Synthesis of 9,9'-[1,2-Ethanediylbis(oxymethylene)]bis-2-amino- 1,9-dihydro-6H-purin-6-one, an impurity of acyclovir

Article abstract of DOI:10.3390/molecules17088735

The synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene)]bis-2-amino-1,9- dihydro- 6H-purin-6-one, a minor impurity of acyclovir, is described. Starting with commercial N-(9-acetyl-6-oxo-1H-purin-2-yl)acetamide, the process uses an acid catalysed phase transfer catalysis (PTC) process to produce the selective alkylation at the 9 position of the guanine ring.

Full text of DOI:10.3390/molecules17088735

Molecules 2012, 17, 8735-8741; doi:10.3390/molecules17088735
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of 9,9′-[1,2-Ethanediylbis(oxymethylene)]bis-2-amino-
1,9-dihydro-6H-purin-6-one, an Impurity of Acyclovir
Rosa M. Suárez 1, Maria Paz Matía 1, José Luis Novella 1, Andres Molina 2, Antonio Cosme 2,
Juan José Vaquero 3 and Julio Alvarez-Builla 3,*
1
Fine Chemicals Pilot Plant Facility, University of Alcalá, 28871 Alcalá de Henares, Madrid, Spain
2
Department of Research and Development, Química Sintética S. A., c./Dulcinea s/n,
28805 Alcala de Henares, Madrid, Spain
3
Department of Organic Chemistry, University of Alcalá, 28871 Alcalá de Henares, Madrid, Spain
* Author to whom correspondence should be addressed; E-Mail: julio.alvarez@uah.es;
Tel.: +34-91-885-4606; Fax: +34-91-885-4686.
Received: 16 June 2012; in revised form: 13 July 2012 / Accepted: 16 July 2012 /
Published: 25 July 2012
Abstract: The synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene)]bis-2-amino-1,9-dihydro-
6H-purin-6-one, a minor impurity of acyclovir, is described. Starting with commercial
N-(9-acetyl-6-oxo-1H-purin-2-yl)acetamide, the process uses an acid catalysed phase
transfer catalysis (PTC) process to produce the selective alkylation at the 9 position of the
guanine ring.
Keywords: nucleosides; acyclovir; drug impurities; phase transfer catalysis
1. Introduction
Drug impurities, called in the literature related substances in an active pharmaceutical ingredient
(API), can have a significant impact on the quality and safety of the pharmaceutical preparations.
Therefore, in any API, it is necessary to study the impurity profile, and control it during the
preparation of the pharmaceutical. As indicated in the ICH guidelines, any impurity, formed at a level
of ≥0.10% with respect to the API, should be identified, synthesized and characterized thoroughly [1].
ICH classifies organic impurities as starting materials, by-products, intermediates, degradation
products, reagents, ligands and catalysts. All of them, in addition to determine the quality and

Molecules 2012, 17
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suitability of the API, are related with possible toxic episodes, or can be used to detect the synthetic
method used to obtain the main product [2–6].
Acyclovir (1, Figure 1) [7,8] is a guanosine analogue antiviral prodrug used for the treatment of
herpes simplex and herpes zoster, and is one of the most commonly used antiviral drugs. Different
synthetic approaches have been described to obtain this compound, many of them in patents [9–14]
describing alternatives for the preparation of the product. Detailed chemical strategies and mechanisms
involved have been detailed in a review [15].
Figure 1. Acyclovir and related compounds.
Acyclovir
1
3
Guanosine
2
Of the processes described, those involving the use of 2-benzoyloxyethoxymethylene chloride (7)
as intermediate [9,10] can give rise to the appearance of 3 (Figure 1) as a minor impurity of 1.
Compound 3 has been cited [16] as part of an HPLC study into acyclovir-related compounds. In our
case, the product was obtained in order to supply samples for analysis validation.
The reason for the formation of 3 is outlined in Scheme 1. The synthesis of acyclovir (1) through
the approaches outlined above requires the preparation of 7 [9,10,17] from the ester 6 [17]. Compound 6
is usually obtained by monoacylation of ethylene glycol, which is used in large excess in the process.
Usually, 6 is purified by distillation, which could easily produce—particularly in large scale
preparations—contaminant traces of the starting glycol. On preparing 7 with paraformaldehyde and HCl,
traces of the symmetric compound 8 would be produced, and the rest of the process would yield variable
amounts of 3 as a contaminant of acyclovir. This has been a common problem in the monofunctionalisation
of 1,2-diols [18,19], although recently a variety of catalysts as lanthanide salts, zeolites and tin
derivatives, among others, have been successfully applied to the monofunctionalisation [20].
Compound 3 is not listed as an official impurity by the European Pharmacopea [21].
Scheme 1. Initial steps of the synthesis of acyclovir.
1
paraformaldehyde
5
(34%)
7
Rflx, 72h
(65%)
ClCH2CH2Cl, HCl
0 ºC, 3 hrs
6
3
4
(traces)
8

