New aza-dipeptide analogues as potent and orally absorbed HIV-1 protease inhibitors: Candidates for clinical development

Article abstract of DOI:10.1021/jm970873c

On the basis of previously described X-ray studies of an enzyme/aza- dipeptide complex, aza-dipeptide analogues carrying N-(bis-aryl-methyl) substituents on the (hydroxethyl)hydrazine moiety have been designed and synthesized as HIV-1 protease inhibitors. By using either equally (12) or orthogonally (13) protected dipeptide isosteres, symmetrically and asymmetrically acylated aza-dipeptides can be synthesized. This approach led to the discovery of very potent inhibitors with antiviral activities (ED₅₀) in the subnanomolar range. Acylation of the (hydroxethyl)hydrazine dipeptide isostere with the L-tert-leucine derivative 29 increased the oral bioavailability significantly when compared to the corresponding L-valine or L-isoleucine derivatives. The bis (L-tert-leucine) derivatives CGP 75355, CGP 73547, CGP 75136, and CGP 75176 combine excellent antiviral activity with high blood concentration after oral administration. Furthermore, they show no cross-resistance with saquinavir-resistant strains and maintain activity against indinavir-resistant ones. Consequently they qualify for further profiling as potential clinical candidates.

Full text of DOI:10.1021/jm970873c

J . Med. Chem. 1998, 41, 3387-3401
3387
New Aza -Dip ep tid e An a logu es a s P oten t a n d Or a lly Absor bed HIV-1 P r otea se
In h ibitor s: Ca n d id a tes for Clin ica l Develop m en t
Guido Bold,* Alexander Fa¨ssler, Hans-Georg Capraro, Robert Cozens, Thomas Klimkait, J anis Lazdins,
J u¨rgen Mestan, Bernard Poncioni, J ohannes Ro¨sel, David Stover, Marina Tintelnot-Blomley, Figan Acemoglu,
Werner Beck, Eugen Boss, Martin Eschbach, Thomas Hu¨rlimann, Elvira Masso, Serge Roussel,
Katharina Ucci-Stoll, Dominique Wyss, and Marc Lang
Research Laboratories Cancer and Infectious Diseases, Ciba-Geigy AG, CH-4002 Basel, Switzerland†
Received December 31, 1997
On the basis of previously described X-ray studies of an enzyme/aza-dipeptide complex,8 aza-
dipeptide analogues carrying N-(bis-aryl-methyl) substituents on the (hydroxethyl)hydrazine
moiety have been designed and synthesized as HIV-1 protease inhibitors. By using either
equally (12) or orthogonally (13) protected dipeptide isosteres, symmetrically and asymmetri-
cally acylated aza-dipeptides can be synthesized. This approach led to the discovery of very
potent inhibitors with antiviral activities (ED50) in the subnanomolar range. Acylation of the
(hydroxethyl)hydrazine dipeptide isostere with the L-tert-leucine derivative 29 increased the
oral bioavailability significantly when compared to the corresponding L-valine or L-isoleucine
derivatives. The bis(L-tert-leucine) derivatives CGP 75355, CGP 73547, CGP 75136, and CGP
75176 combine excellent antiviral activity with high blood concentration after oral administra-
tion. Furthermore, they show no cross-resistance with saquinavir-resistant strains and
maintain activity against indinavir-resistant ones. Consequently they qualify for further
profiling as potential clinical candidates.
Ch a r t 1. Aza-Dipeptide Isosteres as Pseudosymmetric
HIV-1 Protease Inhibitors
In tr od u ction
The causative agent for the pathogenesis of the
acquired immunodeficiency disease syndrome (AIDS) is
the human immunodeficiency virus (HIV). For the
maturation of viral particles to a fully infectious virus,
it has been proven that a functional viral protease (HIV
protease), an enzyme that is responsible for the process-
ing of polyproteins to structural proteins and viral
enzymes, is essential.1 This made the HIV protease a
promising target for an effective AIDS therapy. Recent
clinical results from studies with HIV protease inhibi-
tors as single therapy or in combination with reverse
transcriptase inhibitors showed indeed excellent efficacy
in AIDS patients.2 During the past decade, many
different classes of HIV-1 protease inhibitors have been
synthesized, showing excellent profiles.3 Yet it re-
mained a challenge to introduce new potent and orally
bioavailable inhibitors that show activity against mu-
tant strains of HIV to overcome cross-resistance in
patients.4 In this paper we describe aza-dipeptides with
bis-aryl substituents as ligands for the P1′ pocket, which
combine excellent antiviral activity against wild-type
and mutant HIV strains with high bioavailability in
mice after oral administration. Additionally, the syn-
thesis of these aza-dipeptides follows a series of trivial
transformations, which is considered to be a major
advantage compared to available drugs for large-scale
production.
stimulated the design of pseudo-symmetric inhibitors
of the aza-dipeptide structure type (see Chart 1).6
Recently we published on a novel series of aza-dipeptide
analogues as HIV protease inhibitors with oral bioavail-
ability.7 As an example to describe their profile best,
Chart 1 illustrates compound 1, an orally well-absorbed
aza-dipeptide isostere with only slight antiviral activity
(see Table 1), and compound 2, a highly potent inhibitor
of viral replication in cellular assays with poor oral
bioavailability. However, since the ultimate goal of
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,5
† Present address: Research Oncology, NOVARTIS Pharma AG,
CH-4002 Basel, Switzerland.
S0022-2623(97)00873-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/01/1998

3388 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Ta ble 1. Antiviral Activity of the HIV Protease Inhibitorsa
Bold et al.
a
Activity was measured as previously described.7 The enzymatic activity (IC50) was determined with respect to the icosapeptide H-Arg-
Arg-Ser-Asn-Gln-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Asn-Ile-Gln-Gly-Arg-Arg-OH by HPLC method. The antiviral effect of the compounds
is determined as percent reduction of the reverse transcriptase (RT) activity in HIV-1/MN-infected MT-2 cells. HIV-1/MN stocks were
prepared from cell culture supernatants of the permanently infected cell line H9/HIV-1/MN obtained through the AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, NIH. The ED50 indicates the concentration of compound required to inhibit 50%
of RT production in this assay; similarly ED90 reflects 90% inhibition of RT formation. The pharmacokinetic studies were performed in
mice, and drug concentrations in blood samples were analyzed by reversed-phase HPLC 30 min (f c30) and 90 min (f c90) after oral
application of 120 mg/kg in a standardized formulation: 5% DMSO, 19% hydroxypropyl-â-cyclodextrin in water (for details see the
Experimental Section). The data represent averages of at least three determinations.
combining excellent antiviral activity with good oral
bioavailability was not achieved, we evaluated the
available information from our X-ray structure of an
enzyme/aza-dipeptide complex.8 This structural data
suggested that there should be available space for larger
P1′ substituents, thus enabling the design of derivatives
with higher affinity for the enzyme and hopefully
improved physicochemical properties.

New Aza-Dipeptide Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3389
Sch em e 1. Synthesis of Benzylhydrazine Building Blocks 9a -e,g Starting from Aldehydesa
a
Conditions: (a) Mg, THF; (b) 2-bromopyridine, THF, [1,3-bis(diphenylphosphino)propane]nickel(II) chloride, DIBAH; (c) H3O+; (d)
2-bromothiazole, THF, [1,3-bis(diphenylphosphino)propane]nickel(II) chloride; (e) thiazole, Pd(PPh3)4, CH3CO2K, DMA, 12h 150 °C; (f)
NaN3, LiCl, methoxyethanol, 6 h, vV; (g) CH3I, K2CO3, DMF/dioxane; (h) tert-butyloxycarbonylhydrazine, ethanol or 2-propanol, vV; (i) H2,
Pd/C, MeOH; (j) NaCNBH3, TsOH, THF, r.t.
Ch em istr y
anobenzaldehyde (5) gave 4-(tetrazol-5-yl)benzaldehyde
(6) in good yields. Methylation with methyl iodide then
led regioselectively to the 2-methyl-2H-tetrazole deriva-
tive 7e. By heating the aldehydes 7a -e,g with tert-
butyloxycarbonylhydrazine, the hydrazones 8a-e,g were
obtained. Hydrogenation (for 8a ,b,g) or reduction with
sodium cyanoborohydride10 for the more sensitive sub-
strates 8c-e11 provided the benzyl-hydrazine building
blocks 9a -e,g.
The general route to synthesize the aza-dipeptide
isosteres is outlined in Scheme 2. Opening of the N-Boc-
protected epoxide 106b with the 4-substituted benzyl-
hydrazine building blocks 9a -c,e led to symmetrically
acylated aza-dipeptide mimetics, whereas the same
reaction applied to the N-trifluoroacetyl-protected
epoxides 117 and 9a -d ,g opens access to the asym-
metrically acylated series.
The required N-(tert-butyloxycarbonyl)-2(S)-amino-1-
phenyl-3(R)-3,4-epoxybutane (10)6b as a source for the
P1 substituent and the transition-state hydroxyl group
of the dipeptide isostere was prepared according to
known procedures:12 Peterson olefination of N-Boc-
phenylalaninal,13 reintroduction of the Boc protecting
group, and epoxidation gave an 83:17 mixture of the
threo- and erythro-isomers, which could be separated by
crystallization. Nucleophilic attack of 9a -c,e on the
epoxide 10 gave the symmetrically protected dipeptide
isosteres 12a -c,e. Both of the Boc protecting groups
were simultaneously cleaved off by acidic treatment.
The best condition for the deprotection of 12b is cleavage
by HCl in water/THF at 50 °C. The intermediates 14a -
The synthesis of the 4-substituted benzylhydrazine
building blocks 9a -e,g is shown in Scheme 1. Nickel-
(II)-catalyzed coupling of the Grignard reagent prepared
from 4-bromobenzaldehyde dimethyl acetal (3)9 to 2-bro-
mopyridine or 2-bromothiazole, followed by acidic hy-
drolysis, gave the corresponding arylbenzaldehydes
7b,c. Heating of 4-bromobenzaldehyde (4) and thiazole
in the presence of palladium(0) afforded 4-(thiazol-5-
yl)benzaldehyde (7d ). The structure of 7d was con-
firmed by the observation of a 1H NMR signal at 8.22
ppm (s, 1H), which was assigned to the proton on C(4)
of the thiazole ring, whereas an isomeric thiazol-4-yl
derivative should show a signal at ≈7.7 ppm (H-C(5)).
Cycloaddition of azide to the nitrile function of 4-cy-

3390 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Bold et al.
Sch em e 2. Synthesis of Symmetrically Acylated Aza-Dipeptides 22a ,b and 25a -c,e and Asymmetrically Acylated
Analogues 23a -d , 24a ,b,d , 26b, 27b, and 28d a
a
Conditions: (a) 9a -e,g, 2-propanol, vV; (b) formic acid or HCl/dioxane or HCl in THF/H2O, 50 °C; (c) EDC, HOBT, Et3N/DMF; or
b
TPTU, NMM/DMF; or TPTU, Hu¨nig’s base/DMF; or TPTU, Hu¨nig’s base/CH2Cl2; (d) K2CO3, MeOH/H2O, 16 h 80 °C. Yield for 2 steps.
c,e were coupled with N-methoxycarbonyl-L-valine14
or N-methoxycarbonyl-L-tert-leucine (29), respectively,
according to standard peptide synthesis procedures
[N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide hy-
drochloride, 1-hydroxybenzotriazole, triethylamine; or
O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetramethyluro-
nium tetrafluoroborate, N-methylmorpholine or Hu¨nig’s
base] to furnish the target inhibitors 22a ,b and 25a -
c,e. For asymmetrically acylated aza-dipeptide isos-
teres, the known trifluoroacetyl-protected epoxide 117
was used instead, which was prepared by trifluoroacety-
lation of 3(S)-amino-4-phenyl-1-butene12 and oxidation
using 3-chloroperbenzoic acid, providing 11 as an 87:
13 mixture of threo- and erythro-isomers. Without
separation of the isomers, this mixture was carried
through the nucleophilic opening step by 9a -d ,g, lead-
ing to the orthogonally protected dipeptide isosteres
13a -d ,g. Saponification of the trifluoroacetamide
(f15a -d ) and amide bond formation with the required
carbamoylated amino acid derivatives furnished the
N(C-5)-acylated intermediates 16a -d , 17a ,b,d , and
18b. Acidic cleavage of the Boc protecting groups
(f19a -d , 20a ,b,d , and 21b) and again coupling with
carbamoylated amino acid derivatives gave the asym-
metrically acylated aza-dipeptides 23a -d , 24a ,b,d , 26b,
27b, and 28d .
Alternatively, symmetrically acylated aza-dipeptides
could also be synthesized via the orthogonally protected
dipeptide isosteres 13d ,g (Scheme 3): Successive depro-
tection of the trifluoroacetyl and the Boc protection

