A new, efficient entry to non-lipophilic H-phosphonate monoesters -Preparation of anti-HIV nucleotide analogues

Article abstract of DOI:10.2174/157017809789124821

Crystalline ammonium (9H-fluoren-9-yl)methyl H-phosphonate was prepared by a new, simple method and it was used as the reagent of choice for the introduction of an H-phosphonate monoester functionality into non-lipophilic anti-HIV nucleoside analogues.

Full text of DOI:10.2174/157017809789124821

496
Letters in Organic Chemistry, 2009, 6, 496-499
A New, Efficient Entry to Non-Lipophilic H-Phosphonate Monoesters –
Preparation of Anti-HIV Nucleotide Analogues
Joanna Romanowska, Agnieszka Szymaꢀska-Michalak, Maꢁgorzata Pietkiewicz, Michaꢁ Sobkowski,
Jerzy Boryski, Jacek Stawiꢀski and Adam Kraszewski*
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznaꢀ, Poland
Received December 22, 2008: Revised May 14, 2009: Accepted May 17, 2009
Abstract: Crystalline ammonium (9H-fluoren-9-yl)methyl H-phosphonate was prepared by a new, simple method and it
was used as the reagent of choice for the introduction of an H-phosphonate monoester functionality into non-lipophilic
anti-HIV nucleoside analogues.
Keywords: H-phosphonates, phosphonylating agents, nucleoside analogues, nucleoside H-phosphonates, protecting groups,
ꢂ-elimination.
INTRODUCTION
monoester derivatives. Unfortunately, the preparation of
(9H-fluoren-9-yl)methyl H-phosphonic acid is far from sim-
ple and requires hazardous and commercially restricted rea-
gents (e.g. phosphorus trichloride). To overcome these in-
conveniences we developed a new, efficient method for the
synthesis of ammonium (9H-fluoren-9-yl)methyl H-
phosphonate, and used this reagent for the preparation of 5'-
H-phosphonate monoesters derived from nucleoside ana-
logues with established anti-HIV potency.
H-Phosphonate derivatives are very convenient and use-
ful precursors in the synthesis of biologically active phos-
phates and their analogues, e.g. nucleotides [1], or phos-
phorylated sugar and carbohydrates [2]. Although there are
several efficient protocols available for H-phosphonylation,
they usually work well with lipophilic substrates. Typical
example here is synthesis of 5'-O-tritylated nucleoside 3'-H-
phosphonates, that can be performed using a variety of H-
phosphonylating reagents [3-5]. However, problems are fre-
quently encountered during synthesis of H-phosphonate
monoesters derived from polar hydroxylic components, for
example, anti-HIV active nucleoside analogues such as 2',3'-
dideoxyadenosine (ddA) [6], 2',3'-dideoxyinosine (ddI) [7]
or 2',3'-dideoxyuridine (ddU) [8]. In contrast to the tritylated
nucleoside derivatives, aqueous work-up of the reaction mix-
tures after H-phosphonylation of dideoxynucleosides ap-
peared to be troublesome, and the purification process re-
quires a painstaking chromatography. To overcome these
synthetic limitations we set out to develop a new H-
phosphonylation protocol suitable for the introduction of an
H-phosphonate monoester group into polar hydroxylic com-
pounds.
The synthesis of ammonium (9H-fluoren-9-yl)methyl H-
phosphonate was based on commercially available, non-
toxic and cheap phosphonic acid (1), and (9H-fluoren-9-
yl)methanol (3) (Scheme 1). First, phosphonic acid (1) in
pyridine was treated with pivaloyl chloride to produce quan-
titatively (<3 min, 31P NMR) pyridinium H-pyrophosphonate
(2) [10]. To this, (9H-fluoren-9-yl)methanol (3) was added
and the desired (9H-fluoren-9-yl)methyl H-phosphonate (4)
(pyridinium salt) was formed nearly quantitatively [the dis-
appearance of (3), TLC analysis]. After simple work-up of
the reaction mixture, ammonium (9H-fluoren-9-yl)methyl H-
phosphonate (4) was obtained as a crystalline compound in
high yield (~ 90%).
