On the inhibition of HIV-1 protease by hydrazino-ureas displaying the N→C{double bond, long}O interaction

Article abstract of DOI:10.1016/j.bmc.2009.03.059

To create novel HIV-1 protease (HIV PR) inhibitors, we have extended our investigations of the N→C{double bond, long}O interaction as a moiety that reproduces electrostatic properties of the transition state of peptidolysis. Consequently, we prepared a se

Full text of DOI:10.1016/j.bmc.2009.03.059

Bioorganic & Medicinal Chemistry 17 (2009) 3671–3679
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
On the inhibition of HIV-1 protease by hydrazino-ureas displaying
the N?C@O interaction
*
Michael Waibel, Delphine Pitrat, Jens Hasserodt
Laboratoire de Chimie, UMR CNRS 5182, Université de Lyon-ENS, 46 allée d’Italie, 69364 Lyon cedex 07, France
a r t i c l e i n f o
a b s t r a c t
Article history:
To create novel HIV-1 protease (HIV PR) inhibitors, we have extended our investigations of the N?C@O
interaction as a moiety that reproduces electrostatic properties of the transition state of peptidolysis.
Consequently, we prepared a series of compounds with an unusual hydrazino-urea core. In polar protic
media, these adopt solely a cyclic constitution displaying the interaction on one side of the molecule
while offering a urea moiety on the opposite side meant to hydrogen-bond with the enzyme flaps. Each
inhibitor candidate was obtained via a key series of three synthetic steps employing carbonyl-di-imidaz-
ole (CDI). It was thus possible to efficiently fuse two independent building blocks, a hydrazine and a pro-
tected aminoaldehyde in a convergent manner. NMR and UV analysis proved that all compounds, when
dissolved in polar protic media, existed exclusively in the cyclic constitution exhibiting the N?C@O
interaction. In total, five inhibitor candidates were tested with HIV PR for their potency. The one carrying
the least bulk in peripheral substituents showed the highest activity. Its very low molecular weight
(365 g/mol) holds great promise for future improvements in affinity without violating Lipinski’s rule of
remaining within the limit of 500 g/mol.
Received 5 December 2008
Revised 23 March 2009
Accepted 29 March 2009
Available online 5 April 2009
Keywords:
Cyclic urea
HIV-1 protease inhibitors
Transition-state isostere
N?C@O interaction
Preorganization
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Alternatively, rigid heterocyclic cores have been used as tem-
plates for the development of potent agents that have become
Moieties mimicking the transition-state of peptide hydrolysis
have proved efficient in the design of inhibitors for aspartic prote-
ases.1–3 HIV-1 protease (HIV PR) belongs to this family of enzymes
and remains to be one of the major targets in the search for drugs
battling AIDS.4,5 The development of novel agents against HIV is
necessary because of the continued emergence of virus strains that
resist current clinical drugs.6,7 The established approach in design-
ing inhibitors has been to incorporate a transition-state analog (i.e.,
isostere) into a peptidomimetic reproducing the linear shape of the
peptide substrate. This strategy evolved from the discovery that
certain naturally occurring peptides (e.g., pepstatine), containing
a non-proteinogenic amino acid called statine, are generic aspartic
protease inhibitors. The tetrahedral secondary alcohol moiety con-
tained in statine is believed to act as a transition state mimic ever
since it was shown that it interacts with the two aspartic acid res-
idues of the catalytic center.8,9 Many other transition-state mim-
icking moieties have been explored over the years, such as
known as non-peptidic inhibitors. Two well-known representa-
tives are the cyclic urea DMP 450 (1)11 and tipranavir (2)12,13 that
is based on the 5,6-dihydro-4-hydroxy-2-pyrone core (Fig. 1). The
rigid aspect of these cores can lead to higher affinity because a low-
er entropic penalty has to be paid upon binding.11,14,15 It is also
generally maintained that increased rigidity can result in better
oral bioavailability.16 Bioavailability also critically depends on
molecular weight.17 These points illustrate the need for finding
as yet unexplored molecular motifs with superior affinity to their
target site, allowing for reduction of overall inhibitor size. Another
key feature of non-peptidic heterocycles 1 and 2 is the carbonyl
function located opposite to the hydroxyl group(s). While the latter
one(s) interact(s) with the catalytic aspartyl diad of HIV PR, the
carbonyl undergoes hydrogen-bonding with two protein flaps of
the enzyme. This results in the extrusion of a key enzyme bound
water molecule that is habitually found in X-ray crystal structures
of enzyme complexes with linear inhibitors and thus adds to the
favorable entropic effect mentioned earlier.11,18
hydroxyethylamine, hydroxyethylene, reduced amide,
a
-perfluori-
nated ketones,
a-ketocarboxamides, and phosphorus-based
We have been interested10 in an infrequently observed func-
tional group for its capacity to mimic essential geometric and also
electrostatic features of the transition states of peptide hydrolysis
(see transition state structure TS (8)19,20 and examples 4,21 5,22 6,23
7a–c10 in Fig. 2). This molecular moiety results from the intramo-
lecular interaction of a tertiary amine with a ketone or aldehyde
units.10
* Corresponding author. Tel.: +33 472 72 8394; fax: +33 472 72 8860.
E-mail address: jens.hasserodt@ens-lyon.fr (J. Hasserodt).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.059

3672
M. Waibel et al. / Bioorg. Med. Chem. 17 (2009) 3671–3679
-OOCAsp25
-OOCAsp25
'
'
-OOCAsp25
'
Asp25COOH
Asp25COOH
Asp25COOH
δ−
CF3
O
H
OH
O
R1
R2
R4
R5
HO
N
OH
N
H
N
N
N
δ+
S
N
N
O
O
R3
R6
O
O
O
H2N
Ile50-NH
DMP 450 ( )
NH2
Ile50-NH
HN-Ile50'
HN-Ile50'
Ile50-NH
HN-Ile50'
1
2
3
target core ( )
tipranavir ( )
Figure 1. Hydrogen bonding between the catalytic diad (aspartic acid residues) as well as the enzyme flaps (isoleucines) with cyclic urea inhibitor DMP 450 (1), heterocyclic
inhibitor Tipranavir (2), and hypothesized interactions with target structure 3.
Compounds 7a–c are peptidomimetics incorporating the
δ−
N?C@O interaction that mimic the Phe-Pro dipeptide sequence,
one of the preferred cleavage sites of HIV PR. Molecules 7a–c exist
as an equilibrium of the open form, and a cyclized form that dis-
plays the N?C@O interaction. The highest degree of cyclization
in methanol (70%) was achieved with 7a. Mixtures including sev-
eral constitutional isomers are however difficult to analyze. More
importantly, the presence of an open form rules out any explora-
tion as a drug candidate since aldehydes are justly regarded as
too reactive towards nucleophiles in live organisms.
Recently, we have discovered a hydrazino-urea core unit of the
generic structure 3 that exist to 100% in its cyclic, N?C@O inter-
acted, form in polar protic media (Fig. 1).32 In analogy to the potent
heterocyclic inhibitor cores 1 and 2, it also has a carbonyl group
that might undergo hydrogen-bonding with the enzyme flaps. In
this study, we present the synthesis of a number of structural vari-
ations of 3, the analysis of their folding behavior, and the determi-
nation of their inhibitory potency with HIV PR.
