Aza-peptide analogs as potent human immunodeficiency virus type-1 protease inhibitors with oral bioavailability

Article abstract of DOI:10.1021/jm960022p

A series of aza-peptide analogs with a (hydroxyethyl)hydrazine isostere has been synthesized as HIV-1 protease inhibitors using a simple synthetic scheme. Structure-activity studies based on the X-ray of a previously described inhibitor-enzyme complex led to potent inhibitors with antiviral activity in the low-nanomolar range. The S-configuration of the transition- state hydroxyl group was preferred in this series. Small modifications of the P₂P₃ and P₂'P₃' substituents had little effect on enzyme inhibition but greatly influenced the pharmacokinetic profile. As a result of these studies, the symmetrically acylated compound 8a and its close analog 24a bearing a methyl carbamate in P₃ and an ethyl carbamate in P₃' position were identified as potent inhibitors with plasma concentrations exceeding antiviral ED₅₀ values 150-fold following oral application in mice.

Full text of DOI:10.1021/jm960022p

J . Med. Chem. 1996, 39, 3203-3216
3203
Aza -P ep tid e An a logs a s P oten t Hu m a n Im m u n od eficien cy Vir u s Typ e-1
P r otea se In h ibitor s w ith Or a l Bioa va ila bility
Alexander Fa¨ssler,* Guido Bold, Hans-Georg Capraro, Robert Cozens, J u¨rgen Mestan, Bernard Poncioni,
J ohannes Ro¨sel, Marina Tintelnot-Blomley, and Marc Lang
Research Laboratories Cancer and Infectious Diseases, Pharmaceutical Division, Ciba-Geigy AG, CH-4002 Basel, Switzerland
Received J anuary 9, 1996X
A series of aza-peptide analogs with a (hydroxyethyl)hydrazine isostere has been synthesized
as HIV-1 protease inhibitors using a simple synthetic scheme. Structure-activity studies based
on the X-ray of a previously described inhibitor-enzyme complex led to potent inhibitors with
antiviral activity in the low-nanomolar range. The S-configuration of the transition-state
hydroxyl group was preferred in this series. Small modifications of the P2P3 and P2′P3′
substituents had little effect on enzyme inhibition but greatly influenced the pharmacokinetic
profile. As a result of these studies, the symmetrically acylated compound 8a and its close
analog 24a bearing a methyl carbamate in P3 and an ethyl carbamate in P3′ position were
identified as potent inhibitors with plasma concentrations exceeding antiviral ED50 values 150-
fold following oral application in mice.
In tr od u ction
It is widely accepted that pathogenesis of AIDS is
mediated by the human immunodeficiency virus through
a progressive impairment of the immune system. The
different therapeutic strategies for intervention in this
disease have been reviewed recently.1 A critical role in
the viral replication is played by the HIV protease, an
enzyme responsible for the processing of the polypro-
teins to structural proteins and to enzymes essential for
viral replication.2 This pivotal role makes it a promising
target for chemotherapy of AIDS. Since the initial
characterization of this enzyme, rapid progress has been
achieved toward the development of potent and selective
inhibitors.3 Yet it remains a challenge to combine both
antiviral potency and favorable pharmacokinetic prop-
erties, thereby fulfilling one prerequisite for effective
F igu r e 1. Comparison of transition-state dipeptide iso-
steres: pseudosymmetric (A) incorporated into inhibitor CGP
53820, substrate-based (B), and C2-symmetric (C).
antiviral treatment.
The particular C2-symmetry of HIV-1 protease, which
functions as a dimer with each subunit contributing an
amino acid triad Asp-Thr-Gly to the active site,4 stimu-
which is considered a major advantage for any future
lated the design of symmetry-based inhibitors. On the
therapeutic agent in this field. This concept led to
basis of the hydroxyethylene core as a tetrahedral
potent and selective enzyme inhibitors which have been
transition-state replacement for the peptide substrate,
described previously.11 To provide further insight into
various potent and selective C2-symmetric inhibitors
the enzyme-inhibitor interactions, one of the most
have been described.5 They exhibit diamino diols6 and
potent representatives of symmetrically acylated aza-
diamino alcohols,7 as well as sulfones8 or phosphinates,9
peptide analogs (CGP 53820, Figure 1) has been co-
as nonscissile dipeptide isostere units.
crystallized with HIV-1 and HIV-2 protease.12 The
following structure-activity discussion is based on the
X-ray structure obtained from HIV-1 protease. Thus,
Our own molecular modeling studies prompted us to
explore a novel pseudosymmetric type of dipeptide
isostere which can be considered as an analog of an aza-
the inhibitor binds in an extended conformation with
peptide10 (formula A in Figure 1). The carbon atom
the hydroxyl group positioned symmetrically between
bearing the P1′ substituent of a hydroxyethylene (for-
the two active site aspartate residues. The phenyl and
mula B in Figure 1) or a dihydroxyethyl amine isostere
cyclohexyl substituents are accommodated in the P1 and
(formula C in Figure 1) is replaced by a nitrogen atom
P1′ pockets of the enzyme. All HN and CdO functional
of a hydrazine group. This functional group eliminates
groups of the backbone are arranged within hydrogen-
one stereogenic center and allows broad variability of
bonding distances of H-bond donors and acceptors of the
the substituents by acylation of the amino and hy-
enzyme and can therefore explain the high enzyme
drazino groups. More important, the synthesis of these
affinity. However, in a cellular assay the antiviral
inhibitors follows a series of simple transformations
activity was only moderate (ED90 ) 1 µM). Further-
more, in a preliminary pharmacokinetic evaluation in
mice, the compound did not reach plasma concentrations
above the detection limit after oral application. Nev-
* Address for correspondence: Ciba-Geigy PLC, Central Research,
Hulley Rd., Macclesfield, Cheshire SK10 2NX, U.K.
X Abstract published in Advance ACS Abstracts, J uly 1, 1996.
S0022-2623(96)00022-2 CCC: $12.00 © 1996 American Chemical Society

3204 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Fa¨ssler et al.
Ta ble 1. Analytical Data for Hydrazones 2 and Hydrazines 3
hydrazone 2
hydrazine 3
no.
P1′ substituent
yield, %
mp, °C
1H-NMR (ppm) P1′-CHdNa no. yield, % prep methodb 1H-NMR (ppm) P1′-CH2-NHa
2a cyclohexyl
2b isopropyl
2c isobutyl
2d p-MeO-phenyl
2e 4-F-phenyl
99
99
81
94
96
97
80
91
98
99
97
134-135 7.1, d, J ) 7
3a
3b
3c
3d
3e
3f
3g
3h
3i
95
89
96
73
94
97
78
57
74
95
40
A
A
A
D
B
B
B
B
B
B
C
2.58, d, J ) 7
2.55, d, J ) 7
2.78, t, J ) 7
3.83, s
92-93
98-99
7.0, d, J ) 7d
7.27, d, J ) 7
137-138 7.86, s
166-167 7.81, sd
158-160 7.88, sd
3.83, s
2f
4-CN-phenyl
4.02, sd
2g 4-OH-phenyl
2h 4-tolyl
178 dec
167
7.82, s
3.77, s
7.78, sd
7.88, s, brd
8.05, sd
8.1, s
3.95, sd
2i
2k 2,3,4-MeO-phenyl
2l 2-thienyl
4-CF3-phenyl
132
4.05, m, brd
3.94, sd
ndc
3k
3l
192 dec
4.10, s
a
b
Measured at 200 MHz and referenced to CD3OD as the solvent unless stated otherwise. Refers to experimental section. c Not
d
determined. CDCl3 used as solvent.
dride/p-toluenesulfonic acid14 was preferred over cata-
lytic hydrogenation possibly due to catalyst deactivation
especially in the presence of the thienyl moiety. The
required N-(tert-butyloxycarbonyl)-2(S)-amino-1-phenyl-
3(R)-3,4-epoxybutane as a source of the transition-state
hydroxyl group was prepared according to known pro-
cedures:15 Peterson olefination of N-Boc-phenylalani-
nal,16 reintroduction of the Boc protecting group, and
epoxidation gave a 83:17 mixture of the threo and
erythro isomers, which could be separated by crystal-
lization. The enantiomeric excess of the desired threo
isomer was determined by chiral HPLC to be >98%.17
Nucleophilic ring opening with the hydrazines 3 oc-
curred smoothly in refluxing methanol or 2-propanol to
give the crystalline Boc-protected dipeptide isosteres
4a -l in 45-80% yield. Acidic cleavage of the Boc
protecting groups provided the key intermediates 5a -
l. The relative configuration was unambiguously as-
signed in a representative example by transforming 5a
into the corresponding oxazolidinone 6a using phosgene
in toluene at -60 °C (Scheme 1). The observed coupling
constant J AB of 5.1 Hz in the 1H-NMR spectrum was
consistent with the expected trans-configuration.7c In-
termediates 5a -l were coupled with the required car-
bamoylated valine derivatives according to standard
peptide synthesis procedures ((benzotriazol-1-yloxy)tris-
(dimethylamino)phosphonium hexafluorophosphate, 1-hy-
droxybenzotriazole, 4-methylmorpholine or 1-ethyl-3-(3-
diaminopropyl)carbodiimide, 1-hydroxybenzotriazole,
triethylamine) to furnish the target inhibitors 7a -l
(Table 2) and 8a -12a (Table 3).
Optimization of the antiviral activity and modulation
of physicochemical properties of the inhibitors required
the possibility to introduce different acyl substituents
on the amine and hydrazine functions of the dipeptide
isosteres. Therefore trifluoroacetyl and Boc were chosen
as orthogonal protecting groups compatible with the
overall synthesis. Thus, known allylamine 1315a,b was
trifluoroacetylated to give protected olefin 14, which
afforded trifluoroacetyl epoxide 15 as a 87:13 mixture
of threo and erythro isomers after oxidation using
3-chloroperbenzoic acid (Scheme 2).18 This mixture was
carried on to the following step without separation of
the isomers. Nucleophilic opening with the previously
mentioned alkyl tert-butylcarbazates 3a ,d gave the
protected dipeptide isosteres threo-16 and erythro-16.
The predominant threo isomers of 16 were isolated by
chromatography and crystallization and transformed
into inhibitors 24a -29a via subsequent deprotection
and acylation steps as shown in Scheme 2. The epimeric
Sch em e 1. Synthesis of Symmetrically Acylated
Aza-Dipeptide Analogs 7-12a
a
(a) EtOH, 80 °C, 3 h; (b) H2, Pt/C, MeOH, room temperature
(method A), H2, Pd/C, THF, room temperature (method B),
NaBH3CN, TsOH, THF, room temperature (method C or D); (c)
N-(tert-butyloxycarbonyl)-2(S)-amino-1-phenyl-3(R)-3,4-epoxy-
butane,15a,b MeOH, 80 °C, 16 h; (d) 4 N HCl, dioxane, room
temperature, 2 h; (e) P3-Val-OH, BOP, HOBT, NMM, DMF, room
temperature, 16 h (method A), P3-Val-OH, EDCI, HOBT, Et3N,
DMF, room temperature, 16 h (method B); (f) COCl2, toluene, -60
°C.
ertheless, its high enzyme affinity (IC50 ) 8.5 nM) and
its selectivity compared to other aspartic proteases11b
made CGP 53820 an attractive lead for further optimi-
zation of the biological profile. In this paper we present
our structure-activity studies leading to aza-peptide
analogs as HIV-1 protease inhibitors with good antiviral
acitivity and favorable pharmacokinetics following oral
application in mice.
Ch em istr y
Variability of substitution and ease of synthetic
transformations were emphasized in the planning of the
reaction sequence. The synthesis of symmetrically
substituted aza-dipeptides followed the route described
in Scheme 1. Commercially available aldehydes 1a -l
were transformed into the corresponding hydrazones
2a -l using tert-butylcarbazate followed by catalytic
hydrogenation on Pd/carbon or Pt/carbon to give the
required alkyl tert-butylcarbazates 3a -l bearing the
necessary P1′ substituents as listed in Table 1.13 For
carbazates 3d ,l reduction with sodium cyanoborohy-

