A new scalable synthesis of entecavir

Article abstract of DOI:10.1016/j.tet.2017.12.034

A new synthesis of entecavir from D-glucose in an average total yield of 3.5% was achieved via an intramolecular nitrile oxide cycloaddition (INOC) reaction and a Peterson olefination as key-steps. The present process was designed for industrial application, using widely available raw materials, simple and cheap reagents and avoiding low reaction temperatures, which are very common in the synthetic approaches towards similarly complex structures.

Full text of DOI:10.1016/j.tet.2017.12.034

Accepted Manuscript
A new scalable synthesis of entecavir
Efthymia G. Gioti, Theocharis V. Koftis, Efstratios Neokosmidis, Elli Vastardi,
Stefanos S. Kotoulas, Sakellarios Trakossas, Theodoros Tsatsas, Elizabeth E.
Anagnostaki, Theodoros D. Panagiotidis, Constantinos Zacharis, Evanthia P. Tolika,
Anastasia-Aikaterini Varvogli, Thanos Andreou, John K. Gallos
PII:
S0040-4020(17)31302-9
10.1016/j.tet.2017.12.034
TET 29190
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 19 October 2017
Revised Date: 12 December 2017
Accepted Date: 15 December 2017
Please cite this article as: Gioti EG, Koftis TV, Neokosmidis E, Vastardi E, Kotoulas SS, Trakossas S,
Tsatsas T, Anagnostaki EE, Panagiotidis TD, Zacharis C, Tolika EP, Varvogli A-A, Andreou T, Gallos
JK, A new scalable synthesis of entecavir, Tetrahedron (2018), doi: 10.1016/j.tet.2017.12.034.
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Graphical Abstract
ACCEPTED MANUSCRIPT
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A New Scalable Synthesis of Entecavir
E. G. Gioti, T. V. Koftis, E. Neokosmidis, E. Vastardi, S. S. Kotoulas, S. Trakossas, T. Tsatsas, E. E.
Anagnostaki, T. D. Panagiotidis, C. Zacharis, E. P. Tolika, A.-A. Varvogli, T. Andreou, and J. K. Gallos
Pharmathen SA – R&D API Operations, 9th km Thermi-Thessaloniki, Thessaloniki 57001, Greece, and
Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
1

A New Scalable Synthesis of Entecavir
ACCEPTED MANUSCRIPT
Efthymia G. Gioti,a,b Theocharis V. Koftis,*a Efstratios Neokosmidis,a Elli Vastardi,a Stefanos S.
Kotoulas,a Sakellarios Trakossas,a Theodoros Tsatsas,a Elizabeth E. Anagnostaki,a Theodoros D.
Panagiotidis,a Constantinos Zacharis,a Evanthia P. Tolika,a Anastasia-Aikaterini Varvogli,a Thanos
Andreou,a and John K. Gallos*b
a Pharmathen SA – R&D API Operations, 9th km Thermi-Thessaloniki, Thessaloniki 57001, Greece
b Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
Abstract: A new synthesis of entecavir from D-glucose in an average total yield of 3.5% was achieved via an intramolecular nitrile oxide
cycloaddition (INOC) reaction and a Peterson olefination as key-steps. The present process was designed for industrial application, using
widely available raw materials, simple and cheap reagents and avoiding low reaction temperatures, which are very common in the synthetic
approaches towards similarly complex structures.
Keywords:
entecavir
INOC reaction
Mitsunobu reaction
catalytic hydrogenation
Peterson olefination
Introduction
Hepatitis B is a disease caused by the hepatitis B virus (HBV) and affects the liver.1 The virus is transmitted
through contaminated blood or infected body fluids. Vomiting, yellow skin, dark urine, fatigue and abdominal pain
are typical symptoms of the disease. Most patients with chronic disease are asymptomatic, although ultimately the
patient can be led to the development of liver cirrhosis and liver cancer, which does not respond to chemotherapy,
resulting to a death rate of 15 ̶ 25%.
In recent years, prophylactic vaccination is used, and in many patients presenting mild symptoms of the disease the
body fights it alone. But in patients with chronic hepatitis B there is no cure. In those cases, the use of drugs is the
only way to handle multiplication of the virus and thus to reduce liver damage, in order to increase the life
expectancy of patients. Several antiviral drugs have been developed for this purpose. Among them are entecavir (1,
Baraclude), lamivudine (2, Epivir), telbivudine (3, Tyzeka), adefovir (4, Hepsera) and tenofovir (5, Viread), which
have a modified nucleoside structure and act as reverse transcriptase inhibitors (NRTIs) and inhibit the only known
enzyme target of HBV, the viral polymerase (Figure 1).
Figure 1. Drugs for hepatitis B treatment with a modified nucleoside structure.
* Corresponding author. E-mail address: hkoftis@pharmathen.com (T. V. Koftis) and igallos@chem.auth.gr (J. K. Gallos)
2

Lamivudine (2) and adefovir (4), which were first used, can act quickly and directly, but after prolonged use the
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virus acquires immunity. Entecavir (1), a carbocyclic analogue of 2-deoxyguanosine, originally used for the herpes
virus with disappointing results, was found to be an HBV inhibitor with very low toxicity. It is converted
intracelullarly to the 5-triphosphorylated form and effectively binds the polymerase HBV, resulting in an in vitro
efficacy 30 times greater than that of lamivudine (2) and similar antiviral drugs, whereas patients do not experience
any resistance to the drug after prolonged use, and the virus has not shown mutations. Entecavir exhibits activity
against HBV polymerase in each step involved in the synthesis of viral DNA and, for the moment, it is the most
effective drug against HBV.
As a result, the synthesis of entecavir has attracted considerable attention by synthetic chemists and pharmaceutical
companies. Its first synthesis, completed at Bristol-Myers Squibb in 1992 started from cyclopentadienyl sodium to
prepare entecavir in 15-19% overall yield and >99% ee, and in the following years the same and other research
groups published minor modifications of the original methodology.2a,b,c In 2004, analogous shortened approaches
were published from the laboratories of Bristol-Myers Squibb, starting again from cyclopentadienyl sodium.2d In
2003, Ziegler and Sarpong prepared the carbocyclic sugar core of entecavir from diacetone-D-glucose, and eleven
years later, Jing et al prepared entecavir using this intermediate and introducing guanine base by a Mitsunobu
reaction.2e
A different approach was used by Liotta and Li, who starting from fulvene and chlorosulfonyl isocyanate
developed a short synthesis of entecavir applying palladium chemistry in the key-step.2f By the same time,
researchers from the Korean company Hanmi, reported a new improved synthesis of entecavir in 5% total yield and
99% ee, starting from protected 2-hydroxymethyl-cyclopentenone.2g The same year, Chu et al prepared entecavir
from a cyclopentenone chiral building block and in 2012, Zhou and Li published a new approach, which applied
the intramolecular nitrile oxide cycloaddition (INOC) reaction in order to construct the sugar core, whereas the
chirality was introduced by a Sharpless asymmetric epoxidation reaction.2h,i
Also in 2012, another synthesis of entecavir was reported by Liu et al, which had ten steps and 21% overall yield,
and used a ring closing metathesis (RCM) in order to prepare the carbocyclic ring.2j Finally, one of the most recent
syntheses of entecavir was developed by researchers at the University of Barcelona and Esteve Quimica. They
prepared the protected carbocyclic core already prepared by Ziegler and Sarpong by a different way and introduced
the guanine base by a Mitsunobu reaction.2k,l,m
All the above mentioned processes exhibit some major drawbacks, since many of the reagents employed are
difficult to handle, highly toxic or relatively expensive. Moreover, in many cases, the synthetic steps undertaken
involve highly sensitive manipulations, require low temperatures, harsh conditions or require the use of highly
customized equipment. Since such technical features may significantly hamper the industrial application of the
reported methodologies, there is still a need for an industrially applicable, efficient and safe process for the
production of entecavir.
Results and Discussion
Taking into account the importance of entecavir and also the need for a more convenient synthesis suitable for
industrial scale preparations, we designed a new approach depicted in Scheme 1. According to this retrosynthetic
analysis, entecavir (1) could be prepared from an appropriately protected trihydroxyketone 6 by introduction of
base via a Mitsunobu reaction and olefination of the carbonyl group, whereas 6 is the product of isoxazoline ring
cleavage in 7, which in turn could be made by an intramolecular nitrile oxide cycloaddition (INOC) reaction of
unsaturated oxime 8. This oxime could be prepared from known compound 9, which is prepared from cheap and
commercially available diacetone-D-glucose 10.2e,3 It is interesting to note that, according to a recent publication,
the INOC reactions in oximes analogous to 8 do not need protection of hydroxyl groups, a finding that was
expected to simplify our synthetic approach.4
Commercial diacetone-D-glucose 10 was deoxygenated to give compound 12 (Scheme 2), which was isolated as an
oil in 75% total yield, by a modified Barton-McCombie reaction using a system of Bu3SnH, (Bu3Sn)2O and
polymethylhydrosiloxane (PMHS, HO[MeSi(H)O]nH, n=35) in order to reduce xanthate 11, avoiding thus the use
of explosive AIBN.3d,e,f In the next step, the exocyclic acetonide was hydrolyzed when treated with 40% aqueous
acetic acid for 24 h at 25 °C in 81% yield.2e An attempt to deoxygenate 13, according to the literature method, gave
poor yields (30%) of compound 9.2e However, double mesylation of 13 by standard procedures2n (95%), followed
by treatment with activated Zn and NaI in DME at 90-95 °C for 4 h, gave the desired compound 9 (85% yield),
which easily hydrolyzed with 4% aqueous H2SO4 in THF towards 15, in 93% yield.5,2e
3

