The Metabolic Fate of isoCombretastatin A-4 in Human Liver Microsomes: Identification, Synthesis and Biological Evaluation of Metabolites

Full text of DOI:10.1002/cmdc.201100193

DOI: 10.1002/cmdc.201100193
The Metabolic Fate of isoCombretastatin A-4 in Human Liver Microsomes:
Identification, Synthesis and Biological Evaluation of Metabolites
Mohamed Ali Soussi,[a] Silvio Aprile,[b] Samir Messaoudi,[a] Olivier Provot,[a] Erika Del Grosso,[b] Jꢀrꢁme Bignon,[c]
Joꢂlle Dubois,[c] Jean-Daniel Brion,[a] Giorgio Grosa,*[b] and Mouad Alami*[a]
Combretastatins, naturally occurring stilbenes, were isolated
from Combretum caffrum by Pettit in 1989.[1] Among them,
combretastatin A-4 (CA-4) was shown to strongly inhibit the
polymerization of tubulin by binding to the colchicine binding
site and was the most cytotoxic agent. CA-4 showed toxicity at
a nanomolar level against a variety of human cancer cell lines,
including multiple drug-resistant cancers.[2] CA-4 also exerts
highly selective toxicity in proliferating endothelial cells and, as
a consequence, demonstrates strong suppressive activity on
tumor blood flow leading to cell death. Moreover, in contrast
to colchicine, the antivascular effects of CA-4 are apparent well
below the maximal tolerated dose, offering a wide therapeutic
window. However, there are two major disadvantages with CA-
4 as a drug candidate: its low aqueous solubility limits its effi-
cacy in vivo,[3] and its low chemical stability due to the cis-
double bond isomerization to the more thermally stable but
nonactive trans form during storage and administration. The
first problem was solved by the synthesis of water-soluble pro-
drugs, including the disodium phosphate prodrug CA-4P (1)[4,5]
and its serinamido derivative AVE-8062 (2).[3] Currently, CA-4P,
either as a single agent or in combination therapy, is undergo-
ing several advanced clinical trials worldwide for the treatment
of age-related macular degeneration (AMD)[6] or anaplastic thy-
roid cancer.[7] To overcome the second problem related to the
double bond isomerization, several approaches have been un-
dertaken to rigidify the olefin using a heterocyclic com-
pound.[8]
synthesize[11] on a multigram scale without the need to control
the olefin geometry, and then definitively solves the problem
of the double bond isomerization. Recently, we studied the
metabolic fate of CA-4 in rat and human microsomal prepara-
tions, and several phase I metabolites (resulting from O-deme-
thylation, aromatic hydroxylation and quinones formation), as
well as phase II conjugates,[12] in both cis and trans configura-
tion, were characterized. The role of these metabolites in the
pharmacodynamic activity of CA-4 is still unknown. These con-
siderations together with the structural differences between
CA-4 and isoCA-4, due to the presence of a 1,1-diarylethylene
scaffold, prompted us to examine the metabolic profile of
isoCA-4, identifying and synthesizing the metabolites, and then
evaluating their biological properties. Herein, we report the re-
sults of this study.
In an ongoing project aimed at developing novel tubulin as-
sembly inhibitors,[9] we recently discovered isocombretastatin
A-4 (isoCA-4),[10] a structural isomer of the natural product that
displays biological activities comparable to those of CA-4.
isoCA-4 contains a 1,1-diarylethylene scaffold and is easy to
Possible metabolites of isoCA-4, demethylated/hydroxylated
on the A- and B-rings, hydroxylated on the B-ring, or trans-
formed into p-quinones, were prepared according to
Scheme 1. Hypothetical metabolites 3–6 arising from O-deme-
thylation or hydroxylation of the A- and B-rings were prepared
by the coupling of N-tosylhydrazones with aryl iodides under
palladium catalysis.[13] Then, the crude coupling products were
O-desilylated using a tetrahydrofuran (THF) solution of tetra-n-
butylammonium fluoride (TBAF) to afford the desired phenols
3 and 4, as well as catechols 5 and 6, in good yields. The use
of Fremy’s salt as a biomimetic oxidant with isoCA-4 in a mix-
ture of water and dichloromethane furnished p-quinone 7 in
an acceptable yield (50%). Finally, the reduction of 7 using
sodium borohydride (NaBH4) afforded the putative metabolite
8, which was found to be unstable and oxidizes to p-quinone
7 in air. This compound must be prepared carefully just before
use and stored under an inert atmosphere at À408C.
