Improved P1/P1' substituents for cyclic urea based HIV-1 protease inhibitors: Synthesis, structure-activity relationship, and X-ray crystal structure analysis

Article abstract of DOI:10.1021/jm960839i

We present several novel P1/P1' substituents that can replace the characteristic benzyl P1/P1' moiety of the cyclic urea based HIV protease inhibitor series. These substituents typically provide 5-10-fold improvements in binding affinity compared to the unsubstituted benzyl analogs. The best substituent was the 3,4-(ethylenedioxy)benzyl group. Proper balancing of the molecule's lipophilicity facilitated the transfer of this improved binding affinity into a superior cellular antiviral activity profile. Several analogs were evaluated further for protein binding and resistance liabilities. Compound 18 (IC₉₀ = 8.7 nM) was chosen for oral bioavailability studies based on its log P and solubility profile. A 10 mg/kg dose in dogs provided modest bioavailability with C(max) = 0.22 μg/mL. X-ray crystallographic analysis of two analogs revealed several interesting features responsible for the 3,4-(ethylenedioxy)benzyl-substituted analog's potency: (1) Comparing the two complexes revealed two distinct binding modes for each P1/P1' substituent; (2) The ethylenedioxy moieties are within 3.6 A? of Pro 81 providing additional van der Waals contacts missing from the parent structure; (3) The enzyme's Arg 8 side chain moves away from the P1 substituent to accommodate the increased steric volume while maintaining a favorable hydrogen bond distance between the para oxygen substituent and the guanidine NH.

Full text of DOI:10.1021/jm960839i

J . Med. Chem. 1997, 40, 1465-1474
1465
Im p r oved P 1/P 1′ Su bstitu en ts for Cyclic Ur ea Ba sed HIV-1 P r otea se In h ibitor s:
Syn th esis, Str u ctu r e-Activity Rela tion sh ip , a n d X-r a y Cr ysta l Str u ctu r e
An a lysis
David A. Nugiel,* Kim J acobs, Lyndon Cornelius, Chong-Hwan Chang, Prabhakar K. J adhav, Edward R. Holler,
Ronald M. Klabe, Lee T. Bacheler, Beverly Cordova, Sena Garber, Carol Reid, Kelly A. Logue,
Lorraine J . Gorey-Feret, Gilbert N. Lam, Susan Erickson-Viitanen, Steven P. Seitz
The DuPont Merck Pharmaceutical Company, P.O. Box 80500, Wilmington, Delaware 19880-0500
Received December 13, 1996X
We present several novel P1/P1′ substituents that can replace the characteristic benzyl P1/P1′
moiety of the cyclic urea based HIV protease inhibitor series. These substituents typically
provide 5-10-fold improvements in binding affinity compared to the unsubstituted benzyl
analogs. The best substituent was the 3,4-(ethylenedioxy)benzyl group. Proper balancing of
the molecule’s lipophilicity facilitated the transfer of this improved binding affinity into a
superior cellular antiviral activity profile. Several analogs were evaluated further for protein
binding and resistance liabilities. Compound 18 (IC90 ) 8.7 nM) was chosen for oral
bioavailability studies based on its log P and solubility profile. A 10 mg/kg dose in dogs provided
modest bioavailability with Cmax ) 0.22 µg/mL. X-ray crystallographic analysis of two analogs
revealed several interesting features responsible for the 3,4-(ethylenedioxy)benzyl-substituted
analog’s potency: (1) Comparing the two complexes revealed two distinct binding modes for
each P1/P1′ substituent; (2) The ethylenedioxy moieties are within 3.6 Å of Pro 81 providing
additional van der Waals contacts missing from the parent structure; (3) The enzyme’s Arg 8
side chain moves away from the P1 substituent to accommodate the increased steric volume
while maintaining a favorable hydrogen bond distance between the para oxygen substituent
and the guanidine NH.
In tr od u ction
between the IC90 in a cellular assay and the Ki) and
better overall antiviral activity. In general, the better
antiviral activity resulted mainly from improvement in
whole-cell translation rather than increasing enzyme
affinity.
Advances in acquired immune deficiency syndrome
(AIDS) chemotherapy revealed several promising dis-
coveries used to combat this disease.1 In particular,
recent FDA approvals of Saquinavir, Indinavir, and
Ritonavir show that human immunodeficiency virus
(HIV) protease inhibitors (HIVPR) are valid approaches
to AIDS therapy.2 Despite the potency and good oral
bioavailability of these entities, the emergence of resis-
tant forms of HIV renders their long-term utility in
question.3 Clearly, there is a need for structurally
unique compounds that may overcome these limitations.
Much of the current literature on HIVPR inhibitors
emphasizes structural elements that interact with the
enzyme’s S2/S2′ and S3/S3′ binding domains. Few
reports deal with issues regarding the S1/S1′ pocket.4
Several papers have appeared describing the use of
a novel 7-membered cyclic urea scaffold for the prepara-
tion of inhibitors of HIVPR.5 This research culminated
in the discovery of a promising clinical candidate
DMP450.6 The cyclic urea’s structural rigidity allows
for well-aligned interactions of its substituents with
their corresponding binding elements in the active site
of HIVPR. Typically, the preferred substituent at the
P1/P1′ position was an unsubstituted phenyl ring.
We have since expanded the scope of our investigation
into this class of compounds and have found other P1
substituents that provide an increase in binding affinity.
This culminated in the discovery of a novel P1 substitu-
ent that generated compounds with up to a 10-fold
increase in binding affinity. To our knowledge, this was
the first P1/P1′ substituent that provided such a dra-
matic increase in potency. In addition, we were able to
crystallize two of these analogs in the active site of
HIVPR. This helped determine the source of the
increased binding affinity. This paper describes the
scope and limitations of designing potent P1/P1′ modi-
fied cyclic urea based HIVPR inhibitors and their use
as bioavailable antiviral agents.
Ch em istr y
The P1/P1′-modified compounds were prepared as
shown in Scheme 1. The sequence begins with the key
diepoxide intermediate 1 derived from L-mannonic
γ-lactone. Diepoxide 1 was treated with 4 equiv of an
organocuprate reagent prepared from 3,4-(ethylene-
dioxy)bromobenzene to give the diol 2 in 85% yield. This
diol was converted to diazide 3 using standard Mit-
sunobu methodology8 in 28% yield. This reaction was
plagued by the elimination side reaction of one hydroxyl
group from diol 2 to give an olefin byproduct. This
competing side reaction has been observed previously9
and was especially prevalent when the displaced alcohol
was homobenzylic. The diazide was reduced to the
diamine in 90% yield using LAH. The crude diamine
We recently reported an extensive study of P1/P1′
analogs based on this 7-membered cyclic urea scaffold.7
A few analogs had modestly improved binding affinity
compared to the parent unsubstituted benzyl compound.
By altering the P1/P1′ substitution pattern, we were
also able to modulate the molecule’s physical properties
resulting in improved translation (defined as the ratio
X Abstract published in Advance ACS Abstracts, April 1, 1997.
S0022-2623(96)00839-4 CCC: $14.00 © 1997 American Chemical Society

1466 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Nugiel et al.
Sch em e 1a
a
Reagents and conditions: (a) n-BuLi, CuCN, THF, -78 to 0 °C, 85%; (b) Ph3P, DEAD, (PhO)2P(O)N3, THF, 0-25 °C, 28%; (c) LiAlH4,
THF, room temperature, 90%; (d) CDI, tetrachloroethane, room temperature to reflux, 70%; (e) K+ t-BuO-, BnBr, THF, room temperature,
68%; (f) 20% concentrated HCl, CH3CN, room temperature, 95%.
Sch em e 2a
Ta ble 1
Ki
IC90
compd
R
(nM)a
(nM)a
parent phenyl
3.4
0.44
9.8
8.0
6.4
1.0
1.2
0.7
790
112
3200
>5000
640
6
3,4-(ethylenedioxy)phenyl
â-naphthyl
3,5-dimethoxyphenyl
19
20
21
22
23
24
3,4-dimethoxyphenyl
3,4-(methylenedioxy)phenyl
2,3-dihydro-5-benzofuranyl
4-methyl-3,4-dihydro-6-benzoxazinyl
504
305
218
a
Reagents and conditions: (a) HNO3-SiO2, CH2Cl2, room
temperature, 97%; (b) SnCl2, HCl, 74%; (c) Ac2O, pyridine, quant;
(d) NaOH, MeOH, room temperature, 97%; (e) NaOH, 1,2-
dibromomethane, Aliquat 336, CH2Cl2/CH3CN, room temperature,
90%; (f) KOH, MeOH/H2O, 55 °C, quant; (g) H2CO, NaCNBH3,
CH3CN, 95%.
a
Values determined using methods described in refs 19 and 20
for n ) 2
was then cyclized using 1,1-carbonyldiimidazole in
refluxing 1,1,2,2-tetrachloroethane to give the cyclic
urea 4 in 70% yield. Elevated temperatures were
required for the cyclization due to the presence of the
trans fused acetonide protecting group. The desired P2/
P2′ substituent was introduced using potassium tert-
butoxide and the corresponding alkylating agent. Re-
moval of the acetonide protecting group using 20%
concentrated HCl in methanol gave the target urea 6
in 95% yield.
