Scalable synthesis of cladosporin

Article abstract of DOI:10.1016/j.tetlet.2019.02.012

Cladosporin, a secondary metabolite isolated from fungal sources like Cladosporium cladosporioides and Aspergillus flavus was found to exhibit selective nano-molar activity against malarial parasite Plasmodium falciparum by inhibiting parasitic protein biosynthesis. In addition, this natural product has a broad range of bioactivities including, antiparasitic, antifungal, antibacterial as well as plant growth inhibition. However, it has limited availability from the natural sources for further development. Herein, we report a modified and improved synthetic route which led us to produce this potent natural product in a gram scale. Conversion of the undesired diastereomer to desired one via Mitsunobu inversion of secondary alcohol and carbon monoxide insertion reaction towards the construction of isocoumarin unit are the key features of the present synthesis.

Full text of DOI:10.1016/j.tetlet.2019.02.012

Accepted Manuscript
Scalable Synthesis of Cladosporin
Pronay Das, Yash Mankad, D. Srinivasa Reddy
PII:
S0040-4039(19)30120-0
https://doi.org/10.1016/j.tetlet.2019.02.012
TETL 50606
DOI:
Reference:
Tetrahedron Letters
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Revised Date:
Accepted Date:
11 January 2019
2 February 2019
5 February 2019
Please cite this article as: Das, P., Mankad, Y., Reddy, D.S., Scalable Synthesis of Cladosporin, Tetrahedron
Letters (2019), doi: https://doi.org/10.1016/j.tetlet.2019.02.012
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Tetrahedron Letters
Scalable Synthesis of Cladosporin
Pronay Dasa,b , Yash Mankada and D. Srinivasa Reddya,b
a Organic Chemistry Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India
E-mail: ds.reddy@ncl.res.in
b Academy of Scientific and Innovative Research (AcSIR), New Delhi, 110 025, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Cladosporin, a secondary metabolite isolated from fungal sources like Cladosporium
cladosporioides and Aspergillus flavus was found to exhibit selective nano-molar activity
against malarial parasite Plasmodium falciparum by inhibiting parasitic protein biosynthesis. In
addition, this natural product has a broad range of bioactivities including, antiparasitic,
antifungal, antibacterial as well as plant growth inhibition. However, it has limited availability
from the natural sources for further development. Herein, we report a modified and improved
synthetic route which led us to produce this potent natural product in a gram scale. Conversion
of the undesired diastereomer to desired one via Mitsunobu inversion of secondary alcohol and
carbon monoxide insertion reaction towards the construction of isocoumarin unit are the key
features of the present synthesis.
Available online
Keywords:
Cladosporin
Total Synthesis
Anti-malarials
Mitsunobu
2009 Elsevier Ltd. All rights reserved.
Cladosporin, also known as asperentin is a secondary metabolite
found in diverse fungi including Cladosporium cladosporioides1
and Aspergillus flavus2 with the first isolation documented way
back in 1971 by Scott and Walbeek.1 Structurally it consists of a
THP ring (2,6-disubstituted tetrahydropyran) connected to an
isocoumarin moiety1,3,4 (Figure 1) whose complete
towards drug development demands adequate quantity of the lead
compound (cladosporin), which has limited access from natural
sources. Hence development of an efficient and scalable synthetic
strategy is worth exploring. As of today, one asymmetric total
synthesis4 followed by a formal synthesis3 of the natural product
have been documented by She et. al. and Mohapatra et. al. using
independent and elegant ways, respectively. Following that, we
have documented a synthesis of cladosporin, where we have
adopted a strategy to access different isomers deliberately.11
Although these synthetic routes to access cladosporin were
documented, there is a need for new route which can provide
sufficient material. Here we report a modified approach to access
cladosporin in “gram-scale”.
stereochemical elucidation was reported in 1981 by Springer et.
al.2 Cladosporin was found to illustrate a broad spectrum of bio-
activities (Figure 1) such as antifungal,1 insecticidal, plant growth
inhibition2,5 and antibacterial,6 as well as anti-inflammatory
activity.7 While screening natural product library to identify
inhibitors of Plasmodium falciparum (Pf), which happens to be
the causative pathogen for malaria; Winzeler et. al. identified
cladosporin to display potent antiparasitic activity (~40 nM)
against both blood and liver stage proliferation of the pathogen
by ceasing protein biosynthesis in the parasitic cell through
inhibition of cytosolic lysyl-tRNA synthetase (PfKRS).8,9
Besides, cladosporin was found to be >100 fold selective towards
parasitic KRS as compared to human enzyme (HsKRS).9 Apart
from PfKRS, cladosporin is known to inhibit KRSs from other
species, including helminth parasites such as Loa loa (Ll) and
Schistosomamansoni (Sm).10 By considering promising potential
of cladosporin, we have initiated a program towards it. Recently,
we have accomplished the synthesis of all the possible eight
stereoisomers (cladologs) of cladosporin and in collaboration
with Sharma et. al. we successfully deciphered the
Our synthesis commenced with the known intermediate 1
(prepared through a reported protocol) which was subjected to
epoxidation using mCPBA reagent to its corresponding epoxide
(fragment B) as an inseparable diastereomeric mixture. The
epoxide thus obtained, on Grignard reaction with commercially
available 1-bromo-3,5-dimethoxybenzene (fragment A) afforded
1:1 diastereomeric mixture of alcohols (2 and 3) with excellent
overall yield of 86%. It is worth mentioning that maintaining a
low concentration of Grignard reagent (< 0.5 M) is crucial for the
reaction. Higher concentrations of Grignard reagent results in an
unrequired dimerization product.13 Here, in this case, we were
able to separate both the diastereomers (2 and 3) cleanly using
simple silica gel column chromatography. In this context, we
made interesting observations while analyzing 1H NMR data of
these isomers where identical chemical shifts of all the concerned
stereochemical bases of cladologs’ interaction with PfKRS
through cladolog-PfKRS co-crystallization.11 These interesting
findings and impressive biological profile of cladosporin
undoubtedly make it as a promising candidate towards the
development of novel antimalarials. A proper bio-assessment
———
 Corresponding author. e-mail: ds.reddy@ncl.res.in

