Optimization of peptide-based inhibitors targeting the HtrA serine protease in Chlamydia: Design, synthesis and biological evaluation of pyridone-based and N-Capping group-modified analogues

Article abstract of DOI:10.1016/j.ejmech.2021.113692

The obligate intracellular bacterium Chlamydia trachomatis (C. trachomatis) is responsible for the most common bacterial sexually transmitted infection and is the leading cause of preventable blindness, representing a major global health burden. While C.

Full text of DOI:10.1016/j.ejmech.2021.113692

European Journal of Medicinal Chemistry 224 (2021) 113692
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Optimization of peptide-based inhibitors targeting the HtrA serine
protease in Chlamydia: Design, synthesis and biological evaluation of
pyridone-based and N-Capping group-modified analogues
,
,
Jimin Hwang a 1, Natalie Strange b 1, Matthew J.A. Phillips b, Alexandra L. Krause c,
Astra Heywood d, Allan B. Gamble a **, Wilhelmina M. Huston b ***, Joel D.A. Tyndall a
,
,
, *
a School of Pharmacy, University of Otago, Dunedin, 9054, New Zealand
b School of Life Sciences, Faculty of Science, University of Technology Sydney, New South Wales, Australia
c Department of Microbiology and Immunology, University of Otago, Dunedin, 9054, New Zealand
d Department of Biochemistry, School of Biomedical Sciences, University of Otago, Dunedin, 9054, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
The obligate intracellular bacterium Chlamydia trachomatis (C. trachomatis) is responsible for the most
common bacterial sexually transmitted infection and is the leading cause of preventable blindness,
representing a major global health burden. While C. trachomatis infection is currently treatable with
broad-spectrum antibiotics, there would be many benefits of a chlamydia-specific therapy. Previously, we
have identified a small-molecule lead compound JO146 [Boc-Val-Pro-ValP(OPh)2] targeting the bacterial
serine protease HtrA, which is essential in bacterial replication, virulence and survival, particularly under
stress conditions. JO146 is highly efficacious in attenuating infectivity of both human (C. trachomatis) as
well as koala (C. pecorum) species in vitro and in vivo, without host cell toxicity. Herein, we present our
continuing efforts on optimizing JO146 by modifying the N-capping group as well as replacing the parent
peptide structure with the 2-pyridone scaffold at P3/P2. The drug optimization process was guided by
molecular modelling, enzyme and cell-based assays. Compound 18b from the pyridone series showed
improved inhibitory activity against CtHtrA by 5-fold and selectivity over human neutrophil elastase
(HNE) by 109-fold compared to JO146, indicating that 2-pyridone is a suitable bioisostere of the P3/P2
amide/proline for developing CtHtrA inhibitors. Most pyridone-based inhibitors showed superior anti-
Received 20 May 2021
Received in revised form
4 July 2021
Accepted 5 July 2021
Available online 7 July 2021
chlamydial potency to JO146 especially at lower doses (25 and 50
mM) in C. trachomatis and
C. pecorum cell culture assays. Modifications of the N-capping group of the peptidyl inhibitors did not
have much influence on the anti-chlamydial activities, providing opportunities for more versatile al-
terations and future optimization. In summary, we present 2-pyridone based analogues as a new gen-
eration of non-peptidic CtHtrA inhibitors, which hold better promise as anti-chlamydial drug candidates.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
of currently available antibacterial agents for C. trachomatis, man-
agement of the infection remains challenging with escalating
Chlamydia trachomatis (C. trachomatis) is one of the most com-
mon sexually transmitted bacterial pathogens and a leading cause
of preventable blindness worldwide [1,2]. Despite the effectiveness
global prevalence. According to the latest World Health Organiza-
tion (WHO) reports, the reported prevalence of Chlamydia has
steadily increased with 92, 101, 131, and 153 million incidences in
2009, 2011, 2012 and 2015, respectively [3,4]. C. trachomatis is often
seen as a co-infection with Neisseria gonorrhoeae or Mycoplasma
genitalium, which have already developed resistance to macrolides
and tetracyclines [5e7]. Thus, designing new antibacterial drugs
that specifically target Chlamydia may provide an avenue for
reducing the pressure of antibiotic resistance development in other
pathogens.
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: allan.gamble@otago.ac.nz (A.B. Gamble), Wilhelmina.Huston@
uts.edu.au (W.M. Huston), joel.tyndall@otago.ac.nz (J.D.A. Tyndall).
1
Equal contributions.
https://doi.org/10.1016/j.ejmech.2021.113692
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
In addition, Chlamydia (specifically C. pecorum and
C. pneumoniae) is a dominant threat to the koala (Phascolarctos
cinereus), an iconic marsupial in Australia, that is currently at risk of
extinction [8]. Traditional broad-spectrum antibiotics used to treat
human infections have limited efficacy in koalas, as the antibiotics
are detrimental to their unique gut microbiota that digest toxic
Eucalyptus leaves [9,10]. In addition, chloramphenicol, the main
antibiotic treatment for koala, requires a prolonged course (14e28
days) of daily subcutaneous injections to be effective, and is
decreasing in availability in the market [11]. Therefore, develop-
ment of a new class of antibiotics with a more specified range of
bacterial target is urgently needed for the koala not only to treat the
infection, but also to limit side effects given their unique digestive
system.
and oral bioavailability) associated with peptide-based compounds.
In this study, a variety of pyridone analogues were synthesized with
different combinations of P1, P2, P3 and the capping group along
with a diphenyl phosphonate transition state analogue in a step-
wise optimization method. To evaluate the compatibility of
different N-capping groups in the S4 pocket of CtHtrA, a small pool
of modified groups was also coupled with the new peptidic lead
compounds 2 and 3, which were previously optimized by incor-
porating unnatural residues at P3 and/or P1 (Fig. 1) [31].
2. Results and discussions
2.1. Synthetic chemistry
HtrA (High Temperature Requirement A), an orthologue of E. coli
DegP, is an evolutionarily well-conserved serine protease-
chaperone which plays a vital role in bacterial replication and
survival under stress conditions through protein quality control
[12]. HtrA in Gram-negative bacteria are usually localised to the
periplasm, but are also found on the outer surface of bacteria or in
the extracellular matrix for their direct role in bacterial dissemi-
nation and virulence [12]. The crucial function and accessible
location of bacterial HtrA makes it an attractive target for chemical
inhibition. In fact, earlier studies have shown that HtrA in E. coli
[12], H. pylori [13e15] and C. trachomatis [16] could be targeted with
small molecule inhibitors at a low micromolar range.
2.1.1. 2-Pyridone-based inhibitors
Pyridone analogues were synthesized with different combina-
tions of P1, P2, P3 and capping group residues along with the
diphenyl phosphonate warhead group that is responsible for
forming an irreversible covalent bond with the catalytic serine
residue. Previously, adaptations at the electrophilic warhead group
with non-covalent (N-alkyl methylamides, valinol), and reversible,
covalent (boronates,
a-diketones, a-ketoesters, a-ketoamides and
a
-ketoheterocycles) transition state analogues (TSA) were investi-
gated for optimization of JO146 [33]. Although these warhead
groups have previously been reported to be active against serine
proteases [31,34e39], only a-ketobenzothiazole from the a-keto-
heterocycle series was active with comparable CtHtrA inhibition to
that by JO146 [31]. Therefore, covalent inhibition between the in-
hibitor and the active site of CtHtrA is crucial for anti-chlamydial
activity and diphenyl phosphonate remains the most effective
warhead group against CtHtrA to date.
In our previous studies, inhibition of C. trachomatis HtrA
(CtHtrA) by the lead compound JO146 (1; IC50 ¼ 12.5 2.94
mM;
Fig. 1) during the bacterial mid-replicative phase resulted in the
total loss of inclusion vacuoles and lethality of the human, mouse
and koala Chlamydia strains in vitro and in vivo without causing
cytotoxicity [16,17]. JO146 was selective for CtHtrA over other
The synthesis of the 2-pyridone analogues with modifications of
the capping group, P2 and P1 is outlined in Scheme 1. Commercially
available 3-nitro-2-pyridone 4 was reduced to 3-amino-2-pyridone
5 in the presence of 10% palladium on carbon under H2(g). The Cbz
protection of the aniline in compound 5 was readily achieved by N-
acylation with benzyl chloroformate under the Schotten-Baumann
conditions [40] to give 6. However, due to the low reactivity of the
aryl amine, Boc protection of pyridone 5 with a NaOH in an aqueous
environment was low yielding, regardless of prolonged reaction
time (up to 65 h), temperature (up to 55 ꢀC) and addition of a large
excess of di-tert-butyl dicarbonate (up to 5.3 equiv.). The reaction
yield was lower when conducted independently in methanol and
THF, and triethylamine as base. Therefore, an alternative method
was sought using a combination of 4-dimethylaminopyridine
(DMAP) and triethylamine as an alternative base combination to
NaOH. The starting material 5 was consumed within 4 h using a
previous method by Basel et al. [41] However, owing to the
serine proteases including trypsin (IC50 > 500
trypsin (IC50 > 500 M), but not human neutrophil elastase (HNE;
IC50 2.24 0.12 M) [16]. Although HtrA proteases are ubiqui-
mM) and chymo-
m
m
mM
tous in cells, their structural, functional and mechanistic disparities
between Gram-negative, Gram-positive bacteria and mammalian
cells make HtrAs a more favourable target for selectivity [12,18].
Herein, JO146 was optimized by replacing the P3/P2 backbone
by a non-peptidic 2-pyridone template. The 2-pyridone class and
its derivatives are of particular interest in developing small mole-
cule peptide-mimetic inhibitors against various serine (e.g. HNE
[19], HCV NS3/4 A [20], thrombin [21,22] and tissue factor VIIa
[23e26]) and cysteine proteases (e.g. human rhinovirus 3C [27,28],
COVID-19 Mpro (or 3CLpro) [29] and P. falciparum falcipain-2/3 [30]).
The non-natural pyridine ring, which masks the P3eP2 amide bond
could suppress hydrolytic cleavage by host proteases, potentially
alleviating general pharmacokinetic problems (e.g., poor stability
Fig. 1. Structures of the initial lead compound JO146 (1) from high throughput screening [16] and the newer lead compounds 2 and 3 obtained from optimization of P1 and/or P3
residues [31]. Standard nomenclature for peptide substrates in which amino acid residues are denoted as P3eP1 and their corresponding binding subpockets as S4eS1’ [32]. TSA e
Transition State Analogue. CG e Capping Group.
2

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
Scheme 1. General Route for the Preparation of Pyridone-based inhibitors.
Reagents and conditions: (a) 10% Pd/C, H2(g), DMF/MeOH, rt, 21 h, 95%; (b) benzyl chloroformate, Na2CO3, H2O/acetone, 0e25 ꢀC, 3 h, 86%; (c) di-tert-butyl dicarbonate, NaOH, THF/
H2O, 55 ꢀC, 65 h, 14e21%; (d) di-tert-butyl dicarbonate, DMAP, triethylamine, DCM, rt, 4 h, 22%; (e) ethyl iodopropionate, NaH, DMF, 0e25 ꢀC, 3 h, 74%; (f) ethyl iodoacetate, NaH,
DMF, 0e25 ꢀC, 3 h, 41e81%; (g) Tf2O, 2,6-lutidine, CH2Cl2, 1 h; (h) NaH, THF, 24 h, 63%; (i) LiOH.H2O, THF/H2O, rt, 2 h, 82e98% (j) 11 or 12, DIPEA, HBTU, DMF, rt, 24 h, 43e79%.
formation of impurities such as N-Boc-urea, urea or isocyanate, as
previously reported [41], the yield of 7 remained low (22%).
The N-capped-pyridone-P2 precursors 8a-b and 9 were synthe-
sized according to the method described by Warner et al. [42]
Alanine was incorporated at P2 as a mimic of the Ca-Cb bond in
proline. In addition, glycine was added as an alternative to alanine to
reduce stereochemical complexity arising from the epimerization of
the Ca-P1 chiral centre, which leads to formation of diastereoiso-
meric mixtures in the final compounds. N-alkylation of compound 6
iodoalkyl esters and NaH to produce 8a-b and 9 with moderate to
high yields 41e81%. Due to the high cost of enantiomerically pure
ethyl 2-iodopropionate, a diastereomeric mixture of the reagent was
used in the reaction, this theoretically resulted in compound 8b in
both L- and D-alanine (P2). Alternatively, enantiomerically pure (R)-
lactate 14 was triflated and reacted with compound 6 via SN2 sub-
stitution to produce 16 with an inverted S-configuration at P2 [29].
The L-alanine was selected for P2 as it is evolutionarily more abun-
dant and preferred configuration over the D-enantiomer in natural
and 7 was conducted via an SN2 nucleophilic substitution with
a-
substrates of proteins and proteases.
3

