Antineoplastic agents. 578. synthesis of stilstatins 1 and 2 and their water-soluble prodrugs

Article abstract of DOI:10.1021/np800608c

Efficient syntheses of 3,4-methylenedioxy-4′,5-dimethoxy-2′, 3′-dihydroxy-Z-stilbene (stilstatin 1, 2), 3,4,4′-trimethoxy- 2′,3′,5-trihydroxy-Z-stilbene (stilstatin 2, 5), and respective phosphate prodrugs have been summarized. Both 2 and 5 were accessed via a convergent step synthesis using phosphonium bromides 6 and 21 in Wittig reactions with 2,3- bis(tert-butyldimethylsilyloxy)-4′-methoxybenzaldehyde 14. Deprotection of silyl ethers 15 and 26 with TBAF furnished 2 and 5, respectively. Phosphorylation of 2 and 5 afforded the phosphoric acid intermediates 17 and 28 for prodrug development. These phosphoric acid precursors were employed in parallel series of reactions to produce a selection of metal cation prodrug candidates. The biological activities of stilstatins 1 (2)and2(5) and their respective prodrugs were evaluated against a panel of one murine (P388) and six human cancer cell lines. Compared to combretastatin A-2 (1), stilstatin 1 (2) has an additional vicinal hydroxy group on the B ring, the presence of which was detrimental to the cancer cell line potency; in vivo, however, compound 2 would be predicted to have greater anticancer activity resulting from the o-quinone mechanism of action analogous to that of combretastatin A-1 (4). The substitution of a hydroxy group for a methoxy group on the A ring of combretastatin A-1 (4), resulting in stilstatin 2 (5), gave rise to a modest level of inhibition consistent with that found for 4 against cancer cell lines.

Full text of DOI:10.1021/np800608c

380
J. Nat. Prod. 2009, 72, 380–388
Antineoplastic Agents. 578. Synthesis of Stilstatins 1 and 2 and Their Water-Soluble Prodrugs†
George R. Pettit,* Andrew Thornhill, Noeleen Melody, and John C. Knight
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State UniVersity, P.O. Box 871604,
Tempe, Arizona 85287-1604
ReceiVed September 25, 2008
Efficient syntheses of 3,4-methylenedioxy-4′,5-dimethoxy-2′,3′-dihydroxy-Z-stilbene (stilstatin 1, 2), 3,4,4′-trimethoxy-
2′,3′,5-trihydroxy-Z-stilbene (stilstatin 2, 5), and respective phosphate prodrugs have been summarized. Both 2 and 5
were accessed via a convergent step synthesis using phosphonium bromides 6 and 21 in Wittig reactions with 2,3-
bis(tert-butyldimethylsilyloxy)-4′-methoxybenzaldehyde 14. Deprotection of silyl ethers 15 and 26 with TBAF furnished
2 and 5, respectively. Phosphorylation of 2 and 5 afforded the phosphoric acid intermediates 17 and 28 for prodrug
development. These phosphoric acid precursors were employed in parallel series of reactions to produce a selection of
metal cation prodrug candidates. The biological activities of stilstatins 1 (2) and 2 (5) and their respective prodrugs
were evaluated against a panel of one murine (P388) and six human cancer cell lines. Compared to combretastatin A-2
(1), stilstatin 1 (2) has an additional vicinal hydroxy group on the B ring, the presence of which was detrimental to the
cancer cell line potency; in ViVo, however, compound 2 would be predicted to have greater anticancer activity resulting
from the o-quinone mechanism of action analogous to that of combretastatin A-1 (4). The substitution of a hydroxy
group for a methoxy group on the A ring of combretastatin A-1 (4), resulting in stilstatin 2 (5), gave rise to a modest
level of inhibition consistent with that found for 4 against cancer cell lines.
Some of the natural products we isolated from the African bush
willow, Combretum caffrum (Combretaceae), are characterized by
their remarkable biological activity. Our early research endeavor
with C. caffrum included the isolation, structural elucidation, and
synthesis of combretastatins A-2 (1), A-3, D-1, and D-2, as well
as initial structural modifications.1 Illustrative of the biological
activity are the Z-stilbenes we designated combretastatins A-4 (3,
CA4)2 and A-1 (4, CA1).3 Compounds 3 and 4, along with their
corresponding phosphate prodrug salts (CA4P4 and CA1P,5 re-
spectively), are now of great therapeutic interest, and CA1P and
CA4P are in phase I and III human cancer clinical trials,
respectively.
A comparison of the vicinal diphenol CA1 (4)3 with the
monophenol counterpart CA4 (3)2 revealed a very similar antimi-
totic activity (IC50 2-3 µM) but much lower cancer cell growth
inhibition for CA1 (4) compared to that of CA4 (3, ED50 ∼0.0009
mg/mL, P388 leukemia cell line). Development of both CA1 and
CA4 to the current human cancer clinical trials was accelerated
following syntheses of the phosphate prodrugs and discovery of
their very promising cancer vascular disrupting (VTA) and anti-
angiogenesis effects, as well as aqueous solubility, e.g., for CA4P,
20 mg/mL of H2O. Once administered, the phosphate prodrugs are
converted into the parent drug via nonspecific phosphatases and
then transported intracellularly.4 Both CA1P and CA4P have been
shown to selectively damage tumor neovasculature with induction
of extensive blood flow shutdown in the metastatic tumor compared
to normal tissues.6-8 CA1P (aka Oxi-4503) 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 (formation of an o-quinone intermediate in
ViVo)1,9 of CA1P differs from that of CA4P.
Figure 1. Structures of combretastatins A-2 (1), A-4 (3), and A-1
(4) and stilstatins 1 (2) and 2 (5).
Results and Discussion
In the syntheses of both stilstatin 1 (2) and stilstatin 2 (5), stilbene
formation was based on a Wittig reaction sequence. Synthesis of
the stilstatin 1 A-ring moiety is outlined in Scheme 1. Treatment
of 5-bromovanillin (7) with NaOH furnished benzaldehyde 8.
Subsequent reaction with CH2Br2 gave access to the methylenedioxy
derivative 9. Reduction of 9 with NaBH4 afforded alcohol 10, which
was treated consecutively with PBr3 and triphenylphosphine to
furnish phosphonium bromide 6, the A-ring moiety of 2. The
bromide intermediate 11 was not characterized.
We now report the syntheses of two new and closely related
stilbenes, stilstatin 1 (2) and stilstatin 2 (5), and their phosphate
prodrugs as a next step toward the objective of locating structural
modifications with increased anticancer activity. Compounds 2 and
5 each have an additional phenolic hydroxy group compared to
CA2 (1) and CA1 (4), respectively.
† Dedicated to Dr. David G. I. Kingston of Virginia Polytechnic Institute
and State University for his pioneering work on bioactive natural products.
* To whom correspondence should be addressed. Tel: (480) 965-3351.
Fax: (480) 965-2747. E-mail: bpettit@asu.edu.
The synthesis of the B-ring moiety for both 2 and 5 is outlined
in Scheme 2. Commercially available 2,3,4-trimethoxybenzaldehyde
10.1021/np800608c CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/19/2009

Synthesis of Stilstatins 1 and 2
Journal of Natural Products, 2009, Vol. 72, No. 3 381
Scheme 1. Synthesis of Phosphonium Bromide 6a
Scheme 3. Synthesis of Stilstatin 1 (2) and Its Water-Soluble
Prodrugs 18, 19, and 20a
a Reagents and conditions: (a) NaOH, Cu0, H2O, ∆, 24 h, 97%; (b) CH2Br2,
K2CO3, DMF, 100 °C, 2 h, 88%; (c) NaBH4, EtOH, rt, 3 h, 97%; (d) DCM,
0 °C, PBr3, 8 h to rt, quantitative; (e) toluene, PPh3, ∆, 12 h, 74%.
Scheme 2. Synthesis of Silyl Ether 14a
a Reagents and conditions: (a) (i) n-BuLi, THF, -10 °C to rt, 10 min; (ii)
14, 30 min; (b) THF, 0 °C, TBAF, 2 h, 74%; (c) acetonitrile, CCl4, -10 °C; (d)
DIPEA, DMAP, 1 min, dibenzyl phosphite; (e) DCM, TMSBr, rt, 30 min,
quantitative; (f) 1.0 N KOH in CH3OH, 0 °C, 30 min, 57%.
and the Z/E mix in 50% yield. Further attempts at separation proved
ineffective. Therefore, the isomeric mixture (15) was treated with
TBAF at 0 °C to effect deprotection, yielding stilstatin 1 (Z-2) and
its isomer. However, as was observed with 15, separation of the
isomers proved to be inefficient.
a Reagents and conditions: (a) BCl3, DCM, 26 h, rt, 87%; (b) DMF, DIPEA,
rt, TBDMSCl, 84%.