Table 1 shows several examples (19, 21, and 22)
prepared in similar fashion from commercially available
substituted aryl bromides. Compound 23 was prepared
from the known 5-bromodihydrobenzofuran10 while urea
20 was prepared using an amino acid-based approach
starting with 3,5-dimethoxy-D-phenylalanine as de-
scribed previously.11
The synthesis of 4-methyl-3,4-dihydro-2H-benzo[b]-
[1,4]oxazin-6-yl bromide, used to prepare 24, is shown
in Scheme 2. Selective mononitration of p-bromophenol
proceeded smoothly using nitric acid impregnated silica
gel12 to give the desired 4-bromo-2-nitrophenol 7 in 97%
yield. Reduction of the nitro group using SnCl2 gave
the desired aniline 8 in 74% yield. Bisacylation of the
amino phenol 8 with acetic anhydride followed by
selective removal of the phenolic acetate gave the amide
9 in 97% yield for the two steps. The N-acyl group was
critical for efficient introduction of the ethylene bridging
group in the next step.13 This was accomplished with
1,2-dibromoethane using Aliquat 336 as the base and a
CH2Cl2/CH3CN solvent mixture at room temperature to
give the desired product 10 in 90% yield. A simple two-
step protocol transformed the N-acyl group to the
desired N-methyl group to give the bromide 11 in 95%
overall yield.
Several of the P2 variants shown in Table 2 (25, 26,
and 28) were prepared according to Scheme 1 using
commercially available alkylating agents. The methyl
ketone analogs (13, 30, 35 and 36) were prepared
according to Scheme 3 using 13 as an example. The
urea 4 was alkylated with m-cyanobenzyl bromide
under standard conditions to give compound 12 in 94%
yield. Conversion of the nitrile to the methyl ketone
was accomplished using excess methylmagnesium bro-
mide in THF followed by an acetic acid workup to give
a quantitative yield of the desired ketone. The ac-
etonide protecting group was removed as previously
described to give 13 in 90% overall yield for the two
steps. The p-fluoro methyl ketone analogs 29 and 34
were prepared in a similar fashion from 4-fluoro-3-
cyanobenzyl bromide.14
The m-amino-containing analogs (15, 17, 18, 31, and
32) were prepared according to Scheme 4 using 18 as
an example. Alkylation of urea 4 with m-nitrobenzyl
bromide provided the desired bis-alkylated urea 14 in
84% yield. Reduction to the amine and subsequent
deprotection of the acetonide protecting group was done
in a one-pot procedure and provided 15 in 83% yield for

Cyclic Urea Based HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1467
Ta ble 2
compd
R1
R2
Ki (nM)a
IC90 (nM)a
13
15
17
3,4-(ethylenedioxy)phenyl
m-CH3C(O)C6H4
m-NH2C6H4
R2 ) m-NH2C6H4
R2′ ) m-MeNHC6H4
m-MeNHC6H4
cyclopropyl
â-naphthyl
p-HOCH2C6H4
p-HOC6H4
m-CH3C(O)-p-FC6H4
m-CH3C(O)C6H4
m-NH2C6H4
m-MeNHC6H4
m-CH3C(O)C6H4
m-CH3C(O)-p-FC6H4
m-CH3C(O)C6H4
m-CH3C(O)C6H4
0.04 (0.07)
0.04 (0.23)b
0.11
4.2 (40)
40 (150)b
27
18
25
26
27
28
29
30
31
32
33
34
35
36
0.14 (0.15)
0.27 (2.1)
0.04 (0.31)
0.05 (0.27)
0.03 (0.12)
0.64 (4.4)
0.048
0.11
0.20
0.043
1.5
8.7 (88)
928 (1800)
98 (3900)
83 (136)
107 (32)
84 (860)
10
30.6
61.6
14.7
265
2,3-dihydro-5-benzofuranyl
3,4-(methylenedioxy)phenyl
3,5-dimethoxyphenyl
4-methyl-3,4-dihydro-6-benzoxazinyl
0.4
0.08
46.4
16.4
a
Values determined using methods described in refs 19 and 20 for n ) 2. Values in parentheses correspond to the parent compounds
b
having an unsubstituted phenyl ring at the P1 position. Value for DMP450 taken from ref 6.
Sch em e 3a
a
Reagents and conditions: (a) m-cyanobenzyl bromide, K+t-BuO-, THF, room temperature, 94%; (b) methylmagnesium bromide, THF,
reflux, 95%; (c) HCl, THF:MeOH, room temperature, 88%.
Sch em e 4a
a
Reagents and conditions: (a) m-nitrobenzyl bromide, K+t-BuO-, THF, room temperature, 84%; (b) H2, 5% Pd/C, THF, 94%; (c) 20%
concentrated HCl in MeOH, room temperature, 91%; (d) neat butyl formate, reflux, 94%; (e) BH3-THF, THF, reflux, 88%.
the two steps. Selective monomethyl introduction was
accomplished using a two-step protocol. Formylation
of the aniline nitrogen using butyl formate gave com-
pound 16 in 80% yield. Initial reduction conditions
using borane-THF complex gave a mixture of two
products corresponding to 17 and 18 in a 1:1 ratio. The
desired product could be prepared cleanly by reduction
with LAH in refluxing THF for 15 min to give 18 as the
sole product in 95% yield.
Resu lts a n d Discu ssion
In itia l SAR. Additional oxygen-bearing P1 benzyl
analogs related to compound 6 were prepared to exam-
ine the source of the observed increased binding affinity.
The binding affinity (Ki) and cellular antiviral activity
(IC90) of these analogs are presented in Table 1. Clearly,
the 3,4-(ethylenedioxy)benzyl substituent was unique.
Breaking the ethylene bridge carbon-carbon bond,

1468 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Nugiel et al.
F igu r e 1. Inhibitor 26 bound within the active site of HIVPR. Protein residues located within 4.1 Å from the inhibitor are
shown. Hydrogen bonds between the inhibitor and key enzyme residues are shown as yellow dotted lines.
exemplified by compound 21, eliminates any advanta-
geous binding phenomenon. The binding affinity of 21
was comparable to the parent unsubstituted P1 phenyl
analog, and only a slight improvement was seen in the
whole-cell antiviral activity. The increased binding
affinity does not appear to be related to the P1 phenyl
ring’s electronic character. MOPAC calculations showed
similar electron density coefficients for the P1 phenyl
ring carbons, resulting in similar phenyl ring π distri-
butions for compounds 6 and 21. The order of magni-
tude loss in antiviral activity observed with compound
20 results from poor translation. The source of this loss
in translation is unclear. Both compound 20 and the
parent have identical clogP values.
The piperonyl analogs 22 and 23 also show an
improved binding affinity profile when compared to the
parent. Shortening the bridge between the oxygen
substituents from two carbons to a methylene group
affects the binding compared to 6, but both are still more
potent than the parent. Removing one oxygen atom
from the piperonyl group (23) has little effect on binding
affinity. In addition, the translation improves as indi-
cated by the lower IC90 for 23. It seems that both
oxygen substituents may not be critical for improved
binding affinity.
allows for the formation of water soluble salts, thereby
increasing the chances for good bioavailability.
Pure steric or hydrophobic interactions alone cannot
account for the improved binding affinity. Compound
19 shows that simply filling the hydrophobic S1 pocket
with a large hydrophobic substituent is not sufficient
for increased binding affinity. A combination of both
van der Waals interactions and hydrogen bond interac-
tions seems to be responsible for the improved binding
affinity. These interactions can be seen in the crystal
structures of two analogs 15 and 26 bound in the active
site of HIVPR (Figures 1 and 2).
