Tyrphostin-like compounds with ubiquitin modulatory activity as possible therapeutic agents for multiple myeloma

Article abstract of DOI:10.1016/j.bmc.2011.09.057

With the goal of developing small molecules as novel regulators of signal transduction and apoptosis, a series of tyrphostin-like compounds were synthesized and screened for their activity against MM-1 (multiple myeloma) cells and other cell lines representing this malignancy. Synthesis was completed in solution-phase initially and then adopted to solid-phase for generating a more diverse set of compounds. A positive correlation was noted between compounds capable of inducing apoptosis and their modulation of protein ubiquitination. Further analysis suggested that ubiquitin modulation occurs through inhibition of cellular deubiquitinase activity. Bulky groups on the sidechain near the α,β-unsaturated ketone caused a complete loss of activity, whereas cyclization on the opposite side was tolerated. Theoretical calculations at the B3LYP/LACV3P^** level were completed on each molecule, and the resulting molecular orbitals and Fukui reactivity values for C_β carbon were utilized in developing a model to explain the compound activity.

Full text of DOI:10.1016/j.bmc.2011.09.057

Bioorganic & Medicinal Chemistry 19 (2011) 7194–7204
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tyrphostin-like compounds with ubiquitin modulatory activity as possible
therapeutic agents for multiple myeloma
Zhenghong Peng a, Ashutosh Pal b, Dongmei Han a,b,c,d, Shimei Wang a,b,c,d, David Maxwell a,
Alexander Levitzki c, Moshe Talpaz d, Nicholas J. Donato d, William Bornmann a,
⇑
a Department of Experimental Therapeutics, M. D. Anderson Cancer Center, Houston, TX 77030, United States
b Experimental Diagnostic Imaging Chemistry Section, M. D. Anderson Cancer Center, Houston, TX 77030, United States
c Unit of Cellular Signaling, Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel
d Department of Internal Medicine/Division of Hematology/Oncology, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI 48109, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
With the goal of developing small molecules as novel regulators of signal transduction and apoptosis, a
series of tyrphostin-like compounds were synthesized and screened for their activity against MM-1 (mul-
tiple myeloma) cells and other cell lines representing this malignancy. Synthesis was completed in solu-
tion-phase initially and then adopted to solid-phase for generating a more diverse set of compounds. A
positive correlation was noted between compounds capable of inducing apoptosis and their modulation
of protein ubiquitination. Further analysis suggested that ubiquitin modulation occurs through inhibition
Received 20 April 2011
Revised 22 September 2011
Accepted 28 September 2011
Available online 7 October 2011
Keywords:
of cellular deubiquitinase activity. Bulky groups on the sidechain near the a,b-unsaturated ketone caused
Tyrphostin analogs
Multiple myeloma
Jak2/Stat3
Small molecules
Inhibitors
a complete loss of activity, whereas cyclization on the opposite side was tolerated. Theoretical calcula-
tions at the B3LYP/LACV3P⁄⁄ level were completed on each molecule, and the resulting molecular orbitals
and Fukui reactivity values for Cb carbon were utilized in developing a model to explain the compound
activity.
Synthesis
Quantum chemical calculations
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Signaling proteins are key components of the cellular circuitry
that link internal and external stimuli to changes in cell morphology
and gene expression and are highly regulated in normal cells. In
pathologies, including cancer, regulation of signaling proteins is
disrupted by gene mutations and chromosomal translocations
resulting in unregulated growth and survival, tumor metastates
and blocked differentiation. Reducing expression or returning
signaling proteins to their inactive state reverses many of the
characteristics associated with cancer and these proteins serve as
effective targets for cancer and other therapies.
AG490 (Fig. 1) was the first small molecule found to inhibit Jak2/
Stat3 signaling.1 Targeted inhibition of the Jak/Stat pathway with
AG490 inhibits tumor cell growth and increases sensitivity to apop-
totic stimuli; thus, inhibitors of this pathway represent potential
therapeutics for cancer and possibly other diseases.2–4 Because the
cytokine IL-6 promotes survival and proliferation of certain cancer-
ous cell lines through the phosphorylation of STAT3,5–7 kinase inhib-
itors similar to AG490 have potential as anti-cancer drugs.
Compound AG490 is structurally classified as a tyrphostin. US Patent
Figure 1. Chemical structures of several commonly named tyrphostins discussed in
paper.
WO9528922,8 WO2003068157,9 and WO200505882910 describe
compounds that have structural similarity with AG490. Unfortu-
nately, AG490 has limited activity in animal studies due to pharma-
cokinetic properties and must be used at high concentrations (ꢀ50
to 100 lM). Also, it is difficult to always document the ability of
AG490 to inhibit Jak2/Stat3 signaling in vivo.1,11,12 We therefore
sought to modify AG490 in order to achieve more drug-like
⇑
Corresponding author. Tel.: +1 713 563 0081; fax: +1 713 563 1878.
E-mail address: wbornmann@mdanderson.org (W. Bornmann).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.057

Z. Peng et al. / Bioorg. Med. Chem. 19 (2011) 7194–7204
7195
Table 1a
Anti-tumor activity of compounds against multiple myeloma cell lines
R5
O
R1
R2
X
R9
R8
N
H
R6
CN
R4
R3
R7
Compd
X
R1
R2
R3
R4
R5
R6
R7
R8
R9
IC50 lM
MM-1
U266
OCI-My4
AG490
AG1801
WP1130
WP1066
5
CH
CH
N
N
N
OH
H
Br
Br
Br
Br
OH
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
nPr
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
OMe
H
H
H
H
H
H
>10
7.5
1.0
1.2
1.5
1.2
>12.5
9.0
1.3
1.5
1.8
>12.5
12.0
1.5
1.8
2.0
6
N
H
1.4
1.8
H
H
O
Br
N
N
7
8
N
2
2.5
ND
N
H
CN
O
Br
N
1.5
1.9
ND
N
H
CN
9
N
N
N
N
N
N
N
N
N
N
N
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
CH2OH
Et
Me
Me
CF3
Me
Me
Me
CH2COOCH3
Me
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
CF3
H
CF3
H
Cl
H
H
H
Cl
H
H
F
H
H
H
OMe
H
H
H
H
CF3
H
1.6
2.8
1.1
2.4
2.1
9.6
1.3
1.2
1.3
3.1
1.8
2.0
3.5
1.4
2.8
2.3
11.1
1.6
1.4
1.5
3.3
2.0
2.3
3.9
1.7
3.1
2.4
ND
1.8
ND
ND
ND
2.4
10
11
12
13
14
15
16
17
18
19
H
H
H
H
H
20
21
22
23
24
N
Br
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.1
2.5
>10
1.4
>10
1.3
2.8
>10
ND
ND
1.6
2.9
ND
ND
ND
CH
CH
N
Cl
N
Br
H
H
CH
Hexanyl-
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
N
N
Br
Br
H
H
H
NO2
H
H
H
H
NO2
NO2
H
H
Cl
H
OH
OH
OH
H
NO2
H
H
H
H
H
H
H
NO2
H
H
H
H
H
H
H
H
NO2
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
Me
H
nPr
nPr
nPr
nPr
nPr
nPr
nPr
nPr
nPr
CH2OCOCH3
Me
Ph
Bn
CH2OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>10
>10
2.94
>10
6.25
1.73
1.58
>10
>10
8.2
>10
2.6
>10
5
2.5
>10
ND
ND
3.6
ND
7.2
2.1
1.8
ND
ND
9.3
ND
2.8
ND
5.8
2.7
ND
ND
ND
ND
ND
ND
2.2
1.9
ND
ND
9.7
ND
2.9
ND
6.2
2.9
ND
CH
C-NO2
CH
CH
CH
C-NO2
CH
CH
CH
N
H
NO2
Br
Br
NO2
Br
Br
H
CH
N
N
H
Cl
CH
H
O
O
N
N
41
42
>10
>10
ND
ND
ND
ND
H
CN
H
O
O
F
N
H
CN
F
O

7196
Z. Peng et al. / Bioorg. Med. Chem. 19 (2011) 7194–7204
Table 1b
contained an extra-annular double bond added to the existing con-
Anti-tumor activity of compounds against MM-1 cell lines
jugated system showed submicromolar inhibition against acute
lymphocytic leukemia (ALL) cancer cell growth.15 Our initial work
H
O
R1
with the MM-1 cell line showed low
lM apoptotic and signal
O
N
H
transduction inhibitory activity for WP1066 and WP1130. Our
aim was to develop analogs of the WP compounds in order to ex-
plore the SAR of the tyrphostin core and its mechanism of action
in MM-1 cells. This was accomplished through solution phase syn-
thesis, which was later adapted to solid phase synthesis to expand
the molecular diversity of the library and increase the throughput.
