molecules
Review
Inhibitors of the Hydrolytic Enzyme
Dimethylarginine Dimethylaminohydrolase
(DDAH): Discovery, Synthesis and Development
Rhys B. Murphy † , Sara Tommasi † , Benjamin C. Lewis and Arduino A. Mangoni *
Department of Clinical Pharmacology, School of Medicine, Flinders University, Bedford Park, Adelaide 5042,
Australia; [email protected] (R.B.M.); [email protected] (S.T.);
[email protected] (B.C.L.)
* Correspondence: [email protected]; Tel.: +61-8-8204-7495; Fax: +61-8-8204-5114
† These authors contributed equally to this work.
Academic Editors: Jean Jacques Vanden Eynde and Sylvain Rault
Received: 9 February 2016; Accepted: 4 May 2016; Published: 11 May 2016
Abstract: Dimethylarginine dimethylaminohydrolase (DDAH) is a highly conserved hydrolytic
enzyme found in numerous species, including bacteria, rodents, and humans. In humans, the
DDAH-1 isoform is known to metabolize endogenous asymmetric dimethylarginine (ADMA) and
monomethyl arginine (L-NMMA), with ADMA proposed to be a putative marker of cardiovascular
disease. Current literature reports identify the DDAH family of enzymes as a potential therapeutic
target in the regulation of nitric oxide (NO) production, mediated via its biochemical interaction with
the nitric oxide synthase (NOS) family of enzymes. Increased DDAH expression and NO production
have been linked to multiple pathological conditions, specifically, cancer, neurodegenerative disorders,
and septic shock. As such, the discovery, chemical synthesis, and development of DDAH inhibitors
as potential drug candidates represent a growing field of interest. This review article summarizes
the current knowledge on DDAH inhibition and the derived pharmacokinetic parameters of the
main DDAH inhibitors reported in the literature. Furthermore, current methods of development and
chemical synthetic pathways are discussed.
Keywords: dimethylarginine dimethylaminohydrolase; arginine; nitric oxide; asymmetric
dimethylarginine; monomethyl arginine; enzyme inhibitors; organic synthesis
1. Introduction
Dimethylarginine dimethylaminohydrolase (DDAH) is a modulator of the nitric oxide (NO)
pathway and is responsible for the metabolism of methylated arginines [1]. DDAH is conserved
throughout many species and has been demonstrated to pre-date evolution of the Nitric Oxide
Synthase (NOS) family [2], suggesting an alternative function for methylated arginines independent to
their role as NOS regulatory molecules. Two different isoforms of human DDAH have been identified,
with DDAH-1 and DDAH-2 sharing 62% homology. DDAH-1 is primarily expressed in different areas
of the brain [3], the dorsal root ganglia and the spinal dorsal horn [4]. DDAH-2 appears prevalent in
pancreatic islet cells [5] and infected tissues [6]. In addition, Altman et al. reported a study showing
high expression of DDAH-2 in porcine immune tissues [7]. The two isoforms are both expressed in
kidney [8–10], heart [11,12], adipose tissue [13], placenta and trophoblasts [14], and the liver [15–18].
Phylogenetic analyses indicate DDAH-1 was generated by duplication of the genomic segment located
on chromosome 6 comprising DDAH-2 [2,19]. Although hydrolysis of asymmetrically methylated
arginines appears to be the primary function of DDAH-1, alternate roles for both DDAH isoforms
are evident when they independently interact with other enzymes [20,21]. For example, DDAH-1 is
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Molecules 2016, 21, 615
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reported to bind the tumour suppressor protein neurofibromin, resulting in increased phosphorylation
of neurofibromin
by
levels
of
Molecules 2016,
2016, 21,
21,
615protein kinase A (PKA) [20] and is additionally reported to regulate the
of
31
Molecules
615
22 of
31
phosphorylated protein kinase B (p-Akt) by increasing Ras activity via a mechanism independent to
of neurofibromin
neurofibromin
by Similarly,
protein kinase
kinase
A (PKA)
(PKA)
[20] and
and to
is additionally
additionally
reported
to regulate
regulate
the
levels of
of
by
protein
A
is
reported
to
levels
the NOS
pathway [22].
DDAH-2
is [20]
known
interact with
PKA causing
thethe
induction
of
phosphorylated
protein
kinase
B
(p-Akt)
by
increasing
Ras
activity
via
a
mechanism
independent
of phosphorylated
proteinfactor
kinase(VEGF)
B (p-Akt)via
byphosphorylation
increasing Ras activity
viaSp1
a mechanism
independent
vascular
endothelial growth
of the
transcription
factor [21].
to the
the NOS
NOS pathway
pathway [22].
[22]. Similarly,
Similarly, DDAH-2
DDAH-2 is
is known
known to
to interact
interact with
with PKA
PKA causing
causing the
the induction
induction of
of
to
Asymmetric dimethylarginine (ADMA, 1), monomethylarginine (L-NMMA, 2), and symmetric
vascular
endothelial
growth
factor
(VEGF)
via
phosphorylation
of
the
Sp1
transcription
factor
[21].
vascular endothelial growth factor (VEGF) via phosphorylation of the Sp1 transcription factor [21].
dimethylarginine
(SDMA,
3) are endogenous
methylated
arginines (Figure
1).2),They
are formed by
Asymmetric dimethylarginine
dimethylarginine (ADMA,
(ADMA, 1),
1), monomethylarginine
monomethylarginine ((LL-NMMA,
-NMMA, 2),
and symmetric
symmetric
Asymmetric
and
two biochemical
processes:
post-translational
methylation
of1).
protein
arginine
dimethylarginine
(SDMA,(1)
3)the
are initial
endogenous
methylated arginines
arginines
(Figure
1).
They are
are
formedresidues
by
dimethylarginine
(SDMA,
3)
are
endogenous
methylated
(Figure
They
formed
by
which
is
catalyzed
by
the
protein
arginine
methyltransferases
(PRMT’s);
and
(2)
the
subsequent
two
biochemical
processes:
(1)
the
initial
post-translational
methylation
of
protein
arginine
residues
two biochemical processes: (1) the initial post-translational methylation of protein arginine residues
release
from
their respective
methylated
protein(s) by proteolysis
[23].
Once
released
into the
which
is catalyzed
catalyzed
by the
the protein
protein
arginine methyltransferases
methyltransferases
(PRMT’s); and
and
(2) the
the
subsequent
release
which
is
by
arginine
(PRMT’s);
(2)
subsequent
release
+
fromtransport
their respective
respective
methylated
protein(s)
by proteolysis
proteolysis
[23].
Once released
released
into
the
cytosol,
cytosol,
and efflux
of theprotein(s)
methylated
arginines is
mediated
by theinto
y the
cationic
amino
from
their
methylated
by
[23].
Once
cytosol,
++ cationic
transport and
and efflux
efflux of
of the
the methylated
methylated
arginines
is mediated
mediated
by the
the
cationic amino
amino
acid
(CAT-1)
is
by
(CAT-1)
acid transport
(CAT-1)
transporter
[24].
ADMA arginines
and L-NMMA
inhibit
allyymembers
of theacid
NOS
family of
transporter
[24].
ADMA
and Lcompetes
L-NMMA
-NMMA inhibit
inhibit
all
members aof
ofNOS
the NOS
NOS
family of
of enzymes
enzymes
[25–30],
transporter
[24].
ADMA
and
members
the
family
[25–30],
enzymes
[25–30],
while
SDMA
with Lall
-arginine,
substrate,
for
transport
by the y+
while SDMA
SDMA competes
competes with
with LL-arginine,
-arginine, aa NOS
NOS substrate,
substrate, for
for transport
transport by
by the
the yy++ cation
cation transporter
transporter [31].
[31].
while
cation transporter [31]. Different enzymes play a role in the metabolism of methylated arginines,
Different enzymes
enzymes play
play aa role
role in
in the
the metabolism
metabolism of
of methylated
methylated arginines,
arginines, particularly
particularly alanine-glyoxylate
alanine-glyoxylate
Different
particularly alanine-glyoxylate aminotransferase 2 (AGXT2) [32–34]. However, ADMA and L-NMMA,
aminotransferase 22 (AGXT2)
(AGXT2) [32–34].
[32–34]. However,
However, ADMA
ADMA and
and LL-NMMA,
-NMMA, but
but not
not SDMA,
SDMA, are
are primarily
primarily
aminotransferase
but not
SDMA,
primarily
converted
by dimethylamine
DDAH-1 to L(5)
-citrulline
(4) and dimethylamine
converted
byare
DDAH-1
to LL-citrulline
-citrulline
(4) and
and
or monomethylamine
monomethylamine
(6), respectively
respectively(5) or
converted
by
DDAH-1
to
(4)
dimethylamine (5)
or
(6),
monomethylamine
[35] (Scheme
(Scheme 1).
1). (6), respectively [35] (Scheme 1).
[35]
Figure 1. The endogenous methylated arginines, asymmetric dimethylarginine (ADMA 1),
Figure
Theendogenous
endogenous methylated
methylated arginines,
dimethylarginine
(ADMA
1), 1),
Figure
1. 1.The
arginines, asymmetric
asymmetric
dimethylarginine
(ADMA
-NMMA 2),
2), and
and symmetric
symmetric dimethylarginine
dimethylarginine (SDMA
(SDMA 3)
3) act
act as
as direct
direct or
or
monomethylarginine ((LL-NMMA
monomethylarginine
monomethylarginine (L-NMMA 2), and symmetric dimethylarginine (SDMA 3) act as direct or indirect
L
-NMMA
(2)
are
substrates
for
indirect
inhibitors
of
the
NOS
family
of
enzymes.
ADMA
(1)
and
indirect inhibitors of the NOS family of enzymes. ADMA (1) and L-NMMA (2) are substrates for
inhibitors
of the NOS family of enzymes. ADMA (1) and L-NMMA (2) are substrates for human DDAH-1.
human DDAH-1.
human DDAH-1.
Scheme 1. ADMA and L-NMMA are substrates for DDAH-1 and are converted to L-citrulline (4) and
Scheme 1. ADMA and L-NMMA are substrates for DDAH-1 and are converted to L-citrulline (4) and
Scheme
1. ADMA and L-NMMA are substrates for DDAH-1 and are converted to L-citrulline (4) and
an amine
amine species
species 55 or
or 6.
6.
an
an amine species 5 or 6.
The role
role of
of ADMA
ADMA accumulation
accumulation and
and impaired
impaired DDAH
DDAH expression
expression and/or
and/or activity
activity in
in the
the
The
pathophysiology
of several
several
diseases is
is well
well
documented
[36–44].
Increased
plasma
or serum
serum
ADMA
The
role of ADMA
accumulation
anddocumented
impaired [36–44].
DDAHIncreased
expression
and/or
activity
in the
pathophysiology
of
diseases
plasma
or
ADMA
concentrations
have
been
associated
with
coronary
events
[45–47],
renal
disease
[29,37],
atherosclerosis
concentrations
have
been
associated
with
coronary
events
[45–47],
renal
disease
[29,37],
atherosclerosis
pathophysiology of several diseases is well documented [36–44]. Increased plasma or serum
[48],concentrations
and cerebrovascular
cerebrovascular
disease
[49,50]. Up-regulation
Up-regulation
of DDAH
DDAH
expression
and
activity
might,
[48],
and
[49,50].
of
expression
activity
might,
ADMA
havedisease
been associated
with coronary
events
[45–47],and
renal
disease
[29,37],
therefore,
represent
a
good
strategy
in
the
treatment
of
the
aforementioned
pathologies
to
increase
therefore, represent
a good
strategy in the
treatment
of the Up-regulation
aforementioned of
pathologies
to increase and
atherosclerosis
[48], and
cerebrovascular
disease
[49,50].
DDAH expression
ADMA metabolism
metabolism and lower
lower its plasma concentrations.
concentrations.
ADMA
activity
might, therefore,and
representitsaplasma
good strategy in the treatment of the aforementioned pathologies
In
contrast,
reduced
ADMA
concentrations
have been
been demonstrated
demonstrated in
in other
other disease
disease states,
states, such
such as
as
In contrast, reduced ADMA concentrations have
to increase
ADMA
metabolism
and
lower
its sclerosis
plasma [43],
concentrations.
amyotrophic
lateral
sclerosis
[51],
multiple
Alzheimer’s
disease
and
dementia
[52,53].
amyotrophic lateral sclerosis [51], multiple sclerosis [43], Alzheimer’s disease and dementia [52,53].
In
contrast,
reduced
ADMA
have been
demonstrated
in
other disease
states,
ADMA
has also
also
been shown
shown to
toconcentrations
have neuroprotective
neuroprotective
effects
in aa model
model of
of Parkinson’s
Parkinson’s
disease
[54]. such
ADMA
has
been
have
effects
in
disease
[54].
as amyotrophic lateral sclerosis [51], multiple sclerosis [43], Alzheimer’s disease and dementia [52,53].
Molecules 2016, 21, 615
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ADMA has also been shown to have neuroprotective effects in a model of Parkinson’s disease [54].
Furthermore, DDAH overexpression in tumours enhances their metastatic potential [42,55,56]. Several
reports also show that inhibition of NO synthesis results in significant anti-angiogenic effects both
in vitro and in vivo, with potential beneficial effects in cancer [57–61]. Moreover, excessive NO
concentrations play a key role in the pathophysiology of septic shock [62–65] (an early therapeutic
approach for the treatment of septic shock involved the administration of 546C88 (L-NMMA) to inhibit
NOS and reduce NO synthesis, however the increase in mortality in patients receiving L-NMMA caused
the termination of the clinical trial in phase III [66]. Further analysis of the data revealed that mortality
was associated with elevated L-NMMA concentrations; conversely, lower L-NMMA concentrations
were associated with improved survival [36]. In this context a more controlled, indirect modulation of
methylated arginine concentrations through pharmacological DDAH-1 inhibition (DDAH-2 knockout
affects outcome in models of polymicrobial sepsis [44]) might represent an alternative strategy, and
appears particularly promising in a rat model of septic shock [67]). In these circumstances, DDAH
inhibition could exert a controlled inhibition of the NO synthesis by increasing ADMA concentrations
and offering a new approach in the treatment of these diseases.
Therefore, alterations in DDAH and NOS activity might exert either beneficial or toxic effects,
according to specific experimental models and disease conditions. The potential of DDAH as a
molecular target in the treatment of these pathological conditions has led several groups to study
the endogenous mechanisms involved in the modulation of the expression and activity of human
DDAH-1 and DDAH-2 [68]. The elucidation of these mechanisms may identify new strategies for the
pharmacological activation or inhibition of these enzymes to restore physiological homeostasis after
pathological imbalance. Since the discovery of the DDAH family of enzymes in 1987 [35], a number of
research groups have identified inhibitors with the aim of developing such therapeutics. While no
‘selective’ DDAH inhibitor has entered clinical trials, numerous compounds have been synthesized
that act as prototypic in vitro inhibitors. Herein, we discuss all published DDAH inhibitors according
to their chemical structure (‘substrate-like’ and ‘non-substrate-like’), and summarize the details of
their synthetic pathways as well as how they alter the pharmacokinetic parameters for L-citrulline
formation by DDAH.
2. Endogenous Modulation of DDAH Activity
The production of specific gene products, both in terms of RNA and proteins, is physiologically
regulated by a plethora of mechanisms and can be triggered or silenced by various stimuli and factors.
Of particular note, DDAH activity is reported to be inhibited by divalent transition metals [69,70]
and it is known that elevated zinc(II) concentrations are associated with oxidative stress in numerous
pathological conditions [71–73].
S-Nitrosylation of the enzyme’s cysteine residues is a further important post-translational
regulatory mechanism [74] and is generally associated with increased expression of inducible NOS
(iNOS). The induction of iNOS generates excessive NO that can bind covalently (but reversibly) to the
thiol (SH) moiety of each DDAH cysteine [74]. The apparent S-nitrosylation of the catalytic DDAH
cysteine residue results in DDAH inhibition. This in turn causes the subsequent accumulation of
DDAH substrates, ADMA and L-NMMA, which eventually reach concentrations that reversibly inhibit
the NOS enzymes [75].
Oxidative stress additionally modulates DDAH activity. Factors associated with oxidative stress,
including shear stress [76], tumour necrosis factor α (TNF-α), oxidized low density lipoprotein
(LDL) [76], and homocysteine [77] each suppresses DDAH expression and activity. In contrast,
antioxidant mediators such as probucol [78], estradiol [79], and interleukin-1β (IL-1β) [80] up-regulate
DDAH expression. Moreover, nicotine and cigarette smoking have been shown to decrease DDAH
expression and activity in endothelial cells, with consequential accumulation of ADMA, NOS
inhibition, and endothelial damage [81,82]. Furthermore, it is known that selective down-regulation of
DDAH-1 and up-regulation of DDAH-2 occurs when angiotensin II binds to the type 1 angiotensin
receptor (AT1 -R), linking DDAH transcription to angiotensin-induced inflammation [8]. In addition,
epigenetic modifications are known to alter DDAH-2 regulation at the promoter. For example, DNA
Molecules 2016, 21, 615
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hypermethylation has the ability to suppress DDAH-2 transcription, while histone acetylation has the
ability to up-regulate DDAH-2 transcription [83].
Molecules 2016, 21, 615
4 of 31
3. Pharmacological Induction of DDAH
hypermethylation has the ability to suppress DDAH-2 transcription, while histone acetylation has
As
resulttoofup-regulate
diminishedDDAH-2
DDAH transcription
activity and/or
theaability
[83].accumulation of ADMA and L-NMMA in various
disease states, several studies have investigated the effects of restoring or increasing DDAH expression.
Pharmacological
Induction
of DDAH
Statin3.treatment
is associated
with
a significant reduction in ADMA concentrations [84–86], and an
increase in
DDAH-1
and DDAH-2
expression
and activity
[87–89].
class
of compounds
Asboth
a result
of diminished
DDAH
activity and/or
accumulation
of Another
ADMA and
L-NMMA
in
various
disease
states,
several
studies
have
investigated
the
effects
of
restoring
or
increasing
DDAH
enhancing ADMA metabolism via DDAH up-regulation is represented by the farnesoid X receptor
treatment
is associated
with
a significant
in ADMA
concentrations
(FXR)expression.
agonists.Statin
GW4064
upregulates
both
DDAH-1
andreduction
the cationic
transporter
CAT-1,[84–86],
leading to
and an
increase
in both
DDAH-1
DDAH-2 and
expression
and serum
activityADMA
[87–89].concentrations
Another class of
increased
ADMA
cellular
uptake
and and
metabolism,
decreased
[15,90].
compounds
enhancing
ADMA
metabolism
via
DDAH
up-regulation
is
represented
by
the
farnesoid
Another FXR agonist, INT-747, can increase DDAH-1 expression [91,92], however this is not associated
X receptor (FXR) agonists. GW4064 upregulates both DDAH-1 and the cationic transporter CAT-1,
with significant changes in ADMA and NO concentrations [91]. The lipid lowering drug probucol
leading to increased ADMA cellular uptake and metabolism, and decreased serum ADMA
also lowers ADMA concentrations [93] via up-regulation of both DDAH expression and activity, and
concentrations [15,90]. Another FXR agonist, INT-747, can increase DDAH-1 expression [91,92],
down-regulation
ofnot
PRMT-1
expression
[78,94]. changes
Anotherinlipid
lowering
fenofibrate,
however this is
associated
with significant
ADMA
and NOdrug,
concentrations
[91].decreases
The
ADMA
concentrations
by
restoring
DDAH
activity
in
conditions
of
oxidative
stress
[95].
lipid lowering drug probucol also lowers ADMA concentrations [93] via up-regulation of bothAdditionally,
DDAH
the oral
antidiabetic
drug pioglitazone
and theofβPRMT-1
blocker[78,94].
nebivolol
both
decrease
serum
expression
and activity,
and down-regulation
expression
Another
lipid
lowering
1 receptor
drug,
fenofibrate, decreases
ADMA
concentrations
by restoring
ADMA
concentrations
by increasing
DDAH-2
expression
[96–99]. DDAH activity in conditions of
oxidative stress [95]. Additionally, the oral antidiabetic drug pioglitazone and the β1 receptor blocker
4. Pharmacological
Inhibition
DDAH
nebivolol both decrease
serumofADMA
concentrations by increasing DDAH-2 expression [96–99].
Contrary
to the comments
inof
Section
4. Pharmacological
Inhibition
DDAH3 above, there is a growing body of evidence to suggest that
the modulation of DDAH enzyme activity may be utilized in conditions characterized by excessive
Contrary to the comments in Section 3 above, there is a growing body of evidence to suggest
NO production [36,67]. The remainder of this review will focus on highlighting the current knowledge
that the modulation of DDAH enzyme activity may be utilized in conditions characterized by
associated with the main DDAH inhibitors reported in the literature to date.
excessive NO production [36,67]. The remainder of this review will focus on highlighting the current
knowledge associated with the main DDAH inhibitors reported in the literature to date.
4.1. Substrate-Like Inhibitors
4.1. Substrate-Like Inhibitors
4.1.1. S-Nitroso-L-Homocysteine (HcyNO)
4.1.1. S-NitrosoL-Homocysteine
(HcyNO)
Since
the identification
of S-nitrosylation
as an important mechanism of DDAH regulation,
the discovery
ofidentification
L -homocysteine’s
ability as
toaninhibit
DDAH-1
ledof to
the regulation,
development
Since the
of S-nitrosylation
important
mechanism
DDAH
the of
S-nitrosoL -homocysteine
(HcyNO,
9), an
endogenous
S-nitrosothiol
and a source
of endogenous
discovery
of L-homocysteine’s
ability
to inhibit
DDAH-1
led to the development
of S-nitrosoLhomocysteine
(HcyNO,
9), an
endogenous S-nitrosothiol
source ofDDAH
endogenous
NO [100–102].
