Bridgehead Modifications of Englerin A Reduce TRPC4 Activity and Intravenous Toxicity but not Cell Growth Inhibition

Article abstract of DOI:10.1021/acsmedchemlett.0c00186

Modifications at the bridgehead position of englerin A were made to explore the effects of variation at this site on the molecule for biological activity, as judged by the NCI 60 screen, in which englerin A is highly potent and selective for renal cancer cells. Replacement of the isopropyl group by other, larger substituents yielded compounds which displayed excellent selectivity and potency comparable to the natural product. Selected compounds were also evaluated for their effect on the ion channel TRPC4 as well as for intravenous toxicity in mice, and these had lower potency in both assays compared to englerin A.

Full text of DOI:10.1021/acsmedchemlett.0c00186

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Letter
Bridgehead Modifications of Englerin A Reduce TRPC4
Activity and Intravenous Toxicity but not Cell Growth Inhibition
Zhenhua Wu, Jean-Simon Suppo, Sarka Tumova, Jonathan Strope, Fernando
Bravo, Melody Moy, Ethan S. Weinstein, Cody J. Peer, William D. Figg,
William J. Chain, Antonio M. Echavarren, David J. Beech, and John A. Beutler
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00186 • Publication Date (Web): 03 Aug 2020
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Bridgehead Modifications of Englerin A Reduce TRPC4 Activity and Intravenous
Toxicity but not Cell Growth Inhibition
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Zhenhua Wua,f, Jean-Simon Suppob,f, Sarka Tumovac,f, Jonathan Stroped,f, Fernando
Bravob, Melody Moya, Ethan S. Weinsteina, Cody J. Peerd, William D. Figgd, William J.
Chaina, Antonio M. Echavarrenb, David J. Beechc and John A. Beutlere*
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aDepartment of Chemistry & Biochemistry, University of Delaware, 163 The Green,
Newark DE 19716 USA
bInstitute of Chemical Research of Catalonia (ICIQ), 43007 Tarragona Spain
cSchool of Medicine, University of Leeds, Leeds, LS2 9JT UK
dGenitourinary Malignancies Branch, Center for Cancer Research, National Cancer
Institute, Bethesda, MD 20892
eMolecular Targets Program, Center for Cancer Research, National Cancer Institute,
Frederick, MD 21702 USA
fThese authors contributed equally to this work.
Abstract
Modifications at the bridgehead position of englerin A were made to explore the effects
of variation at this site on the molecule for biological activity, as judged by the NCI 60
screen, in which englerin A is highly potent and selective for renal cancer cells.
Replacement of the isopropyl group by other, larger substituents yielded compounds
which displayed excellent selectivity and potency comparable to the natural product.
Selected compounds were also evaluated for their effect on the ion channel TRPC4 as
well as for intravenous toxicity in mice, and these had lower potency in both assays
compared to englerin A.
Keywords: Englerin, kidney cancer, ion channels, , TRPC4, mice
*correspondence: beutlerj@mail.nih.gov; 301-846-1942
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Englerin A (1) was discovered in extracts of the root and stem bark of the Tanzanian
tree Phyllanthus engleri on the basis of its nanomolar activity against most renal cancer
cell lines and concomitant inactivity against most other cell types in the NCI 60 cell
screen.1 Total syntheses of the molecule established its absolute configuration and
have provided multiple synthetic pathways to the natural product.2-6 Further work in
several groups has explored structure activity relationships.7-15 Plausible molecular
mechanisms of action for englerin A have been proposed, including agonism of protein
kinase C θ,16 and agonism of the ion channels transient receptor potential canonical
(TRPC) 4 and 5.17-18 In the case of TRPC4/5, it appears that englerin A may bind to an
allosteric site on the ion channel complex, thereby facilitating entry of sodium and/or
calcium ions into the cell.19-21 Two recent reviews have summarized progress.22-23 One
barrier to development of englerin A as a drug candidate has been its potent
intravenous toxicity and lethality, observed by both our group and the Novartis group.17
A recent report identified TRPC4 and TRPC5 ion channels as mediating this adverse
response to englerin A.20
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Modifications of the bridgehead position at C-7 of englerin A have not been extensively
explored. A single study found that trimming the isopropyl group to ethyl or methyl led to
a drastic loss of potency in cancer cell growth inhibition.9 We report here on a program
to examine the effects of expanding the size of the isopropyl group on biological activity,
including NCI 60 cell growth inhibition and selectivity, TRPC4/5 agonism, and
intravenous toxicity. We have previously reported only NCI 60 data for the initial
members of this class, compounds 2, 3, 4 and 5 (Table 1, Figure 1).10 Recently, we
