Isolation and synthesis of (-)-(5S)-2-Imino-1-methylpyrrolidine-5- carboxylic acid from cliona tenuis: Structure revision of pyrostatins

Article abstract of DOI:10.1021/ol062087k

(-)-(5S)-2-Imino-1-methylpyrrolidine-5-carboxylic acid (1), previously reported as the N-acetyl-β-D-glucosaminidase inhibitor pyrostatin B, has been isolated from the organic extracts of the burrowing sponge Cliona tenuis. The structure of 1, including its absolute stereochemistry, was characterized from its spectral data and chemical transformations and confirmed by total synthesis. The synthesis of 1 reveals that the structure of pyrostatin B has been incorrectly assigned. Comparison of NMR spectral data strongly suggests that pyrostatins A and B are identical to 5-hydroxyectoine and ectoine, respectively.

Full text of DOI:10.1021/ol062087k

ORGANIC
LETTERS
2006
Vol. 8, No. 21
4967-4970
Isolation and Synthesis of
)-(5S)-2-Imino-1-methylpyrrolidine-5-
(−
carboxylic Acid from Cliona tenuis:
Structure Revision of Pyrostatins
Leonardo Castellanos,† Carmenza Duque,† Sven Zea,‡ Alfonso Espada,§
|
|
Jaime Rodr´ıguez, and Carlos Jime´nez*,
Departamento de Qu´ımica, UniVersidad Nacional de Colombia,
AA 14490 Bogota´, Colombia, Departamento de Biolog´ıa y Centro de Estudios en
Ciencias del Mar, UniVersidad Nacional de Colombia, Cerro Punta de Bet´ın,
AA 10-16 (INVEMAR), Santa Marta, Colombia, Analytical Technologies DCR&T
Alcobendas, Lilly S.A., AVda. de la Industria 30, 28108-Alcobendas/Madrid, Spain, and
Departamento Qu´ımica Fundamental, Facultad de Ciencias, UniVersidad de A Corun˜a,
A Corun˜a E-15071, Spain
carlosjg@udc.es
Received August 23, 2006
ABSTRACT
(−)-(5S)-2-Imino-1-methylpyrrolidine-5-carboxylic acid (1), previously reported as the N-acetyl-â-D-glucosaminidase inhibitor pyrostatin B, has
been isolated from the organic extracts of the burrowing sponge Cliona tenuis. The structure of 1, including its absolute stereochemistry, was
characterized from its spectral data and chemical transformations and confirmed by total synthesis. The synthesis of 1 reveals that the
structure of pyrostatin B has been incorrectly assigned. Comparison of NMR spectral data strongly suggests that pyrostatins A and B are
identical to 5-hydroxyectoine and ectoine, respectively.
Although substituted pyrrolidines can be found in numerous
natural products and pharmaceutically active compounds,1
2-iminopyrrolidines are not very common. Two antibacterial
compounds bearing a new 2-iminopyrrolidine carboxylic acid
structure were isolated recently from Burkholderia plantarii,
a bacterial pathogen of rice.2 Furthermore, recent studies have
shown that a series of substituted 2-iminopyrrolidines are
potent and selective inhibitors of human inducible nitric oxide
synthases (NOS), and they were postulated as potential
therapeutic agents.3
Continuing our search for biologically active secondary
metabolites and the possible role that these compounds play
in the chemical defense of marine sponges,4 we focused our
attention on the Caribbean sponge Cliona tenuis because it
aggressively undermines and displaces live coral tissue and
because its organic extracts showed a potent lethal activity
against corals Madracis mirabilis, Montrastea caVernosa,
and Siderastrea siderea in laboratory and field assays.5
(3) (a) Hagen, T. J.; Bergmanis, A. A.; Kramer, S. W.; Fok, K. F.;
Schmelzer, A. E.; Pitzele, B. S.; Swenton, L.; Jerome, G. M.; Kornmeier,
G. M.; Moore, W. M.; Branson, L. F.; Connor, J. R.; Manning, P. T.; Currie,
M. G.; Hallinan, E. A. J. Med. Chem. 1998, 41, 3675-3683. (b) Tsymbalov,
S.; Hagen, T. J.; Moore, W. M.; Jerome, G. M.; Connor, J. R.; Manning,
P. T.; Pitzele, B. S.; Hallinan, E. A. Bioorg. Med. Chem. Lett. 2002, 12,
3337-3339. (c) Benati, L.; Bencivenni, G.; Leardini, R.; Nanni, D.; Minozzi,
M.; Spagnolo, P.; Scialpi, R.; Zanardi, G. Org. Lett. 2006, 8, 2499-2502.
