Selective protection of 2',2'-difluorodeoxycytidine (gemcitabine)

Article abstract of DOI:10.1021/jo9911140

Gemcitabine (1) is a promising new anticancer agent used in pancreatic cancer. Improvement in the selective targeting of compound 1 and other cytotoxic agents to solid tumors may be enhanced by conjugation to ligands that target peripheral benzodiazepine receptors (PBRs) located on mitochondria and known to be overexpressed in human brain tumors. Development of such chemical conjugates requires selective protection on 4-NH₂, 3'-OH, and 5'-OH of compound 1. All three monoprotected and three diprotected gemcitabine derivatives (2 to 7) were synthesized in good yield by employing a single commonly used protecting reagent, di-tert-butyl dicarbonate, under different conditions. Consequently, the three mono-ligand-gemcitabine conjugates coupled at 4-NH₂, 3'-OH, and 5'-OH respectively (14 to 16) were synthesized in high yield using the PBR ligand PK11195. This selective protection/deprotection strategy offers a relatively straightforward means to modify other nucleosides.

Full text of DOI:10.1021/jo9911140

J . Org. Chem. 1999, 64, 8319-8322
8319
Selective P r otection of 2′,2′-Diflu or od eoxycytid in e (Gem cita bin e)
Zhi-wei Guo and J ames M. Gallo*
Department of Pharmacology Fox Chase Cancer Center 7701 Burholme Avenue,
Philadelphia, Pennsylvania 19111
Received J uly 12, 1999
Gemcitabine (1) is a promising new anticancer agent used in pancreatic cancer. Improvement in
the selective targeting of compound 1 and other cytotoxic agents to solid tumors may be enhanced
by conjugation to ligands that target peripheral benzodiazepine receptors (PBRs) located on
mitochondria and known to be overexpressed in human brain tumors. Development of such chemical
conjugates requires selective protection on 4-NH2, 3′-OH, and 5′-OH of compound 1. All three
monoprotected and three diprotected gemcitabine derivatives (2 to 7) were synthesized in good
yield by employing a single commonly used protecting reagent, di-tert-butyl dicarbonate, under
different conditions. Consequently, the three mono-ligand-gemcitabine conjugates coupled at 4-NH2,
3′-OH, and 5′-OH respectively (14 to 16) were synthesized in high yield using the PBR ligand
PK11195. This selective protection/deprotection strategy offers a relatively straightforward means
to modify other nucleosides.
In tr od u ction
PBR ligands may be covalently coupled to compound
1 at 4-NH2, 3′-OH, and 5′-OH positions directly or via a
linker. All conjugates linked at these different positions
are potentially interesting because they may possess
different PBR binding affinities and cytotoxicity. Many
different protection/deprotection strategies of nucleoside
monomers have been established.4 In those strategies,
however, multiple protecting reagents were employed to
reach the goal. We disclose here that the tert-butoxycar-
bonyl (Boc) group serves as an useful selective protecting
group in gemcitabine derivative synthesis. All three
monoprotected and three diprotected gemcitabine deriva-
tives (2-7) were prepared, respectively, in good yield by
employing one single commonly used protecting reagent
di-tert-butyl dicarbonate (DBDC) under different condi-
tions. The usefulness of this strategy was demonstrated
by the synthesis of three PBR ligand-gemcitabine con-
jugates coupled at 4-NH2, 3′-OH, and 5′-OH, respectively
(14 to 16), in high yield and in a relatively simple
manner.
Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) (1) is
a promising antineoplastic drug approved for use in
pancreatic cancer. In comprehensive preclinical and
clinical studies,1 it has shown activity against a wide
spectrum of human solid tumors including nonsmall cell
lung, pancreatic, colon, breast, bladder, ovarian, head and
neck, cervical and hepatocellular cancers. The hydrochlo-
ride of compound 1 (GEMZAR) (1a ) is now marketed in
many countries.1
Selective drug delivery to solid tumors continues to be
a problem that can be addressed by the development of
chemical conjugates that bind receptors overexpressed
in tumors. Peripheral benzodiazepine receptors (PBRs)
located on the outer membrane of mitochondria may
serve as such a target as they are overexpressed in brain
tumors relative to normal brain.2 A host of PBR ligands
are known that can bind to human PBRs (i.e., PK11195)
and rat PBR (i.e., Ro 5 4864 and PK11195).2 Compound
1 is an attractive candidate for development as a PBR
ligand drug conjugate since its uptake into brain is
expected to be limited because of its hydrophilicity. An
immunoglobulin conjugate of compound 1 has been
reported.3 The PBR ligand-drug conjugates are unique
in that they are of low molecular weight and target an
intracellular receptor as opposed to the more common cell
surface receptor sought in other drug delivery systems.
