Synthesis of valganciclovir hydrochloride congeners

Article abstract of DOI:10.1080/00397911.2011.651249

Valganciclovir hydrochloride (1) is used for the treatment of cytomegalovirus (CMV) retinitis in patients with weakened immune systems. Valganciclovir hydrochloride is a hydrochloride salt of L-valyl ester of ganciclovir (2) that exists as a mixture of two diastereomers. According to the U.S. Food and Drug Administration specifications, the diastereomeric ratio of valganciclovir hydrochloride 1 should be maintained in the range 55:45 to 45:55. According to the U.S. Food and Drug Administration specifications, the diastereomeric ratio of valganciclovir hydrochloride 1 should be maintained in the range 55:45 to 45:55. During the process development of valganciclovir hydrochloride, six related substances (impurities) were observed along with the final active pharmaceutical ingredient. Among these six impurities, ganciclovir (2) and guanine (3) are the key starting materials and degraded impurities of ganciclovir, respectively. The remaining four impurities were identified as isovalganciclovir hydrochloride (4), methoxymethylguanine (5), O-acetoxy ganciclovir (6), and isovalarylganciclovir (7). The present work describes the synthesis and characterization of these four impurities.

Full text of DOI:10.1080/00397911.2011.651249

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Synthesis of Valganciclovir Hydrochloride
Congeners
K. Srihari Babu a b , M. Raghavendar Rao a , G. Goverdhan a , P.
Srinivas a , P. Pratap Reddy a , G. Venkateswarlu b & R. Vijaya Anand
a
a Research and Development, Integrated Product Development, Dr.
Reddy's Laboratories Limited, Bachupally, Qutubullapur, Andhra
Pradesh, India
b Department of Chemistry, Nizam College, Osmania University,
Hyderabad, Andhra Pradesh, India
Accepted author version posted online: 17 May 2012.Version of
record first published: 04 Apr 2013.
To cite this article: K. Srihari Babu , M. Raghavendar Rao , G. Goverdhan , P. Srinivas , P. Pratap
Reddy , G. Venkateswarlu & R. Vijaya Anand (2013): Synthesis of Valganciclovir Hydrochloride
Congeners, Synthetic Communications: An International Journal for Rapid Communication of Synthetic
Organic Chemistry, 43:13, 1751-1758
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Synthetic Communications1, 43: 1751–1758, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2011.651249
SYNTHESIS OF VALGANCICLOVIR HYDROCHLORIDE
CONGENERS
K. Srihari Babu,1,2 M. Raghavendar Rao,1 G. Goverdhan,1
P. Srinivas,1 P. Pratap Reddy,1 G. Venkateswarlu,2 and
R. Vijaya Anand1
1Research and Development, Integrated Product Development, Dr. Reddy’s
Laboratories Limited, Bachupally, Qutubullapur, Andhra Pradesh, India
2Department of Chemistry, Nizam College, Osmania University, Hyderabad,
Andhra Pradesh, India
GRAPHICAL ABSTRACT
Abstract Valganciclovir hydrochloride (1) is used for the treatment of cytomegalovirus
(CMV) retinitis in patients with weakened immune systems. Valganciclovir hydrochloride
is a hydrochloride salt of L-valyl ester of ganciclovir (2) that exists as a mixture of two
diastereomers. According to the U.S. Food and Drug Administration specifications, the dia-
stereomeric ratio of valganciclovir hydrochloride 1 should be maintained in the range 55:45
Received November 5, 2011.
DRL-IPD Communication No. IPDO-IPM-00231.
Address correspondence to K. Srihari Babu, Research and Development, Integrated Product
Development, Dr. Reddy’s Laboratories Ltd., Bachupally, Qutubullapur, Ranga Reddy District
500072, Andhra Pradesh, India. E-mail: karrothusriharibabu@gmail.com
1751

