 1H+2H),
5.53–5.60 (m, 1H), 6.15 (br s, 1H), 6.33 (br s, 1H), 6.46 (d, 1H), 6.68
(t, 1H), 6.90–7.24 (m, 5H), 7.28–7.38 (m, 5H). MS (TOF) m/
z = 696.772 [M+H]+, 718.777 [M+Na]+, 734.805 [M+K]+. Analytical
HPLC: tR = 29.62 min (system A), 20.98 min (system B).
5.1.3.17. (R)-N-(2,2-Dimethyl)propyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-
2-propionylamino-2-phenyl]acetylamino-3,3-dimethyl}butanoyl-
amino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazo-
lidine-4-carboxamide
(EtCO-Phg-Tle-Apns-Dmt-NHtPe,
5.1.3.22. (R)-N-(2,2-Dimethyl)propyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-
2-(3-methyl)butanoylamino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide (iBuCO-Phg-Tle-Apns-Dmt-
NHtPe, 8hc). Compound 8hc was prepared from isovaleric acid
and compound 8ac by general method A. Yield: 60%. 1H NMR
(CDCl3) d = 0.75–0.80 (m, 3ꢁ 3H), 0.82–0.93 (m, 3ꢁ 3H), 1.12–
1.21 (m, 2ꢁ 3H), 1.4–1.9 (br s, 1H), 1.49–1.59 (m, 2ꢁ 3H), 2.42–
2.50 (m, 1H), 2.56–2.80 (m, 2H), 2.81–3.31 (m, 2H), 3.94–5.00
(m, 4ꢁ 1H+2H), 5.42–5.48 (m, 1H), 6.08 (d, 1H), 6.17 (d, 1H),
6.70 (t, 1H), 6.72 (d, 1H), 6.90–7.26 (m, 5H), 7.28–7.39 (m, 5H).
MS (TOF) m/z = 725.082 [M+H]+, 747.071 [M+Na]+, 763.032
[M+K]+. Analytical HPLC: tR = 32.16 min (system A), 27.39 min
(system B).
8cc). Compound 8cc was prepared from propionic acid and com-
pound 8ac by general method A. Yield: 58%. 1H NMR (CDCl3)
d = 0.75–0.80 (m, 3ꢁ 3H), 0.81–0.91 (m, 3ꢁ 3H), 1.16 (t, 3H),
1.47–1.59 (m, 2ꢁ 3H), 2.2–2.5 (br s, 1H), 2.24–2.36 (m, 2H),
2.60–3.29 (m, 2ꢁ 2H), 3.97–5.01 (m, 4ꢁ 1H+2H), 5.51 (d, 1H),
6.17 (d, 1H), 6.27 (d, 1H), 6.69 (t, 1H), 6.84 (d, 1H), 6.90–7.23 (m,
5H), 7.28–7.38 (m, 5H). MS (TOF) m/z = 732.917 [M+Na]+,
748.958 [M+K]+. Analytical HPLC: tR = 30.59 min (system A),
22.63 min (system B).
5.1.3.18. (R)-N-(2,2-Dimethyl)propyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-
2-butyrylamino-2-phenyl]acetylamino-3,3-dimethyl}butanoyl-
amino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazo-
lidine-4-carboxamide (nPrCO-Phg-Tle-Apns-Dmt-NHtPe,
8dc). Compound 8dc was prepared from n-butyric acid and com-
pound 8ac by general method A. Yield: 63%. 1H NMR (CDCl3)
d = 0.75–0.85 (m, 3ꢁ 3H), 0.86–0.96 (m, 4ꢁ 3H), 1.4–2.0 (br s,
1H), 1.49–1.59 (m, 2ꢁ 3H), 1.63–1.70 (m, 2H), 2.20–2.29 (m, 2H),
2.55–2.80 (m, 2H), 2.84–3.30 (m, 2H), 3.95–4.99 (m, 4ꢁ 1H+2H),
5.50 (d, 1H), 6.10 (d, 1H), 6.18 (d, 1H), 6.68 (t, 1H), 6.78 (d, 1H),
6.90–7.25 (m, 5H), 7.27–7.40 (m, 5H). MS (TOF) m/z = 725.065
[M+H]+, 747.012 [M+Na]+, 763.009 [M+K]+. Analytical HPLC:
tR = 32.06 min (system A), 24.40 min (system B).
5.1.3.23. (R)-N-[(R)-1,2-Dimethyl]propyl-5,5-dimethyl-1,3-thiazo-
lidine-4-carboxamide [H-Dmt-NHCH(Me)iPr, 5d]. Compound 5d
was prepared from (R)-3-methyl-2-butylamine and Boc-Dmt-OH
by general methods A and B. Yield: 98%. 1H NMR (CDCl3) d = 0.89
(d, 2ꢁ 3H), 1.00 (d, 3H), 1.41 (s, 3H), 1.62–1.74 (m, 1H+3H), 3.2–
3.6 (br s, 1H), 3.75–3.90 (m, 1H), 4.25 (s, 1H), 4.48 (s, 2H), 6.86
(d, 1H). MS (LC) m/z = 230.89 [M+H]+. Analytical HPLC:
tR = 17.38 min (system A).