In a typical experiment, a nucleoside analogue of type (5)
was reacted with ammonium (9H-fluoren-9-yl)methyl H-
phosphonate (4) in CH2Cl2/pyridine 95:5 (v/v) [11] using
PvCl [12, 13] as an activator. After ca 60 min the reaction
was complete as indicated by 31P NMR spectroscopy and
TLC analysis. The excess of nucleosides (5) and polar re-
mainings from the condensing agent were removed by sim-
ple extraction of the organic solution with water to afford
crude H-phosphonate diesters (6), free from unreacted sub-
strates (TLC, 31P NMR; Table 1).
RESULTS AND DISCUSSION
To this end, we searched for an H-phosphonylating rea-
gent with a lipophilic handle, that would permit simple
aqueous work-up of the reaction mixtures. As a viable can-
didate for this purpose we considered (9H-fluoren-9-
yl)methyl H-phosphonic acid, synthesized by Z. W. Yang et
al. [9] and used for the phosphonylation of 5'-O-tritylated
nucleosides. Preliminary experiments showed that, indeed,
the (9H-fluoren-9-yl)methyl group can provide the required
lipophilicity during a multistep synthesis of H-phosphonate
Purity of the obtained (9H-fluoren-9-yl)methyl nucleo-
side H-phosphonate diesters (6) was in each case high and
permited direct removal of the (9H-fluoren-9-yl)methyl
group without recourse to chromatographic purification of
intermediate (6). To this end, crude product (6) was treated
with triethylamine in anhydrous acetonitrile [14] to furnish
within 20 min at room temperature a clean formation of nu-
*Address correspondence to this author at the Institute of Bioorganic Chem-
istry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznaꢀ,
Poland; Tel: +48 61 852 85 03; Fax: +48 61 852 05 32;
E-mail: adam.kraszewski@ibch.poznan.pl
1570-1786/09 $55.00+.00
© 2009 Bentham Science Publishers Ltd.

Synthesis of Anti-HIV Non-Lipophilic Nucleoside H-Phosphonates
Letters in Organic Chemistry, 2009, Vol. 6, No. 6
497
CH2
OH
O
P
O
P
O
P
3
O
P
PvCl
Py
+HPy -O
O
O- PyH+
HO
OH
O- NH4
+
CH2
O
H
H
H
H
2
4
1
HO
B
O
PvCl
5
X
NEt3
O
P
O
H
NEt3 /CH3CN
Et3NH+
-O
O
CH2
O
P
O
B
B
O
O
H
H
CH2
X
X
7
6
5-7a, B = Ade, X = H
5-7b, B = Hyp, X = H
5-7c, B = Thy, X = N3
5-7d, B = Ura, X = H
PvCl = pivaloyl chloride
Py = pyridine
Scheme 1. Synthesis of ammonium (9H-fluoren-9-yl)methyl H-phosphonate (4) and its use for H-phosphonylation of nucleosides (5).
Table 1. The 31P NMR Data for Intermediates and the Final Products
Cpds
ꢀP (ppm)
JH-P (Hz)
Cpds
ꢀP (ppm)
JH-P (Hz)
4
3.85
1JH-P 613.4; 3JH-P 7.3 (dt)
1JH-P 711.5; 3JH-P 8.3 (two dm)
1JH-P 710.5; 3JH-P 8.3 (two dm)
1JH-P 711.4; 3JH-P 8.3 (two dm)
1JH-P 711.4; 3JH-P 8.3 (two dm)
7a
7b
7c
7d
2.40
2.32
2.83
3.17
1JH-P 617.2; 3JH-P 6.4 (dt)
1JH-P 601.6; 3JH-P 7.3 (dt)
1JH-P 616.3; 3JH-P 6.4 (dt)
1JH-P 615.4; 3JH-P 6.4 (dt)
6a
8.65, 8.08a
8.54, 8.09a
8.62, 8.04a
8.73, 8.14a
6b
6c
6d
aTwo diastereoisomers.
