Development of an Efficient Intermediate, α-[2-(Trimethylsilyl)ethoxy]-2-N-[2-(trimethylsilyl)ethoxycarbonyl]folic Acid, for the Synthesis of Folate (γ)-Conjugates, and Its Application to the Synthesis of Folate-Nucleoside Conjugates

Full text of DOI:10.1021/jo000132a

5016
J . Org. Chem. 2000, 65, 5016-5021
Develop m en t of a n Efficien t In ter m ed ia te,
r-[2-(Tr im eth ylsilyl)eth oxy]-2-N-
[2-(tr im eth ylsilyl)eth oxyca r bon yl]folic
Acid , for th e Syn th esis of F ola te
(γ)-Con ju ga tes, a n d Its Ap p lica tion to th e
Syn th esis of F ola te-Nu cleosid e
Con ju ga tes†
Makoto Nomura,‡,§ Satoshi Shuto,‡ and
Akira Matsuda*,‡
Graduate School of Pharmaceutical Sciences,
Hokkaido University, Kita-12, Nishi-6, Kita-ku,
Sapporo 060-0812, J apan, and Hanno Research Center,
Taiho Pharmaceutical Co. Ltd., 1-27 Misugidai, Hanno,
Saitama 357-8527, J apan
Received February 2, 2000
In tr od u ction
F igu r e 1.
Folic acid (1, Figure 1) is transported into cells by a
folate receptor via receptor mediated endocytosis, a
process called potocytosis.1,2 Consequently, the folate
receptor is attractive as a potential drug delivery vehicle,
since covalently bound folate drug conjugates are also
transported into cells. Therefore, toxin,3a,b liposome con-
taining DOX3c,e or antisense oligodeoxynucleotide (ODN),3d
ODN,3j,k antibody,3i or [111In]DTPA3g,h have all been
conjugated with folate, and their biological effects have
been studied. Antitumor agents are likely to be the most
suitable for conjugating to folate, since the folate receptor
is known to be overexpressed on epithelial malignancies
such as ovarian, colorectal, and breast cancer, whereas
in normal tissue it is expressed at very low levels.2
Sch em e 1
* To whom correspondence and reprint requests should be ad-
dressed. Phone: +81-11-706-3228. Fax: +81-11-706-4980. E-mail:
matuda@pharm.hokudai.ac.jp.
† This paper constitutes part 196 of Nucleosides and Nucleotides;
for part 195 in the series, see: Fukuoka, M.; Shuto, S.; Minakawa, N.;
Ueno, Y, Matsuda, A. J . Org. Chem., in press.
‡ Hokkaido University.
§ Taiho Pharmaceutical Co. Ltd.
(1) Rothberg, K. G.; Ying, Y.; Kolhouse, J . F.; Kamen, B. A.;
Anderson, R. G. W. J . Cell Biol. 1990, 110, 637-649.
Although the biological importance of folic acid and its
derivatives has been widely recognized, an efficient
general method for preparing folate conjugates has not
yet been developed. Until now, drugs have usually been
conjugated to an unprotected folate using a condensing
agent such as DCC to produce an often inseparable
mixture of R-conjugate 4 and γ-conjugate 5 (Scheme
1).3a-f,i-l,4 A practical method for preparing γ-conjugate
5 would therefore be extremely useful, since only γ-con-
jugate can be recognized by the folate receptor.3f Most
recently, Fuchs et al. reported an effective method for
preparing the γ-conjugate 5 via folate (γ)-methyl ester 6
(Figure 2), which was successfully utilized for the syn-
thesis of DTPA-folate conjugates.3g,h Although their
method is suitable for certain kinds of folate conjugates,
further development of the method is needed for prepar-
ing the γ-conjugate since (1) the key intermediate, methyl
ester 6, is almost insoluble in organic solvents, which is
(2) (a) Campbell, I. G.; J ones, T. A.; Foulkes, W. D.; Trowsdale, J .
Cancer Res. 1991, 51, 5329-5338. (b) Weitman, S. D.; Lark, R. H.;
Coney, L. R.; Fort, D. W.; Frasca, V.; Zurawski, J r., V. R.; Kamen, B.
A. Cancer Res. 1992, 52, 3396-3401. (c) Ross, J . F.; Chaudhuri, P. K.;
Ratnam, M. Cancer 1994, 73, 2432-2443. (d) Franklin, W. A.;
Waintrub, M.; Edwards, D.; Christensen, K.; Prendegrast, P.; Woods,
J .; Bunn, P. A.; Kolhouse, J . F. Int. J . Cancer 1994, Suppl. 8, 89-95.
(e) Mantovani, L. T.; Miotti, S.; Me´nard, S.; Canevari, S.; Raspagliesi,
F.; Bottini, C.; Bottero, F.; Colnaghi, M. I. Eur. J . Cancer 1994, 30A,
363-369.
(3) (a) Leamon, C. P.; Low, P. S. J . Biol. Chem. 1992, 267, 24966-
24971. (b) Leamon, C. P.; Pastan, I.; Low, P. S. J . Biol. Chem. 1993,
268, 24847-24854. (c) Lee, R. J .; Low, P. S. J . Biol. Chem. 1994, 269,
3198-3204. (d)Wang, S.; Lee, R. J .; Cauchon, G.; Gorenstein, D. G.;
Low, P. S. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3318-3322. (e) Lee,
R. J .; Low, P. S. Biochim. Biophys. Acta 1995, 1233, 134-144. (f) Wang,
S.; Lee, R. J .; Mathias, C. J .; Green, M. A.; Low, P. S. Bioconjugate
Chem. 1996, 7, 56-62. (g) Luo, J .; Smith, M. D.; Lantrip, D. A.; Wang,
S.; Fuchs, P. L. J . Am. Chem. Soc. 1997, 119, 10004-10013. (h) Wang,
S.; Luo, J .; Lantrip, D. A.; Waters, D. J .; Mathias, C. J .; Green, M. A.;
Fuchs, P. L.; Low, P. S. Bioconjugate Chem. 1997, 8, 673-679. (i) Cho,
B. K.; Roy, E. J .; Patrick, T. A.; Kranz, D. M. Bioconjugate Chem. 1997,
8, 338-346. (j) Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998,
15, 1540-1545. (k) Habus, I.; Xie, J .; Iyer, R. P.; Zhou, W.-Q.; Shen,
L. X.; Agrawal, S. Bioconjugate Chem. 1998, 9, 283-291. (l) Gabizon,
A. G.; Horowitz, A. T.; Goren, D.; Tzemach, D.; Mandelbaum-Shavit,
F.; Qazen, M. M.; Zalipsky, S. Bioconjugate Chem. 1999, 10, 289-298.
