Novel Resveratrol-Based Aspirin Prodrugs: Synthesis, Metabolism, and Anticancer Activity

Article abstract of DOI:10.1021/acs.jmedchem.5b00536

(Graph Presented). Regular aspirin use has been convincingly shown to reduce the risk of colorectal cancer. However, long-term use of aspirin leads to gastrotoxicity. Herein, we designed and synthesized a novel class of resveratrol-based aspirin prodrugs to simultaneously release aspirin and resveratrol to attenuate the side effects caused by aspirin. Prodrug RAH exerted enhanced anticancer activities which are better than a physical mixture of aspirin and resveratrol as well as each individually. Metabolism of RAH in mice showed that the majority of RAH is decomposed to release resveratrol and aspirin or salicylic acid either in the intestine or after absorption. Mechanistic studies demonstrate RAH inhibits cell cycle arrest through downregulation of cyclins and induces apoptosis by activation of caspase-3 in cancer cells. These findings highlighted the improved anticancer properties of resveratrol-based aspirin prodrugs. RAH may represent novel and safe alternatives of aspirin for the purpose of daily use in the future.

Full text of DOI:10.1021/acs.jmedchem.5b00536

Page 1 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
Novel Resveratrol-based Aspirin Prodrugs: Synthesis, Metabolism,
and Anticancer Activity
2
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
3
4
5
Yingdong Zhu,† ,1 Junsheng Fu,†, ‡,1 Kelly L. Shurlknight,† Dominique N. Soroka,† Yuhui Hu,§
Xiaoxin Chen,§ and Shengmin Sang*,†
6
7
†Center for Excellence in PostꢀHarvest Technologies, North Carolina Agricultural and Technical
State University, North Carolina Research Campus, 500 Laureate Way, Kannapolis, NC 28081,
United States
8
9
10
11
12
13
14
15
16
‡College of Life Sciences, Fujian Agriculture and Forestry University, No.15 Shangxiadian
Road, Cangshan District, Fuzhou City, Fujian Province, 350002, P. R. China
§Cancer Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute,
North Carolina Central University, 700 George Street, Durham, NC 27707, United States
1 These authors contributed equally to this work.
Running Title: Novel Resveratrol-based Aspirin Prodrugs
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 47
1
2
3
4
5
6
17
18
ABSTRACT
Regular aspirin use has been convincingly shown to reduce the risk of colorectal cancer.
However, longꢀterm use of aspirin leads to gastrotoxicity. Herein, we designed and synthesized a
novel class of resveratrolꢀbased aspirin prodrugs to simultaneously release aspirin and resveratrol
to attenuate the side effects caused by aspirin. Prodrug RAH exerted enhanced anticancer
activities which are better than a physical mixture of aspirin and resveratrol as well as each
individual. Metabolism of RAH in mice showed that the majority of RAH is decomposed to
release resveratrol and aspirin or salicylic acid either in the intestine or after absorption.
Mechanistic studies demonstrate RAH inhibits cell cycle arrest through downregulation of
cyclins, and induces apoptosis by activation of caspaseꢀ3 in cancer cells. These findings
highlighted the improved anticancer properties of resveratrolꢀbased aspirin prodrugs. RAH may
represent novel and safe alternatives of aspirin for the purpose of daily use in the future.
7
8
9
19
20
21
22
23
24
25
26
27
28
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
2
ACS Paragon Plus Environment

Page 3 of 47
Journal of Medicinal Chemistry
1
2
3
29
4
INTRODUCTION
5
6
7
8
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Colorectal cancer (CRC) is the third most common form of cancer and the fourth most frequent
cause of cancer deaths worldwide.1 Most cases of CRC are sporadic, and genetic and
environmental factors play important roles. Aspirin (ASA) is a classic wonder drug and has been
widely used for the treatment of inflammation, fever, and pain over a century.2 Recently, ASA
has received another boost due to its potential benefits in the prevention/treatment of cancers and
cancer prevention.3ꢀ5 The main targets of ASA are cyclooxygenase (COX) isoforms, COXꢀ1 and
COXꢀ2, which catalyze the rateꢀlimiting step in the conversion of arachidonic acid to
prostaglandins (PGs).6 PGs play a key role in the generation of the inflammatory response. COXꢀ
1, expressed constitutively in most tissues, is the dominant source of PGs that have a protective
effect on gastric mucosa.7 Inhibition of COXꢀ1 impairs the synthesis of PGs, and adverse effects
occur as a result of effects on the constitutively expressed COXꢀ1.8 COXꢀ2, induced by
inflammatory stimuli, hormones, and growth factors, is mainly responsible for inflammatory
reactions and tumor developments.9 Longꢀterm use of ASA is limited because of its lack of
COXꢀ2 selectivity, which is generally considered as the main drawback of ASA and accounts for
ASA’s main side effects, such as gastric ulceration.8 Gastrointestinal toxicity can be reduced by
selective inhibitors of COXꢀ2,10 but chronic use leads to an increased risk of heart attack and
stroke.11 Other than COXꢀ2 inhibitors, significant efforts have been devoted to develop nitric
oxideꢀreleasing ASA derivatives in order to better control side effects of nonsteroidal antiꢀ
inflammatory drugs (NSAIDs). Nitric oxide (NO) is an important gaseous messenger that
mediates a variety of physiological actions, including gastroprotetction.12 Release of NO in the
gastrointestinal tract (GIT) would lead to a local relaxation of the blood vessel with lower
adhesion of leukocytes, therefore improving protection of the gastric mucosa and reducing
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 47
1
2
3
4
5
6
7
8
9
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
haemorrhagic effects.13 Positive in vitro and in vivo results are available,14ꢀ16 but longꢀterm use of
organic nitrate derivatives of NOꢀASA can lead to nitrate tolerance, as occurs with
nitroglycerine.17 In addition, some newꢀdeveloped NOꢀASA derivatives were demonstrated to
release NO with expulsion of formaldehyde,18 the latter has been identified as a human
carcinogen recently.19 Hence, the advancement of new ASA derivatives with more favorable and
safer profiles would be a welcome development indeed.
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
Various plant polyphenols have been shown to have a therapeutic potential for the effective
treatment of peptic ulcers.20 Resveratrol (RES) is without a doubt the most famous dietary
polyphenol present in peanuts, various berries, grapes and red wine, and has been reported to be
capable of protecting the gastric mucosa against the NSAIDꢀinduced side effects.21, 22 Besides its
promising activities against inflammation, heart disease and aging,23 numerous preclinical
findings consider RES as an upꢀandꢀcoming natural arsenal for cancer prevention and
treatment.24, 25 Clinic trials of RES in CRC patients further added RES reduces tumor cell
proliferation.26 Daily oral administration of a high dose of RES, up to 750 mg/kg, to rats for 90
days demonstrated the nontoxicity of this polyphenol 27, 28
Moreover, recent clinical trials
.
evidenced that no adverse events were reported to resveratrol supplementation at doses < 0.5
g/day when the duration was short.29 Thus, RES could be an ideal additive to the clinic use of
ASA to ameliorate the side effects caused by ASA.
A combination of ASA and RES can be an attractive strategy to retain anticancer and antiꢀ
inflammatory properties of ASA while reducing gastric toxicity. Major concerns in joint use
include drug interactions, the definitive exposure to the desired targets, and individual
pharmacokinetics and biodistribution parameters. These difficulties could be overcome by
constructing a single prodrug containing these two moieties, which is able to be decomposed to
4
ACS Paragon Plus Environment

Page 5 of 47
Journal of Medicinal Chemistry
1
2
3
75
4
release corresponding individuals after administration. However, little is known for the
formation of natural productꢀbased ASA derivatives with gastroprotection so far. With these
challenges in mind, we constructed a class of RESꢀbased ASA derivatives (4-10) (Scheme 1),
with the ability to release ASA and RES for their respective biological properties.
5
6
7
8
76
77
78
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
RESULTS AND DISCUSSION
Chemical Synthesis. Acylation of RES (1) by Oꢀacetylsalicyloyl chloride in the presence of
Et3N at 0 oC produced a RESꢀASA hybrid (RAH) 4 and its regioisomer 5 with a ratio of around
2.3:1. Likewise, coupling reaction of pterostilbene (2), a dimethylated RES, with Oꢀ
acetylsalicyloyl chloride afforded a pterostilbeneꢀASA hybrid 6 in 96% yield. Hydrolysis of 6 by
dilute HCl solution led to a deacetylated product 7. Prudent treatment of 6 by BBr3 in CH2Cl2 at ꢀ
78 oC produced prodrug 8 in 3% yield, otherwise demethylated products 9 and 10 were harvested
as major products at 0 oC (Scheme 1).
In order to validate the biological properties of the molecular skeleton composed of ASA and
RES, two RAHꢀlike molecules 11 and 12, which have the molecular skeleton consisted of ASA
residue and RES moiety, were synthesized (Scheme 2). RAH resemblances, 11 and 12, are more
structurally stable, insusceptible to breakage, and most likely exerting their own biological
effects as whole molecules. Analogue 11 was synthesized as outlined in Scheme 2A. In brief, 4ꢀ
bromobenzyl bromide was first refluxed with PPh3, and a subsequent Wittig reaction with 3,5ꢀ
dimethoxybenzaldehyde in the presence of NaOBuꢀt provided 4ꢀbromostilbenoid 14 in 66%
overall yield. Bromostilbene 14 was treated with salicylaldehyde in the presence of of nꢀBuLi to
give additive product 15. Orthoꢀhydroxy assisted and copperꢀcatalyzed oxidation of compound
15 in DMF produced ketone 16,30 which was converted to the target compound 11 by BBr3 in
62% yield. The synthetic route for resemblance 12 was shown in Scheme 2B. In detail, reductive
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 47
1
2
3
4
5
6
7
8
9
98
99
coupling of 4ꢀbromobenzyl bromide with Oꢀacetylsalicyloyl chloride in the presence of
Pd(PPh3)4 and zinc produced coupling product 19. Removal of acetyl group in 19 led to
deacetylated product 20 in 96% yield.31 A Heck reaction between 20 and 18 in the presence of
Pd(PPh3)4 and Et3N provided coupling product 21 in 14% yield.32 Deacetylation of 19 is the key
process to succeed in the preparation of intermediate 21, otherwise acetyl group would be further
involved in an intermolecular aldol reaction (data not shown). Finally, demethylation of 21 took
place using BBr3 to furnish the target compound 12.
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
In Vitro Cytotoxic Activity. RESꢀbased ASA derivatives 4-10 were evaluated for their antiꢀ
cancer activities against human colon cancer cells HCTꢀ116 and HTꢀ29 using MTT assay and
were compared with the reference compounds RES (1), pterostilbene (2) and ASA (3). As shown
in Figure 1, prodrugs 4-10 were more active than the individual drugs 1-3. In particular, RAH 4
displayed the most potential in both cancer cell lines, and followed by deacetyl RAH (DAꢀRAH)
10. RAH 4 was more active than dimethylates 6 and 7, as well as monoꢀmethylates 8 and 9,
indicating that the two free hydroxyl groups at Cꢀ3 and Cꢀ5 in the ring A of RES moiety play an
important role in the observed activity. In addition, RAH 4 exhibited a better activity than its
regioisomer 5, indicating the manner in which the two moieties are linked in molecule 4 favors
the anticancer activity.
As the most potent RESꢀASA hybrid, RAH 4 was further compared to an equimolar mixture
of ASA and RES (ASA+RES) using colony formation assay (Figure 2). Our results showed that
RAH is much more active than ASA+RES in both human colon cancer cells in a doseꢀdependent
manner (Figure 2A). These observations indicate that the molecular skeleton composed of ASA
and RES possesses antiproliferative properties. In order to validate the hypothesis mentioned
above, we further evaluated the anticancer properties of two RAHꢀlike molecules 11 and 12. Our
6
ACS Paragon Plus Environment