Resu lts a n d Discu ssion
When DBDC was added to a solution of 1a in dioxane-
aqueous NaOH, three products formed in 10 min, and
the product profile changed over time. The structures of
the three products were determined in separate experi-
ments as 3′-O-Boc-gemcitabine (2), 3′-O-5′-O-di-Boc-gem-
citabine (3), and 5′-O-Boc-gemcitabine (4). Thus, various
conditions and procedures were studied to achieve selec-
tive protection of 1 in good yield. The results are shown
in Scheme 1. Under condition a with 1 equiv of DBDC, 2
was obtained in 85% isolated yield, and the same product
* Corresponding author. Tel: 215 728-2461. Fax: 215 728-4333.
E-mail: jm_gallo@fccc.edu.
(1) (a) Hertel, L. W.; Kroin, J . S.; Misner, J . W.; Tustin, J . M. J .
Org. Chem. 1988, 53, 3: 2406-2409. (b) Postmus, P. E.; Schramel, F.
M.; Smit, E. F. Semin. Oncol. 1998, 25 (4 Suppl 9), 79-82. (c)
Carmichael, J . Br. J . Cancer 1998, 78 (Suppl 3), 21-25. (d) Graziadei,
I.; Kelly, T.; Schirmer, M.; Geisen, F. H.; Vogel, W.; Konwalinka, G. J .
Hepatol. 1998, 28, 504-509. (e) Aapro, M. S.; Martin C.; Hatty, S. Anti-
Cancer Drugs 1998, 9, 191-201.
(2) (a) Starosta-Rubinstein, S.; Ciliax, B. J .; Penney, J . B.; McKeever,
P.; Young, A. B. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 891-895. (b)
Black, K.; Ikezaki, K.; Santori, E.; Becker, D.; Vinters, H. Cancer 1990,
65, 93-97. (c) Kupczyk-Subotkowska, L.; Siahaan, T. J .; Basile, A. S.;
Friedman, H. S.; Higgins, P. E.; Song, D.; Gallo, J . M. J . Med. Chem.
1997, 40, 1726-1730. (d) Guo, Z. W.; Ma, J . G.; Adams, A.; Gallo, J .
M. Proc. Am. Assoc. Cancer Res. 1999, 40, 585.
(4) (a) Greene, T. W. Protective groups in organic synthesis; J ohn
Wiley & Sons: New York; pp 50-71 and 223-248. (b) Bhat, B.;
Sanghvi, Y. S. Tetrahedron Lett. 1997, 38, 8811-8814. (c) Sinha, N.
D.; Davis, P.; Schultze, L. M.; Upadhya, K. Tetrahedron Lett. 1995,
36, 9277-9280. (d) Hayakawa, Y.; Kato, H.; Uchiyama, M.; Kajino,
H.; Noyori, R. J . Org. Chem. 1986, 51, 2400-2402. (e) Ti, G. S.; Gaffney,
B. L.; J ones, R. A. J . Am. Chem. Soc. 1982, 104, 1316-1319. (f) Ishido,
Y.; Nakazaki, N.; Sakairi, N. J . Chem. Soc., Perkin Trans. 1 1979,
2088-2098. (g) Beaucage, S. L.; Ogilvie, K. K. Tetrahedron Lett. 1977,
20, 1691-1694.
(3) Koppel, G. A.; Kennedy, G. D.; Armour, H. K.; Scott, W. L. Eur.
Patent 0272891A2, 1987.
