Design and synthesis of novel P2 substituents in diol-based HIV protease inhibitors

Article abstract of DOI:10.1016/j.ejmech.2009.09.038

The synthesis and SAR of HIV-1 protease inhibitors containing novel P2 structural elements are presented. The inhibitors were designed having hydrogen bond accepting P2 substituents to probe potential favorable interactions to Asp-29/Asp-30 of the HIV-1 protease backbone utilizing inhibitor 3 as a model template. Several inhibitors were synthesized from an l-Val methyl amide P2 motif by appending hydrogen bonding moieties from either the isopropyl side-chain or from the methyl amide portion. The most promising inhibitors 4a and 4e displayed K_i values of 1.0 nM and 0.7 nM respectively and EC₅₀ values in the MT4 cell-based assay of 0.17 μM and 0.33 μM respectively, a slight loss in potency compared to lead inhibitor 3. These inhibitors were also tested against an HIV protease inhibitor resistant strain carrying the M46I, V82F, and I84V mutations. Inhibitors 4a and 4e displayed a 3 and 4 fold change respectively compared with HIV wild type, whereas lead inhibitor 3 showed a higher 9 fold change. This study further demonstrate the chemical tractability of the approach where various P2 substituents can be introduced in just one chemical step from lactone 21 enabling facile modifications of the overall properties in this inhibitor class.

Full text of DOI:10.1016/j.ejmech.2009.09.038

European Journal of Medicinal Chemistry 45 (2010) 160–170
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of novel P2 substituents in diol-based HIV
protease inhibitors
,
Jenny Adrian Meredith a, Hans Wallberg b, Lotta Vrang b, Stefan Oscarson a c, Kevin Parkes b,
,b,
Anders Hallberg d, Bertil Samuelsson a
*
a Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
b Medivir AB, P.O. Box 1086, SE-141 22 Huddinge, Sweden
c Centre for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
d Department of Medicinal Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and SAR of HIV-1 protease inhibitors containing novel P2 structural elements are
presented. The inhibitors were designed having hydrogen bond accepting P2 substituents to probe
potential favorable interactions to Asp-29/Asp-30 of the HIV-1 protease backbone utilizing inhibitor 3 as
a model template. Several inhibitors were synthesized from an L-Val methyl amide P2 motif by
appending hydrogen bonding moieties from either the isopropyl side-chain or from the methyl amide
Received 7 March 2009
Received in revised form
3 September 2009
Accepted 25 September 2009
Available online 6 October 2009
portion. The most promising inhibitors 4a and 4e displayed Ki values of 1.0 nM and 0.7 nM respectively
and EC50 values in the MT4 cell-based assay of 0.17 mM and 0.33 mM respectively, a slight loss in potency
Keywords:
compared to lead inhibitor 3. These inhibitors were also tested against an HIV protease inhibitor resistant
strain carrying the M46I, V82F, and I84V mutations. Inhibitors 4a and 4e displayed a 3 and 4 fold change
respectively compared with HIV wild type, whereas lead inhibitor 3 showed a higher 9 fold change. This
study further demonstrate the chemical tractability of the approach where various P2 substituents can be
introduced in just one chemical step from lactone 21 enabling facile modifications of the overall prop-
erties in this inhibitor class.
HIV
HIV–PI
PI
Aspartic protease
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
PR inhibitors in combination with nucleoside analogues, which
targets the HIV-1 reverse transcriptase (RT), has emerged as one of
Over two decades ago the human immunodeficiency virus (HIV)
was identified as the etiologic agent of AIDS [1,2] and in 2008 it was
estimated by UNAIDS [3] that 33 million people were afflicted by
this disease. The HIV-1 genome encodes an aspartic protease [4,5]
(HIV-1 PR) that is essential for the production of structural and
functional viral proteins by processing of the gag- and gag-pol viral
gene products [4,6]. Inhibition of the HIV-1 PR leads to the
production of immature virus particles [7,8] and in clinical practice
HIV-1 PR inhibitors has demonstrated high efficacy which has led
to the development of efficacious treatment regimens.
the two major Highly Active Antiretroviral Therapy, HAART, treat-
ment regimens [20,21].
Fig. 1. Compound 1, Darunavir.
Today ten HIV-1 protease inhibitors are approved by the FDA;
Saquinavir, [9] Ritonavir, [10] Indinavir, [11,12] Nelfinavir, [13]
Amprenavir, [14] Fosamprenavir, [15] Lopinavir, [16] Atazanavir,
[17] Tiprinavir [18] and Darunavir [19] (compound 1, Fig. 1). HIV-1
Current drug discovery efforts are now focusing on once daily
HIV-1 PR inhibitors displaying improved efficacy, minor cross
resistance towards clinical mutant resistant strains, high genetic
barrier, low drug–drug interactions and an advantageous side effect
profile. Cost is also a major issue with current HIV therapies and
globally only w20% of the HIV infected population is receiving
adequate treatment [3].
* Corresponding author. Department of Organic Chemistry, Arrhenius Laboratory,
Stockholm University, S-106 91 Stockholm, Sweden. Tel.: þ46 8 54683100; fax: þ46
8 54683199.
E-mail address: bertil.samuelsson@medivir.se (B. Samuelsson).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.09.038

J. Adrian Meredith et al. / European Journal of Medicinal Chemistry 45 (2010) 160–170
161
We have previously reported on the discovery of novel potent
Commercially available Cbz-L-Asp-OMe 8 was converted into its
HIV–1 PR inhibitors, i.e. 2 [22] and 3 [23] (Fig. 2), displaying
a C2-symmetric diol-type central scaffold carrying P1/P10 benzyloxy
residues. In the C2-symmetrical inhibitor 2 there are two amino-
indanol P2/P20 residues whereas in inhibitor 3 one of the amino-
corresponding b-tert-butyl ester (Scheme 2) using isobutylene and
a catalytic amount of H2SO4 [25] furnishing 9 [26] in 82% yield.
Compound 9 was methylated [27,28] using LiHMDS and iodo-
methane to generate 10a/10b [29] as an inseparable mixture of
diastereomers in a 3:1 ratio in 89% yield. Selective hydrolysis [25]
with NaOH (1 M) rendered the acids 11a/11b as an inseparable
mixture in 95% yield. The carboxylic acids 11a and 11b were acti-
vated with ethyl chloroformate and then reduced with sodium
borohydride to generate the alcohols 12a [30] and 12b [30] that
were easily separated by column chromatography in 53% and 19%
yield, respectively.
indanols has been replaced by L-Val methyl amide.
Fig. 2. Lead inhibitors 2 and 3.
We now report on the synthesis and anti-HIV activities of
a series of L-Val methyl amide analogues as P2 residues in inhibitor
structure 3, (i.e. 4a–h, Fig. 3) containing potentially hydrogen
interacting or accepting elements such as fluorine, methoxy groups
and oxetane, with the aim to form positive interactions between
these P2 residues and the N–H moiety in Asp-29 or Asp-30 of the
HIV-1 protease backbone. The most promising inhibitor structures
4a and 4e displayed Ki values of 1.0 nM and 0.7 nM, respectively
and EC50 values in the MT4 cell-based assay of 0.17 mM and 0.33 mM,
Scheme 2. Reagents and conditions: i: Isobutylene, H2SO4 (cat), CH2Cl2; ii: HMDS,
BuLi, MeI, THF, 0 ꢀC; iii: NaOH 1 M, MeOH; iv: a) Ethyl chloroformate, NMM, THF; b)
NaBH4, H2O.
respectively, a slight loss in potency compared to lead inhibitor 3.
Interestingly, these inhibitors displayed a 3 and 4 fold change in
EC50 value, respectively, over HIV wild type when tested against an
HIV PI multiple resistant strain, whereas inhibitor 3 showed
a higher 9 fold change.
Commercially available L-homoserine 13 (Scheme 3) was Cbz
and Bn protected [31] using NaHCO3 and benzyl chloroformate,
followed by treatment with NaOH and benzyl bromide to generate
compound 12c in 50% yield over two steps.
Scheme 3. Reagents and conditions: i: benzyl chloroformate, NaHCO3; ii: NaOH,
benzyl bromide.
The free hydroxyl groups of 12a–c were converted into the
corresponding methyl ethers 14a–c (Scheme 4) in 44–56% yield
using methyl triflate [32] and 2,6-di-tert-butyl-4-methylpyridine.
Fig. 3. Target structures 4a–h.
2. Results and discussion
2.1. Chemistry
For the synthesis of the Cbz protected amines 5a–c (Scheme 1)
commercially available Cbz-L-Val-O-succinimide 6 was coupled
with amines 7a–c to generate 5a–c [24] in 25–84% yield [22].
Scheme 4. Reagents and conditions: i: MeOTf, 2,6-di-tert-butyl-4-methylpyridine,
CH2Cl2; ii: TFA, TES, CH2Cl2; iii: NaOH (aq), THF/MeOH (13:10); iv: PyBOP, DIPEA,
MeNH2, CH2Cl2.
