Potent HIV-1 protease inhibitors incorporating meso-bicyclic urethanes as P2-ligands: Structure-based design, synthesis, biological evaluation and protein-ligand X-ray studies

Article abstract of DOI:10.1039/b809178a

Recently, we designed a series of novel HIV-1 protease inhibitors incorporating a stereochemically defined bicyclic fused cyclopentyl (Cp-THF) urethane as the high affinity P2-ligand. Inhibitor 1 with this P2-ligand has shown very impressive potency again

Full text of DOI:10.1039/b809178a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Potent HIV-1 protease inhibitors incorporating meso-bicyclic urethanes as
P2-ligands: structure-based design, synthesis, biological evaluation and
protein–ligand X-ray studies†
Arun K. Ghosh,*a Sandra Gemma,a Jun Takayama,a Abigail Baldridge,a Sofiya Leshchenko-Yashchuk,a Heather
B. Miller,a Yuan-Fang Wang,b Andrey Y. Kovalevsky,b Yashiro Koh,c Irene T. Weberb and Hiroaki Mitsuyac,d
Received 30th May 2008, Accepted 4th July 2008
First published as an Advance Article on the web 11th August 2008
DOI: 10.1039/b809178a
Recently, we designed a series of novel HIV-1 protease inhibitors incorporating a stereochemically
defined bicyclic fused cyclopentyl (Cp-THF) urethane as the high affinity P2-ligand. Inhibitor 1 with
this P2-ligand has shown very impressive potency against multi-drug-resistant clinical isolates. Based
upon the 1-bound HIV-1 protease X-ray structure, we have now designed and synthesized a number of
meso-bicyclic ligands which can conceivably interact similarly to the Cp-THF ligand. The design of
meso-ligands is quite attractive as they do not contain any stereocenters. Inhibitors incorporating
urethanes of bicyclic-1,3-dioxolane and bicyclic-1,4-dioxane have shown potent enzyme inhibitory and
antiviral activities. Inhibitor 2 (Ki = 0.11 nM; IC50 = 3.8 nM) displayed very potent antiviral activity in
this series. While inhibitor 3 showed comparable enzyme inhibitory activity (Ki = 0.18 nM) its antiviral
activity (IC50 = 170 nM) was significantly weaker than inhibitor 2. Inhibitor 2 maintained an antiviral
potency against a series of multi-drug resistant clinical isolates comparable to amprenavir. A
protein–ligand X-ray structure of 3-bound HIV-1 protease revealed a number of key hydrogen bonding
interactions at the S2-subsite. We have created an active model of inhibitor 2 based upon this X-ray
structure.
Introduction
The proteolytic enzyme HIV-1 protease is essential for viral
assembly and maturation.1 As a consequence, the design of specific
inhibitors for HIV-1 protease has become the subject of immense
interest. In 1996, protease inhibitors (PIs) were introduced in
combination with reverse transcriptase inhibitors to become a
highly active antiretroviral therapy (HAART).2 This treatment
regimen significantly increased life expectancy, improved quality
of life and decreased mortality and morbidity among HIV/AIDS
patients. Despite these notable advances, the emergence of drug-
resistant HIV-1 variants is severely limiting the efficacy of HAART
treatment regimens. Therefore, the development of new broad-
spectrum antiretroviral drugs that produce minimal adverse effects
remains an important therapeutic objective for the treatment
of HIV/AIDS.3 We have recently reported our structure-based
design and development of a series of novel HIV-1 protease
inhibitors including darunavir,4,5 TMC-126,6 and GRL-06579A
(1, Fig. 1).7 These inhibitors were designed with specific features
Fig. 1 Structure of inhibitors 1–3.
aDepartments of Chemistry and Medicinal Chemistry, Purdue University,
West Lafayette, IN 47907, USA. E-mail: akghosh@purdue.edu; Fax: +1
765 4961612; Tel: +1 765 4945323
bDepartment of Biology, Molecular Basis of Disease, Georgia State Univer-
to help combat drug resistance. They have exhibited marked po-
tency in enzyme inhibitory and cell-culture assays. Furthermore,
these inhibitors have shown impressive activity against a broad-
spectrum of HIV isolates including a variety of multi-PI-resistant
clinical strains. Darunavir has been recently approved for the
therapy of HIV/AIDS patients who are harboring drug-resistant
HIV and do not respond to other antiretroviral drugs.
sity, Atlanta, Georgia 30303, USA
cKumamoto University School of Medicine, Kumamoto 860-8556, Japan and
Department of Hematology and Infectious Diseases
dExperimental Retrovirology Section, HIV and AIDS Malignancy Branch,
National Cancer Institute, Bethesda, Maryland 20892, USA
† Electronic supplementary information (ESI) available: HPLC and
HRMS data of inhibitors 2–3 and 26–30; crystallographic data collection
and refinement statistics. See DOI: 10.1039/b809178a
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One of our design principles to combat drug resistance is to
maximize the ligand-binding interactions in the active site and
particularly to promote extensive hydrogen bonding with the
active site protein backbone. Indeed, inhibitor 1 incorporates
a stereochemically defined bicyclic cyclopentanyltetrahydrofuran
(Cp-THF) as the P2-ligand in the hydroxylethylsulfonamide
isostere. The protein–ligand X-ray structure of inhibitor 1 revealed
extensive hydrogen bonding interactions with the backbone atoms
throughout the enzyme active site.8 The cyclic ether oxygen is
involved in hydrogen bonding with the backbone NH of Asp29.
The presence of this oxygen is critical for its superb antiviral
properties, especially against drug resistant HIV strains. Based
upon further examination of the protein–ligand X-ray structure
of 1-bound HIV-1 protease, we subsequently speculated that a
simplified meso-hexahydrocyclopenta-1,3-dioxolane ligand could
conceivably maintain similar interactions with respect to the Cp-
THF ligand in inhibitor 1. Particularly, it appears that one of the
oxygens of this meso ligand can hydrogen bond with the Asp29
NH. Since the Cp-THF ligand in inhibitor 1 contains three chiral
centers, incorporation of a meso ligand as shown in inhibitor 2
would remarkably simplify the synthesis compared to the bicyclic
Cp-THF ligand. Furthermore, we speculated that the second
oxygen atom in the meso-P2-ligand could conceivably engage
in further interactions at the S2-subsite. Herein, we report the
design, synthesis and biological investigation of a series of protease
inhibitors that incorporate structure-based designed symmetrical
meso-bicyclic 1,3-dioxolane and 1,3-dioxane derivatives as the P2-
ligands. Inhibitors (2 and 3) incorporating these ligands have
shown exceedingly potent enzyme inhibitory potency as well as
antiviral activity. Furthermore, we evaluated the drug-resistance
profile of inhibitor 2 against multi-drug-resistant clinical isolates
and it was shown to maintain tremendous potency. The protein–
ligand X-ray structure of 3-bound HIV-1 protease has been
determined and this structure has provided molecular insight into
the ligand-binding site interactions.
Scheme 1 Synthesis of alcohols 9–12.
resistant strains. Accordingly, both intermediates 7 and 8 were
deprotected using tetrabutylammonium fluoride (TBAF) in THF
to provide the anti-alcohols 9 and 10. Compounds 9 and 10
were subsequently subjected to Mitsunobu inversion to afford the
corresponding syn-alcohols 11 and 12.
For the preparation of inhibitors 3 and 29, alcohols 15 and 16
were synthesized as described in Scheme 2. Diol 6 was heated under
reflux in toluene in the presence of dibutyltin oxide with azeotropic
removal of water. The resulting stannylene acetal intermediate
was treated with chloroethanol to obtain the monoalkylated
derivative 13 in 68% overall yield.10 Subsequently, the primary
Chemistry
The hexahydrocyclopenta-1,3-dioxolan-5-ol (11), required for
the synthesis of 2, was prepared as described in Scheme 1.
Commercially available 1,6-heptadien-4-ol 4 was protected as the
corresponding t-butyldiphenylsilyl ether using sodium hydride
as the base in THF. The resulting diene was subjected to a
ring closing metathesis reaction using second generation Grubbs’
catalyst to afford the protected cyclopenten-1-ol 5 in 94% overall
yield. Osmium tetroxide-promoted dihydroxylation of olefin 5
was accomplished using a catalytic amount of osmium tetroxide
and NMO and pyridine to afford diol 6 as a 6 : 1 mixture of
anti- and syn-isomers which were easily separated by column
chromatography. The anti- isomer 6 was subsequently treated
with paraformaldehyde, preliminarily cracked with aqueous hy-
drochloric acid in chloroform under reflux,9 affording the cyclic
acetal 7 in good yield. Along with the desired compound 7, the
trioxepane 8 was also isolated from the reaction mixture in a 1 :
1 ratio. We therefore decided to incorporate the tetrahydro-5aH-
cyclopenta[f ][1,3,5]trioxepan-7-yl-moiety as a P2-ligand (resulting
in inhibitors 27–28, Table 1) because the higher flexibility of the
trioxepane ring could allow an improved adaptability to enzyme
amino acid mutations, leading to better activity against HIV-
Scheme 2 Synthesis of alcohols 15 and 16.
