Stereocontrolled synthesis and biological activity of two diastereoisomers of the potent HIV-1 protease inhibitor saquinavir

Article abstract of DOI:10.1016/j.bmc.2007.10.020

A general enantioselective synthesis of new syn-hydroxyethylamine isosteres has been developed. The approach, based on the controlled opening of functionalized optically active 2,3-epoxy amines, can be conveniently used for the preparation of new peptidom

Full text of DOI:10.1016/j.bmc.2007.10.020

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 902–908
Stereocontrolled synthesis and biological activity
of two diastereoisomers of the potent HIV-1
protease inhibitor saquinavir
a
b,
c
Giuliana Righi,a,* Simona Ciambrone, Carlo Bonini and Pietro Campaner
*
a
`
Istituto di Chimica Biomolecolare-Sezione di Roma, c/o Dipartimento di Chimica, Universita ‘La Sapienza’,
p.le A. Moro 5, 00185 Roma, Italy
b
`
Dipartimento di Chimica, Universita della Basilicata, via N. Sauro 85, 85100 Potenza, Italy
Dipartimento di Scienze Chimiche, Universita di Trieste, via Giorgieri 1, 34127 Trieste, Italy
c
`
Received 23 April 2007; revised 25 September 2007; accepted 9 October 2007
Available online 12 October 2007
Abstract—A general enantioselective synthesis of new syn-hydroxyethylamine isosteres has been developed. The approach, based on
the controlled opening of functionalized optically active 2,3-epoxy amines, can be conveniently used for the preparation of new pep-
tidomimetics with various residues. Finally the total synthesis of two diastereoisomer analogues of HIV-Protease inhibitor Saquin-
avir has been achieved and their biological activity evaluated.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
of HIV-PR, which could overcome the virus resistant
mutagenesis.3,4
The human immunodeficiency virus (HIV), isolated for
the first time in 1983 at the Institute Pasteur in France
and a year later in USA, is the causative agent of the ac-
quired immunodeficiency syndrome (AIDS). The phar-
maceutical industry and a great number of researchers
all over the world are in continuous search for new and
more potent drugs to suppress or slow down the lethal
infection.1 Essential to replication, maturation, and
infectivity of HIV are three viral enzymes: reverse trans-
criptase (RT), integrase (IN) and protease (PR). Inhibi-
tion of each of them can be used against HIV infection
through an antiretroviral therapy.2 Actually, synergy of
the two RT and PR inhibitors represents the most effica-
cious therapy for the treatment of this disease, called
Highly Active Antiretroviral Therapy (HAART).
As well as for other aspartyl proteases, the guideline
adopted to design drugs to block the HIV-PR has fo-
cused on synthetic peptidic analogues which mimic the
transition state (TS) of the catalytic reaction.5,6
Possible TS mimetics for inclusion in new potential
inhibitors0 may be all non-hydrolyzable central cores
wðP1 ꢀ P1 Þ depicted in Figure 1, where it is always rec-
ognizable vicinal amino alcoholic sequence.
The first HIV-PR inhibitor to be marketed in 1995 was
Saquinavir,7 (Ro 31-8959), which showed excellent
activity against HIV-PR with IC50 value of 0.4 nM,
and synergy with other HIV agents, such as RT inhibi-
tors. However the insurgence of resistant viruses to
Saquinavir as well as to other drug,8 led to the need of
developing new HIV-PR inhibitors, both peptido and
non-peptidomimetics.9
In spite of the good results so far obtained, the wide-
spread diffusion of the disease and the development of
numerous mutant resistant viruses to the used therapy
justify the research towards new and selective inhibitors
R
O
R
R'
R
OH O
N
H
N
H
N
H
N
H
OH R'
OH
O
OH R'
Keywords: Peptidomimetic; HIV-1 proteasi inhibitor; Saquinavir; Syn
hydroxyethylamine isoster; Epoxy amine.
hydroxyethylamine
dihydroxyethylene
hydroxyethylene
*
Corresponding authors. Tel.: +39 6490422; fax: +39 649913628
(G.R.); e-mail: giuliana.righi@uniroma1.it
Figure 1. TS mimetics for HIV-PR.
