Upregulation of p53 through induction of MDM2 degradation: Amino acid prodrugs of anthraquinone analogs

Article abstract of DOI:10.1016/j.bmcl.2019.126786

Previously, we reported a class of MDM2-MDM4 dimerization inhibitors that upregulate p53 and showed potent anticancer activity in animal models. However, water solubility hinders their further development. Herein we describe our effort to develop a prodru

Full text of DOI:10.1016/j.bmcl.2019.126786

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Bioorganic & Medicinal Chemistry Letters
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Upregulation of p53 through induction of MDM2 degradation: Amino acid
prodrugs of anthraquinone analogs
Abiodun Anifowose1, Zhengnan Yuan1, Xiaoxiao Yang⁎, Zhixiang Pan, Yueqin Zheng,
Zhongwei Zhang, Binghe Wang⁎
Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Petit Science Center, 100 Piedmont Ave, Atlanta, GA 30303, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Previously, we reported a class of MDM2-MDM4 dimerization inhibitors that upregulate p53 and showed potent
anticancer activity in animal models. However, water solubility hinders their further development. Herein we
describe our effort to develop a prodrug approach that overcomes the solubility problem. The prodrug of BW-
AQ-238, a potent anthraquinone analog, was made by esterification of the hydroxyl group with various natural
amino acids. Cytotoxicity of these compounds toward Hela and EU-1 cells, their aqueous solubility, and the
release kinetics of these prodrugs in buffer and in the presence of hydrolytic enzymes were studied. The results
demonstrate that the amino acid prodrug approach significantly improved the water solubility while main-
taining the potency of the parent drug.
Anticancer agent
Anthraquinone
Amino acid prodrug
Esterase
Kallikrein
Introduction
xenograft model using our lead compound, BW-AQ-101 (Fig. 1).17
Since its discovery in 1979.1–3 the tumor suppressor protein p53 has
been firmly established as a critical transcription factor responsible for
the regulation of cell cycle, apoptosis, DNA repair, and senescence.4,5
Dysregulation of p53 functions takes on two forms: deletion or muta-
tion of p53, and overexpression of its inhibitory proteins.6,7 Approxi-
mately 50% of human cancer has p53 mutation, resulting in inactiva-
tion or loss of p53 protein functions.8 Even in cancer with wild-type
p53, its function is ultimately inhibited, leading to accelerated cancer
development and growth.6,9 The latter scenario is the result of the
overexpression of its negative regulator, the murine double minute 2
(MDM2) protein, which masks the p53 transactivation domain, im-
pairing nuclear import of the p53 protein, and catalyzes the ubiquiti-
nation, leading to the proteasomal degradation of the p53 protein.10,11
Activating p53 in cancer by either down-regulating MDM2 or inter-
cepting MDM2/p53 interaction has proven to be a promising approach
for cancer therapy. Therefore, many efforts have been devoted to tar-
geting the MDM2-p53 interaction by small molecules.12–15 and pep-
tides.16 for the reactivation of p53 for the treatment of cancer that re-
tains wild-type p53.
Complete remission was achieved after treatment with 20 mg/kg/day
three doses per week for two weeks followed by 150 days of observa-
tion; whereas all control mice died within 45 days as expected. Pre-
liminary results also showed minimal or no toxicity to normal cells at
therapeutic concentration. Studies in the EU-1 ALL cells revealed that
BW-AQ-101 inhibited MDM2 but not MDM4, induced activation of
p53, and its target proteins p21 and PUMA in a dose- and time-de-
pendent manner.
The results thus far clearly demonstrated the emerging potential of
this class of anthraquinones. However, the poor water solubility of the
active anthraquinone analogs posed a serious developability problem.
