A new cyclopamine glucuronide prodrug with improved kinetics of drug release

Article abstract of DOI:10.1039/c1ob06081c

We prepared a new glucuronide prodrug of cyclopamine designed to target selectively the Hedgehog signalling pathway of cancer cells. This prodrug includes a novel self-immolative linker bearing a hydrophilic side chain that can be easily introduced via "click chemistry". With this design, the prodrug exhibits reduced toxicity compared to the free drug on U87 glioblastoma cells. However, in the presence of β-glucuronidase, the prodrug conducts to the quick release of cyclopamine thereby restoring its antiproliferative activity. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c1ob06081c

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PAPER
A new cyclopamine glucuronide prodrug with improved kinetics of drug
release†
Brigitte Renoux,a Thibaut Legigan,a Souheyla Bensalma,b Corinne Chade´neau,b Jean-Marc Mullerb and
Se´bastien Papot*a
Received 4th July 2011, Accepted 19th September 2011
DOI: 10.1039/c1ob06081c
We prepared a new glucuronide prodrug of cyclopamine designed to target selectively the Hedgehog
signalling pathway of cancer cells. This prodrug includes a novel self-immolative linker bearing a
hydrophilic side chain that can be easily introduced via “click chemistry”. With this design, the prodrug
exhibits reduced toxicity compared to the free drug on U87 glioblastoma cells. However, in the presence
of b-glucuronidase, the prodrug conducts to the quick release of cyclopamine thereby restoring its
antiproliferative activity.
Introduction
Recently, aberrant activation of the Hedgehog (Hh) signalling
pathway1 has been observed in a wide range of malignancies such
as breast,2 prostate,3 gastric,4 lung5 and brain6 tumours. Ever since
then, many efforts have been devoted to the discovery of small-
molecule Hh inhibitors for cancer chemotherapy.7 Cyclopamine
1 is a natural alkaloid isolated from Veratrum californicum
which was the first Hh inhibitor to be identified (Fig. 1).8 This
compound is a potent antagonist of the Hh pathway which
inactivates Smoothened (Smo) by binding to its heptahelical
bundle.9 Cyclopamine already demonstrated antitumor activity
in the course of preclinical and clinical evaluations.10
Although this Hh inhibitor is a promising chemotherapeutic
agent, cyclopamine could induce serious damage in normal tissues
since somatic stem cells are also Hh-dependent. Moreover, the use
of compound 1 in vivo is limited by its poor aqueous solubility.11
In order to circumvent these drawbacks, our group12 and others13
have proposed to develop water-soluble prodrugs programmed
to deliver cyclopamine selectively in the vicinity of the tumour.
Within this framework, we studied the glucuronide prodrug 2a
(Fig. 1, R H) designed to release cyclopamine in the presence
of b-glucuronidase, an enzyme that has been detected at high
level in necrotic areas of numerous tumours.14 This specificity
of the tumor microenvironment has been already exploited to
activate enzyme-responsive glucuronide prodrugs exclusively in
malignant tissues. To date, several glucuronide prodrugs led to
Fig. 1 b-Glucuronidase-catalysed drug release mechanism.
superior therapeutic efficacy compared to standard treatment
demonstrating the validity of this targeting strategy.15
In our previous study, derivatisation of cyclopamine in the
form of prodrug 2a resulted in a non-toxic compound. However,
incubation of the glucuronide 2a with b-glucuronidase triggered
the clean release of the drug through the mechanism depicted
in Fig. 1 thereby restoring its antiproliferative activity towards
U87 human glioblastoma cells. In our design, we included a
nitrobenzylphenoxy carbamate linker16 between the glucuronide
and the bulky cyclopamine in order to allow a good recognition
of the carbohydrate substrate by the enzyme. As expected, in
the presence of b-glucuronidase the glycosidic bond was rapidly
cleaved to generate the phenol 3a. The latter underwent a 1,6-
elimination followed by a spontaneous decarboxylation leading
to the full expulsion of cyclopamine within 28 h. During this
aUniversite´ de Poitiers, UMR-CNRS 6514, 4 rue Michel Brunet, BP
633, 86022, Poitiers, France. E-mail: sebastien.papot@univ-poitiers.fr;
Fax: (+33) 549453501
bUniversite´ de Poitiers, UMR CNRS 6187, 40 Avenue du Recteur Pineau,
86022, Poitiers, France
† Electronic supplementary information (ESI) available: See DOI:
10.1039/c1ob06081c
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experiment, we observed the precipitation of the linker-drug
intermediate 3a as soon as it was produced by the enzymatic
hydrolysis. Therefore, the kinetics of drug release was limited by
the gradual solubilisation of 3a in aqueous media.
bromo-glucuronide 719 under Koenigs-Knorr conditions in the
presence of silver carbonate as the catalyst (66%). The resulting
b-glucuronide 8 was then treated with tert-butyldimethylsilyl
chloride and imidazole to produce the silyl ether 9 in 91% yield.
