Design and synthesis of several small-size HTLV-I protease inhibitors with different hydrophilicity profiles

Article abstract of DOI:10.1016/j.bmcl.2011.02.066

The human T cell leukemia/lymphotropic virus type 1 (HTLV-I) is clinically associated with adult T cell leukemia/lymphoma, HTLV-I associated myelopathy/tropical spastic paraparesis, and a number of other chronic inflammatory diseases. To stop the replication of the virus, we developed highly potent tetrapeptidic HTLV-I protease inhibitors. In a recent X-ray crystallography study, several of our inhibitors could not form co-crystal complexes with the protease due to their high hydrophobicity. In the current study, we designed, synthesized and evaluated the HTLV-I protease inhibition potency of compounds with hydrophilic end-capping moieties with the aim of improving pharmaceutic and pharmacokinetic properties.

Full text of DOI:10.1016/j.bmcl.2011.02.066

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2425–2429
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of several small-size HTLV-I protease inhibitors
with different hydrophilicity profiles
⇑
Jeffrey-Tri Nguyen, Keiko Kato, Koushi Hidaka, Henri-Obadja Kumada, Tooru Kimura, Yoshiaki Kiso
Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The human T cell leukemia/lymphotropic virus type 1 (HTLV-I) is clinically associated with adult T cell
leukemia/lymphoma, HTLV-I associated myelopathy/tropical spastic paraparesis, and a number of other
chronic inflammatory diseases. To stop the replication of the virus, we developed highly potent
tetrapeptidic HTLV-I protease inhibitors. In a recent X-ray crystallography study, several of our inhibitors
could not form co-crystal complexes with the protease due to their high hydrophobicity. In the current
study, we designed, synthesized and evaluated the HTLV-I protease inhibition potency of compounds with
hydrophilic end-capping moieties with the aim of improving pharmaceutic and pharmacokinetic
properties.
Received 20 January 2011
Revised 10 February 2011
Accepted 15 February 2011
Available online 18 February 2011
Keywords:
Human T cell lymphotropic virus
HAM/TSP
Adult T cell leukemia
Aspartic protease
Inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
The human T cell leukemia/lymphotropic virus type 1 (HTLV-I) is
the first identified human retrovirus1 and the only known retrovirus
to cause human cancer, adult T cell leukemia/lymphoma (ATL).2
HTLV-I infection may also lead to a chronic progressive myelopathy3
and several inflammatory diseases as well as opportunistic infec-
tions.4 There is currently no cure for the estimated 20–30 million
HTLV-I infected patients5 living in endemic regions around the
world.6 To develop a cure for HTLV-I infection, we are targeting
HTLV-I protease, which is an enzyme responsible for processing pre-
cursor proteins to new viral components.7 HTLV-I protease assumes
a C2 symmetry homodimer fold consisting of 125-residue per chain,
and grossly resembling the HIV-1 protease. The narrow tunnel-like
active site where the precursor protein, substrate or inhibitor is
accommodated spans from the symmetrical S5 to S05 subsites
HTLV-I protease inhibitors with different hydrophilicity profiles
without jeopardizing the inhibition potency against HTLV-I prote-
ase. We selected KNI-10562 (1) as the reference compound, be-
cause of its small size and potent inhibition profile against HTLV-
I protease (Table 1).
HTLV-I protease has a high degree of specificity for substrates
over that of HIV-1 protease9 and bovine leukemia virus (BLV) pro-
tease.10 Similar to the substrate studies, we reported HTLV-I prote-
ase has a high degree of specificity for inhibitors.7 This means
structural requirements at subsites are more stringent for HTLV-I
protease binding and inhibition than for HIV-1 protease and BLV
protease. The HTLV-I11 and HIV-112 protease inhibition profiles in
the current study support this hypothesis, because although the
synthesized compounds ranged from low to high HTLV-I protease
inhibition (8–93% inhibition at 50 nM), all exhibited potent HIV-1
protease inhibition (72–100% inhibition at 50 nM). Of the subsites,
general observations on substrate specificity studies on retroviral
aspartic proteases13 along with our experience with HTLV-I prote-
ase inhibitor specificity14 suggest distant subsites from the cata-
(Fig. 1). Tucked between the S1 and S0 subsites, our central P1 inhib-
1
itory unit, a hydroxymethylcarbonyl isostere, is based on the transi-
tion state formed during hydrolysis of peptide bonds.