NACCEPTED MANUOSCRIPT
O N
O
N
OH
OH
HO
HO
NH
NH2
N
HO
HO
HO
1
6
7
HO
HO
N
O
O
O
OH
O
O
O
O
O
HO
10
8
9
Scheme 1. Retrosynthetic analysis of entecavir
The respective oxime was then successfully prepared from 15 by reaction with hydroxylamine hydrochloride in
pyridine (94%) or in methanol using NaHCO3 as a base (99%).4 As expected, product 16 is a suitable substrate for
INOC reactions, which could give intermediate 7 (Scheme 1). Usually, sugar derived polyhydroxylated substrates
for INOC reactions need protection of the hydroxyl groups, since a free hydroxyl group may react with the
electrophilic nitrile oxide carbon under basic conditions used in such reactions. Shing et al reported that using
chloramine-T as oxidant in the presence of silica gel, and thus generate a slight acidic environment, oximes
analogues to 16 give high yields of the respective cycloaddition products without formation of side-products.4
Scheme 2. Reagents and conditions: (i) NaH, CS2, MeI, THF, rt, 2 h; (ii) Bu3SnH, (Bu3Sn)2O, PMHS, 1-BuOH, toluene, 135 oC, 24 h (75%
0
o
for two steps); (iii) 40% CH3COOH, H2O, rt, 24 h (81%); (iv) MsCl, Et3N, DCM, 0-5 C, 1 h (95%); (v) NaI, Zn act, glyme, 90-95 C, 4 h
(85%); (vi) 4% H2SO4, THF, 65-70 oC, 1h (93%); (vii) NH2OH.HCl, NaHCO3, MeOH, rt, 3 h (99%).
However and despite many attempts, we were unable to isolate the desired compound 7, among the several
products generated in the oxidation of oxime 8. A great variety of conditions were tried, regarding oxidants
(chloramine-T, NaOCl, NCS, PhI(OAc)2), solvent (ethanol, isopropanol, DMF, water, pyridine, dioxane),
temperature and reactants ratio, but either the oxime remained unchanged or several products were formed, and the
desired product was never obtained.4,6
These results indicated that a hydroxyl protection was necessary. Thus, changing the initial plans, compound 9
gave 17 by methanolysis, which was then benzylated by standard procedures (Scheme 3).7 Further acetal hydrolysis
with 60% aqueous acetic acid resulted in the formation of lactol 19 in 76% yield from 9, better than that reported in
the literature (68%).7
4

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o
o
Scheme 3. Reagents and conditions: (i) cat. H2SO4, MeOH, 65-70 C, 4 h (90%); (ii) BnBr, NaH, THF, 65-70 C, 4h (95%); (iii) 60%
CH3COOH, cat. H2SO4, 70-75 C, 24 h (89%); (iv) NH2OH.HCl, NaHCO3, MeOH, rt, 2 h (99%); (v) Chloramine-T trihydrate, silica gel,
o
EtOH, rt , 67% 7:1 mixture of 21/22 (for 23 see text).
This substrate was converted to the respective oxime upon treatment with NaHCO3 in methanol and oxidation of
the later gave the desired fused isoxazoline 21, together with diastereoisomer 22.4 After several attempts, the best
results in this step were obtained when chloramine-T in ethanol was used as oxidant at 8-10 °C for 2 h. In this case,
the mixture of 21/22 was obtained in 67% yield in a 7:1 ratio, but their separation was laborious. Pure isoxazolines
21 and 22 were obtained by careful chromatographic separation of a small amount of mixture of 21/22, in order to
1
unequivocally assign their structures using H-NMR, 13C-NMR, DEPT135, COSY, HMBC, HSQC and NOESY
1D techniques. In all oxidation reactions, a small amount of oximolactone 23 was observed, but its characterization
was difficult, since it is eluted together with the sulfonamide reduction product of chloramine-T.
Aiming to separate the mixture of diastereoisomers at a more favourable stage, we proceeded using the
mixture of 21/22 in a classical isoxazoline cleavage by Raney Ni hydrogenolysis in the presence of boric
acid. It is known that these reaction conditions lead to the formation of β-hydroxyketones with
suppression of side reactions and epimerizations. Unfortunately, in our case a mixture of deoxygenated
diastereoisomers 26 was obtained (Scheme 4) when the isoxazoline ring cleavage of 21/22 was attempted,
although many different conditions were queried (Raney Ni/H2/H3BO3, Pd/C/H2).8 Evidently, an
elimination reaction occurs in the initial cleavage product with subsequent hydrogenation of the double
bond formed. Analogous examples have been reported in the literature,8g,h,i when the cleavage of an
isoxazoline fused with hydroxylated cyclopentane ring was attempted. On the other hand, when the
isoxazoline ring cleavage of 21/22 was attempted with O3, an unidentified mixture of products was
obtained.
Scheme 4. Reagents and conditions: (i) Raney Ni/H2/H3BO3, 37% or Pd/C/H2, 43%.
It was thus clear that protection of the free hydroxyl group in 21/22 with a proper protecting group, which reduces
its leaving ability, could prevent such a reaction pathway. To this end, we decided to introduce several protecting
groups in order to find the best one (Scheme 5). Silylation of 21/22 with TBS chloride gave the respective protected
isoxazolines 27a (82%) and 28a (14%), which were easily separated chromatographically. Similarly, the p-
nitrobenzoylation of 21/22 led to the respective compounds 27b (73%) and 28b (6%), in lower yields. By
5

protecting 21/22 with trityl, MOM and TBDPS groups, there were isolated only the major isomer products 27c-e.
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This could be attributed either to the lesser reactivity of more hindered hydroxyl group of 22 or to the loss of
protected minor products during the chromatographic separation.
Scheme 5. Reagents and conditions: (i) a) TBSCl, imidazole, DMAP, DCM, rt, 2 h (82% for 27a); b) p-NO2BzCl, Et3N, DMAP, DCM, rt, 2
h (73% for 27b); c) TrCl, DMAP, pyridine, 100oC, 12 h (58% for 27c); d) MOMCl, DIPEA, DCM, rt, 2 h (63% for 27d); e) TBDPSCl,
DMAP, imidazole, DCM, rt, 2 h (79% for 27e); (ii) H2, B(OH)3, 5% Pd/C, MeOH/H2O, 1.5 h, rt (74%).
In the next step all products 27a-e were subjected to Pd/C catalytic hydrogenation in the presence of boric acid at 0
°C (Scheme 5). The desired product 29a was now isolated from the reaction of 27a in 33%, unfortunately together
with the side-products 25 and 26 in a 1:1:1 ratio. Similarly, isoxazolines 27c and 27e gave the respective 29c and
29e products together with the side-products 25 and 26, but in the less favorable ratio 1:2:2. Finally, none of the
desired products 29 was isolated from the reactions of 27b and 27d, giving mixtures of side-products 25 and 26, in
a 1:1 ratio.
Since the TBS protected isoxazoline 27a gave the best results, we decided to proceed with this compound,
attempting firstly to improve the yield for the formation of 29a. Trying a plethora of conditions, regarding catalyst,
solvent, temperature, catalyst ratio and reaction time, the best results were obtained when 5% Pd/C w/w was used
in a 10% ratio at 25 °C in methanol/water (5:1) as solvent with 15.2 equiv. of boric acid as additive (Scheme 6).8l In
this case, the desired product 29a was isolated in 74% yield, together with the side-products 25 and 26, both in 12%
yield. It is worth mentioning that reaction and yields were reproducible even at 40 g scale.
6

