Noncovalent Protein Arginine Deiminase (PAD) Inhibitors Are Efficacious in Animal Models of Multiple Sclerosis

Article abstract of DOI:10.1021/acs.jmedchem.7b01102

Peptidyl arginine deiminases have been shown to be hyperactive in neurodegenerative diseases including multiple sclerosis. An α-amino acid-based core structure, derived from a hydantoin core, with unique heterocycles on the side chains were synthesized as potential noncovalent inhibitors of PAD enzymes. Among the various heterocycles investigated, compound 23, carrying an imidazole moiety, exhibited the highest potency in this series with some selectivity for PAD2, and was further investigated in vivo. Pharmacokinetics in mice suggested the C_max to be 12.0 ± 2.5 μg/mL and 170 ± 10 ng/mL in the serum and brain, respectively, when compound 23 was administered at 50 mg/kg via single dose ip. At the same dose, compound 23 also reversed physical disability and cleared the brain of T-cell infiltration in an EAE mouse model of multiple sclerosis (MS). This novel series of compounds show promise for further development as disease modifying agents for the potential treatment of MS.

Full text of DOI:10.1021/acs.jmedchem.7b01102

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Article
Non-Covalent Protein Arginine Deiminase (PAD) Inhibitors
Are Efficacious in Animal Models of Multiple Sclerosis
Elizabeth J. Curiel Tejeda, Angelica M Bello, Ewa Wasilewski,
Adam Koebel, Shannon Dunn, and Lakshmi P Kotra
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01102 • Publication Date (Web): 18 Oct 2017
Downloaded from http://pubs.acs.org on October 20, 2017
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Non-Covalent Protein Arginine Deiminase (PAD) Inhibitors Are Efficacious in Animal Models of
Multiple Sclerosis
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Elizabeth J. Curiel Tejeda,1 Angelica M. Bello,1,2 Ewa Wasilewski1,2, Adam Koebel,1,2 Shannon
Dunn,2,3 and Lakshmi P. Kotra1,2,4
*
1Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto,
Ontario, Canada, M5S 3M2
2Centre for Molecular Design and Preformulations, Toronto General Hospital Research Institute,
University Health Network, Toronto, Ontario, Canada, M5G 1L7
3Department of Immunology, University of Toronto, Medical Sciences Building, 1 King's College
Circle, Toronto, Ontario, Canada M5S 1A8
4Multi-Organ Transplant Program, Toronto General Hospital, 200, Elizabeth Street, Toronto, Ontario,
Canada, M5G 2C4
KEYWORDS: Protein arginine deiminases, multiple sclerosis, non-covalent inhibitors, EAE, L-
homoserine.
RECEIVED DATE (to be automatically inserted)
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Abstract.
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Peptidyl arginine deiminases have been shown to be hyperactive in neurodegenerative diseases
including multiple sclerosis. An -amino acid based core structure, derived from a hydantoin core, with
unique heterocycles on the side chains were synthesized as potential non-covalent inhibitors of PAD
enzymes. Amongst the various heterocycles investigated, compound 23 carrying an imidazole moiety,
exhibited the highest potency in this series with some selectivity for PAD2, and was further
investigated in vivo. Pharmacokinetics in mice suggested the Cmax to be 12.0 ± 2.5 μg/mL and 170 ± 10
ng/mL in the serum and brain, respectively, when compound 23 was administered at 50 mg/kg via
single dose i.p. At the same dose, compound 23 also reversed physical disability and cleared the brain
of T-cell infiltration in an EAE mouse model of multiple sclerosis (MS). This novel series of
compounds show promise for further development as disease modifying agents for the potential
treatment of MS.
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Introduction.
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Citrullination is a post-translational modification that consists of the deimination of peptidyl
arginine residues into peptidyl citrulline (Scheme 1), and is known to play an essential role in
physiological processes such as skin keratinization, insulation of neuronal axons, gene regulation, and
maintenance of plasticity of the central nervous system (CNS).1,2 Citrullination is a process mainly
regulated by the Ca2+-dependent amidinotransferase family of enzymes known as protein arginine
deiminases (PADs).3 Five PAD isozymes in humans, PAD 1-4 and PAD6, with well-conserved
primary structure exhibit tissue-specific expression patterns.3 The deregulation of this posttranslational
modification has been associated with neurodegenerative and immunopathological conditions such as
rheumatoid arthritis (RA),4 Alzheimer's disease (AD),5 and multiple sclerosis (MS).6,7,8,9
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Scheme 1. Citrullination (deimination) of peptidyl Arg residue by PAD enzymes.
Due to their potential role in disease pathogenesis, the development of inhibitors towards PADs for
therapeutic use has become of high importance. In the past decade, a series of known drugs were
evaluated and compounds such as taxol, streptomycin, and several tetracycline derivatives have shown
reversible inhibitory properties towards PAD enzymes, albeit with relatively poor potency and
selectivity.10,11 To date a number of PAD inhibitors, primarily covalent modifiers, were identified
which contain a terminal haloacetamidine warhead.12-22 These inhibitors have been observed as good
modulators of NET trap formation, transcriptional regulation in cancer, and remyelination in MS
animal models.12,23 Despite their high potency, concerns remain regarding potential nonspecific
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reactivity of these electrophilic covalent modifiers and their suitability as potential drugs.
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PAD enzymes and their inhibitors have been studied in relation to myelin modification, relevant to
MS as well as the myelin oligodendrocyte glycoprotein induced experimental autoimmune
encephalomyelitis (MOG-EAE) in mice.23,24,25,26 This is routinely used acute EAE model for MS,
which is a CD4(+) T cell-driven model, induced with the immunodominant 35-55 peptide of myelin
oligodendrocyte glycoprotein (pMOG35-55) and was used to test whether citrullination of a T cell
epitope can contribute to disease etiopathology.27,28 PAD2 and PAD4 enzymes are significantly
upregulated in the inflamed CNS in this animal model. T cells that responded specifically to the
citrullinated pMOG could not initiate the EAE lesion, but these cells could provoke exacerbation of
pathology if transferred into mice with an ongoing EAE. A similar study using the peptides from
myelin basic protein (MBP) epitopes indicated that self-antigens could potentially trigger the disease in
susceptible individuals carrying citrullinated peptide epitopes.29,30 It is also noted that citrulline is more
frequently identified in the brains of patients with early onset MS than in healthy subjects using
magnetic resonance spectroscopy.31 This study and others have established the direct correlation
between hypercitrullination and disease progression in MS.32
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Our group took an in silico approach to identify non-covalent inhibitors by exploiting cationic,
anionic, and hydrophobic binding interactions within the active site pocket of PAD4 enzyme.33 From
this library of inhibitors, a hydantoin derivative 1 (Figure 1) showed key binding interactions between
the active site residues Asp350, Asp473, and the piperazine moiety, as well as hydrogen bonding
between the hydantoin moiety and the surface Arg374 residue that stabilized binding interactions and
potentially led to higher potency (Ki = 131 μM).
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Figure 1. Hydantoin series of non-covalent PAD inhibitors.
Recently, we published a structure-activity relationship investigation that evaluated the activity of
further hydantoin derivatives tethered via a two-carbon, instead of single carbon, linker with a variety
of nitrogen-rich heterocycles.34 Interestingly, increasing the linker length was sufficient to reduce the
binding affinity of piperazinyl derivatives. Instead, compounds 2 and 3 with 5-membered heterocycles
were found to be more suitably accommodated within the binding pocket of PADs, presumably due to
a reduction in steric bulk and the optimal linker length (2-carbon chain) (Figure1).34,35 These
hydantoins were evaluated in vivo in the MOG-EAE mouse model of MS demonstrating moderate
improvement in clinical scores as well as reduction in CD3+ cells in the treated brain samples in
comparison to controls.33,34 These investigations provided us an insight into the design of non-covalent
inhibitors for PAD enzymes, and for their potential applications for the treatment of multiple sclerosis.