Page 7 of 47
Journal of Medicinal Chemistry
1
2
3
121
4
results showed that both RAHꢀlike molecules 11 and 12 exert comparable antiꢀproliferative
activities to that of RAH in both cell lines (Figures 1 and 2B). Interestingly, molecule 11 is more
potent in inhibiting the formation of colonies than its resemblance 4 in both cells, while molecule
12 elicits parallel ability suppressing the growth of colonies to RAH 4 in HTꢀ29. These findings
further validated the molecular skeleton consisted of ASA and RES has anticancer properties. As
a consequence, we demonstrated that 1) free hydroxyl groups in the ring A of the RAH moiety
play an important role in activity; 2) RAH is more active than an equimolar mixture of ASA and
RES, individual ASA, or individual RES; and 3) the molecular skeleton composed of ASA and
RES has anticancer properties.
5
6
7
8
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
Metabolism of RAH in Cancer Cells. We have identified RAH 4 as the most promising
anticancer prodrug containing a combination of ASA and RES, its ability to release RES and
ASA was studied in cancer cells HCTꢀ116 using HPLCꢀcoulometric electrode array detector
(CEAD) (Figure 3). Our results illustrated that RAH was gradually hydrolyzed into deacetyl
RAH (DAꢀRAH) 10 in the first 2 h of incubation, and then DAꢀRAH was accumulated
correspondingly to a maximum at around 4 h. Next, DAꢀRAH was gradually decomposed to
release RES and salicylic acid (overlapped by background) over 24 h. This indicated that
antiproliferative activity of RAH in cancer cells may reasonably be attributed to intact RAH and
DAꢀRAH as well as individual RES and salicylic acid.
Major Metabolites and Distribution of RAH in Mice. The metabolism and biodistribution
of RAH 4 in mice was further investigated using LCꢀESI tandem mass spectrometry. As a result,
fourteen metabolites (1, 10, and 22−33) were identified in mouse GIT contents, colon tissue,
plasma, urine or feces after intragastric RAH administration (Table 1 and Figure 4). Compounds
1, 4, 10, and 22−24 were fully identified by direct comparison with the available standards. The
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 47
1
2
3
4
5
6
7
8
9
144
145
146
147
148
149
150
151
152
rest of metabolites were tentatively identified according to their MS/MS spectra (Table 1). In
brief, compound 25 had a molecular ion at m/z 307 [M − H]− (227 + 80), indicating 25 is the
sulfate metabolite of RES. This was further confirmed by observing m/z 227 [M − 80 − H]− (loss
of one sulfate moiety from m/z 307) as the major product ion in its MS/MS spectrum (Table 1).
This was also supported by nonꢀconjugated RES at m/z 227 [M − H]− found in mouse urine after
enzymatic hydrolysis (Figure 4). Peak 26, with a molecular ion at m/z 579 [M − H]− (227 + 176 ×
2), had a major MS/MS fragment at m/z 403 [M − 176 − H]− (loss of one glucuronide moiety
from m/z 579), suggesting 26 is RES diglucuronide. Peak 27, with a molecular ion at m/z 581 [M
− H]− (229 + 176 × 2) which is 2 units higher than 26, had a major MS/MS fragment at m/z 405
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
153
154
155
156
157
158
159
160
161
162
163
164
165
166
[M − 176 − H]− (loss of one glucuronide moiety from m/z 581), indicating 27 is DHꢀRES
diglucuronide. In a similar manner, peak 28 was identified as RES sulfoglucuronide, 29 as DHꢀ
RES sulfoglucuronie, 30 as RES glucuronide, 31 as DHꢀRES glucuronide, and 32 as DHꢀRES
sulfate. Peak 33 (Rt 34.3 min), having an identical MS/MS spectrum but a different retention
time to that of 1 (Rt 30.4 min), was tentatively identified as cisꢀRES.33 As a matter of fact,
conjugates 25−32 have been previously identified as the phase II metabolites of RES in pigs after
intragastric RES administration.33
As shown in Figure 4, RAH was found to be well tolerated in the acidic environment of
stomach and only small amounts of DAꢀRAH 10 appeared in mouse stomach contents in 1 h
after administration (Figure 4C). After 2 h, most RAH was transited out of the stomach (Figure
4D). In contrast, an increasing amount of RAH was found in mouse intestine contents from 1 h
to 2 h after administration (Figures 4F and 4G). Interestingly, RAH was found to be relatively
abundant in the mouse feces collected in 24 h after oral administration (Figure 4H). Particularly,
intact RAH was also detected in the mouse colon tissue collected at 1 h and 2 h after
8
ACS Paragon Plus Environment

Page 9 of 47
Journal of Medicinal Chemistry
1
2
3
167
4
administration (Figures 4I and 4J). Our observations suggested that certain amount of RAH is
able to bear the acidic environment of stomach, avoid enzymatic hydrolysis in the small intestine,
reach the colon, and then incorporated into the colon tissue as an intact molecule, thereby
contributing its potential chemopreventive activity in colon cancer, like RES reported
previously.34
5
6
7
8
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
On the other hand, salicylic acid 23 and RES sulfate 25 were detected as the major
metabolites of RAH in the mouse plasma collected at 2 h after administration (Figure 4K and 4L).
The metabolites found in the colon tissue at 2 h were salicylic acid 23, RES 1 and RES sulfate 25
(Figures 4I and 4J). In addition, four RES conjugates (sulfate 25, diglucuronide 26,
sulfoglucuronide 28, and glucuronide 30), salicyluric acid 22, and salicylic acid 23 were excreted
into the mouse urine collected in 24 h after administration (Figure 4M). These observations
indicated that the majority of RAH is capable of rapidly releasing RES and ASA or salicylic acid
after incorporation into intestinal enterocyte. Whereas, the arise of RES 1, salicyluric acid 22 and
salicylic acid 23 in the intestine contents (Figures 4F and 4G) also suggested certain amount of
RAH collapses in the intestine and subsequently releases RES and salicylic acid. The released
RES and salicylic acid in the intestine may be reabsorbed into blood circulation later. This
hypothesis is supported by the presence of microbiotaꢀderived metabolite DHꢀRES 24 in the
mouse feces (Figure 4H) and four DHꢀRES conjugates (diglucuronide 27, sulfoglucuronide 29,
glucuronide 31, and sulfate 32) in the mouse urine (Figure 4M).33 As a result, RAH is capable of
releasing RES and ASA or salicylic acid for their respective biological effects either in the
intestine or after absorption via intestinal enterocyte. Thus, ASA or its metabolite salicylic acid
displays antiꢀinflammatory and antiꢀcancer properties while RES showing gastroprotective
effects and cancer prevention. At this point, it is worth emphasizing that ASA is not able to be
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 47
1
2
3
4
5
6
7
8
9
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
directly detected in mice, probably due to its rapid hydrolysis to salicylic acid in vivo. The
reported biologic halfꢀlife of ASA is very short, approximately 15 to 20 min in normal adults.35
As a result, our study on the metabolic fate and distribution of RAH in RAHꢀtreated mice
demonstrated 1) RAH is well tolerated in the stomach; 2) RAH is capable of releasing RES and
ASA or salicylic acid for their respective biological effects either in the intestine or after
absorption via intestinal enterocyte; and 3) certain amount of RAH could reach the target sites of
the colon and thereby contribute its enhanced chemoprevention activities as an intact molecule.
RAH Inhibits Human Colon Cancer Cell Cycle Arrest via Down-regulation of Cyclin
D1 and Cyclin E. Cell proliferation is known to follow the orderly progression of the cell cycle,
which is governed by protein complexes composed of cyclins such as cyclins D1 and E, and
cyclinꢀdependent kinases.36, 37 To determine whether the cytotoxic effects induced by the
treatment of RAH are due to cell cycle arrest, we performed flow cytometry analysis. HCTꢀ116
and HTꢀ29 cells were treated with RAH at concentrations of 0, 20, 40, and 60 µM for the
duration of 24 h, respectively. Our results showed that RAH could doseꢀdependently induce cell
cycle arrest at the G1 phase in HCTꢀ116 cells and G1 and S phases in HTꢀ29 cells (Figure 5A).
Further Western blot analysis showed that the expression of cyclins D1 and E in both cancer
cells was significantly decreased in a doseꢀdependent manner after RAH treatment (Figure 5B).
These results indicate that RAH can cause cell cycle arrest through downꢀregulation of cyclins
D1 and E expression.
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
RAH Induces Cancer Cell Apoptosis through Mitochondrial Pathway by Activation of
Caspase-3. We further investigated the potential of RAH to induce apoptosis in HCTꢀ116 and
HTꢀ29 cells using flow cytometry (Figure 6A). We observed that RAH doseꢀdependently
induced apoptosis in both HCTꢀ116 and HTꢀ29 cancer cells (Figure 6A). It is well documented
10
ACS Paragon Plus Environment