5.1.3.24. (R)-N-[(R)-1,2-Dimethyl]propyl-3-[(2S,3S)-3-amino-2-
hydroxy-4-phenyl]butanoyl-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide [H-Apns-Dmt-NHCH(Me)iPr, 6d]. Compound 6d
was prepared from compound 5d and Boc-Apns-OH by general
methods A and B. Yield: 43%. 1H NMR (CDCl3) d = 0.80–0.95 (m,
2ꢁ 3H), 1.00–1.10 (m, 3H), 1.39–1.51 (m, 2ꢁ 3H), 1.63–1.74 (m,
1H), 1.8–2.2 (br s, 1H), 2.90–3.22 (m, 2H), 3.70–3.90 (m, 2ꢁ 1H),
4.25–4.86 (m, 2ꢁ 1H+2H), 6.65–6.75 (m, 1H), 7.20–7.33 (m, 5H).
NH2 not observed. MS (TOF) m/z = 407.926 [M+H]+. Analytical
HPLC: tR = 22.55 min (system A).
5.1.3.19. (R)-N-(2,2-Dimethyl)propyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-
2-cyclopropanecarbonylamino-2-phenyl]acetylamino-3,3-di-
methyl}butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-di-
methyl-1,3-thiazolidine-4-carboxamide (cPrCO-Phg-Tle-Apns-
Dmt-NHtPe, 8ec). Compound 8ec was prepared from cyclopro-
panecarboxylic acid and compound 8ac by general method A.
Yield: 60%. 1H NMR (CDCl3) d = 0.76–0.99 (m, 2ꢁ 2H+6ꢁ 3H),
1.47–1.57 (m, 1H+2ꢁ 3H), 2.5–3.0 (br s, 1H), 2.58–3.29 (m,
2ꢁ 2H), 4.02–5.04 (m, 4ꢁ 1H+2H), 5.54 (d, 1H), 5.61 (d, 1H),

M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 6880–6890
6887
5.1.3.25. (R)-N-[(R)-1,2-Dimethyl]propyl-3-{(2S,3S)-3-[(2S)-2-
amino-3,3-dimethyl]butanoylamino-2-hydroxy-4-phenyl}-
butanoyl-5,5-dimethyl-1,3-thiazolidine-4-carboxamide [H-Tle-
Apns-Dmt-NHCH(Me)iPr, 7d]. Compound 7d was prepared from
Boc-Tle-OH and compound 6d by general methods A and B. Yield:
64%. 1H NMR (CDCl3) d = 0.75–1.05 (m, 6ꢁ 3H), 1.30–1.52 (m, 2ꢁ
3H), 1.60–1.71 (m, 1H), 2.0–2.4 (br s, 1H), 2.80–3.15 (m, 2H),
3.50–3.83 (m, 2ꢁ 1H), 4.25–5.12 (m, 3ꢁ 1H+2H), 6.05–6.30 (m,
1H), 7.10–7.26 (m, 5H), 7.75–7.92 (m, 1H). NH2 not observed. MS
(TOF) m/z = 520.486 [M+H]+, 543.446 [M+Na]+, 559.196 [M+K]+.
Analytical HPLC: tR = 22.71 min (system A).
762.872 [M+K]+. Analytical HPLC: tR = 32.00 min (system A),
23.19 min (system B).
5.1.3.30. (R)-N-[(R)-1,2-Dimethyl]propyl-3-{{(2S,3S)-3-{(2S)-2-
[(2S)-2-cyclopropanecarbonylamino-2-phenyl]acetylamino-3,-
3-dimethyl}butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,
5-dimethyl-1,3-thiazolidine-4-carboxamide [cPrCO-Phg-Tle-
Apns-Dmt-NHCH(Me)iPr, 8ed]. Compound 8ed was prepared
from cyclopropanecarboxylic acid and compound 8ad by general
method A. Yield: 32%. 1H NMR (CDCl3) d = 0.75–0.80 (m, 3ꢁ 3H),
0.82–0.92 (m, 3ꢁ 3H), 0.95–1.08 (m, 2ꢁ 2H), 1.46–1.73 (m, 2ꢁ
1H+2ꢁ 3H, partly covered by H2O), 2.54–2.80 (m, 2H), 3.80–5.02
(m, 5ꢁ 1H+2H), 5.40–5.49 (m, 1H), 5.68 (d, 1H), 6.06 (br s, 1H),
6.20 (d, 1H), 6.36 (d, 1H), 6.88–7.26 (m, 5H), 7.30–7.42 (m, 5H).
OH not observed. MS (TOF) m/z = 723.101 [M+H]+, 745.055
[M+Na]+, 761.039 [M+K]+. Analytical HPLC: tR = 31.18 min (system
A), 23.04 min (system B).
