Proline-based hydroxamates targeting the zinc-dependent deacetylase LpxC: Synthesis, antibacterial properties, and docking studies

Article abstract of DOI:10.1016/j.bmc.2019.03.056

The Zn²⁺-dependent deacetylase LpxC is an essential enzyme in Gram-negative bacteria, which has been validated as antibacterial drug target. Herein we report the chiral-pool synthesis of novel D- and L-proline-derived 3,4-dihydroxypyrrolidine hydroxamates and compare their antibacterial and LpxC inhibitory activities with the ones of 4-monosubstituted and 3,4-unsubstituted proline derivatives. With potent antibacterial activities against several Gram-negative pathogens, the L-proline-based tertiary amine 41g ((S)-N-hydroxy-1-(4-{[4-(morpholinomethyl)phenyl]ethynyl}benzyl)pyrrolidine-2-carboxamide) was found to be the most active antibacterial compound within the investigated series, also showing some selectivity toward EcLpxC (K_i = 1.4 μM) over several human MMPs.

Full text of DOI:10.1016/j.bmc.2019.03.056

Accepted Manuscript
Proline-based Hydroxamates Targeting the Zinc-Dependent Deacetylase LpxC:
Synthesis, Antibacterial Properties, and Docking Studies
Dmitrii V. Kalinin, Oriana Agoglitta, Hélène Van de Vyver, Jelena Melesina,
Stefan Wagner, Burkhard Riemann, Michael Schäfers, Wolfgang Sippl, Bettina
Löffler, Ralph Holl
PII:
S0968-0896(19)30235-4
https://doi.org/10.1016/j.bmc.2019.03.056
BMC 14848
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
9 February 2019
28 March 2019
29 March 2019
Please cite this article as: Kalinin, D.V., Agoglitta, O., Van de Vyver, H., Melesina, J., Wagner, S., Riemann, B.,
Schäfers, M., Sippl, W., Löffler, B., Holl, R., Proline-based Hydroxamates Targeting the Zinc-Dependent
Deacetylase LpxC: Synthesis, Antibacterial Properties, and Docking Studies, Bioorganic & Medicinal Chemistry
(
2019), doi: https://doi.org/10.1016/j.bmc.2019.03.056
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Proline-based Hydroxamates Targeting the Zinc-Dependent Deacetylase LpxC:
Synthesis, Antibacterial Properties, and Docking Studies
Dmitrii V. Kalinin,a,b,c,d Oriana Agoglitta,a,b,e Hélène Van de Vyver, Jelena Melesina,g
d,f
h
h
d,h,i
g
Stefan Wagner, Burkhard Riemann, Michael Schäfers,
Wolfgang Sippl, Bettina
j
a,b,
Löffler, and Ralph Holl *
aInstitute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6,
0146 Hamburg, Germany
2
bGerman Center for Infection Research (DZIF), partner site Hamburg-Lübeck-Borstel-
Riems
cInstitute of Pharmaceutical and Medicinal Chemistry, University of Münster,
Corrensstr. 48, 48149 Münster, Germany
dCells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University of Münster,
Germany
eNRW Graduate School of Chemistry, University of Münster, Germany
fInstitute of Medical Microbiology, University Hospital Münster, Domagkstr. 10, 48149
Münster, Germany
g
,
Martin Luther University of Halle-Wittenberg, Wolfgang-
Langenbeck Str. 4, 06120 Halle (Saale), Germany
hDepartment of Nuclear Medicine, University Hospital Münster, Albert-Schweitzer-
Campus 1, Building A1, 48149 Münster, Germany
iEuropean Institute for Molecular Imaging, University of Münster, Waldeyerstraße 15,
4
8149 Münster, Germany
1

jInstitute of Medical Microbiology, University Hospital Jena, Am Klinikum 1, 07747
Jena, Germany
*To whom correspondence should be addressed. Tel.: +49-40-42838-2825; Fax: +49-
4
0-42838-4325; E-mail: ralph.holl@chemie.uni-hamburg.de
2

Graphical abstract
Key words
proline derivatives; chiral pool synthesis; antibacterials; LpxC inhibitors; structure-
activity relationships; molecular docking studies; MMP inhibitors
3

Abstract
2+
The Zn -dependent deacetylase LpxC is an essential enzyme in Gram-negative
bacteria, which has been validated as antibacterial drug target. Herein we report the
chiral-pool synthesis of novel D- and L-proline-derived 3,4-dihydroxypyrrolidine
hydroxamates and compare their antibacterial and LpxC inhibitory activities with the
ones of 4-monosubstituted and 3,4-unsubstituted proline derivatives. With potent
antibacterial activities against several Gram-negative pathogens, the L-proline-based
tertiary
morpholinomethyl)phenyl]ethynyl}benzyl)pyrrolidine-2-carboxamide) was found to be
the most active antibacterial compound within the investigated series, also showing
amine
41g
((S)-N-hydroxy-1-(4-{[4-
(
some selectivity toward EcLpxC (K
i
= 1.4 μM) over several human MMPs.
4