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
Compounds 8a-b, 9 and 16 were then converted to free acids by
base-catalyzed hydrolysis prior to the P2eP3 peptide coupling with
P1-TSA [31] (11 and 12) to successfully synthesize 13a-f and 18a-b.
Our previous studies of P1 optimization showed that replacement
of valine by isoleucine resulted in improved anti-CtHtrA potency
and selectivity over HNE [31]. Thus, these two hydrophobic resi-
dues were investigated in the pyridone series.
12 were then coupled with N-capped-P3-P2 dipeptides 27a-c and
purified by silica gel flash column chromatography to obtain the
final compounds 28a-e in diastereomeric mixtures that were bio-
logically evaluated.
2.2. Enzyme assays
All synthesized compounds were characterized by 1H, 13C NMR
and mass spectrometry analysis, and produced agreeable spectro-
scopic data, which were in full accordance with their depicted
structures. Diastereomers of the final compounds were further
characterized by 31P NMR spectroscopy.
Protease inhibition assays were conducted for compound ac-
tivity and off-target characterization of the final compounds 13a-f
and 18a-b. IC50 values were measured to assess the inhibitory ac-
tivities of compounds against CtHtrA and HNE (Table 1). The IC50
values varied in different batches of the CtHtrA assays (Table S4A
and Table S4B), hence relative potency and selectivity of com-
pounds were determined compared to JO146 for evaluating their
biological activities (Tables 1 and 2) [31]. We believe this variation
stems from the requirement for activation of HtrA for optimal
enzymatic activity, where recombinant protein may be co-purified
with short activating peptides from the expression host which can
vary between batches. The assessment of the relative selectivity
over HNE compared to JO146 was particularly important as selec-
tivity between HNE and CtHtrA proteases was unable to be inferred
from the IC50 derived from the two different assay platforms [44].
Replacement of P2/P3 peptidic backbone with the 2-pyridone
template generally resulted in a decrease in the CtHtrA inhibitory
potencies relative to JO146, as indicated by the diminished IC50
values of 13a-f (Table 1). The analogues with a Boc capping group
(13c and 13f) elicited weaker inhibition of CtHtrA than those with
the Cbz capping group (13b and 13e), demonstrating the lack of
compatibility of the pyridone scaffold with the Boc capping group.
These results indicate that concerted modifications, which are
conducted with a step-wise approach, at different sites of the lead
compounds is necessary to accommodate the three-dimensional
change in the inhibitor structure resulting from the insertion of a
non-peptidic moiety. Although compounds 13a and 13d, both
containing glycine at P2, showed weaker inhibition of CtHtrA than
JO146, their relative selectivity over elastase was improved by
approximately 5- and 4-fold, respectively, since the reduction in
potency against HNE was greater than CtHtrA. For instance,
analogue 13a reduced inhibitory potency against CtHtrA by half
compared to JO146, but the IC50 difference in elastase assay was
significantly greater, by more than 12-fold, resulting in approxi-
mately 5-fold improvement in the selectivity relative to JO146
(Table 1).
2.1.2. Capping group modification of the peptidic inhibitors
The peptidic lead compounds JO146, 2 and 3 were synthesized
according to the procedure reported by Agbowuro et al. [31] The
tripeptide analogues 28a-28e were synthesized by a series of
solution-phase peptide coupling steps using HBTU coupling re-
agent in the presence of DIPEA base, and a base-catalyzed hydro-
lysis of the ester groups (Scheme 2). Cbz protection of tert-leucine
19 to provide 20 was achieved by N-acylation with benzyl chlor-
oformate under Schotten-Baumann conditions [40]. A 2:1 M ratio
of Na2CO3 to NaHCO3 was employed to replace the traditional use of
a strong base (NaOH). Using the former provided improved buff-
ering capabilities, hence were preferred to maintain the optimal
reaction pH of 8e9 that is required to neutralize the hydrochloric
acid formed as a by-product. Neutralizing the acid prevents
degradation of the benzyl carbamate in 20, while avoiding epi-
merization at the a-amino group with excessive alkalinity.
Replacement of the Boc capping group with tert-butyl amide
and tert-butyl urea was carried out by Boc deprotection of dipeptide
23 using TFA in DCM, and the resulting N-uncapped dipeptide 24
was acylated with tert-butyl isocyanate and trimethylacetyl chlo-
ride to provide 25 and 26, respectively. Formation of the tert-butyl
urea and tert-butyl amide capping groups was confirmed by the
upfield peak shift of the tert-butyl group from 1.41 ppm (23) to
1.29 ppm (25) and 1.19 ppm (26), in accordance with the order of
electronegativity of the O, N and C atoms (see NMR spectra in
supporting information). Given the epimerization-free nature of the
Boc deprotection and isocyanate-mediated asymmetric urea for-
mation, duplication of 1H NMR peaks in the tert-butyl urea-capped
dipeptide ester (25) was speculated to be caused by rotamerization.
In addition, the replacement of the oxygen in the Boc group by
eNH- of the urea accentuates the ability of the amide group to form
resonance structures, giving more defined separation of the
rotamer peaks of the tert-butyl group found in 25 than that in 26.
Rotamerization was verified by the coalescence of deuterated peaks
with the NMR solvent change from deuterated chloroform to
acetonitrile and variable temperature (Fig. S1).
Despite the general reduction of anti-CtHtrA inhibition by the
pyridone analogues relative to JO146, compound 13e (IC50 19.76
1.19
mM) demonstrated slightly improved inhibitory activity to its
corresponding peptide analogue 28e (IC50 24.88 1.18
mM), which
comprises a matching capping group and P1 residue (Table 2). In
addition, since the activity of 13e against HNE decreased by about
20-fold compared to JO146, its selectivity towards CtHtrA over HNE
was 11-fold greater than JO146 (Table 1).
Given that 13e showed the most promising inhibitory and
selectivity profile from the pyridone series, compound 18b was
The key intermediate Cbz-protected
a-aminoalkyl diphenyl
phosphonates (11 and 12) were prepared by the previously
described Mannich-type ligation reaction [43] with either 2-methyl
butyraldehyde or isobutyraldehyde for valine or isoleucine at P1,
respectively [31]. During this reaction, the imine intermediate
formed between the benzylcarbamate and aldehyde permits an SN1
nucleophilic attack by the triphenylphosphite, resulting in both S
synthesized with L-alanine at P2 and its inhibitory activity against
CtHtrA was evaluated separately from the rest of the final com-
pounds (Table S4B). Compound 18b markedly improved the
inhibitory activity against CtHtrA by 5-fold relative to JO146 (8-fold
increase in the relative potency compared to 13e), implicating a
single digit micromolar potency when JO146 displays the reference
and R configurations at the a-aminoalkyl side-chain and the sub-
sequent elimination of a phenoxide group. In the case where 2-
methyl butyraldehyde was reacted, an additional chiral centre at
Cb of the aminoalkyl sidechain formed both threo (RR and SS) and
erythro (RS and SR) diastereomers, which were detected by two
distinct peaks in the 31P NMR spectrum. The acid-catalyzed Cbz
deprotection of N-Cbz-aminoalkyl diphenyl phosphonates by
IC50 value of 12.5
109-fold improvement in selectivity towards CtHtrA over HNE
relative to JO146 indicates that -alanine is critical at P2 for bio-
mM reported in literature [16]. Furthermore, its
L
logical recognition of CtHtrA in the pyridone series. The marked
improvement in the selectivity profile could suggest reduction in
off-target toxicity in vivo. These results together indicated that
hydrobromide in acetic acid afforded hydrobromide salts of
a-
aminoalkyl diphenyl phosphonates 11 and 12. Phosphonates 11 and
4

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
Scheme 2. Functionalization of the N-capping group.
Reagents and conditions: (a) benzyl chloroformate, Na2CO3, NaHCO3, H2O/acetone, rt, overnight, 82%; (b) L-proline methyl ester.HCl, DIPEA, HBTU, DMF, rt, 24 h, 91e100%; (c) TFA,
DCM, rt, 2 h, 100%; (d) tert-butyl isocyanate, DMF, rt, overnight, 45%, (e) trimethylacetyl chloride, NaHCO3, H2O/acetone, rt, overnight, 82%; (f) LiOH.H2O, THF/H2O, rt, 2 h, 91e100%;
(g) 11 or 12, HBTU, DIPEA, DMF, rt, 24 h, 52e82%.
pyridone is a suitable bioisostere for P3/P2 amide, and careful
design of pyridone analogues with an optimal combination of the
capping group, P2 and P1 could improve binding affinity and
selectivity of the parent peptide inhibitors to the active site of
CtHtrA.
Variations of the N-capping group had previously been
employed in the development of HCV NS3/4A serine protease in-
hibitors, which allowed improvement in the enzymatic inhibition
and selectivity against HNE [45]. Here, modifications of the capping
group displayed similar anti-CtHtrA activity to the lead compounds
(JO146, 2 and 3; Table 2). Of the three analogues with a Tle-Pro-Val
peptide backbone, the compounds with a Boc (2) and Cbz (28d)
protecting group performed equally well, closely followed by tert-
butyl urea capping group (28b). Compounds with Ile at P1 generally
decreased anti-CtHtrA potency compared to those with Val at P1
with matching capping groups. Among compounds with Ile at P1,
replacement of the Boc capping group by tert-butyl urea (28c) or
Cbz (28e) did not compromise the inhibitory activity against CtHtrA
compared to their corresponding lead compound 3. Analogue 28a
with a tert-butyl amide capping group, on the other hand, increased
the anti-CtHtrA activity approximately 3-fold and the relative
selectivity to almost 18-fold relative to JO146 (Table 2). Inhibitor
28a was also still slightly better in relative potency (more than 2-
fold) and selectivity (1.3-fold) compared to 3, which share the
5

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
Table 1
Inhibitory activities (IC50) of pyridone inhibitors 13a-f and 18a-b in CtHtrA and HNE enzyme assays.
Cmpd
R
P2
P1
IC50 SEM (
m
M)
Relative selectivityc
CtHtrA (Relative inhibition against CtHtrAa)
HNE (Relative inhibition against HNEb)
JO146
13a
13b
13c
13d
13e
13f
Boc
Cbz
Cbz
Boc
Cbz
Cbz
Boc
Cbz
Cbz
Pro
Gly
Val
Val
Val
Val
Ile
Ile
Ile
Val
Ile
12.47 1.11 (1)
28.98 1.21 (0.43)
41.77 1.31 (0.30)
76.98 1.23 (0.16)
48.34 1.18 (0.26)
19.76 1.19 (0.63)
128.5 1.16 (0.10)
NA
1.15 0.10 (1)
1
12.81 0.54 (0.09)
3.60 0.36 (0.32)
6.64 0.22 (0.17)
16.48 2.25 (0.07)
21.47 3.97 (0.05)
13.37 0.67 (0.09)
6.01 0.48 (0.19)
24.97 4.72 (0.05)
4.79
0.93
0.94
3.70
11.8
1.03
NA
L,D-Ala
L,D-Ala
Gly
L,D-Ala
L,D-Ala
L-Ala
L-Ala
18a
18b
(5.07)d
109
IC50 data presented as mean values for triplicates (n ¼ 3).
NA-not assessed.
a
Relative inhibition against CtHtrA is the ratio of IC50 CtHtrA(JO146) to IC50 CtHtrA (compound).
Relative inhibition against HNE (rounded to 2 decimal place) is the ratio of IC50 HNE (JO146) to IC50 HNE (compound).
Relative selectivity (rounded to 3 significant figure) towards CtHtrA over HNE is the ratio of the relative potency against CtHtrA to relative potency against HNE compared
b
c
to that of JO146.
d
Table S4B.
Table 2
Inhibitory activities (IC50) of compounds 28a-e in CtHtrA and HNE enzyme assays.
Cmpd
R
P1
IC50 SEM (
m
M)
Relative selectivityc
CtHtrA (Relative inhibition against CtHtrAa)
HNE (Relative inhibition against HNEb)
JO146
2
3
28a
Boc
Boc
Boc
Val
Val
Ile
12.47 1.11 (1)
1.15 0.10 (1)
1
9.30 1.10 (1.34)
10.26 1.17 (1.22)
4.57 1.21 (2.73)
3.02 0.18 (0.38)
13.51 0.81 (0.09)
7.69 1.12 (0.15)
3.52
14.28
18.23
Ile
28b
28c
Val
Ile
10.65 1.17 (1.17)
14.09 0.89 (0.89)
1.17 0.17 (0.98)
5.66 1.08 (0.03)
1.20
4.44
28d
28e
Val
Ile
9.30 1.18 (1.34)
24.88 0.50 (0.50)
1.35 0.25 (0.11)
2.47 0.11 (0.47)
1.74
1.11
IC50 data presented as mean values for triplicates (n ¼ 3).
a
Relative inhibition against CtHtrA (rounded to 2 decimal place) is the ratio of IC50 CtHtrA(JO146) to IC50 CtHtrA (compound).
Relative inhibition against HNE (rounded to 2 decimal place) is the ratio of IC50 HNE (JO146) to IC50 HNE (compound).
Relative selectivity (rounded to 3 significant figure) towards CtHtrA over HNE is the ratio of the relative potency against CtHtrA to relative potency against HNE compared
b
c
to that of JO146.
same P1 and P3 amino acid residues. Based on the assay results, the
the parent Boc capping group is not necessary for interacting with
oxygen atom placed between the carbonyl and tert-butyl group in
the target peptide backbone, hence removing it (28a) or replacing it
6

