Structure-activity analysis of peptidic Chlamydia HtrA inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.07.049

Chlamydia trachomatis high temperature requirement A (CtHtrA) is a serine protease that performs proteolytic and chaperone functions in pathogenic Chlamydiae; and is seen as a prospective drug target. This study details the strategies employed in optimizi

Full text of DOI:10.1016/j.bmc.2019.07.049

Journal Pre-Proof
Structure-Activity Analysis of Peptidic Chlamydia HtrA Inhibitors
Ayodeji A. Agbowuro, Jimin Hwang, Emma Peel, Rami Mazraani, Alexandra
Springwald, James W. Marsh, Laura McCaughey, Allan B. Gamble, Wilhelmina
M. Huston, Joel D.A. Tyndall
PII:
S0968-0896(19)30333-5
https://doi.org/10.1016/j.bmc.2019.07.049
BMC 15033
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 February 2019
25 July 2019
29 July 2019
Please cite this article as: Agbowuro, A.A., Hwang, J., Peel, E., Mazraani, R., Springwald, A., Marsh, J.W.,
McCaughey, L., Gamble, A.B., Huston, W.M., Tyndall, J.D.A., Structure-Activity Analysis of Peptidic
Chlamydia HtrA Inhibitors, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.
2019.07.049
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will
undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing
this version to give early visibility of the article. Please note that, during the production process, errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Ltd.

JOURNAL PRE-PROOF
Structure-Activity Analysis of Peptidic Chlamydia HtrA
Inhibitors
Ayodeji A. Agbowuro,a Jimin Hwang,a Emma Peel,b Rami Mazraani,c Alexandra
Springwald,a James W. Marsh,d Laura McCaughey,d,e Allan B. Gamble,a
Wilhelmina M. Huston,*,c Joel D. A. Tyndall*,a
a School of Pharmacy, University of Otago, Dunedin, 9054, New Zealand
b School of Life and Environmental Sciences, The University Sydney, Sydney, NSW 2006,
Australia
c School of Life Sciences, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007,
Australia
d The iThree Institute, University of Technology Sydney, 15 Broadway, Ultimo NSW 2007,
Australia
e Department of Biochemistry, University of Oxford, South Park road, Oxford OX1 3QU, United
Kingdom
*Corresponding authors: joel.tyndall@otago.ac.nz, Wilhelmina.Huston@uts.edu.au
1

JOURNAL PRE-PROOF
S configuration
(R for phosphonates)
Specificity pocket.
is optimal for activity
Valine at P1 produces
the best anti-chlamydial activity
S1
P1
P4
Prime side binding
enhances potency and
O
O
H
R
O
N
S4
N
N
X
selectivity of reversible
inhibitors
S1'
H
O
P3
S3
Only covalently binding
transition state analogues
exhibit anti-chlamydial activity
Tertiary leucine
at P3 produces
the best anti-
chlamydial
activity
ABSTRACT: Chlamydia trachomatis high temperature requirement A (CtHtrA) is a serine
protease that performs proteolytic and chaperone functions in pathogenic Chlamydiae; and
is seen as a prospective drug target. This study details the strategies employed in optimizing
the irreversible CtHtrA inhibitor JO146 [Boc-Val-Pro-ValP(OPh)2] for potency and
selectivity. A series of adaptations both at the warhead and specificity residues P1 and P3
yielded 23 analogues, which were tested in human neutrophil elastase (HNE) and CtHtrA
enzyme assays as well as Chlamydia cell culture assays. Trypsin and chymotrypsin
inhibition assays were also conducted to measure off-target selectivity. Replacing the
phosphonate moiety with α-ketobenzothiazole produced a reversible analogue with
considerable CtHtrA inhibition and cell culture activity. Tertiary leucine at P3 (8a) yielded
approximately 33-fold increase in CtHtrA inhibitory activity, with an IC50 = 0.68 ± 0.02
µM against HNE, while valine at P1 retained the best anti-chlamydial activity. This study
provides a pathway for obtaining clinically relevant inhibitors.
2

JOURNAL PRE-PROOF
1. Introduction
Members of the phylum Chlamydiae are a unique obligate intracellular bacteria, responsible for a
host of diseases with the most notable being Chlamydia (C.) trachomatis and C. pneumoniae.1-2
C. trachomatis parasitizes only humans, causing complications including ectopic pregnancy,
infertility, reversible blindness and infant pneumonia.3-4 C. pneumoniae is the major cause of
respiratory infections in children and adults, leading to pneumonia, bronchitis, sinusitis, and it is
also a risk factor for atherosclerosis.5-6 C. pneumoniae infects other animals including koalas and
horses.
The koala is an iconic Australian marsupial currently listed as vulnerable, with the potential for
extinction. It is often plagued by illnesses as a result of C. pneumoniae and C. pecorum infections.7-
8 C. pecorum causes distressing ocular and urogenital site infections in the animal,8 while the (less
frequently observed) clinical outcomes of C. pneumoniae infection in koala are respiratory in
nature and characterized by coughing, sneezing and purulent discharge.9
Despite the availability of efficacious antibacterial agents, the management of chlamydiosis
remains a challenge. Human chlamydia infections are estimated to have a global incidence of
approximately 130 million10 and clinical treatment failures have been reported11-12 leading to
concern over the effectiveness of current treatment regimens. C. trachomatis is often present as a
co-infection (e.g. with Neisseria gonorrhoeae or Mycoplasma genitalium) and in these pathogens,
resistance to the commonly used anti-chlamydial therapies (macrolides, tetracyclines) is
13-15
increasingly common,
hence a drug targeted to Chlamydia may reduce the selection of
antibiotic resistance in the other pathogens.
We have previously identified the C. trachomatis high temperature requirement A (CtHtrA)
protein as a serine protease that is expressed at elevated levels under Chlamydia stress conditions,
which possesses both proteolytic and chaperone capabilities.16 CtHtrA substrate-specificity studies
3

