Construction and Biological Evaluation of Small Libraries Based on the Intermediates within the Total Synthesis of Uvaretin

Full text of DOI:10.1002/cmdc.202001010

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Construction and Biological Evaluation of Small Libraries
Based on the Intermediates within the Total Synthesis of
Uvaretin
Shashika Perera,[a] Asantha Fernando,[a] Johnathan Dallman,[a] Chamitha Weeramange,[a]
Ashabha Lansakara,[a] Thi Nguyen,[a] and Ryan J. Rafferty*[a]
Introduction
Natural products (NPs) have played a vital role in the treatment
of diseases, and are the cornerstone for modern drug discovery,
since 2600 B.C. in which oils from Cupressus semperviren and
Commiphora were used in the treatment of coughs, colds, and
inflammation.[1] From individual therapeutic agent discovery to
inspiration for the construction of non-natural bioactive agents,
their impact is immeasurable.[2] While NPs constituted the major
pipeline for therapeutic agent discovery for decades, the
introduction of high-throughput screening (HTS) in the late
1980s allowed for the discovery of drug lead scaffolds via
chemical screening libraries (CSLs); initially solely populated by
NPs, NP fragments, and NPs analogs.[3] To answer the call for
the population of CSL in a rapid fashion the industry turned
towards the use of sp2–sp2 metal catalyzed coupling reactions
to gain access to tens to hundreds of thousands of small
molecules for HTS, as means of identifying new drug lead
scaffolds.[4] While numerous FDA approved drugs have been
discovered in this fashion, these compounds tend to lack in the
chemical diversity, complexity, and inherent biological activity
of natural products.[4] To address the diversity of CSLs, multiple
research programs have undertaken various approaches to
construct new CSLs that either possess structural complexity,
rapid small-molecule construction, biological activity-based
design, and others.[5] All of these approaches, as expected, have
their pros and cons associated with them. Noting the historical
importance of NPs, the development of CSLs based upon NPs
should deliver CSLs with not only biological activity, but
chemical diversity in terms of structural complexity, biological
modulating properties, and rich physicochemical properties.
Highlighting the importance of NPs, both in bioactivity and
in chemical complexity, and acknowledging that each NP has
been designed by nature to elicit a specific and discrete
biological affect, our laboratory proposes that CSLs constructed
from the intermediates of total synthesis campaigns will give
access to new bioactive compounds with tunable chemical
complexity. Previously, we have disclosed a new total synthesis
route to the chalcone based uvaretin family of natural
products.[6] To explore our concept of CSL construction from
intermediates of a total synthesis campaign, this route served
as the “proof-of-concept” to test feasibility. A chalcone based
natural product was chosen for this given their well reported
diverse biological activity. From this total synthesis route, and
the intermediates within (1 and 2, shown for example),
numerous small molecules (3–5 and 6–7, shown for example)
were constructed and found to possess diverse and interesting
biological activity (Figure 1). Furthermore, many of the various
phenolic protected variants (MOM or methyl protected) were
elaborated onto the carbon framework of uvaretin as analogs,
in which the trimethylated 8 was shown to have moderate
activity while its allyl protected o-hydroxybenzyl (6) precursor
possessing single-digit micromolar cytotoxicity.
Over 45 compounds were synthesized through these efforts,
which were then investigated for sole agent cytotoxicity and as
possible small molecule potentiators. Intermediate 1 was used
as a starting point for the construction of a 12-member library
differing about the alkyl substitution of the phenol positions in
ring A. Sole cytotoxicity of these compounds was variable,
ranging from 2.2�0.6 to >20 μM IC50 values. Interestingly, it
was found that many of these varied alkyl substituted phenolic
chalcones possessed mild to good potentiating properties with
the clinical prescribed acute lymphocytic leukemia drug 6-
thiopurine (6TP, Figure 1). 6TP is not commonly used in the
treatment of other cancers, due to limited cytotoxicity. How-
ever, it was shown to be active in the pancreatic cancer cell line
MIA PaCa-2 when used in combination with 4. The specific
alkylation patterns upon the phenolic positions in ring A did
reveal interesting potentiation properties. However the alkyl
substitutions explored were limited to methyl and meth-
oxymethyl ethers. Elaboration of 1 onto its benzylated counter-
part, ring C inclusion, revealed compounds with single digit μM
cytotoxicity, but no potentiation properties were observed.
Building above these findings, additional investigations into the
role of each of the rings and the alkyl effects upon the phenolic
central B-ring was explored and disclosed herein.
[a] S. Perera, A. Fernando, J. Dallman, Dr. C. Weeramange, Dr. A. Lansakara,
Dr. T. Nguyen, Prof. R. J. Rafferty
Department of Chemistry
Kansas State University
1212 Mid-Campus Drive North
Manhattan, KS 66506 (USA)
E-mail: rjraff@ksu.edu
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202001010
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Figure 1. Previously disclosed total synthesis route to uvaretin and its unsaturated enone variant, token examples of small-molecule libraries constructed
around intermediates 1 and 2, and biological assessment.
Results and Discussion
sulfate in acetone affording 10 in 89% yield. Previously, no
potentiation properties were observed with either 6TP or
doxorubicin with the aldol condensation product of 10.
Demethylation was then performed with AlCl3 in chlorobenzene
to give access to demethylated 11 and monomethylated 12 in
27 and 31% yield, respectively. Introduction of diethyl group
upon the free phenols of 12 followed by aldol condensation
with benzaldehyde afforded chalcone 13 in 68% yield over
two-steps.[6,8] In a similar fashion, the free phenol within 11 was
ethylated and transformed into its corresponding chalcone 14
in 75% yield over two-steps. The triethylated phenol species
was accessed from 9 through ethylation followed by aldol
condensation to afford 15 in 70% yield over two-steps. A single
MOM group was installed upon 11, previously no single MOM
protected chalcones were constructed and investigated for
potentiation properties, in 84% yield and was transformed to
its corresponding chalcone 16 in 85% yield.
Given the observed potentiation properties of 3–5, and the
moderately strong synergistic combination of 4 with 6TP,
investigations into the substitution effects with the polyphe-
nolic A ring of the chalcone small library were undertaken. No
direct correlation between the number of MOM protected
phenol groups was observed in the previously constructed
small library. Furthermore, no correlation could be extrapolated
on the overall effects of the MOM groups on the observed
potentiation activity. In the effort to probe the substituent(s)
effects upon this phenolic core, four new chalcones were
constructed (Scheme 1). Acetylation of phloroglucinol with
acetic anhydride and boron trifluoride etherate gave access to 9
in 74% yield,[7] which was then trimethylated with dimethyl
With these four analogs of the original chalcone small
library set in hand, biological evaluation was then pursued in
the same cancerous cell lines as our original study, in which
cellular viability was assessed by Alamar Blue quantification:[9]
cervical (HeLa), lymphoma (U937), lung (A549), and pancreatic
(MIA PaCa-2). Our previously disclosed MOM-chalcone CSL
possessed single to double digit μM IC50 activity in both HeLa
and U937, but showed little to no active in both the A549 and
MIA PaCa-2 cancerous cell lines. The diethylated/monometh-
ylated chalcone 13, possessing greater hydrophobic character
than its corresponding di-MOM protected/monomethylated 4
counterpart previous disclosed, possessed 0.9–2.4 μM cytotox-
icity in all cell lines screened. Cytotoxicity decreased for the
monoethylated/dimethylated chalcone 14, marginally within
HeLa and U937 but drastically in A549 and MIA PaCa-2. The
triethylated chalcone 15 had a drastic decrease in cytotoxicity,
all values hovering at 20 μM. (Table 1). A common trend
observed within the alkyl chalcone compounds 13, 14, and 16
is that moderate increase in the hydrophobic character trans-
lates to decreased cytotoxicity, as observed with higher IC50
values in 15, the triethylated chalcone, to the diethylated/
monomethylated chalcone 13. However, the monoethylated/
dimethylated chalcone 14 was shown to possess weaker
Scheme 1. Construction of various ethyl, methyl, and MOM chalcones.