In a recent work, the X-ray diffraction crystallography struc-
tures of several of our tetrapeptidic HTLV-I protease inhibitors
in complex with a des-(117-125)-[Ile40]HTLV-I protease were
solved.8 In the study, only six out of 11 inhibitors formed co-
crystals with the protease, because significant precipitation oc-
curred in the preparation mixtures despite extensive screening
for other crystallization conditions. The co-crystallized inhibitors
were by and large more hydrophilic than those precipitated out
of solution. The aim of the current study is to discover small-size
lytic S1–S0 subsites have lower specificity. This means modifying
1
the two ends of reference inhibitor 1, namely the P01-cap and
P3-cap moieties, has a lower risk for negatively impacting the
HTLV-I protease inhibition potency.
In the current study, using computer-assisted molecular model-
ing,15 we designed novel analogues of reference HTLV-I protease
inhibitor 1 with different hydrophilic groups to the P01-cap and
P3-cap moieties. We foresaw the hydrophilic group could either
have a direct or water-mediated hydrogen bond interaction with
the protease, or otherwise a hydration shell could form at the
⇑
Corresponding author. Tel.: +81 75 595 4635; fax: +81 75 591 9900.
E-mail address: kiso@mb.kyoto-phu.ac.jp (Y. Kiso).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.066

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J.-T. Nguyen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2425–2429
Table 1
HTLV-I protease inhibition potency of P01 analogues
Compounds
R1
HTLV-I protease
HIV-1 protease
inhibition at
50 nM12(%)
inhibition11
(IC50, nM)
1
2
KNI-10562
KNI-10608
7.2 0.6
99
99
Figure 1. Cross-section view with a black slice plane of the active site of HTLV-I
protease in complex with inhibitor 5. Red spheres represent proximal water
molecules. Hydrogen bond interactions with the backbone of the inhibitor occur
above and below the plane of the page.
39.1 5.1
3
KNI-10605
10.4 0.6
99
modified end of the inhibitor and trap the compound inside the ac-
tive site. In our computer models, we observed backbone hydrogen
bond interactions from the P0 -cap to the P3-cap moiety were sim-
1
ilar in all inhibitors with differences where applicable (Fig. 2). We
synthesized the designed compounds (Fig. 3)16 and screened for
greater than 50% HTLV-I protease inhibition at 50 nM. The IC50 va-
lue was determined for any inhibitor passing the screening assay.
Lastly, the relative hydrophilicity was evaluated for highly potent
inhibitors.
Using reference inhibitor 1 as a starting point, the P01-cap
neopentylamine moiety was replaced by more hydrophilic ana-
logues in compounds 2 and 3. Inhibitor 2 was designed for a hydro-
gen bond between its distal hydroxyl group and the oxygen atom
of Met37B. Compared with reference inhibitor 1, the lower HTLV-I
protease inhibition in inhibitor 2 suggests the S02 pocket prefers
hydrophobic groups. The distal protonated hydrazine nitrogen of
the P01-cap t-butylhydrazide moiety in inhibitor 3 was designed
to form a hydrogen bond with Leu57B’s oxygen atom, across from
the S02 pocket. Inhibitor 3 exhibited slightly lower HTLV-I protease
inhibition than reference inhibitor 1.
