The preparation of 3H-labeled acyclic nucleoside phosphonates and study of their stability

Article abstract of DOI:10.1135/cccc2010020

9-(2-Phosphonomethoxyethyl)-2,6-diamino-[8-³H]purine (4), 9-(2-phosphonomethoxyethyl)- [8-³H]guanine (6) and (R)-9-(2-phosphonomethoxypropyl)-[8-³H]adenine (11) with specific activities of 10.9, 7.9 and 16 Ci/mmol, respectively, were prepared by a catalytic dehalogenation of the corresponding 8-bromo derivatives 1, 2 and 9. The rate of the exchange of the tritium label on C-8 of the purine ring in title compounds with the hydrogen of water under physiological pH at 20 °C was studied using ³H NMR. The loss of ³H-label attained 7% in [8-³H]tenofovir (11), 10% in [8-³H]PMEDAP (4) and 12% in [8-³H]PMEG (6) after the period of 3 weeks. Storage at a temperature of -196 °C in liquid nitrogen ensured a better than 97% radiochemical purity of the prepared labeled compounds even after a six-month period.

Full text of DOI:10.1135/cccc2010020

Preparation of 3H-Labeled Acyclic Nucleoside Phosphonates
757
3
THE PREPARATION OF H-LABELED ACYCLIC NUCLEOSIDE
PHOSPHONATES AND STUDY OF THEIR STABILITY
1,
2
3
Tomáš ELBERT *, Petra BŘEHOVÁ and Antonín HOLÝ
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i.,
1
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: elbert@uochb.cas.cz,
2
3
brehova@uochb.cas.cz, holy@uochb.cas.cz
Received March 5, 2010
Accepted June 14, 2010
Published online July 30, 2010
9-(2-Phosphonomethoxyethyl)-2,6-diamino-[8-3H]purine (4), 9-(2-phosphonomethoxyethyl)-
[8-3H]guanine (6) and (R)-9-(2-phosphonomethoxypropyl)-[8-3H]adenine (11) with specific
activities of 10.9, 7.9 and 16 Ci/mmol, respectively, were prepared by a catalytic dehalogen-
ation of the corresponding 8-bromo derivatives 1, 2 and 9. The rate of the exchange of the
tritium label on C-8 of the purine ring in title compounds with the hydrogen of water un-
der physiological pH at 20 °C was studied using 3H NMR. The loss of 3H-label attained 7%
in [8-3H]tenofovir (11), 10% in [8-3H]PMEDAP (4) and 12% in [8-3H]PMEG (6) after the pe-
riod of 3 weeks. Storage at a temperature of –196 °C in liquid nitrogen ensured a better than
97% radiochemical purity of the prepared labeled compounds even after a six-month period.
Keywords: Tritium; 3H NMR; Acyclic nucleotide analogues; 3H-label stability; Tenofovir;
Adefovir; Radioactive labelling.
Acyclic nucleoside phosphonates (ANPs) exhibit a broad spectrum of antivi-
ral and cytostatic activities1. Among them, (R)-9-(2-phosphonomethoxy-
propyl)adenine ((R)-PMPA, tenofovir) (10) is active against retroviruses. Its
prodrug tenofovir disoproxil fumarate was approved for the treatment of
the human immunodeficiency virus (HIV) infection (Viread) as well as its
fixed combinations with emtricitabine (Truvada), emtricitabine and
efavirenz2 (Atripla). Tenofovir was also approved for the treatment of the
hepatitis B virus (HBV) infection3 in 2008. 9-(2-Phosphonomethoxyethyl)-
2,6-diaminopurine (PMEDAP) (3) shows potent in vitro and in vivo antiviral
activity against both DNA viruses and retroviruses4. 9-(2-Phosphono-
methoxyethyl)guanine (PMEG) (5) has also antiviral activity, but the inter-
est in PMEG has focused on its anticancer activity. It is an active compound
of GS-9219, a potent antineoplastic agent, targeting preferentially lym-
phoid cells5.
