Analogues of the Herbicide, N-Hydroxy- N-isopropyloxamate, Inhibit Mycobacterium tuberculosis Ketol-Acid Reductoisomerase and Their Prodrugs Are Promising Anti-TB Drug Leads

Article abstract of DOI:10.1021/acs.jmedchem.0c01919

New drugs to treat tuberculosis (TB) are urgently needed to combat the increase in resistance observed among the current first-line and second-line treatments. Here, we propose ketol-acid reductoisomerase (KARI) as a target for anti-TB drug discovery. Twenty-two analogues of IpOHA, an inhibitor of plant KARI, were evaluated as antimycobacterial agents. The strongest inhibitor of Mycobacterium tuberculosis (Mt) KARI has a Ki value of 19.7 nM, fivefold more potent than IpOHA (Ki = 97.7 nM). This and four other potent analogues are slow- and tight-binding inhibitors of MtKARI. Three compounds were cocrystallized with Staphylococcus aureus KARI and yielded crystals that diffracted to 1.6-2.0 ? resolution. Prodrugs of these compounds possess antimycobacterial activity against H37Rv, a virulent strain of human TB, with the most active compound having an MIC90 of 2.32 ± 0.04 μM. This compound demonstrates a very favorable selectivity window and represents a highly promising lead as an anti-TB agent.

Full text of DOI:10.1021/acs.jmedchem.0c01919

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Article
Analogues of the Herbicide, N‑Hydroxy‑N‑isopropyloxamate, Inhibit
Mycobacterium tuberculosis Ketol-Acid Reductoisomerase and Their
Prodrugs Are Promising Anti-TB Drug Leads
Ajit Kandale, Khushboo Patel, Waleed M. Hussein, Shun Jie Wun, Shan Zheng, Lendl Tan,
Nicholas P. West, Gerhard Schenk, Luke W. Guddat, and Ross P. McGeary*
Cite This: J. Med. Chem. 2021, 64, 1670−1684
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ABSTRACT: New drugs to treat tuberculosis (TB) are urgently needed to
combat the increase in resistance observed among the current first-line and
second-line treatments. Here, we propose ketol-acid reductoisomerase
(KARI) as a target for anti-TB drug discovery. Twenty-two analogues of
IpOHA, an inhibitor of plant KARI, were evaluated as antimycobacterial
agents. The strongest inhibitor of Mycobacterium tuberculosis (Mt) KARI has a
Ki value of 19.7 nM, fivefold more potent than IpOHA (Ki = 97.7 nM). This
and four other potent analogues are slow- and tight-binding inhibitors of
MtKARI. Three compounds were cocrystallized with Staphylococcus aureus
KARI and yielded crystals that diffracted to 1.6−2.0 Å resolution. Prodrugs of
these compounds possess antimycobacterial activity against H37Rv, a virulent
strain of human TB, with the most active compound having an MIC90 of 2.32
0.04 μM. This compound demonstrates a very favorable selectivity window
and represents a highly promising lead as an anti-TB agent.
ethyl group (isomerization) followed by an NADPH-mediated
reduction of the resulting alpha-keto acid (Scheme 1).6,7 For
the isomerization step, two Mg2+ ions and NADPH (or
NADH) are required, while the reduction step can be achieved
by any of Mg2+, Mn2+, or Co2+, along with NADPH as the
preferred hydride donor.8 The two Mg2+ ions and NADPH
bind independently to KARI before the binding of the
substrate (either (S)-2-acetolactate or (S)-2-aceto-2-hydrox-
ybutyrate).9 KARI then converts (S)-2-acetolactate to (2R)-
2,3-dihydroxyisovalerate, leading to the synthesis of valine and
leucine. (S)-2-Aceto-2-hydroxybutyrate is converted by KARI
to (2R,3R)-2,3-dihydroxy-3-methylvalerate, a precursor of
isoleucine.6,7
INTRODUCTION
■
Tuberculosis (TB) is one of the oldest diseases to afflict
mankind and remains a major cause of morbidity and
mortality, resulting in excess of 1.5 million fatalities each
year.1 It is possible to treat TB using combinations of four first-
line drugs, isoniazid, rifampicin, ethambutol, and pyrazinamide,
and in severe cases, second-line drugs (e.g., fluoroquinolones,
kanamycin, amikacin, and capreomycin) can be included in
regimens.2 However, antimicrobial therapy is prolonged, and
patients may suffer from serious side effects, making this
collection of chemotherapeutics less than ideal. Increasing
occurrences of extensively drug-resistant strains of Mycobacte-
rium tuberculosis (Mt), the bacteria responsible, and totally
drug-resistant TB are also of growing concern.3 Thus, there is
an urgent need to discover new anti-TB drugs, especially those
that work by new modes of action.
A potential new target for TB drug discovery is the
branched-chain amino acid (BCAA) biosynthesis pathway.
This has been suggested because Mt is heavily reliant on the
endogenous supply of these metabolites and the alveoli have a
limited supply of available BCAAs.4 Across plants, bacteria, and
fungi, the biosynthesis of the BCAAs requires seven enzymes
to produce leucine, isoleucine, and valine from either two
molecules of pyruvate or one molecule of pyruvate and one
molecule of 2-ketobutyrate.5 Ketol-acid reductoisomerase
(KARI) (EC 1.1.1.86) is the second enzyme in this pathway.
It catalyzes two reactions: an alkyl migration of a methyl or
Based on the lengths of their amino acid sequences, KARIs
are classified into two categories, a short form (Class-I, ∼340
amino acid residues) found in fungi and most bacteria, for
example, Mt, and a long form (Class-II, ∼490 amino acid
residues) found in plants and some bacteria.10
Received: November 5, 2020
Published: January 29, 2021
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Scheme 1. Alkyl Migration and Reduction Reactions
Catalyzed by KARI, and Structures of Experimental
Herbicides, IpOHA, and Hoe 704, Which are Known to
Inhibit Plant KARIs
Table 1. Ki Values of the Synthesized Compounds for
MtKARI and DSs with SeKARI
a
Since the BCAAs are synthesized by plants, fungi, and
bacteria but not by mammals, inhibitors of the first enzyme of
this pathway, acetohydroxyacid synthase (AHAS), have proven
to be effective herbicides and fungicides with low toxicity,11,12
and two studies4,13 have shown that AHAS inhibitors can
prevent the growth of Mt in infected mice. Thus, inhibition of
other enzymes from the BCAA pathway may also be an
effective strategy to develop novel biocides. Compounds such
as N-hydroxy-N-isopropyloxamate (IpOHA) and 2-dimethyl-
phosphinoyl-2-hydroxyacetic acid (Hoe 704) (Scheme 1) are
potent substrate analogue inhibitors of plant KARI.14 IpOHA
only weakly inhibits the growth of three Mt strains at
concentrations in the range 60−1000 μM.4 In contrast,
IpOHA potently inhibits Escherichia coli KARI by slow- and
tight-binding with an initial Ki of 160 nM, decreasing to 22 pM
(final steady-state Ki).14
Previously, we have reported that IpOHA has a Ki value of
97.7 nM for MtKARI15 and thus represents an excellent
starting point for anti-TB drug discovery. Here, we report the
syntheses of twenty-two IpOHA analogues, their inhibition of
MtKARI, and the structure determination of three of the most
potent inhibitors in complex with Staphylococcus aureus (Sa)
KARI, an enzyme which is 56% identical and 76% homologous
to MtKARI.16,17 Prodrugs of these compounds were also tested
against avirulent (H37Ra) and virulent (H37Rv) strains of Mt.
RESULTS AND DISCUSSION
■
Inhibitor Design. Initially, a series of IpOHA analogues
(22 analogues, Table 1) was designed and evaluated by the
docking of trial compounds into the active site of Slackia exigua
(Se) KARI.18 The structure of SaKARI was not available at the
time, and the structure of MtKARI16 was not considered
suitable since the cofactor, NADPH, is not visible. However,
the active site residues of SeKARI are identical to those of
MtKARI, and its structure was solved in the presence of
IpOHA and NADPH and so was considered to be a better
structure for use in docking calculations. The chemical
structures and docking scores (DSs) of the trial compounds
are shown in Table 1. The DS of 5b is −7.8, which matches the
DS of IpOHA. Compounds 4d, 5a, 5c, 5d, and 5e also have
excellent DSs ranging from −5 to −8. Since the binding poses
and interactions of all docked molecules are similar to those
observed for the SeKARI·Mg2+·NADPH·IpOHA complex,
a
Note: ND = post docking energy minimization of the enzyme−
inhibitor complex did not converge. *Scores obtained by docking the
compounds in the SaKARI active site (see below).
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a
Scheme 2. Synthesis of IpOHA Analogues 5a−e
a
Reagents and conditions: (a) NH2OH·HCl, NaOH, EtOH, H2O, 60 °C, 3 h; (b) NaCNBH3, 4 M HCl-dioxane, MeOH, rt, 3 h; (c) ClCOCO2Et,
DIPEA, THF, 4 °C, 3 h; and (d) KOH, THF, H2O, Δ, 1 h.
there is a strong suggestion that these compounds would also
inhibit MtKARI.
Chemistry. The designed compounds were synthesized by
coupling hydroxylamines or amines with ethyl oxalyl chloride
(EOC) in the presence of N,N-diisopropylethylamine
(DIPEA) (Schemes 2−4). Hydrolysis using aqueous KOH
e) which were then saponified using aqueous KOH.19 Finally,
the piperidine-hydroxamate 1220 and the β-carboxyhydrox-
amate 14 were synthesized as shown in Scheme 4.
Inhibition Studies. An initial study was performed to
assess whether the IpOHA analogues exhibit time-dependent
binding to MtKARI by monitoring the progress of the
interaction over 1 h (exemplified for 5b in Figure 1A).
Based on the observed slow binding, all subsequent assays were
performed by first incubating the assay mixture (MtKARI,
NADPH, Mg2+, and IpOHA) for 30 min. The reaction was
then initiated by adding (S)-2-acetolactate (longer incubation
times did not affect the magnitude of the determined Ki
values). In the first round of screening, the inhibitory effects
of the analogues were tested at a concentration of 100 μM;
4a−e and 5a−e were identified as strong inhibitors of
MtKARI. In contrast, the compounds devoid of an N-hydroxyl
group (7a−c and 8a−e) are very weak inhibitors (Table 1).
Compound 11 bearing a Boc-protected piperidine has
negligible inhibition. This is attributed to the large size of
this protecting group preventing it from entering the active
site. Upon removal of the Boc-protecting group (12),
inhibition was observed, supporting this hypothesis. The
malonate derivatives 13 and 14 exhibited poor inhibition
compared to their homologues 4b and 5b, showing the
importance of the α-ketoacid moiety for activity.
a
Scheme 3. Synthesis of Oxamic Acids 8a−e
a
Reagents and conditions: (a) ClCOCO2Et, DIPEA or pyridine, THF
or CH2Cl2 and (d) KOH, THF, H2O.
Scheme 4. Synthesis of Piperidine-hydroxamate, 12, and β-
a
carboxyhydroxamate, 14
Determination of Ki Values. Ki values were determined
by fitting inhibition data to either the Morrison equation (for
tight-binding inhibitors 4b−d, 5a, and 5b−d) or the standard
inhibition equation (for weaker inhibitors 4a, 4e, 5e, and 12)
against MtKARI21 (Table 1). Compound 5b showed a fivefold
increase in potency (Ki of 19.7
1.4 nM) compared to
IpOHA (Ki = 97.7 8.3 nM)15 and is the most active in the
series (Figure 1).