Herein we describe the synthesis of a novel class of PAD inhibitors, based on an amino acid
backbone, in which similar nitrogen-rich heterocycles were appended via a two-carbon linker to an -
amino acid side chain (Table 1). The structure-activity relationship contained within probed non-
covalent binding interactions between the various heterocyclic side chains and active site residues, as
well as N- and C-termini substitutions. In vitro PAD inhibition assays were performed to assess their
enzyme inhibitory activities, and selectivity towards PAD isozymes. Further, in vivo efficacy studies
using the MOG EAE mouse model were conducted to demonstrate the potential of this class of non-
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covalent inhibitors as new avenues for developing disease modifying therapeutics (DMTs) targeting
PAD enzymes.
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Table 1. Enzyme inhibition kinetics for non-covalent compounds hPAD1, mPAD2, and hPAD4 and
CHO cell toxicity for synthesized non-covalent inhibitors. N/A refers to “data not available”.
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#
R1
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(IC50, M)
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Bn 112 ± 17
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RESULTS AND DISCUSSION.
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Synthesis and Structure-Activity Relationships. A retrosynthetic strategy to afford the chiral target
molecules (11-15, 22-23 and 29-30) indicated that enantiopure L-homoserine (4) was the ideal
backbone for this series of molecules as it set the required linker length to accommodate selected
heterocycles at R2. From here, functional group interconversions and amidations with commercially
available reagents could be utilized to scan surface-protein interactions at R1 and R3.
Complications with SN2 substitutions at R2 owing to a lack of nucleophilicity for the selected N-
heterocycles necessitated the employment of multiple approaches to synthesize the target compounds.
Synthesis of the N-methyl piperazine or imidazolyl derivates (11-15) is according to Scheme 2. First,
protection of the primary alcohol of L-homoserine (4) with TBDMSCl was followed by amine
protection with Fmoc carbamate to afford compound 6 according to published procedures.36 Treatment
of 6 with isopropylamine and HATU afforded intermediate 7 which was subsequently treated with the
appropriate acyl chloride to generate the corresponding intermediates 8a-c. Desilylation followed by
mesylation and SN2 substitution with N-methyl piperazine or imidazoles yielded compounds 11-15
(Scheme 2). However, N-imidazolyl substitution using the mesylate derivative of 18 did not yield the
desired products, 22-23, due to the formation of elimination byproducts.
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Scheme 2. Synthesis of compounds 11-15.a
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aReagents and conditions: (a) DBU (1.0 eq), TBDMSCl (1.5 eq), anhyd ACN, N2 atm, rt, 16 h; (b)
Fmoc-O-Succinate (1.1 eq), Et3N (1.5 eq), 50:50 ACN: H2O, rt, 4 h; (c) HATU (1.1 eq), DIPEA (3.0
eq), isopropylamine (10.0 eq), anhyd DMF, N2 atm, 0 °C→rt, 16 h; (d) R1COCl (1.0 eq), DIPEA (3.0
eq), anhyd CH2Cl2, N2 atm, 0 °C→rt, 2-5 h; (e) TBAF (1.0 eq), anhyd THF, N2 atm, 0 °C→rt, 2 hr; (f)
MsCl (1.0 eq), DIPEA (3.0 eq), anhyd CH2Cl2, N2 atm, 0 °C, 2 h; (g) R2-H (1.0 eq), DIPEA (1.5 eq),
anhyd DMF, N2 atm, 0 °C→rt, 16 h.
Thus, a slightly modified procedure was then utilized to afford the target derivatives 22-23 (Scheme
3). Compound 16 was first obtained by amino protection of intermediate 5 with t-butoxy carbamate,
which exhibited improved stability during the subsequent desilylation step. Coupling of 16 with
benzylamine afforded the amide 17. Desilylation and bromination via Appel reaction37 afforded 19.
Subsequent substitution with N-imidazolyl moiety was optimized when 1H-imidazole dissolved in
anhyd DMF was first treated with DIPEA (1 eq) and added dropwise to intermediate 19 to afford 20 in
good yields. Boc deprotection with TFA afforded 21 which was reacted with appropriate acyl chloride
or carboxylic acid to afford compounds 22-23. The synthesis of compounds 28-30 was carried out via
the cyclization of the azide intermediate 24 (Scheme 4), as direct substitutions onto compound 19 with
1H,2,3-triazole were unsuccessful. Intermediate 18 was instead converted into azide 24, followed by
1H,2,3-triazole formation with TMS-acetylene in the presence of copper catalyst to yield compound 25.
Desilytation followed by decarbamoylation yielded intermediate 27, which was coupled with the
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appropriate acyl chlorides or carboxylic acids to afford compounds 28-30.
Scheme 3. Synthesis of N-imidazole substituted compounds.a
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aReagents and conditions: (a) Boc2O (1.1 eq), Et3N (1.5 eq), 50:50 ACN:H2O, rt, 4 hr; (b) HATU (1.10
eq), DIPEA (3.0 eq), benzylamine (1.1 eq), anhyd DMF, N2 atm, 0 °C→rt, 16 hr; (c) TBAF (1.0 eq),
anhyd THF, N2 atm, 0 °C→rt, 2 hr; (d) CBr4 (1.5 eq), PPh3 (1.5 eq), anhyd CH2Cl2, N2 atm, 0 °C→rt,
2.5 hr; (e) imidazole (2.5 eq), DIPEA (1.5 eq), anhyd DMF, N2 atm, 0°C→rt, 16 hr; (f) 30% TFA,
anhyd CH2Cl2 , N2 atm, 0 °C, 45 min; (g) for R1COCl: DIPEA (3.0 eq), CH2Cl2 , N2 atm, 0 °C, 4 hr; and
for R1COOH: HATU (1.1 eq), DIPEA (3.0 eq), ACN, N2 atm, 0 °C→rt, 16 hr.
Scheme 4. Synthesis of 1H,2,3-triazole substituted non-covalent inhibitors.a
aReagents and conditions: a) MsCl (1.1 eq), DIPEA (3.0 eq), anhyd CH2Cl2, N2 atm, 0 °C, 45 min; b)
NaN3 (4.0 eq), anhyd DMF, N2 atm, 60 °C, 2.5 h; c) CuI (1.0 eq), TMEDA (1.0 eq), Et3N (1.0 eq),
TMS-acetylene (2.5 eq), anhyd THF, N2 atm, rt, 18 h; d) TBAF (1.5 eq), anhyd THF, 10% AcOH, N2
atm, 0 °C→rt, 16 h; e) 30% TFA, anhyd CH2Cl2, N2 atm, 0 °C, 45 min; f) for R1COCl: DIPEA (3.0
eq), anhyd CH2Cl2, N2 atm, 0 °C, 4 h; for R1COOH: HATU (1.1 eq), DIPEA (3.0 eq), anhyd ACN, N2
atm, 0 °C→rt, 16 h.
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All synthesized compounds were evaluated for their inhibitory activities against PAD1, PAD2 and
PAD4 (Table 1). N-Methyl piperazine derivatives (11-13), did not inhibit any of the PAD isozymes.
These results were consistent with our previous investigations on hydantoins appended to piperazine
motifs via a similar 2-carbon linker,34 suggesting that the active sites of PADs cannot adequately
accommodate non-covalent inhibitors of this size despite the strong basicity of the side chain.
Compounds 14, 15, 23, 28 and 29 carrying either imidazoles or a triazole appended to the side chain
showed inhibitory activities against at least one PAD isozyme (Ki <400 µM). Among these compounds,
23 and 28 carrying 1-methylpyrroles at R1 (N-terminal) were the most potent compounds with
inhibition constants of 60 and 71 μM, respectively, against PAD2 enzyme. Substitution of the pyrrole
with a furan at R1, compounds 22 and 29, reduced the potency for these compounds (PAD2: Ki >10,000
and 282 μM). Compound 30 did not exhibit any significant inhibition of PAD activities either.
Interestingly, furoyl derivative 14 demonstrated good affinity and greater than 6-fold selectivity for
PAD4 (Ki = 88 μM) over PAD1 and PAD2; however, further in vivo profiling of 14 to investigate the
effects of PAD4 inhibiton was contraindicated by a preliminary toxicity screen which showed that
compound 14 was moderately cytotoxic to CHO cell lines (IC50 = 546 μM). Increasing ring size and
introducing ring saturation at R1 to piperidine resulted in a PAD2 selective reduction in affinity by
more than an order of magnitude relative to the 1-methylpyrrole derivatives.