Aza-Peptides as Potent HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3205
Ta ble 2. Analytical Data, in Vitro Potency, and Antiviral Activity for HIV Protease Inhibitors 7a -m : Modification of the P1′
Substituent
config
prep
FAB-MS
no.
P1′ substituent
of OH yield, % method (M + H)+
formulab
IC50,i nM ED50, µM ED90, µM
7a
7b
7c
7d
7e
7f
7g
7h
7i
cyclohexyl
isopropyl
isobutyl
S
S
S
S
S
S
S
S
S
S
S
R
R
67
70
28
63
47
63
39
47
15
71
66
31
A
A
B
A
A
A
A
B
B
B
B
A
606
566
580
630
618
625
616
636a
668
690
606
606
C31H51N5O7‚0.2H2Oc
84
41
16
16
42
62
18
19
33
0.018
0.023
0.0027
0.009
0.049
0.068
0.150
0.0062
0.017
0.028
0.017
ndj
0.1
1
0.03
0.03
1
C
C
C
C
C
C
28H47N5O7‚0.6 H2O
29H49N5O7
4-MeO-phenyl
4-F-phenyl
4-CN-phenyl
4-OH-phenyl
4-tolyl
4-CF3-phenyl
2,3,4-MeO-phenyl
2-thienyl
32H47N5O8
31H44N5O7F‚0.7H2Od
32H44N6O7‚0.8H2Oe
31H45N5O8
1
1
C32H47N5O7‚0.5H2O
0.03
0.1
0.1
0.1
ndj
0.01
C32H44N5O7F3‚0.5H2Of
g
7k
7l
7m
C
C
C
34H51N5O10
29H43N5O7S
31H51N5O7
34
17
1060
h
cyclohexyl
Ro 31-895919
6.3
0.0045
a
b
d
(M + Na)+. Elemental analysis within (0.4% of the calculated values except where noted. c C, H; N: calcd, 11.49; found, 10.90. H,
N; C: calcd, 59.05; found, 54.43. e H, N; C: calcd, 60.15; found, 58.91. f N, C: calcd, 56.81; found, 58.3. H: calcd, 6.70; found, 7.2. HR-
g
FAB MS (M + H)+ calcd 690.3714, obsd 690.3751. HR-FAB MS (M + H)+ calcd 606.3866, obsd 606.3848. i IC50 values had to be determined
h
at suboptimal conditions at pH 6 in order to insure solubility of inhibitors with different physicochemical properties. They are expected
j
to be 1 order of magnitude lower when determined as described in ref 19. Inclusion criteria for further evaluation in cellular assays is
IC50 < 1 µM.
Ta ble 3. Analytical Data, in Vitro Potency, and Antiviral Activity for HIV Protease Inhibitors 8a -29a : Modification of the P3P2 and
P2′P3′ Substituents
c
yield, prep
IC50
nM
,
ED50
µM
,
ED90,
µM
no.
P3P2 substituent
P2′P3′ substituent
Val-COMe
Val-COOEt
Val-COOCH2CHCH2
Val-COO(CH2)2OMe
%
method
formulaa
33H55N5O7
CGP 5382012 MeOC-Val
8.5 0.3
177
51
164
67
1
1
1
0.3
8a
9a
10a
11a
12a
24a
25a
26a
EtOOC-Val
CH2CHCH2OOC-Val
MeO(CH2)2OOC-Val
92
41
60
B
A
A
A
B
B
B
B
B
B
B
C
0.055
0.083
0.079
C35H55N5O7‚0.2H2O
C35H59N5O9‚0.4H2O
C39H67N5O11‚0.5H2O
MeO(CH2)2O(CH2)2OOC-Val Val-COO(CH2)2O(CH2)2OMe 40
0.041 10
MeOOC-NMe-Val
MeOOC-Val
MeOOC-Val
EtOOC-Val
EtOOC-Val
NMe-Val-COOMe
Val-COOEt
Val-COO(CH2)2OMe
Val-COOMe
Val-COO(CH2)2OMe
Val-COOMe
Val-COOEt
37
69
27
40
29
41
32
C
C
33H55N5O7‚H2Ob
>1000
ndd
nd
32H53N5O7
56
67
0.029
0.028
0.072
0.015
0.014
0.024
0.1
0.1
1
0.1
0.1
0.1
C33H55N5O8‚0.3H2O
C
32H53N5O7‚0.3H2O
76
150
62
27a
28a
29a
C34H57N5O8
MeO(CH2)2OOC-Val
MeO(CH2)2OOC-Val
C33H55N5O8‚0.3H2O
C34H57N5O8
134
Ro-31-895919
6.3 0.0045 0.01
a
b
Elemental analysis within (0.4% of the calculated values except where noted. C, H; N: calcd, 10.74; found, 10.04. c See footnote i
d
in Table 2. Inclusion criteria for further evaluation in cellular assays is IC50 < 1 µM.
alcohol erythro-16a as the minor component was sepa-
rated by chromatography in small quantities and used
to synthesize inhibitor 7m with the R-configuration of
the transition-state hydroxyl group. As the epimer of
compound 7a , it was distinctly different by analytical
HPLC.
The same sequence of acylations (first acylation of the
amino function followed by acylation of the hydrazine
moiety) was initially applied to the synthesis of inhibi-
tors 32-36 containing a quinolinoyl-asparaginyl (Quin-
Asn)19 side chain in P2P3 position (Scheme 3). However,
commonly used procedures to cleave the Boc protecting
group (anhydrous HCl or trifluoroacetic acid in dichlo-
romethane) readily applicable for the synthesis of
compounds 5 and 21-23 were not compatible with the
functional groups of intermediate 30. Instead, formic
acid, when used as the reaction solvent, achieved
cleavage of the Boc protecting group in good yields at

3206 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Fa¨ssler et al.
Sch em e 2. Synthesis of Acylated Aza-Dipeptide Analogs
7m and 24a -29a a
Sch em e 3. Synthesis of Unsymmetrically Acylated
Aza-Dipeptide Analogs 32a -36a a
a
(a) Quin-Asn-OH, DCC, 3-hydroxy-1,2,3-benzotriazin-4(3H)-
one or HOBT, NMM, THF; (b) HCOOH, room temperature, 7-22
h; (c) Boc-Val-OH, HBTU, NMM, CH3CN or MeOOC-Val-OH,
EDCl, HOBT, NMM, DMF or Cbz-Val-OH, HBTU, NMM, DMF;
(d) ClCOOR, K2CO3, dioxane/H2O.
Sch em e 4. Synthesis of Unsymmetrically Acylated
Aza-Dipeptide Analogs 32b and 33ba
a
(a) (CF3CO)2O, pyridine, CH2Cl2, 0 °C, 2 h; (b) m-CPBA,
CH2Cl2, room temperature, 20 h; (c) N-(tert-butyloxycarbonyl)-N-
2-alkylhydrazine 3, MeOH, 80 °C, 16 h; (d) K2CO3, MeOH/H2O,
80 °C, 16 h; (e) P3-Val-OH, EDCI, HOBT, Et3N, DMF, room
temperature, 16 h; (f) 4 N HCl, dioxane, room temperature, 2 h;
(g) P3′-Val-OH, EDCI, HOBT, Et3N, DMF, 16 h, room temperature
(method B).
room temperature. Acylation of the less reactive hy-
drazine moiety of Quin-Asn-substituted intermediates
31 with valine derivatives turned out to be a particu-
larly sluggish and low-yielding reaction compared to
valine-substituted intermediates 21-23. This observa-
tion prompted us to change the synthetic scheme and
to prepare the (Cbz-valyl)alkylhydrazine 40b first, with
the P1′P2′ substituents already in place (Scheme 4). The
Quin-Asn substituent was subsequently introduced by
acylation of the more reactive amine terminus as the
last reaction step to provide inhibitors 32b and 33b.
a
(a) Cbz-Val-OH, EDCl, HOBT, NMM, EtOAc; (b) 2 N HCl,
dioxane, room temperature; (c) isobutyric aldehyde, EtOH, 60 °C,
4 h; (d) NaBH3CN, p-TsOH, THF, room temperature, 1 h; (e)
N-(tert-butyloxycarbonyl)-2(S)-amino-1-phenyl-3(R)-3,4-epoxy-
butane,15a,b EtOH, 80 °C, 16 h; (f) HCOOH, room temperature, 16
h; (g) Quin-Asn-OH, EDCl, HOBT, NMM, THF; (h) H2, Pd/C (10%),
MeOH; (i) ClCOOMe, K2CO3, dioxane/H2O.
Resu lts a n d Discu ssion
in aza-peptide-derived inhibitors is in sharp contrast to
hydroxyethylamine-, (hydroxyethyl)urea, and sulfon-
amide-derived inhibitors which also have the P1′ sub-
stituents attached to a nitrogen atom. In those com-
pounds a distinct preference for the R-stereochemistry
at the hydroxyl group has been reported.20,21 It is
important to note that according to the X-ray structure
the alkylated hydrazine nitrogen bearing the P1′ sub-
stituent is tetrahedral with inverted conformation com-
As a close analog of the previous lead compound CGP
53820, the bis[(methoxycarbonyl)valyl] derivative 7a
had already improved antiviral activity while maintain-
ing high selectivity.11c Inhibitor 7m with R-configura-
tion of the alcohol served to investigate the influence of
the configuration of the transition-state hydroxyl group.
It inhibited the enzyme more than 10-fold weaker
compared to its epimer 7a as shown in Table 2. This
observation of preferred S-configuration of the alcohol

Aza-Peptides as Potent HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3207
Ta ble 4. Analytical Data, in Vitro Potency, and Antiviral Activity for HIV Protease Inhibitors 32-36: Modification of the P1′ and
P2′P3′ Substituents
h
no.
P1′ substituent
P2′P3′ substituent
formulaa
yield, %
mp, °C
IC50
,
nM
ED50, µM
ED90, µM
b
c
d
e
32a
32b
32d
33a
33b
33d
36a
cyclohexyl
isopropyl
4-MeO-phenyl
cyclohexyl
isopropyl
Val-COOMe
Val-COOMe
Val-COOMe
Val-Cbz
Val-Cbz
Val-Cbz
C38H51N7O7
37
84
37
59
56
77
48
nd
27
23
31
41
37
49
126
6.3
0.018
0.063
0.118
0.0098
0.012
0.015
0.0094
0.0045
0.1
0.3
1
0.03
0.03
0.03
0.03
0.01
C
C
C
C
C
C
35H47N7O7
39H47N7O8
44H55N7O7
41H51N7O7
45H51N7O8
39H53N7O7
178-180
160-162
nd
208-210
nd
f
4-MeO-phenyl
cyclohexyl
g
Val-COOEt
nd
Ro 31-895919
a
b
Elemental analysis within (0.4% of the calculated values except where noted. HR-FAB MS (M + H)+ calcd 718.3928, obsd 718.3918.
c HR-FAB MS (M + H)+ calcd 678.3615, obsd 678.3644. HR-FAB MS (M + H)+ calcd 742.3564, obsd 742.3547. e C, H; N: calcd, 12.35;
d
found, 11.93. f HR-FAB MS (M + H)+ calcd 818.3877, obsd 818.3871. g HR-FAB MS (M + H)+ calcd 732.4085, obsd 732.4114. See footnote
h
i in Table 2.
pared to the R-carbon of a peptide substrate or the
hydroxyethylene-based inhibitor CGP 53437.12,22 Fur-
ther examination of the X-ray structure of the lead
compound revealed that the P1′ subsite could accom-
modate lipophilic substituents of diverse size other than
cyclohexyl. Decreasing the steric bulk of the P1′ sub-
stituent by replacing the cyclohexyl moiety with isopro-
pyl slightly increased the affinity to the enzyme but did
not show a significant effect on the antiviral ED50 values
(compounds 7a ,b in Table 2). Elongation of the side
chain to an isobutyl substituent, on the other hand,
resulted in a significant increase in affinity and provided
compound 7c with excellent antiviral potency (ED50 and
ED90 values). The following compounds listed in Table
2 demonstrate that 4-substituted phenyl groups as P1′
side chains were well tolerated in the enzymatic assay.
While the related compounds 7d ,g-i exhibited a similar
degree of enzyme inhibition, only the 4-methoxy deriva-
tive 7d and the 4-tolyl derivative 7h maintained high
activity in the antiviral assay and displayed ED50 values
in the low-nanomolar range. Compound 7g with the
free phenol group had greatly diminished antiviral
potency, presumably due to reduced lipophilicity.23
Subsequently the antivirally most active compounds in
this series (7c,d ,h ) were evaluated in a preliminary
pharmacokinetic model in mice following oral applica-
tion of a formulation containing DMSO/(hydroxypropyl)-
â-cyclodextrin. As shown in Table 5, plasma concen-
trations in the low-micromolar range were measured for
7c at three out of four time points. Unfortunately
neither 7d nor 7h reached plasma concentrations
exceeding the detection limit after oral application in
mice.
Ta ble 5. Preliminary Pharmacokinetic Evaluation of Selected
Compounds after Oral Application in Mice: Plasma
Concentrations (µM) after 30, 60, 90, and 120 mina
conctn (µM) at time given (min)
coefficient
no. ED50, µM
30
60
90
120
C120/ED50
7c
7d
7h
8a
0.0027
0.009
0.0062
0.055
0.083
0.079
0.029
0.028
0.072
0.015
0.014
0.024
0.015
<0.2
<0.2
<0.2
6.7
0.6
nd
<0.2
6.7
1.2
1.1
2.2
2.8
0.4
2.2
<0.5
<0.2
0.2
0.4
<0.2
<0.2
7.8
0.9
<0.5
5.8
3.0
1.2
1.0
<0.5
<0.2
0.8
0.3
<0.2
<0.2
8.7
1.0
<0.5
6.0
3.1
1.4
1.5
<0.5
<0.2
1.1
111
nd
nd
158
12
nd
207
111
19
100
nd
9a
0.8
10a
24a
25a
26a
27a
28a
29a
33a
2.7
3.8b
1.0
3.2b
1.0
<0.5
<0.2
0.3
nd
73
a
The compounds were administered by gavage to female
BALB/c mice as a suspension in DMSO/(hydroxypropyl)-â-cyclo-
dextrin at an average dose of 120 mg/kg. The limit of detection
was 0.2 µM except for compounds 10a and 28a (0.5 µM). Values
indicated in the table represent the mean between four animals.
b
High variability, standard error (1.9.
acyl side chains did not show a pronounced effect on
enzyme affinity as anticipated (Table 3). The bis(allyl
carbamate) 9a and the bis[(methoxyethoxy)ethyl car-
bamate] 11a exhibited IC50 values in the same range
as 7a , whereas 8a and 10a were about 2-fold less potent
in vitro. The antiviral potency was generally dimin-
ished in this series of compounds, the effect being most
significant for 11a . Its dose-response curve showed a
reduced slope, such that 90% inhibition was achieved
only at 10 µM concentration despite of an ED50 of 41
nM. N-Methylation of the valine residues as in 12a
completely abolished inhibitory potency, possibly due to
the loss of two hydrogen bonds to the enzyme. Therefore
we did not examine whether reducing the number of
potential intramolecular hydrogen bonds to the enzyme
might be beneficial for oral absorption.25 Most interest-
ingly, the minor change of 7a to 8a , although slightly
reducing the antiviral potency, resulted in a signifi-
cantly improved pharmacokinetic profile with maximal
plasma concentrations in mice exceeding ED50 more
than 150-fold. Replacement of two methyl carbamates
with two ethyl carbamates resulted in an increase of
As the next step in the optimization program, the role
of the P2P3 and P2′P3′ substituents was investigated
while the cyclohexyl substituent was kept in P1′ position.
Previous findings24 allowed the conclusion that the
hydrogen bonds of the two carbonyl functions of the
acylated valine residues were more important for en-
zyme affinity than the lipophilic interactions with the
P3 and P3′ subsites. Therefore the variations presented
here are restricted to acyl residues or carbamates, which
might serve as a handle to modify the physical proper-
ties in order to improve the pharmacokinetic profile of
the inhibitors made so far. Parallel elongation of the

