New inhibitors of the malaria aspartyl proteases plasmepsin I and II

Article abstract of DOI:10.1016/S0968-0896(03)00312-2

New inhibitors of plasmepsin I and II, the aspartic proteases of the malaria parasite Plasmodium falciparum, are described. From paralell solution phase chemistry, several reversed-statine type isostere inhibitors, many of which are aza-peptides, have been prepared. The synthetic strategy delivers the target compounds in good to high overall yields and with excellent stereochemical control throughout the developed route. The final products were tested for their plasmepsin I and II inhibiting properties and were found to exhibit modest but promising activity. The best inhibitor exhibits K_i values of 250 nM and 1.4 μM for Plm I and II, respectively.

Full text of DOI:10.1016/S0968-0896(03)00312-2

Bioorganic & Medicinal Chemistry 11 (2003) 3423–3437
New Inhibitors of the Malaria Aspartyl Proteases
Plasmepsin I and II
Anders Dahlgren,a Ingemar Kvarnstrom,a Lotta Vrang,b Elizabeth Hamelink,b
Anders Hallberg,c Asa Rosenquista,b,* and Bertil Samuelssonb,d,
*
aDepartment of Chemistry, Linkoping University, S-581 83 Linkoping, Sweden
¨
¨
bMedivir AB, Lunastigen 7, S-141 44 Huddinge, Sweden
cDepartment of Organic Pharmaceutical Chemistry, BMC, Uppsala University, Box 596, S-751 24 Uppsala, Sweden
dDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
Received 13 December 2002; accepted 8 May 2003
Abstract—New inhibitors of plasmepsin I and II, the aspartic proteases of the malaria parasite Plasmodium falciparum, are descri-
bed. From paralell solution phase chemistry, several reversed-statine type isostere inhibitors, many of which are aza-peptides, have
been prepared. The synthetic strategy delivers the target compounds in good to high overall yields and with excellent stereochemical
control throughout the developed route. The final products were tested for their plasmepsin I and II inhibiting properties and were
found to exhibit modest but promising activity. The best inhibitor exhibits Ki values of 250 nM and 1.4 mM for Plm I and II,
respectively.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
process of hemoglobin degradation.1b,5,6 Today at least
10 aspartic protease genes have been identified in the P.
falciparum genome, potentially complicating the picture
of target selection and target redundancy.7 Up until
now, only the parasitic cysteine proteases falcipain 1–3
and the aspartic proteases plasmepsin I and II (Plm I
and II) have been studied in detail, and inhibitors of
falcipains and plasmepsins have shown efficacy in cell
and animal models of malaria, suggesting that
these enzymes may be suitable molecular tagets for drug
discovery.5a,8
Malaria is considered as one of the most serious infec-
tious diseases in the world, affecting approximately 500
million people yearly. It is estimated that the annual
mortality from malaria is 2 million people, and that
more than 40% of the world’s population is at risk of
infection.1 The disease is spread by the Anopheles mos-
quito, mostly found in the tropical regions of the world,
although not all species of the mosquito transmit
malaria.2 There are four major species of the malaria
parasite, that is Plasmodium falciparum, P. vivax, P.
malariae, and P. ovale, of which P. falciparum is
responsible for more than 95% of malaria-related mor-
bidity and mortality.3 The malaria parasite degrades
hemoglobin as a source of nutrients in the erythrocytic
stage, where it invades the host red blood cells.4 The
hemoglobin degradation occurs in an acidic food
vacuole formed by the parasite, and can result in up to
70% of host hemoglobin being consumed.1b The food
vacuole contains aspartic, cysteine, and metallo pro-
teases, which are all considered to play a role in the
Previous studies have shown that the hydroxy-
ethylamine core I, and the statine derived core II (Fig. 1)
found in the naturally occurring peptide aspartic pro-
tease inhibitor pepstatin A, can function as useful tem-
plates for the development of potent plasmepsin
inhibitors.9,10 We have recently reported on the novel
reversed-statine type isosteres (R,R)-III and (S,S)-III
(Fig. 1), where replacement of the N-terminus of the
hydroxyethylamine core I with a C-terminus isostere
was investigated. These compounds were shown to
exhibit modest but promising activity against Plm I and
II.11
*Corresponding author. Tel.: +46-8-608-3104; fax: +46-8-608-3199;
e-mail: asa.rosenquist@medivir.se (A. Rosenquist); bertil.samuelsson
@medivir.se (B. Samuelsson).
We now report on further refinement and extensions of
these inhibitors, resulting in the isosteres (R,R)-IV and
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00312-2

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A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
synthesis of the four epoxides 6–9 (Scheme 1). An R,R
configuration of the central core was selected based on
previous results.11 The p-bromobenzylated methyl ester
111,13 (R,R) was hydrolyzed with LiOH in dioxane/H2O
at 0 ꢀC, and then coupled with a selected set of
amines,9,14 using standard coupling conditions, that is
N0 - (3 - dimethylaminopropyl) - N - ethylcarbodiimide
hydrochloride (EDC), 1-hydroxybenzotriazole (HOBt),
and Et3N in DMF, to deliver the amides 2–5 in 81–93%
overall yields. Deprotection of the isopropylidene group
in compounds 2–5 was achieved using Dowex H+ in
MeOH for amides with non-basic side chains, and
p-TsOH in MeOH for amides with basic side chains.
Finally, the epoxides were formed using Mitsunobu
conditions, that is Ph3P and diethylazodicarboxylate
(DEAD) in refluxing CHCl3,15 to provide the epoxides
6–9 in 70–89% yield over two steps (Scheme 1).
Figure 1. The structures of the hydroxyethylamine core (I) and the
statine-derived core (II). (R,R)-III and (S,S)-III are reversed-statine
based Plm inhibitors.
(R,R)-V (Fig. 2). To increase affinity to Plm I and II, a
benzyl substituent designed to interact with the S10
pocket of the plasmepsins was appended to the amino
terminus nitrogen [cf. isosteres (R,R)-III and (S,S)-III].
To further explore this site of the Plm inhibitor struc-
tures, a hydrazine functionality was introduced, provid-
ing the N-benzyl substituent with increased directional
flexibility to fit into the S10 pocket. This approach has
previously successfully been applied in the development
of potent and orally bioavailable HIV-1 protease inhi-
bitors.12
The epoxides 6–9 were opened with BnNH2 in MeOH at
50 C16 to afford the secondary amine target compounds
10–13 in 82–93% yield (Scheme 2). Only one regioi-
somer was observed in each case according to NMR
analysis. Subsequently, 10–13 were reacted with both di-
tert-butyl dicarbonate (Boc2O) and benzyl chlor-
oformate (Z-Cl) in CH2Cl2 containing Et3N to give the
target carbamates 14, 18, 22, 26 and 15, 19, 23, 27 in 86–
99% yield, and with Boc-Val or Cbz-Val using O-(7-
azabenzotriazol-1-yl)-N,N,N0,N0 -tetramethyluronium-
hexafluorophosphate (HATU) and N,N-diisopropy-
lethylamine (DIEA) in DMF to give the target tertiary
amides 16, 20, 24, 28 and 17, 21, 25, 29 in 93–97% yield
(Scheme 2).
An efficient synthesis for both classes of compounds has
been developed, starting from carbohydrate-derived
precursors and resulting in well-optimized high yield
reactions. Furthermore, the influence of chirality on
inhibitor activity has been determined from synthesis
and comparison of the four stereoisomers (regarding the
central core) of the promising aza-based inhibitor 32,
which exhibits an inhibition of 68 and 35% for Plm I
and II, respectively, at 5 mM.
In order to investigate the combined effect of increased
directional flexibility of the N-benzyl side chain and the
effect of elongation of the core, the N-benzyl hydrazines
30–45 were prepared (Scheme 3). Hydrazines
CbzNHNHBn and BocNHNHBn17,18 were reacted with
Of the two sets of compounds synthesized, that is the
N-benzyl- and the aza-benzyl core compounds, the
N-benzyl derivatives were essentially inactive whereas
the aza-benzyl derivatives exhibited promising activity
towards both Plm I and II, for example 32 and 33
(Table 1). From synthesis and evaluation of the stereo-
isomers of the aza-benzyl inhibitor 32, substantial
potency enhancements were seen with lead inhibitor 57
(R,S stereochemistry) exhibiting Ki values of 250 nM
and 1.4 mM for Plm I and II, respectively (Table 2).
i
epoxides 6–9 in refluxing PrOH-MeOH,19 affording the
target carbamate hydrazines 30–37 in 39–85% yield.
The inclusion of methanol was essential to increase
solubility. From NMR analysis, only one regioisomer
was detected in each case. Lowest yields were obtained
from the pyridin-3-yl-methyl-containing epoxide, that is
epoxide 9, where notable side reactions occurred.20 The
Boc-protected hydrazines 34–37 were reacted with
TFA/CH2Cl2, yielding the free hydrazines, which were
coupled with Boc-Val or Cbz-Val, vide supra, delivering
the target hydrazides 38–45 in 49–96% yield over the
deprotection and the coupling step (Scheme 3).
Results and Discussion
Chemistry
The Cbz-hydrazines 30–33 could not be prepared by the
route starting from the Boc-hydrazines 34–37 (Scheme 3),
that is Boc group cleavage and coupling with Z-Cl21 due
to the preferential formation of the corresponding six-
membered ring-closed oxa-diazinanones.22
The synthetic route to compounds of the general struc-
ture (R,R)-IV and (R,R)-V (Fig. 2) commenced by
For the synthesis of all three additional stereoisomers of
compound 32 (R,R), the stereoisomers of compound 1
(R,R) were used as starting materials. The syn ester 48
was prepared, starting with inversion of the hydroxyl
group in the isopropylidene protected methyl
ester 4611,13,23 (R,R) using Mitsunobu conditions with
Figure 2. The reversed-statine based inhibitors presented in this
report.

A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
3425
Table 1. Biological activities of the most potent target compounds with R,R configuration at the central core
Compd
Structure
Inh, Plm I (%)a
Inh, Plm II (%)a
Ki, Plm I (mM)b
Ki, Plm II (mM)b
23
25
40
ND
ND
25
27
29
31
32
33
40
45
35
36
44
48
68
85
57
48
43
54
64
25
35
39
49
50
ND
ND
ND
ND
>2
ND
ND
ND
ND
>4.7
ND
ND
ND
1.3
ND
ND
aInhibition at an inhibitor concentration of 5 mM.
bND, not determined.
Biological data
p-NO2-benzoic acid, followed by cleavage of the
obtained p-NO2-benzoate ester with NaOMe to give the
syn ester 47 (S,R) in 94% yield (Scheme 4). Subsequent
alkylation with p-BrBnBr, using KI as catalyst, afforded
the p-bromobenzylated syn ester 48 (S,R) in 98% yield.
The p-bromobenzylated ester 50 (R,S) was prepared
from commercially available methyl 3,4-O-iso-
propylidene-l-threonate (49) (R,S) in 96% yield, vide
supra.
All final compounds were tested for their plasmepsin inhi-
bitory properties, and the data of the most potent inhibitors
are outlined in Tables 1 and 2. From analysis of the inhibi-
tion data, it appears that of the two sets of compounds
synthesized, that is the N-benzyl- and the aza-benzyl core
compounds, the N-benzyl derivatives were essentially inac-
tive whereas the aza-benzyl derivatives, for example 32 and
33, exhibited promising activity towards both both Plm I
and II. Additionally, the presence of the benzylcarbamate
group, either alone or coupled with valine, in the P20 posi-
tion seems to be beneficial in terms of potency. Regarding
the the P2 substituents, the basic piperidine- and pyridine-
substituents (C and D in Scheme 1) are incorporated in the
majority of the most potent inhibitors.
The alkylated esters 48 (S,R), 50 (R,S), and 5111 (S,S)
were hydrolyzed and coupled with 1-(2-aminoethyl)-
piperidine to give the amides 52 (S,R), 55 (R,S), and 58
(S,S) in 86–91% yield over two steps (Scheme 5). The
subsequent deprotection and epoxide formation steps
afforded the epoxides 53 (S,R), 56 (R,S), and 59 (S,S) in
82–87% overall yields. Finally, ring-opening with
CbzNHNHBn yielded the target carbamate inhibitors
54 (S,R), 57 (R,S), and 60 (S,S) (73–89%).
In order to assess the influence of chirality on inhibitor
activity, the potent aza-based inhibitor (32) was selected

3426
A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
Table 2. Biological activities of compound 32 and its stereoisomers
Compd
Structure
Inh, Plm I (%)a
Inh, Plm II (%)a
Ki, Plm I (mM)b
Ki, Plm II (mM)b
32
68
35
>2
>4.7
54
57
60
55
98
57
35
74
20
0.82
0.25
>2
3.0
1.4
>4.7
aInhibition at an inhibitor concentration of 5 mM.
bND, not determined.
Scheme 1. (i) LiOH, H2O/dioxane, 0 ꢀC; (ii) RNH2, EDC, HOBt,
TEA, DMF; (iii) Dowex H+, MeOH; (iv) p-TsOH, MeOH; (v)
DEAD, Ph3P, CHCl3, reflux.
Scheme 2. (i) BnNH2, MeOH, 50 ꢀC; (ii) Boc2O or Z-Cl, TEA,
CH2Cl2; (iii) Boc-Val or Z-Val, HATU, DIEA, DMA.

