Dimerization Inhibitors of HIV-1 Protease Based on a Bicyclic Guanidinium Subunit

Article abstract of DOI:10.1021/jm030871u

Original inhibitors of HIV-1 protease based on a chiral bicyclic guanidinium scaffold linked to short peptidic mimics of the terminal protease sequences and to a lipophilic group were designed. These inhibitors prevent dimerization of the native protease

Full text of DOI:10.1021/jm030871u

5196
J . Med. Chem. 2003, 46, 5196-5207
Dim er iza tion In h ibitor s of HIV-1 P r otea se Ba sed on a Bicyclic Gu a n id in iu m
Su bu n it
Perla Breccia,# Nicole Boggetto,§ Ruth Pe´rez-Ferna´ndez,# Michiel Van Gool,# Masayuki Takahashi,‡ Lo¨ıc Rene´,|,†
Pilar Prados,# Bernard Badet,| Miche`le Reboud-Ravaux,§ and J avier de Mendoza*,#
Departamento de Qu´ımica Orga´nica, Universidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain,
Institut J acques Monod, CNRS-Universite´ Paris 6 & 7, 2, Place J ussieu-Tour 43, 75251 Paris Cedex 05, France,
FRE2230 CNRS & Universite´ de Nantes, F-44322 Nantes Cedex 3, France, and Institut de Chimie des Substances
Naturelles-UPRS 2301 CNRS, 91198 Gif-sur-Yvette Cedex, France
Received April 11, 2003
Original inhibitors of HIV-1 protease based on a chiral bicyclic guanidinium scaffold linked to
short peptidic mimics of the terminal protease sequences and to a lipophilic group were designed.
These inhibitors prevent dimerization of the native protease by an interfacial structure at the
highly conserved antiparallel â-strand involving both the N and C termini that substantially
account for dimerization. The preorganized guanidinium spacer introduces additional electro-
static hydrogen-bonding interactions with the C-terminal Phe-99 carboxylate. Lipophilic
residues linked to side chains and the guanidinium scaffold are essential for dimerization
inhibition as ascertained by Zhang kinetics (4, Kid ) 290 nM; 6 or 6′, Kid ) 150 nM; 8, Kid
)
400 nM) combined with a circular dichroism study on the enzyme thermal stability. Remarkably,
less hydrophobic compounds result in mixed dimerization (1a and 3) or active site inhibitors
(5). Removal of the guanidinium hydrophobic groups leads to less active or inactive ligands.
In tr od u ction
To target the â-sheet, agents may be generated that
competitively block the assembly of the homodimer or
disrupt the dimer interface.7 Indeed, kinetic studies
demonstrated that peptides corresponding to native N
or C termini inhibit enzyme activity.7a Inhibitors based
on peptides different from the native sequence were also
analyzed, most exhibiting genuine dimerization inhi-
bition.7g,8 Interface peptides, however, may also be
partly active-site-directed, since terminal segments are
cleavage segments of the protease. Rather small changes
in structure or ionic strength can induce a switch in
binding mode. As a result, a lipophilic N-terminal
blocking group, such as palmitoyl, not only improves the
inhibitory potency of such peptides but also acts as an
interface directing group.9 Also, cross-linked interfacial
peptides that mimic the dimerization interface have
been described.10 Finally, some synthetic compounds
consisting of two tripeptide strands attached to more
rigid aromatic scaffolds (pyridine and naphthalene) were
shown to inhibit dimerization of HIV-1 PR at the
submicromolar range.11
An essential step in the maturation of the human
immunodeficiency virus (HIV) is the processing of the
gag and gag/pol viral proteins by HIV-1 protease (PR).
The crucial role of this enzyme in the production of
infective virus particles makes it a prime target for
therapeutic intervention in AIDS.1 In its active form,
HIV-1 PR is a 22 000 Da homodimeric aspartic protease
consisting of two 99-residue polypeptide chains that self-
assemble to form an approximately 45 × 23 × 25 Å
dimer. The active site is shaped at the interface of the
two subunits, each contributing one catalytic aspartate
residue.2 Preventing or disrupting the self-assembly of
the monomers would be a unique means of inhibiting
protease activity, since most active site inhibitors used
in AIDS therapy have led to resistance by rapid muta-
tion of the virus.3 The protease homodimer is mainly
stabilized by a four-stranded antiparallel â-sheet involv-
ing both the N and C termini of each monomer (H-Pro-
Gln-Ile-Thr and Cys-Thr-Leu-Asn-Phe-OH, respectively)
(Figure 1), which has been found to be highly conserved
in HIV-1 isolates and most HIV-2 isolates.4 More than
50% of the hydrogen bonds along the dimer interface
are provided by terminal residues 1-4 and 96-99 at
the antiparallel â-sheet.5 The contacts in the area
centered around N and C termini account for greater
than 75% of the free energy of dimerization.6 This
suggests that using peptides as competitive inhibitors
of â-sheet formation may efficiently inhibit dimer
formation and hence reduce protease activity.
The novelty of the dimerization inhibitors presented
in this paper is the use of a lipophilic bicyclic guani-
dinium subunit as the central tether between a chain
reproducing the C-terminal native strand, to interact
with both C and N termini of the monomer, and an
additional hydrophobic group targeting the dimer in-
terface. Formation of a salt bridge involving two well-
oriented zwitterionic hydrogen bonds makes bicyclic
guanidinium a useful subunit for the extraction of
carboxylates or phosphates and, thus, an ideal comple-
ment for the C-terminal carboxylate (Phe-99).12 By use
of mutant proteases comprising deletions of either one
of the terminal regions (residues 1-4 or 96-99) or both,
it was demonstrated that the inner C-terminal strands
are absolutely essential for dimer formation.5b We
* To whom correspondence should be addressed. Phone: +34 91 397
4710. Fax: +34 91 397 3966. E-mail: javier.demendoza@uam.es.
#
Universidad Auto´noma de Madrid.
§ CNRS-Universite´ Paris 6 & 7.
‡ CNRS & Universite´ de Nantes.
| Institut de Chimie des Substances Naturelles-UPRS 2301 CNRS.
† Deceased February 2000.
10.1021/jm030871u CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/23/2003

Guanidinium Inhibitors of HIV-1 Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5197
F igu r e 1. (a) HIV-1 PR dimer structure. (b) Antiparallel â-sheet involving N- and C-termini.
terminus derived sequence, was used as a control,
whereas compounds 4-7 incorporate the mercap-
toacetylglycine followed by diverse protected or depro-
tected C-terminus related sequences. Since the carbox-
ylate C-terminal group of Phe-99 can freely rotate, it is
unlikely that the configuration of the guanidinium
moiety influences its binding properties. To confirm this
prediction, an R,R-guanidinium scaffold was used in 6′
instead of the S,S-configuration employed for the rest
of inhibitors. Finally, the silyl substituent of 6 was
replaced by another bulky, nonaromatic, lipophilic
residue, such as the thiocholesteryl substituent in
compound 8. This allows for evaluation of the influence
of aromatic residues in the lipophilic tail that could
participate through stacking or edge-to-face contacts
with the hydrophobic interface. On the other hand,
ursolic acid, a polycyclic triterpene, acts as a mixed
(dimerization/active site) inhibitor.13
Molecular modeling was employed to evaluate the
suitability of the mercaptoacetylglycine spacer between
the guanidinium central core and the complementary
peptide chain. To build up the initial structures, atomic
coordinates for the monomer were taken from the solid-
state structure of HVP PR.2 A bicyclic guanidinium was
placed in front of the Phe-99 carboxylate (an optimized
guanidinium-acetate complex was used for a correct
docking), and suitable fragments were selected as
linking subunits. Initial minimization was performed
in vacuo, with a dielectric constant ꢀ ) 1 that is non-
distance-dependent. The resulting structure was soaked
in a box of water, and optimization was performed again
(Figure 3).
F igu r e 2. Dimerization inhibition scheme using a carboxylate
binding subunit as a scaffold.
hypothesized that these features included in the inhibi-
tors, preformed into a binding competent conformation,
should substantially contribute to overcoming the sta-
bilization of the protease dimer, favoring the monomer-
inhibitor complex (Figure 2).
Compounds 1-8 were designed, differing in linkers
and peptides attached to the guanidinium (Chart 1).
Thus, compounds 1a ,b-2a ,b contain an adipate chain
attached to pentapeptide Ala-Thr-Leu-Asn-Phe-OMe
through an ester function. Adipate contains about the
right number of atoms necessary to link the peptide to
the guanidinium without causing distortion to the
hydrogen-bonded scaffold. The native peptide chain was
slightly modified for stabilization purposes and also to
facilitate its synthesis. Thus, glutamine Gln-2 and
cysteine Cys-95 were both replaced by Ala. Finally,
methyl or benzyl esters were employed instead of free
acids in the peptide chains to prevent inhibitor self-
dimerization. An S,S-configuration for the guanidinium
was employed throughout.
Assuming an extended conformation for the hydro-
carbon chain of the adipate linker, replacement of
adipate for mercaptoacetylglycine should result in a
more preorganized spacer, since (i) two carbons of the
adipate skeleton are replaced by a conformationally
more rigid amide and (ii) the overall chain contains one
atom less, thus providing increasingly restricted con-
formational freedom. The two longer S-C bonds keep
the length of the chain almost unchanged. Therefore, a
second class of potential inhibitors (3-7) was developed,
all containing the mercaptoacetylglycine spacer and a
lipophilic silyl group. Compound 3, containing an N-
Finally, it is emphasized that lateral chains of Ser
and Tyr were benzylated in most of the inhibitors to
enhance the hydrophobicity of the compounds.
Resu lts a n d Discu ssion
Syn th esis. Silyl protected guanidinium ester or
amide derivatives bearing one peptide chain and an
adipate spacer (1a and 1b) were easily prepared from
alcohol 914 or amine 10, respectively, by successive
coupling to adipic acid and Ala-Thr-Leu-Asn-Phe methyl
ester (Scheme 1). Deprotection of the silyl ether afforded
the corresponding alcohol derivatives (2a ,b).
Compounds 3-7 were conveniently prepared in excel-
lent yields from mesylate 13 via acid 14 by alkylation

5198 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Ch a r t 1. HIV-1 PR Guanidinium-Based Inhibitors
Breccia et al.
Sch em e 1. Synthesis of Compounds 1a ,b and 2a ,ba
a
Reagents and conditions: (i) adipic acid, CDI, DMF; (ii) Ala-Thr-Leu-Asn-Phe-OMe, CDI, DMF; (iii) HF-Py 70%, THF; (iv) 30%
HCl-CH3CN.
HPLC. A similar strategy was followed to prepare
inhibitor 8 in four steps from mesylate 13 (Scheme 2).
All peptides were synthesized in solution phase, using
Boc-protected amino acids, and the compounds were
purified by preparative HPLC.
Kin etic An a lysis of En zym e In h ibition . In vitro
HIV-1 PR inhibition by compounds 1-8 was evaluated
kinetically at pH 4.7 and 30 °C by means of a fluori-
metric assay as reported in the literature.11 All mol-
ecules were tested at their solubility limit. The results
are summarized in Table 1. Compound 2b (at 28 µM),
endowed with an amide-linked adipate and a free OH,
was inactive. However, a weak inhibitory effect was
observed with the related structure 2a , where the amide
linker has been replaced by an ester. The superior
inhibitory effect observed for compounds whose guani-
dinium subunit was attached to the spacer through an
ester linkage over the same scaffolds attached through
an amide linkage is also apparent by comparing amide
1b (a weak inhibitor) with ester 1a (an efficient one).
F igu r e 3. Optimized structure of PR-6 complex in water.
The CH2-O-silyl group has been replaced by a methyl.
with sodium mercaptoacetate, followed by coupling to
the corresponding peptides by Castro’s reagent (BOP).
The resulting products were purified by preparative

Guanidinium Inhibitors of HIV-1 Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5199
Sch em e 2. Synthesis of Compounds 3-8a
in the inhibitory activity (i.e., 7-fold decrease in IC50 for
7 with respect to 6). This is likely due to self-association
of the inhibitor by intermolecular carboxylate-guani-
dinium ion-pairing, which decreases the effective inhibi-
tor concentration. This is clearly demonstrated by
comparing the NMR spectra of compounds 6 and 7.