Page 11 of 47
Journal of Medicinal Chemistry
1
2
3
213
4
that mitochondria plays a key role in the apoptotic process,38, 39 and proꢀapoptotic members of
the Bclꢀ2 family proteins can induce the release of cytochrome c into the cytosol which, once
there, activates caspases by triggering mitochondrial membrane permeabilization, leading to cell
death.40ꢀ42 In order to determine how RAH induces apoptosis in colon cancer cells, we performed
a Western blot analysis for Bclꢀ2 family proteins Bim, Bax, Bad, BclXL, Bcl2 and Bak, and
cytochrome c as well as cleaved caspaseꢀ3 (Figures 6B and C). Our results showed that RAH upꢀ
regulated the expression of Bim, Bax, Bad, and Bak while decreasing the levels of Bcl2 and
BclXL in both cell lines in a doseꢀdependent manner (Figure 6B). Increased expression of
cytochrome c was clearly observed in both cell lines (Figure 6B). In addition, we also found the
expression of cleaved caspaseꢀ3 increased significantly with increasing doses of RAH (Figure
6C). These observations indicate that RAH induces the release of cytochrome c into cytoplasm to
activate downstream caspaseꢀ3, thereby leading to cell death in human colon cancer cells HCTꢀ
116 and HTꢀ29.
5
6
7
8
214
215
216
217
218
219
220
221
222
223
224
225
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
226
227
228
229
230
231
232
233
234
235
CONCLUSION
In summary, a novel class of RESꢀbased ASA prodrugs have been designed, synthesized, and
characterized for the first time, and their anticancer properties have also been validated. We
identified RAH 4 as the most potent anticancer agent, which is more active than a physical
mixture of ASA and RES, individual ASA or individual RES. We further validated the
antiproliferative properties of the molecular skeleton composed of ASA and RES by using stable
synthetic RAH resemblances 11 and 12. Stability of RAH in cancer cells revealed that RAH was
hydrolyzed into DAꢀRAH and subsequently decomposed to release RES and salicylic acid over
24 h of incubation. RAH was further found to be well tolerated in the stomach in mice after
intragastric RAH administration, far beyond the common sense of susceptibility of an ester in the
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 47
1
2
3
4
5
6
7
8
9
236
237
238
239
240
241
242
243
244
245
246
247
acidic environment. The majority of RAH is capable of releasing RES and ASA or salicylic acid
for their respective biological effects either in the intestine or after absorption via intestinal
enterocyte, while certain amount of RAH could reach the target sites of the colon and thereby
contribute its enhanced chemoprevention activities as an intact molecule. Further mechanistic
studies demonstrated RAH inhibits cancer cell cycle arrest through downꢀregulation of cyclin D1
and E, and induces cancer cell apoptosis through mitochondrial pathway by activation of
caspaseꢀ3. As a combination of NSAIDs and dietary polyphenol, RAH represents a class of
novel and safer alternatives of ASA, with the ability to release simultaneously RES and ASA,
which effectively retain the broad spectrum of effects for ASA while reducing the side effect of
gastrointestinal ulceration. RAH may be developed as a pharmaceutical for the purpose of
chronic use in the prophylaxis and/or treatment of colon cancer and other diseases in clinical
trials in the future.
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
248
249
250
251
252
253
254
255
256
257
258
EXPERIMENTAL SECTION
Chemistry. General Methods. Anhydrous reactions were carried out in ovenꢀdried glassware
under a nitrogen atmosphere unless otherwise noted. Reactions were monitored by analytical
thinꢀlayer chromatography (TLC) on 250 ꢀm silica gel plates (GF254) (Merck) and visualized
under UV light. The products were isolated and purified by either preparative TLC on 2000 ꢀm
silica gel plates (GF254) (Sorbent Technologies, catalog no. 1617124) or column
1
chromatography (CC) using silica gel (Sorbent Technologies, catalog no. 3093Mꢀ25). H, 13C
NMR, and twoꢀdimensional (2ꢀD) NMR spectra were recorded on a Bruker AVANCE 600 MHz
or 700 MHz spectrometer (Brucker, Inc., Silberstreifen, Rheinstetten, Germany) using TMS as
an internal standard. Chemical shifts (δ) are expressed in ppm. Coupling constants (J) are
expressed in Hz, and multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet),
12
ACS Paragon Plus Environment

Page 13 of 47
Journal of Medicinal Chemistry
1
2
3
259
4
and br (broad). The 13C NMR spectra are proton decoupled. Resveratrol was obtained from Mega
Resveratrol (Danbury, CT, USA). Aspirin was purchased from MP Biomedicals (Solon, OH).
Salicylic acid was bought from Avantor Performance Materials (Phinllipsburg, NJ). Salicyluric
acid was obtained from Sigma (St. Louis, MO). Sulfatase and βꢀglucuronidase were acquired
from Sigma (St. Louis, MO). Other chemicals were purchased from SigmaꢀAldrich (St. Louis,
MO) and were used without further purification. All synthesized compounds were >95% pure.
HPLC Analysis. An HPLCꢀCEAD system (ESA, Chelmsford, MA) consisting of an ESA
model 584 HPLC pump, an ESA model 542 autosampler, an ESA organizer, and an ESA
coularray detector coupled with two ESA model 6210 four sensor cells was used in the current
study. A Gemini C18 column (150 mm × 4.6 mm, 5 ꢀm; Phenomenex, Torrance, CA) was used
to analyze at a flow rate of 1.0 mL/min. The mobile phases consisted of solvent A (30 mM
sodium phosphate buffer containing 1.75% acetonitrile and 0.125% tetrahydrofuran, pH 3.35)
and solvent B (15 mM sodium phosphate buffer containing 58.5% acetonitrile and 12.5%
tetrahydrofuran, pH 3.45). The gradient elution had the following profile: 20% B from 0 to 3
min; 20ꢀ55% B from 3 to 11 min; 55ꢀ60% B from 11 to 12 min; 60ꢀ65% B from 12 to 13 min;
65ꢀ100% B from 13 to 40 min; 100% B from 40 to 45 min; and 20% B from 45.1 to 50 min. The
cells were then cleaned at a potential of 1000 mV for 1 min. The injection volume of the sample
was 10 µL. The eluent was monitored by the Coulochem electrode array system (CEAS) with
potential settings at ꢀ100, 0, 100, 200, 300, 400, and 500 mV.
5
6
7
8
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
LC/MS Analysis. LC/MS analysis was carried out with a ThermoꢀFinnigan Spectra System
which consisted of an Accela high speed MS pump, an Accela refrigerated autosampler, and an
LCQ Fleet ion trap mass detector (Thermo Electron, San Jose, CA, USA) incorporated with an
electrospray ionization (ESI) interface. A Kinetex C18 column (150 mm × 4.6 mm i.d., 2.6 ꢀm,
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 47
1
2
3
4
5
6
7
8
9
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
Phenomenex) was used for separation at a flow rate of 0.3 mL/min. The column was eluted with
100% A (5% aqueous methanol with 0.2% acetic acid (AA) for 5 min, followed by linear
increases in B (95% aqueous methanol with 0.2% AA) to 30% from 5 to 10 min, to 50% from 10
to 35 min, then to 100% B from 35 to 40 min, and then with 100% B from 40 to 45 min. The
column was then reꢀequilibrated with 100% A for 5 min. The injection volume was 10 µL for
each sample. The LC eluent was introduced into the ESI interface. The negative ion polarity
mode was set for the ESI source with the voltage on the ESI interface maintained at
approximately 3.0 kV. Nitrogen gas was used as the sheath gas and auxiliary gas. Optimized
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
o
source parameters, including capillary temperature (297 C), sheath gas flow rate (20 arbitrary
units), auxiliary gas flow rate (10 units), and tube lens (ꢀ80 V), and capillary voltage ꢀ24 V, were
tuned using RES in methanol (10 µg/mL). The collisionꢀinduced dissociation (CID) was
conducted with an isolation width 2 Da and normalized collision energy of 35 for MS/MS. The
mass range was measured from 100 to 800 m/z. Data acquisition was performed with Xcalibur
version 2.0 (Thermo Electron, San Jose, CA, USA).
General Procedure A. To a solution of stilbenes (1.0 eq.) and Et3N (3.0 eq.) in DCM at 0 oC
was added a solution of Oꢀacetylsalicyloyl chloride (1.0 eq.ꢀ2.0 eq.) in DCM dropwise. After
addition, the mixture was stirred at 0 oC for 30 min and allowed to warm up to rt for another 2 h.
The reaction was quenched with water and extracted with ethyl acetate (EA). The organic phase
was washed with water (× 2) and brine (× 1), dried over Na2SO4, and filtered. The filtrate was
evaporated in vacuo, and the residue was subjected to column chromatography (CC) to give the
desired compounds.
General Procedure B. To a solution of methyl ether derivatives (1.0 eq.) in DCM at 0 oC or
o
ꢀ78 C was added a solution of BBr3 in DCM (1.0 M, 2.0ꢀ3.0 eq.) dropwise. The mixture was
14
ACS Paragon Plus Environment