1
. Introduction
Zinc plays an essential role in all types of life, being required for growth, development
and differentiation of microorganisms, animals, and plants.1-2 In the human body, after
iron, zinc is the second most abundant transition metal and about 10% of the human
proteome is potentially zinc-binding.2-3 Zinc enzymes constitute the largest number of
2+
metalloproteins, with their Zn -ions fulfilling structural as well as catalytic roles, thus
being in most cases an essential cofactor for the biological function of these enzymes.2,
4
2+
Due to the diverse and essential biological roles of Zn -dependent enzymes, which
are among others involved in nucleic acid synthesis, the transmission of genetic
information, cell growth and development, signal transduction, apoptosis, and protein
degradation, their dysregulation is associated with a number of ailments, like
inflammatory, cardiovascular, metabolic, neurodegenerative, and infectious diseases
as well as with cancer.1, 4-6 Thus, Zn -dependent enzymes are highly promising targets
2+
2+
for drug discovery. Inhibitors of several Zn -dependent enzymes like carbonic
anhydrases, the angiotensin-converting enzyme (ACE), and human histone
deacetylases (HDACs) have been successfully approved as drugs, being utilized for
the treatment of glaucoma, hypertension, and cancer, respectively.7-9 Additionally,
2+
inhibitors of other clinically relevant Zn -dependent enzymes like the insulin-degrading
enzyme,10 matrix metalloproteinases (MMPs),11 and bacterial proteases12 are under
development.
2+
The Zn -dependent deacetylase LpxC is an essential enzyme in Gram-negative
bacteria and has been validated as antibacterial drug target.13-15 In E. coli, the enzyme
catalyzes the deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine
(
1), the first irreversible step of lipid A biosynthesis (Scheme 1, a)).16-17 Lipid A
5

constitutes the hydrophobic membrane anchor of lipopolysaccharides (LPS), being the
main component of the outer monolayer of the outer membrane of most Gram-negative
18
bacteria, and is thus essential for growth and viability of these bacteria. As LpxC is
highly conserved among Gram-negative bacteria, showing no homology to any
mammalian protein, and due to its central role in lipid A biosynthesis, this deactetylase
represents a promising target for the development of antibiotics, selectively combating
Gram-negative bacteria.15
The deacetylase LpxC comprises two domains, each of them exhibiting two α-helices,
which pack against a five-stranded β-sheet, thus leading to an overall “β-α-α-β
sandwich” fold.19-20 The catalytic Zn -ion, which in E. coli LpxC is coordinated by
His79, His238, and Asp242, resides at the bottom of the enzyme’s active site cleft,
which is located at the interface of the two domains, at one side of the sandwich.20-21
A crystal structure of E. coli LpxC in complex with the deacetylated product 2 (Figure
2+
1
) provided insight into interactions being important for substrate recognition within the
active site of the enzyme.22 Whereas the hexose ring of UDP-3-O-[(R)-3-
hydroxymyristoyl]glucosamine (2) binds upon a highly conserved hydrophobic patch
consisting of Phe192 and Phe194, the 2-amino group as well as the 6’-OH group of
the glucosamine moiety form hydrogen bonds with Leu62 and Lys239, respectively.21-
22
The pyrophosphate moiety of 2 is recognized by Lys239 and undergoes a water-
mediated contact with Lys143, being part of a basic patch, which is formed by residues
22
Lys143, Lys239, Lys262, and His265. The 3-O-[(R)-3-hydroxymyristoyl] substituent
of the deacetylated product 2 binds to a hydrophobic tunnel, which leads out of the
active site.21-22
6

Figure 1: Molecular surface of E. coli LpxC (active site) near the deacetylated
22
product 2 (PDB: 4MDT ), which is shown as green stick model. Amino acid residues
in proximity to the surface are depicted as grey stick models. Oxygen, nitrogen, sulfur
and phosphorus atoms are colored in red, blue, yellow, and orange, respectively. The
2+
Zn -ion is depicted as gray sphere. The surface is colored as follows: lipophilic
regions are in orange, hydrophilic in blue, and neutral in white.
The LpxC-catalyzed deacetylation is supposed to proceed via a general acid/base
mechanism, in which Glu78 and His265 act as a general acid-base catalyst pair
Scheme 1, b)).23-25 According to the proposed mechanism, a Zn -bound water
2+
(
molecule is deprotonated by Glu78 and the resulting hydroxide attacks the carbonyl
2+
carbon of the substrate, which is coordinated to the Zn -ion via its carbonyl oxygen
2+
atom. The resulting gem-diolate interacts with Thr191 and the Zn -ion, stabilizing the
tetrahedral intermediate. Finally, the products are formed via the protonation of the
7

