Heck- And Suzuki-coupling approaches to novel hydroquinone inhibitors of calcium ATPase

Article abstract of DOI:10.3762/bjoc.15.94

In this study, we explored Heck- and Suzuki-coupling methodology to modify the template 2,5-di-tert-butylhydroquinone (BHQ, 2), an inhibitor of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). We found that by utilizing Suzuki coupling, we c

Full text of DOI:10.3762/bjoc.15.94

Heck- and Suzuki-coupling approaches to novel
hydroquinone inhibitors of calcium ATPase
Robert J. Kempton*1, Taylor A. Kidd-Kautz1, Soizic Laurenceau1 and Stefan Paula2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 971–975.
1Department of Chemistry and Biochemistry, Northern Kentucky
University, Nunn Drive, Highland Heights, KY 41099, USA and
2Department of Chemistry, Purdue University, Oval Drive, West
Lafayette, IN 47907, USA
doi:10.3762/bjoc.15.94
Received: 18 January 2019
Accepted: 15 April 2019
Published: 24 April 2019
Email:
Robert J. Kempton* - kempton@nku.edu
Associate Editor: I. R. Baxendale
* Corresponding author
© 2019 Kempton et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
calcium ATPase inhibitors; Heck- and Suzuki-coupling reactions;
hydroquinones; prostate cancer; tethered amino acid
Abstract
In this study, we explored Heck- and Suzuki-coupling methodology to modify the template 2,5-di-tert-butylhydroquinone (BHQ,
2), an inhibitor of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). We found that by utilizing Suzuki coupling,
we could successfully attach a six-carbon tether to BHQ that terminated in a leucine moiety to obtain target 14. Similar to related
compounds based on the structure of the natural product thapsigargin, 14 displayed inhibitory potency against SERCA activity.
This makes 14 a suitable candidate for the future attachment of a deactivating peptide to convey specificity for prostate cancer cells.
Introduction
Sarco/endoplasmic reticulum calcium ATPase (SERCA) is an making TG a highly potent but nonselective cytotoxic agent.
integral protein that resides in the membrane of the sarco- The problem of concomitant toxicity to healthy cells has been
plasmic reticulum (SR) within muscle cells. It transfers Ca2+ circumvented by attaching a short peptide (His-Ser-Ser-Lys-
from the cytosol to the lumen of the SR at the expense of ATP Leu-Gln-Leu) to a tether at TG’s C-8 position (1b). This modi-
hydrolysis. Specific inhibitors of SERCA are of significance to fication renders the inhibitor inactive [3]. Prostate cancer cells
human health because of their well-documented value as produce on their surface the serine protease PSA (prostate-spe-
research tools and their potential as novel anticancer agents cific antigen) that is capable of cleaving the peptide bond be-
[1,2]. The natural product thapsigargin (TG, 1a, Figure 1) is one tween Gln and Leu, thereby producing an active TG analogue
of the most frequently used SERCA inhibitors because of its that can enter the cancer cell and kill it by triggering apoptosis.
high specificity and potency. Both cancerous and healthy cells Apoptosis occurs as the result of elevated cytosolic calcium
undergo apoptosis after exposure to low concentrations of TG, levels, which are caused by inhibiting SERCA and preventing it
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Beilstein J. Org. Chem. 2019, 15, 971–975.
Figure 1: Thapsigargin- and hydroquinone-based SERCA inhibitors.
from loading intracellular calcium stores. No other major selective transesterification of the ester group at C-8 of TG,
proteases share the specificity of PSA, which prevents prema- replacing the natural butanoyl group at that position with an
ture inhibitor activation in healthy cells. Moreover, as PSA is ester moiety terminating in a primary amine.
deactivated by inhibitors present in the blood serum, potential
detrimental effects on other tissues are avoided. Compound 1b Here, we describe our efforts to use transition metal cross-cou-
was found to be selectively toxic to PSA-producing prostate pling reactions (Suzuki [10] and Heck [11]) to modify BHQ
cancer cells as well as in animal studies at submicromolar con- with a side chain terminating with a free primary amine that
centrations [3].
could serve as an attachment point for a peptide group (see 1b
above). We had anticipated that the availability and versatility
Unfortunately, TG has been difficult to obtain: extraction yields of cross-coupling reactions would allow for the introduction of
from natural sources are low and the total synthesis of TG re- side chains of varying lengths and functionalities [10]. In this
quired 42 synthetic steps [4]. More recently, two shorter, scal- paper, we report on the synthesis of 14, a BHQ analogue and
able total syntheses of TG have been reported [5,6].
active SERCA inhibitor containing a side chain terminating in a
leucine moiety.