The tert-butyl esters 14a and 14b were cleaved using TFA [33] to
render 15a and 15b in 88% and 74% yield respectively. The benzyl
ester of 14c was cleaved [34] with NaOH (0.59 M) to generate 14c in
58% yield. The methylamides 16a–c were obtained in 62–88% yield
from 15a–c using PyBOP [35], DIPEA and methylamine.
Scheme 1. Reagents and conditions: i: NMM, 7a, b or c, THF.

162
J. Adrian Meredith et al. / European Journal of Medicinal Chemistry 45 (2010) 160–170
For the synthesis of the Cbz protected amines 17a and 17b
Due to the low yields obtained a modified method using 2 equiv
of 2-hydroxypyridine and DIPEA as solvent was used to open
lactone 21 with the amines of 16b, 17a and 17b (Scheme 9)
furnishing products 4e, 4g and 4h in improved 32–65% yield.
(Scheme 5) a racemic mixture of oxetanylglycine methyl ester
18a/18b [36] was Cbz protected and separated by HPLC to
generate the protected amines 19a and 19b. The methyl ester was
converted to the corresponding methyl amide using methylamine
in ethanol (33%) to generate 17a and 17b in 86% and 49% yield
respectively.
Scheme 5. Reagents and conditions: i: MeNH2 33% in ethanol.
In order to confirm the stereochemistry of 17a, the methyl ester
of 19a was cleaved to generate the acid 20a in 32% yield (Scheme 6).
The Cbz-group was removed by hydrogenolysis over a catalytic
amount of palladium on charcoal to yield (þ)-3-oxetanylglycine in
78% yield, measurement of the optical rotation confirmed the
stereochemistry [36].
Scheme 9. Reagents and conditions: i: H2, Pd/C, MeOH; ii: 21, DIPEA, 2-hydroxypyr-
idine, 70ꢀ, 16 h.
Based on the promising results obtained from inhibitor 4e, the
synthesis of g-fluorinated amino acid analogues from amino acids
12b (Scheme 10) were attempted. A range of fluorination reagents
were examined e.g. DAST [38] Deoxofluor [39] and perfluoro-1-
butanesulfonyl fluoride [40] (PBSF) together with triethylamine
trihydrofluoride (TREAT) and TEA without success with only the
lactonized by-product 25b [41] isolated.
Scheme 6. Reagents and conditions: i: NaOH (1 M), MeOH; ii: H2, Pd/C, MeOH.
The monolactone 21 (Scheme 7) was synthesized in three steps
from
L-mannaric acid g-lactone 22 according to the published
method [37].
Scheme 10. Reagents and conditions: i: DAST, Deoxofluor or PBSF, TEA and TREAT,
DCM or THF.
12a was converted into its corresponding mesylate [42] 26a
(Scheme 11), which was reacted with e.g. TBAF, CsF and KF with 18-
crown-6 in aprotic solvents without providing the desired product.
Scheme 7. Reagents and conditions: i: HNO3, H2O; ii: benzyl-2,2,2-tri-
chloroacetimidate, TfOH, 1,4-dioxane; iii: (1S,2R)-1-amino-2-indanol, 2-hydroxypyr-
idine, CH2Cl2.
The Cbz protection group of 5a–c, 16a–c and 17a and 17b was
removed by hydrogenolysis over a catalytic amount of palladium on
charcoal (Schemes 8 and 9), delivering the corresponding amines
which were used without further purification in the subsequent
step. The amines of 5a–c, 16a and 16c were coupled with the
lactone 21 in refluxing 1,2-dichloroethane to give the products
4a–d and 4f in 15–28% yield (Scheme 8).
Scheme 11. Reagents and conditions: i: MsCl, TEA, DMAP, 0 ꢀC, ii: TBAF, CsF or KF and
18-crown-6.
2.2. Structure activity relationships
The well known amino-indanol moiety is a remarkably efficient
P2/P20 substituent [12,22,43] very neatly filling out the S2/S20
pockets and forming a hydrogen bond to the backbone of Asp-29/
129 in the HIV–PR. It is however highly susceptibility to oxidative
metabolism, mainly mediated by the 3A4 isozyme of the hepatic
cytochrome P-450 system, resulting in rapid hepatic clearance and
poor oral bioavailability, [44] which has stimulated the search for
more drugable P2–P3 amines. We were intrigued by the observa-
tion that whilst the P2 residue in inhibitor 3 does not occupy the S2
pocket to the same extent as the amino-indanol in inhibitor 2, it is
still a highly potent inhibitor. It was thus considered that modifi-
cations in the P2 position of inhibitor 3 might provide scope for
additional potency and overcoming some of the shortcomings of
the amino-indanol moiety.
Scheme 8. Reagents and conditions: i: H2, Pd/C, MeOH; ii: 21, 1,2-dichloroethane,
reflux, 16 h.

J. Adrian Meredith et al. / European Journal of Medicinal Chemistry 45 (2010) 160–170
163
Table 1 (continued)
4
5
4c
2.3
0.25
1.2
5
4d
18
3.5 >10
>2
6
7
4e
4f
0.7
0.33
1.8
1.3
4
3
Fig. 4. 1EBZ PDB X-ray crystal structure of inhibitor 3 co-crystallized with the HIV-1
protease. The flap residues (Met46 – Ile54 and Met146 – Ile154) have been deleted to
improve visibility of the inhibitor binding and the surface color coded by lipophilic
potential.
10
6.9
Inhibitors containing a ‘‘bis-THF’’ like P2 motif, e.g. inhibitor 1 [19]
(Fig. 1), display high potency and moreover, broad-spectrum activity
against multi-PI resistant HIV-1 variants. A 3D structure of inhibitor
1 bound to the HIV protease [45] display a tight binding between the
‘‘bis-THF’’ residue and the main chains of the protease active site
aminoacids (Asp-29and Asp-30). Asthemainchains arelesssensitive
to mutations this binding is postulated to be a key attributing factor
to maintaining potency against multi-drug-resistant HIV-1 [46].
From examining the 3D structure of inhibitor 3 bound to HIV-1
protease (Fig. 4) [47] two design alternatives were selected: a)
building from the methyl group of the methyl amide which is
5.07 Å from the backbone nitrogen of Asp-29, but significantly
further (6.17 Å) from that of Asp-30 and b) extending from the
isopropyl group which is somewhat closer to both potential H-bond
donors, 4.05 Å from the backbone nitrogen of Asp-30 and 4.27 Å
from that of Asp-29.
Three compounds, 4a–c, (Scheme 8) were designed to interact
with Asp-29 and five compounds, 4d–h, with Asp-30 (Schemes 8
and 9). The inhibitors were evaluated for their antiviral potency
against the HIV protease (Ki) and anti-HIV activity in an MT4
cell-based assay (EC50) against both wild type virus and an HIV
protease inhibitor resistant strain carrying the M46I, V82F, and
I84V mutations (Table 1).
8
9
4g
4h
99
10
>10
>10
–
5.4 >10
>1.6
Inhibitors 4a–c all displayed similar potencies (Ki values of 1.0,
1.9 and 2.3 nM, respectively) and antiviral activity (EC50 values of
0.17, 0.15 and 0.25 M, respectively) and with slightly less loss in
cell-based activity against a resistant mutant strain than the lead
inhibitor 3 (Table 1). Inhibitors 4a and 4b were also evaluated for
their apparent permeability (Papp) in a Caco-2 cell assay, and
compared with inhibitor 3 they displayed a somewhat increased
permeability (Table 2).
m
Table 2
HIV-1 protease inhibitor permeability in a Caco-2 cell assay.
Entry
Compound
Papp(Caco-2), 10ꢁ6 cm/s
1
2
3
3
4a
4b
1.4
1.6
2.9
Table 1
HIV-1 protease inhibitory activities.
Log P and the topological polar surface area (PSA) (Table 3) were
calculated for compounds 4a–h where the increase in calculated
log P for compounds 4a and 4b compared to inhibitor 3 supports
the slight increase in permeability observed. Inhibitors 4d–f each
Entry Compound
R]
Ki
(nM)
EC50
M)
EC50 resistant clone EC50
(
m
(m
M)
ratio
Table 3
Calculated log P and topological polar surface areas for the inhibitors.
1
3
0.2
0.14
1.2
9
Entry
Compound
Log P
PSA
1
2
3
4
5
6
7
8
9
3
2.68
2.84
3.66
2.52
1.86
1.86
1.36
1.22
1.22
166
166
166
176
176
176
176
176
176
4a
4b
4c
4d
4e
4f
2
3
4a
4b
1.0
1.9
0.17
0.15
0.44
0.92
3
5
4g
4h

164
J. Adrian Meredith et al. / European Journal of Medicinal Chemistry 45 (2010) 160–170
have a 2-methoxy-ethyl P2 residue, where 4e having a 1R-methyl
substituent on the P2 residue is the most potent, displaying a Ki
value of 0.7 nM and antiviral activity EC50 value 0.33. Finally, the
smaller size and more rigid oxetane compounds 4g–h lost
considerable in potencies both on enzyme and in the cell-based
anti-HIV assay.
