Discovery of N-(4-Aminobutyl)-N′-(2-methoxyethyl)guanidine as the First Selective, Nonamino Acid, Catalytic Site Inhibitor of Human Dimethylarginine Dimethylaminohydrolase-1 (hDDAH-1)

Article abstract of DOI:10.1021/acs.jmedchem.9b01230

N-(4-Aminobutyl)-N′-(2-methoxyethyl)guanidine (8a) is a potent inhibitor targeting the hDDAH-1 active site (K_i = 18 μM) and derived from a series of guanidine- A nd amidine-based inhibitors. Its nonamino acid nature leads to high selectivities

Full text of DOI:10.1021/acs.jmedchem.9b01230

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Brief Article
Discovery of N-(4-Aminobutyl)-N’-(2-Methoxyethyl)guanidine as
the First Selective, Non-Amino Acid, Catalytic Site Inhibitor of
human dimethylarginine dimethylaminohydrolase-1 (hDDAH-1)
Ina Lunk, Felix-A. Litty, Sven Hennig, Ingrid R Vetter, Jürke Kotthaus, Karin S. Altmann,
Gudrun Ott, Antje Havemeyer, Carmen Carrillo García, Bernd Clement, and Dennis Schade
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01230 • Publication Date (Web): 16 Dec 2019
Downloaded from pubs.acs.org on December 17, 2019
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Journal of Medicinal Chemistry
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Discovery of N-(4-Aminobutyl)-N’-(2-Methoxyethyl)guanidine as the
First Selective, Non-Amino Acid, Catalytic Site Inhibitor of human di-
methylarginine dimethylaminohydrolase-1 (hDDAH-1)
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Ina Lunk,1‡ Felix-Alexander Litty,1‡ Sven Hennig,3 Ingrid R. Vetter,2 Jürke Kotthaus,1 Karin S. Altmann,1
Gudrun Ott,1 Antje Havemeyer,1 Carmen Carrillo García,1 Bernd Clement,1 Dennis Schade1,2,4
*
1
Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University Kiel, Gutenbergstr. 76, D-24118
Kiel (Germany)
2 Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Str. 11, D-44227 Dortmund (Germany)
3 Department of Chemistry & Pharmaceutical Sciences, VU University Amsterdam, De Boelelaan 1108 (The Netherlands)
4 Partner site Kiel, DZHK, German Center for Cardiovascular Research, D-24105 Kiel (Germany)
DDAH, nitric oxide, guanidine, amidine, prodrug
small-sized substrate binding pocket with restricted ligand ac-
cess could be possible reasons. The very first described candi-
ABSTRACT: N-(4-aminobutyl)-N’-(2-methoxyethyl)guani-
dine (8a) is as a potent inhibitor targeting the hDDAH-1 active
site (Ki = 18 µM) and derived from a series of guanidine- and
amidine-based inhibitors. Its non-amino acid nature leads to
dates (2005) were the promiscuous, covalently reactive chloro-
acetamidine as well as S-nitroso-L-homo-citrulline.9, 10 In the
same year, the Leiper group disclosed the currently most widely
high selectivities towards other enzymes of the nitric oxide-
used, potent, reversible and selective hDDAH-1 inhibitor L-257
modulating system. Crystallographic data of 8a-bound
(1).11 This compound served as a great starting point for further
hDDAH-1 illuminated a unique binding mode. Together with
its developed N-hydroxyguanidine prodrug 11, 8a will serve as
a most widely applicable, currently available pharmacological
tool to target DDAH-1-associated diseases.
development and subsequently advanced to the recently re-
ported ZST-316 (2).12 Here, bioisosteric replacement of the
-carboxy group by an acylsulfonamide improved potency by
13-fold (Ki = 1 µM) and showed efficacy in breast cancer mod-
els in vitro.13
Within our own long-standing efforts in designing small mol-
ecule inhibitors of hDDAH-1, we synthesized and tested several
amidine- and guanidine-based inhibitors and identified N5-(1-
iminobut-3-enyl)-L-ornithine (V-NIO, 3, Ki = 2 µM) as a highly
potent candidate.14 Later, the Fast group explored the amidines
toward NOS/DDAH dual inhibitory profiles and characterized
their (reversible) binding modes by X-ray crystallography.15
Building on these structure-activity relationships (SARs), irre-
versible inhibitors were designed with a chloroacetamidine as
the covalent warhead (e.g., Cl-NIO, 4).16, 17 Only few more
chemotypes have been reported for hDDAH inhibition such as
4-chloropyridine, ebselen, 4-hydroxy-2-nonenal or PD-404182,
all with questionable selectivities.18-20
INTRODUCTION
Human
dimethylarginine
dimethylaminohydrolase-1
(hDDAH-1) has gained considerable attention as a drug target
for pharmacological intervention of several diseases in recent
years. The enzyme catalyzes the hydrolytic degradation of
Nω-methylated L-arginines such as monomethyl-L-arginine
(NMMA) and Nω,Nω-dimethyl-L-arginine (ADMA).1, 2 These
methylarginines can act as endogenous inhibitors of all three
isoforms of nitric oxide synthase (NOS) thereby reducing the
formation of nitric oxide (NO).