Page 15 of 47
Journal of Medicinal Chemistry
1
2
3
305
4
stirred at certain temperature for 0.5 hꢀ3 h and quenched with water. The resulting mixture was
extracted with EA. The organic solution was washed with water (× 2) and brine (× 1), dried over
Na2SO4, and filtered. The filtrate was evaporated in vacuo, and the residue was purified by CC to
give the desired compounds.
5
6
7
8
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
General Procedure C. A mixture of triphenylphosphine (TPP) (1.0 eq.) and halides (1.0 eq.)
in toluene was refluxed under N2 overnight. The mixture was cooled down to rt, and hexane was
added. The solid was filtered and washed with hexane/EA (10:1) to give the corresponding
alkylphosphoniums. To a stirred solution of 3,5ꢀdimethoxybenzaldehyde (1.2 eq.) in THF was
o
added alkylphosphoniums (1.0 eq.). The resulting mixture was then cooled down to 0 C and
NaOBuꢀt (1.2 eq.) was added. After addition, the mixture was allowed to warm up to rt and
stirred for 6 hꢀ18 h under N2. The reaction was quenched by saturated NH4Cl aqueous. The
mixture was extracted with EA. The organic solution was washed with water (× 2) and brine (×
1), dried over Na2SO4, and filtered. The filtrate was evaporated in vacuo, and the residue was
purified by CC to give the desired compounds.
General Procedure D. A mixture of bromide (1.0 eq.), 1,3ꢀdimethoxyꢀ5ꢀvinylbenzene (1.2
eq.), Pd(PPh3)4 (0.05 eq.), and TEA (2.0 eq) in dry DMF was heated to 100 oC and stirred for 18
hꢀ24 h under N2. The dark mixture was distributed between EA and 10% HCl aqueous. The
organic layer was separated and washed with water (× 2) and brine (× 1), dried over Na2SO4, and
filtered. The filtrate was evaporated in vacuo, and the residue was purified by preꢀTLC to give
the desired compounds.
(E)ꢁ4ꢁ(3,5ꢁDihydroxystyryl)phenyl 2ꢁacetoxybenzoate (4) and (E)ꢁ3ꢁ(4ꢁHydroxystyryl)ꢁ5ꢁ
hydroxyphenyl 2ꢁacetoxybenzoate (5). General procedure A was followed using RES 1 (57 mg,
0.25 mmol, 1.0 eq.), Oꢀacetylsalicyloyl chloride (50 mg, 0.25 mmol, 1.0 eq.) and Et3N (0.14 mL,
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 47
1
2
3
4
5
6
7
8
9
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
0.75 mmol, 3.0 eq.) in DCM (5 mL). The residue was purified by prepꢀTLC (C/M = 15:1, and
10:1) to afford 4 (28 mg, yield: 78%) as a white solid and 5 (10 mg, yield: 28%) as a white solid.
4: 1H NMR (600 MHz, CD3OD) δ 6.50 (2H, d, J = 2.0 Hz, Hꢀ2/6), 6.21 (1H, t, J = 2.0 Hz, Hꢀ4),
6.99 (1H, d, J = 16.3 Hz, Hꢀ7), 7.06 (1H, d, J = 16.3 Hz, Hꢀ8), 7.58 (2H, d, J = 8.6 Hz, Hꢀ10/14),
7.15 (2H, d, J = 8.6 Hz, Hꢀ11/13), 7.22 (1H, d, J = 8.0 Hz, Hꢀ19), 7.69 (1H, dt, J = 8.0, 1.6 Hz,
Hꢀ20), 7.43 (1H, t, J = 7.8 Hz, Hꢀ21), 8.18 (1H, dd, J = 7.8, 1.6 Hz, Hꢀ22), and 2.26 (3H, s,
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
13
CH3C=O); C NMR (150 MHz, CD3OD) δ 140.6 (s, Cꢀ1), 106.2 (d, Cꢀ2/6), 159.8 (s, Cꢀ3/5),
103.3 (d, Cꢀ4), 130.5 (d, Cꢀ7), 128.3 (d, Cꢀ8), 137.0 (s, Cꢀ9), 128.5 (d, Cꢀ10/14), 123.0 (d, Cꢀ
11/13), 151.3 (s, Cꢀ12), 164.6 (s, Cꢀ16), 124.0 (s, Cꢀ17), 152.4 (s, Cꢀ18), 125.1 (d, Cꢀ19), 135.9
(d, Cꢀ20), 127.4 (d, Cꢀ21), 133.0 (d, Cꢀ22), 171.3 (s, CH3C=O), and 21.0 (q, CH3C=O ); negative
1
ESIMS, m/z 449 [M + CH3COOH − H]− and 389 [M − H]−. 5: H NMR (600 MHz, CD3OD) δ
6.80 (1H, brs, Hꢀ2), 6.47 (1H, t, J = 2.0 Hz, Hꢀ4), 6.86 (1H, brs, Hꢀ6), 6.89 (1H, d, J = 16.3 Hz,
Hꢀ7), 7.05 (1H, d, J = 16.3 Hz, Hꢀ8), 7.38 (2H, d, J = 8.6 Hz, Hꢀ10/14), 6.76 (2H, d, J = 8.6 Hz,
Hꢀ11/13), 7.23 (1H, d, J = 8.0 Hz, Hꢀ19), 7.70 (1H, dt, J = 8.0, 1.6 Hz, Hꢀ20), 7.44 (1H, t, J =
8.0 Hz, Hꢀ21), 8.17 (1H, dd, J = 8.0, 1.6 Hz, Hꢀ22), and 2.29 (3H, s, CH3C=O); 13C NMR (150
MHz, CD3OD) δ 141.9 (s, Cꢀ1), 111.5 (d, Cꢀ2), 153.2 (s, Cꢀ3), 108.6 (d, Cꢀ4), 159.8 (s, Cꢀ5),
111.8 (d, Cꢀ6), 125.8 (d, Cꢀ7), 130.8 (d, Cꢀ8), 130.1 (s, Cꢀ9), 129.1 (d, Cꢀ10/14), 116.5 (d, Cꢀ
11/13), 158.7 (s, Cꢀ12), 164.6 (s, Cꢀ16), 124.1 (s, Cꢀ17), 152.4 (s, Cꢀ18), 125.1 (d, Cꢀ19), 135.9
(d, Cꢀ20), 127.4 (d, Cꢀ21), 133.0 (d, Cꢀ22), 171.4 (s, CH3C=O), and 21.3 (q, CH3C=O ); negative
ESIMS, m/z 449 [M + CH3COOH − H]− and 389 [M − H]−.
(E)ꢁ4ꢁ(3,5ꢁDimethoxystyryl)phenyl 2ꢁacetoxybenzoate (6). General procedure A was
followed using pterostilbene 2 (256 mg, 1.0 mmol, 1.0 eq.), Oꢀacetylsalicyloyl chloride (397 mg,
2.0 mmol, 2.0 eq.) and Et3N (0.42 mL, 3.0 mmol, 3.0 eq.) in DCM (10 mL). The residue was
16
ACS Paragon Plus Environment

