Small Molecule Inhibitors of Cyclophilin D to Protect Mitochondrial Function as a Potential Treatment for Acute Pancreatitis

Article abstract of DOI:10.1021/acs.jmedchem.5b01801

Opening of the mitochondrial permeability transition pore (MPTP) causes mitochondrial dysfunction and necrosis in acute pancreatitis (AP), a condition without specific drug treatment. Cyclophilin D (CypD) is a mitochondrial matrix peptidyl-prolyl isomerase that regulates the MPTP and is a drug target for AP. We have synthesized urea-based small molecule inhibitors of cyclophilins and tested them against CypD using binding and isomerase activity assays. Thermodynamic profiles of the CypD/inhibitor interactions were determined by isothermal titration calorimetry. Seven new high-resolution crystal structures of CypD-inhibitor complexes were obtained to guide compound optimization. Compounds 4, 13, 14, and 19 were tested in freshly isolated murine pancreatic acinar cells (PACs) to determine inhibition of toxin-induced loss of mitochondrial membrane potential (δΨ_m) and necrotic cell death pathway activation. Compound 19 was found to have a K_d of 410 nM and a favorable thermodynamic profile, and it showed significant protection of δΨ_m and reduced necrosis of murine as well as human PACs. Compound 19 holds significant promise for future lead optimization.

Full text of DOI:10.1021/acs.jmedchem.5b01801

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES
Article
Small Molecule Inhibitors of Cyclophilin D to Protect Mitochondrial
Function as a Potential Treatment for Acute Pancreatitis
Emma Shore, Muhammad Awais, Neil Kershaw, Robert Gibson, Sravan Pandalaneni, Dianne Latawiec,
Li Wen, Muhammad Ahsan Javed, David Criddle, Neil Berry, Paul O'Neill, Lu-Yun Lian, and Robert Sutton
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01801 • Publication Date (Web): 07 Mar 2016
Downloaded from http://pubs.acs.org on March 8, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Small Molecule Inhibitors of Cyclophilin D to Protect Mitochondrial Function
as a Potential Treatment for Acute Pancreatitis
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
2
1
3
Emma R. Shore , Muhammad Awais , Neil M. Kershaw , Robert R. Gibson , Sravan
3
2
2
2
2
Pandalaneni , Diane Latawiec , Li Wen , Muhammad A. Javed , David N. Criddle ,
1
1*
3*
2
Neil Berry , Paul M. O’Neill , LuꢀYun Lian and Robert Sutton
1
Department of Chemistry, University of Liverpool, Crown Street, Liverpool L69 7ZD,
NIHR Liverpool Pancreas Biomedical Research Unit, Institute of Translational
2
Medicine, University of Liverpool, Royal Liverpool University Hospital, Daulby Street,
3
Liverpool L7 8XP and Institute of Integrative Biology, Biosciences Building,
University of Liverpool, Crown Street, Liverpool L69 7ZD, United Kingdom
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
Opening of the mitochondrial permeability transition pore (MPTP) causes
mitochondrial dysfunction and necrosis in acute pancreatitis (AP), a condition without
specific drug treatment. Cyclophilin D (CypD) is a mitochondrial matrix peptidylꢀprolyl
isomerase that regulates the MPTP and is a drug target for AP. We have
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
synthesised ureaꢀbased small molecule inhibitors of cyclophilins and tested them
against CypD using binding and isomerase activity assays. Thermodynamic profiles
of the CypD/inhibitor interactions were determined by isothermal titration calorimetry.
Seven new highꢀresolution crystal structures of CypDꢀinhibitor complexes were
obtained to guide compound optimisation. Compounds 4, 13, 14 and 19 were tested
in freshly isolated murine pancreatic acinar cells (PACs) to determine inhibition of
toxinꢀinduced loss of mitochondrial membrane potential (∆Ψ
death pathway activation. Compound 19 was found to have a K
favourable thermodynamic profile, showed significant protection of ∆Ψ
m
) and necrotic cell
of 410 nM and
and reduced
d
m
necrosis of murine as well as human PACs. Compound 19 holds significant promise
for future lead optimisation.
ACS Paragon Plus Environment

Page 3 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
Acute pancreatitis (AP) is a common pancreatic disease predominantly caused by
gallstones or excessive alcohol intake. Pancreatic necrosis, systemic inflammatory
response syndrome, multiple organ failure and sepsis are characteristic of severe
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
, 2
AP, which results in the death of one in four patients.
Despite approximately 300
randomised clinical trials in human AP and about 700 publications evaluating agents
in preclinical studies, there is still no internationally validated specific drug therapy for
3
human AP.
Mitochondrial dysfunction is central to the pathogenesis of AP as well as other
diseases including ischemiaꢀreperfusion injury of the heart, brain and kidney,
4ꢀ10
muscular dystrophies and neuroꢀdegeneration.
Mitochondrial dysfunction is the
result of a sudden increase in permeability of the inner mitochondrial membrane
(IMM), via persistent opening of a multiꢀprotein channel known as the mitochondrial
6
permeability transition pore (MPTP). This allows uncontrolled proton flow across the
IMM and unregulated flux of water, ions and solutes up to 1.5 kDa into and out of the
mitochondrial matrix. A resultant loss of mitochondrial membrane potential (∆Ψ
m
)
essential for ATP production, coupled with disruption of calcium homeostasis,
6, 11, 12
activates the necrotic cell death pathway.
In 1984 Fischer et al. found a protein that accelerated efficiently the cis-trans
13
isomerization of prolyl peptide bonds in short oligopeptides. This protein was
named peptidylꢀprolyl cisꢀtrans isomerase (PPIase) which catalyses a 180 degree
14
rotation about the CꢀN linkage of the peptide bond preceding proline. Later, this
was shown to be the protein called cyclophilin which binds very strongly to
15
Cyclosporine A (CsA) (Figure 1).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
6
Cyclophilin D (CypD) is the mitochondrial matrix protein with PPIase activity. It
16ꢀ18
is best recognised as an important regulator of the MPTP.
We recently reported
that MPTP opening is critical to multiple forms of AP, causing diminished ATP
production, defective autophagy, zymogen activation, cytokine release and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
necrosis. Pharmacological or genetic MPTP inhibition in murine or human PACs
m
preserved ∆Ψ , ATP production, autophagy and prevented necrosis in response to
3
pancreatitis toxinꢀinduced calcium release via inositol trisphosphate receptor (IP R)
4
and ryanodine receptor (RyR) calcium channels. This mechanism was confirmed in
four in vivo models of AP. Thus, characteristic local and systemic pathological
responses were greatly reduced or abolished in CypD knockout mice and in wild
type mice treated with MPTP inhibitors, confirming that mitochondrial dysfunction
through MPTP opening is a fundamental pathological mechanism in AP, which can
4, 16
be ameliorated by inhibition of CypD.
Cyclosporin A (CsA), a lipophilic cyclic peptide (Figure 1), has nanomolar binding
19, 20
affinity for cyclophilins (Cyps), most notably CypA, CypB and CypD.
CsA is an
immunosuppressant and is widely used as an antiꢀrejection drug in solid organ
transplantation. The interaction of CsA with cytosolic CypA generates a complex that
has an ability to bind to, and inhibit calcineurin. As a consequence, the calcineurin
substrate, phosphoꢀnuclear factor of activated Tꢀcells (pNFAT), is unable to
21
translocate to the nucleus and initiate an immune response.
Nonꢀ
immunosuppressant semiꢀsynthetic analogues of CsA such as Debio 025 and
20
NIM811 (Figure 1) maintain inhibition of Cyps but do not bind to calcineurin.
However, these inhibitors have unfavourable drugꢀlike characteristics with high
22
molecular weights, limited solubility and poor bioavailability. Small molecule
ACS Paragon Plus Environment

