Paracyclophanes: A novel class of water-soluble inhibitors of HIV proteinase

Article abstract of DOI:10.1021/jm950641i

A versatile synthesis of functionalized para- and metacyclophanes (macrocycles with one or more aromatic rings incorporated; ansa-compounds) has been developed. Cyclophanes constitute a novel building block for potent human immunodeficiency virus (HIV) protease inhibitors. The synthesis of the macrocyclic ring system was achieved by regio- and stereospecific ring opening of N-protected 4-amino-2,3-epoxy-5-phenylpentanoates with appropriate α,ω-diamines and consecutive ring closure under high dilution conditions. The resulting macrocyclic building blocks enabled further broad and flexible derivation. Paracyclophanes, containing oxyethylene substructures, were found to dissolve in phosphate-buffered saline at concentrations as high as 3 mg/mL at physiological pH. Several derivatives with K(i) values lower than 10 nM and antiviral activities in the range of 15-50 nM have been obtained. The influence of the ring size and of the substitution pattern of the cyclophane moiety on enzyme inhibition, antiviral activity, and water solubility are discussed. Preliminary data on oral bioavailability in mice are given for selected compounds.

Full text of DOI:10.1021/jm950641i

J . Med. Chem. 1996, 39, 3291-3299
3291
P a r a cyclop h a n es: A Novel Cla ss of Wa ter -Solu ble In h ibitor s of HIV P r otein a se†
Peter Ettmayer,* Andreas Billich, Peter Hecht, Brigitte Rosenwirth, and Hubert Gstach
Sandoz Forschungsinstitut Ges.m.b.H., Department of Antiretroviral Therapy, Brunnerstrasse 59, A-1235 Vienna, Austria
Received August 29, 1995X
A versatile synthesis of functionalized para- and metacyclophanes (macrocycles with one or
more aromatic rings incorporated; ansa-compounds) has been developed. Cyclophanes
constitute a novel building block for potent human immunodeficiency virus (HIV) protease
inhibitors. The synthesis of the macrocyclic ring system was achieved by regio- and
stereospecific ring opening of N-protected 4-amino-2,3-epoxy-5-phenylpentanoates with ap-
propriate R,ω-diamines and consecutive ring closure under high dilution conditions. The
resulting macrocyclic building blocks enabled further broad and flexible derivation. Paracy-
clophanes, containing oxyethylene substructures, were found to dissolve in phosphate-buffered
saline at concentrations as high as 3 mg/mL at physiological pH. Several derivatives with Ki
values lower than 10 nM and antiviral activities in the range of 15-50 nM have been obtained.
The influence of the ring size and of the substitution pattern of the cyclophane moiety on enzyme
inhibition, antiviral activity, and water solubility are discussed. Preliminary data on oral
bioavailability in mice are given for selected compounds.
In tr od u ction
Recently, we reported on the synthesis of 2-hetero-
substituted 4-amino-3-hydroxy-5-phenylpentanoic acid
as a novel building block for the design of HIV protein-
ase (HIV PR) inhibitors.1 Stereochemical control and
convergent synthesis of the core-unit of the inhibitors
together with maximum synthetic flexibility for deriva-
tion allowed the preparation of different types of PR
inhibitors (Figure 1). Compound 1, a representative of
numerous potent derivatives, was obtained by a novel
synthetic route. Ki values for inhibition of HIV-1 pro-
teinase by chemotype 1 were typically below 5 nM, and
antiviral activity with IC50 values below 30 nM was
demonstrated.1 The characteristic structural feature of
inhibitors of type 1 based on a 2-heterosubstituted
statine moiety is a benzimidazole substituent covering
the P3′-subsite.2 However, compounds of this type lack
oral bioavailability in different animal species.3 Oral
uptake in several animal species of compounds of type
1 was achieved by truncation of the C-terminus of the
inhibitor, and by introduction of 1(S)-amino-2(R)-hy-
droxyindane as P2′-substituent, which yielded PR in-
hibitor 2.4 This compound, designated SDZ PRI 053,
exhibits exceptionally high oral bioavailability in mice,
rats, and dogs.4 However, the antiviral activity of SDZ
F igu r e 1. Chemical structures of HIV PR inhibitors 1-3.
† Abbreviations: The abbreviations for the natural amino acids
(three-letter code) are in accord with the recommendations of the
IUPAC-IUB J oint Commission on Biochemical Nomenclature (Eur. J .
Biochem. 1984, 138, 9-37). In addition: AcN (acetonitrile), BOP (1-
(benzotriazolyloxy)tris(dimethylamino)phosphonium hexafluorophos-
phate, Boc (tert-butyloxycarbonyl; 1,1-dimethylethoxycarbonyl), DCC
(dicyclohexylcarbodiimide), DMAP (4-(dimethylamino)pyridine), DMF
(dimethylformamide), DMSO (dimethyl sulfoxide), EDC‚HCl (N-ethyl-
N ′-(3-(dimethylamino)propyl)carbodiimide hydrochloride), HOBt (1-
hydroxy-benzotriazole), HODhbt (3,4-dihydro-3-hydroxy-4-oxo-1,2,3-
benzotriazine), HPTLC (high performance thin-layer chromatography),
NMM (N-methylmorpholine), Ph (phenyl), PyBOP (1-(benzotriazoly-
loxy)tripyrrolidinophosphonium hexafluorophosphate), RP-HPLC (re-
versed-phase high-performance liquid chromatography), rt (room
temperature), tert-leucine ((2S)-amino-3,3-dimethylbutanoic acid), THF
(tetrahydrofuran), TLC (thin-layer chromatography), Tle (tert-leucine;
(2S)-amino-3,3-dimethylbutanoic acid), TsCl (4-methylbenzenesulfonyl
chloride), TsOH (4-methylbenzenesulfonic acid), Z (benzyloxycarbonyl).
* To whom correspondence should be addressed.
PRI 053 (2) was somewhat lower when compared to
inhibitor 1 (Table 5).
Therefore, we have investigated strategies to improve
antiviral activity while retaining oral bioavailablity of
the compounds.4b,5 One of our approaches was prompted
by reports that increased aqueous solubility of inhibitors
facilitated oral absorption. This statement was origi-
nally based on an analysis of the bioavailability of
sparingly water soluble, albeit potent, peptidomimetic
PR inhibitors.6,7 Few nonpeptidic HIV PR inhibitors
have been reported to be significantly water-soluble.
Furthermore, their antiviral activities are poor com-
pared to peptide-based inhibitors.8 We chose to design
X Abstract published in Advance ACS Abstracts, J uly 15, 1996.
S0022-2623(95)00641-8 CCC: $12.00 © 1996 American Chemical Society

3292 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Ettmayer et al.
Sch em e 1. Synthesis of the Diamines 11 and 12a
peptidomimetic inhibitors containing ether-type mac-
rocycles (3) which would (i) increase the hydrophilicity
of the compounds by introduction of polar structural
elements in positions of the inhibitors which do not
interfere with the hydrophobic active site of the HIV
PR, and (ii) would at least partially fill the S3′ pocket
that had remained unoccupied by 2. Various examples
for cyclic inhibitors of renin have been described.9 In
addition, two reports of macrocyclic, peptide-based
inhibitors of HIV PR were published while this work
was in progress.10 These macrocyclic inhibitors offer the
further advantages of improved binding affinity to the
enzyme resulting from entropic energy gain of confor-
mationally constrained structures, greater stability
against proteolytic enzymes, and enhanced specificity
for the target enzyme. Herein we report the design and
synthesis of a series of cyclophane-containing HIV PR
inhibitors. The structural parameters determining
enzyme inhibition, anti HIV activity, oral bioavailabil-
ity, and aqueous solubility of this novel chemotype of
an HIV PR inhibitor are also discussed.
aConditions: (a) 2-(2-aminoethoxy)ethanol, HOBt, EDC‚HCl,
DMF, rt; (b) p-TsCl, pyridine, CH2Cl2, rt; (c) hydroxybenzaldehyde,
K2CO3, tetrabutylammonium iodide, DMSO, 70 °C; (d) NH2OH‚HCl,
NaOAc, H2O, EtOH, 60 °C; (e) 10% Pd/C, H2, MeOH, 4% HCOOH,
rt.