X-r a y Cr ysta l Str u ctu r e An a lysis. Figure 1 shows
all the structural elements of inibitor 26 interacting
with their corresponding binding domains. The urea
carbonyl oxygen creates a hydrogen bond with both flap
residues Ile 50 and 150. The diol oxygens create two
hydrogen bonds with both catalytic aspartic acid resi-
dues Asp 25 and 125. The P2 naphthyl substituent fills
the S2 pocket and has several van der Waals contacts
with residues Ile 28, Asp 30, Val 32, and Gly 48. The
P2′ substituent has the same contacts mentioned above
in addition to Ile 47′ and Ile 84′. The 3,4-(ethylene-
dioxy)benzyl P1 substituent fills the S1 pocket. The
ethylenedioxy moiety’s para oxygen atom is within 3.6
Å of Pro 81. This interaction contributes additional van
der Waals contacts missing from the parent structure.
In addition, the meta-substituted oxygen lies within 3.7
Å of the Arg 8 guanidine NH creating a weak hydrogen
bond interaction also not present in the parent. An
efficient network of hydrogen bond and van der Waals
interactions contribute to the excellent binding affinity
observed for this analog (Ki ) 0.04 nM).
This is further exemplified by compound 24. Replac-
ing the meta oxygen with an N-methyl nitrogen shows
a slight decrease in Ki compared with 23. This suggests
the meta oxygen lacks critical interactions with S1
pocket residues. In addition, the N-methyl group’s
increased lipophilicity relative to the oxygen atom
further improves the translation of compound 24 with
antiviral activity now 3.5-fold greater than the parent.
Replacing the oxygen atom with an N-methyl group has
an added advantage. The additional basic nitrogen site
Figure 2 shows an overlap of the crystal structures
of DMP450 and its close analog 15. The crystal struc-

Cyclic Urea Based HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1469
F igu r e 2. Overlap of inhibitor 15 and DMP450 in the active site of HIVPR. For 15, carbon atoms are colored gray, oxygen
atoms are red, and nitrogen atoms are blue. All atoms of DMP450 are colored yellow.
ture of DMP450 is shown in yellow, and the overlapped
structure of 15 is colored by element type. This figure
allows us to analyze how the enzyme accommodates the
structurally related inhibitors in different ways. Little
movement is seen in the enzyme’s CR trace when
comparing the two structures.15 Since the P2/P2′ m-
aminobenzyl substituents of DMP450 and 15 show little
variance, they were Z-clipped out of the image for
clarity. Both substituents have similar spatial orienta-
tions of their perspective phenyl rings. In addition, the
hydrogen bond trajectory between the meta amino group
and the side chain carboxylate of Asp 29 is very similar.
The hydrogen bond distance differs by only 0.5 Å.
The S1 pocket shows a bit more diversity. The P1
substituent’s orientation of 15 has rotated 180° com-
pared to 26. This demonstrates the enzyme’s flexibility
at accommodating the same substituent in different
conformations. The same interaction between the eth-
ylenedioxy group and Pro 81 was observed as shown
previously in Figure 1 except here both carbon and
oxygen atoms contribute to the interaction. In addition,
the Arg 8 side chain has now moved away from the P1
substituent compared to DMP450. This movement
accommodates the increased steric bulk of the substi-
tuted P1 phenyl ring while maintaining a weak hydro-
gen bond (4.1 Å) between the para oxygen substituent
and the guanidine moiety. These additional interac-
tions contribute to the improved binding affinity ob-
served for compound 15 (Ki ) 0.04) compared to that
for DMP450 (Ki ) 0.23).
comparison, the parent structure’s values for both Ki
and IC90 are presented in parentheses. In almost every
case, a superior binding affinity profile was observed
compared to the parent structures having an unsubsti-
tuted P1 phenyl ring. It was essential to have the
correct polarity balance between the P1 and P2 sub-
stituents for good antiviral activity. Compounds 15, 27,
and 28 have P2 substituents that are relatively polar.
Despite their excellent binding affinities, they do not
translate well and have mediocre antiviral properties.
Lipophilic substituents at P2 complement the slightly
polar nature of the 3,4-(ethylenedioxy)benzyl substitu-
ent. J udicious choice of the P2 substituent’s character-
istics leads to good translation of the increased binding
affinity. The substituents that provided the best overall
profile are exemplified by compounds 13, 18, 30, 33, and
36. Meta-substituted N-methylamines and methyl ke-
tones gave the most potent compounds in this series.
An attempt at increasing the translation of compounds
13 and 33 by introducing a para fluoro substituent (see
29 and 34) failed. The fluorine atom disrupts the
hydrogen-bonding capability of the carbonyl group. This
resulted in a substantial increase in these analogs’ Ki
values. Nonetheless, they were still 3-7-fold better in
binding affinity compared to the corresponding parent.
The five best compounds in this series were further
evaluated. This entailed determining the compounds’
resistance profiles and protein binding characteristics.
The information is presented in Table 3. The selected
I84V mutant strain is used in our primary resistance
screen due to the severe susceptibility of some members
of the cyclic urea class of HIVPR inhibitors to this
mutant. In addition, this mutation plays a critical role
in the development of cross-resistance to a structurally
diverse set of HIVPR inhibitors.16 The effects of resis-
An tivir a l P oten cy Op tim iza tion a n d in Vivo
Stu d ies. To further improve this series’ binding affinity
and overall antiviral activity, we introduced P2 substit-
uents having additional hydrogen bond generating
capabilities. These examples are shown in Table 2. For

1470 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Ta ble 3. Adjusted IC90 Values for Selected Compounds
Nugiel et al.
distilled from sodium benzophenone ketyl. 1H NMR (300 MHz)
spectra were recorded using tetramethylsilane as an internal
standard. Melting points are uncorrected. TLC was per-
formed on E. Merck 15719 silica gel plates. Flash chroma-
tography was carried out using EM Science silica gel 60 (230-
400 mesh). All final targets were obtained as noncrystalline
amorphous solids unless specified otherwise. Elemental analy-
sis was performed by Quantitative Technologies, Inc., Bound
Brook, NJ . For compounds where analysis was not obtained,
HPLC analysis was used, and purity was determined to be
>98%. Analytical data for compounds 19 and 22 were
disclosed previously.7
1,6-Did eoxy-1,6-bis(3,4-(eth ylen ed ioxy)p h en yl)-3,4-O-
isop r op ylid en e-L-m a n n itol (2). To a solution of 3,4-(ethyl-
enedioxy)bromobenzene (27.75 g, 129 mmol) in THF (150 mL)
at -78 °C was added n-butyllithium (51.5 mL, 129 mmol, 2.5
M in hexane). After 10 min the reaction was allowed to reach
-20 °C to which copper(I) cyanide (5.77 g, 64.4 mmol) was
added in one portion. After complete dissolution of the solid,
the reaction was warmed to 0 °C and diepoxide 1 (3.0 g, 16.1
mmol) in THF (10 mL) was added via cannula. The reaction
was allowed to reach room temperature, and stirring was
continued for 15 min. The reaction was quenched with
saturated NH4Cl solution (25 mL), diluted with ether (600 mL),
and washed with saturated NH4Cl solution (2 × 200 mL). The
organic layer was separated and dried (MgSO4) and the solvent
removed at reduced pressure. Chromatography (silica gel, 30-
70% EtOAc/hexane) gave diol 2 as a foam (6.30 g, 85%). 2:
NMR (CDCl3) δ 6.8 (m, 4 H), 6.7 (m, 2 H), 4.25 (s, 8 H), 3.75
(bs, 4 H), 3.05 (m, 2 H), 2.95 (bs, 2 H), 2.6 (m, 2 H), 1.4 (s, 6
H); CIMS (NH3) m/z 459 (M + H+, 34).
(2R,3S,4S,5R)-1,6-Bis(3,4-(et h ylen ed ioxy)p h en yl)-2,5-
d ia zid o-3,4-O-isop r op ylid en eh exa n ed iol (3). To a solution
of diol 2 (6.3 g, 13.7 mmol) and triphenylphosphine (9.0 g, 34.3
mmol) in THF (30 mL) at 0 °C was added slowly diethyl
azodicarboxylate (5.40 mL, 34.3 mmol). After 10 min diphenyl
phosphorazidate (7.40 mL, 34.3 mmol) was added to the
reaction slowly. The reaction was allowed to reach room
temperature and stirred for 2.5 h. The reaction was quenched
with methanol (10 mL) at 0 °C. The mixture was then stirred
at room temperature for 10 min and the solvent removed at
reduced pressure. Chromatography (silica gel, benzene to 1%
EtOAc/benzene) gave the diazide 3 as an oil (1.95 g, 28%). 3:
NMR (CDCl3) δ 6.8 (d, J ) 8.0 Hz, 2 H), 6.75 (m, 2 H), 6.7 (m,
2 H), 4.25 (s, 8 H), 4.1 (bs, 2 H), 3.25 (m, 2 H), 2.9 (m, 4 H),
1.5 (s, 6 H).
