Protocols and Methods Received 26 March 2013,
Revised 17 April 2013,
Accepted 24 April 2013
Published online 16 July 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3064
An alternative and robust synthesis of [13C4] BaracludeW (entecavir) John A. Easter,a* Richard C. Burrell,a and Samuel J. Bonacorsi Jrb Stable isotope-labeled [13C4]entecavir (1) was prepared in 11 steps. Commercially available [13C]guanidine hydrochloride and diethyl[1,2,3-13C3]malonate were condensed to yield 2-amino[2,4,5,6-13C4]pyrimidine-4,6-diol (8). This was converted to the desired purine (7) in five steps. Introduction of the chiral epoxide was followed by subsequent deprotection to give [13C4]entecavir (1), in an overall yield of 5.7% from labeled precursors. The chemical purity of the title compound was determined to be >99% by HPLC. The isotopic distribution was determined by mass spectrometry to be 282[M + 4], 98.4%; 281[M + 3], 1.6%; and 278[M + 0], 99% by HPLC. This material was cochromatographed with a known material.7 MS [M + H]+ = 282. 1H NMR (400 MHz, DMSO-d6) 10.62 (br s, 1H), 7.68 (dd, J = 10.8, 4.8 Hz, 1H), 6.45 (br s, 2H), 5.37 (t, J = 7.8 Hz, 1H), 5.11 (t, J = 2.1 Hz, 1H), 4.93–4.82 (m, 2H), 4.57 (t, J = 2.1 Hz, 1H), 4.24 (br s, 1H), 3.54 (t, J = 5.9 Hz, 2H), 3.18 (d, J = 5.3 Hz, 1H), 2.29–2.15 (m, 1H), 2.13–1.98(m, 1H). 13C NMR (101 MHz, DMSO-d6) d 156.2 (dd, J = 87.1, 6.9 Hz), 152.8 (d, J = 2.3 Hz), 150.7 (dd, J = 61.7, 7.7 Hz), 115.4 (ddd, J = 64.0, 60.9, 2.3 Hz).
Results and discussion There are several possibilities for introducing isotopic labeling into entecavir. A retrosynthetic look at the molecule reveals a convenient point for disconnection supporting labeling of the purine ring system or the chiral sugar.
Although isotopic labels could be incorporated in either of these structures, labeling the purine would minimize the synthetic challenges associated with preparing chiral sugar derivatives and maximize labeled yields of entecavir. Labeling the purine ring would also allow us to link up with wellestablished chemistry by using an available chiral intermediate.7 By installing the chiral sugar later in the synthesis, the number of manipulations of the fragile sugar moiety is minimized. In our experience, early introduction of the ribose ring has led to variable yields and tedious manipulations. Given the economics of labeled synthesis, strategies that rely on iterative synthetic runs to overcome low yields are not desirable. An initial synthetic pathway to the labeled purine is outlined in Scheme 1. This strategy supports the introduction of up to four carbon-13 and three nitrogen-15 labels by using labeled guanidine and labeled ethyl-2-cyanoacetate, and was piloted using unlabeled precursors. Condensing unlabeled guanidine and unlabeled ethyl-2-cyanoacetate gave 2 in good yield. Nitrosation of 2 at the 5 position with sodium nitrite and glacial acetic acid afforded the nitroso compound 3.8 The nitroso group was then reduced to an amino group to give 4, and the purine
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 632–636
J. A. Easter et al. OH NH H2N
O
NH2
+
O
N
a
CN
OH
H2N
NH2
N
NO
N
b
H2N
N
2
OH N
c
N
H2N
N
HN
d
NH2
4
N
N
e
H2N
N H
N
H2N
OBn
Cl
O NH2
NH2
3
N H
N
5
N
N H2N
N H
N 7
6
Scheme 1. Synthesis of labeled purine 7: a, sodium methoxide, ethanol, 85 C; b, NaNO2, water, acetic acid, 80 C; c, ammonium sulfide, water, 50 C; d, formamide, 225 C; and e, phosphorus oxychloride, 145 C.
