Synthesis and Antiviral Evaluation of TriPPPro-AbacavirTP, TriPPPro-CarbovirTP, and Their 1′,2′-cis-Disubstituted Analogues

Article abstract of DOI:10.1002/cmdc.201800361

Herein we describe the synthesis of lipophilic triphosphate prodrugs of abacavir, carbovir, and their 1′,2′-cis-substituted carbocyclic analogues. The 1′,2′-cis-carbocyclic nucleosides were prepared by starting from enantiomerically pure (1R,2S)-2-((benzyloxy)methyl)cyclopent-3-en-1-ol by a microwave-assisted Mitsunobu-type reaction with 2-amino-6-chloropurine. All four nucleoside analogues were prepared from their 2-amino-6-chloropurine precursors. The nucleosides were converted into their corresponding nucleoside triphosphate prodrugs (TriPPPro approach) by application of the H-phosphonate route. The TriPPPro compounds were hydrolyzed in different media, in which the formation of nucleoside triphosphates was proven. While the TriPPPro compounds of abacavir and carbovir showed increased antiviral activity over their parent nucleoside, the TriPPPro compounds of the 1′,2′-cis-substituted analogues as well as their parent nucleosides proved to be inactive against HIV.

Full text of DOI:10.1002/cmdc.201800361

Accepted Article
Title: Synthesis and Antiviral Evaluation of TriPPPro-AbacavirTP,
TriPPPro-CarbovirTP and their 1',2'-cis-disubstituted Analogues
Authors: Simon Weising, Vicente Sterrenberg, Dominique Schols, and
Chris Meier
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201800361
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800361
A Journal of
www.chemmedchem.org

10.1002/cmdc.201800361
ChemMedChem
FULL PAPER
Synthesis and Antiviral Evaluation of TriPPPro-AbacavirTP, TriPPPro-
CarbovirTP and their 1’,2’-cis-disubstituted Analogues
Simon Weising,[a] Vincente Sterrenberg,[a] Dominique Schols,[b] and Chris Meier*[a]
[a]
S. Weising, V. Sterrenberg, D. Schols, C. Meier
University of Hamburg, Faculty of Sciences, Department Chemistry
Organic Chemistry
Martin-Luther-King Platz 6, 20146 Hamburg, Germany
E-mail: chris.meier@chemie.uni-hamburg.de
D. Schols
[b]
Katholieke Universiteit Leuven
Rega Institute for Medical Research
Herestraat 49, 3000 Leuven, Belgium
Supporting information for this article is given via a link at the end of the document.
Abstract: Herein we describe the synthesis of lipophilic triphosphate
prodrugs of abacavir, carbovir and their 1’,2’-cis-substituted
carbocyclic analogues. The 1’,2’-cis-carbocyclic nucleosides were
prepared starting from enantiomerically pure (1R,2S)-2-
((benzyloxy)methyl)cyclopent-3-en-1-ol by a microwave assisted
Mitsunobu-type reaction with 2-amino-6-chloropurine. All four
nucleoside analogues were prepared from their 2-amino-6-
chloropurine precursors. The nucleosides were converted into their
corresponding nucleoside triphosphate prodrugs (TriPPPro-
approach) by application of the H-phosphonate route. The TriPPPro
compounds were hydrolyzed in different media, in which the
formation of nucleoside triphosphates was proven. While the
TriPPPro-compounds of abacavir and carbovir showed increased
antiviral activity compared to their parent nucleoside, the TriPPPro-
compounds of the 1’,2’-cis-substituted analogues as well as their
parent nucleosides proved to be inactive against HIV.
In order to bypass metabolic hurdles in the phosphorylation,
nucleotide prodrugs have been introduced (e.g. phosphor-
amidates[11], cycloSal-[12] and DiPPro-compounds[13]).
Another life-threating virus is HIV which causes AIDS. Although
until today no complete cure is available for a HIV infection, with
a combined antiretroviral therapy (cART) the viral load was
found to be below detection limit. One component in the cART
are nucleotidic reverse transcriptase inhibitors (NRTIs) which
are used as substrates during the viral DNA elongation, which
lead to an immediate or delayed chain termination of the viral
DNA synthesis. In most cases a lack of the 3’-hydroxyl group is
responsible for an immediate chain termination, whereas
modified pentose scaffolds and nucleobases induce a delayed
chain termination.[14]
Introduction
Since the discovery of viruses one of the most challenging tasks
is to cure viral infections as well as to prevent infections. Every
year a huge number of people dies due to viral infections, e.g.
1.45 million from hepatitis (WHO, 2013). For the treatment of
Figure 1. Antiviral nucleosides Abacavir 1 and Carbovir 2 (1’,4’-cis-carbocyclic
analogues).
hepatitis
B
(HBV) infections the L-nucleoside L-dT
(Telbivudine)[1] and the carbocyclic nucleoside analogue
Entecavir[2,3] were approved in 2006 and 2005, respectively. In
1989, hepatitis C virus (HCV) was characterized, but a complete
cure wasn’t possible until very recently.[4] HCV targets
hepatocytes and a chronic HCV infection leads often to liver
cirrhosis and therefore a liver transplant is needed. The standard
of care was until recently the combination of ribavirin and
pegylated IFN-α, but this is limited to about 50% of the patients
due to toxic side effects and the ineffectiveness of the therapy to
Abacavir (ABC) 1 is a carbocyclic nucleoside analogue which
belongs to these NRTIs. This nucleoside analogue comprises a
carbocyclic dideoxydidehydro core and
a
6-cyclopropyl-
aminopurine nucleobase, which overcomes some disadvantages
of its guanine derivative (carbovir, CBV) 2. A series of these
‘carbovirs’ was developed by the group of Vince et al., that first
discovered the advantage of the 6-substitution with alkylamino
groups.[15–24] In 1998, Abacavir 1 was approved by the FDA for
the treatment of HIV infections.[25] Intracellularly ABC is
phosphorylated to its monophosphate (ABCMP) followed by a
replacement of the alkylamino group by a hydroxyl group to form
carbovir monophosphate (CBVMP). This latter step is crucial
because this process avoids the toxic effects associated with the
parent nucleoside CBV. CBVMP is further phosphorylated to
form the finally active carbovir triphosphate (CBVTP).[26,27]
Recently, we developed the TriPPPro-approach with which all
three intracellular phosphorylation steps can be bypassed. It
various
viral
genotypes.[5]
In
2013,
Sofosbuvir,
a
phosphoramidate prodrug of PSI-6206, was approved by the
FDA which in combination with ribavirin and peg-IFN led to a
cure rate of up to >90% of HCV genotypes 1-4 within 12-24
weeks.[6–8] The mode of action of all these nucleos(t)ides is the
inhibition of the viral replication catalyzed by RNA- or DNA-
polymerases. However, first an intracellular conversion of the
nucleosides into the biologically active triphosphate form by host
kinases is essential.[9] This kinase-catalyzed phosphorylation
proved to be quite inefficient in some cases (e.g. PSI-6206).[10]
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800361
ChemMedChem
FULL PAPER
was proven that the TriPPPro-compounds released the active
triphosphate form after intracellular enzymatic cleavage.[13,28,29]
With this in hand we wanted to examine the antiviral activity of
TriPPPro-compounds of ABC, CBV and their 1’,2’-cis-
carbocyclic counterparts. In the past, few 1’,2’-cis-substituted
carbocyclic nucleoside analogues were found to exhibit some
but only weak antiviral activity against HIV which may have to be
related to an unsufficient phosphorylation into the
triphosphates.[30,31]
The monophosphates of nucleosides 7-10 were prepared by the
method of Sowa and Ouchi (Scheme 3).[41] The corresponding
nucleotides 11-14 were obtained in yields between 72-77%,
after ion exchange to tetrabutylammonium cations.
Results and Discussion
The synthesis of carbocyclic nucleosides analogues can be
achieved on a linear or a convergent route. In the linear
synthesis the nucleobase is formed starting from an amino
function present at the cyclopentyl core. The convergent
approach involves the coupling of the preformed cyclopentyl
core and the complete nucleobase.[32,33] The synthesis for the
1’,2’-cis-substituted analogues started from enantiomerically
pure (1R,2S)-2-((benzyloxy)methyl)cyclopent-3-en-1-ol 3, which
was first used in the field of carbocyclic nucleoside analogues by
Biggadike et al.[34] In our case this cyclopentenol 3 was coupled
with 2-amino-6-chloropurine by an earlier reported microwave-
Scheme 3. Synthesis of nucleoside monophosphates 11-14. Reagents and
conditions: a) i) POCl3, pyridine, H2O, CH3CN, 0 °C; ii) water, NH4(H)CO3, 0 °C.