Page 5 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
inhibitors are needed that exhibit high selectivity for CypD over other Cyps, have
improved pharmacokinetic/pharmacodynamic (PK/PD) properties and do not exhibit
immunosuppression, to treat acute pancreatitis and other diseases in which
mitochondrial dysfunction has a major role.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A number of compounds have previously been identified as potent inhibitors of
23ꢀ26
Cyps, several with nanomolar activities.
We synthesised and tested compounds
1
-4 shown in Figure 2. In our hands, the poorly soluble analogues 1-3 gave variable
results in both PPIase and calcium retention capacity (data not shown) assays
making them unsuitable for further optimisation. On the other hand, compound 4
gave consistent results when tested for PPIase inhibition in the presence and
absence of the detergent NP40. These preliminary data accord with those of
Colliandre et al., who have disclosed a range of Cyp inhibitors based on a urea
24, 25
template in two separate patents.
They identified compound 4 to exhibit
moderate binding selectivity for CypB and CypD over CypA with an IC50 of 6.1, 6.2
and 16.8 ꢁM, respectively. The authors also determined the Xꢀray structure of
compound 4 in complex with CypD (PDB: 3RDC).
CypD has two adjacent binding pockets (S1’ and S2, Figure 3a), both of which
might be exploited. Based on the crystal structures of CypA and peptides/proteins
(
PDB: 1FGL, 1AK4, 1AWR), the S1' site is where the proline binds (Figure 3b).
Residues around the S2 pocket interact with peptide residues ꢀ2 and ꢀ3 relative to
the native proline substrate. In the complexes of CsA with CypA and D (PDB: 1IKF,
2Z6W), Mva (NꢀmethylꢀLꢀvaline) occupies the S1’ pocket and superimposes well with
the proline residue of the peptide ligand. Aba (Lꢀαꢀaminobutyric acid) is located in the
S2 site and the Bmt ((4R)ꢀ4[(E)ꢀ2ꢀbutenyl]ꢀ4,NꢀdimethylꢀLꢀthreonine) binding defines
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the S1 site (Figure 3c). Compound 4 binds in the S2 and S1’ binding pockets (PDB:
3RDC), and is schematically depicted in Figure 3d.
Here we report the development of ureaꢀbased inhibitors of CypD starting from
compound 4. We aimed to identify an optimal template for the S2 and S1' sites by
first addressing S2 to allow for further development in a rational manner. The
interactions between the synthesised compounds and sites S2 and S1’ of CypD
were systematically characterised using Xꢀray crystallography to obtain highꢀ
resolution structures of the complexes and isothermal titration calorimetry (ITC) to
derive the thermodynamic signature of the interactions. PPIase assays were
performed for selected compounds and the most promising analogues were tested in
freshly isolated murine and human PACs for their ability to inhibit pancreatitis toxinꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
m
induced loss of ∆Ψ and necrotic cell death pathway activation. Through the
synthesis of enantiomerically pure compounds, we were able to resolve which
enantiomer affords optimal potency. The outcome of these studies demonstrated
that the enthalpyꢀentropy compensation poses a challenge in the rational structureꢀ
based design of potent inhibitors, even though highꢀresolution Xꢀray crystal
structures of the complexes were available at each stage of the design process.
RESULTS AND DISCUSSION
In-vitro inhibition studies of compounds 1-4. Compounds 1 and 2 were
identified and reported by Ni et al. to have IC50 values of 1.52 nM and 159 nM,
23
respectively, in a chymotrypsinꢀcoupled PPIase assay with CypA. Notably, 1 was
highlighted as being a more potent CypA inhibitor than CsA. Guichou et al. described
a range of diarylureas as CypA inhibitors, identifying 3 to be the most promising lead
26
with an IC50 of 14 nM in a chymotrypsinꢀcoupled PPIase assay. Compound 4, a
ACS Paragon Plus Environment

Page 7 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
urea based template, was identified by Colliandre et al. Although the activity was
modest in comparison with those reported for compounds 1ꢀ3, some desirable
24, 25
selectivity for CypD over CypA was reported.
The same group deposited an Xꢀ
ray crystal structure of 4 in the active site of CypD (PDB: 3RDC), enabling structureꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
based drug design approaches to be adopted.
We performed PPIase assays against CypD with compounds 1-4 in the absence
and presence of a detergent to identify nonꢀspecific inhibitors. Disappointingly, 1ꢀ3
did not exhibit any reproducible activity against CypD, most likely due to
promiscuous behaviour as their interaction with the protein involved aggregation of
2
7, 28
the compounds.
and Xꢀray crystallography for further analyses. However, 4 had a K
detergent, which was only slightly perturbed upon addition of detergent. Furthermore
using ITC, 4 bound to CypD with a K of 10.0 ꢁM, consolidating the result observed
Additionally, extremely low solubilities precluded the use of ITC
i
of 2.9 ꢁM without
d
in the PPIase assay. On the basis of these results, 4 was taken forward for further
investigations.
Molecular docking of compound 4 and analogues. An undesirable feature of
29
4 is the aniline functional group that is a known metabolic alert; hence, the
chemical space around the aniline was probed to determine other substituents that
might be tolerated. Additionally, as the ester is a metabolically labile functional
group, amide replacements were investigated to improve chemical and metabolic
30
stability. Molecular modelling was used to predict both the mode and strength of
compound binding. A docking protocol was developed that reproduced the binding
pose of 4 with CypD (RMSD, 0.53Å) accurately. Once validated, this protocol was
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
employed to dock the compounds in Table 1. All compounds bound in a very similar
fashion to 4. The ChemPLP binding scores were very similar to 4 (see Supporting
Information S1), providing confidence in the synthesis of a library of compounds,
representative examples of which are shown in Table 1. ITC, PPIase inhibition and
Xꢀray crystallographic studies were undertaken for these compounds.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Chemical synthesis of compound 4 analogues. Compounds 5ꢀ9 were
synthesised by reaction of the appropriate amine with ethyl isocyanatoacetate
(Scheme 1A). An alternative strategy was employed for the synthesis of 10ꢀ19
wherein 4ꢀaminobenzylamine was first converted to the corresponding CDIꢀadduct.
This was then coupled to a variety of amino esters and amides to afford the requisite
ureas (Scheme 1B). Table 1 summarises the chemical structures of these
compounds.
Guided by the Colliandre patent, which demonstrates high potency for 2ꢀaryl
pyrrolidine analogues, we sought to define the absolute stereochemical requirements
for good inhibition of CypD as well as to determine the thermodynamic
24, 25
characteristics and binding pose.
The chiral orthoꢀsubstitutedꢀ2ꢀarylpyrrolidine
motif present in 12, 13, 18 and 19 represented a significant synthetic challenge.
Whilst the synthesis of chiral 2ꢀarylpyrrolidines via asymmetric hydrogenation of
cyclic enamines is well established, orthoꢀsubstituents on the aromatic ring are
31, 32
poorly tolerated.
Instead, a strategy relying on asymmetric reduction of an
acyclic precursor was devised and is shown in Scheme 2. Addition of the appropriate
Grignard reagent to commercially available NꢀBocꢀ2ꢀpyrrolidinone afforded the
protected amino ketone 20. Enantioselective reduction using the CBSꢀ
ACS Paragon Plus Environment

Page 9 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
oxazaborolidine reagent established the necessary stereocentre in 21 with good
enantiomeric ratios. Conversion of the alcohol to the mesylate gave 22, amine
deprotection and cyclisation gave the desired 2ꢀarylpyrrolidines 23a and 23b in
excellent yield. 23c was synthesised in an analogous fashion using the enantiomeric
CBSꢀoxazaborolidine reagent. 23 was then coupled to BocꢀGlyꢀOH or BocꢀMetꢀOH
to yield 24 and 25, respectively.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Isothermal titration calorimetry (ITC). Figure 4 shows a subset (compounds 4,
10, 12, 14, 11 and 19) of ITC profiles for the binding of synthesised compounds. The
experimental data were analysed using a singleꢀsite binding model and in all cases
the compounds bound with a stoichiometry of 1:1, as is also observed in the
respective crystal structures.
(i) Compounds 4-9 (S2 site). Of all the compounds tested to probe the S2 site,
compound 4 displayed the best affinity. Modifications of the aniline portion of the
molecule indicated that this portion of the binding site is highly sensitive to
modification. The data summarised in Table 1 and Figure 5a show 4 binding in an
enthalpicallyꢀdriven manner with ∆G ~ ꢀ6.8 kcal/mol, a ∆H ~ ꢀ13.7 kcal/mol and ꢀT∆S
~ 6.9 kcal/mol, similar to CsA. In the CypDꢀ4 crystal complex, a water molecule
forms a network of hydrogen bonds involving the amine group of the aniline moiety
and residues A101, G109 and Q111, whereas with 5ꢀ9 these hydrogen bonds are
not formed. Work is currently in progress to more fully explore the structureꢀactivity
relationships of this site.
(ii) Compounds 10-13 (S1' site). The thermodynamic parameters for 10-13 are
shown in Figure 5b. The pyrrolidine in 10 sits in the active site (PDB 4J58) at the
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
same position as the proline residue in the ‘natural’ substrate. The significant
improvement in entropic contributions to binding of 10 over 4 is negated by a
33
compensatory loss of enthalpy. In 4, a hydrogen bond is formed between the ether
oxygen of the ethyl ester and Arg55. The absence of this oxygen in 10 results in the
loss of this hydrogen bond, hence, a much smaller observed ∆H value. Addition of a
phenyl group to the 2ꢀposition of the pyrrolidine ring, compound 11, resulted in an
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
d
improved K of 5.4 ꢁM.
The Cꢀ2ꢀaryl analogues reported by Colliandre et al. had undefined
24, 25
stereochemistry in the majority of cases.
We established that the Rꢀstereoisomer
is the preferred form for binding to CypD. Compound 12 binds with a higher affinity
than 10, obtained through an enthalpy gain due to an extra hydrogen bond between
Arg55 and the carbonyl oxygen of the urea backbone (PDB 4J5B). Compound 13 is
analogous to 12 with the orthoꢀthiomethyl replaced by an orthoꢀbromo and binds with
a K of 1.2 ꢁM. The results for 11-13 suggest that the phenyl group on the pyrrolidine
d
ring markedly affects how the compound binds in the S1' site, in addition to the
orthoꢀsubstituent playing an important role in binding.
(iii) Compounds 14-17 (S1 site). Substitutions at the Rꢀ position (Scheme 1 and
Table 1) aim to probe the possibility of mimicking the Bmt group of CsA (Figure 3). In
the CypDꢀCsA complex, (PDB: 2Z6W) the Aba2ꢀBmt1ꢀMva11 group makes a
primary hydrophobic and hydrogen bond interaction along the Cyp active site
34
groove. The data in Table 1 shows that the (S)ꢀmethionine side chain in 14 confers
the best affinity and was therefore retained in the final compound investigated,
compound 19.
ACS Paragon Plus Environment