5.1.3.26. (R)-N-[(R)-1,2-Dimethyl]propyl-3-{{(2S,3S)-3-{(2S)-2-
[(2S)-2-amino-2-phenyl]acetylamino-3,3-dimethyl} butanoylamino-
2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazolidine-
4-carboxamide [H-Phg-Tle-Apns-Dmt-NHCH(Me)iPr, 8ad]. Compound
8ad was prepared from Boc-Phg-OH and compound 7d and by gen-
eral methods A and B. Yield: 63%. 1H NMR (CDCl3) d = 0.55–0.80 (m,
3ꢁ 3H), 0.82–1.05 (m, 3ꢁ 3H), 1.40–1.56 (m, 2ꢁ 3H), 1.60–1.70 (m,
1H), 1.8–2.3 (br s, 1H), 2.50–2.85 (m, 2H), 3.72–4.87 (m, 5ꢁ
1H+2H), 5.15–5.40 (m, 1H), 6.27–6.38 (m, 1H), 6.70–6.85 (m,
1H), 6.90–6.98 (m, 1H), 7.10–7.26 (m, 5H), 7.27–7.58 (m, 5H).
NH2 not observed. MS (TOF) m/z = 676.939 [M+Na]+. Analytical
HPLC: tR = 24.99 min (system A), 13.42 min (system B).
5.1.3.31. (R)-N-[(R)-1,2-Dimethyl]propyl-3-{{(2S,3S)-3-{(2S)-2-
[(2S)-2-isobutyrylamino-2-phenyl] acetylamino-3,3-dimethyl}
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide [iPrCO-Phg-Tle-Apns-Dmt-NHCH
(Me)iPr, 8fd]. Compound 8fd was prepared from isobutyric acid
and compound 8ad by general method A. Yield: 35%. 1H NMR
(CDCl3) d = 0.75–1.20 (m, 8ꢁ 3H), 1.46–1.75 (m, 1H+2ꢁ 3H), 1.9–
2.3 (br s, 1H), 2.40–2.50 (m, 1H), 2.58–2.99 (m, 2H), 3.73–5.00
(m, 5ꢁ 1H+2H), 5.38–5.47 (m, 1H), 5.71 (d, 1H), 6.15 (d, 1H),
6.30 (d, 1H), 6.75 (d, 1H), 6.88–7.25 (m, 5H), 7.26–7.41 (m, 5H).
MS (TOF) m/z = 746.58 [M+Na]+, 762.56 [M+K]+. Analytical HPLC:
tR = 31.58 min (system A), 24.24 min (system B).
5.1.3.27. (R)-N-[(R)-1,2-Dimethyl]propyl-3-{{(2S,3S)-3-{(2S)-2-
[(2S)-2-acetylamino-2-phenyl]acetylamino-3,3-dimethyl}butanoyl-
amino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazo-
lidine-4-carboxamide
[Ac-Phg-Tle-Apns-Dmt-NHCH(Me)iPr,
8bd]. Compound 8bd was prepared from acetic acid and com-
pound 8ad by general method A. Yield: 33%. 1H NMR (CDCl3)
d = 0.72–0.79 (m, 3ꢁ 3H), 0.85–0.92 (m, 2ꢁ 3H), 0.99–1.07 (m,
3H), 1.45–1.73 (m, 1H+2ꢁ 3H, partly covered by H2O), 2.00–2.07
(m, 3H), 2.53–2.77 (m, 2H), 3.77–5.00 (m, 5ꢁ 1H+2H), 5.47 (t,
1H), 5.63 (d, 1H), 6.09 (t, 1H), 6.32 (d, 1H), 6.80 (br s, 1H), 6.85–
7.26 (m, 5H), 7.30–7.40 (m, 5H). OH not observed. MS (TOF) m/
z = 696.859 [M+H]+, 718.847 [M+Na]+, 734.898 [M+K]+. Analytical
HPLC: tR = 28.35 min (system A), 20.08 min (system B).
5.1.3.32. (R)-N-[(R)-1,2-Dimethyl]propyl-3-{{(2S,3S)-3-{(2S)-2-
[(2S)-2-pivaloylamino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,
3-thiazolidine-4-carboxamide [tBuCO-Phg-Tle-Apns-Dmt-NHCH
(Me)iPr, 8gd]. Compound 8gd was prepared from pivalic acid and
compound 8ad by general method A. Yield: 34%. 1H NMR (CDCl3)
d = 0.66–0.79 (m, 3ꢁ 3H), 0.84–0.98 (m, 2ꢁ 3H), 1.00–1.09 (m,
3H), 1.20–1.23 (m, 3ꢁ 3H), 1.45–1.73 (m, 1H+2ꢁ 3H, partly cov-
ered by H2O), 2.57–2.83 (m, 2H), 3.69–5.03 (m, 5ꢁ 1H+2H),
5.30–5.41 (m, 1H), 5.61–5.69 (m, 1H), 6.00–6.13 (m, 1H), 6.27–
6.38 (m, 1H), 6.83–6.93 (m, 1H), 7.03–7.26 (m, 5H), 7.27–7.40
(m, 5H). OH not observed. MS (TOF) m/z = 761.084 [M+Na]+,
777.135 [M+K]+. Analytical HPLC: tR = 32.99 min (system A),
26.71 min (system B).
5.1.3.28. (R)-N-[(R)-1,2-Dimethyl]propyl-3-{{(2S,3S)-3-{(2S)-2-
[(2S)-2-propionylamino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide [EtCO-Phg-Tle-Apns-Dmt-
NHCH(Me)iPr, 8cd]. Compound 8cd was prepared from propi-
onic acid and compound 8ad by general method A. Yield: 33%.