IC90
PB
I84V
adj IC90
(nM)d
compd
(nM)a
potency shiftb
resistance shiftc
13
18
30
33
36
4.2
8.7
10
14.7
16.4
30
30
17
9
>50
10
10
31
10
10
1260
2610
5270
1323
>8200
a
Values determined using methods described in ref 20 for n )
b
2. Corresponds to the fold shift in IC90 due to protein binding.
c Corresponds to the fold shift in IC90 due to an I84V mutation.
d
Adj IC90 ) IC90 × PB potency shift × I84V resistance shift.
tance and protein binding are expressed as the fold shift
in the original IC90 value determined from Table 2. A
final value is expressed as the adjusted IC90 which is
the product of the original IC90, the protein binding
shift, and the I84V resistance shift. This value allows
us to evaluate three key issues simultaneously, present-
ing a more accurate antiviral potency profile. The
results show that these compounds are also susceptible
to the I84V mutant of HIVPR and are substantially
protein bound, in effect limiting the compound’s anti-
viral activity. In particular, compound 36 was highly
protein bound with its initial promising antiviral activ-
ity (IC90 ) 16 nM) completely negated. By designing
into the molecule a basic site for salt formation, with
the hope of increased water solubility, we inadvertently
increased its protein binding.
On the basis of a superior aqueous solubility and log
P profile, compound 18 was chosen for further in vivo
evaluation. The compound was dosed in both rats and
dog to determine its bioavailability. At 10 mg/kg the
compound failed to attain any measurable blood levels
in rats. At the same dose in dogs, a measure of
bioavailability was seen with a Cmax ) 0.22 µg/mL and
a F% ) 4.6. The iv data indicated the compound was
cleared quite rapidly from circulation with a half-life of
0.8 h. No additional studies were conducted to deter-
mine the source of the high clearance values observed.
Con clu sion
(4R,5S,6S,7R)-Hexa h yd r o-5,6-O-isop r op ylid en e-4,7-bis-
((3,4-(eth ylen ed ioxy)p h en yl)m eth yl)-2H-1,3-d ia za p in -2-
on e (4). A solution of 3 (1.95 g, 3.83 mmol) in THF (30 mL)
was cooled to 0 °C and was treated with a solution of LAH
(11.5 mL, 11.5 mmol, 1.0 M in THF). The cooling bath was
removed and the reaction reached room temperature. After
20 min the reaction was recooled to 0 °C and quenched with 1
N NaOH, diluted with EtOAc (50 mL), dried (Na2SO4), and
filtered (Celite). The solvent was removed at reduced pressure,
and the crude diamine was used without further purification.
A solution of the diamine in tetrachloroethane (130 mL) was
treated with carbonyldiimidazole (0.62 g, 3.80 mmol) in one
portion. After 15 min the reaction was added via cannula to
tetrachloroethane (10 mL) at reflux. After 15 min the reaction
was cooled and chromatographed directly (silica gel, 2:1 to 1:1
EtOAc/hexane to 2.5% MeOH/CH2Cl2 to 5% MeOH/CH2Cl2) to
give urea 4 as a white solid (1.18 g, 70%). 4: mp 242-243
°C; NMR (CDCl3) δ 6.8 (d, J ) 8.0 Hz, 2 H), 6.75 (m, 2 H), 6.7
(m, 2 H), 4.9 (m, 2 H), 4.25 (s, 8 H), 3.5 (m, 4 H), 2.95 (m, 2 H),
2.7 (m, 2 H) 1.5 (s, 6 H); CIMS (NH3) m/z 483 (M + H+, 100).
Anal. (C26H30N2O7) C, H, N.
(4R,5S,6S,7R)-Hexa h yd r o-5,6-O-isop r op ylid en e-1,3-bis-
(ph en ylm eth yl)-4,7-bis((3,4-(eth ylen ed ioxy)ph en yl)m eth -
yl)-2H-1,3-d ia za p in -2-on e (5). To a solution of 4 (0.1 g, 0.21
mmol) in dry THF (2 mL) at room temperature was added
potassium tert-butoxide (0.62 mL, 0.62 mmol, 1 M in THF).
After 5 min benzyl bromide (0.08 mL, 0.62 mmol) was added
and the reaction stirred at room temperature for 1 h. The
reaction was quenched with H2O, diluted with ether (30 mL),
washed with water (10 mL), dried (MgSO4), and concentrated.
We presented several novel P1/P1′ substituents that
provide increased binding affinity for HIVPR. The most
potent substituent found was the 3,4-ethylenedioxy
group that consistently provided a 5-10-fold improve-
ment in binding affinity. This improvement in binding
affinity frequently translated into an improved antiviral
profile provided the P2/P2′ substituents are carefully
chosen. Several potent analogs were further evaluated
for their resistance and protein binding profiles. These
compounds were found to be highly protein bound,
limiting their antiviral efficacy. The most promising
analog 18 was further evaluated in vivo. The compound
did not attain any detectable blood levels in rats. A 10
mg/kg dose resulted in low blood levels in dogs with a
Cmax ) 0.22 µg/mL. X-ray crystallographic analysis of
two analogs bound in the active site of HIVPR revealed
several interesting features. Both hydrophobic interac-
tions and the presence of a weak hydrogen bond were
responsible for this substituent’s improved potency
profile. Further evaluation of these P1/P1′ substituents
is ongoing and will be reported in due course.
Exp er im en ta l Section
All reactions were carried out with continuous stirring under
an atmosphere of dry nitrogen. Commercial reagents were
used as received without additional purification. THF was

Cyclic Urea Based HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1471
Chromatography (silica gel, 20% EtOAc/hexane) gave urea 5
as a foam (93 mg, 68%). 5: NMR (CDCl3) δ 7.3 (m, 6 H), 7.2
(m, 4 H), 6.8 (d, J ) 8.0 Hz, 2 H), 6.65 (m, 2 H), 6.55 (m, 2 H),
5.0 (d, J ) 14.0 Hz, 2 H), 4.3 (s, 8 H), 3.8 (bs, 2 H), 3.7 (m, 2
H), 3.2 (d, J ) 14.0 Hz, 2 H), 2.8 (m, 4 H), 1.3 (s, 6 H); CIMS
(NH3) m/z 663 (M + H+, 100).
for 18 h. The reaction mixture was filtered, and the solids
were washed with ether. The combined filtrates were con-
centrated at reduced pressure. Chromatography (silica gel,
50% ethyl acetate/hexane) gave the desired product as an oil
(2.8 g, 90%). 10: NMR (CDCl3) δ 7.17 (d, J ) 8.8 Hz, 1 H),
6.80 (d, J ) 8.8 Hz, 1 H), 4.30-4.26 (m, 2 H), 3.91 (m, 2 H),
2.33 (s, 3 H); CIMS (NH3) m/z 273/274 (M + NH4+, 100/98),
256/258 (M + H+, 96/90).
(4R,5S,6S,7R)-Hexa h yd r o-5,6-d ih yd r oxy-1,3-bis(p h en -
ylm et h yl)-4,7-b is((3,4-(et h ylen ed ioxy)p h en yl)m et h yl)-
2H-1,3-d ia za p in -2-on e (6). To a solution of urea 5 (93 mg,
0.14 mmol) in THF (2 mL) at room temperature was added
20% concentrated HCl in methanol (1.0 mL). After 15 min
the reaction was poured into saturated NaHCO3 (4 mL) and
extracted with ethyl acetate (20 mL). The organic layer was
separated, dried (MgSO4), and concentrated. The residue was
crystallized from ether to give 6 as a white solid (83 mg, 95%).
6: mp 195-197 °C; NMR (CDCl3) δ 7.3 (m, 4 H), 7.25 (m, 2
H), 7.2 (m, 4 H), 6.8 (d, J ) 8.1 Hz, 2 H), 6.65 (m, 2 H), 6.6
(dd, J ) 8.1, 2.0 Hz, 2 H), 4.95 (d, J ) 14.2 Hz, 2 H), 4.3 (s, 8
H), 3.6 (m, 4 H), 3.25 (d, J ) 14.2 Hz, 2 H), 2.9 (m, 4 H), 2.1
(bs, 2 H); CIMS (NH3) m/z 623 (M + H+, 100); HRMS calcd
for C37H39N2O7 (M + H+) 623.2757; found 623.2758. Anal.