NO [100–102].
Synthesis
and
characterisation
of HcyNOand
as aa bovine
inhibitor
was reported
Synthesis
characterisation
of HcyNO
as a bovineinDDAH
inhibitor wasyield
reported
Knipp etreaction
al.
by Knipp
et al.and
(2005)
[102]. HcyNO
was synthesized
near quantitative
in aby
two-step
(2005)
[102].
HcyNO
was
synthesized
in
near
quantitative
yield
in
a
two-step
reaction
(Scheme
2)
(Scheme 2) via alkaline hydrolysis of commercially available L-homocysteine thiolactone hydrochloride
via alkaline hydrolysis of commercially available L-homocysteine thiolactone hydrochloride (7) to
(7) to L-homocysteine (8) [103], followed by S-nitrosylation of L-homocysteine with sodium nitrite
L-homocysteine (8) [103], followed by S-nitrosylation of L-homocysteine with sodium nitrite (NaNO2)
(NaNO2 ) and acidification to afford HcyNO (9) [104]. Alternatively, S-nitrosylation can be undertaken
and acidification to afford HcyNO (9) [104]. Alternatively, S-nitrosylation can be undertaken directly
directly
on commercially
available
L -homocysteine (8). Mild reaction conditions are required, and
on commercially
available
L-homocysteine (8). Mild reaction conditions are required, and excellent
excellent
yields
HcyNO
to prepare
a large scale.
yields
makemake
HcyNO
simple simple
to prepare
on a largeon
scale.
Scheme 2. Synthetic scheme for generating HcyNO (9). Reagents and Conditions: (i) 5 M NaOH, 37 °C, ˝
Scheme
2. Synthetic scheme for generating HcyNO (9). Reagents and Conditions: (i) 5 M NaOH, 37 C,
5 min, 96%; (ii) NaNO2, 2 M HCl, 37˝°C, 15 min, >99%.
5 min, 96%; (ii) NaNO2 , 2 M HCl, 37 C, 15 min, >99%.
HcyNO irreversibly inhibits DDAH by covalently binding to the putative catalytic cysteine
HcyNO
irreversibly
inhibits
by[105].
covalently
binding to the
putative
catalytic
with an inhibitory
constant
(Ki) DDAH
of 690 μM
Mass spectrometry
analyses
using
differentcysteine
isotopic with
15NO and HcyN18O) and oxygen-18 labelled water (H218O)
forms of S-nitroso(Hcy
an inhibitory
constantL-homocysteine
(Ki ) of 690 µM
[105].
Mass spectrometry analyses using different isotopic
15 NO and HcyN
18 O) with
HcyNO forms (Hcy
a N-thiosulfoximide
adduct
cysteine 273labelled
(Cys273)water
of bovine
formsdemonstrated
of S-nitroso-that
L -homocysteine
and oxygen-18
(H2 18 O)
DDAH-1.
The
authors’
proposed
mechanism
of
inhibition
requires
the
lone
pair
of
electrons
of
the
demonstrated that HcyNO forms a N-thiosulfoximide adduct with cysteine 273 (Cys273) of bovine
Molecules 2016, 21, 615
5 of 32
Molecules
2016,authors’
21, 615
5 of 31
DDAH-1.
The
proposed mechanism of inhibition requires the lone pair of electrons
of the
Cys273 SH to attack the S-nitroso group in HcyNO to eliminate a hydroxyl anion (Scheme 3). This
Cys273
the S-nitroso group in HcyNO to eliminate a hydroxyl anion (Scheme 3).5This
Molecules SH
2016,to
21,attack
615
of 31
mechanism
is
likely
stabilized by neighbouring amino acids, namely His172. A further hydroxyl
anion
mechanism is likely stabilized by neighbouring amino acids, namely His172. A further hydroxyl anion
originating
from
water
attacks
the
cationic
sulfur
of
the
conjugated
intermediate,
and
subsequently
originating
water
the group
cationic
of to
theeliminate
conjugated
intermediate,
subsequently
Cys273 SH from
to attack
theattacks
S-nitroso
insulfur
HcyNO
a hydroxyl
anionand
(Scheme
3). This
rearranges
to give
the the
covalent
thiosulfoximide
adduct
[102].
rearranges
to
covalent
adduct
[102].
mechanism
is give
likely
stabilized
bythiosulfoximide
neighbouring amino
acids,
namely His172. A further hydroxyl anion
originating from water attacks the cationic sulfur of the conjugated intermediate, and subsequently
rearranges to give the covalent thiosulfoximide adduct [102].
Scheme 3. Schematic of the reaction mechanism proposed by Knipp et al. [102] for the covalent
Scheme 3. Schematic of the reaction mechanism proposed by Knipp et al. [102] for the covalent
inhibition of DDAH-1 by HcyNO (9) at Cys273. Adapted with permission from [102]. Copyright 2005
inhibition
of DDAH-1 by
HcyNO (9) at Cys273. Adapted with permission from [102]. Copyright 2005
American
Society.
Scheme 3.Chemical
Schematic
of the reaction mechanism proposed by Knipp et al. [102] for the covalent
American Chemical Society.
inhibition of DDAH-1 by HcyNO (9) at Cys273. Adapted with permission from [102]. Copyright 2005
4.1.2.American
2-Chloroacetamidine
and N-But-3-ynyl-2-chloroacetamidine
Chemical Society.
4.1.2. 2-Chloroacetamidine
and N-But-3-ynyl-2-chloroacetamidine
Stone et al. [106] identified 2-chloroacetamidine (11) as a nonspecific inhibitor of two enzymes of
4.1.2. 2-Chloroacetamidine and N-But-3-ynyl-2-chloroacetamidine
the amidinotransferases
superfamily,
Pseudomonas (11)
aeruginosa
DDAH (PaDDAH)
human
Stone
et al. [106] identified
2-chloroacetamidine
as a nonspecific
inhibitor and
of two
enzymes
peptydilarginine
deaminase
(PAD).
2-Chloroacetamidine
(11,
SchemeDDAH
4) inhibitor
is a commercially
available
Stone et al. [106]
identified
2-chloroacetamidine
(11) as
a nonspecific
of two enzymes
of
of the
amidinotransferases
superfamily,
Pseudomonas
aeruginosa
(PaDDAH)
and human
compound
bearing a 2-chloromethylene
chain and an aeruginosa
amidinium DDAH
group, which
partially
resembles
the
amidinotransferases
superfamily,
Pseudomonas
(PaDDAH)
and
human
peptydilarginine deaminase (PAD). 2-Chloroacetamidine (11, Scheme 4) is a commercially available
arginine.
It can bedeaminase
synthesized(PAD).
via reaction
of chloroacetonitrile
(10) with4)sodium
methoxide available
followed
peptydilarginine
2-Chloroacetamidine
(11, Scheme
is a commercially
compound bearing a 2-chloromethylene chain and an amidinium group, which partially resembles
by
treatment
with ammonium
chloride [107,108].
as apartially
weak irreversible
compound
bearing
a 2-chloromethylene
chain and 2-Chloroacetamidine
an amidinium group,acts
which
resembles
arginine.
It can be synthesized
reaction
chloroacetonitrile
(10) with to
sodium
methoxide
followed
amidinotransferase
inhibitorvia
with
greaterof
affinity
for PaDDAH,
PAD4
(Table followed
1). Data
arginine. It can be synthesized
via reaction
of
chloroacetonitrile
(10)relative
with sodium
methoxide
by treatment
with
ammonium
chloride
[107,108].
2-Chloroacetamidine
acts
as
a
weak
irreversible
acquired
by electrospray
ionization
mass
spectrometry
revealed the formation
an irreversible
by treatment
with ammonium
chloride
[107,108].
2-Chloroacetamidine
acts as a of
weak
irreversible
amidinotransferase
inhibitor
with
affinity
for PaDDAH,
PaDDAH,
relative
PAD4
(Table
1). Data
thiouronium-enzyme
adduct
whereby
the loss
of chlorine
from therelative
inhibitor
observed
[106]
amidinotransferase
inhibitor
withgreater
greater
affinity
for
to isto
PAD4
(Table
1).. Data
acquired
by electrospray
ionization
revealedthe
theformation
formation
of irreversible
an irreversible
acquired
by electrospray
ionizationmass
massspectrometry
spectrometry revealed
of an
thiouronium-enzyme
adduct
whereby
fromthe
theinhibitor
inhibitor
is observed
[106].
.
thiouronium-enzyme
adduct
wherebythe
theloss
lossof
ofchlorine
chlorine from
is observed
[106]
Scheme 4. Synthetic scheme for generating 2-chloroacetamidine (11) (the conditions described afford
the hydrochloride salt). Reagents and Conditions: (i) NaOMe, dry MeOH, overnight; (ii) AcOH, NH4Cl,
reflux,
h, Synthetic
89%.
Scheme6 4.
scheme for generating 2-chloroacetamidine (11) (the conditions described afford
Scheme 4. Synthetic scheme for generating 2-chloroacetamidine (11) (the conditions described afford
the hydrochloride salt). Reagents and Conditions: (i) NaOMe, dry MeOH, overnight; (ii) AcOH, NH4Cl,
the hydrochlorideTable
salt).1.Reagents
Conditions:
(i) NaOMe,
dry MeOH, overnight;
(ii) AcOH, NH4 Cl,
Reportedand
inhibitory
constants
for 2-chloroacetamidine
(11) [106].
reflux, 6 h, 89%.
reflux, 6 h, 89%.
Enzyme
Ki (mM)
Kinact (min−1)
Table 1. Reported inhibitory constants for 2-chloroacetamidine (11) [106].
PaDDAH
3.1
±
0.8
1.2 ± 0.1
Table 1. Reported inhibitory constants for 2-chloroacetamidine
(11) [106].
PAD4
±5
0.7 (min
± 0.1 −1)
Enzyme
K20
i (mM)
Kinact
PaDDAH
3.1 (mM)
± 0.8
1.2
± 0.1(min´1 )
Enzyme
K
Kinact
i
The addition of an N-but-3-ynyl
PAD4 substituent
20 ±to5 the acetamidine
0.7 ± 0.1 group of 2-chloroacetamidine
PaDDAH
3.1 ˘
0.8
1.2 ˘ 0.1
led to formation of the human
DDAH-1 probe,
N-but-3-ynyl-2-chloro-acetamidine
(18, Scheme 5)
PAD4
20 ˘ 5
0.7 ˘ 0.1
[109].The
N-But-3-ynyl-2-chloro-acetamidine
binds within
putative active-site,
while the N-alkynyl
addition of an N-but-3-ynyl substituent
to thethe
acetamidine
group of 2-chloroacetamidine
substituent
is reacted
with
biotin-PEO
3-azide
under
copper catalysis via click chemistry.
led to formation
of the
human
DDAH-1
probe,
N-but-3-ynyl-2-chloro-acetamidine
(18, Successful
Scheme 5)
conjugation
can
detected in
immunoblots
using
anti-biotin
antibody
[109].the
Synthesis
of led
[109].addition
N-But-3-ynyl-2-chloro-acetamidine
binds to
within
theanputative
active-site,
while
N-alkynyl
The
ofbe
aneasily
N-but-3-ynyl
substituent
the
acetamidine
group
of 2-chloroacetamidine
the
N-but-3-ynyl-2-chloro-acetamidine
probe
was
achieved
in five
steps via
using
twochemistry.
modules
(Scheme
5).[109].
substituent
is reacted
with
biotin-PEO
3-azide
under
copper
catalysis
click
Successful
to formation
of the
human
DDAH-1
probe,
N-but-3-ynyl-2-chloro-acetamidine
(18, Scheme
5)
Amine
module
15 easily
was prepared
inimmunoblots
threewithin
steps: using
activation
of the
terminal
group
of
conjugation
can be
detected in
an anti-biotin
antibodyhydroxyl
[109]. Synthesis
N-But-3-ynyl-2-chloro-acetamidine
binds
the
putative
active-site,
while
the
N-alkynyl
3-butyn-1-ol
(12) through mesylation probe
to afford
13
in 62% in
yield,
followed
bytwo
conversion
to
amine 15
the
N-but-3-ynyl-2-chloro-acetamidine
was
achieved
five
steps
using
modules
(Scheme
5).
substituent is reacted with biotin-PEO3 -azide under copper catalysis via click chemistry. Successful
Amine module 15 was prepared in three steps: activation of the terminal hydroxyl group of
conjugation can be easily detected in immunoblots using an anti-biotin antibody [109]. Synthesis of the
3-butyn-1-ol (12) through mesylation to afford 13 in 62% yield, followed by conversion to amine 15
N-but-3-ynyl-2-chloro-acetamidine probe was achieved in five steps using two modules (Scheme 5).
Molecules 2016, 21, 615
6 of 32
Amine module 15 was prepared in three steps: activation of the terminal hydroxyl group of 3-butyn-1-ol
Molecules 2016,
21, 615
of 31azide
(12) through
mesylation
to afford 13 in 62% yield, followed by conversion to amine 15 6via
intermediate 14 in 49% yield across two steps. The imidic ester module 17 was obtained in moderate
via azide2016,
intermediate
14 in 49% yield across two steps. The imidic ester module 17 was obtained
21, 615 48%)
6 of in
31
yieldsMolecules
(approximately
by reacting chloroacetonitrile (10) with anhydrous ethanol (16)
in the
moderate yields (approximately 48%) by reacting chloroacetonitrile (10) with anhydrous ethanol (16)
presence
of
dry
hydrochloric
acid
gas.
Final
coupling
between
amine
15
and
the
imidic
ester
17
in
presence
of dry hydrochloric
acid
gas. two
Finalsteps.
coupling
between
amine
15 and
the imidic
ester
viathe
azide
intermediate
14 in 49% yield
across
The imidic
ester
module
17 was
obtained
in
proceeds
under
basic
aqueous
conditions
to afford
probe
N-but-3-ynyl-2-chloro-acetamidine
(18) in
17
proceeds
under
basic aqueous
conditions
to afford
probe
N-but-3-ynyl-2-chloro-acetamidine
moderate
yields
(approximately
48%)
by reacting
chloroacetonitrile
(10) with anhydrous ethanol (18)
(16)
reasonable
yields
(approximately
63%).
To
date,
no
data
are
available
regarding
the
selectivity
of
in reasonable
yields
(approximately
63%).gas.
To Final
date, no
data are
available
regarding
of
the presence
of dry
hydrochloric acid
coupling
between
amine
15 andthe
theselectivity
imidic ester
N-but-3-ynyl-2-chloro-acetamidine
human
DDAH-1
and
the
potential
N-but-3-ynyl-2-chloro-acetamidine
for
human
DDAH-1
and
the
potentialcross
crossreactivity
reactivityofofthis
thisprobe
17 proceeds under basic aqueousfor
conditions
to
afford
probe
N-but-3-ynyl-2-chloro-acetamidine
(18)
with PaDDAH.
with probe
PaDDAH.
in
reasonable
yields (approximately 63%). To date, no data are available regarding the selectivity of
N-but-3-ynyl-2-chloro-acetamidine for human DDAH-1 and the potential cross reactivity of this
NH.HCl
probe with PaDDAH.
(iv)
Cl
Cl
N + HO
16
10
Cl
(iv)
N + HO
16
10
O
17
NH.HCl
Cl
O
HCl.H2N
HO
17
Cl
N
H
NH
18
N
H
18
HCl.H2N
(i)
12
MsO
(v)
NH
Cl
15
12
HO
(v)
(i)
13
15(iii)
(ii)
(iii)
N3
(ii)
14
Scheme 5. SyntheticMsO
scheme for generating Nthe
DDAH-1 biochemical probe N-but-3-ynyl-2-chloro3
Scheme
5. Synthetic scheme
for
generating the
DDAH-1
biochemical probe N-but-3-ynyl-2-chloro13 Conditions: (i) DCM,14
acetamidine (18). Reagents and
TEA, DMAP, MsCl, 0 °C for 2 h then 25 °C for 3 h,
acetamidine (18). Reagents and Conditions: (i) DCM, TEA, DMAP, MsCl, 0 ˝ C for 2 h then 25 ˝ C for 3 h,
°C, 3.5 h; the
(iii) Et2O, Ph3biochemical
P, 0 °C for 2 h, addition
of H2O, then 25 °C ˝
62%; (ii) anhydrous
NaN3, 70
5. SyntheticDMF,
N-but-3-ynyl-2-chloro˝ C,
62%; Scheme
(ii) anhydrous
DMF,scheme
NaN3 ,for
70 generating
3.5 h; (iii) DDAH-1
Et2 O, Ph3 P, 0 ˝ C for 2probe
h, addition
of H2 O, then 25 C
0 °C
for 1TEA,
h, stop
HCl(g)MsCl,
, then 00 °C
°C for
for 24 h
h then
and RT
overnight
for
20 h, 49%(18).
(twoReagents
steps); (iv)
HCl(g) at(i)
acetamidine
anddry
Conditions:
DCM,
DMAP,
25 °C
for 3 h,
˝ C for 1 h, stop HCl , then 0 ˝ C for 4 h and
for 2048%–76%;
h, 49% (two
steps);
(iv)
dry
HCl
at
0
RT overnight
(g)3 h, 25 °C for 2 h, neutralized,
(g) RT for 1.5 days, 62%. Adapted
2
O,
pH
=
10,
0
°C
for
(v)
H
3, 70 °C, 3.5 h; (iii) Et2O, Ph3P, 0 °C for 2 h, addition of H2O, then 25 °C
62%; (ii) anhydrous DMF, NaN
˝ C for 2 h, neutralized, RT for 1.5 days, 62%. Adapted
48%–76%;
(v) H O, from
pH =[109].
10, 0 ˝ C for 3 h,
25American
with
Chemical
at 0 °C
for 1 h, stop
HCl(g)Society.
, then 0 °C for 4 h and RT overnight
for 20permission
h, 49%2(two
steps); (iv)Copyright
dry HCl(g)2009
with 48%–76%;
permission
Copyright
American
Society.
pH = 10,
0 °C for 2009
3 h, 25
°C for 2 h,Chemical
neutralized,
RT for 1.5 days, 62%. Adapted
(v)from
H2O,[109].
4.1.3.with
(S)-2-Amino-4-(3-Methylguanidino)butanoic
Acid
Analogues
permission from [109]. Copyright 2009 American Chemical Society.
4.1.3. (S)-2-Amino-4-(3-Methylguanidino)butanoic
Acid
Screening experiments with a library comprising
26Analogues
analogues of ADMA (1) and L-NMMA (2)
4.1.3. (S)-2-Amino-4-(3-Methylguanidino)butanoic Acid Analogues
identified (S)-2-amino-4-(3-methylguanidino)butanoic
(4124W, 21),ofasADMA
a mammalian
Screening
experiments with a library comprisingacid
26 analogues
(1) andDDAH
L -NMMA
inhibitor
[110].
The
original
synthesis
of
4124W
(21),
first
described
by
Ueda
et
al.
inL-NMMA
2003 [80](2)
is
Screening
experiments
with
a
library
comprising
26
analogues
of
ADMA
(1)
and
(2) identified (S)-2-amino-4-(3-methylguanidino)butanoic acid (4124W, 21), as a mammalian DDAH
shown
in
Scheme
6,
and
was
achieved
by
coupling
N,S-dimethylthiopseudouronium
iodide
(19)
identified (S)-2-amino-4-(3-methylguanidino)butanoic acid (4124W, 21), as a mammalian DDAH
inhibitor
[110]. The original synthesis of 4124W (21), first described by Ueda et al. in 2003 [80] is shown
with
the copper
complex
of synthesis
2,4-diamino-n-butyric
acid
dihydrochloride
(20) [32,111].
4124W
(21)
inhibitor
[110]. The
original
of 4124W (21),
first
described by Ueda
et al. in 2003
[80]
is
in Scheme
6,
and
was
achieved
by
coupling
N,S-dimethylthiopseudouronium
iodide
(19)
with
the
was
finally
crystallized
as the
salt.
shown
in Scheme
6, and
washydrochloride
achieved by coupling
N,S-dimethylthiopseudouronium iodide (19)
copper
complex of 2,4-diamino-n-butyric acid dihydrochloride (20) [32,111]. 4124W (21) was finally
with the copper complex of 2,4-diamino-n-butyric acid dihydrochloride (20) [32,111]. 4124W (21)
crystallized
as the
hydrochloride
salt.
was finally
crystallized
as the hydrochloride
salt.
Scheme 6. Synthetic scheme for generating (S)-2-amino-4-(3-methylguanidino)butanoic acid (4124W), as
reported by Ueda et al. [80] using the methods of Ogawa et al. [32] and Corbin et al. [111]. Reagents and
Conditions:
(i) (1) 2,4-diamino-n-butyric
acid
dihydrochloride (20), copper acetate, 1:1
NH(4124W),
4OH/H2O;
Scheme 6. Synthetic
scheme for generating
(S)-2-amino-4-(3-methylguanidino)butanoic
acid
as
Scheme
6. Synthetic scheme for generating
(S)-2-amino-4-(3-methylguanidino)butanoic
acid (4124W),
4+ [111].
form),Reagents
4124W (21)
(2)
N,S-dimethylthiopseudouronium
iodide (19)
24 h;et(3)
reported
by Ueda et al. [80] using the methods
of RT,
Ogawa
al.Dowex
[32] and50WX4
Corbin(NH
et al.
and
as reported
by Ueda as
et al.
using the methods
of Ogawa et al. [32] and Corbin et al. [111]. Reagents and
was
crystallized
the[80]
hydrochloride
salt.
