S.M. Watkins, et al.
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126660
Scheme 1. Reagents and conditions: a) RNH
. NaOH, H O reflux, 2. HCl 65–99% yield.
2
, EtOH, reflux, 30–88% yield; b)
1
2
Burkholderia thailandensis (Bt), Pseudomonas aeruginosa (Pa), Bacillus
cereus (Bc), Escherichia coli (Ec), Mycobacterium smegmatis (Ms),
Klebsiella
pneumoniae
(Kp),
Corynebacterium
xerosis
(Cx),
Corynebacterium pseudodiptheriticum (Cp), and Micrococcus luteus (Ml).
The compounds were tested for binding to IspF using a fluorescence-
based thermal shift (FTS) assay of Q151E BpIspF. This mutant retains
functional activity similar to the highly stable (T
m
> 95 °C) wild-type,
yet reduces the T
m
to allow for use in the FTS assay. The results are
given in Tables 1 and 2.
While not all compounds displayed potent antimicrobial activity
through bacteriostatic action, none of the nine organisms were com-
pletely immune to every compound. Each organism was susceptible to
at least one compound in the series. Also, only one compound failed to
produce positive antimicrobial results against any organism, compound
2
4.
The compounds with the greatest range of antimicrobial activity
were 5, 22, and 39, affecting the growth of seven different organisms.
They were all effective to some extent against B. thailandensis, B. cereus,
E. coli, M. smegmatis, and K. pneumoniae, but varied on P. aeruginosa, C.
xerosis, and C. pseudodiptheriticum. None of these three compounds
displayed activity against M. luteus. Structurally, there is no clear pat-
tern that may lead to a broadly effective compound. Compounds 5 and
22 had a carboxylic acid substituent in the 5-position of the pyrimidine,
while 39 had an ethyl ester in this position. Compounds 5 and 22 also
contained the halogens bromine and chlorine, respectively, but the
position of the halogens varied as did the length of the linker between
the phenyl ring and the pyrimidine ring. However, bond rotation may
make it possible for the two compounds to achieve similar binding if
antibacterial activity is proceeding through the same mechanism for
both compounds. Compound 39 has a 5-indazole substituent and may
be achieving antibacterial activity through a different route.
Fig. 1. Proposed mechanism for the MEcPP formation catalyzed by IspF.
active site is composed of four regions: the ribose pocket (Pocket I), the
lipophilic flexible loop (Pocket II), the cytidine-binding pocket (Pocket
III), and the cation region, where two divalent cations are vital for
2
+
2+
2+
catalysis. The cations vary but are often Zn
and Mg
or Zn
and
2
+ 9,11,12
2+
Mn
.
The Zn
is held in place by an aspartic acid and two
2+ 2+
histidine residues, while the Mg
substrate phosphate groups.
or Mn
ions interact with the
Enzyme inhibitors regularly incorporate metal-binding groups
MBGs) that coordinate active site ions. For example, HIV integrase
(
2
+
13,14
inhibitors target Mg in the active site of HIV integrase.
Here, a de
novo design of ligands that target the zinc ion in the active site of BpIspF
was pursued. Zinc-binding groups (ZBGs) are well-studied based on the
numerous important enzymes containing zinc. The benchmark zinc-
binding group is the hydroxamic acid, which chelates zinc in either a
B. thailandensis, a biosafety level 2 organism (BSL2), was used in
place of B. pseudomallei (BSL3) and is widely accepted as a surrogate for
B. pseudomallei. Both were derived from a common ancestor, having
1
5,16
tetrahedral or trigonal bipyramidal geometry.
Unfortunately, the
1
9
8
5% gene conservation and highly syntenic genomes. Of the 40
majority of hydroxamic acid compounds fail in clinical trials due to
issues such as toxicity, selectivity, metabolism by glucuronidation and
compounds tested, 26 compounds showed antibacterial activity against
B. thailandensis (Tables 1&2). The most potent of these compounds often
contained halogens on a ring attached to the amino group. The position
of the halogen on the ring did not make a significant difference, al-
though this is likely due to the unhindered bond rotation in the linker.
Likewise, the length of the linker between the nitrogen external to the
pyrimidine ring and the phenyl ring varied, the most potent compounds
containing all three lengths used: 0, 1, or 2 methylenes. Assuming IspF
is successfully being targeted, the hydroxyl and carboxylic acid or ethyl
ester substituents on the pyrimidinyl ring are hypothesized to bind the
4
,16
sulfation, and low oral availability.
However, cyclic isosteres of
hydroxamic acid that target matrix metalloproteinases (MMP), classic
zinc-containing enzymes, possessed higher affinity for zinc due to their
1
5
greater stability. ZBGs have been found to bind to zinc in mono-
dentate, bidentate, and a monodentate-bidentate intermediate in
1
5–17
crystal structures.
Observing the binding of salicylic acid and methyl salicylate16 as
1
8
well as the displacement of cytosine analogs by 2-aminopyridines,
a
2
+
de novo design was conceived to bind to Zn . The phenyl ring of the
salicylic acid was replaced with a pyrimidine isostere. This scaffold
consisted of a 2-amino-4-hydroxypyrimidine-5-carboxylate, with the
anticipated bidentate binding interaction with IspF’s active site zinc ion
through the 4-hydroxyl oxygen and the 5-carboxylate carbonyl oxygen,
while accumulating additional interactions with the lipophilic flexible
loop through substituents on the 2-amino nitrogen. The thioether 1 was
reacted with a variety of amines to yield the 2-aminopyrimidine ana-
logs 2, subsequent ester hydrolysis with NaOH followed by acidification
with HCl provided the acids 3 in generally good yields (Scheme 1).
The antibacterial activity of the synthesized compounds was eval-
uated by Kirby Bauer (KB) disk diffusion assays against nine organisms:
2
+
Zn
in the IspF active site, while the amino substituents extend into
and interact with additional active site residues. A carboxylic acid
group in the 5-position of the pyrimidine was generally more effective
than the ethyl ester, as shown by the comparison between 26 and 27,
3
0 and 31, 32 and 33, 40 and 41, and marginally for 22 and 23. This
trend does not hold out for every set of carboxylic acids versus ethyl
esters, showing that the amino group is having an impact on these re-
sults. While the binding of salicylic acid to zinc in a small molecule
crystal structure showed binding to zinc through the carboxylic acid
oxygens, methyl salicylate showed binding to zinc through the phenolic
oxygen and carbonyl oxygen.16 Therefore, the interactions or steric
2