In our patent application, WO2008005954, we provided a descrip-
tion of the compounds and results in the MM-1 cell line, but did
not discuss SAR for each series nor elaborate on the reason for
the differences in activity. In this paper, we sought an explanation
for the difference in observed activity based on a hypothesis that it
was related to nucleophilic attack of the Cb carbon atom. Our re-
cent analysis shows that the more active tyrphostin derivatives
function as deubiquitinase (DUB) inhibitors through a Michael
acceptor reaction with the cysteine residue within the DUB active
site.16,17
CN
R4
R2
R3
O2N
Compd
R1
R2
H
R3
H
R4
IC50
>10
(lM)
43
H
44
45
46
47
Et
H
H
H
H
Cl
H
H
H
H
H
H
H
Me
H
>10
>10
>10
>10
OMe
H
O
O
O
N
H
CH3
48
CN
>10
O2N
2. Chemistry of tyrphostin analogs
The solution phase synthesis of tyrphostin analogs were pre-
pared using the procedure shown in Scheme 1. The procedures used
were based on tyrphostin compounds developed by Levitzki et al.18
First equal molar of benzylamine was coupled with cyanoacetic
methyl ester quantitatively to form the N-benzylcyanoacetamide
as an intermediate, then Knoevenagel condensation with the alde-
hyde furnished the final product. We synthesized 48 tyrphostin like
compound via solution phase. We expanded on those compounds
by adapting the solution phase synthesis to solid support as shown
in Scheme 2. The modified procedures were based on those of sim-
ilar compounds described in the literature.19,20 First primary
amines were attached to BAL-PG-PS resin (1) via a reductive amina-
tion reaction. This reaction works much more consistently than di-
rect coupling of amine to bromo-Merrifield resin. The attachment
was confirmed by positive control of purple color from ninhydrin
test due to the presence of the secondary amine of the attached
BAL resin (2). Cyano acetic acid was attached to secondary amine
BAL resin (2) via a DICPDI/DIEA coupling step. A negative ninhydrin
color test confirmed the completion of the reaction. TFA cleavage at
this step afforded a N-benzylcyanoacetamide intermediate, sup-
ported by Mass Spectrometry (MS) and NMR analysis. Knoevenagel
condensation with benzaldehyde and cyanoacetamide resin (3)
afforded tyrphostin compounds attached to the BAL resin (4). The
compounds. The first lead was AG1801 from the Levitzki laboratory,
which showed higher potency than AG490 (Table 1a and b). Based
on this lead the WP compounds were generated.
WP1066, is a novel inhibitor structurally related to AG490 but
significantly more potent and active in suppressing Jak2/Stat3 sig-
naling and inducing apoptosis in human malignant glioma U87-
MG and U373-MG cells in vitro and in vivo. IC50 values for
WP1066 were 5.6 lM in U87-MG cells and 3.7 lM in U373-MG
cells, which represents 18-fold and 8-fold increases in potency,
respectively, over that of AG490 in cellular studies. WP1066-med-
iated apoptosis was associated with activation of Bax, and suppres-
sion of c-myc, Bcl-XL and Mcl-1 expression. Systemic
intraperitoneal administration of WP1066 in mice significantly
inhibited the growth of subcutaneous malignant glioma.13
WP1130 is a close analog of WP1066 that shows marginal in-
creased potency at the micromolar level against chronic myeloge-
nous leukemia (CML) cells. Compound WP1130 specifically and
rapidly down-regulates both wild-type and T315I mutant Bcr/Abl
mutant protein and suppresses the growth of K562 heterotrans-
planted tumors as well as both wild-type Bcr/Abl and T315I mu-
tant Bcr/Abl—expressing BaF/3 cells transplanted into nude
mice.14 Another published tyrphostin-like compound, CR4, that
Scheme 1. General procedure for the synthesis of tyrphostins analogs.

Z. Peng et al. / Bioorg. Med. Chem. 19 (2011) 7194–7204
7197
Scheme 2. Solid phase synthesis of tyrphostin analogs.
final compounds were cleaved from BAL resin using 95% TFA. Com-
3. Biological activity
pound WP1130 was first successfully synthesized manually on BAL
resin and BAL mimotope Lantern (initial specified loading:
750 lmol). Both resin and lantern gave similar yield and purity. Fi-
The synthesized compounds were tested for their ability to
inhibit Jak2/Stat3 signal transduction as previously described. Com-
pounds were also assessed for their ability to inhibit cell growth and
induce apoptosis as previously described. In those experiments,
multiple myeloma (MM-1) was chosen as model for primary screen-
ing. Two additional myeloma cell lines (U266, OCI-My4) were used
to confirm activity. In a typical experiment, cell lines were incubated
with a range of compound concentrations for 72 h to determine the
concentration required to inhibit cell growth or induce apoptosis by
50% (IC50) as determined by MTT assays previously described.16,17
The IC50 values for the compounds against MM-1 cells are listed in
nally, a chemical library of variants of the tyrphostin compound
incorporating variability at two sites within the molecule, fifteen
at the aldehyde site and six at the amine site (Fig. 2) to make a total
of 90 compounds. TLC was used to confirm the presence of major
products and MS was tested randomly to confirm the molecular
weight of the final tyrphostin compounds. HPLC analyses revealed
that the purity of the compounds ranged from 50% to 95% (Supple-
mentary Fig. 1). The synthesized library was screened against the
MM-1 cell line without further purification.
Figure 2. Building blocks used for solid phase library synthesis of tyrphostin analogs.

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Z. Peng et al. / Bioorg. Med. Chem. 19 (2011) 7194–7204
Table 1a and b. Similar IC50 values were obtained for U266 and OCI-
My4 cell lines treated with these compounds.
explored modifications to the stereocenter of WP1130, to see if
replacing the propyl group (Pr) with either smaller or larger groups
would lead to better activity. There appeared to be no general trend
regarding size since all modifications had decreased or equivalent
activity when compared to WP1130. However, substitution with a
4. Results and discussion
4.1. Study of SAR
phenyl group (38) resulted in a 5
lM inhibitor whereas Bn substitu-
tion (39) retained activity at 2.5
lM. It is interesting to note that
replacing the methyl in WP1066 with an isosteric CF3 led to a much
larger decrease in activity. Adding a group capable of hydrogen-
bond accepting (–CH2COOCH3, –CH2OCOCH3) as well as donating
and accepting (–CH2OH) led to a small decrease in activity. Cyclizing
the RHS (7, 8) to tie up the stereocenter led to a slight decrease in
activity; however, this may be balanced by its ability to provide an
alternative structural framework for adding other functionality.
We found that changing from the configuration (S) for the cyclopro-
pyl group at the R5 position (23) to the racemic structure (20) did not
affect activity. Hydrophobic substitutions at phenyl group from R6 to
R9 are tolerated; however, an amino substitution removes activity
completely. The furan, indole, quinoline structure abolished activity
completely. In summary, the left side of tyrphostin compounds is
more sensitive than the right side. This makes sense if our hypothe-
sis is true that compoundactivityis relatedto the nucleophilicattack
at the Cb carbon, since the left side is directly conjugated. In expand-
ing the scope of the tyrphostin analogs, a chemical library of 90 com-
pounds was synthesized from solid phase. No significant increase of
activity was observed from the solid phase libraries and it indicates
that a plateau has been reached for the current tyrphostin template.
In addition, it appears that the phenyl or pyridine heterocycles at the
left side (Table 1a and b) are crucial to retain bioactivities in cell line
assays.
For discussion purposes, series 1 refers to compounds with
X = CH; series 2 refers to compounds with X = N and R1 = Br, and
series 3 refers to all others. Even though WP1066 and WP1130
had better activity when compared to either AG490 or AG1801, we
chose not to bias our search towards either a phenyl or brominated
pyridine for the left side. Series 1: The activity of the parent AG490
and AG1801 were modified by changing both the position and type
of substituent on the phenyl ring. In two cases, the activity was
nearly the same as that for series 2. Cyclization of the phenyl ring
to make a 1,4-benzoxazine or 1,3-benzodioxole (41, 42) seemed to
destroy the activity. Replacing the left side with a nitrophenylfuran
group (43–48) also removed any evidence of activity. Series 2: We
Table 2
Theoretical LUMO energies and Fukui fNN values for Cb carbon calculated at the B3LYP/
LACV3P⁄⁄ level
Compound
LUMO
Activity
Series
Fukui LUMO fNN
AG1801
AG490
WP1066
WP1130
5
6
7
8
9
10
11
12
13
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
ꢁ0.128
ꢁ0.087
ꢁ0.110
ꢁ0.107
ꢁ0.111
ꢁ0.106
ꢁ0.108
ꢁ0.107
ꢁ0.114
ꢁ0.107
ꢁ0.107
ꢁ0.106
ꢁ0.111
ꢁ0.108
ꢁ0.112
ꢁ0.115
ꢁ0.109
ꢁ0.106
ꢁ0.107
ꢁ0.118
ꢁ0.098
ꢁ0.107
ꢁ0.088
ꢁ0.104
ꢁ0.108
ꢁ0.126
ꢁ0.137
ꢁ0.112
ꢁ0.115
ꢁ0.118
ꢁ0.112
ꢁ0.085
ꢁ0.119
ꢁ0.093
ꢁ0.110
ꢁ0.139
ꢁ0.109
ꢁ0.108
ꢁ0.118
ꢁ0.075
ꢁ0.100
ꢁ0.109
ꢁ0.108
ꢁ0.113
ꢁ0.109
ꢁ0.111
ꢁ0.109
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
2
2
2
1
2
1
1
1
2
2
1
2
1
2
1
1
2
2
2
2
2
2
2
2
2
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
2
1
2
2
1
1
1
1
1
1
1
1
1
2
1
2
2
1
3
3
3
3
3
3
3
3
0.135
0.290
0.236
0.210
0.216
0.209
0.213
0.211
0.241
0.212
0.210
0.209
0.216
0.222
0.238
0.223
0.212
0.210
0.211
0.168
0.081
0.211
0.274
0.207
0.211
0.130
0.069
0.075
0.146
0.167
0.013
0.284
0.000
0.274
0.214
0.113
0.214
0.212
0.170
0.277
0.261
0.147
0.145
0.169
0.152
0.158
0.151
Figure 3. Plot of activity versus Fukui LUMO fNN. 95% confidence levels are shown
as dashed lines.
