Two-step total synthesis of an anti-MRSA and myosin-inhibiting marine natural product pentabromopseudilin via Suzuki-Miyaura coupling of a MIDA boronate ester

Article abstract of DOI:10.1016/j.tetlet.2017.07.057

A marine natural product isolated from Pseudoalteromonas sp., pentabromopseudilin, with promising anti-MRSA and myosin ATPase inhibition activities was synthesized in two steps using recently developed MIDA boronate Suzuki-Miyaura coupling technology. Add

Full text of DOI:10.1016/j.tetlet.2017.07.057

Accepted Manuscript
Two-step Total Synthesis of an Anti-MRSA and Myosin-inhibiting Marine
Natural Product Pentabromopseudilin via Suzuki-Miyaura Coupling of a MIDA
Boronate Ester
Dong-Young Kum, Mohamad Nazari, Kerry L. McPhail, Christina S. Cooper,
Takashi L. Suyama
PII:
S0040-4039(17)30912-7
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.07.057
TETL 49139
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 June 2017
9 July 2017
14 July 2017
Please cite this article as: Kum, D-Y., Nazari, M., McPhail, K.L., Cooper, C.S., Suyama, T.L., Two-step Total
Synthesis of an Anti-MRSA and Myosin-inhibiting Marine Natural Product Pentabromopseudilin via Suzuki-
Miyaura Coupling of a MIDA Boronate Ester, Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.
2017.07.057
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Two-step Total Synthesis of an Anti-MRSA and Myosin-inhibiting Marine Natural Product
Pentabromopseudilin via Suzuki-Miyaura Coupling of a MIDA Boronate Ester
Dong-Young Kum,† Mohamad Nazari,‡ Kerry L. McPhail,‡ Christina S. Cooper,† and Takashi L. Suyama†,§,*
† Department of Health Sciences, Corban University, 5000 Deer Park Dr. SE, Salem, OR 97317, United
States
‡ College of Pharmacy, Oregon State University, Corvallis, OR 97331
§ Physical Science Department, Linn Benton Community College, 6500 SW Pacific Blvd, Albany, OR
97321, United States
* Corresponding Author. Tel.: 1-541-231-3528; fax: 1-541-917-4755; email: suyamat@linnbenton.edu
Abstract:
A marine natural product isolated from Pseudoalteromonas sp., pentabromopseudilin, with promising
anti-MRSA and myosin ATPase inhibition activities was synthesized in two steps using recently
developed MIDA boronate Suzuki-Miyaura coupling technology. Additionally, bromination was shown to
be necessary for the antimicrobial activity of pentabromopseudilin.
Graphical Abstract:
Br
HN
2 steps
Br
Br
N
I
B
Br
+
O
O
O
O
OH
N
OH
Br
pentabromopseudilin
Boc
Keywords:
Suzuki-Miyaura;
Boronic acid MIDA ester;
MRSA;
Brominated pyrrole alkaloid
Introduction
Many medicinally important natural products contain a 2-substituted pyrrole unit, owing to
their biosynthetic origin in proline.1 As such, transition metal-catalyzed carbon-carbon coupling
reactions to append pyrrole to another synthon would be a useful tool.2 However, there have not been
many cases where this coupling strategy was realized in the context of 2-aryl pyrrole natural product
synthesis. As noted by Burke and co-workers, N-Boc-pyrrole-2-boronic acid (1) is a very labile substrate
and often presents difficulties in Suzuki-Miyaura type couplings.3 When compound 1 has been
successfully used in a natural product synthesis, extensive experimentation to investigate different
conditions and ligands was necessary.4
Since the introduction of the N-methyliminodiacetic acid (MIDA) boronic acid esters by Burke
and co-workers, many such derivatives have become commercially available. However, only a handful of
examples in natural product syntheses have emerged in the literature.4 This may be partially attributed
to the exquisite stability of MIDA boronate; in many attempted reactions, the boronate ester is too slow
to carry out the coupling reaction with mild bases that are typically used for Suzuki-Miyaura coupling
(SMC), such as K2CO3.5

