The total syntheses of JBIR-94 and two synthetic analogs and their cytotoxicities against A549 (CCL-185) human small lung cancer cells

Article abstract of DOI:10.1016/j.tetlet.2019.151360

We here disclose the total syntheses of the natural polyphenol JBIR-94 and two nonnatural analogs, whose structures are of interest for their bioactivity potential as radical scavengers. Although we initially attempted this by dually acylating both of putrecine's amine nitrogens in a single pot, our endeavors with this method (which has been successfully reported by other groups) proved ineffectual. We accordingly opted for the lengthier approach of acylating each amine individually, which gratuitously prevailed and also aligns with separate literature precedent. Moreover, we here share our analysis of these target compounds’ cytotoxicities and IC₅₀ values against A549 (CCL-185) human small lung cancer cells.

Full text of DOI:10.1016/j.tetlet.2019.151360

Journal Pre-proofs
The Total Syntheses of JBIR-94 and Two Synthetic Analogs and Their Cyto-
toxicities Against A549 (CCL-185) Human Small Lung Cancer Cells
Cathy L. Mangum, Mica B. Munford, Alyssa B. Sam, Sandra K. Young, Jeremy
T. Beales, Yagya Prasad Subedi, Chad D. Mangum, Tanner J. Allen, Miranda
S. Liddell, Andrew I. Merrell, Diana I. Saavedra, Becky J. Williams, Nicole
Evans, Joseph L. Beales, Michael A. Christiansen
PII:
S0040-4039(19)31151-7
DOI:
https://doi.org/10.1016/j.tetlet.2019.151360
TETL 151360
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 September 2019
28 October 2019
2 November 2019
Please cite this article as: Mangum, C.L., Munford, M.B., Sam, A.B., Young, S.K., Beales, J.T., Subedi, Y.P.,
Mangum, C.D., Allen, T.J., Liddell, M.S., Merrell, A.I., Saavedra, D.I., Williams, B.J., Evans, N., Beales, J.L.,
Christiansen, M.A., The Total Syntheses of JBIR-94 and Two Synthetic Analogs and Their Cytotoxicities Against
A549 (CCL-185) Human Small Lung Cancer Cells, Tetrahedron Letters (2019), doi: https://doi.org/10.1016/j.tetlet.
2019.151360
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The Total Syntheses of JBIR-94 and Two Synthetic Analogs and Their
Cytotoxicities Against A549 (CCL-185) Human Small Lung Cancer
Cells
Cathy L. Manguma, Mica B. Munforda, Alyssa B. Sama, Sandra K. Younga, Jeremy T. Bealesa,
Yagya Prasad Subedib, Chad D. Manguma, Tanner J. Allena, Miranda S. Liddella, Andrew I.
Merrella, Diana I. Saavedrab, Becky J. Williamsa, Nicole Evansa, Joseph L. Bealesa, and
Michael A. Christiansena,*
a
Utah State University, Uintah Basin Campus: 320 North Aggie Blvd, Vernal, UT 84078,
U.S.A.
b
Utah State University, Logan Campus: Department of Chemistry & Biochemistry, Logan,
UT 84322-0300, U.S.A.
Brigham Young University, Department of Chemistry & Biochemistry, Provo, UT 84602,
c
U.S.A
*
Corresponding author. Tel./fax: +1 435 722 1761
E-mail address: m.christiansen@usu.edu (M. A. Christiansen)
Abstract
We here disclose the total syntheses of the natural polyphenol JBIR-94 and two nonnatural
analogs, whose structures are of interest for their bioactivity potential as radical scavengers.
Although we initially attempted this by dually acylating both of putrecine’s amine nitrogens in a
single pot, our endeavors with this method (which has been successfully reported by other
groups) proved ineffectual. We accordingly opted for the lengthier approach of acylating each
amine individually, which gratuitously prevailed and also aligns with separate literature precedent.
Moreover, we here share our analysis of these target compounds’ cytotoxicities and IC50 values
against A549 (CCL-185) human small lung cancer cells.
Keywords
Radical oxygen
JBIR
Natural products
Putrecine
Introduction
In the human body, the unavoidable exposure of living tissues to excess free radicals, such as
reactive oxygen species, can result in oxidative modification of biomolecules.1 These events
contribute to various disorders that include age-related degenerative conditions, mutagenesis,
neural dysfunction, cancer, heart disease, and compromised immune health.2-5 The human body
possesses enzymatic and nonenzymatic antioxidative machinery to scavenge and eliminate free
radicals, which include the dynamic 1-5% of radical molecular oxygen that human cells naturally
produce.1,5 However, the efficacy of these defense mechanisms decreases with age.1
1