Figure 4. Plot of LUMO fNN versus LUMO energies. Active compounds are solid blue
and inactive compounds are outlined in red. Each series is represented by
different shape: rectangle for series 1 with X = C; circle for series 2 with X = N,
2 = Br; triangle for series 3. Lines were placed at x = ꢁ0.106 and y = 0.120. The
patterned area suggests location of inactive compounds.
a
Complete set of associated energies, HOMO, and electrophilicity may be found in
Supplementary data.
R

Z. Peng et al. / Bioorg. Med. Chem. 19 (2011) 7194–7204
7199
Figure 5. LUMO orbitals mapped onto the electron density surface for (top-left) WP1130 (top-right) AG490 (bottom-left) compound 30 (bottom-right) compound 39. A
custom coloring ramp was used that contained red for extremes of negative and positive and white for all other areas.
4.2. Theoretical analysis of reactivity towards nucleophile
of the LUMO fNN for Cb is less (0.146) when compared to WP1130
due to the additional reactive areas ortho- and para- to the nitro
group. Changing from a propyl in WP1130 to a much bulkier group
in compound 39 has virtually no effect on the reactivity values
The LUMO energies and Fukui function, fNN, calculated at the Cb
carbon for each compound is listed in Table 2. Structures with IC50
values 610
l
M in the MM-1 cell line had a moderate correlation
(Cb = 0.212, C = 0.180) and the graphical representation is nearly
identical for both.
a
(r2 = 0.50, F = 26.17, p = 0.0001) observed between the experimen-
tal ꢁlogIC50 values listed in Table 1a and b and the Fukui function,
fNN, calculated at the Cb carbon as shown in Figure 3. The same
analysis done with the U266 and OCI-My4 cell lines showed a cor-
relation of r2 = 0.51 and r2 = 0.40, respectively. Plotting the
ꢁlogIC50 values for MM-1 cell line versus local electrophilicity,
5. Conclusion
In conclusion, by expanding upon the SAR of the tyrphostin
compounds and analysis of the reactivity of compounds through
theoretical calculations, we have found the following:
xk, also showed a
moderate correlation (r2 = 0.44, F = 20.57,
p = 0.0001). This was primarily due to the Fukui function, fNN, since
the global electophilicity index does not correlate well with the
ꢁlogIC50. A combination of LUMO energies and LUMO fNN values
may be used to separate active and inactive compounds. A plot
containing this data is shown in Figure 4, with active compounds
colored blue and inactive compounds colored red. Generally, if
the LUMO is greater than ꢁ0.106 or fNN is lower than 0.120, then
the structures were found to be inactive. There are a few excep-
tions, most of which are in series 3, which we would attribute to
steric effects not considered in the Fukui and orbital energy calcu-
lations. There are two active compounds shown in the inactive re-
1. Identified compounds with equivalent activity to WP series, but
were not able to identify any with improved activity in the MM-
1 cell line.
2. Identified compounds with better activity in Series 1, specifi-
cally compounds 21, 27, 30, and 31.
3. Cyclizing the right-hand side of Series 2 was well tolerated, but
did not improve the activity.
4. Changes to the left-hand side of either series resulted in loss of
activity.
5. The local electrophilicity and Fukui function, fNN, was moder-
ately correlated with the IC50, suggesting that nucleophilic
attack on the Cb carbon may involved in explaining the activity
differences of such compounds.
6. A general model for separating inactive from active compounds
arises from a combination of LUMO energies and Fukui LUMO
fNN values.
7. There is an excellent match of the numerical Fukui LUMO fNN
values with the graphical representation of reactivity (LUMO
mapped onto electron density).
gion; however, they have values closer to 10 lM.
The area’s most susceptible to nucleophilic attack may also be
described graphically, by mapping the LUMO onto the electron
density. This was completed for several molecules as shown in
Figure 5. The custom color ramp utilized red-white-red to keep
the positive and negative lobes of the orbital the same color and
emphasize the most reactive areas. As expected, there is an excel-
lent match of the reactive areas with the LUMO fNN values, espe-
cially at the Cb and C carbons. In all compounds, the LUMO fNN
a
for Cb is always higher than the value on C . For WP1130, the
a
LUMO fNN for Cb and C carbons are 0.210 and 0.181, respectively.
This may be compared with AG490, which has a higher Cb = 0.290
a
6. Experimental section
6.1. Chemical procedures
(C = 0.153), and this same difference may be seen in the combina-
a
tion plot. However, AG490 is not active, which is most likely due to
the higher LUMO energy level being mismatched to the nucleo-
phile. Two other actives from Series 1 and Series 2 are also shown
in Figure 5. Compound 30 is a molecule in the same series as
AG490 having activity similar to WP1130. The overall magnitude
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

7200
Z. Peng et al. / Bioorg. Med. Chem. 19 (2011) 7194–7204
Varian Prostar system, with a Varian Microsorb-MW C18 column
m) using the following solvent system
A = H2O/0.l% TFA and B = acetonitrile/0.1% TFA. Varian Prepstar
3.82 (s, 1H), 1.60 (d, 3H, J = 6.8 Hz); 13C NMR (75 MHz, CDCl3) d
159.56, 158.79, 151.33, 148.64, 139.52, 134.50, 131.02, 127.80,
125.78, 114.63, 109.95, 55.72, 50.09, 21.94; MS (ESI) m/e (rel inten-
sity): 386.3 (16), 388.3 (16).
(250 ꢂ 4.6 mm;
5 l
preparative system equipped with a Prep Microsorb-MW C18 col-
umn (250 ꢂ 41.4 mm; 6
l; 60 Å) was used for preparative HPLC
with the same solvent systems. Program A: Gradient: 0–5 min.
30% B. 5–35 min. 95% B. 30–35 min. 95% B. Program B: Gradient:
0–5 min. 10% B. 5–35 min. 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 Perkin Elmer Lambda 25 UV/Vis spectrometer.
Solid phase synthesis was performed on apptec apex396 combina-
tory synthesizer. IR was measured on Perkin Elmer Spectra One
FT-IR spectrometer. 1H NMR and 13C 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 13C NMR. Chemical
shifts (d) are determined relative to methanol-d4 (referenced to
3.34 ppm (d) for 1H NMR and 49.86 ppm for l3C NMR). Proton–
proton coupling constants (J) are given in Hertz and spectral split-
ting 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
(254 nm) and by staining with potassium permanganate or ceric
ammonium molybdate.
6.2.4. (R,E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-(2,3-dihydro-
1H-inden-1-yl)acrylamide (7)
1H NMR (500 MHz, CDCl3) d 8.28 (s, 1H), 7.58–7.69 (m, 3H), 7.28
(m, 4H), 6.74 (d, 1H, J = 7.8 Hz), 5.62 (m, 1H), 3.07 (m 1H), 2.94 (m,
1H), 2.70 (m, 1H), 1.95 (m, 1H); 13C NMR (125 MHz, CDCl3) d
159.22, 150.96, 148.42, 143.44, 142.51, 141.88, 139.16, 130.68,
128.48, 127.08, 125.42, 125.03, 124.06, 115.67, 109.50, 55.97,
33.82, 30.31; MS (ESI) m/e (rel intensity): 368.2 (30), 370.2 (30).
6.2.5. (R,E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-(1,2,3,4-
tetrahydronaphthalen-1-yl)acrylamide (8)
1H NMR (500 MHz, CDCl3) d 8.29 (s, 1H), 7.59–7.69 (m, 2H), 7.58
(d, 1H, J = 8.5 Hz), 7.19 (m, 2H), 7.13 (d, 1H, J = 7.4 Hz), 6.74 (d, 1H,
J = 8.1 Hz), 5.31 (m, 1H), 2.88 (m 1H), 2.79 (m, 1H), 2.15 (m, 1H),
1.90 (m, 3H); 13C NMR (125 MHz, CDCl3) d 158.76, 151.00,
148.40, 142.49, 139.15, 137.65, 135.31, 130.64, 129.43, 128.47,
127.78, 126.54, 125.35, 115.64, 109.69, 48.99, 29.99, 29.13,
20.02; MS (ESI) m/e (rel intensity): 382 (50), 384 (50).