In h ibitor Design
To support the concept that compounds like 3 could
represent novel HIV PR inhibitors, modeling studies
were performed. The conformation of the 2-aminoben-
zylstatine-containing compound 1, docked in the active
site of the HIV-1 PR,11 was used as a template for the
construction of various macrocyclic inhibitors incorpo-
rating oligo ethylene glycol ether linkages between P1′
and P3′. The benzimidazole moiety in 1 was replaced
by the appropriate linkers which were attached both in
para and meta position to the benzene ring in P1′.
Energy minimization12 of these structures was followed
by the prediction of biological activity (enzyme inhibi-
tion) using the 3D QSAR model described recently.11
The calculated Ki value (40 to 50 nM) for paracyclophane
3 encouraged us to develop a synthetic route to these
type of macrocycle to further investigate their physico-
chemical properties.
Sch em e 2. Epoxide Opening and Cyclizationa
The crucial step in the synthesis of the cyclophanes
is the regio- and stereoselective nucleophilic ring-open-
ing of oxirane 131 (Scheme 2) with diamines. The
straightforward synthesis of the diamines 11, 12 is
outlined in Scheme 1. The appropriate amino alcohol
was coupled to N-carbobenzyloxy valine using EDC‚-
HCl and HOBt. The resulting alcohol 5 was converted
to the tosylate 6 which was subsequently introduced in
a phenolic O-alkylation of m- or p-hydroxybenzaldehyde.
The best yields of crystalline aldehydes 7 and 8 were
obtained when the reaction was carried out in DMSO
with K2CO3 and 10% tetrabutylammonium iodide.
DMSO was superior to DMF since fewer side products
were formed.13 Upon treatment of the aldehydes (7, 8)
with hydroxylamine and subsequent catalytic hydroge-
nation of the corresponding oximes (9, 10; 10% Pd/C in
methanol containing 4% formic acid), the carbobenzy-
loxy protecting group was simultaneously removed to
give the desired diamines 11 and 12.
aConditions: (a) 11 or 12, EtOH, 80 °C; (b) (i) 0.4 M LiOH,
dioxane, rt, (ii) 4 equiv of HODhbt, 4 equiv of EDC‚HCl, 1 mM in
THF, rt; (c) 6 N HCl, dioxane, rt; (d) Z-Val, HODhbt, EDC‚HCl,
dioxane, rt; (e) (+)-3(S)-hydroxytetrahydrofuran, phosgene in
toluene, pyridine, CH2Cl2, rt.
â-hydroxy esters (14, 15). The products were obtained
in a diasteriomerically pure form after chromatographic
workup. The regioselectivity of the nucleophilic attack
and the inversion of configuration at the C-2 chiral
center were determined previously with other examples
from the 2-heterosubstituted statine ester series.1 A 9:1
mixture of the diasteromeric oxiranes 13a and 13b is
readily available via a three-step procedure starting
from N-tert-butyloxycarbonyl phenylalaninol.1 As shown
previously, this mixture can be used in the next step
without further purification.
Synthesis of the seco acid esters 14 and 15 and sub-
sequent cyclization follows the route described in Scheme
2. Upon treatment of oxiranes 13a ,b1 with a diamine
(11, 12) in ethanol, only the sterically less hindered
more nucleophilic nitrogen atom of the diamine reacts
with the 2,3-epoxy ester to give the R-alkylamino

Macrocyclic Inhibitors of HIV Proteinases
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3293
Ta ble 1. Macrocyclization: Yields of 16 as Determined by
RP-HPLC
Biologica l Resu lts a n d Discu ssion
For all cyclophane derivatives reported here, the
inhibition constant (Ki) was measured for HIV-1 pro-
teinase. Antiviral activity (IC50) was determined in an
assay observing the HIV-1 strain IIIB induced cyto-
pathic effect in de novo infected MT4 cells. Aqueous
solubility was evaluated in phosphate-buffered saline
at physiological pH and for selected compounds in the
pH profile 4-8 (see Experimental Section for details).
First, we studied the effects of variation of the ring
size of para- and metacyclophanes on biological activity.
For each macrocycle, both the derivatives spanning the
S3 to S3′ receptor binding site (“type P3-P3′”, R ) Cbz-
Val; 3, 22-24 and 28, 20, 29) and their respective
precursors with the tert-butyloxycarbonyl group occupy-
ing the P2 pocket (“type P2-P3′”, R ) Boc; 16, 25-27
and 30, 17, 31) were tested (Tables 2 and 3).
Increasing the ring size from a [13]- to a [20]-
cyclophane and modifying the substitution pattern on
the aromatic ring from para to meta (Table 2 and 3: 3,
22-24 and 28, 20, 29) did not effect the Ki values (6.6
to 9.4 nM) to a significant extent for the “type P3-P3′”
inhibitors. These findings corroborate the modeling
studies which had suggested that the linkers between
the benzylamine in P1′ and P3′ would mediate contact
to the solvent outside of the active site, and that the
overall conformation of the residues occupying the S1′
and S2′ pockets should not be altered by the size of the
rings.
Whereas, a slight increase of the antiviral activity was
observed in the paracyclophane series when the ring
size was altered from [13] (22) to [17] (23) (Table 2),
the metacyclophane series activity dropped by a factor
of approximately seven (Table 3: 28, 20, 29). Even
though Ki and IC50 values were nearly unaffected by
the variation of the ring size and the substitution
pattern, a larger difference between meta- and paracy-
clophanes was observed with regard to water solubility.
The [14]-paracyclophane (3) is at least 50-fold more
soluble than the [14]-metacyclophane (20). Contrary to
expectation, an increasing number of oxyethylene moi-
eties did not increase aqueous solubility. The [20]-
paracyclophane (24) was found to be 70-fold less soluble
than the [14]-paracyclophane 3 at physiological pH.
c[mM]
solvent seco acid
yielda
% 16
variations
solvent
reagent
DMF
AcN
EtOAc
1.3
1.0
1.3
1.3
2.7
2.7
1.3
1.3
1.3
1.0
1.0
1.0
HODhbt/EDC‚HClb
HODhbt/EDC‚HClb
HODhbt/EDC‚HClb
HODhbt/EDC‚HClb
HODhbt/EDC‚HClb
HODhbt/EDC‚HClb
HOBt/EDC‚HClc
HODhbt/DCCd
45
54
5
solvent and seco THF
acid concn
67
37
29
55
77
28
71
49
72
THF
CH2Cl2
THF
THF
THF
THF
THF
THF
reagent
HOBt/DCCe
BOP/DMAPf
NMMg
PyBOP/DMAPh
a
The kinetics of the macrocyclization was monitored by RP-
HPLC: RP18 Column 150 × 3.9 mm, 10 mM ammonium acetate
(pH ) 7)/AcN 1:1 (v/v), flow rate 1 mL/min, rt, UV-detection at
210 nm. The yields represent the maximum turnover obtained
b
after approximately 30 h.