JOURNAL PRE-PROOF
revealed a preference for hydrophobic amino acid residues at prime and nonprime positions, with
a preference for small hydrophobic residues such as valine (Val) at P1, giving the enzyme an
elastase-like specificity.17
We have previously demonstrated that CtHtrA is essential for Chlamydia survival using two
selective irreversible CtHtrA inhibitors, JO146 1 [Boc-Val-Pro-ValP(OPh)2; IC50 = 12.5 µM] and
JCP83 2 [Boc-Ala-Pro-ValP(OPh)2; IC50 = 47.19 µM; Figure 1] in in vivo18 and ex vivo19 chlamydia
models. JO146 was also found to be active against koala Chlamydia species (C. pecorum and C.
pneumoniae) with negligible toxicity on koala tissue19 thus validating CtHtrA as a viable drug
target for the development of novel anti-chlamydial drugs.
P3
P2
P1
TSA
O
O
O
O
H
H
O
O
O
N
O
N
N
N
P O
O
N
N
P O
O
H
H
O
O
1
Boc-Val-Pro-ValP(OPh)2
2
Boc-Ala-Pro-ValP(OPh)2
Figure 1. Structures of JO146 (1) and JCP83 (2) and the P3-P1 protease nomenclature.18 TSA –
Transition State Analogue.
JO146 (1, IC50 = 12.5 µM)18 is a suitable lead molecule for developing a clinically relevant protease
inhibitor for targeting Chlamydia. However, it is currently not potent enough, and may be
susceptible to chemical and enzymatic degradation due to its peptidic nature. This paper details
the strategies employed in optimizing the potency and selectivity of JO146 (1). Herein, we describe
the design, syntheses and structure activity relationships (SAR) of 23 analogues of JO146 (1) tested
against both human and koala Chlamydia species. The compounds span a series of diphenyl
phosphonates as well as reversible transition state analogues; namely α-ketoheterocycles, α-
4

JOURNAL PRE-PROOF
diketones, N-methylamides, valinol, α-ketoamide, and boronic acid. In addition, the P1 and P3
valine residues of JO146 (1) were systematically replaced with other natural and non-natural amino
acids with the aim of optimizing binding at the respective subpockets of CtHtrA.
2. Results and discussion
2.1 Development of new CtHtrA inhibitors
The syntheses of targeted tripeptide inhibitors containing various transition state analogues are
described in Schemes 1-3, S1 and S2. The diphenyl phosphonates JO146 (1) and 8a-h (Table1)
were synthesized in order to identify the optimal subsite specificity for P3 and P1. Compounds 1,
8a-h were synthesized according to the procedure reported by Winiarski et al.20 Cbz-protected P1
precursors 5a-d were synthesised via an α-amidoalkylation-type reaction using the appropriate
alkyl aldehyde 4a-d. They were subsequently deprotected to yield the hydrobromide salts 6a-d
using 33% HBr in acetic acid. The final compounds (1, 8a-h) were obtained by coupling 6a-d to
pre-synthesized dipeptide precursors 7a-d in an HBTU-promoted solution phase peptide coupling.
The products were obtained as diastereomeric mixtures and biologically evaluated as such.
Table 1. Peptidyl diphenyl phosphonate inhibitors of the form Boc-P3-Pro-P1-(OPh)2
P1
O
O
H
O
O
N
N
N
P O
O
H
P3
O
P3
P1
JO146 (1)
Val
Ala
Tle
Nva
Ile
Val
Val
Val
Val
Val
2
8a
8b
8c
5

JOURNAL PRE-PROOF
8d
8e
8f
Val
Val
Val
Ile
Ile
Nva
Tle
Ile
8g
8h
Tle
Ile
*A derivative of 1 with Leu at P1 has previously shown no inhibition (data not shown)
6

JOURNAL PRE-PROOF
Scheme 1. Synthesis of the peptidyl diphenyl phosphonates (8a-h)a
P1
O
Br
H3N
O
P1
O
P O
O
P
3
O
O
N
P O
a
b
H
O
3
5a-d
6a-d
O
O
4a
=
O
H
N
c
Boc
Boc
N
OH
P3
4b
4c
=
=
O
O
7a-d
O
O
P1
H
N
O
N
N
P O
O
H
P3
4d =
O
8a-h
aReagents and conditions: (a) Benzylcarbamate, 4a-d, 80-90 °C, 2 h, 15-72%; (b) 33% HBr/AcOH,
25 °C, 2 h, 92-98%; (c) HBTU, DIEA, DMF, 25 °C, 20 h, 25-77%.
The potential risk of toxicity associated with irreversible inhibition via the diphenyl phosphonate
group in JO146 led to the decision to investigate reversible transition state analogues as
alternatives. This entailed an exchange of the phosphonate moiety with less reactive groups
including α-ketoheterocycles (α-ketothiazole, α-ketobenzothiazole and α-keto-N-methyl
imidazole), α-diketones, α-ketoamide, boronic acid, and non-reactive N-alkyl methylamides and
valinol derivatives.
Potent peptidyl α-ketoheterocycle inhibitors of the human neutrophil elastase (HNE) as well as
thrombin have been reported.20-21 They possess the ability to form reversible covalent bonds as
well as making key interactions with the enzyme active site around P1'. Peptidyl α-ketoheterocycles
7

JOURNAL PRE-PROOF
14a-e (Table 2), were prepared via Weinreb amides 13a-e by first coupling the tripeptides 11a-e
to N,O-dimethyl-hydroxylamine hydrochloride 12 using HBTU as the reagent (Scheme 2). This
was followed by reacting 13a-e with the respective lithiated heterocycles. The final products 14a-e
1
13
were obtained as isomeric mixtures, characterized by additional peaks in the H and C NMR
spectra and two distinct peaks in the HPLC trace (see SI), and were likely the result of an n-
butyllithium-induced epimerization at the alpha-carbon (relative to the heterocycle).
Table 2. Peptidyl α-ketoheterocycle inhibitors
Compound
14a
P3
P1
Val
Nva
Val
Ile
Heterocycle
Thiazole
Val
Val
Val
Tle
Val
14b
Thiazole
14c
Benzothiazole
Benzothiazole
N-methylimidazole
14d
14e
Val
8

