Design and synthesis of HIV-1 protease inhibitors. Novel tetrahydrofuran P2/P2′-groups interacting with Asp29/30 of the HIV-1 protease. Determination of binding from X-ray crystal structure of inhibitor protease complex

Article abstract of DOI:10.1016/S0968-0896(02)00535-7

A series of HIV-1 protease inhibitors having new tetrahydrofuran P2/P2′ groups have been synthesised and tested for protease inhibition and antiviral activity. Six novel 4-aminotetrahydrofuran derivatives were prepared starting from commercially available

Full text of DOI:10.1016/S0968-0896(02)00535-7

Bioorganic & Medicinal Chemistry 11 (2003) 1107–1115
Design and Synthesis of HIV-1 Protease Inhibitors. Novel
Tetrahydrofuran P2/P20-Groups Interacting with Asp29/30 of the
HIV-1 Protease. Determination of Binding from X-ray Crystal
Structure of Inhibitor Protease Complex
Karin Oscarsson,a Martina Lahmann,a Jimmy Lindberg,b Jussi Kangasmetsa,c
Torsten Unge,b Stefan Oscarson,a Anders Hallbergd and Bertil Samuelssona,c,*
aDepartment of Organic Chemistry, Arrhenius Laboratory, Floor 6, Stockholm University, S-106 91 Stockholm, Sweden
bDepartment of Cell and Molecular Biology, BMC, Uppsala University, Box 596, S-75124 Uppsala, Sweden
cMedivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden
dDepartment of Organic Pharmacutical Chemistry, BMC, Uppsala University, Box 574, S-75123 Uppsala, Sweden
Received 2 August 2002; accepted 18 August 2002
Abstract—A series of HIV-1 protease inhibitors having new tetrahydrofuran P2/P20 groups have been synthesised and tested for
protease inhibition and antiviral activity. Six novel 4-aminotetrahydrofuran derivatives were prepared starting from commercially
available isopropylidene-a-d-xylofuranose yielding six symmetrical and six unsymmetrical inhibitors. Promising sub nanomolar
HIV-1 protease inhibitory activities were obtained. The X-ray crystal structure of the most potent inhibitor (23, Ki 0.25 nM) co-
crystallised with HIV-1 protease is discussed and the binding compared with inhibitors 1a and 1b.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
regimes is a major concern. Moreover, the high costs of
treatment hinder the widespread use of the currently
approved HIV-1 protease inhibitors. Not withstanding
the major advancements made with current HIV-1 pro-
tease inhibitors there is a vast need for improved and
cost-effective protease inhibitors.17,18 We now report on
the design, synthesis, and antiviral activity of HIV-1
inhibitors containing a C2-symmetrical core structure
exemplified by lead compounds 1a and 1b (Fig. 1). It
has been shown13,19,20 that the conformationally con-
strained P2/P20 amino indanol of 1a20 and 1b21 effec-
tively occupies the lipophilic S2/S20 pockets of the HIV-
1 protease and also forms a hydrogen bond with the
Asp 29 backbone of the enzyme. This concept has
resulted in numerous very potent inhibitors and in the
clinically approved drug indinavir. However, the amino
indanol group is susceptible to hCYP450 metabolism,
that is, 3A4, which limits oral bioavailability of inhibi-
tors having this group.22 To overcome the potential lia-
bility of aromatic and benzylic CYP450 oxidation and
to gain affinity from hydrogen bond formation to both
of Asp29 and Asp 30 in the HIV-1 proteases we have
designed and synthesised new non-aromatic P2/P20
Since the discovery of the human immunodeficiency
virus (HIV) as the etiologic agent of AIDS, almost two
decades ago, several drug targets have been identified
from the HIV-1 genome.1,2 The pol gene of HIV-1
encodes for an aspartic protease,3,4 which was shown to
be essential for delivery of structural and functional
proteins by proteolytic processing of the gag- and gag-
pol viral gene products.3,5 Inhibition of this aspartic
protease results in the production of non-infectious vir-
ions and thus constitutes an ideal drug target.6ꢀ9 Today
six protease inhibitors are approved by the FDA for
treatment of HIV-1 infection: saquinavir,10 ritonavir,11
indinavir,12,13 nelfinavir,14 amprenavir,15 and lopina-
vir.16 Due to the rapid turnover of HIV-1 and the high
frequency of mutations in the genome, selection of
mutant strains conferring clinical resistance to the
HAART (Highly Active Anti Retroviral Therapy)
*Corresponding author. Tel.: +46-8-6083104; fax: +46-8-6083199;
e-mail: bertil.samuelsson@medivir.se
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00535-7

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K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 1107–1115
novel aminotetrahydrofurans have been synthesised
from which both symmetrical and unsymmetrical pro-
tease inhibitors have been prepared and tested for
enzyme inhibition and antiviral activity. The most
potent compound (23) in this new series was co-crystal-
lised with HIV-1 PR and the X-ray structure was deter-
mined. The result was compared with the X-ray
structures of 1a and 1b to support the structure activity
relationship.
Results and Discussion
Chemistry
2(S)-Hydroxymethylene-4(S)-tert-butoxycarbonylamino-
tetrahydrofuran (10) was identified as the pivotal inter-
mediate for the synthesis of protected amines 11–13, 15,
and 16, and a straightforward and short synthesis of 10
was developed (Schemes 1 and 2). Intermediate 9 was
prepared from 5 according to Bolon et al.24 but mod-
ified resulting in improvements of the overall yield from
28 to 78%.
Figure 1. Lead compounds 1a and 1b containing the C2-symmetrical
core structure.
tetrahydrofurans and coupled these to the C2-symmetric
l-mannaric P1/P10 template.20 From molecular model-
ling we envisaged that 2-alkoxymethyl-4-aminotetrahy-
drofurans should form two hydrogen bonds, one to
each of the backbones of Asp29/30. Previous work on
for example, ‘bis THFs’23 has shown that potent inhi-
bitors can result from utilising these hydrogen bond
interactions.