(4) Nomura, Y.; Ueno, Y.; Matsuda, A. Nucleic Acids Res. 1997, 25,
2784-2791.
10.1021/jo000132a CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/14/2000

Notes
J . Org. Chem., Vol. 65, No. 16, 2000 5017
gated with various bases, i.e., Et3N, tetramethylguani-
dine,3g iPr2NEt, and N-methyl-1,5,9-triazabicyclo[4.4.0]-
decene (MTBD).3g While the condensations with Et3N or
i-Pr2NEt were unsuccessful, the desired product 9 was
obtained in 47% yield using tetramethylguanidine as a
base. When MTBD3g was used, the best result was
observed; treatment of 9 with 12 (1.5 equiv) in the
presence of MTBD (3 equiv) in DMSO at room temper-
ature gave the protected folate unit 7 in 71% yield, which
was stable enough to be purified by normal silica gel
column chromatography. As expected, it was also soluble
in organic solvents.
Syn t h esis of t h e F ola t e-Cyt osin e Nu cleosid e
Con ju ga tes. To confirm the usefulness of the folate unit
7, the folate-cytidine and -deoxycytidine conjugates
were first synthesized with the mixed-anhydride method.
The protected folate 7 was therefore successively treated
with ClCO2i-Bu/Et3N and cytidine or 2′-deoxycytidine in
dimethylformamide (DMF) at room temperature. These
reactions gave the desired condensation products 13 and
14 in 67 and 70% yield, respectively, in which the
γ-carboxyl group was conjugated at the N-4-position of
the cytosine moiety. The protected folate-cytosine nu-
cleoside conjugates 13 and 14 were readily purified by
silica gel column chromatography. The silyl protecting
groups of these conjugates were simultaneously removed
by treatment with TBAF in THF-DMSO. The reactions
resulted in the target folate-cytosine nucleoside conju-
gates as the tetrabutylammonium salts, which were
subsequently treated with NaOAc in a mixed solvent of
MeOH/EtOH to give the sodium salts of the folate-
nucleoside conjugates 15 and 16 as yellow precipitates.
Using this method, we easily obtained the analytically
pure conjugates 15 and 16 in excellent yields without
further purification. We have thus developed an efficient
method for preparing folate-cytosine nucleoside conju-
gates.
In our studies, DMDC 26 proved to be a potent
antitumor nucleoside, which significantly inhibited the
growth of various human solid tumor cells both in vitro
and in vivo. On the basis of these data, we designed a
folate conjugate of DMDC (3) as a highly tumor selective
antimetabolite. We expected the folate conjugate 3 to
show an even more remarkable antitumor effect than the
parent drug 2, since 3 could be delivered selectively to
tumor tissues due to its affinity for the folate receptor
overexpressed on tumor cells. Therefore, the procedure
used with folate unit 7 was successfully applied to the
synthesis of the target folate-DMDC conjugate 3 via the
protected intermediate 17, as shown in Scheme 3.
Syn t h esis of t h e F ola t e-Con ju ga t ed Nu cleosid e
P h osp h or a m id ite Un it for ODN Syn th esis. Antisense
ODNs have shown great efficacy in the selective inhibi-
tion of gene expression of tumor cells.8 However, the
therapeutic applications of such antisense ODN are often
F igu r e 2.
often troublesome for routine organic reactions and
purification, and (2) the reactivity of the (γ)-methyl ester
may be insufficient for the reaction with relatively weak
nucleophiles, such as aromatic amines including the
4-amino group of cytosine nucleosides. In this report, we
describe a new and efficient method for preparing folate
(γ)-drug conjugates using a novel protected folate unit 7
(Figure 2) and its application to the synthesis of folate
(γ)-conjugate of an antitumor nucleoside 3, 1-(2-deoxy-
2-methylene-â-D-erythro-pentofuranosyl)cytosine (DMDC,
2). The folate-conjugated nucleoside phosphoramidite
unit 18 designed for ODN synthesis was also synthesized
using this method.
Design a n d Syn th esis of th e P r otected F ola te
Un it 7. We designed the γ-carboxylate-free folate deriva-
tive 7 as a novel unit for conjugating drugs to folate, in
which the 2-amino group of the pteroic acid moiety and
the R-carboxylate of the glutamic acid moiety were
protected with a 2-(trimethylsilyl)ethoxycarbonyl (Teoc)
group5 and a 2-(trimethylsilyl)ethyl (TMSEt) group,
respectively. We expected the two lipophilic silyl-protect-
ing groups, which could be simultaneously deprotected
with TBAF, to improve the solubility of the molecule in
organic solvents. We also thought that the protection of
the 2-amino group would effectively decrease the nucleo-
philicity of the nitrogen and thus avoid side reactions.
The protected folate unit 7 was prepared from the 2-N-
Teoc-pteroic acid derivative and the R-carboxyl-protected
glutamic acid, as shown in Scheme 2. Enzymatic hy-
drolysis of folic acid (1) with carboxypeptidase G gave
pteroic acid (8),3g which, without purification, was suc-
cessively treated with carbonyldiimidazole (CDI) and
2-trimethylsilylethanol in dimethyl sulfoxide (DMSO) to
give 1-(2-N-Teoc-pteroyl)imidazole (9) in 50% yield from
folic acid (1). This compound was soluble in organic
solvents such as CHCl3-MeOH and stable at room
temperature and could be purified by normal silica gel
column chromatography.
N-Boc-L-Glu (OBn)-OH (10) was also successively
treated with CDI and 2-trimethylsilylethanol in CH2Cl2
to give the fully protected glutamate, which was hydro-
genated with Pd-C to give in turn the γ-carboxyl-free
glutamate 11 in 87% yield. The N-Boc group of 11 was
deprotected with TsOH and afforded the crystalline R-(2-
TMS-ethyl) glutamate 12 in 57% yield.