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2. Results and Discussion
The method for the preparation of 3 is described in Scheme 2, and starts with ethylene glycol,
converted with paraformaldehyde and hydrogen chloride without solvent, to the dichloro derivative 8 [22].
Compound 8 was subsequently converted into the diacetate 9. This product was then treated in
Dowtherm Q at 150 °C with 2,9-diacetylguanine (10), in the presence of TsOH as acid catalyst and
tetrabutylammonium bromide (TBAB) as phase transfer catalyst. In the absence of TBAB, no
transformation took place.
Scheme 2. Synthesis of impurity 3.
anh NaOAc
(CH2O)n
(2 equiv)
O
(2 equiv)
O
Cl
O
O
CH3
O H
Cl
O
H3C
O
O
HO
90 ºC, 2 h
HCl gas,
rt, 2 h
O
90%
9
5
8
56%
O
N
O
O
H
TsOH, TBAB
Dowtherm Q
+
H
H
H
N
N
N
N
N
H
O
O
N
O
N
+
9
H3C
N
H
N
H3C
N
H
N
150 ºC, 15 h
CH3
N
N
O
H3C
R
N
H
CH3
10
10
O
O
12
O
-(Ac)2O
O
H3C
H3C
R
H3C
O
O
+
O
O
9
11
O
N
N
N
O
N
N
O
N
O
H
HO
N
H
N
H
O
N
N
NH2
N
H
H
N
O
O
N
O
N
N
H
H3C
N
N
N
N
H
N
O
CH3
H
H
H2O, reflux 10h
55%
13
O
77%
3
H
The process indicated in Scheme 2 produced the diacetyl derivative 13, yielding acetic anhydride as
a subproduct. The method is based in an unusual acid catalysed PTC process described in the 70s by
Landini et al., who used it to convert primary alcohols into alkyl chlorides using aqueous HCl [23], to
cleave ethers and esters with aqueous HBr, with conversion of the resulting aliphatic alcohols into
bromides [24], or to add hydrohalogenic acids to alkenes, using aqueous solutions of the corresponding
acid [25]. The method has been more recently improved by Goverdhan et al., with the use of
microwaves, to cleave cyclic ethers to halo alcohols, lactones to haloacids or diols to haloalcohols [26].
Having in mind those results, in our method, the acid, associated to the phase transfer catalyst by a
hydrogen bond, as indicated by Landini, should be transferred to the organic solvent (Figure 2), thus
activating the basic nitrogen of the imidazole moiety, and making the 1-acetyl group more reactive
(Scheme 2). Thus, the acetyl group should be transferred to 9, yielding the oxonium intermediate 11,
which is attacked by the basic nitrogen of 12, displacing acetic anhydride and yielding 13 as the main
product. The last step to deacylate 13, has been extensively described for acyclovir [11], being ammonia
in methanol the most common method [11, 27,28], or alternatively, hot aqueous methylamine [29,30].
In our case, the best yields were obtained by adapting the methylamine procedures by treatment of 13