Thus, we were interested in examining the possibility of using a pro-
drug strategy to solve this solubility problem. Amino acids have been
widely used as a prodrug strategy to improve the delivery of those with
poor solubility and permeability.20–22 Some amino acid prodrugs were
also shown to enable transporter-mediated drug transport in intestinal
epithelial cells.23 Herein, we report the development of amino acid-
based prodrugs for this class of anthraquinones. These prodrugs were
found to have improved water solubility and release the parent drug
under near physiologic conditions upon esterase-mediated hydrolysis
(porcine liver esterase and porcine pancreas kallikrein). The cytotoxi-
city of these prodrugs was evaluated by the water-soluble tetrazolium
salt (WST) assay in Hela and EU-1 cell lines. The valine prodrug was
Along this line, our lab has synthesized a series of anthraquinone-
based compounds capable of inducing MDM2 degradation, p53 upre-
gulation, and thus apoptosis.17–19 The efficacy of such compounds has
been proven in treating acute lymphocytic leukemia (ALL) in a mouse
⁎ Corresponding authors.
E-mail addresses: xyang20@gsu.edu (X. Yang), wang@gsu.edu (B. Wang).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2019.126786
Received 25 July 2019; Received in revised form 22 October 2019; Accepted 24 October 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:AbiodunAnifowose,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.126786

A. Anifowose, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 1. Lead compound derived from Rhein.
Fig. 2. Western blot showed the downregulation of MDM2 and upregulation
p53 by BW-AQ-238 in dosage- (left panel) and time-dependent (right panel)
manner in EU-1 leukemia cells. GAPDH was blotted as the loading control.
found to have comparable potency along with significantly increased
aqueous solubility.
In order to prepare ester prodrugs of the parent drug, a hydroxyl
handle is needed. BW-AQ-101 has two phenolic hydroxyl groups,
which could conceivably be used for prodrug derivatization. However,
phenol esters are generally not stable. Therefore we designed and
synthesized a new analog by installing a 2-hydroxyl ethoxy group on
each of the phenolic hydroxyl positions to afford BW-AQ-238. This
design was based on our earlier findings that the phenolic hydroxyl
groups can be modified with alkyls, alkyxoyalkyl groups without
abolishing the activity of BW-AQ-101.19 The two hydroxyl groups are
intended as sites for prodrug derivatization.
dependent manner and therefore upregulated p53 level in EU-1 cells
(Fig. 2). Specifically, at the 15-h time point, BW-AQ-238 at a dosage of
0.8 μM significantly down-regulated cytoplasmic MDM2 and increased
the p53 level. Down-regulation of MDM2 could be detected after 4-h
treatment with 1 μM BW-AQ-238 and proceeded to 16 h; accordingly,
the p53 level was elevated after 4 h of treatment and maintained at a
high level after 8 h. Such results are consistent with that of the lead
compound BW-AQ-101.17 and the preliminary SAR result we re-
ported.19
The synthesis of BW-AQ-238 started with the methyl ester of rhein
(1),18 followed by the installation of the tert-butyldimethylsilyl (TBS)
protected ethyl alcohol using potassium carbonate as a base and po-
tassium iodide as a catalyst to obtain compound 2. Hydrolysis of the
ester in compound 2 using lithium hydroxide in water afforded the
carboxylic acid derivative (3) in about 65% yield. The resulting car-
boxylic acid group was converted to an azido group using a literature
procedure to afford compound 4. Then the azido moiety was subjected
to Curtius rearrangement to yield compound 5, which was converted to
the corresponding amide (6) through reaction with chloroacetyl
chloride. Deprotection of 6 with tetrabutylammonium fluoride (TBAF)
yielded compound 7 (BW-AQ-238) as a bright yellowish solid (Scheme
1, see Supporting Information for detail).
With BW-AQ-238 in hand, the preparation of water-soluble amino
acid was achieved by coupling with twelve amino acids choosing from
the simplest glycine to hydrophobic phenylalanine to arginine with an
ionizable side chain (Scheme 2, Table 1). First, coupling of compound 7
with the corresponding N-protected amino acid using 1-ethyl-3-(3-di-
methylaminopropyl)carbo-diimide (EDC) as a coupling reagent and 4-
dimethylamino pyridine (DMAP) as a catalyst in acetonitrile afforded
compounds 8a–l. Subsequent deprotection with 2 M hydrogen chloride
in diethyl ether using dichloromethane (DCM) as a co-solvent led to the
formation of corresponding hydrogen chloride salts of amino acid
prodrugs, 9a–l, in quantitative yield.
