Antineoplastic agents. 552. Oxidation of combretastatin A-1: Trapping the o-quinone intermediate considered the metabolic product of the corresponding phosphate prodrug

Article abstract of DOI:10.1021/np800179g

The very unstable (<10 min at rt) o-quinone 5 derived from the vicinal diphenol anticancer drug combretastatin A-1 (1) has been obtained by careful oxidation with NaIO₄ and tetrabutylammonium bromide in water/dichloromethane. Immediate reaction

Full text of DOI:10.1021/np800179g

J. Nat. Prod. 2008, 71, 1561–1563
1561
Antineoplastic Agents. 552. Oxidation of Combretastatin A-1: Trapping the o-Quinone
Intermediate Considered the Metabolic Product of the Corresponding Phosphate Prodrug1
George R. Pettit,* Andrew J. Thornhill, Bryan R. Moser,† and Fiona Hogan
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State UniVersity, P.O. Box 872404, Tempe, Arizona 85287
ReceiVed March 21, 2008
The very unstable (<10 min at rt) o-quinone 5 derived from the vicinal diphenol anticancer drug combretastatin A-1 (1)
has been obtained by careful oxidation with NaIO4 and tetrabutylammonium bromide in water/dichloromethane. Immediate
reaction with phenylenediamine (6) allowed o-quinone 5 to be trapped as the stable phenazine derivative 7. For further
confirmation, 5 was also captured as a dimethoxyphenylenediamine-derived phenazine (11). Both phenazines 7 and 11
significantly inhibited (ED50 ∼0.2 µg/mL) growth of the murine P388 lymphocytic leukemia cell line and provided a
new SAR insight in the combretastatin series of naturally occurring anticancer drugs.
In 1987 we reported2 the isolation and synthesis of combretastatin
A-1 (1, Figure 1), a reasonably abundant (0.9% w/w yield)
antineoplastic constituent of the South African tree Combretum
caffrum. While initial in vivo anticancer evaluations gave encourag-
ing results, it soon became apparent that 1 was unstable relative to
the lead anticancer constituent of C. caffrum, combretastatin A-4
(2, Figure 1).3 An extended series of experiments aimed at
uncovering practical prodrugs of the sparingly soluble (in water) 2
and the unstable 1 culminated in the syntheses of sodium combre-
tastatin A-4 phosphate (3, Zybrestat, Figure 1),4 presently in phase
II human cancer clinical trials5 with phase III trials under way,
and of sodium combretastatin A-1 diphosphate (4, aka OXI-4503
as the potassium salt, Figure 1),6a for which clinical development
is under way.6b
Figure 1. Structures of combretastatins A-1 (1) and A-4 (2) and
the phosphate prodrugs of A-4 (3) and A-1 (4).
Selection of 3 and 4 (as the potassium salt) for development to
clinical trials was based on extensive preclinical experiments that
pointed to both as outstanding cancer antiangiogenic/vascular
disrupting anticancer drug candidates combined with a number of
other promising properties that indicated considerable mechanistic
and metabolic pathway differences.7 For example, in SCID mice
given the MHEC5-T hemangioendothelioma and a single dose of
4 at 100 mg/kg, tumor blood flow was reduced by 50% in 1 h and
by 24 h was decreased over 90% with only partial recovery
(27-35%) at 72 h. Very significantly, by 12 h, the tumor blood
vessels were completely destroyed. Indeed, at 1-3 h, morphological
changes and initiation of apoptosis had already begun, accompanied
by an increase in vascular permeability favoring an increase in the
retention time of companion drug(s). The terminal result as with 3
is massive tumor cell necrosis.7 As indicated in the example first
noted, 4 has shown excellent potential in preclinical studies, leading
to tumor regression as a single agent that also attacks the remaining
tumor rim cancer cells.5,7 The mechanism of action differs (as was
originally presumed, 4 forms an o-quinone intermediate in vivo)7a
from that of 3, and both have been shown to improve the
effectiveness of, for example, Avastin and other anticancer drug
combinations. A phase Ib clinical trial of the 3/Avastin combination
began in early 2006. The first phase I clinical trial of 4 (as the
potassium salt) was initiated in 2005, supported by the use of
extensive blood studies and MRI and PET scans to gain further
insights into the mechanism of action.7
Figure 2. Synthesis of phenazine 7 from CA1 (1).