Page 11 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
iv) Compound 19. Colliandre et al. reported their best compound to have an
IC50 of 0.66 ꢁM against CypD and this molecule is presumed to be a racemic mixture
25
of 4 diastereoisomers. Here, an asymmetric synthesis of their lead compound was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
undertaken to give 2 diastereoisomers; compounds 18 and 19. Compound 19 has a
K
d
of 0.41 ꢁM and a K
stereochemistry required for the aryl group on the pyrrolidine. Compound 19 (Figure
c) compared to 4 shows a significant entropy gain that is larger than the enthalpy
i d
of 99 nM whereas the K for 18 is 82 ꢁM making R the
5
loss, resulting in ∆G (19) of ꢀ8.7 kcal/mol and ∆G (4) of ꢀ6.8 kcal/mol. The entropic
gain is due to an increase in hydrophobic interactions between hydrophobic residues
lining the S1' site (Ile57, Ile60, Met61, Phe102 and Leu122) and the oꢀthiomethyl
phenyl group. There is an enthalpy loss on binding of 19 to CypD compared to 4.
Nevertheless the crystal structure showed that the important hydrogen bonds are
preserved between the CypDꢀcomplex of 4 and 19, therefore this enthalpy loss must
be due to another factor (vide infra).
The marked enthalpyꢀentropy compensation effect observed for the series of
35
compounds described is not uncommon for many biomolecular interactions,
particularly in the binding of structurally similar ligands and when interactions are
weak. The enthalpyꢀentropy compensation here is most likely attributed to the
inherent conformational flexibility and dynamics of the protein structure, as observed
36, 37
for the analogous protein, CypA.
occurs in some cases (vide infra).
In addition a reorganisation of water molecules
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
X-ray crystallography. In this study, the crystal structures (PDB: 5CBT, 5CBU,
5
CBV, 5CCN, 5CBW, 5CCS, 5CCR) were determined to resolutions of 1.4ꢀ2.1Å.
Apart from some variations in the unstructured regions Asp66ꢀGly74 and Ser144ꢀ
Ile156, the most notable feature observed from the various structures is how the
orientations of Arg82 and Arg55 sideꢀchains affect the topology of the binding site.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In either the free protein or the aniline compound complexes (Table 1), the Arg82
sideꢀchain forms one side of the S2 pocket, creating a more compact pocket (Figure
6
a). When other groups replace the aniline moiety (compounds 5-9), Arg82
consistently moves away, leading to a more open S2 pocket (Figure 6b and 6c).
Although the aniline group provides capacity for hydrogen bonding, this alone is not
sufficient to determine the orientation of the Arg82 sideꢀchain; complexes with 10,
1
2, 13 and 14 show Arg82 adopting both the 'open' and 'closed' conformations. Thus
'R' and 'X' substitutions (Table 1) appear to have subtle effects that alter the
orientation of the Arg82 sideꢀchain. The flexibility of Arg82 appears to enable the
protein to bind to larger compounds; interestingly, Arg82 adopts the open
conformation in the CypDꢀCsA complex (PDB 2Z6W). This suggests that Arg82 may
have a role in interactions with specific protein partners since this residue is not
conserved across CypA, CypB, CypC or CypE in what is an otherwise highly
38
conserved binding site.
The S1’ binding pocket also exhibits structural variation, centred on Arg55. The
binding conformation of the ester in 4-9 is conserved, primarily with hydrogen bonds
between Arg55ꢀNω and the ether oxygen atom of the ethyl ester. The interaction is
reinforced though a hydrogen bonding network between Arg55 and Gln63
ACS Paragon Plus Environment

Page 13 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
throughout this initial set of structures (Figure 6d). In the complex with 10 the
absence of the Arg55ꢀNω to ester oxygen interꢀmolecular hydrogen bond results in a
41.3° rotation of the guanidinium moiety out of the pocket, and subsequent hydrogen
bonding to two water molecules. This is further exemplified by 11, 12, 13, 18 and 19
where steric hindrance from the aromatic ring substituents force the Arg55 sideꢀ
chain outwards (Figure 6d), forming a hydrogen bond with the urea carbonyl and
causing a 24.4° puckering to the substituted pyrrole ring with respect to the
unsubstituted pyrrole ring of 10.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The most promising compound, 19, is bound to the active site of CypD through
a large number of cooperative nonꢀcovalent interactions (Figure 7). The amino group
forms a hydrogen bond network (coloured red) via coꢀcrystallised water molecules to
the protein, involving residues Thr107 to Gln111. Aromatic πꢀπ stacking interactions
(orange) are observed between the aniline ring and Asn102 and Gln111. The central
urea motif forms multiple hydrogen bonding contacts that include hydrogen bond
donation and hydrogen bond donorꢀπ interactions (yellow) with Asn102 (red) whilst
the carbonyl functionality forms bonding interactions via a crystallised water
molecule with His54, Gly72 and Arg55 (the interaction with the latter is a cationꢀ
dipole interaction). The enhanced strength of the binding of 19 may be attributed to
the large number of van der Waals interactions that are made between the orthoꢀ
substituted pyrrolidine with Arg55, Ile57, Phe60, Phe113, Leu122 and His126 and
also an edge to face πꢀπ interaction of the thiomethyl substituted ring with Phe60.
The conformation of Arg55 is different for 19 compared with compound 4 due to the
large thiomethyl ortho substitution on the benzene ring. With 19 the Arg55 is
involved in hydrogen bonding with the carbonyl group of the urea and also a cationꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
dipole interaction with a crystallographic water – both of these interactions are not
observed in initial compound 4.
Cellular assays using freshly isolated PACs. Informed by the in vitro data
obtained for the series, we selected 4, 13, 14 and 19 to obtain proof of principle
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
39
studies in freshly isolated PACs, the initial site of injury in AP. We evaluated the
ability of the CypD inhibitors 4, 13, 14 and 19 to inhibit necrotic cell death pathway
activation in freshly isolated murine PACs in the presence of a pancreatitis toxin,
taurolithocholate acid 3ꢀsulphate (TLCS, 500 µM), and compared the effect of the
inhibitors with CsA in a concentration dependent manner. TLCS depolarises
4, 40-42
mitochondria and induces necrosis in PACs from loss of ATP production.
Parallel experiments were conducted in which PACs were exposed to CsA, 4, 13, 14
and 19 without TLCS, to determine primary cytotoxic effects of these compounds. As
shown in Figure 8a, CsA and 19 were tested at 0.1, 1.0 and 10 ꢁM while 4, 13 and
1
4 were tested at 1 and 10 ꢁM. CsA provided significant protection with each
concentration used. Compound 4 did not reduce necrosis significantly at 1.0 and 10
M; compounds 13 and 14 provided significant protection at 10 ꢁM but not 1 ꢁM.
ꢁ
Compound 19 provided significant protection from necrotic cell death pathway
activation at both 1 and 10 ꢁM, likely due to its high binding affinity and strong
inhibitory effect on CypD enzymatic activity (Table 1) (although some of the necrosis
inhibition and membrane potential protection do not directly correlate with the in-vitro
affinity assays and this could be due to differences in the stability and/or cell
permeability). None of the compounds except 13 (10 ꢁM) showed significant primary
cytotoxicity in this assay (S5). To confirm that the reduction of necrosis in PACs
resulted from the maintenance of mitochondrial function by preventing the opening of
ACS Paragon Plus Environment