(C37H38N2O7) C, H, N.
2-Nitr o-4-br om op h en ol (7). Silica gel 60 (EM Science)
was added to nitric acid (69-71%, 150 mL) in one portion and
the mixture stirred at room temperature for 2 h. The mixture
was then filtered and air dried on a Buchner funnel. The solid
was left to air dry in a crystallizing dish for 3 days. The
loading level was determined by titration with 0.1 M NaOH
to be 3.03 mmol g-1 nitric acid. This nitric acid impregnated
silica gel (57.8 mmol, 19.1 g) was suspended in dichlo-
romethane (450 mL) and cooled to 0 °C. The mixture was
treated with 4-bromophenol (5.0 g, 23.0 mmol) in several
portions. After 5 min the mixture was filtered, and the silica
gel cake was washed with dichloromethane. The concentrated
filtrate gave the desired product of sufficient purity for use in
the next step (12.3 g, 97%). 7: NMR (CDCl3) δ 10.5 (s, 1 H),
8.26 (d, J ) 2.6 Hz, 1 H), 7.67 (dd, J ) 6.6, 2.6 Hz, 1 H), 7.09
(d, J ) 8.8 Hz, 1 H); GC/MS (CH4) m/z 218 (M + H+, 20), 200
(100).
4-Meth yl-3,4-d ih yd r o-2H-ben zo[b][1,4]oxa zin -6-yl Br o-
m id e (11). A solution of 10 (2.0 g, 7.81 mmol) and potassium
hydroxide (2.8 g, 50 mmol) in methanol (10 mL) and water (5
mL) was heated at 55 °C for 30 min before the reaction mixture
was poured onto crushed ice and extracted with ether (2 × 75
mL). The combined extracts were dried (MgSO4) and concen-
trated to give the deacylated product which was carried on to
the next step without further purification (1.6 g, quantitative).
This product (0.20, 0.93 mmol) was added to a mixture of 37%
aqueous formaldehyde (0.15 g, 1.86 mmol) and acetonitrile (15
mL) at room temperature. The mixture was treated with
sodium cyanoborohydride (0.10 g, 1.5 mmol) in one portion and
stirred for 15 min before acetic acid (3 drops) was added. The
reaction mixture was stirred for 30 min, poured into ether (30
mL), and washed with 1 M sodium hydroxide (5 mL) and
saturated sodium chloride (5 mL). The organic layer was
separated, dried (Na2SO4), and concentrated at reduced pres-
sure. Chromatography (silica gel, 30% ether/hexane) gave the
product as a yellow oil (0.20 g, 95%): NMR (CDCl3) δ 6.73-
6.70 (m, 2 H), 6.61 (d, J ) 7.2 Hz, 1 H), 4.27-4.24 (m, 2 H),
3.28-3.25 (m, 2 H), 2.87 (s, 3 H); CIMS (NH3) m/z 228/230 (M
+ H+, 95/100).
(4R,5S,6S,7R)-Hexah ydr o-5,6-dih ydr oxy-1,3-bis(3-acetyl-
ben zyl)-4,7-bis((3,4-(et h ylen ed ioxy)p h en yl)m et h yl)-2H-
1,3-d ia za p in -2-on e (13): mp 200-201 °C; NMR (CDCl3) δ
7.85 (d, J ) 7.7 Hz, 2 H), 7.8 (s, 2 H), 7.4 (m, 4 H), 6.8 (d, J )
8.8 Hz, 2 H), 6.5 (m, 4 H), 4.9 (d, J ) 14.3 Hz, 2 H), 4.25 (s, 8
H), 3.7 (bs, 2 H), 3.55 (m, 2 H), 3.4 (d, J ) 14.3 Hz, 2 H), 2.95
(m, 2 H), 2.8 (m, 2 H), 2.6 (s, 6 H); CIMS (NH3) m/z 724 (M +
NH4+, 100); HRMS calcd for C41H43N2O9 (M + H+) 707.2969,
found 707.2969. Anal. (C41H42N2O9) C, H, N.
2-Am in o-4-br om op h en ol (8). A mixture of stannous
chloride dihydrate (24.8 g, 0.11 mmol) and concentrated
hydrochloric acid (55 mL) in methanol (100 mL) was cooled to
15 °C and treated with 4-bromo-2-nitrophenol (5.0 g, 23.0
mmol) in one portion. After the addition was complete, the
cooling bath was removed and the reaction stirred at room
temperature for 16 h. The reaction mixture was diluted with
ethyl acetate (200 mL) and the pH adjusted to 7 with saturated
sodium bicarbonate solution. The mixture was filtered (Celite)
and the filter cake washed with ethyl acetate. The organic
phase was separated, the aqueous phase was extracted with
ethyl acetate (100 mL), and the combined extracts were dried
(MgSO4) and concentrated to give the desired product as a
white solid (3.5 g, 74%). 8: NMR (DMSO-d6) δ 9.28 (s, 1 H),
6.70 (s, 1 H), 6.57-6.47 (m, 2 H), 4.80 (s, 2 H); CIMS (CH4)
m/z 188 (M + H+, 100).
N-Acetyl-2-a m in o-4-br om op h en ol (9). 2-Amino-4-bro-
mophenol (3.5 g, 18.6 mmol) in acetic anhydride (100 mL)
containing several drops of pyridine was heated on a steam
bath for 5 min and then at room temperature for another 5
min before the mixture was cooled in ice. The solid was
collected on a Buchner funnel, washed with n-hexane, and air-
dried to give the bisacetylated product. This solid was added
to 1 M sodium hydroxide (50 mL) and stirred at room
temperature until the mixture became homogeneous. The
reaction mixture was then poured into a mixture of crushed
ice (25 g) and 6 M hydrochloric acid (10 mL). The solid was
collected on a Buchner funnel and air-dried to give the desired
product as a tan solid (4.1 g, 97%). 9: NMR (CD3OD) δ 7.96
(d, J ) 2.2 Hz, 1 H), 7.05 (dd, J ) 8.8, 2.2 Hz, 1 H), 6.74 (d, J
) 2.2 Hz, 1 H), 2.14 (s, 3 H). CIMS (NH3) m/z 247/249 (M +
NH4+, 97/98), 230/232 (M + H+, 100/99).
(4R,5S,6S,7R)-Hexa h yd r o-5,6-O-isop r op ylid en e-1,3-bis-
((3-n it r op h e n yl)m e t h yl)-4,7-b is((3,4-(e t h yle n e d ioxy)-
p h en yl)m eth yl)-2H-1,3-d ia za p in -2-on e (14). To a solution
of 4 (0.1 g, 0.21 mmol) in dry THF (2 mL) at room temperature
was added potassium tert-butoxide (0.62 mL, 0.62 mmol, 1.0
M in THF). After 5 min 3-nitrobenzyl bromide (0.08 mL, 0.62
mmol) was added and the reaction stirred at room temperature
for 1 h. The reaction was quenched with H2O diluted with
ether (30 mL), washed with water (10 mL), dried (MgSO4),
and concentrated. Chromatography (silica gel, 20% EtOAc/
hexane) gave urea 14 as a foam (93 mg, 68%). 14: NMR
(CDCl3) δ 7.3 (m, 6 H), 7.2 (m, 4 H), 6.8 (d, J ) 8.0 Hz, 2 H),
6.65 (m, 2 H), 6.55 (m, 2 H), 5.0 (d, J ) 14.0 Hz, 2 H), 4.3 (s,
8 H), 3.8 (bs, 2 H), 3.7 (m, 2 H), 3.2 (d, J ) 14.0 Hz, 2 H), 2.8
(m, 4 H), 1.3 (s, 6 H); CIMS (NH3) m/z 663 (M + H+, 100).