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 7, pp. 757–766
© 2010 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2010020

758
Elbert, Břehová, Holý:
Because of the importance of these compounds their biological activities
are still being further explored, the high biological potency is dependent on
their transport across the cellular membrane and on their stability inside
3
the cells6. The above mentioned ANPs were labeled by the H radionuclide
at position 8 of the purine ring to facilitate their detection in the biochemi-
cal assays monitoring their cellular uptake and metabolism in the cell. It
has previously been reported in the literature7 that the tritium label at posi-
tion 8 of the purine ring can under certain conditions undergo an exchange
of the hydrogen with the solvent. We have thus studied the stability of the
3
tritium label in water and phosphate buffer (pH 7.4) solutions by H NMR.
RESULTS AND DISCUSSION
3
Syntheses of H-labeled Acyclic Nucleoside Phosphonates
As the 8-bromo derivatives of PMEDAP (1) and PMEG (2) have recently been
prepared8, a reductive catalytic dehalogenation has been chosen as the
method of the introduction of tritium into the title compounds (Scheme 1).
SCHEME 1
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Preparation of 3H-Labeled Acyclic Nucleoside Phosphonates
759
8-Bromo tenofovir (9) was prepared using the same procedure as has been
published for derivatives 1 and 2, i.e. the bis(2-propyl) (R)-9-(2-phosphono-
methoxypropyl)adenine (7) was first brominated to yield bis(2-propyl)
8-bromo-(R)-9-(2-phosphonomethoxypropyl)adenine (8). The deprotection
of the phosphonate group of derivative 8 was then accomplished by a bro-
motrimethylsilane procedure (Scheme 2).
SCHEME 2
The 5% palladium oxide on the barium sulfate was used as a catalyst,
triethyl amine was used as the base and the dehalogenation was performed
in water. In Table I, the yields of the crude products, the radiochemical
purities after the purification on radio-HPLC and the specific activities as-
sayed by two independent methods have been summarized.
The low yield of crude [8-3H]PMEDAP (4) was caused by 1 ml of acetic
acid having been added to the reaction mixture in an attempt to prevent
the exchange of the tritium label in alkaline milieu. Unfortunately, the sol-
ubility of the product was suppressed and it remained absorbed on the cata-
lyst. During the preparation of [8-3H]PMEG (6), the addition of acetic acid
was omitted, which improved the yield. However, even though the attained
specific activities for [8-3H]PMEDAP (4) and [8-3H]PMEG (6) were sufficient
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 7, pp. 757–766

760
Elbert, Břehová, Holý:
for the planned biochemical experiments, it seemed that higher specific ac-
tivities should have been obtained. We evaluated the weak spots of the ex-
perimental procedure. The reaction of the 8-bromo derivatives 1 and 2 with
gaseous tritium was performed by the service laboratory. The removal of
the catalyst by centrifugation was preceded by the removal of the labile ac-
tivity (by successive evaporations of the three 3-ml water portions from the
reaction mixture). We concluded that there is probably a backward ex-
change of the tritium with the water in the presence of the catalyst during
the procedure of the removal of the labile activity. During the preparation
of [8-3H]tenofovir, the catalyst was removed by filtration through a 0.45-µ
teflon syringe filter, prior to the labile activity removal. The much higher
specific activity of [8-3H]tenofovir (11) (see Table I) in comparison with
[8-3H]PMEDAP (4) and [8-3H]PMEG (6) supports the above-mentioned idea
of the backward exchange with water in the presence of the catalyst. The
structures of the tritiated compounds 4, 6 and 11 were confirmed by their
1H NMR spectra. The distinctive position of the label at C-8 of the purine
3
ring in all the three compounds was confirmed by H NMR spectra.