Page 17 of 47
Journal of Medicinal Chemistry
1
2
1
3
351
4
purified by CC (H/E = 20:1, 10:1, and 6:1) to give 6 (400 mg, yield: 96%) as a white solid. H
NMR (700 MHz, CDCl3) δ 6.66 (2H, d, J = 2.0 Hz, Hꢀ2/6), 6.39 (1H, t, J = 2.0 Hz, Hꢀ4), 6.99
(1H, d, J = 16.1 Hz, Hꢀ7), 7.07 (1H, d, J = 16.1 Hz, Hꢀ8), 7.54 (2H, d, J = 8.4 Hz, Hꢀ10/14),
7.16 (2H, d, J = 8.4 Hz, Hꢀ11/13), 7.17 (1H, m, Hꢀ19), 7.64 (1H, dt, J = 7.7, 1.4 Hz, Hꢀ20), 7.38
(1H, t, J = 7.7 Hz, Hꢀ21), 8.21 (1H, dd, J = 7.7, 1.4 Hz, Hꢀ22), 2.30 (3H, s, CH3C=O), and 3.80
5
6
7
8
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
13
(6H, s, OMeꢀ3/5); C NMR (175 MHz, CDCl3) δ 139.2 (s, Cꢀ1), 104.6 (d, Cꢀ2/6), 161.0 (s, Cꢀ
3/5), 100.1 (d, Cꢀ4), 129.1 (d, Cꢀ7), 128.1 (d, Cꢀ8), 135.3 (s, Cꢀ9), 127.7 (d, Cꢀ10/14), 122.0 (d,
Cꢀ11/13), 150.0 (s, Cꢀ12), 163.0 (s, Cꢀ16), 122.5 (s, Cꢀ17), 151.2 (s, Cꢀ18), 124.1 (d, Cꢀ19),
134.7 (d, Cꢀ20), 126.3 (d, Cꢀ21), 132.3 (d, Cꢀ22), 169.8 (s, CH3C=O), 21.1 (q, CH3C=O), and
55.4 (q, OMeꢀ3/5); negative ESIMS, m/z 477 [M + CH3COOH − H]− and 417 [M − H]−.
(E)ꢁ4ꢁ(3,5ꢁDimethoxystyryl)phenyl 2ꢁhydroxybenzoate (7). To a solution of (E)ꢀ4ꢀ(3,5ꢀ
dimethoxystyryl)phenyl 2ꢀacetoxybenzoate 6 (100 mg, 0.24 mmol, 1.0 eq.) in THF (5 mL) at rt
was added a deluted HCl aq. solution (3.0 M, 5 mL). After addition, the mixture was stirred at rt
o
for 24 h and then heated to 60 C for another 6 h. The reaction was cooled back to rt and
distributed between EA (10 mL) and water (20 mL). The organic solution was separated, washed
with water (10 mL × 2) and brine (10 mL × 1), dried over Na2SO4, and filtered. The filtrate was
evaporated in vacuo, and the residue was purified by CC (H/E = 10:1) to give 7 (74 mg, yield:
82%) as a yellow solid. 1H NMR (600 MHz, CDCl3) δ 6.67 (2H, d, J = 2.2 Hz, Hꢀ2/6), 6.41 (1H,
t, J = 2.2 Hz, Hꢀ4), 7.02 (1H, d, J = 16.3 Hz, Hꢀ7), 7.09 (1H, d, J = 16.3 Hz, Hꢀ8), 7.57 (2H, d, J
= 8.6 Hz, Hꢀ10/14), 7.21 (2H, d, J = 8.6 Hz, Hꢀ11/13), 7.04 (1H, d, J = 8.6 Hz, Hꢀ19), 7.54 (1H,
dt, J = 8.6, 1.6 Hz, Hꢀ20), 6.97 (1H, t, J = 8.0 Hz, Hꢀ21), 8.07 (1H, dd, J = 8.0, 1.6 Hz, Hꢀ22),
3.83 (6H, s, OMeꢀ3/5), and 10.48 (1H, s, OHꢀ18); 13C NMR (150 MHz, CDCl3) δ 139.1 (s, Cꢀ1),
104.7 (d, Cꢀ2/6), 161.0 (s, Cꢀ3/5), 100.2 (d, Cꢀ4), 129.3 (d, Cꢀ7), 128.0 (d, Cꢀ8), 135.5 (s, Cꢀ9),
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 47
1
2
3
4
5
6
7
8
9
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
127.6 (d, Cꢀ10/14), 121.9 (d, Cꢀ11/13), 149.5 (s, Cꢀ12), 168.9 (s, Cꢀ16), 111.8 (s, Cꢀ17), 162.2
(s, Cꢀ18), 117.9 (d, Cꢀ19), 136.5 (d, Cꢀ20), 119.5 (d, Cꢀ21), 130.4 (d, Cꢀ22), and 55.4 (q, OMeꢀ
3/5); negative ESIMS, m/z 435 [M + CH3COOH − H]− and 375 [M − H]−.
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
(E)ꢁ4ꢁ(3ꢁHydroxyꢁ5ꢁmethoxystyryl)phenyl 2ꢁacetoxybenzoate (8). General procedure B was
followed using (E)ꢀ4ꢀ(3,5ꢀdimethoxystyryl)phenyl 2ꢀacetoxybenzoate 6 (209 mg, 0.5 mmol, 1.0
o
o
eq.) and BBr3 (1.0 mmol, 2.0 eq.) in DCM (10 mL) at ꢀ78 C. The mixture was stirred at ꢀ78 C
for 1.0 h. The residue was subjected to CC (H/E = 5:1, 4:1, 3:1, and 2:1) and further purified by
preꢀTLC (H/E = 3:1) to yield 8 (6.0 mg, yield: 3%) as a white solid. 1H NMR (600 MHz, CDCl3)
δ 6.63 (1H, brs, Hꢀ2), 6.33 (1H, t, J = 2.2 Hz, Hꢀ4), 6.58 (1H, brs, Hꢀ6), 6.95 (1H, d, J = 16.3
Hz, Hꢀ7), 7.05 (1H, d, J = 16.3 Hz, Hꢀ8), 7.52 (2H, d, J = 8.5 Hz, Hꢀ10/14), 7.16 (2H, d, J = 8.5
Hz, Hꢀ11/13), 7.17 (1H, d, J = 8.0 Hz, Hꢀ19), 7.64 (1H, dt, J = 8.0, 1.5 Hz, Hꢀ20), 7.39 (1H, t, J
= 8.0 Hz, Hꢀ21), 8.22 (1H, dd, J = 8.0, 1.6 Hz, Hꢀ22), 2.31 (3H, s, CH3C=O), and 3.81 (3H, s,
OMeꢀ3); 13C NMR (150 MHz, CDCl3) δ 139.5 (s, Cꢀ1), 105.0 (d, Cꢀ2), 161.2 (s, Cꢀ3), 101.1 (d,
Cꢀ4), 156.8 (s, Cꢀ5), 105.9 (d, Cꢀ6), 128.7 (d, Cꢀ7), 128.3 (d, Cꢀ8), 136.2 (s, Cꢀ9), 127.7 (d, Cꢀ
10/14), 124.1 (d, Cꢀ11/13), 150.0 (s, Cꢀ12), 163.0 (s, Cꢀ16), 123.1 (s, Cꢀ17), 151.5 (s, Cꢀ18),
122.0 (d, Cꢀ19), 134.7 (d, Cꢀ20), 126.2 (d, Cꢀ21), 132.2 (d, Cꢀ22), 55.4 (q, OMeꢀ3), 169.8 (s,
CH3C=O), and 21.0 (q, CH3C=O); negative ESIMS, m/z 463 [M + CH3COOH − H]− and 403 [M
391
392
393
394
395
396
− H]−.
(E)ꢁ4ꢁ(3ꢁHydroxyꢁ5ꢁmethoxystyryl)phenyl
2ꢁhydroxybenzoate
(9)
and
(E)ꢁ4ꢁ(3,5ꢁ
Dihydroxystyryl)phenyl 2ꢁhydroxybenzoate (10). General procedure B was followed using (E)ꢀ4ꢀ
(3,5ꢀdimethoxystyryl)phenyl 2ꢀacetoxybenzoate 6 (400 mg, 0.96 mmol, 1.0 eq.) and BBr3 (1.91
mmol, 2.0 eq.) in DCM (10 mL) at 0 oC. The mixture was stirred at 0 oC for 30 min. The residue
was purified by CC (H/E = 10:1, 6:1, and 5:1; and then C/M = 50:1) to give 9 (110 mg, yield:
18
ACS Paragon Plus Environment

Page 19 of 47
Journal of Medicinal Chemistry
1
2
1
3
397
4
31%) as a white solid and 10 (65 mg, yield: 20%) as a white solid. 9: H NMR (600 MHz,
CDCl3) δ 6.64 (1H, brs, Hꢀ2), 6.35 (1H, t, J = 2.0 Hz, Hꢀ4), 6.60 (1H, brs, Hꢀ6), 6.96 (1H, d, J =
16.3 Hz, Hꢀ7), 7.06 (1H, d, J = 16.3 Hz, Hꢀ8), 7.55 (2H, d, J = 8.5 Hz, Hꢀ10/14), 7.20 (2H, d, J
= 8.5 Hz, Hꢀ11/13), 7.04 (1H, d, J = 8.0 Hz, Hꢀ19), 7.54 (1H, m, Hꢀ20), 6.97 (1H, m, Hꢀ21),
8.07 (1H, dd, J = 8.0, 1.5 Hz, Hꢀ22), 3.81 (3H, s, OMeꢀ3), and 10.48 (1H, s, OHꢀ18); 13C NMR
(125 MHz, CDCl3) δ 139.4 (s, Cꢀ1), 105.1 (d, Cꢀ2), 161.2 (s, Cꢀ3), 101.2 (d, Cꢀ4), 156.8 (s, Cꢀ5),
106.0 (d, Cꢀ6), 129.0 (d, Cꢀ7), 128.2 (d, Cꢀ8), 135.5 (s, Cꢀ9), 127.7 (d, Cꢀ10/14), 121.9 (d, Cꢀ
11/13), 149.5 (s, Cꢀ12), 168.9 (s, Cꢀ16), 111.8 (s, Cꢀ17), 162.2 (s, Cꢀ18), 117.9 (d, Cꢀ19), 136.6
(d, Cꢀ20), 119.5 (d, Cꢀ21), 130.4 (d, Cꢀ22), and 55.4 (q, OMeꢀ3); negative ESIMS, m/z 421 [M +
CH3COOH − H]− and 361 [M − H]−. 10: 1H NMR (600 MHz, CD3OD) δ 6.51 (2H, d, J = 2.0 Hz,
Hꢀ2/6), 6.21 (1H, t, J = 2.0 Hz, Hꢀ4), 6.99 (1H, d, J = 16.3 Hz, Hꢀ7), 7.07 (1H, d, J = 16.3 Hz,
Hꢀ8), 7.60 (2H, d, J = 8.5 Hz, Hꢀ10/14), 7.22 (2H, d, J = 8.5 Hz, Hꢀ11/13), 7.02 (1H, m, Hꢀ19),
7.56 (1H, dt, J = 7.8, 1.6 Hz, Hꢀ20), 7.00 (1H, m, Hꢀ21), and 8.07 (1H, dd, J = 7.9, 1.4 Hz, Hꢀ
22); 13C NMR (125 MHz, CD3OD) δ 140.6 (s, Cꢀ1), 106.2 (d, Cꢀ2/6), 159.8 (s, Cꢀ3/5), 103.4 (d,
Cꢀ4), 130.6 (d, Cꢀ7), 128.3 (d, Cꢀ8), 137.2 (s, Cꢀ9), 128.5 (d, Cꢀ10/14), 123.1 (d, Cꢀ11/13), 151.0
(s, Cꢀ12), 170.0 (s, Cꢀ16), 113.2 (s, Cꢀ17), 163.3 (s, Cꢀ18), 118.7 (d, Cꢀ19), 137.6 (d, Cꢀ20),
120.7 (d, Cꢀ21), and 131.5 (d, Cꢀ22); negative ESIMS, m/z 407 [M + CH3COOH − H]− and 347
5
6
7
8
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
414
415
416
417
418
419
[M − H]−.
(E)ꢁ1ꢁ(3,5ꢁDimethoxystyryl)ꢁ4ꢁbromobenzene (14). General procedure C was followed using
(4ꢀbromobenzyl)triphenylphosphonium bromide (2.2 g, 4.3 mmol, 1.0 eq.) [Prepared from 4ꢀ
bromobenzyl bromide 13 (1.24 g, 5.0 mmol, 1.0 eq.) and PPh3 (1.31 g, 5.5 mmol, 1.1 eq.) in
toluene (10 mL)], 3,5ꢀdimethoxybenzaldehyde (863 mg, 5.2 mmol, 1.2 eq.) and NaOBuꢀt (499
mg, 5.2 mmol, 1.2 eq.) in THF (20 mL). The mixture was stirred at rt for 6 h. The residue was
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 47
1
2
1
3
4
5
6
7
8
9
420
421
422
423
purified by CC (H/E = 60:1 and 40:1) to give 14 (0.9 g, yield: 66%) as a white solid. H NMR
(700 MHz, CDCl3) δ 6.65 (2H, d, J = 2.2 Hz, Hꢀ2/6), 6.40 (1H, t, J = 2.2 Hz, Hꢀ4), 7.02 (1H, d,
J = 16.2 Hz, Hꢀ7), 6.99 (1H, d, J = 16.2 Hz, Hꢀ8), 7.47 (2H, d, J = 8.5 Hz, Hꢀ10/14), 7.36 (2H,
d, J = 8.5 Hz, Hꢀ11/13), and 3.82 (6H, s, OMeꢀ1/3); negative ESIMS, m/z 317 [M − H]−, and 319
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
[M + 2 − H]−.
(E)ꢁ2ꢁ((4ꢁ(3,5ꢁDimethoxystyryl)phenyl)(hydroxy)methyl)phenol (15). To a solution of (E)ꢀ1ꢀ
(4ꢀbromostyryl)ꢀ3,5ꢀdimethoxybenzene 14 (700 mg, 2.20 mmol, 1.5 eq.) in dry THF (5 mL) at ꢀ
o
78 C was added nꢀBuLi (1.06 mL, 2.5 M in hexane, 2.64 mmol, 1.8 eq.). One hour after the
addition, salicylaldehyde (179 mg, 1.47 mmol, 1.0 eq.) in THF (2 mL) was added. The reaction
o
mixture was stirred at ꢀ78 C for another 2 h and water (10 mL) was added to quench the
reaction. The resulting mixture was extracted with EA (10 mL × 3). The extracts were combined
and dried over Na2SO4. Evaporation of solvent followed by CC (H/E = 20:1, 10:1, 5:1 and 3:1)
1
to afford 15 (449 mg, yield: 66%) as yellow oil. H NMR (700 MHz, CDCl3) δ 6.34 (2H, d, J =
2.2 Hz, Hꢀ2/6), 6.29 (1H, t, J = 2.2 Hz, Hꢀ4), 6.68ꢀ6.62 (2H, m, Hꢀ7/8), 7.48 (2H, d, J = 8.3 Hz,
Hꢀ10/14), 7.36 (2H, d, J = 8.3 Hz, Hꢀ11/13), 5.95 (1H, s, Hꢀ15), 6.79ꢀ6.76 (1H, m, Hꢀ18), 7.20ꢀ
7.15 (1H, m, Hꢀ19), 6.88ꢀ6.83 (1H, m, Hꢀ20), 7.06ꢀ7.02 (1H, m, Hꢀ21), and 3.81 (6H, s, OMeꢀ
1/3); negative ESIMS, m/z 361 [M − H]−.
(E)ꢁ(4ꢁ(3,5ꢁDimethoxystyryl)phenyl)(2ꢁhydroxyphenyl)methanone (16). A mixture of (E)ꢀ2ꢀ
((4ꢀ(3,5ꢀdimethoxystyryl)phenyl)(hydroxy)methyl)phenol 15 (228 mg, 0.63 mmol, 1.0 eq.),
CuCl2·2H2O (11 mg, 0.063 mmol, 0.1 eq) and K2CO3 (261 mg, 1.89 mmol, 3.0 eq.) in DMF (8
mL) was stirred at 60 oC for 18 h under air atmosphere. The mixture was neutralized to pH ~7.0
by adding aq. 10% HCl solution and extracted with EA (10 mL × 3). The organic extracts were
washed with water (10 mL × 2) and brine (10 mL × 1), dried over Na2SO4, and filtered. The
20
ACS Paragon Plus Environment