The key intermediate in the formation of the phosphate prodrugs
was accessed by phosphorylation of 2 as the Z/E mixture, using
dibenzyl phosphite for in situ generation of phosphoryl chloride.
Column chromatography led to better separation of the Z- and
E-isomers and provided Z-16 in 31% yield. As expected, a Z/E
mixture was also obtained (44%), along with the pure E (14%)
product. Because the Z- and E-stilbenes had nearly identical Rf
values by TLC, Z-16 was analyzed by HPLC to establish its purity.
Next, removal of the benzyl groups from Z-16 was carried out
with TMSBr. The intermediate phosphoric acid 17 was not isolated,
although FTIR analysis indicated the presence of the phosphoryl
hydroxy groups. Immediate dissolution in CH3OH and addition of
1.0 N KOH solution in CH3OH gave access to the potassium
(12) was selectively demethylated at the ortho and meta positions
with BCl3 to yield the vicinal phenol 13, which was protected as
silyl ether 14. The preceding reaction sequence was based on our
earlier synthesis of combretastatin A-1.3
Next, phosphonium bromide 6 was treated with n-butyllithium
to generate the phosphonium ylide at -10 °C (Scheme 3). Addition
of aldehyde 14 at room temperature yielded stilbene 15 in a Z/E
ratio of 5:1 on a small scale (less than 1.0 g) and 2:1 on larger
scales. Geometrical isomers were determined by NMR spectros-
copy. Separation of the Z- and E-stilbenes (15) was achieved with
column chromatography, which gave the Z-isomer in 10% yield

382 Journal of Natural Products, 2009, Vol. 72, No. 3
Scheme 4. Synthesis of Phosphonium Bromide 21a
Pettit et al.
a Reagents and conditions: (a) DMF, TBDMSCl, rt, 2 h; (b) NaH, 30 min,
CH3I, rt, 45%; (c) LiAlH4, THF, rt, 89%; (d) DCM, 0 °C, PBr3, 10 min,
quantitative; (e) PPh3, toluene, rt (10 min), ∆ (2 h), rt (24 h), 66%.
prodrug 18. The sodium (19) and lithium (20) prodrugs were
obtained from 18 via ion-exchange chromatography using DOWEX-
50W. The resin was charged with the appropriate 1.0 N metal
hydroxide solution, and the salts were eluted in H2O.
In order to prepare stilstatin 2 (5), phosphonium bromide 21 was
first synthesized (Scheme 4). Selective silyl protection of the C-3
hydroxy group of methyl gallate (22) followed by methylation of
the 4,5-dihydroxy functionality with CH3I furnished 23 as the major
product (45% yield). Reduction of ester 23 with LiAlH4 gave
alcohol 24 in high yield, and reaction with PBr3 gave the
intermediate bromide 25. Initial attempts to prepare the phospho-
nium bromide 21 in acceptable yield proved problematic owing to
cleavage of the silyl ether unit under extended reflux conditions. It
was found that a reflux period of 45 min followed by stirring with
cooling over 24 h gave 21 in 45% yield. With 2 h at reflux and a
24 h period with stirring at room temperature, the yield increased
to 66%. However, a reflux time of greater than 2 h resulted in loss
of the silyl ether group and degradation of 21.
Treatment of 21 with n-butyllithium at -10 °C yielded the ylide,
which reacted with aldehyde 14 at room temperature to give an
isomeric mixture of stilbenes (26, Scheme 5), separation of which
was problematic. Initial attempts via column chromatography with
gradient elutions failed to separate the Z- and E-isomers, and
therefore 26 was subjected to medium-pressure liquid chromatog-
raphy, with 4:5 DCM-hexanes as eluant. This technique led to
Z-26 (14%), a Z/E mixture (67%), and E-26 (3%) in 84% total
yield. Deprotection of each isomer with TBAF at 0 °C furnished 5
and its E-isomer in 76% and 71% yields, respectively. In another
experiment, the stilbene mixture 26 was not separated but was
deprotected as an isomeric mixture to give a mixture of 5 and its
isomer, which was carried forward without separation and treated
with dibenzyl phosphite and CCl4 to give a mixture of tri- (27a,c)
and diphosphorylated (27b,d) products. Chromatographic separation
of this mixture (gradient elution) led to 27a in 26% yield, along
with a mixture of 27a and 27b (11%) and a mixture of 27c and
Figure 2. Phosphorylated products: * denotes interchangeable
assignments.
27d (37%). Since 27a was the necessary precursor for the target
phosphate prodrug, it was apparent that the Z- and E-isomers (using
careful chromatography) could be more conveniently separated at
this stage.
Deprotection of 27a with TMSBr provided the free phosphoric
acid intermediate 28 in quantitative yield, and this was immediately
treated with NaOCH3 to give sodium prodrug 29. Potassium (30)
and lithium (31) prodrugs were similarly prepared using KOH and
LiOH, respectively, and each was further purified on a DOWEX-
50W ion-exchange column. The piperidine (32) and morpholine
prodrugs (33) were obtained from 30 via an ion-exchange procedure.
Treatment of phosphoric acid 28 with RbHCO3 gave rubidium salt
34, and calcium prodrug 35 was obtained using calcium hydroxide.
The remaining divalent metal prodrugs (36-38) were synthesized
via treatment with the corresponding metal acetate.
Biological Evaluation. The cancer cell growth inhibitory
activities of stilstatin 1 (2), stilstatin 2 (5), related stilbenes, and
phosphorylated prodrugs are summarized in Table 1. The additional
vicinal hydroxy group on ring B of 2 proved to be detrimental, as
the cancer cell line activity dropped some 100-fold in comparison
to CA2 (1). Interestingly, the phosphate prodrugs (18-20) of 2
show improved activity (∼10-fold) over that of the parent com-
pound, perhaps by increasing its bioavailability. The activity of
stilstatin 2 (5) showed the presence of a hydroxy group on ring A

Synthesis of Stilstatins 1 and 2
Journal of Natural Products, 2009, Vol. 72, No. 3 383
Scheme 5. Synthesis of Stilstatin 2 (5) and Its Water-Soluble Prodrugs 29-38a
a Reagents and conditions: (a) (i) n-BuLi, THF, -10 °C, 14; (b) THF, TBAF (1.0 M in THF); (c) CCl4, -10 °C, acetonitrile; (d) DIPEA, DMAP, dibenzyl
phosphite; (e) TMSBr, DCM, rt, quantitative; (f) NaOCH3/XOH/RbHCO3, CH3OH, rt, 30 min; (g) Ca(OH)2 or X′(OAc)2, CH3OH, rt, 30 min.
unless otherwise stated. When appropriate, crude products were
separated by flash column chromatography (230-400 mesh ASTM
silica from E. Merck). Ion-exchange column chromatography was
carried out on Dowex ion-exchange resin (Dowex 50 × 4-400, Sigma).
Melting points are uncorrected and were determined with an
electrothermal 9100 apparatus. The infrared spectra were obtained using
a Thermo Nicolet AVATAR 360 system with a SMART MIRacle single
to have a moderate effect, and the derived phosphate prodrugs again
improved the activity, giving a similar overall 10-fold increase. Very
importantly, both stilstatins 1 (2) and 2 (5) can be predicted to
provide impressive anticancer activity in ViVo owing to the
o-quinone mechanism of action established recently for the
analogous vicinal phenol system of combretastatin A-1 prodrug
(CA1P, OXi-4503).9
1
reflection HATR. The H and 13C NMR spectra were recorded with
Varian Gemini 300, Varian Unity 400, or Varian Unity 500 instruments
using a deuterated solvent and were referenced to either TMS or the
solvent. The reported figures are given as ppm. Phosphorus NMR
spectra were referenced against a standard of 85% H3PO4. HRMS data
were recorded with a Jeol LCmate mass spectrometer and Jeol GCmate.
Elemental analyses were determined by Galbraith Laboratories, Inc.,
Knoxville, TN.
Experimental Section
General Experimental Procedures. All solvents and reagents were
obtained from Sigma-Aldrich (Milwaukee,WI) or Acros Organics
(Fischer Scientific, Pittsburgh, PA) and used as purchased unless
otherwise stated. Reactions were monitored by TLC using Analtech
silica gel GHLF uniplates visualized under long-wave and short-wave
UV irradiation, along with staining with phosphomolybdic acid/heat,
I2, or KMnO4. Solvent extracts were dried over anhydrous MgSO4
3,4-Dihydroxy-5-methoxybenzaldehyde (8). Preparation of com-
pound 8 was repeated as originally described10 from 5-bromovanillin

384 Journal of Natural Products, 2009, Vol. 72, No. 3
Pettit et al.