Results and Discussion
Early (ca. 1990) in our chemical studies of 1, it seemed likely that
the easily observed color changes and rapid degradation of this vicinal
phenol were due to oxidation reactions leading to the corresponding,
and quite unstable, o-quinone 5.8 In order to better understand the in
vivo metabolism of 1, we have first succeeded in preparing and trapping
the very transient 5 as a phenazine derivative (Figure 2). Prior to
oxidation of 1, a number of oxidative reagents were investigated using
a more readily available diphenol. Thus, catechol was subjected to
oxidative procedures using NaIO4/DCM/Bu4NBr,9 Ag2O/EtOH,10
Ag2O/EtOH/sonication,10,11a and NaIO4/H2O.11b-d The products from
these reactions suggested that the NaIO4/DCM/Bu4NBr procedure
1
might be useful with 1. The H NMR analysis of the crude product
from catechol indicated the presence of the o-quinone with signals at
δ 5.88 and 3.07 as multiplets, along with other products.
Oxidation of 1 with NaIO4/DCM/Bu4NBr gave evidence of 5 in
solution, but it proved to be too unstable to isolate. Although its
existence was indicated by a blue color, this rapidly changed to
* To whom correspondence should be addressed. Tel: 480-965-3461.
Fax: 480-965-2747. E-mail: bpettit@asu.edu.
† Current affiliation: United States Department of Agriculture, Agricul-
tural Research Service, National Center for Agricultural Utilization Research,
Peoria, IL 61604.
10.1021/np800179g CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/27/2008

1562 Journal of Natural Products, 2008, Vol. 71, No. 9
Pettit et al.
sulfate. Where appropriate, the crude products were purified by silica
gel chromatography using gravity (70-230 mesh ASTM) silica gel
from E. Merck (Darmstadt, Germany).
3-Methoxy-6[2-(3,4,5-trimethoxyphenyl)-Z-ethylene]-[1,2]benzo-
quinone (5). To a stirred solution of vicinal phenol 1 (0.10 g, 0.301
mmol) in DCM (5 mL) at rt was added a solution of NaIO4 (83.8 mg,
0.361 mmol) in H2O (1.0 mL) and tetrabutylammonium bromide (10
mg, 0.03 mmol). After 10 min the reaction mixture turned blue. After
separation of the phases, the organic phase was dried and concentrated
in vacuo. The blue coloration over a period of 5-10 min changed to
black, indicating decomposition of 5.
1-Methoxy-4-[(3,4,5-trimethoxyphenyl)-(Z)-vinyl]phenazine (7).
The preceding oxidation was repeated with diphenol 1 (0.10 g, 0.301
mmol) in DCM (5 mL) with an aqueous solution (l mL) of NaIO4 (83.8
mg, 0.361 mmol) and tetrabutylammonium bromide (10 mg, 0.03
mmol). The reaction mixture turned blue over 10 min. FT-IR analysis
yielded a characteristic carbonyl stretch at 1739 cm-1, indicating
formation of 5. The phases were separated, the organic phase was
concentrated in vacuo, and the resultant residue was immediately
dissolved in methanol (10 mL) and stirred at 0 °C. At that point 1,2-
phenylenediamine (6, 50.0 mg, 0.46 mmol) was added. After stirring
for an additional 2 h, the condensation was terminated by addition of
NaHCO3 (10 mL, sat. aq.) and the product was extracted with EtOAc
(3 × 30 mL). The combined organic extract was dried and concentrated
in vacuo, and the oily residue was purified twice by silica gel
chromatography (gravity; 1:1 EtOAc/n-hexane) to afford phenazine 7,
which crystallized from acetone/n-hexane as a red solid (30 mg, 25%
from 1): mp 116-118 °C; IR (neat) νmax 2927, 2854, 1623, 1586, 1503,
Figure 3. Synthesis of phenazine 11.
black, as observed with the catechol oxidation. To confirm
formation of 5, trapping it as a pyrazine derivative11c,d proved to
be very useful. By this route, 1 was oxidized to 5 by use of the
NaIO4 technique (Figure 2).9 After 10 min, diamine 6 was added.
Separation of the crude reaction mixture led to pyrazine 7 in 25%
yield. For added confirmation that 5 had been prepared, albeit in
transient fashion, the oxidation of 1 was repeated a number of times
and the resulting o-quinone allowed to react with diamine 10
prepared (via 9, Figure 3) from veratrole (8).12 As expected,
compound 11 was formed. Additional combretastatin SAR informa-
tion was derived from the o-quinone experiments when phenazines
7 and 11 were found to inhibit significantly (ED50 ∼0.2 µg/mL)
growth of the P388 lymphocytic leukemia cell line.
