Stereoselective synthesis and biological evaluation as inhibitors of hepatitis C virus RNA polymerase of GSK3082 analogues with structural diversity at the 5-position

Article abstract of DOI:10.1016/j.ejmech.2019.03.019

GSK3082 – a hepatitis C virus RNA polymerase inhibitor – and a series of analogues with structural diversity at the 5-position were prepared from a 2,2,4,5-tetrasubstituted pyrrolidine obtained with a well-defined stereochemistry from the 1,3-dipolar cycl

Full text of DOI:10.1016/j.ejmech.2019.03.019

Accepted Manuscript
Stereoselective synthesis and biological evaluation as inhibitors of hepatitis C virus
RNA polymerase of GSK3082 analogues with structural diversity at the 5-position
José A. Gálvez, Rafael Clavería-Gimeno, Juan J. Galano-Frutos, Javier Sancho,
Adrian Velázquez-Campoy, Olga Abian, María D. Díaz-de-Villegas
PII:
S0223-5234(19)30230-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.019
EJMECH 11189
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 December 2018
Revised Date: 6 March 2019
Accepted Date: 6 March 2019
Please cite this article as: José.A. Gálvez, R. Clavería-Gimeno, J.J. Galano-Frutos, J. Sancho, A.
Velázquez-Campoy, O. Abian, Marí.D. Díaz-de-Villegas, Stereoselective synthesis and biological
evaluation as inhibitors of hepatitis C virus RNA polymerase of GSK3082 analogues with structural
diversity at the 5-position, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.03.019.
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ACCEPTED MANUSCRIPT
OMe
N
OMe
structural
R
HO C
2
N
N
HO C
2
S
modification
O
O
tBu
tBu
MeO
MeO
GSK3082
N
CO Et
2
O
,
O
O
R =
,
, CONH , CH OMe,
2 2
O
O
S
CO Me, Et, CH OAc, CH OAllyl, CH OMoc, CH F, CH N , CH NHAc
2
2
2
2
2
2
3
2

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Stereoselective Synthesis and Biological Evaluation as Inhibitors of Hepatitis C
virus RNA polymerase of GSK3082 Analogues with Structural Diversity at the 5-
Position
a,
b,c
c,d,e
c,d,e
José A. Gálvez *, Rafael Clavería-Gimeno , Juan J. Galano-Frutos , Javier Sancho , Adrian
c,f,g
b,c,d,g
a,
Velázquez-Campoy , Olga Abian
, and María D. Díaz-de-Villegas *
a
Instituto de Síntesis Química y Catálisis Homogénea (ISQCH), CSIC – Universidad de Zaragoza,
Departamento de Química Orgánica, Pedro Cerbuna 12, 50009 Zaragoza, Spain.
b
Instituto Aragonés de Ciencias de la Salud (IACS), 50009 Zaragoza, Spain.
c
Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Units: BIFI-IQFR (CSIC) and
GBsC-CSIC, University of Zaragoza, Zaragoza 50018, Spain.
d
Aragon Institute for Health Research (IIS Aragon), 50009 Zaragoza, Spain.
e
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de
Zaragoza, Zaragoza 50009, Spain
f
Fundación ARAID, Government of Aragon, Zaragoza, Spain.
g
Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y
Digestivas (CIBERehd), Barcelona, Spain.
Corresponding Author: jagl@unizar.es, loladiaz@unizar.es
Abstract:
GSK3082 – a hepatitis C virus RNA polymerase inhibitor – and a series of analogues with structural
diversity at the 5-position were prepared from a 2,2,4,5-tetrasubstituted pyrrolidine obtained with a
well-defined stereochemistry from the 1,3-dipolar cycloaddition of the chiral imino ester derived from
leucine tert-butyl ester and (R)-2,3-O-isopropylideneglyceraldehyde with methyl acrylate. The chiral
2
,2-dimethyl-1,3-dioxolane moiety provided by the glyceraldehyde served as a synthetic equivalent for
different substituents and functional groups and these transformations usually required mild reaction
conditions and simple work-up procedures. The inhibitory activity of the resulting GSK3082 analogues
was studied in vitro in a cell-based assay of the subgenomic HCV RNA replication system. Some of the

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analogues showed good inhibitory activity with IC50 values in the nanomolar concentration range.
Key words: Hepatitis C virus (HCV), N-Acylpyrrolidines, HCV replication inhibitors, Structure-
Activity relationshiphs
1
. Introduction
Hepatitis C virus (HCV) is a biological pathogenic agent and it is considered to be the most common
cause of chronic liver disease worldwide [1]. The progressive destruction and regeneration of the liver
parenchyma caused by chronic liver disease is associated with the development of fibrosis, liver
cirrhosis, hepatocellular carcinoma and eventually liver failure and death [2]. HCV infection
constitutes a major public health problem: a large number of people – about 325 million worldwide in
2
015 – are carriers of hepatitis B or C virus infections, each year 1.75 million people newly acquire
hepatitis C virus infection and in 2015 alone viral hepatitis caused 1.34 million deaths, most of which
were due to chronic liver disease and primary liver cancer [3].
Inhibition of HCV NS5B polymerase, a key enzyme for HVC replication with RNA-dependent RNA
polymerase function [4], has proven to be a goal of paramount importance for the development of
specifically targeted antiviral therapy for HCV [5]. The potent antiviral effect of polymerase inhibitors
has led to the development of nucleos(t)ide and non-nucleoside analogues for the treatment of chronic
hepatitis C patients [6].
Figure 1. General structure of N-acylpyrrolidines identified as of HCV NS5B polymerase inhibitors.
In the above context, N-acylpyrrolidines with the general structure A (Figure 1) have been identified as
reversible, non-competitive inhibitors of HCV NS5B polymerase [7]. Structural analyses have shown
that compounds from the N-acylpyrrolidine series bind in the palm pocket I of NS5B near the

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active site of the polymerase [6h,8].
Optimization of the structure based on the analysis of structure-activity relationships (Glaxo-
SmithKline) led to the identification of GSK3082 [7c], a potent inhibitor of RNA-dependent RNA
polymerase (NS5B) in enzymatic assays that also inhibits viral RNA replication in cell-based replicon
assays. These SAR studies showed that (i) the presence of a bulky non-polar group in the 2-position
increases activity, (ii) aryl groups with lipophilic electron-withdrawing groups in the acyl moiety
enhance activity, with the 4-(tert-butyl)-3-methoxybenzoyl group being optimal, and (iii) that the
substituent at the 5-position can be widely varied.
1
,3-Dipolar cycloaddition has proven to be a flexible and versatile synthetic strategy to gain access to
the pyrrolidine core of biologically active compounds [8]. Previously described non-stereoselective [7],
diastereoselective [9] and enantioselective [10] syntheses of antiviral compounds with the general
structure A have been performed using 1,3-dipolar cycloaddition of iminoglycinates and acrylates as
the key step. Substituents R
1 3
and R are provided by the amino acid and the aldehyde from which the
iminoglycinate is obtained and substituent R is generated from the carboxylate moiety in the acrylate
2
(
Scheme 1).
Scheme 1. Strategy for synthesis of N-acylpyrrolidines.
One of our research goals is the synthesis of versatile chiral intermediates from imines derived from
glyceraldehyde – which is readily accessible from inexpensive D-mannitol – and the development of
synthetic routes to obtain bioactive compounds in enantiomerically pure form from these chiral
intermediates [11]. With this aim in mind, we studied the behaviour of chiral iminoesters derived from
glyceraldehyde acetonide in azomethine ylide cycloadditions and developed a stereoselective and
flexible methodology for the synthesis of 2,2,4,5-tetrasubstituted pyrrolidines with a well-defined
stereochemistry [12]. We presume that this methodology would be amenable to the synthesis of HCV
NS5B polymerase inhibitors and related structural analogues. In the work reported here, we synthesised
a series of novel N-acylpyrrolidines – in which the substituent at 5-position in GSK3082 was varied –

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starting from a common precursor. The target compounds are potential HCV polymerase inhibitors and
their inhibitory activities were evaluated in vitro in a cell-based assay of the subgenomic HCV RNA
replication system.
2
. Results and discussion
2
.1. Chemistry
Based on our previous experience we envisaged that the pyrrolidine core of structural analogues of
GSK3082 with different substituents in the 5-position could be obtained by the 1,3-dipolar
cycloaddition between an acrylate and an iminoester derived from
D-glyceraldehyde acetonide and
leucine using silver acetate for metallation. In this way the 2,2,4,5-tetrasubstituted pyrrolidine with the
appropriate configuration would be obtained. The chiral 2,2-dimethyl-1,3-dioxolane moiety provided
by the glyceraldehyde has a double role: (i) to control the stereochemical course of the reaction and (ii)
to serve as a synthetic equivalent for different substituents and functional groups (Scheme 2). The tert-
butylimino ester 1 and methyl acrylate were selected as reagents to obtain an orthogonally protected
pyrrolidine.
Scheme 2. Strategy for synthesis GSK3082 analogues.
Starting imino ester 1 was prepared by reaction of (S)-leucine tert-butyl ester with (R)-2,3-O-
isopropylideneglyceraldehyde in ether at room temperature for 2 h in the presence of anhydrous
MgSO as a dehydrating agent. The crude imino ester was directly submitted to a 1,3-dipolar
4
cycloaddition reaction with methyl acrylate without any purification. Reaction using silver acetate (20
mol%) as the catalyst and DBU (20 mol%) as the base in toluene at room temperature for 24 h led to an

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8
5/15 mixture of endo adducts 2a/2b in 95% yield, in which compound 2a with the desired (2S,4S,5R)
configuration was in excess. The absolute configuration of the major endo adduct was unambiguously
established by a single-crystal X-ray diffraction study on compound 4a, which was obtained from 2a as
shown below. Acylation of the mixture with 4-(tert-butyl)-3-methoxybenzoyl chloride provided the
corresponding N-acyl pyrrolidines 3a/3b as an 85/15 mixture of diastereoisomers in 96% yield
(
Scheme 3).
Scheme 3. Synthesis of N-acylpyrrolidine 3a.
The synthesis of GSK3082 was completed as shown in Schemes 4 and 5. Reduction of the methyl ester
was achieved with a combination of NaBH -MeOH (1:2) in the presence of a catalytic amount of
4
3 4
NaB(OAc) H (2.5 mol % relative to NaBH ) [10a]. After stirring 3a/3b in THF for 4 days at room
temperature the corresponding 4-hydroxymethylpyrrolidines were obtained as an 85/15 diastereomeric
mixture, from which 4a and 4b were isolated as diastereomerically pure compounds in 81% and 12%
yield, respectively.
The O-methylation of 4a with an excess of methyl iodide in the presence of potassium tert-butoxide in
THF at room temperature was complete after 18 h and provided compound 5 in 88% yield. The 2,2-
dimethyl-1,3-dioxolane moiety was converted into the required thiazol-2-yl substituent at the 5-position
3
by first converting the acetonide 5 into the aldehyde 7. Chemoselective deprotection using BiCl in dry
acetonitrile provided diol 6 in 81% yield [13] and this was subsequently converted into the aldehyde 7
in 94% yield by oxidation with sodium periodate.