(6) (a) Takenuki, K.; Matsuda, A.; Ueda, T.; Sasaki, T.; Fujii, A.;
Yamagami, K. J . Med. Chem. 1988, 31, 1063-1064. (b) Matsuda, A.;
Takenuki, K.; Tanaka, M.; Sasaki, T.; Ueda, T. J . Med. Chem. 1991,
34, 812-819. (c) Yamagami, K.; Fujii, A.; Arita, M.; Okumoto, T.;
Sakata, S.; Matsuda, A.; Ueda, T.; Sasaki, T. Cancer Res. 1991, 51,
2319-2323. (d) Ono, T.; Fujii, A.; Hosoya, M.; Okumoto, T.; Sakata,
S.; Matsuda, A.; Sasaki, T. Biochem. Pharmacol. 1996, 52, 1279-1285.
(e) Miwa, M.; Eda, H.; Ura, M.; Ouchi, K. F.; Keith, D. D.; Foley, L.
H.; Ishitsuka, H. Clin. Cancer Res. 1998, 4, 493-497. (f) Eda, H.; Ura,
M.; Foley, L. H.; Ouchi, K. F.; Tanaka, Y.; Miwa, M.; Ishitsuka, H.
Cancer Res. 1998, 58, 1165-1169.
Condensation between 1-(2-N-Teoc-pteroyl)imidazole
(9) and R-(2-TMSEt) glutamate (12) was next investi-
(5) Carpino, L. A.; Tsao, J .-H. J . Chem. Soc., Chem. Commun. 1978,
358-359.
(7) Boral, A. L.; Dessain, S.; Chabner, B. A. Cancer Chemother.
Pharmacol. 1998, 42 (Suppl), S3-S21.

5018 J . Org. Chem., Vol. 65, No. 16, 2000
Notes
Sch em e 2
Sch em e 3
limited by their low cellular uptake.3d,j,k,8 Conjugation of
folic acid to antisense ODN at the γ-carboxyl group may
enhance their intracellular delivery due to binding to the
folate receptor overexpressed on tumor cells. Such folate-
conjugated antisense ODNs may show significant anti-
tumor effects; consequently, they have been extensively
studied.3j,k A folate-conjugated nucleoside phosphoramid-
ite unit, which is applicable to the usual DNA synthe-
sizer, would be very efficient for these antisense studies.
We therefore designed a folate-conjugated 2′-deoxy-
nucleoside derivative 18 (Scheme 4) as an amidite unit
for folate-conjugated antisense DNA synthesis and planned
to investigate its synthesis using the silyl-protected folate
unit 7 described above. The silyl protecting groups on the
folate moiety of 18 were expected to be stable under the
usual conditions for DNA synthesis by the phosphora-
midite method. They would be removed postsynthetically.
The silyl-protected folate 7 was first converted to the
activated ester 19 by treatment with N-hydroxysuccin-
imide and EDC in DMSO. The thymidine derivative 204
bearing an aminohexyl tether at the 5-position was
selected as a 2′-deoxynucleoside unit for conjugating to
folate. The reaction of 19 with 20 (1.2 equiv) was
performed in the presence of Et3N in CH2Cl2 at room
temperature to give the corresponding condensation
product 21 in 83% yield, after purification by silica gel
column chromatography. The phosphoramidyl group was
introduced at the 3′-position of thymidine moiety by the
usual procedure8 to complete the desired folate-conju-
gated amidite unit 18. The synthesis of folate-conjugated
ODNs using the amidite unit 18 is now under investiga-
tion.
Con clu sion s
For synthesizing folate-drug conjugates, we have de-
veloped a novel protected folate unit, R-[2-(trimethylsilyl)-
ethoxy]-2-N-[2-(trimethylsilyl)ethoxycarbonyl]folic acid
(7), which is soluble in organic solvents and readily
purified by silica gel chromatography. This unit 7 can
be conjugated to various drugs having an amino or a
hydroxyl group at the γ-carboxyl of folate. It was suc-
cessfully applied to the synthesis of the antitumor
cytosine nucleoside-folate conjugate 3 as well as to the
folate-conjugated nucleoside phosphoramidite unit 18.
We have thus shown that this method can be applied to
various types of folate-drug conjugate syntheses.
Exp er im en ta l Section
Melting points were not corrected. 1H and 13C spectra were
recorded at 400 or 270 MHz, and chemical shifts are reported
in parts per million downfield from Me4Si. All exchangeable
(8) Sinha, N. D.; Biernat, J .; Ko¨ster, H. Tetrahedron Lett. 1983, 24,
5843-5846.

Notes
J . Org. Chem., Vol. 65, No. 16, 2000 5019
Sch em e 4
protons were detected by disappearance on the addition of D2O.
Mass spectra were obtained by fast atom bombardment (FAB)
methods. Thin-layer chromatography was done on Merck coated
plate 60F254. Silica gel or neutralized silica gel chromatography
was done on Merck silica gel 5715 or ICN silica 60A, respectively.
Reactions were carried out under an argon atmosphere.
(s, 9H); 13C NMR (CDCl3) 177.88, 172.15, 155.35, 80.08, 64.04,
52.85, 30.11, 28.30, 27.78, 17.41, -1.48. Anal. Calcd for C15H29
-
NO6Si: C, 51.84; H, 8.42; N, 4.03. Found: C, 51.50; H, 8.23; N,
3.99.
