Synthesis and Anti-HIV Activity of Guanine Modified Fluorinated Acyclic Nucleoside Phosphonate Derivatives

Article abstract of DOI:10.1002/cbdv.201800532

The preparation of an unprecedented series of nucleobase modified 3-fluoro-2-(phosphonomethoxy)propyl (FPMP) acyclic nucleosides in both their (R) and (S) enantiomerically pure forms is described. The synthesis focuses on a Mitsunobu alkylation reaction t

Full text of DOI:10.1002/cbdv.201800532

Title: Synthesis and anti-HIV activity of guanine modified fluorinated
acyclic nucleoside phosphonates derivatives
Authors: Min Luo, Elisabetta Groaz, Steven De Jonghe, Dominique
Schols, and Piet Herdewijn
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10.1002/cbdv.201800532
Chemistry & Biodiversity
Synthesis and anti-HIV activity of guanine modified fluorinated acyclic nucleoside
phosphonates derivatives
Min Luo,a Elisabetta Groaz,a Steven De Jonghe,b Dominique Schols,b and Piet Herdewijn*a
aMedicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49, 3000
Leuven, Belgium, e-mail: piet.herdewijn@kuleuven.be
bLaboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven,
Herestraat 49 bus 1043, 3000 Leuven, Belgium
Abstract
The preparation of an unprecedented series of nucleobase modified 3-fluoro-2-
(phosphonomethoxy)propyl (FPMP) acyclic nucleosides in both their (R) and (S)
enantiomerically pure forms is described. The synthesis focuses on a Mitsunobu alkylation
reaction to construct the C-N9 bond between a chiral fluorinated side-chain residue and 6- or
7-modified guanine analogues. Prodrugs of FPMP-7-deazaguanine were also synthesized by
derivatization of the corresponding phosphonic acid functionality with (bis)diamyl aspartate
amidate groups, leading to moderate activity against human immunodeficiency virus type 1
(HIV-1).
Keywords
acyclic nucleoside phosphonates, Mitsunobu reaction, prodrugs, HIV, antiviral activity
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10.1002/cbdv.201800532
Chemistry & Biodiversity
Introduction
Nucleoside analogues belonging to the family of acyclic nucleoside phosphonates (ANPs) have
proven to be a particularly valuable source of potent antiviral agents with a broad spectrum
antiviral activity, especially against DNA viruses and retroviruses.[1] The chemical structure
common to all ANPs includes a nucleobase attached to an aliphatic side chain enclosing a
phosphonomethoxy (P-C-O) residue. The presence of such motif imparts a greater metabolic
stability to the resulting molecules compared to the naturally occurring isosteric
phosphoxymethyl functionality (P-O-C).[2] Indeed, contrary to phosphates, the phosphonate
moiety is not susceptible to phosphodiesterase and phosphatase hydrolysis, but can likewise
undergo enzymatic phosphorylation.
3-Fluoro-2-(phosphonomethoxy)propyl (FPMP) derivatives are a subclass of ANPs
characterized by the presence of a fluorine atom in the acyclic side chain,[3] which exhibited
moderate antiviral activity limited to HIV and HBV.[4, 5] Up to now, limited efforts have been
done on the synthesis and antiviral screening of FPMP derivatives with non-canonical
nucleobases, with the exception of a FPMP analogue bearing a 2,6-diaminopurine (DAP)
moiety (Figure 1).[4]
Figure 1. Examples of purine modified acyclic nucleoside analogues relevant to this study.
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10.1002/cbdv.201800532
Chemistry & Biodiversity
Here, we describe an unprecedented combination of structural variations obtained by
reacting under Mitsunobu conditions a chiral fluorinated acyclic pseudosugar with 6- and 7-
modified guanine analogues. The target functionalized purine moieties were chosen based on
the knowledge available in the literature from structure-activity studies. For instance, 6-arylthio
substituted guanine containing acyclic nucleoside analogues have been described as potent
antiviral agents against HBV (Figure 1).[6] The presence of a 7-deazapurine nucleobase
(systematic name: pyrrolo[2,3-d]pyrimidine) often confers antiviral activity to cyclic
nucleosides,[7-12] and a 7-deazaguanine acyclic nucleoside that inhibits HIV replication (IC50 =
3 µM) has been reported (Figure 1).[13]
Thus, 2-amino-6-arylthiopurine and 7-deazaguanine were selected as prime nucleobase
candidates for the synthesis and anti-HIV evaluation of base modified FPMPs. Additional 7-
deazaguanine analogues bearing an electron-withdrawing substituent at the C7 position were
also investigated in order to determine the effect of such modification on the antiviral activity
of the resulting FPMP derivatives as well as the efficiency of the key Mitsunobu alkylation.
Results and discussion
Chemistry
The chiral pool strategy has been frequently applied in the synthesis of ANPs.[14] Various types
of reactions are possible (alkylation, epoxide ring opening, Mitsunobu condensation) between
the heterocyclic base moiety and a chiral side-chain residue, which is preferably
prefunctionalized with an esterified phosphonate functionality. Recently, we and others have
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10.1002/cbdv.201800532
Chemistry & Biodiversity
employed phosphonate esters of fluorohydrin precursors 2a/b (Scheme 1) as convenient chiral
starting materials in the synthesis of enantiomerically pure FPMPs bearing natural
nucleobases.[15, 16] Likewise, 6-chloropurine 1 was reacted under Mitsunobu conditions (Ph3P,
DIAD) with precursors 2a/b to provide compounds 3a/b along with the concomitant formation
of the corresponding triphenylphosphine adducts. However, by refluxing the crude reaction
mixtures for 24 h in water, the undesired adducts were successfully hydrolyzed leading to the
isolation of compounds 3a/b in excellent yields over two steps. The replacement of the chlorine
atom at the 6-position of the nucleobase with an arylthio group was achieved by treatment of
3a/b with 4-methoxythiophenol in the presence of triethylamine in DMF, affording 4a/b in 87
and 67% yield, respectively. TMSBr-promoted hydrolysis of the phosphonate diester group
furnished target 2-amino-6-methoxyphenylthio purine containing acyclic nucleoside
phosphonates 5a/b in good yields (Scheme 1).
Scheme 1. Synthesis of (R)/(S)-fluorinated acyclic phosphonate acids 5a/b. Reagents and
conditions: (a) Ph3P, DIAD, THF, rt, 24 h; (b) THF/H2O, reflux, 24 h; (c) Et3N, DMF, 100 °C,
4 h; (d) TMSBr, 2,6-lutidine, CH3CN, rt, 12 h.
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10.1002/cbdv.201800532
Chemistry & Biodiversity
Subsequent efforts were directed towards the synthesis of FPMPs bearing 7-deazaguanine
and its 7-substituted structural analogues. Commercially available 2-amino-6-chloro-7-
deazapurine 6 was protected as a pivaloyl amide to afford compound 7,[17] which in turn served
as substrate for the preparation of 4-chloro-5-fluoro-2-pivaloylamino-7H-pyrrolo[2,3-
d]pyrimidine 8, as illustrated in Scheme 2. Specifically, Selectfluor was preferred as
fluorinating reagent based on a method described by Chittepu et al.[18] Under the previously
established condensation conditions (Ph3P, DIAD), nucleobases 7 and 8 were coupled with 2a/b
to provide two enantiomeric sets consisting of nucleoside phosphonate diethyl esters 9a/b and
12a/b. The presence of fluorine at the 7-position led to lower yields of isolated products (25-
33%) compared to those of their unsubstituted counterparts 9a/b (78-79%). Next, compounds
10a/b and 13a/b were obtained from 9a/b and 12a/b, respectively, after the one-pot sequential
displacement of the chlorine atom by a hydroxyl group in the presence of DABCO/K2CO3,
followed by the removal of the pivaloyl group upon methanolic ammonia treatment. After
standard removal of the phosphonate ester groups, the fluorinated acyclic phosphonate acids
11a/b and 14a/b were obtained in 50-70% yields.
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10.1002/cbdv.201800532
Chemistry & Biodiversity
Scheme 2. Synthesis of (R)/(S)-fluorinated acyclic phosphonate acids 11a/b and 14a/b.
Reagents and conditions: (a) PivCl, Pyr, rt, 3 h; (b) Selectfluor, MeCN, 50 °C, 30 min; (c) Ph3P,
DIAD, 2a/b, THF, rt, 24 h; (d) (i) DABCO, K2CO3, dioxane/H2O, 90 °C, 3 h; (ii) NH3/MeOH,
rt, 12 h; (e) TMSBr, 2,6-lutidine, CH3CN, rt, 12 h.