Compounds 4b, 4d, 5a, 5b, and 5d are more potent than
IpOHA against MtKARI. 5b is the most potent with an
approximately fivefold decrease in Ki compared to IpOHA.
The ethyl ester analogue (4b) of 5b has a Ki of 32.2 nM. It is
possible that 4b might undergo ester hydrolysis under assay
conditions, thus converting it to its corresponding carboxylic
acid derivative (5b), which may be the main contributor to the
observed inhibition of MtKARI. To investigate this hypothesis,
the compounds present in the assay mixture were monitored
by mass spectrometry. If the ethyl ester group of 4b was being
hydrolyzed to the corresponding carboxylic acid in the assay,
the presence of 5b with a mass of 172.06 amu for the M−H
ion in the negative mode was expected. However, we only
observed a peak at 224.10 amu, corresponding to the [M +
Na]+ ion of the starting ester 4b. This confirmed that the ester
was stable under the assay conditions for 30 min of incubation
and the enzyme inhibition obtained was solely due to 4b itself.
a
Reagents and conditions: (a) (i) NH2OH·HCl, NaOAc, AcOH (ii)
NaCNBH3, MeOH, rt, 16 h; (b) ClCOCO2Et, DIPEA, THF, 3 h; (c)
4 M HCl-dioxane, THF, rt, 1 h; (d) ClCOCH2CO2Me, DIPEA, THF,
4 °C, 3 h; and (e) KOH, THF, H2O, Δ, 1 h.
to cleave the ester side chains, in all cases, gave the
corresponding carboxylic acid derivatives. The hydroxylamines
(3a−e) needed for the synthesis of 5a−e were prepared by the
reaction of the respective ketones (1a−e) with hydroxylamine
hydrochloride to obtain oximes (2a−e), followed by reduction
to the corresponding N-alkylhydroxylamines (3a−e) using
NaCNBH3.
The amines 6a−e required for the syntheses of 8a−e were
purchased from Sigma-Aldrich. Amides 8a−e were prepared as
shown in Scheme 3. Coupling of amines 6a−e with EOC with
either pyridine or DIPEA as a base gave the ethyl esters (7a−
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Figure 1. Inhibition of MtKARI by IpOHA analogues (5a−c). (A) Progress curves illustrating time-dependent inhibition of MtKARI by 5b. (B−D)
Inhibition plots for 5a, 5b, and 5c, respectively, fitted to the Morrison equation.22
a
Table 2. Data Collection and Refinement Statistics for SaKARI in Complex with Mg2+, NADPH, and 5a−c
5a
5b
5c
Data Collection
a = 64.48, b = 80.39, c = 67.30
α = γ = 90, β = 90.13
P21
unit cell length (Å)
unit cell angle (deg)
space group
a = 65.72, b = 79.03, c = 69.64
α = γ = 90, β = 101.39
P21
a = 65.82, b = 78.91, c = 69.37
α = γ = 90, β = 101.10
P21
unique reflections [I > 0σ(I)] 41,023 (2928)
79,180 (3915)
595,692 (27,606)
99.8 (97.0)
0.081 (0.740)
0.034 (0.322)
13.2 (1.9)
56,182 (3161)
193,605 (10,382)
98.8 (87.4)
0.063 (0.476)
0.045 (0.345)
9.1 (1.7)
total observations [I > 0σ(I)]
completeness (%)
Rmerge
140,268 (9792)
98.9 (90.9)
0.070 (0.519)
0.052 (0.388)
8.7 (1.6)
Rpim
⟨I⟩/⟨σ(I)⟩
subunits per asymmetric unit
2
2
2
Refinement
resolution range (Å)
Rwork
43.61−2.09
0.170 (0.197)
0.209 (0.234)
33.08
46.63−1.67
43.50−1.88
0.162 (0.165)
0.207 (0.209)
30.23
0.154 (0.157)
0.192 (0.196)
20.31
Rfree
average B-factor (Å2)
RMS Deviation from Ideal
0.010
bond length (Å)
bond angle (deg)
0.003
0.564
0.017
1.474
1.185
Ramachandran Plot Statistics
97.53%
favored
outliers
97.84%
0%
96.89%
0%
0%
residues not visible in density
1 and 328−334 in both subunits 1 and 328−334 in both subunits 1 and 323−334 (chain A), 1 and 328−334 (chain B)
a
Values in parentheses are for the outer resolution shell. Rmerge
= ∑hkl∑i|Ii(hkl) − (I(hkl))|/∑hkl∑iIi(hkl).
Ä
É
Rpim = ∑hkl
Ñ
1/2∑ |Ii(hkl) − (I(hkl))|/∑ ∑ I(hkl) where Ii(hkl) is the observed intensity and I(hkl) is the average intensity
Å
Ñ
Å
1
Ñ
Å
Å
Å
Ñ
i
i
Ñ
i
hkl
[N(hkl) − 1]
Å
Ñ
Å
Ç
Ñ
Ö
obtained from multiple observations of symmetry-related reflections.
The strong binding of compounds 5a−e to MtKARI is likely
due to the presence of an α-carboxyhydroxamate group that
can bind to the two Mg2+ ions and also the type of substituents
on the nitrogen atom which fits inside the hydrophobic pocket
of the active site. Compounds with smaller alicyclic rings or
aliphatic chains, as seen in 5b and 5d, showed maximum
inhibition. Increasing the ring size to cyclohexane (5a) or the
length of the aliphatic side chain (4-methyl-2-pentyl
substitution in 5e) attached to the nitrogen atom led to a
decrease in activity. Replacing the aliphatic functionality on the
nitrogen atom with an aromatic moiety (2-phenylethyl in 5c)
also lowered the activity compared to 5b.
SaKARI Inhibition Assays. Attempts to crystallize these
compounds in complex with MtKARI were unsuccessful.
Hence, we chose SaKARI for these experiments. The active
site residues in SaKARI and MtKARI are highly conserved
(Supporting Information, Figure S1). To also confirm the
functional similarity of these two enzymes, the inhibitors 5a−c
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were shown also to be potent inhibitors of SaKARI. The Ki
value of 5b is twofold lower for SaKARI than for MtKARI (8.5
SaKARI·Mg2+·NADPH·IpOHA complex which also showed
the presence of the reduced form of IpOHA.17
The electron density maps showed that the nicotinamide
ring of NADPH is puckered (and likely reduced) when in
complex with 5b and 5c and planar (and likely oxidized) when
in complex with 5a (Figures 3A and 4B). When the intact
1.0 nM vs 19.7
1.4 nM), while for 5c, the Ki value is
twofold higher for SaKARI than for MtKARI (193 8.4 nM vs
97.7 8.3 nM). However, the Ki of 5a for SaKARI is ∼25-fold
higher (1.0
0.4 μM) compared to MtKARI (39.6 nM), a
difference that may be due to the presence of isoleucine
instead of valine in position 248 of SaKARI (Supporting
Information, Figure S1), reducing the size of the active site
binding pocket.
Crystal Structures of SaKARI in Complex with 5a−c.
The diffraction data for the SaKARI·Mg2+·NADPH in complex
with 5a, 5b, and 5c were collected to be 2.09, 1.67, and 1.88 Å,
respectively. The data collection and refinement statistics are
shown in Table 2. In the asymmetric unit, a dimer of SaKARI
is present with the two active sites at the interface of the two
subunits. The electron density of the polypeptide is continuous
from the N- to C-termini apart from the first N-terminal
residue and last seven C-terminal residues of each subunit. The
overall fold of the dimer is similar (rmsd: 0.15−0.54 Å) to that
of the SaKARI·Mg2+·NADPH·IpOHA complex.17
The electron density maps after fitting 5a, 5b, and 5c with
full occupancy showed negative density where the N-hydroxy
group is located and additional spherical positive density
nearby (Figure 2). This suggests that the N−O bonds in a
Figure 3. active site of SaKARI in complex with Mg2+, NADPH, and
the IpOHA analogues. (A) 2Fo−Fc electron density map for
nicotinamide of NADPH in complex with 5b contoured at 2σ. (B)
2Fo−Fc electron density map for nicotinamide of NADPH in complex
with 5a contoured at 2σ. (C) 5a and (D) its deoxy form RC5a shown
as yellow sticks. (E) 5b and (F) its deoxy form RC5b are shown as
pink sticks. (G) 5c and (H) its deoxy form RC5c shown as dark green
sticks (PDB codes: 5a 6C55, 5b 6C5N, and 5c 6BUL). The metals
and water molecules are shown as cyan and red spheres, respectively,
and active site residues are shown as white sticks. The metal ligand
bonds are shown in yellow, and hydrogen bonds are shown in green.
Figure 2. Electron density maps for 5b in the SaKARI.
Mg2+.NADPH.5b complex. (A) 2Fo−Fc (blue: 2σ) and difference
maps (red: −3σ and green: 3σ) after fitting 5b at 100% occupancy.
(B,C) 2Fo−Fc map after fitting 5b and RC5b at an occupancy of 0.44
and 0.56, respectively (PDB code: 6C5N). Cyan spheres represent the
Mg2+ ions, and the red sphere represents water.
fraction of these inhibitors were cleaved to amide products. Re-
refinements using combinations of intact (5a, 5b, or 5c) and
deoxy forms (RC5a, RC5b, or RC5c) of the inhibitors with
variable occupancies confirmed that both were present in the
active site. Figure 2 shows 5b as an example. When RC5b is
bound, a water molecule is present to complete the
coordination sphere of Mg2+. This water molecule and the
hydroxyl oxygen of the intact compound (5b) would be only
1.7 Å apart, so their simultaneous existence in the active site is
implausible. A high-resolution mass spectrum of the sample of
5b used for crystallization was obtained to confirm its purity,
and no peaks corresponding to the deoxy form were observed.
Therefore, the reduction must have occurred during
crystallization. These observations are in accordance with our
previous results obtained for the crystal structure of the
inhibitors are bound in the active site, the N−OH group
interacts with Mg2+(II), resulting in a near perfect octahedral
geometry. The hydroxyl oxygen is ∼2.4 Å from the carboxylate
oxygen of E230, suggesting a hydrogen bond, with either E230
or the hydroxyl being the proton donor (Figure 3C,E,G).
When the hydroxyl group is cleaved from these inhibitors, an
additional water molecule is observed. This does not
superimpose with the hydroxyl but fills a nearby space where
it also forms a hydrogen bond with E230. This difference
results in a distortion of the octahedral geometry at the
Mg2+(II) site. Irrespective of whether the intact or cleaved
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inhibitor is present in the crystal structures, the Mg2+(I) site is
unaltered with the positions of the six ligands conserved in an
octahedral arrangement (Figure 3). The carboxyl groups of
each inhibitor make important interactions, with one of the
oxygen atoms forming a hydrogen bond with the amide of
S251 and the other oxygen atom ligating the Mg2+(I) (Figure
3). The cyclohexane ring in 5a has a chair conformation,
whereas the cyclopentane ring in 5b adapts a half-chair
conformation.
forms a hydrophobic interaction with the side chain of A254
(Figure 4C). The active site pockets in Figure 4A,C are
expanded by ∼1−2 Å compared to those in Figure 4B,D. The
cyclohexane ring of 5a and the positioning of the methyl group
of 5c require more space in the active site. The orientation of
the methyl group in the second conformation of 5c results in
the phenyl ring moving deeper into the pocket, leading to a
contraction of the active site (Figure 4D).