6.2.6. (S,E)-3-(6-Bromopyridin-2-yl)-N-(1-(4-chlorophenyl)
ethyl)-2-cyanoacrylamide (9)
1H NMR (500 MHz, CDCl3) d 8.20 (s, 1H), 7.65 (t, 1H, J = 7.5 Hz),
7.58 (m, 2H), 7.25 (m, 2H), 7.17 (d, 2H, J = 8.0 Hz), 6.75 (d, 1H,
J = 7.3 Hz), 5.20 (m 1H), 2.34 (s, 3H), 1.60 (d, 3H, J = 6.9 Hz); 13C
NMR (125 MHz, CDCl3) d 158.44, 150.95, 148.36, 142.47, 139.13,
139.07, 137.53, 130.62, 129.55, 126.07, 125.40, 115.73, 109. 56,
50.03, 21.67, 21.07; MS (ESI) m/e (rel intensity): 370.2 (47), 372.2
(50).
6.2. General method for the preparation of tyrphostin analogs
(Scheme 1)
In step 1, a mixture of methyl cyanoacetate (25 mmol) and the
amine (25 mmol) was stirred vigorously for overnight. The result-
ing solid was triturated with 8 mL 95% ethanol and the product fil-
tered. The amide from the step1 reaction (5.0 mmol), aldehyde
(5 mmol), and piperidine (five drops) were stirred in anhydrous
ethanol (10 mL). Ethanol was evaporated and solid was triturated
with water and dried under high vacuum to give the desired
product.
6.2.7. (R,E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-(2-hydroxy-1-
phenylethyl)acrylamide (10)
1H NMR (500 MHz, DMSO-d6) d 8.82 (d, 1H, J = 7.9 Hz), 8.10 (s,
1H), 7.95 (t, 1H, J = 7.7 Hz), 7.89 (d, 1H, J = 7.3 Hz), 7.80 (d, 1H,
J = 7.8 Hz), 7.38 (d, 2H, J = 7.2 Hz), 7.34 (m, 2H), 7.26 (t, 1H,
J = 7.2 Hz), 5.02 (t, 1H, J = 5.7 Hz), 4.97 (m 1H), 3.68 (m, 2H); 13C
NMR (125 MHz, DMSO-d6) d 161.26, 151.59, 146.67, 141.67,
141.20, 140.77, 130.97, 128.70, 127.56, 127.43, 126.66, 115.53,
111.34, 64.63, 56.94,; MS (ESI) m/e (rel intensity): 372.3 (89),
374.2 (100).
6.2.1. N-(Cyanoacetyl)-1-(40-aminophenyl)ethyl amide
A mixture of methyl cyanoacetate (2.47 g, 25 mmol) and 1-(40-
aminophenyl)ethylamine (3.40 g 25 mmol) was stirred vigorously
for overnight. The resulting solid was triturated with 8 mL 95% eth-
anol and the product filtered as a white solid (4.08 g, 80% yield). 1H
NMR (600 MHz, CD3OD) d 7.12 (dt, 2H, J = 2.9, 8.3 Hz), 6.71 (dt, 2H,
J = 2.9, 8.3 Hz), 4.94 (q, 1H, J = 6.7 Hz), 3.2 (s, 2H), 1.43 (d, 3H,
6.4 Hz); 13C NMR d 162.4, 146.9, 132.7, 127.0, 127.0, 115.6, 115.6,
49.4, 20.9.
6.2.8. (S,E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-
(1-phenylpropyl)acrylamide (11)
1H NMR (500 MHz, CDCl3) d 8.19 (s, 1H), 7.65 (dd, 1H, J = 5.0,
4.3 Hz), 7.57 (d, 2H, J = 4.7 Hz), 7.27–7.38 (m, 3H), 6.78 (d, 1H,
J = 4.8 Hz), 4.99 (m 1H), 1.94 (m, 1H), 0.95 (d, 3H, J = 5.4 Hz); 13C
NMR (125 MHz, CDCl3) d 158.67, 150.92, 148.26, 142.48, 141.01,
139.12, 130.64, 128.84, 127.76, 126.58, 125.41, 115.81, 109.54,
56.34, 29.09, 10.69; MS (ESI) m/e (rel intensity): 370.1 (90), 372.1
(100).
6.2.2. (S,E)-N-(1-(4-Bromophenyl)ethyl)-3-(6-bromopyridin-
2-yl)-2-cyanoacrylamide (5)
1H NMR (500 MHz, CDCl3) d 8.18 (s, 1H), 7.66 (m, 1H), 7.57 (m,
2H), 7.48 (d, 2H, J = 8.4 Hz), 7.24 (d, 2H, J = 8.4 Hz), 6.78 (d, 1H,
J = 6.5 Hz), 5.18 (m 1H),1.59 (d, 3H, J = 11.8 Hz); 13C NMR
6.2.9. (S,E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-
(1-(3-methoxyphenyl)ethyl)acrylamide (12)
1H NMR (500 MHz, CDCl3) d 8.23 (s, 1H), 7.68 (t, 1H, J = 7.8 Hz),
7.60 (m, 2H), 7.31 (t, 1H, J = 7.9 Hz), 6.96 (d, 1H, J = 8.0 Hz), 6.91
(m, 1H), 6.85 (dd, 1H, J = 8.2, 2.5 Hz), 6.79 (d, 1H, J = 7.6 Hz), 5.23
(m 1H), 3.84 (s, 3H), 1.62 (d, 3H, J = 6.9 Hz); 13C NMR (125 MHz,
CDCl3) d 159.99, 158.51, 150.91, 148.34, 143.68, 142.47, 139.14,
130.66, 129.97, 125.43, 118.30, 115.74, 113.03, 112.12, 109.47,
55.27, 50.23, 21.707; MS (ESI) m/e (rel intensity): 386.2 (50),
388.2 (53).
(125 MHz, CDCl3)
130.76, 127.91, 125.56, 49.78, 21.63; MS (ESI) m/e (rel intensity):
433.8 (50), 435.9 (100), 437.8 (50).
d 158.61, 150.83, 148.47, 139.16, 131.95,
6.2.3. (S,E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-
(1-(4-methoxyphenyl)ethyl)acrylamide (6)
1H NMR (300 MHz, CDCl3) d 8.22 (s, 1H), 7.63 (m, 3H), 7.29 (m,
2H), 6.91 (d, 2H, J = 8.6 Hz), 6.73 (d, 1H, J = 6.8 Hz), 5.22 (m 1H),

Z. Peng et al. / Bioorg. Med. Chem. 19 (2011) 7194–7204
7201
6.2.10. (S,E)-3-(6-Bromopyridin-2-yl)-N-(1-(4-
140.66, 133.57, 133.05, 131.62, 130.97, 128.85, 127.94, 127.24,
126.71, 115.99, 107.83, 59.15, 16.69, 4.26, 4.20; MS (EI) m/e (rel
intensity): 382.2 (60).
chlorophenyl)ethyl)-2-cyanoacrylamide (13)
1H NMR (500 MHz, CDCl3) d 8.19 (s, 1H), 7.65 (m, 1H), 7.58 (m,
2H), 7.33 (d, 2H, J = 8.6 Hz), 7.27 (d, 2H, J = 8.6 Hz), 6.75 (d, 1H,
J = 6.5 Hz), 5.20 (m 1H), 1.59 (d, 3H, J = 6.9 Hz); 13C NMR
6.2.18. (E)-3-(4-Chloro-3-nitrophenyl)-2-cyano-N-
(125 MHz, CDCl3)
139.15, 133.56, 130.76, 129.00, 127.56, 125.53, 115.72, 109.24,
49.70, 21.66; MS (ESI) m/e (rel intensity): 390 (70), 392 (85).
d
158.61, 150.80, 148.46, 142.50, 140.67,
(cyclopropyl(phenyl)methyl)acrylamide (21)
1H NMR (600 MHz, CDCl3) d 8.33 (d, 1H, J = 1.8 Hz), 8.28 (s, 1H),
8.08 (dd, 1H, J = 8.4, 2.4 Hz), 7.69 (d, 1H, J = 8.4 Hz), 7.36–7.40 (m,
4H), 7.31 (m, 1H), 6.80 (d, 1H, J = 7.8 Hz), 4.50(t, 1H, J = 8.4 Hz),
1.30 (m, 1H), 0.68 (m, 2H), 0.51 (m, 1H), 0.44 (m, 1H); 13C NMR
6.2.11. (R,E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-
(2,2,2-trifluoro-1-phenylethyl)acrylamide (14)
(600 MHz, CDCl3) d 158.22, 148.96, 140.66, 133.57, 133.05,
131.62, 130.97, 128.85, 127.94, 127.24, 126.71, 115.99, 107.83,
59.15, 16.69, 4.26, 4.20; MS (EI) m/e (rel intensity): 382.2 (60).
1H NMR (300 MHz, CDCl3) d 8.25 (s, 1H) 7.69 (m, 1H), 7.45 (s,
5H), 7.61 (m, 2H), 7.16 (d, 1H, J = 9.5 Hz), 5.80 (m, 1H); MS (ESI)
m/e (rel intensity): 410.2 (94), 412.2 (100), 429.1 (28).