54% yield.8 Chlorination of 9 with phosphorous oxychloride and triethylmethylammonium chloride afforded 10 in 49% yield.11 Amination of 10 with ammonium hydroxide in a sealed tube led cleanly to 11. Formation of the purine ring was accomplished by reacting 11 with formic acid and triethyl orthoformate to produce 12. Yields of 11 and 12 were over quantitative because of the presence of inorganic salts. Benzylation of 12 yielded the desired 7 in 55% yield.13 Linking up with established chemistry, the chiral portion of the molecule was introduced via an epoxide ring opening of 13 with lithium hydroxide in the presence of 7 to yield 14 in 36% yield.7 The necessary olefin functionality was introduced by an Eastwood olifination of 14 in the presence of butylhydroxytoluene, triisopropylorthoformate, and trifluoroacetic acid to give a mixture of dioxolanes. Without isolation, the dioxolanes were reacted with acetic acid and acetic anhydride to give the methanesulfonic acid salt of 15 in 47% yield.7 A protodesilylation/debenzylation of 15 in the presence of methanesulfonic acid and trifluoromethanesulfonic acid in
ring was formed through condensation with formamide to yield 5.9 Unfortunately, all attempts to chlorinate 5 by refluxing in dimethylformamide and phosphorous oxychloride did not result in the desired product 6 but rather unreacted 5.10 Although the chemistry shown in Scheme 1 did not lead to 7, it did point out a weakness in the overall approach. This led us to consider if introducing the chlorine earlier in the synthesis would improve reactivity and allow for the preparation of 7. The literature revealed that diaminopyrimidine-1,2-diols similar to compound 9 can be chlorinated using a solution of phosphorous oxychloride containing a quaternary ammonium chloride and subsequently cyclized to the chloro purine.11,12 This new route to purine 7 is outlined in Scheme 2 and was shown to be reproducible for the preparation of purine 7. We condensed [13C]guanidine hydrochloride and diethyl[1,2,3-13C3]malonate in absolute ethanol and sodium ethoxide to yield 2-amino [2,4,5,6-13C4]pyrimidine-4,6-diol (8).8 The second amine group was introduced by reacting 8 with sodium nitrite in glacial acetic acid followed by reduction with sodium dithionite to give 9 in a OH NH
O
* NH2 + EtO *
H2N
O
a N
* * OEt H2N
*
* N
OH * * NH2 N * * H2N OH N
b
* * OH
8
Cl N H2N
*
*
N
* *
NH2
d
Cl
H2N
10
N *
Cl *
* NH2 *
N
9
e
N H2N
NH2
*
Cl *
* N
OBn O
OH 13
N
H2N
*
N * N H 7
OH SiMe2Ph
h, i
j, k
OBn H2N * N * N
f
12
SiMe2Ph
SiMe2Ph
OBn * * N N
N * N H
11
g
c
OH OH
H2N
* N * N
HN *
* N * OBn
OH
OBn
* N
H2N * N * N HN * N * O
O
14
15
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org
635
J. Label Compd. Radiopharm 2013, 56 632–636
1
Scheme 2. Synthesis of [ C4]entecavir, 10: a, NaOMe, ethanol, 85 C; b, NaNO2, water, 75 C, Na2S2O4; c, phosphorus oxychloride, Et3MeN+Cl , 105 C; d, NH4OH, 105 C; e, formic acid, triethylorthoformate, 100 C; f, benzyl alcohol, KOH, 100 C; g, LiOH, CH2Cl2, 90 C; h, butylhydroxytoluene, trifluoroacetic acid, triisopropylorthoformate, room temperature; i, acetic anhydride, acetic acid, methanesulfonic acid, 120 C; j, methanesulfonic acid, trifluoromethylsulfonic acid, CH2Cl2; and k, Na2CO.31.5H2O2, water, 100 C. 13
J. A. Easter et al. dichloromethane, followed by oxidation of the resulting silyl ether, gave crude 1.7 The crude sample was purified by anion exchange chromatography and semi-preparative HPLC to yield 239 mg of 1 having a purity of 99% as determined by HPLC. The isotopic distribution was determined by mass spectrometry to be 282 [M + 4], 98.4%; 281[M + 3], 1.6%; 278[M + 0],