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Table 1. Evaluation of chalcones 13–16 for cytotoxicity and potentiation properties.
Cmpd
Observed IC50 [μM][a]
HeLa
C.I.[b]
6TP
U937
A549
MIA PaCa-2
Dox
4
9.7�1.3
2.4�0.8
5.9�2.1
18.5�3.2
8.3�3.5
5.9�1.1
0.9�0.9
8.2�3.0
15.3�4.7
7.1�1.4
4.2�1.9
1.5�1.0
>20
>20
16.8�3.6
2.2�0.6
1.1�3.7
>20
N.T.
1.29
1.01
1.84
0.31
N.T.
1.76
1.82
1.99
0.98
13
14
15
16
19.1�2.3
>20
[a] Cytotoxicity was evaluated in 384-well plates over a 72 h treatment. Adherent cells were seeded at 1500 cells/well, whereas suspension cells at 2200 cells/
well. Cellular viability was evaluated via Alamar Blue. Doxorubicin was used as the positive cell death control, and wells with only cells as the alive control. [b]
Cytotoxicity evaluation for combinational index (C.I.) studies were the same as for sole agent cytotoxicity, and the C.I. was determined based on the Chou-
Talalay protocol.[10] N.T.: not tested and n=three biological replicates.
Table 2. Evaluation of C-benzylated chalcones 18–21 for cytotoxicity
properties.
cytotoxicity than 13. This suggests that the tuning of the
Cmpd
Observed IC50 [μM][a]
hydrophobic character of the phenolic A ring could alter and
enhance the cytotoxic activity of the chalcone system. It was
observed that with the inclusion of a single MOM group in the
dimethylated chalcone, a general increase in cytotoxicity was
observed with respect to the previous MOM-chalcone small
library. In terms of small molecule potentiation with 6TP and
doxorubicin, it was observed that the presence of at least one
MOM group is essential for synergistic activity. Assessing the
synergist properties of these compounds, and all other
compounds disclosed herein, with either 6TP or doxorubicin
was performed in a 3x5 matrix format. Two concentrations of
the small molecule screened were used that would induce 10–
20% cell death within a 72-h period. Concentrations of either
6TP or doxorubicin were chosen to result in 40–60% cell death
within the same time period. Combinational effects of cell
death verses concentration were then tabulated, and from
these values the effects of potentiation were determined by
their combination index (C.I.), a value calculated by employing
the Chou-Talalay protocol.[10] C.I. values closer to 0 represent
strong synergism, 0.5 mild synergism, 1 an additive effect, and
values greater than 1 denote antagonism in this method.
Synergy was shown with 16 and both 6TP and doxorubicin with
C.I. of 0.31 and 0.98, respectively. Based upon these inves-
tigations a general trend of the effects of hydrophobicity of the
phenolic core was observed; decreased synergy with increased
hydrophobic character.
HeLa
U937
A549
MIA PaCa-2
6
7
18
19
20
21
5.8�0.5
2.7�0.6
1.2�0.9
0.8�0.7
9.7�1.1
2.7�1.9
7.5�1.1
4.2�1.4
0.8�0.5
0.9�1.0
11.7�2.8
4.7�1.8
8.5�0.6
9.3�1.1
1.4�1.2
1.1�0.7
15.4�3.5
3.1�1.8
9.4�1.3
4.1�0.3
9.7�2.1
4.1�1.8
>20
10.7�4.3
[a] Cytotoxicity was evaluated in 384-well plates over 72 h of treatment.
Adherent cells were seeded at 1500 cells/well, suspension cells at 2200
cells/well. Cellular viability was evaluated with Alamar Blue. Doxorubicin
was used as the positive cell death control, and wells with only cells as the
alive control (n=three biological replicates).
Incorporation of a ortho-hydroxybenzyl substituent upon
the phenolic ring gives rise to the uvaretin family of natural
products. We previously disclosed that 6 and 7 (Figure 1), the
trimethylated phenol and allyl protected o-hydroxybenzyl
group/allyl deprotected free phenol, possessed strong sole
agent cytotoxicity (Table 2). From this, it was envisioned to
incorporate 13, the diethyl/monomethyl chalcone into this
uvaretin carbon framework for cytotoxicity investigations.
Subjecting 13 to our reported FriedelÀ Crafts alkylation con-
ditions with 17 gave access to 18 in 41% yield, and treatment
with Pd(PPh3)4 and potassium carbonate in methanol depro-
tected the allyl group to afford 19 in 96% yield (Scheme 2). In
parallel, the free phenol analog was prepared from 3 by
FriedelÀ Crafts alkylation with 17, with subsequent MOM
deprotection with methanolic HCl furnished 20 in 55%.
Subsequent allyl deprotection with Pd(PPh3)4 and potassium
Scheme 2. Elaboration of chalcones 13 and 3 onto their C-benzylated
variants.
carbonate in methanol gave access to 21 in 90% yield.
Screening of 18–21 against the four cancerous cell line panel
revealed interesting patterns. In both substitution patterns of
the phenolic core, greater cytotoxicity was observed in allyl
deprotected 19 and 21. Overall, the more hydrophobic phenol
core showed greater cytotoxicity. No potentiation properties
with 6TP and 18–21 were observed.
Previously, o-allyloxy benzyl alcohol (17) was employed in
our total synthesis of the natural product uvaretin, as well as in
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the uvaretin framework of chalcone analogs disclosed herein.
Keeping in mind the overarching goals of this project,
construction of new small chemical screening libraries, efforts
were placed towards modifications upon the benzyl (left ring)
of the trimethylated chalcone uvaretin system to explore what
effects, if any, they had upon cytotoxicity. Initially, para-allyloxy
benzyl alcohol was attached onto 22 via our FriedelÀ Crafts
alkylation protocol for the construction of its corresponding
uvaretin carbon core 23 in 67% yield, which was subsequently
transformed into the free phenol under our standard allyl
deprotection condition to access 24 in 33% yield (Figure 2). The
FriedelÀ Crafts alkylation worked well for both the ortho and
para substituted allyloxy benzyl alcohols, however, the meta
system, 25, failed to give any desired product. Lower yields
were obtained with the FriedelÀ Crafts alkylations of the ortho
(26) and para (27) chloro benzyl alcohols, 28 and 25% yields,
respectively. Treating 22 with 2,3-methylenedioxy benzyl alco-
hol afforded 28 in 29% yield, further supporting carbocation
stabilization. No product was observed when benzyl alcohol
(29) or 2-(hydroxymethyl)benzaldehyde (30) were attempted.