Molecules 2012, 17
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with 2-aminoethanol, which produced the final product 3 with the best purity and yield. The method
has been scaled up for the preparation of up to 10 g of the target compound.
Figure 2. TBAB ion pair with the associated acid catalyst.
3. Experimental
General
N-(9-acetyl-6-oxo-1H-purin-2-yl)acetamide, was obtained from Zhejiang Oma Pharmaceuticals. All
other chemicals and solvents were purchased from the usual commercial sources. Melting points were
determined on a Stuart Scientific SMP3 melting point apparatus using open capillary tubes and are
1
uncorrected. IR spectra were recorded on a Perkin–Elmer FTIR 1725X spectrophotometer. H- and
13C-NMR spectra were recorded on either Varian Gemini (200 MHz), Unity (300 or 500 MHz)
spectrometers, using TMS as internal reference. Mass data were collected on an Agilent 6210
Time-of-Flight LC/MS.
1,2-Bis-chloromethoxyethane (8) [16]. HCl gas was bubbled for 2 h into a suspension of
paraformaldehyde (77.4 g, 2.58 mol) in ethylene glycol (71.9 mL, 1.29 mol). After that, the reaction
mixture became transparent and two phases were observed. The mixture was poured into a separation
funnel, the lower phase was separated and the upper phase was extracted with dichloromethane
(70 mL). The combined organic phases were dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The product was isolated by distillation under reduced pressure (20 mbar) and
different fractions were separated. The desired product 8 distilled between 95–110 °C as a colourless
transparent liquid (119.81 g, 58% yield). 1H-NMR (500 MHz, CDCl3) ppm: δ 3.88 (s, 4H), 5.50 (s, 4H).
13C-NMR (125 MHz, CDCl3) ppm: δ 68.5 (2CH2), 82.6 (2CH2).
1,2-Bis(diacetoxy-methyloxy)ethane (9) [16]. Compound 8 (59.96 g, 0.397 mol) was placed in a
200 mL flask equipped with a mechanical stirrer. The flask was cooled with an ice-water bath and
anhydrous NaOAc (67.74 g, 0.826 mol) was added in four portions over 1 h. On completion of the
addition, the mixture was heated at 90 °C for 1 h 30 min and then allowed to cool to room temperature.
The reaction mixture was filtered and the solid (sodium chloride) was washed with dichloromethane
(3 × 70 mL). The combined organic phases were concentrated under reduced pressure. The product
was purified by distillation under reduced pressure (150 °C, 20 mmHg) to give 9 as a colourless
1
transparent liquid (73.40 g, 90% yield). IR (NaCl film): 1744 cm−1 (C=O). H-NMR (300 MHz,

Molecules 2012, 17
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CDCl3) ppm: δ 2.07 (s, 3H), 3.78 (s, 2H), 5.27 (s, 2H). 13C-NMR (125 MHz, CDCl3) ppm: δ 21.0
(2CH3), 69.3 (2CH2), 89.2 (2CH2), 170.5 (2C).
N-{9-[2-(2-Acetylamino-6-oxo-1,6-dihydropurin-9-ylmethoxy)-ethoxymethyl]-6-oxo-6,9-dihydro-1H-
purin-2-yl}acetamide (11) [16]. Compound 10 (1 g, 4.325 mmol), p-toluenesulfonic acid (0.02 g),
tetrabutylammonium bromide (0.03 g) and Dowtherm Q (13 mL) as solvent, were placed in a
three-necked flask equipped with a distillation system and a N2 inlet. The mixture was heated under an
inert atmosphere at 110–120 °C and a solution of 9 (0.97 g, 4.7 mmol) in Dowtherm Q (1 mL) was
added in three portions over 1 h. Vacuum was applied (100 mmHg) and the flask was heated at 150 °C
to distil off acetic anhydride. The reaction mixture became orange and, after 15 h heating, compound 9
had been consumed. The mixture was allowed to cool to room temperature and acetone (20 mL) was
added. The mixture was stirred and cooled in an ice-water bath for 1 h 30 min. A solid appeared that
was filtered and washed with acetone (2 × 15 mL) to give 11 as a white solid. The solid was suspended
in DMF (3 mL) and the mixture was heated to 100 °C for 30 s [31]. The solid was filtered and dried
(1.05 g, 52% yield; 92% purity by HPLC; mp 301.9 °C). 1H-NMR (500 MHz, DMSO-d6) ppm: δ 2.16
(s, 6H), 3.56 (s, 4H), 5.40 (s, 4H), 8.08 (s, 2H), 11.71 (s, 2H). 13C-NMR (125 MHz, DMSO-d6) ppm: δ
23.1 (2CH3) + 30.0 (2CH2), 67.2 (2CH2), 72.0 (2CH2), 119.6 (2C), 139.4 (2CH), 147.5 + 148.2 (2C),
154.3 (2C), 172.9 (2C), 205.7 (2C). HRMS (FAB+): Calc. for C18H20N10O6 [M++1] = 473.1646; found
[M++1] = 473.1647.
9,9'-[1,2-Ethanediylbis(oxymethylene)]bis-2-amino-1,9-dihydro-6H-purin-6-one (3). Compound 11
(0.8 g, 1.7 mmol) was suspended in distilled water (8 mL) and ethanolamine (0.8 mL,
0.08 mol) was added. The mixture was heated under reflux for 10 h and then allowed to cool to room
temperature. Aqueous HCl (10%) was added to give pH 7 and the mixture was heated at 90 °C for
15 min. The solid was filtered off and dried in a vacuum oven at 40 °C. The solid was dissolved in
DMSO (8 mL) and heated to 80–90 °C for 15 min. The solution was filtered and water (10 mL) was
added dropwise. The mixture was stirred at room temperature for 30 min. A white solid formed and the
suspension was filtered, the residue suspended in DMSO (8 mL) and heated to 80–90 °C for 15 min.
Then, the solution was filtered and water was added dropwise (~10 mL) until the liquid became
cloudy. The mixture was then stirred at room temperature for 30 min. The resulting white solid was
filtered and dried in a vacuum oven at 100 °C. Compound 3 was obtained as a white powder (0.51 g,
77% yield; 95% purity by HPLC, Mp > 400 °C). 1H-NMR (500 MHz, DMSO-d6) ppm: δ 3.54 (s, 4H),
13
5.30 (s, 4H), 6.51 (s, 4H), 7.78 (s, 2H), 10.63 (s, 2H). C-NMR (125 MHz, DMSO-d6) ppm: δ 40.6
(CH2), 56.9 (CH2), 67.0 (2CH2), 71.4 (2CH2), 116.0 (2C), 137.1 (2CH), 150.9 (2C), 153.3 (2CH),
156.2 (2C). HRMS (FAB+): Calc. for C14H16N10O4 [M+1] = 389.3464; found [M++1] = 389.3466.
Higher purity can be obtained by repeating the DMSO/water crystallisation process.
4. Conclusions
Validation of analytical methods of active pharmaceutical ingredients (API) requires the availability
of reference samples of impurities eventually formed in the synthetic processes of the corresponding
API, to be used as standards. In this case, a rare impurity of acyclovir has been obtained through an
acid catalysed PTC process which ensures regioselective alkylation of 2,9-diacetylguanine (10).