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
by a hydrogen bond donor (28b-c) did not affect the anti-CtHtrA
potency. In fact, since the S4 pocket is shallow and exposed to the
solvent environment, it could accommodate longer and bulkier
capping groups such as the carboxybenzyl group (28d-e). This
supports the observation that the Cbz group was indeed an
appropriate capping group for the 2-pyridone inhibitors. In addi-
tion, these results showed that the capping group could be an
opportunistic site to add further functionality with different
physicochemical properties to the Boc group, which might be able
to alleviate the solubility issue inherent with the hydrophobic in-
hibitor JO146 [46].
2.3. Anti-bacterial and cytotoxicity cell assays
2.3.1. JO146 has a narrow anti-bacterial specificity
Despite HtrA proteases being widely expressed in bacteria, the
domain architecture varies between Gram-negative and Gram-
positive bacteria [18]. JO146 and the new lead compounds 2 and
3 completely lacked inhibitory activity against Pseudomonas aeru-
ginosa (at 50 and 100
mM), Staphylococcus aureus and Escherichia
coli (at 50 M), supporting that this class of covalent inhibitors is
m
specific for Chlamydial HtrA or at least have a very narrow spec-
trum of bacterial target even within one Gram stain grouping
(Figs. S4 and S5 and Tables S5 and S6). These results indicate that
although HtrA is ubiquitous in bacteria, structural and functional
differences between species could allow fine-tuning of target
specificity. Identification and development of such pathogen-
specific antibacterial reagents that target only one or a small set
of species would be highly valuable in clinical use for their potential
in minimizing disturbance of the host microbiome and slowing the
spread of resistance [47].
Fig. 2. In vitro testing of Chlamydia trachomatis and C. pecorum growth inhibition by
the lead compound JO146 and pyridone analogues 13a-f at 25, 50 and 100 mM. Dashed
line indicates the limit of detection for inclusion forming units (IFU). Error bars
represent the SEM (n ¼ 3). *p-value <0.05, **p-value <0.001, ***p-value <0.0001
compared to DMSO control as measured by two-way ANOVA.
2.3.2. 2-pyridone inhibitors
The in vitro potency of all compounds was assessed by their
ability to significantly reduce the number of inclusion-forming
units (IFU/mL; Tables S1 and S2) of C. trachomatis and C. pecorum
isolates in cell culture, which is a traditional quantitative measure
of the pathogen's infectivity or transmissibility [48]. It is note-
worthy that the bacterial titre of the control group was about 104
lower in C. pecorum assays than C. trachomatis as the former isolate
is more cytotoxic to host cells in vitro, which limits the number of
infectious progeny able to be cultured.
Pyridone analogues, including those that showed decreased
CtHtrA activities relative to JO146, showed overall improvement in
the anti-bacterial activities compared to JO146 especially at 25 mM
in both C. trachomatis and C. pecorum isolates (Fig. 2). These results
could suggest that the pyridone analogues might have a different or
secondary mechanism of anti-chlamydial action other than inhib-
iting the CtHtrA protease.
Compounds 13d-f, which share isoleucine at P1 and inhibited
C. trachomatis more effectively at 25 mM than compounds 13a-c
with valine at P1. Compounds 13d-f on average resulted in
approximately 3.7-log further reduction in the IFU/mL count than
JO146 at 25
chlamydial activities at 50 and 100
showed a good dose-response relationship and decreased the
bacterial titre below the lower limit of detection at 100 M. In
mM. On the other hand, 13a-c displayed stronger anti-
mM doses. In particular, 13c
m
C. pecorum assays, the pyridone analogues were more effective in
reducing the bacterial titre compared to JO146. All compounds
diminished the infectious progeny close to the limit of detection
even at 25 mM except 13c.
Compounds 18a and 18b were more potent than JO146 against
C. trachomatis by approximately a log and 1.6-log difference in IFU/
Fig. 3. In vitro testing of Chlamydia trachomatis and C. pecorum growth inhibition by
the lead compound JO146 and pyridone analogues 18a and 18b at 25, 50 and 100
Dashed line indicates the limit of detection for inclusion forming units (IFU). Error bar
represents the SEM (n 3). ***p-value <0.0001 compared to DMSO control as
measured by two-way ANOVA.
mM.
¼
7

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
mL, respectively, at 25
obliterated the infectious progeny to the limit of detection even at
25 M (Fig. 3).
Overall, the replacement of the traditional peptidic structure by
mM. In C. pecorum assays, both compounds
m
the new pyridone scaffold produced compounds that exhibited
improved inhibitory action against Chlamydia, therefore further
optimization of this class of inhibitors is worthwhile.
Cytotoxicity of the pyridone analogues on the human epithelial
(HEp-2) cells was assessed by MTS cell viability and LDH cytotox-
icity assays, which represent the level of cellular metabolism and
proliferation (as markers of cell viability), and cellular integrity (as a
marker of cell lysis and death), respectively (Fig. 4). All pyridone-
based analogues resulted in a non-cytotoxic relative reduction in
MTS absorbance of ꢁ25% at 25 and 100
mM concentration (Fig. 4a).
Standardised international guidelines for testing cytotoxicity sug-
gest that cell viabilities <70% are indicative of cytotoxic potential;
none of the compounds tested affected cell viability to this degree
[49,50]. Compound 13f had a statistically significant effect on host
cell integrity at 100 mM concentration, however, this was a relative
decrease in cell integrity of <10% (Fig. 4b). These results altogether
demonstrated that pyridone analogues were non-cytotoxic to HEp-
2 cells and their anti-bacterial efficacy was specific to Chlamydia.
2.3.3. Capping group-modified peptidic inhibitors
All compounds from the capping group series demonstrated
improved anti-chlamydial activities relative to JO146, except 28e at
100 mM (Fig. 5). Compounds 28b and 28d containing valine at P1
displayed similar anti-chlamydial activities to their lead compound
2. Despite showing the highest anti-CtHtrA activity (Table 2) within
the capping group series, 28a was the least effective in reducing the
Fig. 5. In vitro testing of Chlamydia trachomatis and C. pecorum growth inhibition by
the lead compounds JO146, 2 and 3, and capping group-modified peptide analogues
28a-e at 25, 50 and 100 mM. Dashed line indicates the limit of detection for inclusion
forming units (IFU). Error bar represents the SEM (n ¼ 3). *p-value <0.05, ***p-value
<0.0001 compared to DMSO control as measured by two-way ANOVA.
bacterial titre at 25 mM with approximately 3.6-log increase in the
IFU/mL count of C. trachomatis compared to the lead compound 3
with a matching P1 valine. Complementing the enzymatic assay
(Table 2), 28e showed the weakest anti-chlamydial potency at
100 mM against both C. pecorum and C. trachomatis owing to a poor
dose-response correlation. This indicated that the Cbz capping
group and Ile at P1 are not complementary to each other in the
tripeptidic analogues. The compounds were all effective against
C. pecorum, reducing the infectious progeny close to the limit of
detection (104 IFU/mL) at the three doses. Modifications of the N-
capping group did not significantly affect the anti-chlamydial po-
tency in C. pecorum, similar to the pattern observed in the CtHtrA
assays (Fig. 5).
All compounds of the capping group series (28a-e) were non-
cytotoxic to HEp-2 cells at 25
mM but had a statistically signifi-
cant effect on cell metabolism when added at 100
m
M concentration
(Fig. 6a). It is noteworthy that compound 28e, which demonstrated
a poor inhibitory activity against CtHtrA and a lack of dose-
dependent efficacy in Chlamydial cell inhibition assays, reduced
relative MTS absorbance by approximately 22% even at 25
mM.
These biological results suggest that the Cbz capping group is not
favoured with a peptidyl inhibitor containing isoleucine at P1.
Nevertheless, cell integrity was not impacted as indicated by the
level of extracellular LDH release being ꢁ3.4% for all compounds at
both doses (Fig. 6b). The slightly contrasting results of the cyto-
toxicity profile of the capping group analogues at 100 mM between
the MTS and LDH assays can be explained by the difference in the
cellular processes or markers the assays measure. Given that MTS
assay measures the level of tetrazolium reduction to formazan in
actively metabolizing or proliferating cells, MTS reduction does not
directly translate to cell death and can even be reversible upon
elimination of the inhibitors. In contrast, the level of cytosolic LDH
leakage indicates the loss of cell integrity and cell lysis, which
Fig. 4. Viability and integrity of HEp-2 cells treated with pyridone-based analogues.
(top) The percentage of MTS reduction compared to cells treated with 1% DMSO.
(bottom) The percentage of extracellular LDH detected compared to maximum release
controls. Error bars represent the SEM (n ¼ 3). *p-value <0.05 compared to DMSO
controls as measured by two-way ANOVA.
8

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
Fig. 6. Viability and integrity of HEp-2 cells treated by lead compounds JO146, 2 and 3,
and capping group-modified peptide analogues 28a-e (analogues of the structure R-
Tle-Pro-P1P(OPh)2). Error bars represent the SEM (n ¼ 3). (top) The percentage of MTS
reduction compared to cells treated with 1% DMSO. (bottom) The percentage of
extracellular LDH detected compared to maximum release controls. *Significantly
different (p-value <0.05) from DMSO controls as measured by two-way ANOVA.
Fig. 7. Docking of 2-pyridone analogues in comparison to JO146 (green) at the active
site of CtHtrA protease. (top) 2-pyridone-based compounds (13a-f; those with alanine
at P2 were docked with L-configuration) and JO146 (green) were docked into the
active site of CtHtrA. Two H-bond constraints were used in the docking. (bottom) Side
view from the N-terminus of JO146 (green), 13b (cyan) and 13c (purple) docked with L-
configuration of alanine (P2) into the active site of CtHtrA. The outer wall of the S3
pocket (Val216-Ser219) is highlighted in magenta.
indicates irreversible cell death. Therefore, the results all together
point towards the fact that these compounds were not cytotoxic to
HEp-2 cells but affected aspects of cellular metabolism at 100
the point of time the measurements were taken.
mM at
eNH of pyridone and the C]O of Ile215 of the protease, preventing
the formation of a hydrogen bond that is important for binding at
the active site (Table S7). This result may explain the drastic
2.4. Molecular modelling
reduction of the anti-CtHtrA activity of 13f (128.5
14.21 mM),
indicating that the Boc N-capping group contributes to the sub-
optimal binding affinity to the active site. In contrast, the flexible
methylene spacer of the Cbz allows for the benzyl group to move
towards the S4 pocket, maintaining hydrophobic interactions with
Val216 at the S4 pocket (Fig. 7b). These results complement the IC50
assays (Table 1) that 13b and 13e with the Cbz capping group
demonstrating superior anti-CtHtrA activities over 13c and 13f
with the Boc group.
2.4.1. In silico docking studies of 2-pyridone-based inhibitors
The 2-pyridone-based analogues were covalently docked into
the homology model of CtHtrA [51] and compared to the lead
compound JO146. For the ligands synthesized as racemic mixtures
of alanine at the P2 position, the
L-enantiomer was docked and
analysed. Compounds with glycine and
L-alanine at P2 bound to the
active site of CtHtrA with high topological similarity to JO146,
indicating that the 2-pyridone scaffold was a successful isosteric
replacement of the traditional peptidic structures. In addition, the
methylene bridge of glycine at the P2 position of 13a and 13d was
perfectly aligned with the superimposed peptide backbone of
JO146 (Fig. 7a). These results were expected as previous X-ray
crystallography studies showed that 2-pyridone inhibitors of serine
proteases were capable of forming a stable, tetrahedral, hemiketal
adduct covalently linked to the catalytic serine, and inducing an
3. Conclusions
In comparison to peptide-based inhibitors that are generally
characterized with poor pharmacokinetic profiles due to their
inherent instability, peptidomimetics are considered advantageous
in reducing susceptibility to proteolytic degradation as well as
resistance development in bacteria [53]. Herein, 2-pyridone-based
peptidomimetics were designed to replace the P2/P3 peptidic
structure of the lead compound JO146, which inhibits HtrA serine
protease in Chlamydia. The lack of anti-bacterial activity of JO146
against E. coli, P. aeruginosa and S. aureus highlighted its merit of
being a narrow spectrum anti-bacterial, which would reduce se-
lection for anti-bacterial resistance and the detrimental effect upon
the host microbiome. Compound 18b from the pyridone series
improved inhibitory activity against CtHtrA by 5-fold and selec-
tivity over HNE by over 100-fold relative to JO146. Pyridone-based
inhibitors with L,D-alanine at P2 generally elicited lower anti-
CtHtrA activities than JO146, underlining the significance of L-
extended
b-strand conformation while retaining hydrogen bond
interactions present in the proteases’ natural substrates [25,42,52].
There was slight discrepancy in the position of the N-Boc
capping group between 2-pyridone ligands and JO146. Compared
to the peptidic analogues exemplified by JO146, which positioned
the Boc group deep into the S4 pocket, the N-Boc group of the
pyridone analogues was positioned towards the outer wall (Fig. 7b)
of the S4 pocket. This can be explained by the fact that the sp2-
hybridised C3 of the pyridone (in contrast to the sp3-hybridised Ca
in a peptidic backbone) positioned the eNH amide group in a
planar orientation, away from the S4 pocket (Fig. 7b). The altered
positioning of the N-Boc group increased the distance between the
9