4 equiv of HODhbt, 4 equiv of
EDC‚HCl. c 4 equiv of HOBt, 4 equiv of EDC‚HCl. 4 equiv of
d
HODhbt, 4 equiv DCC. e 4 equiv of HOBt, 4 equiv DCC. f 2 equiv
g
h
of BOP, 3 equiv of DMAP. 2 equiv of BOP, 3 equiv of NMM.
equiv of PyBOP, 3 equiv of DMAP.
2
14 and 15 were saponified, and the macrocyclic amide
bond was formed upon treatment with 4 equiv of EDC‚-
HCl/HODhbt in dry THF. This macrocyclization step
was optimized for the paracyclophane 16. Although the
seco-acid precursor of 16 hardly dissolves in THF, this
solvent was superior to DMF, AcN, EtOAc, and dichlo-
romethane (Table 1). The optimal concentration of the
seco acid was found to be 1.3 mM. Doubling the
concentration lowered the yield by a factor of two. After
testing several coupling reagents it was found that
HODhbt/DCC, HODhbt/EDC‚HCl, and PyPOB/DMAP
gave almost the same yields (67-77%).
Removal of the Boc protecting group of the cyclo-
phanes 16 and 17 using 6 N HCl in dioxane yielded the
central building blocks 18 and 19 for further derivation
in P3 and P2. Coupling of the N-terminal 3(S)-hydrox-
ytetrahydrofuran moiety with the deprotected cyclo-
phane 18 was performed by prereacting a solution of
phosgene with the appropriate alcohol. Addition of 18
to tetrahydrofuranyl chloroformate prepared in situ
yielded the paracyclophane 21 in 80% yield.
Ta ble 2. Paracyclophanes: Inhibition of HIV-1 Protease, Antiviral Activity against HIV-1, IIIB, in MT4 Cells, and Aqueous
Solubility at pH 7.4
solubility
(µM)
no.
R
R′
ring size
Ki (nM)a
IC50 (nM)b
formulac
C40H53N5O7
22
3
Z-Val
Z-Val
Z-Val
Z-Val
Boc
Boc
Boc
Boc
(CH2)4
(CH2)2O(CH2)2
(CH2)2O(CH2)2O(CH2)2
(CH2)2O(CH2)2O(CH2)2O(CH2)2
(CH2)4
(CH2)2O(CH2)2
(CH2)2O(CH2)2O(CH2)2
(CH2)2O(CH2)2O(CH2)2O(CH2)2
[13]
[14]
[17]
[20]
[13]
[14]
[17]
[20]
6.6
7.9
6.9
7.5
17
14
12.6
23.2
27 ( 6 (6)
21 ( 5 (6)
16 ( 8 (6)
52 ( 3 (4)
155
336
38
C40H53N5O8‚0.8H2O
C42H57N5O9‚0.9H2O
C44H61N5O10‚0.6H2O
C32H46N4O6‚0.6H2O
C32H46N4O7‚0.9H2O
C34H50N4O8‚0.5H2O
C36H54N4O9‚0.8H2O
23
24
25
16
26
27
5
1810 (1)
788
1667
1049
1421
450 ( 213 (5)
169 ( 91 (4)
1170 ( 14 (2)
a
b
Usual standard deviation (20%. Mean value ( standard deviation. Number of determinations given in parentheses. c Satisfactory
elemental analyses within (0.4% of the calculated value for C, H, N were obtained.

3294 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Ettmayer et al.
Ta ble 3. Metacyclophanes: Inhibition of HIV-1 Protease, Antiviral Activity against HIV-1, IIIB, in MT4 Cells, and Aqueous
Solubility at pH 7.4
solubility
(µM)
no.
R
R′
ring size
Ki (nM)a
IC50 (nM)b
formulac
28
20
29
30
17
31
Z-Val
Z-Val
Z-Val
Boc
Boc
Boc
(CH2)4
(CH2)2O(CH2)2
(CH2)2O(CH2)2O(CH2)2
(CH2)4
(CH2)2O(CH2)2
[13]
[14]
[17]
[13]
[14]
[17]
9.4
7.6
7.7
16.1
68.8
207.7
29 ( 17 (6)
60 ( 30 (5)
211 ( 116(5)
335 ( 110 (5)
935 ( 432 (6)
3000 (1)
1
6
5
48
323
607
C40H53N5O7‚0.6H2O
C40H53N5O8
C42H57N5O9‚0.8H2O
C32H46N4O6
C32H46N4O7
C34H50N4O8‚0.5H2O
(CH2)2O(CH2)2O(CH2)2
a
b
Usual standard deviation (20%. Mean value ( standard deviation. Number of determinations given in parentheses. c Satisfactory
elemental analyses within (0.4% of the calculated value for C, H, N were obtained.
remarkable antiviral potency (IC50 ) 170 nM) and
aqueous solubility (1 mM) at physiological pH.
To summarize the results of the variation of ring size
in para- and metacyclophanes, the paracyclophanes
clearly proved superior to the metacyclophanes with
regard to antiviral activity and aqueous solubility.
Modifying the ring size in the paracyclophanes had only
little effect on enzyme inhibition and antiviral activity,
whereas the aqueous solubility decreased when the ring
size was larger than 14. On the basis of the observation
that multiple oxyethylene moieties decreased aqueous
solubility, and the fact that the tetramethylene linker-
containing paracyclophane 22 is >100 times more
soluble than 1 or 2 at physiological pH, we conclude that
the favorable hydrophilic characteristic is an inherent
feature of the paracyclophane. A possible explanation
could be the better accessibility of the polar atoms of
the ring structure by the surrounding water molecules
compared to the acyclic structures 1 and 2.
Consequently, papracyclophanes 3, 16, and 26 were
further modified in P3 and P2 (Table 4). Replacement
of valine in P2 in 3 by tert-leucine yielded a [14]-
paracyclophane (32) with almost the same solubility and
affinity to the enzyme, but a slightly increased antiviral
F igu r e 2. pH-dependency of the water solubility of the
activity. Substitution of the N-terminal benzyloxycar-
bonyl group in 3 by the more polar triazolylthioacetyl
moiety (33) led to the expected enhancement of the
aqueous solubility by a factor of five. While this
compound still is a good HIV PR inhibitor (Ki ) 11 nM),
it failed to inhibit virus growth in a cellular assay at
concentrations as high as 3 µM. This finding illustrates
again that antiviral activity is determined not only by
inhibitory potency against the proteinase, but also by
other poorly understood factors such as cellular uptake,
stability, or intracellular distribution.1
paracyclophanes 3, 22, 23, 24 as well as 1 and 2 in Merck
standard buffers (see Experimental Section for details).
When the aqueous solubilities of the [13], [14], [17], and
[20]-paracyclophanes were measured at different pH in
the range of 5 to 8, the same order 3 > 22 > 23 > 24
was observed (see Figure 2). At pH 4, where protona-
tion of the secondary amine in the ring strongly con-
tributes to solubility, 3 still is more soluble than 22, 23,
and 24, which are 20-fold more soluble than 1 and 2.