6.2.19. (E)-2-Cyano-N-(cyclopropyl(phenyl)methyl)-3-
(3-nitro-4-(piperidin-1-yl)phenyl)acrylamid (22)
6.2.12. (S,E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-
(1-(3-(trifluoromethyl)phenyl)ethyl)acrylamide (15)
1H NMR (500 MHz, CDCl3) d 8.22 (s, 1H), 7.69 (m, 1H), 7.60 (m,
2H), 7.51 (m, 1H), 6.88 (d, 1H, J = 8.4 Hz), 5.31(m 1H), 1.66(d, 3H,
J = 8.4 Hz); 13C NMR (125 MHz, CDCl3) d 158.74, 150.75, 148.60,
143.26, 142.50, 139.16, 130.80, 129.53, 129.37, 125.61, 124.62,
123.02, 122.99, 122.89, 115.70, 109.10, 49.94, 21.68; MS (EI) m/e
(rel intensity): 422.0 (53), 424.0 (67).
1H NMR (600 MHz, CDCl3) d 8.24 (s, 1H), 8.15 (s, 1H), 8.08 (d,
1H, J = 9.0 Hz), 7.35–7.40 (m, 4H), 7.29 (t, 1H, J = 7.2 Hz), 7.10 (d,
1H, J = 9.0 Hz), 6.72 (d, 1H, J = 7.8 Hz), 4.50 (t, 1H, J = 8.4 Hz), 3.22
(m, 4H), 1.72 (m, 4H), 1.68 (m, 2H), 1.58 (s, 2H), 1.27 (m, 1H),
0.66 (m, 2H), 0.51 (m, 1H), 0.43 (m, 1H);
13C NMR (600 MHz, CDCl3) d 159.81, 150.54, 148.55, 141.16,
139.18, 134.16, 131.05, 128.74, 127.72, 126.73, 121.62, 119.97,
117.94, 101.13, 58.72, 51.87, 25.64, 23.80, 16.77, 4.22, 4.11;MS
(ESI) m/e (rel intensity): 431.3 (100).
6.2.13. (S,E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-
(1-(4-fluorophenyl)ethyl)acrylamide (16)
1H NMR (500 MHz, CDCl3) d 8.20 (s, 1H), 7.69 (m, 1H), 7.60 (m,
2H), 7.36 (m, 2H), 7.08 (m, 2H), 6.78 (d, 1H, J = 8.4 Hz), 5.24(t 1H,
J = 8.4 Hz), 1.62(d, 3H, J = 8.4 Hz); 13C NMR (125 MHz, CDCl3) d
163.28, 161.32, 158.62, 150.91, 148.49, 142.57, 139.22, 137.98,
130.80, 127.98, 127.91, 125.57, 115.87, 115.80, 115.70, 109.41,
49.70, 21.77; MS (EI) m/e (rel intensity): 374.3 (11), 376.3 (11).
6.2.20. (S,E)-3-(6-Bromopyridin-2-yl)-2-cyano-
N-(cyclopropyl(phenyl)methyl)acrylamide (23)
MS (EI) m/e (rel intensity): 382.3 (42), 384.3 (31), 399.2 (19),
401.2 (22), 414.3 (45).
6.2.21. (E)-2-Cyano-N-(cyclopropyl(phenyl)methyl)-3-
(4-hexylphenyl)acrylamide (24)
6.2.14. (S,E)-N-(1-(3,5-Bis(trifluoromethyl)phenyl)ethyl)-3-
(6-bromopyridin-2-yl)-2-cyanoacrylamide (17)
1H NMR (600 MHz, CDCl3) d 8.24 (s, 1H), 7.91 (d, 1H, J = 9.0 Hz),
7.35–7.41 (m, 4H), 7.29 (m, 1H), 6.96 (d, 1H, J = 9.0 Hz), 6.73 (d, 1H,
J = 7.8 Hz), 4.51 (t, 1H, J = 8.4 Hz), 4.03 (m, 2H), 1.80 (m, 2H.), 1.46
(m, 2H), 1.35 (m, 4H), 1.26 (m, 1H), 0.91 (t, 1H, J = 6.6 Hz), 0.66 (m,
2H), 0.51 (m, 1H), 0.44 (m, 1H).
1H NMR (500 MHz, CDCl3) d 8.18 (s, 1H), 7.80 (s, 1H), 7.60 (m,
2H), 7.67 (m, 2H), 7.57 (m, 1H), 6.92 (d, 1H, J = 8.4 Hz), 5.31(m
1H), 1.67 (d, 3H, J = 8.4 Hz); 13C NMR (125 MHz, CDCl3) d 158.74,
150.75, 148.60, 143.26, 142.50, 139.16, 130.80, 129.53, 129.37,
125.61, 124.62, 123.02, 122.99, 122.89, 115.70, 109.10, 49.94,
21.68; MS (EI) m/e (rel intensity): 490.1 (86), 492.1 (100).
6.2.22. (S,E)-N-(1-(4-Aminophenyl)ethyl)-3-(6-bromopyridin-
2-yl)-2-cyanoacrylamide (25)
The amide from the previous reaction (1.2 g, 5.2 mmol), 6-bro-
mo-pyridinecarboxyldehyde (0.97 g, 5.2 mmol) and piperidine
(five drops) were stirred in anhydrous ethanol (10 mL). Ethanol
was evaporated and solid was triturated with water and dried un-
der high vacuum to give (1.7 g, 82% yield) of the desired product as
a white solid; 1H NMR (600 MHz, DMSO-d6) d 8.72 (d, 1H,
J = 7.8 Hz), 8.57 (s, 1H), 8.15 (d, 1H, J = 7.6 Hz), 7.91 (t, 1H,
J = 7.8 Hz), 7.79 (d, 1H, 7.8 Hz), 7.36 (m, 4H), 4.93(p, 1H,
J = 7.2 Hz), 3.68 (s, 1H), 1.38 (d, 3H, J = 7.0 Hz); 13C NMR d 162.1,
159.5, 156.1, 149.5, 143.8, 142.1, 141.2, 130.7, 127.8, 122.3,
121.7, 117.1, 49.2, 26.3, 23.0; MS (C17H15BrN4O) estimated
370.04 found, 371.2 and 373.2 (M+H).
6.2.15. (S,E)-Methyl 3-(3-(6-bromopyridin-2-yl)-2-
cyanoacrylamido)-3-phenylpropanoate (18)
1H NMR (300 MHz, CDCl3) d 8.20 (s, 1H), 7.76 (d, 1H, J = 7.8 Hz),
7.62 (m, 3H), 7.32 (m, 5H), 5.52 (m, 1H), 3.70 (s, 1H), 2.98 (m, 2H);
13C NMR (125 MHz, CDCl3) d 171.03, 158.84, 150.90, 148.51,
142.49, 139.56, 139.17, 130.70, 128.96, 128.09, 126.24, 125.39,
115.55, 109.48, 52.11, 50.82, 39.73.
6.2.16. (S,E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-(1-(2-
fluorophenyl)ethyl)acrylamide (19)
1H NMR (500 MHz, CDCl3) d 8.19 (s, 1H), 7.66 (m, 1H), 7.58 (m,
2H), 7.31 (m, 2H), 7.14 (t, 1H, J = 7.5 Hz), 7.08 (m, 1H), 6.99 (d, 1H,
J = 7.0 Hz), 5.41(m 1H, J = 8.4 Hz), 1.61(d, 3H, J = 7.0 Hz); 13C NMR
6.2.23. (E)-N-(3-Aminobenzyl)-3-(6-bromopyridin-2-yl)-
2-cyanoacrylamide (26)
(150 MHz, CDCl3)
d 161.45, 159.82, 158.47, 150.88, 148.40,
142.45, 139.18, 130.68, 129.43, 129.37, 129.16, 129.07, 127.87,
127.84, 125.47, 124.51, 124.49, 116.16, 116.02, 115.65, 109.38,
46.53, 21.29.
1H NMR (600 MHz, DMSO-d6) d 8.74 (t, 1H, J = 5.6 Hz), 8.57 (s,
1H), 7.15 (d, 1H, J = 10.1 Hz), 7.91 (t, 1H, J = 7.8 Hz), 7.84 (d, 1H,
J = 7.8 Hz), 7.35 (s, 4H), 4.32 (d, 2H, J = 5.8 Hz), 3.7 (s, 2H); MS
(C16H13BrN4O) estimated 356.03 found, 355.3 and 357.3 (M+H).
6.2.17. (E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-
(cyclopropyl(phenyl)methyl)acrylamide (20)
6.2.24. (S,E)-2-Cyano-3-(4-nitrophenyl)-N-
1H NMR (600 MHz, CDCl3) d 8.20 (s, 1H), 7.67 (t, 1H, J = 7.8 Hz),
7.58 (m, 2H), 7.38 (m, 4H), 7.30 (m, 1H), 6.99 (d, 1H, J = 7.8 Hz),
4.51 (t, 1H, J = 8.4 Hz), 1.29 (m, 1H), 0.68 (m, 2H), 0.50 (m, 1H),
0.44 (m, 1H); 13C NMR (600 MHz, CDCl3) d 158.22, 148.96,
(1-phenylbutyl)acrylamide (27)
1H NMR (600 MHz, CDCl3) d 9.08 (d, 1H, 2.1 Hz), 8.73 (s, 1H), 8.60
(dd, 1H, J = 6.0, 2.1 Hz), 7.32 (m, 5H), 6.6 (d, 2H, J = 8.1 Hz), 5.08 (q,
1H, J = 7.5 Hz), 1.90 (m, 2H), 1.46 (m, 2H), 0.96 (t, 3H, J = 7.5 Hz); 13
C

7202
Z. Peng et al. / Bioorg. Med. Chem. 19 (2011) 7194–7204
NMR d 157.1, 151.2, 148.7, 140.9, 134.4, 132.2, 128.9, 128.9, 128.5,
127.9, 126.6, 126.6, 120.9, 115.4, 110.0, 54.9, 38.0, 19.5, 13.7; MS
(C20H19N3O3) estimated 349.14 found, 350.2 (M+H).