New Aza-Dipeptide Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3391
Sch em e 3. Synthesis of Symmetrically Acylated Aza-Dipeptides 22d and 25d ,ga
a
Conditions: (a) K2CO3, MeOH/H2O, 16 h, 80 °C; (b) HCl/dioxane, DMF; (c) EDC, HOBT, Et3N/DMF; or TPTU, NMM/DMF; or TPTU,
b
Hu¨nig’s base/DMF; (d) formic acid. Yield for 2 steps.
group transferred 13g to the intermediate 14g, which
gave 25g in good yields via double acylation with
N-methoxycarbonyl-L-tert-leucine (29). Coupling of 19d
and 20d with carbamoylated amino acid derivatives
furnished 22d or 25d , respectively.
that combine high oral bioavailability and antiviral
potency. Some of the previously described leads7 (e.g.,
1 or 2, see Table 1) showed either good oral pharmaco-
kinetics or good antiviral activity, but none of them were
satisfactory in both aspects. CAMM studies based on
X-ray data from the enzyme/inhibitor complex of the
aza-dipeptide CGP 538208 prompted us to examine
larger substituents at the P1′ position: Derivatives with
4-substituted benzyl moieties in this position were
predicted to have the potential to make additional
favorable interactions with Arg8 and/or Phe153, Gly148,
and Gly149 of the enzyme. The prevalent opinion15 that
maximizing the number of nonbonded, especially van
der Waals, interactions with the enzyme decreases the
chances of drug resistance through enzyme mutation
led us to suppose that N-(bis-aryl-methyl) aza-dipeptide
analogues would also be favorable in this respect.
Enzymatic inhibition data for the biphenyl derivative
22a indeed proved that conformationally rigid bulkier
residues are well-tolerated. More interestingly, 22a
showed increased potency on the cellular level. Re-
placement of either one or both of the valine substitu-
ents by tert-leucine increased the cellular antiviral
activity even further, leading to 23a , 24a , and 25a (CGP
75355).
The 4-(2-tert-butyl-2H-tetrazol-5-yl)benzyl derivative
25f was synthesized via a different route, outlined in
Scheme 4, a sequence that opens access to the sym-
metrically and asymmetrically acylated aza-dipeptide
series: Schotten-Baumann acylation of L-tert-leucine
(f29), followed by a N-ethyl-N′-(3-(dimethylamino)-
propyl)carbodiimide hydrochloride/1-hydroxybenzotri-
azole-mediated coupling to tert-butyloxycarbonylhydra-
zine gave the L-tert-leucinylhydrazine derivative 30 in
quantitative yield. Acidic deprotection (f31) and con-
densation with 4-(tetrazol-5-yl)benzaldehyde (6) fur-
nished hydrazone 32. Acid-catalyzed addition of iso-
butene to the tetrazole functionality then provided 33
regioselectively, which by reduction with sodium cy-
anoborohydride/p-toluenesulfonic acid10 gave the hy-
drazine moiety 34. Nucleophilic attack at the epoxide
function of 10 afforded the N(N-2)-acylated aza-dipep-
tide isostere 35. Selective acidic cleavage of the Boc
protecting group by HCl in THF/water (f36) and
acylation with carbamoylated amino acid derivatives
then led to asymmetrically or symmetrically acylated
aza-dipeptide isosteres, like 25f.
Additionally to the higher potency, these tert-leucine
derivatives showed very interesting plasma levels after
oral administration. These effects of the tert-leucine on
oral bioavailability motivated us to increase the hydro-
philicity of the inhibitors by replacement of the 4-bi-
phenyl unit by 4-heterocyclyl-phenyl substituents in
order to enhance the solubility properties in biologically
suitable vehicles, hopefully without affecting the favor-
able pharmacokinetic profile observed so far. In the
4-(pyridin-2-yl)phenyl series, the bis-valinyl derivative
22b already showed oral bioavailability. Indeed, sub-
stitution of either one (f23b, 24b) or, even better, both
Resu lts a n d Discu ssion
To minimize the chance of AIDS treatment failure due
to induction of HIV strains resistant to a given chemo-
therapeutic agent, effective blood concentrations of the
inhibitor must be maintained.4a This prerequisite for
a successful treatment calls for drugs that show good
pharmacokinetics after oral administration and exert
excellent antiviral activity. Therefore, the goal of this
optimization program was to find aza-dipeptide isosteres

3392 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Bold et al.
Sch em e 4. Synthesis of 4-(2-tert-Butyl-2H-tetrazol-5-yl)phenyl Derivative 25fa
a
Conditions: (a) methyl chloroformate, NaOH, dioxane/H2O, 18 h, 60 °C; (b) tert-butyloxycarbonylhydrazine, EDC, HOBT, NMM/
EtOAc; (c) HCl, dioxane; (d) 4-(tetrazol-5-yl)benzaldehyde (6), 2-propanol, 18 h, 90 °C; (e) isobutene, MeSO3H, toluene, 1 h, 110 °C; (f)
NaCNBH3, TsOH, THF, r.t.; (g) epoxide 10, 2-propanol, 16 h, 90 °C; (h) HCl in THF/H2O, 50 °C; (i) 29, TPTU, NMM/DMF.
(f25b, CGP 73547) of the valine residues by tert-leucine
increased potency and, more dramatically, oral absorp-
tion even further (CGP 73547: c90 ) 31.8 µM). Efforts
to replace N-methoxycarbonyl-L-tert-leucine by the less
expensive isomeric building blocks N-methoxycarbonyl-
L-isoleucine (f26b) or N-ethoxycarbonyl-L-valine (f27b)
failed to lead to derivatives with a comparable phar-
macokinetic profile. This tells us once more that oral
bioavailability can hardly be controlled by simple modu-
lation of the lipophilicity of the substituents. The bis-
tert-leucine derivatives displayed the best combination
of antiviral potency and oral bioavailability in the
thiazol-2-yl (f25c, CGP 75136) and thiazol-5-yl (f25d )
series too.
A rough idea about the size of the pocket in the
enzyme is given by the 4-(2-tert-butyl-2H-tetrazol-5-yl)-
phenyl derivative 25f, which still is a potent enzyme
inhibitor (IC50 ) 72 nM). In respect to cellular antiviral
activity, 25f belongs to the most potent inhibitors of this
series (ED90 ) 3 nM). The enzymatically more potent
4-(2-methyl-2H-tetrazol-5-yl)phenyl derivative 25e (CGP
75176; IC50 ) 27 nM) is equally potent in respect to its
ED50 but appears to be slightly weaker if we consider
the ED90. However, its blood plasma level after oral
administration is again excellent (c30 ) 20.3 µM). The
4-(diethylamino)phenyl derivative 25g exerts good an-
tiviral activity too, demonstrating that the 4-arylphenyl
substituent directing to the P1′ pocket of the enzyme
can be replaced by readily available alkylanilines.
However, preliminary pharmacokinetic studies in mice
indicated metabolic instability of 25g: Even though the
parent compound appears in high concentration after
oral administration, rapid formation of metabolites can
be observed. The major metabolite, appearing in con-
centrations close to those of the parent compound, was
identified by LC-MS analysis to be the 4-monoethyl-
aniline derivative. Therefore the 4-diethylaniline de-
rivative 25g was not pursued further.
As a conclusion of our work, most of the described new
compounds have equal or better antiviral activity than
the standard HIV protease inhibitor Ro 31-8959
(saquinavir). Their pharmacokinetic profile in mice
after oral administration is clearly superior to that of
Ro 31-8959. As the pharmacokinetic evaluations were
performed in a formulation which is not acceptable for
clinical use, we investigated the bioavailability of the
mesylate salt of CGP 73547 in mice and dogs: These
experiments confirmed the good oral bioavailability of
the compound. In mice a dose of 120 mg/kg dissolved
in 3% citric acid resulted in plasma concentrations at
30, 60, 90, and 120 min after administration of 11.5,
13.9, 15.6, and 17.0 µM, respectively. In beagle dogs
the mesylate salt (given at a dose equivalent to 90 mg
of free base/kg in 3% citric acid) resulted in peak plasma
levels of 6.1 µM at 2.4 h and an AUC(0-∞) of 38.6 µM.
The symmetrically acylated bis(L-tert-leucine) deriva-
tives show a biological profile equal to or better than
that of the asymmetrically acylated derivatives, and in
addition, their synthesis is simpler. Therefore, bis(L-
tert-leucine) derivatives qualify best for further evalu-
ation. Out of the two symmetrically acylated thiazolyl
derivatives CGP 75136 and 25d , the thiazol-2-yl deriva-
tive CGP 75136 was preferred, since the heteroatoms
in a thiazol-2-yl fragment are considered to be sterically

New Aza-Dipeptide Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3393
Ta ble 2. Antiviral Activity against HIV-1 Protease Inhibitor-Resistant Strains of the HI virusa
effective dose (µM)
saquinavir
resistantb
indinavir
resistantc
CGP 61755
resistantd
CGP 61755
resistante
HI virus
wild-type
CGP 75355
CGP 73547
CGP 75136
CGP 75176
saquinavir
indinavir
0.01
0.01
0.01
0.01-0.03
0.03
0.1
0.01
0.004-0.01
0.004
0.01
0.1-0.3
0.03-0.1
0.1
0.03-0.1
0.1
0.1-0.3
0.1
0.3-0.9
0.3
0.3-0.9
0.3
0.9
0.9
0.9
0.3
ndf
ndf
>2.7
2.7
g0.9
a
The indicated effective dose (µM) is the lowest dose at which virus replication (measured as supernatant reverse transcriptase activity)
was totally inhibited until day 10 of the experiment (for details see the Experimental Section). The data represent averages of at least
three determinations. Mutant: G48V, L90M. c Mutant: L10R, M46I, L63P, V82T, I84V. Mutant: K45I, A71Tmix, L76F, I84Vmix.
b
d
e Mutant: V32I, M46I, L63P, I84A. f nd, not done.
Ch a r t 2. Bis(L-tert-leucine) Derivatives of Aza-Dipeptide Isosteres Selected for Further Profiling
virus-containing supernatant (MOI 0.001-0.005) for 4 h at 37
°C (CO2). After washing with medium, the cells were dis-
pensed in 96-well tissue culture plates at 20 000 cells/100 µL/
well followed by addition of serial 3-fold dilutions of the test
compounds (in 100 µL/well) in triplicates (final volume: 200
µL/well). Plates then were incubated at 37 °C (5% CO2,
incubator). On day 4 postinfection, 10-µL supernatant samples
were taken from each well, frozen, and stored (-20 °C) for
reverse transcriptase (RT) activity determination. Cells were
resuspended, and 50 µL/well of the cell suspension was
transferred to new 96-well plates containing 150 µL/well fresh
medium with test compound. The final test compound con-
centrations were the same as on day 0. Plates were incubated
at 37 °C (5% CO2, incubator). On day 7, again 10-µL RT
samples were taken from each well, frozen, and stored at -20
°C. This time, 150 µL of the culture supernatants was
removed and replaced by 150 µL of fresh medium with test
compound. Again, the final concentrations of the test com-
pounds were the same as on day 0. Plates were incubated at
37 °C (5% CO2, incubator). On day 10, 10-µL RT samples were
taken from each well, frozen, and stored at -20 °C, and the
experiment was terminated. All RT samples from days 4, 7,
and 10 were simultaneously assayed for RT activity. As
controls, infected but untreated cells as well as noninfected
cells were included in each assay.
more hindered and therefore less prone for interactions
with cytochrome P450 isozymes than in the thiazol-5-yl
derivative 25d . On the basis of its excellent pharma-
cokinetic properties, CGP 75176 was chosen as a
representative of the tetrazolyl derivatives.
Consequently, the four bis(L-tert-leucine) derivatives,
CGP 75355, CGP 73547, CGP 75136, and CGP 75176,
presented in Chart 2 were selected for further profiling
against drug-resistant strains of the HI-virus (see Table
2). They all show good antiviral activity against resis-
tant strains raised in the presence of saquinavir and
show antiviral activity at submicromolar concentrations
against resistant strains selected in vitro in the presence
of our Phe-c-Phe hydroxyethylene type HIV protease
inhibitor CGP 61755.16 A description of the resistant
strains and further results will be published in due
course. Their excellent antiviral potency against a
range of HIV strains as well as saquinavir- and indi-
navir-resistant mutants combined with oral bioavail-
ability qualifies these promising compounds for further
characterization toward determining their potential as
candidates for clinical evaluation.
Bioa va ila bility. The compounds were dissolved in DMSO
to a concentration of 240 mg/mL. The stock solution was
diluted 1:20 with an aqueous solution of 20% hydroxypropyl-
â-cyclodextrin. After brief, low-power sonication a milky,
homogeneous suspension was obtained. Then the formulated
compounds were given to female Balb/c mice that had free
Exp er im en ta l Section
Assa y for An tivir a l Activity a ga in st HIV-1 P r otea se
In h ibitor -Resista n t HIV Str a in s. CEM-SS cells were in-
fected with different HIV-1 protease inhibitor-resistant phe-
noypes of HIV. The cells were incubated in a small volume of