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alcohol was selectively tosylated with p-toluenesulfonyl chloride
in the presence of pyridine. Exposure of the resulting compound to
sodium hydride resulted in an intramolecular substitution reaction
leading to the corresponding cyclization compound 14. TBAF-
mediated deprotection furnished the target anti-alcohol 15 in
good overall yield. The syn-alcohol 16 was then obtained after
Mitsunobu inversion of 15 as described above.
The synthesis of the active carbonates required for the synthesis
of the various inhibitors is shown in Scheme 3. Alcohol 9
was converted to the succinimidyl-derivative 17 by treatment
with N,N¢-succinimidylcarbonate in the presence of Et3N as
described previously.11 Alcohols 10–12, 15 and 16 were activated
by conversion to the corresponding p-nitrophenylcarbamates 18–
22 (81–95% yield) by using p-nitrophenylchloroformate and N-
methylmorpholine in THF. The general procedure for the synthesis
of inhibitors 2, 3 and 26–30 is outlined in Scheme 4. Epoxide
2312 was converted into intermediate 24 following our previously
reported procedure.8 Deprotection of 24 by using trifluoroacetic
acid followed by reaction with the activated alcohols 17–22
furnished inhibitors 2, 3 and 26–29 in 43–85% yields.
Scheme 4 Synthesis of inhibitors 2, 3 and 26–30.
Results and discussion
The inhibitory potencies of the synthetic inhibitors were evaluated
using the assay protocol of Toth and Marshall,13 and the results
are shown in Table 1. As can be seen, inhibitor 2 has shown an
enzyme inhibitory potency of 0.11 nM. It appears that the bicyclic
1,3-dioxolane ring can be accommodated by the S2-subsite of
HIV-1 protease. Inhibitor 26 with a meso ligand containing a
trans-bicyclic-1,3-dioxolane ring is 2.5-fold less potent than the
syn-isomer 2. We have examined the effect of both syn and anti-
trioxepane rings as P2-ligands in inhibitors 27 and 28. The syn-
isomer 28 is significantly more potent (Ki = 0.51 nM) than the anti-
isomer 27. Considering the acid sensitivity of 1,3-dioxolane rings,
we not only speculated that the stable 1,4-dioxane ring may fill the
hydrophobic S2-site, but also that the oxygens on the dioxane ring
may interact with backbone atoms or residues in the active site.
As shown, the meso ligand in inhibitor 3 with a syn-bicyclic-1,4-
dioxane ring has shown an enzyme inhibitory potency of 0.18 nM
(Ki value). Consistent with previous results, the corresponding
anti-isomer 29 is significantly less potent. As reported previously,
the P2-ligand Cp-THF with a P2¢-hydroxymethyl sulfonamide
(inhibitor 1) is significantly more potent than the corresponding
P2¢-methoxybenzene sulfonamide derivative. We have, therefore,
compared the inhibitory potency of inhibitor 30, containing a
P2¢-hydroxymethyl benzene sulfonamide derivative, with inhibitor
2. However, inhibitor 30 did not exhibit this potency enhancing
effect.
We have examined selected compounds for their activity against
HIV-1 using a human CD4+ T-cell line (MT-2 cells). The activity
of inhibitor 2 against a variety of multi-drug-resistant HIV-1
variants was also examined in detail using human peripheral
blood mononuclear cells (PBMCs) as target cells. We employed
two endpoints for the activity against HIV-1: (i) the inhibition
of the HIV-1-elicited cytopathic effect for MT-2 cells and (ii) the
inhibition of HIV-1 p24 production for PBMCs.5
Scheme 3 Synthesis of activated alcohols 17–22.
Finally, inhibitor 30 was synthesized from the known8 amine
25. This amine was reacted with the activated carbonate 17 in the
presence of diisopropylethylamine in THF at 23 ◦C to provide 30.
Inhibitor 30 was obtained in 63% yield.
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Table 1 Enzymatic inhibitory activity of compounds 2, 3, 26–30 and antiviral activity of selected inhibitors against HIV-1LAI
Entry
1
Inhibitor
Ki/nMa
IC50/mMb
0.11 0.01
0.0038 0.0001
2
3
0.40 0.04
nd
5.4 0.22
>1
4
5
0.51 0.01
0.38 0.02
0.21 0.04
0.18 0.03
6
7
0.50 0.04
0.34 0.07
nd
0.0077 0.003
a Values are means of at least two experiments. b MT-2 human T-lymphoid cells exposed to HIV-1LAI; antiviral activity of amprenavir (APV), saquinavir
(SQV) and indinavir (IDV) were 0.03 mM, 0.02 mM and 0.03 mM respectively in this assay. nd: not determined.
When examined in MT-2 cells as the target cells, inhibitor 2
displayed an impressive antiviral IC50 of 3.8 nM (Table 1). Inhibitor
3 showed an antiviral IC50 value in the high nanomolar range
(IC50 = 210 nM, Table 1), while it exhibited a similar Ki to inhibitor
2. We subsequently examined inhibitor 2 for its activity against
a clinical wild-type X4-HIV-1 isolate (HIV-1ERS104pre) along with
various multi-drug-resistant clinical X4- and R5-HIV-1 isolates
(Table 2) using PBMCs as the target cells.5 The activity of inhibitor
2 against HIV-1ERS104pre (IC50 = 29 nM) was comparable to those
of currently available protease inhibitors, SQV, APV, and IDV,
which display IC50 values of 12, 33, and 26 nM, respectively. Of
particular note, the IC50 value of inhibitor 2 in PBMCs (IC50=
29 nM) was nearly 8-fold greater than the IC50 value in MT-2 cells
(IC50 = 3.8 nM). With regard to this difference, considering that 2
is highly potent as examined in human T-cells (MT-2 cells) but its
activity is slightly less in PBMCs, it is possible that relatively higher
concentrations of 2 are required to suppress HIV-1 production
in chronically infected macrophages.14 IDV was not capable of
efficiently suppressing the replication of most of the multi-drug-
resistant clinical isolates examined (HIV-1MDR/MM, HIV-1MDR/JSL
,
HIV-1MDR/C, and HIV-1MDR/A), with IC50 values of >1.0 mM. The
potency of inhibitor 2 against most of the multi-drug-resistant
variants was generally comparable to that of SQV and APV,
although DRV was found to be the most potent among those
tested, including inhibitor 2, against HIV-1ERS104pre as well as all the
multi-drug-resistant variants.
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Table 2 Antiviral activity of inhibitor 2 against clinical HIV-1 isolates in PBMC cells
IC50 valuesa(nM)
Virusb
2
DRVc
SQVd
APVe
IDVf
HIV-1ERS104pre (wild-type: X4)
HIV-1MDR/MM(R5)
HIV-1MDR/JSL (R5)
HIV-1MDR/C (X4)
HIV-1MDR/G (X4)
HIV-1MDR/A (X4)
29
3.5
12
33
26
150 (5)
550 (19)
300 (10)
340 (12)
21 (1)
17 (5)
26 (7)
7 (2)
7 (2)
3 (1)
190 (16)
330 (28)
36 (3)
29 (2)
81 (7)
300 (9)
430 (13)
230 (7)
340 (10)
100 (3)
>1000 (>38)
>1000 (>38)
>1000 (>38)
290 (11)
>1000 (>38)
a Amino acid substitutions identified in the protease-encoding region compared to the consensus type B sequence cited from the Los Alamos database
include L63P in HIV-1ERS104pre; L10I, K43T, M46L, I54V, L63P, A71V, V82A, L90M, and Q92K in HIV-1MDR/MM; L10I, L24I, I33F, E35D, M36I, N37S,
M46L, I54V, R57K, I62V, L63P, A71V, G73S, and V82A in HIV-1MDR/JSL; L10I, I15V, K20R, L24I, M36I, M46L, I54V, I62V, L63P, K70Q, V82A, and
L89M in HIV-1MDR/C; L10I, V11I, T12E, I15V, L19I, R41K, M46L, L63P, A71T, V82A, and L90M in HIV-1MDR/G; and L10I, I15V, E35D, N37E, K45R,
I54V, L63P, A71V, V82T, L90M, I93L, and C95F in HIV-1MDR/A. HIV-1ERS104pre served as a source of wild-type HIV-1. The IC50 values were determined
by employing PHA-PBMC (phytohemagglutinin-activated peripheral blood mononuclear cells) as target cells and the inhibition of p24Gag protein
production as the endpoint. All values were determined in triplicate. b X4 denotes CXCR4-tropic HIV-1 while R5 CCR5-tropic HIV-1. c DRV (darunavir).
d SQV (saquinavir). e APV (amprenavir). f IDV (indinavir).