Recently, we published two different methods to synthesize
TriPPPro-compounds.[13,28,29]
includes the coupling of
The
a
phosphoramidite
preformed (non)symmetric
route
phosphoramidite and a nucleoside diphosphate followed by an
oxidation.[29] An alternative route to prepare TriPPPro-
compounds includes the use of H-phosphonate chemistry to
form a dimasked pyrophosphate unit first, which was next
treated with nucleoside monophosphates after activation.[28]
Here, we chose the H-phosphonate route, in which the phenyl
groups of diphenyl H-phosphonate were displaced by 4-
(hydroxymethyl)-phenyldodecanoate 15[28,42] to form the H-
phosphonate diester 16 in a yield of 57% (Scheme 4). Next,
diester 16 was activated with N-chlorosuccinimide to yield the
assisted
Mitsunobu
reaction.[35,36]
In
this
approach,
cyclopentenol 3 was treated with PPh3, DIAD and 2-amino-6-
chloropurine in CH3CN at 50 °C to give the purine derivative 4 in
59% yield.
phosphorochloridate
followed
by
treatment
with
tetrabutylammonium phosphate. The resulting pyrophosphate 17
was obtained almost quantitatively by a fast centrifuge extraction
from CH2Cl2/1 M aq. NH4OAc (pH 6), which is crucial to remove
the excess of phosphate salts.[28]
Scheme 1. Synthesis of the 2-amino-6-chloropurine derivative 5. Reagents
and Conditions: a) PPh3, DIAD, 2-amino-6-chloropurine, CH3CN, microwave
100 W, 50 °C; b) 1M BCl3 in CH2Cl2, -78 °C to -20 °C.
Next the benzyl protecting group was removed by using BCl3 at
low temperatures leading to derivative 5 (89% yield). CBV 9,
ABC 10 and their 1’,2’-cis-analogues 7 and 8 were synthesized
by substitution of the 6-chloro-atom of 5 or 6, respectively, with
either
1 M
aqueous
sodium
hydroxide[37,38]
or
cyclopropylamine[38–40] (Scheme 2) and compounds 7-10 were
Scheme 4. Synthesis of the masked pyrophosphate 17. Reagents and
conditions: a) diphenyl phosphonate, pyridine, 40 °C; b) i) N-chlorosuccinimide,
CH3CN, rt; ii) 0.4 M H2PO4NBu4, CH3CN, rt.
obtained in yields ranging from 88% to 98%.
The TriPPPro-synthesis (Scheme 5) started with the activation
of pyrophosphate 17 with TFAA followed by the addition of 1-
methylimidazole. For the coupling with the corresponding
nucleoside monophosphates 11-14 it was necessary to add THF
as co-solvent because otherwise incomplete consumption of the
nucleotides 11-14 was observed due to the insolubility of the
resulting TriPPPro-compounds in CH3CN. After purification and
Dowex ion-exchange (ammonium form) the TriPPPro-derivatives
18-21 were obtained in yields ranging from 35 to 41%.
Interestingly, a comparison using d4T monophosphate as
nucleotide using identical reaction conditions led to a yield of
Scheme 2. Synthesis of CBV 9, ABC 10 and their 1’,2’-cis-analogues 7 and 8.
Reagents and conditions: for R = OH: a) 1 M aq. NaOH, reflux; for R = NHcPr:
b) cyclopropylamine, EtOH, reflux.
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800361
ChemMedChem
FULL PAPER
74% for TriPPPro-d4TTP. Therefore, it was assumed that the
significant lower yields are caused by the guanine nucleobase.
The biologically active metabolite is the nucleoside triphosphate
which should be released intracellularly and which then should
be a substrate of HIVs reverse transcriptase. In Figure 2 the
possible cleavage mechanisms are summarized. Whereas
pathways A1 and A2 will lead to a release of the NTP, pathways
B and C, respectively, will lead to the formation of the NDP or
the NMP, respectively.
Scheme 5. Synthesis of TriPPPro-compounds 18-21. Reagents and
conditions: a) i) 17, TFAA, Et3N, CH3CN, 0 °C; ii) 1-methylimidazole, Et3N,
CH3CN, 0 °C to rt; iii) NMP’s 11-14, DMF, CH3CN, THF, rt.
The parent nucleosides 7-10 and their corresponding TriPPPro-
derivatives 18-21 were tested in HIV-1 or HIV-2-infected T-
lymphocyte cell cultures (CEM/0 cells) (Table 1). Whereas the
TriPPPro-CBVTP 20 and -ABCTP 21 showed a 3.5-fold and 4.5-
fold, respectively, increased activity against HIV-1 compared to
their parents, both 1’,2’-cis-substituted nucleosides 7 and 8 or
the corresponding TriPPPro-compounds 18 and 19 did not
shown any activity (>38 to >100 µM) against HIV-1 or HIV-2
infected cells. Nevertheless they also showed no cellular toxicity
(>38 to >100 µM).
Figure 2. Hydrolysis pathways for TriPPPro-compounds.
The TriPPPro-compounds were hydrolyzed in different media
(Table 2). In CEM/0 cell extract TriPPPro-CBVTP 20 showed the
highest stability (t1/2 = 6.5 h), while the half-life of TriPPPro-
ABCTP 21 was surprisingly shorter than 1 h. Both the 1’,2’-cis-
substituted analogues showed similar half-lives (18 3.0 h; 19
2.4 h), but still lower compared to the TriPPPro-d4TTP (4.8 h)
and -CBVTP 20. In chemical hydrolysis studies TriPPPro-d4TTP
and -CBVTP 20 have significant higher half-lives (59.4 h and
29.2 h) compared to derivatives 18 with 12.4 h and 19 as well as
21 which have half-lives in the range of 8.5 h.
Table 1. Antiviral data.
Compound
CEM
HIV-1 (HE)
EC50a (µM)
CEM
HIV-2
(ROD)
EC50a (µM)
CEM TK-
HIV-2
CEM
cellular
toxicity
CC50b (µM)
(ROD)
Table 2. Enzymatic and chemical hydrolysis of TriPPPro compounds
EC50a (µM)
Compound
CEM/0a
[h]
PBS (pH 7.3)a
[h]
PLEa,b
[h]
7
8
>100
>100
>100
>100
>100
>100
>100
>100
18
3.00 ± 0.64
2.42 ± 0.37
6.52 ± 0.59
0.73 ± 0.10
4.77 ± 0.78
12.44 ± 0.44
8.33 ± 0.16
29.15 ± 2.25
8.37 ± 0.12
59.43 ± 1.17
4.98 ± 0.28
1.77 ± 0.06
4.57 ± 0.26
2.17 ± 0.03
3.56 ± 0.32
9
3.5 ± 1.0
5.9 ± 2.5
>38
3.0 ± 0.0
5.2 ± 1.5
>38
3.7 ± 1.1
7.0 ± 1.0
>38
139 ± 20
135 ± 21
38
19
10
18
19
20
21
d4T
20
21
>42
>42
>42
42
TriPPPro-d4T
0.99 ± 0.14
1.3 ± 0.0
0.43 ± 0.25
0.15 ± 0.09
1.2 ± 0.5
1.4 ± 0.3
0.34 ± 0.23
0.12 ± 0.00
1.6 ± 0.5
2.1 ± 1.2
>50
28 ± 3
60 ± 2
>50
[a] half lives in hours; [b] pig liver esterase in PBS buffer (100 U/mL).
The enzymatic cleavage of the masks with pig liver esterase
(PLE) in PBS led to
corresponding nucleoside triphosphates (TriPPPro-d4T: 97%
d4TTP, 20: 95% CBVTP, 18: 90% 1’,2’-cis-CBVTP, 19: 90%
1’,2’-cis-ABCTP, 21: 70% ABCTP).
a predominant formation of the
TriPPPro-
d4T
0.83 ± 0.00
20 ± 3
AZT
0.0086 ±
0.0031
0.0060 ±
0.0017
>1000
>100
The chemical hydrolysis in PBS gave in the case of the
TriPPPro-ABC 21 and -1’,2’-cis-ABCTP 19 a ratio of about 1:1
for NTPs vs. NDPs, whereas the carbovir derivatives 18 and 20
showed a ratio of around 1:2. The TriPPPro-d4TTP led to a ratio
of 6.3:1 in favor of the NTP. The increased formation of NDPs
compared to TriPPPro-d4TTP may be explained by the
exocyclic amino group which seems to lead to an activation of
[a] 50% effective concentration or compound concentration required to inhibit
HIV-induced cytopathogenic effect in CEM or CEM TK- cell cultures. [b] 50%
cytotoxic concentration in CEM T cell cultures.
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800361
ChemMedChem
FULL PAPER
Hereaus Biofuge primo R with 8000 rpm for 4 min at 10 °C. HPLC data
were recorded on a VWR-Hitachi Elite LaChrom system with following
parts: DAD-L2455U, Autosampler-L2200 and Pump-L2130. The
EZChrom Elite software was used and samples were measured with the
following method: CH3CN gradient in 2 mM tetrabutylammonium acetate
buffer (pH 6.0); min 0-25 5-100%, flow 1 mL min-1. The purity of tested
compounds was determined by RP-HPLC using the above mentioned
method (see supporting information).
the β- and γ-phosphorous anhydride bond, so the contribution of
pathway B increased in these cases. In contrast in the CEM/0
cell extract only traces of the NTPs were detected. However, the
NDPs were formed rapidly and within 48 h the corresponding
NMPs and even the parent nucleosides were formed. This
points to a rapid dephosphorylation of the NTPs and the
following metabolites. The antiviral activity of the TriPPPro-
ABCTP 21 may be caused by the direct delivery of ABCTP
which interacts with HIV’s RT as a dATP analogue[26]. However,
we cannot exclude that the activity is also due to an intracellular
dephosphorylation of the formed ABCTP to yield the ABCMP
which can be deaminated to give CBVMP and which then enters
the intracellular phosphorylation pathway to finally yield CBVTP.