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Scheme 4. The synthetic route for key intermediate 7.
Further oxidation of aldehyde 7 with potassium permanganate using a mixture of tert-butanol and
aqueous NaH PO as the reaction medium [14], followed by treatment with of isopropyl chloroformate
2
4
in the presence of N-methylmorpholine in THF at room temperature and subsequent reaction with
ammonium hydroxide gave a 79% yield of formamide 8. Direct conversion of formamide 8 into
thioamide 10 using Lawesson’s reagent was unsuccessful and we evaluated a two-step procedure using
nitrile 9 as the intermediate. Reaction of formamide 8 with POCl in pyridine [15] provided a 51%
3
yield of the nitrile intermediate 9, which was subsequently converted into thioamide 10 (95% yield) by
the action of 40–44% aqueous ammonium sulfide solution in methanol. At this point we evaluated the
direct conversion of aldehyde 7 to nitrile 9. This transformation was successfully achieved in 99% yield
using iodine in ammonia water [16]. In an effort to prevent epimerization, the final thiazol-2-yl
substituent at the 5-position in GSK3082 was prepared via a modified Hantzsch method [15] that
involved the cyclocondensation of thioamide 10 with bromoacetaldehyde in dry dimethoxyethane using
NaHCO as base followed by dehydratation of the hydroxythiazoline intermediate by treatment with
3
trifluoroacetic anhydride–pyridine. In this way compound 11 was obtained in 87% yield with complete
stereocontrol. Finally, hydrolysis of the tert-butyl ester with trifluoroacetic acid in dichloromethane at 0
ºC yielded GSK3082 in 95% yield. GSK3082 was obtained from 1 in 10 steps with a good overall yield
of 38%.

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Scheme 5. The synthetic route for target compound GSK3082.
Cyclocondensation of thioamide 10 with ethyl bromopyruvate using this protocol led to an 88% yield
of compound 12, which upon hydrolysis with trifluoroacetic acid in dichloromethane at 0 ºC afforded
GSK3082 analogue 13 in 95% yield without detectable epimerization (Scheme 6).
Scheme 6. The synthetic route for GSK3082 analogue 13.
Alternatively, the thiazol-2-yl substituent at the 5-position was installed by reaction of thioamide 10
with α-bromoacetaldehyde diethyl acetal in THF under reflux and in the presence of a catalytic amount
of HCl in dioxane [17]. In this way compound 14 – the epimer of 11 at C5 – was obtained in 51%
yield, again with complete stereocontrol. tert-Butyl ester 14 was readily hydrolysed to the
corresponding acid 15 in 96% yield under the same conditions as above (Scheme 7).

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Scheme 7. The synthetic route for GSK3082 analogue 15.
The synthesis of the first set of structural analogues of GSK3082 with substituents with different
structural characteristics in the 5-position was readily achieved from intermediates 5, 6 and 8 as
detailed below. The chemoselective hydrolysis of the tert-butyl ester compound 5 was carried out
under reflux with a methanolic solution of potassium hydroxide and this gave analogue 16 in nearly
quantitative yield. Compound 6 was reacted with 1,1'-carbonyldiimidazole in THF at 60 ºC to produce
carbonate 17 in 90% yield, which upon hydrolysis with trifluoroacetic acid in dichloromethane at 0 ºC
led to analogue 18 in 94% yield. GSK3082 analogue 19 was obtained in 96% yield by treatment of
amide 8 with trifluoroacetic acid in dichloromethane at 0 ºC (Scheme 8).
Scheme 8. The synthetic route for GSK3082 analogues 16, 18 and 19.

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Other GSK3082 analogues were obtained from versatile chiral intermediate 7 by transformation of the
formyl group into methoxymethyl, ethyl and methoxycarbonyl groups, as shown in Scheme 9. Reaction
of compound 7 with sodium borohydride in ethanol reduced the C5 formyl group to a hydroxymethyl
group, which in turn reacted with methyl iodide to give compound 20 in 85% yield over the two steps.
Subsequent hydrolysis of 20 with trifluoroacetic acid in dichloromethane at 0 ºC gave GSK3082
analogue 21 in 98% yield. Synthesis of analogue 23 involved oxidation of the formyl group, which was
performed with potassium permanganate using a mixture of tert-butanol and aqueous NaH PO as a
2
4
reaction medium as above. The resulting carboxylic acid was immediately converted into its methyl
ester using trimethylsilyldiazomethane. Transformation of aldehyde 7 into methyl ester 22 took place to
give 89% yield over the two steps and final hydrolysis with trifluoroacetic acid in dichloromethane at 0
ºC afforded a 97% yield of analogue 23. Wittig reaction of 7 and the unstabilised ylide derived from
triphenylphosphonium methyl bromide gave olefin 24 in 91% yield. Compound 24 was hydrogenated
to 25 (93%) with molecular hydrogen using Pd/C as the catalyst. Hydrolysis of the tert-butyl ester
under the usual acidic conditions afforded analogue 26 in 97% yield.
Scheme 9. The synthetic route for GSK3082 analogues 21, 23 and 26.
Compounds 13, 15, 16, 18, 19, 21, 23 and 26 were tested as inhibitors by mimicking HCV viral
replication in cells using a subgenomic HCV RNA replication system and, on the basis of their in vitro
activity (see below), we prepared a second set of structural analogues of GSK3082 taking into account
the beneficial effect of the presence of a heteromethyl side chain at C5. Firstly, aldehyde 7 was

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efficiently converted into acetate 27, allyl ether 29 or methylcarbonate 31 by reduction 7 with sodium
borohydride in ethanol followed by the immediate dimethylaminopyridine-catalysed acetylation with
acetic anhydride, reaction with allyl bromide in the presence of sodium hydride or treatment with
methyl chloroformate in the presence of a catalytic amount of dimethylaminopyridine. Subsequent
hydrolysis of these compounds with trifluoroacetic acid in dichloromethane at 0 ºC yielded GSK3082
analogues 28, 30 and 32 in 94–95% yields from starting aldehyde 7 (Scheme 10).
Scheme 10. The synthetic route for GSK3082 analogues 28, 30 and 32.
Compound 7 was also converted into GSK3082 analogues 34, 36 and 38 with a fluoromethyl,
azidomethyl or acetylaminometyl side chain at C5 (Scheme 11). Access to the fluoromethyl side chain
was achieved by sequential treatment of aldehyde 7 with sodium borohydride in ethanol and the
fluorinating agent diethylaminosulfur trifluoride (DAST). Compound 33, obtained in 52% yield, was
hydrolysed with trifluoroacetic acid in dichloromethane at 0 ºC to afford analogue 34 in almost
quantitative yield. The intermediate hydroxymethyl derivative obtained in the reduction of 7 with
sodium borohydride in ethanol was immediately reacted first with methanesulfonyl chloride in the
presence of triethylamine and then with sodium azide in the presence of a catalytic amount of sodium
iodide to afford 35. Hydrolysis of this compound under the usual conditions gave analogue 36 in 95%
from aldehyde 7. Catalytic reduction of azide 35 gave the corresponding amine, which was

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immediately acetylated with acetic anhydride-triethylamine to produce acetylamino derivative 37 in
6
5% yield. Subsequent hydrolysis with trifluoroacetic acid in dichloromethane at 0 ºC yielded analogue
3
8 in 95% yield.
Scheme 11. The synthetic route for GSK3082 analogues 34, 36 and 38.
.2. Biological evaluation
2
In order to evaluate the potency and cytotoxicity of the 14 synthesised compounds, viral replication
was assessed by in vitro HCV replication assays with genotype 1b Con1 HCV subgenomic replicons
(
Huh 5-2) [18] and the results were compared to those obtained for GSK3082. All of the compounds
inhibited HCV replicon replication (measured as the luciferase signal) in a dose-dependent manner,
with EC50 values lower than 100 microM (Table 1, Figure 2). The estimated EC50 values for these
compounds ranged from nanomolar to micromolar. The anti-HCV activity was not the result of a
cytostatic effect [19], since the CC50 values for these compounds were significantly higher than the
EC50 values.

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Table 1. Evaluation of the potency and cytotoxicity of the obtained compounds in cell assays
a
b
Compound
GSK3082
R
EC50 (µM)
CC50 (µM)
N
0.1
60
15
60
>120
200
S
N
1
1
1
3
5
6
2
CO Et
S
N
*
200
S
>120
O
O
1
8
2
>120
O
O
O
1
2
2
2
2
3
3
3
3
3
9
1
3
6
8
0
2
4
6
8
CONH
CH OMe
CO Me
Et
CH OAc
CH OAllyl
CH OMoc
CH
CH
2
2
0.6
7
>120
>120
>120
200
2
2
40
30
7.5
30
45
100
15
>120
>120
>120
>120
>120
>120
2
2
2
F
2
N
3
2
CH
NHAc
2
a
b
EC50, effective concentration 50%; CC50, cytotoxic concentration 50%. Relative error in the
parameters is 15%.