X-Ray crystallography
substrate analogs.15 The weaker polar interactions such as C–
H ◊ ◊ ◊ O and water-p interactions can be analyzed accurately in
atomic resolution structures.17,18 Inhibitor 3 also shows a water-
mediated interaction of the p system of the P2¢ aromatic ring
with the amide of Asp29¢, which was also observed for darunavir
and inhibitor 1.19 Furthermore, the P2¢ methoxy group forms a
hydrogen bond to the backbone NH of Asp30¢. Importantly, the
P2 group forms a hydrogen bond interaction with the carbonyl
oxygen of Gly48 and a water-mediated interaction with the amide
of Gly48, similar to the interactions described for several peptide
substrate analogs.15 These interactions of the P2 group confirm
the design strategy of incorporating new polar interactions with
conserved backbone regions of the protease.
In an effort to understand the binding interactions of the
corresponding meso-1,3-dioxolane ligand in the S2-subsite, we
have created an active model of inhibitor 2 (Fig. 3) based upon the
X-ray structure of 3-bound HIV-1 protease. The model suggests
that both dioxolane oxygens may interact with both active site
residues Asp29 and Asp30, as well as Gly48 through the structural
water molecule. In comparison, it appears that the dioxane
oxygens of inhibitor 3 are not within hydrogen bonding distance
of the backbone NH of Asp30. This may explain the marked
difference in antiviral activity of inhibitor 2 compared with inhib-
itor 3.
To obtain molecular insight into the ligand-binding site interac-
tions responsible for the impressive enzyme inhibitory potency of
compound 3, we determined the X-ray structure of 3-bound HIV-
1 protease. The crystal structure was solved and refined to an R
˚
factor of 15.2% at a 1.07 A resolution. The inhibitor binds with
extensive interactions from P2 to P2¢ with the protease atoms,
and most notable are the favorable polar interactions including
hydrogen bonds, as shown in Fig. 2. The transition-state hydroxyl
group forms hydrogen bonds to the side chain carboxylate oxygen
atoms of the catalytic Asp25 and Asp25¢. Of particular interest,
the meso-bicyclic 1,4-dioxane ligand appears to be involved in
hydrogen bonding interactions with the backbone atoms and
residues at the S2-site. One of the dioxane oxygens hydrogen bonds
with the backbone NH of Asp29. The other oxygen makes a water-
mediated hydrogen bond with the carbonyl oxygen of Gly48. These
interactions are described in several peptide substrate analogs.15
However, the design of high affinity ligands incorporating this
interaction with Gly48 has not been previously demonstrated.
The inhibitor also hydrogen bonds with the protease main chain
amide carbonyl oxygen of Gly27, and there are water-mediated
interactions with the amides of Ile50 and Ile50¢ that are conserved
in the majority of protease complexes with inhibitors16 and
Fig. 2 Stereoview of the X-ray structure of inhibitor 3 bound to the active site of wild-type HIV-1 protease.
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Fig. 3 A stereoview of an active model of inhibitor 2 (green) with the X-ray structure of inhibitor 3 (magenta)-bound HIV-1 protease.
eters. Optical rotations were measured using a Perkin-Elmer 341
polarimeter. IR spectra were recorded on a Mattason Genesis II
FT-IR spectrometer.
Conclusions
In summary, a series of novel HIV-1 protease inhibitors were
designed and synthesized by incorporating bicyclic meso-1,3-
dioxolane and 1,4-dioxane derivatives as the P2-ligands. A number
of inhibitors have shown very impressive enzyme inhibitory and
antiviral potency, similar to inhibitor 1 with a stereochemically
defined Cp-THF ligand. The design of meso-1,3-dioxolane and
1,4-dioxane P2-ligands as exemplified in inhibitors 2 and 3, respec-
tively, has remarkably simplified the stereochemical complexity as
well as chemical synthesis over the Cp-THF ligand in inhibitor
1. We have developed efficient synthetic routes to these ligands.
Inhibitor 2 has shown potent antiviral activity in both MT-
2 cells and PBMCs. Inhibitor 2 was profiled against a series
of multi-drug-resistant clinical isolates. While inhibitor 2 is less
potent than darunavir, it is significantly more potent than IDV
and comparable to APV and SQV in suppressing the replic-
4-(tert-Butyldiphenylsilyloxy)-4H-cyclopentene (5)
To a suspension of sodium hydride (60% in mineral oil, 0.92 g,
23 mmol) in THF (10 mL), cooled to 0 ◦C, 1,6-heptadien-4-
ol 4 (1 mL, 7.7 mmol) was added dropwise over 10 min. The
resulting suspension was stirred at 0 ◦C for 30 min and then
tert-butyldiphenylchlorosilane (2 mL, 7.9 mmol) was added. The
◦
reaction mixture was stirred at 23 C for 4 h and then quenched
with a saturated solution of ammonium chloride. The solvent
was removed in vacuo and the aqueous phase was extracted with
CH2Cl2. The organic extracts were dried (Na2SO4), the solvent
removed and the residue purified by flash-chromatography (1 :
10, EtOAc–Hex) to afford 4-(tert-butyldiphenylsilyloxy)hepta-1,6-
diene (2.6 g, 96%) as a colorless oil: IR nmax (NaCl; cm-1) 3066,
2951, 1421, 1103 and 696; dH (300 MHz, CDCl3) 7.70 (4 H, dd, J
1.6, 7.6 Hz, ArH), 7.47–7.38 (6 H, m, ArH), 5.83–5.69 (2 H, m, 2 ¥
ation of multi-drug-resistant isolates MDRMM and MDRJSL
.
A protein–ligand X-ray structure of 3-bound HIV-1 protease
revealed extensive interactions of the inhibitor with the active
site of HIV-1 protease. Most notably, both oxygens of the meso-
P2-ligand are involved in hydrogen bonding interactions with the
protein backbone atoms. In particular, a water-mediated hydrogen
bond to the Gly48 carbonyl is very unique. An active model
of inhibitor 2 indicates similar ligand binding site interactions.
Our design principle of increasing ‘backbone binding’ appears
to maintain key interactions in the enzyme active site leading
to retained potency against multi-drug-resistant variants. Further
design and ligand optimization involving these interactions is in
progress.
=
=
CH CH2), 5.02–4.91 (4 H, m, 2 ¥ CH CH2), 3.87–3.80 (1 H, m,
CHOSi), 2.31–2.12 (4 H, m, 3-H2, 5-H2) and 1.08 [9 H, s, C(CH3)3;
dC (75 MHz, CDCl3) 135.9, 134.7, 134.3, 129.5, 127.5, 117.1, 72.4,
40.5, 27.0 and 19.4; m/z (CI) 351 (M + H, 100); HRMS (M + H)+
calcd for C23H31OSi, 351.2144; found, 351.2146.
To a solution of the above compound (2.0 g, 5.7 mmol) in dry
CH2Cl2 (20 mL), second generation Grubbs’ catalyst (48 mg) was
added and the resulting mixture was heated under reflux for 2 h.
Subsequently, the reaction mixture was cooled to 23 ◦C, the solvent
removed under reduced pressure and the residue purified by flash-
chromatography (1 : 10 EtOAc–Hex) to afford 5 (1.8 g, 98%) as
a colorless oil: IR nmax (NaCl; cm-1) 3067, 2853, 2736, 1428, 1109
and 702; dH (300 MHz, CDCl3) 7.67 (4 H, dd, J 1.8, 7.8, ArH),
7.45–7.34 (6 H, m, ArH), 5.61 (2 H, s, 1-H, 2-H), 4.57–4.51 (1 H,
m, 4-H), 2.47–2.33 (4 H, m, 3-H2, 5-H2) and 1.05 [9 H, s, C(CH3)3];
dC (100 MHz, CDCl3) 135.7, 134.5, 129.5, 128.3, 127.5, 73.5, 42.4,
26.9 and 19.1; m/z (CI) 323 (M + H, 100); HRMS (M + H)+ calcd
for C21H27OSi, 323.1831; found, 323.1834.
Experimental
General. All moisture sensitive reactions were carried out
under a nitrogen or argon atmosphere. Anhydrous solvents were
obtained as follows: THF, diethyl ether, and benzene, distilled
from sodium and benzophenone; dichloromethane, pyridine,
triethylamine, and diisopropylethylamine, distilled from CaH2.