In summary, the rather remarkable instability of 5 derived from
1 that can be attributed to its very extended π-electron system may
certainly account for the complex array of metabolic products
produced in vivo as well as the difference in anticancer activity of
4 in comparison to 3.7 Some of those considerations may apply to
rhinacanthone (12), an o-quinone antineoplastic constitutent of the
traditional medicinal (cancer, heptatitis, and others) plant Rhina-
canthus nasutus.13
1
1462, 1244, 1127, 1006, 762 cm-1; H NMR (300 MHz) δ 3.53 (3H,
s, OCH3), 3.55 (9H, s, 3 × OCH3), 6.41 (1H, d, J ) 2.1 Hz, H-2′),
6.53 (1H, d, J ) 8.1 Hz, H-2), 6.67 (2H, brs, CHdCH), 6.79 (1H, d,
J ) 8.1 Hz, H-3), 6.93 (1H, d, J ) 2.1 Hz, H-6′), 7.77 (2H, m, H-10,
H-11), 8.35 (1H, d, J ) 7.5 Hz, H-9), 8.45 (1H, d, J ) 7.5 Hz, H-6);
13C NMR (500 MHz) δ 154.7, 150.9, 150.7, 142.2, 142.0, 141.7, 140.7,
136.4, 129.8, 129.7, 129.5, 129.3, 129.1, 129.0, 127.4, 125.0, 106.7,
104.1, 103.8, 56.4, 56.2, 56.0, 55.8; HRMS (APCI+) m/z 403.1658
[M + H]+ (calcd for C24H23N2O4, 403.1659); anal. C 71.29%, H 5.79%,
calcd for C24H22N2O4, C 71.63%, H 5.51%.
1,2-Dimethoxy-4,5-dinitrobenzene (9). Concentrated HNO3 (10 mL)
and H2O (10 mL) were combined in a 50 mL three-neck round-bottom
flask equipped with an Ar inlet, condenser, and addition funnel. The
reaction mixture was then cooled to 0 °C, and veratrole (8, 4.34 g,
0.032 mol, 4.0 mL) was added dropwise with vigorous stirring over
1 h to produce a yellow slurry, which was then heated to 60 °C to
liberate NO2. Once gas evolution had ceased (4 h), the yellow solution
was poured onto ice/water (100 mL), and the yellow precipitate was
isolated by vacuum filtration. The solid was then washed with NaHCO3
(2 × 200 mL, sat. aq.) and H2O (2 × 200 mL). Crystallization from
ethanol (hot) provided dinitrobenzene 9 (5.33 g, 75%) as yellow needles:
mp 127-129 °C [lit.12a mp 130-131 °C]; IR (neat) νmax 3072, 2989,
1
1590, 1517, 1370, 1328, 1280, 1231, 1047, 876, 786 cm-1; H NMR
(300 MHz) δ 4.02 (6H, s, 2 × OCH3), 7.34 (2H, s, ArH).
1,2-Dimethoxy-4,5-diaminobenzene (10). To a solution of dini-
trobenzene 9 (1.0 g, 4.4 mmol) in EtOAc/EtOH (9:1, 10 mL) at rt was
added Pt2O (0.1 g) in a heavy-walled hydrogenation flask. The flask
was purged (4×) with H2 followed by hydrogenation at 50 psi for 12 h.
The black suspension was then removed by filtration (vacuum) of the
solution phase through a bed of Celite into a hydrochloric acid-saturated
solution of EtOAc/EtOH (9:1, 20 mL). The solvent was removed in
vacuo and the residue crystallized from ethanol to afford the dihydro-
Experimental Section
General Experimental Procedures. Melting points are uncorrected
and were determined with an Electrothermal 9100 melting point
apparatus. The IR spectra were obtained using a ThermoNicolet Avatar
360 Series FT-IR. The H NMR and 13C NMR spectra were recorded
1
1
chloride salt of 10 (0.32 g, 27%) as colorless needles: H NMR (300
on Varian Gemini 300 and Varian Unity 500 instruments using CDCl3
(TMS internal reference) as solvent. High-resolution APCI+ (atmo-
spheric pressure chemical ionization) mass spectra were obtained with
a Jeol JMS-LCmate mass spectrometer. Elemental analyses were
determined by Galbraith Laboratories, Inc. (Knoxville, TN). Ether refers
to diethyl ether and Ar to argon gas. All solvents were redistilled. All
chemicals were purchased from either Sigma-Aldrich Chemical Co.
(Milwaukee, WI) or Acros Organics (Fisher Scientific, Pittsburgh, PA)
and used as received. Reactions were monitored by thin-layer chro-
matography using Analtech silica gel GHLF Uniplates and were
developed with either phosphomolybdic acid (10% w/w solution in
ethanol) or iodine and visualized employing either long- (366 nm) or
short-wave (254 nm) UV irradiation. Solvent extracts of aqueous
solutions were dried over anhydrous magnesium sulfate or sodium
MHz) δ 3.88 (6H, s, 2 × OCH3), 6.96 (2H, s, ArH).