1H NMR (CDCl3) d = 0.70–0.80 (m, 3ꢁ 3H), 0.83–0.95 (m, 2ꢁ
3H), 0.97–1.07 (m, 3H), 1.16 (t, 3H), 1.46–1.59 (m, 2ꢁ 3H),
1.5–2.0 (br s, 1H), 1.63–1.78 (m, 1H), 2.30 (q, 2H), 2.60–3.00
(m, 2H), 3.65–5.00 (m, 5ꢁ 1H+2H), 5.41–5.48 (m, 1H), 5.65
(d, 1H), 6.05 (d, 1H), 6.30 (d, 1H), 6.75 (d, 1H), 6.83–7.25 (m,
5H), 7.27–7.39 (m, 5H). MS (TOF) m/z = 732.826 [M+Na]+,
748.834 [M+K]+. Analytical HPLC: tR = 31.31 min (system A),
21.79 min (system B).
5.1.3.33. (R)-N-[(R)-1,2-Dimethyl]propyl-3-{{(2S,3S)-3-{(2S)-2-
[(2S)-2-(3-methylbutanoyl)amino-2-phenyl]acetylamino-3,3-
dimethyl}butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide [tBuCO-Phg-Tle-
Apns-Dmt-NHCH(Me)iPr, 8hd]. Compound 8hd was prepared
from isovaleric acid and compound 8ad by general method A.
Yield: 30%. 1H NMR (CDCl3) d = 0.70–1.07 (m, 8ꢁ 3H), 1.46–1.68
(m, 1H+2ꢁ 3H, partly covered by H2O), 2.06–2.13 (m, 1H+2H),
2.54–2.79 (m, 2H), 3.75–5.00 (m, 5ꢁ 1H+2H), 5.42–5.48 (m, 1H),
5.60 (d, 1H), 5.90 (d, 1H), 6.28 (d, 1H), 6.68 (d, 1H), 6.88–7.26
(m, 5H), 7.27–7.41 (m, 5H). OH not observed. MS (TOF) m/
z = 761.194 [M+Na]+, 777.078 [M+K]+. Analytical HPLC:
tR = 31.82 min (system A), 25.07 min (system B).
5.1.3.29. (R)-N-[(R)-1,2-Dimethyl]propyl-3-{{(2S,3S)-3-{(2S)-2-
[(2S)-2-butyrylamino-2-phenyl]acetylamino-3,3-dimethyl}but-
anoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,
3-thiazolidine-4-carboxamide [nPrCO-Phg-Tle-Apns-Dmt-
NHCH(Me)iPr, 8dd]. Compound 8dd was prepared from n-butyric
acid and compound 8ad by general method A. Yield: 32%. 1H NMR
(CDCl3) d = 0.72–0.78 (m, 3ꢁ 3H), 0.80–1.07 (m, 4ꢁ 3H), 1.46–1.70
(m, 1H+2H+2ꢁ 3H), 1.5–1.9 (br s, 1H), 2.24 (t, 2H), 2.56–2.79 (m,
2H), 3.65–5.00 (m, 5ꢁ 1H+2H), 5.41–5.49 (m, 1H), 5.68 (d, 1H),
6.10 (d, 1H), 6.30 (d, 1H), 6.75 (d, 1H), 6.87–7.25 (m, 5H), 7.26–
7.40 (m, 5H). MS (TOF) m/z = 724.896 [M+H]+, 746.818 [M+Na]+,
5.1.3.34. (R)-N-[(R)-1,2-Dimethyl]propyl-3-{{(2S,3S)-3-{(2S)-2-
[(2S)-2-(4-pentenoyl)amino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,
3-thiazolidine-4-carboxamide [CH2@CH(CH2)2CO-Phg-Tle-Apns-
Dmt-NHCH(Me)iPr, 8id]. Compound 8id was prepared from 4-
pentenoic acid and compound 8ad by general method A. Yield:

6888
M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 6880–6890
30%. 1H NMR (CDCl3) d = 0.70–1.09 (m, 6ꢁ 3H), 1.35–1.60 (m, 2ꢁ
3H), 1.62–1.73 (m, 1H), 1.8–2.2 (br s, 1H), 2.26–2.40 (m, 2ꢁ 2H),
2.52–2.80 (m, 2H), 3.75–5.09 (m, 5ꢁ 1H+2ꢁ 2H), 5.35–5.85 (m,
2ꢁ 1H), 6.03–6.09 (m, 1H), 6.10–6.20 (m, 1H), 6.28–6.33 (m, 1H),
6.75–6.93 (m, 1H), 7.03–7.25 (m, 5H), 7.26–7.40 (m, 5H). MS
(TOF) m/z = 758.894 [M+Na]+, 774.911 [M+K]+. Analytical HPLC:
tR = 32.08 min (system A), 25.28 min (system B).
732.927 [M+Na]+, 748.945 [M+K]+. Analytical HPLC:
tR = 30.30 min (system A), 22.23 min (system B).
