Synthesis of amino acid conjugates to 2-imino-3-methylene-5-carboxypyrrolidine and 2-imino-3-methylene-6-carboxypiperidine

Article abstract of DOI:10.1016/j.bmcl.2010.01.128

The four stereomers of 2-imino-3-methylene-5-l(carboxy-l-valyl)pyrrolidine, a bacterial metabolite that is inhibitory to the fire blight bacterium Erwinia amylovora, were synthesised and compared for antibacterial activity. Several alternative amino acid conjugates with l,l-stereochemistry were also prepared, and the synthesis was extended to 3-methylenepiperidine-6-l-carboxylic acid and a selection of 2-imino-3-methylenepiperidine-6-l-carboxy-l-amino acid conjugates. All synthetic amino acid conjugates (l,l-stereomers) were inhibitory to the growth of E. amylovora. The likely participation of the conjugated iminomethylene moiety as a Michael acceptor is implicated.

Full text of DOI:10.1016/j.bmcl.2010.01.128

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1910–1912
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of amino acid conjugates to 2-imino-3-methylene-5-
carboxypyrrolidine and 2-imino-3-methylene-6-carboxypiperidine
*
Robin E. Mitchell
The New Zealand Institute for Plant and Food Research Limited (formerly HortResearch), Private Bag 92169, Auckland 1025, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
The four stereomers of 2-imino-3-methylene-5-L(carboxy-L-valyl)pyrrolidine, a bacterial metabolite that is
Received 4 November 2009
Revised 26 January 2010
Accepted 28 January 2010
Available online 1 February 2010
inhibitory to the fire blight bacterium Erwinia amylovora, were synthesised and compared for antibacterial
activity. Several alternative amino acid conjugates with -stereochemistry were also prepared, and the
synthesis was extended to 3-methylenepiperidine-6- -carboxylic acid and a selection of 2-imino-3-methy-
lenepiperidine-6- -carboxy- -amino acid conjugates. All synthetic amino acid conjugates ( -stereomers)
L,L
L
L
L
L,L
were inhibitory to the growth of E. amylovora. The likely participation of the conjugated iminomethylene
moiety as a Michael acceptor is implicated.
Keywords:
Antibacterial
Iminomethylenepyrrolidine
Iminomethylenepiperidine
Fire blight
Ó 2010 Elsevier Ltd. All rights reserved.
The structures of two new compounds from liquid cultures of
Burkholderia plantarii, a bacterial pathogen on rice, were recently
The synthesis of 2-imino-3-methylenepyrrolidine-5-carboxy-
valine stereomers is summarised in Schemes 1A and 2A. The first
stage (Scheme 1A) inserts the exocyclic 3-methylene group4,5 into
elucidated and reported as 2-imino-3-methylene-5-
L
(carboxy-
L-
valyl)pyrrolidine (1) and 2-imino-3-methylene-5- (carboxy-
L
L
-thre-
each of
uct 2-oxo-3-methylene-pyrrolidine-5-carboxylic acid (3A-
3A- ).
These were then coupled (Scheme 2) to - or D-valine methyl es-
ter (as the free base), resulting in the four lactam analogues (4A) of
the targeted products, confirmed by FAB-MS (MH+ 255) and NMR
(Supplementary data) following purification (HPLC data, Table 1).
Conversion of the lactam carbonyl of these four products to the
imino functionality was accomplished in a two-step reaction
(Scheme 2A). Purified products were base hydrolysed to the final
iminopyrrolidine compounds 5A. Each imino acid product dis-
played an MH+ at 240 (FAB-MS), and had 1H and 13C spectra con-
sistent for the required products (Supplementary data).
L
- and
D
-pyroglutamic acid, resulting in the required prod-
oninyl)pyrrolidine (2) (Fig. 1).1 Both compounds inhibited the
growth of Erwinia amylovora in an agar diffusion assay, the
bacterium responsible for fire blight disease in Rosaceae, in particu-
lar, apple and pear trees. The newly reported 2-imino-3-methylene-
pyrrolidine structural component of these compounds has
considerable potential significance in that past studies2 have shown
that a series of substituted iminopyrrolidines are potent and selec-
tive inhibitors of human inducible nitric oxide synthases, suggesting
possible adaptation to therapeutics. More recently, a product from a
marine sponge was found to be the related compound 2-imino-1-
methylpyrrolidine-5-carboxylic acid and was reported to have
allelopathicactivityagainstcorals.3 Toconfirmthestructuralassign-
ment for 1 and 2, and to further define the nature of the antibacterial
activity, the synthesis and properties of all four stereomers of the va-
line conjugate to the 2-imino-3-methylenepyrrolidine product was
L
and
D
L
The four synthetic stereomers were compared for their antibac-
terial activity against E. amylovora, by agar diffusion assay1 (Table
2). The L,L-stereomer (natural product configuration) inhibited
undertaken, and extended to a number of
L,L
-analogues with differ-
growth at 0.1
l
g per application site (10 mm zone), and gave
ent amino acids. The basis of the structural specificity for the activity
against E. amylovora was further evaluated by the synthesis of the
corresponding six-membered ring homologs of a selection of the
products. This required first the synthesis of 2-oxo-3-methylenepi-
peridine-6-carboxylic acid and then adaptation of parallel synthetic
procedures used for the iminopyrrolidine compounds.
7
7
3
OH
3
H
4
H
4
2
5
1
2
1
5
6 N
H
CO2H
6 N
H
CO2H
N
HN
C
O
N
HN
C
O
H
H
H
H
2
1
* Tel.: +64 649 9257186; fax: +64 649 9257001.
E-mail address: Robin.Mitchell@plantandfood.co.nz.
Figure 1. Natural product structures.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.128