Page 15 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the MPTP, we performed ∆Ψ
positively charged mitochondrial dye tetramethyl rhodamine methyl ester (TMRM).
Figure 8b shows a decrease in ∆Ψ in response to TLCS that was ameliorated in the
presence of 13, 14 and 19, indicating protection of ∆Ψ . The potential applicability of
m
assays in freshly isolated PACs loaded with the
m
m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
19 as a treatment for human AP is demonstrated by its effects in freshly isolated
human PACs (obtained from fully informed and consenting patients undergoing
pancreatic resectional surgery). As shown in Figure 8c, 19 at 10 ꢁM prevented
necrotic cell death pathway activation in human PACs.
CONCLUSION
We have designed, optimized and synthesized a series of analogues and
enantiomers (5-19) of the hit compound 4 to develop competitive inhibitors of CypD
with potential protective activity against cellular necrosis. Compound 19 shows
enzymatic inhibition in the nanomolar range. Thermodynamic profiling of each
interaction together with the highꢀresolution crystal structures of each complex was
integral in optimisation. The structures show the important roles played by the local
flexibility within the CypD structure, reflected in variations in the orientations of sideꢀ
chains Arg55 and Arg82, both of which form direct and indirect hydrogen bonds via
water molecules with the different compounds. Cellular assays using freshly isolated
murine and human PACs showed that cells treated with the highest affinity
m
compound 19 were capable of inhibiting both loss of ∆Ψ and activation of the
necrotic cell death pathway induced by a bile acid. We provide here one of the few
examples of a drug development project where thermodynamic profiling coupled to
molecular modelling and structure determination has been systematically used to
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
provide improvements in the efficacy of the compounds. The urea compound 19
could, therefore, serve as a lead compound for further development of small
molecule agents against AP.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
Chemistry General Information:
All reactions were carried out employing standard chemical techniques under an
inert atmosphere. Solvents used for extraction, washing, and chromatography were
HPLC grade. Chemicals were purchased from Sigma Aldrich, Fluorochem or Apollo
and used without further purification. Purity values for all tested compounds were
found to be above 95% from either combustion analysis (4-7, 9ꢀ11 and 14ꢀ16) or
highꢀperformance liquid chromatography (HPLC) analyses on an Agilent 1200 (8, 12ꢀ
13 and 17ꢀ19). Absolute stereochemistry was determined by analytical HPLC using a
chiral stationary phase. The column utilised was a CHIRALPAK ADꢀH column using
a flow rate of 1.2ml/min eluting with 15% IPA in hexane. Retention times, t , of the
R
products are reported in minutes and (%) purity at 210 or 254 nm. Mass spectra
were recorded on an Agilent QTOF 7200 mass spectrometer. When EI mass
analysis was utilised, formic acid and methanol were used as the solvent systems.
When CI mass analysis was utilised, ammonia was used as the carrier gas. All mass
spectrometry and elemental microanalysis was done within the University of
Liverpool's chemistry department. NMR spectra were recorded using a Bruker MX
400 MHz spectrometer. Chemical shifts are listed as δ values in ppm downfield from
an internal standard of tetramethylsilane. Infrared spectra were recorded using a
PerkinElmer1720ꢀx FTꢀIR spectrometer. Melting points were recorded manually
using a Gallenkamp melting point apparatus.
ACS Paragon Plus Environment

Page 17 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Chemistry General Procedures:
General procedure A (4-9) – Ethyl isocyanatoacetate (1 eq) was dissolved in DMF
0.14 M). The given amine (1 eq) was added and the reaction mixture was stirred at
(
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
room temperature for 2 hours. After this time, the solvent was removed in vacuo to
yield the crude product.
General procedure B (10-13 and 17-19) – The given Bocꢀprotected amino amide
derivative (1eq) was first dissolved in a mixture of dichloromethane and
trifluoroacetic acid, 5:1 (0.1 M) and stirred for 1 hour then concentrated in vacuo to
afford the corresponding amino amide as a trifluoroacetate salt which was used
without further purification. 4ꢀaminobenzylamine (1 eq) was dissolved in DCM (0.1M)
and the reaction mixture was cooled to 0°C. CDI (1.1 eq) and DMAP (0.1 eq) were
added and the reaction mixture was stirred at r.t overnight. After this time, the
reaction mixture was diluted with EtOAc and washed with water. The organic layer
was dried over MgSO , filtered and concentrated in vacuo to yield a yellow solid that
4
required no further purification. Both the amino amide as the trifluoroacetate salt (1
eq) and the CDI adduct (1 eq) were dissolved in acetonitrile (0.1 M) and
triethylamine (2.2 eq) was added. The resulting suspension was stirred at room
temperature for 16 hours. After this time, the solvent was removed in vacuo to yield
the crude product.
General Procedure C (14-16) – The given amino acid ethyl ester as its
hydrochloride salt (1 eq) was dissolved in acetonitrile (0.1 M) and triethylamine (2.2
eq) was added followed by Nꢀ(4ꢀaminobenzyl)ꢀ1Hꢀimidazoleꢀ1ꢀcarboxamide (1 eq) in
a single portion. The resulting suspension was stirred at room temperature for 16
hours. After this time, the solvent was removed in vacuo to yield the crude product.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Ethyl 2-(3-(4-aminobenzyl)ureido)acetate (4). The crude product was triturated
with diethyl ether to yield a white solid (156 mg, 0.62 mmol, 8%). M.p: 164 – 166°C;
ꢀ1
IR νmax (cm ) 1207 (CꢀO), 1564 (NꢀH), 1610 (amide C=O), 1732 (ester C=O), 3348
1
(
NꢀH); H NMR (400MHz; CDCl
3
; Me
4
Si; ppm) δ 7.09 (2H, ap: d, J = 8.4), 6.65 (2H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ap: d, J = 8.4), 4.87 (1H, t, J = 5.4), 4.74 (1H, t, J = 5.4), 4.25 (2H, d, J = 5.4), 4.18
13
(
2H, q, J = 7.1), 3.99 (2H, d, J = 5.4), 3.65 (2H, s), 1.27 (3H, t, J = 7.1); C NMR
(
100 MHz; CDCl
3
; Me
4
Si) δ 171.6, 158.0, 151.8, 129.3, 129.0, 115.6, 61.8, 44.8,
+
4
2.7, 14.6; HRMS (ES+) m/z 274.1159 [M+Na] C12
H
17
N
3
O
3
Na requires 274.1168
(Diff ꢀ3.1 ppm). Anal. Calcd. for C12
17 3 3
H N O : C, 57.36; H, 6.82; N, 16.72. Found C,
57.28; H, 6.89; N, 16.81.
Ethyl 2-(3-benzylureido)acetate (5). The crude product was triturated with diethyl
ꢀ1
ether to yield a white solid (2.18 g, 9.23 mmol, 99%). M.p: 78 – 80°C; IR νmax (cm )
1
1
200 (CꢀO), 1574 (NꢀH), 1622 (amide C=O), 1745 (ester C=O), 3330 (NꢀH); H NMR
(400MHz; CDCl
3 4
; Me Si; ppm) δ 7.32 – 7.22 (5H, m), 5.12 (2H, br s), 4.34 (2H, s),
1
3
4
.13 (2H, q, J = 7.2), 3.95 (2H, s), 1.24 (3H, t, J = 7.2); C NMR (100 MHz; CDCl3;
Me
4
Si) δ 171.83, 158.45, 139.39, 129.01, 127.82, 127.69, 61.78, 44.86, 42.66,
+
1
4.51; HRMS (ES+) m/z 259.1057 [M+Na] C12
H
16
N
2
O
3
Na requires 259.1059 (Diff ꢀ
16 2 3
0.6 ppm); Anal. Calcd. for C12H N O : C, 61.00; H, 6.83; N, 11.86. Found C, 59.66;
H, 6.87; N, 12.04.
Ethyl 2-(3-(4-(pyridin-4-yl)benzyl)ureido)acetate (6). The crude product was
triturated with diethyl ether to yield a white solid (72 mg, 0.23 mmol, 43%). M.p: 156
ꢀ
1
–
157°C; IR νmax (cm ) 1215 (CꢀO), 1574 (NꢀH), 1628 (amide C=O), 1732 (ester
1
C=O), 3305 (NꢀH), 3386 (NꢀH); H NMR (400MHz; CDCl
3
; Me
4
Si; ppm) δ 8.64 (2H,
d, J = 5.3), 7.57 (2H, d, J = 8.1), 7.49 (2H, d, J = 5.5), 7.41 (2H, d, J = 8.1), 5.31 (1H,
t, J = 5.6), 5.27 (1H, t, J = 5.2), 4.45 (2H, d, J = 5.7), 4.19 (2H, q, J = 7.1), 4.03 (2H,
ACS Paragon Plus Environment