(4) (a) Lo´pez-Victoria, M.; Zea, S.; Weil, E. Mar. Ecol. Prog. Ser. 2006,
312, 113-121. (b) Petrichtcheva, N. V.; Duque, C.; Duen˜as, A.; Zea, S.;
Hara, N.; Fujimoto, Y. J. Nat. Prod. 2002, 65, 851-855.
† Departamento de Qu´ımica, Universidad Nacional de Colombia.
‡ Departamento de Biolog´ıa y Centro de Estudios en Ciencias del Mar,
Universidad Nacional de Colombia.
§ Analytical Technologies DCR&T Alcobendas Lilly S.A.
| Departamento de Qu´ımica Fundamental, Universidad de A Corun˜a.
(1) O’Hagan, D. Nat. Prod. Rep. 2000, 17, 435-446.
(2) Mitchell, R. E.; Teh, K. L. Org. Biomol. Chem. 2005, 3, 3540-
3543.
10.1021/ol062087k CCC: $33.50
© 2006 American Chemical Society
Published on Web 09/19/2006

Sponges belonging to the Cliona genus are usually burrowing
organisms that are able to excavate a variety of calcareous
substrates such as rocks, coralline reefs, and oyster shells.
Previous chemical investigations on Cliona species led to
the isolation of different classes of secondary metabolites
such as steroids,6 linear peptides,7 and pyrrole alkaloids.8
bioactive fraction. Finally, RP-HPLC purification on a
Discovery F5 column [using acetonitrile/water (8:2) and
0.05% formic acid] of 80 mg of that fraction yielded 3.5
mg of compound 1, which was slightly contaminated with
some salts. To isolate this compound in a cleaner way, a
portion of the same fraction (100 mg) was methylated using
methanol and thionyl chloride and then purified by HPLC
using acetonitrile/water (1:9) and 0.05% formic acid to afford
3.3 mg of the methyl ester derivative 2.
The prominent (+)-ESIMS pseudomolecular ion [M +
H]+ at m/z 143 and the pair [M + Na]+/[M + K]+ at m/z
165 and 181, respectively, for 1 and the corresponding
[M - H]- at m/z 141 in the (-)-ESIMS are consistent with
a molecular weight of 142 amu. The (+)-HRESIMS of the
pseudomolecular [M + H]+ ion at m/z 143.0819 established
for 1 the molecular formula C6H11N2O2 (calcd 143.0815).
The 13C NMR and DEPT spectra of 1 in D2O (Table 1)
displayed six distinct resonances: two sp2 quaternary carbons
at δ 169.55 ppm (C-2) and δ 177.43 ppm (C-7), suggesting
the presence of a CdN carbon and a carboxylic acid,
respectively; two methylenes at δ 24.29 ppm (C-4) and δ
30.12 ppm (C-3); a methine at δ 70.36 ppm (C-5); and finally
a methyl group at δ 31.74 ppm (C-6). The presence of a
carboxylic acid was also confirmed from the mass peak in
the (+)-ESIMS of 1 at m/z 97, corresponding to the loss of
COOH.
Allelopathic bioassay guided fractionation of the organic
extracts of the sponge allowed us to find a very bioactive
fraction from which we isolated and characterized compound
1. Analysis of the spectral data indicated that its structure is
2-imino-1-methylpyrrolidine-5-carboxylic acid, which was
previously reported as pyrostatin B.9 However, the data were
different from those published for pyrostatin B.9a Because
of this discrepancy, the total synthesis of 2-imino-1-meth-
ylpyrrolidine-5-carboxylic acid was addressed. This allowed
us to confirm our proposed structure for compound 1 and,
consequently, to demonstrate that the reported structure of
pyrostatin B was incorrect. Furthermore, on searching the
literature, we found that the actual NMR data reported for
pyrostatin B matched those of ectoine.10,11
A combination of 1H-1H COSY, HSQC, and long-range
C-H correlations (HMBC) were used to construct the
molecule through quaternary carbons and the nitrogen (Figure
1). On the basis of these data, we assigned compound 1 as
2-imino-1-methylpyrrolidine-5-carboxylic acid.