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1
00
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
0
1,675
3,75
7,5
15
30
60
120
0
0.104 0.208 0.41 0.83 1.67 3.25 7.5 15
30
60 120
GSK3082 (µM)
21 (µM)
Figure 2. Evaluation of viral load reduction (bars) of the HCV replicon system in human hepatoma
cells (Huh5-2) with cell growth (lines) upon increasing the concentration of compound GSK3082
(
green) and 21 (red).*UTC: untreated controls.
2
.3. SAR analysis
To uncover relationships between EC50 activities and structural features or physical-chemical
properties in the compound series, a simple SAR (Structure-Activity Relationship) analysis was
performed. The compound structures were optimized and 36 molecular descriptors were calculated.
Then, we sought for linear correlations between effectiveness of compounds against HCV NS5B
polymerase (-log(EC50)) and any of the previously calculated molecular descriptors. Although this
analysis did not show any high correlation between any of the molecular descriptors selected and the
biological activity (not shown) it provided some clues. It was noticed that GSK3082, the most effective
compound, presented marked differences with most other compounds in regard to properties such as
aromatic ratio (ARR) and reactivity (Re) (Figure 3 panels A and B). According to these plots, only
compounds 15 and 13 differentiated from the group in the same direction as GSK3082. This was
somewhat expected due to their close structural similarity (15 is a GSK3082's estereoisomer and 13
presents the same thiazole ring but ramified with an ethyl carboxylate) but did not allow us to explain
their lower activity compared to that of GSK3082. On the other hand, compound 13 was that with
highest molecular weight (Figure 3 panel C) and displayed one of the lowest activities. Thus, we
thought the activity could be qualitatively correlated with structural features of the complexes formed
by the compounds and the target enzyme. Based on the availability of a 3D structure for the complex
between HCV NS5B polymerase and a ligand structurally close to GSK3082 (PDB code: 2jc1) [7c],
we modeled the binding of all the compounds. As shown in SI Figure S1 (panels B and C) both
compounds 15 and 13 appear to face steric problems (clashes) to fit in the putative binding site. As for

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the remaining compounds, the lower activity of 16, 23, 28, 30, 32 and 38 could also be related to steric
issues (SI Figure S2 panel A), while that of the others (e.g. 18, 19, 21, 26, 34 and 36, SI Figure S2
panel B) appears related to loss of polar and/or hydrophobic interactions with the protein binding
pocket.
Figure 3. Scatter plots of molecular descriptor values versus the assayed biological activities: (A)
aromatic ratio, (B) reactivity and (C) molecular weight values versus the effectiveness of compounds
against HCV NS5B polymerase. Substituent structures (R
3 and 15 are shown.
3
) at the 5-position in compounds GSK3082,
1

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3
. Conclusions
In summary, we have developed a very efficient diastereoselective approach to novel 2,2,4,5-
tetrasubstituted -acyl pyrrolidines with a well-defined stereochemistry starting from a chiral
iminoester derived from leucine tert-butyl ester and ( )-2,3- -isopropylideneglyceraldehyde. The
N
R
O
cycloadduct derived from the 1,3-dipolar cycloaddition with methyl acrylate has served as a common
precursor for the synthesis of GSK3082 and structurally related compounds with different substituents
at C5, with the chiral 2,2-dimethyl-1,3-dioxolane moiety provided by the glyceraldehyde acting as a
synthetic equivalent for structural diversity. The inhibitory activity of the obtained GSK3082 analogues
has been studied in vitro in a cell-based assay of a subgenomic HCV RNA replication system and some
of the analogues have shown a good inhibitory activity with estimated IC50 values ranging from
nanomolar to micromolar.
4
. Experimental Section
4
.1. Chemistry
4
.1.1. General Details
Unless otherwise specified, all reagents were obtained from commercial suppliers and were used as
received without further purification. For anhydrous conditions, reactions were carried out under Ar in
solvents dried using a Solvent Purification System (SPS). TLC was performed on precoated silica gel
6
0 F254 polyester plates and products were visualised using UV light (254 nm) and potassium
permanganate solutions followed by heating. Column chromatography was performed on silica gel (60,
0−63 m) with air pressure. Melting points were determined in open capillary tubes and are not
corrected. FT-IR spectra were recorded as thin films on NaCl plates,
given for the main absorption bands. Optical rotations were measured on a digital polarimeter at
4
µ
–
1
ν
max values expressed in cm are
λ
589
–1
2
–1
nm and 25 ºC in cells with 1 or 10 cm path length, [
α
] values are given in 10 deg·cm ·g and
D
1
19
13
concentrations are given in g/100 mL. H, F and C NMR spectra were acquired on a 400 MHz
spectrometer in the stated deuterated solvent at room temperature unless otherwise stated using a 5-mm
1
3
1
probe. C NMR spectra were acquired with the H broad-band decoupled mode, chemical shifts (δ) are
reported in parts per million (ppm) with the solvent resonance as the internal standard [20], and
coupling constants (J) in hertz (Hz). High-resolution mass spectra were recorded from methanolic
solutions on a MICROTOF-Q (quadrupole time-of-flight) micro instrument using the positive
electrospray ionization mode (ESI+). The X-ray diffraction data were collected at room temperature on
a four-circle diffractometer, using graphite-monochromated Mo-K� radiation (
 = 0.71073 Å).

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4
.1.2. Synthesis of crude (E)-tert-butyl N-{[(S)-2,2-dimethyl-1,3-dioxolan-4-yl]methylidene}-(S)-
leucinate (1)
To a solution of (S)-leucine tert-butyl ester hydrochloride (1,79 g, 8.0 mmol) in water (5 mL) at room
temperature was added a solution of potassium carbonate (2.21 g, 16.0 mmol) in water (5 mL). The
resulting solution was extracted with dichloromethane (3 x 20 mL) and the combined organic layers
were dried over anhydrous MgSO , filtered and evaporated under reduced pressure to give crude tert
-
4
butyl (S)-leucinate. In the meantime to a stirred solution of 1,2:5,6-di-O-isopropylidene-D-mannitol
(
1.31 g, 5.0 mmol) in dichloromethane (20 mL) at room temperature was added saturated aqueous
solution of sodium hydrogen carbonate (1 mL). Then, sodium metaperiodate (2.14 g, 10.0 mmol) was
added with vigorous agitation and the reaction was allowed to proceed for 1 h at 35 ºC. The solids were
removed by filtration and washed with dichloromethane (30 mL). The combined organic solutions were
dried over anhydrous MgSO
crude ( )-2,3- -isopropylideneglyceraldehyde, which was used immediately in the condensation
reaction with tert-butyl ( )-leucinate. To a solution containing crude tert-butyl ( )-leucinate and ( )-
,3- -isopropylideneglyceraldehyde in dry diethyl ether (20 mL) at room temperature was added
4
, filtered and evaporated under reduced pressure at less than 30 ºC to give
R
O
S
S
R
2
O
anhydrous MgSO (1.0 g) and the resulting suspension was stirred for 2 h. The reaction mixture was
4
then filtered and the solid residue was washed with diethyl ether (20 mL). The combined organic
filtrates were evaporated under reduced pressure to afford 2.60 g of crude imine 2 of 65 % purity, as
1
determined by H NMR using 1,3,5-trimethoxybenzene as an internal standard [21]. Crude imine 2 was
1
used without further purification in the next reaction. Oil; H NMR (400 MHz, CDCl
3
)
δ
0.85 (d,
J
=
6
1
.6 Hz, 3H), 0.92 (d,
.63−1.99 (m, 2H), 3.78 (dd,
= 6.9, 5.9, 4.7 Hz, 1H), 7.66 (d, J
J
= 6.6 Hz, 3H), 1.39 (s, 3H), 1.41−1.43 (m, 1H), 1.44 (s, 3H), 1.45 (s, 9H),
= 8.2, 6.2 Hz, 1H), 3.94 (dd, = 8.5, 5.9 Hz, 1H), 4.20 (dd, = 8.5, 6.9
= 4.7 Hz, 1H).
J
J
J
Hz, 1H), 4.65 (ddd,
J
4
2
.1.3. 2-(tert-Butyl) 4-methyl (2S,4S,5R)-5-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-isobutylpyrrolidine-
,4-dicarboxylate (2a)
To a stirred suspension of crude imine 1 of 65% purity (2.60 g, 5.6 mmol) and silver acetate (183 mg,
.1 mmol) in dry toluene (30 mL) under argon at room temperature were sequentially added ethyl
1
acrylate (966 mg, 11.2 mmol) and DBU (171 mg, 1.1 mmol), both of them previously solved in dry
toluene (1 mL). The reaction mixture was stirred in the dark at room temperature for 24 h. The mixture
®
®
was quenched with water (5 mL) and filtered through a short Celite pad. Celite residues were
thoroughly washed with diethyl ether (50 mL) and the combined organic filtrates were dried over

ACCEPTED MANUSCRIPT
anhydrous MgSO , filtered and concentrated under reduced pressure. The crude cycloadducts were
4
purified by flash chromatography (eluent: diethyl ether/hexane, 1/2) to afford 2 (2.05 g, 95% yield) as a
mixture of endo diasteroisomers 2a and 2b (2a/2b = 85/15). Some column chromatography fractions
containing exclusively major cycloadduct 2a were used for characterization purposes. Colourless oil;
2
4
–1 1
[
α
]D = −39.23 (
.83 (d, = 6.4 Hz, 3H), 0.90 (d,
s, 9H), 1.61−1.74 (m, 2H), 1.81 (dd,
c = 1.11 in CHCl ); IR (neat) 3341, 3308, 1734 cm ; H NMR (400 MHz, CDCl ) δ
3
3
0
J
J
= 6.5 Hz, 3H), 1.29 (s, 3H), 1.39 (s, 3H), 1.41−1.45 (m, 1H), 1.46
= 13.7, 7.9 Hz, 1H), 2.48 (dd, = 13.7, 5.5 Hz, 1H), 2.92 (ddd,
= 7.9, 6.9, 5.5 Hz, 1H), 3.00 (bs, 1H), 3.26 (dd, = 6.9, 5.3 Hz, 1H), 3.60 (s, 3H), 3.71 (dd, = 8.0,
.1 Hz, 1H), 3.93 (dd, = 8.0, 6.4 Hz, 1H), 4.12−4.20 (m, 1H); C-APT{ H} NMR (100 MHz, CDCl )
23.7, 24.4, 25.5, 25.5, 26.6, 28.0, 40.9, 46.2, 49.2, 51.8, 63.2, 67.5, 68.7, 75.0, 81.1, 109.3, 173.5,
(
J
J
J
7
δ
1
J
J
1
3
1
J
3
+
+
75.1; HRMS(ESI ) m/z [M+H] calcd for C H NO 386.2538, found 386.2546.
2
0
36
6
4
.1.4. 2-(tert-Butyl) 4-methyl (2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-5-((S)-2,2-dimethyl-1,3-
dioxolan-4-yl)-2-isobutylpyrrolidine-2,4-dicarboxylate (3a)
A mixture of 4-tert-butyl-3-methoxybenzoic acid (1.56 g, 7.5 mmol) and thionyl chloride (12 mL) was
refluxed for 4 hours. Thionyl choride in excess was removed by evaporation under reduced pressure
and the residue was solved in toluene (5 mL). The organic solvent was evaporated under reduced
pressure to remove thionyl chloride traces and the crude 4-tert-butyl-3-methoxybenzoyl chloride was
solved in anhydrous dichloromethane (10 mL). To this stirred solution at 0 ºC was added a solution of
compound 2 as an 85/15 mixture of diastereoisomers 2a and 2b (1.93 g, 5.0 mmol) in dichloromethane
(
10 mL) followed by triethylamine (2.8 mL, 20.1 mmol) and this mixture was stirred at room
temperature for 36 hours. After that, the reaction mixture was washed with saturated aqueous sodium
bicarbonate solution (2 x 20 mL). The organic layer was dried over MgSO , filtered and evaporated
4
st
under reduced pressure. The residue was purified by flash chromatography (1 eluent: diethyl
nd
ether/hexane, 1/3; 2 eluent: diethyl ether/hexane 1/1) to afford 3 (2.76 g, 96% yield) as a mixture of
endo diastereoisomers 3a and 3b (3a/3b = 85/15). Following the same experimental procedure starting
from major diastereoisomer 2a, diastereomerically pure compound 3a was obtained and fully
2
4
–1 1
characterised. Colourless oil; [
NMR (400 MHz, CDCl3) 0.52 (s, 3H), 0.97 (d,
H), 1.32 (s, 9H), 1.52 (s, 9H), 1.72−1.83 (m, 1H), 2.00 (dd,
.9 Hz, 1H), 2.24−2.32 (m, 1H), 2.64 (dd, = 13.4, 13.4 Hz, 1H), 3.28 (dd,
= 13.4, 7.3, 6.9 Hz, 1H), 3.67−3.73 (m, 1H); 3.71 (s, 3H), 3.84 (s, 3H), 4.32−4.42 (m, 1H), 4.73
α]D = 0.65 (c = 1.94 in CHCl ); IR (neat) 1734, 1636, 1567 cm ; H
3
δ
J
= 6.6 Hz, 3H), 1.00 (d,
= 15.1, 4.4 Hz, 1H), 2.25 (dd,
= 7.7, 7.7 Hz, 1H), 3.47
J
= 6.6 Hz, 3H), 1.12 (s,
3
6
J
J
= 13.4,
J
J
(
ddd, J