The TriPPPro-1’,2’-cis-derivatives 18 and 19 as well as their
parent nucleosides 7 and 8 did not show any antiviral activity.
Hydrolysis studies
CEM/0 hydrolysis: To a mixture of 50 µL CEM/0 cell extract and 10 µL
water was added 10 µL of a 6 mM solution of the corresponding
TriPPPro in DMSO. These hydrolysis samples were incubated at 37 °C
and analyzed by RP-HPLC (injection volume 20 µL) at different time.
PBS hydrolysis: To a mixture of 300 µL PBS, 189 µL water and 89 µL
DMSO was added 22 µL of a 25 mM solution of the corresponding
TriPPPro in DMSO. This mixture was incubated at 37 °C and at different
time aliquots of 35 µL were taken and analyzed by RP-HPLC (injection
volume 30 µL).
Conclusions
We successfully synthesized TriPPPro-compounds of ABC, CBV
and their 1’,2’-cis-disubstituted derivatives. The TriPPPro-
ABCTP and -CBVTP derivatives exhibit a superior antiviral
activity compared to their parent nucleosides. Unfortunately, the
1’,2’-cis-derivatives did not show any antiviral activity in their
nucleoside nor in their TriPPPro-form. It cannot be excluded that
the delivered triphosphates were rapidly dephosphorylated and
could not be rephosphorylated from there mono- or diphosphate
species. Interestingly, also the parent nucleosides proved to be
completely inactive. However, it also cannot be excluded that
even if the triphosphates are formed, these are no substrates for
the HIV-RT. This can be examined in further experiments using
the isolated triphosphates together with RT in a primer extension
assay.
PLE hydrolysis: To a mixture of 30 µL PLE solution (100 U/mL in PBS,
pH 7.3), 60 µL DMSO and 400 µL PBS (pH 7.3) was added 40 µL of a
6 mM solution of the corresponding TriPPPro in DMSO. This mixture was
incubated at 37 °C and at different time aliquots of 35 µL were taken and
analyzed by RP-HPLC (injection volume 30 µL).
9-((2R)-2-((Benzyloxy)methyl)cyclopent-3-en-1-yl)-6-chloro-9H-purin-
2-amine (4)
To
a
solution
of cyclopentenol
3
(400 mg, 1.96 mmol),
triphenylphosphine (771 mg, 2.94 mmol) and 2-amino-6-chloropurine
(499 mg, 2.94 mmol) in THF (15 mL) was added DIAD (0.58 mL,
2.94 mmol). The reaction mixture was irradiated at 50 °C (100 W) for 1 h.
The solvent was removed under reduced pressure and the residue was
purified on silica gel (petroleum ether/EtOAc; 1:2 to 1:3, CH2Cl2/CH3OH;
19:1) tp yield 4 (0.414 g, 59%) as a colourless resin. Rf = 0.41 (petroleum
ether/EtOAc, 1:2). [α]D25: -219 (c 0.1, CH3OH). IR (neat): 3313, 3195,
2856, 1604, 1558, 1456, 1402, 1216, 1088, 901, 697 cm-1. 1H NMR
(500 MHz, CDCl3): δ = 7.88 (s, 1H, H-8), 7.31-7.19 (m, 3H, H-Bn), 7.06-
6.98 (m, 2H, H-Bn), 6.08-6.02 (m, 1H, H-4’), 5.84-5.78 (m, 1H, H-3’),
Experimental Section
All experiments involving water- or air-sensitive compounds were carried
out under anhydrous conditions (N2-atmosphere). Reagents were
purchased by various suppliers and used without further purification, if
not otherwise noted. Anhydrous solvents were purchased from Acros
Organics (extra dry over molecular sieves) or dried by a MBraun (MB
SPS-800)-System. Solvents for chromatography were purchased in
technical grade and distilled prior to use. Ultrapure water and CH3CN for
reversed phase chromatography were purified by a Sartorius Aurium pro
apparatus (Sartopore 0.2 µm, UV) and purchased from VWR (HPLC
grade), respectively. Column chromatography on silica gel was
performed by using silica gel MN 60 M (0.04-0.063 mm) from Macherey
Nagel. Thin layer chromatography was performed on pre-coated TLC-
sheets ALUGRAM® Xtra SIL G/UV254 purchased from Macherey Nagel.
Visualisation of non-UV active compounds was done by heat-staining
with vanillin in sulfuric acid, acetic acid and CH3OH. Automated flash
reversed phase chromatography was performed by using MN RS 16 C18
ec, RS 40 C18 ec or RS 120 C18 ec columns on an Interchim Puriflash
430 system. Microwave-assisted reactions were conducted in a CEM
discover system in open- or closed-vessel mode at different
temperatures and power. NMR solvents were purchased from Euroiso-
Top (CDCl3, CD3OD and D2O) and Deutero (DMSO-d6). NMR-spectra
were recorded at room temperature on Bruker instruments Avance I 400,
DRX 500 and Avance III 600. IR- and mass-spectra were recorded on
Bruker Alpha IR-sprectrometer and Agilent 6224 ESI-TOF instrument,
2
5.38-5.30 (m, 1H, H-1’), 5.20 (br s, 2H, NH2), 4.16 (d, 1H, J = 11.6 Hz,
CHHPh), 4.02 (d, 1H, 2J = 11.6 Hz, CHHPh), 3.44-3.36 (m, 1H, H-2’),
3.27 (dd, 2J = 9.7 Hz, 3J = 4.7 Hz, H-6a’), 3.12 (dd, 2J = 9.7 Hz, 3J =
7.7 Hz, H-6b’), 3.05-2.96 (m, 1H, H-5a’), 2.87-2.79 (m, 1H, H-5b’). 13C
NMR (126 MHz, CDCl3): δ = 158.8 (Cq-6), 153.9 (Cq-2), 151.1 (Cq-4),
141.7 (C-8), 137.4 (Cq-Bn), 131.1 (C-3’), 130.4 (C-4’), 128.4 (2x C-Bn),
127.8 (C-Bn), 127.7 (2x C-Bn), 124.5 (Cq-5), 73.4 (CH2Ph), 68.3 (C-6’),
54.6 (C-1’), 49.6 (C-2’), 39.2 (C-5’). HRMS-ESI+: m/z calcd for
C18H18ClN5O (M+H): 356.1273; found: 356.1266.
((1R)-5-(2-amino-6-chloro-9H-purin-9-yl)cyclopent-2-en-1-
yl)methanol (5)
The derivative 4 (489 mg, 1.37 mmol) was dissolved in CH2Cl2 (20 mL)
and cooled to -78 °C. Then a 1M BCl3 solution in CH2Cl2 (6.9 mL,
6.9 mmol) was added dropwise. The reaction mixture was slowly warmed
to – 20 °C and stirred for 1 h at this temperature. Afterwards sat. aq.
NaHCO3 solution (20 mL) was added and the mixture was warmed to
room temperature. All volatiles were removed in vacuo and the resulting
residue was purified on RP18 silica gel with automated flash
chromatography (H2O/CH3CN, 100:0 to 0:100) to obtain 5 (323 mg, 89%;
purity: >98%) as a colourless foam. Rf = 0.26 (CH2Cl2/CH3OH, 95:5).
[α]D19: -292 (c 0.2, CH3OH). IR (neat): 3314, 3199, 2861, 1604, 1559,
respectively. The optical rotations were measured with
a P8000
1
1460, 1395, 1256, 904, 711 cm-1. H NMR (600 MHz, CD3OD): δ = 8.08
polarimeter of A.Krüss Optonic GmbH at indicated temperatures. The
phase separation for the masked pyrophosphate was performed on a
(s, 1H, H-8), 6.10-6.04 (m, 1H, H-4’), 5.89-5.85 (m, 1H, H-3’), 5.34-5.28
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800361
ChemMedChem
FULL PAPER
(m, 1H, H-1’), 3.34-3.29 (m, 1H, H-6a’), 3.25-3.19 (m, 2H, H-6a’, H-2’),
3.05-2.92 (m, 2H, H-5’). 13C NMR (151 MHz, CD3OD): δ = 161.5 (Cq-6),
155.8 (Cq-4), 151.4 (Cq-2), 143.6 (C-8), 132.0 (C-3’), 131.5 (C-4’), 124.6
(Cq-5), 61.3 (C-6’), 56.4 (C-1’), 52.2 (C-2’), 38.7 (C-5’). HRMS-ESI+: m/z
calcd for C11H12ClN5O (M+H): 266.0803; found: 266.0814.
gel (CH2Cl2/CH3OH, 95:5 to 90:10) to afford Abacavir 10 (2.12 g, 98%;
purity: >97%) as a colourless foam. Rf = 0.65 (CH2Cl2/CH3OH, 85:15).