The heterocyclic building block 19 was available in four steps from chloroacetonitrile 15
according to a literature procedure (Scheme 3).[19] Formylation of 15 with methyl formate
resulted in the formation of the unstable chloroaldehyde 16, which was immediately used in the
next step without further purification to afford the cyclocondensation product 17. Then, the
exocyclic amino group of compound 17 was protected using pivaloyl chloride, while the
subsequent chlorination was accomplished with POCl3 in the presence of a phase transfer
catalyst to give the desired chloro-intermediate 19. Starting from 2a/b, the base condensation
under Mitsunobu conditions occurred smoothly to yield nucleoside phosphonates 20a/20b.
Similar to the low yield observed for 13a/b, the catalytic hydrolysis of 20a to 21a in the
presence of DABCO/K2CO3 proceeded also in poor yield,[20] however this step could be
improved by switching to DABCO/NaOAc,[21] which allowed to access the desired products
21a/b in good yield (60% over 2 steps). Cleavage of the phosphonate esters in the presence of
the TMSBr afforded 22a/b in 70 and 75% yield, respectively.
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10.1002/cbdv.201800532
Chemistry & Biodiversity
Scheme 3. Synthesis of (R)/(S)-fluorinated acyclic phosphonate acids 22a/b. Reagents and
conditions: (a) methyl formate, NaH, THF, 0 °C, 5 h; (b) 2,6-diamino-6-hydroxypyrimidine,
NaOAc, H2O, 100 °C, 24 h; (c) PivCl, Pyr, 85 °C, 2 h; (d) POCl3, DMA, BnEt3NCl, MeCN,
90 °C, 1 h; (e) Ph3P, DIAD, 2a/b, THF, rt, 24 h; (f) (i) DABCO, K2CO3, dioxane/H2O, 90 °C, 3
h or DABCO, NaOAc, DMF, rt, 48 h; (ii) NH3/MeOH, rt, 12 h; (g) TMSBr, 2,6-lutidine, CH3CN,
rt, 12 h.
Recently, we disclosed that the prodrug derivatization of the phosphonic acid functionality
of FPMPs with a diamyl aspartate phenoxyamidate group not only produced a dramatic
improvement in antiretroviral potency but also led to compounds endowed with an
unprecedented spectrum of activity against DNA viruses.[15] Therefore, the amyl aspartate
phosphonoamidate and phosphonobisamidate prodrugs 23 and 24 were prepared from the
parent 7-deazaguanine derived FPMP nucleoside (R)-11a in moderate yields using a common
procedure, which utilizes 2,2’-dithiodipyridine and triphenylphosphine as activating agents
(Scheme 4). Compound 23 was isolated as a diastereomeric mixture (ratio: 1/1.4), which was
biologically tested as such.
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10.1002/cbdv.201800532
Chemistry & Biodiversity
Scheme 4. Synthesis of (R)-fluorinated acyclic phosphonamidates 23 and 24. Reagents and
conditions: (a) L-aspartic acid amyl ester HCl salt, PhOH, Et3N, 2,2'-dithiodipyridine, PPh3,
Pyr, 60 °C, 12 h; (b) L-aspartic acid amyl ester HCl salt, Et3N,2,2'-dithiodipyridine, PPh3, Pyr,
60 °C, 12 h.
Anti-HIV activity
All base modified FPMPs 5a/b, 11a/b, 14a/b, and 22a/b as well as two prodrugs, compounds
23 and 24, were evaluated for antiviral activity by luciferase assay in TZM-bl cells[22] infected
with the wild type HIV-1 strains NL4.3 (CXCR4-tropic HIV-1 strain) and BaL (CCR5-tropic
HIV-1 strain). In parallel, their possible toxic effects on the same cell line were also assessed.
Tenofovir (PMPA), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF)
were included as positive controls. Disappointingly, all synthesized base-modified FPMPs
completely lacked anti-HIV activity, as indicated by their EC50 values higher than 100 µM.
However, the fluorinated acyclic aryloxyphosphonomonoamidate 23 and the
phosphonodiamidate prodrug 24 displayed a significant improvement of the anti-HIV activity
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10.1002/cbdv.201800532
Chemistry & Biodiversity
with EC50 values in the 2-10 µM range compared to the parent (R)-FPMP-7-deazaguanine
nucleoside phosphonate 11 (Table 1), most likely due to their increased lipophilicity and
consequential improved cellular permeability. Such prodrugs were also tested for anti-HIV
activity in peripheral blood mononuclear cells (PBMCs), exhibiting moderate anti-HIV-1
activity against CXCR4-using (X4) NL4.3 with IC50 values of 4.80 and 1.81 µM, respectively,
whereas being endowed with weak antiviral activity against the CCR5-using (R5) BaL strain.
However, for both cell lines, the increased cytotoxicity of such prodrugs in combination with
their weak antiviral activity gave rise to unfavorable selectivity indexes.
Table 1. Anti-HIV-1 activity and cytotoxicity of prodrugs 23 and 24 in TZM-bl cells and
(PHA)-stimulated PBMCs.
EC50a (µM) in TZM-bls
HIV-1 X4
EC50a (µM) in PBMCs
HIV-1 X4
b
CC50
(µM)
Compound
CC50b (µM)
HIV-1 R5
HIV-1 R5
(BaL)
7.95
(BaL)
> 20
(NL4.3)
9.28
(NL4.3)
4.80
23
44.35
8.94
31.8
24
> 4
2.62
1.81
> 4
6.86
> 25
> 5
PMPA
TDF
TAF
2.38
0.027
0.003
3.13
>100
39.97
82.75
0.49
1.69
0.02
0.0023
0.0007
0.068
0.0024
0.0033
3.27
[a]
[b]
Compound concentration required to reduce HIV-induced cytopathicity by 50%.
Compound concentration required to reduce cell viability by 50%.
Conclusions
Both enantiomeric series of 3-fluoro-2-(phosphonomethoxy)propyl acyclic nucleoside
phosphonates bearing modified nucleobases including 2-amino-6-arylthiopurine, 7-
deazaguanine and its analogues were synthesized. The presence of an electron-withdrawing
substituent at the C7 position of 7-deazaguanine was found to reduce the efficiency of the
Mitsunobu alkylation. In addition, two representative L-aspartic acid (di)amyl ester
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10.1002/cbdv.201800532
Chemistry & Biodiversity
phosphonoamidate prodrugs of (R)-FPMP-7-deazaguanine were also prepared. All compounds
were tested for their antiviral activity against HIV-1. While the parent phosphonates were
completely devoid of anti-HIV activity, prodrugs 23 and 24 were endowed with moderate
antiviral activity, with EC50 values in the low micromolar range against HIV-1 in both TZM-bl
cells and (PHA)-stimulated PBMCs.
Experimental Section
Unless otherwise specified, all reagents and solvents were obtained from commercial sources
and used as received. NMR spectra were measured on a Bruker Avance 300, 500, or 600 MHz
spectrometer using tetramethylsilane as internal standard or the residual solvent as internal
31
standard, and 85% H3PO4 for P NMR experiments. The numbering system utilized in the
assignment of the NMR peaks is illustrated in Figure 2. High resolution mass spectra (HRMS)
were recorded on a quadruple orthogonal acceleration time-of-flight mass spectrometer (Synapt
G2 HDMS, Waters, Milford, MA). Reactions were monitored by thin-layer chromatography on
silica gel (pre-coated aluminum sheets, 254 nm). Column chromatography was performed on
silica gel (60 Å, 0.035−0.070 mm, Acros Organics). Preparative reverse phase HPLC
purification was performed on a Phenomenex Gemini 110A column (C18, 10 μm, 21.2 mm ×
250 mm, 5 or 8 mL/min) using H2O/CH3CN containing 50 mmol TEAB or H2O/CH3CN as
eluent gradient. The purity of all final compounds was determined to be >95% by analytic
HPLC analysis.
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10.1002/cbdv.201800532
Chemistry & Biodiversity
Figure 2. General numbering system for the assignment of NMR peaks.
(R)-2-Amino-6-chloro-9-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}purine (3a).