5.1.3.40. (R)-N-[(R)-1,2,2-Trimethyl]propyl-3-{{(2S,3S)-3-{(2S)-
2-[(2S)-2-propionylamino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide [EtCO-Phg-Tle-Apns-Dmt-
NHCH(Me)tBu, 8ce]. Compound 8ce was prepared from propi-
onic acid and compound 8ae by general method A. Yield: 35%.
1H NMR (CDCl3) d = 0.74–0.78 (m, 3ꢁ 3H), 0.88–0.92 (m, 3ꢁ
3H), 1.00–1.06 (m, 3H), 1.10–1.18 (m, 3H), 1.45–1.60 (m, 2ꢁ
3H, partly covered by H2O), 2.23–2.38 (m, 2H), 2.50–2.79 (m,
2H), 3.80–5.17 (m, 5ꢁ 1H+2H), 5.41–5.47 (m, 1H), 5.52 (d,
1H), 6.03 (d, 1H), 6.11 (d, 1H), 6.24 (d, 1H), 6.70–7.26 (m, 5H),
7.27–7.43 (m, 5H). OH not observed. MS (TOF) m/z = 746.875
[M+Na]+, 762.813 [M+K]+. Analytical HPLC: tR = 31.26 min (sys-
tem A), 23.75 min (system B).
5.1.3.35. (R)-N-[(R)-1,2,2-Trimethyl]propyl-5,5-dimethyl-1,3-
thiazolidine-4-carboxamide [H-Dmt-NHCH(Me)tBu, 5e]. Compound
5e was prepared from Boc-Dmt-OH and (R)-1,2,2-trimethylpropyl-
amine by general methods A and B. Yield: 98%. 1H NMR (CDCl3)
d = 0.89 (s, 3ꢁ 3H), 1.08 (d, 3H), 1.42 (s, 3H), 1.63 (s, 3H), 3.86–
3.95 (m, 1H), 4.33 (s, 1H), 4.51 (s, 2H), 5.0–5.4 (br s, 1H), 6.85 (d,
1H). MS (LC) m/z = 245.40 [M+H]+. Analytical HPLC: tR = 18.54 min
(system A).
5.1.3.36. (R)-N-[(R)-1,2,2-Trimethyl]propyl-3-[(2S,3S)-3-amino-
2-hydroxy-4-phenyl]butanoyl-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide [H-Apns-Dmt-NH NHCH(Me)tBu, 6e]. Compound
6e was prepared from Boc-Apns-OH and compound 5e by general
methods A and B. Yield: 45%. 1H NMR (CDCl3) d = 0.85–0.95 (m, 3ꢁ
3H), 0.98–1.09 (m, 3H), 1.35–1.51 (m, 2ꢁ 3H), 2.3–2.8 (br s, 1H),
2.61–2.94 (m, 2H), 3.06–3.25 (m, 1H), 3.81–3.92 (m, 1H), 4.30–
4.54 (m, 1H+2H), 4.71–4.88 (m, 1H), 6.65–6.75 (m, 1H), 7.26–
7.37 (m, 5H), 7.9–8.3 (br s, 2H). MS (TOF) m/z = 421.477 [M+H]+.
Analytical HPLC: tR = 23.16 min (system A).
5.1.3.41. (R)-N-[(R)-1,2,2-Trimethyl]propyl-3-{{(2S,3S)-3-{(2S)-
2-[(2S)-2-butyrylamino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide [n PrCO-Phg-Tle-Apns-Dmt-
NHCH(Me)tBu, 8de]. Compound 8de was prepared from n-bu-
tyric acid and compound 8ae by general method A. Yield: 31%.
1H NMR (CDCl3) d = 0.73–0.78 (m, 3ꢁ 3H), 0.85–0.95 (m, 4ꢁ
3H), 0.96–1.07 (m, 3H), 1.45–1.70 (m, 2H+2ꢁ 3H), 1.5–1.8 (br
s, 1H), 2.18–2.29 (m, 2H), 2.53–2.73 (m, 2H), 3.78–5.05 (m,
5ꢁ 1H+2H), 5.46–5.50 (m, 1H), 5.65 (d, 1H), 6.01 (d, 1H), 6.19
(d, 1H), 6.26 (d, 1H), 6.70–7.24 (m, 5H), 7.28–7.42 (m, 5H).
MS (TOF) m/z = 760.828 [M+Na]+, 776.946 [M+K]+. Analytical
HPLC: tR = 32.70 min (system A), 26.80 min (system B).
5.1.3.37. (R)-N-[(R)-1,2,2-Trimethyl]propyl-3-{(2S,3S)-3-[(2S)-2-
amino-3,3-dimethyl]butanoylamino-2-hydroxy-4-phenyl}butanoyl-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide [H-Tle-Apns-
Dmt-NHCH(Me)tBu, 7e]. Compound 7e was prepared from Boc-
Tle-OH and compound 6e by general methods A and B. Yield:
62%. 1H NMR (CDCl3) d = 0.80–1.15 (m, 7ꢁ 3H), 1.35–1.60 (m, 2ꢁ
3H), 2.8–4.0 (br s, 1H), 2.80–3.20 (m, 2H), 3.40–3.60 (m, 1H),
3.70–3.85 (m, 1H), 4.25–5.14 (m, 3ꢁ 1H+2H), 5.95–6.05 (m, 1H),
6.15–6.25 (m, 1H), 7.15–7.26 (m, 5H), 7.5–7.7 (br s, 2H). MS
(TOF) m/z = 535.28 [M+H]+. Analytical HPLC: tR = 23.95 min (sys-
tem A), 11.46 min (system B).