[a] M. A. Soussi,+ Dr. S. Messaoudi, Dr. O. Provot, Prof. Dr. J.-D. Brion,
Dr. M. Alami
Univ Paris-Sud, Facultꢀ de Pharmacie, CNRS, BioCIS-UMR 8076
Laboratoire de Chimie Thꢀrapeutique
5 Rue J.-B. Clꢀment, Chꢁtenay-Malabry 92296 (France)
E-mail: mouad.alami@u-psud.fr
[b] Dr. S. Aprile,+ Dr. E. Del Grosso, Dr. G. Grosa
Dipartimento di Scienze Chimiche Alimentari
Farmaceutiche e Farmacologiche and Drug and Food Biotechnology Center
Universitꢂ degli Studi del Piemonte Orientale “A. Avogadro”
Largo Donegani 2, 28100 Novara (Italy)
E-mail: Giorgio.Grosa@pharm.unipmn.it
[c] Dr. J. Bignon, Dr. J. Dubois
isoCA-4 was incubated in human liver microsomal (HLM)
fractions in the presence of a NADPH regenerating system.
isoCA-4 and its metabolites were separated by HPLC using a
C18 reverse-phase column and a mixture of water and acetoni-
trile acidified with 1% formic acid as the eluent. The LC/MS
Institut de Chimie des Substances Naturelles, UPR 2301, CNRS
Avenue de la Terrasse, 91198 Gif-sur-Yvette (France)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100193.
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193 in M1 and M2 MS2 spectra
suggests that O-demethylation
occurs on both the A- and B-
rings of isoCA-4 during microso-
mal biotransformation (Table 1).
Furthermore, the LC/MS traces
and retention times of synthetic
compounds 5 and 3 completely
matched those of metabolites
M1 and M2, respectively. Based
on these data, the structures of
2-hydroxy-5-[1-(3,4,5-trimethoxy-
phenyl)ethenyl]phenol (5), and
2-methoxy-5-[1-(4-hydroxy-3,5-
dimethoxyphenyl)ethenyl]phenol
(3) were assigned to M1 and M2,
respectively. M3 showed the
same fragmentation pattern as
M2, indicating that O-demethyla-
tion occurs on the A-ring and al-
lowing us to assign the structure
of
2-methoxy-5-[1-(5-hydroxy-
3,4-dimethoxyphenyl) ethenyl]-
phenol (4).
Monitoring the ion at m/z 331
(Figure 1c), one peak was re-
vealed (M4), with a retention
time and MS2 spectra identical
to those of synthetic quinone 7.
Finally, monitoring the ion at m/
z 333 (Figure 1d), three peaks
were found: M5, M6 and M7.
The retention time and MS2
spectra of metabolite M7 com-
pletely match those of synthetic
compound 8, as such, the struc-
ture of 4-hydroxy-2-methoxy-
Scheme 1. Synthesis of metabolites 3–8. Reagents and conditions: a) Pd2(dba)3 (10 mol%), Xphos (20 mol%),
tBuOLi (2.5 equiv), dioxane, 758C, 3 h; b) TBAF (3.0 equiv), THF, RT, 2 h, 32% (3), 50% (4), 80% (5), 53% (6);
c) K2[NO(SO3)2] (3.5 equiv), Aliquat 336 (1.3 equiv), NaH2PO4·H2O (8.5 equiv), H2O/CH2Cl2 (4:1 v/v), RT, 3 h, 50%;
d) NaBH4 (2.0 equiv), THF, RT, 10 min, 99%.
analysis of the HLM incubation extracts revealed that isoCA-4
was metabolized in microsomes to almost the same extent (~
5%) as found for CA-4 microsomal metabolism producing a
complex metabolic pattern made by several metabolites that
were not found in the control incubation performed without
the NADP(H) regenerating system. The full-scan analysis re-
vealed the presence of several ions at m/z 303 ([MÀ14]), 331
([M+14]), and 333 ([M+16]), hence LC/MS2 analyses were per-
formed monitoring these ions, and the chromatograms
(Figure 1) and mass spectra were acquired (see Supporting In-
formation).