Revised 17 April 2013,
Accepted 24 April 2013
Published online 16 July 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3064
An alternative and robust synthesis of [13C4] BaracludeW (entecavir) John A. Easter,a* Richard C. Burrell,a and Samuel J. Bonacorsi Jrb Stable isotope-labeled [13C4]entecavir (1) was prepared in 11 steps. Commercially available [13C]guanidine hydrochloride and diethyl[1,2,3-13C3]malonate were condensed to yield 2-amino[2,4,5,6-13C4]pyrimidine-4,6-diol (8). This was converted to the desired purine (7) in five steps. Introduction of the chiral epoxide was followed by subsequent deprotection to give [13C4]entecavir (1), in an overall yield of 5.7% from labeled precursors. The chemical purity of the title compound was determined to be >99% by HPLC. The isotopic distribution was determined by mass spectrometry to be 282[M + 4], 98.4%; 281[M + 3], 1.6%; and 278[M + 0], 99% by HPLC. This material was cochromatographed with a known material.7 MS [M + H]+ = 282. 1H NMR (400 MHz, DMSO-d6) 10.62 (br s, 1H), 7.68 (dd, J = 10.8, 4.8 Hz, 1H), 6.45 (br s, 2H), 5.37 (t, J = 7.8 Hz, 1H), 5.11 (t, J = 2.1 Hz, 1H), 4.93–4.82 (m, 2H), 4.57 (t, J = 2.1 Hz, 1H), 4.24 (br s, 1H), 3.54 (t, J = 5.9 Hz, 2H), 3.18 (d, J = 5.3 Hz, 1H), 2.29–2.15 (m, 1H), 2.13–1.98(m, 1H). 13C NMR (101 MHz, DMSO-d6) d 156.2 (dd, J = 87.1, 6.9 Hz), 152.8 (d, J = 2.3 Hz), 150.7 (dd, J = 61.7, 7.7 Hz), 115.4 (ddd, J = 64.0, 60.9, 2.3 Hz).
Results and discussion There are several possibilities for introducing isotopic labeling into entecavir. A retrosynthetic look at the molecule reveals a convenient point for disconnection supporting labeling of the purine ring system or the chiral sugar.
Although isotopic labels could be incorporated in either of these structures, labeling the purine would minimize the synthetic challenges associated with preparing chiral sugar derivatives and maximize labeled yields of entecavir. Labeling the purine ring would also allow us to link up with wellestablished chemistry by using an available chiral intermediate.7 By installing the chiral sugar later in the synthesis, the number of manipulations of the fragile sugar moiety is minimized. In our experience, early introduction of the ribose ring has led to variable yields and tedious manipulations. Given the economics of labeled synthesis, strategies that rely on iterative synthetic runs to overcome low yields are not desirable. An initial synthetic pathway to the labeled purine is outlined in Scheme 1. This strategy supports the introduction of up to four carbon-13 and three nitrogen-15 labels by using labeled guanidine and labeled ethyl-2-cyanoacetate, and was piloted using unlabeled precursors. Condensing unlabeled guanidine and unlabeled ethyl-2-cyanoacetate gave 2 in good yield. Nitrosation of 2 at the 5 position with sodium nitrite and glacial acetic acid afforded the nitroso compound 3.8 The nitroso group was then reduced to an amino group to give 4, and the purine
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 632–636
J. A. Easter et al. OH NH H2N
O
NH2
+
O
N
a
CN
OH
H2N
NH2
N
NO
N
b
H2N
N
2
OH N
c
N
H2N
N
HN
d
NH2
4
N
N
e
H2N
N H
N
H2N
OBn
Cl
O NH2
NH2
3
N H
N
5
N
N H2N
N H
N 7
6
Scheme 1. Synthesis of labeled purine 7: a, sodium methoxide, ethanol, 85 C; b, NaNO2, water, acetic acid, 80 C; c, ammonium sulfide, water, 50 C; d, formamide, 225 C; and e, phosphorus oxychloride, 145 C.