5.1.3.42. (R)-N-[(R)-1,2,2-Trimethyl]propyl-3-{{(2S,3S)-3-{(2S)-
2-[(2S)-2-cyclopropanecarbonylamino-2-phenyl]acetylamino-
3,3-dimethyl}butanoylamino-2-hydroxy-4-phenyl}}butanoyl-
5,5-dimethyl-1,3-thiazolidine-4-carboxamide [cPrCO-Phg-Tle-
Apns-Dmt-NHCH(Me)tBu, 8ee]. Compound 8ee was prepared
from cyclopropanecarboxylic acid and compound 8ae by general
method A. Yield: 33%. 1H NMR (CDCl3) d = 0.71–1.07 (m, 2ꢁ
2H+7ꢁ 3H), 1.45–1.58 (m, 1H+2ꢁ 3H), 1.6–2.0 (br s, 1H), 2.60–
2.77 (m, 2H), 3.79–5.08 (m, 5ꢁ 1H+2H), 5.47 (t, 1H), 5.63 (d, 1H),
6.12 (d, 1H), 6.19 (d, 1H), 6.28 (d, 1H), 6.85–7.25 (m, 5H), 7.27–
7.40 (m, 5H). MS (TOF) m/z = 758.873 [M+Na]+, 774.895 [M+K]+.
Analytical HPLC: tR = 32.08 min (system A), 25.10 min (system B).
5.1.3.38. (R)-N-[(R)-1,2,2-Trimethyl]propyl-3-{{(2S,3S)-3-{(2S)-
2-[(2S)-2-amino-2-phenyl]acetylamino-3,3-dimethyl}butanoyl-
amino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazo-
lidine-4-carboxamide [H-Phg-Tle-Apns-Dmt-NHCH(Me)tBu,
8ae]. Compound 8ae was prepared from Boc-Phg-OH and com-
pound 7e by general methods A and B. Yield: 65%. 1H NMR (CDCl3)
d = 0.60–0.73 (m, 3ꢁ 3H), 0.81–1.03 (m, 4ꢁ 3H), 1.40–1.55 (m, 2ꢁ
3H), 2.4–2.8 (br s, 1H), 2.53–2.80 (m, 2H), 3.63–4.95 (m, 5ꢁ
1H+2H), 5.22–5.41 (m, 1H), 6.50–6.65 (m, 1H), 6.70–6.80 (m,
1H), 6.83–6.93 (m, 1H), 7.05–7.20 (m, 5H), 7.37–7.60 (m, 5H).
NH2 not observed. MS (TOF) m/z = 668.096 [M+H]+, 690.374
[M+Na]+, 706.47 [M+K]+. Analytical HPLC: tR = 25.99 min (system
A), 15.63 min (system B).
5.1.3.43. (R)-N-[(R)-1,2,2-Trimethyl]propyl-3-{{(2S,3S)-3-{(2S)-
2-[(2S )-2-isobutyrylamino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide [iPrCO-Phg-Tle-Apns-Dmt-
NHCH(Me)tBu, 8fe]. Compound 8fe was prepared from isobutyric
acid and compound 8ae by general method A. Yield: 33%. 1H NMR
(CDCl3) d = 0.71–1.07 (m, 2ꢁ 2H+7ꢁ 3H), 1.45–1.58 (m, 1H+2ꢁ
3H), 1.6–2.0 (br s, 1H), 2.60–2.77 (m, 2H), 3.79–5.08 (m, 5ꢁ
1H+2H), 5.47 (t, 1H), 5.63 (d, 1H), 6.12 (d, 1H), 6.19 (d, 1H), 6.28
(d, 1H), 6.85–7.25 (m, 5H), 7.27–7.40 (m, 5H). MS (TOF) m/
z = 758.873 [M+Na]+, 774.895 [M+K]+. Analytical HPLC:
tR = 32.08 min (system A), 25.10 min (system B).
5.1.3.39. (R)-N-[(R)-1,2,2-Trimethyl]propyl-3-{{(2S,3S)-3-{(2S)-
2-[(2S)-2-acetylamino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide
[Ac-Phg-Tle-Apns-Dmt-
NHCH(Me)tBu, 8be]. Compound 8be was prepared from ace-
tic acid and compound 8ae by general method A. Yield: 32%.
1H NMR (CDCl3) d = 0.75–0.80 (m, 3ꢁ 3H), 0.85–0.93 (m, 3ꢁ
3H), 1.00–1.06 (m, 3H), 1.45–1.64 (m, 2ꢁ 3H, partly covered
by H2O), 2.03–2.06 (m, 3H), 2.50–2.77 (m, 2H), 3.79–5.06
(m, 5ꢁ 1H+2H), 5.45–5.50 (m, 1H), 5.59 (d, 1H), 5.98 (d, 1H),
6.09 (d, 1H), 6.28 (d, 1H), 6.78–7.25 (m, 5H), 7.29–7.42 (m,
5H). OH not observed. MS (TOF) m/z = 710.872 [M+H]+,
5.1.3.44. (R)-N-[(R)-1,2,2-Trimethyl]propyl-3-{{(2S,3S)-3-{(2S)-
2-[(2S)-2-pivaloylamino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide [t BuCO-Phg-Tle-Apns-Dmt-
NHCH(Me)tBu, 8ge]. Compound 8ge was prepared from piva-
lic acid and compound 8ae by general method A. Yield: 32%.