As an alternative to TG, the structurally much less complex in-
hibitor 2,5-di-tert-butylhydroquinone (BHQ, 2, Figure 1) has a Results and Discussion
somewhat lower inhibitory potency than TG (400 nM versus The starting materials for the syntheses were the halides 4a,b
low nanomolar range), but offers advantages such as ease of (Scheme 1). Bromide 4a is commercially available. We investi-
synthesis and greatly reduced cost. Recent synthetic work from gated several ways to prepare the corresponding iodide 4b from
our group has shown that the tert-butyl groups of BHQ can be 1,4-dimethoxybenzene, among them iodine in the presence of
replaced by a variety of alkyl and cycloalkyl groups with trichlorocyanuric acid and silica gel [9], I2/periodic acid [10],
minimal reduction in potency [7-9]. In fact, the 2,5-bis(1- I2/silfen [11], N-iodosuccinimide [12], and potassium iodide/
methylcyclopentyl) analogue of BHQ 3 has an IC50 value of
500 nM, which is comparable to BHQ’s value of 400 nM.
Having available tethered SERCA inhibitors that are not based
on the structure of TG could be beneficial for future in vivo
trials because of the greater flexibility afforded by the struc-
turally less complex BHQ template. Furthermore, using a BHQ
template could be a valuable alternative to TG-based com-
pounds, should the latter encounter problems such as low
bioavailability or drug resistance.
The first step towards the development of BHQ-based com-
pounds of therapeutic value is the establishment of a synthetic
route to BHQ analogues with a tether that could serve as the
basis for future efforts aimed at the attachment of deactivating
peptides. The rationale is analogous to the Denmeade group’s
Scheme 1: Friedel–Crafts alkylation of 4.
strategy for circumventing TG’s toxicity to healthy cells by
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Beilstein J. Org. Chem. 2019, 15, 971–975.
potassium iodate [13]. In our hands the best yields of 4b were hydride by itself as well as with added aluminium chloride or
obtained using iodine in the presence of silfen [11]. Alkylation samarium iodide produced only trace amounts of an amine (e.g.,
of 4 under typical Friedel–Crafts conditions gave 5. In the case 9). When samarium iodide was used as the reagent, the only
of 5a, the 1H NMR of the crude product, while clearly showing discernible product (not fully characterized) appeared to be the
that it was the desired 4-substituted isomer (1H singlets at saturated derivative of 6 (2H triplets at δ 2.61 and 2.92;
δ 6.89 and 7.02), also exhibited an impurity (doublets at δ 6.83 J = 7.3 Hz in the 1H NMR spectrum; signal at 2245 cm−1 in the
and 7.12, J = 3.0 Hz) suggesting the presence of a small amount IR spectrum).
of the 3-tert-butyl isomer. The corresponding iodo analogue 5b
was prepared following the procedure of Hayashi [14]. Al- We next turned our attention to Suzuki coupling [10]. The reac-
though both halides 5a and 5b were now available to us, subse- tion of commercially available 5-hexenenitrile with 9-BBN-H,
quent experiments determined that the iodo analogue offered us followed by addition of bromide 5a, potassium phosphate and
no advantage over the more accessible bromo analogue 5a in PdCl2(dppf)2 in DMF at 85 °C for 3.5 h gave the coupled prod-
cross-coupling reactions.
uct 10 in 47–63% yields (Scheme 3). In contrast to the case of
the Heck coupling products (6 and 8, Scheme 2), nitrile 10
Heck cross coupling [15] of 4a and 5a with acrylonitrile in the underwent facile reduction with lithium aluminium hydride.
presence of Pd2(dba)3, [(t-Bu)3PH]BF4 and Cy2NCH3 in The product amine was not isolated. Instead, it was immedi-
dioxane, following the procedure of Fu [16,17], gave the ately reacted with the N-hydroxysuccinimidyl ester of Boc-
coupled products 6 and 8, respectively, in yields of 89% and leucine to give 12a in yields of 56–80% overall for two steps.
