Vinylogous amide analogues of diaminopimelic acid (DAP) as inhibitors of enzymes involved in bacterial lysine biosynthesis.

Article abstract of DOI:10.1021/ol000271e

[reaction: see text] Vinylogous amides 5 and 6 have been synthesized from L-propargyl glycine and tested against diaminopimelate (DAP) enzymes involved in bacterial lysine biosynthesis. Both are reversible inhibitors of DAP D-dehydrogenase and DAP epimerase with IC(50) values in the 500 microM range. Compound 5 shows competitive inhibition against the L-dihydrodipicolinate (DHDP) reductase with a K(i) value of 32 microM, which is comparable to the planar dipicolinate 16 (K(i) = 26 microM), the best known inhibitor of the enzyme.

Full text of DOI:10.1021/ol000271e

ORGANIC
LETTERS
2000
Vol. 2, No. 24
3857-3860
Vinylogous Amide Analogues of
Diaminopimelic Acid (DAP) as Inhibitors
of Enzymes Involved in Bacterial Lysine
Biosynthesis
Jennifer F. Caplan,† Renjian Zheng,‡ John S. Blanchard,‡ and
,†,§
John C. Vederas*
Department of Chemistry, UniVersity of Alberta, Edmonton,
Alberta, Canada T6G 2G2, and Department of Biochemistry,
Albert Einstein College of Medicine, Bronx, New York 10461
john.Vederas@ualberta.ca
Received September 14, 2000
ABSTRACT
Vinylogous amides 5 and 6 have been synthesized from L-propargyl glycine and tested against diaminopimelate (DAP) enzymes involved in
bacterial lysine biosynthesis. Both are reversible inhibitors of DAP D-dehydrogenase and DAP epimerase with IC values in the 500 µM range.
50
Compound 5 shows competitive inhibition against the L-dihydrodipicolinate (DHDP) reductase with a K value of 32 µM, which is comparable
i
to the planar dipicolinate 16 (K ) 26 µM), the best known inhibitor of the enzyme.
i
The search for new broad spectrum antimicrobial agents has
encouraged extensive studies on enzymes and inhibitors of
the diaminopimelic acid (DAP) pathway to L-lysine (Scheme
1).1 The meso isomer of DAP (1) is involved in the cross-
linking of the peptidoglycan cell wall layer of Gram negative
bacteria, whereas its biosynthetic product, L-lysine, has an
analogous function in many Gram positive organisms.2 Since
mammals lack this pathway and require a dietary source of
lysine, inhibitors of DAP enzymes may act as antibiotics
with low mammalian toxicity. Several enzymes in the DAP
pathway either utilize substrates having an imine bond or
have transient intermediates wherein the R-carbon in the
amino acid moiety becomes planar. Among these are DAP
D-dehydrogenase, DAP epimerase, and dihydrodipicolinate
(DHDP) reductase.1 The X-ray crystal structures of these
enzymes have been reported.3-5 The proposed mechanism
of DAP D-dehydrogenase involves generation of an acyclic
imine intermediate,5 whereas that of DAP epimerase creates
anionic character at the R-center of the substrate, with one
active site cysteine thiolate acting as the base and another
cysteine thiol as the acid to deliver the proton from the
opposite face.6 The natural substrate of DHDP reductase is
an imine. On this basis, a number of successful inhibitors of
† University of Alberta.
‡ Albert Einstein College of Medicine.
§ Tel 780-492-5475. FAX 780-492-8231.
(1) For reviews, see: (a) Cox, R. J. Nat. Prod. Rep. 1996, 13, 29-43.
(b) Scapin, G.; Blanchard, J. S. AdV. Enzymol. Relat. Areas Mol. Biol. 1998,
72, 279-364. (c) Cox, R. J.; Sutherland, A.; Vederas, J. C. Bioorg. Med.
Chem. 2000, 8, 843-872.
(2) For selected reviews, see: (a) Bugg, T. D. H.; Walsh, C. T. Nat.
Prod. Rep. 1992, 9, 199-215. (b) Ward, J. B. Antibiotic Inhibitors of
Bacterial Cell Wall Biosynthesis; Tipper, D. J., Ed.; Pergamon Press: New
York, 1987; pp 1-43. (c) Ghuysen, J. M. Int. J. Antimicrob. Agents 1997,
8, 45-60. (d) Van Heijenoort, J. Cell. Mol. Life Sci. 1998, 54, 300-304.
(e) Wong, K. K.; Pompliano, D. L. AdV. Exp. Med. Biol. 1998, 456, 197-
217.
(3) Scapin, G.; Reddy, S. G.; Blanchard, J. S. Biochemistry 1996, 35,
13540-13551.
(4) Cirilli, M.; Zheng, R.; Scapin, G.; Blanchard, J. S. Biochemistry 1998,
37, 16452-16458.
(5) (a) Scapin, G.; Blanchard, J. S.; Sacchettini, J. C. Biochemistry 1995,
34, 3502-3512. (b) Reddy, S. G.; Scapin, G.; Blanchard, J. S. Biochemistry
1996, 35, 13294-13302.
10.1021/ol000271e CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/10/2000