Visual inspections of the 4d docks reveal the top pose to be
a miss-dock with disrupted interactions with the catalytic aspar-
tates. Some of the lower ranked docks, and pose 3 in particular,
show a weak H-bond to Asp-30 (3.41 Å) and a much more tenuous
one to Asp-29 (4.00 Å) (Fig. 6). Unfortunately this is achieved at the
expense of a close contact (3.46 Å) between the unsubstituted P2
methyl group and the flap Ile-37 side-chain which may account for
the low potency observed. All five docks of 4e show that the
methoxy group can be accommodated along the binding channel
towards bulk solvent, but fail to make either of the desired polar S2
interactions. This result is compatible with the minor effect on
activity resulting from this substitution.
2.3. Modeling
Docking of compounds 4a and 4b in the crystal structure of 1EBZ
gave high scoring poses (entries 2 and 3, Table 4), although further
inspection indicated that neither 4a nor 4b would achieve polar
interactions with Asp-29 or Asp-30. For these two inhibitors the
substituent added to the terminal methyl amide group lies in the
solvent channel and directed away from the polar groups in the S2
pocket (Fig. 5).
Fig. 6. Docked pose of inhibitor 4e (magenta) overlaid with that of inhibitor 3 (orange)
from the 1EBZ crystal structure presented as in Fig. 3.
The significantly lower biological activity of 4f is puzzling
since this inhibitor should be able to adopt a conformation similar
to that of 4e. Despite the slightly lower docking score, this possibly
reflects the likely entropic cost of constraining a highly mobile
side-chain.
Fig. 5. Docked structure of inhibitor 4a (magenta) overlaid with that of inhibitor 3
(orange) from the 1EBZ crystal structure presented as in Fig. 3.
The oxetane moiety in 4g and 4h is a small, rigid oxygen con-
taining ring in the equivalent position to the methoxy substituents
in 4d–f. It is also a conformationally constrained analogue of the
The introduction of fluorine lead to a minor increase in calcu-
lated log P (entry 1, Table 3), which is reflected in a corresponding
slight improvement in Caco-2 permeability (entry 2, Table 2)
relative to inhibitor 3 (entry 1, Table 2). The effect of fluorination on
permeability is more marked in the trifluorinated analogue 4b
(entry 3, Table 2).
Docking of 4d and 4e gave similar high scoring top poses of
which the highest ranked are disclosed in Table 4. Furthermore,
the subsequent four docks for 4d have markedly lower scores
(90.19–92.95) which are in contrast to the docks for 4e, where
the scores (95.37–96.48) are comparable with the top pose
(Table 4).
g-methoxy-L-Val found in 4d and 4e.
However the activities of these analogues were found to be
disappointingly low. This is in agreement with the markedly lower
docking scores (Table 4) and is perhaps unsurprising for the
‘‘unnatural’’ enantiomer 4g and could also be rationalized for 4h on
inspection of the modeled structure where the effect of the ring
system is to ‘‘tie back’’ the oxygen atom away from both the Asp-29
and Asp-30 N–Hs and from solvent.
These inhibitors were also tested against an HIV protease
inhibitor resistant strain carrying the M46I, V82F, and I84V muta-
tions (Table 1). Inhibitors 4a (entry 2, Table 1) and 4e (entry 6, Table
1) in particular displayed substantially lower losses of activity
against the resistant clone than does lead inhibitor 3 (entry 1, Table
1). However, whilst the I84V mutation is on the edge of the S2
pocket and might influence the binding, there is no clear overall
pattern in the fold changes observed for these inhibitors against the
mutant clone.
Table 4
GoldScores of the highest ranked poses.
Entry
Compound
GoldScore
1
2
3
4
5
6
7
8
9
3
96.45
99.05
101.51
102.11
98.45
97.34
94.90
81.58
85.72
4a
4b
4c
4d
4e
4f
3. Conclusion
4g
4h
Eight novel inhibitors have been synthesized where inhibitor 4a
and inhibitor 4e furnished the most potent inhibition of the HIV-1

J. Adrian Meredith et al. / European Journal of Medicinal Chemistry 45 (2010) 160–170
165
protease and where inhibitors 4a and 4b were roughly equipotent
to lead 3 (EC50 ¼ 0.1 M) in regards to anti-HIV activity in the MT4
the automatic genetic algorithm parameters and the GoldScore
fitness function. Because of the symmetric nature of the enzyme
H-bond constraints from the ligand to the conserved water were
applied to ensure that all docking solutions were bound in
a consistent orientation. From each run the five highest ranked
poses were retained for assessment both on the basis of the docking
score and by visual inspection.
m
cell-based assay. Noteworthy, the introduction of one or more
fluorines, i.e. inhibitors 4a and 4b, increases the cell permeability,
as expected and supported by Caco-2 data and calculated log P data
and this may contribute to the overall favorable anti-HIV activity.
Inhibitors 4d, 4e, 4g and 4h, have extensions from the P2
isopropyl group and inhibitor 4f have a P2 methoxyethyl group. Of
these compounds inhibitor 4e is believed to interact favorably with
Asp-30. However, extensions with polar groups from the isopropyl
side-chain of the P2 Val appear to be less well accommodated in the
S2 pocket.
Calculations of log P and topological polar surface area were
performed with ChemAxon’s calculators [53,54].
4.4. General methods
Attempts to make a fluorine analogue of 4e to evaluate a smaller
electronegative substituent and its interactions in the S2 pocket
were not successful. When tested against a multi resistant clone, in
particular two of the analogues prepared, 4a and 4e, displayed
favorable lower fold changes compared with lead inhibitor 3.
The advantageous cellular anti-HIV activities for the fluorinated
compounds 4a and 4b as well as the short synthesis routes from the
appropriate P2 amino acids (one chemical step) warrants further
explorations of fluorinated P2-substituents.
All glassware was dried over an open flame before use in
connection with an inert atmosphere. Concentrations were
performed under reduced pressure at <40 ꢀC (bath tempera-
ture). Thin layer chromatography was performed using Merck
silica gel 60 F-254 plates with detection by UV, charring with 8%
sulphuric acid or ammonium molybdate (100 g): Ce(IV)sulphate
(2 g): sulphuric acid (10%, 2 L). Column chromatography was
performed on silica (0.035–0.070 mm). NMR spectra was recor-
ded at 25 ꢀC on a Varian 300 MHz or 400 MHz instrument using
the solvent residual peak (CDCl3 1H
d
¼ 7.26 ppm and 13C
4. Experimental section
d
¼ 77.16 ppm or CD3OD 1H
d
¼ 3.31 and 13C
d
¼ 49.0 ppm) as
standard. Optical rotations were measured on a Perkin–Elmer
241 polarimeter using a 10 cm, 1 mL cell. LC–MS was performed
on a Waters (micro mass ZQ) preparative LC–MS, using a reverse
4.1. HIV-1 protease inhibition
HIV-1 protease was cloned and heterologously expressed in
Escherichia coli [48] and Ki-values were determined using a fluoro-
metric assay [49].
phase column: ACE C-8, 21 ꢂ100 mm, 5
mm particles, 100A from
ACE (UK) and the mobile phases were based on water/acetoni-
trile containing 0.1% TFA. Mass spectra were recorded with
a
micrOTOF 125 spectrometer. Unless stated otherwise, all
4.2. In vitro anti-HIV activity
materials were obtained from commercial suppliers and used
without further purification. CH2Cl2 and THF were distilled from
CaH2 before use.
The anti-HIV activity was measured in an HIV cytopathic
assay in MT-4 cells where the effect was quantified using vital
dye XTT [50]. The mutant virus was generated in MT4 cells when
HIV, IIIb was replicating in the presence of increasing concen-
trations of Ritonavir. Resistant virus containing M46I, V82F, and
I84V was isolated after 20 passages and used for cross resistance
studies.
Briefly, test compounds in 5-fold serial dilutions were added to
MT4 cells (2 ꢂ104 cells/well) in a micro plate and at the same time
was added 20–50 tissue culture infectious doses (TCID) of wild type
or mutant HIV, IIIb.
4.5. Synthetic experiments
4.5.1. General method for the preparation of compounds 5a–c
To a cooled, ꢁ15 C, stirred suspension of Cbz-Val-OSu (1 equiv)
in dry THF (5 mL) was added 4-methylmorpholine (3 equiv). After
careful addition of the amine (1.5 equiv) the stirred solution was
allowed to attain room temperature and left for 16 h. The solvent
was removed and the crude product purified by silica gel
chromatography (light petroleum (40–60)–ethyl acetate, gradient
3:1 – 1:1) to yield the title compounds 5a–c.