While in compound 6 the guanidinium NH signals
appear at 7.41-7.64 ppm (typical for non-hydrogen-
bonded guanidiniums),12 the same signals are strongly
deshielded by ca. 2.5 ppm (9.95 and 10.08 ppm) in
compound 7, revealing a strong guanidinium-carboxy-
late interaction. Finally, it should be noted that control
molecules such as peptides Gly-Ala-Thr(Bn)-Leu-Asn-
Phe-OBn (see Supporting Information) and MeO2C(CH2)4-
CO-Ala-Thr-Leu-Asn-Phe-OMe (20) and guanidinium
salts lacking peptide chains (9, 14, or the synthetic
intermediate 21) did not lead to inhibition.
The mechanism of inhibition was characterized ki-
netically using the method developed by Zhang.7a Thus,
initial rates vi were determined at constant initial
substrate concentrations using different enzyme con-
centrations [E]0 for various inhibitor concentrations.
Plots of [E]0/xvi vs xvi, with xvi ) kexp[S]0, were
constructed. Typical parallel lines for dimerization
inhibition were found for molecules 4, 6, 6′, and 8
(Figure 4). Patterns found for compounds 1a and 3 were
consistent with a mixed inhibition mechanism in which
the inhibitors could bind not only to the interfacial
region but also to the active site. Convergent lines were
obtained for compound 5, demonstrating that this
molecule acts as a competitive inhibitor without affinity
for the monomer. The values of the inhibition constants
Kic and Kid were calculated using the equations
a
Reagents and conditions: (i) sodium mercaptoacetate, t-BuOK,
MeOH-THF; (ii) peptide, BOP, HOBt, NMM, DMF; (iii) H2, Pd-
C, MeOH, cat. AcOH; (iv) (a) GlyOMe, BOP, HOBt, NMM, DMF,
(b) 1 N NaOH, THF; (v) thiocholesterol, t-BuOK, MeOH-THF;
(vi) HF-Py 70%, THF; (vii) Ms2O, NMM, THF; (viii) Gly-Ala-
Thr(Bn)-Leu-Asn-Phe-OBn, BOP, HOBt, NMM, DMF.
Ta ble 1. Inhibition of HIV-1 Protease by Guanidinium
Hairpins
compound
IC50 (µM)a
Kid (µM)b
Kic (µM)c
1a
1b
2a
2b
3
4
5
1.9
Id
0.55
5.25
Ie
NIf
5
3.3
5
6.50
0.29
7.00
6.00
6 (S,S)
6′ (R,R)
7
8
1.6
2
11
3
0.15
0.15
[I]0
Kic ) a0
a - a0
0.40
0.40
and
a
b
Standard errors: e10%. Dimerization inhibition. c Competi-
tive inhibition. A 28% inhibition (I) was observed at 28 µM. e A
d
28% inhibition (I) at 14 µM. f No inhibition (NI) detected at the
[I]0
solubility limit of the compound tested.
Kid ) b0
b - b0
The presence of a lipophilic group attached to the
guanidinium strongly enhances HIV-1 PR inhibitor
activity. Thus, 1a and 3-8 were found to be quite
efficient inhibitors (IC50 ) 1.6-5 µM), whereas in
compounds lacking the TBDPS group the inhibitory
effect is abolished (compare 2b with 1b) or decreased
(compare 2a with 1a ). As anticipated, the configuration
of the guanidinium scaffold (S,S or R,R) does not
influence its binding properties toward HIV protease
(compare 6 and 6′). However, replacing the silyl group
of compound 6 with another lipophilic derivative such
as the cholesteryl tail of 8, which does not contain any
aromatic ring, led to a 2-fold decrease of the IC50 values.
It is likely that the phenyl groups of TBDPS could
contribute to the binding to the hydrophobic interface,
although this hypothesis requires further experiments
to be fully validated. Either inhibitors with adipate (1a ,
1b, or 2a ) or mercaptoacetate spacers (3-8) were active.
Thus, attachment of a hydrophobic moiety to the peptide
strongly enhances inhibitory power, as was previously
pointed out by Schramm.9
respectively, where a and a0 are the slopes and b and
b0 are the intercepts on the y axis in the presence (a, b)
or absence (a0, b0) of inhibitor.
Stereoisomers 6 and 6′ (Kid ) 0.15 µM) are thus
inhibitors acting exclusively by inhibiting dimerization
of HIV-1 PR. Replacement of the TBDPS group by a
cholesteryl moiety (8 vs 6) decreases the inhibitory
potency by 2.6-fold while maintaining the dissociative
mechanism. For ursolic acid, dimerization inhibition
was reported (Kid ) 3.4 µM) with a slight competitive
inhibitory component (Kic ≈ 130 µM).13 Our results with
8 confirm that triterpenes may fit into the hydrophobic
interface of PR monomers. However, guanidinium de-
rivative 8 not only is a more potent inhibitor than ursolic
acid but also has the advantage of acting as a pure
dimerization inhibitor. Interestingly, compound 4 in-
hibits HIV-1 PR through a dissociative mechanism with
a Kid value of 0.29 µM whereas the structurally related
5 acts as a poor active site inhibitor (Kic ) 16 µM). Thus,
removal of the hydrophobic benzyl protecting groups at
the serine and tyrosine residues of compound 4 switched
the inhibition mechanism and caused the molecule to
Replacement of the terminal methyl or benzyl ester
for a free carboxylic acid caused a noticeable decrease

5200 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Breccia et al.
F igu r e 5. Thermal denaturation of HIV-1 PR (3.5 µM) in the
absence (blue) and presence of compound 6 (red) and acetylpep-
satin (black) at pH 4.7 in 100 mM sodium acetate, 1 mM
EDTA, and 1 M NaCl containing 0.2% DMSO (v/v): temper-
ature range, 30-75 °C (2 °C/min); [6] ) 2 µM; [acetylpepstatin]
) 0.2 µM; λ ) 230 nm. Melting was followed by far-ultraviolet
circular dichroism at 230 nm.
change in the secondary structure of protein and sup-
porting our model. The enzyme was thermally denatur-
ated in 100 mM sodium acetate buffer, pH 7.0, and
monitored in the far-UV CD in the absence and in the
presence of active site inhibitor acetylpepstatin and
dimerization inhibitor 6 (Figure 5). The averages Tm
values of 59.5, 59.5, and 61.5 °C were obtained at a ramp
rate of 2 °C/min. This indicates that a significant
stabilization was obtained in the acetylpepstatin com-
plex, which had a 2 °C increase in Tm when compared
to native enzyme. Conversely, no stabilization was
observed by addition of compound 6, in agreement with
a different binding mode. In this case, the destabiliza-
tion of PR by dimer interface disruption6 is probably
compensated by the putative stabilization due to the
formation of the monomer-inhibitor complex, thus
leading to identical Tm values for native and treated
enzymes. The importance of PR stability of interactions
implicating the C-terminal strands has been empha-
sized.5b
F igu r e 4. Plots of [E]0/xvi vs xvi for hydrolysis of the
fluorogenic substrate by HIV-1 PR at pH 4.7 and 30 °C in the
absence (b) and presence of 2.5 µM (2), 1 µM (9) of compound
6 (A) and compound 6′ (B) and presence of 12 µM (2) and 4.5
µM (9) of compound 3 (C).
Con clu sion s a n d Ou tlook
become a weak active site inhibitor. The same effect may
be evoked to explain the mixed mechanism observed for
1a (the threonine residue is not protected). Neverthe-
less, the presence of a protecting group is not sufficient
to switch the mechanism from a mixed to a pure
dimerization inhibition when the peptide strand mimics
the N terminus (3) instead of the C terminus (com-
pounds 4, 6, 6′, 8). By use of small peptides, more
efficient dimerization inhibitors were obtained with
peptides mimicking the C rather than the N terminus.8
Cir cu la r Dich r oism . To further probe the mecha-
nism of inhibition, we have examined the structure and
thermal stability of PR alone or complexed with inhibi-
tors by measurements of the CD signal at far-UV. An
active site inhibitor should certainly stabilize the dimer
form and thus the thermal stability of PR, while a
dimerization inhibitor might not. The tested inhibitors
(active site inhibitor acetylpepstatin and dimerization
inhibitor 6) did not affect the CD signal, indicating no
Guanidinium derivatives are novel dimerization in-
hibitors of HIV-PR. Contrary to cross-linked peptides
bridged by a flexible tether,10 the compounds described
in the present report display a rigid and positively
charged scaffold linked simultaneously to a peptidic
strand and a hydrophobic bulky group (TBDPS, choles-
teryl) (1a ,b, 3-8). The rigid spacer likely contributes
to reduction of the entropic cost of the complexation by
allowing the preorganization of both peptidic and hy-
drophilic arms, while the positively charged guani-
dinium likely provides an additional and strong inter-
action with the carboxylate of Phe-99. It was previously
reported that scaffolds such as 2,6-pyridine, 2,7-
naphthalene, and dibenzofuryl linked to two peptide
strands led to dimerization inhibitors. For example, Kid
) 0.56 µM for the 2,7-naphthalenediol spacer11a and Kid
) 5.40 µM for the dibenzofuryl spacer.11b The combina-
tion of both the steric and electronic effects within the
bicyclic guanidinium scaffold explains the increase of

Guanidinium Inhibitors of HIV-1 Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5201
lens (J asco Europe, Italy). The temperature was controlled by
a Peltier effect device. The spectra were averaged over four
scans (bandwidth of 2 nm, response time of 2 s, and scan rate
of 50 nm/min). The thermal denaturation of protein was
studied by monitoring the change in CD signal at 230 nm
(bandwidth of 10 nm, response time of 8 s) upon elevation of
temperature (rate: 2 °C/min) from 30 °C.
Molecu la r Mod elin g. InsightII/Discover program modules
were used, as implemented on a Silicon Graphics Indigo
workstation, using the cvff (consistence valence force field)
force field.17 Initial minimizations were performed in vacuo,
with a dielectric constant ꢀ ) 1 that is non-distance-dependent.
Then, the most promising complexes were soaked in a box of
water and structures were optimized again.
Syn th esis. All commercially available reagents were used
without any further purification unless specified. The solvents
were dried and distilled as described in the literature.18 All
reactions were performed under Ar atmosphere unless speci-
fied. The peptides were synthesized in solution phase using a
Boc-based strategy (experimental data available in Supporting
Information). Chiral bicyclic guanidinium derivatives were
synthesized in S,S configuration with exception of compound
6′ (R,R configuration; same synthetic procedure as reported
for 6). The coupling reaction with the peptides were followed
by analytical HPLC (column C18 Scharlau; flow ) 1 mL/min;
UV detector, Waters dual band with λ ) 230 and 254 nm).
For preparative HPLC analysis a radial column PrepLC
(Waters) (flow ) 20 mL/min) was used, while semipreparative
HPLC was performed using an LC 18 column (Phenomenex)
(flow ) 6 mL/min). All solvents used in HPLC contained 0.05%
TFA.
the inhibitory potency observed for the new inhibitors
presented here (Kid ) 0.15 µM).15
Other hydrophobic subunits for more efficient contacts
with the PR monomer interface are currently under
investigation. Also, it must be emphasized that binding
to the monomer and therefore inhibition could be
substantially optimized by modification of the peptide
sequences employed. Both rational and combinatorial
approaches can be envisioned for this purpose. Finally,
use of peptoids or nonnatural amino acids would provide
a new generation of inhibitors resistant to in vivo
hydrolysis by proteases. We are currently working on
these modifications.
Exp er im en ta l Section
Abbr evia tion s. γ-Abu, γ-amino butyric acid; Adip, adipic;
Ar, aromatic; Bn, benzyl; Boc, tert-butyloxycarbonyl; BOP, 1-H-
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexaflu-
orophosphate; Chol, cholesteryl; CDI, N,N′-carbonyldiimida-
zole; DABCYL, 4-(4′-dimethylaminophenylazo)benzoyl; DCC,
1,3-dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EDANS, 5-[(2-
aminoethyl)amino]naphthalene-1-sulfonic acid; EDTA, ethyl-
enedinitrilotetraacetic acid disodium salt; Guan, guanidinium;
HOBt, 1-hydroxybenzotriazole; NMM, N-methylmorpholine;
MES, 2-(N-morpholine)ethanesulfonic acid; Ms, mesylate; Ph,
phenyl; TBDPS, tert-butyldiphenylsilyl; TFA, trifluoroacetic
acid; τR, retention time in HPLC analysis.
