ActiVation of Prodrugs of Camptothecins
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4353
1
1
1
9.64, 27.30, 31.80, 52.66, 56.09, 66.57, 73.02, 95.03, 97.30,
12.11, 117.86, 123.18, 132.04, 134.28, 136.70, 141.63, 144.65,
General Procedure for the Isolation of Intermediates I and
I′. To a solution of compound 1, 2, 5, or 6 (50 mg) in anhydrous
DMF (6 mL) was added 1.5 molar equiv of triethylamine at room
temperature. The reaction was monitored by HPLC. After prodrugs
completely disappeared, solvents were evaporated under vacuum
at room temperature. The residues were washed with diethyl ether
to give the expected intermediates (i.e., predominantly I′).
46.96, 149.19, 150.41, 156.23, 157.70, 174.27; MS (EI) m/z 523
+
(M
+ H).
General Procedure for Synthesis of DB-67 Esters. A solution
of 7 (1 equiv), N-tert-butoxycarbonylamino acid (3 equiv), and
DMAP (0.6 equiv) in anhydrous CH Cl was cooled to 0 °C,
followed by the addition of EDCI (3 equiv). The reaction mixture
was allowed to warm to room temperature and stirred overnight.
The resulting solution was washed with HCl (0.1 N) and water
and then extracted with CH
MgSO , filtered, and concentrated under vacuum. The crude product
was purified by column chromatography on silica gel to afford the
corresponding protected compound (8 or 9), which was then
dissolved in CH
temperature for 0.5-2 h (monitored by TLC). Solvents were
removed under vacuum and the title compound 5 or 6 was
2
2
I′-1: 1H NMR (400 MHz, DMSO-d
6
) δ 0.93 (t, J ) 7.2, 3H,
H-18), 2.02-2.40 (m, 2H, H-19), 3.33-3.49 (m, 2H, H-23), 4.85
(A of AB system, JAB ) 16.2, H-17A), 5.05 (B of AB system, JAB
) 16.2, H-17B), 5.22 (s, 2H, H-5), 7.23 (s, 1H, H-14), 7.67-7.71
(m, 1H, H-10), 7.83-7.87 (m, 1H, H-11), 7.93 (s, 1H, 22-OH),
2
Cl2. The organic layer was dried over
4
13
8.09-8.20 (m, 2H, H-9, H-12), 8.64 (s, 1H, H-7); C NMR (75
MHz, DMSO-d ) δ 7.96, 32.62, 45.72, 47.88, 49.97, 57.35, 81.85,
6
Cl
2 2
and treated with 1 molar equiv of TFA at room
100.23, 107.44, 127.29, 127.67, 128.08, 128.30, 128.78, 129.60,
130.11, 131.27, 142.38, 147.68, 150.46, 152.52, 158.55, 167.93;
MS (FAB) m/z: 406 (M + H).
I′-5: 1H NMR (400 MHz, DMSO-d
) δ 0.64 (s, 6H, Si(CH
recrystallized from MeOH-CH
2
Cl
2
.
6
3 2
) ),
1
0-Methoxymethyl-DB-67-20(S)-N-tert-butoxycarbonyl-
0.94-1.02 (m, 12H, Si[C(CH ) ], H-18), 2.00-2.38 (m, 2H, H-19),
3 3
1
glycinate (8): yield 90%; H NMR (400 MHz, CDCl
3
) δ 0.68 (s,
], H-18), 1.41 (s,
H, BOC), 2.10-2.34 (m, 2H, H-19), 3.54 (s, 3H, CH OCH O),
.02-4.25 (ABX system, 2H, H-23), 5.22 (m, 4H, H-5, CH OCH O),
.39 (A of AB system, JAB ) 17.2, 1H, H-17A), 5.69 (B of AB
system, JAB ) 17.2, 1H, H-17B), 7.22 (s, 1H, H-14), 7.48-7.51
dd, J ) 9.2, 2.4, 1H, H-9), 7.92 (d, J ) 2.4, 1H, H-11), 8.14 (d,
3.28-3.48 (m, 2H, H-23), 4.73 (A of AB system, JAB ) 15.8, 1H,
H-17A), 5.12 (B of AB system, JAB ) 15.8, 1H, H-17B), 5.17 (s,
2H, H-5), 7.13 (s, 1H, H-14), 7.36-7.40 (m, 1H, H-9), 7.55-7.56
(m, 1H, H-11), 7.88 (s, 1H, 22-OH), 8.15 (d, J ) 4.4, 1H, H-12),
6
9
4
5
H, Si(CH
3
2 3 3
) ), 0.97-1.00 (m, 12H, Si[C(CH )
3
2
3
2
+
10.31 (s, 1H, 10-OH); MS (MALDI/TOF) m/z: 536 (M + H).