Scheme 6. Reagents and conditions: (i) TBSCl, imidazole, DMAP, DCM, rt, 30 min (85%); (ii) TMSCH2MgCl 1.0M in Et2O, rt, 1 h (90%);
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(iii) H2, 10% Pd/C, THF, rt, 2 h (80%); (iv) KHMDS, THF, rt, 1 h (70%); (v) 6-chloro-2-aminopurine or 6-chloro-2-boc-aminopurine or 6-
iodo-2-boc-aminopurine, Ph3P, DEAD, THF (64-85%); (vi) 2N aqueous HCl, THF (80-90%).
Having the carbocyclic ring of entecavir in quantity, we then protected the primary hydroxyl group of 29a as a TBS
ether towards 31, in 85% yield. Due to the basic conditions used, an elimination product was isolated in 10% yield,
which was found to be the TBS ether 30 (Figure 2). The Wittig reaction is the most general process for the
introduction of exomethylene group in the laboratory. However, it is problematic for an industrial synthesis,
because of the triphenylphosphine oxide by-product and the problems associated with its separation and also the
low temperature needed for the ylide generation. Thus, we decided to apply the Peterson olefination reaction,
which overcomes these problems: the reaction can be performed at ambient temperature and the by-products are
volatile and easily driven off.
Furthermore, the Peterson reaction seems ideal for our case: the intermediate β-hydroxysilane can be isolated and at
this stage, we could debenzylate the secondary hydroxyl group, by hydrogenolysis. Indeed, the addition of
TMSCH2MgCl to ketone 30 resulted in the formation of 32 in high yield, which was then reductively debenzylated
to give good yields of 33.9 We did not identify the configuration of the newly formed stereocenter in either
compound 32 or 33, since it is going to be destroyed in the next step. Thus, we proceeded into the second step of
the Peterson reaction to generate the double bond and found that the best conditions were treatment of a solution of
33 in THF with 5 equiv. of 0.5M solution of KHMDS at 0 °C.10 The expected compound 34 was the sole product
(70%).
The conversion of 34 into entecavir (1) has been reported in the literature.2j,11 We introduced the base by the
standard Mitsunobu reaction, using 6-chloro- or 6-iodo-purine, with or without protection of the amino group in 64-
85% yields. The final deprotection with 2N aqueous HCl in THF resulted in the formation of entecavir in 80-90%
yield.
All above reactions were carried out in gram scale and all products were separated chromatographically in order to
be fully characterized. To our delight, these reactions gave repeatable results in 100 g scale and products were of
accepted purity and ready for use directly in the next step without chromatographic separation. Thus, starting
compound 10 was converted to the oily intermediate 12 and then to solid 13, which was purified by
recrystallization. In the next steps, intermediate products 14, 9, 17-19 were of acceptable purity and after their
work-up, they were used in the following reactions as they were, to give finally crystals of oxime 20, which
purified again by recrystallization. Since mixture 21/22 was inseparable, chromatography is needed for the
purification of compound 27a. Compounds 29a and 31 being relatively unstable were used to next reactions
without further purification. The later was converted to solids 33 and 34, which as in the following intermediates
were easily purified by recrystallization.
In conclusion, entecavir (1) was synthesized in eighteen steps from D-glucose in an average total yield of 3.5%
through compounds 35a, 35b or 35c. Although compound 34 has already been prepared through different
processes, it may be now synthesized from D-glucose through an INOC reaction and a Peterson olefination.2d,2i,2j
The present process was designed for industrial application, using widely available raw materials, simple and cheap
reagents and avoiding low reaction temperatures, which are very common in the synthetic approaches towards
similarly complex structures. These advantages12 have been notable during the technology transfer and scale up of
the initial laboratory synthesis. The novel approach described herein has, therefore, evolved into a process with
robustness and reproducibility, which are features of significant importance for industrial manufacturing.
Experimental Section
General
All reagents are commercially available and were used without further purification. Solvents were dried
by standard methods. The progress of reactions was checked by thin layer chromatography (TLC) on
Merck silica gel 60F254 glass plates (0.25 mm). The spots were visualised by heat staining with
anisaldehyde in ethanol/sulfuric acid. Column chromatography was performed with Merck silica gel 60
(0.063–0.200 mm). Melting points were determined with a Kofler hot-stage microscope. 1H and 13C NMR
spectra were recorded at 300 or 500 MHz and 75 or 126 MHz, respectively. 1H NMR chemical shifts (δ)
are reported in ppm relative to tetramethylsilane (δ = 0.0 ppm), using the residual solvent signal as an
internal standard (δ = 7.26, singlet, for CDCl3), or using tetramethylsilane itself. The proton resonances
are annotated as: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br.,
13
broad), coupling constant (J [Hz]), and integration. C NMR chemical shifts are reported in ppm, and
7

spectra were calibrated using the central line of the triplet at δ = 77.0 ppm for CDCl3. IR spectra were
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recorded with an FTIR instrument as indicated. High-resolution mass spectra (HRMS) were obtained
using the electrospray technique, positive mode.
(2R,4S)-2-(Benzyloxy)-4-hydroxyhex-5-enal oxime (20)
To a solution of compound 19 (3.4 g, 15.4 mmol) in methanol (225 mL), NaHCO3 (2.594 g, 30.87 mmol) and
NH2OH.HCl (1.605 g, 23.1 mmol) were added and the mixture was stirred for 2 h at rt. Then, the solvent was
evaporated off and EtOAc (140 mL) was added to the residue. Solids were removed by filtration and filtrate was
evaporated to give a residue, which was purified by silica gel column chromatography (petroleum ether/ethyl
acetate 7:3) to afford compound 20 (3.587 g, 99%) as a colorless oil. FTIR (film) 3273, 3089, 3032, 2921, 1497,
1
1455, 1425, 1395, 1334, 1272, 1208, 1087 cm-1. H NMR (300 MHz, CDCl3, mixture of isomers ca. 3:7): δ 9.03
(br, 0.3H) and 8.63 (br, 0.7H) for NOH, 7.32 (m, 5H), 7.37 (d, J = 8.0 Hz, 0.7H) and 6.82 (d, J = 6.5 Hz, 0.3H) for
1-H, 5.82 (m, 1H), 5.26-5.06 (m, 2H), 4.94 (m, 0.3H), 4.63-4.19 (m, 3.7H), 4.45 (d, J = 11.5 Hz, 1H), 4.42 (d, J =
11.5 Hz, 2.5H), 4.34-4.29 (m, 3.5H), 3.22 (br, 1H, OH), 1.81–1.74 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 151.3,
139.9, 137.2, 128.6, 128.1, 128.0, 115.1, 75.2, 71.1, 70.7 (major isomer) and δ 153.5, 139.7, 137.1, 128.6, 128.1,
128.0, 115.0, 72.1, 71.0, 70.8 (minor isomer). LC-MS (ESI positive) m/z: 258 [M+Na]+ HRMS m/e: C13H17NNaO3
[(M+Na)+] calcd: 258.11006, found: 258.10923.
(3aR,4S,6R)-6-(benzyloxy)-3a,4,5,6-tetrahydro-3H-cyclopenta[c]iso-xazol-4-ol (21) and (3aS,4S,6R)-6-
(benzyloxy)-3a,4,5,6-tetrahydro-3H-cyclopenta[c]isoxazol-4-ol (22)
To a solution of compound 20 (4.95 g, 21 mmol) in EtOH, silica gel (19.8 g) was added and to this slurry a solution
of (8.87 g, 31.5 mmol) chloramine-T trihydrate in EtOH (100 mL) was added dropwise during a period of 2 h,
o
while temperature was kept at 8-10 C. The mixture was then left to warm at room temperature, solids were
removed by filtration, the solvent was evaporated off and the residue was separated by column chromatography
(petroleum ether/ethyl acetate 9:1) to give the mixture of diastereomers 21 and 22 (3.27g, 67%), as a colorless oil.
Small pure amounts of 21 and 22 were obtained by chromatographic separation of a small amount of this mixture
under the same conditions.
Compound 21: FTIR (film) 3423, 3088, 3062, 3032, 2992, 2933, 2881, 1735, 1708, 1648, 1384, 1374, 1267, 1207,
1
1159, 1067, 1028 cm-1. H NMR (300 MHz, CDCl3): δ 7.34 (m, 5Η), 4.67 (d, J = 10.0 Hz, 1H), 4.66 (d, J = 11.5
Hz, 1H), 4.49 (d, J = 11.5 Hz, 1H), 4.43 (dd, J = 6.5, 4.0 Hz, 1H), 4.05-3.92 (m, 3H), 2.77 (dt, J = 14.5, 7.0 Hz,
1H), 2.38 (br., 1H), 2.17 (ddd, J = 14.5, 6.5, 4.0 Hz, 1H). 13C NMR (126 MHz, CDCl3): δ 165.5, 137.0, 128.5,
128.2, 128.0, 74.2, 72.7, 71.5, 70.4, 60.0, 45.4. LC-MS (ESI positive) m/z: 256 [M+Na]+ HRMS m/e:
C13H15NNaO3 [(M+Na)+] calcd: 256.09441, found: 256.09463.
ο
Compound 22: M.p. 104.3-108.4 C. FTIR (film, cm-1) 3404, 3387, 3362, 3111, 3088, 3032, 2949, 2909, 2863,
1
1653, 1397, 1348, 1320, 1286, 1279, 1239, 1210, 1192, 1160, 1105, 1067, 1030. H-NMR (300 MHz, CDCl3) δ
7.37-7.27 (m, 5Η), 4.93 (d, J=12.0, Hz, 1H), 4.64 (d, J=12.0 Hz, 1H), 4.55 (d, J=8.0 Hz, 1H), 4.41 (dd, J=10.5, 8.0
Hz, 1H), 4.24 (dd, J=12.5, 8.0 Hz, 1H), 4.13 (t, J=4.0 Hz, 1H), 3.72 (dt, J=11.5, 4.0 Hz, 1H), 2.61 (br., 1H), 2.55
(ddd, J = 15.0, 8.0, 4.0 Hz, 1H), 2.34 (d, J = 15.0 Hz, 1H). 13C NMR (126 MHz, CDCl3): δ 170.1, 137.1, 128.4,
128.2, 128.0, 72.0, 70.1, 68.7, 67.6, 61.1, 47.1. LC-MS (ESI positive) m/z: 256 [M+Na]+ HRMS m/e:
C13H15NNaO3 [(M+Na)+] calcd: 256.09441, found: 256.09468.
(3aR,4S,6R)-6-(Benzyloxy)-4-(tert-butyldimethylsilyloxy)-3a,4,5,6-tetrahydro-3H-cyclopenta[c]isoxazole (27a) and
(3aS,4S,6R)-6-(benzyloxy)-4-(tert-butyldimethylsilyloxy)-3a,4,5,6-tetrahydro-3H-cyclopenta[c]isoxazole (28a)
Imidazole (6.073 g, 89.18 mmol) and TBSCl (10.08 g, 66.89 mmol) were added to a solution of the inseparable
mixture of compounds 21 and 22 (10.4 g, 44.59 mmol) in DCM (93 mL), and the mixture was stirred at room
temperature for 3 h. Then, water (40 mL) was added, the layers were separated and the aqueous layer was extracted
with DCM (40 mL). Combined organic extracts were dried over Na2SO4, the solvent was then evaporated off and
the residue was separated by column chromatography (petroleum ether/ethyl acetate 9:1) to give firstly compound
27a (12.7 g, 82%) followed by compound 28a (3.27 g, 14%), both as colorless oils.
Compound 27a: FTIR (film) 3089, 3065, 3032, 2992, 2930, 2881, 1646, 1384, 1374, 1253, 1208, 1090, 1029, 1006
cm-1. 1H NMR (500 MHz, CDCl3): δ 7.37-7.27 (m, 5H), 4.68 (d, J = 11.5 Hz, 1H), 4.60 (dd, J = 10.5, 8.0 Hz, 1H),
4.50 (d, J = 11.5 Hz, 1H,), 4.39 (d, J = 6.0 Hz, 1H), 4.04 (dd, J = 10.5, 8.0 Hz, 1H), 3.96 (m, 1H), 3.89 (dd, J =
15.5, 8.0 Hz, 1H), 2.83 (dt, J = 14.0, 7.5 Hz, 1H), 2.20 (ddd, J = 14.0, 8.5, 5.0 Hz, 1H), 0.87 (s, 9H), 0.05 (s, 3H),
13
0.03 (s, 3H). C NMR (126 MHz, CDCl3): δ 164.5, 137.3, 128.4, 1281, 127.8, 74.0, 73.3, 71.3, 69.6, 60.1, 46.0,
25.7, 17.9, -4.7, -4.8. LC-MS (ESI positive) m/z: 370 [M+Na]+. HRMS m/e: C19H29NNaO3Si [(M+Na)+] calcd:
370,18089, found: 370.18076.
8

