Synthesis and Structure-Activity Relationships of N-(4-Benzamidino)-Oxazolidinones: Potent and Selective Inhibitors of Kallikrein-Related Peptidase 6

Article abstract of DOI:10.1002/cmdc.201900536

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins. Aberrant expression of KLK6 has been found in different cancers and neurodegenerative diseases, and KLK6 is currently studied as a potential target in these pathologies. We report a novel series of KLK6 inhibitors discovered in a high-throughput screen within the European Lead Factory program. Structure-guided design based on docking studies enabled rapid progression of a hit cluster to inhibitors with improved potency, selectivity and pharmacokinetic properties. In particular, inhibitors 32 ((5R)-3-(4-carbamimidoylphenyl)-N-((S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) and 34 ((5R)-3-(6-carbamimidoylpyridin-3-yl)-N-((1S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) have single-digit nanomolar potency against KLK6, with over 25-fold and 100-fold selectivities against the closely related enzyme trypsin, respectively. The most potent compound, 32, effectively reduces KLK6-dependent invasion of HCT116 cells. The high potency in combination with good solubility and low clearance of 32 make it a good chemical probe for KLK6 target validation in vitro and potentially in vivo.

Full text of DOI:10.1002/cmdc.201900536

Accepted Article
Title: Synthesis and structure–activity relationships of N-(4-
benzamidino)-oxazolidinones–potent and selective inhibitors of
kallikrein-related peptidase 6
Authors: Elena De Vita, Niels Smits, Helma van den Hurk, Elizabeth
M. Beck, Joanne Hewitt, Gemma Baillie, Emily Russell,
Andrew Pannifer, Véronique Hamon, Angus Morrison, Stuart
P. McElroy, Philip Jones, Natalia Ignatenko, Nikolas Gunkel,
and Aubry Kern Miller
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900536
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900536
A Journal of
www.chemmedchem.org

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
Synthesis and structure–activity relationships of N-(4-
benzamidino)-oxazolidinones–potent and selective inhibitors of
kallikrein-related peptidase 6
Elena De Vita,[a,b] Niels Smits,[c] Helma van den Hurk,[c] Elizabeth M. Beck,[d] Joanne Hewitt,[d] Gemma
Baillie,[d] Emily Russell,[d] Andrew Pannifer,[d] Véronique Hamon,[d] Angus Morrison,[d] Stuart P.
McElroy,[d] Philip Jones,[d] Natalia A. Ignatenko,[e,f] Nikolas Gunkel,[a,g] and Aubry K. Miller*[a,g]
[a]
Dr. E. De Vita, 0000-0003-4707-8342, Dr. N. Gunkel, Dr. A. K. Miller, 0000-0002-1761-4143
Cancer Drug Development Group, German Cancer Research Center (DKFZ)
Im Neuenheimer Feld 280, 69120, Heidelberg, Germany
E-mail: aubry.miller@dkfz-heidelberg.de
[b]
Dr. E. De Vita
Faculty of Biosciences, University of Heidelberg
Heidelberg 69120, Germany
[c]
[d]
Mr. N. Smits, Dr. H. van den Hurk
Pivot Park Screening Centre, Kloosterstraat 9, 5349 AB Oss, The Netherlands
Dr. J. Hewitt, Dr. E. M. Beck, Dr. G. Baillie, Dr. A. Pannifer, Dr. P. Jones, Dr. V. Hamon, Dr. S. P. McElroy, Dr. A. Morrison, Ms. E. Russell
European Screening Centre Newhouse (ESC)
Biocity Scotland, Bo´ness Road, ML15UH, Newhouse, Scotland, UK
Dr. N. A. Ignatenko
University of Arizona Cancer Center, University of Arizona, Tucson, AZ, USA
Dr. N. A. Ignatenko
[e]
[f]
Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA
Dr. N. Gunkel, Dr. A. K. Miller
[g]
German Cancer Consortium (DKTK)
Heidelberg 69120, Germany
Abstract: Kallikrein-related peptidase 6 (KLK6) is a secreted serine
protease that belongs to the family of tissue kallikreins. Aberrant
expression of KLK6 has been found in different cancers and
neurodegenerative diseases, and KLK6 is currently studied as a
potential target in these pathologies. We report a novel series of KLK6
inhibitors discovered in a high-throughput screen within the European
Lead Factory program. Structure-guided design based on docking
studies enabled rapid progression of a hit cluster to inhibitors with
improved potency, selectivity and pharmacokinetic properties. In
particular, inhibitors 32 and 34 have single digit nanomolar potency
against KLK6, with over 25-fold and 100-fold selectivities against the
closely related enzyme trypsin, respectively. The most potent
compound, 32, effectively reduces KLK6-dependent invasion of
HCT116 cells. The high potency in combination with good solubility
and low clearance of 32 make it a good chemical probe for KLK6
target validation in vitro and potentially in vivo.
P1 residues, preferably arginine. Broader sequence requirements
have been reported for the flanking residues (P2, P3, P1’, and
P2’).[6] Relevant endogenous substrates of KLK6 have been
identified in vitro and include protease-activated receptors
(PARs),[7] -synuclein,[7-8] and myelin basic protein.[9]
Secreted proteases (e.g. KLKs and matrix-metalloproteinases)
are investigated as potential therapeutic drug targets due to their
role in extracellular signaling via proteolysis-mediated production
of small signaling molecules or proteolytic activation of membrane
receptors.[10] KLK6 can activate PARs, and this signaling pathway
has been found to be dysregulated in cutaneous malignant
melanoma.[11] In this cancer, KLK6 was found to be secreted by
the keratinocytes surrounding the tumor cells in response to
stimuli from the tumor, and acts in a paracrine fashion to activate
PAR-1 receptors, which are overexpressed on melanocytes. This
signaling cascade was found to have an effect on tumor migration
and invasiveness in vitro[11] and is considered to contribute to
recurrence and metastasis in melanoma patients that undergo
surgery.[12] KLK6-promoted migration was also observed in colon
cancer, where knockdown of KLK6 reduced migration and
invasion of HCT116 cells in vitro.[13] Furthermore, in an orthotopic
colon cancer mouse model, mice injected with KLK6 positive
HCT116 cells had significantly more metastases and worse
survival than mice injected with shKLK6 HCT116 clones.[13]
Nevertheless, the role of KLK6 needs further investigation in
these and other types of cancers, as its role is clearly tumor-
dependent. In head-and-neck cancer for example, high levels of
KLK6 have been associated with a better prognosis for the
patients, resulting in reduced aggressiveness of the disease.[14]
Introduction
Kallikrein-related peptidase 6 (KLK6), previously known as
protease M, zyme, neurosin, or myelencephalon specific
protease,[1] is a secreted serine protease that belongs to the
family of tissue kallikreins (KLKs).[2] Like all 15 KLKs, KLK6 is
released into the extracellular matrix as a zymogen and activated
upon cleavage of a pro-peptide, a process which can be mediated
by other proteases such as KLK5,[3] plasmin,[4] urokinase (uPA),[4]
and MMP-20.[5] Removal of the pro-peptide generates mature
KLK6, a trypsin-like enzyme with cleavage specificity after basic
1
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
In addition to malignancies, KLK6 is studied in the central nervous
system (CNS), where its physiological abundance might imply an
important role for KLK6 in the maintenance of homeostasis in
these organs. In neurodegenerative diseases such as multiple
sclerosis, Alzheimer’s[15] and Parkinson’s,[16] as well as spinal cord
injury,[17] aberrant levels of KLK6 have been reported. Potential
therapeutic approaches targeting KLK6 have been investigated
but require further validation, particularly with high-quality KLK6
chemical probes.[9]
To date, few accounts of reversible KLK6 inhibitors have been
reported, the most relevant being two sets of small molecules
(e.g. 1 and 2, Figure 1) discovered by in silico high-throughput
screening (HTS) supported by X-ray crystallography,[18] and a
series of pseudopeptides (e.g. 3), which were reported to be
highly selective over the closely related KLK5.[19] Covalent
coumarin-based suicide inhibitors (e.g 4)[20] as well as transient
quiescent affinity labelers (e.g. DKFZ-251), our first disclosure of
KLK6 inhibitors,[21] have also been reported.
curve analysis (Figure 2). These substances were tested at seven
concentrations from 20 M to 20 nM, resulting in 312 entities with
a pIC50 > 4.7. A selectivity screen against trypsin, thrombin and
factor Xa, as previously reported,[21] reduced the number to 226
preliminary hits. Further validation of these hits was performed via
surface plasmon resonance (SPR), resulting in 61 likely reversible
KLK6 binders. LC-MS analysis eliminated 4 impure substances,
and a qualified hit list (QHL) of 50 compounds was generated.
These were sorted into ten structural clusters with fifteen
singletons. Many of the hits contained a highly basic moiety such
as amidine or guanidine, functional groups that are often found in
trypsin-like serine protease inhibitors and sometimes associated
with poor cellular permeability. Nevertheless, we chose to further
advance with such compounds because KLK6 is a secreted
protease, mindful that permeability might need to be considered
at an early stage.
Given the growing interest in KLK6 as a drug target and the
potential benefit of being able to control its enzymatic activity to
validate current biological hypotheses, we set out to find a novel
series of selective reversible KLK6 inhibitors.
NH2
S
O
HN
N
H2N
O
N
O
S
NH
N
O
N
MeO
S
O
N
1: IC50 = 1.8 M
2: IC50 = 2.9 M
Figure 2. Visualization of the HTS triage process to generate the QHL.
H
H2N
O
N
Cl
O
NH
Compounds containing an oxazolidinone benzamidine were the
largest and most promising cluster with nine members. All of the
compounds in the cluster were potent enough to generate a pIC50
in the primary dose-response assay (Table 1). Each substance
also gave pKD values in the SPR assay which were similar to the
pIC50 values. Furthermore, hits from this cluster exhibited on/off
binding kinetics in the SPR assay consistent with a reversible
mechanism of action (data not shown). In addition, hints of
structure–activity relationships (SAR) could be gleaned. For
example, the most potent hit (5) is the only compound in the
cluster with an amide directly linked to the oxazolidinone.
Compounds with an amide extended by one carbon (6) or
containing other functional groups (7 and 8) are significantly less
active. In addition, 5 had by far the best selectivity profile relative
to trypsin, thrombin and factor Xa (vide infra).
O
H
N
Cl
O
Cl
N
H
NH2
O
O
O
Cl
3: Ki = 1.5 M
4: kinact/Ki = 131 ± 13 M–1s–1
Me
HN
O
Me
H
N
O
N
O
N
Me
DKFZ-251: kinact/Ki = 1883 M-1s-1
Figure 1. Published KLK6 inhibitors.
Results and Discussion
High-throughput screening and validation of a promising hit
Using our previously published KLK6 assay format,[21] we
performed a HTS with the European Lead Factory (ELF),[22] and
tested ~350,000 substances at 10 M for their ability to reduce
KLK6-catalyzed hydrolysis of the fluorogenic Boc-Phe-Ser-Arg-
AMC peptide. About 8,000 actives were identified and re-tested.
After applying a threshold cutoff of 25% inhibition, and discarding
compounds with inherent high fluorescence (>3 times the
background), 1026 compounds were selected for dose-response
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
with the target (Figure 3A). As expected, the highest scoring
poses predict that the amidine group makes critical hydrogen
bonds (H-bonds) with Asp189 and Ser190 in the S1 pocket and is
likely responsible for a significant amount of the binding energy.
A secondary H-bond network is also formed by the amide: the
carbonyl of the amide interacts with the backbone of Gly193 in the
anionic hole and the amide NH interacts with His57 of the catalytic
triad. The more solvent exposed S1´/S2´regions are occupied by
the lipophilic -methylbenzyl amine substituent. Close inspection
suggested that binding could be increased through more effective
filling of the S1’/S2’ pockets lined by Leu40, Leu41, and Phe151.
Comparison of our KLK6 model and X-ray structures of trypsin,[23]
KLK4,[24] and KLK8[25] suggested that by exploiting subtle
differences between the enzymes’ S1’ and S2’ pockets, we could
modulate selectivity for KLK6. For example, the S1’ pocket
appears more compact in KLK6 with Lys60, Leu41, and the
Cys42/Cys58 disulfide bond forming a tight lipophilic space. In
trypsin, the S1’ pocket features Lys60, Phe41 and Cys42/58;
however, the Lys60 side chain appears pulled back by an intra-
residue H-bond with Tyr39, creating a more open pocket which
may prefer to accommodate larger groups (Figure 3B). We also
predicted that it could be possible to improve binding by adding
an additional peptide bond to the inhibitor scaffold, thereby adding
an H-bond to the Leu41 backbone carbonyl and the Gln192 side
chain (Figure 3C). On the basis of our modeling, initial medicinal
chemistry efforts were invested into increasing affinity toward the
S1´/S2´ pockets, which showed the highest potential for
exploration and structural expansion. Furthermore, the model was
tested by synthesizing and testing substances which lacked
specific features that were predicted to be key for binding.
Table 1. Four members of the top hit cluster.
KLK6
pIC50[a]
Cmpd
Structure
[b]
pKD
5
7.2
7.0
6
7
5.0
5.8
4.9
4.7
8
5.1
5.1
[a] pIC50 measured in the enzymatic inhibition assay. [b] pKD measured in the
SPR binding assay.
Determination of the active stereochemical series
Hit 5 was resynthesized along with all three of its stereoisomers
9–11. The activity of 5 against KLK6 was confirmed, although a
consistently lower pIC50 (6.6 vs. 7.2) was found relative to the
same substance in the HTS library (Table 2). Encouragingly, the
other three stereoisomers showed poor activity against KLK6,
suggesting only the (R,S) configuration of the scaffold allows for
productive molecular interactions between the inhibitor and KLK6.
Synthesis of N-benzamidine-oxazolidinone derivatives
Compounds for this study were synthesized as depicted in
Scheme 1A, beginning with conversion of 4-aminobenzonitrile
(12) to the corresponding benzyl carbamate 14. The key
enantiomerically enriched oxazolidinone 17 was prepared by
treating commercially available (R)-glycidyl butyrate ((R)-16) with
the lithium salt of 14. This cascade transformation begins with
opening of the epoxide by the lithio-carbamate, followed by
oxazolidinone ring closure from the resulting alkoxide.
Subsequent transesterification of the butyrate ester with benzyl
alkoxide provides 17. Primary alcohol 17 was oxidized in one step
to carboxylic acid 19 with TEMPO/PhI(OAc)2, and then coupled
with various amines to give amides 21a–21m. In some cases,
chiral racemic amines were used in the amide coupling step, and
the resulting diastereomers were separated by chromatography.
To obtain non-commercial enantioenriched chiral amines 39a–
39c for the above amide coupling, we utilized Ellman´s t-
butylsulfinamide chemistry (Scheme 1B)[26]. Conversion of the
cyano group in 21a–21m to an amidine was performed in a one-
pot, three-step process: addition of hydroxylamine, yielding an
amidoxime, which was acetylated and then reduced with zinc dust
to give 5, 11, and 23–33. Compounds 9 and 10 (not depicted in
Scheme 3) were synthesized in analogy to 5 and 11, but using
(S)-glycidyl butyrate ((S)-16) in the second step. Compound 34
was prepared similarly, but starts with 2-cyano-5-aminopyridine
(13) instead of 12. Benzyl amine 35 was prepared from the
corresponding nitrile via hydrogenation with Raney nickel.
Table 2. Testing of the four stereoisomers of 5 indicate that 5 is the active
stereoisomer.
Cmpd
Structure
KLK6
pIC50[a]
5
6.6
O
N
O
H
H2N
HN
9
<4.7
<4.7
(R)
N
OMe
(S)
O
Me
10
O
N
O
H2N
HN
H
N
(R)
11
<4.7
OMe
(R)
O
Me
[a] pIC50 measured in the enzymatic inhibition assay.