One requirement for the success of a prodrug strategy is the ability
to release the parent drug under physiologic conditions. Therefore, we
examined the chemical and enzyme-mediated hydrolysis of 9a–l in PBS
at pH 7.4 and 37 °C using HPLC. Specifically, porcine liver esterase
(PLE) and porcine pancreas kallikrein (PPK) were chosen as model
enzymes for the parent drug release studies. PLE has been extensively
used by others24 and us25–27 in the study of esterase-sensitive prodrugs.
PPK was used in this study to resemble human tissue kallikrein (KLKs).
KLKs is a class of serine protease with diverse roles in a variety of
human tissues and is found to be upregulated in several cancers in-
cluding prostate, ovarian, and breast. Several KLKs members have been
identified as cancer markers including prostate and breast tissues.28
Among them, human glandular kallikrein 2 has been used to develop a
peptide-based prodrug targeting prostate cancer.29 Since PPK has been
found to be able to hydrolyze the amino acid ester,30 it was included in
this study to explore other specific metabolic release of the prodrug
besides esterase. The chemical hydrolysis stability and enzyme specific
release of the prodrugs were examined in PBS solution by monitoring
the formation of BW-AQ-238 and the disappearance of the respective
prodrug. The metabolic profiles are shown in Figs. S1–S3 (Supporting
information), and the half-life values are shown in Table 2. In PBS so-
lution the ester bond of the prodrug can be chemically hydrolyzed. The
release rate was relatively slow except for 9a with the least steric
HeLa cell line and an in-house established wild-type p53 leukemia
cell line (EU-1)17 were used for cytotoxicity assessment using the WST
assay. The IC50 values of BW-AQ-238 were determined to be 3.76 µM
and 0.89 µM in HeLa and EU-1 leukemia cells, respectively (Table 3, see
Supporting information for detail). In comparison, IC50 values of BW-
AQ-101 were determined to be 2.21 µM and 0.83 µM in HeLa and EU-1
leukemia cells, respectively. Western blot studies also showed that BW-
AQ-238 induced downregulation of MDM2 in a dose- and time-
Scheme 1. Synthesis of BW-AQ-238. a) (TBS)OC2H4Cl, K2CO3, KI, DMF,
120 °C, 4 h; b) LiOH in H2O, THF, r.t., 2 h; c) DPPA, TEA, DMF, r.t., 2 h; d) (i)
dioxane, reflux, 2 h (ii) H2O, 60 °C, 1 h; e) chloroacetyl chloride, dioxane, r.t., 5
mins; g) TBAF, THF, r.t., 4 h.
Scheme 2. Synthesis of 9 a–l. a) N-Boc protected amino acids, EDC, DMAP,
ACN, 0–21 °C, 4–6 h; b) 2 M HCl in diethyl ether, DCM, 0 °C-r.t.
2

A. Anifowose, et al.
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Table 1
Table 3
Prodrug intermediates and the amino acid prodrugs.a
Cytotoxicity of amino acid prodrugs.
R1
R2
Compound No.
Amino Acid
IC50 (μM)a
HeLa cell
8a
8b
8c
8d
8e
8f
Boc-Gly-
9a
9b
9c
9d
9e
9f
Gly-
Ala-
Val-
Leu-
Ile-
EU-1 ALL
Boc-Ala-
BW-AQ-238
–
3.76 (3.39–4.49)
3.66 (3.44–3.90)
8.21 (7.44–9.04)
4.15 (3.27–5.27)
2.80 (2.51–3.13)
5.75 (5.48–6.02)
5.64 (5.52–5.76)
5.40 (4.74–6.16)
10.7 (9.60–11.8)
13.0 (11.4–14.6)
12.3 (9.70–15.5)
9.11 (7.89–10.5)
7.07 (6.52–7.66)
0.89 (0.86–0.92)
1.10 (1.04–1.16)
1.66 (1.51–1.83)
1.08 (0.96–1.20)
1.17 (1.03–1.31)
1.14 (1.03–1.27)
1.23 (1.16–1.30)
1.27 (1.00–1.60)
1.28 (1.48–1.90)
2.07 (1.91–2.25)
1.45 (1.29–1.63)
3.21 (2.94–4.10)
1.24 (1.16–1.32)
Boc-Val-
9a
9b
9c
9d
9e
9f
Gly
Ala
Val
Leu
Ile
Boc-Leu-
Boc-Ile-
Boc-Phe-
Phe-
Tyr-
Lys-
Arg-
Gln-
Glu-
Met-
8g
8h
8i
Boc-Tyr(OtBu)-
Boc-Lys(Boc)-
Boc-Arg(Boc)-
Boc-Gln(Trt)-
Boc-Glu(tBu)-
Boc-Met-
9g
9h
9i
Phe
Tyr
Lys
Arg
Gln
Glu
Met
9g
9h
9i
8j
9j
8k
8l
9k
9l
9j
a
9k
9l
All amino acids are L-amino acids; ester bond was formed with carboxylic
acid group of the amino acid; detailed structure is listed in Supporting in-
formation (Table S1).