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Comparable to the hydantoin series, exemplified by 2 and 3, PAD enzymes similarily tolerated 5-
membered heteroaromatics appended via a 2-carbon linker to the side chain of a core amino acid.
Among the peptidomimetic inhibitors, those possessing 1-methylpyrrole subsitutents at R1
demonstrated the most favorable affinity. The most potent compound in this series, 23 — having
demonstrated the highest potentcy and selectivity towards PAD2 — was further investigated in vivo to
understand its potential in a disease model of MS.
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weight of EAE mice treated with either PBS or compound 23, (B) The progression of EAE in
C57BL/6 female mice treated with PBS (n=5) or 23 (n=7) — black arrow signals start of treatment,
(C) Survival graph showing the mortality rates in PBS-treated and compound 23-treated mice.
Efficacy Studies. Compound 23 was evaluated in a mouse MOG-EAE model. EAE is an immune-
mediated mouse model that mimics the CNS inflammation and lesioning commonly observed in MS
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patients, and has been extensively used to evaluate drugs targeting the disease.38,39 EAE was induced
by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Daily treatment with
compound 23 at a dose of 50 mg/kg i.p. was initiated once the mice reached a clinical disease score of
2 and was continued for the duration of the study, up to 60 days. EAE clinical scores were recorded for
each mouse in the treatment group, and compared to those in the control group, receiving PBS. Body
weight was recorded daily for animals in both the treatment and vehicle group as a measure of general
toxicity and health, with no difference observed between groups (Figure 2A). Physical disability in the
PBS treated mice peaked around the scores of 2-2.5 and remained relatively constant throughout the
duration of the study (Figure 2B). In comparison, mice treated with compound 23 showed a significant
reduction in EAE clinical scores to the point that nearly complete amelioration of disease symptoms
was observed following day 35. The data is also suggestive of a trend towards improved survival rates
in the treated vs control group (100% vs 80%); although follow up studies with larger sample sizes
would be necessary to reach definitive conclusions regarding this outcome measure (Figure 2C).
Both treated and healthy mice of equal age were sacrificed following completion of the study. The
brains and spinal cords were collected to analyze for infiltrating T-cells and the status of myelin (Figure
3). We found that by 60 days, there was not as much inflammation in the spinal cord, but we did see
some in the brain that differed in extent between the PBS and compound 23-treated groups. Since the
inflammation became more focused in the brain, we analyzed the inflammation in the brain. Thus,
these brain sections were subjected to hematoxylin and eosin (H&E) stain, as a general means to detect
hypercellularity, as well as Luxol-Fast Blue (LFB) staining to visualize myelin. The number and
severity of lesions in the brains of mice treated with PBS were greater than those treated with
compound 23, and the most marked changes were observed in the cerebellum (Figures 3A-C), olfactory
tract (Figures 3D-F) and internal capsule (Figures 3G-I). Brain tissue sections, obtained from the mice
treated with compound 23, have the tissue morphology similar to those of healthy mice, and are in
complete contrast with those from vehicle treated mice.
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Figure 3. Cerebellum of healthy (A) and EAE affected mice (B and C). EAE control animals received
PBS and drug treated animals were injected (i.p.) with 50 mg/kg of 23 daily. Yellow arrows point to
lessions (perivascular cuffs) formed in the cerebellum of the sick mice. The PBS receiving mice had a
higher number of more pronounced lesions as compared to 23 treated mice. Lateral olfactory tract area
(Figure D-F) was often observed to be affected by lesions. (E) Shown is a lesion and lymphocytic
leptomeningitis in PBS treated mouse. The corresponding areas (indicated by arrows) in healthy and 23
treated mouse are clear. Perivascular leukoencepahlitis, lymphocytic and neutrophilic inflammation
along the internal capsule. (G) healthy control, (H) PBS control, (I) compound 23 treated.
Magnification 20x. Scale bar indicates 100 µm. Magnification 20x (ImageScope viewer).
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Further immunohistochemical analysis was conducted using anti-CD3 antibodies to investigate the
presence of infiltrating T-cells within the mouse brain sections (Figures 4 and 5). The analysis across
all stained brain sections suggested that CD3+ T-cell counts were highest in the PBS group, and were
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reduced in mice treated with compound 23 (Figure 4).
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Corroborating the patterns in the LFB stained brain sections (Figure 3), CD3 markers saw the
greatest reduction in the cerebellum (Figure 5A-C) and internal capsule (Figure 5D-F) regions of the
brains of the mice treated with 23. Although this is an animal model of MS, and primarily considered
as an inflammation-driven MS model, it is well-characterized that disease pathology such as lesions
appearance is localized to cerebellum and internal capsule areas of the brain.40,41,42
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Figure 4. Immunohistochemistry of EAE affected mice brains. The tissue was probed with
CD3+ve antibody to detect T lymphocytes in the CNS of healthy and sick mice. The sick
animals were treated with PBS (n = 5) or compound 23 (n =7).
8e-5
6e-5
4e-5
2e-5
0
23
KP-302
Healthy
PBS
We conducted a single dose pharmacokinetic analysis in healthy mice to determine the levels of
compound 23 in the serum and brain (Table 2 and Figure 6). Thus, compound 23 was administered to
C57BL/6 female mice i.p. (50 mg/kg, single dose), and the serum and brain concentrations were
profiled. The maximum concentration (Cmax) was determined to be 12.0 ± 2.5 μg/mL and 0.17 ± 0.01
μg/mL in the serum and brain, respectively. The Cmax was observed at 15 min after the administration
of 23, in both serum and brain. The elimination rate constant (ke) in the serum was 0.02 ± 0.008 min−1
and 0.018 ± 0.009 min−1 in the brain. The half-life of 23 (t1/2) was 34.8 min and 38.5 min in the plasma
and brain, respectively. AUC(0-360) (µg·min/mL) for 23 in the serum and brain were 507.8, and 6.3,
respectively. These data indicate the presence of the test compound in the brain with respectable half-
life, albeit lower concentrations than those in the serum. Drug metabolism studies and oral
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bioavailability, among others are under investigation to understand the behavior of the drug, and its
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potential fate in vivo.
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Figure 5. CD3+ staining for T-cells lymphocytes. Shown is the inflammation in cerebellum (A-
C) and along the internal capsule (D-F). The inflammation as indicated by the number of
infiltrating proinflammatory T cells (yellow arrows) was in general more severe in the PBS
receiving mice. The tissue collected from 23-treated mice showed milder lesions and scattered T
cells not clearly associated with any specific lesion. Magnification 20x (ImageScope viewer).
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Figure 6. The time-concentration profile for compound 23 in (A) serum and (B) brain of healthy
C57BL/6 mice (n=27), measured post-administration.
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Compound 23, when compared to compounds 1 and 2 — which are earlier generation non-covalent
PAD inhibitors — is more potent in vivo (dose of 50 mg/kg vs 100 mg/kg). Despite the reduction in
dose, the complete recovery of the animals and almost complete suppression of the disease symptoms
are noteworthy. Pharmacokinetic studies indicated the presence of compound 23 in the brain, although
further studies are required to shed light on the pharmacodynamics and dose-response relationships.
The concentration of compound 23 in the brain is low, hence complete abrogation of PAD activities in
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the brain may not occur. It is also possible that there may be peripheral effects of compound 23.
However, PAD inhibitors such as 2 exhibit excellent efficacy in cuprizone-mediated demyelinating
mouse model of MS that does not involve peripheral inflammation similar to the EAE mouse model.34
Thus, the efficacy due to 23 is through the moderate inhibition of citrullination in the brain.
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Table 2. The pharmacokinetics profile of
compound 23 (single dose, 50 mg/kg) in healthy
C57BL/6 female mice (n = 27).
Parameter
Serum
Brain
Rate = ke (min-1) 0.02 ± 0.008 0.018 ± 0.009
t1/2 (min)
34.8
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Cmax (g/mL)
Tmax (min)
AUC(0-360)
507.8
6.3
(µg min /mL)
While compound 23 exhibited only minimal selectivity towards PAD2 and may not be among the
most potent PAD inhibitors identified to date, its in vivo efficacy is impressive. It appears that the
inhibition of PAD4, in addition to PAD2, may be necessary for the treatment of complex diseases such
as multiple sclerosis, where inflammatory and neurodegenerative components exist. Likewise, its
possible that complete abrogation of isozyme specific PAD activities and hence the application of
highly-potent and isozyme selective compounds, is not essential nor warranted for treatment. Rather
multitargeted PAD inhibitors capable of modestly modulating PAD hyperactivity is sufficient to
achieve therapeutic effecicacy. Some evidence can be gleaned from PAD knock-out studies.