3208 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Fa¨ssler et al.
DRX-500 (500 MHz), Bruker AM-360 (360 MHz), Varian
Gemini-300 (300 MHz) or Varian Gemini-200 (200 MHz
spectrometer); chemical shifts of signals are expressed in parts
per million (ppm) and referenced to the deuterated solvents
used. Coupling constants are recorded in hertz (Hz). IR
spectra: Bruker FT-IR-spectrometer IFS 48. MS spectra:
FAB-ZAB, HF (VG analytical spectrometer). HPLC chro-
matography: stationary phase, Nucleosil C18, 5 µm analytical
column (125 × 4.6 mm); mobile phase, 20% acetonitrile/0.1%
trifluoroacetic acid (TFA) in water/0.1% TFA f 100% aceto-
nitrile/0.1% TFA during 20 min + 10 min 100% acetonitrile/
0.1% TFA; tR refers to the retention time in minutes. Elemen-
tal analyses were performed by the Ciba Analytical Department
and are within (0.4% of the calculated values unless noted
otherwise. Abbreviations: BOP, (benzotriazol-1-yloxy)tris-
(dimethylamino)phosphonium hexafluorophosphate; DCC, N,N′-
dicyclohexylcarbodiimide; HBTU, O-benzotriazol-1-yl-N,N,N′,N′-
tetramethyluroniumhexafluorophosphate; HOBT, 1-hydroxy-
benzotriazole hydrate; EDCI, 1-ethyl-3-(3-diaminopropyl)car-
bodiimide hydrochloride; NMM, 4-methylmorpholine; p-TsOH,
4-toluenesulfonic acid.
Cycloh exylm eth a n on e (ter t-Bu tyloxyca r bon yl)h yd r a -
zon e (2a ) (Gen er a l syn th esis of h yd r a zon es 2a -l). To a
solution of 133 g (1.359 mol) of N-(tert-butyloxycarbonyl)-
hydrazine in 3 L of absolute ethanol was added 200 mL (1.495
mol) of cyclohexanecarboxaldehyde at ambient temperature.
Upon completion of the addition, the temperature rose to 34
°C, and the mixture was heated to reflux for 3 h. About 1.2 L
of solvent was removed by distillation, and the residue was
diluted with 500 mL of water. After cooling to 46 °C, 1 L of
water was added. Crystallization of the product was completed
by slow addition of 3 L of water during 1 h at ambient
temperature. The resulting suspension was filtered; the filter
cake was washed with 3 L of water and dried at 60 °C to give
300 g (100%) of 2a homogeneous by TLC (80:20 hexane:ethyl
acetate): mp 134-135 °C, 1H-NMR (CDCl3) δ 7.65 (s, br, 1H),
7.0 (d, J ) 6, 1H), 2.26 (m, 1H), 1.83-1.55 (m, 5H), 1.45 (s,
9H), 1.3-1.08 (m, 5H); HPLC tR 14.2 min; FAB MS (M + H)+
) 227.
lipophilicity by 1 log P unit (log P: 3.61 for 8a vs 2.55
for 7a ), which we consider one factor contributing to oral
absorption.23
This result indicated that even more conservative
changes might influence the overall profile, and there-
fore the acyl substituents at the amine and hydrazine
terminus needed to be varied independently. Therefore
the constitutional isomers 24a and 26a with only one
ethyl carbamate were evaluated. Interestingly, only
24a showed a clear superiority in all respects (IC50,
ED50, C90). This compound exhibited the best overall
profile so far by yielding plasma concentrations more
than 200-fold the ED50. Compounds containing one
(methoxyethoxy)ethyl carbamate maintained their high
degree of antiviral activity in the nanomolar range, but
their blood levels in mice were clearly inferior to those
of 24a .
Structure-activity studies that led to Ro 31-895919
revealed that the hydrophilic amino acid asparagine was
well tolerated in P2 position. As a consequence, the
quinolinoyl-asparagine side chain has become a con-
stituent of various potent HIV protease inhibitors with
different central units.26 Inhibitors containing this
Quin-Asn side chain generally maintained a high degree
of enzyme affinity (Table 4). The ED50 values of
compounds 32a -33d show a trend that antiviral po-
tency is improved by increasing lipophilicity of either
the P1′ or P2′P3′ substituents. Thus, combination of a
cyclohexyl group in P1′ with the lipophilic Cbz-Val
residue in P2′P3′ culminated in the best antiviral potency
in this series (33a ). However, none of these inhibitors
achieved plasma concentrations exceeding 1 µM in mice.
Su m m a r y
N-1-(ter t-Bu t yloxyca r b on yl)-N-2-(cycloh exylm et h yl)-
h yd r a zin e (3a ) (Red u ction a ccor d in g to m eth od A). To
a solution of 300 g (1.32 mol) of hydrazone 2a in 3 L of MeOH
was added 60 g of 5% Pt on carbon, and the mixture was
hydrogenated during 8 h at room temperature and atmo-
spheric pressure of hydrogen. Upon theoretical uptake of
hydrogen, the catalyst was filtered, and the filtrate was
concentrated under reduced pressure. The crude product was
diluted with 2 L of methylene chloride and washed with 2 L
of water. The organic layer was filtered through a pad of
cotton wool and evaporated under reduced pressure to give
288 g (95 %) of 3a as a colorless oil: 1H-NMR (CDCl3) δ 6.20
(s, br, 1H), 3.85 (s, br, 1H), 2.63 (d, J ) 7, 2H), 1.78-1.65 (m,
6H), 1.42 (s, 9H), 1.3-1.05 (m, 3H), 1.0-0.82 (m, 2H); IR
(dichloromethane) cm-1 3440, 3040, 2990, 2920, 2850, 1710,
1450.
N -1-(t er t -Bu t yloxyca r b on yl)-N -2-[(4-flu or op h e n yl)-
m eth yl]h yd r a zin e (3e) (Red u ction a ccor d in g to m eth od
B). To a solution of 55 g (231 mmol) of hydrazone 2e in 500
mL of THF was added 5.5 g of 5% Pd on carbon, and the
mixture was hydrogenated during 30 min at room temperature
and atmospheric pressure of hydrogen. Upon theoretical
uptake of hydrogen, the catalyst was filtered, and the filtrate
was concentrated under reduced pressure to give 52 g (94%)
of 3e as an amorphous, white solid: 1H-NMR (MeOD) δ 7.35
(dd, J ) 8, 5, 2H), 7.05 (t, J ) 8, 2H), 3.86 (s, 2H), 1.42 (s,
9H).
Aza-peptide analogs containing a (hydroxyethyl)-
hydrazine isostere led to potent HIV-1 protease inhibi-
tors with high antiviral activity. The central dipeptide
isostere was acylated either in a symmetric fashion with
valine substituents or subsequently with different valine
or asparagine substituents. Inhibitors of this type
showed a distinct preference for the S-configuration of
the transition-state hydroxyl group. Subtle changes of
the carbamoylated valine residues in P2P3 and P2′P3′
positions, which had little effect on enzyme affinity,
greatly influenced the pharmacokinetic profile. Thus,
replacement of one or both methyl carbamates on the
valine substituents with an ethyl carbamate produced
a substantial increase in plasma concentrations upon
oral application in mice. As a result, 8a and 24a were
identified as inhibitors with the best overall profile.
They combined both good antiviral potency with favor-
able pharmacokinetics in mice upon oral application of
a DMSO/(hydroxypropyl)-â-cyclodextrin formulation with
plasma concentrations exceeding ED50 by 150-fold.
Exp er im en ta l Section
Ch em istr y. All reactions with air- or moisture-sensitive
reactants and solvents were carried out under nitrogen
atmosphere. Reagents and solvents were used as purchased
without further purification. Analytical thin layer chroma-
tography was performed on silica F254 glass plates (E. Merck).
Components were visualized by UV light of λ 254 nm or
spraying with phosphomolybdic acid. Column flash chroma-
tography was performed on silica gel 60 (230-400 mesh
N-1-(ter t-Bu tyloxyca r bon yl)-N-2-(2-th ien ylm eth yl)h y-
d r a zin e (3l) (Red u ction a ccor d in g to m eth od C). To a
suspension of 50 g (221 mmol) of hydrazone 2l in 220 mL of
THF was added 16.3 g of sodium cyanoborohydride at ambient
temperature. The pH of the reaction mixture was adjusted
to 3.5 by dropwise addition of a solution of 42 g (221 mmol) of
p-TsOH in 220 mL of THF and kept constant at this value for
72 h using a pH-controlled addition valve. The reaction
mixture was then diluted with 1 L of ethyl acetate and
subsequently washed with 1 L of brine, 1 L of saturated
ASTM, E. Merck) under
a positive nitrogen pressure of
approximately 20 psi. Melting points were determined in an
open capillary and are not corrected. 1H-NMR spectra: Bruker