At this stage, the protecting groups of the carbohydrate moiety
were modified through a three step strategy to give the fully allyl
protected derivative 12. This choice was motivated by a recent
study described by Schmidt and co-workers who demonstrated
that both allyl ester and carbonates are compatible with the
presence of either alkali- or acid-sensitive anticancer drugs.20
Furthermore, the entire deprotection of the glucuronide can be
achieved in a one step procedure under mild conditions at the end
of the synthesis. Thus, this synthetic strategy will limit the number
of steps after the introduction of the expensive cyclopamine 1 on
the linker unit. The O-acetyl groups of compound 9 were first
removed using catalytic amount of MeONa to afford the hydroxyl
free derivative 10 (72%). Transesterification of the methyl ester
with sodium allylate produced the allyl ester 11 in 97% yield. The
three allyl carbonates were then introduced in the presence of 30
equivalents of allyl chloroformate employing pyridine as solvent.
After 3 days under these conditions, the fully allyl protected
glucuronide 12 was obtained in an excellent yield of 94%. Cleavage
of the silyl ether was undertaken with HF/pyridine to furnish the
alcohol 13 which was subsequently activated in the form of the
4-nitrophenyl carbonate 14 (92%).
The slow release of cyclopamine from the phenol 3a could be
problematic in the course of a tumour-activated prodrug therapy.
Indeed, in such an approach it is well admitted that the liberation
of the active compound has to occur quickly after the enzymatic
activation step in order to avoid the diffusion of the linker-drug
intermediate outside of the tumour site.16 In this context, it seems
worthwhile to develop new cyclopamine glucuronide prodrugs
with improved kinetics of drug release. Thus, we decided to pursue
our investigations by the study of the prodrug 2b composed of a
glucuronide trigger, the potent cyclopamine and a self-immolative
linker bearing a glycosylated poly(ethylene glycol) side chain17
(Fig. 1). With this design, we anticipated that enzymatic hydrolysis
of 2b will yield the phenol 3b which will be readily water soluble
thanks to the presence of the hydrophilic side chain. Under such
circumstances, cyclopamine should be eliminated faster from the
intermediate 3b than from its weakly soluble analogue 3a.
Results and discussion
Prodrug 2b was prepared in ten steps starting from a racemic
mixture of the readily accessible nitrophenol 618 (Scheme 1).
First, stereoselective glycosylation of 6 was carried out with the
Coupling between carbonate 14 and cyclopamine 1 gave the
clickable derivative 15 in 85% yield (Scheme 2). The “click
chemistry” reaction was then carried out at room temperature in
the presence of the azide 1621 using Cu(CH3CN)4PF6 as a catalyst.
Under these conditions, the triazole 17 was prepared in 81% yield
after purification by flash column chromatography. The prodrug
2b was finally obtained as a mixture of two diastereoisomers by the
Scheme 1 Synthesis of the carbonate 14.
Scheme 2 Synthesis of prodrug 2b from carbonate 14.
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cleavage of protecting groups using catalytic amount of Pd(PPh3)4
and two equivalents of aniline (80%).
First, the stability of 2b was examined in phosphate buffer (0.02
M, pH = 7.2) at 37 ◦C. No decomposition of prodrug 2b was
detected after 24 h under these conditions. Enzymatic hydrolysis
was then conducted with E. coli b-glucuronidase and monitored
by HPLC/MS (Fig. 2).
Fig. 3 Viability of U87 cells treated during 5 days with prodrug 2b in
the absence or presence of b-glucuronidase (b-glu, 40 U/mL) or with
cyclopamine 1. Values were obtained from two independent experiments,
each performed in hexaplicate and are expressed as mean SEM.
as previously observed in the course of enzymatic hydrolysis
experiments.
Conclusions
In summary, we prepared a novel non-toxic prodrug of cy-
clopamine bearing a hydrophilic side chain introduced via “click
chemistry” on the self-immolative linker. When activated by b-
glucuronidase, this prodrug exhibits improved kinetics of drug
release compared to its previous analogue which included a less
water soluble linker. Furthermore, incubation of the prodrug in
the presence of the activating enzyme restores its antiproliferative
activity on U87 glioblastoma cells. All these results suggest that
this new glucuronide prodrug possesses the necessary prerequisites
for further in vivo investigation in the course of a tumor targeting
strategy.