Hence, targeting DDAH is an alternative strategy to indi-
rectly affect NO formation in situations where elevated NO lev-
els contribute to pathophysiology, e.g. septic shock, certain
forms of pain (e.g., migraine), pulmonary fibrosis, inflamma-
tory and neurodegenerative disorders or tumor angiogenesis.2-5
In the latter context, increased expression and/or activity of
DDAH-1 has been found in different cancer types and linked to
tumor aggressiveness, for example in prostate cancer.6, 7 Spe-
cific targeting of the DDAH-1 is attractive as this isoform is not
much expressed in immune cells (e.g., macrophages) and endo-
thelial cells, and thus, does not significantly contribute to vas-
cular NO production which has been largely attributed to
DDAH-2.2
Figure 1. Chemical structures and Ki values of known sub-
strate-based hDDAH-1 inhibitors harboring either guanidine
(1, 2) or amidine functionalities (3, 4).
Given its clinical relevance, it comes as a surprise that rather
few DDAH inhibitor chemotypes have been reported to date,
with the majority representing substrate analogs.8 A relatively
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Thus, there is a great need for novel modalities with lead-like
Page 2 of 12
Scheme 1. Synthesis of guanidine-based inhibitors (8) (A),
including the N-hydroxylated prodrug candidate 11 (B).a
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8
qualities which we addressed in the present study. Since ami-
dine 3 did not exhibit a desirable selectivity over other key en-
zymes of the NO regulating system, i.e. NOSs and arginase,21
but guanidine 1 did, we revisited the structural basis for this
phenomenon. Here, for the first time, we could show that the
-carboxy group is dispensable for potent hDDAH-1 inhibition.
This finding potentially opens avenues to the design of lead-like
candidates which we exemplified with a viable prodrug ap-
proach.
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RESULTS AND DISCUSSION
Chemistry. Diversely substituted guanidine-based inhibitor
candidates were accessible via previously reported methods
(Scheme 1A).22, 23 Briefly, preparation of Cbz-protected thiou-
reas (6) followed by EDCI-mediated desulfurization and reac-
tion with desired amines furnished guanidines (8) after final
deprotection with TFA/thioanisol in moderate to good overall
yields (30-88%). The N-hydroxylated prodrug candidate of
guanidine 8a was prepared in a similar fashion (Scheme 1B).
From O-THP-protected 2-methoxyethylthiourea 9, the N-hy-
droxyguanidine scaffold was built up with tert-butyl(4-amino-
butyl)carbamate (10) in the presence of EDCI, followed by sim-
ultaneous deprotection of both THP- and Boc-protecting groups
with HCl/dioxane.
The synthesis of novel amidine-based DDAH inhibitors
largely built on strategies described for the preparation of 3 and
similar NOS inhibitor candidates.24, 25 As depicted in Scheme 2,
this chemistry employed various imidates (14) that were reacted
with the desired amines (15). A final deprotection step of ei-
ther/or Boc- and tBu-ester groups with TFA in dichloromethane
furnished amidines 16 in moderate to good overall yields
(30-76%).
Inhibition profiles: DDAH-1, NOSs, arginase. The most
potent hDDAH-1 inhibitors belong to the large group of sub-
strate mimetics, especially Nω-substituted L-arginine analogues
and N5-(1-iminoalk(en)yl)-L-ornithine derivatives.8 As outlined
in the introduction, specific targeting of hDDAH-1 represents
an attractive option to perturb NO production in a tissue-selec-
tive fashion while not interfering with physiological NO func-
tions. To keep this potential pharmacological asset, activities on
revelant inhibitor off-targets need to be minimized. This mainly
includes selectivities over NOSs and arginases, both of which
share very similar substrates, inhibitors and active site topolo-
gies.1, 15 The distinct selectivity profiles of 1 and 3 on theses
enzymes were deemed a promising starting point. Interestingly,
L-arginine analogue 1 (Ki (hDDAH-1) = 13 µM) already exhibits an
excellent selectivity profile over the other arginine converting
enzymes. A key responsible feature seems to be the Nω-(2-
methoxyethyl)-substituent that is not well-tolerated by NOSs
due to size-restrictions in the guanidinium-binding pocket.15 In
sharp contrast, amidine analogue 3 is even more potent against
hDDAH-1 (Ki = 2 µM) but completely lacks selectivity (Ta-
ble 1).21 The question was which pharmacophoric elements
contribute most significantly to potency and selectivity? The
2-methoxyalkyl-substituent, a guanidine- or an amidine-scaf-
fold? Therefore, systematic combinations of N-(2-methoxy-
alkyl)- and alkenyl-groups with either guanidine- (Table 1) or
amidine-based (Table 2) compounds were tested. At the same
time, modifications of the amino acid side chain were realized
to revisit the influence of the α-amino acid moiety.
a Reagents: A) (i) N-benzyloxycarbonyl isothiocyanate, (ii) R2-NH2 7, (iii)
TFA/thioanisole; B) (iv) tert-butyl(4-aminobutyl)carbamate 10, (v) HCl,
dioxane, R3 = 2-methoxyethyl.
Scheme 2. Synthesis of amidine-based inhibitors (16).a
a Detailed information on the synthesis of novel imidates 14 and amidines
16 can be found in Supporting Information.