R-[2-(Tr im eth ylsilyl)eth oxy]-L-glu ta m ic Acid (12). A mix-
ture of 11 (10.4 g, 30.0 mmol) and p-TsOH‚H2O (8.58 g, 45.0
mmol) in water (18 mL) and dioxane (54 mL) was stirred at 60
°C for 3 h. The reaction mixture was neutralized by Dowex 1 ×
1-[2-N -[2-(T r im e t h y ls ily l)e t h o x y c a r b o n y l]p t e r o y l]-
im id a zole (9). A mixture of folic acid (1, 17.7 g, 40.0 mmol),
ZnCl2 (48 mg, 0.35 mmol), and carboxypeptidase G (Sigma, 7
mg, 20 units) dissolved in Tris-HCl buffer (pH 7.3, 0.1 M, 780
mL) was stirred at 30 °C for 2 weeks. The pH of the mixture
was adjusted to 4.0 with 3 N HCl, and the resulting yellow
precipitate was filtered and dried to give crude pteroic acid 8
(12.1 g). The suspension of the crude pteroic acid obtained (5.0
g), Et3N (8.92 mL, 64.0 mmol), and CDI (10.4 g, 64.0 mmol) in
DMSO (80 mL) was stirred at room temperature for 3.5 h. To
the resulting solution was added 2-(trimethylsilyl)ethanol (18.3
mL, 128 mmol), and the whole was stirred at the same temper-
ature for 5 h. The reaction mixture was poured into a mixture
of water (550 mL), AcOH (16 mL), and Et2O (320 mL). The
resulting yellow precipitate was collected by filtration, dried, and
purified on a silica gel column with 10% MeOH in CHCl3 to give
a yellow solid, which was washed with Et2O to give 9 (4.13 g,
50%, as a yellow powder) in a pure form: mp > 300 °C; FAB-
-
8 (HCO3 form, 18 g), which was filtered off, and the filtrate
was evaporated and purified on a silica gel column with 20-
33% MeOH in CHCl3. Crystallization from MeOH afforded 12
in a pure form (4.20 g, 57%, as colorless needles): mp 122-123
°C; FAB-MS m/z 248 (MH+); 1H NMR (DMSO-d6) 4.09-4.14 (m,
2H), 3.31 (dd, 1H, J ) 4.9, 8.3 Hz), 2.22-2.31 (m, 2H), 1.77-
1.85 (m, 1H), 1.55-1.64 (m, 1H), 0.92-0.96 (m, 2H), 0.02 (s, 9H);
13C NMR (DMSO-d6) 174.61, 174.06, 62.26, 53.15, 30.84, 29.13,
16.90, -1.43. Anal. Calcd for C10H21NO4Si‚1/4H2O: C, 47.40; H,
8.55; N, 5.53. Found: C, 47.57; H, 8.15; N, 5.29.
R-[2-(Tr im e t h ylsilyl)e t h oxy]-2-N -[2-(t r im e t h ylsilyl)-
eth oxyca r bon yl]folic Acid (7). A mixture of 9 (1.52 g, 3.0
mmol), 12 (1.11 g, 4.5 mmol), and MTBD (1.29 mL, 9.0 mmol)
in DMSO (15 mL) was stirred at room temperature for 21 h.
The resulting mixture was poured into a mixture of aqueous
AcOH (1 M, 600 mL), MeOH (250 mL), and CHCl3 (600 mL),
and the whole was partitioned. The organic layer was washed
with aqueous AcOH (1 M)-MeOH (1:1, 400 mL) and then twice
with H2O-MeOH (2:1, 600 mL). The organic layer was dried
(Na2SO4), filtered, and evaporated. The residue was purified on
a silica gel column with CHCl3-MeOH-AcOEt-AcOH (17:1:2:
0.08) and then CHCl3-MeOH-AcOH (9:1:0.025) to give a yellow
solid, which was washed with CHCl3-Et2O to give 7 (1.47 g,
71% as a yellow powder): mp 137-138 °C; FAB-HRMS calcd
for C30H44N7O8Si2 m/z 686.2790, found m/z 686.2790 (MH+); 1H
NMR (DMSO-d6) 11.84 (br s, 2H), 8.81 (s, 1H), 8.29 (br d, 1H, J
) 6.4 Hz), 7.63 (d, 2H, J ) 8.8 Hz), 7.02 (t, 1H, J ) 6.1 Hz),
6.64 (d, 2H, J ) 8.8 Hz), 4.57 (d, 2H, J ) 6.1 Hz), 4.31-4.35 (m,
1H), 4.24-4.29 (m, 2H), 4.08-4.12 (m, 2H), 2.30 (t, 2H, J ) 7.2
Hz), 1.87-2.04 (m, 2H), 1.00-1.05 (m, 2H), 0.89-0.94 (m, 2H),
0.05 (s, 9H), 0.00 (s, 9H); 13C NMR (DMSO-d6) 173.65, 172.12,
166.23, 159.31, 154.80, 154.40, 151.92, 150.54, 149.05, 148.94,
129.85, 128.93, 121.15, 111.13, 64.59, 62.43, 51.96, 45.96, 30.24,
25.83, 17.06, 16.84, -1.43, -1.45. Anal. Calcd for C30H43N7O8-
Si2‚1/2H2O: C, 51.85; H, 6.38; N, 14.11. Found: C, 51.60; H, 6.26;
N, 14.04.
1
MS m/z 507 (MH+); H NMR (DMSO-d6) 11.74 (br s, 2H), 8.87
(s, 1H), 8.15 (s, 1H), 7.68 (t, 1H, J ) 6.1 Hz), 7.61 (d, 2H, J )
8.8 Hz), 7.60 (s, 1H), 7.09 (s, 1H), 6.77 (d, 2H, J ) 8.8 Hz), 4.65
(d, 2H, J ) 6.1 Hz), 4.26-4.30 (m, 2H), 1.01-1.07 (m, 2H), 0.05
(s, 9H); 13C NMR (DMSO-d6) 164.87, 159.39, 159.23, 154.90,
154.65, 153.05, 151.36, 149.18, 137.99, 132.72, 130.06, 129.67,
118.71, 117.61, 111.70, 64.62, 45.63, 17.00, -1.54. Anal. Calcd
for C23H26N8O4Si: C, 54.53; H, 5.17; N, 22.12. Found: C, 54.43;
H, 5.21; N, 21.92.