JOURNAL PRE-PROOF
Scheme 2. Synthesis of the peptidyl α-ketoheterocycles 14a-ea
O
O
O
O
P1
O
O
P1
H
H
N
H
N
b
OH
N
O
a
Boc
N
OH
Boc
N
N
Boc
N
N
H
H
P3
P3
O
P3
O
7a-b
11a-e
10a-e
+
O
H
H
O
N+
Cl-
c
H2N
O
12
P1
9a-c
O
O
P1
O
O
P1
H
H
N
Het
N
N
d
Boc
N
N
Boc
N
N
O
H
H
P3
O
P3
O
13a-e
14a-e
aReagents and conditions: (a) HBTU, DIEA, DMF, 25 °C, 20 h, 45-54%; (b) LiOH.H2O, THF,
H2O, 25 °C, 2 h, 78-90%; (c) HBTU, DIEA, DMF, 25 °C, 20 h, 28-63%; (d) heterocycle, n-BuLi,
THF, -78 °C, 28-54%. P3 = Val or Tle; P1 = Val, Nva or Ile
Compounds with a 1,2-dicarbonyl moiety such as α-ketoamides and α-diketones, are very good
serine traps and have been incorporated in potent and safe hepatitis C NS3 protease inhibitors used
clinically.22-24 Previous studies into CtHtrA specificity suggest a preference for hydrophobic
residues at P1',16-17 identifying glycine and isoleucine as the most favored residues. In the present
study, we utilized the ability of α-diketones to interact reversibly with the S1' pocket of CtHtrA
and the α-diketones 18a-c were synthesized with methyl, ethyl and cyclopropyl substituents at P1'.
The synthesis of compounds in the α-diketone series (Scheme 3) was conducted as reported by
Han et al.22 The aldehyde 15 was generated according to the method described by Dekhane and
Dodd.25 A solution of the Weinreb amide 13a in THF was treated with LiAlH4 under nitrogen,
which formed a stable metal complex, restricting further hydride addition that otherwise produces
the unwanted alcohol product. Upon a mild acidic workup, the complex dissociated, yielding the
9

JOURNAL PRE-PROOF
desired aldehyde 15. After column purification, 15 was recovered as a diastereomeric mixture, as
indicated by the additional peaks in the 1H and 13C NMR spectra. Peptide aldehydes are reported
to epimerize during purification, and this occurs irrespective of the purification method
employed.26 Compound 15 was then used as the starting material for the synthesis of the 1,2-diols
17a-c by pinacol cross coupling of 15 with the aliphatic aldehydes 16a-c as reported by
Freudenberger et al.27 Oxidation of the 1,2-diols with Dess-Martin periodinane (DMP) afforded
the peptidyl α-diketones 18a-c. Compounds 17a-c and 18a-c were all obtained as diastereomeric
mixtures by virtue of their enantiomerically impure aldehyde precursor (15).
10

JOURNAL PRE-PROOF
Scheme 3. Synthesis of the peptidyl α-diketones (18a-c)a
O
O
O
O
H
O
R
H
a
N
H
N
N
+
Boc
N
N
H
Boc
N
N
O
H
H
O
O
16a-c
15
13a
a
R
R
R
=
=
=
b
b
c
O
O
O
O
O
OH
R
c
H
H
N
N
Boc
N
N
R
Boc
N
N
H
H
O
OH
18a-c
17a-c
aReagents and conditions: (a) LiAlH4, THF, -30 °C, 1 h, 83%; (b) VCl3(THF)3, Zn, DCM, 25 °C,
2 h, 36-56%; (c) Dess-Martin periodinane (DMP), DCM, 25 °C, 20 min, 20-67%.
The peptidic N-methylamides 24a-d, and the valinol derivative 28 were synthesized as
noncovalent binders of CtHtrA. Compounds 24a-d were specifically designed with methyl, ethyl,
cyclopropyl and allyl side chains for exploring P1'. The N-methylamide series (24a-d) were
synthesized as outlined in Scheme S1 with a view to rapidly explore P1'. The N-alkylamide
precursors, 22a-d were prepared by coupling Boc-L-valine 19 to the appropriate N-methylamine
hydrochloride 21a-d via HATU-promoted amide coupling. Compounds 22a-d were then subjected
to Boc deprotection using TFA, followed by treatment with 10% KOH solution to liberate the free
amines 23a-d. Finally, 24a-d were obtained by coupling 23a-d to the pre-synthesized dipeptide
7a using HBTU. Conformational isomerization about the amide bond in the N-methyl amide series
24a-d was observed by NMR spectroscopy, thus purity of the final compounds was confirmed by
HPLC (see SI).
11

JOURNAL PRE-PROOF
The peptidic valinol derivative 28 was also synthesized as a replacement for the phosphonate
moiety (Scheme S2). Benzyl protected L-valinol 26 was coupled to 7a using HBTU to afford 27,
which was converted to the final compound 28 by hydrogenolysis using Pd catalysis. In CDCl3,
compound 28 was observed as an isomeric mixture, with additional 1H and 13C NMR peaks evident
in the spectra. However, HPLC analysis revealed only a single peak at tr = 32.5 min, indicative of
a conformation isomer, and not diastereomers.
Compounds 29 and 30 with a boronic acid and α-ketoamide transition state analogue respectively
(both present in protease inhibitors used clinically) were purchased and tested to investigate their
applicability in this system.28-29
2.2 Enzyme inhibition and anti-chlamydial activity
We set out to optimize the binding affinity of the inhibitor at the nonprime positions of JO146 by
stepwise replacement of the P1 and P3 valine residues (Table 1) with norvaline (Nva), isoleucine
and tertiary leucine (Tle). Compounds 8a-f, 14a-c, and 18a-c were thus synthesized and tested for
their biological activity. A bio-assessment of the compounds was conducted via protease inhibition
assays initially against human neutrophil elastase (HNE) and CtHtrA along with off-target
assessment carried out against the human proteases trypsin and chymotrypsin (Table 3). HNE
possesses a similar substrate specificity to CtHtrA where substrates consist of mainly small
hydrophobic residues such as valine and alanine, particularly at P1. Activity against HNE provides
a good prediction for anti-CtHtrA activity and this was used as an indicator of CtHtrA activity.30-
31
The in vitro susceptibility of C. trachomatis and koala C. pecorum isolates to all compounds
was measured by their ability to significantly reduce the pathogens’ infectious progeny, measured
as inclusion forming units (IFU/mL; Table 3; Figures 2 and 3).
12