[α]D19: -49 (c 0.2, CH3OH). IR (neat): 3307, 3194, 2866, 1586, 1478, 1386,
1351, 1205, 1027, 788, 640 cm-1. 1H NMR (500 MHz, DMSO-d6): δ =
7.60 (s, 1H, H-8), 7.26 (br s, 1H, NH), 6.13-6.07 (m, 1H, H-3’), 5.90-5.84
(m, 1H, H-2’), 5.81 (br s, 2H, NH2), 5.42-5.35 (m, 1H, H-1’), 4.73 (t, 1H, 3J
=
5.3 Hz, OH), 3.49-3.41 (m, 2H, H-6’), 3.11-2.97 (m, 1H, CH-
1’,2’-cis-Carbovir (7)
cyclopropyl), 2.91-2.82 (m, 1H, H-4’), 2.65-2.55 (m, 1H, H-5a’), 1.63-1.54
(m, 1H, H-5b’), 0.69-0.53 (m, 4H, CH2-cyclopropyl). 13C NMR (126 MHz,
DMSO-d6): δ = 160.0 (Cq-6), 155.9 (Cq-2), 151.0 (Cq-4), 137.9 (C-3’),
134.8 (C-8), 130.0 (C-2’), 113.6 (Cq-5), 64.1 (C-6’), 58.1 (C-1’), 47.7 (C-
4’), 34.3 (C-5’), 23.8 (CH-cyclopropyl), 6.4 (2x CH2-cyclopropyl). HRMS-
ESI+: m/z calcd for C14H18N6O (M+H): 287.1615; found: 287.1622.
The derivative 5 (300 mg, 2.07 mmol) was suspended into 1 M aq. NaOH
solution (20 mL) and heated to reflux for 4 h. Purification on RP18 silica
gel with automated flash chromatography (H2O/CH3CN, 100:0 to 0:100)
afforded 7 (246 mg, 88%; purity: >98%) after freeze drying (H2O/CH3CN,
1:1) as a colourless cotton. Rf = 0.23 (CH2Cl2/CH3OH, 85:15). [α]D19: -221
(c 0.2, CH3OH). IR (neat): 3370, 3185, 2870, 2728, 1679, 1629, 1603,
1386, 1175, 783, 577 cm-1. 1H NMR (600 MHz, DMSO-d6): δ = 10.5 (br s,
1H, NH), 7.61 (s, 1H, H-8), 6.44 (br s, 2H, NH2), 6.00-5.96 (m, 1H, H-4’),
General procedure monophosphate synthesis
3
5.87-5.83 (m, 1H, H-3’), 5.03-4.97 (m, 1H, H-1’), 4.43 (t, 1H, J = 4.8 Hz,
Freshly distilled POCl3 (412 µL, 4.40 mmol) was dissolved in CH3CN
(1 mL) and cooled to 0 °C. After subsequent dropwise addition of pyridine
(392 µL, 4.80 mmol) and H2O (50 µL, 2.8 mmol) the mixture was stirred
for 10 min. Then the appropriate nucleoside (1.00 mmol) was added and
the reaction was stirred at 0 °C until full conversion. The reaction mixture
was poured into ice cold H2O and hydrolysed for 1 h. The pH value was
set to 8 with solid NH4HCO3 and afterwards the volatiles were removed
by freeze drying. The residue was purified on RP18 silica gel with
automated flash chromatography (H2O/CH3CN, 100:0 to 0:100). The
OH), 3.12-3.07 (m, 1H, H-6a’), 3.02-2.93 (m, 2H, H-2’, H-6b’), 2.88-2.79
(m, 2H, H-5’). 13C NMR (151 MHz, DMSO-d6): δ = 156.8 (Cq-6), 153.5
(Cq-4), 151.5 (Cq-2), 135.9 (C-8), 131.6 (C-3’), 129.8 (C-4’), 116.1 (Cq-5),
59.9 (C-6’), 53.6 (C-1’), 50.6 (C-2’), 37.5 (C-5’). HRMS-ESI+: m/z calcd
for C11H13N5O2 (M+H): 248.1142; found: 248.1147.
1’,2’-cis-Abacavir (8)
ammonia
form
was
dissolved
in
H2O/CH3CN,
then
The derivative 5 (311 mg, 1.17 mmol) was dissolved in ethanol (5 mL)
and cyclopropylamine (0.81 mL, 12 mmol) was added. The reaction
mixture was heated to reflux with microwave irradiation (150W) until full
conversion. All volatiles were removed in vacuo and the residue was
purified on RP18 silica gel with automated flash chromatography
(H2O/CH3CN, 100:0 to 0:100) to afford 8 (325 mg, 97%; purity: >99%)
after freeze drying (H2O/CH3CN, 1:1) as a colourless cotton. Rf = 0.60
(CH2Cl2/CH3OH, 85:15). [α]D19: -238 (c 0.2, CH3OH). IR (neat): 3321,
3201, 2861, 1593, 1478, 1391, 1265, 1029, 789, 640 cm-1. 1H NMR
(400 MHz, DMSO-d6): δ = 7.60 (s, 1H, H-8), 7.29 (br s, 1H, NH), 6.02-
5.97 (m, 1H, H-4’), 5.91-5.79 (m, 3H, NH2, H-3’), 5.09-5.01 (m, 1H, H-1’),
4.50 (br s, 1H, OH), 3.11-2.77 (m, 6H, H-6’, H-2’, CH-cyclopropyl, H-5’),
0.69-0.55 (m, 4H, CH2-cyclopropyl). 13C NMR (101 MHz, DMSO-d6): δ =
160.1 (Cq-6), 155.9 (Cq-2), 152.0 (Cq-4), 135.7 (C-8), 131.7 (C-3‘), 129.9
(C-4‘), 113.0 (), 59.9 (C-6‘), 53.4 (C-1‘), 50.9 (C-2‘), 37.5 (C-5‘), 23.8
(CH-cyclopropyl), 6.4 (CH2-cyclopropyl), 6.3 (CH2-cyclopropyl). HRMS-
ESI+: m/z calcd for C14H18N6O (M+H): 287.1615; found: 287.1626.
tetrabutylammoniumhydroxide was added and the solvents were
removed under reduced pressure. The resulting resin was purified on
RP18 silica gel with automated flash chromatography (H2O/CH3CN, 100:0
to 0:100) to afford the monophosphate.
1’,2’-cis-Carbovir-monophosphate (11)
1
Yield: 11 (627 mg, 77%) as a colourless resin. H NMR (400 MHz, D2O):
δ = 7.86 (s, 1H, H-8), 6.08-6.03 (m, 1H, H-4’), 5.98-5.93 (m, 1H, H-3’),
5.16-5.08 (m, 1H, H-1’), 3.55-3.47 (m, 1H, H-6b’), 3.39-3.25 (m, 2H, H-2’,
H-6b’), 3.21-3.10 (m, 16H, NBu4), 3.05-2.94 (m, 1H, H-5a’), 2.82-2.73 (m,
1H, H-5b’), 1.68-1.55 (m, 16H, NBu4), 1.39-1.26 (m, 16H, NBu4), 0.91 (t,
3JH,H = 7.4 Hz, NBu4). 13C NMR (101 MHz, D2O): δ = 159.0 (Cq-6), 153.8
(Cq-2), 151.5 (Cq-4), 138.7 (C-8), 131.2 (C-3’), 130.0 (C-4’), 115.4 (Cq-5),
2
3
62.5 (d, JP,C = 4.8 Hz, C-6’), 58.1 (CH2-NBu4), 54.3 (C-1’), 49.7 (d, JP,C
= 6.9 Hz, C-2’), 38.8 (C-5’), 23.1 (CH2-NBu4), 19.1 (CH2-NBu4), 12.8
(CH3-NBu4). 31P NMR (162 MHz, D2O): δ = 3.59 (s). HRMS-ESI-: m/z
calcd for C27H49N6O5P (M-H)-: 567.3429; found: 567.3430.
Carbovir (9)
1’,2’-cis-Abacavir-monophosphate (12)
The 6-chloropurine 6 (1.20 g, 4.52 mmol) suspended into 0.5 M aq.