(4R,5S,6S,7R)-Hexa h yd r o-5,6-d ih yd r oxy-1,3-bis((3-a m i-
n op h en yl)m et h yl)-4,7-b is((3,4-(et h ylen ed ioxy)p h en yl)-
m eth yl)-2H-1,3-d ia za p in -2-on e (15). A solution of urea 14
(0.1 g, 0.13 mmol) in THF (2 mL) was treated with 10%
palladium on carbon (20 mg) in one portion and stirred under
a atmosphere of hydrogen. After 16 h a solution of 20%
concentrated HCl in methanol (2 mL) was added in one portion
and the reaction stirred at room temperature for 1 h. The
reaction mixture was filtered (Celite) and the filtrate poured
into saturated NaHCO3 solution (5 mL). The mixture was
extracted with EtOAc (10 mL), the organic layer was separated
and dried (Na2SO4), and the solvent was evaporated. Chro-
matography (silica gel, 5% MeOH/CH2Cl2) gave the desired
product as a white solid (74 mg, 88%). 15: mp 140-142 °C;
NMR (CD3OD) δ 7.05 (m, 2 H), 6.8 (d, J ) 8.3 Hz, 2 H), 6.65
(m, 2 H), 6.6 (m, 4 H), 6.55 (m, 2 H), 6.5 (d, J ) 8.9 Hz, 4 H),
4.7 (d, J ) 14.2 Hz, 2 H), 4.2 (s, 8 H), 3.5 (m, 4 H), 2.95 (d, J
) 14.2 Hz, 2 H), 2.9 (m, 4 H); ESMS m/z 653 (M + H+, 14),
327 (M + 2H+, 100); HRMS calcd for C37H41N4O7 (M + H+)
653.2975, found 653.2979. Anal. (C37H40N4O7) C, H, N.
(4R,5S,6S,7R)-Hexah ydr o-5,6-dih ydr oxy-1,3-bis((3-(car -
boxya m in o)p h en yl)m eth yl)-4,7-bis((3,4-(eth ylen ed ioxy)-
1-(6-Br om o-3,4-d ih yd r o-2H-b en zo[b][1,4]oxa zin -4-yl)-
1-eth a n on e (10). A solution of 9 (2.8 g, 12.2 mmol), 1.2-
dibromoethane (4.2 mL, 49.0 mmol), sodium hydroxide (2.0 g,
49.0 mmol), and Aliquat 336 (0.6 g) in dichloromethane (25
mL) and acetonitrile (15 mL) was stirred at room temperature

1472 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Nugiel et al.
p h en yl)m eth yl)-2H-1,3-d ia za p in -2-on e (16). Urea 15 (0.13
g, 0.2 mmol) was suspended in butyl formate (2 mL) and
heated to reflux for 1 h. All solids dissolve after 5 min. The
mixture was cooled and the butyl formate removed at reduced
pressure. The residue was chromatographed (silica gel, 5-7%
MeOH/CH2Cl2) to give the desired product (0.12 g, 85%). The
NMR spectrum in MeOH-d4 shows a multiplicity of signals
due to the presence of amide bond rotomers: ESMS m/z 709
(M + H+, 100).
H), 5.0 (d, J ) 14 Hz, 2 H), 4.7 (t, J ) 7.5 Hz, 4 H), 3.7 (bs, 2
H), 3.6 (m, 2 H), 3.3 (m, 6 H), 3.0 (m, 4 H); CIMS m/z 591 (M
+ H+, 100); HRMS calcd for C37H39N2O5 (M + H+) 591.2859,
found 591.2866. Anal. (C37H38N2O5) C, H, N.
(4R,5S,6S,7R)-Hexa h yd r o-5,6-d ih yd r oxy-1,3-bis(p h en -
ylm eth yl)-4,7-bis((4-m eth yl-3,4-dih ydr o-2H-ben zo[b][1,4]-
oxa zin -6-yl)m et h yl-2H -1,3-d ia za p in -2-on e (24): NMR
(CDCl3) δ 7.33 (m, J ) 7.7 Hz, 1H), 7.17 (d, J ) 4.1), 7.10 (dd,
J ) 7.7, 10.6 Hz, 3 H), 6.80 (d, J ) 3.7 Hz, 1H), 6.80 (d, 8.42,
1H), 6.38 (m, 2H), 4.93 (d, J ) 14.7 Hz, 1H), 4.28 (m, 2H),
3.60 (bs, 1H), 3.49 (m, 1 H), 3.25 (m, 2H), 3.17 (d, J ) 14.2
Hz, 1H), 2.84 (m, 1H), 2.83 (s, 3H), 2.67 (bs, 1H); ESI-MS m/z
874.3 ((M + H)+, 12.1), 424.3 ((M + 2H)2+, 100); HRMS calcd
for C45H46N6O7S2 (M + H+) 847.2939, found 847.2948.
(4R,5S,6S,7R)-Hexa h yd r o-5,6-d ih yd r oxy-1,3-bis(cyclo-
pr opylm eth yl)-4,7-bis((3,4-(eth ylen edioxy)ph en yl)m eth yl)-
2H-1,3-d ia za p in -2-on e (25): mp 125-130 °C; NMR (CDCl3)
δ 6.7 (d, J ) 9.5 Hz, 2 H), 6.65 (m, 2 H), 6.6 (m, 2 H), 4.25 (s,
8 H), 3.95 (bs, 2 H), 3.55 (m, 2 H), 2.9 (m, 4 H), 2.5 (bs, 2 H),
2.1 (m, 2 H), 0.85 (m, 2 H), 0.35 (m, 4 H), 0.05 (m, 4 H); CIMS
(NH3) m/z 551 (M + H+, 100); HRMS calcd for C31H39N2O7 (M
+ H+) 551.2757, found 551.2749. Anal. (C31H38N2O7) C, H,
N.
(4R,5S,6S,7R)-H exa h yd r o-5,6-d ih yd r oxy-1-((3-a m in o-
p h en yl)m eth yl)-3-((3-(m eth yla m in o)p h en yl)m eth yl)-4,7-
bis((3,4-(eth ylen ed ioxy)p h en yl)m eth yl)-2H-1,3-d ia za p in -
2-on e (17). A solution of compound 16 (0.12 g, 0.17 mmol) in
THF (2 mL) was cooled to 0 °C and treated with BH3‚THF
(0.51 mL, 0.51 mmol, 1.0 M in THF) dropwise over 10 min.
The reaction mixture was allowed to reach room temperature
over 1 h and then heated at reflux for 16 h. The mixture was
cooled and quenched by the addition of MeOH (1 mL).
Concentrated HCL (1 mL) was added, and the mixture was
stirred at room temperature for 30 min. The solvent was
removed at reduced pressure, and the residue was taken up
in EtOAc (10 mL) and washed with saturated NaHCO3 (5 mL),
the organic layer was separated and dried (Na2SO4), and the
solvent was evaporated. Chromatography (silica gel, 5%
MeOH/CH2Cl2) gave a higher running Rf product which
corresponded to the monomethylated product 17 (60 mg, 53%).
Further elution gave the symmetrical di-N-methylurea 18 (40
mg, 35%). 17: NMR (CD3OD) δ 7.2 (m, 2 H), 6.9-6.5 (m, 12
H), 4.8 (dd, J ) 14.0, 3.7 Hz, 2 H), 4.3 (s, 8 H), 3.6 (m, 6 H),
3.4 (s, 3 H), 3.0 (m, 4 H), 2.8 (s, 3 H); ESMS m/z 667 (M + H+,
100); HRMS calcd for C38H43N4O7 (M + H+) 653.2975, found
653.2979.
(4R,5S,6S,7R)-Hexah ydr o-5,6-dih ydr oxy-1,3-bis(2-n aph -
th ylm eth yl)-4,7-bis((3,4-(eth ylen ed ioxy)p h en yl)m eth yl)-
2H-1,3-d ia za p in -2-on e (26): mp 232-234 °C; NMR (CDCl3)
δ 7.85 (m, 6 H), 7.55 (s, 2 H), 7.5 (m, 6 H), 6.8 (d, J ) 8.1 Hz,
2 H), 6.7 (m, 2 H), 6.6 (dd, J ) 8.1, 1.8 Hz, 2 H), 5.15 (d, J )
14.3 Hz, 2 H), 4.3 (s, 8 H), 3.6 (m, 4 H), 3.4 (d, J ) 14.3 Hz, 2
+
H), 3.0 (m, 4 H), 2.2 (bs, 2 H); CIMS (NH3) m/z 740 (M + NH4
,
100); HRMS calcd for C45H43N2O7 (M + H+) 723.3070, found
723.3051. Anal. (C45H42N2O7) C, H, N.
(4R ,5S ,6S ,7R )-H e xa h yd r o-5,6-d ih yd r oxy-1,3-b is((3-
(m et h yla m in o)p h en yl)m et h yl)-4,7-b is((3,4-(et h ylen ed i-
oxy)p h en yl)m eth yl)-2H-1,3-d ia za p in -2-on e (18). A solu-
tion of compound 16 (0.12 g, 0.17 mmol) in THF (3 mL) was
cooled to 0 °C and treated with a solution of LAH (0.5 mL, 0.5
mmol, 1.0 M in THF) dropwise over 10 min. The cooling bath
was removed and the reaction mixture heated at reflux for 30
min. The reaction mixture was cooled to 0 °C and quenched
with 1 N NaOH (4 drops). After quenching, the reaction was
allowed to reach room temperature and diluted with EtOAc.