To a solution of diamine 10 dihydrochloride (2.0 g, 8.3 mmol) in
methanol (100 mL) at rt was added K2CO3 (2.29 g, 16.6 mmol). The
mixture changed in color from pink to yellow. The solution was filtered
(under Ar) and evaporated to dryness. The resultant orange solid was
triturated with methanol (3×) to furnish diamine 10 as an orange solid,
which was used immediately without characterization to avoid
decomposition.
1,7,8-Trimethoxy-4-[2-(3,4,5-trimethoxyphenyl)-(Z)-vinyl]phena-
zine (11). Diphenol 1 (0.10 g, 0.30 mmol) in DCM (5 mL) was oxidized
to 5 with NaIO4 (83.8 mg, 0.34 mmol) and tetrabutylammonium
bromide (10 mg, 0.03 mmol) in water as described above. As usual,
after 10 min, the reaction mixture turned blue and FT-IR analysis

Antineoplastic Agents
Journal of Natural Products, 2008, Vol. 71, No. 9 1563
yielded the expected carbonyl stretch at 1739 cm-1. As summarized
above (see 7), the organic phase was concentrated in vacuo, the residue
was immediately dissolved in methanol (10 mL) and stirred at 0 °C,
and diamine 10 (77 mg, 0.46 mmol) was added. Two hours later, the
condensation was terminated by addition of NaHCO3 (10 mL, sat. aq.)
and the mixture was extracted with EtOAc (3 × 30 mL). The combined
organic extract was dried and concentrated in vacuo, and the oily residue
was separated (2×) by silica gel chromatography (gravity; 1:1 EtOAc/
n-hexane) to yield phenazine 11 as crystals from acetone/n-hexane (0.04
g, 29% from 1): mp 141-142 °C; IR (neat) νmax 2928, 2851, 1625,
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1
1589, 1506, 1466, 1241, 1123, 1009, 764 cm-1; H NMR (300 MHz)
δ 3.55 (3H, s, OCH3), 3.57 (9H, s, 3 × OCH3), 3.66 (6H, s, 2 × OCH3),
6.49 (1H, d, J ) 2.1 Hz, H-2′), 6.63 (1H, d, J ) 8.1 Hz, H-2), 6.77
(2H, brs, CHdCH), 6.89 (1H, d, J ) 8.1 Hz, H-3), 6.99 (1H, d, J )
2.1 Hz, H-6′), 7.79 (1H, s, H-9), 7.84 (1H, s, H-6) ppm; 13C NMR
(500 MHz) δ 156.8, 156.7, 145.4, 150.9, 150.7, 140.0, 139.0, 138.9,
138.7, 138.4, 129.5, 129.0, 127.6, 126.0, 106.9, 105.2, 105.0, 104.1,
103.8, 56.7, 56.5, 56.4, 56.2, 56.0, 55.8; HRMS (APCI+) m/z 463.1797
[M + H]+ (calcd for C26H27N2O6, 463.1869); anal. C 67.41%, H 5.79%,
calcd for C26H26N2O6, C 67.52%, H 5.67%.
Acknowledgment. We are pleased to thank for financial assistance
grants R01 CA90441-01-05 and 2R56 CA090441-06A1 awarded by
the Division of Cancer Treatment and Diagnosis, National Cancer
Institute, DHHS; the Arizona Disease Control Research Commission;
the Robert S. Dalton Endowment Fund; Dr. A. D. Keith; the J. W.
Kieckhefer Foundation; the Margaret T. Morris Foundation; and the
Caitlin Robb Foundation. Other very helpful assistance was provided
by Drs. J. C. Knight, J.-C. Chapuis, and M. Hoard and by F.
Craciunescu and M. Dodson.
References and Notes
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(1) Contribution 552 in the series Antineoplastic Agents; for part 551,
see: Pettit, G. R.; Numata, A.; Iwamoto, C.; Usami, Y.; Yamada, T.;
Ohishi, H.; Cragg, G. M. J. Nat. Prod. 2006, 69, 332–337.
(2) Pettit, G. R.; Singh, S. B.; Niven, M. L.; Hamel, E.; Schmidt, J. M. J.
Nat. Prod. 1987, 50, 119–131.
(3) (a) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.;
Garcia-Kendall, D. Experientia 1989, 45, 209–211. (b) Pettit, G. R.;
NP800179G