Fig. 2 Enzymatic hydrolysis of the two diastereoisomers of prodrug 2b
with E. coli b-glucuronidase in phosphate buffer (0.02 M, pH 7.0) at 37 ◦C.
a) t = 0 min; b) t = 10 min; c) t = 120 min.
The glycosidic bond of prodrug 2b was rapidly cleaved (within
30 min) showing that the glucuronide trigger is readily accessible
by the enzyme, despite the presence of bulky moieties such as
cyclopamine and the glycosylated poly(ethylene glycol) side chain
attached on the linker unit. Only ten minutes after the addition
of b-glucuronidase, cyclopamine 1 and the intermediate 3b were
detected in the medium (Fig. 2b). The latter totally disappeared
in less than two hours leading to the clean release of the drug
along with the formation of the benzyl alcohol 5b (Fig. 2c). All
together, these results confirmed that the disassembly of prodrug
2b proceed through the self-immolative mechanism illustrated in
Fig. 1. Cyclopamine was expelled significantly faster from prodrug
2b than from our previous glucuronide 2a (<2 h versus 28 h). In
contrast with its analogue 3a, the phenol 3b did not precipitate
in the reaction mixture. As expected, this intermediate was fairly
water soluble thanks to the hydrophilic side chain thereby allowing
the quick release of cyclopamine.
Prodrug 2b was then tested for its anti-proliferative activity on
U87 glioblastoma cells after a 5-day treatment (Fig. 3). When
incubated alone in the culture medium, prodrug 2b did not affect
viability of cells whereas the free drug was highly toxic with an
IC50 value of 16.5 mM. This result indicated that derivatisation
of cyclopamine in the form of prodrug 2b markedly reduced
its anti-proliferative activity. On the other hand, addition of b-
glucuronidase in the culture medium induced a dramatic anti-
proliferative effect with an IC50 value close to that obtained
for cyclopamine (IC50 = 24.5 mM). This can be unambiguously
attributed to the release of the drug in the culture medium
Experimental
Preparation of compound 8
A
solution of 1,1,4,7,10,10-hexamethyltriethylenetetramine
(HMTTA) (6.3 mL, 23.1 mmol) and Ag2CO3 (33.7 g, 122.3 mmol)
in anhydrous CH3CN (33 mL) was stirred during 2 h at room
temperature. Nitrophenol 6 (4.56 g, 22 mmol) and bromo-
glucuronide 7 (11.31 g, 33 mmol) were added at 0 ◦C, and the
solution mixture was stirred for 4 h at room temperature. The
reaction was quenched with water and extracted with ethyl acetate
(3 ¥ 20 mL). The combined organic layers were washed with
HCl 1 M, dried over MgSO4, filtered and concentrated under
reduced pressure. The resulting crude material was purified by
column chromatography over silica gel (petroleum ether/AcOEt
6/4, 5/5, 4/6) to afford 8 (7.62 g, 0.84 mmol, 66%) as a mixture
of two diastereoisomers (pale yellow solid). Rf 0.56 (petroleum
ether/AcOEt 50/50). mp = 67.3 ◦C. 1H NMR (400 MHz, CDCl3)
d 2.04 (s, 6H), 2.10 (s, 4H), 2.62 (m, 2H), 3.38 (bs, 1H), 3.74 (s,
3H), 4.28 (d, 1H, J = 9 Hz), 4.92 (m, 1H), 5.25–5.32 (m, 4H),
7.34 (d, 1H, J = 8.6 Hz), 7.60 (m, 1H), 7.87 (d, 1H, J = 2.1 Hz);
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13C NMR (100 MHz, CDCl3) d 20.5, 29.2, 53.1, 68.7, 70.1, 70.6,
71.1, 71.8, 72.3, 79.8, 99.5, 119.4, 122.7, 131.5, 139.1, 140.7, 148.0,
166.8, 169.5, 170.1; HRMS (ESI) [M+Na]+ m/z 546.1228 (calcd
for C23H25NO13Na: 546.1224); [M+K]+ m/z 562.0997 (calcd for
C23H25NO13K: 562.0963).