Fortuitously, diets rich in fresh fruits and vegetables correlate with lowered frequency of the
aforementioned maladies, presumably because such foods contain high concentrations of
antioxidative molecules: in particular, of polyphenolic compounds, which often possess
antimutagenic, anti-inflammatory, antimicrobial, and free-radical-oxygen-scavenging properties.6-
15
It follows, then, that long-term healthy eating might partly offset the negative health effects in
play as we age. Beyond its relevance to health, radical oxidation is also associated with food
decomposition, as it “affects food safety, color, flavor, and texture” by contributing to the
deterioration of lipids.6 Thus, antioxidants might also enhance and preserve food quality.6,16-17
With our research focus on synthesizing small, bioactive natural molecules with potential
implications for disease prevention, we took note of two polyphenolic compounds, JBIR-94 and
JBIR-125 (1 and 2, Figure 1), which were discovered in 2012 by Takagi and Shin-ya as secondary
metabolites of the bacterium Streptomyces R56-07.18 According to their report, when molecules
1 and 2 were tested in an α-diphenyl-β-picrylhydrazyl (DPPH) assay, they exhibited free-radical-
scavenging IC50 potencies of 11.4 μM and 35.1 μM, respectively.18 By comparison, α-tocopherol,
a potent, radical-scavenging form of vitamin E,19-20 gave an IC50 value of 9.0 μM in the same
assay.18 Thus, the more active natural product, JBIR-94 (1), manifested radical-scavenging
potency comparable to that of Vitamin E, a well-known dietary antioxidant.20
Figure 1. JBIR-94 (1), JBIR-125 (2), and synthetic analogs 3 and 4.
As often occurs in the field of natural products, both 1 and 2 were isolated in scant amounts from
their natural source: 1.3 mg (1) and 2.3 mg (2), respectively, from 2 L of bacterial fermentation
broth.18 Thus, large-scale access to either compound for more extensive study would be most
efficiently achieved through total synthesis. As molecule 1 was the more potent of the two, we
focused on its assembly, which we reasoned would be facile, given its symmetry. Because
polyphenols are often reported to be potent radical scavengers,7-15 we hypothesized that the
hydroxyl groups in 1 would be necessary for bioactivity but that the methoxy groups would not.
Thus, analog 3 (Figure 1), which retains the hydroxyl moieties of 1 but lacks its methoxy groups,
might theoretically exert similar antioxidative potency, while analog 4, which lacks both, would
not. With the goal of providing efficient access to these molecules for later analysis, we sought to
develop their first total syntheses.
Despite finishing this work in mid-2016,21 time and resource constraints precluded us from
an earlier disclosure. Moreover, as we initially raced toward its completion, elegant syntheses of
1 and 2,22 as well as a separate synthesis of 4,23 were published by other groups in late 2015,
thereby diminishing the impact of our research. Nevertheless, that we are aware, analog 3 has
2

never been previously reported. Thus, this paper presents the first disclosed synthesis of 3.
Additionally, we share here the results of a cytotoxicity study of 1, 3, and 4, which should aid
further efforts in developing bioactivity profiles for these molecules.
Results and discussion
At the outset, we envisioned a highly efficient one-pot, three-molecule assembly of the JBIR core,
for which some related transformations were known.24 Thus, trans-ferulic acid (5) was treated with
H2 gas and 5% Pd/C to quantitatively furnish 6 (Scheme 1).25 At this point, we divergently
converted 6 into differentially protected analogs 7a-c26-28 (yields shown in Scheme 1). We then
individually coupled molecules 6 and 7a-c with putrecine (8) in a 2:1 ratio in anticipation of forming
hydrocarbon-tethered diamides 1 (from 6) or 9a-c (from 7a-c), respectively. Coupling parameters
were screened under Conditions A-D (Scheme 1, bottom-left inset). Unfortunately, all of these
attempts failed and instead gave only complex mixtures, unreacted starting materials, or
unidentifiable byproducts. This contrasts with the work of Sorensen and Taj, who reported using
EDCI (Condition B) to successfully couple 7a with 8, forming 9a in 63% yield.22
Scheme 1. Attempted three-molecule/one-pot assemblies of the JBIR-94 core.
Although this setback’s exact cause was uncertain, we suspected detrimental interaction with our
protecting groups—for instance, an amine displaing the acetyl appendage in 7a—or undesired
cross-reactivity between chlorine and silicon when silyl ether 7b is treated with oxalyl chloride
(Condition D).
Thus, as an alternative approach (Scheme 2), we treated molecules 7a-b (7c was not
attempted) with N-hydroxysuccinimide (10) and DCC to quantitatively form intermediates 11a-b,
whose succinimidyl-functionalization has been reported elsewhere for assembling diamides.29
However, our efforts to obtain 11a-b in pure form were plagued with difficulties and proved
unsuccessful. Nevertheless, we individually reacted our impure intermediates 11 with putrecine
(8) in a 2:1 molar ratio in anticipation of forming products 1 or 9a-b. Unfortunately, these
endeavors were unsuccessful. Alternatively, we converted carboxylic acids 6 and 7a-b into their
methyl ester derivatives (not shown), which we then reacted with 8 under conditions reported by
McCluskey et al.30 These attempts also failed, which effectively ended our campaign to synthesize
the JBIR core through a one-pot, three-molecule strategy.
3