The 1,2-O-Isopropylidene-a-d-xylofuranose 5 was pro-
tected at the primary hydroxyl group giving the toloyl
ester 6 as a crystalline solid.25 Deoxygenation of the 3-
hydroxyl group according to Barton–McCombie condi-
tions by reduction of the corresponding 3-O-imidazol
thiocarbonyl ester using AIBN/Bu3SnH, gave 7 in
almost quantitative yield.24 Compound 7 was then
For the synthesis of the 2-alkoxymethyl-4-aminote-
trahydrofurans d-xylose was used as starting material.
The synthesised aminotetrahydrofurans were coupled to
the C2-symmetric P1/P10 backbone using either the
activated l-mannaric acid 2 or the bislactone 3 (Fig. 2),
delivering symmetrical inhibitors.20
Previous work has highlighted that in contrast to the
synthesis of symmetrical inhibitors, unsymmetrical
inhibitors are not readily available using this general-
methodology20 but could be prepared using solid sup-
port methodology.21 We now report on a new solution
phase synthesis of the key intermediate monolactone 4
(Fig. 2), from readily available 3, which provides facile
access to these unsymmetrical inhibitors. In summary,
Scheme 1.
Figure 2. The activated l-mannaric acid, the bislactone and mono-
lactone used for preparation of the symmetrical and unsymmetrical
inhibitors.
Scheme 2.

K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 1107–1115
1109
directly converted into the dideoxy compound 8 using
Et3SiH and BF3.OEt2 at 0 ꢁC in 85% yield.24,26 Inver-
sion of configuration at C-2 was subsequently achieved
employing Mitsunobu conditions,27 using triphenyl-
phosphine, diisopropyl azodicarboxylate (DIAD) and
diphenylphosphonic azide (DPPA) giving the azido
derivative 9 in quantitative yield. Reduction of the azide
9 with Pd/C (H2) in the presence of Boc2O followed by
removal of the toluoate under Zemplen conditions gave
10 in 74% yield.28 Thus the synthesis of target inter-
mediate tetrahdrofuran 10 could be achieved in an
overall yield of 57% from compound 5.
methodology was developed for the preparation of
monolactone 4 and the subsequent transformation to
unsymmetrical inhibitors 23–28. The mono-opening
reaction of bislactone 3 could be achieved with (1S, 2R)-
(ꢀ)-1-amino-2-indanol in dichloromethane utilising 2-
hydroxypyridine21 as catalyst resulting in amide 4 (Fig.
1), which was isolated in 40% yield. The major side
reactions, b-elimination and aminolysis of the second
lactone, was surpressed by selection of the appropriate
solvent and catalyst. The ammonium salts of 10–13, 15
and 16 (Scheme 4) were coupled with the monolactone
4 in refluxing dichloroethane in the presence of trie-
thylamine to give the unsymmetrical products 23
(29%), 24 (34%), 25 (28%), 26 (47%), 27 (44%), and 28
(49%).
Employing Williamson conditions,29 the alkylated deri-
vatives 11 (78%), 12 (87%), and 13 (87%) were
obtained (Scheme 2). Attempted oxidation of 10 using
the Dess–Martin reagent failed to give the correspond-
ing aldehyde 14. However, Swern oxidation of 10
employing oxalyl chloride in DMSO delivered the alde-
hyde albeit in a moderate yield, 51%. Grignard alkyl-
ations of aldehyde 14 provided derivatives 15 and 16
(Scheme 2).30 Initially, the Grignard reactions were
performed at ꢀ60 ꢁC to+10 ꢁC in THF, which only
resulted in recovered starting material. However, adding
the reagents at 0 ꢁC in Et2O and allowing the mixture to
attain room temperature overnight furnished com-
pounds 15 (59%) and 16 (48%) as diastereomeric mix-
tures (approximately 1:1), no chelation is observed in
this case.31
Anti-HIV-1 activity and 3-D structure
Notably from examination of Tables 1 and 2 there are
some very potent unsymmetrical inhibitors with enzyme
inhibition values in the sub nanomolar range; i.e., 23 Ki
0.25 nM, 25 Ki 0.74 nM and 27 Ki 0,54 nM. In contrast
to the unsymmetrical inhibitors, the symmetrical inhi-
bitors (17–22) are considerable less potent, one expla-
nation being that the lipophilic P2 indanol moiety, for
this P1/P10 core template, gives an excellent fit to the S2
pocket of the enzyme which cannot be achieved for the
less lipophilic tetrahydrofurans. This is confirmed by
examination of the X-ray crystal structures, vide infra.
It is interesting to note that in inhibitor 23, the modelled
hydrogen bonds to Asp29 and Asp30 were observed in
the X-ray crystal structure. However, the added affinity
from hydrogen bond formation from inhibitor hydro-
xymethylene group to backbone NH of Asp30 in the
HIV-1 PR is offset by not optimally filling of the S2
pocket. In spite of the high potency at the enzyme level,
the cellular antiviral activities observed were surpris-
ingly low, even for the most potent unsymmetrical inhi-
bitors. For example compound 23 exhibited an ED50
value of 1.1 mM and was consequently considerable less
potent than inhibitors 1a and 1b, both showing an ED50
value of 0.1 mM.
For the preparation of symmetrical inhibitors 17–22
(Scheme 3) the Boc group in compounds 10–13, 15 and
16 was deprotected using HCl (concd) in EtOAc. The
resulting ammonium salts were immediately coupled
with disuccinimidylester 220 producing the iso-
propylidene protected l-mannaric bisamides. After
cleavage of the isopropylidene group the symmetrical
inhibitors 17 (47%), 18 (37%), 19 (77%),20 (77%), 21
(62%), and 22 (47%) were obtained.