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
configuration of alanine. Compounds 13a and 13d were designed
with replacement of alanine by glycine at P2 in an attempt to
reduce stereochemical complexity of the inhibitors. Although
General procedure for peptide coupling to a-aminoalkyl
phosphonate diphenyl esters (13a-f, 18a-b, 28a-e). To the solution
of a hydrobromide salt of 1-aminoalkyl phosphonate diphenyl ester
11 or 12 (1.5 equiv.) in anhydrous DMF was added DIPEA (2.5
equiv.), and the reaction mixture was stirred for 10 min until
complete dissolution was achieved. The acid precursor (1.0 equiv.)
in anhydrous DMF was then added dropwise, followed by the
addition of HBTU (1.2 equiv.). The reaction mixture was stirred at
room temperature for 24 h. When the reaction was complete, the
mixture was extracted with EtOAc (3 ꢃ 25 mL) and the combined
organic layers were washed with saturated aqueous NaHCO3
(3 ꢃ 25 mL), MilliQ water (3 ꢃ 25 mL) and then brine (3 ꢃ 25 mL).
The organic layer was dried over MgSO4(s), filtered, concentrated in
vacuo then purified by flash column chromatography (3:2
Hex:EtOAc).
glycine at P2 was less favourable than L-alanine, it was still
compatible with the pyridone structure with improvement of
selectivity over HNE relative to JO146. The pyridone analogues
generally exhibited greater anti-chlamydial activity on whole cells
especially at 25 and 50 mM, supporting 2-pyridone as a suitable and
biologically active template for targeting Chlamydia. The pyridone
inhibitors were non-cytotoxic to HEp-2 cells unlike the peptide-
based analogues, all of which significantly reduced the relative
MTS levels at 100 mM. Modifications of the capping group of the
parent peptidic structures suggested flexibility of the S4 pocket in
accommodating more diverse structures, providing avenues to
improve physicochemical properties of the lipophilic inhibitors in
future optimization. Overall, the N-Cbz-2-pyridone moiety was
found to be a potential bioisostere for the traditional ‘N-capped-P3-
P2’ peptide structure, which provides an alternative approach for
future drug optimization in developing anti-chlamydial drugs.
Phenyl N-[1-({[1-(diphenoxyphosphoryl)-2-methylpropyl]car-
bamoyl}methyl)-2-oxo-1,2-dihydropyridin-3-yl]carbamate (13a).
Compound 11 (0.24 g, 0.63 mmol), 10a (0.19 g, 0.63 mmol), DIPEA
(270 mL, 1.57 mmol), HBTU (0.29 g, 0.76 mmol) in DMF (2 mL) were
reacted and purified as described in the general procedure above
4. Experimental
using to yield a colourless oil 13a as a racemic mixture (0.30 g,
21
0.50 mmol, 79%, Rf 0.31, 1:1 Hex:EtOAc). [
a
]
¼ ꢂ2.38 (c ¼ 0.6,
589
4.1. Chemistry
CHCl3). 1H NMR (400 MHz, CDCl3)
d
8.08 (d, J ¼ 7.6 Hz, 1H), 7.80 (s,
1H), 7.57 (d, J ¼ 10.0 Hz, 1H), 7.38e7.05 (m, 15H), 6.98 (dd, J ¼ 6.8,
1.6 Hz, 1H), 6.28 (t, J ¼ 7.2 Hz, 1H), 5.17 (s, 2H), 4.71e4.63 (m, 2H),
4.53e4.49 (m, 1H), 2.45e2.35 (m, 1H), 1.08e1.01 (m, 6H). 13C NMR
4.1.1. General methods
All solvents and reagents were commercially procured (Sigma-
Aldrich, Merck, AK Scientific, Thermo Scientific, Acros Organics,
BDH, and Cambridge Isotope) and used without further purifica-
tion. Organic solvent extracts were dried with MgSO4(s) and sub-
jected to rotary evaporation, and finally dried at 10ꢂ1 mbar using a
high vacuum pump. Silica gel 60 (0.040e0.063 mm, 200e400
mesh) was used for flash column chromatography with all solvent
systems expressed as volume to volume (v/v) ratios. Solids were
recrystallized from a minimum amount of hot solvent and collected
by vacuum filtration. Analytical thin layer chromatography (TLC)
was performed on Merck TLC aluminium plates coated with
0.2 mm silica gel 60 F254. Spots were generally detected by UV
(254 nm) and/or permanganate staining. 1H, 13C and 31P NMR
spectra were recorded at room temperature on Varian 400 or
500 MHz spectrometers. Samples were prepared in deuterated
(101 MHz, CDCl3)
d
167.0 (d, JC-P ¼ 6.1 Hz), 157.3, 153.2, 150.1 (d, JC-
¼ 10.5 Hz), 149.9 (d, JC-P ¼ 9.5 Hz), 135.7, 129.8 (d, JC-P ¼ 0.9 Hz),
P
129.7 (d, JC-P ¼ 0.8 Hz), 129.50, 128.6, 128.4, 128.3, 125.4 (d, JC-
¼ 1.1 Hz),125.2 (d, JC-P ¼ 1.0 Hz),121.1,120.6 (d, JC-P ¼ 4.1 Hz),120.3
P
(d, JC-P ¼ 4.4 Hz), 107.7, 67.2, 54.0, 51.4 (d, JC-P ¼ 154.4 Hz), 50.6, 29.1
(d, JC-P ¼ 3.7 Hz), 20.4 (d, JC-P ¼ 13.4 Hz), 17.9 (d, JC-P ¼ 4.8 Hz). 31
P
NMR (162 MHz, CDCl3)
d 16.49. HRMS-ESI calculated for
C
31H32N3NaO7P [M þ Naþ] 612.1870, found m/z 612.1842. Analytical
RP-HPLC (Method I) tR ¼ 27.57 min.
Phenyl N-[1-(1-{[1-(diphenoxyphosphoryl)-2-methylpropyl]
carbamoyl}ethyl)-2-oxo-1,2-dihydropyridin-3-yl]carbamate
(13b). Compound 12 (0.27 g, 0.88 mmol), 10b (0.25 g, 0.80 mmol),
DIPEA (350
mL, 2.00 mmol), HBTU (0.36 g, 0.96 mmol) in DMF
(2 mL) were reacted and purified as described in the general pro-
cedure above to yield a colourless oil 13b in a diastereomeric
solvents, chloroform
49.00 ppm) or acetonitrile (
respective 1H and 13C chemical shifts shown in brackets. Chemical
shifts ( ) are expressed in parts per million (ppm) and coupling
(d
7.26, 77.16 ppm), methanol
(d 3.31,
d
1.95, 118.69 and 1.72 ppm) with the
mixture (0.30 g, 0.50 mmol, 62%, Rf 0.42, 1:1 Hex:EtOAc).
21
[
a
]
¼ ꢂ1.64 (c ¼ 0.6, CHCl3). 1H NMR (400 MHz, CDCl3)
d 8.07 (d,
589
d
J ¼ 7.2 Hz, 0.5H), 7.98 (d, J ¼ 7.2 Hz, 0.5H), 7.89 (s, 0.5H),7.81 (s,
0.5H), 7.70e7.62 (m, 1H), 7.41e7.04 (m, 15H), 6.99e6.96 (m, 0.5H),
6.94e6.91 (m, 0.5H), 6.34 (t, J ¼ 7.2 Hz, 0.5H), 6.22 (t, J ¼ 7.2 Hz,
0.5H), 5.85 (q, J ¼ 7.2 Hz, 0.5H), 5.56 (q, J ¼ 7.2 Hz, 0.5H), 5.20 (s,1H),
5.13 (s,1H), 4.69e4.60 (m,1H), 2.44e2.30 (m,1H),1.66 (d, J ¼ 7.2 Hz,
1.5H),1.54 (d, J ¼ 7.2 Hz,1.5H),1.18e1.09 (m, 4H), 0.94e0.91 (m,1H),
constants (J) in Hertz (Hz), both measured against the residual
solvent peak. High resolution mass spectrometry was recorded on a
Bruker microTOF-Q spectrometer with an electrospray ionization
(ESI) source. The purity of compounds was determined by reverse-
phase high performance liquid chromatography (HPLC) carried out
on an Agilent HPLC with a Gemini 5
m
m C18 110 column (250 Å
0.81e0.79 (m, 1H). 13C NMR (101 MHz, CDCl3)
d 169.6 (d, JC-
~4.6 mm, Phenomenex, New Zealand). Compounds were eluted
using solvent A: 0.1% trifluoroacetic acid (TFA) in water and solvent
B: 0.1% TFA in acetonitrile (ACN) over a linear gradient according to
the methods specified (supporting information). Compounds were
detected at 254/210 nm with a flow rate of 1.0 mL/min. Optical
rotation was recorded by using an Autopol IV polarimeter (Rudolph
Research Analytical, USA). Final compounds including those ob-
tained as isomeric mixtures, are >95% pure, see supporting infor-
mation for HPLC traces of the compounds.
¼ 6.1 Hz), 157.5, 157.4, 153.5,153.3,150.6 (d, JC-P ¼ 9.6 Hz), 150.5 (d,
P
JC-P ¼ 9.8 Hz), 150.2 (d, JC-P ¼ 9.6 Hz), 150.0 (d, JC-P ¼ 9.3 Hz), 149.9
(d, JC-P ¼ 10.2 Hz), 135.97, 135.92, 130.0 (d, JC-P ¼ 1.0 Hz), 129.90,
129.85 (d, JC-P ¼ 0.7 Hz), 129.8 (d, JC-P ¼ 1.1 Hz), 129.74 (d, JC-
¼ 0.9 Hz), 129.22, 129.16, 128.8, 128.7, 128.6, 128.5, 128.38, 128.35,
P
125.5 (d, JC-P ¼ 1.0 Hz), 125.42 (d, JC-P ¼ 1.0 Hz), 125.38 (d, JC-
¼ 0.8 Hz), 125.34 (d, JC-P ¼ 1.0 Hz), 125.30 (d, JC-P ¼ 0.7 Hz), 125.26
P
(d, JC-P ¼ 0.9 Hz), 125.2, 125.1, 120.8 (d, JC-P ¼ 4.1 Hz), 120.73 (d, JC-
¼ 4.2 Hz), 120.69 (d, JC-P ¼ 4.3 Hz), 120.6 (d, JC-P ¼ 5.2 Hz), 120.46
P
(d, JC-P ¼ 4.3 Hz), 120.45 (d, JC-P ¼ 4.4 Hz), 107.74, 107.72, 67.4, 67.2,
53.9, 53.7, 51.6 (d, JC-P ¼ 153.0 Hz), 51.5 (d, JC-P ¼ 155.7 Hz), 29.23 (d,
JC-P ¼ 3.7 Hz), 29.19 (d, JC-P ¼ 3.8 Hz), 20.7 (d, JC-P ¼ 12.9 Hz), 20.7 (d,
JC-P ¼ 12.9 Hz), 20.4 (d, JC-P ¼ 13.6 Hz), 18.4 (d, JC-P ¼ 5.3 Hz), 17.7 (d,
4.1.2. Synthesis and characterization of final compounds 13a-f,
18a-b, 28a-e
Synthetic procedures and characterization of precursor com-
pounds (8e10, 15e17, 20, 21, 23e27) are reported in the supporting
information.
JC-P ¼ 4.8 Hz), 15.7, 15.3. 31P NMR (162 MHz, CDCl3)
d 17.22 (13.5%),
16.29 (86.5%). HRMS-ESI calculated for C32H34N3NaO7P [M þ Naþ]
10