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.020

G. Righi et al. / Bioorg. Med. Chem. 16 (2008) 902–908
903
X-ray structures of HIV-PR co-crystallized with Saquin-
avir demonstrated that the hydroxy group of the drug is
located between the catalytic aspartic acids and that the
R configuration at the hydroxyl-bearing carbon atom is
preferred.10 This hydroxyl group is in an antirelation-
ship (see Fig. 2) with the vicinal amino group. The
[3S-(4aS, 8aS)]-N-(tert-butyl)-decahydro-3-isoquinolin-
ca0rboxy amide (DIQ) occupies almost the entire subsite
S10, while the tert-butyl amide group tightly fits into the
S2 subsite. Finally, the S,S,S stereochemistry of the
DIQ ring is optimal for good inhibitory activity.11
different side chains or modifying the stereochemistry
at the CHOH of the core,12 and several efforts have
been made to synthesize HEA in a stereocontrolled
manner with the correct relative and absolute
stereochemistry.
Considering our experience in the stereo- and regiocon-
trolled preparation of such backbones via epoxides and
aziridines chemistry, 13 we focused our attention on pep-
tidomimetics synthesis with a hydroxyethylamine isoster
core (syn-HEA) (see Fig. 3). In this paper, we report a
highly stereocontrolled synthesis of two new Saquinavir
analogues, with modification in the relative and absolute
configuration at the amino alcohol core. Preliminary
biological activity as HIV-PR potential inhibitors has
been also evaluated (see Fig. 4).
The potency of Saquinavir prompted further design
and synthesis of its analogues as candidates to HIV-
PR inhibition. Numerous derivatives have been syn-
thesized making structural changes, like incorporating
Ph
H
O
H
N
H
anti hydroxyethylamine isostere
N
N
N
O
H
O
OH
NH2
NHtBu
O
Saquinavir
Figure 2. Saquinavir: A peptididomimetic with an antiHEA core.
P2'-P3'
P2'-P3'
P3-P2
P3-P2
Ph
H
Ph
H
O
H
O
H
R
S
R
N
H
N
H
S
N
N
N
O
N
N
N
O
H
H
O
OH
O
OH
NH2
NH2
NHtBu
NHtBu
1
2
O
O
P1-P1'
P1-P1'
O
HN
Ph
N
OH
syn HEA
Figure 3. Epimers of Saquinavir and syn-HEA.
Figure 4. Variation of fluorescence at different concentration of substrate.

904
G. Righi et al. / Bioorg. Med. Chem. 16 (2008) 902–908
2. Chemistry
H
H
N
Br
O
Our general synthetic approach, which will be described
initially for known compound 114 (see retrosynthetic
Scheme 1), relays on the stereo- and regioselective open-
ing of the properly prepared epoxy amine 6, having the
(S,S,S)-decahydroisoquinoline-3-carboxylamide residue
(DIQ) already introduced. The key epoxy amine 6 in
turn can be easily obtained from the known (2R,3R)-
epoxy alcohols 4.
a
Ph
H
Ph
H
N
O
OH
7
6
NH
t
Bu
NHtBu
O
b
H
H
N
N3
NH2
2.1. Synthesis of epoxy amine 6
c
Ph
H
Ph
H
R
R
N
O
The enantiomerically pure (2R,3R)-4-phenyl-2, 3-epoxy-
butanol 4 was promptly prepared by known procedure,
starting from alcohol 3.15 Then the DIQ residue was
readily introduced via mesylate derivative 5, affording
the epoxyamine 6 with an overall yield of 77.4% from
3 (Scheme 2).
OH
OH
8
9
NHtBu
NHtBu
O
Scheme 3. Reagents and conditions: (a) LiBr/Amberlyst 15, CH3CN,
ꢀ20 °C, 81%; (b) NaN3, 18-crown-6, DMSO/THF 1/1, rt, 72%; (c) H2,
Pd/C, MeOH, rt, 86%.
2.2. Synthesis of the syn HEA isoster 9
hol 8 was obtained as single diastereoisomer in satisfac-
tory chemical yield (72%).
The syn-relationship of the amino alcohol fragment was
introduced following an already reported stereocon-
trolled opening of epoxide by halide and subsequent
substitution of the halogen by azide.16
The subsequent catalytic hydrogenation afforded the
syn(R,R)-hydroxyethylene isostere 9, as epimer of the
anti(S,R) Saquinavir core (Scheme 3).
The regio- and stereoselective opening of the epoxy
amine 6 was performed utilizing the LiBr/Amberlyst
15 reagent;17,18 the bromo derivative 7 was thus ob-
tained with good chemical yield and at satisfactory reg-
ioisomeric ratio (92:8), as determined by spin–spin
decoupling experiments on the acetylate derivative.