The viability of the cells was determined with XTT after five days
of incubation and the 50% effective concentrations (EC50) were
calculated from dose response curves of the percent cytoprotection
for individual compound concentrations.
4.5.1.1. (S)-benzyl 1-(2-fluoroethylamino)-3-methyl-1-oxobutan-2-yl
carbamate (5a). The compound 5a was prepared from 2-fluorethyl
amine, according to general procedure, vide supra, in 84% yield
4.3. Modeling
(250 mg, 0.84 mmol) as a crystalline white solid. [
a]D – 12.1 (c 0.66,
CHCl3); 1H NMR (400 MHz, CDCl3,)
d
7.29 (m, 5H), 5.76 (d, 1H,
Ligand building, and general modeling and visualization with
performed with Sybyl 8.0 [51]. The docking receptor was
prepared from the 1EBZ RCSB PDB file in Sybyl by addition of
hydrogen atoms and charges and the deletion of the ligand and
of all water molecules with the exception of the one between the
flaps which H-bonds to the ligand. Receptor residues were
ionized as appropriate for pH 7.2 and in particular the two
catalytic aspartyl residues were both ionized. The ligand mole-
cules were sketched in 2D and protonated and converted to 3D
with Concord and minimized in Sybyl with MMFF94S force field
using the default parameters.
J ¼ 8.0 Hz), 5.05 (s, 2H), 4.49 (t, 1H, J ¼ 4.5, 9.0 Hz), 4.37 (t, 1H,
J ¼ 4.5, 9.0 Hz), 3.89 (dd,1H, J ¼ 6.7, 9.0 Hz), 3.48 (m, 2H), 2.55 (d,1H
J ¼ 7.2 Hz), 2.02 (m, 1H), 0.90 (dd, 6H, J ¼ 6.8, 11.3 Hz). 13C NMR
(100 MHz, CDCl3,)
d 172.2, 156.8, 136.2, 128.6, 128.3, 128.0, 83.2,
81.6, 67.1, 60.5, 40.1, 31.9, 31.2, 19.2, 18.0; MS (ESI) m/z 319.1
([M þ Na]þ calcd for C15H21FN2NaOþ3 319.1).
4.5.1.2. (S)-benzyl 3-methyl-1-oxo-1-(2,2,2-trifluoroethylamino)
butan-2-ylcarbamate (5b). The compound 5b was prepared from
2,2,2-trifluorethyl amine according to general procedure, vide
supra, in 83% yield (300 mg, 0.84 mmol) as a crystalline white solid.
20
Docking calculations were performed with GOLD v4.0 [52]. The
binding site was defined as receptor atoms within 10 Å of the
oxygen atom of the inter-flap water and the GOLD cavity detection
algorithm applied. The genetic algorithm was run 25 times using
[a] d 8.07 (bs,
D – 8.5 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3,)
1H), 7.25 (m, 5H), 5.00 (s, 2H), 3.95 (m, 1H), 3.88 (d, 1H, J ¼ 6.7 Hz),
1.94 (m, 1H), 0.90 (dd, 6H, J ¼ 6.8, 9.6 Hz); 13C NMR (100 MHz,
CDCl3,)
d 172.8, 156.8, 136.1, 128.5, 128.2, 127.9, 125.4, 122.7, 67.0,

166
J. Adrian Meredith et al. / European Journal of Medicinal Chemistry 45 (2010) 160–170
20
60.2, 40.6, 40.2, 31.2, 18.8, 17.7; MS (ESI) m/z 355.1 ([M þ Na]þ calcd
4-hydroxybutanoate (12b). [
a]
D þ 62.7 (c 1.00, CHCl3); 13C NMR
for C15H19F3N2NaOþ3 355.1).
(75 MHz, CDCl3,) d 171.2, 157.7, 136.0, 128.7, 128.4, 128.3, 82.6,
67.5, 64.0, 54.2, 38.9, 28.1, 9.9. MS (ESI) m/z 346.2 ([M þ Na]þ
calcd for C17H25NNaOþ5 346.2). 1H NMR data were in agreement
with published values [30].
4.5.1.3. (S)-benzyl 1-(2-methoxyethylamino)-3-methyl-1-oxobutan-
20
2-ylcarbamate (5c). [a] D – 8.3 (c 1.00, MeOH); MS (ESI) m/z 331.2
([M þ Na]þ calcd for C16H24N2NaO4þ 331.2); 1H NMR data and 13C
NMR data were in agreement with published values [24].
4.5.2. General method for preparation of compounds 14a–c
The alcohol (1 equiv) was dissolved in dry CH2Cl2 (3 mL) and
stirred under argon when 2,6-di-tert-butyl-4-methylpyridine
(3 equiv) was added. The mixture was cooled on an ice bath and
MeOTf (3 equiv) was added. The reaction mixture was allowed to
reach room temperature and was then left stirring for 16 h. After
addition of NaHCO3 (aq., sat., 3 mL), the organic phase was sepa-
rated and the aqueous phase was extracted with CH2Cl2 (3 ꢂ 3 mL).
The organic phases were combined and washed with brine, dried
(MgSO4), concentrated and purified by silica column chromatog-
raphy (toluene–ethyl acetate, gradient 100:1–20:1) to give the title
compounds 14a–c.
4.5.1.4. (S)-1-tert-butyl
4-methyl
2-(benzyloxycarbonylamino)
succinate (9), 8. (15 g, 53.3 mmol) was dissolved in CH2Cl2
(200 mL) in a 500 mL three necked round bottom flask that was
equipped with a gas inlet and a cooling finger. After treatment with
H2SO4 (conc., 0.80 mL) the solution was cooled to ꢁ78 ꢀC and iso-
butene (694 mmol, 66 mL) was condensed into the mixture. The
reaction was sealed, allowed to reach room temperature and then
left stirring for 16 h. After neutralisation with NaHCO3 (aq., sat.), the
organic phase was washed with brine, dried over Na2SO4
and concentrated. After purification by silica gel chromatography
(toluene–ethyl acetate, gradient 50:1–30:1) compound 9 was
obtained as a clear oil in 82% yield (14.65 g, 43.4 mmol). MS
(ESI) m/z 374.2 ([M þ Na]þ calcd for C18H25NNaO6þ 374.2); 1H
NMR data and 13C NMR data were in agreement with published
values [26].
4.5.2.1. (2S,3R)-tert-butyl 2-(benzyloxycarbonylamino)-4-methoxy-
3-methylbutanoate (14a). The compound 14a was prepared from
12a, according to general procedure, vide supra, in 52% yield
20
(53 mg, 0.16 mmol).
(300 MHz, CDCl3,)
[a
]
D þ 2.5 (c 1.00, CHCl3); 1H NMR
d
7.38–7.29 (m, 5H), 5.69 (d, 1H, J ¼ 8.5 Hz), 5.10
4.5.1.5. (2S,3R)-3-(benzyloxycarbonylamino)-4-tert-butoxy-2-
methyl-4-oxobutanoic acid (11a) and (2S,3S)-3-(benzyloxy
carbonylamino)-4-tert-butoxy-2-methyl-4-oxobutanoic acid (11b).
To a stirred mixture of 10a and 10b (2.13 g, 6.06 mmol) in MeOH
(10 mL) was added NaOH (1 M, 6.7 mL). After stirring for 2 h in
room temperature the mixture was concentrated to approx. 4 mL
thereafter diluted with H2O (15 mL). The aqueous solution was
extracted with Et2O (2 ꢂ15 mL) before being transferred to
a round bottom flask and Et2O (15 mL) was added. During
vigorous stirring of the mixture, the pH was set to w2 using HCl
(conc.). The phases were separated and the aqueous phase was
extracted with Et2O (15 mL). The pooled organic phases were
washed with brine, dried (Na2SO4), concentrated and filtered
through a short silica gel column (toluene–ethyl acetate, gradient
10:1–1:1) to yield the crude product as a clear syrup (1.94 g,
5.75 mmol, 95%), which was used in the next step without further
purification.
(s, 2H), 4.27 (dd, 1H, J ¼ 4.2, 8.8 Hz), 3.36–3.24 (m, 4H). 2.34 (m,
1H), 1.45 (s, 9H), 1.00 (d, 3H, J ¼ 7.1 Hz); 13C NMR (75 MHz, CDCl3,)
d
171.0, 156.6, 136.6, 128.6, 128.2, 81.9, 74.6, 66.9, 59.0, 57.7, 35.9,
28.1, 14.4; MS (ESI) m/z 360.2 ([M þ Na]þ calcd for C18H27NNaOþ5
360.2).
4.5.2.2. (2S,3S)-tert-Butyl 2-(benzyloxycarbonylamino)-4-methoxy-
3-methylbutanoate (14b). The compound 14b was prepared from
12b, according to general procedure, vide supra, in 56% yield
20
(100 mg, 0.29 mmol).