Aza-Peptides as Potent HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3209
NaHCO3, and 1 L of brine. The aqueous layers were re-
extracted with 1 L of ethyl acetate, and the combined organic
layers were dried with Na2SO4 and evaporated under reduced
pressure. The resulting crude product was purified by column
chromatography using 1 kg of silica gel (eluent: 20:1 dichlo-
romethane:ether) and suspended in 265 mL of 1 N aqueous
NaOH. After stirring during 2.5 h at ambient temperature,
the mixture was extracted twice with 200 mL of dichlo-
romethane. The organic extracts were filtered through a pad
of cotton wool and evaporated under reduced pressure to give
19.95 g (40%) of 3l as a colorless oil homogeneous by TLC (20:1
dichloromethane:ether): 1H-NMR (CDCl3) δ 7.3 (dd, J ) 5, 1,
1H), 6.95 (m, 2H), 4.10 (s, 2H), 0.95 (s, 9H); HPLC tR 9.6 min;
FAB MS (M + H)+ ) 229.
N-1-(ter t-Bu tyloxyca r bon yl)-N-2-[(4-m eth oxyp h en yl)-
m eth yl]h yd r a zin e (3d ) (Red u ction a ccor d in g to m eth od
D). A solution of 86.5 g (502 mmol) of p-TsOH in 400 mL of
THF was added dropwise in 30 min to a stirred solution of
125.7 g (502 mmol) of 2d and 31.6 g (502 mmol) of NaBH3CN
in 1.6 L of THF. After stirring for 4 h at room temperature,
the resulting suspension was concentrated under reduced
pressure. This material was redissolved in ethyl acetate and
washed subsequently with saturated NaHCO3 and brine. The
aqueous layers were re-extracted twice with ethyl acetate; the
organic layer was dried (Na2SO4), filtered, and concentrated
in vacuo. The resulting yellow mass was taken up in 1.5 L of
1 N NaOH and stirred at ambient temperature for 90 min.
Then the reaction mixture was neutralized with 2 N HCl and
extracted three times with dichloromethane. The organic
layers were washed with brine, dried (Na2SO4), filtered, and
concentrated in vacuo. Vigorous stirring in diisopropyl ether
yielded 66.4 g of crystalline 3d . The concentrated filtrate
afforded upon medium pressure chromatography (Lichroprep;
0% f 40% ethyl acetate in hexane) another 26.6 g (a total of
73%) of 3d : mp 145-147 °C; TLC (2:1 hexane:ethyl acetate)
Rf ) 0.36; 1H-NMR (MeOD) δ 7.27 and 6.87 (2d, J ) 8, 2, 2H),
3.83 (s, 2H), 3.77 (s, 3H), 1.44 (s, 9H). Anal. (C13H20N2O3) C,
H, N.
1-Cycloh exyl-5(S)-2,5-bis[(ter t-bu tyloxyca r bon yl)a m i-
n o]-4(S)-h ydr oxy-6-ph en yl-2-azah exan e (4a) (Gen er al pr o-
ced u r e for th e syn th esis of com p ou n d s 4a -l). A solution
of 54.6 g (239.3 mmol) of 3a and 60 g (227.9 mmol) of N-(tert-
bu t yloxyca r bon yl)-2(S )-a m in o-1-ph en yl-3(R)-3,4-epoxy-
butane15a,b in 500 mL of absolute MeOH was heated to reflux
temperature during 16 h. Thereafter the solvent was evapo-
rated and the crude product precipitated by addition of 200
mL of diisopropyl ether. The solid was filtered and recrystal-
lized by dissolving in 50 mL of dichloromethane and adding
hexane to the solution to give 54.2 g of 4a as a white solid.
The mother liquor was purified by chromatography on silica
gel (eluent: 3:1 hexane:ethyl acetate) to give another 7.92 g
of 4a homogeneous by TLC (1:1 hexane:ethyl acetate), com-
bined yield 62.12 g (55.4%): mp 156-157 °C; 1H-NMR (MeOD)
δ 7.3-7.15 (m, 5H), 3.7 (m, 1H), 3.62 (m, 1H), 2.97-2.7 (m,
2H), 2.65-2.35 (m, 4H), 1.95-0.8 (m, br, 11H), 1.40 (s, 9H),
1.36 (s, 9H); HPLC tR 19.2 min; EI MS m/ z 491, 391, 362,
335, 241, 185, 141.
1-Cycloh exyl-5(S)-2,5-d ia m in o-4(S)-h yd r oxy-6-p h en yl-
2-a za h exa n e Hyd r och lor id e (5a ) (Gen er a l p r oced u r e for
th e syn th esis of com p ou n d s 5a -l). A solution of 16.4 g
(33.4 mmol) of 4a in 90 mL of 4 N HCl(g) in dioxane was stirred
at room temperature. After 1 h a white precipitate formed,
and the reaction mixture was diluted with 30 mL of 4 N HCl-
(g) in dioxane. TLC (5:1 chloroform:MeOH) indicated comple-
tion of the reaction after 6 h at ambient temperature. The
mixture was diluted with 100 mL of dioxane to give a solution
which was then lyophilized overnight and yielded 12.02 g (90%)
of 5a as an amorphous white solid: 1H-NMR (MeOD) δ 7.45-
7.27 (m, 5H), 3.95 (m, 1H), 3.65 (m, 1H), 3.15-2.86 (m, 4H),
2.75-2.5 (m, 2H), 1.95-0.75 (m, 11H); HPLC tR 7.8 min; FAB
MS (M + H)+ ) 291.
reaction mixture was allowed to warm to room temperature
and stirred for an additional 30 min. It was diluted with
dichloromethane and washed with saturated NaHCO3. The
organic layer was filtered through a pad of cotton wool and
evaporated to dryness. The resulting oil (200 mg) was purified
by column chromatography on silica gel (eluent: 15:1 dichlo-
romethane:MeOH) and evaporated to dryness to give 30 mg
(19%) of 6a as a colorless oil: 1H-NMR (MeOD) δ 7.4-7.15
(m, 5H), 4.59 (dd, J ) 6.1, 5.2, 1H), 3.91 (dd, J ) 6.4, 5.1, 1H),
2.90 (d, J ) 6.4, 1H), 2.63 (dd, J ) 13.2, 6.1, 1H), 2.52 (dd, J
) 13.2, 6.1, 1H), 2.30 (d, J ) 7.1, 1H), 1.86-1.55 (m, 4H), 1.55-
1.35 (m, 1H), 1.35-1.03 (m, 4H), 1.0-0.72 (m, 2H); HPLC tR
9.98 min.
1-Cycloh exyl-5(S)-2,5-b is[[2-N-(m et h oxyca r b on yl)-L-
va lin yl]a m in o]-4(S)-h yd r oxy-6-p h en yl-2-a za h exa n e (7a )
(Gen er a l syn th esis of com p ou n d s 7a ,b,d -g a n d 9a -11a
u sin g cou p lin g m eth od A). To form an active ester, 1.47 g
(8.4 mmol) of (methoxycarbonyl)-L-valine, 1.13 g (8.4 mmol)
of HOBT, and 3.71 g (8.4 mmol) of BOP were dissolved in 54
mL of a 0.3 M solution of NMM in DMF. After 15 min 1.12 g
(2.8 mmol) of 5a was added at ambient temperature, and the
mixture was stirred overnight. The solvents were evaporated
at 50 °C under reduced pressure, and the residue was dissolved
in 300 mL of dichloromethane. This solution was subsequently
washed twice with 300 mL of saturated NaHCO3
and 300 mL
of brine. The aqueous layers were re-extracted with 200 mL
of dichloromethane. The combined organic layers were filtered
through a pad of cotton wool and evaporated under reduced
pressure. The resulting crude product was purified by column
chromatography on silica gel (eluent: 15:1 dichloromethane:
MeOH) and precipitated twice by dissolving in 5 mL of
dichloromethane and adding diisopropyl ether. After lyo-
philization from dioxane, there was obtained 1.14 g (67%) of
7a which was homogenous by TLC (15:1 dichloro-
methane:MeOH):
1H-NMR (MeOD) δ 7.2 (m, 5H), 4.11 (ap-
parent q, 1H), 3.9-3.68 (m, 3H), 3.65 (2s, 6H), 2.95-2.75 (m,
2H), 2.65-2.4 (m, 4H), 1.95 (m, 3H), 1.66 (br, 4H), 1.18-0.75
(m, 6H), 0.92 (d, J ) 7, 3H), 0.90 (d, J ) 7, 3H), 0.80 (d, J )
7, 6H); HPLC tR 14.9 min; FAB MS (M + H)+ ) 606. Anal.
(C31H51N5O7) C, H; N: calcd, 11.49; found, 10.90.
1-(4-Tolyl)-5(S)-2,5-bis[[2-N-(m eth oxyca r bon yl)-L-va li-
n yl]am in o]-4(S)-h ydr oxy-6-ph en yl-2-azah exan e (7h ) (Gen -
er a l syn th esis of com p ou n d s 7c,h -l, 8a , a n d 12a u sin g
cou p lin g m eth od B). To a stirred solution of 582 mg (3.32
mmol) of (methoxycarbonyl)-L-valine, 1.18 g (6.15 mmol) of
EDCI, and 500 mg (3.70 mmol) of HOBT in 20 mL of DMF
was added 1.05 mL (7.53 mmol) of triethylamine. After 30
min 502 mg (1.23 mmol) of 5h was added, and the mixture
was allowed to stir at room temperature for 16 h. The solvent
was evaporated under reduced pressure and the residue
dissolved in 100 mL of dichloromethane. This solution was
subsequently washed with 100 mL of 10% aqueous citric acid,
100 mL of saturated NaHCO3, and 100 mL of brine. The
aqueous layers were re-extracted twice with 100 mL of
dichloromethane. The combined organic layers were filtered
through a pad of cotton wool and evaporated under reduced
pressure. The residue was crystallized by dissolving in 5 mL
of dichloromethane and adding diisopropyl ether. Lyophiliza-
tion from dioxane gave 355 mg of 7h (47%) as a white,
amorphous solid which was homogeneous by TLC (95:5
dichloromethane:MeOH):
1H-NMR (MeOD) δ 7.25-7.05 (m,
9H), 4.10 (apparent q, br, 1H), 3.96-3.55 (m, 5H), 3.63 (2s,
6H), 2.98-2.75 (m, 3H), 2.60 (d, br, J ) 10, 1H), 2.27 (s, 3H),
1.85 (m, 1H), 1.67 (m, 1H), 0.77 (2d, J ) 7, 2 × CH3) 0.65 (d,
J ) 7, 3H), 0.60 (d, J ) 7, 3H); HPLC tR 14.1 min; FAB MS
(M + Na)+ ) 636. Anal. (C32H47N5O7‚0.5H2O) C, H, N.
1-Cycloh exyl-5(S)-2,5-b is[[2-N-(m et h oxyca r b on yl)-L-
va lin yl]a m in o]-4(R)-h ydr oxy-6-p h en yl-2-a za h exa n e (7m ).
Deprotection of erythro-17a using 4 N HCl in dioxane at room
temperature for 3 h followed by lyophilization of the reaction
solution gave a quantitative yield of crude 1-cyclohexyl-5(S)-
2,5-diamino-4(R)-hydroxy-6-phenyl-2-azahexane hydrochloride
(erythro-5a ). Coupling of the crude product with (methoxy-
carbonyl)-L-valine using method A as described for 7a yielded
12 mg (31%) of 7m : 1H-NMR (MeOD) δ 7.2 (m, 5H), 4.08 (m,
1H), 3.76 (d, J ) 7, 1H), 3.64 (s, 6H), 3.6 (m, 2H), 3.20-2.94
4-Ben zyl-5-(3-cycloh exyl-2-a m in o-2-a za p r op yl)oxa zo-
lid in -2-on e (6a ). To a solution of 200 mg (0.499 mmol) of 5a
in 5 mL of dichloromethane, which was cooled to -60 °C, were
added 350 mL (2.49 mmol) of NEt3 and 247 mL of a 20%
solution of phosgene in toluene. After 1 h at -60 °C the