O
H
δ+
N
δ+
N
H
O
δ−
N
OH
4
HO
H
O
(-) sarain A (5)
δ−
O
δ+
N
δ−
δ+
P2HN
O
N
H
O
COP2'
8
TS ( )
6
O
δ−
H
Ph
O
H
δ+
COR2
Ph
R1
R1HN
N
N
2. Results and discussion
2.1. Chemical synthesis
N
H
R2OC
7a: R1 = Alloc, R2= OMe
: R1 = CbzVal, R2= NHtBu
7b
7c: R1 = CbzVal, R2= ValNHtBu
Scheme 1 illustrates the convergent assembly of two examples
of the generic structure in 3 that are derived from phenylalanine
and proline. The synthesis of the hydrazine building block started
with the amination of (S)-proline using freshly prepared p-cyano-
phenyl oxaziridine to furnish N-Boc protected hyrazino proline
9.33,34
Figure 2. A selection of compounds (4–7), displaying the N?C@O interaction, and
2nd hydrolysis TS (8) of the preferred dipeptide sequence Phe-Pro of HIV PR.
function (N?C@O interaction), and has been observed initially in a
rare class of alkaloids as a transannular contact across medium-
sized rings.24,25 The N?C@O interaction has been described as a
through-space homoconjugation where electron density is shifted
from the n nitrogen orbital to the carbonyl p* orbital,26 giving rise
to an enhanced dipole moment27 and a hypsochromic shift of the
carbonyl UV absorption.10,28 It has been shown that the interaction
is favored in polar protic media, and when incorporated into con-
formationally restricted frameworks.10,21,23,29–31 Recently, the
N?C@O moiety has featured prominently in the total synthesis
of the marine natural product (ꢀ) sarain A (5).22 Its attractive fea-
tures for this project are: (a) a carbon with a transient configura-
tion between sp2 and sp3; (b) an N–C bond order between 0 and
1; (c) a C–O bond order between 1 and 2; and (d) partial charges
on the oxygen and the nitrogen, resulting in an enhanced dipole
moment.27 This strongly polarized unit may thus form particularly
strong hydrogen bonds and electrostatic interactions with the cat-
alytic diad of the two aspartate residues.
Hydrazinopeptides 10 and 11 were obtained by reacting com-
pound 9 with H2N-tBu, and H-Ile-Val-NH-tBu, respectively, using
EDC and HOBt as coupling reagents.35 The latter building block
was initially prepared by peptide coupling of Cbz-Ile-OH and H-
Val-NH-tBu, followed by Pd/C-catalyzed hydrogenolysis. A subse-
quent attempt to remove the Boc group from the hydrazine unit
according to a reported procedure (HClaq in dioxane)36 gave unsat-
isfactory results. These may be the consequence of reactions
involving the highly nucleophilic hydrazine during water removal
in the workup procedure at slightly elevated temperatures. On the
other hand, exposure of 10 and 11 to neat TFA for two hours fol-
lowed by solvent removal at room temperature, gave the desired
hydrazine intermediate as the only product. This was immediately
treated with benzaldehyde, followed by reduction of the resulting
hydrazone with NaCNBH3 and an excess of acetic acid to produce
12 and 13, respectively. The overall yields of deprotection, hydra-
zone formation and reduction were 83% and 80%, respectively,

M. Waibel et al. / Bioorg. Med. Chem. 17 (2009) 3671–3679
3673
R
N
HN
R
O
b,c,d
a
O
CO2H
N
N
NHBoc
BocHN
9
10 R= NHtBu (74%)
11 R= IleValNHtBu (78%)
12
13
R= NHtBu (83%, 3 steps)
R= IleValNHtBu (80%, 3 steps)
R
O
R
O
N
H
N
N
MeO
MeO
MeO
MeO
H
N
N
N
NH2
e,f,g
h
O
O
O
(S)-14
15 R= NHtBu (66%, 3 steps)
16 R= IleValNHtBu (72%, 3 steps)
17 R= NHtBu (83%)
18 R= IleValNHtBu (78%)
Scheme 1. Synthesis of target compounds 17 and 18. Reagents and conditions: (a) RNH2, EDC, HOBt, CH2Cl2, rt, 2 h; (b) TFA, rt, 2 h; (c) PhCHO, MeOH, rt, 12 h; (d) NaCNBH3,
HOAc, MeOH, rt, 2 h; (e) CDI, THF, rt, 15 min; (f) MeI, MeCN, rt, 12 h; (g) 12 (for 15) or 13 (for 16), NEt3, CH2Cl2, rt, 12 h; (h) TMSCl, NaI, MeCN, rt, 1 h.
while purification by column chromatography was only necessary
following the last step. The hydrazine building block can thus be
prepared quite efficiently from commercially available (S)-proline.
Carbonyl-di-imidazole (CDI) was used as coupling reagent to con-
nect 12 and 13 with the readily available building block (S)-14 to
furnish unsymmetrical ureas 15 and 16. Replacement of only one
imidazole unit of CDI by (S)-14 is achieved in only 15 min at room
temperature. The resulting intermediate is much less reactive and
does not react with 12 or 13. By contrast, when the imidazole
nitrogen is quaternized with iodomethane,37 the corresponding,
highly reactive imidazolium salt reacted smoothly with the hydra-
zine building blocks under mild conditions. The overall yields of
CDI reaction with the amine part, activation and reaction with
the hydrazine were 66% and 72%, respectively.
Deprotection of dimethyl acetals 15 and 16 with in situ gener-
ated TMSI gave target compounds 17 and 18.38 Scheme 2 shows
the synthesis of a different example of the generic structure in 3
in which the stereocenter of the phenylalanine part is reversed.
Readily available benzyl hydrazine 19, and non natural amino acid
derivative (R)-14 were coupled with CDI as described above to pro-
duce unsymmetrical urea (R)-20. Subsequent deprotection gave
target compound (R)-21 in excellent yield.
2.2. Configurational and constitutional analysis
Prior to in vitro tests with HIV PR, it was important to explore
the tendency of the new compounds to exist in their cyclized form.
NMR and UV spectroscopy are an appropriate means to prove the
existence of the N?C@O interaction.10,21,23,29–31 The proton and
carbon NMR spectra in chloroform of compounds 17, 18 and (R)-
21 all exhibit only one set of signals including one resonance cor-
responding to the aldehyde group. By contrast, in methanol all sig-
nals corresponding to the aldehyde forms are no longer present. In
effect, the 13C NMR spectra now show two independent signal sets
of equal intensity, including two characteristic resonances (101.4
and 100.0 ppm in 17, Fig. 3), right in the middle of the region be-
tween the olefinic/aromatic and the aliphatic region. These signals
may be ascribed to the newly formed pseudo-tetrahedral methine
carbon, adopting a hybridization state between those of sp2 and
sp3. The former prochiral aldehyde group has thus been turned into
a new stereogenic unit resulting in the formation of two diastereo-
mers without any particular stereoselectivity; similar observations
were made in a previous study.23 The characteristics of the carbon
spectra are reflected by those of the proton ones, in that the signals
of the aldehyde protons have disappeared, while new resonances
were observed in the shift interval between 4.61 and 4.46 ppm
that can be attributed to the methine protons of the folded
forms. These combined observations agree with literature data,
according to which the N?C@O interaction is favored in polar
protic media.10,21,23,30,31
In our previous work, compounds 7a–c adopted only the linear
aldehydic form in chloroform, while in methanol they existed as a
mixture of varying proportions between the open and the cyclized
forms.10 In fact, the cyclization tendency of 7a–c decreased with
the increase in length of the peptide side chain, shifting from
70% cyclic 7a to 55% cyclic 7b and 45% cyclic 7c. This was explained
with the hydrophobic nature of the peripheral chains that may fold
back onto each other in order to minimize exposure to the polar
solvent, resulting in the rupture of the N?C@O interaction. In
the present work, cyclization is complete for all compounds. This
demonstrates the pronounced preorganizing effect of the planar
urea moiety, and thus corroborates our molecular design.