82%, respectively (Scheme 2). In both cases, the products were We also obtained the corresponding Z-protected analogue 12b
a mixture of E- and Z-isomers, with an E/Z ratio of ca. 85:15 in 43% overall yield for two steps. Both 10 and 12a could be
based on 1H NMR. The pure E-isomers could be obtained oxidized with CAN to the corresponding quinones 11 and 13,
by recrystallization from hexane. 1H NMR spectra of the recrys- respectively. Numerous unsuccessful attempts were made to
tallized products exhibited doublets at δ 6.03 and 7.61 convert the dimethoxy compound [12] or one of its precursors
(J = 16.9 Hz) and 6.01 and 7.57 (J = 17.0 Hz) for 6 and 8, re- into a hydroquinone (e.g., 14), either in one step (BBr3·S(CH3)2
spectively, confirming the E-stereochemistry. Attempts to [18] or (CH3)3SiCl/NaI [19]), or sequentially (CAN followed
convert the two methoxy groups of 6 and 8 to hydroxy groups by Na2S2O4 or H2/Pd). What finally proved effective was the
(e.g., 7) in a single step using BBr3·S(CH3)2 [18] or two step sequence of oxidation of 12a with CAN followed by
(CH3)3SiCl/NaI [19] were unsuccessful. Instead, 7 was ob- treatment of the resulting quinone 13 with sodium borohydride
tained in good yield by the two-step sequence of oxidation em- in dry methanol – a sequence that gave 14 (which upon standing
ploying ammonium cerium(IV) nitrate (CAN) [20] to afford the readily re-oxidizes back to 13) in 34% overall yield from 12a.
corresponding quinone (not shown), followed by reduction with
sodium hydrosulfite [21]. With the nitrile in hand, we next A standard coupled ATPase activity assay showed that 14 was
attempted to reduce that functional group to an amine to which an active SERCA inhibitor with an IC50 value of 23 ± 7 μM
a leucine moiety could be appended. Unfortunately, reactions of (4 trials). This observation demonstrated that despite their con-
6 and 8 utilizing catalytic hydrogenation, lithium aluminium siderable size in relation to the active BHQ entity, the tether and
Scheme 2: Heck cross-coupling reactions.
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Beilstein J. Org. Chem. 2019, 15, 971–975.
Scheme 3: Suzuki approach to a tethered hydroquinone.
the protected leucine residue did not abolish inhibitory activity, cytosolic calcium levels and on the viability of both cancer and
although they caused an approximately 50-fold reduction in po- healthy cells.
tency. This finding was significant because cleavage of a
peptide carrier by PSA would result in a compound structurally
Supporting Information
similar to 14 (its deprotected version) that needs to be an active
SERCA inhibitor to be of use against prostate cancer cells.
Supporting Information File 1
Even though the Boc group of 14 would not be present in the
final cleavage product, it has been demonstrated for TG-based
inhibitors that its presence did not affect inhibitory potency
[22]. In support of this observation, X-ray crystallography
revealed the location of the Boc group in a solvent-exposed area
on the surface of SERCA where it did not undergo major inter-
actions with the enzyme [22]. Therefore, conducting the inhibi-
tion assay with the Boc-protected BHQ derivative facilitates a
convenient direct comparison with inhibition results for TG-an-
alogues that have been characterized with Boc groups present
[23].
Experimental.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-15-94-S1.pdf]
Supporting Information File 2
NMR spectra.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-15-94-S2.pdf]
Acknowledgements
This work was supported in part by grants from the National
Conclusion
Institutes of Health (Institutional Development Award
Using Heck- and Suzuki-coupling reactions, we have de- 5P20GM103436 and Academic Research Enhancement Award
veloped a synthetic route that provides a BHQ template teth- 1R15GM084431-02 to S.P.). S.L. would like to thank Northern
ered to a leucine residue (14). Similar to its TG-based counter- Kentucky University's STEM International Research & Schol-
parts, 14 is an active SERCA inhibitor, which is a requirement arly Exchange Program for its financial support.
for its potential use as an antiprostate cancer agent. To achieve
ORCID® iDs
Robert J. Kempton - https://orcid.org/0000-0003-3631-7395
the latter, the leucine residue will need to be extended to yield
the full-length His-Ser-Ser-Lys-Leu-Gln-Leu peptide which can
Soizic Laurenceau - https://orcid.org/0000-0002-5893-0919
serve as a substrate for PSA. In future work, such a compound
Stefan Paula - https://orcid.org/0000-0003-4100-1467
would need to be evaluated in living cells to assess its effects on
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Beilstein J. Org. Chem. 2019, 15, 971–975.
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