Since isoCA-4 showed, in its positive MS2 spectrum, two di-
agnostic fragment ions at m/z 149 and 193 arising from ring B
and ring A, respectively, LC/MS2 data may effectively assist with
the assignment of the structures of the metabolites. Monitor-
ing the ion at m/z 303, three peaks (M1, M2 and M3) were
found, while the peak at 8.26 min is matrix related (Figure 1b).
It is worth mentioning that the chromatographic conditions do
not permit the separation of M1 and M2 metabolites. The pres-
ence of the positive fragment ions at m/z 135, 149, 179 and
5’[1-(3,4,5-trimethoxyphenyl)ethenyl]phenol was assigned. The
M5 peak may be attributed to a derivative that is further hy-
droxylated on the B-ring, since its fragmentation pathway is
identical to that of metabolite M7. However, mass spectral
data do not allow the determination of the exact position (C-6’
or C-3’) of hydroxylation on the B-ring; different retention
times were measured for M5 (8.36 min) and 6 (13.39). Since
the structure of metabolite M7 was previously attributed to 8,
and the retention time of synthetic compound 6 does not
match with that of M5, we can conclude that hydroxylation oc-
curred at C-3’ and assign the structure of 3-hydroxy-2-me-
thoxy-5’[1-(3,4,5-trimethoxyphenyl)ethenyl] phenol to M5. Due
to the intrinsic instability of the cathecolic function for hydrox-
ylation at C-6’, the formation of the C-3’-hydroxylated metabo-
lite appears more likely.
Structural assignment of M6 was more difficult; indeed, its
product ion spectrum at m/z 333 showed a different fragmen-
tation pattern lacking the typical fragment ions of isoCA4 (m/z
149 and 193, Figure 1d). On the contrary, two fragment ions
arising from a loss in mass of 18 Da were observed suggesting
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Figure 1. Representative LC/MS chromatograms of isoCA-4 (HLM or S9) incubation extracts performed in positive product ion scan mode (MS2): a) full mass,
b) m/z=303, c) m/z=331, d) m/z=333, e) m/z=491, f) m/z=395.
the structure of isoCA-4 epoxide for M6. However, M6 shows
good stability under our incubation conditions since the for-
mation of the corresponding diolic derivative was not ob-
served in LC/MS chromatograms (data not shown). Moreover,
when the incubation was performed in the presence of mer-
captoethanol (MSH), no adduct was found by LC/MS analysis.
Finally, our efforts to synthetize isoCA-4 epoxide using different
approaches were unsuccessful. Taken together, these observa-
tions do not allow the confident assignment of the structure
of the epoxide to metabolite M6.
formed from metabolite M1 (Figure 3a). Indeed, despite the
absence of this reactive species in the LC/MS2 trace (m/z 301),
the presence of the adduct with MSH made clear its formation
in the incubation mixture. One can note that all our attempts
to oxidize synthetic catechol 5 into its corresponding o-qui-
none failed, confirming the high instability of such compounds
as previously reported by Pettit et al.[14]
To study phase II metabolism, isoCA-4 was incubated in the
presence of uridine-5’-diphosphoglucuronic acid trisodium salt
(UDPGA) and 3’-phosphoadenosine-5’-phosphosulfate (PAPS)
cofactors. As expected, the LC/MS analysis of the incubation
extracts (Figure 2e and f) revealed that metabolism of isoCA-4
produces the corresponding glucuronide and sulfate metabo-
lites, which were not found in the control incubation per-
formed without UDPGA or PAPS. The formation of the conju-
gated metabolites of isoCA-4 is in agreement with that previ-
ously observed for CA-4. Scheme 2 summarizes the isoCA-4
phase I and II metabolism in human liver fractions.