The binding of 5a−c to SaKARI causes rotation of the N-
terminal domains of the dimer (Figure 5A). In addition, there
is a movement of the helix (P132−E142) by 1.72 Å. Binding of
5a and 5c in the conformation shown in Figure 4C perturbs
the position of P132, E230, and I234 (Figure 5B). This
confirms that the active site is flexible. In contrast, S251 and
D190 remain in the same position in all structures. The
potencies of these compounds correlate with the degree to
which the active site is contracted. The most potent inhibitor
5b (Ki = 8.5 nM for SaKARI) binds in a way that the active site
is contracted, and the binding of the least potent 5a (Ki = 1
μM) keeps the active site comparatively expanded. The activity
of 5c (Ki = 193 nM) is intermediate between 5a and 5b,
consistent with the observation that there are two alternative
conformations in the active site (Figure 4C,D).
The rings of 5a, 5b, and 5c make hydrophobic interactions
with the surrounding residues P132, L198, I234, and I250
(Figure 4). In the first conformation of 5c, its methyl group
Comparison of the Molecular Docking Results with
the Crystal Structures. Since we determined the crystal
structures of 5a−c in complex with SaKARI·Mg2+·NADPH, we
were able to compare these experimental data with results
predicted from in silico docking. The predicted docking
orientations of 5a and 5b were consistent with the crystal
structures (Figure 6). The cyclohexane ring in 5a is in a chair
conformation in both experimental and docked structures. The
confirmation of 5b in the crystal structure matches well with
the pose predicted from the docking, other than a slight
rotational offset of the cyclopentane ring away from S251-
(Figure 6B). However, the lengths of the metal−oxygen
coordination bonds predicted by docking were shorter (by
0.1−0.3 Å) than those observed in the crystal structures.
When either the R or S enantiomer of 5c was docked into
the active site of SeKARI, the software could not predict any
Figure 4. Hydrophobic interactions for 5a−5c at the active site. The
active site residues are shown as green sticks, and the compounds are
shown in different shades of purple sticks. The hydrophobic
interactions are shown as black dashed lines. Metals are shown as
cyan spheres.
Figure 5. Comparison of the overall fold and active site of the SaKARI in complex with the newly synthesized inhibitors. 5a (yellow), 5b
(magenta), conformation 1 of 5c (light green), and conformation 2 of 5c (dark green) (PDB codes: 5a 6C55, 5b 6C5N, and 5c 6BUL). Metals are
shown as cyan spheres. (A) Black arrows indicate rotation of the domain associated with the active site and substrate binding. (B) Orange arrows
indicate movement of amino acids in the active site.
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the docking of both enantiomers of 5c into the SaKARI·Mg2+·
NADPH·5c structure was performed (Figure 6). Scores of
−7.8 and −7.7 for the R and S isomers, respectively, were
obtained, suggesting that these isomers could have similar
potencies. However, the prediction made by Glide showed that
the mode of binding of the S isomer was different to that
observed in the crystal structure. Instead of the carbonyl
oxygen of (S)-5c bridging the metal ions as seen in the crystal
structure, a carboxylate oxygen performs this role in the
docked structure (Figure 6C). The predicted docking of the R
isomer of 5c shows a binding mode similar to that observed in
the crystal structure of the SaKARI·Mg2+·NADPH·5c complex
(i.e., the carbonyl oxygen bridges the metal ions, Figure 6D).
The fact that the crystal structure of SaKARI with 5c has only
the S isomer bound suggests that it favors the S isomer. Thus,
the true Ki value of the S-enantiomer of 5c may be less than the
97.7 nM obtained for the racemate.
Antimycobacterial Activity of MtKARI Inhibitors.
Although several of the 22 IpOHA analogues are potent
inhibitors of MtKARI, none exhibited MIC90 values <200 μM
(data not shown). Figure 7A shows, as an example, the
inhibition of H37Rv growth due to compound 5b. A likely
reason for their lack of activity is that their hydrophilicity
prevented them from crossing the complex membrane of Mt.
To investigate this possibility, prodrugs were synthesized by
esterifying the carboxylate group of the most potent inhibitor,
Figure 6. Comparison of the docking and crystal structure results.
The crystal structures of all the compounds are shown in light pink.
The docked structure for 5a is in yellow, 5b is in magenta, and 5c is in
light green for the R isomer and dark green for the S isomer. 5a and
5b were docked using the SeKARI·Mg2+·NAPDH·IpOHA complex.
The 5c enantiomers were docked using the SaKARI·Mg2+·NAPDH·
IpOHA complex.
valid poses due to failure in postdocking energy minimization.
This could be due to insufficient space in the SeKARI active
site. However, our experimental data show that 5c has Ki
values of 97.7 nM for MtKARI and 193 nM for SaKARI,
confirming its potent inhibition. Thus, to investigate further,
Figure 7. Antimycobacterial activity of the prodrugs of 5b. (A) Inhibition of H37Rv growth by 5b. (B−F) Inhibition of H37Ra growth by 23−27
in the absence (black) and in the presence (blue) of BCAAs. (G−I) Inhibition of H37Rv growth by 24−26 in the absence (black) and in the
presence (red) of BCAAs.
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a
Scheme 5. Synthesis of Esters 23−27
a
Reagents and conditions: (a) ROH, CH2Cl2, 0 °C, 14 h and (b) 5b, DIPEA, THF, 0 °C, 14 h.
5b, adding long-chain lipophilic structures of various lengths,
giving derivatives 23−27 (Scheme 5). Prodrugs derived from
5b, esterified with octanol (24), decanol (25), and dodecanol
(26), exhibited excellent MIC90 values in the range of 3−6 μM
for H37Ra and 2−3 μM for H37Rv strains (Figure 7D−I),
while derivatives 23 and 27 (Figure 7 B,C), derived from
hexanol and octadecanol, respectively, showed lower activity
with MIC90 values >30 μM. To confirm that the
antimycobacterial activity of these compounds was not due
to the prodrug attachments themselves, the long-chain alcohols
were all tested in Mt assays (Supporting Information, Figure
S3). All these have MIC90 values >30 μM. Thus, it appears that
these prodrugs can cross the membrane of Mt and then be
hydrolyzed by cellular esterases to release the KARI inhibitor
(5b).
CONCLUSIONS
■
Twenty-two analogues of IpOHA were designed, synthesized,
and assayed against MtKARI. The Ki values of these
compounds were as low as 19 nM (5b). Cell susceptibility
assays showed that the parent compounds were inactive but
lipophilic prodrugs of 5b successfully inhibited the growth of
Mt with MIC90 values as low as 2.3 μM. Supplementation of
the growth media with BCAAs demonstrated that the target
was the BCAA pathway. Moreover, the prodrugs were still
effective against Mt even at high BCAA concentrations. Crystal
structures of several IpOHA analogues (5a−c) in complex with
SaKARI·Mg2+·NADPH demonstrated a conserved mechanism
of inhibitor binding, resulting in closure of the active site
(Figures 4 and 5). This is the first comprehensive study that
demonstrates that a designed KARI inhibitor is a promising
drug lead to combat TB.
To confirm that the target of the active compounds is the
BCAA pathway, the assays were repeated but with media
supplemented with 1 mM each of leucine, isoleucine, and
valine, concentrations much higher than expected in vivo
(typical concentrations in the alveoli range from 100 to 200
μM23). After the addition of the BCAAs, the activities of the
compounds were reduced by three- to ninefold, confirming the
BCAA pathway as their target (Figure 7D−I). Furthermore,
24−26 have very similar MIC90 values against H37Rv,
regardless of which ester group was attached (Figure 7G−I).
Interestingly, the reduction in the inhibitory activity of 24−26
in H37Rv (six- to ninefold) upon the addition of the BCAAs
was more pronounced than that observed in H37Ra (three- to
ninefold).
EXPERIMENTAL SECTION
■
Materials. All reagents and solvents required for the synthesis
were purchased from Sigma-Aldrich, Merck, Novachem, Chembridge
chemicals, and AK scientific. Silica gel (0.040−0.063 mm) for flash
chromatography was purchased from Merck. MtKARI16 and
SaKARI17,24 were expressed and purified as described previously.
NADPH was purchased from Sigma-Aldrich. (S)-2-Acetolactate was
synthesized by alkaline hydrolysis of the methyl ester of 2-hydroxy-2-
methyl-3-oxobutyrate purchased from Sigma-Aldrich. Although a
racemic mixture is obtained as a result of hydrolysis, only (S)-2-
acetolactate is a substrate for KARI. The concentration of the (S)-
isomer was determined by measuring the consumption of NADPH at
340 nm until all of the (S)-2-acetolactate was depleted. Small aliquots
were stored at −80 °C until use. Purity of synthesized compounds is
1
>95%, as determined by elemental analyses and H NMR spectros-
Cytotoxicity of Compound 25 in Mammalian Cell
Lines. To access cytotoxicity, the most potent prodrug, 25,
was assessed using an MTT assay in three mammalian cell lines
(RAW 264.7, HEK 293, and SW620). The compound was not
toxic up to 50 μM concentration, which is about 16-fold higher
than the MIC90 in H37Rv cells (Figure 8).
copy.
Chemical Synthesis. Synthesis of Oximes 2a−e. Hydroxylamine
hydrochloride (0.074 mol, 5.13 g) and sodium hydroxide (0.148 mol,
5.92 g) were dissolved in a sufficient distilled water and ethanol
mixture (3:1) in a 500 mL round bottom flask. The ketone 1a−e
(0.05 mol) was added to this solution. More ethanol was added if
necessary to solubilize the ketone completely. The mixture was heated
at 60 °C. The reaction was monitored by thin-layer chromatography
(TLC) and continued until complete disappearance of the initial
ketone (approximately 3 h). After the reaction, the ethanol was
evaporated under reduced pressure and the aqueous layer was
extracted with CH2Cl2 (3 × 30 mL). The combined organic layers
were washed with water, dried over anhydrous sodium sulfate, filtered,
and evaporated under reduced pressure to give the crude oxime. The
product was recrystallized from petroleum ether and dichloromethane
(DCM) (9:1).
Cyclohexanone Oxime (2a). From 0.05 mol (4.9 g) of
cyclohexanone (1a) in ethanol−water (20 and 60 mL), a light
brown solid was obtained (1.82 g, 32%); mp 86−88 °C (lit.25 mp 87−
89 °C); TLC (Rf = 0.80, petroleum ether and ethyl acetate, 9:1).
Cyclopentanone Oxime (2b). From 0.05 mol (4.2 g) of
cyclopentanone (1b) in ethanol−water (20 and 60 mL), a light
brown solid was obtained (2.2 g, 45%); mp 57−58 °C (lit.26 mp 57−
58.5 °C); TLC (Rf = 0.74, petroleum ether and ethyl acetate, 8:2).
Acetophenone Oxime (2c). From 0.25 mol (30 g) of
acetophenone (1c) in ethanol−water (100 and 300 mL), a light
Figure 8. MTT assay of 25 against three mammalian cell lines. RAW
264.7 cells are viable up to 100 μM, and HEK293 and SW620 cells are
viable up to 50 μM concentration of the compound.
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brown solid was obtained (30.4 g, 90%); mp 59−60 °C (lit.27 mp 59−
60 °C); TLC (Rf = 0.88, petroleum ether and ethyl acetate, 7:3).