TABLE I
The yields, radiochemical purities, specific activities and stability data of the tritiated com-
pounds
Parameter
[3H]PMEDAP (4)
[3H]PMEG (6)
[3H]Tenofovir (11)
25.3
112.3
226
Yield of the crude product, mCi
Radiochemical purity after
HPLC, %
>99
10.9
10.7
>99
7.9
7
>98
16
Speccific activity (UV), Ci/mmol
Specific activity (1H NMR),
Ci/mmol
15.7
Radiochemical purity after
5 months of storagea, %
97.1
96.1
97.5
97.7
97.1
Radiochemical purity after
11 months of storagea, %
96.33
a
The tritiated compounds were stored at a concentration of 2 mCi/ml of water at –196 °C
in liquid nitrogen.
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Preparation of 3H-Labeled Acyclic Nucleoside Phosphonates
761
3
The Stability of H-labeled Acyclic Nucleoside Phosphonates
To mimic the physiological conditions, the stability of the tritium label on C-8
of the purine ring of [3H]PMEDAP (4), [3H]PMEG (6) and [3H]tenofovir (11)
was studied in 50 mM phosphate buffer, pH 7.4. For comparison, the loss of
3
tritium was followed in neutral water solution for all three H-labeled deriv-
atives and in alkaline solution for [3H]tenofovir (11). The percentage of tri-
tium label on C-8 was calculated from integrals of intensity of NMR signals
3
3
of H on C-8 and in water (no other H signals appeared in spectra during a
60-day period). The results are summarized in Fig. 1. Under neutral condi-
tions, the tritium label on C-8 was reasonably stable and after 3 weeks there
was still more than 95% of the tritium bound in the purine ring. At pH 7.4,
3
the exchange rate was higher and after 3 weeks the content of H-label in
guanosine derivative 6 dropped by 12%, in diaminopurine derivative 4 and
in adenine derivative 11 by 10 and 7%, respectively. The highest rate of ex-
change in guanosine derivative 6 is in accordance with the literature
data7a,7b. On the other hand, our results suggest higher dependence of the
exchange rate on the pH in nucleotide analogues 6, 4 and 11 in compari-
son with the free bases and the nucleosides. The increase in the rate of the
exchange with increasing pH supports the idea of abstraction of tritium
from C-8 by hydroxyl anion as a rate determining step.
The results of the radiochemical purity checks of the samples kept in liq-
3
uid nitrogen are summarized in Table II. In the H NMR spectra of all three
of the labeled compounds stored in liquid nitrogen for periods indicated in
3
Table II, no H signal of water was detected.
FIG. 1
The exchange of 3H-label on C-8 with water at different pH and 20 °C (the detailed conditions
are given in Experimental)
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Elbert, Břehová, Holý:
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Preparation of 3H-Labeled Acyclic Nucleoside Phosphonates
763
CONCLUSIONS
[8-3H]PMEDAP (4), [8-3H]PMEG (6) and [8-3H]tenofovir (11) with high spe-
cific activities and excellent radiochemical purities were prepared. Using
3
3H NMR spectroscopy, we observed the exchange of the H-label on C-8 of
purine ring of these labeled compounds with water at room temperature.
The rate of the exchange increased with increasing pH of the solution.
At pH 7.4, i.e. under the conditions of biochemical experiment on plant
cells, the loss of 3H-label attained 7% in [8-3H]tenofovir (11), 10% in
[8-3H]PMEDAP (4) and 12% in [8-3H]PMEG (6) after the period of 3 weeks.
This exchange must be taken into account when interpreting the results of
biochemical experiments with cultivation periods longer than 1 week. The
storage of the compounds labeled by tritium at the temperature –196 °C
in liquid nitrogen proved to be a very efficient method of conserving suf-
ficient radiochemical purity for more than 6 months.