E n zym e a n d In h ib it ion Assa ys. HIV-1 PR was kindly
supplied by H. J . Schramm, Max-Planck Institut fu¨r Biochemie
(Martinsried, Germany). It was produced by bacterial expres-
sion in E. coli using the plasmid pET9c-PR as decribed by
Schramm8 and Billich.16 The stock solution protease (34 µM)
was stored at -80 °C in 5 mM MES, pH 6.0, 1 mM EDTA, 1
mM DTT, 0.5 M NaCl, 5% v/v glycerol. The fluorogenic
substrate DABCYL-γ-abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-
EDANS was purchased from Bachem (Voisins-le-Bretonneux,
France). Other reagents and solvents were purchased from
commercial sources. The fluorescence and absorbance mea-
surements were performed using a spectrofluorometer Perkin-
Elmer LS 50B and a Uvikon 941 spectrophotometer, respec-
tively.
Protease activity was routinely determined fluorometrically
using DABCYL-γ-abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS
in 100 mM sodium acetate, 1 mM EDTA, and 1 M NaCl at pH
4.7 and 30 °C according to the method described.11 Excitation
and emission wavelengths were 340 and 490 nm, respectively.
The substrate and the compounds were previously dissolved
in DMSO, with the final concentration kept constant at 3%
(v/v). In a typical inhibition experiment, the enzyme and
substrate concentrations were 7.5 nM and 5.2 µM, respectively.
The IC50 values (inhibitor concentration leading to 50% inhibi-
tion) were obtained from a plot of the percent inhibition vs
inhibitor concentration (0.5-28 µM) by fitting to the equation
Melting points were measured with a Gallenkamp ap-
paratus. The optical rotations were determined on a Perkin-
Elmer 241 MC polarimeter using a cell (1 dm) at 20 °C (NaD,
589 nm). 1H NMR spectra were recorded on Bruker AC-300
(300 MHz), AMX-300 (300 MHz), and DRX-500 (500 MHz)
spectrometers, and 13C NMR spectra were performed with the
same apparatus using magnetic fields at 75 and 125 MHz. In
NMR assignments, the H and C of the bicyclic guanidinium
derivatives are named as in the literature.19 Mass spectra were
recorded on a VG AutoSpec spectrometer using a FAB tech-
nique and on a HP1100MSD using an API-ES technique.
Elemental analyses were performed on a Perkin-Elmer 2400
CHN and 2400 CHNS apparatus.
Syn th esis of (2S,8S)-2-(ter t-Bu tyld ip h en ylsila n yloxy-
m et h yl)-8-(a m in om et h yl)-3,4,6,7,8,9,-h exa h yd r o-2H -p y-
r im ido[1,2-a ]pyr im idin -1-iu m Hexaflu or oph osph ate (10).
The mesylate derivative 13 (PF6-) (301 mg, 0.46 mmol) was
dissolved in a mixture of MeOH (10 mL) and 30% aqueous
NH3 (4 mL). The resulting solution was stirred for 30 min at
room temperature. Then the solvent was removed and the
crude was dissolved in CH2Cl2 and washed with 0.1 N NH4-
PF6. After filtration over cotton and evaporation of the organic
phase, the product was precipitated with CH3CN, resulting
in 10 (PF6-) (308 mg, 92%) as a white solid. Mp 81 °C. [R]25
D
1
+31 (c 0.5, CHCl3). H NMR (300 MHz, acetone-d6): δ 7.73-
7.71 (m, 4H, PhSi), 7.51-7.48 (m, 6H, PhSi), 7.18 (s, 1H, NH),
7.13 (s, 1H, NH), 4.13-4.07 (m, 2H, CH2OSi), 3.84 (m, 2H,
CHR), 3.69-3.59 (m, 4H, CH2γ), 2.61-2.52 (m, 2H, CH2NH2),
2.37-2.15 (m, 4H, CH2â), 1.09 (s, 9H, (CH3)3C). 13C NMR (75
MHz, acetone-d6): δ 151.3 (C-guan), 135.9, 133.3, 130.5, 128.4
(ArC, ArCH), 66.2 (CH2OSi), 51.7 (CH2NH2), 50.6, 47.6 (CHR),
45.5, 44.6 (CH2γ), 26.7 [(CH3)3C], 23.3, 22.6 (CH2â), 19.1
[(CH3)3C]. FAB/LSIMS m/z: 437.3 [(M - PF6-)+, 100%]. HRMS
[I]0
percent inhibition ) 100 ×
[I]0 + IC50
The mechanism of inhibition and the corresponding kinetic
constants Kic (competitive inhibition) or/and Kid (dimerization
inhibition) were determined using Zhang’s method.7a At least
seven different concentrations of enzyme ranging from 1.88
to 28 nM were tested for a given inhibitor concentration ([S]0
) 5.2 µM). Inhibitor concentrations were 3.15 and 6.3 µM
(compound 1a ), 4.5 and 12 µM (compound 3), 1.5 and 4.5 µM
(compound 4), 2.5, 5, and 7.5 µM (compound 5), 1 and 2.5 µM
(compounds 6 and 6′), and 1.5 and 4.5 µM (compound 8).
+
for [C25H37N4OSi] 437.2736; found 437.2734.
Gen er a l P r oced u r e for Com p ou n d s 11 a n d 12. A
mixture of adipic acid and CDI in dry DMF was stirred for 30
min. Then a solution of compound 9 or 10 in dry DMF was
added and the reaction mixture was stirred at room temper-
ature overnight. After removal of the solvent, the crude was
dissolved in CH2Cl2 and washed with a solution of 1 N HCl,
water, and brine. The organic solution was dried over Na2-
SO4, filtered, and concentrated to dryness. Purification was
performed by precipitation.
Cir cu la r Dich r oism . CD was measured in a J -710 spec-
trometer (J asco, J apan) using a 1 cm path length minicuvette
(Hellma, Germany) on which the light was focused by a quartz

5202 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Breccia et al.
Syn th esis of 2-(ter t-Bu tyld ip h en ylsila n yloxym eth yl)-
8-(5-ca r boxyp en ta n oyloxym eth yl)-3,4,6,7,8,9-h exa h yd r o-
2H-p yr im id o[1,2-a ]p yr im id in -1-iu m Ch lor id e (11). Adipic
acid (198 mg, 1.35 mmol), CDI (153 mg, 0.95 mmol), DMF (10
mL), 9 (Cl-) (212 mg, 0.45 mmol), DMF (1 mL). Precipitation
with a mixture of diethyl ether-CH2Cl2 (10:1) afforded 11 (Cl-)
21.8 (CH3-Leu), 20.4 (CH3-Ala), 20.0 [(CH3)3C], 17.6 (CH3-
Thr). FAB/LSIMS m/z: 1126.7 [(M - Cl-)+, 100%]. HRMS for
[C58H84N9O12Si+] 1126.6009; found 1126.6034.
Syn th esis of Com p ou n d 1b. A mixture was prepared that
contained compound 12 (Cl-) (65 mg, 0.108 mmol), CDI (18
mg, 0.111 mmol), and THF (1 mL). Then peptide (75 mg, 0.108
mmol), NMM (15 µL, 0.108 mmol), and DMF (1 mL) were
added. Reaction time was 48 h at room temperature. Precipi-
tation by dissolving in MeOH and adding some drops of water
afforded 1b (Cl-) (94 mg, 74%) as a white solid. HPLC
(gradient 10-100% CH3CN-H2O in 20 min; τR ) 18.5 min;
(206 mg, 76%) as a white solid. Mp 80 °C. [R]25 +43 (c 0.5,
D
1
CHCl3). H NMR (300 MHz, CDCl3): δ 8.69 (s, 1H, NH), 8.60
(s, 1H, NH), 7.67-7.63 (m, 4H, PhSi), 7.49-7.36 (m, 6H, PhSi),
4.21 (dd, J ) 11.0, 5.0 Hz, 1H, CH2COOR), 4.14 (dd, J ) 11.0,
8.0 Hz, 1H, CH2COOR), 3.82-3.56 (m, 4H, CH2OSi, CHR),
3.40-3.28 (m, 4H, CH2γ), 2.53-2.43 (m, 2H, CH2COOH),
2.43-2.35 (m, 2H, OCOCH2), 2.29-1.78 (m, 4H, CH2â), 1.76-
1.57 (m, 4H, (CH2)2), 1.09 (s, 9H, (CH3)3C). 13C NMR (75 MHz,
CDCl3): δ 176.5, 172.8 (CO), 151.0 (C-guan) 135.4, 132.5,
129.8, 127.7 (ArC, ArCH), 65.1 (CH2O), 50.0, 47.0 (CHR), 44.8,
44.6 (CH2γ), 33.9, 33.2 (CH2CO), 26.7 [(CH3)3C], 23.9 (CH2),
22.6, 22.4 (CH2â), 19.0 [(CH3)C]. FAB/LSIMS m/z: 566.4 [(M
- Cl-)+, 100%]. Anal. Calcd for C31H44N3O5SiCl: C, 61.8; H,
7.4; N, 7.0. Found: C, 61.0; H, 7.1; N, 7.3.
purity 97%). Mp 186 °C. [R]25 +18 (c 0.3, CHCl3). 1H NMR
D
(300 MHz, COSY, MeOH-d4): δ 7.66-7.52 (m, 4H, PhSi),
7.47-7.40 (m, 6H, PhSi), 7.27-7.07 (m, 5H, ArH-Phe), 4.71-
4.45 (m, 1H, CHR-Asn), 4.35-4.08 (m, 4H, CHR-Phe, Leu,
Thr, Ala), 3.98-3.85 (m, 2H, CH2OSi), 3.68-3.41 (m, 2H,
CHR-guan), 3.59 (s, 3H, OCH3), 3.40-3.35 (m, 4H, CH2γ-
guan), 3.11-2.90 (m, 2H, CH2-Phe), 2.65-2.48 (m, 2H, CH2-
Asn), 2.41-2.28 (m, 2H, CH2CO-adip), 2.20-2.10 (m, 2H,
CH2CO-adip), 2.01-1.70 (m, 4H, CH2â-guan), 1.63-1.46 (m,
7H, (CH2)2-adip, CH2â-Leu, CHγ-Leu), 1.37 (d, 3H, J ) 7.2
Hz, CH3-Ala), 1.19 (d, J ) 6.2 Hz, 3H, CH3-Thr), 0.98 (s,
9H, (CH3)3C), 0.81 (d, J ) 6.0 Hz, 3H, CH3-Leu), 0.78 (d, J )
6.0 Hz, 3H, CH3-Leu). 13C NMR (75 MHz, MeOH-d4): δ 181.8,
176.7, 175.7, 174.8, 173.0, 171.8, 171.5 (CO), 152.3 (C-guan),
135.1, 134.8, 133.9, 131.5, 130.8, 130.2, 129.8, 129.2, 127.7
(ArC, ArCH), 67.6 (CHO-Thr), 65.0, 64.9 (CH2O), 58.1 (CHR-
Thr), 54.9 (CHR-Phe), 52.5, 52.1, 51.8 (CHR-Asn, Leu, Ala),
51.7 (OCH3), 49.9, 47.6 (CHR-guan), 46.9, 46.6 (CH2γ-guan),
46.2 (CH2NH), 43.9 (CH2-Leu), 38.1 (CH2-Asn), 36.6 (CH2-
Phe), 34.5 (CH2-adip), 34.4 (CH2-adip), 26.2 [(CH3)3C)], 25.5
(CH2-adip), 24.8 (CH-Leu), 24.2, 23.9 (CH2â-guan), 23.7
(CH3-Leu), 20.0 [CH3-Ala, (CH3)3C)], 17.3 (CH3-Thr). FAB/
LSIMS m/z: 1125.7 [(M - Cl-)+, 100%].