Page 21 of 47
Journal of Medicinal Chemistry
1
2
3
443
4
filtrate was removed under reduced pressure, and the residue was purified by CC (H/E = 20:1
5
6
7
8
1
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
and 10:1) to give 16 (138 mg, yield: 61%) as a yellow solid. H NMR (600 MHz, CDCl3) δ 6.71
(2H, d, J = 2.2 Hz, Hꢀ2/6), 6.45 (1H, t, J = 2.2 Hz, Hꢀ4), 7.18 (1H, d, J = 16.3 Hz, Hꢀ7), 7.14
(1H, d, J = 16.3 Hz, Hꢀ8), 7.69 (2H, d, J = 8.2 Hz, Hꢀ10/14), 7.63 (2H, d, J = 8.2 Hz, Hꢀ11/13),
7.07 (1H, d, J = 8.3 Hz, Hꢀ18), 7.53ꢀ7.48 (1H, m, Hꢀ19), 6.86 (1H, t, J = 8.0 Hz, Hꢀ20), 7.64
(1H, d, J = 8.0 Hz, Hꢀ21), and 3.85 (6H, s, OMeꢀ1/3); negative ESIMS, m/z 359 [M − H]−.
(E)ꢁ(4ꢁ(3,5ꢁDihydroxystyryl)phenyl)(2ꢁhydroxyphenyl)methanone (11). General procedure B
was followed using (E)ꢀ(4ꢀ(3,5ꢀdimethoxystyryl)phenyl)(2ꢀhydroxyphenyl)methanone 16 (138
mg, 0.38 mmol, 1.0 eq.) and BBr3 (1.34 mmol, 2.0 eq.) in DCM (10 mL). The mixture was
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
o
stirred at ꢀ78 C for 1 h and then allowed to warm up to rt for another 2 h. The residue was
subjected to preꢀTLC (C/M = 10:1) and further purified by LHꢀ20 (EtOH) to give 11 (79 mg,
1
yield: 62%) as a yellow solid. H NMR (600 MHz, CD3OD) δ 6.53 (2H, d, J = 2.0 Hz, Hꢀ2/6),
6.23 (1H, t, J = 2.0 Hz, Hꢀ4), 7.18 (1H, d, J = 16.3 Hz, Hꢀ7), 7.13 (1H, d, J = 16.3 Hz, Hꢀ8),
7.66 (2H, d, J = 8.3 Hz, Hꢀ10/14), 7.70 (2H, d, J = 8.3 Hz, Hꢀ11/13), 7.01 (1H, d, J = 8.3 Hz, Hꢀ
18), 7.51 (1H, dt, J = 8.3, 1.5 Hz, Hꢀ19), 6.93 (1H, t, J = 8.0 Hz, Hꢀ20), and 7.58 (1H, dd, J =
8.0, 1.4 Hz, Hꢀ21); 13C NMR (150 MHz, CD3OD) δ 140.2 (s, Cꢀ1), 106.4 (d, Cꢀ2/6), 159.9 (s, Cꢀ
3/5), 103.9 (d, Cꢀ4), 133.0 (d, Cꢀ7), 128.2 (d, Cꢀ8), 143.1 (s, Cꢀ9), 127.3 (d, Cꢀ10/14), 131.1 (d,
Cꢀ11/13), 137.8 (s, Cꢀ12), 201.7 (t, Cꢀ15), 121.7 (s, Cꢀ16), 163.0 (s, Cꢀ17), 118.9 (d, Cꢀ18),
136.7 (d, Cꢀ19), 120.0 (d, Cꢀ20), and 133.9 (d, Cꢀ21); negative ESIMS, m/z 331 [M − H]−.
1,3ꢁDimethoxyꢁ5ꢁvinylbenzene (18). General procedure
C
was followed using
methyltriphenylphosphonium iodide (4.3 g, 10.6 mmol, 1.0 eq.) [Prepared from
triphenylphosphine (2.62 g, 10.0 mmol) and iodomethane (1.42 g, 10.0 mmol) in toluene (20
mL)], 3,5ꢀdimethoxybenzaldehyde 17 (2.1 g, 12.8 mmol, 1.2 eq.) and NaOBuꢀt (1.2 g, 12.8
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 47
1
2
3
4
5
6
7
8
9
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
mmol, 1.2 eq.) in THF (20 mL). The mixture was stirred at rt for 18 h. The residue was purified
1
by CC (H/E = 60:1, 40:1, and 20:1) to give 18 (1.58 g, yield: 75%) as a colorless oil. H NMR
(600 MHz, CDCl3) δ 6.57 (2H, d, J = 2.2 Hz, Hꢀ2/6), 6.39 (1H, t, J = 2.2 Hz, Hꢀ4), 6.65 (1H, dd,
J = 17.5, 10.8 Hz, Hꢀ7), 5.74 (1H, d, J = 17.5 Hz, Hꢀ8a), 5.25 (1H, d, J = 10.8 Hz, Hꢀ8b), and
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
13
3.81 (6H, s); C NMR (150 MHz, CDCl3) δ 160.9 (s, Cꢀ1/3), 100.1 (d, Cꢀ2), 104.3 (d, Cꢀ4/6),
139.6 (s, Cꢀ5), 136.8 (d, Cꢀ7), 114.3 (t, Cꢀ8), and 55.3 (q, OMeꢀ1/3); negative ESIMS, m/z 163
[M − H]−.
2ꢁ(2ꢁ(4ꢁBromophenyl)acetyl)phenyl acetate (19). To a suspension of Pd(PPh3)4 (578 mg, 0.5
mmol, 0.05 eq.) and zinc powder (1.3 g, 20 mmol, 2.0 eq.) in dry THF (50 mL) was added 4ꢀ
bromobenzyl bromide 13 (2.48 g, 10 mmol, 1.0 eq.) and Oꢀacetylsalicyloyl chloride (2.08 g, 10.5
mmol, 1.05 eq.) at rt under N2. The mixture was stirred at rt for 4 h and filtered. The filtration
was concentrated under reduced pressure, and the residue was subjected to CC (H/E = 20:1, 10:1,
1
and 5:1) to give 19 (1.0 g, yield: 30%) as a white solid. H NMR (600 MHz, CDCl3) δ 7.45 (2H,
d, J = 8.4 Hz, Hꢀ3/5), 7.09 (2H, d, J = 8.4 Hz, Hꢀ2/6), 4.16 (2H, s, Hꢀ7), 7.13 (1H, dd, J = 8.0,
1.5 Hz, Hꢀ11), 7.54 (1H, dt, J = 8.0, 1.5 Hz, Hꢀ12), 7.32 (1H, dt, J = 8.0, 1.5 Hz, Hꢀ13), 7.79
(1H, dd, J = 8.0, 1.5 Hz, Hꢀ14), and 2.35 (3H, s, CH3CO); 13C NMR (150 MHz, CDCl3) δ 133.0
(s, Cꢀ1), 131.4 (d, Cꢀ2/6), 131.7 (d, Cꢀ3/5), 121.1 (s, Cꢀ4), 47.2 (t, Cꢀ7), 196.9 (s, Cꢀ8), 130.5 (s,
Cꢀ9), 149.0 (s, Cꢀ10), 124.0 (d, Cꢀ11), 133.5 (d, Cꢀ12), 126.1 (d, Cꢀ13), 129.8 (d, Cꢀ14), 169.5
(s, CH3CO), and 21.1 (q, CH3CO); negative ESIMS, m/z 331 [M − H]− and 333 [M + 2 − H]−.
2ꢁ(4ꢁBromophenyl)ꢁ1ꢁ(2ꢁhydroxyphenyl)ethanone (20). General procedure B was followed
using 2ꢀ(2ꢀ(4ꢀbromophenyl)acetyl)phenyl acetate 19 (200 mg, 0.6 mmol, 1.0 eq.) and BBr3 (0.89
o
mmol, 2.0 eq.) in DCM (5 mL). The mixture was stirred at ꢀ78 C for 15 min. The residue was
1
purified by preꢀTLC (H/E = 10:1) to give 20 (168 mg, yield: 96%) as a white solid. H NMR
22
ACS Paragon Plus Environment