3394 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Bold et al.
access to food and water throughout the experiments. The
mice received an average dose of 120 mg of compound/kg by
gavage. At allotted times four mice were sacrificed, heart
blood was collected into heparinized tubes, and plasma was
prepared by centrifugation. The plasma was either analyzed
immediately or stored frozen at -20 °C. For analysis, the
heparinized plasma samples were deproteinated by the addi-
tion of an equal volume of acetonitrile. After thorough mixing,
the tubes were allowed to stand for 20-30 min at r.t., and the
precipitated protein was removed by centrifugation (10 000g,
5 min). The supernatant was collected and analyzed by
reversed-phase HPLC: 100 µL of the supernatant was injected
onto a Nucleosil C18, 5-µm analytical column (125 × 4.6 mm);
mobile phase 20% acetonitrile/0.05% trifluoroacetic acid (TFA)
in water/0.05% TFA f 100% acetonitrile/0.05% TFA during
20 min + 5 min 100% acetonitrile/0.05% TFA. Compounds
were detected by UV absorbance and identified on the chro-
matograms by retention time and UV spectrum compared to
control plasma spiked with compound. Quantitation was by
the external standard method using peak heights to quantitate
the amounts by reference to a calibration curve. The calibra-
tion curve was constructed by the analysis of plasma samples
containing known amounts of the compound under evaluation
which had been processed as described above. The limit of
quantitation was 0.1-0.5 µmol/L (compound-dependent) under
these conditions.
[1,3-bis(diphenylphosphino)propane]nickel(II) chloride (38 g,
70 mmol), and diisobutylaluminum hydride (330 mL, 20% in
hexane). The Grignard reagent was added at 15-20 °C during
45 min; the mixture was stirred for 90 min at r.t. and then
poured into a mixture of ice (10 kg), HCl (1.5 L, 37%), and
citric acid (1.5 kg). After adding Hyflo super cel (1 kg), the
mixture was stirred for 1 h and filtered. The solid was washed
with water (2 L), 2 × toluene (2 L), and 2 × 1 N HCl (2 L).
The first filtrate and the washing water were combined; the
aqueous layer was separated and extracted with the two
toluene filtrates. The resulting organic layers were washed
then with the two HCl filtrates. After toluene (6 L) was added
to the combined aqueous layers, NaOH (4.6 L, 30% in water)
was added (pH ≈ 8-9). The mixture was filtered through
Celite and the aqueous layer separated and extracted twice
with toluene (2 L). The organic layers were washed twice with
water, dried (Na2SO4), and treated with charcoal. Addition
of 0.5 kg of silica gel, stirring, filtration, and concentration in
vacuo yielded 1820 g (90%) of 7b: TLC Rf(hexane/ethyl acetate,
2:1) ) 0.25; 1H NMR (CDCl3) δ 10.1 (s, HCO), 8.77 (d, 1H),
8.20 (d, 2H), 8.00 (d, 2H), 7.81 (m, 2H), 7.31 (q, 1H); HPLC tR
6.7 min.
4-(Th ia zol-2-yl)ben za ld eh yd e (7c). To magnesium (9.2
g, 378 mmol; activated with iodine) in THF (84 mL) at 60 °C
was added a solution of 4-bromobenzaldehyde dimethyl acetal9
(3; 82.6 g, 357 mmol) in THF (677 mL) dropwise. After stirring
for 40 min at 65 °C, the Grignard reagent was transferred into
the dropping funnel of a second apparatus, containing a red
suspension of 2-bromothiazole (31.7 mL, 356 mmol) in THF
(1680 mL) and [1,3-bis(diphenylphosphino)propane]nickel(II)
chloride. The Grignard reagent was added during 30 min, and
the mixture stirred for 12 h at r.t. After the reaction was
quenched with water (840 mL), the mixture was partially
concentrated in vacuo. Then ethyl acetate (1 L) and 2 N HCl
(340 mL) were added, and stirring was continued for 90 min.
The aqueous phase was separated and extracted twice with
ethyl acetate (0.5 L). The organic layers were washed with 2
N HCl (2 × 250 mL), water, saturated NaHCO3, water, and
brine, dried (Na2SO4), and concentrated in vacuo. Filtration
through silica gel (1 kg; ethyl acetate/hexane, 1:4) and washing
with hexane yielded 26.3 g (39%) of 7c: TLC Rf(hexane/ethyl
Ch em istr y. All reactions with air- or moisture-sensitive
reactants and solvents were carried out under nitrogen
atmosphere. In general, reagents and solvents were used as
purchased without further purification. THF was freshly
distilled from sodium/benzophenone. Analytical thin-layer
chromatography was performed on silica F254 glass plates (E.
Merck). Components were visualized by UV light of 254 nm
or by spraying with phosphomolybdic acid. Column flash
chromatography was performed on silica gel 60 (230-400 mesh
ASTM, E. Merck) under
a positive nitrogen pressure of
approximately 0.4 atm. Melting points were determined in
an open capillary and are not corrected. 1H NMR spectra:
Bruker DRX-500 (500 MHz), Bruker AM-360 (360 MHz),
Varian Gemini-300 (300 MHz), or Varian Gemini-200 (200
MHz); chemical shifts of signals are expressed in parts per
million (ppm) and are referenced to the deuterated solvents
used. MS spectra: FAB-ZAB, HF (VG Analytical). HPLC
chromatography: stationary phase Nucleosil C18, 5-µm ana-
lytical column (125 × 4.6 mm); mobile phase 20% acetonitrile/
0.1% TFA in water/0.1% TFA f 100% acetonitrile/0.1% TFA
during 20 min + 10 min 100% acetonitrile/0.1% TFA; tR refers
to the retention time. Elemental analyses were performed by
the Ciba Analytical Department and are within (0.4% of the
calculated values.
Abbr evia tion s: EDC, N-ethyl-N′-[3-(dimethylamino)pro-
pyl]carbodiimide hydrochloride; HBTU, O-benzotriazol-1-yl-
N,N,N′,N′-tetramethyluronium hexafluorophosphate; HOBT,
1-hydroxybenzotriazole hydrate; NMM, N-methylmorpholine;
r.t., room temperature; p-TsOH, 4-toluenesulfonic acid mono-
hydrate; TPTU, O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tet-
ramethyluronium tetrafluorborate.
4-(Tetr a zol-5-yl)ben za ld eh yd e (6). 4-Cyanobenzaldehyde
(5) (54.3 g, 0.414 mol), lithium chloride (26.3 g, 0.621 mol),
and sodium azide (26.9 g, 0.414 mol) in methoxyethanol (0.4
L) were heated for 6 h to reflux temperature. The suspension
then was poured into a mixture of ice (1.3 kg) and HCl (37%,
130 mL), stirred, and filtered. Washing with water afforded
66.5 g (92%) of 6: mp 182-184 °C; 1H NMR (DMSO-d6) δ 10.11
(s, 1H), 8.29 (d, 2H), 8.15 (d, 2H).
4-(P yr id in -2-yl)ben za ld eh yd e (7b). To magnesium (317
g, 13.0 mol) in THF (3.5 L) was added iodine (11 g) followed
by 200 g of 4-bromobenzaldehyde dimethyl acetal9 (3). When
the reaction (eventually slightly heating) had started, another
2540 g (total amount: 2740 g, 11.8 mol) of 3 in toluene (3.5 L)
was added dropwise (temperature 25-30 °C; 1 h). After
stirring for 1 h at 25-30 °C the Grignard reagent was
transferred into the dropping funnel of a second apparatus,
containing 2-bromopyridine (1750 g, 11.0 mol) in THF (3.3 L),
1
acetate, 3:1) ) 0.21; H NMR (CDCl3) δ 10.05 (s, HCO), 8.15
(d, 2H), 7.95 (m, 3H), 7.45 (d, 1H); MS (M)+ ) 189. Anal.
(C10H7NOS) C, H, N, S.
4-(Th ia zol-5-yl)ben za ld eh yd e (7d ). 4-Bromobenzalde-
hyde (4) (28.0 g, 150 mmol), thiazole (65.3 g, 767 mmol),
potassium acetate (22.3 g, 227 mmol), and tetrakis(triphen-
ylphosphine)palladium(0) (8.7 g, 7.5 mmol) in dimethyl acet-
amide (376 mL) were heated in a sealed tube for 12 h at 150
°C. The brown suspension was filtered and the filtrate
concentrated in vacuo. The resulting residue was redissolved
in ethyl acetate/water and the aqueous phase separated and
extracted twice with ethyl acetate. The organic layers were
washed with water and brine, dried (Na2SO4), and concen-
trated. Column chromatography (toluene/acetone, 9:1) and
crystallization from diethyl ether/hexane gave 17.3 g (61%)
1
7d : TLC Rf(toluene/acetone, 9:1) ) 0.28; H NMR (CDCl3) δ
10.02 (s, HCO), 8.85 (s, 1H), 8.22 (s, 1H), 7.94 (d, 2H), 7.75 (d,
2H); MS (M)+ ) 189.
4-(2-Meth yl-2H-tetr a zol-5-yl)ben za ld eh yd e (7e). A so-
lution of 6 (75.5 g, 0.433 mol) in 550 mL of DMF/dioxane (1:1)
was added dropwise to an ice-cooled mixture of potassium
carbonate (180 g, 1.3 mol) and DMF/dioxane (1:1) (200 mL).
After 30 min of stirring, methyl iodide (40 mL, 0.64 mol) was
added, and stirring was continued for 3 h at 0 °C and for 15 h
at r.t. The suspension then was poured into ice water (2.8 L),
stirred, and filtered. Washing with water afforded 78 g (95%)
of 7e: mp 137-139 °C; 1H NMR (CD3OD/CDCl3 mixture) δ
10.06 (s, 1H), 8.29 (d, 2H), 8.03 (d, 2H), 4.45 (s, 3H).
N-1-(ter t-Bu tyloxyca r bon yl) N-2-(4-Bip h en ylylm eth -
ylid en e) Hyd r a zon e (8a ). Preparation from 7a as described
for 8b yielded 126 g (quantitative) of 8a : mp 169 °C; 1H NMR
(CD3OD) δ 7.95 (s, 1H), 7.77 (d, 2H), 7.63 (m, 4H), 7.40 (m,
3H), 1.55 (s, 9H).