A marine natural product isolated from Pseudoalternomonas sp., pentabromopseudilin (2)
possesses just such a difficult aryl-pyrrole connection.6 It has been shown to possess impressive
antimicrobial activity against MRSA at a level comparable to those of last resort drugs such as
vancomycin (IC50 of 0.1 μM for 2 and 0.91 μM for vancomycin, respectively).7 A more recent study has
shown that it is a selective and potent myosin ATPase inhibitor (IC50 of 25 μM for class-2 myosins).8
Naturally, compound 2 has attracted much attention from the organic synthesis community and a
variety of unique and elegant syntheses have been described.8,9 Moreover, the biosynthesis of 2 has
recently been described in detail by Moore and co-workers.10 However, all of the previous syntheses
involve seven or more steps.8,9 Herein, we report a two-step total synthesis of pentabromopseudilin (2)
through the use of the MIDA boronic ester technology.
In all previous syntheses of 2, the pyrrole ring was constructed en route, involving many steps to
install the necessary functional groups for cyclization. In the example by Correia and co-workers, a
pyrroline ring was installed via Heck coupling onto a protected phenol and subsequent oxidation was
necessary to form the desired pyrrole.9a Inspired by Schmidt and co-workers unprotected halophenol
SMC11 and Weinreb and co-workers SMC of 1 with a decorated heterocycle in the synthesis of
ageladine,4a a retrosynthetic dissection was made at the aryl-pyrrole carbon-carbon bond without a
protecting group for the phenol.
Br
OH
HN
B
Br
Br
N
OH
Boc
Br
OH
1
Br
2
Figure 1. Labile boronic acid 1 and 2-aryl pyrrole marine natural product, pentabromopseudilin 2.
Results and Discussion
When 2-iodophenol 3 and 1 were selected as the initial SMC partners with Pd/C as a
heterogeneous catalyst only rapid decomposition and oxidative dimerization of 1 were observed.
Attributing this to the instability of 1, we turned our attention to the MIDA boronic ester substrate 4.
Firstly, the choice of an appropriate aqueous base was investigated by comparing K2CO3 and K3PO4. As
previously reported,3 the more basic K3PO4 was required for sufficiently rapid hydrolysis of the boronate
ester, as indicated by the observation that 4 remained intact after many hours of reflux when K2CO3 was
employed.
Br
R
HN
pyrHBr3
6 eq
N
N
I
conditions
Br
Br
B
+
O
O
O
O
Br
see Table 1
N
EtOH
60%
OH
OH
Boc
OH
3
Br
4
R=H
R=Boc 6
5
2
Scheme 1. Two-step total synthesis of pentabromospeudilin 2.
Entry Pd catalyst
Solvent
Base Yield 5 (%)b
K3PO4 0%
1
2
3
4
5
Pd/C, CyJohnPhos THF/H2O
PdCl2, CyJohnPhos THF/H2O
KOH
K3PO4 15%
KOH
64%a
31%
Sphos Pd G2
Sphos Pd G2
Sphos Pd G2
THF/H2O
THF/H2O
MeCN/H2O K3PO4 trace