gem-diolate’s nitrogen atom by general acid His265, leading to the cleavage of the C-
N bond.15
Scheme 1: a) LpxC catalyzed deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-
acetylglucosamine (1); b) proposed catalytic reaction mechanism of the LpxC-
catalyzed deacetylation.15, 25-26
Several classes of LpxC inhibitors have been described in the literature.15, 27 For
instance, the N-aroyl-L-threonine hydroxamic acids CHIR-090 (3) and LPC-009 (4)
represent two of the most potent LpxC inhibitors reported so far (Figure 2).19, 28 Inspired
by the glycosidic nature of the natural substrate of LpxC, we have developed a series
of conformationally restricted C-glycosides, like e.g. dihydroxytetrahydrofuran
derivative 5 (Figure 2).29-31 However, these compounds exhibited considerably
reduced antibacterial as well as LpxC inhibitory activity compared to CHIR-090 (3)
(Table 1). The poor activity of C-furanoside 5 might be due to its structure, lacking an
amide moiety, which in case of the N-aroyl-L-threonine hydroxamic acids was shown
to be involved in important interactions within the active site of LpxC.19, 32 In order to
find new and effective LpxC inhibitors, the tetrahydrofuran ring of the C-glycosides
8

should be replaced by another conformationally restrained five-membered heterocycle,
which allows the attachment of the lipophilic side chain via an amide moiety. As the
pyrrolidine ring meets the desired requirements, proline derivatives of general formula
6
should be synthesized and tested for their antibacterial and LpxC inhibitory activities.
Like the C-furanosidic compound 5, the (3S,4R)-3,4-dihydroxy-L-proline derivatives 6
being described in this article bear hydroxy groups in positions 3 and 4 of their central
heterocycle. As in case of the previously reported C-glycoside 5, the inversion of the
configuration of the stereocenter in α-position of the hydroxamate moiety (7) as well as
the acetalization of the two hydroxy groups (8) had led to an increase in antibacterial
30
activity in the disc diffusion assay against E. coli D22 (Table 1), the respective proline
derivatives 9 and 10 were synthesized and tested, which is also described in this
article. Additionally, in case of the dihydroxytetrahydrofuran-derived LpxC inhibitor 5,
the removal of one of the two hydroxy groups (11) had increased the inhibitory activity
towards LpxC (Table 1).30 Therefore, the proline- and 4-hydroxyproline-derived
hydroxamic acids 12, ent-12, 13 and 14, which lack one or both hydroxy groups at the
33
pyrrolidine ring and which were disclosed in one of our previous publications, were
tested for their antibacterial and LpxC inhibitory activities. To learn more about the role
of the carbonyl oxygen atom of the compounds’ amide moiety as well as the basicity
of the nitrogen atom of their pyrrolidine ring for binding to LpxC, the antibacterial
33
properties of the reported proline-derived tertiary amines 15 were also investigated.
The structure of the lipophilic side chain, which addresses the hydrophobic tunnel of
LpxC, generally plays a crucial role for the affinity of LpxC inhibitors toward the
enzyme.15, 27, 29, 34 Thus, within the investigated series of proline-based hydroxamates,
the effect of different substituents (R) at the terminal phenyl ring of the
diphenylacetylene moiety was studied, too.
9

Figure 2: Structures of reported and envisaged hydroxamic acids.
10

2
2
. Results and discussion
.1. Chemistry
Scheme 2: Synthesis of 3,4-dihydroxyproline-derived hydroxamic acids 6 and 9.
Reagents and conditions: (a) LiAlH
MsCl, pyridine, CH Cl , 0 °C → rt, 20 88%, 21 69%; (d) benzylamine, toluene,
microwave irradiation, 22 84%, 23 75%; (e) 80% aqueous acetic acid, 50 °C, 24
2%, 25 74%; (f) H , Pd/C, Boc O, MeOH, rt, 26 93%, 27 71%; (g) 1. NaIO , MeOH,
rt, 2. AgNO , KOH, H O/THF, rt (28) or NaClO , 2-methylbut-2-ene, NaH PO , t-
BuOH/H O, rt (29), 3. CH I, K CO , DMF, rt, 28 74%, 29 56%; (h) HCl (gas), MeOH,
Δ, 30 79%, 31 34%; (i) 4-iodobenzoyl chloride, NEt , DMAP, CH Cl , 0 °C → rt, 32
3%, 33 60%; (j) Pd(PPh , CuI, NEt , ACN, rt, 35g 99%, 35h 96%, 36g 79%; (k)
NOH·HCl, NaOMe, MeOH, rt, 9g 81%, 9h 15%, 6g 48%.
4
, THF, rt, 18 91%; (b) LiAlH , THF, rt, 19 95%; (c)
4
2
2
5
2
2
4
3
2
2
2
4
2
3
2
3
3
2
2
7
)
3 4
3
H
2
11