R. E. Mitchell / Bioorg. Med. Chem. Lett. 20 (2010) 1910–1912
1911
Table 1
HPLC characteristics of stereomersa
N
Stereomer
Elution times (min)
Iminopyrrolidine 5
(CH2)n
H
(CH2)n
H
(CH2)n
H
i, ii
iii
Lactam 4
O
O
t
t
N
N
O
CO2 Bu
CO2 Bu
N
CO2H
4.97
5.23
4.29
4.54
L,
L- and
D
L
,D
-
-
C-OtBu
C-OtBu
H
D,L
- and
,D
O
O
a
HPLC used an ODS Hypersil C18 column, 250 Â 0.4 mm, flow rate 0.8 ml minÀ1
,
and isocratic elution with solvent compositions of CH3CN/H2O/MeOH + 0.05% TFA,
70:25:5 for 4, 50:45:5 for 5.
(CH2)n
H
(CH2)n
v
iv
H
Table 2
O
O
N
t
N
Antibacterial activitya of iminopyrrolidine stereomers
CO2H
CO2 Bu
H
C-OtBu
Stereomer
Zone size (mm) at loading of
O
3A, n=1
3B, n=2
0.1
l
g
1.0
l
g
10
lg
100 lg
10
—
19
6
L
,
,
L-
A: n=1
B: n=2
10
—
19
10
—
L
D-
D
,L
-
Scheme 1. Reagents and conditions: (i) tBuOAc, HClO4; (ii) (tBuO)2CO, DMAP,
MeCN, 0 °C; (iii) HC[N(CH3)2]3, CH3OCH2CH2OCH3, 72 °C, 88%; (iv)
[(CH3)2CHCH2]2AlH, THF, À78 °C, 61%; (v) TFA/CH2Cl2, 10 M, 72%.
D
,D-
—
a
Agar diffusion assay, samples applied in 10 lL water to 4 mm diameter wells.
strong inhibition at 1
natural product. In contrast, the
at loadings up to 100
g (i.e., >104 less active). The
reomers showed intermediate degrees of inhibition:
loading was comparable to - at 0.1 g, and - at 100
comparable to - at 0.1 g. The estimated differentials in concen-
trations for -: -: -: - to give comparable inhibitions there-
fore are 1: 102: 103: >104. The activity observed for the
stereomer was concluded to be real rather than from the presence
of a low concentration of -stereomer. HPLC analysis of -stere-
omer spiked with ca. 1% of the -product clearly showed this as a
resolved ‘pimple’ not present in the parent, indicating that the
stereomer did not contain the -stereomer. If there were a pres-
ence of -stereomer at a concentration below HPLC detectability
this would be significantly below the concentration to account
for the activity observed for the -stereomer.
The natural product 2-imino-3-methylene-5-
valyl)pyrrolidine was found to contain an -valine and was pro-
posed to have the -stereochemistry at the 5-position of the imino-
pyrrolidine ring.1 In the present study, the HPLC characteristics of
synthetic -stereomer were the same as those of the natural prod-
uct, and differed from the
l
g (19 mm zone), comparable to that of the
-stereomer gave no inhibition
- and -ste-
- at a 10
g was
The same methodology outlined was also used to prepare sev-
eral additional 2-imino-3-methylene-5- -carboxy? -amino acid
D
,D
L
L
l
L,
D
D
,L
conjugates for comparative antibacterial testing. Each was purified
to homogeneity, as evaluated by HPLC, and their identity con-
firmed by MS. The amino acid component of these included gly-
cine, alanine, b-alanine, 2-methyl alanine, leucine, phenylalanine,
proline, and the dipeptide valylleucine. These products generally
displayed an antibacterial activity similar to that of the threonine-
and valine-containing natural products, although some (b-alanine,
glycine, val-leu) were active to a lesser degree (2.5-, 5-, 20-fold,
respectively).
L,
D
lg
L
,L
l
D,L
l
L
,
L
L
l
D,D
L
,L
,
D
D
,L
L,D-
L
,L
L,D
L,L
L,D
-
Synthesis of the six-membered ring homologs started with 2-
L,L
oxopiperidine-6-
tained from the cyclisation of 2-L
L
-carboxylic acid (Scheme 1B), which was ob-
L
,
L
-aminoadipic acid.6 Introduction
of the exocyclic methylene lead to 3B, 3-methylenepiperidine-6-
carboxylic acid (Scheme 1B). This was then coupled with various
amino acids (as their methyl ester) followed by conversion of the
lactam carbonyl to the 2-imino functionality, and base hydrolysis
to the free carboxylic acid (Scheme 2B). Three amino acid-coupled
L,D
L
(carboxy-L-
L
L
products were prepared (L-Val, L-Ile, L-Thr); in all cases only low
L
,L
overall yields were achieved, but sufficient material was obtained
for characterisation (HPLC data and FAB-MS, and for the Val conju-
gate, full NMR analysis) and antibacterial testing. Each of the com-
D,L-stereomer, and therefore the natural
product is confirmed to be the
L,L
-stereomer.1
(CH2)n
H
(CH2)n
i
ii
H
3
O
H3CO
N
N
N
H
CO2CH3
H
C
O
N
H
CO2CH3
H
C
O
H
4
(CH2)n
H
(CH2)n
H
iii
iv
HN
N
HN
N
N
H
CO2H
H
C
O
N
CO2CH3
H
C
O
H
H
H
5A, n=1
5B, n=2
A: n=1
B: n=2
Scheme 2. Reagents and conditions: (i) DCCD, MeCN, then ValOMe(base), 75%; (ii) Me3O+BF4À, CH2Cl2 2 h, product used without purification; (iii) NH4Cl, MeOH, 70 °C 16 h,
19% from i; (iv) aq 0.1 M NaOH, 62%.