1752
K. S. BABU ET AL.
to 45:55. According to the U.S. Food and Drug Administration specifications, the diaster-
eomeric ratio of valganciclovir hydrochloride 1 should be maintained in the range 55:45 to
45:55. During the process development of valganciclovir hydrochloride, six related sub-
stances (impurities) were observed along with the final active pharmaceutical ingredient.
Among these six impurities, ganciclovir (2) and guanine (3) are the key starting materials
and degraded impurities of ganciclovir, respectively. The remaining four impurities were
identified as isovalganciclovir hydrochloride (4), methoxymethylguanine (5), O-acetoxy
ganciclovir (6), and isovalarylganciclovir (7). The present work describes the synthesis
and characterization of these four impurities.
Keywords Antiviral; CMV retinitis inhibitor; related substances; valganciclovir
INTRODUCTION
The presence of impurities, also called related substances, in an active pharmaceu-
tical ingredient (API) can have a significant impact on the quality and safety of the drug
products. Therefore, it is necessary to study the impurity profile of any API and control
the impurity during the manufacturing of a drug product. As per the International Con-
ference of Harmonization (ICH) guidelines, any impurity that forms at a level of
ꢀ0.10% with respect to an API should be identified, synthesized, and characterized thor-
oughly.[1] Valganciclovir hydrochloride (1)[2] [Valcyte], which is used for the treatment of
cytomegalovirus (CMV) retinitis in patients with weakened immune systems,[3] is the
latest antiviral medication[4] approved by the U.S. Food and Drug Administration
(FDA) for the treatment of cytomegalovirus infections (Fig. 1).[5] After oral administra-
tion, intestinal and hepatic esterases hydrolyze both diastereomers to ganciclovir (2),
which inhibits replication of human cytomegalovirus in vitro and in vivo. It is evident
from the literature that valganciclovir hydrochloride (1) is a highly recognized substrate
of the intestinal peptide transporter PEPT1, which instigates the 10-fold greater bioa-
vailability of ganciclovir after valganciclovir compared to oral ganciclovir administra-
tion.[6] During the process for the synthesis of 1, four impurities were observed along
with ganciclovir (2) and guanine (3). However, as far as we are aware, syntheses of
two impurities (4 and 7) have not been reported so far. Herein, we discuss identification,
synthesis, and characterization of these four impurities of 1.
Very recently, we have described a novel and efficient synthesis of valgan-
ciclovir hydrochloride (1).[7] During the synthesis of 1, we came across many process-
related impurities, and some of them were captured in our prior report.[7] To
comprehend the complete impurity profile of valganciclovir hydrochloride (1) and
to compare the extent of contamination of the impurities in 1, we have decided to
synthesize all the possible impurities. The high-performance liquid chromatography
(HPLC) chromatogram,[8] Fig. 2, shows the impurity profile of 1.
Figure 1. Molecular framework of valganciclovir hydrochloride, ganciclovir, and guanine.

VALGANCICLOVIR HYDROCHLORIDE CONGENERS
1753
Figure 2. HPLC chromatogram of valganciclovir hydrochloride.
Figure 3. Related substances of valganciclovir hydrochloride 1.
Initially, 1 was subjected to liquid chromatography–mass spectrometry
(LCMS) to learn about the number of contaminants associated with it. Apart from
the molecular ion peak of 1, six more peaks with distinct molecular ions were
observed in LCMS. Based on the molecular ion peaks, the four structures 4–7 were
proposed (Fig. 3). All the impurities 4–7 are process-related impurities, and synthesis
of impurities 5 and 6 were reported,[9,10] whereas the other 4 and 7 impurities have
not been reported elsewhere. In this report, syntheses and characterization of all
the four impurities of 1 are discussed in detail.
RESULTS AND DISCUSSION
The impurity 4, also known as isovalganciclovir hydrochloride, is assumed to
originate from the ganciclovir, which contained isoganciclovir (13) as an impurity.
The isoganciclovir (13), which is present in ganciclovir, reacts similarly to ganciclovir
and forms the impurity 4 as a regioisomer of valganciclovir hydrochloride. Hence,
isoganciclovir (13) was prepared starting from allyl alcohol (8) and was then used
for the preparation of impurity 4 as shown in Scheme 1.

