Supplemental Text File 1. Chemistry Experimental Procedures
All chemicals and solvents were obtained from Sigma-Aldrich (Milwaukee, WI) of
Fisher Scientific (Pittsburg, PA) and used without further purification. Analytical HPLC
was performed on a Varian Prostar system, with a Varian Microsorb-MW C18 column
(250 x 4.6 mm; 5 ) using the following solvent system A= H2O/0.l % TFA and
B=acetonitrile/0.1 % TFA. Varian Prepstar preparative system equipped with a Prep
Microsorb-MW C18 column (250 x 41.4 mm; 6 ; 60 Å) was used for preparative HPLC
with the same solvent systems. Program A: Gradient: 0-5 min. 30 % B. 5-35min. 95 %
B. 30-35 min. 95% B. Program B: Gradient: 0-5 min. 10 % B. 5-35min. 95% B. 30-40
min. 95% B. Mass spectra (ionspray, a variation of electrospray, unless otherwise noted)
were acquired on an Applied Biosystems Q-trap 2000 LC-MS-MS. UV was measured on
a Perkin Elmer Lambda 25 UV/Vis spectrometer. IR was measured on a Perkin Elmer
Spectra One FT-IR spectrometer.
1
H-NMR and
13
C-NMR spectra were recorded on a
Brucker Biospin spectrometer with a B-ACS 60 autosampler: 600.13 MHz for 1H-NMR
and 150.92 MHz for
13
C-NMR.
Chemical shifts (δ) are determined relative to d4-
methanol (referenced to 3.34 ppm (δ) for 1H-NMR and 49.86 ppm for
l3
C-NMR).
Proton-proton coupling constants (J) are given in Hertz and spectral splitting patterns are
designated as singlet (s), doublet (d), triplet (t), quadruplet (q), multiplet or overlapped
(m), and broad (br). Flash chromatography was performed using Merck silica gel 60
(mesh size 230-400 ASTM) or using an Isco (Lincoln, NE) combiFlash Companion or
SQ16x flash chromatography system with RediSep columns (normal phase silica gel
(mesh size 230-400ASTM)) and Fisher Optima TM grade solvents.
Thin-layer
chromatography (TLC) was performed on Merck (Darmstadt, Germany) silica gel F-254
aluminum-backed plates with visualization under UV (254nm) and by staining with
potassium permanganate or ceric ammonium molybdate.
Chemical synthesis of ON012380.
ON012380 was synthesized following the general procedure described in patent WO
03/0720621 (supplement figure 1).
Synthesis of 4-Methoxv-3-Nitro benzylbromide(1) :
A solution of 4-methyl-2-nitroanisole (10g, 60 mmol), N-bromosuccinimide (10.6 g, 60
mmol) and benzoyl peroxide (14.5 g, 60 mmol) in carbontetrachloride (240 mL) was
heated at reflux for 18h. The reaction mixture was then poured into water and solid
separated was filtered. The aqueous layer was extracted with carbontetrachloride (3 x 100
mL) and the organic phase was separated and evaporated to give a solid product. The
solid products were combined and recrystallized from ethyl acetate-hexane to give a
crystalline product of 3-nitro-4-methoxy benzyl bromide. m.p.110-112 oC, yield 70-75%.
1
H NMR (DMSO-d6)  8.02 (1H, d, J = 1.8 Hz), 7.77 (1H, dd, J = 9, 1.8 Hz), 7.38 (1H,
d, J = 8.4 Hz), 4.76 (2H, s), 3.95 (3H, s).
13
C NMR (DMSO-d6)  151.81, 138.81,
135.41, 130.53, 125.57, 114.70, 56.91, 32.65.
Synthesis of 4-Methoxv-3-Nitrobenzylthioacetic acid (2):
To a cold solution of sodium hydroxide (2.7 g, 81 mmol) in methanol (80 mL),
thioglycollic
acid
(3
g,
40
mmol)
was
added
slowly over
30
minutes.
Sodium thioglycollate precipitated was dissolved by stirring and warming up the solution.