Whereas the previously described proline-derived hydroxamic acids 12, ent-12, 13, 14,
and 15 had been obtained in chiral pool syntheses starting from the methyl esters of
(
2S,4R)-4-hydroxyproline, L- and D-proline,33 the envisaged 3,4-dihydroxyproline-
derived LpxC inhibitors were synthesized from sugar derivatives (Scheme 2). The
3S,4R)-3,4-dihydroxy-D-proline hydroxamates 9g and 9h were obtained from D-
(
31
mannose derived bisacetonide 16. The (3S,4R)-3,4-dihydroxy-L-proline derivative 6g
was synthesized from D-talono-1,4-lactone 17, which can be accessed from the
35
respective D-mannose derivative via a one-pot sequence. In both cases, the desired
pyrrolidine ring was established according to a reported procedure.36-38 Thus, at first,
lactol 16 and lactone 17 were reduced with LiAlH
4
to yield diol 18 and its diastereomer
1
9, respectively. Subsequently, both hydroxy groups of diols 18 and 19 were
36
mesylated. The resulting bis-mesylates 20 and 21 were subjected to a microwave-
assisted cyclization with benzylamine, involving two S 2 reactions, thus yielding
N
pyrrolidine derivatives 22 and 23 with an inverted configuration in position 2 of the
pyrrolidine ring. In the next reaction steps, the side chain acetonide of bisacetonides
2
2 and 23 was selectively cleaved using 80% acetic acid, the benzyl group was
38
hydrogenolytically removed and in situ replaced by a Boc protective group. The glycol
moiety of the resulting Boc protected pyrrolidine derivatives 26 and 27 was cleaved
with sodium periodate, the intermediately formed aldehydes were oxidized with silver
nitrate or via a Pinnick oxidation, and the resulting carboxylic acids were finally
transformed into methyl esters 28 and 29 with methyl iodide and potassium carbonate
in DMF.30 When pyrrolidine derivatives 28 and 29 were heated to reflux in HCl-
saturated methanol, the remaining acetonide moiety as well as the Boc protective
group were cleaved, yielding compounds 30 and 31, respectively. The secondary
amines were acylated with 4-iodobenzoyl chloride to obtain amides 32 and 33.
Sonogashira couplings of aryl iodides 32 and 33 with terminal alkynes 34g and 34h
12

gave diphenylacetylene derivatives 35g, 35h, and 36g, which were transformed into
hydroxamic acids 9g, 9h, and 6g via aminolyses with hydroxylamine.29
Additionally, the (3S,4R)-3,4-dihydroxy-D-proline derived acetonide 10g was
synthesized (Scheme 3). Starting from diol 32, the hydoxamic acid was obtained in
three consecutive steps. At first, diol 32 was reacted with acetone in the presence of
p-toluenesulfonic acid, yielding acetonide 37, which was subjected to a Sonogashira
coupling with morpholinomethyl-substituted phenylacetylene 34g to give
diphenylacetylene derivative 38g. The reaction of ester 38g with hydroxylamine finally
yielded hydroxamic acid 10g.
Scheme 3: Synthesis of acetonide 10g.
Reagents and conditions: (a) pTsOH, acetone, Δ, 71%; (b) Pd(PPh
3 4
)
, CuI, NEt ,
3
ACN, rt, 93%; (c) H NOH·HCl, NaOMe, MeOH, rt, 66%.
2
13

2
.2. Biological evaluation
To characterize the antibacterial properties of the synthesized proline derivatives,
different bioassays were performed. On the one hand, agar diffusion tests against E.
coli BL21, the defective E. coli D22 stain, which due to an impaired lipid A biosynthesis
is more sensitive towards several antibiotics including LpxC inhibitors, as well as
several clinically important Gram-negative bacteria were performed. Additionally, MIC-
values were determined. On the other hand, an in vitro enzyme inhibition assay was
carried out to provide evidence that the tested compounds exert their antibacterial
properties via the inhibition of the deacetylase LpxC.
14

Table 1: Antibacterial and LpxC inhibitory activities of tetrahydrofuran- and pyrrolidine-based hydroxamates. * n.d. – not determinable.
cmpd.
zone of inhibition [mm]
MIC [µM]
enzyme assay
R1
R2
config.
R
E. coli BL21
E. coli D22
E. coli BL21
E. coli D22
IC50 [µM]
i
K [µM]
5
7
8
OH
OH
OH
OH
(2S)
(2R)
(4S)
(2S)
<6
<6
<6
<6
13.2
>256
>256
>256
>256
16
8
>200
10 ± 1
>200
>27.6
1.4 ± 0.2
>27.6
22.1 ± 1.8
14.7 ± 0.6
15.3 ± 0.6
128
16
11
H
OH
87 ± 31
12 ± 4
6g
OH
OH
(2S)
<6
18.0 ± 1.7
128
16
2.5 ± 0.6
0.34 ± 0.08
15

