1,1-Diarylalkenes as anticancer agents: Dual inhibitors of tubulin polymerization and phosphodiesterase 4

Article abstract of DOI:10.1016/j.bmc.2011.08.068

A series of 1,1-diarylalkene derivatives were prepared to optimize the properties of CC-5079 (1), a dual inhibitor of tubulin polymerization and phosphodiesterase 4 (PDE4). By using the 3-ethoxy-4-methoxyphenyl PDE4 pharmacophore as one of the aromatic ri

Full text of DOI:10.1016/j.bmc.2011.08.068

Bioorganic & Medicinal Chemistry 19 (2011) 6356–6374
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,1-Diarylalkenes as anticancer agents: Dual inhibitors of tubulin
polymerization and phosphodiesterase 4
Alexander L. Ruchelman a, , Hon-Wah Man a, Roger Chen a, Wei Liu a, Ling Lu a, Dorota Cedzik a,
⇑
Ling Zhang a, Jim Leisten b, Alice Collette b, Rama Krishna Narla b, Heather K. Raymon b, George W. Muller a
a Drug Discovery Department, Celgene Corporation, 86 Morris Avenue, Summit, NJ 07901, USA
b Department of Pharmacology, Celgene Signal Research, 4550 Towne Centre Court, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,1-diarylalkene derivatives were prepared to optimize the properties of CC-5079 (1), a dual
inhibitor of tubulin polymerization and phosphodiesterase 4 (PDE4). By using the 3-ethoxy-4-methoxy-
phenyl PDE4 pharmacophore as one of the aromatic rings, a significant improvement in PDE4 inhibition
was achieved. Compound 28 was identified as a dual inhibitor with potent PDE4 (IC50 = 54 nM) and anti-
Received 27 June 2011
Revised 24 August 2011
Accepted 30 August 2011
Available online 5 September 2011
tubulin activity (HCT-116 IC50 = 34 nM and tubulin polymerization IC50 ꢀ1
lM). While the nitrile group
at the alkene terminus was generally required for potent antiproliferative activity, its replacement was
tolerated if there was a hydroxyl or amino group on one of the aryl rings. Conveniently, this group could
also serve as a handle for amino acid derivatization to improve the compounds’ solubility. The glycinam-
ide analog 45 showed significant efficacy in the HCT-116 xenograft model, with 64% inhibition of tumor
growth upon dosing at 20 mg/kg qd.
Keywords:
Anticancer
Antitubulin
Combretastatin A-4 (CA-4)
Phosphodiesterase 4 (PDE4)
Ó 2011 Elsevier Ltd. All rights reserved.
Tumor necrosis factor-
HCT-116
a (TNF-a)
1. Introduction
Unlike colchicine, vinblastine, and paclitaxel, 1 was equipotent in
parental cancer cells and their multi-drug resistant variants, indi-
Microtubules are a crucial component of the intracellular
machinery responsible for mitosis and cell division. They consist
cating that this novel analog can evade P-gp-mediated drug resis-
tance seen with other chemotherapeutic agents in tested cell lines.
In addition to its activity as a tubulin inhibitor, 1 also potently
inhibits phosphodiesterase 4 (PDE4) enzymatic activity. PDE4
inhibitors are commonly known for their general anti-inflammatory
and bronchodilatory properties.32–34 Inhibition of PDE4 results in
accumulation of the second messenger cAMP, which attenuates
multiple pro-inflammatory processes in neutrophils, eosinophils,
mast cells, basophils, lymphocytes, monocytes, and macrophages.35
Notably, inhibition of PDE4 results in decreased production of the
of polymerized chains of
a-tubulin and b-tubulin heterodimers;
compounds that perturb the polymerization dynamics of
a
,b-tubu-
lin dimers cause mitotic arrest and can trigger cell death.1,2 For this
reason, microtubules are an important target in cancer therapy.3–5
Moreover, recent literature reports indicate that by disruption of
the endothelial cytoskeleton, certain tubulin-binding agents can
block the blood supply to tumors.6–9 Combretastatin A-4 (CA-4)
(Fig. 1) is a cis stilbene natural product that binds at the colchicine
site of tubulin and exhibits potent antineoplastic and antivascular
activity by virtue of its microtubule destabilizing activity. Its phos-
phate derivative is currently undergoing clinical trials.10–12 Since
the initial reports describing CA-4’s isolation13–15 and biological
activity,16,17 extensive structure–activity studies have investigated
related stilbenes as well as modifications of the double bond.18–27
For example, phenstatin is an analog wherein the double bond of
CA-4 is replaced by a carbonyl.28,29 Previously, we have disclosed
the structure and biological activity of CC-5079 (1, Fig. 1).30,31 The
diarylacrylonitrile 1 has significant cytotoxic effects on fast-prolif-
erating cells but not on quiescent cells, and was shown to inhibit
tubulin polymerization by binding to the colchicine-binding site.
inflammatory cytokine tumor necrosis factor-a (TNF-a) in mono-
cytes.36 Inhibitors of PDE4 have most frequently been investigated
within the context of respiratory and inflammatory diseases such
as asthma and chronic obstructive pulmonary disease, but there is
evidence indicating that it might also be an useful target in oncol-
ogy. PDE4 is the principal enzyme responsible for cAMP hydrolysis
in 41 of 60 human tumor cell lines evaluated, and lymphoid tumors
such as chronic lymphocytic leukemia cells appear to be susceptible
to PDE4 inhibitor-induced apoptosis.37–39 PDE4 has also been iden-
tified as a target for brain tumor therapy.40,41 In addition, there have
been reports of PDE4 inhibitors having anti-angiogenic activity.42
PDE4 inhibitors have direct apoptotic effects on CLL, ALL, and some
NHL cells, and while the direct antiproliferative properties of PDE4
inhibitors against solid tumors is very modest, they could have
⇑
Corresponding author. Tel.: +1 908 673 9587; fax: +1 908 673 2792.
E-mail address: aruchelman@celgene.com (A.L. Ruchelman).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.068

A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
6357
O
O
O
NHAc
O
O
O
OR
O
O
O
Combretastatin A-4 (CA4) R = H
Combretastatin A-4 phosphate (CA4P) R = PO3Na2
Colchicine
CN
O
O
O
OH
O
O
O
O
O
O
1: CC-5079
Phenstatin
Figure 1. Structures of colchicine, combretastatin A-4 and its phosphate prodrug, phenstatin, and 1.
antitumor effects by virtue of their antiinflammatory activity. For
these reasons, we hypothesize that dual inhibitors of PDE4 and
tubulin polymerization might have superior therapeutic value in
the treatment of some forms of cancer. The combination of these
two independent pharmacological activities into a single small mol-
ecule is a new concept in anticancer therapy. The additional biolog-
ical target also serves to differentiate 1 from other known tubulin
interactive agents, such as CA-4 and paclitaxel.
In common with CA-4, the diarylalkene 1 has a simple structure
and impressive antineoplastic potency. Unfortunately, 1 also pos-
sesses poor aqueous solubility and metabolic stability, detriments
also observed for CA-4.43 In order to both optimize PDE4 and tubu-
lin inhibitory activities as well as improve upon physicochemical
properties, we have undertaken a detailed study of the struc-
ture–activity relationships (SAR). In this report, we describe results
of an exploratory project to study the SAR of this novel series of
anti-tumor agents, which were discovered as an offshoot of our
Apremilast PDE4 inhibitor program.44 A full description of chemi-
cal synthesis and SAR is provided. The results of xenograft studies
for selected analogs are also presented.
depicted in Scheme 2. 4-Bromo-2-ethoxy-1-methoxybenzene was
converted to the corresponding aryllithium and then allowed to react
with various benzaldehydes. The resultant alcohol intermediates 52a–
d were oxidized (MnO2) to benzophenones and then HWE olefination
provided the targeted analogs. The benzophenone intermediates
could be accessed directly by reaction of 2-ethoxy-4-lithio-1-
methoxybenzene with the appropriate Weinreb amide.45
The synthesis of analogs bearing other alkene terminus substitutions
was performed as outlined in Scheme 3. Methyl acrylate 13 was pre-
pared by HWE olefination of the benzophenone 49a with methyl
diethylphosphonoacetate, and saponification provided acrylic acid 14.
Methyl ketone 15 was obtained by a two-step sequence involving Wittig
olefination of 3,5-dimethoxybenzaldehyde to provide the benzalace-
tone 55, followed by Heck coupling under Jeffery’s ligand-free condi-
tions.46,47 Intermediate 51a reacted with phosphorus ylides to provide
alkyl-, aminoalkyl-, and methoxy-substituted alkenes 18–21, as well
the unsubstituted analog 13. Intermediate 51a was also converted to
enyne 22 using the Peterson olefination reaction with 1,3-bis(trimethyl-
silyl)propyne.48,49 The synthesis of phenol-containing analogs started
with TBS protection of 4-bromo-2-hydroxyphenol and by way of the
analogous synthetic route, the targeted analogs were obtained upon
TBS deprotection (Scheme 4). For the synthesis of anilines, 4-meth-
oxy-3-nitrobenzaldehyde was added to a solution of the appropriate
Grignard reagent (Scheme 5). Subsequent synthetic manipulation pro-
vided nitro intermediates, which were then reduced using either stan-
nous chloride or catalytic hydrogenation. Aniline 35 was treated with
2,5-dimethoxytetrahydrofuran in refluxing THF to provide the corre-
sponding pyrrole 39. For the synthesis of dimethylamino analog 38, 4-
dimethylaminobenzaldehyde was added to a solution of 3-ethoxy-4-
methoxyphenyllithium. Surprisingly, treating the resultant benzyl alco-
hol 63 with excess manganese dioxide resulted in partial demethylation
of the aniline; a separable mixture of mono- and dimethylamino benz-
ophenones was obtained in 23% and 17% yields, respectively, after flash
column chromatographic purification. The individual products were
subsequently carried forward to diarylacrylonitrile analogs 37 and 38.
The anilines 11 and 36 were converted to amide derivatives as shown
in Scheme 6. Treatment of 11 with acetyl chloride in refluxing THF
provided 44. Aniline 36 was coupled with Boc-protected glycine
and alanine (DCC, HOBt) prior to acidic deprotection. Scheme 7 de-
picts the synthesis of carbamate- and ester-containing analogs.
Reaction of 35 with N,N-dimethylcarbamyl chloride provided di-
methyl carbamate 47. The phenol 35 was also converted to amino
acid esters by DCC coupling with Boc-protected glycine, alanine,
and valine, followed by deprotection of the Boc group.
2. Results and discussion
2.1. Chemistry
The general method for the synthesis of 1 and related 3,3-diarylacr-
ylonitrile analogs, as well as the 3,3-diarylacrylamide 14 is outlined in
Scheme 1. Friedel–Crafts reaction of commercially available acid chlo-
rides with veratrole provided benzophenone intermediates, which
were then subjected to Horner–Wadsworth–Emmons (HWE) alkeny-
lation with diethyl cyanomethylphosphonate. The products were typ-
ically obtained as a ꢀ1:1 mixture of geometric isomers. The E- and Z-
isomers of 1, compounds 1E and 1Z, were separated by preparative
HPLC. The stereochemical assignment was made based on NOE, as
shown in Figure 2. Compound 1E, with the nitrile trans to the 3,5-
dimethoxyphenyl ring, displays NOE between the alkene proton and
the ortho protons of the 3,5-dimethoxyphenyl ring, while compound
1Z, which has the nitrile cis to the 3,5-dimethoxyphenyl ring, shows
NOE between the alkene proton and the ortho protons on the 3,4-
dimethoxyphenyl ring. Compound 1 could be converted to acrylamide
14 upon treatment with potassium trimethylsilanolate in toluene.
Commercially available benzophenones were also converted to diary-
lacrylonitriles 3–9 using HWE conditions. Related procedures were
used to prepare analogs possessing a 3-ethoxy-4-methoxyphenyl ring,

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A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
O
O
R1
R2
R1
Cl
+
a
O
R2
O
R3
O
R3
O
51a-c
O
CN
NH2
R1
R2
R1
R2
c
b
O
O
R3
O
R3
O
1;
R1=R3=OCH3; R2=H
14
; R1=R3=OCH3; R2=H
2; R1=R2=OCH3; R3=H
30
; R1=OCH2CH3; R2=OCH3; R3=H
CN
O
R1
R2
R1
R2
R6
R5
R6
b
R5
R3
R3
R4
R4
3; R1=R3=R5=OCH3; R2=R4=R6=H
4
5
; R1=R2=R3= R4=R5=R6=OCH3
; R2=R4=R5=OCH3; R1=R3=R6=H
6; R1=R4=R5=OCH3; R2=R3=R6=H
7
8
; R1=R3=R4=OCH3; R2=R5=R6=H
; R1=R3=R5=OCH3; R2=R4=R6=H
9; R1=R3=OCH3; R2=R4=R5=R6=H
Scheme 1. Synthesis of 3,3-diarylacrylonitriles and 3,3-diarylacrylamide 14. Reagents and conditions: (a) AlCl3, CH2Cl2; (b) (EtO)2P(O)CH2CN, LiHMDS or NaH, THF; (c)
KOSi(CH3)3, PhCH3.
Table 1 summarizes the biological data for inhibition of HCT-
116 cell growth and inhibition of PDE4 and TNF-a. Data obtained
with CA-4 are also presented for reference. The lead analog 1 po-
tently inhibited the growth of HCT-116 cells, with an IC50 of
H
CN
H
NC
H
O
H
O
O
O
18 nM. It also inhibited PDE4 and TNF-
crease in potency (IC50 values for PDE4 and TNF-
a
, albeit with ꢀ10-fold de-
a
were 350 and
H
O
H
O
270 nM, respectively). When the individual isomers were tested,
1Z was about 10-fold more potent in the HCT-116 assay, and about
O
O
1E
1Z
five-fold more potent in the PDE4 and TNF-a assays. The greater
activity of the 1Z isomer is in accord with results reported for a
previous series of analogs.52 To facilitate rapid synthesis and
screening, subsequent analogs were tested as the ꢀ1:1 E/Z mix-
tures obtained in the HWE reactions. The 3,5-dimethoxyphenyl
moiety was found to be important for potent antiproliferative
activity; its replacement with a 3,4-dimethoxyphenyl group (i.e.,
2) resulted in a nearly 10-fold loss of antiproliferative potency in
the HCT-116 assay, but had no effect on PDE4 inhibitory activity.
On the other hand, if this ring was replaced with the 3,4,5-trime-
thoxyphenyl ring found in CA-4 (i.e., 3), then good antiproliferative
potency was maintained, but PDE4 inhibitory potency dropped six-
fold. Substitution of both phenyl rings with 3,4,5-trimethoxyl (4)
resulted in reduced activity in all assays. Analogs 5–8 are trimeth-
oxy analogs, and among them 6–8 result from deletion of a single
methoxyl from the structure of 1. Data from this series indicates
that the 3,5-dimethoxyphenyl moiety is required for potent anti-
proliferative activity and so is at least one methoxyl on the other
ring. The 3,4-dialkoxyphenyl motif, a well-known PDE4 pharmaco-
phore,53,54 was essential for PDE4 inhibitory activity.
Figure 2. Key NOE correlations for 1E and 1Z.
2.2. Biological evaluation
All analogs were evaluated for in vitro cell growth inhibition
against the human colon carcinoma cell line HCT-116 to evaluate
antitumor activity. Additionally, PDE4 enzyme inhibition was mea-
sured in a cell-free cAMP hydrolysis assay.50 To quantify the down-
stream effect of PDE4 inhibition, TNF-a inhibition was measured in
lipopolysaccharide (LPS)-stimulated human peripheral blood
mononuclear cells (hPBMC).51 Since this is a phenotypic assay
using human cells, it may have greater clinical relevance than inhi-
bition of purified PDE4. However, care should be taken in interpret-
ing the results of the TNF-
also result in the inhibition of TNF-
pound which is toxic to hPBMC would probably interfere with the
a
data, as other cellular phenomena can
a
release. For example, a com-
measurable physiological response (release of TNF-
inhibiting the PDE4 enzyme.
a), without