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Data shown in Table 1 revealed for N-(2-methoxyethyl)-
a conserved overall structure of hDDAH-1, that is comparable
to all known structures (Fig. 2A, Fig. S4). Superposition of 8a
(this work, pdb 6szp)- and 1-bound hDDAH-1 (pdb 2jaj) shows
almost identical binding modes of both inhibitors (Fig. 2B).
A detailed comparison of guanidine- (1 and 8a), amidine-
(e.g., L-IPeO, pdb 3p8p)26 and urea (i.e., natural catalytic
DDAH product L-citrulline, pdb 2jai)3-based DDAH-ligands
provided interesting insights into distinct binding modes (Fig.
2C): All ligands have the primary amine of the original -amino
acid moiety locked into place by tight interactions with the side
chain of Asp73 and backbone interactions via carbonyl oxygens
of Leu30 and Val268. This underlines the particular relevance
of the -NH3+ group for potent binding and is likely due to op-
timal geometric orientation of all participating H-bond partners.
However, the -carboxy groups in both L-IPeO and L-citrulline
point towards Arg145, whereas -COOH of 1 is twisted away
from Arg145 which in turn is rotated outwards not undergoing
one interaction anymore. The exact same twisting of Arg145 is
observed for bound 8a. Given the fact that the butyl chains in 1
and 8a align very well within the hDDAH-1 binding pocket, it
appears that a quite specific and significant orientation of these
two inhibitors originates from their guanidine moieties. Even
the energetically less favoured gauche conformation of the bu-
tyl chain is tolerated due to the strong binding via the guanidino
and -amino groups. In contrast, butyl chains in amidine-based
inhibitors adopt anti conformations suggesting weaker binding
contributions. This putative guanidine-triggered butyl chain ori-
entation seemed to make an interaction with Arg145 dispensa-
ble, and thus, the necessity of an -COOH group for potent
binding.
Following this hypothesis, we questioned which contribu-
tions of the guanidine are responsible for this phenomenon and
how does this binding pattern differ from other ligands? The
crystal structure of 1- and 8a-bound hDDAH-1 showed that the
outwards pointing NH in 1/8a is interacting with the side chain-
carboxylate of Asp79 (Fig. 2C). The aminobutyl-substituted
guanidino-NH additionally contacts Asp79. The second set of
interactions holds the methoxyethyl-substituted (distal) guani-
dino-NH on the opposite side in place via side chain- and back-
bone carbonyl-oxygens of Asp269 (Fig. 2C, Fig. S6/7). Thus,
Asp79 and Asp269 together form two clamps causing a tight
fixation of the guanidino group (Fig. 2D). As a result, the rela-
tive orientations of both substituents (i.e., 2-methoxyethyl and
4-aminobutyl) are well-predestined. This is in sharp contrast to
the L-IPeO and L-citrulline binding modes, where the respec-
tive amidino or urea group cannot be fixated by Asp79/269 in
the same fashion (Fig. 2C). The consequence is greater flexibil-
ity of both the pentyl chain (L-IPeO) as well as the amino acid
side chain (L-IPeO, L-citrulline). We believe that the high ba-
sicity of a guanidine, compared to an amidine or urea, further
contributes to tight binding due to stronger electrostatic interac-
tion within this acidic site (see Fig. S8). This strong positioning
of the guanidino group within the two Asp clamps induces a
rotation of His173 out of its apo-position, eventually causing an
outward twist of Arg145. This effect can neither be observed
for L-IPeO nor L-citrulline (Fig. 2C).
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3
4
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7
8
guanidine derivatives 8a-8d that the amino acid group cannot
be replaced by methyl- (8c) or isopropyl (8d) as such modifi-
cations led to massively decreased potency or lack of activity,
respectively. However, removal of the carboxylic acid moiety
(8a, Ki = 18 ± 6 µM) was tolerated and retained potent DDAH
inhibition in about the same range as the original amino acid 1
(Ki = 13 ± 2 µM). Most importantly, both compounds showed
comparable characteristics regarding selectivity over NOS iso-
enzymes and arginase. This finding was surprising and indi-
cated that the N-(2-methoxyethyl)-guanidine group contributes
significantly to DDAH binding affinity. To underline the im-
portance of the 2-methoxyethyl-substituent, we replaced it with
a similarly sized butenyl-residue (8e,f), mimicking the potent
amidine N5-(1-iminobutenyl)-ornithine (3). Both the amino acid
(8f, Ki = 57 µM)21 and decarboxylated (8e, Ki = 59 µM) com-
pounds showed decent and comparable inhibitory activities on
hDDAH-1. These results confirmed that the -carboxylic acid
is indeed dispensable for DDAH-1 inhibition. However, the
overall selectivity profiles of 8e,f were inferior to 1 and 8a,
highlighting the value of the 2-methoxyalkyl group. Still, it
should be noted that both decarboxylated 8a and 8e tended to
be more selective than their amino acid congeners.
Next, we attempted transferring these findings to an amidine-
based scaffold (Table 2). Since the 2-methoxyalkyl-group
turned out essential, we systematically searched for a ‘sweet
spot’ in length of this substituent (see 16a-f). Both the amino
acid as well as decarboxylated analogues were synthesized and
tested to recapitulate results with the guanidino compound se-
ries 8. As shown in Table 2, all -amino acids derivatives were
moderate to highly potent inhibitors of DDAH-1, with 16c (Ki
= 9 µM) representing the best candidate. In contrast to guani-
dines 1 and 8, omitting the -carboxy group in amidines 16 cor-
rupted DDAH inhibition by 1-2 orders of magnitude in all cases.