Table 1. Human Cancer Cell Line (GI50 µg/mL) and Murine P388 Lymphocytic Leukemia Cell Line Inhibitory Acitvity (ED50 µg/mL)
of Stilbenes 1-5 and Their Phosphate Prodrugs 15-38
murine
leukemia
P388
pancreas
BXPC-3
breast
MCF-7
CNS
SF-268
lung
NCI-H460
colon
KM2012
prostate
DU-145
thyroid
KAT-4
thyroid
SW1736
compound
3, CA4
CA4P
4, CA1
CA1P
1, CA2
CA2P
15
2
18
19
20
0.0026
0.0029
0.3
0.39
0.23
4.4
>10
0.014
2.1
>10
4.9
5.3
>10
>10
>10
1.8
>0.01
0.0006
0.0004
0.74
3.3
0.043
0.41
>10
0.97
1.4
2.8
2.7
>10
1.5
0.34
0.0008
0.0007
0.17
2.7
0.0054
0.053
>10
>10
0.93
2.5
0.3
>10
0.47
3.8
>10
>10
>10
>10
>10
>10
1.9
0.016
0.0250
>10
2.0
0.17
1.4
0.3
>100
1.7
0.0042
0.045
>10
0.16
0.39
3.4
1.2
>10
1.6
>10
0.63
0.65
2.8
2.4
>10
1.6
9.6
4.7
2.1
>10
1.8
26
5
29
30
31
34
35
36
37
38
0.14
0.12
0.028
0.26
0.14
0.15
0.064
0.59
1.2
0.75
5.8
>10
3.6
>10
5.9
>10
0.3
0.34
0.3
0.76
4.3
0.46
4.5
0.63
3.6
0.30
0.30
0.35
3.3
0.35
3.5
3.1
2.9
6.1
>10
5.2
>10
< 10
>10
0.26
0.22
0.82
3.9
0.37
4.6
0.15
0.36
3.9
0.38
3.5
0.5
>10
>10
>10
1.3
>10
>10
1.7
0.32
0.51
0.42
>10
(7). Additional NMR and IR data are provided here: colorless solid
that crystallized from toluene (97% yield); mp 131-133 °C [lit.10 mp
132-133 °C]; Rf 0.5 (1:1 EtOAc-hexanes); IR (neat) νmax 3305, 1674,
1587, 1434, 1289, 1156, 1045, 678 cm-1; 1H NMR (CDCl3, 300 MHz)
δ 3.96 (3H, s, OCH3), 5.49 (1 H, s, OH), 5.99 (1 H, s, OH), 7.08 (1 H,
d, J ) 1.5 Hz, ArH), 7.14 (1H, d, J ) 1.5 Hz, ArH), 9.78 (1H, s,
CHO); 13C NMR (CDCl3, 100 MHz) δ 56.61, 107.99, 108.15, 130.01,
130.09, 147.65, 148.87, 189.67.
mmol) was added via cannula. After 2 h the second equivalent of BCl3
(200 mL, 200 mmol) was added and the dark reaction mixture was
allowed to stir at rt for 24 h. The resultant mixture was carefully poured
onto ice-cold 10% NaHCO3 solution (1700 mL) and reacidified to pH
1 with concentrated HCl. The CH2Cl2 layer was separated and the
aqueous phase extracted with EtOAc (3 × 500 mL). The combined
organic phases were dried and the solvent was removed in Vacuo.
Purification was achieved with crystallization from 1:1 hexanes-EtOAc,
which furnished the title compound as a colorless solid in 87% (28.9
g) yield: mp 115-116 °C [lit.3 mp 116-117 °C, lit.14 mp 118-119
°C]; Rf 0.22 (1:1 EtOAc -hexanes); IR (neat) νmax 3365, 2944, 1645,
3,4-Methylenedioxy-5-methoxybenzaldehyde (9).11a,b A solution
of 8 (29.0 g, 0.17 mol), CH2Br2 (35.46 g, 0.20 mol, 14.55 mL), and
K2CO3 (28.19 g, 0.204 mol) in anhydrous DMF (350 mL) was gradually
heated to 100 °C and stirred for 2 h. After cooling to rt, H2O (500 mL)
was added, the reaction mixture was extracted with EtOAc (2 × 100
mL) and dried, and the solvent was removed in Vacuo to furnish a
brown oil. The crude product was purified by flash chromatography,
eluting in 7:3 hexanes-EtOAc, to give the title compound in quantita-
tive yield as a white solid that crystallized from 1:1 EtOAc-hexanes
as a colorless solid (26.8 g, 88%): mp 132-133 °C [lit.11c mp 130-131
°C]; Rf 0.36 (7:3 hexanes-EtOAc); IR (neat) νmax 3100, 2844, 1688,
1459, 1321, 1274, 1125, 1039, 970 cm-1; 1H NMR (CDCl3, 300 MHz)
δ 3.96 (3H, s, OCH3), 6.09 (2H, s, O-CH2-O), 7.05 (1H, d, J ) 2.3
Hz, ArH), 7.13 (1H, d, J ) 2.3 Hz, ArH), 9.78 (1H, s, CHO); 13C
NMR (100 MHz CDCl3) δ 56.23, 95.75, 103.63, 110.07, 133.13,
140.96, 147.76, 153.17, 189.73. HRMS (APCI+) m/z 181.0509 [M +
H]+ (calcd for C9H9O4, 181.0508).
3,4-Methylenedioxy-5-methoxybenzyl Alcohol (10). A solution of
9 (3.0 g, 16.67 mmol) in EtOH (40.0 mL) was stirred for 10 min, and
NaBH4 (0.75 g, 20.0 mmol) was added in portions over 30 min.12 After
stirring for 3 h at rt, solid NaHCO3 was added until the effervescing
ceased. The solution was filtered, and the solvent was removed in Vacuo,
which furnished a white foam. The foam was redissolved in EtOAc
and washed with H2O (2 × 50 mL). The organic phase was dried, the
solvent removed in Vacuo, and the colorless solid crystallized from
1:1 hexanes-EtOAc to yield the title compound in 97% (2.9 g) yield:
mp 64-66 °C [lit.11b mp 66-67 °C]; Rf 0.25 (7:3 hexanes-EtOAc);
IR (neat) νmax 3100, 2844, 1688, 1459, 1321, 1274, 1125, 1039, 970
cm-1; 1H NMR (CDCl3, 300 MHz) δ 3.40 (1H, brs, OH), 3.81 (3H, s,
OCH3), 4.44 (2H, s, CH2OH), 5.87 (2H, s, O-CH2-O), 6.44 (1H, s,
ArH), 7.13 (1H, s, ArH); 13C NMR (100 MHz CDCl3) δ 56.44, 65.15,
101.21, 101.36, 106.34, 134.49, 135.52, 143.53, 148.80; GC MS (EI+)
m/z 182.0593 [M]+ (calcd for C9H10O4 182.0579).
1
1507, 1453, 1277, 1104, 1025 cm-1; H NMR (CDCl3, 300 MHz) δ
3.98 (3H, s, OCH3), 5.44 (1H, s, OH-3), 6.62 (1H, d, J ) 9 Hz, ArH),
7.12 (1H, d, J ) 9 Hz, ArH), 9.74 (1H, s, CHO), 11.09 (1H, s, OH-2);
13C NMR (CDCl3, 100 MHz) δ 56.58, 103.86, 116.32, 126.28, 133.25,
149.29, 153.23, 195.38; HRMS (APCI+) m/z 169.0554 [M + H]+
(calcd for C8H9O4, 169.0500).
2,3-Bis(tert-butyldimethylsilyloxy)-4-methoxybenzaldehyde (14).
DIPEA (68.5 g, 0.53 mol, 92.3 mL) was added to a stirred solution of
13 (28.9 g, 0.17 mol) in anhydrous DMF (200 mL) at rt. tert-
Butyldimethylsilyl chloride (57.04 g, 0.37 mol) was added (in portions)
over 5 min, and the reaction mixture was allowed to stir at rt for 3 h.
Water (300 mL) was added to terminate the reaction, and the mixture
was extracted with EtOAc (3 × 300 mL). The organic phase was
washed consecutively with 10% NaHCO3 (200 mL) and H2O (200 mL).
The organic phase was dried and the solvent was removed in Vacuo.
Purification by crystallization from CH3OH furnished 14 in 84% (57.4
g) yield as a colorless solid: mp 75-76 °C [lit.3 mp 74.5-76 °C]; Rf
0.76 (1:1 hexanes-EtOAc); IR (neat) νmax 2932, 2859, 1683, 1585,
1
1455, 1297, 1099 cm-1; H NMR (CDCl3 300 MHz) δ 0.12 (12H, s,
2 × (CH3)2Si)), 0.98 (9H, s, t-Bu), 1.02 (9H, s, t-Bu), 3.82 (3H, s,
OCH3), 6.61 (1H, d, J ) 8.9 Hz, ArH), 7.47 (1H, d, J ) 8.9 Hz, ArH),
10.21 (1H, s, CHO); 13C NMR (CDCl3, 100 MHz) δ -3.92, -3.84,
18.55, 18.74, 26.02, 26.17, 55.17, 105.40, 115.21, 120.75, 121.37,
123.36, 136.80, 150.99, 157.53, 189.26; HRMS (APCI+) m/z 397.2234
[M + H]+ (calcd for C20H37O4Si2, 397.2230).