Sponge specimens (7 kg) were collected from the Rosario
Islands in the Colombian Caribbean and extracted with
MeOH and CH2Cl2. The methanol and dichloromethane
extracts (93 g) were combined, evaporated in vacuo, and then
partitioned between CH2Cl2 and H2O. The bioactive aqueous
layer was evaporated and further partitioned between water
and n-BuOH saturated with water, with the activity again
remaining in the aqueous layer. After evaporation of the
solvent in vacuo, the residue was loaded onto XAD-4 resin,
which was washed with distilled water, then with methanol,
and finally acetone. The fraction eluted with water, which
was found to be the most active, was evaporated under
vacuum. Slow addition of methanol to this fraction allowed
the sequential precipitation of inorganic media components.
Filtration and evaporation of the methanol filtrate to dryness
yielded a residue (14 g), which was chromatographed on
Sephadex LH-20 (10% MeOH/H2O) to afford 4.7 g of a
Figure 1. Selected COSY and HMBC correlations observed for 1
in D2O.
(5) Unpublished results.
(6) Fattorusso, E.; Taglialatela-Scafati, O.; Petrucci, F.; Bavestrello, G.;
Calcinai, B.; Cerrano, C.; Di Meglio, P.; Ianaro, A. Org. Lett. 2004, 6,
1633-1635.
The structure of compound 2 was deduced on the basis of
MS and NMR data and the correlation of these data to those
of 1. The high-field shift of the carbonyl carbon to δ 172.35
ppm and the additional resonance at δ 53.40 ppm, corre-
sponding to a methyl group in the 13C NMR spectrum of
compound 2, indicated the formation of the methyl ester
derivative of 1. Furthermore, 2D NMR spectra, including
an HMBC spectrum, showed the same set of correlations as
compound 1. This, in conjunction with the MS data that
showed a mass 14 amu higher than the respective parent
compound, defines 2 as methyl 2-imino-1-methylpyrrolidine-
5-carboxylate. This was confirmed by the pseudomolecular
(7) (a) Andersen, R. J. Tetrahedron Lett. 1978, 2541-2544. (b) Andersen,
R. J.; Stonard, R. J. Can. J. Chem. 1979, 57, 2325-2338. (c) Stonard, R.
J.; Andersen, R. J. J. Org. Chem. 1980, 45, 3687-3691. (d) Stonard, R. J.;
Andersen, R. J. Can. J. Chem. 1980, 58, 2121-2126. (e) Palermo, J. A.;
Rodr´ıguez, M. F.; Cabezas, E.; Balzaretti, V.; Seldes, A. M. J. Nat. Prod.
1998, 61, 488-490.
(8) Palermo, J. A.; Rodr´ıguez, M. F.; Seldes, A. M. Tetrahedron 1996,
52, 2727-2734.
(9) (a) Aoyama, T.; Kojima, F.; Imada, C.; Muraoka, Y.; Naganawa,
H.; Okami, Y.; Takeuchi, T.; Aoyagi, T. J. Enzyme Inhib. 1995, 8, 223-
232. (b) Imada, C. Antonie Van Leeuwenhoek 2005, 87, 59-63.
(10) Inbar, L.; Lapidot, A. J. Biol. Chem. 1988, 263, 16014-16022.
(11) Inbar, L.; Frolow, F.; Lapidot, A. Eur. J. Biochem. 1993, 214, 897-
906.
4968
Org. Lett., Vol. 8, No. 21, 2006

1
Table 1. 13C NMR (125 MHz) and H NMR (500 MHz) Spectral Data of Natural and Synthetic 1 and Their Methyl Ester Derivatives
(2) in D2O
methylated natural
methylated synthetic
position/mult
natural compound 1
synthetic compound 1
compound 1 (2)
compound 1 (2)
δC
δH mult (J Hz)
δC
δH mult (J Hz)
δC
δH, mult (J)
δC
δH, mult (J)
2
3
4
C
CH2
CH2
169.55
30.12
24.29
170.11
29.68
23.59
170.30
29.64
23.41
170.27
29.53
23.32
2.95 m
2.12 m
2.51 m
4.37 dd
(9.5, 4.2)
3.04 s
2.95 m
2.23 m
2.55 m
4.60 dd
(9.8, 3.6)
3.05 s
3.05 m
2.63 m
2.35 m
4.7a
3.03 m
2.62 m
2.35 m
4.69 dd
(9.8, 3.2)
3.15 s
5
CH
70.36
67.72
67.49
67.38
6
7
OMe
CH3
C
CH3
31.74
177.43
31.77
174.01
31.84
172.35
53.40
3.15 s
3.88 s
31.74
172.27
53.29
3.88 s
a Under the solvent signal.