All other solvents were HPLC grade. Column chromatography
was performed with Whatman 240–400 mesh silica gel under low
pressure (5–10 psi). TLC was carried out with E. Merck silica
(1a,2a,4b)-4-(tert-Butyldiphenylsilyloxy)-1,2-cyclopentanediol (6)
A mixture of 5 (5.1 g, 15.8 mmol), osmium tetroxide (2.5 wt%
solution in tert-butanol, 4 mL), N-methylmorpholine-N-oxide
(2.6 g, 22.2 mmol), and pyridine (1.3 mL, 15.8 mmol) in a
gel 60 F254 plates. H and 13C NMR spectra were recorded on
Varian Mercury 300 and Bruker Avance 400 and 500 spectrom-
1
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3 : 2 : 1 mixture of tert-butanol, THF, and water (80 mL) was heated
under reflux for 4 h. The reaction mixture was cooled to 23 ◦C and
treated with a 20% aqueous solution of sodium bisulfite (10 mL).
The organic solvents were removed under reduced pressure and
the aqueous phase was extracted with EtOAc. The organic extracts
were washed with 1 N hydrochloric acid, water and brine, and dried
(Na2SO4). The solvent was removed in vacuo and the residue was
purified by flash-chromatography (1 : 1 EtOAc–Hex) to yield diol
6 (5.3 g, 94%) as a colorless oil: IR nmax (NaCl; cm-1) 3006, 2676,
1427, 1112 and 702; dH (300 MHz, CDCl3) 7.62 (4 H, dd, J 1.8,
7.5, ArH), 7.45–7.33 (6 H, m, ArH), 4.84–4.42 (1 H, m, 4-H), 4.30–
4.29 (2 H, m, 1-H, 2-H), 2.22 (2 H, br. s, 2 ¥ OH), 1.99–1.80 (4 H,
m, 3-H2, 5-H2) and 1.04 [9 H, s, C(CH3)3]; dC (100 MHz, CDCl3)
135.6, 134.0, 129.6, 127.6, 72.4, 71.2, 41.9, 26.8 and 19.0; m/z (EI)
356 (M, 100); HRMS (M)+ calcd for C21H28O3Si, 356.1808; found,
356.1803.
and 1.61–1.51 (2 H, m, 3-H¢¢, 5-H¢¢); dC (75 MHz, CDCl3) 94.1,
78.9, 70.8 and 40.6; m/z (CI) 129 (M - H, 100); HRMS (M - H)+
calcd for C6H9O3, 129.0552; found, 129.0556.
(5aa,7b,8aa)-7-Hydroxytetrahydrocyclopenta[f ]-1,3,5-trioxepane
(10)
The title compound was obtained as described for 9 in 83%
yield. Flash-chromatography was performed using EtOAc: IR
nmax (NaCl; cm-1) 3036, 2649, 1424, 1118 and 930; dH (300 MHz,
CDCl3) 5.15 (2 H, d, J 7.2, 2-H¢, 4-H¢), 4.67 (2 H, d, J 7.2, 2-H¢¢,
4-H¢¢), 4.47–4.40 (3 H, m, 5a-H, 7-H, 8a-H), 2.07–2.02 (4 H, m,
6-H2, 8-H2) and 1.86 (1 H, br. s, OH); dC (75 MHz, CDCl3) 96.1,
82.3, 70.0 and 40.8; m/z (EI) 160 (M, 100); HRMS (M)+ calcd for
C7H12O4,160.0736; found, 160.0738.
(1b,2b,4b)-4-Hydroxy-1,2-(methylenedioxy)cyclopentane (11)
(1b,2b,4a)-4-(tert-Butyldiphenylsilyloxy)-1,2-(methylenedioxy)-
cyclopentane (7) and (5aa,7b,8aa)-7-(tert-butyldiphenylsilyloxy)-
tetrahydrocyclopenta[f ]-1,3,5-trioxepane (8)
To a mixture of 9 (100 mg, 0.77 mmol), p-nitrobenzoic acid
(250 mg, 1.5 mmol), and triphenylphosphine (450 mg, 1.5 mmol),
was added diisopropylazodicarboxylate (300 mL, 1.5 mmol)
dropwise and the resulting mixture was stirred at 23 ◦C. After
16 h, the solvent was removed under reduced pressure and the
residue purified by flash-chromatography (1 : 2 EtOAc–Hex). The
resulting ester was dissolved in a 3 : 2 : 1 mixture of THF, methanol,
and water (10 mL) and LiOH·H2O (162 mg, 3.8 mmol) was added.
The yellow mixture was stirred at 23 ◦C for 5 h and then the solvent
was removed in vacuo. The residue was diluted with water and the
aqueous phase extracted with Et2O. The organic extracts were
dried (Na2SO4) and the solvent was evaporated. Purification of
the residue by flash-chromatography (1 : 1 EtOAc–Hex) afforded
11 (57 mg, 57%) as a colorless oil: IR nmax (NaCl; cm-1) 3052,
2804, 2577, 1164, 1096, 1011 and 924; dH (300 MHz, CDCl3) 5.17
(1 H, s, OCHHO), 4.68 (1 H, s, OCHHO), 4.61 (2 H, d, J 4.8,
1-H, 2-H), 4.27 (1 H, t, J 4.7, 4-H), 2.33 (1 H, br. s, OH), 2.21 (2
H, d, J 15.3, 3-H¢, 5-H¢) and 1.85–1.77 (2 H, m, 3-H¢¢, 5-H¢¢); dC
(75 MHz, CDCl3) 94.7, 81.5, 74.0 and 41.0; m/z (EI) 129 (M -
H, 100); HRMS (M - H)+ calcd for C6H9O3, 129.0611; found,
129.1012.
A mixture of paraformaldehyde (0.77 g, 25.7 mmol) and con-
centrated hydrochloric acid (2 mL) in CHCl3 (2 mL) was stirred
at 23 ◦C until a clear solution was formed (6 h) and then a
solution of 6 (0.2 g, 0.54 mmol) in CHCl3 (2 mL) was added.
The resulting mixture was heated under reflux overnight and the
aqueous phase was extracted with CHCl3. The organic extracts
were dried (Na2SO4) and evaporated under reduced pressure to
yield 7 (0.18 g, 86%) after flash-chromatography (1 : 10, EtOAc–
Hex): IR nmax (NaCl; cm-1) 2791, 1589, 1471, 1428, 822 and 699;
dH (300 MHz, CDCl3) 7.64 (4 H, d, J 6.3, ArH), 7.45–7.35 (6 H,
m, ArH), 4.78 (1 H, s), 4.60 (1 H, s), 4.51 (2 H, d, J 5.4, 1-H,
2-H), 4.47–4.39 (1 H, m, 4-H), 1.99 (2 H, dd, J 6.0, 13.8, 3-H¢,
5-H¢), 1.77–1.68 (2 H, m, 3-H¢¢, 5-H¢¢) and 1.04 [9 H, s, C(CH3)3];
dC (75 MHz, CDCl3) 135.6, 134.0, 129.7, 127.6, 94.0, 78.8, 72.7,
41.0, 26.9 and 19.1; m/z (EI) 368 (M, 100). After further elution of
the column 8 (0.5 g, 5%) was obtained: IR nmax (NaCl; cm-1) 2827,
2726, 1427, 1113 and 703; dH (300 MHz, CDCl3) 7.66–7.62 (4 H,
m, ArH), 7.46–7.35 (6 H, m, ArH), 5.17 (2 H, d, J 7.8, 2-H¢, 4-H¢),
4.70 (2 H, d, J 7.8, 2-H¢¢, 4-H¢¢), 4.52–4.43 (3 H, m, 5a-H, 7-H,
8a-H), 2.15–2.08 (2 H, m, 6-H¢, 8-H¢), 1.93–1.85 (2 H, m, 6-H¢¢, 8-
H¢¢) and 1.06 [9 H, s, C(CH3)3]; dC (75 MHz, CDCl3) 135.6, 133.8,
129.7, 127.7, 96.1, 82.5, 71.7, 41.1, 26.9 and 19.1; m/z (CI) 397
(M - H, 100); HRMS (M - H)+ calcd for C23H29O4Si, 397.1832;
found, 397.1832.
(5aa,7a,8aa)-7-Hydroxytetrahydrocyclopenta[f ]-1,3,5-trioxepane
(12)
The title compound 12 was obtained as described for 11 in 69%
yield. Flash-chromatography was performed using EtOAc: IR nmax
(NaCl; cm-1) 3044, 2832, 2633, 1481, 1116 and 928; dH (300 MHz,
CDCl3) 5.18 (2 H, d, J 7.2, 2-H¢, 4-H¢), 4.67 (2 H, d, J 7.2, 2-H¢¢,
4-H¢¢), 4.31–4.25 (2 H, m, 5a-H, 8a-H), 4.18–4.13 (1 H, m, 7-H),
2.40 (1 H, br. s, OH), 2.17–2.08 (2 H, m, 6-H¢, 8-H¢) and 2.03–1.96
(2 H, m, 6-H¢¢, 8-H¢¢); dC (75 MHz, CDCl3) 95.3, 82.8, 71.0 and
41.1; m/z (CI) 161 (M + H, 100); HRMS (M + H)+ calcd for
C7H12O4, 161.0814; found, 161.0814.