Raijmakers et al. had shown that PAD2 knockout mice developed EAE despite a lack of PAD2
mediated MBP citrullination, and assumed citrullination did not facilitate a role in EAE initiation.43
Later, it was confirmed that significant levels of citrullinated MBP were infact present in the PAD2
knockout mice.44 The citrullination of MBP was due to the upregulated activity but not overexpression
of PAD4 in the brain and spinal cord, with levels of PAD4 found to be similar between the PAD2
knockout and wild type mice.44 In a recent study, Coudane et al also demonstrated that the absence of
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PAD2 in PAD2 (-/-) mice did not have a significant affect on protein citrullination following wound
induction/inflammation.45 Thus, the near complete suppression of EAE clinical scores and the
reflection of this efficacy in the reversal of brain tissue pathology in the treated animals warrant further
investigations into the utility of multi-targeted PAD inhibitors, in particular compound 23 as a DMT for
the potential treatment of MS.
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Here, we disclosed a novel set of -amino acid-based designer molecules carrying unique
heterocycles, especially compound 23, as non-covalent inhibitors to PAD enzymes with a potential for
optimization as preclinical lead compounds. These findings and the structural core revealed in this
work lend themselves to further development of this class of compounds as potential DMTs for MS.
EXPERIMENTAL SECTION.
Chemistry. General. All reagents were purchased from Sigma-Aldrich and Bachem chemicals
unless otherwise noted. The compounds were purified by column chromatography using silica gel (60
Å, 70-230 mesh) and reverse-phase (C18) silica cartridges. NMR spectra were recorded on a Bruker
1
spectrometer (400 MHz for H, 100 MHz for 13C and 376.62 MHz for 19F). Chemical shifts δ are
1
reported in ppm with tetramethylsilane (TMS) as a reference standard for H, and trifluoroacetic acid
19
(TFA) as an external reference for F, and are reported as s (singlet), brs (broad singlet), d (doublet),
dd (doublet of doublet), t (triplet), q (quadruplet), and m (multiplet). UV-Vis and fluorescence spectra
were recorded on a SpectraMax plate reader (GE Healthcare) in dioxane or PBS as a solvent. LC-MS
analyses were conducted using a Waters™ system equipped with a Waters™ 2545 binary gradient
module system and Waters™ 3100 mass detector. All compounds evaluated in biological assays were
found to be more than 95% pure by two methods of HPLC analysis, and the details are presented in the
Supporting Materials.
O-(tert-Butyldimethylsilyl)-L-Homoserine (5). A solution of L-homoserine, 4 (1 g, 8.4 mmol) and
DBU (1.32 mL, 8.8 mmol) in anhyd ACN (20 mL) was treated with t-butyldimethysilyl chloride (1.32
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g, 8.8 mmol) in ACN (20 mL) at 0 °C. The reaction mixture was stirred at rt for 16 h. after which a
white precipitate formed. The suspension was filtered, washed with cold ACN (200 mL), cold H2O
(200 mL) and Et2O (200 mL), and dried under reduced pressure to afford 5 as a white powder (yield:
6
7
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1.45 g, 74%). m.p: 148-153 °C. H NMR (CD3OD) δ 3.87 (t, J = 6 Hz, 2H), 3.71-3.68 (m, 1H), 2.21-
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1.93 (m, 2H), 0.93 (s, 9H), 0.12 (s, 6H). 13C NMR (CD3OD) δ 61.9, 54.7, 34.4, 26.4, -5.4;.
N-((9[H]-Fluoren-9-yl) methoxy carbonyl)-O-(tert-butyldimethylsilyl)-L-homoserine (6). To a
solution of 5 (1.45 g, 6.2 mmol) in 100 mL of 5% NaHCO3 (w/v) in 50% ACN/H2O, Fmoc-O-
succinimide (2.3 g, 6.8 mmol) was added. The reaction mixture was stirred at rt for 12 h, the solvent
was partially removed under reduced pressure, and the remaining suspension was acidified to pH 2.0
with 10% citric acid. Aqueous layer was extracted using EtOAc (3 × 200 mL) and dried over MgSO4.
Combined organic layers were concentrated under reduced pressure and the crude was purified by
silica gel column chromatography (5-40% EtOAc gradient in hexanes) to afford 6 as a white semi-solid
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(yield: 2.41 g, 85%). H NMR (CDCl3) δ 7.57 (d, J = 7.6 Hz, 2H), 7.60 (t, J = 6 Hz, 2H), 7.39 (t, J =
7.6 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 6.15 (d, J = 7.2 Hz, 2H), 4.21 (t, J = 6.8 Hz, 2H), 3.82 (t, 2H),
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2.18-2.05 (m, 2H), 0.91 (s, 9H), 0.09 (s, 6H). C NMR (CDCl3) δ 156.4, 143.9, 141.4, 127.8, 127.2,
125.2, 120.1, 67.3, 60.7, 53.1, 47.3, 33.5, 25.9, 18.3, -5.5.
(S)-2-Amino-4-tert-butyldimethylsilyloxy-N-isopropylbutanamide (7). Compound 6 (1.64 g, 3.6
mmol) was dissolved in anhyd DMF (5 mL) to which DIPEA (1.9 mL, 1.8 mmol) and HATU (1.51 g,
3.6 mmol) were added and stirred at 4 °C. Isopropylamine (4.56 mL, 36.0 mmol) was added drop wise
to the reaction mixture and was further stirred at 4 °C. Upon completion of the reaction, solvent was
evaporated and the crude was dissolved in CH2Cl2 (15 mL), washed with H2O (200 mL), brine (200
mL), and dried (MgSO4). All organic layers were combined and were purified by silica gel column
chromatography (30-100% EtOAc gradient in hexanes) to afford 7 as an yellow oil (yield: 390 mg,
40%). 1H NMR (CDCl3) δ 6.68 (m, 1H, NH), 6.15 (m, 1H, NH), 4.10 (m, 1H), 4.03 (septet, 1H), 3.75
(m, 2H), 1.97 (m, 1H), 1.95 (m, 1H), 1.13 (d, J = 6 Hz, 6H), 0.91 (s, 9H), 0.075 (d, J = 4 Hz, 6H). 13C
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NMR (CDCl3) δ 170.3, 59.5, 51.1, 40.6, 38.2, 30.7, 25.8, 22.2, 22.1, -5.4.
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(S)-N-(4-tert-Butyldimethylsilyloxy-1-isopropylamino-1-oxobutan-2-yl)-furan-2-carboxamide
6
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(8a). Compound 7 (360 mg, 1.3 mmol) was dissolved in anhyd CH2Cl2 (5 mL) to which DIPEA (762
µL, 4.4 mmol) was added at 0 °C, followed by 2-furoyl carbonyl chloride (215 µL, 2.2 mmol) dropwise
and the reaction mixture was stirred at 0 °C for 3 h. Solvent was evaporated, the crude was dissolved in
CH2Cl2 (30 mL), washed with sat. NaHCO3 solution (200 mL), H2O (until pH was neutralized), brine
(200 mL), and dried (MgSO4). Resulting crude was purified by silica gel column chromatography (0-
5% MeOH:CH2Cl2) to afford 8a as a yellow resin (yield: 140 mg, 29%). 1H NMR (CDCl3) δ 7.77 (d, J
= 8 Hz, 1H), 7.12 (d, J = 4 Hz, 1H), 6.70 (d, J = 8 Hz, 1H), 6.50 (brs, 1H, NH), 4.73 (m, 1H), 4.09
(septet, 1H), 3.92 (m, 1H), 3.81 (m, 1H), 2.16 (m, 1H), 2.06 (m, 1H), 1.12 (d, J = 4 Hz, 6H), 0.93 (s,
9H), 0.105 (d, J = 4 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 170.0, 158.3, 147.7, 144.1, 114.6, 112.2,
61.0, 51.7, 41.6, 34.4, 26.0, 22.6, 18.4, -5.3.