2.3. Synthesis of saquinavir analogue 1
Finally, the coupling between the known dipeptide
Quin-Asn-OH of the P3–P2 portion of Saquinavir and
the HEA isostere 9 afforded the target Saquinavir ana-
logue 1 characterized by the novel syn-HEA isostere
(Scheme 4).
The following displacement of bromine by azide ion was
not a trivial one, because of unwanted elimination reac-
tions which lowered the overall yield. After several at-
tempts in various reaction conditions (solvent,
temperature, azide reagent), by employing NaN3 in
DMSO/THF = 1/1 as solvent, in the presence of 18-
crown-6 at room temperature,19 the desired azido alco-
2.4. Synthesis of saquinavir analogue 2
The same synthetic sequence (starting from ent-4,
Scheme 5) was then applied to the preparation of the
new Saquinavir analogue 2, with the S configuration
NH2
O
H
H
H
Ph
Ph
O
N
N
Ph
1
OH
OH
O
4
O
H
HN
HN
6
Scheme 1. Retrosynthetic pathway.
H
N
O
O
O
O
Ph
H
b
a
c
Ph
Ph
Ph
OH
OH
OMs
3
4
5
6
NHtBu
Scheme 2. Reagents and conditions: (a) TBHP, (ꢀ)DET, Ti(i-OPr)4, CH2Cl2, ꢀ20 °C, 88%; (b) MsCl, Et3N, DMAP, CH2Cl2, rt, 90%; (c) DIQ,
DMSO, 45 °C, rt, 86%.

G. Righi et al. / Bioorg. Med. Chem. 16 (2008) 902–908
905
Ph
O
H
H
N
a
R
R
9
+
Quin-Asn-OH
H
N
N
H
N
O
O
OH
NH2
NHtBu
1
O
Scheme 4. Reagents and condition: (a) EDC, HOBt, Et3N, THF, rt, 72%.
H
H
N
Br
O
Ph
H
O
a
Ph
H
N
O
Ph
3
OH
10
OH
11
ent-4
NHtBu
NH
t
Bu
O
Ph
H
H
N
H
N
O
O
N3
NH2
H
N
S
S
H
Ph
H
Ph
H
S
S
N
N
N
O
H
O
OH
OH
12
OH
13
NH2
NHtBu
NHtBu
2
NHtBu
O
O
Scheme 5. Reagents and conditions: (a) TBHP, (+)DET, Ti(i-OPr)4, CH2Cl2, ꢀ20 °C, 88%.
at the CH(OH) in the syn HEA core. The reaction yields
of the single steps were found to be identical to the pre-
vious preparation of compound 1 (see Schemes 2–4),
and the spectroscopical data are reported in Section 4.
Both 1 and 2 show a clear activity against HIV-PR, with
IC50 values in the nanomolar range (483 and 290 nM,
respectively), but they are less active than Saquinavir
which shows a lower IC50 (Table 1).
The synthesis of the target compounds 1 and 2 was thus
performed in total stereo- and regiocontrolled fashion,
with satisfactory overall yield and total control of stere-
oselectivity with respect to previous preparation (ꢁ 20%,
nine steps from the commercially available
phenylacetaldehyde).20
3. Conclusion
In conclusion we developed a general strategy for the
synthesis of peptidomimetics with syn-hydroxyethylene
isosteres, where the key step was the regio- and stereo-
controlled opening of an optically active 2,3-epoxy
amine. A distinct feature of this approach is the possibil-
ity of 0 introducing different side chains as P2–Pn and
2.4.1. Biological activity. For both 1 and 2 the extent of
inhibition of the activity of the HIV-1 aspartic protease
was determined as previously reported for other series of
mono- and dihydroxy pseudopeptide inhibitors21
through ‘intramolecular fluorescence resonance energy
transfer’.22 IC50 values were evaluated on the inhibition
of a recombinant wild-type HIV-PR (purchased from
Bioczech, Prague, Czech Republic) by measuring the ini-
tial velocities of hydrolysis of the fluorogenic hexapepti-
dic substrate Abz-Thr-Ile-Nle-Phe(p-NO2)-Gln-Arg-
NH2 in absence and with variable concentrations of
the two epimers of Saquinavir.
0
P2 –Pn residues. The final Saquinavir analogues, ob-
tained in overall good yield and total stereocontrol, were
also evaluated as HIV-PR inhibitors, showing a modest
activity compared to the original Saquinavir.