From the retrosynthetic perspective, it seemed that the
vinylogous amide functionality could be made by reductive
ring opening of an isoxazole ring.9 Thus, protection of
L-propargyl glycine (7) using standard protocols gives methyl
N-(benzyloxycarbonyl)-L-propargyl glycinate (8)10 in 95%
yield (Scheme 2). Treatment of 8 with ethyl chlorooximi-
Scheme 1. Biosynthesis of L-Lysine via the DAP Pathway
Scheme 2. Synthesis of Vinylogous Amide 6a
DAP enzymes have been prepared which have a planar
R-carbon, and some show antimicrobial activity.1c For
example, DAP dehydrogenase is inhibited by isoxazoline 2
(Ki ) 23 µM)7 and unsaturated derivatives 3 and 4 (Ki )
5.3 µM and 44 µM, respectively).8 Since enzymes in the
DAP pathway tend to have relatively strict substrate specific-
ity and excellent stereochemical recognition at the distal
(nonreacting) sites, the design of potent inhibitors is often a
challenging task that requires stereospecific incorporation of
numerous similar functionalities into a small molecule.1c In
the present study, we describe the preparation and testing of
vinylogous amides 5 and 6 as potential inhibitors of DAP
dehydrogenase, DAP epimerase, and DHDP reductase en-
zymes.
a Reagents and conditions: (a) SOCl2 in dry MeOH, 4 equiv,
10 h, 25 °C; (b) 1.2 equiv of Boc2O, 1.2 equiv of Et3N, MeCN, 3
h, 25 °C; (c) 3.0 equiv of EtO2CC(Cl)dNOH 9, ether, 25 °C, then
3.0 equiv of Na2CO3 in H2O, via syringe pump, 3 h; (d) 2.1 equiv
of LiOH, MeCN/H2O (1:1), 17 h, 25 °C; (e) 10 equiv of TFA,
CH2Cl2, 1 h, 25 °C; (f) H2/Pd-C(10%), H2O, 2 h, 25 °C; (g) 2
equiv of Na, 1 equiv of tert-butyl alcohol, NH3 (liq), 1 h, - 78 °C;
(h) 0.65 equiv of Mo(CO)6, 1 equiv of H2O, MeCN, 80 °C; (i) 10
equiv of TFA, CH2Cl2, 1 h, 25 °C; (j) 2.1 equiv of LiOH, MeCN/
H2O (1:1), 17 h, 25 °C.
(6) (a) Wiseman, J. S.; Nichols, J. S. J. Biol. Chem. 1984, 259, 8907-
8914. (b) Koo, C. W.; Blanchard, J. S. Biochemistry 1999, 38, 4416-4422.
(c) Koo, C. W.; Sutherland, A.; Vederas, J. C.; Blanchard, J. S. J. Am.
Chem. Soc. 2000, 122, 6122-6123.
(7) (a) Abbott, S. D.; Lane-Bell, P.; Sidhu, K. P. S.; Vederas, J. C. J.
Am. Chem. Soc. 1994, 116, 6513-6520. (b) Scapin, G.; Cirilli, M.; Reddy,
S. G.; Gao, Y.; Vederas, J. C.; Blanchard, J. S. Biochemistry 1998, 37,
3278-3285.
(8) (a) Sutherland, A.; Caplan, J. F.; Vederas, J. C. J. Chem. Soc., Chem.
Commun. 1999, 555-556. (b) Cirilli, M.; Scapin, G.; Sutherland, A.;
Vederas, J. C.; Blanchard, J. S. Protein Sci., in press.
doacetate 9 under basic conditions affords the 1,3-dipolar
cycloaddition product,11 isoxazole 10, as a single regioisomer
(9) Bu¨chi, G. H.; Vederas, J. C. J. Am. Chem. Soc. 1972, 94, 9128-
9132.
3858
Org. Lett., Vol. 2, No. 24, 2000

