Synthesis and Antileukemic Activities of Piperlongumine and HDAC Inhibitor Hybrids against Acute Myeloid Leukemia Cells

Article abstract of DOI:10.1021/acs.jmedchem.6b00772

Synergistic-to-additive antileukemic interactions of piperlongumine (PL) and HDAC inhibitor (HDACi) SAHA (Vorinostat) provide a compelling rationale to construct PL-HDACi hybrids, such as 1-58, which recapitulated the synergism between the parental compounds in high-risk and chemoresistant AML cells. Both PL and HDACi components, either in combination or in hybrid molecules, are essential for inducing significant DNA damage and apoptosis. Introducing C2-chloro substituent to 1-58 yielded 3-35 with increased cytotoxicity but decreased selectivity in noncancerous MCF-10A cells; eliminating C7-C8 olefin of PL obtained 3-31/3-98 scaffolds which were still more active than PL or SAHA in AML and were well-tolerated by MCF-10A cells. The HDACi function was crucial for modulating expression of DNA repair and apoptosis-related proteins. Collectively, PL and SAHA hybrids are potent, multifunctional anti-AML agents, acting in part, by interfering cellular GSH defense, suppressing expression of DNA repair and pro-survival proteins, and inducing expression of pro-apoptotic proteins.

Full text of DOI:10.1021/acs.jmedchem.6b00772

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Article
Synthesis and Antileukemic Activities of Piperlongumine and
HDAC Inhibitor Hybrids against Acute Myeloid Leukemia Cells
Yi Liao, Xiaojia Niu, Bailing Chen, Holly Edwards, Liping Xu, Chengzhi
Xie, Hai Lin, Lisa Anne Polin, Jeffrey W. Taub, Yubin Ge, and Zhihui Qin
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 09 Aug 2016
Downloaded from http://pubs.acs.org on August 9, 2016
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Synthesis and Antileukemic Activities of Piperlongumine and HDAC Inhibitor Hybrids
against Acute Myeloid Leukemia Cells
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Yi Liao1,#, Xiaojia Niu2,3,#, Bailing Chen1, Holly Edwards4,5, Liping Xu1, Chengzhi Xie4,5, Hai
Lin6, Lisa Polin4,5, Jeffrey W. Taub2,5,7, Yubin Ge2,4,5*, Zhihui Qin1,5*
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1Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health
Sciences, Wayne State University, 259 Mack Ave., Detroit, MI 48201, USA;
2 Department of Pediatrics, Wayne State University School of Medicine, Detroit, MI 48201, USA;
3School of Life Sciences, Jilin University, Changchun 130012, P.R. China;
4Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201, USA;
5Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute;
6 Department of Hematology and Oncology, The First Hospital of Jilin University, Changchun,
P.R. China;
7Division of Pediatric Hematology/Oncology, Children’s Hospital of Michigan, Detroit, MI
48201, USA;
#These authors contributed equally to this work
*Corresponding Authors
Zhihui Qin: email, zqin@wayne.edu; phone, 1-313-577-1567.
Yubin Ge: email, gey@karmanos.org; phone, 1-313-578-4285.
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ABSTRACT
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Synergistic-to-additive antileukemic interactions of piperlongumine (PL) and HDAC inhibitor
(HDACi) SAHA (Vorinostat) provide a compelling rationale to construct PL-HDACi hybrids,
such as 1-58 which recapitulated the synergism between the parental compounds in high-risk and
chemoresistant AML cells. Both PL and HDACi components, either in combination or in hybrid
molecules are essential for inducing significant DNA damage and apoptosis. Introducing C2-
chloro substituent to 1-58 yielded 3-35 with increased cytotoxicity but decreased selectivity in
non-cancerous MCF-10A cells; eliminating C7-C8 olefin of PL obtained 3-31/3-98 scaffolds
which were still more active than PL or SAHA in AML and were well tolerated by MCF-10A
cells. The HDACi function was crucial for modulating expression of DNA repair and apoptosis-
related proteins. Collectively, PL and SAHA hybrids are potent, multi-functional anti-AML
agents, acting in part, by interfering cellular GSH defense, suppressing expression of DNA repair
and pro-survival proteins, and inducing expression of pro-apoptotic proteins.
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INTRODUCTION
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Acute myeloid leukemia (AML) remains a devastating disease with poor outcomes,
especially in elderly patients.1 In 2015, the disease was projected to affect 20,830 new cases and
to cause 10,460 deaths in the US.2 Although the prognosis of AML in younger patients has been
steadily improved, long term disease-free survival of elderly patients remains poor, only 10-15%.
Except acute promyelocytic leukemia, which can be treated with all-trans retinoic acid and
arsenic trioxide, the mainstay of initial treatment for all other subtypes of AML using a
combination of cytosine arabinoside (Ara-C, also known as cytarabine) and an anthracycline
(e.g., daunorubicin) has not been changed for more than three decades. Treatment failure is
mainly due to resistance to chemotherapy and disease relapse arising from leukemic stem cells
(LSCs).3, 4 LSCs are defined as CD34+CD38-CD123+ cells and are the primary origin of AML
growth and relapse.5 The discovery of innovative therapeutic agents for AML treatment
represents an urgent and essential medical need.
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Histone deacetylase inhibitors (HDACis), such as suberoylanilide hydroxamic acid
(SAHA, Scheme 1), display diverse mechanisms of action that are attractive for AML therapy in
preclinical models.6 DNA damage is one of the mechanisms contributing to HDACi-induced
growth arrest and apoptosis in both AML cell lines and patient blasts.7-9 HDACis may cause
DNA damage and subsequent leukemia cell death through induction of reactive oxygen species
(ROS),7, 10 down-regulation and/or impairment of the function of DNA repair enzymes (e.g.,
Ku70, RAD51, CHK1, etc.),10-14 modulation of apoptotic pathways (such as upregulation of pro-
apoptotic genes and down-regulation of pro-survival genes),14 and disruption of chaperone
function of Hsp90, resulting in degradation of pro-growth/pro-survival client proteins (Bcr-Abl,
mutant FLT-3, c-Raf, AKT, etc.).15 Recent evidence also supports the role of HDACi in
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targeting LSCs, e.g. using drug combinations of HDACi and CHK116 or Wee117 inhibitors to
suppress primitive leukemia cell populations. On the other hand, clinical efficacy of single-agent
HDACis, such as SAHA, in AML patients remains modest,18, 19 indicating that the ultimate role
of HDACi for AML treatment may lie in combinatorial approaches.20
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Piperlongumine (PL, Scheme 1) is a natural product reported to selectively kill cancer
cells over non-malignant cells in vitro and in vivo.21 PL induces primary myeloid leukemia cell
death22 and causes severe cytotoxicity against CD34+ AML cells from patient specimens, which
may be attributed to its electrophilic GSH-depleting properties.23 Its selective toxicity to LSCs
relative to hematopoietic stem and progenitor cells (EC50: 380nM vs. 4900nM) was also
reported.24 By using a DNA repair-deficient DT40 cell line as genotoxic model, PL was found to
induce DNA double strand breaks (DSBs) and to suppress homologous recombination (HR)
DNA repair mechanism.25 PL-induced DNA damage, e.g., increased levels of 8-OHdG and DNA
strand breaks, was also observed in pancreatic cancer cell lines and in the corresponding in vivo
mouse model.26
Genetic alterations resulting in intrinsic DNA DSBs and defective DNA repair
mechanisms have been reported in AML cells,27 which makes targeting genomic integrity and
DNA damage response an attractive anti-AML strategy that can be therapeutically exploited to
induce synthetic lethality in AML cells.28 Given the observations that PL is able to induce DNA
DSBs and to inhibit the HR DNA repair mechanism,25 and HDACis directly cause DNA damage
meanwhile interfering with DNA repair processes at multiple levels, such as down-regulating
DNA repair proteins, inhibiting both HR and NHEJ repair29 and disrupting cell cycle check
points,9 we envisioned that HDACi and PL can cooperatively cause DNA damage in AML cells
and synergistically induce AML cell death.
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Clinical resistance to SAHA in leukemia patients has been correlated with overexpression
of antioxidant defense enzymes,18 and cellular GSH-depleting agent phenethyl isothiocyanate
(PEITC) was able to significantly enhance cytotoxicity of HDACi in drug resistant AML cells
and in primary leukemia cells.30 Because of the α,β-unsaturated imide pharmacophore,
electrophilic PL could compromise GSH-related antioxidant capacity by depleting the cellular
GSH pool via direct conjugation and/or inhibiting critical GSH-detoxification pathway enzymes,
such as GSTP1.21, 23 PL treatment has been shown to significantly reduce total GSH level and to
subsequently induce cell death in CD34+ primitive AML cells.23 Therefore, besides the
aforementioned DNA damage, electrophilic PL and PL analogues could also act synergistically
with HDACis by interfering with cellular GSH levels and GSH regulatory pathways. It is
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noteworthy that attenuated GSH defense could also lead to elevated DNA damage,31,
contributing to the suppression of AML cells.