NaOH solution (60 mL) and heated to reflux for 4 h. The crude product
was adsorbed on silica gel and afterwards purified on silica
(CH2Cl2/CH3OH, 95:5 to 80:20) to afford 9 (1.09 g, 97%) as a colourless
solid. Rf = 0.17 (CH2Cl2/CH3OH, 85:15). [α]D19: -68 (c 0.2, CH3OH). IR
(neat): 3326, 3127, 2850, 2664, 1680, 1601, 1473, 1385, 1176, 1030,
781, 688, 568 cm-1. 1H NMR (400 MHz, DMSO-d6): δ = 10.6 (br s, 1H,
NH), 7.59 (s, 1H, H-8), 6.44 (br s, 2H, NH2), 6.14-6.07 (m, 1H, H-3’),
Yield: 12 (622 mg, 73%) as a yellowish resin. 1H NMR (400 MHz, D2O): δ
= 7.87 (s, 1H, H-8), 6.09-6.03 (m, 1H, H-4’), 5.99-5.94 (m, 1H, H-3’),
5.15-5.08 (m, 1H, H-1’), 3.51-3.42 (m, 1H, H-6a’), 3.36-3.27 (m, 1H, H-2’),
3.22-3.08 (m, 17H, H-6b’, NBu4), 3.03-2.92 (m, 1H, H-5a’), 2.85-2.74 (m,
2H, CH-cyclopropyl, H-5b’), 1.68-1.53 (m, 16H, NBu4), 1.39-1.25 (m, 16H,
NBu4), 0.96-0.82 (m, 26H, NBu4, CH2-cyclopropyl), 0.66-0.58 (m, 2H,
CH2-cyclopropyl). 13C NMR (101 MHz, D2O): δ = 160.1 (Cq-6), 156.3 (Cq-
2), 150.4 (Cq-4), 138.3 (C-8), 131.3 (C-3’), 130.0 (C-4’), 112.7 (Cq-5),
3
5.89-5.83 (m, 1H, H-2’), 5.38-5.29 (m, 1H, H-1’), 4.72 (t, 1H, J = 5.3 Hz,
OH), 3.49-3.39 (m, 2H, H-6a’), 2.91-2.80 (m, 1H, H-4’), 2.64-2.53 (m, 1H,
H-5a’), 1.62-1.51 (m, 1H, H-5b’). 13C NMR (101 MHz, DMSO-d6): δ =
156.9 (Cq-6), 153.4 (Cq-2), 150.8 (Cq4), 138.3 (C-3‘), 135.1 (C-8), 129.7
(C-2‘), 116.7 (Cq-5), 64.0 (C-6’), 58.5 (C-1’), 47.7 (C-4’), 34.4 (C-5’).
HRMS-ESI+: m/z calcd for C11H13N5O2 (M+H): 248.1142; found:
248.1146.
2
3
62.5 (d, JP,C = 4.9 Hz, C-6’), 58.1 (CH2-NBu4), 54.0 (C-1’), 49.5 (d, JP,C
= 7.2 Hz, C-2’), 38.7 (C-5’), 23.2 (CH-cyclopropyl), 23.1 (CH2-NBu4), 19.1
(CH2-NBu4), 12.8 (CH3-NBu4), 6.8 (CH2-cyclopropyl), 6.7 (CH2-
cyclopropyl). 31P NMR (162 MHz, D2O): δ = 3.65 (s). HRMS-ESI-: m/z
calcd for C14H18N6O4P (M-H)-: 365.1133; found: 365.1140.
Abacavir (10)
Carbovir-monophosphate (13)
1
The 6-chloropurine 6 (2.00 g, 7.53 mmol) was dissolved in ethanol
(15 mL) and cyclopropylamine (5.2 mL, 75 mmol) was added. The
reaction mixture was heated to 70 °C overnight. The volatiles were
removed under reduced pressure and the residue was purified on silica
Yield: 13 (614 mg, 76%) as a colourless resin. H NMR (400 MHz, D2O):
δ = 7.83 (s, 1H, H-8), 6.27-6.21 (m, 1H, H-3’), 5.88-5.82 (m, 1H, H-2’),
5.42-5.34 (m, 1H, H-1’), 3.82-3.74 (m, 1H, H-6’), 3.21-3.04 (m, 17H, NBu4,
H-4’), 2.79-2.67 (m, 1H, H-5a’), 1.67-1.52 (m, 17H, NBu4, H-5b’), 1.38-
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800361
ChemMedChem
FULL PAPER
1.24 (m, 16H, NBu4), 0.90 (t, 24H, 3J = 7.4 Hz, NBu4). 13C NMR
(101 MHz, D2O): δ = 158.7 (Cq-6), 153.5 (Cq-2), 150.9 (Cq-4), 138.6 (C-
evaporated and the residue was dissolved in CH2Cl2 (15 mL). The
organic phase was washed with 1 M aq. NH4OAc and H2O (phase
separation via centrifuge) followed by drying over Na2SO4. The solvent
was removed and the resulting pyrophosphate 17 (867 mg, 87%) was
used without further purification (absence of phosphate salt was
controlled by 31P-NMR under Schlenk conditions).
2
3‘), 138.2 (C-8), 129.1 (C-2‘), 115.9 (Cq-5), 67.1 (d, JP,C = 4.8 Hz, C-6’),
3
59.7 (C-1‘), 58.1 (CH2-NBu4), 46.0 (d, JP,C = 7.5 Hz, C-4’), 34.4 (C-5’),
23.1 (CH2-NBu4), 19.1 (CH2-NBu4), 12.8 (CH3-NBu4). 31P NMR (162 MHz,
D2O): δ = 3.13 (s). HRMS-ESI-: m/z calcd for C27H49N6O5P (M-H)-:
567.3429; found: 567.3404.
The pyrophosphate (1 eq) was dissolved in CH3CN and cooled to 0 °C
and then a 0 °C cold solution of TFAA (5 eq.) and triethylamine (8 eq) in
CH3CN was added. The mixture was stirred for 10 min at 0 °C. After
evaporation the residue was coevaporated with CH3CN and afterwards
dissolved in CH3CN. Then triethylamine (5 eq) and 1-methylimidazole
(3 eq) were added at 0 °C and the reaction mixture was stirred for 10 min.
Then THF and the appropriate nucleoside monophosphate (0.5 eq)
dissolved in DMF were added. The reaction progress was controlled by
RP-HPLC. After full conversion all volatiles were removed and the
residue was purified on RP18 silica gel with automated flash
Abacavir-monophosphate (14)
Yield: 14 (611 mg, 72%) as a yellowish resin. 1H NMR (400 MHz, D2O): δ
= 7.85 (s, 1H, H-8), 6.30-6.24 (m, 1H, H-3’), 5.88-5.82 (m, 1H, H-2’),
5.44-5.35 (m, 1H, H-1’), 3.80-3.67 (m, 2H, H-6’), 3.22-2.99 (m, 17H, NBu4,
H-4’), 2.86-2.67 (m, 2H, CH-cyclopropyl, H-5a’), 1.67-1.48 (m, 17H, NBu4,
H-5b’), 1.40-1.20 (m, 16H, NBu4), 0.96-0.78 (m, 26H, NBu4, CH2-
cyclopropyl), 0.63-0.55 (m, 2H, CH2-cyclopropyl). 13C NMR (101 MHz,
D2O): δ = 159.9 (Cq-6), 156.2 (Cq-2), 149.8 (Cq-4), 138.8 (C-3‘), 137.6 (C-
2
chromatography (H2O/CH3CN, 90:10 to 0:100) followed by ion exchange
8), 129.0 (C-2‘), 113.3 (Cq-5), 67.1 (d, JP,C = 4.8 Hz, C-6’), 59.3 (C-1‘),
+
3
(NH4
) and purification on RP18 silica gel with automated flash
58.0 (CH2-NBu4), 46.0 (d, JP,C = 7.5 Hz, C-4’), 34.6 (C-5’), 23.2 (CH-
chromatography (H2O/CH3CN, 90:10 to 0:100).
cyclopropyl), 23.1 (CH2-NBu4), 19.1 (CH2-NBu4), 12.8 (CH3-NBu4), 6.7
(CH2-cyclopropyl), 6.7 (CH2-cyclopropyl). 31P NMR (162 MHz, D2O): δ =
3.65 (s). HRMS-ESI-: m/z calcd for C14H18N6O4P (M-H)-: 365.1133;
found: 365.1112.
TriPPPro-1’,2’-cis-CBVTP (ammonium salt) (18)
The reaction was carried out with pyrophosphate 17 (453 mg, 455 µmol)
in CH3CN (6 ml) and TFAA (323 µL, 2.27 mmol) and triethylamine
(504 µL, 3.64 mmol) in CH3CN (4 mL). Afterwards triethylamine (315 µL,
2.27 mmol) and 1-methylimidazole (109 µL, 1.36 mmol) in CH3CN (4 mL)
were used. Then THF (6 mL) was added followed by monophosphate 11
(184 mg, 227 µmol) in DMF (2 mL). Yield: 18 (87.2 mg, 35%; purity:
>98%) as a colourless cotton. 1H NMR (600 MHz, CD3OD): δ = 7.97 (s.