To a solution of 1 (120 mg, 0.74 mmol), 2a (150 mg, 0.61 mmol), and Ph3P (300 mg, 1.20
mmol) in anhydrous THF (5 mL) was added DIAD (0.24 mL, 1.20 mmol) dropwise at room
temperature. The reaction mixture was stirred for 24 h, then H2O (5 mL) was added. The
resulting mixture was refluxed for 24 h in order to remove the triphenylphosphine adducts. The
solvent was removed under reduced presseure, and the crude residue was purified by silica gel
chromatography (DCM/MeOH, 50:1 to 25:1, v/v) to give 3a (200 mg). Colorless oil. Yield: 83%
1H NMR (300 MHz, CDCl3): 7.91 (s, H-C(8)), 5.50 (s, NH2), 4.71-4.35 (m, H-C(3′), Ha-C(1′)),
4.26-3.92 (m, Hb-C(1′), H-C(2′), 2 x CH2CH3, Ha-C(4′)), 3.83 (dd, J = 13.9, 8.8, Hb-C(4′)), 1.35-
1.26 (m, 2 x CH3); 13C NMR (75 MHz, CDCl3): 159.5 (C(2)), 154.1 (C(4)), 151.5 (C(6)), 143.5
(C(8)), 125.1 (C(5)), 82.0 (d, J = 173.9, C(3′)), 78.4 (dd, J = 19.5, 9.6, C(2′)), 64.7 (d, J = 167.1
Hz, C(4′)), 62.9, 62.8 (CH2CH3), 43.8 (d, J = 8.0 Hz, C(1′)), 16.7, 16.6 (CH3); 31P NMR (121
MHz, CDCl3): 20.5; HR-MS: 396.1002 ([M+H]+, C13H21ClFN5O4P+, calc. 396.0998).
(S)-2-Amino-6-chloro-9-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}purine (3b).
Compound 3b was synthesized according to the procedure used for the preparation of 3a,
starting from 1 (250 mg, 1.47 mmol), 2b (300 mg, 1.23 mmol), Ph3P (640 mg, 2.46 mmol), and
DIAD (0.48 mL, 2.46 mmol) in anhydrous THF (10 mL). The crude residue was purified by
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10.1002/cbdv.201800532
Chemistry & Biodiversity
silica gel chromatography (DCM/MeOH, 50:1 to 25:1, v/v). Colorless oil. Yield: 70%. 1H NMR
(300 MHz, CDCl3): 7.90 (s, H-C(8)), 5.40 (s, NH2), 4.71-4.34 (m, H-C(3′), Ha-C(1′)), 4.25-3.79
13
(m, Hb-C(1′), H-C(2′), 2 x CH2CH3, H-C(4′)), 1.35-1.24 (m, 2 x CH3); C NMR (75 MHz,
CDCl3): 159.5 (C(2)), 154.2 (C(4)), 151.5 (C(6)), 143.6 (C(8)), 125.1 (C(5)), 82.0 (d, J = 173.7,
C(3′)), 78.4 (dd, J = 19.7, 9.5, C(2′)), 64.8 (d, J = 167.0, C(4′)), 62.9, 62.8 (CH2CH3), 43.8 (d,
J = 7.8, C(1′)), 16.7, 16.6 (CH3); 31P NMR (121 MHz, CDCl3): 20.5; HR-MS: 396.0998
([M+H]+, C13H21ClFN5O4P+, calc. 396.0998).
(R)-2-Amino-6-(4-methoxyphenylthio)-9-{3-fluoro-2-
[(diethylphosphoryl)methoxy]propyl}purine (4a). 4-Methoxythiophenol (0.12 mL, 0.96
mmol) was added to a mixture of 3a (190 mg, 0.48 mmol) and triethylamine (0.07 mL, 0.48
mmol) in anhydrous DMF (10 mL) at room temperature, and the mixture was stirred at 100 °C
for another 4 h. The solution was diluted with EtOAc (100 mL) and washed with saturated aq.
NaHCO3 (50 mL) and brine (50 mL). The organic layer was dried over Na2SO4 and
concentrated. The residue was then purified by silica gel chromatography (DCM/MeOH, 60:1
to 40:1, v/v) to give 4a (210 mg). Colorless oil. Yield: 87%. 1H NMR (600 MHz, CDCl3): 7.76
(s, H-C(8)), 7.52 (d, J = 8.9, 2 arom. H), 6.94 (d, J = 8.9, 2 arom. H), 4.88 (s, NH2), 4.64-4.38
(m, H-C(3′)), 4.33-4.28 (m, Ha-C(1′)), 4.19-4.04 (m, Hb-C(1′), H-C(2′), 2 x CH2CH3), 3.91 (dd,
J = 13.9, 8.7, 1H, Ha-C(4′)), 3.85-3.77 (m, OCH3, Hb-C(4′)), 1.34-1.27 (m, 2 x CH3); 13C NMR
(150 MHz, CDCl3): 161.7 (C(2)), 160.4 (arom. C), 159.1 (C(6)), 150.9 (C(4)), 141.0 (C(8)),
137.1 (arom. C), 124.6 (C(5)), 118.0 (arom. C), 114.5 (arom. C), 82.0 (d, J = 173.6, C(3′)), 78.3
(dd, J = 19.2, 9.9, C(2′)), 64.5 (d, J = 166.9, C(4′)), 62.5, 62.4 (d, J = 6.4 , CH2CH3), 55.3
(OCH3), 43.1 (d, J = 7.9, C(1′)), 16.4, 16.3 (CH3); 31P NMR (121 MHz, CDCl3): 19.9; HR-MS:
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10.1002/cbdv.201800532
Chemistry & Biodiversity
500.1526 ([M+H]+, C20H28FN5O5PS+, calc. 500.1527).
(S)-2-Amino-6-(4-methoxyphenylthio)-9-{3-fluoro-2-
[(diethylphosphoryl)methoxy]propyl}purine (4b). Compound 4b was synthesized according
to the procedure used for the preparation of 4a, starting from 3b (190 mg, 0.48 mmol), 4-
methoxythiophenol (0.12 mL, 0.96 mmol), and triethylamine (0.07 mL, 0.48 mmol) in
anhydrous DMF (10 mL). The crude residue was purified by silica gel chromatography
(DCM/MeOH, 60:1 to 40:1, v/v). Colorless oil. Yield: 67%.1H NMR (300 MHz, CDCl3): 7.77
(s, H-C(8)), 7.53 (d, J = 8.8, 2 arom. H), 6.95 (d, J = 8.8, 2 arom. H), 5.03 (s, 1H, NH2), 4.70-
4.30 (m, H-C(3′), Ha-C(1′)), 4.22-3.98 (m, Hb-C(1′), H-C(2′), 2 x CH2CH3), 3.93-3.77 (m, H-
C(4′), OCH3), 1.35-1.26 (m, 2 x CH3); 13C NMR (75 MHz, CDCl3): 162.1 (C(2)), 160.9 (arom.
C), 159.0 (C(6)), 151.0 (C(4)), 141.7 (C(8)), 137.5 (arom. C), 124.9 (C(5)), 118.0 (arom. C),
115.0 (arom. C), 82.3 (d, J = 173.7, C(3′)), 78.7 (dd, J = 19.5, 9.9, C(2′)), 64.9 (d, J = 166.9,
C(4′)), 62.9, 62.8 (CH2CH3), 55.6 (OCH3), 43.6 (d, J = 8.1, C(1′)), 16.8, 16.7 (CH3); 31P NMR
(121 MHz, CDCl3): 20.5; HR-MS: 500.1518 ([M+H]+, C20H28FN5O5PS+, calc. 500.1527).
(R)-2-Amino-6-(4-methoxyphenylthio)-9-[3-fluoro-2-(phosphonomethoxy)propyl]
purine (5a). To a solution of 4a (200 mg, 0.40 mmol) and 2,6-lutidine (0.74 mL, 6.40 mmol)
in anhydrous acetonitrile (10 mL) was added bromotrimethylsilane (0.43 mL, 3.20 mmol)
dropwise at 0 °C. The reaction mixture was then set aside in the dark for 12 h at room
temperature. The reaction was quenched with 1.0 M TEAB buffer (1 mL), and the solvent was
removed under reduced pressure. The residue was first purified by silica gel chromatography
(acetone/Et3N/H2O, 6:1:1, v/v/v), followed by further preparative reverse phase HPLC (linear
gradient, 2-98% CH3CN in a 0.05 M TEAB solution) to give 5a (120 mg). White foam. Yield:
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10.1002/cbdv.201800532
Chemistry & Biodiversity
70%. 1H NMR (300 MHz, D2O): 7.93 (s, H-C(8)), 7.19 (d, J = 8.8, 2 arom. H), 6.73-6.70 (m, 2
arom. H), 4.60-4.21 (m, H-C(3′)), 4.18-4.08 (m, H-C(1′)), 3.89-3.81 (m, H-(2′)), 3.61 (s, OCH3),
3.59-3.41 (m, H-(4′)); 13C NMR (75 MHz, D2O): 161.1 (C(2)), 160.0 (arom. C), 159.3 (C(6)),
150.1 (C-4), 142.8 (C-8), 136.8 (arom. C), 123.0 (C(5)), 116.8 (arom. C), 114.8 (arom. C), 82.1
(d, J = 168.0, C(3′)), 77.4 (dd, J = 18.9, 11.2, C(2′)), 66.9 (d, J = 154.8, C(4′)), 55.3 (OCH3),
42.7 (d, J = 7.1, C(1′)); 31P NMR (121 MHz, D2O): 14.1; HR-MS: 442.0752 ([M-H]-,
C16H18FN5O5PS-, calc. 442.0756).
