Advance of Seriniquinone Analogues as Melanoma Agents

Article abstract of DOI:10.1021/acsmedchemlett.8b00391

Seriniquinone, a marine natural product, displayed potent cytotoxicity and selectivity against melanoma cancer cells. This selectivity, combined with a novel mode of action (MOA), prompted studies to translate a pharmacologically relevant lead. Herein, we report on structure-activity relationships (SARs), and provide a strategy to prepare analogues that retain activity and offer an improved water solubility and isomeric purity. From intermediates made on a gram-scale, derivatives were prepared and evaluated for their antiproliferation activity and melanoma selectivity. Overall these studies provide methods to install side chain motifs that demonstrate a common, and yet unique, biological profile.

Full text of DOI:10.1021/acsmedchemlett.8b00391

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Advance of Seriniquinone Analogues as Melanoma Agents
Justin C. Hammons,† Lynnie Trzoss,† Paula C. Jimenez,‡ Amanda S. Hirata,§ Leticia V. Costa-Lotufo,§
James J. La Clair,*,∥ and William Fenical*,†
†Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La
Jolla, California 92093-0204, United States
‡Instituto do Mar, Universidade Federal de Sao
̃
Paulo, Santos, Sao
̃
Paulo 11070-100, Brazil
Paulo, Sao Paulo 05508-900, Brazil
§
̂
́
Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao
∥Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California
92093-0358, United States
̃
̃
̃
S
* Supporting Information
ABSTRACT: Seriniquinone, a marine natural product, displayed potent cytotoxicity and selectivity against melanoma cancer
cells. This selectivity, combined with a novel mode of action (MOA), prompted studies to translate a pharmacologically relevant
lead. Herein, we report on structure−activity relationships (SARs), and provide a strategy to prepare analogues that retain
activity and offer an improved water solubility and isomeric purity. From intermediates made on a gram-scale, derivatives were
prepared and evaluated for their antiproliferation activity and melanoma selectivity. Overall these studies provide methods to
install side chain motifs that demonstrate a common, and yet unique, biological profile.
KEYWORDS: Drug discovery, melanoma, marine natural products, medicinal chemistry, seriniquinone, dermcidin
alignant melanoma, classified by genetic defects within
treatment options for cancer. Seriniquinone (1), a marine
Mpigment-producing melanocytes, is attributed to the natural product isolated from a rare Gm+ (Gram-positive)
largest number of skin-related cancer deaths.1,2 Its incidence
bacterium, demonstrated potent activity and selectivity toward
melanoma cell lines.11 Preliminary cancer biology studies
and mortality have been increasing steadily over the last 50
indicated that 1 (Figure 2) inhibits cell proliferation marked by
autophagocytosis and induces cell death due to caspase-9
dependent apoptosis. Furthermore, mode of action studies
suggested that 1 targets dermcidin (DCD),11 a small protein
whose role in cancer proliferation has been recently
recognized.12,13 To the best of our knowledge, no other
small molecule has been demonstrated to bind this protein.
Thus, the investigations into 1 would facilitate better
understanding of the biological function and significance of
dermcidin. Combined, these findings suggest 1 as a potential
new pharmacophore against melanoma. However, the trans-
lation of 1 into a chemotherapeutic agent is complex. It
requires significant medicinal chemistry efforts to generate a
large number of derivatives to improve its “druglike”
years. The unique epidemiological concerns for the generation
of such tumors and the lack of curative treatment options place
melanoma as one of the most dangerous cancers.3,4 Once
melanoma metastasizes, prognosis is poor, and the therapeutic
options are limited. Although protective measures can be
taken, the aggressive and rapid metastatic properties of
melanoma-based tumors continue to challenge clinical treat-
ments. The discovery of prevalent proto-oncogene protein B-
raf (BRAF) mutations in at least 50% of melanoma tumors led
to the development of BRAF inhibitors.5 The recently
approved BRAF inhibitors (Figure 1) for the treatment of
metastatic melanoma have made a significant impact on patient
survival, though the results were shadowed by the appearance
of drug resistance.6,7 Thus, selective antimelanoma leads with
novel modes of action (MOAs) are still of great interest.8−10
Nature has provided a diverse cornucopia of bioactive
substructures and molecular scaffolds for the development of
new drugs, and these natural products continue to provide vital
Received: October 27, 2018
Accepted: January 29, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00391
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ACS Medicinal Chemistry Letters
Letter
Scheme 1. Synthesis of Core Analogues 2 and 3
Figure 1. Exemplary structures of FDA approved drugs for melanoma
including Dabrafenib (BRAF inhibitor),14 Vemurafenib (BRAF
inhibitor),15 Trametinib (Mitogen-activated protein kinase (MEK)
inhibitor),16 Cobimetinib (MEK inhibitor),17 and Dacarbazine (DNA
alkylator).18,19 Other agents (not shown) including Encorafenib
(BRAF inhibitor) and Binimetinib (MEK inhibitor) have ended phase
III, and NDA requests are filed.