“Type P2-P3′” paracyclophanes (R ) Boc, Table 2: 16,
25-27) still show notable protease inhibition with Ki
values in the range of 12.6 to 23.2 nM. As before, the
ring size can be regarded as essentially irrelevant to
the thermodynamics of enzyme binding. This is not the
case for the “type P2-P3′” metacyclophanes where the
Ki value of the 17-membered ring is 12-fold higher than
that of the 13-membered ring (Table 3: 31, 30). Though
the antiviral activity of these “type P2-P3′” inhibitors
is significantly lower than with the “type P3-P3′”
compounds, the [17]paracyclophane (26) exhibits a
Recently, it has been reported that 3(S)-tetrahydro-
furanol is a good mimic for the P2-amino acid of selected
protease inhibitors.14 Encouraged by the potency of the
paracyclophanes with a tert-butoxycarbonyl group in P2,
we decided to replace this protecting group in 16 and
26 by the more hydrophilic tetrahydrofuran group. We
expected that this replacement would result in even
higher solubility. Indeed, with aqueous solubilities of
1.5 mg/mL and 2.1 mg/mL at pH 7.4, 14-paracyclophane
21 and [17]-paracyclophane 34 displayed a 1.5 and 3
times higher aqueous solubility compared to 16 and 26.

Macrocyclic Inhibitors of HIV Proteinases
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3295
Ta ble 4. Paracyclophanes Modified in P3-P2: Inhibition of HIV-1 Protease, Antiviral Activity against HIV-1, IIIB, in MT4 Cells, and
Aqueous Solubility at pH 7.4
aUsual standard deviation (20%. bMean value ( standard deviation. Number of determinations given in parentheses. cSatisfactory
elemental analyses within (0.4% of the calculated value for C, H, N were obtained unless otherwise noted. dN: calcd, 14.61; found,
13.29.
Ta ble 5. Activity of Aminobenzylstatine-Containing Inhibitors against HIV-1, HIV-2, Human Cathepsin D and against HIV-1, IIIB,
and HIV-2, EHO Replication in MT4 Cells and Aqueous Solubility at pH 7.4, and Oral Bioavailability
HIV-1
IC50 (nM)b
HIV-2
IC50 (nM)b
460
IC50 (nM)
solubility
(µM)
AUC(0f∞)
(µM‚h)
no.
Ki (nM)a
Ki (nM)a
human cathepsin D
1
2
3
23
32
3.4
9.5
7.9
6.9
8.0
29 ( 19 (18)
242 ( 114 (10)
21 ( 5 (6)
16 ( 8 (6)
15 ( 9 (6)
120
50
19
27
16
380
>10000
>10000
>10000
>10000
<10
<10
336
38
<0.4
82.5
2.2
<0.4
4.7
843 ( 335 (3)
20 ( 2 (3)
37 ( 22(2)
53 ( 7 (2)
323
a
b
Usual standard deviation (20%. Mean value ( standard deviation. Number of determinations given in parentheses.
The beneficial effect of this mimic of the P2-amino acid
on enzyme inhibition and antiviral activity14 was not
observed for these paracyclophanes to a significant
extent.
Paracyclophanes 3, 23, and 32 were administered
orally to mice and plasma levels of the compounds were
analyzed by HPLC (Table 5). At a dose of 125 mg/kg,
the compounds showed only low oral bioavailability in
mice compared to compound 2, despite better aqueous
solubility.
A selection of paracyclophanes (3, 23, and 32) was
characterized in more detail (see Table 5). The com-
pounds do not inhibit the mammalian aspartic protein-
ase cathepsin D which was affected by earlier com-
pounds like 1 that span the P3 to P3′ subsites. The
paracyclophanes are 2- to 4-fold weaker inhibitors of
HIV-2 proteinase than of the HIV-1 enzyme. The
difference in inhibition of the two enzymes is less
pronounced than with other aminobenzylamino-substi-
tuted statine derivatives (e.g., 1; Table 5). Inhibition
of HIV-2 replication in MT4 cells is inhibited at similar
(compound 3) or 2- to 4-fold higher (23, 32) concentra-
tions than that of HIV-1.
Compound 23 was also tested against HIV in primary
cells. In primary T4-lymphocytes, the paracyclophane
showed IC50 ) 3.1 nM for inhibition of HIV-1, strain
IIIB, and IC50 ) 17 nM in the case of HIV-2, strain MS.
For inhibition of a monocytotropic variant of HIV-1,
namely strain BaL, an IC50 value of 40 nM in primary
monocytes was measured. These results may be com-
pared with IC50 values for Saquinavir () Ro31-895915)
of 4.3 and 4.9 nM for inhibition of HIV-1, IIIB and BaL,
respectively, obtained in parallel experiments.
Su m m a r y
We have designed a versatile synthesis of HIV-
proteinase inhibitors which contain a cyclophane ring
system as the characteristic structural feature covering
the S1′-S3′ subsites2 of the enzyme. This central part
of the inhibitors is available by regio- and stereospecific
ring opening of N-protected 4-amino-2,3-epoxy-5-phe-
nylpentanoic acid ester with appropriate R,ω-diamines
followed by macrocyclization, and allows flexible deriva-
tion. Substitution of this building block with Z protected
amino acids as P2-P3 substituent provided HIV-inhibi-
tors which, in terms of anti-HIV activity, are comparable
to Saquinavir () Ro31-895915). Remarkably, the ring
size of the macrocycle has very little influence on
enzyme inhibition and antiviral activity in both series
of cyclophanes (meta and para). In terms of antiviral
activity, the paracyclophanes turned out to be more
active compared to their meta-substituted congeners.
Besides high anti-HIV activity of several derivatives
synthesized, the most striking feature of this novel type

3296 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Ettmayer et al.
vacuo. Ethyl acetate and 1 N aqueous sodium hydroxide
were added, and the organic layer was dried over Na2SO4
and concentrated. After addition of n-hexane the product
crystallized: yield 42.5 g of 7 (83%) (chromatographic purifica-
tion (ethyl acetate/n-hexane ) 1/1) of the mother liquid yielded
additional 6.5 g of 7; overall yield: 96%); mp 115-118 °C; 1H-
NMR (250 MHz; CDCl3) δ 9.89 (s, 1H); 7.83 and 7.00 (2d, J )
9 Hz, 4H); 7.37-7.30 (m, 5H); 6.31 (bs, 1H); 5.41 (d, J ) 9 Hz,
1H); 5.09 (s, 2H); 4.20-4.16 (m, 2H); 3.96 (dd, J ) 6, 9 Hz,
1H); 3.84-3.81 (m, 2H); 3.64-3.45 (m, 4H); 2.09 (oct, J ) 7
Hz, 1H); 0.94 and 0.90 (2d, J ) 7 Hz, 6H). Anal. (C24H30N2O6‚
0.3H2O) C, H, N.
of inhibitor is the good solubility in water at physiologi-
cal pH. Solubilities at pH 7.4 as high as 2.1 mg/mL
could be achieved while still inhibiting virus growth
with an IC50 of 157 nM. Three protease inhibitors from
this class (3, 23, and 32), were selected for their antiviral
activity and were tested for oral bioavailability. Con-
trary to expectation, enhanced aqueous solubility (at
least 3-10 times higher than 2) did not result in
enhanced systemic uptake in mice.
Exp er im en ta l Section
3-[2-[2-[[N-[(Ben zyloxy)ca r bon yl]va lyl]a m in o]eth oxy]-
eth oxy]ben za ld eh yd e (8). Preparation was as described for
Ch em istr y. 1H-NMR spectra were recorded with a Bruker
WC-250 or AMX-500 spectrometer at 300 K unless otherwise
noted; chemical shifts are reported in ppm (δ) relative to
internal Me4Si. All J values are given in hertz (Hz). Elemen-
tal analyses were performed by Analytical Department, Sandoz
Basle, Switzerland, and Mikroanalytisches Laboratorium,
Institut fu¨r Physikalische Chemie der Universita¨t Wien, and
are within 0.4% of the theoretical value. All compounds gave
correct FAB-MS spectra. Analytical thin-layer chromatogra-
phy was performed on silica gel 60 F254 glass plates (HPTLC,
Merck). Preparative column chromatography was performed
on silica gel (40-63 µm) under pressure (∼0.2 mPa). Solvents
were AR grade and were used without further purification.
