α-Amino-β-carboxymuconate-?-semialdehyde Decarboxylase (ACMSD) Inhibitors as Novel Modulators of de Novo Nicotinamide Adenine Dinucleotide (NAD+) Biosynthesis

Article abstract of DOI:10.1021/acs.jmedchem.7b01254

NAD⁺ has a central function in linking cellular metabolism to major cell-signaling and gene-regulation pathways. Defects in NAD⁺ homeostasis underpin a wide range of diseases, including cancer, metabolic disorders, and aging. Although the beneficial effects of boosting NAD⁺ on mitochondrial fitness, metabolism, and lifespan are well established, to date, no therapeutic enhancers of de novo NAD⁺ biosynthesis have been reported. Herein we report the discovery of 3-[[[5-cyano-1,6-dihydro-6-oxo-4-(2-thienyl)-2-pyrimidinyl]thio]methyl]phenylacetic acid (TES-1025, 22), the first potent and selective inhibitor of human ACMSD (IC₅₀ = 0.013 μM) that increases NAD⁺ levels in cellular systems. The results of physicochemical-property, ADME, and safety profiling, coupled with in vivo target-engagement studies, support the hypothesis that ACMSD inhibition increases de novo NAD⁺ biosynthesis and position 22 as a first-class molecule for the evaluation of the therapeutic potential of ACMSD inhibition in treating disorders with perturbed NAD⁺ supply or homeostasis.

Full text of DOI:10.1021/acs.jmedchem.7b01254

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Article
#-Amino-#-carboxymuconate-#-semialdehyde decarboxylase
(
ACMSD) Inhibitors as Novel Modulators of de novo
+
Nicotinamide Adenine Dinucleotide (NAD) Biosynthesis
Roberto Pellicciari, Paride Liscio, Nicola Giacchè, Francesca De Franco, Andrea Carotti,
Janet Robertson, Lucia Cialabrini, Elena Katsyuba, Nadia Raffaelli, and Johan Auwerx
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01254 • Publication Date (Web): 18 Jan 2018
Downloaded from http://pubs.acs.org on January 18, 2018
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αꢀAminoꢀβꢀcarboxymuconateꢀεꢀsemialdehyde
decarboxylase (ACMSD) Inhibitors as Novel
Modulators of de novo Nicotinamide Adenine
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Dinucleotide (NAD ) Biosynthesis
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Roberto Pellicciari,* Paride Liscio, Nicola Giacchè, Francesca De Franco, Andrea Carotti,
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Janet Robertson, Lucia Cialabrini, Elena Katsyuba, Nadia Raffaelli and Johan Auwerx*
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TES Pharma S.r.l., ITꢀ06073, Corciano, Perugia, Italy
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Department of Pharmaceutical Sciences, University of Perugia, ITꢀ06123 Perugia, Italy
3
Department of Agricultural, Food and Environmental Sciences, Polytechnic University of Marche,
ITꢀ60131 Ancona, Italy
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Laboratory of Integrative and Systems Physiology, Interfaculty Institute of Bioengineering, École
Polytechnique Fédérale de Lausanne CHꢀ1015, Switzerland
*
Address correspondence to: R.P. rpellicciari@tespharma.com ; J.A. : johan.auwerx@epfl.ch
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KEYWORDS: de novo NAD Biosynthesis, Kynurenine Pathway, ACMSD, NAD .
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ABSTRACT: NAD has a central function in linking cellular metabolism to major cell signaling
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and gene regulation pathways. Defects in NAD homeostasis underpin a wide range of diseases,
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including cancer, metabolic disorders, and aging. Whilst the beneficial effects of NAD boosting
are well established in mitochondrial fitness, metabolism, and lifespan, to date, no therapeutic
+
enhancers of de novo NAD biosynthesis have been reported. Herein we report the discovery of 3ꢀ
[
[[5ꢀcyanoꢀ1,6ꢀdihydroꢀ6ꢀoxoꢀ4ꢀ(2ꢀthienyl)ꢀ2ꢀpyrimidinyl]thio]methyl]phenylacetic acid (TESꢀ
1
025, 22) the first potent and selective inhibitor of human ACMSD (IC50 = 0.013 µM) that increase
+
NAD levels in cellular systems. Results of physicochemical properties, ADME, and safety
profiling, coupled with in vivo target engagement studies, support the hypothesis that ACMSD
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inhibition increases de novo NAD biosynthesis and position 22 as a first in class molecule for
evaluation of the therapeutic potential of ACMSD inhibition in treating disorders with perturbed
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NAD supply and/or homeostasis.