(S)-2-Amino-6-(4-methoxyphenylthio)-9-[3-fluoro-2-(phosphonomethoxy)propyl]
purine (5b). Compound 5b was synthesized according to the procedure used for the preparation
of 5a, starting from compound 4b (150 mg, 0.30 mmol), bromotrimethylsilane (0.32 mL, 2.40
mmol), and 2,6-lutidine (0.56 mL, 4.80 mmol) in anhydrous acetonitrile (10 mL). The crude
residue was purified by silica gel chromatography (acetone/Et3N/H2O, 6:1:1, v/v/v). White
1
foam. Yield: 65%. H NMR (300 MHz, D2O): 7.95 (s, H-C(8)), 7.22 (d, J = 8.4, 2 arom. H),
6.82-6.65 (m, 2 arom. H), 4.63-4.24 (m, H-C(3′)), 4.20-4.10 (m, H-C(1′)), 3.93-3.85 (m, H-
13
(2′)), 3.64 (s, OCH3), 3.61-3.45 (m, H-C(4′)); C NMR (75 MHz, D2O): 161.1 (C(2)), 160.0
(arom. C), 159.3 (C(6)), 150.1 (C(4)), 142.8 (C-8), 136.8 (arom. C), 123.0 (C(5)), 116.8 (arom.
C), 114.8 (arom. C), 82.1 (d, J = 168.1 Hz, C(3′)), 77.5 (dd, J = 18.9, 11.1, C(2′)), 66.7 (d, J =
155.5, C(4′)), 55.3 (OCH3), 42.8 (d, J = 7.1, C(1′)); 31P NMR (121 MHz, D2O): 14.3; HR-MS:
442.0734 ([M-H]-, C16H18FN5O5PS-, calc. 442.0756).
(R)-4-Chloro-7-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}-2-pivaloylamino-
7H-pyrrolo[2,3-d]pyrimidine (9a). DIAD (0.24 mL, 1.20 mmol) was added dropwise to a
mixture of 7[17] (180 mg, 0.74 mmol), 2a (150 mg, 0.61 mmol), and Ph3P (300 mg, 1.20 mmol)
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10.1002/cbdv.201800532
Chemistry & Biodiversity
in anhydrous THF (10 mL) at room temperature. After stirring for 24 h, the solvent was
removed under reduced pressure. The resultant residue was purified by silica gel column
chromatography (DCM/MeOH, 60:1 to 30:1, v/v) to give 9a (220 mg). Colorless oil. Yield:
78%. 1H NMR (300 MHz, CDCl3): 8.16 (s, NHCO), 7.29 (dd, J = 0.9, H-C(6)), 6.53 (dd, J =
3.6, 0.9, H-C(5)), 4.74-4.31 (m, H-C(3′), H-C(1′)), 4.18-3.86 (m, H-C(2′), 2 x CH2CH3, H-
13
C(4′)), 1.36-1.27 (m, (CH3)3C, 2 x CH2CH3); C NMR (150 MHz, CDCl3): 175.7 (NHCO),
152.3 (C(4), C(7a)), 151.6 (C(2)), 130.1 (C(6)), 114.2 (C(4a)), 100.1 (C(5)), 82.9 (d, J = 173.2,
C(3′)), 79.4 (dd, J = 19.1, 10.9, C(2′)), 64.9 (d, J = 166.9, C(4′)), 62.7, 62.6 (CH2CH3), 44.9 (d,
31
J = 8.5, C(1′)), 40.5 (C(CH3)3), 27.7 (C(CH3)3), 16.7, 16.6 (CH2CH3); P NMR (121 MHz,
CDCl3): 20.9; HR-MS: 479.1617 ([M+H]+, C19H30ClFN4O5P+, calc. 479.1621).
(S)-4-Chloro-7-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}-2-pivaloylamino-7H-
pyrrolo[2,3-d]pyrimidine (9b). Compound 9b was synthesized according to the procedure
used for the preparation of 9a, starting from 7[17] (180 mg, 0.74 mmol), 2b (150 mg, 0.61 mmol),
Ph3P (300 mg, 1.20 mmol), and DIAD (0.24 mL, 1.20 mmol) in anhydrous THF (10 mL). The
crude residue was purified by silica gel chromatography (DCM/MeOH, 60:1 to 30:1, v/v).
Colorless oil. Yield: 79%. 1H NMR (600 MHz, CDCl3): 8.13 (s, NHCO), 7.30 (dd, J = 3.6, 2.0,
H-C(6)), 6.53 (dd, J = 3.6, 2.5, H-C(5)), 4.69-4.59 (m, Ha-C(3′)), 4.55-4.51 (m, Ha-C(1′)), 4.46-
4.33 (m, Hb-C(3′), Hb-C(1′)), 4.15-4.08 (m, H-C(2′), 2 x CH2CH3), 4.00-3.96 (m, Ha-C(4′)),
3.92-3.88 (m, Hb-C(4′)), 1.35-1.29 (m, (CH3)3C, 2 x CH2CH3); 13C NMR (150 MHz, CDCl3):
175.5 (NHCO), 152.1 (C(4), C(7a)), 151.3 (C(2)), 129.8 (C(6)), 113.9 (C(4a)), 99.8 (C(5)), 82.5
(d, J = 173.0, C(3′)), 79.1 (dd, J = 19.0, 11.1, C(2′)), 64.5 (d, J = 167.2, C(4′)), 62.5, 62.4
(CH2CH3), 44.5 (d, J = 8.5, C(1′)), 40.2 (C(CH3)3), 27.4 (C(CH3)3), 16.4 (CH2CH3); 31P NMR
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10.1002/cbdv.201800532
Chemistry & Biodiversity
(121 MHz, CDCl3): 20.9; HR-MS: 479.1624 ([M+H]+, C19H30ClFN4O5P+, calc. 479.1621).
(R)-2-Amino-3,4-dihydro-7-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}-7H-
pyrrolo[2,3-d]pyrimidin-4-one (10a). Asolution of 9a (200 mg, 0.42 mmol), DABCO (46 mg,
0.42 mmol), and K2CO3 (58 mg, 0.42 mmol) in a mixture of dioxane and H2O (6 mL, 5:1, v/v)
was stirred at 90 °C for 3 h. The solvent was then removed under reduced pressure to give crude
residue that was redissolved in satd NH3 in MeOH (20 mL) and stirred at room temperature for
12 h. After removal of all the volatiles, the residue was purified by silica gel chromatography
1
(DCM/MeOH, 20:1 to 10:1, v/v) to give 10a (100 mg). Colorless oil. Yield: 64%. H NMR
(300 MHz, CDCl3): 11.01 (s, NHCO), 6.59 (dd, J = 3.5, 0.9, H-C(6)), 6.50 (dd, J = 3.5, 0.9, H-
C(5)), 6.23 (s, NH2), 4.67-4.28 (m, H-C(3′)), 4.23-4.10 (m, H-C(1′), 2 x CH2CH3), 4.05-3.91
(m, H-C(2′), Ha-C(4′)), 3.85-3.77 (m, Hb-C(4′)), 1.37-1.31 (m, 2 x CH3); 13C NMR (75 MHz,
CDCl3): 160.7 (C(4)), 152.8 (C(2)), 151.1 (C(7a)), 121.6 (C(6)), 102.6 (C(4a)), 101.3 (C(5)),
82.2 (d, J = 173.4, C(3′)), 79.6 (dd, J = 18.7, 11.5, C(2′)), 64.7 (d, J = 168.3, C(4′)), 62.3, 62.2
(CH2CH3), 44.5 (d, J = 8.0, C(1′)), 16.7, 16.6 (CH3); 31P NMR (121 MHz, CDCl3): 21.0; HR-
MS: 377.1387 ([M+H]+, C14H23FN4O5P +, calc. 377.1384).