Page 19 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
3
d, J = 5.3), 1.27 (3H, t, J = 7.1); C NMR (100 MHz; CDCl
3 4
; Me Si) δ 171.7, 158.2,
1
49.9, 149.0, 141.1, 137.1, 128.7, 127.6, 122.0, 61.8, 44.5, 42.7, 14.6; HRMS (ES+)
+
m/z 314.1502 [M+H] C17
20 3 3
H N O requires 314.1505 (Diff ꢀ0.8 ppm); Anal. Calcd. for
17 19 3 3
C H N O
: C, 65.16; H, 6.11; N, 13.41. Found C, 64.91; H, 6.07; N, 13.30.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Ethyl 2-(3-(3-(pyridin-3-yl)benzyl)ureido)acetate (7). The crude product was
purified by flash column chromatography eluting with 70% EtOAc in hexane to 100%
ꢀ1
EtOAc to yield a white solid (183 mg, 0.58 mmol, 67%). M.p: 78 ꢀ 80°C; IR νmax (cm )
1
1
209 (CꢀO), 1568 (NꢀH), 1626 (amide C=O), 1736 (ester C=O), 3330 (NꢀH); H NMR
(400MHz; CDCl
3 4
; Me Si; ppm) δ 8.76 (1H, s), 8.56 (1H, d, J = 4.9), 7.83 (1H, ap: dt,
J = 7.9, 2.0), 7.45 (1H, ap: t, J = 1.8), 7.41 (1H, ap: dt, J = 7.8, 1.8), 7.37 (1H, ap: t, J
=
5
1
7.4), 7.34 (1H, dd, J = 7.4, 4.8), 7.30 (1H, ap: dt, J = 7.4, 1.6), 5.72 (1H, t, J = 5.9),
.54 (1H, t, J = 5.5), 4.41 (2H, d, J = 5.8), 4.13 (2H, q, J =7.2), 3.96 (2H, d, J = 5.4),
13
3 4
.22 (3H, t, J = 7.1); C NMR (100 MHz; CDCl ; Me Si) δ 171.8, 158.6, 148.8,
148.6, 140.8, 138.4, 136.8, 134.9, 129.7, 127.6, 126.5, 126.3, 124.0, 61.7, 44.6,
+
4
2.7, 14.6; HRMS (ES+) m/z 336.1314 [M+Na] C H N O Na requires 336.1324
17
19
3
3
19 3 3
(Diff ꢀ3.0 ppm); Anal. Calcd. for C17H N O : C, 65.16; H, 6.11; N, 13.41. Found C,
64.68; H, 6.09; N, 13.29.
Ethyl 2-(3-(3-(3-aminopyridin-4-yl)benzyl)ureido)acetate (8). The crude product
was purified by flash column chromatography eluting with 100% EtOAc to 10%
MeOH in EtOAc to yield a hygroscopic cream foam (304 mg, 0.93 mmol, 92%). IR
ꢀ1
ν
max (cm ) 1191 (CꢀO), 1552 (NꢀH), 1634 (amide C=O), 1736 (ester C=O), 3326 (Nꢀ
1
H); H NMR (400MHz; CDCl
3
; Me
4
Si; ppm) δ 8.12 (1H, s), 8.02 (1H, d, J = 4.8), 7.42
(1H, ap: t, J = 7.9), 7.37 (1H, ap: t, J = 1.4), 7.33 (2H, ap: dd, J = 7.8, 1.4), 7.00 (1H,
d, J = 4.9), 5.39 (1H, t, J = 5.5), 5.27 (1H, s), 4.42 (2H, d, J = 5.4), 4.16 (2H, q, J =
13
7
.2), 3.98 (2H, d, J = 3.3), 1.26 (3H, t, J = 7.2); C NMR (100 MHz; CDCl ; Me Si) δ
3 4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
171.7, 158.3, 140.7, 140.3, 140.3, 138.3, 137.6, 133.9, 129.8, 127.7, 127.6, 127.6,
+
1
24.6, 61.8, 44.7, 42.7, 14.5; HRMS (ES+) m/z 329.1616 [M+H] C17
H
21
N
4
O
3
requires 329.1614 (Diff 0.7 ppm).
Ethyl ((4-acetamidobenzyl)carbamoyl)glycinate (9). The crude product was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
purified by flash column chromatography eluting with 50% EtOAc in hexane to 100%
EtOAc to yield a beige foam (255 mg, 0.87 mmol, 96%). M.p: 171 ꢀ 173°C; IR νmax
ꢀ1
(
cm ) 1203 (CꢀO), 1533 (NꢀH), 1581 (NꢀH), 1622 (amide C=O), 1685 (amide C=O),
1
1
739 (ester C=O), 3321 (NꢀH), 3354 (NꢀH), 3379 (NꢀH); H NMR (400MHz; DMSO;
4
Me Si; ppm) δ 9.89 (1H, s), 7.49 (2H, d, J = 8.5), 7.15 (2H, d, J = 8.5), 6.61 (1H, t, J
= 6.0), 6.26 (1H, t, J = 6.0), 4.14 (2H, d, J = 6.0), 4.08 (2H, q, J = 7.1), 3.77 (2H, d, J
1
3
=
1
4
6.0), 2.02 (3H, s), 1.19 (3H, t, J = 7.1); C NMR (100 MHz; DMSO; Me Si) δ
71.2, 168.1, 157.9, 137.9, 135.2, 127.4, 118.9, 60.2, 42.5, 41.6, 23.9, 14.1; HRMS
+
(
ES+) m/z 316.1263 [M+Na] C14
H
19
N
3
O
4
Na requires 316.1273 (Diff ꢀ3.2 ppm); Anal.
19 3 4
Calcd. for C14H N O : C, 57.33; H, 6.53; N, 14.33. Found C, 57.45; H, 6.74; N,
13.45.
1-(4-Aminobenzyl)-3-(2-oxo-2-(pyrrolidin-1-yl)ethyl)urea (10). The crude product
was purified by flash column chromatography eluting with 50% EtOAc in hexane to
ꢀ
1
1
00% EtOAc to yield an off white foam (140 mg, 0.61 mmol, 79%) IR νmax (cm )
1
1638, 1655, 3299; H NMR (400MHz; (CD
3
)
2
SO; Me
4
Si; ppm) δ 6.81 (2H, d, J = 8.4
Hz), 6.43ꢀ6.39 (3H, m), 5.91 (1H, br t, J = 5.0 Hz), 4.82 (2H, br s), 3.92 (2H, d, J =
5.2 Hz), 3.71 (2H, d, J = 4.8 Hz), 3.29ꢀ3.19 (4H, m), 1.79 (2H, quintet, J = 6.8 Hz),
13
1
3 2 4
.67 (2H, quintet, J = 6.8Hz); C NMR (100 MHz; (CD ) SO; Me Si) δ 169.2, 158.3,
143.2, 139.4, 136.2, 125.6, 50.7, 44.6, 43.2, 28.9; HRMS (ES+) m/z 277.1655
+
[
M+H] C14
H
21
N
4
O
2
requires 277.1659. Anal. Calcd. for C14
H
20
N
4
O
2
: C, 60.85; H,
7.30; N, 20.28. Found C, 59.95; H, 7.12; N, 19.74.
ACS Paragon Plus Environment