(300 MHz, CDCl3,)
[a]
D þ 6.7 (c 1.00, CHCl3); 1H NMR
d
7.39–7.29 (m, 5H), 5.51 (d,1H, J ¼ 8.7 Hz), 5.11 (s,
2H), 4.27 (dd, 1H, J ¼ 2.7, 8.9 Hz), 3.35–3.18 (m, 4H). 2.30 (m,
1H), 1.46 (s, 9H), 0.88 (d, 3H, J ¼ 7.1 Hz); 13C NMR (75 MHz,
CDCl3,)
d 171.0, 156.3, 136.6, 128.6, 128.2, 82.1, 74.9, 67.0, 59.0, 56.3,
36.6, 28.1, 12.3; MS (ESI) m/z 360.2 ([M þ Na]þ calcd for
C18H27NNaOþ5 360.2).
4.5.2.3. (S)-Benzyl 2-(benzyloxycarbonylamino)-4-methoxybuta
noate (14c). The compound 14c was prepared from 12c, according
4.5.1.6. (2S,3R)-tert-butyl 2-(benzyloxycarbonylamino)-4-hydroxy-
3-methylbutanoate (12a) and (2S,3S)-tert-butyl 2-(benzyloxy
carbonylamino)-4-hydroxy-3-methylbutanoate (12b). To a cooled
(ꢁ10 ꢀC) stirred solution of 11a and 11b (717 mg, 2.12 mmol) in dry
to general procedure, vide supra, in 44% yield (321 mg, 0.90 mmol).
20
[a
]
D ꢁ 11.8 (c 1.00, MeOH); 1H NMR (300 MHz, CDCl3,)
d 7.40–7.29
(m, 10H), 5.77 (d, 1H, J ¼ 7.8 Hz), 5.17 (s, 2H), 5.11 (s, 2H), 4.53 (m,
THF (4 mL) was added NMM (240
m
l, 2.14 mmol). After an addi-
1H), 3.48–3.32 (m, 2 H), 3.23 (s, 3H), 2.20–1.93 (m, 2H); 13C NMR
tional 3 min ethyl chloroformate (205
ml, 2.14 mmol) was added
(75 MHz, CDCl3,)
d 172.1, 156.1, 136.5, 135.6, 128.7, 128.6, 128.5,
drop wise. The mixture was allowed to reach room temperature
during 15 min and was then filtered. The filtrate was added drop
wise during 30 min to a cooled (3 ꢀC) solution of NaBH4 (176 mg,
4.66 mmol) in H2O (2 mL). The mixture was allowed to slowly reach
room temperature. After 3 h, the mixture was again cooled on an
ice bath, and acidified to pH w2 with HCl (conc.). After extraction
with ethyl acetate (3 ꢂ 7.5 mL), the organic phases was washed
with brine, dried (Na2SO4) and concentrated to yield the crude
product. After silica gel chromatography (toluene–ethyl acetate,
gradient 100:1–40:1) 12b was obtained (130 mg, 0.40 mmol, 19%)
followed by 12a (362 mg, 1.12 mmol, 53%). tert-Butyl (2S,3R)-2-
128.4, 128.2, 69.0, 67.2, 67.0, 58.9, 52.6, 31.9; MS (ESI) m/z 380.1
([M þ Na]þ calcd for C20H23NNaOþ5 380.1).
4.5.3. General method for preparation of compounds 15a and 15b
The tert-butyl ester (1 equiv) was dissolved in dry CH2Cl2
(32 equiv) and while stirring TFA (13 equiv) was added followed by
Et3SiH (2.5 equiv). After 45 min of stirring in room temperature the
reaction mixture was concentrated and toluene was then co-
evaporated three times from the residue, which was then purified
on a short silica gel column (toluene–ethyl acetate, gradient: 10:1–
1:1) to yield compounds 15a and 15b which were used without
further purification in the next step.
[(benzyloxycarbonyl)amino]-3-methyl-4-hydroxybutanoate (12a).
20
[a]
D þ 2.5 (c 1.00, CHCl3); 13C NMR (75 MHz, CDCl3,)
d 171.3,
156.7, 136.3, 128.6, 128.3, 128.2, 82.5, 67.2, 64.4, 57.0, 38.8, 28.1,
13.8. MS (ESI) m/z 346.2 ([M þ Na]þ calcd for C17H25NNaOþ5
346.2). 1H NMR data were in agreement with published values
[30]. tert-Butyl (2S,3S)-2-[(benzyloxycarbonyl)amino]-3-methyl-
4.5.3.1. (2S,3R)-2-(benzyloxycarbonylamino)-4-methoxy-3-methyl-
butanoic acid (15a). The compound 15a was prepared from 14a,
according to general procedure, vide supra, in 88% yield (158 mg,

J. Adrian Meredith et al. / European Journal of Medicinal Chemistry 45 (2010) 160–170
167
20
0.56 mmol). [
d
a
]
D þ 13.7 (c 0.2, CHCl3); 1H NMR (400 MHz, CDCl3,)
4.5.4.3. (S)-benzyl 4-methoxy-1-(methylamino)-1-oxobutan-2-ylcar
bamate (16c). The compound 16c was prepared from 15c, accord-
7.39–7.29 (m, 5H), 5.92 (d,1H, J ¼ 7.1 Hz), 5.12 (s, 2H), 4.41 (bs,1H),
3.56 (s, 1H) 3.40–3.23 (m, 5H). 2.44 (bs, 1H), 1.05 (d, 3H, J ¼ 7.1 Hz);
ing to general procedure, vide supra, in 88% yield (115 mg,
13C NMR (100 MHz, CDCl3,)
d
156.8, 136.4, 128.7, 128.3, 128.3, 75.2,
0.41 mmol).
[
a
]
D ꢁ 14.1 (c 1.0, MeOH); 1H NMR (400 MHz,
20
67.2, 59.3, 35.5, 29.8, 14.0; MS (ESI) m/z 304.1 ([M þ Na]þ calcd for
CD3OD,) d 7.38–7.23 (m, 5H), 5.09 (m, 2H), 4.86 (s, 1H), 4.19 (dd, 1H,
C14H19NNaOþ5 304.1).
J ¼ 4.9, 8.9 Hz), 3.42 (dd, 1H, J ¼ 5.2, 6.8 Hz), 3.29 (s, 3H), 2.73 (s,
3H). 2.02 (m, 1H), 1.83 (m, 1H); 13C NMR (75 MHz, CD3OD,)
172.8,
(2S,3S)-2-(benzyloxycarbonylamino)-4-methoxy-3-methyl-
butanoic acid (15b)
d
156.5, 136.0, 128.4, 128.1, 127.9, 69.1, 66.9, 58.6, 32.1, 25.9; MS (ESI)
The compound 15b was prepared from 14b, according to general
m/z 303.1 ([M þ Na]þ calcd for C14H20N2NaOþ4 303.1).
20
procedure, vide supra, in 74% yield (62 mg, 0.22 mmol). [
a
]
D ꢁ 4.1
(c 1.00, MeOH); 1H NMR (400 MHz, CD3OD,)
d
7.40–7.27 (m, 5H),
4.5.4.4. (R)-Methyl 2-(benzyloxycarbonylamino)-2-(oxetan-3-yl ace
20
5.10 (s, 2H), 4.43 (d, 1H, J ¼ 3.9 Hz), 3.29–3.20 (m, 5H). 2.36 (m, 1H),
tate) (19a). [
d
a]
D þ 14.4 (c 1.0, MeOH); 1H NMR (400 MHz, CDCl3,)
0.89 (d, 3H, J ¼ 7.1 Hz); 13C NMR (100 MHz, CD3OD,)
d
158.8, 138.2,
7.40–7.29 (m, 5H), 5.50 (d,1H, J ¼ 7.5 Hz), 5.13 (s, 2H), 4.78–4.51 (m,
129.4, 129.0, 128.8, 75.5, 67.6, 59.0, 49.2, 36.8, 12.2; MS (ESI) m/z
304.1 ([M þ Na]þ calcd for C14H19NNaOþ5 304.1).
5H), 3.76 (s, 3H), 3.36 (q,1 H, J ¼ 6.9 Hz); 13C NMR (100 MHz, CDCl3,)
d
171.4, 156.4, 136.1, 128.7, 128.4, 128.3, 73.7, 73.4, 67.5, 55.1, 52.8.
37.7; MS (ESI) m/z 302.1 ([M þ Na]þ calcd for C14H17NNaO5þ 302.1).