N-(ter t-Bu t oxyca r b on yl)-R-[2-(t r im et h ylsilyl)et h yoxy]-
L-glu ta m ic Acid (11). After a mixture of CDI (8.11 g, 50 mmol)
and 10 (16.9 g, 50.0 mmol) in CH2Cl2 (230 mL) was stirred at
room temperature for 1 h, 2-(trimethylsilyl)ethanol (7.17 mL,
50 mmol) was added, and the mixture was further stirred at
room temperature for 18 h. Water (150 mL) was added, and the
resulting mixture was partitioned. The organic layer was dried
(Na2SO4), filtered, and evaporated. The residue was purified on
a silica gel column with 25% EtOAc in hexane to give a colorless
oil. The oil was dissolved in EtOH (230 mL) and 10% Pd/C (3.0
g) was added, and the whole was stirred at room temperature
for 2 h under a hydrogen atmosphere. The mixture was filtered
through a Celite pad, and the filtrate was evaporated. The
residue was purified on a silica gel column with 10% MeOH in
CHCl3 to give 11 (15.2 g, 87%, as a colorless oil): FAB-MS m/z
Ger er a l P r oced u r e for t h e Cou p lin g 7 w it h Cyt osin e
Nu cleosid es. After a mixture of Et3N (46.0 µL, 0.33 mmol),
ClCO2i-Bu (42.8 µL, 0.33 mmol), and 7 (206 mg, 0.30 mmol) in
DMF (2.0 mL) was stirred at room temperature for 12 min, a
nucleoside (0.60 mmol) was added to the mixture, which was
further stirred at room temperature for 2 h. The reaction mixture
was poured into a mixture of aqueous AcOH (0.5 M, 60 mL),
1
348 (MH+); H NMR (CDCl3) 5.17 (br d, 1H, J ) 7.3 Hz), 4.31
(m, 1H), 4.20-4.25 (m, 2H), 2.38-2.53 (m, 2H), 2.16-2.20 (m,
1H); 1.88-1.97 (m, 1H), 1.44 (s, 9H), 0.98-1.05 (m, 2H), 0.05

5020 J . Org. Chem., Vol. 65, No. 16, 2000
Notes
MeOH (60 mL), and CHCl3 (80 mL), and then the whole was
partitioned. The organic layer was washed twice with a mixture
of water (60 mL) and MeOH (50 mL), dried (Na2SO4), filtered,
and evaporated. The residue was purified on a silica gel column
with CHCl3-MeOH-AcOEt-AcOH (9:1:1:0.04 to 6:1:1:0.03) to
afford a yellow foam. The foam was dissolved in DMSO (5 mL)
and poured into an aqueous AcOH (0.1 M, 150 mL), and the
resulting yellow powder was collected by filtration to give the
corresponding coupling product.
1-(â-D-Ribofu r an osyl)-4-N-[γ-[R-[2-(tr im eth ylsilyl)eth oxy]-
2-N-[2-(tr im eth ylsilyl)eth oxyca r bon yl]folyl]]cytosin e (13).
Cytidine (146 mg, 0.6 mmol) was used as a nucleoside to give
13 (182 mg, 67% as a yellow powder): mp 214-217 °C (decomp.);
FAB-HRMS calcd for C39H55N10O12Si2 m/z 911.3540, found m/z
911.3536 (MH+); 1H NMR (DMSO-d6) 11.75, 11.66 (each br s,
each 1H), 10.84 (s, 1H), 8.83 (s, 1H), 8.40 (d, 1H, J ) 7.6 Hz),
8.21 (d, 1H, J ) 7.6 Hz), 7.64 (d, 2H, J ) 8.8 Hz), 7.17 (d, 1H,
J ) 7.6 Hz), 7.01 (t, 1H, J ) 6.1 Hz), 6.65 (d, 2H, J ) 8.8 Hz),
5.76 (d, 1H, J ) 2.7 Hz), 5.44 (d, 1H, J ) 4.9 Hz), 5.13 (t, 1H, J
) 4.9 Hz), 5.02 (d, 1H, J ) 5.6 Hz), 4.58 (d, 2H, J ) 6.1 Hz),
4.30-4.34 (m, 3H), 4.27-4.29 (m, 2H), 3.93-3.98 (m, 2H), 3.87-
3.90 (m, 1H), 3.74 (ddd, 1H, J ) 2.9, 4.9, 12.2 Hz), 3.59 (ddd,
1H, J ) 3.2, 4.9, 12.2 Hz), 2.52 (t, 2H, J ) 7.6 Hz), 2.04-2.14
(m, 1H), 1.90-1.98 (m, 1H), 1.02-1.06 (m, 2H), 0.90-0.94 (m,
2H), 0.05 (s, 9H), -0.01 (s, 9H); 13C NMR (DMSO-d6) 172.91,
172.06, 166.24, 162.05, 159.18, 154.58, 154.48, 151.92, 150.53,
149.09, 148.79, 145.23, 129.85, 128.96, 121.20, 111.12, 95.14,
90.09, 84.12, 74.45, 68.62, 64.60, 62.46, 59.89, 51.92, 45.96, 40.41,
room temperature for 10 h. After an additon of AcOH (1.25 mL/
0.1 mmol of the starting material), the mixture was poured into
a mixture of CHCl3 and AcOEt (4:1, 25 mL/0.1 mmol of the
starting material), and the precipitated yellow powder (a tet-
rabutylammonium salt) was collected by filtration. To a solution
of the yellow powder obtained in EtOH-MeOH (1:1, 7 mL/0.1
mmol starting material) was added a solution of NaOAc in
MeOH (13 mg/mL, 1.6 mL/0.1 mmol starting material), and the
resulting mixture was centrifuged and decanted. MeOH and/or
EtOH was added to the yellow precipitate, centrifuged, and
decanted (×3) to afford the corresponding 4-N-(γ-folyl)cytosine
nucleoside sodium salt (15, 16, and 3).