Compound 28a: FTIR (film) 3065, 3032, 2952, 2857, 1472, 1463, 1389, 1361, 1318, 1255, 1211, 1159, 1119, 1006
ACCEPTED MANUSCRIPT
cm-1. 1H NMR (500 MHz, CDCl3): δ 7.32 (m, 5H), 4.94 (d, J = 12.0 Hz, 1H), 4.64 (d, J = 12.0 Hz, 1H), 4.60 (dd, J
= 9.0, 3.0 Hz, 1H), 4.34 (dd, J = 11.0, 7.5 Hz, 1H), 4.23 (dd, J = 11.5, 7.5 Hz, 1H), 4.19 (m, 1H), 3.77 (dt, J = 11.5,
5.0 Hz, 1H), 2.65 (ddd, J = 15.0, 9.0, 4.5 Hz, 1H), 2.27 (d, J = 15.0 Hz, 1H), 0.89 (s, 9H), 0.07 (s, 3H), 0.04 (s,
3H). 13C NMR (126 MHz, CDCl3): δ 169.4, 137.9, 128.2, 127.9, 127.5, 71.6, 70.3, 68.3, 67.2, 60.3, 47.3, 25.7,
18.1, -4.5, -5.0. LC-MS (ESI positive) m/z: 370 [M+Na]+. HRMS m/e: C19H29NNaO3Si [(M+Na)+] calcd:
370,18089, found: 370.18077.
(3aR,4S,6R)-6-(Benzyloxy)-3a,4,5,6-tetrahydro-3H-cyclopenta[c]iso-xazol-4-yl 4-nitrobenzoate (27b) and
(3aS,4S,6R)-6-(benzyloxy)-3a,4,5,6-tetrahydro-3H-cyclopenta[c]isoxazol-4-yl 4-nitrobenzoate (28b)
Α solution of inseparable mixture of compounds 21 and 22 (1.00 g, 4.29 mmol), DMAP (0.005 g, 0.043 mmol) and
Et3N (0.84 ml, 6.0 mmol) in dry DCM (11.6 mL), was stirred at 0oC for 10 min and then a solution of p-
nitrobenzoyl chloride (0.955g, 5.14mmol) in dry DCM (5.1 ml) was added dropwise at the same temperature. The
mixture was allowed to warm to rt and stirring was continued for an additional 2 h. Saturated aqueous NH4Cl (13
mL) was carefully added while stirring for 15 more minutes. The organic layer was separated, washed with water
(10 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the residue was purified by
flash column chromatography (petroleum ether/ethyl acetate 9:1) to afford compound 27b (1.5 g, 73%) colorless
crystals and compound 28b (0.1 g, 6%), as a colorless oil.
ο
Compound 27b: M.p. 107-107.9 C. FTIR (KBr) 2931, 2872, 1609, 1528, 1457, 1447, 1408, 1368, 1351, 1337,
1
1319, 1293, 1278, 1240, 1206, 1176, 1166, 1126, 1104, 1096, 1075, 1026, 1015, 1001 cm-1. H NMR (300 MHz,
CDCl3): δ 8.29 (d, J = 8.5 Hz, 2H), 8.19 (d, J = 8.5 Hz, 2H), 7.37-7.32 (m, 5H), 4.98 (dd, J = 15.0, 7.0 Hz, 1H),
4.77 (t, J = 10.0 Hz, 1H), 4.69 (d, J= 11.5 Hz, 1H), 4.57-4.51 (m, 2H), 4.35 (dd, J = 11.0, 9.5 Hz, 1H), 4.14 (td, J =
11.0, 7.0 Hz, 1H), 3.09 (dt, J = 15.0, 7.0 Hz, 1H), 2.54 (ddd, J = 15.0, 7.0, 3.0 Hz, 1H). 13C NMR (126 MHz,
CDCl3): δ 164.5, 164.0, 150.8, 136.9, 134.6, 130.8, 128.5, 128.2, 128.1, 123.6, 75.4, 75.1, 71.5, 69.3, 57.9, 42.1.
HRMS m/e: C20H18N2NaO6 [(M+Na)+] calcd: 405.35649, found: 405.35664.
1
Compound 28b: H NMR (300 MHz, CDCl3): δ 8.21 (d, J = 8.5 Hz, 2H), 8.16 (d, J = 8.5 Hz, 2H), 7.37-7.26 (m,
5H), 5.44 (t, J = 4.3 Hz, 1H), 5.01 (d, J = 11.5 Hz, 1H), 4.71 (d, J = 8.5 Hz, 1H), 4.68 (d, J = 11.5 Hz, 1H), 4.49
(dd, J = 10.0, 8.0 Hz, 1H), 4.08 (ddd, J =12.0, 10.0, 4.5 Hz, 1H), 4.01 (dd, J =12.0, 8.0 Hz, 1H), 2.85 (ddd, J =15.5,
8.5, 4.5 Hz, 1H), 2.61 (d, J = 15.5 Hz, 1H). 13C NMR (126 MHz, CDCl3): δ 169.0, 163.9, 150.8, 137.2, 134.7,
131.0, 128.4, 128.2, 128.0, 123.6, 72.2, 70.9, 69.5, 68.6, 59.0, 44.6. HRMS m/e: C20H18N2NaO6 [(M+Na)+] calcd:
405.35649, found: 405.35656.
(3aR,4S,6R)-6-(Benzyloxy)-4-(trityloxy)-3a,4,5,6-tetrahydro-3H-cyclo-penta[c]isoxazole (27c)
To a solution of of isoxazolines mixture 21 and 22 (0.495 g, 2.1 mmol) in pyridine (14 mL), 4-
dimethylaminopyridine (0.007 g, 0.057 mmol) and tritylchloride (1.42 g, 5.1 mmol) were successively added argon
o
atmosphere and the resulting suspension was left to stir overnight at elevated temperature (100 C). The reaction
mixture was left to cool to room temperature and then stirred with H2O (20 mL) for 10 minutes and extracted with
EtOAc (2 x 25 mL). Combined organic layers were dried over Na2SO4, the solvent was removed under reduced
pressure and the residue was purified with flash column chromatography (petroleum ether/ethyl acetate 9:1) to
1
afford 0.57g of the title compound (58% yield), as a colorless oil. H NMR (300 MHz, CDCl3): δ 7.45 (d, J = 7.5
Hz, 6H), 7.35 (d, J = 4.5 Hz, 4H), 7.35-7.23 (m, 10H), 4.62 (d, J = 11.5 Hz, 1H), 4.42 (d, J = 11.5 Hz, 1H), 4.17
(dd, J = 6.5, 4.0 Hz, 1H), 4.05–3.94 (m, 2H), 3.86 (dd, J = 14.0, 7.0 Hz, 1H), 3.28 (dd, J = 10.5, 7.5 Hz, 1H), 2.30–
2.19 (m, 2H). HRMS m/e: C32H29NNaO3 [(M+Na)+] calcd: 498.20451, found: 498.20464.
(3aR,4S,6R)-6-(Benzyloxy)-4-(methoxymethoxy)-3a,4,5,6-tetrahydro-3H-cyclopenta[c]isoxazole (27d)
To a solution of of isoxazolines mixture 21 and 22 (0.146 g, 0.63 mmol) in dry DCM (6 mL), methoxymethyl
chloride (0.48 mL) and DIPEA (2.15 mL) were added and the reaction mixture was left to stir at room temperature
for 2 h. Water (10 mL) was added and the mixture was stirred for an additional 10 min. The organic layer was
separated and the aqueous layer was extracted with DCM (2 x 25 mL). The combined organic solution was dried
over Na2SO4, the solvent was evaporated off and the residue was purified with flash column chromatography
1
(petroleum ether/ethyl acetate 9:1) to afford 0.11 g of the title compound in 63% yield, as an oil. H NMR (500
MHz, CDCl3): δ 7.38-7.27 (m, 5H), 4.69 (m, 4H), 4.67-4.62 (m, 1H), 4.60 (d, J = 7.0 Hz, 1H), 4.51 (d, J = 11.5 Hz,
1H), 4.40 (ddd, J = 7.0, 4.5, 1.0 Hz, 1H), 4.11 (dd, J = 11.0, 8.5 Hz, 1H), 4.03-3.96 (m, 1H), 3.74 (q, J = 8.0 Hz,
1H), 3.35 (s, 1H), 2.93 (dt, J = 15.0, 7.5 Hz, 1H), 2.27 (ddd, J = 14.5, 8.5, 4.5 Hz, 1H). 13C NMR (126 MHz,
CDCl3): δ 164.8, 137.3, 129.7, 128.6, 128.3, 128.0, 127.3, 96.5, 78.3, 74.8, 71.5, 69.2, 58.7, 55.7, 43.3. HRMS m/e:
C15H19NNaO4 [(M+Na)+] calcd: 300.12118, found: 300.12131.
9