In addition to co-administration, combined PL and HDACi activities could also be
realized by constructing hybrid molecules that contain both pharmacophores (Scheme 1). This
single-molecule “hybrid drug” approach more efficiently co-localizes PL and HDACi activities
in the same cell and could greatly simplify the optimization of drug-like properties, PK and dose-
toxicity profiles33 at a later drug discovery and development stage. Because of complementary
and overlapped anti-AML properties, we hypothesized that when combined together, either in a
drug combination or hybridized in a single molecule, PL could potentiate HDACi activities
leading to potent antileukemic effects. In the present study, we investigated in vitro anti-AML
properties of PL and SAHA either in combination or in hybrid molecules (PL-HDACis). PL and
SAHA combinatorial treatment synergistically induced apoptosis in phenotypically distinct AML
cells and this synergy is maintained by PL-HDACis, such as compounds 1-58 and 3-35 (Scheme
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1). Similar to drug combination, prototype PL-HDACis displayed potent anti-AML activities, in
part, by interfering cellular GSH defense, inducing DNA damage (e.g., DSBs), suppressing DNA
repair and pro-survival genes, and up-regulating the expression of pro-apoptotic genes in AML
cells.
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RESULTS AND DISCUSSION
1. Chemistry. In order to incorporate both PL and HDACi activities into a single
chemical entity, the prototype hybrid molecule 1-58 was designed by introducing a seven-carbon
linker at the 4’ position of PL. The linker was further connected to the hydroxamic acid moiety, a
zinc binding group (ZBG), to mimic the partial structure of SAHA (Scheme 1). The modified PL
structure served as a “cap” that could bind at the surface of HDAC enzymes. Two electrophilic
olefins are present in the PL structure; published PL structure-activity relationship (SAR) studies
suggested that the C2-C3 olefin was responsible for GSH conjugation via Michael addition and
was vital for ROS induction. The C2 substituent could potentially affect the chemical reactivity
of C2-C3 olefin and impact anticancer potency of PL. For instance, electron-withdrawing C2-
chloride increases, while C2-methyl group decreases cytotoxicity of PL to cancer cells.34, 35 After
conjugation with cellular GSH, close interactions of PL-GSH conjugate with GSH-binding
proteins may increase the availability of electrophilic C7-C8 olefin to protein thiol groups and
therefore promote C7-C8 olefin-caused covalent protein modification, this process was also
referred as cellular glutathionylation because of the attachment of PL-GSH conjugate to a
cellular protein.35 Elimination of the C7-C8 double bond has been shown to reduce
cytotoxicity,35 which might be a consequence of decreased PL covalent protein binding. To
investigate how the C2 substituent and the C7-C8 olefin affect the overall anti-AML properties
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of 1-58, compounds 3-35 with a C2-Cl substituent and 3-31/3-98 (Scheme 1) of which the
internal C7-C8 double bond was eliminated, were also synthesized and tested in cell culture
models. Although just containing partial PL structure, we hypothesized that the C2-C3 olefin of
3-31 or 3-98 may still work cooperatively with the HDACi functional group to achieve improved
anti-AML activities compared to parental drug, either SAHA or PL. Instead of having both
HDACi and PL pharmacophores, compounds 27, 28, 35 and 36 are close structural analogues of
1-58 or 3-35 with just one active pharmacophore (Scheme 1), e.g., ZBG of 1-58 or 3-35 was
replaced by a methyl ester in 27 and 28 respectively, and the electrophilic C2-C3 olefin of 1-58
was blocked by either a C3-methyl substituent (compound 35) or by hydrogen saturation
(compound 36). These compounds were used to make comparisons with 1-58 and 3-35 to
demonstrate both PL and HDACi pharmacophores contribute to the observed superior anti-AML
properties of PL-HDACis.
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The synthesis of 1-58/3-35 and 3-31/3-98 scaffold were started from commercially
available sinapinic acid 3 and syringic acid 5, respectively (Scheme 2). Carboxylic acids 3 and 5
were first converted to corresponding methyl esters 4 and 6 followed by the alkylation of phenol
hydroxyl groups with compound 2, a t-butyl ester of 7-bromoheptanoic acid to afford 7 and 8
respectively. Free carboxylic acids 9 and 10 were obtained via basic hydrolysis of methyl esters
7 and 8, while the t-butyl esters of these molecules were intact. In order to conduct SAR studies
of PL-like pharmacophores, α,β-unsaturated lactams 1136, 1234 which has a C2-Cl substituent,
and 1337 with a C3-methyl group were synthesized according to published procedures and
compound 14, δ-valerolactam, was purchased. Different lactams 11-14 were de-protonated by n-
BuLi and then coupled with pivaloyl chloride or oxalyl chloride-activated 9 or 10 respectively, to
afford 15-20. In following steps, t-butyl esters were cleaved using TFA and the obtained acids
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21-26 were coupled to THP-protected hydroxyl amine. Final acidic de-protection using TFA in
MeOH gave hydroxamic acid-containing 1-58, 3-35, 3-31, 3-98, 35, and 36. Methyl esters 27
and 28 were prepared from 21 and 22 by using diazomethane as esterification reagent
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respectively. Synthetic intermediates and final products were characterized using H, C NMR
and mass spectrometry; for the biological studies, purity of the compounds was further
confirmed using HPLC analysis.
2. Anti-leukemic interactions between SAHA and PL. To investigate if PL could
enhance the anti-AML activity of SAHA, U937 (p53 null) and MOLM-13 (p53 wt, FLT3-ITD)
cells were treated with either SAHA or PL, alone or in combination, at the indicated
concentrations for 24 h. The presence of FLT3/ITD in MOLM-13 cells represents an aggressive
AML disease phenotype.38 After drug treatment, cell death was assessed by Annexin V–FITC/PI
(propidium iodide) staining and flow cytometry analysis. In U937 cells, the combination of
SAHA with PL at 2 µM and 4 µM (1:1 ratio), respectively, resulted in significant induction of
cell death compared to individual drug treatment (Figure 1A). Furthermore, for U937 cells the
interaction of PL and SAHA was synergistic, as determined by calculating CI (combination
Index) values (CI < 0.9), while the interaction observed in MOLM-13 cells was additive (0.9 ≤
CI ≤ 1.1, Figure 1A). Drug-induced cell death was accompanied by cleavage of PARP-1 and
caspase-3, demonstrating that the cells underwent apoptosis (Figure 1B). The synergistic to
additive antileukemic interactions between PL and SAHA provide a compelling rationale to
construct PL-HDACi hybrid molecules against AML cells.
3. Anti-leukemic activity of the PL-HDACi hybrid 1-58 in AML cell lines. On the
basis of favorable anti-AML interactions between PL and SAHA, we explored a “hybrid drug”
approach to discover a single chemical entity possessing both HDACi and PL-related activities.
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In both U937 and MOLM-13 cell lines, the prototype hybrid compound 1-58 triggered
concentration-dependent apoptotic cell death, as indicated by a significant increase of Annexin
V-positive cells (Figure 1C) and cleavage of PARP and caspase-3 (Figure 1D). As shown in
Table 1, apoptosis-inducing activity of 1-58 recapitulated those of the PL and SAHA
combinations (1:1 ratio) at all tested concentrations (1, 2, 4 µM). EC50s of 1-58 and the
combination of PL and SAHA were also very similar, significantly lower than those of single
agents, indicating improved anti-AML potency (Figure 1E). 1-58 was further tested in other
AML cell lines representing various disease subtype and status, for instance, in pediatric AML
cell lines CMS, CMK, and CMY and in Ara-C-resistant HL60/ARA-C and CMK/ARA-C cells
(Ara-C IC50s > 8 M in CMY, HL60/ARA-C, and CMK/ARA-C cells, unpublished data). MTT
assay was conducted (72h treatment), IC50s of 1-58 were sub-micromolar for the tested cell lines
and were equivalent or substantially lower than combined PL and SAHA treatment (Table 2),
even in the Ara-C-resistant cell lines (Table 2). Collectively, 1-58 was effective against Ara-C-
resistant AML cells and showed a broad spectrum of anti-AML activities regardless of many
disease-specific molecular characteristics, such as p53 and FLT3-ITD mutational status.