A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
6359
OH
R1
R2
CHO
Br
R1
R2
+
a
O
O
R3
O
O
R3
52a-d
NC
O
R1
R2
R1
R2
b
c
O
O
O
R3
O
R3
53a-d
28
; R1=R3=OCH3; R2=H
29; R1=OCH2CH3; R2=OCH3; R3=H
30; R1=CH3; R2=OCH3; R3=H
31
; R1=OCH3; R2=R3=H
NC
O
O
R1
R2
O
Br
R1
R1
R2
N
+
a
c
O
O
R2
O
R3
O
O
O
54a-b
33
34;
; R1=F; R2=OCH3
R1=H; R2=SO2CH3
Scheme 2. Synthesis of diarylacrylonitriles with a 4-ethoxy-3-methoxyphenyl ring. Reagents and conditions: (a) n-BuLi or Mg, THF; (b) MnO2, CH2Cl2; (c) (EtO)2P(O)CH2CN,
LiHMDS, THF.
Compound 10, which possesses the 3-hydroxy-4-methoxy-
phenyl ring found in CA-4, was very potent in the HCT-116 assay
(IC50 ꢀ2 nM), as were the amino-substituted analogs 11 and 12.
These analogs suffered substantial loss of PDE4 inhibitory activity;
ally reminiscent of the recently reported isocombretastatins.55,56
Like the other hydroxyl-substituted diarylacrylonitrile 10 without
a 3,4-alkoxyphenyl moiety, these analogs were not active against
PDE4. When the alkene was disubstituted (25) or substituted with
a phenyl ring (26), activity dropped substantially. Use of an amino
group in lieu of the hydroxyl (e.g., 27) was also found to restore
activity in the HCT-116 assay. It is unclear why the presence of po-
lar functionality on the aromatic ring seems to compensate for the
loss of the nitrile group, but its requirement is in accord with pre-
viously disclosed SAR for the structurally related phenstatins28,29
and isocombretastatins.55,56
nevertheless, they did inhibit the release of TNF-
a from hPBMC.
This may be a result of toxicity to hPBMC or it may be due to effects
on another signaling pathway. Combretastatin A-4 was extremely
potent in the HCT-116 assay (IC50 of 0.7 nM), but was not active
as a PDE4 inhibitor. Although CA-4 did inhibit the release of TNF-
a
from hPBMC, it evidently did so by a mechanism other than inhi-
bition of PDE4.
The role of the group at the alkene terminus was explored in a
series of analogs shown in Table 2. In general, the nitrile proved to
be critical for potent antiproliferative and PDE4 inhibitory activity.
Its removal provided 13, which showed a 5-fold and 20-fold drop
in potency in the PDE4 and TNF-a assays, and was entirely inactive
against HCT-116 cells. Replacement of the nitrile with amide (14),
The 3,4-dialkoxyphenyl moiety is a key known pharmacophore
for PDE4 inhibitors. Generally, a methoxy group at the 4-position is
optimal, and a bulky group such as cyclopentoxy at the 3-position
leads to more potent analogs in a majority of reported series of
PDE4 inhibitors which contain the 3,4-dialkoxyphenyl group.53,54
Previously, we have shown that an ethoxy group is sufficient to
confer the same potency boost as a cyclopentoxy at the 3-position
in some series of PDE4 inhibitors.44 In order to enhance potency for
PDE4 inhibition, therefore, diarylacrylonitrile analogs bearing a 3-
ethoxy-4-methoxyphenyl ring were prepared and evaluated for
activity (Table 3). Use of this ring system in conjunction with a
3,5-dimethyoxyphenyl ring provided 28, and resulted in potent
activity in all three assays, with all three IC50 values ꢀ50 nM. In
this case, substitution of ethoxy for methoxy (i.e., 28 vs 1) provided
a sevenfold improvement in PDE4 activity, with no loss of HCT-116
antiproliferative potency. Use of the 3-ethoxy-4-methoxy substitu-
tion motif on both phenyl rings (29) resulted in a 10-fold drop-off
in HCT-116 antiproliferative activity while PDE4 activity remained
unchanged relative to 28. Analogs 29–34, with various mono- or
3,4-disubstitutions, displayed reduced antiproliferative activity
methyl ester (15), or carboxylic acid (16) resulted in a loss of both
antiproliferative activity (IC50 >10
>1 M). Compound 17, which possesses a methyl ketone at this
site, retained activity in the PDE4 and TNF- assays, but was not
lM) and PDE4 inhibition (IC50
l
a
active against HCT-116 cells. Analogs 18–22, variously substituted
with alkyl, aminoalkyl, methoxyl, and ethyne groups, all showed
reduced activity relative to 1. The only one of these analogs to dis-
play any activity against HCT-116 was the methyl analog 18, with
an IC50 of 480 nM. Interestingly, when the 3-hydroxy-4-methoxy-
phenyl ring found in CA-4 was used instead of 3,4-dimethoxy-
phenyl, antiproliferative activity was restored. Compound 23,
which is unsubstituted at the alkene terminus, and compound
24, which possesses a methyl group at this site, both displayed
similar activity against HCT-116 as 1. These analogs are structur-

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A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
CO2CH3
CO2H
O
O
O
O
O
O
O
O
O
O
a
b
51a
15
16
O
O
O
O
O
O
Br
O
O
CHO
O
O
O
c
d
O
55
17
O
O
O
R
O
O
O
O
O
O
O
e
O
O
51a
13
; R=H
18
; R=CH3
19; R=CH2CH3
20
21
; R=CH2CH2N(CH3)2
; R=OCH3
TMS
TMS
O
O
O
O
O
O
Li
f
O
O
O
51a
22
Scheme 3. Synthesis of analogs featuring nitrile replacements. Reagents and conditions: (a) (EtO)2P(O)CH2CO2CH3, LiHMDS, THF; (b) KOH, H2O, CH3OH;(c) Ph3P@CHCOCH3,
CH3CN; (d) Pd(OAc)2, NaOAc, Bu4NBr, DMF; (e) Ph3P@CHR, THF; (f) THF.
R1
R2
CHO
OH
R1
R2
OTBS
O
R3
b
O
O
a
c
Br
OH
Br
OTBS
R3
56
57a-b
R4
R5
R4
R5
O
R1
R2
OTBS
R1
R2
OTBS
R1
R2
OH
O
e
d
O
O
R3
R3
R3
58a-b
59a-h
10
23
; R1=R3=OCH3; R2=R4=H; R5=CN
; R1=R3=OCH3; R2=R4=R5=H
24; R1=R3=OCH3; R2=R4=H; R5=CH3
25
26
; R1=R3=OCH3; R2=H; R4=R5=CH3
; R1=R3=OCH3; R2=R4=H; R5=Ph
35; R1=OCH2CH3; R2=OCH3; R3=R4=H; R5=CN
40
41
R1=OCH2CH3; R2=OCH3; R3=R4=R5=H
; R1=OCH2CH3; R2=OCH3; R3=H; R4=R5=F
Scheme 4. Synthesis of hydroxy-substituted 3,3-diarylacrylonitriles. Reagents and conditions: (a) TBSCl, DIEA, DMF; (b) n-BuLi, THF; (c) MnO2, CH2Cl2; (d) (EtO)2P(O) CHR4R5
or XPh3PCHR4R5, LiHMDS, THF (X = Br or I); (e) TBAF, THF.

A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
6361
OH
O
R1
R2
Br
CHO
R1
R2
R1
R2
+
a
b
O
O
O
R3
NO2
R3
NO2
R3
NO2
60a-c
61a-c
R4
R4
R1
R2
R1
R2
c
d
O
O
R3
35
O
NO2
R3
11; R1=R3=OCH3; R2=H; R4=CN
12
NH2
62a-f
; R1=R2=R3=OCH3; R4=CN
27; R1=R3=OCH3; R2=R4=H
36; R1=OCH2CH3; R2=OCH3; R3=H; R4=CN
42
; R1=OCH2CH3; R2=OCH3; R3=R4=H
43; R1=OCH2CH3; R2=OCH3; R3=H; R4=CH3
CN
CN
e
O
O
O
O
O
NH2
O
N
39
OH
Br
OHC
+
f
g
O
N
O
N
O
O
63
O
O
+
O
NH
O
N
64
65
O
c
c
CN
CN
O
NH
O
N
O
O
37
38
Scheme 5. Synthesis of pyrrole- and amino-substituted 3,3-diarylacrylonitriles. Reagents and conditions: (a) Mg, cat. I2, THF; (b) MnO2, CH2Cl2 or PCC, CH2Cl2; (c) (EtO)2P(O)
CH2CN or BrPh3PCH2R4, LiHMDS, THF; (d) SnCl2, EtOH, EtOAc or H2, Pd/C, EtOAc; (e) 2,5-dimethoxytetrahydrofuran, AcOH; (f) n-BuLi, THF; (g) MnO2, CH2Cl2
but similar PDE4 activity to 28. However, the inclusion of a small
polar group such as hydroxyl or amino was again found to have
a beneficial effect on antiproliferative potency. Use of the 3-hydro-
xy-4-methoxyphenyl ring found in CA-4 resulted in 35, which had
similar activity as 28. The corresponding amino analog 26 exhib-
ited comparable activity, with IC50 values in the range of 10–
50 nM across the three different assays. Use of a mono- or dimeth-
ylamino group at the para-position provided compounds 37–38,
which also had IC50 values ꢀ50 nM in all three assays. Incorpora-
tion of the amino group into a pyrrole provided 39, and had no sig-
nificant impact upon activity. Since these data indicate that the 3-
ethoxy-4-methoxyphenyl group is compatible with the 3-hydroxy-
4-methoxyphenyl, compounds des-nitrile analogs 40–43 were pre-
pared. Unlike des-nitrile analogs 23–25 and 27, which retained
antiproliferative activity but not PDE4 inhibitory activity, these
des-nitrile analogs inhibited PDE4 as well as HCT-116, although

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A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
CN
CN
O
O
a
O
O
O
NH2
O
HN
11
44
O
CN
CN
CN
b
c
O
O
O
O
O
O
O
O
O
NH2
O
HN
O
HN
36
R
NHBoc
R
NH3Cl
6a-b6
45; R=H
46; R=CH3
Scheme 6. Synthesis of amide-containing 3,3-diarylacrylonitriles. Reagents and conditions: (a) AcCl, THF; (b) BocHNCH(R)CO2H, DCC, HOBt, DMF; (c) HCl, CH2Cl2.
CN
CN
a
O
O
O
O
O
O
OH
O
O
35
N
47
CN
CN
CN
b
c
O
O
O
O
O
O
O
O
O
OH
O
O
O
O
35
R
NHBoc
R
NH3Cl
67a-c
48; R=H
49; R=CH3
50; R=CH(CH3)2
Scheme 7. Synthesis of carbamate- and ester-containing 3,3-diarylacrylonitriles. Reagents and conditions: (a) ClCON(Me)2, THF; (b) BocHNCH(R)CO2H, DCC, 4-pyrrolidin-1-
yl-pyridine, CH2Cl2; (c) HCl, CH2Cl2.
they still were ꢀ10-fold more potent in the HCT-116 assay. Re-
moval of the nitrile from 35 provided 40, which had similar activity
to the lead compound 1. Difluorination at the alkene terminus pro-
vided 41, and had no effect on activity. The des-nitrile aniline 42
had very similar activity to phenol analog 40, while addition of a
methyl to the alkene terminus of 42 provided 43, which had sim-
ilar activity against HCT-116 and PDE4.
In order to improve on physicochemical properties, some of the
more potent agents were derivatized as amides and esters (Ta-
ble 4). The effect of acylation upon activity was first examined by
acetylation of 11. Disappointingly, acetamide 44 was ꢀ50-fold less
active against HCT-116, though the IC50 was still a reasonable
92 nM. Neither 11 nor 44 inhibited PDE4 at submicromolar con-
decrease in HCT-116 and PDE4 activity, with a 2–3-fold decline
in activity for alanine derivative 46, and a slightly greater dropoff
for glycine analog 45. Among derivatives of phenol 35, the carba-
mate 47 was inactive against HCT-116 but retained good inhibitory
activity against PDE4, while the amino acid esters (48–50) had no
noticeable loss of activity, with very similar potency to the parent
compound. Considering that many of the analogs in Table 4 exhibit
similar activity to their respective parent compounds, it is possible
that some hydrolysis of these analogs may occur during the assays
to release the parent active agents 35 or 36; no efforts were made
to detect 35 or 36 in the assay media. It is therefore not possible to
say whether the activity observed in these assays is intrinsic to the
analogs themselves or if it is due to hydrolysis to active agents.
However, the stability of 45–50 was tested in human plasma to
determine whether they can function as prodrugs of 35 and 36.
The ester analogs 48–50 were highly unstable in human plasma,
centrations, although both inhibited TNF-a release. For the prepa-
ration of more water-soluble analogs, 36 was derivatized with
small amino acids. These analogs 45–46 displayed a modest