To study the reactivity of isoCA-4 metabolites toward nucleo-
philic thiols, MSH was added to the incubations and the ions
at m/z 379 and 409 were monitored. The LC/MS chromato-
grams reported in Figure 2 highlight the presence of two addi-
tional peaks (M8m and M4m). Further confirmation of M4 reac-
tivity was the presence of the M4m peak in the chromatogram
of compound 7 after treatment with MSH. The fragment ions
at m/z 333 and 211 found in the MS2 spectrum of metabolite
M8m suggest that a reactive and unstable o-quinone species is
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isoCA-4 as
a reference com-
Table 1. MS2 fragment ions, retention times (tR) and proposed structures of isoCA-4 metabolites.
pound (Table 2). Compared to
the parent compound isoCA-4,
hydroxylated metabolite 8 and
p-quinone 7 showed decreased
cytotoxicity, revealing that addi-
tional hydroxylation and subse-
quent oxidation of the B-ring is
not tolerated in terms of biologi-
cal activity. Similarly, O-demethy-
lated compounds 5 and 3 on
the B- and A-rings, respectively,
also led to a significant loss in
activity. However, in contrast to
compound 3, synthetic com-
pound 4 (M3), resulting from O-
demethylation on the C-3 posi-
tion of the A-ring, exhibited
potent cytotoxicity with an IC50
value of 34 nm against HCT-116
cells. Next, all synthesized me-
tabolites were evaluated for
their ability to inhibit tubulin as-
sembly in vitro. Except for cate-
chol 5 (M1), isoCA-4 metabolites
inhibited tubulin assembly at
concentrations comparable or
better to that of the parent mol-
ecule isoCA-4. It is interesting to
Metabolite Compd tR [min] [M+H]+
MS2 fragment ions
Proposed structure
Parent
isoCA-4
Phase I
M1
15.91
317
302, 286, 285, 193, 149
5
3
12.87
12.87
303
303
288, 272, 193, 135
288, 271, 179, 149
M2
M3
M4
4
7
13.31
14.32
303
331
288, 271, 179, 149
316, 303, 299, 298
note that p-quinone
7 (M4),
which displayed the highest an-
titubulin activity (IC50 =1.0 mm),
is only a weak cytotoxic agent,
suggesting that this molecule
may be a lead compound for
the development of antimitotic
agents with diminished side ef-
fects.
M5
M6
M7
–
–
8
8.36
9.76
333
333
333
315, 301, 255, 193, 165
315, 297, 279
unknown
In conclusion, the metabolic
profile of the synthetic drug
isoCA-4 was achieved with
human liver microsomal frac-
tions. We successfully identified
seven phase I metabolites result-
ing mainly from O-demethyla-
tion and hydroxylation of the ar-
omatic rings. For further struc-
ture confirmation, five of these
metabolites were also synthe-
sized and evaluated. Compound
4, resulting from O-demethyla-
tion on the A-ring, was found to
be the most potent, exhibiting a
significant cytotoxicity against
10.76
315, 301, 193, 165
Phase II
isoCA-4G
9.20
9.43
491
395
473, 315, 300, 175
isoCA-4S
380, 315, 300
All the synthesized isoCA-4 metabolites 3, 4, 5, 7 and 8 were
first assessed in vitro for their ability to inhibit cell proliferation
against the human colon carcinoma HCT-116 cell line with
HCT116 cancer cell line with an IC50 value of 34 nm. Compound
7, resulting from hydroxylation of the B-ring followed by p-qui-
none formation, exhibited comparable antitubulin activity to
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Figure 2. Representative LC/MS chromatograms of isoCA-4 incubation (HLM) extract performed in positive product ion mode scan (MS2): a) m/z=379 (with
MSH), b) m/z=379 (without MSH), c) m/z=409 (with MSH), d) m/z=409 (without MSH).
gel 60 (40–63 mm, 230–400 mesh ASTM) at medium pressure
(200 mbar). All solvents were distilled before use and stored over
isoCA-4 but was 100-fold less cytotoxic. Further experiments to
evaluate the potential of 7 as an anticancer agent are now un-
derway in our laboratory.
4 ꢅ molecular sieves. All reagents were obtained from commercial
suppliers unless otherwise stated. In general, organic extracts were
dried over Na2SO4.
General procedure for the preparation of compounds 3–6: A so-
lution of Pd2(dba)2 (0.1 mmol, 10 mol%), Xphos (0.2 mmol,
20 mol%), N-tosylhyrazone (1.0 mmol), tBuOLi (2.5 mmol) and aryl
iodide (1.0 mmol) in dioxane (5.0 mL) were heated in a sealed tube
for 3 h at 758C. The resulting suspension was cooled to RT and fil-
tered through a pad of Celite, rinsing with EtOAc, to remove the
inorganic salts. The solvents were removed in vacuo, and the
crude residue was redissolved in THF (2.0 mL). A 1m solution of
tetra-n-butylammonium fluoride (TBAF) in THF (3.0 mL, 3.0 mmol)
was added, and the reaction mixture was stirred for 2 h at RT. The
mixture was diluted with EtOAc, washed with H2O, and extracted.