Based on these results, it can be concluded that electron
donating groups are required in the ortho and para positions to
stabilize the induced carbocation from the boron trifluoride
complexation with the benzyl alcohol. Screening of 26 and 27
showed >20 μM cytotoxicity, and no potentiation properties
with either 6TP or doxorubicin. Unexpectedly, both the allyl
(23) and free phenol (24) uvaretin carbon core analogs showed
cytotoxicity low double digit μM cytotoxicity, with no potentiat-
ing properties observed. Limited cytotoxicity was observed
from 28, IC50 of 18.5�3.8 μM in U937, but had strong synergism
with doxorubicin possessing a combination index of 0.33. From
this, it can be speculated that the substitution pattern upon the
left benzyl ring plays a critical role in the cytotoxicity of uvaretin
based small libraries.
ality is not only responsible for cytotoxicity, but also potentia-
tion. To explore this further, the enone in 22 was reduced to 31
in 99% yield (Scheme 3). Noting the single digit μM cytotoxicity
observed with 22, the complete loss of activity for 31 was
unexpected. Uvaretin, the parent natural product that spurred
these investigations, is a 2.0–3.7 μM cytotoxic compound that
possesses no enone functional group. Given that uvaretin does
not possess an enone group, we speculate that the o-hydroxyl
benzyl group along with the phenolic central ring is responsible
for its cytotoxicity. To probe the effects of the reduced enone
portion of the chalcone, noting that no sole cytotoxicity was
observed, potentiating studies were undertaken. A mild syner-
gism, combinational index of 0.72, was observed with 31 and
6TP in MIA PaCa-2 suggesting that the reduced enone portion
could be responsible for potentiation character or transport.
To exploit the possibility that the enone and o-hydroxy
benzyl group are responsible for cytotoxicity and the reduced
enone responsible for potentiation and transport properties,
our focus shifted towards the construction of a hybrid trimeth-
ylated uvaretin analog possessing all of these attributes.
Diacetylation of phloroglucinol was accomplished with acetic
anhydride and boron trifluoride etherate accessing 32 in 52%
yield, which was subsequently trimethylated with dimethyl
sulfate in acetone to afford 33 in quantitative yield (Scheme 4).
Aldol condensation upon 33 with excess equivalences of
benzylaldehyde and potassium hydroxide furnished chalcone
34 in 82% yield. Subjecting 34 to FriedelÀ Crafts alkylation
conditions with 17 furnished the dienone/o-allyloxybenzyl
chalcone 35 in 46% yield, followed by allyl deprotection with
Pd(PPh3)4 and potassium carbonate in methanol to afford 36 in
90% yield. Full reduction of the enones within 34 gave access
to 37 in quantitative yield, subjection to FriedelÀ Crafts
alkylation conditions with 17 afforded 38 in 29% yield. Allyl
deprotection upon 38 was troublesome, as such, reduction of
the enones in 36 was accomplished to give access to 39.
Biological assessment of these compounds, discussed below,
lead to the construction of a hybrid chalcone possessing both
an enone and reduced enone. Accessing the enone/reduced
enone system was undertaken by treating 33 to Aldol
Condensation with benzaldehyde (0.9 equiv.) to access 40 in
69% yield. Reduction of the enone was accomplished with
palladium on carbon in 71% yield followed by Aldol Condensa-
tion to afford 41 in 45% yield. Unfortunately, all attempts to
include the o-allyloxy benzyl alcohol upon 41 failed.
Previously, we disclosed that the trimethylated chalcone 22
was shown to have 4.3–6.7 μM activity in HeLa, U937, and A549
cancerous cells lines, with no activity observed in MIA PaCa-2.[6]
Varying the groups upon the phenolic oxygens with a mixture
of MOM, ethyl, and methyl groups gave rise to different small-
molecule potentiators with varying micromolar cytotoxicity.
Based on this, it could be concluded that the enone function-
Screening 34–41 for sole agent cytotoxicity against the four
panel cancerous cell lines used in this work was then under-
taken (Table 3). The di-enone chalone (34) possessed similar IC50
values in comparison to its mono-enone chalcone counterpart
22, suggesting that the presence of a second enone does not
Figure 2. FriedelÀ Crafts alkylation attempts upon 22 with varied substituted
benzyl alcohol systems.
Scheme 3. Enone reduction for potentiation and cytotoxicity comparisons.
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Scheme 4. Construction of hybrid chalcone and reduced enone chalcones.
Table 3. Evaluation of chalcones 34–41 for cytotoxicity and potentiation
properties.
6TP in MIA Paca-2 was shown to have mild to strong synergism,
while also possessing sole cytotoxic properties.
Cmpd
Observed IC50 [μM][a]
C.I.[b]
6TP
HeLa
U937
A549
MIA PaCa-2
22
34
35
36
37
38
39
41
5.2�1.4
4.7�2.4
1.1�0.5
1.0�0.9
19.7�5.1
13.4�5.0
11.8�2.1
4.7�2.0
6.7�0.8
5.4�0.7
1.7�1.2
2.0�0.8
>20
16.7�6.2
15.8�4.8
4.8�3.1
4.3�0.9
3.7�1.1
0.8�0.9
1.5�1.5
18.0�2.4
11.8�1.9
12.0�3.7
3.7�2.9
>20
N.T.
1.51
1.78
1.67
N.T.
1.18
1.04
0.42
18.4�5.8
12.4�8.2
13.5�4.7
>20
13.9�2.5
15.0�3.2
19.2�7.3
Conclusion
The construction of new small-molecule chemical libraries
based upon the intermediates of our total synthesis route
towards the uvaretin class of natural products is disclosed.
Building from our previous efforts, further structure–activity
relationship (SAR) studies were performed upon the sole
chalcone core, possessing no benzylated system. Previously, we
found interesting potentiation properties for the MOM pro-
tected chalcone system 3–5, of which 4 was shown to have
good synergistic properties towards 6TP in the pancreatic cell
line MIA PaCa-2; 6TP has no reported activity in this cell line. In
the efforts to optimize the potentiation properties of these
chalcones, we constructed 13–16 possessing various states of
methyl, ethyl, and single MOM group inclusion upon the
triphenol ring. It was found that increasing the hydrophobic
character upon the triphenol ring led to an overall decrease in
cytotoxicity. Unexpectedly, the dimethylated mono-MOM pro-
tected 16, while showing moderating cytotoxicity, was found to
possess strong synergistic properties with both 6TP and
doxorubicin in comparison to its more hydrophobic counter-
parts 13–15. Through our previous efforts, and the ones
disclosed herein, the role of the benzyl group was explored.
The triethylated chalcone (15) was observed to have little to no
cytotoxicity, however when incorporation of the o-hydroxyben-
zyl to form 18/19 gave access to cytotoxic activity. Thus,
suggesting that the o-hydroxybenzyl group possess cytotoxicity
in its own right, thus helping to explain the cytotoxic activity of
uvaretin. Through these efforts, the identification of the enone
as the bioactive portion of the chalcone was further confirmed,
and through the libraries constructed and evaluated it was
observed that the reduced enone systems, while possessing
marginal cytotoxicity, possess potentiation properties. In the
efforts to merge these two characteristics, we set forth to
construct our final library set of eight compounds, 34–41. Sole
[a] Cytotoxicity was evaluated in 384-well plates over a 72 h treatment.
Adherent cells were seeded at 1500 cells/well, whereas suspension cells at
2200 cells/well. Cellular viability was evaluated via Alamar Blue.