3210 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Fa¨ssler et al.
(m, 2H), 2.8-2.5 (m, 4H), 2.05-0.7 (m, 25H); HPLC tR 14.7
min; HR-FAB MS (C31H51N5O7) (M + H)+ calcd 606.3866, obsd
606.3848.
erythro-16a : TLC (2:1 toluene:ethyl acetate) Rf ) 0.57; 1H-NMR
(MeOD) δ 7.2 (m, 5H), 4.10 and 3.68 (2m, 2H), 3.18 (dd, J )
14, 4, 1H), 2.82-2.4 (m, 5H), 2.03-0.8 (m, 11H), 1.45 (s, 9H);
FAB MS (M + H)+ ) 488.
N-(Tr iflu or oa cetyl)-3(S)-a m in o-4-p h en yl-1-bu ten e (14).
To a solution of 117.8 g (0.794 mol) of 3(S)-amino-4-phenyl-
1-butene (13)15a,b in 2 L of dichloromethane was added 691 mL
of pyridine at room temperature. The resulting yellow solution
was cooled to -5 °C, and 167 mL (1.198 mol) of trifluoroacetic
anhydride was added within 15 min. The resulting mixture
was allowed to stir at 0 °C during 1 h and then diluted with
1.2 L of dichloromethane. The organic layer was subsequently
washed with 3 L of 1 N aqueous HCl (3×), 3 L of water, and
3 L of brine, dried with Na2SO4, and evaporated under reduced
pressure to give 170.4 g (88%) of 14 as an oil: 1H-NMR (CDCl3)
δ 7.4-7.1 (m, 5H), 6.2 (s, br, 1H), 5.85 (m, 1H), 5.22 (m, 2H),
4.82 (q, J ) 7, 1H), 2.95 (dd, J ) 6.6, 1.9, 2H); HPLC tR 14.0
min; FAB MS (M + H)+ ) 244. Anal. (C12H12NF3O) C, H, N,
F.
N-(Tr iflu or oa cet yl)-2(S)-a m in o-1-p h en yl-3(R)-3,4-ep -
oxybu ta n e (15). To a solution of 255 mg (1.05 mmol) of 14
in 5 mL of dichloromethane was added 1.06 g (5.2 mmol) of
3-chloroperbenzoic acid at 0 °C. The resulting mixture was
allowed to stir at ambient temperature during 20 h. The
resulting suspension was diluted with 15 mL of ether and
poured into 50 mL of 10% aqueous sodium sulfite. The organic
layer was washed subsequently with 50 mL of 10% aqueous
sodium sulfate, 50 mL of saturated NaHCO3, and 50 mL of
brine, dried with Na2SO4, and evaporated under reduced
pressure. The resulting crude product was purified by column
chromatography on silica gel (eluent: 19:1 dichloromethane:
MeOH) to give 270 mg (90%) of 15 as a 7:1 mixture of the
threo and erythro isomers: 1H-NMR (CDCl3, 7:1 mixture of
threo-15:erythro-15) δ 7.3-7.15 (m, 5H), 6.2 (s, br, 1H), 4.43
(qd, J ) 7.3, 1.7, 0.85H, threo isomer), 4.08 (q, br, J ) 7, 0.15H,
erythro isomer), 3.15 (m, 1H), 3.02 (dd, J ) 7, 1.7, 2H), 2.76
(t, J ) 4.1, 1H), 2.53 (dd, J ) 4.2, 2.7, 1H); HPLC tR 12.3 min
(no separation of isomers); FAB MS (M + H)+ ) 260. Anal.
(C12H12NF3O2) H, N; C: calcd, 55.60; found, 56.25. F: calcd,
21.99; found, 21.35.
1-Cycloh exyl-2-[(ter t-b u t yloxyca r b on yl)a m in o]-4(S)-
h yd r oxy-5(S)-a m in o-6-p h en yl-2-a za h exa n e (th r eo-17a ).
To a solution of 60 g (0.123 mol) of threo-16a in 2 L of methanol
was added 615 mL of a 1 N aqueous solution of K2CO3. The
resulting suspension was heated to reflux for 16 h. The solvent
was evaporated under reduced pressure and the residue
dissolved in 2 L of dichloromethane. This solution was washed
with 1 L of water and 1.5 L of brine, and the aqueous layers
were re-extracted with 500 mL of dichloromethane. The
organic layers were combined, filtered through a pad of cotton
wool, and concentrated. Crystallization of the crude product
by dissolving in 100 mL of dichloromethane and adding 600
mL of diisopropyl ether gave 45.6 g (95%) of threo-17a as a
white solid: 1H-NMR (MeOD) δ 7.32-7.12 (m, 5H), 3.5 (m,
1H), 2.95-2.4 (m, 7H), 2.0 (d, br, 1H), 1.85-0.8 (m, 10H), 1.43
(s, 9H); HPLC tR 13.5 min.
1-Cycloh exyl-2-[(ter t-b u t yloxyca r b on yl)a m in o]-4(R)-
h yd r oxy-5(S)-a m in o-6-p h en yl-2-a za h exa n e (er yth r o-17a ).
Preparation was as described above for threo-17a . Data for
erythro-17a : yield 202 mg (77%); 1H-NMR (MeOD) δ 7.27 (m,
5H), 3.54 and 3.12 (2m, 2H), 2.96-2.4 (m, 6H), 2.05-0.8 (m,
11H), 1.42 (s, 9H).
1-(4-Meth oxyph en yl)-2-[(ter t-bu tyloxycar bon yl)am in o]-
4(S)-h yd r oxy-5(S)-a m in o-6-p h en yl-2-a za h exa n e (th r eo-
17d ). A 1 N aqueous solution (975 mL) of K2CO3 was added
dropwise to a solution of 50 g (97.6 mmol) of threo-16d in 1 L
of methanol at 70 °C. The solution was heated to reflux for
16 h. The solvent was evaporated under reduced pressure and
the residue dissolved in dichloromethane. This solution was
washed with water and brine, and the aqueous layers were
re-extracted with two portions of dichloromethane. The
organic layers were dried (Na2SO4), filtered, and concentrated
in vacuo. Crystallization of the crude oil by vigorous stirring
in hexane gave 35.6 g (87%) of threo-17d : mp 113-115 °C;
TLC (dichloromethane:methanol) Rf ) 0.37; 1H-NMR (MeOD)
δ 7.25 (m, 7H), 6.84 (d, J ) 8, 2H), 3.77 (s, 3H), 3.86-3.64 (m,
3H), 3.56 (m, 1H), 3.00-2.55 (m, 4H), 1.31 (s, 9H). Anal.
(C23H33N3O4) C, H, N.
1-Cycloh exyl-2-[(ter t-b u t yloxyca r b on yl)a m in o]-4(S)-
h yd r oxy-5(S)-[[N-(m eth oxyca r bon yl)-L-va lin yl]a m in o]-6-
p h en yl-2-a za h exa n e (18a ). To a stirred solution of 32.2 g
(0.183 mol) of (methoxycarbonyl)-L-valine, 66.1 g (0.435 mol)
of EDCI, and 31 g (0.228 mol) of HOBT in 500 mL of DMF
was added 32 mL (0.230 mol) of triethylamine. After 30 min
a solution of 32.2 g (0.183 mol) of threo-17a in 1 L of DMF
was added and the mixture allowed to stir at room tempera-
ture for 16 h. The solvent was evaporated under reduced
pressure and the residue dissolved in 1 L of dichloromethane.
This solution was subsequently washed with 1 L of 10%
aqueous citric acid, 1 L of saturated NaHCO3, and 1 L of brine,
and the aqueous layers were re-extracted with 500 mL of
dichloromethane. The combined organic layers were filtered
through a pad of cotton wool and evaporated under reduced
pressure. The crude product was crystallized by dissolving
in 100 mL of methanol and adding 700 mL of diisopropyl ether
to give 50.1 g (79%) of 18a as a white solid homogeneous by
TLC (97:3 dichloromethane:MeOH): 1H-NMR (MeOD) δ 7.3-
7.12 (m, 5H), 4.03 (t, br, J ) 8, 1H), 3.86 (d, J ) 8, 1H), 3.7-
3.55 (m, 1H), 3.66 (s, 3H), 3.0-2.75 (m, 2H), 2.65-2.33 (m,
4H), 1.95 (m, 2H), 1.85-0.76 (m, 10H), 1.40 (s, 9H), 0.86 (2d,
J ) 7, 6H); HPLC tR 17.5 min.
1-Cycloh exyl-2-[(ter t-b u t yloxyca r b on yl)a m in o]-4(S)-
h yd r oxy-5(S)-[(tr iflu or a cetyl)a m in o]-6-p h en yl-2-a za h ex-
a n e (th r eo-16a ). A solution of 60 g (0.232 mol) of a (7:1 threo:
erythro) mixture of N-(trifluoroacetyl)-2(S)-amino-1-phenyl-
3(R)-3,4-epoxybutane (15) and 52.8 g (0.232 mol) of N-1-(tert-
butyloxycarbonyl)-N-2-(methylcyclohexyl)hydrazine (3a ) in 700
mL of methanol was heated to reflux for 16 h. The solvent
was evaporated under reduced pressure to give a yellow oil.
Crystallization was induced by adding 300 mL of dichlo-
romethane to give 60.6 g (54%) of threo-16a as a white solid
homogenous by TLC and HPLC: mp 144-145 °C; TLC (2:1
1
toluene:ethyl acetate) Rf ) 0.66; H-NMR (MeOD) δ 7.25 (m,
5H), 4.18 (t, br, J ) 7, 1H), 3.72 (m, 1H), 3.02 (dd, J ) 7, 13,
1H), 2.95-2.8 (m, 1H), 2.58 (m, 2H), 2.47 (t, J ) 7, 2H), 2.01-
0.9 (m, 11H), 1.43 (s, 9H); HPLC tR 17.8 min. Anal.
(C24H36N3F3O4) C, H, N.
1-(4-Meth oxyph en yl)-2-[(ter t-bu tyloxycar bon yl)am in o]-
4(S)-h ydr oxy-5(S)-[(tr iflu or acetyl)am in o]-6-ph en yl-2-aza-
h exa n e (th r eo-16d ). A solution of 79.7 g (0.307 mol) of a (7:1
threo:erythro) mixture of N-(trifluoroacetyl)-2(S)-amino-1-
phenyl-3(R)-3,4-epoxybutane (15) and 77.6 g (0.307 mol) of
N-1-(tert-butyloxycarbonyl)-N-2-[(4-methoxyphenyl)methyl]hy-
drazine (3d ) in 1 L of ethanol was heated to 80 °C for 16 h.
The solvent was evaporated under reduced pressure, and the
resulting crystalline residue was triturated with diisopropyl
ether to give 83.6 g of pure threo-16d . The concentrated
filtrate afforded upon flash chromatography (1:1 hexane:ethyl
acetate) another 10 g (a total of 59%) of threo-16d : mp 170-
171 °C; TLC (2:1 hexane:ethyl acetate) Rf ) 0.51; 1H-NMR
(MeOD) δ 7.23 (m, 7H), 6.84 (d, J ) 8, 2H), 4.20 (m, 1H), 3.77
(s, 3H), 3.75 (m, 3H), 2.99 (dd, J ) 13, 6, 1H), 2.85 (m, 1H),
2.65 (m, 2H), 1.33 (s, 9H). Anal. (C25H32N3F3O5 ) C, H, N.
1-Cycloh exyl-2-[(ter t-b u t yloxyca r b on yl)a m in o]-4(R)-
h yd r oxy-5(S)-[(tr iflu or a cetyl)a m in o]-6-p h en yl-2-a za h ex-
a n e (er yth r o-16a ). Preparation was as described for threo-
16a . Flash chromatography (toluene:ethyl acetate, 10:1)
yielded 61% of threo-16a and 7% of erythro-16a . Data for
1-Cycloh exyl-2-[(ter t-b u t yloxyca r b on yl)a m in o]-4(S)-
h yd r oxy-5(S)-[[N-(et h oxyca r b on yl)-L-va lin yl]a m in o]-6-
p h en yl-2-a za h exa n e (19a ). Preparation was as described
for 18a . Yield was 1.207 g of 19a (84%) homogeneous by TLC
(95:5 dichloromethane:MeOH): 1H-NMR (MeOD) δ 7.20 (m,
5H), 4.10 (q, J ) 7, 2H), 4.08 (m, 1H), 3.86 (d, J ) 8, 1H), 3.62
(d, br, 1H), 3.0-2.75 (m, 2H), 2.6-2.33 (m, 4H), 1.98 (m, 2H),
1.8-0.88 (m, 10H), 1.40 (s, 9H), 1.25 (t, J ) 7, 3H), 0.86 (2d,
J ) 7, 6H); HPLC tR 17.7 min.
1-Cycloh exyl-2-[(ter t-b u t yloxyca r b on yl)a m in o]-4(S)-
h yd r oxy-5(S)-[[N-[(m eth oxyeth oxy)ca r bon yl]-L-va lin yl]-