10.1021/jo9911140 CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/06/1999

8320 J . Org. Chem., Vol. 64, No. 22, 1999
Guo
Sch em e 1a
a
(a) Dioxane-water (4/1), Na2CO3, DBDC (1-5 equiv); (b) dioxane-1 M aqueous KOH (1/1), DBDC (excess); (c) dioxane, DBDC (excess),
then 1 M aqueous KOH (1/1 to dioxane); (d) MeOH-1 M aqueous Na2CO3 (1/1); (e) dioxane, TEA, DMAP, DBDC (excess); (f) dioxane,
DBDC (excess), 37 °C.
Ta ble 1. Ch em ica l Sh ift Da ta (p p m )a
Sch em e 2a
5
6
1′
3′
4′
5′A
5′B
1
2
3
4
5
6
7
8
5.98
5.96
5.98
6.01
7.27
7.23
7.27
7.28
7.85
7.75
7.62
7.62
8.04
8.31
8.20
8.02
6.25
6.33
6.34
6.33
6.32
6.27
6.37
6.38
4.35
5.32
5.28
4.33
4.36
4.54
5.36
5.32
3.95
4.21
4.42
4.17
4.24
4.02
4.30
4.50
3.95
3.95
4.52
4.53
4.54
4.02
4.00
4.55
3.82
3.82
4.45
4.38
4.42
3.86
3.86
4.50
a
The 600 MHz 1H NMR spectra were obtained in acetone-d6
with 2% D2O. The position numbers are as indicated in Scheme
1.
profile was obtained with 5 equiv of DBDC based on TLC
analysis. Excess DBDC was used for all the following
experiments. A 1:1 mixture of 2 and 3 was obtained under
condition b with excess DBDC, and the crude product
mixture was converted into 3 under condition c to give
90% overall yield. Compound 3 was partially deprotected
under condition d to give 4 in 85% isolated yield and
recovered 1 in 10%. When 1a was treated under forced
condition e with excess DBDC, a mixture of two nonpolar
products formed based on TLC analysis. The structure
of the major product was determined in a separate
experiment as 4-N-3′-O-5′-O-tri-Boc-gemcitabine (8). The
minor product was less polar than 8 by TLC, and its
structure was not determined. If pyridine was used
instead of dioxane, a similar product profile was obtained
based on TLC analysis. The product mixture was par-
tially deprotected under condition d to give 4-N-5′-O-di-
Boc-gemcitabine (5) in 48% isolated yield plus 4-N-Boc-
gemcitabine (6) in 41% isolated yield. Finally, 4-N-3′-O-
Boc-gemcitabine (7) was obtained in 95% isolated yield
by using 2 as the starting material under condition f.
Interestingly, compound 8 was not detected by TLC
analysis under this condition. If triethylamine or diiso-
propylethylamine was added, a mixture of 7 and 8
formed. When 1a was used instead of 2 under condition
f, no reaction occurred after 48 h based on TLC analysis,
most likely because 1a is practically insoluble in dry
dioxane. The structure determination of compounds 2
a
(a) Succinic anhydride, DIEA; (b) DCC, 1-hydroxybenzotria-
zole; (c) TFA; (d) NaHCO3; (e) DCC, DMAP.
PK11195 is a model human PBR ligand5 and served
as the PBR ligand to synthesize conjugates 14, 15, and
16 by coupling compound 10 with di-protected gemcit-
abine derivatives (3, 5, and 7) followed by deprotection
using TFA as shown in Scheme 2. Compound 10 was
synthesized from succinic anhydride and compound 9,
which was prepared as described in the literature.5 To
assign the 1H NMR spectrum of compound 10, it was
compared to the spectrum of an authentic sample of
PK11195 [Research Biochemical, Natick, MA 01760].
Both spectra indicated slow rotation about the amide
C-N bond. The structure determination of conjugates 14,
1
15, and 16 was based on H 1D and 2D NMR, and FAB
(5) (a) Newman, A. H.; Lueddens, H. W. M.; Skolnick, P.; Rice, K.
C. J . Med. Chem. 1987, 30, 1901-1905. (b) Walsh, D. A.; Sancilio, L.
F.; Reese, D. L. J . Med. Chem. 1978, 21, 582-585.