cleoside 5'-H-phosphonates (7) [ꢀ-elimination of the (9H-
fluoren-9-yl)methyl group]. Products (7) were isolated by
simple silica gel filtration using a stepwise gradient of
methanol [0 – 30%] in methylene chloride containing Et3N
(3%). The yields of H-phosphonate monoesters (7a-d) were
invariably high (72 - 90%) and purity of the isolated prod-
ucts was >98% (1H, 13C, and 31P NMR spectroscopy, and
HPLC analysis).
ammonium (9H-fluoren-9-yl)methyl H-phosphonate (4), a
simple and efficient procedure was also developed.
EXPERIMENTAL
1H, 13C and 31P NMR spectra were recorded on 300 MHz
or 400 MHz machines. The 31P NMR experiments were car-
ried out in 5 mm tubes using 0.1 M solutions of the phospho-
rus-containing compound. 31P NMR chemical shifts are re-
ported in ppm relative to 85% H3PO4 in water (external stan-
In conclusion, we have developed an efficient protocol
for the introduction of an H-phosphonate monoester function
into non-lipophilic nucleosides and this can be considered as
a general method for phosphonylation of various types of
polar hydroxylic compounds. It makes use of an H-
phosphonylating reagent (4) with a lipophilic (9H-fluoren-9-
yl)methyl handle that permits an aqueous work-up of the
intermediate (9H-fluoren-9-yl)methyl H-phosphonate di-
esters (6). The efficacy of the developed method was demon-
strated in the synthesis of various important anti-HIV active
nucleotide analogues (7a-d), which were isolated in good
yields. For the preparation of a phosphonylating reagent,
dard). Mass spectra were recorded with liquid secondary ion
+
mass technique (LSIMS) using Cs (12 keV) for ionisation.
The amount of water in solvents was measured with Karl
Fisher coulometric titration. Methylene dichloride was dried
over P2O5, distilled, and kept over molecular sieves 4 Å until
the amount of water was less than 10 ppm. Pyridine was
stored over molecular sieves 4 Å until the amount of water
was below 20 ppm. Triethylamine was distilled and stored
over CaH . For column chromatography silica gel 60
2

498 Letters in Organic Chemistry, 2009, Vol. 6, No. 6
Romanowska et al.
+
(Merck) was used. For TLC analysis, the precoated plates
(Merck silica gel 60 F254) were used.
exch. D2O, NH4 ), 7.39 (t, J = 7.4 Hz, 2H, ArH), 7.31 (t, J =
7.4 Hz, 2H, ArH), 6.59 (d, JH-P = 635.4 Hz, 1H, H-P), 4.15
(t, J = 7.2 Hz, 1H, 9-CH), 3.93 (dd, 3JH-P = 7.2 Hz, JH-H = 7.2
Hz, 2H, CH2); 13C NMR (D2O) ꢁ 143.75 (C-10 & C-13),
140.72 (C-11 & C-12), 127.6 (C-1 & C-8), 127.1 (C-4 & C-
5), 124.95 (C-2 & C-7), 119.81 (C-3 & C-6), 65.52 (CH2),
Synthesis of Ammonium (9H-fluoren-9-yl)methyl
H-phosphonate (4)
+ -
Phosphonic acid (4.1 g, 50 mmol) was rendered anhy-
drous by repeated evaporation of the added pyridine. The
residue was dissolved in the same solvent (~ 150 mL) and
treated with PvCl (3.5 mL, 0.55 molar equiv.) for 5 min. To
the generated pyridinium H-pyrophosphonate, (9H-fluoren-
9-yl)methanol (4.9 g, 25 mmol) was added, the reaction mix-
ture was concentrated to ꢀ of its initial volume, and was left
at room temperature for 48 h (over weekend) or for 24 h at
47.84 (C-9); HRMS [M –NH4 ] m/z 259,0502, calculated
for C14H12O3P 259,0524.