Doxorubicin was used as the positive cell death control, and wells with
only cells as the alive control. [b] Cytotoxicity evaluation for combinational
index (C.I.) studies were the same as for sole agent cytotoxicity, and the C.I.
was determined based on the Chou-Talalay protocol.[10] N.T.: not tested and
n=three biological replicates.
increase the cytotoxic character of the chalcone. Incorporation
of the o-allyloxy benzyl group did translate into an across-the-
board increase in cytotoxicity. However, deprotection of the
allyl group did not significantly affect cytotoxic properties. A
near complete loss of cytotoxicity was observed in the reduced
di-enone 37, o-allyloxy benzyl incorporation (38) did show a
marginal increase in cytotoxicity relative to 37. Like with 35 and
36, de-allylated 39 showed non-significant changes in cytotox-
icity relative to allylated 38. Investigating possible potentiating
properties of 34–39 with 6TP in MIA PaCa-2 were concluded to
investigate, and elucidate, the possible role of the enone and
reduced enone. All enone containing compounds (34–36) were
found to possess antagonism, non-synergist relationships. In
contrast, the reduced enones (38 and 39) were shown to
possess borderline mild synergism. These biological investiga-
tions led support to our initial theory that both the enone and
o-allyloxy and hydroxy benzyl groups tend to be responsible for
sole agent cytotoxicity and the reduced enone supports
potentiation properties. Evaluation of the enone/reduced enone
hybrid 41 for sole agent cytotoxicity revealed cytotoxicity
similar to 34 and 22. Most excitingly, potentiation of 34 with
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(6 equiv.), and Pd(PPh3)4 (0.02 equiv.) in MeOH (final concentration
relative to chalcone 16 mM) and allowed to stir at reflux for 2 h.
The reaction mixture was filtered through a short silica plug, rinsed
with MeOH (1 vol.equiv. to initial MeOH), and concentrated to
afford the desired product.
agent cytotoxicity was observed with dienones 34–37, and
potentiation properties with the dual reduced enones 37–39.
Construction of the hybrid enone/reduced enone 41 was
accomplished and was shown to possess single digit μM
cytotoxicity in all cell lines screened, except MIA PaCa-2, and
retained strong synergistic properties with 6TP in MIA PaCa-2.
We can conclude that small chemical library construction, as
well as SAR studies upon said libraries, has led to the
identification of new and interesting small molecules with
diverse biological activities.
Enone reduction. To a flamed dried RBF under an argon atmosphere
was added the enone material (1.0 equiv.) and 10% palladium over
carbon in MeOH (2.3 M final concentration relative to the enone). A
hydrogen balloon was introduced and the mixture was left to stir at
°
25 C for 2 h, filtered through Celite, and rinsed with MeOH or
CHCl3. The filtrate was concentration to afford the ketone species.
Compound synthesis
Experimental Section
2,4,6-Trihydroxyacetophenone (9):[11] BF3·OEt2 (7.5 mL, 59.4 mmol)
and acetic anhydride (1.9 mL, 19.8 mmol) followed by phlorogluci-
nol (2.5 g, 19.8 mmol) was added to a RBF under argon, and the
mixture was stirred at room temperature (RT). After 15 h, the
reaction mixture was poured onto a 10% NaOAc solution (70 mL)
and left to stir overnight (o/n) under ambient atmosphere. The
precipitate was filtered off and washed with water to obtain 9
Chemistry and general methods
All reagents were commercially available and used without
purification unless otherwise stated. NMR spectra were recorded
with a Varian and Bruker 400 MHz instrument. The chemical shifts
are given in parts per million (ppm) relative to residual CHCl3 at δ
7.26 ppm or DMSO at δ 2.50 ppm for proton spectra and relative to
CDCl3 at δ 77.23 ppm or DMSO at δ 39.52 ppm for carbon spectra,
unless otherwise noted. Low-resolution mass spectra were obtained
using a Waters Xevo-TQD via direct injection; samples were
dissolved in methanol, filtered, and the supernatant injected. Flash
column chromatography was performed with silica gel grade 60
(230–400 mesh). Dichloromethane (CH2Cl2), and methanol (CH3OH)
were all degassed with argon and passed through a solvent
purification system containing alumina or molecular sieves. All
commercially available reagents were used as received. All
procedures including anhydrous solvents were performed with
rigorously dried glassware under inert atmosphere.
1
(2.9 g) in 88% yield. H NMR (400 MHz, CD3OD) δ 5.79 (s, 2H), 2.59
(m, 3H). 13C NMR (101 MHz, CD3OD) δ 204.56, 166.32, 165.90,
105.60, 95.59, 32.71.
2,4,6-Trimethyoxyacetophenone (10):[12] Compound
9
(0.5 g,
2.98 mmol), K2CO3 (2.0 g, 14.9 mmol), and anhydrous acetone
(10 mL) were added to a flamed RBF under argon. The reaction
°
mixture was stirred at 25 C for 15 h, and then brought to reflux for
an additional 4 h. The reaction was quenched by the addition of
saturated ammonium chloride, and the mixture was extracted with
EtOAc (×3). The organic layers were combined, washed with brine,
dried over sodium sulfate and concentrated under reduced
pressure. The crude material was purified by flash silica gel
chromatograph (Hex/EtOAc 1:1) to afford the 10 (556 mg) in 89%
1
yield. H NMR (CDCl3, 400 MHz) δ (ppm): 6.10 (s, 2H), 3.82 (s, 3H),
General procedures
3.79 (s, 6H), 2.46 (s, 3H). 13C NMR (CDCl3, 101 MHz) δ (ppm): 202.1,
Aldol condensation. To a round bottom flask (RBF) was added the
162.5, 158.6, 113.8, 90.7, 56.0, 55.7, 32.8.
acetophenone species (1.0 equiv.), benzaldehyde (1.0 equiv., unless
2,4-Dimethoxy-6-hydroxy-acetephone (11) and 2-Methoxy-4,6-dihy-
droxy-acetophenone (12): To a flamed-dried RBF was added 10
(0.5 g, 2.38 mmol) and PhCl (24 mL) followed by AlCl3 (0.64 g,
4.8 mmol), and the mixture was left to stir o/n at RT. The mixture
°
otherwise noted), and EtOH (20 mL) and brought to 0 C. Once at
temperature, a solution of KOH (17 equiv.) in water (25 mM final
concentration) was added dropwise, after which the mixture was
°
brought to 25 C and left to stir for 14 h. The reaction was diluted
°
was then brought to 70 C and stirred for an additional 24 h. The
with EtOAc, washed with water, acidified with 1 M HCl, and
extracted with EtOAc (×2). The organic layers were combined,
washed with brine, dried over sodium sulfate and concentrated to
afford the crude material. Specific purification protocols are given
with each specific reaction.
reaction was quenched with saturated Rochelle Salt, and the
mixture was stirred for 30 min, then extracted with EtOAc (×3). The
organic layers were combined, dried over sodium sulfate, and
concentrated under reduced pressure. The crude material was
purified by flash silica gel chromatography (Hex/EtOAc 7:3) to
afford 11 (126 mg) in 27% yield and 12 (134 mg) in 31% yield.