A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
3427
and all stereoisomers (regarding the central core) of this
inhibitor were synthesized. The plasmepsin inhibiting
properties of compound 32 (R,R) together with the stereo-
isomeric inhibitors 54, 57, and 60 are presented in Table
2. Ki values were determined for all three compounds 54
(Ki=820 nM for Plm I and 3.0 mM for Plm II), 57
(Ki=250 nM for Plm I and 1.4 mM for Plm II), and for
60 (Ki>2 mM for Plm I and >4.7 mM for Plm II).
Changing the stereochemistry at the central core of
inhibitor 32 proved to be beneficial with regard to plas-
mepsin affinity. Furthermore, there seems to be a trend
towards a preference for Plm I over Plm II regarding the
inhibitors in general in this report, and this observation
is evidently even more significant for 54, 57, and 60.
Somewhat surprisingly, the best inhibitor (57) in the
whole series of target compounds presented in this
report has the (R,S) syn configuration in the central part
of the molecule. This fact gives rise to the opportunity
to further optimize the present series of Plm I and II
inhibitors.
Scheme 3. (i) CbzNHNHBn, iPrOH, MeOH, reflux; (ii)
BocNHNHBn, iPrOH, MeOH, reflux; (iii) TFA, CH2Cl2; (iv) Boc-Val
or Z-Val, HATU, DIEA, DMF.
Scheme 4. (i) p-NO2-Benzoic acid, DEAD, Ph3P, THF; (ii) NaOMe,
MeOH; (iii) p-BrBnBr, Ag2O, KI, toluene.
Scheme 5. Compare the synthesis of 32 (Schemes 1 and 3): (i) LiOH, H2O/dioxane, 0 ꢀC; (ii) 1-(2-aminoethyl)-piperidine, EDC, HOBt, TEA, DMF;
i
(iii) p-TsOH, MeOH; (iv) DEAD, Ph3P, CHCl3, reflux; (v) CbzNHNHBn, PrOH, MeOH, reflux.

3428
A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
Conclusions
USA) and hydrolysis was recorded as the increase in
fluorescence intensity over a 10-min time period, during
which the rate increased linearly with time. Stock solu-
tions of inhibitors in DMSO were serially diluted in
DMSO and added directly before addition of substrate,
giving a final DMSO concentration of 1%.
We have identified new small lead inhibitors of the
aspartic Plm I and II proteases based on carbohydrate-
derived reversed-statine templates. The synthetic strat-
egy allows for excellent control of the stereochemical
properties of the intermediate compounds as well as the
target compounds throughout the route. Additionally,
the set of target compounds synthesized can easily be
diversified using this methodology, which offers the
opportunity to further optimize the plasmepsin inhibiting
properties of the compounds presented. Some of the tar-
gets show promising activity against the plasmepsins, with
the best inhibitor 57 having an in vitro activity of 98%
inh@5 mM for Plm I and 74% inh@5 mM for Plm II, and
with Ki=250 nM for Plm I and 1.4 mM for Plm II.
IC50 values were obtained by assuming competitive
inhibition and fitting a Langmuir isotherm [vi/vo=1/
(1+[I]/IC50)] to the dose–response data (Grafit), where
vi and vo are the initial velocities for the inhibited and
uninhibited reaction, respectively, and [I] is the inhibitor
concentration.25 The Ki was subsequently calculated by
using Ki=IC50/(1+[S]/Km)26 and a Km value deter-
mined according to Michaelis–Menten.
Synthetic experimentals
Unless otherwise noted, the compounds below were
obtained as colorless solids.
Experimental
General methods
Hydrolysis of methyl ester followed by peptide coupling
to give the amides 2–5, typical procedure: To an icecold
solution of the methyl ester (1.15 mmol) in dioxane/
H2O 1:1 (20 mL) was added aqueous LiOH (1 M, 1.50
mL, 1.50 mmol) dropwise during 5 min. The solution
was stirred at 0 ꢀC for an additional 45 min, after which
the pH was adjusted to approximately 3. Saturation
with NH4Cl, several extractions with CH2Cl2, followed
by drying, filtration, and evaporation of the organic
phases provided the corresponding carboxylic acid
which was used without further purification. The acid
(1.15 mmol) was dissolved in DMF (10 mL) and the
amine (1.72 mmol), 1-hydroxybenzotriazole (HOBt)
(1.81 mmol), and Et3N (3.45 mmol) were added. The
temperature was lowered to 0 ꢀC and N0-(3-dimethyla-
minopropyl)-N-ethylcarbodiimide hydrochloride (EDC)
(1.72 mmol) was added. The mixture was stirred at 0 ꢀC
for 1 h and then at rt overnight. The solvent was eva-
porated and the crude product was purified by flash
column chromatography.
NMR spectra were recorded on a Varian 300 MHz
instrument using CDCl3 or MeOH-d4 with TMS as an
internal standard. Optical rotations were measured in
CHCl3 solutions on a Perkin-Elmer 141 polarimeter.
TLC was carried out on Merck precoated 60 F254 plates
using UV-light and charring with EtOH/H2SO4/HOAc/
p-anisaldehyde 90:3:1:2 for visualisation. Column chro-
matography was performed using silica gel 60 (0.040–
0.063 mm, Merck). Organic phases were dried over
anhydrous magnesium sulfate. Concentrations were
performed under diminished pressure (1–2 kPa) at a
bath temperature of 40 ꢀC. The intermediate com-
pounds 2–9, 47, 48, 50, 52, 53, 55, 56, 58, and 59 were
subjected to MALDI-TOF analysis, using a Voyager-
DE STR Biospectrometry Workstation with a-cyano-4-
hydroxycinnamic acid as reference. The LC–MS spectra
of the target compounds were obtained using a Waters-
micromass instrument equipped with a Waters 2790
pump, a Waters 996 diode array detector and a Waters
ZMD MS detector. Column: Phenomenex Synergy
max-rp 50ꢁ4.6 mm, C18, 4 mm, 80 A. Eluent: Gradient
using acqueous NH4HCO3 (10 mM) and CH3CN.
Flow: 1 mL/min. Wavelength: 254 nm.
(2R,3R)-2-(4-Bromobenzyloxy)-3,4-dihydroxy-N-(furan-
2-yl-methyl)-3,4-O-isopropylidene-butyramide (2). Com-
pound 2 was synthesized in 93% yield from 1 (synthe-
sized according to the methods described in refs 11 and
13) and furfurylamine. Chromatography eluent: tolu-
Plasmepsin assay measurements
1
ene/ethyl acetate 4:1. 2: [a]2D2 +37.5 (c 0.8, CHCl3); H
Pro-plasmepsin II was generously provided from
Helena Danielson (Department of Biochemistry,
Uppsala University, Uppsala, Sweden). The expression
and purification of plasmepsin I will be published else-
where.24 The activities of Plm I and Plm II were mea-
sured essentially as described previously,9 using a total
reaction volume of 100 mL. The concentration of pro-
Plm II was 3 nM and the amount of Plm I was adjusted
to give similar catalytic activity. The pro-sequence of
Plm II was cleaved off by preincubation in an assay
reaction buffer [100 mM sodium acetate buffer (pH 4.5),
10% glycerol and 0.01% Tween 20] at room tempera-
ture for 40 min. The reaction was initiated by the addi-
tion of 3 mM substrate (DABCYL-Glu-Arg-Nle-Phe-
Leu-Ser-Phe-Pro-EDANS, AnaSpec Inc, San Jose, CA,
NMR (CDCl3, 300 MHz): d 1.35 (s, 3H), 1.40 (s, 3H),
3.92 (dd, J=6.9, 8.2 Hz, 1H), 3.96 (dd, J=6.9, 8.2 Hz,
1H), 4.10 (d, J=3.3 Hz, 1H), 4.38 (dd, J=5.6, 16.0 Hz,
1H), 4.47 (dd, J=6.1, 16.0 Hz, 1H), 4.51 (ddd, J=3.3,
6.9, 6.9 Hz, 1H) 4.59 (d, J=11.7 Hz, 1H), 4.69 (d,
J=11.7 Hz, 1H), 6.18 (dd, J=0.8, 3.3 Hz, 1H), 6.32 (dd,
J=1.9, 3.3 Hz, 1H), 6.86 (m, 1H), 7.17 (d, J=8.5 Hz,
2H), 7.35 (dd, J=0.8, 1.9 Hz, 1H), 7.46 (d, J=8.5 Hz,
2H); 13C NMR (CDCl3, 75.5 MHz): d 25.3, 26.3, 36.1,
64.6, 73.6, 76.9, 79.2, 107.7, 109.9, 110.6, 122.4, 129.9,
131.9, 136.0, 142.5, 150.9, 169.1. MS calcd for
C19H22BrNO5Na (M+Na)+: 446.06. Found: 446.01.
(2R,3R)-2-(4-Bromobenzyloxy)-3,4-dihydroxy-N-((1S,2R)-
2-hydroxy-indan-1-yl)-3,4-O-isopropylidene-butyramide

