M + 4 stable isotope labeling of levovirin and M + 7 and carbon-14 labeling of levovirin valinate pro-drug

Article abstract of DOI:10.1002/jlcr.1138

[M + 4]-labeled levovirin 5 (231 mg) was synthesized as an MS reference compound from [M + 4] triazole ester 2. [M + 7]-labeled levovirin valinate 6 (127 mg) was synthesized as a comparison MS reference compound from [M + 6] triazole ester 3. [¹⁴

Full text of DOI:10.1002/jlcr.1138

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 1223–1236.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1138
Research Article
M+4 stable isotope labeling of levovirin and M+7 and
carbon-14 labeling of levovirin valinate pro-drug
Steve A. de Keczer1,*, Mohammad R. Masjedizadeh1, Shao-Yong Wu1,
Teresa Lara-Jaime1, Kate Comstock1, Charles Dvorak1, Yu-Ying Liu2 and
Walter Berger2
1 Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
2 Hoffmann- La Roche, 340 Kingsland Street, Nutley, NJ 07110, USA
Summary
[M+4]-labeled levovirin 5 (231 mg) was synthesized as an MS reference compound
from [M+4] triazole ester 2. [M+7]-labeled levovirin valinate 6 (127 mg) was
synthesized as a comparison MS reference compound from [M+6] triazole ester 3.
[14C]-Levovirin 7 and [14C]-levovirin valinate 8 were synthesized to support
metabolism studies. The synthesis of 7 was accomplished in 33% overall yield
(35.4 mCi, 57 mCi/mmol) from Ba14CO3 and 8 was synthesized in 41% yield
(12.5 mCi, 57 mCi/mmol) from 7. An efficient metallation/carbonation reaction was
developed to synthesize [14C]-triazole ester 4. Copyright # 2006 John Wiley & Sons,
Ltd.
Received 13 July 2006; Revised 28 August 2006; Accepted 11 September 2006
Key Words: carbon-13; carbon-14; nitrogen-15; stable isotope; levovirin; levovirin
valinate; pro-drug
Introduction
Levovirin is the L-enantiomer of ribovirin. Like ribovirin, levovirin is intended
for use in the treatment of patients with chronic hepatitis C. The
immunomodulatory activity of levovirin is thought to be the underlying
mechanism of action. Levovirin has similar potency to ribovirin in pre-clinical
immunomodulation models, but has the advantage of a potentially more
specific mode of action. However, because of a reduced clearance time,
levovirin has reduced bioavailability. The problem of relatively low bio-
availability of levovirin was ameliorated by the development of an appropriate
*Correspondence to: Steve A. de Keczer, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304,
USA. E-mail: Steve.de keczer@roche.com
Copyright # 2006 John Wiley & Sons, Ltd.

1224
S. A. DE KECZER ET AL.
pro-drug. The increased bioavailability would ultimately result in a lower
required dose, less frequent administration of the drug, lower costs of
treatment, and higher exposures of the active drug component enabling
enhanced antiviral response.1 To test the bioavailability of the levovirin
metabolically released from the pro-drug, levovirin valinate, compared to
levovirin in its native form in the same individual, two isotopically labeled
(M+4 5 and M+7 23) differentiable forms of levovirin were synthesized. The
M+7 isotopic form was further elaborated to the M+7 pro-drug levovirin
valinate 6. The reason for the relatively large mass displacements was to
enhance the mass spectral signature of the labeled drugs. The 14C-labeled
forms of levovirin and levovirin valinate were synthesized for ADME studies.
Results and discussion
To simplify our syntheses, we chose to prepare both the M+4 and M+7
stable labeled and the 14C-radiolabeled versions of levovirin via the common
1H-[1,2,4]-triazole-3-carboxylic acid methyl ester intermediates (2, 3, and 4).2–6
The condensation of these intermediates with 1,2,3,5-tetra-O-acetyl-l-ribose
1 was based on the modification of a method reported by Ramasamy.7 Further
elaboration afforded: [13C3,15N]-levovirin 5, [13C3,15N4]-levovirin valinate 6,
[14C]-levovirin 7, and [14C]-levovirin valinate 8 (Figure 1).