The organic phase was separated, dried (Na2SO4), filtered and con-
centrated in vacuo. Purification by flash chromatography on silica
gel gave the desired compounds 3–6.
Experimental Section
Chemistry
General: Melting points (mp) were recorded on a Bꢄchi B-450 ap-
paratus and are uncorrected. NMR experiments were performed on
a Bruker AVANCE 300 (1H: 300 MHz; 13C: 75 MHz) in CDCl3, unless
otherwise stated. Chemical shifts (d) are reported in ppm, and the
following abbreviations are used: singlet (s), doublet (d), triplet (t),
multiplet (m). Mass spectrometry (MS) was performed on a Bruker
Esquire electrospray ionization (ESI) apparatus. Thin-layer chroma-
tography (TLC) was performed on silica gel 60 plates with a fluo-
rescent indicator and visualized under a UVP Mineralight UVGL-58
lamp (254 nm). Flash chromatography was performed using silica
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Figure 3. Product ion scan spectra (MS2), (m/z 379, and 409) of M8m and M4m metabolites from isoCA-4 incubations in the presence of MSH.
Scheme 2. Metabolism of isoCA-4 in HLM fractions. The structure in brackets is an unstable metabolite.
Compound 3 (M2): Yield=32% (brown solid); mp: 172–1738C;
Rf =0.5 (EtOAc/c-hexane, 5:5); 1H NMR (300 MHz, CDCl3): d=6.98
(d, 1H, J=1.2 Hz), 6.86–6.79 (m, 2H), 6.57 (s, 2H), 5.60 (s, 1H), 5.55
(s, 1H), 5.33 (d, 1H, J=1.3 Hz), 5.28 (d, 1H, J=1.3 Hz), 3.90 (s, 3H),
3.85 ppm (s, 6H); 13C NMR (75 MHz, CDCl3): d=149.7, 146.7 (2C),
146.5, 145.3, 135.1, 134.6, 133.2, 120.4, 114.6, 112.4, 110.2, 105.5
(2C), 56.5, 56.1 ppm (2C); IR (neat): n˜ =1511, 1446, 1419, 1180,
1114, 1030, 822 cmÀ1; MS (ESI+): m/z (%): 303.1 (66) [M+H]+.
Compound 4 (M3): Yield=50% (pale yellow solid); mp: 133–
1358C; Rf =0.25 (EtOAc/c-hexane, 3:7); 1H NMR (300 MHz, CDCl3):
d=6.96 (d, 1H, J=1.9 Hz), 6.84 (dd, 1H, J=1.9, 8.3 Hz), 6.81 (d,
1H, J=8.3 Hz), 6.61 (d, 1H, J=1.9 Hz), 6.45 (d, 1H, J=1.9 Hz), 5.73
(s, 1H), 5.57 (s, 1H), 5.34 (d, 1H, J=1.1 Hz), 5.31 (d, 1H, J=1.1 Hz),
3.92 (s, 3H), 3.91 (s, 3H), 3.81 ppm (s, 3H); 13C NMR (75 MHz,
CDCl3): d=152.0, 149.4, 148.9, 146.5, 145.3, 138.0, 135.3, 134.9,
120.4, 114.7, 113.1, 110.3, 108.5, 104.7, 61.1, 56.1, 56.0 ppm; IR
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brown oil (99%; unstable) of sufficient purity for biological evalua-
tions. Rf =0.4 (CH2Cl2/c-hexane, 5:5); 1H NMR (300 MHz,
CD3COCD3): d=7.10 (s, 1H), 7.02 (s, 1H), 6.64 (s, 2H), 6.59 (s, 1H),
6.54 (s, 1H), 5.64 (d, 1H, J=1.5 Hz), 5.28 (d, 1H, J=1.5 Hz), 3.81 (s,
3H), 3.76 (s, 6H), 3.73 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d=
153.5 (2C), 147.4, 147.0, 145.4, 139.2, 135.5, 119.0, 115.9, 115.6,
104.4 (2C), 99.7, 60.9, 56.3 (2C), 56.1 ppm (1C unaccounted for); IR
(neat): n˜ =3000, 1643, 1593, 1577, 1510, 1328, 1235, 1128, 994, 856,
830, 714 cmÀ1; MS (ESI+): m/z (%): 333.1 (100) [M+H]+.
