Discovery and Pharmacokinetics of Sulfamides and Guanidines as Potent Human Arginase 1 Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00508

We designed and synthesized a series of arginase inhibitors as derivatives of the well-known 2-(S)-amino-6-boronohexanoic acid (ABH) with basic and neutral side chains in the α-position relative to the amino acid group. In an effort to improve the pharmacokinetic profile of literature examples and retain potent enzymatic activity, sulfamido moieties were introduced to generate hydrogen bond interaction with the aspartic acid residue in the arginase active site. The compounds with basic guanidine-containing side chains were even more potent arginase inhibitors. Both groups of compounds, as designed, demonstrated low clearance in their pharmacokinetic profile. The most active inhibitor 15aa showed high nanomolar potency with IC₅₀ = 32 nM toward human arginase 1 and demonstrated low clearance (4.2 mL/min/kg), long t_1/2, and moderate volume of distribution in rat pharmacokinetic studies.

Full text of DOI:10.1021/acsmedchemlett.9b00508

pubs.acs.org/acsmedchemlett
Letter
Discovery and Pharmacokinetics of Sulfamides and Guanidines as
Potent Human Arginase 1 Inhibitors
Roman Blaszczyk,* Joanna Brzezinska, Barbara Dymek, Paulina S. Stanczak, Marcin Mazurkiewicz,
Jacek Olczak, Julita Nowicka, Karolina Dzwonek, Agnieszka Zagozdzon, Jakub Golab,
and Adam Golebiowski
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ABSTRACT: We designed and synthesized a series of arginase
inhibitors as derivatives of the well-known 2-(S)-amino-6-
boronohexanoic acid (ABH) with basic and neutral side chains
in the α-position relative to the amino acid group. In an effort to
improve the pharmacokinetic profile of literature examples and
retain potent enzymatic activity, sulfamido moieties were
introduced to generate hydrogen bond interaction with the
aspartic acid residue in the arginase active site. The compounds
with basic guanidine-containing side chains were even more potent
arginase inhibitors. Both groups of compounds, as designed,
demonstrated low clearance in their pharmacokinetic profile. The
most active inhibitor 15aa showed high nanomolar potency with
IC50 = 32 nM toward human arginase 1 and demonstrated low
clearance (4.2 mL/min/kg), long t1/2, and moderate volume of distribution in rat pharmacokinetic studies.
KEYWORDS: Arginase inhibitors, arginases, boronic acids, immuno-oncology, cancer immunotherapy, amino acids
rginase (ARG) is a manganese-dependent enzyme that
Ahydrolyzes arginine to ornithine and urea. Two isoforms
of this protein are known (ARG1 and ARG2) and both
catalyze the same reaction, but the occurrence of enzyme
isoforms in cellular environments is different. ARG1 is a
cytosolic protein, and ARG2 is localized in the mitochondrial
matrix. Disorders related to arginases activity have been
observed in patients with asthma, pulmonary hypertension,
hypertension, T-cell dysfunction, erectile dysfunction, athero-
sclerosis, renal disease, ischemia-reperfusion injury, neuro-
degenerative disease, inflammatory disease, and fibrotic
disease.1−3 The role of ARG1 as a target in cancer
immunotherapy is under investigation.4 The myeloid-derived
suppressor cells (MDSCs) secrete arginase 1 which depletes
the local arginine concentration.5 Depletion of arginine
represents an important mechanism of immunosuppression,
and high arginase activity in plasma and tumors demonstrated
in patients with a wide spectrum of cancers correlates with a
poor prognosis. Arginase promotes the immune escape of
cancer cells by decreasing L-arginine concentration, which is
required for proliferation and activation of cytotoxic T and NK
cells.4−12 Moreover, some cancer cells release ARG1-
containing exosomes further suppressing antitumor immun-
ity.13 Restoration of arginine levels in the tumor microenviron-
ment by ARG inhibitors induces T-cell activation and
proliferation leading to T-cell-mediated antitumor re-
sponses.13−17 The primary objective of our drug discovery
program was to identify novel, highly potent and efficacious
arginase inhibitors that could be used in cancer immunother-
apy. The clinical relevance of arginase inhibitors in cancer
immunotherapy is under investigation in ongoing phase 1/2
trials with the use of CB-1158 (compound 1, Figure 1), as well
as our molecule OATD-02 (phase 1 to be started mid-
2020).4,18
Boronic acids are well-known pharmacophore fragments of
arginase inhibitors.19−33 Many simple α-substituted ABH (2-
(S)-amino-6-boronohexanoic acid (2)) analogs exhibit potent
inhibitory activity toward ARG1.22,23,28 Conformationally
restricted cyclic ABH analogs are even more active inhibitors
(Figure 1).24,27,29−33 Unfortunately, almost all of these
compounds suffer from weak pharmacokinetics (PK), in
particular, fast clearance and/or low oral bioavailability, due
to their very polar zwitterionic nature (high PSA, negative
logD).23,24 On the one hand, the presence of a basic amino
Received: November 5, 2019
Accepted: March 12, 2020
https://dx.doi.org/10.1021/acsmedchemlett.9b00508
© XXXX American Chemical Society
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Sulfonamidyl and sulfamidyl are well-known as hydrogen
bond-donor/acceptor functional groups with strong H-bond
interaction and tetrahedral shape. Highly basic, planar
guanidines also have very high affinity to H-bond formation.