Anhydrous sodium sulfate was added directly to the mixture
with continued stirring for 20 min. The reaction was then
filtered (Celite), and the remaining salts were washed with
additional EtOAc. The filtrate was evaporated and the residue
chromatographed (silica gel, 2.5-5% MeOH/CH2Cl2) to give
the desired product (0.11 g, 95%). 18: NMR (CD3OD) δ 7.2
(t, J ) 6.5 Hz, 2 H), 6.9 (d, J ) 7.5 Hz, 2 H), 6.8 (m, 2 H), 6.75
(m, 2 H), 6.6 (m, 2 H), 6.55 (m, 2 H), 6.5 (bs, 2 H), 5.0 (d, J )
14 Hz, 2 H), 4.4 (s, 8 H), 3.8 (bs, 2 H), 3.65 (m, 4 H), 3.2 (d, J
) 14 Hz, 2 H), 3.0 (m, 4 H), 2.8 (s, 6 H), 2.5 (bs, 2 H); ESMS
m/z 681 (M + H+, 100); HRMS calcd for C39H45N4O7 (M + H+)
681.3288, found 681.3289.
(4R,5S,6S,7R)-Hexa h yd r o-5,6-d ih yd r oxy-1,3-bis(p h en -
ylm eth yl)-4,7-bis((3,5-d im eth oxyp h en yl)m eth yl)-2H-1,3-
d ia za p in -2-on e (20): mp 210-212 °C; NMR (CD3OD) δ 7.3
(m, 4 H), 7.25 (m, 2 H), 7.2 (d, J ) 6 Hz, 4 H), 6.4 (m, 2 H),
6.25 (s, 4 H), 4.75 (d, J ) 14 Hz, 2 H), 3.8 (s, 12 H), 3.6 (m, 4
H), 3.1 (d, J ) 14 Hz, 2 H), 2.95 (d, J ) 8.5 Hz, 2 H), 2.85 (m,
2 H); CIMS m/z 627 (M + H+, 100); HRMS calcd for C37H43N2O7
(M + H+) 627.3070, found 627.3078. Anal. (C37H42N2O7) C,
H, N.
(4R,5S,6S,7R)-Hexa h yd r o-5,6-d ih yd r oxy-1,3-bis(p h en -
ylm eth yl)-4,7-bis((3,4-d im eth oxyp h en yl)m eth yl)-2H-1,3-
d ia za p in -2-on e (21): mp 204-206 °C; NMR (CD3OD) δ 7.3
(m, 6 H), 7.2 (d, J ) 5.5 Hz, 4 H), 6.9 (d, J ) 5.5 Hz, 2 H), 6.65
(m, 4 H), 4.7 (d, J ) 14 Hz, 2 H), 3.85 (s, 6 H), 3.8 (s, 6 H), 3.6
(m, 4 H), 3.1 (d, J ) 14 Hz, 2 H), 2.95 (m, 2 H), 2.85 (m, 2 H);
CIMS m/z 627 (M + H+, 100); HRMS calcd for C37H43N2O7 (M
+ H+) 627.3070, found 627.3080. Anal. (C37H42N2O7) C, H,
N.
(4R,5S,6S,7R)-Hexa h yd r o-5,6-d ih yd r oxy-1,3-bis(p h en -
ylm eth yl)-4,7-bis(2,3-d ih yd r oben zo[b]fu r a n -5ylm eth yl)-
2H-1,3-d ia za p in -2-on e (23): mp 194-196 °C; NMR (CDCl3)
δ 7.4 (m, 6 H), 7.25 (m, 4 H), 6.9 (m, 4 H), 6.8 (d, J ) 7 Hz, 2
(4R,5S,6S,7R)-Hexa h yd r o-5,6-d ih yd r oxy-1,3-bis((4-(h y-
d r oxym et h yl)p h en yl)m et h yl)-4,7-b is((3,4-(et h ylen ed i-
oxy)p h en yl)m eth yl)-2H-1,3-d ia za p in -2-on e (27): mp 127-
130 °C; NMR (CDCl3) δ 7.3 (d, J ) 8.1 Hz, 4 H), 7.2 (d, J ) 8.1
Hz, 4 H), 6.8 (d, J ) 8.3 Hz, 2 H), 6.55 (dd, J ) 8.3, 2.0 Hz, 2
H), 6.4 (m, 2 H), 4.8 (d, J ) 14.1 Hz, 2 H), 4.6 (s, 4 H), 4.25 (s,
8 H), 3.6 (bs, 2 H), 3.5 (m, 2 H), 3.3 (d, J ) 14.1 Hz, 2 H), 2.9
(m, 2 H), 2.8 (m, 2 H); CIMS (NH3) m/z 700 (M + NH4+, 100);
HRMS calcd for C39H43N2O9 (M + H+) 683.2969, found
683.2970. Anal. (C39H42N2O9) C, H, N.
(4R,5S,6S,7R)-Hexa h yd r o-5,6-d ih yd r oxy-1,3-bis((4-h y-
dr oxyph en yl)m eth yl)-4,7-bis((3,4-(eth ylen edioxy)ph en yl)-
m eth yl)-2H-1,3-d ia za p in -2-on e (28): mp 228-230 °C; NMR
(CD3OD) δ 7.1 (d, J ) 8.1 Hz, 4 H), 6.8 (m, 6 H), 6.7 (s, 2 H),
6.65 (m, 2 H), 4.75, (d, J ) 14.0 Hz, 2 H), 4.35 (s, 8 H), 3.6 (m,
2 H), 3.55 (bs, 2 H), 3.1 (d, J ) 14.0 Hz, 2 H), 2.95 (m, 2 H),
2.9 (m, 2 H); CIMS (NH3) m/z 672 (M + NH4+, 100); HRMS
calcd for C37H39N2O9 (M + H+) 655.2656, found 655.2662.
Anal. (C37H38N2O9) C, H, N.
(4R,5S,6S,7R)-Hexah ydr o-5,6-dih ydr oxy-1,3-bis(3-acetyl-
4-flu or oben zyl)-4,7-bis((3,4-(eth ylen ed ioxy)p h en yl)m eth -
yl)-2H-1,3-diazapin -2-on e (29): mp 197-199 °C; NMR (CDCl3)
δ 7.7 (m, 2 H), 7.45 (m, 2 H), 7.05 (m, 2 H), 6.8 (d, J ) 8.1 Hz,
2 H), 6.45 (m, 4 H), 4.7 (d, J ) 14.2 Hz, 2 H), 4.25 (s, 8 H),
3.75 (bs, 2 H), 3.5 (m, 4 H), 2.95 (m, 2 H), 2.7 (m, 2 H), 2.6 (d,
J ) 4.6 Hz, 6 H), 2.3 (bs, 2 H); CIMS (NH3) m/z 743 (M + H+,
100); HRMS calcd for C41H41F2N2O9 (M + H+) 743.2780, found
743.2800. Anal. (C41H40N2O9F2) C, H, N.
(4R,5S,6S,7R)-Hexah ydr o-5,6-dih ydr oxy-1,3-bis(3-acetyl-
b en zyl)-4,7-b is(2,3-d ih yd r ob en zo[b]fu r a n -5-ylm et h yl)-
2H-1,3-d ia za p in -2-on e (30): mp 237-240 °C; NMR (DMSO-
d6) δ 7.9 (d, J ) 5 Hz, 2 H), 7.7 (s, 2 H), 7.5 (m, 4 H), 6.6 (m,
6 H), 4.6 (d, J ) 14 Hz, 2 H), 4.5 (t, J ) 7.5 Hz, 4 H), 3.45 (m,
4 H), 3.2 (d, J ) 14 Hz, 2 H), 3.1 (t, J ) 7.5 Hz, 4 H), 2.9 (m,
2 H), 2.7 (m, 2 H), 2.55 (s, 6 H); CIMS m/z 675 (M + H+, 100);
HRMS calcd for C41H43N2O7 (M + H+) 675.3070, found
675.3088. Anal. (C41H42N2O7) C, H, N.
(4R,5S,6S,7R)-Hexa h yd r o-5,6-d ih yd r oxy-1,3-bis((3-a m i-
n op h en yl)m eth yl)-4,7-bis(2,3-d ih yd r oben zo[b]fu r a n -5-yl-
m eth yl)-2H-1,3-d ia za p in -2-on e (31): mp 232-235 °C; NMR
(CD3OD) δ 7.0 (t, J ) 6 Hz, 2 H), 6.9 (s, 2 H), 6.85 (m, 2 H),
6.7 (d, J ) 6 Hz, 2 H), 6.6 (m, 2 H), 6.5 (m, 4 H), 4.7 (d, J ) 14
Hz, 2 H), 4.5 (t, J ) 6.5 Hz, 4 H), 3.6 (m, 4 H), 3.3 (d, J ) 14
Hz, 2 H), 3.2 (t, J ) 6.5 Hz, 4 H), 2.9 (m, 4 H); CIMS m/z 621

Cyclic Urea Based HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1473
Ta ble 4. Crystallographic Refinement Statistics
constraints were applied to maintain identity between the two
protease monomers. Standard geometry of the inhibitor was
based on the single crystal structure of a cyclic urea. Statistics
of the crystallographic refinement are detailed in Table 4.