6
7
8
Sphos Pd G2
Sphos Pd G2
Xphos Pd G2
MeCN/H2O KOH
H2O
trace
21%
K3PO4 0%
THF/H2O
KOH
Table 1. Optimization of the SMC between 4 and 3. Reagents and conditions: catalyst (10 mol%), 4 M
aqueous base (8 equiv), 65 °C unless otherwise specified. (a) 64% yield at 70, 65, and 55 °C, but 21%
yield at rt (b) isolated yield.
When Pd/C alone or in conjunction with the CyJohnPhos ligand was used as the catalyst, no
desired coupled product was observed (not shown). However, when PdCl2 and CyJohnPhos were used as
the catalyst, a low yield of the coupled product without the Boc group (5) was obtained. Interestingly,
we were not able to isolate the Boc-protected aryl-pyrrole 6 in any of the reactions. Aqueous base
hydrolysis of pyrrole Boc groups has been previously observed, especially during SMC which often
require heating.12 This finding is not surprising because the pyrrole ring is able to stabilize a formal
negative charge on nitrogen, aiding the decarboxylative cleavage of the Boc group without acidic
activation of the nitrogen atom. This presented an advantage to us since it negates the deprotection
step and makes our synthesis of 2 a two-step synthesis from commercially available starting materials,
which is significantly more concise compared to the previous syntheses.
Three palladium catalysts that were available to us were investigated for their effectiveness
(Table 1). The Buchwald 2nd generation precatalyst (Sphos Pd G2) was more effective than PdCl2 or
Xphos Pd G2 (Entries 2, 4, and 8). The stronger the aqueous base, the better yield was obtained, owing
to the stability of the boronate ester group in 4. In our hands, the SMC product, pseudilin (5), was
obtained after purification as a white solid that turns reddish-brown after several hours at rt.
Comparison of the known bromination protocol using pyridinium tribromide7a,b,c and molecular bromine
in AcOH revealed that the latter treatment oxidatively opened the pyrrole ring to produce a carbonyl-
containing product, as evidenced by the IR stretch at 1724 cm-1.13 The known protocol gratifyingly
produced pentabromopseudilin 2 in 60% yield.8,9a,9b Our synthetic material 2 possessed virtually identical
1H and 13C NMR chemical shifts to those of the authentic sample as reported by Laatsch14 and co-
workers and the synthetic material reported by Knölker and co-workers.8
Freshly prepared 5 and 2 were tested against Staphylococcus epidermidis, a non-pathogenic
relative of MRSA, using a disc diffusion assay. Both compounds were used at concentrations ranging
from 50 μM to 0.05 µM to determine their relative activity against S. epidermidis. Compound 5 showed
no activity against S. epidermidis growth. However, compound 2 resulted in clear zones of inhibition at
50 and 5 µM. Previously, 5 was likewise shown to have no inhibitory activity on myosin-2 while 2
showed potent inhibition through a novel allosteric binding pocket.15 However, to the best of our
knowledge, this is the first time that bromination of 5 was shown to be necessary for
pentabromopseudilin’s antimicrobial activity.
Conclusion
An efficient total synthesis of the antibiotic marine natural product, pentabromopseudilin 2 was
achieved through SMC in 2 steps with a 38% overall yield.16 Application of this synthesis to the
biosynthetically related tricyclic natural product, 2,3,5,7-tetrabromobenzofuro[3,2-b]pyrrole is now
under investigation.
Acknowledgements:
The authors thank Prof. Carlisle Chambers at George Fox University for his assistance with preliminary
NMR analysis. Some equipment and supplies from Prof. Sarah Comstock at Corban University and from