The synthesis of three additional substances is depicted in
Scheme 2. In the first example, 12 and 2-methylenesuccinic acid
(40) were combined to make lactam 41 as a racemate, which was
Docking study predicts key binding interactions
Compound 5 was docked into an X-ray crystal structure of KLK6
(i.e. PDB ID 4D8N), allowing us to model the main interactions
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
Figure 3. (A) Docking model of 5 (coral) bound to KLK6. (B) Docking model of 5 (coral) overlaid on trypsin (PDB ID 1GJ6). (C) Docking model of 28 (green) bound
to KLK6. Both KLK6 models are derived from PDB ID 4D8N.
A
O
O
OH
H
N
b.
BuLi
n-
NH2
a. CbzCl
N
c. TEMPO
R
Cbz
then
PhI(OAc)2
NC
X
NC
X
O
NC
X
O
n-Pr
17: X = C
18: X = N
14: X = C
15: X = N
O
12: X = C
13: X = N
(R)-16
R1
R3
O
O
HN
O
e. NH2OH•HCl;
R2
N
Ac2O, AcOH;
Zn or
Pd/C, H2
H2N
X
H2N
R1
NH
R1
O
O
O
O
5, 11, 23–33: X = CH
34: X=N, R1 = 1-naphthyl, R2= Et, R3 = H
OH
HN
O
R2 R3
R3
d.
R2
N
N
O
HATU
R1
R3
O
NC
X
NC
X
O
HN
O
R2
19: X = C
20: X = N
21a–21m: X = C
H2 (50 bar)
f.
N
22: X = N; R1 = 1-naphthyl,
H2N
R2 = Et, R3 = H
Ra-Ni
55 °C
35: R1 = 1-naphthyl, R2 = Et, R3 = H
B
NH2
36
S
O
H
N
g.
O
h. EtMgBr
i. HCl
H2N
R
N
R
R
S
S
H
R
Et
O
O
Et
37a: R = 3-OMePh
37b: R = 1-naphthyl
37c: R = 4-quinolyl
38a: R = 3-OMePh
38b: R = 1-naphthyl
38c: R = 4-quinolyl
39a: R = 3-OMePh
39b: R = 1-naphthyl
39c: R = 4-quinolyl
Scheme 1. Reagents and conditions: (a) Benzyl chloroformate, Et3N, CH2Cl2; (b) n-BuLi, THF, –78 °C, then 16; (c) TEMPO, PhI(OAc)2, 1:1 MeCN/H2O; (d) amine,
HATU, i-Pr2NEt, DMF; (e) NH2OH•HCl, i-Pr2NEt, EtOH, 100 °C, wave, 60 min, then concentrate; Ac2O, AcOH, 20 h, then add Zn, 4 h or concentrate then 10%
Pd/C, H2 (10 bar), 1:1 MeOH/EtOAc; (f) H2 (50 bar), Raney-Ni, EtOH, 55 °C; (g) 36, 5 mol% PPTS, MgSO4 (5 equiv.), CH2Cl2; (h) EtMgBr, CH2Cl2, –40 °C; (i) 4M
HCl in dioxane, MeOH.
advanced to 43 via amide coupling with 42 followed by amidine
formation. Amide 43 was separated from the diastereomer arising
from the undesired enantiomer contained in rac-41 via
chromatography. The synthesis of 47 began with an SNAr reaction
between 4-fluorobenzonitrile (44) and methyl (R)-pyrrolidine-3-
carboxylate (45) to give pyrrolidine 46, which was advanced to 47
as above. Compound 49 was synthesized starting with
conversion of alcohol 17 to amine 48, via activation of 17 as a
mesylate and displacement with amine 42. Conversion of the
cyano group in 48 to an amidine gave 49.
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
b.
H2N
OMe
OMe
a.
O
O
Me
42
HN
NH2
O
OH
i) 160°C, wave
N
Me
OH
N
HATU
+
HO
O
O
NC
ii) TFA, 55 °C
c.
NH2OH•HCl;
H2N
O
NC
Ac2O, AcOH;
Zn
43
12
40
41
NH
b.
H2N
OMe
OH
OMe
F
d. DMSO, 85 °C
e. LiOH
HN
OMe
Me
N
HN
O
42
HATU
NH2OH•HCl;
O
+
N
Me
NC
O
NC
44
45
H2N
c.
46
Ac2O, AcOH;
Zn
47
NH
OMe
O
O
48: R = CN
O
c. NH2OH•HCl;
Ac2O, AcOH;
Zn
OH
O
HN
f. MsCl
N
N
Me
NH
2
g.
49: R =
NH
NC
H2N
R
OMe
17
Me
42
Scheme 2. Reagents and conditions: (a) i. neat, 160 °C; ii. TFA, 55 °C; (b) 40, HATU, i-Pr2NEt, DMF; (c). NH2OH•HCl, i-Pr2NEt, EtOH, 100 °C, wave, 60 min, then
concentrate; Ac2O, AcOH, 20 h then add Zn, 4 h; (d) DMSO, 85 °C; (e) LiOH, H2O/THF; (f) methanesulfonyl chloride, Et3N, CH2Cl2; (g) 40, DMF, 120 °C, wave.
Structure-activity relationship studies
pocket of trypsin and KLK8. The appropriate stereochemistry is
essential to direct the P1’ substituent towards the S1´ pocket, as
testified by the loss of potency of diastereoisomer 11.
Interestingly, a dimethyl group, which presumably still directs one
methyl group to the S1´ pocket, is also tolerated and appears to
be beneficial for selectivity (cmpd. 26).
Guided by the docking studies, we set out to establish SAR by
dissecting the main molecular features of the compound class. In
addition to trypsin, thrombin, and factor Xa, we included KLK4,
KLK7, and KLK8 as additional targets for selectivity analysis
(Table 3). Having already determined the active stereoisomer 5,
we assessed the importance of the oxygen atoms in the
oxazolidinone ring by testing lactam 43 and pyrrolidine 47. While
43 showed similar potency and selectivity toward KLK6 relative to
5, 47 slightly lost potency and selectivity against KLK6, indicating
some binding role for the carbonyl. A much larger effect was
observed with 49, which lacks the amide carbonyl and results in
a 40-fold KLK6 potency loss. This data is largely consistent with
the docking model, where the amide makes H-bonds, while the
oxazolidinone heteroatoms make no strong interactions with
surrounding residues.
We next examined variations of the amide N-substituents, which
are hypothesized to bind in the S1´/S2´pockets. As mentioned
above, docking models suggested these were regions where
subtle differences between KLK6 and the related proteases could
be harnessed to boost selectivity. In particular, the small methyl
group of 5 was predicted to fit into the S1´ pocket. This pocket is
shallower in KLK6 than in related proteases, but our model
suggested that it could accommodate a slightly larger group,
strengthening this interaction. Consistent with the model, we
found that removal of this methyl group (cmpd. 23) is detrimental
for activity, while replacement with an ethyl group (cmpd. 24)
results in an almost 10-fold gain in potency for KLK6 and an
increase in selectivity. A larger cyclopropane substituent (cmpd.
25) does not improve the potency against KLK6 relative to 24, and
is deleterious for selectivity against trypsin and KLK8. It may be
that the cyclopropyl group can better interact with the larger S1´
The SAR of the P2´ substituent of the inhibitor class was
investigated next. Compound 28 was synthesized to validate the
docking prediction, which was indeed supported by an increase
in potency compared to parent compound 27, suggesting that the
additional amide moiety might form further H-bonds with the
active site of KLK6. Moreover, 28 exhibited excellent selectivity
over KLK4 and trypsin. In parallel, larger lipophilic substituents
were introduced at P2’. Compound 29, with a 1-naphthyl P2’, was
more potent than 28 (7.9 vs. 7.5 pIC50). Aiming to minimize the
peptidic-nature of the compounds, we continued building on 29.
Interestingly, the 1-naphthyl regioisomer (29) was preferred over
the 2-naphthyl (30), which showed a 10-fold potency loss. While
a dimethyl P1’ substituent showed promise in the case of 26 the
gem-dimethyl derivative 31 was ~6 fold less potent than 29.
Fortunately, the optimal P1’ (ethyl) and P2’ (1-naphthyl)
substituents showed additive effects, with compound 32
approaching the potency limit of the KLK6 assay (pIC50 = 8.6).
This compound retained a similar selectivity profile to the original
hit 5, while having almost 100-fold higher potency against KLK6.
Interestingly, introduction of a nitrogen atom in the naphthalene
bicycle resulted in a dramatic loss of potency as measured for
compound 33, further highlighting the steep SAR observed within
the P2´ substituent.
With a potent compound such as 32 in hand, we examined the
possibility of lowering the basicity of the P1 amidino group, which
could have an effect on permeability. Previous attempts to
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
Table 3. Activity of synthesized inhibitors against KLK6 and related proteases
Cmpd
Structure
KLK6a
KLK4a
KLK7 a
< 4.7
KLK8 a
Trypsin a
Thrombin a
Factor Xa a
5
6.6
5.1
5.0
5.3
< 4.7
< 4.7
11
4.8
–
< 4.7
< 4.7
< 4.7
< 4.7
< 4.7
43
47
49
6.5
6.1
5.0
5.1
5.9
–
< 4.7
< 4.7
< 4.7
< 4.7
5.3
5.3
< 4.7
< 4.7
< 4.7
< 4.7
< 4.7
< 4.7
5.1
< 4.7
< 4.7
23
24
5.4
7.5
–
< 4.7
< 4.7
< 4.7
< 4.7
< 4.7
5.6
< 4.7
5.3
5.8
< 4.7
< 4.7
25
26
7.5
6.8
–
–
< 4.7
6.1
6.6
5.0
< 4.7
< 4.7
–
< 4.7
–
–
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
27
28
6.1
7.5
4.4
5.8
< 4.7
< 4.7
< 4.7
< 4.7
< 4.7
–
–
5.2
–
–
29
30
7.9
6.2
< 4.7
6.0
6.1
< 4.7
< 4.7
7.0
7.1
5.5
< 4.7
5.0
5.4
5.3
< 4.7
< 4.7
31
–
–
< 4.7
–
–
O
N
O
H2N
H
N
32
8.6*
6.9
8.3
6.8
< 4.7
6.7
< 4.7
6.3
7.2
5.2
6.2
< 4.7
< 4.7
HN
O
Et
33
34
–
–
–
–
–
–
–
–
35
6.5
5.3
–
5.0
5.3
–
–
a All values are reported as pIC50. *8.6, i.e. 2.5 nM, represents the assay limit in the KLK6 assay. SAR highlight of each compound is given by the dashed orange
box. An entry of “–“ means the compound was not measured in that assay.
introduce less basic functionalities, e.g. benzylamine, amide and
aminoisoquinoline within the 5 scaffold, resulted in significant to
complete loss of activity against the target protease (data not
shown). When the benzylamine replacement was made on the
improved scaffold of 32, it resulted in ~100-fold potency loss
(cmpd 35). However, good activity was still detected against KLK6
(pIC50 = 6.5) with an altogether unchanged selectivity profile,
which provides a good starting point for further development of
amidine-free KLK6 inhibitors. Interestingly, introduction of a
nitrogen atom in the benzamidine ring (cmpd 34), which is
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
predicted to reduce the pKa of the amidine from ~11 to ~9,
resulted in a compound of slightly reduced potency for KLK6, but
an improved selectivity profile against the related enzymes. An
increase in selectivity over trypsin was also observed when the
indole of DKFZ-251 was replaced with a 7-azaindole.[21] The
structural reasons for these effects and whether they are
connected is currently under further investigation.
the 3A4 isozyme was inhibited with an IC50 of 4.8 M. The
compound showed no instability in mouse plasma over 3 h, and
was found to bind protein plasma to an extent of 89%.
Table 4. ADME Profiling of compound 32•TFA.
ALogP
3.26
When analyzing the compounds in a lipophilic efficiency (LipE)
plot (LipE = pIC50 – logP), a number of observations could be
made (Figure 4). The value of an S1’ ethyl vs. methyl is clear when
comparing compounds 5 (LipE = 4.79) and 24 (LipE = 5.16). The
two highly potent compounds 32 and 34 show improved LipE
values of 5.34 and 5.76, respectively. Interestingly, 28 shows the
highest LipE value of 6.20, due to its relatively low lipophilicity.
While the additional amide in 28 might be expected to pose a
problem for cell permeability, particularly in conjunction with an
amidine moiety in the same molecule, further medicinal chemistry
optimization could focus on mimicking the amide with
heterocycles or other H-bond donors/acceptors.
pKa
11.3
Kinetic Solubility
Thermodynamic Solubility
Mouse CLint
Human CLint
Mouse Hep Cl
>250 M
2.3 mM
2.4 mL/min/g
3.1 mL/min/g
2.3 mL/min/g
>10 / >10 / 7.9 / >10 / 4.8 M
CYP Inhibition IC50
1A2 / 2C9 / 2C19 / 2D6 / 3A4
Protein plasma (mouse) binding
(fraction bound)
89%
Mouse Plasma Stability (t1/2
)
>180 min
Compound 32 reduces invasion of HCT116 cells
We have previously shown that knockdown of KLK6 reduces
migration and invasion of HCT116 cells in vitro.[13] In order to test
whether this effect could be recapitulated with small molecule
KLK6 inhibitors, we measured the ability of 32 to reduce invasion
of HCT116 cells. Because the enantiomer of 32 was never
prepared, compound 9, the enantiomer of hit 5, was used as an
inactive control substance. While compound 9 had no significant
effect on the invasion of HCT116 cells after 24 h at multiple
concentrations, compound 32 induced a significant reduction in
invasion at both 50 nM and 500 nM (Figure 5). HCT116 cell
growth and viability was not altered upon treatment with
compounds 9 and 32 at the tested concentrations (data not
shown).
Conclusions
Figure 4. Lipophilicity plot of the compounds in this manuscript. The size of
each dot represents selectivity over trypsin, with a larger dot indicating better
selectivity. Trypsin data was used for this plot because trypsin data was
generated for each compound and because trypsin activity varied significantly
between derivatives. ALogP was calculated using Pipeline Pilot. Blue = hit
substance. Yellow = stereoisomers of the hit. Red = potential leads.
After a high throughput screen of the European Lead Factory
compound collection, we discovered a validated hit cluster of N-
(4-benzamidino)-oxazolidinones that showed consistent inhibitory
activity against KLK6. Docking-guided optimization of this
scaffold, with a focus on potency against KLK6 and selectivity
against up to six different related proteases resulted in
compounds with single digit nanomolar potency and good to
excellent selectivity. Compound 32 was found to be the most
potent inhibitor, while compounds 28 and 34 exhibited the highest
selectivity against trypsin. ADME profiling of 32 showed that it has
reasonable properties for pre-clinical biological experiments.
Finally, 32 was found to reduce invasion of HCT116 cells in a
dose-dependent manner, while 9, a control substance from the
inactive enantiomeric series, showed no such effect even at the
highest tested concentrations.
Lead compound pharmacokinetic properties
The most potent inhibitor, 32, was tested in a number of
computational and experimental ADME profiling assays (Table 4).
The free base of 32 was calculated to have an ALogP of 3.26, and
its conjugate acid a pKa of 11.3. The trifluoroacetate salt of 32
showed excellent solubility in both kinetic and thermodynamic
assay formats, presumably a result of the basic amidine.