Attempts to couple the amines directly to the benzylated
bislactone 3 failed, resulting in b-elimination of 3 and
unreacted starting material (TLC examinations). A new
Scheme 3.
Scheme 4.

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K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 1107–1115
Table 2. HIV-1 protease inhibitory activities for compounds 23–28
Table 1. HIV-1 protease inhibitory activities for compounds 17–22
Comp.
R1
R2
Ki (nM)
ED50 (mM)
Compd
R1
R2
Ki (nM)
ED50 (mM)
23
24
25
26
27
28
H
Allyl
Me
Et
H
H
H
H
H
H
Me
i-Pr
0.25
11.30
0.74
3.00
0.54
1.60
1.12
5.45
3.10
4.60
6.10
3.75
17
18
19
20
21
22
H
Allyl
Me
Et
H
H
H
H
H
H
Me
i-Pr
4.80
>5000
103
>5000
208
1590
>10
>10
>10
>10
>10
>10
Inhibitor 23 was co-crystallised with HIV-1 PR, and the
structure was determined. The binding to the HIV-1 PR
was then compared with inhibitors 1a,20 and 1b.32
group could be observed (Fig. 3). However, the three
different P2 substituents adopt binding modes distinct
from each other. Mainly the size and charge distribution
and the lining residues of the S2-subsite guide the posi-
tion of the P2-substituents. The P2 indanol in the sym-
metric compound 1a occupies a major part of the S2-
subsite volume with optimal vdW contacts to Ala28,
Val32, Ile47 and Leu76. In contrast, 1b and 23 exploit
the S2-subsite less effective, due to the smaller P2 sub-
stituents, which reduce the number of vdW contacts to
the S2 pocket. The reduced number of non-polar con-
tacts of compound 23 is compensated by formation of
an additional hydrogen bond from the free hydroxyl
group to the backbone nitrogen of Asp30 (3.0 A) (Fig. 4).
Similarly, the valine amide of 1b is involved in a hydro-
gen bond to the backbone carbonyl of Gly48 (2.9 A).
When associated to the active site, all three adopt a
similar overall binding conformation. This is clearly
apparent upon a structural superimposition of the three,
where only minor differences in position of the central
diols, P1/P10 benzyloxy substituents and the P20 indanol
All three compounds form a hydrogen bond with the
backbone nitrogen of Asp29 from either the indanol
hydroxyl, amide carbonyl or the furan ring oxygen (1a:
3.0A; 1b: 2.9 A; 23: 3.1 A). Small changes in position of
the side chains in the S2-subsite accommodated these
substantial differences in size and charge distribution of
the P2-substituent in these compounds.
Figure 3. Superimposition of compounds 1a (blue), 1b (gold) and 23
(green) bound to the active site of HIV-1 PR. The image was compiled
using Swiss-PDB viewer version 3.5133 and 3-D-rendered with POV-
ray version 3.1.34
Figure 4. Stereo view of the three compounds 1a (blue), 1b (gold) and 23 (green) bound to the S2-subsite of HIV-1 PR. This orientation visualizes
the interactions between the different substituents of the P2 arm and key residues in the S2-subsite. The hydrophobic contribution to the binding is
reflected in how effective the different substituent are filling out the S2-subsite. The image was compiled using Swiss-PDB viewer version 3.5133 and
3-D-rendered with POV-ray version 3.1.34

K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 1107–1115
1111
Furthermore, all three complexes reveal that one of the
hydroxyl group of the central diol points toward the
Asp25/125 residues in the active site and is hydrogen
bonded to both carboxyl oxygens. The other hydroxyl
group points away from the active site but is still
hydrogen bonded to one of the Asp residue. This
asymmetric interaction pattern of the diol is known for
this class of compounds.20
were performed under reduced pressure at <40 ꢁC (bath
temperature). Thin layer chromatography was per-
formed using silica gel 60 F-254 plates with detection by
UV and charring with 8% sulfuric acid. Silica gel
(0.040–0.063 mm) was used for column chromato-
graphy. Me4Si (0.0 ppm) was used as an internal stan-
dard in 1H NMR and Me4Si or CDCl3 (77.0 ppm) were
used in 13C NMR. Optical rotations were measured on
a Perkin–Elmer 241 polarimeter. Unless stated other-
wise, all materials were obtained from commercial sup-
pliers and used without further purification.
Conclusion
In summary, we have developed new tetrahydrofuran
P2 analogues exhibiting potent HIV-1 protease inhibit-
ing properties. Examination of the X-ray crystal struc-
ture of inhibitor 23 co-crystallized with HIV-1 PR
reveals that the hydroxymethyl group forms the pre-
dicted hydrogen bond to the backbone NH of Asp 30
accounting for the high potency of this inhibitor and
close analogues. In spite of the promising HIV-1 pro-
tease inhibition of this series of compounds, the anti-
viral activity in cell culture (ED50) was not improved
over inhibitor 1a and 1b (Fig. 1).20,21 However, our
results strongly support that optimisation of inhibitor
potency can be achieved by formation of hydrogen
bonds from inhibitor to NH groups of the HIV-1 pro-
tease backbone. The X-ray crystal structure of inhibitor
23 will provide the basis towards further optimisation
of this new inhibitor series with the aim to fully uti-
lise the S2/S20 pockets and to achieve high anti-viral
activity.
2R-O-Benzyl-4-[1R-O-benzyl-2N-(2R-hydroxy-1S-inda-
nyl)]ethylcarbamoyl-3S-hydroxy-1,4R-ꢀ-lactone
(4).