1
thru 8 was based on H NMR and ESI MS. The chemical
shift data are listed in Table 1.

Selective Protection of 2′,2′-Difluorodeoxycytidine
J . Org. Chem., Vol. 64, No. 22, 1999 8321
brine, dried over Na2SO4, and then concentrated to dryness.
Flash chromatography on a short column (CH2Cl2-acetone 4:1)
gave a nonpolar product mixture. To a stirred solution of the
above mixture in 20 mL of MeOH was added 20 mL of aqueous
1 M Na2CO3 at 24 °C. The reaction progress was followed by
TLC, and after 4 h was completed. A similar extraction
procedure as stated above gave a mixture of two products 5
and 6 that were subjected to flash chromatography (CH2Cl2-
acetone-EtOH 2:1:0 to 1:1:0.02) to give compounds 5 [(66 mg,
48%), homogeneous by TLC (CH2Cl2-acetone-EtOH 5:4:1).
1H NMR data are shown in Table 1 except two singlets (δ 1.51,
9H; 1.49, 9H). ESI MS m/z 486 [positive, (M + Na)], 462
[negative, (M - H)]] and 6 [(45 mg, 41%), homogeneous by
TLC (CH2Cl2-acetone-EtOH 5:4:1). 1H NMR data are shown
in Table 1 except a singlet (δ 1.51, 9H). ESI MS m/z 386
[positive, (M + Na)], 362 [negative, (M - H)]].
HRMS. All NMR spectra of compounds 11-16 also
indicated slow rotation about the amide bond and are
available as Supporting Information with detailed peak
assignment. The three mono-PBR ligand-gemcitabine
conjugates were obtained in high yield, demonstrating
the usefulness of the selective protection/deprotection
strategy disclosed here. This strategy may be applied to
other PBR ligand-nucleoside conjugates, and generally
to other types of chemical conjugates involving nucleo-
sides in which a selective linkage is required.
Exp er im en ta l Section
Gemcitabine hydrochloride (1a ) was obtained from Eli Lilly
and Co. All other reagents were commercially available.
ACROS silica gel (35-70 µm) was used for flash chromatog-
4-N-3′-O-Bis(ter t-Bu toxyca r bon yl)gem cita bin e (7). To
a stirred solution of 2 (73 mg, 0.2 mmol) in 8 mL of dioxane
was added DBDC (436 mg, 2 mmol). The reaction mixture was
maintained at 37 °C, 250 rpm, in a rotary shaker for 70 h at
which time the solvent was removed under reduced pressure.
The residue was washed with 2 mL of water. The solids were
dried and subjected to flash chromatography (CH2Cl2-acetone
9:1 to 4:1) to give 7 (88 mg, 95%), homogeneous by TLC (CH2-
1
1
raphy. 600 MHz 1D H, 2D H-1H COSY NMR spectra were
obtained in acetone-d6 with 2% D2O. Coupling constants are
reported in hertz.
3′-O-(ter t-Bu toxyca r bon yl)gem cita bin e (2). To a stirred
mixture of 1a (60 mg, 0.2 mmol) and Na2CO3 (106 mg), in 4
mL of dioxane and 1 mL of water was added DBDC (44 mg,
0.2 mmol), and the resulting mixture stirred at 24 °C for 48
h. After 2 mL of water was added, the mixture was extracted
with 2 × 30 mL of EtOAc. The organic extracts were washed
with water (5 mL) and brine (5 mL), dried over Na2SO4, and
concentrated to dryness under reduced pressure. The residue
was subjected to flash chromatography (CH2Cl2-acetone-
EtOH 1:1:0.02) to give 2 (62 mg, 85%), homogeneous by TLC
(CH2Cl2-acetone-EtOH 5:4:1). 1H NMR data are shown in
Table 1 except a singlet (δ 1.49, 9H). ESI MS m/z 364 [positive,
(M + H)], 362 [negative, (M - H)].