A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
6363
Table 1
Inhibitory effect of 1 and related 3,3-diarylacrylonitrile analogs on the growth of HCT-116 cells, PDE4 activity, and TNF-
a
release from hPBMC
CN
CN
NC
R1
R2
R6
R5
O
O
O
O
O
O
O
O
R3
R4
1-12
1E
1Z
Compd
R1
R2
H
R3
R4
R5
R6
HCT-116a
(lM)
PDE4a
(lM)
TNF-aa
(lM)
130
1E
1Z
2
3
4
5
6
7
8
OCH3
OCH3
OCH3
OCH3
H
0.018 0.001
0.11 0.012
0.011 0.0003
0.14 0.0085
0.041 0.0044
>10
0.024 0.0016
0.31 0.080
0.015 0.0005
0.0012 0.0005
0.97 0.14
0.0025 0.0013
0.0014 0.0005
0.0018 0.0003
0.00074 0.0002
0.35 0.20
0.73 0.24
0.15 0.032
0.31 0.0088
1.8 0.26
2.4 0.61
0.19 0.042
0.16 0.037
3.4 0.55
3.8 1.2
0.27 0.036
0.65 0.23
0.093 0.0006
0.6
0.54 0.15
0.87 0.093
0.35 0.010
0.66 0.13
0.29 0.14
0.44 0.11
2.4 0.81
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
OH
NH2
NH2
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH3
H
OCH3
OCH3
OCH3
H
H
OCH3
H
H
H
H
H
H
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
9
1.9 0.56
2.4 0.87
9.8 1.73
>10
10
11
12
CA-4
0.0012 0.001
0.011 0.024
0.2 0.007
0.011 0.004
H
OCH3
H
H
>10
a
IC50 values are the means of at least three independent determinations.
Table 2
Inhibitory effect of analogs bearing nitrile replacements on the growth of HCT-116 cells, PDE4 activity, and TNF-
a
release from hPBMC
R1
R2
O
O
O
R3
Compd
R1
R2
R3
HCT-116a
(lM)
PDE4a
(l
M)
TNF-aa
(lM)
130
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
CN
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
0.018 0.001
>10
>10
>10
>10
>10
0.48 0.12
>10
>10
>10
>10
0.021
0.030 0.002
0.97 0.15
>10
0.35 0.20
2.2 0.55
6.7 0.67
1.3 0.22
>10
0.14 0.025
1.5 0.34
2.0 0.30
>10
3.6 3.1
3.5
>10
>10
>10
0.27 0.036
4.8 0.63
5.5 2.2
5.4 0.62
>10
0.75 0.16
2.6 0.29
>10
CONH2
CO2CH3
CO2H
COCH3
CH3
CH2CH3
CH2CH2N(CH3)2
OCH3
C„CH
H
>10
6.3 1.8
7.2 3.1
3.0 1.2
0.15 0.15
7.0 1.6
>10
CH3
CH3
Ph
H
OH
OH
OH
NH2
>10
>10
0.013 0.0002
0.33 0.046
a
IC50 values are the means of at least three independent determinations.
completely falling apart to the parent phenol 35 in under 20 min.
On the other hand, amide analogs 45 and 46 were quite stable in
human plasma, with half-lives of 56 h and 27 h, respectively, and
the formation of aniline 36 was not observed. The modifications
did result in improved solubility. For example, the solubility of
of tubulin polymerization. As shown in Table 5, the new analogs
inhibited tubulin polymerization as expected. In the tubulin inhibi-
tion assay, CA-4 was somewhat more potent than the diarylalkene
analogs, in accord with its greater potency against HCT-116. These
findings strongly suggest that the compounds’ cytotoxicity is based
on the same mechanism as that of 1, namely inhibition of tubulin
polymerization.
We have docked compound 28 into the colchicine binding site
of the tubulin crystal structure (PDB code 1SA0)57 using MOE soft-
ware.58 Considering the greater activity of 1Z compared to 1E, the
molecule was docked in the Z-configuration. Figure 3 shows the
docking results for compound 28 overlaid upon the crystal struc-
ture of colchicine. Due to the relatively large binding pocket, two
36 (<3
its conversion to 45 (356
7.4) or 46 (1,045 g/mL at pH 5.0 and 405
l
g/mL at both pH 5.0 and 7.4) was greatly improved by
g/mL at pH 5.0 and 205 g /mL at pH
g/mL at pH 7.4).
l
l
l
l
It has been shown previously that the cytotoxicity of 1 is based
on binding to tubulin at the colchicine binding site, thereby inhib-
iting its polymerization.33 To confirm that the same mechanism
underlies the cytotoxic activity of the new analogs described here-
in, selected compounds were assayed at 5 and 10 lM for inhibition

6364
A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
Table 3
Inhibitory effect of analogs with a 3-ethoxy-4-methoxyphenyl ring on the growth of HCT-116 cells, PDE4 activity, and TNF-
a release from hPBMC
R4
R5
R3
R2
O
O
R1
Compd
R1
R2
R3
R4
R5
HCT-116a
(
lM)
PDE4a
(
lM)
TNF-aa
(lM)
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
OCH3
OCH2CH3
OCH3
CH3
OCH3
F
H
OH
NH2
H
H
Pyrrol-1-yl
OH
OH
NH2
NH2
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
0.034 0.0029
0.44 0.0077
0.20 0.083
0.087 0.019
0.45 0.014
0.13 0.0011
>10
0.015 0.0018
0.019 0.0048
0.055 0.011
0.031 0.0063
0.049 0.0054
0.036 0.0014
0.074 0.012
0.038 0.0075
0.11 0.026
0.054 0.011
0.070 0.015
0.083 0.020
0.082 0.0005
0.11 0.0099
0.07 0.009
0.20 0.0064
0.091 0.030
0.047 0.0099
0.041 0.005
0.052 0.0095
0.070 0.014
0.38 0.048
0.36 0.058
0.68 0.10
0.043 0.014
0.048 0.010
0.18 0.019
0.10 0.032
0.23 0.058
0.031 0.040
0.13 0.057
0.044 0.0056
0.031 0.0066
0.14 0.026
0.13 0.057
0.067 0.033
0.029 0.0017
0.054 0.046
0.38 0.24
OCH3
OCH3
OCH3
H
OCH3
SO2CH3
OCH3
OCH3
NHCH3
N(CH3)2
OCH3
OCH3
OCH3
OCH3
OCH3
F
H
CH3
H
H
0.59 0.090
2.8 0.43
a
IC50 values are the means of at least three independent determinations.
Table 4
Inhibitory effect of ester- and amide-containing analogs on the growth of HCT-116 cells, PDE4 activity, and TNF-
a
release from hPBMC
CN
O
CN
O
NH
O
Ar
O
O
O
44
45-50
M)
Compd
Ar
HCT-116a
(l
PDE4a
>10
(lM)
TNF-aa
(lM)
44
0.092 0.021
0.066 0.026
H
N
NH3Cl
NH3Cl
45
46
0.093 0.0018
0.4 0.072
0.031 0.020
O
O
O
H
N
0.04 0.0014
0.15 0.016
0.027 0.014
O
O
N
47
48
49
>10
0.12 0.01
0.049 0.007
0.069
0.24 0.062
0.058 0.014
0.057 0.0054
O
O
O
O
NH3Cl
NH3Cl
0.019 0.001
0.017 0.0017
O
O
O
O
O
O
NH3Cl
50
0.022 0.0033
0.12 0.010
0.067 0.0030
O
a
IC50 values are the means of at least three independent determinations.

A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
6365
Table 5
Tubulin polymerization inhibition data and inhibition of HCT-116
CN
R1
R2
R6
R5
R3
R4
Compd
R1
R2
R3
R4
R5
R6
Tubulin inhib% at 5
lM
Tubulin inhib% at 10
lM
HCT-116a
(lM)
1
7
OCH3
OCH3
OCH3
OCH3
OCH3
OCH2CH3
OCH3
H
H
H
H
H
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
OH
NH2
OCH2CH3
NH2
NHC(O)CH3
NHC(O)CH2NH3Cl
OCH3
H
H
H
H
H
H
H
H
H
65
67
77
76
48
65
46
59
88
84
88
N/D
N/D
59
76
74
70
N/D
0.018
0.015
0.0025
0.0014
0.034
0.019
0.092
0.023
0.00074
10
11
28
36
44
45
CA-4
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH2CH3
OCH3
a
% inhibition values are the means of at least two independent determinations; N/D: not determined.
Figure 3. Docked models of 28 (green) overlaid on the crystal structure of colchicine (white). A and B display two different binding modes.
different binding modes were possible and these are shown in Fig-
ure 3A and B. The first binding mode, shown in Figure 3A, shows
good overlay of the aromatic rings of compound 28 with colchicine.
The nitrile moiety resides in a similar region as the acetamide of
colchicine. The decision to lock the ligand in the Z-configuration re-
sulted in a reversal of the 3,4- and 3,5-dialkoxy rings relative to the
corresponding rings in colchicine. This is reasonable considering
that results from the SAR study have shown that the 3,4,5-dime-
thoxy substitution is not critical in this series; for example,
replacement of the 3,4,5-trimethoxyphenyl with a 4-methoxy-
phenyl ring (as in compounds 3 and 5) resulted in comparable
activity against HCT-116. In this binding mode, all of the contacts
are nonpolar, forming a fairly large lipophilic pocket. The other
binding mode, shown in Figure 3B, has the 3,4- and 3,5-dialkoxy
rings in the analogous positions to the corresponding rings in col-
chicine. Although the nitrile has no overlay with colchicine, it does
engage in an H-bonding interaction with Ala250 in the tubulin pro-
tein backbone.
control in the study was Camptosar (irinotecan), a topoisomerase I
inhibitor with good efficacy in this model.59,60 As anticipated, Cam-
2500
Vehicle
Cmpd 28; 20 mg/kg, qd, i.p.
Cmpd 45; 20 mg/kg, qd, i.p.
Camptosar; 5 mg/kg, q4d, i.p.
2000
(-16%)
*p<0.001
**p<0.01
1500
1000
500
0
*
*
*
*
(-64%)
(-85%)
**
*
**
*
*
2.3. HCT-116 xenograft study of selected analogs
5
10
15
20
25
30
Day After Innoculation
Compounds 28 and 45 were selected for further evaluation in
the HCT-116 xenograft model. The compounds were administered
intraperitoneally (ip) to mice bearing subcutaneous tumors at
20 mg/kg once daily (qd). Figure 4 shows the compounds’ effect
on HCT-116 tumor mouse xenografts, and Figure 5 shows the com-
pounds’ effect on the body weights of treated groups. The positive
Figure 4. Effect of 28 and 45 on the growth of human colorectal cancer (HCT-116)
mouse xenograft. HCT-116 cells (2 ꢁ 106) were injected s.c. into SCID mice. When
the tumors reached ꢀ100 mm3, the mice were treated ip with vehicle control, 28
(20 mg/kg once daily), 45 (20 mg/kg once daily), or positive control Camptosar57,58
(5 mg/kg once every four days). Data Points, mean tumor volumes plotted against
time; bars, SE.⁄, p <0.001; ⁄⁄, p <0.01.

6366
A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
ples in preclinical and clinical development. The diarylalkenes de-
22
20
18
16
14
12
10
scribed herein are distinguished by the introduction of PDE4
inhibitory activity, and it is to be hoped that this additional mech-
anism of action will result in enhanced clinical efficacy. The suc-
cess of this strategy will be determined in future studies.
*
4. Experimental section
*p<0.05
*
*
Commercial reagents and solvents were used as received
without further purification. Reactions were run under nitrogen
atmosphere, unless noted otherwise. Column chromatography
refers to flash chromatography using Isco brand prepacked silica
gel cartridges. Proton (1H NMR) and carbon (13C NMR) nuclear
magnetic resonance were recorded on a Brucker 250 spectrome-
ter. NMR spectra (250 MHz 1H and 62.5 MHz 13C) were recorded
in the deuterated solvent indicated with chemical shifts reported
in d units downfield from tetramethylsilane (TMS). Coupling con-
stants are reported in hertz (Hz). Elemental analyses and were
obtained from QTI Intertek, Whitehouse, NJ. Elemental analyses
were within 0.4% of theory. Purity of compounds was also ana-
lyzed by reverse phase HPLC using the solvent systems indi-
cated. All compounds submitted for pharmacological testing
possessed purity P95% as evidenced by the results of elemental
analysis and HPLC analysis. Melting points were obtained from
QTI Intertek, Whitehouse, NJ. CA-4 was prepared according to
the literature procedure.15
**p<0.01
**
**
Vehicle
Cmpd 28; 20 mg/kg, qd, i.p.
Cmpd 45; 20 mg/kg, qd, i.p.
Camptosar; 5 mg/kg, q4d, i.p.
5
10
15
20
25
30
Day After Innoculation
Figure 5. Effect of 28 and 45 on body weights of mice from HCT-116 colorectal
cancer mouse xenograft study. Mice were treated ip with vehicle control, 28
(20 mg/kg once daily), 45 (20 mg/kg once daily), or positive control Camptosar57,58
(5 mg/kg once every four days).Data points, mean body weights plotted against
time; bars, SE.⁄, p <0.01; ⁄⁄, p <0.05.
ptosar administration resulted in efficacious anti-cancer effect,
with 85% inhibition of tumor growth compared to the vehicle
group. Compound 45 demonstrated significant efficacy, with a
64% tumor growth inhibition over the vehicle group. Overall, the
compound was well tolerated. The mice in this group showed a
15% decrease in body weight, with a majority of the body weight
loss in this group occurring after day 19 of the study. Tumor vol-
ume in mice treated with compound 28 was reduced by only
16% with respect to the vehicle-treated mice. For this compound,
it is likely that poor solubility was partly responsible for the lack
of efficacy observed in the study.
4.1. Preparation of 3,3-diarylacrylonitriles by Horner–Wadsworth–
Emmons reaction. General procedure A. (E/Z)-3-(3,4-Dimeth-
oxyphenyl)-3-(3,5-dimethoxyphenyl)acrylonitrile (1)30,31
To
a stirred solution of diethylcyanomethylphosphonate
(0.42 mL, 2.5 mmol) in THF (15 mL) was added a 1.3 M solution
of LiHMDS in THF (1.9 mL, 2.5 mmol) at 0 °C. The solution was al-
lowed to warm to room temperature and was stirred for 30 min,
and then a mixture of 51a (0.7 g, 2.3 mmol) in THF (20 mL) was
added. The reaction mixture was refluxed overnight and was then
cooled and quenched with water (100 mL). The resulting mixture
was extracted with methylene chloride (2 ꢁ 50 mL). The combined
extracts were washed with water (100 mL), dried (MgSO4), and
concentrated under vacuum. The residue was chromatographed
in 99:1 CH2Cl2/EtOAc to provide 0.66 g (81% yield) of a mixture
of the E and Z isomers as a white solid: mp 88–90 °C. 1H NMR
(CDCl3) d 3.77 (s, 3H), 3.80 (s, 3H), 3.84–3.87 (m, 3H), 3.91–3.94
(m, 3H), 5.61–5.66 (m, 1H), 6.40–6.61 (m, 3H), 6.80–7.10 (m,
3H). 13C NMR (CDCl3) d 55.4, 55.5, 55.9, 56.0, 93.1, 93.4, 102.0,
102.1, 107.0, 107.6, 110.7, 110.7, 110.9, 112.6, 118.0, 118.2,
122.1, 123.2, 129.2, 131.1, 138.9, 141.3, 148.5, 148.8, 150.6,
151.1, 160.6, 160.7, 162.5, 162.7. Anal. Calcd for Cl9H19NO4: C,
70.14; H, 5.89; N, 4.30. Found: C, 70.33; H, 5.89; N, 4.03.
3. Conclusion
The novel analogs reported herein, dual inhibitors of tubulin
polymerization and PDE4, were designed to elicit an antineoplastic
effect by means of two independent molecular mechanisms. The
SAR for antitubulin activity was similar to that of other combre-
tastatin analogs, with the additional requirement of the nitrile
group at the alkene terminus. No other alkene substitution was
found to restore antitubulin activity in the absence of polar func-
tionality on the ring. However, the nitrile could be replaced or
eliminated in the presence of an appropriate hydroxyl or amino
phenyl substitution without loss of antiproliferative activity. The
presence of a 3,4-dialkoxy motif in these analogs did provide
PDE4 inhibition. The use of 3-ethoxy-4-methoxyphenyl PDE4
pharmacophore resulted in a boost in PDE4 inhibition relative to
the 3,4-dimethoxy analogs such as 1. The critical structural motifs
were combined in compounds 35 and 36, which possess the 3-eth-
oxy-4-methoxyphenyl PDE4 pharmacophore and another ring
bearing a hydroxyl or amino respectively. These analogs inhibited
HCT-116 with IC50 ꢀ20 nM and PDE4 with IC50 ꢀ50 nM. Their
respective amino acid ester or amide derivatives, prepared for im-
proved solubility, showed comparable in vitro activity. One such
analog, the glycinamide analog 45 also displayed significant
in vivo efficacy in the HCT-116 xenograft study, and merits further
study to identify the active isomer and develop a stereoselective
synthesis.
4.2. (E)-3-(3,4-Dimethoxyphenyl)-3-(3,5-dimethoxyphenyl)-
acrylonitrile (1E)
The geometric isomers of compound 1 were separated on a
Waters Prep LC 4000 System, eluting with a 2:3 mixture of aceto-
nitrile–water, providing the product as a white solid: mp 114–
116 °C. 1H NMR (DMSO-d6) d 3.74–3.83 (m, 12H), 6.21 (s, 1H),
6.49 (d, J = 2.2 Hz, 2H), 6.63 (t, J = 2.1 Hz, 1H), 6.93 (dd, J = 8.1 Hz,
J = 1.8 Hz, 1H), 6.99 (d, J = 1.8 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H). 13C
NMR (DMSO-d6) d 55.4, 55.5, 55.6, 94.0, 95.2, 100.5, 102.0, 106.7,
111.4, 112.6, 118.4, 122.5, 129.0, 140.4, 148.2, 150.1, 160.3,
161.4. Anal. Calcd for Cl9H19NO4: C, 70.14; H, 5.89; N, 4.30. Found:
C, 69.88; H, 5.88; N, 4.40.
In recent years, a multitude of tubulin interactive anti-cancer
agents have been reported in the literature, with scattered exam-