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(S)-N-(4-tert-Butyldimethylsilyloxy-1-isopropylamino-1-oxobutan-2-yl)morpholine-4-
carboxamide (8b). Compound 7 (330 mg, 1.20 mmol) was dissolved in anhyd CH2Cl2 (5 mL) and
DIPEA (1 mL, 6 mmol) was added at 0 °C. Morpholine carbonyl chloride (210 µL, 1.8 mmol) was
added dropwise to the reaction at 0 °C. The reaction was monitored as per the procedure described for
compound 8a. Compound 8b was obtained as a colorless resin (yield: 245 mg, 53%). 1H NMR (CDCl3)
δ 6.71 (brs, 1H, NH), 6.02 (m, 1H, NH), 4.41 (q, 1H), 4.04 (dq, 1H), 3.85 (m, 1H), 3.75 (m, 1H), 3.67
(m, 4H), 3.37 (m, 4H), 1.99 (m, 1H), 1.92 (m, 1H), 1.15 (d, J = 4 Hz, 6H), 0.91 (s, 9H), 0.085 (d, J = 4
Hz, 6H). 13C NMR (CDCl3) δ 171.4, 157.5, 66.5, 61.4, 53.0, 44.5, 44.1, 35.3, 26.1, 22.8, 18.5, -5.1.
(S)-N-(4-tert-Butyldimethylsilyloxy-1-isopropylamino-1-oxobutan-2-yl)-cyclohexane carboxamide
(8c). Compound 7 (330 mg, 1.2 mmol) was dissolved in anhyd CH2Cl2 (5 mL)and DIPEA (1 mL, 6
mmol) was added at 0 °C. Cyclohexyl carbonyl chloride (240 µL, 1.8 mmol) was added dropwise at 0
°C. The reaction was monitored as per procedure described for 8a to obtain the target compound.
1
Compound 8c was obtained as a white solid (yield: 200 mg, 52%). m.p: decomp. at 310 °C; H NMR
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(CDCl3) δ 6.61 (brs, 1H, NH), 5.85 (brs, 1H, NH), 4.40 (q, 1H), 4.05 (dq, 1H), 3.89 (m, 1H), 3.77 (m,
1H), 3.33 ( m, 4H), 2.03 (m, 1H), 1.91 (m, 1H), 1.59-1.54 (m, 6H), 1.12 (d, J = 4 Hz, 6H), 0.91 (s, 9H),
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0.085 (d, J = 4 Hz, 6H). C NMR (CDCl3) δ 171.6, 157.3, 61.4 53.0 47.8 44.8 41.2 34.8 25.9 25.5
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(S)-N-(4-Hydroxy-1-isopropylamino-1-oxobutan-2-yl) furan-2-carboxamide (9a). Compound 8a
(140 mg, 380 µmol) was dissolved in 5 mL of anhyd THF and TBAF (165 µL, 570 µmol) was added at
0 °C for 30 min. Reaction solvent was evaporated and resulting crude purified by silica gel column
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chromatography (0-10% MeOH:CH2Cl2) to yield 9a as a yellow resin (yield: 75 mg, 78%). H NMR
(CDCl3) δ 7.53 (d, J = 7.2 Hz, 1H), 7.45 (m, 1H, NH), 7.13 (d, 1H), 6.75 (d, J = 7.3 Hz, 1H), 6.51 (m,
1H, NH), 4.75 (m, 1H), 4.08 (septet, 1H), 3.72 (m, 2H), 2.09 (m, 1H), 1.83 (m, 1H), 1.16 (d, J = 4 Hz,
6H).
(S)-N-(4-Hydroxy-1-isopropylamino-1-oxobutan-2-yl)-morpholine-4-carboxamide
(9b).
Compound 9b was synthesized from 8b (245 mg, 632 µmol) following reaction procedure described
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for 9a. Compound 9b was obtained as a white solid (yield: 103 mg, 60%). m.p: 210 °C decomp. H
NMR (CDCl3) δ 6.04 (d, 1H, NH), 5.75 (m, 1H, NH), 4.45 (septet, 1H), 4.21(brs, 1H, OH), 4.05 (m,
1H) 3.69 (m, 6H), 3.34 (m, 4H), 1.97 (m, 1H), 1.65 (m, 1H), 1.17 (d, J = 4 Hz, 6H). 13C NMR (CDCl3)
δ 171.4, 157.5, 66.5, 61.4, 53.0, 44.5, 44.1, 35.3, 26.1, 22.8, 18.5, -5.1.
(S)-N-(4-Hydroxy-1-isopropylamino-1-oxobutan-2-yl) cyclohexanecarboxamide (9c). Compound
9c was synthesized from 8c (200 mg, 520 µmol) following procedure described for synthesis of 9a.
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Compound 9c was afforded as a white resin (yield: 110 mg, 78%). H NMR (CDCl3) δ 6.61 (d, 1H,
NH), 5.85 (d, 1H, NH), 4.40 (q, 1H), 4.05 (dq, 1H), 3.89 (m, 1H), 3.77 (m, 1H), 3.33 (m, 4H), 2.03 (m,
1H), 1.91 (m, 1H), 1.59-1.54 (m, 6H), 1.12 (d, J = 4 Hz, 6H). 13C NMR (CDCl3) δ 171.6, 157.3, 61.40,
53.0, 47.8, 44.8, 41.2, 34.9, 25.9, 25.5, 24.3, 22.6.
General procedure for the preparation of the L-N-R1-homoserine-OMs (10a-c). Compound 9a or
9b or 9cwas dissolved in approximately 5 mL of anhyd CH2Cl2 to which DIPEA (3.0 eq) was added at
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0 °C. Methanesulfonyl chloride (2.5 eq) was added dropwise to the reaction mixture at 0 °C with
continued stirring, and the reaction was quenched with methanol (5 mL) after 2 hrs. Solvent was
evaporated, crude was dissolved in 10 mL of CH2Cl2, washed with dilute NH4Cl solution (150 mL),
brine (200 mL), and dried (MgSO4). Solvent was evaporated to obtain the corresponding mesylate,
10a-c. Intermediates were used immediately for the next reaction without further purification.
(S)-N-(1-(Isopropylamino)-4-(4-methylpiperazin-1-yl)-1-oxobutan-2-yl)furan-2-carboxamide
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(11). Compound 10a (50 mg, 150 µmol) was dissolved in 2 mL of anhyd DMF and stirred at 0 °C. 1-
methylpiperazine (18.15 µL, 164 µmol) was added drop wise at 0 °C and reaction was allowed to warm
up 60 °C and stir for 2 hr. Upon reaction completion, solvent was removed in vacuo and resulting crude
was dissolved in CH2Cl2 (50 mL) and washed with H2O (150 mL), brine (150 mL) and dried over
MgSO4. Crude was purified by silica gel column chromatography in a gradient of 0-10% MeOH in
CH2Cl2 to afford 11 as a clear semi-solid (yield: 19 mg, 38%). 1H NMR (CDCl3) δ 7.89 (brs, 1H, NH),
7.49 (d, J = 8 Hz, 1H), 7.16 (d, J = 4 Hz, 1H) 6.78 (m, 1H, NH), 6.52 (dd, J = 8 Hz, J = 4 Hz, 1H)
4.47 (m, 1H), 4.28 (m, 1H), 3.69 (m, 4H), 3.39 (m, 4H), 2.79 (m, 2H), 2.20 (m, 2H), 2.12 (s, 3H), 1.21
+
(d, J = 4 Hz, 6H); MS (ESI) m/z: [M + H]+ calcd for C17H29N4O3 337.22, found 337.19.