New Aza-Dipeptide Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3395
N-1-(ter t-Bu tyloxyca r bon yl) N-2-[4-(P yr id in -2-yl)ben -
zylid en e] Hyd r a zon e (8b). A solution of 7b (1770 g, 9.67
mol) and tert-butyl carbazate (1220 g, 9.2 mol) in ethanol (12.5
L) was heated to reflux temperature during 4 h. The solution
was cooled to 40 °C, and then ice (6 kg) was added. Filtration,
washing with water (6 L), and drying (50 °C; in vacuo) afforded
2554 g (93%) of 8b: TLC Rf(hexane/ethyl acetate, 1:2) ) 0.38;
1H NMR (CDCl3) δ 8.70 (d, 1H), 8.02 (m, 3H), 7.87 (s, 1H),
7.81 (s, 1H), 7.76 (m, 3H), 7.25 (m, 1H), 1.55 (s, 9H).
N-1-(ter t-Bu tyloxyca r bon yl) N-2-[4-(Th ia zol-2-yl)ben -
zylid en e] Hyd r a zon e (8c). Preparation from 7c as described
for 8b yielded 30 g (73%) of 8c: 1H NMR (CDCl3) δ 8.06 (s,
HN), 7.97 (d, 2H), 7.87 (m, 2H), 7.75 (d, 2H), 7.36 (d, 1H), 1.55
(s, 9H).
N-1-(ter t-Bu tyloxyca r bon yl) N-2-[4-(Th ia zol-5-yl)ben -
zylid en e] Hyd r a zon e (8d ). Preparation from 7d as de-
scribed for 8b yielded 25.4 g (92%) of 8d : 1H NMR (CD3OD)
δ 8.98 (s, 1H), 8.23 (s, 1H), 7.93 (s, 1H), 7.73 (m, 4H), 1.54 (s,
9H).
N-1-(ter t-Bu tyloxyca r bon yl) N-2-[4-(2-Meth yl-2H-tet-
r a zol-5-yl)ben zylid en e] Hyd r a zon e (8e). Preparation from
7e as described for 8b yielded 61 g (51%) of 8e: 1H NMR
(DMSO-d6) δ 11.05 (s, 1H), 8.10 (m, 3H), 7.80 (d, 2H), 4.45 (s,
3H), 1.49 (s, 9H). Anal. (C14H18N6O2) C, H, N.
N-1-(ter t-Bu tyloxyca r bon yl)-N-2-[4-(2-m eth yl-2H-tetr a -
zol-5-yl)ben zyl]h yd r a zin e (9e). NaCNBH3 (17.6 g, 85%
purity, 238 mmol) was added to 8e (60 g, 198 mmol) in THF
(700 mL); then a solution of p-TsOH (45.2 g, 238 mmol) in THF
(350 mL) was added dropwise (f precipitation). After 2 h,
the solid was filtered off, washed with ethyl acetate, and
discarded. Water and ethyl acetate were added to the filtrate;
the aqueous phase was separated off and extracted twice with
ethyl acetate. The organic phases were washed with saturated
NaHCO3 solution, water, and brine, dried (Na2SO4), and
concentrated by evaporation. The resulting crystals were
taken up in methanol (834 mL) and THF (416 mL), and cooled
by an ice bath, and then a solution of K2B4O7‚4H2O (254 g,
0.83 mol) in H2O (834 mL) was added dropwise (f foam
production). The mixture was stirred at r.t. overnight, poured
into water (4.4 L), and extracted three times with ethyl acetate.
The organic phases were washed with saturated NaHCO3
solution, water, and brine, dried (Na2SO4), and concentrated
by evaporation. Filtration through silica gel using dichlo-
romethane/THF (10:1) as the eluant, concentration by evapo-
ration to a residual volume of about 0.1 L, and addition of
diisopropyl ether (150 mL) yielded 47.9 g (79%) of crystalline
1
9e: mp 100-102 °C; H NMR (CD3OD) δ 8.06 (d, 2H), 7.53
(d, 2H), 4.42 (s, 3H), 4.00 (s, 2H), 1.44 (s, 9H). Anal.
(C14H20N6O2) C, H, N.
N-1-(ter t-Bu t yloxyca r b on yl) N-2-[4-(Diet h yla m in o)-
ben zylid en e] Hyd r a zon e (8 g). Preparation from 7g as
described for 8b yielded 149.5 g (91%) of 8g: 1H NMR (CDCl3)
δ 7.68 (s, 1H), 7.65 (s, 1H), 7.53 (d, 2H), 6.62 (d, 2H), 3.38 (q,
4H), 1.53 (s, 9H), 1.18 (t, 6H).
N-1-(ter t-Bu tyloxyca r bon yl)-N-2-[4-(d ieth yla m in o)ben -
zyl]h yd r a zin e (9g). Preparation from 8g as described for 9b
yielded 72 g (89%) of 9g: 1H NMR (CDCl3) δ 7.19 (d, 2H), 6.65
(d, 2H), 6.1 (sb, HN), 4.08 (sb, HN), 3.87 (s, 2H), 3.36 (q, 4H),
1.48 (s, 9H), 1.16 (t, 6H).
N-1-(ter t-Bu tyloxyca r bon yl)-N-2-(4-bip h en ylylm eth yl)-
h yd r a zin e (9a ). Preparation from 8a as described for 9b
yielded 58.7 g (62%) of 9a : mp 84-85 °C; H NMR (CD3OD)
δ 7.6 (m, 4H), 7.4 (m, 5H), 3.95 (s, 2H), 1.44 (s, 9H).
1-(4-Bip h en ylyl)-5(S)-2,5-bis[(ter t-bu tyloxyca r bon yl)-
a m in o]-4(S)-h yd r oxy-6-p h en yl-2-a za h exa n e (12a ). Prepa-
ration from 9a as described for 12b yielded 95 g (86%) of 12a :
1
1
mp 184-185 °C; H NMR (CD3OD) δ 7.7-7.1 (m, 14H), 3.90
(s, 2H), 3.7 (m, 2H), 3.0-2.5 (m, 4H), 1.34 (s, 9H), 1.32 (s, 9H);
N-1-(ter t-Bu tyloxyca r bon yl)-N-2-[4-(p yr id in -2-yl)ben -
zyl]h yd r a zin e (9b). A suspension of 8b (1655 g, 5.57 mol)
in methanol (12 L) was hydrogenated in the presence of Pd/C
(10%, 166 g). The catalyst was filtered off, the filtrate
concentrated under reduced pressure, and the oily residue
crystallized from hexane (3 L). Filtration of the crystallisate,
washing twice in hexane (2.5 L), and drying yielded 1478 g
(89%) of 9b: TLC Rf(hexane/ethyl acetate, 1:2) ) 0.3; mp 74-
77 °C; 1H NMR (DMSO-d6) δ 8.64 (d, 1H), 8.26 (sb, HN), 8.02
(d, 2H), 7.93 (d, 1H), 7.85 (dd, 1H), 7.42 (d, 2H), 7.32 (dd, 1H),
FAB MS (M + H)+ ) 562. Anal. (C33H43N3O5) C, H, N.
1-[4-(P yr id in -2-yl)p h en yl]-5(S)-2,5-b is[(ter t-b u t yloxy-
ca r b on yl)a m in o]-4(S )-h yd r oxy-6-p h e n yl-2-a za h e xa n e
(12b). A solution of N-(tert-butyloxycarbonyl)-2(S)-amino-1-
phenyl-3(R)-3,4-epoxybutane (10)6b,12 (1185 g, 4.5 mol) and 9b
(1230 g, 4.1 mol) in 2-propanol (14 L) was heated to reflux
temperature during 16 h. Then ice (15 kg) and water (10 L)
were added portionwise. After stirring for 2 h the precipitate
was filtered off and washed with water (6 L). The crude
product was twice suspended in diethyl ether (5 L), again
filtered, and washed with diethyl ether (2 L) each time and
finally with diethyl ether/tert-butylmethyl ether (1:1) (2 L).
Drying in vacuo at 40 °C yielded 1583 g (69%) of 12b: TLC
4.80 (m, HN), 3.92 (d, 2H), 1.38 (s, 9H); FAB MS (M + H)+
300. Anal. (C17H21N3O2) C, H, N.
)
N-1-(ter t-Bu tyloxycar bon yl)-N-2-[4-(th iazol-2-yl)ben zyl]-
h yd r a zin e (9c). NaCNBH3 (16.9 g, 95% purity, 256 mmol)
was added to an ice-cooled solution of 8c (77.6 g, 255 mmol)
in THF (450 mL). Then a solution of p-TsOH (49.6 g, 0.26
mol) in THF (450 mL) was added dropwise. After 17 h of
stirring at r.t., another portion of NaCNBH3 (3.38 g) and
p-TsOH (9.9 g) was added, and stirring was continued for 20
h. Then the reaction mixture was diluted with ethyl acetate
and water and the aqueous layer separated and extracted
twice with ethyl acetate. The organic layers were washed with
brine, saturated NaHCO3, and again brine, dried (Na2SO4),
and concentrated in vacuo. The resulting oil was diluted with
dichloroethane (306 mL), then 1 N NaOH in water (306 mL)
was added (cooling), and the biphasic mixture was heated for
3.5 h to reflux temperature. After dichloromethane and water
were added, the aqueous layer was separated and extracted
two times with dichloromethane. The organic phases were
dried (Na2SO4) and concentrated. Filtration through silica gel
(1 kg; toluene/acetone, 9:1) and washing with hexane yielded
60.3 g (77%) of 9c: TLC Rf(hexane/ethyl acetate 3:2) ) 0.30;
1H NMR (CDCl3) δ 7.93 (d, 2H), 7.87 (d, 1H), 7.43 (d, 2H), 7.33
(d, 1H), 6.10 (s, HN), 4.25 (s, HN), 4.03 (d, 2H), 1.47 (s, 9H).
Rf(hexane/ethyl acetate, 1:2) ) 0.45; mp 185-186 °C; [R]D
)
1
-12° (c ) 0.5, CHCl3); H NMR (CD3OD) δ 8.60 (d, 1H), 7.88
(m, 4H), 7.50 (d, 2H), 7.36 (m, 1H), 7.25 (m, 4H), 7.18 (m, 1H),
3.93 (m, 2H), 3.70 (m, 2H), 3.0-2.6 (m, 4H), 1.33 (s, 9H), 1.30
(s, 9H); HPLC tR 13.9 min; FAB MS (M + H)+ ) 563. Anal.
(C32H42N4O5‚0.14H2O) C, H, N, H2O.
1-[4-(Th ia zol-2-yl)p h en yl]-5(S)-2,5-b is[(ter t-b u t yloxy-
ca r b on yl)a m in o]-4(S )-h yd r oxy-6-p h e n yl-2-a za h e xa n e
(12c). Preparation from 9c as described for 12b yielded 28.7
g (74%) of 12c: TLC Rf(hexane/acetone, 3:2) ) 0.30; 1H NMR
(CDCl3) δ 7.92 (d, 2H), 7.86 (d, 1H), 7.40 (d, 2H), 7.32 (d, 1H),
7.25 (m, 5H), 5.29 (s, 1H), 5.10 (d, 1H), 4.51 (s, 1H), 4.01 (d,
1H), 3.88 (d, 1H), 3.63 (m, 2H), 2.93 (d, 2H), 2.82 (m, 1H), 2.46
(m, 1H), 1.38 (s, 9H), 1.34 (s, 9H).
1-[4-(2-Meth yl-2H-tetr azol-5-yl)ph en yl]-5(S)-2,5-bis[(ter t-
bu tyloxyca r bon yl)a m in o]-4(S)-h yd r oxy-6-p h en yl-2-a za -
h exa n e (12e). Preparation from 9e as described for 12b
yielded 55.6 g (78%) of 12e: mp 175-178 °C; 1H NMR (CDCl3/
CD3OD mixture) δ 8.07 (d, 2H), 7.43 (d, 2H), 7.21 (m, 5H),
4.39 (s, 3H), 4.01 (d, 1H), 3.87 (d, 1H), 3.62 (m, 2H), 2.92 (d,
2H), 2.78 (m, 1H), 2.47 (m, 1H), 1.36 (s, 9H), 1.30 (s, 9H); FAB
MS (M + H)+ ) 568. Anal. (C29H41N7O5) C, H, N.
N-1-(ter t-Bu tyloxycar bon yl)-N-2-[4-(th iazol-5-yl)ben zyl]-
h yd r a zin e (9d ). Preparation from 8d as described for 9c
yielded 20.6 g (80%) of 9d : mp 93-95 °C; 1H NMR (CDCl3) δ
8.75 (s, 1H), 8.07 (s, 1H), 7.56 (d, 2H), 7.41 (d, 2H), 6.03 (s,
HN), 4.25 (s, HN), 4.03 (m, 2H), 1.47 (s, 9H); FAB MS (M +
H)+ ) 306. Anal. (C15H19N3O2S) C, H, N, S.
1-(4-Biph en ylyl)-2-[(ter t-bu tyloxycar bon yl)am in o]-4(S)-
h yd r oxy-5(S)-[(t r iflu or oa cet yl)a m in o]-6-p h en yl-2-a za -
h exa n e (13a ). A suspension of N-(trifluoroacetyl)-2(S)-amino-
1-phenyl-3(R)-3,4-epoxybutane (11)7 (7:1 threo:erythro mixture;