Table 2. Cytotoxic activity of isoCA-4 metabolites against HCT-116 cells
and inhibition of tubulin polymerization (ITP).
Compd (Metabolite)
HCT-116 IC50[a] [nm]
ITP IC50[b] [mm]
3 (M2)
4 (M3)
5 (M1)
7 (M4)
8 (M7)
isoCA-4[c] (parent)
400Æ55
34Æ3
3.5Æ0.6
5.0Æ0.2
65Æ5
100Æ15
300Æ50
2000Æ150
2.0Æ0.2
1.0Æ0.1
6.4Æ0.5
1.9Æ0.3
[a] The IC50 value is defined as the compound concentration required to
decrease cell growth by 50% following treatment for 72 h; values repre-
sent the average ÆSEM of three experiments. [b] Inhibition of tubulin
polymerization (ITP); the IC50 value is defined as the compound concen-
tration required to decrease the rate of microtubule assembly by 50%;
values represent the average ÆSEM of three experiments. [c] Both IC50
values (cytotoxicity and ITP) were experimentally determined for isoCA-4
in this study.
Biology
Cell culture and proliferation assay: Cancer cell lines were obtained
from the American Type Culture Collection (Rockville, MD, USA)
and were cultured according to the supplier’s instructions. Human
HCT116 colorectal carcinoma cells were grown in RPMI 1640 con-
taining 10% fetal calf serum (FCS) and 1% glutamine. All cell lines
were maintained at 378C in a humidified atmosphere containing
5% CO2. Cell viability was assessed using CellTiter-Blue reagent
(Promega) according to the manufacturer’s instructions. Cells were
seeded in 96-well plates (5ꢆ103 cells/well) containing 50 mL
growth medium. After 24 h culture, cells were treated with test
compound (50 mL) dissolved in DMSO (less than 0.1% in each
preparation). After 72 h incubation, 20 mL of resazurin was added
and the cells were incubated for a further 2 h before recording
fluorescence (lex =560 nm, lem =590 nm) using a Victor microtiter
plate fluorometer (Perkin–Elmer,USA). The IC50 value corresponds
to the concentration of test compound that causes a decrease of
50% in fluorescence of drug treated cells compared with untreated
cells. Experiments were performed in triplicate.
(neat): n˜ =2940, 1720, 1581, 1508, 1357, 1253, 1103, 908, 730 cmÀ1
MS (APCI+): m/z (%): 303.0 (100) [M+H]+.
;
Compound 5 (M1): Yield=80% (yellow oil); Rf =0.5 (EtOAc/c-
1
hexane, 5:5); H NMR (300 MHz, CDCl3): d=6.87 (d, 1H, J=1.2 Hz),
6.80 (m, 2H), 6.54 (s, 2H), 5.96 (s, 2H), 5.34 (d, 1H, J=1.1 Hz), 5.27
(d, 1H, J=1.1 Hz), 3.87 (s, 3H), 3.76 ppm (s, 6H); 13C NMR (75 MHz,
CDCl3): d=152.7 (2C), 149.5, 143.9, 143.4, 137.9, 137.4, 134.3, 121.1,
115.4, 115.0, 112.8, 105.8 (2C), 61.1, 56.3 ppm (2C); IR (neat): n˜ =
1616, 1580, 1503, 1412, 1344, 1289, 1264, 1237, 1126, 999, 954,
846, 788, 735, 701 cmÀ1; MS (ESI+): m/z (%): 300.9 (100) [M+H]+.