[M + H]+ ion at m/z 157.0974 in the (+)-HRESIMS of 2
corresponding to the molecular formula C6H11N2O2 (calcd
157.0972).
and then N-methylated to give tert-butyl N-methyl (S)-
pyroglutamate (3).14 Treatment of 3 with chlorosulfonyl
isocyanate (CSI) and the addition of MeOH afforded tert-
butyl (S)-5-(methoxysulfonylimino)-1-methylpyrrolidin-2-
carboxylate (4) in 77% yield. The polarity of this intermediate
facilitates its purification by silica gel chromatography. In
contrast, treatment of 3 with CSI and subsequent hydrolysis
with aqueous sodium hydroxide, following the methodology
developed by Sattur et al., gave 1 along with the correspond-
ing salts, a situation that made isolation difficult. Finally,
removal of the protecting and methoxysulfonyl groups was
achieved by treatment of 4 with TFA to give 1 in quantitative
yield (Scheme 1).
The spectral data for the synthetic product are practically
identical to those of compound 1 (Table 1). The NMR
chemical shift data for the C-5 and C-7 positions differ
slightly from each other due to the strong pH dependence
of the chemical shifts of amino acids in D2O solution.15
Furthermore, the synthetic product and compound 1 coeluted
when a mixture of both compounds was analyzed by HPLC
under the isolation conditions described before. Treatment
of synthetic compound 1 with thionyl chloride in MeOH gave
the corresponding methyl ester 2 in quantitative yield
(Scheme 1). Once again, the NMR spectral data for the
synthetic compound are identical to those of 2 (Table 1),
showing even better agreement than the comparison between
1 and the corresponding synthetic compound. This is due to
the presence of a methyl ester in 2 as opposed to a free
carboxylic acid in 1.
The structure of 2-imino-1-methylpyrrolidine-5-carboxylic
acid (1) was previously assigned to pyrostatin B, a compound
purified from the culture broth of Streptomyces sp. SA-3501
isolated from a marine environment.9 Pyrostatin B and its
hydroxylated derivative (pyrostatin A) have been reported
and patented as useful therapeutics due to their inhibition of
N-acetylglucosaminidase.12 However, the spectral data ob-
tained for compound 1 did not match those reported for
pyrostatin B.
To confirm our proposed structure for compound 1, we
decided to carry out the synthesis of 2-imino-1-methylpyr-
rolidine-5-carboxylic acid. For the synthesis of 1, we
employed a modification of the methodology developed by
Sattur et al. for the preparation of 2-imino-1-methylazacar-
bocycles by using chlorosulfonyl isocyanate (Scheme 1).13
Scheme 1
The absolute stereochemistry of the asymmetric center at
C-5 of the iminopyrrolidine ring was deduced by comparison
of the optical rotation of compound 2 {[R]D of -10.1°
(c 0.1, H2O)} to that of the synthetic methyl ester deriva-
tive obtained from (S)-pyroglutamatic acid {[R]D of -7.5°
(13) (a) Rama Rao, K.; Nageswar, Y. V. D.; Srinivasan, T. N.; Sattur,
P. B. Synth. Commun. 1988, 18, 877-880. (b) Rama Rao, K.; Nageswar,
Y. V. D.; Srinivasan, T. N.; Satur, P. B. Indian J. Chem. 1990, 29B, 1041-
1043.
The synthesis started from the commercial (S)-pyro-
glutamatic acid, which was protected as the tert-butyl ester
(14) (a) Kolasa, T.; Miller, M. J. J. Org. Chem. 1990, 55, 1711-1721.
(b) Itoh, T.; Miyazaki, M.; Ikeda, S.; Nagata, K.; Yokoya, M.; Matsuya,
Y.; Enomoto, Y.; Ohsawa, A. Tetrahedron 2003, 59, 3527-3536.
(15) Winkler, T. Magn. Reson. Chem. 2006, 44, 571-572.