2',3'-Dideoxyandenosin-5'-yl H-phosphonate Triethylam-
monium Salt (7a)
Yield 90%. 1H NMR (D2O) ꢁ 8.28 (s, 1H, H-8), 8.05 (s,
1H, H-2), 6.61 (d, J = 637.8 Hz, H-P), 6.19 (dd, J = 6.8 Hz, J
= 3.2 Hz, 1H, 1'-H), 4.46-4.39 (m, 1H, 4'-H), 4.11-4.06 &
3.96-3.90 (2m, 2H, 5',5''-H2), 3.15 (q, J = 7.2 Hz, 6H,
CH2CH3), 2.65-2.55 & 2.47-2.41 (2m, 2H, 3',3''-H2), 2.27-
2.19 & 2.13-2.03 (2m, 2H, 2',2''-H2), 1.24 (t, J = 7.2 Hz, 9H,
CH2CH3). 13C NMR (D2O) ꢁ 154.50 (C-4), 151.55 (C-2),
147.84 (C-6), 139.65 (C-8), 118.22 (C-5), 84.78 (C-1'),
80.74 (C-5'), 64.44 (C-4'), 46.57 (CH2CH3), 31.57 (C-2'),
25.65 (C-3'), 8.16 (CH2CH3). HRMS [M – Et3NH+]- m/z
298.0719, calculated for C10H13N5O4P 298.0705.
o
40 C (overnight). After cooling down the reaction flask on
ice-bath, water (10 mL) was added, and upon standing for 10
min, the solvents were removed under reduced pressure. The
viscous oil was dissolved in dichloromethane (150 mL),
washed with brine (2 x 50 mL), and the organic layer was
separated, dried over Na2SO4 anh., and evaporated. The (9H-
fluoren-9-yl)methyl pyridinium H-phosphonate was con-
verted into the ammonium salt by addition of propan-2-ol
(50 mL) containing aqueous ammonia (5% v/v). Repeated
evaporation of the added propan-2-ol caused spontaneous
precipitation of (4) as a white solid. After filtration and dry-
ing, product (4) (ammonium salt, 6.2 g) was obtained as non-
2',3'-Dideoxyinosin-5'-yl H-phosphonate Triethylammo-
nium Salt (7b)
Yield 72%. 1H NMR (D2O) ꢁ 8.34 (s, 1H, 8-H), 8.18 (s,
1H, 2-H), 6.63 (d, J = 637.8 Hz, 1H, H-P), 6.34 (dd, J = 6.6
Hz, J = 2.7 Hz, 1-H, 1'-H), 4.14-4.08 (m, 1H, 4'-H), 4.02-
3.90 (m, 2H, 5',5''-H2), 3.21 (q, J = 7.2 Hz, 6H, CH2CH3),
2.68-2.62 & 2.60-2.54 (2m, 2H, 3',3''-H2), 2.30-2.23 & 2.3-
2.14 (2m, 2H, 2',2''-H2), 1.29 (t, J = 7.2 Hz, 9H, CH2CH3).
13C NMR (D2O) ꢁ 158.10 (C-4), 147.96 (C-6), 145.78 (C-8),
139.36 (C-2), 123.42 (C-5), 85.16 (C-5'), 81.02 (C-1'), 64.16
(C-4'), 46.60 (CH2CH3), 31.66 (C-2'), 23.71 (C-3'), 8.20
(CH2CH3); HRMS [M – Et3NH+]- m/z 299.0517, calculated
for C10H12N4O5P 299.0545.
o
hygroscopic micro-crystals (m. p. 174-176 C). 31P NMR
data are given in Table 1.