Clearance measurements with both mouse and human
microsomes was relatively low. Clearance in mouse hepatocytes
correlated well with the microsomal clearance (2.3 vs 2.4
mL/min/g), suggesting no significant contribution from phase II
pathways. Measurement of cytochrome P450 (CYP) activity with
five different isozymes showed no significant inhibition by 32. Only
These compounds show promise as useful chemical probes for
the study of KLK6 biology. One particular benefit is the availability
of inactive enantiomeric control compounds. While the
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
selection of 226 compounds showing KLK6 pEC50 > 6.0 OR 5.0 < KLK6
pEC50 < 6.0 selectivity >0.1 OR 4.7 <KLK6 pEC50 <5.0 selectivity >1. Upon
testing of these compounds using SPR and LC-MS a qualified hit list (QHL)
was registered containing 50 compounds. For SPR, Biacore T200 was
used and KLK6 was immobilized onto a CM5 chip. 226 test compounds
were screened at 4 concentrations: 20, 4, 0.8 and 0.16 μM in running
buffer: 50 mM Tris-HCl, 1 mM EDTA pH 7.5, 150 mM NaCl, 0.05% Tween
20, 3% DMSO at 25°C. Control injections of 5 μM 1 were used throughout
the screen to ensure that KLK6 was stable and had not been blocked by
potential irreversible binders/denaturants. An 8 point 1 in 3 dilution series
of 1 (50 μM to 0.02 μM) was also applied at the beginning and end of each
screening day.
enantiomers of 28, 32, and 34 were not synthesized as part of this
ELF project, their synthesis is straightforward and will be made
available upon request to interested scientists in the future.
Docking Studies: The Schrodinger Suite was used to prepare the KLK6
structure (PDB ID 4D8N) and to perform the docking experiments (Glide).
Molecules were standardized and 3D conformers generated with the
Ligprep module from the Schrodinger Suite using standard settings. Input
stereochemistry was retained. The docking was performed using the Extra
Precision (XP) method and the other parameters are assigned to their
default values. A post-docking refinement step was applied using the MM-
GBSA module that approximates the free energy of binding.
Chemistry: (General) Chemicals and solvents were from commonly used
suppliers and were used without further purification. Silica gel 60 F254
analytical thin layer chromatography (TLC) plates were from Merck
(Darmstadt, Germany) and visualized under UV light and/or with KMnO4
stain. Automated chromatography was performed with a Biotage Isolera
Purification system (Uppsala, Sweden). Deuterated solvents were
obtained from Cambridge Isotopes. All NMR spectra were recorded using
a Bruker Avance 400 MHz spectrometer, and the residual solvent peak
was used as internal reference (1H NMR: CHCl3 (7.26 ppm); DMSO (2.50
ppm); MeOH (3.31 ppm); 13C NMR: CHCl3 (77.16 ppm); DMSO (39.52
ppm); MeOH (49.00 ppm)). Chemical shifts () are reported in ppm and
coupling constants (J) in Hz. The following abbreviations were used to for
multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, p=pentet,
m=multiplet, br=broad. Preparative HPLC was carried out on a Waters
HPLC comprising a Waters 2767 Sample Manager, Waters 2545 Binary
Gradient Module, Waters Systems Fluidics Organiser, Waters 515 ACD
pump, Waters 2998 Photodiode Array Detector, using a Waters XBridge
Prep OBD C18, 5 μm, 19 mm x 50mm i.d. column and a flow rate of 20 mL
/ min. The general method used a gradient of 5% acetonitrile / 95% water
to 100% acetonitrile (with 0.1% trifluoroacetic acid in both phases). UV
detection e.g. 254 nM is used for the collection of fractions from HPLC. All
final compounds were found to have ≥ 95% purity, controlled by analytical
(LC/MS) and confirmed by 1H NMR.
Figure 5. HCT116 cells were treated with 9 or 32 at the indicated concentrations
and plated on Matrigel®-coated filters. The upper chamber contained serum-
free medium, while the lower chamber contained 10% fetal bovine serum. After
24 h, the number of invading cells was measured optically. (A) This figure is one
representative example of four biological replicates, each with similar outcomes.
Error bars are SEM. Technical replicates: DMSO and 9 at 5 nM (n=3); 9 at 50
and 500 nM and 32 at all concentrations (n=6). Statistical significance was
calculated using an unpaired two-tailed t-test. n.s = p ≥ 0.05; ** = p < 0.01; ***
= p < 0.001. (B) Representative images (Scale bar, 400 mm) of stained invaded
cells outside of the inserts 24 h hours after treatment with 9, 32, or DMSO
solvent control at the indicated concentrations. Phase contrast images were
taken with EVOS FL Cell imagine system FL Auto microscope (Bio-Rad).
Method A (amide coupling): To a mixture of carboxylic acid (1 equiv) and
HATU (1.5 equiv) in DMF is added amine (1.2 equiv) and i-Pr2NEt (3.0
equiv). After stirring for 24 h, the mixture is partitioned between EtOAc and
brine/water. The layers are separated and the aqueous phase is extracted
with EtOAc (2 x). The combined organics are washed with brine (3 x), dried
(Na2SO4), filtered, concentrated under reduced pressure, and purified by
chromatography.
Experimental Section
Method B (nitrile to amidine conversion): A mixture of benzonitrile (1
equiv), NH2OH•HCl (5 equiv), and i-Pr2NEt (5 equiv) in EtOH is heated to
100 °C in a microwave for 60 min. After cooling, the mixture is diluted with
MeOH and concentrated under reduced pressure. The residue is dissolved
in AcOH and Ac2O (5 equiv), stirred at rt for 20 h, diluted with MeOH, and
concentrated under reduced pressure. The residue is then dissolved in
AcOH and Zn powder (10 equiv) is added with stirring for 2.5 h. The
mixture is filtered, rinsing with MeOH, and the filtrate is concentrated,
followed by azeotroping with heptane (x3). Purification by HPLC yields the
product as a trifluoroacetate salt.
High Throughput Screen: The European Lead Factory library of
~350,000 compounds was used for high throughput screening at the Pivot
Park Screening Center facilities in Oss, The Netherlands. HighRes
Biosolutions robotic infrastructure using Cellario software was
programmed to perform screening of 281 1536-well plates. Using the
primary KLK6 fluorescent intensity (FI) assay (see below), 7794
compounds having a Z-score ≤ -4 were selected for further follow-up. An
additional set of compounds was added based on Bayesian modeling to
include potential false negatives in the hit confirmation. Of the tested 8706
compounds, 1026 showed >25 % inhibition when re-tested in the KLK6 FI
assay. When tested in serial dilutions (20 µM to 20 nM; 7-points 10
diluted), 312 compounds showed a pEC50 ≥ 4.7. This set was subsequently
tested in Trypsin, Thrombin and FXa assays (see below) resulting in a
Method
C (sulfinyl imine formation): To a solution of (R)-(+)-2-
methylpropane-2-sulfinamide (36) (1 equiv) in CH2Cl2 is added pyridinium
p-toluenesulfonate (PPTS) (50 mol%), MgSO4•H2O (5 equiv), and
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
aldehyde (2 equiv). After 2 d, the resulting mixture is filtered, rinsed with
CH2Cl2, and the filtrate is concentrated and purified by chromatography.
allowed to warm to rt and stirred overnight. The reaction was quenched
with saturated aqueous NH4Cl and extracted with EtOAc twice. The
combined organic extracts were washed with brine, dried (Na2SO4),
filtered and concentrated under reduced pressure. Chromatography (Telos
20g, EtOAc in CH2Cl2, 0 to 75%) gave 0.72 g (74%) of 17: 1H NMR
(CD3OD) : 7.76–7.85 (m, 2H), 7.69–7.76 (m, 2H), 4.73–4.81 (m, 1H), 4.17
(t, J = 9.2 Hz, 1H), 3.97 (dd, J = 9.0, 6.3 Hz, 1H), 3.87 (dd, J = 12.6, 3.3
Hz, 1H), 3.70 (dd, J = 12.6, 3.3 Hz, 1H) ppm.
Method D (Grignard addition to sulfinyl imine): To a solution of 37a or
37c (1 equiv) in CH2Cl2 is added a solution of EtMgBr (2 equiv) in Et2O at
–78 °C under argon. After 4 h, the mixture is allowed to warm to rt. After
18 h, the reaction is quenched with H2O, diluted with EtOAc, and the two
layers are separated. The aqueous layer is further extracted with EtOAc (2
x), the combined organics are washed (brine), dried (MgSO4), filtered,
concentrated, and purified by chromatography.
(5R)-5-(5-(hydroxymethyl)-2-oxooxazolidin-3-yl)picolinonitrile (18):
To a cooled (–78 °C) solution of carbamate 15 (0.30 g, 1.2 mmol) in dry
THF (12 mL) was added a solution of n-BuLi (0.57 mL, 2.5 M in hexanes,
1.43 mmol) dropwise. After stirring at –78 °C for 45 min, ((2R)-oxiran-2-
yl)methyl butanoate (16) (0.17 mL, 1.2 mmol) was added dropwise. The
resulting mixture was stirred at –78 °C for 2 h and then allowed to warm to
rt and stirred overnight. The reaction was quenched with saturated
aqueous NH4Cl and extracted with EtOAc (x2). The combined organic
extracts were washed with brine, dried (Na2SO4), filtered and concentrated
under reduced pressure. Chromatography (ZIP-30g, EtOAc in heptane, 0–
100%, then MeOH in CH2Cl2) gave a material that was triturated with EtOH
and a white solid collected by filtration, further rinsed with cold EtOH then
dried on the filter pad to give 100 mg (39%) of 18: 1H NMR (DMSO-d6) :
8.96 (d, J = 2.5 Hz, 1H), 8.21 (dd, J = 8.7, 2.6 Hz, 1H), 8.06 (d, J = 8.7 Hz,
1H), 5.25 (t, J = 5.4 Hz, 1H), 4.81 (qd, J = 6.2, 3.5 Hz, 1H), 4.14–4.22 (m,
1H), 3.93 (dd, J = 9.0, 6.0 Hz, 1H), 3.65–3.75 (m, 1H), 3.53–3.64 (m, 1H)
ppm. LCMS m/z = 220.0 (M+H)+.
(5R)-3-(4-carbamimidoylphenyl)-N-[(S)-1-(3-methoxyphenyl)ethyl]-2-
oxooxazolidine-5-carboxamide (5): Method B with amide 21a (78 mg,
0.21 mmol), NH2OH•HCl (71 mg, 1.0 mmol), i-Pr2NEt (0.18 mL, 1.0 mmol)
and EtOH (2.9 mL). Then AcOH (2.0 mL) and Ac2O (0.1 mL, 1.1 mmol).
Then AcOH (2.0 mL) and Zn (0.14 g, 2.1 mmol). HPLC purification yielded
70 mg (66%) of 5•TFA: 1H NMR (DMSO-d6) : 9.25 (br s, 2H), 9.09 (br s,
2H), 8.97 (d, J = 8.1 Hz, 1H), 7.85–7.91 (m, 2H), 7.77–7.83 (m, 2H), 7.24
(t, J = 8.1 Hz, 1H), 6.88–6.93 (m, 2H), 6.81 (ddd, J = 8.3, 2.5, 1.1 Hz, 1H),
5.17 (dd, J = 9.3, 5.6 Hz, 1H), 4.89–5.00 (m, 1H), 4.36 (t, J = 9.3 Hz, 1H),
4.08 (dd, J = 9.3, 5.7 Hz, 1H), 3.73 (s, 3H), 1.40 (d, J = 7.0 Hz, 3H) ppm.
13C NMR (DMSO-d6) : 166.8, 164.6, 159.3, 158.2 (q, JC-F = 30 Hz), 153.6,
145.5, 142.7, 129.4, 129.2, 122.3, 118.2, 117.4, 112.1, 111.8, 70.6, 55.0,
48.3, 47.5, 22.1 ppm. HRMS (ESI) m/z: (M+H)+ calcd for C20H23N4O4
:
+
383.1714; found: 383.1712.
(5R)-3-(4-carbamimidoylphenyl)-N-[(R)-1-(3-methoxyphenyl)ethyl]-2-
(5R)-3-(4-cyanophenyl)-2-oxo-oxazolidine-5-carboxylic acid (19): To a
cooled (0 °C) solution of alcohol 17 (0.72 g, 3.3 mmol) in MeCN/water (1:1,
18 mL) was added iodobenzene diacetate (2.7 g, 8.2 mmol) followed by
TEMPO (0.10 g, 0.66 mmol). The resulting mixture was stirred at rt for
18 h, then partitioned between a saturated aqueous solution of NaHCO3
and EtOAc. The layers were separated and the organic layer was further
washed with a saturated aqueous solution of NaHCO3. The aqueous
extract was acidified with 5 M HCl and extracted with EtOAc (x3). The
combined acidic organic extracts were dried (Na2SO4), filtered and
concentrated under reduced pressure to give 730 mg (96%) of 19: 1H NMR
(DMSO-d6), : 13.78 (br s, 1H), 7.87 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 8.8
Hz, 2H), 5.24 (dd, J = 9.8, 5.3 Hz, 1H), 4.40 (dd, J = 9.8, 9.4 Hz, 1H), 4.18
(dd, J = 9.4, 5.3 Hz, 1H) ppm.
oxooxazolidine-5-carboxamide (11): Method B with amide 21b (81 mg,
0.22 mmol), NH2OH-HCl (74 mg, 1.1 mmol), i-Pr2NEt (0.19 mL, 1.1 mmol)
and EtOH (3.0 mL). Then AcOH (2.0 mL) and Ac2O (0.10 mL, 1.1 mmol).
Then AcOH (2.0 mL) and Zn (0.14 g, 2.2 mmol). HPLC purification yielded
65 mg (59%) of 11•TFA: 1H NMR (DMSO-d6) d: 9.24 (m, 3H), 8.96 (d, J =
5.8 Hz, 1H), 7.93–7.84 (m, 2H), 7.83–7.76 (m, 2H), 7.24 (t, J = 8.1 Hz, 1H),
6.95–6.89 (m, 2H), 6.81 (ddd, J = 8.3, 2.5, 1.1 Hz, 1H), 5.21–5.12 (m, 1H),
4.95 (app p, J = 7.1 Hz, 1H), 4.35 (t, J = 9.6 Hz, 1H), 4.03 (dd, J = 9.3, 5.8
Hz, 1H), 3.74 (s, 3H), 1.41 (d, J = 7.0 Hz, 3H) ppm. 13C NMR (DMSO-d6)
: 166.8, 164.7, 159.3, 153.6, 145.5, 142.7, 129.4, 129.2, 122.3, 118.3,
117.4, 112.1, 112.0, 70.5, 55.0, 48.3, 47.5, 22.2 ppm. HRMS (ESI) m/z:
(M+H)+ calcd for C20H23N4O4+: 383.1714; found: 383.1713.
Benzyl N-(4-cyanophenyl)carbamate (14): To
a solution of 4-
(5R)-3-(6-cyanopyridin-3-yl)-2-oxooxazolidine-5-carboxylic acid (20):
aminobenzonitrile (12) (1.0 g, 8.5 mmol) in THF (17 mL) was added a
solution of K2CO3 (2.3 g, 17 mmol) in water (17 mL), followed by slow
addition of benzyl chloroformate (1.4 mL, 10 mmol). The resulting mixture
was stirred at rt for 2.5 d, then partitioned between EtOAc and water. The
two layers were separated and the organic layer was further washed with
brine, dried (Na2SO4), filtered and concentrated under reduced pressure.
The product was purified by flash chromatography (Telos 25 g, CH2Cl2
100%) to provide 2.0 g (95%) of 14: 1H NMR (400 MHz, CDCl3),  7.58–
7.65 (m, 2H), 7.49–7.57 (m, 2H), 7.35–7.47 (m, 5H), 6.91 (br s, 1H), 5.24
(s, 2H) ppm.
To
a cooled (0 °C) solution of alcohol 18 (99 mg, 0.45 mmol) in
MeCN/water (1:1, 2.4 mL) was added iodobenzene diacetate (0.36 g,
1.1 mmol) followed by TEMPO (14 mg, 90 µmol). The resulting mixture
was stirred at rt for 48 h then partitioned between a saturated aqueous
solution of. NaHCO3 and EtOAc. The organic extracts were further washed
with a saturated aqueous solution of NaHCO3. The aqueous extract was
acidified with 5 M HCl and extracted with EtOAc (x3). The combined acidic
organic extracts were dried (Na2SO4), filtered and concentrated under
reduced pressure to give 100 mg (97%) of 20: 1H NMR (CD3OD) : 8.91–
9.00 (m, 1H), 8.14–8.27 (m, 1H), 7.82–7.93 (m, 1H), 5.25 (dd, J = 9.8, 5.3
Hz, 1H), 4.41–4.53 (m, 1H), 4.22–4.33 (m, 1H) ppm.