hydrolyzed with IRC-50 acidic resin, filtrated and concentrated in
vacuo. The crude product was purified by column chromatography
over silica gel (CH2Cl2/MeOH 98/2) to give 11 (399 mg, 97%) as
a mixture of two diastereoisomers (pale yellow solid). Rf 0.75
(CH2Cl2/MeOH 95/5). mp = 55.9 ◦C. 1H NMR (400 MHz,
CDCl3) d -0.04 (2 s, 3H), 0.10 (2 s, 3H), 0.89 (2 s, 9H), 1.99
(2t, 1H, J = 2.6 Hz), 2.49–2.60 (m, 2H), 3.50–3.81 (m, 2H), 3.95
(m, 1H), 4.07 (2d, 1H, J = 9.6 Hz), 4.75 (m, 2H), 4.83 (t, 1H, J =
6.4 Hz), 4.96 (d, 1H, J = 6.8 Hz), 5.30 (m, 1H), 5.38 (m, 1H), 5.97
(m, 1H), 7.38 (2d, 1H, J = 8.6 Hz), 7.60 (m, 1H), 7.90 (d, 0.5H,
J = 2.1 Hz), 7.93 (d, 0.5H, J = 2.1 Hz); 13C NMR (100 MHz,
CDCl3) d -4.8, -4.7, 18.3, 25.8, 30.9, 66.7, 70.9, 71.3, 72.2, 73.0,
74.6, 74.9, 80.3, 103.2, 118.8 and 119.0, 119.5, 123.3, 131.0, 132.1,
140.0, 149.6, 168.3; HRMS (ESI) [M+Na]+ m/z 560.1922 (calcd
for C25H35NO10SiNa: 560.19224); [M+K]+ m/z 576,1664 (calcd for
C25H35NO10SiK: 576.16618).
Preparation of compound 9
Imidazole (2.4 g, 35.2 mmol) and TBDMSCl (5.3 g, 35.2 mmol)
were dissolved in dry DMF (4 mL). The solution was stirred for
0.5 h and a solution of 8 (9.23 g, 17.6 mmol) in DMF (14 mL) was
added. After stirring at room temperature for 20 h, water (100 mL)
was added; the layers were separated and the aqueous layer
was extracted three times with dichloromethane. The combined
organic layers were dried with anhydrous MgSO4, filtered and
concentrated in vacuo. The crude product was purified by column
chromatography over silica gel (CH2Cl2/petroleum ether 50/50;
75/25) to give 9 (10.32 g, 91%) as a mixture of two diastereoisomers
(pale◦yellow solid). Rf 0.60 (petroleum ether/AcOEt 60/40). mp =
Preparation of compound 12
1
59.5 C. H NMR (400 MHz, CDCl3) d -0.04 (s, 3H), 0.09 (2 s,
11 (1.3 g, 2.42 mmol) was dissolved in dry pyridine (12 mL). The
mixture was cooled at 0 ◦C and allyl chloroformiate (7.07 mL,
72.54 mmol) was added dropwise. The mixture was stirred 72 h
at room temperature and was hydrolyzed with aqueous 1 M HCl
(40 mL). The mixture was extracted three times with ethyl acetate
and the combined organic layers were dried over MgSO4, filtrated
and concentrated in vacuo. The crude product was purified by
column chromatography over silica gel (petroleum ether/AcOEt
80/20) to give 12 (1.80 g, 94%) as a mixture of two diastereoisomers
(pale◦yellow solid). Rf 0.84 (petroleum ether/AcOEt 60/40). mp =
3H), 0.89 (2 s, 9H), 1.99 (s, 1H), 2.06 (s, 6H), 2.13 (s, 3H), 2.42–2.62
(m, 2H), 3.74 (s, 3H), 4.22 (m, 1H), 4.82 (t, 1H, J = 7.0 Hz), 5.19–
5.23 (m, 1H), 5.30–5.37 (m, 3H), 7.33 (2d, 1H, J = 8.7 Hz), 7.55
(m, 1H), 7.80 (d, 0.5H, J = 2.1 Hz), 7.84 (d, 0.5 H, J = 2.1 Hz); 13
C
NMR (100 MHz, CDCl3) d -4.8, -4.6, 18.3, 20.7, 25.8, 30.9, 53.2,
68.9, 70.3, 71.3, 72.1, 72.2, 72.7, 80.3, 99.9 and 100.0, 119.4 and
119.9, 122.8, 131.3 and 131.4, 140.3 and 140.4, 141.0, 148.4, 166.9,
169.4, 169.5, 170.2; HRMS (ESI) [M+Na]+ m/z 660.2083 (calcd
for C29H39NO13SiNa: 660.20829); [M+K]+ m/z 676.1819 (calcd for
C29H39NO13SiK: 676.18223).
