Vinyl fluoride as an isoelectronic replacement for an enolate anion: Inhibition of type II dehydroquinases

Article abstract of DOI:10.1039/b205105m

A vinyl fluoride analogue of the intermediate in the reaction catalysed by type II dehydroquinase enzymes has been synthesized over seven steps from (-)-quinic acid and shown to be a potent enzyme inhibitor.

Full text of DOI:10.1039/b205105m

Vinyl fluoride as an isoelectronic replacement for an enolate anion:
Inhibition of type II dehydroquinases
Martyn Frederickson,a John R. Cogginsb and Chris Abell*a
a University Chemical Laboratory, Lensfield Road, Cambridge, UK CB2 1EW. E-mail: ca26@cam.ac.uk
b Division of Biochemistry and Molecular Biology, IBLS, Glasgow University, Glasgow, UK G12 8QQ
Received (in Cambridge, UK) 27th May 2002, Accepted 8th July 2002
First published as an Advance Article on the web 26th July 2002
A vinyl fluoride analogue of the intermediate in the reaction
catalysed by type II dehydroquinase enzymes has been
synthesized over seven steps from (2)-quinic acid and shown
to be a potent enzyme inhibitor.
The shikimate1,2 and quinate3 pathways (the enzymatic routes
from acyclic carbohydrates to essential aromatics utilized by
micro-organisms, fungi and the higher plants) continue to elicit
Fig. 1
major research efforts especially since the recent discovery4 of
the shikimate pathway in apicomplexan parasites including the
most virulent of the malarial parasites (Plasmodium falci-
parum).
Our search for even more effective inhibitors focused on
derivatives in which the true geometric and electronic features
of enolate 2 were more accurately mimicked, particularly the
electron distribution and high electron density generated at
C(3). Despite the relative rarity of the vinyl fluoride function-
ality we chose to replace the enolate oxyanion with its closest
chemical equivalent, the isosteric and isoelectronic fluorine
atom. Herein we describe the use of the vinyl fluoride moiety as
an isosteric and isoelectronic replacement for an enolate anion
and report the syntheses of fluorovinyl 6 and difluoro 712
acids.
Dehydroquinase [3-dehydroquinate dehydratase; E.C.
4.2.1.10] which catalyses the reversible conversion of 3-dehy-
droquinate (DHQ) 1 to 3-dehydroshikimate (DHS) 3 is uniquely
common to both pathways. It occurs in two distinct forms each
with differing chemical and biochemical properties. Type I
enzymes are heat labile protein dimers5 that catalyse a syn-
elimination6 of water through Schiff’s base intermediates7
involving a conserved lysine. Type II dehydroquinases are more
thermally robust dodecameric species8 that effect an anti-
dehydration via an enzyme stabilized enolate 29 (Scheme 1).
Acids 6 and 7 were prepared over seven steps from
(2)-quinic acid 8 (Scheme 2). Esterification of 8 was
Scheme 1
The mechanistic differences of the two enzyme types led us
to design inhibitors that were selective for the type II proteins
which are found in a number of clinically important bacterial
strains including Mycobacterium tuberculosis. Based upon the
idea that compounds which mimicked the enolate intermediate
2 were likely to show such selectivity we prepared derivatives
which, when compared to DHQ, possessed either increased sp2
character at C(2) and C(3) (alkene 4) or had superior hydrogen
bonding potential at C(3) (oxime 5) (Fig. 1).
We were thus able to demonstrate10 that 4 and 5 were potent
and highly selective (up to 1000 fold) competitive reversible
inhibitors of type II dehydroquinases. Recent crystallographic
studies11 on the complex formed between alkene 4 and the type
II dehydroquinase from Streptomyces coelicolor (PDB acces-
sion code : 1GU1) has allowed its mode of action to be fully
determined.
Scheme
2 Reagents and conditions: i, MeC(OMe)2C(OMe)2Me,
HC(OMe)3, CSA, MeOH, 65 °C, Ar, 16 h; ii, RuCl3, KIO4, K2CO3, H2O,
CHCl3, 20 °C, 48 h; iii, CH2(OMe)2, P2O5, CHCl3, 20 °C, 16 h; iv, DAST,
DME, 80 °C, Ar, 6 h; v, TFA, H2O, 60 °C, 2 h; vi, NaOH, H2O, 20 °C, 30
min; vii, Amberlite IR-120(H), H2O, 20 °C, 10 min.
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accomplished concomitantly with protection of the 4,5-trans-
diol as the cyclic bis-ketal 9 as described previously.13
Ruthenium(III) catalysed periodate oxidation to the C(3) ketone
10 followed by MOM protection of the tertiary alcohol at C(1)
afforded the fully protected 3-dehydroquinate 11.14 Incorpora-
tion of fluorine at C(3) proved to be somewhat capricious. It was
ultimately reproducibly achieved15 upon treatment of ketone 11
