Stereochemical basis for the anti-chlamydial activity of the phosphonate protease inhibitor JO146

Article abstract of DOI:10.1016/j.tet.2017.10.031

JO146, a mixture of two diastereomers of a peptidic phosphonate inhibitor for Chlamydial HtrA (CtHtrA), has reported activity against Chlamydia species in both human and koala. In this study we isolated the individual diastereomers JO146-D1 and JO146-D2 (

Full text of DOI:10.1016/j.tet.2017.10.031

Accepted Manuscript
Stereochemical basis for the anti-chlamydial activity of the phosphonate protease
inhibitor JO146
Ayodeji A. Agbowuro, Rami Mazraani, Laura C. McCaughey, Wilhelmina M. Huston,
Allan B. Gamble, Joel D.A. Tyndall
PII:
S0040-4020(17)31046-3
10.1016/j.tet.2017.10.031
TET 29034
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 26 June 2017
Revised Date: 8 October 2017
Accepted Date: 11 October 2017
Please cite this article as: Agbowuro AA, Mazraani R, McCaughey LC, Huston WM, Gamble AB, Tyndall
JDA, Stereochemical basis for the anti-chlamydial activity of the phosphonate protease inhibitor JO146,
Tetrahedron (2017), doi: 10.1016/j.tet.2017.10.031.
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Stereochemical Basis for the Anti-Chlamydial
Activity of the Phosphonate Protease Inhibitor
JO146
Leave this area blank for abstract info.
Ayodeji A. Agbowuroa, Rami Mazraanib, Laura C. McCaugheyc,d, Wilhelmina M. Hustonb, Allan B. Gamblea*,
Joel D. A. Tyndalla*
a School of Pharmacy, University of Otago, Dunedin, 9054, New Zealand
b School of Life Sciences, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007, Australia
c The iThree Institute, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007, Australia
d Department of Biochemistry, University of Oxford, South Park road, Oxford OX1 3QU, United Kingdom

1
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Tetrahedron
journal homepage: www.elsevier.com
Stereochemical Basis for the Anti-Chlamydial Activity of the Phosphonate Protease
Inhibitor JO146
Ayodeji A. Agbowuroa, Rami Mazraanib, Laura C. McCaugheyc,d, Wilhelmina M. Hustonb, Allan B.
Gamblea*, Joel D. A. Tyndalla*
a School of Pharmacy, University of Otago, Dunedin, 9054, New Zealand
b School of Life Sciences, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007, Australia
c The iThree Institute, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007, Australia
d Department of Biochemistry, University of Oxford, South Park road, Oxford OX1 3QU, United Kingdom
JO146, a mixture of two diastereomers of a peptidic phosphonate inhibitor for Chlamydial HtrA
(CtHtrA), has reported activity against Chlamydia species in both human and koala. In this study
we isolated the individual diastereomers JO146-D1 and JO146-D2 (in ≥ 90% purity) and
assessed their individual inhibitory activity against the serine protease human neutrophil elastase
(HNE) which is structurally and functionally related to CtHtrA, as well as in Chlamydia
trachomatis cell culture. JO146-D2 [S,S,R-Boc-Val-Pro-ValP(OPh)
2
], the isomer with the
, had an approximate 2.5 – fold increase in in vitro HNE
inhibition potency over JO146-D1 [S,S,S-Boc-Val-Pro-ValP(OPh)
] and greater than 100 – fold
increase in cellular anti-chlamydial activity compared to JO146-D1 which possesses the
unnatural valine at P . JO146 and the individual diastereomers had excellent selectivity for the
ARTICLE INFO
physiologically relevant valine at P
1
2
1
serine protease HNE over the potential off-target serine proteases trypsin and chymotrypsin.
Docking studies supported the biological data with a geometrically unfavoured interaction
observed between the P
1
valine residue of JO146-D1 and the enzyme S sub-pocket.
1
Article history:
Received
Received in revised form
Accepted
JO146, a mixture of two diastereomers of a peptidic phosphonate inhibitor for Chlamydial HtrA
(CtHtrA), has reported activity against Chlamydia species in both human and koala. In this study
we isolated the individual diastereomers JO146-D1 and JO146-D2 (in ≥ 90% purity) and
assessed their individual inhibitory activity against the serine protease human neutrophil elastase
(HNE) which is structurally related to CtHtrA, as well as in Chlamydia trachomatis cell culture.
JO146-D2 [S,S,R-Boc-Val-Pro-ValP(OPh)2], the isomer with the physiologically relevant valine
at P1, had an approximate 2.5 – fold increase in in vitro HNE inhibition potency over JO146-D1
[S,S,S-Boc-Val-Pro-ValP(OPh)2] and greater than 100 – fold increase in cellular anti-chlamydial
activity compared to JO146-D1 which possesses the unnatural valine at P1. JO146 and the
individual diastereomers had excellent selectivity for the serine protease HNE over the potential
off-target serine proteases trypsin and chymotrypsin. Docking studies supported the biological
data with a geometrically unfavoured interaction observed between the P1 valine residue of
JO146-D1 and the enzyme S1 sub-pocket.
Available online
Keywords:
Chlamydia
HtrA inhibition
Protease
JO146
Diastereomers
2009 Elsevier Ltd. All rights reserved
.
survival and virulence of the Chlamydia pathogen.6 Thus, it is an
attractive target for small molecule therapeutics. JO146 [Boc-
Val-Pro-ValP(OPh)2], a mixture of two diastereomers with R-/S-
Val at P1, was previously identified as a covalent inhibitor of
CtHtrA (IC50 = 12.5 µM) in a screen against a library of 1090
serine protease inhibitors. The N-terminal Boc group is retained
as a hydrophobic P4 mimic and is necessary for activity. Further
evaluation in different biological models revealed its selective
toxicity against human and koala pathogenic Chlamydia
species,6-7 presenting the molecule as a viable lead towards the
development of anti-chlamydial drugs with organism specificity
and a different mode of action from current antibiotics.