O
O
O
O
15 N
15N
13C
OAc
OAc
13C
13C
14C
N
13C
13
13C
OMe
OMe
OMe
C
15N 15
N
H
N
H
N
N
N
OAc
OAc
H
1
2
3
4
O
O
13C
15
15
N
N
13C
13
C
13C
O
13 C
13
C
Cl
15NH2
NH2
15N 15
H3N+
O
N
N
O
N
O
HO
HO
OH
HO
OH
6
5
O
14C
O
14C
N
N
Cl
O
NH2
NH2
H3N+
O
N
N
O
N
N
O
HO
HO
OH
HO
OH
7
8
Figure 1. Key products and intermediates
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1223–1236
DOI: 10.1002/jlcr

LABELING OF LEVOVIRIN AND LEVOVIRIN VALINATE PRO-DRUG
1225
The synthesis of the M+4 stable labeled methyltriazole involved the
preparation of [13C2,15N]-thioacetamide 11 from commercially available
[13C2,15N]-acetonitrile 9. A potentially unreliable route (40–70%) requiring
use of H2S was avoided by using 2 equivalents of odorless diphenyl-
dithiophosphonic acid 102, affording pure labeled thioacetamide 11 in 93%
yield. [13C]-Formic hydrazine 14 was prepared by treating commercially
available ethyl [13C]-formate 12 with excess hydrazine hydrate 13 in toluene
for 16 h at ambient temperature. Removal of volatiles afforded pure
[13C]-formic hydrazine 14 in 96% yield. The two labeled compounds 11
and 14 were coupled and cyclized by heating in toluene for 16 h at 888C.4
The crude [13C3,15N]-methyltriazole 15 obtained by aqueous work-up was
oxidized by the overnight reaction with aqueous KMnO4 at 908C.5 Solids
were removed by centrifuge, and the supernatant was acidified with conc.
HCl to precipitate the [13C3,15N]-triazole carboxylic acid 16 in 71% yield
from 11. The labeled acid 16 was stirred in HCl-saturated methanol at
ambient temperature for 64 h, and the HCl salt of [13C3,15N]-methyltriazole-
carboxylate 2 was obtained by crystallization from the reaction mixture in
72% yield (300 mg).
Similarly, the M+6 stable labeled methyltriazole (leading to M+7 labeled
levovirin) was prepared via the [13C2,15N]-thioacetamide 11. However, to
increase the isotopic substitution, ethyl [13C]-formate 12 was treated with
[15N2]-hydrazine, converted to the free base from [15N2]-hydrazine sulfate. The
elaboration to [13C3,15N3]-methyltriazole carboxylate 3 was accomplished in a
similar fashion as described above, except that the intermediate [M+6]-
methyltriazole was purified before the oxidation step. This additional
purification step resulted in an improved isotopic distribution as further
discussed below.
S
13C
S
H
H
13C
Ph
15 NH2
11
P
2
SH
H13C13
C
15N
H
10
H
H
9
H2N NH2
O
13C
H
13C
15 N
13C
15 N
H
H
O
13C
KMnO4
13C
13
13C
11
13C
H
13CO2Et
12
OH
NH2
N
H
N
H
N
H
N
H
N
16
14
15
O
13C
O
O
15 N
15 N
OAc
13C
13C
13C
N
AcO
13C
13C
N
NH2
O
O
13C
15
N
AcO
OAc
N
N
O
O
13
HCl 13C
C
1
NH3
AcO
O
HO
16
N
H
N
MeOH
AcO
OAc
MeOH
HO
OH
5
2
17
Scheme 1. [13C3, 15N]-levovirin
Copyright # 2006 John Wiley & Sons, Ltd.
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S. A. DE KECZER ET AL.
The 14C-labeled triazole carboxylic acid methyl ester 4 was prepared in a
three step, 1-pot procedure from readily prepared 1-pyrrolidin-1-ylmethyl-1H-
[1,2,4]-triazole, 27.8 A THF solution of protected triazole 27 was treated first
with n-BuLi followed by 14CO2 (from Ba14CO3) to afford the protected
triazole 14C-carboxylate. Volatiles were removed by vacuum transfer and
methanolic HCl was injected onto the residue. After stirring overnight,
volatiles were again removed and the crude de-protected methyl ester paste
was neutralized with aqueous NH4OH. Chromatography afforded 168 mCi
(80% yield) of 99.9% radio-TLC pure 4.
S
13C
S
H
H
13C
H
15NH2
11
Ph
P
2
SH
H13
H
C
13C 15N
H
10
9
H125N15NH2
O
H
13C
O
C
13C
15N
13C
H
H
15N
13C
KMnO4
13C
13
15 NH2
11
13C
15N15N
18
OH
15
H13CO2Et
15N15
H
N
N
H
H
H
12
19
20
21
O
15N
13C
O
OAc
13C
15N
OAc
13C
OMe
15N
O
15N
13C
O
13C
OAc
OAc
OAc
13C
HC
l
OMe
1
21
15N15
N
MeOH
H
OAc
OAc
3
22
O
O
15N
13C
13C
O 15
13C
15NH2
O
O
O
HO
13C15
N15
N
O
N
N
O
O
HO
15NH3
MeOH
15N
13C
O
15N 13C
15NH2
HO
OH
25
O
O
23
24
O
O
H
N
O
O
H3N+
13C15
O
15N
13C
13C
15N
N
O
O
15N
O
13C
O
O
aq HCl
15N 13C
15N 13C
15NH2
15NH2
i-PrOH/tol
O
O
HO
OH
6
26
Scheme 2. [13C3,15N4]-levovirin and [13C3,15N4]-levovirin valinate and 3-
methyltriazole-[13C3,15N3]
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

LABELING OF LEVOVIRIN AND LEVOVIRIN VALINATE PRO-DRUG
1227
The remainders of the syntheses to labeled levovirin, 5 and 7, and labeled
levovirin valinate, 6 and 8, were formally similar from all three of the labeled
methyltriazole carboxylates, 2, 3, or 4. However, in the case of [13C3,15N4]-
levovirin valinate 6 the isomer substitution was increased from M+6 to M+7
by substituting commercially available [15N]-ammonia (Scheme 2) in place of
unlabeled ammonia (Scheme 1) at the amination step.