(1S,2R)-(ꢀ)-1-amino-2-indanol (0.5 g, 3.36 mmol) and
2-hydroxypyridine (27 mg, 0.28 mmol) were added to a
solution of 3 (1.0 g, 2.82 mmol) in CH2Cl2 (100 mL)
under N2 and refluxed for 15 h. The solution was con-
centrated to 20 mL and the residue was purified by silica
gel column chromatography (1,2-dichloroethane–
MeOH 49:1) to provide compound 4 as a white solid
(0.57 g, 1.13 mmol, 40%). [a]2D0 ꢀ33.1 (c 1.09, CH2Cl2)
1H NMR (300 MHz, CDCl3) d 7.42–7.04 (m, 14H), 5.29
(dd, 1H), 4.91–4.49 (m, 8H), 4.11 (s, 1H), 3.09 (dd, 1H),
2.90 (dd, 1H); 13C NMR (75 MHz, CDCl3) d 173.0,
169.8, 140.6, 139.9, 136.5, 136.4 128.7, 128.5, 128.4,
128.4, 127.1, 125.3, 124.3, 79.7, 77.2, 74.4, 74.3, 73.0,
72.7, 68.2, 57.6, 39.4.
3-Deoxy-1,2-O-isopropylidene-5-O-(4-methylbenzoyl)-ꢁ-
D-erythro-pentofuranose (7). A solution of 6 (20 g, 65
mmol) in dry dichloroethane (400 mL) was brought to
reflux. 1,10-Thiocarbonyldiimidazole (20 g, 112 mmol)
was added to the solution and the refluxing was con-
tinued for 1.5 h. The solvent was evaporated and the
residue was purified by silica gel column chromato-
graphy (toluene; toluene–EtOAc 2:1) to provide the
thiocarbonyl ester (26.6 g, 62.4 mmol, 96%) as a light
yellow solid. [a]2D0 ꢀ26.1 (c 0.56, CH2Cl2); 13C NMR
(75 MHz, CDCl3) d 184.6, 166.2, 144.4, 137.2, 131.6,
130.0, 129.4, 117.9, 113.0, 105.1, 84.5, 83.0, 76.9, 61.2,
26.8, 26.4, 21.9.
Experimental
Crystallography
The details of the crystallisation and structure determi-
nation will be published elsewhere. Briefly, the complex
of HIV-1 PR together with 23, 1a and 1b were crystal-
lised in space group P21212 and determined to 2.0 A
resolution with R-values of 0.19, 0.18 and 0.18 respec-
tively (Rfree 0.23, 0.20 and 0.21).
To a refluxing solution of Bu3SnH (36 mL, 134 mmol)
and a catalytic amount of AIBN in toluene (2000 mL), a
solution of thiocarbonyl ester (26 g, 62.1 mmol) in tol-
uene (400 mL) was added dropwise over a period of 1 h.
The reaction mixture was stirred at reflux for 1 h and
then at room temperature overnight. The solvent was
evaporated and the residue was purified by silica gel
column chromatography (toluene; toluene–EtOAc 6:1)
to provide 7 (18.2 g, 62.1 mmol, 100%) as a colourless
oil. 13C NMR (75 MHz, CDCl3) d 166.6, 144.0, 130.0,
129.3, 111.5, 106.0, 80.5, 76.1, 65.4, 35.7, 27.0, 26.4,
21.9.
HIV-1 protease inhibition
(Tables 1 and 2: column 4) HIV-1 protease was cloned
and heterologously expressed in Escherichia coli35 and
Ki-values were determined using a fluorometric assay.36
In vitro anti-HIV activity
(Tables 1 and 2; column 5) The anti-HIV activity was
measured in a HIV cytopathic assay in MT-4 cells
where the effect was quantified using vital dye XTT.37
The 50% inhibitory concentrations (ED50) were calcu-
lated from the percent cytoprotection for individual
compounds.
2(S)-(O-4-Methylbenzoyl-methyl)-tetrahydrofuran-4(R)-ol
(8). To a solution of 7 (6.0 g, 20.5 mmol) in CH2Cl2 (80
mL) at 0 ꢁC were added Et3SiH (30 mL, 187 mmol) and
.
BF3 OEt2 (23.6 mL, 186 mmol) and the reaction was
General
stirred at 0 ꢁC for 1 h. The reaction was quenched by
addition of ice cold saturated NaHCO3/H2O (60 mL).
The aqueous layer was extracted (CH2Cl2, 3ꢂ50 mL)
All glassware was dried over an open flame before use in
connection with an inert atmosphere. Concentrations

1112
K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 1107–1115
and the combined organic phase was washed (brine) and
dried (MgSO4). Purification by silica gel column chro-
matography (toluene–EtOAc 1:1) gave 8 (4.1 g, 17.4
mmol, 85%) as a white solid. [a]2D0 +18.6 (c 0.57,
(C10H19NO4): C, 55.3; H, 8.8; N, 6.5. Found: C, 54.7;
H, 8.7; N, 6.3.
General method for the preparation of compounds 11–13.
Compound 10 (200 mg, 0.92 mmol) in THF (2 mL) was
added to a stirred solution of NaH (27 mg, 1.1 mmol) in
THF (6 mL) at 0 ꢁC followed by addition of the alkyl-
halide (9.2 mmol). The reaction was allowed to warm to
room temperature. TLC showed completion of the
reaction after 0.5–18 h. The reaction was quenched by
addition of MeOH and the solvent was evaporated.
Purification by silica gel column chromatography (tolu-
ene–EtOAc 1:1) gave the target compounds 11, 12 and
13.