1
53.7 C. H NMR (400 MHz, CDCl3) d -0.05 (s, 3H), 0.09 (2 s,
3H), 0.89 (2 s, 9H), 1.98 (2t, 1H, J = 2.6 Hz), 2.41–2.61 (m, 2H),
4.31–4.34 (m, 1H), 4.59–4.70 (m, 6H), 4.74–4.72 (m, 2H), 4.81 (2t,
1H, J = 6.5 Hz), 5.24–5.40 (m, 12H), 5,82–6,00 (m, 4H), 7.31 (2d,
1H, J = 8.5 Hz), 7.55 (2t, 1H, J = 8.9 Hz), 7.84 (d, 0.5H, J =
2.1 Hz), 7.86 (d, 0.5H, J = 2.1 Hz); 13C NMR (100 MHz, CDCl3)
d -4.8, -4.7, 18.2, 25.8, 30.8, 67.0, 69.2, 69.3, 69.6, 71.2, 72.0 and
72.1, 72.3, 72.5, 74.0, 75.1, 80.3, 99.8, 118.7, 119.0, 119.1, 119.3,
119.4, 123.0, 130.9, 131.0, 131.1, 131.3, 131.4, 140.1, 140.2, 140.6,
140.7, 148.4 and 148.5, 153.5, 154.0, 165.7; HRMS (ESI) [M+Na]+
m/z 812.2556 (calcd for C37H47NO16SiNa: 812.2556), [M+K]+ m/z
828.2930 (calcd for C37H47NO16SiK: 828.2957).
Preparation of compound 10
9 (2.6 g, 4.08 mmol) was dissolved in THF (48 mL) and methanol
(96 mL). The mixture was cooled at 0 ◦C and sodium methoxyde
(220 mg, 4.08 mmol) was added. Sodium methoxide (110 mg,
2.04 mmol) was added twice after 1 h and 2 h of stirring. The
mixture was stirred again for 1 h and was hydrolyzed with IRC-
50 acidic resin. The mixture was then filtrated and concentrated in
vacuo. The crude product was purified by column chromatography
over silica gel (CH2Cl2, MeOH 99/1; 98/2; 97/3) to give 10
(1.51 g, 72%) as a mixture of two diastereoisomers (white solid).
◦
1
Rf 0.33 (CH2Cl2/MeOH 95/5). mp = 62.9 C. H NMR (400
MHz, CDCl3) d -0.05 (2 s, 3H), 0.09 (2 s, 3H), 0.89 (2 s, 9H),
1.98 (m, 1H), 2.42–2.62 (m, 2H), 3.76–3.85 (m, 5H), 3.94 (t, 1H,
J = 9.7 Hz), 4.10 (2d, 1H, J = 9.7 Hz), 4.81 (2t, 1H, J = 6.3 Hz),
5.01 (2d, 1H, J = 7.3 Hz), 7.34 (2d, 1H, J = 8.7 Hz), 7.59 (dd,
1H, J = 2.1 Hz, J = 8.7 Hz), 7.88 (d, 0.5H, J = 2.1 Hz), 7.90 (d,
0.5H, J = 2.1 Hz); 13C NMR (100 MHz, CDCl3) d -4.8, -4.6, 18.3,
25.8, 30.8, 53.2, 71.0, 71.3, 72.1 and 72.2, 73.0, 74.7, 75.0, 80.4,
102.8 and 102.9, 118.3 and 118.6, 123.3, 132.1 and 132.2, 139.7
and 139.8, CD3OD 140.0, 149.6 and 149.7, 169.1; HRMS (ESI)
[M+Na]+ m/z 534.1766 (calcd for C23H33NO10SiNa: 534.17659);
[M+K]+ m/z 550.1503 (calcd for C23H33NO10SiK: 550.15053).