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
626.2027, found m/z 626.2002. Analytical RP-HPLC (Method IV)
tR ¼ 34.89, 36.34 min.
7.46e6.79 (m, 17H), 6.33 (t, J ¼ 7.2 Hz, 0.5H), 6.24e6.17 (m, 0.5H),
5.82e5.79 (m, 0.5H), 5.59e5.51 (m, 0.5H), 5.23e5.14 (m, 2H),
4.85e4.76 (m, 0.5H), 4.71e4.61 (m, 0.5H), 2.20e2.14 (m, 0.5H),
1.86e1.78 (m, 0.5H), 1.65e1.63 (m, 1.5H), 1.55e1.52 (m, 1.5H),
1.38e1.25 (m, 2H), 1.15e1.05 (m, 2H), 0.99e0.91 (m, 2H), 0.87e0.81
Diphenyl {1-[2-(3-{[(tert-butoxy)(hydroxy)methyl]amino}-2-
oxo-1,2-dihydropyridin-1-yl)propanamido]-2-methylpropyl}
phosphonate (13c). Compound 11 (0.12 g, 0.30 mmol),10c (84.0 mg,
0.30 mmol), DIPEA (1.30 mL, 0.75 mmol), HBTU (0.14 g, 0.36 mmol)
in DMF (2 mL) were reacted and purified as described in the general
procedure above to yield a colourless oil 13c in a diastereomeric
(m, 1H), 0.79e0.74 (m, 1H). 13C NMR (101 MHz, CDCl3)
d 169.5 (d, JC-
¼ 6.5 Hz), 169.4 (d, JC-P ¼ 6.5 Hz), 157.48, 157.45, 153.5, 153.2,
P
150.64 (d, JC-P ¼ 9.7 Hz), 150.56 (d, JC-P ¼ 9.0 Hz), 150.2 (d, JC-
mixture (0.12 g, 0.21 mmol, 71%, Rf 0.30, 3:2 in a diastereomeric
¼ 9.7 Hz), 150.0 (d, JC-P ¼ 9.1 Hz), 149.9 (d, JC-P ¼ 10.3 Hz), 135.94,
P
mixture, Hex:EtOAc). [
(400 MHz, CDCl3)
a
]
589
¼ ꢂ2.96 (c ¼ 0.5, CHCl3). 1H NMR
135.92, 129.94 (d, JC-P ¼ 1.1 Hz), 129.93 (d, JC-P ¼ 1.1 Hz), 129.88 (d,
JC-P ¼ 0.7 Hz), 129.8 (d, JC-P ¼ 0.9 Hz), 129.7 (d, JC-P ¼ 0.9 Hz), 129.23,
129.21, 129.18, 128.77, 128.75, 128.56, 128.55, 128.53, 128.38, 128.3,
125.53 (d, JC-P ¼ 1.2 Hz), 120.50 (d, JC-P ¼ 1.2 Hz), 125.41 (d, JC-
21
d
8.02 (d, J ¼ 7.6 Hz, 0.5H), 7.93 (d, J ¼ 7.6 Hz,
0.5H), 7.64 (s, 0.5H), 7.57 (s, 0.5H), 7.35e7.00 (m, 11H), 6.88e6.85
(m, 1H), 6.32 (t, J ¼ 7.6 Hz, 0.5H), 6.18 (t, J ¼ 7.2 Hz, 0.5H), 5.78 (q,
J ¼ 7.2 Hz, 0.5H), 5.55 (q, J ¼ 7.2 Hz, 0.5H), 4.69e4.60 (m, 1H),
2.46e2.29 (m, 1H), 1.64 (d, J ¼ 7.2 Hz, 1.5H), 1.55 (d, J ¼ 7.2 Hz, 1.5H),
1.51e1.48 (m, 9H),1.13e1.10 (m, 3H), 0.93e0.91 (m,1.5H), 0.81e0.79
¼ 1.0 Hz), 125.35 (d, JC-P ¼ 1.0 Hz), 125.3 (d, JC-P ¼ 1.5 Hz), 125.1,
P
125.04, 125.02, 120.8 (d, JC-P ¼ 4.3 Hz), 120.73 (d, JC-P ¼ 3.6 Hz),
120.72 (d, JC-P ¼ 4.0 Hz), 120.55 (d, JC-P ¼ 4.0 Hz), 120.47 (d, JC-
(m, 1.5H). 13C NMR (101 MHz, CDCl3)
d
169.6 (d, JC-P ¼ 6.6 Hz), 169.5
¼ 4.3 Hz), 120.46 (d, JC-P ¼ 4.2 Hz), 120.4, 107.8, 107.73, 107.72,
P
(d, JC-P ¼ 5.0 Hz), 157.63, 157.61, 152.9,152.7, 150.6 (d, JC-P ¼ 10.0 Hz),
107.69, 67.4, 67.2, 53.84, 53.76, 53.6, 53.5, 51.4 (d, JC-P ¼ 154.6 Hz),
49.5 (d, JC-P ¼ 152.6 Hz), 49.4 (d, JC-P ¼ 155.9 Hz), 36.00 (d, JC-
150.2 (d, JC-P ¼ 9.3 Hz), 150.0 (d, JC-P ¼ 8.9 Hz), 149.9 (d, JC-
¼ 10.6 Hz), 130.0 (d, JC-P ¼ 1.0 Hz), 129.9 (d, JC-P ¼ 0.8 Hz), 129.8 (d,
¼ 4.1 Hz), 35.95 (d, JC-P ¼ 3.9 Hz), 35.5 (d, JC-P ¼ 3.9 Hz), 35.4 (d, JC-
P
P
JC-P ¼ 1.0 Hz), 129.7 (d, JC-P ¼ 1.0 Hz), 125.5 (d, JC-P ¼ 1.2 Hz), 125.38
(d, JC-P ¼ 1.2 Hz), 125.36 (d, JC-P ¼ 1.1 Hz), 124.4, 124.3, 120.8 (d, JC-
¼ 3.8 Hz), 27.5 (d, JC-P ¼ 14.9 Hz), 27.2 (d, JC-P ¼ 14.9 Hz), 25.2 (d, JC-
P
¼ 5.4 Hz), 24.7(d, JC-P ¼ 5.2 Hz), 16.6 (d, JC-P ¼ 10.7 Hz), 16.4 (d, JC-
P
¼ 4.0 Hz), 120.7 (d, JC-P ¼ 4.1 Hz), 120.6 (d, JC-P ¼ 4.5 Hz), 120.5 (d,
¼ 11.3 Hz), 15.5 (d, JC-P ¼ 4.4 Hz), 15.4 (d, JC-P ¼ 3.4 Hz), 15.3, 15.2,
P
P
JC-P ¼ 4.5 Hz), 120.1, 119.9, 107.9, 81.2, 81.0, 53.8, 53.4, 51.5 (d, JC-
15.0 (d, JC-P ¼ 3.2 Hz), 11.8 (d, JC-P ¼ 1.2 Hz), 11.6 (d, JC-P ¼ 1.2 Hz),
¼ 152.5 Hz), 51.4 (d, JC-P ¼ 155.1 Hz), 29.3 (d, JC-P ¼ 3.5 Hz), 29.2 (d,
11.5, 11.4. 31P NMR (162 MHz, CDCl3)
d 17.61 (7.2%), 17.12 (5.8%),
P
JC-P ¼ 3.8 Hz), 28.40, 28.39, 20.7 (d, JC-P ¼ 12.8 Hz), 20.4 (d, JC-
16.77 (49.8%), 16.35 (37.1%). HRMS-ESI calculated for
¼ 13.4 Hz), 18.3 (d, JC-P ¼ 5.2 Hz), 17.7 (d, JC-P ¼ 4.4 Hz), 15.3, 15.02.
C
33H36N3NaO7P [M þ Naþ] 640.2183, found m/z 640.2135. Analyt-
P
31P NMR (162 MHz, CDCl3)
d
16.86 (35.6%), 16.30 (64.4%). HRMS-ESI
ical RP-HPLC (Method IV) tR ¼ 39.11, 39.97, 40.68 min.
calculated for C29H36N3NaO7P [M þ Naþ] 592.2183, found m/z
592.2187. Analytical RP-HPLC (Method II) tR ¼ 34.65, 35.90 min.
Phenyl N-[1-({[1-(diphenoxyphosphoryl)-2-methylbutyl]car-
bamoyl}methyl)-2-oxo-1,2-dihydropyridin-3-yl]carbamate (13d).
Compound 12 (73.4 mg, 0.23 mmol), 10a (69.4 mg, 0.23 mmol),
DIPEA (0.10 mL, 0.58 mmol), HBTU (0.10 g, 0.28 mmol) in DMF
(2 mL) were reacted and purified as described in the general pro-
cedure above to yield a colourless oil 13d in a diastereomeric
Diphenyl {1-[2-(3-{[(tert-butoxy)(hydroxy)methyl]amino}-2-
oxo-1,2-dihydropyridin-1-yl)propanamido]-2-methylbutyl}phos-
phonate (13f). Compound 12 (74.4 mg, 0.23 mmol), 10c (65.8 mg,
0.23 mmol), DIPEA (150 mL, 0.58 mmol), HBTU (0.11 g, 0.28 mmol) in
DMF (2 mL) were reacted and purified as described in the general
procedure above to yield a colourless oil 13f in a diastereomeric
mixture (58.0 mg, 0.21 mmol, 43%, Rf 0.33, 3:2 Hex:EtOAc).
21
[a]
¼ ꢂ1.53 (c ¼ 1.0, CHCl3). 1H NMR (400 MHz, CDCl3)
d 8.02 (d,
589
mixture (68 mg, 0.11 mmol, 48%, Rf 0.34, 3:2 Hex:EtOAc).
J ¼ 7.6 Hz, 0.5H), 7.94 (br s, 0.5H), 7.64 (s, 0.5H), 7.57 (s 0.5H),
7.34e6.98 (m, 11H), 6.91e6.84 (m, 1H), 6.34e6.30 (m, 0.5H),
6.21e6.14 (m, 0.5H), 5.79e5.74 (m, 0.5H), 5.58e5.50 (m, 0.5H),
4.85e4.75 (m, 0.5H), 4.71e4.61 (m, 0.5H), 2.21e2.04 (m, 0.5H),
1.85e1.79 (m, 0.5H), 1.68e1.62 (m, 1.5H), 1.55e1.53 (m, 1.5H),
1.51e1.47 (m, 9H), 1.38e1.26 (m, 1H), 1.14e0.92 (m, 4.5H), 0.89e0.84
21
[
a
]
¼ ꢂ1.75 (c ¼ 0.6, CHCl3). 1H NMR (400 MHz, CDCl3)
d 8.07 (br
589
s,1H), 7.81 (s,1H), 7.47e7.03 (m,16H), 6.97e6.93 (m,1H), 6.29e6.24
(m, 1H), 5.17 (s, 2H), 4.86e4.78 (m, 0.5H), 4.73e4.63 (m, 0.5H),
4.60e4.47 (m, 2H), 2.18e2.09 (m, 0.5H), 1.81e1.75 (m, 0.5H),
1.43e1.32 (m, 1H), 1.27e1.17 (m, 1H), 1.08e1.03 (m, 3H), 0.91e0.86
(m, 3H). 13C NMR (101 MHz, CDCl3)
d
167.0 (d, JC-P ¼ 5.9 Hz), 166.9
(m,1H), 0.80e0.76 (m,1.5H). 13C NMR (101 MHz, CDCl3)
d 169.5 (d, JC-
(d, JC-P ¼ 6.2 Hz), 157.4, 153.2, 150.19 (d, JC-P ¼ 10.2 Hz), 150.17 (d, JC-
¼ 6.6 Hz), 169.4 (d, JC-P ¼ 6.5 Hz), 157.63, 157.60, 152.9, 152.7, 150.7
P
¼ 10.2 Hz), 150.98 (d, JC-P ¼ 9.3 Hz), 149.97 (d, JC-P ¼ 9.4 Hz),
(d, JC-P ¼ 9.8 Hz), 150.6 (d, JC-P ¼ 9.8 Hz), 150.2 (d, JC-P ¼ 9.4 Hz), 150.1
(d, JC-P ¼ 9.0 Hz), 149.9 (d, JC-P ¼ 10.8 Hz), 130.0 (d, JC-P ¼ 1.1 Hz),
129.94 (d, JC-P ¼ 1.1 Hz), 129.89 (d, JC-P ¼ 0.8 Hz), 129.76 (d, JC-
P
135.73, 135.71, 129.73 (d, JC-P ¼ 1.0 Hz), 129.70 (d, JC-P ¼ 1.0 Hz),
129.65 (d, JC-P ¼ 0.8 Hz), 129.53, 129.51, 129.4, 129.3, 128.6, 128.4,
128.3, 125.4 (d, JC-P ¼ 1.3 Hz), 125.3 (d, JC-P ¼ 1.1 Hz), 125.2 (d, JC-
¼ 1.6 Hz), 129.75 (d, JC-P ¼ 1.7 Hz), 129.72 (d, JC-P ¼ 1.0 Hz), 125.54
P
¼ 0.8 Hz), 120.9, 120.60 (d, JC-P ¼ 4.2 Hz), 120.56 (d, JC-P ¼ 4.2 Hz),
(d, JC-P ¼ 1.3 Hz), 125.50 (d, JC-P ¼ 1.2 Hz), 125.38, 125.35, 125.33,
124.42, 124.36, 124.3,120.8 (d, JC-P ¼ 4.0 Hz), 120.74 (d, JC-P ¼ 4.4 Hz),
120.72 (d, JC-P ¼ 4.0 Hz), 120.6 (d, JC-P ¼ 4.6 Hz), 120.50 (d, JC-
P
120.33 (d, JC-P ¼ 4.3 Hz), 120.31 (d, JC-P ¼ 4.4 Hz), 107.6, 67.2, 54.1,
51.3 (d, JC-P ¼ 154.2 Hz), 49.4 (d, JC-P ¼ 154.4 Hz), 35.9 (d, JC-
¼ 3.5 Hz), 35.4 (d, JC-P ¼ 3.5 Hz), 27.2 (d, JC-P ¼ 14.9 Hz), 24.8 (d, JC-
¼ 4.4 Hz), 120.46 (d, JC-P ¼ 4.5 Hz), 120.1, 119.9, 107.9, 81.2, 81.0,
P
P
¼ 5.5 Hz),16.4 (d, JC-P ¼ 11.8 Hz),15.1 (d, JC-P ¼ 3.2 Hz),11.6,11.4. 31
P
53.82, 53.76, 53.41, 53.35, 51.4 (d, JC-P ¼ 153.0 Hz), 49.5 (d, JC-
P
NMR (162 MHz, CDCl3)
d
16.99 (59.3%), 16.55 (40.7%). HRMS-ESI
¼ 153.0 Hz), 49.3 (d, JC-P ¼ 155.7 Hz), 36.0 (d, JC-P ¼ 4.2 Hz), 36.0 (d,
P
calculated for C32H34N3NaO7P [M þ Naþ] 626.2027, found m/z
626.1982. Analytical RP-HPLC (Method IV) tR ¼ 34.91, 35.60 min.
JC-P ¼ 4.2 Hz), 35.5 (d, JC-P ¼ 3.5 Hz), 35.4 (d, JC-P ¼ 3.8 Hz), 28.4, 27.5
(d, JC-P ¼ 14.9 Hz), 27.2 (d, JC-P ¼ 15.0 Hz), 25.2 (d, JC-P ¼ 5.7 Hz), 24.7
(d, JC-P ¼ 5.4 Hz),16.6 (d, JC-P ¼ 6.6 Hz),16.4 (d, JC-P ¼ 10.6 Hz), 16.4 (d,
JC-P ¼ 11.3 Hz),15.34 (d, JC-P ¼ 5.5 Hz),15.4 (d, JC-P ¼ 3.6 Hz),15.3 (d, JC-
Phenyl
N-[1-(1-{[1-(diphenoxyphosphoryl)-2-methylbutyl]
carbamoyl}ethyl)-2-oxo-1,2-dihydropyridin-3-yl]carbamate
(13e). Compound 12 (0.17 g, 0.53 mmol), 10b (0.15 g, 0.48 mmol),
DIPEA (0.20 mL, 1.21 mmol), HBTU (0.22 g, 0.58 mmol) in DMF
(2 mL) were reacted and purified as described in the general pro-
cedure above to yield a colourless oil 13e in a diastereomeric
¼ 5.6 Hz), 15.14, 15.13, 15.0 (d, JC-P ¼ 3.2 Hz), 11.8 (d, JC-P ¼ 1.2 Hz),
P
11.7 (d, JC-P ¼ 1.0 Hz), 11.5, 11.4. 31P NMR (162 MHz, CDCl3)
d 17.44
(14.5%), 16.93 (12.3%), 16.82 (40.2%), 16.37 (33.0%). HRMS-ESI calcu-
lated for C30H38N3NaO7P [M þ Naþ] 606.2340, found m/z 606.2359.
Analytical RP-HPLC (Method IV) tR ¼ 39.18, 40.00, 40.67 min.
mixture (0.17 g, 0.29 mmol, 59%, Rf 0.31, 3:2 Hex:EtOAc).
21
[
a
]
¼ ꢂ1.57 (c ¼ 0.6, CHCl3). 1H NMR (400 MHz, CDCl3)
d
8.07 (d,
Benzyl
N-{1-[(1S)-{[1-(diphenoxyphosphoryl)-2-
589
J ¼ 7.2 Hz, 0.5H), 7.97 (br s, 0.5H), 7.89 (s, 0.5H), 7.80e7.79 (m, 0.5H),
methylpropyl]carbamoyl}ethyl]-2-oxo-1,2-dihydropyridin-3-yl}
11