1. Introduction
Chlamydia is the world’s most common sexually transmitted
bacterial infection. The largely asymptomatic infection often
leads to complications such as ectopic pregnancy, blindness,
infertility, and pneumonia,1-4 particularly in areas where there is
limited access to quality healthcare. The ever increasing global
prevalence of this infection5 is a result (at least in part) of the
inadequacies of present management options and necessitates the
adoption of better strategies. Central to this need is the
development of drugs with good resistance profiles.
Chlamydia are obligate intracellular bacterial pathogens, and
here we focus on Chlamydia (C.) trachomatis. The Chlamydia
high temperature requirement A (CtHtrA) protease is
It is well known that diastereomers interact differently with
biological systems and often produce different biological
effects.8-9 As a result, it has become mandatory to have
a
multimeric, multidomain serine protease that is crucial for the

2
Tetrahedron
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diastereomers properly investigated for potential therapeutic
and proton, we were able to assign the beta (β) and gamma (γ)
and toxicological variation in the course of drug development.10
In this study, we report the synthesis of JO146, 5, and the
isolation and characterization of its two diastereomers JO146-D2,
6 and JO146-D1, 7 (Figure 1). Of the diastereomers, JO146-D2 6
displayed the best inhibitory activity against human neutrophil
elastase (HNE), and C. trachomatis cell culture, providing strong
evidence that it is the diastereomer with the physiologically
relevant valine residue. For peptidyl phosphonate compounds,
protons of the P1-valine, and subsequently, the protons of the P3-
valine. This was important, as it enabled us to establish the
influence of stereochemistry at the P1-valine on the proton
chemical shifts of the P2-proline and P3-valine residues (vide
infra).
Scheme 1. Synthesis of JO146 5 (A and B) and mechanism of racemate
formation (C).a
the physiologically relevant
L-amino acid at P1 has an R-
configuration (instead of the usual S-configuration) due to a
substitution of a phosphonate group for the carbonyl, causing a
reversal of the direction in assignment priority. 1H NMR
spectroscopic analysis of the change in chemical shift for protons
of the P2-proline and P3-valine residue on 6 vs 7 indicated
shielding/deshielding effects that may enable differentiation of
diastereomeric
mixtures
of
α-aminoalkylphosphonate
diaryl/diphenyl esters in the future.
aReagents and conditions: (a) Proline methyl ester, HBTU, DIEA, DMF,
25 °C, 20 h, 57%; (b) LiOH.H2O, THF, H2O, 25 °C, 2 h, 90%; (c) Benzyl
carbamate, isobutyraldehyde, 80-90 °C, 2 h, 72%; (d) 33% HBr/AcOH, 25
°C, 20 h, 75%; (e) 2, HBTU, DIEA, DMF, 25 °C, 20 h, 90%.
Figure 1. Structures of JO146 5 and the isolated diastereomers 6 and 7.
2. Results
2.2. Protease activity
2.1. Synthesis
Next, a preliminary assessment of the bioactivity of the
diastereomers 6 and 7 alongside JO146 5 was carried out. The
initial protease inhibition assays were performed against human
neutrophil elastase (HNE), a human serine protease with similar
substrate specificity as CtHtrA, and one that has been well
studied as a target for phosphonate inhibitors.12-14 The substrate
specificity of HNE is largely dependent on the structural makeup
of its S1 substrate-binding pocket, and has been reported to
consist mainly of hydrophobic residues15 that enable it to bind
preferentially to substrates with small hydrophobic amino acids
such as valine and alanine at P1.16 The substrate specificity of
CtHtrA is similar to this as it also preferentially cleaves
substrates with small hydrophobic residues including Val, Pro,
Ala and Ile at P1.17-18 As a result, activity against HNE would
normally provide a good prediction for potential anti-CtHtrA
activity. We therefore used HNE as a frame of reference. An in
vitro off-target assessment of the inhibitors was also conducted
using the abundant digestive serine proteases trypsin and
chymotrypsin.
The synthesis of JO146 5 was carried out over five steps
(Scheme 1A and Scheme 1B). Boc-Val-Pro-OH
2 was
synthesized via saponification of Boc-Val-Pro-OMe 1, which
was formed by HBTU-promoted peptide coupling of Boc-Val-
OH to Pro-OMe (Scheme 1A). Concurrently, racemic Val-
phosphonate 4 (isolated as the HBr salt) was synthesized in two
steps via an α-amidoalkylation-type reaction, followed by
deprotection of the benzylcarbamate (CBz) group (Scheme 1B).
Finally, HBTU-promoted coupling of 4 with 2 provided JO146 5
(Scheme 1B) as a mixture of JO146-D1:JO146-D2 (46:54, See
Table S1). The two diastereomers were isolated via silica gel
column chromatography JO146-D2 (R-P1-Val) 6 and JO146-D1
(S-P1-Val) 7, in sufficient amounts and purity (≥90%, Table S1
and HPLC traces in supporting information) for biological
testing. The formation of the racemic CBz-protected Val-
phosphonate 3 with R/S configuration at the residue, which
becomes the P1-Val, occurs during the α-amidoalkylation-type
reaction. The condensation of isobutyraldehyde with
benzylcarbamate in acetic acid generates an imine intermediate
that permits both frontside and backside attack by the incoming
nucleophilic triphenyl phosphite (P(OPh)3),11 thereby generating
an enantiomeric mixture (Scheme 1C).