her collaborators at Oregon Health Science University are gratefully acknowledged. This research did not
receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
References and Notes:
1. Walsh, C. T.; Garneau-Tsodikova, S.; Howard-Jones, A. R. Nat. Prod. Rep. 2006, 26, 517.
2. Young, I. S.; Thornton, P. D.; Thompson, A. Nat. Prod. Rep. 2010, 27, 1801.
3. Knapp, D. M.; Gillis, E. P.; Burke, M. D. J. Am. Chem. Soc. 2009, 131, 6961.
4. Meketa, M. L.; Weinreb, S. M. Org. Lett. 2006, 8, 1443.
5. Riley, A. P.; Day, V. W.; Navarro, H. A.; Prisinzano, T. E. Org. Lett. 2013, 15, 5936.
6. (a) Burkhold, P. R.; Pfister, R. M.; Leitz, F. H. Appl. Microbiol. 1966, 14, 649. (b) Lovell, F. M. J.
Am. Chem. Soc. 1966, 88, 4510.
7. Fehér, D.; Barlow, R.; McAtee, J.; Hemscheidt, T. K. J. Nat. Prod. 2010, 73, 1963.
8. Martin, R.; Risachar, C.; Barthel, A.; Jäger, A.; Schmidt, A. W.; Richter, S.; Böhl, M.; Chinsalapudi,
K.; Manstein, D. J.; Gutzeit, H. O.; Knölker, H. J. Eur. J. Org. Chem. 2014, 4487.
9. (a) Schwalm, C. S.; de Castro, I. B. D.; Ferrari, J.; de Oliveira, F. L.; Aparicio, R.; Correia, C. R. D.
Tetrahedron Lett. 2012, 53, 1660. (b) Xu, Z.; Lu, X. J. Org. Chem. 1998, 63, 5031. (c) Hanessian, S.;
Kaltenbronn, J. S. J. Am. Chem. Soc. 1966, 88, 4509.
10. (a) Agarwal, V.; El Gamal, A. A.; Yamanaka, K.; Poth, D.; Kersten, R. D.; Schorn, M.; Allen, E. E.;
Moore, B. S. Nat. Chem. Biol. 2014, 10, 640. (b) El Gamal, A.; Vinayak Agarwal, V.; Diethelm, S.;
Rahman, I.; Schorn, M. A.; Sneed, J. M.; Louie, G. V.; Whalen, K. E.; Mincer, T. J.; Noel, J. P.; Paul,
V. J.; Moore, B. S. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 3797. (c) El Gamal, A.; Agarwal, V.;
Rahman, I.; Moore, B. S. J. Am. Chem. Soc. 2016, 138, 13167.
11. Schmidt, B.; Riemer, M. J. Org. Chem. 2014, 79, 4104.
12. Handy, S. T.; Bregman, H.; Lewis, J.; Zhang, X. L.; Zhang, Y. N. Tetrahedron Lett. 2003, 44, 427.
13. ApSimon, J. W.; Durham, D. G.; Rees, A. H. J. Chem. Soc. Perkin Trans. I 1978, 1588.
14. Hanefeld, U.; Floss, H. G.; Laatsch, H. J. Org. Chem. 1994, 59, 3604.
15. Martin, R.; Jäger, A.; Böhl, M.; Richter, S.; Fedorov, R.; Manstein, D. J.; Gutzeit, H. O.; Knölker, H.
J. Angew. Chem. Int. Ed. 2009, 48, 1.
16. Experimental Procedures: Materials and Methods. The SPhos Pd G2 and XPhos Pd G2 catalysts,
N-Boc-pyrrole-2-boronic acid MIDA ester, iodophenol, and CyJohnPhos were purchased from
Sigma-Aldrich. Other reagents and solvents were obtained from various commercial sources. All
solvents were deoxygenated by passing a stream of nitrogen gas immediately before use. All
reactions were carried out under a nitrogen atmosphere introduced via evacuation by an
aspirator and a nitrogen balloon. MS data were obtained on an ABSciex 3200 QTrap mass
spectrometer. 1H and 13C NMR spectra were obtained on Bruker Avance III 500 MHz
spectrometer equipped with a 5mm BBO probe in CDCl3. Chemical shifts are reported relative to
residual protonated chloroform (δ 7.26) for 1H NMR and chloroform (δ 77.16) for 13C NMR.
Infrared spectra were obtained on Thermo-Fischer Nicolet iS10 FT-IR spectrometer. Thin layer
chromatography analysis was performed using EMD Millipore silica gel 60 F254 aluminum-backed
TLC plates.
Representative Protocol for synthesis of 2-phenyl-1H-pyrrole (5).
The small conical vial containing SPhos Pd G2 (4.5 mg, 0.0062 mmol) was charged with THF (0.5
mL). Upon stirring with a magnetic stirrer for 10 minutes at rt under N2, 2-iodophenol (13 mg,
0.059 mmol), N-Boc-pyrrole-2-boronic acid MIDA ester (20 mg, 0.062 mmol), and an aqueous 4
M KOH solution (0.124 mL, 0.496 mmol) were added successively and the vial sealed with a
septum lid. Immediately, the conical vial was placed in a sand bath preheated to 55 °C. While
the temperature of the reaction mixture climbed to the sand bath temperature, excessive
pressure was relieved twice by puncturing the septum with a needle connected to a balloon