3′,5′-O-Bis(ter t-Bu toxyca r bon yl)gem cita bin e (3). To a
stirred solution of 1a (600 mg, 2 mmol) in 40 mL of 1 M
aqueous KOH was added DBDC (4.36 g, 20 mmol) dropwise
to 40 mL of dioxane over 20 min. The reaction mixture was
then stirred at 24 °C for an additional 40 min and extracted
with EtOAc (3 × 80 mL). The organic extracts were washed
with brine (2 × 10 mL), dried over Na2SO4, and concentrated
to dryness under reduced pressure. The residue was a mixture
of 2 and 3 (about 1:1 by TLC). To a stirred clear solution of
the above residue and DBDC (4.36 g) in 40 mL of dioxane at
24 °C was added 40 mL of 1 M aqueous KOH. The reaction
progress was followed by TLC. After 30 min, the reaction was
nearly complete to give only one major product 3 that was
extracted by a similar procedure as described above. Flash
chromatography (CH2Cl2-acetone 1:1) gave 3 (833 mg, 90%),
homogeneous by TLC (CH2Cl2-acetone-EtOH 5:4:1). 1H NMR
data are shown in Table 1 except two singlets (δ 1.50, 9H; 1.47,
9H). ESI MS m/z 464 [positive, (M + H)], 462 [negative, (M -
H)].
1
Cl2-acetone-EtOH 5:4:1). H NMR data are shown in Table
1 except two singlets (δ 1.51, 9H; 1.50, 9H). ESI MS m/z 464
[positive, (M + H)], 462 [negative, (M - H)].
4-N-3′-O-5′-O-Tr is(ter t-Bu toxycar bon yl)gem citabin e (8).
In a separate experiment under similar condition as the first
step for the preparation of 5 and 6, 8 was isolated from the
product mixture by flash chromatography (CH2Cl2-acetone
9:1), homogeneous by TLC (CH2Cl2-acetone 4:1). 1H NMR data
are shown in Table 1 except three singlets (δ 1.51, 9H; 1.50,
9H; 1.49, 9H). ESI MS m/z 564 [positive, (M + H)], 562
[negative, (M - H)].
(()-1-(2-Ch lor p h en yl)-N-(1-m et h ylp r op yl)-N-[2-[N-(2-
h yd r oxyca r bon yl-eth ylca r bon yl)a m in oeth yl]]-3-isoqu in -
olin eca r boxya m id e (10). To a solution of dihydrochloride of
9 (1.36 g, 3 mmol) in CH2Cl2 (60 mL) and DIEA (2.09 mL),
was added succinic anhydride (600 mg, 6 mmol), and the
reaction mixture was stirred at 24 °C for 1 h. An additional
60 mL of CH2Cl2 was added to the mixture that was then
washed with 1 M HCl (2 × 20 mL) and brine (20 mL), dried
over MgSO4, and concentrated to dryness. Flash chromatog-
raphy (CH2Cl2-acetone-HOAc 9:1:0 to 3:1:0.04) produced
compound 10 (1.20 g, 83%), homogeneous by TLC (CH2Cl2-
1
acetone-HOAc 3:1:0.1). H NMR δ 8.20-8.10 (m, 2H), 7.90-
7.86 (m, 1H), 7.73-7.55 (m, 6H), 4.1-3.9 (m, 1H), 3.7-3.2 (m,
4H), 2.6-2.3 (m, 4H), 2.0-1.4 (m, 2H), 1.37-1.21 (m, 3H),
0.97-0.73 (m, 3H). 2D NMR was in agreement with the
structure. ESI MS m/z (relative intensity) 482/484 [3/1, posi-
tive, (M + H)], 480/482 [3/1, negative, (M - H)].
3′-O-5′-O-Bis(ter t-Bu toxyca r bon yl)-4-N-[2-[2-[N-(1-m e-
th ylp r op yl),N-[1-(2-ch lor op h en yl)-isoqu in olin e-3-ca r bo-
n y l ]a m i n o ]e t h y l a m i n o c a r b o n y l ]e t h y l c a r b o n y l ]-
gem cita bin e (11). A reaction mixture of 3 (93 mg, 0.2 mmol),
10 (96 mg, 0.2 mmol), DCC (83 mg, 0.4 mmol), and 1-hydroxy-
benzotriazole hydrate (27 mg, 0.2 mmol) in CH2Cl2 (10 mL)
was stirred at 24 °C for 20 h. After an additional 50 mL of
CH2Cl2 was added, it was washed with water (10 mL) and
brine (2 × 10 mL), dried over Na2SO4, and concentrated to
dryness. The residue was stirred in 20 mL acetone for 1 h.