3396 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Bold et al.
40.0 g, 154 mmol) and 9a (46.0 g, 154 mmol) in 2-propanol
(800 mL) was heated to 80 °C for 16 h. After concentration of
the solution to a volume of ≈0.3 L and cooling to r.t., the
product crystallized. Filtration and washing with cold 2-pro-
and HCl (93 mL, 4 N in water) was stirred for 8 h at 50 °C.
The mixture was concentrated in vacuo, and the residue was
four times diluted with ethanol and concentrated, yielding 15.5
g (quantitative) of crystalline 14e: mp 227-230 °C (recrystal-
lized from 2-propanol/diisopropyl ether). Anal. (C19H25N7O‚
2 HCl‚0.20H2O) C, H, N, Cl, H2O.
1-[4-(Dieth yla m in o)p h en yl]-5(S)-2,5-d ia m in o-4(S)-h y-
d r oxy-6-p h en yl-2-a za h exa n e Tr ih yd r och lor id e (14g). To
an ice-cold solution of 15g (57 g, 125 mmol) in DMF (200 mL)
was added 4 N HCl in dioxane (950 mL). After 4 h at ambient
temperature, the mixture was concentrated in vacuo. The
residue was diluted with dioxane and toluene and again
concentrated, giving 58 g (quantitative) of 14g: FAB MS (M
+ H)+ ) 367.
1
panol and hexane yielded 54 g (62%) of 13a : mp 173 °C; H
NMR (CD3OD) δ 7.57 (m, 4H), 7.42 (m, 5H), 7.25 (m, 5H), 4.25
(m, 1H), 3.90 (m, 2H), 3.80 (m, 1H), 3.03 (dd, 1H), 2.88 (m,
1H), 2.73 (m, 2H), 1.32 (s, 9H); HPLC tR 18.6 min; FAB MS
(M + H)+ ) 558. Anal. (C30H34N3O4F3) C, H, N.
1-[4-(P yr id in -2-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon yl)-
am in o]-4(S)-h ydr oxy-5(S)-[(tr iflu or oacetyl)am in o]-6-ph en -
yl-2-a za h exa n e (13b). Preparation from 9b as described for
1
13a yielded 16 g (60%) of 13b: mp 194 °C; H NMR (DMSO-
d6) δ 9.12 (d, HN), 8.64 (d, 1H), 8.14 (s, HN), 8.00 (d, 2H), 7.92
(d, 1H), 7.85 (t, 1H), 7.43 (d, 2H), 7.33 (m, 1H), 7.23 (m, 4H),
7.16 (m, 1H), 4.89 (s, HO), 4.21 (m, 1H), 3.94 (AB, 2H), 3.69
(m, 1H), 2.90 (dd, 1H), 2.77 (m, 2H), 2.69 (dd, 1H), 1.28 (s,
9H); HPLC tR 12.8 min; FAB MS (M + H)+ ) 559.
1-[4-(Th ia zol-2-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon yl)-
am in o]-4(S)-h ydr oxy-5(S)-[(tr iflu or oacetyl)am in o]-6-ph en -
yl-2-a za h exa n e (13c). Preparation from 9c as described for
13a yielded 5.1 g (73%) of 13c: mp 167-168 °C; 1H NMR
(CDCl3) δ 7.92 (d, 2H), 7.87 (d, 1H), 7.40 (d, 2H), 7.33 (d, 1H),
7.24 (m, 5H), 6.92 (d, 1H), 5.31 (s, 1H), 4.81 (s, 1H), 4.0 (m,
3H), 3.69 (m, 1H), 3.00 (m, 2H), 2.66 (m, 1H), 2.48 (m, 1H),
1.34 (s, 9H); FAB MS (M + H)+ ) 565. Anal. (C27H31N4O4F3-
S) C, H, N, F, S.
1-[4-(Th ia zol-5-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon yl)-
am in o]-4(S)-h ydr oxy-5(S)-[(tr iflu or oacetyl)am in o]-6-ph en -
yl-2-a za h exa n e (13d ). Preparation from 9d as described for
13a yielded 14.4 g (52%) of 13d : 1H NMR (CDCl3) δ 8.77 (s,
1H), 8.07 (s, 1H), 7.55 (d, 2H), 7.36 (d, 2H), 7.25 (m, 5H), 6.87
(d, 1H), 5.30 (s, 1H), 4.78 (s, 1H), 3.98 (m, 3H), 3.67 (m, 1H),
2.99 (m, 2H), 2.63 (m, 1H), 2.45 (m, 1H), 1.35 (s, 9H); FAB
MS (M + H)+ ) 565.
1-[4-(Dieth ylam in o)ph en yl]-2-[(ter t-bu tyloxycar bon yl)-
am in o]-4(S)-h ydr oxy-5(S)-[(tr iflu or oacetyl)am in o]-6-ph en -
yl-2-a za h exa n e (13g). Preparation from 9g as described for
13a yielded 79 g (55%) of 13g: mp 159-161 °C; 1H NMR (CD3-
OD) δ 7.22 (m, 4H), 7.16 (m, 1H), 7.11 (d, 2H), 6.62 (d, 2H),
4.16 (m, 1H), 3.73 (m, 1H), 3.69 (s, 2H), 3.33 (q, 4H), 2.97 (m,
1H), 2.81 (m, 1H), 2.66 (m, 1H), 2.59 (m, 1H), 1.32 (s, 9H),
1.11 (t, 6H); FAB MS (M + H)+ ) 553. Anal. (C28H39N4O4F3)
C, H, N, F.
1-(4-Biph en ylyl)-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 (15a ). To a
solution of 13a (54.0 g, 96.8 mmol) in boiling methanol (1.5 L)
was added a 1 N aqueous solution of K2CO3 (484 mL) dropwise.
The resulting suspension was heated at reflux temperature
for 3 h (f solution). The solvent was partially evaporated
under reduced pressure and the residue dissolved in ethyl
acetate (2 L). This solution was washed with water (0.5 L)
and brine (1 L), and the aqueous layers were reextracted with
ethyl acetate. Drying (Na2SO4) of the organic layers, concen-
tration, and washing with hexane gave 42.6 g (95%) of 15a :
1
mp 134-136 °C; H NMR (CD3OD) δ 7.58 (m, 4H), 7.5-7.15
(m, 10H), 3.91 (m, 2H), 3.60 (dt, 1H), 3.1-2.7 (m, 4H), 2.63
(dd, 1H), 1.31 (s, 9H); FAB MS (M + H)+ ) 462. Anal.
(C28H35N3O3) C, H, N.
1-[4-(P yr id in -2-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon 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
(15b). Prepared from 13b as described for 15a (4.1 g, 75%):
1H NMR (CD3OD) δ 8.59 (d, 1H), 7.88 (m, 3H), 7.83 (d, 1H),
7.50 (d, 2H), 7.35 (m, 1H), 7.3-7.15 (m, 5H), 3.94 (s, 2H), 3.59
(dt, 1H), 3.02 (m, 1H), 2.90 (m, 2H), 2.81 (m, 1H), 2.66 (dd,
1H), 1.29 (s, 9H); FAB MS (M + H)+ ) 463.
1-[4-(Th ia zol-2-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon 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
(15c). Prepared from 13c as described for 15a (2.4 g, 91%):
HPLC tR 11.9 min; FAB MS (M + H)+ ) 469.
1-[4-(Th ia zol-5-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon 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
(15d ). Prepared from 13d as described for 15a (4.4 g, 93%):
HPLC tR 11.5 min. Anal. (C25H32N4O3S‚0.53H2O) C, H, N, S,
H2O.
1-[4-(Dieth ylam in o)ph en yl]-2-[(ter t-bu tyloxycar bon 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
(15g). Prepared from 13g as described for 15a (58 g, 90%):
mp 97-98 °C. Anal. (C26H40N4O3) C, H, N.
1-(4-Biph en ylyl)-5(S)-2,5-diam in o-4(S)-h ydr oxy-6-ph en -
yl-2-a za h exa n e Dih yd r och lor id e (14a ). A solution of 12a
(95.2 g, 169.5 mmol) in 4 N HCl in dioxane (300 mL) was
stirred for 3 h at r.t. and then concentrated in vacuo. Lyo-
philization from dioxane yielded 74 g (quantitative) of 14a :
FAB MS (M + H)+ ) 362.
1-(4-Biph en ylyl)-2-[(ter t-bu tyloxycar bon yl)am 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 (16a ). To a solution of N-(methoxy-
carbonyl)-L-valine14 (25.9 g, 148 mmol), EDC (53.1 g, 277
mmol), and HOBT (25 g, 185 mmol) in DMF (500 mL) was
added triethylamine (77 mL, 554 mmol). The suspension was
stirred for 30 min; then a solution of 15a (42.6 g, 92.2 mmol)
in DMF (300 mL) was added dropwise. The ice bath was
removed, and stirring was continued at r.t. for 3 h. The
reaction mixture then was concentrated in vacuo and the
residue taken up in ethyl acetate (2.5 L) and water (1.8 L).
For complete dissolution of the remaining solids, methanol (0.2
L) and triethylamine (150 mL) were added optionally. The
organic phase was separated and washed three times with
water, saturated NaHCO3 solution, water, and brine. The
aqueous layers were extracted with ethyl acetate; the organic
phases were dried (Na2SO4) and concentrated in vacuo. Stir-
ring in diisopropyl ether yielded 45 g (79%) of crystalline 16a :
mp 201 °C; 1H NMR (CDCl3) δ 7.54 (m, 4H), 7.42 (m, 2H), 7.34
(m, 3H), 7.21 (d, 4H), 7.15 (m, 1H), 6.37 (m, HN), 5.28 (sb,
HN), 5.09 (m, HN), 4.57 (sb, HO), 4.00 (m, 1H), 3.93 (m, 3H),
3.66 (s, 3H), 3.60 (m, 1H), 2.94 (m, 1H), 2.89 (m, 1H), 2.73 (m,
1H), 2.38 (m, 1H), 1.97 (m, 1H), 1.33 (s, 9H), 0.81 (d, 3H), 0.74
(d, 3H); HPLC tR 17.9 min; FAB MS (M + H)+ ) 619. Anal.
(C35H46N4O6‚0.33H2O) C, H, N.
1-[4-(P yr id in -2-yl)p h en yl]-5(S)-2,5-d ia m in o-4(S)-h y-
d r oxy-6-p h en yl-2-a za h exa n e Tr ih yd r och lor id e (14b). A
solution of 12b (1465 g, 2.6 mol) in THF (12 L) and HCl (4 L,
4 N in water) was stirred for 4 h at 50 °C. From the resulting
biphasic mixture the aqueous layer was separated and con-
centrated in vacuo. The residue was diluted with ethanol (4
L), concentrated, again diluted with ethanol/toluene (1:1) (4
L), concentrated, diluted with another 4 L of ethanol, and
finally concentrated. Stirring in diisopropyl ether (9 L),
filtration, and drying (40 °C, in vacuo) gave 1303 g (quantita-
tive) of crystalline 14b. Anal. (C22H26N4O‚3.15HCl‚1.10H2O)
C, H, N, Cl, O, H2O.
1-[4-(Th ia zol-2-yl)p h e n yl]-5(S)-2,5-d ia m in o-4(S)-h y-
d r oxy-6-p h en yl-2-a za h exa n e (14c). A solution of 12c (21.7
g, 38.5 mmol) in formic acid (1 L) was stirred for 4 h at r.t.
and then concentrated in vacuo. The residue was redissolved
in dichloromethane and saturated NaHCO3 solution. The
aqueous layer was separated and extracted twice with dichlo-
romethane. The organic layers were washed with brine, dried
(Na2SO4), and concentrated, yielding 14.6 g of 14c, which was
used without further purification for the next step.
1-[4-(2-Meth yl-2H-tetr azol-5-yl)ph en yl]-5(S)-2,5-diam in o-
4(S)-h yd r oxy-6-p h en yl-2-a za h exa n e Dih yd r och lor id e
(14e). A solution of 12e (20 g, 35.2 mmol) in THF (280 mL)

New Aza-Dipeptide Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3397
1-[4-(P yr id in -2-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon yl)-
a m in o]-4(S)-h yd r oxy-5(S)-{[N-(m eth oxyca r bon yl)-L-va l-
in yl]am in o}-6-ph en yl-2-azah exan e (16b). Preparation from
15b as described for 16a yielded 5.1 g (92%) of 16b: 1H NMR
(CD3OD) δ 8.59 (d, 1H), 7.88 (m, 3H), 7.83 (d, 1H), 7.48 (d,
2H), 7.34 (m, 1H), 7.22 (m, 4H), 7.14 (m, 1H), 4.10 (m, 1H),
3.91 (s, 2H), 3.83 (d, 1H), 3.69 (m, 1H), 3.63 (s, 3H), 2.95 (dd,
1H), 2.83 (dd, 1H), 2.76 (m, 1H), 2.57 (m, 1H), 1.87 (m, 1H),
1.30 (s, 9H), 0.78 (d, 3H), 0.76 (d, 3H); HPLC tR 11.9 min; FAB
MS (M + H)+ ) 620.
for 3 h at r.t. and then concentrated in vacuo. Lyophilization
from dioxane then yielded 56.4 g (quantitative) of 19a : FAB
MS (M + H)+ ) 519; HPLC tR 12.7 min.
1-[4-(P yr id in -2-yl)p h en yl]-2-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 Dih yd r och lor id e (19b). Prepared from 16b as
described for 19a (4.0 g, quantitative): FAB MS (M + H)+
520; HPLC tR 8.0 min.
)
1-[4-(Th ia zol-2-yl)p h en yl]-2-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 (19c). Deprotection of 16c by formic acid as described
for 14c (552 mg, 70%): FAB MS (M + H)+ ) 526; HPLC tR
10.6 min.
1-[4-(Th ia zol-5-yl)p h en yl]-2-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 (19d ). Deprotection of 16d by formic acid as described
for 14c (903 mg): FAB MS (M + H)+ ) 526; HPLC tR 10.0
min.
1-(4-Bip h en ylyl)-2-a m in o-4(S)-h yd r oxy-5(S)-{[N-(m eth -
oxyca r bon yl)-L-ter t-leu cin yl]a m in o}-6-p h en yl-2-a za h ex-
a n e Hyd r och lor id e (20a ). Deprotection of 17a as described
for 19a (820 mg, 97%): FAB MS (M + H)+ ) 533; HPLC tR
13.2 min.
1-[4-(P yr id in -2-yl)p h en yl]-2-a m in o-4(S)-h yd r oxy-5(S)-
{[N-(m eth oxyca r bon yl)-L-ter t-leu cin yl]a m in o}-6-p h en yl-
2-a za h exa n e Dih yd r och lor id e (20b). Prepared from 17b
as described for 19a (4.4 g, ≈ quantitative): FAB MS (M +
H)+ ) 534; HPLC tR 8.5 min.
1-[4-(Th ia zol-2-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon yl)-
a m in o]-4(S)-h yd r oxy-5(S)-{[N-(m eth oxyca r bon yl)-L-va l-
in yl]am in o}-6-ph en yl-2-azah exan e (16c). Preparation from
15c as described for 16a yielded 2.7 g (86%) of 16c: mp 178-
1
179 °C; H NMR (CDCl3) δ 7.92 (d, 2H), 7.87 (d, 1H), 7.40 (d,
2H), 7.33 (d, 1H), 7.23 (m, 5H), 6.38 (d, 1H), 5.31 (s, 1H), 5.12
(m, 1H), 4.60 (s, 1H), 4.02 (m, 1H), 3.93 (m, 3H), 3.66 (s, 3H),
3.59 (m, 1H), 2.93 (m, 2H), 2.73 (m, 1H), 2.39 (m, 1H), 1.94
(m, 1H), 1.33 (s, 9H), 0.80 (d, 3H), 0.73 (d, 3H); HPLC tR 15.9
min; FAB MS (M + H)+ ) 626.
1-[4-(Th ia zol-5-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon yl)-
a m in o]-4(S)-h yd r oxy-5(S)-{[N-(m eth oxyca r bon yl)-L-va l-
in yl]am in o}-6-ph en yl-2-azah exan e (16d). Preparation from
15d as described for 16a yielded 4.5 g (75%) of 16d : mp 114-
1
115 °C; H NMR (CDCl3) δ 8.75 (s, 1H), 8.07 (s, 1H), 7.53 (d,
2H), 7.36 (d, 2H), 7.22 (m, 5H), 6.40 (d, 1H), 5.31 (s, 1H), 5.12
(m, 1H), 4.58 (s, 1H), 4.02 (m, 1H), 3.93 (m, 3H), 3.68 (s, 3H),
3.61 (m, 1H), 2.93 (m, 2H), 2.73 (m, 1H), 2.37 (m, 1H), 1.97
(m, 1H), 1.35 (s, 9H), 0.82 (d, 3H), 0.74 (d, 3H); HPLC tR 15.1
min; FAB MS (M + H)+ ) 626. Anal. (C32H43N5O6S‚0.27H2O)
C, H, N, S, H2O.
1-[4-(Th ia zol-5-yl)p h en yl]-2-a m in o-4(S)-h yd r oxy-5(S)-
{[N-(m eth oxyca r bon yl)-L-ter t-leu cin yl]a m in o}-6-p h en yl-
2-a za h exa n e (20d ). Deprotection of 17d by formic acid as
described for 14c (882 mg, quantitative): FAB MS (M + H)+
) 540.
1-(4-Biph en ylyl)-2-[(ter t-bu tyloxycar bon yl)am in o]-4(S)-
h ydr oxy-5(S)-{[N-(m eth oxyca r bon yl)-L-ter t-leu cin yl]a m i-
n o}-6-p h en yl-2-a za h exa n e (17a ). Preparation as described
for 16a starting from 29 yielded 900 mg (90%) of 17a : TLC
Rf(dichloromethane/methanol, 19:1) ) 0.57; 1H NMR (CD3OD)
δ 7.58 (m, 4H), 7.43 (m, 5H), 7.23 (m, 4H), 7.18 (m, 1H), 4.10
(m, 1H), 3.89 (s, 3H), 3.70 (m, 1H), 3.65 (s, 3H), 2.9 (m, 2H),
2.76 (m, 1H), 2.55 (m, 1H), 1.31 (s, 9H), 0.85 (s, 9H); HPLC tR
18.2 min; FAB MS (M + H)+ ) 633.
1-[4-(P yr id in -2-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon yl)-
a m in o]-4(S)-h yd r oxy-5(S)-{[N-(m eth oxyca r bon yl)-L-ter t-
leu cin yl]a m in o}-6-p h en yl-2-a za h exa n e (17b). Preparation
from 15b and 29 as described for 16a yielded 4.4 g (81%) of
17b: 1H NMR (CD3OD) δ 8.59 (d, 1H), 7.88 (m, 3H), 7.83 (d,
1H), 7.48 (d, 2H), 7.35 (m, 1H), 7.21 (m, 4H), 7.13 (m, 1H),
4.10 (m, 1H), 3.91 (s, 2H), 3.87 (s, 1H), 3.70 (m, 1H), 3.64 (s,
3H), 2.94 (dd, 1H), 2.83 (dd, 1H), 2.78 (m, 1H), 2.58 (m, 1H),
1.30 (s, 9H), 0.85 (s, 9H); HPLC tR 12.5 min; FAB MS (M +
H)+ ) 634. Anal. (C35H47N5O6) C, H, N.
1-[4-(P yr id in -2-yl)p h en yl]-2-a m in o-4(S)-h yd r oxy-5(S)-
{[N-(m eth oxyca r bon yl)-L-isoleu cin yl]a m in o}-6-p h en yl-2-
a za h exa n e d ih yd r och lor id e (21b). Prepared from 18b as
described for 19a (1.6 g, quantitative): FAB MS (M + H)+
534; HPLC tR 8.8 min.
)
1-(4-Bip h en ylyl)-5(S)-2,5-bis{[N-(m eth oxyca r bon yl)-L-
valin yl]am in o}-4(S)-h ydr oxy-6-ph en yl-2-azah exan e (22a).
To an ice-cooled suspension of N-(methoxycarbonyl)-L-valine14
(48.4 g, 276 mmol) and EDC (101.8 g, 530.9 mmol) in DMF (1
L) was added HOBT (43 g, 318.5 mmol), followed by triethyl-
amine (82.9 mL, 594.6 mmol). After 20 min of stirring, 1-(4-
biphenylyl)-5(S)-2,5-diamino-4(S)-hydroxy-6-phenyl-2-azahex-
ane dihydrochloride (14a ) (46 g, 106 mmol) was added
portionwise. The ice bath was removed, and stirring was
continued overnight. The reaction mixture then was concen-
trated in vacuo and the residue redissolved in dichloromethane
and washed with aqueous solutions of NaHCO3, citric acid,
and NaCl. The aqueous layers were extracted three times with
dichloromethane; the organic phases were dried (Na2SO4) and
concentrated in vacuo. Recrystallization from methanol/
diisopropyl ether yielded 43.8 g (61%) of 22a : TLC Rf(ethyl
acetate) ) 0.37; mp 214-216 °C; 1H NMR (CD3OD) δ 7.57 (m,
2H), 7.52 (d, 2H), 7.43 (d, 2H), 7.41 (t, 2H), 7.31 (t, 1H), 7.21
(m, 4H), 7.13 (m, 1H), 4.13 (m, 1H), 3.98 (m, 1H), 3.88 (m,
1H), 3.79 (d, 1H), 3.73 (m, 1H), 3.64 (s, 3H), 3.63 (m, 1H), 3.61
(s, 3H), 2.93 (dd, 1H), 2.84 (m, 2H), 2.64 (d, 1H), 1.85 (m, 1H),
1.67 (m, 1H), 0.77 (d, 3H), 0.75 (d, 3H), 0.64 (d, 3H), 0.60 (d,
3H); HPLC tR 15.5 min; FAB MS (M + H)+ ) 676. Anal.
(C37H49N5O7) C, H, N.
1-[4-(P yr id in -2-yl)p h en yl]-5(S)-2,5-b is{[N-(m et h oxy-
ca r bon 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 (22b). Preparation from 14b as described for 22a
yielded 295 mg (44%) of 22b: mp 210-212 °C; 1H NMR
(DMSO-d6) δ 9.06 (s, HN), 8.64 (d, 1H), 7.97 (d, 2H), 7.90 (d,
1H), 7.85 (td, 1H), 7.50 (d, 1H), 7.41 (d, 2H), 7.32 (m, 1H), 7.18
(m, 4H), 7.12 (m, 1H), 7.04 (d, 1H), 6.96 (d, 1H), 5.00 (s, 1H),
4.00 (m, 1H), 3.96 (d, 1H), 3.90 (d, 1H), 3.73 (t, 1H), 3.57 (m,
2H), 3.50 (s, 6H), 2.78 (dd, 1H), 2.70 (m, 2H), 2.61 (d, 1H),
1.77 (m, 1H), 1.59 (m, 1H), 0.68 (d, 3H), 0.63 (d, 3H), 0.55 (d,
3H), 0.48 (d, 3H); FAB MS (M + H)+ ) 677. Anal. (C36H48N6O7‚
0.6H2O) C, H, N, H2O.
1-[4-(Th ia zol-5-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon yl)-
a m in o]-4(S)-h yd r oxy-5(S)-{[N-(m eth oxyca r bon yl)-L-ter t-
leu cin yl]a m in o}-6-p h en yl-2-a za h exa n e (17d ). Preparation
from 15d and 29 as described for 16a yielded 2.2 g (76%) of
17d : 1H NMR (CD3OD) δ 8.96 (s, 1H), 8.15 (s, 1H), 7.60 (d,
2H), 7.45 (d, 2H), 7.2 (m, 5H), 4.11 (m, 1H), 3.89 (m, 3H), 3.7
(m, 1H), 3.66 (s, 3H), 3.0-2.7 (m, 3H), 2.56 (m, 1H), 1.30 (s,
9H), 0.83 (s, 9H); HPLC tR 16.0 min; FAB MS (M + H)+
640.
)
1-[4-(P yr id in -2-yl)p h en yl]-2-[(ter t-bu tyloxyca r bon yl)-
a m in o]-4(S)-h yd r oxy-5(S)-{[N-(m eth oxyca r bon yl)-L-iso-
leu cin yl]a m in o}-6-p h en yl-2-a za h exa n e (18b). Preparation
as described for 16a , starting from 15b and N-(methoxycar-
bonyl)-L-isoleucine14 yielded 1.6 g (72%) of 18b: 1H NMR (CD3-
OD) δ 8.58 (d, 1H), 7.88 (m, 3H), 7.83 (d, 1H), 7.48 (d, 2H),
7.34 (m, 1H), 7.23 (m, 4H), 7.14 (m, 1H), 4.12 (m, 1H), 3.91
(m, 2H), 3.87 (d, 1H), 3.70 (m, 1H), 3.63 (s, 3H), 2.95 (m, 1H),
2.87-2.75 (m, 2H), 2.58 (m, 1H), 1.67 (m, 1H), 1.34 (m, 1H),
1.29 (s, 9H), 1.05 (m, 1H), 0.79 (t, 3H), 0.73 (d, 3H); HPLC tR
12.5 min; FAB MS (M + H)+ ) 634.
1-(4-Bip h en ylyl)-2-a m in o-4(S)-h yd r oxy-5(S)-{[N-(m eth -
oxycar bon yl)-L-valin yl]am in o}-6-ph en yl-2-azah exan e Hy-
d r och lor id e (19a ). A solution of 16a (57.2 g, 92.4 mmol) in
4 N HCl in dioxane (200 mL) and methanol (20 mL) was stirred