9
g
h
OH
OH
OH
OH
(2R)
(2R)
(4R)
(2S)
(2S)
(2S)
(2S)
(2S)
(2S)
(2S)
(2R)
<6
<6
<6
>256
>256
>256
>256
>256
>256
256
>256
128
256
64
>200
>200
>27.6
>27.6
>27.6
>27.6
4.1 ± 2.2
-
9
8.3 ± 1.2
10.0 ± 1.7
14.3 ± 0.6
19.5 ± 0.6
12.3 ± 1.5
20.0 ± 2.6
13.3 ± 2.1
12.0 ± 0
10g
9.0 ± 0.8
<6
>200
13a
H
H
H
H
H
H
H
H
OH
OH
H
H
>200
13g
11.3 ± 0.6
7.5 ± 0.7
7.8 ± 1.9
<6
16
29.5 ± 15.7
n.d.*
39a
NH
NH
F
2
128
64
39g
2
n.d.*
-
4
0a
2a
H
H
>256
>256
64
128
128
16
65.9 ± 15.4
>200
9.1 ± 2.1
>27.6
6.3 ± 2.0
8.7 ± 3.8
1
H
<6
12g
H
13.2 ± 0.3
11.3 ± 0.6
20.0 ± 1.0
24.7 ± 1.2
45.6 ± 14.3
63.4 ± 27.5
ent-12g
H
>256
8
16

CHIR-090 (3)
24.6 ± 1.9
30.3 ± 2.5
1
0.032
0.12 ± 0.05
17

The biological evaluation of the dihydroxyproline derivatives revealed that the (2S)-
configured hydroxamic acid 6g is a more potent antibiotic, causing a larger zone of
inhibition in the disc diffusion assay against the defective E. coli D22 strain and
exhibiting a lower MIC value when tested against E. coli BL21, than the respective
dihydroxytetrahydrofuran derivative 5 (Table 1). Especially in the enzyme assay, L-
proline derivative 6g was found to be a considerably more potent LpxC inhibitor than
C-glycoside 5, exhibiting a K-value of 0.34 µM. However, although the compound only
i
differs from CHIR-090 (3) by a rigidified scaffold and the presence of an additional
hydroxy group, L-proline derivative 6g is significantly less potent than the L-threonine
derivative. In contrast to the C-furanosides, the inversion of configuration in α-position
of the hydroxamate moiety leading to D-proline derivative 9g caused a complete loss
of antibacterial and inhibitory activity. Interestingly, when the oxygen atom of the
morpholine ring of the inactive (2R)-configured hydroxamate 9g was replaced by a
sulfur atom, leading to thiomorpholinomethyl derivative 9h, a slight increase in
antibacterial activity against E. coli D22 was observed. Similarly to what was observed
in case of C-glycosides 5 and 8, the acetalization of the two hydroxy groups of 9g
caused an increase in antibacterial activity in the disc diffusion test against E. coli D22.
Also when being tested against E. coli BL21, acetonide 10g was able to slightly inhibit
bacterial growth. However, none of the synthesized (3S,4R)-3,4-dihydroxy-D-proline-
derived hydroxamates was able to inhibit the enzymatic activity of LpxC at
concentrations up to 200 μM.
The removal of the hydroxy group in position 3 of the pyrrolidine ring of 6g or even
both of its hydroxy groups, leading to the (4S)-configured 4-hydroxy-L-proline
derivative 13g and the 3,4-unsubstituted L-proline-derived hydroxamic acid 12g,
respectively, caused a more than tenfold decrease in the LpxC inhibitory activities of
18

the compounds (13g: K
i
= 4.1 μM, 12g: K = 6.3 μM), whereas the antibacterial
i
properties remained unaffected or were even slightly increased, as indicated by the
performed disc diffusion assays.
Further variations of the substituent in position 4 of the pyrrolidine ring yielded the (4S)-
configured 4-aminoproline derivatives 39a and 39g, which demonstrated antibacterial
activities being comparable to the ones of their 4-hydroxyproline analogs 13a and 13g.
Both compounds were found to inhibit the growth of E. coli D22 as well as of E. coli
BL21 in the performed disc diffusion assays. Although the introduction of a fluorine
atom in 4-position of the pyrrolidine ring, leading to the (4S)-configured L-proline
derivative 40a, did not result in improved antibacterial activities in the disc diffusion
assays as well as the MIC tests, the fluorinated compound 40a exhibited superior LpxC
inhibitory activity (K
i
= 9.1 μM) compared to its 4-hydroxyproline analog 13a as well as
> 27.6 μM, each).
the 3,4-unsubstituted compound 12a (K
i
Surprisingly, in case of the 3,4-unsubstituted proline-derived hydroxamic acids, the
configuration in position 2 of the pyrrolidine ring has apparently only a minor effect on
the biological activity of the compounds, as 12g and its enantiomer ent-12g only slightly
differed in their antibacterial and LpxC inhibitory properties.
19

