Thiocolchicine-Podophyllotoxin Conjugates
1
1: purified by chromatography (CH
4%; R 0.69 (CH Cl /MeOH 20:1); [R]
H NMR (CDCl , 300 MHz) δ 6.67 (s, 1H), 6.56 (s, 1H), 6.04-
2
Cl
2
/MeOH 50:1); yield
crude compound that was directly used for the next step. 18: yield
1
7
f
2
2
D
) -0.1 (CHCl
3
, c ) 1);
3
71%; H NMR (CDCl , 300 MHz) δ 7.92 (s, 1H), 7.41 (d, J ) 9
1
3
Hz, 1H), 7,23 (d, J ) 9 Hz, 1H), 6,61 (s, 1H), 4,97-4,81 (m, 1H),
3.97 (s, 3H), 3.87 (s, 3H), 3.65 (s, 3H), 2.75-2.61 (m, 2H), 2.43
5
.85 (m, 3H), 4.61 (d, J ) 2.7 Hz, 1H), 4.38 (t, J ) 10.8 Hz, 1H),
.18 (t, J ) 10.7 Hz, 1H), 3.81 (s, 3H), 3.77 (s, 6H), 3.07-2.71
4
(s, 3H), 2.40-2.25 (m, 2H), 1.98-1.86 (m, 4H), 1.31-1.19 (m,
13
(
m, 6H); 13C NMR (CDCl
3
, 75 MHz) δ 173.5, 172.0, 152.6 (2C),
4H); C NMR (CDCl
3
, 75 MHz): δ 182.7, 174.7, 170.9, 158.9,
1
1
47.5 (2C), 136.7, 134.7, 132.3, 127.9, 109.7, 108.1 (2C), 106.9,
01.6, 74.1, 71.2, 60.7, 56.2 (2C), 45.5, 43.7, 38.6, 34.0, 32.9; ESI
153.9, 152.4, 151.1, 141.7, 139.4, 135.6, 134.3, 128.6, 128.0, 125.4,
107.5, 61.6, 61.3, 56.1, 51.9, 37.3, 36.3, 34.9, 34.0, 33.8, 30.0, 15.0.
Preparation of 13 by Direct Condensation of 18 with
Podophyllotoxin 3. A solution of 18 (0.18 mmol), DMAP (0.18
mmol), DCC (0.26 mmol), and podophyllotoxin (0.18 mmol) in
+
positive MS calcd for C50
025.23886.
Formation of Heterodimer: Base-Catalyzed Equilibration.
A solution of 8 (or 9) (0.012 mmol) and 10 (or 11) (0.012 mmol)
Cl (or acetone, CH CN, THF) (10 mL)
H
50
O
18
S
2
+ Na 1025.23308, found
1
2 2
CH Cl (10 mL) was stirred at room temperature for 3 days.
and TEA (50 µL) in CH
2
2
3
Filtration through a Celite layer and evaporation of the solvent
afforded a crude that was purified by column chromatography.
Target-Assisted Equilibration. A solution of 9 (0.004 mmol),
11 (0.004 mmol), TEA (50 µL), and target (subtilisin A from
Bacillus sp. or bovine albumin) (2 mg) in acetone (10 mL) was
stirred for a period longer than 20 days. The equilibrium’s state
was quantitatively monitored by HPLC (RP18, CH CN/H O 6:4,
was stirred at 37 °C for 20 days. The solvent was removed and the
mixture submitted to flash-chromatography column. The equilib-
rium’s state was monitored qualitatively by TLC (eluent CH
MeOH 20:1) and quantitatively by HPLC (RP18, CH CN/H O 6:4,
,8 mL/min). In acetone: 8 (40%), 10 (39%), 12 (17%); For 9, 11,
and 13, see the text and Table 2. Base-Catalyzed Equilibration
with the Addition of benzylmercaptan. A solution of 8 (or 9)
2 2
Cl /
3
2
0
3
2
0.8 mL/min).
(
0.012 mmol) and 10 (or 11) (0.012 mmol), TEA (50 µL), and
Equilibration of Homodimers 8-11. Samples (150 µL) of the
four solutions obtained by dissolving 0.0012 mmol of each the
compounds 8, 9, 10, and 11 in acetone (1 mL) were mixed in the
presence of 1 µL of a solution (1 mg/100 mL of acetone) of
benzylmercaptan. The mixture was stirred at room temperature for
120 h. The mixture was directly submitted to ESI MS (see Figure
4 and Table 3).
-
4
benzylmercaptan (0.07 × 10 mmol, 1%) in CH
2 2
Cl (10 mL) was
stirred at 37 °C for 120 h. The solvent was removed and the mixture
submitted to HPLC analysis. Dithiothreitol-Induced Equilibra-
tion. A solution of 8 (or 9) (0.060 mmol) and dithiothreitol (0.18
2 2
mmol) in CH Cl (15 mL) was stirred at room temperature for 2
days. Then, 10 (or 11) (0.049 mmol) and TEA (400 µL) were added,
and the mixture was stirred for 100-120 h.