(4a,1b,2b)-4-Hydroxy-1,2-(methylenedioxy)cyclopentane (9)
A mixture of 7 (0.47 g, 1.3 mmol) and n-Bu4N+F- (1.0 M solution
in THF, 1.4 mL, 1.4 mmol) in dry THF (10 mL) was stirred at
23 ◦C for 16 h. To the reaction mixture was added a saturated
solution of NaHCO3, the solvent was removed in vacuo and the
aqueous phase extracted with Et2O. The organic extracts were
dried (Na2SO4) and evaporated and the residue was purified by
flash-chromatography (1 : 1 EtOAc–Hex) to yield 9 (0.16 g, 96%)
as a colorless oil: IR nmax (NaCl; cm-1) 3044, 2792, 2602, 1065, 821
and 602; dH (300 MHz, CDCl3) 4.89 (1 H, s, OCHHO), 4.59 (1
H, s, OCHHO), 4.50 (2 H, d, J 6.0, 1-H, 2-H), 4.41–4.32 (1 H, m,
4-H), 3.13 (1 H, br. s, OH), 2.09 (2 H, dd, J 5.6, 14.0, 3-H¢, 5-H¢)
( )-(1b,2b,4a)-2-(2¢-Hydroxyethoxy)-4-(tert-
butyldiphenylsilyloxy)cyclopentane-1-ol (13)
A mixture of 6 (1.4 g, 3.9 mmol) and dibutyltin oxide (0.94 g,
3.9 mmol) in dry toluene (130 mL) was heated under reflux with
azeotropic removal of water. After 5 h, the reaction mixture was
concentrated to half the initial volume and chloroethanol (2.5 mL,
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39 mmol) and n-Bu4N+I- (1.4 g, 3.9 mmol) were added. The
resulting mixture was heated under reflux for 19 h. Afterwards
the solvent was evaporated and the residue was purified by flash-
chromatography (10 : 1 EtOAc–MeOH) to afford 13 (1.3 g, 86%) as
a colorless oil: IR nmax (NaCl; cm-1) 3102, 2604, 1589, 1471, 1062,
823 and 612; dH (300 MHz, CDCl3) 7.62 (4 H, d, J 8.7, ArH),
7.44–7.33 (6 H, m, ArH), 4.45–4.40 (1 H, m, 4-H), 4.33–4.28 (1
H, m, 2-H), 4.04–3.98 (1 H, m, 1-H), 3.76–3.71 (2 H, m, CH2O),
3.66–3.55 (2H, m, CH2O), 3.01 (2 H, br. s, 2 ¥ OH), 1.97–1.80 (4
H, m, 3-H2, 5-H2) and 1.04 (s, 9H); dC (75 MHz, CDCl3) 135.6,
134.1, 129.6, 127.6, 80.7, 71.1, 71.0, 70.8, 61.7, 42.3, 39.0, 26.9 and
14.2; m/z (ESI) 423 (M + Na, 100).
(1b,2b,4b)-4-Hydroxy-1,2-(ethylendioxy)cyclopentane (16)
Starting from 15 the title compound 16 was obtained as described
for 11 in 83% yield as a colorless oil. Flash-chromatography was
performed using EtOAc: IR nmax (NaCl; cm-1) 3014, 2571, 1135,
1081 and 875; dH (300 MHz, CDCl3) 4.22–4.16 (1 H, m, 4-H), 4.01
(2 H, t, J 4.2, 1-H, 2-H), 3.88–3.80 (2 H, m, CH2O), 3.63–3.55 (2
H, m, CH2O), 2.57 (1 H, br. s, OH) and 2.10–1.93 (4 H, m, 3-H2,
5-H2); dC (75 MHz, CDCl3) 76.0, 71.4, 62.3 and 37.5; m/z (EI)
144 (M, 100).
(1b,2b,4b)-1,2-(Methylenedioxy)cyclopent-4-yl
succinimidylcarbonate (17)
To a solution of 9 (67 mg, 0.52 mmol) in dry acetonitrile
(2 mL), N,N¢-disuccinimidyl carbonate (198 mg, 0.77 mmol) and
triethylamine (145 mL, 1.0 mmol) were added and the resulting
mixture was stirred at 23 ◦C. After 8 h the solvent was removed,
the residue was taken up in a saturated solution of NaHCO3
and the aqueous phase was extracted with EtOAc. The organic
extracts were dried (Na2SO4) and the solvent was removed in
vacuo. Purification of the residue by flash-chromatography (10 :
1 CHCl3–MeOH) yielded 17 (58 mg, 55%): IR nmax (NaCl; cm-1)
2759, 1787, 1740, 1210, 1090; dH (300 MHz, CDCl3) 5.27 (1 H,
t, J 7.2, 4-H), 4.97 (1 H, s, OCHHO), 4.69 (1 H, s, OCHHO),
4.61–4.59 (2 H, m, 1-H, 2-H), 2.82 (4 H, s, CH2CH2), 2.38 (2 H,
dd, J 6.2, 14.2, 3-H¢, 5-H¢) and 1.99–1.89 (2 H, m, 3-H¢¢, 5-H¢¢);
dC (100 MHz, CDCl3) 168.6, 150.8, 94.5, 81.2, 78.1, 37.3 and 25.4;
m/z (CI) 270 (M - H, 100); HRMS (M - H)+ calcd for C11H12NO7,
270.0614; found, 270.0607.
(1b,2b,4a)-4-(tert-butyldiphenylsilyloxy)-1,2-(ethylenedioxy)-
cyclopentane (14)
A mixture of 13 (1.2 g, 3.0 mmol), p-toluenesulfonyl chloride
(1.3 mg, 6.6 mmol), pyridine (1.2 mL, 15 mmol) and a catalytic
amount of N,N-dimethylaminopyridine in CH2Cl2 (40 mL) was
◦
stirred at 23 C for 24 h. The reaction mixture was treated with
1 N HCl and the aqueous phase was extracted with CH2Cl2. The
organic extracts were dried (Na2SO4) and the solvent removed.
Purification of the residue by flash-chromatography (1 : 1 EtOAc–
Hex) afforded the tosylated alcohol (990 mg, 60%) as a colorless
oil: IR nmax (NaCl; cm-1) 3104, 2992, 2691, 1598, 1359, 1177, 923
and 705; dH (300 MHz, CDCl3) 7.76 (2 H, d, J 8.4, ArH), 7.61
(4 H, d, J 7.8, ArH), 7.42–7.26 (8 H, m, ArH), 4.53–4.25 (1 H,
m, CHO), 4.15–4.09 (3 H, m, CHO, CH2O), 3.96–3.91 (1 H, m,
CHO), 3.68–3.62 (2 H, m, CH2O), 2.41 (3 H, s, CH3), 1.89–1.75
(4 H, m, 3-H2, 5-H2) and 1.03 [9 H, s, C(CH3)3]; dC (100 MHz,
CDCl3) 135.6, 134.0, 132.8, 129.8, 129.6, 127.9, 127.8, 127.6, 80.7,
71.3, 71.0, 69.0, 68.6, 67.0, 42.1, 38.7, 26.8, 21.6 and 18.9. To a
solution of the above product (150 mg, 0.27 mmol) in dry THF
(12 mL), NaH (60% in mineral oil, 22 mg, 0.54 mmol) was added
and the resulting suspension was heated under reflux for 30 min.
(5aa,7b,8aa)-7-(4-nitrophenoxycarbonyloxy)-
tetrahydrocyclopenta[f ]-1,3,5-trioxepane (18)
To
a solution of 10 (15 mg, 0.094 mmol) and N-
methylmorpholine (31 mL, 0.28 mmol) in dry THF (3 mL), p-
nitrophenylchloroformate (57 mg, 0.28 mmol) was added and the
resulting mixture was stirred at 23 ◦C. After 1 h, water was added,
the solvent was removed under reduced pressure and the aqueous
phase was extracted with CHCl3. The organic extracts were dried
(Na2SO4) and the solvent evaporated. The residue was purified by
flash-chromatography (1 : 4 EtOAc–CHCl3) to afford 18 (31 mg,
95%) as a pale yellow viscous oil: IR nmax (NaCl; cm-1) 2831, 2598,
1766, 1529, 1350, 1116 and 859; dH (300 MHz, CDCl3) 8.27 (2 H,
d, J 8.7, ArH), 7.38 (2 H, d, J 8.7, ArH), 5.34–5.31 (1 H, m, 7-H),
5.19 (2 H, d, J 6.9, 2-H¢, 4-H¢), 4.77 (2 H, d, J 6.9, 2-H¢¢, 4-H¢¢),
4.51–4.47 (2 H, m, 5a-H, 8a-H) and 2.38–2.26 (4 H, m, 6-H2, 8-
H2); dC (75 MHz, CDCl3) 155.3, 126.2, 125.3, 121.7, 115.6, 95.5,
81.2, 78.5 and 37.6; m/z (EI) 325 (M, 100).