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Tetrahedron Letters
14
3
10
Figure 1: Spectrum of Cladosporin bio-activities.
Hydrogen bonding
Energy minimized conformation
Compound 3
2.79
Compound 2
2.73
2.71
2.62 2.61
2.60
2.78
2.80
2.82
2.58
1H NMR
benzylic CH2 splitting
2.85
2.80
2.75
2.70
2.65
2.60
2.55
2.75
2.70
2.65
2
1
1
ORTEP of Cladosporin
Scheme 1: Gram scale synthesis of cladosporin

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proton signals were observed for both the compounds except the benzylic protons, which showed significant chemical shift
difference for both. This difference in the1HNMR pattern could probably be explained using energy minimized conformer of both
the compounds using Gaussian. Unlike compound 2, compound 3 was found to exhibit a hydrogen bonding between the secondary
alcohol and the tetrahydropyran oxygen which might be the probable cause for a constrained conformation of compound 3 and this
in turn resulted in a separate splitting pattern for both the benzylic protons (Scheme 1). To make use of undesired diastereomer,
compound 3 was subjected to Mitsunobu reaction protocol followed by ester hydrolysis which led to the complete inversion of the
secondary alcohol center in 3 to afford the required diastereomeric alcohol 2 in good yields. Next we focused to construct the six-
membered lactone present in the natural product which was envisioned using palladium catalyzed carbon monoxide insertion
reaction. Accordingly, the alcohol 2 was converted to its corresponding iodo-compound 4a in excellent yields by using N-
iodosuccinimide (NIS) and catalytic amount of pTSA in chloroform solvent. The iodo-compound 4a was treated with Pd(PPh3)2Cl2,
potassium carbonate and 1,10-phenanthroline in presence in DMF at 100 °C under blanket of carbon monoxide to obtain desired
compound 5 in 70% yield.15 Demethylation of compound 5 was achieved through aluminium triiodide mediated exhaustive
demethylation16 to furnish the natural product cladosporin. The spectral data of the synthesized compound was in complete
agreement with the reported data.3,4 Besides, structure and relative stereochemistry of the synthesized compound was further
confirmed by single crystal X-ray diffraction analysis for an unambiguous assignment of its stereocenters. Herein, we would also
like to document alternate ways/attempts to form the six-membered lactone ring of cladosporin, in particular using bromo