Scheme 2. Alternative approach to the JBIR-94 core via intermediates 11a-b.
At this point, we turned to the more conservative, stepwise approach shown in Scheme 3. With
concerns about potential amine cross-reactivity with acetyl or silyl protecting groups—and
knowing of benzyl ethers’ general robustness, putative ease of deprotection, and unreactivity
toward amines31—we focused exclusively on substrate 7c from this juncture. To avoid prior
obstacles with the diamine functionality of putrecine (8), we opted for commercially available, t-
Boc-protected analog 12. Subsequent treatment of its free amine with 7c, EDCI, Et3N, and
catalytic DMAP, followed by overnight recrystallization from 1:1 CH2Cl2/hexanes, furnished 13
cleanly, in 88% yield. Deprotection was facilitated by exposure to TFA in toluene (Scheme 3).
Extensive trial and error revealed that this reaction had to be quenched by adding enough 2M
aqueous NaOH to bring the precipitated suspension to pH > 10. After extensive extraction (20x)
with CH2Cl2, drying over MgSO4, concentration, and overnight recrystallization from 90%
EtOAc/hexanes, product 14 was obtained in pure form in 82% yield. Renewed subjection to EDCI
coupling with 7c then gave intermediate 9c in 85% yield (recrystallized from CH2Cl2), and Pd-
catalyzed hydrogenation concomitantly removed both benzyl groups to furnish JBIR-94 (1) in 84%
yield (52-54% overall yield from 5).
Scheme 3. Completed synthesis of JBIR-94 (1).
We next turned to analogs 3 and 4. Thus, commercial reactant 15a was converted over two steps
to 15c, the methoxy-free surrogate of 7c (Scheme 4). Next, EDCI coupling with 12 provided 16
quantitatively, and TFA-mediated deprotection formed 17 in 78% yield. Renewed coupling with
15c then gave 18 in 53% yield, and subsequent deprotection afforded 3 in 92% yield (26% overall
yield from 15a).
4

Scheme 4. Completed synthesis of JBIR-94 analog 3.
The synthesis of diamide 4 was carried out by coupling 3-phenylpropionic acid (19) with 12 to
afford 20 quantitatively (Scheme 5). Deprotection, followed by subsequent coupling with 19, then
gave target molecule 4 in 98% over two steps from 20.
Scheme 5. Completed synthesis of JBIR-94 analog 4.
Cytotoxicity Assay
The cytotoxicities of JBIR-94 and analogs 3-4 were evaluated through a colorometric assay
against A549 (CCL-185) (human small lung cancer) cells using MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) dye. For this, 200 μL of cell suspension in growth medium with
5000 cells was seeded to the wells of a 96-well culture plate and incubated for 24 hours. The
medium was removed, and the compounds were added to the cells across a concentration
gradient (0.01, 0.1, 1.0, 10, and 100 µM) in growth medium. Our assay also included a control
with only medium and a control with 1% 100X Triton in medium. Cells were then incubated for 48
hours, and 20 μL of MTT dye (5 mg/mL stock) was added to each well and incubated for another
5 hours. The medium was removed, the cells were washed with PBS (pH = 7.4), and the reduced
MTT dye was dissolved in 100 µL of DMSO, with absorption being measured at 570 nm and 650
nm. This experiment was performed in triplicate of triplicate, with mean cytotoxicities presented
in Figure 2. Our results indicated that compound 4 has relative cell viability at around 55% at 100
μM, whereas JBIR-94 and analog 3 are relatively more toxic, with cell viabilities at around 60%,
even at 10 µM concentration. Moreover, JBIR-94 and compounds 3-4 have IC50 values of 52.88
± 11.69 µM, 78.92 ± 8.92 µM, and >100 µM, respectively, showing mild toxicity to human lung
cells.
5

JBIR-94
3
4
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90
60
30
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M
M
M
M


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

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.01
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ito
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100
Figure 2. Gradient cytotoxicities of JBIR-94 and analogs 3-4 towards A549 (CCL-185) cells.
Conclusions
In conclusion, we disclose here the total syntheses of the natural product JBIR-94 (1), as well as
nonnatural analogs 3 and 4. Although syntheses of 1 and 4 have been reported previously in the
literature,22-23 compound 3 has not. Additionally, we measured the cytotoxicities and IC50 values
of 1, 3, and 4 against A549 (CCL-185) human small lung cancer cells using an MTT colormetric
assay. Our results indicated that analog 4 has relative cell viability at around 55%, whereas 1 and
3 are relatively more toxic, with cell viabilities at around 60%, even at 10 µM concentration.
Separately, 1, 3, and 4 have IC50 values of 52.88 ± 11.69 µM, 78.92 ± 8.92 µM, and >100 µM,
respectively, showing mild toxicity to human lung cells.
Acknowledgments
The authors would like to thank Professors Steven L. Castle and Merritt Andrus from BYU, as well
as Dr. Tom Chang from USU, for their suggestions and contributions during difficult junctures of
this project. The authors gratefully acknowledge funding from the Uintah Basin Impact Mitigation
Fund, the Utah State University (USU) Uintah Basin Summer Internship Program, and the USU
Department of Chemistry and Biochemistry.
Supplementary data
6

Supplementary data associated with this article, which include complete experimental procedures
and spectral characterization (1H and 13C NMR), are available in the online version at.
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Highlights:
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Cancer Research
Small Molecule synthesis
Organic Chemistry
Medicinal Chemistry
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