1
CH2Cl2); H NMR (300 MHz, CDCl3) d 7.95–7.92 (d,
2H), 7.27–7.15 (d, 2H), 4.54 (m, 2H), 4.41 (dd, 1H,
J=3.5, 11.9), 4.27 (dd, 1H, J=6.2, 11.4 Hz), 4.00 (dd,
1H, J=4.1, 9.8 Hz) 3.82 (dd, 1H, J=1.4, 9.8 Hz), 3.0
(1H, OH), 2.39 (s, 3H), 2.07 (m, 1H), 1.89 (m, 1H); 13C
NMR (75 MHz, CDCl3) d 166.6, 143.8, 129.7, 129.1,
75.8, 75.7, 72.0, 66.3, 37.7, 21.6. Anal. calcd.for
(C13H16O4): C, 66.1; H, 6.8. Found: C, 65.7; H, 6.8.
4(S)-Azido-2(S)-(O-4-methylbenzoyl-methyl)-tetrahydro-
furan (9). DIAD (4.2 mL, 21.3) was added dropwise to a
cooled (ꢀ5 ꢁC) solution of Ph3P (5.3 g, 20.1 mmol) in
THF (80 mL). After 30 min, a solution of 8 (3.9 g, 16.5
mmol) in THF (30 mL) was added. After additional10
min diphenyl phosphorazidate (DPPA; 4.2 mL, 19.5
mmol) was added and the reaction mixture was allowed
to warm to room temperature. After stirring over night,
the solvent was evaporated and the residue was purified
by silica gel column chromatography (toluene; toluene–
EtOAc 50:1) to provide 9 (4.3 g, 16.5 mmol, 100%) as a
white solid. [a]D20 +33.7 (c 1.32, CH2Cl2); 1H NMR
(300 MHz, CDCl3) d 7.95–7.92 (d, 2H), 7.24–7.21 (d,
2H), 4.47–4.26 (m, 4H), 4.15 (m, 1H), 3.96 (m, 1H), 3.85
(m, 1H), 2.45–2.32 (m, 4H), 1.89 (m, 1H); 13C NMR
(75 MHz, CDCl3) d 166.7, 144.0, 130.0, 129.3, 76.8,
72.9, 66.2, 61.1, 34.5, 21.9. Anal. calcd for
(C13H15N3O4): C, 59.8; H, 5.8; N, 16.1. Found: C, 59.7;
H, 5.8; N, 15.8.
2(S)-Allyloxymethyl-4(S)-tert-butoxycarbonylamino-tetra-
hydrofuran (11). The title compound was prepared
according to the general procedure, vide supra, using
allyl bromide (700 mL, 9.2 mmol) with stirring for 18 h,
and was isolated as a colourless syrup in 78% yield (185
mg, 0.72 mmol). [a]2D0 ꢀ32.5 (c 0.97, CH2Cl2); 1H NMR
(300 MHz, CDCl3) d 5.92 (m, 1H), 5.74 (bd, 1H), 5.27
(m, 1H), 5.17 (m, 1H), 4.29 (bs, 1H), 4.10 (m, 1H), 4.00
(m, 1H), 3.80 (d, 1H, J=9.7 Hz), 3.71–3.61 (m, 2H),
3.43 (dd, 1H, J=3.3, 10.5 Hz), 2.29 (m, 1H), 1.70 (m,
1H), 1.40 (s, 9H); 13C NMR (75 MHz, CDCl3) d 155.4,
134.1, 117.6, 78.8, 77.0, 74.9, 72.6, 72.0, 51.1, 34.2, 28.4.
2(S)-Methoxymethyl-4(S)-tert-butoxycarbonylamino-tet-
rahydrofuran (12). The title compound was prepared
according to the general procedure, vide supra, using
MeI (570 mL, 9.2 mmol) with stirring for 30 min, and
was isolated as a colourless syrup in 87% yield (185 mg,
0.80 mmol). [a]D20ꢀ24.8 (c 0.80, CH2Cl2); 1H NMR
(300 MHz, CDCl3) d 5.52 (bd, 1H), 4.12 (bs, 1H), 4.10
(m, 1H), 3.72 (d, 1H, J=9.0 Hz), 3.64 (dd, 1H, J=6.0,
10.5 Hz), 3.50 (m, 1H), 3.33 (s, 3H), 3.30 (dd, 1H,
J=6.0, 12.0 Hz), 2.23 (m, 1H), 1.58 (m, 1H), 1.37 (s,
9H); 13C NMR (75 MHz, CDCl3) d 155.4, 79.0, 77.17,
74.7, 74.4, 59.2, 51.1, 34.2, 28.3.
2(S)-Hydroxymethyl-4(S)-tert-butoxycarbonylamino-tet-
rahydrofuran (10). A catalytic amount of Pd/C (10%)
was added to a solution of 9 (3.6 g, 13.8 mmol) and tert-
butoxycarbonyl anhydride (Boc2O; 3.2 g, 14.7 mmol) in
EtOAc (150 mL). Hydrogen was added at atmospheric
pressure and the reaction mixture was stirred at ambient
temperature overnight. The suspension was filtered
trough Celite and the solvent was evaporated. The resi-
due was purified by silica gel column chromatography
(toluene–EtOAc 6:1) to provide the protected amine
(4.3 g, 12.9 mmol, 93%) as a white solid. [a]2D0 +8.5 (c
0.58, CH2Cl2); 13C NMR (75 MHz, CDCl3) d 166.8,
155.6, 144.0, 129.9, 129.4, 127.3, 79.8, 76.5, 74.1, 66.3,
51.6, 35.5, 28.6, 28.5, 21.9.