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Tetrahedron Letters
compound 4b. Alcohol 2 upon treatment with N-bromo succunimide (NBS) in CH2Cl2 afforded bromo compound 4b in excellent
yield. The bromo compound thus obtained treated with nBuLi, LiHMDS to generate corresponding dianion which was subsequently
quenched with electrophiles like triphosgene, carboxydiimidazole (CDI),methylchloroformate and ethyl chloroformate which did
not give any fruitful results(Scheme 2). We also tried to insert copper in the carbon halogen bond followed by protodecupration to
furnish the required lactone.14Although we were successful in obtaining the required product 5 in a moderate yield of 60%, we were
unable to reproduce comparable yields at higher scale. As an alternative, we also prepared the required lactone 5 by following She’s
protocol.4
In short, we have accomplished the scalable total synthesis of cladosporin natural product. Gram-scale operations, Mitsunobu
inversion to convert undesired alcohol to required one and palladium-catalyzed carbon monoxide insertion reaction to construct six-
membered lactone ring are the highlights of present work. Now we have more than two grams of material in hand which is
sufficient for further profiling such as in-depth assessment of the pharmacokinetics and pharmacodynamics.
Acknowledgments
We would like to acknowledge University Grants Commission (UGC) for providing research fellowship to PD. We would also like
to express our gratitude to Dr. Amit Sharma (ICGEB, New Delhi) for goading us into this project. Besides we thank Dr. Rambabu
Dandela for X-ray crystallographic analysis of
Scheme 2: Alternatives towards lactonization
cladosporin and Tamal Das for conducting the energy minimization of two diastereomers. This work is a part of the project proposal
submitted to SERB, New Delhi, India, under the special call “Theme-Based Call for Proposals Initiated by the Program Advisory
Committee of Organic Chemistry” (Reference EMR/2016/004301/OC). This proposal is currently under evaluation.
References and notes
[1] P. M. Scott, W. Van Walbeek, W. M. Maclean, J. Antibiot. 24 1971) 747.
[2] J. P. Springer, H. G. Cutler, F. G. Crumley, R. H. Cox, E. E. Davis, J. E. Thean, J. Agric. Food Chem. 29 (1981) 853.
[3] D. K. Mohapatra, S. Maity, T. S. Rao, J. S. Yadav, B. Sridhar, Eur. J. Org. Chem. 2013 (2013) 2859.
[4] H. Zheng, C. Zhao, B. Fang, P. Jing, J. Yang, X. Xie, X. She, J. Org. Chem. 77 (2012) 5656.
[5] Y. Kimura, N. Shimomura, F. Tanigawa, S. Fujioka, A. Shimada, Z Naturforsch C. 67 (2012) 587.
[6] H. Anke, J. Antibiot. (Tokyo) 32 (1979) 952.
[7] J. D. Miller, M. Sun, A. Gilyan, J. Roy, T. G. Rand, ChemBiol Interact. 183 (2010) 113.
[8] D. Hoepfner, C. W. McNamara, C. S. Lim, C. Studer, R. Riedl, T. Aust, S. L. McCormack, D. M. Plouffe, S. Meister, S. Schuierer, U. Plikat, N.
Hartmann, F. Staedtler, S. Cotesta, E. K. Schmitt, F. Petersen, F. Supek, R. J.Glynne, J. A. Tallarico, J. A. Porter, M. C. Fishman, C. Bodenreider,
T. T. Diagana, N. R. Movva, E. A. Winzeler, Cell Host Microbe. 11 (2012) 654..

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[9] S. Khan, A. Garg, N. Camacho, J. Van Rooyen, A. Kumar Pole, H. Belrhali, L. Ribas de Pouplana, V. Sharma, A. Sharma, ActaCrystallogr.,
Sect. D: Biol. Crystallogr. 69 (2013) 785.
[10] A. Sharma, M. Sharma, M. Yogavel, A. Sharma, PLoS Neglected Trop. Dis. 10 (2016) 1.
[11] P. Das, P. Babbar, N. Malhotra, M. Sharma, G. R. Jachak, R. G. Gonnade, D. Shanmugam, K. Harlos, M. Yogavel, A. Sharma, D. S. Reddy, J.
Med. Chem. 61 (2018) 5664.
[12] A. S. Legera, C. Sinadinosa, L. R. Pouplana, Bioengineered 7 (2016) 60.
[13] Formation of dimeric compound (tentative structure shown below) at higher concentrations of Grignard reagent was observed.
[14] U. Nookraju, E. Begari, P. Kumar, Org. Biomol. Chem. 12 (2014) 5973.
[15] B. T. V. Srinivas, A. R. Maadhur, S. Bojja, Tetrahedron, 70 (2014) 8161.
[16] J. Tian, D. Sang, ARKIVOC, 2015 (2015) 446.
Supplementary Material
Experimental details, copies of NMR spectra are provided. Supplementary data of this article can be found online at….

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Tetrahedron Letters
Graphical Abstract
Scalable Synthesis of Cladosporin
Pronay Dasa,b, Yah Mankada and D. Srinivasa Reddya,b
Leave this area blank for abstract info.
MITSUNOBU INVERSION
CO INSERTION
Fragment A
Fragment B
RAM SCLALE
G
1.45 grams
2 grams

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Highlights:
-
-
-
Developed a scalable synthetic route to
cladosporion, a potent antimalarial natural
product.
Mitsunobu inversion and carbon monoxide
insertion are the key steps of the present
synthesis.
The target natural product cladosporin has
been synthesized in gram scale for the first
time.