At the P3-cap region of reference inhibitor 1, X-ray diffraction
crystallography data8 indicate the nitrogen of the P3-cap amide
forms a hydrogen bond with the side-chain of Asp36A, and the oxy-
gen of the amide has a hydrogen bond interaction with the amide
NH of Leu57A. Our past study reveals the S4 subsite can accommo-
date for several hydrophobic moieties of different length and
bulk.17 Considering the P3-cap and P4 moieties sit at the entrance
to the active site, a hydration shell should form with any hydro-
philic group introduced in the vicinity. In the current study, the
P3-cap methyl carbamate moiety in reference inhibitor 1 was first
substituted with P4 glycine as exemplified by inhibitor 4 that was
significantly less potent than reference inhibitor 1 (Table 2).
Extending the P4 residue to b-alanine afforded a slightly more po-
Figure 2. A typical hydrogen bond interaction pattern observed in inhibitors 1, 4–9
and HTLV-I protease. A cation-
p interaction is present between the inhibitor’s P3
phenyl group and the protease’s Arg10B (arrow). For simplicity, intra- and
intermolecular hydrogen bond interactions between the two protease chains are
not depicted.
possible hydrogen bond interactions found in inhibitors 4 and 5.
Compound 8 exhibited low inhibitory potency against HTLV-I pro-
tease. Compound 9 was designed with similar interactions in mind
and it too presented with low HTLV-I protease inhibition. Steric
hindrance is not likely a problem because the protease can accom-
modate different hydrophobic bulk in the S4 subsite.17,18 It is
conceivable hydrogen bond interactions at the S4 subsite disrupt
the favorable interactions elsewhere in the inhibitor. Indeed, our
recent X-ray diffraction study on HTLV-I protease inhibitors indi-
cates stronger interactions in the peripheral subsites can pull the
tips of the two flaps apart to the extent that two water molecules
are bound between the flaps.8
Computer-assisted modeling experiments were performed with
compounds 4–9 in extended pose to correlate the P3-cap/P4 struc-
ture with HTLV-I protease inhibition potency (Fig. 4). The proton-
ated amino group of inhibitor 5’s P4 b-alanine is maximally
hydrated by three water molecules (Fig. 4D). One of the water mol-
ecules mediates a hydrogen bond interaction with Ser55A. Another
water molecule bridged by a secondary water molecule links inhib-
itor 5’s amino group with Asp36A to the hydrogen bond network.
Thus, inhibitor 5’s potent inhibition against HTLV-I protease could
tent HTLV-I protease inhibitor (5). Further elongation to
c-
aminobutyric acid (GABA) led to inhibitor 6 exhibiting lower
HTLV-I protease inhibition potency than reference inhibitor 1.
Hence, the potency order from least to most potent for the P4-res-
idue is glycine, GABA and b-alanine. Reference inhibitor 717 with a
P3-cap n-butyramide moiety is more structurally similar to P4 b-
alanine inhibitor 5 than P3-cap methylcarbamate reference inhibi-
tor 1. Inhibitor 5 is slightly more potent or equipotent to reference
inhibitor 7, which suggests hydration shell formation is more likely
than direct hydrogen bond interaction with the protease.
To explore the effect of two hydrophilic moieties at the P4 posi-
tion on HTLV-I protease inhibition, a
L-2,3-diaminopropionic acid
residue was introduced in compound 8 to compare with the

J.-T. Nguyen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2425–2429
2427
Figure 3. A typical synthetic route for exemplified compounds 5 and 10.16 Reaction conditions: (a) 1.2 equiv carboxylic acid, 1.3 equiv BOP, DMF, 1.5 equiv Et3N, rt, overnight;
(b) 4 N HCl in dioxane, rt, 30 min; (c) 1.2 equiv EDC, 1.3 eq HOBt, DMF, rt, 30 min, 1.1 equiv carboxylic acid in DMF, dropwise, then overnight; (d) (pre-activation: 1.2 equiv
carboxylic acid, 1.3 equiv CDI, DMF, 1.5 equiv Et3N, rt, 1 h), overnight.