Attempts were made to improve the coupling step with each individual
isotopic synthesis. In the first coupling, tetra-O-acetyl-l-ribose 1 and labeled
triazole methyl ester 2 were combined with a catalytic amount of bis(4-
nitrophenyl)phosphate.7 This neat mixture was heated for 35 min at 1508C
under vacuum and the evolved acetic acid was trapped. Following an aqueous
NaHCO3 workup and chromatography, a 35% yield of coupling product 17
was obtained. In the second coupling, excess tetra-O-acetyl-l-ribose 1 and a
catalytic amount of bis(4-nitrophenyl)phosphate were added in two portions
to labeled triazole methyl ester 3 contained in a rounded bottom flask to avoid
‘bumping’. This neat mixture was heated for 1 h at 105–1108C under vacuum
and the evolved acetic acid was trapped. The melt was directly purified by
chromatography affording a 77% yield of nearly pure coupling product.
Further purification by crystallization from methanol afforded a 57% yield of
pure product 22. In the coupling of 14C-labeled triazole methyl ester 4, an
O
14C
O
N
OAc
AcO
O
14C
C
OMe
N
1. n-BuLi
N
2. 14CO2 /THF
O
N
N
N
AcO
OMe
OAc
AcO
N
N
1
N
N
H
3. HCl/MeOH
4. NH4OH
( one pot )
AcO
OAc
27
4
28
O
O
14C
O
N
N
O
HO
O
C
NH2
N
N
N
O
O
N
HO
O
O
O
N
NH3
14C
NH2
25
28
O
MeOH
HO
O
OH
O
7
29
Cl
H3N +
O
H
N
O
O
O
N
N
O
O
O
N
O
O
N
14C
NH2
N
14C
N
HCl
NH2
HO
O
OH
O
30
8
Scheme 3. [14C]-levovirin and [14C]-levovirin valinate
Copyright # 2006 John Wiley & Sons, Ltd.
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S. A. DE KECZER ET AL.
attempt to improve the yield by mixing the components in methanol followed
by removal of the methanol as the toluene azeotrope did not increase the yield
(Scheme 3).
The isotopic distribution of the M+4 labeled levovirin was unexpected.
Based on the purities of the isotopically labeled intermediates, we expected 5
90% M+4 isotopic substitution. Instead, we obtained 71.2% M+4, and
27.8% M+3. Though this distribution was adequate for our purposes, we
attempted to discover the source of the apparent isotope dilution to M+3.
Mass spectral distributions were run of each of the intermediates to determine
the source of the dilution. The dilution was found to occur during the KMnO4
oxidation step. We could not rationalize this based on the chemistry at that
step. We attributed the dilution related to the impure intermediates used at
that step. In the subsequent M+6 synthesis a purification was performed on
compound 20 to give ꢀ80% purity by NMR. After the KMnO4 oxidation the
isotopic distribution was somewhat improved to 87.2% M+7 and 12.6%
M+6 for [13C3,15N4]-levovirin valinate 6. The dilution of isotopic incorpora-
tion is still not fully understood.
Experimental
Proton spectra were run on a Brucker 300 MHz NMR spectrometer. Mass
spectrometric analyses were carried out on a Finnegan LCQ ion trap mass
spectrometer equipped with an electrospray ionization (ESI) source. The
operating conditions for the ESI source were as follows: capillary temperature:
2208C, capillary voltage: 32 V. The sample was flow injected via a HP model
1100 HPLC system with 30% solvent ‘A’ ( H2O with 0.5% acetic acid ) and
70% solvent ‘B’ (Acetonitrile) at a flow rate of 200 ml/min. LC/MS data were
recorded from an Agilent 1100 Series LC/MS. The identity of intermediates
was confirmed by MS and 1H-NMR. All spectral data were consistent with the
proposed structures. The specific activity was determined using a Beckman LS
6000IC scintillation counter and confirmed by MS. TLC plates (silica gel
250 mm) were obtained from Analtech and scanned using a Bioscan System 200
Imaging Scanner. The small molecules were reagent-grade, used as is from
various sources. [13C2,15N]-acetonitrile and ethyl [13C]-formate were acquired
from Isotec. [15N2]-hydrazine sulfate was obtained from Cambridge Isotope
Laboratories, Inc. Anhydrous ammonia 99.9% and [15N]-ammonia were
purchased from Aldrich. Tetra-O-acetyl-l-ribose 1 was obtained from
Hoffman-La Roche, Nutley, NJ. For the preparation of 1 from l-ribose see
Liu and Burger3.