evaluated these and other englerin analogues in an assay18 for TRPC4 agonism. While
compound 3, the cyclopropyl bridgehead analogue of englerin A, was very similar to
englerin A in its TRPC4 agonist properties, the cyclohexyl and phenyl bridgehead
analogues 4 and 5, showed much reduced ability to open the ion channel.24-25
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Thus, we embarked on a collaborative program of analogue synthesis and biological
testing using the NCI 60 assay as the primary measure. The synthesis protocols were
based on the total syntheses published by the Chain6, 11 and Echavarren groups.3, 10
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Analogues 2-5 had been prepared following a simplified route that provides 50% of an
unwanted and unreactive diastereomer, and which was therefore less efficient in terms
of overall yield, but simplified the scale up of the chemical steps. Those analogues
possessed an unnatural 4,5 double bond,10 therefore going forward we chose to
prepare the corresponding saturated natural analogues 6 and 7 by the route in Scheme
1. Compound 6 showed particularly good potency. The synthesis of 6 and 7 by the
previously described protocols6 was straightforward but suffered diminished yields of
most steps of the overall sequence due to the increased size of the bridgehead C7-
substituent (diversity point R1, Scheme 1). For example, the Michael adducts 19 and 26
resulting from the combination of the furanones 17 and 25 with the aldehyde 18 were
isolated in 68% and 49% yield, respectively. The diastereoselectivity of these reactions
(1:1.1 dr and 1:2.2 dr [desired: others], respectively) was modest but did not present a
problem for analogue production (see Supporting Information).6 Later, the samarium(II)
iodide-mediated reductive cyclization to give the ketoalcohols 20 and 27 proceeded in
19% and 7% yield, respectively (see Supporting Information).6 Fortunately, steps
downstream of these events were not significantly impacted; acylations to give a variety
of ester substitution at C6 (diversity point R2, Scheme 1) proceeded in 48–99% yield.
The resultant analogues 8-11 with modifications of the cinnamate moiety also
possessed the desired potency and selectivity (Table 1). In all cases, the final three-
step reduction and glycolate installation sequences were straightforward and proceeded
in 15–52% overall yield.
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Table 1. NCI 60 selected renal cancer cell growth inhibition data for englerin A (1) and
analogues (GI50 values in nM). n is the number of NCI 60 five dose tests.
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Compound
n
Pearson Correlation to
Englerin A
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Cell lines
A498
UO-31 RXF 393
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510
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1.00
0.94
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n/a
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---b
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0a
0a
1
---
---
---
---
---
---
n/a
2,200
---
48
3,300
---
0.78
n/a
0a
---
aCompound did not pass one dose NCI 60 test. See SI Section VII for one dose data.
bAssay failed for this cell line.
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H
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OH
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OH
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R1 = cyclohexyl, R2 = phenyl
R1 = phenyl, R2 = phenyl
R1 - cyclohexyl, R2 = cyclohexyl
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R = isopropyl
R = cyclopropyl
R = cyclohexyl
R = phenyl
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R = CH3
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R = phenyl
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R = H
R = (CO)CH3
Figure 1. Structures of englerin A and analogues tested in this work.
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Scheme 1.
Next, to determine if nitrogen could be accommodated in the bridgehead cyclohexyl,
compounds 12 and 13 were synthesized by means of a gold(I)-catalyzed cascade
reaction of 1,6-enyne 44, which was prepared by an aldol reaction between the enolate
N-Boc protected 1-(piperidin-4-yl)ethan-1-one with aldehyde 43 (Scheme 2).3,10 The
gold(I)-catalyzed cyclization of 44 (as a1:1 mixture of epimeric alcohols), followed by
desilylation, gave rise to key intermediate 46 in low yield, although as a single
stereoisomer. After protection of the secondary alcohol, 47 was converted into 48 by
allylic rearrangement and subsequent epoxidation.3,10 Reduction of 48 with WCl6 and n-
BuLi provided 49, which furnished 52 by stepwise esterification of both secondary
alcohols and removal of the N-Boc protecting group. Finally, intermediate 52 was
transformed into 12 and 13 by standard procedures.
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Scheme 2.