The results obtained from the HNE inhibition assay revealed
JO146-D2 6 to be ~2.5 – fold more potent than JO146-D1 7
(Table 1). No significant protease inhibition of trypsin or
chymotrypsin was observed for either isomer (Table 1, IC50
>
Analysis of 2D NMR experiments run on JO146 5 (see
supporting information), allowed for the complete assignment of
the P1 to P3 residue protons on 6 and 7 in their respective 1D
NMR spectra. The doublet in the 13C NMR spectra centered at δ
51.3 and δ 51.1 for 6 and 7 respectively, had a 13C-31P coupling
constant (J) of ~155 Hz, and was assigned as the P1-valine alpha
(α) carbon. Using the chemical shift of the P1-valine α carbon
500 µM). Based on previous X-ray crystallography studies,19-21
and those reported by Winiarski et al.,14 which confirmed that the
P1-(R)-diastereomers of phosphonate inhibitors are better
inactivators of HNE-type serine proteases, our HNE inhibition
studies in conjunction with our molecular docking and cell-based
C. trachomatis data (vide infra), suggest that JO146-D2 6 is the

3
isomer containing the (R)-valine at P1.In an attempt to
ACCEPTED MANUSCRIPT
unequivocally confirm the stereochemical assignment of 6 and 7,
2D-NOESY experiments were carried out (Figures S23 and S24).
However, no distinct NOE correlations between the α-proton of
the P1-valine and the neighbouring α-proton on the P2-proline
were observed. Any distinct NOE correlations between the β-
and γ-protons of the P1-valine and the P2-P3 residues were not
evident, and as a result, stereochemical differentiation of both
diastereomers could not be achieved using this technique.
However, a comparison of the 1H NMR spectra of 6, 7 and the N-
Boc protected tripeptide S,S,S-Boc-Val-Pro-Val-OMe 8 with a C-
terminal L-valine residue (Figures S14, S17 and S25), revealed
close similarity between JO146-D2 6 and 8. While peaks
corresponding to the stereogenic P1-Val-αH, and P2-Pro-αH
resonated at the same approximate frequency (δ 4.78 – 4.69) in 7,
both peaks are clearly separated in 6 (δ 4.77 – 4.69 and δ 4.46)
and 8 (δ 4.64 – 4.54 and δ 4.42), suggesting similar
stereochemistry at P1 for both of these structures. This further
supports 6 as having the P1-levorotatory valine (R-configuration)
isomer.
Table 1. Protease Inhibition Dataa for Compounds 5, 6 & 7
against Human Neutrophil Elastase (HNE), Trypsin and
Chymotrypsin
Compound
HNE
Chymotrypsin
IC50 (µM)
> 500
Trypsin
IC50 (µM)
> 500
IC50 (µM)
0.83 ± 0.01
0.67 ± 0.01
1.65 ± 0.11
5
6
7
Figure 2. Binding poses of 7, JO146-D1 (A) and 6, JO146-D2 (B) docked
> 500
> 500
into a homology model of CtHtrA (solid surface) showing the different
orientations of the respective P1 isopropyl group relative to the S1 pocket
binding I242 residue (cyan).
> 500
> 500
aData are presented as the mean ± SEM of triplicate
experiments.
2.4. Cellular activity
2.3. Molecular modelling
Based on our HNE inhibition data and the docking
experiments, we hypothesized that the stereochemical difference
between the diastereomers 6 and 7 would affect the cellular
activity of the inhibitors against C. trachomatis. JO146 5 has
been previously identified to be most effective when added to C.
trachomatis cell culture at 16 h post infection (PI)6 which
corresponds to the mid-replicative phase of the C. trachomatis
developmental cycle. When 10 ꢀM of JO146 5 was added at this
time point, it was highly effective against Chlamydia cells with
about 1.5 log loss of infectious progeny (inclusion forming units;
IFU/mL). In the present study, treatment of the cell culture with
50 µM concentration of 5, 6 and 7 at 16 h PI, resulted in
appreciable loss of infectious progeny that ranged between 101
and 103 relative to the negative DMSO control (Figure 3). JO146-
D2 6 was most effective with over 1000 – fold reduction in
IFU/mL relative to DMSO, and approx. 100 – fold difference in
activity compared to JO146-D1 7 and JO146 5 which had
approximately 101 and 101.5 reduction in IFU/mL, respectively.
At a concentration of 10 ꢀM, 7 had no detectable anti-chlamydial
activity while 6 still produced a significant 101 reduction in
IFU/mL (Figure 3).
To give an indication of the binding mode to CtHtrA which
would be the desired target in the cell culture assays,
diastereomers
6
and
7
were docked (GOLD v5.2
https://www.ccdc.cam.ac.uk/solutions/csd-
discovery/components/gold/) into a homology model of CtHtrA
based on the crystal structure of Escherichia coli DegP (PDB ID:
3OTP).22-23 The ligands were prepared and docked as phosphonic
acids without the aryl portion as formation of the covalent bond
is accompanied by the loss of both phenoxy groups as observed
in several crystal structures of phosphonate based inhibitors e.g.
PDB IDs 4MVN, 3UFA, 1P12, 1CGH and 1MAX.24-27 The
highest ranked binding mode and interactions for both
diastereomers were viewed and analyzed. Similar interactions
were observed in both cases (Figure 2); individual residues of the
inhibitors occupy the corresponding enzyme S1-S4 sub-pockets.
JO146-D2 6 was however found to be more stereochemically
favored, interacting with the S1 specificity pocket by virtue of its
(R)-configuration, which enables the isopropyl group of valine to
make significant van der Waals interactions with I242 of the
pocket.28 JO146-D1 7 on the other hand had a weakened
interaction at this point as the (S)-configuration orients the
isopropyl group away from I242 (Figure 2). This structural
difference is thought to be responsible for the observed difference
in in vitro potency against HNE.

4
Tetrahedron
determine if the shielding/deshielding effect at P3 is due to the
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conformational restriction imposed by the residues at P1 and P2.
Table 2. Observed chemical shift (δ) differences for protons
on residues P2 and P3 of JO146-D2 6 and JO146-D1 7.
Residue/Proton
(R)-6 (ppm)
(S)-7 (ppm)
ꢀδRS
(ppm)
P2-Pro-αH
P2-Pro-δH
4.46 (dd)
3.64 (m)a
3.61(m)a
4.30 (dd)
2.01 (m)a
0.99 (d)
4.74 (m)a
3.71 (m)a
3.48 (m)a
4.24 (dd)
1.82 (m)a
0.86 (d)
-0.28
-0.07
+0.13
P3-Val-αH
+0.06
+0.19
+0.13
+0.10
P3-Val-βH
2 × P3-Val-γH’s
0.93 (d)
0.83 (d)
aCentre of multiplet (and not range) reported.