The
solution
was
cooled
to
room
temperature
and
4-methoxy-3-
nitrobenzyl chloride (8 g, 40 mmol) was added in portions to reduce the intensity of
exothermic reaction. The reaction mixture was then refluxed for 4 hours, cooled and
poured onto crushed ice (1 Kg) containing hydrochloric acid (11 mL). The precipitate
formed was filtered, washed with ice cold water and dried under vacuum. (8 g, 95 %
yield) m.p.130-132 oC. . MS 275.2 (M+NH4).
Synthesis of 4-Methoxy-3-Nitrobenzylsulfonylacetic acid (3):
4-methoxy-benzylthioacetic acid (8 g, 31 mmol) was dissolved in glacial acetic acid (65
mL) and 30 % hydrogen peroxide (16 mL) was added in one portion and the mixture was
stirred at room temperature for 10 hours. The contents of the flask were cooled and
poured on to the crushed ice (400 g). The yellow precipitate formed, filtered, washed
with cold water and dried (55 % yield). Recrystallization from hot water yielded crystals
4-methoxy-3-nitrobenzylsulfonyl acetic acid. m. p. 96-98 oC. MS 307.2 (M+NH4).
1
H NMR (DMSO-d6)  7.93 (1H, d, J = 2.0 Hz), 7.69 (1H, dd, J = 8.4, 1.8 Hz), 7.43
(1H, d, J = 7.8 Hz), 4.69 (2H, s), 4.23 (2H,s), 3.90 (2H,s), 3.93 (2H, s), 3.84 (3H, s), 13C
NMR (DMSO-d6)  164.45, 152.39, 138.78, 137.29, 127.48, 120.11, 114.60, 57.28,
56.57, 55.52.
Synthesis of (E)-2,4,6-Trimethoxvstyrvl-4-Methoxy-3-Nitrobenzylsulfone (4):
A solution of 4-methoxy-3-nitrobenzylsulfonylacetic acid (7g, 24.2 mmol) in 50 mL of
glacial acetic acid was treated with 2,4,6-trimethoxybenzaldehyde (4.84 g, 24.2 mmol) in
the presence of catalytic amounts of benzylamine (1 mL). The reaction mixture was
refluxed for 6 hours and acetic acid was removed under vacuum. The gummy material
obtained was treated with 2-propanol to yield a solid product which was recrystallized
from a mixture of acetic acid and 2propanol. Yield 28%, m. p.186-187 oC. MS 424.2
(M+H).
1
H NMR (DMSO-d6)  7.89(1H, d, J = 2.0 Hz), 7.65 (1H, dd, J = 8.4, 1.8 Hz), 7.52
(1H,d, J = 15Hz), 7.52 (1H,d, J = 15 Hz), 7.38(1H, d, J = 8.4Hz), 7.10(1H, d, J = 15
Hz), 6.29(s,1H), 4.60 (2H, s), 3.93 (3H,s), 3.85 (9H, s), 13C NMR (DMSO-d6)  163.80,
160.96, 152.04, 138.56, 137.13, 134.01, 127.32, 122.66, 121.82, 114.25, 102.37, 90.88,
58.27, 56.72, 56.01, 55.60.
Synthesis of (E)-2, 46-Trimethoxvstvrvl-4-Methoxy-3-Nitrobenzylsulfone (5):
A solution of (E)-2, 4, 6-trimethoxystyryl-4-methoxy-3-nitrobenzylsulfone (4) (2 g, 4.72
mmol) (4) in acetone-water 60 ml (10:5) was heated to 50oC. After 30 min, sodium
hydrosulfite (Na2S204) (19.3g, 95 mmol) was added slowly, and the mixture was heated at
reflux (50 C,1 h. ), cooled to room temperature and water was added. The product was
rinsed withNaHC03, and then isolated by extraction with ethyl acetate. The organic layer
was
dried
over
anhydrous
Na2SO4.
The
solvent
was
removed under reduced pressure and the crude product was recrystallized from 2propanol, m. p.148-150 oC. . MS 394.3 (M+H).
1
H NMR (DMSO-d6)  7.58 ( 1H, d, J = 15 Hz), 7.08 (1H, J = 15 Hz), 6.75 (1H,d, J =
8Hz), 6.63 (1H, d, J = 2Hz), 6.50 (1H,dd, J =7.8,2 Hz), 6.29(s,1H), 4.84 (2H, br), 4.21
(s, 2H), 3.84 (9H,s), 3.82 (3H, s).