FriedelÀ Crafts alkylation. To a flamed dried RBF under an argon
atmosphere was added chalcone (1.0 equiv.) and dry dioxane (final
concentration of 46 mM) and stirred at RT. In a second flamed dried
RBF was added 17[6] (1.2 equiv.) in dry dioxane (final concentration
of 0.22 M), which after stirring for 5 min at RT was added to the
chalcone mixture. To a separate flamed dried RBF under an argon
atmosphere was added BF3 ·Et2O (3.05 equiv.) and dioxane (final
concentration of 0.56 M), this solution was added to the previously
prepared RBF in four portions 10 min apart. The reaction was then
1
Compound 8: H NMR (CDCl3, 400 MHz) δ (ppm): 5.90 (1H, d, J=
2.1 Hz), 3.84 (3H, s), 3.80 (3H, s), 2.60 (3H, s). 13C NMR (CDCl3,
101 MHz) δ (ppm): 203.97, 166.83, 166.41, 162.85, 105.60, 92.14,
1
32.98. Compound 9: H NMR (CDCl3, 400 MHz) δ (ppm): 8.30 (s, 1H),
6.03 (d, J=2.0 Hz, 1H), 5.99 (d, J=2.0 Hz, 1H), 3.91 (s, 3H), 2.59 (s,
3H). 13C NMR (CDCl3, 101 MHz) δ (ppm): 204.0, 166.7, 165.2, 164.6,
106.5, 96.8, 92.0, 56.0, 33.0.
(E)-1-(2,4-Diethoxy-6-methoxyphenyl)-3-phenylprop-2-en-1-one (13):
The general procedure for aldol condensation was followed using
12a (0.3 g, 1.26 mmol). The crude material was purified by flash
silica gel chromatography (Hex/EtOAc 7:3) to give 13 (382 mg,
°
brought to 60 C and stirred for 3 h. The reaction was cooled,
diluted with EtOAc, washed with water (×2), and extracted with
EtOAc (×2). The combined organic layers were combined, washed
with brine, dried over sodium sulfate, and concentrated under
reduced pressure. Specific purification protocols are given with
each specific reaction.
1
pale-white solid) in 93% yield. H NMR (CDCl3, 400 MHz) δ (ppm):
7.67–7.58 (m, 3H), 7.55–7.44 (m, 3H), 7.30 (d, J=15.0 Hz, 1H), 6.56
(dd, J=15.6, 2.0 Hz, 2H), 4.39 (q, J=5.9 Hz, 2H), 4.06 (q, J=5.9 Hz,
2H), 3.91 (s, 3H), 1.65 (t, J=5.9 Hz, 3H), 1.34 (t, J=5.9 Hz, 3H). 13C
NMR (CDCl3, 101 MHz) δ (ppm): 192.6, 162.4, 162.1, 161.9, 142.9,
Allyl deprotection. To
a flamed dried RBF under an argon
atmosphere was added chalcone species (1.0 equiv.), K2CO3
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135.5, 129.8, 129.0, 128.6, 126.1, 112.1, 95.4, 92.8, 65.2, 63.9, 56.0,
14.7, 14.2. LRMS (ESI): m/z calcd for C20H23O4+: [M+H]+ 327.15,
found 327.16.
130.2, 129.8, 129.0, 128.6, 128.3, 126.9, 126.0, 120.9, 115.8, 97.4,
+
68.5, 65.0, 56.2, 22.9, 15.0, 14.5. LRMS (ESI): m/z calcd for C27H29O5
:
[M+H]+ 433.12, found 433.12.
(E)-1-(2-Ethoxy-4,6-dimethoxyphenyl)-3-phenylprop-2-en-1-one (14):
The general procedure for aldol condensation was followed using
11a (0.37 g, 1.65 mmol). The crude material was purified by flash
silica gel chromatography (Hex/EtOAc 7:3) to give 14 (423 mg,
(E)-1-(3-(2-(Allyloxy)benzyl)-2,4,6-trihydroxyphenyl)-3-phenylprop-2-en-
1-one (20): The general procedure for FriedelÀ Crafts alkylation was
followed using 3 (0.3 g, 0.77 mmol). The crude material was purified
by flash silica gel chromatography (Hex/EtOAc 7:3) to give 20
(171 mg, white solid) in 55% yield. 1H NMR (CDCl3, 400 MHz) δ
(ppm): 7.70–7.65 (m, 3H), 7.63 (dt, J=15.0, 0.9 Hz, 1H), 7.54–7.35 (m,
5H), 7.00 (td, J=7.5, 2.1 Hz, 1H), 6.95 (td, J=7.4, 2.2 Hz, 1H), 6.83
(dd, J=7.4, 2.2 Hz, 1H), 6.27 (s, 1H), 6.05 (ddt, J=16.3, 10.1, 6.2 Hz,
1H), 5.58 (ddt, J=13.9, 10.1, 1.1 Hz, 1H), 5.33 (ddt, J=16.7, 13.7,
0.9 Hz, 1H), 4.57 (dt, J=6.2, 1.1 Hz, 2H), 3.90 (d, J=1.1 Hz, 2H). 13C
NMR (CDCl3, 101 MHz) δ (ppm): 193.5, 164.1, 162.4, 160.6, 156.2,
142.1, 135.5, 133.0, 131.0, 129.8, 129.3, 129.0, 128.6, 128.2, 126.0,
121.6, 117.8, 114.0, 107.1, 105.3, 98.2, 69.2, 23.1. LRMS (ESI): m/z
calcd for C25H23O5+: [M+H]+ 403.15, found 403.14.
1
white solid) in 82% yield. H NMR (CDCl3, 400 MHz) δ (ppm): 7.67–
7.60 (m, 3H), 7.54–7.43 (m, 6H), 7.29 (d, J=15.1 Hz, 1H), 6.50 (s, 2H),
4.39 (q, J=5.9 Hz, 2H), 3.91 (s, 3H), 3.80 (s, 3H), 1.65 (t, J=5.9 Hz,
3H). 13C NMR (CDCl3, 101 MHz) δ (ppm): 192.6, 165.4, 162.6, 162.0,
142.9, 135.5, 129.8, 129.0, 128.6, 126.1, 111.2, 95.9, 93.8, 65.2, 56.0,
14.2. LRMS (ESI): m/z calcd for C20H21O4+: [M+H]+ 313.14, found
313.14.
(E)-3-Phenyl-1-(2,4,6-triethoxyphenyl)prop-2-en-1-one (): The general
procedure for aldol condensation was followed using 9a (0.28 g,
1.11 mmol). The crude material was purified by flash silica gel
chromatography (Hex/EtOAc 7:3) to give 15 (332 mg, white solid)
(E)-3-Phenyl-1-(2,4,6-trihydroxy-3-(2-hydroxybenzyl)phenyl)prop-2-en-
1-one (21): The general procedure for allyl deprotection was
followed using 20 (88 mg, 0.22 mmol) to give 21 (71 mg, pale oil) in
90% yield. 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.67–7.43 (m, 7H),
7.20 (ddt, J=7.5, 2.1, 1.1 Hz, 1H), 7.12 (td, J=7.4, 2.0 Hz, 1H), 6.91
(td, J=7.5, 2.0 Hz, 1H), 6.78 (dd, J=7.4, 2.0 Hz, 1H), 6.22 (s, 1H), 3.90
(d, J=1.1 Hz, 2H). 13C NMR (CDCl3, 101 MHz) δ (ppm): 193.5, 164.1,
162.4, 160.6, 154.2, 142.1, 135.5, 130.2, 129.8, 129.0, 128.6, 128.3,
126.8, 126.0, 120.9, 115.8, 107.1, 105.3, 98.2, 22.8. LRMS (ESI): m/z
calcd for C22H19O5+: [M+H]+ 363.12, found 363.13.