EXPERIMENTAL
The NMR spectra (δ, ppm) were recorded with a Bruker Avance 400 spectrometer (1H at 400,
13C at 100.6 MHz) in DMSO-d6 or D2O. Chemical shifts in DMSO-d6 were referenced to the
solvent signal for 1H δ 2.5 and for 13C δ 39.7. Chemical shifts in D2O were referenced to
1,4-dioxane for 1H δ 3.75 and for 13C δ 67.19. The 3H and 1H NMR spectra of the labeled
compounds were measured on a Bruker Avance II 300 MHz spectrometer in D2O, with the
signal of water at 4.7 ppm taken as an internal standard. Mass spectra were measured on
a LCQ classic spectrometer using electrospray ionization (ESI). HPLC was performed on
a system consisting of a WATERS Delta 600 Pump and Controller, a WATERS 2487 UV detec-
tor and a RAMONA radio chromatographic detector from Raytest (Germany) with inter-
changeable fluid cells. For preparative runs, the cell with a single small crystal of solid
scintillator was used; for analytical runs, the column effluent was mixed with a Zinsser
Quickszint Flow 302 cocktail at a 1:3 ratio. The data were collected and processed using
Empower 2.0 software. Radioactivities were measured on a Perkin–Elmer Tri-Carb 2900
liquid scintillation counter (LSC) in a Zinsser Quicksafe A cocktail. The evaporations were
done using a CentriVap concentrator from Labconco. The 5% PdO on the BaSO4 catalyst
and the triethyl amine were purchased from Sigma–Aldrich.
Bis(2-propyl) 8-Bromo-(R)-9-(2-phosphonomethoxypropyl)adenine (8)
Bis(2-propyl) (R)-9-(2-phosphonomethoxypropyl)adenine (7) prepared as described in ref.9
(1.6 g, 3.6 mmol) in DMF (10 ml) was treated with 0.7 M bromine solution in CCl4 at room
temperature for 48 h. The resulting mixture was diluted with CHCl3 and washed with satu-
rated Na2S2O3 and brine, with the organic extract subsequently dried over MgSO4. Flash
chromatography (CHCl3/MeOH) afforded diester 8 as a white solid (1.08 g, 56%), m.p.
111 °C. ESI MS: 450.1 (85) [M + H]+; 472.1 (100) [M + Na]+. 1H NMR (DMSO-d6): 8.11 s, 1 H
(H-2); 7.39 bs, 2 H (NH2); 4.45 dh, J(H-C-O-P) = 7.6, J(CH-CH3) = 6.2, 1 H and 4.36 dh,
J(H-C-O-P) = 7.7, J(CH-CH3) = 6.2, 1 H (CH ipr.); 4.05–4.19 m, 3 H (H-1’, H-2’); 3.75 dd,
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 7, pp. 757–766

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Elbert, Břehová, Holý:
Jgem = 13.9, J(H-C-P) = 8.9, 1 H and 3.56 dd, Jgem = 13.9, J(H-C-P) = 9.3, 1 H (PCH2O);
1.17 d, J(3’-2’) = 6.4, 3 H (H-3’); 1.16 d, 3 H, 1.11 d, 3 H, 1.08 d, 3 H, 1.03 d, 3 H,
J(CH3-CH) = 6.2 (CH3 ipr.). 13C NMR (DMSO-d6): 154.99 (C-6); 152.94 (C-2); 151.19 (C-4);
127.17 (C-8); 119.17 (C-5); 74.98 d, J(2’-P) = 12.0 (C-2’); 70.35 d, J(C-O-P) = 6.4 and 70.22 d,
J(C-O-P) = 6.3 (CH ipr.); 62.72 d, J(C-P) = 164.4 (PCH2O); 48.69 (C-1’); 23.65–23.99 m (CH3
ipr.); 16.94 (C-3’).
8-Bromo-(R)-9-(2-phosphonomethoxypropyl)adenine (9)
Diester 8 (540 mg, 1.2 mmol) in acetonitrile (10 ml) was treated with Me3SiBr (1 ml) at
room temperature overnight. The volatiles were removed under reduced pressure and the
residue was co-distilled with water. The crude product was purified by the preparative HPLC
(a gradient of MeOH (0–100%) in H2O). Crystallization from ethanol afforded a white crys-
talline product (150 mg, 35%), whose m.p. is not reached below 250 °C, decomposition.