All reagents were obtained from commercial suppliers and
were used without further purification. Evaporations were
carried out in vacuo with a rotary evaporator. Melting points
were determined with a thermovar apparatus (Reichert-J ung)
or capillary melting point apparatus (neoLab) and are not
corrected.
1
7: yield 3.97 g of 8 (45%); mp 91-94 °C; H-NMR (250 MHz;
CDCl3) δ 9.97 (s, 1H); 7.47-7.41 (m, 2H); 7.36-7.27 (m, 6H);
7.26-7.18 (m, 1H); 6.37 (bs, 1H); 5.42 (d, J ) 9 Hz, 1H); 5.09
(s, 2H); 4.19-4.15 (m, 2H); 3.98 (dd, J ) 6, 9 Hz, 1H); 3.84-
3.80 (m, 2H); 3.63-3.47 (m, 4H); 2.09 (oct, J ) 7 Hz, 1H); 0.96
and 0.90 (2d, J ) 7 Hz, 6H). Anal. (C24H30N2O6‚0.1H2O) C,
H, N.
4-[2-[2-[[N-[(Ben zyloxy)ca r bon yl]va lyl]a m in o]eth oxy]-
eth oxy]ben za ld eh yd e Oxim e (9). 7 (48.4 g, 0.109 mol) was
dissolved in 200 mL of ethanol (gentle warming) and treated
with hydroxyammonium chloride (11.4 g, 0.164 mol) and 13.5
g (0.165 mol) sodium acetate dissolved in 100 mL of water.
The reaction mixture was stirred at 60 °C, for 90 min, the
solvent was removed in vacuo, ethyl acetate and water were
added, and the organic layer was washed with saturated
aqueous sodium hydrogen carbonate and brine, dried over Na2-
SO4, and concentrated. The title compound precipitates after
addition of n-hexane: yield 50.0 g of 9 (quantitative); mp 71-
73 °C; 1H-NMR (250 MHz; CDCl3) δ 9.70 (s, 1H); 8.08 (s, 1H);
7.49 and 6.87 (2d, J ) 9 Hz, 4H); 7.38-7.23 (m, 5H); 6.45 (bs,
1H); 5.59 (d, J ) 9 Hz, 1H); 5.13 and 5.06 (AB, J ) 12 Hz,
2H); 4.12-4.08 (m, 2H); 3.96 (dd, J ) 7, 9 Hz, 1H); 3.82-3.79
(m, 2H); 3.68-3.40 (m, 4H); 2.09 (oct, J ) 7 Hz, 1H); 0.90 and
0.87 (dd, J ) 7 Hz, 6H). Anal. (C24H31N3O6) C, H, N.
3-[2-[2-[[N-[(Ben zyloxy)ca r bon yl]va lyl]a m in o]eth oxy]-
eth oxy]ben za ld eh yd e Oxim e (10). Preparation was as
Representative methods for all compounds synthesized
according to Scheme 1 are described. 2-[2-(2-Aminoethoxy)-
ethoxy]ethanol and 2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-
ethanol were prepared from tri- and tetraethylene glycol by
monotosylation and aminolysis with aqueous ammonia.16
2-[2-[[N-[(Ben zyloxy)ca r b on yl]va lyl]a m in o]et h oxy]-
eth a n ol (5). 2-(2-Aminoethoxy)ethanol (22.5 g, 0.213 mol),
HOBt (27.7 g, 0.181 mol), and EDC‚HCl (37.5 g, 0.196 mol)
were added to an ice-cooled solution of Z-Val (49.1 g, 0.195
mol) in 500 mL of DMF. The reaction mixture was stirred for
24 h at room temperature. After evaporation of the solvent,
ethyl acetate and water were added. The organic layer was
washed with 1 N HCl, 1 N NaOH, and brine, dried, and
concentrated in vacuo. Addition of cyclohexane precipitated
1
described for 9: yield 3.1 g of 10 (86%); mp 114-117 °C; H-
NMR (250 MHz; CDCl3) δ 9.61 (s, 1H); 8.15 (s, 1H); 7.49 (s,
1H); 7.35-7.22 (m, 6H); 7.04 (d, J ) 8HZ, 1H); 6.96-6.92 (m,
2H); 5.78 (d, 1H); 5.14 and 5.07 (AB, J ) 12 Hz, 2H); 4.17-
4.14 (m, 2H); 3.98 (dd, J ) 7, 9 Hz, 1H); 3.86-3.53 (m, 5H);
3.38-3.23 (m, 1H); 2.06 (oct, J ) 7 Hz, 1H); 0.94 and 0.93 (dd,
J ) 7 Hz, 6H). Anal. (C24H31N3O6 ) C, H, N.
1
54.8 g of 5 (83%) as a white solid: mp 123-125 °C; H-NMR
(250 MHz; CDCl3) δ 7.36-7.27 (m, 5H); 6.94 (bs, 1H); 5.62 (d,
J ) 9 Hz, 1H); 5.09 (bs, 2H); 3.95 (dd, J ) 7, 9 Hz, 1H); 3.71-
3.68 (m, 2H); 3.55-3.35 (m, 6H); 3.18 (bs, 1H); 2.10 (oct, J )
7 Hz, 1H); 0.96 and 0.93 (2d, J ) 7 Hz, 6H).
4-[2-[2-[(Va lyl)a m in o]eth oxy]eth oxy]ben zyla m in e (11).
A solution of 9 (49.5 g, 0.108 mol) in 1 L of methanol and 60
mL of formic acid was treated with 4 g of 10% Pd/C and stirred
under atmospheric pressure of hydrogen for 3 h. After
filtration over Celite the solvents were removed in vacuo. The
oily residue was suspended in dichloromethane and sufficient
1 N NaOH added to maintain in the aqueous phase at pH >
8. The organic layer was dried over Na2SO4, the solvent
was removed, and the residue was chromatographed on silica
gel (eluent: dichloromethane/methanol/aqueous ammonia )
10/1/0.1): yield 15.1 g of 11 (44%) as a waxlike solid; 1H-NMR
(250 MHz; CDCl3) δ 7.50 (bs, 1H); 7.21 and 6.87 (2d, J ) 9
Hz, 4H); 4.12-4.08 (m, 2H); 3.82-3.79 (m, 4H); 3.65-3.60 (m,
2H); 3.51-3.44 (m, 2H); 3.18 (d, J ) 4 Hz, 1H); 2.24 (dhept, J
) 4, 7 Hz, 1H); 1.53 (bs, 4H); 0.96 and 0.80 (2d, J ) 7 Hz,
6H). Anal. (C16H27N3O3‚0.4H2O) C, H, N.
3-[2-[2-[(Va lyl)a m in o]eth oxy]eth oxy]ben zyla m in e (12).
Preparation was as described for 11: yield 1.43 g of 12 (48%);
1H-NMR (250 MHz; CDCl3) δ 7.52 (bs, 1H); 7.22 (t, J ) 8 Hz,
1H); 6.94-6.83 (m, 3H); 6.78 (dd, J ) 2, 8 1Hz, 1H); 4.13-
4.09 (m, 2H); 3.82 (s, 2H), 3.82-3.78 (m, 2H); 3.67-3.55 and
3.55-3.40 (2m, 3H); 3.16 (d, J ) 4 Hz, 1H); 2.22 (dhept, J )
7 Hz, 4Hz, 1H); 1.58 (bs, 4H); 0.94 and 0.79 (2d, J ) 7 Hz,
6H). Anal. (C16H27N3O3‚0.44H2O) C, H, N.