2(S)-Ethoxymethyl-4(S)-tert-butoxycarbonylamino-tetra-
hydrofuran (13). The title compound was prepared
according to the general procedure, vide supra, using
EtI (700 mL, 9.2 mmol) with stirring for 7 h, and was
isolated as a colourless syrup in 87% yield (197 mg, 0.81
mmol). [a]2D0 ꢀ34.0 (c 1.33, CH2Cl2); 1H NMR
(300 MHz, CDCl3) d 6.01 (bd, 1H), 4.18–4.02 (m, 2H),
3.76 (d, 1H, 10.8), 3.65–3.40 (m, 4H), 3.34 (dd, 1H,
J=1.8, 7.5 Hz), 2.22 (m, 1H), 1.68 (m, 1H), 1.35 (s, 9H);
13C NMR (75 MHz, CDCl3) d 155.5, 78.7, 77.2, 75.1,
72.4, 67.1, 51.0, 34.1, 28.4, 14.9.
To a stirred solution of the protected amine (3.8 g, 11.3
mmol) in MeOH (50 mL), a catalytic amount of sodium
was added and the mixture was stirred at ambient tem-
perature. After 6 h Dowex1 HCR-W2 (H+) ion
exchange resin was added to neutralise the solution. The
resin was filtered off and the solvent was evaporated.
The residue was purified by silica gel column chroma-
tography (toluene; toluene–EtOAc 1:1) to provide 10
(1.9 g, 8.7 mmol, 77%) as a colourless syrup, which
solidified upon standing. [a]2D0ꢀ24.6 (c 0.61, CH2Cl2);
1H NMR (300 MHz, CDCl3) d 5.50 (d, 1H, J=7.8 Hz),
4.21 (bs, 1H), 4.04 (m, 1H), 3.82–3.71 (m, 3H), 3.50 (dd,
1H, J=3.6, 11.7), 3.00 (bs, 1H), 2.35 (m, 1H), 1.65 (m,
1H), 1.40 (s, 9H); 13C NMR (75 MHz, CDCl3) d 155.7,
78.9, 74.8, 64.1, 51.6, 34.2, 28.6. Anal. calcd for
2(S)-(10-Hydroxy)ethyl-4(S)-tert-butoxycarbonylamino-
tetrahydrofuran (15). Oxalyl chloride (550 mL, 6.4
mmol) in CH2Cl2 (12 mL) was added to a solution of
DMSO (790 mL, 11.1 mmol) in CH2Cl2 (5 mL) at
ꢀ70 ꢁC under argon. After stirring for 5 min, alcohol 10
(1.0 g, 4.6 mmol) dissolved in CH2Cl2 (2 mL) was added
dropwise. Stirring was continued for an additional 15
min. NEt3 (3.0 mL, 22.5 mmol) was added and the
reaction mixture was stirred during 1.5 h while the
reaction mixture slowly was allowed to attain room

K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 1107–1115
1113
temperature. Dilution with H2O, extraction of the aqu-
eous layer with CH2Cl2 (3x), drying (MgSO4) and the
solvent was evaporated. Purification by silica gel col-
umn chromatography (toluene–EtOAc 1:1) gave the
aldehyde 14 (500 mg, 2.32 mmol, 51%) as a colourless
oil. [a]2D0 ꢀ15.1 (c 0.97, CH2Cl2); 13C NMR (75 MHz,
CDCl3) d 202.2, 155.4, 82.1, 74.5, 51.2, 34.8, 28.6.
dure, vide supra, using 10. [a]2D0 ꢀ11.3 (c 1.15, CH2Cl2);
13C NMR (75 MHz, CDCl3) d 171.2, 137.2, 128.7,
128.5, 128.4, 81.1, 79.0, 74.9, 73.8, 71.4, 63.6, 50.2, 33.3.
Anal. calcd for (C30H40N2O10): C, 61.2; H, 6.9; N, 4.8.
Found: C, 60.8; H, 6.9; N, 4.8.
N1,N6-Di[2(S)-allyloxymethyl-tetrahydrofuran-4(S)-yl]-
(2R,3R,4R,5R)-2,5-dibenzyloxy-3,4-O-dihydroxyhexane-
diamide (18). The title compound was prepared in 37%
yield (26 mg, 0.039 mmol) according to general proce-
dure, vide supra, using 11. [a]2D0 ꢀ6.24 (c 0.85, CH2Cl2);
13C NMR (75 MHz, CDCl3) d 171.9, 137.1, 134.6, 128.8,
128.6, 128.5, 118.0, 79.2, 77.7, 76.8, 74.8, 73.9, 72.7, 71.8,
71.1, 49.9, 34.2. Anal. calcd for (C36H48N2O10): C, 64.7;
H, 7.2; N, 4.2. Found: C, 64.7; H, 7.4; N, 4.3.
The crude aldehyde 14 (140 mg, 0.66 mmol) in diethyl
ether (1 mL) was added to a stirred solution of methyl-
magnesium bromide (3 M in THF, 1 mL, 3 mmol) in
diethyl ether (2 mL) at 0 ꢁC. The reaction was stirred at
0 ꢁC for 10 min and 3 h. at room temperatureThe reac-
tion was quenched with saturated NH4Cl. The aqueous
layer was extracted (diethyl ether, 3ꢂ50 mL) and the
combined organic phase was washed (H2O) and dried
(MgSO4). Purification by silica gel column chromato-
graphy (toluene; toluene–EtOAc 1:1) gave 15 (90 mg,
0.39 mmol, 59%) as a diasteroemeric mixture (approx.
1:1). 13C NMR (75 MHz, CDCl3) d 82.7, 82.4, 74.8,
74.3, 70.2, 67.6, 51.6, 51.2, 35.6, 31.3, 28.6, 19.9, 18.9.