Benzyl (6-cyanopyridin-3-yl)carbamate (15): To a suspension of 5-
aminopyridine-2-carbonitrile (13) (1.0 g, 8.4 mmol) and K2CO3 (1.7 g,
13 mmol) in THF (69 mL) was added benzyl chloroformate (1.8 mL,
13 mmol). The resulting mixture was stirred at rt for 2.5 days, then filtered
rinsing with EtOAc. The filtrate was concentrated under reduced pressure,
then purified by flash chromatography (ZIP 30 g, EtOAc in CH2Cl2, 0 to
10%) to give 1.4 g (66%) of 15: 1H NMR (CDCl3), : 8.55 (d, J = 2.5 Hz,
1H), 8.21 (dd, J = 8.4, 2.1 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.33–7.49 (m,
5H), 7.02 (br s, 1H), 5.27 (s, 2H) ppm.
(5R)-3-(4-cyanophenyl)-N-[(1S)-1-(3-methoxyphenyl)ethyl]-2-oxo-
oxazolidine-5-carboxamide (21a): Method
A
with 19 (75 mg,
0.32 mmol), HATU (0.18 g, 0.48 mmol), DMF (2.0 mL), (S)-1-(3-
methoxyphenyl)ethan-1-amine (42) (60 µL, 0.39 mmol), and i-Pr2NEt
(0.17 mL, 0.97 mmol). Chromatography: (ZIP-10g, EtOAc in heptane, 0–
60% then ZIP-10g, EtOAc in CH2Cl2, 0–10%) gave 92 mg (78%) of 21a:
1H NMR (CD3OD) : 7.69–7.82 (m, 4H), 7.21 (t, J = 7.9 Hz, 1H), 6.85–6.94
(m, 2H), 6.78 (dd, J = 8.3, 1.8 Hz, 1H), 5.14 (dd, J = 9.5, 5.8 Hz, 1H), 5.05
(p, J = 7.0 Hz, 1H), 4.35 (t, J = 9.3 Hz, 1 H), 4.14 (dd, J = 9.2, 5.9 Hz, 1H),
3.75 (s, 3H), 1.50 (d, J = 7.0 Hz, 3H) ppm.
4-[(5R)-5-(hydroxymethyl)-2-oxo-oxazolidin-3-yl]benzonitrile (17): To
a cooled (–78 °C) solution of carbamate 14 (1.1 g, 4.4 mmol) in dry THF
(44 mL) a solution of n-BuLi (2.1 mL, 2.5 M in hexanes, 5.25 mmol) was
added dropwise. After stirring the reaction mixture at –78 °C for 45 min,
((2R)-oxiran-2-yl)methyl butanoate (16) (0.62 mL, 4.4 mmol) was added
dropwise. The resulting mixture was stirred at –78 °C for 2 h and then
(5R)-3-(4-cyanophenyl)-N-[(1R)-1-(3-methoxyphenyl)ethyl]-2-
oxooxazolidine-5-carboxamide (21b): Method
A with 19 (75 mg,
0.32 mmol), (R)-1-(3-methoxyphenyl)ethan-1-amine (59 mg, 0.39 mmol),
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
HATU (0.18 g, 0.48 mmol), DMF (2.5 mL), and i-Pr2NEt (0.17 mL,
0.97 mmol). Chromatography (ZIP-10g, EtOAc in heptane, 0–60%; then
ZIP-10g, EtOAc in CH2Cl2, 0–10%) gave 96 mg (81%) of 21b: 1H NMR
(400 MHz,CD3OD) : 7.69–7.83 (m, 4H) 7.24 (t, J = 8.0 Hz, 1H) 6.89–6.97
(m, 2H) 6.75–6.86 (m, 1H) 5.11 (dd, J = 9.5, 6.0 Hz, 1H) 5.05 (q, J = 7.0
Hz, 1H) 4.35 (t, J = 9.4 Hz, 1H) 4.12 (dd, J = 9.3, 6.0 Hz, 1H) 3.79 (s, 3H)
1.50 (d, J = 7.0 Hz, 3H) ppm.
7.67–7.73 (m, 2H), 7.43–7.50 (m, 2H), 7.22–7.32 (m, 2H), 7.04–7.12 (m,
1H), 5.15 (dd, J = 9.5, 6.0 Hz, 1H), 4.34 (t, J = 9.4 Hz, 1H), 4.11–4.16 (m,
1H), 1.62 (s, 3H), 1.61 (s, 3H) ppm.
(5R)-3-(4-cyanophenyl)-N-((S)-1-(naphthalen-1-yl)ethyl)-2-
oxooxazolidine-5-carboxamide (21i): Method
A with 19 (50 mg,
0.22 mmol), (1S)-1-(1-naphthyl)ethanamine (40 µL, 0.26 mmol), HATU
(0.12 g, 0.32 mmol), DMF (1.3 mL), and i-Pr2NEt (0.11 mL, 0.65 mmol).
Chromatography (ZIP-10g, EtOAc in heptane, 0–100%) gave 79 mg,
(95%) of 21i: 1H NMR (DMSO–d6) : 9.11 (d, J = 8.0 Hz, 1H), 8.10 (d, J =
8.3 Hz, 1H), 7.92–7.98 (m, 1H), 7.82–7.89 (m, 3H), 7.72–7.79 (m, 2H),
7.47–7.61 (m, 4H), 5.70–5.81 (m, 2H), 5.16 (dd, J = 9.3, 5.5 Hz, 1H), 4.35
(t, J = 9.3 Hz, 1H), 4.04–4.11 (m, 1H), 1.56 (d, J = 7.0 Hz, 3H) ppm.
(5R)-3-(4-cyanophenyl)-N-(3-methoxybenzyl)-2-oxooxazolidine-5-
carboxamide (21c): Method
A with 19 (69 mg, 0.30 mmol), (3-
methoxyphenyl)methanamine (50 µL, 0.36 mmol), HATU (0.17 g,
0.45 mmol), DMF (1.5 mL), and i-Pr2NEt (0.16 mL, 0.89 mmol).
Chromatography (ZIP-10g, EtOAc in heptane, 0–100%) gave 64 mg (60%)
of 21c: 1H NMR (DMSO–d6)  9.00 (t, J = 5.9 Hz, 1H), 7.83–7.91 (m, 2H),
7.74–7.83 (m, 2H), 7.18–7.29 (m, 1H), 6.75–6.90 (m, 3H), 5.18 (dd, J =
9.4, 5.6 Hz, 1H), 4.36 (t, J = 9.3 Hz, 1H), 4.31 (d, J = 6.0 Hz, 2H), 4.10 (dd,
J = 9.3, 5.8 Hz, 1H), 3.72 (s, 3H) ppm.
(5R)-3-(4-cyanophenyl)-N-((S)-1-(naphthalen-2-yl)ethyl)-2-
oxooxazolidine-5-carboxamide (21j): General Method A with 19 (0.10 g,
0.43 mmol), (1S)-1-(2-naphthyl)ethanamine (88 mg, 0.52 mmol), HATU
(0.24 g, 0.65 mmol), DMF (1.5 mL), and i-Pr2NEt (0.23 ml, 1.3 mmol).
Chromatography (Telos 4 g, 0-100% EtOAc in DCM) gave a solid which
was triturated with MeOH, filtered, and rinsed with cold EtOH. The solid
(5R)-3-(4-cyanophenyl)-N-[(1S)-1-(3-methoxyphenyl)propyl]-2-oxo-
oxazolidine-5-carboxamide (21d): Method
A
with 19 (50 mg,
0.22 mmol), 39a•HCl (52 mg, 0.26 mmol), HATU (0.12 g, 0.32 mmol),
DMF (1.3 mL), and i-Pr2NEt (0.15 mL, 0.86 mmol). Chromatography (ZIP-
10g, EtOAc in heptane, 0–75%) gave 75 mg (94%) of 21d: 1H NMR
(CD3OD) : 7.69–7.81 (m, 4H), 7.15–7.27 (m, 1H), 6.84–6.92 (m, 2H),
6.73–6.82 (m, 1H), 5.16 (dd, J = 9.4, 5.9 Hz, 1H), 4.79 (t, J = 7.5 Hz, 1H),
4.35 (t, J = 9.3 Hz, 1H), 4.05–4.15 (m, 2H), 3.75 (s, 3H), 1.79–1.92 (m,
2H), 1.24 (t, J = 7.1 Hz, 1H), 0.93 (t, J = 7.4 Hz, 3H) ppm.
was further dried under vacuum at 50 °C to give 0.14 g (83%) of 21j: 1
H
NMR (DMSO-d6) 9.04 (d, J = 7.8 Hz, 1H), 7.81–7.93 (m, 6H), 7.75–7.81
(m, 2H), 7.44–7.57 (m, 3H), 5.08–5.21 (m, 2H), 4.34 (t, J = 9.3 Hz, 1H),
4.03 (dd, J = 9.3, 5.8 Hz, 1H), 1.52 (d, J = 7.0 Hz, 3H) ppm.
(5R)-3-(4-cyanophenyl)-N-(2-(naphthalen-1-yl)propan-2-yl)-2-
oxooxazolidine-5-carboxamide (21k): Method
A with 19 (70 mg,
0.30 mmol), 2-(naphthalen-1-yl)propan-2-amine (67 mg, 0.36 mmol),
HATU (0.17 g, 0.45 mmol), DMF (1.8 mL), and i-Pr2NEt (0.21 mL,
1.2 mmol). Chromatography (ZIP-10g, EtOAc in heptane, 0–65%) gave
0.10 g (87%) of 21k: 1H NMR (CD3OD) : 8.35–8.46 (m, 1H), 7.79–7.88
(m, 1H), 7.57–7.79 (m, 6H), 7.27–7.48 (m, 3H), 5.01 (dd, J = 9.5, 5.3 Hz,
1H), 4.18 (t, J = 9.3 Hz, 1H), 3.71–3.80 (m, 1H), 1.96 (s, 3H), 1.93 (s, 3H)
ppm.
(5R)-3-(4-cyanophenyl)-N-((S)-cyclopropyl(3-
methoxyphenyl)methyl)-2-oxooxazolidine-5-carboxamide
(21e):
Method with 19 (0.10 g, 0.43 mmol), (±)-cyclopropyl(3-
A
methoxyphenyl)methanamine•HCl (110 mg, 0.52 mmol), HATU (0.25 g,
0.65 mmol), DMF (4.0 mL), and i-Pr2NEt (0.30 mL, 1.7 mmol).
Chromatography twice (Telos-20g, EtOAc in heptane, 0–50%) to obtain 54
mg (32%) of the desired diastereoisomer 21e (second eluting spot). The
stereochemistry of 21e was assigned by advancing both diastereomers to
final products and determining which was the active inhibitor.1H NMR
(DMSO-d6) : 9.09 (d, J = 8.3 Hz, 1H), 7.82–7.92 (m, 2H), 7.72–7.82 (m,
2H), 7.17–7.30 (m, 1H), 6.90–6.99 (m, 2H), 6.76–6.87 (m, 1H), 5.17 (dd, J
= 9.5, 5.8 Hz, 1H), 4.31–4.42 (m, 1H), 4.19 (t, J = 8.8 Hz, 1H), 4.02 (dd, J
= 9.3, 5.8 Hz, 1H), 3.68–3.77 (m, 3H), 1.13–1.31 (m, 2H), 0.45–0.58 (m,
2H), 0.27–0.43 (m, 2H) ppm.
(5R)-3-(4-cyanophenyl)-N-((S)-1-(naphthalen-1-yl)propyl)-2-
oxooxazolidine-5-carboxamide (21l): Method
A with 19 (70 mg,
0.30 mmol), 39b•HCl (80 mg, 0.36 mmol), HATU (0.17 g, 0.45 mmol),
DMF (1.3 mL), and i-Pr2NEt (0.21 mL, 1.2 mmol). Chromatography (Telos-
20g, EtOAc in heptane, 0–50%) gave 92 mg (76%) of 21l: 1H NMR
(CD3OD) : 8.13 (d, J = 8.5 Hz, 1H) 7.82–7.91 (m, 1H) 7.78 (d, J = 8.3 Hz,
1H), 7.65–7.75 (m, 4H) 7.37–7.58 (m, 4H) 5.70 (dd, J = 8.7, 5.9 Hz, 1H)
5.19 (dd, J = 9.2, 5.8 Hz, 1H) 4.36 (t, J = 9.2 Hz, 1H) 4.11 (dd, J = 9.3, 6.0
Hz, 1H) 1.94–2.14 (m, 2H) 1.06 (t, J = 7.4 Hz, 3H) ppm.
(5R)-3-(4-cyanophenyl)-N-(2-(3-methoxyphenyl)propan-2-yl)-2-
oxooxazolidine-5-carboxamide (21f): Method
A with 19 (0.14 g,
0.60 mmol), 2-(3-methoxyphenyl)propan-2-amine[27] (0.12 g, 0.72 mmol),
HATU (0.34 g, 0.90 mmol), DMF (3.6 mL) and i-Pr2NEt (0.42 mL,
2.4 mmol). Chromatography twice (ZIP-10g, EtOAc in heptane, 0–75%)
gave 100 mg (44%) of 21f: 1H NMR (CD3OD) : 7.69–7.83 (m, 4H), 7.20
(t, J = 8.0 Hz, 1H), 6.87–7.00 (m, 2H), 6.76 (dd, J = 8.0, 2.1 Hz, 1H), 5.10
(dd, J = 9.3, 5.8 Hz, 1H), 4.32 (t, J = 9.3 Hz, 1H), 4.03–4.13 (m, 1H), 3.74
(s, 3H), 1.69 (s, 3H), 1.67 (s, 3H) ppm.
(5R)-3-(4-cyanophenyl)-2-oxo-N-((S)-1-(quinolin-4-
yl)propyl)oxazolidine-5-carboxamide (21m): Method A with 19 (70 mg,
0.30 mmol), 39c•HCl (80 mg, 0.36 mmol), HATU (172 mg, 0.45 mmol),
DMF (1.3 mL), and i-Pr2NEt (0.21 mL, 1.2 mmol). Chromatography (Telos-
10g, EtOAc in heptane, 20–100%) gave 77 mg (37%) of 21m: 1H NMR
(CD3OD) : 8.82 (d, J = 4.5 Hz, 1H), 8.26 (dt, J = 8.4, 0.7 Hz, 1H), 8.02–
8.09 (m, 1H), 7.69–7.81 (m, 5H), 7.65 (ddd, J = 8.4, 6.9, 1.2 Hz, 1H), 7.57
(d, J = 4.5 Hz, 1H), 5.71 (dd, J = 9.0, 5.5 Hz, 1H), 5.17 (dd, J = 9.5, 5.8 Hz,
1H), 4.34 (t, J = 9.4 Hz, 1H), 4.14 (dd, J = 9.3, 5.8 Hz, 1H), 1.92–2.10 (m,
2H), 1.06 (t, J = 7.4 Hz, 3H) ppm.
(5R)-3-(4-cyanophenyl)-2-oxo-N-(2-phenylpropan-2-yl)oxazolidine-5-
carboxamide (21g): Method
A with 19 (50 mg, 0.22 mmol), 2-
phenylpropan-2-amine (40 µL, 0.26 mmol), HATU (0.12 g, 0.32 mmol),
DMF (1.3 mL), and i-Pr2NEt (0.11 mL, 0.65 mmol). Chromatography (ZIP-
10g, EtOAc in heptane, 0–60%) gave 58 mg (78%) of 21g: 1H NMR
(DMSO–d6) : 8.63 (s, 1H), 7.82–7.92 (m, 2H), 7.70–7.82 (m, 2H), 7.24–
7.39 (m, 4H), 7.14–7.22 (m, 1H), 5.13 (dd, J = 9.2, 5.6 Hz, 1H), 4.25–4.37
(m, 1H), 3.96 (dd, J = 9.2, 5.6 Hz, 1H), 1.61 (s, 3H), 1.60 (s, 3H) ppm.