54% yield.8 Chlorination of 9 with phosphorous oxychloride and triethylmethylammonium chloride afforded 10 in 49% yield.11 Amination of 10 with ammonium hydroxide in a sealed tube led cleanly to 11. Formation of the purine ring was accomplished by reacting 11 with formic acid and triethyl orthoformate to produce 12. Yields of 11 and 12 were over quantitative because of the presence of inorganic salts. Benzylation of 12 yielded the desired 7 in 55% yield.13 Linking up with established chemistry, the chiral portion of the molecule was introduced via an epoxide ring opening of 13 with lithium hydroxide in the presence of 7 to yield 14 in 36% yield.7 The necessary olefin functionality was introduced by an Eastwood olifination of 14 in the presence of butylhydroxytoluene, triisopropylorthoformate, and trifluoroacetic acid to give a mixture of dioxolanes. Without isolation, the dioxolanes were reacted with acetic acid and acetic anhydride to give the methanesulfonic acid salt of 15 in 47% yield.7 A protodesilylation/debenzylation of 15 in the presence of methanesulfonic acid and trifluoromethanesulfonic acid in
ring was formed through condensation with formamide to yield 5.9 Unfortunately, all attempts to chlorinate 5 by refluxing in dimethylformamide and phosphorous oxychloride did not result in the desired product 6 but rather unreacted 5.10 Although the chemistry shown in Scheme 1 did not lead to 7, it did point out a weakness in the overall approach. This led us to consider if introducing the chlorine earlier in the synthesis would improve reactivity and allow for the preparation of 7. The literature revealed that diaminopyrimidine-1,2-diols similar to compound 9 can be chlorinated using a solution of phosphorous oxychloride containing a quaternary ammonium chloride and subsequently cyclized to the chloro purine.11,12 This new route to purine 7 is outlined in Scheme 2 and was shown to be reproducible for the preparation of purine 7. We condensed [13C]guanidine hydrochloride and diethyl[1,2,3-13C3]malonate in absolute ethanol and sodium ethoxide to yield 2-amino [2,4,5,6-13C4]pyrimidine-4,6-diol (8).8 The second amine group was introduced by reacting 8 with sodium nitrite in glacial acetic acid followed by reduction with sodium dithionite to give 9 in a OH NH
O
* NH2 + EtO *
H2N
O
a N
* * OEt H2N
*
* N
OH * * NH2 N * * H2N OH N
b
* * OH
8
Cl N H2N
*
*
N
* *
NH2
d
Cl
H2N
10
N *
Cl *
* NH2 *
N
9
e
N H2N
NH2
*
Cl *
* N
OBn O
OH 13
N
H2N
*
N * N H 7
OH SiMe2Ph
h, i
j, k
OBn H2N * N * N
f
12
SiMe2Ph
SiMe2Ph
OBn * * N N
N * N H
11
g
c
OH OH
H2N
* N * N
HN *
* N * OBn
OH
OBn
* N
H2N * N * N HN * N * O
O
14
15
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org
635
J. Label Compd. Radiopharm 2013, 56 632–636
1
Scheme 2. Synthesis of [ C4]entecavir, 10: a, NaOMe, ethanol, 85 C; b, NaNO2, water, 75 C, Na2S2O4; c, phosphorus oxychloride, Et3MeN+Cl , 105 C; d, NH4OH, 105 C; e, formic acid, triethylorthoformate, 100 C; f, benzyl alcohol, KOH, 100 C; g, LiOH, CH2Cl2, 90 C; h, butylhydroxytoluene, trifluoroacetic acid, triisopropylorthoformate, room temperature; i, acetic anhydride, acetic acid, methanesulfonic acid, 120 C; j, methanesulfonic acid, trifluoromethylsulfonic acid, CH2Cl2; and k, Na2CO.31.5H2O2, water, 100 C. 13
J. A. Easter et al. dichloromethane, followed by oxidation of the resulting silyl ether, gave crude 1.7 The crude sample was purified by anion exchange chromatography and semi-preparative HPLC to yield 239 mg of 1 having a purity of 99% as determined by HPLC. The isotopic distribution was determined by mass spectrometry to be 282 [M + 4], 98.4%; 281[M + 3], 1.6%; 278[M + 0],