+
14: ESI positive MS calcd for C H O S N + Na 929.22405,
45
50 10
4
2
12. The pooled equilibration reactions of 8 and 10 have been
found 929.22199.
+
submitted to chromatography (CH
sample of heterodimer: R 0.18 (CH
, c ) 1); H NMR (400 MHz, CDCl
H), 6.80 (s, 1H), 6.53 (m, 2H), 6.38 (m, 2H), 6.05-5.85 (m, 3H),
.80-4.15 (m, 4H), 3.94 (s, 3H), 3.90 (s, 3H), 3.82 (s, 3H), 3.75
2
Cl
2
/MeOH 35:1) to give a pure
/MeOH 30:1); [R] ) -1.1
) δ 8.00-7.05 (m,
15: ESI positive MS calcd for C H O S + Na 1011.21743,
49
48 18 2
f
2
Cl
2
D
found 1011.22305.
16 and 17: ESI positive MS calcd for C H O S N + Na
1
+
(
4
4
CHCl
3
3
4
7
49 14 3
970.22074, found 970.22226.
In Vitro Studies. The human tumor cell lines used in this study
included NCI-H460, a human lung large cell carcinoma cell line
(ATCC HTB 177). All of the cell lines were cultured in RPMI-
1640 containing 10% fetal calf serum. Cytotoxicity was assessed
by growth inhibition assay after 1 h of drug exposure. Briefly, cells
in the logarithmic phase of growth were harvested and seeded in
duplicate into six-well plates. Twenty-four hours after seeding, cells
were exposed to the drug and harvested 72 h after exposure and
counted with a Coulter counter. IC50 is defined as the inhibitory
drug concentration causing a 50% decrease of cell growth over
that of untreated control. All compounds are insoluble in water and
were dissolved in DMSO prior to dilution into the biological assay.
(s, 6H), 3.61 (s, 3H), 3.00-1.80 (m, 10H), 2.48 (s, 3H); ESI positive
+
MS calcd for C46
3. The pooled equilibration reactions of 9 and 11 have been
submitted to chromatography (CH Cl /MeOH 35:1) to give a pure
sample of heterodimer: R 0.18 (CH Cl /MeOH 30:1); [R] ) -1.6
, c ) 1); H NMR (400 MHz,CDCl ) δ 7.1 (d, J ) 10 Hz,
H), 7.49 (d, J ) 8 Hz, 1H), 7.42 (s, 1H), 7.34 (d, J ) 10 Hz, 1H),
47 14 3
H O S N + Na 956.20509, found 956.20598.
1
2
2
f
2
2
D
1
(
CHCl
3
3
1
6
5
1
2
.79 (s, 1H), 6.56 (s, 1H), 6.53 (s, 1H), 6.41 (s, 2H), 5.98 (m, 2H),
.95 (d, J ) 8 Hz, 1H), 4.75-4.63 (m, 1H), 4.62 (d, J ) 3.5 Hz,
H), 4.43 (dd, J ) 10.0, 7.0 Hz, 1H), 4.22 (t, J ) 10.0 Hz, 1H),
.91 (m, 1H), 2.78 (m, 1H), 2.62-2.51 (m, 1H), 2.48 (s, 3H), 2.47
-
2.38 (m, 1H), 2.34-2.18 (m, 1H), 1.98-1.68 (m, 1H); 13C NMR
(100 MHz, CD
3
OD) δ 182.4, 173.7, 158,3, 153.5, 151.2 (2C), 141.7,
Acknowledgment. We are grateful for the financial support
provided by Ministero dell’Istruzione, dell’Universit a` e della
Ricerca (MIUR) COFIN 2001-2003 Program “Sostanze Natu-
rali ed Analoghi Sintetici con Attivit a` Antitumorale”. G.A.
expresses her gratitude to INDENA S.p.A. (Milano) for the
Ph.D. fellowship.
1
1
4
38.3, 137.2, 135.3, 134.9, 128.6, 126.6, 109.7, 108.2 (2C), 1075.5,
07.1, 101.6, 74.0, 71.4, 61.6, 61.4, 60.7, 56.2, 56.1, 52.1, 45.5,
4.4, 38.7, 36.8, 34.4, 34.1, 33.6, 30.0, 15.1; ESI positive MS calcd
+
for C48
H
51
O
14
S
3
N + Na 984.23639, found 984.23254.
Preparation of 18. A solution of dithiodipropionic acid (1.01
mmol), DMAP (0.34 mmol), DCC (1.01 mmol), and deacetyl-
2 2
thiocolchicine (0.67 mmol) in CH Cl (30 mL) was stirred at room
temperature for 2 h and then filtered through a Celite layer. The
removal of the solvent gave a crude mixture that was dissolved in
Supporting Information Available: NMR spectra for new
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
CH
2
Cl
2
and extracted with NH
4
OH (sol 3.5%). The aqueous phase
Cl to give a
was treated with HCl (1 N) and extracted with CH
2
2
JO052677G
J. Org. Chem, Vol. 71, No. 7, 2006 2853