[1,2-13C,15N]-thioacetamide 11
The labeled acetonitrile 9 (440 mg, 10.0 mmol) was combined in a 250 cm3
stopcock flask with 6.37 g (25.5 mmol) diphenyldithiophosphonic acid 10 in
Copyright # 2006 John Wiley & Sons, Ltd.
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LABELING OF LEVOVIRIN AND LEVOVIRIN VALINATE PRO-DRUG
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50 ml anhydrous isopropanol. The sealed flask was heated and stirred in a
588C bath for 38 h. After cooling to ambient temperature, the suspension was
filtered, and the filtrate was cooled to À208C and decanted from the bulk of
the remaining phosphonic anhydride by-product. This mixture was concen-
trated to an oil and the [1,2-13C,15N]-thioacetamide 11 (725 mg, 93% yield)
was isolated by flash chromatography (silica gel; 1% NH4OH, 9% MeOH in
CH2Cl2). A second batch of [1,2-13C,15N]-thioacetamide 11 was prepared for
the M+7 synthesis by the method described here for the M+4 synthesis.
[13C]-formic acid hydrazide 14
The 13C-labeled ethyl formate (1.0 g, 13mmol) 12 was added dropwise to a
solution of 1.5 ml (26.5 mmol) hydrazine hydrate in 10ml toluene. The biphasic
mixture was stirred for 14h at ambient temperature, then volatiles were
removed under vacuum for 2 h at 508C. After cooling, the residual white crystals
(0.758g, 96% yield) were identified as pure [13C]-formic acid hydrazide 14.
3-[13C]-methyl-[3,5-13C, 4-15N]-1H-[1,2,4]-triazole 15
[1,2-13C,15N]-thioacetamide 11 (302.3 mg, 3.87 mmol) and [13C]-formic acid
hydrazide 14 (300.9 mg, 4.93 mmol) were combined with 5 ml anhydrous
toluene in a 20 cm3 tube connected to a pair of traps, each containing 10 ml
10% NaOH solutions to capture the evolved H2S gas. The tube was heated for
26 h at 988C, then cooled to ambient temperature and the toluene extracted
with a total of 5 ml water. The combined aqueous phases, containing [M+4]-
methyltriazole 15, were used as is in the next step.
[3,5-13C, 4-15N]-1H-[1,2,4]-triazole-3-[13C]-carboxylic acid 16
The aqueous solution from the previous step (4.9 of 5.0 ml, ca. 3.8 mmol from
3) was added dropwise via an addition funnel to a 40 cm3 tube containing a
908C stirred solution of 1.9 g (12 mmol) KMnO4 in 14 ml water. The addition
funnel was replaced with a condenser and the deep purple solution was stirred
for 15 h at 908C. Following removal of the spin bar from the deep brown
suspension, the tube was centrifuged at 2 K rpm. The clear colorless aqueous
phase was removed and the brown pellet was washed twice with 15 ml
portions of water at 2.5 K rpm. The water was removed from the combined
aqueous solutions by rotary evaporation and the white crystalline solid was
re-dissolved and rinsed into a 20 cm3 tube using a total of 2 ml water. This
solution was titrated to pH 1–2, while cooled in an ice bath, with a total
of 0.45 ml conc. HCl. The creamy suspension was centrifuged, decanted and
the pellet washed twice by centrifugation with 1.0 ml portions of water. The
solid 16 was dried to constant weight as a snow white powder (317 mg, 81%
yield based on 11).
Copyright # 2006 John Wiley & Sons, Ltd.
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[3,5-13C, 4-15N]-1H-[1,2,4]-triazole-3-[13C]-carboxylic acid methyl ester 2
The labeled triazole acid 16 (303.4 mg, 2.62 mmol) was added to an ice bath
cooled solution of HCl (2.4 g) in 3 ml MeOH. The suspension was stirred for
62 h and was re-cooled in a 08C refrigerator for 2 h. The supernatant was
removed and the fine white powder was washed once with À208C MeOH
affording a first crop 216 mg HCl salt of the methyl ester. After concentration
of the mother liquor, a second crop (88 mg) and a third crop (18 mg) were
similarly obtained. The combined yield of [3,5-13C, 4-15N]-1H-[1,2,4]-triazole-
3-[13C]-carboxylic acid methyl ester 2 HCl salt was 322 mg (72%), as a fine,
snow-white powder.