Since activity appeared to drop with longer methoxyalkyl-resi-
dues, compound 16g was designed and tested. Here, we tested
whether methylene-extension in the methoxyalkyl-residue can
be compensated by shortening the amino acid side chain (i.e.,
nor-L-ornithine). No DDAH inhibition was detected for 16g
which is in general agreement with previous work on nor-argi-
nine or nor-ornithine analogues.11 This data further emphasized
the particular importance of an intact -amino acid group-cen-
tred binding mode of amidine-based compounds. To complete
this SAR aspect, we also tested a decarboxylated version of 3
(i.e., 16h) which was almost 400-fold less potent in direct com-
parison (Ki (hDDAH-1) = 768 µM).
Together, our DDAH inhibition data suggested that omitting
the -carboxy group is tolerated only by guanidines but not by
amidines. It also implied a predominant role for a 2-methoxy-
ethyl-substituent. In this regard, 2-methoxyethyl-amidine 16c
performed similar to 1 as a hDDAH-1 inhibitor, but turned out
much less selective towards NOSs and arginase (see Table 2).
Promiscuous inhibition of these enzymes was a general obser-
vation for all amidines presented herein.
hDDAH-1 crystal structure and binding mode. Systematic
exploration of distinct substitution patterns of guanidines 8 and
amidines 16 revealed important structural elements that contrib-
uted to high affinity for DDAH-1. A surprising finding was that
the -COOH appeared only dispensable for the guanidine-type
of inhibitors as exemplified with pairs 1/8a or 8f/e compared to
amidines 16c/d. Thus, we determined the crystal structures for
ligand-free apo-hDDAH-1 (solved at 2.41 Å, pdb 6szq) as well
as hDDAH-1 in complex with the best inhibitor candidate 8a
(solved at 1.76 Å, pdb 6szp). Overlay of both structures showed
9
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Lacking the guanidine group-mediated pre-orientation of the
two alkyl-substituents in an amidine-based inhibitor seems to
create a higher degree of conformational freedom enabling in-
teraction of the -carboxy group with Arg145. Here, this op-
portunity becomes vital to ensure potent binding and – once lost
due to a missing COOH group – results in loss of activity
against hDDAH-1 (see Table 2, all amidine-based inhibitors).
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Table 1. Selectivity profiles of guanidine-based DDAH inhibitors.
Page 4 of 12
1
2
3
4
5
6
hDDAH-1
Ki (µM)
Arginase nNOS
eNOS
iNOS
entry
R1
R2
% inhibition at 1 mM
7
8
1a
13 ± 2
29 ± 6
12 ± 8
10 ± 5
0 ± 13
9
10
11
12
13
14
15
16
17
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22
23
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8a
8b
8c
18 ± 6
11 ± 6
35 ± 4
15 ± 4
0 ± 8
5 ± 4
0 ± 8
13 ± 5
13 ± 4
0 ± 4
0 ± 13
2 ± 6
489 ± 16
2815 ± 946
21 ± 4
8d
2763 ± 714
20 ± 2
0 ± 1
19 ± 10
4 ± 12
8fa
8e
57 ± 9
59 ± 3
70 ± 21
0 ± 12
14 ± 4
5 ± 5
35 ± 5
40 ± 8
23 ± 6
35 ± 17
Ki -values are mean values of at least two independent experiments ± SD. Inhibition data at 1 mM derived from at least three inde-
pendent experiments (mean ± SD). a Values taken from Kotthaus et al. (2012)21
Table 2. Selectivity profiles of amidine-based inhibitors
hDDAH-1
Arginase nNOS
eNOS
iNOS
entry
R1
R2
Ki (µM)
73 ± 6
% inhibition at 1 mM
16a
22 ± 2
21 ± 2
43 ± 1
22 ± 2
20 ± 1
14 ± 2
93 ± 2
78 ± 1
65 ± 1
19 ± 23
35 ± 13
7 ± 13
89 ± 4
44 ± 19
54 ± 18
28 ± 6
29 ± 19
36 ± 6
75 ± 5
42 ± 8
50 ± 7
16 ± 5
26 ± 13
15 ± 4
983 ± 19
9 ± 1
16b
16c
16d
16e
16f
1446 ± 91
156± 9
n.d.b
n.d.b
n.d.
n.d.
n.d.
n.d.
16g
3a
2 ± 1
76± 10
6 ± 5
100 ± 1
99 ± 1
88 ± 3
56 ± 8
100 ± 1
100 ± 1
16h
768 ± 4
Ki -values are mean values of at least two independent experiments ± SD. Inhibition data at 1 mM derived from at least three inde-
pendent experiments (mean ± SD).a Values taken from Kotthaus et al. (2012)21; b n.d. = not determined, when inhibition values were
below 20% at 1 mM.
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Future studies will have to address the particular importance
1
2
3
4
5
6
of the 2-methoxyethyl-substituent as there did not seem to be
direct contacts in close proximity or water-mediated interac-
tions. The closest possible contact would be Cys274 in ca. 3.6 Å
distance (Fig 2D). Moreover, it would also be interesting to see
whether conformational restrictions within the butyl chain can
rescue potency and selectivity of amidine analogs.