N1,N6-Di[2(S)-methoxymethyl-tetrahydrofuran-4(S)-yl]-
(2R,3R,4R,5R)-2,5-dibenzyloxy-3,4-O-dihydroxyhexane-
diamide (19). The title compound was prepared in 77%
yield (26 mg, 0.042 mmol) according to general proce-
dure, vide supra, using 12. [a]2D0 ꢀ4.6 (c 0.70, CH2Cl2);
13C NMR (75 MHz, CDCl3) d 172.0, 137.2, 128.8,
128.7, 128.5, 79.2, 77.6, 75.1, 74.7, 74.1, 71.2, 59.5, 49.8,
34.0. Anal. calcd for (C32H44N2O10): C, 62.3; H, 7.2; N,
4.5. Found: C, 62.8; H, 7.5; N, 4.4.
2(S)-(10-Hydroxy-20-methyl)propyl-4(S)-tert-butoxycarbo-
nylamino-tetrahydrofuran (16). The crude aldehyde 14
(250 mg, 1.16 mmol) (prepared as described above) in
diethyl ether (4 mL) was added to a stirred solution of
isopropylmagnesium bromide (2 M in diethyl ether, 5.5
mL, 11 mmol) in diethyl ether (10 mL) at 0 ꢁC. The
reaction was stirred at 0 ꢁC for 10 min and then at room
temperature overnight. The reaction was quenched with
saturated NH4Cl. The aqueous layer was extracted
(diethyl ether, 3ꢂ50 mL) and the combined organic
phase was washed (H2O) and dried (MgSO4). Purifica-
tion by silica gel column chromatography (toluene; tol-
uene–EtOAc 3:1) gave 16 (145 mg, 0.56 mmol, 48%) as
a diasteroemeric mixture (approx. 1:1). 13C NMR
(75 MHz, CDCl3) d 155.5, 79.8, 78.7, 78.4, 76.8, 74.6,
74.3, 51.4, 35.7, 31.6, 31.3, 28.5, 19.6, 19.5, 18.9, 18.4.
N1,N6-Di[2(S)-ethoxymethyl-tetrahydrofuran-4(S)-yl]-
(2R,3R,4R,5R)-2,5-dibenzyloxy-3,4-O-dihydroxyhexane-
diamide (20). The title compound was prepared in 77%
yield (27 mg, 0.042 mmol) according to general proce-
dure, vide supra, using 13. [a]2D0 ꢀ4.1 (c 0.75, CH2Cl2);
13C NMR (75 MHz, CDCl3) d 172.0, 137.1, 128.8,
128.7, 128.5, 79.2, 77.7, 74.8, 73.9, 72.4, 71.1, 67.2, 49.9,
34.4, 15.1. Anal. calcd for (C34H48N2O10): C, 63.3; H,
7.5; N, 4.3. Found: C, 63.0; H, 7.3; N, 4.6.
N1,N6-Di[2(S)-(10-hydroxy)ethyl-tetrahydrofuran-4(S)-
yl]-(2R,3R,4R,5R)-2,5-dibenzyloxy-3,4-O-dihydroxyhex-
anediamide (21). The title compound was prepared in
47% yield (32 mg, 0.051 mmol) according to general
procedure, vide supra, using 15 as a diasteroemeric
mixture (approx. 1:1). 13C NMR (75 MHz, CDCl3) d
171.7, 171.4, 137.1, 128.8–128.4, 82.5, 82.3, 80.5, 80.0,
74.5, 74.3, 74.0, 73.9, 71.5, 71.4, 69.8, 69.8, 67.4, 50.0, 49.7,
35.1, 30.6, 20.1, 19.4 Anal. calcd for (C32H44N2O10): C,
62.3; H, 7.2; N, 4.5. Found: C, 62.4; H, 7.3; N, 4.5.
General method for the preparation of compounds 17–22.
Compounds 10–13, 15, and 16 (2.0 equiv) were dissolved
in EtOAc (3 mL) and HCl (concd, 1 mL) and stirred at
room temperature until TLC showed completion of the
reaction (1 h). The solvent was evaporated. The residue
was taken up in toluene (5 mL) and revaporated to give
the amine salts. The amine salts were dissolved in
CH2Cl2 (3 mL) and Et3N (2.0 equiv) was added fol-
lowed by addition of disuccinimidylester (1.0 equiv) The
reaction mixture was stirred at room temperature for 16
h. The solvent was removed and the crude product
purified by column chromatography (CH2Cl2; CH2Cl2–
MeOH 20:1). The purified l-mannaric amides were dis-
solved in 5 mL of 4% HCl in MeOH, and the reaction
mixture was stirred at ambient temperature for 16 h.
The mixture was partitioned between CH2Cl2 and satu-
rated aqueous NaHCO3, dried, concentrated, and pur-
ified by silica gel column chromatography (CH2Cl2–
MeOH 20:1) to give the title compounds 17–22.
N1,N6-Di[2(S)-(10-hydroxy-20-methyl)propyl-tetrahydro-
furan-4(S)-yl]-(2R,3R,4R,5R)-2,5-dibenzyloxy-3,4-O-di-
hydroxyhexanediamide (22). The title compound was
prepared in 62% yield (46 mg, 0.068 mmol) according
to general procedure, vide supra, using 16 as a diaster-
oemeric mixture (approx. 1:1). 13C NMR (75 MHz,
CDCl3) d 171.8, 171.5, 137.3, 128.8, 128.7, 128.5, 128.4,
80.3, 79.9, 79.7, 79.5, 78.8, 78.5, 77.0, 74.7, 74.2, 71.5,
71.4, 50.0, 49.8, 35.4, 31.7, 31.6, 30.9, 19.6, 19.5, 19.1,
18.9. Anal. calcd for (C36H52N2O10): C, 64.3; H, 7.8; N,
4.2. Found: C, 64.3; H, 7.9; N, 4.2.
N1,N6-Di[2(S)-hydroxymethyl-tetrahydrofuran-4(S)-yl]-
(2R,3R,4R,5R)-2,5-dibenzyloxy-3,4-O-dihydroxyhexane-
diamide (17). The title compound was prepared in 47%
yield (21 mg, 0.036 mmol) according to general proce-
General method for the preparation of compounds 23–28.