4. Experimental
1H and 13C NMR spectra were recorded at 200 and
50.3 MHz, respectively. Reactions were monitored by
TLC using Merck silica gel 60 F-254 plates with UV
indicator; spots were also visualized with phosphomo-
lybdic acid (10% solution in EtOH). Flash column chro-
matography on silica gel was normally used for
purification of the reaction mixtures. ESI–MS analyses
were performed using a commercial API 365 triple-
quadrupole mass spectrometer from Perkin Elmer Sciex
Instruments, equipped with an ESI source and a syringe
Table 1. IC50 values
Compound
IC50 (nM)
Saquinavir
0.4
1
2
483
290

906
G. Righi et al. / Bioorg. Med. Chem. 16 (2008) 902–908
pump. The experiments were conducted in the positive
ion mode. Optical rotations were recorded at the So-
dium D line with a polarimeter at room temperature.
ture was purified by column chromatography (petro-
leum ether/AcOEt 8:2) affording 7 as a soft white
20
D
powder, yield 81%. ½aꢂ ꢀ64.0 (c 2.3, CHCl3), 1H
NMR (200 MHz, CDCl3): d 7.37–7.15 (m, 5H, Ph);
6.10 (bs, 1H, NH); 4.21 (ddd, 1H, J = 2.9, 6.6, 9.5 Hz
CHBr); 3.85 (bq, 1H, J = 6.6 Hz, CHOH); 3.48 (dd,
1H, J = 2.9, 14.6 Hz, CHAPh); 3.00 (dd, 1H, J = 9.5,
14.6 Hz, CHBPh); 2.86 (dd, 1H, J = 4.4, 13.9 Hz,
CHAN); 2.73 (dd, 1H, J = 2.9, 10.2 Hz, CHNDIQ); 2.50
(dd, 1H, J = 6.6, 13.9 Hz, CHBN); 2.4 (dd, 1H,
J = 2.9, 11.7 Hz, CHANDIQ); 2.08–1.22 (m, 14H, DI-
Q + OH); 1.32 (s, 9H, tert-But).13C NMR (50 MHz,
CDCl3): d 173.2, 138.2, 129.2, 128.05, 126.4, 72.2,
69.9, 60.6, 60.3, 59.03, 50.9, 39.9, 35.9, 33.1, 30.7, 30.4,
28.5, 25.9, 25.6, 20.5. HR-MS (ES Q-TOF) Calcd for
C24H38BrN2O2 [M + H]+: 465.2116. Found: 465.2121.
Compounds 3,23 4, and ent-424 are known. Quin-Asn-
OH ((Quinolin-2-ylcarbonyl)-L-asparagine) has been
prepared in according to Ref. 20c.
4.1. (20R,30R)-Methanesulfonic acid 2-[20,30-epoxy-40-
phenyl-butyl]-ester 5
To a solution of 4 (1 mmol) in 1.6 mL of CH2Cl2,
2 mmol of Et3N (0.27 mL) and a catalytic amount of
DMAP were added. After being stirred at 0 °C,
0.08 mL (1 mmol) of methane sulfonyl chloride was
added dropwise. After 2 h (TLC monitoring), the reac-
tion was quenched with ice cold water, the organic lay-
ers were extracted with CH2Cl2, washed with ice cold
HCl 1N, saturated aqueous NaHCO3, and then brine.
Organic extracts were dried over Na2SO4 and then evap-
orated to dryness in vacuo to give 5, which was used
without any purification (90%). 1H NMR (200 MHz,
CDCl3): d 7.36–7.14 (m, 5H, Ph); 4.44 (dd, 1H,
J = 2.9, 12.4 Hz, CHAOMs); 4.08 (dd, 1H, J = 6.2,
12.4 Hz, CHBOMs); 3.18–3.04 (m, 2H, CHOCH); 3.01
(s, 3H, CH3); 2.89 (d, 2H, J = 5.1 Hz, CH2Ph). 13C
NMR (50 MHz, CDCl3): d 136.7, 128.8, 128.4, 126.8,
69.5, 56.4, 54.5, 37.5, 37.3. HR-MS (ES Q-TOF) Calcd
for C11H15O4S [M + H]+: 243.0691. Found: 243.0697.