◦
After cooling to 23 C, the reaction mixture was quenched with
a saturated solution of NH4Cl, the solvent was removed and the
aqueous phase was extracted with EtOAc. The organic extracts
were dried (Na2SO4) and the solvent was removed in vacuo. The
residue was purified by flash-chromatography (1 : 3 EtOAc–Hex)
to afford 14 (82 mg, 80%) as a colorless oil: IR nmax (NaCl; cm-1)
2803, 1427, 1136, 957 and 703; dH (300 MHz, CDCl3) 7.65 (4 H,
d, J 7.8, ArH), 7.46–7.36 (6 H, m, ArH), 4.55–4.48 (1 H, m, 4-H),
4.18 (2 H, t, J 5.1, 1-H, 2-H), 3.70–3.62 (2 H, m, CH2O), 3.53–3.46
(2 H, m, CH2O), 2.16–2.07 (2 H, m, 3-H¢, 5-H¢), 1.82–1.74 (2 H,
m, 3-H¢¢, 5-H¢¢) and 1.06 [9 H, s, C(CH3)3]; dC (75 MHz, CDCl3)
135.6, 134.1, 129.5, 127.6, 75.2, 71.1, 62.2, 37.5, 27.0 and 19.1;
m/z (CI): 383.25 (M + H, 100).
(1b,2b,4b)-4-(4-Nitrophenoxycarbonyloxy)-1,2-
(1b,2b,4a)-4-Hydroxy-1,2-(ethylendioxy)cyclopentane (15)
(methylenedioxy)cyclopentane (19)
The above compound was deprotected as described for 9 to afford
15 in 90% yield as a colorless oil: IR nmax (NaCl; cm-1) 3013, 2797,
2550, 1129 and 664; dH (300 MHz, CDCl3) 4.58–4.51 (1 H, m,
4-H), 4.17 (2 H, t, J 4.8, 1-H, 2-H), 3.78–3.71 (2 H, m, CH2O),
3.58–3.51 (2 H, m, CH2O), 2.34–2.25 (2 H, m, 3-H¢, 5-H¢) and
1.72–1.66 (3 H, m, 3-H¢¢, 5-H¢¢, OH); dC (75 MHz, CDCl3) 75.1,
69.6, 62.3 and 37.2; m/z (EI) 144 (M, 100).
The title compound 19 was obtained from 11 as described for
18 in 81% yield. Flash-chromatography was performed using 1 :
1 EtOAc–Hex: IR nmax (NaCl; cm-1) 2739, 1764, 1527, 1348 and
1204; dH (300 MHz, CDCl3) 8.27 (2 H, d, J 5.1, ArH), 7.38 (2 H,
d, J 5.1, ArH), 5.20–5.16 (2 H, m, OCH2O), 4.83–4.81 (1 H, m,
4-H), 4.68 (2 H, d, J 5.7, 1-H, 2-H), 2.38 (2 H, d, J 14.7, 3-H¢,
5-H¢) and 2.11–2.02 (2 H, m, 3-H¢¢, 5-H¢¢); dC (100 MHz, CDCl3)
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155.4, 126.1, 125.2, 121.8, 115.5, 95.0, 80.7, 80.4 and 38.4; m/z
(CI) 296 (M + H, 100); HRMS (M + H)+ calcd for C13H14NO7,
296.0770; found, 296.0769.
d, J 5.4, 1-H, 2-H), 3.87 (3 H, s, OCH3), 3.79 (2 H, m, CHN,
CHOH), 3.10–2.76 (6 H, m, CH2N, CH2Ph), 2.11–1.80 [5 H, m,
3-H2, 5-H2, CH(CH3)2], 0.90 (3 H, d, J 6.6, CHCH3) and 0.86 (3
H, d, J 6.6, CHCH3); dC (75 MHz, CDCl3) 162.9, 155.3, 137.5,
129.9, 129.6, 129.3, 128.5, 126.4, 114.3, 94.7, 80.5, 74.2, 72.3, 58.8,
55.6, 54.9, 53.8, 38.5, 35.4, 27.3, 20.2 and 19.9; m/z (ES) 563
(M + H, 100); HRMS (M + H)+ calcd For C28H39N2O8S, 563.2427;
found, 563.2406.
(5aa,7a,8aa)-7-(4-Nitrophenoxycarbonyloxy)-
tetrahydrocyclopenta[f ]-1,3,5-trioxepane (20)
The title compound was obtained from 12 as described for 18
in 94% yield. Flash-chromatography was performed using 1 : 6
EtOAc–CHCl3: IR nmax (NaCl; cm-1) 2587, 1765, 1594, 1528, 1349
and 858; dH (300 MHz, CDCl3) 8.25 (2 H, d, J 8.0, ArH), 7.39 (2
H, d, J 8.0, ArH), 5.20 (2 H, d, J 7.5, 2-H¢, 4-H¢), 5.10–5.02 (1
H, m, 7-H), 4.75 (2 H, d, J 7.5, 2-H¢¢, 4-H¢¢), 4.29–4.24 (2 H, m,
5a-H, 8a-H), 2.51–2.41 (2 H, m, 6-H¢, 8-H¢) and 2.25–2.17 (2 H,
m, 6-H¢¢, 8-H¢¢); dC (75 MHz, CDCl3) 155.2, 126.2, 125.2, 121.7,
115.6, 94.6, 80.8, 76.6 and 36.9; m/z (CI) 324 (M - H, 100).
(1¢S,2¢R)-{1¢-Benzyl-2¢-hydroxy-3¢-[isobutyl(4-
methoxybenzenesulfonyl)amino]propyl} carbamic acid
(1b,2b,4a)-1,2-(methylenedioxy)cyclopent-4-yl ester (26)
A solution of 24 (40 mg, 0.079 mmol) in 30% trifluoroacetic acid
in CH2Cl2 (6 mL) was stirred at 23 ◦C for 40 min and then the
solvent was removed under reduced pressure. The residue was
dissolved in CH2Cl2 (4 mL), a solution of 17 (23 mg, 0.1 mmol)
in CH2Cl2 (2 mL) and diisopropylethylamine (140 mL, 0.8 mmol)
were added. After 2 h the organic phase was washed with water,
dried (Na2SO4) and evaporated. The residue was purified by flash-
chromatography (1 : 1 EtOAc–Hex) to afford 26 (34 mg, 76%) as
(1b,2b,4a)-4-(4-Nitrophenoxycarbonyloxy)-1,2-
(ethylenedioxy)cyclopentane (21)
The title compound was obtained from 15 as described for 18
in 81% yield. Flash-chromatography was performed using 1 : 4
EtOAc–CHCl3: IR nmax (NaCl; cm-1) 2655, 1757, 1592, 1503, 1337,
852 and 754; dH (300 MHz, CDCl3) 8.29 (2 H, d, J 7.3, ArH), 7.36
(2 H, d, J 7.3, ArH), 5.22–5.18 (1 H, m, 4-H), 3.86–384 (2 H, m,
1-H, 2-H), 3.78–3.63 (4 H, m, CH2O), 2.38–2.24 (4 H, m, 3-H2, 5-
H2); dC (100 MHz, CDCl3) 161.8, 126.2, 125.3, 121.7, 115.6, 78.1,
74.3, 62.1 and 33.9; m/z (CI) 310 (M + H, 100).
20
a white foam: [a] +3.6 (c 1.3 in CH2Cl2); IR nmax (NaCl; cm-1)
DP
3216, 2801, 2670, 1712, 1597, 1497, 1154 and 755; dH (300 MHz,
CDCl3) 7.70 (2 H, d, J 8.7, ArH), 7.32–7.21 (5 H, m, ArH), 7.00
(2 H, d, J 8.7, ArH), 5.06 (1 H, t, J 7.0, 4-H), 4.93 (1 H, s,
OCHHO), 4.76 (1 H, d, J 8.4, NH), 4.71 (1 H, s, OCHHO), 4.52
(2 H, m, 1-H, 2-H), 3.87 (3 H, s, OCH3), 3.84 (2 H, m, CHN,
CHOH), 3.11 (1 H, dd, J 8.0, 14.8, CHHN), 3.04–2.91 (4 H, m,
CHHN, CH2N, CHHPh), 2.78 (1 H, dd, J 6.7, 13.1, CHHPh),
2.17–2.10 (2 H, m, 3-H¢, 5-H¢), 1.86–1.58 [3 H, m, 3-H¢¢, 5-H¢¢,
CH(CH3)2], 0.91 (3 H, d, J 6.6, CHCH3) and 0.87 (3 H, d, J 6.9,
CHCH3); dC (75 MHz, CDCl3) 162.9, 155.8, 137.6, 129.9, 129.7,
129.4, 128.4, 126.5, 114.3, 94.3, 78.5, 74.5, 72.6, 58.8, 55.6, 54.9,
53.7, 37.8, 35.3, 27.3, 20.2 and 19.9; m/z (ES) 585 (M + Na, 100);
HRMS (M + Na)+ calcd for C28H38N2NaO8S, 585.2247; found,
585.2228.