A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
6367
4.3. (Z)-3-(3,4-Dimethoxyphenyl)-3-(3,5-dimethoxyphenyl)-
acrylonitrile (1Z)
4.8. (E/Z)-3-(3,4-Dimethoxyphenyl)-3-(3-methoxyphenyl)-
acrylonitrile (6)
Compound 1Z was also obtained from the separation that pro-
vided 1E: mp 129–131 °C. 1H NMR (DMSO-d6) d 3.76–3.78 (m,
12H), 6.32 (s, 1H), 6.47 (d, J = 1.8 Hz, 2H), 6.65 (t, J = 2.1 Hz, 1H),
6.74 (dd, J = 8.5 Hz, J = 1.7 Hz, 1H), 6.96 (d, J = 8.5 Hz, 1H), 7.14 (s,
1H). 13C NMR (DMSO-d6) d 55.4, 55.6, 55.6, 93.9, 101.0, 107.2,
110.6, 111.2, 118.3, 122.3, 129.7, 139.2, 148.7, 151.0, 160.3,
161.1. Anal. Calcd for Cl9H19NO4: C, 70.14; H, 5.89; N, 4.30. Found:
C, 69.81; H, 5.87; N, 4.19.
Compound 6 was prepared from (3,4-dimethoxyphenyl)(3-
methoxyphenyl)methanone (2.0 g, 7.3 mmol) using general proce-
dure A. Chromatographic purification using a hexanes/ethyl ace-
tate gradient gave 1.99 g (92% yield) as a pink oil: 1H NMR
(CDCl3) d 3.79–3.94 (m, 9H), 5.61–5.68 (m, 1H), 6.82–7.38 (m,
7H, Ar). 13C NMR (CDCl3) d 55.5, 56.1, 56.1, 56.2, 93.3, 93.8,
110.9, 110.9, 111.2, 112.8, 114.5, 115.0, 115.9, 116.0, 118.2,
118.4, 121.3, 122.2, 122.3, 123.4, 129.5, 129.7, 129.7, 131.4,
138.6, 140.9, 148.8, 149.0, 150.8, 151.3, 159.6, 159.7, 162.7,
162.8. Anal. Calcd for C18H17NO3ꢂ0.25H2O: C, 72.10; H, 5.88; N,
4.67. Found: C, 72.33; H, 6.10; N, 4.60.
4.4. 3,3-Bis(3,4-dimethoxyphenyl)acrylonitrile (2)
Compound 2 was prepared from 51b61,62 (1.51 g, 5.0 mmol)
using general procedure A. Chromatographic purification using
95:5 CH2Cl2/EtOAc gave 1.0 g (61% yield), as a tan glass: 1H NMR
(CDCl3) d 3.84 (s, 3H), 3.87 (s, 3H), 3.92 (s, 3H), 3.94 (s, 3H), 5.57
(s, 1H), 7.95 (br, 6H). 13C NMR (DMSO-d6) d 55.9, 55.9, 56.0,
91.9, 110.7, 110.7, 111.4, 112.7, 118,6, 122.2, 123.2, 129.5,
131.8, 148.5, 148.8, 150.5, 151.0, 162.4. Anal. Calcd for
Cl9H19NO4ꢂ0.325H2O: C, 68.90; H, 5.97; N, 4.23. Found: C, 68.99;
H, 5.84; N, 4.10.
4.9. (E/Z)-3-(3,5-Dimethoxyphenyl)-3-(3-methoxyphenyl)-
acrylonitrile (7)
Compound 7 was prepared from (3,5-dimethoxyphenyl)(3-
methoxyphenyl)methanone (1.02 g, 3.8 mmol) using general pro-
cedure A. Chromatographic purification using a hexanes/ethyl ace-
tate gradient gave 1.06 g (96% yield) as an off-white solid: mp 81–
83 °C. 1H NMR (CDCl3) d 3.76–3.79 (m, 6H), 3.82 (s, 3H), 5.72 (s,
1H), 6.42–7.35 (m, 7H). 13C NMR (CDCl3) d 55.5, 55.6, 55.6, 95.4,
95.4, 102.2, 102.34, 107.0, 107.8, 114.3, 115.0, 115.9, 116.1,
117.8, 117.8, 121.0, 122.1, 129.7, 129.8, 138.2, 138.8, 140.1,
141.0, 159.6, 159.8, 160.8, 160.9, 163.0, 163.0. Anal. Calcd for
4.5. (E/Z)-3-(3,4-dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-
acrylonitrile (3)
Compound
3
was prepared from (3,4-dimethoxyphenyl)
C18H17NO3: C, 73.20; H, 5.80; N, 4.74. Found: C, 73.10; H, 5.72; N,
(30,40,50-trimethoxyphenyl)methanone (3.3 g, 10 mmol) using gen-
eral procedure A. Chromatographic purification using 95:5 CH2Cl2/
EtOAc gave 2.7 g (77% yield) as a white solid: mp 119–121 °C. 1H
NMR (DMSO-d6) d 3.84–3.71 (m, 15H), 6.19–6.25 (m, 1H), 6.66
(d, J = 3 Hz, 2H), 7.00–6.77 (m, 2H), 7.14–7.07 (m, 1H). 13C NMR
(DMSO-d6) d 55.5, 55.5, 55.6, 56.0, 56.0, 60.1, 93.5, 94.2, 106.3,
106.9, 111.0, 111.2, 111.3, 112.7, 118.6, 118.7, 122.4, 122.7,
128.9, 130.0, 132.5, 133.7, 138.4, 139.4, 148.2, 148.6, 150.1,
151.0, 152.6, 161.3, 161.4. Anal. Calcd for C20H21NO5: C, 67.59; H,
5.96; N, 3.94. Found: C, 67.66; H, 5.98; N, 3.88.
4.68.
4.10. (E/Z)-3-(3,5-Dimethoxyphenyl)-3-(4-methoxyphenyl)-
acrylonitrile (8)
Compound 8 was prepared from (3,5-dimethoxyphenyl)(4-
methoxyphenyl)methanone (1.86 g, 6.8 mmol) using general pro-
cedure A. Chromatographic purification using a hexanes/ethyl ace-
tate gradient gave 1.97 g (98% yield) as an off-white solid: mp 79–
81 °C. 1H NMR (CDCl3) d 3.76–3.86 (m, 9H), 5.60–5.65 (m, 1H),
6.42–7.43 (m, 7H). 13C NMR (CDCl3) d 55.5, 55.6, 55.6, 93.0, 93.8,
102.1, 102.2, 107.2, 107.8, 114.0, 114.2, 118.2, 118.4, 129.3,
130.1, 131.0, 131.5, 139.2, 141.7, 160.8, 160.9, 161.2, 161.7,
162.6, 162.8. Anal. Calcd for C18H17NO3: C, 73.20; H, 5.80; N,
4.74. Found: C, 72.90; H, 5.52; N, 4.67.
4.6. 3,3-Bis(3,4,5-trimethoxyphenyl)acrylonitrile (4)
Compound
4
was prepared from bis(3,4,5-trimethoxy-
phenyl)methanone (0.32 g, 0.88 mmol) using general procedure
A. Chromatographic purification using 95:5 CH2Cl2/EtOAc gave
0.25 g (74% yield) as a white solid: mp 146–148 °C. 1H-NMR
(DMSO-d6) d 3.77–3.72 (m, 18H), 6.32 (s, 1H), 6.69 (d, J = 5 Hz,
4H). 13C NMR (DMSO-d6) d 56.1, 56.1, 60.1, 60.1, 95.2, 100.4,
106.2, 107.1, 118.4, 131.9, 133.1, 138.7, 139.6, 152.6, 152.7,
161.3. Anal. Calcd for C21H24NO6: C, 65.44; H, 6.02; N, 3.63. Found:
C, 65.08; H, 5.99; N, 3.45.
4.11. (E/Z)-3-(3,5-Dimethoxyphenyl)-3-phenylacrylonitrile (9)
Compound
9 was prepared from (3,5-dimethoxy-phenyl)
(phenyl)methanone (0.34 g, 1.4 mmol) using general procedure A.
Chromatographic purification using a hexanes/ethyl acetate gradi-
ent gave 0.33 g (89% yield) as a light beige oil: 1H NMR (CDCl3) d
3.76 (s, 3H), 3.79 (s, 3H), 5.73 (s, 1H), 6.41–7.44 (m, 8H). 13C
NMR (CDCl3) d 55.6, 55.7, 55.6, 95.1, 95.3, 102.2, 102.3, 107.0,
107.8, 107.8, 117.9, 128.5, 128.6, 128.8, 129.7, 130.2, 130.57,
137.0, 138.7, 139.0, 141.1, 160.9, 160.9, 163.1, 163.2. Anal. Calcd
for C18H17NO3ꢂ0.25H2O: C, 76.44; H, 5.74; N, 5.24. Found: C,
76.41; H, 5.40; N, 5.18.
4.7. (E/Z)-3-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)-
acrylonitrile (5)
Compound 5 was prepared from (3,4-dimethoxyphenyl)(4-
methoxyphenyl)methanone (2.00 g, 7.3 mmol) using general pro-
cedure A. Chromatographic purification using a hexanes/EtOAc
gradient gave 2.08 g (96% yield) as a pink oil: 1H NMR (CDCl3) d
3.83–3.94 (m, 9H), 5.56 (s, 1H), 6.79–7.42 (m, 7H). 13C NMR (CDCl3)
d 55.5, 55.5, 56.1, 56.1, 56.29, 91.8, 91.9, 110.9, 111.5, 112.9, 114.0,
114.1, 118.8, 118.8, 122.3, 123.2, 129.5, 129.8, 130.3, 131.5, 131.7,
132.1, 148.8, 149.0, 150.7, 151.2, 161.2, 161.6, 162.5, 162.6. Anal.
Calcd for C18H17NO3ꢂ0.3H2O: C, 71.89; H, 5.90; N, 4.66. Found: C,
71.95; H, 6.04; N, 4.54.
4.12. Deprotection of tert-butyldimethylsilyl ethers. General
procedure B. (E/Z)-3-(3,5-Dimethoxyphenyl)-3-(3-hydroxy-4-
methoxyphenyl)acrylonitrile (10)
A 1.0 M solution of tetrabutylammonium fluoride (1.5 mL,
1.5 mmol) was added to a stirred solution of 59a (0.53 g, 1.3 mmol)
in THF (15 mL) at room temperature. After 1 h, ice water (10 mL)