JOURNAL PRE-PROOF
Table 3 CtHtrA Inhibitory activity and selectivity assessment
#
Compound structure
Site
CtHtrA
relative
inhibition
Elastase Trypsin Chymotrypsin
In vitro
Ctrach
Changea
IC50
IC50
IC50 (µM)
(µM)
(µM)
activityb
IFU/mL
4.34 × 102 ±
9.0× 10-4
O
O
1
H
N
1
0.83 ±
0.01
> 500
> 500
O
P O
O
O
N
N
H
(JO146)
O
Control
DMSO
6.2 × 106 ±
8.5× 105
Nonprime Site Optimization
O
O
O
O
O
O
8a
8b
8c
8d
H
N
P3
P3
P3
P1
33.33× more
active
0.68 ±
0.02
> 500
> 500
> 500
> 500
> 500
> 500
1.5 × 103 ±
216
O
P O
O
O
O
O
N
N
N
N
H
O
O
O
H
N
9.34× less
active
0.45 ±
0.09
1.7 × 106 ±
1.5 × 105
O
P O
O
N
H
H
N
14.29× more
active
0.40 ±
0.03
> 500
5.0 × 104 ±
7252
O
P O
O
N
H
20× more
active
0.36 ±
0.02
38.45 ± 0.51
9.2 × 103 ±
2660
O
O
H
N
O
P O
O
O
N
N
H
O
13

JOURNAL PRE-PROOF
8e
P1
NA
NA
0.68 ±
0.02
> 500
> 500
1.5 × 105 ±
30297
O
O
O
O
H
N
O
P O
O
O
O
N
N
N
H
O
O
8f
P1
> 500
> 500
> 500
> 500
2.1 × 104 ±
4220
H
N
O
P O
O
N
H
8g
P1 P3
13.09× less
active
0.84 ±
0.05
1.28 ± 0.06
1.1 × 104 ±
11565
O
O
O
O
H
N
O
P O
O
O
O
N
N
N
H
O
O
8h
P1 P3
NA
2.35 ±
0.22
> 500
0.53 ± 0.04
1.1 × 104 ±
2947
H
N
O
P O
O
N
H
Prime sites
O
O
O
S
S
14a
14b
TSA
TSA
NA
NA
ND
ND
ND
3.31 × 106 ±
3.9 × 105
H
N
O
N
N
N
N
H
O
O
> 150
> 500
> 500
ND
O
N
H
N
O
N
N
H
O
O
N
14c
14d
TSA
TSA
1.06 × more
active
2.60 ±
0.15
> 500
> 500
> 500
> 500
2.5 × 105 ±
4.3 × 104
O
O
S
H
O
N
N
H
O
O
O
NA
4.83 ±
0.08
2.8 × 105 ±
4.6 × 104
O
O
S
H
O
N
N
N
N
H
O
14

JOURNAL PRE-PROOF
O
O
O
O
O
O
O
O
O
O
O
18a
18b
18c
24a
P1’
P1’
P1’
P1’
NA
NA
NA
NA
1.35 ±
0.17
> 500
> 500
> 500
ND
> 500
> 500
> 500
ND
8.6 × 106 ±
1.1 × 105
H
N
O
O
O
N
N
N
N
H
O
O
O
O
O
O
4.71 ±
0.66
5.7 × 106 ±
5.2 × 105
H
N
N
H
ND
5.5 × 106 ±
1.5× 105
H
N
N
H
ND
ND
H
N
O
O
N
N
N
H
O
O
O
O
O
24b
24c
P1’
NA
NA
ND
ND
ND
ND
ND
ND
ND
H
N
N
N
N
N
H
O
O
O
P1’
ND
ND
ND
H
N
O
N
N
H
O
O
24d
O
O
P1’
NA
ND
ND
ND
H
O
N
N
N
N
H
O
O
28
TSA -
valinol
NA
ND
ND
ND
ND
ND
ND
O
O
H
O
N
N
N
H
O
OH
29a
TSA -
Activates
9.9 × 103 ±
1.6 × 103
boronic acid enzyme before
inhibiting not
able to fit
O
O
H
O
N
OH
N
N
B
H
O
OH
curve
15

JOURNAL PRE-PROOF
O
O
O
30
TSA - α-
ketoamide
NA
ND
ND
ND
ND
H
O
N
N
N
NH2
H
O
O
aCompounds differed from 1 by varying either the P3, P1 residue, the transition state analogue or a combination thereof. bC. trachomatis
susceptibility to inhibitors at a dose of 100 µM, 16 h PI. NA = not active in CtHtrA assay (see table S3 for details). ND = not determined,
Data are presented as the mean ± standard error of the mean (SEM) of triplicate readings. TSA= transition state analogue.
16

JOURNAL PRE-PROOF
2.3 P3-P1 optimization
P3 Optimization: Replacing valine with norvaline at P3 (8b) showed better activity at HNE (IC50
= 0.45 ± 0.09 µM; JO146 IC50 = 0.83 ± 0.01 µM) but reduced both CtHtrA (9.34 × less active,
Table 3) and cellular activity (C. trachomatis), with approximately 103.5 IFU reduction compared
with JO146 (1) at 100 µM (Figure 2). Isoleucine at P3 (8c) was more potent against HNE IC50 =
0.40 ± 0.03 µM) and approximately fourteen times more active than JO146 (1) (CtHtrA relative
inhibition; Table 3), but with less cellular activity (C. trachomatis) causing a reduction in IFU/mL
of 102 at 100 µM (Figure 2). Compound 8a with tertiary leucine at P3 showed improved HNE
activity (IC50 = 0.68 ± 0.02 µM) over JO146 (1) and was the most active P3-optimized analogue
yielding a 33-fold increase in relative CtHtrA inhibition compared to JO146 (1) (Table 3). The
cellular potencies of JO146 (1) and 8a were comparable (Figure 2).
C. pecorum displayed greater susceptibility to P3-optimized compounds 8a and 8c compared to C.
trachomatis. Against C. pecorum, 8a remained the most active analogue with approximately 104
reduction in IFU counts at 100 µM relative to the DMSO control. Similarly, 8c produced a
substantial reduction in IFU counts, accounting for 103 loss of infectious progeny at 100 µM.
However, compound 8b with norvaline at P3 showed only marginal activity against C. pecorum at
100 µM.
17