(1b,2b,4b)-4-(4-Nitrophenoxycarbonyloxy)-1,2-
(ethylenedioxy)cyclopentane (22)
The title compound was obtained from 16 as described for 18
in 95% yield. Flash-chromatography was performed using 1 : 4
EtOAc–CHCl3: IR nmax (NaCl; cm-1) 2588, 1725, 1594, 1222, 1109
and 773; dH (400 MHz, CDCl3) 8.27 (2 H, d, J 7.0, ArH), 7.38 (2
H, d, J 7.0, ArH), 5.14–5.10 (1 H, m, 4-H), 3.99 (2 H, t, J 4.6,
1-H, 2-H), 3.91–3.86 (2 H, m, CH2O), 3.64–3.59 (2 H, m, CH2O)
and 2.31–2.18 (4 H, m, 3-H2, 5-H2); dC (100 MHz, CDCl3) 162.5,
126.1, 125.2, 121.7, 115.5, 81.4, 74.3, 62.3 and 32.5; m/z (CI) 310
(M + H, 100).
(1S,2R)-{1¢-Benzyl-2¢-hydroxy-3¢-[isobutyl(4-
methoxybenzenesulfonyl)amino]propyl} carbamic acid
(5aa,7b,8aa)-tetrahydrocyclopenta[f ]-1,3,5-trioxaepan-7-yl ester
(27)
(1¢S,2¢R)-{1¢-Benzyl-2¢-hydroxy-3¢-[isobutyl(4-
methoxybenzenesulfonyl)amino]propyl} carbamic acid
(1b,2b,4b)-1,2-(methylenedioxy)cyclopent-4-yl ester (2)
The title compound was obtained from 24 and 18 as described for
2 in 43% yield. Flash-chromatography was performed with 1 : 4
EtOAc–CHCl3: [a]D20P +5.2 (c 1.7 in CH2Cl2); IR nmax (NaCl; cm-1)
3118, 2825, 2656, 1712, 1596, 1012 and 771; dH (300 MHz, CDCl3)
7.70 (2 H, d, J 9.0, ArH), 7.32–7.21 (5 H, m, ArH), 6.98 (2 H, d,
J 9.0, ArH), 5.15 (2 H, d, J 7.2, 2-H¢, 4-H¢), 5.05 (1 H, br. s, NH),
4.76 (1 H, d, J 8.4, 7-H), 4.68 (2 H, d, J 7.2, 2-H¢¢, 4-H¢¢), 4.32–
4.23 (2 H, m, 5a-H, 8a-H), 3.87 (3 H, s, OCH3), 3.83–3.80 (2 H, m,
CHN, CHOH), 3.10 (1 H, dd, J 8.4, 15.3, CHHN), 3.04–2.88 (4 H,
m, CHHN, CH2N, CHHPh), 2.78 (1 H, dd, J 6.9, 13.5, CHHPh),
2.09–1.94 (4 H, m, 6-H2, 8-H2), 1.86–1.77 [1 H, m, CH(CH3)2],
0.91 (3 H, d, J 6.9, CHCH3) and 0.87 (3 H, d, J 6.3, CHCH3); dC
(75 MHz, CDCl3) 163.0, 155.7, 137.6, 129.8, 129.7, 129.4, 128.4,
126.5, 114.3, 95.4, 81.5, 73.6, 72.7, 58.8, 55.7, 54.9, 53.7, 37.8, 35.4,
27.3, 20.2 and 19.9; m/z (ES) 615 (M + Na, 100); HRMS (M +
Na)+ calcd for C29H40N2NaO9S, 615.2353; found, 615.2361.
A solution of 24 (25 mg, 0.05 mmol) in 30% trifluoroacetic acid
in CH2Cl2 (4 mL) was stirred at 23 ◦C for 40 min and then the
solvent was removed under reduced pressure. The residue was
dissolved in THF (3 mL), a solution of 19 (18 mg, 0.059 mmol)
in THF (1 mL) and diisopropylethylamine (100 mL, 0.6 mmol)
were added. After 24 h the organic phase was diluted with CHCl3,
washed with water, dried (Na2SO4), and evaporated. The residue
was purified by flash-chromatography eluting with a 1 : 1 mixture
of EtOAc and hexanes to afford 2 (20 mg, 74%) as a white solid:
[a]20 +4.5 (c 1.2 in CH2Cl2), mp 68 ◦C (from EtOAc–Hex); IR
DP
nmax (NaCl; cm-1) 3129, 2801, 2660, 1711, 1597, 1497, 1155 and
761; dH (300 MHz, CDCl3) 7.71 (2 H, d, J 8.8, ArH), 7.32–7.19
(5 H, m, ArH), 6.98 (2 H, d, J 8.8, ArH), 5.01 (1 H, s, OCHHO),
4.92 (1 H, br. s, NH), 4.80 (2 H, m, 4-H, OCHHO), 4.57 (2 H,
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H, m, CH(CH3)2], 0.91 (3 H, d, J 6.6, CHCH3) and 0.86 (3 H, d,
J 6.6, CHCH3); dC (75 MHz, CDCl3) 163.1, 156.1, 137.6, 129.8,
129.6, 129.5, 128.5, 126.6, 114.4, 74.6, 73.2, 72.7, 62.2, 58.8, 55.7,
54.9, 53.8, 35.4, 34.3, 34.2, 27.3, 20.2 and 19.9; m/z (ES) 599 (M +
Na, 100); HRMS (M + H)+ calcd for C29H40N2NaO8S, 599.2403;
found, 599.2421.
(1¢S,2¢R)-{1¢-Benzyl-2¢-hydroxy-3¢-[isobutyl(4-
methoxybenzenesulfonyl)amino]propyl} carbamic acid
(5aa,7a,8aa)-tetrahydrocyclopenta[f ]-1,3,5-trioxaepan-7-yl ester
(28)
The title compound was obtained from 24 and 20 as described for
2 in 42% yield. Flash-chromatography was performed with 1 : 1
20
EtOAc–Hex: [a] +7.3 (c 1.7 in CH2Cl2); IR nmax (NaCl; cm-1)
DP
(1¢S,2¢R)-{1¢-Benzyl-2¢-hydroxy-3¢-[isobutyl(4-
3117, 2801, 2707, 1711, 1596, 1260 and 1153; dH (300 MHz,
CDCl3) 7.70 (2 H, d, J 8.7, ArH), 7.31–7.21 (5 H, m, ArH), 6.97
(2 H, d, J 8.7, ArH), 5.14 (2 H, d, J 6.9, 2-H¢, 4-H¢), 4.91 (1 H, d, J
7.8, NH), 4.83–4.78 (1 H, m, 7-H), 4.68 (2 H, d, J 6.9, 2-H¢¢, 4-H¢¢),
4.15–4.10 (2 H, m, 5a-H, 8a-H), 3.87 (3 H, s, OCH3), 3.81–3.83
(2 H, m, CHN, CHOH), 3.12–2.85 (5 H, m, 2 ¥ CH2N, CHHPh),
2.77 (1 H, dd, J 6.9, 13.5, CHHPh), 2.34–2.21 (2 H, m, 6-H¢, 8-H¢),
1.94–1.76 [3 H, m, 6-H¢¢, 8-H¢¢, CH(CH3)2], 0.90 (3 H, d, J 6.6,
CHCH3) and 0.86 (3 H, d, J 6.6, CHCH3); dC (100 MHz, CDCl3)
162.9, 156.1, 137.5, 129.8, 129.5, 129.4, 128.4, 126.4, 114.3, 94.8,
81.0, 72.3, 71.3, 58.6, 55.6, 55.0, 53.6, 37.1, 35.5, 27.1, 20.1 and
19.8; m/z (ES) 615 (M + Na, 100); HRMS (M + Na)+ calcd for
C29H40N2NaO9S, 615.2353; found, 615.2349.