Scheme 2. Synthesis of target compound (R)-21. Reagents and conditions: (a) (R)-
14, CDI, THF, rt, 15 min; (b) MeI, MeCN, rt, 12 h; (c) 19, NEt3, CH2Cl2, rt, 12 h, 73%
(three steps); (d) TMSCl, NaI, MeCN, rt, 1 h, 93%.
It has been previously demonstrated that UV spectroscopy is
an independent means to detect the N?C@O interaction.10,28,32

3674
M. Waibel et al. / Bioorg. Med. Chem. 17 (2009) 3671–3679
Figure 3. 13C NMR spectra (127 MHz) of 17 in CDCl3 (j signal of the open-form aldehydic carbon) and MeOH-d4 (d signals of the cyclized form methine carbons). Expanded
regions illustrate the generation of two diastereomers with no particular selectivity.
The N?C@O group is characterized by an absorption that is
much more intense and appears at a shorter wavelength than
that caused by the aldehyde moiety. Figure 4 shows the UV
spectra of compounds 17, 18, and (R)-21 in chloroform and in
methanol. In chloroform, all spectra have their maximum at
241 nm, corresponding to the absorption of the aldehyde func-
absorption behavior of the N?C@O interaction is now well
established in the literature and perfectly correlates with the
NMR data.10,28,32
2.3. In vitro Inhibition of HIV PR
tion. Their extinction coefficients were calculated to be
e
= 674,
The capacity of compounds 17, 18 and (R)-21 and of previously
synthesized ureas (S)-21 and 22 (see Table 1),32 to inhibit HIV PR
was tested in a well-established continuous FRET (fluorescence
resonance energy transfer) assay.39,40 Pepstatine was tested simul-
taneously as standard. Initially, we determined the kinetic param-
eters of our recombinant source of HIV PR with a commercial FRET
731, and 871 L molꢀ1 cmꢀ1, being in the typical range for alde-
hydes. By contrast, in methanol this band is shifted to 208 nm
and has become much more intense. The corresponding extinc-
tion coefficients were
e
= 20,350, 18,201, and 21,200 L molꢀ1
cmꢀ1 for compounds 17, 18 and (R)-21, respectively. This
Figure 4. UV spectra of compounds 17, 18 and (R)-21 in MeOH (dotted line) and in CHCl3 (continuous line).

M. Waibel et al. / Bioorg. Med. Chem. 17 (2009) 3671–3679
3675
substrate in order to evaluate our assay performance in compari-
son to the literature data. The obtained mean values of KM = 25 M,
and Vmax = 272 nM/min are in the range of those from previous re-
ports.10,39 The potency of the inhibitors was first estimated by
determining their IC50 values by fitting the experimental data to
by methanol. In no instance have we noticed an impairment to
the mutual approach of the two interacting functional groups for
steric reasons. Generally, the new compounds exhibit higher inhib-
itory potency than those from our earlier work (7a–c), conceivably
caused by the combined effects of simultaneous satisfaction of the
di-Asp motif and the binding partners on the protein flaps; this re-
mains of course to be confirmed by further SAR studies concerning
all points of variation on the core and by a complementary X-ray
structure analysis of the corresponding enzyme–inhibitor complex.
It should be stressed that the demonstrated absence of any aldehy-
dic component constitutes a major stride towards the consider-
ation of the N?C@O motif as a future pharmacophore. The most
potent inhibitor in this study is so small that great improvements
in affinity and ADME properties may be obtained by introduction
of additional molecular bulk without violating Lipinski’s rule of
remaining within the limit of 500 g/mol. We feel thus encouraged
to initiate a hit-to-lead transformation17 by a combined computa-
tional and combinatorial tactic.
l
v/v0 = 1/(1 + [I]/IC50) (Table 1). The exact Ki constants were derived
from the same set of experiments.41 For comparison, this table also
includes the inhibition data for compounds 7a–c.10 The IC50 values
of all hydrazino-ureas tested were slightly higher than the Ki val-
ues, which is in accord with the general relation for competitive
inhibitors (IC50 = Ki(1 + [S]/KM) when the substrate concentrations
are low compared to the KM value, as they were in this study.42
The Ki value obtained for pepstatine serves as a positive control
and closely corresponds to the literature values that range from
0.4 to 250 nM depending on the experimental conditions used.39
Matayoshi and Krafft obtained a Ki value of 17 nM with a similar
FRET substrate.39 We have earlier reported an inhibition constant
of Ki = 36 nM using the same substrate and conditions as in the
present work.10 In general, the compounds of the hydrazino-urea
series have better inhibitory potency than the first-generation
inhibitors 7a–c. Among the newly synthesized molecules, the
small (S)-21 and 17 were the best compounds having about one or-
der of magnitude higher affinity than 18, 22 and (R)-21. They were
also approximately three times more effective than the best com-
pound of the earlier series (7c). This increase in performance has to
be appreciated in the light of the considerable reduction in size
(46% drop) when switching from 7c to (S)-21 (see Table 1).
As a structurally novel entity the hydrazino urea motif had to be
examined also in regard to its mode of inhibition. The curve pat-
terns of the graphical representation in Figure 5, obtained from
the experimental data for compound (S)-21, shows that this mole-
cule is a competitive inhibitor.41 Corresponding representations
were obtained for the other four derivatives. The (R)-enantiomer
of 21 had the lowest inhibitory effect of the new compounds with
a Ki about 20-fold higher than that of (S)-21. Clearly an (S)-config-
ured phenylalanine-derived motif is much more tolerated by HIV
PR. In this context, it is interesting to note that subtle configura-
tional changes such as a single stereocenter inversion can have a
huge impact on affinity. In an extreme case a Ki difference of
approximately seven orders of magnitude was reported.3,43,44
Interestingly, the new hydrazino-urea series exhibited an inhib-
itory tendency opposite to our previous series: while inhibitor can-
didates 7a–c showed increasing potency (of more than one order of
magnitude) with growing length of the peripheral chains, our cur-
rent molecule candidate (S)-21, being the smallest, is the best per-
former in the series. While 17 is only marginally less effective,
compounds 18 and 22 exhibit a quick drop in affinity. We are
tempted to speculate that the hydrazino-urea core has appreciable
complementarity with the active site of HIV PR. Since (S)-21 sports
a molecular weight of only 365, there is considerable room for
improvement in affinity and ADME properties while plainly
remaining within the Lipinski limit of 500 g/mol.17 On the other
hand, extending the peptide chain beyond the proline-carbonyl re-
sults in lower potency (22 vs 17). However, further expansion of
this peripheral chain (18 vs 22) recovers some affinity, maybe be-
cause of the increase in surface area available for hydrophobic
interactions.