3,4-Methylenedioxy-4′,5-dimethoxy-2′,3′-bis(tert-butyldimethyl-
silyloxy)-E- and -Z-stilbenes (15). n-Butyllithium (22.17 mmol, 8.87
mL, 2.5 M by titration) was added dropwise to a stirred suspension of
6 (13.19 g, 21.17 mmol) at -10 °C in anhydrous THF (300 mL). After
10 min at rt 14 (7.98 g, 20.16 mmol) was added and stirring continued
at rt. TLC analysis (10% EtOAc-hexanes, stain 2,4-DNPH-PMA) was
used to follow the reaction. After 30 min the reaction was terminated
by the addition of ice water (200 mL). The mixture was extracted with
Et2O (3 × 150 mL). The ethereal phase was rewashed with H2O and
dried, and the solvent was removed (in Vacuo) to furnish crude 2′,3′-
bis(tert-butyldimethylsilyloxy)-E,Z-stilstatin 1 as a yellow oil. Separa-
tion was initially achieved by flash chromatography, eluting with 95:5
3,4-Methylenedioxy-5-methoxybenzyltriphenylphosphonium Bro-
mide (6). Preparation of the bromide 6 was repeated as originally
described:13 colorless solid, 74% yield; mp 241-243 °C [lit.13 mp
241-243 °C].
2,3-Dihydroxy-4-methoxybenzaldehyde (13). A solution of 2,3,4-
trimethoxybenzaldehyde (12, 39.2 g, 200 mmol) in anhydrous CH2Cl2
(1000 mL) was stirred at rt for 10 min, and then BCl3 (200 mL, 200

Synthesis of Stilstatins 1 and 2
Journal of Natural Products, 2009, Vol. 72, No. 3 385
hexanes-EtOAc. Fractions 15-28 were combined and separated again
with a gradient elution of hexane-EtOAc (99:1, 4000 mL; 97:3, 3000
mL; 95:5, 3000 mL). By this means, two main fractions were obtained:
the pure Z-isomer (1.13 g, 10%) as a colorless solid (recrystallized
from DCM-hexanes) and a Z/E mix (5.5 g, 50%) as a colorless solid.
Z-15: mp 63-65 °C; Rf 0.25 (95:5 hexanes-EtOAc); IR (neat) νmax
NMR (CDCl3, 162 MHz) δ -5.06 (d, J ) 3.5 Hz), -4.87 (d, J ) 2.4
Hz); HRMS (APCI+) m/z 837.2247 [M + H]+ (calcd for C45H43O12P2,
837.2230); anal. C 63.34%, H 5.28%, calcd for C45H42O12P2 ·
CH3CH2OCOCH3, C 63.58%, H 5.40%.
Potassium Stilstatin 1 2′,3′-O-Diphosphate (18). TMSBr (152 mg,
0.99 mmol, 130 µL) was added (dropwise) to a solution of Z-16 (208
mg, 0.25 mmol) in anhydrous CH2Cl2 (16 mL), and the mixture stirred
at rt for 30 min. The reaction was terminated by the addition of 10%
Na2S2O7 solution (10 mL) and extracted with n-butanol (3 × 10 mL),
and the solvent was evaporated (in Vacuo) to afford the phosphoric
acid intermediate 17 as a colorless oil. Phosphoric acid 17 was dissolved
in CH3OH (10 mL) and cooled to 0 °C. KOH solution in CH3OH (1.0
N, 5 mL) was added, and the reaction mixture was allowed to stir at 0
°C for 30 min. Et2O (20 mL) was added at 0 °C to allow for
precipitation of the solid salt, which was collected and washed with
further portions of Et2O (3 × 30 mL). After drying under reduced
pressure, the off-white solid was reprecipitated with H2O-acetone. The
resultant phosphate prodrug was then crystallized from EtOH-H2O to
2931, 2857, 1592, 1492, 1442, 1310, 1250, 1101 cm-1 1H NMR
;
(CDCl3, 300 MHz) δ 0.00 (6H, s, (CH3)2Si), 0.06 (6H, s, (CH3)2Si),
0.88 (9H, s, t-Bu), 0.91 (9H, s, t-Bu), 3.60 (3H, s, OCH3), 3.62 (3H, s,
OCH3), 5.80 (2H, s, O-CH2-O), 6.23 (1H, d, J ) 12.3 Hz,
-CHdCH-), 6.27 (1H, d, J ) 8.7 Hz, ArH), 6.40 (1H, s, ArH), 6.44
(1H, d, J ) 12.6 Hz, -CHdCH-), 6.48 (1H, d, J ) 1.2 Hz, ArH),
6.77 (1H, d, J ) 8.7 Hz, ArH); 13C NMR (CDCl3, 100 MHz) δ -3.89,
-3.32, 18.52, 18.72, 26.12, 26.34, 54.90, 56.21, 101.27, 102.81, 104.40,
108.31, 121.83, 123.20, 126.76, 127.76, 132.01, 134.10, 136.86, 143.13,
146.04, 148.45, 151.64; HRMS (APCI+) m/z 545.2754 [M + H]+
(calcd for C29H45O6Si2, 545.2759); anal. C 64.33%, H 8.34%, calcd
for C29H44O6Si2, C 63.93%, H 8.14%.
1
Stilstatin 1 (2). To a solution of Z-15 (0.87 g, 1.60 mmol) in
anhydrous THF (8 mL) was added a solution of TBAF (3.52 mmol,
3.52 mL, 1.0 M solution in THF) at 0 °C. The reaction mixture was
stirred with warming to ambient temperature over 2 h. The reaction
was terminated by addition of ice-cold 6 N HCl (4.82 mL) and extracted
with EtOAc (3 × 50 mL). The organic extract was washed with brine
(2 × 30 mL) and dried, and the solvent was removed (in Vacuo) to
yield a dark brown oil. Separation using flash chromatography and
eluting with 1:1 hexanes-EtOAc furnished 2 as a tan oil in 74% (0.37
g) yield: Rf 0.24 (1:1 hexanes-EtOAc); IR (neat) νmax 3487, 3006,
furnish a colorless solid in 57% (89 mg) yield: mp 152-154 °C; H
NMR (D2O, 300 MHz) δ 3.75 (3H, s, OCH3), 3.68 (3H, s, OCH3),
5.78 (2H, s, O-CH2-O), 6.47 (1H, d, J ) 12.3 Hz, -CHdCH-),
6.48 (2H, s, ArH), 6.50 (1H, d, J ) 12.3 Hz, -CHdCH-), 6.56 (1H,
d, J ) 8.4 Hz, ArH), 6.80 (1H, d, J ) 8.4 Hz, ArH).
General Procedure for the Synthesis of Other Stilstatin 1
Prodrugs by Ion Exchange. The exchange resin Dowex-50W was
washed consecutively with H2O (200 mL), CH3OH (200 mL), H2O
(200 mL), 1 N HCl (until pH 1), H2O (until pH 7), and 1.0 M metal
hydroxide (until pH 14) and rinsed with H2O (until pH 7). The
potassium stilstatin 1 2′,3′-O-phosphate salt (25 mg) was dissolved in
H2O (5 mL) and passed down the resin, eluting with H2O (30 mL).
Fractions were spotted onto TLC plates, and the product-containing
fractions were visualized using both UV light and I2 vapor. The pooled
fractions were reduced to dryness via freeze-drying.
1
2900, 1624, 1508, 1290, 800, 736 cm-1; H NMR (CDCl3,300 MHz)
δ 3.76 (3H, s, OCH3), 3.84 (3H, s, OCH3), 5.45 (2H, brs, 2 × OH),
5.90 (2H, s, O-CH2-O), 6.38 (1H, d, J ) 8.1 Hz), 6.45-6.51 (4H,
m, 2 × -CHdCH-, 2 × ArH), 6.74 (1H, d, J ) 9.0 Hz, ArH); 13C
NMR (CDCl3,100 MHz) δ 56.08, 56.22, 101.31, 102.77, 102.98,
108.41, 117.72, 120.18, 123.65, 130.04, 131.54, 132.57, 134.43, 141.52,
143.21, 146.28, 148.49; HRMS m/z 317.1025 [M + H]+ (calcd for
C17H17O6, 317.1010).