2-Butanone Oxime (2d). From 0.25 mol (18 g) of 2-butanone
(1d) in ethanol−water (100 and 300 mL), a yellow liquid was
obtained (11.3 g, 52%); bp 151−153 °C (lit.27 bp 152 °C); TLC (Rf
= 0.48, petroleum ether and ethyl acetate, 7:3).
4-Methylpentan-2-one Oxime (2e). From 0.25 mol (25 g) of 4-
methylpentan-2-one (1e) in ethanol−water (100 and 300 mL), a pink
solid was obtained (19.5 g, 68%); mp 61.5−63 °C (lit.28 mp 63 °C);
TLC (Rf = 0.64, petroleum ether and ethyl acetate, 8:2).
Reduction of Oximes 2a−e to Hydroxylamines 3a−e. Reduc-
tions were carried out according to the reported procedure.29 To a
methanolic solution of oxime (2a−e) (2 mmol), sodium cyanobor-
ohydride (4 mmol, 0.252 g) and approximately 10 mg of a
bromocresol green indicator were added, followed by the dropwise
addition of 4 M dioxane-HCl under a nitrogen atmosphere to
maintain the yellow color (pH 4) for 30 min. The reaction mixture
was stirred at room temperature for 3 h. The precipitated salt was
filtered off, and the filtrate was evaporated under reduced pressure.
The residue was taken up in 200 mL of water, and 6 M potassium
hydroxide solution was added until alkaline (pH 12). The aqueous
layer was saturated with sodium chloride and extracted with CH2Cl2
(3 × 30 mL). The combined organic extracts were washed with brine,
dried over anhydrous sodium sulfate, and filtered. The filtrate was
evaporated under reduced pressure to obtain the hydroxylamine.
N-Cyclohexylhydroxylamine (3a). From 0.02 mol (2.3 g) of
cyclohexanone oxime (2a) in 50 mL of methanol, a colorless solid was
obtained (2.1 g, 89%); mp 139−140 °C (lit.30 mp 138−140 °C);
TLC (Rf = 0.16, chloroform and methanol, 9:1).
N-Cyclopentylhydroxylamine (3b). From 0.02 mol (2.0 g) of
cyclopentanone oxime (2b) in 50 mL of methanol, a colorless solid
was obtained (1.12 g, 56%); mp 91.5−92.5 °C (lit.31 mp 92−93 °C);
TLC (Rf = 0.49, chloroform and methanol, 9:1).
N-(1-Phenylethyl)hydroxylamine (3c). From 0.02 mol (2.7 g) of
acetophenone oxime (2c) in 20 mL of methanol, a colorless solid was
obtained (0.90 g, 33%); mp 69−71 °C (lit.32 mp 69−71 °C); TLC
(Rf = 0.38, petroleum ether and ethyl acetate, 7:3).
N-(But-2-yl)hydroxylamine (3d). From 0.04 mol (3.5 g) of 2-
butanone oxime (2d) in 40 mL of methanol, a colorless solid was
obtained (1.96 g, 55%); mp 96−97.5 °C (lit.30 mp 97 °C); TLC (Rf =
0.37, petroleum ether and ethyl acetate, 1:1).
(CH2CH3), 55.3 (C5H10CH), 28.5, 25.2, 24.8, 13.9; HRMS (ESI/
micrOTOF-Q) m/z: [M + Na]+ calcd for C10H17NO4Na, 238.1050;
found, 238.1048; Anal. Calcd for C10H17NO4: C, 55.80; H, 7.96; N,
6.51. Found: C, 55.76; H, 8.00; N, 6.49.
Ethyl N-Cyclopentyl-N-hydroxyoxamate (4b). From 0.01 mol
(1.01 g) of N-cyclopentylhydroxylamine (3b) in 20 mL of anhydrous
THF, a brown solid was obtained (830 mg, 41%); mp 47.0−49.2 °C;
1
TLC (Rf = 0.38, petroleum ether and ethyl acetate, 9:1); H NMR
(300 MHz, CDCl3, 25 °C): δ 8.00 (bs, 1H, N−OH), 4.67−4.79 (m,
1H, C4H8CH−N), 4.36 (q, 2H, CH3CH2, J = 7.3), 1.41−2.03 (m,
8H, C4H8CH−N), 1.34−1.38 (t, 3H, CH3CH2, J = 7.3 Hz); 13C
NMR (75 MHz, CDCl3, 25 °C): δ 163.5 (COO), 160.3 (CON), 62.5
(CH2CH3), 56.6 (C4H8CH), 29.4, 28.2, 25.0, 24.7, 13.9; HRMS
(ESI/micrOTOF-Q) m/z: [M + Na]+ calcd for C9H15NO4Na,
224.0893; found, 224.0890; Anal. Calcd for C9H15NO4: C, 53.72; H,
7.51; N, 6.96. Found: C, 53.58; H, 7.45; N, 6.94.
Ethyl N-(1-Phenylethyl)-N-hydroxyoxamate (4c). From 0.003 mol
(0.41 g) of N-(1-phenylethyl)hydroxylamine (3c) in 20 mL of
anhydrous THF, a colorless solid was obtained (250 mg, 35%); mp
77.6−78.8 °C (lit.33 mp 78 °C); TLC (Rf = 0.42, petroleum ether and
ethyl acetate, 7:3); 1H NMR (300 MHz, CDCl3, 25 °C): δ 7.29−7.43
(m, 5H, C6H5), 6.92 (bs, 1H, N−OH), 5.69 (q, 1H, J = 6.0,
CHCH3), 4.39 (q, 2H, CH3CH2, J = 7.0 Hz), 1.65 (d, 3H, CHCH3, J
= 6.0), 1.39 (t, 3H, CH3CH2, J = 7.0); 13C NMR (75 MHz, CDCl3,
25 °C): δ 162.8 (COO), 160.3 (CON), 138.2, 128.7, 127.6, 126.9,
62.4 (CH2CH3), 54.3 (CHCH3), 15.6, 13.9; HRMS (ESI/micrO-
TOF-Q) m/z: [M + Na]+ calcd for C12H15NO4Na, 260.0893; found,
260.0894: [M + H]+ calcd for C12H16NO4, 238.1074; found,
238.1079; Anal. Calcd for C12H15NO4: C, 60.75; H, 6.37; N, 5.90.
Found: C, 60.56; H, 6.36; N, 5.90.
Ethyl N-Isobutyl-N-hydroxyoxamate (4d). From 0.005 mol (0.45
g) of N-(isobutyl)hydroxylamine (3d) in 20 mL of anhydrous THF,
an orange liquid was obtained (580 mg, 59%); TLC (Rf = 0.75,
petroleum ether and ethyl acetate, 1:1); 1H NMR (300 MHz, CDCl3,
25 °C): δ 4.35 (q, 2H, CH3CH2, J = 7.0 Hz), 4.18−4.29 (m, 1H,
CH3CH2CHCH3), 1.49−1.60 (m, 2H, CH3CH2CHCH3), 1.18−1.41
(m, 6H, CH3CH2CHCH3), 0.93 (t, 3H, CH3CH2, J = 7.0); 13C NMR
(75 MHz, CDCl3, 25 °C): δ 169.8 (COO), 163.5 (CON), 62.9
(CH2CH3), 53.5 (CH3CH2CHCH3), 25.9, 16.7, 13.9, 10.6; HRMS
(ESI/micrOTOF-Q) m/z: [M + Na]+ calcd for C8H15NO4Na,
212.0893; found, 212.0898: [M + H]+ calcd for C8H16NO4, 190.1074;
found, 190.1077; Anal. Calcd for C8H15NO4: C, 50.78; H, 7.99; N,
7.40. Found: C, 50.77; H, 7.84; N, 7.07.
N-(4-Methylpentan-2-yl)hydroxylamine (3e). From 0.04 mol (4.6
g) of 4-methylpentan-2-one oxime (2e) in 40 mL of methanol, a
yellow solid was obtained (2.88 g, 62%); mp 52−53.5 °C (lit.28 mp 63
Ethyl N-(4-Methyl-pent-2-yl)-N-hydroxyoxamate (4e). From 0.01
mol (1.17 g) of N-(4-methyl-pent-2-yl)hydroxylamine (3e) in 20 mL
of anhydrous THF, a yellow liquid was obtained (1.45 g, 66%); TLC
1
°C); TLC (Rf = 0.24, petroleum ether and ethyl acetate, 8:2). H
NMR (300 MHz, CDCl3, 25 °C): δ 5.52 (bs, 2H, NHOH), 2.99−
3.10 (m, 1H, CH3CHNH), 1.59−1.72 (m, 1H, CH3CHCH3), 1.13−
1.43 (m, 2H, CHCH2CH), 1.08−1.10 (d, 3H, J = 6.0 Hz,
CH3CHNH), 0.89−0.89 (d, 6H, J = 6.0 Hz, CH3CHCH3); 13C
NMR (75 MHz, CDCl3, 25 °C): δ 55.3 (CH3CHNH), 43.0
(CHCH2CH), 24.9 (CH3CHCH3), 23.2 (CH3CHCH3), 22.4
(CH3CHCH3), 18.0 (CH3CHNH).
1
(Rf = 0.61, petroleum ether and ethyl acetate, 6:4); H NMR (300
MHz, CDCl3, 25 °C): δ 4.53−4.60, m, 1H, (CH3)2CHCH2CHCH3,
4.37, q, 2H, CH3CH2, J = 7.0 Hz, 1.80−1.89, m, 1H,
(CH3)2CHCH2CHCH3, 1.33−1.70, m, 6H, (CH3)2CHCH2CHCH3
and CH3CH2, 1.24, dd, 2H, J = 6.0, 6.0, (CH3)2CHCH2CHCH3,
0.86−0.97, m, 6H, (CH3)2CH; 13C NMR (75 MHz, CDCl3, 25 °C):
δ 164.3 (COO), 160.4 (CON), 62.5 (CH2CH3), 50.0 (N−CHCH3),
41.7 ((CH3)2CHCH2), 24.9 ((CH3)2CHCH2CHCH3), 22.8 and 22.2
((CH3)2CHCH2CHCH3), 17.3 ((CH3)2CHCH2CHCH3), 13.9
(CH3CH2); HRMS (ESI/micrOTOF-Q) m/z: [M + Na]+ calcd for
C10H19NO4Na, 240.1206; found, 240.1209: [M + H]+ calcd for
C10H20NO4, 218.1387; found, 218.1391; Anal. Calcd for C10H19NO4:
C, 55.28; H, 8.81; N, 6.45. Found: C, 55.18; H, 8.51; N, 6.07.
Ethyl N,N-Dicyclohexyloxamate (7a). From 0.01 mol (1.81 g) of
dicyclohexylamine (6a) in 20 mL of anhydrous THF, a colorless solid
was obtained (1.46 g, 52%); mp 88.9−90.1 °C; TLC (Rf = 0.94,
petroleum ether and ethyl acetate, 1:1); 1H NMR (300 MHz, CDCl3,
25 °C): δ 4.32, q, 2H, CH3CH2, J = 7.3 Hz, 2.98−3.21, m, 2H,
2(C5H10CH), 1.02−2.47, m, 20H, 2(C5H10CH), 1.35, t, 3H,
CH3CH2, J = 7.3; 13C NMR (75 MHz, CDCl3, 25 °C): δ 163.6
(COO), 161.9 (CON), 61.4 (CH2CH3), 59.2 (C5H10CH), 55.7
(C5H10CH), 31.0, 29.4, 26.4, 25.7, 25.1, 25.0, 14.0 (CH3); HRMS
(ESI/micrOTOF-Q) m/z: [M + Na]+ calcd for C16H27NO3Na,
304.1883; found, 304.1878: [M + H]+ calcd for C16H28NO3,
Synthesis of Hydroxamates 4a−e and Amides 7a−b. To a
solution of hydroxylamines (3a−e) or amines (6a−c) (1−10 mmol)
in 20 mL of anhydrous tetrahydrofuran (THF) was added DIPEA (2
equiv). To this solution, EOC (1.1 equiv) was added dropwise at 4 °C
during 15 min under an atmosphere of nitrogen. After the addition,
the ice bath was removed, the reaction mixture was allowed to warm
to the room temperature, and stirring was continued for 3 h. The
precipitated salt was filtered off, and the filtrate was evaporated under
reduced pressure to get the crude ester, which was purified by flash
chromatography, eluting with petroleum ether and ethyl acetate (7:3).