interactions with Ser55A are disfavored, because the interactions
pull the protease’s two flaps apart. Glycine inhibitor 4 shares a sim-
Table 2
HTLV-I protease inhibition potency of P3-cap and P4 analogues
ilar water-mediated hydrogen bonding pattern with Asp36A and
Ser55A as b-alanine inhibitor 5 (Fig. 4C). However, the water mole-
cule mediating a hydrogen bond interaction with Ser55A also asso-
ciates with the side-chain of Gln62A, thus making the overall
interactions at the P4 position stronger in glycine inhibitor 4 than
b-alanine inhibitor 5. In the case of GABA inhibitor 6, a hydration
shell is present (Fig. 4E). The lack of direct or strong water-medi-
Compounds
R1
HTLV-I
HIV-1 protease
inhibition at
50 nM12 (%)
ated hydrogen bond interaction with the protease contributes to
inhibitor 6’s higher inhibition potency than compounds 4, 8 and
9. However, the weaker association with the protease at the S4 sub-
site renders inhibitor 6 less potent than inhibitor 5. Taking to-
gether, we assume hydrophobic bulk is a minor factor17,18 and
propose HTLV-I protease inhibition potency at the S4 subsite is
determined by a hydrogen bond balance between strong interac-
tions in compounds 4, 8 and 9 and weak interactions in inhibitor
6. The more potent inhibitor 5 has well balanced hydrogen bond
interactions. However, from the same line of reasoning, reference
inhibitor 7 should exhibit low potency because of the absence of
hydrogen bond interactions at the P4 position, yet the hydrophobic
compound is quite potent against HTLV-I protease (Fig. 4F). The
current study demonstrates P4 hydrophilic groups, on the other
hand, significantly affect protease inhibition from 8% (compound
8) to 90% (compound 5) at 50 nM compound concentrations.
Hence, we speculate hydrophobic moieties are generally preferred
at the P4 position over hydrophilic moieties, unless there are P4
hydrogen bond interactions anchoring the inhibitor in the active
site without pulling the protease’s flaps apart.
protease
inhibition11
(IC50
)
4
5
6
7
KNI-10856
41.1 11.2 nM
5.7 0.4 nM
25.4 7.7 nM
6.4 1.5 nM
94
99
99
99
KNI-10838
KNI-10857
KNI-10635
8
9
KNI-10859
KNI-10858
8% at 50 nM
72
86
18% at 50 nM
In the previously reported X-ray diffraction crystallography
study, tetrapeptidic HTLV-I protease inhibitors with a free P3 ami-
no group formed co-crystals with the mutant protease.8 Hence, the
P3-cap truncated analogue of reference inhibitor 1,17 namely com-
pound 10, is expected to also form co-crystals with the protease. It
is noteworthy reference inhibitor 10 is less potent than reference
inhibitor 1 because of a missing important hydrogen bond interac-
tion with the protease’s Leu57A. The P3 amino group forms an
important hydrogen bond with the protease’s Asp36A. Appending
a methyl group in inhibitor 11 or an ethyl group in inhibitor 12
seems to successively improve the inhibition profiles. However,
the presence of a benzyl moiety as the P3-cap moiety in compound
13 is less favored by the protease than ethyl-capped inhibitor 12.
During the synthesis of inhibitors 11 and 12, the respective disub-
stituted compounds 14 and 15 were isolated after a nucleophilic
substitution reaction. Although the tertiary amino group in com-
pounds 14 and 15, once protonated to a quartenary salt, can have
hydrogen bond interactions with Asp36A, the inhibition profiles are
10
11
KNI-10680
KNI-10752
37.0 5.3 nM
31.4 3.8 nM
>99
>99
12
13
14
15
KNI-10767
KNI-10756
KNI-10753
KNI-10768
25.5 4.6 nM
44% at 50 nM
38% at 50 nM
39% at 50 nM
99
92
99
98
be attributed to two water-mediated hydrogen bond interactions.