(hydroxymethyl)benzenesulfonyl)amino]propyl} carbamic acid
(1b,2b,4b)-1,2-(methylenedioxy)cyclopent-4-yl ester (30)
To a solution of 258 (40 mg, 0.1 mmol) and diisopropylethylamine
(150 mL, 0.9 mmol) in THF (3 mL), a solution of 17 (30 mg,
0.11 mmol) was added and the resulting mixture was stirred at
23 ◦C. After 48 h, the organic phase was diluted with CHCl3,
washed with water, dried (Na2SO4) and evaporated. The residue
was purified by flash-chromatography (2 : 1 EtOAc–Hex) to afford
20
DP
30 (35 mg, 63%) as an amorphous solid: [a] +7.8 (c 1.3 in CHCl3);
IR nmax (NaCl; cm-1) 3042, 2996, 2707, 1710, 1530, 1334, 1156 and
755; dH (400 MHz, CDCl3) 7.77 (2 H, d, J 8.1, ArH), 7.52 (2 H,
d, J 8.1, ArH), 7.32–7.21 (5 H, m, ArH), 5.00 (1 H, s, NH), 4.92
(1 H, m, 4-H), 4.82–4.80 (4 H, m, OCH2O, CH2OH), 4.58–4.57
(2 H, m, l-H, 2-H), 3.81–3.79 (2 H, m, CHN, CHOH), 3.11–2.83
(6 H, m, 2 ¥ CH2N, CH2Ph), 6H), 2.10–1.82 [5 H, m, 3-H2, 5-H2,
CH(CH3)2], 0.91 (3 H, d, J 6.6, CHCH3) and 0.83 (3 H, d, J 6.6,
CHCH3); dC (100 MHz, CDCl3) 156.2, 146.2, 137.5, 137.1, 129.5,
128.5, 127.5, 127.1, 126.5, 94.6, 80.5, 75.8, 72.2, 64.0, 58.5, 55.1,
53.5, 38.4, 35.4, 27.1, 20.0 and 19.8; m/z (ES) 585 (M + Na, 100);
HRMS (M +Na)+ calcd for C28H38N2NaO8S, 585.2247; found,
585.2246.
(1¢S,2¢R)-{1¢-Benzyl-2¢-hydroxy-3¢-[isobutyl(4-
methoxybenzenesulfonyl)amino]propyl} carbamic acid
(1b,2b,4b)-1,2-(ethylenedioxy)cyclopent-4-yl ester (3)
The title compound was obtained from 24 and 22 as described for
2 in 40% yield. Flash-chromatography was performed with 1 : 1
20
EtOAc–Hex: [a] +6.9 (c 0.7 in CH2Cl2); IR nmax (NaCl; cm-1)
DP
3120, 2788, 2656, 2542, 1712, 1596, 1259, 1154 and 755; dH
(500 MHz, CDCl3) 7.70 (2 H, d, J 9.0, ArH), 7.31–7.22 (5 H,
m, ArH), 6.97 (2 H, d, J 9.0, ArH), 4.90–4.86 (2 H, m, NH, 4-H),
3.87 (3 H, s, OCH3), 3.85–3.79 (7 H, m, 2 ¥ CH2O, 1-H, 2-H,
OH), 3.57–3.54 (2 H, m, CHN, CHOH), 3.11 (1 H, dd, J 8.2, 14.7,
CHHN), 3.03–2.88 (4 H, m, CHHN, CH2N, CHHPh), 2.78 (1 H,
dd, J 6.7, 13.2, CHHPh), 2.17–2.08 (2 H, m, 3-H¢, 5-H¢), 1.98–
1.95 (2 H, m, 3-H¢¢, 5-H¢¢), 1.90 [1 H, dt, J 5.2, 15.0, CH(CH3)2],
0.91 (3 H, d, J 6.5, CHCH3) and 0.86 (3 H, d, J 6.5, CHCH3); dC
(75 MHz, CDCl3) 163.0, 156.2, 137.6, 129.8, 129.6, 129.5, 128.5,
126.5, 114.3, 74.5, 73.2, 72.5, 71.8, 62.5, 62.3, 58.8, 55.6, 55.0, 53.8,
35.5, 33.8, 33.5, 27.3, 20.2 and 19.9; m/z (ES) 599 (M + Na, 100);
HRMS (M + Na)+ calcd for C29H40N2NaO8S, 599.2403; found,
599.2394.
X-Ray crystallography. The HIV-1 protease construct with the
substitutions Q7K, L33I, L63I, C67A, and C95A to optimize
protein stability,20 was expressed and purified as described.21
Crystals were grown by the hanging drop vapor diffusion method
using a 1 : 15 molar ratio of protease at 2.0 mg mL-1 and the
inhibitor dissolved in dimethylsulfoxide. The reservoir contained
0.1 M sodium acetate buffer (pH = 4.2) and 1.5 M NaCl. Crystals
were transferred into a cryoprotectant solution containing the
reservoir solution and 20–30% (v/v) glycerol, mounted on a nylon
loop and flash-frozen in liquid nitrogen. X-ray diffraction data
were collected on the SER-CAT beamline of the Advanced Photon
Source, Argonne National Laboratory. Diffraction data were pro-
cessed using HKL200022 resulting in a Rmerge value of 7.0% (41.8%)
˚
for 90 315 unique reflections between 50 and 1.07 A resolution with
(1¢S,2¢R)-{1¢-Benzyl-2¢-hydroxy-3¢-[isobutyl(4-
a completeness of 88.1% (51.3%), where the values in parentheses
are for the final highest resolution shell. Data were reduced in space
methoxybenzenesulfonyl)amino]propyl} carbamic acid
(1b,2b,4a)-1,2-(ethylenedioxy)cyclopent-4-yl ester (29)
˚
˚
group P21212 with unit cell dimensions of a = 58.00 A, b = 86.34 A
˚
The title compound was obtained from 24 and 21 as described for
2 in 40% yield. Flash-chromatography was performed with 1 : 1
and c = 45.83 A with one dimer in the asymmetric unit. The
structure was solved by molecular replacement using the CPP4i
suite of programs,23,24 with the structure of the D30N mutant
of HIV protease in complex with GRL-98065 (2QCI)19 as the
starting model. The structure was refined using SHELX9725 and
refitted manually using the molecular graphics programs O26 and
COOT.27 Alternate conformations were modeled for the protease
residues when obvious in the electron density maps. Anisotropic
atomic displacement parameters (B-factors) were refined for all
atoms including solvent molecules. Hydrogen atoms were added
at the final stages of the refinement. The identity of ions and
other solvent molecules from the crystallization conditions was
20
EtOAc–Hex: [a] +8.2 (c 1.0 in CH2Cl2); IR nmax (NaCl; cm-1)
DP
3121, 2706, 1711, 1596, 1260, 1154 and 757; dH (500 MHz, CDCl3)
7.70 (2 H, d, J 8.7, ArH), 7.31–7.28 (2 H, m, ArH), 7.24–7.22 (3
H, m, ArH), 6.98 (2 H, d, J 8.7, ArH), 5.09 (1 H, br. s, NH), 4.74
(1 H, d, J 8.0, 4-H), 4.06–4.01 (2 H, m, 1-H, 2-H), 3.87 (3 H, s,
OCH3), 3.82–3.81 (2 H, m, CH2O), 3.75–3.71 (2 H, m, CH2O),
3.55–3.51 (2 H, m, CHN, CHOH), 3.10 (1 H, dd, J 15.0, 8.5,
CHHN), 3.03–2.86 88 (4 H, m, CHHN, CH2N, CHHPh), 2.78 (1
H, dd, J 13.5, 6.5, CHHPh), 2.32–2.23 (2 H, m, 3-H¢, 5-H¢), 1.81
(1 H, q, J = 6.5, 3-H¢¢), 1.79–1.68 (1 H, m, 5-H¢¢), 1.62–1.53 [1
3712 | Org. Biomol. Chem., 2008, 6, 3703–3713
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deduced from the shape and peak height of the 2Fo–Fc and Fo–Fc
electron density, the hydrogen bond interactions and interatomic
distances. The solvent structure was refined with one sodium ion,
three chloride ions, and 203 water molecules including partial
occupancy sites. The final Rwork was 15.2% and RBfree was 17.7%
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˚
for all data between 10 and 1.07 A resolution. The rmsd values
˚
˚
from ideal bonds and angle distances were 0.015 A and 0.034 A,
2
˚
respectively. The average B-factor was 13.1 and 18.2 A for
protease main chain and side chain atoms, respectively, 12.5 A for
inhibitor atoms and 24.0 A for solvent atoms. The X-ray crystal
2
˚
2
˚
structure of the inhibitor 3 complex with the HIV-1 protease has
been deposited in the Protein Databank (PDB)28 with an access
code of 3DKJ.
Acknowledgements
The research was supported by grants from the National Institutes
of Health (GM53386, AKG, and GM62920, IW). This work
was also supported by the Intramural Research Program of the
Center for Cancer Research, National Cancer Institute, National
Institutes of Health and in part by a Grant-in-aid for Scientific Re-
search (Priority Areas) from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (Monbu Kagakusho), a
Grant for Promotion of AIDS Research from the Ministry of
Health, Welfare, and Labor of Japan (Kosei Rohdosho: H15-
AIDS-001), and a Grant to the Cooperative Research Project
on Clinical and Epidemiological Studies of Emerging and Re-
emerging Infectious Diseases (Renkei Jigyo: No. 78, Kumamoto
University) of Monbu-Kagakusho.
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This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 3703–3713 | 3713
©