4. Conclusion
Figure 3. Inhibition of C. trachomatis by JO146 5 and its diastereomers 6
In conclusion, significantly higher anti-chlamydial activity
was observed for the P1-Val levorotatory JO146-D2 6 over the
P1-Val dextrorotary JO146-D1 7. Therefore, future inhibition
assays in the series should be conducted with phosphonates
having the physiologically relevant (R)-stereochemistry at the P1
residue. Our data also suggest that phosphonate ester inhibitors
have selectivity for HNE and the structurally related CtHtrA over
abundantly available serine proteases (e.g. trypsin and
chymotrypsin), an important feature as we develop our inhibitors
for future in vivo pre-clinical studies.
and 7 during mid-replicative phase of the pathogen’s developmental cycle.
Infectious yield counts (IFU/mL) were determined upon completion of
developmental cycle. Black bars are JO146-D2 6, light gray bars are JO146-
D1 7 and dark gray bars are the JO146 5. Error bars indicate SEM obtained
from experimental replicates (minimum of 5). ****P < 0.0001
3. Discussion
While peptidyl α-aminoalkylphosphonate diaryl/diphenyl
esters have been extensively studied as serine protease inhibitors,
the absolute configurations of isomers obtained in previous
studies were determined by X-ray crystallography .19-20, 24 We did
not obtain the absolute stereochemistry using these techniques,
but our inhibition data, supported by previous HNE activity
assays by Winiarski et al.14 in which they identified the P1-(R)-
diastereomers for similar P1-substituted tripeptide phosphonates
as better inactivators of HNE compared to their P1-(S)-
counterparts, suggests that JO146-D2 6 and JO146-D1 7 contain
5. Experimental section
5.1. General
All solvents and reagents were of reagent grade and used
without further purification except where stated. All reactions
were conducted in standard glassware and in air or under
nitrogen gas unless otherwise stated. Organic solvent extracts
were dried with MgSO4 and subjected to rotary evaporation, and
finally dried at 10-1 mbar using a high vacuum pump.
the (R)-L-Val and (S)-D-Val at P1, respectively. The better
inhibition of HNE and CtHtrA by 6 is also supported by our
molecular docking study (Figure 2). Upon careful inspection of
the 1H and 13C NMR spectra for JO146 5, 6, and 7, we identified
some key differences in the respective chemical shifts for the P2-
Pro and P3-Val residues (Table 2). The most notable change was
for the protons on the P3-Val residue, which were relatively more
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 and/or
permanganate staining. Flash column chromatography was
performed using silica gel 60 (0.040 – 0.063 mm, 200 – 400
mesh) 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.
1
shielded in the H NMR spectrum of 7. Also of note, and to the
best of our knowledge, the 13C NMR spectra of small peptidyl α-
aminoalkylphosphonate diaryl/diphenyl esters has not been fully
reported. Interestingly, the 13C NMR for 6 and 7, both contain
eight doublets in the aromatic region (due to 13C-31P coupling),
indicating that the two phenyl groups are not in an identical
chemical space; i.e. they are diastereotopic, and as a result
High performance liquid chromatography (HPLC) was carried
out on an Agilent HPLC. Samples of 20 ꢀL volume were injected
onto a Gemini 5 µm C18 110 Å column (250 by 4.6 mm,
Phenomenex, New Zealand). The compounds were eluted using
solvent A: 0.1% trifluoroacetic acid (TFA) in water and solvent
B: 0.1% TFA in acetonitrile (ACN) in a linear gradient manner.
At a flow rate of 1.0 mL/min, compounds were detected at the
wavelengths of 254/210 nm.
magnetically
non-equivalent.
While
preliminary,
the
interpretation of the H and 13C NMR spectra suggests that the
bulky P1-valine residue and the P2-proline residue could be
playing a role in the observed ꢁδRS for P2-Pro and P3-Val on 6
and 7. The observed chemical shift differences provide spatial
information that may be useful for determining the absolute
configurations of both molecules. The general application of this
phenomenon could be further investigated as a potential method
for rapid stereochemical identification of future inhibitors in the
diphenyl/diaryl phosphonate series. By substituting the proline
residue at P2 with a non-cyclic amino acid and utilizing a less
bulky P1-substituent (e.g. alanine), it may be possible to
1
Proton (1H) and carbon-13 (13C) nuclear magnetic resonance
(NMR) spectra were acquired on Varian 400 or 500 MHz
spectrometers. Samples were prepared in deuterated solvent,
1
chloroform (δ 7.26, 77.16 ppm) with the respective H and 13C
chemical shifts of the solvent shown in brackets. Chemical shifts
(δ) are expressed in parts per million (ppm) and coupling
constants (J) in Hertz (Hz), both measured against the internal

5
5.2.3. Diphenyl [(1R,1S)-1-[(benzyloxy)amino] -2-
methylpropyl]phosphonate (3)
standard. Multiplicities are reported as either singlet (s), doublet
(d), doublet of doublets (dd), triplet (t), quartet (q), multiplet (m)
or broad signal (br). Compound 5 was fully characterized using
two-dimensional NMR experiments, Homonuclear Correlation
Spectroscopy (COSY), Heteronuclear Single Quantum
Coherence (HSQC) and Heteronuclear Multiple Bond
Correlation (HMBC).
ACCEPTED MANUSCRIPT
Carboxybenzyl (Cbz)-protected 1-Aminoalkyl-phosphonate
diphenyl ester 3 was synthesized using a literature method.32
Triphenyl phosphite (0.933 g, 3.007 mmol) was dissolved in
glacial acetic acid (8 mL), and isobutyraldehyde (0.239 g, 3.308
mmol) and benzyl carbamate (0.500 g, 3.308 mmol) were added.