While slight renal selectivity was seen in piperidine 12 in the NCI 60 single dose test
(Supplemental information), the piperidine acetamide 13 was essentially inactive. Thus,
no further testing was conducted on the pair.
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Finally, recapitulation of our previous strategy to replace the glycolate ester with an
amide11 led to the bridgehead cyclohexyl aza-analogue 14 and bridgehead phenyl aza-
analogue 15 as shown in Scheme 1. These scaffolds were prepared as described
above from ketoesters 21 and 28, and incorporation of the nitrogen atom proceeded
exactly as previously described11 with no modifications required and no loss of reaction
efficiency. Compound 14 was weakly active and renal selective, while 15 was nearly
inactive. This and the results for the piperidine analogues 12 and 13 may indicate that
the englerin pharmacophore does not tolerate nitrogen in the context of either a
glycolamide or a bridgehead piperidine.
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Based on all of the above results, we chose to carefully test compounds 1, 4, 5, 6, and
10 in a cellular TRPC4-TRPC1 assay utilizing Tet+ HEK 293 cells with an endpoint of
Ca+2 response via Fura-2.17-18 These selected compounds had excellent NCI-60
potency as seen in Table 1, and were available in amounts sufficient for further testing
in mice. Their Pearson correlations to englerin A were all >0.8, indicating a very similar
pattern of cell line selectivity to the natural product.
In the TRPC4-TRPC1 assay, however, all of these analogues had lower potency at the
ion channel when compared to 1. At a concentration of 1 μM, the order of potency was
5 < 4 <6 < 10 < 1 (Figure 2A). At 100 nM, only 1 displayed any detectable activity
(Figure 2B). This is quantified in Figure 3.
Furthermore, all responses were completely inhibited by the TRPC4/5 antagonist Pico
145 (Figure S1A).26 Experiments with the same set of compounds in a similar TRPC5
assay at 10 nM concentration confirmed 1 to be highly active but only compound 10 of
the four analogues fully opened the channel (Figure S1B).
The same four analogues were tested in mice to estimate the maximum tolerated
intravenous dose using the “Up-and-Down” protocol, which is designed to minimize the
number of mice used for both ethical and economic reasons.27 As reported previously,11
1 was tolerated only at the very low dose of 50 μg/kg. Compounds 4, 6 and 10 were
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tolerated at 500 μg/kg, while 5 was tolerated at 200 μg/kg. Hence, 4, 6, and 10 are
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essentially equipotent with 1 in the NCI 60 screen, but 10-fold weaker in both the
TRPC4 assay and intravenous toxicity. Detailed results are found in the Supplemental
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Information.
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Figure 2.
Activation of TRPC4/TRPC1 channels. (a, b) The [Ca2+] responses were measured in
TRPC4/TRPC1 expressing HEK293 Tet cells after stimulation with compound 1, 4, 5, 6
or 10 at 1 µM (a) or 0.1 µM (b) concentration. The traces represent mean responses +/-
SEM obtained from (n/N=4/22) measurements for all compounds in (a), or (n/N=4/20)
measurements in (b), except for the compound 4 (n/N=3/18). Vehicle control was 0.1 %
DMSO.
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Figure 3:
Quantification of the [Ca2+] responses triggered by the different compounds. The
maximum response was measured in TRPC4/TRPC1 (A) and TRPC5 (B) cells after
addition of vehicle or compound at final concentration of 0.1 or 1 µM (A) or 10 nM (B).
The values were calculated as mean±standard error of mean from the data presented in
figure 1 and S1B. At each concentration (0.1 µM and 1 µM for TRPC4/TRPC1 and 10
nM for TRPC5), the differences between the effects of the compounds 1, 4, 5, 6 and 10
were assessed by one-way ANOVA followed by post-hoc Bonferroni test for means
comparison. The lowercase letters above the bars indicate statistical significance within
each group; the means labelled with different letters are significantly different from each
other (p<0.05 in the Bonferroni test) while those denoted with common letter are not
significantly different (p>0.05).