Conditions:
(i) (1) 2,4-diamino-n-butyric
acid dihydrochloride (20), copper acetate, 1:1 NH4OH/H2O;
Conditions: (i) (1) 2,4-diamino-n-butyric acid dihydrochloride (20), copper acetate,
1:1 NH4 OH/H2 O;
(21)
(2) N,S-dimethylthiopseudouronium iodide (19) RT, 24 h; (3) Dowex 50WX4 (NH4+ form), 4124W
+ form), 4124W (21)
(2) N,S-dimethylthiopseudouronium
iodide
(19)
RT,
24
h;
(3)
Dowex
50WX4
(NH
High
chemical
and
structural
similarity
between
4124W
and
the
DDAH
substrate
L-NMMA exists
4
was crystallized as the hydrochloride salt.
(compound
21, Scheme
6, and compound
was
crystallized
as the hydrochloride
salt.2, Figure 1), and although exhibiting weak DDAH inhibition
(IC50 High
= 416 chemical
μM) [110],
this
prompted
Rossiterbetween
et al. [112]
to synthesize
a further
34 compounds
and
structural
similarity
4124W
and the DDAH
substrate
L-NMMAbased
exists
on
the
4124W
structure.
Furthermore,
Rossiter
et
al.
[112]
reported
an
alternative
synthetic
approach,
(compound
21,
Scheme
6,
and
compound
2,
Figure
1),
and
although
exhibiting
weak
DDAH
inhibition
High chemical and structural similarity between 4124W and the DDAH substrate L-NMMA exists
which
although
requiring
three steps,
is synthetically
versatile
and awas
applied
to developbased
their
(IC50 = 416
[110],
Rossiter
et al. 1),
[112]
to although
synthesize
further
34weak
compounds
(compound
21, μM)
Scheme
6,this
andprompted
compound
2, Figure
and
exhibiting
DDAH inhibition
library
of 4124W
analogues.
Rossiter’s strategy
protected
intermediates
to avoid
complications
on the 4124W
structure.
Furthermore,
Rossiterutilized
et al. [112]
reported
an alternative
synthetic
approach,
(IC50 associated
= 416 µM)with
[110],
this
prompted
Rossiter et al.
[112] to synthesize
a further
34 compounds
based
the
purification
polarisamino
acids. Intermediate
24 was
(Scheme
7) istogenerated
which although
requiring
threeofsteps,
synthetically
versatile and
applied
develop from
their
on the
4124W structure.
Furthermore,
Rossiter et al. [112] reported an alternative
approach,
commercially
available
N,N′-bis-tert-butoxycarbonylpyrazole-1H-carbox-amidine
(22)synthetic
by
Mitsunobu
library of 4124W
analogues.
Rossiter’s strategy utilized protected intermediates to avoid
complications
which
although
requiring
three steps,
is amino
synthetically
versatile and
was applied
to develop
associated
with
the purification
of polar
acids. Intermediate
24 (Scheme
7) is generated
from their
library
of 4124W analogues.
Rossiter’s strategy utilized protected intermediates to
avoid
complications
commercially
available N,N′-bis-tert-butoxycarbonylpyrazole-1H-carbox-amidine
(22)
by Mitsunobu
associated with the purification of polar amino acids. Intermediate 24 (Scheme 7) is generated from
Molecules 2016, 21, 615
7 of 32
Molecules 2016,
21, 615
7 of 31
commercially
available
N,N1 -bis-tert-butoxycarbonylpyrazole-1H-carbox-amidine (22) by Mitsunobu
reaction with alcohol 23 containing the desired R1 group, followed by reaction with (S)-tert-butyl
reaction
with21,alcohol
23 containing the desired R1 group, followed by reaction with (S)-tert-butyl
Molecules 2016,
615
7 of 31
4-amino-2-((tert-butoxycarbonyl)amino)butanoate (25) to afford the protected amino acid analogue 26
4-amino-2-((tert-butoxycarbonyl)amino)butanoate (25) to afford the protected amino acid analogue
(Scheme
7). Cleavage
of the
protecting
isR1achieved
with a 4by
M solutionwith
of HCl
in 1,4-dioxane
reaction
with
23Boc
containing
the groups
desiredgroups
group,
followed
(S)-tert-butyl
26
(Scheme
7).alcohol
Cleavage
of
the Boc protecting
is achieved
withreaction
a 4 M solution
of HCl in
and produced
4124W
(21)
in
moderate
yields
(approximately
44%)
or
analogues
27
in
moderate
4-amino-2-((tert-butoxycarbonyl)amino)butanoate
to afford
the protected
amino
acid analogue
1,4-dioxane
and produced 4124W (21) in moderate(25)
yields
(approximately
44%)
or analogues
27 in to
goodmoderate
yields
(12%–67%).
26
(Scheme
7). Cleavage
of the Boc protecting groups is achieved with a 4 M solution of HCl in
to good
yields (12%–67%).
1,4-dioxane and produced 4124W (21) in moderate yields (approximately 44%) or analogues 27 in
moderate to good yields (12%–67%).
Scheme 7. Synthetic scheme for generating (S)-2-amino-4-(3-methylguanidino)butanoic acid analogues.
Scheme 7. Synthetic scheme for generating (S)-2-amino-4-(3-methylguanidino)butanoic acid analogues.
Reagents and Conditions: (i) DEAD, PPh3, THF, 0 °C-RT, 3–16 h; (ii) DIPEA, CH3CN, RT, 24 h; (iii) 4M
Reagents and Conditions: (i) DEAD, PPh3 , THF, 0 ˝ C-RT, 3–16 h; (ii) DIPEA, CH3 CN, RT, 24 h; (iii)
Scheme 7. Synthetic
generating
(S)-2-amino-4-(3-methylguanidino)butanoic
analogues.
HCl/1,4-dioxane,
RT, scheme
24–72 h,for
12%–67%
overall;
(iv) SOCl2, 0 °C for 30 min, reflux for 1 h,acid
RT overnight,
˝ C for 30 min, reflux for 1 h, RT
4M HCl/1,4-dioxane,
RT,
24–72
h,
12%–67%
overall;
(iv)
SOCl
,
0
2
3
,
THF,
0
°C-RT,
3–16
h;
(ii)
DIPEA,
CH
3
CN,
RT,
24
h; (iii) 4M
Reagents
and
Conditions:
(i)
DEAD,
PPh
44%–84%. Adapted with permission from [112]. Copyright 2005 American Chemical Society.
overnight,
44%–84%. RT,
Adapted
with
permission
[112].
Copyright 2005 American Chemical Society.
HCl/1,4-dioxane,
24–72 h,
12%–67%
overall;from
(iv) SOCl
2, 0 °C for 30 min, reflux for 1 h, RT overnight,
Additionally,
Rossiter
et al. from
developed
a synthetic
method
for Society.
N,N-disubstituted
44%–84%. Adapted
with permission
[112]. Copyright
2005 American
Chemical
alkylguanidinobutanoic
acids
and
N-arylguanidines
(Scheme
8).
This
involved
N,N’-bisAdditionally, Rossiter et al.
developed a synthetic method forreacting
N,N-disubstituted
Additionally, Rossiter et al. developed
a synthetic
method for ester
N,N-disubstituted
tert-butoxycarbonylpyrazole-1H-carboxamidine
(22)
with
Boc-homoserine-tert-butyl
(30),
whereby
alkylguanidinobutanoic acids and N-arylguanidines (Scheme 8). This involved reacting
N,N’-bisalkylguanidinobutanoic
and N-arylguanidines
8). Thiswith
involved
N,N’-bisthe
pyrazole group in theacids
intermediate
31 is displaced (Scheme
by the reaction
amine reacting
32 containing
the
tert-butoxycarbonylpyrazole-1H-carboxamidine
(22) with Boc-homoserine-tert-butyl ester (30),
tert-butoxycarbonylpyrazole-1H-carboxamidine
(22) with Boc-homoserine-tert-butyl ester (30), whereby
desired
R1 and R2 groups.
whereby
the pyrazole
group
the intermediate
31 is displaced
the reaction
with32
amine
32 containing
the pyrazole
group
in theinintermediate
31 is displaced
by the by
reaction
with amine
containing
the
1
2
the desired
R2 groups.
groups.
desiredRR1 and
and R
Scheme 8. Synthetic scheme for generating N,N-disubstituted alkylguanidinobutanoic acid and
N-arylguanidine analogues. Reagents and Conditions: (i) DEAD, Ph3P, THF, 0 °C-RT, 3–16 h; (ii) DIPEA,
Scheme RT,
8. Synthetic
for generating N,N-disubstituted
alkylguanidinobutanoic
acid and
CH
24 h; (iii) 4scheme
M HCl/1,4-dioxane,
h, 0.29%–41% overall.
Adapted with permission
Scheme3CN,
8. Synthetic
scheme
for generating24–72
N,N-disubstituted
alkylguanidinobutanoic
acid and
3–16
h;
(ii)
DIPEA,
N-arylguanidine
analogues.
Reagents and
Conditions:
(i) DEAD, Ph3P, THF, 0 °C-RT,
from
[112]. Copyright
2005 American
Chemical
Society.
N-arylguanidine analogues. Reagents and Conditions: (i) DEAD, Ph3 P, THF, 0 ˝ C-RT, 3–16 h; (ii) DIPEA,
CH3CN, RT, 24 h; (iii) 4 M HCl/1,4-dioxane, 24–72 h, 0.29%–41% overall. Adapted with permission
CH3 CN, RT, 24 h; (iii) 4 M HCl/1,4-dioxane, 24–72 h, 0.29%–41% overall. Adapted with permission
from [112]. Copyright 2005 American Chemical Society.
from [112]. Copyright 2005 American Chemical Society.
Molecules 2016, 21, 615
8 of 32
Deprotection of the amino acid analogues 33 is carried out using the same acidic conditions
described
in the synthesis of Rossiter’s 4124W analogues in Scheme 7 and provides analogues
34
Molecules 2016, 21, 615
8 of 31
in low to moderate yields (0.29%–41%). Each analogue’s inhibitory potential was assessed based
Deprotection
of the of
amino
acid analogues
is carried out
the same
acidic conditions
on the reduced
conversion
[14 C]-NMMA
to [1433
C]-citrulline
byusing
rat DDAH.
Screening
experiments
described
in the synthesis of to
Rossiter’s
4124W
analogues
Scheme 7 and provides
analogues
34 in
revealed
N-monosubstitution
be more
effective
thanindisubstitution,
and that
a R1 substituent
low
to
moderate
yields
(0.29%–41%).
Each
analogue’s
inhibitory
potential
was
assessed
based
on
the 27a;
comprising a N-2-methoxyethyl group increased inhibitor binding affinity (viz. compound
reduced conversion of [14C]-NMMA to [14C]-citrulline by rat DDAH. Screening experiments revealed
IC50 = 189 µM; Scheme 7). Furthermore, a series of N-2-methoxyethyl guanidinobutanoate esters [112]
N-monosubstitution to be more effective than disubstitution, and that a R1 substituent comprising a
was synthesized via thionyl chloride promoted esterification with alcohol containing the desired R3
N-2-methoxyethyl group increased inhibitor binding affinity (viz. compound 27a; IC50 = 189 μM;
group
(Scheme 7). The generated benzyl ester 29a resulted in substantial improvement in DDAH-1
Scheme 7). Furthermore, a series of N-2-methoxyethyl guanidinobutanoate esters [112] was synthesized
inhibition
(IC50chloride
= 27 µM).
A series
of amidewith
analogues
based on the
structures
(structures
3 group
(Schemeomitted)
7).
via thionyl
promoted
esterification
alcohol containing
desired R29
exhibited
poor DDAH-1
potentials.
The generated
benzylinhibitory
ester 29a resulted
in substantial improvement in DDAH-1 inhibition (IC50 =
27 μM). A series of amide analogues based on structures 29 (structures omitted) exhibited poor
4.1.4.DDAH-1
NG -SubstitutedinhibitoryL-Arginine
potentials. Analogues
Rossiter
et al. [112] also synthesized a library of N-substituted arginine analogues. A significant
4.1.4. NG-Substituted-L-Arginine Analogues
increase in inhibitor binding affinity was obtained for NG -(2-methoxyethyl)-L-arginine (commonly
et al. [112] also39;
synthesized
library
of N-substituted
analogues.
A significant
known asRossiter
L-257 (compound
IC50 = 22aµM;
Scheme
9) and itsarginine
corresponding
methyl
ester (also
G
increase in inhibitor binding affinity was obtained for N -(2-methoxyethyl)-L-arginine (commonly
known as L-291 (compound 40; IC50 = 20 µM; Scheme 9), relative to compound 27a (Scheme 7) that
known as L-257 (compound 39; IC50 = 22 μM; Scheme 9) and its corresponding methyl ester (also known
contains one less carbon within the main-chain. Interestingly, the benzyl ester of L-257 did not increase
as L-291 (compound 40; IC50 = 20 μM; Scheme 9), relative to compound 27a (Scheme 7) that contains one
DDAH-1
inhibition
as the
wasmain-chain.
observed with
compound
29a. These
suggest
that
arginine
analogues
less carbon
within
Interestingly,
the benzyl
ester data
of L-257
did not
increase
DDAH-1
may inhibition
bind differently
within
the
putative
DDAH-1
active-site.
Moreover,
neither
the
corresponding
as was observed with compound 29a. These data suggest that arginine analogues may bind
methyl
amide ofwithin
L-257,the
norputative
the (R)-isomer
L-257 (structures
omitted)
in DDAH-1methyl
inhibition.
differently
DDAH-1ofactive-site.
Moreover,
neitherresulted
the corresponding
amidesynthesis
of L-257, nor
(R)-isomer
L-257 (structures
DDAH-1 inhibition.
The
of the
L-257
(39) isofrepresented
in omitted)
Scheme resulted
9. Thein reaction
sequence follows a
The synthesis
of described
L-257 (39) is
in Scheme
9. The reaction sequence follows a similar
similar approach
to that
inrepresented
the synthesis
of the (S)-2-amino-4-(3-methylguanidino)butanoic
to that
described
in the synthesis
the (S)-2-amino-4-(3-methylguanidino)butanoic
acid
acid approach
analogues,
using
Boc protected
aminoof acid
analogues. Firstly, 2-methoxyethyl pyrazole
analogues,
using
Boc
protected
amino
acid
analogues.
Firstly,
2-methoxyethyl
pyrazole
carboxamidine (36) is prepared under Mitsunobu conditions [113] from 2-methoxyethanol (35) and
carboxamidine (36) is prepared under Mitsunobu conditions [113] from 2-methoxyethanol (35) and
N,N1 -bis-tert-butoxycarbonylpyrazole-1H-carboxamidine (22). This intermediate 36 is reacted with
N,N′-bis-tert-butoxycarbonylpyrazole-1H-carboxamidine (22). This intermediate 36 is reacted with
Boc-Orn-OBut (37)
to introduce the N-alkyl-guanidino group to compound 38. Final deprotection of
Boc-Orn-OBut (37) to introduce the N-alkyl-guanidino group to compound 38. Final deprotection of
38 with
4
M
HCl
in
afforded
in 44%
44%overall
overallyield.
yield.
L-257
be subsequently
38 with 4 M HCl1,4-dioxane
in 1,4-dioxane
affordedL-257
L-257 (39)
(39) in
L-257
cancan
be subsequently
esterified
to
afford
L-291
(40)
in
yields
of
approximately
80%.
esterified to afford L-291 (40) in yields of approximately 80%.
Scheme 9. Synthetic scheme for generating L-257 (39) and L-291 (40). Reagents and Conditions: (i)
Scheme
9. Synthetic scheme for generating L-257 (39) and L-291 (40). Reagents and Conditions: (i) DEAD,
DEAD, Ph˝3P, THF, 0 °C-RT, 3–16 h; (ii) DIPEA, CH3CN, RT, 24 h; (iii) 4 M HCl/1,4-dioxane, 24–72 h,
Ph3 P, THF, 0 C-RT, 3–16 h; (ii) DIPEA, CH3 CN, RT, 24 h; (iii) 4 M HCl/1,4-dioxane, 24–72 h, 44%
44% overall; (iv) SOCl2, 0 °C for 30 min, reflux for 1 h, RT overnight, 80%. Adapted with permission
overall; (iv) SOCl2 , 0 ˝ C for 30 min, reflux for 1 h, RT overnight, 80%. Adapted with permission
from [112]. Copyright 2005 American Chemical Society.
from [112]. Copyright 2005 American Chemical Society.
Both L-257 (39) and L-291 (40) inhibit DDAH-1 in vivo [112]. They are non-cytotoxic and selective
for
DDAH-1
against
the three
isoforms
of in
NOS
and are
arginase
[115], an and
enzyme
Both L-257 (39)
and L-291
(40) main
inhibit
DDAH-1
vivo[112,114]
[112]. They
non-cytotoxic
selective
responsible
for
the
metabolism
of
L
-arginine
into
urea
and
ornithine
[112,114,115].
L-257
(39)
was
for DDAH-1 against the three main isoforms of NOS [112,114] and arginase [115], an enzyme responsible
utilized
by Kotthaus
al. [114] as
a validated
active
‘parent’[112,114,115].
compound for L-257
determining
viable
lead by
for the
metabolism
of Let
-arginine
into
urea and
ornithine
(39) was
utilized
compounds, and to obtain initial structure-activity relationships (SARs) for those leads. In
Kotthaus et al. [114] as a validated active ‘parent’ compound for determining viable lead compounds,
Molecules 2016, 21, 615
9 of 32
Molecules 2016, 21, 615
9 of 31
and to obtain initial structure-activity relationships (SARs) for those leads. In particular, the role
of
the N-substituent in DDAH inhibition was investigated as the guanidino moiety is considered to
particular, the role of the N-substituent in DDAH inhibition was investigated as the guanidino moiety
be important in the proposed catalytic mechanism and binding to the enzyme [116]. In the work
is considered to be important in theGproposed catalytic mechanism and binding to the enzyme [116].
undertaken by Kotthaus et al., the N -(2-methoxyethyl) side-chain was varied along with the degree of
In the work undertaken by Kotthaus et al., the NG-(2-methoxyethyl) side-chain was varied along with
guanidine substitution. Similarly, Tommasi et al. synthesized further L-257 derivatives with differing
the degree of guanidine substitution. Similarly,
Tommasi et al. synthesized further L-257 derivatives
side-chains [117]. In both studies, the NG -(2-methoxyethyl) side-chain
conferred inhibitors with activity
with differing side-chains [117]. In both studies, the NG-(2-methoxyethyl) side-chain conferred
toward DDAH-1. N-monosubstituted examples are represented in Table 2.
inhibitors with activity toward DDAH-1. N-monosubstituted examples are represented in Table 2.
Table 2. Inhibitory parameters for the N-monosubstituted L-arginine analogues with varied
Table 2. Inhibitory parameters for the N-monosubstituted L-arginine analogues with varied
N-substituents (R groups) [114,117].
N-substituents (R groups) [114,117].
Entry
Inhibition at
IC50
(µM)
Kii (μM)
(µM)
50 (μM)
Entry
R–R–
Inhibition
at11mM
mM(%)
(%)
IC
K
* CH
83
31
13
1 11 1
* CH
3OCH
2CH
2–
83
31
13
3 OCH
2 CH
2–
1
CH
NHCH
CH
–
29
2
1
3
2 2–
2
2 1
CH3NHCH
2CH
29
-(CH
)
NHCH
CH
–
14
3
3
2
2
2
31 1
(CH3)2NHCH2CH2–
14
CH3 SHCH2 CH2 –
80
408
4
4 15 1
CH3SHCH
2CH2–
80
408
-FCH2 CH
67
379
2–
5 16 1
FCH
2
CH
2
–
67
379
-BnCH2 –
55
866
2
1
CH
CH
CH
–
60
283
90
7
6
BnCH
55
866
3
2– 2
2
CH
70
207
58
2 CHCH
2–
7 28
CH
3CH
2CH2–
60
283
90
2
CH2 CHCH2 CH2 –
71
189
57
8 29 2
CH2CHCH2–
70
207
58
CHCCH2 –
83
55
17
10
9 211 2
CH2CHCH
2CH2–
71
189
57
F3 CCH
41
2–
2 2
10 12
CHCCH
83
55
17
O2 N–2–
6
2 2
11 13
F23CCH
2–2 CH2 –
41
-NH
COCH
43
12 2 L-257 (39) (Scheme
O2N–9); 1 Measured by Ultra Performance
6
* Denotes
Liquid Chromatography-Mass
Spectrometry;
2 Measured by colorimetric assay.
13 2
NH2COCH2CH2–
43
-
* Denotes L-257 (39) (Scheme 9);
1
Measured by Ultra Performance Liquid Chromatography-Mass
4.1.5.Spectrometry;
NG -(2-Methoxyethyl)-Arginine
Carboxylate
Bioisosteres
2 MeasuredLby
colorimetric
assay.
The use of bioisosteric replacements is a well-known strategy adopted by medicinal
4.1.5.
NG-(2-Methoxyethyl)L-Arginine Carboxylate Bioisosteres
chemists
to improve the
pharmacokinetic and pharmacodynamic properties of certain
compounds,
including
butreplacements
not limited istoa inhibition
potency,
pKa , andchemists
metabolic
The use of
bioisosteric
well-known
strategylipophilicity,
adopted by medicinal
to
stability
[118].
Tommasi
et
al.
[117]
reported
the
synthesis
and
pharmacological
evaluation
improve the pharmacokinetic and pharmacodynamic properties of certain compounds, including
of five
carboxylate
bioisosteres
of L-257
(compounds
44a–c;
Scheme
10, [118].
49a–b;Tommasi
Schemeet11).
but
not limited
to inhibition
potency,
lipophilicity,
pKa, and
metabolic
stability
al.