Page 23 of 47
Journal of Medicinal Chemistry
1
2
3
489
4
(600 MHz, CDCl3) δ 7.47 (2H, d, J = 8.3 Hz, Hꢀ3/5), 7.14 (2H, d, J = 8.3 Hz, Hꢀ2/6), 4.26 (2H,
s, Hꢀ7), 6.99 (1H, d, J = 8.0 Hz, Hꢀ11), 7.48 (1H, t, J = 8.0 Hz, Hꢀ12), 6.91 (1H, t, J = 8.0 Hz,
Hꢀ13), 7.82 (1H, d, J = 8.0 Hz, Hꢀ14), and 12.10 (1H, s, OHꢀ10); 13C NMR (150 MHz, CDCl3) δ
132.8 (s, Cꢀ1), 131.2 (d, Cꢀ2/6), 131.9 (d, Cꢀ3/5), 118.9 (s, Cꢀ4), 44.4 (t, Cꢀ7), 203.2 (s, Cꢀ8),
121.3 (s, Cꢀ9), 162.9 (s, Cꢀ10), 118.8 (d, Cꢀ11), 136.8 (d, Cꢀ12), 119.1 (d, Cꢀ13), and 130.2 (d,
Cꢀ14); negative ESIMS, m/z 289 [M − H]− and 291 [M + 2 − H]−.
5
6
7
8
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
2ꢁ(4ꢁ(3,5ꢁDimethoxystyryl)phenyl)ꢁ1ꢁ(2ꢁhydroxyphenyl)ethanone (21). General procedure D
was followed using 2ꢀ(4ꢀbromophenyl)ꢀ1ꢀ(2ꢀhydroxyphenyl)ethanone 20 (168 mg, 0.58 mmol,
1.0 eq.), 1,3ꢀdimethoxyꢀ5ꢀvinylbenzene 18 (114 mg, 0.7 mmol, 1.2 eq.), TEA (117 mg, 1.16
mmol, 2.0 eq.), and Pd(PPh3)4 (34 mg, 0.029 mmol, 0.05 eq.) in dry DMF (3 mL). The mixture
o
was heated to 100 C and stirred for 18 h. The residue was purified by preꢀTLC (H/E = 10:1) to
1
give 21 (32 mg, yield: 15%) as a white solid. H NMR (600 MHz, CDCl3) δ 6.65 (1H, d, J = 2.1
Hz, Hꢀ2/6), 6.39 (1H, t, J = 2.1 Hz, Hꢀ4), 7.00 (1H, d, J = 15.8 Hz, Hꢀ7), 7.05 (1H, d, J = 15.8
Hz, Hꢀ8), 7.48 (2H, d, J = 8.0 Hz, Hꢀ10/14), 7.25 (2H, d, J = 8.0 Hz, Hꢀ11/13), 4.30 (2H, s, Hꢀ
15), 6.98 (1H, d, J = 8.0 Hz, Hꢀ19), 7.52 (1H, m, Hꢀ20), 6.90 (1H, t, J = 8.0 Hz, Hꢀ21), 7.86
(1H, d, J = 8.0 Hz, Hꢀ22), 3.82 (6H, s, OMeꢀ1/3), and 12.19 (1H, s, OHꢀ18); 13C NMR (150
MHz, CDCl3) δ 203.7, 162.9, 161.0, 139.3, 136.6, 136.2, 133.4, 130.4, 129.8, 129.7, 128.9,
128.6, 127.3, 127.2, 127.0, 119.0, 118.7, 55.4, and 44.9; negative ESIMS, m/z 373 [M − H]−.
2ꢁ(4ꢁ(3,5ꢁDihydroxystyryl)phenyl)ꢁ1ꢁ(2ꢁhydroxyphenyl)ethanone (12). General procedure B
was followed using (E)ꢀ2ꢀ(4ꢀ(3,5ꢀdimethoxystyryl)phenyl)ꢀ1ꢀ(2ꢀhydroxyphenyl)ethanone 21 (32
mg, 0.086 mmol, 1.0 eq.) and BBr3 (0.34 mmol, 2.0 eq.) in DCM (5 mL). The mixture was
o
stirred at 0 C for 1 h and then allowed to warm up to rt for another 2 h. The residue was
subjected to preꢀTLC (C/M = 10:1) and further purified by LHꢀ20 (EtOH) to give 12 (20 mg,
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 47
1
2
1
3
4
5
6
7
8
9
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
yield: 67%) as a white solid. H NMR (600 MHz, CD3OD) δ 6.47 (2H, d, J = 2.0 Hz, Hꢀ2/6),
6.19 (1H, t, J = 2.0 Hz, Hꢀ4), 6.98 (1H, d, J = 16.3 Hz, Hꢀ7), 7.02 (1H, d, J = 16.3 Hz, Hꢀ8),
7.47 (2H, d, J = 8.1 Hz, Hꢀ10/14), 7.27 (2H, d, J = 8.1 Hz, Hꢀ11/13), 4.36 (2H, s, Hꢀ15), 6.93
(1H, t, J = 8.3 Hz, Hꢀ19), 7.49 (1H, m, Hꢀ20), 6.95 (1H, d, J = 8.3 Hz, Hꢀ21), and 8.02 (1H, dd,
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
13
J = 8.3, 1.3 Hz, Hꢀ22); C NMR (150 MHz, CD3OD) δ 140.8 (s, Cꢀ1), 106.1 (d, Cꢀ2/6), 159.8
(s, Cꢀ3/5), 103.2 (d, Cꢀ4), 130.0 (d, Cꢀ7), 129.0 (d, Cꢀ8), 137.6 (s, Cꢀ9), 127.7 (d, Cꢀ10/14),
130.9 (d, Cꢀ11/13), 135.3 (s, Cꢀ12), 45.9 (t, Cꢀ15), 205.9 (s, Cꢀ16), 120.5 (s, Cꢀ17), 163.8 (s, Cꢀ
18), 119.1 (d, Cꢀ19), 137.5 (d, Cꢀ20), 120.2 (d, Cꢀ21), and 132.2 (d, Cꢀ22); negative ESIMS, m/z
345 [M − H]−.
Biological Assays. Materials and Methods. Human colon cancer cells HCTꢀ116 and HTꢀ29
were obtained from American Type Tissue Culture (Manassas, VA). McCoy’s 5A medium was
purchased from Thermo Fisher Scientific (Waltham, MA). Supplements of Fetal Bovine Serum
(FBS) and penicillin/streptomycin were purchased from Gemini BioꢀProducts (West Sacramento,
CA). Crystal violet, glutaraldehyde and propidium iodide were obtained from Thermo Fisher
Scientific (Waltham, MA). Primary antibodies against Bim, Bax, Bid, Bad, Bak, BclXL, and
Bclꢀ2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cytochrome c, Cleaved
Caspase 3, βꢀactin, as well as secondary antibodies conjugated to HRP (Horseradish Peroxydase)
against mouse and rabbit were purchased from Cell Signaling Technology (Beverly, MA).
Metabolism of RAH in Human Colon Cancer Cells. Cancer cells HCTꢀ116 (1.0 × 106)
o
were plated in 6ꢀwell culture plates and allowed to attach for 24 h at 37 C in a 5% CO2
incubator. RAH in DMSO was added to McCoy’s 5A medium (containing 10% fetal bovine
serum, 1% penicillin/streptomycin, and 1% glutamine) to reach a final concentration of 20 ꢀM
and then incubated. At different time pints (0, 0.5, 1, 2, 4, 8, and 24 h), 100 ꢀL samples of
534
24
ACS Paragon Plus Environment

