Chlorizidine, a cytotoxic 5H-pyrrolo[2,1-a]isoindol-5-one-containing alkaloid from a marine Streptomyces sp.

Article abstract of DOI:10.1021/ol303374e

Cultivation of an obligate marine Streptomyces strain has provided the cytotoxic natural product chlorizidine A. X-ray crystallographic analysis revealed that the metabolite is composed of a chlorinated 2,3-dihydropyrrolizine ring attached to a chlorinated 5H-pyrrolo[2,1-a]isoindol-5-one. The carbon stereocenter in the dihydropyrrolizine is S-configured. Remarkably, the 5H-pyrrolo[2,1-a]isoindol-5-one moiety has no precedence in the field of natural products. The presence of this ring system, which was demonstrated to undergo facile nucleophilic substitution reactions at the activated carbonyl group, is essential to the molecule's cytotoxicity against HCT-116 human colon cancer cells.

Full text of DOI:10.1021/ol303374e

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Chlorizidine, a Cytotoxic
5H‑Pyrrolo[2,1‑a]isoindol-5-one-
Containing Alkaloid from a Marine
Streptomyces sp.
Xavier Alvarez-Mico, Paul R. Jensen, William Fenical, and Chambers C. Hughes*
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography,
University of California;San Diego, La Jolla, California 92093-0204, United States
chughes@ucsd.edu
Received December 10, 2012
ABSTRACT
Cultivation of an obligate marine Streptomyces strain has provided the cytotoxic natural product chlorizidine A. X-ray crystallographic analysis
revealed that the metabolite is composed of a chlorinated 2,3-dihydropyrrolizine ring attached to a chlorinated 5H-pyrrolo[2,1-a]isoindol-5-one.
The carbon stereocenter in the dihydropyrrolizine is S-configured. Remarkably, the 5H-pyrrolo[2,1-a]isoindol-5-one moiety has no precedence in
the field of natural products. The presence of this ring system, which was demonstrated to undergo facile nucleophilic substitution reactions at the
activated carbonyl group, is essential to the molecule’s cytotoxicity against HCT-116 human colon cancer cells.
There are a substantial number of chemotherapeutic
drugs on the market that are based on the scaffolds of
actinomycete-derived natural products.1 For instance, che-
mical studies of terrestrial Streptomyces bacteria led to the
discovery of actinomycin D,2a doxorubicin,2b bleomycin,2c
carzinophilin,2d chromomycin A3,2e mithramycin,2f mito-
mycin C,2g sarkomycin,2h streptozocin,2i and neocarzi-
nostatin.2f No truly novel actinomycete-derived natural
product structure has led to the development of a drug
in recent years,1 which at least suggests that terrestrial
actinomycetes are no longer a viable source for new lead
compounds. Studies of marine actinomycete bacteria, how-
ever, continue to yield unique chemical structures with
anticancer activity.3 For example, salinosporamide A from
Salinispora tropica is poised to enter phase II clinical trials.4
In an effort to identify new chemotypes for therapeutic
development, Streptomyces sp. strain CNH-287 was culti-
vated in a seawater-based medium (20 ꢀ 1 L).5 Notably,
the strain required seawater for growth. Amberlite resin
(XAD-18) was added after the first day of cultivation. The
resin was filtered and extracted with acetone after seven
days, and the crude material was then fractionated on silica
(1) (a) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311–335.
(b) Butler, M. S. Nat. Prod. Rep. 2005, 22, 162–195. For a microbial
perspective, see: (c) Berdy, J. J. Antibiot. 2005, 58, 1–26. (d) Demain,
A. L.; Sanchez, S. J. Antibiot. 2009, 62, 5–16.
(2) (a) Waksman, S. A.; Woodruff, H. B. Proc. Soc. Exper. Biol. Med.
1940, 45, 609–614. (b) Arcamone, F.; Cassinelli, G.; Fantini, G.; Grein,
A.; Orezzi, P.; Pol, C.; Spalla, C. Biotechnol. Bioeng. 1969, 11, 1101–
1110. (c) Umezawa, H.; Maeda, K.; Takeuchi, T.; Okami, Y. J. Antibiot.