The mixture was heated for 2 hours at 80-90 °C, and the solvent
evaporated. The crude product was subjected to column
chromatography (Hexane/EtOAc, 5:1 v/v) This yielded 3 as a
High resolution mass spectra were recorded on a Brucker
microTOF-Q spectrometer with an electrospray ionization (ESI)
source.
1
colorless oil (0.950 g, 72%). H NMR (400 MHz, CDCl3) δ 7.38
5.2. Synthetic Details
– 6.98 (m, 15H, 10 × Phenoxy-H & 5 × phenyl-H), 5.22 (dd, J =
10.9, 2.5 Hz, 1H, 1 × Val-NH), 5.15 – 5.01 (m, 2H, 2 × benzylic-
H), 4.45 – 4.34 (m, 1H, 1 × Val-αH), 2.39 (m, 1H, 1 × Val-βH),
1.08 (dd, J = 6.8, 1.3 Hz, 6H, 6 × Val-γH). 13C NMR (101 MHz,
CDCl3) δ 156.2 (d, JC-P = 7.1 Hz), 150.2 (d, JC-P = 10.1 Hz),
149.9 (d, JC-P = 9.6 Hz), 136.0, 129.7, 129.6, 128.5, 128.2,
128.1, 125.3, 125.1, 120.6 (d, JC-P = 4.1 Hz), 120.4 (d, JC-P = 4.1
Hz), 67.4, 53.3 (d, JC-P = 161.6 Hz), 29.1 (d, JC-P = 5.3 Hz), 20.3
(d, JC-P = 14.1 Hz), 17.6 (d, JC-P = 4.2 Hz). ESI-MS (m/z): calcd
for C24H26NNaO5P (M + Na) m/z 462.1446 found 462.1400.
5.2.1. Methyl(2S)-1-[(2S)-2-{[(tert-
butoxy)carbonyl]amino}-3-
methylbutanoyl]pyrrolidine-2-carboxylate (1)
Compound 1 was synthesized using a modified literature
procedure of solution-phase peptide coupling.29 L-Proline methyl
ester hydrochloride (0.605 g, 3.65 mmol) was dissolved in 5 mL
dimethylformamide (DMF). To this was added Boc-Val-OH
(0.833 g, 3.84 mmol), O-(Benzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium hexafluoro-phosphate (HBTU) (1.662 g,
4.38 mmol) and N,N-Diisopropylethylamine (DIEA) (1.3 mL,
7.30 mmol). The reaction mixture was stirred under nitrogen
overnight. The reaction was diluted with ethyl acetate (EtOAc;
30 mL) and an equal amount of sodium bicarbonate, washed with
brine (6 × 30 mL), dried (MgSO4) and concentrated. The crude
product was chromatographed using flash silica gel column
chromatography (Hexane/EtOAc, 7:3 v/v). This yielded 1 on
vacuum concentration as a light yellow oil (0.723 g, 57%), which
was spectroscopically identical to that previously reported.29-30 1H
NMR (400 MHz, CDCl3) δ 5.19 (d, J = 9.4 Hz, 1H, -NH), 4.54 –
4.48 (m, 1H, 1 × Pro-αH), 4.26 (dd, J = 9.3, 6.5 Hz, 1H, 1 × Val-
αH), 3.80 – 3.73 (m, 1H, 1 × Pro-δH), 3.72 (s, 3H, OCH3), 3.68 –
3.61 (m, 1H, 1 × Pro-δH), 2.25 – 2.14 (m, 1H, 1 × Pro-βH), 2.00
(m, 4H, 1 × Pro-βH, 2 × Pro-γH and 1 × Val-βH), 1.41 (s, 9H,
tButyl), 1.01 (dd, J = 6.8, 1.2 Hz, 3H, 3 × Val-γH), 0.92 (dd, J =
6.8, 1.2 Hz, 3H, 3 × Val-γH). 13C NMR (101 MHz, CDCl3) δ
172.4, 171.2, 155.8, 79.4, 58.8, 56.8, 52.1, 47.1, 31.3, 29.0, 28.3,
25.0, 19.2, 17.3. ESI-MS (m/z): calcd for C16H28N2NaO5 (M +
Na) m/z 351.1896 found 351.1877.
5.2.4. Diphenyl [(1R,1S)-1-amino-2-
methylpropyl]phosphonate (4)
Compound 4 was obtained by dissolving compound 3 (0.425
g, 0.967 mmol) in 33% HBr/AcOH solution (3 mL). The reaction
was carried out at room temperature for 2 hours. The volatile
components of the mixture were removed under reduced
pressure. The residue was dissolved in 1 mL methanol, and 80
mL ether was added. Compound 4 was left to crystallize at 4 °C
to yield 0.28 g of the hydrobromide salt, a white solid. The
amount of compound 4 thus obtained was 0.221 g, 75%. No
further recrystallization was necessary. 1H NMR (400 MHz,
CDCl3) δ 9.07 (s, 2H, 2 × NH2), 7.35 – 7.04 (m, 10H, 10 ×
Phenoxy-H), 3.82 (brd, J = 14.2 Hz, 1H, 1 × Val-αH), 2.75 –
2.58 (m, 1H, Val-βH), 1.29 (dd, J = 6.8, 4.5 Hz, 6H, 6 × Val-γH).
13C NMR (101 MHz, CDCl3) δ 149.42 (d, JC-P = 9.6 Hz), 149.39
(d, JC-P = 9.9 Hz), 129.7, 125.6, 125.5, 120.9 (d, JC-P = 4.3 Hz),
120.6 (d, JC-P = 4.5 Hz), 53.5 (d, JC-P = 155 Hz), 28.4 (d, JC-P
=
2.0 Hz), 19.9 (d, JC-P = 9.1 Hz), 19.1 (d, JC-P = 4.0 Hz). ESI-MS
(m/z): calcd for C16H20NNaO3P (M + Na) m/z 328.1078 found
328.1045.