1H NMR (CDCl3) d = 0.72–0.78 (m, 3ꢁ 3H), 0.85–0.90 (m, 3ꢁ
3H), 0.97–1.06 (m, 3H), 1.22 (s, 3ꢁ 3H), 1.47–1.57 (m, 2ꢁ

M. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 6880–6890
6889
3H), 1.7–2.0 (br s, 1H), 2.50–2.73 (m, 2H), 3.77–5.10 (m, 5ꢁ
1H+2H), 5.35–5.45 (m, 1H), 5.64 (d, 1H), 6.05 (d, 1H), 6.18 (d,
1H), 6.26 (d, 1H), 6.85–7.19 (m, 5H), 7.26–7.42 (m, 5H). MS
(TOF) m/z = 775.167 [M+Na]+, 791.149 [M+K]+. Analytical
HPLC: tR = 34.16 min (system A), 28.38 min (system B).
8df was prepared from n-butyric acid and compound8af by general
method A. Yield: 50%. 1H NMR (CDCl3) d = 0.75–0.99 (m, 6ꢁ 3H),
1.30–1.73 (m, 1H+2ꢁ 2H+2ꢁ 3H), 2.25 (t, 2H), 2.60–2.85 (m, 2H),
3.10–3.42 (m, 2H), 3.95–5.00 (m, 4ꢁ 1H+2H), 5.44–5.54 (m, 1H),
6.10 (d, 1H), 6.18 (d, 1H), 6.56 (t, 1H), 6.72 (d, 1H), 6.90–7.26 (m,
5H), 7.27–7.42 (m, 5H). OH not observed. MS (TOF) m/
z = 724.845 [M+H]+, 746.461 [M+Na]+, 762.532 [M+K]+. Analytical
HPLC: tR = 31.79 min (system A), 24.22 min (system B).
5.1.3.45. (R)-N-[(R)-1,2,2-Trimethyl]propyl-3-{{(2S,3S)-3-{(2S)-
2-[(2S)-2-(3-methylbutanoyl)amino-2-phenyl]acetylamino-3,3-
dimethyl}butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide [iBuCO-Phg-Tle-
Apns-Dmt-NHCH(Me)tBu, 8he]. Compound 8he was prepared
from isovaleric acid and compound 8ae by general method A. Yield:
31%. 1H NMR (CDCl3) d = 0.70–1.04 (m, 9ꢁ 3H), 1.45–1.55 (m, 2ꢁ
3H), 1.7–2.2 (br s, 1H), 2.00–2.20 (m, 1H+2H), 2.52–2.78 (m, 2H),
3.75–5.07 (m, 5ꢁ 1H+2H), 5.51–5.59 (m, 1H), 5.83 (d, 1H), 6.13 (d,
1H), 6.27 (d, 1H), 6.38 (d, 1H), 6.80–7.25 (m, 5H), 7.27–7.40 (m,
5H). MS (TOF) m/z = 775.008 [M+Na]+, 791.11 [M+K]+. Analytical
HPLC: tR = 33.71 min (system A), 28.44 min (system B).
5.1.3.51. (R)-N-(3-Methyl)butyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-2-(3-
methylbutanoyl)amino-2-phenyl]acetylamino-3,3-dimethyl}-
butanoylamino-2-hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-
1,3-thiazolidine-4-carboxamide (iBuCO-Phg-Tle-Apns-Dmt-
NHiPe, 8hf). Compound 8hf was prepared from isovaleric acid
and compound 8af by general method A. Yield: 51%. 1H NMR
(CDCl3) d = 0.73–0.98 (m, 7ꢁ 3H), 1.30–1.60 (m, 1H+2H+2ꢁ 3H),
2.07–2.20 (m, 1H+2H), 2.58–2.85 (m, 2H), 3.07–3.42 (m, 2H),
4.00–5.00 (m, 4ꢁ 1H +2H), 5.53 (t, 1H), 6.14–6.29 (m, 1H), 6.30–
6.40 (br s, 1H), 6.60 (t, 1H), 6.75 (d, 1H), 6.90–7.26 (m, 5H),
7.27–7.40 (m, 5H). OH not observed. MS (TOF) m/z = 738.641
[M+H]+, 760.501 [M+Na]+, 776.499 [M+K]+. Analytical HPLC:
tR = 32.75 min (system A), 25.83 min (system B).