4. Structure−Activity Relationships (SAR) of 1-58
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4.1. Cellular HDACi activities. Designed as multifunctional anticancer agents, hybrid
molecules were expected to retain HDACi activities. Since HDACs remove acetyl groups from
lysine residues of histone and non-histone proteins, the acetylation status of nuclear histone H4
and cytosolic α-tubulin have been frequently used as markers of cellular HDACi activity on
nuclear HDACs and on cytosolic HDAC6 respectively. In our experiments, U937 cells were
treated with 1 µM PL-HDACis and related analogues for 4 h and cell lysates were analyzed by
Western blotting. Like SAHA (the positive control), hybrid molecules 1-58, 3-35, 3-31 and 3-98
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induced hyper-acetylation of histone H4 and α-tubulin (Figure 2), indicating strong pan-HDACi
activity. Comparisons between 1-58/3-35 and 3-31/3-98 demonstrated that the absence of C7-C8
olefin in 3-31 and 3-98 did not change cellular HDACi efficacy in U937 cells. As expected, PL
itself did not provoke an increase and had no effect on SAHA-induced acetylation of H4 and α-
tubulin. Likewise, 27 and 28, which lack the hydroxamic acid functional group, didn’t affect
acetylation of H4 and α-tubulin. The cellular HDACi activity of compound 35 with C3-methyl
group was better than that of compound 36 in which C2-C3 olefin was saturated. Compared to 1-
58, structural modification of the 5,6-dihydro-2(1H)-pyridinone ring of PL, by introducing a C3-
methyl group (compound 35) or saturation of C2-C3 olefin (compound 36), decreased cellular
HDACi activities. On the contrary, C2-Cl substitution and elimination of C7-C8 olefin did not.
These results suggested that the PL or certain partial PL scaffold was an acceptable “cap” group
to render effective HDACi activities to the hybrid molecules.
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HDACs are critical regulators of gene expression by changing acetylation status of
histones and modifying chromatin structure. In the following studies, we further demonstrated
that the HDACi function in drug combination or in the chimeric molecules was responsible for
the modulation of DNA repair and apoptotic pathways in AML cells and resulted in improved
anti-AML effects.
4.2. Impact of HDACi and PL moieties. First, to clarify the contributions of HDACi and
PL moieties to the overall anti-AML effects of 1-58, close structural analogues, i.e. compounds
27, 35 and 36, were tested in U937 cells. Hydroxamic acid, the ZBG of 1-58, was replaced by a
methyl ester in 27 to eliminate HDACi activity. On the other hand, compounds 35 and 36
retained the ZBG but the electrophilic C2-C3 olefin of PL was blocked by using C3-methyl
substituent in compound 35 or via hydrogen saturation in compound 36. In contrast to 1-58, 27
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did not induce apoptosis in U937 cells (Figure 3A). Without an active C2-C3 olefin, compounds
35 and 36 also failed to induce significant apoptosis (Figure 3A), which was likely due to
reduced cellular HDACi activity compared to 1-58 (Figure 2) and the lack of C2-C3 olefin-
provoked PL-like cellular activities, such as GSH conjugation and/or protein alkylation. The
impact of functional C2-C3 olefin can also be emphasized by the comparisons of cellular HDACi
potency and the apoptotic EC50s of 1-58 and SAHA: although the two compounds caused similar
histone H4 and α-tubulin hyperacetylation, a reflection of close cellular HDACi activity (Figure
2), the apoptotic EC50s of 1-58 in AML cells are significantly lower than those of SAHA (Figure
1E). Nevertheless, these results suggest that the integration of intact HDACi and PL moieties
into one chemical entity is necessary for the observed superior anti-AML effects, blocking either
pharmacophore in close structural analogues significantly decreased their apoptosis induction
activity. Once getting into AML cells, 1-58 may have a dual action mode: some drug molecules
act as HDAC inhibitors, and others may induce PL-like effects. Synergism of HDACi and PL
structural components is reflected by the dramatically enhanced apoptotic effects of 1-58
compared to PL or SAHA alone (Figure 1A).
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Next we examined if the chemical reactivity of the C2-C3 olefin plays a role in the
biological properties of hybrid molecules. C2 chloro-substituted PL was reported to be more
cytotoxic to cancer cells and a C2-chloro PL analogue was well tolerated in an in vivo lung
cancer mouse model.34 To test if the introduction of an electron-withdrawing group at C2
position could increase electrophilicity of the C2-C3 olefin and therefore results in more potent
PL-HDACis, 3-35 was designed as an analogue of 1-58 with a C2-chloro substituent. We found
that 3-35 was much more potent than 1-58 in inducing apoptosis in U937 AML cells. While 29.9%
and 49.2% apoptosis were detected with 0.25 and 0.5 µM 3-35 treatments, respectively, 1-58 was
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not active at these concentrations (Figure 3A). The cooperative actions of PL and HDACi
moieties was also observed by comparing compounds 3-35 and 28: 28 is a 3-35 analogue with a
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methyl ester instead of a ZBG which is critical for HDAC inhibition. At 1 µM, 28 induced 29.7%
apoptosis whereas 3-35, having both C2-chloro PL and HDACi functionalities, induced 88.0%
apoptosis. Although 28 is a potent apoptosis inducer at 2 µM, its effect was still inferior to that
of 3-35 which caused almost 100% apoptotic cell death (Figure 3A). Furthermore, 3-35
treatment resulted in concentration-dependent apoptosis in two primary AML patient samples
(Figure 3C). Again, 3-35-induced apoptosis in U937 cells and in primary AML patient samples
was further verified by the detections of cleavage of PARP-1 and caspase-3 via Western blots
(Figure 3B&D). These results demonstrate that introducing an electron-withdrawing group at
the C2 position of PL enhanced the potency of hybrid molecules, which could be attributed to the
electron-deficiency of C2-C3 olefin that facilitates the conjugation with cellular nucleophiles.
We then looked at the effects of eliminating the C7-C8 olefin of PL. 3-31 and 3-98 were
synthesized as 1-58 and 3-35 analogues without the C7-C8 double bond. It has been reported that
disrupting the electrophilicity/reactivity of the C7-C8 olefin by saturation, steric blockade or
cyclization into a heterocycle could substantially diminish PL’s cytotoxicity and covalent
modification of GSH-binding proteins.35 Interestingly, these modifications did not interfere with
PL’s ROS production and GSH conjugation properties as long as C2-C3 olefin was intact.
Indeed, although 3-31/3-98 had similar cellular HDACi activities (Figure 2) as 1-58/3-35, they
induced less apoptotic cell death than 1-58/3-35 at 2 µM, likely due to the lack of the C7-C8
olefin (Figure 3A). It’s noteworthy that 3-31 and 3-98 are still more active than SAHA or PL at
inducing U937 cell death. After 24 h drug treatment at 2 µM, SAHA induced 25% apoptosis
(Figure 1A), whereas 3-31 and 3-98 induced approximately 40% (Figure 3A). Because 3-31 and
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3-98 showed similar cellular HDAC inhibition effects as SAHA (Figure 2), the increased
cytotoxic effects are due to the cellular actions of the remaining PL-related structures, i.e. the
imide bond and the dihydropyridinone ring. Interestingly, 28 consistently outperformed 3-98 at 1
and 2 µM, demonstrating that the presence of C7-C8 olefin, which is related to covalent protein
glutathionylation (i.e. cross-linking of drug-GSH conjugate with cellular proteins35), contributed
more to the overall cytotoxicity of 3-35 than HDAC inhibition. In contrast, without the C2-
chloro substituent, the HDACi functional group became more critical than the internal C7-C8
olefin: removal of ZBG from 1-58 resulted in a complete loss of apoptotic effect, as
demonstrated by compound 27. The C2-chloro substituent seems to rely on C7-C8 olefin to
enhance cytotoxicity of PL-HDACis: without C7-C8 olefin, cellular apoptotic effects of 3-31 and
3-98 were comparable; on the contrary, in the presence of C7-C8 olefin, 3-35 with C2-chloro
substituent was much more potent than 1-58 (Scheme 3A).
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4.3. Cytotoxicity vs. selectivity. Since both cytotoxicity and selectivity (i.e., sparing non-
cancer cells) are important for an anticancer compound, we further evaluated PL, SAHA, PL plus
SAHA, and hybrid compounds 1-58, 3-31, 3-35 and 3-98 in non-cancer cells. PL has been shown
to be non-toxic to MCF-10A, a non-cancerous breast epithelial cell line, at concentrations up to
15 µM in 24 h treatment,21 which makes it an interesting model for comparison. Although
normal human blood cells would be more relevant to leukemia treatment, because of limited
availability of clinical samples and the sensitivities of a few triple negative breast cancer cell
lines to 1-58, e.g., 1-58 showed IC50 of 382.4 ± 12.9nM in MDA-MB-231 cells (MTT assay,
activities of PL-HDACis in solid tumor cell lines will be described separately), we started our
selectivity evaluation using MCF-10A cells to rank order drug candidates for further studies.