JOURNAL PRE-PROOF
Figure 2 C. trachomatis susceptibility to representative inhibitors (100 µM, 16 h PI) of different
covalent and noncovalent transition state analogues during mid-replicative phase of the pathogen’s
developmental cycle. Inclusion forming units counts (IFU/mL) were determined upon completion
of developmental cycle. Green (P3 variants); brown (P1 variants); purple (α-ketoheterocyclic
variants); beige (α-diketone variants); light blue and lilac (P3 and P1 variants); grey (P3 and P1 α-
ketoheterocycle). Error bars indicate the standard error of the mean obtained from experimental
replicates (minimum of 5).*Note in this assay 103 is the threshold for detection and can be
equivalent to too few to accurately quantify.
2.4 P1 Optimization: Of the compounds synthesized for P1 optimization, the analogue with
isoleucine at P1 (8d) showed potent HNE activity (IC50 = 0.36 ± 0.02 µM) and produced the highest
CtHtrA relative inhibition; exhibiting twenty-fold increase in potency compared to JO146 (1,Table
3). However, this activity was not extended to the cellular assays of the molecule at C. trachomatis
(Figure 2), as it only produced approximately 103 loss in infectious progeny at 100 µM, which was
less potent than JO146 (1). In addition, it also showed chymotrypsin inhibition (IC50 = 38.45 ±
0.51 µM) which is not surprising due to the preference of chymotrypsin for larger hydrophobic
18

JOURNAL PRE-PROOF
residues such as isoleucine at P1.32 The replacement of valine at P1 with norvaline (8e) and tertiary
leucine (8f), on the other hand, was completely devoid of any CtHtrA inhibition activity and the
latter also failed to inhibit HNE (Table 3). However, both 8e and 8f displayed promising cellular
activity at 100 µM. Compound 8f produced ~102.5 reduction in inclusion forming units (IFU)
counts against C. trachomatis while 8e yielded approximately 101.5 reduction in IFU counts
relative to the untreated DMSO control (Figure 2). C. pecorum was generally less susceptible to
compounds with P1 modification, yielding appreciably lower reduction in IFU counts for all three
compounds relative to their activity against C. trachomatis. Although 8e produced a surprising 103
reduction in C. pecorum IFU counts at 100 µM (Figure 3), against the 101.5 reduction recorded at
C. trachomatis (Figure 2). The lack of in vitro CtHtrA activity by 8e and 8f but with both molecules
displaying considerable cellular activity, suggests they may act via a different mechanism of
action.
Figure 3: Susceptibility of C. pecorum to CtHtrA inhibitors (100 µM; 16 h PI) during mid-
replicative phase of the pathogen’s developmental cycle. Infectious yield counts (IFU/mL) were
determined upon completion of developmental cycle. Colours the same as above in Figure 2. Error
19

JOURNAL PRE-PROOF
bars indicate the standard error of the mean obtained from experimental replicates (minimum of
5).
2.5 Replacement of Diphenylphosphonate
The effects of substituting the phosphonate moiety in JO146 (1) with reversible analogues were
evaluated by testing compounds 14a-e, 18a-c, 24a-d, and 28-30 for in vitro CtHtrA and HNE
inhibition, and trypsin and chymotrypsin to assess off-target selectivity (Table 3).
Among the reversible derivatives of JO146 (1), only the benzothiazole α-ketoheterocycle transition
state analogue (14c) showed viable anti-chlamydial activity when simply replacing the diphenyl
phosphonate group. The compound displayed CtHtrA relative inhibition comparable or slightly
better than JO146 (1, 1.06 × more active; Table 3) It was less active at the closely related HNE
(IC50 = 2.60 ± 0.15 µM; JO146 (IC50 = 0.83 ± 0.01)) implying improved selectivity over HNE
which is desirable. The inhibitor produced some cellular activity against C. trachomatis. Against
C. pecorum it produced a considerably greater reduction with approximately 4 log loss of
infectious bodies at 100 µM (Figure 3). The lack of anti-chlamydial activity displayed by the
thiazole (14a) and N-methyl imidazole (14e; data not shown) derivatives, is traceable to their
inability to sufficiently activate the ketone carbonyl carbon towards a nucleophilic attack by the
catalytic Ser247 of CtHtrA. These results align with previous studies by Edwards et al.33 which
identified peptidyl ketoheterocycle HNE inhibitors with imidazoles as incapable of forming a
covalent adduct and supports the necessity of a covalent interaction between the inhibitor and the
active site of CtHtrA for anti-chlamydial activity.
To understand the structural basis for the activity exhibited by the 14c benzothiazole α-
ketoheterocycle compared to 14a, both molecules were docked into a homology model of CtHtrA.
20

JOURNAL PRE-PROOF
The docking results (Figure 4) revealed more interaction between the benzothiazole moiety of 14c
and the S1' pocket of CtHtrA, with the heterocycle engaging in van der Waals interactions with
Val125 and Val144 that line the walls of the pocket, as well as a potential edge-to-face pi-stacking
interaction with His143. Compound 14a lacks the benzene extension and is therefore unable to
make these interactions. Any possible hydrogen bonding between the heterocyclic (thiazole)
nitrogen and the catalytic His143 of CtHtrA was revealed to be unfavourable, as the orientation of
the imidazole ring of His143 relative to the inhibitor thiazole ring makes it unlikely for hydrogen
bonding to occur. This may also contribute to the difference in the anti-chlamydial activity between
compounds 14a and 14c.
Figure 4. Binding poses of 14c, magenta (A) and 14a, cyan (B) to CtHtrA (transparent surface).
Oxygen is shown in red, nitrogen blue and sulphur in yellow.
2.6 P1' Optimization: In addition to replacing the phosphonate group, expansion and optimization
of P1' could be probed using α-diketone based inhibitors. Three inhibitors were synthesized (18a-
c) containing a methyl, ethyl or cyclopropyl group at P1’and showed no CtHtrA inhibition or
cellular activity (Table 3; Figures 2 and 3). This result is surprising given both 18a and 18b showed
inhibition against HNE albeit modest (IC50 = 1.35 ± 0.17 and IC50 = 4.71 ± 0.66 µM respectively)
and the α-diketone is present is some clinical HCV protease inhibitors.
21