6.2.31. (S,E)-2-Cyano-3-(4-hydroxy-3-nitrophenyl)-N-(1-
phenylbutyl)acrylamide (34)
1H NMR (600 MHz, CDCl3) d 8.25 (s, 1H), 7.20 (d, 1H, J = 9.0 Hz),
7.60 (s, 1H), 7.49 (d, 1H, J = 8.4 Hz), 7.33 (m, 5H), 6.65 (d, 1H,
J = 7.8 Hz), 5.07 (q, 1H, J = 7.2 Hz), 1.90 (m, 2H), 1.44 (m, 2H),
0.97 (t, 3H, J = 7.5 Hz); 13C NMR d 158.1, 154.9, 149.8 141.1,
139.9, 134.8, 132.9, 131.6, 130.8, 128.9, 128.9, 127.8, 126.5,
126.5, 126.0, 121.9, 120.9, 115.8, 109.2, 54.8, 38.1, 19.5, 13.7; MS
(C20H19N3O4) estimated 365.137 found, 366.5 (M+H).
6.2.25. (S,E)-2-Cyano-3-(2,4-dinitrophenyl)-N-(1-phenylbutyl)
acrylamide (28)
1H NMR (600 MHz, CDCl3) d 9.11 (d, 1H, J = 1.0 Hz), 8.76 (s, 1H),
8.64 (dd, 1H, J = 4.5, 1.2 Hz), 7.98 (d, 1H, J = 4.2 Hz), 7.35 (m, 5H),
6.58 (d, 1H, J = 3.9 Hz), 5.10 (q, 1H, J = 7.5 Hz), 1.93 (m, 2H), 1.39
(m, 2H), 0.99 (t, 3H, J = 7.5 Hz); 13C NMR d 157.1, 149.1, 148.7,
140.9, 134.4, 132.0, 128.9, 128.9,128.5, 128.0, 127.9, 126.6, 126.6,
120.9, 114.6, 112.3, 54.9, 38.0, 19.5, 13.7; MS (C20H18N4O5) esti-
mated 394.12 found, 393.2 (MꢁH).
6.2.32. (S,E)-3-(3-Bromo-4-hydroxyphenyl)-2-cyano-N-(1-
phenylbutyl)acrylamide (35)
1H NMR (600 MHz CDCl3) d 8.13 (s, 1H), 8.03 (s, 1H), 7.20 (d, 1H,
J = 8.4 Hz), 7.30 (m, 5H), 7.05 (dd, 1H, J = 8.4, 0.6 Hz), 6.59 (d, 1H,
J = 7.8 Hz), 5.07 (q, 1H, J = 7.2 Hz), 1.90 (m, 2H), 1.44 (m, 2H),
0.97 (t, 3H, J = 7.5 Hz); 13C NMR d 156.6, 153.6, 141.1, 137.7,
132.9, 130.6, 130.4, 130.4, 128.9, 127.8, 126.5, 126.5, 123.5,
123.5, 122.3, 122.2, 120.6, 116.9, 114.8 54.9, 38.2, 19.5, 13.7; MS
(C20H19BrN2O2) estimated 398.06 found, 399.4 and 401.2 (M+H).
6.2.26. (S,E)-2-Cyano-3-(2-nitrophenyl)-N-(1-phenylbutyl)
acrylamide (29)
1H NMR (600 MHz, CDCl3) d 8.71 (d, 1H, J = 2.0 Hz), 8.23 (dd, 1H,
J = 4.5, 1.2 Hz), 7.75 (d, 2H, J =4.2 Hz), 7.65 (m, 1H), 7.35 (m, 4H),
7.29 (m, 1H), 6.68 (m, 1H), 5.09 (q, 1H, J =7.5 Hz), 1.89 (m, 2H),
1.37 (m, 2H), 0.93 (t, 3H, J = 7.5 Hz); 13C NMR d 158.1, 151.2,
147.5, 141.3, 134.4, 131.8, 130.4, 128.8, 128.8, 128.7, 127.7,
126.7, 126.7, 125.4, 115.4, 110.0, 54.7, 38.1, 19.5, 13.8; MS
(C20H19N3O3) estimated 349.14 found, 348.5 (MꢁH).
6.2.33. (R,E)-2-(3-(6-Bromopyridin-2-yl)-2-cyanoacrylamido)-2-
phenylethyl acetate (36)
1H NMR (600 MHz, CDCl3) d 8.20 (s, 1H), 7.67 (m, 1H), 7.59 (m,
2H), 7.38 (m, 5H), 7.28 (d, 1H, J = 8.4 Hz), 6.50 (d, 1H, J = 7.2 Hz),
5.39 (m 1H), 4.44 (m 2H), 2.08 (s, 3H); 13C NMR d 171.0, 159.1,
150.7, 148.6, 142.5, 139.2, 137.3, 130.8, 129.0, 129.0, 128.4,
126.7, 126.7, 125.6, 115.6, 109.2, 65.8, 53.7, 20.8; MS
(C19H16BrN3O3) estimated 413.037 found, 414.1 and 416.1 (M+H).
6.2.27. (S,E)-2-Cyano-3-(3-nitrophenyl)-N-(1-phenylbutyl)
acrylamide (30)
1H NMR (600 MHz CDCl3) d 8.68 (d, 1H, J = 1.8 Hz), 8.36 (m, 2H),
8.24 (d, 1H, J = 7.8 Hz), 7.70 (t, 1H, J = 7.8 Hz), 7.33 (m, 5H), 6.62 (d,
1H, J = 7.8 Hz), 5.08 (q, 1H, J = 7.2 Hz), 1.90 (m, 2H), 1.38 (m, 2H),
0.96 (t, 3H, J = 7.2 Hz); 13C NMR d 158.3, 150.1, 148.7, 141.2,
135.1, 133.4, 130.4, 128.9, 128.9, 127.8, 126.6, 126.5, 125.2,
116.0, 107.7, 54.8, 38.1, 19.5, 13.7; MS (C20H19N3O3) estimated
349.14 found, 348.3 (MꢁH).
6.2.34. (S,E)-2-Cyano-3-(3,4-dinitrophenyl)-N-(1-phenylethyl)
acrylamide (37)
1H NMR (600 MHz, CDCl3) d 8.23 (s, 1H), 8.16 (s, 1H), 8.08 (d,
1H, J = 9.0 Hz), 7.37 (m, 5H), 7.10 (d, 1H, J = 9.0 Hz), 5.23 (d, 1H,
J = 7.2 Hz), 1.59 (d, 3H, J = 7.2 Hz; MS (C18H14N4O5) estimated
366.096 found, 367.2 (M+H).
6.2.28. (S,E)-3-(4-Chloro-3-nitrophenyl)-2-cyano-N-
(1-phenylbutyl)acrylamide (31)
1H NMR (600 MHz, CDCl3) d 8.32 (d, 1H, J = 2.4 Hz), 8.26 (s, 1H),
8.07 (dd, 1H, J = 6.0, 1.8 Hz), 7.68 (d, 1H, J = 8.4 Hz), 7.31 (m, 5H),
6.58 (d, 1H, J = 7.8 Hz), 5.08 (q, 1H, J = 7.2 Hz), 1.90 (m, 2H), 1.38
(m, 2H), 0.96 (t, 3H, J = 7.2 Hz); 13C NMR d 158.1, 148.7 147.5,
141.1, 133.5, 132.9, 131.6, 130.8, 128.9, 128.9, 127.8, 127.1 126.5,
126.5, 115.9, 107.8, 54.8, 38.1, 19.5, 13.7; MS (C20H18ClN3O3) esti-
mated 383.10 found, 384.2 (M+H).
6.2.35. (E)-N-Benzhydryl-3-(6-bromopyridin-2-yl)-2-
cyanoacrylamide (38)
1H NMR (600 MHz, CDCl3) d 8.26 (s, 1H), 7.67 (m, 1H), 7.59 (m,
2H), 7.38 (m, 5H), 7.33 (m 11H), 6.50 (d, 1H, J = 7.2 Hz); 13C NMR d
158.8, 150.8, 148.8, 142.5, 140.3, 139.2, 130.8, 129.1, 129.0 (4C),
128.3 (2C), 128.0, 127.4 (4C), 126.9, 125.7, 115.7, 109.1, 58.2; MS
(C22H16BrN3O) estimated 417.047 found, 418.1 and 420.1 (M+H).