A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
3429
(3). Compound 3 was synthesized in 81% yield from 1
and (1S,2R)-1-amino-2-indanol. Chromatography elu-
ent: toluene/ethyl acetate 2:1. 3: [a]2D2 +23.1 (c 0.3,
matography. (B) Compounds with basic side chains: to
a solution of the isopropylidene protected amide (0.59
mmol) in MeOH was added p-TsOH (0.83 mmol) and the
mixture was stirred at rt for 4 h. The solution was neu-
tralized with Et3N and evaporated and the crude material
was filtered through a short silica plug. The epoxide for-
mation was then achieved as described above.
1
CHCl3); H NMR (CDCl3, 300 MHz): d 1.36 (s, 3H),
1.45 (s, 3H), 2.64 (br s, 1H), 2.92 (dd, J=2.1, 16.6 Hz,
1H), 3.15 (dd, J=5.2, 16.6 Hz, 1H), 4.00 (dd, J=6.8,
8.0 Hz, 1H), 4.04 (dd, J=6.8, 8.0 Hz, 1H), 4.14 (d,
J=3.3 Hz, 1H), 4.50 (ddd, J=3.3, 6.8, 6.8 Hz, 1H), 4.61
(ddd, J=2.2, 5.2, 5.2 Hz, 1H); 4.65 (s, 2H), 5.14 (dd,
J=5.2, 8.5 Hz, 1H), 7.09–7.28 (m, 5H), 7.18 (d, J=8.2
Hz, 2H), 7.43 (d, J=8.2 Hz, 2H); 13C NMR (CDCl3,
75.5 MHz): d 25.4, 26.3, 39.7, 57.4, 65.2, 73.1, 73.2, 77.0,
79.3, 110.0, 122.3, 124.3, 125.5, 127.3, 128.5, 129.8,
131.8, 135.9, 140.2, 140.3, 169.9. MS calcd for
C23H26BrNO5Na (M+Na)+: 498.09. Found: 498.05.
(2R,3R)-2-(4-Bromobenzyloxy)-3,4-epoxy-N-(furan-2-yl-
methyl)-butyramide (6). Compound 6 was synthesized
in 70% yield from 2. Chromatography eluent: toluene/
ethyl acetate 2:1. 6: [a]2D2 +40.7 (c 0.6, CHCl3); 1H
NMR (CDCl3, 300 MHz): d 2.70 (dd, J=4.1, 5.2 Hz,
1H), 2.83 (dd, J=2.5, 5.2 Hz, 1H), 3.42 (ddd, J=2.0,
2.5, 4.1 Hz, 1H), 4.21 (d, J=2.0 Hz, 1H), 4.35 (dd,
J=5.5, 15.7 Hz, 1H) 4.48 (dd, J=6.0, 15.7 Hz, 1H),
4.49 (d, J=11.8 Hz, 1H), 4.59 (d, J=11.8 Hz, 1H), 6.19
(dd, J=0.8, 3.3 Hz, 1H), 6.32 (dd, J=1.9, 3.3 Hz, 1H),
6.86 (m, 1H), 7.14 (d, J=8.2 Hz, 2H), 7.34 (dd, J=0.8,
1.9 Hz, 1H), 7.45 (d, J=8.2 Hz, 2H); 13C NMR (CDCl3,
75.5 MHz): d 36.1, 43.4, 52.2, 73.3, 77.1, 107.6, 110.5,
122.3, 129.8, 131.8, 135.8, 142.4, 150.8, 168.7. MS calcd
for C16H17BrNO4 (M+H)+: 366.03. Found: 366.05.
(2R,3R)-2-(4-Bromobenzyloxy)-3,4-dihydroxy-3,4-O-iso-
propylidene-N-(2-piperidin-1-yl-ethyl)-butyramide
(4).
Compound 4 was synthesized in 84% yield from 1 and
1-(2-aminoethyl)-piperidine. Chromatography eluent:
toluene/ethyl acetate 1:1+1.5% Et3N. 4: [a]2D2 +34.1 (c
1
1.1, CHCl3); H NMR (CDCl3, 300 MHz): d 1.28 (s,
3H), 1.32–1.53 (m, 6H) 1.37 (s, 3H), 2.20–2.39 (m, 4H),
2.34 (t, J=6.0 Hz, 2H), 3.13–3.37 (m, 2H), 3.87 (dd,
J=6.9, 8.1 Hz, 1H), 3.93 (dd, J=6.9, 8.1 Hz, 1H), 4.03
(d, J=3.3 Hz, 1H), 4.47 (ddd, J=3.3, 6.9, 6.9 Hz, 1H),
4.56 (d, J=11.8 Hz, 1H), 4.64 (d, J=11.8 Hz, 1H), 7.10
(br s, 1H), 7.19 (d, J=8.2 Hz, 2H), 7.41 (d, J=8.2 Hz,
2H); 13C NMR (CDCl3, 75.5 MHz): d 24.3, 25.1, 25.9,
26.2, 35.6, 54.2, 56.9, 64.4, 73.1, 76.8, 79.1, 109.5, 121.9,
129.5, 131.6, 136.3, 168.9. MS calcd for C21H32BrN2O4
(M+H)+: 455.16. Found: 455.12.
(2R,3R)-2-(4-Bromobenzyloxy)-3,4-epoxy-N-((1S,2R)-2-
hydroxy-indan-1-yl)-butyramide (7). Compound 7 was
synthesized in 89% yield from 3. Chromatography elu-
ent: ethyl acetate/toluene 2:1. 7: [a]2D2 +26.3 (c 0.8,
CHCl3); 1H NMR (CDCl3, 300 MHz): d 2.74 (br s, 1H),
2.76 (dd, J=4.4, 5.0 Hz, 1H), 2.88 (dd, J=2.8, 5.0 Hz,
1H), 2.94 (dd, J=1.9, 16.6 Hz, 1H), 3.14 (dd, J=5.2,
16.6 Hz, 1H), 3.40 (ddd, J=1.9, 2.8, 4.4 Hz, 1H), 4.30
(d, J=1.9 Hz, 1H), 4.62 (m, 3H), 5.32 (dd, J=5.0, 8.8
Hz, 1H), 7.05–7.16 (m, 3H), 7.19 (d, J=8.2 Hz, 2H),
7.21–7.30 (m, 2H), 7.46 (d, J=8.2 Hz, 2H); 13C NMR
(CDCl3, 75.5 MHz): d 39.5, 44.2, 52.4, 57.7, 72.9, 73.1,
78.1, 122.4, 124.1, 125.5, 127.2, 128.5, 129.7, 131.9,
135.7, 139.9, 140.6, 169.0. MS calcd for C20H21BrNO4
(M+H)+: 418.07. Found: 418.12.
(2R,3R)-2-(4-Bromobenzyloxy)-3,4-dihydroxy-3,4-O-iso-
propylidene-N-(pyridin-3-yl-methyl)-butyramide
(5).
Compound 5 was synthesized in 86% yield from 1 and
3-aminomethylpyridine. Chromatography eluent: ethyl
acetate/toluene 2:1+1.5% Et3N. 5: [a]2D2 +34.6 (c 0.9,
1
CHCl3); H NMR (CDCl3, 300 MHz): d 1.28 (s, 3H),
1.33 (s, 3H), 3.85 (dd, J=6.9, 8.0 Hz, 1H), 3.92 (dd,
J=6.9, 8.0 Hz, 1H), 4.06 (d, J=3.3 Hz, 1H), 4.33 (dd,
J=6.2, 14.8 Hz, 1H), 4.41 (dd, J=8.8, 14.8 Hz, 1H),
4.46 (ddd, J=3.3, 6.9, 6.9 Hz, 1H), 4.53 (d, J=11.8 Hz,
1H), 4.63 (d, J=11.8 Hz, 1H), 7.05–7.21 (m, 2H), 7.09
(d, J=8.2 Hz, 2H), 7.36 (d, J=8.2 Hz, 2H), 7.45–7.52
(m, 1H), 8.19–8.25 (m, 2H); 13C NMR (CDCl3,
75.5 MHz): d 25.0, 26.2, 40.4, 64.6, 73.3, 76.7, 79.0,
109.7, 122.2, 123.5, 129.7, 131.7, 133.6, 135.3, 135.9,
148.9, 149.1, 169.3. MS calcd for C20H24BrN2O4
(M+H)+: 435.09. Found: 435.04.
(2R,3R)-2-(4-Bromobenzyloxy)-3,4-epoxy-N-(2-piperidin-
1-yl-ethyl)-butyramide (8). Compound 8 was synthe-
sized in 86% yield from 4. Chromatography eluent:
ethyl acetate/MeOH 5:1+1% Et3N. 8: [a]2D2 +24.6 (c
1
1.0, CHCl3); H NMR (CDCl3, 300 MHz): d 1.31–1.55
(m, 6H), 2.23–2.40 (m, 4H), 2.37 (t, J=6.0 Hz, 2H), 2.67
(dd, J=4.1, 5.2 Hz, 1H), 2.83 (dd, J=2.5, 5.2 Hz, 1H),
3.17–3.38 (m, 2H), 3.41 (ddd, J=1.9, 2.5, 4.1 Hz, 1H),
4.15 (d, J=1.9 Hz, 1H), 4.50 (d, J=11.8 Hz, 1H), 4.59
(d, J=11.8 Hz, 1H), 7.07–7.20 (m, 1H), 7.18 (d, J=8.2
Hz, 2H), 7.43 (d, J=8.2 Hz, 2H); 13C NMR (CDCl3,
75.5 MHz): d 24.4, 26.1, 35.8, 43.4, 52.5, 54.4, 57.0, 73.2,
77.2, 122.1, 129.6, 131.8, 136.3, 168.7. MS calcd for
C18H26BrN2O3 (M+H)+: 397.11. Found: 397.09.
Isopropylidene group deprotection followed by epoxide
formation to give the epoxides 6–9, typical procedure:
(A) Compounds with non-basic side chains: to a solu-
tion of the isopropylidene protected amide (0.36 mmol)
in MeOH (5 mL) was added a suspension of Dowex H+
(50ꢁ8, 20–50 mesh) in MeOH (3 mL) and the mixture
was stirred at rt for 5 h. After filtration and evaporation
the obtained product was dissolved in CHCl3 (10 mL),
and Ph3P (0.38 mmol) and diethylazodicarboxylate
(DEAD) (0.38 mmol) were added. The solution was
heated at reflux for 2 h, after which it was evaporated
and the remainder was purified by flash column chro-
(2R,3R)-2-(4-Bromobenzyloxy)-3,4-epoxy-N-(pyridin-3-
yl-methyl)-butyramide (9). Compound 9 was synthe-
sized in 85% yield from 5. Chromatography eluent:
ethyl acetate+1% Et3N. 9: [a]2D2 +37.5 (c 0.4, CHCl3);
1H NMR (CDCl3, 300 MHz): d 2.73 (dd, J=4.1, 5.2 Hz,
1H), 2.83 (dd, J=2.5, 5.2 Hz, 1H), 3.43 (ddd, J=1.9,
2.5, 4.1 Hz, 1H), 4.25 (d, J=1.9 Hz, 1H), 4.44 (d, J=6.3
Hz, 2H), 4.50 (d, J=11.5 Hz, 1H), 4.62 (d, J=11.5 Hz,

3430
A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
1H), 6.98 (m, 1H), 7.14 (d, J=8.2 Hz, 2H), 7.20–7.28
(m, 1H), 7.44 (d, J=8.2 Hz, 2H), 7.48–7.60 (m, 1H),
8.49 (m, 2H); 13C NMR (CDCl3, 75.5 MHz): d 40.6,
43.5, 52.2, 73.5, 77.2, 122.5, 123.7, 129.8, 131.9, 133.6,
135.5, 135.8, 149.2, 149.3, 169.0. MS calcd for
C18H26BrN2O3 (M+H)+: 377.05. Found: 377.03.
(2R,3R)-4-Benzylamino-2-(4-bromobenzyloxy)-3-hydroxy-
N-(pyridin-3-yl-methyl)-butyramide (13). Compound 13
was synthesized in 82% yield from 9. Chromatography
eluent: CHCl3/EtOH (99.5%) 9:1+2% MeOH satd
1
with NH3. 13: [a]D22 +24.0 (c 1.0, CHCl3); H NMR
(CDCl3, 300 MHz): d 2.67 (dd, J=4.7, 12.4 Hz, 1H),
2.75 (dd, J=6.6, 12.4 Hz, 1H), 3.28 (br s, 1H), 3.64 (d,
J=13.2 Hz, 1H), 3.72 (d, J=13.2 Hz, 1H), 3.96 (d,
J=4.4 Hz, 1H), 4.03 (ddd, J=4.4, 4.7, 6.6 Hz, 1H), 4.35
(d, J=6.0 Hz, 2H) 4.45 (d, J=11.8 Hz, 1H), 4.52 (d,
J=11.8 Hz, 1H), 7.05 (d, J=8.5 Hz, 2H), 7.12–7.32 (m,
8H), 7.35 (d, J=8.5 Hz, 2H), 7.46–7.52 (m, 1H), 8.38–
8.42 (m, 2H); 13C NMR (CDCl3, 75.5 MHz): d 40.3,
50.0, 53.6, 70.8, 72.8, 81.3, 122.0, 123.5, 127.0, 128.1,
128.3, 129.6, 131.6, 133.8, 135.4, 136.0, 139.7, 148.6,
148.8, 170.9. MS (M+H)+: 484. Purity: 96%.
Epoxide opening with BnNH2 to give compounds 10–
13, typical procedure: To a solution of the epoxide (0.88
mmol) in MeOH (15 mL) was added BnNH2 (3.60
mmol) and the mixture was stirred at 50 C for 4 h. After
evaporation the crude product was purified by flash
column chromatography.
(2R,3R)-4-Benzylamino-2-(4-bromobenzyloxy)-N-(furan-
2-yl-methyl)-3-hydroxy-butyramide (10). Compound 10
was synthesized in 90% yield from 6. Chromatography
eluent: CHCl3/EtOH (99.5%) 97:3+2% MeOH satd
Carbamate formation to give compounds 14, 15, 18, 19,
22, 23, 26, and 27, typical procedure: To a solution of
the secondary amine (0.13 mmol) in CH2Cl2 (3 mL)
were added di-tert-butyl dicarbonate (Boc2O) (0.14
mmol) or benzyl chloroformate (Z-Cl) (0.14 mmol) and
Et3N (0.33 mmol) and the mixture was stirred at rt for 1
h. The solvent was evaporated and the crude product
was purified by flash column chromatography.
1
with NH3. 10: [a]D22 +35.9 (c 1.3, CHCl3); H NMR
(CDCl3, 300 MHz): d 2.64–2.78 (m, 2H), 2.90 (br s, 2H),
3.68 (d, J=13.2 Hz, 1H), 3.77 (d, J=13.2 Hz, 1H), 4.00
(m, 2H), 4.34 (dd, J=5.3, 15.5 Hz, 1H), 4.46 (dd,
J=6.0, 15.5 Hz, 1H), 4.50 (d, J=11.5 Hz, 1H), 4.57 (d,
J=11.5 Hz, 1H), 6.15 (dd, J=0.8, 3.3 Hz, 1H), 6.29 (dd,
J=1.9, 3.3 Hz, 1H), 6.95–7.05 (m, 1H), 7.06 (d, J=8.2
Hz, 2H), 7.18–7.35 (m, 6H), 7.41 (d, J=8.2 Hz, 2H);
13C NMR (CDCl3, 75.5 MHz): d 36.0, 49.7, 53.7, 70.8,
73.2, 81.0, 107.5, 110.5, 122.1, 127.0, 128.1, 128.4, 129.7,
131.7, 136.0, 139.8, 142.2, 150.8, 170.7. MS (M+H)+:
473. Purity: 99%.
Peptide coupling to give compounds 16, 17, 20, 21, 24,
25, 28, and 29, typical procedure: To a solution of the
secondary amine (0.10 mmol) in DMF (3 mL) were
added Boc-Val (0.14 mmol) or Cbz-Val (0.14 mmol)
and N,N-diisopropylethylamine (DIEA) (0.30 mmol).
The temperature was lowered to 0 ꢀC and O-(7-aza-
benzotriazol-1-yl)-N,N,N0,N0 -tetramethyluroniumhexa-
fluorophosphate (HATU) (0.11 mmol) was added. The
mixture was stirred at 0 ꢀC for 1 h and at rt for 1 h, after
which the solvent was evaporated and the crude product
was purified by flash column chromatography.
(2R,3R)-4-Benzylamino-2-(4-bromobenzyloxy)-3-hydroxy-
N-((1S,2R)-2-hydroxy-indan-1-yl)-butyramide
(11).
Compound 11 was synthesized in 93% yield from 7.
Chromatography eluent: CHCl3/EtOH (99.5%)
97:3+2% MeOH satd with NH3. 7: [a]2D2 +12.3 (c 0.5,
1
CHCl3); H NMR (CDCl3, 300 MHz): d 2.69–2.82 (m,
2H), 2.86 (dd, J=1.9, 16.6 Hz, 1H), 3.06 (dd, J=5.3,
16.6 Hz, 1H), 3.50 (br s, 2H), 3.65 (d, J=13.2 Hz, 1H),
3.72 (d, J=13.2 Hz, 1H), 3.93 (d, J=3.6 Hz, 1H), 3.96–
4.05 (m, 1H), 4.47–4.53 (m, 1H), 4.49 (d, J=11.8 Hz,
1H), 4.58 (d, J=11.8 Hz, 1H), 5.25 (dd, J=4.9, 8.8 Hz,
1H), 7.11 (d, J=8.5 Hz, 2H), 7.16–7.33 (m, 9H), 7.40 (d,
J=8.5 Hz, 2H); 13C NMR (CDCl3, 75.5MHz): d 39.3,
50.3, 53.5, 57.6, 71.1, 72.3, 72.5, 81.9, 122.1, 124.0, 125.3,
127.0, 127.1, 128.2, 128.3, 128.4, 129.6, 131.7, 135.8, 139.3,
140.0, 140.7, 170.9. MS (M+H)+: 525. Pu rity: 99%.
(2R,3R)-2-(4-Bromobenzyloxy)-4-(N-tert-butyloxycarbo-
nylbenzylamino)-N-(furan-2-yl-methyl)-3-hydroxy-butyr-
amide (14). Compound 14 was synthesized in 99%
yield from 10 and Boc2O. Chromatography eluent:
CHCl3. 14: [a]D22 ꢂ2.8 (c 0.9, CHCl3); 1H NMR (CDCl3,
300 MHz): d 1.42 (s, 9H), 3.05–3.18 (m, 2H), 3.62 (br s,
1H), 3.92 (d, J=4.4 Hz, 1H), 4.15–4.23 (m, 2H), 4.36
(dd, J=5.5, 15.7 Hz, 1H), 4.45 (dd, J=6.0, 15.7 Hz,
1H), 4.47–4.65 (m, 3H), 6.15 (dd, J=0.8, 3.3 Hz, 1H),
6.26 (dd, J=1.9, 3.3 Hz, 1H), 6.97 (m, 1H), 7.09–7.21
(m, 2H), 7.14 (d, J=8.2 Hz, 2H), 7.24–7.36 (m, 4H),
7.43 (d, J=8.2 Hz, 2H); 13C NMR (CDCl3, 75.5 MHz):
d 28.6, 36.2, 49.7, 52.3, 73.0, 73.3, 81.2, 81.6, 107.6,
110.6, 122.3, 127.4, 128.7, 128.8, 129.9, 131.9, 132.0,
138.1, 142.4, 151.0, 156.9, 169.7. MS (M+H)+: 573.
Purity: 96%.
(2R,3R)-4-Benzylamino-2-(4-bromobenzyloxy)-3-hydroxy-
N-(2-piperidin-1-yl-ethyl)-butyramide (12). Compound
12 was synthesized in 86% yield from 8. Chromato-
graphy eluent: CHCl3/EtOH (99.5%) 9:1+2% MeOH
satd with NH3. 12: [a]22 +29.8 (c 1.8, CHCl3); 1H NMR
(CDCl3, 300 MHz): dD1.35–1.55 (m, 6H), 2.22–2.41 (m,
6H), 2.75 (dd, J=5.8, 12.1 Hz, 1H), 2.81 (dd, J=4.1,
12.1 Hz, 1H), 3.29–3.39 (m, 2H), 3.72 (d, J=13.2 Hz,
1H), 3.81 (d, J=13.2 Hz, 1H), 3.92–4.01 (m, 2H), 4.52
(d, J=11.8 Hz, 1H), 4.58 (d, J=11.8 Hz, 1H), 7.04 (m,
1H), 7.17 (d, J=8.2 Hz, 2H), 7.20–7.37 (m, 5H), 7.44 (d,
J=8.2 Hz, 2H); 13C NMR (CDCl3, 75.5 MHz): d 24.3,
25.9, 35.7, 50.3, 53.9, 54.3, 57.3, 71.2, 72.8, 80.9, 122.0,
127.0, 128.2, 128.4, 129.6, 131.6, 136.2, 140.2, 171.0. MS
(M+H)+: 504. Purity: 98%.
(2R,3R)-4-(N-Benzyloxycarbonylbenzylamino)-2-(4-bro-
mobenzyloxy)-N-(furan-2-yl-methyl)-3-hydroxy-butyr-
amide (15). Compound 15 was synthesized in 89%
yield from 10 and Z-Cl. Chromatography eluent:
CHCl3. 15: [a]D22 +4.1 (c 1.2, CHCl3); 1H NMR (CDCl3,
300 MHz): d 3.19–3.43 (m, 2H), 3.65 (br s, 1H), 3.80–
3.98 (m, 2H), 4.14–4.27 (m, 1H), 4.27–4.70 (m, 5H), 5.16
(s, 2H), 6.15 (dd, J=0.8, 3.3 Hz, 1H), 6.28 (dd, J=1.9,