Table 1. Amorphous Solubility (S) and Activity Data (IC50
a
Values)
IC50 values (μM)
S, μM
Malme-3M
SK-MEL-28
HCT-116
1
2
3
4
0.06
0.09
0.4
0.5
5.5
5.1
2.7
3.3
2.1
0.05
1.11
2.03
0.10
0.11
0.28
0.001
0.009
0.001
0.22
0.06
0.06
0.50
0.36
3.47
4.30
2.15
0.25
0.74
0.15
0.34
0.46
0.33
1.80
7.00
3.0
1.29
4.08
4.24
0.54
1.18
0.73
0.06
0.48
0.53
1.45
0.75
8.16
0.48
9a/9b
11a/11b
12a/12b
13a/13b
14a/14b
23
Figure 2. Structures of seriniquinone (1) and core analogues 2−4.
properties. Herein, we report the design and synthesis of
seriniquinone derivatives that aim to investigate their
structure−activity relationship (SAR) profile and provide a
solution to pharmacological optimization.
0.4
24.6
8.2
24
25
doxorubicin
A preliminary SAR study was initiated to identify the
pharmacophore of 1 (Figure 2). We began by preparing
furanyl- and pyrrolyl-derivatives 2 and 3, respectively, to
understand the role of the central core (Figure 2). As
illustrated in Scheme 1, the synthesis of 2 started from 5,20
which, upon treatment with aqueous NaOH solution, gave rise
to dihydroxylated 6. Compound 6 was then converted to 2
under acidic conditions. The preparation of 3 began with
treatment of 721 with ammonia followed by cyclization,
affording pyrrolyl-derivative 3. We also prepared quinoline
analogue 4 (Scheme S3) to explore the functional tolerance
within the aryl substituents.
Samples of 2−4 were screened across three cell lines (Table
1). Furan 2 was 3−22-fold less active than 1. The pyrrolyl-
analogue 3 proved to be even less active, while 4 displayed
activity comparable to or better than 1 (Table 1). While 1 was
readily accessed synthetically,11 its low solubility in both
aqueous and organic media directed our attention to explore
analogues with modifications in the aryl rings. Here, our goal
was to prepare agents with comparable activity profile to 1 but
with an improved solubility and associated pharmacological
properties.
a
The data for 9−12 was collected with a 2:1 mixture of regioisomers
a:b (Figure 3). S was determined at pH 6.5 for 1−4, pH 6.5 for 9a/
9b−14a/14b, or pH 7.2 for 23−25. IC50 values were reported as
averages and were within 5% deviation over two repetitions via the
MTT assay (see Table S1). Compound 9a/9b and 10a/10b would be
the same in buffer, and hence only one was screened.
Beginning with α-myrcene, we were able to access acid 9a as
a mixture with regioisomer 9b in gram-scale through a nine-
step sequence.11 Derivatization of this acid as a sodium salt
10a/10b or amides 11a/11b−14a/14b provided materials that
allowed us to confirm that the activity was maintained in terms
of both its potency and melanoma selectivity (Table 1). While
these materials provided improved solubility, the lack of an
effective procedure to separate isomers a and b (right, Figure
3) proved problematic. This was further complicated by the
inability to deliver a regioselective approach to either
regioisomer in 11−14. Based on these issues, we turned our
attention to explore an alternate route that would enable
preparation of a single regioisomer (a or b, Figure 3).
B
DOI: 10.1021/acsmedchemlett.8b00391
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with Pd(PPh3)4 and K2CO3 in warm MeOH, afforded 12.2 g of
23 in 54% yield from 21 as a light brown solid. This material
was then converted to carbamate 24 by treatment with n-
butylisocyanate in DMF containing EtNiPr2 and methyl ether
25 by methylation with MeI in DMF containing K2CO3.
Overall, this route provided effective access to the single
regioisomeric products 23−25.
We then assessed the activity of 23−25 and observed
comparable activity of 23 and 24 to compound 1 (Table 1;
Figure 4 for hydrolysis of 24 to 23) across three cell lines.
Figure 3. Structures of the synthetic analogues 9a/9b, acid salt 10a/
10b, and amides 11a/11b−14a/14b. These materials, prepared at
milligram-scale for testing, were prepared as inseparable mixtures of
regioisomers a and b.
Figure 4. Hydrolytic release. Carbamate 24 hydrolyses to phenol 23
in aqueous media. Samples from DMSO stocks at 1.0 mg/mL (A, B)
or 0.5 mg/mL (C, D) of 24 in PBS pH 7.2 undergo hydrolysis to
purple salt 23 when diluted 20-fold and stored for 24 h at 23 °C.
Our attention was drawn to commercially available 7-
methoxy-1-tetralone (15) as a vehicle to address the issue
associated with regioselectivity (Scheme 2). We began by
Scheme 2. Gram-Scale Synthesis of Analogues 23−25
While 25 was slightly less active to HCT-116 and SK-MEL-28,
all three compounds, 23, 24, and 25, shared similar enhanced
activity in the melanoma cell line Malme-3M. The solubility
displayed by 25 was comparable to that observed for 9a/9b−
14a/14b, while 24 was 5−10 times less soluble (Table 1).