Ethyl N-Cyclohexyl-N-hydroxyoxamate (4a). From 0.005 mol
(0.58 g) of N-cyclohexylhydroxylamine (3a) in 20 mL of anhydrous
THF, a yellowish orange solid was obtained (1.02 g, 94%); mp 81.3−
82.1 °C (lit.33 mp 82 °C); TLC (Rf = 0.80, petroleum ether and ethyl
1
acetate 1:1); H NMR (300 MHz, CDCl3, 25 °C): δ 4.37 (q, 2H,
CH3CH2, J = 7.3 Hz), 4.03−4.07 (m, 1H, C5H10CH−N), 1.10−1.89
(m, 10H, C5H10CH−N), 1.6 (t, 3H, CH2CH3, J = 7.3 Hz); 13C NMR
(75 MHz, CDCl3, 25 °C): δ 163.7 (COO), 160.3 (CON), 62.4
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1
282.2064; found, 282.2057; Anal. Calcd for C16H27NO3: C, 68.29; H,
9.67; N, 4.98. Found: C, 68.33; H, 9.77; N, 4.97.
52%); H NMR (300 MHz, DMSO-d6, 25 °C): δ 8.70 (bs, 1H, N−
OH), 4.28−4.33, m, 1H, (CH3)2CHCH2CHCH3, 1.50−1.59, m, 2H,
1.07−1.17, m, 4H, 0.81−0.90, m, 6H; 13C NMR (75 MHz, DMSO-d6,
25 °C): δ 161.9 (COO), 157.3 (CON), 52.5 (N−CHCH3), 42.1
(CH3)2CHCH2CHCH3, 24.5 (CH3)2CHCH2CHCH3, 23.5 and 21.9
(CH3)2CHCH2CHCH3, 18.2 (CH3)2CHCH2CHCH3; HRMS (ESI/
micrOTOF-Q) m/z: [M − H]− calcd for C8H14NO4, 188.0928;
found, 188.0934: [M + Na − 2H]− calcd for C8H13NO4Na, 210.0748;
found, 210.0747.
Ethyl N,N-Diethyloxamate (7b).34 From 0.01 mol (0.73 g) of
diethylamine (6b) in 20 mL of anhydrous THF, a yellow liquid was
obtained (1.54 g, 89%); TLC (Rf = 0.92, petroleum ether and ethyl
1
acetate, 1:1); H NMR (300 MHz, CDCl3, 25 °C): δ 4.34 (q, 2H,
OCH2CH3, J = 7.0 Hz), 3.36, q, 4H, N(CH2CH3)2, J = 7.3, 1.37, t,
3H, OCH2CH3, J = 7.0 Hz, 1.21, t, 6H, N(CH2CH3)2, J = 7.3; 13C
NMR (75 MHz, CDCl3, 25 °C): δ 163.2 (COO), 161.4 (CON), 61.8
(OCH2CH3), 42.4 (NCH2CH3), 39.0 (NCH2CH3), 14.1, 14.0, 12.5;
N,N-Dicyclohexyloxamate (8a). From 0.001 mol (0.28 g) of
compound 7a in 10 mL of THF and 5 mL of water, a colorless solid
was obtained (182 mg, 72%); mp 126.5−128.0 °C; H NMR (300
MHz, DMSO-d6, 25 °C): δ 3.04−3.21, m, 2H, 2(C5H10CH), 1.01−
2.31, m, 20H, 2(C5H10CH); 13C NMR (75 MHz, DMSO-d6, 25 °C):
δ 165.6 (COO), 163.3 (CON), 59.3 (C5H10CH), 54.5 (C5H10CH),
30.1, 29.6, 26.0, 25.7, 25.4, 24.8; HRMS (ESI/micrOTOF-Q) m/z:
[M − H]− calcd for C14H22NO3, 252.1605; found, 252.1612; Anal.
Calcd for C14H23NO3: C, 66.37; H, 9.15; N, 5.53. Found: C, 66.45;
H, 9.23; N, 5.29.
HRMS (ESI/micrOTOF-Q) m/z: [M
+
Na]+ calcd for
1
C8H15NO3Na, 196.0944; found, 196.0945: [M + H]+ calcd for
C8H16NO3, 174.1125; found, 174.1123; Anal. Calcd for C8H15NO3:
C, 55.47; H, 8.73; N, 8.09. Found: C, 55.13; H, 8.40; N, 7.87.
Hydrolysis of Esters 4a−e and 7a−b. The esters (4a−e and 7a−
b) (0.001−0.003 mol) were saponified by dissolving them in 10 mL
of THF and 5 mL of water and then adding 6 M aq KOH (2 mL).
The resulting solution was heated under reflux for 1 h, maintaining
the pH at 10, if needed, by the addition of additional 6 M KOH
solution. After cooling to room temperature, the mixture was acidified
to pH 3 by the addition of 10% aq HCl solution. The solvent was
evaporated completely under reduced pressure, and the residue was
extracted with EtOAc (3 × 10 mL). The organic layer was dried over
anhydrous sodium sulfate, filtered, and evaporated again under
reduced pressure to obtain the final hydroxyoxamate/oxamate
compounds (5a−e and 8a−b). No further purification was required.
N-Cyclohexyl-N-hydroxyoxamate (5a). From 0.003 mol (0.65 g)
of compound 4a, a brown solid was obtained (265 mg, 47%); mp
N,N-Diethyloxamate (8b). From 0.003 mol (0.52 g) of compound
7b in 10 mL of THF and 5 mL of water, a brown solid was obtained
(133 mg, 31%); mp 90.0−91.2 °C; H NMR (300 MHz, DMSO-d6,
1
25 °C): δ 3.26, q, 4H, N(CH2CH3)2, J = 7.3 Hz, 1.07, t, 6H,
N(CH2CH3)2, J = 7.3 Hz; 13C NMR (75 MHz, DMSO-d6, 25 °C): δ
165.6 (COO), 162.8 (CON), 42.2 (NCH2CH3), 38.2 (NCH2CH3),
14.5, 12.9; HRMS (ESI/micrOTOF-Q) m/z: [M − H]− calcd for
C6H10NO3, 144.0666; found, 144.0667; Anal. Calcd for C6H11NO3:
C, 49.65; H, 7.64; N, 9.65. Found: C, 49.67; H, 7.44; N, 9.51.
Ethyl N-Benzyloxamate (7c). To a vigorously stirring solution of
benzylamine 6c (0.03 mol, 3.22 g) in anhydrous THF (20 mL) at 5
°C was added a solution of EOC (0.01 mol, 1.37 g) in anhydrous
THF (5 mL) dropwise during 15 min. The cooling bath was removed,
and the mixture was stirred for 3 h under an atmosphere of nitrogen.
The precipitated salt was filtered, and the solvent was evaporated
under reduced pressure to give a white solid which was purified by
flash chromatography, eluting with petroleum ether and ethyl acetate
(7:3). Yield 65%, 1.34 g; mp 50.1−51.3 °C (lit.35 mp 50−51 °C);
1
114.6−115.5 °C; H NMR (300 MHz, DMSO-d6, 25 °C): δ 10.11
(bs, N−OH, 1H), 3.91−4.01 (m, 1H, C5H10CH−N), 0.95−1.77 (m,
10H, C5H10CH−N); 13C NMR (75 MHz, DMSO-d6, 25 °C): δ 165.3
(COO), 162.4 (CON), 54.3 (C5H10CH), 28.9, 25.2, 25.1; HRMS
(ESI/micrOTOF-Q) m/z: [M − H]− calcd for C8H12NO4, 186.0772;
found, 186.0773: [M + Na − 2H]− calcd for C8H11NO4Na, 208.0591;
found, 208.0591; Anal. Calcd for C8H13NO4: C, 51.33; H, 7.00; N,
7.48. Found: C, 51.12; H, 7.01; N, 7.56.
N-Cyclopentyl-N-hydroxyoxamate (5b). From 0.002 mol (0.4 g)
of compound 4b, a brown solid was obtained (325 mg, 94%); mp
1
TLC (Rf = 0.80, petroleum ether and ethyl acetate, 1:1); H NMR
1
118.2−119.0 °C; H NMR (300 MHz, DMSO-d6, 25 °C): δ 10.10
(300 MHz, CDCl3, 25 °C): δ 7.28−7.36 (m, 5H, C6H5), 4.53 (d, 2H,
C6H5CH2, J = 6.0 Hz), 4.36 (q, 2H, CH2CH3, J = 7.3 Hz), 1.40 (t,
3H, CH2CH3, J = 7.3 Hz); 13C NMR (75 MHz, CDCl3, 25 °C): δ
160.5 (COO), 156.4 (CON), 136.7 (C6H5CCH2), 128.8, 128.0,
127.9, 63.3 (CH2CH3), 43.9 (C6H5CCH2), 13.9 (CH2CH3); HRMS
(ESI/micrOTOF-Q) m/z: [M + Na]+ calcd for C11H13NO3Na,
230.0788; found, 230.0782: [M + H]+ calcd for C11H14NO3,
208.0968; found, 208.0967; Anal. Calcd for C11H13NO3: C, 63.76;
H, 6.32; N, 6.76. Found: C, 63.84; H, 6.32; N, 6.87.
(bs, 1H, N−OH), 4.53−4.63 (m, 1H, C4H8CH−N), 1.42−1.73 (m,
8H, C4H8CH−N); 13C NMR (75 MHz, DMSO-d6, 25 °C): δ 165.3
(COO), 162.7 (CON), 55.6 (C4H8CH), 28.3, 24.9; HRMS (ESI/
micrOTOF-Q) m/z: [M − H]− calcd for C7H10NO4, 172.0615;
found, 172.0616: [M + Na − 2H]− calcd for C7H9NO4Na, 194.0435;
found, 194.0435; Anal. Calcd for C7H11NO4: C, 48.55; H, 6.40; N,
8.09. Found: C, 48.54; H, 6.41; N, 8.41.
N-(1-Phenylethyl)-N-hydroxyoxamate (5c). From 0.001 mol (0.24
g) of compound 4c, a brownish solid was obtained (950 mg, 45%);
N-Benzyloxamate (8c). The ester 7c was hydrolyzed using aq
KOH and following the same procedure described in previous
sections for the hydrolysis of esters.