filled with N2. After heating and stirring overnight, the reaction mixture was cooled to rt under
N2. Upon diluting with diethyl ether, aqueous 0.5 M HCl was added to acidify the mixture. After
phase separation, the aqueous layer was washed twice with diethyl ether (4 mL x2) and the
combined organic layers dried over Na2SO4. After removal of the solvents in vacuo, the crude
mixture was purified by a silica gel column with a gradient mobile phase ranging from 100%
hexanes to 3:7 EtOAc/hexanes. After removal of the solvents in vacuo, a white solid (6.0 mg,
0.038 mmol, 64% yield) was obtained, which turned slightly reddish brown upon standing in air
at rt. TLC Rf = 0.20 (1:4 EtOAc/hexanes), visualized by UV (spot turns purple after a while on
TLC). IR (film) νmax 3433 (br), 2932, 2854, 1587, 1495, 1465, 1407, 1331, 1291, 1244, 1181, 1101,
1050, 1036, 922, 817, 796, 750, 724, 658 cm-1. 1H NMR (500 MHz, CDCl3) δ 9.39 (br s, 1H), 7.54
(dd, J = 1.6, 7.8 Hz, 1H), 7.10 (dt, J = 1.7, 7.7 Hz, 1H), 6.97 (dt, J = 1.2, 7.6 Hz, 1H), 6.91-6.90 (m,
1H), 6.85 (dd, J = 1.1, 8.0 Hz, 1H), 6.58-6.57 (m, 1H), 6.33-6.31 (m, 1H), 5.36 (br s, 1H) ppm. 13C
NMR (175 MHz, CDCl3) δ 151.1, 128.8, 127.4, 127.2, 121.6, 119.8, 118.7, 116.4, 109.4, 106.4
ppm. LRMS (ESI+): calcd for C10H10NO (M+H): 160.08, found 160.1.
Synthesis of pentabromopseudilin (2).
To a solution of pseudilin (5) (15 mg, 0.094 mmol) in EtOH (2.3 mL) at rt under N2 was added
pyridinium tribromide (201 mg, 0.566 mmol, 6 eq). The reaction mixture was stirred under N2
for 3 days. Upon removal of the solvent in vacuo, the residues were purified by silica gel
chromatography with dichloromethane/hexanes (1:1). A slightly pink-purple solid (31 mg, 0.056
mmol, 60%) was obtained upon removal of solvents in vacuo. TLC Rf = 0.34 (1:1 CH2Cl2/hexanes)
and Rf = 0.65 (chloroform), visualized by UV. IR (film) νmax 3473 (br), 3405 (br), 3064, 2980, 1724,
1591, 1470, 1426, 1343, 1320, 1230, 1158, 1128, 996, 976, 856, 749, 689 cm-1. 1H NMR (500
MHz, CDCl3) δ 9.49 (br s, 1H), 8.11 (d, J = 2.2 Hz, 1H), 7.58 (d, J = 2.2 Hz, 1H), 6.05 (s, 1H) ppm.
13C NMR (175 MHz, CDCl3) δ 147.4, 133.3, 131.0, 125.2, 119.5, 113.4, 112.1, 104.0, 101.1, 99.5.
ppm. LRMS (ESI-): calcd for C10H3Br5NO (M-H): 547.61, found 547.5.
Antibacterial Assay.
The Kirby-Bauer method was followed for the disk diffusion assay. Sterile filter paper disks
(Fisher Scientific, Porosity: Fine, Flow Rate: Slow, cut to 7.5 mm) were embedded with 5 μL of
pseudilin, pentabromopseudilin, or DMSO as a negative control. Pre-made antibiotic disks of
ciprofloxacin (5 μg), chloramphenicol (30 μg), and sulfamethoxazole with trimethoprim (5 μg; all
obtained from Carolina Biological Supply Company) were used as positive controls. Both
pseudilin (5) and pentabromopseudilin (2) were used at the following concentrations: 50 μM, 5
μM, 0.5 μM, 0.05 μM. After adding the compounds to the filter paper disks, they were
incubated overnight at 4 °C. The Staphylococcus epidermidis at a concentration of 1.5 x 108
CFU/ml was spread onto Mueller-Hinton agar plates (made from dehydrated medium from
Carolina Biological Supply company and poured 4mm deep) using a glass rod. The embedded
filter paper disks were added to the Mueller-Hinton agar plates and incubated at 37 °C for 21 hr.
A clear zone of inhibition around the filter paper disk indicated activity against S. epidermidis.
Supplementary Material
The supplementary material contains copies of 1H and 13C NMR spectra of 5 and 2 (PDF).

•
•
•
Two-step total synthesis of pentabromopseudilin (previously seven steps)
MIDA boronate ester Suzuki-Miyaura coupling
Bromination shown to be necessary for antimicrobial activity