4.5.3.2. (S)-2-(benzyloxycarbonylamino)-4-methoxybutanoic acid
(15c). To a stirred solution of 14c (370 mg, 1.04 mmol) in THF
(5 mL) was added NaOH (1 M, 3.13 mL) and MeOH (2 mL). After
stirring at room temperature for 25 min. the THF solution was
reduced in volume on a rotavapor to 2 mL, diluted with H2O
(5 mL) and extracted with Et2O. The pH was set to w2 using HCl
(1 M), before extraction with CH2Cl2 (4 ꢂ 5 mL). The combined
organic phases were washed with brine, dried (Na2SO4),
concentrated and purified on a short silica gel column (toluene–
ethyl acetate gradient 10:1–1.1) to obtain 17c in 58% yield
4.5.4.5. (S)-methyl 2-(benzyloxycarbonylamino)-2-(oxetan-3-yl)ace-
20
tate (19b). [
a]
D ꢁ 19.5 (c 1.0, MeOH); 1H NMR (400 MHz, CDCl3,)
d
7.39–7.29 (m, 5H), 5.50 (d, 1H, J ¼ 8.2 Hz), 5.13 (s, 2H), 4.78–4.52
(m, 5H), 3.73 (s, 3H), 3.36 (q, 1 H, J ¼ 7.3 Hz); 13C NMR (100 MHz,
CDCl3,) d 171.4, 156.4, 136.1, 128.7, 128.4, 128.3, 73.7, 73.4, 67.5, 55.1,
52.8. 37.7; MS (ESI) m/z 302.1 ([M þ Na]þ calcd for C14H17NNaOþ5
302.1).
4.5.5. General method for preparation of compounds 17a and 17b
To a stirred solution of the ester (1 equiv) in EtOH (1 mL) was
added MeNH2 (33% in EtOH) (7.5 equiv). The reaction mixture was
left stirring in room temperature overnight before the solvent was
removed. The product was recrystallised from EtOH and diethyl
ether to obtain the title compounds.
(164 mg, 0.61 mmol). 1H NMR (300 MHz, CDCl3,)
d 10.72 (bs, 1H),
7.39–7.26 (m, 5H), 5.94 (d, 1H, J ¼ 7.6 Hz), 5.12 (s, 2H), 4.49 (m,
1H), 3.50 (m, 2H), 3.30 (s, 3H). 2.25–1.91 (m, 2H); 13C NMR
(75 MHz, CDCl3,)
d 176.2, 156.4, 136.2, 128.6, 128.2, 128.2, 69.2,
67.1, 58.8, 52.3, 31.4.
4.5.4. General method for preparation of compounds 16a–c
The acid (1 equiv) was dissolved in dry CH2Cl2 (2 mL) and pyBOP
(1 equiv) was added followed by DIEA (1 equiv). After 45 min of
stirring in room temperature, methylamine (1.1 equiv, 2 M in THF)
was added to the mixture, followed by DIEA (1 equiv). The reaction
mixture was stirred for another 2 h, and was then diluted with
CH2Cl2 (5 mL), washed with NaHCO3 (aq., sat.) (5 ꢂ 5 mL), and
NH4Cl (1 ꢂ5 mL). The aqueous layers were extracted with CH2Cl2
(3ꢂ), and the pooled organic phases were washed with brine, dried
(MgSO4), concentrated and purified on a silica gel column (CH2Cl2)
to yield compounds 19a–c.
4.5.5.1. (R)-benzyl 2-(methylamino)-1-(oxetan-3-yl)-2-oxoethylcar
bamate (17a). The title compound was prepared from 19a
according to general procedure, vide supra, in 86% yield (86 mg,
20
0.31 mmol) and obtained as a white solid. [
a
]
D þ 8.0 (c 1.0,
MeOH); 1H NMR (400 MHz, CDCl3,)
d
7.36–7.30 (m, 5H), 6.61 (s, 1H),
5.76 (d, 1H, J ¼ 8.3 Hz), 5.10 (s, 2H), 4.75 (m, 2H), 4.49 (m, 3H), 3.36
(m, 1H), 2.75 (d, 3 H, J ¼ 4.6 Hz); 13C NMR (100 MHz, CDCl3,)
d 170.9,
157.0, 135.9, 128.7, 128.5, 128.1, 73.9, 67.5, 56.5, 37.3, 26.3; MS (ESI)
m/z 301.1 ([M þ Na]þ calcd for C14H18N2NaOþ4 301.1).
4.5.5.2. (S)-benzyl 2-(methylamino)-1-(oxetan-3-yl)-2-oxoethylcar
bamate (17b). The title compound was prepared from 19b
4.5.4.1. Benzyl (2S,3R)-4-methoxy-3-methyl-1-(methylamino)-1-oxo
butan-2-ylcarbamate (16a). The compound 16a was prepared from
15a, according to general procedure, vide supra, in 66% yield
according to general procedure, vide supra, in 49% yield (49 mg,
20
0.18 mmol) and obtained as a white solid. [
a
]
D ꢁ 8.5 (c 1.0, MeOH);
1H NMR (400 MHz, CD3OD)
d
7.39–7.27 (m, 5H), 5.11 (dd, 2H, J ¼ 12.5,
20
(110 mg, 0.37 mmol). [
CD3OD,)
a
]
D þ 0.7 (c 1.0, MeOH); 1HNMR (400 MHz,
11.0 Hz), 4.57 (t, 1H, J ¼ 6.4 Hz), 4.50 (t, 1H, J ¼ 6.3 Hz), 4.45 (d, 1H,
d
7.39–7.26 (m, 5H), 5.08 (dd, 2H, J ¼ 10.0, 12.4 Hz) 4.06 (d,
J ¼ 9.5 Hz), 2.70 (s, 3 H), 2.63 (bs, 1H); 13C NMR (100 MHz, CDCl3,)
1H, J ¼ 6.7 Hz), 3.34 (d, 1H, J ¼ 4.8 Hz), 3.28 (s, 3H), 2.73 (s, 3H). 2.67
d 170.9,157.0,135.9,128.7,128.5,128.2, 73.9, 67.6, 56.5, 37.3, 26.3; MS
(bs, 1H), 2.17 (m, 1H), 0.98 (d, 3H, J ¼ 7.0 Hz) 13C NMR (100 MHz,
(ESI) m/z 279.1 ([M þ H]þ calcd for C14H19N2Oþ4 279.1).
CDCl3,) d 174.5, 158.4, 138.1, 129.5, 129.1, 129.0, 75.5, 67.8, 59.5, 59.3,
36.8, 26.2, 14.7; MS (ESI) m/z 317.1 ([M þ Na]þ calcd for
4.5.6. General method for preparation of compounds 4a–d and 4f
The benzyl carbamate (1 equiv.) was dissolved in MeOH (3 mL)
under an argon atmosphere and palladium on charcoal (w5%) was
added followed by pressure reduction. The reaction mixture was left
stirring under an H2-atmosphere at 1 atm overnight. The Pd/C was
filtered off and the filtrate concentrated to yield the free amine
which was used in the next step without further purification.
The amine (1.5 equiv) was dissolved in 2–5 mL 1,2-dichloroethane
and lactone 21 (1.0 equiv) was added. The reaction mixture was
stirred at reflux for 16 h. The solvent was removed and the residue
purified by silica gel chromatography (CH2Cl2–MeOH 40:1) to yield
compounds 4a–d and 4f. Compounds 4d and 4f were purified on LC–
MS and 4a–c were re-crystallized from n-hexane and ethyl acetate.
C15H22N2NaOþ4 317.1).
4.5.4.2. Benzyl (2S,3S)-4-methoxy-3-methyl-1-(methylamino)-1-oxo
butan-2-ylcarbamate (16b). The compound 16b was prepared from
15b, according to general procedure, vide supra, in 62% yield
20
(40 mg, 0.14 mmol).
(400 MHz, CDOD3,)
[
a
]
D þ 12.0 (c 0.10, CHCl3); 1H NMR
d
7.39–7.27 (m, 5H), 5.09 (dd, 2H, J ¼ 12.4,
13.6 Hz), 4.23 (d,1H, J ¼ 5.4 Hz), 3.29–3.19 (m, 5H), 2.73 (s, 3H), 2.69
(s, 1H), 2.25 (m, 1H), 0.89 (d, 3H, J ¼ 7.0); 13C NMR (100 MHz,
CDOD3,) d 174.6,158.6,138.2, 129.4,129.1,128.9, 75.6, 67.8, 59.1, 58.1,
37.1, 26.3, 12.6; MS (ESI) m/z 317.1 ([M þ Na]þ calcd for
C15H22N2NaOþ4 317.1).