Synthesis
of
(E)-2,
4,
6-Trimethoxystyryl-3-amino-substituted-4-
methoxvbenzyrlsulfone derivatives (6):
To a stirred solution of methyl bromoacetate (0.7 ml, 6.35 mmol) and sodium acetate
(0.53 g, 6.35 mmol) in methanol (20 mL) was added 2,4, 6-trimethoxy-3-amino-4methoxy- benzysulfone (5) (0.5 g, 1.27 mmol) and stirring was continued under reflux
temperature for 12- 15 h. The contents of the flask were cooled and poured onto the ice.
The resultant ester obtained (85 % yield) was filtered (m. p. 182-185°C). MS 480.3
(M+H).
Crystal structure was obtained for the compound 6. The compound is racemic around
C20A. (Fig. 2).
1
H NMR (DMSO-d6)  7.55(1H, d, J = 15 Hz), 7.09 (1H, dd=, J = 15 Hz), 6.80 (1H,d, J
= 8.4Hz), 6.60 (1H,dd, J =8.4, 1.8 Hz), 6.44(1H, d, J = 1.8 Hz), 6.29(2H, s), 4.98 (1H,
d, J =7.4 Hz), 4.27 (2H, s), 4.02 (1H, br), 3.85 (9H, s), 3.84 (3H,s), 3.61 (3H, s), 1.35
(3H, d, J = 7.0 Hz).
13
C NMR (DMSO-d6)  174.28, 163.57, 160.89, 146.44, 135.92,
132.73, 125.58, 121.31, 119.67, 112.38, 109.58, 102.53, 90.87, 60.26, 55.99, 55.59,
55.39, 51.87, 50.66, 18.1.
Synthesis of ON012380 2:
A solution of ester (6) (100 mg, 0.2 mmol) in 10 ml DCM was added NaOSiMe3 (34 mg,
0.3 mmol) and the mixture was stirred at r.t. overnight. Remove of the solvent and added
1N HCl aqueous solution and the mixture was extracted with DCM. The organic layer
was separated, washed with water and dried over anhydrous sodium sulfate. Volatiles
were removed under vacuum and the product. MS 466.3 (M+H). 1H NMR (DMSO-d6) 
7.55 (1H, d, J = 15 Hz), 7.11 (1H, d, J = 15 Hz), 6.80 (1H, d, J = 7.8 Hz), 6.59 (1H, dd,
J = 7.8, 1.8 Hz), 6.48 (1H, d, J = 1.8 Hz), 6.29 (2H, s), 4.27 (2H,s), 3.90 (1H,q, J = 7.2
Hz), 3.85 (6H, s), 3.84 (3H, s), 3.79 (3H, s), 1.31 (3H, d, J = 7.2 Hz).
C NMR 
13
175.19, 172.05, 163.6, 160.9, 146.4, 135.99, 132.76, 123.64, 121.38, 119.41, 112.33,
109.57, 102.58, 90.93, 60.33, 56.04,55.63,55.45, 50.47, 21.07, 18.29.
Reference:
1.
PREMKUMAR, R.; RAMANA, R.; STANLEY, B. AMINO-SUBSTITUTED
(E)-2. 6-DIALKOXYSTYRYL 4-SUBSTITUTEDBENZYLSULFONES FOR TREATING PROLIFERATIVE DISORDERS
WO03072062 2003-09-04 2003.
2.
Laganis, E. D.; Chenard, B. L., Metal Silanolates: Organic Soluble Equivalents
for O-2. Tetrahedron Letters 1984, 25, (51), 5831-5834.
Experimental
Data Collection
A colorless plate crystal of C23H29NO8S having approximate dimensions of 0.44
x 0.25 x 0.10 mm was mounted on a Mylar loop. All measurements were made on a
Rigaku RAXIS SPIDER imaging plate area detector with graphite monochromated CuK radiation.
Indexing was performed from 4 oscillations that were exposed for 60 seconds.
The crystal-to-detector distance was 127.40 mm.