7
8
9
Bioactivation of guanidine-prodrug candidate 11. Com-
pound 8a exemplified that the -carboxy group is dispensable
for potent DDAH-1 inhibition while conveying the molecule
with high selectivity over other enzymes of the NO modulating
system. These features highlight prime qualities for lead opti-
mization. In this regard, an opportunity for oral administration
is generally desirable. Due to the zwitterionic structure of 1, ab-
sorption would only be mediated by active uptake via amino
acid transporters. The preferred route of absorption after oral
intake would be by passive diffusion. Although this is feasible
for the primary amine of 8a, its guanidine functionality would
be permanently charged under physiological conditions, pre-
venting significant membrane transport by passive diffusion.
Therefore, we applied a well-established prodrug approach and
prepared N-hydroxyguanidine 11. We and others have demon-
strated that such N-hydroxylated guanidines, similar to amidox-
imes as prodrugs of amidines, are effectively bioactivated by
the mARC-containing N-reductive enzyme system in vitro and
in vivo.27
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11
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Figure 3. In vitro bioactivation of N-hydroxyguanidine 11 to 8a.a
a
Data represent means of biological triplicates ± SD. OMV, outer mito-
chondrial membrane vesicles.
Here, we could show that N-hydroxyguanidine 11 is very ef-
ficiently converted to the active principle 8a (Fig. 3). System-
atic exploration of distinct subcellular liver fractions revealed
highest reduction rates of 11 in outer mitochondrial membrane
vesicles (OMV) followed by mitochondria and liver homoge-
nate. Furthermore, incubations with reconstituted, heterolo-
gously expressed human mARC-1 and -2 isoenzymes con-
firmed that 11 is a very good substrate of this N-reductive en-
zyme system. The determined specific activities are in a similar
range as other reported substrates, such as benzamidoxime
(served as positive control),28 sulfamethoxazole hydroxylamine
29 or the orphan drug upamostat.30 Based on this data, there is a
Figure 2. Binding mode analysis of guanidine- and amidine-
types of hDDAH-1 inhibitors. A) Superposition of crystal struc-
tures of ligand-free (apo) or 1- or 8a-bound hDDAH-1; B)
Binding modes of superimposed 8a and 1, dashed lines indicate
interacting residues; C) 8a-bound hDDAH-1 with superim-
posed 1 (= guanidines), L-citrulline (= urea) and L-IPeO (= am-
idine). D) Schematic view on 8a binding mode, illustrating con-
tributions by active site amino acids.
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high probability for decent peroral bioavailability of 11. A gen- RP-18 silica gel; eluent: 0.1 % TFA in water; ninhydrine posi-
eral concern is chemical stability of N-hydroxyguanidines.1
However, we found that 11 was very stable at different pHs over
24 hours at 37°C (see Fig. S3). Finally, both the guanidine 8a
and its prodrug 11 revealed an excellent profile regarding cell
toxicity/viability (see Supp Info 2.4), further underlying their
utility as a pharmacological toolset.
tive fractions were pooled and lyophilized. Yield: 234 mg of a
colourless oil (99%); Rf = 0.22 (i-PrOH/H2O/AcOH, 8:2+0.5);
1H NMR (DMSO-d6): δ/ppm = 1.54 (m, 4H, N-CH2-CH2-CH2),
2.80 (m, 2H, N-CH2), 3.14 (m, 2H, N-CH2), 3.27 (s, 3H, O-
CH3), 3.32, 3.42 (2 × t, 4H, N-CH2-CH2-O), 7.47 (br s, 2H,
NH2), 7.61 (br t, 1H, NH), 7.71 (br t, 1H, NH), 7.87 (br s, 3H,
1
2
3
4
5
6
7
8
+
+
NH3 ); 13C NMR (DMSO-d6): δ/ppm = 24.1 (CH2-CH2-NH3 ),
+
25.4 (CH2-CH2-CH2-NH3 ), 38.3 (N-CH2), 40.3 (N-CH2), 40.7
CONCLUSION
(N-CH2), 58.0 (O-CH3), 70.0 (O-CH2), 155.9 (C=N); MS (ESI):
m/z = 189 [M + H]+; Anal. calcd for C8H20N4O·2.5 CF3COOH
(473.33): C 32.99, H 4.79, N 11.84; C 32.86, H 4.80, N 11.50.
N-Hydroxy-N´-(4-aminobutyl)-N´´-(2-methoxy-
9
The human DDAH-1 has emerged as an attractive drug target
in the past two decades, and recently gained particular attention
in the context of tumor angiogenesis and progression. It has
been discussed that indirect regulation of NO production via
DDAH-1 inhibition would represent a safer option compared to
the use of NOS inhibitors. However, only few agents with lead-
like qualities have been described to date. Through rational de-
sign, synthesis, biochemical profiling and crystallization, we
dissected key structural requirements for high affinity-binding
to hDDAH-1 while ensuring a desired selectivity towards other
key enzymes of the NO-modulating system.