168
J. Adrian Meredith et al. / European Journal of Medicinal Chemistry 45 (2010) 160–170
4.5.6.1. (2R,3R,4R,5R)-2,5-bis(benzyloxy)-N1-((S)-1-(2-fluoroethyl
amino)-3-methyl-1-oxobutan-2-yl)-3,4-dihydroxy-N6-((1S,2R)-2-
hydroxy-2,3-dihydro-1H-inden-1-yl)hexanediamide (4a). The title
4.5.6.5. (2R,3R,4R,5R)-2,5-bis(benzyloxy)-3,4-dihydroxy-N1-((1S,2R)-
2-hydroxy-2,3-dihydro-1H-inden-1-yl)-N6-((S)-4-methoxy-1-(methyl-
amino)-1-oxobutan-2-yl)hexanediamide (4f). The title compound
compound was prepared from 5a according to general procedure,
was prepared from 16c according to general procedure, vide supra,
20
20
vide supra, in 28% yield (23 mg, 34
m
mol). [
a
]
D ꢁ 11.6 (c 0.88,
in 28% yield (23 mg, 35
(400 MHz, CDCl3,)
m
mol). [
a
]
D ꢁ 3.0 (c 0.15, CHCl3); 1H NMR
CHCl3); 1H NMR (400 MHz, CDCl3,)
d
7.43–7.16 (m, 15H), 5.34 (dd,
d
8.28 (d, 1H, J ¼ 7.9 Hz), 7.37–7.10 (m, 14 H), 5.34
1H, J ¼ 4.9, 8.5 Hz), 4.73–4.62 (m, 5H), 4.44 (m, 1H), 4.31 (m, 2H),
4.19 (m, 2H), 4.14 (m, 2H), 3.49 (m, 1H), 3.42 (m, 1H), 3.11 (dd, 1 H,
J ¼ 5.7, 16.6 Hz), 2.93 (dd, 1 H, J ¼ 1.8, 16.6 Hz). 2.41 (m, 1H), 0.93 (d,
3H, J ¼ 6.9 Hz), 0.84 (d, 3H, J ¼ 6.9 Hz); 13C NMR (100 MHz, CDCl3,)
(m 1H), 5.17 (bs, 1H), 5.01 (bs, 1H), 4.71–4.55 (m 5H), 4.18 (s, 1H),
4.15 (s, 2H), 4.03 (d, 1H, J ¼ 2.3 Hz), 3.34 (m, 2H), 3.19–3.09 (m, 4H)
2.97 (d, 1H, J ¼ 16.7 Hz), 2.75 (d, 3H, J ¼ 4.7 Hz), 2.10 (m, 1H), 2.02
(m,1H); 13C NMR (100 MHz, CDCl3,)
d 172.5,172.0,170.7,141.1,139.6,
d
172.1, 171.9, 170.8, 141.0, 139.8, 136.7, 136.6, 128.9, 128.8, 128.8,
136.7, 136.7, 128.9, 128.8, 128.6, 128.5, 128.3, 128.3, 127.2, 125.6,
124.0, 82.6, 82.2, 77.4, 73.6, 73.6, 73.5, 73.0, 72.4, 70.6, 59.0, 58.5,
52.9, 39.4, 30.5, 26.4; HRMS (ESI) m/z 650.3054 ([M þ H]þ calcd for
C35H44N3Oþ9 650.3072).
128.6, 128.5, 128.5, 128.3, 128.2, 127.2, 125.5, 124.1, 83.6, 81.6, 81.4,
81.3, 73.5, 73.4, 72.9, 72.6, 72.4, 58.4, 58.1, 40.2, 39.9, 39.4, 29.2 19.7
17.2; HRMS (ESI) m/z 666.3173 ([M þ H]þ calcd for C36H45FN3Oþ8
666.3185).
4.5.7. General method for preparation of compounds 4e, 4g and 4h
The benzyl carbamate (1 equiv) was dissolved in MeOH (3 mL)
under an argon atmosphere and palladium on charcoal (w5%) was
added followed by pressure reduction. The reaction mixture was
left stirring under an H2-atmosphere at 1 atm overnight. The Pd/C
was filtered off and the filtrate concentrated to yield the free amine
which was used in the next step without further purification. The
amine (2 equiv) was solved in diisopropyl ethyl amine (DIPEA)
(1 mL). The lactone 21 (1 equiv) and 2-hydroxypyridine (2 equiv)
was added. The round bottom flask was mounted with a cooler and
the reaction mixture was warmed (70 ꢀC) and left stirring for 16 h.
The solvent was removed and the residue purified by silica gel
chromatography (CH2Cl2–MeOH 40:1) to yield compounds 4e, 4g
and 4h which were further purified on LC–MS.
4.5.6.2. (2R,3R,4R,5R)-2,5-bis(benzyloxy)-3,4-dihydroxy-N1-((1S,2R)-
2-hydroxy-2,3-dihydro-1H-inden-1-yl)-N6-((S)-3-methyl-1-oxo-1-
(2,2,2-trifluoroethylamino)butan-2-yl)hexanediamide (4b). The title
compound was prepared from 5b according to general procedure,
20
vide supra, in 15% yield (12 mg, 17
m
mol). [
a
]
D ꢁ 12.3 (c 0.22,
CHCl3); 1H NMR (400 MHz, CDCl3)
d
7.95 (m, 1H), 7.38–7.13 (m,
14H), 5.31 (dd, 1H, J ¼ 5.3, 8.8 Hz), 4.71–4.55 (m, 5H), 4.24 (dd, 1H,
J ¼ 5.4, 9.0 Hz), 4.16–4.03 (m, 4H), 3.77 (m, 2H), 3.11 (dd, 1H, J ¼ 5.3,
16.5 Hz), 2.91 (dd, 1 H, J ¼ 1.5, 16.5 Hz), 2.28 (m, 1H), 0.90 (d, 3H,
J ¼ 6.9 Hz), 0.82 (d, 3H, J ¼ 6.9 Hz); 13C NMR (100 MHz, CDCl3,)
d
172.7, 172.2, 170.8, 141.0, 139.5, 136.4, 136.4, 129.0, 128.9, 128.8,
128.7, 128.7, 128.4, 128.2, 127.3, 125.6, 124.1, 82.0, 81.3, 77.4, 73.7,
73.5, 73.3, 72.7, 72.5, 58.5, 58.4, 58.2, 40.8, 40.3, 39.3, 29.0,19.6,17.0;
HRMS (ESI) m/z 724.2810 ([M þ Na]þ calcd for C36H42F3N3NaOþ8
724.2816).
4.5.7.1. (2R,3R,4R,5R)-2,5-bis(benzyloxy)-3,4-dihydroxy-N1-((1S,2R)-
2-hydroxy-2,3-dihydro-1H-inden-1-yl)-N6-((2S,3R)-4-methoxy-3-
methyl-1-(methylamino)-1-oxobutan-2-yl)hexanediamide (4e). The
title compound was prepared from 16b according to general
4.5.6.3. (2R,3R,4R,5R)-2,5-bis(benzyloxy)-3,4-dihydroxy-N1-((1S,2R)-
2-hydroxy-2,3-dihydro-1H-inden-1-yl)-N6-((S)-1-(2-methoxy
ethylamino)-3-methyl-1-oxobutan-2-yl)hexanediamide (4c). The title
procedure, vide supra, in 32% yield (9 mg, 13 mmol) and obtained as
20
a white solid. [
a
]
D þ 40.3 (c 0.3, MeOH); 1H NMR (400 MHz,
compound was prepared from 5c according to general proce-
CDCl3)
d
7.87 (d,1H, J ¼ 8.6 Hz), 7.39–7.18 (m,14H), 7.14 (m,1H), 6.92
20
dure, vide supra, in 18% yield (51 mg, 75
m
d
mol). [
a
]
D ꢁ 11.8 (c
(m,1H), 5.34 (dd,1H, J ¼ 4.9, 8.6 Hz), 4.70–4.63 (m 5H), 4.50 (dd,1H,
J ¼ 3.6, 8.7 Hz), 4.18–4.12 (m, 3H), 4.06 (d, 1H, J ¼ 3.0 Hz), 3.31 (d,
2H, J ¼ 6.4 Hz), 3.22 (s, 3H), 3.12 (dd, 1H, J ¼ 5.4, 16.5 Hz), 2.95 (dd, 1
H, J ¼ 1.3, 16.5 Hz), 2.71 (d, 3H, J ¼ 4.7 Hz), 2.37 (m, 1H) 1.80 (bs, 2H),
0.38, CDOD3); 1H NMR (400 MHz, CDCl3)
7.48–7.13 (m, 14H),
5.36 (d, 1H, J ¼ 5.1 Hz), 4.69–4.55 (m, 6H), 4.23 (m, 2H), 4.14 (m,
3H), 3.45–3.32 (m, 4H), 3.29 (s, 3H), 3.16 (dd, 1H, J ¼ 5.4,
16.4 Hz), 2.92 (dd, 1 H, J ¼ 1.4, 16.4 Hz), 2.13 (m, 1H), 0.95 (d, 3H,
J ¼ 6.8 Hz), 0.91 (d, 3H, J ¼ 6.8 Hz); 13C NMR (75 MHz, CDOD3,)
0.98 (d, 3H, J ¼ 7.1 Hz); 13C NMR (100 MHz, CDCl3)
d 172.4, 171.8,
170.5, 141.1, 139.7, 136.7, 136.6, 128.9, 128.8, 128.6, 128.5, 128.3,
128.3, 127.2, 125.6, 124.1, 82.3, 81.9, 77.4, 75.6, 73.6, 73.6, 73.3, 72.7,
72.5, 59.1, 58.3, 55.8, 39.4, 35.5, 26.3, 13.5; HRMS (ESI) m/z 686.3036
([M þ Na]þ calcd for C36H45N3NaOþ9 686.3048).
d
173.8, 173.7, 173.5, 142.0, 141.8, 138.8, 138.7 129.5, 129.4, 129.3,
129.3, 129.0, 129.0, 129.0, 127.9, 126.2, 125.4, 81.6, 81.4, 74.0,
73.8, 73.6, 72.1, 72.1, 71.8, 59.9, 58.9, 58.6, 40.7, 40.2, 31.7, 19.8,
18.4; HRMS (ESI) m/z 678.3372 ([M þ H]þ calcd for C37H48N3Oþ9
678.3385).