(3aR,4S,6R)-6-(Benzyloxy)-4-(tert-butyldiphenylsilyloxy)-3a,4,5,6-tetrahydro-3H-cyclopenta[c]isoxazole (27e)
ACCEPTED MANUSCRIPT
To a solution of isoxazolines mixture 21 and 22 (0.146 g, 0.63 mmol) in dry DCM (6 mL), imidazole (0.128 g),
DMAP (0.007 g) and TBDPSCl (0.25 mL) were added and the reaction mixture was left to stir at room temperature
for 2 h. Water (10 mL) was then added and the mixture was stirred for an additional 10 min. The organic layer was
separated and the aqueous layer was extracted with DCM (2 x 25 mL). The combined organic solution was dried
over Na2SO4, the solvent was evaporated off and the residue was purified with flash column chromatography
1
(petroleum ether/ethyl acetate 9:1) to afford 0.232 g of the title compound in 79% yield, as an oil. H NMR (500
MHz, CDCl3): δ 7.54-7.22 (m, 15H), 4.56 (d, J = 11.5 Hz, 1H), 4.38 (d, J = 11.5 Hz, 1H), 4.22-4.14 (m, 1H), 4.07-
3.96 (m, 2H), 3.92 (dd, J = 18.5, 10.5 Hz, 1H), 3.81 (dd, J = 15.5, 7.5 Hz, 1H), 3.25-3.16 (m, 1H), 2.22-2.32 (m,
1H), 0.99 (s, 9H). HRMS m/e: C29H33NNaO3Si [(M+Na)+] calcd: 494.21274, found: 494.21290.
(2R,3S,5R)-5-(Benzyloxy)-3-(tert-butyldimethylsilyloxy)-2-(hydroxyl-methyl)cyclopentanone (29a), (R)-5-
(benzyloxy)-2-(hydroxymethyl)-cyclopent-2-enone (25) and (R)-2-(benzyloxy)-5-(hydroxymethyl)-cyclopentanone
(26)
To a solution of of compound 27a (1 g, 2.88 mmol) in MeOH (91 mL) and H2O (18.1 mL), B(OH)3 (2.704 g, 43.74
mmol) and 5% Pd/C (0.1 g) (50%) were added and the mixture was stirred at room temperature, under hydrogen
atmosphere for 1.5 h. The reaction mixture was filtered through a celite pad and celite pad was washed with DCM.
The filtrates were treated with saturated aqueous solution of NaHCO3 (22 mL), the organic layers are separated and
the aqueous layer was extracted with DCM (2x110 mL). Combined organic extracts were dried over Na2SO4 and
the solvent was evaporated off to give a residue, which was purified with flash column chromatography (petroleum
ether/ethyl acetate 7:3) to afford 0.735 g of compound 29a (74% yield), 0.078 g of compound 25 (12% yield) and
0.079 g of compound 26 (12% yield), all of them as oils.
Compound 29a: 1H NMR (500 MHz, CDCl3): δ 7.42-7.27 (m, 5H), 4.85 (d, J = 12.0 Hz, 1H), 4.69 (d, J = 12.0 Hz,
1H), 4.23 (dd, J = 16.0, 9.0 Hz, 1H), 4.02 (dd, J = 11.0, 2.5 Hz, 1H), 3.75 (ddd, J = 15.0, 11.0, 6.0 Hz, 2H), 2.63-
2.55 (m, 1H), 2.39-2.33 (m, 1H), 2.0 (br, 1H), 1.83 (q, J = 6.0 Hz, 1H), 0.90 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H). 13C
NMR (126 MHz, CDCl3): δ 214.6, 137.6, 128.6, 128.1, 128.0, 79.8, 72.6, 66.0, 59.1, 58.8, 37.9, 25.8, 18.0, -4.3, -
4.7. HRMS m/e: C19H30NaO4Si [(M+Na)+] calcd: 373.18111, found: 373.18122.
Compound 25: FTIR (film) 3419, 3088, 3063, 3032, 2927, 2875, 2868, 1705, 1634, 1497, 1455, 1432, 1394, 1331,
1290, 1262, 1207, 1161, 1120, 1001 cm-1. 1H NMR (500 MHz, CDCl3) δ 7.43–7.38 (m, 6H), 4.93 (d, J = 12.0 Hz,
1H), 4.75 (d, J = 12.0 Hz, 1H), 4.36 (s, 2H), 4.09 (dd, J=6.5, 2.5 Hz, 1H), 2.94-2.88 (m, 1H), 2.57-2.52 (m, 1H),
2.10 (br, 1H). 13C NMR (126 MHz, CDCl3): δ 206.8, 155.4, 143.3, 137.5, 128.5, 128.1, 127.9, 77.1, 72.3, 57.4,
34.3. LC-MS (ESI positive) m/z: 241 [M+Na]+, m/z: 459 [2M+Na]+. HRMS m/e: C13H14NaO3 [(M+Na)+] calcd:
241.08352, found: 241.08355, C26H28NaO6 [(2M+Na)+] calcd: 459.17781, found: 459.17800.
Compound 26: FTIR (film) 3460, 3088, 3063, 3031, 2943, 2875, 1747, 1497, 1455, 1393, 1313, 1209, 1123, 1066,
1
1028 cm-1. H NMR (500 MHz, CDCl3): δ 7.38-7.27 (m, 5H), 4.83 and 4.79 (d and d, J = 12.0 and 12.0 Hz, 1H),
4.69 and 4.64 (d and d, J = 12.0 and 12.0 Hz, 1H), 3.92-3.66 (m, 3H) 2.45-1.60 (m, 5H). 13C NMR (126 MHz,
CDCl3): δ 217.8/217.7, 137.6/137.5, 128.4, 128.0/127.9, 127.9/127.8, 80.9/79.7, 72.2/71.8, 62.8/61.7, 48.8/47.0,
28.1/27.8, 21.2/20.2. LC-MS (ESI positive) m/z: 243 [M+Na]+, m/z: 463 [2M+Na]+. HRMS m/e: C13H16NaO3
[(M+Na)+] calcd: 243.09971, found: 243.09906, C26H32NaO6 [(2M+Na)+] calcd: 463.20911, found: 463.20931.
(2R,3S,5R)-5-(Benzyloxy)-3-(tert-butyldimethylsilyloxy)-2-((tert-butyl-dimethylsilyloxy)methyl)cyclopentanone (31)
A stirred solution of compound 29a (24.2 g, 69.04 mmol) in DCM (90.8 mL) was cooled at 0-5oC and TBSCl
(31.217 g, 207.4 mmol), imidazole (18.790 g, 276.16 mmol) and DMAP (0.674 g, 5.52 mmol) were added, under
argon atmosphere. The solution was left to warm at rt and stirred for 30 more min. Then, the mixture was cooled
o
again at 0-5 C, DCM (412 mL) was added slowly followed by addition of saturated aqueous solution of NH4Cl
o
(412 mL), while the temperature was kept below 10 C. The reaction mixture was stirred for 10 more minutes at
that temperature and then layers were separated. The aqueous layer was extracted with DCM (412 mL) and
combined organic layer were washed with H2O (97 mL) and saturated aqueous solution of NaCl (97 mL), dried
over Na2SO4, and solvents were evaporation off. The resulting residue was purified with flash column
chromatography (petroleum ether/ethyl acetate 9:1) to afford 27.275 g of compound 31 (85% yield) and 2.29 g of
compound 30 (85% yield), both as colorless oils.
Compound 31: 1H NMR (500 MHz, CDCl3): δ 7.38-7.28 (m, 5H), 4.87 (d, J = 12.0 Hz, 1H), 4.69 (d, J = 12.0 Hz,
1H), 4.38 (ddd, J = 10.0, 8.0, 6.5 Hz, 1H), 4.02 (dd, J = 10.0, 2.0 Hz, 1H), 3.73–3.67 (m, 2H), 2.55 (ddd, J = 11.5,
8.0, 6.4 Hz, 1H), 2.16 (dt, J = 8.0, 2.0 Hz, 1H), 1.83–1.76 (m, 1H), 0.89 (s, 9H), 0.81 (s, 9H), 0.09 (s, 3H), 0.08 (s,
3H), 0.02 (s, 3H), -0.01 (s, 3H). 13C NMR (126 MHz, CDCl3): δ 214.0, 137.6, 128.4, 128.0, 127.8, 80.6, 72.3, 65.1,
10