4. Experimental
4.1. Materials and general methods
Reagents and solvents were purchased from Aldrich, Acros and
Alfa Aesar. THF was distilled from Na/benzophenone, and dichloro-
methane was dried using CaCl2 prior to distillation. Triethylamine
was distilled from CaH2. Compounds 9, (R)-14, (S)-14, and 19 were
prepared according to literature protocols.32,33 All reactions were
performed in anhydrous solvents under argon atmosphere in dried
glassware. Column chromatography was performed using Merck
silica gel Si 60 (40–63 l
m). 1H and 13C NMR spectra were recorded
on a Varian Unity 500 spectrometer (499.83 MHz, 126.7 MHz,
respectively), or a Bruker DPX 200 instrument (200.13 MHz,
50.13 MHz, respectively). Chemical shifts (d) are reported in ppm
(s = singlet, d = doublet, t = triplet, m = multiplet, br = broad) and
referenced from tetramethyl silane or from solvent references.45
NMR coupling constants (J) are reported in hertz. HRMS, MS, and
elemental analyses were carried out by the Service Central d’Ana-
lyse du CNRS, Solaize, France. UV spectra were recorded on a JASCO
V550 UV–vis spectrophotometer using 10 mm path length cells.
Optical rotations were measured on a JASCO P1010 polarimeter
with a 100 mm path length cell thermostated at 25 °C. Melting
point (mp) determinations were carried out on a Büchi melting
point B-540 apparatus and are uncorrected.
4.2. Synthetic protococls
4.2.1. (S)-(2-tert-Butylcarbamoyl-pyrrolidin-1-yl)-carbamic
acid tert-butyl ester (10)
Compound 9 (320 mg, 1.04 mmol) was dissolved in CH2Cl2
(10 mL) and EDC (280 mg, 1.46 mmol), then HOBt (1.56 mL of a
1 N solution in N-methylmorpholine, 1.56 mmol) were added.
After 5 min freshly distilled tBuNH2 (330
lL, 3.12 mmol) was
added, and the reaction was stirred overnight. NaHCO3 (satd aque-
ous solution, 10 mL) was added in order to quench the reaction and
the mixture was extracted with CH2Cl2 (3 ꢁ 10 mL). The combined
organic extracts were dried over Na2SO4 and the solvent was evap-
orated under vacuum. Purification by flash chromatography (33%
EtOAc/CH2Cl2) gave 10 as a white solid (219 mg, 74%). Mp
3. Conclusion
207 °C; ½a 2D5
ꢂ
ꢀ43.1 (c 1.02, CHCl3); 1H NMR (500 MHz, CDCl3) d
This study broadens our exploration of the N?C@O motif as a
potential isostere that reproduces key stereoelectronic properties
of the transition states of peptide hydrolysis. In contrast to our
first-generation inhibitors, only one constitutional component is
present in all molecule candidates in a protic medium provided
7.87 (br s, 1H), 5.53 (br s, 1H), 3.40–3.35 (m, 1H), 3.14 (dd,
J = 10.1, 4.6 Hz, 1H), 2.65–2.59 (m, 1H), 2.27–2.19 (m, 1H), 1.90–
1.78 (m, 3H), 1.45 (s, 9H), 1.35 (s, 9H); 13C NMR (50 MHz, CDCl3)
d 172.1, 155.7, 80.1, 69.8, 56.2, 50.3, 28.8, 28.5, 28.2, 21.9; HRMS
(ESI) calcd for C14H28N3O3 [M+H]+ 286.2132, found 286.2138.

3676
M. Waibel et al. / Bioorg. Med. Chem. 17 (2009) 3671–3679
Table 1
Inhibition data of hydrazino-ureas and related inhibitors towards HIV PR
Compound
IC50
(lM)
Ki (l
M)a
MW (g/mol)
Lineweaver and Burk
Hanes
26.2
Dixon
34.8
- O
δ
δ+
N
N
HN
(S)-21
37.2
26.2
365
O
O
δ-
HN
δ+
N
N
(R)-21
543
461.0
461.0
453.9
365
O
CONHtBu
- O
δ
δ+
N
17
44.5
34.3
34.3
43.9
450
HN
N
O
CO-Val-NHtBu
- O
δ
δ+
N
22
341
292.0
292.0
286.2
549
HN
N
O
O
CO-Ile-Val-NHtBu
δ-
HN
δ+
N
18
169
148.6
148.6
137.8
662
388
N
O
COOMe
- O
δ
7ab
>1000
—
—
—
Ph
+
N
δ
AllocHN
CONHtBu
δ-
O
7bb
545
204
574
99
618
96
577
96
578
677
Ph
+
N
δ
Z-Val-HN
CO-Val-NHtBu
- O
δ
7cb
Ph
+
N
δ
Z-Val-HN
OH
O
Iva-Val-Val-HN
Ala-Sta-OH
Pepstatine
0.041
0.038
0.038
0.050
686
a
Ki values were obtained by use of three different methods.
b
Data are from Gautier et al.10

M. Waibel et al. / Bioorg. Med. Chem. 17 (2009) 3671–3679
3677
29.5, 29.4, 28.6, 28.4, 24.8, 23.1, 19.4, 17.9, 15.7, 11.2; HRMS
(ESI) calcd for C25H48N5O5 [M+H]+ 498.3658, found 498.3659.
4.2.3. (S)-1-Benzylamino-pyrrolidine-2-carboxylic acid tert-
butylamide (12)
Compound 10 (770 mg, 2.70 mmol) was dissolved in TFA
(60 mL) and stirred for 2 h at rt. TFA was evaporated and the resi-
due was dried under vacuum. The yellow oil was redissolved in
anhydrous MeOH (20 mL), then benzaldehyde (329 mg,
3.11 mmol) was added, and the reaction was stirred overnight.
Acetic acid (4 mL) and NaCNBH3 (850 mg, 13.50 mmol) were added
and stirring was continued for 2 h. Most part of the solvent was re-
moved under vacuum, then a pH value around 8 was adjusted by
adding NaHCO3 (satd aq solution, 10 mL) and NaOH (aq solution),
and the mixture was extracted with CH2Cl2 (3 ꢁ 10 mL). The com-
bined organic extracts were dried over Na2SO4 and the solvent was
evaporated under vacuum. After purification by flash chromatogra-
phy (5% MeOH/CH2Cl2), 12 was obtained as a yellow oil (620 mg,
Figure 5. Inhibition of HIV PR by (S)-21 at pH 4.7 and 37 °C: diagram according to
the method of Lineweaver and Burk41 with inhibitor concentrations (from bottom
to top) [I] = 0 lM, 5 lM, 10 lM, 22 lM, 52 lM, 74 lM, 104 lM.
83%). ½a 2D5
ꢂ
ꢀ45.5 (c 1.02, CHCl3); 1H NMR (500 MHz, CDCl3) d
7.59 (br s, 1H), 7.40–7.31 (m, 5H), 6.96 (br s, 1H), 4.04 (d,
J = 16.1 Hz, 1H), 3.97 (d, J = 16.1 Hz, 1H), 3.60–3.55 (m, 1H), 3.17
(dd, J = 9.6, 5.5 Hz, 1H), 2.47–2.43 (m, 1H), 2.21–2.17 (m, 1H),
1.87–1.77 (m, 3H), 1.25 (s, 9H); 13C NMR (50 MHz, CDCl3) d
173.2, 138.6, 128.7, 128.3, 127.1, 70.3, 56.5, 54.1, 49.9, 28.6, 28.4,
22.3; HRMS (ESI) calcd for C16H26N3O [M+H]+ 276.2077, found
276.2086.