Page 21 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
tert-Butyl (4-(2-bromophenyl)-4-oxobutyl)carbamate (20a). A flame dried, Ar filled
flask was charged with iPrMgCl.LiCl (0.68 ml, 0.89 mmol, 1.05 eq) and cooled to ꢀ
15°C. 2ꢀbromoiodobenzene (0.11 ml, 250 mg, 0.88 mmol, 1 eq) was added and the
reaction mixture was allowed to stir for 30 mins at ꢀ10°C. Tertꢀbutyl 2ꢀoxopyrrolidineꢀ
1ꢀcarboxylate (179 mg, 0.97 mmol, 1.1 eq) was added and the reaction mixture was
stirred at 0°C for 2 hrs. After this time, the reaction mixture was quenched with sat.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NH
4
Cl (10 ml) and extracted with EtOAc (3 x 10 ml). The combined organic layers
, filtered and
were washed with water (10 ml) and brine (10 ml), dried over MgSO
4
concentrated in vacuo. The crude product was purified by flash column
chromatography eluting with 5% EtOAc in hexane to 10% EtOAc in hexane to yield a
1
colourless oil (91 mg, 0.27 mmol, 30%). H NMR (400MHz; CDCl
3
; Me
4
Si; ppm) δ
7.60 (1H, d, J = 7.9), 7.39 (1H, dd, J = 7.9, 2.5), 7.35 (1H, dd, J = 7.7, 1.1), 7.30 (1H,
dd, J = 7.9, 2.6), 4.93 (1H, s, minor rotamer), 4.71 (1H, s, major rotamer), 3.37 –
3.33 (2H, m, minor rotamer), 3.22 (2H, q, J = 6.1, major rotamer), 2.96 (2H, t, J =
7.0), 1.93 (2H, quintet, J = 7.0), 1.45 (9H, s, minor rotamer), 1.43 (9H, s, major
13
rotamer); C NMR (100 MHz; CDCl
3
; Me
4
Si) δ 203.8, 156.1, 155.7, 141.7, 135.4,
133.6, 131.5, 129.3, 128.3, 127.4, 123.5, 118.5, 79.2, 39.9, 38.8, 30.9, 28.4, 28.3,
+
79
2
3
4.3; LRMS (ES+) m/z 364.1 [M+Na] C15H20NO BrNa requires 364.1 (99%), m/z
+
81
3
366.0 [M+Na] C15H20NO BrNa requires 366.1 (100%);
tert-Butyl (4-(2-(methylthio)phenyl)-4-oxobutyl)carbamate (20b and 20c).
Magnesium (1.00 g, 41.20 mmol, 10 eq) and iodine (single crystal) were stirred
overnight under Ar in a flame dried flask at r.t. After this time, THF (10.3 ml, 0.4 M)
was added to the activated magnesium. 2ꢀbromothioanisole (0.55 ml, 837 mg, 4.12
mmol, 1 eq) was added at 0°C and the reaction mixture was allowed to warm to r.t.
after 15 mins and refluxed for 2 hrs. After this time, the reaction mixture was allowed
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to cool to r.t. Tertꢀbutyl 2ꢀoxopyrrolidineꢀ1ꢀcarboxylate (555 mg, 3.00 mmol, 1 eq)
was dissolved in THF (21 ml, 0.1 M) and cooled to ꢀ78°C. The aryl grignard (9 ml,
3.60 mmol, 1.2 eq) was added and allowed to stir for 15 mins before warming to 0°C
and stirred for 30 mins. The reaction mixture was then warmed to r.t. and stirred
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
overnight. After this time, the reaction mixture was quenched with sat. NH Cl (40 ml)
4
and extracted with EtOAc (3 x 40 ml). The combined organic layers were dried over
4
MgSO , filtered and concentrated in vacuo. The crude product was purified by flash
column chromatography eluting with 100% hexane to 10% EtOAc in hexane to yield
1
a white semiꢀsolid (959 mg, 3.10 mmol, 36%). H NMR (400MHz; CDCl
3 4
; Me Si;
ppm) δ 7.81 (1H, d, J = 7.7), 7.47 (1H, t, J = 7.7), 7.33 (1H, d, J = 7.9), 7.19 (1H, t, J
=
7
1
7.5), 3.22 (2H, q, J = 7.0), 3.01 (2H, t, J = 7.0), 2.43 (3H, s), 1.94 (2H, quintet, J =
13
3 4
.0), 1.42 (9H, s); C NMR (100 MHz; CDCl ; Me Si) δ 200.9, 156.1, 134.5, 129.5,
32.2, 130.1, 125.2, 123.6, 77.2, 40.1, 37.2, 28.4, 24.6, 16.0; HRMS (ES+) m/z
+
332.1298 [M+Na] C16
H
23NO
3
SNa requires 332.1296 (Diff 0.5 ppm).
(
S)-tert-Butyl (4-(2-bromophenyl)-4-hydroxybutyl)carbamate (21a). A solution of
tertꢀbutyl (4ꢀ(2ꢀbromophenyl)ꢀ4ꢀoxobutyl)carbamate (2.90 g, 8.48 mmol, 1 eq) in THF
12.7 ml, 0.67 M) was added dropwise to (R)ꢀ2ꢀmethylꢀCBSꢀoxazaborolidine (0.25
ml, 236 mg, 0.85 mmol, 10 mol%) in 1M BH ꢀDMS (8.48 ml, 8.48 mmol, 1 eq). The
(
3
reaction mixture was stirred at r.t. for 3 hrs. After this time, the reaction mixture was
quenched with 1M HCl (60 ml) and extracted with EtOAc (2 x 60 ml). The combined
4
organic layers were dried over MgSO , filtered and concentrated in vacuo. The crude
product was purified by flash column chromatography eluting with 10% EtOAc in
hexane to 15% EtOAc in hexane to yield a colourless oil (2.81 g, 8.19 mmol, 97%).
1
3 4
H NMR (400MHz; CD OD; Me Si; ppm) δ 7.56 (1H, dd, J = 7.8, 1.5), 7.50 (1H, dd, J
=
8.0, 1.0), 7.33 (1H, td, J = 7.7, 0.9), 7.12 (1H, td, J = 7.9, 1.7), 5.10 – 5.07 (1H, m),
ACS Paragon Plus Environment

Page 23 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4.63 (1H, br s), 3.28 – 3.23 (2H, m, minor rotamer), 3.19 – 3.11 (2H, m, major
rotamer), 2.58 (1H, br s), 1.83 – 1.78 (4H, m, minor rotamer), 1.72 – 1.64 (4H, m,
13
3 4
major rotamer), 1.43 (9H, s); C NMR (100 MHz; CDCl ; Me Si) δ 156.2, 143.8,
132.6, 128.8, 127.7, 127.3, 121.8, 79.3, 72.6, 40.3, 34.4, 28.4, 26.5; HRMS (ES+)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
79
m/z 366.0675 [M+Na] C H NO BrNa requires 366.0681 (100%) (Diff ꢀ1.6 ppm),
15
22
3
+
81
m/z 368.0657 [M+Na] C15
H
3
22NO BrNa requires 368.0660 (98.5%) (Diff ꢀ0.9 ppm).
HPLC on a chiral stationary phase revealed a 95:5 er with the first eluting peak at t
6.14 min (major enantiomer) corresponding to (S)ꢀ21a, the second eluting peak at
= 6.97 min (minor enantiomer) corresponding to (R)ꢀ21a
(S)-tert-Butyl (4-hydroxy-4-(2-(methylthio)phenyl)butyl)carbamate (21b).
R
=
t
R
A
solution of tertꢀbutyl (4ꢀ(2ꢀ(methylthio)phenyl)ꢀ4ꢀoxobutyl)carbamate (825 mg, 2.67
mmol, 1 eq) in THF (4 ml, 0.67 M) was added dropwise to (R)ꢀ2ꢀmethylꢀCBSꢀ
3
oxazaborolidine (1 M in toluene) (0.27 ml, 0.27 mmol, 10 mol%) in 1M BH ꢀDMS
(2.67 ml, 2.67 mmol, 1 eq). The reaction mixture was stirred at r.t. for 3 hrs. After this
time, the reaction mixture was quenched with 1M HCl (40 ml) and extracted with
4
EtOAc (2 x 40 ml). The combined organic layers were dried over MgSO , filtered and
concentrated in vacuo. The crude product was purified by flash column
chromatography eluting with 10% EtOAc in hexane to 20% EtOAc in hexane to yield
25.4
D
1
a colourless oil (761 mg, 2.45 mmol, 92%). [α]
3
ꢀ41.6 (c 0.01, CH OH); H NMR
(
400MHz; CDCl ; Me Si; ppm) δ 7.48 (1H, d, J = 7.4), 7.25 – 7.23 (2H, m), 7.20 (1H,
3
4
dt, J = 7.6, 2.9), 5.13 (1H, dd, J = 7.6, 4.4), 4.67 (1H, s), 3.23 – 3.13 (2H, m), 2.46
13
(
3 4
3H, s), 1.78 – 1.58 (4H, m), 1.43 (9H, s); C NMR (100 MHz; CDCl ; Me Si) δ
156.1, 143.0, 135.3, 127.9, 126.3, 125.6, 125.5, 79.1, 70.4, 40.3, 34.7, 28.4, 26.5,
+
1
6.4; LRMS (ES+) m/z 334.1 [M+Na] C16
H
25NO
3
SNa requires 334.1. HPLC on a
chiral stationary phase revealed a 95:5 er with the first eluting peak at t = 8.87 min
R
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
R
(major enantiomer) corresponding to (S)ꢀ21b, the second eluting peak at t = 10.66
min (minor enantiomer) corresponding to (R)ꢀ21c.(R)-tert-Butyl (4-hydroxy-4-(2-
(methylthio)phenyl)butyl)carbamate (21c).
A
solution of tertꢀbutyl (4ꢀ(2ꢀ
(methylthio)phenyl)ꢀ4ꢀoxobutyl)carbamate (825 mg, 2.67 mmol, 1 eq) in THF (4 ml,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.67 M) was added dropwise to (S)ꢀ2ꢀmethylꢀCBSꢀoxazaborolidine (1 M in toluene)
0.27 ml, 0.27 mmol, 10 mol%) in 1M BH ꢀDMS (2.67 ml, 2.67 mmol, 1 eq). The
(
3
reaction mixture was stirred at r.t. for 3 hrs. After this time, the reaction mixture was
quenched with 1N HCl (40 ml) and extracted with EtOAc (2 x 40 ml). The combined
4
organic layers were dried over MgSO , filtered and concentrated in vacuo. The crude
product was purified by flash column chromatography eluting with 10% EtOAc in
hexane to 15% EtOAc in hexane to yield a colourless oil (728 mg, 2.34 mmol, 88%).
25.1
D
1
[
α]
3 3 4
+26.0 (c 0.01, CH OH); H NMR (400MHz; CDCl ; Me Si; ppm) δ 7.48 (1H, d,
J = 7.5), 7.25 – 7.23 (2H, m), 7.20 (1H, dt, J = 7.5, 3.1), 5.13 (1H, dd, J = 7.5, 4.5),
1
3
4.64 (1H, s), 3.25 – 3.13 (2H, m), 2.47 (3H, s), 1.78 – 1.61 (4H, m), 1.43 (9H, s);
C
NMR (100 MHz; CDCl ; Me Si) δ 156.1, 143.0, 135.3, 127.9, 126.4, 125.6, 125.6,
3
4
+
7
9.1, 70.5, 40.3, 34.7, 28.4, 26.6, 16.4; LRMS (ES+) m/z 334.1 [M+Na]
SNa requires 334.1. HPLC on a chiral stationary phase revealed a 95:5 er
with the first eluting peak at t = 8.73 min (minor enantiomer) corresponding to (S)ꢀ
21b, the second eluting peak at t = 10.47 min (major enantiomer) corresponding to
16 3
C H25NO
R
R
(
(
(
R)ꢀ21c.
S)-1-(2-Bromophenyl)-4-((tert-butoxycarbonyl)amino)butyl methanesulfonate
22a). (S)ꢀtertꢀbutyl (4ꢀ(2ꢀbromophenyl)ꢀ4ꢀhydroxybutyl)carbamate (2.81 g, 8.19
3
mmol, 1 eq) was dissolved in DCM (81.9 ml, 0.1 M) and cooled to 0°C. NEt (4.57
ml, 3.31 g, 32.76 mmol, 4 eq) and methanesulfonyl chloride (1.90 ml, 2.81 g, 24.57
mmol, 3 eq) were added to the reaction mixture and stirred at 0°C for 15 mins before
ACS Paragon Plus Environment