(5R)-3-(6-cyanopyridin-3-yl)-N-((S)-1-(naphthalen-1-yl)propyl)-2-
oxooxazolidine-5-carboxamide (22): Method
A with 20 (50 mg,
0.21 mmol), 39b•HCl (60 µL, 0.26 mmol), HATU (0.12 g, 0.32 mmol), DMF
(1.3 mL), and i-Pr2NEt (0.15 mL, 0.86 mmol). Preparative HPLC gave 58
mg (67%) of 22: 1H NMR (DMSO-d6) : 9.07 (d, J = 8.03 Hz, 1H), 8.95 (d,
J = 2.5 Hz, 1H), 8.11–8.27 (m, 2H), 8.07 (d, J = 8.8 Hz, 1H), 7.95 (d, J =
7.8 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.45–7.63 (m, 4H), 5.51–5.63 (m,
1H), 5.26 (dd, J = 9.3, 5.8 Hz, 1H), 4.33–4.47 (m, 1H), 4.06–4.18 (m, 1H),
1.81–2.00 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H) ppm.
(5R)-3-(4-cyanophenyl)-N-(2-methyl-1-oxo-1-(phenylamino)propan-2-
yl)-2-oxooxazolidine-5-carboxamide (21h): Method A with 19 (70 mg,
0.30 mmol),
2-amino-2-methyl-N-phenyl-propanamide
(64
mg,
0.36 mmol), HATU (0.14 g, 0.36 mmol), DMF (2.8 mL), and i-Pr2NEt
(0.21 mL, 1.2 mmol). Chromatography (ZIP-10g, EtOAc in heptane, 0–
100%) gave 0.11 g (93%) of 21h: 1H NMR (CD3OD) : 7.73–7.79 (m, 2H),
(5R)-3-(4-carbamimidoylphenyl)-N-(3-methoxybenzyl)-2-
oxooxazolidine-5-carboxamide (23): Method
B with 21c (62 mg,
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
0.18 mmol), NH2OH•HCl (59 mg, 0.85 mmol), i-Pr2NEt (0.15 mL,
0.85 mmol) and EtOH (2.9 mL). Then AcOH (2.0 mL) and Ac2O (83 µL,
0.88 mmol). Then AcOH (2.0 mL) and Zn (0.15 g, 1.8 mmol). HPLC
purification yielded 32 mg (38%) of 23•TFA: 1H NMR (DMSO-d6) : 9.26
(s, 2H), 9.14 (s, 2H), 9.05 (d, J = 6.9 Hz, 1H), 7.96–7.85 (m, 2H), 7.85–
7.77 (m, 2H), 7.24 (t, J = 8.1 Hz, 1H), 6.88–6.80 (m, 3H), 5.25–5.17 (m,
1H), 4.43–4.35 (m, 1H), 4.32 (d, J = 6.0 Hz, 2H), 4.12 (dd, J = 9.3, 5.7 Hz,
1H), 3.72 (s, 3H) ppm. 13C NMR (DMSO) : 167.9, 164.7, 159.3, 158.0 (q,
2.8 mmol). Preparative HPLC gave 48 mg (33%) of 28•TFA: 1H NMR
(DMSO–d6) : 9.50 (s, 1H), 9.26 (s, 2H), 9.18 (s, 2H), 8.58 (s, 1H), 7.93–
7.86 (m, 2H), 7.80 (d, J = 9.0 Hz, 2H), 7.60–7.49 (m, 2H), 7.31–7.21 (m,
2H), 7.06–7.00 (m, 1H), 5.26–5.13 (m, 1H), 4.38 (t, J = 9.4 Hz, 1H), 4.09–
3.98 (m, 1H), 1.50 (s, 3H), 1.49 (s, 3H) ppm. 13C NMR (DMSO-d6) : 172.1,
167.3, 164.7, 158.5 (q, JC-F = 33 Hz), 153.6, 142.7, 139.1, 129.3, 128.4,
123.3, 122.3, 120.3, 117.4, 116.9 (q, JC-F = 297 Hz), 70.3, 56.9, 47.6, 24.8,
24.6 ppm. HRMS (ESI) m/z: (M+H)+ calcd for C21H24N5O4+: 410.1823;
found: 410.1823.
J
C-F = 30 Hz), 153.5, 142.7, 140.2, 129.4, 129.2, 122.4, 119.5, 117.5, 117.2
(q, JC-F = 300 Hz), 113.0, 112.4, 70.6, 55.0, 47.6, 42.2 ppm. HRMS (ESI)
m/z: (M+H)+ calcd for C19H21N4O4+: 369.1557; found: 369.1557.
(5R)-3-(4-carbamimidoylphenyl)-N-((S)-1-(naphthalen-1-yl)ethyl)-2-
oxooxazolidine-5-carboxamide (29): Method B with amide 21i (78 mg,
0.20 mmol), NH2OH•HCl (67 mg, 0.97 mmol), i-Pr2NEt (0.17 mL,
0.97 mmol) and EtOH (2.7 mL). Then AcOH (2.0 mL) and Ac2O (0.10 mL,
1.0 mmol). Then AcOH (2.0 mL) and Zn (0.13 g, 2.0 mmol). Preparative
HPLC gave a solid which was triturated with cold EtOH to give 32 mg
(30%) of 29•TFA: 1H NMR (DMSO-d6) : 9.24 (s, 2H), 9.15 (d, J = 7.8 Hz,
1H), 8.89 (br s, 2H), 8.11 (d, J = 8.0 Hz, 1H), 7.91–7.99 (m, 1H), 7.75–7.91
(m, 4H), 7.46–7.63 (m, 4H), 5.76 (p, J = 7.0 Hz, 1H), 5.19 (dd, J = 9.3, 5.8
Hz, 1H), 4.37 (t, J = 9.2 Hz, 1H), 4.10 (dd, J = 9.3, 5.8 Hz, 1H), 1.56 (d, J
= 7.0 Hz, 3H) ppm. LCMS m/z = 403.0 [M+H]+.
(5R)-3-(4-carbamimidoylphenyl)-N-[(1S)-1-(3-methoxyphenyl)propyl]-
2-oxo-oxazolidine-5-carboxamide (24): Method B with 21d (70 mg, 0.18
mmol), NH2OH•HCl (61 mg, 0.89 mmol), i-Pr2NEt (0.15 mL, 0.89 mmol)
and EtOH (3.3 mL). Then AcOH (3.0 mL), and Ac2O (90 µL, 0.92 mmol).
Then AcOH (3.0 mL) and Zn (0.24 g, 3.7 mmol). HPLC purification gave a
solid which was triturated with EtOH to give 52 mg (56%) of 21d•TFA: 1
H
NMR (DMSO-d6) : 9.20 (s, 4H), 8.93 (d, J = 8.4 Hz, 1H), 7.92–7.84 (m,
2H), 7.83–7.78 (m, 2H), 7.27–7.21 (m, 1H), 6.94–6.87 (m, 2H), 6.83–6.77
(m, 1H), 5.20 (dd, J = 9.3, 5.7 Hz, 1H), 4.70 (td, J = 8.3, 6.5 Hz, 1H), 4.37
(t, J = 9.3 Hz, 1H), 4.05 (dd, J = 9.3, 5.7 Hz, 1H), 3.73 (s, 3H), 1.82–1.66
(m, 2H), 0.85 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (DMSO-d6) : 167.1, 164.7,
159.3, 158.5 (q, JC-F = 31 Hz), 153.6, 144.5, 142.7, 129.3, 129.2, 122.3,
118.8, 117.4, 117.2 (q, JC-F = 300 Hz), 112.3, 112.1, 70.6, 55.0, 54.5, 47.6,
28.9, 11.0 ppm. HRMS (ESI) m/z: (M+H)+ calcd for C21H25N4O4
397.1870; found: 397.1870.
(5R)-3-(4-carbamimidoylphenyl)-N-[(1S)-1-(2-naphthyl)ethyl]-2-oxo-
oxazolidine-5-carboxamide (30): Method B with 21j (60 mg, 0.16
mmol), NH2OH•HCl (52 mg, 0.75 mmol), i-Pr2NEt (0.13 ml, 0.75 mmol)
and EtOH (2.5 mL). Then AcOH (2.0 mL) and Ac2O (70 µl, 0.78 mmol).
Then AcOH (2.0 mL) and Zn (0.10 mg, 1.6 mmol). Preparative HPLC
gave 26 mg (41%) of 30•TFA: 1H NMR (DMSO–d6) 9.24 (s, 2H), 9.08
(d, J = 8.0 Hz, 1H), 8.90 (br s , 2H), 7.84–7.93 (m, 5H), 7.76–7.84 (m,
3H), 7.43–7.57 (m, 3H), 5.11–5.23 (m, 2H), 4.37 (t, J = 9.4 Hz, 1H), 4.10
(dd, J = 9.4, 5.6 Hz, 1H), 1.52 (d, J = 7.0 Hz, 3H) ppm. LCMS m/z =
403.2 [M+H]+.
+
:
(5R)-3-(4-carbamimidoylphenyl)-N-((1S)-cyclopropyl(3-
methoxyphenyl)methyl)-2-oxooxazolidine-5-carboxamide
(25):
Method B with 21e (52 mg, 0.13 mmol), NH2OH•HCl (44 mg, 0.64 mmol),
i-Pr2NEt (0.11 mL, 0.64 mmol) and EtOH (2.5 mL). Then AcOH (2.0 mL)
and Ac2O (60 µL, 0.66 mmol). Then AcOH (2.0 mL) and Zn (87 mg,
1.3 mmol). HPLC purification gave 35 mg (50%) of 25•TFA: 1H NMR
(DMSO-d6) : 9.24 (s, 2H), 9.12 (d, J = 8.3 Hz, 1H), 8.95 (s, 2H), 7.85–
7.92 (m, 2H), 7.77–7.85 (m, 2H), 7.20–7.29 (m, 1H), 6.92–6.99 (m, 2H),
6.82 (ddd, J = 8.3, 2.4, 1.1 Hz, 1H), 5.20 (dd, J = 9.4, 5.6 Hz, 1H), 4.34–
4.42 (m, 1H), 4.19 (t, J = 8.7 Hz, 1H), 4.05 (dd, J = 9.3, 5.8 Hz, 1H), 3.69–
3.77 (m, 3H), 1.19–1.29 (m, 1H), 0.52 (td, J = 7.5, 5.1 Hz, 2H), 0.26–0.43
(m, 2H) ppm. LCMS m/z = 409.2 [M+H]+.
(5R)-3-(4-carbamimidoylphenyl)-N-(2-(naphthalen-1-yl)propan-2-yl)-
2-oxooxazolidine-5-carboxamide (31): Method B with 21k (0.10 g, 0.26
mmol), NH2OH•HCl (85 mg, 1.2 mmol), i-Pr2NEt (0.21 mL, 1.2 mmol), and
EtOH (4.6 mL). Then AcOH (3.9 mL) and Ac2O (0.12 mL, 1.3 mmol). Then
AcOH (3.0 mL) and Zn (0.33 g, 5.1 mmol). Preparative HPLC gave a solid
which was triturated with cold EtOH to give 62 mg (46%) of 31•TFA: 1H
NMR (DMSO–d6) : 9.22 (s, 2H), 9.06 (s, 1H), 8.89 (br s, 2H), 8.47–8.57
(m, 1H), 7.87–7.96 (m, 1H), 7.74–7.87 (m, 3H), 7.69 (d, J = 9.0 Hz, 2H),
7.56 (d, J = 7.0 Hz, 1H), 7.39–7.50 (m, 3H), 5.10 (dd, J = 9.3, 5.3 Hz, 1H),
4.26 (t, J = 9.3 Hz, 1H), 3.68–3.80 (m, 1H), 1.85 (s, 3H), 1.84 (s, 3H) ppm.
LCMS m/z = 417.2. [M+H]+.
(5R)-3-(4-carbamimidoylphenyl)-N-(2-(3-methoxyphenyl)propan-2-
yl)-2-oxooxazolidine-5-carboxamide (26): Method B with 21f (0.10 g,
0.26 mmol), NH2OH•HCl (88 mg, 1.3 mmol), i-Pr2NEt (0.22 mL, 1.3 mmol)
and EtOH (4.8 mL). Then AcOH (4.0 mL) and Ac2O (0.12 mL, 1.3 mmol).
Then AcOH (4.0 mL) and Zn (0.34 g, 5.3 mmol). HPLC purification gave a
solid which was triturated with cold EtOH and cold MeOH to give 49 mg
(37%) of 26•TFA: 1H NMR (DMSO–d6) : 9.09 (br s, 4H), 8.65 (s, 1H),
7.74–7.93 (m, 4H), 7.21 (t, J = 8.0 Hz, 1H), 6.84–6.98 (m, 2H), 6.77 (dd, J
= 8.0, 2.0 Hz, 1H), 5.16 (dd, J = 9.2, 5.4 Hz, 1H), 4.34 (t, J = 9.3 Hz, 1H),
3.98 (dd, J = 9.2, 5.6 Hz, 1H), 3.72 (s, 3H), 1.61 (s,3H), 1.58 (s, 3H) ppm.
LCMS m/z = 397.2 [M+H]+.
(5R)-3-(4-carbamimidoylphenyl)-N-((S)-1-(naphthalen-1-yl)propyl)-2-
oxooxazolidine-5-carboxamide (32): Method
B with 21l (90 mg,
0.22 mmol), NH2OH•HCl (75 mg, 1.1 mmol), i-Pr2NEt (0.19 mL, 1.1 mmol)
and EtOH (2.5 mL). Then AcOH (2.0 mL) and Ac2O (0.11 mL, 1.1 mmol).
Then AcOH (2.0 mL) and Zn (0.15 g, 2.2 mmol). Preparative HPLC gave
50 mg (42%) of 32•TFA: 1H NMR (DMSO-d6) : 9.12 (br m, 4H), 9.10 (d, J
= 8.3 Hz, 1H), 8.18–8.11 (m, 1H), 7.94 (dd, J = 7.7, 2.0 Hz, 1H), 7.89–7.82
(m, 3H), 7.82–7.76 (m, 2H), 7.59–7.48 (m, 4H), 5.61–5.50 (m, 1H), 5.23
(dd, J = 9.3, 5.6 Hz, 1H), 4.44–4.34 (m, 1H), 4.06 (dd, J = 9.3, 5.6 Hz, 1H),
1.96–1.86 (m, 2H), 0.97 (t, J = 7.3 Hz, 3H) ppm. 13C NMR (DMSO-d6) :
167.3, 164.6, 158.2 (q, JC-F = 32 Hz), 153.6, 142.7, 138.7, 133.4, 130.5,
129.3, 128.8, 127.5, 126.3, 125.7, 125.5, 123.0, 122.9, 122.3, 117.4, 117.2
(q, JC-F = 300 Hz), 70.6, 50.4, 47.7, 28.5, 11.3 ppm. HRMS (ESI) m/z:
(M+H)+ calcd for C24H25N4O3+: 417.1921; found: 417.1921.
(5R)-3-(4-carbamimidoylphenyl)-2-oxo-N-(2-phenylpropan-2-
yl)oxazolidine-5-carboxamide (27): Method B with 21g (58 mg, 0.17
mmol), NH2OH•HCl (56 mg, 0.80 mmol), i-Pr2NEt (0.14 mL, 0.18 mmol)
and EtOH (2.3 mL). Then AcOH (2.0 mL) and Ac2O (80 µL, 0.84 mmol).
Then AcOH (2.0 mL) and Zn (0.11 g, 1.7 mmol). Preparative HPLC gave
61 mg (76%) of 27•TFA: 1H NMR (DMSO–d6) : 9.24 (br s, 2H), 8.94 (br
s, 2H), 8.67 (s, 1H), 7.76–7.92 (m, 4H), 7.24–7.42 (m, 4H), 7.14–7.24 (m,
1H), 5.16 (dd, J = 9.2, 5.6 Hz, 1H), 4.34 (t, J = 9.2 Hz, 1H), 3.99 (dd, J =
9.3, 5.8 Hz, 1H), 1.63 (s, 3H), 1.59 (s, 3H) ppm. LCMS m/z = 367 [M+H]+.