Sodium Stilstatin 1 2′,3′-O-Diphosphate (19). Recrystallization
from H2O-acetone and H2O-EtOH yielded the sodium salt as a
1
colorless solid (14.6 mg) in 58% yield: mp 176-177 °C; H NMR
3,4-Methylenedioxy-4′,5-dimethoxy-2′,3′-O-di[bis(benzyl)phos-
phory]-E- and -Z-stilbenes (16). A mixture of DIPEA (1.67 g, 12.95
mmol, 2.25 mL), DMAP (0.07 g, 0.63 mmol), and dibenzyl phosphite
(2.51 g, 9.57 mmol, 2.12 mL, added dropwise over 5 min) was added
to a cooled (-10 °C) solution prepared in order from acetonitrile (5
mL), E,Z -2 (1.0 g, 3.19 mmol), and CCl4 (4.91 g, 31.9 mmol, 3.08
mL) that had stirred for 10 min. After 3 h at -10 °C the reaction was
treated with KH2PO4 (0.5 M, 50 mL), stirred for a further 10 min, and
extracted with EtOAc (3 × 100 mL). The organic phase was dried and
the solvent removed (in Vacuo) to furnish a yellow oil. The products
were separated by flash chromatography in hexanes-EtOAc (gradient
elution: 3:1, 1000 mL; 7:3, 1000 mL; 1:1, 3000 mL). The result was
three main fractions: pure Z-isomer (0.81 g, 31%) as a yellow oil; a
Z/E mixture (1.16 g, 44%); and pure E-isomer (0.38 g, 14%) as a
colorless solid.
(D2O, 300 MHz) δ 3.57 (3H, s, OCH3), 3.68 (3H, s, OCH3), 5.77 (2H,
s, O-CH2-O), 6.41 (1H, s, ArH), 6.46 (1H, d, J ) 12.3 Hz,
-CHdCH-), 6.48 (1H, s, ArH), 6.50 (1H, d, J ) 12.3 Hz,
-CHdCH-), 6.56 (1H, d, J ) 8.1 Hz, ArH), 6.79 (1H, d, J ) 8.1 Hz,
ArH).
Lithium Stilstatin 1 2′,3′-O-Diphosphate (20). Recrystallization
from H2O-acetone and H2O-EtOH yielded the lithium salt as a
1
colorless solid (16.0 mg) in 70% yield: mp 208-209 °C; H NMR
(D2O, 300 MHz) δ 3.56 (3H, s, OCH3), 3.67 (3H, s, OCH3), 5.76 (2H,
s, O-CH2-O), 6.40 (1H, s, ArH) 6.46 (1H, s, ArH), 6.49 (1H, d, J )
10.4 Hz, -CHdCH-), 6.52 (1H, d, J ) 10.4 Hz, -CHdCH-), 6.57
(1H, d, J ) 7.2 Hz, ArH), 6.78 (1H, d, J ) 8.4 Hz, ArH).
3-tert-Butyldimethylsilyloxy-4,5-dimethoxybenzoate (23). A solu-
tion of anhydrous DMF (1000 mL), t-BDMSCl (72.0 g, 0.47 mol),
and DIPEA (59.45 g, 0.46 mol, 80.0 mL) was stirred at rt for 10 min,
and methyl gallate (22, 80.0 g, 0.43 mol) was added portionwise. After
the mixture had stirred at rt for 2 h, NaH (60%, 56.0 g, 1.4 mol) was
added, followed 30 min later by CI4 (204.4 g, 1.44 mol, 90.0 mL).
After 4 h the reaction was terminated by the addition of H2O (500
mL) and extracted with hexanes (4 × 300 mL), the organic phase was
dried, and the solvent was evaporated in Vacuo. The residue was
separated by flash chromatography with 98:2 hexanes-EtOAc (2000
mL) followed by 95:5 hexanes-EtOAc (4000 mL) as eluant to furnish
the title compound in 45% (63.1 g) yield as a colorless oil: Rf 0.20
(95:5 hexanes-EtOAc); IR (neat) νmax 2953, 2857, 1721, 1584, 1347,
Z-16: Rf 0.25 (1:1 EtOAc-hexanes); IR (neat) νmax 2955, 1609, 1502,
1
1453, 1286, 1212, 1013, 739 cm-1; H NMR (CDCl3, 400 MHz) δ
3.65 (3H, s, OCH3), 3.76 (3H, s, OCH3), 5.06-5.09 (4H, m, 2 ×
CH2Ph), 5.16-5.18 (4H, m, 2 × CH2Ph), 5.87 (2H, s, O-CH2-O),
6.44 (2H, s, ArH), 6.47 (1H, d, J ) 11.6 Hz, -CHdCH-), 6.59 (1H,
d, J ) 11.6 Hz, -CHdCH-), 6.67 (1H, d, J ) 8.7 Hz, ArH), 7.02
(1H, d, J ) 9.0 Hz, ArH), 7.20-7.30 (20H, m, 2 × Ph); 13C NMR
(CDCl3, 100 MHz) δ 56.21, 56.27, 69.65, 69.70, 69.86, 69.91, 101.29,
102.94, 108.52, 109.29, 123.75, 124.30, 126.61, 127.74-128.50,
131.17, 134.38, 135.57, 135.64, 135.84, 135.92, 143.28, 148.46, 151.45;
31P NMR (CDCl3, 162 MHz) δ -4.91 (d, J ) 3.2 Hz), -4.85 (d, J )
2.9 Hz); HRMS (APCI+) m/z 837.2228 [M + H]+ (calcd for
C45H43O12P2, 837.2230).
E-16:mp145-146°C(EtOAc-hexanes);Rf0.20(1:1EtOAc-hexanes);
IR (neat) νmax 2895, 1610, 1507, 1454, 1293, 1041, 737 cm-1; 1H NMR
(CDCl3, 400 MHz) δ 3.80 (3H, s, OCH3), 3.83 (3H, s, OCH3), 5.04
(4H, m, CH2Ph), 5.21 (4H, m, CH2Ph), 5.97 (2H, s, O-CH2-O), 6.59
(1H, d, J ) 1.2 Hz, ArH), 6.66 (1H, d, J ) 1.2 Hz, ArH), 6.84 (1H,
d, J ) 16.4 Hz, -CHdCH-), 6.84 (1H, d, J ) 10.0 Hz, ArH),
7.16-7.31 (11H, m, 2 × Ph, -CHdCH-), 7.41 (1H, d, J ) 8.8 Hz,
ArH); 13C NMR (CDCl3, 100 MHz) δ 56.34, 56.42, 69.75, 69.79, 70.09,
100.14, 101.47, 106.83, 109.82, 121.12, 122.11, 127.79-129.09,
132.47, 135.03, 135.45, 135.52, 135.88, 135.94, 143.56, 149.13; 31P
1
1222, 1115, 834 cm-1; H NMR (CDCl3, 300 MHz) δ 0.15 (6H, s,
(CH3)2Si), 0.96 (9H, s, t-Bu), 3.79 (3H, s, OCH3), 3.83 (3H, s, OCH3),
3.84 (3H, s, OCH3), 7.14 (1H, d, J ) 3.0 Hz, ArH), 7.18 (1H, d, J )
3.0 Hz, ArH); 13C NMR (CDCl3, 100 MHz) δ -4.50, 18.15, 25.82,
52.27, 56.23, 60.55, 106.94, 116.06, 125.28, 144.87, 149.31, 153.61,
166.87; GC MS (CI+) m/z 327.1654 [M + H]+ (calcd for C16H27O5Si,
327.1628).
3-tert-Butyldimethylsilyloxy-4,5-dimethoxybenzyl alcohol (24). A
suspension of LiAlH4 (4.44 g, 0.11 mol) in anhydrous THF (200 mL)
was stirred at rt for 10 min. A solution of 23 (28.5 g, 0.09 mol) in
anhydrous THF (20 mL) was next added dropwise over 30 min. TLC
evaluation indicated the presence of starting material, and therefore a
further portion of LiAlH4 (2.0 g, 0.05 mol) was added and the mixture

386 Journal of Natural Products, 2009, Vol. 72, No. 3
Pettit et al.
stirred at rt for 12 h. The reaction mixture was cooled to 0 °C and
terminated by careful addition of H2O (20 mL) and brine (300 mL).
The solvent and suspension was passed through a bed of Celite and
the filtrate extracted with EtOAc (3 × 100 mL). The organic phase
was dried, and the solvent was removed in Vacuo to furnish the crude
product. Purification was achieved by chromatography, and elution with
3:2 hexanes-EtOAc gave access to the title compound as a clear oil
in 89% (23.9 g) yield: Rf 0.05 (9:1 hexanes-EtOAc); IR (neat) νmax
3427, 2953, 2858, 1586, 1427, 1113 cm-1; 1H NMR (CDCl3, 300 MHz)
δ 0.00 (6H, s, (CH3)2Si), 0.83 (9H, s, t-Bu), 1.5 (1H, s, OH), 3.58 (3H,
s, OCH3), 3.67 (3H, s, OCH3), 4.39 (2H, s, CH2OH), 6.32 (1H, d, J )
3.0 Hz, ArH), 6.43 (1H, d, J ) 3.5 Hz, ArH); 13C NMR (CDCl3, 100
MHz) δ -4.67, 18.28, 25.66, 55.91, 60.34, 65.30, 104.04, 112.59,
136.37, 139.71, 149.43, 153.82; HRMS (APCI+) m/z 281.1581 [M +
H - H2O]+ (calcd for C15H25O3Si, 281.1573).