Both MCF-10A and U937 cells were treated with escalating concentrations (0.5-5µM) of PL,
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SAHA, PL plus SAHA (1:1), 1-58, 3-31, 3-35 and 3-98 for 24 h and viable cells were measured
using MTT assays. As expected, SAHA and PL were well tolerated in MCF-10A cells in 24h
treatments (Figure S1). Interestingly, although 1-58 and PL + SAHA (1:1) had similar effects in
U937 cells, 1-58 had little to no effect on MCF-10A cells, suggesting that 1-58 was more
selective than PL + SAHA (Figure 4A). Similar to 1-58, 3-31 and 3-98 also showed selectivity
between the two cell lines (Figure 4B). Strikingly different than the previous compounds, 3-35
caused significant reduction of viable cells in both MCF-10A and U937 cells (Figure 4C).
Distinct from 1-58, the C2-C3 olefin of 3-35 is more electron deficient and we envisioned
that the increased electrophilicity of 3-35 was the major reason leading to the non-selective
cytotoxicity. To test this hypothesis, we incubated 3-35 and 1-58 (50µM) with and without GSH
(5mM) in phosphate buffer (pH 7.4) at 37°C up to 4h, and analyzed the incubation mixture by
HPLC. Both compounds remained intact in the absence of GSH; however, whereas 1-58 was still
stable in the presence of GSH, 3-35 was consumed and gave new peaks (Figure S2A).
Interestingly, we also observed reaction of 1-58 and GSH in the presence of U937 cell lysate
(Figure S2B). These results suggest that 3-35 may efficiently cause cellular GSH conjugation
and trigger the subsequent massive protein glutathionylation which is responsible for the non-
selective toxicity. In support of this assumption, we observed that 3-98 was still able to react with
GSH in the absence of cell lysate (Figure S2A), however, due to the lack of the
glutathionylation-required C7-C8 olefin, it was tolerable in MCF-10A cells (Figure 4C). On the
other hand, 1-58-caused GSH conjugation/glutathionylation may occur at a lower extent
compared to 3-35, either due to the relatively low intrinsic chemical reactivity of C2-C3 olefin,
or because the GSH conjugation caused by 1-58 might be an enzymatic process and therefore
could be affected by the distinct expression levels of the responsible enzyme(s) (such as certain
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GST enzyme) in U937 and in MCF-10A cells. Further studies (such as comparing drug-induced
cellular glutathionylations etc.) are needed to understand the more detailed mechanisms of the
selectivity for PL-HDACi hybrid molecules. Nonetheless, the comparison of 1-58 and 3-35 in
U937 and in MCF-10A cells suggest that 1-58 is more suitable than 3-35 for further research.
To build a connection between cellular GSH consumption and apoptosis induction, we
conducted an N-acetyl cysteine (NAC) rescue experiment. Pre-incubation of NAC (3 mM), a
well-known GSH precursor and antioxidant, with U937 cells was conducted (3 h, followed by
PBS wash to remove excess NAC in culture media) to increase intracellular free thiol/GSH
levels. We found that NAC rescued U937 cells from SAHA, PL, SAHA plus PL, 1-58 or 3-35-
induced apoptosis at indicated concentrations (Figure 4D). The result emphasized the role of
GSH consumption and/or ROS induction played in the overall apoptotic effects. It is interesting
to note that compared to the partial rescue observed in SAHA-related treatments (SAHA or
SAHA+PL), NAC completely rescued U937 cells from 1-58 and 3-35 treatments. This
observation could be explained by the conjugation between PL-HDACis and cellular GSH/NAC:
once the PL moiety is conjugated, the resulted bulky and highly polar conjugate cannot serve as
an effective “cap” group binding at the surface of HDAC enzymes, which leads to a complete
inactivation of both PL and HDACi activities. In the case of SAHA, because of the pleiotropic
anticancer characteristics,6 although its ROS induction/GSH consumption effects could be
neutralized by NAC treatment, SAHA can still elevate apoptosis levels through other pathways.
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5. DNA damage caused by PL and HDACi treatments in U937 cells. Since
both
PL25, 26 and HDACis, such as SAHA,7 are able to induce DNA damage and inhibit DNA repair
mechanisms, we investigated their impact on the integrity of DNA in U937 cells. We first treated
U937 cells with PL and SAHA, alone or in combination, for 20 h and then assessed DNA
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damage using alkaline comet assay. The alkaline comet assay is a single cell gel electrophoresis
assay to analyze DNA damage ranging from DNA single strand breaks, DSBs and alkali-labile
sites in the DNA.39 This assay allows the direct observation of the “comet tail” which is
correlated with the damaged DNA content. In our experiment, PL (2µM) and SAHA (2µM)
combination caused more prominent DNA damage than each drug alone (Figure 5A). At 2 µM,
although the damaged DNA via SAHA or PL treatments were undetectable, their combinatorial
effect was significant; moreover, the DNA content in the tail induced by 1-58 (2 µM) treatment
was comparable to that of combined SAHA and PL (Figure 5B).
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To further confirm drug-induced DNA damage and to exploit the mechanisms behind the
increased DNA-damaging effects, protein levels of γH2AX and a few DNA repair-related
proteins, CHK1 and Rad51, were analyzed by Western blots. As shown in Figure 5C, SAHA (2
µM) treatment resulted in increased expression of γH2AX, an established biomarker of DNA
double-strand breaks.40 Although PL itself didn’t cause increased γH2AX, it substantially
potentiated SAHA’s effects (2.5-fold) and resulted in a 4.1-fold increase of γH2AX in
combinatorial treatment. Compound 1-58, which has both HDACi and PL-related properties,
acted comparably to the drug combination and induced a 4.5-fold increase of γH2AX.
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Furthermore, Rad51, an essential protein in the HR DNA repair pathway, was up-regulated
1.4-fold by PL (2 µM), which could be a cellular adaptive reaction to the PL-induced DNA
damage. Strikingly, this increase was abolished by combination with SAHA or via the hybrid
compound 1-58 (Figure 5C). Meanwhile, the protein levels of CHK1, a checkpoint regulator
that participates in multiple pathways to protect cells from DNA damages,42 were also reduced
by HDACi treatments to 0.6, 0.8 and 0.7-fold in SAHA, SAHA plus PL and 1-58 treatments,
respectively. Our experiments suggest that 1-58 treatment-associated CHK1 and/or Rad51
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reduction was related to its HDACi activities. Current literature gave a few possibilities to
understand the observed CHK1/Rad 51 downregulation, such as HDACi-induced
downregulation of E2F1 transcriptional factor14 and HDAC inhibition-mediated induction of
microRNA miR-182 which was linked to time- and dose-dependent decreases in the levels of
Rad51.43 Nonetheless, the reduction of Rad51 and CHK1 contribute to the observed significant
DNA damage induced by SAHA and PL combination and their hybrid molecule 1-58.
6. Apoptotic proteins affected by PL-HDACi hybrid molecule. The balance between
the expression/activity of the pro- and anti-apoptotic pathways could ultimately determine the
fate of a given cell. It is well established that HDACis can induce apoptosis by up-regulating
pro-apoptotic proteins (e.g., Bim, Bax, Puma, Noxa, etc) and down-regulating pro-survival
proteins (e.g., survivin, XIAP).44 Among the Bcl-2 and IAP (inhibitors of apoptosis) families,
our Western blotting analysis revealed up-regulation of pro-apoptotic Bim (EL isoform, 1.8 fold)
and down-regulation of anti-apoptotic protein XIAP (to 0.6 fold) by 1-58 (Figure 5D). Because
PL itself didn’t change the expression of Bim and XIAP (Figure 5D), the observed modulations
were mainly attributed to the retained HDACi activity of 1-58. PL and SAHA combination, as
well as SAHA alone, also showed similar effects.
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BH3-only protein Bim plays a role in initiating apoptosis and has been shown to be up-
regulated by different HDACis, for instance, by belinostat in AML and acute lymphoblastic
leukemia cells;45 HDACi-mediated transcriptional activation of Bim may occur through histone
H4 hyperacetylation within the gene promoter region.46 Similarly, the expression of short-lived
anti-apoptotic proteins XIAP can also be modulated by HDACis; our results are consistent with a
previous report which demonstrated that XIAP proteins were reduced by SAHA in U937 cells.47
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In our experiments, HDACi activities of SAHA, in the PL plus SAHA drug combination,
or PL-HDACi hybrid modulated the expression of proteins involved in DNA repair and
apoptotic pathways, which collectively favored enhanced DNA damage and apoptotic cell death.