Page 25 of 47
Journal of Medicinal Chemistry
1
2
3
535
4
supernatant were taken and transferred to vials. An equal volume of acetonitrile was added to the
samples before centrifugation. The supernatant was harvested, and the samples were analyzed by
HPLCꢀCEAD.
5
6
7
8
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
MTT Assay. Human colon cancer cells HCTꢀ116 and HTꢀ29 were plated in 96ꢀwell
microtiter plates with 3000 cells/well and allowed to attach for 24 h at 37 ºC. The test
compounds (in DMSO) were added to cell culture medium to the desired final concentrations
(final DMSO concentrations for control and treatments were 0.1 %). After the cells were cultured
for 24 h, the medium was aspirated and cells were treated with 200 ꢀL fresh medium containing
2.41 mmol/L 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazoliumomide (MTT). After
incubation for 3 h at 37 ºC, the medium containing MTT was aspirated, 100 ꢀL of DMSO was
added to solubilize the formazan precipitate, and plates were shaken gently for 1 h at room
temperature. Absorbance values were derived from the plate reading at 550 nm on a Biotek
microtiter plate reader (Winooski, VT). The reading reflected the number of viable cells and was
expressed as a percentage of viable cells in the control. Both HCTꢀ116 and HTꢀ29 cells were
cultured in McCoy’s 5A medium. The media used above was supplemented with 10 % fetal
bovine serum, 1 % penicillin/streptomycin, and 1 % glutamine, and the cells were kept in a 37 ºC
incubator with 95% humidity and 5% CO2. The IC50 values were conducted by using GraphPad
Prism software (version 5).
Colony Formation Assay. Human colon cancer cells HCTꢀ116 or HTꢀ29 (1,000 cells per
well) were seeded in 6ꢀwell culture plates for 24 h and then incubated with the compounds in
DMSO in a 37 °C incubator with 5% CO2. After 2 weeks, colonies were washed with phosphateꢀ
buffered saline (PBS), then stained with a mixture of 6.0% glutaradehyde and 0.5% crystal violet
for 30 min at room temperature, rinsed in water, airꢀdried, and then photographed.
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 47
1
2
3
4
5
6
7
8
9
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
Apoptosis and Cell Cycle Analysis. Apoptosis and cell cycle were determined by FACS
analysis of propidium iodide (PI)ꢀstained cells. In brief, cells were trypsinized, washed with cold
phosphateꢀbuffered saline (PBS), fixed in iceꢀcold 70% ethanol for 1 h, and then resuspended in
2 mL PBS supplemented with 10 µL RNase (100 mg/ml) and incubated at 37°C for 30 min.
After incubation, DNA was stained with 1 mg/mL PI in PBS. Cell staining was analyzed using a
Cell Lab QuantaTM SC flow cytometer (Beckman Coulter, Danvers, MA) and data were
processed using FlowJo vesion 7.6.1 software (Tree Star, San Carlos, CA). The percentage of
cell cycle distribution in each sample was determined based on the G1, S and G2 peaks detected
in Watson model. The percentage of apoptotic cells in each sample was determined based on the
sub G1 peaks detected in monoparametric histograms.
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
Western Blot Analysis. Cell lysates were prepared in iceꢀcold RIPA lysis buffer [25 mM
TrisꢀHCl (pH 7.6), 150 mM NaCl, 1% NPꢀ40, 1% sodium deoxycholate, 0.1% SDS, Thermo
Fisher Scientific] supplemented with a protease inhibitor cocktail (AEBSF, aprotinin, bestatin, Eꢀ
64, leupeptin and pepstatin A in DMSO with EDTA, Thermo Fisher Scientific). Protein content
was measured by a Pierce BCA Assay Kit (Thermo Fisher Scientific). Protein contents of cell
lysates (30 µg protein/lane) were resolved by SDSꢀPAGE. Proteins were then electroꢀtransferred
onto PVDF membranes and blots were blocked for one hour at room temperature in 1 × TBS
with 1% Casein (BioꢀRad Laboratories, Berkeley, CA). Blots were then incubated overnight at 4
°C with the desired primary antibody diluted in TBS with 0.5% Tweenꢀ20. Blots were then
washed with TBSꢀTween 20 and probed for 1 h with the appropriate secondary antibody
(1:1000). Protein bands were visualized with chemiluminescence using West Femto maximum
detection substrate (Thermo Fisher Scientific). To confirm equal protein loading in each lane,
26
ACS Paragon Plus Environment

Page 27 of 47
Journal of Medicinal Chemistry
1
2
3
580
4
immunoblots were stripped and reꢀprobed for βꢀactin. Protein foldꢀinduction was calculated by
normalizing the intensity of the band of interest to βꢀactin first, and then to control lanes.
Animals Study. Animals and Study Design. Experiments with mice were carried out
according to protocols approved by the Institutional Review Board for the Animal Care and
Facilities Committee at North Carolina Central University. Female C57BL/6J mice were
purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and allowed to acclimate for at
least 1 week prior to the start of the experiment. The mice were housed 5 per cage and
5
6
7
8
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
9
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
maintained in airꢀconditioned quarters with a room temperature of 20
± 2 ºC, relative humidity of
50 10 %, and an alternating 12ꢀh light/dark cycle. Mice were fed Rodent Chow #5001
±
(LabDiet) and water, and were allowed to eat and drink ad libitum. RAH in DMSO was
administered to mice by oral gavage (200 mg/kg). Stomach contents, intestine contents, and
colon tissue were collected at 1 h and 2 h, respectively, after administration of vehicle (control
group, n = 5), and RAH (treated group, n = 5). Urine and fecal samples were collected in
metabolism cages (5 mice per cage) in 24 h after administration. Blood samples were collected
by cardiac puncture and plasma was obtained after centrifugation at 17,000 × g for 5 min at 4 oC.
The samples were stored at ꢀ80 ºC until analysis.
Sample Preparation. For the metabolic profile, mouse urine samples (50
ꢀL for each group)
597
598
599
600
601
602
were added to 950 L methanol to precipitate proteins. After centrifugation at 17,000 × g for 5
ꢀ
min, the supernatant was transferred into vials for LC/MS analysis. Enzymatic deconjugation of
mouse urine was performed as described previously with slight modification.43 In brief, triplicate
samples were prepared in the presence of βꢀglucuronidase (250 U) and sulfatase (3 U) for 24 h at
o
37 C. After incubation, the medium was diluted 5 times by adding methanol containing 0.2%
AA. The resulting solution was centrifuged at 17,000 g for 5 min, and 10
ꢀ
L of supernatant was
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 47
1
2
3
4
5
6
7
8
9
603
604
605
606
607
608
609
610
611
612
613
614
analyzed directly by LC/MS. Fecal samples (4 pieces) were selected and placed in 1.0 mL of
MeOH (0.2% AA). Samples were homogenized for 5 min by an Omni Bead Ruptor
Homogenizer (Kennesaw, GA) and then centrifuged at 17,000 g for 10 min. The supernatant
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
(250 ꢀL) was collected and diluted 5 times for analysis. Stomach contents or small intestine
contents (5 mg), along with 500 µL methanol was sonicated until homogenous. Next the samples
were centrifuged at 17,000 g for 10 min at 4 oC. Supernatant was filtered thru 0.22 µM filter and
stored in vial for analysis. Colon tissues in 750 µL 60% methanol (1% AA) were placed in ꢀ20
o
oC for 10 min. Next, samples were homogenized for 5 min and then centrifuged at 4 C for 10
min. 600 µL of supernatant was removed and dried out by vacufuge. Samples were redissolved
in 200 µL 80% methanol (0.2% AA) and transferred to vials for analysis. Plasma samples (50
µL) were added to 950 ꢀL methanol to precipitate proteins. After centrifugation at 17,000 × g for
5 min, the supernatant was transferred into vials for LC/MS analysis.
615
616
617
ASSOCIATED CONTENT
Supporting Information
NMR spectra (1H NMR, 13C NMR and 2ꢀD NMR).
618
619
620
621
622
623
AUTHOR INFORMATION
Corresponding Authors
*Eꢀmail: ssang@ncat.edu or shengminsang@yahoo.com. Phone: 704ꢀ250ꢀ5710. Fax: 704ꢀ250ꢀ
5709.
Notes
The authors declare no competing financial interest.
624
ACKNOWLEDGMENTS
28
ACS Paragon Plus Environment

Page 29 of 47
Journal of Medicinal Chemistry
1
2
3
625
4
This investigation was supported by grant CA159353 (S. Sang) from the National Cancer
Institute and grant 2011ꢀBRGꢀ1203 (S. Sang) from North Carolina Biotechnology Center.
5
6
626
7
8
9
627
628
629
630
631
632
633
ABBREVIATIONS USED
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
AA, acetic acid; ASA, aspirin; ASA+RES, an equimolar mixture of aspirin and resveratrol; CC,
column chromatography; CEAD, coulometric electrode array detector; COX, cyclooxygenase;
CRC, colorectal cancer; DHꢀRES, dehydroresveratrol; EA, ethyl acetate; GIT, gastrointestinal
tract; MTT, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazoliumomide; NO, nitric oxide;
NSAIDs, nonsteroidal antiꢀinflammatory drugs; PGs, prostaglandins; RAH, resveratrol−aspirin
hybrid; RES, resveratrol; TPP, triphenylphosphine.
29
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 30 of 47
1
2
3
4
5
6
7
8
9
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
REFERENCES
(1) Weitz, J.; Koch, M.; Debus, J.; Hohler, T.; Galle, P. R.; Buchler, M. W. Colorectal cancer.
Lancet 2005, 365, 153ꢀ165.
10
11
12
13
14
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
49
50
51
52
53
54
55
56
57
58
59
60
(2) Scarpignato, C.; Hunt, R. H. Nonsteroidal antiinflammatory drugꢀrelated injury to the
gastrointestinal tract: clinical picture, pathogenesis, and prevention. Gastroenterol. Clin. North
Am. 2010, 39, 433ꢀ464.
(3) Elwood, P. C.; Gallagher, A. M.; Duthie, G. G.; Mur, L. A.; Morgan, G. Aspirin,
salicylates, and cancer. Lancet 2009, 373, 1301ꢀ1309.
(4) Thorat, M. A.; Cuzick, J. Role of aspirin in cancer prevention. Curr. Oncol. Rep. 2013, 15,
533ꢀ540.
(5) Thun, M. J.; Jacobs, E. J.; Patrono, C. The role of aspirin in cancer prevention. Nat. Rev.
Clin. Oncol. 2012, 9, 259ꢀ267.
(6) Ricciotti, E.; FitzGerald, G. A. Prostaglandins and inflammation. Arterioscler. Thromb.
Vasc. Biol. 2011, 31, 986ꢀ1000.
(7) Dubois, R. N.; Abramson, S. B.; Crofford, L.; Gupta, R. A.; Simon, L. S.; Van De Putte, L.
B.; Lipsky, P. E. Cyclooxygenase in biology and disease. FASEB J. 1998, 12, 1063ꢀ1073.
(8) Wolfe, M. M.; Lichtenstein, D. R.; Singh, G. Gastrointestinal toxicity of nonsteroidal
antiinflammatory drugs. N. Engl. J. Med. 1999, 340, 1888ꢀ1899.
(9) Brown, J. R.; DuBois, R. N. COXꢀ2: a molecular target for colorectal cancer prevention. J.
Clin. Oncol. 2005, 23, 2840ꢀ2855.
(10) Everts, B.; Wahrborg, P.; Hedner, T. COXꢀ2ꢀSpecific inhibitorsꢀꢀthe emergence of a new
class of analgesic and antiꢀinflammatory drugs. Clin. Rheumatol. 2000, 19, 331ꢀ343.
30
ACS Paragon Plus Environment