Table 2: Antibacterial and LpxC inhibitory activities of 3,4-unsubstituted L-proline derivatives. * n.d. – not determinable.
cmpd.
zone of inhibition [mm]
MIC [µM]
enzyme assay
R
H
E. coli BL21
E. coli D22
E. coli BL21
E. coli D22
IC50 [µM]
K [µM]
i
1
1
1
1
1
2a
2b
2e
2g
2h
<6
12.0 ± 0
>256
>256
>256
64
128
>200
>27.6
-
NH
2
7.5 ± 0.7
7.0 ± 0
14.3 ± 0.6
18.7 ± 0.6
20.0 ± 1.0
18.0 ± 1.0
17.0 ± 1.0
128
128
16
8
n.d.*
>200
>27.6
13.2 ± 0.3
12.7 ± 1.2
10.7 ± 0.8
45.6 ± 14.3
45.2 ± 4.3
17.1 ± 5.8
6.3 ± 2.0
6.2 ± 0.6
2.4 ± 0.8
128
1
2i
>256
64
20

When investigating the biological activities of the hydroxamic acids, possessing a 3,4-
unsubstituted L-proline scaffold, all of the tested compounds from this series showed
antibacterial properties in the disc diffusion assay against E. coli D22 and were found
to exhibit MIC values between 8 μM and 128 μM against this strain (Table 2). Moreover,
most of the compounds exhibited some activity in the disc diffusion assay against E.
coli BL21 and hydroxamates 12g and 12h were shown to inhibit the growth of E. coli
BL21 with MIC values of 64 μM and 128 μM, respectively. The substituent at the distal
phenyl ring of the lipophilic side chain significantly influences the antibacterial
properties of the compounds. Generally, all of the investigated substituents in para-
position of the distal phenyl ring resulted in enhanced antibacterial properties. Whereas
the insertion of a primary amino group (compound 12b) or a piperidin-1-yl-methyl
moiety (compound 12e) resulted only in a slight increase in antibacterial activity, the
cyclic
and
polar
morpholinomethyl,
thiomorpholinomethyl
and
1,1-
dioxothiomorpholinomethyl substituents of compounds 12g, 12h, and 12i led to even
larger zones of inhibition and significantly lower MIC-values against both E. coli strains.
The results of the LpxC enzyme assay additionally confirmed these trends, indicating
that in the series of 3,4-unsubstituted L-proline derivatives the transition from
no/small/unpolar cyclic substituents in para-position of the distal phenyl ring of the
compounds to cyclic and polar substituents (e.g. a morpholinomethyl moiety) causes
an increase in inhibitory activity. Consequentially, compounds 12g, 12h, and 12i
bearing
a
morpholinomethyl,
a
thiomorpholinomethyl
and
a
1,1-
dioxothiomorpholinomethyl substituent, respectively, were found to be most active in
the LpxC enzyme assay, inhibiting the enzyme with K-values between 2.4 μM and 6.3
i
μM.
21

Table 3: Antibacterial and LpxC inhibitory activities of 4-hydroxy-L-proline-derived hydroxamic acids. * n.d. – not determinable.
cmpd.
zone of inhibition [mm]
MIC [µM]
enzyme assay
config.
R
H
E. coli BL21
E. coli D22
E. coli BL21
E. coli D22
IC50 [µM]
K [µM]
i
1
4a
3a
(4R)
<6
14.0 ± 1.7
>256
>256
>256
>256
>256
>256
>256
128
103 ± 36
14.2 ± 5.0
1
(4S)
(4R)
(4R)
(4S)
(4R)
(4S)
H
F
<6
<6
<6
<6
<6
<6
14.3 ± 0.6
10.7 ± 0.6
9.7 ± 2.1
<6
64
256
64
>200
>200
n.d.*
n.d.*
n.d.*
n.d.*
>27.6
1
4j
>27.6
14k
13k
14b
13b
OCH
3
3
-
-
-
-
OCH
128
128
128
NH
2
2
12.3 ± 1.5
13.0 ± 1.0
NH
22

1
1
1
1
4c
3c
3d
3e
(4R)
(4S)
(4S)
(4S)
(4S)
(4R)
(4S)
(4S)
(4R)
(4S)
(4S)
N(CH
3
)
)
2
2
<6
<6
14.0 ± 1.7
13.0 ± 0.8
14.3 ± 1.2
19.2 ± 1.0
23.7 ± 1.5
20.7 ± 1.5
19.5 ± 0.6
21.3 ± 1.5
8.0 ± 1.4
8.0 ± 1.4
<6
256
256
32
32
n.d.*
-
N(CH
3
16.7 ± 4.7
>200
2.3 ± 0.6
>27.6
<6
>256
>256
64
64
<6
32
54.5 ± 24.1
44.0 ± 5.8
47.9 ± 18.6
29.5 ± 15.7
32.8 ± 14.6
5.9 ± 3.1
>200
7.5 ± 3.3
6.1 ± 0.8
6.6 ± 2.6
4.1 ± 2.2
4.5 ± 2.0
0.81 ± 0.4
>27.6
1
3f
15.7 ± 0.6
7.5 ± 0.7
11.3 ± 0.6
13.0 ± 1.0
<6
4
14g
13g
13h
>256
>256
128
16
16
8
1
4l
>256
>256
>256
16
1
3l
<6
>128
>256
13m
O(CH
2
)
5
F
<6
n.d.*
-
23