Preparation of compound 13
To a solution of 12 (370 mg, 0.47 mmol) in dry THF (5.7 mL)
was added dropwise HF/pyridine 70% (1.8 mL). The mixture
was stirred 1 h at room temperature and poured into 100 mL of
ice-cold saturated aqueous NaHCO3. The mixture was extracted
three times with ethyl acetate. The combined organic layers were
dried over MgSO4, filtered and concentrated in vacuo. The crude
product was purified by column chromatography over silica gel
(petroleum ether/AcOEt 70/30) to afford 13 (272 mg, 85%) as
mixture of two diastereoisomers (white solid). Rf 0.62 (petroleum
ether/AcOEt 60/40). mp = 61.8 ◦C. 1H NMR (400 MHz, CDCl3)
d 2.10 (t, 1H, J = 2.6 Hz), 2.51 (bs, 1H), 2.61–2.64 (m, 2H), 4.32 (d,
1H, J = 9.1 Hz), 4.59–4.69 (m, 6H), 4.72 (m, 2H), 4.90 (t, 1H, J =
6.2 Hz), 5.22–5.39 (m, 12H), 5,81–6,00 (m, 4H), 7.34 (d, 1H, J =
8.6 Hz), 7.57 (m, 1H), 7.87 (d, 0.5H, J = 2.1 Hz), 7.89 (d, 0.5H, J =
2.1 Hz); 13C NMR (100 MHz, CDCl3) d 29.6; 67.0, 69.3, 69.4, 69.7,
Preparation of compound 11
To a solution of 10 (390 mg, 0.76 mmol) in allylic alcohol
(12 mL) was added dropwise a solution of sodium allylate 0.125 M
(1.15 mL, 0.145 mmol). The mixture was stirred 40 min and was
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70.8, 72.1, 72.4, 72.5, 74.0, 75.0, 79.5, 99.8, 119.2, 119.3, 119.4,
119.5, 122.9 and 123.0, 131.0, 131.1, 131.2, 131.3, 131.4, 138.8,
140,9 and 141.0, 148.7, 153.6, 154.1, 165.7; HRMS (ESI) [M+Na]+
m/z 698.1692 (calcd for C31H33NO16Na: 698.16915); [M+K]+ m/z
714.1430 (calcd for C31H33NO16K: 714.14309).
tetrakis(acetonitrile)copper(I) hexafluorophosphonate (1 eq.,
0.14 g). The resulting mixture was stirred at room temperature
for 20 h. After removing the volatiles under reduced pressure, the
crude material was purified by flash column chromatography on
silica gel (CH2Cl2/MeOH 90/10) to afford 17 (0.47 g, 70%) as a
mixture of two diastereoisomers. Rf 0.21 (CH2Cl2/MeOH 90/10).
mp = 133.5 C. H NMR (400 MHz, CD3OD, 313 K) d 0.65–
1.3 (m, 15H), 1.4–2.4 (m, 19H), 3.1–3.4 (m, 24H), 4.49 (m, 1H),
4.78 (m, 15H), 5.5 (m, 12H), 6.11 (m, 5H), 7.60 (m, 1H), 7.77
(m, 1H), 7.96 (m, 2H); 13C NMR (100 MHz, CD3OD) d 10.89,
13.99, 15.58, 19.07, 19.76, 19.93, 20.58, 21.21, 21.40, 25.69, 28.51,
29.28, 29.95, 30.77, 32.09, 33.44, 37.74, 37.87, 38.09, 39.49, 42.58,
43.44, 49.93, 50.12, 50.58, 53.39, 61.13, 62.80, 64.12, 67.87, 68.06,
68.48, 69.71, 70.11, 70.30, 70.39, 71.40, 71.67, 72.48, 72.66, 72.86,
73.36, 73.99, 75.03, 75.54, 76.76, 78.0, 86.81, 89.83, 100.06, 104.52,
119.04, 119.31, 119.56, 122.72, 127.58, 132.68, 132.73, 132.82,
132.90, 141.77, 143.15, 143.29, 144.55, 148.53, 149.83, 150.05,
155.01, 155.22, 155.29, 155.47, 158.14, 167.32, 167.67; HRMS
(ESI) [M+Na]+: m/z 1472.6115 (calcd. for C71H95N5O27Na:
1472.61066).
◦
1
Preparation of compound 14
To a solution of 13 (262 mg, 0.38 mmol) in dry dichloromethane
(4 mL) were added para-nitrophenol chloroformiate (198 mg,
0.77 mmol) and pyridine (77 mL, 0.96 mmol). The mixture
was stirred 2 h at room temperature and saturated aqueous
NaHCO3 was added. The layers were separated and aqueous layer
was extracted three times with dichloromethane. The combined
organic layers were washed three times with saturated aqueous
NaHCO3, dried over MgSO4, filtrated and concentrated in vacuo.