3398 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Bold et al.
1-[4-(Th ia zol-5-yl)p h en yl]-5(S)-2,5-bis{[N-(m eth oxyca r -
bon yl)-L-valin yl]am in o}-4(S)-h ydr oxy-6-ph en yl-2-azah ex-
a n e (22d ). Preparation as described for 23b starting from
N-(methoxycarbonyl)-L-valine14 and 1-[4-(thiazol-5-yl)phenyl]-
2-amino-4(S)-hydroxy-5(S)-{[N-(methoxycarbonyl)-L-valinyl]-
amino}-6-phenyl-2-azahexane (19d ) yielded 124 mg (36%) of
22d : mp 207-208 °C; 1H NMR (CDCl3) δ 8.75 (s, 1H), 8.05 (s,
1H), 7.50 (d, 2H), 7.36 (d, 2H), 7.2 (m, 5H), 6.90 (s, 1H), 6.60
(d, 1H), 5.15 (m, 2H), 4.83 (s, 1H), 4.2-3.8 (m, 4H), 3.68 (s,
3H), 3.63 (s, 3H), 3.6 (m, 2H), 2.95 (d, 2H), 2.83 (m, 1H), 2.56
(m, 1H), 2.01 (m, 1H), 1.82 (m, 1H), 0.84 (d, 3H), 0.74 (d, 3H),
0.67 (d, 3H), 0.63 (d, 3H); HPLC tR 13.3 min; FAB MS (M +
H)+ ) 683. Anal. (C34H46N6O7S‚0.66H2O) C, H, N, S, H2O.
1-(4-Bip h en ylyl)-2-{[N-(m eth oxyca r bon yl)-L-ter t-leu ci-
n 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 (23a ). A solution of
N-(methoxycarbonyl)-L-tert-leucine (29) (18.04 g, 95.3 mmol)
and TPTU (28.4 g, 95.3 mmol) in DMF (180 mL) was stirred
for 15 min. Then a solution of 1-(4-biphenylyl)-2-amino-4(S)-
hydroxy-5(S)-{[N-(methoxycarbonyl)-L-valinyl]amino}-6-phen-
yl-2-azahexane hydrochloride (19a ) (52.8 g, 95.3 mmol) and
NMM (31.4 mL, 286 mmol) in DMF (560 mL) was added
dropwise. After stirring for 16 h, the reaction mixture was
poured into water (7 L) and the resulting precipitate filtered
off and washed with water. The crude product was redissolved
in ethyl acetate (2.5 L) and washed with water and brine. The
aqueous layers were extracted with ethyl acetate and the
organic phases dried (Na2SO4) and concentrated to a volume
of ≈1 L. Crystallization by adding hexane (2 L), filtration,
and recrystallization from methanol afforded 33 g (50%) of
23a : mp 206-207 °C; [R]D ) -42° (c ) 1, EtOH); 1H NMR
(DMSO-d6) δ 8.82 (s, 1H), 7.58 (d, 2H), 7.50 (d, 2H), 7.44 (t,
2H), 7.40 (t, 2H), 7.30 (m, 2H), 7.19 (m, 4H), 7.12 (m, 1H),
6.57 (m, 1H), 6.35 (m, 1H), 4.78 (s, 1H), 4.05 (q, 1H), 3.95 (m,
2H), 3.77 (dd, 1H), 3.66 (d, 1H), 3.63 (m, 1H), 3.53 (s, 3H),
3.51 (s, 3H), 2.84 (dd, 1H), 2.74 (m, 3H), 1.85 (oct, 1H), 0.75
(d, 3H), 0.72 (d, 3H), 0.70 (s, 9H); HPLC tR 16.7 min; FAB MS
(M + H)+ ) 690. Anal. (C38H51N5O7) C, H, N.
1-[4-(P yr id in -2-yl)p h en yl]-2-{[N-(m eth oxyca r bon yl)-L-
ter t-leu cin yl]a m in o}-4(S)-h yd r oxy-5(S)-{[N-(m et h oxy-
ca r bon yl)-L-va lin yl]a m in o}-6-p h en yl-2-a za h exa n e (23b).
To an ice-cooled solution of 29 (152 mg, 0.80 mmol), EDC (287
mg, 1.5 mmol), and HOBT (135 mg, 1.0 mmol) in DMF (3 mL)
was added triethylamine (0.49 mL, 3.5 mmol). The suspension
was stirred for 10 min; then a solution of 1-[4-(pyridin-2-yl)-
phenyl]-2-amino-4(S)-hydroxy-5(S)-{[N-(methoxycarbonyl)-L-
valinyl]amino}-6-phenyl-2-azahexane dihydrochloride (19b)
(296 mg, 0.50 mmol) in DMF (3 mL) was added. The ice bath
was removed, and stirring was continued at r.t. for 3 h. The
reaction mixture then was concentrated in vacuo and the
residue redissolved in dichloromethane/water. The aqueous
phase was separated and extracted twice with dichloromethane.
The organic layers were washed with saturated NaHCO3
solution, water, and brine, dried (Na2SO4), and concentrated
in vacuo. Medium-pressure liquid chromatography (SiO2,
ethyl acetate/hexane, 4:1) and stirring in diisopropyl ether
yielded 105 mg (30%) of 23b: TLC Rf(ethyl acetate) ) 0.35;
1H NMR (CD3OD) δ 8.58 (d, 1H), 7.87 (m, 3H), 7.82 (d, 1H),
7.51 (d, 2H), 7.34 (m, 1H), 7.22 (m, 4H), 7.13 (m, 1H), 4.13 (m,
1H), 3.99 (m, 2H), 3.79 (d, 1H), 3.74 (m, 1H), 3.68 (s, 1H), 3.62
(s, 3H), 3.59 (s, 3H), 2.93 (dd, 1H), 2.84 (m, 2H), 2.67 (m, 1H),
1.86 (m, 1H), 0.76 (d, 3H), 0.74 (d, 3H), 0.70 (s, 9H); HPLC tR
11.1 min; FAB MS (M + H)+ ) 691.
1-[4-(Th ia zol-5-yl)p h en yl]-2-{[N-(m eth oxyca r bon yl)-L-
ter t-leu cin yl]a m in o}-4(S)-h yd r oxy-5(S)-{[N-(m et h oxy-
ca r bon yl)-L-va lin yl]a m in o}-6-p h en yl-2-a za h exa n e (23d ).
Preparation from 19d as described for 23b yielded 67 mg (9%)
of 23d : mp 200-201 °C; 1H NMR (CD3OD) δ 8.95 (s, 1H), 8.15
(s, 1H), 7.58 (d, 2H), 7.47 (d, 2H), 7.23 (m, 4H), 7.15 (m, 1H),
4.13 (m, 1H), 3.96 (m, 2H), 3.79 (d, 1H), 3.75 (m, 1H), 3.66 (s,
1H), 3.64 (s, 3H), 3.61 (s, 3H), 2.87 (m, 3H), 2.67 (m, 1H), 1.86
(m, 1H), 0.76 (d, 3H), 0.74 (d, 3H), 0.70 (s, 9H); HPLC tR 14.0
min; FAB MS (M + H)+ ) 697.
1-(4-Biph en ylyl)-2-{[N-(m eth oxycar bon yl)-L-valin yl]am i-
n o}-4(S)-h ydr oxy-5(S)-{[N-(m eth oxycar bon yl)-L-ter t-leu ci-
n yl]a m in o}-6-p h en yl-2-a za h exa n e (24a ). Preparation as
described for 23a starting from N-(methoxycarbonyl)-L-valine14
and 20a yielded 564 mg (61%) of 24a : 1H NMR (CD3OD) δ
7.6-7.1 (m, 14H), 4.15 (m, 1H), 3.96 (m, 2H), 3.88 (s, 1H), 3.76
(d, 1H), 3.65 (s, 3H), 3.62 (s, 3H), 3.6 (m, 1H), 2.9 (m, 3H),
2.67 (m, 1H), 1.70 (oct, 1H), 0.84 (s, 9H), 0.66 (d, 3H), 0.60 (d,
3H); HPLC tR 16.5 min; FAB MS (M + H)+ ) 690. Anal.
(C38H51N5O7‚0.21H2O) C, H, N, H2O.
1-[4-(P yr id in -2-yl)p h en yl]-2-{[N-(m eth oxyca r bon yl)-L-
valin yl]am in o}-4(S)-h ydr oxy-5(S)-{[N-(m eth oxycar bon yl)-
L-ter t-leu cin yl]am in o}-6-ph en yl-2-azah exan e (24b). Prepa-
ration as described for 23b starting from N-(methoxycarbonyl)-
L-valine14 and 20b yielded 310 mg (30%) of 24b: TLC Rf(ethyl
acetate) ) 0.35; 1H NMR (CD3OD) δ 8.58 (d, 1H), 7.87 (m, 3H),
7.81 (d, 1H), 7.50 (d, 2H), 7.35 (m, 1H), 7.21 (m, 4H), 7.12 (m,
1H), 4.15 (m, 1H), 4.02 (d, 1H), 3.94 (d, 1H), 3.85 (s, 1H), 3.75
(m, 1H), 3.63 (m, 4H), 3.60 (s, 3H), 2.93 (dd, 1H), 2.86 (m, 2H),
2.68 (m, 1H), 1.69 (m, 1H), 0.83 (s, 9H), 0.66 (d, 3H), 0.60 (d,
3H); HPLC tR 10.9 min; FAB MS (M + H)+ ) 691. Anal.
(C37H50N6O7‚0.59H2O) C, H, N, H2O.
1-[4-(Th ia zol-5-yl)p h en yl]-2-{[N-(m eth oxyca r bon yl)-L-
valin yl]am in o}-4(S)-h ydr oxy-5(S)-{[N-(m eth oxycar bon yl)-
L-ter t-leu cin yl]am in o}-6-ph en yl-2-azah exan e (24d). Prepa-
ration as described for 23a starting from N-(methoxycarbonyl)-
L-valine14 and 20d yielded 174 mg (43%) of 24d : mp 134-135
°C; 1H NMR (CD3OD) δ 8.96 (s, 1H), 8.15 (s, 1H), 7.59 (d, 2H),
7.47 (d, 2H), 7.22 (m, 4H), 7.17 (m, 1H), 4.15 (m, 1H), 3.96 (m,
2H), 3.86 (s, 1H), 3.75 (m, 1H), 3.65 (s, 3H), 3.62 (s, 3H), 3.57
(m, 1H), 2.9 (m, 3H), 2.67 (m, 1H), 1.70 (m, 1H), 0.83 (s, 9H),
0.68 (d, 3H), 0.62 (d, 3H); HPLC tR 14.0 min; FAB MS (M +
H)+ ) 697. Anal. (C35H48N6O7S) C, H, N, S.
1-(4-Bip h en ylyl)-5(S)-2,5-bis{[N-(m eth oxyca r bon yl)-L-
ter t-leu cin yl]a m in o}-4(S)-h yd r oxy-6-p h en yl-2-a za h ex-
a n e (25a , CGP 75355). Preparation from 14a as described
for 25b yielded 45.1 g (44%) of 25a (CGP 75355): mp 210-
1
211 °C; [R]D ) -58° (c ) 1, EtOH); H NMR (CD3OD) δ 7.56
(d, 2H), 7.51 (d, 2H), 7.44 (d, 2H), 7.41 (t, 2H), 7.31 (t, 1H),
7.20 (m, 4H), 7.12 (m, 1H), 4.13 (t, 1H), 3.96 (m, 2H), 3.85 (s,
1H), 3.75 (d, 1H), 3.69 (s, 1H), 3.63 (s, 3H), 3.60 (s, 3H), 2.92
(dd, 1H), 2.85 (m, 2H), 2.66 (d, 1H), 0.83 (s, 9H), 0.71 (s, 9H);
HPLC tR 12.1 min; FAB MS (M + H)+ ) 704. Anal.
(C39H53N5O7‚0.22H2O) C, H, N, O, H2O.
1-[4-(P yr id in -2-yl)p h en yl]-5(S)-2,5-b is{[N-(m et h oxy-
ca r bon yl)-L-ter t-leu cin yl]a m in o}-4(S)-h yd r oxy-6-p h en yl-
2-a za h exa n e (25b, CGP 73547). To an ice-cooled suspension
of N-(methoxycarbonyl)-L-tert-leucine (29) (567 g, 3.0 mol) and
TPTU (891 g, 3.0 mol) in dichloromethane (3 L) was added
dropwise N-ethyldiisopropylamine (775 g, 6 mol). Stirring for
20 min produced a solution. Then a suspension of 1-[4-
(pyridin-2-yl)phenyl]-5(S)-2,5-diamino-4(S)-hydroxy-6-phenyl-
2-azahexane trihydrochloride (14b) (472 g, 1.0 mol) in dichlo-
romethane (3 L) was added portionwise at 0-5 °C. The ice
bath was removed, and stirring was continued overnight. The
reaction mixture then was washed with water (10 L), saturated
NaHCO3 solution (10 L), and brine (5 L). The aqueous layers
were extracted twice with dichloromethane (5 L); the organic
phases were dried (Na2SO4) and concentrated in vacuo. The
residue was dissolved in ethyl acetate (6 L) and filtered
through silica gel (500 g); the column was eluted with ethyl
acetate (6 L) and the eluate concentrated under reduced
pressure. The resulting material was stirred for 1 h in boiling
diisopropyl ether with 2% ethanol (9 L). Cooling to r.t.,
1-[4-(Th ia zol-2-yl)p h en yl]-2-{[N-(m eth oxyca r bon yl)-L-
ter t-leu cin yl]a m in o}-4(S)-h yd r oxy-5(S)-{[N-(m et h oxy-
ca r bon yl)-L-va lin yl]a m in o}-6-p h en yl-2-a za h exa n e (23c).
Preparation from 19c as described for 23b yielded 95 mg (39%)
of 23c: 1H NMR (CD3OD) δ 7.88 (d, 2H), 7.85 (d, 1H), 7.60 (d,
1H), 7.52 (d, 2H), 7.2 (m, 5H), 4.15 (m, 1H), 4.00 (s, 2H), 3.77
(m, 3H), 3.64 (s, 3H), 3.61 (s, 3H), 3.0-2.6 (m, 4H), 1.86 (m,
1H), 0.78 (d, 3H), 0.75 (d, 3H), 0.71 (s, 9H); HPLC tR 14.5 min;
FAB MS (M + H)+ ) 697. Anal. (C35H48N6O7S‚0.7H2O) C, H,
N, S, H2O.