in boiling anhydrous DME under argon with multiple portions
of DAST to afford a mixture of two chromatographically
results together with details of X-ray crystallographic studies of
complexes of 6 with type II enzymes will be reported
elsewhere.
Fluorine has been widely utilized to alter both the chemical
and biochemical properties of many compounds, chiefly as a
replacement for H, OH or neutral oxygen atoms (as part of a
CHF of CF2 group); those pertinent to the shikimate pathway
include 2-fluoro,18 6-fluoro19 (both H replacements), 3-fluoro20
and 3,5-difluoroshikimates21 (OH groups). This is the first
report of fluorine as a replacement for an oxyanion, which it
resembles more closely than a hydroxy group, by being both
isosteric and isoelectronic.
separable products 12 and 13. Extensive NMR analysis (1H, 13
C
and 19F in deuteriochloroform) showed the less polar compound
to be the desired vinyl fluoride 12 (dF 2115.9 coupled to dC
159.6). Determination of the structure and relative ster-
eochemistry of the more polar, crystalline product 13 (dF
2113.3 and 2103.3 coupled to dC 119.7) proved possible by X-
ray crystallographic analysis16,17 (Fig. 2). The product was
thereby unequivocally shown to be the corresponding C(3) gem-
difluoride.
We thank Neil Feeder for the X-ray crystallographic analysis
of difluoride 13, John Greene and David Robinson for protein
purification, and the BBSRC for postdoctoral support (MF).
Notes and references
1 E. Haslam, Shikimic acid: Metabolism and Metabolites, John Wiley and
Sons, Chichester, 1993.
2 C. Abell, in Comprehensive Natural Products Chemistry, ed. U.
Sankawa, Elsevier, Amsterdam, 1999, vol. 1, p. 573.
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Coggins, G. H. Coombs, W. K. Milhous, S. Tzipori, D. J. P. Ferguson,
D. Chakrabarti and R. McLeod, Nature, 1998, 393, 801.
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6 B. W. Smith, M. J. Turner and E. Haslam, J. Chem. Soc., Chem.
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8 A. R. Hawkins, W. R. Reinhert and N. H. Giles, Biochem. J., 1982, 203,
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Abell, J. Org. Chem., 1999, 64, 2612.
11 A. W. Roskaz, D. A. Robinson, T. Krell, I. S. Hunter, M. Frederickson,
C. Abell, J. R. Coggins and A. J. Lapthorn, Structure, 2002, 10, 493.
12 The corresponding 3,3-difluoro derivative of DHS has been described
previously: S. Jiang, G. Singh, D. J. Boam and J. R. Coggins,
Tetrahedron Asym., 1999, 10, 4087.
13 J.-L. Montchamp, F. Tian, M. E. Hart and J. W. Frost, J. Org. Chem.,
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7373.
Fig. 2 Single crystal X-ray structure of 13.
15 For optimal results, recrystallized ketone 11 [from hexane and diethyl
ether] was used with fresh samples of DAST and anhydrous DME.
16 Crystal data for 13: C16H26F2O8, M = 384.37, tetragonal, a = b =
19.184 (3), c = 10.165 (4) Å, U = 3740.9 (17) Å3, T = 180 (2) K, space
group P41212 (no. 92), Z = 8, m (Mo-Ka) = 0.12 mm21, 3845
reflections measured, 3296 unique (Rint = 0.028) which were used in all
calculations. The final wR(F2) were 0.047 [I > 2s(I)] and 0.074 (all
data).
17 Full crystallographic data including lists of bond length, bond angles,
hydrogen coordinates and thermal parameters have been deposited at the
Cambridge Crystallographic Data Centre, CCDC 184932. See http://
www.rsc.org/suppdata/cc/b2/b205105m/ for crystallographic files in
.cif or other electronic format.
18 R. H. Rich and P. A. Bartlett, J. Org. Chem., 1996, 61, 3916.
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The desired product 12 was the minor of the two (12 13%, 13
45%). Attempted dehydrofluorination of 13 to afford the
required fluoride 12 proved to be unsuccessful under a variety
of basic conditions both with and without the addition of silver
salts (added to encourage fluoride precipitation). Nonetheless,
three step deprotection of 12 and 13 (acid catalysed bis-ketal
and MOM deprotection, saponification and ion-exchange)
afforded the fluorovinyl 6 and difluoro 7 acids (63% and 96%
respectively).
The fluorovinyl acid 6 was assayed against both type I and
type II dehydroquinases and was shown to be highly selective
for the type II proteins. Specifically 6 has a Ki of 10 mM against
the dehydroquinase from Mycobacterium tuberculosis, making
it the most potent inhibitor reported for this enzyme. Sig-
nificantly, 4 which has a hydrogen instead of a fluorine at C(3),
has a Ki of 200 mM against the same enzyme. Full biochemical
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