Microtubule assembly assay: Sheep brain tubulin was purified ac-
cording to the method of Shelanski[15] by two cycles of assembly–
disassembly and then dissolved in the assembly buffer containing
0.1m 2-(N-morpholino)ethanesulfonic acid (MES), 0.5 mm MgCl2,
1 mm ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic
acid (EGTA), and 1 mm guanosine 5’-triphosphate (GTP), pH 6.6;
the concentration of tubulin was about 2–3 mgmLÀ1. Tubulin as-
sembly was monitored and recorded continuously by turbidimetry
at 350 nm in a UV spectrophotometer equipped with a thermostat-
ed cell set at 378C. All samples were dissolved in DMSO. The test
compound (1 mL) was added to the microtubular solution (150 mL),
and the solution was incubated at 378C for 10 min and at 08C for
5 min before evaluation of the tubulin assembly rate. The IC50
value is defined as the concentration of test compound that de-
creases the maximum assembly rate of tubulin by 50% compared
to the rate in the absence of test compound. The IC50 values for all
compounds were compared to the IC50 of isoCA-4 and measured
the same day under the same conditions.
Compound 6: Yield=53% (white solid); mp: 111–1138C; Rf =0.4
(EtOAc/c-hexane, 5:5); 1H NMR (300 MHz, CDCl3): d=6.69 (d, 1H,
J=8.5 Hz), 6.58 (s, 2H), 6.51 (d, 1H, J=8.6 Hz), 5.67 (s, 1H), 5.50–
5.25 (m, 3H), 3.92 (s, 3H), 3.86 (s, 3H), 3.81 ppm (s, 6H); 13C NMR
(75 MHz, CDCl3): d=153.0 (2C), 147.0, 145.6, 141.7, 138.0, 136.6,
132.8, 121.7, 121.2, 115.5, 104.4 (2C), 103.0, 60.9, 56.1 ppm (3C); IR
(neat): n˜ =3548, 1628, 1578, 1506, 1481, 1464, 1413, 1348, 1291,
1274, 1235, 1204, 1171, 1115, 1092, 941, 913, 841, 761 cmÀ1; MS
(ESI+): m/z (%): 333.2 (7) [M+H]+; 355.0 (100) [M+Na]+
Compound 7 (M4): A mixture of Aliquat 336 (530 mg, 1.2 mmol)
and NaH2PO4·H2O (948 mg, 7.9 mmol) in H2O (60 mL) was treated
with a solution of isoCA-4 (295 mg, 0.933 mmol) in CH2Cl2 (15 mL).
Fremy’s salt (K2[NO(SO3)2]; 875 mg, 3.26 mmol) was then added,
and the mixture was stirred at RT for 3 h (color change from
mauve to red). The reaction was extracted with CH2Cl2 (3ꢆ20 mL),
and the combined organic layers were dried (Na2SO4), filtered and
concentrate in vacuo. Purification of the crude by flash chromatog-
raphy (EtOAc/c-hexane, 5:5) gave 7 as an orange solid (154.1 mg,
50%); mp: 135–1378C; Rf =0.5 (EtOAc/c-hexane, 5:5); 1H NMR
(300 MHz, CDCl3): d=6.64 (s, 1H), 6.45 (s, 2H), 5.97 (s, 1H), 5.73 (d,
1H, J=0.8 Hz), 5.63 (d, 1H, J=0.8 Hz), 3.86 (s, 3H), 3.85 (s, 3H),
3.83 ppm (s, 6H); 13C NMR (75 MHz, CDCl3): d=186.4, 182.5, 158.7,
153.4 (2C), 147.4, 143.0, 138.4, 135.2,132.2, 121.4, 108.4, 104.7 (2C),
61.1, 56.5, 56.3 ppm (2C); IR (neat): n˜ =2161, 1673, 1647, 1581,
1508, 1265, 1129, 732 cmÀ1; MS (ESI+): m/z (%): 331.1 (100) [M+
H]+.
Acknowledgements
We thank the Association pour la Recherche sur le Cancer (ARC)
for their financial support of this research (doctoral fellowship to
MS).
Keywords: anticancer
isocombretastatin A-4 · drug metabolism · tubulin
agents
·
cytotoxicity
·
Compound 8 (M7): A solution of 7 (1.0 mmol) in THF (10 mL) was
treated with NaBH4 (2.0 equiv). The reaction was stirred for 10 min
at RT and then carefully hydrolyzed with H2O. The reaction was ex-
tracted with Et2O (20 mL), and the combined organic extracts were
dried (Na2SO4), filtered and concentrated in vacuo to give 8 as a
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Published online on July 11, 2011
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ChemMedChem 2011, 6, 1781 – 1788