The white solids were removed by filtration after cooled in an
ice-bath. The filtrate was concentrated and subjected to flash
chromatography (CH2Cl2-acetone 4:1 to 2:1) to give 11 (158
mg, 85%), homogeneous by TLC (EtOAc). 1H NMR δ 8.23-
8.11 (m, 2H), 8.03 (d, 7.6, 1H), 7.86 (m, 1H), 7.73-7.54 (m,
6H), 7.42 (m, 1H), 6.38 (m, 1H), 5.33 (m, 1H), 4.6-4.4 (m, 3H),
4.2-3.9 (m, 1H), 3.8-3.2 (m, 4H), 2.9-2.5 (m, 4H), 2.0-1.4
(m, 2H, overlap with the following two singlets), 1.50 (s, 9H),
1.47 (s, 9H), 1.37-1.21 (m, 3H), 0.96-0.73 (m, 3H). ESI MS
m/z (relative intensity) 927/929 [3/1, positive, (M + H)], 925/
927 [3/1, negative, (M - H)].
5′-O-(ter t-Bu toxyca r bon yl)gem cita bin e (4). To a stirred
solution of 3 (833 mg) in 40 mL of MeOH was added 40 mL of
1 M aqueous Na2CO3 at 24 °C. The reaction progress was
followed by TLC. After 4 h, the reaction was complete to give
one major product 4 and a small amount of 1. A similar workup
procedure as stated above for 2 gave 4 (555 mg, 85%),
homogeneous by TLC (CH2Cl2-acetone-EtOH 5:4:1). 1H NMR
data are shown in Table 1 except a singlet (δ 1.47, 9H). ESI
MS m/z 364 [positive, (M + H)], 362 [negative, (M - H)]. The
aqueous portions during the extraction were combined and
dried under reduced pressure at 50 °C. The residue was treated
with acetone (2 × 20 mL). The acetone filtrate was then
concentrated. Flash chromatography on a short column (CH2-
1
Cl2-acetone-EtOH 1:2:0.1) gave 1 (48 mg, 10%), and the H
NMR data are shown in Table 1.
4-N-5′-O-Bis(ter t-Bu toxyca r bon yl)gem cita bin e (5) a n d
4-N-(ter t-Bu toxyca r bon yl)gem cita bin e (6). To a stirred
solution of 1a (90 mg, 0.3 mmol) and DMAP (5 mg) in 5 mL of
dioxane and 5 mL of TEA was added DBDC (655 mg, 3 mmol).
The reaction mixture was stirred at 24 °C for 18 h. Solvents
were removed under reduced pressure. The residue was
treated with 100 mL of EtOAc, washed with 5% NaHCO3 and
4-N-5′-O-Bis(ter t-Bu toxyca r bon yl)-3′-O-[2-[2-[N-(1-m e-
th ylp r op yl),N-[1-(2-ch lor op h en yl)isoqu in olin e-3-ca r bo-

8322 J . Org. Chem., Vol. 64, No. 22, 1999
Guo
n y l ]a m i n o ]e t h y l a m i n o c a r b o n y l ]e t h y l c a r b o n y l ]-
gem cita bin e (12). To a stirred solution of 5 (46 mg, 0.1 mmol),
10 (48 mg, 0.1 mmol), and DCC (42 mg, 0.2 mmol) in 5 mL of
CH2Cl2 was added 2 mg of DMAP, and the resulting mixture
was stirred at 24 °C for 2 h. After an additional 55 mL of CH2-
Cl2 was added, it was washed with 20 mM, pH 7 phosphate
buffer (2 × 20 mL) and brine (2 × 20 mL), dried over Na2SO4,
and concentrated to dryness. The residue was stirred in 12
mL acetone for 1 h. The white solids were removed by filtration
after being cooled in an ice-bath. The filtrate was concentrated
and subjected to flash chromatography (EtOAc-acetone 1:0
to 9:1) to give 12 (82 mg, 88%), homogeneous by TLC (EtOAc).