ACCEPTED MANUSCRIPT
(
dd,
J
= 9.3, 7.3 Hz, 1H), 6.92 (dd,
J
= 7.9, 1.6 Hz, 1H), 7.04 (d,
H); C-APT{ H} NMR (100 MHz, CDCl ) 24.4, 24.7, 24.9, 25.3, 25.7, 28.2, 29.6, 35.0, 36.1, 41.6,
4.6, 52.3, 55.1, 65.3, 66.4, 69.3, 75.2, 81.5, 108.6, 112.6, 118.5, 125.7, 137.0, 140.4, 158.8, 169.7,
J = 1.6 Hz, 1H), 7.18 (d, J = 7.9 Hz,
1
3
1
1
4
1
δ
3
+
+
70.8, 172.8; HRMS(ESI ) m/z [M+H] calcd for C H NO 576.3531, found 576.3518.
32
50
8
4
4
.1.5. tert-Butyl (2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-5-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-
-(hydroxymethyl)-2-isobutylpyrrolidine-2-carboxylate (4a)
To a stirred solution of compound 3 as an 85/15 mixture of diastereoisomers 3a and 3b (2.30 g, 4.0
mmol) in dry THF (40 mL) were sequentially added NaBH (606 mg, 16.0 mmol) and NaB(OAc) H
4
3
(
172 mg, 0.8 mmol). To the white slurry under argon at 0 ºC dry methanol (1.3 mL, 32.1 mmol) was
added and the mixture was stirred at room temperature for 4 days until complete disappearance of the
starting material was observed by TLC. The resulting reaction mixture was quenched with saturated
aqueous NH Cl solution (20 mL) and the organic solvents were evaporated under reduced pressure.
4
The aqueous layer was extracted with dichloromethane (3 x 20 mL) and the combined organic layers
were dried over anhydrous MgSO , filtered and concentrated under reduced pressure. The crude
4
st
nd
product was purified by flash chromatography (1 eluent: diethyl ether/hexane, 1/1; 2 eluent: diethyl
2
4
ether) to provide diastereomerically pure 4a (1.78 g, 81% yield) as a white solid. M.p. = 204 ºC; [α]D
–
1 1
=
−3.07 (
c
= 0.99 in CHCl ); IR (nujol) 3379, 1730, 1593, 1563 cm ; H NMR (400 MHz, CDCl )
3
3
δ 0.73 (s, 3H), 0.97 (d,
J
= 6.6 Hz, 3H), 1.00 (d,
J
= 6.6 Hz, 3H), 1.11 (s, 3H), 1.32 (s, 9H) 1.50 (s,
9
1
2
4
H), 1.77−1.89 (m, 1H), 1.93 (dd,
2.9, 6.8 Hz, 1H), 2.25 (dd, = 14.9, 6.0 Hz, 1H), 2.68 (bs, 1H), 2.80−2.94 (m, 1H), 3.48−3.63 (m,
H), 3.67 (dd, = 8.8, 8.8 Hz, 1H), 3.83 (s, 3H), 3.98 (dd, = 8.8, 6.5 Hz, 1H), 4.14−4.23 (m, 1H),
= 7.9, 1.7 Hz, 1H), 7.01 (d, = 1.7 Hz, 1H), 7.18 (d, = 7.9
24.5, 24.9, 25.0, 25.7, 28.2, 29.7, 35.0, 37.1, 41.9,
J = 12.9, 12.9 Hz, 1H), 2.01 (dd, J = 14.9, 4.4 Hz, 1H), 2.14 (dd, J =
J
J
J
.61 (dd,
J
= 7.5, 7.5 Hz, 1H), 6.93 (dd,
J
J
J
1
3
1
Hz, 1H); C-APT{ H} NMR (100 MHz, CDCl )
δ
3
4
1
3.4, 55.2, 61.1, 63.9, 66.9, 69.7, 75.5, 81.2, 108.0, 112.0, 118.5, 125.9, 137.0, 140.3, 158.7, 170.0,
+
+
7
73.2; HRMS(ESI ) m/z [M+H] calcd for C31H50NO 548.3582, found 548.3598.
4
2
.1.6. tert-Butyl (2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-5-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-
-isobutyl-4-(methoxymethyl)pyrrolidine-2-carboxylate (5)
To a stirred solution of compound 4a (1.64 g, 3.0 mmol) in dry THF (30 mL) under argon at 0 ºC was
t
added KO Bu (673.3 mg, 6.0 mmol) and the mixture was stirred at room temperature for 15 min. Then,
iodomethane (0.94 mL, 15.1 mmol) was added and the slurry was stirred for 18 hours at room

ACCEPTED MANUSCRIPT
temperature. The resulting reaction mixture was quenched with saturated aqueous NH Cl solution (20
4
mL) and the organic solvent was evaporated under reduced pressure. The aqueous layer was extracted
4
with dichloromethane (3 x 20 mL) and the combined organic layers were dried over anhydrous MgSO ,
filtered and concentrated under reduced pressure. The crude product was purified by flash
chromatography (eluent: diethyl ether/hexane: 1/1), to provide compound 5 (1.78 g, 88% yield) as a
2
4
–1 1
white solid. M.p. = 109 ºC; [
α
]
D
= −13.72 (
c
= 1.45 in CHCl
= 6.6 Hz, 3H), 1.00 (d,
.32 (s, 9H), 1.50 (s, 9H), 1.81−1.99 (m, 1H), 1.93−2.05 (m, 2H), 2.13 (dd,
= 14.8, 6.5 Hz, 1H), 2.83−2.96 (m, 1H), 3.17 (dd, = 9.7, 9.2 Hz, 1H), 3.30 (s, 3H), 3.34 (dd,
.7, 5.1 Hz, 1H), 3.58 (dd, = 8.6, 7.4 Hz, 1H), 3.83 (s, 3H), 3.87 (dd, = 8.6, 6.4 Hz, 1H), 4.26 (ddd,
= 9.3, 7.4, 6.4 Hz, 1H), 4.55 (dd, = 9.3, 6.9 Hz, 1H), 6.92 (dd, = 8.0, 1.7 Hz, 1H), 7.06 (d, = 1.7
= 8.0 Hz, 1H); C-APT{ H} NMR (100 MHz, CDCl 24.4, 24.7, 25.0, 25.4,
5.7, 28.2, 29.7, 34.9, 36.8, 40.9, 41.8, 55.1, 58.8, 65.2, 67.0, 69.2, 71.2, 75.3, 81.0, 108.1, 112.7,
3
); IR (nujol) 1729, 1612 cm ; H NMR
= 6.6 Hz, 3H), 1.15 (s, 3H),
= 12.8, 6.8 Hz, 1H), 2.28
(
400 MHz, CDCl3) δ 0.54 (s, 3H), 0.96 (d,
J
J
1
J
(
dd,
J
J
J =
9
J
J
J
J
J
J
1
3
1
Hz, 1H), 7.15 (d,
J
3
) δ
2
1
5
+
+
18.8, 125.6, 137.5, 140.1, 158.7, 170.1, 173.4; HRMS(ESI ) m/z [M+H] calcd for C H NO
7
3
2
52
62.3739, found 562.3751.
4
4
.1.7. tert-Butyl (2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-5-((S)-1,2-dihydroxyethyl)-2-isobutyl-
-(methoxymethyl)pyrrolidine-2-carboxylate (6)
Compound 5 (1.40 g, 2.5 mmol) and bismuth(III) chloride (79 mg, 0.25 mmol) were mixed in wet
acetonitrile (50 mL) and the mixture was stirred at room temperature for 4 hours. The resulting
reaction mixture was quenched with saturated aqueous sodium bicarbonate solution (20 mL) and the
organic solvent was evaporated under reduced pressure. The aqueous layer was extracted with
dichloromethane (3 x 20 mL) and the combined organic layers were dried over anhydrous MgSO4,
filtered and concentrated under reduced pressure. The crude product was purified by flash
st
nd
chromatography chromatography (1 eluent: diethyl ether/hexane, 1/1; 2 eluent: diethyl ether) to
2
4
provide compound 6 (1.06 g, 81% yield) as colourless oil. [
α
]D = −17.34 (
c = 0.88 in CHCl ); IR
3
–
1 1
(
nujol) 3579, 3411, 1737, 1699, 1639, 1566 cm ; H NMR (400 MHz, CDCl ) δ 0.99 (d,
J
= 6.6 Hz,
= 14.8, 5.0 Hz,
= 14.8, 6.0 Hz, 1H),
.83−2.98 (m, 1H), 3.02−3.14 (m, 1H), 3.20−3.29 (m, 1H), 3.30 (s, 3H), 3.33−3.42 (m, 3H), 3.57−3.66
3
3
1
2
H), 1.03 (d,
J
= 6.6 Hz, 3H), 1.34 (s, 9H), 1.53 (s, 9H), 1.78−1.90 (m, 1H), 1.95 (dd,
= 13.0, 13.0 Hz, 1H), 2.20 (dd, = 13.0, 6.9 Hz, 1H), 2.37 (dd,
J
H), 2.00 (dd,
J
J
J
(
m, 1H), 3.83 (s, 3H), 4.36 (dd,
J
= 6.7, 6.7 Hz, 1H), 5.26 (d,
J
= 5.4 Hz, 1H), 6.93 (dd,
J
= 7.9, 1.7 Hz,
24.5,
13
1
1
H), 7.04 (d,
J
= 1.7 Hz, 1H), 7.21 (d,
J
= 7.9 Hz, 1H); C-APT{ H} NMR (100 MHz, CDCl
3
)
δ