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INTRODUCTION
Originally described as an enzyme cofactor, Nicotinamide Adenine Dinucleotide (NAD ) is a key
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cellular factor linking cellular metabolism to major cell signaling and gene regulation pathways.
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The medical importance of NAD was initially established in 1937 when Conrad Elvehjem and his
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team discovered that the devastating disease “pellagra” could be cured by nicotinic acid and
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nicotinamide, both NAD precursors. Today deficits or disturbances in NAD homeostasis are
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associated with a wide range of diseases , including kidney dysfunction, metabolic disorders,
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2,12ꢀ14
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muscle diseases, neurodegeneration , and aging.
In humans, NAD is synthesized from four
dietary sources: nicotinic acid, nicotinamide, and nicotinamide riboside (collectively known as
vitamin B3) through metabolic pathways that salvage the preformed pyridine ring, and tryptophan
+
2,11
(
Trp) through the de novo NAD Biosynthesis / Kynurenine Pathway (KP).
More than 95% of dietary tryptophan undergoes metabolism by the KP through the production of
15ꢀ17
a large array of metabolites with various bioactivities.
To date, indoleamine 2,3ꢀdioxygenaseꢀ1
and 2 (IDO1 and 2), kynurenine 3ꢀmonooxygenase (KMO) and kynurenine aminotransferase I and
1
8ꢀ21
II (KATI and KATII) are the most widely studied enzymes as potential druggable targets
in this
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23ꢀ24
16,25ꢀ27
pathway for cancer, immune dysfunction
, and central nervous system (CNS) disorders.
28ꢀ29
30ꢀ32
Our early contributions in the discovery of potent and selective KMO
and KATII
inhibitors
(
Figure 1A) provided excellent chemical tools, instrumental for the development of more advanced
33ꢀ35
preclinical candidate compounds (Figure 1B).
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a
O
NH2
CO H
O
NH2
CO
Cl
Cl
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H
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CO H
2
O
S
O
NO2
O
1
, UPF-648
2, m-NBA
KMO IC50 = 0.9 µM
3, (S)-ESBA
KATII IC50 = 71 µM
KMO IC50 = 0.02 µM
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O
Cl
Cl
CO H
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NH2
CO H
2
4, GSK18033
KMO IC50 = 0.006 µM
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, PF-0485998935
6
KMO IC50 = 0.0006 µM
KATII IC50 = 0.023 µM
Figure 1. Early (a) and recently (b) disclosed KMO and KATII inhibitors.
In this context and as a continuation of our interest in the field, we have recently moved our
attention towards unexplored branches of the KP focusing on the discovery of novel inhibitors of αꢀ
aminoꢀβꢀcarboxymuconateꢀεꢀsemialdehyde decarboxylase (ACMSD, EC 4.1.1.45), a critical
+
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enzyme in de novo NAD biosynthesis. (Figure 2)
The enzyme catalyzes the decarboxylation of 2ꢀamino 3ꢀcarboxymuconate 6ꢀsemialdehyde
(
ACMS), an intermediate of the KP, to 2ꢀaminomuconateꢀ6ꢀsemialdehyde (AMS). AMS can either
undergo spontaneous closure of the pyridine ring to form picolinic acid (PIC) or be oxidized to 2ꢀ
aminomuconate, which is further metabolized to enter the tricarboxylic acid (TCA) cycle (Figure
2). When the enzymatic capacity of ACMSD is exceeded then ACMS can also cyclize
+
spontaneously to quinolinic acid (QUIN), which is further converted to the coenzyme NAD
(
Figure 2).
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L-Tryptophan
K yy nurenine pp athway
OHC
CO H
CO H
2
2
Nonenzymatic
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NAD+
H N
CO H
N
CO H
2
2
2
ACMS
QUIN
ACMSD
OHC
Nonenzymatic
N
CO H
H N
CO H
2
2
2
PIC
AMS
TCA Cycle
Figure 2. The role of ACMSD as the branching point in the Kynurenine Pathway leading to de
+
novo NAD biosynthesis.
Since the cyclization of ACMS into QUIN is a spontaneous reaction, the amount of ACMS
+
undergoing this conversion, and therefore leading to a production of NAD , is primarily determined
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by the activity of ACMSD. Thus, inhibition of ACMSD would channel ACMS towards de novo
+
+
+
NAD biosynthesis, providing a novel way to boost NAD levels and to reꢀestablish NAD
homeostasis.