Notably, the tested 4-hydroxy-L-proline-derived hydroxamic acids showed antibacterial
properties, generally being comparable to the ones of their 4-unsubstituted analogs
(Table 3). Hence, with the exception of methyl ether 13k and the fluorinated derivative
1
3m, all 4-hydroxy-L-proline derived hydroxamic acids were able to inhibit the growth
of the defective E. coli D22 strain in the performed disc diffusion assays. Among these
hydroxamic acids, with a diameter of the halo of inhibition of 23.7 mm and a MIC value
of 4 μM, the (4-hydroxypiperidin-1-yl)methyl-substituted compound 13f was found to
possess the highest antibacterial activity against E. coli D22. In the disc diffusion
assays against E. coli BL21, only four of the 4-hydroxy-L-proline derivatives exhibited
zones of inhibition exceeding the diameter of the filter disc. Again, 4-hydroxypiperidine
derivative 13f exhibited the highest antibacterial activity.
The substituent in para-position of the distal phenyl ring as well as the configuration of
the stereocenter in 4-position of the pyrrolidine ring were found to influence the
biological activity of the compounds. Whilst small substituents like a methoxy
(
compounds 13k and 14k) or an amino group (compounds 13b and 14b) at the distal
phenyl ring were found to have little to no effect on the antibacterial properties of the
-hydroxyproline derivatives, the introduction of a N,N-dimethylamino group, a n-pentyl
4
moiety as well as cyclic polar substituents resulted in enhanced antibacterial activities
and/or higher inhibitory potency toward LpxC compared to unsubstituted compounds
1
3a and 14a. Consequently, the n-pentyl and N,N-dimethylamino-substituted
compounds 14l and 13c were found to be low micromolar (13c: K = 2.3 μM) or even
nanomolar (14l: K = 0.81 μM) inhibitors of LpxC, whereas compounds 13a and 14a,
bearing no substituent in para-position of the distal phenyl ring, exhibited from no (13a:
> 27.6 μM) to low (14a: K = 14.2 μM) inhibitory activity toward LpxC. Also the
introduction of a heterocycle resulted in improved inhibitory (K = 4.1 – 7.5 μM) and
i
i
K
i
i
i
24

antibacterial activities, with only the pyrrolidin-1-ylmethyl-substituted compound 13d
being inactive in the enzyme assay.
When comparing different pairs of diastereomeric 4-hydroxyproline derivatives,
ambiguous effects were observed. In case of the (4R)-configured LpxC inhibitors 14a
(K
i
= 14.2 μM) and 14l (K = 0.81 μM), possessing no substituent or an n-pentyl residue
i
in para-position of the distal phenyl ring, the inversion of the stereocenter in position 4
of the pyrrolidine ring, leading to the (4S)-configured compounds 13a and 13l, was
detrimental for the inhibitory activity of the hydroxamic acids. In contrast, this effect
was not observed in case of the morpholinomethyl-substituted diastereomers 14g and
1
3g, exhibiting similar inhibitory activities toward LpxC (K = 6.6 μM vs. 4.1 μM).
i
Furthermore, in case of the primary amines 13b and 14b, the dimethylamine
derivatives 13c and 14c, and the morpholinomethyl-substituted compounds 13g and
1
4g, the respective diastereomers did not substantially differ in their ability to inhibit
the growth of E. coli BL21 and E. coli D22 in the disc diffusion tests as well as in the
MIC assays. Although being the most potent LpxC inhibitor in this series of 4-
hydroxyproline derivatives, the n-pentyl-substituted compound 14l was found to
possess only low antibacterial activity, which might be due to unfavorable
physicochemical properties, reducing the compound’s ability to penetrate the Gram-
negative cell envelope.
25

Table 4: Antibacterial and LpxC inhibitory activities of amines 41g, 41h, 42g, and 43.
cmpd.
zone of inhibition [mm]
MIC [µM]
enzyme assay
R2
H
config.
R
E. coli BL21
E. coli D22
E. coli BL21
E. coli D22
IC50 [µM]
K [µM]
i
4
4
4
1g
1h
2g
15.0 ± 1.7
25.0 ± 3.0
128
64
4
10.5 ± 5.1
1.4 ± 0.7
1.1 ± 0.4
8.6 ± 1.9
>27.6
H
OH
F
10.7 ± 1.5
12.3 ± 0.6
<6
17.7 ± 1.2
21.0 ± 1.0
10.7 ± 2.1
1
8
8.1 ± 3.2
62.1 ± 14.0
>200
(4R)
(4S)
>256
>256
4
3a
H
128
26