1966, 19, 200–209. (d) Hata, T.; Koga, F.; Sano, Y.; Kanamori, K.;
Matsumae, A.; Sugawara, R.; Hoshi, T.; Shima, T.; Ito, S.; Tomizawa,
S. J. Antibiot. 1954, 7, 107–112. (e) Sato, K.; Okamura, N.; Utagawa, K.;
Ito, Y.; Watanabe, M. Sci. Rep. Res. Inst. Tohoku Univ. Med. 1960, 9,
224–232. (f) Julian, P. E.; Schenck, J. R. Antibiotics and Chemotherapy
1953, 3, 1218–1220. (g) Hata, T.; Hoshi, T.; Kanamori, K.; Matsumae,
A.; Sano, Y.; Shima, T.; Sugawara, R. J. Antibiot. 1956, 9, 141–146.
(h) Umezawa, H.; Takeuchi, T.; Nitta, K.; Yamamoto, T.; Yamaoka, S.
J. Antibiot. 1953, 6, 101. (i) Vavra, J. J.; Deboer, C.; Dietz, A.; Hanka,
L. J.; Sokolski, W. T. Antibiot. Annu. 1959, 7, 230–235. (f) Ishida, N.;
Miyazaki, K.; Kumagai, K.; Rikimaru, M. J. Antibiot. 1965, 18, 68–76.
(3) (a) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.;
Prinsep, M. R. Nat. Prod. Rep. 2013, 30, 237–323. (b) Blunt, J. W.; Copp,
B. R.; Keyzers, R. A.; Munro, M. H.; Prinsep, M. R. Nat. Prod. Rep.
2012, 29, 144–222. (c) Fenical, W.; Jensen, P. R. Nat. Chem. Biol. 2006, 2,
666–673.
(4) Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C. A.;
Jensen, P. R.; Fenical, W. Angew. Chem., Int. Ed. 2003, 42, 355–357.
(5) Strain CNH-287 (GenBank accession no. bankit JX680598) was
obtained from a marine sediment sample collected at the intertidal zone
near San Clemente, CA.
r
10.1021/ol303374e
XXXX American Chemical Society

gel. One fraction displayed significant cytotoxicity against
HCT-116 human colon cancer. A cytotoxic metabolite
with a prominent UV/vis profile was isolated from this
fraction using C18 reversed-phase HPLC.
It proved difficult to obtain a pure compound sufficient
for full spectroscopic analysis. During concentration from
either organic or aqueous solutions, extensive degradation
occurred. Much less decomposition was observed when
solutions were kept cool and dried under a stream of
nitrogen. In addition, the metabolite could be stored in
dilute solutions away from light and air.
literature. Various synthetic accounts of the heterocycle,
however, are well documented.7 The pyrroloisoindolone is
connected to a dichlorinated 2,3-dihydro-1H-pyrrolizine
at C-7.8 In the solid state, the congested region about the
sp2ꢁsp3 bond between the two ring systems forces the
molecule to adopt a twisted conformation. In solution,
though, 1 does not appear to exhibit atropisomerism.9
Table 1. 1H, 13C, and HMBC NMR Spectral Data for
Chlorizidine A (1) (CD3CN)
A nonzero optical rotation, [R]D ꢁ35 (c 0.50, CH3CN),
indicated that the natural product was optically active, and
a strong IR stretch at 1721 cmꢁ1 revealed the presence of
a carbonyl group. Mass spectrometry data for the natural
product [HRESI-FT-MS m/z (M þ H)þ = 442.9511,
444.9481, 446.9452, 448.9422] showed a molecular ion
cluster consistent with molecular formulas that include
Cl2Br or Cl4. With 13 degrees of unsaturation, however,
only C18H10Cl4N2O3 agreed with proton and carbon
NMR data.
δH, mult. (J, Hz)b
HMBCb
a
C no.
δC
2, 3, 5a, 14, 15c
1
101.9
163.0
163.9
113.3d
157.5
108.2
135.5d
132.7d
53.0
6.55, br s
5
6
7
8
9
6.42, s
5, 7, 9a, 9b
9a
9b
10
11
5.80, dde
2.84, m
2.54, m
3.08, m
2.90, m
6ꢁ8, 11
We initially attempted to solve the structure of the
natural product using 1D and 2D NMR (COSY, HSQC,
HMBC) experiments (Table 1). The numbering of the
molecule is shown in Figure 1. Low-field signals at δH
6.55 (δC 101.9) and 6.42 (δC 108.2) were conspicuous, in
addition to two overlapping signals at δH 5.80 (δC 99.3 and
δC 53.0). A spin system including a proton at δH 5.80 and
the remaining upfield methylene protons at δH 3.08, 2.90,
32.5
7, 12, 12a
6ꢁ8, 10, 12, 12a
10, 11, 12a, 13
10, 11, 12a, 13
12
25.4
12a
13
136.8
99.3
5.80, se
12a, 14
a 75 MHz. b 500 MHz. c δC = 116.9, 113.0d, 109.7, 106.0, 105.9.
d Signals may be switched. e Overlapping signals.