(S)-N-(1-(Isopropylamino)-4-(4-methylpiperazin-1-yl)-1-oxobutan-2-yl)morpholine-4-
carboxamide (12). Compound 12 was synthesized from 10b (140 mg, 398 µmol) following reaction
1
protocol described for 11. Compound 12 was obtained as a clear semi-solid (yield: 56 mg, 40%). H
NMR (CDCl3) δ 4.68 (m, 1H), 4.06 (septet, 1H), 2.73 (m, 2H), 2.43 (s, 3H), 2.37 (m, 8H), 1.12 (d, J =
+
4 Hz, 6H); MS (ESI) m/z: [M + H]+ calcd for C17H34N5O3 356.27, found 356.37.
(S)-N-(1-(Isopropylamino)-4-(4-methylpiperazin-1-yl)-1-oxobutan-2yl) cyclohexanecarboxamide
(13). Compound 13 was synthesized from 10c (50 mg, 144 µmol) following the procedure described
for the synthesis of 11. Compound 13 was obtained as a white solid (yield: 6 mg, 12%). m.p.: 418-420
1
°C; H NMR (CDCl3) δ 6.13 (d, 1H, NH), 4.54 (m, 1H), 4.19 (m, 1H), 4.125 (q, 2H), 2.85 (m, 1H),
2.17 (m, 1H), 2.14 (s, 3H), 1.91-1.37 (m, 10H), 1.23 (d, J = 4 Hz, 6H); MS (ESI) m/z: [M + H]+ calcd
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for C19H37N4O2 353.29, found 353.31.
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(S)-N-(4-(1[H]-Imidazol-1-yl)-1-(isopropylamino)-1-oxobutan-2-yl) furan-2-carboxamide
(14). A solution of imidazole (54 mg, 793 µmol) and DIPEA (61 mg, 473 µmol) in anhyd DMF (2 mL)
was stirred at rt for 30 min. Compound 10a (100 mg, 301 µmol) was dissolved in 1 mL of anhyd DMF
and added dropwise to the reaction mixture, at 0 °C. Upon completion of the reaction, solvent was
evaporated in vacuo and the resulting crude was dissolved in EtOAc (50 mL) and washed with H2O
(100 mL), brine (100 mL), and dried over MgSO4. Resulting crude was purified by silica gel column
chromatography (30-100% EtOAc gradient in hexanes) to afford 14 as a yellow semi-solid (yield: 25
mg, 27%). 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 7.70 (s, 1H), 7.49 (d, J= 7.2 Hz, 1H),7.19 (d, J
= 4 Hz, 1H), 7.14 (d, J= 7.3 Hz, 1H), 7.03 (d, 1H, NH), 6.78 (m, 1H, NH), 6.52 (dd, J= 7.2 Hz, J= 7.3
Hz, 1H ), 6.15 (d, 1H, NH), 4.59 (m, 1H), 4.11 (m, 2H) 4.08 (m, 1H), 2.41 (m, 1H), 2.21 (m, 1H), 1.18
(d, J = 4 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 171.3, 161.3, 137.8, 133.1, 128.7, 128.1, 126.0,
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+
120.6, 113.3, 111.8, 50.3, 43.2, 38.5, 35.8; MS (ESI) m/z: [M + H]+ calcd for C15H21N4O3 305.16,
found 305.16.
(S)-N-(4-(4-(Cyanomethyl)-1[H]-imidazol-1-yl)-1-(isopropylamino)-1-oxobutan-2-yl)furan-2-
carboxamide (15). Compound 15 was synthesized from 10a (140 mg, 421 µmol) following procedure
1
described for 14, and the product was obtained as a yellow-brown semi-solid (yield: 35 mg, 24%). H
NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 7.17 (d, J = 7.2 Hz, 1H), 7.07 (d, 1H, NH), 7.03 (m, J = 4 Hz,
1H), 6.54 (dd, J = 7.2 Hz, J = 4 Hz, 1H), 4.55 (m, 1H), 4.05 (m, 2H), 3.74 (m, 1H), 3.67 (s, 2H), 2.34
+
(m, 2H), 2.18 (m, 2H), 1.15 (d, J = 4 Hz, 6H); MS (ESI) m/z: [M + H]+ calcd for C17H22N5O3 344.17,
found 344.27.
N-(tert-Butoxycarbonyl)-O-(tert-butyldimethylsilyl)-L-homoserine (16). Compound 5 (2.36 g, 10.1
mmol) was dissolved in 50% aqueous acetone (50 mL), triethyl amine (2.1 mL, 15.1 mmol) and Boc-
anhydride (2.4 g, 11.0 mmol) were added and the reaction mixture was stirred at rt for 6 h. Solvent was
evaporated under reduced pressure, and the aqueous layer was acidified to pH 2.0 with 10% citric acid.
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The aqueous layer was extracted with EtOAc (3 x 200 mL) and dried (MgSO4). Compound 16 was
obtained as a viscous clear oil (yield: 2.72 g, 81%). 1H NMR (CDCl3) δ 5.92 (d, 1H, NH), 4.33 (q, 1H),
3.81 (m, 2H), 2.07 (m, 2H), 1.441 (s, 9H), 0.910 (s, 9H), 0.08 (d, J = 4 Hz, 6H).
8
9
tert-Butyl (S)-(1-(benzylamino)-4-(tert-butyldimethylsilyloxy)-1-oxobutan-2-yl)carbamate (17).
Compound 16 (2.72 g, 8.16 mmol) was dissolved in anhyd ACN (15 mL) and DIPEA (4.25 mL, 24.4
mmol) and HATU (3.41 g, 8.97 mmol) were added at 4 °C. Benzylamine (980 µL, 8.97 mmol) was
then added dropwise to the reaction mixture and continued stirred at 4 °C for 6 h. Reaction solvent was
evaporated in vacuo, crude was dissolved in EtOAc (15 mL), washed with H2O (150 mL), brine (150
mL), and dried (MgSO4). Crude was purified by silica gel column chromatography (5-30% EtOAC:
hexanes) to afford 17 as a clear oil (yield: 2.29 g, 66%). 1H NMR (CDCl3) δ 7.37 (m, 5H), 7.10 (d, 1H,
NH), 6.20 (d, 1H, NH), 4.55 (dd, J = 4 Hz, 2H), 4.41 (m, 1H), 3.85 (m, 2H), 2.12 (m, 2H), 1.52 (s,
9H), 0.99 (s, 9H), 0.15 (d, J = 4 Hz, 6H).
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tert-Butyl (S)-(1-(benzylamino)-4-hydroxy-1-oxobutan-2-yl)carbamate (18). Compound 17 (1.13 g,
2.67 mmol) was dissolved in anhyd THF (10 mL) and stirred at 0 °C. TBAF (1M solution in THF, 2.67
mL, 2.67 mmol) was added drop wise to reaction mixture and further stirred at 0 °C for 30 min.
Solvent was removed and the crude was purified by silica gel column chromatography (10-80%
EtOAc: hexanes) to afford 18 as a white solid (yield: 680 mg, 83%).1H NMR (CDCl3) δ 7.59 (d, 1H,
NH), 7.30-7.20 (m, 5H), 5.86 (d, 1H, NH), 4.38 (dd, J = 4 Hz, 2H), 4.31 (m, 1H), 3.60 (m, 2H), 1.98-
1.69 (m, 2H), 1.40 (s, 9H). m.p.: 330-333 °C.
tert-Butyl (S)-(1-(benzylamino)-4-bromo-1-oxobutan-2-yl)carbamate (19). Compound 18 (250 mg,
811 µmol) and carbon tetrabromide (318 mg, 959 μmol) were dissolved anhyd CH2Cl2 (10 mL) while
stirring at 0 °C and triphenylphosphine (403 mg, 1.54 mmol) dissolved in anhyd CH2Cl2 (5 mL) was
added dropwise to the reaction mixture over 5 min. The stirring was continued for 4 h, the solvent was
evaporated and the crude was purified by silica gel column chromatography (10-40% EtOAc:hexanes)
to afford compound 19 as a white powder (yield: 173 mg, 58%). 1H NMR (CDCl3) δ 7.59 (d, 1H, NH),
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7.22 (m, 5H), 6.09 (d, 1H, NH), 4.41 (dd, J = 4 Hz, 2H), 4.31 (m, 1H), 3.39 (m, 2H), 2.42-2.14 (m,
2H), 1.40 (s, 9H). m.p.: 328-330 °C.