4.2.2. (S,S,S)-{2-[1-(1-tert-Butylcarbamoyl-2-methyl-
propylcarbamoyl)-2-methyl-butylcarbamoyl]-pyrrolidin-1-yl}-
carbamic acid tert-butyl ester (11)
Cbz-Ile-Val-NH-tBu: Cbz-Ile-OH (2.23 g, 8.43 mmol) was dis-
solved in CH2Cl2 (60 mL), and EDC (2.27 g, 11.77 mmol), then HOBt
(12.65 mL of a 1 N solution in N-methylmorpholine, 12.65 mmol)
were added. After 5 min, a suspension of HValNHtBuꢃHCl (1.75 g,
8.43 mmol) and triethylamine (1.17 mL, 8.43 mmol) in CH2Cl2
(20 mL) was added and the reaction was stirred overnight. NaHCO3
(satd aqueous solution, 80 mL) was added and the mixture was ex-
tracted with CH2Cl2 (3 ꢁ 100 mL). The combined organic extracts
were dried over Na2SO4 and the solvent was evaporated under vac-
uum. After purification by flash chromatography (50% EtOAc/cyclo-
hexane), Cbz-Ile-Val-NH-tBu was obtained as a white solid (3.35 g,
95%). Mp 175 °C; 1H NMR (500 MHz, CDCl3) d 7.33–7.29 (m, 5H),
6.68 (br d, J = 8.5 Hz, 1H), 5.89 (br s, 1H), 5.54 (br d, J = 8.5 Hz,
1H), 5.08 (s, 2H), 4.14–4.03 (m, 2H), 2.12–2.03 (m, 1H), 1.85–1.77
(m, 1H), 1.52–1.44 (m, 1H), 1.32 (s, 9H), 1.15–1.07 (m, 1H), 0.91–
0.87 (m, 12H); 13C NMR (50 MHz, CDCl3) d 171.9, 170.3, 156.4,
136.5, 128.3, 127.9, 127.8, 66.6, 59.6, 59.5, 51.2, 37.6, 30.4, 28.5,
24.9, 19.1, 18.5, 15.3, 11.2; MS (ESI) m/z [M++H] 420 (100).
H-Ile-Val-NH-tBu: 10% Pd–C (840 mg) was added to a solution of
Cbz-Ile-Val-NH-tBu (3.32 g, 7.92 mmol) in MeOH (100 mL). The
solution was purged with hydrogen and stirred under a hydrogen
atmosphere (1 bar) for 1.5 h. The catalyst was filtered off and the
filtrate was evaporated to dryness. The residue was redissolved
in CH2Cl2 and dried over Na2SO4. The solvent was removed under
vacuum to give pure dipeptide H-Ile-Val-NH-tBu as a white solid
(2.26 g, 100%); Mp 118 °C; 1H NMR (500 MHz, CDCl3) d 7.87 (br
d, J = 8.6 Hz, 1H), 5.91 (br s, 1H), 4.00 (dd, J = 8.6, 7.4 Hz, 1H),
3.37 (d, J = 3.9 Hz, 1H), 2.10–1.81 (m, 3H), 1.33 (s, 9H), 1.20–1.09
(m, 1H), 0.95–0.90 (m, 12H); 13C NMR (50 MHz, CDCl3) d 174.3,
170.5, 59.8, 58.8, 51.1, 37.9, 30.8, 28.6, 23.7, 19.3, 18.4, 16.0,
11.7; MS (ESI) m/z [M++H] 286 (100).
4.2.4. (S,S,S)-1-Benzylamino-pyrrolidine-2-carboxylic acid [1-
(1-tert-butylcarbamoyl-2-methyl-propylcarbamoyl)-2-methyl-
butyl]-amide (13)
As described in the preparation of 12, compound 11 (2.30 g,
4.62 mmol) was dissolved in TFA (100 mL) and stirred for 2 h at
rt. After solvent evaporation, the dried residue was reacted with
benzaldehyde (564 mg, 5.32 mmol) in methanol (35 mL). Subse-
quent treatment with NaCNBH3 (1.46 g, 23.10 mmol) and acetic
acid (7 mL) gave 13 as a colorless oil (1.80 g, 80%) after purification
by flash chromatography (5% MeOH/CH2Cl2). ½a D25
ꢀ39.6 (c 1.07,
ꢂ
CHCl3); 1H NMR (500 MHz, CDCl3) d 7.63 (br s, 1H), 7.38–7.26
(m, 6H), 6.69 (br d, J = 8.6, 1H), 5.91 (br s, 1H), 4.21 (dd, J = 7.9,
6.9 Hz, 1H), 4.03–3.98 (m, 3H), 3.53–3.49 (m, 1H), 3.25 (dd,
J = 9.4, 5.6 Hz, 1H), 2.32–2.15 (m, 4H), 1.91–1.76 (m, 3H), 1.45–
1.28 (m, 1H), 1.32 (s, 9H), 1.01–0.86 (m, 13H); 13C NMR (50 MHz,
CDCl3) d 174.1, 171.3, 170.0, 138.1, 128.6, 128.2, 127.2, 69.5,
59.2, 57.5, 56.0, 54.6, 51.1, 36.7, 29.9, 28.5, 27.9, 24.7, 22.2, 19.1,
17.9, 15.5, 11.1; HRMS (ESI) calcd for C27H46N5O3 [M+H]+
488.3603, found 488.3608.
4.2.5. (S,S)-1-[1-Benzyl-3-(1-benzyl-2,2-dimethoxy-ethyl)-
ureido]-pyrrolidine-2-carboxylic acid tert-butylamide (15)
CDI (390 mg, 2.40 mmol) was suspended in anhydrous THF
(3 mL), and a solution of amine (S)-14 (425 mg, 2.18 mmol) in
THF (2 mL) was added dropwise at rt. The reaction was stirred
for 15 min, then the solvent was evaporated. The residue was
redissolved in CH2Cl2 (10 mL) and washed with water (2 ꢁ 5 mL).