1
1
2.84, and 2.54 was apparent in the Hꢁ H COSY spec-
trum. Interestingly, the upfield proton signals from δ
2.54ꢁ3.08 exhibited complex splitting patterns due to
the flexibility in the molecule (vide infra). The paucity of
hydrogen atoms and the plethora of quaternary carbon
atoms made complete structural elucidation by NMR
problematic.
The phenolic substituents at C-6 and C-8 of chlorizidine
A (1) could be readily functionalized. Treatment of 1 with
acetic anhydride/triethylamine gave 2, and methylation
with dimethyl sulfate provided 3 (Scheme 1). Acetate 2 was
a stable chemical entity much less prone to degradation
than 1. Interestingly, like the natural product, its proton
NMR spectrum showed evidence of slow C-7/C-10 bond
rotation relative to the NMR time scale. The well-resolved
proton signals at C-1, C-9, C-10, and C-13 in 1 were now
“doubled” in 2. The diacetate structure was confirmed
using X-ray crystallography. The crystal was composed
of two low-energy “twisted” conformers (see Supporting
Information (SI)).
The structure of chlorizidine A (1) was finally deter-
mined using X-ray crystallographic techniques (Figure 2).
Slow evaporation of a concentrated solution of 1 in
benzene provided X-ray quality crystals. A molecule of
benzene was incorporated into the crystal lattice.6 Addi-
tionally, the lone tertiary carbon stereocenter was assigned
an S-configuration [Flack parameter ꢁ0.02(2)].
Chlorizidine A (1) displays an unprecedented structure
involving a nitrogen-containing carbon skeleton. The dis-
covery of a naturally occurring 5H-pyrrolo[2,1-a]isoindol-
5-one ring system has not been previously described in the
(6) On the inclusion of solvent molecules in the crystal structures of
€
organic molecules, see: Gorbitz, C. H.; Hersleth, H.-P. Acta Crystallogr.
2000, B56, 526–534.
(7) For the synthesis of 5H-pyrrolo[2,1-a]isoindol-5-ones, see:
(a) Marsili, A.; Scartoni, V.; Morelli, I.; Pierangeli, P. J. Chem. Soc.,
Perkin Trans. 1 1977, 959–965. (b) Itahara, T. J. Chem. Soc., Chem.
Commun. 1981, 254–255. (c) Maruyama, K.; Kubo, Y. J. Org. Chem.
1981, 46, 3612–3622. (d) Crabb, T. A.; Patel, A.; Newton, R. F.; Price,
B. J.; Tucker, M. J. J. Chem. Soc., Perkin Trans. 1 1982, 2783–2786.
(e) Danheiser, R. L.; Kwasigroch, C. A.; Tsai, Y.-M. J. Am. Chem. Soc.
1985, 107, 7233–7235. (f) Grigg, R.; Sridharan, V.; Stevenson, P.;
Sukirthalingam, S.; Worakun, T. Tetrahedron 1990, 46, 4003–4018. (g)
Yavari, I.; Islami, M. R. J. Chem. Res. (S) 1998, 166–167. (h) Kaden, S.;
Figure 1. (ꢁ)-(S)-Chlorizidine A (1).
(8) For related plant-derived pyrrolizidine alkaloids, see: Smith,
L. W.; Culvenor, C. C. J. J. Nat. Prod. 1981, 44, 129–152.
(9) The conformers could not be resolved using either achiral or
chiral column chromatography.
€
Reissig, H.-U.; Brudgam, I.; Hartl, H. Synthesis 2006, 1351–1359.
(i) McNab, H.; Tyas, R. G. J. Org. Chem. 2007, 72, 8760–8769.
B
Org. Lett., Vol. XX, No. XX, XXXX

Scheme 1. Acylation and Methylation of the Phenolic Groups in
Chlorizidine A (1)
Figure 2. ORTEP plot of 1 with benzene (cocrystallized).
The semisynthesis of bulkier phenolic esters;isobuty-
rate 4, pivalate 5, and benzoate 6;was undertaken in
an attempt to produce an atropiosmeric mixture, but this
approach was not successful (see Scheme 1).6 Analysis of
these derivatives was much more complex, as the ester
functionalities revealed their own conformational prefer-
ences. The proton NMR spectra showed the simultaneous
presence of several confomers with distinct chemical shifts
(see SI).