In addition, both groups possess drug-like properties, well-
known for decades, which may explain the huge number of
drugs incorporating these motifs.37−41
First, sulfamide derivatives were prepared. Alkylation of N-
protected cyanoacetate 5 with pinacol-4-bromobutylboronate
6 afforded quaternary boronic cyanoaminoesters 7 that were
further subjected to subsequent methylation, followed by
reduction, or direct reduction to afford free primary amines 9.
Sulfamoylation of 9, followed by hydrolysis of such formed
sulfamides 10a−c, gave the desired boronic acids 11a−c. N-
Terminally alkylated analogs 11d and 11e were obtained from
10a using appropriate alcohol in Mitsunobu reaction and
subsequent deprotection of such formed sulfamides 10d and
10e (Scheme 1).42,43
Figure 1. Properties (inhibitory activity toward human ARG1,
molecular weight, clearance and oral bioavailability in rats) of
literature examples of ARG inhibitors.23,24,34
side chain increases the activity (2 vs 3; 1200 nM vs 223 nM;
Figure 1) via interaction with aspartic acid Asp 183, but on the
other hand, it has a negative influence on the PK profile (CL
and F).24
a
Scheme 1. Synthesis of Sulfamides 11
As shown in Figure 1, compound 3 (MW = 286 g/mol) is
relatively flexible and has a moderate oral bioavailability and
clearance; however, it is probably not active enough for studies
in humans. Compound 4 with a high molecular weight (443.2
g/mol) and rigid construction has a fast clearance and very low
oral bioavailability.24 It is noteworthy that cyclic compound 1
with low molecular weight and high potency shows unusually
good oral bioavailability. Structurally, 1 differs from the others.
Being a dipeptide-like compound it could be actively
transported from the intestine via a peptide transporter system
(e.g., PEPT1/2) rather than a simple amino acids transporter
system (e.g., OATs, OCTs, CATs). However, it has a
moderately fast clearance and because of that, this compound
must be administered twice a day to humans. There is still a
need to find compounds that are potent arginase inhibitors but
with low clearance and high oral bioavailability.
As part of our arginase program, we designed and
synthesized two groups of inhibitors: one with a nonbasic
side chain and the second with a basic side chain, low
molecular weight, and simple linear structure (Figure 2). We
a
Reagents and conditions: (a) NaH, DMF; (b) NaH, DMF, then
MeI; (c) NaBH4, NiCl2·6H2O, MeOH; (d) ClSO2NCO, t-BuOH,
TEA, DCM, 0 °C (for 10a); (tert butoxycarbonyl)((4-
(methyliminio)pyridin-1(4H)-yl)sulfonyl)amide, TEA, DCM, RT,
(for 10b); ClSO2NMe2, TEA, DCM, 0 °C (for 10c); (e) TPP,
DIAD, THF, then MeOH; (f) TPP, DIAD, THF, then BnOH; (g)
HCl/dioxane; (h) HCl(aq)
.
Figure 2. Compounds designed and synthesized in this work.