(5R)-3-(4-carbamimidoylphenyl)-2-oxo-N-((1S)-1-(quinolin-4-
yl)propyl)oxazolidine-5-carboxamide (33): A mixture of 21m (0.14 g,
0.34 mmol), NH2OH•HCl (0.11 g, 1.6 mmol) and i-Pr2NEt (0.28 mL,
1.6 mmol) in EtOH (2.0 mL) was heated to 100 °C in a microwave reactor
for 60 min. After cooling, the reaction mixture was concentrated and
purified by preparative HPLC to give 90 mg (71%) of the corresponding
amidoxime ((5R)-3-(4-(N-hydroxycarbamimidoyl)phenyl)-2-oxo-N-((S)-1-
(quinolin-4-yl)propyl)oxazolidine-5-carboxamide) as a TFA salt: LCMS m/z
(5R)-3-(4-carbamimidoylphenyl)-N-(2-methyl-1-oxo-1-
(phenylamino)propan-2-yl)-2-oxooxazolidine-5-carboxamide
Method B with 21h (0.11 g, 0.28 mmol), NH2OH•HCl (93 mg, 1.3 mmol), i-
Pr2NEt (0.23 mL, 1.3 mmol) and EtOH (2.0 mL). Then AcOH (2.0 mL), and
Ac2O (0.13 mL, 1.4 mmol). Then AcOH (2.0 mL) and Zn (0.18 g,
(28):
12
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
= 434.2 [M+H]+. This material (40 mg, 60 mol) was mixed with AcOH (1.0
mL) and Ac2O (30 L, 0.31 mmol). After 18 h, the reaction mixture was
concentrated, azeotroping with heptane. The residue was redissolved in a
mixture of MeOH/EtOAc (8.0 mL, 1:1) and subjected to hydrogenation
0.47 g (38%) of 37c: 1H NMR (CDCl3),  9.23 (s, 1H), 9.11 (d, J = 4.5 Hz,
1H), 8.78–8.87 (m, 1H), 8.20–8.28 (m, 1H), 7.80–7.89 (m, 2H), 7.72 (ddd,
J=8.5, 7.0, 1.4 Hz, 1H), 1.34–1.39 (m, 9H) ppm.
using an H-cube apparatus (Pd/C, rt, 10 bar,
1 mL/min). After
(R)-N-[(1S)-1-(3-methoxyphenyl)propyl]-2-methyl-propane-2-
sulfinamide (38a): Method D with 37a (0.17 g, 0.69 mmol), CH2Cl2 (4.1
mL), and 3 M EtMgBr (0.46 mL). Chromatography (ZIP-10g, CH2Cl2,
100%, then EtOAc in CH2Cl2, 0–75%) gave 0.16 g (86%) of 38a as a ~10:1
mixture of diastereomers, which was used without further purification. 1H
NMR (CDCl3) : 7.23–7.34 (m, 2H), 6.76–6.98 (m, 3H), 4.20–4.36 (m, 1H),
3.74–3.88 (m, 3H), 3.39 (br s, 1H), 1.72–1.94 (m, 2H), 1.14–1.34 (m, 9H),
0.75–0.93 (m, 3H) ppm.
concentration, HPLC purification gave 14 mg (29%) of 33•TFA: 1H NMR
(DMSO-d6) : 9.20–9.34 (m, 3H), 8.98 (d, J = 4.8 Hz, 3H), 8.34 (d, J = 8.0
Hz, 1H), 8.11 (dd, J = 8.5, 1.0 Hz, 1H), 7.83–7.90 (m, 3H), 7.76–7.83 (m,
2H), 7.73 (ddd, J = 8.4, 6.9, 1.2 Hz, 1H), 7.66 (d, J = 4.8 Hz, 1H), 5.57–
5.66 (m, 1H), 5.25 (dd, J = 9.4, 5.6 Hz, 1H), 4.37 (t, J = 9.3 Hz, 1H), 4.03
(dd, J = 9.4, 5.6 Hz, 1H), 1.85–1.96 (m, 2H), 0.94–1.04 (m, 3H) ppm.
LCMS m/z = 418.2 [M+H]+.
(5R)-3-(6-carbamimidoylpyridin-3-yl)-N-((1S)-1-(naphthalen-1-
yl)propyl)-2-oxooxazolidine-5-carboxamide (34): A mixture of 22 (53
mg, 0.13mmol), NH2OH•HCl (44.15 mg, 0.64 mmol) and i-Pr2NEt (0.11 ml,
0.64 mmol) in EtOH (2.4 mL) was heated with microwave irradiation at
100 °C for 60 min. The mixture was concentrated and taken up in AcOH
(2 mL) and Ac2O (0.06 ml, 0.66 mmol). After 18 h at rt, additional Ac2O
(0.06 ml, 0.66 mmol) was added. After another 24 h, the acetylation
reaction was not complete and the mixture was concentrated under
reduced pressure, azeotroping with heptane (x3). Remaining “starting
material” amidoxime was separated from the acetylated amidoxime
product via preparative HPLC. The acetylated fractions were
concentrated, dissolved in 1:1 EtOH/EtOAc (10 mL) and subjected to
hydrogenation using an H-cube apparatus (Pd/C, rt, 10 bar, 1 mL/min).
After concentration, preparative HPLC gave 34•TFA: 1H NMR (DMSO-d6)
: 9.44 (br s, 2H), 9.19 (br s, 4H), 8.21–8.35 (m, 2H), 8.16 (d, J = 8.0 Hz,
1H), 7.95 (d, J = 7.5 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.41–7.63 (m, 4H),
5.51–5.63 (m, 1H), 5.29 (dd, J = 9.2, 5.9 Hz, 1H), 4.44 (t, J = 9.4 Hz, 1H),
4.16 (dd, J = 9.2, 5.9 Hz, 1H), 1.84–1.99 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H)
ppm. LCMS m/z = 418.2 [M+H]+.
(R)-2-methyl-N-[(S)-1-(naphthalen-1-yl)propyl]propane-2-sulfinamide
(38b): To a cooled (0 °C) solution of sulfinamide 37b (0.10 g, 0.40 mmol)
in toluene (4.0 mL) was added dropwise 3 M EtMgBr (0.27 mL). The
resulting mixture was stirred for 1 h, then quenched with a saturated
aqueous solution of NH4Cl and partitioned between EtOAc and brine. The
aqueous phase was further extracted with EtOAc, and the combined
organic extracts were dried (Na2SO4), filtered and concentrated under
reduced pressure. The mixture was purified by flash chromatography (ZIP-
10g, EtOAc in CH2Cl2 0–30%). The residue was taken up in DMSO/MeOH
then purified by preparative HPLC to yield 0.12 g (74%) of 38b•TFA as a
~5:1 mixture of diastereomers, which was used without further purification.
1H NMR (CDCl3, major diastereomer)  8.18–8.28 (m, 1H), 7.86–7.93 (m,
1H), 7.81 (d, J = 8.0 Hz, 1H), 7.44–7.61 (m, 4H), 5.15 (t, J = 6.7 Hz, 1H),
3.47–3.63 (m, 1H), 1.98–2.22 (m, 2H), 1.20 (s, 9H), 0.93 (t, J = 7.4 Hz, 3H)
ppm.
(R)-2-methyl-N-((1S)-1-(quinolin-4-yl)propyl)propane-2-sulfinamide
(38c): Method D with 37c (0.43 mL, 1.8 mmol), CH2Cl2 (11.0 mL) and 3 M
EtMgBr (1.3 mL). Chromatography (Telos 50 g, EtOAc in heptane, 0–
100%) gave 0.36 g (68%) of 38: 1H NMR (CDCl3)  8.91 (s, 1H), 8.10–
8.20 (m, 2H), 7.73 (ddd, J = 8.4, 6.9, 1.2 Hz, 1H), 7.54–7.65 (m, 1H), 7.42–
7.49 (m, 1H), 5.11–5.22 (m, 1H), 3.69 (d, J = 4.3 Hz, 1H), 2.06–2.17 (m,
2H), 1.22–1.25 (m, 9H), 0.83–0.92 (m, 3H) ppm.
(5R)-3-(4-(aminomethyl)phenyl)-N-[(1S)-1-(naphthalen-1-yl)propyl]-2-
oxooxazolidine-5-carboxamide (35): A solution of amide 21l (50 mg,
0.13 mmol) in MeOH/dioxane (1:1, 6.5 mL) was subjected twice to
hydrogenation using H-cube (50 bar) at 55 °C with a Raney-Nickel column
and a flow of 1 mL/min. The eluting solution was concentrated under
reduced pressure, and the residue was purified by preparative HPLC to
yield 20 mg (31%) of 35•TFA: 1H NMR (DMSO-d6) : 9.08 (d, J = 8.3 Hz,
1H), 8.20 (br s, 3H), 8.16 (d, J = 8.3 Hz, 1H), 7.95 (dd, J = 8.2, 1.4 Hz, 1H),
7.84 (d, J = 7.8 Hz, 1H), 7.58–7.65 (m, 2H), 7.41–7.58 (m, 5H), 5.51–
5.61 (m, 1H), 5.19 (dd, J = 9.4, 5.6 Hz, 1H), 4.33 (t, J = 9.2 Hz, 1H), 3.96–
4.05 (m, 3H), 1.86–1.98 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H) ppm. LCMS m/z
= 388.4 [M + H+–NH3]+.
(1S)-1-(3-methoxyphenyl)propan-1-amine hydrochloride (39a): To a
solution of 38a (0.16 g, 0.59 mmol) in MeOH (0.60 mL), was added HCl
(0.30 mL, 4 M in dioxane) under argon. The resulting mixture was stirred
at rt for 30 min, after which the mixture was concentrated under reduced
pressure. Recrystallisation from MeCN gave 54.4 mg (45%) of 39a•HCl:
1H NMR (CD3OD) 7.32–7.44 (m, 1H), 6.93–7.04 (m, 3H), 4.06–4.17 (m,
1H), 3.83 (s, 3H), 1.85–2.10 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H) ppm.
(1S)-1-(quinolin-4-yl)propan-1-amine (39c): To a solution of 38c (0.36 g,
1.2 mmol) in MeOH (1.0 mL) was added HCl (0.62 mL, 4 M in dioxane).
After 30 min, the mixture was concentrated. The residue was triturated with
MeCN to yield 0.25 g (79%) of 39c•2HCl: 1H NMR (CD3OD)  8.17 (d, J
= 8.5 Hz, 1H), 7.94–8.01 (m, 2H), 7.52–7.69 (m, 3H), 5.19 (t, J = 7.3 Hz,
1H), 2.07–2.25 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H) ppm.
(R)-N-(3-methoxybenzylidene)-2-methylpropane-2-sulfinamide (37a):
Method C with 36 (0.50 g, 4.1 mmol), PPTS (52 mg, 0.21 mmol),
MgSO4•H2O (2.5 g, 21 mmol), 3-methoxybenzaldehyde (1.01 ml, 8.25
mmol), and CH2Cl2 (7.0 mL). Chromatography (Telos 40 g, EtOAc in
heptane, 0–30%) gave 0.88 g (89%) of 37a: 1H NMR (CDCl3), : 8.58 (s,
1H), 7.43 (m, 3H), 7.04–7.14 (m, 1H), 3.89 (s, 3H), 1.23–1.36 (m, 9H) ppm.
1-(4-cyanophenyl)-5-oxopyrrolidine-3-carboxylic
acid
(41):
A
(R)-2-methyl-N-(naphthalen-1-ylmethylene)propane-2-sulfinamide
(37b): Method C with 36 (0.20 g, 1.6 mmol), PPTS (21 mg, 80 µmol),
MgSO4∙H2O (0.99 g, 8.2 mmol) and naphthalene-1-carbaldehyde
(0.45 mL, 3.3 mmol) in CH2Cl2 (2.6 mL). Chromatography (Telos 20 g,
EtOAc in heptane, 0–30%) gave 0.31 g (72%) of 37b: 1H NMR (CDCl3), 
9.19 (s, 1H), 9.06 (d, J = 8.5 Hz, 1H), 8.00–8.14 (m, 2H), 7.90–7.99 (m,
1H), 7.52–7.72 (m, 3H), 1.31–1.40 (m, 9H) ppm.
microwave vial containing a mixture of 4-aminobenzonitrile (12) (2.0 g,
17 mmol) and 2-methylenebutanedioic acid (40) (2.2 g, 17 mmol) was
heated to 160 °C for 1.5 h, then allowed to cool to rt. The resulting gel was
sonicated in 2 M NaOH and filtered to remove a pink solid. The filtrate was
acidified with 5 M HCl and extracted with EtOAc (x3). The combined
organic extracts were dried (Na2SO4), filtered and concentrated under
reduced pressure. The residue was taken up in TFA (10 mL) and stirred at
rt for 3 h. The mixture was heated to 55 °C for 18 h, then quenched with a
saturated aqueous solution of NaHCO3. 2 M NaOH was added and the
aqueous phase was extracted using CH2Cl2, then acidified with 5 M HCl,
and extracted with EtOAc (x4). The combined organic extracts were dried
(Na2SO4), filtered and concentrated under reduced pressure. The oily
residue was triturated with EtOAc/Et2O to produce a sticky solid, which
upon trituration with CH2Cl2 produced a filterable off-white solid. This was
filtered, washed with CH2Cl2, and dried in vacuo to give 1.3 g (33%) of 41:
(R)-2-methyl-N-(quinolin-4-ylmethylene)propane-2-sulfinamide (37c):
To a solution of quinoline-4-carbaldehyde (0.75 g, 4.8 mmol) and 36 (0.58
g, 4.8 mmol) in anhydrous THF (4.0 mL) under argon was added Ti(OEt)4
(1.6 g, 7.2 mmol). The resulting mixture was stirred at rt for 20 h, then
quenched with brine (2.0 mL) for 10 min. The resulting suspension was
filtered and concentrated under reduced pressure. The filtrate was purified
by flash chromatography (Telos 50 g, EtOAc in heptane, 0–85%) to give
13
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
1H NMR (DMSO–d6) : 12.86 (br s, 1H), 7.82–7.91 (m, 4H), 4.05–4.13 (m,
1H), 3.97–4.04 (m, 1H), 3.30–3.43 (m, 1H), 2.79–2.89 (m, 1H), 2.71–2.79
(m, 1H) ppm. LCMS m/z = 229.0 [M–H]–.
3H), 3.18 (dt, J = 14.9, 7.6 Hz, 1H), 2.18–2.29 (m, 1H), 1.99–2.11 (m, 1H),
1.35 (d, J = 7.0 Hz, 3H) ppm. LCMS m/z = 367.2 [M+H]+.