1H NMR δ 8.2-8.02 (m, 3H), 7.88 (m, 1H), 7.80-7.54 (m, 6H),
7.28 (d, 7.6, 1H), 6.40 (m, 1H), 5.48 (m, 1H), 4.6-4.4 (m, 3H),
4.1-3.9 (m, 1H), 3.8-3.2 (m, 4H), 2.8-2.4 (m, 4H), 2.0-1.4
(m, 2H, overlap with the following two singlets), 1.51 (s, 9H),
1.47 (s, 9H), 1.37-1.21 (m, 3H), 0.96-0.73 (m, 3H). ESI MS
m/z (relative intensity) 949/951 [3/1, positive, (M + Na)], 925/
927 [3/1, negative, (M - H)].
4-N-3′-O-Bis(ter t-Bu toxyca r bon yl)-5′-O-[2-[2-[N-(1-m e-
th ylp r op yl),N-[1-(2-ch lor op h en yl)isoqu in olin e-3-ca r bo-
n y l ]a m i n o ]e t h y l a m i n o c a r b o n y l ]e t h y l c a r b o n y l ]-
gem cita bin e (13). To a stirred solution of 7 (46 mg, 0.1 mmol),
10 (48 mg, 0.1 mmol), and DCC (42 mg, 0.2 mmol) in 5 mL of
CH2Cl2 was added 2 mg of DMAP. The resulting mixture was
stirred at 24 °C for 2 h. After an additional 55 mL of CH2Cl2
was added, it was washed with 20 mM, pH 7 phosphate buffer
(2 × 20 mL) and brine (2 × 20 mL), dried over Na2SO4, and
concentrated to dryness. The residue was stirred in 12 mL of
acetone for 1 h. The white solids were removed by filtration
after cooled in an ice-bath. The filtrate was concentrated and
subjected to flash chromatography (CH2Cl2-acetone 9:1 to 4:1)
to give 13 (80 mg, 86%), homogeneous by TLC (CH2Cl2-
acetone 4:1). 1H NMR δ 8.22-8.10 (m, 3H), 7.88-7.56 (m, 7H),
7.36-7.28 (m, 1H), 6.39 (m, 1H), 5.38 (m, 1H), 4.61-4.36 (m,
3H), 4.1-3.9 (m, 1H), 3.7-3.2 (m, 4H), 2.8-2.3 (m, 4H), 2.0-
1.4 (m, 2H, overlap with the following two singlets), 1.49 (s,
9H), 1.48 (s, 9H), 1.36-1.20 (m, 3H), 0.95-0.72 (m, 3H). ESI
MS m/z (relative intensity) 949/951 [3/1, positive, (M + Na)],
925/927 [3/1, negative, (M - H)].
4H), 2.1-1.4 (m, 2H), 1.36-1.20 (m, 3H), 0.96-0.72 (m, 3H).
2D NMR was in agreement with the structure. ESI MS m/z
(relative intensity) 727/729 [3/1, positive, (M + H)], 725/727
[3/1, negative, (M - H)]. FAB HRMS [M + H] calcd for C35H38
-
ClF2N6O7 727.2459, found 727.2447.
3′-O-[2-[2-[N-(1-Met h ylp r op yl),N-[1-(2-ch lor op h en yl)-
isoq u in olin e-3-ca r b on yl]a m in o]et h yla m in oca r b on yl]-
eth ylca r bon yl]-gem cita bin e (15). To a stirred solution of
12 (80 mg) in 5 mL of CH2Cl2 at 0 °C, 5 mL TFA was added.
The reaction reached completion in 1.5 h at the same temper-
ature. The solvents were evaporated, and the residue was
treated with EtOAc (50 mL) and 5 mL of 5% aqueous NaHCO3.
The organic extract was washed with 5 mL of NaHCO3 and
brine (2 × 5 mL), dried over Na2SO4, and concentrated.