1754
K. S. BABU ET AL.
Scheme 1. Synthesis of isovalganciclovir hydrochloride (4). Reagents and conditions: (i) HCHO, HCl;
(ii) 10, NaH, DMF; (iii) KMnO4, acetone; (iv) 10% Pd=C, MeOH; (v) S-2-azido-3-methylbutanoic acid,
DCC, DMSO; (vi) 10% Pd=C, MeOH.
The allyl alcohol (8) was chloromethylated using paraformaldehyde and dry
HCl in dichloromethane to afford 9 in good yield.[11] Reacting the chloromethylated
compound 9 with commercially available O-benzyl guanine (10) in the presence of
sodium hydride in dimethylformamide (DMF) afforded 11. The oxidation of 11 with
potassium permanganate in water and acetone gave O-benzyl derivative of isoganci-
clovir (12). Debenezylation of compound 12 using Pd=C gave the isoganciclovir (13).
Coupling of (S)-2-azido-3-methylbutanoic acid[12] with 13 in the presence of
N,N0-dicyclohexylcarbodiimide (DCC) in dimethylsulfoxide (DMSO) gave the ester
14. The reduction of azido group in compound 14 using Pd=C=aqueous HCl at high
pressure afforded isovalganciclovir hydrochloride (4) as a white solid in good yield.
The purity of 4 found to be 99% by HPLC[8]] and its structure was confirmed by
spectral analysis.
The origin of the impurity 5 again may be due to the contamination of ganci-
clovir (2) with methoxymethylguanine. During the synthesis of valganciclovir,
because there is no functionality to react further, the methoxymethylguanine 5
remains as it is and appears as an impurity in valganciclovir hydrochloride API.
As mentioned earlier, it is essential that the physical quantity of the impurity must
be in hand to study the impurity profile of an API. Methoxymethylguanine (5)
was therefore synthesized by condensing the commercially available methoxy methyl
(MOM) chloride with O-benzyl guanine (10). NaH was used as base, and the reac-
tion was conducted in dimethylformamide (DMF) to give the substituted guanine 15.
The debanzylation of guanine derivative 15 using Pd=C in methanol gave the desired
methoxymethylguanine (5) as a white solid (Scheme 2).
To prepare the O-acetoxyganciclovir (6), as shown in Scheme 3, ganciclovir 2
was reacted with trityl chloride in the presence of triethylamine (TEA) in DMF
to afford ditrityl-protected ganciclovir 16,[13] which on acetylation using dicyclo-
hexylcarbodiimide (DCC) coupling methodology gave the O-acetylated ganciclovir
derivative 17. The removal of trityl groups of 17 using TFA in dichloromethane gave
the desired O-acetylganciclovir (6).

VALGANCICLOVIR HYDROCHLORIDE CONGENERS
1755
Scheme 2. Synthesis of methoxymethylguanine (5). Reagents and conditions: (i) MOM chloride, NaH,
DMF; (ii) 10% Pd=C, MeOH.
Scheme 3. Synthesis of O-acetylganciclovir (6). Reagents and conditions: (i) TEA=trityl chloride; (ii) acetic
acid, TEA, DCC; (iii) TFA, DCM.
Scheme 4. Proposed mechanism for the formation of impurity 7.
The synthesis of valganciclovir hydrochloride involved the reduction of azido
group using Pd=C and aqueous HCl in methanol.[7] During the hydrogenation, we
speculate that a small quantity of deamination of L-valine moity in valganciclovir
hydrochloride takes place under acidic and high-pressure conditions (Scheme 4).
The in situ reduction of double bond of 3-methylbutenoic acid ester derivative 18
forms the isovalarylganciclovir 7 as an impurity
The synthesis of isovalarylganciclovir (7) started from ditrityl-protected
ganciclovir 16. The reaction of protected ganciclovir 16 with isovalaric acid
using the DCC coupling method gave trityl-protected O-isovalarylganciclovir
19. The deprotection of trityl groups using TFA in dichloromethane afforded
isovalarylganciclovir (7).
Finally, all the impurities 4–7 were individually co-injected with the API 1 into
the HPLC, and the HPLC data was compared with that of the API 1. As expected,
all the retention times (RTs) and relative retention times (RRTs) of the impurities
matched the impurity profile of valganciclovir hydrochloride 1.