The ultimate contribution of each protein can be further evaluated using ectopic overexpression
or shRNA knockdown methodologies in future studies.
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CONCLUSIONS
We found that PL and HDACi SAHA, either used in combination or hybridized into a
hybrid molecule, synergistically caused apoptotic cell death of AML cells. The IC50s of 1-58 (a
prototype PL-HDACi) in multiple AML cell lines, including aggressive and chemotherapy-
resistant cell models, are significantly lower than those of PL, SAHA and their combination (1:1
ratio). SAR study of 1-58 suggested that both PL and HDACi pharmacophores are required for
superior anti-AML activities. Introducing electron-withdrawing C2-Cl substituent to 1-58
yielded 3-35 with increased cytotoxicity but decreased selectivity in a MCF-10A non-cancerous
cell culture model; eliminating C7-C8 olefin of PL obtained 3-31/3-98 scaffold, which are still
more active than PL or SAHA alone against U937 cells. On the contrary to 3-35, compounds 1-
58, 3-31 and 3-98 were well tolerated by MCF-10A cells, suggesting better selectivity of these
compounds. The difference of 3-35 and 1-58 in AML cells and in MCF-10A cells could be
partially attributed to their distinct reactivity to cellular thiol components, such as GSH. Pre-
treatment with NAC, the precursor of GSH biosynthesis and an antioxidant/thiol-based
nucleophile, rescued U937 cells from drug-induced cytotoxicity, which emphasized the critical
role of GSH conjugation in the overall antiproliferative effects of drug combinations and hybrid
molecules. Revealed by comet assays and Western blots, PL and SAHA demonstrated their
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ability to cooperatively induce DNA damage in U937 cells, either in combination or in hybrid.
The hyperacetylation patterns of histone H4 and cytosolic α-tubulin suggested that PL-HDACis
1-58, 3-35, 3-31 and 3-98 retained SAHA comparable pan-HDACi activities. The HDACi
functions of drug combination or 1-58 were responsible for the modulated expression of proteins
in DNA repair and apoptotic pathways, e.g., downregulation of DNA repair-related CHK1 and
Rad51, upregulation of pro-apoptotic Bim and suppression of pro-survival XIAP. These effects
collectively favor DNA damage and apoptotic cell death. Our current results demonstrate that
PL and HDACi combination or PL-HDACi hybrids are potent antileukemic agents against AML,
they act in part, by interfering cellular GSH defense, suppressing expression of DNA repair and
pro-survival genes, and inducing expression of pro-apoptotic genes. Prototype hybrid compounds
in this study could be a promising starting point to discover more potent and cancer cell selective
PL-HDACis against hematological malignancies.
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EXPERIMENTAL SECTION
1
General Methods for Chemistry. H and 13C NMR spectra were obtained using Varian
Mercury 600MHz and Advance 400 MHz spectrometers. Chemical shifts are reported as δ
values in parts per million (ppm) relative to tetramethylsilane (TMS) for all recorded NMR
spectra. All reagents and solvents were obtained commercially from Acros, Aldrich or Fisher and
were used without purification. Flash column chromatography was performed over 200−300
mesh silica gel. High resolution mass spectral data were collected using a LCT Premier XE
KD128 instrument. All compounds submitted for biological testing were found to be at least 95%
pure by analytical HPLC. Purity check and HPLC analysis of incubations were conducted using
a Phenomenex (LunaR) C18, 3.5 μm, 100 mm × 3.0 mm column on a Shimadzu UFLC
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instrument. HPLC parameters were: solvent A, water containing 0.1% formic acid (FA) and 10%
(v/v) CH3OH; solvent B, 0.1% FA in CH3CN; flow rate 0.5 mL/min; gradient, t = 0 min, 10% B;
t = 1 min, 10% B; t = 18 min, 80% B; t = 20 min, 80% B; t = 21 min, 10% B.
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tert-Butyl 7-bromoheptanoate (2). 7-Bromoheptanoic acid (1.11 g, 5.3 mmol, 1 eq) and
Boc anhydride (1.38g, 6.3mmol, 1.2 eq) were mixed in tert-butyl alcohol (5 mL). After the
mixture was stirred at room temperature for 10 min, DMAP (195 mg, 1.6 mmol, 0.3 eq) was
added and the reaction mixture was stirred for another 2 h. Upon completion, the solvent was
removed under reduced pressure and the residue was re-dissolved in ethyl acetate and was
washed with water and brine, dried over anhydrous Na2SO4. Purification by flash
1
chromatography (Hex/EA, 20/1) afforded product (colorless oil, 570 mg, 40%). H NMR (600
MHz, CDCl3): δ 3.38 (t, J=6.6Hz, 2H), 2.19 (t, J=7.2Hz, 2H), 1.82-1.86 (m, 2H), 1.55-1.60 (m,
2H), 1.40-1.45 (m, 2H), 1.42 (s, 9H), 1.29-1.34 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 173.06,
80.01, 35.39, 33.81, 32.55, 28.16, 28.09, 27.81, 24.83.
(E)-Methyl 3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate (4). Sinapinic acid (1.03 g, 4.6
mmol, 1 eq) was dissolved in methanol (20 mL) followed by addition of several drops of conc.
H2SO4. The reaction mixture was refluxed for 6 h. Upon completion, the reaction mixture was
cooled to ambient temperature and solvent was removed under vacuum. The resulting residue
was diluted with ethyl acetate and washed using sat. aq. NaHCO3, water and brine, and dried
over anhydrous Na2SO4. Purification by flash chromatography (Hex/EA, 2:1) afforded product
1
(yellow oil, 1.11g, quantitative yield). H NMR (600 MHz, CDCl3): δ 7.55 (d, J=15.6Hz, 1H),
6.72 (s, 2H), 6.26 (d, J=15.6Hz, 1H), 5.79 (s, 1H), 3.89 (s, 6H), 3.76(s, 3H). 13C NMR (125 MHz,
CDCl3): δ 167.61, 147.18, 145.15, 137.13, 125.77, 115.41, 105.00, 56.27, 51.62.
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Methyl 3,5-dimethoxy-4-hydroxybenzoate (6). Prepared from syringic acid using a similar
procedure described for compound 4 (quantitative yield). 1H NMR (600 MHz, CDCl3): δ 7.32 (s,
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2H), 6.04 (s, 1H), 3.93 (s, 6H), 3.90 (s, 3H); C NMR (125 MHz, CDCl3): δ 166.85, 146.59,
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139.16, 120.98, 106.57, 56.36, 52.07.
(E)-tert-Butyl 7-(2,6-dimethoxy-4-(3-methoxy-3-oxoprop-1-enyl)phenoxy)heptanoate (7).
Compound 2 (570 mg, 2.1 mmol, 1.1 eq), compound 4 (476 mg, 2 mmol, 1 eq) and K2CO3 (838
mg, 6 mmol, 3 eq) were mixed in DMF (4 mL). The reaction mixture was stirred at 60oC
overnight. The reaction mixture was cooled to room temperature and diluted with water. After
extraction using ethyl acetate three times, the combined organic phase was washed with water
and brine, and dried over anhydrous Na2SO4. Purification by flash chromatography (Hex/EA, 6:1)
afforded product compound 7 (colorless oil, 630 mg, 75%). 1H NMR (600 MHz, CDCl3): δ 7.55
(d, J=15.6Hz, 1H), 6.69 (s, 2H), 6.28 (d, J=15.6Hz, 1H), 3.94 (t, J=6.6Hz, 2H), 3.81(s, 6H), 3.75
(s, 3H), 2.16 (t, J=7.2Hz, 2H), 1.67-1.72 (m, 2H), 1.53-1.68(m, 2H), 1.41-1.46(m, 2H), 1.38 (s,
9H), 1.28-1.33(m, 2H). 13C NMR (125 MHz, CDCl3): δ 173.16, 167.36, 153.60, 144.89, 139.40,
129.57, 116.76, 105.23, 79.84, 73.41, 56.07, 51.61, 35.49, 29.86, 28.80, 28.04, 25.47, 25.01.
HRMS(ESI): calcd for [C23H34O7+H]+, 423.2383; found, 423.2404.