1
in 88% yield. H NMR (CDCl3, 400 MHz) δ (ppm): 7.68–7.59 (m, 3H),
7.56–7.42 (m, 3H), 7.30 (d, J=15.1 Hz, 1H), 6.57 (s, 2H), 4.39 (q, J=
5.9 Hz, 4H), 4.06 (q, J=5.9 Hz, 2H), 1.65 (t, J=5.9 Hz, 6H), 1.34 (t, J=
5.9 Hz, 3H). 13C NMR (CDCl3, 101 MHz) δ (ppm): 192.7, 162.5, 162.0,
142.9, 135.5, 129.8, 129.0, 128.6, 126.1, 113.9, 95.2, 65.2, 63.9, 14.6,
14.2. LRMS (ESI): m/z calcd for C21H24O4+: [M+H]+ 340.17, found
340.17.
(E)-1-(2,4-Dimethoxy-6-(methoxymethoxy)phenyl)-3-phenylprop-2-en-
1-one (16): The general procedure for aldol condensation was
followed using 11b (0.3 g, 1.25 mmol). The crude material was
purified by flash silica gel chromatography (Hex/EtOAc 7:3) to give
1,1’-(2,4,6-Trihydroxy-1,3-phenylene)bis(ethan-1-one) (32): To a flame
dried RBF under an argon atmosphere was added BF3·OEt2
(0.44 mL, 3.56 mmol) and acetic anhydride (0.12 mL, 1.19 mmol)
under argon and left to stir for 30 min at RT. The solution was
added dropwise over 10 min to a flame dried RBF charged with
phloroglucinol (0.1 g, 0.59 mmol) under argon and left to stir at RT
overnight. The reaction was quenched with a 10% sodium acetate
(2.8 mL) solution and left to stir overnight. The mixture was filtered,
rinsed with water and air dried to obtain 32 (64 mg, white solid) in
1
16 (348 mg, white solid) in 85% yield. H NMR (CDCl3, 400 MHz) δ
(ppm): 7.65–7.59 (m, 3H), 7.54–7.44 (m, 3H), 7.31 (d, J=15.1 Hz, 1H),
6.52 (d, J=2.0 Hz, 1H), 6.51 (d, J=1.8 Hz, 1H), 6.02 (s, 2H), 3.91 (s,
3H), 3.80 (s, 3H), 3.24 (s, 3H). 13C NMR (CDCl3, 101 MHz) δ (ppm):
192.6, 165.3, 161.4, 142.9, 135.5, 129.8, 129.0, 128.6, 126.1, 110.8,
96.0, 95.2, 94.4, 56.4. LRMS (ESI): m/z calcd for C19H21O5+: [M+H]+
329.14, found 329.14.
1
52% yield. H NMR (CDCl3, 400 MHz) δ (ppm): 5.82 (s, 1H), 2.73 (s,
(E)-1-(3-(2-(Allyloxy)benzyl)-2,4-diethoxy-6-methoxyphenyl)-3-phenyl-
prop-2-en-1-one (18): The general procedure for FriedelÀ Crafts
alkylation was followed using 13 (0.2 g, 0.61 mmol). The crude
material was purified by flash silica gel chromatography (Hex/EtOAc
8H). LRMS (ESI): m/z calcd for C10H10O5Na+: [M+Na]+ 233.04, found
233.03.
1,1’-(2,4,6-Trimethoxy-1,3-phenylene)bis(ethan-1-one) (33): To
a
1
7:3) to give 18 (118 mg, white solid) in 41% yield. H NMR (CDCl3,
flamed dried RBF under argon was added acetone (3.2 mL), 32
(0.2 g, 0.95 mmol) and potassium carbonate (0.66 g, 4.76 mmol)
followed by dimethyl sulfate (0.9 mL, 9.52 mmol) and left to stir at
RT for 12 h and then brought to reflux for 4 h. The mixture was
allowed to cool to RT and then was quenched with NH4Cl(satd) and
extracted with EtOAc (x2). The organic layers were combined, dried
over sodium sulfate, and concentrated under reduced pressure. The
crude material was purified by flash silica gel chromatography
(Hex/EtOAc 3:1) to afford 33 (239 mg, white solid)in quantitative
400 MHz) δ (ppm): 7.65–7.60 (m, 3H), 7.53–7.44 (m, 3H), 7.26 (d, J=
15.1 Hz, 1H), 7.22 (ddt, J=7.4, 2.0, 1.0 Hz, 1H), 7.00 (td, J=7.5,
2.0 Hz, 1H), 6.90 (td, J=7.4, 2.1 Hz, 1H), 6.81 (dd, J=7.4, 2.0 Hz, 1H),
6.77 (s, 1H), 6.05 (ddt, J=16.3, 10.1, 6.2 Hz, 1H), 5.58 (ddt, J=13.9,
10.1, 1.1 Hz, 1H), 5.33 (ddt, J=16.7, 13.7, 0.9 Hz, 1H), 4.57 (dt, J=
6.2, 1.1 Hz, 2H), 4.34 (q, J=8.0 Hz, 2H), 4.08 (q, J=8.0 Hz, 2H), 3.91
(s, 3H), 3.90 (d, J=1.1 Hz, 2H), 1.64–1.58 (m, 3H), 1.43 (t, J=8.0 Hz,
3H). 13C NMR (CDCl3, 101 MHz) δ (ppm): 192.1, 162.1, 161.5, 160.3,
156.2, 142.1, 135.5, 133.0, 131.0, 129.8, 129.3, 129.0, 128.6, 128.2,
16.0, 121.6, 117.8, 114.0, 113.8, 113.6, 97.4, 69.2, 68.5, 65.0, 56.2,
23.0, 15.0, 14.5. LRMS (ESI): m/z calcd for C30H33O5+: [M+H]+
473.23, found 473.22.
1
yield. H NMR (CDCl3, 400 MHz) δ (ppm): 6.19 (s, 1H), 3.78 (s, 6H),
3.64 (s, 3H), 2.41 (s, 6H). LRMS (ESI): m/z calcd for C13H17O5+: [M+
H]+ 253.10, found 253.10.