Syn th esis of 2-(ter t-Bu tyld ip h en ylsila n yloxym eth yl)-
8-[(5-car boxypen tan oylam in o)m eth yl]-3,4,6,7,8,9-h exah y-
d r o-2H-p yr im id o[1,2-a ]p yr im id in -1-iu m Ch lor id e (12). A
mixture was prepared that contained adipic acid (69 mg, 0.472
mmol), CDI (100 mg, 0.617 mmol), DMF (1 mL), and 10 (Cl-)20
(112 mg, 0.237 mmol). Precipitation with diethyl ether afforded
12 (Cl-) (101 mg, 71%) as a white solid. Mp 88 °C. [R]25 +42
D
(c 0.5, CHCl3). 1H NMR (300 MHz, CDCl3): δ 9.21 (s, 1H, NH-
guan), 8.64 (s, 1H, NH-guan), 7.78 (m, 1H, CONH), 7.68-
7.61 (m, 4H, PhSi), 7.43-7.36 (m, 6H, PhSi), 3.75-3.56 (m,
6H, CH2OSi, CH2NH, CHR), 3.23-3.06 (m, 4H, CH2γ), 2.25
(m, 4H, CH2CO-adip), 2.00-1.94 (m, 4H, CH2â-guan), 1.64
(m, 4H, (CH2)2-adip), 1.09 (s, 9H, (CH3)3C). 13C NMR (75 MHz,
CDCl3): δ 181.1, 173.9 (CO), 151.4 (C-guan), 135.7, 135.5,
132.8, 129.8, 127.8, 127.7 (ArC, ArCH), 65.2 (CH2O-guan),
49.7, 48.7 (CHR), 45.9, 44.3 (CH2γ), 43.2 (CH2CO-adip), 37.4,
36.9 [(CH2)2-adip], 26.7 [(CH3)3C], 25.7, 24.7 (CH2â-guan),
19.7 [(CH3)3C)]. FAB/LSIMS m/z: 565.3 [(M - Cl-)+, 100%)].
Syn th esis of Com p ou n d 2a . To compound 1a (Cl-) (50
mg, 0.054 mmol) in dry THF (2 mL) was added dropwise 70%
HF-Py (0.121 mL) at 0 °C, and the reaction mixture was
stirred at room temperature for 4 h. After neutralization with
Na2CO3, the solvent was removed and the solid was dissolved
in water and washed with diethyl ether and CH2Cl2. The
aqueous phase was concentrated and the product was ex-
tracted from the salt with CH3CN, affording 2a (Cl-) (28 mg,
70%) as a white solid. HPLC (gradient 10-100% CH3CN-H2O
Gen er a l P r oced u r e for Com p ou n d s 1a a n d 1b. A solu-
tion of guanidinium compound and CDI in dry DMF was
stirred at room temperature for 30 min. Then, a solution of
peptide Ala-Thr-Leu-Asn-Phe-OMe (TFA salt) and NMM in
dry DMF was added and the reaction mixture was stirred
overnight. After removal of the solvent, the crude was washed
with CH2Cl2 and precipitated.
in 20 min; τR ) 12.3 min; purity 96%). Mp 168 °C. [R]25 +23
D
(c 0.2, MeOH). 1H NMR (300 MHz, COSY, MeOH-d4): δ 7.45-
7.31 (m, 5H, ArH-Phe), 4.86-4.72 (m, 2H, CHR-Asn, CHR-
Phe), 4.54-4.34 (m, 5H, CHR-Leu, CH2COOR, CHR-Thr,
Ala), 4.18-4.08 (m, 1H, CH2OH-guan), 3.91-3.60 (m, 2H,
CH2OH, CHR-guan), 3.78 (s, 3H, OCH3), 3.60-3.34 (m, 5H,
CH2O, CHR-guan, CH2γ-guan), 3.31-3.11 (m, 2H, CH2-
Phe), 2.89-2.68 (m, 2H, CH2-Asn), 2.61-2.34 (m, 4H, CH2-
CO-adip), 2.28-2.06 (m, 2H, CH2â-guan), 2.04-1.74 (m, 9H,
CH2â-guan, (CH2)2-adip, CHγ-Leu), 1.53-1.49 (m, 2H,
CH2â-Leu), 1.52 (d, J ) 7.0 Hz, 3H, CH3-Ala), 1.32 (d, J )
6.0 Hz, 3H, CH3-Thr), 1.07 (d, J ) 7.0 Hz, 3H, CH3-Leu),
1.04 (d, J ) 6.5 Hz, 3H, CH3-Leu). 13C NMR (75 MHz, MeOH-
d4): δ 176.5, 176.1, 175.1, 174.8, 174.7, 173.4, 173.0, 172.9
(CO), 152.7 (C-guan), 138.2 (ArC-Phe), 130.7, 129.9, 128.2
(ArCH-Phe), 68.5, 67.2, 65.4 (CHO-Thr, CH2O-guan), 60.3,
53.9, 53.0, 52.0, 51.8 (CHR-Ala, Thr, Leu, Asn, Phe), 51.4
(CH3O), 46.8, 46.4 (CHR-guan), 41.5 (CH2γ-guan), 38.7, 38.0,
36.3 (CH2-Leu, Asn, Phe), 34,7 (CH2CO-adip), 26.3, 26.1
(CH2-adip), 25.7 (CH-Leu), 24.0, 23.9 (CH2â-guan), 22.1,
Syn th esis of Com p ou n d 1a . A mixture was prepared that
contained compound 11 (Cl-) (130 mg, 0.216 mmol), CDI (37
mg, 0.226 mmol), DMF (5 mL). Then peptide (150 mg, 0.217
mmol), NMM (14 µL), and DMF (5 mL) were added. Reaction
temperature was 50 °C. Precipitation with MeOH-H2O (8:1)
afforded 1a (Cl-) (196 mg, 78%) as a white solid. HPLC
(gradient 10-100% CH3CN-H2O in 20 min; τR ) 18.5 min;
purity 95%). Mp 150 °C. [R]25 +33 (c 0.5, MeOH). 1H NMR
D
(500 MHz, COSY, MeOH-d4): δ 7.73-7.66 (m, 4H, PhSi),
7.47-7.40 (m, 6H, PhSi), 7.27-7.21 (m, 5H, ArH-Phe), 4.71-
4.66 (dd, J ) 5.6, 1.8 Hz, 1H, CHR-Asn), 4.64-4.60 (m, 1H,
CHR-Phe), 4.39-4.34 (m, 1H, CHR-Leu), 4.31-4.23 (m, 2H,
CH2CO), 4.05-4.01 (m, 2H, CHR-Thr, Ala), 3.72-3.68 (m, 2H,
CH2OSi), 3.65 (s, 3H, OCH3), 3.60-3.53 (m, 4H, CH2O, CHR),
3.39-3.35 (m, 4H, CH2γ), 3.11-3.05 (m, 2H, CH2-Phe), 2.69-
2.63 (m, 2H, CH2-Asn), 2.49-2.41 (m, 4H, CH2CO-adip),
2.28-1.81 (m, 4H, CH2â), 1.79-1.50 (m, 7H, (CH2)2-adip,
CH2â, CHγ-Leu), 1.37 (d, J ) 7.2 Hz, 3H, CH3-Ala), 1.19 (d,
J ) 6.2 Hz, 3H, CH3-Thr), 1.02 (s, 9H, (CH3)3C), 0.94 (d, J )
6.2 Hz, 3H, CH3-Leu), 0.90 (d, J ) 6.2 Hz, 3H, CH3-Leu).
13C NMR (125 MHz, MeOH-d4): δ 176.3, 175.8, 174.5 (CONH),
173.0, 172.7 (COOR), 152.3 (C-guan), 137.9, 136.7 136.6,
135.9, 134.1, 134.0, 131.2, 130.9, 130.3, 130.4, 129.5, 129.0,
128.8, 128.6, 127.9 (ArCH, ArC), 68.2 (CHO-Thr), 67.3, 66.7
(CH2O), 60.1, 55.5, 53.6, 52.7, 51.6 (CHR-Ala, Thr, Leu, Asn,
Phe), 51.5 (OCH3), 51.4 (CHR-guan), 46.3 (CH2γ-guan), 41.2
(CH2-Leu), 38.4 (CH2-Asn), 37.8 (CH2-Phe), 36.1 (CH2-
CONH-adip), 34.5 (CH2CO2-adip) 27.4, 27.2 [(CH3)3C], 26.1
[(CH2)2-adip], 25.8 (CH-Leu), 25.4, 23.8 (CH2â-guan), 23.6,
20.6, 17.8 (CH3-Leu, Ala, Thr). FAB/LSIMS m/z: 888.6 [(M
+
- Cl-)+, 100%]. HRMS for [C42H66N9O12
888.4801.
]
888.4830; found
Syn th esis of Com p ou n d 2b. A solution of compound 1b
(Cl-) (76 mg, 0.065 mmol) in a mixture of 30% HCl-CH3CN
(1:4, 5 mL) was stirred for 4 h. After removal of the solvent,
the crude was dissolved in water and washed with diethyl
ether and CH2Cl2. After evaporation of the water, the crude
was dissolved in CH3COOH and purified by preparative HPLC
(CH3CN-H2O, 60:40), affording 13 mg of 2b with different
anions (analytical gradient 0-100% CH3CN-H2O in 20 min:

Guanidinium Inhibitors of HIV-1 Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5203
2b (Cl-), τR ) 12.8 min; 2b (CH3CO2-), τR ) 15.2 min). 1H NMR
(Cl-) (300 MHz, MeOH-d4): δ 7.21-7.09 (m, 5H, ArH-Phe),
4.61-4.51, (m, 2H, CHR-Asn, Phe), 4.28-4.13 (m, 4H, CHR-
Leu, Thr, Ala), 3.59-3.54 (m, 2H, CH2O-guan), 3.58 (s, 3H,
OCH3), 3.47-3.17 (m, 6H, CHR-guan, CH2γ-guan, CH2N),
3.11-3.05 (m, 2H, CH2-Phe), 2.69-2.56 (m, 2H, CH2-Asn),
2.31 (m, 2H, CH2CO-adip), 2.28 (m, 2H, CH2CO-adip), 2.10-
1.89 (m, 4H, CH2â-guan), 1.85-1.61 (m, 7H, (CH2)2-adip,
CH2â-Leu, CHγ-Leu), 1.37 (d, J ) 7.2 Hz, 3H, CH3-Ala),
1.19 (d, J ) 6.2 Hz, 3H, CH3-Thr), 0.97 (d, J ) 6.0 Hz, 3H,
CH3-Leu), 0.94 (d, J ) 6.0 Hz, 3H, CH3-Leu). 13C NMR (Cl-)
(75 MHz, MeOH-d4): δ 175.9, 175.5, 174.5, 174.2, 173.4, 171.5,
171.0 (CO), 154.3 (C-guan), 137.7, 130.1, 129.3, 127.7 (ArC,
ArCH-Phe), 66.6 (CHO-Thr), 64.8 (CH2O-guan), 59.7 (CHR-
Thr), 55.2, 53.3, 52.4 (CHR-Leu, Asn, Phe), 51.4 (OCH3), 51.3
(CHR-Ala), 50.9 (CHR-guan), 46.3 (CH2γ-guan), 45.8 (CH2-
NH), 40.9 (CH2-Leu), 38.1 (CH2-Asn), 37.5 (CH2-Phe), 35.8
(CH2CO-adip), 34.2 (CH2CO-adip), 25.7, 25.5 [(CH2)2-adip],
25.1 (CH-Leu), 23.5, 21.1 (CH2â-guan), 23.3 (CH3-Leu), 20.0
(CH3-Thr), 17.3 (CH3-Ala). FAB/LSIMS m/z: 887.5 [(M -
Cl-)+, 100%].
reaction mixture was stirred at room temperature and moni-
tored by HPLC. After removal of the solvent, the crude was
purified by preparative HPLC.