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A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
was poured into the reddish solution, followed by addition of ether
(50 mL). The mixture was washed with water (2 ꢁ 80 mL), dried
over MgSO4, filtered and evaporated under vacuum, providing
the product as a foamy solid (0.35 g, 90% yield): mp 43–45 °C. 1H
NMR (DMSO-d6) d 3.78–3.81 (m, 6H), 3.84–3.87 (m, 3H), 6.17–
6.19 (m, 1H), 6.49 (t, J = 2.0 Hz, 2H), 6.65–6.69 (m, 1H), 6.82–6.91
(m, 2H), 7.00–7.07 (m, 1H), 9.28–9.36 (m, 1H). 13C NMR (CDCl3) d
55.6, 56.1, 56.2, 93.5, 94.2, 102.1, 102.2, 107.1, 107.8, 110.4,
110.4, 114.4, 116.0, 118.1, 118.2, 121.3, 122.3, 130.2, 131.9,
139.1, 141.4, 145.5, 145.7, 148.2, 148.7, 160.8, 160.8, 162.6,
162.8. Anal. Calcd for C18H17NO4ꢂ0.08H2O: C, 69.12; H, 5.53; N,
4.48. Found: C, 68.76; H, 5.69; N, 4.22.
(s, 3H), 5.39 (s, 1H), 5.40 (s, 1H), 6.44 (t, J = 2.1 Hz, 1H), 6.51 (d,
J = 2.4 Hz, 2H), 6.81–6.93 (m, 3H). 13C NMR (CDCl3) d 55.5, 56.1,
100.1, 106.8, 110.9, 111.6, 113.4, 121.1, 134.2, 143.9, 148.7,
149.0, 149.8, 160.6. Anal. Calcd for Cl8H20O4: C, 71.98; H, 6.71; N,
0.00. Found: C, 71.65; H, 7.08; N, <0.05.
4.16. (E/Z)-3-(3,4-Dimethoxyphenyl)-3-(3,5-dimethoxyphenyl)-
acrylamide (14)
KOSi(CH3)3 (0.39 g, 3.1 mmol) was added to a stirred solution of
compound 1 (0.50 g, 1.5 mmol) in toluene (10 mL), and the result-
ing mixture was heated to reflux under nitrogen for 20 h. The mix-
ture was cooled and evaporated under vacuum. The residue was
partitioned between water (100 mL) and EtOAc (75 mL), and the
aqueous phase was extracted with EtOAc (2 ꢁ 75 mL). The com-
bined organic phases were washed with water (100 mL) and brine
(100 mL), and were dried (MgSO4) and evaporated, to afford 0.45 g
of the product, in 85% yield: mp 119–121 °C. 1H NMR (CDCl3) d
3.50–4.00 (m, 12H), 6.32–7.24 (m, 9H). 13C NMR (CDCl3) d 55.2,
55.4, 55.4, 55.5, 55.5, 99.2, 100.0, 106.2, 107.5, 110.5, 111.0,
111.3, 113.4, 121.0, 121.3, 122.1, 122.8, 131.0, 133.1, 141.2,
143.8, 147.6, 147.8, 148.5, 149.4, 159.9, 160.3, 167.4, 167.6. Anal.
Calcd for C19H21NO5: C, 66.46; H, 6.16; N, 4.08. Found: C, 66.09;
H, 6.32; N, 3.92.
4.13. Tin (II) chloride reduction of nitro compounds. General
procedure C. (E/Z)-3-(3-Amino-4-methoxyphenyl)-3-(3,5-
dimethoxyphenyl)acrylonitrile (11)
A suspension of 62a (1.5 g, 4.4 mmol) and tin chloride dihydrate
(5.4 g, 23.0 mmol) in ethanol (25 mL) was heated at 70 °C for 1 h.
The mixture was cooled and poured onto ice (200 mL). The pH
was made strongly alkaline by the addition of 10 N NaOH. The mix-
ture was extracted with EtOAc (5x50 mL) and the combined organ-
ic extracts were washed with water (40 mL), brine (40 mL), and
dried (MgSO4). The solvent was removed and the residue was puri-
fied by column chromatography eluting with 75:25 hexane/EtOAc,
to afford the product (0.8 g, 54% yield) as a yellow solid: mp 93–
95 °C. 1H NMR (CDCl3) d 3.73 -3.79 (m, 6H), 3.70–3.87 (br, 2H),
3.87–3.89 (m, 3H), 5.54 (s, 1H), 6.42–6.82 (m, 6H); 13C NMR
(CDCl3) d 55.5, 55.6, 92.7, 93.4, 101.9, 102.0, 107.0, 107.6, 109.8,
109.9, 114.2, 115.8, 119.4, 120.7, 129.5, 131.2, 136.0, 136.2,
139.3, 141.7, 148.8, 149.2, 160.6, 160.6, 162.9, 163.1. Anal. Calcd
for C18H18N2O3: C, 69.66; H, 5.85; N, 9.03. Found: C, 69.60; H,
5.58; N, 8.88.
4.17. (E/Z)-Methyl 3-(3,4-dimethoxyphenyl)-3-(3,5-dimethoxyphenyl)-
acrylate (15)
To a stirred suspension of methyl diethylphosphonoacetate
(1.1 g, 5.4 mmol) in THF (10 mL) was added a 1.0 M solution of
LiHMDS in THF (5.4 mL, 5.4 mmol) at 0 °C. The solution was al-
lowed to warm to room temperature and was stirred for 30 min,
and then a mixture of 51a (1.5 g, 4.9 mmol) in THF (20 mL) was
added, and the mixture was stirred at rt for 4 h. Then, methyl
diethylphosphonoacetate (1.1 g, 5.4 mmol) and 1.0 M LiHMDS in
THF (5.4 mL, 5.4 mmol) were added, and the mixture was heated
to reflux for 6 days. The reaction mixture was cooled to room tem-
perature and quenched with water (100 mL). The resulting mixture
was extracted with methylene chloride (2 ꢁ 50 mL). The combined
extracts were washed with water (80 mL), dried (MgSO4), and con-
centrated under vacuum. The residue was purified using reverse
phase preparative HPLC, running a water–acetonitrile gradient.
The product, a mixture of geometric isomers, was obtained as a
colorless oil, 0.45 g (25% yield): 1H NMR (CDCl3) d 3.62–3.64 (m,
3H), 3.75–3.77 (m, 6H), 3.82–3.84 (m, 3H), 3.89–3.92 (m, 3H),
5.39 (s, 1H), 6.28–6.29 (m, 1H), 6.29–6.49 (m, 3H), 6.72–6.91 (m,
2H). 13C NMR (CDCl3) d 51.3, 51.3, 55.4, 55.4, 55.8, 56.0, 100.2,
101.4, 106.9, 107.2, 110.4, 110.7, 112.8, 115.0, 116.7, 122.1,
122.4, 131.0, 132.9, 140.9, 143.4, 148.3, 148.8, 149.2, 150.5,
156.4, 156.5, 160.3, 160.6, 166.4, 166.6. Anal. Calcd for
4.14. Catalytic hydrogenation of nitro compounds. General
procedure D. (E/Z)-(3-Amino-4-methoxyphenyl)-3-(3,4,5-
trimethoxyphenyl)acrylonitrile (12)
A mixture of 62b (0.9 g, 2.4 mmol) and 10% Pd/C (0.1 g) in EtOAc
(100 mL) was hydrogenated under 50 psi of hydrogen for 16 h. The
mixture was filtered through Celite and the filtrate was concen-
trated. The crude product was purified by flash chromatography
(CH2Cl2–EtOAc 85:15) to afford the product (0.3 g, 41% yield) as a
yellow solid: mp 97–99 °C. 1H NMR (CDCl3) d 3.80–3.91 (m,
14H), 5.51–5.75 (s, 1H), 6.51–6.87 (m, 5H); 13C NMR (CDCl3) d
55.5, 55.6, 56.2, 60.9, 92.1, 92.6, 106.2, 107.2, 109.8, 109.8, 114.4,
115.9, 119.5, 120.8, 129.5, 131.5, 132.6, 135.1, 136.0, 136.2,
139.3, 139.9, 148.8, 149.2, 152.9, 153.0, 163.0, 163.1. Anal. Calcd
for C19H20N2O4: C, 67.05; H, 5.92; N, 8.23. Found: C, 67.08; H,
6.08; N, 7.94.
C
20H22O6ꢂ0.3CH2Cl2: C, 63.52; H, 5.93; N, 0.00. Found: C, 66.02;
4.15. Wittig reaction of benzophenones. General procedure E. 4-
(1-(3,5-Dimethoxyphenyl)vinyl)-1,2-dimethoxybenzene (13)
H, 5.81; N, <0.05.
4.18. (E/Z)-3-(3,4-Dimethoxyphenyl)-3-(3,5-dimethoxyphenyl)-
To a stirred suspension of methyltriphenylphosphonium bro-
mide (2.4 g, 6.6 mmol) in THF (20 mL) was added a 1.0 M solution
of LiHMDS in THF (6.6 mL, 6.6 mmol) at 0 °C. The solution was al-
lowed to warm to room temperature and was stirred for 30 min,
and then a mixture of 51a (1.0 g, 3.3 mmol) in THF (20 mL) was
added. The reaction mixture was heated to reflux for 16 h and
was then quenched with water (100 mL). The resulting mixture
was extracted with methylene chloride (2 ꢁ 50 mL). The combined
extracts were washed with water, dried (MgSO4), and concentrated
under vacuum. The residue was chromatographed using a hexanes/
ethyl acetate gradient, to provide 0.76 g (74% yield) of the product
as a colorless oil: 1H NMR (CDCl3) d 3.78 (s, 6H), 3.84 (s, 3H), 3.90
acrylate (16)
A mixture of 15 (0.76 g, 2.1 mmol), and KOH (2.3 g, 40 mmol) in
4:1 MeOH–water (10 mL) was stirred at room temperature under
nitrogen for 4 h. The mixture was evaporated under vacuum to re-
move the methanol, and the remaining solution was diluted with
water (10 mL). This mixture was washed with ethyl ether
(2 ꢁ 60 mL) and then acidified to pH 2–3 (conc. HCl). The resulting
mixture was extracted with CH2Cl2 (2 ꢁ 100 mL), and the com-
bined extracts were dried (MgSO4) and evaporated, providing the
product as an off-white foam: mp 90–92 °C. 1H NMR (CDCl3) d
3.75–3.92 (m, 12H), 6.24–6.50 (m, 6H), 6.75–6.90 (m, 2H). 13C

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6369
NMR (CDCl3) d 55.5, 55.6, 56.0, 56.1, 100.7, 101.9, 106.1, 107.2,
107.5, 110.6, 110.8, 111.0, 113.2, 114.7, 116.3, 122.5, 122.9,
130.6, 132.9, 140.5, 143.5, 148.4, 148.9, 149.7, 150.9, 158.4,
158.7, 160.5, 160.7, 170.6, 170.9. Anal. Calcd for C19H20O6: C,
66.27; H, 5.85; N, 0.00. Found: C, 66.02; H, 5.81; N, <0.05.
an oil. This oil was dissolved in 4.0 M HCl in 1,4-dioxane (8 mL),
and then evaporated. The residue was triturated in ether, filtered,
and dried under vacuum, providing 0.40 g (37% yield), as an off-
white solid: mp 123–125 °C. 1H NMR (CDCl3) d 2.42–2.46 (m,
2H), 2.68 (s, 6H), 3.16 (m, 2H), 3.70–3.79 (m, 12H), 6.11 (m, 1H),
6.28 (d, J = 2.3 Hz, 1H), 6.38–6.42 (m, 1H), 6.50–6.57 (m, 1H),
6.67–6.70 (m, 1H), 6.85–7.07 (m, 2H), 10.56 (br, 1H). 13C NMR
(CDCl3) d 24.7, 24.7, 41.9, 55.2, 55.4, 55.5, 55.6, 55.7, 55.8, 98.9,
99.1, 105.5, 107.2, 110.1, 111.3, 111.6, 112.9, 120.0, 121.7, 123.7,
131.1, 133.6, 141.2, 142.8, 143.0, 143.8, 148.0, 148.4, 148.5,
160.2, 160.5. Anal. Calcd for C22H30NO4Cl: C, 64.78; H, 7.41; N,
3.43. Found: C, 64.43; H, 7.40; N, 3.36.
4.19. (E/Z)-4-(3,4-Dimethoxyphenyl)-4-(3,5-dimethoxyphenyl)-
but-3-en-2-one (17)
A mixture of Pd(OAc)2 (67 mg, 0.30 mmol) in DMF (1 mL) was
added to a mixture of 5563,64 (2.1 g, 10 mmol), 4-bromoveratrole
(3.2 g, 14.9 mmol), NaOAc (1.4 g, 17 mmol), and Bu4NBr (3.5 g,
11 mmol) in DMF (20 mL). The mixture was heated to 60 °C for
48 h, and then cooled to room temperature. The mixture was par-
titioned between water (150 mL) and EtOAc (50 mL), and the aque-
ous phase was extracted with EtOAc (2 ꢁ 50 mL), brine (50 mL),
dried (MgSO4) and evaporated. The residue was chromatographed
using a hexanes/ethyl acetate gradient. Further purification using
reverse phase preparative HPLC in 38–62 acetonitrile–water pro-
vided 200 mg of the product as an oil, in 6% yield: 1H NMR (CDCl3)
d 1.91 (s, 3H), 3.79 (s, 6H), 3.85 (s, 3H), 3.89 (s, 3H), 6.38 (d,
J = 2.3 Hz, 2H), 6.51–6.53 (m, 2H), 6.77–6.82 (m, 2H), 6.92–6.93
(m, 1H). 13C NMR (CDCl3) d 30.0, 55.4, 55.6, 55.8, 56.1, 100.9,
110.8, 113.2, 122.4, 126.5, 132.9, 141.1, 148.9, 150.7, 153.8,
160.9. Anal. Calcd for C20H22O5: C, 70.16; H, 6.48; N, 0.00. Found:
C, 70.26; H, 6.37; N, <0.05.
4.23. (E/Z)-4-(1-(3,5-Dimethoxyphenyl)-2-methoxyvinyl)-1,2-
dimethoxybenzene (21)
Compound 21 was prepared from 51a (1.0 g, 3.3 mmol) and
(methoxymethyl)triphenylphosphonium
chloride
(2.3 g,
6.6 mmol) using general procedure E. Chromatographic purifica-
tion using a hexanes/ethyl acetate gradient provided 0.77 g as a
colorless oil, in 77% yield: 1H NMR (CDCl3) d 3.75 (m, 9H), 3.82
(s, 3H), 3.88 (s, 3H), 6.38 (m, 3H), 6.66 (m, 1H), 6.86 (m, 2H),
6.97 (m, 1H). 13C NMR (CDCl3) d 55.3, 55.3, 55.8, 55.8, 55.9, 60.6,
98.6, 98.9, 106.4, 108.0, 110.7, 111.0, 111.8, 113.1, 120.1, 120.2,
120.7, 122.5, 130.0, 133.0, 139.6, 142.6, 145.9, 146.0, 147.7,
147.9, 148.3, 148.6, 160.3, 160.6. Anal. Calcd for C19H22O5: C,
69.07; H, 6.71; N, 0.00. Found: C, 69.10; H, 6.85; N, 0.00.
4.20. (E/Z)-4-(1-(3,5-Dimethoxyphenyl)prop-1-en-1-yl)-1,2-
dimethoxybenzene (18)
4.24. (E/Z)-4-(1-(3,5-Dimethoxyphenyl)but-1-en-3-yn-1-yl)-1,2-
dimethoxybenzene (22)
Compound 18 was prepared from 51a (0.96 g, 3.2 mmol) and
ethyltriphenylphosphonium bromide (2.4 g, 6.4 mmol) using gen-
eral procedure E. Chromatographic purification using a hexanes/
EtOAc gradient provided 0.97 g (97% yield) of the product as a pale
yellow oil: 1H NMR (CDCl3) d 1.78–1.74 (m, 3H), 3.74 (s, 3H), 3.78
(s, 3H), 3.83 (s, 3H), 3.86–3.91 (m, 3H), 6.20–6.03 (m, 1H), 6.34–
6.43 (m, 3H), 6.68–6.76 (m, 2H), 6.83–6.89 (m, 1H). 13C NMR
(CDCl3) d 15.8, 15.9, 55.4, 55.5, 56.0, 56.0, 99.0, 99.2, 105.8,
108.2, 110.3, 110.9, 111.0, 113.3, 119.9, 122.6, 122.8, 124.4,
132.5, 135.6, 142.2, 142.3, 142.3, 145.4, 148.0, 148.3, 148.7,
148.7, 160.6, 160.7. Anal. Calcd for C19H22O4: C, 72.59; H, 7.05; N,
0.00. Found: C, 72.43; H, 6.96; N, <0.05.
A solution of prop-1-yne-1,3-diylbis(trimethylsilane) (2.2 g,
12 mmol) in THF (40 mL) was cooled to ꢃ78 °C, and then a 1.7 M
solution of t-BuLi in pentane (7.1 mL, 12 mmol) was added drop-
wise, and stirring proceeded at this temperature for 1 h following
completion of the addition. Intermediate 51a (1.8 g, 6.0 mmol)
was then added and after 5 min the cooling bath was removed.
The reaction flask was allowed to warm to room temperature
and was then heated to reflux for 1 h. The mixture was cooled to
room temperature and then quenched by the addition of water
(20 mL). The mixture stirred at rt for 16 h. The solvent was evapo-
rated, and the residue was partitioned between ethyl acetate
(100 mL) and water (100 mL), and the aqueous phase was ex-
tracted with ethyl acetate (100 mL). The combined organic phases
were washed with water (2 ꢁ 100 mL), 1 N HCl (2 ꢁ 100 mL), and
water (100 mL), were dried (Na2SO4) and evaporated under vac-
uum. The residue was chromatographed using a hexanes/ethyl
acetate gradient, providing 900 mg (47% yield) of the product as
a colorless oil which darkened over time: 1H NMR (CDCl3) d 3.05
(d, J = 2.5 Hz, 1H), 3.73 (s, 3H), 3.75 (s, 3H), 3.81–3.89 (m, 6H),
5.92 (m, 1H), 6.43–6.47 (m, 2H), 6.61–6.62 (m, 1H), 6.79–6.85
(m, 2H), 6.97–7.16 (m, 1H). 13C NMR (CDCl3) d 55.3, 55.8, 55.9,
81.7, 81.8, 82.5, 82.8, 100.6, 100.8, 104.6, 105.3, 106.5, 108.0,
110.4, 110.7, 110.8, 113.2, 121.1, 123.0, 131.1, 131.5, 140.7,
143.5, 148.1, 148.6, 149.1, 149.6, 154.1, 154.2, 160.2, 160.5. Anal.
Calcd for C20H20O4ꢂH2O: C, 70.16; H, 6.48; N, 0.00. Found: C,
70.41; H, 6.14; N, 0.08.
4.21. (E/Z)-4-(1-(3,5-Dimethoxyphenyl)but-1-en-1-yl)-1,2-
dimethoxybenzene (19)
Compound 19 was prepared from 51a (2.0 g, 6.5 mmol) and
propyltriphenylphosphonium bromide (5.0 g, 13 mmol) using gen-
eral procedure E. Chromatographic purification using a hexanes/
EtOAc gradient provided 2.1 g (98% yield) of the product as a color-
less oil: 1H NMR (CDCl3) d 1.04 (t, J = 7.40 Hz, 3H), 2.04–2.16 (m,
2H), 3.74–3.91 (m, 12H), 6.08–5.94 (m, 1H), 6.33–6.43 (m, 3H),
6.67–6.76 (m, 2H), 6.84–6.88 (1H). 13C NMR (CDCl3) d 14.7, 14.8,
23.4, 23.4, 55.4, 26.0, 99.0, 99.2, 105.8, 108.0, 110.3, 110.9, 110.9,
113.2, 120.0, 122.4, 130.3, 132.0, 132.8, 135.5, 140.7, 140.8,
142.5, 145.3, 148.1, 148.4, 148.7, 160.6, 160.7. Anal. Calcd for
C20H24O4: C, 73.15; H, 7.37; N, 0.00. Found: C, 72.98; H, 6.25; N,
<0.05.
4.25. 5-(1-(3,5-Dimethoxyphenyl)vinyl)-2-methoxyphenol (23)
4.22. (E/Z)-4-(3,4-Dimethoxyphenyl)-4-(3,5-dimethoxyphenyl)-
N,N-dimethylbut-3-en-1-aminium chloride (20)
Compound 23 was prepared from 59b (0.6 g, 1.5 mmol) using
general procedure B. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient, providing the
product as an oil (0.4 g, 93% yield): 1H NMR (CDCl3) d 3.78 (s,
6H), 3.88 (s, 3H), 5.36 (d, J = 1.1 Hz, 1H), 5.41 (d, J = 1.1 Hz, 1H),
5.72 (s, 1H), 6.46 (t, J = 2.1 Hz, 1H), 6.52 (d, J = 2.1 Hz, 2H),
Compound 20 was prepared from 51a (0.88 g, 2.9 mmol) and
(3-(dimethylamino)propyl)triphenylphosphonium bromide (2.5 g,
5.8 mmol) using general procedure E. Chromatographic purifica-
tion (CH2Cl2–MeOH–concd NH4OH 98.5:1:0.5) provided 0.37 g as