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tert-Butyl-(S)-(1-(benzylamino)-4-(1[H]-imidazol-1-yl)-1-oxobutan-2-yl)carbamate (20). A solution
of compound 19 (520 mg, 1.40 mmol) in anhyd DMF (3 mL) was treated with a solution of imidazole
(238 mg, 3.50 mmol) and DIPEA (366 µL, 2.10 mmol) in DMF (3 mL) at 0 °C, and the reaction was
continued for 4 h. Solvent was then evaporated under reduced pressure, the resulting crude was
dissolved in EtOAc (100 mL), washed with H2O (300 mL), brine (300 mL) and dried (MgSO4). The
crude product was purified by silica gel column chromatography (0-10% MeOH:CH2Cl2) to yield
compound 20 as a yellow resin (yield: 380 mg, 76%). 1H NMR (CDCl3) δ 8.17 (s, 1H), 7.32 (d, J = 4
Hz, 1H), 7.21 (m, 5H), 6.87 (d, J = 4 Hz, 1H), 5.94 (d, 1H, NH), 4.38 (dd, J = 4 Hz, 2H), 4.22 (m, 1H),
3.97 (m, 2H) 2.24-1.99 (m, 2H), 1.43 (s, 9H).
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(S)-2-Amino-N-benzyl-4-(1[H]-imidazol-1-yl)butanamide (21). Compound 20 (380 mg, 1.06 mmol)
in anhyd CH2Cl2 (15 mL) was treated with 30%TFA (2 mL) and stirred at 0 °C for 1 h. Reaction
mixture was then treated with toluene (5 mL), concentrated under reduced pressure to yield compound
21 as yellow crystalline solid (yield: 356 mg, quant.). This compound was used as such for the next
reaction without further purification.
(S)-N-(1-(Benzylamino)-4-(1[H]-imidazol-1-yl)-1-oxobutan-2-yl)
furan-2-carboxamide
(22).
Compound 21 (95 mg, 368 µmol) was dissolved in 3 mL of anhyd CH2Cl2 to which DIPEA (320 µL,
1.84 mmol) was added and stirred at 0 °C. 2-Furoyl chloride (36 µL, 366 µmol) was added dropwise at
0 °C to the reaction mixture and the reaction was stirred at 0 °C for 30 min. The reaction was then
allowed to warm up to rt and continued stirring for an additional 12 h. Reaction solvent was evaporated
and resulting crude was dissolved in 15 mL of EtOAc and washed with sat. NaHCO3 (200 mL), H2O
(until pH was neutralized), brine (200 mL), and dried over MgSO4. Resulting crude was purified by
silica gel column chromatography in a 0-5% MeOH gradient in CH2Cl2 to afford 21 as a yellow semi-
solid (yield: 25 mg, 19%). 1H NMR (CDCl3) δ 8.41(d, J = 4 Hz, 2H), 7.69 (s, 1H), 7.47(s, 1H), 7.28
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(m, 5H), 7.09 (d, J = 4 Hz, 1H), 6.92 (d, 1H), 6.50 (dd, J = 4 Hz, 1H), 5.53 (d, 1H, NH) 4.72 (m, 1H),
4.40 (dd, J = 4 Hz, 2H), 3.97 (m, 2H) 2.24-1.99 (m, 2H); MS (ESI) m/z: [M + H]+ calcd for
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7
+
C19H21N4O3 353.16, found 353.25.
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(S)-N-(1-(Benzylamino)-4-(1[H]-imidazol-1-yl)-1-oxobutan-2-yl)-1-methyl-1[H]-pyrrole-2-
carboxamide (23). N-Methylpyrrole-2-carboxylic acid (46 mg, 368 µmol) was dissolved in 2 mL of
anhyd DMF to which DIPEA (320 µL, 1.84 mmol) and HATU (139 mg, 366 µmol) were added and
stirred at 0 °C for 20 min. Compound 21 (95 mg, 368 µmol) dissolved in anhyd DMF (1 mL) was
added to the reaction mixture and stirred for an additional hour at 0 °C, and then stirred for 12 h at rt.
Solvent was evaporated and crude was dissolved in EtOAc (50 mL), washed with H2O (100 mL) and
brine (100 mL), and dried over MgSO4. Resulting crude was purified by silica gel column
chromatography in a 0-15% MeOH gradient in CH2Cl2 to afford 23 as a white semi-solid (yield: 35
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mg, 26%). H NMR (CDCl3) δ 8.18 (s, 1H), 7.53 (d, J = 8 Hz, 1H), 7.35 (s, 1H), 7.34 (d, J = 4 Hz,
1H), 7.27 (m, 5H), 7.08 (d, J = 4 Hz, 1H), 6.92 (s, 1H), 6.74 (s, 1H), 6.09 (d, 1H, NH), 5.54 (d, 1H),
4.63 (m, 1H), 4.40 (dd, J = 4 Hz, 2H), 4.00 (m, 2H), 3.88 (s, 3H), 2.24-1.99 (m, 2H). 13C NMR
(CDCl3) δ 179.4, 171.1, 162.2, 138.1, 128.8, 127.7, 127.6, 113.1, 107.7, 50.2, 43.62, 3.91, 34.3; MS
+
(ESI) m/z: [M + H]+ calcd for C20H24N5O2 366.19, found 366.19.
tert-Butyl (S)-(4-azido-1-(benzylamino)-1-oxobutan-2-yl)carbamate (24). A solution of compound
18 (1.75 g, 5.68 mmol) in anhyd CH2Cl2 (5 mL) was treated with DIPEA (3 mL, 17.2 mmol) and mesyl
chloride (483 µL, 6.24 mmol) at 0 °C and the reaction mixture was stirred at rt for 16 h. Reaction
mixture was concentrated under reduced pressure, the crude was dissolved in EtOAc (30 mL), washed
with dilute NH4Cl (150 mL) and brine (150 mL), and dried (MgSO4). Combined organic layers were
concentrated and the resulting yellow solid was dissolved in anhyd DMF (5 mL) in a reaction vessel.
Sodium azide (1.47 g, 22.6 mmol) was then added to reaction and stirred at 60 °C for 2.5 h under N2
atmosphere. Reaction was allowed to cool to rt, concentrated under reduced pressure and resulting
crude was dissolved in EtOAc (30 mL). The crude was then washed with H2O (150 mL), brine (150
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mL), dried over MgSO4, and purified by silica gel column chromatography (10-60% EtOAc: hexanes)
to yield compound 24 as a clear resin (yield: 1.04 g, 55%). 1H NMR (CDCl3) δ 7.32-7.24 (m, 5H), 6.68
(brs, 1H, NH), 5.26 (d, 1H, NH), 4.40 (dd, J = 4 Hz, 2H), 4.21 (septet, 1H), 3.41 (m, 2H), 2.09-2.01
(m, 1H), 1.96-1.92 (m, 1H), 1.42 (s, 9H).
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tert-Butyl (S)-(1-(benzylamino) -1-oxo-4-(4-(trimethylsilyl) -1[H]-1,2,3-triazol-1-yl) butan-2-yl)
carbamate (25). To a suspension of CuI (245 mg, 1.29 mmol) in anhyd THF (5 mL), TMEDA (200 µL
1.3 mmol), Et3N (180 µL, 1.29 mmol) and TMS-acetylene (950 µL, 6.86 mmol) were added and stirred
at rt. Compound 24 (860 mg, 2.58 mmol) was dissolved anhyd THF (10 mL) and immediately added to
the reaction mixture and allowed to stir at rt for 18 h. Reaction was quenched with H2O and extracted
using EtOAc (60 mL). Organic layer was then washed with brine (150 mL), dried (MgSO4) and
concentrated under reduced pressure. Resulting crude was purified by silica gel column
chromatography (30-60% EtOAc:hexanes) to afford compound 25 as a colorless resin (yield: 600 mg,
54%). 1H NMR (CDCl3) δ 7.57 (s, 1H), 7.51 (m, 1H, NH), 7.28-7.19 (m, 5H), 5.87 (d, 1H, NH), 4.47
(dd, J = 4 Hz, 2H), 4.39 (m, 2H), 4.30 (septet, 1H), 2.47-2.39 (m, 1H), 2.30-2.25 (m, 1H), 1.40 (s, 9H),
0.29 (s, 9H).
tert-Butyl (S)-(1-(benzylamino) -1-oxo-4-(1[H]-1,2,3-triazol-1-yl) butan-2-yl) carbamate (26). A
solution of compound 25 (700 mg, 1.62 mmol) in anhyd THF (10 mL) and 1M glacial AcOH (1 mL),
1.0 M TBAF in THF (1.62 mL, 1.62 mmol) was added at 0 °C. The reaction was allowed to warm up
to rt and was stirred for 16 h. Reaction was concentrated under reduced pressure and the crude was
purified by silica gel column chromatography (0-5% MeOH:CH2Cl2) to yield compound 26 as a
colorless resin (yield: 435 mg, 75%). 1H NMR (CDCl3) δ 7.65 (s, 1H), 7.56 (s, 1H), 7.32-7.26 (m, 5H),
6.74 (brs, 1H, NH), 5.32 (d, 1H, NH), 4.47 (dd, J = 4 Hz, 2H), 4.43 (m, 2H), 4.11 (m, 1H), 2.47-2.2.40
(m, 1H), 2.33-2.26 (m, 1H), 1.42 (s, 9H).