Page 25 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
warming to r.t. and stirred for 3 hrs. After this time, the reaction mixture was
concentrated in vacuo. The residue was dissolved in EtOAc (60 ml) and washed with
1N HCl (50 ml), NaHCO
3
(50 ml), water (50 ml) and brine (50 ml). The organic layer
was dried over MgSO , filtered and concentrated in vacuo. The crude product was
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
purified by flash column chromatography eluting with 1% EtOAc in hexane to 5%
1
EtOAc in hexane to yield a pale yellow oil (1.97 g, 4.68 mmol, 57%). H NMR
(400MHz; CDCl
3 4
; Me Si; ppm) δ 7.51 (1H, dd, J = 7.9, 1.0), 7.25 (1H, t, J = 7.4), 7.14
(1H, d, J = 7.8), 7.08 (1H, t, J = 7.4), 5.22 (1H, d, J = 7.5, minor rotamer), 5.12 (1H,
dd, J = 7.8, 4.4, major rotamer), 3.71 – 3.60 (2H, m, major rotamer), 3.55 – 3.49 (2H,
m, minor rotamer), 2.44 – 2.32 (1H, m), 1.92 – 1.85 (3H, m, major rotamer), 1.82 –
1.75 (3H, m, minor rotamer), 1.60 (3H, s, minor rotamer), 1.46 (9H, s, minor
13
rotamer), 1.44 (3H, s, major rotamer), 1.18 (9H, s, major rotamer); C NMR (100
MHz; CDCl
3 4
; Me Si) δ 154.3, 143.8, 132.8, 132.6, 129.6, 128.6, 128.0, 128.0, 127.3,
126.5, 121.9, 79.3, 52.6, 47.3, 36.6, 34.0, 28.4, 28.1, 23.0; HRMS (ES+) m/z
+
79
3
48.0569 [MꢀOMs+Na] C H NO BrNa requires 348.0575 (100%) (Diff ꢀ1.8 ppm),
15
20
2
+
81
m/z 350.0548 [MꢀOMs+Na] C15
H
2
20NO BrNa requires 350.0555 (99.7%) (Diff ꢀ1.9
ppm).
(
S)-4-((tert-Butoxycarbonyl)amino)-1-(2-(methylthio)phenyl)butyl
methanesulfonate (22b). (S)ꢀtertꢀbutyl (4ꢀhydroxyꢀ4ꢀ(2ꢀ
methylthio)phenyl)butyl)carbamate (761 mg, 2.45 mmol, 1 eq) was dissolved in
DCM (25 ml, 0.1 M) and cooled to 0°C. NEt (1.37 ml, 992 mg, 9.80 mmol, 4 eq) and
(
3
methanesulfonyl chloride (0.57 ml, 842 mg, 7.35 mmol, 3 eq) were added to the
reaction mixture and stirred at 0°C for 15 mins before warming to r.t. and stirred for 3
hrs. After this time, the reaction mixture was concentrated in vacuo. The residue was
dissolved in EtOAc (40 ml) and washed with 1N HCl (30 ml), NaHCO (30 ml), water
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
(30 ml) and brine (30 ml). The organic layer was dried over MgSO , filtered and
concentrated in vacuo. The crude product was purified by flash column
chromatography eluting with 1% EtOAc in hexane to 5% EtOAc in hexane to yield a
1
colourless oil (542 mg, 1.39 mmol, 57%). H NMR (400MHz; CDCl
3
; Me
4
Si; ppm) δ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7.23 (1H, s), 7.19 (1H, d, J = 8.4), 7.14 (1H, d, J = 8.4), 7.10 (1H, s), 5.16 (1H, s),
3.67 – 3.60 (2H, m), 2.47 (3H, s), 2.35 – 2.28 (1H, m), 1.86 – 1.77 (3H, m), 1.46 (3H,
13
3 4
s), 1.17 (9H, s). C NMR (100 MHz; CDCl ; Me Si) δ 154.5, 143.3, 135.0, 127.0,
126.6, 125.2, 124.9, 79.1, 58.6, 50.9, 47.1, 34.1, 28.1, 23.1, 16.6; LRMS (ES+) m/z
+
3
2
16.1 [MꢀOMs+Na] C16H23NO SNa requires 316.1.
(R)-4-((tert-Butoxycarbonyl)amino)-1-(2-(methylthio)phenyl)butyl
methanesulfonate (22c). (R)ꢀtertꢀbutyl (4ꢀhydroxyꢀ4ꢀ(2ꢀ
methylthio)phenyl)butyl)carbamate (728 mg, 2.34 mmol, 1 eq) was dissolved in
DCM (23 ml, 0.1 M) and cooled to 0°C. NEt (1.30 ml, 947 mg, 9.36 mmol, 4 eq) and
(
3
methanesulfonyl chloride (0.54 ml, 804 mg, 7.02 mmol, 3 eq) were added to the
reaction mixture and stirred at 0°C for 15 mins before warming to r.t. and stirred for 3
hrs. After this time, the reaction mixture was concentrated in vacuo. The residue was
dissolved in EtOAc (40 ml) and washed with 1N HCl (30 ml), NaHCO
3
(30 ml), water
4
, filtered and
(30 ml) and brine (30 ml). The organic layer was dried over MgSO
concentrated in vacuo. The crude product was purified by flash column
chromatography eluting with 2% EtOAc in hexane to 4% EtOAc in hexane to yield a
1
colourless oil (505 mg, 1.30 mmol, 55%). H NMR (400MHz; CDCl
3
; Me
4
Si; ppm) δ
7.23 (1H, s), 7.19 (1H, d, J = 8.2), 7.13 (1H, d, J = 8.2), 7.10 (1H, s), 5.16 (1H, s),
3.67 – 3.59 (2H, m), 2.47 (3H, s), 2.35 – 2.28 (1H, m), 1.87 – 1.77 (3H, m), 1.46 (3H,
13
3 4
s), 1.17 (9H, s). C NMR (100 MHz; CDCl ; Me Si) δ 154.5, 143.3, 135.0, 127.0,
ACS Paragon Plus Environment