5.2.2. (2S)-1-[(2S)-2-{[(tert-
5.2.5. Tert-butylN-[(2S)-1-[(2S)-2-{[(1R,1S)-1-
(diphenoxyphosphoryl)-2-
butoxy)carbonyl]amino}-3-
methylbutanoyl]pyrrolidine-2-carboxylic acid (2)
methylpropyl]carbamoyl}pyrrolidin-1-yl]-3-methyl-
1-oxobutan-2-yl] carbamate (5)
Compound 2 was produced by alkaline hydrolysis of 1 using a
modified literature procedure.31 1 (1.372 g, 4.18 mmol) was
dissolved in a 3:1 mixture of tetrahydrofuran (THF) and water
(60 mL + 20 mL respectively). LiOH.H2O (0.878 g, 20.89 mmol)
was added and the reaction mixture was stirred for 2 hours at
room temperature. The reaction was quenched by acidification
with 50 mL 0.5 M HCl, and the mixture was extracted with
EtOAc (4 × 60 mL). The combined organic fraction was washed
with brine (1 × 100 mL), dried over anhydrous MgSO4 and the
solvents evaporated under reduced pressure to yield a white solid
Compound 2 (0.119 g, 0.570 mmol) was dissolved in 3 mL
DMF. To this was added HBTU (0.173 g, 0.457 mmol) and
DIEA (0.17 mL, 0.953 mmol) to generate the activated ester.
After 10 minutes, compound 4 (0.22 g, 0.721 mmol) was added
and the reaction mixture was stirred under nitrogen overnight.
The reaction was diluted with EtOAc (25 mL) and an equal
amount of sodium bicarbonate, washed with brine (6 × 25 mL),
dried (MgSO4) and concentrated in vacuo. The crude product was
subjected to column chromatography (100% EtOAc). This
yielded 5 as a mixture of diastereomers, as a colorless solid
(0.205 g, 90%) which formed a white solid foam under reduced
pressure. >99% purity for diastereomers by HPLC (Gemini 5 µm
C18 110 Å, gradient elution, 40% acetonitrile/water with 0.1%
TFA to 85% acetonitrile in 45 minutes; Retention time (tr) =
33.690 and 34.713 min). ESI-MS (m/z): calcd for
C31H44N3NaO7P (M + Na) m/z 624.2815 found 624.2772. Full
NMR characterization of the pure diastereomers 6 and 7 is
provided below.
1
(1.186 g, 90%). H NMR (400 MHz, CDCl3) δ 9.37 (br s, 1H,
carboxylic acid proton), 5.46 (d, J = 9.4 Hz, 1H, -NH), 4.55 (dd,
J = 8.1, 4.8 Hz, 1H, 1 × Pro-αH), 4.26 (dd, J = 9.4, 6.8 Hz, 1H, 1
× Val-αH), 3.83 – 3.59 (m, 2H, 2 × Pro-δH), 2.25 – 1.86 (m, 5H,
t
2 × Pro-βH, 2 × Pro-γH and 1 × Val-βH,), 1.40 (s, 9H, Butyl),
0.98 (d, J = 6.7 Hz, 3H, 3 × Val-γH), 0.91 (d, J = 6.7 Hz, 3H, 3 ×
Val-γH). 13C NMR (101 MHz, CDCl3) δ 174.5, 172.4, 155.9,
79.6, 59.1, 57.0, 47.5, 31.2, 28.5, 28.3, 24.8, 19.2, 17.6. ESI-MS
(m/z): calcd for C15H26N2NaO5 (M + Na) m/z 337.1739 found
337.1734.
5.2.6. Separation of Diastereomers.

6
Tetrahedron
methylbutanoyl]pyrrolidin-2-yl] formamido}-3-
Single diastereomers of 5 were obtained by column
ACCEPTED MANUSCRIPT
methylbutanoate (8)
chromatography on silica gel using CHCl3/EtOAc, 4:1 v/v.
Compounds 6 and 7 were thus obtained.
Compound 2 (0.450 g, 1.43 mmol) was dissolved in 4 mL
DMF. To this was added HBTU (0.619 g, 1.63 mmol) and DIEA
(0.48 mL, 2.73 mmol) to generate the activated ester. After 10
minutes, L-Valine methyl ester hydrochloride (0.23 g, 1.37
mmol) was added and the reaction mixture was stirred under
nitrogen overnight. The reaction was diluted with EtOAc (20
mL) and an equal amount of sodium bicarbonate, washed with
brine (6 × 20 mL), dried (MgSO4) and concentrated in vacuo.
The crude product was subjected to column chromatography
(Hexane/EtOAc, 2:3 v/v) yielding 8 as a white solid (0.26 g,
45%). 1H NMR (400 MHz, CDCl3) δ 7.21 (d, J = 8.4 Hz, 1H, 1 ×
P1-Val-NH), 5.25 (d, J = 9.3 Hz, 1H, 1 × P3-Val-carbamate-NH),
4.64 – 4.54 (m, 1H, 1 × P1-Val-αH), 4.42 (dd, J = 8.4, 5.1 Hz,
1H, 1 × P2-Pro-αH), 4.29 (dd, J = 9.4, 6.2 Hz, 1H, 1 × P3-Val-
αH), 3.72 (s, 3H, OCH3), 3.78 – 3.66 (m, 1H, 1 × P2-Pro-δH),
3.64 – 3.54 (m, 1H, 1 × P2-Pro-δH), 2.40 – 2.28 (m, 1H, 1 × P1-
Val-βH), 2.19 – 2.03 (m, 2H, 2 × P2-Pro-βH), 2.01 – 1.91 (m, 2H,
1 × P3-Val-βH and 1 × P2-Pro-γH), 1.88 – 1.75 (m, 1H, 1 × P2-
Pro-γH), 1.42 (s, 9H, tButyl), 0.98 (d, J = 6.8 Hz, 3H, 3 × P1-Val-
γH), 0.94 – 0.87 (m, 9H, 3 × P1-Val-γH and 6 × P3-Val-γH). 13C
NMR (101 MHz, CDCl3) δ 172.58, 172.12, 170.92, 155.80,
79.56, 59.92, 57.54, 56.77, 52.04, 47.68, 31.43, 31.09, 28.32,
27.20, 25.15, 19.55, 18.94, 17.86, 17.40. ESI-MS (m/z): calcd for
C21H37N3NaO6 (M + Na) m/z 450.2580 found 450.2540.