4-N-(γ-F olyl)-1-(â-D-r ibofu r a n osyl)cytosin e Sod iu m Sa lt
(15). From 13 (128 mg, 0.14 mmol), the sodium salt 15 (98 mg,
90%, as a yellow powder) was obtained: mp 240-245 °C (dec);
FAB-HRMS calcd for C28H30N10NaO10 m/z 689.2044, found m/z
689.2027 (MH+); 1H NMR (DMSO-d6) 11.20 (s, 1H), 8.60 (s, 1H),
8.38 (d, 1H, J ) 7.6 Hz), 7.57 (d, 1H, J ) 6.1 Hz), 7.52 (d, 2H,
J ) 8.6 Hz), 7.28 (br s, 2H), 7.18 (d, 1H, J ) 7.6 Hz), 6.90 (t, 1H,
J ) 5.9 Hz), 6.62 (d, 2H, J ) 8.6 Hz), 5.75 (d, 1H, J ) 2.0 Hz),
5.54 (br s, 1H), 5.18 (br s, 1H), 5.10 (br s, 1H), 4.45 (d, 2H, J )
5.9 Hz), 3.91-4.01 (m, 3H), 3.85-3.88 (m, 1H), 3.72 (br d, 1H,
J ) 12.2 Hz), 3.58 (br d, 1H, J ) 12.2 Hz), 2.39 (t, 2H, J ) 7.4
Hz), 1.86-2.04 (m, 2H);13C NMR (DMSO-d6) 174.44, 173.88,
165.12, 162.16, 154.97, 154.60, 150.40, 148.25, 147.93, 144.99,
128.31, 127.79, 122.09, 111.24, 95.29, 90.09, 84.13, 74.49, 68.57,
59.88, 53.20, 45.97, 33.49, 28.04. Anal. Calcd for C28H29N10
-
NaO10‚EtOH‚H2O: C, 47.87; H, 4.95; N, 18.61. Found: C, 47.76;
H, 4.89; N, 18.23.
32.94, 25.52, 17.05, 16.81, -1.47. Anal. Calcd for C39H54N10O12
-
Si2‚1/2H2O: C, 50.91; H, 6.02; N, 15.22. Found: C, 50.69; H, 5.91;
N, 14.83.
1-(2-Deoxy-â-D-r ibofu r a n osyl)-4-N-(γ-folyl)cytosin e So-
d iu m Sa lt (16). From 14 (125 mg, 0.14 mmol), the sodium salt
16 (80 mg, 75%, as a yellow powder) was obtained: mp 230-
235 °C (dec); FAB-HRMS calcd for C28H30N10NaO9 m/z 673.2095,
found m/z 673.2081 (MH+); 1H NMR (DMSO-d6) 11.21 (br s, 1H),
8.61 (s, 1H), 8.27 (d, 1H, J ) 7.6 Hz), 7.58 (br s, 1H), 7.51 (d,
2H, J ) 8.3 Hz), 7.22 (br s, 2H), 7.20 (d, 1H, J ) 7.6 Hz), 6.90
(t, 1H, J ) 5.6 Hz), 6.62 (d, 2H, J ) 8.3 Hz), 6.08 (t, 1H, J ) 6.3
Hz), 5.26 (d, 1H, J ) 3.9 Hz), 5.06 (t, 1H, J ) 5.1 Hz), 4.45 (d,
2H, J ) 5.6 Hz), 4.17-4.21 (m, 1H), 3.94-3.99 (m, 1H), 3.81-
3.84 (m, 1H), 3.51-3.62 (m, 2H), 2.39 (t, 2H, J ) 7.6 Hz), 2.22-
2.28 (m, 1H), 1.85-2.03 (m, 3H); 13C NMR (DMSO-d6) 174.29,
173.78, 165.02, 162.03, 161.89, 156.04, 154.89, 154.28, 150.33,
148.17, 147.83, 144.50, 128.22, 127.73, 122.06, 111.18, 95.29,
87.76, 85.95, 69.78, 60.86, 53.20, 45.95, 40.84, 33.49, 28.06. Anal.
Calcd for C28H29N10NaO9‚EtOH‚H2O: C, 48.91; H, 5.06; N, 19.01.
Found: C, 48.89; H, 5.05; N, 18.71.
1-(2-Deoxy-â-D-r ibofu r a n osyl)-4-N-[γ-[R-[2-(tr im eth ylsi-
lyl)eth oxy]-2-N-[2-(tr im eth ylsilyl)eth oxyca r bon yl]folyl]]-
cytosin e (14). 2′-Deoxycytidine (136 mg, 0.6 mmol) was used
as a nucleoside to give 14 (187 mg, 70% as a yellow powder):
mp 217-219 °C (dec); FAB-HRMS calcd for C39H55N10O11Si2 m/z
895.3591, found m/z 895.3581 (MH+); 1H NMR (DMSO-d6) 11.75,
11.66 (each br s, each 1H), 10.83 (s, 1H), 8.83 (s, 1H), 8.31 (d,
1H, J ) 7.6 Hz), 8.21 (d, 1H, J ) 7.6 Hz), 7.64 (d, 2H, J ) 8.8
Hz), 7.19 (d, 1H, J ) 7.6 Hz), 7.02 (t, 1H, J ) 5.9 Hz), 6.64 (d,
2H, J ) 8.8 Hz), 6.08 (t, 1H, J ) 6.3 Hz), 5.24 (d, 1H, J ) 4.2
Hz), 5.03 (t, 1H, J ) 5.1 Hz), 4.58 (d, 2H, J ) 5.9 Hz), 4.27-
4.34 (m, 3H), 4.18-4.22 (m, 1H), 4.07-4.11 (m, 2H), 3.85 (q, 1H,
J ) 3.7 Hz), 3.60 (ddd, 1H, J ) 3.7, 5.1, 12.0 Hz), 3.56 (ddd, 1H,
J ) 3.7, 5.1, 12.0 Hz), 2.52 (t, 2H, J ) 7.3 Hz), 2.23-2.30 (m,
1H), 2.06-2.14 (m, 1H), 1.89-2.03 (m, 2H), 1.02-1.06 (m, 2H),
0.90-0.94 (m, 2H), 0.05 (s, 9H), -0.01 (s, 9H); 13C NMR (DMSO-
d6) 172.91, 172.11, 166.26, 162.02, 159.20, 154.67, 154.59, 154.28,
151.95, 150.55, 149.12, 148.83, 144.86, 129.89, 128.99, 121.19,
111.14, 95.20, 87.83, 86.08, 69.89, 64.64, 62.49, 60.91, 51.94,
45.97, 40.89, 32.94, 25.53, 17.07, 16.82, -1.44. Anal. Calcd for
1-(2-Deoxy-2-m eth ylen e-â-D-er yth r o-p en tofu r a n osyl)-4-