Aza-Peptides as Potent HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3211
a m in o]-6-p h en yl-2-a za h exa n e (20a ). Preparation was as
described for 18a . Yield was 1.236 g of 20a (81%) homoge-
neous by TLC (95:5 dichloromethane:MeOH): 1H-NMR (MeOD)
δ 7.22 (m, 5H), 4.18 (t, br, J ) 7, 2H), 4.05 (t, br, J ) 8, 1H),
3.87 (d, J ) 7, 1H), 3.61 (t, J ) 7, 2H), 3.38 (s, 3H), 3.0-2.78
(m, 2H), 2.65-2.36 (m, 4H), 2.0 (m, 2H), 1.8-0.82 (m, 10H),
1.39 (s, 9H), 0.87 (apparent t, J ) 7, 6H); HPLC tR 17.1 min.
1-Cycloh exyl-2-a m in o-4(S)-h yd r oxy-5(S)-[[N-(m eth ox-
yca r bon yl)-L-va lin yl]a m in o]-6-p h en yl-2-a za h exa n e Hy-
d r och lor id e (21a ). A solution of 50 g (91.1 mmol) of 18a in
400 mL of 4 N HCl(g) in dioxane was stirred at room
temperature for 2 h. The solvent was evaporated under
reduced pressure (gas adsorber filled with sodium hydroxide)
and the resulting crude product suspended in 600 mL of
dioxane followed by evaporation to dryness. This latter
procedure was repeated with 600 mL of diisopropyl ether to
give 61 g (99%) of 21a as a white amorphous solid: 1H-NMR
(MeOD) δ 7.3-7.15 (m, 5H), 4.25 (m, 1H), 4.05 (m, 1H), 3.75
(s, 3H), 3.7-3.55 (m, 1H), 3.12-2.6 (m, 6H), 2.05-0.76 (m,
12H), 0.8 (2d, J ) 7, 6H); HPLC tR 11.0 min; FAB MS (M +
H)+ ) 449.
saturated NaHCO3 and brine. The aqueous layers were re-
extracted twice with ethyl acetate, and the organic layers were
dried (Na2SO4), filtered, and concentrated in vacuo. Flash
chromatography (ethyl acetate) afforded 10.21 g (76%) of 30a
as a white solid: TLC (90:10:1 dichloromethane:ethanol:NH3,
concentrated) Rf ) 0.61; 1H-NMR (MeOD) δ 8.48 (d, J ) 9,
1H), 8.20 (2d, J ) 8, 2H), 8.02, 7.86, and 7.70 (3m, 3H), 7.3-
6.95 (m, 5H), 4.93 (t, J ) 6, 1H), 4.08 (m, 1H), 3.60 (d, br, J )
8, 1H), 3.0-2.8 (m, 4H), 2.7-2.3 (m, 4H), 1.37 (s, 9H), 2.0-
0.6 (m, 11H); HPLC tR 16.4 min. Anal. (C36H48N6O6) H, N; C:
calcd, 65.43; found, 65.01.
1-(4-Meth oxyph en yl)-2-[(ter t-bu tyloxycar bon yl)am in o]-
4(S)-h yd r oxy-5(S)-[[N-(q u in olin -2-ylca r b on yl)-L-a sp a r -
a gin yl]a m in o]-6-p h en yl-2-a za h exa n e (30d ). To an ice-cold
stirred solution of 10.00 g (24.0 mmol) of threo-17d and 8.55
g (26.4 mmol) of (quinolin-2-ylcarbonyl)-L-asparagine hydro-
chloride in 600 mL of THF were added 2.9 mL (26.4 mmol) of
NMM, 5.45 g (26.4 mmol) of DCC, and finally 3.56 g (26.4
mmol) of HOBT. After 15 min at 0 °C, the mixture was stirred
for 18 h at room temperature and then filtered. The filtrate
was concentrated in vacuo; the residue was diluted with ethyl
acetate and subsequently washed with two portions of satu-
rated NaHCO3 and brine. The aqueous layers were re-
extracted twice with ethyl acetate, and the organic layers were
dried (Na2SO4), filtered, and concentrated in vacuo. Medium
pressure chromatography (Lichroprep; 9:1 dichloromethane:
methanol) yielded, after trituration with diisopropyl ether, 11.0
g (67%) of 30d : mp 135-137 °C; TLC (9:1 dichloromethane:
1-Cycloh exyl-2-a m in o-4(S)-h yd r oxy-5(S)-[[N-(eth oxy-
ca r b on yl)-L-va lin yl]a m in o]-6-p h en yl-2-a za h exa n e H y-
d r och lor id e (22a ). Preparation was as described for 21a .
Data for 22a : yield 1.14 g (98%); 1H-NMR (MeOD) δ 7.23 (m,
5H), 4.3-4.05 (m, 2H), 4.13 (q, J ) 7, 2H), 3.80 (d, J ) 7, 1H),
3.05-2.7 (m, 6H), 1.98-0.85 (m, 12H), 1.25 (t, J ) 7, 3H), 0.80
(d, J ) 7, 6H); HPLC tR 11.7 min.
1
methanol) Rf ) 0.7; H-NMR (MeOD + CDCl3) δ 8.42 (d, J )
1-Cycloh exyl-2-a m in o-4(S)-h yd r oxy-5(S)-[[N-(m eth ox-
yet h oxyca r b on yl)-L-va lin yl]a m in o]-6-p h en yl-2-a za h ex-
a n e Hyd r och lor id e (23a ). Preparation was as described for
9, 1H), 8.20 and 8.16 (2d, J ) 9, 2H), 7.97 (d, J ) 9, 1H), 7.83
and 7.68 (2m, 2H), 7.25-6.95 (m, 7H), 6.79 (d, J ) 8, 2H),
4.92 (t, J ) 6, 1H), 4.06 (m, 1H), 3.75 (s, 3H), 3.64 (m, 1H),
3.2-2.7 (m, 5H), 2.52 (m, 1H), 1.9-1.5 (m, 2H), 1.27 (s, 9H);
HPLC tR 14.4 min. Anal. (C37H44N6O7) C, H, N.
1
21a . Data for 23a : yield 1.132 g (98%); H-NMR (MeOD) δ
7.25 (m, 5H), 4.20 (m, 4H), 3.77 (d, J ) 8, 1H), 3.65 (s, 3H),
3.57 (m, 2H), 3.63-3.55 (m, 6H), 2.0-0.8 (m, 12H), 0.78 (d, J
) 7, 6H); HPLC tR 10.9 min.
1-Cycloh exyl-2-a m in o-4(S)-h yd r oxy-5(S)-[[N-(qu in olin -
2-ylca r bon yl)-L-a sp a r a gin yl]a m in o]-6-p h en yl-2-a za h ex-
a n e (31a ). A solution of 7.00 g (10.6 mmol) of 30a in 48 mL
of formic acid was stirred for 17 h at room temperature. The
solvent was evaporated under reduced pressure and the
resulting solid partitioned between ethyl acetate and saturated
NaHCO3. The layers were separated, and the aqueous layer
was extracted twice with ethyl acetate. The combined organic
extracts were washed with brine, dried (Na2SO4), filtered, and
concentrated in vacuo, yielding 31a as a white solid (6.2 g,
quantitative): TLC (90:10:1 dichloromethane:ethanol:NH3,
concentrated) Rf ) 0.40; 1H-NMR (MeOD) δ 8.48 (d, J ) 9,
1H), 8.18 (m, 2H), 8.02, 7.84, and 7.72 (3m, 3H), 7.3-6.9 (m,
5H), 4.97 (t, J ) 7, 1H), 4.13 and 3.88 (2m, 2H), 3.0-2.2 (m,
8H), 1.9-0.7 (m, 11H); HPLC tR 11.7 min.
1-(4-Met h oxyp h en yl)-2-a m in o-4(S)-h yd r oxy-5(S)-[[N-
(qu in olin -2-ylca r bon yl)-L-a sp a r a gin yl]a m in o]-6-p h en yl-
2-a za h exa n e (31d ). Preparation was as described for 31a .
The crude product was precipitated from dichloromethane by
addition of diisopropyl ether to give 2.9 g (64%) of 31d as a
white solid which was used in the next step (synthesis of 32d )
without further purification and characterization.
1-Cycloh exyl-2-N-[[(m eth oxycar bon yl)-L-valin yl]am in o]-
4(S)-h yd r oxy-5(S)-[[N-(q u in olin -2-ylca r b on yl)-L-a sp a r -
a gin yl]a m in o]-6-p h en yl-2-a za h exa n e (32a ). Preparation
was as described below for 33a yielded 541 mg (37%) of 32a
as a white solid: TLC (47:3 ethyl acetate:ethanol) Rf ) 0.46;
1H-NMR (MeOD) δ 8.49 (d, J ) 9, 1H), 8.22 (d, J ) 9, 1H),
8.18 (d, J ) 8, 1H), 8.02 (d, J ) 8, 1H), 7.85 and 7.72 (2m,
2H), 7.25-6.9 (m, 5H), 4.9 (m, 1H), 4.07 (m, 1H), 3.75 (d, J )
7, 1H), 3.64 (s, 3H), 3.6 (m, 1H), 2.9-2.4 (m, 8H), 2.0-0.7 (m,
18H); HPLC tR 14.6 min; HR-FAB MS (C38H51N7O7) (M + H)+
calcd 718.3928, obsd 718.3918.
2-Met h yl-4-[[N-(m et h oxyca r b on yl)-L-va lin yl]a m in o]-
6(S)-h yd r oxy-7(S)-[[N-(q u in olin -2-ylca r b on yl)-L-a sp a r -
a gin yl]a m in o]-8-p h en yl-4-a za octa n e (32b). To a mixture
of 0.10 g (0.161 mmol) of 2-methyl-4-(L-valinylamino)-6(S)-
hydroxy-7(S)-[[N-(quinolin-2-ylcarbonyl-L-asparaginyl]amino]-
8-phenyl-4-azaoctane (43b) and 223 mg (1.6 mmol) of K2CO3
in 1.4 mL of dioxane and 0.7 mL of water was added 12 µL
(0.16 mmol) of methyl chloroformate at 0 °C. After 16 h of
stirring at room temperature, an additional 6 µL of methyl
1-Cycloh exyl-2-[[N-(eth oxyca r bon yl)-L-va lin yl]a m in o]-
4(S)-h yd r oxy-5(S)-[[N-(m eth oxyca r bon yl)-L-va lin yl]a m i-
n o]-6-p h en yl-2-a za h exa n e (24a ) (Gen er a l syn th esis of
com p ou n d s 24a -29a u sin g cou p lin g m eth od B). To a
stirred solution of 27.5 g (0.146 mol) of (ethoxycarbonyl)-L-
valine, 52.3 g (0.273 mol) of EDCI, and 24.5 g (0.182 mol) of
HOBT in 700 mL of DMF was added 127 mL (0.91 mol) of
triethylamine. After 30 min a solution of 60 g (91 mmol) of
21a in 300 mL of DMF was added and the mixture allowed to
stir at room temperature for 16 h. The solvent was evaporated
under reduced pressure and the residue dissolved in 1 L of
dichloromethane. This solution was subsequently washed
with 1 L of 10% aqueous citric acid, 1 L of saturated NaHCO3,
and 1 L of brine, and the aqueous layers were re-extracted
with 500 mL of dichloromethane. The combined organic layers
were filtered through a pad of cotton wool and evaporated
under reduced pressure. The crude product was crystallized
by dissolving in 100 mL of dichloromethane and adding 700
mL of diisopropyl ether. Recrystallization by dissolving in 200
mL of hot methanol and adding 600 mL of diisopropyl ether
upon cooling to room temperature gave 39 g of 24a (79%) which
was homogeneous by TLC (95:5 dichloromethane:MeOH): 1H-
NMR (MeOD) δ 7.20 (m, 4H), 7.13 (m, 1H), 4.08 (m, 3H), 3.83
(d, J ) 7, 1H), 3.76 (d, J ) 7, 1H), 3.65 (s, 3H), 3.62 (m, br,
1H), 2.95-2.78 (m, 2H), 2.68-2.43 (m, 4H), 1.93 (m, 3H), 1.70
(m, 4H), 1.41 (m, 1H), 1.23 (t, J ) 7, 3H), 1.18 (s, br, 3H), 0.90
(apparent t, J ) 7, 6H), 0.85 (d, br, J ) 7, 2 × CH3 + 2H);
HPLC tR 15.7 min; FAB MS (M + H)+ ) 620. Anal.
(C32H53N5O7) C, H, N.
1-Cycloh exyl-2-[(ter t-b u t yloxyca r b on yl)a m in o]-4(S)-
h y d r o x y -5(S )-[[N -(q u in o lin -2-y lc a r b o n y l)-L -a s p a r a -
gin yl]a m in o]-6-p h en yl-2-a za h exa n e (30a ). To a cooled
(-10 °C) stirred suspension of 8.00 g (20.4 mmol) of 1-cyclo-
hexyl-2-[(tert-butyloxycarbonyl)amino]-4(S)-hydroxy-5(S)-amino-
6-phenyl-2-azahexane (threo-17a ) and 7.28 g (22.4 mmol) of
(quinolin-2-ylcarbonyl)-L-asparagine hydrochloride27 in 200 mL
of THF were added 3.6 mL (32.7 mmol) of NMM, 4.64 g (22.4
mmol) of DCC, and finally 3.67 g (22.4 mmol) of 3-hydroxy-
1,2,3-benzotriazin-4(3H)-one. After 2 h at -10 °C, the mixture
was stirred for 20 h at room temperature. The mixture was
diluted with ethyl acetate and subsequently washed with