These efforts culminated in the discovery of 8a which po-
tently inhibited hDDAH-1 (Ki = 18 µM) without significantly
affecting NOSs and arginase. Its guanidine enabled a unique
binding mode that obviates the need for the -carboxy group,
which was not the case for corresponding amidine analogs. Im-
portantly, these features opened an opportunity for designing a
prodrug candidate of 8a, i.e. N-hydroxyguanidine 11, that even-
tually complemented a unique toolset of chemical modalities
for experimental pharmacology with lead-like qualities for fur-
ther development. Future directions should be devoted to fur-
ther optimization of 8a as a lead with the aim to improve po-
tency towards the low µM range. Our structure-based data sug-
gests that conformational restraints in the butyl chain might add
to binding affinity, potentially regaining potency (and selectiv-
ity) for amidine-based inhibitors. Moreover, the acylsulfona-
mide-site of interaction postulated for ZST316 could represent
an attractive option for further improvements despite the rather
tight ligand binding pocket of DDAH.
10
11
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ethyl)guanidine Dihydrosulphate (11). 400 mg (1 mmol) of
the protected precursor (S4) were dissolved in 15 ml of a mix-
ture from water and dioxane (2:1). 3 ml H2SO4 96% were added
and the solution was stirred at room temperature for 3 hours.
The pH was adjusted to 6 with NaHCO3 and the mixture was
dried in vacuum. The residue was dissolved in methanol and
inorganic salts were removed by filtration. The mixture was
dried in vacuum and the residue further purified by RP18 silica
gel flash chromatography and eluted with Aqua bidest. Yield:
277 mg, colourless foam (89%). Rf = 0.46 (i-PrOH/H2O/AcOH,
6:3:1); 1H NMR (D2O): δ/ppm = 1.74 (m, 4H, 2´,3´-CH2), 3.06
(t, 3J = 6.7 Hz, 2H, 4´-CH2), 3.30 (t, 3J = 6.1 Hz, 2H, 1´-CH2),
3.42 (s, 3H, O-CH3), 3.46 (t, 3J = 5.0 Hz, 2H, 1´´-CH2), 3.66 (t,
3J = 5.0 Hz, 2H, 2´´-CH2); 13C-NMR (D2O): δ/ppm = 24.0 (3´-
CH2), 25.0 (2´-CH2), 39.0 (4´-CH2), 40.3 (1´-CH2), 40.8 (1´´-
CH2), 58.3 (O-CH3), 70.3 (2´´-CH2), 157.6 (C=N); ; MS (ESI):
m/z = 205 [M + H]+. Anal. calcd for C8H22N4O6S·1.0 H2O
(320.37): C 29.99, H 7.55, N 17.49, S 10.01; found: C 29.99, H
7.89, N 17.12, S 10.64.
General procedure for the synthesis of amidine deriva-
tives (16). Synthesis of amidine derivates (16) started with im-
idates (e.g., 14b) as building blocks which were were prepared
from the appropriate nitrile (e.g., S8) according to the literature
with minor changes.24 Briefly, imidates (e.g., 14b) were added
to 100 ml of cold, dry Et2O to give a precipitate which has been
(in most cases) collected by filtration, dried in vacuo (phospho-
rous pentoxide) and stored under argon at -20 °C. All imidates
were white solids with yields between 90 % and 95 %. Two
methods were employed for the preparation of the protected am-
idine precursors (e.g., S10c). Method B was used for the syn-
thesis of amidine 16c: Nα-Boc-, O-tBu-protected L-ornithine
(15a) (1 mmol) was dissolved in 15 ml MeOH at 0ꢀ°C and the
imidate 14b (3 mmol), DIPEA (1 ml) and DMAP (1 crystal)
added. The mixture was stirred for 8 h at 0ꢀ°C and then overnight
at room temperature. The solvent was evaporated and the prod-
ucts purified by column chromatography (SiO2, EtOAc/MeOH,
6:1). For deprotection, amidines (0.5 mmol) were dissolved in
5 ml TFA/CH2Cl2 (1:1) and stirred for 30 min at room temper-
ature. The mixture was concentrated in vacuo, diluted with 5 ml
water and washed twice with Et2O. The aqueous phase was
evaporated and the compounds further purified by flash column
chromatography (RP18) with water as the eluent.
EXPERIMENTAL SECTION
All final test compounds were purified to >95% as
determined by combustion analysis or LC/MS. The analytical
methods, general chemistry, experimental information, and
syntheses of all other compounds are provided in the
Supporting Information.
General procedure for the synthesis of guanidine
derivatives (8). Protected carbamoylguanidines were prepared
according to previously described protocols.22, 23 Briefly, 1.5 eq
DIPEA, the respective amine (e.g., 7a) and EDCI were reacted
with 0.5 mmol thiourea (e.g., 6a) in 10 ml of dry CH2Cl2. Unless
noted otherwise, reactions were complete after stirring
overnight. The organic phase was diluted with 10 ml of CH2Cl2
and washed with small amounts of 1% aqueous HCl, water and
brine. The resulting oils were purified by column
chromatography on silica gel. For deprotection, the
intermediates (e.g., S3a) were stirred in 10 ml of TFA and 3 ml
thioanisole overnight. TFA was evaporated, 5 ml water, 15 ml
Et2O added, the organic phase extracted (2x) with 5 ml water
and the combined aqueous phases washed once with Et2O. The
aqueous phase was concentrated and the crude products purified
by chromatography.