1
mp 105.2−106.0 °C; H NMR (300 MHz, DMSO-d6, 25 °C): δ
10.44 (bs, 1H, COOH), 7.23−7.44 (m, 5H, C6H5), 7.32 (bs, 1H, N−
OH), 5.44 (q, 1H, CHCH3, J = 6.0 Hz), 1.48 (d, 3H, CHCH3, J =
6.0); 13C NMR (75 MHz, DMSO-d6, 25 °C): δ 165.2 (COO), 162.9
(CON), 140.5, 128.7, 127.8, 127.4, 53.7 (CHCH3), 17.3 (CH3);
HRMS (ESI/micrOTOF-Q) m/z: [M − H]− calcd for C10H10NO4,
208.0615; found, 208.0618: [M + Na − 2H]− calcd for C10H9NO4Na,
230.0435; found, 230.0437; Anal. Calcd for C10H11NO4: C, 57.41; H,
5.30; N, 6.70. Found: C, 57.19; H, 5.28; N, 6.42.
From 0.002 mol (0.41 g) of compound 7c, a colorless solid was
obtained (160 mg, 45%); mp 117.0−119.0 °C (lit.36 mp 118−120
1
°C); H NMR (300 MHz, DMSO-d6, 25 °C): δ 9.34 (t, 1H, J = 9.0
Hz), 7.20−7.34 (m, 5H), 4.31 (d, 2H, J = 9.0 Hz); 13C NMR (75
MHz, DMSO-d6, 25 °C): δ 162.6 (COO), 158.9 (CON), 139.0
(C6H5CCH2), 128.7, 127.7, 127.4, 42.8 (C6H5CCH2); HRMS (ESI/
micrOTOF-Q) m/z: [M − H]− calcd for C9H8NO3, 178.0510; found,
178.0510: [M + Na − 2H]− calcd for C9H7NO3Na, 200.0329; found,
200.0331; Anal. Calcd for C9H9NO3: C, 60.33; H, 5.06; N, 7.82.
Found: C, 60.36; H, 5.15; N, 7.58.
N-Isobutyl-N-hydroxyoxamate (5d). From 0.002 mol (0.38 g) of
compound 4d, an orange solid was obtained (166 mg, 51%); mp
75.3−77.0 °C; 1H NMR (300 MHz, DMSO-d6, 25 °C): δ 4.07−4.18
(m, 1H, CH3CH2CHCH3), 1.34−1.46 (m, 2H, CH3CH2CHCH3),
0.73−1.14 (m, 6H, CH3CH2CHCH3); 13C NMR (75 MHz, DMSO-
d6, 25 °C): δ 165.5 (COO), 163.2 (CON), 52.5 (CH3CH2CHCH3),
25.9, 17.4, 11.1; HRMS (ESI/micrOTOF-Q) m/z: [M − H]− calcd
for C6H10NO4, 160.0615; found, 160.0615: [M + Na − 2H]− calcd
for C6H9NO4Na, 182.0435; found, 182.0437; Anal. Calcd for
C6H11NO4: C, 44.72; H, 6.88; N, 8.69. Found: C, 45.09; H, 7.23;
N, 8.42.
N-Isopropyloxamate (8d).37 EOC (0.010 mol, 1.38 g) was added
dropwise to a solution of pyridine (0.01 mol, 0.8 g) and
isopropylamine 6d (0.030 mol, 1.8 g) in CH2Cl2 (20 mL) at 4 °C.
The reaction mixture was stirred for 2 h at this temperature, allowed
to warm to room temperature, and then washed with 1 M HCl (3 ×
10 mL). The organic phase was shaken with 10 mL of 6 M aq KOH
for 15 min. The aqueous phase was separated and acidified with 5 M
HCl. Ether extraction and evaporation gave 8d as a solid (473 mg,
36%); mp 107.5−108.7 °C (lit.37 mp 110−113 °C); H NMR (300
1
N-(4-Methyl-pent-2-yl)-N-hydroxyoxamate (5e). From 0.002 mol
(0.43 g) of compound 4e, an orange liquid was obtained (197 mg,
MHz, DMSO-d6, 25 °C): δ 7.15 (bs, 1H, NH), 4.01−4.16 (m, 1H,
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CH3CHCH3), 1.26 (d, 6H, CH3CHCH3, J = 6.0); 13C NMR (75
MHz, DMSO-d6): δ 162.8 (COO), 157.9 (CON), 41.5
(CH3CHCH3), 22.2 (CH3); HRMS (ESI/micrOTOF-Q) m/z: [M
− H]− calcd for C5H8NO3, 130.0510; found, 130.0510: [M + Na −
2H]− calcd for C5H7NO3Na, 152.0329; found, 152.0330; Anal. Calcd
for C5H9NO3: C, 45.80; H, 6.92; N, 10.68. Found: C, 46.15; H, 7.11;
N, 10.97.
starting material (approximately 1 h). After the completion of the
reaction, the solvent was evaporated completely under reduced
pressure to give 12 as a white solid (yield 59%, 75 mg); mp 193.0−
1
193.7 °C; H NMR (300 MHz, D2O, 25 °C): δ 4.33−4.50 (m, 1H,
CHNOH), 4.26−4.34 (q, 2H, CH2CH3, J = 7.0 Hz), 3.02−3.50 (m,
4H, CH2NHCH2), 1.94−2.03, m, 4H, CH2(CHNOH)CH2, 1.21−
1.26, t, 3H, CH2CH3, J = 7.0 Hz; 13C NMR (75 MHz, D2O): δ 163.1
(COO), 161.8 (CON), 63.8 (CH2CH3), 50.7 (CHNOH), 42.8
(CH2NCH2), 24.4 (CH2(CHNOH)CH2), 13.1 (CH2CH3); HRMS
(ESI/micrOTOF-Q) m/z: [M + H]+ calcd for C9H17N2O4, 217.1183;
found, 217.1178; Anal. Calcd for C9H17ClN2O4: C, 42.78; H, 6.78; N,
11.09. Found: C, 42.59; H, 6.75; N, 10.78.
N-Isobutyloxamate (8e). This compound was prepared in an
analogous manner to 8d, except that isobutyamine was used in place
of isopropylamine. From 0.005 mol of EOC in CH2Cl2 (20 mL), a
colorless solid was obtained (710 mg, 49%); mp 104.4−105.3 °C
(lit.38 mp 106 °C); H NMR (300 MHz, DMSO-d6, 25 °C): δ 8.78
1
Methyl N-Cyclopentyl-N-hydroxymalonate (13). To a solution of
the N-cyclopentylhydroxylamine (3b) (0.01 mol, 1.01 g) and DIPEA
(0.01 mol, 1.29 g) in anhydrous THF (20 mL) at 4 °C was added
methyl malonyl chloride (0.015 mol, 2.05 g) dropwise during 15 min
under an atmosphere of nitrogen. The reaction mixture was allowed
to warm to the room temperature and stirred for 3 h. The precipitated
salt was filtered off, and the solvent was evaporated under reduced
pressure to give crude methyl N-hydroxy-N-cyclopentylmalonate
which was purified by flash chromatography, eluting with petroleum
ether and ethyl acetate (3:7) to give 13 as a pale yellow liquid (0.40 g,
(bs, 1H, NH), 2.92, d, 2H, (CH3)2CHCH2, J = 6.0, 1.70−1.83, m,
1H, (CH3)2CHCH2, 0.82, d, 6H, (CH3)2CHCH2, J = 6.0 Hz; 13C
NMR (75 MHz, DMSO-d6, 25 °C): δ 162.8 (COO), 158.8 (CON),
46.8 (CH3)2CHCH2, 28.2 (CH3)2CHCH2, 20.4 (CH3)2CHCH2;
HRMS (ESI/micrOTOF-Q) m/z: [M − H]− calcd for C6H10NO3,
144.0666; found, 144.0666: [M + Na − 2H]− calcd for C6H9NO3Na,
166.0486; found, 166.0486; Anal. Calcd for C6H11NO3: C, 49.65; H,
7.64; N, 9.65. Found: C, 49.70; H, 7.75; N, 9.77.
N-(N-Boc-piperidin-4-yl)hydroxylamine (10).20 To a solution of
N-Boc-piperidin-4-one (9) (0.04 mol, 8.0 g) in methanol (100 mL)
was added acetic acid (0.07 mol, 4.2 g), sodium acetate (0.048 mol,
4.00 g), hydroxylamine hydrochloride (0.040 mol, 2.72 g), and
sodium cyanoborohydride (0.12 mol, 7.68 g). The mixture was stirred
overnight at room temperature. The solvent was removed under
reduced pressure, and the residue was washed with saturated aqueous
sodium bicarbonate solution. The aqueous layer was extracted with
CH2Cl2 (3 × 100 mL). The combined organic phase was dried over
anhydrous sodium sulfate, filtered, and evaporated under reduced
pressure. The crude product was recrystallized from petroleum ether
and chloroform (9:1) to give 10 as a cream solid (4.3 g, 50%); mp
117.2−118.4 °C; TLC Rf = 0.80, petroleum ether and ethyl acetate,
1
20%); TLC (Rf = 0.58, petroleum ether and ethyl acetate, 3:7); H
NMR (300 MHz, CDCl3, 25 °C): δ 3.77 (s, 3H, CH3), 3.61 (s, 2H,
COCH2CO), 3.50−3.57, m, 1H, CH2CH(NOH)CH2, 1.55−2.18, m,
8H, CH2CH2CH2CH2; 13C NMR (75 MHz, CDCl3): δ 170.5
(COO), 165.5 (CON), 56.5 (CH2CH(NOH)CH2), 52.8 (CH3),
41.3 (COCH2CO), 28.2, 24.8; HRMS (ESI/micrOTOF-Q) m/z: [M
+ Na]+ calcd for C9H15NO4, 224.0899; found, 224.0900; Anal. Calcd
for C9H15NO4: C, 53.72; H, 7.51; N, 6.96. Found: C, 53.71; H, 7.44;
N, 6.64.
N-Cyclopentyl-N-hydroxymalonate (14). From 0.001 mol (0.20
g) of compound 13 in THF (10 mL), an orange liquid was obtained
(yield 74%, 140 mg); 1H NMR (300 MHz, DMSO-d6, 25 °C): δ 9.59
(bs, 1H, NOH), 3.91 (s, 2H, COCH2CO), 3.34−3.70, m, 1H,
CH2CH(NOH)CH2, 1.43−1.69 (m, 8H, CH2CH2CH2CH2); 13C
NMR (75 MHz, DMSO-d6): δ 169.0 (COO), 166.81 (CON), 55.5
(CH2CH(NOH)CH2), 40.9 (COCH2CO), 28.0, 24.5; HRMS (ESI/
micrOTOF-Q) m/z: [M + Na]+ calcd for C8H13NO4Na, 210.0742;
found, 210.0731; Anal. Calcd for C8H13NO4: C, 51.33; H, 7.00; N,
7.48. Found: C, 51.43; H, 7.04; N, 7.35.
1
3:7. H NMR (300 MHz, CDCl3, 25 °C): δ 5.37 (bs, 2H, NHOH),
4.05−4.09 (m, 2H, HCH(N-Boc)HCH), 2.98−3.08 (m, 1H,
CH2CH(NH−OH)CH2), 2.78−2.86 (m, 2H, HCH(N-Boc)HCH),
1.24−1.92 (m, 4H, CH2CH(NH−OH)CH2), 1.45, s, 9H,
(CH3)3CCOO; 13C NMR (75 MHz, CDCl3, 25 °C): δ 154.7
(CH3)3CCOO, 79.6 (CH3)3CCOO, 58.7 (CHNHOH), 42.1
(CH2NCH2), 29.1 (CH2(CHNOH)CH2), 28.4 ((CH3)3CCOO).