1756
K. S. BABU ET AL.
Scheme 5. Synthesis of isovalarylganciclovir (7). Reagents and conditions: (i) Isovalaric acid, TEA, DCC;
(ii) TFA, DCM.
EXPERIMENTAL
1H NMR spectra were recorded at 400 MHz on a Varian FT-NMR spec-
trometer. The chemical shifts are reported in d ppm relative to tetramethylsilane
(TMS). The infrared (IR) spectra were obtained using a Perkin–Elmer, Spectrum
One FTIR spectrophotometer, with substances being pressed in a KBr pellet. The
mass analyses have been performed on AB-4000 Q-trap LC-MS=MS mass spec-
trometer (MDS Sciex, Applied Biosystems, California, USA). All the solvents and
reagents were used without further purification.
An in-house HPLC gradient method was developed for the separation of all
possible related substances of valganciclovir hydrochloride. Waters HPLC system
equipped with 515 pump and 2998 photodiode-array (PDA) detector was used for
better separation. The buffer solution used for the preparation of mobile phases A
and B consisted of 0.001 M aqueous potassium dihydrogen phosphate monohydrate
and 1.0 mL of TEA, and its pH was adjusted to 2.8 with diluted phosphoric acid.
Mobile phase A was buffer solution; mobile phase B was prepared in the ratio of
10:90 (v=v) of water and acetonitrile. Vertisep AQS C-18, 250 mm ꢁ 4.6 mm, 3m
particle size column was used with a time gradient program of T (min)=%B (v=v).
Initial gradient started with 10% of B and at 5 min it was 25%. The ratio was
continued up to 35 min; at 40 min it was 60%, which was continued to 45 min; at
50 min it was 90%, which was continued up to 55 min; at 60 min it was 10%, which
was continued to 65 min. Flow rate was 1.0 ml=min, column oven temperature was
30 ꢂC, and column eluent was monitored by PDA detector at 254 nm.
Synthesis of Isovalganciclovir Hydrochloride (4)
To a stirred solution of compound 14 (2.0 g, 0.005 mol) in methanol (40 mL)
was added 10% Pd=C (0.2 g), followed by 33% aqueous hydrochloric acid (1.7 mL,
0.0057 mol). The resulting mixture was hydrogenated in autoclave at 4 kg=cm2 press-
ure for 2 h. The reaction mass was filtered through Celite and washed with methanol
(10 mL). The solvent was removed under reduced pressure, and the residue was
recrystallized from isopropanol (22 mL) to give the title compound 4 as a white solid
1
(0.45 g, 90%). IR (KBr): 3324 cmꢃ1; H NMR (400 MHz, DMSO-d6) d 0.90–0.94
(d, J ¼ 6.8 Hz, 6H), 2.09–2.17 (m,1H), 3.81–3.83 (m, 1H), 3.97–3.99 (d, J ¼ 5.2 Hz,