(2E,2’E)-1,1’-(2,4,6-Trimethoxy-1,3-phenylene)bis(3-phenylprop-2-en-1-
one) (34): To a RBF was added 33 (0.20 g, 0.79 mmol), benzylalde-
hyde (0.16 mL, 1.58 mmol), and ethanol (10.5 mL). The mixture was
(E)-1-(2,4-Diethoxy-3-(2-hydroxybenzyl)-6-methoxyphenyl)-3-phenyl-
prop-2-en-1-one (19): The general procedure for allyl deprotection
was followed using 18 (93 mg, 0.19 mmol) to give 19 (82 mg, pale
°
cooled to 0 C to which a solution of KOH (1.51 g, 2.55 mmol) in
1
yellow oil) in 96% yield. H NMR (CDCl3, 400 MHz) δ (ppm): 7.68–
water (3.16 mL) was added dropwise and left to stir overnight while
warming to RT. The reaction mixture was quenched by acidification
with 1 M HCl and extracted with EtOAc (x3). The organic layers
were combined, dried over sodium sulfate, and concentrated under
reduced pressure. The crude material was purified by flash silica gel
chromatography (Hex/EtOAc 3:1) to afford 34 (277 mg, yellow
solid) in 82% yield. 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.61–7.53 (m,
7.63 (m, 3H), 7.54–7.44 (m, 3H), 7.35 (d, J=15.1 Hz, 1H), 7.28 (ddt,
J=7.4, 2.0, 1.0 Hz, 1H), 7.14 (td, J=7.5, 2.0 Hz, 1H), 6.93 (td, J=7.5,
2.0 Hz, 1H), 6.85 (dd, J=7.5, 2.0 Hz, 1H), 6.71 (s, 1H), 4.34 (q, J=
5.9 Hz, 2H), 4.08 (q, J=5.9 Hz, 2H), 3.91 (s, 3H), 3.90 (d, J=1.1 Hz,
2H), 1.61 (t, J=5.9 Hz, 3H), 1.43 (t, J=5.9 Hz, 3H). 13C NMR (CDCl3,
101 MHz) δ (ppm): 192.1, 162.1, 161.5, 160.3, 154.2, 142.1, 135.5,
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4H), 7.50–7.38 (m, 7H), 7.05 (d, J=16.1 Hz, 2H), 6.37 (s, 2H), 3.89 (s,
6H), 3.71 (s, 3H). 13C NMR (CDCl3, 101 MHz) δ (ppm): 193.6, 159.6,
159.5, 157.1, 145.2, 134.6, 130.6, 128.9, 128.6, 128.5, 116.2, 91.1,
63.4, 56.1. LRMS (ESI): m/z calcd for C27H25O5+: [M+H]+ 429.17,
found 429.18.
3.81 (s, 2H), 3.78 (s, 3H), 3.77 (s, 3H), 3.11 (t, J=8.2 Hz, 2H), 3.02 (t,
J=7.6 Hz, 2H). LRMS (ESI): m/z calcd for C34H34O6Na+: [M+Na]+
561.23, found 561.23.
(E)-3-Phenyl-1-(2,4,6-trimethoxy-3-(3-phenylpropanoyl)phenyl)prop-2-
en-1-one (41): The general procedure for enone reduction was
followed using 40 (70 mg, 0.21 mmol) to give afford the reduced
enone in 71% yield. The general procedure for aldol condensation
was followed using the formed reduced enone (50 mg, 0.19 mmol).
The crude material was purified by flash silica gel chromatography
(gradient Hex/EtOAc: 100:0, 5:1, 7:3, 3:2, to 1:1) to give 38 in 45%
(2E,2’E)-1,1’-(5-(2-Hydroxybenzyl)-2,4,6-trimethoxy-1,3-phenylene)bis
(3-phenylprop-2-en-1-one) (35): The general procedure for
FriedelÀ Crafts alkylation was followed using 34 (0.1 g, 0.23 mmol).
The crude material was purified by flash silica gel chromatography
(gradient Hex/EtOAc: 100:0, 9:1, 5:1, to 7:3) to afford 35 (60 mg,
1
1
white solid) in 46% yield. H NMR (CDCl3, 400 MHz) δ (ppm): 7.47–
yield. H NMR (CDCl3, 400 MHz) δ (ppm): 7.50–7.44 (m, 2H), 7.33–
7.42 (m, 6H), 7.33 (d, J=16.1 Hz, 2H), 7.30–7.26 (m, 4H), 6.96 (d, J=
16.0 Hz, 1H), 6.83–6.62 (m, 3H), 6.07–5.97 (m, 1H), 5.40 (dq, J=17.3,
1.7 Hz, 1H), 5.20 (dt, J=10.6, 1.6 Hz, 1H), 4.53 (dt, J=5.0, 1.7 Hz, 2H),
3.94 (s, 2H), 3.76 (s, 3H), 3.72 (s, 3H), 3.44 (s, 3H). 13C NMR (CDCl3,
101 MHz) δ (ppm): 194.6, 160.6, 158.0, 157.1, 156.3, 144.7, 134.9,
133.8, 133.0, 130.3, 129.8, 129.6, 128.89, 128.84, 127.8, 126.5, 120.9,
120.4, 117.7, 116.8, 116.5, 116.4, 114.2, 114.2, 111.5, 111.2, 91.5,
68.8, 68.7, 62.9, 62.2, 56.0, 55.8, 22.8. LRMS (ESI): m/z calcd for
C37H35O6+: [M+H]+ 575.24, found 575.24.
7.29 (m, 4H), 7.23–7.14 (m, 3H), 7.14–7.06 (m, 2H), 6.92 (d, J=
16.0 Hz, 1H), 6.20 (s, 1H), 3.76 (s, 3H), 3.74 (s, 3H), 3.55 (s, 3H), 3.09–
3.01 (m, 2H), 3.00–2.92 (m, 2H). 13C NMR (CDCl3, 101 MHz) δ (ppm):
202.8, 193.4, 159.4, 158.8, 156.5, 145.2, 141.2, 134.6, 130.6, 129.6,
128.9, 128.6, 128.50, 128.46, 128.4, 128.3, 126.0, 118.2, 116.3, 91.1,
63.8, 56.1, 56.0, 46.4, 29.8. LRMS (ESI): m/z calcd for C27H27O5+: [M+
H]+ 431.18, found 431.18.
Biological
(2E,2’E)-1,1’-(5-(2-Hydroxybenzyl)-2,4,6-trimethoxy-1,3-phenylene)bis
(3-phenylprop-2-en-1-one) (36): The general procedure for allyl
deprotection was followed using 35 (26 mg, 0.05 mmol) to afford
Cell culture information. The cancerous cell lines used in these
investigations were purchased directly from the American Type
Culture Collection. Cell were grown in media supplemented with
fetal bovine serum (FBS) and antibiotics (100 μg/mL penicillin and
100 U/mL streptomycin). Specifically, experiments were performed
using the following cell lines and media compositions: HeLa, U937,
Reh (RPMI-1640+10% FBS), Mia PaCa-2 (DMEM+10% FBS), A549
(F-12K +10% FBS), and HCT-116 (McCoy 5A+10% FBS). Cells were
1
36 (60 mg, white solid) in 46% yield. H NMR (CDCl3, 400 MHz) δ
(ppm): 7.72–7.63 (m, 3H), 7.59–7.42 (m, 6H), 7.41–7.30 (m, 6H), 7.08
(td, J=7.7, 1.8 Hz, 2H), 6.99 (d, J=16.1 Hz, 2H), 6.86–6.75 (m, 2H),
3.91 (s, 3H), 3.79 (s, 3H), 3.77 (s, 3H). 13C NMR (CDCl3, 101 MHz) δ
(ppm): 194.1, 159.6, 157.3, 157.2, 155.8, 154.9, 145.5, 134.7, 13.0,
132.0, 131.98, 131.96, 131.6, 130.5, 128.9, 128.5, 128.33, 128.29,
127.8, 125.7, 119.7, 116.3, 116.10, 116.08, 114.02, 114.00, 92.1, 63.6,
56.1, 55.9, 29.7, 24.4. LRMS (ESI): m/z calcd for C34H30O6Na+: [M+
Na]+ 557.19, found 557.19.
°
incubated at 37 C in a 5% CO2, 95% humidity atmosphere for all
experiments.
IC50 value determination for adherent cells using Alamar Blue.