Crystallization from EtOAc gave 15 (55 mg, 88%), homoge-
neous by TLC (CH2Cl2-acetone-EtOH 5:4:1). 1H NMR δ
8.24-8.11 (m, 2H), 7.90-7.80 (m, 2H), 7.75-7.56 (m, 6H), 6.35
(m, 1H), 6.03 (d, 7.4, 1H), 5.49 (m, 1H), 4.26-3.75 (m, 4H),
3.7-3.2 (m, 4H), 2.8-2.3 (m, 4H), 2.0-1.42 (m, 2H), 1.37-
1.19 (m, 3H), 0.96-0.73 (m, 3H). 2D NMR was in agreement
with the structure. ESI MS m/z (relative intensity) 727/729
[3/1, positive, (M + H)], 725/727 [3/1, negative, (M - H)]. FAB
HRMS [M + H] calcd for C35H38ClF2N6O7 727.2459, found
727.2443.
5′-O-[2-[2-[N-(1-Met h ylp r op yl),N-[1-(2-ch lor op h en yl)-
isoq u in olin e-3-ca r b on yl]a m in o]et h yla m in oca r b on yl]-
eth ylca r bon yl]gem cita bin e (16). To a stirred solution of 13
(68 mg) in 5 mL of CH2Cl2 at 0 °C was added 5 mL of TFA.
The reaction reached completion in 12 h at the same temper-
ature. The solvents were evaporated, and the residue was
treated with EtOAc (50 mL) and 5 mL of 5% aqueous NaHCO3.
The organic extract was washed with 5 mL of NaHCO3 and
brine (2 × 5 mL), dried over Na2SO4, and concentrated.
Crystallization from EtOAc gave 16 (48 mg, 90%), homoge-
neous by TLC (CH2Cl2-acetone-EtOH 5:4:1). 1H NMR δ
8.24-8.11 (m, 2H), 7.91-7.56 (m, 8H), 6.30 (brs, 1H), 6.11-
5.90 (m, 1H), 4.55-4.3 (m, 3H), 4.14 (m, 1H), 4.08-3.92 (m,
1H), 3.6-3.3 (m, 4H), 2.7-2.4 (m, 4H), 1.96-1.42 (m, 2H),
1.37-1.19 (m, 3H), 0.97-0.72 (m, 3H). 2D NMR was in
agreement with the structure. ESI MS m/z (relative intensity)
727/729 [3/1, positive, (M + H)], 725/727 [3/1, negative, (M -
H)]. FAB HRMS [M + H] calcd for C35H38ClF2N6O7 727.2459,
found 727.2461.
4-N-[2-[2-[N-(1-Met h ylp r op yl),N-[1-(2-ch lor op h en yl)-
isoq u in olin e-3-ca r b on yl]a m in o]et h yla m in oca r b on yl]-
eth ylca r bon yl]-gem cita bin e (14). To a stirred solution of
11 (130 mg) in 10 mL of CH2Cl2 at 0 °C was added 10 mL of
TFA. The reaction reached completion in 1 h at the same
temperature. The solvents were evaporated, and the residue
was treated with EtOAc (2 × 30 mL) and 10 mL of 5% aqueous
NaHCO3. The organic extract was washed with 10 mL of
NaHCO3 and brine (2 × 10 mL), dried over Na2SO4, and
concentrated. Crystallization from EtOAc gave 14 (85 mg, 84%)
in two portions, homogeneous by TLC (CH2Cl2-acetone-EtOH
5:4:1). 1H NMR δ 8.31 (d, 7.6, 1H), 8.23-8.12 (m, 2H), 7.87
(m, 1H), 7.75-7.55 (m, 6H), 7.48 (m, 1H), 6.27 (t, 7.3, 1H),
4.43 (m, 1H), 4.15-3.70 (m, 4H), 3.6-3.2 (m, 4H), 2.9-2.5 (m,
Ack n ow led gm en t. NMR spectra obtained by Fox
Chase Cancer Center’s Spectroscopy Support Facility.
Partial funding provided by NIH Core grant CA-06927.
Su p p or tin g In for m a tion Ava ila ble: 1D 1H NMR spectra
of compounds 2-8 and 10-16, and 2D 1H-1H NMR spectra
of compounds 10 and 14-16. This material is available free
of charge via the Internet at http://pubs.acs.org.
J O9911140