(S)-2-Amino-3,4-dihydro-7-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}-7H-
pyrrolo[2,3-d]pyrimidin-4-one (10b). Compound 10b was synthesized according to the
procedure used for the preparation of 10a, starting from 9b (100 mg, 0.21 mmol), DABCO (23
mg, 0.21 mmol), and K2CO3 (29 mg, 0.21 mmol) in a mixture of dioxane and H2O (6 mL, 5:1,
v/v), followed by treatment with NH3/MeOH (20 mL). The crude residue was purified by silica
gel chromatography (DCM/MeOH, 20:1 to 10:1, v/v). Colorless oil. Yield: 64%. 1H NMR (300
MHz, CDCl3): δ 11.04 (s, NHCO), 6.60 (d, J = 3.4, H-C(6)), 6.49 (d, J = 3.5, H-C(5)), 6.21 (s,
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10.1002/cbdv.201800532
Chemistry & Biodiversity
NH2), 4.67-4.29 (m, H-C(3′)), 4.22-4.11 (m, H-C(1′), 2 x CH2CH3), 4.04-3.93 (m, H-C(2′), Ha-
13
C(4′)), 3.86-3.78 (m, Hb-C(4′)), 1.36-1.30 (m, 2 x CH3); C NMR (75 MHz, CDCl3): 160.6
(C(4)), 152.7 (C(2)), 150.7 (C(7a)), 121.6 (C(6)), 102.6 (C(4a)), 101.2 (C(5)), 82.2 (d, J = 173.6,
C(3′)), 79.6 (C(2′)), 64.7 (d, J = 168.3, C(4′)), 62.3, 62.2 (CH2CH3), 44.6 (d, J = 8.0 Hz, C(1′)),
16.7, 16.6 (CH3); 31P NMR (121 MHz, CDCl3): 21.0; HR-MS: 377.1384 ([M+H]+,
C14H23FN4O5P +, calc. 377.1384).
(R)-2-Amino-3,4-dihydro-7-[3-fluoro-2-(phosphonomethoxy)propyl]-7H-pyrrolo[2,3-
d]pyrimidin-4-one (11a). Compound 11a was synthesized according to the procedure used for
the preparation of 5a, starting from compound 10a (100 mg, 0.26 mmol), bromotrimethylsilane
(0.28 mL, 2.12 mmol), and 2,6-lutidine (0.50 mL, 4.24 mmol) in anhydrous acetonitrile (10
mL). The crude residue was purified by silica gel chromatography (acetone/Et3N/H2O, 4:1:1,
v/v/v). White foam. Yield: 70%. 1H NMR (300 MHz, D2O): 6.65 (d, J = 3.7, H-C(6)), 6.17 (d,
J = 3.6, H-C(5)), 4.50-4.04 (m, H-C(3′)), 4.95-3.93 (m, H-C(1′)), 3.83-3.70 (m, H-C(2′)), 3.41
(d, J = 9.2, H-C(4′)); 13C NMR (75 MHz, D2O): 160.8 (C(4)), 152.0 (C(2)), 150.3 (C(7a)), 123.1
(C(6)), 100.9 (C(5)), 99.5 (C(4a)), 82.3 (d, J = 167.3, C(3′)), 78.0 (dd, J = 18.4, 10.7, C(2′)),
31
67.6 (d, J = 151.9, C(4′)), 43.4 (d, J = 7.3, C(1′)); P NMR (121 MHz, D2O): 12.3; HR-MS:
319.0611 ([M-H]-, C10H13FN4O5P-, calc. 319.0612).
(S)-2-Amino-3,4-dihydro-7-[3-fluoro-2-(phosphonomethoxy)propyl]-7H-pyrrolo[2,3-
d]pyrimidin-4-one (11b). Compound 11b was synthesized according to the procedure used for
the preparation of 5a, starting from compound 10b (50 mg, 0.13 mmol), bromotrimethylsilane
(0.14 mL, 1.06 mmol), and 2,6-lutidine (0.25 mL, 2.12 mmol) in anhydrous acetonitrile (5 mL).
The crude residue was purified by silica gel chromatography on (acetone/Et3N/H2O, 4:1:1,
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10.1002/cbdv.201800532
Chemistry & Biodiversity
1
v/v/v). White foam. Yield: 70%. H NMR (300 MHz, D2O): δ 6.73 (d, J = 3.5, H-C(6)), 6.25
(d, J = 3.5, H-C(5)), 4.56-4.11 (m, H-C(3′)), 4.09-3.98 (m, H-C(1′)), 3.90-3.78 (m, H-C(2′)),
3.42 (d, J = 9.2, H-C(4′)); 13C NMR (75 MHz, D2O): 166.0 (C(4)), 156.5 (C(2)), 151.1 (C(7a)),
122.3 (C(6)), 100.6 (C(4a), C(5)), 82.4 (d, J = 167.0, C(3′)), 78.0 (dd, J = 18.2, 10.4, C(2′)),
31
68.1 (d, J = 150.0, C(4′)), 43.2 (d, J = 7.2, C(1′)); P NMR (121 MHz, D2O): 12.6; HR-MS:
319.0598 ([M-H]-, C10H13FN4O5P-, calc. 319.0612).
(R)-4-Chloro-5-fluoro-7-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}-2-
pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine (12a). Compound 12a was synthesized
according to the procedure used for the preparation of 9a, starting from 8 (200 mg, 0.74 mmol),
2a (150 mg, 0.61 mmol), Ph3P (300 mg, 1.20 mmol), and DIAD (0.24 mL, 1.20 mmol) in
anhydrous THF (10 mL). The crude residue was purified by silica gel chromatography
(DCM/MeOH, 80:1 to 40:1, v/v). Colorless oil. Yield: 25%. 1H NMR (300 MHz, CDCl3): 8.13
(s, NHCO), 7.10 (d, J = 2.6, H-C(6)), 4.72-3.96 (m, H-C(3′), H-C(1′), H-C(2′), 2 x CH2CH3),
4.00 (dd, J = 13.8, 8.7, Ha-C(4′)), 3.88 (dd, J = 13.8, 9.2, Hb-C(4′)), 1.34-1.28 (m, (CH3)3C, 2 x
CH2CH3); 13C NMR (125 MHz, CDCl3): 175.6 (NHCO), 152.0 (C(2)), 150.9 (d, J = 4.2, C(4)),
147.9 (C(7a)), 141.6 (d, J = 252.5, C(5)), 112.5 (d, J = 26.5, C(6)), 103.6 (d, J = 14.9, C(4a)),
82.6 (d, J = 173.3, C(3′)), 79.5, 79.4, 79.2, 79.1 (C(2′)), 64.8 (d, J = 166.9, C(4′)), 62.7, 62.6
(CH2CH3), 44.4 (d, J = 8.5, C(1′)), 40.5 (C(CH3)3), 27.6 (C(CH3)3), 16.6, 16.5 (CH2CH3); 31P
NMR (121 MHz, CDCl3): 20.9; HR-MS: 497.1523 ([M+H]+, C19H29ClF2N4O5P+, calc.
497.1527).
(S)-4-Chloro-5-fluoro-7-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}-2-
pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine (12b). Compound 12b was synthesized
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10.1002/cbdv.201800532
Chemistry & Biodiversity
according to the procedure used for the preparation of 9a, starting from 8 (200 mg, 0.74 mmol),
2b (150 mg, 0.61 mmol), Ph3P (300 mg, 1.20 mmol), and DIAD (0.24 mL, 1.20 mmol) in
anhydrous THF (10 mL). The crude residue was purified bysilica gel chromatography
1
(DCM/MeOH, 80:1 to 40:1, v/v). Colorless oil. Yield: 33%. H NMR (600 MHz, CDCl3): δ
8.13 (s, NHCO), 7.10 (d, J = 2.5, H-C(6)), 4.67-4.25 (m, H-C(3′), H-C(1′)), 4.18-4.02 (m, H-
C(2′), 2 x CH2CH3), 4.00 (dd, J = 13.8, 8.7, Ha-C(4′)), 3.89 (dd, J = 13.8, 9.2, Hb-C(4′)), 1.34-
1.30 (m, (CH3)3C, 2 x CH2CH3); 13C NMR (125 MHz, CDCl3): 175.5 (NHCO), 151.8 (C(2)),
150.7 (C(4)), 147.6 (C(7a)), 141.3 (d, J = 252.3, C(5)), 112.4 (d, J = 26.3, C(6)), 103.4 (d, J =
14.9, C(4a)), 82.4 (d, J = 173.3, C(3′)), 79.2, 79.0 (C(2′)), 64.5 (d, J = 167.0, C(4′)), 62.5
(CH2CH3), 44.1 (d, J = 8.5, C(1′)), 40.3 (C(CH3)3), 27.4 (C(CH3)3), 16.4 (CH2CH3); 31P NMR
(121 MHz, CDCl3): 20.8; HR-MS: 497.1521 ([M+H]+, C19H29ClF2N4O5P+, calc. 497.1527).