in 70% yield. Hydrolysis of the ester functionalities using
lithium hydroxide, followed by deprotection of the amino
moiety, generates the bis acid 11. However, attempts to
transform the isoxazole ring into the target vinylogous amide
using standard conditions, such as hydrogenation or reduction
with sodium in liquid ammonia,9,12 produce exclusively the
saturated alcohol 12 as a mixture of four diastereoisomers.
Formation of the vinylogous amide moiety is possible by
treatment of isoxazole 10 with a catalytic amount of
molybdenum hexacarbonyl and 1 equiv of water in refluxing
acetonitrile.13 Subsequent exposure of 13 to excess trifluoro-
acetic acid (TFA) in dichloromethane for 1 h generates a
1:1 mixture of cyclic and open-chain vinylogous amides 14
and 15 in 90% yield. The yield of 15 can be improved to
80% by using 5 equiv of TFA and careful monitoring of the
reaction by TLC. The cyclic derivative 14 could arise from
Michael addition of the nitrogen liberated by removal of the
tert-butoxycarbonyl moiety onto the R,â-unsaturated alkene
followed by subsequent loss of ammonia. Hydrolysis of
diester 14 using lithium hydroxide followed by workup gives
5 in 90% yield. Compound 15 is unstable at room temper-
ature and is therefore best transformed immediately into the
dilithium salt 6 (85% yield). Acidification of 6 leads rapidly
to formation of the cyclized derivative 5; therefore this
compound was stored as its dilithium derivative. Vinylogous
amide 5 is an interesting compound in terms of functional
group density and structural fragments (ketone, primary
amine, primary enamine) that would be incompatible or
would decompose in water were it not for the stabilization
afforded by the extended conjugation.
With vinylogous amides 5 and 6 available, DAP D-
dehydrogenase was purified from Bacillus spaericus IFO
3525 as previously reported.7a,14 In addition, DAP epimerase
was isolated from an Escherichia coli mutant BL21(DE3)
pLysS using a modified procedure.6a,15 DAP D-dehydrogenase
assay at pH 7.8 employs the reverse reaction, wherein
NADP+ oxidatively deaminates the D-amino acid center of
meso-DAP (1) to L-tetrahydrodipicolinate (L-THDP) with
generation of NADPH, thereby allowing continuous spec-
trophotometric assay at 340 nm.7a,15 The stability of 6 during
the enzyme assays can be monitored by TLC which reveals
that cyclization begins to be detectable after 3 h in the buffer
solution. Hence, each assay employed freshly dissolved
inhibitor. No isomerization of the double bond geometry of
6 could be detected. Compounds 5 and 6 are not substrates
for DAP D-dehydrogenase and show only poor reversible
inhibition of DAP D-dehydrogenase, with IC50 values in the
range of 400-450 µM. This is surprisingly weak binding
given that these molecules possess much of the functionality
present in the substrates (THDP and meso-DAP) and putative
imine intermediate. Although the presence of an extra oxygen
at the center of 6 could generate unfavorable steric interac-
tions with the enzyme, a more likely cause for failure of
effective binding to DAP dehydrogenase is the coplanarity
of the vinylogous amide system. Crystallographic studies
reveal that the isoxazoline 2 and the unsaturated analogue 3
bind in the active site of the DAP D-dehydrogenase with
conformations which for analogues 5 and 6 could require
bond rotation in the coplanar portions.7b,8b
Inhibition studies with DAP epimerase involve a coupled
enzyme assay at pH 7.8, whereby meso-DAP (1) generated
by the epimerase from LL-DAP is transformed by DAP
D-dehydrogenase to produce L-THDP and NADPH, which
is followed spectrophotometrically.15 The results show that,
as expected, 5 is a very poor inhibitor of DAP epimerase.
Disappointingly, the acyclic vinylogous amide 6 is also a
weak competitive inhibitor (IC50 of 500 µM) of this enzyme
despite having most of its atoms in locations that might be
expected to mimic the transition state or intermediate DAP-
derived R-anion. Although a crystal structure of active DAP
epimerase with a substrate analogue in the active site is not
yet available,16 the poor binding to DAP epimerase may be
again be due to the planarity of the vinylogous amide system
as well as consequent reduced basicity of the amino group
on the sp2 carbon. Initially it seemed that an enzyme-induced
conformational twist around the C-3 to C-4 bond in 6 or
around the C-2 to C-3 bond in its tautomer 6a could generate
reactive functionality and trigger addition of an active site
thiol group, but the required tautomerization has not been
observed (Scheme 3).
Scheme 3. Potential Tautomerization of 6 with Attack by
DAP Epimerase
Fortunately, the cyclic vinylogous amide 5, which shows
considerable structural similarity to L-DHDP proved to be a
good inhibitor of L-DHDP reductase. This enzyme, which
was isolated from E. coli, catalyzes the transfer of a hydrogen
from NADPH to the γ-position of L-DHDP to give L-
tetrahydrodipicolinic acid (THDP).17 Inhibition studies dem-
onstrate that 5 is a reversible competitive inhibitor of the
DHDP reductase with respect to L-DHDP with an inhibition
constant (Ki) of 32 µM. Hence 5 is comparable to the most
potent competitive inhibitor of DHDP reductase reported thus
far, the fully planar dipicolinic acid (16) (Ki 26 µM). Since
(10) Collet, S.; Bauchat, P.; Danion-Bougot, R.; Danion, D. Tetrahe-
dron: Asymmetry 1998, 9, 2121-2131.
(11) Kozikowski, A. P.; Adamczyk, M. J. Org. Chem. 1983, 48, 366-
372.
(12) Alberola, A.; Andres, C.; Gonzalez Ortega, A.; Pedrosa, R. J.
Heterocycl. Chem. 1984, 21, 1575-1576.
(13) (a) Nitta, M.; Kobayashi, T. J. Chem. Soc., Chem. Commun. 1982,
877-878. (b) Baraldi, P. G.; Barco, A.; Benetti, S.; Guarneri, M.;
Manfredini, S.; Pillini, G. P.; Simoni, D. Tetrahedron Lett. 1985, 26, 5319-
5322.
(14) Misono, H.; Soda, K. J. Biol. Chem. 1980, 255, 10599-10605.
(15) Song, Y. H.; Niederer, D.; Lane-Bell, P.; Lam, L. K. P.; Crawley,
S.; Palcic, M. M.; Pickard, M. A.; Pruess, D. L.; Vederas, J. C. J. Org.
Chem. 1994, 59, 5784-5793.
(16) The reported crystal structure of DAP epimerase (ref 4) is of an
inactive form wherein the two thiols are linked as a disulfide.
(17) Scapin, G.; Blanchard, J. S.; Sacchettini, J. C. Biochemistry 1995,
34, 3502-3512.
Org. Lett., Vol. 2, No. 24, 2000
3859