1912
R. E. Mitchell / Bioorg. Med. Chem. Lett. 20 (2010) 1910–1912
pounds displayed strong inhibition of E. amylovora in the agar dif-
fusion assay, comparable to their iminopyrrolidine analogues.
The five- and six-membered 2-imino-3-methylenelactam ring
structures, when coupled to various amino acids through the car-
boxyl group, provide a diverse family of compounds with antibacte-
rial activity. New and unusual chemistry is represented in the
iminomethylene lactams, and their bioactivity may well be attribut-
able to the conjugated iminomethylene moiety acting as a Michael
acceptor. The conjugation to amino acids is a phenomenon not
uncommon in biology, and probably represents some biophysical
requirement for functionality. It is feasible that various further
chemical modifications may provide scope for the development of
new antibacterial products. Given the known nitric oxide synthase
activity of related compounds, there would appear to be consider-
able potential for discovery of additional bioactivities in these
compounds.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.01.128.
References and notes
1. Mitchell, R. E.; Teh, K. L. Org. Biomol. Chem. 2005, 3, 3540.
2. Hagen, T. J.; Bergmanis, A. A.; Kramer, S. W.; Fok, K. F.; Schmelzer, A. E.; Pitzele,
B. S.; Swenton, L.; Jerome, G. M.; Kornmeier, C. 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.
3. Castellanos, L.; Duque, C.; Zea, S.; Espada, A.; Rodriguez, J.; Jimenez, C. Org. Lett.
2006, 8, 4967.
4. August, R. A.; Khan, J. A.; Moody, C. M.; Young, D. W. J. Chem. Soc., Perkin Trans. 1
1996, 507.
5. Kolasa, T.; Miller, M. J. J. Org. Chem. 1990, 55, 1711.
6. Greenstein, J. P.; Winitz, M. In Chemistry of the Amino Acids. John Wiley & Sons:
New York, London, 1961; Vol. 3, Chapter 43, p 2411.
Acknowledgments
Provision of NMR data by Michael Walker and Michael Schmitz
of Auckland University Chemistry Department is gratefully
acknowledged.