NMR (CDCl3, 300 MHz) δ 0.02 (6H, s, (CH3)2Si), 0.04 (6H, s,
(CH3)2Si), 0.10 (6H, s, (CH3)2Si), 0.90 (9H, s, t-Bu), 0.93 (9H, s, t-Bu),
0.99 (9H, s, t-Bu), 3.69 (3H, s, OCH3), 3.69 (3H, s, OCH3), 3.77 (3H,
s, OCH3), 6.46 (1H, d, J ) 8.1 Hz, ArH), 6.51 (1H, d, J ) 16.5 Hz,
-CHdCH-), 6.62 (1H, s, ArH), 6.68 (1H, s, ArH), 7.10 (1H, d, J )
8.7 Hz, ArH), 7.17 (1H, d, J ) 16.50 Hz, -CHdCH-); 13C NMR
(CDCl3, 100 MHz) δ -4.67, -3.85, -3.52, 18.30, 18.61, 18.75, 25.70,
26.11, 26.46, 54.96, 55.75, 60.42, 102.64, 105.12, 112.73, 117.38,
123.81, 123.89, 126.06, 133.63, 136.89, 139.60, 145.43, 149.39, 151.72,
153.68; HRMS (APCI+) m/z 661.3771 [M + H]+ (calcd for
C35H61O6Si3, 661.3776); anal. C 63.35%, H 9.77%, calcd for
C35H60O6Si3, C 63.59%, H 9.15%.
Z-Stilstatin 2 (Z-5). To a cooled (0 °C) solution of Z-26 (2.0 g,
3.03 mmol) in anhydrous THF (10 mL) was added TBAF (9.99 mL,
9.99 mmol, 1.0 M solution in THF), and the reaction mixture was stirred
at 0 °C for 90 min. Ice-cold 6 N HCl (13.6 mL) was added, and the
mixture was extracted with EtOAc (4 × 50 m). The organic phase
was dried and the solvent removed (in Vacuo) to afford a brown oil.
Separation via column chromatography with 1:1 acetone-hexanes as
eluant provided the title compound, which crystallized from EtOAc-
hexanes in 76% yield (0.73 g): mp 147-148 °C; Rf 0.46 (49.5:49.5:1
EtOAc-hexanes-HOAc); IR (neat) νmax 3434, 2933, 1625, 1583, 1508,
1464, 1288, 1091, 997, 912, 731 cm-1; 1H NMR (CDCl3, 300 MHz) δ
3.62 (3H, s, OCH3), 3.87 (6H, s, 2 × OCH3), 5.34 (2H, brs, 2 × OH),
5.67 (1H, s, OH), 6.39 (1H, d, J ) 9 Hz, ArH), 6.41 (1H, s, ArH),
6.51 (1H, d, J ) 12.0 Hz, -CHdCH-), 6.54 (1H, s, ArH), 6.58 (1H,
d, J ) 12.6 Hz, -CHdCH-), 6.76 (1H, d, J ) 9 Hz, ArH); 13C NMR
(CDCl3, 100 MHz) δ 55.54, 56.09, 60.92, 102.94, 104.72, 108.71,
117.74, 120.28, 124.17, 130.00, 132.57, 133.01, 134.73, 141.58, 146.32,
148.83, 151.80; HRMS (APCI+) m/z 319.1238 [M + H]+ (calcd for
C17H19O6, 319.1181); anal. C 63.25%, H 5.83%, calcd for C17H18O6 ·1/
2CH3CH2OCOCH3, C 62.97%, H 6.12%.
3-tert-Butyldimethylsilyloxy-4,5-dimethoxybenzyltriphenylphos-
phonium Bromide (21). To a cooled (0 °C) solution of 24 (26.3 g,
88.22 mmol) in anhydrous CH2Cl2 (340 mL) was added dropwise a
solution of PBr3 (11.94 g, 44.11 mmol, 4.14 mL) in anhydrous CH2Cl2
(120 mL). After stirring at 0 °C for 10 min, the reaction was terminated
by careful addition of saturated NaHCO3 (20 mL). The organic phase
was dried and the solvent evaporated (in Vacuo). The bromide
intermediate (25) was dissolved in toluene (370 mL), and a solution of
Ph3P (27.8 g, 105.86 mmol) in toluene (200 mL) was added. The
reaction mixture was stirred at room temperature for 10 min, heated at
reflux for 2 h, and then allowed to cool with stirring over 24 h. The
resultant precipitate was collected via filtration, dried under reduced
pressure, and crystallized from EtOH as a colorless solid in 66% (36.0
g) yield: mp 249-250 °C; Rf 0.59 (9:1 CH2Cl2-CH3OH); IR (neat)
1
νmax 2955, 1585, 1435, 1111, 730 cm-1; H NMR (CDCl3, 300 MHz)
δ 0.00 (6H, s, (CH3)2Si), 0.89 (9H, s, t-Bu), 3.73 (3H, s, OCH3), 5.37
(2H, d, J ) 14.1 Hz, CH2P), 6.11 (1H, t, J ) 2.1 Hz, ArH), 6.79 (1H,
t, J ) 2.1 Hz, ArH), 7.9 (15H, m, 3 × Ph); 13C NMR (CDCl3, 100
MHz) δ -4.97, 18.00, 25.39, 30.53 (d, J ) 47.2 Hz), 56.16, 60.22,
109.98 (d, J ) 6.1 Hz), 115.78 (d, J ) 5.3 Hz), 117.23, 118.08, 122.20
(d, J ) 9.2 Hz), 129.89-134.79, 140.22 (d, J ) 4.5 Hz), 149.15 (d, J
) 3.8 Hz), 153.67 (d, J ) 3.0 Hz); 31P NMR(CDCl3, 162 MHz) δ
23.62; HRMS (APCI+) m/z 543.2487 [M - Br]+ (calcd for
C33H40O3PSi, 543.2484).
2′,3,3′-Tris(tert-butyldimethylsilyloxy)-4,4′,5-trimethoxy-E- and
-Z-stilbenes (26). To a stirred suspension of 21 (37.24 g, 59.76 mmol)
at -10 °C in anhydrous THF (1000 mL) was added (dropwise) n-BuLi
(62.62 mmol, 25.04 mL, 2.5 M by titration). After 10 min, 14 (22.54
g, 56.92 mmol) was added to the red solution at rt. TLC analysis (1:3
EtOAc-hexanes, stain 2,4-DNPH/PMA) was used to follow the
reaction, which was terminated (after 30 min) by the addition of ice
water (1400 mL). The mixture was extracted with Et2O (3 × 500 mL).
The ethereal phase was rewashed with H2O (2 × 250 mL) and dried
and the solvent removed (in Vacuo) to furnish crude 2′,3,3′-tris(tert-
butyldimethylsilyloxy)-E- and -Z-stilstatin 2 as a yellow oil. Separation
was done by flash chromatography using a gradient elution beginning
with hexanes (2 L) and continuing with hexanes-EtOAc (97:3, 3 L;
95:5, 3 L). Fractions 13-19 were combined and further separated using
medium-pressure liquid chromatography [LOBAR size B (310-25)
Si60, 3 × 1-inch columns in series, 1.0 g injections]. By this procedure
three main fractions were obtained: pure Z (5.45 g, 14%) as a colorless
solid recrystallized from CH2Cl2-hexanes; a mixture of Z and E (25.12
g, 67%) as a colorless solid; and pure E (1.16 g, 3%) as a colorless
solid.