5.2.6.1. Tert-butyl-N-[(2S)-1-[(2S)-2-{[(1R)-1-
(diphenoxyphosphoryl)-2-
methylpropyl]carbamoyl}pyrrolidin-1-yl]-3-methyl-
1-oxobutan-2-yl] carbamate (6)
A colorless solid (0.03 g) which formed a white solid foam
under reduced pressure. 96% purity by HPLC, the second
diastereomer accounts for the remaining 4% (Gemini 5 µm C18
110 Å, gradient elution, 40% acetonitrile/water with 0.1% TFA
to 85% acetonitrile in 45 min; tr = 33.557 min), see full HPLC
trace on page 17. Retention factor (Rf) = 0.34 (CHCl3/EtOAc, 4:1,
1
v/v). H NMR (400 MHz, CDCl3) δ 7.33-7.14 (m, 11H, 10 ×
Phenoxy-H and 1 × P1-Val-NH), 5.24 (d, J = 9.2 Hz, 1H, 1 × P3-
Val-carbamate-NH), 4.77 – 4.69 (m, 1H, 1 × P1-Val-αH), 4.46
(dd, J = 8.4, 3.2 Hz, 1H, 1 × P2-Pro-αH), 4.30 (dd, J = 9.6, 6.4
Hz, 1H, 1 × P3-Val-αH), 3.77 – 3.51 (m, 1H, 1 × P2-Pro-δH),
3.63-3.58 (m, 1H, 1 × P2-Pro-δH), 2.44 – 2.34 (m, 1H, 1 × P1-
Val-βH), 2.24 – 2.09 (m, 2H, 2 × P2-Pro-βH), 2.08 – 1.94 (m, 2H,
1 × P3-Val-βH and 1 × P2-Pro-γH), 1.88 – 1.79 (m, 1H, 1 × P2-
Pro-γH), 1.43 (s, 9H, tButyl), 1.11 (d, J = 7.2 Hz, 3H, 3 × P1-Val-
γH), 1.04 (dd, J = 6.8, 1.2 Hz, 3H, 3 × P1-Val-γH), 0.99 (d, J =
6.4 Hz, 3H, 3 × P3-Val-γH), 0.93 (d, J = 6.8 Hz, 3H, 3 × P3-Val-
γH). 13C NMR (101 MHz, CDCl3) δ 172.6, 171.2 (d, JC-P = 6.1
Hz), 155.8, 150.4 (d, JC-P = 10.1 Hz), 150.1 (d, JC-P = 10.1 Hz),
129.8 (d, JC-P = 1.0 Hz), 129.7 (d, JC-P = 0.6 Hz), 125.3 (d, JC-P
=
1.3 Hz), 125.1 (d, JC-P = 0.9 Hz), 120.7 (d, JC-P = 4.1 Hz), 120.4
(d, JC-P = 4.4 Hz), 79.6, 60.0, 56.8, 51.3 (d, JC-P = 154.2 Hz),
5.3. Protease Assays
47.7, 31.4, 29.3 (d, JC-P = 3.9 Hz), 28.3, 27.4, 25.3, 20.3 (d, JC-P
13.6 Hz), 19.6, 18.0 (d, JC-P = 4.7 Hz), 17.4. ESI-MS (m/z): calcd
for C31H44N3NaO7P (M + Na) m/z 624.2815 found 624.2763.
=
The inhibition of human neutrophil elastase (HNE), trypsin
and chymotrypsin by compounds 5, 6 and 7 was also carried out.
This was conducted by modifying an existing method.33 The
activity of the enzymes was measured at 37 °C over a period of 5
min using AAPV-pNA (Sigma-Aldrich M4765), Nα-Benzoyl-
DL-R-pNA (Sigma-Aldrich B4875) and Suc-AAPF-pNA
(Sigma-Aldrich S7388) as HNE, trypsin and chymotrypsin
substrates respectively. Absorbance readings were measured at
405 nm and monitored at 15 sec intervals. The solubilization of
the substrates which is critical for obtaining reliable results was
achieved by dissolving 5 mg in 50 µL of dimethyl sulphoxide
(DMSO) and making up to 5 mL with the assay buffer (0.10 M
Tris-HCl, pH 8.1, 0.02 M CaCl2). For Nα-Benzoyl-DL-R-pNA,
100% DMSO was required.