Tolu en e-4-su lfon ic Acid 2-[2-[[N-[(Ben zyloxy)ca r bon -
yl]va lyl]a m in o]eth oxy]eth yl Ester (6). An ice-cooled solu-
tion of 5 (54.8 g, 0.162 mol) and pyridine (65 mL) in 550 mL
of dichloromethane was treated with toluene-4-sulfonic acid
chloride (30.9 g, 0.162 mol) in 50 mL of dichloromethane and
stirred for 12 h while gradually warming to room temperature.
The reaction mixture was washed with water, 1 N HCl,
saturated aqueous sodium hydrogen carbonate, and brine. The
organic layer was dried over Na2SO4 and concentrated in
vacuo. The product crystallized after trituration with diethyl
ether: yield 61 g of 6 (76%) (chromatographic purification
(eluent: ethyl acetate/n-hexane ) 2/1) of the mother liquid
yielded additional 6.3 g of 6; overall yield 84%): mp 74-75
°C; 1H-NMR (250 MHz; CDCl3) δ 7.17 (d, J ) 8 Hz, 2H); 7.36-
7.27 (m, 7H); 6.44 (bs, 1H); 5.49 (d, J ) 9 Hz, 1H); 5.11 (s,
2H); 4.19-4.15 (m, 2H); 4.02 (dd, J ) 6, 9 Hz, 1H); 3.65-3.61
(m, 2H); 3.50-3.42 (m, 4H); 2.44 (s, 3H); 2.13 (oct, J ) 7 Hz,
1H); 0.95 and 0.91 (2d, J ) 7 Hz, 6H). Anal. (C24H32N2-
O7S‚0.3H2O) C, H, N.
4-[2-[2-[[N-[(Ben zyloxy)ca r bon yl]va lyl]a m in o]eth oxy]-
eth oxy]ben za ld eh yd e (7). A solution of 4-hydroxybenzal-
dehyde (14 g, 0.115 mol) and 6 (56.5 g, 0.115 mol) in 500 mL
of dimethyl sulfoxide was treated with K2CO3 (38.6 g, 0.279
mol) and 4.2 g (11.4 mmol) of tetrabutylammonium iodide.
After stirring for 4 h at 70 °C, the solvent was removed in
(2R,3S,4S)-4-[[(1,1-Dim eth yleth oxy)ca r bon yl]a m in o]-
3-h yd r oxy-2-[4-[2-[2-[(va lyl)a m in o]eth oxy]eth oxy]ben zy-
la m in o]-5-p h en ylp en ta n oic Acid Eth yl Ester (14).
A
solution of 11 (14.5 g, 46.9 mmol) and 131 (11.78 g, 35.1 mmol)
in 400 mL of ethanol was kept at 80 °C for 24 h. The solvent

Macrocyclic Inhibitors of HIV Proteinases
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3297
was evaporated in vacuo, and the residue was purified by silica
gel chromatography (eluent: ethyl acetate/n-hexane ) 1/1):
9, 6 Hz, 2H); 3.04-2.98 (m, 1H); 1.81 (dhept, J ) 7, 4 Hz, 1H);
0.76 and 0.70 (2d, J ) 7 Hz, 6H). Anal. (C27H40Cl2N4O5‚2H2O)
C, H, N.
1
yield 11.55 g of 14 (51%); H-NMR (250 MHz; CDCl3) δ 7.49
(bs, 1H); 7.26 and 6.85 (2d, J ) 7 Hz, 4H); 7.30-7.18 (m, 5H);
4.91 (d, J ) 9 Hz, 1H); 4.23-4.13 (m, 2H); 4.10-4.08 (m, 2H);
4.05 (bq, J ) 8 Hz, 1H); 3.80 (dd, J ) 3, 4 Hz, 2H); 3.70 and
3.52 (AB, J ) 12 Hz, 2H); 3.68 (d, J ) 8 Hz); 3.65-3.58 (m,
2H); 3.52-3.45 (m, 2H); 3.29 (d, J ) 8 Hz, 1H); 3.18 (d, J ) 6
Hz, 1H); 2.95-2.80 (m, 2H); 2.23 (m, 1H); 1.37 (s, 9H); 1.26 (t,
J ) 7 Hz, 3H); 0.96 and 0.82 (2d, J ) 7 Hz, 6H). Anal.
(C34H52N4O8‚0.5H2O) C, H, N.
(2R,3S,4S)-4-[[(1,1-Dim eth yleth oxy)ca r bon yl]a m in o]-
3-h yd r oxy-2-[3-[2-[2-[(va lyl)a m in o]et h oxy]et h oxy]b en -
zyla m in o]-5-p h en ylp en t a n oic Acid E t h yl E st er (15).
Preparation was as described for 14: yield 1.05 g of 15 (55%);
1H-NMR (250 MHz; CDCl3) δ 7.55 (bs, 1H); 7.30-7.06 (m, 6H);
6.90-6.70 (m, 3H); 5.10 (d, J ) 8 Hz, 1H); 4.05-4.18 (m, 5H);
3.79 (dd, J ) 3 Hz, 5Hz, 2H); 3.70-3.40 (m, 8H); 3.27 (d, J )
7 Hz, H); 3.16 (d, J ) 4 Hz, H); 2.90-2.80 (m, 2H); 2.20 (dhept,
J ) 4 Hz, 7Hz, H); 2.00 (bs, 5H); 1.35 (s, 9H); 1.23 (t, J ) 7
Hz, H); 0.94 and 0.79 (2d, J ) 7 Hz, 6 H).
(1′S,2′S,9S,12R)-12-[2′-[[(1,1-Dim eth yleth oxy)car bon yl]-
am in o]-1′-h ydr oxy-3′-ph en ylpr op-1′-yl]-9-(1-m eth yleth yl)-
7,10,13-tr iaza-1,4-dioxa-8,11-dioxo[14]par acycloph an e (16).
14 (13.2 g, 20.5 mmol) was dissolved in 240 mL of dioxane
and treated with aqueous LiOH (76.7 mL of a 0.4 M solution,
30.7 mmol). The reaction mixture was stirred for 16 h at room
temperature (completeness of reaction was judged by HPTLC
analysis), treated with 1 N HCl (30.7 mL), and freeze-dried.
The freeze-dried seco acid (7 g, 10.29 mmol) was suspended
in 7.5 L of dry tetrahydrofuran, cooled in an ice-bath, and
treated with HODhbt (6.72 g, 41.2 mmol) and DCC (8.49 g,
41.2 mmol). After stirring for 5 days at room temperature,
the solvent was removed in vacuo and the residue was
dissolved in ethyl acetate. The suspension was filtered, and
the filtrate was washed with 1 N NaOH and brine, dried over
Na2SO4, and concentrated in vacuo. Pure 16 was obtained as
an amorphous solid by chromatography on silica gel (eluent:
gradient ethyl acetate/methanol ) 1/0 to 1/0.05): yield 3.51 g
(57%); mp 109-114 °C; 1H-NMR (500 MHz; DMSO-d6; 373 K)
δ 7.34 (bs, 1H); 7.29-7.24 (m, 4H); 7.19-7.16 (m, 1H); 6.98
and 6.78 (2d, J ) 8 Hz, 4H); 6.66 (d, J ) 8 Hz, 1H); 5.85 (bs,
1H); 4.30 (q, J ) 8 Hz, 1H); 4.23-4.15 (m, 3H); 3.95 (dd, j )
5, 8 Hz, 1H); 3.83 and 3.37 (AB, J ) 15 Hz, 2H); 3.75-3.61
(m, 4H); 3.52-3.48 (m, 1H); 3.17 (bt, 1H); 2.92 (d, J ) 10 Hz,
1H); 2.87-2.77 (m, 3H); 1.86 (dhept, J ) 5, 7 Hz, 1H); 1.41 (s,
9H); 0.79 and 0.78 (2d, J ) 7 Hz, 6H). Anal. (C32H46N4O7‚
0.9H2O) C, H, N.