Methyl 4-(7-tert-butoxy-7-oxoheptyloxy)-3,5-dimethoxybenzoate (8). A flask was charged
with compound 6 (1.76 g, 8.23 mmol) and compound 2 (2.4 g, 9.06 mmol). DMF (10 mL) and
potassium carbonate (3.41 g, 24.70 mmol) were added at room temperature. The reaction was
o
allowed to undergo at 80 C overnight. Once completed, the resulting mixture was washed with
water and extracted with DCM three times, and then the organic phase was dried over Na2SO4
for 30 min. Purification done by flash chromatography (Hex/EA, 8:1) produces compound 8
1
(colorless oil, 3.7 g, 94%). H NMR (600 MHz, CDCl3): δ 7.26 (s, 2H), 3.99 (t, J=7.2Hz, 2H),
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3.87 (s, 3H), 3.86 (s, 6H), 2.18 (t, 7.2Hz, 2H), 1.70-1.75 (m, 2H), 1.55-1.60 (m, 2H), 1.42-1.47
(m, 2H), 1.41 (s, 9H), 1.31-1.36 (m, 2H).13C NMR (125 MHz, CDCl3): δ 173.20, 166.75, 153.12,
141.43, 124.88, 106.74, 79.88, 73.37, 56.16, 52.16, 35.52, 29.89, 28.82, 28.08, 25.48, 25.03.
HRMS (ESI): calcd. for [C21H32O7+Na]+, 419.2046; found, 419.2046.
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(E)-3-(4-(7-tert-Butoxy-7-oxoheptyloxy)-3,5-dimethoxyphenyl)acrylic
acid
(9).
Compound 7 (588 mg, 1.4 mmol, 1 eq) was dissolved in THF/H2O (5 mL/5 mL) followed by the
addition of LiOH (40 mg, 1.7 mmol, 1.2 eq). The reaction mixture was stirred at room
temperature overnight. Upon completion, 2 N HCl was added to acidify the reaction and the
solution was extracted with ethyl acetate three times. The collected organic phase was washed
with water and brine, and dried over anhydrous Na2SO4. Evaporation of solvent afforded product
compound 9 (yellow oil, 600 mg, 100%) which was used in the next step without further
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purification. H NMR (600 MHz, CDCl3): δ 7.68 (d, J=15.6Hz, 1H), 6.75 (s, 2H), 6.33 (d,
J=15.6Hz, 1H), 3.98 (t, J=6.6Hz, 2H), 3.85 (s, 6H), 2.19 (t, J=7.2Hz, 2H), 1.71-1.75 (m, 2H),
1.56-1.61 (m, 2H), 1.43-1.48 (m, 2H), 1.42 (s, 9H), 1.33-1.37 (m, 2H). 13C NMR (125 MHz,
CDCl3): δ 173.31, 172.21, 153.66, 147.04, 139.82, 129.26, 116.31, 105.57, 79.97, 73.50, 56.13,
35.54, 29.89, 28.83, 28.09, 25.49, 25.04.
4-(7-tert-Butoxy-7-oxoheptyloxy)-3,5-dimethoxybenzoic acid (10). Compound 10 was
made from 8 (3.7g, 7.7mmol) as described for compound 9. Product was obtained as yellow oil
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(1.7g, 47%). H NMR (600 MHz, CDCl3): δ 7.37 (s, 2H), 4.05(t, J=7.2Hz, 2H), 3.90(s, 6H),
2.23(t, J=7.8Hz, 2H), 1.74-1.79(m, 2H), 1.59-1.64(m, 2H), 1.46-1.51(m, 2H), 1.44 (s, 9H), 1.35-
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1.39(m, 2H); C NMR (125 MHz, CDCl3): δ 173.33, 171.37, 153.17, 142.18, 124.04, 107.34,
79.99, 73.44, 56.18, 35.53, 29.90, 28.82, 28.08, 25.47, 25.04.
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(E)-tert-Butyl 7-(2,6-dimethoxy-4-(3-oxo-3-(2-oxo-5,6-dihydropyridin-1(2H)-yl)prop-1-
enyl)phenoxy)heptanoate (15). Step 1: compound 9 (540 mg, 1.3 mmol, 1 eq) was dissolved in
DCM (12 mL) followed by addition of oxalyl chloride (0.56 mL, 6.5 mmol, 5 eq) and catalytic
amount of DMF (two drops). The reaction mixture was stirred at room temperature for 1 h after
which the solvent was removed under reduced pressure. The residue was dissolved in THF (3
mL) and directly used in the next step without purification. Step 2: compound 11 (150 mg, 1.56
mmol, 1.2 eq) was dissolved in anhydrous THF (5 mL) and n-BuLi (2.5 M in hexane, 0.68 mL,
1.7 mmol, 1.3 eq) was added dropwise via syringe at -78oC. The reaction mixture was stirred for
15 min at the same temperature followed by the dropwise addition of the THF solution in the
first step. TLC showed the completion of the reaction within about 30 min at -78°C. Sat. aq.
NH4Cl was added to quench this reaction and the resulting solution was extracted with ethyl
acetate three times. The combined organic phase was washed with water and brine, and dried
over anhydrous Na2SO4. Flash chromatography (Hex/EA, 4:1) purification afforded product as
yellow oil (358mg, 56%). 1H NMR (600 MHz, CDCl3): δ 7.66 (d, J=15.6Hz, 1H), 7.39 (d,
J=15.6Hz, 1H), 6.91-6.94 (m, 1H), 6.77(s, 2H), 6.02 (m, 1H), 4.02 (t, J=6.6Hz, 2H), 3.96 (t,
J=6.6Hz, 2H), 3.85 (s, 6H), 2.45-2.47 (m, 2H), 2.19 (t, J= 7.8Hz, 2H), 1.70-1.75 (m, 2H), 1.56-
1.61(m, 2H), 1.48-1.44 (m, 2H), 1.42 (s, 9H), 1.32-1.37 (m, 2H). 13C NMR (125 MHz, CDCl3): δ
173.24, 168.90, 165.84, 153.55, 145.49, 143.94, 139.31, 130.37, 125.83, 120.83, 105.55, 79.90,
73.46, 56.15, 41.63, 35.56, 29.91, 28.86, 28.10, 25.52, 25.07, 24.79. HRMS (ESI): calcd. for
[C27H37NO7+Na]+, 510.2468; found, 510.2455.
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(E)-tert-Butyl 7-(4-(3-(3-chloro-2-oxo-5,6-dihydropyridin-1(2H)-yl)-3-oxoprop-1-enyl)-
2,6-dimethoxyphenoxy)heptanoate (16). Compound 16 was synthesized from compounds 9
(360mg, 0.88mmol) and 12 (139mg, 1.1mmol) following the procedure described for compound
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15, except 9 was activated using pivaloyl chloride (0.18mL, 0.97mmol) in the presence of TEA
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(0.24mL, 1.8mmol) in THF (5mL) at -10°C. Flash chromatography (Hex/EA, 4:1) purification
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afforded product as colorless oil (150mg, oil, 33%). H NMR (600 MHz, CDCl3): δ 7.68 (d,
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J=15.0Hz, 1H), 7.38 (d, J=15.6Hz, 1H), 7.07 (t, J=4.8Hz, 1H), 6.77 (s, 2H), 4.06 (t, J=6.6Hz,
2H), 3.99 (t, J=6.6Hz, 2H), 3.85 (s, 6H), 2.53-2.56 (m, 2H), 2.19 (t, J=7.2Hz, 2H), 1.71-1.74 (m,
2H), 1.57-1.59 (m, 2H), 1.43-1.63 (m, 2H), 1.41 (s, 9H), 1.33-1.36 (m, 2H). 13C NMR (125 MHz,
CDCl3): δ 173.21, 168.50, 161.37, 153.59, 145.12, 141.10, 139.58, 130.09, 128.20, 119.99,
105.69, 79.89, 73.46, 56.20, 41.77, 35.55, 29.90, 28.85, 28.09, 25.51, 25.28, 25.05. HRMS (ESI):
calcd. for [C27H36ClNO7+Na]+, 544.2078; found, 544.2054.