Molecules 2012, 17
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Acknowledgments
The authors are grateful to Química Sintética (Chemo Group) for providing financial support.
References
1. International Conference on Harmonization (ICH) Guidelines, Q3A (R) Impurities in New Drug
Substances, February 2002.
2. Gorog, S. The role of impurity profiling in drug research, development and production.
Progr. Pharm. Biomed. Anal. 2000, 4, 38–47.
3. Gorog, S. Identification, structure elucidation and determination of related organic impurities 2.1.
Strategies in impurity profiling. Progr. Pharm. Biomed. Anal. 2000, 4, 67–83.
4. Biba, M.; Welch, C.J.; da Silva, J.O.; Mathre, D.J. Impurity isolation in support of pharmaceutical
process research. Am. Pharm. Rev. 2005, 8, 68–72.
5. Grekas, N. Organic impurities in chemical drug substances. Pharm. Tech. Eur. 2005, 24–32.
6. Glazkov, I.N.; Bochkareva, N.L.; Revel’skii, A. Determination of organic impurities in
pharmaceutical preparations. J. Anal. Chem. 2005, 60, 107–118.
7. Schaeffer, H.J.; Beauchamp, L.; De Miranda, P.; Elion, G.B.; Bauer, D.J.; Collins, P.
9-(2-Hydroxyethoxymethyl)guanine activity against viruses of the herpes group. Nature 1978,
272, 583–585.
8. De Clercq, E.; Field, H.J. Antiviral prodrugs—The development of successful prodrug strategies
for antiviral chemotherapy. Br. J. Pharmacol. 2006, 147, 1–11.
9. Schaeffer, H.J. Substituted purines. DE 2539963, 1976.
10. Schaeffer, H.J. Compositions for treating viral infections and guanine acyclic nucleosides.
U.S. Patent 4199574, 1980.
11. Matsumoto, H.; Kaneko, C.; Yamada, K.; Takeuchi, T.; Mori, T.; Mizuno, Y. A convenient
synthesis of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir) and related compounds.
Chem. Pharm. Bull. 1988, 36, 1153–1157.
12. Schaeffer, H.J. Esters of 9-(2-hydroxyethoxymethyl)guanine and 2-amino-9-(2-hydroxyethoxy-
methyl)adenine and pharmaceutical compositions containing them. GB 1567671, 1976.
13. Schloemer, G.C.; Han, Y.K.; Harrington, P.J. Preparation of acyclovir using 1,3 dioxolane.
US 5567816, 1996.
14. Hansen, H.; Scheuplein, S.W.; Stuhltraeger, K.; Bauer, K. Improved procedure for the preparation
of acyclovir. DE 19536164, 1996.
15. Gao, H.; Mitra, A.K. Synthesis of acyclovir, ganciclovir and their prodrugs: A review. Synthesis
2000, 329–351.
16. Ashton, D.S.; Beddell, C.; Ray, A.D.; Valko, K. Quantitative structure-retention relationships of
acyclovir esters using immobilized albumin high-performance liquid chromatography and
reversed-phase high-performance liquid chromatography. J. Chromatogr. A 1995, 707, 367–372.
17. Martin, J.C.; Dvorak, C.A.; Smee, D.F.; Matthews, T.R.; Verheyden, J.P.H. 9-(1,3-Dihydroxy-2-
propoxymethyl)guanine: A new potent and selective antiherpes agent. J. Med. Chem. 1983, 26,
759–761.