4-((S)-5-((((S)-1-(3-methoxyphenyl)ethyl)amino)methyl)-2-
(5R)-1-(4-carbamimidoylphenyl)-N-[(1S)-1-(3-methoxyphenyl)ethyl]-
5-oxopyrrolidine-3-carboxamide (43): Step 1: Method A with racemic 41
(0.15 g, 0.65 mmol), 42 (0.12 g, 0.78 mmol), HATU (0.37 g, 0.98 mmol),
DMF (3.0 mL) and i-Pr2NEt (0.34 mL, 1.9 mmol). Chromatography (Telos-
12g, EtOAc in heptane, 0–100%; then Telos-25g, EtOAc in CH2Cl2, 0–
100%) gave 84 mg (36%) of the desired diastereomer, which was
ultimately identified by its activity against KLK6 after conversion to
compound 43. 1H NMR (CD3OD) : 8.58 (d, J = 7.8 Hz, 1H), 7.75–7.91 (m,
4H), 7.17–7.27 (m, 1H), 6.74–6.93 (m, 3H), 4.90 (p, J = 7.1 Hz, 1H), 4.06
(t, J = 9.2 Hz, 1H), 3.93 (dd, J = 9.8, 5.8 Hz, 1H), 3.75 (s, 3H), 2.81 (dd, J
= 17.2, 9.5 Hz, 1H), 2.60 (dd, J = 17.2, 6.6 Hz, 1H), (m, 3H), 1.36 (d, J =
7.0 Hz, 3H) ppm. Step 2: Method B with the above amide (82 mg,
0.23 mmol), NH2OH•HCl (75 mg, 1.1 mmol), i-Pr2NEt (0.19 mL,
1.1 mmol), and EtOH (3.4 mL). Then AcOH (3.0 mL) and Ac2O (0.11 mL,
1.1 mmol). Then AcOH (3.0 mL) and Zn (0.15 g, 2.3 mmol). Preparative
HPLC gave 58 mg (52%) of 43•TFA: 1H NMR (DMSO-d6) : 9.25 (s, 2H),
9.16 (s, 2H), 8.62 (d, J = 8.0 Hz, 1H), 7.89–7.94 (m, 2H), 7.82–7.88 (m,
2H), 7.24 (ddd, J = 8.1, 7.3, 0.7 Hz, 1H), 6.85–6.94 (m, 2H), 6.80 (ddd, J
= 8.2, 2.5, 1.1 Hz, 1H), 4.90 (app p, J = 7.1 Hz, 1H), 4.09 (dd, J = 9.9, 8.5
Hz, 1H), 3.96 (dd, J = 9.9, 5.8 Hz, 1H), 3.75 (s, 3H), 3.29–3.34 (m, 1H),
2.83 (dd, J = 17.1, 9.5 Hz, 1H), 2.61 (dd, J = 17.1, 6.7 Hz, 1H), 1.36 (d, J
= 7.0 Hz, 3H) ppm. 13C NMR (DMSO) : 173.6, 171.5, 165.2, 159.8, 159.0
(q, JC-F = 31 Hz), 158.8, 158.5, 146.6, 144.2, 129.9, 129.5, 122.9, 119.0,
118.6, 117.7 (q, JC-F = 300 Hz), 112.4, 112.2, 55.5, 51.0, 48.6, 36.6, 35.8,
23.0 ppm. HRMS (ESI) m/z: (M+H)+ calcd for C21H25N4O3+: 381.1921;
found: 381.1921.
oxooxazolidin-3-yl)benzonitrile (48): To a solution of 17 (100 mg, 0.46
mmol) in THF (1.1 mL) was added Et3N (0.08 ml, 0.6 mmol) followed by
MsCl (0.05 ml, 0.6 mmol). A suspension resulted which was stirred at rt for
16 h. The resulting mixture was quenched with H2O and concentrated. The
residue was taken up in CH2Cl2 and passed through a hydrophobic phase
separator, further rinsing with CH2Cl2. The filtrate was concentrated under
reduced pressure. The residue was taken up in DMF (0.5 mL) and
transferred to a microwave vial containing 42 (104 mg, 0.69 mmol). The
resulting mixture was subjected to microwave irradiation at 120 °C for 60
min and then 130 °C for 90 min. Thermal heating was then used at 100 °C
for 16 h. The resulting mixture was partitioned between EtOAc and
brine/water. The aqueous phase was further extracted with EtOAc and the
combined organic extracts washed with brine (x2), dried (Na2SO4), filtered
and concentrated, and purified by column chromatography (Telos 12 g,
EtOAc in DCM, 0-80%), then (SNAP Ultra-10g, EtOAc in DCM, 0-50% over
12CV then up to 65%) to give 80 mg of 48, which taken on directly in the
next step.
4-((S)-5-((((S)-1-(3-methoxyphenyl)ethyl)amino)methyl)-2-
oxooxazolidin-3-yl)benzimidamide (49): Method B with 48 (159 mg,0.45
mmol), NH2OH•HCl (150.9 mg, 2.17 mmol), i-Pr2NEt (0.38 ml, 2.17 mmol)
in EtOH (7.8 mL). Then AcOH (5.2 mL) and Ac2O (0.21 ml, 2.26 mmol).
Then AcOH (5.2 mL) and Zn (592 mg, 9.05 mmol). Preparative HPLC gave
144.5 mg (66%) of 49•TFA: 1H NMR (CD3OD) : 7.74–7.90 (m, 4H) 7.40
(t, J = 7.9 Hz, 1H) 6.95–7.14 (m, 3H) 5.02–5.14 (m, 1H) 4.48 (q, J = 6.9
Hz, 1H) 4.30 (t, J = 9.2 Hz, 1H) 3.79–3.89 (m, 4H) 3.40 (dd, J = 13.5, 2.5
Hz, 1H) 3.22 (dd, J = 13.5, 10.0 Hz, 1H) 1.72 (d, J = 6.8 Hz, 3H) ppm.
LCMS m/z = 369.2 [M+H]+.
(3R)-1-(4-cyanophenyl)pyrrolidine-3-carboxylic
acid
(46):
A
suspension of 44 (0.26 g, 2.2 mmol), 45 (0.28 g, 2.2 mmol) and Na2CO3
(804.2 mg, 7.59 mmol) in DMSO (3.0 mL) was heated to 85 °C overnight
in a sealed vial. The reaction mixture was diluted with EtOAc and filtered
through celite. The combined organic extracts were washed with a
saturated aqueous solution of NaHCO3 and brine, dried (Na2SO4) and
concentrated under reduced pressure. The crude material was purified by
flash column chromatography (Biotage-10g, EtOAc in n-heptane, 0–65%)
to give 390 mg (69%) of the desired ester. To a solution of this ester (0.30
g, 1.3 mmol) in THF (5.0 mL) at 0 °C was added a solution of LiOH·H2O
(57 mg, 1.3 mmol) in H2O (1.0 mL). The resulting mixture was stirred for
1.5 h, then acidified with 1 M HCl and concentrated under reduced
pressure. The residue was extracted with EtOAc (x2), and the combined
organic extracts were dried (Na2SO4), filtered and concentrated under
reduced pressure to give 0.22 g (79%) of 46: 1H NMR (CDCl3) 7.45–
7.53 (m, 2H), 6.48–6.59 (m, 2H), 3.60–3.72 (m, 2H), 3.49–3.56 (m, 1H),
3.38–3.49 (m, 1H), 3.27–3.38 (m, 1H), 2.33–2.45 (m, 2H) ppm.
Enzyme inhibition assays: All chemicals were purchased from Sigma
Aldrich (Gillingham, UK) unless otherwise stated. All experiments were
conducted in 384 well black, low volume microplates (Greiner 784076) with
a final volume of 20 µL and compounds were dispensed into assay plates
using an ECHO 550 acoustic dispenser (Labcyte). KLK6 (5 nM) (prepared
as in [21]) was incubated with test compounds for 30 min at room
temperature in assay buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 0.05% tween 20, 0.1% bovine serum albumin (BSA), 1 mM
dithiothreitol (DTT)) before the reaction was started by addition of 20 µM
Boc-Phe-Ser-Arg-AMC substrate (Bachem). Fluorescence intensity was
read at ex/em 355/460 following incubation for 2 h in the dark at room
temperature.
Assays using KLK7 and KLK8 (2B scientific) were conducted in the same
manner as for KLK6 with the following exceptions. Both KLK7 and KLK8
required activation before assaying. KLK7 was combined with an equal
volume of 20 µg/ml thermolysin (R&D systems) in activation buffer (50 mM
Tris, pH 7.5, 10 mM CaCl2, 150 mM NaCl and 0.05% Brij-L23) and
incubated at 37 °C for 2 h, the reaction was stopped with the addition of
100 mM EDTA in stop solution (50 mM Tris, 150 mM NaCl, pH 8.5). KLK8
was combined with an equal volume of 0.4 mU/mL lysyl endopeptidase
(Alpha laboratories) in activation buffer (50 mM Tris, pH 8.0) and incubated
at 37 °C for 30 min. Activated KLK7 (40 nM) and KLK8 (25 nM) were
assayed with 40 µM Mca-RPKPVE-Nval-WRK(Dnp)-NH2 fluorogenic
MMP substrate (R&D systems) and 25 µM Boc-V-P-R-AMC substrate
(R&D systems), respectively.
(3R)-1-(4-carbamimidoylphenyl)-N-[(1S)-1-(3-
methoxyphenyl)ethyl]pyrrolidine-3-carboxamide (47): Step 1: Method
A with 46 (80 mg, 0.37 mmol), 42 (60 µL, 0.41 mmol), HATU (0.21 g,
0.55 mmol), DMF (1.5 mL) and i-Pr2NEt (0.19 mL, 1.1 mmol).
Chromatography (Telos-10g, EtOAc in heptane, 0–100%) gave 120 mg,
(91%) of the desired amide: 1H NMR (DMSO–d6) : 8.48 (d, J = 8.03 z,
1H), 7.57–7.48 (m, 2H), 7.28–7.13 (m, 1H), 6.91–6.85 (m, 2H), 6.79 (ddd,
J = 8.2, 2.4, 1.1 Hz, 1H), 6.62–6.56 (m, 2H), 4.95–4.84 (m, 1H), 3.77–3.72
(m, 3H), 3.55–3.47 (m, 1H), 3.44–3.35 (m, 2H), 3.30–3.25 (m, 1H), 3.16
(p, J = 7.5 Hz, 1H), 2.21 (dtd, J = 12.1, 7.3, 7.3, 4.6 Hz, 1H), 2.10–2.00 (m,
1H), 1.35 (d, J = 7.0 Hz, 3H) ppm. Step 2: Method B with the above amide
(60 mg, 0.17 mmol), NH2OH•HCl (57 mg, 0.82 mmol), i-Pr2NEt (0.14 mL,
0.82 mmol) and EtOH (2.9 mL). Then AcOH (2.0 mL) and Ac2O (80 µL,
0.86 mmol). Then AcOH (2.0 mL) and Zn (0.11 g, 1.7 mmol). Preparative
HPLC gave 35 mg (42%) of 47•TFA: 1H NMR (DMSO–d6) : 8.82 (s, 2H),
8.50 (d, J = 8.0 Hz, 1H), 8.39 (s, 2H), 7.72 (d, J = 9.0 Hz, 2H), 7.21–7.28
(m, 1H), 6.86–6.91 (m, 2H), 6.80 (ddd, J = 8.2, 2.6, 1.0 Hz, 1H), 6.66 (d, J
= 9.0 Hz, 2H), 4.90 (p, J = 7.2 Hz, 1H), 3.52–3.60 (m, 1H), 3.30–3.49 (m,
Human thrombin was purchased from Biopur. Human trypsin was
purchased from Polymun Scientific. Human Factor Xa was purchased from
Enzo Life Sciences. Thrombin (20 nM), trypsin (0.000015%) or Factor Xa
(10 nM) were assayed in a similar manner to the KLKs but were incubated
with compound for 20 min at room temperature in assay buffer (50 mM Tris
HCl, pH 7.6, 100 mM NaCl, 1 mg/ml CHAPS, 0.05% bovine serum albumin
(BSA), 1 mM dithiothreitol (DTT)) before the addition of 5 µM, 2.5 µM or
1.5 µM Rhodamine 110 substrate (Cambridge bioscience ltd) respectively.
14
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
Fluorescence intensity was then read at ex/em 485/535 following
incubation for 1 h at room temperature (thrombin and factor Xa) or 20 min
(trypsin).
Fluorescence-based CYP Inhibition using recombinantly expressed
CYP bactosomes: Fluorescence CYP inhibition studies were conducted
at 37 °C in 96-well, flat-bottom, clear polystyrene plates. Incubation
mixtures containing EasyCYP bactosomes (1000 pmoles/mL, 10 mg/mL
Cypex TM), fluorigenic substrate (Cypex TM) and 50 mM potassium
phosphate buffer (pH 7.4) were prepared at the following final
concentrations: CYP1A2, 5 pmol/mL + 0.5 µM Ethoxyresorufin (ER);
CYP2C9, 10 pmol/mL + 30µM 7-methoxy-4-(trifluoromethyl)-coumarin
(MFC); CYP2C19, 5pmol/mL + 25µM 3-Cyano-7-Ethoxycoumarin (CEC);
Kinetic aqueous solubility: The aqueous solubility of the test compounds
was measured using laser Nephelometry. Compounds were subjected to
serial dilution from 10 mM to 0.5 mM in DMSO. An aliquot was then mixed
with Milli-Q water to obtain an aqueous dilution plate with a final
concentration range of 250–12 µM, with a final DMSO concentration of
2.5%. Triplicate aliquots were transferred to a flat bottomed polystyrene
plate which was immediately read on the NEPHELOstar (BMG Lab
Technologies). The amount of laser scatter caused by insoluble
particulates (relative nephelometry units, RNU) was plotted against
compound concentration using a segmental regression fit, with the point
of inflection being quoted as the compounds aqueous solubility (µM).
CYP2D6, 10 pmol/mL
+ 6µM 7-methoxy-4-(aminomethyl)-coumarin
(MAMC); CYP3A4, 10 pmol/mL + 1µM Diethoxyfluorescein (DEF) & 10
pmol/mL + 15 µM 7-Benzyloxyquinoline (BQ). Bactosome control protein
was included in reactions to give a final concentration of 0.025 mg/mL.
Reaction times were verified to be within the limits of kinetics linearity. For
each isoform, 220 µL of incubation mix was added to each well of a 96-
well, flat-bottom, clear polystyrene plate. Test compounds were prepared
as 5 mM solutions in DMSO and serially diluted 1 in 3.03, 1 in 3.3
alternatively in a v-bottomed 96 well plate to give a concentration range of
5, 1.65, 0.5, 0.165, 0.05, 0.0165, 0.05 mM. Positive control inhibitor,
Miconazole, was prepared as a 0.5 mM solution in DMSO and similarly
diluted. 5 µL of each concentration was mixed with 220 µL of the incubation
Thermodynamic solubility: Test compound (3 mg) is accurately weighed
into a Micronics tube. Extra tubes for each control compound, Warfarin
(high solubility control) and Cinnarizine (low solubility control) were also
accurately weighed. To each tube is added 1 mL of Milli-Q water. All
samples are vortexed for 20 s and placed in a bench top incubator shaker
set at 1300 rpm at 37 ˚C for 5 h. After the incubation, samples are
centrifuged at 10,000 rpm for 3 min. 100 µL aliquots of the supernatant in
duplicate were placed in a 96-well plate. All samples, calibration standards
and QC samples are analysed using a validated HPLC method on the
Dionex Ultramate 3000 HPLC system. The concentration determined from
the supernatant was then reported as the solubility of each compound in
µg/mL.
mix and pre-incubated at 37 °C for
5 min (final test compound
concentration range: 100, 33, 10, 3.3, 1.0, 0.33, 0.1 or 10, 3.3, 1.0, 0.33,
0.1, 0.033, 0.01 and 0 µM, Miconazole 10, 3.3, 1.0, 0.33, 0.1, 0.033, 0.01
and 0 µM). Reactions were initiated by the addition of 25 µL regenerating
cofactor solution (23 mM Glucose-6-Phosphate, 2.2 mM NADP, 6 Units
per ml Glucose-6-Phosphate Dehydrogenase (from Baker's yeast S.
cerevisiae) in 2% w/v NaHCO3, Sigma) and subsequent production of
fluorescence metabolite measured at 1 min intervals over a 10 min period
using a BMG Optima fluorescence detector (ER: Exc 530 nm, Em 590 nm,
MFC: Exc 410 nm, Em 530 nm, CEC: Exc 410 nm, Em 530 nm, MAMC:
Exc 405 nm, Em 460 nm, DEF: Exc 405 nm, Em 560 nm, 7BQ: Exc 485
nm, Em 530 nm). Fluorescence responses were adjusted to a percentage
of uninhibited control before plotting against compound concentration and
deriving the IC50 by fitting to a 4 parameter logistic regression model.