4.4. (ꢀ)-(3S, 4aS, 8aS, 20R, 30R)-2-[30-Azido-20-hydroxy-
40-phenyl-butyl]-N-tert-butyldecahydro-isoquinoline-3-
carboxamide 8
To a solution of 7 (1 mmol) in the mixed solvent
DMSO/THF = 1/1 (4 mL), NaN3 260 mg (4 mmol) and
18-crown-6 (264 mg, 1 mmol) were added, and the mix-
ture was stirred at room temperature for 48 h. Then the
reaction was diluted with EtOAc (8 mL), washed several
times with water and brine. The combined organic ex-
tracts were dried over Na2SO4 and concentrated in va-
cuo. The crude mixture was purified by flash
chromatography (petroleum ether/EtOAc, 7:3) to afford
20
D
4.2. (ꢀ)-(3S, 4aS, 8aS, 20R, 30R)-2-[20,30-Epoxy-40-
phenyl-butyl]-N-tert-butyldecahydro-isoquinoline-3-
carboxamide 6
8 yield 72%. ½aꢂ 121.3 (c 1.1, CHCl3), 1H NMR
(200 MHz, CDCl3): d 7.40–7.16 (m, 5H, Ph); 6.15 (s,
1H, NH); 3.71 (bt, 1H, J = 7.3 Hz, CHOH); 3.47 (bt,
1H, J = 7.3 Hz, CHN3); 3.11 (bd, 2H, J = 7.3, CH2Ph);
2.62 (dd, 1H, J = 8.0; 12.4 Hz, CHAN); 2.58–2.39 (m,
2H, CHNDIQ + CHANDIQ); 2.19 (dd, 1H, J = 6.6,
12.4 Hz, CHBN); 2.11–1.99 (m, 1H, CHBNDIQ); 1.81–
1.08 (m, 13H, DIQ + OH); 1.32 (s, 9H, tert-But). 13C
NMR (50 MHz, CDCl3): d 173, 4; 137, 3; 129, 1; 128,
6; 126, 7; 70, 6; 68, 7; 62, 9; 58, 8; 58, 6; 50, 7; 36, 1;
35, 5; 33, 1; 30, 8; 30, 5; 28, 5; 26, 0; 25, 7; 20, 4. HR-
MS (ES Q-TOF) Calcd for C24H38N5O2 [M + H]+:
428.3025. Found: 428.3031.
To a solution of 5 (1 mmol) in 1 mL di DMSO, 2 mmol
of DIQ was added. The reaction mixture was stirred at
50 °C overnight. Then the reaction was diluited with
EtOAc (8 mL), washed several times with water and
brine, dried over Na2SO4 and concentrated in vacuo.
The crude mixture was purified by flash chromatogra-
phy (petroleum ether/AcOEt 6:4) affording 6 as a light
20
1
brown oil in 86% yield; ½aꢂ ꢀ103.9 (c 1.7, CHCl3); H
D
NMR (200 MHz, CDCl3): d 7.38–7.13 (m, 5H, Ph);
6.50 (bs, 1H, NH); 3.11–2.6 (m, 5H, CH2Ph + CH
N
DIQ + CHAOCHB); 2.58–2.48 (m, 2H, CH2NDIQ);
4.5. (ꢀ)-(3S, 4aS, 8aS, 20R, 30R)-2-[30-Ammino-20-
hydroxy-40-phenyl-butyl]-N-tert-butyldecahydro-iso-
quinoline-3-carboxamide 9
2.18–1.99 (m, 2H, CH2N); 1.78–1.02 (m, 12H, DIQ);
1.33 (s, 9H, tert-But). 13C NMR (50 MHz, CDCl3): d
173.6, 137.1, 128.7, 128.5, 126.5, 69.4, 59.6, 56.8, 56.2,
55.7, 50.3, 38.2, 35.8, 33.0, 30.8, 30.6, 28.5, 26.3, 25.6,
20.3. HR-MS (ES Q-TOF) Calcd for C24H37N2O2
[M + H]+: 385.2855. Found: 385.2861.