A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
3431
3.3 Hz, 1H), 6.90–7.08 (m, 1H), 7.14 (d, J=8.2 Hz, 2H),
7.18–7.37 (m, 11H), 7.43 (d, J=8.2 Hz, 2H); 13C NMR
(CDCl3, 75.5 MHz): d 35.9, 49.7, 51.6, 67.7, 72.4, 73.1,
81.3, 107.4, 110.4, 122.2, 127.4, 127.9, 128.1, 128.4,
128.5, 128.6, 129.1, 129.7, 129.8, 131.7, 137.2, 142.2,
150.6, 158.0, 169.5. MS (M+H)+: 607. Purity: 98%.
CHCl3. 19: [a]D22 ꢂ3.8 (c 0.4, CHCl3); 1H NMR (CDCl3,
300 MHz): d 1.38 (br s, 1H), 2.79–3.17 (m, 2H), 3.29–
3.53 (m, 3H), 3.84–3.98 (m, 1H), 4.07–4.75 (m, 6H),
5.15, (s, 2H), 5.30 (dd, J=5.0, 8.6 Hz, 1H), 6.90–7.62
(m, 19H); 13C NMR (CDCl3, 75.5 MHz): d 39.4, 50.1,
51.9, 57.8, 67.8, 71.9, 72.6, 72.7, 82.1, 122.3, 124.0,
125.4, 127.1, 127.5, 127.8, 128.0, 128.2, 128.3, 128.5,
128.6, 128.7, 129.7, 131.8, 135.8, 137.4, 139.8, 140.7,
157.8, 170.4. MS (M+H)+: 659. Purity: 98%.
(2R,3R)-2-(4-Bromobenzyloxy)-4-[((S)-2-N-tert-butyloxy-
carbonylamino-3-methyl-butyryl)-benzylamino]-N-(furan-
2-yl-methyl)-3-hydroxy-butyramide (16). Compound 16
was synthesized in 95% yield from 10 and Boc-Val.
Chromatography eluent: CHCl3. 16: [a]2D2 ꢂ18.8 (c 0.9,
(2R,3R)-2-(4-Bromobenzyloxy)-4-[((S)-2-N-tert-butyloxy-
carbonylamino-3-methyl-butyryl)-benzylamino]-3-hydroxy-
1
CHCl3); H NMR (CDCl3, 300 MHz) (mixture of rota-
N-((1S,2R)-2-hydroxy-indan-1-yl)-butyramide
(20).
mers): d 0.82–1.00 (m, 6H), 1.40 (s, 9H), 1.83–2.03 (m,
1H), 3.02–3.50 (m, 2H), 3.85–4.09 (m, 2H), 4.17–5.10
(m, 9H), 6.07–6.15 (m, 1H), 6.22–6.27 (m, 1H), 6.85–
6.96 (m, 1H), 7.08–7.38 (m, 8H), 7.39–7.45 (m, 2H); 13C
NMR (CDCl3, 75.5 MHz) (mixture of rotamers): d 17.4,
18.3, 19.3, 19.5, 28.3, 31.3, 31.8, 35.9, 48.0, 48.2, 48.9,
55.3, 55.4, 69.9, 72.6, 73.3, 73.7, 79.6, 80.3, 81.5, 81.8,
107.4, 110.3, 110.4, 122.1, 122.3, 127.4, 127.9, 128.4,
128.5, 128.8, 129.7, 130.1, 131.7, 135.6, 135.7, 135.9,
137.2, 142.1, 150.7, 157.0, 169.0, 169.4, 173.1, 175.2. MS
(M+H)+: 672. Purity: 99%.
Compound 20 was synthesized in 96% yield from 11
and Boc-Val. Chromatography eluent: CHCl3. 20: [a]D22
ꢂ6.6 (c 1.0, CHCl3); 1H NMR (CDCl3, 300 MHz)
(mixture of rotamers): d 0.80–1.01 (m, 6H), 1.40 (s, 9H),
1.85–2.01 (m, 1H), 2.10 (br s, 1H), 2.85–2.96 (m, 1H),
3.04–3.15 (m, 1H), 3.17–3.65 (m, 2H), 3.26–3.30 (m,
1H), 3.83–3.94 (m, 1H), 4.04–4.82 (m, 7H), 5.16–5.21
(m, 1H), 7.00–7.46 (m, 15H); 13C NMR (CDCl3,
75.5 MHz) (mixture of rotamers): d 17.2, 17.4, 19.6,
19.8, 28.4, 31.6, 39.4, 40.0, 48.2, 48.5, 55.3, 57.6, 57.9,
69.7, 71.6, 72.0, 72.4, 72.6, 80.4, 81.8, 82.2, 122.3, 123.9,
124.1, 125.4, 127.1, 127.3, 127.5, 127.9, 128.0, 128.4,
128.5, 128.7, 128.9, 129.7, 131.7, 131.8, 135.7, 136.1,
137.1, 139.9, 140.8, 155.7, 157.0, 169.8, 170.2, 172.9. MS
(M+H)+: 724. Purity: 99%.
(2R,3R)-4-[((S)-2-N-Benzyloxycarbonylamino-3-methyl-
butyryl)-benzylamino]-2-(4-bromobenzyloxy)-N-(furan-2-
yl-methyl)-3-hydroxy-butyramide (17). Compound 17
was synthesized in 97% yield from 10 and Z-Val.
Chromatography eluent: CHCl3. 17: [a]2D2 ꢂ12.7 (c 0.5,
(2R,3R)-4-[((S)-2-N-Benzyloxycarbonylamino-3-methyl-
butyryl)-benzylamino]-2-(4-bromobenzyloxy)-3-hydroxy-
1
CHCl3); H NMR (CDCl3, 300 MHz) (mixture of rota-
mers): d 0.78–1.01 (m, 6H), 1.85–2.04 (m, 1H), 2.07 (br
s, 1H), 3.02–3.65 (m, 2H), 3.78–5.75 (m, 11H), 6.07–6.15
(m, 1H), 6.22–6.29 (m, 1H), 6.87–7.01 (m, 1H), 7.05–
7.46 (m, 15H); 13C NMR (CDCl3, 75.5 MHz) (mixture
of rotamers): d 17.2, 17.9, 19.4, 19.5, 31.3, 31.7, 35.9,
47.9, 48.1, 48.8, 52.3, 55.9, 56.0, 66.9, 67.1, 69.9, 72.4,
73.2, 73.7, 81.5, 81.7, 107.4, 110.3, 110.4, 122.1, 122.3,
127.3, 127.4, 127.9, 128.0, 128.1, 128.4, 128.5, 128.8,
129.7, 130.0, 131.6, 131.7, 135.6, 135.7, 135.8, 136.1,
137.0, 142.1, 150.6, 156.2, 157.2, 169.0, 169.4, 172.7,
174.5. MS (M+H)+: 706. Purity: 98%.
N-((1S,2R)-2-hydroxy-indan-1-yl)-butyramide
(21).
Compound 21 was synthesized in 94% yield from 11
and Z-Val. Chromatography eluent: CHCl3. 21: [a]D22
ꢂ5.1 (c 0.5, CHCl3); 1H NMR (CDCl3, 300 MHz)
(mixture of rotamers): d 0.80–1.22 (m, 6H), 1.88 (br s,
1H), 1.90–2.02 (m, 1H), 2.87–2.99 (m, 1H), 3.07–3.17
(m, 1H), 3.18–3.60 (m, 2H) 3.27–3.38 (m, 1H), 3.83–4.78
(m, 8H), 5.02–5.23 (m, 2H), 5.25–5.42 (m, 1H), 6.99–
7.45 (m, 20H); 13C NMR (CDCl3, 75.5 MHz) (mixture
of rotamers): d 17.1, 17.3, 19.6, 19.8, 31.6, 38.8, 40.0,
48.1, 48.4, 49.2, 52.6, 55.9, 56.1, 57.6, 57.9, 67.1, 67.4,
71.8, 72.1, 72.4, 72.6, 72.7, 81.7, 82.2, 121.0, 122.2,
122.3, 123.9, 124.1, 125.5, 127.1, 127.2, 127.6, 128.0,
128.1, 128.2, 128.3, 128.4, 128.6, 128.8, 129.0, 129.5,
129.7, 129.8, 131.8, 131.8, 135.7, 135.8, 136.0, 137.0,
139.9, 140.7, 151.9, 156.3, 157.4, 169.7, 170.2, 172.5,
174.3. MS (M+H)+: 758. Purity: 97%.
(2R,3R)-2-(4-Bromobenzyloxy)-4-(N-tert-butyloxycarbo-
nylbenzylamino)-3-hydroxy-N-((1S,2R)-2-hydroxy-indan-
1-yl)-butyramide (18). Compound 18 was synthesized in
97% yield from 11 and Boc2O. Chromatography eluent:
CHCl3. 18: [a]D22 ꢂ5.6 (c 1.2, CHCl3); 1H NMR (CDCl3,
300 MHz): d 1.41 (s, 9H), 1.86 (br s, 1H), 2.92 (dd,
J=1.9, 16.8 Hz, 1H), 3.12 (dd, J=5.5, 16.8 Hz, 1H)
3.21–3.38 (m, 2H), 3.45–3.61 (m, 1H), 3.91 (d, J=3.3
Hz, 1H), 4.11–4.67 (m, 6H), 5.12 (dd, J=5.0, 8.5 Hz,
1H), 7.05 (d, J=8.5 Hz, 1H), 7.09–7.36 (m, 9H), 7.15 (d,
J=8.2 Hz, 2H), 7.43 (d, J=8.2 Hz, 2H); 13C NMR
(CDCl3, 75.5 MHz): d 28.5, 39.4, 50.0, 52.3, 57.9, 72.6,
72.7, 73.0, 81.0, 82.2, 122.1, 124.0, 125.5, 127.1, 127.4,
127.6, 128.4, 128.6, 129.7, 131.8, 135.9, 138.1, 139.9,
140.8, 157.8, 170.4. MS (M+H)+: 625. Purity: 99%.
(2R,3R)-2-(4-Bromobenzyloxy)-4-(N-tert-butyloxycarbo-
nylbenzylamino)-3-hydroxy-N-(2-piperidin-1-yl-ethyl)-bu-
tyramide (22). Compound 22 was synthesized in 99%
yield from 12 and Boc2O. Chromatography eluent:
CHCl3/EtOH (99.5%) 97:3+2% MeOH satd with
1
NH3. 22: [a]22 +1.3 (c 0.8, CHCl3); H NMR (CDCl3,
300 MHz): dD1.33–1.55 (m, 6H), 1.50 (s, 9H), 2.23–2.40
(m, 6H), 3.08–3.40 (m, 4H), 3.50–3.64 (m, 1H), 3.80–
3.92 (m, 1H), 4.09–4.63 (m, 5H), 6.95–7.11 (m, 1H),
7.14–7.38 (m, 7H), 7.42 (d, J=8.5 Hz, 2H); 13C NMR
(CDCl3, 75.5 MHz): d 24.4, 25.9, 28.5, 35.8, 49.4, 51.9,
54.4, 57.3, 72.6, 72.8, 80.5, 81.7, 122.1, 127.3, 127.5,
128.6, 129.7, 131.7, 136.4, 138.3, 167.4, 170.0. MS
(M+H)+: 604. Purity: 100%.
(2R,3R)-4-(N-Benzyloxycarbonylbenzylamino)-2-(4-bro-
mobenzyloxy)-3-hydroxy-N-((1S,2R)-2-hydroxy-indan-1-
yl)-butyramide (19). Compound 19 was synthesized in
86% yield from 11 and Z-Cl. Chromatography eluent:

3432
A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
(2R,3R)-4-(N-Benzyloxycarbonylbenzylamino)-2-(4-bro-
mobenzyloxy)-3-hydroxy-N-(2-piperidin-1-yl-ethyl)-buty-
ramide (23). Compound 23 was synthesized in 94%
yield from 12 and Z-Cl. Chromatography eluent:
CHCl3/EtOH (99.5%) 97:3+2% MeOH satd with
2H); 13C NMR (CDCl3, 75.5 MHz): d 28.5, 40.6, 49.8,
52.3, 72.7, 73.0, 81.0, 81.4, 122.3, 123.8, 127.5, 128.7,
128.8, 129.9, 131.9, 134.0, 135.8, 136.0, 138.0, 148.7,
148.9, 157.8, 170.1. MS (M+H)+: 584. Purity: 99%.
1
NH3. 23: [a]2D2 +5.6 (c 0.5, CHCl3); H NMR (CDCl3,
(2R,3R)-4-(N-Benzyloxycarbonylbenzylamino)-2-(4-bro-
mobenzyloxy)-3-hydroxy-N-(pyridin-3-yl-methyl)-butyra-
mide (27). Compound 27 was synthesized in 94% yield
from 13 and Z-Cl. Chromatography eluent: CHCl3/
EtOH (99.5%) 97:3+2% MeOH satd with NH3. 27:
300 MHz): d 1.32–1.55 (m, 6H), 2.20–2.41 (m, 6H),
3.20–3.52 (m, 4H), 3.54–3.65 (m, 1H), 3.75–3.91 (m,
1H), 4.08–4.71 (m, 5H), 5.17 (s, 2H), 6.78–7.51 (m,
15H); 13C NMR (CDCl3, 75.5 MHz): d 24.3, 25.8, 35.7,
49.8, 51.6, 54.3, 57.3, 67.7, 72.2, 72.9, 81.6, 122.1, 127.4,
127.5, 128.0, 128.2, 128.5, 128.6, 128.8, 129.7, 131.7,
136.5, 137.8, 157.9, 170.0. MS (M+H)+: 638. Purity:
99%.
1
[a]2D2 ꢂ2.5 (c 1.0, CHCl3); H NMR (CDCl3, 300 MHz):
d 3.18–3.45 (m, 2H), 3.52–3.63 (m, 1H), 3.84–3.95 (m,
1H), 4.19–4.25 (m, 1H), 4.30–4.62 (m, 6H), 5.16 (s, 2H),
6.93–7.58 (m, 17H), 8.42–8.52 (m, 2H); 13C NMR
(CDCl3, 75.5 MHz): d 40.5, 50.0, 51.8, 67.9, 72.3, 73.0,
81.3, 122.3, 123.7, 127.0, 127.5, 128.0, 128.2, 128.6,
128.7, 129.8, 131.8, 133.8, 135.7, 136.1, 137.2, 148.6,
148.8, 158.0, 170.0. MS (M+H)+: 618. Purity: 100%.
(2R,3R)-2-(4-Bromobenzyloxy)-4-[((S)-2-N-tert-butyloxy-
carbonylamino-3-methyl-butyryl)-benzylamino]-3-hydroxy-
N-(2-piperidin-1-yl-ethyl)-butyramide (24). Compound
24 was synthesized in 93% yield from 12 and Boc-Val.
Chromatography eluent: CHCl3/EtOH (99.5%)
97:3+2% MeOH satd with NH3. 24: [a]2D2 ꢂ11.9 (c 1.0,
(2R,3R)-2-(4-Bromobenzyloxy)-4-[((S)-2-N-tert-butylox-
ycarbonylamino - 3 - methyl - butyryl) - benzylamino] - 3 -
hydroxy-N-(pyridin-3-yl-methyl)-butyramide (28). Com-
pound 28 was synthesized in 93% yield from 13 and
Boc-Val. Chromatography eluent: CHCl3/EtOH
(99.5%) 97:3+2% MeOH satd with NH3. 28: [a]D22
ꢂ18.3 (c 0.9, CHCl3); 1H NMR (CDCl3, 300 MHz)
(mixture of rotamers): d 0.81–1.01 (m, 6H), 1.40 (s, 9H),
1.82–2.01 (m, 1H), 3.05–3.25 (m, 2H), 3.67–3.80 (m,
1H), 3.87–4.05 (m, 2H), 4.20–4.82 (m, 7H), 7.03–7.57
(m, 13H), 8.41–8.52 (m, 2H); 13C NMR (CDCl3,
75.5 MHz) (mixture of rotamers): d 17.5, 18.3, 19.5,
19.6, 28.4, 31.4, 31.9, 40.5, 48.5, 48.6, 49.3, 52.6, 55.7,
70.1, 72.5, 73.2, 73.6, 80.2, 80.5, 81.6, 81.8, 122.3, 122.5,
123.8, 127.5, 127.6, 128.1, 128.5, 128.7, 129.0, 129.9,
130.1, 131.8, 131.9, 133.9, 134.0, 135.9, 137.3, 148.6,
148.7, 148.8, 156.0, 157.2, 169.7, 167.0, 173.3, 174.5. MS
(M+H)+: 683. Purity: 99%.
1
CHCl3); H NMR (CDCl3, 300 MHz) (mixture of rota-
mers): d 0.82–0.99 (m, 6H), 1.22–1.53 (m, 15H), 1.82–
2.01 (m, 1H), 2.18–2.40 (m, 6H), 3.02–3.49 (m, 4H),
3.60–3.71 (m, 1H), 3.80–4.02 (m, 2H), 4.16–4.23 (m,
1H), 4.42–4.63 (m, 4H), 7.00–7.10 (m, 1H), 7.14–7.35
(m, 8H), 7.40–7.45 (m, 2H); 13C NMR (CDCl3,
75.5 MHz) (mixture of rotamers): d 17.4, 18.0, 19.4,
19.6, 24.3, 25.8, 28.4, 31.5, 32.0, 35.7, 48.3, 48.4, 52.2,
54.3, 55.4, 57.2, 70.0, 72.3, 73.0, 73.1, 80.1, 82.0, 122.1,
122.2, 127.5, 128.0, 128.6, 128.9, 129.7, 130.0, 131.7,
136.0, 136.3, 137.4, 156.0, 156.9, 169.6, 169.9, 173.3,
174.6. MS (M+H)+: 703. Purity: 99%.
(2R,3R)-4-[((S)-2-N-Benzyloxycarbonylamino-3-methyl-
butyryl)-benzylamino]-2-(4-bromobenzyloxy)-3-hydroxy-
N-(2-piperidin-1-yl-ethyl)-butyramide (25). Compound
25 was synthesized in 95% yield from 12 and Z-Val.
Chromatography eluent: CHCl3/EtOH (99.5%)
97:3+2% MeOH satd with NH3. 25: [a]2D2 ꢂ7.6 (c 0.8,
(2R,3R)-4-[((S)-2-N-Benzyloxycarbonylamino-3-methyl-
butyryl)-benzylamino]-2-(4-bromobenzyloxy)-3-hydroxy-
N-(pyridin-3-yl-methyl)-butyramide (29). Compound 29
was synthesized in 97% yield from 13 and Z-Val.
Chromatography eluent: CHCl3/EtOH (99.5%)
97:3+2% MeOH satd with NH3. 29: [a]2D2 ꢂ12.4 (c 0.9,
1
CHCl3); H NMR (CDCl3, 300 MHz) (mixture of rota-
mers): d 0.80–1.00 (m, 6H), 1.27–1.52 (m, 6H), 1.83–
2.02 (m, 1H), 2.17–2.41 (m, 6H), 3.03–3.37 (m, 4H),
3.58–3.66 (m, 1H), 3.78–4.01 (m, 2H), 4.15–4.24 (m,
1H), 4.42–4.87 (m, 4H), 4.98–5.17 (m, 2H), 6.98–7.09
(m, 1H), 7.11–7.43 (m, 15H); 13C NMR (CDCl3,
75.5 MHz) (mixture of rotamers): d 17.3, 17.7, 19.3,
19.6, 24.3, 25.8, 31.6, 31.9, 35.7, 38.7, 48.2, 48.4, 52.1,
54.2, 56.0, 57.2, 57.3, 67.0, 67.1, 70.0, 72.2, 73.0, 81.9,
122.1, 122.2, 127.5, 127.6, 128.1, 128.2, 128.3, 128.6,
129.0, 129.7, 129.9, 131.7, 135.9, 136.2, 136.3, 137.3,
156.5, 157.1, 169.7, 169.8, 173.0, 174.3. MS (M+H)+:
737. Purity: 99%.
1
CHCl3); H NMR (CDCl3, 300 MHz) (mixture of rota-
mers): d 0.82–1.00 (m, 6H), 1.84–2.03 (m, 1H), 3.07–
3.25 (m, 2H), 3.62–3.75 (m, 1H), 3.88–4.06 (m, 2H),
4.21–4.82 (m, 7H), 4.98–5.20 (m, 2H), 7.05–7.55 (m,
18H), 8.41–8.50 (m, 2H); 13C NMR (CDCl3, 75.5 MHz)
(mixture of rotamers): d 17.3, 17.9, 19.3, 19.6, 31.4, 31.8,
40.5, 40.6, 48.4, 49.3, 52.6, 56.1, 56.2, 67.1, 67.3, 70.1,
72.4, 73.2, 73.6, 81.6, 122.4, 122.5, 123.8, 127.4, 127.7,
128.0, 128.1, 128.2, 128.4, 128.5, 128.6, 128.8, 129.1,
129.9, 130.1, 131.8, 131.9, 133.9, 135.8, 135.9, 136.0,
136.3, 137.2, 148.6, 148.8, 156.4, 156.4, 169.7, 170.0,
173.0, 174.8. HRMS calcd for C37H42BrN4O6
(M+H)+: 717.2287. Found: 717.2282. Purity: 100%.
(2R,3R)-2-(4-Bromobenzyloxy)-4-(N-tert-butyloxycarbo-
nylbenzylamino)-3-hydroxy-N-(pyridin-3-yl-methyl)-buty-
ramide (26). Compound 26 was synthesized in 98%
yield from 13 and Boc2O. Chromatography eluent:
CHCl3/EtOH (99.5%) 97:3+2% MeOH satd with
Epoxide opening with CbzNHNHBn or BocNHNHBn
to give compounds 30–37, typical procedure: To a
1
NH3. 26: [a]22 ꢂ5.5 (c 0.6, CHCl3); H NMR (CDCl3,
300 MHz): dD1.42 (s, 9H), 3.07–3.22 (m, 2H), 3.49–3.62
(m, 1H), 3.92 (d, J=3.9 Hz, 1H), 4.13–4.22 (m, 1H)
4.24–4.61 (m, 6H), 7.07–7.58 (m, 12H), 8.42–8.52 (m,
solution of the epoxide (0.13 mmol) in PrOH (0.5 mL)
and MeOH (1 mL) was added CbzNHNHBn (0.18
mmol) or BocNHNHBn (0.18 mmol), and the mixture
i