3212 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Fa¨ssler et al.
chloroformate was added to complete the conversion (20 h).
The solution was partitioned between dichloromethane and
saturated NaHCO3. The aqueous layer was separated and
extracted twice with dichloromethane. The organic extracts
were washed with brine, dried (Na2SO4), filtered, and evapo-
rated under reduced pressure. Vigorous stirring in diisopropyl
ether yielded 92 mg (84%) of crystalline 32b: mp 178-180
droxy-7(S)-amino-8-phenyl-4-azaoctane (42b), 1.18 g (6.15
mmol) of EDCI, and 0.83 g (6.15 mmol) of HOBT. After 60
min at 5 °C, the mixture was allowed to stir at room
temperature for 16 h. The suspension was filtered, the filtrate
concentrated under reduced pressure, and the residue dis-
solved in ethyl acetate. This solution was subsequently
washed with saturated NaHCO3 and brine, and the aqueous
layers were re-extracted twice with ethyl acetate. The com-
bined organic layers were dried (Na2SO4), filtered, and con-
centrated in vacuo. The crude product was triturated with
diisopropyl ether, filtered, and redissolved in a small amount
of DMF. It was precipitated again by addition of diisopropyl
ether to give 2.37 g (56%) of 33b: mp 208-210 °C; TLC (9:1
dichloromethane:methanol) Rf ) 0.35; 1H-NMR (MeOD) δ 8.48
(d, J ) 8, 1H), 8.21 and 8.17 (2d, J ) 8, 2H), 8.00 (m, 1H),
7.84 (dd, J ) 8, 1H), 7.70 (dd, J ) 8, 1H), 7.30 (m, 5H), 7.17
(m, 2H), 7.05 (m, 2H), 6.92 (m, 1H), 5.08 (AB, J ) 14, 2H), 4.9
(1H), 4.08 (t, J ) 7, 1H), 3.76 (d, J ) 7, 1H), 3.63 (d, br, J )
9, 1H), 2.9-2.3 (m, 8H), 1.94 and 1.66 (2m, 2H), 0.86 (d, J )
7, 12H); HPLC tR 15.3 min; FAB MS (M + H)+ ) 754. Anal.
(C41H51N7O7) C, H, N.
1-(4-Meth oxyp h en yl)-2-[[N-(ben zyloxyca r bon yl)-L-va li-
n yl]am in o]-4(S)-h ydr oxy-5(S)-[[N-(qu in olin -2-ylcar bon yl)-
L-a sp a r a gin yl]a m in o]-6-p h en yl-2-a za h exa n e (33d ). To a
stirred solution of 1.10 g (1.88 mmol) of 31d in 18.2 mL of 0.3
M NMM in DMF were added 0.52 g (2.07 mmol) of Cbz-L-valine
and 0.784 g (2.07 mmol) of HBTU. After stirring for 17 h at
room temperature, the reaction mixture was concentrated
under reduced pressure. The residue was dissolved in ethyl
acetate and washed with saturated NaHCO3, water, and brine.
The aqueous layers were re-extracted twice with ethyl acetate;
the organic layers were dried (Na2SO4), filtered, and evapo-
rated in vacuo. Crystallization from dichloromethane/diiso-
propyl ether afforded 1.18 g (77%) of 33d : TLC (9:1 dichlo-
romethane:methanol) Rf ) 0.58; 1H-NMR (MeOD) δ 8.46 (d, J
) 9, 1H), 8.18 and 8.15 (2d, J ) 9, 2H), 8.00 (d, J ) 9, 1H),
7.83 and 7.70 (2m, 2H), 7.4-6.7 (m, 14H), 5.06 (s, 2H), 4.92
(m, 1H), 4.08 (m, 1H), 3.72 (s, 3H), 4.0-3.6 and 3.0-2.6 (2m,
10H), 1.67 (m, 1H), 0.64 and 0.60 (2d, J ) 7, 6H); HPLC tR
15.3 min; HR-FAB MS (C45H51N7O8) (M + H)+ calcd 818.3877,
obsd 818.3871.
1-Cycloh exyl-2-[[N-(ter t-bu tyloxyca r bon yl)-L-va lin yl]-
a m in o]-4(S)-h yd r oxy-5(S)-[[N-(qu in olin -2-ylca r bon yl)-L-
a sp a r a gin yl]a m in o]-6-p h en yl-2-a za h exa n e (34a ). To a
stirred solution of 5.93 g (10.6 mmol) of 31a in 244 mL of 0.125
M solution of NMM in acetonitrile were added 4.40 g (11.6
mmol) of HBTU and 2.52 g (11.6 mmol) of Boc-L-valine. After
stirring for 17 h, the precipitated crude product (5 g) was
filtered and washed with acetonitrile. Further washing with
hot acetonitrile, ether, and hexane yielded 4.15 g (52%) of 34a .
A 3 g portion of the product was purified by flash chromatog-
raphy (49:1 dichloromethane:methanol) to give 2.32 g of pure
34a : TLC (9:1 dichloromethane:methanol) Rf ) 0.6; 1H-NMR
(MeOD + CDCl3) δ 8.37 (d, J ) 8, 1H), 8.20 and 8.14 (2d, J )
8, 2H), 7.92 (d, J ) 8, 1H), 7.80 (dd, J ) 8, 1H), 7.64 (dd, J )
8, 1H), 7.2-6.9 (m, 5H), 4.87 (t, J ) 7, 1H), 4.02 (m, 1H), 3.69
(m, 1H), 3.53 (m, 1H), 2.9-2.4 (m, 8H), 1.40 (s, 9H), 2.0-0.7
(m, 18H); HPLC tR 16.4 min; FAB MS (M + H)+ ) 760. Anal.
(C41H57N7O7) C, H, N.
1-Cycloh exyl-2-[[N-(eth oxyca r bon yl)-L-va lin yl]a m in o]-
4(S)-h yd r oxy-5(S)-[[N-(q u in olin -2-ylca r b on yl)-L-a sp a r -
a gin yl]a m in o]-6-p h en yl-2-a za h exa n e (36a ). Preparation
as described for 33a , using ethyl chloroformate instead of
benzyl chloroformate, yielded 67 mg (48%) of 36a as a white
solid: TLC (19:1 dichloromethane:methanol) Rf ) 0.2; 1H-NMR
(MeOD + CDCl3) δ 8.43 (d, J ) 9, 1H), 8.22 and 8.17 (2d, J )
8, 2H), 7.97 (d, J ) 8, 1H), 7.84 and 7.71 (2m, 2H), 7.3-6.8
(m, 5H), 4.9 (m, 1H), 4.07 (m, 3H), 3.75 and 3.60 (2m, 2H),
3.0-2.4 (m, 8H), 2.0-0.7 (m, 21H); HPLC tR 15.3 min; HR-
FAB MS (C39H53N7O7) (M + H)+ calcd 732.4085, obsd 732.4114.
[(Ben zyloxyca r b on yl)-L-va lin yl]h yd r a zin eca r b oxylic
Acid ter t-Bu tyl Ester (37). To a stirred solution of 15.0 g
(59.7 mmol) of (benzyloxycarbonyl)-L-valine and 7.9 mL (71.6
mmol) of NMM in 200 mL of ethyl acetate were added 12.6 g
(65.7 mmol) of EDCI and 8.87 g (65.7 mmol) of HOBT. After
10 min 8.7 g (65.8 mmol) of hydrazinecarboxylic acid tert-
1
°C; TLC (9:1 dichloromethane:methanol) Rf ) 0.46; H-NMR
(MeOD) δ 8.49 (d, J ) 8, 1H), 8.21 and 8.17 (2d, J ) 8, 2H),
8.02 (d, J ) 8, 1H), 7.86 (dd, J ) 8, 1H), 7.70 (dd, J ) 8, 1H),
7.3-6.9 (m, 5H), 4.9 (1H), 4.08 (t, J ) 7, 1H), 3.75 (m, 1H),
3.65 (m, 1H), 3.62 (s, 3H), 2.95-2.4 (m, 8H), 1.9 and 1.65 (2m,
2H), 0.88 (m, 12H); HPLC tR 13.2 min; HR-FAB MS
(C35H47N7O7) (M + H)+ calcd 678.3615, obsd 678.3644.
1-(4-Meth oxyp h en yl)-2-[[N-(m eth oxyca r bon yl)-L-va li-
n yl]am in o]-4(S)-h ydr oxy-5(S)-[[N-(qu in olin -2-ylcar bon yl)-
L-a sp a r a gin yl]a m in o]-6-p h en yl-2-a za h exa n e (32d ). To a
stirred solution of 385 mg (2.20 mmol) of N-(methoxycarbonyl)-
L-valine in 13.3 mL of a 0.3 M solution of NMM in DMF were
added 421 mg (2.20 mmol) of EDCI and 385 mg (2.85 mmol)
of HOBT. After 15 min 1.17 g (2.0 mmol) of 31d was added,
and the reaction mixture was stirred for 18 h. The solution
was concentrated in vacuo and the residue partitioned between
ethyl acetate and saturated NaHCO3. The aqueous layer was
separated and extracted twice with ethyl acetate. The organic
extracts were washed with water and brine, dried (Na2SO4),
filtered, and evaporated under reduced pressure. Precipitation
from dichloromethane with diisopropyl ether afforded 0.56 g
(37%) of crystalline 32d : mp 160-162 °C; TLC (9:1 dichlo-
romethane:methanol) Rf ) 0.56; 1H-NMR (MeOD) δ 8.43 (d, J
) 9, 1H), 8.19 and 8.16 (2d, J ) 9, 2H), 7.98 (d, J ) 9, 1H),
7.83 and 7.68 (2m, 2H), 7.3-6.7 (m, 9H), 4.92 (m, 1H), 4.08
(m, 1H), 3.73 and 3.62 (2s, 2 × CH3), 4.0-3.5 and 3.0-2.6 (2m,
10H), 1.67 (m, 1H), 0.63 and 0.59 (2d, J ) 7, 6H); HPLC tR
13.2 min; HR-FAB MS (C39H47N7O8) (M + H)+ calcd 742.3564,
obsd 742.3547.
1-Cycloh exyl-2-[[N-(ben zyloxyca r bon yl)-L-va lin yl]a m i-
n o]-4(S)-h yd r oxy-5(S)-[[N-(q u in olin -2-ylca r b on yl)-L-a s-
p a r a gin yl]a m in o]-6-p h en yl-2-a za h exa n e (33a ). A solution
of 302 mg (0.397 mmol) of 34a in 2 mL of formic acid was
stirred for 22 h at room temperature. The solvent was
evaporated under reduced pressure and the resulting solid
partitioned between dichloromethane and saturated NaHCO3.
The layers were separated, and the aqueous layer was
extracted twice with dichloromethane. The organic extracts
were washed with brine, dried (Na2SO4), filtered, and concen-
trated in vacuo, yielding 0.28 g (quantitative) of 1-cyclohexyl-
2-(L-valinylamino)-4(S)-hydroxy-5(S)-[[N-(quinolin-2-ylcarbonyl)-
L-asparaginyl]amino]-6-phenyl-2-azahexane (35a ) as a white
solid which was used without further purification and char-
acterization.
Crude 35a was dissolved in a mixture of 2.9 mL of dioxane
and 2.7 mL of water. Then 528 mg (3.8 mmol) of K2CO3 was
added followed by a solution of 126 µL (0.79 mmol) of benzyl
chloroformate in 1.5 mL of dioxane. After stirring for 2 h at
room temperature, the reaction mixture was partitioned
between dichloromethane and saturated NaHCO3. The aque-
ous layer was separated and extracted twice with dichlo-
romethane. The organic extracts were washed with brine,
dried (Na2SO4), filtered, and evaporated under reduced pres-
sure. Flash chromatography (3% f 10% methanol in dichlo-
romethane) yielded 186 mg (59%) of 33a as a white solid: TLC
(17:3 dichloromethane:ethanol) Rf ) 0.42; 1H-NMR (MeOD) δ
8.48 (d, J ) 9, 1H), 8.21 and 8.18 (2d, J ) 8, 2H), 8.02 (d, J )
8, 1H), 7.86 (dd, J ) 8, 1H), 7.70 (dd, J ) 8, 1H), 7.4-6.9 (m,
10H), 5.09 (s, 2H), 4.9 (m, 1H), 4.08 (m, 1H), 3.77 (d, J ) 7,
1H), 3.63 (m, 1H), 2.9-2.4 (m, 8H), 2.0-0.6 (m, 18H); HPLC
tR 16.5 min; FAB MS (M + H)+ ) 794. Anal. (C44H55N7O7) C,
H; N: calcd, 12.35; found, 11.93.
2-Meth yl-4-[[N-(ben zyloxyca r bon yl)-L-va lin yl]a m in o]-
6(S)-h yd r oxy-7(S)-[[N-(q u in olin -2-ylca r b on yl)-L-a sp a r -
a gin yl]a m in o]-8-p h en yl-4-a za octa n e (33b). To a stirred
suspension of 1.99 g (6.15 mmol) of (quinolin-2-ylcarbonyl)-L-
asparagine hydrochloride and 1.0 mL (9 mmol) of NMM in 100
mL of THF at 5 °C were added 2.71 g (5.59 mmol) of 2-methyl-
4-[[N -(ben zyloxyca r bon yl)-L -va lin yl]a m in o]-6(S )-h y-