Ethyl N-Hydroxy-N-(N-Boc-piperidin-4-yl)oxamate (11). To a
solution of N-(N-Boc-piperidin-4-yl)hydroxylamine (10) (0.015 mol,
3.24 g) in THF (20 mL) at 4 °C under an atmosphere of nitrogen was
added DIPEA (0.075 mol, 9.7 g) followed by the dropwise addition of
EOC (0.030 mol, 4.1 g) during 15 min. The reaction mixture was
allowed to warm to room temperature and stirred for 3 h, and then,
the solvent was evaporated under reduced pressure. The residue was
dissolved in petroleum ether and ethyl acetate (8:2), and the tertiary
amine and its salt were removed by running the crude solution
through a silica plug. The product was further purified by flash
column chromatography, eluting with petroleum ether and ethyl
acetate (1:1) to give 11 as a colorless solid (2.40 g, 51%); mp 141.8−
142.5 °C; TLC (Rf = 0.54, petroleum ether and ethyl acetate, 1:1); 1H
NMR (300 MHz, CDCl3, 25 °C): δ 8.16 (bs, 1H, NOH), 4.37 (q,
2H, CH2CH3, J = 7.0), 4.30−4.37 (m, 1H, CHNOH), 4.21 (bs, 2H),
2.71−2.88 (m, 2H), 1.71−2.13, m, 4H, CH2(CHNOH)CH2, 1.46, s,
9H, (CH3)3CCOO, 1.37−1.42, t, 3H, CH2CH3, J = 7.0; 13C NMR
(75 MHz, CDCl3, 25 °C): δ 162.7 (COO), 161.0 (CON), 154.7
(CH3)3CCOO, 80.4 (CH3)3CCOO, 62.3 (CH2CH3), 57.7
(CHNOH), 42.8 (CH2NCH2), 29.1 (CH2(CHNOH)CH2), 28.4
((CH3)3CCOO), 27.7 (CH2(CHNOH)CH2), 13.9 (CH2CH3);
Preparation of n-Alkyl Esters (23−27) of 5b. The synthesis of
compound 23 is described below as an example for this series of
compounds.
(i) Monoesterification of oxalyl chloride
A solution of n-hexanol (63 μL, 51 mg, 0.5 mmol) in dry DCM (1
mL) was added dropwise to a solution of oxalyl chloride (0.25 mL,
370 mg, 2.9 mmol, 5.83 equiv) in dry DCM (2 mL) at 0 °C under a
nitrogen atmosphere. The solution was stirred at room temperature
for 14 h. The excess oxalyl chloride and solvent were removed by
evaporation, and the oily product was used directly for the next step.
(In the case of stearyl alcohol, the alcohol (333 μL, 271 mg, 1 mmol)
was solubilized in dry DCM (1 mL) by warming the solution in a
water bath at 50 °C. The dropping funnel was also heated gently using
a heat gun to liquefy the dropping solution.)
(ii) Coupling of N-cyclopentylhydroxylamine (3b) with hexoxy
oxalyl chloride
To a solution of the N-cyclopentylhydroxylamine (3b) (51 mg, 0.5
mmol) in anhydrous THF (3 mL), DIPEA (174 μL, 129 mg, 1 mmol,
2 equiv) was added. To this solution, hexoxy oxalyl chloride (96 mg,
0.5 mmol, 1 equiv) in anhydrous THF (2 mL) was added dropwise at
0 °C for 15 min under an atmosphere of nitrogen. After the addition,
the ice bath was removed and the reaction mixture was allowed to
warm to the room temperature and stirred for 14 h. The precipitated
salt was filtered off using Celite as a filter aid, and the filtrate was
evaporated under reduced pressure to get the desired ester compound
hexyl N-hydroxy-N-cyclopentyloxamate (23) in the crude form as a
brown oil which was further purified by flash chromatography using
hexane and ethyl acetate (8:2).
HRMS (ESI/micrOTOF-Q) m/z: [M
+
Na]+ calcd for
C14H24N2O6Na, 339.1527; found, 339.1529; Anal. Calcd for
C14H24N2O6: C, 53.15; H, 7.65; N, 8.86. Found: C, 53.47; H, 7.68;
N, 8.66.
Ethyl N-Piperidin-4-yl-N-hydroxyoxamate Hydrochloride (12).
To a solution of ethyl N-hydroxy-N-(N-Boc-piperidin-4-yl)oxamate
(11) (0.158 g, 0.50 mmol) in anhydrous THF (5 mL) was added 4 M
dioxane-HCl (5 equiv). The reaction mixture was monitored by TLC
and stirred at room temperature until the disappearance of the
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Hexyl N-Cyclopentyl-N-hydroxyoxamate (23). From 0.5 mmol
(51 mg) of N-cyclopentyl hydroxylamine (3b) in 3 mL of anhydrous
THF, a brown oil was obtained (yield 58%, 75 mg); TLC (Rf = 0.38,
hexane and ethyl acetate, 8:2); 1H NMR (500 MHz, CDCl3, 25 °C):
δ 8.30 (bs, 1H, N−OH), 4.71 and 4.50 (2× quintet, 0.4H and 0.5H,
C4H8CH−N, J = 7.2 and 7.5 Hz), 4.24 and 4.21 (2× t, 1.1H and
0.8H, CH2CH2O, J = 6.8 and 6.8 Hz), 1.98−1.65 (m, 8H, C4H8CH−
N), 1.53−1.51 (m, 2H, CH2CH2O), 1.38−1.25 (m, 6H,
CH3C3H6CH2CH2O), 0.85 (t, 3H, CH3CH2, J = 7.0 Hz); 13C
NMR (125 MHz, CDCl3, 25 °C): δ 163.9 (COO), 160.7 (CON),
154.5, 66.8, 66.4 (OCH2), 60.7, 56.5 (C4H8CH), 31.24, 31.21, 29.3,
28.16, 28.11, 25.3, 25.2, 25.0, 24.7, 22.4, 13.9; HRMS (ESI/
0.5H, CH2CH2O, J = 6.8 and 6.8 Hz), 2.02−1.67 (m, 8H, C4H8CH−
N), 1.56−1.53 (m, 2H, CH2CH2O), 1.35−1.18 (m, 30H,
CH3C15H30CH2CH2O), 0.85 (t, 3H, CH3CH2, J = 7.0 Hz); 13C
NMR (125 MHz, CDCl3, 25 °C): δ 163.7 (COO), 160.5, 160.3
(CON), 153.9, 66.9, 66.5 (OCH2), 60.5, 56.6 (C4H8CH), 31.9, 29.7,
29.64, 29.61, 29.52, 29.45, 29.40, 29.3, 29.1, 28.3, 28.2, 25.72, 25.66,
25.1, 24.8, 22.7, 14.1; HRMS (ESI/micrOTOF-Q) m/z: [M − H]−
−
calcd for C25H46NO4 , 424.3432; found, 424.3424; and [M + Na]+
+
calcd for C25H47NNaO4 , 448.3397; found, 448.3393; Anal. Calcd for
C25H35NO4: C, 70.54; H, 11.13; N, 3.29. Found: C, 70.16; H, 11.07;
N, 3.33.
Inhibition Assays. All analogues (100 μM) were incubated for 30
min with 0.27 μM MtKARI in the assay buffer (50 μM NADPH, 50
mM Mg2+ and 0.1 M Tris−HCl, pH 8) at 25 °C. The reaction was
initiated by the addition of 200 μM 2-acetolactate, and the decrease in
absorbance at 340 nm was measured for 5 min. To determine the Ki
of the compounds that showed more than 80% inhibition in this
preliminary screening, the abovementioned assay was employed but
with varying concentrations of inhibitors ranging from 1 nM to 100
μM. The Ki values of the most potent compounds were calculated by
fitting the experimental data to the Morrison equation15,22 using
GraphPad Prism 6.0.39
Crystallization and Structure Determination of SaKARI·
Mg2+·NADPH in Complex with 5a, 5b, and 5c. To prepare the
samples for crystallization, 0.5 mM SaKARI was incubated with 5 mM
NADPH, 0.75 mM inhibitor, and 5 mM MgCl2 for 30 min at room
temperature. Crystals of SaKARI·Mg2+·NADPH in complex with 5b
were grown in a well solution containing 0.1 M imidazole (pH 8.0)
and 17.5% PEG 8000, and crystals of SaKARI·Mg2+·NADPH in
complex with 5a and 5c were grown in a well solution containing 0.2
M sodium acetate (pH 8.0) and 22.5% PEG 3350 by the hanging
drop method. The plates were incubated at 16 °C for 7 days.
Subsequently, the crystals were harvested and cryocooled using 20%
glycerol as the cryoprotectant. The data were collected by remote
access on beam line MX1 for 5b and MX2 for 5a and 5c and
processed using XDS.40 Molecular replacement was performed using
5W3K (SaKARI·Mg2+·NADPH·CPD complex) as a template in
Phenix41 followed by model building using Coot 0.8.42 The
coordinates have been deposited in the Protein Data Bank with
access codes 6C55 (5a), 6C5N (5b), and 6BUL (5c).
+
micrOTOF-Q) m/z: [M + H]+ calcd for C13H24NO4 , 258.1700;
+
found, 258.1701; and [M + Na]+ calcd for C13H23NNaO4 , 280.1519;
found, 280.1520; Anal. Calcd for C13H23NO4: C, 60.68; H, 9.01; N,
5.44. Found: C, 60.40; H, 9.11; N, 5.15.
Octyl N-Cyclopentyl-N-hydroxyoxamate (24). From 0.5 mmol
(51 mg) of N-cyclopentyl hydroxylamine (3b) in 3 mL of anhydrous
THF, a brown oil was obtained (yield 31%, 88 mg); TLC (Rf = 0.31,
hexane and ethyl acetate, 8:2); 1H NMR (500 MHz, CDCl3, 25 °C):
δ 7.67 (bs, 1H, N−OH), 4.76 and 4.60 (2× quintet, 0.3H and 0.6H,
C4H8CH−N, J = 7.1 and 7.0 Hz), 4.26 and 4.24 (t and m, 1.3H and
0.6H, CH2CH2O, J = 6.8), 2.00−1.68 (m, 8H, C4H8CH−N), 1.56−
1 . 5 3 ( m , 2 H , C H 2 C H 2 O ) , 1 . 3 5 − 1 . 1 8 ( m , 1 0 H ,
CH3C5H10CH2CH2O), 0.85 (t, 3H, CH3CH2, J = 6.9 Hz); 13C
NMR (125 MHz, CDCl3, 25 °C): δ 163.7 (COO), 160.5 (CON),
160.3, 154.0, 66.9, 66.5 (OCH2), 60.5, 56.6 (C4H8CH), 31.7, 29.4,
29.08, 29.07, 28.3, 28.2, 25.7, 25.6, 25.0, 24.7, 22.6, 14.0; HRMS
+
(ESI/micrOTOF-Q) m/z: [M + Na]+ calcd for C15H27NNaO4 ,
308.1832; found, 308.1834; Anal. Calcd for C15H27NO4: C, 63.13; H,
9.54; N, 4.91. Found: C, 62.72; H, 9.61; N, 5.21.