Syn th esis of Com p ou n d 3. A mixture was prepared that
contained compound 14 (Cl-) (51 mg, 0.093 mmol), HOBt (17
mg, 0.093 mmol), BOP (66.5 mg, 0.102 mmol), and DMF (2
mL). Then Ala-Ile-Thr(Bn)-Leu-Trp-OMe (TFA salt, 75 mg,
0.093 mmol), NMM (36 µL, 0.186 mmol), and DMF (1 mL) were
added. The reaction was monitored by HPLC, and the reaction
time was 2 h. Purification was by preparative HPLC (H2O-
CH3CN 30:70, 45:65 for purification; analytical gradient
0-100% CH3CN-H2O in 20 min; τR ) 19.5 min; purity >99%),
affording 3 (Cl-) (106 mg, 92%) as a white solid. Mp 180-
1
182°C. [R]25 +25 (c 0.2, DMSO). H NMR (500 MHz, COSY,
D
DMSO-d6): δ 10.88 (s, 1H, NH-guan), 8.36 (d, J ) 7.0 Hz,
1H, NH-Trp), 8.19 (d, J ) 6.2 Hz, 1H, NH-Ala), 8.04 (t, J )
7.6 Hz, 1H, NH-Leu), 7.90 (d, J ) 8.6 Hz, 1H, NH-Ile), 7.77
(d, J ) 8.5 Hz, 1H, NH-Thr), 7.69 (d, J ) 4.0 Hz, 1H, ArH),
7.63-7.61 (m, 4H, ArH), 7.56-7.39 (m, 6H, PhSi), 7.34-7.24
(m, 8H, ArH), 7.12 (s, 1H, NH), 7.06 (t, J ) 7.5 Hz, 1H, ArH-
Trp), 6.96 (t, J ) 8.0 Hz, 1H, ArH-Trp), 4.56 (m, 1H, OCH2-
Ph), 4.50-4.37 (m, 5H, CHR-Ile, Trp, Ala, Thr, OCH2Ph), 4.30
(t, J ) 7.5 Hz, 1H, CHR-Leu), 3.95-3.68 (m, 1H, CHâ-Thr),
3.65-3.48 (m, 7H, CH2OSi, CHR-guan, OCH3), 3.44-3.42 (m,
4H, CH2γ-guan), 3.23 (s, 2H, SCH2CO), 3.09-3.01 (m, 2H,
CH2-Trp), 2.84-2.55 (m, 2H, CH2S), 1.99-1.90 (m, 2H,
CH2â-guan), 1.80-1.72 (m, 3H, CH2â-guan, CHγ-Leu),
1.63-1.57 (m, 2H, CH2-Leu), 1.44-1.42 (m, 3H, CH-Ile,
CH2-Ile), 1.16 (d, J ) 7.0 Hz, 3H, CH3-Ala), 1.05 (d, J ) 6.0
Hz, 3H, CH3-Thr), 1.03 (s, 9H, (CH3)3C), 0.98-0.93 (m, 3H,
CH3-Ile), 0.86-0.80 (m, 9H, CH3-Leu, Ile). 13C NMR (125
MHz, DEPT, DMSO-d6): δ 174.5, 172.9, 169.9, 168.9, 167.9
(CONH), 153.9 (C-guan), 142.2, 139.5, 136.0, 135.9 (ArCH,
ArC), 135.4, 134.9, 133.3, 131.0, 130.9 (PhSi), 128.9, 128.86,
128.4, 128.1, 127.9 (ArC, ArCH), 121.8, 109.0 (ArC, ArCH-
Trp), 77.3 (CHâ-Thr), 71.4 (OCH2Ph), 67.1 (CH2OSi), 61.1
(CH3O), 57.8 (CHR-Leu), 56.7 (CHR-Trp), 52.6 (CHR-Ala),
50.8 (CHR-Leu), 49.0 (CHR-Thr), 48.5, 48.4 (CHR-guan),
48.1, 47.0 (CH2γ-guan), 46.8 (CH2), 42.2 (CH2â-Leu), 37.3
(CH2S), 35.1 (SCH2CO), 27.5 [(CH3)3C], 27.4 (CH-Ile), 25.9
(CH2â-guan), 24.9, 23.9 (CH3-Leu), 22.5 (CH3-Ile), 19.7
[(CH3)3C], 19.2 (CH3-Ala), 17.1 (CH3-Thr), 16.1 (CH3-Ile),
11.9 (CH2-Ile). FAB/LSIMS m/z: 1200.0 [(M - Cl- )+, 100%].
HRMS for [C65H90N9O9SSi+] 1200.6352; found 1200.6360.
Syn th esis of (2S,8S)-2-(ter t-Bu tyld ip h en ylsila n yloxy-
m et h yl)-8-m et h a n esu lfon yloxym et h yl-3,4,6,7,8,9,-h exa -
h yd r o-2H-p yr im id o[1,2-a ]p yr im id in -1-iu m Hexa flu or o-
p h osp h a te (13). To a stirred solution of guanidinium salt 914
-
20
(PF6
)
(1.15 g, 1.98 mmol) and Et3N (1.5 mL, 10.8 mmol) in
dry THF (40 mL) was added at 0 °C a solution of methane-
sulfonic anhydride (774 mg, 4.31 mmol) in dry THF (10 mL).
The reaction mixture was stirred at this temperature for 1 h.
Then the solvent was removed, and after addition of CH2Cl2,
the organic phase was washed with 0.1 N NH4PF6, filtered
over cotton, and concentrated and the crude was purified by
silica gel column chromatography (2% MeOH-CH2Cl2), af-
fording 13 (PF6-) (1.32 g, 98%) as a white solid. Mp 60-62°C.
1
[R]25 +43 (c 0.5, CHCl3). H NMR (300 MHz, CDCl3): δ 7.63
D
(m, 4H, PhSi), 7.62 (m, 6H, PhSi), 6.24 (s, 1H, NH), 6.08 (s,
1H, NH), 4.30 (m, 1H, CH2OMs), 4.17 (m, 1H, CH2OMs), 3.80
(m, 1H, CHR), 3.65 (m, 2H, CH2OSi), 3.57 (m, 1H, CHR), 3.33
(m, 4H, CH2γ), 3.08 (s, 3H, CH3SO3), 2.05-1.89 (m, 4H, CH2â),
1.06 (s, 9H, (CH3)3C). 13C NMR (75 MHz, CDCl3): δ 150.6 (C-
guan), 135.5, 132.5, 130.0, 128.9 (ArCH, ArC), 69.5 (CH2OMs),
66.2 (CH2OSi), 50.1, 47.7 (CHR), 45.3, 44.9 (CH2γ), 37.1 (CH3-
SO3), 26.7 [(CH3)3C], 22.4, 21.9 (CH2â), 19.1 [(CH3)3C]. FAB/
LSIMS m/z: 516.2 [(M - PF6-)+, 100%]. HRMS for [C26H38N3O4-
SSi]+ 516.2352; found 516.2354.
Syn th esis of Com p ou n d 6. A mixture was prepared that
contained compound 14 (Cl-) (97 mg, 0.176 mmol), HOBt (32
mg, 0.176 mmol), BOP (100 mg, 0.194 mmol), and DMF (4 mL).
Gly-Ala-Thr(Bn)-Leu-Asn-Phe-OBn (TFA salt) (183 mg, 0.176
mmol), NMM (60 µL, 0.352 mmol), and DMF (2 mL) were then
added. The reaction was monitored by HPLC (H2O-CH3CN,
0-100% in 20 min). Reaction time was 2 h. Purification was
by preparative HPLC (H2O-CH3CN 30:70, 40:60 for purifica-
tion; analytical gradient 0-100% CH3CN-H2O; τR ) 20.5 min;
purity >99%), affording 6 (Cl-) as a white solid (210 mg, 89%).
Mp 214-216 °C. [R]25D +31 (c 0.1, DMSO). 1H NMR (500 MHz,
COSY, DMSO-d6): δ 8.27 (t, J ) 5.7 Hz, 1H, NH-Gly), 8.17-
8.13 (m, 3H, NH-Phe, Asn, Ala), 8.04 (d, J ) 8.9 Hz, 1H, NH-
Leu), 7.67 (d, J ) 8.2 Hz, 1H, NH-Thr), 7.64-7.62 (m, 5H,
PhSi, NH-guan), 7.50-7.41 (m, 7H, PhSi, NH-guan), 7.36-
7.27 (m, 8H, ArH), 7.26-7.19 (m, 6H, ArH), 7.16 (d, J ) 6.6
Hz, 1H, ArH), 6.90 (s, 2H, NH2), 5.05-5.04 (d, J ) 5.0 Hz,
2H, CO2CH2-Phe), 4.57 (dd, J ) 13.2, 7.7 Hz, 1H, CHR-Asn),
4.51 (d, J ) 12.0 Hz, 1H, OCH2-Phe), 4.50-4.36 (m, 4H,
CHR-Phe, Ala, Thr, Leu), 4.43 (d, J ) 12.0 Hz, 1H, OCH2-
Phe), 4.00-3.96 (m, 1H, CHâ-Thr), 3.80 (dd, J ) 17.0, 6.0
Hz, 1H, CH2-Gly), 3.73 (dd, J ) 17.0, 6.0 Hz, 1H, CH2-Gly),
3.64-3.42 (m, 4H, CH2OSi, CHR-guan), 3.34-3.27 (m, 4H,
CH2γ-guan), 3.26 (s, 2H, SCH2CO), 2.98 (d, J ) 8.0 Hz, 2H,
CH2â-Phe), 2.82 (dd, J ) 14.0, 6.0 Hz, 1H, CH2S), 2.72 (dd, J
) 14.0, 6.0 Hz, 1H, CH2S), 2.36 (dd, J ) 15.0, 8.0 Hz, 2H, CH2-
Asn), 2.02-1.95 (m, 2H, CH2â-guan), 1.81-1.75 (m, 2H,
CH2â-guan), 1.57-1.55 (m, 1H, CHγ-Leu), 1.42-1.40 (m, 2H,
CH2â-Leu), 1.24 (d, J ) 7.0 Hz, 3H, CH3-Ala), 1.10 (d, J )
6.0 Hz, 3H, CH3-Thr), 1.03 (s, 9H, (CH3)3C), 0.82 (d, J ) 6.5
Syn th esis of (2S,8S)-8-(ter t-Bu tyld ip h en ylsila n yloxy-
m eth yl)-2-(car boxym eth ylsu lfan ylm eth yl)-3,4,6,7,8,9,-h exa-
h yd r o-2H-p yr im id o[1,2-a ]p yr im id in -1-iu m Ch lor id e (14).
To a stirred solution of 13 (PF6-) (680 mg, 1.03 mmol) in dry
THF (50 mL) was added a solution of sodium mercaptoacetic
acid (363 mg, 3.08 mmol) and t-BuOK (341 mg, 2.88 mmol) in
MeOH (20 mL). The resulting mixture was stirred for 4 h at
room temperature. The solvent was removed, and the solid
residue was dissolved in CH2Cl2 (100 mL), washed with 1 N
NaHCO3, water, and HCl (1 N, 100 mL each), and then dried
over Na2SO4. Filtration and evaporation of the solvent gave a
crude that was triturated with ethyl acetate to afford 14 (Cl-)
(571 mg, 82%) as a white solid. Mp 166-168 °C. [R]25 +50 (c
D
1
0.5, MeOH). H NMR (300 MHz, CDCl3): δ 8.27 (s, 1H, NH),
8.16 (s, 1H, NH), 7.65-7.60 (m, 4H, PhSi), 7.41-7.39 (m, 6H,
PhSi), 3.77 (dd, J ) 11.0, 4.0 Hz, 1H, CH2OSi), 3.59 (m, 3H,
CH2OSi, CHR), 3.43 (d, J ) 15.0 Hz, 1H, SCH2CO), 3.36 (d, J
) 15.0 Hz, 1H, SCH2CO), 3.28-3.23 (m 4H CH2γ), 2.95-2.71
(m, 2H, CH2S), 2.12-1.85 (m, 4H, CH2â), 1.07 [s, 9H, (CH3)3C)].
13C NMR (75 MHz, CDCl3): δ 171.9 (CO), 150.9 (C-guan),
135.5, 135.4, 132.6, 129.8, 127.8 (ArCH, ArC), 65.3 (CH2O),
49.3, 48.0 (CHR), 45.1, 44.7 (CH2γ), 36.7 (CH2S), 34.5 (SCH2-
CO), 26.7 [(CH3)3C)], 24.8, 22.5 (CH2â), 19.1 [(CH3)3C)]. FAB/
LSIMS m/z: 512.4 [(M - Cl-)+, 100%]. Anal. Calcd for
C27H38N3O3SSiCl‚H2O: C, 57.1; H, 7.1; N, 7.4; S, 5.6. Found:
C, 57.5; H, 6.8; N, 7.3; S, 5.5.
Gen er a l P r oced u r e for Com p ou n d s 3-6, 8, a n d 20. To
a solution of guanidinium compound, HOBt, and BOP in DMF
was added a solution of peptide and NMM in DMF. The

5204 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Breccia et al.