Cell constants and an orientation matrix for data collection corresponded to a
primitive triclinic cell with dimensions:
a =
b =
c =
V =
7.5873(8) Å
9.5354(10) Å
17.614(2) Å
1155.5(2) Å3
 = 97.696(8)o
 = 96.712(8)o
 = 111.541(7)o
For Z = 2 and F.W. = 479.54, the calculated density is 1.378 g/cm3. Based on a statistical
analysis of intensity distribution, and the successful solution and refinement of the
structure, the space group was determined to be:
P-1 (#2)
The data were collected at a temperature of -100 + 1oC to a maximum 2 value of
136.4o. A total of 136 oscillation images were collected. A sweep of data was done using
 scans from 20.0 to 200.0o in 5.0o step, at =0.0o and  = 0.0o. The exposure rate was
36.0 [sec./o]. A second sweep was performed using  scans from 30.0 to 120.0o in 5.0o
step, at =54.0o and  = 0.0o. The exposure rate was 36.0 [sec./o]. Another sweep was
performed using  scans from 30.0 to 100.0o in 5.0o step, at =54.0o and  = 90.0o. The
exposure rate was 36.0 [sec./o]. Another sweep was performed using  scans from 20.0
to 180.0o in 5.0o step, at =54.0o and  = 180.0o. The exposure rate was 36.0 [sec./o].
Another sweep was performed using  scans from 20.0 to 200.0o in 5.0o step, at =54.0o
and  = 270.0o. The exposure rate was 36.0 [sec./o]. The crystal-to-detector distance was
127.40 mm. Readout was performed in the 0.100 mm pixel mode.
Data Reduction
Of the 5103 reflections that were collected, 921 were unique (Rint = 0.027);
equivalent reflections were merged.
The linear absorption coefficient, , for Cu-K radiation is 16.740 cm-1. An
empirical absorption correction was applied which resulted in transmission factors
ranging from 0.682 to 0.846. The data were corrected for Lorentz and polarization
effects.
Structure Solution and Refinement
The structure was solved by direct methods1 and expanded using Fourier
techniques2. Most non-hydrogen atoms were refined anisotropically; one pair of
disordered carbon atoms was refined isotropically. Hydrogen atoms were used in
calculated positions, except for the amine H atom, which was refined. The final cycle of
full-matrix least-squares refinement3 on F2 was based on 3857 observed reflections and
306 variable parameters and converged (largest parameter shift was 0.01 times its esd)
with unweighted and weighted agreement factors of:
R1 =  ||Fo| - |Fc|| /  |Fo| = 0.0367
wR2 = [  ( w (Fo2 - Fc2)2 )/  w(Fo2)2]1/2 = 0.0962
The standard deviation of an observation of unit weight4 was 1.13. Unit weights
were used. The maximum and minimum peaks on the final difference Fourier map
corresponded to 0.27 and -0.32 e-/Å3, respectively.
Neutral atom scattering factors were taken from Cromer and Waber5. Anomalous
dispersion effects were included in Fcalc6; the values for f' and f" were those of
Creagh and McAuley7. The values for the mass attenuation coefficients are those of
Creagh and Hubbell8. All calculations were performed using the CrystalStructure9,10
crystallographic software package except for refinement, which was performed using
SHELXL-9711.
The structure is racemic; both enantiomers exist in the unit cell, related by the
inversion center in space group #2 (P-1). In addition, there is a slight disorder at each site
such that the enantiomers are disordered. In the final model, atoms C20 and C21 are
disordered 85.8% in the major orientation (and therefore 14.2% in the minor orientation).
The existence of the racemic mixture was verified by solving the structure in the chiral
space group P1 (#1). In space group P1, there are two independent molecules in the
asymmetric unit, and they represent both enantiomers. If the substance was
enantiomerically pure, both of the independent molecules in space group P1 would be the
same enantiomer.
References
(1) SIR92: Altomare, A., Cascarano, G., Giacovazzo, C., Guagliardi, A., Burla, M.,
Polidori, G., and Camalli, M. (1994) J. Appl. Cryst., 27, 435.
(2) DIRDIF99: Beurskens, P.T., Admiraal, G., Beurskens, G., Bosman, W.P., de Gelder,
R., Israel, R. and Smits, J.M.M.(1999). The DIRDIF-99 program system, Technical
Report of the Crystallography Laboratory, University of Nijmegen, The Netherlands.
(3) Least Squares function minimized: (SHELXL97)
w(Fo2-Fc2)2
where w = Least Squares weights.