General Procedure for the Synthesis of Nucleoside
H-phosphonate Monoesters of Type (7)
Nucleoside analogue of type (5) (1.05 mmol) and ammo-
nium (9H-fluoren-9-yl)methyl -H-phosphonate (4) (1.0
mmol, 1 molar equiv.) were made anhydrous by repeated
evaporation of the added pyridine. After this, the residue was
dissolved in 10 mL of CH2Cl2/pyridine (95:5, v/v) and PvCl
(1.5 molar excess) was added. H-Phosphonylation of nucleo-
sides (5) was complete (31P NMR, TLC) after ca 60 min. The
reaction mixture was diluted with the same volume of meth-
ylene chloride, and the excess of nucleoside (5) and polar
remainings from the condensing agent, were removed by
extraction of the organic solution with water (one third of the
total volume). The organic layer was separated, dried over
Na2SO4 anh., and the volatiles were removed by evaporation.
The remaining oily residue was dissolved in CH3CN/Et3N [2
: 1, v/v; 10 mL per 1 mmol of (6)] and kept 20 min at room
temperature to effect a quantitative elimination of the (9H-
fluoren-9-yl)methyl group. The reaction mixture was evapo-
rated and products (7) were isolated by a silica gel filtration
using a stepwise gradient of methanol (0 – 30% v/v) in
methylene chloride containing Et3N (3% v/v). Fractions con-
taining pure product (7) were collected and evaporated, to
furnish colourless, crispy foams. 31P NMR data of products
(7) are listed in Table 1.
3'-Azido-3'-dideoxythymidin-5'-yl H-phosphonate Triethy-
lammonium Salt (7c)
Yield 85%. 1H NMR (D2O) ꢁ 7.71 (s, 1H, H-6), 6.80 (d, J
= 638.2 Hz, 1H, H-P), 6.26 (t, 1H, J = 6.6 Hz, 1H, 1'-H),
4.53-4.49 (m, 1H, 3'-H), 4.21-4.18 (m, 1H, 4'-H), 4.14-4.09
(m, 2H, 5',5''-H2), 3.21 (q, J = 7.2 Hz, 6H, CH2CH3), 2.51 (t,
J = 6.4 Hz, 2H, 2',2''-H2), 1.92 (s, 3H, 5-CH3), 1.30 (t, J =
7.2 Hz, 9H, CH2CH3). 13C NMR (D2O) ꢁ 166.39 (C-4),
151.54 (C-2), 137.19 (C-6), 111.57 (C-5), 84.92 (C-1'),
82.89 (C-4'), 64.31 (C-5'), 60.48 (C-3'), 46.65 (CH2CH3),
36.27 (C-2'), 11.61 (5-CH3), 8.41 (CH2CH3); HRMS [M –
Et3NH+]- m/z 330.0576, calculated for C10H13N5O6P
330.0603.
2',3'-Dideoxyuridin-5'-yl H-phosphonate Triethylammo-
nium Salt (7d)
Yield 88%. 1H NMR (D2O) ꢁ 7.94 (d, J = 8.0 Hz, 1H, 6-
H), 6.76 (d, J = 637.4 Hz, 1H, H-P), 6.10 (dd, 1H, J = 7.2
Hz, J = 3.2 Hz, 1H, 1'-H), 5.88 (d, J = 8.0 Hz, 1H, 5-H) 4.36-
4.33 (m, 1H, 4'-H), 4.18-4.13 & 4.02-3.97 (2m, 2H, 5',5''-
H2), 3.20 (q, J = 7.2 Hz, 6H, CH2CH3), 2.48-2.42 (m, 1H, 3'-
or 3''-H), 2.20-2.09 (m, 2H, 2', 2''-H2), 2.02-1.92 (m, 1H, 3'-
or 3''-H), 1.30 (t, J = 7.2 Hz, 9H, CH2CH3). 13C NMR (D2O)
ꢁ 166.26 (C-4), 151.55 (C-2), 142.10 (C-6), 101.69 (C-5),
86.39 (C-1'), 80.50 (C-5'), 64.31 (C-4'), 46.62 (CH2CH3),
1H, 13C NMR and HRMS Data for Reagent (4) and
Nucleoside H-phosphonate Monoesters (7a-d)
Ammonium (9H-fluoren-9-yl)methyl H-phosphonate (4)
1
Yield 90%. H NMR (DMSO-d6) ꢁ 7.86 (d, J = 7.4 Hz,
2H, ArH), 7.67 (d, J = 7.4 Hz, 2H, ArH), 7.42 (br s, 4H,

Synthesis of Anti-HIV Non-Lipophilic Nucleoside H-Phosphonates
Letters in Organic Chemistry, 2009, Vol. 6, No. 6
499
[4]
[5]
[6]
Jankowska, J.; Sobkowski, M.; Stawinski, J.; Kraszewski, A.