N5-(1-Imino-3-methoxypropyl)-ornithine bis(trifluoro-
acetate) (16c). Yield: 134 mg colourless oil (67ꢀ%). Rf = 0.10
(i-PrOH/H2O/AcOH, 8:2+0.5); 1H-NMR (DMSO-d6): δ/ppm =
3
1.53-1.90 (m, 4H, 3,4-CH2), 2.65 (t, J = 6.1 Hz, 2H, CH2-
3
C=N), 3.23 (t, J = 6.2 Hz, 2H, 5-CH2), 3.25 (s, 3H, CH3-O),
3.64 (t, 3J = 6.1 Hz, 2H, O-CH2), 3.80 (t, 3J = 6.2 Hz, 1H, CH),
8.80, 9.31, 9.77 (3 x br s, 1H, NH); 13C-NMR (DMSO-d6):
δ/ppm = 23.1 (4-CH2), 27.2 (3-CH2), 33.0 (CH2-C=N), 40.9 (5-
CH2), 51.6 (CH), 58.9 (CH3), 67.9 (O-CH2), 164.0 (C=OtBu),
N-(4-Aminobutyl)-N'-(2-methoxyethyl)guanidine bis(tri-
fluoroacetate) (8a). Purification by flash chromatography on
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Journal of Medicinal Chemistry
170.8 (C=N); MS (ESI): m/z = 218 [M + H]+ ; Anal. calcd for
1.
Schade, D.; Kotthaus, J.; Clement, B., Modulating the NO
C9H19N3O3 . 1.5 CF3COOH . 0.8 H2O (402.72): C 35.79, H
5.53, N 10.43, found: C 35.76, H 5.38, N 10.54.
generating system from a medicinal chemistry perspective: current
trends and therapeutic options in cardiovascular disease. Pharmacol.
Ther. 2010, 126 (3), 279-300.
1
2
3
4
5
6
7
8
9
Biochemical assays for hDDAH-1, NOSs and arginase.
Assays were performed according to our described procedures
and can be found in the Supporting Information file.14, 21
In vitro bioactivation. Incubations of prodrug 11 with dis-
tinct subcellular porcine liver enzyme sources and the reconsti-
tuted human mARC-1 and -2 systems were performed as previ-
ously described.31 Guanidine 8a was quantified by HPLC using
a precolumn derivatization protocol (o-phthalaldehyde).
hDDAH-1 crystal structures and binding mode analysis.
Protein expression and purification of hDDAH-1 was done as
described previously.14 Details on crystallization and structure
solution can be found in the Supporting Information file.
Atomic coordinates are published in the RCSB Protein Data
Bank (apo-hDDAH-1 pdb: 6szq, hDDAH-1 8a complex pdb:
6szp).
2.
Leiper, J.; Nandi, M., The therapeutic potential of targeting
endogenous inhibitors of nitric oxide synthesis. Nat. Rev. Drug
Discov. 2011, 10 (4), 277-291.
3.
Malaki, M.; O'Hara, B.; Rossiter, S.; Anthony, S.; Madhani, M.;
Selwood, D.; Smith, C.; Wojciak-Stothard, B.; Rudiger, A.;
Stidwill, R.; McDonald, N. Q.; Vallance, P., Disruption of
methylarginine metabolism impairs vascular homeostasis. Nat. Med.
2007, 13 (2), 198–203.
Leiper, J.; Nandi, M.; Torondel, B.; Murray-Rust, J.;
10
11
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13
14
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4.
Pullamsetti, S. S.; Savai, R.; Dumitrascu, R.; Dahal, B.
K.; Wilhelm, J.; Konigshoff, M.; Zakrzewicz, D.; Ghofrani, H. A.;
Weissmann, N.; Eickelberg, O.; Guenther, A.; Leiper, J.; Seeger,
W.; Grimminger, F.; Schermuly, R. T., The role of dimethylarginine
dimethylaminohydrolase in idiopathic pulmonary fibrosis. Sci. Transl.
Med. 2011, 3 (87), 87ra53.
5.
Wang, Y.; Hu, S.; Gabisi, A. M., Jr.; Er, J. A.; Pope, A.;
Burstein, G.; Schardon, C. L.; Cardounel, A. J.; Ekmekcioglu, S.;
Fast, W., Developing an irreversible inhibitor of human DDAH-1, an
enzyme upregulated in melanoma. ChemMedChem 2014, 9 (4), 792-
797.
ASSOCIATED CONTENT
Supporting Information. Synthesis and characterization of all
intermediates and final compounds; Description of biochemical as-
says for hDDAH-, NOS- and arginase-inhibition, and bioactivation
studies; HPLC method for the quantification of 8a and 11 (Fig. S1-
S2); stability assessment of 11 (Fig. S3); X-ray data collection and
refinement statistics (Table S1); hDDAH-1 crystal structures and
augmented reality sessions (Fig. S4-S7). Molecular Formula
Strings are available. This material is available free of charge via
the Internet at http://pubs.acs.org.
6.
Kostourou, V.; Robinson, S. P.; Cartwright, J. E.;
Whitley, G. S., Dimethylarginine dimethylaminohydrolase I enhances
tumour growth and angiogenesis. Br. J. Cancer 2002, 87 (6), 673-
680.
7.