Hz, 3H, CH3-Leu), 0.78 (d, J ) 6.5 Hz, 3H, CH3-Leu). 13C
NMR (125 MHz, DEPT, DMSO-d6): δ 173.4, 172.5, 172.1,
171.9, 170.1, 170.0, 169.1 (CO), 151.2 (C-guan), 139.5, 137.6,
136.5 (ArC), 136.0, 135.9 (ArCH), 133.5 (ArC), 133.3 (ArC-
Phe), 131.0, 130.9 (ArCH), 129.9 (ArCH-Phe), 129.2, 129.16,
128.9, 128.8, 128.7, 128.3, 128.1, 127.5 (ArCH, ArC), 75.5
(CHâ-Thr), 71.3 (OCH2Ph-Thr), 66.9 (CO2CH2Ph), 66.7 (CH2-
OSi), 57.8 (CHR-Leu), 54.7 (CHR-Phe), 51.5 (CHR-Ala), 50.3
(CHR-guan), 50.2 (CHR-Asn), 48.9 (CHR-Thr), 48.1 (CHR-
guan), 45.3 (CH2γ-guan), 42.8 (CH2-Gly), 42.2 (CH2â-Leu),
37.8 (CH2-Asn), 37.5 (CH2-Phe), 37.2 (CH2S), 35.1 (SCH2-
CO), 27.5 [(CH3)3C], 25.6 (CH2â-guan), 24.8 (CH3-Leu), 23.9
(CH3-Leu), 19.7 [(CH3)3C], 19.2 (CH3-Ala), 17.2 (CH3-Thr).
FAB/LSIMS m/z: 1296.0 [(M - Cl-)+, 100%]. HRMS for
[C69H91N10O11SSi]+ 1295.6358; found 1295.6372.
mmol), HOBt (24 mg, 0.170 mmol), DMF (12 mL), and NMM
(21 µL, 0.038 mmol). Reaction time was 12 h. The residue was
triturated with MeOH and filtered and the solid was purified
by semipreparative HPLC (gradient CH3CN-H2O 75:25 to 60:
40; flow 6 mL/min; τR ) 23.6 min; purity 93%) to afford
compound 4 (PF6-) (191 mg, 72%) as a white solid. Mp 200
°C. [R]25 +10 (c 0.5, DMSO). 1H NMR (500 MHz, COSY,
D
DMSO-d6): δ 8.27-8.23 (m, 2H, NH-Gly, Tyr), 8.14 (d, J )
8.0 Hz, 1H, NH-Ser), 8.07 (d, J ) 7.7 Hz, 1H, NH-Ile), 7.95-
7.92 (m, 2H, NH-Asn, Leu), 7.64-7.61 (m, 4H, PhSi), 7.60 (s,
1H, NH-guan), 7.51-7.25 (m, 16H, ArH), 7.12 (d, J ) 8.7 Hz,
2H, ArHm-Tyr), 6.93 (s, 1H, NH-guan), 6.83 (d, J ) 8.7 Hz,
2H, ArHo-Tyr), 5.02 (s, 2H, OCH2Ph-Tyr), 4.62-4.48 (m, 5H,
CH-Tyr, Ser, Asn, OCH2Ph-Ser), 4.30-4.26 (m, 2H, CH-
Leu, Ile), 3.84-3.77 (m, 2H, CH2-Gly), 3.67-3.63 (m, 2H,
CH2O-Ser), 3.62 (s, 3H, OCH3), 3.61-3.53 (m, 2H, CHR),
3.52-3.42 (m, 2H), 3.33-3.22 (m, 4H, CH2γ), 2.99-2.95 (m,
1H, CH2-Asn), 2.84-2.69 (m, 3H, CH2S, CH2-Asn), 2.55-
2.37 (m, 2H, CH2Ph-Tyr), 2.01-1.95 (m, 2H, CH2â), 1.85-
1.73 (m, 2H, CH2â), 1.72-1.46 (m, 4H, CH-Ile, CH2-Leu,
CH-Leu), 1.43-1.35 (m, 2H, CH2-Ile), 1.03 (s, 9H, (CH3)3C),
0.87 (d, J ) 6.6 Hz, 3H, CH3-Leu), 0.83 (d, J ) 6.5 Hz, 3H,
CH3-Leu), 0.78-0.75 (m, 6H, CH3-Ile). 13C NMR (125 MHz,
DEPT, DMSO-d6): δ 173.6, 172.1, 171.9, 171.7, 171.3, 169.9,
169.8, 169.3 (CO), 151.2 (C-guan), 138.9, 138.1 (ArC), 136.0,
135.9 (ArCH), 133.5, 133.3, 131.2 (ArC), 131.0, 130.9, 130.5,
129.3, 129.1, 128.9, 128.8, 128.6, 128.4, 128.2 (ArCH), 115.1
(ArCo-Tyr), 73.2, 72.9, 70.0, 61.1 (CH2O), 57.5, 54.7, 53.5 (CH),
52.7 (CH3O), 51.2, 50.3, 50.2, 48.1 (CH), 45.3, 45.2, 42.9, 40.2
(CH2), 39.9, 38.0 (CH), 37.6, 37.2, 35.1 (CH2), 31.5 (CH), 27.5
[(CH3)3C], 25.6, 25.0 (CH2), 23.6 (CH), 23.0 (CH2), 22.2 (CH),
19.7 [(CH3)3C], 16.1, 11.9 (CH3). FAB/LSIMS m/z: 1353.3 [(M
- PF6-)+, 100%]. HRMS for [C72H97N10O12SSi]+ 1353.6777;
found 1353.6739.
Syn th esis of Com p ou n d 7. To a solution of compound 6
(Cl-) (20 mg, 0.014 mmol) in MeOH (5 mL) with few drops of
acetic acid was added 10% Pd-C (15 mg, 0.014 mmol), and
the mixture was kept under H2 atmosphere for 5 h. The
reaction mixture was filtered over Celite and evaporated,
affording 7 (Cl-) (13 mg, 80%) as a white solid. Analytical
HPLC (gradient 10-100% CH3CN-H2O; τR ) 19.1 min; purity
96%). Mp 220°C. [R]25D +38 (c 0.1, DMSO). 1H NMR (300 MHz,
DMSO-d6): δ 10.08 (s, 1H, NH-guan), 9.95 (s, 1H, NH-guan),
8.51 (m, 1H, NH-Gly), 8.33 (m, 1H, NH-Phe), 8.05 (m, 1H,
NH-Asn), 7.64-7.60 (m, 5H, PhSi, NH-Ala), 7.50-7.41 (m,
7H, PhSi, NH-Leu), 7.31-7.17 (m, 5H, ArH, NH-Thr), 7.12-
7.02 (m, 6H, ArH), 6.83 (s, 2H, NH2), 4.57-4.20 (m, 7H, CHR-
Asn, OCH2, CHR-Phe, Ala, Thr, Leu), 4.00-3.96 (m, 1H,
CHâ-Thr), 3.80-3.20 (m, 12H, CH2-Gly, CH2OSi, CHR-
guan, CH2γ-guan, SCH2CO), 2.98-2.10 (m, 6H, CH2-Phe,
CH2S, CH2-Asn), 2.02-1.65 (m, 4H, CH2â-guan), 1.57-1.15
(m, 6H, CHγ-Leu, CH2â-Leu, CH3-Ala), 1.06 (d, J ) 6.0 Hz,
3H, CH3-Thr), 1.00 (s, 9H, (CH3)3C), 0.77 (d, J ) 6.5 Hz, 3H,
CH3-Leu), 0.68 (d, J ) 6.5 Hz, 3H, CH3-Leu). FAB/LSIMS
m/z: 1250.6 [(M - Cl-)+, 100%].
Syn th esis of Com p ou n d 15. To a solution of 14 (Cl-) (50
mg, 0.090 mmol), glycine methyl ester hydrochloride (12 mg,
0.09 mmol), HOBt (12 mg, 0.090 mmol), and BOP (44 mg,
0.100 mmol) in DMF (2 mL) was added NMM (22 µL, 0.190
mmol). The resulting mixture was stirred overnight. The
solvent was evaporated under reduced pressure, and the solid
residue dissolved in CH2Cl2 (30 mL) was washed successively
with a saturated solution of NaHCO3 and then with a solution
of HPF6 (0.01 N). The organic phase was dried, filtered, and
concentrated. Purification by silica gel column chromatography
with a solvent mixture of 4% MeOH in CH2Cl2 afforded a white
solid (58 mg, 90%). To a solution of the resulting solid (38 mg,
0.052 mmol) in THF (1 mL) was added a solution of NaOH (1
N, 0.10 mL). The mixture was stirred for 4 h at room
temperature. The solvent was evaporated, and the solid
residue was dissolved in CH2Cl2 (20 mL) and washed with 0.1
N NH4PF6. The organic layer was dried (Na2SO4), filtered, and
concentrated. The resulting solid residue was triturated with
diethyl ether and dried to afford compound 15 (PF6-) (28 mg,
95%) as a white solid. Mp 94-95 °C. [R]25D +42 (c 0.2, CHCl3).
1H NMR (500 MHz, COSY, CDCl3): δ 10.05 (s, 1H, NH), 9.98
(s, 1H, NH), 7.82 (t, J ) 4.6 Hz, 1H, NHCO), 7.65-7.62 (m,
4H, PhSi), 7.46-7.38 (m, 6H, PhSi), 3.91 (dd, J ) 13.0, 4.5.0
Hz, 1 H, NHCH2CO), 3.82 (dd, J ) 4.5, 17.5 Hz, 1H, NHCH2-
CO), 3.77 (d, J ) 6.0 Hz, 1 H, CH2OSi), 3.58 (m, 3H, CH2OSi,
CHR), 3.37-3.20 (d, J ) 15.1 Hz, 2H, SCH2CO), 3.24-3.12
(m, 4H CH2γ), 2.84 (dd, J ) 14, 5 Hz, 1H, CH2S), 2.78 (dd, J
) 14, 6.5 Hz, 1H, CH2S), 2.05-1.90 (m, 4H, CH2â), 1.06 (s,
9H, (CH3)3C). 13C NMR (125 MHz, DEPT, CDCl3): δ 175.1
(CONH), 168.9 (CO), 151.4 (C-guan), 135.5, 132.8, 128.3,
127.8 (ArCH, ArC), 65.3 (CH2O), 48.9, 48.4 (CHR), 45.3 (CH2N),
44.5, 44.0 (CH2γ), 36.8, 36.5 (CH2S, SCH2CO), 26.8 [(CH3)3C],
25.0, 22.7 (CH2â), 19.2 [(CH3)3C]. FAB/LSIMS m/z: 569.1 [(M+,
100%].
Syn th esis of Com p ou n d 5. 5 was prepared according to
the general procedure. A mixture was prepared that contained
compound 15 (PF6-) (30 mg, 0.05 mmol), Ile-Ser-Tyr-Asn-Leu-
OMe (TFA salt) (39 mg, 0.05 mmol), BOP (26 mg, 0.06 mmol),
HOBt (7 mg, 0.05 mmol), DMF (12 mL), and NMM (6 µL, 0.058
mmol). Reaction time was 12 h. The product was precipitated
with MeOH-H2O and triturated with AcOEt and toluene to
afford compound 5 (PF6-) (54 mg, 77%; HPLC gradient CH3-
CN-H2O 10-100% in 20 min; τR ) 17.8 min; purity 94%) as
1
a white solid. Mp 178 °C. [R]25 +15 (c 0.2, DMSO). H NMR
D
(500 MHz,COSY, DMSO-d6): δ 9.12 (s, 1H, OH), 8.27 (br t,
1H, NH-Gly), 8.17 (d, J ) 6.5 Hz, 1H, NH), 8.04-7.80 (m,
4H, NH), 7.64-7.62 (m, 4H, PhSi), 7.48-7.30 (m, 6H, PhSi),
6.98 (d, J ) 8.4 Hz, 2H, CH-Tyr), 6.92 (s, 1H, NH), 6.59 (d, J
) 8.4 Hz, 2H, CH-Tyr), 4.58-4.54 (m, 2H, CH-Asn, Leu),
4.42-4.26 (m, 3H, CH-Ser, Tyr, Ile), 3.81-3.79 (m, 2H, CH2-
Gly), 3.72-3.41 (m, 7H, CH2O, OCH3), 3.40-3.27 (m, 9H, CHR,
SCH2CO, CH2γ), 3.18-2.68 (m, 4H, CH2S, CH2-Asn), 2.41-
2.36 (m, 2H, CH2-Ph), 2.22-1.86 (m, 4H, CH2â), 1.80-1.24
(m, 4H, CH-Ile, Leu, CH2-Leu), 1.03 (s, 9H, (CH3)3C), 0.89
(d, J ) 6.5 Hz, 3H, CH3-Leu), 0.85 (d, J ) 6.5 Hz, 3H, CH3-
Leu), 0.78-0.75 (m, 6H, CH3-Ile). 13C NMR (125 MHz, DEPT,
CDCl3): δ 178.7, 170.6 (CONH), 156.6, 151.8 (C-guan), 141.8,
136.0, 135.9, 133.3, 130.9, 128.9 (ArCH, ArC), 115.7 (Co-Tyr),
73.2, 67.4, 63.9, 61.1 (CH2O), 57.0, 56.5, 56.3, 53.6, 52.7, 50.0,
49.1, 47.3, 45.5, 43.0, 38.6, 38.0, 37.9, 37.5 (CH2S), 32.2, 31.6,
27.5 [(CH3)3C], 24.9, 24.7 (CH2â), 23.6, 23.0, 22.1, 19.7 [(CH3)3C],
16.1, 11.8 (CH3). HRMS for [C58H85N10O12SSi]+ 1173.5838;
found 1173.5853.