(S)-2-Amino-N-benzyl-4-(1[H]-1, 2,3-triazol-1-yl) butanamide (27). A solution of compound 26
(430 mg, 1.2 mmol) in anhyd CH2Cl2 (10 mL) was treated with 30% TFA and stirred at 0 °C for 45
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min. Reaction was diluted with toluene (3 mL) and concentrated to afford compound 27 as a yellow
resin, which was used for next reaction without further purification (crude yield: 620 mg, quant.).
(S)-N-(1-(Benzylamino)-1-oxo-4-(1[H]-1,2,3-triazol-1-yl)butan-2-yl)-1-methyl-1[H]-pyrrole-2-
carboxamide (28). N-Methylpyrrole-2-carboxylic acid (147 mg, 1.18 mmol) was dissolved in 3 mL of
anhyd DMF to which HATU (492 mg, 1.29 mmol) and DIPEA (615 µL, 3.53 mmol) were added, and
stirred at 0 °C for 45 min. Compound 27 (305 mg, 1.18 mmol) was dissolved in DMF (2 mL) and
added dropwise to the mixture at 0 °C, and the reaction mixture was stirred for 16 h at rt. Solvent was
evaporated in vacuo and the resulting crude was dissolved in EtOAc (30 mL), washed with H2O (200
mL), brine (200 mL), and dried over anhyd MgSO4. The combined solvents were concentrated and the
crude was purified by silica gel column chromatography (0-10% MeOH gradient in CH2Cl2) to yield 28
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as a white semi-solid (yield: 120 mg, 28%). H NMR (CDCl3) δ 7.97 (s, 1H), 7.76 (d, J = 7 Hz, 1H),
7.63 (s, 1H), 7.47 (d, J = 4 Hz, 1H), 7.29-7.19 (m, 5H), 6.77 (d, J = 7 Hz, 1H, NH), 6.05 (d, J = 4 Hz,
1H), 5.30 (d, 1H, NH), 4.62 (septet, 1H), 4.52 (dd, J = 4 Hz, 2H), 4.40 (m, 2H), 3.86 (s, 3H), 2.91-2.79
+
(m, 1H), 1.83-1.67 (m, 1H); MS (ESI) m/z: [M + H]+ calcd for C19H23N6O2 367.19, found 367.29.
(S)-N-(1-(Benzylamino)-1-oxo-4-(1[H]-1,2,3-triazol-1-yl)butan-2-yl)furan-2-carboxamide
(29).
Compound 27 (305 mg, 1.18 mmol) and DIPEA (615 µL, 3.53 mmol) were dissolved in 7 mL of anhyd
CH2Cl2 and stirred at 0 °C for 45 min. 2-Furoyl chloride (116 µL, 1.18 mmol) was added to the
reaction mixture dropwise at 0 °C and the reaction mixture was stirred for an additional 16 h at rt.
Solvent was evaporated and resulting crude was dissolved in 20 mL of EtOAc and washed sat NaHCO3
(200 mL), H2O (until pH was neutralized), brine (200 mL), and dried over MgSO4. Resulting crude was
purified by silica gel column chromatography (0-10% MeOH gradient in CH2Cl2) to afford 29 as a light
1
brown semi-solid (yield: 75 mg, 18 %). H NMR (CDCl3) δ 7.77 (m, 1H, NH), 7.70 (d, J = 7.2 Hz,
1H), 4.56 (s, 1H), 7.49 (d, J = 4 Hz, 1H), 4.45 (s, 1H), 7.29-7.21 (m, 5H), 7.03 (dd, J =7.2 Hz, J = 4
Hz, 1H), 6.48 (brs, 1H, NH), 4.70 (m, 1H), 4.49 (dd, J = 4 Hz, 2H), 4.45 (m, 2H), 2.38-2.33 (m, 1H),
13
2.43-2.36 (m, 1H). C NMR (CDCl3) δ 170.6, 157.9, 146.9, 143.8, 136.9, 129.2, 128.8, 126.7, 112.1,
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111.1, 55.7, 46.8, 43.2, 30.1; MS (ESI) m/z: [M + H]+ calcd for C18H20N5O3 354.16, found 354.22.
(S)-N-(1-(Benzylamino)-1-oxo-4-(1[H]-1,2,3-triazol-1-yl)butan-2-yl)-4-fluorobenzamide (30). p-
Fluorobenzoic acid ( 148 mg, 1 mmol) was dissolved in 5 mL of anhyd DMF to which HATU (403 mg,
1 mmol) and DIPEA (510 µL, 2.892 mmol) were added and stirred at 0 °C for 45 min. Compound 27
(250 mg, 0.96 mmol) was dissolved in 2 mL of DMF and added dropwise to the mixture at 0 °C.
Reaction mixture was stirred for an additional 16 h, solvent was evaporated, the resulting crude was
dissolved in EtOAc (30 mL), washed with H2O (200 mL), brine (200 mL), and dried over MgSO4.
Resulting crude was purified by silica gel column chromatography in a 0-10% MeOH gradient in
CH2Cl2 to yield 30 as a white solid (yield: 125 mg, 34%). m.p. 180-185 °C. 1H NMR (CDCl3) δ 7.99 (t,
1H), 7.95 (t, 1H), 7.77 (td, 2H), 7.65 (d, 1H), 7.53 (s, 1H), 7.27-7.19 (m, 5H), 7.01 (td, 2H), 4.79
+
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13
(septet, 1H), 4.46 (dd, J = 4 Hz, 2H), 4.38 (d, 2H), 2.55-2.48 (m, 1H), 2.45-2.39 (m, 1H). C NMR
(CDCl3) δ 170.9, 166.7, 163.7, 137.7, 129.7, 128.6, 127.5, 115.7, 115.4, 51.1, 46.9, 43.6, 33.3. 19F
NMR (CDCl3) δ -107.14 (s); MS (ESI) m/z: [M + H]+ calcd for C20H21FN5O2 382.17, found 382.31.
+
Enzyme inhibition assays. Human PAD1 and PAD4, and mouse PAD2 were purchased from
SignalChem (Richmond, BC). Benzoyl arginine ethyl ester (BAEE), DTT, diacetyl monoxime,
thiosemicarbazide, and ammonium iron (III) sulfate were purchased from Sigma-Aldrich (Oakville,
ON). Tris buffer (100 mM Tris, 100 mM NaCl, 10 mM CaCl2, 5 mM DTT and pH 7.5) was employed
as “assay buffer” to conduct the assays. Color reagent was prepared by mixing one volume of reagent
A (80 mM diacetyl monoxime, 2 mM thiosemicarbazide) and three volumes of reagent B (17.35 % v/v
H3PO4, 33.7 % v/v H2SO4, and 0.765 mg/mL ammonium iron (III) sulfate). The inhibition studies of
hPAD1, hPAD4, and mPAD2 were performed at 37 °C. All test compounds were dissolved in DMSO
to prepare the stock solutions and were diluted with the assay buffer. Final concentration of DMSO in
all samples for incubation was ca. 5%. Control reaction contained no inhibitor and the final substrate
concentration was 5 mM in each reaction well. Final concentration of hPAD1, hPAD4 in the enzyme
reaction mixture was 50 nM, and for mPAD2, it was 100 nM. The inhibitors were tested at the final
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