The organic phase was dried over Na2SO4 and the solvent was
evaporated under vacuum. The residue was dissolved in anhydrous
As described in the synthesis of 10, usage of
9 (1.94 g,
8.43 mmol), EDC (2.26 g, 11.77 mmol), HOBt (12.65 mL of a 1 N
solution in N-methylmorpholine, 12.65 mmol) and H-Ile-Val-
NH-tBu (2.40 g, 8.43 mmol) in CH2Cl2 (80 mL) gave 11 as colorless
oil (3.27 g, 78%) after purification by flash chromatography (50%
acetonitril (4 mL), and iodomethane (527 lL, 8.45 mmol) was
added. The reaction was stirred overnight at rt, then the solvent
was evaporated and the yellow oil was dried under vacuum. The
residue was redissolved in anhydrous CH2Cl2 (22 mL), and hydra-
CH2Cl2/EtOAc). ½a D25
ꢂ
ꢀ38.4 (c 1.09, CHCl3); 1H NMR (500 MHz,
CDCl3) d 8.42 (br s, 1H), 6.68 (br d, J = 8.5 Hz, 1H), 6.04 (br s, 1H),
5.94 (br s, 1H), 4.20 (dd, J = 7.9, 6.9 Hz, 1H), 3.99 (dd, J = 8.5,
6.9 Hz, 1H), 3.50 (dd, J = 10.0, 4.8 Hz, 1H), 3.41–3.37 (m, 1H),
2.84–2.78 (m, 1H), 2.21–1.86 (m, 6H), 1.47–1.27 (m, 2H), 1.44 (s,
9H), 1.31 (s, 9H), 0.93–0.83 (m, 12H); 13C NMR (50 MHz, CDCl3) d
174.2, 171.3, 170.1, 155.7, 80.8, 68.3, 59.4, 58.5, 56.0, 51.1, 35.9,
zine 12 (600 mg, 2.18 mmol) and triethylamine (300 lL,
2.18 mmol) were added. After stirring overnight at rt, the reaction
was quenched by adding NaHCO3 (satd aq solution, 10 mL) and the
mixture was extracted with CH2Cl2 (3 ꢁ 10 mL). The combined or-
ganic extracts were dried over Na2SO4 and the solvent was re-
moved under vacuum. Compound 15 was obtained as a white

3678
M. Waibel et al. / Bioorg. Med. Chem. 17 (2009) 3671–3679
solid (714 mg, 66%) after purification by flash chromatography
451.2711. Anal. Calcd for C26H34N4O3: C, 69.31; H, 7.61; N, 12.43.
Found: C, 69.23; H, 7.64; N 12.41.
(33% EtOAc/CH2Cl2). Mp 112 °C; ½a D25
ꢂ
ꢀ66.7 (c 1.05, CHCl3); 1H
NMR (500 MHz, CDCl3) d 7.33–7.10 (m, 10H), 6.55 (br s, 1H), 5.35
(br s, 1H), 4.76 (d, J = 16.2 Hz, 1H), 4.40–4.29 (m, 1H), 4.26–4.10
(m, 2H), 3.46 (s, 1H), 3.44 (s, 1H), 3.24–3.12 (m, 1H), 3.06 (dd,
J = 14.1, 4.4 Hz, 1H), 2.75 (dd, J = 14.1, 10.1 Hz, 1H), 2.64–2.55 (m,
1H), 2.52–2.41 (m, 1H), 1.95–1.66 (m, 4H), 1.23 (s, 9H); 13C NMR
(50 MHz, CDCl3) d 170.9, 158.5, 139.6, 138.7, 129.1, 128.3, 128.0,
126.9, 126.7, 125.8, 105.8, 63.7, 55.8, 55.3, 52.7, 50.7, 49.2, 43.2,
34.6, 28.3, 28.1, 21.5; HRMS (ESI) calcd for C28H41N4O4 [M+H]+
497.3130, found 497.3133.
4.2.8. (S,S,S,S)-1-[1-Benzyl-3-(1-benzyl-2-oxo-ethyl)-ureido]-
pyrrolidine-2-carboxylic acid [1-(1-tert-butylcarbamoyl-2-
methyl-propylcarbamoyl)-2-methyl-butyl]-amide (18)
As described in the preparation of 17, acetal 16 (99 mg,
0.14 mmol) was treated with NaI (53 mg, 0.35 mmol) and TMSCl
(45 lL, 0.35 mmol) in anhydrous acetonitrile (3 mL). After purifica-
tion by flash chromatography (75% EtOAc/cyclohexane), 18 was
obtained as a colorless oil (72 mg, 78%). ½a D25
ꢀ53.7 (c 1.03, CHCl3);
ꢂ
1H NMR (500 MHz, CDCl3) d 9.62 (s, 1H), 7.32–7.13 (m, 11H), 6.50
(br s, 1H), 6.38 (br s, 1H), 5.76 (br s, 1H), 4.71 (d, J = 16.2 Hz, 1H),
4.52–4.45 (m, 1H), 4.33 (d, J = 16.2 Hz, 1H), 4.27 (dd, J = 8.8,
7.1 Hz, 1H), 3.93 (dd, J = 8.8, 7.1 Hz, 1H), 3.50–3.41 (m, 1H), 3.22
(dd, J = 14.0, 5.6 Hz, 1H), 2.96 (dd, J = 14.0, 9.0 Hz, 1H), 2.82–2.73
(m, 1H), 2.69–2.60 (m, 1H), 2.02–1.93 (m, 2H), 1.78–1.65 (m,
4H), 1.30 (s, 9H), 1.27–1.20 (m, 1H), 1.09–0.98 (m, 1H), 0.92–0.79
(m, 12 H); 13C NMR (50 MHz, CDCl3) d 201.7, 172.6, 170.8, 169.7,
158.6, 139.1, 136.9, 129.3, 128.6, 128.4, 127.2, 127.1, 126.7, 63.7,
60.4, 59.4, 57.4, 51.5, 50.0, 44.1, 37.6, 35.1, 30.7, 29.5, 28.5, 24.8,
22.4, 19.0, 18.3, 15.3, 11.2; 1H NMR (500 MHz, MeOH-d4), (1:1 mix-
ture of two diasteromers) d 7.35–7.18 (m, 16H), 7.11 (d, J = 7.4 Hz,
2H), 7.06 (d, J = 7.5 Hz, 2H), 4.58–4.46 (m, 6H), 4.25–4.19 (m, 2H),
4.09–3.98 (m, 4H), 3.69–3.65 (m, 2H), 3.11 (dd, J = 13.8, 4.3 Hz, 1H),
3.02 (dd, J = 13.7, 5.7 Hz, 1H), 2.86–2.64 (m, 6H), 2.17–1.96 (m, 4H),
1.88–1.69 (m, 8H), 1.56–1.45 (m, 2H), 1.36 (s, 9H), 1.34 (s, 9H),
1.20–1.07 (m, 2H), 0.99–0.90 (m, 24H); 13C NMR (127 MHz,
MeOH-d4) d 175.7, 175.6, 174.3, 174.2, 173.28, 173.25, 161.5,
142.0, 141.7, 141.1, 141.0, 131.5, 131.4, 130.3, 130.2, 130.2,
130.1, 128.6, 128.5, 128.09, 128.05, 127.87, 127.85, 101.5, 100.0,
65.0, 64.5, 61.6, 61.4, 60.5, 60.1, 58.2, 58.0, 53.1, 53.0, 52.5, 52.1,
45.3, 45.0, 38.8, 38.6, 38.0, 37.8, 32.7, 32.0, 30.8, 30.4, 29.6, 27.1,
27.0, 24.0, 23.5, 20.5, 19.9, 19.8, 16.8, 16.7, 12.3, 12.2; HRMS
(ESI) calcd for C37H54N6O5Na [M+Na]+ 685.4056, found 685.4059.
Anal. Calcd for C37H54N6O5: C, 67.04; H, 8.21; N, 12.68. Found: C,
66.94; H, 8.23; N 12.64.