ACCEPTED MANUSCRIPT
2
5.1, 25.7, 28.1, 29.7, 35.0, 37.8, 41.6, 41.7, 55.3, 59.0, 62.0, 64.9, 70.0, 70.4, 70.8, 83.3, 112.1, 119.1,
+
+
1
26.0, 136.2, 140.4, 158.6, 171.2, 176.6; HRMS(ESI ) m/z [M+Na] calcd for C H NNaO 544.3245,
29 47 7
found 544.3256.
4
.1.8.
tert-Butyl
(2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-5-formyl-2-isobutyl-4-
(
methoxymethyl)pyrrolidine-2-carboxylate (7)
To a stirred solution of compound 6 (1.04 g, 2.0 mmol) in THF (30 mL) at room temperature was
added water (6 mL) followed by sodium metaperiodate (1.71 g, 8.0 mmol) and the mixture was
vigorously stirred at 35 ºC for 16 hours. After that, the solvents were evaporated under reduced
pressure and the residue was partitioned between a mixture of dichloromethane (30 mL) and water (10
mL). The aqueous layer was extracted with dichloromethane (20 mL) and the combined organic layers
were dried over anhydrous MgSO , filtered and concentrated under reduced pressure. The crude
4
product was purified by flash chromatography (eluent: diethyl ether/hexane, 1/1) to afford compound 7
2
4
(
921 mg, 94% yield) as a white solid. M.p. = 175 ºC; [
α
]D = −3.55 (
727, 1728, 1630, 1563 cm ; H NMR (400 MHz, CDCl ) 1.03 (d,
= 14.9, 3.7 Hz, 1H), 2.17 (dd,
= 14.9, 8.2 Hz, 1H), 2.88−2.99 (m, 1H), 3.19 (s, 3H), 3.24 (dd,
c
= 1.01 in CHCl ); IR (nujol)
3
–
1 1
2
1
δ
J
= 6.5 Hz, 6H), 1.32 (s, 9H),
3
.53 (s, 9H), 1.77−1.91 (m, 1H), 2.07 (dd,
= 13.1, 13.1 Hz, 1H), 2.35 (dd,
= 9.8, 4.8 Hz, 1H), 3.43 (dd, = 9.8, 5.2 Hz, 1H), 3.80 (s, 3H), 4.38 (dd,
J
J = 13.1, 7.6 Hz, 1H), 2.26
(
dd,
J
J
J
J
J
J
= 9.1, 3.7 Hz, 1H), 6.75 (d,
1
3
= 1.7 Hz, 1H), 6.77 (dd,
J
= 7.8, 1.7 Hz, 1H), 7.22 (d,
J
= 7.8 Hz, 1H), 9.67 (d, J = 3.7, 1H); C-
1
APT{ H} NMR (100 MHz, CDCl
3
)
δ
24.0, 24.7, 25.8, 28.1, 29.6, 35.1, 38.0, 41.8, 43.2, 55.1, 58.7,
+
6
9.6, 70.2, 70.9, 81.7, 109.6, 117.9, 127.0, 135.0, 140.6, 158.8, 169.5, 172.7, 200.1; HRMS(ESI ) m/z
+
[
M+H] calcd for C H NO 490.3164, found 490.3149.
28 44 6
4
.1.9.
tert-Butyl
(2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-5-carbamoyl-2-isobutyl-4-
(
methoxymethyl)pyrrolidine -2-carboxylate (8)
A solution of compound 7 (146.9 mg, 0.3 mmol) in tert-butanol (12 mL) was diluted with an aqueous
.25M sodium phosphate buffer (4 mL) and the resulting mixture was treated with an aqueous 1M
1
potassium permanganate solution (1.8 mL, 1.8 mmol) at room temperature and vigorously stirred for 2
hours. Then, saturated aqueous sodium sulfite solution (4 mL) was added and the pH of the mixture
was adjusted to 3 with 1M hydrochloric acid. The reaction mixture was extracted with dichloromethane
(
3 x 20 mL) and the combined organic extracts were dried over MgSO and evaporated under reduced
4
pressure to afford a residue containing the crude acid. To a stirred solution of the resulting crude acid

ACCEPTED MANUSCRIPT
and
N
-methylmorpholine (30.5 mg, 0.3 mmol) in anhydrous THF (10 mL) at room temperature was
added a 1M solution of isopropyl chloroformate in toluene (0.45 mL, 0.45 mmol) and the mixture
stirred for 30 minutes. Then, 28% ammonium hydroxide solution (1 mL) was added and the reaction
mixture was stirred for 2 hours. The organic solvent was removed under reduced pressure and the
resulting aqueous solution was extracted with dichloromethane (3 x 20 mL). The combined organic
extracts were dried over anhydrous MgSO , filtered and evaporated under reduced pressure. The
4
residue was purified by flash chromatography (eluent: diethyl ether/hexane, 1/1) to afford compound 8
2
4
(
119.5 mg, 79% yield) as colourless oil. [
α
]
D
= 7.83 (
1.00 (d,
= 13.1, 13.1 Hz, 1H), 2.08−2.19 (m, 2H), 2.41 (dd,
= 9.6, 8.4 Hz, 1H), 3.29 (s, 3H), 3.59 (dd, = 9.6,
= 9.0 Hz, 1H), 5.58 (bs, 1H), 6.86 (d, = 1.7 Hz, 1H), 6.89 (dd,
= 7.9 Hz, 1H), 9.25 (bs, 1H); C-APT{ H} NMR (100 MHz, CDCl )
c
= 1.01 in CHCl
3
); IR (neat) 3324, 3189, 1697,
–
1 1
1
650, 1563 cm ; H NMR (400 MHz, CDCl
3
)
δ
J
= 6.6 Hz, 3H), 1.02 (d,
J
= 6.7 Hz, 3H), 1.32
(
s, 9H), 1.55 (s, 9H), 1.76−1.88 (m, 1H), 2.02 (dd, J
J
= 13.1, 6.4 Hz, 1H), 2.82−2.99 (m, 1H), 3.14 (dd,
.9 Hz, 1H), 3.80 (s, 3H), 4.44 (d,
.9, 1.7 Hz, 1H), 7.22 (d,
J
J
4
7
2
1
5
J
J
J
=
1
3
1
J
δ
3
4.3, 25.0, 25.7, 28.1, 29.6, 35.0, 39.0, 41.3, 41.7, 55.1, 59.2, 65.9, 71.4, 72.3, 83.6, 109.8, 118.1,
+
+
45 2 6
26.8, 134.7, 140.4, 158.5, 169.8, 173.0, 175.8; HRMS(ESI ) m/z [M+H] calcd for C28H N O
05.3273, found 505.3290.
4
.1.10.
tert-Butyl
(2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-5-cyano-2-isobutyl-4-
(
methoxymethyl)pyrrolidine -2-carboxylate (9)
Method A: Phosphorus oxychloride (192 mg, 1.25 mmol) was added to a stirred solution of compound
(252 mg, 0.5 mmol) in pyridine (3 mL) at 0 ºC and the solution was stirred for 6 hours at the same
8
temperature. The resulting solution was poured into ice, diluted with water (3 mL) and extracted with
diethyl ether (3 x 10 mL). The combined organic extracts were dried over anhydrous MgSO , filtered
4
and evaporated under reduced pressure. The residue was purified by flash chromatography (eluent:
diethyl ether/hexane, 1/2) to yield compound 9 (124 mg, 51% yield) as colourless oil.
Method B: Iodine (1.27 g, 5.0 mmol) was added to a stirred solution of compound 7 (245 mg, 0.5
mmol) in aqueous 28% ammonium hydroxide solution (20 mL) and THF (5 mL) at room temperature
and the reaction mixture was stirred for 16 hours. After that, the resulting dark mixture was treated
with saturated aqueous sodium sulfite solution (5 mL), followed by extraction with dichloromethane (2
4
x 20 mL). The combined organic extracts were dried over anhydrous MgSO , filtered and evaporated
under reduced pressure. The residue was purified by flash chromatography (eluent: diethyl
2
4
ether/hexane, 1/2) to afford compound 9 (240.9 mg, 99% yield) as colourless oil. [α]D = 32.95 (c =