A second metabolite that was prone to degradation,
chlorizidine B (7), was isolated from culture extracts of
CNH-287 (Scheme 2). A stable diacetate adduct 8 was
constructed using acetic anhydride. An additional aromatic
NMR signal corresponding to C-7 was noticeable. Pre-
sumably, pyrrole 7 arises from hydrolysis and decarboxyla-
tion of chlorizidine A (1). The notion that 7 is an artifact
of the culturing process was substantiated by treating 1
with a pH 10 buffer composed of K2B4O7, K2CO3, and
KOH.10 Under these conditions, the C-7 carboxylic acid
was observed [LRESI-MS m/z (MꢁH)ꢁ = 459, 461,
463].11 Notably, many other compounds are formed from
the degradation of chlorizidine (1) under basic conditions
(see SI).
The central electrophilic carbonyl group of the 5H-
pyrrolo[2,1-a]isoindol-5-one moiety engages sulfur-, oxy-
gen-, and amine-containing nucleophiles in a substitution
reaction, whereby the electron-poor dichloropyrrole func-
tions as a leaving group (see Scheme 2).5g When subjected
to N-acetylcysteamine and potassium carbonate, thioester
9 was produced. Likewise, treatment of 1 with benzylamine
gave amide 10 and treatment with potassium carbonate in
methanol afforded ester 11. Unlike 9 and 10, ester 11 was
not suitably stable for complete purification and analysis.
Peracetylation of 9ꢁ11 furnished stable derivatives 12ꢁ14.
A more efficient method for the synthesis of 14 was
accomplished by treating diacetate 2 with aqueous NaOH
in CH3OH followed by acetylation. Certainly, the isolation
ofpure chlorizidine A (1) ishindered, in part, bythe lability
of the pyrroloisoindolone moiety toward nucleophiles.
Chlorizidine A (1) and acylated derivatives 2, 4, and 5
exhibit noteworthy activity in a colon cancer cytotoxicity
bioassay (Table 2).12 Against the HCT-116 adenocarcino-
ma cell line,13 1 showed an IC50 of 3.2ꢁ4.9 μM. Compounds
2, 4, and 5 showed similar activity. Irreversible methylation
of the phenolic functionality in 1, yielding 3, rendered the
compound completely inactive. Any of the series of deriva-
tives lacking the key pyrroloisoindolone ring system (7ꢁ14)
had no measurable activity, strongly suggesting that this
moiety is a crucial part of the metabolite’s pharmacophore.
Chlorizidine A diacetate (2) was tested against the NCI’s
panel of 60 tumor cell lines.14 It was modestly selective
in terms of its cytotoxicity (see SI). However, against
SK-MEL-5 and SK-MEL-2 melanoma cancer cells, 2
showed a pronounced LC50 of 3.6 and 11 μM, respectively.
Against MDA-MB-231/ATCC breast cancer cells, an
LC50 of 11 μM was also determined.
Chlorizidine A (1) is structurally similar to marinopyr-
role A (15), a secondary metabolite from marine-derived
Streptomyces sp. CNQ-418 (Scheme 3).15 Biosynthetic
precursor 16 is derived from a mixed NRPS-PKS pathway,
whereby proline is loaded onto a peptidyl carrier protein,
oxidized, chlorinated by an FADH2-dependent halogen-
ase, and subsequently extended by the PKS machinery.16
Cyclization/aromatization provides monodeoxypyolute-
orin (17).17 1,30-Bipyrrole 15 is then formed via a novel
(12) The therapeutic properties of other 5H-pyrrolo[2,1-a]isoindol-5-
one-containing compounds have been described. For dihydropyridine-
substituted 5H-pyrrolo[2,1-a]isoindol-5-ones and their use, see: Kuhl, A.
D.E. Patent 10,034,265, 2005. For 5H-pyrrolo[2,1-a]isoindol-5-ones that
function as Cdk4 inhibitors, see: Honma, T.; Hayashi, K.; Aoyama, T.;
Hashimoto, N.; Machida, T.; Fukasawa, K.; Iwama, T.; Ikeura, C.; Ikuta, M.;
Suzuki-Takahashi, I.; Iwasawa, Y.; Hayama, T.; Nishimura, S.; Morishima,
H. J. Med. Chem. 2001, 44, 4615–4627.
(13) Brattain, M. G.; Fine, W. D.; Khaled, F. M.; Thompson, J.;
Brittain, D. E. Cancer Res. 1981, 41, 1751–1756.