1H, H-8), 7.40-7.37 (m, 4H, H-Bn), 7.06-7.02 (m, 4H, H-Bn), 6.00-5.93 (m,
2H, H-4’, H-3’), 5.24-5.19 (m, 1H, H-1’), 5.14 (dd, 4H, 3J = 8.3 Hz, 2.8 Hz,
2x CH2Ph), 3.88 (ddd, 1H, 2J = 10.4 Hz, 3J = 6.9 Hz, 5.6 Hz, H-6a’), 3.68-
3.62 (m, 1H, H-6b’), 3.41-3.36 (m, 1H, H-2’), 2.94-2.84 (m, 2H, H-5’),
2.57 (t, 4H, 3J = 7.4 Hz, CH2-alkyl), 1.77-1.70 (m, 4H, CH2-alkyl), 1.47-
1.24 (m, 32H, CH2-alkyl), 0.90 (t, 6H, 3J = 7.0 Hz, CH3-alkyl). 13C NMR
(151 MHz, CD3OD): δ = 173.8 (2x Cq-acyl), 163.3 (Cq-6), 155.4 (Cq-2),
4-(Hydroxymethyl)-phenyldodecanoate (15)
To a solution of 4-hydroxybenzyl alcohol (10.0 g, 80.6 mmol) and 4-
(dimethylamino)pyridine (1.08 g, 8.86 mmol) in THF (150 mL)
triethylamine (12.5 mL, 90.2 mmol) was added. The mixture was cooled
to 0 °C followed by addition of dodecanoyl chloride (17.8 mL, 76.5 mmol)
in THF (50 mL) over a period of 30 min. The reaction was warmed to
room temperature and stirred for 1.5 h at this temperature. The resulting
suspension was filtrated and the solvent was removed under reduced
pressure. The residue was purified on silica gel (petroleum ether/EtOAc,
3:1 to 2:1) to yield 15 (17.4 g, 74%) as an amorphous solid. Rf = 0.61
(petroleum ether/EtOAC, 70:30). The analytical data is identical to those
reported in the literature.[42]
3
153.0 (Cq-4), 152.3 (2x Cq-Bn), 138.6 (C-8), 135.1 (d, JC,P = 7.3 Hz, 2x
Cq-Bn), 132.3 (C-3’), 131.1 (C-4’), 130.5 (4x CH-Bn), 122.8 (4x CH-Bn),
Bis-(4-dodecanoyloxybenzyl)-phosphonate (16)
2
2
114.8 (Cq-5), 70.3 (d, JC,P = 6.0 Hz, 2x CH2Ph), 65.6 (d, J = 6.6 Hz, C-
6’), 56.1 (C-1’), 50.7 (d, 3J = 7.9 Hz, C-2’) 39.1 (C-5’),, 35.0 (2x CH2-alkyl),
33.1 (2x CH2-alkyl), 30.7 (4x CH2-alkyl), 30.6 (2x CH2-alkyl), 30.5 (2x
CH2-alkyl), 30.4 (2x CH2-alkyl), 30.2 (2x CH2-alkyl), 26.0 (2x CH2-alkyl),
23.7 (2x CH2-alkyl), 14.4 (2x CH3-alkyl). 31P NMR (243 MHz, CD3OD): δ
= -11.6 (d, 2J = 20.1 Hz), -13.3 (d, 2J = 16.9 Hz), -23.8 (dd, 2J = 20.1 Hz,
16.9 Hz). HRMS-ESI-: m/z calcd for C49H71N5O15P3 (M-H)-: 1062.4165;
found: 1062.4117.
The dodecanoate 15 (6.74 g, 22.0 mmol) was coevaporated two times
with pyridine (each 5 mL) and then dissolved in pyridine (20 mL). Then
diphenyl phosphonate (1.92 mL, 10.0 mmol) was added and the reaction
was stirred for 4 h at 40 °C. The solvent was evaporated and the residue
was coevaporated three times with toluene and once with CH2Cl2. The
resulting residue was recrystallized from CH3OH to yield 16 (3.75 g, 57%)
as an amorphous solid. 1H NMR (600 MHz, CDCl3): δ = 7.36 (d, 4H, 3J =
2
8.5 Hz, H-Bn), 7.08 (d, 4H, 3J = 8.5 Hz, H-Bn), 6.93 (d, 1H, JH,P
=
TriPPPro-1’,2’-cis-ABCTP (ammonium salt) (19)
708 Hz, P-H), 5.09-4.99 (m, 4H, 2x CH2Ph), 2.55 (t, 4H, 3J = 7.5 Hz, CH2-
alkyl), 1.78-1.71 (m, 4H, CH2-alkyl), 1.44-1.21 (m, 32H, CH2-alkyl), 0.88 (t,
6H, 3J = 7.0 Hz, CH3-alkyl). 13C NMR (151 MHz, CDCl3): δ = 172.3 (2x
Cq-acyl), 151.1 (2x Cq-Bn), 133.1 (d, 3JC,P = 6.1 Hz, 2x Cq-Bn), 129.4 (4x
The reaction was carried out with pyrophosphate 17 (442 mg, 443 µmol)
in CH3CN (6 ml) and TFAA (315 µL, 2.22 mmol) and triethylamine
(492 µL, 3.59 mmol) in CH3CN (4 mL). Afterwards triethylamine (307 µL,
2.22 mmol) and 1-methylimidazole (106 µL, 1.33 mmol) in CH3CN (4 mL)
were used. Then THF (6 mL) was added followed by monophosphate 12
(188 mg, 222 µmol) in DMF (2 mL). Yield: 19 (90.7 mg, 36%) as a
2
CH-Bn), 122.1 (4x CH-Bn), 66.8 (d, JC,P = 5.8 Hz, 2x CH2Ph), 34.5 (2x
CH2-alkyl), 32.0 (2x CH2-alkyl), 29.7 (4x CH2-alkyl), 29.6 (2x CH2-alkyl),
29.5 (2x CH2-alkyl), 29.4 (2x CH2-alkyl), 29.2 (2x CH2-alkyl), 25.0 (2x
CH2-alkyl), 22.8 (2x CH2-alkyl), 14.3 (2x CH3-alkyl). 31P NMR (243 MHz,
CDCl3): δ = 7.71 (s). HRMS-ESI+: m/z calcd for C38H59NaO7P (M+Na):
681.3891 found: 681.3866.
1
colourless cotton. H NMR (600 MHz, DMSO-d6): δ = 7.80 (s, 1H, H-8),
7.38 (d, 4H, 3J = 8.1 Hz, H-Bn), 7.26 (br s, 3H, NH, NH2), 7.04 (d, 4H, 3J
= 8.0 Hz, H-Bn) 6.00-5.95 (m, 1H, H-4’), 5.85-5.74 (m, 1H, H-3’), 5.21-
5.00 (m, 5H, H-1’, 2x CH2Ph), 3.85-3.18 (m, 3H, H-6’, H-2’), 2.92-2.71 (m,
2H, H-5’), 2.58-2.51 (CH2-alkyl, CH-cyclopropyl), 1.67-1.58 (m, 4H, CH2-
General procedure TriPPPro synthesis
3
alkyl), 1.40-1.17 (m, 32H, CH2-alkyl), 0.85 (t, 6H, J = 6.8 Hz, CH3-alkyl),
The phosphonate 16 (659 mg, 1.00 mmol) was dissolved under heating
in CH3CN (25 mL) and N-chlorosuccinimide (334 mg, 2.50 mmol) was
added. The reaction mixture was stirred overnight at room temperature
(full conversion was controlled by 31P-NMR). Afterwards the mixture was
added dropwise to a solution of 0.4 M tetrabutylammonium phosphate
monobasic (7.5 mL, CH3CN) in CH3CN (10 mL). After 1 h the solvent was
0.77-0.52 (m, 4H, 2x CH2-cyclopropyl). 13C NMR (152 MHz, DMSO-d6):
δ = 13C NMR (152 MHz, DMSO-d6): δ = 171.7 (2x Cq-acyl), 150.2 (2x Cq-
3
Bn), 133.9 (d, JC,P = 7.7 Hz, 2x Cq-Bn), 130.3 (C-3‘), 130.2 (C-4’), 129.0
2
(4x CH-Bn), 121.6 (4x CH-Bn), 67.8 (d, JC,P = 5.3 Hz, 2x CH2Ph), 63.4
(C-6’), 53.4 (C-1‘), 49.3 (C-2’), 38.5 (C-5’), 33.5 (2x CH2-alkyl), 31.3 (2x
CH2-alkyl), 29.0 (4x CH2-alkyl), 28.9 (2x CH2-alkyl), 28.7 (2x CH2-alkyl),
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800361
ChemMedChem
FULL PAPER
28.7 (2x CH2-alkyl), 28.4 (2x CH2-alkyl), 24.3 (2x CH2-alkyl), 22.1 (2x
CH2-alkyl), 13.9 (2x CH3-alkyl), 6.30 (2x CH2-cyclopropyl). (Cq-6, Cq-2,
Cq-4, C-8, Cq-5 and CH-cyclopropyl) could not be detected due to micelle
formation. 31P NMR (243 MHz, DMSO-d6): δ = -12.7 (m), -13.1 (d, 2J =
were used. Then THF (4 mL) was added followed by d4T
monophosphate (63.0 mg, 80.0 µmol) in DMF (1 mL). Yield: (60.3 mg,
1
74%; purity: >98%) as a colourless cotton. H NMR (600 MHz, CD3OD):
δ = 7.67 (q. 1H, 4J = 1.2 Hz, H-6), 7.42-7.36 (m, 4H, H-Bn), 7.07-7.01 (m,
4H, H-Bn), 6.94-6.91 (m, 1H, H-1’), 6.47 (dt, 1H, 3J = 6.0 Hz, 4J = 1.8 Hz,
17.9 Hz), -23.9 (t, 2J
=
19.8 Hz). HRMS-ESI-: m/z calcd for
C52H76N6O14P3 (M-H)-: 1101.4638; found: 1101.4686.