Aza-Peptides as Potent HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3213
butyl ester was added. The reaction mixture was stirred for
18 h and then was diluted with ethyl acetate and saturated
NaHCO3. The aqueous layer was separated and extracted
twice with ethyl acetate. The organic extracts were washed
with water and brine, dried (Na2SO4), filtered, and evaporated
under reduced pressure. Vigorous stirring in diisopropyl ether
afforded 20.7 g (95%) of crystalline 37: mp 142-143 °C; TLC
layers were separated, and the aqueous layer was extracted
twice with ethyl acetate. The organic extracts were washed
with water and brine, dried (Na2SO4), filtered, and concen-
trated in vacuo. Medium pressure chromatography (Lichro-
prep; 0% f 8% methanol in dichloromethane) yielded 4.00 g
(68%) of 42b as an oil: TLC (9:1 dichloromethane:methanol)
Rf ) 0.3; 1H-NMR (MeOD) δ 7.4-7.15 (m, 10H), 5.09 (AB, J )
13, 2H), 3.78 (d, J ) 7, 1H), 3.53 and 3.00 (2m, 2H), 2.93-
2.73 (m, 3H), 2.69-2.56 (m, 2H), 2.43 (dd, J ) 13, 8, 1H), 1.98
(oct, J ) 7, 1H), 1.70 (non, J ) 7, 1H), 0.93 and 0.90 (2d, J )
7, 12H); HPLC tR 14.5 min. Anal. (C27H40N4O4‚0.41H2O) C,
H, N.
1
(9:1 dichloromethane:methanol) Rf ) 0.57; H-NMR (MeOD)
δ 7.35 (m, 5H), 5.08 (s, 2H), 3.95 (m, 1H), 2.05 (m, 1H), 1.44
(s, 9H), 1.00 and 0.96 (2d, J ) 7, 6H). Anal. (C18H27N3O5) C,
H, N.
[(Ben zyloxyca r bon yl)-L-va lin yl]h yd r a zin e (38). A solu-
tion of 20.7 g (56.6 mmol) of 37 in 260 mL of dioxane and 142
mL of 4 N HCl(g) in dioxane was stirred for 28 h. The reaction
mixture was lyophilized. The white powder was dissolved in
dichloromethane and saturated NaHCO3. The aqueous layer
was separated and extracted twice with dichloromethane. The
organic extracts were washed with saturated NaHCO3 and
brine, dried (Na2SO4), filtered, and evaporated in vacuo.
Vigorous stirring in diisopropyl ether yielded 13.9 g (92%) of
crystalline 38: mp 178-180 °C; TLC (9:1 dichloromethane:
methanol) Rf ) 0.50; 1H-NMR (DMSO-d6) δ 9.13 (s, 1H), 7.35
(m, 6H), 5.03 (s, 2H), 4.25 (s, 2H), 3.75 (t, J ) 8, 1H), 1.90 (m,
1H), 1.44 (s, 9H), 0.86 and 0.83 (2d, J ) 7, 6H). Anal.
(C13H19N3O3) C, H, N.
2-Me t h yl-4-(L-va lin yla m in o)-6(S )-h yd r oxy-7(S )-[[N -
(q u in olin -2-ylca r bon yl-L-a sp a r a gin yl]a m in o]-8-p h en yl-
4-a za octa n e (43b). A solution of 1.00 g (1.33 mmol) of 33b
in 50 mL of methanol and 0.25 g of 10% Pd/C was stirred under
1 atm of H2 for 6 h. The mixture was filtered through Celite,
and the filtrate was concentrated in vacuo. Recrystallization
by dissolving in dichloromethane and adding diisopropyl ether
afforded 0.77 g (93%) of 43b: mp 157-160 °C; TLC (9:1
dichloromethane:methanol) Rf ) 0.3; 1H-NMR (MeOD) δ 8.50
(d, J ) 8, 1H), 8.22 and 8.18 (2d, J ) 8, 2H), 8.03 (d, J ) 8,
1H), 7.86 (dd, J ) 8, 1H), 7.72 (dd, J ) 8, 1H), 7.30-6.95 (m,
5H), 4.9 (m, 1H), 4.10 (t, J ) 7, 1H), 3.62 (m, 1H), 3.0-2.4 (m,
9H), 1.84 and 1.68 (2m, 2H), 0.88 (m, 12H); HPLC tR 10.8 min;
FAB MS (M + H)+ ) 620. Anal. (C33H45N7O5‚H2O) C, H, N.
N-(Meth oxyca r bon yl)-L-va lin e (Gen er a l m eth od for
th e syn th esis of ca r ba m a te d er iva tives of L-va lin e). To
a solution of 7.0 g (60 mmol) of L-valine in a mixture of 100
mL of 2 N aqueous NaOH and 30 mL of dioxane was slowly
added at 0 °C 5.67 g (60 mmol) of methyl chloroformate. Upon
completion of the addition, the mixture was warmed to room
temperature and allowed to stir during 16 h. The reaction
mixture was then extracted with 100 mL of dichloromethane,
and the aqueous layer was acidified to pH 2 with 4 N aqueous
HCl. It was then extracted twice with 100 mL of dichlo-
romethane. Filtration of the organic layer through a pad of
cotton wool followed by evaporation to dryness gave 5.01 g
(48%) of N-(methoxycarbonyl)-L-valine as a white amorphous
solid: mp 108-109 °C; 1H-NMR (MeOD) δ 4.05 (d, J ) 5, 1H),
2-Meth yl-1-p r op a n on e[(Ben zyloxyca r bon yl)-L-va lin yl]-
h yd r a zon e (39b). A solution of 13.9 g (52.4 mmol) of 38 and
4.2 mL (46 mmol) of isobutyric aldehyde in 250 mL of ethanol
was heated for 4 h at 60 °C. After cooling to room temperature,
the white precipitate was filtered and washed with diisopropyl
ether, yielding 12.8 g (87%) of crystalline 39b: mp 151-153
1
°C; TLC (9:1 dichloromethane:methanol) Rf ) 0.83; H-NMR
(MeOD) δ 7.32 (m, 6H), 5.07 (s, 2H), 3.86 (d, J ) 8, 1H), 2.53
(sept, J ) 7, 1H), 2.04 (m, 1H), 1.10 (d, J ) 7, 6H), 0.97 and
0.95 (2d, J ) 7, 6H). Anal. (C17H25N3O3) C, H, N.
N-1-[(Ben zyloxyca r b on yl)-L-va lin yl]-N-2-isob u t ylh y-
d r a zin e (40b). A solution of 7.17 g (41.6 mmol) of p-TsOH
in 100 mL of THF was added dropwise in 15 min to a stirred
solution of 12.8 g (41.6 mmol) of 39b and 2.61 g (41.6 mmol)
of NaBH3CN in 150 mL of THF. Stirring was continued for 1
h at room temperature. The resulting suspension was diluted
with ethyl acetate and washed subsequently with brine,
saturated NaHCO3, and brine. The aqueous layers were re-
extracted twice with ethyl acetate; the organic layer was dried
(Na2SO4), filtered, and concentrated under reduced pressure.
The resulting yellow wax was taken up in 125 mL of 1 N NaOH
and stirred at ambient temperature for 90 min. Then the
white suspension was neutralized with 2 N HCl and extracted
three times with dichloromethane. The organic layers were
washed with brine, dried (Na2SO4), filtered, and concentrated
in vacuo. Vigorous stirring in diisopropyl ether afforded 11.75
g (87%) of crystalline 40b: mp 152 °C; TLC (2:1 hexane:ethyl
3.65 (s, 3H), 2.18 (m, 1H), 0.97 (t, J ) 7, 6H). Anal. (C7H13
NO4) C, H, N.
-
(Qu in olin -2-ylca r bon yl)-L-a sp a r a gin e ter t-Bu tyl Ester .
To an ice-cooled stirred solution of 123.3 g (0.66 mol) of
L-asparagine tert-butyl ester in 3.7 L of THF was added 162.26
g (0.786 mol) of DCC. After stirring for 10 min, 106.1 g (0.786
mol) of HOBT and 125.03 g (0.72 mol) of quinoline-2-carboxylic
acid were added. Stirring was continued for a further 18 h;
then the reaction mixture was filtered. The filtrate was
concentrated in vacuo and the resulting solid partially redis-
solved in 2.5 L of ethyl acetate; 2 L of saturated NaHCO3 was
added, and the heterogeneous mixture was filtered again. The
aqueous layer of the filtrate was separated and extracted twice
with 1 L of ethyl acetate. The organic layers were washed
with 2 L of saturated NaHCO3, water, and brine (2×), dried
(Na2SO4), filtered, and concentrated under reduced pressure
to afford a brown oil. Crystallization from 0.3 L of warm
dichloromethane and 0.39 L of hexane yielded 204 g (90%) of
(quinolin-2-ylcarbonyl)-L-asparagine tert-butyl ester: TLC (3:1
ethyl acetate:hexane) Rf ) 0.15; 1H-NMR (MeOD) δ 8.47 (d, J
) 9, 1H), 8.19 and 8.17 (2d, J ) 9, 2H), 8.00 (d, J ) 8, 1H),
7.84 (q, J ) 8, 1H), 7.69 (dd, J ) 8, 1H), 4.9 (t, J ) 6, 1H),
3.03 (dd, J ) 16, 6, 1H), 2.88 (dd, J ) 16, 6, 1H), 1.49 (s, 9H);
FAB MS (M + H)+ ) 344.
1
acetate) Rf ) 0.44; H-NMR (MeOD) δ 7.35 (m, 5H), 5.09 (s,
2H), 3.83 (d, J ) 8, 1H), 2.71 (d, J ) 7, 2H), 2.02 (oct, J ) 7,
1H), 1.84 (non, J ) 7, 1H), 0.95 (d, J ) 7, 12H); HPLC tR 14.5
min.
2-Meth yl-4-[[N-(ben zyloxyca r bon yl)-L-va lin yl]a m in o]-
6(S)-h ydr oxy-7(S)-[(ter t-bu tyloxycar bon yl)am in o]-8-ph en -
yl-4-a za octa n e (41b). A suspension of 5.5 g (17.1 mmol) of
40b and 4.5 g (17.1 mmol) of N-(tert-butyloxycarbonyl)-2(S)-
amino-1-phenyl-3(R)-3,4-epoxybutane15a,b in 150 mL of ethanol
was stirred at 80 °C for 16 h. The resulting solution was
concentrated under reduced pressure. Crystallization from 1:1
diisopropyl ether:hexane yielded 8.0 g (80%) of 41b: mp 195-
198 °C; TLC (2:1 hexane:ethyl acetate) Rf ) 0.3; 1H-NMR
(MeOD) δ 7.4-7.1 (m, 10H), 5.09 (AB, J ) 14, 2H), 3.76 (d, J
) 7, 1H), 3.70 and 3.62 (2m, 2H), 2.85-2.38 (m, 6H), 1.97 (oct,
J ) 7, 1H), 1.69 (non, J ) 7, 1H), 1.33 (s, 9H), 0.90 (m, 12H);
HPLC tR 17.3 min. Anal. (C32H48N4O6) C, H, N.
2-Meth yl-4-[[N-(ben zyloxyca r bon yl)-L-va lin yl]a m in o]-
6(S)-h yd r oxy-7(S)-a m in o-8-p h en yl-4-a za octa n e (42b). A
mixture of 7.0 g (11.9 mmol) of 41b in 80 mL of formic acid
was stirred for 16 h at ambient temperature. The solvent was
evaporated under reduced pressure and the resulting solid
partitioned between ethyl acetate and saturated NaHCO3. The
(Qu in olin -2-ylca r bon yl)-L-a sp a r a gin e Hyd r och lor id e.27
To a stirred solution of 203 g (0.59 mol) of (quinolin-2-
ylcarbonyl)-L-asparagine tert-butyl ester in 2.03 L of dioxane
was added 2.03 L of 4 N HCl(g) in dioxane at 10 °C. The
solution was stirred for 3 h at 45 °C and then cooled to room
temperature. The resulting precipitate was separated, resus-
pended in 1.5 L of ether, and stirred vigorously. Filtration
afforded 168 g (88%) of crystalline (quinolin-2-ylcarbonyl)-L-
asparagine hydrochloride: mp 122-125 °C; 1H-NMR (MeOD)
δ 8.50 (d, J ) 8, 1H), 8.21 and 8.17 (2d, J ) 8, 2H), 8.00 (d, J
) 8, 1H), 7.83 (q, J ) 8, 1H), 7.68 (q, J ) 8, 1H), 5.12 (t, J )
6, 1H), 3.02 (d, J ) 6, 2H).

3214 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Fa¨ssler et al.
Biology. HIV p r otea se in h ibition a ssa y: HIV-1 protease
was expressed and purified according to the method described
by Billich et al.28 A peptide cleavage assay was performed
using the icosapeptide H-Arg-Arg-Ser-Asn-Gln-Val-Ser-Gln-
Asn-Tyr-Pro-Ile-Val-Gln-Asn-Ile-Gln-Gly-Arg-Arg-OH as de-
scribed by Richards et al.29 and monitored by HPLC. The
inhibitors were dissolved in DMSO, diluted with 20 mM
â-morpholinoethanesulfonic acid (MES) buffer pH 6.0, and
added to the icosapeptide (122 µM) in 20 mM MES buffer, pH
6.0. DMSO concentration did not exceed 5% in the assay
system. The reaction was initiated by adding 10 µL of protease
solution and was terminated after an incubation time of 1 h
at 37 °C by the addition of 10 µL of 0.3 M HClO4. The samples
were centrifuged at 10000g during 5 min, and 20 mL of the
supernatant was injected onto a reversed phase HPLC column
(Nucleosil C18, 5 µm; Macherey & Nagel). The cleavage
products were analyzed after elution with a water/acetonitrile
gradient containing 0.1% TFA, a flow rate of 1 mL/min, and
detection at 215 nm. Ro 31-895919 served as a reference
compound in this assay with an IC50 of 6.3 nM. The standard
error was determined to be (13%.
An tivir a l a ssa y: The dose-response curves for HIV pro-
tease inhibitors were determined in MT-2 cells, seeded in 96-
well round bottom plates (Nunc, Kamstrup, Denmark) at 2 ×
104/well in 50 µL of complete medium. Serial dilutions from
DMSO stock solutions of compound were freshly prepared in
complete medium prior to tests. The compound was added in
a volume of 50 µL and HIV-1/MN (2800 TCID50/well) in a
volume of 100 µL. The assays were performed in triplicates.
After 4 days of incubation at 37 °C in a fully humidified
atmosphere containing 5% CO2, 10 µL samples of the culture
supernatants were collected, and virus production was mea-
sured as virus-associated reverse transcriptase (RT) activity
in these samples. The antiviral effect of the compound was
determined as percent RT reduction as compared to the virus
controls. The ED50 was determined as the concentration of
compound required to inhibit 50% of RT production in this
assay, similarly ED90 as 90% inhibition of RT formation. The
assays were performed in triplicates using Ro 31-895919 as a
reference compound.
Mrs. S. Maggio, and Mr. D. Wyss for their skillful
experimental work in chemistry and Mrs. B. Bohler,
Mrs. P. Frei, Mr. G. Goutte, Mr. P. Hauser, and Mrs.
C. Kowalik for their capable technical assistance in
biological testing.
Su p p or tin g In for m a tion Ava ila ble: 1H-NMR (200 MHz)
data for compounds 4b-l, 5b-l, 7e,f,i, and 12a and elemental
analysis data, mass spectra data, and HPLC data for com-
pounds 7-43 (5 pages). Ordering information is given on any
current masthead page.
Refer en ces
(1) De Clercq, E. Toward Improved Anti-HIV Chemotherapy: Thera-
peutic Strategies for Intervention with HIV Infections. J . Med.
Chem. 1995, 38, 2491-2517.
(2) (a) Darke, P. L.; Huff, J . R. HIV Protease as an Inhibitor Target
for the Treatment of AIDS. Adv. Pharmacol. 1994, 25, 399-454.
(b) Lang, M.; Ro¨sel, J . HIV-1 Protease Inhibitors: Development,
Status, and Potential Role in the Treatment of AIDS. Arch.
Pharm. (Weinheim, Ger.) 1993, 326, 921-924. (c) Martin, J . A.
Recent advances in the design of HIV proteinase inhibitors.
Antiviral Res. 1992, 17, 265-278. (d) Debouck, C. The HIV-1
Protease as a Therapeutic Target for AIDS. AIDS Res. Human
Retroviruses 1992, 8, 153-164. (e) Huff J . R. HIV Protease: A
Novel Chemotherapeutic Target for AIDS. J . Med. Chem. 1991,
34, 2305-2314.
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P h a r m a cok in etic stu d ies: All compounds were adminis-
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(hydroxypropyl)-â-cyclodextrin (Wacker Chemie, Munich, FRG)/
5% DMSO in water. Twenty-eight day old female Balb/c mice
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Each mouse received 200 µL resulting in an average dose of
120 mg of compound/kg of body weight. Four mice were used
per time point (30, 60, 90, and 120 min). Mice were sacrificed,
and heart blood was collected into heparinized tubes at given
time points after administration. The blood samples were
centrifuged (10000g, 5 min); the plasma was removed and
mixed with an equal volume of acetonitrile. The protein
precipitate was removed by centrifugation (10000g, 5 min) and
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ether fractions were pooled and dried under vacuum. The
residue was dissolved in 50% acetonitrile/water prior to
analysis by reversed phase HPLC. Chromatographic analysis
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compound under investigation. The flow rate was 1 mL/min.
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pounds. Furthermore, transport into the cell is suggested to

3216 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Fa¨ssler et al.
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