The racemic guanidine derivatives 15a−c were obtained in a
similar manner via guanidinylation of amines 9, as depicted in
Scheme 2.44
postulate that the only possible mechanisms of absorption in
such polar, transition state analog inhibitors are active
transport and/or paracellular transport and that these strongly
hydrophilic compounds are not likely to undergo extensive
first-pass metabolism. Moreover, reabsorption of the com-
pounds, preventing renal clearance, must run via active
transport predominantly. For these two mechanisms, the
most important factors are molecular weight (<300 g/mol)
and similarity to the naturally transported substances (amino
acids) rather than polarity (log D < 0 is favorable).35,36
The asymmetric route was also developed. The synthesis of
enantiomerically enriched guanidine 15aa is illustrated in
Scheme 3. Asymmetric, diastereoselective aza-Henry reaction
led to the formation of nitro amino ester 19 that was subjected
to hydroboration and subsequent reduction to obtain boronate
21.45 Amine 21 was next guanidylated and deprotected to give
a desired amino acid 15aa.44 Enantiomeric purity of 15aa was
>98% ee based on the diastereomeric excess of the product
after guanylation of 21.
B
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Letter
a
Scheme 2. Synthesis of Guanidines 15
Table 1. In Vitro Activity of Sulfamoylo Derivatives
Cmpd
x
n
R1
R2
R3
ARG1 IC50 (μM)
1
2
Reference compound
Reference compound
0.12
1.2
0.33
2.3
0.73
1.0
1.4
a
Reagents and conditions: (a) tert-butyl (((tert-butoxycarbonyl)-
amino)(1H-pyrazol-1-yl)methylene)carbamate, MeCN (for 14a and
14b); tert-butyl (((tert-butoxycarbonyl)-imino)-(1H-pyrazol-1-yl)-
methyl)(methyl)carbamate, MeCN (for 14c); (b) HCl/dioxane
and/or 6 M HClaq.
11a
11b
11c
11d
11e
12
NH
NH
NH
NH
NH
NH
CH2
1
1
1
1
1
2
1
H
Me
H
H
H
H
H
Me
H
H
H
H
Me
Me
Bn
H
Scheme 3. Asymmetric Synthesis of Guanidine 15aa
H
H
H
H
2.8
1.1
13
H
Table 2. In Vitro Activity of Guanidine Analogs
Cmpd
n
1
R1
R2
H
ARG1 IC50 (nM)
15a
15aa
15ab
15b
15c
16
H
67
32
6800
78
233
7000
R-enantiomer
S-enantiomer
1
1
2
Me
H
H
H
Me
H
compounds (11c−e). N,N-Dimethylsulfamoyl derivative
(11c) was the most active of them (0.73 μM). In turn,
bulkier, benzyl substituent as R3 (in 11e) was the least
tolerated. We also found that potency decreases together with
an extension of the spacer to two carbon atoms, and so simple
ethylene-linked unsubstituted sulfamide homologue 12 ex-
hibited substantially lower activity than 11a. Interestingly,
sulfonamide 13 demonstrated a clear impairment (3.3-fold) in
potency compared to compounds being sulfamide 11a. It
clearly shows that NH in the methylene-bridge position is
more favorable than the CH2 group, likely due to its H-donor
character. The most active compound in this series (11a) was
nevertheless almost 3-fold weaker ARG1 inhibitor than 1.
From the guanidine series presented in Table 2 the
compounds with methylene-linker (15a−15c) showed a clear
improvement over ABH and sulfamides 11 in the inhibitory
potency toward arginase. In this context, the replacement of
the sulfamide moiety in molecule 11a into guanidine in
inhibitor 15a resulted in the formation of a 5-fold more active
compound (15a) with IC50 = 67 nM. This presumably results
from a more optimal and stronger binding interaction with Asp
181 and Asp 183 in the ARG1 active site by dual-ionic and
hydrogen-bond interaction induced by guanidine. However,
other interactions are also possible (especially with Ser137,
Glu186, Gly142, Thr136, His126, and Asn130).22−24
a
Reagents and conditions: (a) (S)-(−)-2-methyl-2-propanesulfina-
mide, Ti(OEt)4, DCM; (b) TBAF, MeNO2; (c) pinacolborane,
[Ir(cod)Cl]2, DPPE, DCM; (d) NaBH4, NiCl2·6H2O, MeOH; (e)
tert-butyl (((tert-butoxycarbonyl)amino)(1H-pyrazol-1-yl)methylene)
carbamate, Et3N, MeCN; (f) 6M HClaq.