The less potent HTLV-I protease inhibition profiles for compounds
8
(Fig. 4B) and 9 (Fig. 4A) suggest direct hydrogen bond

2428
J.-T. Nguyen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2425–2429
Figure 4. Hydrogen bond interactions observed at the P4 position in the extended pose of compounds (A) 9, (B) 8, (C) 4, (D) 5, (E) 6 and (F) 7. For simplicity, only water
molecules interacting with the compound at the S4 subsite are depicted. Water molecules are red spheres, protease residues are green, and the compound is gray.
as expected less potent than their primary and secondary amine
analogues (10–12). As previously mentioned, the occupancy in
the S4 subsite by a hydrophobic group is a minor determinant of
HTLV-I protease inhibition potency17,18 and the moderate changes
in the inhibition profiles of compounds 10–15 (38–62% at 50 nM)
support this generalization.
Compounds 1, 3, 5 and 7 were selected for relative hydrophi-
licity profiling based on their high potency against HTLV-I prote-
ase with IC50 values less than 15 nM. Their hydrophilicity
profiles were estimated using three calculation methods examin-
ing their calculated log P (log of the octanol/water partition coef-
ficient including implicit hydrogens),15 S log P (log P calculation
that accounts for acidic protonation state)19 and log S (log of
the aqueous solubility [mol/L])20 values. The relative hydrophilic-
ity for the compounds was experimentally determined by their
mobility in an HPLC gradient mixture of acetonitrile and aqueous
0.1% trifluoroacetic acid (Fig. 5). The HPLC chart suggests S log P
is more accurate than calculated log P and log S at predicting the
Figure 5. HPLC chart of a mixture of pure compounds 1, 3, 5 and 7. In this reverse
phase HPLC running with a linear 20–100% gradient of acetonitrile (30 min, 2 min
delay) in 0.1% aqueous trifluoroacetic acid, the more hydrophilic compounds have
shorter retention time.
relative hydrophilicity order of the compounds—from most to
least hydrophilic, compounds 3, 5, 1 and 7. Because of its
N-terminal carbamate nitrogen, compound
1 is sufficiently
hydrophilic to form co-crystal complexes with HTLV-I protease.8
In addition to the carbamate group, compound 3 has a P01-cap
hydrazide nitrogen that can be protonated under the experimen-
tal acidic aqueous conditions to render it more hydrophilic than
its reference compound 1. Similarly the P4 b-alanine amino
group in compound 5 is protonated under experimental condi-
tions to make the compound more hydrophilic than its reference
compound 7. As described in our X-ray crystal study,8 significant
precipitation was observed in mixtures of HTLV-I protease and a
hydrophobic inhibitor resembling compound 7, and despite
extensive screening for several crystallization conditions, crystals
were not formed. That being said, we also expect similar difficul-
ties with hydrophobic compound 7. On the other hand, several

J.-T. Nguyen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2425–2429
2429
structurally similar hydrophilic compounds with a P3 free amino
References and notes
group can form co-crystal complexes with the protease.8 Hence,
we anticipate the formation of co-crystal HTLV-I protease com-
plexes with hydrophilic compounds 3 and 5. In addition, these
compounds with different hydrophilicity properties should also
facilitate future pharmaceutics and drug formulation studies.
From a pharmacokinetic perspective, the hydrophilicity of a
compound plays a significant role in its absorption in the gastro-
intestinal tract, subsequent metabolism in the gut wall and liver,
and clearance. One should note the P3-cap methylcarbamate
1. Poiesz, B. J.; Ruscetti, F. W.; Reitz, M. S.; Kalyanaraman, V. S.; Gallo, R. C. Nature
1981, 294, 268.
2. Takatsuki, K. Retrovirology 2005, 2, 16.
3. Jacobson, S. J. Infect. Dis. 2002, 186, 187.
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5. Nicot, C. Am. J. Hematol. 2005, 78, 232.