a
Experiment was done in triplicate and 95% confidence intervals of IC50 are
shown in parentheses.
Table 2
Prodrug degradation half-life.
greater than the solubility of parent drug BW-AQ-238 (60.0 µg/ml).
Comparing with the solubility result BW-AQ-101 (8.5 μg/ml), the im-
provement was even more striking (Table S2, Supporting information).
Therefore, it is reasonable to assume that the solubility of the prodrugs
was greatly improved.
No.
Half-life (mins)a
PBS (pH 7.4)
PLE
PPK
9a
9b
9c
9d
9e
9f
31.5 (26.1–39.6)
10.1 (9.2–11.1)
6.2 (5.9–6.5)
35.9 (35.5–36.3)
24.7 (24.1–25.2)
126.3 (120.9–132.3)
39.6 (34.6–46.2)
48.4 (44.7–52.8)
6.5 (6.1–6.8)
100.9 (93.8–109.1)
147.7 (87.4–123.5)
127.7 (73.4–111.5)
154.5 (123.4–206.5)
73.7 (64.8–85.5)
With success in demonstrating improved solubility and the ability to
release the parent drug, we were interested in examining whether the
prodrugs would be able to deliver a sufficient quantity of the parent
drug to cells for the desired cytotoxicity. Therefore, the cytotoxicity of
these prodrugs was tested in Hela and EU-1 leukemia cell lines using the
WST-8 assay. The IC50 values of these prodrugs ranged from 2.8 to
13 μM in Hela cells and 1.08 to 3.5 μM in EU-1 cells (Table 3). The IC50
of 9d in Hela cells is marginally lower than that of the parent. However,
the small difference makes it hard to interpret whether there is any
mechanistic significance.
25.3 (23.3–27.5)
2.5 (2.4–2.7)
24.3 (23.1–25.7)
1.0 (0.9–1.2)
9g
9h
9i
70.6 (60.3–85.1)
1.7 (1.6–1.7)
6.5 (6.3–6.8)
140.8 (132.8–149.8)
187.1 (176.8–198.6)
137.1 (127.7–148.2)
268.6 (264.4–272.9)
44.8 (41.2–48.9)
44.2 (42.9–45.6)
63.0 (61.1–65.0)
73.0 (71.5–74.5)
117.9 (116.2–119.6)
3.7 (3.3–4.2)
2.4 (2.3–2.5)
0.2 (0.1–0.3)
9j
66.9 (63.8–70.4)
110.0 (104.9–115.6)
12.0 (11.3–12.8)
9k
9l
a
Experiment was done in triplicate and 95% confidence intervals are shown
To further examine the prodrug activation in cell culture, com-
pounds 9a and 9k were selected to test the stability in the culture
media. Their half-lives turned out to be 19.3 and 156.1 min respectively
(Fig. S4), which are shorter than their chemical hydrolysis in PBS
(31.5 min for 9a and 268.6 min for 9k). Therefore, one can assume that
prodrug activation is due to the combined effect of enzymatic and
chemical hydrolysis, with enzymatic activation likely playing a domi-
nant role.
in parentheses.
hindrance with a glycine promoiety. This is consistent with reported
ester prodrugs.31 The hydrolysis rate was boosted by either PLE or
kallikrein, with half-lives ranging from 0.2 to 118 min. These results
clearly showed that the aromatic amino acid promoiety such as Phe and
Tyr are moret susceptible to PLE with half-lives within 2 min. PLE also
showed its preference to other hydrophobic amino acids, with the order
of decreasing stability being Val ≈ Ile > Gly > Ala > Met > Leu.