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
carbamate (18a). Compound 11 (0.15 g, 0.38 mmol), 17 (0.10 g,
0.32 mmol), DIPEA (140 mL, 0.79 mmol), HBTU (0.14 g, 0.38 mmol)
in DMF (4 mL) were reacted and purified as described in the general
phosphonate (28a). Compound 12 (0.12 g, 0.38 mmol), 27c (78 mg,
0.25 mmol), DIPEA (0.10 mL, 0.63 mmol), HBTU (0.11 g, 0.30 mmol)
in DMF (2 mL) were reacted and purified as described in the general
procedure above to yield a colourless oil 18a (47 mg, 0.078 mmol,
procedure above to yield a colourless oil 28a (0.13 mg, 0.21 mmol,
21
21
25%, Rf ¼ 0.23 in 7:3 PE:EtOAc). [
a
]
¼ ꢂ1.10 (c ¼ 0.6, CHCl3). 1H
82%, Rf 0.33, 0.21 in 1:1 Hex:EtOAc). [
a
]
d
¼ ꢂ73.40 (c ¼ 0.5,
589
589
NMR (400 MHz, CDCl3)
d
7.98 (d, J ¼ 7.2 Hz, 0.5H), 7.88 (d, J ¼ 7.6 Hz,
CH3OH). 1H NMR (400 MHz, CD3OD)
7.40e7.09 (m, 10H),
0.5H), 7.81 (s, 0.5H), 7.71 (s, 0.5H), 7.40e6.76 (m, 17H), 6.24 (t,
J ¼ 7.2 Hz, 0.5H), 6.11 (t, J ¼ 7.2 Hz, 0.5H), 5.72 (q, J ¼ 7.2 Hz, 0.5H),
5.47 (q, J ¼ 7.2 Hz, 0.5H), 5.10 (s, 1H), 5.04 (s, 1H), 4.67e4.49 (m, 1H),
2.35e2.19 (m, 1H), 1.55 (d, J ¼ 7.2 Hz, 1.5H), 1.44 (d, J ¼ 7.2 Hz, 1.5H),
6.78e6.75 (m, 1H), 4.96e4.88 (m, 1H), 4.72e4.63 (m, 2.5H),
4.54e4.48 (m, 0.5H), 3.94e3.84 (m, 1H), 3.78e3.68 (m, 1H),
2.31e2.25 (m, 0.5H), 2.17e2.05 (m, 2H), 2.03e1.92 (m, 1H),
1.90e1.73 (m, 1.5H), 1.49e1.41 (m, 1H), 1.38e1.28 (m, 1H), 1.28e1.17
(m, 11H), 1.12e1.09 (m, 1H), 1.03e0.98 (m, 9H), 0.96e0.92 (m, 3H).
1.04e1.00 (m, 3H), 0.82 (d, J ¼ 6.8 Hz, 1.5H), 0.72e0.69 (m, 1.5H). 13
C
NMR (101 MHz, CDCl3)
153.3, 150.6 (d, JC-P ¼ 9.8 Hz), 150.2 (d, JC-P ¼ 9.2 Hz), 150.0 (d, JC-
d
169.6 (d, JC-P ¼ 6.6 Hz), 157.5, 157.4, 153.5,
13C NMR (101 MHz, CD3CN)
d
178.31, 178.30, 173.1 (d, JC-P ¼ 5.5 Hz),
172.88 (d, JC-P ¼ 5.7 Hz),172.85 (d, JC-P ¼ 5.9 Hz),171.83,171.80,151.4
(d, JC-P ¼ 10.1 Hz), 151.22 (d, JC-P ¼ 8.4 Hz), 151.21 (d, JC-P ¼ 8.9 Hz),
151.17 (d, JC-P ¼ 10.3 Hz), 151.15 (d, JC-P ¼ 10.4 Hz), 130.9 (d, JC-
¼ 9.3 Hz), 149.9 (d, JC-P ¼ 10.5 Hz), 136.0, 135.9, 129.94 (d, JC-
P
¼ 0.9 Hz), 129.89 (d, JC-P ¼ 0.7 Hz), 129.8 (d, JC-P ¼ 1.0 Hz), 129.7 (d,
P
JC-P ¼ 0.9 Hz), 129.22, 129.18, 128.81, 128.77, 128.74, 128.64, 128.61,
128.56, 128.52, 128.38, 128.37, 125.5 (d, JC-P ¼ 1.1 Hz), 125.40 (d, JC-
¼ 1.2 Hz),130.83 (d, JC-P ¼ 1.2 Hz),130.75 (d, JC-P ¼ 1.1 Hz),130.7 (d,
P
JC-P ¼ 1.0 Hz), 126.38 (d, JC-P ¼ 1.4 Hz), 126.36 (d, JC-P ¼ 1.5 Hz),
126.33 (d, JC-P ¼ 1.1 Hz), 126.28 (d, JC-P ¼ 1.4 Hz), 122.1 (d, JC-
¼ 1.3 Hz), 125.38 (d, JC-P ¼ 0.9 Hz), 125.3 (d, JC-P ¼ 1.2 Hz), 125.1,
P
120.8 (d, JC-P ¼ 4.3 Hz), 120.7 (d, JC-P ¼ 4.2 Hz), 120.58, 120.6, 120.46
(d, JC-P ¼ 4.2 Hz),120.45 (d, JC-P ¼ 4.4 Hz), 107.7, 67.4, 67.2, 53.8, 53.6,
51.6 (d, JC-P ¼ 152.7 Hz), 51.5 (d, JC-P ¼ 154.9 Hz), 29.23 (d, JC-
¼ 3.9 Hz),121.8 (d, JC-P ¼ 4.2 Hz),121.7 (d, JC-P ¼ 4.0 Hz),121.5 (d, JC-
P
¼ 4.4 Hz), 61.21, 61.18, 60.91, 60.87, 57.4, 51.7 (d, JC-P ¼ 153.4 Hz),
P
51.2 (d, JC-P ¼ 153.2 Hz), 49.4, 48.9 (d, JC-P ¼ 154.1 Hz), 39.4, 37.1 (d,
JC-P ¼ 4.6 Hz), 36.9 (d, JC-P ¼ 4.4 Hz), 36.7 (d, JC-P ¼ 4.4 Hz), 36.4 (d,
JC-P ¼ 4.0 Hz), 30.2, 30.1, 29.5, 29.4, 28.0 (d, JC-P ¼ 15.0 Hz), 27.77,
27.76, 27.6 (d, JC-P ¼ 15.3 Hz), 26.8, 26.7, 25.94, 25.87, 25.73, 25.66,
16.9 (d, JC-P ¼ 11.5 Hz), 16.7 (d, JC-P ¼ 10.9 Hz), 16.0 (d, JC-P ¼ 3.4 Hz),
15.8 (d, JC-P ¼ 2.8 Hz), 12.03, 12.02, 11.7, 11.6. 31P NMR (162 MHz,
¼ 3.6 Hz), 29.17 (d, JC-P ¼ 3.7 Hz), 20.7 (d, JC-P ¼ 11.9 Hz), 20.4 (d, JC-
P
¼ 12.3 Hz), 18.4 (d, JC-P ¼ 5.2 Hz), 17.7 (d, JC-P ¼ 4.9 Hz), 15.6, 15.3.
P
31P NMR (162 MHz, CDCl3)
d
17.06 (17.4%), 16.28 (82.6%). HRMS-ESI
calculated for
604.2189. Analytical RP-HPLC (Method IV) tR ¼ 34.70, 36.14 min.
Benzyl N-{1-[(1S)-1-{[1-(diphenoxyphosphoryl)-2-
C
32H35N3O7P [M
þ
Hþ] 604.2207, found m/z
CD3OD)
d 18.76 (26.3%), 18.32 (10.3%), 18.24 (32.2%), 17.99 (31.2%).
methylbutyl]carbamoyl}ethyl]-2-oxo-1,2-dihydropyridin-3-yl}
carbamate (18b). Compound 12 (39.6 mg, 0.12 mmol), 17 (32.6 mg,
0.10 mmol), DIPEA (45 mL, 0.26 mmol), HBTU (47.0 mg, 0.12 mmol)
HRMS-ESI calculated for C33H48N3NaO6P [M þ Naþ] 636.3173,
found m/z 636.3159. Analytical RP-HPLC (Method III) tR ¼ 40.17,
40.95, 41.22, 43.03 min.
in DMF (2 mL) were reacted and purified as described in the general
procedure above to yield a colourless oil 18b (27 mg, 0.044 mmol,
Diphenyl (1-{[(2S)-1-[(2S)-2-[(tert-butylcarbamoyl)amino]-
3,3-dimethylbutanoyl]pyrrolidin-2-yl]formamido}-2-
methylpropyl)phosphonate (28b). Compound 11 (46.1 mg,
0.15 mmol), 27b (49.3 mg, 0.15 mmol), DIPEA (65 mL, 0.38 mmol),
HBTU (68.6 mg, 0.18 mmol) in DMF (2 mL) were reacted and pu-
21
42%, Rf ¼ 0.26 in 7:3 PE:EtOAc). [
a
]
¼ ꢂ0.20 (c ¼ 0.6, CHCl3). 1H
589
NMR (400 MHz, CDCl3)
d
8.07 (d, J ¼ 7.2 Hz, 0.5H), 7.97 (br s, 0.5H),
7.89 (s, 0.5H), 7.81e7.79 (m, 0.5H), 7.41e6.87 (m, 17H), 6.33 (t,
J ¼ 7.2 Hz, 0.5H), 6.24e6.17 (m, 0.5H), 5.82e5.79 (m, 0.5H),
5.59e5.51 (m, 0.5H), 5.23e5.14 (m, 2H), 4.85e4.76 (m, 0.5H),
4.71e4.61 (m, 0.5H), 2.20e2.04 (m, 0.5H), 1.88e1.78 (m, 0.5H),
1.68e1.60 (m, 1.5H), 1.54e1.50 (m, 1.5H), 1.39e1.25 (m, 2H),
1.15e1.05 (m, 2H), 0.97e0.91 (m, 2H), 0.88e0.82 (m, 1H), 0.80e0.75
rified as described in the general procedure above to yield a col-
ourless oil 28b (53.3 mg, 0.087 mmol, 57%, Rf 0.21 in 1:1
21
Hex:EtOAc). [
a
]
¼ ꢂ57.60 (c ¼ 0.5, CHCl3). 1H NMR (400 MHz,
589
CDCl3)
d
8.00 (s, 0.5H), 7.89 (d, J ¼ 10.4, 0.5H), 7.35 (d, J ¼ 10.4, 0.5H),
7.30e7.07 (m, 10H), 6.86e6.82 (m, 0.5H), 5.63 (bd, J ¼ 9.6 Hz, 0.5H),
5.14 (d, J ¼ 9.6 Hz, 0.5H), 4.83e4.67 (m, 2H), 4.58 (d, J ¼ 9.6 Hz,
0.5H), 4.49 (d, J ¼ 9.2 Hz, 0.5H), 3.93e3.84 (m, 1H), 3.74e3.69 (m,
0.5H), 3.62e3.57 (m, 0.5H), 2.45e2.38 (m, 0.5H), 2.36e2.30 (m,
0.5H), 2.21 (br, 0.5H), 2.10e2.03 (m, 0.5H), 1.95e1.78 (m, 3H),
1.29e1.24 (m, 9H), 1.11e1.04 (m, 6H), 0.97e0.93 (m, 9H). 13C NMR
(m, 1H). 13C NMR (101 MHz, CDCl3)
d
169.5 (d, JC-P ¼ 6.3 Hz), 169.4
(d, JC-P ¼ 6.3 Hz),157.47,157.43,153.5,153.2,150.63 (d, JC-P ¼ 9.9 Hz),
150.57 (d, JC-P ¼ 9.8 Hz), 150.2 (d, JC-P ¼ 9.7 Hz), 150.0 (d, JC-
¼ 9.0 Hz), 149.9 (d, JC-P ¼ 10.6 Hz), 135.94, 135.91, 129.94 (d, JC-
P
¼ 1.1 Hz), 129.93 (d, JC-P ¼ 1.1 Hz), 129.88 (d, JC-P ¼ 0.7 Hz), 129.77
P
(d, JC-P ¼ 1.0 Hz), 129.73 (d, JC-P ¼ 1.0 Hz), 129.70 (d, JC-P ¼ 0.8 Hz),
129.22, 129.21, 129.19, 129.16, 128.8, 128.7, 128.6, 128.54, 128.52,
128.4, 128.3, 125.53 (d, JC-P ¼ 1.2 Hz), 120.50 (d, JC-P ¼ 1.2 Hz), 125.41
(d, JC-P ¼ 1.0 Hz), 125.35 (d, JC-P ¼ 1.0 Hz), 125.3 (d, JC-P ¼ 1.6 Hz),
125.14, 125.08, 125.04, 120.8 (d, JC-P ¼ 4.2 Hz), 120.72 (d, JC-
(101 MHz, CDCl3)
d
173.8, 172.9 (d, JC-P ¼ 8.1 Hz), 172.8, 172.1 (d, JC-
¼ 6.1 Hz), 157.4, 157.1, 150.5 (d, JC-P ¼ 10.1 Hz), 150.4 (d, JC-
P
¼ 10.1 Hz), 150.2 (d, JC-P ¼ 10.1 Hz), 150.1 (d, JC-P ¼ 10.1 Hz), 129.91,
P
129.89 (d, JC-P ¼ 0.8 Hz), 129.84 (d, JC-P ¼ 0.7 Hz), 125.48 (d, JC-
¼ 1.1 Hz), 125.46 (d, JC-P ¼ 0.9 Hz), 125.34 (d, JC-P ¼ 0.7 Hz), 125.29
P
¼ 3.9 Hz), 120.70 (d, JC-P ¼ 4.0 Hz), 120.57, 120.55, 120.48 (d, JC-
(d, JC-P ¼ 0.8 Hz), 120.9 (d, JC-P ¼ 4.0 Hz), 120.8 (d, JC-P ¼ 4.1 Hz),
120.55 (d, JC-P ¼ 4.1 Hz), 120.48 (d, JC-P ¼ 4.4 Hz), 60.5, 59.8, 57.5,
57.3, 51.3 (d, JC-P ¼ 155.6 Hz) 51.1 (d, JC-P ¼ 153.6 Hz), 50.4, 50.2, 48.9,
48.8, 36.1, 35.4, 29.73, 29.67, 29.6, 29.44 (d, J CeP ¼ 4.6 Hz), 29.40 (d,
P
¼ 4.3 Hz), 120.46 (d, JC-P ¼ 4.4 Hz), 120.45 (d, JC-P ¼ 4.4 Hz), 120.4,
P
107.71, 107.68, 67.4, 67.2, 53.83, 53.76, 53.6, 53.5, 51.4 (d, JC-
¼ 154.0 Hz), 49.5 (d, JC-P ¼ 153.1 Hz), 49.4 (d, JC-P ¼ 156.6 Hz), 36.0
P
(d, JC-P ¼ 4.2 Hz), 35.9 (d, JC-P ¼ 3.8 Hz), 35.5 (d, JC-P ¼ 3.6 Hz), 35.4
(d, JC-P ¼ 3.8 Hz), 27.4 (d, JC-P ¼ 14.5 Hz), 27.2 (d, JC-P ¼ 14.9 Hz), 25.2
(d, JC-P ¼ 5.9 Hz), 24.7 (d, JC-P ¼ 5.3 Hz), 16.6 (d, JC-P ¼ 10.3 Hz), 16.4
(d, JC-P ¼ 11.7 Hz), 15.6 (d, JC-P ¼ 5.8 Hz), 15.4 (d, JC-P ¼ 3.1 Hz), 15.34,
J
CeP ¼ 4.3 Hz), 29.0, 28.3, 26.6, 26.5, 25.4, 25.2, 20.7 (d, JC-
¼ 13.1 Hz), 20.4 (d, J CeP ¼ 13.2 Hz), 18.4 (d, J CeP ¼ 5.9 Hz), 18.2 (d,
P
J
CeP ¼ 4.9 Hz). 31P NMR (162 MHz, CDCl3)
d
17.90 (52.7%), 17.04
Naþ]
(47.3%). HRMS-ESI calculated for
C
32H47N4NaO6P [M
þ
15.25, 15.0 (d, JC-P ¼ 1.1 Hz), 11.8 (d, JC-P ¼ 1.0 Hz), 11.6, 11.5, 11.4. 31
P
637.3125, found m/z 637.3121. Analytical RP-HPLC (Method I)
tR ¼ 27.22, 27.36 min.
NMR (162 MHz, CDCl3)
d 17.60 (9.3%), 17.16 (7.3%), 16.78 (52.9%),
16.37 (30.5%). HRMS-ESI calculated for C33H36N3NaO7P [M þ Naþ]
640.2183, found m/z 640.2164. Analytical RP-HPLC (Method IV)
tR ¼ 38.92, 39.76, 40.44, min.
Diphenyl (1-{[(2S)-1-[(2S)-2-[(tert-butylcarbamoyl)amino]-
3,3-dimethylbutanoyl]pyrrolidin-2-yl]formamido}-2-
methylbutyl)phosphonate (28c). Compound 12 (49.4 mg,
Diphenyl (1-{[(2S)-1-[(2S)-2-(2,2-dimethylpropanamido)-3,3-
dimethylbutanoyl]pyrrolidin-2-yl]formamido}-2-methylpropyl)
0.15 mmol), 27b (50.0 mg, 0.15 mmol), DIPEA (66
mL, 0.38 mmol),
HBTU (69.6 mg, 0.18 mmol) in DMF (2 mL) were reacted and
12