ACCEPTED MANUSCRIPT
–
1 1
1
.01 in CHCl
Hz, 3H), 1.01 (d,
3.1 Hz, 1H); 2.05 (dd,
Hz, 1H), 2.83−2.96 (m, 1H), 3.33 (s, 3H), 3.47 (dd,
3
); IR (neat) 2250, 1734, 1657, 1565 cm ; H NMR (400 MHz, CDCl
= 6.6 Hz, 3H), 1.36 (s, 9H), 1.55 (s, 9H), 1.70−1.82 (m, 1H), 2.00 (dd,
= 15.0, 3.4 Hz, 1H), 2.20 (dd, = 13.1, 6.5 Hz, 1H), 2.39 (dd, = 15.0, 8.8
= 9.6, 9.6 Hz, 1H), 3.58 (dd, = 9.6, 5.0 Hz, 1H),
= 7.9, 1.7 Hz, 1H), 6.97 (d, = 1.7 Hz, 1H), 7.30 (d,
23.5, 24.4, 25.7, 28.1, 29.6, 35.1, 37.4, 41.2,
3
)
δ
0.98 (d,
J
= 6.6
J
J = 13.1,
1
J
J
J
J
J
3
.86 (s, 3H), 4.76 (d,
J
= 7.2 Hz, 1H), 6.92 (dd,
J
J
J
1
3
1
=
7.9 Hz, 1H); C-APT{ H} NMR (100 MHz, CDCl
3
)
δ
4
1.7, 55.1, 55.1, 59.4, 70.2, 72.0, 82.4, 109.3, 116.1, 117.3, 127.1, 134.7, 140.5, 158.7, 169.4, 171.9;
+
+
HRMS(ESI ) m/z [M+H] calcd for C H N O 487.3167, found 487.3171.
2
8
43
2
5
4
.1.11.
tert-Butyl
(2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-5-carbamothioyl-2-isobutyl-4-
(
methoxymethyl)pyrrolidine -2-carboxylate (10)
Compound 9 (195.7 mg, 0.4 mmol) was solved in methanol (6 mL) and aqueous 40-44% ammonium
sulfide solution (3 mL) was added. The reaction mixture was stirred at 90 ºC for 4 days in an autoclave.
The resulting mixture was concentrated under reduced pressure and the residue was partitioned
between dichloromethane (20 mL) and water (20 mL). The aqueous layer was further extracted with
dichloromethane (2 x 20 mL) and the combined organic extracts were dried over anhydrous MgSO4,
filtered and evaporated under reduced pressure. The residue was purified by flash chromatography
(
eluent: diethyl ether/hexane, 1/2) to yield compound 10 (197.9 mg, 95% yield) as a white solid. M.p. =
2
4
–
1
58 ºC; [α]D = −27.95 (c = 1.25 in CHCl ); IR (nujol) 3302, 3138, 1743, 1700, 1653, 1565, 1500 cm
3
1
1
;
H NMR (400 MHz, CDCl
3
)
δ
1.00 (d,
= 13.2, 13.2 Hz, 1H), 2.11 (d,
3.2, 6.4 Hz, 1H), 2.85−2.98 (m, 1H), 3.12 (dd, = 9.5, 9.2 Hz, 1H), 3.28 (s, 3H), 3.74 (dd,
= 9.1 Hz, 1H), 6.79 (d,
J
= 6.6 Hz, 3H), 1.01 (d,
J
= 6.6 Hz, 3H), 1.32 (s, 9H), 1.55
= 5.7 Hz, 2H), 2.42 (dd,
= 9.5, 4.6
= 7.9, 1.7 Hz, 1H),
24.3,
(
s, 9H), 1.75−1.90 (m, 1H), 1.99 (dd,
J
J
J =
1
J
J
Hz, 1H), 3.81 (s, 3H), 5.06 (d,
J
J
= 1.7 Hz, 1H), 6.85 (dd, J
1
3
1
7
2
1
.21 (d,
J
= 8.0 Hz, 1H), 7.62 (bs, 1H), 10.66 (bs, 1H); C-APT{ H} NMR (100 MHz, CDCl
3
)
δ
5.0, 25.7, 28.0, 29.6, 35.0, 39.0, 41.2, 41.6, 55.2, 59.1, 70.9, 72.3, 72.8, 83.9, 109.9, 118.2, 126.7,
+
+
45 2 5
34.2, 140.4, 158.5, 170.0, 176.6, 205.2; HRMS(ESI ) m/z [M+H] calcd for C28H N O S 521.3044,
found 521.3058.
4
.1.12. tert-Butyl (2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-2-isobutyl-4-(methoxymethyl)-5-
thiazol-2-yl)pyrrolidine -2-carboxylate (11)
To a solution of thioamide 10 (52.1 mg, 0.1 mmol) and crude bromoacetaldehyde with a 21.5 % purity
22] (228.7 mg, 0.4 mmol) in dry dimethoxyethane (2 mL) was added sodium hydrogen carbonate
(
[

ACCEPTED MANUSCRIPT
(
67.2 mg, 0,8 mmol) and the mixture was stirred at room temperature for 18 hours. The reaction
mixture was concentrated under reduced pressure and the residue was partitioned between
dichloromethane (10 mL) and water (10 mL). The aqueous layer was further extracted with
dichloromethane (2 x 10 mL) and the combined organic extracts were dried over anhydrous MgSO4,
filtered and evaporated under reduced pressure. The residue was solved in dry dimethoxiethane (1mL)
and cooled to 0 ºC. A solution of trifluoroacetic anhydride (84.1 mg, 0,4 mmol) in dry
dimethoxyetnane (0.5 mL) was added followed by a solution of pyridine (63.3 mg, 0.8 mmol) in dry
dimethoxyetane (0.5 mL). The reaction mixture was stirred at room temperature for 24 hours. After
that, the resulting mixture was concentrated under reduced pressure and the residue was partitioned
between dichloromethane (20 mL) and water (10 mL). The aqueous layer was further extracted with
dichloromethane (2 x 10 mL) and the combined organic extracts were dried over anhydrous MgSO4,
filtered and evaporated under reduced pressure. The residue was purified by flash chromatography
(
eluent: diethyl ether/hexane, 1/1) to afford compound 11 (47.4 mg, 87% yield) as a brown solid. M.p.
2
4
–1
1
=
144 ºC; [
α
]D = 66.42 (
1.06 (d,
.91−2.02 (m, 1H), 2.10 (dd,
.95 (dd,
= 9.4, 6.6 Hz, 1H), 3.02−3.14 (m, 1H), 3.06 (s, 3H), 3.55 (s, 3H), 5.46 (d,
.31 (d,
H), 7.48 (d,
c = 0.55 in CHCl ); IR (neat) 3113, 1713, 1608, 1567, 1502 cm ; H NMR
3
(
400 MHz, CDCl3)
δ
J
= 6.6 Hz, 3H), 1.07 (d,
J
= 6.6 Hz, 3H), 1.27 (s, 9H), 1.58 (s, 9H),
= 9.4, 7.1 Hz, 1H),
= 8.5 Hz, 1H),
= 7.9 Hz, 1H), 7.19 (d, = 3.2 Hz,
24.6, 25.3, 25.6, 28.3, 29.6,
1
2
6
1
3
1
5
J
= 14.6, 7.0 Hz, 1H), 2.25−2.35 (m, 3H), 2.81 (dd, J
J
J
J
= 1.7 Hz, 1H), 6.62 (dd,
J
= 7.9, 1.7 Hz, 1H), 7.09 (d,
J
J
1
3
1
J
= 3.2 Hz, 1H); C-APT{ H} NMR (100 MHz, CDCl )
δ
3
4.9, 37.3, 42.7, 42.8, 54.8, 59.0, 63.9, 70.9, 72.0, 81.9, 109.4, 117.1, 119.8, 126.4, 135.9, 139.2, 141.7,
+
+
58.3, 169.5, 169.8, 172.2; HRMS(ESI ) m/z [M+H] calcd for C H N O S 545.3044, found
3
0
45
2
5
45.3061.
4
.1.13.
(2S,4S,5R)-1-(4-(tert-Butyl)-3-methoxybenzoyl)-2-isobutyl-4-(methoxymethyl)-5-(thiazol-2-
yl)pyrrolidine -2-carboxylic acid (GSK3082)
To a solution of compound 11 (27.2 mg, 0.05 mmol) in dichloromethane (0.6 mL) at 0 ºC was added
trifluoroacetic acid (0.2 mL) and the resulting solution was stirred at 0 ºC for 14 hours. The reaction
mixture was diluted with dichloromethane (15 mL), neutralised with saturated aqueous sodium
hydrogen carbonate solution and carefully acidified at pH 1 with 1M hydrochloric acid. The organic
layer was separated and the aqueous layer was extracted with dichloromethane (2 x 10 mL). The
combined organic extracts washed with water (10 mL), dried over anhydrous MgSO , filtered and
4
evaporated under reduced pressure to give compound 1 (23.1 mg, 95% yield) as a brown solid. All

ACCEPTED MANUSCRIPT
7
c
physical and spectroscopic data are in agreement with those previously described in the literature.
4
.1.14. tert-Butyl (2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-5-(4-(ethoxycarbonyl)thiazol-2-yl)-2-
isobutyl-4-(methoxymethyl)pyrrolidine -2-carboxylate (12)
To a solution of thioamide 10 (52.1 mg, 0.1 mmol) and ethyl bromopyruvate 85% (91.8 mg, 0.4 mmol)
in dry dimethoxyethane (2 mL) was added sodium hydrogen carbonate (67.2 mg, 0,8 mmol) and the
mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under
reduced pressure and the residue was partitioned between dichloromethane (10 mL) and water (10 mL).
The aqueous layer was further extracted with dichlorometane (2 x 10 mL) and the combined organic
4
extracts were dried over anhydrous MgSO , filtered and evaporated under reduced pressure. The
residue was solved in dry dimethoxyethane (1 mL) and cooled to 0 ºC. A solution of trifluoroacetic
anhydride (84.1 mg, 0,4 mmol) in dry dimethoxyetnane (0.5 mL) was added followed by a solution of
pyridine (63.3 mg, 0.8 mmol) in dry dimethoxyetane (0.5 mL). The reaction mixture was stirred at
room temperature for 24 hours. After that, the resulting mixture was concentrated under reduced
pressure and the residue was partitioned between dichloromethane (20 mL) and water (10 mL). The
aqueous layer was further extracted with dichlorometane (2 x 10 mL) and the combined organic
extracts were dried over anhydrous MgSO , filtered and evaporated under reduced pressure. The
4
residue was purified by flash chromatography (eluent: diethyl ether/hexane, 1/1) to afford compound
2
4
1
2 (54.2 mg, 88% yield) as a brown solid. M.p. = 114 ºC; [
α
]D = 53.75 (
nujol) 3092, 1730, 1653, 1591 cm ; H NMR (400 MHz, CDCl ) 1.07 (d,
= 14.8, 7.3 Hz, 1H), 2.28 (dd,
= 13.0, 6.3 Hz, 1H), 2.78 (dd, = 9.2,
= 9.2, 5.4 Hz, 1H), 3.58 (s, 3H), 4.26−4.41
c = 1.05 in CHCl ); IR
3
–
1 1
(
δ
J = 6.5, 6H), 1.25 (s, 9H),
3
1
1
7
.34 (t,
3.0, 13.0 Hz, 1H), 2.28 (dd,
.5 Hz, 1H), 3.02−3.12 (m, 1H), 3.06 (s, 3H), 3.16 (dd,
J
= 7.1 Hz, 3H), 1.59 (s, 9H), 1.89−2.00 (m, 1H), 2.07 (dd,
J
J =
J
= 14.8, 3.9 Hz, 1H), 2.37 (dd,
J
J
J
(
m, 2H), 5.59 (d,
J
= 8.7 Hz, 1H), 6.37 (d,
J
= 1.7 Hz, 1H), 6.64 (dd,
J
= 7.9, 1.7 Hz, 1H), 7.09 (d,
J =
1
3
1
7
2
1
.9 Hz, 1H), 8.04 (d,
J
= 0.7 Hz, 1H); C-APT{ H} NMR (100 MHz, CDCl
3
)
δ
14.5, 24.6, 25.3, 25.6,
8.3, 29.5, 34.9, 37.6, 42.5, 42.7, 54.9, 59.0, 61.5, 64.0, 71.1, 71.8, 82.2, 109.3, 117.3, 126.5, 128.4,
+
+
35.4, 139.6, 146.0, 158.2, 161.2, 169.6, 171.0, 172.3; HRMS(ESI ) m/z [M+H] calcd for
C H N O S 617.3255, found 617.3272.
33
49
2
7
4
4
.1.15. (2S,4S,5R)-1-(4-(tert-Butyl)-3-methoxybenzoyl)-5-(4-(ethoxycarbonyl)thiazol-2-yl)-2-isobutyl-
-(methoxymethyl)pyrrolidine-2-carboxylic acid (13)
To a solution of compound 12 (30.8 mg, 0.05 mmol) in dichloromethane (0.6 mL) at 0 ºC was added