(R)-2-Amino-5-fluoro-3,4-dihydro-7-{3-fluoro-2-
[(diethylphosphoryl)methoxy]propyl}-7H-pyrrolo[2,3-d]pyrimidin-4-one
(13a).
Compound 13a was synthesized according to the procedure used for the preparation of 10a,
starting from 12a (60 mg, 0.12 mmol), DABCO (14 mg, 0.12 mmol) and K2CO3 (17 mg, 0.12
mmol) in a mixture of dioxane and H2O (6 mL, 5:1, v/v), followed by treatment with
NH3/MeOH (20 mL). The crude residue was purified by silica gel chromatography
(DCM/MeOH, 18:1 to 10:1, v/v). Colorless oil. Yield: 30%. 1H NMR (300 MHz, CDCl3): 6.42
(d, J = 2.3, H-C(6)), 5.99 (s, NH2), 4.66-3.83 (m, H-C(3′), H-C(1′), H-C(2′), 2 x CH2CH3, H-
13
C(4′)), 1.37-1.32 (m, 2 x CH3); C NMR (75 MHz, CDCl3): 158.4 (d, J = 2.6, C(4)), 152.3
(C(2)), 146.7 (C(7a)), 145.4 (d, J = 248.6, C(5)), 103.6 (d, J = 26.7, C(6)), 90.6 (d, J = 13.9,
C(4a)), 81.9 (d, J = 173.2, C(3′)), 79.0 (dd, J = 19.0, 10.8, C(2′)), 64.2 (d, J = 167.5, C(4′)),
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10.1002/cbdv.201800532
Chemistry & Biodiversity
62.4, 62.3 (CH2CH3), 43.5 (d, J = 7.9, C(1′)), 16.1, 16.0 (CH3); 31P NMR (121 MHz, CDCl3):
20.3; HR-MS: 395.1284 ([M+H]+, C14H22F2N4O5P+, calc. 395.1290).
(S)-2-Amino-5-fluoro-3,4-dihydro-7-{3-fluoro-2-
[(diethylphosphoryl)methoxy]propyl}-7H-pyrrolo[2,3-d]pyrimidin-4-one
(13b).
Compound 13b was synthesized according to the procedure used for the preparation of 10a,
starting from 12b (90 mg, 0.18 mmol), DABCO (20 mg, 0.18 mmol), and K2CO3 (25 mg, 0.18
mmol) in a mixture of dioxane and H2O (6 mL, 5:1, v/v), followed by treatment with
NH3/MeOH (20 mL). The crude residue was purified by silica gel chromatography
(DCM/MeOH, 18:1 to10:1, v/v). Colorless oil. Yield: 35%. 1H NMR (300 MHz, CDCl3): δ 6.41
(d, J = 2.4, H-C(6)), 6.00 (s, NH2), 4.66-3.82 (m, H-C(3′), H-C(1′), H-C(2′), 2 x CH2CH3, H-
C(4′)), 1.36-1.31 (m, 2 x CH3); 13C NMR (75 MHz, CDCl3):159.0 (C(4)), 152.9 (C(2)), 147.3
(C(7a)), 146.1 (d, J = 248.6, C(5)), 104.2 (d, J = 26.5, C(6)), 91.2 (d, J = 13.7, C(4a)), 82.5 (d,
J = 173.1, C(3′)), 79.6 (dd, J = 19.2, 11.1, C(2′)), 64.8 (d, J = 167.6, C(4′)), 63.0, 62.9 (CH2CH3),
44.2 (d, J = 7.9, C(1′)), 16.7, 16.6 (CH3); 31P NMR (121 MHz, CDCl3): 20.9; HR-MS: 395.1286
([M+H]+, C14H22F2N4O5P+, calc. 395.1290).
(R)-2-Amino-5-fluoro-3,4-dihydro-7-[3-fluoro-2-(phosphonomethoxy)propyl]-7H-
pyrrolo[2,3-d]pyrimidin-4-one (14a). Compound 14a was synthesized according to the
procedure used for the preparation of 5a, starting from compound 13a (20 mg, 0.05 mmol),
bromotrimethylsilane (0.05 mL, 0.40 mmol), and 2,6-lutidine (0.09 mL, 0.81 mmol) in
anhydrous acetonitrile (5 mL). The crude residue was purified by silica gel chromatography
acetone/Et3N/H2O, 4:1:1, v/v/v). White foam. Yield: 50%. 1H NMR (300 MHz, D2O): 6.56 (d,
J = 2.1, H-C(6)), 4.59-4.16 (m, H-C(3′)), 4.00-3.98 (m, H-C(1′)), 3.90-3.81 (m, H-C(2′)), 3.49
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10.1002/cbdv.201800532
Chemistry & Biodiversity
(d, J = 9.1, H-C(4′)); 13C NMR (75 MHz, D2O): 159.5 (d, J = 2.8, C(4)), 152.3 (C(2)), 146.0 (d,
J = 3.0, C(7a)), 144.6 (d, J = 246.3, C(5)), 105.1 (d, J = 26.6, C(6)), 90.0 (d, J = 14.4, C(4a)),
82.4 (d, J = 167.2, C(3′)), 78.1 (dd, J = 18.3, 10.4, C(2′)), 67.2 (d, J = 153.2, C(4′)), 43.2 (d, J
31
= 7.4, C(1′)); P NMR (121 MHz, D2O): 13.8; HR-MS: 337.0503 ([M-H]-, C10H12F2N4O5P-,
calc. 337.0519).
(S)-2-Amino-5-fluoro-3,4-dihydro-7-[3-fluoro-2-(phosphonomethoxy)propyl]-7H-
pyrrolo[2,3-d]pyrimidin-4-one (14b). Compound 14b was synthesized according to the
procedure used for the preparation of 5a, starting from compound 13b (20 mg, 0.05 mmol),
bromotrimethylsilane (0.05 mL, 0.40 mmol), and 2,6-lutidine (0.09 mL, 0.81 mmol) in
anhydrous acetonitrile (5 mL). The crude residue was purified by silica gel chromatography
(acetone/Et3N/H2O, 4:1:1, v/v/v). White foam. Yield: 60%. 1H NMR (300 MHz, D2O): 6.52 (d,
J = 2.2, H-C(6)), 4.57-4.09 (m, H-C(3′)), 4.07-3.92 (m, H-C(1′)), 3.89-3.77 (m, H-C(2′)), 3.43
(d, J = 9.1, H-C(4′)); 13C NMR (75 MHz, D2O): 167.1 (C(4)), 159.1 (C(2)), 147.4 (C(7a)), 144.6
(d, J = 244.3, C(5)), 103.5 (d, J = 27.2, C(6)), 91.2 (d, J = 14.4, C(4a)), 82.4 (d, J = 166.9,
31
C(3′)), 77.9 (dd, J = 18.2, 10.3, C(2′)), 68.1 (d, J = 150.1, C(4′)), 42.7 (d, J = 7.2, C(1′)); P
NMR (121 MHz, D2O): 12.7; HR-MS: 337.0522 ([M-H]-, C10H12F2N4O5P-, calc. 337.0519).
(R)-4-Chloro-5-cyano-7-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}-2-
pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine (20a).
Compound 20a was synthesized according to the procedure used for the preparation of 9a,
starting from 19 (150 mg, 0.54 mmol), 2a (120 mg, 0.49 mmol), Ph3P (260 mg, 0.98 mmol),
and DIAD (0.19 mL, 0.98 mmol) in anhydrous THF (10 mL). The crude residue was purified
1
by silica gel chromatography (DCM/MeOH, 50:1 to 30:1, v/v). Colorless oil. Yield: 80%. H
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10.1002/cbdv.201800532
Chemistry & Biodiversity
NMR (300 MHz, CDCl3): 8.24 (s, NHCO), 7.91 (s, H-C(6)), 4.74-4.29 (m, H-C(3′), H-C(1′)),
4.17-3.96 (m, H-C(2′), 2 x CH2CH3, Ha-C(4′)), 3.81 (dd, J = 13.9, 9.0, Hb-C(4′)), 1.34-1.25 (m,
(CH3)3C, 2 x CH2CH3); 13C NMR (75 MHz, CDCl3): 175.8 (NHCO), 153.4, 153.2, 152.3 (C(4),
C(7a), C(2)), 138.2 (C(6)), 113.5 (C(5)), 112.1 (C(4a)), 86.0 (CN), 82.3 (d, J = 174.0, C(3′)),
78.7 (dd, J = 19.2, 9.8, C(2′)), 64.7 (d, J = 167.1, C(4′)), 62.9, 62.8 (CH2CH3), 45.6 (d, J = 8.4,
31
C(1′)), 40.6 (C(CH3)3), 27.6 (C(CH3)3), 16.7 (CH2CH3); P NMR (121 MHz, CDCl3): 20.7;
HR-MS: 504.1567 ([M+H]+, C20H29ClFN5O5P+, calc. 504.1573).