The crude product was purified by column chromatography over
silica gel (petroleum ether/AcOEt 70/30) to afford 14 (293 mg,
92%) as a mixture of two diastereoisomers (white solid). Rf 0.79
◦
1
(petroleum ether/AcOEt 60/40). mp = 52.6 C. H NMR (400
MHz, CDCl3) d 2.09 (t, 1H, J = 2.6 Hz), 2.82–2.97 (m, 2H), 4.35 (d,
1H, J = 8.65 Hz), 4.68–4.70 (m, 6H), 4.72 (d, 2H, J = 5.8 Hz), 5.24–
5.39 (m, 12H), 5.80 (t, 1H, J = 6.6 Hz), 5.83–6.00 (m, 4H), 7.36–
7.40 (m, 3H), 7.63 (dd, 1H, J = 2.2 Hz, J = 8.6 Hz), 7.95 (d, 1H,
J = 2.2 Hz), 8.27 (d, 2H, J = 8.6 Hz); 13C NMR (100 MHz, CDCl3)
d 26.2, 67.0, 69.2, 69.4, 69.6, 72.2, 72.3, 72.5, 73.9, 74.9, 77.2,
77.6, 99.3, 119.0, 119.1, 119.4, 119.5, 121.8, 123.9, 125.4, 130.9,
131.0, 131.1, 131.2, 132.5, 133.4, 140.7, 145.6, 149.6, 151.6, 153.5,
154.0, 155.2, 165.5; HRMS (ESI) [M+Na]+ m/z 863.1755 (calcd
for C38H36N2O20Na: 863.17536), [M+K]+ m/z 879.1478 (calcd for
C38H36N2O20K: 879.1493).
Preparation of compound 2b
To a solution of 17 (0.21 g, 0.147 mmol) in MeOH/CH2Cl2 (10/90,
5
mL) was added tetrakis(triphenylphosphine)palladium(0)
(17 mg, 0.0147 mmol) and aniline (0.03 mL, 0.29 mmol). Total
deprotection was observed after stirring at room temperature for
24 h (HPLC analysis, Method A). Solvents were removed under
reduced pressure. The resulting solid was washed three times in
CH2Cl2 and collected by filtration (0.136 g, 80%), (85% purity
HPLC analysis). High degree of purity for compound 2b was
obtained using preparative-reverse phase HPLC (0.067 g, purity
> 95%). Retention times for the two diastereoisomers of 2b are
13.57 and 13.83 min (for HPLC conditions see HPLC analysis).
Preparation of compound 15
Anhydrous pyridine (0.06 mL, 1.5 eq.) was added dropwise to
a solution of 14 (0.41 g, 0.48 mmol) and cyclopamine (0.2 g,
0.48 mmol) in DMF (5.6 mL). The mixture was stirred for 20
h at room temperature and the crude mixture was concentrated
in vacuo. Purification by flash chromatography on silica gel
(CH2Cl2/MeOH 98/2) afforded 15 (0.46 g, 85%) as a mixture
of two diastereoisomers. Rf 0.77 (CH2Cl2/MeOH 98/2). mp =
◦
1
mp = 149.5 C. H NMR (400 MHz, CD3OD) d 0.73–1.05 (m,
13H), 1.13–1.38 (m, 8H), 1.38–1.90 (m, 20H), 2.10–2.35 (m, 8H),
2.70 (m, 1H), 3.1 (m, 1H), 3.3 (m, 1H), 3.36 (m, 1H), 3.47–3.75 (m,
14H),3.84–3.87 (m, 4H), 3.99 (m, 1H), 4.30 (dd, 1H, J = 7.7 Hz,
J = 2.2 Hz), 4.51 (sl, 2H), 5.10 (m, 1H), 5.37 (sl, 1H), 6 (m, 1H),
7.50 (m, 1H), 7.58 (m, 1H), 7.70 (s, 0.5H), 7.76 (s, 0.5H), 7.78
(m, 1 H). 13C NMR (100 MHz, CD3OD) d 10.60, 10.72, 13.83,
18.99, 21.27, 25.66, 29.33, 29.89, 30.79, 32.02, 32.08, 33.41, 37.74,
39.46, 42.56, 42.78, 43.47, 51.37, 53.41, 62.80, 69.71, 70.45, 71.44,
71.66, 72.50, 73.29, 74.51, 75.08, 76.39, 77.03, 78.03, 86.86, 102.33,
104.45, 122.69, 125.38, 127.57, 133.04, 141.51, 143.17, 144.58,
151.22, 158.24; HRMS (ESI) [M - H]- m/z 1156.5195 (calcd.
for C56H78N5O21: 1156.51948).