(1′S,2′S,9S,12R)-12-(2′-Am in o-1′-h ydr oxy-3′-ph en ylpr op-
1′-yl)-9-(1-m eth yleth yl)-7,10,13-tr iaza-1,4-dioxa-8,11-dioxo-
[14]m eta cyclop h a n e (19). Preparation was as described for
18. The free base was obtained by treating a solution of the
dihydrochloride of 19 in
a small amount of water with
saturated aqueous K2CO3 solution, and extraction of the free
1
base with ethyl acetate: yield 200 mg of 19 (100%); H-NMR
(250 MHz; D2O) δ 8.20-8.11 (m, 1H); 7.49-7.28 (m, 6H); 7.14-
7.07 (m, 2H); 6.91 (s, 1H); 4.48 and 4.12 (AB, J ) 14 Hz, 2H);
4.26 (t, J ) 6 Hz, 1H); 4.16 (d, J ) 5 Hz, 1H); 4.28-4.15 (m,
2H); 3.86 (d, J ) 6 Hz, 1H); 3.91-3.64 (m, 6H); 3.12 and 2.94
(ABX; J ) 14, 9, 5 Hz, 2H); 3.14-3.02 (m, 1H); 1.96 (oct, J )
7 Hz, 1H); 0.88 and 0.82 (2d, J ) 7 Hz, 6H). Anal.
(C27H38N4O5‚1.7H2O) C, H, N.
(1′S,2′S,2′′S,9S,12R)-12-[2′′-[[N-[(Ben zyloxy)ca r bon yl]-
va lyl]a m in o]-1′-h yd r oxy-3′-p h en ylp r op -1′-yl]-9-(1-m eth -
yle t h yl)-7,10,13-t r ia za -1,4-d ioxa -8,11-d ioxo[14]p a r a -
cyclop h a n e (3). An ice-cooled solution of 18‚2HCl (100 mg,
0.175 mmol) in 10 mL of dioxane was treated with Z-Val (93
mg, 0.351 mmol), HODhbt (57 mg, 0.349), EDC‚HCl (67 mg,
0.35 mmol), and triethylamine (147 µL, 1.05 mmol). After
stirring overnight at room temperature, the solvent was
removed in vacuo. The residue was dissolved in ethyl acetate
and 0.1 M HCl, and the organic layer was washed with
saturated aqueous sodium hydrogen carbonate and brine, dried
over Na2SO4, and concentrated. Purification by silica gel
chromatography (dichloromethane/methanol ) 10/1) yielded
the title compound as an amorphous solid: yield: 65 mg of 3
1
(50%); mp 107-110 °C; H-NMR (500 MHz; CDCl3; 330 K) δ
7.35-7.34 (m, 3H); 7.34-7.26 (m, 2H); 7.26-7.25 (m, 4H);
7.19-7.14 (m, 1H); 7.03 and 6.83 (2d, J ) 9 Hz, 4H); 6.56 (d,
J ) 8 Hz, 1H); 6.33 (d, J ) 9 Hz, 1H); 5.31-5.26 (m, 1H); 5.26
(d, J ) 8 Hz, 1H); 5.17-5.10 (AB, J ) 12 Hz, 2H); 4.65 (q, J
) 8 Hz, 1H); 4.20 (t, J ) 3 Hz, 2H); 3.87 (d, J ) 8 Hz, 1H);
3.80-3.59 (m, 9H); 3.51-3.48 (m, 1H); 3.03 (d, J ) 9 Hz, 1H);
3.00 and 2.93 (AB-part of ABX, J ) 14, 8, 2 Hz, 2H); 2.90-
2.85 (m, 1H); 1.97 (dhept, J ) 7, 5 Hz, 1H); 0.98 (s, 9H); 0.78
and 0.75 (2d, J ) 7 Hz, 6H). Anal. (C41H55N5O8‚1.2H2O) C,
H, N.
(1′S,2′S,2′′S,9S,12R)-12-[2′′-[[N-[(Ben zyloxy)ca r bon yl]-
va lyl]a m in o]-1′-h yd r oxy-3′-p h en ylp r op -1′-yl]-9-(1-m eth -
yle t h yl)-7,10,13-t r ia za -1,4-d ioxa -8,11-d ioxo[14]m e t a -
cyclop h a n e (20). Preparation was as described for 3: yield
158 mg of 20 (45%); mp 86-92 °C; 1H-NMR (500 MHz; CDCl3;
330 K) δ 6.83 (d, J ) 8 Hz, 1H); 7.36-7.23 (m, 10H); 7.20-
7.17 (m, 2H); 7.06 (t, J ) 2 Hz, 1H); 6.83 (d, J ) 8 Hz, 1H);
6.80-6.78 (m, 1H); 6.42 (d, J ) 9 Hz, 1H); 6.20 (bs, 1H); 5.11
and 5.08 (AB, J ) 12Hz, 2H); 5.03 (d, J ) 8 Hz, 1H); 4.70 (bs,
1H); 4.49 (q, J ) 8 Hz, 1H); 4.26 (dd, J ) 5, 9 Hz, 1H); 4.21-
4.19 (m, 2H); 3.94 (dd, J ) 1, 10 Hz, 1H); 3.90 (dd, J ) 5, 8
Hz, 1H); 3.78-3.72 (m, 2H); 3.68-3.64 (m, 2H); 3.52-3.46 (m,
3H); 3.22 (d, J ) 9.5 Hz, 1H); 3.10-3.02 (m, 1H); 3.00 and
2.96 (AB-part of ABX, J ) 14, 8 Hz, 2H); 2.24 (2dh, J ) 5, 7
Hz, 2H); 0.90 and 0.85 and 0.79 and 0.73 (4d, J ) 7 Hz, 12H).
Anal. (C40H53N5O8) C, H, N.
(1′S ,2′S ,3′′S ,9S ,12R )-12-[2′-[[(3′′-Te t r a h yd r ofu r a n yl-
oxy)ca r bon yl]a m in o]-1′-h yd r oxy-3′-p h en ylp r op -1′-yl]-9-
(1-m et h ylet h yl)-7,10,13-t r ia za -1,4-d ioxa -8,11-d ioxo[14]-
p a r a cyclop h a n e (21). A cooled solution (0 °C) of phosgene
(420 µL, 1.93 M solution in toluol) and pyridine (64 µL) in 5
mL of dichloromethane were treated with a solution of (+)-
3(S)-hydroxytetrahydrofuran (54 µL, 0.61 mmol in 5 mL of
dichloromethane) via a syringe pump at a rate of 2.5 mL/h.