All final compounds described in this work were obtained
and tested as hydrochloride salts. The novel sulfamide and
guanidine analogs were evaluated in a human recombinant
arginase 1 colorimetric enzymatic assay, measuring the
inhibition of arginase-induced urea production.46 The results
from these experiments are listed in Tables 1 and 2, where the
IC50 of synthesized compounds and reference inhibitors 1 and
2 are shown.
The introduction of the unsubstituted sulfamoylomethylene
group in the α-position of ABH scaffold led to 11a with more
than 3.5-fold higher potency than ABH (0.33 vs 1.2 μM). It is
worth noting that 11a (MW = 283 g/mol) is much more active
than unsubstituted basic primary aminomethylene analog (IC50
= 2 μM, lit. data)22 and is the most active linear ABH analog
with nonbasic side chain with the MW < 300 g/mol described
so far.22,25
As expected, the small highly polar enzyme active site
accommodates only amino acids with the natural R-stereo
configuration such as 15aa. Unnatural amino acids with S-
To improve the potency of 11a simple methyl substituent
was introduced in the R1 position, but instead, a significant
decrease in the inhibitory activity of 11b was observed.
Modification of the sulfamide moiety by the introduction of
terminal N-alkyl substituents also generated less active
configuration, such as 15ab, are much less active with IC50
=
6.8 μM. Enantiomerically enriched guanidine 15aa is the most
active hARG1 inhibitor with such a low molecular weight
C
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(246.1 g/mol) as well as with the linear, noncyclic structure
described to date. Because the α-amino group of the arginase
inhibitors is involved in numerous important interactions with
protein, only a limited choice of substituents at this group is
possible. Interestingly, as seen with 15b, incorporation of a
simple methyl substituent at position R1 has no high influence
on the inhibitory potency. On the other hand, the methyl
group on guanidine (in 15c) resulted in decreased activity. The
elongation of the carbonic linker in the guanidine series to two
carbon atoms gave 16 with substantially lower activity. Thus,
homologue 16 was the least active compound described in this
work, with IC50 = 7 μM.
Transforming an α position on the scaffold by the introduction
of sulfamides and guanidines led to increasing activity
providing compounds with low molecular weight and linear
structure. Synthesized inhibitors demonstrated an improved
pharmacokinetic profile after intravenous administration
compared to the other known inhibitors; however, they
suffered from low oral bioavailability.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00508.
Next, the most potent racemic inhibitors from both series
(11a and 15a) were screened in mouse PK (Table 3).
Chemistry and synthetic schemes, synthetic procedures
and compound characterization, description of bio-
chemical assays, PK (PDF)
Table 3. Pharmacokinetic Parameters of 11a, 15a, and 15aa
Compound
11a (mouse)
15a (mouse)
15aa (rat)
AUTHOR INFORMATION
Corresponding Author
■
AUC,n0‑inf(i.v. kg·h/L)
C0orCmax(i.v.mg/L)
CL (i.v.mL/min/kg)
4.13
34.59
4.0
3.26
0.2
1.66
18.83
10.0
0.62
0.16
0.26
<1
3.92
21.22
4.2
32.71
3.04
11.91
4
Roman Blaszczyk − OncoArendi Therapeutics, 02-089 Warsaw,
Poland; orcid.org/0000-0001-7348-9837;
Email: r.blaszczyk@oncoarendi.com
t
1/2(i.v. h)
Vss (i.v.L/kg)
MRT (i.v. h)
F (p.o. %)
0.81
7
Authors
Joanna Brzezinska − OncoArendi Therapeutics, 02-089
Warsaw, Poland
Barbara Dymek − OncoArendi Therapeutics, 02-089 Warsaw,
Poland
Paulina S. Stanczak − OncoArendi Therapeutics, 02-089
Warsaw, Poland
Marcin Mazurkiewicz − OncoArendi Therapeutics, 02-089
Warsaw, Poland
Jacek Olczak − OncoArendi Therapeutics, 02-089 Warsaw,
Poland; orcid.org/0000-0001-9932-2373
Julita Nowicka − OncoArendi Therapeutics, 02-089 Warsaw,
Poland
Karolina Dzwonek − OncoArendi Therapeutics, 02-089
Warsaw, Poland
Agnieszka Zagozdzon − OncoArendi Therapeutics, 02-089
Warsaw, Poland
Jakub Golab − Department of Immunology, Medical University
of Warsaw, 02-097 Warsaw, Poland
Adam Golebiowski − OncoArendi Therapeutics, 02-089
Warsaw, Poland; orcid.org/0000-0001-5960-2995
Following intravenous administration in mice, compounds in
this series generally exhibited low to moderate clearance, low
volume of distribution, and low to moderate half-life. When
compared to mouse PK of 1 the novel inhibitors 11a and 15a
have over 5- and 2-fold slower clearance, respectively.34 The
bioavailability for oral dosing was low. Because of a low oral
bioavailability in mice, antitumor efficacy studies in mouse
tumor models have not been performed.