A solution of 8 (1 mmol) in MeOH (2 mL) was hydroge-
nated at atmospheric pressure over 10% Pd/C (51 mg)
for 3 h at room temperature; then the solution was fil-
tered on a Celite pad and concentrated in vacuo. Flash
4.3. (ꢀ)-(3S, 4aS, 8aS, 20R, 30S)-2-[30-Bromo-20-hydro-
xy-40-phenyl-butyl]-N-tert-butyldecahydro-isoquinoline-
3-carboxamide 7
chromatography (chloroform/methanol, 9:1) furnished
20
D
9 yield 86%. ½aꢂ ꢀ50.9 (c 1.0, CHCl3), 1H NMR
(200 MHz, CD3OD): d 7.9 (s, 1H, NH); 7.42–7.20 (m,
5H, Ph); 3.82 (dt, 1H, J = 8.0, 1.5, CHOH); 3.65 (dt,
1H, J = 8.8, 1.5 Hz, CHNH2); 3.11 (dd, 1H, J = 13.2,
8.8 Hz, CHAPh); 2.96 (dd, 1H, J = 13.2, 5.9 Hz,
CHBPh); 2.74 (dd, 1H, J = 10.2, 2.9 Hz, CHAN); 2.37–
2.22 (m, 3H, CHBN + CHNDIQ + CHANDIQ); 2.19-
2.07 (m, 2H, CHBNDIQ + NH); 2.0–1.09 (m, 13H,
DIQ + NH); 1.34 (s, 9H, tert-But). 13C NMR
(50 MHz, CDCl3): d 174, 6; 136, 7; 129, 4; 129, 0; 127,
To a stirred solution of 6 (1 mmol) in CH3CN (10 mL)
at ꢀ20 °C, LiBr (348 mg, 4 mmol) and Amberlyst15
(217 mg, 1 mmol) were added. The mixture was stirred
for 6 h (TLC monitoring) and filtered. The filtrate solu-
tion, diluted with EtOAc, was washed with saturated
aqueous NaHCO3 and brine; the organic layer, dried
over Na2SO4 was concentrated in vacuo. The crude mix-

G. Righi et al. / Bioorg. Med. Chem. 16 (2008) 902–908
907
1; 69, 3; 66, 2; 58, 2; 56, 8; 55, 8; 52, 1; 33, 5; 35, 6; 35, 3;
29, 6; 29, 2; 28, 7; 26, 2; 25, 6; 20, 1. HR-MS (ES Q-
TOF) Calcd for C24H40N3O2 [M + H]+: 402.3120.
Found: 402.3126.
CHAPh); 3.04 (dd, 1H, J = 8.8, 14.6 Hz, CHBPh);
2.89–2.52 (m, 3H, CH2NDIQ + CHNDIQ); 2.47 (dd,
1H, J = 2.9, 12.4 Hz, CHAN); 2.27 (dd, 1H, J = 2.9,
11.7 Hz, CHBN); 1.99–1.12 (m, 12H, DIQ); 1.37 (s,
9H, t-Bu).13C NMR (50 MHz, CDCl3): d 173.4, 138.0,
129.1, 128.1, 126.5, 69.8, 69.1, 59.9, 58.9, 58.3, 50.9,
40.4, 35.7, 33.2, 30.9, 30.7, 28.6, 26.2, 25.3, 20.1. HR-
MS (ES Q-TOF) Calcd for C24H38BrN2O2 [M + H]+:
465.2116. Found: 465.2119.
4.6. (ꢀ)-(3S, 4aS, 8aS, 20R, 30R)-2-[20-Hydroxy-40-
phenyl-30[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]-
butyl]-N-tert-butyldecahydro-isoquinoline-3-carboxamide 1
To
a
solution of 1 mmol Quin-Asn-OH, EDC
(1.1 mmol, 210 mg) and HOBt (1.1 mmol, 148 mg) in
THF (15 mL), Et3N was (2 mmol, 0.27 mL) was added
at 0 °C. After 30 min 9 (1 mmol) was added and the mix-
ture was allowed to stir at room temperature overnight.
Then the mixture was filtered and evaporated under re-
duced pressure. The residue was diluted with EtOAc and
was washed with 10 mL of 10% aqueous citric acid,
10 mL of saturated NaHCO3and brine. The organic
layer was dried over Na2SO4 and concentrated in vacuo.