◦
1
121.5 C. H NMR (400 MHz, CDCl3) d 0.95–1.45 (m, 14H),
1.50–2.45 (m, 21H), 2.76 (m, 1H), 2.89 (m, 1H), 3.06 (m, 1H),
3.27 (m, 1H), 3.57 (m, 2H), 4.33 (m, 1H), 4.72 (m, 8H), 5.37
(m, 14H), 5.91 (m, 5H), 7.35 (m,1H), 7.56 (m, 1H), 7.88 (m,
1H); 13C NMR (100 MHz, CDCl3) d 10.54, 13.55, 14.20, 17.46,
18.67, 19.29, 20.64, 21.06, 24.59, 25.35, 26.42, 29.03, 31.07, 31.36,
32.56, 36.54, 36.96, 38.14, 38.31, 41.55, 41.77, 41.9, 49.2, 51.99,
59.65, 60.41, 63.10, 66.93, 69.16, 69.27, 69.52, 69.61, 71.8, 71.92,
72.3, 72.92, 73.85, 74.88, 78.47, 85.14, 99.56, 119.01, 119.24,
119.30, 119.32, 119.41, 119.46, 121.82, 122.63, 123.47, 126.33,
130.84, 130.94, 131.05, 131.16, 135.67, 140.77, 141.58, 143.41,
148.84, 149.07, 153.46, 153.95, 156.35, 165.58, 165.62; HRMS
(ESI) [M+Na]+: m/z 1135.4625 (calcd. for C59H72N2O19Na:
1135.46215).
HPLC analysis
Analytical HPLC was carried out using a Dionex Ultimate 3000
System with UV variable wavelength detector. Compounds 17,
2b, cyclopamine analysis and enzymatic hydrolysis analysis were
performed on a reverse phase column chromatography (Method A:
-1
˚
R
Acclaim ꢀ 120, C18, 250 ¥ 4,6 mm, 5 mm, 120 A; Flow 1 mL min .;
mobile phase CH3CN, H2O+0.2% TFA, 20/80: 100). Retention
time for compounds 17, 2b and cyclopamine 1 are 22.88, 13.57–
13.83 and 14.3 respectively. Peak area and calibration curves were
obtained with Dionex Chromeleon software.
Preparation of compound 17
To
a
solution of 15 (0.46 g, 0.41 mmol) and 16
(0.14 g, 1 eq.) in anhydrous CH2Cl2 (14.7 mL) was added
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Stability
Notes and references
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Compound 2b (0.1 mg, 0.8 mmol) was incubated in 1 mL of
phosphate buffer (0.02 M, pH 7.0) at 37 ◦C. Stability was
monitored by analytical HPLC using Method A. HPLC analysis
showed no detectable degradation of compound 2b during 24 h
under these conditions.
4 D. M. Berman, S. S. Karhadkar, A. Maitra, R. M. De Oca, M. R.
Gerstenblith, K. Briggs, A. R. Parker, Y. Shimada, J. R. Eshleman, D.
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and S. B. Baylin, Nature, 2003, 422, 313–317.
Enzymatic hydrolysis
Enzymatic hydrolysis was carried out with commercially available
b-glucuronidase from Escherichia coli (purchased from Sigma
Aldrich ref. G8162). Prodrug 2b (1 mmol) was incubated with
Escherichia coli (133 U/mL) in phosphate buffer (0.02 M, pH 7)
at 37 ◦C and sample were analyzed by HLPC/MS.
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Cell Culture
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The U87 human glioblastoma cell line was maintained in Dul-
becco’s modified Eagle’s medium (DMEM) with GlutaMAXTM
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and sodium pyruvate (Invitrogen), supplemented with 10% fetal
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Cell proliferation
R
Cell viability was evaluated using the CellTiter 96ꢀ Aqueous
One Solution Cell Proliferation Assay (Promega). U87 cells
were plated in 96-well plates at a density of 400 cells/well
in 100 ml medium. After 24 h of incubation, medium was
replaced by medium containing the prodrug b-glucuronidase
(40 U/mL) or cyclopamine. Control cells were incubated in
the presence of DMSO (used for prodrug or cyclopamine
solubilization) b-glucuronidase. Cell viability was determined
R
after 5 days of treatment by adding 20 ml of CellTiter 96ꢀ
Aqueous One Solution Reagent into each well 3 h before
measuring the optical density. Metabolically active cells con-
vert 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-ulfophenyl)-2H-tetrazolium (MTS) into a coloured formazan
product that was measured in a spectrophotometric microplate
reader at 490 nm. The OD of control cells was considered as the
100 value.
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8464 | Org. Biomol. Chem., 2011, 9, 8459–8464
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The Royal Society of Chemistry 2011
©