Tetrahedron Lett., 1994, 35(20), 3355-3358.
Garegg, P. J.; Regberg, T.; Stawinski, J.; Stromberg, R. Chem. Scr.,
1985, 25, 280-282.
Hao, Z.; Cooney, D. A.; Hartman, N. R.; Perno, C.-F.; Fridland, A.;
DeVico, A. L.; Sarngadharan, M. G.; Broder, S.; Johns, D. G. Mol.
Pharmacol., 1988, 34, 431-435.
Morse, G. D.; Shelton, M. J.; O'Donnell, A. M. Clin.
Pharmacokinet., 1993, 24(2), 101-123.
Zelphati, O.; Deglos, G.; Loughrey, H.; Leserman, L.; Pompon, A.;
Puech, F.; Maggio, A.-F.; Imbach, J.-L.; Gosselin, G. Antiviral
Res., 1993, 21, 181-195.
Yang, Z.-W.; Xu, Z.-S.; Shen, N.-Z.; Fang, Z.-Q. Nucleoside
Nucleotide, 1995, 14(1&2), 167-173.
31.24 (C-2'), 26.50 (C-3'), 8.40 (CH2CH3); HRMS [M –
Et3NH+]- m/z 275.0408, calculated for C9H12N2O6P
275.0433.
ACKNOWLEDGEMENTS
[7]
[8]
We are grateful to Dr. Y. Hagiwara and Dr. K. Izawa
from Ajinomoto Co., Inc., Kawasaki, Japan for a gift of ddA
and ddI. A financial support from the government of the Pol-
ish Republic (project No. PBZ-MNiSW - 07/I/2007; 2008-
2010) is gratefully acknowledged.
[9]
[10]
[11]
[12]
[13]
[14]
Stawinski, J.; Thelin, M. Nucleoside Nucleotide, 1990, 9(1), 129-
135.
Cieslak, J.; Sobkowski, M.; Kraszewski, A.; Stawinski, J.
Tetrahedron Lett., 1996, 37(26), 4561-4564.
Froehler, B. C.; Matteucci, M. D. Tetrahedron Lett., 1986, 27(4),
469-472.
Garegg, P. J.; Lindh, I.; Regberg, T.; Stawinski, J.; Stromberg, R.;
Henrichson, C. Tetrahedron Lett., 1986, 27(34), 4055-4058.
Adamiak, R. W.; Biala, E.; Grzeskowiak, K.; Kierzek, R.;
Kraszewski, A.; Markiewicz, W. T.; Okupniak, J.; Stawinski, J.;
Wiewiorowski, M. Nucleic Acids Res., 1978, 5(6), 1889-1905.
REFERENCES
[1]
Cieslak, J.; Sobkowski, M.; Jankowska, J.; Wenska, M.; Szymczak,
M.; Imiolczyk, B.; Zagorowska, I.; Shugar, D.; Stawinski, J.;
Kraszewski, A. Acta Biochim. Pol., 2001, 48(2), 429-442.
Yashunsky, D. V.; Nikolaev, A. V. J. Chem. Soc. Perkin Trans. 1
2000, 1195-1198.
[2]
[3]
Marugg, J. E.; Burik, A.; Tromp, M.; Van der Marel, G. A.; Van
Boom, J. H. Tetrahedron Lett., 1986, 27(20), 2271-2274.