59.1, 58.6, 37.6, 25.74, 25.73, 18.1, 18.0, -4.5, -4.8, -5.68, -5.73. HRMS m/e: C25H44NaO4Si2 [(M+Na)+] calcd:
ACCEPTED MANUSCRIPT
487.26758, found: 487.26770.
Compound 30: FTIR (film): 3065, 3032, 2954, 2929, 2884, 2857, 1713, 1639, 1472, 1463, 1400, 1361, 1295, 1256,
1
1205, 1160, 1120, 1003 cm-1. H NMR (500 MHz, CDCl3): δ 7.41-7.33 (m, 5H), 7.31-7.28 (m, 1H), 4.93 (d, J =
12.0 Hz, 1H), 4.75 (d, J = 12.0 Hz, 1H), 4.36 (s, 2H), 4.09 (dd, J = 6.5, 2.5 Hz, 1H), 2.90 (ddd, J = 18.5, 6.5, 2.5
Hz, 1H), 2.50-2.55 (m, 1H), 0.92 (s, 9H), 0.08 (s, 6H). 13C NMR (126 MHz, CDCl3): δ 205.8, 154.4, 144.6, 137.8,
128.6, 128.2, 128.0, 77.7, 72.3, 58.2, 34.3, 26.0, 18.4, -5.3, -5.3. LC-MS (ESI positive) m/z: 355 [M+Na]+. HRMS
m/e: C19H28NaO3Si [(M+Na)+] calcd: 355.16999, found: 355.16973.
(2R,3S,5R)-5-(Benzyloxy)-3-(tert-butyldimethylsilyloxy)-2-((tert-butyl-dimethylsilyloxy)methyl)-1-
((trimethylsilyl)methyl)cyclopentanol (32)
A 1.0 M solution of TMSCH2MgCl in Et2O (234.7 mL, 234.7 mmol) was added dropwise in a solution of
compound 31 (27.275 g, 58.7mmol) in DCM (314 mL). The reaction mixture was stirred at ambient temperature
till reaction completion (checked by TLC) and then saturated aqueous solution of NH4Cl (90 mL) and DCM (65
mL) were added. The organic layer was separated and the aqueous layer was extracted with DCM (90mL).
Combined organic extracts were washed with a 20% w/v aqueous solution of NaCl (20 mL), dried over Na2SO4,
the solvent was evaporated off and the residue was purified by column chromatography (petroleum ether/ethyl
acetate 9:1) to afford 29.2 g of compound 32 (90% yield, as an oil. FTIR (film): 3480, 3091, 3032, 2954, 2930,
2898, 2858, 1472, 1387, 1362, 1340, 1316, 1285, 1256, 1209, 1094, 1042, 1006 cm-1. 1H NMR (500 MHz, CDCl3):
δ 7.36-7.26 (m, 5H), 4.56 (d, J = 12.0 Hz, 1H), 4.49 (td, J = 8.5, 5.5 Hz, 1H), 4.38 (d, J = 12.0 Hz, 1H), 4.35 (s,
1H), 4.07 (dd, J = 10.5, 2.5 Hz, 1H), 3.98 (d, J = 10.5, 1H), 3.59 (m, 1H), 2.50 (ddd, J = 14.5, 8.5, 6.5 Hz, 1H),
1.75 (d, J = 8.5 Hz, 1H), 1.63-1.59 (m, 1H), 1.29 (d, J = 14.5 Hz, 1H), 0.99 (d, J = 14.5 Hz, 1H), 0.91 (s, 9H), 0.89
(s, 9H), 0.14 (s, 3H), 0.11 (s, 3H), 0.06 (s, 6H), 0.04 (s, 9H). 13C NMR (126 MHz, CDCl3): δ 138.9, 128.2, 127.3,
127.2, 84.4, 84.3, 70.6, 69.9, 59.6, 55.6, 38.6, 25.9, 25.7, 22.9, 18.0, 17.9, 0.6, -4.3, -4.8, -5.7, -5.8. LC-MS (ESI
positive) m/z: 575 [M+Na]+. HRMS m/e: C29H56NaO4Si3 [(M+Na)+] calcd: 575.33841, found: 575.33752.
(2R,4S,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyl-oxy)methyl)-1-
((trimethylsilyl)methyl)cyclopentane-1,2-diol (33)
A solution of compound 32 (25.2 g, 45.57 mmol) in THF (1.0 L), was stirred at rt for 2 h under hydrogen
atmosphere in the presence of 10% Pd/C (115,74 g, 50% wet). Catalyst was filtered off through a celite pad and the
solvent was removed. To the semi-solid residue MeOH was added and the solid separated by filtration, dried under
o
25
vacuum to afford 16.87 g of compound 33 (80% yield), as a white solid, M.p. 183-185 C, [α]D +7.8o (c 1.0,
THF). FTIR (KBr) 3434, 2955, 2931, 2904, 2859, 1472, 1386, 1361, 1326, 1305, 1255, 1206, 1150, 1106, 1090,
1
1068, 1041, 1005 cm-1. H NMR (500 MHz, CDCl3): δ 4.33 (td, J = 7.0, 4.0 Hz, 1H), 4.07 (s, 1H), 4.04 (dd, J =
10.5, 3.5 Hz, 1H), 3.92 (dd, J = 10.5, 3.5 Hz, 1H), 3.80 (dd, J = 6.0, 4.5 Hz, 1H), 2.57-2.48 (m, 1H), 1.99 (d, J =
6.5 Hz, 1H), 1.78 (dt, J = 6.5, 3.0 Hz, 1H), 1.50 (ddd, J = 14.5, 4.0, 2.0 Hz, 1H), 1.16 (d, J = 15.0 Hz, 1H), 1.04 (d,
13
J = 15.0 Hz, 1H), 0.90 (s, 9H), 0.88 (s, 9H), 0.12 (s, 3H), 0.10 (s, 3H), 0.08 (s, 9H), 0.05 (s, 6H). C NMR (126
MHz, CDCl3): δ 84.9, 78.7, 72.3, 60.4, 56.8, 42.1, 25.8, 25.7, 24.0, 17.9, 0.5, -4.5, -4.9, -5.7, -5.8. LC-MS (ESI
positive) m/z: 485 [M+Na]+ HRMS m/e: C22H50NaO4Si3 [(M+Na)+] calcd: 485.29146, found: 485.29095.
(1R,3R,4S)-4-((tert-butyldimethylsilyl)oxy)-3-(((tert-butyldimethyl-silyl)oxy)methyl)-2-methylenecyclopentanol (34)
A 0.5 M solution of KHMDS in toluene (300 mL, 150 mmol) was diluted with dry THF (122 mL) and compound
33 (13.9 g, 30 mmol) was added drop wise to this mixture during a period of 30 min at rt. The mixture was stirred
for another 30 min and then cooled to 0-5 oC, before the addition of MTBE (1390 mL) and 1 N HCl (150 mL) and
further stirring for 10 more min. The organic layer was separated and the aqueous layer was extracted with MTBE
(400 mL). The combined organic phase was washed with saturated aqueous solution of NaCl (3 x 2300 mL), and
dried over Na2SO4. The solvent was evaporated off and the residue was purified with flash column chromatography
(petroleum ether/ethyl acetate 9:1) to afford 8,89 g of compound 34 in 47% yield, with analytical and spectroscopic
25
data identical to those reported in the literature. M.p. 69-71 oC (Lit.2j m.p. 63-66oC) [α]D -37.8o (c 1.004, CHCl3)
25
20
[lit.2i [α]D -37o (c 0.035, CHCl3)] [Lit.2j [α]D -34o (c 0.44, CHCl3)]. FTIR (film) 3367, 2957, 2856, 1472, 1257,
1123, 1078 cm-1. H NMR (500 MHz, CDCl3): δ 5.38 (s, 1H), 5.12 (s, 1H), 4.35 (m, 2H), 3.56 (dd, J = 10.5, 5.0
1
Hz, 1H), 3.31 (dd, J = 10.0, 8.5 Hz, 1H), 2.75 (br, 1H), 1.98 (ddd, J = 13.5, 5.5, 4.5 Hz, 1H), 1.82 (dd, J = 13.5, 1.5
Hz, 1H), 0.89 (s, 9H), 0.88 (s, 9H), 0.09 (s, 6H), 0.04 (s, 3H), 0.03 (s, 3H). 13C NMR (126 MHz, CDCl3): δ 154.3,
111.7, 75.5, 75.4, 64.7, 55.0, 42.1, 25.9, 25.8, 18.3, 17.9, -4.8, -4.9, -5.5, -5.6. LC-MS (ESI positive) m/z: 395
[M+Na]+. HRMS m/e: C19H40NaO3Si2 [(M+Na)+] calcd: 395,24027, found: 395,24073. Alternatively, the product
was crystallysed as a white solid from the reaction mixture by addition of CH3CN (7.78 g; yield 70%).
11