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It has been established through experiments in knockout models that TRPC4 and
TRPC5 are both involved in the intravenous lethality and morbidity of englerin A in the
mouse.20 Here we have sought to identify chemical modifications which would prevent
the TRPC-mediated lethal effect. TRPC channels, including TRPC4 and TRPC5, are
known to form heterotetrameric complexes, with an impact on their pharmacological
properties and functionality28-29 Because of its ubiquitous expression, TRPC1 is
expected to participate widely in the heteromer formation in vivo; indeed heteromeric
channels containing combinations of TRPC1, TRPC4 and TRPC5 subunits have been
detected,30 and formation of novel TRPC channels by complex subunit interactions has
been detected in embryonic brain.30-31 For englerin A, TRPC4/TRPC1 channels in
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particular have been shown to mediate the toxicity in synovial sarcoma cells;19 therefore
we have used a cell line expressing a TRPC4-TRPC1 concatemer to determine the
compound’s ability to activate this specific channel. The results were further validated in
TRPC5 expressing cells to confirm the effects.
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We have shown here that modification of the 7-isopropyl substituent of the englerin core
can substantially reduce the TRPC4 and TRPC5 agonism of the compounds, while
having a limited impact on potency and selectivity of cell growth inhibition, as measured
by the NCI 60 assay. Furthermore, the best analogues have a 10-fold reduced liability
for acute intravenous toxicity in mice.
These modifications may provide a template for development of englerins as human
cancer therapeutics. It is important to consider that many oncology drugs are
administered by continuous intravenous infusion to prevent adverse effects. However,
the acute mouse tail vein model we utilized is only partially informative about the
potential clinical hazards of englerins, where slow infusion would likely reduce the acute
effects. Furthermore, it is possible that the antitumor activity we observed for englerin A
in kidney and prostate cancer xenografts16 may not be due to either PKCθ or TRPC4/5
agonism. Indeed, a compound with parallel activity against both targets, tonantzitlolone
A32-33 has been found to also affect kinesin-5 function.34 Englerin A has not been tested
for this activity.
Recently obtained cryo-EM structures of TRPC4 may also inform the relationship
between englerin A, its analogues, and the ion channels.35-36 Coupled with data from
mutational analysis of TRPC5 function in relation to englerin agonism,21 it may
eventually be possible to rationally design englerin analogues with reduced clinical
liabilities.
Acknowledgments
Thanks to Jason Evans for the COMPARE analysis. Research supported in part by the
Intramural Program of the Center for Cancer Research, National Cancer Institute, under
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1Z1A BC011470-07 (J.A.B.) and 1ZIABC010547-17 (W.D.F.). Support for the
Echavarren lab was from MINECO/FEDER, UE (CTQ2016-75960-) and AGAUR (2017
SGR 1257). S.T. was funded by a Medical Research Council Confidence in Concept
Award to D.J.B. W.J.C. gratefully acknowledges financial support from the University of
Delaware and the National Institutes of Health (R01CA163287 and
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P20GM104316). Spectral data at UD was acquired on instruments obtained with the
assistance of NSF and NIH funding (NSF CHE0421224, CHE0840401, CHE1229234,
CHE1048367; NIH S10 OD016267-01, S10 RR026962-01, P20GM104316,
P30GM110758).
Supplementary Material
Detailed synthetic procedures, NMR spectra, ion channel, NCI 60 and mouse toxicity
details and data.
Abbreviations
GI50, Growth Inhibition 50 Percent; TRPC1, Transient Receptor Canonical 1; TRPC4,
Transient Receptor Canonical 4; TRPC5, Transient Receptor Canonical 5.
References and notes
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Ratnayake, R.; Covell, D. G.; Ransom, T. T.; Gustafson, K. R.; Beutler, J. A., Englerin A, a Selective
Total Synthesis and Absolute Configuration of the Guaiane Sesquiterpene Englerin A. Angew. Chem. Int.
Ed. Engl. 2009, 48, 9105-9108.
3.
Angew. Chem. Int. Ed. Engl. 2010, 49, 3517-3519.
4. Xu, J.; Caro-Diaz, E. J.; Theodorakis, E. A., Enantioselective Formal Synthesis of (-)-Englerin A via a
Rh-Catalyzed [4 + 3] Cycloaddition Reaction. Org. Lett. 2010, 12, 3708-3711.
5. Zhou, Q.; Chen, X.; Ma, D., Asymmetric, Protecting-Group-Free Total Synthesis of (-)-Englerin A.
Angew. Chem. Int. Ed. Engl. 2010, 49, 3513-3515.
6. Li, Z.; Nakashige, M.; Chain, W. J., A Brief Synthesis of (-)-Englerin A. J. Am. Chem. Soc. 2011, 133,
6553-6556.
Molawai, K.; Delpont, N.; Echavarren, A. M., Enantioselective Synthesis of (-)-Englerins A and B.
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