4.5.7.2. N1-[(1R,2S)-2-Hydroxy-2,3-dihydro-1H-indenyl]-N6-
[N(methyl)-2-oxetan-amide]-(2R,3R,4R,5R)-2,5-dibenzyloxy-3,4-
O-dihydroxyhexanediamide (4g). The title compound was prepared
4.5.6.4. (2R,3R,4R,5R)-2,5-bis(benzyloxy)-3,4-dihydroxy-N1-((1S,2R)-
2-hydroxy-2,3-dihydro-1H-inden-1-yl)-N6-((2S,3S)-4-methoxy-3-
methyl-1-(methylamino)-1-oxobutan-2-yl)hexanediamide (4d). The
from 17a according to general procedure, vide supra, in 61% yield
20
(33 mg, 50
m
mol) and obtained as white solid. [
a
]
D þ 25.5 (c 0.1,
title compound was prepared from 16a according to general
MeOH); 1H NMR (400 MHz, CD3OD,)
d 7.40–7.15 (m, 14H), 5.37 (d,
20
procedure, vide supra, in 15% yield (6 mg, 9
m
mol). [
d
a
]
D þ 5.7
1H, J ¼ 5.1 Hz), 4.78 (m, 2H), 4.71–4.53 (m, 7H), 4.49 (m, 1H), 4.21
(m, 2H), 4.14 (m, 3H) 3.65 (m, 1H), 3.39 (m, 1H), 3.17 (dd, 1H, J ¼ 5.2,
16.5 Hz), 2.94 (dd, 1 H, J ¼ 1.4, 16.4 Hz), 2.69 (s, 3H); 13C NMR
(c 0.35, CHCl3); 1H NMR (300 MHz, CDCl3,)
8.50 (d, 1H,
J ¼ 8.4 Hz), 7.44–7.12 (m, 14 H), 5.34 (dd, 1H, J ¼ 5.1, 8.8 Hz), 5.27
(s, 1H), 5.09 (bs, 1H), 4.71–4.57 (m 4H), 4.46 (m, 1H), 4.20 (s,
1H), 4.16 (s, 2H), 4.03 (s, 1H), 3.55 (dd, 1H, J ¼ 2.6, 9.8 Hz), 3.30
(dd, 1 H, J ¼ 2.8, 9.8 Hz), 3.21–3.09 (m, 4H), 2.97 (d, 1H,
J ¼ 16.5 Hz), 2.76 (d, 3H, J ¼ 4.7 Hz), 2.63 (m, 1H), 1.11 (d, 3H,
(100 MHz, CD3OD),
d 174.5, 173.9, 172.7, 142.0, 141.8, 138.6, 129.5,
129.5, 129.4, 129.3, 129.1, 129.0, 127.9, 126.2, 125.4, 82.0, 80.7, 75.7,
75.4, 73.9, 73.5, 72.7, 71.6, 58.6, 55.9, 40.8, 38.2, 26.3; HRMS (ESI) m/
z 670.2721 ([M þ Na]þ calcd for C35H41N3NaOþ9 670.2735).
J ¼ 7.3 Hz); 13C NMR (75 MHz, CDCl3,)
d 172.6, 172.6, 170.6, 141.2,
139.5, 136.6, 136.5, 128.9, 128.8, 128.7, 128.6, 128.5, 128.3, 128.2,
127.2, 125.7, 124.0, 82.8, 82.5, 77.4, 75.2, 74.1, 73.7, 73.5, 73.3,
72.4, 59.4, 58.6, 58.2, 39.3, 33.4, 26.3, 15.3; HRMS (ESI) m/z
664.3213 ([M þ H]þ calcd for C36H46N3Oþ9 664.3229).
4.5.7.3. N1-[(1R,2S)-2-Hydroxy-2,3-dihydro-1H-indenyl]-N6-
[N(methyl)-2-oxetan-amide]-(2R,3R,4R,5R)-2,5-dibenzyloxy-3,4-
O-dihydroxyhexanediamide (4h). The title compound was prepared
from 17b according to general procedure, vide supra, in 35% yield

J. Adrian Meredith et al. / European Journal of Medicinal Chemistry 45 (2010) 160–170
169
20
L. Ruiz, W.M. Kati, H.L. Sham, T. Robins, K.D. Stewart, A. Hsu, J.J. Plattner,
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P.I. Graham, J.H. Condra, L. Gotlib, M.K. Holloway, J. Lin, I.-W. Chen, K. Vastag,
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(10 mg, 15
m
mol) and obtained as an oil. [
a]
D þ 3.7 (c 0.2, MeOH);
1H NMR (400 MHz, CD3OD,)
d
7.40–7.16 (m, 14H), 5.37 (d, 1H,
J ¼ 5.1 Hz), 4.81–4.75 m (2H), 4.72–4.54 (m, 7H), 4.48 (t, 1H,
J ¼ 6.4 Hz), 4.21 (m, 1H), 4.13 (m, 3H) 3.65 (m, 1H), 3.41 (m, 1H), 3.17
(dd, 1H, J ¼ 5.1, 16.4 Hz), 2.93 (dd, 1 H, J ¼ 1.4, 16.4 Hz), 2.70 (s, 3H),
1.61 (bs, 1H); 13C NMR (125 MHz, CD3OD,)
d 174.3, 173.8, 172.7,
142.0, 141.8, 138.8, 138.7, 129.5, 129.4, 129.3, 129.1, 129.0, 127.9,
126.2, 125.4, 81.5, 81.4, 75.7, 75.5, 74.0, 73.8, 73.6, 72.5, 72.1, 58.6,
56.1, 40.8, 38.2, 26.3; HRMS (ESI) m/z 648.2921 ([M þ H]þ calcd for
C35H42N3Oþ9 648.2916).
[13] V. Kalish, S. Kaldor, B. Shetty, J. Tatlock, M. Davies, M. Hammond, B. Derssman,
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(20a). To a stirred mixture of 19a (80 mg, 0.30 mmol) in MeOH
(2 mL) was added NaOH (1 M, 0.40 mL). After stirring for 2 h in
room temperature the mixture was concentrated to approx. 1 mL
thereafter diluted with H2O (4 mL). The aqueous solution was
extracted with Et2O (2 ꢂ 5 mL) before being transferred to a round
bottom flask and Et2O (5 mL) was added. During vigorous stirring of
the mixture, the pH was set to w2 using HCl (1 M.). The phases
were separated and the aqueous phase was extracted with Et2O
(5 mL). The pooled organic phases were washed with brine, dried
(Na2SO4), concentrated and purified on a Sep-Pak C18 column
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20
33% yield (26 mg, 0.098 mmol) as a white solid. [
a
]
D þ 30.4 (c 1.0,
MeOH); 1H NMR (400 MHz, CD3OD,)
d
7.38–7.27 (m, 5H), 5.10 (s,
2H), 4.46 (t, 1H, J ¼ 8.7 Hz), 4.30 (d, 1H, J ¼ 11.0 Hz), 4.14 (t, 1H,
J ¼ 9.4 Hz), 3.75–3.62 (m, 2H), 2.76 (m, 1H); 13C NMR (100 MHz,
CDCl3)
d 177.2, 158.5, 137.9, 129.5, 129.4, 129.1, 128.9, 128.7, 69.4,
67.9, 60.6, 52.9, 44.9.
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4.5.7.5. (þ)-3-Oxetanylglycine. The benzyl carbamate 20a (26 mg,
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sphere and palladium on charcoal (w5%) was added followed by
pressure reduction. The reaction mixture was left stirring under an
H2-atmosphere at 1 atm overnight. The Pd/C was filtered off and
the filtrate concentrated to obtain the title compound as a white
20
solid in 78% yield (10 mg, 0.076 mmol). [
a
]
D þ 18.2 (c 1.0, H2O)
this is in accordance with published data [36].
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Acknowledgement
We would like to thank Medivir AB for financial support and
biological testing. We would like to thank Dr Kurt Benkestock for
help with LC–MS purifications. We would also like to thank Medivir
for kindly providing us with substances 19a and 19b.
ꢀ
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