The crude mixture was purified by flash chromatogra-
4.9. (ꢀ)-(3S, 4aS, 8aS, 20S, 30S)-2-[30-Azido-20-hydroxy-
40-phenyl-butyl]-N-tert-butyldecahydro-isoquinoline-3-
carboxamide 12
20
D
½aꢂ ꢀ93.6 (c 1.1, CHCl3). 1H NMR (200 MHz, CDCl3):
d 7.47–7.16 (m, 5H, Ph); 6.01 (s, 1H, NH); 3.81 (ddd,
1H, J = 2.9, 5.9, 10.2 Hz, CHOH); 3.41 (bs, 1H, OH);
3.26–3.13 (m, 1H, CHN3); 3.12–2.98 (m, 2H, CH2Ph);
2.93–2.62 (m, 3H, CH2NDIQ + CHNDIQ); 2.26–2.11
(dd, 1H, J = 2.9, 11.7 Hz, CHAN); 2.09–1.91 (m, 1H,
CHBN); 1.94–1.18 (m, 12H, DIQ); 1.32 (s, 9H, tert-
Bu). 13C NMR (50 MHz, CDCl3): d 173, 0; 137, 6;
129, 1; 128, 7; 126, 8; 70, 2; 68, 3; 65.1; 58, 5; 58, 1;
50, 9; 36, 7; 35, 8; 33, 3; 30, 8; 30, 5; 28, 6; 26, 3; 25,
6; 20, 3. HR-MS (ES Q-TOF) Calcd for C24H38N5O2
[M + H]+: 428.3025. Found: 428.3029.
phy (CHCl3/MeOH, 95:5) to afford 1 as a brown oil.
20
D
½aꢂ ꢀ10.1 (c 0.7, CHCl3), 1H NMR (200 MHz, CDCl3):
d 8.42–8.08 (m, 3H, Quin); 7.89 (d, 1H, J = 8.0 Hz,
Quin); 7.85–7.71 (m, 1H, Quin); 7.66 (d, 1H,
J = 8.0 Hz, Quin); 7.45–7.02 (m, 5H, Ph); 6.15 (bs, 2H,
NH2Asn; 5.87 (bs, 1H, NHQuin-Asn); 5.56 (bs, 1H,
NHDIQ); 5.17 (m, 2H, CHAsn + NH); 4.13–3.86 (m,
1H, CHOH); 3.77–3.70 (m, 1H, CHCH2Ph); 3.11–2.55
(m, 5H, CH2Ph þ CHNDIQ þ CH2Asn); 2.49–2.11 (m,
4H, CH2N + CH2NDIQ); 1.92–0.80 (m, 13H, DI-
Q + OH); 1.14 (s, 9H, tert-But ). 13C NMR (50 MHz,
CDCl3): d174.8; 173.1; 170.5; 164.8; 152.2; 149.0;
137.4; 137.3; 130.5; 130.1; 129.4; 128.5; 128.1; 127.6;
126.3; 125.7; 118.7; 69.4; 67.2; 58.9; 60.6; 53.1; 50.7;
50.0; 36.9; 35.5; 32.9; 31.8; 30.3; 29.6; 28.4; 25.8;
25.6; 20.8. HR-MS (ES Q-TOF) Calcd for
C38H51N6O5 [M + H]+: 671.3921. Found: 671.3896.
Calcd for C38H50N6O5Na [M + Na]+: 693.3740. Found:
693.3736.
4.10. (ꢀ)-(3S, 4aS, 8aS, 20R, 30R)-2-[30-Ammino-20-
hydroxy-40-phenyl-butyl]-N-tert-butyldecahydro-iso-
quinoline-3-carboxamide 13
20
D
½aꢂ ꢀ61.3 (c 0.63, CHCl3), 1H NMR (200 MHz,
CDCl3): d: 7.34–7.19 (m, 5H, Ph); 6.06–5.50 (bs, 2H,
OH + NHDIQ); 3.97–3.82 (m, 1H, CHOH); 3.69–3.51
(m, 1H, CHNH2); 3.49–3.21 (m, 3H, CH2Ph + NH);
3.21–2.85 (m, 3H, NH + CHANDIQ + CHNDIQ); 2.84–
2.66 (m, 1H, CHAN); 2.66–2.49 (m, 1H, CHBNDIQ);
2.21–1.12 (m, 13H, DIQ + CHBN); 1.34 (s, 9H, t-Bu).
13C NMR (50 MHz, CDCl3): d 172.4; 137.5; 129, 3;
128, 6; 126, 6; 69, 0; 67, 0; 60.7; 59, 6; 56, 6; 51, 1;
39.1; 35, 6; 32.5; 30.6; 30.0; 28, 5; 26, 2; 25, 2; 20, 1.
HR-MS (ES Q-TOF) Calcd for C24H40N3O2
[M + H]+: 402.3120. Found: 402.3128.