Molecules 2012, 17
8741
18. Robinson, B. Non-catalytic alcoholysis of nitriles. J. Chem. Soc. 1963, 2417–2419.
19. Senkus, M. Reaction of some cyclic acetals with acid anhydrides. J. Am. Chem. Soc. 1946, 68,
734–736.
20. Zaragoza Dörwald, F. Side Reactions in Organic Synthesis; John Wiley & Sons: Weinheim,
Germany, 2005; p. 336.
21. European Pharmacopeia 6.0; Council of Europe: Strasbourg, France, 2007; Volume 2, p. 1107.
22. Yang, G.Y.; Oh, K.-A.; Park, N.-J.; Jung, Y.-S. New oxime reactivators connected with
CH2O(CH2)nOCH2 linker and their reactivation potency for organophosphorus agents-inhibited
acetylcholinesterase. Bioorg. Med. Chem. 2007, 15, 7704–7710.
23. Landini, D.; Montanari, F.; Rolla, F. Conversion of primary alcohols to alkyl chlorides using
aqueous hydrochloric acid in the presence of phase-transfer catalysts. Synthesis 1974, 37–38.
24. Landini, D.; Montanari, F.; Rolla, F. Cleavage of dialkyl and aryl alkyl esters with hydrobromic
acid in the presence of phase-transfer catalysts. Synthesis 1978, 771–773.
25. Landini, D.; Rolla, F. Addition of hydrohalogenic acids to alkenes in aqueous-organic, two-phase
systems in the presence of catalytic amounts of onium salts. J. Org. Chem. 1980, 45, 3527–3529.
26. Goverdhan, L.K.; Irvinder, K.; Monica, B.; Jasvinder, S.; Jasamrit, K. Functional Group
Transformations of Diols, Cyclic Ethers, and Lactones Using Aqueous Hydrobromic Acid and
Phase Transfer Catalyst under Microwave Irradiation. Org. Proc. Res. Dev. 2003, 7, 339–340.
27. Radl, S.; Zikan, V. A new method of preparation of the antiviral agent acyclovir. Cesk. Farm.
1987, 36, 58–60.
28. Saleh, A.; D’Angelo, J.G.; Morton, M.D.; Quinn, J.; Redden, K.; Mielguz, R.W. Pavlik, C.;
Smith, M.B. Synthesis of 3-Guaninyl- and 3-Adeninyl-5-hydroxymethyl-2-pyrrolidinone
Nucleosides. J. Org. Chem. 2011, 76, 5574–5583.
29. Schaeffer, H.J. Compositions for treating viral infections and guanine acyclic nucleosides.
U.S. Patent 4146715, 1979.
30. Madre, M.A.; Zhuk, R.A.; Lidak, M.J. Analogs of purine nucleosides. I. Methods for synthesis of
9-(2-hydroxyethoxymethyl)guanine-acycloguanosine. Khim.-Farm. Zh. 1985, 19, 1371–1375.
31. It is important to obtain 11 pure, because 3 is more difficult to purify. Compound 10 can be
separated from 11 by addition of DMF (3 mL) to a sample of 1 g of the product and heating the
mixture to 100 °C for 30 s. In this way, up to 100 mg of 6 can be dissolved and remains in
solution even at room temperature, while 11 is insoluble. Thus, the mixture can be filtered, the
solid recovered and dried.
Sample Availability: Samples of the compounds are available from the authors.
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