JOURNAL PRE-PROOF
2.7 Combined optimization
Based on the in vitro HNE and CtHtrA relative inhibition results above, where isoleucine and
tertiary leucine were identified as optimal residues at P1 and P3 respectively, compounds retaining
the diphenyl phosphonate combining P3 and P1 optimisation 8g, 8h along with an α-
ketoheterocycle transition state analogue 14d were synthesized. Compound 8h produced
approximately 102.5 reduction in IFU counts at 100 µM (C. trachomatis) and a 103 reduction in
IFU counts against C. pecorum at 100 µM (Figure 3).
The benzothiazole analogue 14d failed to show in vitro CtHtrA inhibition with only moderate
HNE inhibition, but showed some cellular activity against C. trachomatis (Figure 2), and a slightly
improved activity at C. pecorum (103 reduction in IFU 100 µM; Figure 3). However, in contrast
to the phosphonate compounds 8g and 8h which exhibited high chymotrypsin inhibition with IC50
values of 1.28 ± 0.06 µM and 0.53 ± 0.04 µM respectively (Table 2); 14d showed no chymotrypsin
inhibition (IC50> 500 µM) despite differing only at the transition state analogue (Table 3).
As a measure of off-target effect, no inhibition of trypsin and chymotrypsin (IC50 > 500 µM) was
observed for most of the active compounds except 8d, 8g and 8h which have isoleucine at P1 and
the latter compound showing appreciable activity such to exclude it from further study. Compound
8g showed higher potency compared to 8h against HNE (IC50 = 0.84 ± 0.05 µM; IC50 = 2.35 ±
0.22µM) but only at a similar level to JO146.
The dicarbonyl-containing compounds 18a-c and 30 (α-diketone and α-ketoamide respectively)
exhibited an unexpected lack of biological activity. Both molecules lacked in vitro CtHtrA
inhibition. This was extended to negligible cellular performance of the peptidyl α-ketoamide 30
against C. trachomatis.
22

JOURNAL PRE-PROOF
Unusual activity was observed for the boronic acid inhibitor 29. The compound activated CtHtrA
at doses less than 50 µM, but displayed enzyme inhibition at higher doses (Figure S1). At 100 µM,
it produced a 3 log reduction in IFU count against C. trachomatis (data not shown). Whilst the
precise mechanism for the initial CtHtrA activation is unclear, the presence of the boronic acid
moiety mimics a carboxylate terminus of a peptide substrate, which would bind to the PDZ1
domain of CtHtrA and thus activating it towards proteolysis.17
Generally, the noncovalent binders (peptidyl N-methylamides 24a-d, and the peptidyl valinol 28)
lacked both biochemical (IC50 > 500µM) and cellular activity, indicating a covalent interaction
between the inhibitor and the active site of CtHtrA is crucial for optimal activity.
3. Conclusion
We have found that inhibitors are required to interact with CtHtrA via a covalent interaction, either
through reversible or irreversible transition state analogues. This is evident in both in vitro and in
vivo data. The most potent compound identified was 8a (CtHtrA relative inhibition) which has
retained the diphenylphosphonate moiety with the related compound 8d showing the highest
activity against HNE. The identification of the α-ketobenzothiazole moiety as a selective reversible
transition state analogue with in vitro activity against both human and koala pathogenic Chlamydia
species, opens up another possible avenue to obtain more “drug-like” inhibitors of CtHtrA. This
moves away from the previously identified irreversible diphenylphosphonate inhibitors. It also
indicates that P1' optimization via the use of α-ketoheterocycles is a potential strategy for improved
potency and selectivity.
Optimization of binding affinity at CtHtrA subpockets via modification to inhibitor residues P1
and P3 produced considerable improvement in potency and/or selectivity. Selectivity was obtained
23

JOURNAL PRE-PROOF
over, trypsin and chymotrypsin and likely HNE, in addition to retaining cellular activity
comparable with JO146 (1), when tertiary leucine was tested as the P3 residue (8a). This is also a
viable means to obtain molecules with favourable pharmacokinetic properties, given that tertiary
leucine is a non-natural amino acid, and may avoid recognition by degradative enzymes, thus
potentially enhancing inhibitor performance in vivo. After testing numerous derivatives, valine
remains the best residue at P1 for in vivo activity. Outside of selectivity, there seems no explanation
as to why the α-diketoamide transition state analogues and the related α-diketone derivatives
lacked activity given the use of the former in clinical protease inhibitors. It may be related to the
more polar character as compared to the diphenyl phosphonates.
CtHtrA is a complicated enzyme, which makes simple comparisons difficult. The enzyme is
known to exist in at least 3 oligomeric states, an inactive hexamer and, following activation of the
PDZ domain by a peptide terminal carboxylate, active 12-mers and 24-mers.16-17, 34 In addition,
the covalent nature of the inhibitors complicates the use of explicit IC50 values hence the use of
relative values.35 The allosteric activation of multiple monomers in a hexamer to then
accommodate inhibitor binding adds further complexity. This is illustrated with the behaviour of
the boronic acid derivative (29) which acted as both an activator and inhibitor (Figure S1).
JO146 remains the most active inhibitor in terms of cellular performance, however potent
analogues with malleable structural features and promising pharmacokinetic prospects have been
identified. Further optimisation at P2 (proline) and P4 (Boc) to increase potency and reduce peptidic
character is the next obvious step in moving towards clinically relevant protease inhibitors for the
treatment of human and animal chlamydiosis.
24

JOURNAL PRE-PROOF
4. Experimental
4.1. Compound Synthesis
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 purification. 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.
Silica gel 60 (0.040-0.063 mm, 200-400 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 and 13C NMR spectra were recorded at room temperature on Varian 400 or 500 MHz
spectrometers. Samples were prepared in deuterated solvents, chloroform (δ 7.26, 77.16 ppm) or
acetonitrile (δ 1.95, 118.69 and 1.72 ppm) with the respective 1H and 13C chemical shifts shown
in brackets. Chemical shifts (δ) are expressed in parts per million (ppm) and coupling constants
(J) in Hertz (Hz), both measured against the residual solvent peak or internal standard
tetramethylsilane (TMS). High resolution mass spectrometry were 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 µm C18 110 Å column
(250 × 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
25