related derivatives such as isophthalic acid (17), a racemic
mixture of trans-piperidine dicarboxylic acids (18), cis-
piperidine dicarboxylic acid (19), pipecolic acid (20), and
picolinic acid (21) are poor inhibitors, additional crystal-
show only weak inhibition of DAP dehydrogenase and DAP
epimerase, 5 is an excellent competitive inhibitor of DHDP
reductase. Synthesis of other DAP analogues and evaluation
of their interactions with DAP enzymes is currently underway
in our laboratory.
Acknowledgment. Financial support was provided by the
Natural Sciences and Engineering Research Council of
Canada and the National Institutes of Health (AI-33696). We
gratefully acknowledge Bruce Malcolm (Schering Plough)
for a gift of E. coli BL21 (DE3) pLysS which overexpresses
DAP epimerase. We also thank Michael Pickard and Richard
Mah (Microbiology Department, University of Alberta) for
large scale fermentations of bacterial strains.
lographic studies of DHDP reductase bound to 5 should help
to clarify the substrate-enzyme interactions at the stereo-
genic center of DHDP. This would potentially lead to other
cyclic derivatives with even greater inhibition.
In summary, we have described an efficient synthesis of
highly functionalized vinylogous amides 5 and 6 in six steps
starting from L-propargyl glycine. Although these compounds
Supporting Information Available: Experimental pro-
cedures and spectroscopic data for compounds 5, 6, 8, and
11-15. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL000271E
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