Bin d in g Affin ity Assa y. Inhibition of HIV protease was
measured by assay of the cleavage of a fluorescent peptide
substrate using HPLC.19
Wh ole Cell An tivir a l Assa y. The antiviral potency of
compounds was assessed by measuring their effect on the
accumulation of viral RNA transcripts three days after infec-
tion of MT-2 cells with HIV-1 RF.20
15
26
R factora
0.196
0.173
10.0-2.25
resolution range (Å)
no. of reflections used
no. of protein atoms
no. of inhibitor atoms
rms deviations from ideal values
bond lengths (Å)
10.0-2.1
6197
7032
1514
54
1514
48
0.019
3.88
0.019
3.71
bond angles (deg)
a
R factor ) ∑||Fo| - |Fc||/∑|Fo|, where |Fo| and |Fc| are the
I84V Mu ta n t An tivir a l Assa y. The I84V mutant virus
was selected using continuous passage of HIV-RF in PBMC’s
in the presence of 1 µg/mL of DMP323. The yield reduction
plaque assay was determined as described previously.21
observed and calculated structure factor amplitudes, respectively.
(M + H+, 100); HRMS calcd for C37H41N4O5 (M + H+) 621.3077,
found 621.3070. Anal. (C37H40N4O5) C, H, N.
Ack n ow led gm en t. We thank William A. Nugent
and J ohn E. Feaster, J r., for providing D-3,5-dimethox-
yphenylalanine.
(4R ,5S ,6S ,7R )-H e xa h yd r o-5,6-d ih yd r oxy-1,3-b is((3-
(m eth yla m in o)p h en yl)m eth yl)-4,7-bis(2,3-d ih yd r oben zo-
[b]fu r a n -5-ylm eth yl)-2H-1,3-d ia za p in -2-on e (32): mp 222-
226 °C; NMR (CD3OD) δ 7.0 (t, J ) 6 Hz, 2 H), 6.9 (s, 2 H),
6.85 (m, 2 H), 6.7 (d, J ) 6 Hz, 2 H), 6.6 (m, 2 H), 6.5 (m, 4 H),
4.7 (d, J ) 14 Hz, 2 H), 4.5 (t, J ) 6.5 Hz, 4 H), 3.6 (m, 4 H),
3.3 (d, J ) 14 Hz, 2 H), 3.2 (t, J ) 6.5 Hz, 4 H), 2.9 (m, 4 H),
2.8 (s, 6 H); CIMS m/z 649 (M + H+, 100); HRMS calcd for
C39H45N4O5 (M + H+) 649.3390, found 649.3389.
(4R,5S,6S,7R)-Hexah ydr o-5,6-dih ydr oxy-1,3-bis(3-acetyl-
ben zyl)-4,7-bis((3,4-(m eth ylen ed ioxy)p h en yl)m eth yl)-2H-
1,3-d ia za p in -2-on e (33): NMR (CDCl3) δ 7.8 (m, 4 H), 7.5-
7.3 (m, 4 H), 6.7 (d, J ) 7 Hz, 2 H), 6.5 (m, 4 H), 5.9 (m, 4 H),
4.8 (d, J ) 14 Hz, 2 H), 3.7 (bs, 2 H), 3.55 (bd, 2 H), 3.4 (d, J
) 14 Hz, 2 H), 3.0 (m, 2 H), 2.8 (t, J ) 6 Hz, 4 H), 2.5 (s, 6 H);
CIMS m/z 679 (M + H+, 100); HRMS calcd for C39H39N2O9 (M
+ H+) 679.2656, found 679.2649.
(4R,5S,6S,7R)-Hexah ydr o-5,6-dih ydr oxy-1,3-bis(4-flu or o-
3-a cet ylb en zyl)-4,7-b is((3,4-(m et h ylen ed ioxy)p h en yl)-
m eth yl)-2H-1,3-d ia za p in -2-on e (34): NMR (CD3OD) δ 7.7
(m, 2 H), 7.4 (m, 2 H), 7.0 (t, J ) 8.4 Hz, 2 H), 6.7 (d, J ) 7
Hz, 2 H), 6.4 (m, 4 H), 6.0 (d, J ) 6.5 Hz, 4 H), 4.6 (d, J ) 14
Hz, 2 H), 3.8 (bs, 2 H), 3.5 (m, 4 H), 3.0 (m, 2 H), 2.75 (m, 2
H), 2.6 (d, J ) 4 Hz, 6 H), 2.5 (m, 2 H); CIMS m/z 715 (M +
H+, 100); HRMS calcd for C39H37N2O9F2 (M + H+) 715.2467,
found 715.2462.
(4R,5S,6S,7R)-Hexah ydr o-5,6-dih ydr oxy-1,3-bis(3-acetyl-
ben zyl)-4,7-bis((3,5-d im eth oxyp h en yl)m eth yl)-2H-1,3-d i-
a za p in -2-on e (35): NMR (CD3OD) δ 7.9 (m, 2 H), 7.8 (bs, 2
H), 7.45 (m, 4 H), 6.3 (m, 2 H), 6.2 (m, 4 H), 4.7 (d, J ) 14 Hz,
2 H), 3.7 (s, 12 H), 3.65 (m, 4 H), 3.4 (d, J ) 14 Hz, 2 H), 3.0
(m, 2 H), 2.8 (m, 2 H), 2.6 (s, 6 H); CIMS m/z 711 (M + H+,
100); HRMS calcd for C41H47N2O9 (M + H+) 711.3282, found
711.3296.
(4R,5S,6S,7R)-Hexah ydr o-5,6-dih ydr oxy-1,3-bis(3-acetyl-
b en zyl)-4,7-b is((4-m et h yl-3,4-d ih yd r o-2H -b en zo[b][1,4]-
oxazin -6-yl)m eth yl)-2H-1,3-diazapin -2-on e (36): NMR (CD3-
OD) δ 7.8 (d, J ) 7.7 Hz, 2 H), 7.7 (s, 2 H), 7.5-7.4 (m, 2 H),
6.7 (d, J ) 8.4 Hz, 2 H), 6.4-6.3 (m, 4 H), 4.9 (d, J ) 14.2 Hz,
2 H), 4.3 (m, 4 H), 3.6 (bs, 2 H), 3.6-3.5 (m, 2 H), 3.3 (bs, 2 H),
3.3-3.2 (m, 6 H), 2.9 (d, J ) 6 Hz, 2 H), 2.9-2.75 (m, 2 H), 2.8
(s, 6 H), 2.58 (s, 6 H); CIMS m/z 733 (M + H+, 100); HRMS
calculated for C43H49N4O7 (M + H+) 733.3605, found 733.3601.
X-r a y Cr ysta llogr a p h y. The complexes of 15 and 26 with
HIV-1 protease were crystallized as described previously.17 The
unit cell dimensions of both complexes are a ) b ) 63.3 Å and
c ) 84.2 Å. The diffraction data were collected with a R-AXIS
II imaging plate mounted on a RU200 Rigaku rotating anode
generator operating at 50 kV and 100 mA. For 15, a total of
25 974 reflections were collected resulting in 14 838 unique
reflection and Rsym ) 7.2%. Although the crystal diffracts up
to 1.8 Å, the data can be considered at 2.1 Å resolution with
the criteria of F2 >) 1.0σF2, and 80% complete data. For 26,
15 938 unique reflections from 36 874 observations were
obtained with Rsym ) 9.4%. Using the same criteria, the data
can be considered having 2.25 Å resolution and contains 88%
complete data. The difference maps calculated with the
protein coordinates of a related cyclic urea structure revealed
the corresponding inhibitor position.5a The structures were
refined using the simulated annealing method, XPLOR.18 No
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J M960839I