The free base was obtained by neutralizing 154 mg (0.92 mmol) of 2, to pH
ꢀ7 with 100 ml conc. NH4OH, and then adding 4 ml of a solution of 1%
NH4OH/20% MeOH/CH2Cl2 to a final pH 8–9. The clear colorless super-
natant was removed and was concentrated to dryness giving 96 mg of 2 (80%
yield from the HCl salt).
1-(3,4-diacetoxy-5-acetoxymethyl-tetrahydro-furan-2-yl)-[3,5-13C,4-15N]-1H-
1,2,4]triazole-3-[13C]-carboxylic Acid methyl ester 17
Labeled triazole methyl ester 2 (81 mg, 0.62 mmol) and tetra-O-acetyl-l-ribose
1 (203 mg, 0.64 mmol) were combined with a catalytic amount of bis(4-
nitrophenyl)phosphate (4.8 mg, 0.014 mmol) in a 20 cm3 tube. An ice bath
cooled trap was attached to contain the HOAc evolved and the system was
evacuated to 30 mmHg. The evacuated reaction flask was lowered into a 1508C
bath. The initial vigorous effervescence diminished and completely ceased after
35 min, at which time the brown melt was allowed to cool to ambient
temperature. The brown solid was completely dissolved in 2 ml EtOAc by
heating at reflux. After cooling, 1.0 ml saturated aqueous NaHCO3 was added,
and the organic phase was removed and the aqueous phase was washed with
2 ml EtOAc. The combined organic phase was sequentially extracted with
2.0 ml NaHCO3, 1.0 ml water, and 2.0 ml brine, and then dried with Na2SO4,
filtered, and concentrated to a brown oil. The product was isolated by
Chromatotron chromatography (2 mm silica gel rotor; 0.33% NH4OH, 33%
MeOH, CH2Cl2), affording 85 mg (35% yield) of coupling product 17 as a
clear, pale, nearly colorless film.
1-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-[3,5-13C,4-15N]-1H-
[1,2,4]-triazole-3-[13C]-carboxylic acid amide 5, (levovirin)
The labeled methyl ester 17 (79 mg, 0.20 mmol) was rinsed with a total of 2 ml
of 20% MeOH/CH2Cl2 into a 5 cm3 stopcock flask, concentrated with a
nitrogen sweep, and then pumped to dryness. The partially crystallized glass
was re-dissolved in 1.5 ml refluxing methanol, cooled with an ice bath, and
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LABELING OF LEVOVIRIN AND LEVOVIRIN VALINATE PRO-DRUG
1231
immediately (before crystallization of 17 from the cold methanol could begin)
bubbled with ammonia gas. Moderate bubbling was continued for 10 min, and
then the flask was sealed. After warming to ambient temperature, the stopcock
was ‘cracked’ to allow minor degassing to reduce the pressure. The sealed
reaction was stirred for 18 h at ambient temperature, and then the stopcock
was carefully opened allowing the reaction to degas (ꢀ15 s). The crude
product was purified by Chromatotron chromatography (2 mm silica gel rotor;
20–33% MeOH/CH2Cl2) affording 41.3 mg (83% yield) of a clear brittle
colorless glass which appeared to be pure by TLC. In order to transfer the
product, it was re-dissolved in 2 ml MeOH at reflux and transferred to a 4 cm3
vial. On cooling to ambient temperature, the bulk of the levovirin 5
crystallized from solution. This first crop was pumped to constant weight
(23.7 mg), the mother liquor was concentrated and a second crop (15.0 mg)
was obtained. The isotopic distribution by MS was 68.3% M+4, 28.3%
M+3, and 1.4% M+2.
[13C,15N2]-formic hydrazide 19
[15N2]-hydrazine sulfate (5 g, 37.9 mmol) was added to a solution prepared
from Ba(OH)2 Á 8H2O (11.95 g, 37.9 mmol) in H2O (60 ml). The mixture
was stirred at room temperature for 2.5 h, filtered and the filtrate,
containing [15N2]-hydrazine 18 as a free base, was mixed with toluene
(30 ml). To this mixture, [13C]-ethyl formate (12, 2.84 g, 37.9 mmol) was
added dropwise and the mixture stirred at room temperature overnight.
The solvent was removed under vacuum and the residue was purified by
passing through a short silica gel cartridge eluting with a mixture of
NH4OH/MeOH/CH2Cl2 at an initial ratio of 0.1:1:9, slowly changing to a
final ratio of 0.3:3:7. After removal of solvent, the product was obtained
as a wax-like solid. 2.24 g, 94% yield. TLC showed the same Rf as that of
a non-labeled reference sample.
[13C3,15N3]-3-methyltriazole 20
The preparation of [13C3,15N3]-3-methyltriazole 20 was similar to that
described for the synthesis of [13C3,15N]-3-methyltriazole 15, except stoichio-
metric amounts of the reagents were used, the toluene phase was discarded,
and the crude solid product was purified by silica gel chromatography
(MeOH/CH2Cl2) in 70% yield. The structure was confirmed by LCMS
(M+ 84.2).