Additionally, the biological activity of tertiary amines 41g, 41h, 42g, and 43a was
investigated to study the effect of the formal reduction of the amide moiety (Table 4).
Surprisingly, although lacking the carbonyl oxygen atom, which was considered to be
an important pharmacophore, amines 41g, 41h, and 42g exhibited inhibitory activity
toward LpxC as well as notable antibacterial properties. The 3,4-unsubstituted L-
proline derivatives 41g and 41h were found to be able to inhibit LpxC in the low
micromolar range (41g: K
i
= 1.4 μM, 41h: K
i
= 1.1 μM), being more potent LpxC
= 8.6 μM). The comparison
inhibitors than (4R)-4-hydroxy-L-proline derivative 42g (K
i
of the two morpholine derivatives 41g and 42g revealed that the superior inhibitory
activity of the 3,4-unsubstituted L-proline derivative 41g toward LpxC is translated into
higher antibacterial activities in the MIC tests as well as in the disc diffusion assays.
Whereas tertiary amines 41g and 41h exhibited higher LpxC inhibitory activity than
their amide-based analogs 12g and 12h, the (4S)-configured 4-fluoroproline-derived
amine 43a was found to be a less potent LpxC inhibitor that the corresponding amide
4
0a.
27

Table 5: Antibacterial activities of the investigated hydroxamates against clinically
relevant Gram-negative bacterial strains.
diameter of the zones of inhibition [mm]
cmpd.
9
9
g
h
<6
<6
8
8
7
<6
<6
<6
8
<6
<6
8
<6
<6
8
<6
<6
<6
7
<6
<6
11
8
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
10g
12a
12b
12e
12g
8
<6
<6
13
15
11
18
11
<6
<6
<6
<6
<6
7
8
<6
<6
12
15
9
<6
<6
10
12
9
7
<6
10
13
9
<6
11
9
7
9
8
9
12
12
14
<6
7
ent-12g
8
<6
10
10
7
1
2h
11
<6
7
14
10
<6
<6
8
14
8
12
9
1
2i
13a
14k
13k
14b
13b
14c
13c
13d
13e
<6
<6
<6
<6
<6
10
9
7
7
7
8
7
7
<6
7
9
8
7
<6
<6
<6
8
<6
<6
<6
<6
<6
13
<6
<6
9
<6
<6
8
8
8
8
8
9
8
8
9
<6
9
<6
9
<6
<6
<6
<6
8
<6
10
9
<6
11
11
9
<6
9
1
3f
<6
9
<6
<6
8
<6
10
12
14
1
1
1
4g
3g
3h
16
15
18
10
15
11
10
11
14
9
28

1
1
4l
3l
<6
<6
7
7
8
<6
<6
7
<6
7
9
9
7
<6
<6
7
7
<6
7
8
39a
39g
41g
41h
42g
7
8
<6
<6
12
<6
<6
<6
40
7
9
13
13
11
<6
<6
33
<6
20
14
<6
<6
36
11
18
13
<6
<6
34
7
8
10
<6
<6
<6
<6
29
20
14
7
18
8
16
13
<6
<6
33
<6
<6
36
DMSO
<6
36
CHIR-090 (3)
Besides E. coli, the deacetylase LpxC is present in virtually all Gram-negative bacteria.
Thus, some of the investigated compounds were also tested against several other
clinically relevant Gram-negative bacterial strains (Table 5). Among the
dihydroxyproline derivatives, only acetonide 10g exhibited weak antibacterial
properties against some of the investigated Gram-negative bacteria, with Citrobacter
freundii being most susceptible toward the compound (diameter of the zone of
inhibition: 11 mm). However, among the 3,4-unsubstituted L-proline derivatives as well
as the 4-hydroxy-L-proline derived hydroxamic acids more potent antibacterial
compounds were found. Similarly to what was observed in the disc diffusion assays
against the two E. coli strains, the substituent and the configuration in position 4 of the
pyrrolidine ring as well as the substituent at the terminal phenyl ring influenced the
antibacterial properties of the compounds against the other investigated Gram-
negative bacteria. Generally, the thiomorpholinomethyl and morpholinomethyl
substituted compounds were most active. Thus, the (4S)-4-hydroxy-L-proline derivative
1
3h and its 3,4-unsubstituted L-proline-derived analog 12h were found to inhibit the
growth of seven out of eight tested Gram-negative pathogens, with K. pneumonia, E.
cloacae, E. aerogenes, and C. freundii being most susceptible toward these
29