(12) Takeuchi, T.; Aoyanagi, T.; Okami, Y.; Osanawa, H.; Muraoka,
Y.; Imada, C.; Aoyama, T. Jpn. Kokai Tokkyo Koho 1995, 12 pp. JP
07291924 A2 19951107 Heisei.
Org. Lett., Vol. 8, No. 21, 2006
4969

(c 0.3, H2O)}. On the basis of these data, the absolute
structure of 1 was thus established as (-)-(5S)-2-imino-1-
methylpyrrolidine-5-carboxylic acid.
a new unreported biological activity for these compounds
as N-acetyl-â-D-glucosaminidase (NAG) inhibitors. The role
of N-acetyl-â-D-glucosaminidase in the degradation of ex-
tracellular matrix components suggested the usefulness of
hexosaminidase inhibitors as potential antiinvasive antitumor
agents.18 Furthermore, it was found that the activity of this
enzyme is increased markedly in patients with diabetes,
leukemia, or cancer and their inhibitors can help to elucidate
the mechanism of these diseases.9,19
In summary, we have isolated and characterized 2-imino-
1-methylpyrrolidine-5-carboxylic acid from a very active
allelopathic fraction obtained from the sponge Cliona tenuis.
The structure of this compound was previously reported for
the natural N-acetylglucosaminidase inhibitor named pyro-
statin B. The synthesis of this compound from (S)-pyro-
glutamatic acid indicated that the published structure does
not correspond to that of the natural product. Comparison
of NMR spectral data strongly suggests that pyrostatins A
and B are identical with 5-hydroxyectoine and ectoine,
respectively. An investigation into the allelopathic activity
of 1 and 2 against corals is underway.
Careful analysis of the NMR data reported for pyrostatin
B showed that some chemical shifts are incompatible with
the proposed structure. For example, the carbon chemical
shift of the N-methyl group at δ 18.9 ppm is unexpected
because this value is usually higher. Taking into account the
data reported for pyrostatin B, a literature search for other
possible structures showed that its reported NMR data match
those of 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic
acid, named as ectoine or THP(B). This metabolite was first
isolated from extremely halophilic and phototrophic species
of the bacterial genus Ectothiorhodospira.16 (S)-Ectoine is
now commercially available and is sold for its osmopro-
tectant activities against a wide variety of organisms, for its
protective effects in Escherichia coli during drying and
strorage, and as a stabilizer for enzymes.17 The perfect
agreement of the NMR chemical shifts reported for pyrostatin
B with those of ectoine is evident from the data in Table 2.
Acknowledgment. This work was financially supported
by Grants from Xunta de Galicia (PGIDIT05RMA10302PR),
Ministerio de Educacio´n y Ciencia (CQT2005-00793), and
COLCIENCIAS from Colombia (grant 1101-09-13544). L.C.
thanks Program Alâan, the European Union Program of High
Level Scholarships for Latin America for a scholarship
(E04D033392CO), and Universidad Nacional de Colombia
for the leave of absence to perform doctoral studies.
1
Table 2. Reported 13C NMR and H NMR Spectral Data for
Pyrostatin B and Ectoine [THP(B)]
pyrostatin B9a
δC (mult) δH mult (J Hz)
161.2 (C)
ectoine
C
C
δC (mult)10 δH mult (J Hz)11
2
3
2 161.5 (C)
38.0 (CH2)
3.30 ddd
(14.0, 8.6, 5.0)
3.46 ddd
6
38.0 (CH2)
3.28 ddd
(13.5, 8.4, 4.8)
3.44 ddd
(14.0, 5.6, 5.6)
2.07-2.19 m
4.07 dd
(5.6, 5.6)
2.24 s
(13.5, 5.4, 5.4)
2.11 m
4.06 dd
(5.4, 5.4)
2.22 s
Supporting Information Available: Experimental details
and spectral data for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
4
5
22.1 (CH2)
53.9 (CH)
5
4
23.6 (CH2)
53.6 (CH)
6
7
18.9 (CH3)
177.4 (C)
2′ 19.1 (CH3)
4′ 177.0 (C)
OL062087K
(16) Galinski, E. A.; Pfeiffer, H.-P.; Tru¨per, H. G. Eur. J. Biochem. 1985,
149, 135-139.
(17) Sigma (E 2271) and Fluka (81619) catalogues.
Furthermore, the NMR data reported for pyrostatin A also
matched those of 5-hydroxyectoine [THP(A)], which is
produced by Streptomyces parVulus.10,11 This finding suggests
(18) Woynarowska, B.; Wikiel, H.; Sharma, M.; Carpenter, N.; Fleet,
G. W. J.; Bernacki, R. J. Anticancer Res. 1992, 12, 161-166.
(19) Muzzarelli, R. A. A. EXS 1999, 87, 235-247 (CAN 132: 104447).
4970
Org. Lett., Vol. 8, No. 21, 2006