4.2.6. (S,S,S,S)-1-[1-Benzyl-3-(1-benzyl-2,2-dimethoxy-ethyl)-
ureido]-pyrrolidine-2-carboxylic acid [1-(1-tert-butylcarbamoyl-
2-methyl-propylcarbamoyl)-2-methyl-butyl]-amide (16)
As in the preparation of 15, compound (S)-14 (642 mg,
3.29 mmol) was initially reacted with CDI (588 mg, 3.62 mmol)
in THF (7.5 mL). The resulting residue was treated with iodometh-
ane (795
tion with hydrazine 13 (1.60 g, 3.29 mmol) and triethylamine
(460 L, 3.29 mmol) in CH2Cl2 (35 mL) to give 16 as a white solid
lL, 12.77 mmol) in acetonitril (6 mL), followed by reac-
l
(1.68 g, 72%) after purification by flash chromatography (75%
EtOAc/cyclohexane). Mp 156 °C; ½a D25
ꢂ
ꢀ59.5 (c 1.06, CHCl3); 1H
NMR (500 MHz, CDCl3) d 7.25–7.11 (m, 10H), 6.81 (br s, 1H), 6.52
(br s, 1H), 6.27 (br s, 1H), 5.77 (br s, 1H), 4.68 (d, J = 16.2, 1H),
4.36–4.28 (m, 1H), 4.23–4.14 (m, 3H), 3.96 (dd, J = 8.8, 7.1 Hz,
1H), 3.44 (s, 1H), 3.42 (s, 1H), 3.38–3.32 (m, 1H), 3.06 (dd,
J = 14.0, 4.3 Hz, 1H), 2.74 (dd, J = 14.0, 10.3 Hz, 1H), 2.69–2.61 (m,
1H), 2.58–2.50 (m, 1H), 2.08–1.89 (m, 2H), 1.82–1.59 (m, 4H),
1.46–1.37 (m, 1H), 1.30 (s, 9H), 1.07–0.98 (m, 1H), 0.91–0.79 (m,
12H); 13C NMR (50 MHz, CDCl3) d 172.8, 171.0, 169.9, 158.5,
139.4, 138.9, 129.2, 128.3, 128.0, 126.8, 126.7, 125.7, 105.2, 63.4,
59.1, 57.7, 56.2, 55.0, 52.6, 51.1, 50.1, 44.4, 37.1, 34.7, 30.4, 29.3,
28.4, 24.8, 22.5, 19.0, 18.2, 15.3, 11.2; HRMS (ESI) calcd for
C39H61N6O6 [M+H]+ 709.4656, found 709.4662.
4.2.7. (S,S)-1-[1-Benzyl-3-(1-benzyl-2-oxo-ethyl)-ureido]-
pyrrolidine-2-carboxylic acid tert-butylamide (17)
To a solution of compound 15 (64 mg, 0.13 mmol) in anhydrous
acetonitrile (3 mL) were added NaI (49 mg, 0.33 mmol) and TMSCl
4.2.9. (R)-1-Benzyl-3-(1-benzyl-2,2-dimethoxy-ethyl)-1-
piperidin-1-yl-urea ((R)-20)
(42
l
L, 0.33 mmol). The reaction was stirred at rt for 1 h, and was
According to the preparation of 15, (R)-14 (300 mg, 1.54 mmol)
was first reacted with CDI (274 mg, 1.69 mmol) in THF (3.5 mL).
then quenched by adding NaHCO3 (satd aq solution, 5 mL). The mix-
ture was extracted with CH2Cl2 (3 ꢁ 10 mL) and the combined or-
ganic extracts were washed with Na2S2O3 (satd aq solution,
10 mL), and then with water (10 mL). The organic phase was dried
over Na2SO4 and the solvent was evaporated under vacuum. After
purification by flash chromatography (25% EtOAc/CH2Cl2), 17 was
The resulting residue was treated with iodomethane (372
5.98 mmol) in acetonitril (3 mL), followed by reaction with hydra-
zine 19 (293 mg, 1.54 mmol) and triethylamine (214 L,
lL,
l
1.54 mmol) in CH2Cl2 (15 mL) to give (R)-20 as a colorless oil
(461 mg, 73%) after purification by flash chromatography (33%
obtained as colorless oil (49 mg, 83%). ½a D25
ꢂ
ꢀ63.4 (c 1.05, CHCl3);
EtOAc/cyclohexane). ½a D25
ꢂ
+28.7 (c 1.09, CHCl3); HRMS, 1H and 13C
NMR spectra were identical to those reported for the (S)
1H NMR (500 MHz, CDCl3) d 9.67 (s, 1H), 7.33–7.20 (m, 11H), 5.17
(br s, 1H), 4.90 (d, J = 16.1, 1H), 4.49–4.43 (m, 1H), 4.28 (d, J = 16.1,
1H), 3.26–3.14 (m, 2H), 2.96 (dd, J = 14.0, 8.9 Hz, 1H), 2.83–2.76
(m, 1H), 2.71–2.63 (m, 1H), 1.97–1.87 (m, 1H), 1.85–1.64 (m, 3H),
1.23 (s, 9H); 13C NMR (50 MHz, CDCl3) d 201.0, 171.2, 158.8, 139.7,
136.7, 129.4, 128.7, 128.5, 127.4, 127.2, 126.8, 64.2, 60.6, 51.2,
49.5, 43.4, 35.2, 28.7, 28.5, 21.9; 1H NMR (500 MHz, MeOH-d4),
(1:1 mixture of two diasteromers) d 7.35–7.19 (m, 16H), 7.15 (d,
J = 7.5, 2H), 7.08 (d, J = 7.5, 2H), 4.58–4.46 (m, 6H), 4.08–4.02 (m,
2H), 3.50–3.46 (m, 2H), 3.10 (dd, J = 13.8, 4.4 Hz, 1H), 3.02 (dd,
J = 13.6, 5.7 Hz, 1H), 2.85–2.65 (m, 6H), 2.08–1.98 (m, 2H), 1.92–
1.69 (m, 6H), 1.33 (s, 9H), 1.31 (s, 9H); 13C NMR (127 MHz, MeOH-
d4) d 175.2, 174.7, 161.7, 141.9, 141.8, 141.2, 141.1, 131.43,
131.40, 130.31, 130.25, 130.17, 130.15, 128.63, 128.61, 128.53,
128.47, 128.07, 128.05, 101.4, 100.0, 65.5, 65.3, 58.3, 58.1, 52.93,
52.90, 52.2, 51.7, 44.9, 44.8, 38.1, 37.9, 30.6, 30.2, 29.8, 29.7, 23.71,
23.3; HRMS (ESI) calcd for C26H35N4O3 [M+H]+ 451.2711, found
enantiomer.32
4.2.10. (R)-1-Benzyl-3-(1-benzyl-2-oxo-ethyl)-1-piperidin-1-yl-
urea ((R)-21)
As described in the preparation of 17, (R)-20 (193 mg,
0.47 mmol) was treated with NaI (178 mg, 1.18 mmol) and TMSCl
(151 lL, 1.18 mmol) in anhydrous acetonitrile (10 mL). After puri-
fication by flash chromatography (50% EtOAc/cyclohexane), (R)-21
was obtained as a colorless oil (160 mg, 93%). ½a D25
ꢂ
+ 30.6 (c 1.09,
CHCl3); Anal. Calcd for C22H27N3O2: C, 72.30; H, 7.45; N, 11.50.