A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
3433
was refluxed overnight. The solvents were evaporated
and the remainder was purified by flash column chro-
matography.
CHCl3); 1H NMR (CDCl3, 300 MHz): d 2.50 (br s, 1H),
2.63–2.75 (m, 1H), 3.00 (dd, J=10.2, 12.4 Hz, 1H), 3.92
(d, J=13.6 Hz, 1H), 4.01 (d, J=13.6 Hz, 1H), 4.06–4.18
(m, 2H) 4.42 (dd, J=6.1, 15.1 Hz, 1H), 4.50 (dd, J=6.0,
15.1 Hz, 1H), 4.52–4.70 (m, 2H), 5.02 (d, J=12.4 Hz,
1H), 5.07 (d, J=12.4 Hz, 1H), 5.82 (br s, 1H), 7.11 (d,
J=8.2 Hz, 2H), 7.16–7.36 (m, 13H), 7.42 (d, J=8.2 Hz,
2H), 8.46–8.60 (m, 2H); 13C NMR (CDCl3, 75.5 MHz):
d 40.4, 58.4, 62.7, 67.2, 69.5, 73.1, 80.6, 122.2, 124.1,
127.9, 128.0, 128.3, 128.6, 129.4, 129.8, 129.9, 131.7,
131.9, 135.0, 135.6, 136.1, 137.1, 147.2, 147.6, 156.6,
170.4. HRMS calcd for C32H34BrN4O5 (M+H)+:
633.1712. Found: 633.1707. Purity: 98%.
(2R,3R)-4-(N-Benzyl-N0-benzyloxycarbonyl-hydrazino)-
2-(4-bromobenzyloxy)-N-(furan-2-yl-methyl)-3-hydroxy-
butyramide (30). Compound 30 was synthesized in 80%
yield from 6 and CbzNHNHBn. Chromatography elu-
ent: toluene/ethyl acetate 2:1. 30: [a]2D2 +14.2 (c 0.6,
CHCl3); 1H NMR (CDCl3, 300 MHz): d 2.40 (br s, 1H),
2.67 (dd, J=2.5, 12.2 Hz, 1H), 3.00 (dd, J=10.2, 12.2
Hz, 1H), 3.83–4.05 (m, 2H) 4.07–4.13 (m, 1H), 4.14–
4.22 (m, 1H), 4.36 (dd, J=5.2, 15.5 Hz, 1H), 4.46 (dd,
J=6.1, 15.5 Hz, 1H), 4.55 (d, J=11.5 Hz, 1H), 4.67 (d,
J=11.5 Hz, 1H), 5.01 (d, J=12.1 Hz, 1H), 5.08 (d,
J=12.1 Hz, 1H), 5.80 (br s, 1H), 6.19 (dd, J=0.8, 3.3
Hz, 1H), 6.32 (dd, J=1.9, 3.3 Hz, 1H), 6.92–7.03 (m,
1H), 7.12 (d, J=8.2 Hz, 2H), 7.13–7.33 (m, 10H), 7.35
(dd, J=0.8, 1.9 Hz, 1H), 7.43 (d, J=8.2 Hz, 2H); 13C
NMR (CDCl3, 75.5 MHz): d 36.0, 58.1, 62.7, 67.2, 69.5,
73.2, 80.6, 107.5, 110.5, 122.1, 125.3, 127.9, 128.0, 128.5,
129.0, 129.3, 129.9, 131.7, 135.3, 135.9, 136.2, 142.2,
151.0, 156.7, 169.6. MS (M+H)+: 622. Purity: 96%.
(2R,3R)-4-(N-Benzyl-N0-tert-butyloxycarbonyl-hydrazino)-
2-(4-bromobenzyloxy)-N-(furan-2-yl-methyl)-3-hydroxy-
butyramide (34). Compound 34 was synthesized in 73%
yield from 6 and BocNHNHBn. Chromatography elu-
ent: toluene/ethyl acetate 2:1. 34: [a]2D2 +18.0 (c 0.6,
1
CHCl3); H NMR (CDCl3, 300 MHz): d 1.35 (m, 9H),
2.55–2.66 (m, 1H), 2.97 (dd, J=10.2, 12.4 Hz, 1H), 3.17
(br s, 1H), 3.80–4.03 (m, 2H), 4.08–4.22 (m, 2H), 4.36
(dd, J=5.8, 15.4 Hz, 1H), 4.46 (dd, J=6.0, 15.4 Hz,
1H), 4.57 (d, J=11.5 Hz, 1H), 4.70 (d, J=11.5 Hz, 1H),
5.45 (br s, 1H), 6.19 (dd, J=0.8, 3.3 Hz, 1H), 6.32 (dd,
J=1.9, 3.3 Hz, 1H), 6.94–7.07 (m, 1H), 7.13 (d, J=8.2
Hz, 2H), 7.18–7.36 (m, 6H), 7.43 (d, J=8.2 Hz, 2H);
13C NMR (CDCl3, 75.5 MHz): d 28.3, 36.1, 58.1, 63.0,
69.6, 73.3, 80.4, 80.8, 107.5, 110.6, 122.1, 127.8, 128.5,
129.4, 130.0, 131.7, 135.8, 136.4, 142.3, 151.1, 156.4,
169.7. MS (M+H)+: 588. Purity: 100%.
(2R,3R)-4-(N-Benzyl-N0-benzyloxycarbonyl-hydrazino)-
2-(4-bromobenzyloxy)-3-hydroxy-N-((1S,2R)-2-hydroxy-
indan-1-yl)-butyramide (31). Compound 31 was synthe-
sized in 83% yield from 7 and CbzNHNHBn. Chroma-
tography eluent: toluene/ethyl acetate 1:1. 31: [a]D22
1
+19.9 (c 0.7, CHCl3); H NMR (CDCl3, 300 MHz): d
2.39 (br s, 1H), 2.77–2.88 (m, 1H), 2.96 (dd, J=1.9, 12.5
Hz, 1H), 3.03–3.18 (m, 2H), 3.88–4.12 (m, 4H), 4.52–
4.69 (m, 3H), 5.04 (s, 2H), 5.36 (dd, J=4.7, 8.8 Hz, 1H),
5.85 (br s, 1H), 7.06 (d, J=8.8 Hz, 1H), 7.10–7.37 (m,
16H), 7.44 (d, J=8.2 Hz, 2H); 13C NMR (CDCl3,
75.5 MHz): d 39.2, 58.1, 59.2, 62.7, 67.4, 69.9, 72.6, 72.7,
81.2, 122.3, 124.1, 125.4, 125.6, 127.2, 128.1, 128.2,
128.4, 128.6, 128.7, 129.2, 129.6, 129.8, 131.9, 135.1,
136.0, 139.9, 141.1, 157.1, 170.4. MS (M+H)+: 674.
Purity: 95%.
(2R,3R)-4-(N-Benzyl-N0-tert-butyloxycarbonyl-hydrazino)-
2-(4-bromobenzyloxy)-3-hydroxy-N-((1S,2R)-2-hydroxy-
indan-1-yl)-butyramide (35). Compound 35 was synthe-
sized in 85% yield from 7 and BocNHNHBn. Chroma-
tography eluent: toluene/ethyl acetate 1:1. 35: [a]D22
1
+16.9 (c 0.6, CHCl3); H NMR (CDCl3, 300 MHz): d
1.33 (s, 9H), 2.72–3.20 (m, 4H), 3.33 (br s, 1H), 3.80–
4.13 (m, 3H), 4.01 (d, J=1.9 Hz, 1H), 4.54–4.71 (m,
3H), 5.36 (dd, J=8.8, 14.0 Hz, 1H), 5.58 (br s, 1H),
7.05–7.38 (m, 12H), 7.44 (d, J=8.2 Hz, 2H); 13C NMR
(CDCl3, 75.5 MHz): d 28.2, 39.2, 58.0, 59.1, 62.6, 69.9,
72.6, 72.7, 80.6, 81.3, 122.2, 124.1, 125.5, 127.1, 127.9,
128.5, 129.5, 130.2, 131.8, 132.1, 135.6, 136.1, 140.0,
141.0, 156.5, 170.4. MS (M+H)+: 640. Purity: 98%.
(2R,3R)-4-(N-Benzyl-N0-benzyloxycarbonyl-hydrazino)-
2-(4-bromobenzyloxy)-3-hydroxy-N-(2-piperidin-1-yl-
ethyl)-butyramide (32). Compound 32 was synthesized
in 84% yield from 8 and CbzNHNHBn. Chromato-
graphy eluent: ethyl acetate/MeOH 5:1+1% Et3N. 32:
[a]2D2 +10.5 (c 0.5, CHCl3); 1H NMR (CDCl3,
300 MHz): d 1.37–1.59 (m, 6H), 2.23–2.45 (m, 6H), 2.59
(br s, 1H), 2.67–3.07 (m, 2H), 3.25–3.39 (m, 2H), 3.85–
4.21 (m, 4H), 4.50–4.71 (m, 2H), 5.00 (d, J=12.4 Hz,
1H), 5.06 (d, J=12.4 Hz, 1H), 5.85 (br s, 1H), 7.07–7.39
(m, 13H), 7.43 (d, J=8.2 Hz, 2H); 13C NMR (CDCl3,
75.5 MHz): d 24.2, 25.7, 35.6, 54.3, 57.2, 58.0, 62.7, 67.0,
69.8, 72.9, 80.9, 121.9, 127.8, 128.0, 128.5, 128.6, 129.3,
129.5, 129.7, 131.6, 136.0, 136.3, 136.8, 156.5, 169.8.
HRMS calcd for C33H42BrN4O5 (M+H)+: 653.2338.
Found: 653.2333. Purity: 99%.
(2R,3R)-4-(N-Benzyl-N0-tert-butyloxycarbonyl-hydrazino)-
2-(4-bromobenzyloxy)-3-hydroxy-N-(2-piperidin-1-yl-ethyl)-
butyramide (36). Compound 36 was synthesized in 78%
yield from 8 and BocNHNHBn. Chromatography elu-
ent: ethyl acetate/MeOH 9:1+1% Et3N. 36: [a]2D2 +12.7
1
(c 1.2, CHCl3); H NMR (CDCl3, 300 MHz): d 1.32 (s,
9H), 1.35–1.59 (m, 6H), 2.25–2.43 (m, 6H), 2.52 (br s,
1H), 2.60–2.70 (m, 1H), 2.98 (dd, J=9.6, 12.4 Hz, 1H),
3.22–3.39 (m, 2H), 3.80–4.04 (m, 2H), 4.05–4.20 (m,
2H), 4.57 (d, J=11.8 Hz, 1H), 4.69 (d, J=11.8 Hz, 1H),
5.51 (br s, 1H), 7.18 (d, J=8.2 Hz, 2H), 7.20–7.31 (m,
6H), 7.42 (d, J=8.2 Hz, 2H); 13C NMR (CDCl3,
75.5 MHz): d 24.3, 25.9, 28.2, 35.7, 54.3, 57.2, 58.1, 62.8,
69.8, 73.0, 80.4, 81.0, 121.9, 127.7, 128.5, 129.5, 129.7,
131.6, 136.0, 136.7, 156.3, 169.8. MS (M+H)+: 619.
Purity: 97%.
(2R,3R)-4-(N-Benzyl-N0-benzyloxycarbonyl-hydrazino)-
2-(4-bromobenzyloxy)-3-hydroxy-N-(pyridin-3-yl-methyl)-
butyramide (33). Compound 33 was synthesized in 39%
yield from 9 and CbzNHNHBn. Chromatography elu-
ent: ethyl acetate+1% Et3N. 33: [a]2D2 +7.1 (c 0.4,