Syn th esis of 8-(ter t-Bu tyld ip h en ylsila n yloxym eth yl)-
2-[17-(1,5-d im et h ylh exyl)-10,13-d im et h yl-2,3,4,7,8,9,10,-
11,12,13,14,15,16,17-t et r a d eca h yd r o-1H -cyclop en t a [a ]-
p h en a n th r en -4-ylsu lfa n ylm eth yl]-3,4,6,7,8,9-h exa h yd r o-
2H-pyr im ido[1,2-a ]pyr im idin -1-iu m Hexaflu or oph osph ate
(16). To a stirred solution of 13 (PF6-) (460 mg, 0.70 mmol) in
dry THF (40 mL) was added a solution of thiocholesterol (420
mg, 1.04 mmol) and t-BuOK (115 mg, 0.97 mmol) in THF-
MeOH (1:1) (20 mL). The resulting mixture was stirred for 4
h at room temperature. The solvent was removed, and the
residue was dissolved in CH2Cl2 and washed with a solution
Syn th esis of Com p ou n d 4. 4 was prepared according to
the general procedure. A mixture was prepared that contained
compound 15 (100 mg, 0.170 mmol), Ile-Ser(Bn)-Tyr(Bn)-Asn-
Leu-OMe (TFA salt) (161 mg, 0.170 mmol), BOP (86 mg, 0.180

Guanidinium Inhibitors of HIV-1 Protease
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5205
of 0.1 N NH4PF6. The organic phase was filtered over cotton
and concentrated in vacuo. Purification by silica gel column
chromatography (0.02% and 1% MeOH-CH2Cl2) afforded 16
1H, NH), 7.09 (s, 1H, NH), 5.36 (s, 1H, CH-chol), 4.32-4.17
(m, 2H, CH2OMs), 3.60-3.35 (m, 6H, CHR, CH2γ), 3.15 (s, 3H,
CH3OSO2), 2.88-2.63 (m, 2H, CH2S), 2.60-2.57 (m, 1H, CHS-
chol), 2.35-2.10 (m, 4H, CH2CH-chol, CH2â), 2.04-1.86 (m,
10H, CH2CH-chol, CH2-chol, CH2â), 1.68-1.43 (m, 9H, CH2-
chol, CH-chol), 1.22-1.07 (m, 5H, CH2, CH), 0.99 (s, 3H, CH3),
0.91 (d, J ) 6.4 Hz, 3H, CH3), 0.87 (d, J ) 6.6 Hz, 3H, CH3),
0.86 (d, J ) 6.6 Hz, 3H, CH3), 0.67 (s, 3H, CH3). 13C NMR
(125 MHz, CDCl3): δ 150.7 (C-guan), 141.2 (CdCH), 121.4
(CHdC), 69.8 (CH2O), 56.7 (CH-chol), 56.2 (CH-chol), 50.2,
49.0 (CHR), 47.5 (CH-chol), 45.6, 45.2 (CH2γ), 44.8, 42.3 (CH-
chol), 39.8, 39.7 (CH2-chol), 39.5, 39.4 (CH2), 37.2 (CH3), 36.8,
36.2 (CH2), 35.8 (CH), 34.1, 31.8 (CH2), 31.78, 29.9, 29.7 (CH),
28.2, 28.0, 25.0, 24.3, 23.8 (CH2), 22.8, 22.5 (CH3), 21.0 (CH2),
19.3, 18.7, 11.8 (CH3). FAB/LSIMS m/z: 662.3 [(M - PF6-)+,
100%]. HRMS for [C37H64N3O3S2]+ 662.4389; found 662.4388.
(PF6-) (560 mg, 83%) as a white solid. Mp 82 °C. [R]25 +16 (c
D
1
0.5, CHCl3). H NMR (500 MHz, COSY, CDCl3): δ 7.66-7.64
(m, 4 H, PhSi), 7.48-7.42 (m, 6H, PhSi), 6.17 (s, 1H, NH), 6.00
(s, 1H, NH), 5.38-5.37 (m, 1H, CH-chol), 3.70-3.68 (dd, J )
9.9, 4.7 Hz, 2H, CH2OSi), 3.57-3.45 (m, 2H, CHR), 3.37-3.29
(m, 4H, CH2γ), 2.80 (dd, J ) 14.1, 6.3 Hz, 1H, CH2S), 2.69
(dd, J ) 14.1, 6.3 Hz, 1H, CH2S), 2.63-2.56 (m, 1H, CHS-
chol), 2.34-2.25 (m, 2H, CH2CH-chol), 2.63-2.56 (m, 1H,
CHS-chol), 2.34-2.25 (m, 2H, CH2CH-chol), 2.22-2.16 (m,
2H, CH2â), 1.94-1.80 (m, 5H, CH2â, CH-chol, CH2-chol),
1.70-1.67 (m, 1H, CH2), 1.63-1.25 (m, 17H, CH2-chol, CH-
chol), 1.20-1.10 (m, 4H, CH2C-chol, CH2CH), 1.06 (s, 9H,
(CH3)3C), 1.00 (s, 3H, CH3-chol), 1.00-0.90 (m, 1H, CH-chol),
0.93 (d, J ) 6.5 Hz, 3H, CH3), 0.87 (d, J ) 6.6 Hz, 3H, CH3),
0.86 (d, J ) 6.6 Hz, 3H, CH3), 0.66 (s, 3H, CH3). 13C NMR
(125 MHz, DEPT, CDCl3): δ 150.4 (C-guan), 141.0 (CdCH),
135.5, 132.5, 130.0, 128.0 (ArC, ArCH), 121.5 (CHdC), 65.4
(CH2O), 56.7, 56.1 (CH-chol), 50.2, 50.1 (CHR), 49.0 (CH-
chol), 45.6, 45.3 (CH2γ), 45.1, 42.3 (CH-chol), 39.8, 39.7 (CH2-
chol), 39.5, 39.4 (CH2), 36.8 (CH), 36.2 (CH2), 35.8 (CH), 34.0,
31.8 (CH2), 31.7 (CH), 29.8 (CH2), 28.2 (CH), 28.0 (CH2), 26.8
[(CH3)3C], 25.1, 24.3, 23.8 (CH2), 22.8 (CH3), 22.6 (CH2), 22.5
(CH3), 20.9 (CH2), 19.3 (CH3), 19.1 [(CH3)3C], 18.7, 11.8 (CH3).
ESI+ m/z: 822.7 [(M - PF6-)+, 100%]. HRMS for [C52H80N3-
OSSi]+ 822.5791; found 822.5779.
Syn th esis of 8-Ca r boxym eth ylsu lfa n ylm eth yl-2-[17-
(1,5-d im et h ylh exyl)-10,13-d im et h yl-2,3,4,7,8,9,10,11,12,-
13,14,15,16,17-tetr a d eca h yd r o-1H-cyclop en ta [a ]p h en a n -
t h r e n -4-ylsu lfa n ylm e t h yl]-3,4,6,7,8,9-h e xa h yd r o-2H -
p yr im id o[1,2-a ]p yr im id in -1-iu m Hexa flu or op h osp h a t e
(19). To a stirred solution of 18 (PF6-) (70 mg, 0.09 mmol) in
dry THF (10 mL) was added a solution of sodium mercap-
toacetic acid (31 mg, 0.26 mmol) and t-BuOK (29 mg, 0.24
mmol) in THF-MeOH (1:1) (20 mL). The resulting mixture
was stirred for 4 h at room temperature. After removal of the
solvent, the solid residue was dissolved in CH2Cl2 and washed
successively with NaHCO3, H2O, and 0.1 N HPF6 and then
dried over Na2SO4. Evaporation of the solvent gave a crude
that was purified by silica gel column chromatography (5%
MeOH-CH2Cl2) to afford 19 (PF6-) (53 mg, 77%) as a white
Syn th esis of 2-[17-(1,5-Dim eth ylh exyl)-10,13-d im eth yl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetr a d eca h yd r o-1H-cy-
clop en ta [a ]p h en a n th r en -4-ylsu lfa n ylm eth yl]-8-h yd r oxy-
m eth yl)-3,4,6,7,8,9-h exa h yd r o-2H-p yr im id o[1,2-a ]p yr im i-
d in -1-iu m Hexa flu or op h osp h a te (17). To a solution of 16
(PF6-) (420 mg, 0.43 mmol) in dry THF (45 mL) was added
dropwise HF-Py (70%, 2.25 mL) at 0 °C. The resulting
mixture was stirred overnight at room temperature. The
solvent was evaporated and the residue was purified two times
by silica gel column chromatography (3% and 5% MeOH-CH2-
solid. Mp 179 °C. [R]25 +27 (c 0.204, CHCl3). 1H NMR (500
D
MHz, CDCl3): δ 6.57 (s, 1H, NH), 6.28 (s, 1H, NH), 5.28-5.23
(m, 1H, CH-chol), 3.62-3.20 (m, 6H, CHR, CH2γ), 3.19 (s, 2H,
SCH2), 2.90-2.48 (m, 5H), 2.30-2.10 (m, 6H), 1.96-1.80 (m,
8H), 1.50-0.90 (m, 19H), 0.91 (s, 3H, CH3), 0.84 (d, J ) 6.5
Hz, 3H, CH3), 0.80 (d, J ) 6.6 Hz, 3H, CH3), 0.79 (d, J ) 6.6
Hz, 3H, CH3), 0.60 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3):
δ 174.3 (CO), 150.5 (C-guan), 141.2 (CdCH), 121.4 (CHdC),
56.7 (CH-chol), 56.2 (CH-chol), 50.2 (CH-chol), 49.7, 48.0
(CHR), 45.6, 45.4 (CH2γ), 44.9 (CH-chol), 42.3 (CH-chol), 39.8
(CH2-chol), 39.7, 39.5, 39.4, 37.3, 36.2 (CH2), 35.8 (CH), 34.4
(CH2), 34.1 (SCH2CO), 31.4 (CH2), 31.8 (CH), 29.8, 28.2 (CH2),
28.0 (CH), 25.4, 25.3, 24.3, 23.9 (CH2), 22.8, 22.6 (CH3), 20.9
(CH2), 19.3, 18.7, 11.8 (CH3). FAB/LSIMS m/z: 658.2 [(M -
PF6-)+, 100%]. HRMS for [C38H64N3O2S2]+ 658.4439; found
658.4458.
Cl2), affording 17 (PF6-) (297 mg, 94%) as a white solid. Mp
1
243 °C. [R]25 +50 (c 0.4, CHCl3). H NMR (500 MHz, COSY,
D
CDCl3): δ 6.79 (s, 1H, NH), 6.57 (s, 1H, NH), 5.35 (d, J ) 5.1
Hz, 1H, CH-chol), 3.80 (br s, 1H, OH), CH2OH), 3.58 (dd, J )
18.7, 7.6 Hz, 2H, CH2OH), 3.49-3.41 (m, 2H, CHR), 3.40-
3.20 (m, 4H, CH2γ), 2.76 (dd, J ) 13.5, 6.6 Hz, 1H, CH2S),
2.70 (dd, J ) 13.5, 7.2 Hz, 1H, CH2S), 2.57 (m, 1H, CHS-
chol), 2.28-2.14 (m, 4H, CH2CH-chol), 2.01-1.77 (m, 12H,
CH2â, CH2-chol), 1.55-1.20 (m, 11H, CH2CH-chol, CH2-chol,
CH-chol), 1.18-1.09 (m, 1H, CH-chol), 1.00-0.92 (m, 4H,
CH2-chol, CH-chol), 0.98 (s, 3H, CH3), 0.91 (d, J ) 6.5 Hz,
3H, CH3), 0.87 (d, J ) 6.6 Hz, 3H, CH3), 0.86 (d, J ) 6.6 Hz,
3H, CH3), 0.66 (s, 3H, CH3). 13C NMR (125 MHz, DEPT,
CDCl3): δ 150.7 (C-guan), 141.1 (CdCH), 121.4 (CHdC), 64.6
(CH2O), 56.7 (CH-chol), 56.2 (CH-chol), 50.2 (CHR), 48.5
(CH-chol), 45.6, 45.4 (CH2γ), 44.8, 42.3 (CH-chol), 39.8, 39.7
(CH2-chol), 39.5, 39.4 (CH2), 36.8 (CH), 36.2 (CH2), 35.8 (CH),
34.6, 31.8 (CH2), 31.7 (CH), 29.8 (CH2), 28.2 (CH), 28.0, 25.5,
24.3, 23.8 (CH2), 22.7 (CH3), 22.5 (CH2), 22.46 (CH3), 20.9, 19.3
(CH2), 18.7, 11.8 (CH3). ESI+ m/z: 584.5 [(M - PF6-)+, 100%].