In compounds
44a–c
(Scheme
the carboxylicevaluation
acid moiety
is modified
by bioisosteres
introducingof
anL-257
acyl
[117]
reported the
synthesis
and10),
pharmacological
of five
carboxylate
methyl sulfonamide
44a (2-amino-5-(3-(2-methoxyethyl)guanidino)-N-(methylsulfonyl)-pentanamide;
(compounds
44a–c; Scheme
10, 49a–b; Scheme 11). In compounds 44a–c (Scheme 10), the carboxylic acid
ZST316),
O-methyl-hydroxamate
a hydroxamate
The replacement is introduced
moiety
is an
modified
by introducing an44b
acyland
methyl
sulfonamide44c.
44a (2-amino-5-(3-(2-methoxyethyl)
on Z-ornithine(Boc)-OH (41) in moderate to good
yieldsan
(51%–86%)
to afford 42, followed
by the
guanidino)-N-(methylsulfonyl)-pentanamide;
ZST316),
O-methyl-hydroxamate
44b and
a
quantitative
cleavage
of
Boc
protected
42
with
4
M
HCl
in
1,4-dioxane.
Guanidination
of
42
with
hydroxamate 44c. The replacement is introduced on Z-ornithine(Boc)-OH (41) in moderate to good
2-methoxyethylpyrazole
carboxamidine
undertaken
as described
et al. [112]
(see
yields
(51%–86%) to afford
42, followed(36)
by isthe
quantitative
cleavage by
of Rossiter
Boc protected
42 with
Sections
4.1.3
and
4.1.4
Schemes
7
and
9).
Deprotection
of
the
Bocand
Z-appended
arginine
derivatives
4 M HCl in 1,4-dioxane. Guanidination of 42 with 2-methoxyethylpyrazole carboxamidine (36) is
43a–c is achieved
in a single
usingetTFMSA
and(see
TFASections
[117,119]4.1.3
andand
produces
undertaken
as described
bystep
Rossiter
al. [112]
4.1.4, compounds
Schemes 7 44a–c.
and 9).
Bioisosteric
replacement
the carboxylic
acid moiety
in L-257
withisO-methyl
44b
Deprotection
of the
Boc- andofZ-appended
arginine
derivatives
43a–c
achievedhydroxamate
in a single step
and
hydroxamate
44c
resulted
in
a
decrease
in
DDAH-1
inhibition
(IC
=
230
and
131
µM,
respectively).
using TFMSA and TFA [117,119] and produces compounds 44a–c. 50
By contrast,
introduction
of anofacyl
sulfonamidic
moiety
in ZST316
(44a) led hydroxamate
to significant
Bioisosteric
replacement
the methyl
carboxylic
acid moiety
in L-257
with O-methyl
improvements
in pharmacokinetic
(98%ininhibition
1 mM, IC50
Ki 131
= 1 µM).
44b
and hydroxamate
44c resultedparameters
in a decrease
DDAH-1atinhibition
(IC=503 =µM
230and
and
μM,
Compound
ZST316
(44a)
is
the
most
potent
human
DDAH-1
inhibitor
with
‘substrate-like’
structure
respectively). By contrast, introduction of an acyl methyl sulfonamidic moiety in ZST316 (44a) led to
reported inimprovements
the literature toindate.
significant
pharmacokinetic parameters (98% inhibition at 1 mM, IC50 = 3 μM and
Ki = 1 μM). Compound ZST316 (44a) is the most potent human DDAH-1 inhibitor with ‘substrate-like’
structure reported in the literature to date.
Molecules 2016, 21, 615
10 of 32
Molecules 2016, 21, 615
10 of 31
Molecules 2016, 21, 615
10 of 31
Scheme 10. Synthetic scheme for generating NG-(2-methoxyethyl)-L-arginine carboxylate bioisosteres.
Scheme 10. Synthetic scheme for generating NG -(2-methoxyethyl)-L-arginine carboxylate bioisosteres.
Reagents and Conditions: (i) CDI, DBU, methanesulfonamide,
THF, RT, 7 h, 51%; (ii) HATU, DIPEA,
Scheme
Synthetic scheme
for generating
NG-(2-methoxyethyl)-L-arginine
carboxylate
bioisosteres.
Reagents
and10.
Conditions:
(i) CDI,
DBU, methanesulfonamide,
THF, RT,
7 h, 51%;
(ii) HATU,
O-methylhydroxylamine hydrochloride, THF, 0 °C-RT, 16 h, 86%; (iii) HATU, DIPEA,
Reagents
and
Conditions:
(i)
CDI,
DBU,
methanesulfonamide,
THF,
RT,
7
h,
51%;
(ii)
HATU,
DIPEA,
˝
DIPEA,
O-methylhydroxylamine
hydrochloride,
THF,
0
C-RT,
16
h,
86%;
(iii)
HATU,
DIPEA,
O-benzylhydroxylamine, THF, 0 °C-RT, 16 h, 57%; (iv) 4 M HCl/1,4-dioxane, DCM, RT, 30 min, 99%;
O-methylhydroxylamine hydrochloride,
0 °C-RT, 16 h, 86%; (iii) HATU, DIPEA,
˝ C-RT, 16 h,THF,
O-benzylhydroxylamine,
THF,
0
57%;
(iv)
4
M
HCl/1,4-dioxane,
DCM,
RT,
30
min,
(v) DIPEA, DCM, RT, 24 h, 43a 42%, 43b 22%, 43c 56%; (vi) TFMSA/TFA, RT, 1 h, 99%. Reproduced 99%;
O-benzylhydroxylamine, THF, 0 °C-RT, 16 h, 57%; (iv) 4 M HCl/1,4-dioxane, DCM, RT, 30 min, 99%;
(v) DIPEA,
RT, 24
h,permission
43a 42%, 43b
22%,
43cSociety
56%; (vi)
TFMSA/TFA, RT, 1 h, 99%. Reproduced in
in partDCM,
from [117]
with
of the
Royal
of Chemistry.
(v) DIPEA, DCM, RT, 24 h, 43a 42%, 43b 22%, 43c 56%; (vi) TFMSA/TFA, RT, 1 h, 99%. Reproduced
part from [117] with permission of the Royal Society of Chemistry.
in part from [117] with permission of the Royal Society of Chemistry.
Scheme 11. Synthetic scheme for generating NG-(2-methoxyethyl)-L-arginine carboxylate bioisosteres.
Reagents and Conditions: (i) i-BuCOCl, N-methylmorpholine, NH4OH (30%), THF, −10 °C-RT, 3 h,
Scheme 11. Synthetic scheme for generating NGG-(2-methoxyethyl)-L-arginine carboxylate bioisosteres.
Scheme
scheme
generating
N (iii)
-(2-methoxyethyl)L-arginine
bioisosteres.
99%;11.
(ii)Synthetic
TFAA, TEA,
THF, for
0 °C-RT,
16 h, 47%;
4 M HCl/1,4-dioxane,
DCM, carboxylate
RT, 30 min, 99%;
(iv)
Reagents and Conditions: (i) i-BuCOCl, N-methylmorpholine, NH4OH (30%), THF, −10 ˝°C-RT, 3 h,
Reagents
and
Conditions:
(i)
i-BuCOCl,
N-methylmorpholine,
NH
OH
(30%),
THF,
´10
C-RT,
3 h, 99%;
16 h, 30%; (vi) (1) NH2OH·HCl,
DIPEA, DCM, RT, 24 h, 40%; (v) NaN3, ZnBr2, H2O/2-propanol, reflux,
4
99%; (ii) TFAA, TEA,
THF, 0 °C-RT, 16 h, 47%; (iii) 4 M HCl/1,4-dioxane, DCM, RT, 30 min, 99%; (iv)
˝ C-RT,
(ii) TFAA,
TEA,
THF, 0reflux,
4 THF,
M HCl/1,4-dioxane,
DCM,
30 min, 99%;
(iv)RT,
DIPEA,
NaHCO
3, DMSO,
1616
h; h,
(2)47%;
CDI, (iii)
DBU,
reflux, 30 min, 73%;
(vii)RT,
TFMSA/TFA,
DCM,
DIPEA, DCM, RT, 24 h, 40%; (v) NaN3, ZnBr2, H2O/2-propanol, reflux, 16 h, 30%; (vi) (1) NH2OH·HCl,
30RT,
min,2499%.
Reproduced
in part
from2 ,[117]
with permission reflux,
of the Royal
Society
of
Chemistry.
DCM,NaHCO
h,
40%;
(v)
NaN
,
ZnBr
H
O/2-propanol,
16
h,
30%;
(vi)
(1)
NH
OH¨
HCl,
3
2
2
3, DMSO, reflux, 16 h; (2) CDI, DBU, THF, reflux, 30 min, 73%; (vii) TFMSA/TFA, DCM, RT,
NaHCO
,
DMSO,
reflux,
16
h;
(2)
CDI,
DBU,
THF,
reflux,
30
min,
73%;
(vii)
TFMSA/TFA,
DCM,
RT,
3
30 min, 99%. Reproduced in part from [117] with permission of the Royal Society of Chemistry.
Other
inhibitors
in this
series
the tetrazole
1-[4-amino-4-(1H-tetrazol-5-yl)
30 min,
99%.noteworthy
Reproduced
in part from
[117]
withinclude
permission
of the Royal
Society of Chemistry.
butyl]-3-(2-methoxyethyl)guanidine
(ZST086,
49a,
Scheme
11)
and
the 5-oxo-1,2,4-oxadiazole
Other noteworthy inhibitors in this series include the tetrazole 1-[4-amino-4-(1H-tetrazol-5-yl)
1-[4-amino-4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)butyl]-3-(2-methoxyethyl)guanidine
(ZST152, 49b,
butyl]-3-(2-methoxyethyl)guanidine
(ZST086, 49a, Scheme 11) and the 5-oxo-1,2,4-oxadiazole
Other
noteworthy
inhibitors
inofthis
series
include
theazole
tetrazole
1-[4-amino-4-(1H-tetrazol-5-yl)
Scheme
11)
[117].
The
synthesis
these
inhibitors
with
bioisosteric
groups
is
achieved49b,
via
1-[4-amino-4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)butyl]-3-(2-methoxyethyl)guanidine (ZST152,
butyl]-3-(2-methoxyethyl)guanidine
(ZST086,
49a,
Schemeamide
11) 45
and
the 5-oxo-1,2,4-oxadiazole
conversion
of
the
terminal
carboxylic
acid
in
41
to
primary
in
quantitative
yields
using
Scheme 11) [117]. The synthesis of these inhibitors with azole bioisosteric groups is achieved via
1-[4-amino-4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)butyl]-3-(2-methoxyethyl)guanidine
(ZST152,
isobutylchloroformate,
N-methylmorpholine,
andto aqueous
amideyields
45 isusing
then
conversion
of the terminal
carboxylic acid in 41
primary ammonia.
amide 45 inPrimary
quantitative
converted11)
to nitrile
using
trifluoroacetic
anhydride
and with
triethylamine
in
47% yield.
Compound
46
49b, Scheme
[117]. 46
The
synthesis
of these
inhibitors
azole bioisosteric
groups
isobutylchloroformate,
N-methylmorpholine,
and aqueous
ammonia.
Primary
amide
45 is
is achieved
then
is
guanylated
[112]
(see
Sections
4.1.3
and
4.1.4,
Schemes
7
and
9)
to
afford
compound
47,
from
via conversion
of
the
terminal
carboxylic
acid
in
41
to
primary
amide
45
in
quantitative
yields
converted to nitrile 46 using trifluoroacetic anhydride and triethylamine in 47% yield. Compound 46using
which
the heterocycle
is constructed
from
the4.1.4,
nitrile.
The tetrazole
is9)introduced
by treating
nitrile
47 then
isobutylchloroformate,
N-methylmorpholine,
andSchemes
aqueous
ammonia.
Primary
amide
is
is
guanylated
[112] (see
Sections 4.1.3
and
7 and
to afford
compound
47,45
from
with
sodium
azide
in
the
presence
of
zinc(II)
bromide
to
afford
compound
48a,
using
the
click
which to
thenitrile
heterocycle
is constructed
from the
nitrile. Theand
tetrazole
is introduced
by treating
47
converted
46 using
trifluoroacetic
anhydride
triethylamine
in 47%
yield.nitrile
Compound
chemistry
procedure
by Demko
and Sharpless
[120].
Although the use48a,
of zinc(II)
bromide
sodium
azide (see
inreported
the
presence
of zinc(II)
bromide
to afford
using the
click
46 is with
guanylated
[112]
Sections
4.1.3
and 4.1.4
Schemes
7 andcompound
9) to afford compound
47, from
in the synthesis
of Boc-amino
tetrazoles
derived
from α-amino
acids
has been
reported
[121],bromide
the use
procedure
reported
by Demko
and nitrile.
Sharpless
[120].
Although
the use
of zinc(II)
whichchemistry
the
heterocycle
isresult
constructed
from
the
The
tetrazole
is introduced
byAmmonium
treating nitrile
of
a
Lewis
acid
may
in
partial
deprotection
of
compound
48a
during
the
reaction.
in the synthesis of Boc-amino tetrazoles derived from α-amino acids has been reported [121], the use
47 with
sodium
azide in the
presence
of zinc(II)
bromide
to afford compound
48a, usingfrom
the click
chloride
represents
alternative
reagent
[122]. The
5-oxo-1,2,4-oxadiazole
48b
is synthesized
of
a Lewis
acid may an
result
in partial
deprotection
of compound
48a during the
reaction.
Ammonium
chemistry
procedure
reported
by
Demko
and
Sharpless
[120].
Although
the
use
of
zinc(II)
bromide
nitrile 47represents
using hydroxylamine
and5-oxo-1,2,4-oxadiazole
sodium bicarbonate in
followed
by in
chloride
an alternative hydrochloride
reagent [122]. The
48bDMSO,
is synthesized
from
the synthesis
ofusing
Boc-amino
tetrazoles
derived
fromand
α-amino
acids
has been
[121],
thebyuse of
treatment
CDI
and DBU
[123].
Acidic
deprotection
of 48a–b
is achieved
asreported
described
previously
nitrile
47 with
hydroxylamine
hydrochloride
sodium
bicarbonate
in
DMSO,
followed
[117,119].
Despite
obtaining
lower
yields
for
heterocyclic
adducts
49a–b
(7%–13%)
relative
to
a Lewis
acid
may
result
in
partial
deprotection
of
compound
48a
during
the
reaction.
Ammonium
treatment with CDI and DBU [123]. Acidic deprotection of 48a–b is achieved as described previously
bioisosteres
44a–c
(19%–32%),
the
inhibitory
potential
of
5-oxo-1,2,4-oxadiazole
ZST152
(49b)
was
chloride
represents
an
alternative
reagent
[122].
The
5-oxo-1,2,4-oxadiazole
48b
is
synthesized
[117,119]. Despite obtaining lower yields for heterocyclic adducts 49a–b (7%–13%) relative tofrom
nitrilebioisosteres
47 using hydroxylamine
hydrochloride
and
sodiumofbicarbonate
in DMSO, followed
by treatment
44a–c (19%–32%),
the inhibitory
potential
5-oxo-1,2,4-oxadiazole
ZST152 (49b)
was
with CDI and DBU [123]. Acidic deprotection of 48a–b is achieved as described previously [117,119].
Despite obtaining lower yields for heterocyclic adducts 49a–b (7%–13%) relative to bioisosteres 44a–c
Molecules 2016, 21, 615
11 of 32
(19%–32%), the inhibitory potential of 5-oxo-1,2,4-oxadiazole ZST152 (49b) was significant (95% at
21, 615K = 7 µM), while tetrazole ZST086 (49a) displayed 91% inhibition at 111
of 31 with
1 mM,Molecules
IC50 =2016,
18 µM,
mM,
i
Molecules
2016,
21,K
615
11 of 31
IC50 =
34 µM
and
i = 14 µM [117].
significant (95% at 1 mM, IC50 = 18 μM, Ki = 7 μM), while tetrazole ZST086 (49a) displayed 91%
50 = 34 μM and Ki = 14 μM [117].
inhibition
at
1 mM,
IC
(95%
at 1with
mM,
IC
50 = 18 μM,
Ki = 7 μM), while tetrazole ZST086 (49a) displayed 91%
4.1.6.significant
N5 -(1-Iminoalk(en)yl)L -Ornithine
Derivatives
inhibition at 1 mM, with IC50 = 34 μM and Ki = 14 μM [117].
4.1.6. N5-(1-Iminoalk(en)yl)L-Ornithine
Derivatives
Amidines
are commonly known
bioisosteres
of the guanidine moiety [124]. N5 -(1-iminoalk(en)yl)-L5 -(1-imino-3-butenyl)- L -ornithine (L-VNIO, 54a, Figure 2), are reported to
5-(1-Iminoalk(en)yl)ornithines
asareNcommonly
4.1.6. Amidines
Nsuch
L-Ornithine
Derivatives
known bioisosteres
of the guanidine moiety [124]. N5-(1-iminoalk(en)yl)-L5
inhibit
NOS
by
competing
with
L
-arginine
for
binding
at moiety
the Figure
enzyme
catalytic-site
[125–127].
ornithines
such
ascommonly
N -(1-imino-3-butenyl)L-ornithine
(L-VNIO,
54a,
reported to inhibit
Amidines
are
known bioisosteres
of the guanidine
[124].2),
N5are
-(1-iminoalk(en)yl)LTherefore,
of this
class of
have
been
extensively
studied
as DDAH
L-arginine
for compounds
binding
at the
enzyme
catalytic-site
[125–127].
Therefore,
NOS byderivatives
competing
with
ornithines
such as N5-(1-imino-3-butenyl)L-ornithine
(L-VNIO,
54a,
Figure
2), are
reported
to inhibit
derivatives
of synthetic
this class
compounds
have
extensively
asL-ornithine
DDAH
inhibitors.
The 54
inhibitors.
approach
to binding
affordbeen
N5the
-(1-iminoalk(en)yl)derivatives
L-arginine
for
at
enzyme studied
catalytic-site
[125–127].
Therefore,
NOS
by The
competing
with of
5
synthetic
approach
to afford
N
-(1-iminoalk(en)yl)-ornithine
derivatives
(Scheme
12) [127,128],
(Scheme
12) [127,128],
similar
approach
to Lthe
N-but-3-ynyl-2-chloro-acetamidine
probe
derivatives
of this utilized
class
of acompounds
have been
extensively
studied as54DDAH
inhibitors.
The (18)
utilized4.1.2,
aapproach
similar
approach
to
the
N-but-3-ynyl-2-chloro-acetamidine
probe
(18)
Section
4.1.2,ethyl
synthetic
to afford
N5-(1-iminoalk(en)yl)54 to
(Scheme
12)
[127,128],
(see Section
Scheme
5) [109].
The
R1 furnishedL-ornithine
nitrile 50 derivatives
is converted
its(see
corresponding
Scheme a5)similar
[109]. The
R1 furnished
nitrile
50 is converted to its corresponding
ethyl
imidic
ester 4.1.2,
51 by
utilized
approach
to
the
N-but-3-ynyl-2-chloro-acetamidine
probe
(18)
(see
Section
imidic ester 51 by bubbling dry hydrochloric acid gas through a solution of anhydrous ethanol (16).
bubbling
hydrochloric
acid gas
through
a solution to
of its
anhydrous
ethanol
(16).imidic
The ethyl
1
Scheme
5) dry
[109].
The
nitrile
50 is
converted
corresponding
ethyl
esterimidic
51
by 53.
The ethyl
imidic
ester
51Ris furnished
then reacted
with
Nα
-Boc-L-ornithine
(52) to afford
the protected
adduct
α-Boc-L-ornithine (52) to afford the protected adduct 53. Deprotection
ester
51
is
then
reacted
with
N
bubbling dry hydrochloric acid gas through a solution of anhydrous ethanol
(16). The ethyl imidic
5
Deprotection
is obtained
by treatment
with
M achieve
HCl to achieve
the N -(1-iminoalk(en)yl)L-ornithine
5-(1-iminoalk(en)yl)is obtained
byreacted
treatment
6 M
HCl6 to
the Nthe
-ornithine
54 in
ester
51 is then
withwith
Nα-BocL-ornithine
(52) to afford
protected adductL53.
Deprotection
54 in excellent
excellentoverall
overall
yields
(84%–94%).
yields (84%–94%).
5
is obtained by treatment with 6 M HCl to achieve the N -(1-iminoalk(en)yl)-L-ornithine 54 in
excellent overall yields (84%–94%).
Scheme 12. Synthetic scheme for generating N5-(1-iminoalk(en)yl)-L-ornithine derivatives. Reagents
Scheme 12. Synthetic scheme for generating N5 -(1-iminoalk(en)yl)-L-ornithine derivatives. Reagents
and Conditions:
(i) dry scheme
HCl(g) atfor
0 °C for 1 h, stop
HCl(g), then 0 °C for-ornithine
4 h and RT
overnight; Reagents
(ii) H2O,
Scheme
12.(i)
Synthetic
N5-(1-iminoalk(en)yl)and Conditions:
dry HCl
0 ˝ Cgenerating
for 1 h, stop
HCl(g) , then 0 ˝ CLfor
4 h andderivatives.
RT overnight;
(ii) H2 O,
(g) at
5 °C,
pH = 10, 1.5
h, adjust
pH
= °C
7, overnight,
Dowex
50WX8-400
(H24Ohthen
10%
aqueous pyridine);
and
Conditions:
(i)
dry
HCl
(g)
at
0
for
1
h,
stop
HCl
(g)
,
then
0
°C
for
and
RT
overnight;
(ii)
H2O,
5 ˝ C, (iii)
pH 6= M
10,HCl,
1.5 h,
adjust
pH
=
7,
overnight,
Dowex
50WX8-400
(H
O
then
10%
aqueous
pyridine);
2 with permission from [127].