(S)-4-Chloro-5-cyano-7-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}-2-
pivaloylamino-7H-pyrrolo[2,3-d]pyrimidine (20b).
Compound 20b was synthesized according to the procedure used for the preparation of 9a,
starting from 19 (150 mg, 0.54 mmol), 2b (120 mg, 0.49 mmol), Ph3P (260 mg, 0.98 mmol),
and DIAD (0.19 mL, 0.98 mmol) in anhydrous THF (10 mL). The crude residue was purified
1
by silica gel chromatography (DCM/MeOH, 50:1 to 30:1, v/v). Colorless oil. Yield: 60%. H
NMR (300 MHz, CDCl3): 8.24 (s, NHCO), 7.92 (s, H-C(6)), 4.74-4.30 (m, H-C(3′), H-C(1′)),
4.15-3.96 (m, H-C(2′), 2 x CH2CH3, Ha-C(4′)), 3.81 (dd, J = 13.9, 9.0, Hb-C(4′)), 1.35-1.26 (m,
(CH3)3C, 2 x CH2CH3); 13C NMR (75 MHz, CDCl3): 175.7 (NHCO), 153.2, 153.0, 152.1 (C(4),
C(7a), C(2)), 138.1 (C(6)), 113.4 (C(5)), 111.9 (C(4a)), 85.8 (CN), 82.1 (d, J = 174.0, C(3′)),
78.5 (dd, J = 19.2, 9.9, C(2′)), 64.5 (d, J = 167.0, C(4′)), 62.8-62.6 (CH2CH3), 45.4 (d, J = 8.6,
31
C(1′)), 40.5 (C(CH3)3), 27.5 (C(CH3)3), 16.6, 16.5 (CH2CH3); P NMR (121 MHz, CDCl3):
20.7; HR-MS: 504.1578 ([M+H]+, C20H29ClFN5O5P+, calc. 504.1573).
(R)-2-Amino-5-cyano-3,4-dihydro-7-{3-fluoro-2-
[(diethylphosphoryl)methoxy]propyl}-7H-pyrrolo[2,3-d]pyrimidin-4-one
(21a).
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10.1002/cbdv.201800532
Chemistry & Biodiversity
Compound 21a was synthesized according to the procedure used for the preparation of 10a,
starting from 20a (220 mg, 0.44 mmol), DABCO (33 mg, 0.30 mmol) and K2CO3 (61 mg, 0.44
mmol) in a mixture of dioxane and H2O (6 mL, 5:1, v/v), followed by treatment with
NH3/MeOH (20 mL). The residue was purified by silica gel chromatography (DCM/MeOH,
1
20:1 to 10:1, v/v). Colorless oil. Yield: 30%. H NMR (300 MHz, CDCl3): 10.89 (s, NHCO),
7.30 (d, J = 1.1, H-C(6)), 6.16 (s, NH2), 4.71-4.29 (m, H-C(3′), Ha-C(1′)), 4.21-3.93 (m, Hb-
C(1′), H-C(2′), 2 x CH2CH3, Ha-C(4′)), 3.83 (dd, J = 13.8, 9.1, Hb-C(4′)), 1.38-1.32 (m, 2 x
13
CH3); C NMR (75 MHz, CDCl3): 159.2 (C(4)), 154.0 (C(2)), 152.2 (C(7a)), 130.9 (C(6)),
115.5 (C(5)), 99.6 (C(4a)), 86.7 (CN), 82.4 (d, J = 173.6, C(3′)), 79.1 (dd, J = 19.1, 10.4, C(2′)),
64.8 (d, J = 167.7, C(4′)), 62.9, 62.8 (CH2CH3), 45.2 (d, J = 8.1, C(1′)), 16.8, 16.7 (CH3); 31P
NMR (121 MHz, CDCl3): 20.7; HR-MS: 402.1324 ([M+H]+, C15H22FN5O5P+, calc. 402.1336).
(S)-2-Amino-5-cyano-3,4-dihydro-7-{3-fluoro-2-[(diethylphosphoryl)methoxy]propyl}-
7H-pyrrolo[2,3-d]pyrimidin-4-one (21b). A mixture of 20b (150 mg, 0.30 mmol), Et3N (0.12
mL, 0.89 mmol), NaOAc (73 mg, 0.89 mmol), and DABCO (33 mg, 0.30 mmol) in DMF (5
mL) was stirred at room temperature for 40 h. Subsequently, H2O (5 mL) was added and the
reaction mixture was stirred for another 1 h. The mixture was extracted with EtOAc (2 x 25
mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated under
reduced pressure to give a crude product. The residue was then resolved in satd NH3 in MeOH
(20 mL) and stirred at room temperature for 12 h. After removal of all the volatiles, the residue
was purified by silica gel chromatography (DCM/MeOH, 20:1 to 10:1, v/v) to give 21b (80
mg). Colorless oil. Yield: 67%. 1H NMR (300 MHz, CDCl3): 10.89 (s, NHCO), 7.30 (d, J = 1.1,
H-C(6)), 4.71-4.28 (m, H-C(3′), Ha-C(1′)), 4.21-3.93 (m, Hb-C(1′), H-C(2′), 2 x CH2CH3, Ha-
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10.1002/cbdv.201800532
Chemistry & Biodiversity
13
C(4′)), 3.87-3.80 (m, Hb-C(4′)), 1.38-1.32 (m, 2 x CH3); C NMR (75 MHz, CDCl3): 159.2
(C(4)), 154.0 (C(2)), 152.2 (C(7a)), 130.9 (C(6)), 115.5 (C(5)), 99.5 (C(4a)), 86.6 (CN), 82.4
(d, J = 173.5, C(3′)), 79.1 (dd, J = 19.2, 10.4, C(2′)), 64.8 (d, J = 167.2, C(4′)), 63.0, 62.9
(CH2CH3), 45.2 (d, J = 8.2, C(1′)), 16.8, 16.7 (CH3); 31P NMR (121 MHz, CDCl3): 20.7; HR-
MS: 402.1329 ([M+H]+, C15H22FN5O5P+, calc. 402.1336).
(R)-2-Amino-5-cyano-3,4-dihydro-7-[3-fluoro-2-(phosphonomethoxy)propyl]-7H-
pyrrolo[2,3-d]pyrimidin-4-one (22a). Compound 22a was synthesized according to the
procedure used for the preparation of 5a, starting from compound 21a (50 mg, 0.13 mmol),
bromotrimethylsilane (0.13 mL, 1.00 mmol), and 2,6-lutidine (0.23 mL, 2.00 mmol) in
anhydrous acetonitrile (5 mL). The crude residue was purified by silica gel chromatography
(acetone/Et3N/H2O, 5:1:1, v/v/v). White foam. Yield: 70%. 1H NMR (300 MHz, D2O): 7.51 (s,
H-C(6)), 4.60-4.21 (m, H-C(3′)), 4.10-4.08 (m, H-C(1′)), 3.90-3.83 (m, H-C(2′)), 3.44 (d, J =
9.1, H-C(4′)); 13C NMR (75 MHz, D2O): 162.0 (C(4)), 155.6 (C(2)), 151.1 (C(7a)), 132.6 (C(6)),
116.2 (C(5)), 99.3 (C(4a)), 84.3 (CN), 82.2 (d, J = 167.1, C(3′)), 77.3 (dd, J = 18.6, 10.5, C(2′)),
31
68.0 (d, J = 149.9, C(4′)), 44.1 (d, J = 7.0, C(1′)); P NMR (121 MHz, D2O): 12.5; HR-MS:
344.0573 ([M-H]-, C11H12FN5O5P-, calc. 344.0565).