6. Proietti, F. A.; Carneiro-Proietti, A. B.; Catalan-Soares, B. C.; Murphy, E. L.
Oncogene 2005, 24, 6058.
7. Nguyen, J. T.; Kiso, Y. In Viral Proteases and Antiviral Protease Inhibitor Therapy
Proteases in Biology and Disease; Lendeckel, U., Hooper, N. M., Eds.; Springer
Science: Dordretch, 2009. Vol. 8, pp 83–100.
8. Satoh, T.; Li, M.; Nguyen, J.-T.; Kiso, Y.; Gustchina, A.; Wlodawer, A. J. Mol. Biol.
2010, 401, 626.
group of compound
1 might be degraded in vivo under
acid–base conditions to produce less potent inhibitor 10. Similar
problems might arise from compound 3. Computational pharma-
cokinetics suggest oral bioavailability is likely for compounds
with log P values between ꢀ0.4 and 5.6.20 The calculated log P
values for compounds 1, 3, 5 and 10 fall within range (from
3.8 to 5.1) with hydrophobic compound 7 at the borderline
(log P = 5.7). Being hydrophobic, compound 7 has a higher risk
for unwanted binding with untargeted biological substances,
such as plasma proteins, that delay and prevent the compound
from reaching the target site of action. Conversely, high water
solubility compounds have higher renal clearance from the body.
However, high renal clearance is not expected in compounds 1,
3, 5, 7 and 10, because they are not highly hydrophilic. On the
other hand, diverse types of metabolic reactions can occur with
compounds 3, 5 and 10 at the respective nitrogen, such as glu-
curonide formation, that may deactivate and assist in their re-
moval from the body.21 Nevertheless, the P01-cap hydrazide free
nitrogen in compound 3 should have relatively lower metabolic
reactivity because of steric bulk from the adjacent t-butyl group.
Overall, in designing our compounds, we looked not only at po-
tent inhibition against HTLV-I protease but also at a series of
compounds with a large spread in their hydrophilicity profiles.
Now, having established P01-cap t-butyl hydrazide and P4
b-alanine moieties increase hydrophilicity without greatly affect-
ing HTLV-I protease inhibition potency, we could evaluate com-
pounds with several P01-cap bulky hydrazide groups and various
P3-cap amido or P4 b-alanine moieties in future studies to obtain
a larger selection of potent HTLV-I protease inhibitors with a
wide range of hydrophilicity property.
9. Kádas, J.; Weber, I. T.; Bagossi, P.; Miklóssy, G.; Boross, P.; Orozslan, S.; Tözsér, J.
J. Biol. Chem. 2004, 279, 27148.
10. Tözsér, J. Viruses 2010, 2, 147.
11. Recombinant L40I mutation HTLV-I protease percent inhibition potency at
50 nM of the test compound was evaluated as single determinations using
previously reported procedures.8 IC50 values were calculated from the sigmoid
plot derived by percent inhibition data at 1, 5, 10, 20, 50 and 100 nM of the test
compound, as a single determination at each concentration, using Synergy
Software’s KaleidaGraph (Supplementary data). The error range for IC50 values
was calculated from the root mean square deviation (RMSD) of the plot, that is,
50% RMSD inhibition.
12. Recombinant HIV-1 protease percent inhibition potency at 50 nM of the test
compound was evaluated as single determinations using previously reported
procedures.22
13. Tözsér, J.; Zahuczky, G.; Bagossi, P.; Copeland, J. M.; Oroszlan, S.; Harrison, R.
W.; Weber, I. T. Eur. J. Biochem. 2000, 267, 6287.
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Hayshi, Y.; Kiso, Y. Bioorg. Med. Chem. Lett. 2007, 17, 3276.