JOURNAL PRE-PROOF
gradient (40% ACN to 85% ACN over 45 min). Compounds were detected at 254/210 nm with a
flow rate of 1.0 mL/min. The purity of compound 28 was determined on a Shimadzu HPLC with
a Gemini 5 µm C18 110 Å column (250 × 4.6 mm, Phenomenex, New Zealand). The compound
was eluted using solvent A: 0.1% formic acid in water and solvent B: 0.1% formic acid in ACN in
a linear gradient from 30 to 60% ACN over 60 min. The flow rate was 1.2 mL/min and compounds
were detected at the wavelength of 224 nm. Final compounds including those obtained as isomeric
mixtures, are >95% pure unless otherwise stated, see supporting information for HPLC traces of
the compounds.
4.1.2. General Procedure for Synthesis of peptidyl phosphonates (1, 8a-h)20
Compounds 7a-d (1 equiv.) were dissolved in 3 mL DMF. To this O-(Benzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluoro-phosphate (HBTU) (1.2 equiv.) and N,N-
Diisopropylethylamine (DIEA) (2.5 equiv.) were added to generate the activated esters. After 10
min, compounds 6a-d (1.5 equiv.) were added and the reaction mixtures were stirred under
nitrogen overnight. The mixtures were diluted with EtOAc (25 mL) and an equal amount of sodium
bicarbonate (NaHCO3), washed with brine (6 × 25 mL), dried (MgSO4) and concentrated in vacuo.
The crude products were purified by column chromatography (100% EtOAc). This yielded 1, 8a-
13
h, as diastereomeric mixtures with characteristic C-31P coupling. We have previously reported
the full NMR and mass spectrometric characterization of JO146 1, as part of the stereochemical
investigation of its two diastereomers.36 The analogues 8a-h are characterized as follows with 13C-
31P J values reported where clearly evident.
26

JOURNAL PRE-PROOF
Tert-butyl-N-[(2S)-1-[(2S)-2-{[1-(diphenoxyphosphoryl)-2-
methylpropyl]carbamoyl}pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamate (8a)
Using general procedure above, 0.17 g (0.52 mmol)
of 7b was coupled to 0.30 g (0.78 mmol) of 6a to
yield a brown oil, which formed a brown solid foam
under reduced pressure (0.08 g, 25%). ~94% purity
O
O
H
O
P O
O
O
N
N
N
H
O
by HPLC; tr = 38.234 and 38.651 min. 1H NMR (400 MHz, CDCl3) δ 7.37 – 7.24 (m, 5H), 7.20 –
7.11 (m, 5H), 5.44 (d, J = 9.9 Hz, 0.5H), 5.25 (d, J = 9.9 Hz, 0.5H), 4.80 – 4.70 (m, 1H), 4.55 –
4.48 (m, 1H), 4.35 – 4.26 (m, 1H), 3.83 – 3.51 (m, 2H), 2.48 – 2.33 (m, 2H), 2.10 – 1.81 (m, 3H),
13
1.44 – 1.40 (m, 9H), 1.15 – 1.04 (m, 6H), 1.00 – 0.91 (m, 9H). C NMR (101 MHz, CDCl3) δ
172.9, 171.9, 171.7 (d, JC-P = 14.0 Hz), 171.4 (d, JC-P = 5.8 Hz), 155.72, 155.65, 150.4 (d, JC-P
=
10.0 Hz), 150.2, 150.1, 150.0, 130.9, 129.8 (d, JC-P = 1.0 Hz), 129.71 (d, JC-P = 1.0 Hz), 129.65 (d,
JC-P = 0.9 Hz), 129.60 (d, JC-P = 0.9 Hz), 125.24, 125.14 (d, JC-P = 1.1 Hz), 125.07 (d, JC-P = 0.8
Hz), 120.8 (d, JC-P = 4.0 Hz), 120.64 (d, JC-P = 4.4Hz), 120.63 (d, JC-P = 4.0 Hz), 120.4 (d, JC-P
=
4.3 Hz), 79.7, 79.5, 60.4, 60.1, 58.3, 51.2 (d, JC-P = 154.8 Hz), 51.0 (d, JC-P = 155.6 Hz), 48.61,
48.58, 35.7, 35.5, 35.2, 29.7, 29.33 (d, JC-P = 3.9 Hz), 29.25 (d, JC-P = 3.9 Hz), 28.37, 28.35, 28.3,
27.9, 27.6, 26.4, 26.3, 26.2, 25.4, 25.0, 20.57 (d, JC-P = 13.9 Hz), 20.28 (d, JC-P = 13.8 Hz), 18.0
(d, JC-P = 4.7 Hz), 17.9 (d, JC-P = 4.4 Hz). ESI-MS (m/z): calcd for C32H46N3NaO7P (M + Na) m/z
638.2971 found 638.2918.
27