E-Stilstatin 2 (E-5). To a cooled (0 °C) solution of E-26 (1.0 g,
1.51 mmol) in anhydrous THF (5 mL) was added TBAF (4.98 mmol,
4.98 mL, 1.0 M solution in THF). After 90 min at 0 °C, ice-cold 6
N HCl (6.77 mL) was added, and the mixture was extracted with
EtOAc (4 × 50 mL). The dried extract was evaporated (in Vacuo)
to a brown oil. Separation of the oil using column chromatography
and elution with 1:1 hexanes-EtOAc gave a solid, which crystallized
from EtOAc-hexanes in 71% (0.34 g) yield: mp 178-180 °C; Rf
0.35 (1:1 EtOAc-hexanes); IR (neat) νmax 3445, 1585, 1511, 1291,
1
1096, 910, 734 cm-1; H NMR (CDCl3, 300 MHz) δ 3.81 (3H, s,
OCH3), 3.82 (3H, s, OCH3), 3.83 (3H, s, OCH3), 5.62 (1H, brs,
OH), 5.79 (1H, s, OH), 5.85 (1H, s, OH), 6.42 (1H, d, J ) 9.0 Hz,
ArH), 6.57 (1H, d, J ) 1.8 Hz, ArH), 6.71 (1H, d, J ) 1.8 Hz,
ArH), 6.94 (1H, d, J ) 16.5 Hz, -CHdCH-), 6.97 (1H, d, J ) 8.7
Hz, ArH), 7.17 (1H, d, J ) 16.5 Hz, -CHdCH-); 13C NMR
(CDCl3, 100 MHz) δ 55.88, 56.16, 61.05, 102.29, 103.04, 106.05,
117.76, 118.34, 122.98, 127.59, 132.25, 134.46, 134.95, 142.08,
146.17, 149.27, 152.36; HRMS (APCI+) m/z 319.1238 [M + H]+
(calcd for C17H19O6, 319.1181).
2′,3,3′-O-Tri[bis(benzyl)phosphoryl]-4,4′,5-trimethoxy-E- and -Z-
stilbenes (27a,c) and 2′,3-O-Di[bis(benzyl)phosphoryl]-3′-hydroxy-
4,4′,5-trimethoxy-E- and -Z-stilbenes (27b,d). A solution prepared
from DIPEA (6.32 g, 48.92 mmol, 8.52 mL), DMAP (0.30 g, 2.40
mmol), and dibenzyl phosphite (9.68 g, 36.92 mmol, 8.15 mL) in
that order was added (dropwise) over 5 min to a cooled (-10 °C)
solution prepared from acetonitrile (13 mL), Z-, E-stilstatin 2 (Z-,
E-5, 2.54 g, 8.00 mmol), and CCl4 (18.46 g, 120.0 mmol, 11.59
mL) in that order and stirred for 10 min. After 3 h at -10 °C the
mixture was treated with KH2PO4 (0.5 M, 100 mL), stirred for a
further 10 min, and extracted with EtOAc (3 × 150 mL). The organic
phase was dried and the solvent evaporated (in Vacuo) to a yellow
oil. The products were separated by flash chromatography using a
gradient elution sequence of 1:1 (4000 mL) to 1:3 (1400 mL) to
1:4 (2000 mL) hexanes-EtOAc, resulting in three main fractions:
a mixture of the di- (27b) and triphosphorylated (27a) Z-isomers
(0.94 g, 11%) as a yellow oil, pure 27a (2.52 g, 26%) as a yellow
oil, and a mixture of the di- (27d) and triphosphorylated (27c)
E-isomers (3.31 g, 37%) as a white solid.
Z-26: mp 135-137 °C; Rf 0.45 (95:5 hexanes-EtOAc); IR (neat)
1
νmax 2931, 2857, 1574, 1497, 1449, 1250, 1103, 837 cm-1; H NMR
(CDCl3, 300 MHz) δ 0.04 (12H, s, 2 × (CH3)2Si), 0.07 (6H, s,
(CH3)2Si), 0.85 (9H, s, t-Bu), 0.88 (9H, s, t-Bu), 0.93 (9H, s, t-Bu),
3.53 (3H, s, OCH3), 3.61 (3H, s, OCH3), 3.65 (3H, s, OCH3), 6.22
(1H, d, J ) 12.3 Hz, -CHdCH-), 6.24 (1H, d, J ) 8.4 Hz, ArH),
6.42 (2H, s, ArH), 6.47 (1H, d, J ) 12.0 Hz, -CHdCH-), 6.80 (1H,
d, J ) 8.7 Hz, ArH); 13C NMR (CDCl3, 100 MHz) δ -4.75, -3.94,
-3.34, 18.18, 18.51, 18.68, 25.63, 26.08, 26.33, 54.86, 55.88, 60.33,
104.33, 105.93, 114.39, 122.01, 123.16, 126.90, 127.44, 132.78, 136.71,
139.24, 146.08, 148.97, 151.55, 153.03; HRMS (APCI+) m/z 661.3771
[M + H]+ (calcd for C35H61O6Si3, 661.3776); anal. C 62.68%, H 9.69%,
calcd for C35H60O6Si3 ·1/2H2O, C 62.78%, H 9.11%.
2′,3,3′-O-Tri[bis(benzyl)phosphoryl]-4,4′,5-trimethoxy-Z-stil-
bene (27a): Rf 0.18 (7:3 EtOAc-hexanes); IR (neat) νmax 3050, 2923,
E-26: mp 147-149 °C; Rf 0.44 (95:5 hexanes-EtOAc); IR (neat)
2852, 1502, 1453, 1281, 1100, 999, 735 cm-1 1H NMR (CDCl3,
;
1
νmax 2931, 2857, 1574, 1497, 1449, 1331, 1250, 1103, 837 cm-1; H
300 MHz) δ 3.50 (3H, s, OCH3), 3.72 (3H, s, OCH3), 3.81 (3H, s,

Synthesis of Stilstatins 1 and 2
Journal of Natural Products, 2009, Vol. 72, No. 3 387
OCH3), 5.06-5.19 (12H, m, CH2Ph), 6.42 (1H, d, J ) 11.4 Hz,
-CHdCH-), 6.64 (3H, d, J ) 9.9 Hz, 2 × ArH, -CHdCH-),
6.76 (1H, s, ArH), 6.98 (1H, d, J ) 8.7 Hz, ArH); 13C NMR (CDCl3,
100 MHz) δ 55.91, 56.36, 61.08, 69.69, 69.73, 69.77, 69.82, 69.91,
69.96, 109.36, 109.83, 114.57, 124.17, 124.93, 126.87, 130.56,
132.11, 135.54-135.87, 139.52, 143.80, 149.32, 151.57, 152.37,
153.15; 31P NMR (CDCl3, 162 MHz) δ -5.68, -5.04 (d, J ) 3.7
Hz), -4.76 (d, J ) 2.4 Hz); HRMS (APCI+) m/z 1099.2961 [M +
H]+ (calcd for C59H58O15P3, 1099.2988).
tation from H2O-acetone, the prodrug was obtained as a tan solid
1
(25 mg, 75%): mp 145-147 °C; H NMR (D2O, 300 MHz) δ 1.51
(12H, m, -CH2CH2CH2-), 1.62 (24H, m, CH2CH2CH2), 3.00 (24H,
t, J ) 5.5 Hz, -CH2N-), 3.67 (6H, s, 2 × OCH3), 3.77 (3H, s,
OCH3), 6.46 (1H, d, J ) 11.8 Hz, -CHdCH-), 6.57 (1H, d, J )
9.0 Hz, ArH), 6.57 (1H, s, ArH), 6.60 (1H, d, J ) 11.8 Hz,
-CHdCH-), 6.81 (1H, d, J ) 8.5 Hz, ArH), 6.83 (1H, s, ArH).
Morpholine Stilstatin 2 2′,3,3′-O-Triphosphate Prodrug (33). The
morpholine prodrug was obtained from the potassium prodrug (30
mg) via ion exchange on morpholine-loaded resin. After eluting in
H2O (30 mL), the prodrug was freeze-dried and precipitated from
H2O-acetone as an off-white solid (25 mg, 73%): mp 148-149
°C; 1H NMR (D2O, 300 MHz) δ 3.14 (24H, t, J ) 4.8 Hz, 6 ×
(CH2)2) 3.65 (3H, s, OCH3), 3.66 (3H, s, OCH3), 3.67 (3H, s, OCH3),
3.79 (24H, t, J ) 5.2 Hz, 6 × (CH2)2), 6.45 (1H, d, J ) 12.4 Hz,
-CHdCH-), 6.55 (1H, d, J ) 8.8 Hz, ArH), 6.64 (1H, d, J ) 12.0
Hz, -CHdCH-), 6.76 (1H, brs, ArH), 6.85 (1H, d, J ) 9.2 Hz,
ArH) 6.90 (1H, brs, ArH).
Rubidium Stilstatin 2 2′,3,3′-O-Triphosphate Prodrug (34). The
rubidium prodrug was precipitated from H2O-acetone, collected,
and dried under vacuum. The procedure was repeated three times.