Reddy, K. R. K.; Dasari, C.; Duscharla, D.; Supriya, B.;
Ram, N. S.; Surekha, M. V.; Kumar, J. M.; Ummanni, R.,
Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is frequently
upregulated in prostate cancer, and its overexpression conveys tumor
growth and angiogenesis by metabolizing asymmetric
dimethylarginine (ADMA). Angiogenesis 2018, 21 (1), 79-94.
8.
A., Inhibitors of the hydrolytic enzyme dimethylarginine
dimethylaminohydrolase (DDAH): Discovery, synthesis and
development. Molecules 2016, 21 (5), pii: E615.
AUTHOR INFORMATION
Corresponding Author
*For D.S.: phone, +49 431 8801176; e-mail, schade@pharma-
zie.uni-kiel.de. ORCID-ID: 0000-0002-5515-1821.
Murphy, R. B.; Tommasi, S.; Lewis, B. C.; Mangoni, A.
9.
Stone, E. M.; Schaller, T. H.; Bianchi, H.; Person, M. D.;
Fast, W., Inactivation of two diverse enzymes in the
amidinotransferase superfamily by 2-chloroacetamidine:
dimethylargininase and peptidylarginine deiminase. Biochemistry
2005, 44 (42), 13744-13752.
Notes
The authors decare no competing financial interest.
Author Contributions
10.
Knipp, M.; Braun, O.; Vasak, M., Searching for DDAH
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script. ‡These authors contributed equally.
inhibitors: S-nitroso-L-homocysteine is a chemical lead. J. Am. Chem.
Soc. 2005, 127 (8), 2372-2373.
11.
H.; Leiper, J. M.; Vallance, P.; Selwood, D. L., Selective substrate-
based inhibitors of mammalian dimethylarginine
dimethylaminohydrolase. J. Med. Chem. 2005, 48 (14), 4670–4678.
Rossiter, S.; Smith, C. L.; Malaki, M.; Nandi, M.; Gill,
ACKNOWLEDGMENT
We thank Dr. U. Girreser for analytical service and M. Zietz and S.
Wichmann for excellent technical assistance. We acknowledge
support by the beamline staff of the X10SA SLS (Paul Scherrer I.,
Villigen), the P11 at PETRA synchrotron (Hamburg), as well as
our colleagues at MPI Dortmund for help with the data collection.
We thank Dr. M. P. Müller for assistance with the AR-protocol.
12.
Tommasi, S.; Zanato, C.; Lewis, B. C.; Nair, P. C.;
Dall'Angelo, S.; Zanda, M.; Mangoni, A. A., Arginine analogues
incorporating carboxylate bioisosteric functions are micromolar
inhibitors of human recombinant DDAH-1. Org. Biomol. Chem. 2015,
13 (46), 11315–11330.
13.
Hulin, J.-A.; Tommasi, S.; Elliot, D.; Mangoni, A. A.,
Small molecule inhibition of DDAH1 significantly attenuates triple
negative breast cancer cell vasculogenic mimicry in vitro. Biomed.
Pharmacother. 2019, 111, 602–612.
ABBREVIATIONS
L-IPeO, N5-(1-iminopentyl)-L-ornithine; L-VNIO, N5-(1-imino-
3-butenyl)-L-ornithine; mARC, mitochondrial amidoxime re-
ducing component; NOS, nitric oxide synthase.
14.
Kotthaus, J.; Schade, D.; Muschick, N.; Beitz, E.;
Clement, B., Structure-activity relationship of novel and known
inhibitors of human dimethylarginine dimethylaminohydrolase-1:
Alkenyl-amidines as new leads. Bioorg. Med. Chem. 2008, 16 (24),
10205–10209.
PDB ID CODES
15.
Wang, Y.; Monzingo, A. F.; Hu, S.; Schaller, T. H.;
pdb 2jaj (compound: 1), pdb 3p8p (compound: L-IPeO), pdb
2jai (compound: L-citrulline), pdb 6szp (compound: 8a), pdb
6szq (apo-hDDAH-1). Authors will release the atomic coordi-
nates and experimental data upon article publication.
Robertus, J. D.; Fast, W., Developing dual and specific inhibitors of
dimethylarginine dimethylaminohydrolase-1 and nitric oxide
synthase: Toward a targeted polypharmacology to control nitric oxide.
Biochemistry 2009, 48 (36), 8624–8635.
16.
Wang, Y.; Hu, S.; Gabisi, A. M.; Er, J. A. V.; Pope, A.;
Burstein, G.; Schardon, C. L.; Cardounel, A. J.; Ekmekcioglu, S.;
Fast, W., Developing an irreversible inhibitor of human DDAH-1, an
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Figure 2. Binding mode analysis of guanidine- and amidine-types of hDDAH-1 inhibitors. A) Superposition
of crys-tal structures of ligand-free (apo) or 1- or 8a-bound hDDAH-1; B) Binding modes of superimposed
8a and 1, dashed lines indicate interacting residues; C) 8a-bound hDDAH-1 with superimposed 1 (=
guanidines), L-citrulline (= urea) and L-IPeO (= amidine). D) Schematic view on 8a binding mode,
illustrating contributions by active site amino acids.
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Figure 3. In vitro bioactivation of N-hydroxyguanidine 11 to 8a.
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