N-(γ-folyl)cytosin e Sod iu m Sa lt (3). From 17 (91 mg, 0.10
mmol), the sodium salt 3 (60 mg, 80%, as a yellow powder) was
obtained: mp 231-235 °C (dec); FAB-MS m/z 685 (MH+); FAB-
HRMS calcd for
C29H30N10NaO9 m/z 685.2095, found m/z
C
39H54N10O11Si2‚1/2H2O: C, 51.81; H, 6.13; N, 15.49. Found: C,
685.2078; 1H NMR (DMSO-d6) 11.24 (br s, 1H, DMDC 4-NH),
8.61 (s, 1H), 8.04 (d, 1H, J ) 7.6 Hz), 7.57 (br s, 1H), 7.52 (d,
2H, J ) 8.0 Hz), 7.29 (br, 2H), 7.19 (d, 1H, J ) 7.6 Hz), 6.90 (t,
1H, J ) 5.6 Hz), 6.63 (d, 2H, J ) 8.0 Hz), 6.53 (s, 1H), 5.72 (d,
1H, J ) 5.6 Hz), 5.33, 5.30 (each s, each 1H), 5.07 (t, 1H, J )
5.1 Hz), 4.48-4.53 (m, 1H), 4.45 (d, 2H, J ) 5.6 Hz), 3.96-4.00
(m, 1H), 3.58-3.74 (m, 3H), 2.39 (t, 2H, J ) 7.5 Hz), 1.85-2.04
(m, 2H); 13C NMR (DMSO-d6) 174.21, 173.93, 165.10, 162.32,
161.86, 154.85, 154.70, 150.40, 150.22, 148.25, 147.96, 145.65,
128.28, 127.79, 122.10, 111.56, 111.24, 96.03, 84.98, 84.79, 69.28,
60.22, 53.17, 45.98, 33.50, 28.02; Anal. Calcd for C29H29N10NaO9‚
EtOH‚H2O: C, 49.73; H, 4.98; N, 18.71. Found: C, 50.09; H,
4.98; N, 18.74.
51.56; H, 6.11; N, 15.18.
1-(2-Deoxy-2-m eth ylen e-â-D-er yth r o-p en tofu r a n osyl)-4-
N-[γ-[R-[2-(t r im et h ylsilyl)et h oxy]-2-N-[2-(t r im et h ylsilyl)-
eth oxyca r bon yl]folyl]]cytosin e (17). DMDC (2) (144 mg, 0.6
mmol) was used as a nucleoside to give 17 (180 mg, 66% as a
yellow powder): mp 219-222 °C (dec); FAB-HRMS calcd for
C
40H55N10O11Si2 m/z 907.3590, found m/z 907.3577 (MH+); 1H
NMR (DMSO-d6) 11.75, 11.66 (each br s, each 1H), 10.88 (s, 1H),
8.83 (s, 1H), 8.20 (d, 1H, J ) 7.9 Hz), 8.09 (d, 1H, J ) 7.6 Hz),
7.64 (d, 2H, J ) 8.6 Hz), 7.18 (d, 1H, J ) 7.6 Hz), 7.02 (t, 1H, J
) 5.9 Hz), 6.64 (d, 2H, J ) 8.6 Hz), 6.53 (s, 1H), 5.66 (d, 1H, J
) 5.9 Hz), 5.33, 5.30 (each s, each 1H), 5.01 (t, 1H, J ) 4.9 Hz),
4.58 (d, 2H, J ) 5.9 Hz), 4.47-4.53 (br, 1H), 4.26-4.32 (m, 3H),
4.06-4.12 (m, 2H), 3.54-3.76 (m, 3H), 2.52 (t, 2H, J ) 7.1 Hz),
1.86-2.13 (m, 2H), 1.01-1.07 (m, 2H), 0.89-0.95 (m, 2H), 0.05
(s, 9H), -0.01 (s, 9H); 13C NMR (DMSO-d6) 172.98, 172.10,
166.26, 162.22, 159.21, 154.60, 151.95, 150.55, 150.22, 149.12,
148.81, 145.94, 129.90, 128.98, 121.19, 111.55, 111.14, 95.85,
85.00, 84.87, 69.28, 64.64, 62.49, 60.21, 51.88, 45.96, 32.93, 25.48,
17.07, 16.81, -1.43. Anal. Calcd for C40H54N10O11Si2‚1/2H2O: C,
52.44; H, 6.05; N, 15.29. Found: C, 52.04; H, 6.04; N, 14.92.
4-N-(γ-F olyl)cytosin e Nu cleosid e Sod iu m Sa lt (Gen er a l
P r oced u r e). A mixture of a protected folate-cytosine nucleoside
conjugate (13, 14, 17) and TBAF (1 M in THF, 10 equiv) in
DMSO (1 mL/0.1 mmol of the starting material) was stirred at
R-[2-(Tr im e t h ylsilyl)e t h oxy]-2-N -[2-(t r im e t h ylsilyl)-
eth oxyca r bon yl]folic Acid γ-(N-Hyd r oxysu ccin im id e) Es-
ter (19). A mixture of 7 (617 mg, 0.90 mmol), N-hydroxysuc-
cinimide (125 mg, 1.1 mmol), and EDC‚HCl (173 mg, 0.90 mmol)
in DMF (4.5 mL) was stirred at room temperature for 18 h. The
reaction mixture was poured into water (300 mL), and the
resulting yellow precipitate was collected by filtration to give
19 (647 mg, 87%, as a yellow powder): FAB-MS m/z 783 (MH+);
1H NMR (DMSO-d6) 11.69 (br s, 2H), 8.83 (s, 1H), 8.31 (d, 1H,
J ) 7.1 Hz), 7.64 (d, 2H, J ) 8.8 Hz), 7.05 (t, 1H, J ) 6.1 Hz),
6.65 (d, 2H, J ) 8.8 Hz), 4.58 (d, 2H, J ) 6.1 Hz), 4.36-4.42 (m,
1H), 4.26-4.31 (m, 2H), 4.10-4.14 (m, 2H), 2.72-2.86 (m, 6H),
2.02-2.13 (m, 2H), 1.01-1.06 (m, 2H), 0.90-0.94 (m, 2H), 0.05

Notes
J . Org. Chem., Vol. 65, No. 16, 2000 5021
(s, 9H), -0.01 (s, 9H). Anal. Calcd for C34H46N8O10Si2‚3H2O: C,
57.70; H, 6.58; N, 11.05. Found: C, 57.99; H, 6.20; N, 10.84.