The solid was reprecipitated from H2O-EtOH as a tan solid (195
mg, 83%): mp 188-190 °C; 1H NMR (D2O, 300 MHz) δ 3.42 (3H,
s, OCH3), 3.63 (3H, s, OCH3), 3.68 (3H, s, OCH3), 6.40 (1H, d, J
) 12.4 Hz, -CHdCH-), 6.46 (1H, d, J ) 8.8 Hz, ArH), 6.61 (1H,
s ArH), 6.75 (1H, d, J ) 12.4 Hz, -CHdCH-), 6.86 (1H, d, J )
8.8 Hz, ArH), 6.93 (1H, s, ArH).
2′,3,3′-O-Tri[bis(benzyl)phosphoryl]-4,4′,5-trimethoxy-E-stil-
bene (27c): Rf 0.13 (7:3 EtOAc-hexanes); IR (neat) νmax 3033, 2839,
1578, 1504, 1453, 1280, 1100, 999, 731 cm-1 1H NMR (CDCl3,
;
300 MHz) δ 3.70 (3H, s, OCH3), 3.80 (3H, s, OCH3), 3.85 (3H, s,
OCH3), 5.00-5.10 (4H, m, CH2Ph), 5.15-5.21 (8H, m, CH2Ph),
6.78 (1H, d, J ) 16.0 Hz, -CHdCH-), 6.84 (1H, d, J ) 9.5 Hz,
ArH), 6.84 (1H, s, ArH), 6.92 (1H, s, ArH), 7.13-7.35 (31H, m, 6
× Ph, 1 × ArH), 7.40 (1H, d, J ) 9.0 Hz, ArH); 13C NMR (CDCl3,
100 MHz) δ 55.85, 56.28, 61.12, 69.72, 69.76, 69.86, 69.91, 70.05,
70.09, 106.11, 109.80, 112.83, 112.85, 122.18, 122.40, 124.08,
127.74, 127.85, 127.96, 128.24, 128.31, 128.36, 128.47, 132.92,
133.18, 135.36, 135.41, 135.50, 135.56, 135.79, 135.85, 139.92,
139.97, 143.96, 144.02, 151.70, 153.72; 31P NMR (CDCl3, 162 MHz)
δ -5.63, -5.12 (d, J ) 2.7 Hz), -4.91 (d, J ) 2.7 Hz); HRMS
APCI+ m/z 1099.2961 [M + H]+ (calcd for C59H58O15P3, 1099.2988).
Sodium Stilstatin 2 2′,3,3′-O-Triphosphate Prodrug (29). To a
solution of 27a (300 mg, 0.27 mmol) in anhydrous CH2Cl2 (15 mL)
was added TMSBr (267 mg, 1.74 mmol, 230 µL), and the reaction
mixture stirred at rt for 30 min. After addition of H2O (10 mL), the
phases were separated and the aqueous layer extracted with EtOAc
(3 × 20 mL). The combined organic extracts were dried and
concentrated (in Vacuo) to provide the phosphoric acid intermediate
(28). The resulting oil was dissolved in EtOH (10 mL), and NaOMe
(94.0 mg, 1.74 mmol) was added. An immediate precipitate formed
and after 30 min was collected and washed with EtOAc (3 × 20
mL) and hexanes (3 × 20 mL). After drying under reduced pressure
the off-white solid was reprecipitated with H2O-acetone. The
resultant solid crystallized from EtOH-H2O as an off-white solid
Calcium Stilstatin 2 2′,3,3′-O-Triphosphate Prodrug (35). The
solid that separated over 30 min was collected and washed
successively with hexanes (3 × 30 mL) and Et2O (3 × 30 mL).
Reprecipitation from H2O-acetone led to an off-white solid (55
mg, 36%): mp 201-203 °C; IR (solid) νmax 3630, 3362, 3005, 1680,
1615, 1405, 1107, 873, 621 cm-1
.
Zinc Stilstatin 2 2′,3,3′-O-Triphosphate Prodrug (36). The mixture
was stirred for 30 min, and the resulting solution was evaporated to
dryness. The solid residue was washed with hexanes (3 × 30 mL),
Et2O (3 × 30 mL), and hexanes (3 × 30 mL) to afford a tan solid
(134 mg) in 72% yield: mp 185-187 °C; IR (solid) νmax 3594, 2955,
1
(134 mg, 71% yield): mp 185-187 °C; H NMR (D2O, 300 MHz)
1541, 1455, 1263, 1105, 1024 cm-1
.
δ 3.44 (3H, s, OCH3), 3.64 (3H, s, OCH3), 3.68 (3H, s, OCH3),
6.51 (1H, d, J ) 11.9 Hz, -CHdCH-), 6.58 (1H, d, J ) 10 Hz,
ArH), 6.62 (1H, d, J ) 11.9 Hz, -CHdCH-), 6.62 (1H, s, ArH),
6.81 (1H, s, ArH), 6.84 (1H, d, J ) 10 Hz, ArH).
Magnesium Stilstatin 2 2′,3,3′-O-Triphosphate Prodrug (37).
After the mixture was stirred for 30 min, the solvent was evaporated
to dryness. The resulting solid was washed with hexanes (3 × 30
mL), Et2O (3 × 30 mL), and hexanes (3 × 30 mL). This method
gave an off-white solid (125 mg) in 92% yield: mp 157-158 °C;
General Procedure for the Synthesis of Stilstatin 2 Phosphate
Prodrugs. Each of the metal cation-containing salts was obtained
by the procedure outlined above for the preparation of the sodium
salt. The metal counterions were introduced by treatment of the
phosphoric acid intermediate using the corresponding hydroxide (Li,
K, and Ca), hydrogen carbonate (Rb), or acetate (Zn, Mg, and Mn).
The alkali metal salts were further purified by ion-exchange
chromatography as follows. Dowex-50W was treated in succession
with H2O (200 mL), CH3OH (200 mL), H2O (200 mL), 1 N HCl
(until pH 1), H2O (until pH 7), and 1.0 M metal hydroxide (until
pH 14) and rinsed with H2O (until pH 7). The phosphate prodrug
(25 mg) was dissolved in H2O (5 mL) and passed down the resin,
eluting in H2O (30 mL). Fractions were spotted onto TLC plates,
and the product-containing fractions were visualized using both UV
light and I2 vapor. The pooled fractions were reduced to dryness
via freeze-drying.
IR (solid) νmax 3447, 3326, 2961, 1543, 1418, 1262, 1102, 806 cm-1
.
Manganese Stilstatin 2 2′,3,3′-O-Triphosphate Prodrug (38). The
manganese prodrug was prepared as summarized above for the
magnesium salt and provided an off-white solid (100 mg) in 72%
yield: mp 156-157 °C; IR (solid) νmax 3474, 2960, 2921, 1550,
1395, 1262, 1101, 1022, 803 cm-1
.
Cancer Cell Line Bioassay Evaluations. Cancer cell growth
inhibitory data were obtained using the National Cancer Institute’s
standard sulforhodamine B assay as described earlier.15,16
Acknowledgment. We are pleased to acknowledge the very
necessary financial support provided by grants RO1 CA 90441-01-
05, 2R56-CA 09441-06A1, and 5RO1 CA 090441-07 from the
Division of Cancer Treatment and Diagnosis, NCI, DHHS; The
Arizona Disease Control Research Commission (presently, ABRC);
Dr. Alec D. Keith; J. W. Kieckhefer Foundation; Margaret T. Morris
Foundation; the Robert B. Dalton Endowment Fund; Dr. William
Crisp; and Anita Crisp. For other helpful assistance we thank Drs.
J.-C. Chapuis, F. Hogan, J. C. Knight, M. Hoard, D. Doubek, and
C. Herald as well as M. Dodson.
Potassium Stilstatin 2 2′,3,3′-O-Triphosphate Prodrug (30).
Precipitation from H2O-acetone followed by H2O-EtOH provided
the potassium salt as a colorless solid (68 mg, 74%): mp 193-194
1
°C; H NMR (D2O, 300 MHz) δ 3.41 (3H, s, OCH3), 3.67 (6H, s,
2 × OCH3), 6.48 (1H, d, J ) 12.8 Hz, -CHdCH-), 6.55 (1H, s,
ArH), 6.58 (1H, d, J ) 7.5 Hz, ArH), 6.83 (1H, d, J ) 7.5 Hz,
ArH), 6.83 (1H, d, J ) 12.8 Hz, -CHdCH-), 7.01 (1H, s, ArH).
Lithium Stilstatin 2 2′,3,3′-O-Triphosphate Prodrug (31). Pre-
cipitation (2×) from H2O-acetone and H2O-EtOH provided the
lithium salt as a yellow solid (63 mg, 39%): mp 174-176 °C; IR
(solid) νmax 3712, 3058, 2949, 2850, 1752, 1576, 1437, 1342, 1108,
References and Notes
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(2) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-
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(4) Pettit, G. R.; Rhodes, M. Anti-Cancer Drug Des. 1998, 13, 183–191.
609 cm-1
.
Piperidine Stilstatin 2 2′,3,3′-O-Triphosphate Prodrug (32). The
piperidine prodrug was obtained via ion exchange from the potassium
prodrug (32.9 mg), the resin being loaded with piperidine. After
eluting in H2O (30 mL), followed by freeze-drying and reprecipi-

388 Journal of Natural Products, 2009, Vol. 72, No. 3
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NP800608C