9-((1S,3R,4S)-4-(tert-Butyldimethylsilyloxy)-3-((tert-butyldimethyl-silyloxy)methyl)-2-methylenecyclopentyl)-6-
ACCEPTED MANUSCRIPT
chloro-9H-purine-2-amine (35a)
A mixture of Ph3P (1.054 g, 4.02 mmol), 6-chloro-2-aminopurine (0.682 g, 4.02 mmol) and compound 34 (1 g,
2.68 mmol) in THF (44.7 mL) was stirred for 30 min at -15 oC, and then DEAD (0.84 mL, 5.36 mmol) was added
o
at the same temperature drop wise during a period of 10 min. The mixture was left to warm at 25 C and after
consumption of starting material (checked by TLC), water (14 mL) and DCM (160 mL) were added and the
resulting mixture was stirred for 15 min. Layers were separated, aqueous layer was extracted with DCM (44 mL),
and the combined organic layers were dried over Na2SO4. The solvent was then evaporated off and the residue was
purified by column chromatography (petroleum ether/ethyl acetate 8:2) to afford 0.92 g of compound 35a (64%
yield). M.p. 38-39 °C (lit.2j m.p. 37-39 °C), [α]D25 33.0 (c = 0.2, CHCl3), [lit.2j [α]D25 33.1 (c = 0.15, CHCl3)], with
spectra identical to those reported in the literature.2j 1H NMR (500 MHz, CDCl3): δ 7.83 (s, 1H), 5.52 (t, J = 8.5
Hz, 1H), 5.18 (s, 1H), 4.83 (s, 1H), 4.43 (s, 1H), 3.84-3.74 (m, 2H), 2.67 (br, 1H), 2.30-2.24 (m, 1H), 2.23-2.14 (m,
1H), 1.29 (m, 1H), 0.92 (s, 9H), 0.90 (s, 9H), 0.10 (s, 6H), 0.08 (s, 6H). 13C NMR (126 MHz, CDCl3): δ 158.9,
153.9, 151.2, 148.9, 141.8, 125.4, 111.4, 72.4, 64.1, 56.3, 54.7, 40.4, 26.0, 25.9, 18.5, 18.1, -4.6, -4.7, -5.3, -5.4.
LC-MS (ESI positive) m/z: 546 [M+Na]+.
9-((1S,3R,4S)-4-(tert-butyldimethylsilyloxy)-3-(tert-butyldimethyl-silyloxymethyl)-2-methylenecyclopentyl)-6-
chloro-9H-purine-2-carbamic acid tert-butyl ester (35b)
A mixture of Ph3P (1.054 g, 4.02 mmol), 4-chloro-1H-imidazo[4,5-c]pyridin-6-yl tert-butylcarbamate (chloro-Boc
purine) (1.085 g, 4.02mmol) and compound 34 (1 g, 2.68 mmol) in THF (44.7 mL) was stirred for 30 min at -15
oC, and then DEAD (0.84 mL, 5.36 mmol) was added at the same temperature drop wise during a period of 10 min.
The mixture was left to warm at 25 oC and after consumption of starting material (checked by TLC), water (14 mL)
and DCM (160 mL) were added and the resulting mixture was stirred for 15 min. Layers were separated, aqueous
layer was extracted with DCM (44 mL), and the combined organic layers were dried over Na2SO4. The solvent was
then evaporated off and the residue was purified by column chromatography (petroleum ether/ethyl acetate 8:2) to
afford 1.42 g of compound 35b (85% yield) as a solid, m.p. 105-109 oC, with spectra identical to those reported in
the literature.11 FTIR (film) 3453, 3125, 3085, 2956, 2930, 2895, 2858, 1758, 1608, 1571, 1520, 1487, 1463, 1393,
1
1368, 1280, 1257, 1155, 1091 cm-1. H NMR (500 MHz, CDCl3): δ 8.04 (s, 1H), 7.42 (s, 1H), 5.65 (t, J = 8.0 Hz,
1H), 5.22 (s, 1H), 4.83 (s, 1H), 4.46 (d, J = 4.0 Hz, 1H), 3.82-3.86 (m, 2H), 2.67 (br, 1H), 2.35 (ddd, J = 13.5, 8.5,
13
5.0 Hz, 1H), 2.20-2.27 (m, 1H), 1.54 (s, 9H), 0.92 (s, 9H), 0.90 (s, 9H), 0.09 (s, 6H), 0.08 (s, 6H). C NMR (126
MHz, CDCl3): δ 152.9, 152.2, 151.1, 150.1, 148.8, 143.9, 127.9, 111.8, 81.6, 72.3, 63.8, 56.8, 54.7, 40.6, 28.3,
26.0, 25.9, 18.5, 18.1, -4.6, -4.7, -5.3, -5.4. LC-MS (ESI positive) m/z: 646 [M+Na]+ HRMS m/e:
C29H50ClN5NaO4Si2 [(M+Na)+] calcd: 646.29821, found: 646.29790.
9-((1S,3R,4S)-4-(tert-butyldimethylsilyloxy)-3-(tert-butyldimethylsilyl-oxymethyl)-2-methylenecyclopentyl)-6-iodo-
9H-purine-2-carbamic acid tert-butyl ester (35c)
A mixture of Ph3P (1.003 g, 3.82 mmol), 4-iodo-1H-imidazo[4,5-c]pyridin-6-yl tert-butylcarbamate (iodo-Boc
purine) (1.381 g, 3.82mmol) and compound 34 (0.95 g, 2.55 mmol) in THF (42.5 mL) was stirred for 30 min at -15
oC, and then DEAD (0.80 mL, 5.10 mmol) was added at the same temperature drop wise during a period of 10 min.
The mixture was left to warm at 25oC and after consumption of starting material (checked by TLC), water (13 mL)
and DCM (152 mL) were added and the resulting mixture was stirred for 15 min. Layers were separated, aqueous
layer was extracted with DCM (40 mL), and the combined organic layers were dried over Na2SO4. The solvent was
then evaporated off and the residue was purified by column chromatography (petroleum ether/ethyl acetate 8:2) to
afford 1.28 g of compound 35c (70% yield), as colorless oil, with spectra identical to those reported in the
literature.11 1H NMR (500 MHz, CDCl3): δ 8.01 (s, 1H), 7.43 (s, 1H), 5.63 (t, J = 8.0 Hz, 1H), 5.20 (s, 1H), 4.80 (s,
1H), 4.45 (d, J = 3.5 Hz, 1H), 3.82 (d, J = 5.5 Hz, 2H), 2.66 (s, 1H), 2.43-2.31 (m, 1H), 2.31-2.19 (m, 1H), 1.53 (s,
9H), 0.92 (s, 9H), 0.90 (s, 9H), 0.10 (s, 6H), 0.09 (s, 3H), 0.08 (s, 3H). 13C NMR (126 MHz, CDCl3): δ 151.8,
150.1, 149.0, 148.8, 143.2, 135.0, 121.9, 111.6, 81.4, 72.3, 63.8, 56.7, 54.7, 40.5, 28.2, 26.0, 25.8, 18.4, 18.0, -4.6,
-4.7, -5.4, -5.5. LC-MS (ESI positive) m/z: 694 [M+Na]+. HRMS m/e: C29H50IN5NaO4Si2 [(M+Na)+] calcd:
738.23437, found: 738.23428.
2-Amino-9-(4-hydroxy-3-hydroxymethyl-2-methylenecyclopentyl)1,9-dihydro-purin-6-one (1)
From 35a: To a solution of compound 35a (2.0 g, 3.8 mmol) in THF (57 mL), 2N HCl (57 mL) was added and the
mixture was heated for 20-24 h at 60-65 oC. THF was then removed under vacuum and pH was adjusted to 6.8-7.0
by adding NaOH 10% (53 mL). A solid was precipitated and stirred for 10-12 h at room temperature, then cooled
o
to 0-5 C and collected with filtration to give 0.85 g of desired product in 80% yield. From 35b: To a solution of
compound 35b (2.0 g, 3.2 mmol) in THF (48 mL), 2N HCl (48 mL) was added and the mixture was heated for 20-
12