HRMS for [C36H62N3OS]+ 584.4613; found 584.4633.
Syn th esis of Com p ou n d 8. 8 was prepared according to
the general procedure. A mixture was prepared that contained
compound 19 (PF6-) (11 mg, 0.014 mmol), Gly-Ala-Thr(Bn)-
Leu-Asn-Phe-OBn (TFA salt) (10 mg, 0.014 mmol), BOP (7 mg,
0.015 mmol), HOBt (2 mg, 0.014 mmol), DMF (1 mL), and
NMM (2 µL, 0.015 mmol). Reaction time was 12 h. The crude
was triturated with MeOH, and the obtained solid was purified
by semipreparative HPLC (CH3CN-H2O 75:25; flow 5 mL/min;
τR ) 21.8 min; purity 91%) to afford compound 8 (PF6-) (6 mg,
27%) as a white solid. Mp 207 °C. [R]25 +24 (c 0.3, DMSO).
D
1H NMR (300 MHz, DMSO-d6): δ 8.82-8.47 (m, 3H, NH-Gly,
Phe, Asn), 8.15-7.94 (m, 2H, NH-Ala, Trp), 7.77-7.64 (m,
18H, NH-Leu, NH-guan, ArH), 7.38 (br s, 2H, NH2), 5.81
(br s, 1H, CH-chol), 5.49 (s, 2H, CO2CH2Ph), 5.04-4.84 (m,
4H, CHR-Asn, CHâ-Thr, OCH2Ph), 4.51-4.39 (m, 4H, CHR-
Phe, Ala, Thr, Leu), 4.25-4.19 (m, 2H, CH2-Gly), 4.04-3.90
(m, 3H, CHR, SCH2), 3.79-2.52 (m, 17H), 2.45-1.29 (m, 55H),
1.11 (s, 3H, CH3). FAB/LSIMS m/z: 1441.4 [(M - PF6-)+,
100%]. HRMS for [C80H117N10O10S2]+ 1441.8395; found
1441.8413.
Syn th esis of 2-[17-(1,5-Dim eth ylh exyl)-10,13-d im eth yl-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetr a d eca h yd r o-1H-cy-
clopen ta[a ]ph en an th r en -4-ylsu lfan ylm eth yl]-8-m eth an e-
su lfon yloxym eth yl-3,4,6,7,8,9-h exah ydr o-2H-pyr im ido[1,2-
a ]pyr im idin -1-iu m Hexaflu or oph osph ate (18). To a solution
of 17 (PF6-) (100 mg, 0.140 mmol) and NMM (60 µL, 0.540
mmol) in dry THF (10 mL) was added Ms2O (61 mg, 0.340
mmol) in dry THF (15 mL). The reaction mixture was stirred
for 4 h at room temperature. After evaporation of the solvent,
the resulting crude was dissolved in CH2Cl2 and washed with
a 0.1 N NH4PF6 solution. The organic layer was filtered over
cotton and concentrated in vacuo. Purification by silica gel
column chromatography (4% MeOH-CH2Cl2) afforded 18
Syn th esis of 5-[1-(1-{1-[2-Ca r boxy-1-(1-m eth oxyca r bo-
n yl-2-p h en yleth ylca r ba m oyl)eth ylca r ba m oyl]-3-m eth yl-
b u t ylca r b a m oyl}-2-h yd r oxyp r op ylca r b a m oyl)e t h yl-
ca r b a m oyl] Met h ylp en t a n oa t e (20). 20 was prepared
according to a general procedure. A mixture was prepared that
contained adipic acid methyl ester (8.2 µL, 0.05 mmol), Ala-
Thr-Leu-Asn-Phe-OMe (TFA salt) (38.0 mg, 0.048 mmol), BOP
(PF6-) (107 mg, 97%) as a white solid. Mp 183 °C. [R]25 +21
D
1
(c 0.2, CHCl3). H NMR (500 MHz, COSY, CDCl3): δ 7.22 (s,

5206 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Breccia et al.
15591-15594. (b) Schramm, H. J .; Nakashima, H.; Schramm,
W.; Wakayama, H.; Yamamoto, N. HIV-1 Reproduction Is
Inhibited by Peptides Derived from the N- and C-Termini of
HIV-1 Protease. Biochem. Biophys. Res. Commun. 1991, 179,
847-851. (c) Schramm, H. J .; Breipohl, G.; Hansen, J .; Henke,
S.; J aeger, E.; Meichsner, C.; Riess, G.; Ruppert, D.; Rucknagel,
K. P.; Schafer, W. Inhibition of HIV-1 Protease by Short Peptides
Derived from the Terminal Segments of the Protease. Biochem.
Biophys. Res. Commun. 1992, 184, 980-985. (d) Franciskovich,
J .; Houseman, K.; Mueller, R.; Chmielewski, J . A Systematic
Evaluation of the Inhibition of HIV-1 Protease by Its C- and
N-Terminal Peptides. Bioorg. Med. Chem. Lett. 1993, 3, 765-
768. (e) Schramm, H. J .; Billich, A.; J aeger, E.; Rucknagel, K.;
Arnold, G.; Schramm, W. The Inhibition of HIV-1 Protease by
Interface Peptides. Biochem. Biophys. Res. Commun. 1993, 194,
595-600. (f) Valente, S.; Gobbo, M.; Licini, G.; Scarso, A.;
Scrimin, P. Allosteric Regulation of an HIV-1 Protease Inhibitor
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For a recent review, see the following. Boggetto, N.; Reboud-
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(34 mg, 0.06 mmol), HOBt (15.0 mg, 0.05 mmol), NMM (14
µL, 0.10 mmol), and DMF (1 mL). Reaction time was 12 h.
Purification was by precipitation with EtOAc-MeOH to afford
20 (32 mg, 92%). 1H NMR (300 MHz, MeOH-d4): δ 8.12-8.00
(m, 3H, NH), 7.74 (d, J ) 9.0 Hz, 1H, NH), 7.68 (d, J ) 6.0
Hz, 1H, NH), 7.30-7.16 (m, 7H, ArH, NH2-Asn), 6.92 (s, 1H,
NH), 5.00-3.99 (m, 6H, CHR, CHOH), 3.59 (s, 3H, OCH3), 3.57
(s, 3H, OCH3), 2.93 (m, 2H, CH2CO-Asn), 2.10-2.00 (m, 4H
COCH2-adip), 1.63-1.43 (m, 7H, (CH2)2-adip, CH2, CH-
Leu), 1.18 (d, J ) 7.1 Hz, 3H, CH3-Ala), 1.00 (d, J ) 6.5 Hz,
3H, CH3-Thr), 0.83 (d, J ) 8.2 Hz, 3H, CH3-Leu), 0.80 (d, J
) 6.5 Hz, 3H, CH3-Leu). FAB/LSIMS m/z: 721.4 [(M + H)+,
42%], 743.4 [(M + Na+), 5%]. HRMS for C34H53N6O11 721.3772;
found 721.3771.
Syn th esis of 2-(ter t-Bu tyld ip h en ylsila n yloxym eth yl)-
8-m e t h oxyca r b on ylm e t h ylsu lfa n ylm e t h yl-3,4,6,7,8,9-
h exa h yd r o-2H-p yr im id o[1,2-a ]p yr im id in -1-iu m Ch lor id e
(21). To a stirred solution of 13 (PF6-) (250 mg, 0.466 mmol)
in MeOH (10 mL) was added at room temperature a solution
of methyl mercaptoacetate (0.17 mL, 1.864 mmol) and Cs2CO3
(500 mg, 3.723 mmol). The reaction mixture is kept refluxing
for 6 h, and after addition of water (6 mL), the solvent is
removed. The crude is dissolved in CH2Cl2 and washed with
NH4Cl and water. The organic layer was dried over Na2SO4,
the solvent was eliminated, and the residue was purified by
silica gel column chromatography (8% MeOH-CH2Cl2) to
(8) Schramm, H. J .; Boetzel, J .; Buttner, J .; Fritsche, E.; Gohring,
W.; J aeger, E.; Konig, S.; Thumfart, O.; Wenger, T.; Nagel, N.
E.; Schramm, W. The Inhibition of Human Immunodeficiency
Virus Proteases by Interface Peptides. Antiviral Res. 1996, 30,
155-170.
(9) (a) Schramm, H. J .; de Rosny, E.; Reboud-Ravaux, M.; Buttner,
J .; Dick, A.; Schramm, W. Lipopeptides as Dimerization Inhibi-
tors of HIV-1 Protease. Biol. Chem. 1999, 380, 593-596. (b)
Dumond, J .; Boggetto, N.; Schramm, H. J .; Schramm, W.;
Takahashi, M.; Reboud-Ravaux, M. Thyroxine-Derivatives of
Lipopeptides: Bifunctional Dimerization Inhibitors of Human
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(11) (a) Bouras, A.; Boggetto, N.; Benatalah, Z.; de Rosny, E.; Sicsic,
S.; Reboud-Ravaux, M. Design, Synthesis and Evaluation of
Conformationally Constrained Tongs, New Inhibitors of HIV-1
Protease Dimerization. J . Med. Chem. 1999, 42, 957-962. (b)
Song, M.; Rajesh, S.; Hayashi, Y.; Kiso, Y. Design and Synthesis
of New Inhibitors of HIV-1 Protease Dimerization with Confor-
mationally Constrained Templates. Bioorg. Med. Chem. Lett.
2001, 11, 2465-2468.
afford 21 (Cl-) (134 mg, 58%) as a white solid. Mp 62 °C. [R]25
D
+54 (c 0.5, CHCl3). H NMR (Cl-) (300 MHz, CDCl3): δ 9.19
1
(s, 1H, NH), 8.74 (s, 1H, NH), 7.64-7.59 (m, 4H, PhSi), 7.44-
7.35 (m, 6H, PhSi), 3.81-3.74 (m, 1 H, CH2OSi), 3.72 (s, 3H,
OCH3), 3.63-3.44 (m, 3H, CH2OSi, CHR), 3.34 (m, 1H, SCH2-
CO), 3.29-3.14 (m 4H, SCH2CO, CH2γ), 2.99-2.93 (m, 1H,
CH2S), 2.78-2.70 (m, 1H, CH2S), 2.18-1.82 (m, 4H, CH2â),
1.05 (s, 9H, (CH3)3C). 13C NMR (Cl-) (75 MHz, CDCl3): δ 170.7
(CO), 151.2 (C-guan), 135.6, 135.5, 132.6, 129.9, 127.9 (C-
PhSi), 65.2 (CH2O), 52.5 (OCH3), 49.1, 48.0 (CHR), 45.2, 44.6
(CH2γ), 37.0 (CH2S), 34.3 (SCH2CO), 26.8 [(CH3)3C], 24.8, 22.7
(CH2â), 19.2 [(CH3)3C]. FAB/LSIMS m/z: 526.3 [(M + H)+,
100%].
Ack n ow led gm en t. This research was supported by
a BIOMED 2 Networ (Grant BMH4-CT96-0823). We are
grateful to J . Dumond and F. Ainoun for their help in
some kinetic experiments.
(12) For selected examples, see the following. (a) Echavarren, A. M.;
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P.; Salvatella, X.; Sa´nchez-Quesada, J .; de Mendoza, J .; Giralt,
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Guanidinium Inhibitors of HIV-1 Protease
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