EtOAc,
84%–94%
from
Nα-Boc-Dowex
L-ornithine (52). Adapted
5 °C, pH = 10, 1.5
h, adjust
pH = 7,
overnight,
50WX8-400 (H2O then
10% aqueous pyridine);
α -Boc- L
(iii) 6Copyright
M HCl, EtOAc,
84%–94%
from N
-ornithine
(52).
Adapted
with permission from [127].
1999
American
Chemical
Society.
(iii) 6 M HCl, EtOAc, 84%–94% from Nα-Boc-L-ornithine (52). Adapted with permission from [127].
Copyright 1999 American Chemical Society.
Copyright 1999 American Chemical Society.
Figure 2. N5-(1-Iminoalk(en)yl)-L-ornithine derivatives, L-VNIO (54a), L-IPO (54b), and Cl-NIO (54c),
as synthesized
by Kotthaus et al.
[114] and Wang et al. [128,129].
Figure
2. N5-(1-Iminoalk(en)yl)L-ornithine derivatives, L-VNIO (54a), L-IPO (54b), and Cl-NIO (54c),
Figure 2. N5 -(1-Iminoalk(en)yl)-L-ornithine derivatives, L-VNIO (54a), L-IPO (54b), and Cl-NIO (54c),
as synthesized by Kotthaus et al. [114] and Wang et al. [128,129].
5-(1-iminoalk(en)yl)as synthesized
by Kotthaus
et al. similarity
[114] and Wang
[128,129].
The significant
structural
of theetNal.
L-ornithine derivatives with
DDAH
(1) and
L-NMMA
groups to study
this class
of compounds
Thesubstrates,
significantADMA
structural
similarity
of (2)
theled
N5different
-(1-iminoalk(en)yl)L-ornithine
derivatives
with
5-(1-iminoalk(en)yl)-L5
as
potential
DDAH
inhibitors
[114,128].
Kotthaus
et
al.
[114]
examined
five
N
DDAH
substrates,structural
ADMA (1) similarity
and L-NMMA
(2) led
groups to study
this class ofderivatives
compounds with
The significant
of the
N different
-(1-iminoalk(en)yl)L -ornithine
ornithines
with
varying
side-chain
and bond
saturation
for theirfive
ability
to inhibit L-citrulline
as
potential
DDAH
inhibitors
Kotthaus
et al.
[114]different
examined
N5-(1-iminoalk(en)yl)LDDAH
substrates,
ADMA
(1) [114,128].
andlengths
L-NMMA
(2)
led
groups
to
study this class
of
formation
by
human
DDAH-1
using
both
high
performance
liquid
chromatography
and colorimetry.
ornithines
with
varying
side-chain
lengths
and
bond
saturation
for
their
ability
to
inhibit
L
-citrulline
compounds as potential DDAH inhibitors [114,128].
Kotthaus et al. [114] examined five
L-VNIO (54a,
FigureDDAH-1
2) emerged
the high
best performance
DDAH-1 inhibitor
of this series of derivatives
(97%
formation
by human
usingasboth
liquid chromatography
and colorimetry.
N5 -(1-iminoalk(en)yl)L -ornithines with varying side-chain lengths and bond saturation for their
inhibition
at
1
mM,
IC
50 = 13 μM, Ki = 2 μM). However, increasing the main-chain length by one
L-VNIO (54a, Figure 2) emerged as the best DDAH-1 inhibitor of this series of derivatives (97%
ability
to inhibit
L -citrulline
formation
by human
DDAH-1
using
both high performance liquid
carbon
atom
lysine
DDAH
inhibitory
potential.
inhibition
at 1from
mM,ornithine
IC50 = 13toμM,
Ki reduced
= 2 μM).itsHowever,
increasing
the main-chain length by one
chromatography
and colorimetry. L-VNIO (54a, Figure 2) emerged as the best DDAH-1 inhibitor of
carbon atom from ornithine to lysine reduced its DDAH inhibitory potential.
this series of derivatives (97% inhibition at 1 mM, IC50 = 13 µM, Ki = 2 µM). However, increasing the
main-chain length by one carbon atom from ornithine to lysine reduced its DDAH inhibitory potential.
Molecules 2016, 21, 615
12 of 32
Wang et al. [128] investigated this class of compounds as dual DDAH-1 and NOS
5
inhibitors,
Moleculesreporting
2016, 21, 615 the synthesis and kinetic parameters for four N -(1-iminoalkyl)- L -ornithines.
12 of 31
5
N -(1-Iminopropyl)-L-ornithine (L-IPO 54b, Figure 2) was identified as the best compound exhibiting
et human
al. [128]DDAH-1
investigated
compounds
as dual
DDAH-1 and NOS inhibitors,
inhibitionWang
of both
(Ki this
= 52class
µM) of
and
rat nNOS (K
i = 3 µM). Wang et al. [129] additionally
reporting the synthesis and kinetic parameters5 for four N5-(1-iminoalkyl)-L-ornithines.
developed
an irreversible human DDAH-1 inhibitor, N -(1-imino-2-chloroethyl)-L-ornithine (Cl-NIO,
N5-(1-Iminopropyl)-L-ornithine (L-IPO 54b, Figure 2) was identified as the best compound
54c, Figure 2), whose chemical structure reflects a combination of both L-IPO (54b) and
exhibiting inhibition of both human DDAH-1 (Ki = 52 μM) and rat nNOS (Ki = 3 μM). Wang et al.
2-chloroacetamidine
(11). Theansynthesis
Cl-NIO
(54c) inhibitor,
is similar
to that described for the
[129] additionally developed
irreversibleofhuman
DDAH-1
N5-(1-imino-2-chloroethyl)5
α
N -(1-iminoalk(en)yl)L -ornithine
derivatives
(Scheme
12). reflects
Here, aNcombination
-Boc-L-ornithine
is reacted
L-ornithine (Cl-NIO,
54c, Figure 2),
whose chemical
structure
of both L-IPO
with (54b)
2-chloro-1-ethoxyethanimine,
intermediate
is deprotected
with 4.6
M HCl
and 2-chloroacetamidine (11).and
Thethe
synthesis
of Cl-NIOobtained
(54c) is similar
to that described
for the
L-ornithine
(Scheme
12). Here,
NαThe
-Boc-inhibition
L-ornithine potency
is reactedofwith
N5-(1-iminoalk(en)yl)in dioxane
to generate Cl-NIO
(54c,derivatives
Figure 2) in
low yields
(10%).
Cl-NIO
and assay
the intermediate
obtained
deprotected
with 4.6 M
HCl in
(54c) 2-chloro-1-ethoxyethanimine,
was assessed via colorimetric
and exhibited
Ki =is1.3
µM. In summary,
several
of the
5
dioxane
to
generate
Cl-NIO
(54c,
Figure
2)
in
low
yields
(10%).
The
inhibition
potency
of
Cl-NIO
N -(1-iminoalk(en)yl)-L-ornithine derivatives (54a–c, Figure 2) are potent human DDAH-1 inhibitors.
(54c) was assessed via colorimetric assay and exhibited Ki = 1.3 μM. In summary, several of the
Off-target
effects on the broader NO pathway (NOSs, arginases, AGTX-2) may either represent an
N5-(1-iminoalk(en)yl)-L-ornithine derivatives (54a–c, Figure 2) are potent human DDAH-1 inhibitors.
advantage (strong NO suppression), or limit their application (loss of NO physiological functions) as
Off-target effects on the broader NO pathway (NOSs, arginases, AGTX-2) may either represent an
potential
therapeutics.
advantage (strong NO suppression), or limit their application (loss of NO physiological functions) as
potential therapeutics.
4.2. Non-Substrate-Like Inhibitors
4.2. Non-Substrate-Like Inhibitors
4.2.1. Pentafluorophenyl (PFP) Sulfonate Esters
4.2.1. Pentafluorophenyl
(PFP) Sulfonate
Esters
Vallance
et al. [130] identified
pentafluorophenyl
(PFP) sulfonate esters 61 as nonspecific PaDDAH
and bacterial
arginine
(ADI)pentafluorophenyl
inhibitors, after highlighting
their
structural
similarity with
Vallance
et al.deaminase
[130] identified
(PFP) sulfonate
esters
61 as nonspecific
PaDDAH
and bacterial
deaminase
(ADI)
afterbehighlighting
structural
the cysteine
protease
familyarginine
of enzymes.
This class
of inhibitors,
inhibitor can
obtained intheir
moderate
to good
similarity
with
the
cysteine
protease
family
of
enzymes.
This
class
of
inhibitor
can
be
obtained
in
yields (44%–75%) via the 1,3-dipolar cycloaddition between nitrone 60 and the electron deficient
moderate
to
good
yields
(44%–75%)
via
the
1,3-dipolar
cycloaddition
between
nitrone
60
and
the
vinyl-appended PFP sulfonate 57 (Scheme 13) [131]. The shelf-stable PFP vinylsulfonate 57 is
electron in
deficient
vinyl-appended
57 (Scheme (55)
13) and
[131].2-chloroethane-1-sulfonyl
The shelf-stable PFP
synthesized
moderate
yield (59%) PFP
fromsulfonate
pentafluorophenol
vinylsulfonate 57 is synthesized in moderate yield (59%) from pentafluorophenol (55) and
chloride (56) [132], while various nitrones 60 can be generated from their corresponding aldehydes 58
2-chloroethane-1-sulfonyl chloride (56) [132], while various nitrones 60 can be generated from their
by reaction
with N-methyl hydroxylamine (59) under mild conditions and in good yield (for examples,
corresponding aldehydes 58 by reaction with N-methyl hydroxylamine (59) under mild conditions
pleaseand
seein[133,134]).
good yield (for examples, please see [133,134]).
Scheme 13. Synthetic scheme for generating PFP sulfonate esters 61. Reagents and Conditions: (i) DCM,
Scheme 13. Synthetic scheme for generating PFP sulfonate esters 61. Reagents and Conditions: (i) DCM,
TEA, 0 °C, 59%; (ii) Method A: NaHCO3, anhydrous MgSO4, DCM, reflux, 24 h, 73%–90% [133];
TEA, 0 ˝ C, 59%; (ii) Method A: NaHCO3 , anhydrous MgSO4 , DCM, reflux, 24 h, 73%–90% [133];
Method B: NaOAc, EtOH, RT, 90% [134]; (iii) dry toluene, reflux, 1–24 h, 44%–75%. Reproduced in part
Method
B: NaOAc, EtOH, RT, 90% [134]; (iii) dry toluene, reflux, 1–24 h, 44%–75%. Reproduced in part
from [130–133] with permission of the Royal Society of Chemistry and the American Chemical Society.
from [130–133] with permission of the Royal Society of Chemistry and the American Chemical Society.
The electron deficiency of the vinyl PFP sulfonate dipolarophile 57 affords the reversed
regioselective
of the
in compound 57
61,affords
contrarytheto reversed
the
The electron 4C-substituted
deficiency of adduct
the vinyl
PFPisoxazolidine
sulfonate dipolarophile
5C-substituted
adduct typically
observed
with isoxazolidine
olefins. Although
was previously
observed for
regioselective
4C-substituted
adduct
of the
inthis
compound
61, contrary
to the
electron deficient dipolarophiles [135–138], Caddick et al. [131] reported the 4C-substituted
5C-substituted adduct typically observed with olefins. Although this was previously observed
isoxazolidine 61 is increasingly favoured at higher temperatures. Each of the nitrones 60 reported by
for electron
deficient dipolarophiles [135–138], Caddick et al. [131] reported the 4C-substituted
Caddick et al. [131] participates in cycloaddition with the vinyl PFP sulfonate 57, including C-arylisoxazolidine
61 is increasingly
higher temperatures.
Each of the nitrones
60 reported
and C-alkyl-N-methyl
speciesfavoured
with both at
electron-donating
and electron-withdrawing
substituents
in by
Caddick
et
al.
[131]
participates
in
cycloaddition
with
the
vinyl
PFP
sulfonate
57,
including
C-aryladdition to poly- and hetero-aromatic substituents. This robust synthetic methodology provides the
and C-alkyl-N-methyl
with
both electron-donating
andesters
electron-withdrawing
substituents
ability to generate a species
structurally
diverse
library of PFP sulfonate
61 with moderate ease
and high in
yields
PFP sulfonate esters
61 have beenThis
screened
forsynthetic
their activity
as PaDDAH provides
and ADI the
addition
to[130].
poly-Several
and hetero-aromatic
substituents.
robust
methodology
ability to generate a structurally diverse library of PFP sulfonate esters 61 with moderate ease and
high yields [130]. Several PFP sulfonate esters 61 have been screened for their activity as PaDDAH and
Molecules 2016, 21, 615
13 of 32
ADI inhibitors
at two concentrations (500 µM and 50 µM), with five compounds emerging as13relatively
Molecules 2016, 21, 615
of 31
potent PaDDAH inhibitors (IC50 = 16–58 µM, see [130] for structures). Vallance et al. revealed the
modeinhibitors
of inhibition
competitive
and reversibility
experiments
at twowas
concentrations
(500utilising
μM andtime-dependence
50 μM), with five compounds
emerging
as relatively[130].
potent PaDDAH
50 = 61
16–58
μM,
seeproposed
[130] for structures).
et al. revealed
the
Interestingly,
the PFP inhibitors
sulfonate (IC
esters
have
been
as potentialVallance
antibacterial
agents, however
mode
inhibition
was identifying
competitive utilising
time-dependence
and reversibility
experiments
[130].
no data
hasofbeen
reported
if PaDDAH
inhibition modulates
bacterial
growth or
viability.
Interestingly,
theutilizing
PFP sulfonate
61 have
have been
been reported
proposedidentifying
as potential
antibacterial
agents,
Likewise,
no studies
humanesters
DDAH
whether
the PFP
sulfonate
however no data has been reported identifying if PaDDAH inhibition modulates bacterial growth or
esters exhibit specificity for bacterial PaDDAH. Non-specific PFP sulfonate ester binding to both human
viability. Likewise, no studies utilizing human DDAH have been reported identifying whether the
DDAH and PaDDAH would be counterproductive for their proposed application as antibiotics.
PFP sulfonate esters exhibit specificity for bacterial PaDDAH. Non-specific PFP sulfonate ester binding
to both human DDAH and PaDDAH would be counterproductive for their proposed application as
4.2.2. Indolylthiobarbituric Acid Derivatives
antibiotics.
The indolylthiobarbituric acid derivatives 68 [139] were identified as PaDDAH inhibitors by
4.2.2. Indolylthiobarbituric
Acid Derivatives
a combination
of virtual screening
using a compound library comprising 308,000 structures and
subsequent
screening. acid
Among
the 10968highest
scoring
compounds,
90 were
commercially
Thebiological
indolylthiobarbituric
derivatives
[139] were
identified
as PaDDAH
inhibitors
by a
combination
screening
usingactivity
a compound
comprising 308,000
structures
and
available
and usedofinvirtual
colorimetric
enzyme
assays.library
Three compounds
consisting
of two chemical
subsequent
biological
screening.
Among
the
109
highest
scoring
compounds,
90
were
commercially
scaffolds were identified as potential PaDDAH inhibitors. One of these scaffolds, indolylthiobarbituric
available
and used
in colorimetric
activity 68b–c
assays. identified
Three compounds
of two
chemical
acid (68a),
together
with
additionalenzyme
compounds
from aconsisting
similarity
search,
emerged
scaffolds were identified as potential PaDDAH inhibitors. One of these scaffolds, indolylthiobarbituric
as promising inhibitors of PaDDAH (IC50 = 2–16.9 µM). The most potent inhibitor of this series was
acid (68a), together with additional compounds 68b–c identified from a similarity search, emerged
SR445 (68c, IC50 = 2 µM), and no member of this class of compounds showed any human DDAH
as promising inhibitors of PaDDAH (IC50 = 2–16.9 μM). The most potent inhibitor of this series was
inhibition
[114].
SR445 (68c, IC50 = 2 μM), and no member of this class of compounds showed any human DDAH
This
class[114].
of inhibitors is comprised of thiobarbituric acid module 64 and indolyl module 67
inhibition
(Scheme 14).
The
moduleof64thiobarbituric
is synthesized
using
a modified
versionmodule
of the method
This classthiobarbituric
of inhibitors isacid
comprised
acid
module
64 and indolyl
67
described
by
Fisher
and
Mering
in
1903,
by
reaction
of
N-substituted
2-thiourea
63
diethyl
(Scheme 14). The thiobarbituric acid module 64 is synthesized using a modified version of thewith
method
described
by Fisher The
and indolyl
Mering module
in 1903, 67
by is
reaction
of N-substituted
2-thiourea
63 with diethyl
malonate
(62) [140,141].
alkylated
by deprotonation
of indole-3-carbaldehyde
malonate
(62)with
[140,141].
The indolyl module 67 is alkylated by deprotonation
of indole-3-carbaldehyde
(65) and
reaction
2-chloro-N-furan-2-ylmethyl-acetimide
(66) using a method
previously reported
and[142].
reaction
with
2-chloro-N-furan-2-ylmethyl-acetimide
(66) using a67method
previously reported
(yield(65)
83%)
The
aldehyde
substituent of the indolyl derivative
is subsequently
reacted with
(yield 83%) [142]. The aldehyde substituent of the indolyl derivative 67 is subsequently reacted with
the thiobarbituric acid derivative 64 under acidic conditions to afford 68 (yields not reported) [139,143].
the thiobarbituric acid derivative 64 under acidic conditions to afford 68 (yields not reported)
This produced a mixture of (E)- and (Z)-isomers about the linking double bond.
[139,143]. This produced a mixture of (E)- and (Z)-isomers about the linking double bond.
Scheme 14. Synthetic scheme for generating indolylthiobarbituric acid derivatives 68. Reagents and
Scheme 14. Synthetic scheme for generating indolylthiobarbituric acid derivatives 68. Reagents and
Conditions: (i) NaOMe, MeOH, 60 °C, 6 h, yields not reported; (ii) NaH, DMF, RT, overnight, 83%;
Conditions: (i) NaOMe, MeOH, 60 ˝ C, 6 h, yields not reported; (ii) NaH, DMF, RT, overnight, 83%;
(iii) 6 M HCl, EtOH, RT, 2–3 h, yields not reported.
(iii) 6 M HCl, EtOH, RT, 2–3 h, yields not reported.
4.2.3. Ebselen
4.2.3. Ebselen
Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one, 73), a selenazole heterocyclic compound
with
well-known
antioxidant properties [144], was recently
as a potent
DDAH-1
inhibitor. with
Ebselen
(2-phenyl-1,2-benzisoselenazol-3(2H)-one,
73),identified
a selenazole
heterocyclic
compound
This
was
achieved
by
utilizing
a
high
throughput
screening
(HTS)
assay
of
two
commercial
libraries
well-known antioxidant properties [144], was recently identified as a potent DDAH-1
inhibitor.
comprising 2446 compounds [145]. The HTS identified each compound’s ability to inhibit the
This was achieved by utilizing a high throughput screening (HTS) assay of two commercial libraries
conversion of the synthetic substrate, S-methyl-L-thiocitrulline (SMTC) to citrulline and methanethiol
comprising 2446 compounds [145]. The HTS identified each compound’s ability to inhibit the
by PaDDAH. Compounds exhibiting PaDDAH inhibitory potentials were validated for their ability
conversion
of the synthetic substrate, S-methyl-L-thiocitrulline (SMTC) to citrulline and methanethiol
to inhibit human DDAH-1 using ADMA as the substrate. The quantification of inhibition was
by PaDDAH. Compounds exhibiting PaDDAH inhibitory potentials were validated for their ability to
inhibit human DDAH-1 using ADMA as the substrate. The quantification of inhibition was measured
using derivatized citrulline formation and colorimetric detection. Characterisation of PaDDAH and
Molecules 2016, 21, 615
Molecules 2016, 21, 615
14 of 32
14 of 31
human
DDAH-1
inhibition
by ebselen
was
subsequently
determined
by dose-response,
reversibility,
measured
using
derivatized
citrulline
formation
and colorimetric
detection.
Characterisation
of
and ESI-MS
experiments.
PaDDAH
and human DDAH-1 inhibition by ebselen was subsequently determined by dose-response,
reversibility,
and
ESI-MS
experiments.available, it can be prepared via two main methods [146–148].
While
ebselen
(73)
is commercially
While
ebselen
(73)
is
commercially
available, is
it can
be prepared
main methods
[146–148].
The method described in Scheme
15 [147,148]
preferred
due via
to two
its shorter
reaction
times and
The
method
described
in
Scheme
15
[147,148]
is
preferred
due
to
its
shorter
reaction
times
elementary design. This method involves the treatment of benzoic acid (69) with thionyl and
chloride
elementary design. This method involves the treatment of benzoic acid (69) with thionyl chloride
and aniline to afford benzanilide (70) followed by ortho-lithiation to dianion 71 using n-butyl lithium.
and aniline to afford benzanilide (70) followed by ortho-lithiation to dianion 71 using n-butyl lithium.
Selenium is then inserted into the carbon-lithium bond of 71, forming dianion intermediate 72, whose
Selenium is then inserted into the carbon-lithium bond of 71, forming dianion intermediate 72,
cyclization
to ebselen to
(73)
is promoted
by copper(II)
bromide
oxidant.
whose cyclization
ebselen
(73) is promoted
by copper(II)
bromide
oxidant.