VALGANCICLOVIR HYDROCHLORIDE CONGENERS
1757
1H), 4.07–4.09 (d, J ¼ 5.2 Hz, 2H), 4.16–4.20 (d, J ¼ 5.2 Hz, 2H), 5.39 (s, 2H), 6.74 (s,
2H), 8.03 (s, 1H), 8.49 (s, 2H), 10.92 (s, 1H); 13C NMR (400 MHz, DMSO-d6) d 17.1,
17.4, 17.9, 29.2, 57.2, 57.3, 64.6, 66.6, 69.5, 69.6, 72.4, 115.1, 137.6, 151.1, 154.2,
156.2, 168.5: MS m=z (DIP): 355.1 (Mþ þ 1). CHN analysis calcd. for
C16H22N6O5: C, 43.02; H, 5.93; N, 21.50. Found: C, 42.82; H, 5.22; N, 30.0.
Synthesis of Methoxy Methyl Guanine (5)
To the stirred solution of compound 15 (4.0 g) in methanol (100 mL) and
water (40 mL) in autoclave vessel was added 10% Pd=C (0.8 g). Then hydrogen gas
was applied (4 kg=cm2) for 5 h at room temperature using an autoclave machine.
The reaction mass was filtered through celite and washed with methanol (20 mL).
The solvent was removed under reduced pressure to give the crude product which
was then crystallized from acetone to give title compound 5 as a white solid (2.0 g,
1
80%). IR (KBr): 3311, 1690 cmꢃ1; H NMR (400 MHz, DMSO-d6) d 3.23 (s, 3H),
5.26 (s, 2H), 6.50 (s, 2H), 7.80 (s, 1H), 10.62 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) d 56.1, 73.3, 116.6, 138.1, 151.7, 154.0, 157.1; MS m=z (DIP): 194.0
(Mþ þ 1). CHN Analysis calcd. for C16H17N5O5: C, 43.08; H, 4.65; N, 35.88. Found:
C, 43.11; H, 4.72; N, 35.93.
Synthesis of O-Acetoxy Ganciclovir (6)
A solution of trifluoroacetic acid (40 mL, 0.104 mol) in dichloromethane
(50 mL) was added to a solution of 17 (20.0 g, 0.025 mol) in dichloromethane
(100 mL) at 15 ꢂC over a period of 10 min and stirred for 3 h. Methyl t-butyl ether
(240 mL) and n-hexane (240 mL) were added to the reaction mass, and the resulting
mixture was stirred for 20 min. The solvent was decanted, and the compound was
crystallized from ethanol (50 mL) and dried under vacuum at 45 ꢂC to afford title
compound 6 as a white crystalline powder (7.0 g, 92%); FT IR (KBr) 3317 cmꢃ1
;
1H NMR (400 MHz, DMSO-d6) d 1.85 (s, 3H), 3.22–3.43 (m, 3H), 3.78–3.81 (m,
1H), 3.82–3.86 (d, J ¼ 3.6 Hz, 1H), 4.82–4.85 (d, J ¼ 3.6 Hz, 1H), 5.40 (s, 2H), 6.47
(s, 2H), 7.80 (s, 1 H), 10.60 (s, 1H); 13C NMR (400 MHz, DMSO-d6) d 20.4, 60.4,
63.4, 71.5, 76.9, 116.5, 137.9, 151.5, 153.9, 157.0, 169.9; MS m=z (DIP) 279.9
(Mþ þ 1). CHN analysis calcd. for C11H15N5O5: C, 44.44; H, 5.09; N, 23.56. Found:
C, 44.38; H, 5.12; N, 23.61.
Synthesis of Isovalaryl Ganciclovir (7)
A solution of trifluoroacetic acid (40 mL, 0.104 mol) in dichloromethane
(50 mL) was added to a solution of 19 (20.0 g, 0.025 mol) in dichloromethane
(100 mL) at 15 ꢂC over a period of 10 min, and the resulting mixture was stirred
for 3 h. Methyl t-butyl ether (240 mL) and n-hexane (240 mL) were added to the
mass, and the resulting mixture was stirred for 20 min. The solvent was decanted,
and the compound was crystallized from ethanol (50 mL) and dried under vacuum
at 45 ꢂC to afford title compound 7 as a white crystalline powder (7.1 g, 88%); FT
1
IR (KBr) 1693 cmꢃ1; H NMR (400 MHz, DMSO-d6) d 0.81–0.83 (d, J ¼ 6.8 Hz,
6H), 1.79–1.86 (m, 1H), 1.97–1.98 (d, 6.8 Hz, 2H), 3.37–3.42 (m, 1H), 3.44–3.45

1758
K. S. BABU ET AL.
(d, J ¼ 5.6 Hz, 1H), 3.79–3.80 (d, J ¼ 5.2 Hz, 1H), 3.85–3.86 (d, J ¼ 6.8 Hz, 1H),
3.88–3.89 (d, J ¼ 6.8 Hz, 1H), 4.83–4.85 (t, J ¼ 5.2 Hz, 1H), 5.41 (s, 2H), 6.46 (s,
2H), 7.78 (s, 1H), 10.60 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d 22.1, 25.1,
42.4, 60.5, 61.0, 63.2, 71.7, 77.3, 115.2, 137.8, 151.2, 154.1, 156.5, 172.1; MS m=z
(DIP) 340.1 (Mþ þ 1). CHN analysis calcd. for C14H21N5O5: C, 49.55; H, 6.24; N,
20.64. Found: C, 49.62; H, 6.29; N, 20.70.
CONCLUSIONS
We have demonstrated the synthesis and complete characterization of some of
the critical impurities of valganciclovir hydrochloride (1). This investigation not only
helped us to establish the impurity profile of 1 but also allowed us to understand the
formation and control of these impurities.
ACKNOWLEDGMENTS
We greatly appreciate the supportive environment encouraged at Dr. Reddy’s
Laboratories Ltd. We are thankful to the Intellectual Property Management
(IPM), NMR group of Dr. Reddy’s Research Foundation and the analytical group
of Integrated Product Development Organization (IPDO) for their magnanimous
support.
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