(E)-tert-Butyl
7-(2,6-dimethoxy-4-(3-(4-methyl-2-oxo-5,6-dihydropyridin-1(2H)-yl)-3-
oxoprop-1-enyl)phenoxy)heptanoate (17). Compound 17 was synthesized from compounds 9 and
13 following the procedure described for compound 16. 9 (141mg, 0.34mmol) was activated
using pivaloyl chloride (0.068mL, 0.37mmol, 1.1 eq) in the presence of TEA (0.09mL, 2eq) in
THF at -10°C. Compound 13 (44mg, 0.4mmol) was deprotonated by using n-BuLi (2eq) in THF
and then reacted with activated 9 to afford product 17 (24mg, 23%). 1H NMR (600 MHz, CDCl3):
δ7.63 (d, J=15.6Hz, 1H), 7.40 (d, J=15.6Hz, 1H), 6.76 (s, 2H), 5.81 (d, J=1.2Hz, 1H), 4.00 (t,
J=6.6Hz, 2H), 3.96 (t, J=6.6Hz, 2H), 3.84(s, 6H), 2.37 (d, J=6.6Hz, 2H), 2.19 (t, J=7.2Hz, 2H),
1.99 (s, 3H), 1.72 (m, 2H), 1.58 (m, 2H), 1.43-1.48 (m, 2H), 1.41 (s, 9H), 1.33-1.36 (m, 2H). 13C
NMR (125 MHz, CDCl3): δ173.25, 168.87, 166.09, 157.85, 153.54, 143.61, 139.21, 130.46,
121.29, 120.97, 105.50, 79.89, 73.44, 56.13, 41.53, 35.55, 29.90, 29.89, 28.84, 28.09, 25.50,
25.05, 22.97. HRMS (ESI): calcd for [C28H39NO7+H]+, 502.2805; found, 502.2807.
(E)-tert-Butyl
7-(2,6-dimethoxy-4-(3-oxo-3-(2-oxopiperidin-1-yl)prop-1-enyl)phenoxy)
heptanoate (18). Compound 18 was synthesized from compounds 9 and 14 following the
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procedure described for compound 16. 9 (900mg, 2.21mmol) was activated using pivaloyl
chloride (0.44mL, 2.43mmol, 1.1 eq) in the presence of TEA (0.6mL, 2eq) in THF at -10°C.
Compound 14 (263mg, 2.65mmol) was deprotonated by using n-BuLi (2eq) in THF and then
reacted with activated 9 to afford product 18 (760mg, 75%). 1H NMR (600 MHz, CDCl3): δ 7.59
(d, J=15.0Hz, 1H), 7.31 (d, J=15.6Hz, 1H), 6.74 (s, 2H), 3.95 (t, J=6.6Hz, 2H), 3.83(s, 6H), 3.76
(t, J=4.8Hz, 2H), 2.57(t, J=6.6Hz, 2H), 2.18 (t, J=7.2Hz, 2H), 1.84-1.86 (m, 4H), 1.69-1.72 (m,
2H), 1.55-1.58 (m, 2H), 1.42-1.46 (m, 2H), 1.40 (s, 9H), 1.31-1.35 (m, 2H). 13C NMR (125 MHz,
CDCl3): δ173.86, 173.21, 169.59, 153.53, 143.53, 139.23, 130.39, 121.07, 105.48, 79.87, 73.42,
56.12, 44.60, 35.53, 34.92, 29.88, 28.84, 28.08, 25.50, 25.04, 22.54, 20.60. HRMS (ESI): calcd.
for [C27H39NO7+Na]+, 512.2624; found, 512.2625.
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tert-Butyl
7-(2,6-dimethoxy-4-(6-oxo-1,2,3,6-tetrahydropyridine-1-carbonyl)phenoxy)
heptanoate (19). Compound 19 was synthesized from compounds 10 and 11 following the
procedure described for compound 16. 10 (1.0g, 2.62mmol) was activated using pivaloyl
chloride (0.52mL, 2.88mmol, 1.1 eq) in the presence of TEA (0.72mL, 2eq) in THF at -10°C.
Compound 11 (305mg, 3.14mmol) was deprotonated by using n-BuLi (2eq) in THF and then
reacted with activated 10 to afford product 19 (340mg, 28%).1H NMR (600 MHz, CDCl3): δ
6.96-6.99 (m, 1H), 6.85 (s, 2H), 5.98-6.00 (m,1H), 4.01 (t, J=6.6Hz, 2H), 3.96 (t, J=6.6Hz, 2H),
3.84 (s, 6H), 2.59-2.61 (m, 2H), 2.22 (t, J=7.8Hz, 2H), 1.73-1.77 (m, 2H), 1.58-1.63 (m, 2H),
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1.45-1.49 (m, 2H), 1.44 (s, 9H), 1.33-1.39 (m, 2H). C NMR (125 MHz, CDCl3): δ 173.64,
173.22, 165.56, 152.96, 145.48, 140.82, 130.66, 125.71, 106.24, 79.87, 73.39, 56.18, 43.72,
35.54, 29.91, 28.84, 28.08, 25.49, 25.05, 24.88. HRMS (ESI): calcd for [C25H35NO7+H]+,
462.2492; found, 462.2491.
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tert-Butyl
7-(4-(5-chloro-6-oxo-1,2,3,6-tetrahydropyridine-1-carbonyl)-2,6-dimethoxy
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phenoxy)heptanoate (20). Compound 20 was synthesized from compounds 10 and 12 following
the procedure described for compound 16. 10 (1.0g, 2.62mmol) was activated using pivaloyl
chloride (0.52mL, 2.88mmol, 1.1 eq) in the presence of TEA (0.72mL, 2eq) in THF at -10°C.
Compound 12 (413mg, 3.14mmol) was deprotonated by using n-BuLi (1.5eq) in THF and then
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reacted with activated 10 to afford product 20 (350mg, 27%). H NMR (600 MHz, CDCl3): δ
7.11 (t, J=4.2Hz, 1H), 6.83 (s, 2H), 3.99-4.02 (m, 4H), 3.84 (s, 6H), 2.67-2.70 (m, 2H), 2.22 (t,
J=7.8Hz, 2H), 1.73-1.77 (m, 2H), 1.58-1.63 (m, 2H), 1.46-1.48 (m, 2H), 1.44 (s, 9H), 1.34-1.39
(m, 2H). 13C NMR (125 MHz, CDCl3): δ 173.25, 173.15, 161.18, 153.07, 141.19, 140.88, 130.02,
127.75, 106.28, 79.90, 73.47, 56.26, 43.90, 35.55, 29.92, 28.84, 28.09, 25.49, 25.42, 25.06.
HRMS (ESI): calcd. for [C25H34ClNO7+Na]+, 518.1922; found, 518.1922.
(E)-7-(2,6-Dimethoxy-4-(3-oxo-3-(2-oxo-5,6-dihydropyridin-1(2H)-yl)prop-1-enyl)
phenoxy)heptanoic acid (21). Compound 15 (350 mg, 0.72 mmol) was dissolved in a mixture of
DCM/TFA (10 mL/1 mL) and the reaction mixture was stirred at room temperature overnight.
Upon completion shown by TLC, the reaction mixture was diluted with DCM and washed with
water and brine, and dried over anhydrous Na2SO4. Evaporation of solvent afforded product as
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yellow oil (311 mg, quantitative yield) which was used in the next step without purification. H
NMR (600 MHz, CDCl3): δ 7.67 (d, J=15.6Hz, 1H), 7.41 (d, J=15.0, 1H), 6.93-6.95 (m, 1H),
6.79 (s, 2H), 6.04-6.06 (m, 1H), 4.04 (t, J=6.6Hz, 2H), 3.99 (t, J=6.6Hz, 2H), 3.87 (s, 6H), 2.47-
2.49 (m, 2H), 2.36 (t, J=7.2Hz, 2H), 1.73-1.76 (m, 2H), 1.64-1.67 (m, 2H), 1.47-1.51 (m, 2H),
1.39-1.43 (m, 2H).
(E)-7-(4-(3-(3-Chloro-2-oxo-5,6-dihydropyridin-1(2H)-yl)-3-oxoprop-1-enyl)-2,6-
dimethoxyphenoxy)heptanoic acid (22). Compound 22 was synthesized from 16 following the
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DCM (5mL) followed by the addition of TFA (1mL) to afford compound 22 (110mg, 95%). H
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NMR (600 MHz, CDCl3): δ 7.69 (d, J=15.6Hz, 1H), 7.39 (d, J=15.0Hz, 1H), 7.08 (t, J=4.8Hz,
1H), 6.78 (s, 2H), 4.07 (t, J=6.6Hz, 2H), 3.98 (t, J=6.6Hz, 2H), 3.85 (s, 6H), 2.54-2.57 (m, 2H),
2.35 (t, J=7.2Hz, 2H), 1.71-1.76 (m, 2H), 1.62-1.67 (m, 2H), 1.45-1.50 (m, 2H), 1.36-1.41 (m,
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2H). C NMR (125 MHz, CDCl3): δ 179.33, 168.59, 161.41, 153.57, 145.19, 141.15, 139.51,
130.12, 128.19, 119.98, 105.70, 73.38, 56.20, 41.79, 35.85, 29.84, 28.78, 25.44, 25.29, 24.61.