Such results are consistent with literature reports in the hydrophobic
substrate specificity of PLE.32 In the presence of kallikrein, due to its
trypsin-like nature, hydrolysis showed a strong preference for basic
amino acids with the Arg prodrug being hydrolyzed faster than the Lys
counterpart. In addition, cleavage of Phe and Tyr prodrug was also
found with the kallikrein treatment, suggesting a trypsin-like and
chymotrypsin-like dual substrate specificity.33 This could be further
verified by its moderate preference to the methionine prodrug 9l.
The key reason for the preparation of these prodrugs was to improve
solubility. For this, we assessed the water solubility of these prodrugs. It
is important to note that all the amino acid prodrugs could be easily
dissolved in pH 7.4 PBS at a concentration greater than 100 mg/ml,
indicating a significant improvement in solubility compared with the
lead compound BW-AQ-101 and the parent drug BW-AQ-238. In ad-
dition, according to the observations from several reported studies that
the prodrug with phenylalanine promoiety resulted in the lowest so-
lubility among other amino acid-based prodrugs,23 we chose the phe-
nylalanine prodrug 9f as a benchmark to test aqueous solubility and
quantitatively compare with its parent drug. Determined by an HPLC
method.34 (see Supplementary materials for detail), the water solubility
of phenylalanine prodrug was 703 mg/ml, which was over 11,000-fold
In EU-1 leukemia cells, the IC50 values are around 1 μM for most
prodrugs, which are comparable with that of the parent drug. In order
to study prodrug activation by cellular components, we used none-de-
natured cell lysate of EU-1 leukemia cells and the valine prodrug 9c as
an example. It was found that the ester bond was readily cleaved within
15 min to give the parent drug BW-AQ-238 (Fig. S5). This activation
was much faster than chemical hydrolysis (t1/2 of ~148 min) and PLE-
mediated degradation (t1/2 ~ 25.3 min). Such results suggest that the
activation of prodrug 9c can at least partially be attributed to the cy-
tosolic enzymatic degradation.
In conclusion, to address the poor solubility of anthraquinone-based
anti-leukemia agents, twelve amino acid prodrugs of BW-AQ-238 were
synthesized with remarkable improvement in aqueous solubility. The
chemical and enzymatic hydrolytic release of the parent drug from 9a–l
were determined by HPLC. Efficient release of the parent drug was
achieved with both PLE and PPK. For PLE, the aromatic amino acid
promoieties such as Phe and Tyr showed the fastest release rate fol-
lowed by the hydrophobic amino acid such as Leu and Met. On the
other hand, PPK favored the basic amino acids such as Arg and Lys. The
kallikrein metabolic profile also revealed that this peptidase could also
hydrolyze an ester bond. Both valine prodrug 9c and leucine prodrug
9d demonstrated comparable cytotoxicity with the parent drug in HeLa
and EU-1 cell lines. Given that valine has been successfully utilized as
3

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Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
the promoiety of anti-viral compound Zanamivir-L-Val35 and a mar-
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in-vivo anti-cancer therapeutic studies.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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Acknowledgments
19. Draganov AB, Yang X, Anifowose A, et al. Upregulation of p53 through induction of
MDM2 degradation: anthraquinone analogs. Bioorg Med Chem.
Financial support from the National Institutes of Health (CA180519)
is gratefully acknowledged. We thank the Center for Diagnostic and
Therapeutics for providing a CDT fellowship to Abiodun Anifowose and
the Molecular Basis of Disease Program for providing an MBD fellow-
ship to Zhixiang Pan.
2019;27(17):3860–3865.
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Appendix A. Supplementary data
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