ESI MS: 364.1 (38) [M – H]–. 1H NMR (D2O, ref. dioxane): 8.37 s, 1 H (H-2); 4.37 m, 2 H
(H-1’); 4.05–4.12 m, 1 H (H-2’); 3.62 dd, 1 H, J(H-C-P) = 8.90, Jgem = 13.48 and 3.43 dd,
1 H, J(H-C-P) = 8.90, Jgem = 13.47 (OCH2P); 1.28 d, 3 H, J(3’-2’) = 6.29 (H-3’). 13C NMR
(D2O, ref. dioxane): 150.92 (C-6); 150.09 (C-4); 145.95 (C-2); 132.53 (C-8); 119.44 (C-5);
76.63 d, J(2’-P) = 10.27 (C-2’); 65.62 d, J(CH2-P) = 157.73 (OCH2P); 50.31 (C-1’); 17.14
(C-3’). For C9H13BrN5O4P (366.11) calculated: 29.53% C, 3.58% H, 21.83% Br, 19.13% N,
8.46% P; found: 29.36% C, 3.62% H, 21.57% Br, 18.90% N, 8.00% P.
9-(2-Phosphonomethoxyethyl)-2,6-diamino-[8-3H]purine (4)
To 8-bromo PMEDAP 1 (6 mg, 16.3 µmol) in the presence of 27 mg 5% PdO on BaSO4 in a
small flask equipped with a magnetic stirrer, water (1.2 ml) and triethyl amine (0.13 ml)
were added. The reaction mixture was degassed by three successive freeze-thaw cycles under
vacuum. 5 Ci of carrier-free 3H2 were transferred to the reaction flask (with a starting pres-
sure of 640 torr) and the reaction was left to proceed under vigorous agitation at room tem-
perature for 1.5 h. The residual tritium gas was pumped off and the reaction mixture was
transferred to a heart-shaped flask (the reaction flask was washed 3 times with 1 ml of wa-
ter, added to the reaction mixture). Acetic acid (0.5 ml) was added and the solvents were
evaporated in a closed system under vacuum (the solvent trap was cooled by liquid nitro-
gen). The residue was dispersed in 3 ml of water and the mixture was evaporated to dryness.
This procedure was repeated once more. After the last evaporation, the residue was dispersed
in 3 ml of water and the catalyst was separated by centrifugation. The supernatant was
taken off and the catalyst was washed three times with 2 ml of water. The volume of the
unified supernatants was adjusted to 10 ml with water. The total activity of the crude prod-
uct was 25.3 mCi. According to the HPLC analysis of the crude product, there was no start-
ing compound and more than 95% of the activity was in desired product 4. The crude
product was purified on a semi-preparative Synergi 4µ Fusion-RP 80, 250 × 10 mm column
(Phenomenex, USA) in the isocratic mode with water containing 0.25% (v/v) of acetic acid
as the mobile phase. The yield was 17.65 mCi of the product with a radiochemical purity of
>99% (radio HPLC). The 3H NMR (D2O, ref. TDO) was 7.98 s (3H-8). The 1H NMR was
identical to that of the standard with the exception of the integration of the H-8 signal.
From the decrease of the intensity of the 1H-8 signal, the specific activity was calculated as
10.7 Ci/mmol. The UV method of specific activity assay consisted of a measurement of the
volume activity by LSC and an assay of the mass concentration by HPLC at 284 nm. The re-
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Preparation of 3H-Labeled Acyclic Nucleoside Phosphonates
765
sult was 10.9 Ci/mmol. Before the solution was stored in liquid nitrogen, the volume activ-
ity was adjusted to 2 mCi/ml.