New Aza-Dipeptide Analogues
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3399
filtration, and washing with diisopropyl ether (+2% ethanol)
afforded 513 g (73%) of crude 25b: TLC Rf(ethyl acetate) )
0.53; mp 201-203 °C. Redissolving at 70 °C in ethanol (70%
in water), filtration, and crystallization by adding water and
cooling to 5 °C gave pure 25b (CGP 73547) in 67% yield: mp
2.56 (d, 1H), 1.12 (t, 6H), 0.84 (s, 9H), 0.75 (s, 9H); HPLC tR
11.8 min; FAB MS (M + H)+ ) 699. Anal. (C37H58N6O7‚
0.55H2O) C, H, N, H2O.
1-[4-(P yr id in -2-yl)p h en yl]-2-{[N-(m eth oxyca r bon yl)-L-
valin yl]am in o}-4(S)-h ydr oxy-5(S)-{[N-(m eth oxycar bon yl)-
L-isoleu cin yl]a m in o}-6-p h en yl-2-a za h exa n e (26b). Prepa-
ration as described for 23a starting from N-(methoxycarbonyl)-
L-valine14 and 21b yielded 465 mg (45%) of 26b: TLC Rf(ethyl
acetate) ) 0.4; 1H NMR (CD3OD) δ 8.58 (d, 1H), 7.88 (m, 3H),
7.82 (d, 1H), 7.51 (d, 2H), 7.34 (m, 1H), 7.21 (m, 4H), 7.13 (m,
1H), 4.15 (m, 1H), 4.04 (d, 1H), 3.92 (d, 1H), 3.84 (d, 1H), 3.74
(m, 1H), 3.63 (m, 4H), 3.61 (s, 3H), 2.92 (dd, 1H), 2.86 (m, 2H),
2.67 (m, 1H), 1.66 (m, 2H), 1.29 (m, 1H), 1.03 (m, 1H), 0.79 (t,
3H), 0.71 (d, 3H), 0.63 (d, 3H), 0.58 (d, 3H); HPLC tR 11.1 min;
FAB MS (M + H)+ ) 691. Anal. (C37H50N6O7‚0.18H2O) C, H,
N, H2O.
1
207-209 °C; [R]D ) -47° (c ) 1, EtOH); H NMR (CD3OD) δ
8.58 (d, 1H), 7.86 (m, 3H), 7.81 (d, 1H), 7.50 (d, 2H), 7.35 (dd,
1H), 7.20 (m, 4H), 7.12 (m, 1H), 4.15 (t, 1H), 3.99 (m, 2H),
3.85 (s, 1H), 3.76 (d, 1H), 3.68 (s, 1H), 3.63 (s, 3H), 3.59 (s,
3H), 2.93 (dd, 1H), 2.86 (m, 2H), 2.69 (d, 1H), 0.83 (s, 9H),
0.71 (s, 9H); HPLC tR 11.9 min; FAB MS (M + H)+ ) 705.
Anal. (C38H52N6O7‚0.18H2O) C, H, N, O, H2O.
1-[4-(Th iazol-2-yl)ph en yl]-2,5-bis{[N-(m eth oxycar bon yl)-
L-ter t-leu cin yl]a m in o}-4(S)-h yd r oxy-6-p h en yl-2-a za h ex-
a n e (25c, CGP 75136). Preparation from 14c as described
for 25b (solvent: DMF) yielded 17.1 g (62%) of 25c (CGP
75136): mp 134-136 °C; TLC Rf(hexane/ethyl acetate, 1:3) )
1-[4-(P yr id in -2-yl)p h en yl]-2-{[N-(et h oxyca r b on yl)-L-
valin yl]am in o}-4(S)-h ydr oxy-5(S)-{[N-(m eth oxycar bon yl)-
L-va lin yl]a m in o}-6-p h en yl-2-a za h exa n e (27b). Prepara-
tion as described for 23b starting from N-(ethoxycarbonyl)-L-
valine17 yielded 310 mg (45%) of 27b: TLC Rf(ethyl acetate)
1
0.22; [R]D ) -46° (c ) 0.6, EtOH); H NMR (CD3OD) δ 7.86
(d, 2H), 7.84 (d, 1H), 7.58 (d, 1H), 7.51 (d, 2H), 7.20 (m, 4H),
7.12 (m, 1H), 4.15 (m, 1H), 3.98 (s, 2H), 3.84 (s, 1H), 3.75 (m,
1H), 3.68 (s, 1H), 3.63 (s, 3H), 3.60 (s, 3H), 2.92 (dd, 1H), 2.85
(m, 2H), 2.69 (m, 1H), 0.82 (s, 9H), 0.72 (s, 9H); FAB MS (M
+ H)+ ) 711. Anal. (C36H50N6O7S‚0.23H2O) C, H, N, S, H2O.
1
) 0.33; H NMR (CD3OD) δ 8.59 (d, 1H), 7.87 (m, 3H), 7.82
(d, 1H), 7.50 (d, 2H), 7.34 (m, 1H), 7.21 (m, 4H), 7.13 (m, 1H),
4.14 (m, 1H), 4.03 (m, 3H), 3.93 (d, 1H), 3.79 (d, 1H), 3.75 (m,
1H), 3.64 (d, 1H), 3.63 (s, 3H), 2.92 (dd, 1H), 2.85 (m, 2H),
2.67 (m, 1H), 1.85 (m, 1H), 1.68 (m, 1H), 1.20 (t, 3H), 0.76 (d,
3H), 0.74 (d, 3H), 0.65 (d, 3H), 0.59 (d, 3H); HPLC tR 11.1 min;
FAB MS (M + H)+ ) 691. Anal. (C37H50N6O7‚0.25H2O) C, H,
N, H2O.
1-[4-(Th iazol-5-yl)ph en yl]-2,5-bis{[N-(m eth oxycar bon yl)-
L-ter t-leu cin yl]a m in o}-4(S)-h yd r oxy-6-p h en yl-2-a za h ex-
a n e (25d ). Preparation as described for 23a starting from
1-[4-(thiazol-5-yl)phenyl]-2-amino-4(S)-hydroxy-5(S)-{[N-(meth-
oxycarbonyl)-L-tert-leucinyl]amino}-6-phenyl-2-azahexane (20d)
yielded 105 mg (34%) of 25d : mp 207-209 °C; 1H NMR (CD3-
OD) δ 8.96 (s, 1H), 8.15 (s, 1H), 7.59 (d, 2H), 7.47 (d, 2H), 7.2
(m, 5H), 4.15 (m, 1H), 3.97 (s, 2H), 3.85 (s, 1H), 3.75 (m, 1H),
3.69 (s, 1H), 3.64 (s, 3H), 3.62 (s, 3H), 2.9 (m, 3H), 2.67 (m,
1H), 0.82 (s, 9H), 0.72 (s, 9H); HPLC tR 14.7 min; FAB MS (M
+ H)+ ) 711. Anal. (C36H50N6O7S‚0.25H2O) C, H, N, S, H2O.
1-[4-(2-Meth yl-2H-tetr azol-5-yl)ph en yl]-2,5-bis{[N-(m eth -
oxycar bon yl)-L-ter t-leu cin yl]am in o}-4(S)-h ydr oxy-6-ph en -
yl-2-a za h exa n e (25e, CGP 75176). Preparation from 14e
as described for 25b (solvent: DMF) yielded 16 g (64%) of 25e
(CGP 75176): mp 191-192 °C; [R]D ) -46° (c ) 0.5, EtOH);
1H NMR (DCl2C-CCl2D, 80 °C) δ 8.09 (d, 2H), 7.47 (d, 2H),
7.3-7.15 (m, 5H), 6.6 (sb, 1H), 6.24 (d, 1H), 5.13 (m, 2H), 4.39
(s, 3H), 4.08 (m, 2H), 4.01 (d, 1H), 3.75 (d, 1H), 3.8-3.55 (m,
3H), 3.68 (s, 3H), 3.64 (s, 3H), 2.95 (m, 2H), 2.89 (m, 1H), 2.68
(m, 1H), 0.91 (s, 9H), 0.83 (s, 9H); HPLC tR 14.4 min; FAB MS
(M + H)+ ) 710. Anal. (C35H51N9O7) C, H, N, O.
1-[4-(2-ter t-Bu t yl-2H -t et r a zol-5-yl)p h en yl]-2,5-b is{[N-
(m eth oxyca r bon yl)-L-ter t-leu cin yl]a m in o}-4(S)-h yd r oxy-
6-p h en yl-2-a za h exa n e (25f). A solution of N-(methoxycar-
bonyl)-L-tert-leucine (29) (54 mg, 0.283 mmol) and TPTU (84
mg, 0.283 mmol) in DMF (1 mL) was stirred for 10 min. Then
a solution of 1-[4-(2-tert-butyl-2H-tetrazol-5-yl)phenyl]-2-{[N-
(methoxycarbonyl)-L-tert-leucinyl]amino}-4(S)-hydroxy-5(S)-
amino-6-phenyl-2-azahexane hydrochloride (36) (175 mg, 0.283
mmol) and NMM (94 µL, 0.85 mmol) in DMF (2 mL) was
added. After stirring for 16 h, the reaction mixture was poured
into water (40 mL) and extracted with three portions of
dichloromethane. The organic layers were washed with brine,
dried (Na2SO4), and concentrated. Column chromatography
(CH2Cl2/methanol, 25:1) gave 79 mg (37%) of 25f: mp 140 °C
(crystallized from toluene/hexane); [R]D ) -41.5° (c ) 0.9,
EtOH); TLC Rf(CH2Cl2/methanol, 19:1) ) 0.48; 1H NMR (CD3-
OD) δ 8.02 (d, 2H), 7.55 (d, 2H), 7.23 (m, 4H), 7.16 (m, 1H),
4.17 (m, 1H), 4.01 (s, 2H), 3.86 (s, 1H), 3.78 (m, 1H), 3.7 (s,
1H), 3.63 (s, 3H), 3.62 (s, 3H), 2.9 (m, 3H), 2.70 (m, 1H), 1.82
(s, 9H), 0.84 (s, 9H), 0.73 (s, 9H); FAB MS (M + H)+ ) 752.
Anal. (C38H57N9O7) C, H, N, O.
1-[4-(Th ia zol-5-yl)p h en yl]-2-{[N-(et h oxyca r b on yl)-L-
valin yl]am in o}-4(S)-h ydr oxy-5(S)-{[N-(m eth oxycar bon yl)-
L-ter t-leu cin yl]am in o}-6-ph en yl-2-azah exan e (28d). Prepa-
ration as described for 23a starting from N-(ethoxycarbonyl)-
L-valine17 and 20d yielded 229 mg (55%) of 28d : mp 168-169
°C; 1H NMR (CD3OD) δ 8.97 (s, 1H), 8.15 (s, 1H), 7.60 (d, 2H),
7.47 (d, 2H), 7.22 (m, 4H), 7.15 (m, 1H), 4.15 (m, 1H), 4.06 (q,
2H), 3.96 (m, 2H), 3.87 (s, 1H), 3.77 (m, 1H), 3.65 (s, 3H), 3.62
(m, 1H), 2.9 (m, 3H), 2.69 (m, 1H), 1.70 (m, 1H), 1.21 (t, 3H),
0.82 (s, 9H), 0.68 (d, 3H), 0.62 (d, 3H); HPLC tR 14.7 min; FAB
MS (M + H)+ ) 711. Anal. (C36H50N6O7S) C, H, N, S.
N-(Meth oxyca r bon yl)-L-ter t-leu cin e (29). To a solution
of L-tert-leucine (1250 g, 9.5 mol) in dioxane (5 L) and 2 N
aqueous sodium hydroxide (15.7 L) was added methyl chloro-
formiate (1470 mL, 18.9 mol) during 1.5 h. Then the clear
solution was stirred for 18 h at 60 °C, cooled to r.t., and
extracted twice with dichloromethane (15 L). The aqueous
phase was acidified by addition of 4 N HCl (8 L, pH ≈ 2) and
extracted three times with ethyl acetate (10 L). Drying (Na2-
SO4) of the ethyl acetate layers, concentration in vacuo, stirring
in cold hexane (5 L), and filtration afforded 1622 g (90%) 29:
1
mp 108-109 °C; H NMR (CD3OD) δ 7.00 (d, HN), 4.00 (m,
1H), 3.66 (s, 3H), 1.02 (s, 9H). Anal. (C8H15NO4) C, H, N, O.
N-1-(ter t-Bu tyloxycar bon yl)-N-2-[N-(m eth oxycar bon yl)-
L-ter t-leu cin yl]h yd r a zin e (30). To 29 (10 g, 52.8 mmol),
EDC (11.1 g, 58.1 mmol), and HOBT (7.85 g, 58.1 mmol) in
ethyl acetate (130 mL) was added NMM (7.0 mL, 63.7 mmol).
After 30 min, tert-butyl carbazate (7.69 g, 58.1 mmol) was
added, and stirring was continued for 16 h. Then the reaction
mixture was diluted with ethyl acetate (0.3 L) and washed with
saturated NaHCO3 solution, water, and brine. The inorganic
layers were reextracted twice with ethyl acetate; the combined
organic phases were dried (Na2SO4) and concentrated, yielding
16 g of 30 (≈ quantitative): 1H NMR (CD3OD) δ 3.98 (s, 1H),
3.66 (s, 3H), 1.47 (s, 9H), 1.03 (s, 9H); FAB MS (M + H)+
304.
)
[N-(Meth oxyca r bon yl)-L-ter t-leu cin yl]h yd r a zin e (31).
A solution of 30 (16 g, 52.8 mmol) in 4 N HCl in dioxane (100
mL) was stirred for 18 h at r.t. and then concentrated in vacuo.
The residue was diluted with saturated NaHCO3 solution and
extracted four times with dichloromethane (800 mL). The
organic layers were washed with brine, dried (Na2SO4), and
concentrated, yielding 6.5 g (60%) of 31: 1H NMR (CD3OD) δ
1-[4-(Diet h yla m in o)p h en yl]-2,5-b is{[N-(m et h oxyca r -
b on yl)-L-ter t-leu cin yl]a m in o}-4(S)-h yd r oxy-6-p h en yl-2-
a za h exa n e (25g). Preparation from 14g as described for 25b
(solvent: DMF) yielded 69 g (82%) of 25g: mp 155 °C; [R]D
)
1
-54° (c ) 0.55, EtOH); H NMR (CD3OD) δ 7.2 (m, 4H), 7.13
(m, 1H), 7.13 (d, 2H), 6.61 (d, 2H), 4.07 (t, 1H), 3.86 (s, 1H),
3.78 (m, 2H), 3.71 (s, 1H), 3.70 (m, 1H), 3.65 (s, 3H), 3.64 (s,
3H), 3.33 (q, 4H), 2.90 (dd, 1H), 2.84 (dd, 1H), 2.81 (m, 1H),
3.89 (s, 1H), 3.65 (s, 3H), 0.98 (s, 9H); FAB MS (M + H)+
204.
)