Page 27 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
126.6, 125.2, 124.9, 79.1, 58.6, 50.9, 47.1, 34.1, 28.1, 23.1, 16.6; LRMS (ES+) m/z
+
3
16.1 [MꢀOMs+Na] C16
H
23NO
2
SNa requires 316.1.
(R)-2-(2-Bromophenyl)pyrrolidine
(23a).
(S)ꢀ1ꢀ(2ꢀbromophenyl)ꢀ4ꢀ((tertꢀ
butoxycarbonyl)amino)butyl methanesulfonate (985 mg, 2.34 mmol, 1 eq) was
dissolved in DCM:TFA (23.4 ml, 5:1, 0.1 M) and stirred at r.t. for 2 hrs. After this
time, the reaction mixture was concentrated in vacuo. The yellow oil was dissolved in
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
M NaOH (7.02 ml, 7.02 mmol, 3 eq) in MeOH (16.38 ml, 0.1 M total volume). The
reaction mixture was stirred overnight at r.t. After this time, the reaction mixture was
concentrated in vacuo. The residue was neutralised with 1M HCl and washed with
EtOAc (2 x 50 ml). The combined organic layers were dried over MgSO , filtered and
4
concentrated in vacuo to yield a yellow solid that required no further purification (802
1
mg, 3.56 mmol, Quantitative). H NMR (400MHz; CDCl
3
4
; Me Si; ppm) δ 7.54 (2H,
dd, J = 7.7, 3.6), 7.33 – 7.28 (1H, m), 7.16 (1H, t, J = 7.6), 4.88 (1H, t, J = 8.1), 3.41
– 3.35 (1H, m), 3.31 – 3.23 (1H, m), 2.47 – 2.40 (1H, m), 2.20 – 2.13 (1H, m), 2.10 –
13
1
.95 (2H, m); C NMR (100 MHz; CDCl ; Me Si) δ 135.8, 133.2, 130.1, 128.1,
3 4
+
79
1
27.6, 123.9, 61.6, 45.7, 32.0, 24.0; HRMS (ES+) m/z 226.0226 [M+H] C10
H
H
13N Br
+
81
requires 226.0231 (97.4%) (Diff ꢀ2.4 ppm), m/z 228.0206 [M+H]
C
10
13N Br
requires 228.0211 (100%) (Diff ꢀ2.1 ppm).
(R)-2-(2-(Methylthio)phenyl)pyrrolidine (23b). (S)ꢀ4ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ
ꢀ(2ꢀ(methylthio)phenyl)butyl methanesulfonate (542 mg, 1.39 mmol, 1 eq) was
1
dissolved in DCM:TFA (13.9 ml, 5:1, 0.1 M) and stirred at r.t. for 2 hrs. After this
time, the reaction mixture was concentrated in vacuo. The pale yellow oil was
dissolved in 1M NaOH (4.17 ml, 4.17 mmol, 3 eq) in MeOH (9.73 ml, 0.1 M total
volume). The reaction mixture was stirred overnight at r.t. After this time, the reaction
mixture was concentrated in vacuo. The residue was neutralised with 1M HCl and
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 58
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
washed with EtOAc (2 x 40 ml). The combined organic layers were dried over
MgSO
4
, filtered and concentrated in vacuo to yield an orange oil that required no
1
further purification (181 mg, 0.94 mmol, 68%). H NMR (400MHz; CDCl
3
4
; Me Si;
ppm) δ 7.51 (1H, d, J = 7.4), 7.23 (2H, s), 7.14 (1H, t, J = 7.3), 4.56 (1H, t, J = 7.3),
3.20 (1H, q, J = 7.8), 3.05 (1H, q, J = 7.9), 2.46 (3H, s), 2.30 – 2.20 (1H, m), 1.93 –
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
1
1
1
3 4
.85 (2H, m), 1.66 – 1.58 (1H, m); C NMR (100 MHz; CDCl ; Me Si) δ 142.0,
36.6, 127.4, 125.9, 125.7, 125.3, 58.9, 46.7, 32.7, 25.3, 16.2; HRMS (CI+) m/z
+
94.1005 [M+H] C11
H
16NS requires 194.0998 (Diff 3.5 ppm).
(S)-2-(2-(Methylthio)phenyl)pyrrolidine (23c). (R)ꢀ4ꢀ((tertꢀbutoxycarbonyl)amino)ꢀ
1ꢀ(2ꢀ(methylthio)phenyl)butyl methanesulfonate (505 mg, 1.30 mmol, 1 eq) was
dissolved in DCM:TFA (13.0 ml, 5:1, 0.1 M) and stirred at r.t. for 2 hrs. After this
time, the reaction mixture was concentrated in vacuo. The pale orange oil was
dissolved in 1M NaOH (3.90 ml, 3.90 mmol, 3 eq) in MeOH (9.10 ml, 0.1 M total
volume). The reaction mixture was stirred overnight at r.t. After this time, the reaction
mixture was concentrated in vacuo. The residue was neutralised with 1M HCl and
washed with EtOAc (2 x 40 ml). The combined organic layers were dried over
MgSO
4
, filtered and concentrated in vacuo to yield a pale yellow oil that required no
1
further purification (250 mg, 1.29 mmol, 100%). H NMR (400MHz; CDCl
3
4
; Me Si;
ppm) δ 8.14 (1H, br s), 7.51 (1H, d, J = 7.4), 7.22 (2H, s), 7.16 (1H, t, J = 7.3), 4.62
(1H, t, J = 7.4), 3.27 – 3.23 (3H, m), 3.09 (1H, q, J = 7.4), 2.47 (3H, s), 2.34 – 2.26
13
(
3
1H, m), 1.97 – 1.89 (1H, m), 1.73 – 1.64 (1H, m); C NMR (100 MHz; CDCl ;
4
Me Si) δ 141.1, 136.7, 127.6, 126.1, 125.8, 125.4, 59.0, 46.6, 32.6, 25.2, 16.4;
+
LRMS (CI+) m/z 194.1 [M+H] C11
H16NS requires 194.1.
(R)-tert-butyl (2-(2-(2-bromophenyl)pyrrolidin-1-yl)-2-oxoethyl)carbamate (24a).
(R)ꢀ2ꢀ(2ꢀbromophenyl)pyrrolidine (802 mg, 3.56 mmol, 1 eq), BocꢀGlycine (748 mg,
ACS Paragon Plus Environment

Page 29 of 58
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4.27 mmol, 1.2 eq) and HATU (1.76 g, 4.63 mmol, 1.3 eq) were dissolved in DMF
(
17.8 ml, 0.2 M). DIPEA (0.93 ml, 690 mg, 5.34 mmol, 1.5 eq) was added and the
reaction mixture was stirred at room temperature overnight. After this time, the
reaction mixture was diluted with EtOAc (40 ml) and washed with NaHCO (3 x 40
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ml), water (40 ml) and brine (40 ml). The organic layer was dried over MgSO4,
filtered and concentrated in vacuo. The crude product was purified by flash column
chromatography eluting with 15% EtOAc in hexane to 20% EtOAc in hexane to yield
1
a colourless oil (862 mg, 2.26 mmol, 63%). H NMR (400MHz; CDCl
3
; Me
4
Si; ppm) δ
7
.57 (1H, d, J = 8.0, major rotamer), 7.55 (1H, dd, J = 8.0, 0.8, minor rotamer), 7.28
(1H, t, J = 7.5, major rotamer), 7.23 (1H, t, J = 7.5, minor rotamer), 7.15 (1H, t, J =
.8, major rotamer), 7.09 (1H, td, J = 7.8, 1.5, minor rotamer), 7.03 (1H, d, J = 7.6,
7
major rotamer), 6.94 (1H, dd, J = 7.7, 1.2, minor rotamer), 5.42 (1H, dd, J = 8.5, 2.5,
major rotamer), 5.35 (1H, br s), 5.20 (1H, d, J = 8.1, minor rotamer), 4.03 – 4.00 (1H,
m, major rotamer), 3.98 – 3.93 (1H, m, minor rotamer), 3.88 – 3.83 (2H, m, minor
rotamer), 3.79 – 3.70 (2H, m, major rotamer), 3.59 (1H, ap: q, J = 7.9, minor
rotamer), 3.26 (1H, dd, J = 17.3, 3.7, major rotamer), 2.48 – 2.39 (1H, m, major
rotamer),
1H, m, minor rotamer), 2.03 – 1.94 (2H, m), 1.92 – 1.88 (1H, m, major rotamer),
1.87 – 1.81 (1H, m, minor rotamer), 1.43 (9H, s, minor rotamer), 1.40 (9H, s, major
2.36
–
2.29
(
13
rotamer); C NMR (100 MHz; CDCl ; Me Si) δ 168.0, 167.0, 155.9, 155.7, 141.0,
3
4
140.6, 133.6, 133.3, 129.2, 128.5, 127.9, 127.4, 126.1, 125.9, 122.1, 121.9, 79.6,
79.5, 61.2, 60.5, 47.8, 46.9, 43.2, 42.8, 32.3, 28.3, 23.4; HRMS (ES+) m/z 405.0783
+
79
[M+Na] C17
H
23
N
2
O
3
BrNa requires 405.0790 (100%) (Diff ꢀ1.7 ppm), m/z 407.0778
+
81
[
M+Na] C17
H
23
N
2
O
3
BrNa requires 407.0769 (95.9%) (Diff 2.1 ppm).
ACS Paragon Plus Environment