6370
A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
6.77–6.83 (m, 2H), 6.99 (d, J = 2.0 Hz, 1H). 13C NMR (CDCl3) d 55.3,
55.9, 99.8, 106.7, 110.2, 113.3, 114.5, 120.2, 134.6, 143.9, 145.2,
146.4, 149.5, 160.5. Anal. Calcd for C17H18O4ꢂ0.1H2O: C, 70.87; H,
6.37; N, 0.00. Found: C, 70.77; H, 6.17; N, <0.05.
tography using a hexanes/ethyl acetate gradient to provide the
product as a white solid (0.60 g, 89% yield): mp 110–112 °C. 1H
NMR (CDCl3) d 1.41–1.49 (m, 3H), 3.77 (s, 3H), 3.80 (s, 3H), 3.90–
3.92 (m, 3H), 3.99–4.13 (m, 2H), 5.59–5.65 (m, 1H), 6.43–6.55
(m, 3H), 6.82–7.04 (m, 3H). 13C NMR (CDCl3) d 14.8, 55.7, 56.1,
56.2, 64.7, 93.2, 93.8, 102.2, 102.3, 107.2, 107.8, 111.1, 111.2,
112.7, 114.2, 118.2, 122.2, 123.3, 129.33, 131.2, 139.1, 141.6,
148.0, 148.3, 151.1, 151.7, 160.8, 160.9, 162.8, 162.9. Anal. Calcd
for C20H21NO4: C, 70.78; H, 6.24; N, 4.13. Found: C, 70.62; H,
6.25; N, 4.01.
4.26. (E/Z)-5-(1-(3,5-Dimethoxyphenyl)prop-1-en-1-yl)-2-
methoxyphenol (24)
Compound 24 was prepared from 59c (1.0 g, 2.4 mmol) using
general procedure B. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient, eluting the prod-
uct at 25:75 ethyl acetate/hexanes as an oil (0.7 g, 97% yield): 1H
NMR (CDCl3) d 1.71–1.78 (m, 3H), 3.75–3.91 (m, 9H), 5.50–5.57
(m, 1H), 6.05–6.13 (m, 1H), 6.31–6.41 (m, 3H), 6.63–6.77 (m,
2H), 6.82–6.88 (m, 1H). 13C NMR (CDCl3) d 15.6, 15.7, 55.3, 55.9,
56.0, 98.7, 99.0, 105.7, 108.0, 110.2, 110.3, 113.2, 116.3, 118.8,
121.8, 122.7, 124.2, 133.1, 136.1, 141.9, 142.2, 145.1, 145.4,
145.4, 145.6, 160.4, 160.6. Anal. Calcd for C18H20O4: C, 71.98; H,
6.71; N, 0.00. Found: C, 71.72; H, 6.48; N, <0.05.
4.31. 3,3-Bis(3-ethoxy-4-methoxyphenyl)acrylonitrile (29)
Compound 29 was prepared from 53b (7.3 g, 22 mmol) using
general procedure A. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as a white solid (3.92 g, 50% yield): mp 142–144 °C. 1H
NMR (DMSO-d6) d 1.31 (t, J = 7.0 Hz, 6H), 3.79 (s, 3H), 3.83 (s,
3H), 3.94–4.04 (m, 4H), 6.11 (s, 1H), 6.77–7.09 (m, 6H). 13C NMR
(DMSO-d6) d 14.6, 14.6, 55.5, 55.5, 63.8, 92.6, 111.3, 111.5, 112.3,
113.8, 118.9, 122.3, 122.4, 129.3, 130.6, 147.3, 147.8, 150.1,
151.0, 161.4. Anal. Calcd for C21H23NO4: C, 71.37; H, 6.56; N,
3.96. Found: C, 71.42; H, 6.42; N, 3.97.
4.27. 5-(1-(3,5-Dimethoxyphenyl)-2-methylprop-1-en-1-yl)-2-
methoxyphenol (25)
Compound 25 was prepared from 59d (0.85 g, 2.05 mmol) using
general procedure B. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient, eluting the prod-
uct at 25:75 ethyl acetate/hexanes as an oil (0.6 g, 96% yield): 1H
NMR (CDCl3) d 1.77 (s, 3H), 1.80 (s, 3H), 3.75 (s, 6H), 3.86 (s, 3H),
5.53 (s, 1H), 6.28–6.32 (m, 3H), 6.63 (dd, J = 8.3, J = 2.0 Hz, 1H),
6.74–6.78 (m, 2H). 13C NMR (CDCl3) d 22.3, 22.5, 55.3, 55.9, 98.1,
107.9, 110.1, 116.0, 121.3, 130.7, 136.4, 136.5, 144.9, 145.6,
160.3. Anal. Calcd for C19H22O4: C, 72.59; H, 7.05; N, 0.00. Found:
C, 72.59; H, 7.10; N, <0.05.
4.32. (E/Z)-3-(3,4-Dimethoxyphenyl)-3-(3-ethoxy-4-methoxy-
phenyl) acrylonitrile (30)
Compound 30 was prepared from 51c (1.6 g, 5.0 mmol) using
general procedure A. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as a white solid (0.6 g, 35% yield): mp 103–106 °C. 1H
NMR (CDCl3) d 1.36–1.54 (m, 3H), 3.76–4.17 (m, 11H), 5.55 (s,
1H), 6.75–7.10 (m, 6H). 13C NMR (CDCl3) d 14.6, 55.9, 56.0, 64.5,
91.8, 110.7, 110.7, 110.9, 110.9, 111.4, 112.8, 112.9, 114.2, 118.6,
122.0, 122.1, 123.1, 123.2, 129.5, 129.6, 131.7, 131.9, 147.8,
148.1, 148.6, 148.8, 150.5, 150.8, 151.0, 162.5. Anal. Calcd for
4.28. (E/Z)-5-(1-(3,5-Dimethoxyphenyl)-2-phenylvinyl)-2-
methoxyphenol (26)
C20H21NO4: C, 70.78; H, 6.24; N, 4.13. Found: C, 70.62; H, 6.21; N,
Compound 26 was prepared from 59e (1.2 g, 2.6 mmol) using
general procedure B. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient, eluting the prod-
uct at 30:70 ethyl acetate/hexanes as an oil (0.9 g, 96% yield): 1H
NMR (CDCl3) d 3.69 (s, 3H), 3.77 (s, 3H), 3.90–3.91 (m, 3H), 5.54–
5.55 (m, 1H), 6.35–6.49 (m, 3H), 6.69–6.90 (m, 4H), 6.99–7.15
(m, 5H). 13C NMR (CDCl3) d 55.4, 55.9, 56.0, 99.5, 99.9, 106.1,
108.1, 110.2, 113.6, 116.5, 119.5, 122.3, 126.6, 126.7, 126.8,
127.9, 128.1, 129.4, 129.5, 133.2, 136.6, 137.3, 137.4, 141.8,
142.1, 142.3, 145.2, 145.6, 145.9, 146.0, 160.5, 161.0. Anal. Calcd
for C17H18O4ꢂ0.1H2O: C, 70.87; H, 6.37; N, 0.00. Found: C, 70.77;
H, 6.17; N, <0.05.
4.07.
4.33. (E/Z)-3-(3-Ethoxy-4-methoxyphenyl)-3-(4-methoxy-3-
methylphenyl)acrylonitrile (31)
Compound 31 was prepared from 53c (1.2 g, 4.0 mmol) using
general procedure A. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as a white solid (0.97 g, 75% yield): mp: 86–88 °C. 1H
NMR (DMSO-d6) d 1.30 (t, J = 7.0 Hz, 3H), 2.13–2.17 (m, 3H),
3.78–3.86 (m, 3H), 3.82 (s, 3H), 3.93–4.04 (m, 2H), 6.01–6.08 (m,
1H), 6.72–7.26 (m, 6H). 13C NMR (DMSO-d6) d 14.6, 14.6, 16.0,
63.8, 92.1, 92.4, 110.0, 110.2, 111.4, 111.5, 112.3, 113.8, 119.0,
122.3, 122.4, 125.6, 125.9, 128.1, 128.6, 128.9, 129.4, 130.1,
130.4, 130.8, 131.3, 147.4, 147.8, 150.2, 151.0, 158.4, 159.3,
161.5, 161.5. Anal. Calcd for C20H21NO3: C, 74.28; H, 6.55; N,
4.33. Found: C, 74.16; H, 6.48; N, 4.30.
4.29. 5-(1-(3,5-Dimethoxyphenyl)vinyl)-2-methoxyaniline (27)
Compound 27 was prepared from 62c (1.12 g, 3.55 mmol) using
general procedure C. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient, providing the
product as a solid (0.64 g, 63% yield): mp 93–95 °C. 1H NMR (CDCl3)
d 3.77–3.87 (m, 11H), 5.31–5.37 (m, 2H), 6.43–6.73 (m, 6H). 13C NMR
(CDCl3) d 55.36, 55.53, 99.77, 106.7, 109.9, 112.8, 114.9, 118.6, 134.1,
135.6, 144.2, 147.2, 149.9, 160.4. Anal. Calcd for C17H19NO3: C,
71.56; H, 6.71; N, 4.91. Found: C, 71.31; H, 6.76; N, 4.92.
4.34. (E/Z)-3-(3-Ethoxy-4-methoxyphenyl)-3-(3-methoxyphenyl)-
acrylonitrile (32)
Compound 32 was prepared from 53d (1.6 g, 5.5 mmol) using
general procedure A. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as a white solid (1.6 g, 93% yield): mp 96–98 °C. 1H NMR
(DMSO-d6) d 1.31 (t, J = 7 Hz, 3H), 3.77–3.83 (m, 6H), 3.94–4.05
(m, 2H), 6.18–6.29 (m, 1H), 6.72–7.43 (m, 7H). 13C NMR (DMSO-
d6) d 14.5, 14.6, 55.2, 55.3, 55.5, 55.6, 63.8, 63.9, 93.9, 94.9, 111.3,
111.5, 111.8, 113.7, 114.6, 115.0, 115.9, 118.4, 118.5, 121.0,
4.30. (E/Z)-3-(3,5-Dimethoxyphenyl)-3-(3-ethoxy-4-methoxy-
phenyl)acrylonitrile (28)
Compound 28 was prepared from 53a (0.63 g, 2.0 mmol) using
general procedure A. The product was purified by column chroma-