5.2.6.2. Tert-butyl-N-[(2S)-1-[(2S)-2-{[(1S)-1-
(diphenoxyphosphoryl)-2-
methylpropyl]carbamoyl}pyrrolidin-1-yl]-3-methyl-
1-oxobutan-2-yl] carbamate (7)
A colorless solid (0.015 g) which formed a white solid foam
under reduced pressure. 90% purity by HPLC, the second
diastereomer accounts for the remaining 10%. (Gemini 5 µm C18
110 Å, gradient elution, 40% acetonitrile/water with 0.1% TFA
to 85% acetonitrile in 45 min; tr = 34.496 min), see full HPLC
1
trace on page 18. Rf =0.45 (CHCl3/EtOAc, 4:1 v/v). H NMR
(400 MHz, CDCl3) δ 7.36 (brd, J = 10.1 Hz, 1H, 1 × P1-Val-NH),
7.31 – 7.26 (m, 5H, 5 × Phenoxy-H), 7.18 – 7.14 (m, 5H, 5 ×
Phenoxy-H), 5.17 (d, J = 9.6 Hz, 1H, P3-Val-Carbamate-NH),
4.78 – 4.69 (m, 2H, 1 × P1-Val-αH and 1 × P2-Pro-αH), 4.24 (dd,
J = 9.2, 6.0 Hz, 1H, 1 × P3-Val-αH), 3.74 – 3.67 (m, 1H, 1 × P2-
Pro-δH), 3.50 – 3.45 (m, 1H, 1 × P2-Pro-δH), 2.48-2.39 (m, 2H,
1× P1-Val-βH and 1 ×P2-Pro-βH), 2.00-1.91 (m, 3H, 1 × P2-Pro-
βH and 2 x P2-Pro-γH), 1.86 – 1.77 (m, 1H, 1 × P3-Val-βH), 1.42
(s, 9H, tButyl), 1.13 (d, J = 6.8 Hz, 3H, 3 × P1-Val-γH), 1.07 (dd,
J = 6.8 Hz, 1.6 Hz, 3H, 3 × P1-Val-γH), 0.86 (d, J = 6.8 Hz, 3H, 3
× P3-γH), 0.83 (d, J = 6.8 Hz, 3H, 3 × P3-Val-γH).13C NMR (101
MHz, CDCl3) δ 173.5, 171.2 (d, JC-P = 6 Hz), 155.8, 150.3 (d, JC-
Inhibition by compounds 5, 6 and 7 was assessed by
incubating 50 µL of the enzyme solutions in the assay buffer with
1.5 µL of the inhibitors (dissolved in DMSO), all made up to 100
µL in the assay buffer. Incubation was carried out at 37 °C for 15
min before the addition of 50 µL of the substrate which initiated
the reaction to give an intense yellow coloration for controls and
wells without enzyme inhibition.
All dilutions were in triplicate and inhibition was measured as
the percentage of enzyme activity remaining. Data analysis was
conducted using GraphPad prism.
5.4. Cell-based Assays
= 10.0 Hz), 150.0 (d, JC-P = 9.2 Hz), 129.7 (d, JC-P = 0.9 Hz),
P
129.6 (d, JC-P = 0.6 Hz), 125.3 (d, JC-P = 1.1 Hz), 125.1 (d, JC-P
0.8 Hz), 120.7 (d, JC-P = 4.4 Hz), 120.4 (d, JC-P = 4.3 Hz), 79.7,
60.3, 56.8, 51.1 (d, JC-P = 155.6 Hz), 47.7, 31.3, 29.3 (d, JC-P
3.8 Hz), 28.3, 27.3, 25.0, 20.5 (d, JC-P = 13.9 Hz), 19.6, 17.9 (d,
JC-P = 4.3 Hz), 17.3. ESI-MS (m/z): calcd for C31H44N3NaO7P (M
+ Na) m/z 624.2815 found 624.2764.
=
In vitro C. trachomatis cell culture assays were conducted
based on a previously reported method.7 C. trachomatis D
(D/UW-3/Cx) was obtained from the ATCC and routinely
cultured in McCoy B cells on DMEM, 10% fetal calf serum
(FCS) at 37 °C, 5% CO2, 0.1 mg/mL streptomycin, and 0.05
mg/ml gentamycin. Inhibitor experiments were routinely
conducted in 96-well plates seeded with 20,000 host cells per
well 24 h prior to the Chlamydia infection. Infections were
=
5.2.7. Methyl-(2S)-2-{[(2S)-1-[(2S)-2-{[(tert-
butoxy)carbonyl]amino}-3-

7
25. Bone, R.; Sampson, N. S.; Bartlett, P. A.; Agard, D. A., Biochemistry-
Us 1991, 30 (8), 2263-2272.
routinely conducted at a Multiplicity of Infection (MOI) of 0.3.
The Inclusion Forming Units (IFU) were determined from
cultures harvested at the completion of the developmental cycle
during which inhibitor treatment was conducted. Briefly, JO146
(5) at 50 µM dose, the diastereomers (6 and 7) at 50 µM and 10
µM doses alongside a DMSO control (JO146 solvent), were
added at 16 h post infection (PI) and left in the cultures until
completion of the developmental cycle. Harvested cultures were
lysed by vigorous pipetting and serially diluted onto fresh
monolayers, fixed and stained for enumeration of IFU/ml.
ACCEPTED MANUSCRIPT
26. Hof, P.; Mayr, I.; Huber, R.; Korzus, E.; Potempa, J.; Travis, J.; Powers,
J. C.; Bode, W., EMBO J. 1996, 15 (20), 5481-91.
27. Bertrand, J. A.; Oleksyszyn, J.; Kam, C.-M.; Boduszek, B.; Presnell, S.;
Plaskon, R. R.; Suddath, F.; Powers, J. C.; Williams, L. D., Biochemistry-Us
1996, 35 (10), 3147-3155.
28. Gloeckl, S.; Tyndall, J. D. A.; Stansfield, S. H.; Timms, P.; Huston, W.
M., J. Mol. Microb. Biotech. 2012, 22 (1), 10-16.
29. Chen, Y.; Guan, Z., J. Am. Chem. Soc. 2010, 132 (13), 4577-4579.
30. Dahiya, R., Turk. J. Chem. 2008, 32 (2), 205-215.
31. Jacobsen, Ø.; Klaveness, J.; Ottersen, O. P.; Amiry-Moghaddam, M. R.;
Rongved, P., Org. Biomol. Chem. 2009, 7 (8), 1599-1611.
32.Winiarski, Ł.; Oleksyszyn, J. z.; Sieńczyk, M., J. Med. Chem. 2012, 55
(14), 6541-6553.
33. Bennett, S.; Joshua, A.; Russell, P. J., BioTechniques 1997, 23 (1), 70-72.
Acknowledgments
This research was supported by grants from the Otago
Medical Research Foundation and New Zealand Pharmacy
Education Foundation. The authors would like to thank the
Department of Chemistry for use of their NMR and mass spec
facilities.
Supplementary Material
HPLC spectra for JO146 and its diastereomers and NMR spectra
are available in the Supplementary Material.
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