On the other hand, to compare PK profiles of the most
active inhibitor 15aa to other known inhibitors (e.g., 1, 3, and
4, for which the rat PK parameters are known), the PK
parameters of the compound in rats were measured.
The rat PK profile for optically active 15aa was better than
that for reference inhibitors 1, 3, and 4 after i.v. administration
(slow clearance, a moderate volume of distribution, long t1/2).
Its oral bioavailability, however, was still very poor (4%).
Nevertheless, the concentration of 15aa is up to 4-fold higher
than IC50 of human ARG1 for 24 h after compound
administration i.v. as well as p.o. in low doses (Figure 3).
In summary, we have discovered a series of potent inhibitors
of arginase 1, starting from ABH as a lead compound.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.9b00508
Author Contributions
All authors have given approval to the final version of the
manuscript.
Funding
The work was supported by the grant 265503/3/NCBIR/15
(STRATEGMED2) from the National Center for Research
and Development (Poland).
Notes
The authors declare the following competing financial
interest(s): Some of the authors are current employees of
OncoArendi Therapeutics S.A. and own company stocks.
1. CB-1158 also known as INCB001158 was discovered by
Calithera Biosciences and colicensed to Incyte Corporation.
The compound is commercially available from several vendors.
CAS: 2095732-06-0; UNII: IFD73D535A; DB15286.
Figure 3. Pharmacokinetic profile of 15aa in rats. Plasma
concentrations following 3 mg/kg i.v. (orange line) and 10 mg/kg
p.o. (blue line) administrations to male Sprague−Dawley rats.
D
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McEwen-Smith, R. M.; Qureshi, A.; Dazzi, F.; Vyas, P.; Cerundolo, V.
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Cierniak, S.; Koktysz, R.; Elishaev, E.; Gruca, S.; Stefanowicz, A.;
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S.; Dranoff, G.; Hammerman, P. S.; Pecot, C. V.; Wong, K. K.; Akbay,
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pressive environment in colorectal cancer. Oncotarget. 2015, 6,
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(16) Steggerda, S. M.; Bennett, M. K.; Chen, J.; Emberley, E.;
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Marguier, G.; Murray, P. J.; Neou, S.; Pan, A.; Parlati, F.; Rodriguez,
M. L. M.; Van de Velde, L. A.; Wang, T.; Works, M.; Zhang, J.;
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2. Reference inhibitor 1 is compound no. 10 from patent
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salts. ARG1/CB-1158 crystal structure has been resolved.47
ABBREVIATIONS
■
ABH, 2(S)-amino-6-boronohexanoic acid; AUC, area under
curve; Boc, tert-butoxycarbonyl; CL, clearance; Cmax, peak
concentration; cmpd, compound; cod, 1,5-cyclooctadiene;
DCM, dichloromethane; DIAD, diisopropyl azodicarboxylate;
DPPE, ethylenebis(diphenylphosphine); F(%), oral bioavail-
ability; H-bond, hydrogen bond; hARG1, human arginase 1;
i.v., intravenous; LOQ, limit of quantification; MW, molecular
weight; OAT, organic anion transporter; OCT, organic cation
transporter; CAT, cationic amino acid transporter; Pg,
protective group; PK, pharmacokinetics; p.o., per os; PSA,
polar surface area; t1/2, half-life; TBAF, tetra-n-butylammonium
fluoride; TEA, triethylamine; TPP, triphenylphosphine; Vss,
volume of distribution
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