ACCEPTED MANUSCRIPT
trifluoroacetic acid (0.2 mL) and the resulting solution was stirred at 0 ºC for 14 hours. The reaction
mixture was diluted with dichloromethane (15 mL), neutralised with saturated aqueous sodium
hydrogen carbonate solution and carefully acidified at pH 1 with 1M hydrochloric acid. The organic
layer was separated and the aqueous layer was extracted with dichloromethane (2 x 10 mL). The
combined organic extracts were washed with water (10 mL), dried over anhydrous MgSO
4
, filtered and
evaporated under reduced pressure to give compound 13 (26.5 mg, 95% yield) as a brown solid. M.p. =
2
4
–1 1
5
5 ºC; [
α]D = 61.89 (c = 0.64 in CHCl ); IR (nujol) 3500−2400, 1741, 1653, 1607, 1565 cm ; H
3
NMR (400 MHz, CDCl3)
δ
1.09 (d,
.1 Hz, 3H), 1.90−2.00 (m, 1H), 2.17−2.23 (m, 2H), 2.30 (dd,
.1 Hz, 1H), 2.54 (dd, = 10.3, 9.4 Hz, 1H), 3.11 (s, 3H), 3.17−3.30 (m, 1H), 3.33 (dd,
H), 3.56 (s, 3H), 4.44 (q, = 7.1 Hz, 2H), 5.50 (d, = 8.1 Hz, 1H), 6.31 (d, = 1.7 Hz, 1H), 6.61 (dd,
= 7.9, 1.7 Hz, 1H), 7.12 (d, = 7.9 Hz, 1H), 8.06 (s, 1H); C-APT{ H} NMR (100 MHz, CDCl
4.4, 24.2, 24.9, 25.7, 29.6, 35.0, 35.6, 42.6, 43.5, 55.0, 58.9, 62.4, 64.0, 70.8, 71.3, 109.1, 116.7,
J
= 6.6 Hz, 3H), 1.12 (d,
J
= 6.6 Hz, 3H); 1.28 (s, 9H), 1.45 (t,
= 15.0, 4.0 Hz, 1H), 2.40 (dd, = 15.0,
= 9.4, 4.5 Hz,
J =
7
8
1
J
J
J
J
J
J
J
J
1
3
1
J
3
) δ
1
1
+
+
26.6, 128.3, 135.0, 140.2, 145.9, 158.6, 160.4, 169.9, 172.0, 173.6; HRMS(ESI ) m/z [M+Na] calcd
NaO S 583.2449, found 583.2441.
for C29
H
40
N
2
7
4
.1.16. tert-Butyl (2S,4S,5S)-1-(4-(tert-butyl)-3-methoxybenzoyl)-2-isobutyl-4-(methoxymethyl)-5-
thiazol-2-yl)pyrrolidine-2-carboxylate (14)
To a solution of thioamide 10 (156.2 mg, 0.3 mmol) and bromoacetaldehyde diethyl acetal (295.6 mg,
.5 mmol) in dry THF (12 mL) was added HCl/dioxane (4N, 0,1 mL) and the mixture was stirred at 65
(
1
ºC for 24 hours. The reaction mixture was concentrated under reduced pressure and the residue was
purified by flash chromatography (eluent: diethyl ether/hexane, 1/1) to afford compound 14 (83.3 mg,
2
4
–
5
1% yield) as an oil. [
α
]
D
= −28.67 (
1.05 (d,
= 12.9, 10.7 Hz, 1H), 2.39−2.49 (m, 2H), 2.75−2.86 (m, 1H),
= 9.6, 6.4 Hz, 1H), 3.47 (dd, = 9.6, 5.2 Hz, 1H), 3.63 (s, 3H), 5.16 (d, = 8.3
= 3.2 Hz, 1H), 7.10 (d,
25.4, 25.4, 25.5, 28.1,
c
= 0.99 in CHCl
3
); IR (neat) 3082, 1734, 1652, 1610, 1566 cm
1
1
;
H NMR (400 MHz, CDCl
3
)
δ
J
= 6.3 Hz, 3H), 1.07 (d,
J
= 6.4 Hz, 3H), 1.28 (s, 9H), 1.51
(
s, 9H), 2.02−2.11 (m, 2H), 2.20 (dd, J
3
.28 (s, 3H), 3.34 (dd,
J
J
J
Hz, 1H), 6.43 (d,
J
= 1.7 Hz, 1H), 6.76 (dd,
J
= 7.9, 1.7 Hz, 1H), 7.06 (d,
J
J =
13
1
7
2
1
5
.9 Hz, 1H), 7.42 (d,
J
= 3.2 Hz, 1H); C-APT{ H} NMR (100 MHz, CDCl )
δ
3
9.6, 34.9, 38.2, 44.1, 47.4, 54.9, 59.1, 64.8, 71.6, 72.3, 81.5, 109.6, 117.6, 119.2, 126.4, 135.9, 139.3,
+
+
45 2 5
42.1, 158.4, 170.4, 172.0, 172.3; HRMS(ESI ) m/z [M+H] calcd for C30H N O S 545.3044, found
45.3056.

ACCEPTED MANUSCRIPT
4
.1.17.
(2S,4S,5S)-1-(4-(tert-Butyl)-3-methoxybenzoyl)-2-isobutyl-4-(methoxymethyl)-5-(thiazol-2-
yl)pyrrolidine-2-carboxylic acid (15)
To a solution of compound 14 (27.2 mg, 0.05 mmol) in dichloromethane (0.6 mL) at 0 ºC was added
trifluoroacetic acid (0.2 mL) and the resulting solution was stirred at 0 ºC for 14 hours. The reaction
mixture was diluted with dichloromethane (15 mL), neutralised with saturated aqueous sodium
hydrogen carbonate solution and carefully acidified at pH 1 with 1M hydrochloric acid. The organic
layer was separated and the aqueous layer was extracted with dichloromethane (2 x 10 mL). The
combined organic extracts washed with water (10 mL), dried over anhydrous MgSO
4
, filtered and
2
4
evaporated under reduced pressure to give compound 15 (23.5 mg, 96% yield) as a brown oil. [
α
]D
=
–
1 1
−
52.63 (
c = 0.90 in CHCl ); IR (neat) 3500−2400, 1728, 1650, 1607, 1566, 1501 cm ; H NMR (400
3
MHz, CDCl
3
)
δ
1.03 (d,
= 14.0, 6.2 Hz, 1H), 2.42 (dd,
.5 Hz, 1H), 2.87 (dd, = 14.0, 5.8 Hz, 1H); 3.33 (s, 3H), 3.34 (dd,
.8, 5.1 Hz, 1H), 3.61 (s, 3H), 5.26 (d, = 3.3 Hz, 1H), 6.38 (d, = 1.6 Hz, 1H), 6.63 (dd,
= 7.9 Hz, 1H), 7.15 (d, = 3.3 Hz, 1H), 7.60 (d, = 3.3 Hz, 1H), 8.51 (bs, 1H);
C-APT{ H} NMR (100 MHz, CDCl )
J
= 6.6 Hz, 3H), 1.04 (d,
= 13.2, 7.2 Hz, 1H), 2.48−2.58 (m, 1H), 2.83 (dd,
= 9.8, 8.3 Hz, 1H), 3.42 (dd,
= 7.9, 1.6
J
= 6.6 Hz, 3H), 1.29 (s, 9H), 1.79−1.91 (m, 1H),
2
3
9
.20 (dd,
J
J
J
= 13.2,
J
J
J =
J
J
J
Hz, 1H), 7.13 (d,
J
J
J
1
3
1
δ
24.0, 24.8, 25.2, 29.5, 35.0, 36.6, 43.8, 46.0, 55.0, 59.1, 65.6,
3
+
7
2.8, 74.5, 108.8, 117.3, 119.6, 126.7, 135.0, 140.1, 142.3, 158.3, 170.9, 173.9, 174.6; HRMS(ESI )
+
m/z [M+Na] C H N NaO S 511.2238, found 511.2251.
2
6
36
2
5
4
.1.18.
(2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-5-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-2-
isobutyl-4-(methoxymethyl)pyrrolidine-2-carboxylic acid (16)
A solution of compound 5 (112.4 mg, 0.2 mmol) in 10% KOH/methanol (5 mL) was refluxed for 5
days. The resulting mixture was cooled and the solvent evaporated under reduced pressure. The
residue was diluted in water (10 mL) and washed with dichloromethane (5 mL). The aqueous layer was
then acidified with 1M hydrochloric acid and extracted with dichloromethane (2 x 20 mL). The
combined organic extracts were dried over anhydrous MgSO , filtered and concentrated under reduced
4
2
4
pressure to provide compound 16 (100.1 mg, 99% yield) as a white solid. M.p. = 155 ºC; [
α
]D
); IR (nujol) 3400−2200, 1739, 1605, 1558 cm ; H NMR (400 MHz,
= 6.4 Hz, 3H), 0.98 (d, = 6.3 Hz, 3H), 1.15 (s, 3H), 1.22 (s, 3H), 1.34 (s, 9H),
.73−1.85 (m, 2H), 2.18 (dd, = 12.6, 6.5 Hz, 1H), 2.36 (dd, = 15.4, 7.7 Hz, 1H), 2.70−2.82 (m, 1H),
.82 (dd, = 12.6, 12.6 Hz, 1H), 3.24 (dd, = 9.6, 9.6 Hz, 1H), 3.29 (s, 3H), 3.38 (dd, = 8.0, 8.0 Hz,
H), 3.46 (dd, = 9.6, 4.4 Hz, 1H), 3.67 (dd,
= 8.0, 6.0 Hz, 1H), 3.35 (s, 3H), 3.88−3.95 (m, 1H),
=
–
1
1
−
99.59 (c = 1.11 in CHCl
3
CDCl δ 0.96 (d,
3
)
J
J
1
2
1
J
J
J
J
J
J
J