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6371
121.3, 122.2, 122.4, 129.1, 129.7, 129.8, 129.9, 138.6, 139.8, 147.4,
147.8, 150.3, 151.1, 159.0, 161.2, 161.4. Anal. Calcd for C19H19NO3:
C, 73.77; H, 6.19; N, 4.53. Found: C, 73.43; H, 6.13; N, 4.54.
4.39. (E/Z)-3-(3-Ethoxy-4-methoxyphenyl)-3-(4-(methylamino)-
phenyl)acrylonitrile (37)
Compound 37 was prepared from 64 (0.29 g, 1.0 mmol) using
general procedure A. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as an off-white solid (0.26 g, 81% yield): mp 81–83 °C. 1H
NMR (DMSO-d6) d 1.28–1.34 (m, 3H), 2.69–2.74 (m, 3H), 3.79–
3.83 (m, 3H), 3.94–4.02 (m, 2H), 5.77–5.87 (m, 1H), 6.25–6.36
(m, 1H), 6.50–7.19 (m, 7H). 13C NMR (DMSO-d6) d 14.6, 14.6,
29.2, 29.3, 55.5, 55.5, 63.7, 88.3, 89.4, 110.8, 111.1, 111.3, 111.4,
112.8, 113.9, 119.6, 122.2, 123.4, 124.5, 129.8, 129.9, 130.9,
131.6, 147.3, 147.6, 149.8, 150.7, 151.3, 151.9, 161.9, 162.1. Anal.
Calcd for C19H20N2O2: C, 74.00; H, 6.54; N, 9.08. Found: C, 73.67;
H, 6.70; N, 8.81.
4.35. (E/Z)-3-(3-Ethoxy-4-methoxyphenyl)-3-(3-fluoro-4-
methoxyphenyl)acrylonitrile (33)
Compound 33 was prepared from 54a (0.35 mg, 1.2 mmol)
using general procedure A. The product was purified by column
chromatography using a hexanes/ethyl acetate gradient to provide
the product as an off-white solid (0.26 g, 69% yield): mp 122–
124 °C. 1H NMR (DMSO-d6) d 1.31 (t, J = 7 Hz, 3H), 3.78–3.94 (s,
6H), 4.01 (q, J = 7 Hz, 2H), 6.15–6.21 (m, 1H), 6.74 (dd, J =2, 8 Hz,
1H), 6.92–7.36 (m, 5H). 13C NMR (DMSO-d6) d 14.5, 14.56, 55.5,
55.5, 56.1, 56.1, 63.8, 63.8, 93.6, 93.8, 111.4, 111.6, 112.1, 113.8,
115.3, 115.6, 116.7, 116.9, 118.5, 122.2, 122.3, 125.7, 125.7,
126.2, 128.8, 129.6, 129.7, 130.1, 130.8, 130.9, 147.4, 147.9,
148.1, 148.2, 148.9, 149.0, 150.2, 150.8 (d, J = 240), 151.1 (d,
J = 240), 151.2, 160.0, 160.1. Anal. Calcd for C19H18NO3F: C, 69.71;
H, 5.54; N, 4.28. Found: C, 69.55; H, 5.58; N, 4.25.
4.40. (E/Z)-3-(4-(Dimethylamino)phenyl)-3-(3-ethoxy-4-
methoxyphenyl)acrylonitrile (38)
Compound 38 was prepared from 65 (0.22 g, 0.74 mmol) using
general procedure A. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as an off-white solid (0.21 g, 87% yield): mp 81–83 °C. 1H
NMR (DMSO-d6) d 1.28–1.34 (m, 3H), 2.96–2.99 (m, 6H), 3.79–
3.83 (m, 3H), 3.94–4.03 (m, 2H), 5.84–5.93 (s, 1H), 6.68–7.26 (m,
7H). 13C NMR (DMSO-d6) d 14.6, 14.6, 39.6, 39.7, 55.5, 55.5, 63.8,
89.0, 90.0, 111.2, 111.3, 111.4, 111.5, 112.8, 113.9, 119.5, 122.2,
123.7, 124.7, 129.6, 129.8, 130.7, 131.5, 147.3, 147.7, 149.9,
150.8, 151.2, 151.6, 161.7, 161.9. Anal. Calcd for C20H22N2O2: C,
74.51; H, 6.88; N, 8.69. Found: C, 74.42; H, 7.11; N, 8.61.
4.36. (E/Z)-3-(3-Ethoxy-4-methoxyphenyl)-3-(4-(methylsulfon-
yl)phenyl)acrylonitrile (34)
Compound 34 was prepared from 54b (0.8 g, 2.4 mmol) using
general procedure A. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as a white solid (0.53 g, 62% yield): mp, 159–161 °C. 1H
NMR (DMSO-d6) d 1.31 (t, J = 7 Hz, 3H), 3.26–3.37 (m, 3H), 3.78–
3.84 (m, 3H), 3.94–4.07 (m, 2H), 6.30–6.48 (m, 1H), 6.61 (dd,
J = 8.0 Hz, J = 2.0 Hz, 1H), 6.88–7.14 (m, 2H), 7.60–8.08 (m, 4H).
13C NMR (DMSO-d6) d 14.6, 14.6, 55.6, 55.6, 63.9, 64.0, 95.2, 97.3,
111.5, 111.6, 111.7, 113.6, 118.1, 122.4, 122.5, 127.2, 128.5,
129.4, 129.4, 130.2, 141.6, 142.0, 142.4, 143.3, 147.6, 148.1,
150.5, 151.4, 159.8, 159.9. Anal. Calcd for C19H19NO4S: C, 63.85;
H, 5.36; N, 3.92. Found: C, 63.56; H, 5.21; N, 3.73.
4.41. (E/Z)-3-(3-Ethoxy-4-methoxyphenyl)-3-(4-methoxy-3-
(1H-pyrrol-1-yl)phenyl)acrylonitrile (39)
2,5-Dimethoxytetrahydrofuran (0.22 g, 1.7 mmol) was added to
a stirred solution of 36 (0.50 g, 1.5 mmol) in AcOH (10 mL), and the
mixture was heated to reflux for 1 h. The mixture was cooled to
room temperature and diluted with ethyl acetate (100 mL). Satu-
rated aqueous sodium bicarbonate (40 mL) was then carefully
added. The organic phase was washed with water (60 mL) and
brine (60 mL), dried (MgSO4) and evaporated. The residue was
purified using a hexanes/ethyl acetate gradient, providing the
product as a white solid (0.35 g, 60% yield): mp 109–111 °C. 1H
NMR (CDCl3) d 1.45 (m, 3H), 3.91 (m, 6H), 4.05 (m, 2H), 5.57–
5.59 (m, 1H), 6.29–6.32 (m, 2H), 6.81–6.91 (m, 3H), 6.93–7.10
(m, 3H), 7.24–7.27 (m, 1 H), 7.35–7.47 (m, 1H). 13C NMR (CDCl3)
d 14.7, 56.0, 56.1, 64.6, 92.5, 109.2, 109.3, 111.1, 112.7, 112.0,
112.1, 114.0, 118.4, 118.5, 121.9, 122.1, 122.2, 123.1, 125.9,
127.1, 127.9, 129.0, 129.8, 130.1, 131.4, 132.1, 148.3, 148, 151.0,
151.6, 153.9, 154.4, 161.6, 161.7. Anal. Calcd for C23H22N2O3: C,
73.78; H, 5.92; N, 7.48. Found: C, 73.46; H, 5.71; N, 7.35.
4.37. (E/Z)-3-(3-Ethoxy-4-methoxyphenyl)-3-(3-hydroxy-4-
methoxyphenyl)acrylonitrile (35)
Compound 35 was prepared from 59f (0.61 g, 1.4 mmol) using
general procedure B. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as a white solid (0.4 g, 89% yield): mp 63–65 °C. 1H NMR
(CDCl3) d 1.40–1.47 (m, 3H), 3.89–3.94 (m, 6H), 3.98–4.12 (m,
2H), 5.53–5.55 (m, 1H), 5.68 (m, 1H), 6.80–7.09 (m, 6H). 13C NMR
(CDCl3) d 14.7, 55.9, 56.0, 64.5, 92.0, 110.2, 111.0, 112.8, 114.1,
114.7, 116.0, 118.6, 118.7, 121.4, 122.1, 122.2, 123.1, 129.5,
130.4, 131.7, 132.5, 145.3, 145.5, 145.5, 147.9, 148.0, 148.1,
148.4, 150.8, 151.4, 162.4; Anal. Calcd for C19H19NO4: C, 70.14; H,
5.89; N, 4.31. Found: C, 69.93; H, 5.84; N, 4.06.
4.38. (E/Z)-3-(3-Amino-4-methoxyphenyl)-3-(3-ethoxy-4-
methoxyphenyl)acrylonitrile (37)
4.42. 5-(1-(3-Ethoxy-4-methoxyphenyl)vinyl)-2-methoxy-
phenol (40)
Compound 37 was prepared from 62d (3.3 g, 9.7 mmol) using
general procedure C. The product was purified by column chroma-
tography eluting with 7:3 hexanes/ethyl acetate to provide the
product as a yellow solid (1.1 g, 33% yield): mp 141–143oC. 1H
NMR (CDCl3) d 1.40–1.47 (m, 3H), 3.88–3.92 (m, 8H), 3.98–4.12
(m, 2H), 5.49 (m, 1H), 6.64–7.00 (m, 6H). 13C NMR (CDCl3) d 14.7,
55.5, 55.6, 109.8, 110.9, 113.0, 114.2, 114.7, 116.0, 118.8, 118.9,
119.6, 120.7, 122.1, 123.1, 129.8, 129.9, 132.1, 135.9, 136.1,
147.8, 148.0, 146.7, 150.7, 151.2, 163.0. Anal. Calcd for
Compound 40 was prepared from 59g (1.1 g, 2.7 mmol) using
general procedure B. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as a white solid (0.65 g, 83% yield): mp 99–101 °C. 1H
NMR (CDCl3) d 1.42 (t, J = 7.0 Hz, 3H), 3.89 (s, 3H), 3.91 (s, 3H),
4.06 (q, J = 7.0 Hz, 2H), 5.29 (d, J = 1.2 Hz, 1H), 5.32 (d, J = 1.2 Hz,
1H), 5.60 (s, 1H), 6.78–6.98 (m, 6H). 13C NMR (CDCl3) d 14.8,
55.9, 56.0, 64.3, 110.1, 111.0, 112.1, 113.1, 114.6, 120.2, 121.0,
134.5, 135.2, 145.2, 145.3, 147.8, 149.1, 149.2. Anal. Calcd for
C19H20N2O3: C, 70.35; H, 6.21; N, 8.64. Found: C, 70.25; H, 6.21;
C18H20O4: C, 71.98; H, 6.71; N, 0.00. Found: C, 71.90; H, 6.74; N,
N, 8.46.
<0.05.

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A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
4.43. 5-(1-(3-Ethoxy-4-methoxyphenyl)-2,2-difluorovinyl)-2-
methoxyphenol (41)
4.47. Boc deprotection reactions. General procedure F. (E/Z)-2-
Amino-N-(5-(2-cyano-1-(3-ethoxy-4-methoxyphenyl)vinyl)-2-
methoxyphenyl)acetamide hydrochloride (45)
Compound 41 was prepared from 59h (0.80 g, 1.8 mmol) using
general procedure B. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as a white solid (0.5 g, 83% yield); mp 74–76 °C; 1H NMR
(CDCl3) d 1.43 (t, J = 7.0 Hz, 3H), 3.88 (s, 3H), 3.90 (s, 3H), 4.03 (q,
J = 7.0 Hz, 2H), 5.57 (s, 1H), 6.74–6.86 (m, 6H). 13C NMR (CDCl3) d
14.7, 55.9, 64.4, 95.5 (t, J = 19), 110.4, 111.2, 114.3 (t, J = 3.4),
115.76 (t, J = 3.8), 121.5 (t, J = 3.0), 122.3 (t, J = 3.4 Hz), 126.8 (t,
J = 2.6), 127.7 (t, J = 3.0), 145.4, 145.9, 148.0, 148.8, 153.5 (t,
J = 290). Anal. Calcd for C18H18F2O4: C, 64.28; H, 5.39; N, 0.00.
Found: C, 64.27; H, 5.46; N, <0.05.
A solution of 4 N HCl/dioxane (3 mL) was added to a stirred
solution of 66a (1.1 g, 2.4 mmol) in CH2Cl2 (35 mL). The resulting
solution was stirred at room temperature overnight. Ether
(10 mL) was added and the mixture was stirred for 2 h. The slurry
was filtered and was washed with ether to afford the product as a
white solid (0.44 g, 44% yield): mp 208–210 °C. 1H NMR (DMSO-d6)
d 1.31 (t, J = 6 Hz, 3H), 3.78 (s, 2H), 3.83 (s, 3H), 3.90 (s, 3H), 3.99 (q,
J = 7 Hz, 2H), 6.01 (s, 1H), 7.36–6.91 (m, 5H), 7.89 (d, J = 1 Hz, 1H),
8.20 (br, 3H), 9.88 (s, 1H). 13C NMR (DMSO-d6) d 14.6, 41.0, 55.5,
56.1, 63.8, 92.8, 111.5, 113.8, 118.7, 122.4, 125.2, 126.2, 129.2,
130.5 147.4, 150.1, 151.4, 161.4, 165.4. Anal. Calcd for
4.44. 5-(1-(3-Ethoxy-4-methoxyphenyl)vinyl)-2-methoxyani-
line (42)
C
21H24N3O4Clꢂ1.4H2O: C, 56.92; H, 6.10; N, 9.48. Found: C, 56.58;
H, 5.85; N, 9.72.
Compound 42 was prepared from 62e (0.9 g, 2.8 mmol) using
general procedure C. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as a solid (0.4 g, 48% yield): mp 91–93 °C. 1H NMR (CDCl3)
d 1.43 (t, J = 6.8 Hz, 3H), 3.75 (br, 2H), 3.86 (s, 3H), 3.88 (s, 3H), 4.06
(q, J = 7.1 Hz, 2H), 5.28 (d, J = 6.5 Hz, 2H), 6.72–6.91 (m, 6H). 13C
NMR (CDCl3) d 14.8, 55.5, 56.0, 64.3, 109.9, 110.9, 111.5, 113.1,
115.0, 118.7, 120.9, 134.6, 134.7, 135.6, 147.1, 147.7, 149.0,
149.6. Anal. Calcd for C18H21NO3: C, 72.22; H, 7.07; N, 4.68. Found:
C, 72.16; H, 7.10; N, 4.74.
4.48. (S)-(E/Z)-2-Amino-N-(5-(2-cyano-1-(3-ethoxy-4-
methoxyphenyl)vinyl)-2-methoxyphenyl)propan-amide
hydrochloride (46)
Compound 46 was prepared from 66b (2.1 g, 4.4 mmol) using
general procedure F, to afford the product as a white solid (1.4 g,
74% yield): mp 207–209 °C. 1H NMR (DMSO-d6) d 1.31 (t, J = 6 Hz,
3H), 1.41 (m, 3H), 3.79–3.83 (m, 3H), 3.91–3.94 (m, 3H), 3.99 (m,
2H), 4.19 (m, 1H), 6.02–6.16 (m, 1H), 6.75–7.92 (m, 6H), 8.31 (br,
3H), 9.90–9.94 (m, 1H). 13C NMR (DMSO-d6) d 14.6, 14.6, 17.4,
48.6, 55.5, 55.6, 56.0, 56.1, 63.8, 63.8, 92.9, 93.0, 111.3, 111.4,
111.6, 114.5, 112.3, 113.8, 118.7, 118.8, 122.2, 122.4, 122.9,
123.1, 125.6, 126.1, 126.6, 129.1, 129.2, 130.4, 130.5, 147.4,
147.8, 151.0, 151.1, 150.2, 151.9, 161.0, 161.3, 168.8. Anal. Calcd
for C22H26N3O4Clꢂ0.03H2O: C, 61.10; H, 6.07; N, 9.72. Found: C,
60.75; H, 5.84; N, 9.51.
4.45. (E/Z)-5-(1-(3-Ethoxy-4-methoxyphenyl)prop-1-en-1-yl)-2-
methoxyaniline (43)
Compound 43 was prepared from 62f (1.10 g, 3.2 mmol) using
general procedure C. The product was purified by column chroma-
tography using a hexanes/ethyl acetate gradient to provide the
product as a solid (0.73 g, 73% yield): mp 108–110 °C. 1H NMR
(CDCl3) d 1.39–1.46 (m, 3H), 1.72–1.77 (m, 3H), 3.71–3.75 (br,
2H), 3.83–3.90 (m, 6H), 3.99–4.09 (m, 2H), 5.97–6.05 (m, 1H),
6.54–6.61 (m, 6H). 13C NMR (CDCl3) d 14.8, 15.7, 15.7, 55.5, 55.5,
55.9, 56.0, 64.2, 64.3, 109.9, 110.0, 111.0, 111.0, 112.1, 114.1,
114.8, 116.8, 117.5, 119.9, 120.4, 121.8, 122.0, 122.4, 132.9,
132.9, 135.5, 135.6, 136.3, 136.5, 141.9, 142.0, 146.3, 146.5,
147.8, 148.0, 148.3. Anal. Calcd for C19H23NO3: C, 73.82; H, 7.40;
N, 4.47. Found: C, 72.57; H, 7.47; N, 4.40.
4.49. (E/Z)-5-(2-Cyano-1-(3-ethoxy-4-methoxyphenyl)vinyl)-2-
methoxyphenyl dimethylcarbamate (47)
A mixture of 35 (0.57 g, 1.8 mmol), dimethylcarbamyl chloride
(1.5 g, 14 mmol), and DIEA (0.91 g, 7.0 mmol) was heated to reflux
for 5 h. The mixture was cooled and evaporated, and the residue
was purified by column chromatography using a hexanes/ethyl
acetate gradient, to provide the product as an off-white solid
(0.58 g, 84% yield): mp 114–116 °C. 1H NMR (DMSO-d6) d 1.31 (t,
J = 7.0 Hz, 3H), 2.88–3.01 (m, 6H), 3.79–3.85 (m, 6H), 3.95–4.05
(m, 2H), 6.13–6.15 (m, 1H), 6.74–7.35 (m, 6H). 13C NMR (DMSO-
d6) d 14.6, 14.6, 36.1, 36.3, 55.5, 55.6, 55.9, 56.0, 63.8, 93.1, 93.2,
111.4, 111.6, 112.3, 112.5, 112.5, 113.8, 118.7, 118.7, 122.1,
122.2, 123.2, 124.3, 127.0, 127.7, 129.0, 129.1, 130.4, 139.6,
139.9, 147.4, 147.8, 150.2, 151.1, 152.6, 153.3, 153.4, 160.3,
160.4. Anal. Calcd for C22H24N2O5: C, 66.65; H, 6.10; N, 7.07. Found:
C, 66.47; H, 6.20; N, 6.95.
4.46. (E/Z)-N-(5-(2-Cyano-1-(3,5-dimethoxyphenyl)vinyl)-2-
methoxyphenyl)acetamide (44)
A mixture of 11 (0.4 g, 1.3 mmol) and acetyl chloride (0.2 mL) in
THF (50 mL) was refluxed overnight, and then cooled and
quenched with water (100 mL). The resulting mixture was ex-
tracted with methylene chloride (2 ꢁ 50 mL). The combined ex-
tracts were washed with water (100 mL), dried (MgSO4), and
concentrated under vacuum. The crude product was purified by
flash chromatography (hexane-ethyl acetate 1:1) to afford the
product as a white solid (0.3 g, 62% yield): mp 163–165 °C. 1H
NMR (CDCl3) d 2.06 (s, 3H), 3.76 (s, 6H), 3.91 (s, 3H), 6.14–6.22
(m, 1H), 6.46 (d, J = 1.8 Hz, 2H), 6.62 (s, 1H), 7.17 (m, 2H), 7.93 (s,
1H), 9.28 (s, 1H). 13C NMR (CDCl3) d 23.8, 55.4, 55.8, 95.0, 102.0,
106.7, 110.9, 118.3, 122.5, 125.5, 127.3, 128.5, 140.3, 150.5,
160.3, 161.3, 168.7. Anal. Calcd for C20H20N2O4: C, 68.17; H, 5.72;
N, 7.95. Found: C, 68.03; H, 5.54; N, 7.85.
4.50. (E/Z)-5-(2-Cyano-1-(3-ethoxy-4-methoxyphenyl)vinyl)-2-
methoxyphenyl 2-aminoacetate hydrochloride (48)
Compound 48 was prepared from 67a (0.36 g, 0.76 mmol) using
general procedure F, to afford the product as a white solid (0.18 g,
58% yield): mp 190–192 °C. 1H NMR (DMSO-d6) d 1.32 (t, J = 7.0 Hz,
3H), 3.79–3.88 (m, 6H), 3.94–4.04 (m, 2H), 4.10–4.13 (m, 2H),
6.14–6.22 (m, 1H), 6.73–7.48 (m, 6H), 8.54 (br, 3H). 13C NMR
(DMSO-d6) d 14.6, 14.6, 55.5, 55.6, 56.1, 63.8, 93.5, 93.7, 111.4,