[1,2,4-15N-3,5-13C]-1H-[1,2,4]-Triazole-3-[13C]-carboxylic acid 21
The same method was followed as described above for the synthesis of [M+4]-
triazole-carboxylic acid 16 except purified [13C3,15N3]-3-methyl triazole 20 was
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1232
S. A. DE KECZER ET AL.
used instead of crude [13C3,15N3]-3-methyltriazole 15, affording a 67% crude
yield of [M+6]-triazole carboxylic acid 21.
[1,2,4-15N-3,5-13C]-1H-[1,2,4]-triazole-3-[13C]-carboxylic acid methyl ester 3
The same method as described for the synthesis of [M+4]-triazole-carboxylic
acid methyl ester 2 was followed. The product was further purified by silica gel
chromatography (1% NH4OH, 15% MeOH/CH2Cl2) to afford 291 mg (54%
yield) of white solid methyl ester 3.
1-(3,4-diacetoxy-5-acetoxymethyl-tetrahydro-furan-2-yl)-[1,2,4-15N-3,5-13C]-
1H-[1,2,4]-triazole-3-[13C]-carboxylic acid methyl ester 22
The method described above was somewhat improved and used for the
synthesis of the acetoxytetrahydrofuranyl [M+4]-triazole-carboxylic acid
methyl ester 17. The reagents were added in two portions and the reaction
was heated for 50 min at 105–1108C, (instead of 35 min at 1508C). Also, the
eluent used for the silica gel chromatography was changed to 5–10% acetone/
CH2Cl2. These changes resulted in a 77% yield of product which was further
purified by crystallization from methanol to afford 422.6 mg (57% yield) of
pure 22.
1-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-[1,2,4-15N-3,5-13C]-
1H-[1,2,4]-triazole-3-[13C]-carboxylic acid [15N]-amide 23, (levovirin)
The same method was followed as described above for the synthesis of the
[M+4]-levovirin 5 with [15N]-ammonia gas being used as described below.
Product 22 (397.9 mg, 1.017 mmol) was transferred using 5 ml warm
methanol to a 30 cm3 centrifuge tube attached to a wishbone on the vacuum
line. After evacuation at À788C the clear colorless solution was equilibrated
with 335 mg (18.6 mmol) [15N]-ammonia gas contained in a 500 cm3 stopcock
flask. The reaction was warmed to ambient temperature, and stirred overnight.
Chromatography afforded 230.9 mg (90.5% yield) of [M+7]-levovirin 23
having a purity of 99.3% by HPLC. The isotopic distribution by MS was
87.8% M+7, and 10.5% M+6.
1-(3,4-dihydroxycyclopentanoneacetal-5-hydroxymethyl-tetrahydro-furan-2-yl)-
[1,2,4-15N-3,5-13C]-1H-[1,2,4]-triazole-3-[13C]-carboxylic acid [15N]-amide 24
[M+7]-Levovirin 23 (160.5 mg, 0.64 mmol) was dissolved in 2 ml CH3CN in a
100 cm3 pear flask. The sides were rinsed down by mild reflux. To this
suspension was added a solution of 250 ml (2.8 mmol, 4.4 eq) cyclopentanone,
350 ml trimethylorthoformate (3.15 mmol, 4.9 eq.), and 20 mg p-TSA in 0.5 ml
CH3CN. The clear colorless solution was stirred at 358C bath temperature for
3 h. The pH of the reaction mixture was adjusted to 7 with 150 ml 1 N NaOH
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LABELING OF LEVOVIRIN AND LEVOVIRIN VALINATE PRO-DRUG
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and concentrated to dryness. The residue was re-dissolved in 2 ml warm 3%
MeOH/CH2Cl2 and applied to a 1 mm Chromatotron rotor pre-equilibrated
with 3% MeOH/CH2Cl2. Pure product containing fractions were combined
and concentrated affording 149.8 mg (74% yield) 100% pure (by HPLC) of
acetal 24.
2-tert-butoxycarbonylamino-3-methyl-butyric acid 5-(3-[13C,15N]-carbamoyl)-
[1,2,4-15N-3,5-13C]-[1,2,4]-triazol-1-yl)-3,4-dihydroxycyclo pentanoneacetal-
tetrahydro-furan-2-yl methyl ester 26
The [M+7]-levovirin cyclopentylidene 24 (149.8 mg, 0.47 mmol), contained in
a 100 cm3 pear flask, was dissolved in 5 ml anhydrous THF at mild reflux.
After addition of 5 ml of dry toluene the solvents were removed by rotary
evaporation and the process repeated. The residue was re-dissolved in 5 ml
THF, triethylamine (10 ml) and 4-isopropyl-2,5-dioxo-oxazolidine-3-car-
boxylic acid- tert-butyl ester 25 (0.54 g, 2.2 mmol, 4.7 eq.) were added and
the solution was stirred overnight at ambient temperature. The crude reaction
was concentrated to dryness and diluted with 2–3 ml CH2Cl2 and applied
directly to a pre-equilibrated (3% MeOH/CH2Cl2) 1 mm silica gel Chromato-
tron rotor. Pure product fractions were combined to afford 267.2 mg of TLC
pure BOC-valinate 26, which was used directly in the next step.