Phenol 23, however, was insoluble in most media, similar to
the parent seriniquinone (1), indicating that side chain
derivatization was vital to compound solubility.
Next, we evaluated if 1, 9a/9b, and 23 displayed the DNA
binding and redox activity typically ascribed to quinones.22−24
Using the known DNA intercalator, doxorubicin, as a control
(Figure 5), the fluorescence from 1 did not attenuate upon
addition of ct-DNA, whereas doxorubicin demonstrated a
potent quench in fluorescence upon binding. While 9a/9b and
24 were less fluorescent than 1, they also did not interact with
DNA, as evident by the lack of attenuation (Figure 5).
Analogues 23−25 demonstrated remarkable stability in the
solid state, with >99% purity retained at 23 °C for 3 months. In
converting 6-methoxy-1-tetralone (15) to 17 by a two-step
sequence in 82% yield. Samples of 17 were then oxidized with
O2 in a mixture of tBuOK/tBuOH,22 and the resulting product
18 was coupled directly thereafter with 2,3-dichloronaphtha-
lene-1,4-dione (19). Pure 21 was then obtained by
chlorination of crude coupling product 20 followed by flash
chromatographic purification. In one batch, we were able to
convert 16.3 g (0.10 mol) of 17 to 27.7 g (63%) of 21 with 19
as the major impurity.
Dichloride 21 was then treated with Na2S in aq THF at
room temperature (rt) to yield 22, which, after deprotection
Figure 5. Fluorescence emission spectra upon titration of ct-DNA
into 20 μM doxorubicin, 60 μM 1, 60 μM 9a/9b, or 60 μM 24.
C
DOI: 10.1021/acsmedchemlett.8b00391
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Letter
PBS at pH 7.2, solutions of 23 and 25 remained at >98% purity
for 1 month at 23 °C and 48 h (limit of testing) at 37 °C in cell
culture media as determined by monitoring by NMR. On the
other hand, carbamate 24 underwent hydrolysis in aqueous
media, converting to a purple phenolate of 23, suggesting its
potential to provide a means to improve the pharmacological
properties of 24 through pro-drug optimization and activation
strategies.
solubility and comparable activity. Analogues with improved
solubility, while retaining the cytotoxicity and similar biological
profiles, were synthesized. Finally, a new route was presented
that provides a solution to the regioselectivity issue. Overall,
these studies suggest that it is feasible to design melanoma-
specific seriniquinone derivatives with druglike properties.
While carbamate 24 suggests the possibility of pro-drug design,
referring to it as a pro-drug would be premature without in
depth in vivo studies. Efforts are now underway to complete
pharmacokinetic/pharmacodynamic optimization and translate
a lead molecule with viable in vivo activity against melanoma.
Next, we explored the effects of 1, 23, and 24 on DCD
expression to validate that 23 and 24 target the same pathway
as 1.11 As shown in Figure 6, we observed a comparable
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00391.
Experimental methods, spectroscopic data, and copies of
1
select H and 13C NMR spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: jlaclair@ucsd.edu.
*E-mail: wfenical@ucsd.edu.
Figure 6. Relative DCD mRNA expression in HCT-116, Malme-3M,
and SK-MEL-28 cells treated with 1, 23, and 24. Concentrations were
given in HCT-116 by 1 μM (+) and 10 μM (++), and in Malme-3M
or SK-MEL-28 by 30 nM (+) and 100 nM (++). Doxorubicin (D; 0.5
μM) and DMSO (0.5%) were used as positive and negative controls,
respectively. Total RNA was extracted and subjected to quantitative
PCR analysis by a one-step RT-PCR system to detect the mRNA
expression of DCD and RPLPO (large ribosomal protein).
ORCID
James J. La Clair: 0000-0001-6500-4107
William Fenical: 0000-0002-8955-1735
Notes
The authors declare no competing financial interest.
increase in DCD expression in cells treated with 1 and phenol
23. Across all three cell lines, phenol 23 and carbamate 24
demonstrated comparable or improved induction of DCD
expression when compared to 1. This increase in DCD
expression (Figure 6) combined with activity data (Table 1)
indicates that 23 and 24 share similar modes of action as 1.11
Finally, to evaluate for “off-target” redox activity, we resorted
to the CellROX assay25,26 using HCT-116 cells. We did not
detect oxidative stress elicited by these materials, as indicated
by the lack of green fluorescent mitochondria due to the
generation of reactive oxygen species (Figure 7, Figure S6).
This, along with the lack of DNA binding (Figure 5), confirms
that 9a/9b and 24 do not act like quinones24 but, rather,
represent a unique DCD-targeting pharmacophore.
ACKNOWLEDGMENTS
■
This work was supported by the NIH, National Cancer
Institute under Grant CA 044848 to W.F., the Taubman
Foundation to W.F., and FAPESP Grant (processes 2015/
17117-6, 2016/10854-5, and 2018/07661-6) to L.V.C.-L. We
thank Drs. Yongxuan Su (UC San Diego) and Anthony Mrse
(UC San Diego) for assistance with mass spectral analyses and
NMR spectral data, respectively.
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