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
purified as described in the general procedure above to yield a
colourless oil 28c (76 mg, 0.095 mmol, 62%, Rf 0.21 in 1:1 Hex:-
4.47e4.45 (m,1H), 4.39e4.33 (m,1H), 3.80e3.77 (m,1H), 3.68e3.66
(m, 0.5H), 3.62e3.56 (m, 0.5H), 2.36 (br, 0.5H), 2.18e2.04 (m, 2H),
1.98e1.91 (m, 1.5H), 1.86e1.76 (m, 1H), 1.51e1.42 (m, 1H), 1.34e1.23
(m, 1H), 1.18e1.04 (m, 3H), 1.03e0.83 (m, 12H). 13C NMR (101 MHz,
21
EtOAc). [
d
a
]
589
¼ ꢂ67.00 (c ¼ 0.5, CHCl3). 1H NMR (400 MHz, CDCl3)
7.86e7.79 (m, 1H), 7.40e7.09 (m, 11H), 5.57 (d, J ¼ 9.6, 1H),
5.04e4.96 (m, 0.5H), 4.89e4.81 (m, 0.5H), 4.74e4.70 (m, 1H), 4.58
(d, J ¼ 10 Hz, 0.5H), 4.50e4.47 (m, 0.5H), 3.92e3.81 (m, 1H),
3.73e3.69 (m, 0.5H), 3.60e3.56 (m, 0.5H), 2.28 (br, 0.5H), 2.10e2.04
(m, 1.5H), 1.91e1.71 (m, 4H), 1.54e1.39 (m, 1H), 1.29e1.24 (m, 9H),
1.15e1.03 (m, 3H), 0.97e0.77 (m, 12H). 13C NMR (101 MHz, CDCl3)
CDCl3)
d
171.7 (d, JC-P ¼ 5.1 Hz), 171.6, 171.4 (d, JC-P ¼ 6.3 Hz), 156.54,
156.53, 150.6 (d, JC-P ¼ 10.1 Hz), 150.3 (d, JC-P ¼ 9.1 Hz), 150.26 (d, JC-
¼ 10.6 Hz), 136.38, 136.37, 129.92 (d, JC-P ¼ 1.0 Hz), 129.89 (d, JC-
P
¼ 0.9 Hz), 129.88 (d, JC-P ¼ 1.1 Hz), 129.82, 129.77 (d, JC-P ¼ 0.7 Hz),
P
129.75 (d, JC-P ¼ 0.8 Hz), 128.7, 128.3, 128.22, 128.19, 125.43 (d, JC-
d
174.0, 173.9, 172.81, 172.78, 172.7 (d, JC-P ¼ 4.7 Hz), 172.6 (d, JC-
¼ 0.9 Hz), 125.39 (d, JC-P ¼ 1.2 Hz), 125.3 (d, JC-P ¼ 1.3 Hz), 125.2 (d,
P
¼ 6.4 Hz), 171.8 (d, JC-P ¼ 5.4 Hz), 171.7 (d, JC-P ¼ 4.9 Hz), 157.31,
JC-P ¼ 0.8 Hz), 120.9 (d, JC-P ¼ 4.2 Hz), 120.85 (d, JC-P ¼ 4.8 Hz),
P
157.29, 156.99, 156.98, 150.5 (d, JC-P ¼ 10.3 Hz), 150.4 (d, JC-
120.82, 120.81, 120.80, 120.78, 120.77, 120.74, 120.52 (d, JC-
¼ 9.8 Hz), 150.2 (d, JC-P ¼ 8.8 Hz), 150.1 (d, JC-P ¼ 10.0 Hz), 129.91
¼ 4.4 Hz), 120.50 (d, JC-P ¼ 4.4 Hz), 67.1, 60.5, 60.3, 59.2, 51.3 (d, JC-
P
P
(d, JC-P ¼ 1.7 Hz), 129.87 (d, JC-P ¼ 1.6 Hz), 129.80, 129.78, 125.42 (d,
JC-P ¼ 2.3 Hz), 125.37, 125.34, 125.26, 121.0 (d, JC-P ¼ 4.3 Hz), 120.91
(d, JC-P ¼ 2.4 Hz), 120.87 (d, JC-P ¼ 1.4 Hz), 120.8, 120.54 (d, JC-
¼ 153.2 Hz), 51.1 (d, JC-P ¼ 154.5 Hz), 49.4 (d, JC-P ¼ 155.2 Hz), 49.2
P
(d, JC-P ¼ 155.2 Hz), 48.9, 48.8, 48.6, 48.4, 36.33 (d, JC-P ¼ 3.8 Hz),
36.28 (d, JC-P ¼ 5.0 Hz), 35.8 (d, JC-P ¼ 5.4 Hz), 35.7 (d, JC-P ¼ 3.7 Hz),
35.4, 28.1, 27.9, 27.5 (d, JC-P ¼ 14.9 Hz), 27.0 (d, JC-P ¼ 14.9 Hz), 26.34,
26.31, 25.5, 25.2, 25.0 (d, JC-P ¼ 4.6 Hz), 24.9 (d, JC-P ¼ 5.5 Hz),16.6 (d,
JC-P ¼ 12.1 Hz), 16.3 (d, JC-P ¼ 12.1 Hz), 15.3 (d, JC-P ¼ 2.7 Hz), 11.9,
¼ 2.8 Hz), 120.51, 120.47 (d, JC-P ¼ 2.4 Hz), 60.5, 59.7, 59.6, 57.4,
P
57.3, 51.07 (d, JC-P ¼ 153.8 Hz), 51.05 (d, JC-P ¼ 154.9 Hz), 50.5, 50.2,
49.1 (d, JC-P ¼ 155.0 Hz), 49.0 (d, JC-P ¼ 156.1 Hz), 48.9, 48.8, 36.3 (d,
JC-P ¼ 4.1 Hz), 36.1, 35.9 (d, JC-P ¼ 4.1 Hz), 35.7 (d, JC-P ¼ 3.5 Hz), 35.3
(d, JC-P ¼ 3.6 Hz), 29.74, 29.66, 29.6, 28.92, 28.85, 28.0, 27.9, 27.5 (d,
JC-P ¼ 15.1 Hz), 26.9 (d, JC-P ¼ 14.5 Hz), 26.61, 26.6, 26.5, 25.4, 25.3,
25.1, 25.0, 16.6 (d, JC-P ¼ 11.8 Hz), 16.1 (d, JC-P ¼ 12.3 Hz), 15.8 (d, JC-
11.8, 11.7, 11.5. 31P NMR (162 MHz, CDCl3)
d 17.96 (8.8%), 17.66 (5.1%),
17.38 (52.3%), 16.99 (33.8%). HRMS-ESI calculated for
C
36H46N3NaO7P [M
þ
Naþ] 686.2966, found m/z 686.2944.
Analytical RP-HPLC (Method II). tR ¼ 50.99, 51.22, 51.72, 52.41 min.
¼ 3.8 Hz), 15.3 (d, JC-P ¼ 3.0 Hz), 11.92, 11.86, 11.7, 11.3. 31P NMR
P
(162 MHz, CDCl3)
d
18.17 (24.5%), 17.80 (14.6%), 17.55 (34.1%), 17.25
4.2. Biological assays
(26.8%). HRMS-ESI calculated for C33H49N4NaO6P [M þ Naþ]
651.3282, found m/z 651.3269. Analytical RP-HPLC (Method I)
tR ¼ 30.00, 30.42, 30.78 min.
4.2.1. CtHtrA and HNE enzyme assays
CtHtrA recombinant protein purification and assay protocols
have been established as previously outlined [54,55]. Assays were
(Benzyloxy)carbonyl-N-[(2S)-1-[(2S)-2-{[1-(diphenox-
yphosphoryl)-2-methylpropyl]carbamoyl}pyrrolidin-1-yl]-2,2-
dimethyl-1-oxobutan-2-yl]carbamate (28d). Compound 11
performed in 96-well black assay plates, where 2.5
per well was treated against six-dose series of JO146
(0.01e125 M) and equal volumes of DMSO. CtHtrA and inhibitors
mM of CtHtrA
a
(0.13 g, 0.34 mmol), 27a (0.12 g, 0.34 mmol), DIPEA (150
mL,
m
0.85 mmol), HBTU (0.15 g, 0.41 mmol) in DMF (2 mL) were reacted
and purified as described in the general procedure above to yield a
were diluted to an equal total volume in 50 mM Tris, 20 mM MgCl2
at pH 7.0. Plates were warmed at 37 ꢀC for 10 min to allow for
colourless oil 28d (0.14 g, 0.22 mmol, 65%, Rf 0.52 in 1:1 Hex:EtOAc).
CtHtrA activation prior to addition of 10 mL substrate (prepared in
21
[
a
]
¼ ꢂ58.60 (c ¼ 0.5, CHCl3). 1H NMR (400 MHz, CDCl3)
1 mg/mL in a mixture of 1:1 isopropanol:buffer). MeOCoum-
ENLHLPLPIIF-DNP (Mimotopes) was used as the substrate, where
MeOCoum represents an N-terminal 7-methoxycoumarin-4-acetic
589
7.34e7.12 (m, 16H), 5.73 (d, J ¼ 9.6, 0.5H), 5.53 (d, J ¼ 9.6, 0.5H),
5.12e5.02 (m, 2H), 4.79e4.70 (m, 1.5H), 4.52e4.49 (m, 0.5H),
4.40e4.34 (m, 1H), 3.82e3.77 (m, 1H), 3.68e3.64 (m, 0.5H),
3.61e3.57 (m, 0.5H), 2.47e2.33 (m, 1H), 2.15e1.83 (m, 4H),
1.26e1.02 (m, 6H), 0.98e0.92 (m, 9H). 13C NMR (101 MHz, CDCl3)
acid fluorophore and DNP represents
a
C-terminal 2,4-
dinitrophenyl-lysine quencher. Proteolysis was then monitored
over 30 min at 37 ꢀC using an Infinite M200 Plate Reader (Tecan,
Switzerland), taking readings every 30 s with 10 ꢃ flashes per well
at 340 nm excitation, 405 nm emission. Plates were orbitally
shaken for 1 s prior to each reading with 1 mm amplitude, and
multiple reads (3 ꢃ 3) were taken from each well; gain was
calculated based on DMSO control wells. All compounds were
tested in triplicate at each dose, alongside an n ¼ 3 dose series of
JO146 and n ¼ 6 DMSO controls. Data was analysed using a non-
linear regression analysis of the normalized response in Graph-
Pad Prism version 7.04.
d
172.5, 171.8 (d, JC-P ¼ 6.9 Hz), 171.7, 171.6 (d, JC-P ¼ 5.5 Hz), 156.6,
156.5, 150.6 (d, JC-P ¼ 10.1 Hz), 150.3 (d, JC-P ¼ 10.1 Hz), 150.1, 136.4,
136.3, 129.91 (d, JC-P ¼ 0.9 Hz), 129.87 (d, JC-P ¼ 0.8 Hz), 129.83 (d, JC-
¼ 0.6 Hz) 129.77 (d, JC-P ¼ 0.8 Hz), 128.7, 128.32, 128.29, 128.21,
P
128.19, 125.43 (d, JC-P ¼ 1.4 Hz), 125.41 (d, JC-P ¼ 1.5 Hz), 125.3 (d, JC-
¼ 0.8 Hz), 125.2 (d, JC-P ¼ 0.9 Hz), 121.0 (d, JC-P ¼ 4.0 Hz), 120.80 (d,
P
JC-P ¼ 1.7 Hz), 120.75 (d, JC-P ¼ 1.3 Hz), 120.5 (d, JC-P ¼ 4.4 Hz), 67.2,
67.1, 60.6, 60.3, 59.2, 51.4 (d, JC-P ¼ 154.7 Hz), 51.2 (d, JC-
¼ 154.7 Hz), 48.82, 48.79, 35.8, 35.4, 29.5 (d, JC-P ¼ 4.1 Hz), 29.4 (d,
P
JC-P ¼ 3.9 Hz), 28.1, 27.9, 26.4, 26.3, 25.5, 25.2, 20.7 (d, JC-P ¼ 13.7 Hz),
20.4 (d, JC-P ¼ 13.4 Hz), 18.2 (d, JC-P ¼ 4.6 Hz), 18.1 (d, JC-P ¼ 4.6 Hz).
The in vitro elastase (Athens Research and Technology) inhibi-
tion assay was performed in 0.1 M Tris-HCl, pH 8.1, 0.02 M CaCl2.
31P NMR (162 MHz, CDCl3)
d
17.59 (24.7%), 16.92 (75.3%). HRMS-ESI
Each well was incubated with 25
concentration, 186 nM) and 25
m
m
L elastase solution in buffer (final
L inhibitor solution (final con-
M to 31.25
M) for 15 min at 37 ꢀC
L of methoxysuccinyl-Ala-Ala-Pro-Val-pNA
calculated for C35H44N3NaO7P [M þ Naþ] 672.2809, found m/z
672.2783. Analytical RP-HPLC (Method II) tR ¼ 45.94, 46.32 min.
(Benzyloxy)carbonyl-N-[(2S)-1-[(2S)-2-{[1-(diphenox-
centration ranging from 0.12
before addition of 100
m
m
m
yphosphoryl)-2-methylbutyl]carbamoyl}pyrrolidin-1-yl]-3,3-
dimethyl-1-oxobutan-2-yl]carbamate (28e). Compound 12 (0.15 g,
0.47 mmol), 27a (0.15 g, 0.43 mmol), DIPEA (0.20 mL, 1.06 mmol),
HBTU (0.19 g, 0.51 mmol) in DMF (2 mL) were reacted and purified
as described in the general procedure above to yield a colourless oil
(Sigma-Aldrich) substrate. The inhibitor solution was prepared by
diluting the 1 mg/mL stock solution in DMSO with buffer, and the
resulting solution was then serially diluted to acquire eight con-
centration levels. The final concentration of DMSO in the well
containing the highest inhibitor concentration did not exceed 1%.
The 1 mg/mL substrate solution was prepared in 1% DMSO in buffer
to fully solubilize the substrate. All experiments were conducted in
triplicate and the residual activity of the enzyme in each well was
measured by absorbance at 405 nm every 20 s for 10 min. IC50
28e (0.15 g, 0.23 mmol, 55%, Rf 0.47, 0.41 in 1:1 Hex:EtOAc).
21
[
d
a
]
¼ ꢂ58.10 (c ¼ 1.0, CHCl3). 1H NMR (400 MHz, CDCl3)
589
7.34e7.12 (m, 16H), 5.65 (d, J ¼ 9.6 Hz, 0.5H), 5.58e5.52 (br s,
0.5H), 5.13e5.02 (m, 2H), 4.96e4.88 (m, 0.5H), 4.82e4.72 (m, 0.5H),
13