Microsomal Intrinsic clearance (CLint) assay: Test compound (0.5 µM)
was incubated with female CD1 mouse liver microsomes (Xenotech LLC
TM; 0.5mg/mL 50mM potassium phosphate buffer, pH 7.4) and the
reaction started with addition of excess NADPH (8mg/mL 50mM potassium
phosphate buffer, pH 7.4). Immediately, at time zero, then at 3, 6, 9, 15
and 30 min an aliquot (50 µL) of the incubation mixture was removed and
mixed with acetonitrile (100 µL) to stop the reaction. Internal standard was
added to all samples, the samples centrifuged to sediment precipitated
protein and the plates then sealed prior to UPLC-MS/MS analysis using a
Quattro Premier XE (Waters Corporation, USA). XLfit (IDBS, UK) was
used to calculate the exponential decay and consequently the rate
constant (k) from the ratio of peak area of test compound to internal
standard at each timepoint. The rate of intrinsic clearance (CLint) of each
test compound was then calculated using the following calculation: CLint
(mL/min/g liver) = k x V x Microsomal protein yield. Where V (mL/mg
protein) is the incubation volume/mg protein added and microsomal
protein yield is taken as 52.5 mg protein/g liver. Verapamil (0.5 µM) was
used as a positive control to confirm acceptable assay performance.
Plasma protein binding assay: Briefly, a 96 well equilibrium dialysis
apparatus was used to determine the free fraction in plasma for each
compound (HT Dialysis LLC, Gales Ferry, CT). Membranes (12-14 kDA
cut-off) were conditioned in deionised water for 60 minutes, followed by
conditioning in 80:20 deionised water: ethanol for overnight at 4 °C and
then rinsed in isotonic buffer before use. Female CD1 mouse plasma was
removed from the freezer and allowed to thaw on the day of experiment.
Thawed plasma was then centrifuged (Allegra X12-R, Beckman Coulter,
USA), spiked with test compound (final concentration 10 µg/mL), and 150
μL aliquots (n=6 replicate determinations) loaded into the 96-well
equilibrium dialysis plate. Dialysis vs isotonic buffer (150 μL) was carried
out for 5 h in a temperature-controlled incubator at 37 ᵒC (Barworld
scientific Ltd, UK) using an orbital microplate shaker at 100 rpm (Barworld
scientific Ltd, UK). At the end of the incubation period, 50 µL aliquots of
plasma or buffer were transferred to micronic tubes (Micronic B.V., the
Netherlands) and the composition in each tube balanced with control fluid
(50 µL), such that the volume of buffer to plasma is the same. Sample
extraction was performed by the addition of 200 μL of acetonitrile
containing an appropriate internal standard. Samples were allowed to mix
for 1 min and then centrifuged at 3000 rpm in 96-well blocks for 15 min
(Allegra X12-R, Beckman Coulter, USA) after which 150 µL of supernatant
was removed to 50 µL of water. All samples were analysed by UPLC-
MS/MS on a Quattro Premier XE Mass Spectrometer (Waters Corporation,
USA). The unbound fraction was determined as the ratio of the peak area
in buffer to that in plasma.
Hepatocytes Intrinsic clearance (CLint) assay: Cryopreserved vials of
mouse cryopreserved hepatocytes, supplied by Life Technologies, were
thawed according to manufacturer’s instructions and cells re-suspended in
Williams Medium E (WME) containing cell maintenance supplement pack
(CM4000, Life Technologies). Hepatocytes were incubated in suspension
(0.5 million cells/mL) in 48 well non-collagen coated cell culture plates for
10 min at 37 °C, 95% O2 5% CO2. Upon addition of an equal volume of
supplemented WME containing 1 µM test compound, an aliquot of
incubation solution was removed to acetonitrile containing internal
standard (final concentration 0.5 µM test compound and a cell density of
0.25 x 106 cells/mL). Similarly, aliquots were removed at 3, 6, 9, 15, 30,
45, 60, 90 and 120 min. 100 µL of 80:20 H2O/MeCN was added to all
samples and the analysis plate was centrifuged for 10 min at room
temperature prior to injection and analysis of samples by UPLC-MS/MS.
The response (area ratio of test compound to internal standard) was
plotted against time using an exponential decay model and rate of
disappearance calculated. Hepatocyte CLint (mL/min/106 cells) was
scaled to in vivo CLint (mL/min/g liver) using the hepatocellularity scaling
factor of 120 x 106 cells/g of liver.
Plasma stability assay: Test compound (10 µM) was incubated in pre-
warmed plasma at 37 °C (that is buffered to pH 7.4 in ratio of 70:30 plasma
to buffer). Immediately, at time zero, then at 30, 60, 120, and 180 min, a
50 µL aliquot of the incubation mixture was removed and mixed with 200
µL acetonitrile containing Donepezil as the internal standard (50 ng/mL) to
stop the reaction. The samples were centrifuged to sediment the
precipitated protein and the plates then sealed prior to UPLC-MS/MS
15
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
[4]
H. Yoon, S. I. Blaber, D. M. Evans, J. Trim, M. A. Juliano,
I. A. Scarisbrick, M. Blaber Prot. Sci. 2008, 17, 1998–
2007.
M. Blaber, H. Yoon, S. I. Blaber, W. Li, I. A. Scarisbrick
Biol. Chem. 2013, 394, 137–147.
analysis using a Quattro Premier XE (Waters Corporation, USA). XLfit
(IDBS, UK) was used to calculate the exponential decay and consequently
the rate constant (k) from the ratio of peak area of test compound to
internal standard at each time point. The half-life was calculated for each
[5]
[6]
test compound from the rate by using the following calculation: t1/2
0.693/k
=
a) P. F. Angelo, A. R. Lima, F. M. Alves, S. I. Blaber, I. A.
Scarisbrick, M. Blaber, L. Juliano, M. A. Juliano J. Biol.
Chem. 2006, 281, 3116–3126; b) M. Debela, V. Magdolen,
N. Schechter, M. Valachova, F. Lottspeich, C. S. Craik, Y.
Choe, W. Bode, P. Goettig J. Biol. Chem. 2006, 281,
25678–25688; c) H.-X. Li, B.-Y. Hwang, G. Laxmikanthan,
S. I. Blaber, M. Blaber, P. A. Golubkov, P. Ren, B. L.
Iverson, G. Georgiou Protein Sci. 2008, 17, 664–672; d) A.
Magklara, A. A. Mellati, G. A. Wasney, S. P. Little, G.
Sotiropoulou, G. W. Becker, E. P. Diamandis Biochem.
Biophys. Res. Commun. 2003, 307, 948–955.
K. Oikonomopoulou, K. K. Hansen, M. Saifeddine, I. Tea,
M. Blaber, S. I. Blaber, I. A. Scarisbrick, P. Andrade-
Gordon, G. S. Cottrell, N. W. Bunnett, E. P. Diamandis, M.
D. Hollenberg J. Biol. Chem. 2006, 281, 32095–32112.
A. Iwata, M. Maruyama, T. Akagi, T. Hashikawa, I.
Kanazawa, S. Tsuji, N. Nukina Hum. Mol. Genet. 2003,
12, 2625–2635.
S. I. Blaber, B. Ciric, G. P. Christophi, M. J. Bernett, M.
Blaber, M. Rodriguez, I. A. Scarisbrick FASEB J. 2004, 18,
920–922.
M. Cudic, G. B. Fields Curr. Protein Pept. Sci. 2009, 10,
297–307.
S. Krenzer, H. Peterziel, C. Mauch, S. I. Blaber, M. Blaber,
P. Angel, J. Hess J. Invest. Dermatol. 2011, 131, 2281–
2288.
P. J. Heenan, M. Ghaznawie Br. J. Plast. Surg. 1999, 52,
209–213.
E. Sells, R. Pandey, H. Chen, B. A. Skovan, H. Cui, N. A.
Ignatenko Neoplasia 2017, 19, 396–411.
C. H. Schrader, M. Kolb, K. Zaoui, C. Flechtenmacher, N.
Grabe, K. J. Weber, T. Hielscher, P. K. Plinkert, J. Hess
Mol. Cancer 2015, 14, 1–14.
K. Patra, A. Soosaipillai, S. B. Sando, C. Lauridsen, G.
Berge, I. Møller, G. R. Grøntvedt, G. Bråthen, I. Begcevic,
S. Moussaud, L. Minthon, O. Hansson, E. P. Diamandis,
L. R. White, H. M. Nielsen Alzheimer's Res. Ther. 2018,
10, 1–11.
K. Ogawa, T. Yamada, Y. Tsujioka, J. Taguchi, M.
Takahashi, Y. Tsuboi, Y. Ujino, M. Nakajima, T.
Yamamoto, H. Akatsu, S. Mitsui, N. Yamaguchi Psychiatry
Clin. Neurosci. 2000, 54, 419–426.
I. A. Scarisbrick, P. Sabharwal, H. Cruz, N. Larsen, A. G.
Vandell, S. I. Blaber, S. Ameenuddin, L. M. Papke, M. G.
Fehlings, R. K. Reeves, M. Blaber, A. J. Windebank, M.
Rodriguez Eur. J. Neurosci. 2006, 24, 1457–1469.
G. Liang, X. Chen, S. Aldous, S. F. Pu, S. Mehdi, E.
Powers, A. Giovanni, S. Kongsamut, T. Xia, Y. Zhang, R.
Wang, Z. Gao, G. Merriman, L. R. McLean, I. Morize ACS
Med. Chem. Lett. 2012, 3, 159–164.
B. Severino, F. Fiorino, A. Corvino, G. Caliendo, V.
Santagada, D. M. Assis, J. R. Oliveira, L. Juliano, S.
Manganelli, E. Benfenati, F. Frecentese, E. Perissutti, M.
A. Juliano Biol. Chem. 2015, 396, 45–52.
F. Soualmia, E. Bosc, S. A. Amiri, D. Stratmann, V.
Magdolen, D. Darmoul, M. Reboud-Ravaux, C. E. Amri
Biol. Chem. 2018, 399, 1073–1078.
E. De Vita, P. Schüler, S. Lovell, J. Lohbeck, S. Kullmann,
E. Rabinovich, A. Sananes, B. Hessling, V. Hamon, N.
Papo, J. Hess, E. W. Tate, N. Gunkel, A. K. Miller J. Med.
Chem. 2018, 61, 8859–8874.
Invasion assay: The HCT116 colorectal adenocarcinoma cell line
(ATCC® CCL-247™) was maintained in Dulbecco’s Modified Eagle
Medium (DMEM) with 4.5 mg/L glucose, L-glutamine w/o sodium pyruvate,
supplemented with 10% FBS and 1% penicillin/streptomycin. HCT116 cell
invasion was performed in 24-well chambers with Matrigel coated inserts
with a 0.8 µm pore size (Corning Inc., Corning, NY). In the lower chamber
of each well 0.5 mL of media supplemented with 10% FBS was added as
a
chemoattractant. HCT116 cell suspension was prepared at a
[7]
concentration of 0.5 x 106 cells/mL in serum-free medium. Cells in
suspension were pre-treated with different concentrations of test
compounds for 20 min before seeding onto inserts. 200 µL of cell
suspensions (1 x 105 cells) were plated into each insert with six inserts
plated for each condition. The cells were allowed to invade for 24 h in the
incubator at 37 ºC in 5% CO2. After incubation, the media was removed
and inserts were rinsed briefly with 1 X PBS and swabbed gently inside
with a cotton tip to remove non-invading cells. The invading cells outside
the insert were fixed in 100% methanol for 2 min, stained with 1% toluidine
blue in 1% borax for 2 min, rinsed twice in ddH2O, swabbed gently again
inside with a cotton tip and air-dried. The stained membranes were cut
from the inserts and the incorporated toluidine blue was dissolved in 200
μL of 0.1 M citric acid in a 96 well plate while shaking for 5 min on a high-
speed titer plate shaker. The samples were transferred to a new 96 well
plate and read at 560 nm on a Synergy 2 Multi-Detection Microplate
Reader (Bio-Tek Instruments, Inc.,Winooski, VT).
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Acknowledgements
[15]
The research leading to these results was done within the
European Lead Factory and has received support from the
Innovative Medicines Initiative Joint Undertaking under grant
agreement n° 115489, resources of which are composed of
financial contribution from the European Union's Seventh
Framework Programme (FP7/2007-2013) and EFPIA companies’
in-kind contribution.
[16]
[17]
[18]
[19]
Keywords: Drug Discovery • High-throughput screening •
Medicinal Chemistry • Structure-activity relationship • Protease
inhibitors
References:
[1]
a) A. Anisowicz, G. Sotiropoulou, G. Stenman, S. C. Mok,
R. Sager Mol. Med. 1996, 2, 624–636; b) I. A. Scarisbrick,
M. D. Towner, P. J. Isackson J. Neurosci. 1997, 17, 8156–
8168; c) K. Yamashiro, N. Tsuruoka, S. Kodama, M.
Tsujimoto, Y. Yamamura, T. Tanaka, H. Nakazato, N.
Yamaguchi Biochim. Biophys. Acta, Gene Struct.
Expression 1997, 1350, 11–14; d) S. P. Little, E. P. Dixon,
F. Norris, W. Buckley, G. W. Becker, M. Johnson, J. R.
Dobbins, T. Wyrick, J. R. Miller, W. MacKellar, D.
Hepburn, J. Corvalan, D. McClure, X. Liu, D. Stephenson,
J. Clemens, E. M. Johnstone J. Biol. Chem. 1997, 272,
25135–25142.
[20]
[21]
[22]
[23]
F. Giordanetto, P. Jones, A. Nelson, J. Benningshof, G.
Müller, A. Pannifer, S. van Boeckel, D. Tzalis in
Comprehensive Medicinal Chemistry III (Eds.: S.
Chackalamannil, D. Rotella, S. E. Ward), Elsevier, Oxford,
2017, pp. 505–519.
B. A. Katz, P. A. Sprengeler, C. Luong, E. Verner, K.
Elrod, M. Kirtley, J. Janc, J. R. Spencer, J. G.
Breitenbucher, H. Hui, D. McGee, D. Allen, A. Martelli, R.
L. Mackman Chem. Biol. 2001, 8, 1107-1121.
[2]
[3]
a) G. M. Yousef, E. P. Diamandis Endocr. Rev. 2001, 22,
184–204; b) I. Prassas, A. Eissa, G. Poda, E. P.
Diamandis Nat. Rev. Drug Disc. 2015, 14, 183–202.
H. Yoon, G. Laxmikanthan, J. Lee, S. I. Blaber, A.
Rodriguez, J. M. Kogot, I. A. Scarisbrick, M. Blaber J. Biol.
Chem. 2007, 282, 31852–31864.
16
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
[24]
[25]
[26]
M. Debela, V. Magdolen, V. Grimminger, C. Sommerhoff,
[27]
K. Jaudzems, K. Tars, G. Maurops, N. Ivdra, M. Otikovs,
J. Leitans, I. Kanepe-Lapsa, I. Domraceva, I. Mutule, P.
Trapencieris, M. J. Blackman, A. Jirgensons ACS Med.
Chem. Lett. 2014, 5, 373-377.
A. Messerschmidt, R. Huber, R. Friedrich, W. Bode, P.
Goettig J. Mol. Biol. 2006, 362, 1094–1107.
T. Kishi, M. Kato, T. Shimizu, K. Kato, K. Matsumoto, S.
Yoshida, S. Shiosaka, T. Hakoshima J. Biol. Chem. 1999,
274, 4220-4224.
J. A. Ellman, T. D. Owens, T. P. Tang Acc. Chem. Res.
2002, 35, 984–995.
17
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900536
ChemMedChem
FULL PAPER
Entry for the Table of Contents
A successful screen performed within the European Lead Factory
program, followed by docking studies, has resulted in compound
32, a potent and selective N-(4-benzamidino)-oxazolidinone
inhibitor of kallikrein-related peptidase 6 (KLK6). Aberrant levels
of this serine protease have been found in different malignancies
and in neurodegenerative diseases. Good solubility and low
clearance of 32 make it a good probe for KLK6 target validation.
18
This article is protected by copyright. All rights reserved.