Adherent cells were added to 384-well plated (1500 cells/well) in
10 μL of media and were allowed to adhere to 2–3 h. Compounds
were solubilized in DMSO (10 μM stock solutions), added to a 96-
well plate over a range of concentrations (31.6 nM to 200 μM) with
media, 40 μL was added to the 384-well plate in triplicate for each
concentration of compound. After 69 h of continuous exposure,
5 μL of Alamar blue was added to each well and the cells were
allowed to incubate for an additional 3 h. The plates were then
read for fluorescence intensity with an excitation of 560 nm and
1,1’-(2,4,6-Trimethoxy-1,3-phenylene)bis(3-phenylpropan-1-one) (37):
The general procedure enone reduction was followed using 34
(25 mg, 0.058 mmol) to give afford 37 (25 mg, white solid) in
1
quantitative yield. H NMR (CDCl3, 400 MHz) δ (ppm): 7.25–7.04 (m,
10H), 6.12 (s, 1H), 3.71 (s, 6H), 3.46 (s, 3H), 3.01 (ddd, J=8.4, 6.9,
1.7 Hz, 4H), 2.93 (ddd, J=8.6, 6.8, 1.7 Hz, 4H). 13C NMR (CDCl3,
101 MHz) δ (ppm): 202.7, 158.70, 158.67, 155.8, 141.2, 128.50,
128.45, 128.37, 128.32, 128.2, 126.0, 118.29, 118.28, 91.1, 64.4, 55.9,
46.4, 29.8. LRMS (ESI): m/z calcd for C27H29O5+: [M+H]+ 433.20,
found 433.20.
emission of 590 nm on
a BioTek Synergy H1 plate reader.
Doxorubicin and etoposide were both used as positive death
controls and wells with no compounds added as negative death
controls. IC50 values were determined from three or more
independent experiments using GraphPad Prism 7.0. (LaJolla
California)
1,1’-(5-(2-(Allyloxy)benzyl)-2,4,6-trimethoxy-1,3-phenylene)bis(3-phe-
nylpropan-1-one) (38): The general procedure for FriedelÀ Crafts
alkylation was followed using 37 (83 mg, 0.19 mmol). The crude
material was purified by flash silica gel chromatography (gradient
Hex/EtOAc: 100:0, 9:1, 5:1, 7:3, to 1:1) to afford 38 (32 mg, white
solid) in 29% yield. 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.24–6.94 (m,
8H), 6.92–6.57 (m, 4H), 6.11–5.90 (m, 3H), 5.41–5.33 (m, 1H), 5.26–
5.10 (m, 1H), 4.64 (s, 2H), 4.52 (ddt, J=5.3, 3.4, 1.6 Hz, 4H), 3.88 (s,
2H), 3.72 (d, J=2.0 Hz, 3H), 3.67 (s, 3H), 3.37 (s, 3H), 3.09–3.01 (m,
2H), 2.97–2.90 (m, 2H). 13C NMR (CDCl3, 101 MHz) δ (ppm): 204.0,
160.5, 157.3, 156.5, 154.5, 141.5, 134.0, 133.5, 133.0, 132.6, 131.2,
129.7, 129.4, 129.0, 128.5, 127.4, 127.0, 120.9, 120.6, 118.3, 117.7,
116.9, 114.3, 111.7, 111.5, 111.4, 91.4, 68.9, 63.2, 55.8, 46.4, 35.1,
30.0. LRMS (ESI): m/z calcd for C37H39O6+: [M+H]+ 579.27, found
579.27.
IC50 value determination for non-adherent cells using Alamar Blue.
The same procedure for adherent cells was used, with the following
modifications. Cells (2200 cell/well) in media (10 μL) were added
after 40 μL of compound in medium were added to the 384-well
plate. No time was given to allow cells to adhere.
Combination studies: All combinational cell death experiments were
performed in 96-well plates with a total volume of 100 μL. To each
well was added 49 μL of cell media, either 2.5 or 5 μM of 6TP (from
a DMSO stock solution), and compounds independently. To each
well on the plate was added two different concentration of each
compound and five concentrations of 6TP or doxorubicin (both
prepared from 10 mM stock solutions in DMSO), cell media
(adjusted to reach 50 μL volume) and 0.5 μL of DMSO to achieve a
1% DMSO concentration. To each well was then added 50 μL of a
suspension of cells to obtain a final cell density of 4000 cells/well
(adherent cells) and 7000 cells/well (suspension cells). Doxorubicin
and etoposide were both used as positive death controls, and wells
1,1’-(5-(2-Hydroxybenzyl)-2,4,6-trimethoxy-1,3-phenylene)bis(3-phenyl-
propan-1-one) (39): The general procedure for enone reduction was
followed using 36 (8 mg, 0.015 mmol) to afford 39 (7 mg, white
1
solid) in quantitative yield. H NMR (CDCl3, 400 MHz) δ (ppm): 7.86–
7.76 (m, 1H), 7.75–7.65 (m, 2H), 7.63–7.52 (m, 2H), 7.49–7.31 (m, 2H),
7.25–7.02 (m, 4H), 7.29–6.98 (m, 4H), 6.88–6.74 (m, 4H), 3.92 (s, 3H),
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doi.org/10.1002/cmdc.202001010
ChemMedChem
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°
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This work could not have been undertaken without the gracious
financial support from the Johnson Cancer Research Center of
Kansas State University, Startup Capital from Kansas State
University, and NSF (CHE-1848186). Funding for this research was
also provided by the NSF REU program under grant number MPS-
1852182 and CHE-1460898. J. Dallman was supported by the NSF-
REU program.
Conflict of Interest
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Keywords: library construction · medicinal chemistry · natural
products · small-molecule potentiating agents
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Manuscript received: December 30, 2020
Accepted manuscript online: January 25, 2021
Version of record online: ■■■, ■■■■
ChemMedChem 2021, 16, 1–10
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© 2021 Wiley-VCH GmbH
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These are not the final page numbers!

FULL PAPERS
S. Perera, A. Fernando, J. Dallman,
Dr. C. Weeramange, Dr. A.
Lansakara, Dr. T. Nguyen, Prof. R. J.
Rafferty*
1 – 10
Construction and Biological Evalu-
ation of Small Libraries Based on
the Intermediates within the Total
Synthesis of Uvaretin
Discovering therapeutic agents: New
bioactive agents, either as sole or
combinational agents, have been con-
structed through the synthetic manip-
ulation of the intermediates within
the total synthesis of the uvaretin
class of natural products. It was found
that increasing the hydrophobic
bicity of the phenolic core of the
chalcones gives biases: less cytotoxic-
ity with more hydrophobic cores. In
addition, it was observed that the po-
tentiation, evaluated with 6-thiopur-
ine in the pancreatic cancer cell line
MIA PaCa-2, is tunable by the
inclusion of less-hydrophobic
character of the phenolic core corre-
lates to a decrease in sole agent cyto-
toxicity. The synthesis of new, small
chemical screening libraries (CSL) con-
structed from the intermediates of our
total synthesis route of the uvaretin
class of natural products is demon-
strated herein. Numerous chalcone-
based CSLs with various substitution
on the phenolic groups within the
chalcone core were assembled.
character on the phenolic core. The
role of the o-hydroxybenzyl group,
present within the uvaretin family,
was revealed to be cytotoxic in
character. Merging all of the
structure–activity relationship studies
performed on the CSLs constructed in
this effort led to the construction of a
new chalcone hybrid possessing both
a cytotoxic enone group and a small-
molecule-potentiating, reduced
enone group.
Through cytotoxicity investigations, it
was found that the level of hydropho-