I′-6: 1H NMR (400 MHz, DMSO-d
0.90-0.95 (m, 12H, Si[C(CH )
3 3
) δ 0.64 (s, 6H, Si(CH
, H-18), 2.00-2.40 (m, 2H, H-19),
3.41 (A of AB system, JAB ) 12.6, 1H, H-23A), 3.69 (B of AB
system, JAB ) 12.6, 1H, H-23B), 4.73 (A of AB system, JAB
6
3 2
) ),
(
13
J ) 9.2, 1H, H-12); C NMR (75 MHz, CDCl
3
) δ -0.58, 7.88,
1
9
1
1
9.72, 27.38, 28.54, 32.04, 42.64, 52.60, 56.15, 67.39, 77.05, 80.34,
)
5.01, 95.41, 112.05, 119.20, 123.20, 131.81, 134.24, 136.44,
41.66, 144.41, 145.58, 146.78, 148.82, 155.53, 156.12, 157.21,
67.37, 169.52.
16.0, 1H, H-17A), 5.11 (B of AB system, JAB ) 16.0, 1H, H-17B),
5.15 (A of AB system, JAB ) 19.0, 1H, H-5A), 5.17 (B of AB
system, JAB ) 19.0, 1H, H-5B), 7.16 (s, 1H, H-14), 7.36-4.39
(dd, J ) 9.6, 2.4, 1H, H-9), 7.56 (d, J ) 2.8, 1H, H-11), 7.96 (s,
1H, 22-OH), 8.04 (d, J ) 8.8, 1H, H-12), 10.31 (s, 1H, 10-OH);
DB-67-20(S)-glycinate, TFA salt (5): yield 62%; purity >95%
1
(HPLC); H NMR (400 MHz, DMSO-d
6
) δ 0.66 (s, 6H, Si(CH
3
)
2
),
1
3
0
4
.92-0.96 (m, 12H, Si[C(CH
3
)
3
], H-18), 2.10-2.22 (m, 2H, H-19),
6
C NMR (75 MHz, DMSO-d ) δ -1.02, 7.97, 18.90, 27.18, 33.18,
.10 (A of AB system, JAB ) 15.8, 1H, H-23A), 4.31 (B of AB
34.07, 52.02, 54.80, 57.32, 82.03, 99.10, 106.96, 110.70, 121.86,
126.82, 131.33, 133.57, 137.06, 138.36, 142.08, 147.90, 150.50,
155.84, 158.30, 166.42; MS (MALDI/TOF) m/z: 552 (M + H).
system, JAB ) 15.8, 1H, H-23B), 5.22 (A of AB system, JAB
1
5
2
)
+
8.8, 1H, H-5A), 5.26 (B of AB system, JAB ) 18.8, 1H, H-5B),
.52 (s, 2H, H-17), 7.16 (s, 1H, H-14), 7.38-7.41 (dd, J ) 9.2,
Reconversion of Intermediates (I and I′) to Prodrug 1. To
explore the reversibility of the reaction between prodrugs 1 and 2
and their corresponding intermediates I and I′, the intermediate
mixture comprised mainly of I′-1 (1 mg, 2.46 µmol) was dissolved
.4, 1H, H-9), 7.57 (d, J ) 2.4, 1H, H-11), 8.01 (d, 1H, J ) 9.2,
+
13
3
H-12), 8.36 (br s, 3H, NH ), 10.49 (s, 1H, 10-OH); C NMR
(100 MHz, DMSO-d
6
) δ -0.95, 7.53, 7.71, 18.92, 27.12, 27.30,
0.22, 52.24, 66.31, 77.52, 94.00, 110.67, 117.55, 131.23, 133.84,
36.99, 138.69, 142.08, 144.61, 146.22, 147.48, 156.09, 156.20,
66.69, 166.75; MS (MALDI/LDI) m/z 536 (M+ - CF
COOH).
0-Methoxymethyl-DB-67-20(S)-N-tert-butoxycarbonyl-N-
3
1
1
6
in 1 mL of DMSO-d in an NMR tube at room temperature. A
catalytic amount of deuterated TFA was added to the solution, and
1
3
H NMR spectra were recorded (at 25 °C) at regular intervals until
1
I′-1 disappeared completely.
Kinetics. PBS buffers (pH 3.0 and 7.4) having an ionic strength
of 0.159 (adjusted with NaCl) were used for the kinetic studies.