JOURNAL PRE-PROOF
Tert-butyl-N-[(2S)-1-[(2S)-2-{[1-(diphenoxyphosphoryl)-2-methylpropyl]carbamoyl}
pyrrolidin-1-yl]-1-oxopentan-2-yl]carbamate (8b)
Using general procedure above, 0.08 g (0.25 mmol)
of 7c was coupled to 0.15 g (0.38 mmol) of 6a to
yield a brown oil which formed a brown solid foam
under reduced pressure (0.10 g, 67%). HPLC tr =
O
O
H
O
O
N
N
N
P O
O
H
O
33.679 and 34.581 min. 1H NMR (400 MHz, CDCl3) δ 7.56 – 7.44 (m, 1H), 7.34 – 7.09 (m, 10H),
5.27 (d, J = 9.3 Hz, 0.5H), 5.12 (d, J = 9.0 Hz, 0.5H), 4.78 – 4.65 (m, 2H), 4.56 – 4.32 (m, 1H),
3.74 – 3.52 (m, 2H), 3.45 – 3.36 (m, 1H), 2.47 – 2.27 (m, 2H), 2.20 – 2.08 (m, 1H), 2.02 – 1.75
(m, 3H), 1.64 – 1.50 (m, 2H), 1.46 – 1.39 (m, 9H), 1.13 – 0.97 (m, 6H), 0.93 – 0.65 (m, 3H). 13C
NMR (101 MHz, CDCl3) δ 173.9, 173.1, 171.0, 170.9 (d, JC-P = 7.1 Hz), 155.6, 155.5, 150.42 (d,
JC-P = 9.1 Hz), 150.3, 150.2, 150.0 (d, JC-P = 9.1 Hz), 129.83 (d, JC-P = 1.1 Hz), 129.76 (d, JC-P
=
1.1 Hz), 129.73 (d, JC-P = 1.1 Hz), 129.67 (d, JC-P = 0.8 Hz), 129.63, 125.32 (d, JC-P = 2.0 Hz),
125.26 (d, JC-P = 1.3 Hz), 125.10, 120.73 (d, JC-P = 4.1 Hz), 120.72 (d, JC-P = 4.1 Hz), 120.4 (d,
JC-P = 4.4 Hz), 79.66, 59.90, 59.69, 51.1 (d, JC-P = 154.6 Hz), 50.9 (d, JC-P = 130.6 Hz), 47.4,
47.3, 35.6, 35.1, 29.2 (d, JC-P = 4.1 Hz), 28.34, 28.33, 26.83, 26.81, 25.2, 25.0, 20.5 (d, JC-P = 13.8
Hz), 20.3 (d, JC-P = 14.1 Hz), 18.77, 18.75, 17.9 (d, JC-P = 4.6 Hz), 17.8 (d, JC-P = 4.3 Hz), 13.7,
13.4. ESI-MS (m/z): calcd for C31H44N3NaO7P (M + Na) m/z 624.2815 found 624.2784.
Tert-butyl-N-[(2S)-1-[(2S)-2-{[1-(diphenoxyphosphoryl)-2-
methylpropyl]carbamoyl}pyrrolidin-1-yl]-3-methyl-1-oxopentan-2-yl]carbamate (8c)
Using general procedure above, 0.25 g (0.76 mmol)
of 7d was coupled to 0.35 g (1.1 mmol) of 6a to
yield a brown oil which formed a brown solid foam
O
O
H
O
O
N
N
N
P O
O
H
O
under reduced pressure (0.23 g, 49%). HPLC tr =
28

JOURNAL PRE-PROOF
1
37.496 and 37.939 min. H NMR (400 MHz, CDCl3) δ 7.40 (d, J = 10.4 Hz, 0.5H), 7.27 – 7.17
(m, 5H), 7.17 – 7.08 (m, 5.5H), 5.27 (d, J = 9.5 Hz, 0.5H), 5.11 (d, J = 9.5 Hz, 0.5H), 4.84 – 4.64
(m, 2H), 4.51 – 4.43 (m, 1H), 4.31 – 4.13 (m, 1H), 3.78 – 3.50 (m, 2H), 2.42 – 2.04 (m, 2H), 2.00
– 1.63 (m, 3H), 1.60 – 1.47 (m, 2H), 1.43 – 1.32 (m, 9H), 1.11 – 0.97 (m, 6H), 0.92 (m, 2H), 0.85
– 0.76 (m, 3H), 0.71 – 0.65 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 172.6, 171.3, 171.2, 155.7,
155.6, 150.4, 150.3, 150.2, 150.1 (d, JC-P = 9.0 Hz), 150.0, 129.7, 129.62, 129.57, 125.25 (d, JC-P
= 1.0 Hz), 125.20 (d, JC-P = 1.2 Hz), 125.07 (d, JC-P = 0.8 Hz), 125.04 (d, JC-P = 1.1 Hz), 120.7 (d,
JC-P = 4.1 Hz), 120.6 (d, JC-P = 4.0 Hz), 120.42, 120.37, 120.3, 79.6, 79.5, 60.3, 60.0, 56.2, 56.1,
51.3 (d, JC-P = 154.6 Hz), 51.0 (d, JC-P = 154.6 Hz), 48.0, 47.8, 37.83, 37.75, 29.3 (d, JC-P = 3.6
Hz), 29.2 (d, JC-P = 3.8 Hz), 28.30, 28.29, 27.4, 25.2, 25.0, 24.3, 24.0, 20.5 (d, JC-P = 13.3 Hz),
20.2 (d, JC-P = 13.7 Hz), 18.0 (d, JC-P = 5.1 Hz), 17.9 (d, JC-P = 4.6 Hz), 15.6, 15.2, 11.1, 10.9. ESI-
MS (m/z): calcd for C32H46N3NaO7P (M + Na) m/z 638.2971 found 638.2911.
Tert-butyl-N-[(2S)-1-[(2S)-2-{[1-(diphenoxyphosphoryl)-2-
methylbutyl]carbamoyl}pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]carbamate (8d)
Using general procedure above, 0.10 g (0.32 mmol)
O
O
H
O
P O
O
of 7a was coupled to 0.15 g (0.48 mmol) of 6d to
yield a brown oil which formed a brown solid foam
under reduced pressure (0.06 g, 31%). HPLC tr =
O
N
N
N
H
O
37.845, 38.674 and 39.275 min. 1H NMR (400 MHz, Chloroform-d) δ 7.46 – 7.35 (m, 1H), 7.32 –
7.10 (m, 10H), 5.36 (d, J = 9.6 Hz, 0.5H), 5.20 (d, J = 9.4 Hz, 0.5H), 4.96 – 4.68 (m, 2H), 4.52 –
4.44 (m, 1H), 4.32 – 4.21 (m, 1H), 3.79 – 3.41 (m, 2H), 2.47 – 2.37 (m, 0.5H), 2.20 – 2.03 (m,
13
2.5H), 2.02 – 1.74 (m, 4H), 1.49 – 1.36 (m, 9H), 1.16 – 1.02 (m, 3H), 0.99 – 0.78 (m, 9H). C
NMR (101 MHz, CDCl3) δ 173.43, 173.37, 172.44, 172.39, 171.3 (d, JC-P = 6.5 Hz), 171.22 (d,
JC-P = 6.5 Hz), 171.19 (d, JC-P = 5.7 Hz), 171.11, 171.06, 155.83, 155.80, 150.49, 150.47, 150.39,
29