18 (200 mg, 0.40 mmol) was suspended in 5 mL of dichlo-
romethane and 64 µL of pyridine, cooled to 0 °C, and treated
with 7 mL of the above described solution. After stirring for
16 h at room temperature, ethyl acetate and saturated aqueous
hydrogen carbonate were added. The organic layer was
(1′S,2′S,9S,12R)-12-[2′-[[(1,1-Dim eth yleth oxy)car bon yl]-
am in o]-1′-h ydr oxy-3′-ph en ylpr op-1′-yl]-9-(1-m eth yleth yl)-
7,10,13-t r ia za -1,4-d ioxa -8,11-d ioxo[14]m et a cyclop h a n e
(17). Preparation was as described for 16. Instead of DCC,
EDC‚HCl was used: yield 180 mg of 17 (19%); mp 155-156
1
°C; H-NMR (250 MHz; CDCl3/D2O; 373 K) δ 7.29-7.11 (m,
5H); 7.12 (t, J ) 8 Hz, 1H); 6.81-6.78 (m, 3H); 4.17-4.07 (m,
4H); 3.69-3.35 (m, 8H); 2.95-3.10 (m, 2H); 2.77 and 2.72 (AB-
part of ABX, J ) 13 Hz, 2H); 1.95 (oct, J ) 7 Hz, 1H); 1.31 (s,
9H); 0.82 and 0.81 (2d, J ) 7 Hz, 6H). Anal. (C32H46N4O7) C,
H, N.
(1′S,2′S,9S,12R)-12-(2′-Am in o-1′-h ydr oxy-3′-ph en ylpr op-
1′-yl)-9-(1-m eth yleth yl)-7,10,13-tr iaza-1,4-dioxa-8,11-dioxo-
[14]p a r a cyclop h a n e Dih yd r och lor id e (18‚2HCl). 16 (3.5
g, 5.85 mmol) was dissolved in 10 mL of dioxane and treated
with an equal amount of 18% aqueous HCl. The solvents were
immediately evaporated. Completeness was judged by HPTLC
analysis. The oily residue was dissolved in water and lyoph-
ilized (the free base (18) is sparingly soluble in ethyl acetate
and in dichloromethane) to give an amorphous solid: yield 3.34
g of 18‚2HCl (100%); mp 175-180 °C; 1H-NMR (500 MHz; D2O)
δ 7.92 (dd, J ) 8, 4 Hz, 1H); 7.50-7.47 (m, 2H); 7.45-7.42 (m,
1H); 7.37-7.35 (m, 2H); 7.28 and 6.93 (2d, J ) 9 Hz, 4H); 4.53
and 4.00 (AB, J ) 14 Hz, 2H); 4.28 and 4.22 (AB-part of ABXY,
J ) 12, 6, 2 Hz, 2H); 4.18 (t, J ) 5 Hz, 1H); 3.98 (d, J ) 6 Hz,
1H); 3.96-3.89 (m, 2H); 3.89-3.83 (m, 1H); 3.83-3.67 (m, 4H);
3.79 (d, J ) 4Hz, 1H); 3.14 and 2.98 (AB-part of ABX, J ) 14,
washed with brine, dried over Na2
SO4, and concentrated.
Purification by silica gel chromatography (gradient: ethyl
acetate/methanol 20/1 to 10/1) yielded 180 mg of 21 (75%); mp
1
175-178 °C; H-NMR (500 MHz; DMSO-d6; 410 K) δ 7.29-
7.25 (m, 4H); 7.19-7.16 (m, 1H); 7.07 (bs, 1H); 6.99-6.80 (2d,

3298 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17
Ettmayer et al.
J ) 8 Hz, 4H); 6.62 (d, J ) J ) 8 Hz, 1H); 6.09 (d, J ) 9 Hz,
1H); 5.14-5.10 (m, 1H); 4.31 (dq, J ) 7, 2 Hz, 1H); 4.24-4.17
(m, 3H); 3.96 (dd, J ) 8, 5 Hz, 1H); 3.86 and 3.38 (AB, J ) 15
Hz, 2H); 3.82 (q, J ) 8 Hz, 1H); 3.79 (dd, J ) 10, 5 Hz, 1H);
3.76-3.66 (m, 3H); 3.64-3.58 (m, 3H); 3.52-3.48 (m, 1H); 3.28
(bd, 1H); 2.97 (d, J ) 9 Hz, 1H); 2.89 and 2.82 (ABX, J ) 14,
8 Hz, 2H); 2.92-2.83 (m, 1H); 2.43 (bs, 1H); 2.13 (oct, J ) 7
Hz, 1H); 2.93-1.84 (m, 2H); 0.81 and 0.80 (2d, J ) 7 Hz, 6H).
Anal. (C32H44N4O8) C, H, N.
Biology. HIV-1 proteinase was expressed in Escherichia
coli and was purified to homogeneity as described.17 Enzy-
matic activity was measured by following cleavage of the
substrate H-Lys-Ala-Arg-Val-Leu-pNph-Glu-Ala-Nle-NH2, orig-
inally described by Richards et al.18 with the modifications
reported previously.19 Recombinant HIV-2 proteinase was
kindly supplied by P. Strop, Prague, and was assayed at pH
4.7 using the same substrate and similar condition as for
HIV-1 proteinase. All test compounds were dissolved in
dimethyl sulfoxide to 10 mM and diluted into assay buffer;
the concentration of dimethyl sulfoxide did not exceed 5%.
The HIV-1 strain IIIB and the HIV-2 strain EHO have been
described.20,21 Inhibition of virus-induced cytopathic effect was
determined in MT4 cells by measuring the viability of both
HIV- and mock-infected cells essentially as in Pauwels et al.22
Viability was assessed spectrophotometrically via in situ
reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT). Compounds were added postadsorption.
Concentrations of test compounds reducing cell density of mock
infected cultures by 50% (CC50) and those reducing virus-
induced cytopathic effect by 50% (IC50) were determined by
viability staining 5 days after infection.
Primary T4-lymphocytes were purified from human spleens
obtained from healthy donors by using a commercial kit
(“Lympho-Kwik”) as described recently.19 Cells were stimu-
lated with phytohemagglutinin (PHA) and were infected with
HIV-1, IIIB, or HIV-2, MS, as described, and incubated in
medium containing IL-2.19 Test compound was added after
stimulation and virus adsorption. Every 3-4 days half of the
supernatant of the infected cultures was removed and replaced
by fresh medium containing IL-2 and the test compound at
the particular concentration. Preparation and infection of
monocytes were performed essentially as in Perno et al.,
1989,23 with the modifications described.24 Mononuclear cells
were isolated from healthy, HIV negative donors using Ficoll
density separation and adherence to tissue culture plates for
5 days. Preparations obtained by this procedure were >95%
positive for nonspecific esterase; cell viability was always
>95%. The cells were infected with HIV-1, strain BaL, and
were cultivated in the presence of different drug concentra-
tions. Every 3-4 days the supernatant of the infected cultures
was removed and replaced by fresh medium containing the
test compound at the particular concentration. IC50 and IC90
values were calculated by comparing p24 antigen concentra-
tions (determined by ELISA, Coulter) in supernatants of
treated, infected cells to those of untreated, infected cells at
days postinfection, when p24 production was increasing ex-
ponentially.
concentration of the compound was calculated by external
standardization of peak area versus concentration for each
sample. Minimal detectable concentration: 0.1 µg/mL; stan-
dard deviation: (10%.
Ack n ow led gm en t. We thank Dr. R. Datema, Head
of Department of Antiretroviral Therapy, Sandoz For-
schungsinstitut, Vienna, for continous support of this
work. We thank M. Hu¨bner, L. Malacek, E. Mlynar,
E. Pursch, L. Strommer, and G. Winkler for excellent
technical assistance.
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J M950641I