1
methylglycinate (9): yield 90%; H NMR (400 MHz, CDCl
0
3
) δ
.70 (s, 6H, Si(CH ), 1.01 (m, 12H, Si[C(CH ], H-18), 1.45 (d,
3
)
2
3 3
)
J ) 4.8, 9H, BOC), 2.10-2.34 (m, 2H, H-19), 2.96 (s, 3H, H-24),
Low buffer concentrations were used to minimize possible buffer
-
3
.54 (s, 3H, CH
.27-5.32 (m, 4H, H-5, CH
17.2, 1H, H-17A), 5.70 (B of AB system, JAB ) 17.2, 1H,
H-17B), 7.22 (s, 1H, H-14), 7.46-7.51 (m, 1H, H-9), 7.91-7.94
dd, J ) 6.4, 2.4, 1H, H-11), 8.06 (d, J ) 9.2, 1H, H-12); 13
NMR (100 MHz, CDCl ) δ -0.79, 7.74, 19.55, 27.23, 28.43, 31.87,
3
OCH
2
O), 4.05-4.31 (ABX system, 2H, H-23),
catalysis (H
7.4, respectively; HPO
2
PO
4
concentrations were 9 and 3 mM at pH 3.0 and
2-
5
3
OCH O), 5.40 (A of AB system, JAB
2
4
concentrations were 0.001 and 6 mM at
)
pH 3.0 and 7.4, respectively). pH values were measured with a
Beckman Φ40 pH meter with a Ross semimicro w/BNG glass
electrode (Thermo Orion, Beverly, MA) and remeasured before each
experiment. The pH meter was calibrated with standard aqueous
buffer solutions from Fisher Scientific. All concentrated standard
(
C
3
3
1
1
1
5.60, 50.38, 50.83, 52.49, 56.08, 67.23, 76.84, 94.91, 95.75,
12.08, 118.96, 119.16, 123.14, 131.69, 131.99, 134.24, 136.48,
41.43, 141.71, 144.49, 145.97, 146.85, 148.86, 149.05, 155.41,
56.21, 157.31, 167.33, 167.66, 169.15, 169.41.
-
3
stock solutions (2 × 10 M) of 1, 2, I′-1, I′-2, and CPT-lactone
were prepared in dimethyl sulfoxide and stored in the dark at -20
-
4
°C. The stock solution of CPT-carboxylate (4 × 10 M) was
generated in PBS buffer (∼pH 12).
DB-67-20(S)-N-methylglycinate, TFA salt (6): yield 90%;
1
purity >95% (HPLC); H NMR (400 MHz, CD
3
OD) δ 0.73 (s,
Kinetic runs were initiated by injecting 10 µL of a stock solution
in DMSO (or aqueous buffer at pH 12 in the case of CPT-
carboxylate) to the appropriate buffer solution preequilibrated at
6H, Si(CH
3
)
2
), 1.02 (s, 9H, Si[C(CH
3
)
3
]), 1.08 (t, J ) 7.2, 3H,
H-18), 2.18-2.35 (m, 2H, H-19), 2.75 (S, 3H, H-24), 4.30 (A of
-
6
AB system, JAB ) 17.4, 1H, H-23A), 4.38 (A of AB system, JAB
37 ( 0.2 °C. The final substrate concentration was 1 × 10
M
)
)
17.4, 1H, H-23B), 5.35 (s, 2H, H-5), 5.48 (A of AB system, JAB
16.8, 1H, H-17A), 5.63 (B of AB system, JAB ) 16.8, 1H,
and the percentage of DMSO in the buffer solution was 0.25%
(v/v). The reactions were monitored by following the disappearance
of the starting material and the appearance of products.
H-17B), 7.34 (s, 1H, H-14), 7.42-7.45 (dd, J ) 9.2, 1.2, 1H, H-9),
7
.67 (d, J ) 2.8, 1H, H-11), 8.19 (d, J ) 9.2, 1H, H-12); 13C NMR
75 MHz, CD OD) δ -0.42, 8.22, 20.30, 27.91, 31.98, 33.65, 54.30,
Reactant and product concentrations were determined by reversed-
phase HPLC (Waters 2690 separations module) equipped with a
scanning fluorescence detector (Waters 474) and a 150 × 3.9 mm,
5 µm C18 column (Symmetry, Waters). A flow rate of 1 mL/min
was employed with linear mobile phase gradients. Acetonitrile was
(
3
6
7.84, 80.04, 96.59, 112.46, 119.31, 123.90, 132.22, 136.12, 138.29,
42.07, 144.02, 147.50, 148.27, 148.62, 158.20, 158.81; MS
MALDI/LDI) m/z: 551 (M
1
(
+
-
3
CF COOH).