Found: C, 72.21; H, 7.47; N 11.48; HRMS, 1H and 13C NMR spectra
were identical to those reported for the (S) enantiomer.32
4.3. Enzyme assays
HIV PR was purchased from Bachem, and HIV PR substrate 1
(Arg-Glu(EDANS)SerGlnAsnTyr-ProIleValGlnLys-(DABCYL)Arg) was

M. Waibel et al. / Bioorg. Med. Chem. 17 (2009) 3671–3679
10. Gautier, A.; Pitrat, D.; Hasserodt, J. Bioorg. Med. Chem. 2006, 14, 3835.
3679
obtained from Sigma–Aldrich. Enzymatic activity and inhibition
were determined by a fluorogenic assay using a microplate spectro-
fluorimeter (Spectramax Gemini XS, Molecular Devices) and black
384-well plates (NUNCLONE, Nunc Inc.). Hydrolysis of the fluoro-
genic substrate was investigated at pH 4.7, 37 °C in 0.1 M acetate,
1 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 1 mg/mL bovine
serum albumin. The entire time course was recorded by monitoring
the fluorescence at 490 nm with an excitation wavelength set to
340 nm. Each initial rate was the average value of five indepen-
dently performed reactions.
KM determination was performed using a range of fluorogenic
substrate concentrations from 0.5 to 10 lM and an HIV PR concen-
tration of 4 nM. Final solutions contained 10% DMSO.
For determination of IC50 and Ki values, inhibitors were preincu-
bated with HIV PR for 30 min. IC50 determinations were performed
using 10% DMSO solutions containing a final of 5 nM HIV PR and
11. DeLucca, G. V.; EricksonViitanen, S.; Lam, P. Y. S. Drug Discov. Today 1997, 2, 6.
12. Thaisrivongs, S.; Strohbach, J. W. Biopolymers 1999, 51, 51.
13. Hagen, S.; Prasad, J. V. N. V.; Tait, B. D. Adv. Med. Chem. 2000, 5, 159.
14. Mader, M. M.; Bartlett, P. A. Chem. Rev. 1997, 97, 1281.
15. Tyndall, J. D. A.; Nall, T.; Fairlie, D. P. Chem. Rev. 2005, 105, 973.
16. Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D.
J. Med. Chem. 2002, 45, 2615.
17. Bleicher, K. H.; Bohm, H. J.; Müller, K.; Alanine, A. I. Nat. Rev. Drug Disc. 2003, 2,
369.
18. Dunitz, J. D. Science 1994, 264, 670.
19. Okimoto, N.; Tsukui, T.; Hata, M.; Hoshino, T.; Tsuda, M. J. Am. Chem. Soc. 1999,
121, 7349.
20. Piana, S.; Bucher, D.; Carloni, P.; Rothlisberger, U. J. Phys. Chem. B 2004, 108,
11139.
21. McCrindle, R.; McAlees, A. J. J. Chem. Soc., Chem. Commun. 1983, 61.
22. Becker, M. H.; Chua, P.; Downham, R.; Douglas, C. J.; Garg, N. K.; Hiebert, S.;
Jaroch, S.; Matsuoka, R. T.; Middleton, J. A.; Ng, F. W.; Overman, L. E. J. Am.
Chem. Soc. 2007, 129, 11987.
23. Carroll, J. D.; Jones, P. R.; Ball, R. G. J. Org. Chem. 1991, 56, 4208.
24. Kermack, W.; Robinson, R. J. Chem. Soc. 1922, 121, 427.
25. Leonard, N. Rec. Chem. Prog. 1956, 17, 243.
4 lM fluorogenic substrate. Initial rates were determined for at
26. Rademacher, P. Chem. Soc. Rev. 1995, 24, 143.
least five inhibitor concentrations. Ki determinations were per-
formed using 10% DMSO solutions containing a final of 5 nM HIV
PR. Initial rates were determined for at least five inhibitor concen-
trations and four fluorogenic substrate concentrations (2, 3, 4, and
27. Leonard, N. J.; Morrow, D. F.; Rogers, M. T. J. Am. Chem. Soc. 1957, 79, 5476.
28. Leonard, N. J.; Oki, M. J. Am. Chem. Soc. 1955, 77, 6239.
29. Bell, M. R.; Archer, S. J. Am. Chem. Soc. 1960, 82, 151.
30. Kirby, A. J.; Komarov, I. V.; Bilenko, V. A.; Davies, J. E.; Rawson, J. M. Chem.
Commun. 2002, 2106.
5 lM).
31. Pilme, J.; Berthoumieux, H.; Robert, V.; Fleurat-Lessard, P. Chem. Eur. J. 2007, 13,
5388.
32. Waibel, M.; Hasserodt, J. J. Org. Chem. 2008, 73, 6119.
33. Vidal, J.; Damestoy, S.; Guy, L.; Hannachi, J. C.; Aubry, A.; Collet, A. Chem. Eur. J.
1997, 3, 1691.
34. Vidal, J.; Hannachi, J. C.; Hourdin, G.; Mulatier, J. C.; Collet, A. Tetrahedron Lett.
1998, 39, 8845.
35. Li, W. H.; Hanau, C. E.; d’Avignon, A.; Moeller, K. D. J. Org. Chem. 1995, 60, 8155.
36. Raju, B.; Mortell, K.; Anandan, S.; O’Dowd, H.; Gao, H. W.; Gomez, M.;
Hackbarth, C.; Wu, C.; Wang, W.; Yuan, Z. Y.; White, R.; Trias, J.; Patel, D. Bioorg.
Med. Chem. Lett. 2003, 13, 2413.
Acknowledgments
This work was supported by the Rhone-Alpes Region through
research cluster 5, and by the French Research Ministry and the
CNRS. Michael Waibel acknowledges a European Doctoral Fellow-
ship from the Research Ministry.
37. Grzyb, J. A.; Shen, M.; Yoshina-Ishii, C.; Chi, W.; Brown, R. S.; Batey, R. A.
Tetrahedron 2005, 61, 7153.
References and notes
38. Jung, M. E.; Andrus, W. A.; Ornstein, P. L. Tetrahedron Lett. 1977, 4175.
39. Matayoshi, E. D.; Wang, G. T.; Krafft, G. A.; Erickson, J. Science 1990, 247, 954.
40. Gershkovich, A.; Kholodovych, V. J. Biochem. Biophys. Methods 1996, 33, 135.
41. Cornish-Bowden, A. Fundamentals in Enzyme Kinetics; Portland Press Ltd:
London, 1995.
42. Burlingham, B. T.; Widlanski, T. S. J. Chem. Educ. 2003, 80, 214.
43. Schramm, V. L.; Baker, D. C. Biochemistry 1985, 24, 641.
44. Rich, D. H.; Sun, C. Q.; Prasad, J.; Pathiasseril, A.; Toth, M. V.; Marshall, G. R.;
Clare, M.; Mueller, R. A.; Houseman, K. J. Med. Chem. 1991, 34, 1222.
45. Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62, 7512.
1. Lienhard, G. E. Science 1973, 180, 149.
2. Rich, D. H. J. Med. Chem. 1985, 28, 263.
3. Wolfenden, R. Bioorg. Med. Chem. 1999, 7, 647.
4. Erickson, J. W.; Eissenstat, M. A. In Proteinases of Infectious Agents; Dunn, B. M.,
Ed.; Academic Press: San Diego, CA, 1999; p 1.
5. Brik, A.; Wong, C. H. Org. Biomol. Chem. 2003, 1, 5.
6. De Clercq, E. Nat. Rev. Drug Disc. 2007, 6, 1001.
7. Ghosh, A. K.; Chapsal, B. D.; Weber, I. T.; Mitsuya, H. Acc. Chem. Res. 2008, 41,
78.
8. Babine, R. E.; Bender, S. L. Chem. Rev. 1997, 97, 1359.
9. Dunn, B. Chem. Rev. 2002, 102, 4431.