3434
A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
(2R,3R)-4-(N-Benzyl-N0-tert-butyloxycarbonyl-hydrazino)-
2-(4-bromobenzyloxy)-3-hydroxy-N-(pyridin-3-yl-methyl)-
butyramide (37). Compound 37 was synthesized in 48%
yield from 9 and BocNHNHBn. Chromatography elu-
ent: ethyl acetate/MeOH 9:1+1% Et3N. 37: [a]2D2 +10.6
69.8, 73.2, 80.7, 107.5, 110.6, 122.2, 127.9, 128.1, 128.3,
128.5, 128.7, 129.2, 129.6, 129.9, 131.8, 135.7, 136.4,
142.3, 151.1, 156.4, 169.8, 171.0. MS (M+H)+: 721.
Purity: 99%.
(2R,3R)-4-[N-Benzyl-N0 -((S)-2-tert-butyloxycarbonyl-
amino-3-methyl-butyryl)-hydrazino]-2-(4-bromobenzyloxy)-
3-hydroxy-N-((1S,2R)-2-hydroxy-indan-1-yl)-butyramide
(40). Compound 40 was synthesized in 70% yield from
35 and Boc-Val. Chromatography eluent: CHCl3+2%
MeOH satd with NH3. 40: [a]2D2 +7.9 (c 0.9, CHCl3); 1H
NMR (CDCl3, 300 MHz) (mixture of conformational
isomers): d 0.64–1.02 (m, 6H), 1.38–1.42 (m, 9H), 1.85–
2.15 (m, 1H), 2.50 (m, 1H), 2.87–3.18 (m, 4H), 3.62–3.72
(m, 1H), 3.88–4.12 (m, 4H), 4.57 (d, J=11.5 Hz, 1H),
4.58–4.62 (m, 1H), 4.63 (d, J=11.5 Hz, 1H), 5.27–5.38
(m, 1H), 5.71 (br s, 1H), 6.13 (br s, 1H), 7.04–7.38 (m,
10H), 7.15 (d, J=8.2 Hz, 1H), 7.42 (d, J=8.2 Hz, 1H);
13C NMR (CDCl3, 75.5 MHz) (mixture of conforma-
tional isomers): d 17.8, 19.3, 19.4, 20.1, 28.4, 28.5, 30.2,
30.8, 38.7, 39.3, 58.0, 59.3, 62.3, 69.9, 72.4, 72.7, 80.4, 80.7,
81.4, 82.4, 122.2, 124.0, 124.1, 125.6, 127.2, 128.0, 128.5,
128.6, 128.9, 129.4, 129.5, 129.7, 131.7, 131.8, 135.7, 136.1,
140.0, 141.1, 156.3, 157.2, 170.5, 171.0, 171.6. MS
(M+H)+: 739. HRMS calcd for C37H48BrN4O7
(M+H)+: 739.2706. Found: 739.2701. Purity: 99%.
1
(c 0.8, CHCl3); H NMR (CDCl3, 300 MHz): d 1.37 (s,
9H), 2.57–2.67 (m, 1H), 2.97 (dd, J=10.2, 12.2 Hz, 1H),
3.24 (br s, 1H), 3.80–4.01 (m, 2H), 4.05–4.20 (m, 2H),
4.38–4.47 (m, 2H), 4.56 (d, J=11.8 Hz, 1H), 4.66 (d,
J=11.8 Hz, 1H), 5.58 (br s, 1H), 7.10 (d, J=8.5 Hz,
2H), 7.14–7.53 (m, 10H), 8.42–8.54 (m, 2H); 13C NMR
(CDCl3, 75.5 MHz): d 28.3, 40.5, 58.4, 63.0, 69.6, 73.1,
80.3, 80.8, 122.2, 123.8, 127.8, 128.5, 128.7, 129.4, 129.9,
131.9, 133.3, 134.1, 135.8, 148.8, 148.9, 156.6, 170.1. MS
(M+H)+: 599. Purity: 95%.
Deprotection of Boc-protected hydrazine followed by
peptide coupling to give compounds 38–45, typical pro-
cedure: To a solution of the Boc-protected hydrazine
(0.18 mmol) in CH2Cl2 (1 mL) and TFA (1 mL) was
added anisole (0.35 mmol) and the mixture was stirred
at rt for 30 min. The volatiles were evaporated and the
crude product was purified by flash column chromato-
graphy. The obtained deprotected hydrazine was then
coupled to Boc-Val or Cbz-Val using the procedure
described for compounds 16, 17, 20, 21, and so on.
(2R,3R)-4-[N-Benzyl-N0 -((S)-2-tert-butyloxycarbonyl-
amino-3-methyl-butyryl)-hydrazino]-2-(4-bromobenzyloxy)-
N-(furan-2-yl-methyl)-3-hydroxy-butyramide (38). Com-
pound 38 was synthesized in 96% yield from 34 and
Boc-Val. Chromatography eluent: CHCl3+2% MeOH
satd with NH3. 38: [a]22 +12.7 (c 1.4, CHCl3); 1H NMR
(CDCl3, 300 MHz): dD0.67–0.83 (m, 6H), 1.40 (s, 9H),
1.86–2.02 (m, 1H), 2.66–2.80 (m, 1H), 2.98 (dd, J=10.3,
12.4 Hz, 1H) 3.61–3.70 (m, 1H), 3.96 (s, 2H), 4.02–4.18
(m, 2H), 4.34 (dd, J=6.0, 15.4 Hz, 1H), 4.44–4.52 (m,
1H), 4.46 (dd, J=6.2, 15.4 Hz, 1H), 4.54 (d, J=11.5 Hz,
1H), 4.65 (d, J=11.5 Hz, 1H), 4.82–4.92 (m, 1H), 6.18
(dd, J=0.8, 3.3 Hz, 1H), 6.30 (dd, J=1.9, 3.3 Hz, 1H),
6.94–7.05 (m, 1H), 7.11 (d, J=8.2 Hz, 2H), 7.20–7.36
(m, 6H), 7.41 (d, J=8.2 Hz, 2H); 13C NMR (CDCl3,
75.5 MHz): d 17.8, 19.3, 28.4, 30.4, 36.1, 38.7, 58.3, 62.3,
69.7, 73.1, 80.4, 80.8, 107.5, 110.6, 122.2, 127.9, 128.6,
129.3, 129.9, 131.7, 131.8, 136.4, 142.3, 151.2, 157.1,
169.7, 171.4. MS (M+H)+: 687. Purity: 99%.
(2R,3R)-4-[N-Benzyl-N0-((S)-2-benzyloxycarbonylamino-
3-methyl-butyryl)-hydrazino]-2-(4-bromobenzyloxy)-3-
hydroxy-N-((1S,2R)-2-hydroxy-indan-1-yl)-butyramide
(41). Compound 41 was synthesized in 91% yield from
35 and Z-Val. Chromatography eluent: CHCl3+2%
MeOH satd with NH3. 41: [a]2D2 +3.0 (c 0.8, CHCl3); 1H
NMR (CDCl3, 300 MHz) (mixture of conformational
isomers): d 0.62–1.01 (m, 6H), 1.82–2.18 (m, 1H), 2.48–
2.59 (m, 1H), 2.87–3.18 (m, 4H), 3.66–3.78 (m, 1H),
3.84–4.13 (m, 4H), 4.42–4.68 (m, 3H), 4.95–5.17 (m,
2H), 5.13–5.17 (m, 1H), 5.69 (br s, 1H), 6.05 (br s, 1H),
6.98–7.45 (m, 15H), 7.14 (d, J=8.2 Hz, 2H), 7.41 (d,
J=8.2 Hz, 2H); 13C NMR (CDCl3, 75.5 MHz) (mixture
of conformational isomers): d 16.4, 17.8, 19.4, 20.1,
30.3, 31.0, 38.7, 39.3, 58.0, 59.2, 62.3, 67.3, 67.5, 69.9,
72.5, 72.7, 81.5, 82.4, 122.2, 124.1, 125.6, 127.2, 128.1,
128.2, 128.4, 128.5, 128.6, 128.7, 128.9, 129.4, 129.7,
131.8, 136.2, 140.0, 141.0, 156.8, 170.5, 171.3, 173.8. MS
(M+H)+: 773. Purity: 99%.
(2R,3R)-4-[N-Benzyl-N0-((S)-2-benzyloxycarbonylamino-
3-methyl-butyryl)-hydrazino]-2-(4-bromobenzyloxy)-N-
(furan-2-yl-methyl)-3-hydroxy-butyramide (39). Com-
pound 39 was synthesized in 92% yield from 34 and Z-
Val. Chromatography eluent: CHCl3+2% MeOH satd
with NH3. 39: [a]D22 +8.2 (c 0.9, CHCl3); 1H NMR
(CDCl3, 300 MHz): d 1.65–1.84 (m, 6H), 1.96–2.02 (m,
1H), 2.67–2.80 (m, 1H), 3.00 (dd, J=9.6, 12.6 Hz, 1H),
3.15 (br s, 1H), 3.66–3.77 (m, 1H), 3.86–4.14 (m, 4H),
4.33 (dd, J=5.9, 15.4 Hz, 1H), 4.45 (dd, J=6.1, 15.4
Hz, 1H), 4.54 (d, J=11.5 Hz, 1H), 4.66 (d, J=11.5 Hz,
1H), 4.95–5.07 (m, 2H), 5.12–5.22 (m, 1H), 6.18 (dd,
J=0.8, 3.3 Hz, 1H), 6.30 (dd, J=1.9, 3.3 Hz, 1H), 6.95–
7.03 (m, 1H), 7.11 (d, J=8.2 Hz, 2H), 7.20–7.37 (m,
12H), 7.40 (d, J=8.2 Hz, 2H); 13C NMR (CDCl3,
75.5 MHz): d 17.8, 19.3, 30.4, 36.1, 38.7, 58.2, 62.3, 67.2,
(2R,3R)-4-[N-Benzyl-N0 -((S)-2-tert-butyloxycarbonyl-
amino-3-methyl-butyryl)-hydrazino]-2-(4-bromobenzyloxy)-
3-hydroxy-N-(2-piperidin-1-yl-ethyl)-butyramide
(42).
Compound 42 was synthesized in 89% yield from 36
and Boc-Val. Chromatography eluent: ethyl acetate/
MeOH 9:1+1% Et3N. 42: [a]2D2 +8.3 (c 0.8, CHCl3); 1H
NMR (CDCl3, 300 MHz) (mixture of conformational
isomers): d 0.65-0-99 (m, 6H), 1.34–1.63 (m, 15H), 1.86–
2.00 (m, 1H), 2.10 (m, 1H), 2.25–2.54 (m, 6H), 2.81–3.12
(m, 2H), 3.22–3.42 (m, 2H), 3.60–3.71 (m, 1H), 3.93–
4.14 (m, 4H), 4.58 (d, J=11.5 Hz, 1H), 4.65 (d, J=11.5
Hz, 1H), 4.82–4.95 (m, 1H), 6.60 (br s, 1H), 7.03 (br s,
1H), 7.18 (d, J=8.2 Hz, 2H), 7.20–7.35 (m, 5H), 7.43 (d,
J=8.2 Hz, 2H); 13C NMR (CDCl3, 75.5 MHz) (mixture
of conformational isomers): d 17.8, 19.3, 24.3, 25.9,
28.4, 28.5, 30.4, 35.7, 54.3, 54.4, 57.0, 57.3, 58.2, 59.0,