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
values were obtained based on a nonlinear regression of a
normalized variable slope (four-parameter) model [56].
PI, cells were harvested via vigorous pipette-lysis and serially
diluted onto fresh host cell monolayers, which were fixed and
stained at 44 h PI for enumeration of infectious yield.
For data analysis, the percentages of the enzyme activity
remaining at each level of an inhibitor concentration was calculated
in reference to the free enzyme (100% enzyme activity) and media
controls (0% enzyme activity). The concentrations of inhibitors and
the percentages of remaining protease activity calculated are then
inserted into the Prism. Results obtained from these experiments
are presented in the Supporting Information.
4.2.3. Cytotoxicity assessments against human epithelial
monolayers
4.2.3.1. MTS assay for cell metabolism and proliferation. MTS assays
were performed using CellTiter 96® AQueous MTS Reagent Powder
(Promega, Australia) according to the manufacturer's instructions.
MTS powder and phenazine methosulfate (PMS; Sigma, USA) were
solubilised in dPBS and filter sterilised before use.
4.2.2. Anti-bacterial cell assays
4.2.2.1. Pseudomonas aeruginosa assay. P. aeruginosa strain PAO1
HEp-2 cells were cultured at a density of 5000 cells/well and at
24 h post-culture were treated with 1% v/v DMSO and inhibitors (at
was cultured overnight by shaking at 37 ꢀC in fluorescent broth (FB)
and diluted 1/1000 in 10 mL with 60
Separately, 6 and 12 L of 10 mM stock solutions (diluted in DMSO)
of the protease inhibitors JO146, 2, and 3 were added to 1.2 mL of
cellular dilutions, resulting in 50 M and 100 M final concentra-
tions. Controls had 6 L (0.5%) and 12 L (1%) DMSO added.
5 ꢃ 200 L of the 1.2 mL solutions were added to a 96-well black
m
L of 1 M MgSO4 added.
25 mM and 100 mM doses); media only controls were also included,
m
with all treatments performed in triplicate. At 24 h following
treatment, the cells were given fresh supplemented DMEM and
incubated with MTS/PMS solution for 4 h prior to reading the
absorbance at 490 nm. Following absorbance readings, all media
was removed. Data was statistically analysed in GraphPad Prism 7.
m
m
m
m
m
clear bottom plate. Growth (OD 600) and fluorescence ([Ex]
405 nm/[Em] 460 nm) were measured every 15 min for 24 h or
48 h. Pyoverdine, a fluorescent biomolecule, produced by the bac-
teria was analysed as area under the curve (AUC) as a biomarker for
P. aeruginosa growth [57]. FB (pH 7.0) consisted of 10 g/L bacto™
tryptone (peptone 140), 10 g/L bacto protease peptone #3 (peptone
180), 18.6 mM K2HPO4, and 1% (v/v) glycerol.
4.2.3.2. LDH assay for cell integrity. Extracellular lactate dehydro-
genase (LDH) was measured in treated HEp-2 cells using CytoTox
96® Non-Radioactive Cytotoxicity Assay (Promega, Australia) ac-
cording to the manufacturer's instructions. HEp-2 cells were
cultured at a density of 5000 cells/well to match the MTS assay. At
24 h post-culture, cells were treated with 25 mM and 100 mM of
each JO146 analogue, including 1% v/v DMSO and media only
controls, each performed in triplicate. Cells were treated for 8 h
prior to removal of supernatant. Maximum release controls were
also performed 45 min prior to supernatant removal, where un-
treated cells were lysed with 0.8% v/v Triton X-100. Cell superna-
tants were treated with CytoTox reagent for 30 min followed by
stop solution, as per manufacturer's instructions. The absorbance of
each well was then read at 490 nm. Statistical analysis of data was
performed in GraphPad Prism 7.
4.2.2.2. Staphylococcus aureus and Escherichia coli assays.
S. aureus, and E. coli were grown in cation adjusted Mueller Hinton
(MH) broth at 37 ꢀC with shaking (200 rpm). MIC plates were set up
as follows; 150
and 75 l of media was added to the remaining wells of a 96-well
plate. Compound was added to column 1 at a final concentration
of 32 g/mL (approximately 50 M) and serially diluted 2-fold
(75 L transfer) into the neighbouring wells, making sure to
discard 75 L from the last well. Thus, resulting in a serial dilution
of each compound from 32 g/mL to 0.015 g/mL (0.0025 M).
Overnight cultures of bacteria were diluted in fresh Ca-MH before
mL of Ca-MH media was added to column 1 (A-H)
m
m
m
m
m
m
m
m
4.3. Docking studies
adding 75
m
L of culture to each well of the MIC plate, to achieve a
Docking was conducted using Gold v5.2 with a previously
constructed homology model of CtHtrA [51]. Ligands were drawn
using Avogadro® and docked as covalent ligands (to Ser197) using
the CHEMPLP scoring function. Two key hydrogen bonding con-
straints between the enzyme and ligands were pre-defined be-
tween C]O of Thr213 and NH of P1 amide, and between NH of
Ile215 amide and C]O of 2-pyridone, which corresponds to the C]
O of P3 in peptide-based analogues. These constraints were pre-
defined to enable direct comparison with the docking position of
the peptidic lead compound JO146, and as these hydrogen bonds
had been previously verified by X-ray crystallography of HNE
uniform CFU/ml of ~5 ꢃ 105 in the MIC plate. Plates were incubated
at 37 ꢀC with shaking for 24 h before determining the MIC. MIC's
were determined as the lowest concentration at which growth did
not occur. Assays were carried out in technical triplicate.
4.2.2.3. Chlamydia trachomatis and Chlamydia pecorum cell assays.
In vitro C. trachomatis and C. pecorum cell culture assays were
conducted according to our previously reported method [17].
C. trachomatis D (D/UW-3/Cx; CtD) and C. pecorum G (MC/MarsBar;
CpG) isolates were routinely cultured in HEp-2 cells and McCoy B
cells, respectively, in high glucose (4.5 g/L) DMEM (product number
D6546, Sigma-Aldrich) supplemented with heat-inactivated 10%
complexed
with
5-aminopyrimidin-6-one-containing
tri-
fluoromethyl ketones inhibitors (PDB 1EAT) [58].
Declaration of competing interest
The authors have no conflicts of interest.
Acknowledgments
foetal calf serum (Sigma-Aldrich), 4 mM L-Alanyl-
L-glutamine,
100 g/mL streptomycin, and 50 g/mL gentamicin. Inhibition ex-
m
m
periments were routinely conducted in 96-well plates seeded with
20,000 host cells per well 24 h prior to the chlamydial infection. A
high Multiplicity of Infection (MOI) of 12 for C. trachomatis and MOI
of 4 for C. pecorum was used to achieve a high concentration of
infectious yield, to allow for more accurate determination of the
dynamic range of responses to each compound at each dose. In-
fections were synchronised at 500ꢃg/28 ꢀC for 30 min, and infec-
tious media was replaced with fresh supplemented DMEM
containing 1
tures were treated with a dose series of 25
of each compound at 16 h PI, alongside 1% v/v DMSO and media-
only controls. Each treatment was performed in triplicate. At 44 h
This project has been supported by the NSW Government under
the NSW Koala Strategy. Jimin Hwang was supported by a Univer-
sity of Otago Doctoral Scholarship. Natalie Strange was supported
by a UTS Faculty of Science HDR (Higher Degree by Research)
Scholarship. The authors would like to thank the Department of
Chemistry at the University of Otago for use of their NMR and mass
spec facilities.
mg/mL cycloheximide at 2 h post infection (PI). Cul-
m
M, 50 M and 100 mM
m
14

J. Hwang, N. Strange, M.J.A. Phillips et al.
European Journal of Medicinal Chemistry 224 (2021) 113692
M.S. South, J. Med. Chem. 46 (2003) 4696e4701.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113692.
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