H-3’), 5.79 (ddd, 1H, J = 6.1 Hz, 2.5 Hz, J = 1.4 Hz, H-2’), 5.15 (d, 4H,
3J = 8.3 Hz, 2x CH2Ph), 4.96-4.91 (m, 1H, H-4’), 4.29 (ddd, 1H, 2J =
11.6 Hz, 3J = 6.8 Hz, 3.3 Hz, H-5a’), 4.19 (ddd, 1H, 2J = 11.6 Hz, 3J =
5.5 Hz, 3.2 Hz, H-5b’), 2.57 (t, 4H, 3J = 7.4 Hz, CH2-alkyl), 1.89 (d, 3H, 4J
= 1.2 Hz, CH3), 1.78-1.68 (m, 4H, CH2-alkyl), 1.48-1.24 (m, 32H, CH2-
3
4
TriPPPro-CBVTP (ammonium salt) (20)
The reaction was carried out with pyrophosphate 17 (461 mg, 463 µmol)
in CH3CN (6 ml) and TFAA (328 µL, 2.31 mmol) and triethylamine
(513 µL, 3.70 mmol) in CH3CN (4 mL). Afterwards triethylamine (321 µL,
2.31 mmol) and 1-methylimidazole (111 µL, 1.39 mmol) in CH3CN (4 mL)
were used. Then THF (6 mL) was added followed by monophosphate 13
(187 mg, 231 µmol) in DMF (2 mL). Yield: 20 (104 mg, 41%; purity:
>98%) as a colourless cotton. 1H NMR (600 MHz, CD3OD): δ = 8.16 (s,
1H, H-8), 7.39-7.34 (m, 4H, H-Bn), 7.04-7.00 (m, 4H, H-Bn), 6.19-6.15 (m,
1H, H-3’), 5.80-5.77 (m, 1H, H-2’), 5.52-5.48 (m, 1H, H-1’), 5.14 (dd, 4H,
J = 8.3 Hz, 3.6 Hz, 2x CH2Ph), 4.15-4.05 (m, 2H, H-6’), 3.13-3.07 (m, 1H,
H-4’), 2.74-2.67 (m, 1H, H-5a’), 2.56 (t, 4H, 3J = 7.4 Hz, CH2-alkyl), 1.91-
1.86 (m, 1H, H-5b’), 1.76-1.69 (m, 4H, CH2-alkyl), 1.45-1.24 (m, 32H,
CH2-alkyl), 0.90 (t, 6H, 3J = 6.9 Hz, CH3-alkyl). 13C NMR (152 MHz,
CD3OD): δ = 173.7 (2x Cq-acyl), 158.4 (Cq-6), 155.5 (Cq-2), 152.3 (d, J =
3
alkyl), 0.90 (t, 6H, J = 7.1 Hz, CH3-alkyl). 13C NMR (151 MHz, CD3OD):
δ = 173.8 (2x Cq-acyl), 166.5 (Cq-4), 152.8 (Cq-2), 152.3 (2x Cq-Bn),
3
138.7 (C-6), 135.8 (C-3’), 135.0 (d, JC,P = 7.7 Hz, 2x Cq-Bn), 130.5 (d,
5
4JC,P = 5.3 Hz, 4x CH-Bn), 127.1 (C-2’), 122.8 (d, JC,P = 2.5 Hz, 4x CH-
Bn), 112.1 (C-5), 90.8 (C-1’), 87.3 (d, 3J = 9.1 Hz, C-4’), 70.4 (m, 2x
CH2Ph), 67.9 (d, 2J = 6.1 Hz, C-5’), 35.0 (2x CH2-alkyl), 33.1 (2x CH2-
alkyl), 30.7 (4x CH2-alkyl), 30.6 (2x CH2-alkyl), 30.5 (2x CH2-alkyl), 30.4
(2x CH2-alkyl), 30.2 (2x CH2-alkyl), 26.0 (2x CH2-alkyl), 23.7 (2x CH2-
alkyl), 14.4 (CH3-alkyl), 12.5 (CH3). 31P NMR (243 MHz, CD3OD): δ = -
2
2
11.8 (d, J = 19.7 Hz), -13.3 (d, J = 17.8 Hz), -23.9 (t, 18.8 Hz). HRMS-
ESI-: m/z calcd for C48H70N2O17P3 (M-H)-: 1039.3893; found: 1039.3864.
3
1.9 Hz, 2x Cq-Bn), 152.3 (Cq-4), 139.7 (C-3‘), 138.5 (C-8), 134.9 (d, JC,P
Acknowledgements
= 7.3 Hz, 2x Cq-Bn), 130.5 (C-2‘), 130.4 (d, J = 4.1 Hz, 4x CH-Bn), 122.8
(d, J = 1.9 Hz, 4x CH-Bn) 115.2 (Cq-5), 70.3 (d, 2JC,P = 6.3 Hz, 2x CH2Ph),
69.1 (d, 2J = 7.0 Hz, C-6’), 61.4 (C-1‘), 47.5 (d, 3J = 9.1 Hz, C-4’), 35.0
(2x CH2-alkyl), 34.8 (C-5‘), 33.1 (2x CH2-alkyl), 30.7 (4x CH2-alkyl), 30.6
(2x CH2-alkyl), 30.5 (2x CH2-alkyl), 30.4 (2x CH2-alkyl), 30.2 (2x CH2-
alkyl), 26.0 (2x CH2-alkyl), 23.7 (2x CH2-alkyl), 14.4 (2x CH3-alkyl). 31P
NMR (243 MHz, CD3OD): δ = -11.3 (d, 2J = 19.5 Hz), -13.2 (d, 2J =
16.9 Hz), -23.6 (dd, 2J = 19.5 Hz, 16.9 Hz). HRMS-ESI-: m/z calcd for
C49H71N5O15P3 (M-H)-: 1062.4165; found: 1062.4157.
The authors are grateful to Universität Hamburg for financial
support.
Keywords: Abacavir • Carbovir • carbocyclic • TriPPPro •
triphosphate
References:
TriPPPro-ABCTP (ammonium salt) (21)
[1]
[2]
A. Ruiz-Sancho, J. Sheldon, V. Soriano, Expert Opin. Biol. Ther.
2007, 7, 751–761.
The reaction was carried out with pyrophosphate 17 (429 mg, 431 µmol)
in CH3CN (6 ml) and TFAA (306 µL, 2.15 mmol) and triethylamine
(478 µL, 3.45 mmol) in CH3CN (4 mL). Afterwards triethylamine (299 µL,
2.15 mmol) and 1-methylimidazole (103 µL, 1.29 mmol) in CH3CN (4 mL)
were used. Then THF (6 mL) was added followed by monophosphate 14
(183 mg, 215 µmol) in DMF (2 mL). Yield: 21 (93.1 mg, 38%; purity:
>95%) as a colourless cotton. 1H NMR (600 MHz, DMSO-d6): δ = 7.78j
(s, 1H, H-8), 7.39 (d, 4H, 3J = 8.4 Hz, H-Bn), 7.26 (br s, 3H, NH, NH2),
7.04 (d, 4H, 3J = 8.4 Hz, H-Bn) 6.06-6.02 (m, 1H, H-3’), 5.78-5.72 (m, 1H,
H-2’), 5.42-5.34 (m, 1H, H-1’), 5.07 (d, 4H, 3J = 7.4 Hz, CH2Ph), 3.95-
3.840 (m, 2H, H-6’), 3.01-2.94 (m, 1H, H-4’), 2.61-2.51 (m, 6H, H-5a’,
CH-cyclopropyl, CH2-alkyl), 1.76-1.67 (m, 1H, H-5b’), 1.65-1.58 (m, 4H,
G. S. Bisacchi, S. T. Chao, C. Bachard, J. P. Daris, S. Innaimo, G. A.
Jacobs, O. Kocy, P. Lapointe, A. Martel, Z. Merchant, W. S.
Slusarchyk, J. E. Sundeen, M. G. Young, R. Colonno, R. Zahler,
Bioorg. Med. Chem. Lett. 1997, 7, 127–132.
[3]
S. Levine, D. Hernandez, G. Yamanaka, S. Zhang, R. Rose, S.
Weinheimer, R. J. Colonno, Antimicrob. Agents Chemother. 2002,
46, 2525–32.
[4]
[5]
Q. Choo, G. Kuo, A. Weiner, L. Overby, D. Bradley, M. Houghton,
Science 1989, 244, 359–362.
3
CH2-alkyl), 1.39-1.17 (m, 32H, CH2-alkyl), 0.85 (t, 6H, J = 6.9 Hz, CH3-
Y. Tanaka, N. Nishida, M. Sugiyama, M. Kurosaki, K. Matsuura, N.
Sakamoto, M. Nakagawa, M. Korenaga, K. Hino, S. Hige, Y. Ito, E.
Mita, E. Tanaka, S. Mochida, Y. Murawaki, M. Honda, A. Sakai, Y.
Hiasa, S. Nishiguchi, A. Koike, I. Sakaida, M. Imamura, K. Ito, K.