Scheme 15. Synthetic scheme for generating ebselen (73). Reagents and Conditions: (i) (1) DCM,
Scheme
15. Synthetic scheme for generating ebselen (73). Reagents and Conditions: (i) (1) DCM, pyridine,
pyridine, SOCl2, reflux, 2 h; (2) toluene, aniline, reflux, 3 h, 80%; (ii) dry THF, n-BuLi, nitrogen
SOCl2 , reflux, 2 h; (2) toluene, aniline, reflux, 3 h, 80%; (ii) dry THF, n-BuLi, nitrogen atmosphere,
atmosphere, 0 °C, 30 min, 76% (determined via methylation of 72); (iii) same solution as step (ii), Se,
0 ˝ C, 30 min, 76% (determined via methylation of 72); (iii) same solution as step (ii), Se, 0 ˝ C, 30 min;
0 °C, 30 min; (iv) same solution as step (iii), CuBr2, 30 min at −78 °C, then 2 h at RT, 63%. Adapted
(iv) same
solution as step (iii), CuBr2 , 30 min
at ´78 ˝ C, then 2 h at RT, 63%. Adapted with permission
with permission from [147], copyright
1989 American Chemical Society, and [148], copyright 2003
from The
[147],
copyright 1989
American
Chemical Society, and [148], copyright 2003 The Pharmaceutical
Pharmaceutical
Society
of Japan.
Society of Japan.
Ebselen is reported to irreversibly inhibit both PaDDAH (IC50 = 96 ± 2 nM) and human DDAH-1
(IC50 = 330
30 nM), forming
a covalent
selenosulfide
bond with
each
enzyme’s
catalytic
Ebselen
is ±reported
to irreversibly
inhibit
both PaDDAH
(IC50
= 96
˘ 2 nM)respective
and human
DDAH-1
cysteine. In silico docking experiments suggest inhibitor binding is supported by π-stacking
(IC50 = 330 ˘ 30 nM), forming a covalent selenosulfide bond with each enzyme’s respective catalytic
interactions with Phe76 in addition to other polar interactions involving His162. In vitro experiments
cysteine. In silico docking experiments suggest inhibitor binding is supported by π-stacking interactions
demonstrated ebselen exhibits no effect on human DDAH-1 expression [145].
with Phe76
in addition
to other
polar
interactions
His162. In vitrostructure
experiments
demonstrated
Ebselen
is the most
potent
DDAH
inhibitorinvolving
with ‘non-substrate-like’
reported
in the
ebselen
exhibits
no
effect
on
human
DDAH-1
expression
[145].
literature to date and demonstrates an anti-inflammatory response [149]. However, its application as
Ebselen
is the most
potent
with ‘non-substrate-like’
structure
a DDAH inhibitor
is limited
due toDDAH
its poor inhibitor
DDAH selectivity.
A wide range of molecular
targetsreported
are
modulated
by ebselen
NADPH oxidase
[150,151], H+/K+ ATPase
transporter
[152], However,
Ca2+in the
literature
to dateincluding:
and demonstrates
an 2anti-inflammatory
response
[149].
and phospholipid-dependent
protein
[151],
[153],selectivity.
thioredoxin A
reductase
its application
as a DDAH inhibitor
is kinase
limitedC due
to lipoxygenase
its poor DDAH
wide range
+
+ ATPase
[154],
and
metallothionein
[155]).
Moreover,
cellular
toxicity
in
leukocytes
and
hepatocytes
has
of molecular targets are modulated by ebselen including: NADPH oxidase 2 [150,151], H /Kbeen
reported, further reducing
the likelihood of ebselen’s use in clinical practice [156,157].
2+
transporter [152], Ca - and phospholipid-dependent protein kinase C [151], lipoxygenase [153],
thioredoxin
reductase [154], and metallothionein [155]). Moreover, cellular toxicity in leukocytes
4.2.4. 4-Hydroxy-2-Nonenal
and hepatocytes has been reported, further reducing the likelihood of ebselen’s use in clinical
4-Hydroxy-2-nonenal (4-HNE, 76) is a cytotoxic aldehyde generated from lipid peroxidation
practice [156,157].
during inflammation [158,159] and is abundant in the body. 4-HNE is highly reactive toward
nucleophilic groups, such as thiol and imidazole moieties. Nucleophilic attack affords
4.2.4.proteinaceous
4-Hydroxy-2-Nonenal
Michael adducts, whilst Schiff bases are formed with primary amines (e.g., lysine residues) [160–163].
4-Hydroxy-2-nonenal
(4-HNE,
76) for
is athe
cytotoxic
generated
from lipid[164–166].
peroxidation
Different methods have been
reported
synthesisaldehyde
of 4-HNE (76)
and its derivatives
Theinflammation
method reported
by Soulérand
et al. is
[166]
is described
to its4-HNE
simplicity,
versatility
during
[158,159]
abundant
in here
the due
body.
is speed,
highlyand
reactive
toward
(Scheme
16).
The
synthesis
is
completed
in
a
single
cross-metathesis
reaction
between
octen-3-ol
(75)
proteinaceous nucleophilic groups, such as thiol and imidazole moieties. Nucleophilic attack affords
and adducts,
2-propenal
(74) inSchiff
the presence
of Hoveyda-Grubbs
2nd generation
ruthenium
under
a
Michael
whilst
bases are
formed with primary
amines (e.g.,
lysine catalyst
residues)
[160–163].
nitrogen
atmosphere
and
in
dry
oxygen-free
solvent
(Scheme
16).
The
reaction
proceeds
at
room
Different methods have been reported for the synthesis of 4-HNE (76) and its derivatives [164–166].
temperature for 25 h affording the (E)-isomer of 4-HNE (76) in 75% yield.
The method reported by Soulér et al. [166] is described here due to its simplicity, speed, and versatility
(Scheme 16). The synthesis is completed in a single cross-metathesis reaction between octen-3-ol (75)
and 2-propenal (74) in the presence of Hoveyda-Grubbs 2nd generation ruthenium catalyst under
a nitrogen atmosphere and in dry oxygen-free solvent (Scheme 16). The reaction proceeds at room
temperature for 25 h affording the (E)-isomer of 4-HNE (76) in 75% yield.
Molecules 2016, 21, 615
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21, 615
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2016,2016,
21, 615
15 of 32
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15 of 31
Scheme 16. Synthetic scheme for generating 4-hydroxy-2-nonenal (4-HNE, 76). Reagents and
Scheme
16.
scheme
for generating
4-hydroxy-2-nonenal
(4-HNE,(4-HNE,
76). Reagents
Conditions:
Scheme
16.Synthetic
Synthetic
scheme
for generating
4-hydroxy-2-nonenal
76). and
Reagents
and
Conditions: (i) dry oxygen-free DCM, RT, 25 h, 75%, Hoveyda-Grubbs 2nd generation (0.025 eq.).
(i)
dry
oxygen-free
DCM,
RT,
25
h,
75%,
Hoveyda-Grubbs
2nd
generation
(0.025
eq.).
Conditions: (i) dry oxygen-free DCM, RT, 25 h, 75%, Hoveyda-Grubbs 2nd generation (0.025 eq.).
4-HNE is reported to react with cysteine and histidine residues and it was this reactivity that
4-HNE
is reported
to react with
andDDAH-1
histidineinhibitor
residues
andInitForbes’
was this
reactivity that
led Forbes
et al. to investigate
its usecysteine
as a human
[167].
experiments,
DDAHetactivity
was measured
and radioisotope
assays,
withexperiments,
4-HNE
its via
useboth
led Forbes
al. to investigate
investigate
as acolorimetric
human DDAH-1
inhibitor activity
[167]. In
Forbes’
showing
dose-dependent
inhibition
(ICcolorimetric
50 = 50 μM) and
near
complete inhibition
500 μM.
Mass
both
colorimetric
and
radioisotope
4-HNE
DDAH
activity
was measured
via both
and
radioisotope
activity at
assays,
with
4-HNE
spectrometry
experiments
elucidated
the
mechanism
of
inhibition,
whereby
the
formation
of
μM.aMass
showing dose-dependent inhibition (IC50
50 μM)
µM) and
and near
near complete
complete inhibition
inhibition at 500 µM.
50 == 50
Michael
adduct
between
4-HNE
and
His173
of
the
DDAH-1
active-site
occurs.
spectrometryexperiments
experimentselucidated
elucidated
mechanism
of inhibition,
whereby
the formation
of a
spectrometry
thethe
mechanism
of inhibition,
whereby
the formation
of a Michael
Michael
adduct
between
4-HNE
and
His173
of
the
DDAH-1
active-site
occurs.
adduct
between
4-HNE and His173 of the DDAH-1 active-site occurs.
4.2.5.
PD 404182
(PD 404182, 79), is a
4.2.5. PD 6H-6-Imino-(2,3,4,5-tetrahydropyrimido)[1,2-c]-[1,3]benzothiazine
404182
commercially available heterocyclic pyrimidobenzothiazine and a suitable lead compound for SAR
6H-6-Imino-(2,3,4,5-tetrahydropyrimido)[1,2-c]-[1,3]benzothiazine
(PD 404182,
404182,
79),
is a
6H-6-Imino-(2,3,4,5-tetrahydropyrimido)[1,2-c]-[1,3]benzothiazine
studies due to its simple synthetic methodology [168]. PD 404182 (79) is a (PD
compound
listed 79),
in theis
commercially
available
heterocyclic
pyrimidobenzothiazine
and
a suitable
lead
compound
for
heterocyclic
pyrimidobenzothiazine
and
a suitable
lead
compound
for SAR
Library ofavailable
Pharmacologically
Active
Compounds (LOPAC) and
is
reported
to have
antibacterial
SAR
studies
due
to its simple
methodology
PD
(79) is aassay
compound
[168,169]
[170,171]synthetic
activity.
Ghebremariam
et[168].
al.
[61] utilized
toin the
studies
due toand
its antiviral
simple
synthetic
methodology
[168]. PD
404182
(79)404182
is aa colorimetric
compound
listed
screen
a Library
library ofofover
1200Active
compounds
for
theirCompounds
ability to inhibit
conversion
of ADMA
listed
in of
thePharmacologically
Pharmacologically
Active
andtois have
reported
toto have
Library
Compounds
(LOPAC)
and(LOPAC)
is the
reported
antibacterial
L
-citrulline
and
dimethylamine
by
recombinant
human
DDAH-1.
Compounds
with
reasonable
antibacterial
antiviral
[170,171]
activity. Ghebremariam
et al. [61]a utilized
a colorimetric
[168,169] and[168,169]
antiviraland
[170,171]
activity.
Ghebremariam
et al. [61] utilized
colorimetric
assay to
inhibitory
potentials
were
subjected
kinetic characterisation
via fluorimetric
using the synthetic
assay
toascreen
a library
of1200
over
1200 to
compounds
for their
ability
to inhibit
the
conversion
of ADMA
screen
library
of over
compounds
for their
ability
to inhibit
theassay
conversion
of ADMA
to
substrate, S-methylthiocitrulline. Reversibility and ESI-MS studies were additionally undertaken.
to
L -citrulline
and
dimethylaminebybyrecombinant
recombinanthuman
human DDAH-1.
DDAH-1. Compounds
Compounds with
with reasonable
L-citrulline
and
dimethylamine
reasonable
PD 404182 was subsequently evaluated for its ability to reduce angiogenesis and to protect against
inhibitory potentials were subjected
to
kinetic
characterisation
via fluorimetric assay using the synthetic
subjected
to
kinetic
characterisation
synthetic
lipopolysaccharide-induced NO production. PD 404182 (79) was found to be a potent irreversible
substrate,
S-methylthiocitrulline.
Reversibility
and
ESI-MS
studies
were
additionally
undertaken.
S-methylthiocitrulline.
Reversibility
studies
wereasadditionally
undertaken.
competitive
human DDAH-1 inhibitor
(IC50 = 9and
μM)ESI-MS
and showed
promise
an anti-inflammatory
PD 404182
was subsequently
against
and antiangiogenic
agent. evaluated for its ability to reduce angiogenesis and to protect against
lipopolysaccharide-induced
NO production.
production.
PDvia
404182
(79) was
was
found
irreversible
lipopolysaccharide-induced
NO
PD
404182
(79)
found
to approaches
be a potent
potentgenerate
irreversible
The synthesis of PD404182
can be achieved
two routes
[172,173].
Both
competitive
human
DDAH-1
inhibitor over
(IC50
µM)
and showed
promise
as an anti-inflammatory
reasonable
to high
yields (61%–88%)
synthetic
steps. The
first approach
involves the
50several
== 99 μM)
anti-inflammatory
reaction of 2-(2′-haloaryl)-tetrahydropyrimidine
77 with carbon disulfide in the presence of sodium
and antiangiogenic
agent.
hydride,
followed
byPD404182
hydrolysis
ofbethe
derivative
78 [172,173].
andBoth
subsequent
The
synthesis
becarbamodithioate
achieved
via routes
two
routes
Bothtreatment
approaches
synthesis
ofofPD404182
cancan
achieved
via two
[172,173].
approaches
generate
with
cyanogen
bromide
to
afford
79
(Scheme
17;
Route
A).
Alternatively,
the
2-(2′-haloaryl)generate
reasonable
to high
yields (61%–88%)
over
severalsteps.
synthetic
steps.
The first
approach
reasonable
to high yields
(61%–88%)
over several
synthetic
The first
approach
involves
the
tetrahydropyrimidine 77 can1 be reacted with tert-butylisothiocyanate (80) in the presence of sodium
involves
reaction of 2-(2 -haloaryl)-tetrahydropyrimidine
77 with incarbon
disulfide
in the
reaction ofthe
2-(2′-haloaryl)-tetrahydropyrimidine
77 with carbon disulfide
the presence
of sodium
hydride to afford intermediate 81, followed by treatment with trifluoroacetic acid to afford 79
presence
of sodiumbyhydride,
followed
hydrolysis of the
carbamodithioate
derivative
78 and
hydride,
followed
hydrolysis
of the by
carbamodithioate
derivative
78 and subsequent
treatment
(Scheme 17; Route B).
subsequent
treatment
with
afford
79 A).
(Scheme
17; Routethe
A).2-(2′-haloaryl)Alternatively,
with cyanogen
bromide
to cyanogen
afford 79bromide
(Schemeto17;
Route
Alternatively,
the
2-(21 -haloaryl)-tetrahydropyrimidine
77 can
be reacted with tert-butylisothiocyanate
in the
tetrahydropyrimidine
77 can be reacted with
tert-butylisothiocyanate
(80) in the presence (80)
of sodium
presence
of
sodium
hydride
to
afford
intermediate
81,
followed
by
treatment
with
trifluoroacetic
hydride to afford intermediate 81, followed by treatment with trifluoroacetic acid to affordacid
79
to
afford 17;
79 (Scheme
(Scheme
Route B).17; Route B).
Scheme 17. Synthetic scheme for generating PD 404182 (79). Reagents and Conditions: Route A: (i)
DMF, NaH, CS2, argon atmosphere, 80 °C, 12 h, 88%; (ii) (1) 0.1 M NaOH, MeOH/H2O (9:1), reflux, 12
h; (2) anhydrous EtOH, BrCN, argon atmosphere, reflux, 2 h, 61% (two steps). Route B: (iii) DMF,
NaH, argon atmosphere, 80 °C, 2 h, 62%; (iv) TFA, CHCl3, molecular sieves 4 Å, reflux, 1 h, 85%.
Adapted with permission from [172,173]. Copyright 1975, 2010 American Chemical Society.
Scheme 17.
17. Synthetic
Syntheticscheme
schemeforfor
generating
404182
Reagents
and Conditions:
(i)
Scheme
generating
PDPD
404182
(79).(79).
Reagents
and Conditions:
RouteRoute
A: (i) A:
DMF,
˝
2
,
argon
atmosphere,
80
°C,
12
h,
88%;
(ii)
(1)
0.1
M
NaOH,
MeOH/H
2
O
(9:1),
reflux,
12
DMF,
NaH,
CS
NaH, CS2 , argon atmosphere, 80 C, 12 h, 88%; (ii) (1) 0.1 M NaOH, MeOH/H2 O (9:1), reflux, 12 h;
h; (2)
anhydrous
EtOH,
BrCN,
argon
atmosphere,
reflux,
2 h, 61%
steps).
(iii) DMF,
(2)
anhydrous
EtOH,
BrCN,
argon
atmosphere,
reflux,
2 h, 61%
(two (two
steps).
RouteRoute
B: (iii)B:DMF,
NaH,
3, molecular
sieves
4
Å,
reflux,
1
h,
85%.
NaH, argon
atmosphere,
2 h,
62%;
(iv)
TFA,
CHCl
argon
atmosphere,
80 ˝ C, 280h,°C,
62%;
(iv)
TFA,
CHCl
,
molecular
sieves
4
Å,
reflux,
1
h,
85%.
Adapted
3
Adapted
with permission
from [172,173].
2010 American
with
permission
from [172,173].
CopyrightCopyright
1975, 20101975,
American
Chemical Chemical
Society. Society.
Molecules 2016, 21, 615
Molecules 2016, 21, 615
16 of 32
16 of 31
4.2.6. Proton Pump Inhibitors
4.2.6. Proton Pump Inhibitors
+ ATPase pump inhibitors (PPIs) are widely prescribed to inhibit gastric
Molecules 2016,
21, 615
16 of 31 acid
Proton
H+ /K
Proton H+/K+ ATPase pump inhibitors (PPIs) are widely prescribed to inhibit gastric acid
secretion
and
are
characterized
by
a
2-pyridylmethylsulfinylbenzimidazole
scaffold.
The
chemical
secretion and are characterized by a 2-pyridylmethylsulfinylbenzimidazole scaffold. The chemical
4.2.6. Proton Pump Inhibitors
structures
of the
prescribed
inFigure
Figure3.3.
structures
of commonly
the commonly
prescribedPPIs
PPIs82a–f
82a–f are
are shown
shown in
Proton H+/K+ ATPase pump inhibitors (PPIs) are widely prescribed to inhibit gastric acid
secretion and are characterized by a 2-pyridylmethylsulfinylbenzimidazole scaffold. The chemical
structures of the commonly prescribed PPIs 82a–f are shown in Figure 3.
Figure 3. Chemical structures of commonly prescribed proton pump inhibitors (PPIs) 82a–f.
Figure 3. Chemical structures of commonly prescribed proton pump inhibitors (PPIs) 82a–f.
HTS experiments utilising a library of 130,000 compounds and recombinant enzyme identified
HTS
experiments
utilising
a library
of 130,000
compounds
and
recombinant
identified
Figure 3.DDAH-1
Chemical
structures
of
commonly
prescribed
proton
pump
inhibitors using
(PPIs)enzyme
82a–f.
PPIs as
human
inhibitors
[174,175].
Initial
screening
was
undertaken
a colorimetric
assay
with
ADMA
as
the
substrate.
Further
characterization
of
compounds
of
interest
was
achieved
PPIs as human DDAH-1 inhibitors [174,175]. Initial screening was undertaken using a colorimetric
experiments
a library
of 130,000
compounds
and
recombinant
enzyme (SPR)
identified
aHTS
fluorimetric
using S-methylthiocitrulline
[174,175]. of
Surface
plasmonof
resonance
was
assayvia
with
ADMA
asassay
the utilising
substrate.
Further
characterization
compounds
interest
was achieved
PPIs
as human
DDAH-1
inhibitors
[174,175].ofInitial
screening
was undertaken
using
a sensor
colorimetric
additionally
utilized
to
detect
the
binding
PPIs
to
amine-coupled
DDAH-1
(CM5
chip)
via a fluorimetric assay using S-methylthiocitrulline [174,175]. Surface plasmon resonance (SPR) was
assay
ADMA as theavailable
substrate.
Further
of compounds
of interest
achieved
[175]. with
All commercially
PPIs
werecharacterization
found to reversibly
inhibit DDAH-1
(IC50 was
= 51–63
μM).
additionally utilized to detect the binding of PPIs to amine-coupled DDAH-1 (CM5 sensor chip) [175].
via
a
fluorimetric
assay
using
S-methylthiocitrulline
[174,175].
Surface
plasmon
resonance
(SPR)
was
Interestingly, in vitro experiments using human endothelial cells and in vivo experiments in mice
All commercially
available
PPIsthewere
found
to to
reversibly
inhibit
DDAH-1
(IC
= 51–63
µM).
50
additionally
utilized
to
detect
binding
of
PPIs
amine-coupled
DDAH-1
(CM5
sensor
chip)
demonstrated that PPIs increase ADMA concentrations [175] further supporting the evidence that
Interestingly,
inuse
vitro
experiments
using
human
cells
and
indisease
vivo (IC
experiments
in mice
[175].
All commercially
PPIs
were
foundendothelial
to risk
reversibly
inhibit
DDAH-1
50 = 51–63 μM).
prolonged
of PPIs
isavailable
associated
with
an increased
of cardiovascular
[176,177].
demonstrated
that
PPIs
increase
ADMA
concentrations
[175]
further
supporting
the
evidence
Interestingly,
in
vitro
experiments
using
human
endothelial
cells
and
in
vivo
experiments
in
mice
PPIs are comprised of pyridine and benzimidazole moieties with different substituents. Their that
demonstrated
that is
PPIs
increase
ADMA
[175]
further
supporting
the evidence
that
prolonged
use are
of PPIs
associated
with
anconcentrations
increased
of cardiovascular
disease
[176,177].
syntheses
protected
by current
patents
(with therisk
exception
of omeprazole
(82a)).
The original
prolonged
use
of
PPIs
is
associated
with
an
increased
risk
of
cardiovascular
disease
[176,177].
PPIs
pyridine
and benzimidazole
moieties
with different substituents.
patentare
forcomprised
omeprazoleof
(82a)
[178] details
preparation of the
2-(chloromethyl)pyridine
derivative 84.Their
PPIs
comprised
of
pyridine
andwith
benzimidazole
moieties of
with
different
substituents.