9-(2-Phosphonomethoxyethyl)-[8-3H]guanine (6)
8-Bromo PMEG 2 (6.6 mg, 18 µmol) in the presence of 26.5 mg of 5% PdO on BaSO4 and
triethyl amine (0.13 ml) in water (1.2 ml) was treated with 5 Ci of carrier-free 3H2 as de-
scribed for 8-bromo PMEDAP 1 (see above). The work-up was the same with one exception,
i.e. no acetic acid was added before the first evaporation of the reaction mixture. The yield
of the crude product was 112.3 mCi. According to the radio HPLC, some unreacted 8-bromo
PMEG 2 was present; more than 95% of activity was in desired product 6. One half of the
solution of the crude product was evaporated to dryness, dissolved in 1 ml of 0.25% (v/v)
ammonium hydroxide and purified on a semi-preparative HPLC column under the same
conditions as for [8-3H]PMEDAP 4 (see above). The yield was 31.1 mCi of product with
a radiochemical purity of >99% (radio HPLC). The 3H NMR (D2O, ref. TDO) was 7.93 s
(3H-8). The 1H NMR was identical with that of the standard. The specific activity calculated
from the 1H NMR was 7 Ci/mmol, the UV assay yielded 7.9 Ci/mmol. Before the solution
was stored in liquid nitrogen, the volume activity was adjusted to 2 mCi/ml.
(R)-9-(2-Phosphonomethoxypropyl)-[8-3H]adenine (11)
8-Bromo (R)-PMPA 9 (25.4 mg, 69.4 µmol) in the presence of 26.2 mg of 5% PdO on BaSO4
and triethyl amine (0.13 ml) in water (1 ml) was treated with 5 Ci of carrier-free 3H2 as de-
scribed for 8-bromo PMEDAP 1 (see above). The reaction mixture was left in contact with
tritium gas for 3 h. The reaction flask was disconnected from the tritiation manifold and the
reaction mixture was filtered through a 0.45-µ PTFE syringe filter. The reaction flask and fil-
ter were washed with three 1-ml portions of water. The clear colorless reaction mixture was
evaporated to dryness. The solid white residue was dissolved in 4 ml of water and again
evaporated to dryness. The residue was dissolved in 8 ml of water and the activity was veri-
fied. The total activity of the crude product was 226 mCi. More than 95% of the activity was
in the desired product and no starting compound was present according to radio HPLC. Part
of the crude product (56 mCi) was purified on a semi-preparative HPLC column under the
same conditions as for [8-3H]PMEDAP 4 (see above). The yield was 49.8 mCi of the product
with a radiochemical purity of >99% (radio HPLC). The 3H NMR (D2O, NaOD, ref. TDO) was
8.35 s (3H-8). The 1H NMR was identical with that of the standard. The specific activity cal-
culated from 1H NMR was 15.7 Ci/mmol, the UV assay yielded 16 Ci/mmol. Before the solu-
tion was stored in liquid nitrogen, the volume activity was adjusted to 2 mCi/ml.
The Study of the Stability of the Tritiated Compounds 4, 6 and 11
a) The stability of 3H-label at pH 7. The solution of 2 mCi of each of the labeled com-
pounds in 0.6 ml of water containing 10% D2O was placed in an NMR tube and the
3H NMR spectra were taken at specified time intervals. The tubes were kept at 20 °C between
the measurements.
b) The stability of 3H-label at pH 7.4. The aliquotes of water solutions of labeled com-
pounds (2 mCi) were evaporated on a CentriVap to dryness, each residue was dissolved in
a mixture of 540 µl of 0.05 M phosphate buffer, pH 7.4, and 60 µl of deuterated water.
The solutions were filtered through 0.45-µ teflone syringe filter to NMR tubes and 3H NMR
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Elbert, Břehová, Holý:
spectra were taken at specified time intervals. The tubes were kept at 20 °C between the
measurements.
c) The solutions of the labeled compounds 4, 6 and 11 in water at a concentration of
2 mCi/ml were placed in 1.5 ml Nalgene Cryovials protected by Nunc Cryoflex foil and
stored in liquid nitrogen. At the intervals indicated, the vials were withdrawn from the liq-
uid nitrogen, the contents were left to thaw, and 2 µl of the solution were injected on the
analytical radio-HPLC. D2O was added to the concentration of 10% and the sample was
checked by 3H NMR.
This work was performed as a part of a research project of the Institute of Organic Chemistry and
Biochemistry (Z40550506) and was supported by Grant IAA400550801 of the Grant Agency of the
Academy of Sciences of the Czech Republic and by the Centre of New Antivirals and Antineoplastics
1M0508 of the Ministry of Education, Youth and Sports of the Czech Republic.
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