15. Computer-assisted modeling experiments and chemical property calculations
were performed using Molecular Operating Environment 2009.1001 (MOE), and
modeled from PDB 3LIN. When studying the involvement of water (Fig. 4), a
sphere (10 Å) of water was generated around the compound’s amino or hydroxyl
groups using MOE’s Water-Soak function. The system was energy minimized
using the LigX function under MMFF94x force-field: the proximal (8 Å) protease
heavy atom residues were tethered while the compound was flexible.
Visualization of the subsites (Fig. 1) was assisted by UCSF Chimera 1.4.1.
16. The synthesis of reference compounds 1,8 717 and 1017 was previously
described (Fig. 3). Compounds 1–15 were synthesized by standard solution
phase peptide synthesis by which sequential elongation and coupling of an
amine to a carboxylic acid was performed in DMF with benzotriazol-1-yl-oxy-
tris-(dimethylamino)phosphonium hexafluorophosphate (BOP) or N,N0-
carbonyldiimidazole (CDI) as coupling reagent and Et3N as base. Although
BOP was favored over CDI for coupling to the P01 residue, CDI showed higher
coupling reactivity with bulky amines. Peptide coupling to the P1 residue was
accomplished
using
1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide
hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt) as additive, without
a base, to avoid reported side-reactions.23 Attachment of a Boc protection
group to an amino group was achieved with Boc2O and Et3N in DMF, while the
removal of the Boc protection group was achieved with 4 N HCl in dioxane. The
In the current study, we designed and synthesized HTLV-I pro-
tease inhibitors with the aim of retaining high inhibition potency
with a good mix of hydrophilicity profiles. We succeeded in iden-
tifying inhibitors 1, 3, 5 and 7 that have IC50 values less than
15 nM and an adequate spread in hydrophilicity.
hydroxyl group in compounds
2 and 9 was acetyl protected with acetic
anhydride in pyridine, and eventually removed by a 50% v/v mixture of 4 N
NaOH(aq) and MeOH. The N-terminal cap was appended in DMF and Et3N as
base by a reaction with methyl chloroformate for compounds 2 and 3, or with
benzylbromide for compound 13. The nucleophilic substitution reactions in
compounds 11, 12, 14 and 15 was performed with methyl iodide or ethyl
iodide, respectively, to afford a mixture of mono- and disubstituted products.
After preparative HPLC purification, all target compounds (1–15) were >95%
pure by analytical HPLC (Supplementary data). The identities of the
compounds were confirmed by TOF MS and ESI-Q MS.
Acknowledgments
This study was supported in part by the ‘Academic Frontier’
Project for Private Universities, a matching fund subsidy from the
Ministry of Education, Culture, Sports, Science and Technology
(MEXT), Japan. We thank Mr. T. Hamada for performing HIV-1 pro-
tease inhibition assay.
17. Zhang, M.; Nguyen, J.-T.; Kumada, H. O.; Kimura, T.; Cheng, M.; Hayashi, Y.;
Kiso, Y. Bioorg. Med. Chem. 2008, 16, 6880.
18. Zhang, M.; Nguyen, J.-T.; Kumada, H. O.; Kimura, T.; Cheng, M.; Hayashi, Y.;
Kiso, Y. Bioorg. Med. Chem. 2008, 16, 5795.
19. Oprea, T. I.; Davis, A. M.; Teague, S. J.; Leeson, P. D. J. Chem. Inf. Comput. Sci.
2001, 41, 1308.
20. Hou, T. J.; Xia, K.; Zhang, W.; Xu, X. J. J. Chem. Inf. Comput. Sci. 2004, 44, 266.
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Supplementary data
22. Maegawa, H.; Kimura, T.; Arii, Y.; Matsui, Y.; Kasai, S.; Hayashi, Y.; Kiso, Y.
Bioorg. Med. Chem. Lett. 2004, 14, 5925.
23. Hayashi, Y.; Kinoshita, Y.; Hidaka, K.; Kiso, A.; Uchibori, H.; Kimura, T.; Kiso, Y. J.
Org. Chem. 2001, 66, 5537.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.066.