Yano, N. Masaki, F. Sugauchi, N. Izumi, K. Tokunaga, M. Mizokami,
Nat. Genet. 2009, 41, 1105–1109.
alkyl), 0.70-0.56 (m, 4H, 2x CH2-cyclopropyl). 13C NMR (152 MHz,
DMSO-d6): δ = 171.7 (2x Cq-acyl), 150.1 (2x Cq-Bn), 137.7 (C-3‘),133.9
3
(d, JC,P = 8.0 Hz, 2x Cq-Bn), 129.9 (C-2‘), 129.0 (4x CH-Bn), 121.6 (4x
2
CH-Bn), 67.8 (d, JC,P = 5.4 Hz, 2x CH2Ph), 67.1 (C-6’), 58.9 (C-1‘), 45.7
(d, 3J = 7.7 Hz, C-4’), 33.4 (2x CH2-alkyl), 33.2 (C-5‘), 31.3 (2x CH2-alkyl),
29.0 (4x CH2-alkyl), 28.9 (2x CH2-alkyl), 28.7 (2x CH2-alkyl), 28.7 (2x
CH2-alkyl), 28.4 (2x CH2-alkyl), 24.3 (2x CH2-alkyl), 22.1 (2x CH2-alkyl),
13.9 (2x CH3-alkyl), 6.55 (2x CH2-cyclopropyl). (Cq-6, Cq-2, Cq-4, C-8, Cq-
5 and CH-cyclopropyl) could not be detected due to micelle formation.
31P NMR (243 MHz, DMSO-d6): δ = -12.2 (d, 2J = 20.3 Hz), -12.9 (d, 2J =
[6]
[7]
[8]
M. J. Sofia, Antiviral Res. 2014, 107, 119–124.
A. J. Muir, Am. J. Gastroenterol. 2014, 109, 628–635.
E. Lawitz, A. Mangia, D. Wyles, M. Rodriguez-Torres, T. Hassanein,
S. C. Gordon, M. Schultz, M. N. Davis, Z. Kayali, K. R. Reddy, I. M.
Jacobson, K. V. Kowdley, L. Nyberg, M. Subramanian, R. H. Hyland,
S. Arterburn, D. Jiang, J. McNally, D. Brainard, W. T. Symonds, J. G.
McHutchison, A. M. Sheikh, Z. Younossi, E. J. Gane, N. Engl. J.
Med. 2013, 368, 1878–1887.
18.7 Hz), -23.9 (t, 2J
=
16.2 Hz). HRMS-ESI-: m/z calcd for
C52H76N6O14P3 (M-H)-: 1101.4638; found: 1101.4612.
TriPPPro-d4TTP (ammonium salt)
The reaction was carried out with pyrophosphate 17 (169 mg, 170 µmol)
in CH3CN (3 ml) and TFAA (120 µL, 846 µmol) and triethylamine (189 µL,
1.36 mmol) in CH3CN (2 mL). Afterwards triethylamine (118 µL,
851 µmol) and 1-methylimidazole (41 µL, 0.51 mmol) in CH3CN (3 mL)
[9]
E. De Clercq, Nat. Rev. Drug Discov. 2002, 1, 13–25.
M. J. Sofia, D. Bao, W. Chang, J. Du, D. Nagarathnam, S.
Rachakonda, P. G. Reddy, B. S. Ross, P. Wang, H.-R. Zhang, S.
[10]
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800361
ChemMedChem
FULL PAPER
Bansal, C. Espiritu, M. Keilman, A. M. Lam, H. M. Micolochick
1649–1652.
Steuer, C. Niu, M. J. Otto, P. A. Furman, J. Med. Chem. 2010, 53,
7202–7218.
[38]
[39]
[40]
M. T. Crimmins, B. W. King, J. Org. Chem. 1996, 61, 4192–4193.
M. T. Crimmins, W. J. Zuercher, Org. Lett. 2000, 2, 1065–1067.
S. M. Daluge, M. T. Martin, B. R. Sickles, D. A. Livingston, Nucl.
Nucl. Nucl. Acids 2000, 19, 297–327.
[11]
Y. Mehellou, H. S. Rattan, J. Balzarini, J. Med. Chem. 2018, 61,
2211–2226.
[12]
[13]
[14]
[15]
C. Meier, European J. Org. Chem. 2006, 2006, 1081–1102.
C. Meier, Antivir. Chem. Chemother. 2017, 25, 69–82.
E. De Clercq, Nat. Rev. Drug Discov. 2007, 6, 1001–1018.
Y. H. Yeom, R. P. Remmel, S. H. Huang, M. Hua, R. Vince, C. L.
Zimmerman, Antimicrob. Agents Chemother. 1989, 33, 171–175.
W. B. Parker, S. C. Shaddix, R. Vince, L. L. Bennett, Antiviral Res.
1997, 34, 131–136.
[41]
[42]
T. Sowa, S. Ouchi, Bull. Chem. Soc. Jpn. 1975, 48, 2084–2090.
T. Schulz, J. Balzarini, C. Meier, ChemMedChem 2014, 9, 762–775.
[16]
[17]
[18]
[19]
[20]
R. Vince, J. Brownell, S. A. Beers, Nucleosides and Nucleotides
1995, 14, 39–44.
L. L. Bondoc, W. M. Shannon, J. A. Secrist, R. Vince, A. Fridland,
Biochemistry 1990, 29, 9839–9843.
P.-T. Pham, R. Vince, Phosphorus. Sulfur. Silicon Relat. Elem. 2007,
182, 779–791.
R. Vince, J. Kilama, P. T. Pham, S. A. Beers, B. J. Bowdon, K. A.
Keith, W. B. Parker, Nucleosides and Nucleotides 1995, 14, 1703–
1708.
[21]
[22]
R. Vince, M. Hua, J. Brownell, S. Daluge, F. Lee, W. M. Shannon, G.
C. Lavelle, J. Qualls, O. S. Weislow, R. Kiser, P. G. Canonico, R. H.
Schultz, V. L. Narayanan, J. G. Mayo, R. H. Shoemaker, M. R. Boyd,
Biochem. Biophys. Res. Commun. 1988, 156, 1046–1053.
W. B. Parker, S. C. Shaddix, L. M. Rose, P. T. Pham, M. Hua, R.
Vince, Nucleosides, Nucleotides and Nucleic Acids 2000, 19, 795–
804.
[23]
[24]
[25]
R. Vince, M. Hua, J. Med. Chem. 1990, 33, 17–21.
S. Daluge, R. Vince, J. Org. Chem. 1978, 43, 2311–2320.
A. R. Hughes, W. R. Spreen, M. Mosteller, L. L. Warren, E. H. Lai, C.
H. Brothers, C. Cox, A. J. Nelsen, S. Hughes, D. E. Thorborn, B.
Stancil, S. Hetherington, D. Burns, A. Roses, Pharmacogenomics J.
2008, 8, 365–374.
[26]
[27]
S. M. Daluge, S. S. Good, M. B. Faletto, W. H. Miller, M. H. St. Clair,
L. R. Boone, M. Tisdale, N. R. Parry, J. E. Reardon, R. E. Dornsife,
et al., Antimicrob. Agents Chemother. 1997, 41, 1082–1093.
M. B. Faletto, W. H. Miller, E. P. Garvey, M. H. St. Clair, S. M.
Daluge, S. S. Good, Antimicrob. Agents Chemother. 1997, 41,
1099–1107.
[28]
[29]
[30]
[31]
T. Gollnest, T. Dinis de Oliveira, A. Rath, I. Hauber, D. Schols, J.
Balzarini, C. Meier, Angew. Chem. Int. Ed. Engl. 2016, 55, 5255–8.
T. Gollnest, T. D. De Oliveira, D. Schols, J. Balzarini, C. Meier, Nat.
Commun. 2015, 6, DOI 10.1038/ncomms9716.
L. Santana, M. Teijeira, E. Uriarte, J. Balzarini, E. De Clercq, Nucl.
Nucl. Nucl. Acids 1995, 14, 521–523.
O. R. Ludek, T. Krämer, J. Balzarini, C. Meier, Synthesis 2006,
2006, 1313–1324.
[32]
[33]
[34]
V. E. Marquez, M.-I. Lim, Med. Res. Rev. 1986, 6, 1–40.
A. D. Borthwick, K. Biggadike, Tetrahedron 1992, 48, 571–623.
K. Biggadike, A. D. Borthwick, D. Evans, A. M. Exall, B. E. Kirk, S.
M. Roberts, L. Stephenson, P. Youds, J. Chem. Soc. Perkin Trans.
1 1988, 549-554.
[35]
[36]
S. Weising, I. Torquati, C. Meier, Synthesis 2018, 50, 1264–1274.
S. Weising, P. Dekiert, D. Schols, J. Neyts, C. Meier, Synthesis
2018, 50, 2266–2280.
[37]
M. Asami, J. Takahashi, S. Inoue, Tetrahedron: Asymmetry 1994, 5,
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800361
ChemMedChem
FULL PAPER
Entry for the Table of Contents
One option to intracellularly release a nucleoside triphosphate is given by the TriPPPro-approach. In the case of Carbovir and
Abacavir their corresponding TriPPPro-derivatives had a 3.5-fold and 4.5-fold, respectively, superior antiviral activity compared to the
parent nucleosides. In the case of 1’,2’-cis-derivatives of Carbovir and Abacavir neither the nucleosides nor their TriPPPro-derivatives
showed any antiviral activity.
9
This article is protected by copyright. All rights reserved.