3400 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Bold et al.
N-1-[N-(Meth oxyca r bon yl)-L-ter t-leu cin yl] N-2-[4-(Tet-
r a zol-5-yl)ben zylid en e] Hyd r a zon e (32). A solution of
4-(tetrazol-5-yl)benzaldehyde (6) (2.57 g, 14.8 mmol) and 31
(3.0 g, 14.8 mmol) in 2-propanol (30 mL) was heated to reflux
temperature during 18 h. Addition of water (100 mL) and
filtration afforded 4.3 g (80%) of 32: 1H NMR (CD3OD) δ 8.23
(s, 1H), 8.1 (m, 2H), 8.0 (m, 2H), 4.08 (s, 1H), 3.68 (s, 3H),
1.06 (s, 9H); FAB MS (M + H)+ ) 360.
technical assistance in biological testing and Dr. U.
Schneider and Mr. W. Salamin for spectral measure-
ments.
Refer en ces
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N-1-[N-(Met h oxyca r b on yl)-L-ter t-leu cin yl] N-2-[4-(2-
ter t-Bu tyl-2H-tetr a zol-5-yl)ben zylid en e] Hyd r a zon e (33).
In a sealed tube, 32 (3.0 g, 8.34 mmol), toluene (25 mL),
methanesulfonic acid (0.08 g, 0.83 mmol), and isobutene (1.2
g) were heated to 110 °C for 1 h. The reaction mixture was
cooled to r.t. and then diluted with ethyl acetate and saturated
NaHCO3 solution. The aqueous phase was separated and
extracted twice with ethyl acetate. The organic layers were
washed with brine, dried (Na2SO4), and concentrated in vacuo.
Column chromatography (hexane/ethyl acetate, 1:1) gave 2.35
1
g (68%) of 33: TLC Rf(hexane/ethyl acetate, 1:1) ) 0.22; H
NMR (CD3OD) δ 8.21 (s, 1H), 8.17 (m, 2H), 7.94 (m, 2H), 4.07
(m, 1H), 3.68 (s, 3H), 1.82 (s, 9H), 1.08 (s, 9H); FAB MS (M +
H)+ ) 416.
N-1-[N-(Met h oxyca r b on yl)-L-ter t-leu cin yl]-N-2-[4-(2-
ter t-bu tyl-2H-tetr a zol-5-yl)ben zyl]h yd r a zin e (34). NaCN-
BH3 (317 mg, 5.05 mmol) was added to 33 (2.0 g, 4.81 mmol)
in THF (9 mL); then a solution of p-TsOH (915 mg, 4.81 mmol)
in THF (9 mL) was added dropwise. After 18 h, the reaction
mixture was diluted with ethyl acetate. The aqueous layer
was separated and extracted twice with ethyl acetate. The
organic phases were washed with saturated NaHCO3 solution
and brine, dried (Na2SO4), and concentrated by evaporation.
The residue was taken up in water (20 mL) and THF (20 mL),
then K2B4O7‚4H2O (6.1 g, 20 mmol) was added, and the
mixture stirred at r.t. overnight. After dilution with ethyl
acetate and saturated NaHCO3 solution, the aqueous phase
was separated and extracted twice with ethyl acetate. The
organic layers were washed with brine, dried (Na2SO4), and
concentrated by evaporation. Column chromatography (hex-
ane/ethyl acetate, 1:2) gave 1.17 g (58%) of 34: TLC Rf-
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2H), 7.53 (d, 2H), 4.04 (s, 2H), 3.83 (s, 1H), 3.64 (s, 3H), 1.82
(s, 9H), 0.92 (s, 9H); FAB MS (M + H)+ ) 418.
1-[4-(2-ter t-Bu tyl-2H-tetr a zol-5-yl)p h en yl]-2-{[N-(m eth -
oxyca r bon yl)-L-ter t-leu cin yl]a m in o}-4(S)-h yd r oxy-5(S)-
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phenyl-3(R)-3,4-epoxybutane (10)6b,12 (737 mg, 2.8 mmol) and
34 (1.17 g, 2.8 mmol) in 2-propanol (15 mL) was heated to
reflux temperature during 16 h. Addition of water (100 mL)
and filtration gave the crude product. Recrystallization from
CH2Cl2 by addition of diisopropyl ether/hexane afforded 803
mg (42%) of 35: TLC Rf(CH2Cl2/methanol, 30:1) ) 0.34; 1H
NMR (CD3OD) δ 8.01 (d, 2H), 7.55 (d, 2H), 7.23 (m, 4H), 7.15
(m, 1H), 4.00 (s, 2H), 3.74 (m, 2H), 3.68 (s, 1H), 3.59 (s, 3H),
2.8 (m, 4H), 1.80 (s, 9H), 1.30 (s, 9H), 0.72 (s, 9H); FAB MS
(M + H)+ ) 681.
1-[4-(2-ter t-Bu tyl-2H-tetr a zol-5-yl)p h en yl]-2-{[N-(m eth -
oxyca r bon yl)-L-ter t-leu cin 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 Hyd r och lor id e (36). A sus-
pension of 35 (200 mg, 0.294 mmol) in THF (2.3 mL) and HCl
(1.6 mL, 2 N in water) was stirred for 8 h at 50 °C. The
resulting solution was concentrated in vacuo. The residue was
four times diluted with ethanol and again concentrated, giving
182 mg (quantitative) of 36: TLC Rf(CH2Cl2/methanol/H2O/
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a potent inhibitor of the
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CH3COOH, 170:26:3:1) ) 0.28; H NMR (CD3OD) δ 8.03 (d,
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Sonderegger, and Mr. F. Stauffer for their capable

New Aza-Dipeptide Analogues
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