(4) Standard deviation of an observation of unit weight:
[w(Fo2-Fc2)2/(No-Nv)]1/2
where: No = number of observations
Nv = number of variables
(5) Cromer, D. T. & Waber, J. T.; "International Tables for X-ray Crystallography", Vol.
IV, The Kynoch Press, Birmingham, England, Table 2.2 A (1974).
(6) Ibers, J. A. & Hamilton, W. C.; Acta Crystallogr., 17, 781 (1964).
(7) Creagh, D. C. & McAuley, W.J .; "International Tables for Crystallography", Vol C,
(A.J.C. Wilson, ed.), Kluwer Academic Publishers, Boston, Table 4.2.6.8, pages 219-222
(1992).
(8) Creagh, D. C. & Hubbell, J.H..; "International Tables for Crystallography", Vol C,
(A.J.C. Wilson, ed.), Kluwer Academic Publishers, Boston, Table 4.2.4.3, pages 200-206
(1992).
(9) CrystalStructure 3.7.0: Crystal Structure Analysis Package, Rigaku and Rigaku/MSC
(2000-2005). 9009 New Trails Dr. The Woodlands TX 77381 USA.
(10) CRYSTALS Issue 10: Watkin, D.J., Prout, C.K. Carruthers, J.R. & Betteridge, P.W.
Chemical Crystallography Laboratory, Oxford, UK. (1996)
(11) SHELX97: Sheldrick, G.M. (1997).
EXPERIMENTAL DETAILS
A. Crystal Data (Supplement Figure 2)
Empirical Formula
C23H29NO8S
Formula Weight
479.54
Crystal Color, Habit
colorless, plate
Crystal Dimensions
0.44 X 0.25 X 0.10 mm
Crystal System
triclinic
Lattice Type
Primitive
Indexing Images
4 oscillations @ 60.0 seconds
Detector Position
127.40 mm
Pixel Size
0.100 mm
Lattice Parameters
a = 7.5873(8) Å
b = 9.5354(10) Å
c = 17.614(2) Å
 = 97.696(8) o
 = 96.712(8) o
 = 111.541(7) o
V = 1155.5(2) Å3
Space Group
P-1 (#2)
Z value
2
Dcalc
1.378 g/cm3
F000
508.00
(CuK)
16.740 cm-1
B. Intensity Measurements
Diffractometer
Rigaku RAXIS-RAPID
Radiation
CuK ( = 1.54180 Å)
graphite monochromated
Detector Aperture
280 mm x 256 mm
Data Images
136 exposures
Exposure Rate
36.0 sec./o
 oscillation Range (=0.0, =0.0)
20.0 - 200.0o
 oscillation Range (=54.0, =0.0)
30.0 - 120.0o
 oscillation Range (=54.0, =90.0)
30.0 - 100.0o
 oscillation Range (=54.0, =180.0)
20.0 - 180.0o
 oscillation Range (=54.0, =270.0)
20.0 - 200.0o
Detector Position
127.40 mm
Pixel Size
0.100 mm
2max
136.4o
No. of Reflections Measured
Total: 5103
Corrections
Unique: 10146 (Rint = 0.027)
Lorentz-polarization
Absorption
(trans. factors: 0.682 - 0.846)
C. Structure Solution and Refinement
Structure Solution
Direct Methods (SIR92)
Refinement
Full-matrix least-squares on F2
Function Minimized
 w (Fo2 - Fc2)2
Least Squares Weights
w = 1/ [ 2(Fo2) + (0.0430 . P)2
+ 0.2438 . P ]
where P = (Max(Fo2,0) + 2Fc2)/3
2max cutoff
136.4o
Anomalous Dispersion
All non-hydrogen atoms
No. Observations (All reflections)
3857
No. Variables
306
Reflection/Parameter Ratio
12.60
Residuals: R1 (I>2.00(I))
0.0367
Residuals: R (All reflections)
0.0483
Residuals: wR2 (All reflections)
0.0962
Goodness of Fit Indicator
1.126
Max Shift/Error in Final Cycle
0.011
Maximum peak in Final Diff. Map
0.27 e-/Å3
Minimum peak in Final Diff. Map
-0.32 e-/Å3