5-[6-[γ-[R-[2-(Trimethylsilyl)ethoxy]-2-N-[2-(trimethylsilyl)-
e t h o x y c a r b o n y l]fo ly l]a m i n o ]h e x y lc a r b a m o y l]-1-(2-
d e o x y -5-O -(4,4′-d im e t h o x y t r it y l)-â-D -r ib o fu r a n o s y l)-
u r a cil (21). A mixture of 19 (392 mg, 0.47 mmol), 205 (387 mg,
0.575 mmol), and Et3N (0.104 mL, 0.75 mmol) in CH2Cl2 (10
mL) was stirred at room temperature for 3.5 h. The reaction
mixture was diluted with CHCl3 (25 mL) and washed with water
(25 mL × 2). The organic layer was dried (Na2SO4), evaporated,
and purified on a neutralized silica gel column with CHCl3-
AcOEt-MeOH (25:3:1 to 17:2:1) and then 10% MeOH in CHCl3
to give 21 (528 mg, 83%, as a yellow foam): FAB-MS m/z 1340
(MH+); 1H NMR (DMSO-d6) 11.92, 11.74, 11.67 (each br s, each
1H), 8.82 (s, 1H), 8.65 (t, 1H, J ) 5.4 Hz), 8.41 (s, 1H), 8.28 (d,
1H, J ) 7.1 Hz), 7.77 (t, 1H, J ) 5.1 Hz), 7.63 (d, 2H, J ) 8.8
Hz), 7.15-7.25 (m, 9H), 7.01 (t, 1H, J ) 6.3 Hz), 6.86 (d, 4H, J
) 9.0 Hz), 6.64 (d, 2H, J ) 8.8 Hz), 6.05 (t, 1H, J ) 6.5 Hz),
5.32 (d, 1H, J ) 4.4 Hz), 4.57 (d, 2H, J ) 6.3 Hz), 4.23-4.30 (m,
3H), 4.06-4.10 (m, 3H), 3.91 (m, 1H), 3.71 (s, 6H), 3.15-3.23
(m, 4H), 2.95-3.00 (m, 2H), 2.10-2.29 (m, 4H), 1.97-2.05 (m,
1H), 1.83-1.93 (m, 1H), 1.38-1.45 (m, 2H), 1.29-1.35 (m, 2H),
1.15-1.28 (m, 4H), 1.01-1.06 (m, 2H), 0.89-0.93 (m, 2H), 0.04
(s, 9H), -0.02 (s, 9H). Anal. Calcd for C67H85N11O15Si2‚1/2H2O:
C, 51.47; H, 5.98; N, 14.08. Found: C, 51.56; H, 5.98; N, 14.15.
5-[6-[γ-[2-N-[R-[2-(Tr im eth ylsilyl)eth oxy]-2-(tr im eth ylsi-
lyl)et h oxyca r b on yl]folyl]a m in o]h exylca r b a m oyl]-1-[3-O-
[(2-cyan oeth yl)(N,N-diisopr opylam in o)ph osph in yl]-2-deoxy-
5-O-(4,4′-d im eth oxytr ityl)-â-D-r ibofu r a n osyl]u r a cil (18). To
a solution of 21 (380 mg, 0.282 mmol) in CH2Cl2 (4.2 mL) were
added EtNi-Pr2 (246 µL, 1.41 mmol) and 2-(cyanoethyl)-N,N-
diisopropylchlorophosphoramidite (94.4 µL, 0.423 mmol), and the
resulting mixture was stirred at room temperature for 5.5 h.
The reaction mixture was poured into a mixture of CHCl3 (100
mL) and saturated aqueous NaHCO3 (100 mL), and the whole
was partitioned. The organic layer was washed with saturated
aqueous NaHCO3, dried (Na2SO4), and evaporated. The residue
was purified on a neutralized silica gel column with 67% CH3-
CN in CHCl3 and then CHCl3-MeOH-AcOEt (34:1:4 to 17:1:2)
to give 18 (273 mg, 63% as a yellow foam); FAB-HRMS calcd
for C76H103N13O16PSi2 m/z 1540.6922, found m/z 1540.6920
(MH+); 1H NMR (DMSO-d6) 11.94 (br s, 1H), 11.73 (br s, 2H),
8.83 (s, 1H), 8.67 (t, 1H, J ) 5.6 Hz), 8.48 (s, 1/2H), 8.46 (s, 1/2H),
8.28 (d, 1H, J ) 7.3 Hz), 7.78 (t, 1H, J ) 5.6 Hz), 7.65 (d, 2H, J
) 8.8 Hz), 7.18-7.37 (m, 9H), 7.01 (t, 1H, J ) 6.1 Hz), 6.85-
6.88 (m, 4H), 6.65 (d, 2H, J ) 8.8 Hz), 6.09 (t, 1/2H, J ) 6.1 Hz),
6.06 (t, 1/2H, J ) 6.5 Hz), 4.59 (d, 2H, J ) 6.1 Hz), 4.25-4.39
(m, 4H), 4.02-4.13 (m, 3H), 3.73 (s, 6H), 3.40-3.75 (m, 4H),
3.17-3.28 (m, 4H), 2.97-3.02 (m, 2H), 2.75 (t, 2/2H, J ) 5.9
Hz), 2.63 (t, 2/2H, J ) 5.9 Hz), 2.36-2.46 (m, 2H), 2.15-2.20
(m, 2H), 1.98-2.06 (m, 1H), 1.86-1.95 (m, 1H), 1.40-1.47 (m,
2H), 1.30-1.37 (m, 2H), 1.20-1.28 (m, 4H), 0.90-1.12 (m, 16H),
0.06 (s, 9H), 0.01 (s, 9H); 31P NMR (acetone-d6) 150.04, 149.86
(85% H3PO4 as an internal standard).
J O000132A