Compounds 10–13, 15, and 18–4 (1.5 equiv) were
dissolved in EtOAc (3 mL) and HCl (conc., 1 mL), stir-
red at room temperature until TLC showed completion

1114
K. Oscarsson et al. / Bioorg. Med. Chem. 11 (2003) 1107–1115
of the reaction (1 h). The solvent was evaporated. The
residue was taken up in toluene (5 mL) and revaporated
to give the amine salts. The amine salts were dissolved
in dichloroethane (8 mL) and Et3N (1.5 equiv) was
added followed by addition of 4 (1.0 equiv). The reac-
tion mixture was stirred at reflux for 16 h. The solvent
was removed and the crude product purified by silica gel
column chromatography (CH2Cl2–MeOH 40:1) to give
the title compounds 23–28.
0.035 mmol) according to general procedure, vide supra,
using 15 as a diasteroemeric mixture (approx. 1:1). 13C
NMR (75 MHz, CDCl3) d 172.3, 172.2, 171.1, 141.0,
140.1, 137.1, 136.9, 128.9–128.5, 127.3, 125.6, 124.3,
82.6, 82.3, 81.4, 81.3, 80.8, 74.4, 74.3, 73.8, 73.8, 72.8,
71.9, 71.6, 69.7, 67.2, 58.0, 50.1, 49.8, 39.5, 35.0, 29.9,
20.1, 19.5. Anal. calcdfor (C35H42N2O9): C, 66.2; H, 6.7;
N, 4.4. Found: C, 66.2; H, 6.8; N, 4.5.
N1-[(1R,2S)-2-Hydroxy-2,3-dihydroxy-1H-indenyl]-N6-
[2(S)-(10-hydroxy-20-methyl)propyl-tetrahydrofuran-4(S)-
yl]-(2R,3R,4R,5R)-2,5-dibenzyloxy-3,4-O-dihydroxyhex-
anediamide (28). The title compound was prepared in
49% yield (45 mg, 0.068 mmol) according to general
procedure, vide supra, using 16 as a diasteroemeric
mixture (approx. 1:1). 13C NMR (75 MHz, CDCl3) d
172.1, 170.9, 140.9, 140.0, 137.1, 136.8, 128.7–128.3,
127.1, 125.5, 124.2, 81.3, 80.4, 78.6, 78.4, 74.5–71.5,
58.0, 57.9, 50.0, 49.8, 39.4, 35.2, 31.7, 31.5, 19.5, 19.4,
19.0. Anal. (C37H46N2O9): C, 67.1; H, 7.2; N, 4.1.
Found: C, 67.0; H, 7.1; N, 4.2.
N1-[(1R,2S)-2-Hydroxy-2,3-dihydroxy-1H-indenyl]-N6-[2(S)-
hydroxymethyl-tetrahydrofuran-4(S)-yl]-(2R,3R,4R,5R)-
2,5-dibenzyloxy-3,4-O-dihydroxyhexanediamide
(23).
The title compound was prepared in 29% yield (34 mg,
0.055 mmol) according to general procedure, vide supra,
using 10. [a]D20 ꢀ19.7 (c 1.15, CH2Cl2); 13C NMR
(75 MHz, CDCl3) d 171.6, 170.3, 140.5, 139.6, 136.6,
136.5, 128.3, 128.2, 128.0, 127.9, 127.8, 126.7, 125.0, 123.8,
80.8, 80.7, 78.3, 74.3, 73.1, 72.3, 71.5, 71.1, 62.9, 57.5, 49.7,
38.9, 32.7. Anal. calcd for (C34H40N2O9): C, 65.8; H, 6.5;
N, 4.5. Found: C, 65.3; H, 6.5; N, 4.5.
N1-[(1R,2S)-2-Hydroxy-2,3-dihydroxy-1H-indenyl]-N6-[2(S)-
allyloxymethyl-tetrahydrofuran-4(S)-yl]-(2R,3R,4R,5R)-
Acknowledgements
2,5-dibenzyloxy-3,4-O-dihydroxyhexanediamide
(24).
The title compound was prepared in 44% yield (40 mg,
0.061 mmol) according to general procedure, vide supra,
using 11. [a]2D0 ꢀ16.2 (c 1.10, CH2Cl2); 13C NMR
(75 MHz, CDCl3) d 172.2, 171.1, 140.8, 140.1, 136.9,
136.8, 134.5, 128.7–128.3, 127.1, 125.4, 124.1, 117.9,
81.3, 80.5, 77.5, 74.6, 73.7, 72.6, 72.5, 71.5, 57.9, 49.8,
39.6, 33.9. Anal. calcd for (C37H44N2O9): C, 67.3; H,
6.7; N, 4.2. Found: C, 67.1; H, 6.8; N, 4.3.
We gratefully acknowledge Medivir AB, Huddinge,
Sweden for financial support and for the biological
testings.
References and Notes
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methoxymethyl-tetrahydrofuran-4(S)-yl]-(2R,3R,4R,5R)-
2,5-dibenzyloxy-3,4-O-dihydroxyhexanediamide
(25).
The title compound was prepared in 28% yield (25 mg,
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(75 MHz, CDCl3) d 172.0, 170.8, 140.6, 139.8, 136.7,
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81.2, 80.5, 76.5, 74.7, 74.2, 73.7, 73.6, 72.4, 71.3, 59.1, 57.7,
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(75 MHz, CDCl3) d 172.2, 171.2, 140.8, 140.0, 136.9,
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2,5-dibenzyloxy-3,4-O-dihydroxyhexanediamide (27).
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