A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
3435
62.1, 70.0, 72.9, 80.3, 80.9, 81.3, 121.9, 127.9, 128.4,
128.6, 128.7, 128.8, 129.3, 129.4, 129.6, 129.7, 129.8,
131.6, 131.7, 135.8, 136.7, 156.1, 169.9, 171.2, 171.4. MS
(M+H)+: 718. Purity: 95%.
according to the methods desribed in refs 11 and 13)
(0.580 g, 3.05 mmol) in dry THF (10 mL) were added p-
nitrobenzoic acid (0.82 g, 4.91 mmol) and Ph3P (1.27 g,
4.85 mmol). The reaction flask was placed in a water
bath at room temperature, and a solution of diethyl
azodicarboxylate (DEAD) (0.77 mL, 0.860 g, 4.94
mmol) in dry THF (10 mL) was added dropwise to the
stirred solution during half an hour at room tempera-
ture. The reaction solution was stirred overnight at
room temperature and was then evaporated and pur-
ified by flash column chromatography (toluene/EtOAc
6:1). The product was dissolved in MeOH (15 mL) and
was subsequently reacted with NaOMe in MeOH (1 M,
1.5 mL, 1.5 mmol), which was slowly added dropwise
into the stirred solution at room temperature. The
solution was stirred at room temperature for an addi-
tional 30 min, after which it was neutralized with
HOAc, evaporated, and purified by flash column chro-
matography (toluene/EtOAc 6:1). This afforded com-
pound 47 (0.543 g, 94%) as a colorless oil (cf. ref 11).
47: [a]2D2 ꢂ19.1 (c 0.5, CHCl3); 1H NMR (CDCl3,
300 MHz): d 1.23 (s, 3H), 1.30 (s, 3H), 3.09 (br s, 1H),
3.68 (s, 3H), 3.88 (dd, J=6.8, 8.2 Hz, 1H), 3.96 (dd,
J=6.8, 8.2 Hz, 1H), 4.03 (d, J=3.0 Hz, 1H), 4.27 (ddd,
J=3.0, 6.8, 6.8 Hz, 1H); 13C NMR (CDCl3, 75.5 MHz):
d 25.1, 25.9, 52.5, 65.4, 70.3, 76.2, 109.8, 172.4. MS
calcd for C8H15O5 (M+H)+: 191.09. Found: 191.06.
(2R,3R)-4-[N-Benzyl-N0-((S)-2-benzyloxycarbonylamino-
3-methyl-butyryl)-hydrazino]-2-(4-bromobenzyloxy)-3-
hydroxy-N-(2-piperidin-1-yl-ethyl)-butyramide
(43).
Compound 43 was synthesized in 68% yield from 36
and Z-Val. Chromatography eluent: ethyl acetate/
MeOH 9:1+1% Et3N. 43: [a]2D2 +6.4 (c 0.8, CHCl3); 1H
NMR (MeOH-d4, 300 MHz): d 0.64–0.78 (m, 6H), 1.38–
1.62 (m, 6H), 1.72–1.86 (m, 1H), 2.34–2.52 (m, 6H),
2.85–2.99 (m, 2H), 3.30–3.39 (m, 1H), 3.80–4.09 (m,
4H), 4.54 (s, 2H), 4.98 (d, J=12.6 Hz, 1H), 5.04 (d,
J=12.6 Hz, 1H), 7.13–7.38 (m, 12H), 7.45 (d, J=8.5
Hz, 2H); 13C NMR (MeOH-d4, 75.5 MHz): d 17.3, 18.4,
23.9, 25.4, 30.5, 35.7, 54.3, 57.4, 59.1, 59.9, 61.8, 66.5,
69.7, 72.1, 81.6, 121.5, 127.4, 127.6, 127.7, 127.8, 128.1,
128.3, 129.3, 129.9, 131.3, 136.8, 137.2, 156.5, 171.2,
171.9. MS (M+H)+: 752. Purity: 99%.
(2R,3R)-4-[N-Benzyl-N0-((S)-2-tert-butyloxycarbonyla-
mino-3-methyl-butyryl)-hydrazino]-2-(4-bromobenzyloxy)-
3-hydroxy-N-(pyridin-3-yl-methyl)-butyramide
(44).
Compound 44 was synthesized in 51% yield from 37
and Boc-Val. Chromatography eluent: ethyl acetate/
MeOH 9:1+1% Et3N. 44: [a]2D2 +4.5 (c 0.5, CHCl3); 1H
NMR (MeOH-d4, 300 MHz): d 0.63–0.75 (m, 6H), 1.40
(s, 9H), 1.67–1.80 (m, 1H), 2.89–3.00 (m, 2H), 3.59–3.66
(m, 1H), 3.85 (d, J=13.2 Hz, 1H), 3.96 (d, J=13.2 Hz,
1H), 3.99–4.10 (m, 2H), 4.40 (d, J=13.2 Hz, 1H), 4.46
(d, J=13.2 Hz, 1H), 4.52 (d, J=11.8 Hz, 1H), 4.59 (d,
J=11.8 Hz, 1H), 7.16–7.41 (m, 8H), 7.44 (d, J=8.5 Hz,
2H), 7.73–7.79 (m, 1H), 8.38–8.52 (m, 2H); 13C NMR
(MeOH-d4, 75.5 MHz): d 17.2, 18.4, 27.5, 30.7, 40.0,
59.2, 59.5, 61.7, 69.7, 72.0, 80.9, 81.5, 121.5, 124.0,
127.4, 128.1, 129.3, 130.0, 131.3, 135.4, 136.4, 136.8,
137.0, 147.6, 148.2, 156.8, 171.6, 172.0. MS (M+H)+:
698. Purity: 96%.
Methyl
(2S,3R)-2-(4-bromobenzyloxy)-3,4-dihydroxy-
3,4-O-isopropylidene- butyrate (48). To a solution of
compound 47 (0.455 g, 2.39 mmol) in toluene (10 mL)
were added p-bromobenzyl bromide (2.12 g, 8.47
mmol), Ag2O (1.68 g, 7.27 mmol), and KI (67 mg, 0.41
mmol) and the mixture was stirred at room temperature
for 15 min (some heat evolution was observed). The
suspension was filtered, evaporated, and purified by
flash column chromatography (toluene/EtOAc 6:1) to
give compound 48 (0.84 g, 98%). (cf. ref 11). 48: [a]D22
1
ꢂ46.4 (c 2.3, CHCl3); H NMR (CDCl3, 300 MHz): d
1.28 (s, 3H), 1.33 (s, 3H), 3.70 (s, 3H), 3.89 (dd, J=6.0,
8.8 Hz, 1H), 3.92 (d, J=6.0 Hz, 1H) 3.96 (dd, J=6.0,
8.8 Hz, 1H), 4.34 (ddd, J=6.0, 6.0, 6.0 Hz, 1H), 4.40 (d,
J=11.8 Hz, 1H), 4.65 (d, J=11.8 Hz, 1H), 7.18 (d,
J=8.2 Hz, 2H), 7.39 (d, J=8.2 Hz, 2H); 13C NMR
(CDCl3, 75.5 MHz): d 25.2, 26.2, 52.0, 65.4, 71.9, 75.7,
78.7, 109.8, 121.7, 129.6, 131.4, 136.2, 170.2. MS calcd
for C15H19BrO5K (M+K)+: 397.01. Found: 397.05.
(2R,3R)-4-[N-Benzyl-N0-((S)-2-benzyloxycarbonylamino-
3-methyl-butyryl)-hydrazino]-2-(4-bromobenzyloxy)-3-
hydroxy-N-(pyridin-3-yl-methyl)-butyramide (45). Com-
pound 45 was synthesized in 49% yield from 37 and
Z-Val. Chromatography eluent: ethyl acetate/MeOH
1
9:1+1% Et3N. 45: [a]2D2 +2.2 (c 1.1, CHCl3); H NMR
(MeOH-d4, 300 MHz): d 0.69 (d, J=6.9 Hz, 3H), 0.74
(d, J=6.7 Hz, 3H), 1.69–1.82 (m, 1H), 2.85–3.00 (m,
2H), 3.63–3.67 (m, 1H), 3.85 (d, J=13.7 Hz, 1H), 3.95
(d, J=13.7 Hz, 1H), 4.00–4.09 (m, 2H), 4.39 (d, J=13.5
Hz, 1H), 4.45 (d, J=13.5 Hz, 1H), 4.50 (d, J=11.8 Hz,
1H), 4.57 (d, J=11.8 Hz, 1H), 4.97 (d, J=12.4 Hz, 1H),
5.05 (d, J=12.4 Hz, 1H), 7.13–7.46 (m, 13H), 7.42 (d,
J=8.5 Hz, 2H), 7.72–7.79 (m, 1H), 8.38–8.52 (m, 2H);
13C NMR (MeOH-d4, 75.5 MHz): d 17.3, 18.4, 30.5,
40.0, 59.4, 59.9, 61.8, 66.5, 69.7, 72.0, 81.4, 121.5, 124.0,
127.4, 127.6, 127.8, 128.0, 128.2, 128.3, 129.3, 130.0,
131.3, 135.4, 136.4, 136.8, 137.0, 147.6, 148.2, 157.2,
171.6, 171.9. MS (M+H)+: 732. Purity: 97%.
Methyl
(2R,3S)-2-(4-bromobenzyloxy)-3,4-dihydroxy-
3,4-O-isopropylidene- butyrate (50). Compound 50 was
synthesized in 96% yield from methyl 3,4-O-iso-
propylidene-L-threonate (49) according to the proce-
dure for preparing 48. 50: [a]2D2 +47.3 (c 0.7, CHCl3);
NMR data: See compound 48. MS calcd for
C15H19BrO5K (M+K)+: 397.01. Found: 397.07.
(2S,3R)-2-(4-Bromobenzyloxy)-3,4-dihydroxy-3,4-O-iso-
propylidene-N-(2-piperidin-1-yl-ethyl)-butyramide (52).
Compound 52 was synthesized in 86% yield from 48
and 1-(2-aminoethyl)-piperidine according to the proce-
dure for preparing 4. Chromatography eluent: toluene/
ethyl acetate 1:1+1.5% Et3N. 52: [a]2D2 ꢂ30.3 (c 2.3,
Methyl (2S,3R)-3,4-O-isopropylidene-2,3,4-trihydroxy-bu-
tyrate (47). To a solution of compound 46 (synthesized
1
CHCl3); H NMR (CDCl3, 300 MHz): d 1.27 (s, 3H),

3436
A. Dahlgren et al. / Bioorg. Med. Chem. 11 (2003) 3423–3437
1.30–1.49 (m, 6H) 1.35 (s, 3H), 2.20–2.39 (m, 4H), 2.34
(t, J=6.0 Hz, 2H), 3.15–3.37 (m, 2H), 3.73 (d, J=6.2
Hz, 1H), 3.95 (dd, J=6.2 Hz, 8.8 Hz, 1H), 3.98 (dd,
J=6.2 Hz, 8.8 Hz, 1H), 4.22 (ddd, J=6.2, 6.2, 6.2 Hz,
1H), 4.54 (d, J=11.8 Hz, 1H), 4.60 (d, J=11.8 Hz, 1H),
7.14 (m, 1H), 7.20 (d, J=8.2 Hz, 2H), 7.41 (d, J=8.2
Hz, 2H); 13C NMR (CDCl3, 75.5 MHz): d 24.3, 25.5,
26.0, 26.4, 35.6, 54.2, 57.0, 66.1, 72.7, 76.8, 81.0, 109.4,
121.9, 129.5, 131.5, 136.2, 169.3. MS calcd for
C21H32BrN2O4 (M+H)+: 455.16. Found: 455.20.
in 89% yield from 56 and CbzNHNHBn according to
the procedure for preparing 32. Chromatography elu-
ent: ethyl acetate/MeOH 5:1+1% Et3N. 57: [a]2D2 +30.0
(c 1.1, CHCl3); NMR data: See compound 54. HRMS
calcd for C33H42BrN4O5 (M+H)+: 653.2338. Found:
653.2333. Purity: 99%.
(2S,3S)-2-(4-Bromobenzyloxy)-3,4-dihydroxy-3,4-O-iso-
propylidene-N-(2-piperidin-1-yl-ethyl)-butyramide (58).
Compound 58 was synthesized in 91% yield from 51
and 1-(2-aminoethyl)-piperidine according to the proce-
dure for preparing 4. Chromatography eluent: toluene/
ethyl acetate 1:1+1.5% Et3N. 58: [a]2D2 ꢂ34.9 (c 1.1,
CHCl3); NMR data: See compound 4. MS calcd for
C21H32BrN2O4 (M+H)+: 455.16. Found: 455.20.
(2S,3R)-2-(4-Bromobenzyloxy)-3,4-epoxy-N-(2-piperidin-
1-yl-ethyl)-butyramide (53). Compound 53 was synthe-
sized in 82% yield from 52 according to the procedure
for preparing 8. Chromatography eluent: ethyl acetate/
MeOH 5:1+1% Et3N. 53: [a]22 ꢂ7.5 (c 0.7, CHCl3); 1H
NMR (CDCl3, 300 MHz): d D1.34–1.57 (m, 6H), 2.28–
2.44 (m, 4H), 2.42 (t, J=6.0 Hz, 2H), 2.81–2.87 (m, 2H),
3.13–3.20 (m, 1H), 3.21–3.44 (m, 2H), 3.59 (d, J=6.3
Hz, 1H), 4.57 (d, J=11.8 Hz, 1H), 4.72 (d, J=11.8 Hz,
1H), 7.20–7.31 (m, 1H), 7.23 (d, J=8.5 Hz, 2H), 7.45 (d,
J=8.5 Hz, 2H); 13C NMR (CDCl3, 75.5 MHz): d 24.3,
26.0, 35.7, 44.0, 53.2, 54.2, 56.9, 71.7, 81.2, 122.0, 129.5,
131.6, 136.1, 168.3. MS calcd for C18H26BrN2O3
(M+H)+: 397.11. Found: 397.11.
(2S,3S)-2-(4-Bromobenzyloxy)-3,4-epoxy-N-(2-piperidin-
1-yl-ethyl)-butyramide (59). Compound 59 was synthe-
sized in 87% yield from 58 according to the procedure
for preparing 8. Chromatography eluent: ethyl acetate/
MeOH 5:1+1% Et3N. 59: [a]2D2 ꢂ25.2 (c 1.1, CHCl3);
NMR data: See compound 8. MS calcd for
C18H26BrN2O3 (M+H)+: 397.11. Found: 397.11.
(2S,3S)-4-(N-Benzyl-N0 -benzyloxycarbonyl-hydrazino)-
2-(4-bromobenzyloxy)-3-hydroxy-N-(2-piperidin-1-yl-ethyl)-
butyramide (60). Compound 60 was synthesized in 82%
yield from 59 and CbzNHNHBn according to the pro-
cedure for preparing 32. Chromatography eluent: ethyl
acetate/MeOH 5:1+1% Et3N. 60: [a]2D2 ꢂ9.9 (c 1.0,
CHCl3); NMR data: See compound 32. HRMS calcd
for C33H42BrN4O5 (M+H)+: 653.2338. Found:
653.2333. Purity: 98%.
(2S,3R)-4-(N-Benzyl-N0 -benzyloxycarbonyl-hydrazino)-
2-(4-bromobenzyloxy)-3-hydroxy-N-(2-piperidin-1-yl-
ethyl)-butyramide (54). Compound 54 was synthesized
in 73% yield from 53 and CbzNHNHBn according to
the procedure for preparing 32. Chromatography elu-
ent: ethyl acetate/MeOH 5:1+1% Et3N. 54: [a]2D2 ꢂ29.1
1
(c 0.7, CHCl3); H NMR (CDCl3, 300 MHz): d 1.38–
1.67 (m, 6H), 2.26–2.84 (m, 7H), 2.81 (dd, J=5.2, 12.9
Hz, 1H), 2.85–3.16 (m, 1H), 3.18–3.75 (m, 2H), 3.83–
4.17 (m, 4H), 4.26–4.65 (m, 2H), 4.97–5.07 (m, 2H), 6.01
(br s 1H), 7.03–7.40 (m, 13H), 7.43 (d, J=8.2 Hz, 2H);
13C NMR (CDCl3, 75.5 MHz): d 24.0, 25.4, 35.6, 54.4,
57.9, 58.6, 62.4, 67.0, 69.4, 72.6, 80.7, 122.2, 127.8, 128.1,
128.3, 128.6, 129.5, 129.6, 130.0, 131.7, 132.0, 136.1,
136.3, 156.4, 171.2. HRMS calcd for C33H42BrN4O5
(M+H)+: 653.2338. Found: 653.2333. Purity: 99%.
Acknowledgements
We greatly acknowledge Medivir AB for financial sup-
port and for performing the biological testings. We
would also like to thank Susana Ayesa at Medivir AB
and Prof. Hans Boren at the Department of Chemistry
at Linkoping University for valuable assistance during
the LC-MS analyses.
(2R,3S)-2-(4-Bromobenzyloxy)-3,4-dihydroxy-3,4-O-iso-
propylidene-N-(2-piperidin-1-yl-ethyl)-butyramide (55).
Compound 55 was synthesized in 91% yield from 50
and 1-(2-aminoethyl)-piperidine according to the proce-
dure for preparing 4. Chromatography eluent: toluene/
ethyl acetate 1:1+1.5% Et3N. 55: [a]2D2 +31.2 (c 1.3,
CHCl3); NMR data: See compound 52. MS calcd for
C21H32BrN2O4 (M+H)+: 455.16. Found: 455.11.
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