1-[5-(3-[13C,15N]-carbamoyl-)-[1,2,4-15N-3,5-13C]-[1,2,4]-]-triazol-1-yl)-3,4-
dihydroxy tetrahydro-furan-2-ylmethoxycarbonyl]-2-methyl-propyl-ammonium
chloride 6
The [M+7]-BOC-levovirin valinate 26 (200 mg, 0.39 mmol) was dissolved in
1.2 ml toluene at 508C. Isopropanol (0.24 ml) and 6 N aq. HCl (0.27 ml) were
added and the two phase reaction was stirred at ambient temperature
overnight. The reaction was allowed to settle and HPLC evaluation of each
phase indicated about 30% of the starting material remained in the toluene
phase and the product was in the aqueous phase. The aqueous phase was
removed, and the toluene phase was rinsed with two 0.1 ml portions of water.
The toluene layer was mixed with water (0.24 ml) and 3 N HCl (0.27 ml) and
stirred overnight. HPLC of this second reaction showed complete conversion
to nearly pure product 6. The products from the first and second reactions
were crystallized from i-PrOH/H2O. The aqueous extracts were concentrated
in a stream of nitrogen and then re-dissolved in water (0.15 ml) and i-PrOH
(0.5 ml). The solution was then warmed to 508C and treated with i-PrOH
(1.5 ml), which was added dropwise over about 5 min. The clear colorless
solution was stirred for about 5 min until the product began to crystallize and
then 10 additional minutes. The fine white suspension was removed from the
bath and stirred overnight at ambient temperature, compacted by centrifuge,
and the clear colorless supernatant was removed by pipette. The snow white
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S. A. DE KECZER ET AL.
pellet was washed with 1 ml 48C i-PrOH (0.5 ml for the second product) and
dried for 2 h at 0.5 mm Hg. The total dry weight of the combined two pellets
was 127.1 mg (ꢀ85% yield) of [M+7]-levovirin valinate 6, 99% pure by
HPLC. The isotopic distribution by MS was 87.2% M+7 and 12.6% M+6.
1-pyrrolidin-1-ylmethyl-1H-[1,2,4]-triazole 27
[1,2,4]-Triazole (10 g, 145 mmol) was dissolved in 50 ml EtOH. To this solution
was added 13 ml (156 mmol) pyrrolidine and 11.5 ml (153 mmol) 37% aq
HCHO and the solution was stirred under nitrogen at reflux for 6 h. The
cooled reaction was diluted with 50 ml water, extracted three times with 50 ml
portions of CH2Cl2, the combined organic phase was dried with Na2SO4 and
concentrated. The residual oil was vacuum distilled using a Vigreux column
(66–688C head temp at 0.4 mm Hg) affording 16.3 g (74% yield) of NMR pure
product 27.
1H-[1,2,4]-triazole-3-[14C]-carboxylic acid methyl ester 4
Three 100 cm3 septum-side arm stopcock flasks (A, B, and C) all equipped
with spin bars were attached by a ‘tri-bone’ (14CO2 generator flask C via
Drierite tube) to the vacuum line. The system was dried by heat gun under
vacuum. Ba14CO3 (738.5 mg, 3.71 mmol, 57 mCi/mmol, 211 mCi) was weighed
into flask C. Flask A was charged with about 30 ml THF/lithium aluminum
hydride (LAH) as drying agent. Protected triazole 27 (651 mg, 4.28 mmol) was
weighed into flask B. THF (15–20 ml) was vacuum transferred from LAH into
flask B at À788C. After warming to ambient temperature to dissolve the
triazole in the THF, flask B was again cooled to À788C and the vacuum was
released with nitrogen. A solution of n-BuLi (2.70 ml of 1.6 N in hexane,
4.3 mmol) was added slowly, and the white suspension was stirred at À788C
for at least 30 min and then gradually warmed to À158C over 1 h. The mixture
was cooled first with dry ice and then with liquid N2. Flasks B and C were
evacuated and then isolated. Conc. H2SO4 (10 ml) was injected onto the
Ba14CO3. After 15 min all effervescence had ceased and all of the activity had
transferred to the reaction flask (B) as determined by b-probe. The reaction
was warmed to À788C and the milky suspension was allowed to warm to
ambient temperature overnight. Volatiles were vacuum transferred from the
reaction flask B and the white granular free flowing crude lithium carboxylate
salt was used directly in the next step.