6.2.29. (S,E)-3-(2-Chloro-6-nitrophenyl)-2-cyano-N-(1-phenyl-
butyl)acrylamide (32)
6.2.36. (E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-
(1,2-diphenylethyl)acrylamide (39)
1H NMR (600 MHz, CDCl3) d 8.54 (s, 1H), 8.16 (d, 1H, J = 8.4 Hz),
7.82 (d, 1H, J = 7.8 Hz), 7.59 (t, 1H, J = 8.4 Hz), 7.36 (m, 5H), 6.62 (d,
1H, J = 7.8 Hz), 5.10 (q, 1H, J = 7.2 Hz), 1.90 (m, 2H), 1.36 (m, 2H),
0.97 (t, 3H, J = 7.2 Hz); 13C NMR d 157.4, 149.0, 148.3, 141.1,
135.5, 134.7, 131.1, 128.9, 128.9, 128.0, 127.8, 126.7, 126.7,
123.8, 114.2, 113.5, 54.8, 38.1, 19.5, 13.8; MS (C20H18ClN3O3) esti-
mated 383.10 found, 382.2 (MꢁH).
1H NMR (600 MHz, CDCl3) d 8.06 (s, 1H), 7.56 (t, 1H, J = 7.8 Hz),
7.50 (d, 1H, J = 7.8 Hz),), 7.45 (d, 1H, J = 7.8 Hz), 7.20 (m 10H),
5.34 (q, 1H, J = 7.2 Hz), 3.19 (m 2H), 13C NMR d 158.8, 150.8,
148.3, 142.4, 140.8, 139.3, 136.8, 130.7, 129.4 (2C), 128.8 (2C),
128.7 (2C), 127.8,126.9, 126.6 (2C), 125.8, 115.7, 109.1, 56.0, 42.5;
MS (C23H18BrN3O) estimated 431.063 found, 432.2 and 434.2
(M+H).
6.2.30. (S,E)-2-Cyano-3-(4-hydroxyphenyl)-N-(1-phenylbutyl)
acrylamide (33)
6.2.37. (R,E)-3-(4-Chloro-3-nitrophenyl)-2-cyano-N-
(2-hydroxy-1-phenylethyl)acrylamide (40)
1H NMR (600 MHz, CDCl3) d 8.14 (s, 1H), 7.86 (dd, 2H, J = 6.0,
2.1 Hz), 7.32 (m, 5H), 6.7 (d, 2H, J = 8.1 Hz), 6.62 (d, 1H,
J = 7.8 Hz), 5.08 (q, 1H, J = 7.2 Hz), 1.90 (m, 2H), 1.46 (m, 2H),
0.96 (t, 3H, J =7.5 Hz); 13C NMR d 160.9, 157.0, 155.7, 151.2,
144.1, 135.2 132.0, 131.4, 128.9, 128.9, 127.7, 126.5, 126.5, 125.9,
117.0, 111.1, 54.8, 38.3, 19.5, 13.7; MS (C20H20N2O2) estimated
320.15 found, 321.6 (M+H).
1H NMR (600 MHz, DMSO-d6) d 8.32 (d, 1H, J = 8.4 Hz), 8.13 (s,
1H), 8.08 (dd, 1H, J = 9.0, 2.4 Hz), 7.36 (m, 4H), 7.30 (m, 1H), 7.08
(d, 1H, J = 8.4 Hz), 7.04 (d, 1H, J = 6.6 Hz), 5.20 (q, 1H, J = 7.2 Hz),
3.96 (m 2H), 2.08 (s, 3H); 13C NMR d 160.9, 150.7, 148.6, 139.0,
138.3, 134.1, 131.2, 130.8, 129.0, 129.0, 128.2, 126.7, 126.7,
125.6, 121.4, 119.9, 117.3, 100.8, 65.9, 56.5, 20.8; MS
(C18H14ClN3O4) estimated 371.067 found, 372.2 (M+H).

Z. Peng et al. / Bioorg. Med. Chem. 19 (2011) 7194–7204
7203
6.2.38. (S,E)-2-Cyano-3-(4-methyl-3,4-dihydro-2H-benzo[b]
6.2.45. (E)-2-Cyano-N-((5-methylfuran-2-yl)methyl)-3-(5-(3-
[1,4]oxazin-7-yl)-N-(1-phenylethyl)acrylamide (41)
nitrophenyl)furan-2-yl)acrylamide (48)
1H NMR (600 MHz, CDCl3 d 8.06 (s, 1H), 7.47 (d, 1H, J = 1.2 Hz),
7.42 (dd, 1H, J = 9.0, 2.4 Hz), 7.33 (m, 4H), 7.27 (m, 1H), 6.60 (d, 1H,
J = 8.4 Hz), 6.51 (d, 1H, J = 6.6 Hz), 5.20 (p, 1H, J = 7.2 Hz), 4.22 (t,
2H, J = 4.8 Hz), 3.41 (t, 2H, J = 4.8 Hz), 2.99 (s, 3H), 1.56 (d, 3H,
J = 6.6 Hz); 13C NMR d 160.4, 152.7, 143.2, 142.8, 131.7, 128.8
(2C), 128.2, 127.6, 126.2, 126.1 (2C), 120.9, 118.6, 117.2, 110.8,
63.8, 49.9, 48.7, 38.1, 21.5; MS (C21H21N3O2) estimated 347.163
found, 348.3 (M+H).
1H NMR (600 MHz, DMSO-d6) d 8.85 (t, 1H, J = 5.4 Hz), 8.71 (s,
1H), 8.30 (d, 1H, J = 8.4 Hz), 8.25 (d, 1H, J = 8.4 Hz), 8.05 (s, 1H),
7.84 (t, 1H, J = 7.8 Hz), 7.64 (d, 1H, J = 3.6 Hz), 7.49 (d, 1H, 3.6 Hz),
2.24 (s, 3H), 6.16 (s, 1H), 6.00 (s, 1H), 4.35 (d, 2H, J = 5.4 Hz).
6.3. General procedure for reductive amination using
asymmetric aromatic amines with BAL-PG-PS resin
Resin (1 g), NaBH3CN (1.56 g, 25 equiv), DCE (35 mL) 2-phenyl
ethyl amine (3.25 mL, 25 equiv), AcOH (0.38 mL, 4 equiv) was ro-
tated on orbital shaker for 24 h. The resultant secondary amine re-
sin was washed with DCM (10 mL ꢂ 10) and DMF–CH2Cl2 (1:1)
(10 mL ꢂ 10) and dried well. The reaction was checked by the nin-
hydrin test (color shows purple).
6.2.39. (S,E)-2-Cyano-3-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-
N-(1-phenylethyl)acrylamide (42)
1H NMR (600 MHz, CDCl3) d 8.26 (s, 1H), 7.79 (d, 1H, J = 1.2 Hz),
7.82 (d, 1H, J = 7.8 Hz), 7.58 (dd, 1H, J = 8.4, 1.8 Hz), 7.36 (m, 4H),
7.30 (m, 1H), 7.17 (d, 1H, J = 8.4 Hz), 6.56 (d, 1H, J = 6.6 Hz), 5.24
(p, 1H, J = 7.2 Hz), 1.60 (d, 3H, J = 6.6 Hz); 13C NMR d 158.9, 151.5,
146.6, 144.4, 142.1, 131.7, 128.9 (4C), 128.1, 127.9, 126.2 (2C),
116.7, 110.1, 103.9, 50.3, 21.7; MS (C19H14F2N2O3) estimated
356.097 found, 358.1 (M+H).
6.4. General procedure for cyanoacylation of secondary amine
A solution of cyanoacetic acid (3 g, 20 equiv), DIPCDI (20 equiv),
DIPEA (20 equiv) in DMF (30 mL) was added to the above resin and
rotated on an orbital shaker for 7 h. The resin was washed with
DMF (20 mL ꢂ 10) and the acylation was repeated for another
7 h. The resin was washed and dried. The reaction was checked
by ninhydrin test.
6.2.40. (E)-2-Cyano-N-(cyclopropyl(phenyl)methyl)-3-
(5-(3-nitrophenyl)furan-2-yl)acrylamide (43)
1H NMR (600 MHz, CDCl3) d 8.63 (s, 1H), 8.23 (d, 1H, J = 8.4 Hz),
8.19 (d, 1H, J = 7.8 Hz), 8.06 (s, 1H), 7.68 (t, 1H, J = 7.2 Hz), 7.39 (m,
4H), 7.30 (t, 1H, J = 6.6 Hz), 7.20 (s, 1H), 7.05 (d, 1H, J = 2.4 Hz), 6.80
(d, 1H, J = 7.2 Hz), 4.52 (t, 1H, J = 8.4 Hz), 1.28 (m, 1H), 0.68 (m, 2H),
0.52 (m, 1H), 0.44 (m, 1H);13C NMR (600 MHz, CDCl3) d 159.53,
149.03, 156.06, 148.78, 141.03, 136.41, 130.50, 130.45, 128.70,
127.69, 126.65, 123.72, 123.32, 120.91, 119.87, 117.19, 112.54,
110.38, 100.54, 58.70, 16.74, 4.19, 4.03; MS (EI) m/e (rel intensity):
414.6 (100), 431.4 (42).
6.5. General procedure for Knoevenagel condensation
A solution of aldehyde (5 equiv) in anhydrous DMF/EtOH (10:2)
(1.5 mL) and 10 drops of piperidine was added to the above resin in
96 wells and shaken over night. The reaction mixture was drained,
and the resin was washed with DMF (1.5 mL ꢂ 10).