Their
Condensation
[179] of
84
2-mercaptobenzimidazole
derivative
83 affords
the
syntheses
are are
protected
bycompound
current
patents
(with
the exception
omeprazole
(82a)).
The
original
syntheses
are
protected
by
current
patents
(with
the
exception
of
omeprazole
(82a)).
The
original
thioether
precursor (82a)
85, which
subsequently
oxidized
sulfoxide in omeprazole (82a)
using 84.
patent
for omeprazole
[178]isdetails
preparation
of to
thethe
2-(chloromethyl)pyridine
derivative
patent
for omeprazole
(82a) [178]
details preparation
ofcatalyst
the 2-(chloromethyl)pyridine
derivative
84.
conditions
such of
as compound
hydrogen
peroxide
a vanadium
[180] derivative
(Scheme 18;83
Route
A). the thioether
Condensation
[179]
84 withand
2-mercaptobenzimidazole
affords
Condensation [179] of compound 84 with 2-mercaptobenzimidazole derivative 83 affords the
precursor 85, which is subsequently oxidized to the sulfoxide in omeprazole (82a) using conditions
thioether precursor 85, which is subsequently oxidized to the sulfoxide in omeprazole (82a) using
such conditions
as hydrogen
andperoxide
a vanadium
[180]
(Scheme
Route18;
A).
suchperoxide
as hydrogen
and acatalyst
vanadium
catalyst
[180] 18;
(Scheme
Route A).
Scheme 18. Synthetic scheme for generating omeprazole (82a). Reagents and Conditions: Route A: (i)
1 M NaOH (2 eq.), EtOH, RT, 8 h; (ii) catalyst: V2O5, NaVO3, NH4VO3, [(CH3COCH2COCH3)2VO]
(0.0001–0.1 eq.), H2O2: 10%–70% aqueous or organic solution (1–3 eq.), solvent: halogenated
Scheme
Synthetic
forketones,
generating
omeprazole
Reagents0and
Route of
A:the
(i)
temperature:
°C Conditions:
to boiling point
hydrocarbons,
ethers, scheme
alcohols,
nitriles,
or H2O, (82a).
Scheme 18. 18.
Synthetic
scheme
for
generating
omeprazole
(82a).
Reagents
and Conditions:
Route
A:
1solvent,
M NaOH
(2 eq.),
EtOH, RT,Route
8 h; (ii)
V2O5, NaVO
3, NH4VOnot
3, [(CH
3COCHin
2COCH
3)2VO]
0.5–24
h, 89%–93%.
B: catalyst:
(iii) condensation,
conditions
specified
patent;
(iv)
(i) 1 M NaOH (2 eq.), EtOH, RT, 8 h; (ii) catalyst: V2 O5 , NaVO3 , NH4 VO3 , [(CH3 COCH2 COCH3 )2 VO]
(0.0001–0.1
H23O
: 10%–70%
aqueous
solvent:
halogenated
[(CH3COCHeq.),
2COCH
)22VO]
(0.006 eq.),
30% H2or
O2, organic
acetone, solution
0–5 °C for(1–3
1 h eq.),
then 20–22
°C for
1 h, 90%;
(0.0001–0.1 eq.), H2 O2 : 10%–70% aqueous or organic solution (1–3 eq.), solvent: halogenated
temperature:
°Cv/v
to isopropanol/toluene,
boiling point of the
hydrocarbons,
alcohols,
ketones,
nitriles,
or CO
H2O,
(v) 10% NaOH,ethers,
nitrogen
atmosphere,
50 °C,
3 h; (vi)
2, extraction
into01:1
hydrocarbons,
ethers,
alcohols,
ketones,
nitriles,
or H2 O,conditions
temperature:
0 ˝ C toinboiling
of
solvent,
0.5–24
h, 42%
89%–93%.
B: and
(iii)
condensation,
not specified
patent; point
(iv)
reflux, 20–30
min,
(acrossRoute
steps (v)
(vi)).
the solvent,
0.5–24
h,
89%–93%.
Route
B:
(iii)
condensation,
conditions
not
specified
in
patent;
[(CH3COCH2COCH3)2VO] (0.006 eq.), 30% H2O2, acetone, 0–5 °C for 1 h then 20–22 °C for 1 h, 90%;
(iv) [(CH
(0.006 eq.),
30%
H2(vi)
O2 CO
, acetone,
0–5 ˝into
C for1:11 h
20–22 ˝ C for 1 h, 90%;
(v) 10%
NaOH,
nitrogen
atmosphere,
50 °C,
3 h;
2, extraction
v/vthen
isopropanol/toluene,
3 COCH
2 COCH
3 )2 VO]
˝
(v) 10%
NaOH,
C, 3(vi)).
h; (vi) CO2 , extraction into 1:1 v/v isopropanol/toluene,
reflux,
20–30nitrogen
min, 42%atmosphere,
(across steps 50
(v) and
reflux, 20–30 min, 42% (across steps (v) and (vi)).
Molecules 2016, 21, 615
17 of 32
Further development of the synthetic method to yield omeprazole (82a) [180] identified that the
Molecules
2016, 21, the
615 pyridine and benzimidazole moieties can be modified to simplify purification.
17 of 31
coupling
between
Principally, this method avoids the formation of the thioether intermediate 85. Furthermore, the crude
Further development of the synthetic method to yield omeprazole (82a) [180] identified that the
product containing omeprazole (82a) produced from the original synthesis is prone to discolouration
coupling between the pyridine and benzimidazole moieties can be modified to simplify purification.
during the oxidation step [180]. The revised synthesis (Scheme 18; Route B) proceeds via condensation
Principally, this method avoids the formation of the thioether intermediate 85. Furthermore, the
of 2-chloromercaptobenzimidazole
derivative
86 with afrom
primary
amide pyridine
crude product containing omeprazole
(82a) produced
the original
synthesis derivative
is prone to87 to
afford
the
acetamide
thioether
intermediate
88,
which
is
oxidized
to
the
acetamide
sulfoxide 89.
discolouration during the oxidation step [180]. The revised synthesis (Scheme 18; Route B) proceeds
Compound
89 is subsequently
hydrolysed to the carboxylic
acid salt
90 under
alkaline
conditions,
via condensation
of 2-chloromercaptobenzimidazole
derivative
86 with
a primary
amide
pyridine with
derivative 87yielding
to affordomeprazole
the acetamide
thioether
intermediate(82c),
88, which
is oxidized
to the
acetamide 86
decarboxylation
(82a)
or lansoprazole
depending
on the
benzimidazole
sulfoxide
89. Compound
89 isused
subsequently
hydrolysed
to the carboxylic acid salt 90 under alkaline
and the
pyridine
87 derivatives
as the starting
materials.
conditions, with decarboxylation yielding omeprazole (82a) or lansoprazole (82c), depending on the
86 and the pyridine 87 derivatives used as the starting materials.
4.2.7.benzimidazole
1,2,3-Triazoles
The
of 1,2,3-triazoles 95 (Scheme 19) comprised of 4-halopyridine and benzimidazole
4.2.7.synthesis
1,2,3-Triazoles
units have been explored as ‘non-substrate-like’ DDAH-1 inhibitors [117]. The synthesis of these
The synthesis of 1,2,3-triazoles 95 (Scheme 19) comprised of 4-halopyridine and benzimidazole
derivatives was undertaken based on reports that identified binding of both the 4-halopyridine and
units have been explored as ‘non-substrate-like’ DDAH-1 inhibitors [117]. The synthesis of these
benzimidazole
fragments
to human
DDAH-1
[181].
A triazole
linker
wasthe
selected
to connect
derivatives was
undertaken
based on
reports that
identified
binding
of both
4-halopyridine
andthese
fragments
via copper(I)-catalysed
‘click’
cycloaddition
between
thewas
terminal
92 and
benzimidazole
fragments to human
DDAH-1
[181]. A triazole
linker
selectedalkyne
to connect
theseazide
94 (Scheme
19).
alkyne 92 is synthesized
by Sonogashira
of the92alkyne
derivative
fragments
via The
copper(I)-catalysed
‘click’ cycloaddition
between thecoupling
terminal alkyne
and azide
94
with (Scheme
the required
species 91by[182,183].
The
azideof94theisalkyne
synthesized
bywith
reaction
of
19). Thehalogenated
alkyne 92 is synthesized
Sonogashira
coupling
derivative
the
required
halogenated
species
91
[182,183].
The
azide
94
is
synthesized
by
reaction
of
diphenylphosphoryl azide with the required primary alcohol 93 [184].
diphenylphosphoryl
azide with albeit
the required
primary
alcohol 93 [184].
Human
DDAH-1 inhibition,
low (IC
50 = 422 µM, 57%, inhibition at 1 mM), was reported
Human
DDAH-1
inhibition,
albeit
low
(IC
50 = 422 μM, 57%, inhibition at 1 mM), was reported
with triazole 95a. In general, 95a–b failed to show significant inhibitory potential and may be due to
with triazole 95a. In general, 95a–b failed to show significant inhibitory potential and may be due to
the triazole linker not facilitating a productive inhibitory binding conformation of the fragments in the
the triazole linker not facilitating a productive inhibitory binding conformation of the fragments in
enzyme active-site.
the enzyme active-site.
Scheme 19. Synthetic scheme for generating 1,2,3-triazoles 95. Reagents and Conditions: (i) 92a
Scheme 19. Synthetic scheme for generating 1,2,3-triazoles 95. Reagents and Conditions: (i) 92a
(Ph3P)2PdCl2, CuI, TEA, TIPS-acetylene, DMF, 80 °C, 72 h, 30%, 92b (Ph3P)2PdCl2, CuI, TEA, ethynyl-TMS,
(Ph3 P)2 PdCl2 , CuI, TEA, TIPS-acetylene, DMF, 80 ˝ C, 72 h, 30%, 92b (Ph3 P)2 PdCl2 , CuI, TEA,
80 °C, 20 h, 48%; (ii) DPPA, DBU, toluene, RT, 16 h, 94a 55%, 94b 83%; (iii) CuI, sodium ascorbate,
ethynyl-TMS, 80 ˝ C, 20 h, 48%; (ii) DPPA, DBU, toluene, RT, 16 h, 94a 55%, 94b 83%; (iii) CuI, sodium
TEA, DCM, RT, 24 h, 95a 65%, 95b 30%. Reproduced in part from [117] with permission of the Royal
ascorbate,
DCM, RT, 24 h, 95a 65%, 95b 30%. Reproduced in part from [117] with permission of
SocietyTEA,
of Chemistry.
the Royal Society of Chemistry.
5. Conclusions
5. Conclusions
Current evidence suggest that inhibiting the DDAH family of enzymes may be used as a
therapeutic
tool to indirectly
the NOS enzymes
via increasing
of endogenous
Current
evidence
suggestinhibit
that inhibiting
the DDAH
family concentrations
of enzymes may
be used as a
NOS inhibitors, such as ADMA and L-NMMA. Therefore the development and synthesis of DDAH
therapeutic tool to indirectly inhibit the NOS enzymes via increasing concentrations of endogenous
inhibitors is emerging as a promising field of clinical research. Furthermore, engineering compounds
NOS inhibitors, such as ADMA and L-NMMA. Therefore the development and synthesis of DDAH
that target bacterial DDAH may represent a suitable strategy for the development of new
inhibitors
is emerging as a promising field of clinical research. Furthermore, engineering compounds
antimicrobial agents; however further investigations are required. This has led to different classes of
that target
DDAH
represent
a suitable
strategycharacterized
for the development
of new
antimicrobial
DDAHbacterial
inhibitors
beingmay
reported
within
the last decade
by alternative
features
in
agents;
however
further
investigations
are
required.
This
has
led
to
different
classes
of DDAH
terms of chemical structure, potency, enzyme selectivity, and mechanism of action. The importance
inhibitors
reported
within depends
the last on
decade
characterized
features
in terms of
placedbeing
on each
of these features
the end
application of by
the alternative
inhibitor. Table
3 summarizes
the most
potent DDAH
inhibitors
themechanism
literature to date.
Some compounds,
namely
chemical
structure,
potency,
enzymedocumented
selectivity,inand
of action.
The importance
placed
PFPof
sulfonate
esters 61depends
or indolylthiobarbituric
acid derivatives
exhibit Table
significant
PaDDAH the
on each
these features
on the end application
of the 68,
inhibitor.
3 summarizes
most potent DDAH inhibitors documented in the literature to date. Some compounds, namely PFP
Molecules 2016, 21, 615
18 of 32
sulfonate esters 61 or indolylthiobarbituric acid derivatives 68, exhibit significant PaDDAH inhibition.
In particular, selectivity experiments using indolylthiobarbiturates failed to exhibit inhibitory effects
on the human isoenzyme, therefore derivatives of this structural class are well positioned for further
investigation in antibiotic applications. Among classes of molecules which are active on the human
isoenzyme (amino acid derivatives 27, 29, 39, 40, 44, 49, 54, ebselen (73), 4-HNE (76), PD 404182
(79), PPIs 82, 1,2,3-triazoles 95), L-257 (39) and L-291 (40) are highly selective for DDAH-1, while
ornithine derivatives such as L-VNIO (54a) or L-IPO (54b) are known to inhibit not only DDAH, but
also arginase and the NOS family of enzymes. Furthermore, compounds such as ebselen (73) and
4-HNE (76), although very potent DDAH inhibitors, are well known to exhibit a range of off-target
effects, thus are more suitable for experiments in vitro rather than in vivo.
In terms of the varied approaches to chemical synthesis of the DDAH inhibitors discussed here,
many can be synthesized in moderate to excellent overall yields and use reaction sequences that can
be easily modified to enable the design of a large number of analogues. The commercial availability
of several of the compounds determined to be DDAH inhibitors (2-chloroacetamidine (11), ebselen
(73) 4-HNE (76) and the PPIs 82) demonstrates the versatility of some of the synthetic routes for large
scale production.
Based on the in vitro pharmacokinetic and/or enzyme selectivity data compiled in this review,
there appear to be three promising human DDAH-1 inhibitors that have the potential to progress to
clinical trial: ZST316 (Ki = 1 µM), Cl-NIO (Ki = 1.3 µM), and L-257 (Ki = 13 µM) (Table 3). Despite
exhibiting the most potent inhibitory response in vitro, a paucity of data exists with respect to the
in vivo pharmacokinetic and pharmacodynamic profiling of ZST316. Literature reports involving
the enzyme selectivity of Cl-NIO shows considerable promise, with no measurable cross-reactivity
in experiments specifically involving eNOS [129]. However, data are not available regarding this
inhibitor‘s interactions with iNOS, nNOS, or the arginase family of enzymes. Contrary to this, data
reported for L-257 by Kotthaus et al. [115] demonstrates that L-257 is highly selective for DDAH-1.
The administration of L-257 in a rodent model of septic shock not only improved survival, but
additionally improved haemodynamic and organ function [67], indicating a potential clinical role
for L-257.
Interestingly, little is understood about the regulation and pharmacological modulation of
human DDAH-2. As such, the development of improved metabolomic approaches for the sensitive
measurement of DDAH-2 activity in vitro is an important step forward in profiling the endogenous
substrates and biochemical role(s) of this important enzyme in humans.
The continued exploration of DDAH inhibition and the synthesis of new chemical entities with
improved pharmacokinetic parameters are therefore at an exciting junction in the development of
novel medicines.
Molecules 2016, 21, 615
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Molecules 2016, 21, 615
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Chemical
Chemical
Molecules
2016,Chemical
21, 615
Name
19 of 32
Table 3. Summary of the main DDAH inhibitors and their kinetic parameters.
Table 3. Summary of the main DDAH inhibitors and their kinetic parameters.
Table 3. Summary of the main DDAH inhibitors and their kinetic parameters.
Table 3. Summary of the main DDAH inhibitors and their kinetic parameters.
Table 3. Summary of the main DDAH inhibitors and their kinetic parameters.
Chemical
Enzyme
IC5050(μM)
(µM)
Table 3. Summary
of theStructure
main DDAH inhibitors and
their kinetic parameters.
Chemical Name
Chemical
Structure
Enzyme
IC
Chemical
Number
Table 3. Summary
of theStructure
main DDAH inhibitors and
their kinetic parameters.
Chemical
Number Chemical Name
Chemical
Enzyme
IC50 (μM)
Table 3. Summary
of theStructure
main DDAH inhibitors and
their kinetic parameters.
Chemical
Chemical Name
Chemical
Enzyme
IC
50 (μM)
Number
Table 3. Summary
of the
main DDAH inhibitors and
their kinetic parameters.
Chemical
Chemical
Structure
Enzyme
IC50 (μM)
Number Chemical Name
Table 3. Summary
of the
main DDAH inhibitors and
their kinetic parameters.