A 38% solution of HCl in methanol was freshly prepared by bubbling HCl
gas into 10 ml methanol at ice bath temperature. All of this solution was added
to the crude lithium carboxylate salt still in the same flask attached to the
vacuum line (no exotherm was observed). The clear pale yellow solution
was stirred overnight at ambient temperature. Formation of product (ꢀ80%)
Copyright # 2006 John Wiley & Sons, Ltd.
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LABELING OF LEVOVIRIN AND LEVOVIRIN VALINATE PRO-DRUG
1235
was confirmed by TLC (sample neutralized with NH4OH before spotting).
Volatiles were removed to a liquid N2 trap. The cream colored paste
was neutralized with 6 ml 5% aq. NH4OH, and 6 ml methanol was added
(pH=7–8). The crude solution was concentrated and redissolved in a mixture
of 5 ml MeOH, 5 ml CH2Cl2, and 1 ml water. This solution was chromato-
graphed through a 100 cm3 silica gel column pre-equilibrated with 1% aq.
NH4OH in 7% MeOH/CH2Cl2. Pure product fractions were combined and
concentrated to a white solid. The solid was redissolved in 50 ml MeOH.
Purity of 4 by TLC was 99.9%, and total activity was 168 mCi (80% yield).
1-(3,4-diacetoxy-5-acetoxymethyl-tetrahydro-furan-2-yl)-1H-[1,2,4]-triazole-3-
[14C]-carboxylic acid methyl ester 28
The same method as described above for the preparation of acetoxytetra-
hydrofuranyl [M+6]-triazole-carboxylic acid methyl ester 22 was followed
except methanol was added to the reaction mixture to improve mixing and was
then removed as the toluene azeotrope under vacuum. The reaction time was
extended to 2 h with no improvement of yield. Chromatography afforded
48 mCi (57% yield) of coupling product 28 as a clear colorless glass.
1-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-1H-[1,2,4]-triazole-
3-[14C]-carboxylic acid amide 7
The same method was followed as described above for the synthesis of the
[M+4]-levovirin 5 to afford 35.4 mCi (74% yield) of deprotected product 7
having a radiochemical purity of 96% by HPLC.
1-(3,4-dihydroxycyclopentanoneacetal-5-hydroxymethyl-tetrahydro-furan-2-yl)-
1H-[1,2,4]-triazole-3-[14C]-carboxylic acid amide 29
The same method was followed as described above for the [M+7]-
cyclopentanone adduct 24 except the number of equivalents of cyclopentanone
and trimethylorthoformate were reduced to 2.8 and 3.0, respectively.
However, this resulted in an incomplete reaction. To drive the reaction, a
second portion of reagents was added. Following chromatography, this
afforded 22.5 mCi (66% radiochemical yield) of 90% pure acetal 29 as a clear
colorless glass.
2-tert-butoxycarbonylamino-3-methyl-butyric acid 5-(3-[14C]-carbamoyl-[1,2,
4]-triazol-1-yl)-3,4-dihydroxycyclopentanoneacetal-tetrahydro-furan-2-yl-
methylester 30
The same method as described above for the [M+7]-BOC-valinate 26 was
followed, affording 18.5 mCi (88% yield) of radiochemically pure [14C]-BOC-
valinate 30 which was used directly in the next step.
Copyright # 2006 John Wiley & Sons, Ltd.
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S. A. DE KECZER ET AL.
1-[5-(3-[14C]-carbamoyl-[1,2,4]triazol-1-yl)-3,4-dihydroxy-tetrahydro-furan-
2-ylmethoxycarbonyl]-2-methyl-propyl-ammonium Chloride 8 ([14C]-levovirin
valinate
The same method as described above for the [M+7]-valinate 6 was followed,
affording 12.5 mCi [14C]-levovirin valinate 8 with a specific activity of 57 mCi/
mmol. The radiochemical purity by AN-HPLC was 98.75%, with 1.25% [14C]-
levovirin 7.
Conclusion
The seven step synthesis of [M+4]-levovirin was accomplished in 12% overall
yield from commercially available [13C2,15N]-acetonitrile and ethyl [13C]-
formate. The similar seven step synthesis of [M+7]-levovirin was accom-
plished in 14% overall yield from the same stable labeled commercial reagents
and commercially available [15N2]-hydrazine sulfate and [15N]-ammonia. The
three step conversion to the [M+7]-levovirin valinate pro-drug was
accomplished in 69% overall yield from [M+7]-levovirin. The synthesis of
[14C]-levovirin was achieved in 33% overall yield from barium [14C]-carbonate.
This included a one pot procedure for the synthesis of triazole-[14C]-carboxylic
acid methyl ester in 80% yield from barium [14C]-carbonate. The three step
conversion to the [14C]-levovirin valinate pro-drug was accomplished in 41%
overall yield from [14C]-levovirin.
Acknowledgements
We thank Tatyana Voronin and Rong Teng for analytical support and
Yanzhou Liu for NMR analysis.
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Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1223–1236
DOI: 10.1002/jlcr