4.7. (ꢀ)-(3S, 4aS, 8aS, 20S, 30S)-2-[20, 30-Epoxy-40-
phenyl-butyl]-N-tert-butyldecahydro-isoquinoline-3-
carboxamide 10
20
D
½aꢂ ꢀ91.5 (c 0.86, CHCl3); 1H NMR (200 MHz,
4.11. (ꢀ)-(3S, 4aS, 8aS, 20S, 30S)-2-[20-Hydroxy-40-
phenyl-30[N-(2-quinolylcarbonyl)-L-asparaginyl]ammi-
no]butyl]-N-tert-butyldecahydro-isoquinoline-3-carbox-
amide 2
CDCl3): d 7.41–7.22 (m, 5H, Ph); 6.60 (bs, 1H, NH);
3.03–2.74 (m, 5H, CH2Ph + CHNDIQ + CHAOCHB);
2.66–2.50 (m, 2H, CH2NDIQ); 2.34–2.16 (m, 2H,
CH2N); 1.89–1.18 (m, 12H, DIQ); 1.39 (s, 9H, tert-
But). 13C NMR (50 MHz, CDCl3): d 173.2, 136.7, 128.
8, 128.5, 126.6, 69.2, 59.61, 58.6, 58.4, 58.1, 55.6, 50.3,
38.20, 35.92, 33.01, 30.71, 28.52, 26.25, 25.49, 20.21.
HR-MS (ES Q-TOF) Calcd for C24H37N2O2
[M + H]+: 385.2855. Found: 385.2859.
20
D
½aꢂ ꢀ55.9 (c 1.0, MeOH). 1H NMR (200 MHz,
CDCl3): d 8.37–8.05 (m, 3H); 7.93–7.53 (m, 3H); 7.34–
6.89 (m, 5H); 6.72 (bs, 2H); 6.44 (bs, 1H); 5.85 (bs,
1H, NHDIQ); 5.05–4.86 (m, 1H, CHAsn); 4.07–3.88 (m,
1H, CHOH); 3.87–3.70 (bd, 1H, J = 9.5 Hz,
CHNCH2Ph); 2.99–2.43 (m, 7H); 2.20–1.94 (m, 2H);
1.87–1.04 (m, 12H); 1.36 (s, 9H). 13C NMR (50 MHz,
CDCl3): d 174, 0; 172, 9; 171, 0; 164, 7; 149, 8; 148, 8;
137, 9; 137, 3; 130, 1; 130, 0; 129, 3; 129, 1; 128, 2;
128, 0; 127, 5; 126, 1; 118, 6; 69, 5; 68, 1; 59, 8; 58,
1; 54, 2; 50, 9; 50, 1; 37, 8; 37, 0; 35, 7; 33, 1; 30, 9;
30, 7; 28, 6; 26, 1; 25, 4; 20, 2. HR-MS (ES Q-TOF)
Calcd for C38H51N6O5 [M + H+]: 671.3921. Found:
671.3925.
4.8. (ꢀ)-(3S, 4aS, 8aS, 20S, 30R)-2-[30-Bromo-20-hydro-
xy-40-phenyl-butyl]-N-tert-butyldecahydro-isoquinoline-
3-carboxamide 11
20
D
½aꢂ ꢀ99.07 (c 0.94, CHCl3), 1H NMR (200 MHz,
CDCl3): d 7.45–7.15 (m, 5H, Ph); 6.11 (bs, 1H, NH);
4.18 (ddd, 1H, J = 8.8, 5.1, 4.4 Hz CHBr); 3.91–3.72
(m, 2H, CHOH + OH); 3.35 (dd, 1H, J = 4.4, 14.6 Hz,

908
G. Righi et al. / Bioorg. Med. Chem. 16 (2008) 902–908
12. Roberts, N. A. et al. Science 1990, 248, 358.
Acknowledgment
13. (a) Righi, G.; Chionne, A.; Bonini, C. Eur. J. Org. Chem.
2000, 3127; (b) Righi, G.; Ronconi, S.; Bonini, C. Eur. J.
Org. Chem. 2002, 1573; Review (c) Righi, G.; Bonini, C.
Tetrahedron 2002, 58, 4981.
14. Pesenti, C.; Arnone, A.; Arosio, P.; Frigerio, M.; Meille,
S. V.; Panzeri, W.; Viani, F.; Zanda, M. Tetrahedron Lett.
2004, 45, 5125.
We thank MIUR (Ministry of University and Re-
search— Rome) for partial financial support (PRIN
2005: Sintesi stereoselettiva e valutazione biologica di
`
composti mirati all’attivita antivirale).
15. Righi, G.; D’Achille, C.; Pescatore, G.; Bonini, C.
Tetrahedron Lett. 2003, 44, 6999.
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