A. L. Ruchelman et al. / Bioorg. Med. Chem. 19 (2011) 6356–6374
6373
111.6, 112.2, 113.0, 113.2, 113.7, 118.5, 122.1, 122.3, 122.7, 123.6,
127.9, 128.9, 128.9, 129.5, 130.3, 130.8, 137.9, 138.0, 147.5, 147.9,
150.3, 151.1, 151.7, 152.3, 159.9, 160.1, 166.0. Anal. Calcd for
30 °C, terminated by boiling, treated with 1 mg/ml snake venom,
and separated using AG-1X8 ion exchange resin (BioRad). Reac-
tions consumed less than 15% of available substrate. IC50s and
SEM were calculated as described above.
C20H23N2O5Clꢂ0.5H2O: C, 58.95; H, 5.65; N, 6.55. Found: C, 58.57;
H, 5.52; N, 6.35.
4.55. PBMC TNF- production assay
4.51. (S)-(E/Z)-5-(2-Cyano-1-(3-ethoxy-4-methoxyphenyl)vinyl)-
2-methoxyphenyl 2-aminopropanoate hydrochloride (49)
Peripheral blood mononuclear cells (PBMC) were obtained from
leukocyte units (Blood Center of New Jersey, East Orange, NJ) by Fi-
coll Hypaque (Pharmacia, Piscataway, NJ, USA) density centrifuga-
tion. Cells were cultured in R10 medium (RPMI) (Life Technologies,
Grand Island, NY, USA) supplemented with 10% AB+ human serum
Compound 49 was prepared from 67b (0.89 g, 1.8 mmol) using
general procedure F, to afford the product as a white solid (0.52 g,
67% yield): mp 173–175 °C. 1H NMR (DMSO-d6) d 1.32 (t, J = 7.0 Hz,
3H), 1.55–1.59 (m, 3H), 3.74–3.88 (m, 6H), 3.94–4.05 (m, 2H),
4.30–4.39 (m, 1H), 6.14–6.22 (m, 1H), 6.73–7.42 (m, 6H), 8.64
(br, 3H). 13C NMR (DMSO-d6) d 14.6, 14.6, 15.8, 24.4, 25.3, 33.3,
47.5, 47.8, 55.5, 55.6, 56.2, 56.3, 63.8, 93.6, 93.8, 111.4, 111.6,
112.2, 113.0, 113.1, 113.7, 118.5, 122.2, 122.3, 122.5, 123.4,
128.0, 128.9, 129.0, 129.5, 130.2, 130.9, 138.0, 138.2, 147.5,
147.9, 150.3, 151.2, 151.5, 152.3, 159.9, 160.1, 168.3, 168.3. Anal.
Calcd for C20H25N2O5Clꢂ0.6H2O: C, 59.55; H, 5.95; N, 6.31. Found:
C, 59.20; H, 5.83; N, 6.67.
(Gemini Bio-products, Woodland, CA, USA), 2 mM
L-glutamine,
100 U/ml penicillin, and 100 g/ml streptomycin (Life Technolo-
l
gies)). PBMC (2 ꢁ 105 cells) were plated in 96-well flat-bottom Cost-
ar tissue culture plates (Corning, NY, USA) in triplicate. Compounds
were dissolved in DMSO (Sigma) and further dilutions were done in
culture medium immediately before use. The final DMSO concentra-
tion in all sampleswas 0.25%. Compoundswere added to cells 1 h be-
fore stimulation. Cells were stimulated with LPS (from Salmonella
abortus equi, Sigma, St.Louis, MO, USA) at 1 ng/ml in the absence or
presence of compounds. Cells were incubated for 18–20 h at 37 °C
in 5% CO2 and supernatants were then collected, diluted with culture
4.52. (S)-(E/Z)-5-(2-Cyano-1-(3-ethoxy-4-methoxyphenyl)vinyl)-
2-methoxyphenyl 2-amino-3-methylbut-anoate hydrochloride
(50)
medium and assayed for TNF-a levels by ELISA (Endogen, Boston,
MA, USA) according to the manufacturer’s instructions. IC50s and
SEM were calculated as described above.
Compound 50 was prepared from 67c (0.82 g, 1.6 mmol) using
general procedure F, to afford the product as a white solid
(0.51 g, 70% yield): mp 192–194 °C. 1H NMR (DMSO-d6) d 1.07–
1.14 (m, 6H), 1.32 (t, J = 7.0 Hz, 3H), 2.30–2.37 (m, 1H), 3.79–3.88
(m, 6H), 3.94–4.02 (m, 2H), 4.13–4.19 (m, 1H), 6.14–6.22 (m,
1H), 6.74–7.45 (m, 6H), 8.61 (br, 3H). 13C NMR (DMSO-d6) d 14.6,
17.4, 18.0, 29.5, 55.5, 55.6, 56.0, 56.1, 57.3, 63.8, 93.6, 93.8,
111.4, 111.6, 112.2, 113.0, 113.1, 113.7, 118.5, 122.1, 122.3,
122.6, 123.5, 128.0, 128.8, 129.0, 129.5, 130.9, 137.9, 138.0,
147.5, 147.9, 150.3, 151.2, 151.5, 152.2, 159.9, 160.0, 167.0. Anal.
Calcd for C24H29N2O5Clꢂ0.1H2O: C, 62.29; H, 6.39; N, 6.05. Found:
C, 62.07; H, 6.30; N, 6.06.
4.56. Tubulin polymerization assay
Tubulin polymerization was measured in a cell-free spectro-
photometric assay system, as previously described.30
4.57. Solubility determination
Solubility was determined using 0.5 mL of a ꢀ1 mg/mL suspen-
sion of the appropriate samples in pH 5.0 buffer (Tritisol 9882–2)
or pH 7.4 buffer (Metrepak CAS#270–7.40) in a sealed matrix tube
containing a stir flea. Each sample was set on stir plate set at
400 rpm for 4 h. After 4 h, the samples were filtered using a
0.45 mm filter. Samples were analyzed using a Spectramax UV–
vis Plate Reader (200–400 nm) and compared with standard curves
generated (range 0.25–100 mg/mL) for each compound to deter-
mine the concentration in buffer solution. Replicate measurements
were made for buffer 5.0 determinations.
4.53. HCT-116 cell proliferation assay
The human colorectal carcinoma tumor cell line HCT-116 was
purchased from American Type Culture Collection (Manassas,
VA). Cell proliferation was assessed by [3H] thymidine incorpora-
tion assay. Briefly, cells were seeded on 96-well microtiter plates
24 h before addition of compound to allow them to adhere to
plates. Each compound was tested at serial dilutions in triplicate.
Following compound treatment, the cells were incubated at 37 °C
4.58. Human plasma stability
Analogs were incubated in human plasma at 37 °C for 0, 0.5, 1, 2,
3, 4, 6, 8, and 24 h. At each time point, 60
for additional 72 h. [3H]thymidine (1
lCi in 20
lL medium) was
lL of each plasma sample
added to each well for the last 6 h of incubation time. The cells
were then harvested for detection of tritium incorporation with a
TopCountÒ Microplate Scintillation Counter (Packard Instrument
Company, Meriden, CT). Final cumulative half-maximal inhibitory
concentrations (IC50s) were calculated using non-linear regression
and sigmoidal dose–response, constraining the top to 100% and the
bottom to 0% and allowing variable slope, using GraphPad Prism
version 5.01. SEM (standard error of the mean) was calculated from
the individual IC50s of each replicate.
was added into 120 L of acetonitrile containing 0.1% formic acid
and internal standard. After centrifugation at P3000g, the superna-
tants were transferred for LC–MS/MS analysis.
l
A Shimadzu HPLC system interfaced with an Applied System
Sciex 4000 QTrap mass spectrometer was used for analysis of
samples. The LC separation was carried out on a Phenomenex
Onyx Monolithic C18 column (50 ꢁ 2.0 mm) with gradient of
aqueous acetonitrile containing 0.1% formic acid. The optimized
MRM tandem mass spectrometry was used to detect the analytes
and IS. The percentage remaining data of each compound were
calculated using the peak area ratios (analyte/IS) of the samples
at the designated time points and those at time zero.
4.54. PDE4 enzyme assay
PDE4 enzyme was purified from U937 human monocytic cells
by gel filtration chromatography, and phosphodiesterase reactions
were carried as previously described.50 Briefly, reactions were car-
ried out in 96-well deep well plates in 50 mM Tris–HCl pH 7.5,
4.59. Docking study
Compound 28 was docked into the colchicine binding site of the
5 mM MgCl2, 1
l
M cAMP, plus 10 nM [3H]-cAMP for 30 min at
tubulin crystal structure (PDB code 1SA0)57 using MOE software.58

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21. Medarde, M.; Ramos, A.; Caballero, E.; Peláez-Lamamié de Clairac, R.; López, J.
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Pettit, R. K. J. Med. Chem. 2003, 46, 525.
After aligning the ligand with colchicine, the ligand and protein
residues with atoms within 5 Angstroms of the bound inhibitor
were minimized using the standard parameters in MOE/LigX. Res-
idues outside the 5 Angstrom radius remained fixed.
24. Gaukroger, K.; Hadfield, J. A.; Lawrence, N. J.; Nolan, S.; McGown, A. T. Org.
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4.60. Human tumor xenograft model
25. Pettit, G. R.; Rhodes, M. R.; Herald, D. L.; Hamel, E.; Schmidt, J. M.; Pettit, R. K. J.
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26. Monk, K. A.; Siles, R.; Hadimani, M. B.; Mugabe, B. E.; Ackley, J. F.; Studerus, S.
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Female SCID mice (6–8 weeks old) were obtained from the
Charles River Laboratory (Wilmington, MA) and maintained in
microisolator cages under sterile conditions. The right hind legs of
the mice were inoculated s.c. with HCT-116 cells suspended in ster-
ile PBS (2 ꢁ 106 cells per mouse). On day 8, tumors of all mice were
measured with a digital caliper and volumes were calculated with a
formula of W2 ꢁ L / 2, where W is width (short axis) and L is length
(long axis). Mice bearing tumor size ranging between 75 and 125
mm3 were pooled together and randomly distributed into cages.
The mice were then ear tagged and cages were randomly assigned
to treatment groups. On day 8, the tumors were measured and con-
sidered as starting volumes. The mice were then dosed ip with either
vehicle (CMC-Tween: 0.5% Carboxymethylcellulose and 0.25%
Tween 80 in water), Compound 28 (20 mg/kg), Compound 45
(20 mg/kg) or positive control Camptosar (5 mg/kg; Pfizer, Inc.,
New York, NY). Compound 28 and Compound 45 were given once
daily administrations (qd). Camptosar was administered once every
four days (q4d). Mice were monitored daily for health status as well
as tumor growth. Tumors were measured twice weekly.
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