ACCEPTED MANUSCRIPT
4
.49 (dd,
J
= 6.2, 6.2 Hz, 1H), 6.91 (dd,
J
= 7.9, 1.7 Hz, 1H), 7.04 (bs, 1H), 7.26 (d,
J =7.9 Hz, 1H);
1
3
1
C-APT{ H} NMR (100 MHz, CDCl )
δ 23.9, 24.7, 24.9, 25.1, 25.9, 29.5, 35.3, 35.8, 39.2, 42.7, 55.2,
3
5
9.0, 65.1, 66.3, 71.2, 73.0, 73.7, 109.1, 112.1, 119.3, 126.7, 134.1, 142.5, 150.0, 174.0, 175.4;
+
+
HRMS(ESI ) m/z [M+Na] calcd for C H NNaO 528.2932, found 528.2919.
2
8
43
7
4
2
.1.19. tert-Butyl (2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-2-isobutyl-4-(methoxymethyl)-5-((S)-
-oxo-1,3-dioxolan-4-yl)pyrrolidine-2-carboxylate (17)
To a solution of compound 6 (104.3 mg, 0.2 mmol) in THF (5 mL) was added 1,1’-
carbonyldiimidazole (97.3 mg, 0.6 mmol) and the mixture was stirred at 60 ºC for 24 hours. After that,
the solvent was evaporated under reduced pressure and the residue was purified by flash
chromatography (eluent: diethyl ether/hexane, 3/1) to afford compound 17 (98.5 mg, 90% yield) as a
2
4
–
white solid. M.p. = 90 ºC; [
α
]D = −37.76 (
H NMR (400 MHz, CDCl3) δ 0.93−1.00 (m, 3H), 1.03 (d,
H), 1.76−1.90 (m, 1H), 1.91−2.05 (m, 1H), 2.10−2.22 (m, 2H), 2.28−2.42 (m, 1H), 2.86−3.02 (m,
H), 3.15−3.26 (m, 1H), 3.29 (s, 3H), 3.46 (dd, = 10.2, 4.0 Hz, 1H), 3.88 (s, 3H); 4.03−4.15 (m, 1H),
.20−4.31 (m, 1H), 4.55−4.68 (m, 1H), 4.84−4.95 (m, 1H), 6.84 (bd, = 7.9 Hz, 1H), 7.08 (d, = 1.7
24.4, 25.2, 25.6, 28.1, 29.6,
c = 1.01 in CHCl ); IR (nujol) 1815, 1729, 1639, 1565 cm
3
1
1
;
J = 6.6 Hz, 3H), 1.34 (s, 9H), 1.52 (s,
9
1
J
4
J
J
1
3
1
Hz, 1H), 7.17 (d,
J = 7.9 Hz, 1H); C{ H} NMR (100 MHz, CDCl ) δ
3
3
1
5
5.0, 36.5, 40.8, 41.7, 55.1, 59.1, 63.6, 67.3, 69.3, 70.3, 75.7, 77.4, 82.1, 112.4, 117.8, 125.8, 135.8,
+
+
40.4, 153.4, 159.1, 170.5, 173.1; HRMS(ESI ) m/z [M+H] calcd for C H NO 548.3218, found
3
0
46
8
48.3232.
4
.1.20. (2S,4S,5R)-1-(4-(tert-Butyl)-3-methoxybenzoyl)-2-isobutyl-4-(methoxymethyl)-5-((S)-2-oxo-1,3-
dioxolan-4-yl)pyrrolidine-2-carboxylic acid (18)
To a solution of compound 17 (54.7 mg, 0.1 mmol) in dichloromethane (1.2 mL) at 0 ºC was added
trifluoroacetic acid (0.4 mL) and the resulting solution was stirred at 0 ºC for 14 hours. The reaction
mixture was diluted with dichloromethane (15 mL), neutralised with saturated aqueous sodium
hydrogen carbonate solution and carefully acidified at pH 1 with 1M hydrochloric acid. The organic
layer was separated and the aqueous layer was extracted with dichloromethane (2 x 15 mL). The
combined organic extracts washed with water (10 mL), dried over anhydrous MgSO , filtered and
4
evaporated under reduced pressure to give compound 18 (46.1 mg, 94% yield) as a brown solid. M.p. =
2
4
1
94 ºC; [
α
]
D
= −146.28 (
c
= 0.92 in CHCl
3
); IR (nujol) 3700−2500, 1818, 1691, 1653, 1608, 1572
= 6.7 Hz, 3H), 1.02 (d, = 6.7 Hz, 3H), 1.36 (s, 9H),
–
1 1
cm ; H NMR (400 MHz, CDCl
3
)
δ 1.00 (d,
J
J

ACCEPTED MANUSCRIPT
1
.76−1.85 (m, 2H), 2.25 (dd,
13.9, 13.9 Hz, 1H), 3.18 (dd,
s, 3H), 3.95 (dd, = 9.2, 5.8 Hz, 1H), 4.32 (dd,
= 9.9, 8.0, 5.8 Hz, 1H), 6.79 (dd, = 7.9, 1.7 Hz, 1H), 7.10 (d,
.9 Hz, 1H); C{ H} NMR (100 MHz, CDCl ) 23.8, 24.7, 25.1, 29.5, 34.7, 35.5, 39.1, 42.6, 55.3,
9.1, 66.3, 67.0, 69.7, 71.9, 74.1, 112.0, 117.6, 126.6, 133.7, 142.3, 153.1, 159.5, 173.8, 176.1;
J
= 13.9, 7.8 Hz, 1H), 2.41−2.52 (m, 1H), 2.72−2.84 (m, 1H), 2.98 (dd,
J
=
J
= 10.5, 7.8 Hz, 1H), 3.28 (s, 3H), 3.52 (dd,
= 9.2, 8.0 Hz, 1H), 4.45 (dd,
= 1.7 Hz, 1H), 7.26 (d, J =
J
= 10.5, 3.5 Hz, 1H), 3.89
(
J
J
J
= 9.9, 6.2 Hz, 1H),
4
7
5
.58 (ddd,
J
J
J
1
3
1
δ
3
+
+
HRMS(ESI ) m/z [M+Na] calcd for calcd for C H NNaO 514.2412, found 514.2425.
2
6
37
8
4
.1.21.
(2S,4S,5R)-1-(4-(tert-Butyl)-3-methoxybenzoyl)-5-carbamoyl-2-isobutyl-4-
(
methoxymethyl)pyrrolidine-2-carboxylic acid (19)
To a solution of compound 8 (50.4 mg, 0.1 mmol) in dichloromethane (1.2 mL) at 0 ºC was added
trifluoroacetic acid (0.4 mL) and the resulting solution was stirred at 0 ºC for 8 hours. The reaction
mixture was diluted with dichloromethane (15 mL), neutralised with saturated aqueous sodium
hydrogen carbonate solution and carefully acidified at pH 1 with 1M hydrochloric acid. The organic
layer was separated and the aqueous layer was extracted with dichloromethane (2 x 20 mL). The
combined organic extracts were washed with water (10 mL), dried over anhydrous MgSO
4
, filtered and
evaporated under reduced pressure to give compound 19 (43.1 mg, 96% yield) as a brownish solid.
2
4
M.p. = 253 ºC; [α]D = 7.12 (c = 1.01 in CHCl ); IR (nujol) 3600−2200, 3433, 3337, 3209, 1720,
3
–
1 1
1
3
2
3
1
3
1
650, 1608, 1570 cm ; H NMR (400 MHz, CDCl )
δ
1.01 (d,
= 13.4, 13.4 Hz, 1H), 2.16 (dd,
= 15.1, 8.3 Hz, 1H), 2.94−3.14 (m, 1H), 3.28 (s, 3H),
= 8.5 Hz, 1H), 6.45 (bs, 1H), 6.80−6.84 (m, 2H), 7.05 (bs,
J
= 6.7 Hz, 3H), 1.01 (d,
J = 6.7 Hz,
3
H), 1.31 (s, 9H), 1.75−1.89 (m, 1H), 2.01 (dd,
.20 (dd, = 13.4, 6.7 Hz, 1H), 2.34 (dd,
.31−3.40 (m, 2H), 3.77 (s, 3H), 4.64 (d,
J
J
= 15.1, 3.6 Hz, 1H),
J
J
J
1
3
1
H), 7.22 (d,
J
= 8.0 Hz, 1H); C-APT{ H} NMR (100 MHz, CDCl
3
)
δ
23.9, 24.6, 25.7, 29.6, 35.1,
7.0, 41.5, 41.9, 55.2, 59.1, 65.4, 70.6, 71.5, 109.8, 117.6, 126.8, 134.3, 140.6, 158.7, 170.4, 175.4,
+
+
75.4; HRMS(ESI ) m/z [M+Na] calcd for calcd for C H N NaO 471.2466, found 471.2481.
2
4
36
2
6
4
.1.22.
tert-Butyl
(2S,4S,5R)-1-(4-(tert-butyl)-3-methoxybenzoyl)-2-isobutyl-4,5-
bis(methoxymethyl)pyrrolidine-2-carboxylate (20)
A solution of compound 7 (97.9 mg, 0.2 mmol) in ethanol (2 mL) was reacted with sodium
borohydride (75.7 mg, 2 mmol) for 16 hours at room temperature. The reaction was quenched with
saturated aqueous ammonium chloride solution (2 mL) and concentrated under reduced pressure. The
residue was partitioned between dichloromethane (20 mL) and water (10 mL), the organic layer was