(10) Toward the end of the seven-day cultivation, the pH of the
culture broth normally reaches 10.
(14) Shoemaker, R. H. Nat. Rev. Cancer 2006, 6, 813–823.
(11) Since the carboxylic acid compound could not be isolated, the
decarboxylation event was not clearly demonstrated in vitro. For the
facile decarboxylation of 2,4-dihydroxybenzoic acid derivatives, see:
Horper, W.; Marner, F.-J. Phytochemistry 1996, 41, 451–456.
(15) (a) Hughes, C. C.; Prieto-Davo, A.; Jensen, P. R.; Fenical, W.
Org. Lett. 2008, 10, 629–631. (b) Hughes, C. C.; Kauffman, C. A.;
Jensen, P. R.; Fenical, W. J. Org. Chem. 2010, 75, 3240–3250.
Org. Lett., Vol. XX, No. XX, XXXX
C

Scheme 2. Reactivity of the 5H-Pyrrolo[2,1-a]isoindol-5-one in
Chlorizidine A (1)
Scheme 3. Proposed Biosynthetic Relationship of 1 to
Marino-pyrrole A (15)
its simplicity, may be utilized tomake two diverse, complex
structure types is remarkable.
Derived from what appears to represent a new, obligate
marine Streptomyces sp., chlorizidine isthe first example of
a natural product containing a 5H-pyrrolo[2,1-a]isoindol-
5-one ring. Studies are now in progress to examine the
biosynthesis and mechanism of action of these novel
metabolites.
Acknowledgment. Financial support was provided by
the NIH National Cancer Institute under Grant R37-
CA044848. X. A.-M. was supported by the commission
for Universities and Research from the Department of
Innovation, Universities and Enterprises of the Generalitat
de Catalunya (fellowship 2007 BP-A 00042). We thank
the NCI for performing the 60 cell line drug screen and
Drs. Arnold L. Rheingold and Curtis Moore (UCSD) for
providing X-ray diffraction structures. We also thank
Kelley A. Gallagher (SIO) for the phylogenetic tree in
the SI. In support of much of the work in this manuscript,
Prof. Ted Molinski (UCSD) graciously provided labora-
tory space and equipment.
Table 2. Cytotoxicity of 1ꢁ14 (μM)
a
HCT116 (IC50
)
1
3.2ꢁ4.9
0.6ꢁ3.0
NSA
2
3
Supporting Information Available. Phylogenetic analy-
sis of strain CNH-287. Isolation procedures for 1 and 7.
Procedures for the synthesis of 2ꢁ6, 8ꢁ14 including
HRMS data, proton and carbon NMR spectra, and
UV/vis spectra. Crystallographic data for 1 (CCDC
900052) and 2 (CCDC 900051) in CIF format. The
material is available free of charge via the Internet at
http://pubs.acs.org.
4
1.0ꢁ1.2
2.1ꢁ3.7
NSA
5
6ꢁ14
a HCT-116 is a human colon cancer cell line. Positive control:
etoposide (IC50 = 0.49ꢁ4.9 μM). NSA = no significant activity.
atroposelective N,C-pyrrole coupling reaction.18 An alter-
native route using 16 that includes N-acylation and reduc-
tion could yield chlorizidine A (1). The fact that 16, despite
(17) (a) Takeda, R. J. J. Am. Chem. Soc. 1958, 80, 4749–4750.
(b) Takeda, R. J. Bull. Agr. Chem. Soc. Jpn. 1959, 23, 126–130.
(18) Yamanaka, K.; Ryan, K. S.; Gulder, T. A. M.; Hughes, C. C.;
Moore, B. S. J. Am. Chem. Soc. 2012, 134, 12434–12437.
(16) (a) Nowak-Thompson, B.; Chaney, N.; Wing, J. S.; Gould, S. J.;
Loper, J. E. J. Bacteriol. 1999, 181, 2166–2174. (b) Thomas, M. G.;
Burkart, M. D.; Walsh, C. T. Chem. Biol. 2002, 9, 171–184. (c)
Dorrestein, P. C.; Yeh, E.; Garneau-Tsodikova, S.; Kelleher, N. L.;
Walsh, C. T. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 13843–13848.
The authors declare no competing financial interest.
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