(S)-2-Amino-5-cyano-3,4-dihydro-7-[3-fluoro-2-(phosphonomethoxy)propyl]-7H-
pyrrolo[2,3-d]pyrimidin-4-one (22b). Compound 22b was synthesized according to the
procedure used for the preparation of 5a, starting from compound 21b (80 mg, 0.20 mmol),
bromotrimethylsilane (0.21 mL, 1.60 mmol), and 2,6-lutidine (0.37 mL, 3.20 mmol) in
anhydrous acetonitrile (5 mL). The crude residue was purified by silica gel chromatography
(acetone/Et3N/H2O, 5:1:1, v/v/v). White foam. Yield: 75%. 1H NMR (300 MHz, D2O): 7.40 (d,
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10.1002/cbdv.201800532
Chemistry & Biodiversity
J = 1.1 Hz, H-C(6)), 4.70-4.20 (m, H-C(3′)), 4.08-3.93 (m, H-C(1′)), 3.90-3.78 (m, H-C(2′)),
13
3.58-3.42 (m, H-C(4′)); C NMR (75 MHz, D2O): 159.0 (C(4)), 153.3 (C(2)), 150.7 (C(7a)),
132.7 (C(6)), 115.7 (C(5)), 98.7 (C(4a)), 84.6 (CN), 82.2 (d, J = 167.7, C(3′)), 77.7 (dd, J =
18.5, 11.3, C(2′)), 66.7 (d, J = 155.2, C(4′)), 44.3 (d, J = 7.6, C(1′)); 31P NMR (121 MHz, D2O):
14.3; HR-MS: 344.0557 ([M-H]-, C11H12FN5O5P-, calc. 344.0565).
(R)-2-Amino-3,4-dihydro-7-{3-fluoro-2-[phenyloxy-bis(n-amyl-L-
aspartyl)phosphoryl]methoxy]propyl}-7H-pyrrolo[2,3-d]pyrimidin-4-one
(23).
Anhydrous Et3N (0.13 mL, 0.95 mmol) was added to a solution of compound 11a (40 mg, 0.09
mmol, Et3N salt), L-aspartic acid amyl diester HCl salt (50 mg, 0.16 mmol), and phenol (40
mg, 0.42 mmol) in anhydrous pyridine (5 mL). The mixture was stirred at 60 °C under a
nitrogen atmosphere. In a separate flask, 2,2′-dithiodipyridine (150 mg, 0.66 mmol) was mixed
with PPh3 (180 mg, 0.66 mmol) in anhydrous pyridine (2 mL) and the resultant mixture was
stirred for 10-15 min. This light yellow solution was then added to the solution of 11a. The
combined mixture was stirred at 60 °C for 12 h and then concentrated under reduced pressure.
The resultant residue was first purified by silica gel chromatography (DCM/MeOH, 30:1, v/v;
20:1, v/v) and then purified by RP-HPLC (linear gradient, 5-95% CH3CN in water) to afford
compound 23 (60 mg). White powder. Yield: 40%. 1H NMR (300 MHz, CD3CN): δ 10.08 (s,
NHCO), 7.37-7.08 (m, 5 arom. H), 6.72-6.69 (m, H-C(6)), 6.40-6.38 (m, H-C(5)), 5.80, 5.78
(s, NH2), 4.70-4.25 (m, H-C(3′), NHPO, H-α-Asp), 4.16-3.89 (m, H-C(1′), H-C(2′), 2 x
OCH2(CH2)3CH3, H-C(4′)), 2.84-2.57 (m, H-β-Asp), 1.58-1.52 (m, 2 x OCH2CH2(CH2)2CH3),
13
1.32-1.24 (m, 2 x O(CH2)2(CH2)2CH3), 0.91-0.84 (m, 2 x O(CH2)4CH3); C NMR (75 MHz,
CD3CN): 172.9, 172.8, 172.7 (CO-α), 171.6 (CO-β), 160.5 (C(4)), 153.5, 153.4 (C(2)), 152.0,
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10.1002/cbdv.201800532
Chemistry & Biodiversity
151.9 (C(7a)), 130.7 (arom. C), 125.9 (arom. C), 122.4 (C(6)), 121.9, 121.8 (arom. C), 102.7
(C(5)), 101.7 (C(4a)), 83.6 (d, J = 170.3, C(3′)), 83.5 (d, J = 170.0, C(3′)), 80.7, 80.5, 80.4,
80.3, 80.1 (C(2′)), 66.8 (d, J = 156.2, C(4′)), 66.6 (d, J = 154.2 Hz, C(4′)), 66.6, 65.9, 65.8
(OCH2(CH2)3CH3), 51.7, 51.6 (C-α-Asp), 45.1, 45.0, 44.9 (C(1′)), 39.9 (d, J = 3.9, C-β-Asp),
39.7 (d, J = 3.9, C-β-Asp), 29.1, 29.0, 28.8, 28.7 (OCH2(CH2)2CH2CH3), 23.1, 23.0
31
(O(CH2)3CH2CH3), 14.3 (O(CH2)4CH3); P NMR (121 MHz, CD3CN): 23.0, 22.2; HR-MS:
652.2923 ([M+H]+, C31H43FN5O8P+, calc. 652.2905).
(R)-2-Amino-3,4-dihydro-7-{3-fluoro-2-[[N,N’-
bis(n-amyl-L-
(24).
aspartyl)phosphoryl]methoxy]propyl}-7H-pyrrolo[2,3-d]pyrimidin-4-one
Anhydrous Et3N (0.04 mL, 0.40 mmol) was added to a solution of compound 11a (15 mg, 0.04
mmol, Et3N salt) and L-aspartic acid amyl diester HCl salt (44 mg, 0.14 mmol) in anhydrous
pyridine (3 mL).The mixture then was stirred at 60 °C under a nitrogen atmosphere. In a
separate flask, 2,2′-dithiodipyridine (55 mg, 0.25 mmol) was mixed with PPh3 (65 mg, 0.25
mmol) in anhydrous pyridine (2 mL) and the resultant mixture was stirred for 10-15 min. This
light yellow solution was added to the solution of 11a. The combined mixture was stirred at
60 °C for 12 h and then concentrated under reduced pressure. The resultant residue was first
purified by silica gel chromatography (DCM/MeOH, 30:1, v/v; 20:1, v/v) and then purified by
RP-HPLC (linear gradient, 5-95% CH3CN in water) to afford compound 24 (5 mg). White
powder. Yield: 20%. 1H NMR (300 MHz, CD3CN): 9.87 (s, NHCO), 6.63 (d, J = 3.5, H-C(6)),
6.32 (d, J = 3.5, H-C(5)),6.06 (s, NH2), 4.68, 3.74 (m, H-C(3′), 2 x NHPO, 2 x H-α-Asp, H-
C(1′), H-C(2′), 4 x OCH2(CH2)3CH3, H-C(4′)), 2.89-2.73 (m, 2 x H-β-Asp), 1.61-1.53 (m, 4 x
OCH2CH2(CH2)2CH3), 1.33-1.24 (m, 4 x O(CH2)2(CH2)2CH3), 0.91-0.84 (m, 4 x O(CH2)4CH3);
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10.1002/cbdv.201800532
Chemistry & Biodiversity
13C NMR (75 MHz, CD3CN): 173.3, 173.2 (CO-α), 171.9, 171.8 (CO-β), 160.0 (C(4)), 153.7
(C(2)), 151.6 (C(7a)), 122.1 (C(6)), 102.8 (C(5)), 101.8 (C(4a)), 83.1 (d, J = 170.2, C(3′)), 80.13
(dd, J = 18.7, 11.5, C(2′)), 68.1 (d, J = 136.9, C(4′)), 66.6, 66.5, 65.8 (OCH2(CH2)3CH3), 51.1,
50.5 (C-α-Asp), 44.86 (d, J = 8.1, C(1′)), 40.0, 39.9, 39.8 (d, J = 3.9, C-β-Asp), 29.1, 29.0, 28.8
(OCH2(CH2)2CH2CH3), 23.1, 23.0 (O(CH2)3CH2CH3), 14.3 (O(CH2)4CH3); 31P NMR (121
MHz, CD3CN): 21.5; HR-MS: 829.4298 ([M-H]-, C38H63FN6O11P -, calc.829.4282).
Acknowledgments
Min Luo greatly acknowledges the China Scholarship Council (CSC) for funding. We thank
Eric Fonteyn, Sandra Claes and Evelyne Van Kerckhove for excellent technical assistance in
the generation of the anti-HIV-1 data.
Author Contribution Statement
M. L. synthesized the compounds. E. G. wrote the article. P. H.conceived the study. S. D. J.,
and D. S. contributed to the biological evaluation.
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