Decyl N-Cyclopentyl-N-hydroxyoxamate (25). From 0.5 mmol
(51 mg) of N-cyclopentyl hydroxylamine (3b) in 3 mL of anhydrous
THF, a brown oil was obtained (yield 41%, 64 mg); TLC (Rf = 0.48,
1
hexane and ethyl acetate, 75:25); H NMR (500 MHz, CDCl3, 25
°C): δ 8.30 (bs, 1H, N−OH), 4.73 and 4.53 (2× quintet, 0.3H and
0.6H, C4H8CH−N, J = 7.5 and 7.5 Hz), 4.25 and 4.22 (2× t, 1.1H
and 0.8H, CH2CH2O, J = 6.8 and 6.9 Hz), 2.00−1.65 (m, 8H,
C4H8CH−N), 1.55−1.48 (m, 2H, CH2CH2O), 1.38−1.22 (m, 14H,
CH3C7H14CH2CH2O), 0.84 (t, 3H, CH3CH2, J = 7.0 Hz); 13C NMR
(125 MHz, CDCl3, 25 °C): δ 163.9 (COO), 160.65, 160.61 (CON),
154.4, 66.9, 66.4 (OCH2), 60.7, 56.5 (C4H8CH), 31.8, 29.44, 29.42,
29.40, 29.3, 29.2, 29.11, 29.08, 28.2, 28.1, 25.67, 25.60, 25.0, 24.7,
22.6, 14.0; HRMS (ESI/micrOTOF-Q) m/z: [M + H]+ calcd for
Molecular Docking. All of the designed compounds along with
43
̈
IpOHA were docked using the program Glide from Schrodinger.
A
crystal structure of SeKARI·Mg2+·NADPH·IpOHA (PDB code
4KQX) with identical active site residues to MtKARI was used for
docking. A protein file compatible for Glide docking was prepared by
adding hydrogens. The entire protein was energy minimized using the
OPLS 2005 force field. The search cube was limited to 8 Å in all
directions with IpOHA at the center. All inhibitors were prepared for
+
C17H32NO4 , 314.2326; found, 314.2319; and [M + Na]+ calcd for
+
C17H31NNaO4 , 336.2145; found, 336.2138; Anal. Calcd for
C17H31NO4: C, 65.14; H, 9.97; N, 4.47. Found: C, 64.87; H,
10.01; N, 4.30.
̈
docking using the Ligprep module of the Schrodinger Maestro which
Dodecyl N-Cyclopentyl-N-hydroxyoxamate (26). From 0.5 mmol
(51 mg) of N-cyclopentyl hydroxylamine (3b) in 3 mL of anhydrous
THF, a brown oil was obtained (yield 44%, 75 mg); TLC (Rf = 0.51,
hexane and ethyl acetate, 8:2); 1H NMR (500 MHz, CDCl3, 25 °C):
δ 8.40 (bs, 1H, N−OH), 4.71 and 4.49 (2× quintet, 0.3H and 0.5H,
C4H8CH−N, J = 7.3 and 7.5 Hz), 4.23 and 4.19 (2× t, 1.1H and
0.9H, CH2CH2O, J = 6.8 and 6.8 Hz), 1.99−1.62 (m, 8H, C4H8CH−
N), 1.57−1.51 (m, 2H, CH2CH2O), 1.38−1.21 (m, 18H,
CH3C9H18CH2CH2O), 0.83 (t, 3H, CH3CH2, J = 7.0 Hz); 13C
NMR (125 MHz, CDCl3, 25 °C): δ 163.8 (COO), 160.74 (CON),
160.69, 154.59, 66.8, 66.3 (OCH2), 60.8, 56.5 (C4H8CH), 31.81,
31.80, 29.52, 29.50, 29.47, 29.44, 29.37, 29.25, 29.24, 29.08, 29.05,
28.2, 28.1, 25.63, 25.57, 25.0, 24.9, 24.7, 22.6, 14.0; HRMS (ESI/
produces different ionization states, and metal binding states were
̈
generated for pH 6.0−8.0 using the Epic module of Schrodinger
Maestro. All possible stereoisomers were also generated, having
different ionization states, stereoisomers, tautomers, and ring
conformations. Flexible docking was performed by allowing the
side-chain hydroxyl groups to rotate around the bond axis. Extra
precision (XP)43 docking was performed for all the molecules.
Resazurin Microtiter Assay. Compounds were tested using
resazurin microtiter assay (REMA)44 to calculate the minimum
inhibitory concentration (MIC). Both virulent (H37Rv) and avirulent
(H37Ra) strains of Mt were used. The bacteria were grown at 37 °C
in defined minimal media (1 g of KH2PO4, 2.5 g of Na2HPO4, 0.5 g of
L-asparagine, 5 mg of ferric ammonium citrate, 40 μM MgSO4, 4.5
μM CaCl2, 0.45 μM ZnSO4, and 0.1% glycerol in 1 L H2O) until the
OD600 reached 0.4−0.8, which corresponds to the log/exponential
phase. The culture was centrifuged at 800 rpm for 8 min to aggregate
bacterial clumps and generated a single cell suspension. The OD of
the bacterial suspension was measured, and necessary dilutions were
made to reach the final OD of 0.001 for H37Ra and 0.003 for H37Rv.
Serial dilutions (1:2) of the compounds with a starting concentration
of 30 μM were performed in a 96-well plate. Rifampicin was used as
+
micrOTOF-Q) m/z: [M + Na]+ calcd for C19H35NNaO4 , 364.2458;
found, 364.2459; Anal. Calcd for C19H35NO4: C, 66.83; H, 10.33; N,
4.10. Found: C, 66.95; H, 10.38; N, 4.23.
Octadecyl N-Cyclopentyl-N-hydroxyoxamate (27). From 1.0
mmol (101 mg) of N-cyclopentyl hydroxylamine (3b) in 5 mL of
anhydrous THF, a brown oil was obtained (yield 21%, 91 mg); TLC
(Rf = 0.27, hexane and ethyl acetate, 9:1); 1H NMR (500 MHz,
CDCl3, 25 °C): δ 4.77 and 4.61 (2× quintet, 0.3H and 0.6H,
C4H8CH−N, J = 7.2 and 7.5 Hz), 4.27 and 4.23 (2× t, 1.3H and
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the control, and the plate was incubated at 37 °C for 5 days before
adding resazurin. 24 h after the addition of resazurin, the fluorescence
was measured with an excitation wavelength of 530 nm and emission
wavelength at 590 nm. The data were analyzed, and inhibition curves
were produced to obtain MIC90 using the Gompertz equation.45
Succinic Dehydrogenase (MTT) Assay. Three mammalian cells
lines, RAW 264.7, HEK 293, and SW620, were grown to 80%
confluency in DMEM (RAW 267.4 and HEK293) and RPMI
(SW620) media with 10% fetal bovine serum and 1 mM nonessential
amino acids and plated on 96-well plates at 2 × 105 RAW 264.7 cells,
5 × 103 HEK293 cells, and 2 × 103 SW620 cells, respectively, per well.
The plates were incubated for 24−48 h at 37 °C and 5% CO2.
Twofold serial dilutions of 25 were prepared in 20% dimethyl
sulfoxide (DMSO), and 10 μL of the compound was added to the
wells to maintain 1% DMSO through the plate. The untreated cells
and media-only controls were also added with 1% DMSO. The cells
were incubated overnight at 37 °C and 5% CO2. The MTT reagent (5
mg/mL) was added, and the plates were incubated for 3 h. The media
was removed after 3 h, 50 μL of DMSO was added to the plates, and
the contents were mixed by shaking the plates at 50 rpm for 2 min.
Absorbance at 570 nm was measured, and data were plotted.
Shan Zheng − School of Chemistry and Molecular Biosciences,
The University of Queensland, Brisbane, Queensland 4072,
Australia
Lendl Tan − School of Chemistry and Molecular Biosciences,
The University of Queensland, Brisbane, Queensland 4072,
Australia; orcid.org/0000-0002-9955-2440
Nicholas P. West − School of Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane,
Queensland 4072, Australia; orcid.org/0000-0003-0955-
9890
Gerhard Schenk − School of Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane,
Queensland 4072, Australia; Sustainable Minerals Institute
and Australian Institute of Bioengineering and
Nanotechnology, The University of Queensland, Brisbane,
Queensland 4072, Australia
Luke W. Guddat − School of Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane,
Queensland 4072, Australia; orcid.org/0000-0002-8204-
8408
ASSOCIATED CONTENT
■
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01919
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01919.
Author Contributions
Molecular formula strings (CSV)
A.K. and K.P. are joint first authors. A.K. synthesized and
characterized the compounds, other than the prodrugs; he also
determined the Ki values of these compounds. A.K. performed
docking, and K.P. grew the crystals and solved the crystal
structures. W.M.H. synthesized and characterized the prodrug
compounds. K.P., L.T., and S.J.W. performed the REMA
assays, and W.M.H., S.J.W., and K.P. performed the MTT
assays. S.J.W. purified the enzyme, and S.Z. helped in
performing Ki assays. N.P.W., G.S., L.W.G., and R.P.M. were
responsible for the experimental design and overall supervision
of the project. All authors contributed to manuscript
preparation.
Alignment of active site residues of Mt and SaKARI;
inhibition plots of compounds 4d, 5a, 5c, and 5d against
MtKARI; effects of hexanol, octanol, decanol, and
1
dodecanol on the growth of H37Ra cells; and H and
13C NMR spectra of compounds 3e, 4a−e, 5a−e, 7a−c,
8a−e, and 10−14 (PDF)
Compound 5a (PDB)
Compound 5b (PDB)
R isomer of compound 5c (PDB)
S isomer of compound 5c (PDB)
Accession Codes
Notes
Atomic coordinates have been deposited in the Protein Data
Bank (https://rcsb.org) for SaKARI in complex with 5a (PDB
ID: 6C55), 5b (PDB ID: 6C5N), and 5c (PDB ID: 6BUL).
The authors will release the atomic coordinates and
experimental data upon article publication.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Initial crystallographic conditions were determined using the
University of Queensland Remote-Operation Crystallization
and X-ray Diffraction facility (UQROCX). X-ray measure-
ments were performed at the Australian Synchrotron, ANSTO.
This research was undertaken in part using the MX1 and MX2
beamline at the Australian Synchrotron and made use of the
Australian Cancer Research Foundation (ACRF) detector. The
work was funded by the National Health and Medical Research
Foundation, grant number 114729. We thank Dr Thierry
Lonhienne for helpful discussions, Dr Tri Le for assistance
with NMR studies, and Dr Amanda Nouwens for mass
spectrometry measurements.
AUTHOR INFORMATION
Corresponding Author
Ross P. McGeary − School of Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane,
Queensland 4072, Australia; orcid.org/0000-0001-9149-
6264; Email: r.mcgeary@uq.edu.au
■
Authors
Ajit Kandale − School of Chemistry and Molecular Biosciences,
The University of Queensland, Brisbane, Queensland 4072,
Australia
Khushboo Patel − School of Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane,
Queensland 4072, Australia
Waleed M. Hussein − School of Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane,
Queensland 4072, Australia
Shun Jie Wun − School of Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane,
Queensland 4072, Australia
ABBREVIATIONS
■
AHAS, acetohydroxyacid synthase; BCAA, branched-chain
amino acids; DIPEA, N,N-diisopropylethylamine; DS, docking
score; HOE 704, 2-(dimethylphosphoryl)-2-hydroxyacetic
acid; IpOHA, N-hydroxy-N-isoproyloxamic acid; KARI,
ketol-acid reductoisomerase; Mt, Mycobacterium tuberculosis;
Sa, Staphylococcus aureus; Se, Slackia exigua
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H. General approach to reversing ketol-acid reductoisomerase
cofactor dependence from NADPH to NADH. Proc. Natl. Acad. Sci.
U.S.A. 2013, 110, 10946−10951.
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