24 h at 60-65 oC. THF was then removed under vacuum and pH was adjusted to 6.8-7.0 by adding NaOH 10% (53
ACCEPTED MANUSCRIPT
mL). A solid was precipitated and stirred for 10-12 h at room temperature, then cooled to 0-5 oC and collected with
filtration to give 0.80 g of desired product in 90% yield. From 35c: To a solution of compound 35c (2.0 g, 2.8
mmol) in THF (45 mL), 2N HCl (45 mL) was added and the mixture was heated for 20-24 h at 60-65 oC. THF was
then removed under vacuum and pH was adjusted to 6.8-7.0 by adding NaOH 10% (42 mL). A solid was
precipitated and stirred for 10-12 h at room temperature, then cooled to 0-5 oC and collected with filtration to give
0.966 g of desired product in 85% yield. [α]D +26.8o (c 1.00, H2O). IR (ATR): 3445, 3360, 3295, 3175, 3110,
25
1
2950, 2860, 2626, 1710 cm−1. H NMR (500 MHz, DMSO-d6): δ 10.61 (s, 1H), 7.66 (s, 1H), 6.43 (br, 2H), 5.36
(dd, J = 10.0, 8.0 Hz, 1H), 5.10 (s, 1H), 4.89 (d, J = 3.0 Hz, 1H), 4.85 (t, J = 5.5 Hz, 1H), 4.57 (s, 1H), 4.24 (s, 1H),
13
3.54 (t, J = 6.0 Hz, 2H), 2.52 (m, 1H), 2.22 (td, J = 12.5, 4.5 Hz, 1H), 2.05 (dd, J = 12.5, 8.0 Hz, 1H). C NMR
(126 MHz, DMSO-d6) δ 157.3, 154.0, 151.9, 151.7, 136.5, 116.7, 109.8, 70.8, 63.5, 55.6, 54.5, 39.6. HRMS: m/e
C12H16N5O3 [M+H]+ calcd: 278.1253; found: 278.1248.
References and Notes
1. (a) Opio CK, Lee WL, Kirkpatick P. Nat Rev Drug Discov. 2005;4:535;
(b) Langley DR, Walsh AW, Baldick CJ, Eggers BJ, Rose RE, Levine SM, Kapur AJ, Colonno RJ, Tenney DJ. J
Virol. 2007:3992;
(c) Kamiya N, J Antimicrob Chemother. 2003;51:1085.
2. (a) Zahler R, Slusarchyk WA, EP Patent 1992:0481754;
(b) Zahler, R., Slusarchyk WA, US Patent 1993:5206244;
(c) Bisacchi GS, Chao ST, Bachard C, Daris JP, Innaimo S, Jacobs GA, Kocy O, Lapointe P, Martel A, Merchant Z,
Slusarchyk WA, Sundeen JE, Young MJ, Colonno R, Zahler R. Bioorg Med Chem Lett. 1997;7:127;
(d) Pendri YR, Chen C-PH, Patel AS, Evans JM, Liang Z, Kronenthal DR, Powers GL, Prasad SJ, Bien JT,
Zhongping S, Patel RN, YChan YY, Rijhwani SK, Singh AK, Wang A, Stojanovic M, Polniaszek R, Lewis C,
Thottathil J, Krishnamurty D, Zhou MX, Vemishetti P. WO Patent 2004:052310;
(e) Ziegler FE, Sarpong MA, Tetrahedron. 2003;59:9013;
(f) Jing F, Bingkun S, Jianfeng M, Minhua F. Patent CN 2012:102417506A;
(g) Liotta D, Li Y. WO Patent 2009:021114;
(h) Lee J, Park G-S, Kim JH, Lee JE, Park CH, Choi TJ, Park E-J, Kim CK, Lim EJ, Chang Y-K, Lee GS. WO Patent
2010:074534;
(i) Rawal RK, Singh US, Gadthula S, Chu CK. Curr Protoc Nucleic Acid Chem. 2011;47:14.7.1-14.7.17;
(j) Zhou B, Li Y. Tetrahedron Lett. 2012;53:502;
(k) Liu X, Jiao X, Wu Q, Tian C, Li R, Xie P. Tetrahedron Lett. 2012;53:3805;
(l) Bartra M, Berenguer R, Velasco J, Ariza J, Farras J, Garcia J. WO Patent 2012:085209;
(m) Berenguer R, Badia L, Gasanz Y, WO Patent 2013:076236;
(n) Velasco J, Ariza X, Badia L, Bartra M, Berenguer R, Farras J, Gallardo J, Garcia J, Gasanz Y. J Org Chem.
2013;78:5482;
(o) Gillard F, Heissler D, Riehl JJ. J. Chem. Soc. Perkin Trans 1. 1988:229.
3. (a) Barton DHR, McCombie SW., J. Chem. Soc. Perkin Trans 1. 1975:1574;
(b) Stand, Luthe C. Can J Chem. 1980;58:1799;
(c) Hartwig W, Tetrahedron. 1983;39:2609;
(d) Iacono S, Rasmussen JR Org Synth. 1990;7:139;
(e) Lopez RM, Hays DS, Fu GC. J Am Chem Soc. 1997;119:6949;
(f) Tormo J, Fu GC. Org. Synth. 2004;10:240;
(g) Jiang J, Biggins JB, Thorson JS. J Am Chem Soc. 2000;122:6803;
(h) Andersen SM, Ekhart Lundt CI, Stütz AE. Carbohydr Res. 2000;326:22.
4. Shing TKM, Wong WF, Cheng HM, Kwok WS, So KH. Org Lett. 2007;9:753.
5. (a) Roy BL, Deslongchamps P. Can. J. Chem. 1985, 63, 651;
(b) Rokach J, Khanapure SP, Hwang SW, Adiyaman M, Schio L, Fitzgerald GA. Synthesis 1998, 569.
6. (a) Gallos JK, Koumbis AE, Xiraphaki VP, Dellios CC, E. Coutouli-Argyropoulou E. Tetrahedron. 1999;55:15167;
(b) Gallos JK, Koftis TV, Koumbis AE, Moutsos VI, Synlett 1999:1289;
(c) Harding SL, Marcuccio SM, Savage GP, Beilstein J Org Chem. 2012;8:606.
7. (a) Sharma GVM, Kumar KR. Tetrahedron Asymmetry. 2004;15:2323;
(b) Petrignet J, Prathap I, Chandrasekhar S, Singh Yadav J, Gree R, Angew Chem Int Ed. 2007;46:6297.
8. (a) Curran DP. J Am Chem Soc. 1982;104:4024;
(b) Kozikowski AP, Adamczyc M. Tetrahedron Lett. 1982;23:3123;
(c) Curran DP. J Am Chem Soc. 1983;105:5826;
(d) Kozikowski AP, P. D. Stein PD. J Org Chem. 1984;49:2301;
(e) Zhang Y. O'Connor B. Negishi E. J Org Chem. 1988;53:5590;
(f) Tatsuta K, Niwata Y, Umezawa K, Toshima K, Nakata M, Tetrahedron Lett. 1990;31:1171;
13

(g) Nakata M, Akazawa S, Kitamura S, Tatsuta K, Tetrahedron Lett. 1991;32:5363;
ACCEPTED MANUSCRIPT
(h) Kobayashi Y, Miyazaki H, Shiozaki M. J Am Chem Soc. 1992;114:10065;
(i) Kobayashi Y, Miyazaki H, Shiozaki M. J Org Chem. 1994;59:813;
(j) Pulkkinen JT, Vepsalainen JJ, J Org Chem. 1996;61:8604;
(k) Fernandez-Mateos A, Coca GP, Gonzalez RR, Hernandez CT. J Org Chem. 1996;61:9097;
(l) Lohse-Fraefel N, Carreira EM. Chem Eur J. 2009;15:12065.
9. Udodong UE, Fraser-Reid B, J Org Chem. 1989;54:2103.
10. (a) Takao K, Tsujita T, Hara M, Tadano K. J Org Chem 2002;67:6690;
(b) Denmark SE, Baiazitov RY. J Org Chem. 2006;71:593;
(c) Granger K, Snapper ML. Eur J Org Chem. 2012:2308.
11. Z. Zheng, WO Patent 2012:006964.
12. (a) Koftis T, Neokosmidis E, Gioti E, Kotoulas S, Andreou T, Varvogli A-A. WO Patent 2015:051900;
(b) Koftis T, Neokosmidis E, Gioti E, Kotoulas S, Tsatsas T, Menisiou A, Andreou T, Varvogli A-A. WO Patent
2015:051903.
Supplementary Material
Supplementary data related to this article can be found at
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