Chemical
Chemical
Structure
Enzyme
IC50 (μM)
Chemical
Number
L-homocysteine
S-Nitroso-Name
Chemical
Number
Chemical
Name
Chemical
Structure
Enzyme
IC50 (μM)
(9)
Bovine
DDAH-1
75
L
-homocysteine
S-Nitroso(9) Chemical
S-NitrosoL
-homocysteine
(HcyNO)
Bovine
DDAH-1
75
Chemical
Name
Chemical
Structure
Enzyme
IC50 (μM)
Number
L
-homocysteine
S-Nitroso(HcyNO) Name
(9)
Bovine
DDAH-1
Chemical
Chemical
Chemical
Structure
Enzyme
IC5075
(μM)
Number
LName
-homocysteine
S-Nitroso(9)
Bovine
DDAH-1
(HcyNO)
Chemical
Chemical
Structure
Enzyme
IC
5075
(μM)
Number
S-Nitroso(9)
Bovine DDAH-1
75
(HcyNO) L-homocysteine
Number
(9)
Bovine DDAH-1
75
S-Nitroso-L-homocysteine
(HcyNO)
S-Nitroso-L-homocysteine
(HcyNO)
(9)
Bovine DDAH-1
75
1
L-homocysteine
S-Nitroso(9)
Bovine
DDAH-1
75(HcyNO)
(11)
2-Chloroacetamidine
PaDDAH
11
S-Nitroso-L-homocysteine
(9) 2-Chloroacetamidine
Bovine
DDAH-1
75
(HcyNO)
-2-Chloroacetamidine
PaDDAH
(11) (11)
1
(9)
Bovine
DDAH-1
75
(HcyNO)
(11)
2-Chloroacetamidine
PaDDAH1
(HcyNO)
(11)
2-Chloroacetamidine
PaDDAH 1
(11)
2-Chloroacetamidine
PaDDAH 1
N-But-3-ynyl-2-chloro-acetamid
(11)
2-Chloroacetamidine
PaDDAH
(18)
Human
DDAH-1
350
N-But-3-ynyl-2-chloro-acetamid
1
(11)
2-Chloroacetamidine
PaDDAH
N-But-3-ynyl-2-chloro-acetamid
1
ine
(18)
Human
DDAH-1
350
2-Chloroacetamidine
PaDDAH
N-But-3-ynyl-2-chloro-acetamid
1
(18) N-But-3-ynyl-2-chloro-acetamidine
DDAH-1
350
(18) (11)
Human
DDAH-1
350
ine
(11)
2-Chloroacetamidine
PaDDAH
N-But-3-ynyl-2-chloro-acetamid
(18)
Human
DDAH-1
350
ine
NH
NH2
(18)
Human
DDAH-1
350
N-But-3-ynyl-2-chloro-acetamid
ine
NH
NH
2
N-But-3-ynyl-2-chloro-acetamid
ine
(18)
Human DDAH-1
350
(S)-2-Amino-4-(3-methylguanid
NH
NH2 OH
N-But-3-ynyl-2-chloro-acetamid
(18)
Human
DDAH-1
350
ine
(21)
Rat DDAH-1
416
(S)-2-Amino-4-(3-methylguanid
NH2 OH
N NH N
N-But-3-ynyl-2-chloro-acetamid
(18)
Human
DDAH-1
350
ine
(S)-2-Amino-4-(3-methylguanid
ino)butanoic acid (4124W)
(21)
Rat DDAH-1
416
NH NH
NH2
H
OH
N
(18)
Human
DDAH-1
350
ine
(S)-2-Amino-4-(3-methylguanid
(21) (S)-2-Amino-4-(3-methylguanidino)
Rat
DDAH-1
416
ino)butanoic
acid
(4124W)
NH
NH
OH
N
N
O
H
H
2
ine
(S)-2-Amino-4-(3-methylguanid
Rat
416
ino)butanoic acid (4124W)
(21) (21)
RatDDAH-1
DDAH-1
416
NH2O OH
NH NHNH
(21) butanoic
Rat DDAH-1
416
(S)-2-Amino-4-(3-methylguanid
ino)butanoic
acid (4124W)
acid (4124W)
NH2O OH
N NHH
N
H
(S)-2-Amino-4-(3-methylguanid
ino)butanoic
acid (4124W)
(21)
Rat DDAH-1
416
NH
NH
H
H
OH
G
N
N
O
2
NG -(2-Methoxyethyl)L-arginine
(S)-2-Amino-4-(3-methylguanid
(21)
Rat DDAH-1
416 2
ino)butanoic
acid (4124W)
N
N
O OH
H
H
(S)-2-Amino-4-(3-methylguanid
(39)
Human
DDAH-1
31
N
-(2-Methoxyethyl)L-arginine
(21)
Rat DDAH-1
416
ino)butanoic
acid (4124W)
N
N
H
H
G-(2-Methoxyethyl)O OH
N
L-arginine
(21)
Rat DDAH-1
41622
(L-257)
(39)
Human
DDAH-1
31
ino)butanoic
acid (4124W)
N
N
H
H
G-(2-Methoxyethyl)O
N
L
-arginine
(39) G (L-257)
Human DDAH-1
312
ino)butanoic
acid (4124W)
H
H
G
O
N(L-257)
-(2-Methoxyethyl)L-arginine
(39) N -(2-Methoxyethyl)Human DDAH-1
31 2 2
L -arginine
O
Human
31312
NGG-(2-Methoxyethyl)-L-arginine
(L-257)
(39) (39)
HumanDDAH-1
DDAH-1
N GG-(2-Methoxyethyl)-L-arginine
(L-257)
(39) (L-257)
Human DDAH-1
31 2
N
-(2-Methoxyethyl)LL-arginine
(39)
Human
DDAH-1
31 2
(L-257)
N
-(2-Methoxyethyl)-arginine
G
NG-(2-Methoxyethyl)-(2-Methoxyethyl)-LL-arginine
-arginine
(39)
Human
DDAH-1
31
(L-257)
(40)
Rat
DDAH-1
20
N
G-(2-Methoxyethyl)-L-arginine
(39)
Human
DDAH-1
31
(L-257)
N
methyl
ester
(L-291)
(40)
Rat
DDAH-1
20 2
G
(L-257)
Nmethyl
-(2-Methoxyethyl)L-arginine
(40)
Rat DDAH-1
20
ester
(L-291)
G
Nmethyl
-(2-Methoxyethyl)(40)
Rat DDAH-1
20
ester (L-291) L-arginine
(40) NG -(2-Methoxyethyl)Rat DDAH-1
20
NGG-(2-Methoxyethyl)L-arginine
methyl
ester (L-291)
L -arginine
N2-Amino-5-(3-(2-methoxyethyl)
-(2-Methoxyethyl)methyl
ester (L-291) L-arginine
Rat
DDAH-1
2020
(40) (40)
Rat
DDAH-1
G
2-Amino-5-(3-(2-methoxyethyl)
N Gester
-(2-Methoxyethyl)L-arginine
(40) methyl
Rat DDAH-1
20
methyl
ester
(L-291)
(L-291)
2-Amino-5-(3-(2-methoxyethyl)
N
-(2-Methoxyethyl)(44a)
guanidino)-N-(methylsulfonyl)Human
DDAH-1
3
(40)
Rat DDAH-1
20
methyl
ester (L-291) L-arginine
2-Amino-5-(3-(2-methoxyethyl)
(40)
Rat
DDAH-1
20
(44a)
guanidino)-N-(methylsulfonyl)Human
DDAH-1
3
methyl ester (L-291)
2-Amino-5-(3-(2-methoxyethyl)
(44a)
guanidino)-N-(methylsulfonyl)Human DDAH-1
3
methyl
ester (L-291)
pentanamide
(ZST316)
2-Amino-5-(3-(2-methoxyethyl)
(44a)
guanidino)-N-(methylsulfonyl)Human DDAH-1
3
pentanamide (ZST316)
2-Amino-5-(3-(2-methoxyethyl)
(44a) 2-Amino-5-(3-(2-methoxyethyl)
guanidino)-N-(methylsulfonyl)Human DDAH-1
3
pentanamide (ZST316)
2-Amino-5-(3-(2-methoxyethyl)
(44a)
guanidino)-N-(methylsulfonyl)Human
DDAH-1
3
pentanamide
(ZST316)
5
2-Amino-5-(3-(2-methoxyethyl)
N5 -(1-Imino-3-butenyl)guanidino)-N-(methylsulfonyl)Human
DDAH-1
33
pentanamide
(ZST316) L-ornith
(44a) (44a)
guanidino)-N-(methylsulfonyl)Human
DDAH-1
(54a)
Human DDAH-1
DDAH-1
13
-(1-Imino-3-butenyl)N
(44a)
guanidino)-N-(methylsulfonyl)Human
3
pentanamide
(ZST316) LL-ornith
5-(1-Imino-3-butenyl)-ornith
N
(44a) pentanamide
guanidino)-N-(methylsulfonyl)Human
DDAH-1
3
ine
(L-VNIO)
(54a)
Human
DDAH-1
13
(ZST316)
pentanamide
(ZST316) L-ornith
5-(1-Imino-3-butenyl)N
(54a)
Human DDAH-1
13
ine
(L-VNIO)
pentanamide
(ZST316)
5
-(1-Imino-3-butenyl)L-ornith
Npentanamide
(54a)
Human
DDAH-1
13
ine
(L-VNIO)
(ZST316)
(54a)
Human DDAH-1
13
L-ornith
N55-(1-Imino-3-butenyl)ine
(L-VNIO)
L-ornith
N 5-(1-Imino-3-butenyl)(L-VNIO)
(54a) 5 ine
Human DDAH-1
13
5-(1-Imino-3-butenyl)L-ornith
N
(54a) N -(1-Imino-3-butenyl)Human
DDAH-1
13
L -ornithine
ine
(L-VNIO)
N
-(1-Iminopropyl)L-ornithine
5
-(1-Imino-3-butenyl)L-ornith
N5-(1-Iminopropyl)Human
DDAH-1
13
(54a) (54a)
13
ine
(L-VNIO)
(54b)
Human
DDAH-1
N
L
-ornithine
5-(1-Iminopropyl)(54a) (L-VNIO)
Human DDAH-1
DDAH-1
13
ine
(L-VNIO)
N
L-ornithine
(L-IPO)
(54b)
Human
5
ine
(L-VNIO)
N(L-IPO)
-(1-Iminopropyl)L-ornithine
(54b)
Human DDAH-1
5
N(L-IPO)
-(1-Iminopropyl)-L-ornithine
(54b)
Human DDAH-1
(54b)
Human DDAH-1
N55-(1-Iminopropyl)-L-ornithine
(L-IPO)
N 5-(1-Iminopropyl)L-ornithine
(L-IPO)
(54b)
Human
DDAH-1
5-(1-Iminopropyl)N
L-ornithine
(54b) 5 (L-IPO)
Human
DDAH-1
N
-(1-Imino-2-chloroethyl)L-or
5
N5-(1-Imino-2-chloroethyl)-(1-Iminopropyl)L-ornithine
(54b)
Human
-(L-IPO)
L -ornithine
(54c)N -(1-Iminopropyl)Human DDAH-1
DDAH-1
N
L-or
5
(54b)
(54b) (54c)
Human
DDAH-1
(L-IPO)
N
-(1-Imino-2-chloroethyl)L-or
nithine
(Cl-NIO)
Human
-- 5
(L-IPO)
Nnithine
-(1-Imino-2-chloroethyl)L
-or
(54c)(L-IPO)
Human
DDAH-1
(Cl-NIO)
5-(1-Imino-2-chloroethyl)-L-or
Nnithine
(54c)
Human DDAH-1
(Cl-NIO)
(54c)
Human DDAH-1
N55-(1-Imino-2-chloroethyl)L-or
nithine
(Cl-NIO)
N 5-(1-Imino-2-chloroethyl)L-or
nithine
(Cl-NIO)
(54c)
Human DDAH-1
N 5-(1-Imino-2-chloroethyl)L-or
(54c)
Human DDAH-1
nithine
(Cl-NIO)
N -(1-Imino-2-chloroethyl)L-or
(54c)
Human DDAH-1
nithine
(Cl-NIO)
(54c)
Human DDAH-1
nithine (Cl-NIO)
nithine (Cl-NIO)
Ki (µM)
Ki (μM)
Ki (μM)
Ki (μM)
Ki (μM)
Ki (μM)
Ki (μM)
690 690
Ki (μM)
Ki690
(μM)
Ki 690
(μM)
690
690
690
690
3100
6903100
3100
690
3100
3100
3100
3100
3100
3100
- 3100
---- 2
13
13- 22
132
13 2
13 2 13 2
13 2
13 2
13- 2
13-1
11
1
1
1
1 1
12
21
2
2
2
2
2
2 2
52
2
52
52
52
52
52
52
52
1.3
52
52
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
19 of 31
19 of 31
19 of 31
19 of 31
19 of 31
19 of 31
19 of 31
19 of 31
19 ofReference
31
´1 )
Kinact
Reference
Kinact−1Kinact (min
Kinact
)
(min
Reference
Kinact−1−1)
Reference
(min
Kinact
Reference
)
(min
−1)
Reference
Kinact
(min
−1)
Kinact
(min
Reference
−1)
[105]
0.38
K0.38
inact
Reference
(min
−1)
0.38
[105]
K
inact
Reference
(min
−1
0.38−1)
[105]
Reference
(min
0.38 )
[105]
(min
0.38
[105]
0.38
[105]
0.38
[105]
1.2
[106]
0.38
[105]
1.2
1.2 [106]
0.38
[105]
1.2
[106]
1.2
[106]
1.2
[106]
1.2[106]
[109]
1.2
[106]
[109]
1.2
[109]
- [106]
1.2
[106]
[109]
[109]
[109]
-[109]
[110]
[109]
--[110]
[109]
[110]
- [110]
[110]
[110]
[110]
[112]
-[110]
[110]
-[112]
[112]
[112]
- [112]
[112]
[112]
-[112]
[112]
[112]
-[112]
[112]
[112]
[112]
- [112]
[112]
[117]
-[112]
[112]
-[117]
[117]
[117]
[117]
[117]
[117]
[114]
-[117]
[117]
-[114]
[114]
[114]
[114]
[114]
[114]
-- [114]
[128]
[114]
-[128]
[128]
[128]
[128]
[128]
[128]
[128]
0.34
[129]
[128]
0.34
[129]
0.34
[129]
0.34
[129]
0.34
[129]
0.34
[129]
0.34
[129]
0.34
[129]
0.34
[129]
[105]
[106]
[109]
[110]
[112]
[112]
[117]
[114]
[128]
(40)
NG-(2-Methoxyethyl)-L-arginine
methyl ester (L-291)
Rat DDAH-1
20
-
-
[112]
(44a)
2-Amino-5-(3-(2-methoxyethyl)
guanidino)-N-(methylsulfonyl)pentanamide (ZST316)
Human DDAH-1
3
1
-
[117]
Molecules 2016, 21, 615
(54a)
20 of 32
N5-(1-Imino-3-butenyl)-L-ornith
ine (L-VNIO)
N5-(1-Iminopropyl)-L-ornithine
Chemical(54b)
Chemical
Name
(L-IPO)
Number
Molecules 2016, 21, 615
Molecules 2016, 21,5 615 5
N -(1-Imino-2-chloroethyl)L-or
L-ornithine
Molecules 2016, N
21,-(1-Imino-2-chloroethyl)615
(54c) 21, 615
(54c) 2016,
Molecules
(Cl-NIO)
Molecules 2016, 21,
615nithine (Cl-NIO)
Molecules 2016, 21, 615
Human DDAH-1
13
2
-
Chemical Structure
Human
DDAH-1
Enzyme
IC50- (µM)
52K (µM)
i
- K
[128]
´1
inact (min )
Human
Human DDAH-1
--
1.3 1.3
PaDDAH
PaDDAH 11
PaDDAH 11
PaDDAH1
PaDDAH 1
PaDDAH
PaDDAH 1
16–58
16–58
16–58
16–58
16–58
16–58
16–58
-
Indolylthiobarbituric acid
Indolylthiobarbituric acid
68a–c
Indolylthiobarbituric
acid
derivatives
68a–c 68a–c Indolylthiobarbituric
acid derivatives
68a–c
Indolylthiobarbituric
acid
derivatives
Indolylthiobarbituric
acid
derivatives
68a–c
Indolylthiobarbituric acid
68a–c
derivatives
68a–c
derivatives
derivatives
PaDDAH 111
PaDDAH
PaDDAH
PaDDAH 1
PaDDAH 11
PaDDAH 1
PaDDAH
2–17
2–17
2–17
2–17
2–17
2–17
2–17
-
Ebselen
Human DDAH-1
0.33
Ebselen
Human DDAH-1
0.33
Ebselen
Human
0.33
Ebselen
Human DDAH-1
DDAH-1
0.33
Ebselen
Human DDAH-1
0.33
Ebselen
Human DDAH-1
0.33
Ebselen
Human DDAH-1
0.33
4-Hydroxy-2-nonenal (4-HNE)
Human DDAH-1
50
4-Hydroxy-2-nonenal (4-HNE)
Human DDAH-1
50
4-Hydroxy-2-nonenal (4-HNE)
Human DDAH-1
50
4-Hydroxy-2-nonenal
(4-HNE)
Human
5050
4-Hydroxy-2-nonenal
(4-HNE)
HumanDDAH-1
DDAH-1
4-Hydroxy-2-nonenal
(4-HNE)
Human DDAH-1
50
6H-6-Imino-(2,3,4,5-tetrahydropy
4-Hydroxy-2-nonenal
(4-HNE)
Human DDAH-1
50
6H-6-Imino-(2,3,4,5-tetrahydropy
6H-6-Imino-(2,3,4,5-tetrahydropy
rimido)[1,2-c]-[1,3]benzothiazine
Human DDAH-1
9
6H-6-Imino-(2,3,4,5-tetrahydropy
rimido)[1,2-c]-[1,3]benzothiazine
Human DDAH-1
9
6H-6-Imino-(2,3,4,5-tetrahydropy
rimido)[1,2-c]-[1,3]benzothiazine
Human DDAH-1
9
(PD
404182)
6H-6-Imino-(2,3,4,5-tetrahydropy
rimido)[1,2-c]-[1,3]benzothiazine
Human DDAH-1
9
6H-6-Imino-(2,3,4,5-tetrahydropyrimido)
(PD 404182)
rimido)[1,2-c]-[1,3]benzothiazine
Human
99
HumanDDAH-1
DDAH-1
(PD 404182)
rimido)[1,2-c]-[1,3]benzothiazine
Human DDAH-1
9
[1,2-c]-[1,3]benzothiazine
(PD 404182)
(PD
404182)
(PD 404182)
(PD 404182)
Proton H+/K+ ATPase pump
Proton H++/K++ ATPase pump
82a–f
Human DDAH-1
51–63
ATPase pump
Proton H+ /K
82a–f
Human DDAH-1
51–63
inhibitors
(PPIs)
+ ATPase pump
82a–f
Human DDAH-1
51–63
Proton
H +/K
inhibitors
(PPIs)
+ ATPase pump
/K
Proton
H
inhibitors
(PPIs)
82a–f
Human
DDAH-1
51–63
+ ATPase pump
Proton
H++/K
82a–f Proton
Human DDAH-1
51–63
H+ /K
ATPase
pump
inhibitors
(PPIs)
Human
51–63
82a–f 82a–f
HumanDDAH-1
DDAH-1
51–63
inhibitors (PPIs)
1 Pa: Pseudomonas aeruginosa; 2 as determined by Tommasi et al. [117].
inhibitors
(PPIs)
inhibitors
(PPIs)
1 Pa: Pseudomonas aeruginosa; 2 as determined by Tommasi et al. [117].
1 Pa: Pseudomonas aeruginosa; 2 as determined by Tommasi et al. [117].
1 Pa: Pseudomonas aeruginosa; 2 as determined by Tommasi et al. [117].
1 Pa: Pseudomonas aeruginosa; 2 as determined by Tommasi et al. [117].
1 Pa:
2 as 2determined by Tommasi et al. [117].
aeruginosa;
1 Pseudomonas
Pa: Pseudomonas
aeruginosa;
as determined by Tommasi et al. [117].
-
(73)
(73)
(73)
(73)
(73)
(73)
(76)
(76)
(76)
(76) (76)
(76)
(76)
(79)
(79)
(79)
(79)
(79) (79)
(79)
(73)
0.34
-
-
-
-
-
-
-
Reference
20 of 31
20 of 31
20 of 31
20 of 31
20 of 31
0.34 [129] 20 of 31 [129]
Table 3. Cont.
Table 3. Cont.
Table 3. Cont.
Table 3. Cont.
Table 3. Cont.
Table 3. Cont.
Pentafluorophenyl
(PFP)
Pentafluorophenyl
(PFP)(PFP)
Pentafluorophenyl
61
Pentafluorophenyl
(PFP)
61 sulfonate
sulfonate
estersesters
61
Pentafluorophenyl
(PFP)
sulfonate esters
Pentafluorophenyl
sulfonate esters (PFP)
61
Pentafluorophenyl
(PFP)
61
sulfonate esters
61
sulfonate esters
sulfonate esters
61
[114]
Table 3. Cont.
-
- [130]
[130]
[130]
[130]
[130]
[130]
-
[139]
- [139]
[139]
[139]
[139]
[139]
-
[145]
[145]
- [145]
[145]
[145]
[145]
[167]
[167]
[167]
- [167]
[167]
[167]
[61]
[61]
[61]
[61]
- [61]
[61]
-
[175]
[175]
[175]
[175]
[175]
- [175]
[130]
[139]
[145]
[167]
[61]
[175]
Molecules 2016, 21, 615
21 of 32
Acknowledgments: The authors thank Flinders Medical Centre Foundation and Flinders Faculty of Health
Sciences, the Scottish Universities Life Sciences Alliance (SULSA), and the NHS Grampian Endowment Fund for
providing financial support which assisted with reading available evidence and conducting experimental work.
Author Contributions: The manuscript was conceived by A.A.M., R.B.M. and S.T. collected the primary data and
compiled draft manuscripts. B.C.L. and A.A.M. supervised development of the manuscript, and assisted in data
interpretation, manuscript evaluation and editing.
Conflicts of Interest: The authors declare no conflicts of interest.
Abbreviations
The following abbreviations are used in this manuscript:
DDAH
ADMA
L-NMMA
NOS
NO
SDMA
PRMTs
CAT
AGXT2
PKA
Akt
VEGF
iNOS
TNF-α
LDL
IL-1β
AT1 -R
DNA
FXR
HcyNO
NaNO2
NaOH
HCl
Min
Ki
µM
H2 O
Cys
His
PaDDAH
PAD
NaOMe
MeOH
AcOH
NH4 Cl
PEO
DCM
TEA
DMAP
MsCl
Et2 O
RT
NaN3
Dimethylarginine dimethylamino hydrolase
Asymmetric dimethylarginine
Monomethyl arginine
Nitric oxide synthase
Nitric oxide
Symmetric dimethylarginine
Protein arginine methyl transferases
Cationic amino acid transporter
Alanine-glyoxylate amino transferase 2
Protein kinase A
Protein kinase B
Vascular endothelial growth factor
Inducible nitric oxide synthase
Tumour necrosis factor α
Low density lipoprotein
Interleukin-1β
Type 1 angiotensin receptor
Deoxyribonucleic acid
Farnesoid X receptor
S-Nitroso-L-homocysteine
Sodium nitrite
Sodium hydroxide
Hydrochloric acid
Minutes
Inhibition constant
Micromolar
Water
Cysteine
Histidine
Pseudomonas aeruginosa dimethylarginine dimethylamino hydrolase
Peptydilarginine deaminase
Sodium methoxide
Methanol
Acetic acid
Ammonium chloride
Poly(ethylene oxide)
Dichloromethane
Triethylamine
4-Dimethylaminopyridine
Methanesulfonyl chloride
Diethyl ether
Room temperature
Sodium azide
Molecules 2016, 21, 615
DMF
Ph3 P
4124W
Boc
M
DEAD
THF
DIPEA
CH3 CN
SOCl2
SAR
IC50
L-257
L-291
Orn
Bu
Z
TFMSA
TFA
CDI
DBU
h
HATU
mM
DMSO
i-BuCOCl
NH4 OH
TFAA
ZnBr2
NH2 OH.HCl
NaHCO3
L-VNIO
EtOH
EtOAc
L-IPO
Cl-NIO
PFP
NaOAc
ADI
HTS
SMTC
ESI-MS
BuLi
Se
CuBr2
NADPH
H+/K+ ATPase
Ca2+
4-HNE
LOPAC
NaH
22 of 32
N,N-Dimethylformamide
Triphenylphosphine
(S)-2-Amino-4-(3-methylguanidino)butanoic acid
tert-Butyloxycarbonyl
Molar
Diethyl azodicarboxylate
Tetrahydrofuran
N,N-Diisopropylethylamine
Acetonitrile
Thionyl chloride
Structure activity relationship
Inhibition constant
NG -(2-Methoxyethyl)-L-arginine
NG -(2-Methoxyethyl)-L-arginine methyl ester
Ornithine
Butyl
Carboxybenzyl
Trifluoromethanesulfonic acid
Trifluoroacetic acid
1,11 -Carbonyldiimidazole
1,8-Diazabicyclo[5.4.0]undec-7-ene
Hours
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide
hexafluorophosphate
Millimolar
Dimethyl sulfoxide
Isobutyl chloroformate
Ammonium hydroxide
Trifluoroacetic anhydride
Zinc bromide
Hydroxylamine hydrochloride
Sodium bicarbonate
N5 -(1-Imino-3-butenyl)-L-ornithine
Ethanol
Ethyl acetate
N5 -(1-Iminopropyl)-L-ornithine
N5 -(1-Imino-2-chloroethyl)-L-ornithine
Pentafluorophenyl
Sodium acetate
Arginine deaminase
High throughput screening
S-methyl-L-thiocitrulline
Electrospray ionization mass spectrometry
Butyl lithium
Selenium
Copper(II) bromide
Nicotinamide adenine dinucleotide phosphate
Proton/potassium ATPase
Calcium 2+ cation
4-Hydroxy-2-nonenal
Library of Pharmacologically Active Compounds
Sodium hydride
Molecules 2016, 21, 615
CS2
BrCN
Å
PPIs
SPR
CM
(Ph3 P)2 PdCl2
CuI
TIPS
TMS
DPPA
23 of 32
Carbon disulfide
Cyanogen bromide
Angstrom
Proton pump inhibitors
Surface plasmon resonance
Carboxymethylated
Bis(triphenylphosphine)palladium chloride
Copper(I) iodide
Triisopropylsilyl
Trimethylsilane
Diphenylphosphoryl azide
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