7-(2,6-Dimethoxy-4-(6-oxo-1,2,3,6-tetrahydropyridine-1-carbonyl)phenoxy)heptanoic
acid (23). Compound 23 was synthesized from 19 following the procedure described for
compound 21. Compound 19 (340mg, 0.74mmol) was dissolved in DCM (10mL) followed by
the addition of TFA (2mL) to afford compound 23 (242mg, 81%). 1H NMR (600 MHz, CDCl3):
δ 6.99-7.02 (m, 1H), 6.84 (s, 2H), 6.00-6.03 (m, 1H), 4.03 (t, J=6.6Hz, 2H), 3.97 (t, J=6.6Hz,
2H), 3.84 (s, 6H), 2.60-2.63 (m, 2H), 2.36 (t, J=7.2Hz, 2H), 1.73-1.77 (m, 2H), 1.63-1.68 (m,
2H), 1.46-1.51 (m, 2H), 1.36-1.41 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 179.71, 173.77,
166.01, 152.94, 145.99, 140.88, 130.44, 124.97, 106.29, 73.35, 56.19, 43.83, 33.89, 29.80, 28.69,
25.36, 24.85, 24.59.
7-(4-(5-Chloro-6-oxo-1,2,3,6-tetrahydropyridine-1-carbonyl)-2,6-dimethoxyphenoxy)
heptanoic acid (24). Compound 24 was synthesized from 20 following the procedure described
for compound 21. Compound 20 (310mg, 0.63mmol) was dissolved in DCM (10mL) followed
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by the addition of TFA (1.5 mL) to afford compound 24 (275mg, quantitative yield). H NMR
(600 MHz, CDCl3): δ 7.09 (t, J=4.2Hz, 1H), 6.80 (s, 2H), 4.00 (t, J=6.6Hz, 2H), 3.98 (t, J=6.6Hz,
2H), 3.81 (s, 6H), 2.65-2.68 (m, 2H), 2.35 (t, J=7.2Hz, 2H), 1.70-1.75 (m, 2H), 1.61-1.66 (m,
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2H), 1.43-1.48 (m, 2H), 1.34-1.39 (m, 2H). C NMR (125 MHz, CDCl3): δ 180.10, 173.35,
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161.43, 153.01, 141.28, 141.04, 129.97, 127.59, 106.29, 73.47, 56.26, 43.98, 33.89, 29.76, 28.67,
25.37, 25.34, 24.55.
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(E)-7-(2,6-Dimethoxy-4-(3-(4-methyl-2-oxo-5,6-dihydropyridin-1(2H)-yl)-3-oxoprop-1-
enyl)phenoxy)heptanoic acid (25). Compound 25 was synthesized from 17 following the
procedure described for compound 21. Compound 17 (136mg, 0.27mmol) was dissolved in
DCM (10mL) followed by the addition of TFA (1.5 mL) to afford compound 25 (120mg,
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quantitative yield). H NMR (600 MHz, CDCl3): δ 7.63 (d, J=15.6Hz, 1H), 7.37 (d, J=15.6Hz,
1H), 6.77(s, 2H), 5.84 (s, 1H), 3.96-4.00(m, 4H), 3.83(s, 6H), 2.39(t, J=6Hz, 2H), 2.33(t,
J=7.2Hz, 2H), 2.00(s, 3H), 1.69-1.74(m, 2H), 1.61-1.65(m, 2H), 1.43-1.48(m, 2H), 1.34-1.39(m,
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2H). C NMR (125 MHz, CDCl3): δ 179.72, 169.28, 166.85, 159.21, 153.49, 144.23, 139.15,
130.37, 120.78, 120.71, 105.58, 73.41, 56.09, 41.72, 33.90, 29.85, 29.76, 28.74, 25.40, 24.58,
23.03.
(E)-7-(2,6-Dimethoxy-4-(3-oxo-3-(2-oxopiperidin-1-yl)prop-1-enyl)phenoxy)heptanoic
acid (26). Compound 26 was synthesized from 18 following the procedure described for
compound 21. Compound 18 (400mg, 0.82mmol) was dissolved in DCM (10mL) followed by
the addition of TFA (2.0 mL) to afford compound 26 (354mg, quantitative yield). 1H NMR (600
MHz, CDCl3): δ 7.61 (d, J=15.6Hz, 1H), 7.31 (d, J=15.6Hz, 1H), 6.76 (s, 2H), 3.97 (t, J=6.6Hz,
2H), 3.84 (s, 6H), 3.76 (t, J=6.0Hz, 2H), 2.59 (t, J=6.6Hz, 2H), 2.34 (t, J=7.2Hz, 2H), 1.84-1.87
(m, 4H), 1.70-1.75 (m, 2H), 1.61-1.66 (m, 2H), 1.44-1.49 (m, 2H), 1.35-1.40 (m, 2H). 13C NMR
(125 MHz, CDCl3): δ179.54, 174.14, 169.71, 153.52, 143.71, 139.20, 130.39, 121.07, 105.51,
73.34, 56.13, 44.68, 34.88, 33.90, 29.82, 28.77, 25.44, 24.60, 22.52, 20.57.
(E)-Methyl
7-(2,6-Dimethoxy-4-(3-oxo-3-(2-oxo-5,6-dihydropyridin-1(2H)-yl)prop-1-
enyl)phenoxy)heptanoate (27). Compound 21 was dissolved in diethyl ether/MeOH (6 mL/0.6
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mL) and trimethylsilyldiazomethane (2 M in diethyl ether, 0.18 mL, 1.2 eq) was added at 0°C.
The reaction mixture was stirred at room temperature overnight. Solvent was removed under
reduced pressure and the residue was re-dissolved in ethyl acetate, washed with water and brine,
dried over anhydrous Na2SO4. Crude product was purified by flash chromatography (Hex/EA,
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
1
2:1) to afford 27 (90 mg, yellow oil, 64%). H NMR (600 MHz, CDCl3): δ 7.62 (d, J=15.6Hz,
1H), 7.36(d, J=15.6Hz, 1H), 6.88-6.90(m, 1H), 6.75(s, 2H), 5.98 (m,1H), 3.98(t, J=6.6Hz, 2H),
3.93(t, J=6.6Hz, 2H), 3.82(s, 6H), 3.61(s, 3H), 2.41-2.44(m, 2H), 2.27(t, J=7.2Hz, 2H), 1.67-
1.72(m, 2H), 1.58-1.63(m, 2H), 1.41-1.46(m, 2H), 1.30-1.35(m, 2H).
13C NMR (125 MHz,
CDCl3): δ 174.20, 168.85, 165.82, 153.53, 145.53, 143.82, 139.25, 130.38, 125.76, 120.87,
105.51, 73.35, 56.12, 51.42, 41.61, 33.99, 29.86, 28.86, 25.46, 24.88, 24.77. HRMS (ESI): calcd.
for [C24H31NO7+H]+, 446.2179; found, 446. 2188.
(E)-Methyl 7-(4-(3-(3-chloro-2-oxo-5,6-dihydropyridin-1(2H)-yl)-3-oxoprop-1-enyl)-2,6-
dimethoxyphenoxy)heptanoate (28). Compound 28 was synthesized from 22 following the
procedure described for compound 27. Compound 22 (70mg, 0.15mmol) was reacted with
trimethylsilyldiazomethane (2M in diethyl ether, 0.13mL, 1.2eq) in a mixture of diethyl ether
and MeOH (5mL:0.5mL) to afford compound 28 (22mg, colorless oil, 31%). 1H NMR (600 MHz,
CDCl3): δ 7.70 (d, J=15.6Hz, 1H), 7.41(d, J=15.6Hz, 1H), 7.09(t, J=4.2Hz, 1H), 6.80(s, 2H),
4.09(t, J=6.0Hz, 2H), 3.99(t, J=6.0Hz, 2H), 3.88 (s, 6H), 3.67(s, 3H), 2.57(q, J=6.0Hz, 2H),
2.32(t, J=7.2Hz, 2H), 1.73-1.78(m, 2H), 1.63-1.68(m, 2H), 1.46-1.51(m, 2H), 1.35-1.40 (m, 2H).
13C NMR (125 MHz, CDCl3): δ 174.25, 168.51, 161.38, 153.58, 145.11, 141.11, 139.55, 130.11,
128.20, 120.00, 105.69, 73.40, 56.20, 51.45, 41.78, 34.02, 29.88, 28.88, 25.47, 25.29, 24.90.
HRMS (ESI): calcd. for [C24H30ClNO7+H]+, 480.1789; found, 480.1795.
29
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