6.6. General procedure for final cleavage
6.2.41. (S,E)-2-Cyano-3-(5-(3-nitrophenyl)furan-2-yl)-N-
(1-phenylpropyl)acrylamide (44)
All products were cleaved from the resin with 90% TFA in water
for 1.5 h and collected from 96-well deep well blocks. The solvent
was removed in vacuo washed three times with ether and the res-
idues were dissolved in CH3CN and analyzed by LC/MS.
1H NMR (600 MHz, CDCl3) d 8.63 (s, 1H), 8.22 (d, 1H, J = 7.8 Hz),
8.18 (d, 1H, J = 7.2 Hz), 8.05 (s, 1H), 7.68 (t, 1H, J = 7.8 Hz), 7.33 (m,
5H), 7.19 (m, 1H), 7.04 (m, 1H), 6.57 (d, 1H, J = 7.2 Hz), 4.99 (m,
1H), 1.94 (m, 2H), 0.96 (t, 1H, J = 7.2 Hz).
6.7. Preparation of degrasyn using solid support lanterns
6.7.1. General procedure for reductive amination
6.2.42. (E)-N-(2-Chlorobenzyl)-2-cyano-3-(5-(3-nitrophenyl)
furan-2-yl)acrylamide (45)
A-series BAL Lantern (initial specified loading:750
treated with 11 mL of a solution of amine (0.5 M, 5.3 mmol, 7 -
mol equiv) and sodium cyanoborohydride (0.05 M, 530 mol,
0.7 mol equiv) in 1% acetic acid/DMF at 60 °C for 17 h. After cooling
to rt, the reagent solution is decanted and the Lanterns washed
with DMF (3 ꢂ 3 min) and DCM (3 ꢂ 3 min) 20 mL.
lmol) is
1H NMR (600 MHz, CDCl3) d 8.96 (t, 1H, J = 5.4 Hz), 8.72 (s, 1H),
8.31 (d, 1H, J = 6.0 Hz), 8.26 (d, 1H, J = 6.0 Hz), 8.09 (s, 1H), 7.84 (m,
1H), 7.65 (d, 1H, J = 3.6 Hz), 7.52 (d, 1H, J = 3.6 Hz), 7.46 (d, 1H,
J = 7.8 Hz), 7.36 (m, 3H), 4.50 (s, 2H), 3.17 (d, 1H, J = 5.4 Hz).
l
6.2.43. (E)-2-Cyano-N-(4-methylbenzyl)-3-(5-(3-nitrophenyl)
furan-2-yl)acrylamide (46)
6.7.2. General procedure for cyanoacylation of secondary amine
Same as resin.
1H NMR (600 MHz, DMSO-d6) d 8.91 (t, 1H, J = 5.4 Hz), 8.71 (s,
1H), 8.30 (d, 1H, J = 7.8 Hz), 8.25 (d, 1H, J = 7.8 Hz), 8.06 (s, 1H),
7.84 (t, 1H, J = 6.0 Hz), 7.63 (d, 1H, J = 3.6 Hz), 7.49 (d, 1H,
J = 3.6 Hz), 7.20 (d, 2H, J = 7.8 Hz), 7.14 (d, 2H, J = 7.8 Hz), 4.37 (d,
1H, J = 5.4 Hz), 3.17 (d, 1H, J = 4.8 Hz), 2.28 (s, 3H).
6.7.3. General procedure for Knoevenagel condensation
Same as resin.
6.7.4. General procedure for final cleavage
Same as resin.
6.2.44. (E)-2-Cyano-N-(3-methoxybenzyl)-3-(5-(3-
7. Molecular modeling
7.1. Ligand preparation
nitrophenyl)furan-2-yl)acrylamide (47)
1H NMR (600 MHz, DMSO-d6) d 8.94 (t, 1H, J = 5.4 Hz), 8.72
(s, 1H), 8.31 (d, 1H, J = 7.8 Hz), 8.25 (d, 1H, J = 7.8 Hz), 8.07
(s, 1H), 7.84 (t, 1H, J = 7.8 Hz), 7.63 (d, 1H, J = 3.6 Hz), 7.50 (d, 1H,
J = 3.6 Hz), 7.26 (t, 1H, J = 7.8 Hz), 6.89 (s, 2H), 6.83 (d, 1H,
J = 6.0 Hz), 4.40 (d, 1H, J = 6.0 Hz), 3.75 (s, 3H).
Each of the compounds were drawn into ChemDraw, exported in
MOL format, and concatenated into a single SDF formatted file.

7204
Z. Peng et al. / Bioorg. Med. Chem. 19 (2011) 7194–7204
LigPrep21 was used to generate a single 3D structure of each com-
pound. A MacroModel22 conformational search using the mixed tor-
sional/low-mode sampling method was used to generate five
representative low energy starting configurations for the quantum
chemical calculations. The OPLS2005 force field23 was used with
water solvent. The default value for the maximum iterations during
minimization was increased from 500 to 1500 to allow for conver-
gence that is more consistent. The number of steps for conformation
acid and 1 M HCl such that the final concentration of acetic acid
is 2.5% and HCl is 2.5%) into each well and incubated for 6 h. The
OD570 of each sample is determined by using a SPECTRA MAX
M2 plate reader (Molecular Devices). The OD in control and treated
wells is used as an estimate of the effect of compounds on cell
growth and survival.
8.3. Cell lines
search was increased to 1500 steps and redundant conformers were
0
eliminated based on 1.0 ÅA RMSD cutoff. Additionally, the dihedral
C@C–C(@O)–N between double bond and carbonyl was fixed at
180.0° to avoid a bias of sampling configuration near 0.0°, which
was observed during the conformational search, but not observed
as favorable during ab initio calculations.
Multiple myeloma cell lines (MM-1, U266, OCI-My4) were
grown in RPMI 1640 containing 10% heat-inactivated fetal bovine
serum and 2 mM glutamine.
Acknowledgements
7.2. Quantum chemical calculations
The authors thank Cancer Center support grant CA016672 for
the support of the translational chemistry core Facility and NMR
facility at M. D. Anderson Cancer Center. We also gratefully
acknowledge funding support from Callisto Pharmaceuticals (New
York, NY).
Quantum chemical calculations were completed using Jaguar
(Version 7.6, Schrödinger, LLC, New York, NY, 2009). All geometry
optimization were completed using Density Functional Theory
(DFT) and B3LYP functional. As many structures contain a Br atom,
it was necessary to select a basis set with an effective core poten-
tial (ECP). Both the LACVP and LACV3P basis sets utilizes the 6-31G
basis set for all atoms H-Ar, and the LCA pseudopotential for Br.24
Calculation on the five low-energy structures from conformational
search were completed with the LACVP⁄ basis set and the lowest
configuration for each was submitted for full property analysis at
with the LACV3P⁄⁄ level. Molecular orbital surfaces were created
for the LUMO level in each case and these were mapped onto the
electron density to highlight the preferred position of attack of
HOMO of cysteine. The atomic Fukui indices25 were also calculated
for each structure to give a numeric measure of the reactivity. In
particular, we focused on the LUMO FNN value, which is a measure
of the change in electron density when a molecule undergoes reac-
tion with a change in electron density but spin density held con-
stant. This quantity relates the reactivity of each atom towards
an incoming nucleophile.26 This value may also be combined with
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.09.057.
References and notes
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Immunol. 1998, 28, 3523.
the global electrophilicity index
index, k via the equation, k = fNN
index is a more absolute scale of electrophilicity that is indepen-
dent of the nucleophilic partner. It is defined as ; where
2/2
ꢃ (eH L)/2 and
x
, to give the atom electrophilicity
x
x
x.
27 The global electrophilicity
x
=
l
g
l
+
e
g
ꢃ
eL
ꢁ
eH.
8. Biology
8.1. Screening for anti-tumor activity
12. Iwamaru, A.; Szymanski, S.; Iwado, E.; Aoki, H.; Yokoyama, T.; Fokt, I.; Hess, K.;
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2007, 26, 2435.
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All compounds are tested for purity (HPLC, NMR) and the calcu-
lated molecular weight is used to make up a 10 mM stock solution
of each compound (in 100% dimethylsulfoxide or 50% dimethyl-
sulfoxide:50% polyethylene glycol 300). Compounds are diluted
into cell culture media consisting of RPMI1640 with 10% fetal bo-
vine serum to a final starting concentration of 10 lM. [Highest final
DMSO content = 0.1%]. Tumor cells (20,000) are plated into individ-
ual wells of a 96-well culture plate in 0.1 mL of culture media.
Diluted compounds are added to individual wells containing
pre-plated cells (final volume 0.2 mL) and incubated at 37 °C for
24–72 h. Well receive vehicle alone as a control.
8.2. MTT (viability) assay
MTT reagent (20
to the cells and the plates are incubated at 37 °C for another 2 h.
Cells are lysed by adding 100 l of lysis buffer (20% SDS in 50%
N,N-dimethylformamide (Sigma) adjusted to pH 4.7 by 80% acetic
ll of 5 mg/mL stock solution, Sigma) is added
l