5.1.3.46. (R)-N-(3-Methyl)butyl-5,5-dimethyl-1,3-thiazolidine-
4-carboxamide (H-Dmt-NHi Pe, 5f). Compound 5f was prepared
from 3-methylbutylamine and Boc-Dmt-OH by general methods A
and B. Yield: 99%. 1H NMR (CDCl3) d = 0.90 (d, 2ꢁ 3H), 1.30–1.42
(m, 2H+3H), 1.55–1.67 (m, 1H+3H), 3.20–3.45 (m, 2H), 4.20 (s, 1H),
4.46 (s, 2H), 4.3–4.6 (br s, 1H), 6.85–7.05 (m, 1H). MS (LC) m/
z = 230.89 [M+H]+. Analytical HPLC: tR = 18.44 min (system A).
5.2. HIV-1 and HTLV-I protease inhibition assays
Compounds 8aa–8id were evaluated for HIV-1 protease inhibi-
tory activity at 50 nM of the test compound, and HTLV-I protease
inhibitory activity as IC50 values, using previously reported proce-
dures.21 The HTLV-I protease used in the assay was an L40I mutant
of the wild-type protease to prevent autolysis and improve
stability.23
5.1.3.47. (R)-N-(3-Methyl)butyl-3-[(2S,3S)-3-amino-2-hydroxy-
4-phenyl]butanoyl-5,5-dimethyl-1,3-thiazolidine-4-carboxamide
(H-Apns-Dmt-NHiPe, 6f). Compound 6f was prepared from Boc-
Apns-OH and compound 5f by general methods A and B. Yield:
50%. 1H NMR (CDCl3) d = 0.70–0.88 (m, 2ꢁ 3H), 1.30–1.50 (m,
1H+2H+2ꢁ 3H), 2.78–3.10 (m, 2H), 3.12–3.42 (m, 2H), 3.70–3.85
(m, 1H), 4.26–4.48 (m, 1H+2H), 4.92–5.07 (m, 1H), 7.06–7.14 (m,
2H), 7.21–7.40 (m, 5H), 7.9–8.1 (br s, 1H). OH not observed. MS
(TOF) m/z = 407.959 [M+H]+, 430.041 [M+Na]+, 445.92 [M+K]+.
Analytical HPLC: tR = 22.44 min (system A).
5.3. Quantitative structure–activity relationship study
QSAR equations were derived using Microsoft Excel and the
‘least squares’ method to calculate a straight line that best fits
the data. Scores were assigned as described in Section 3.
5.1.3.48. (R)-N-(3-Methyl)butyl-3-{(2S,3S)-3-[(2S)-2-amino-3,3-
dimethyl]butanoylamino-2-hydroxy-4-phenyl}butanoyl-5,5-
dimethyl-1,3-thiazolidine-4-carboxamide (H-Tle-Apns-Dmt-
NHiPe, 7f). Compound 7f was prepared from Boc-Tle-OH and com-
pound6f by general methods A and B. Yield: 66%. 1H NMR (CDCl3)
d = 0.68–1.00 (m, 5ꢁ 3H), 1.30–1.60 (m, 1H+2H+2ꢁ 3H), 2.77–3.85
(m, 2ꢁ 1H+2ꢁ 2H), 4.20–5.20 (m, 2ꢁ 1H+2H), 6.5–6.7 (br s, 1H),
7.03–7.27 (m, 5H), 8.0–8.2 (br s, 1H). OH and NH2 not observed.
MS (TOF) m/z = 521.049 [M+H]+, 543.142 [M+Na]+, 559.076
[M+K]+. Analytical HPLC: tR = 23.55 min (system A).
5.4. Computer-assisted docking experiments
Computer-assisted docking experiments of an HTLV-I protease
inhibitor with an HTLV-I protease L40I mutant were performed
as previously described,22 except that the target inhibitor is com-
pound 8dc.
Acknowledgments
This research was supported in parts by The Frontier Research
Program, The 21st Century COE Program from The Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan; and Japan
Society for the Promotion of Science’s Post-Doctoral Fellowship
for Foreign Researchers. We are grateful to Mr. T. Hamada for mass
spectrometry and HIV-1 enzymatic assay determinations.
5.1.3.49. (R)-N-(3-Methyl)butyl-3-{{(2S,3S)-3-{(2S)-2-[(2S)-2-
amino-2-phenyl]acetylamino-3,3-dimethyl}butanoylamino-2-
hydroxy-4-phenyl}}butanoyl-5,5-dimethyl-1,3-thiazolidine-4-
carboxamide (H-Phg-Tle-Apns-Dmt-NHiPe, 8af). Compound 8af
was prepared from Boc-Phg-OH and compound 7f by general
methods A and B. Yield: 65%. 1H NMR (CDCl3) d = 0.60–0.90 (m,
5ꢁ 3H), 1.25–1.63 (m, 1H+2H+2ꢁ 3H), 2.54–3.50 (m, 2ꢁ 2H),
3.80–5.32 (m, 5ꢁ 1H+2H), 6.35–6.45 (m, 1H), 6.60–6.70 (m, 1H),
6.75–6.85 (m, 1H), 6.90–7.00 (m, 1H), 7.07–7.26 (m, 5H), 7.28–
7.60 (m, 5H). OH and NH2 not observed. MS (TOF) m/z = 653.911
[M+H]+, 676.319 [M+Na]+, 692.083 [M+K]+. Analytical HPLC:
tR = 25.24 min (system A), 13.94 min (system B).
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