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2
ACMSD is a Zn ꢀdependent amidohydrolase conserved in eukaryotes and several bacterial
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species. In mammals, the expression of the enzyme is restricted to the kidney, liver, and brain,
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with an expression ratio of 1,300:30:1. The murine enzyme has been shown to be under nutritional
4
0ꢀ41
and hormonal control, being upregulated by high protein diets
and downregulated by dietary
42
43
polyunsaturated fatty acids
and cholesterol.
Notably, the enzyme is upregulated in
41
streptozotocinꢀinduced diabetic rats, whilst insulin treatment suppresses this elevation. As
expected, changes in the ACMSD activity are readily reflected in the serum levels of QUIN and
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PIC
and in the rate of tryptophan to NAD conversion, confirming the role of the enzyme in
regulating the production of metabolites whose levels are known to impact on neuronal and
metabolic functions. Indeed, increased QUIN levels have been associated with several neurological
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diseases. On the other hand, it is widely documented that enhanced NAD biosynthesis has both
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protective and therapeutic effects on a large spectrum of disorders, including metabolic,
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neurodegenerative, muscular, cardiac, and renal dysfunctions. Thus, inhibition of ACMSD
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activity might be of therapeutic potential, since it should raise cellular NAD content.
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Work carried out by Fukuwatari et al. identified phthalate monoesters, such as mono(2ꢀ
ethylhexyl)phthalate (MEHP, 7, Figure 3), as the first examples of mammalian ACMSD inhibitors.
Previous efforts in understanding the mechanism of recognition and binding of ligands to the active
site of the human enzyme resulted in the release of the first coꢀcrystal complex of the enzyme (PDB
47
code 2WM1) with 1,3ꢀdihydroxyacetonephosphate (DHAP, 8, Figure 3). More recently, a second
complex of hACMSD with a competitive inhibitor (PDB code 4IH3), namely pyridineꢀ2,6ꢀ
dicarboxylic acid (PDC, 9, Figure 3) was disclosed, refining the previous findings and highlighting
48
the homoꢀdimeric nature of the enzyme.
HO
O
O
O
OH
O
O
O
HO
P
HO C
N
CO H
2
2
HO
7
, MEHP
8, DHAP,
PDB Code: 2WM1)
9, PDC,
(
(PDB Code: 4IH3)
Figure 3. Early ACMSD inhibitors.
With only limited evidence of ACMSD inhibitors available in literature to date, the quest for
potent and selective inhibitors is of great interest to clarify the role of this unexplored enzyme in
physiopathological conditions and to define its therapeutic potential. In this work, we describe our
efforts spanning from hit identification to early chemical optimization, concluding with in vivo
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proofꢀofꢀconcept studies that our compounds enhanced de novo NAD biosynthesis via ACMSD
target engagement.
RESULTS AND DISCUSSION
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Hit Identification. Hit identification efforts started with the construction of an in silico model of
the enzyme binding site guided by the available structural information from the two crystal
complexes of human recombinant ACMSD (hACMSD) with 8 (DHAP) and 9 (PDC) (Figure 3).
The molecular modeling workflow was first tested with a limited set of known binders (7ꢀ9) and
then applied to the TES internal database of compounds. The selected compounds were tested for
their inhibitory effect on the catalytic activity of hACMSD by using an enzymeꢀcoupled
49
spectrophotometric assay. The virtual screening campaign enabled the identification of 3ꢀ(5ꢀ
cyanoꢀ6ꢀoxoꢀ4ꢀphenylꢀ1,6ꢀdihydroꢀpyrimidinꢀ2ꢀylsulfanylmethyl)ꢀbenzoic acid (10) as the first low
nanomolar hACMSD inhibitor (IC50 = 0.045 µM, Figure 4). Despite showing a widely used 2ꢀ
thiopyrimidoneꢀ5ꢀcarbonitrile core, the metaꢀcarboxyphenyl moiety of compound 10 confers
structural novelty to this class of derivatives and encouraged us to further investigate this structural
class of drugꢀlike small molecule ACMSD inhibitors.
Preliminary observation of the InducedꢀFit docking pose of 10 into the hACMSD catalytic site
coming from the 4IH3 Xꢀray (Figure 5) suggested that the majority of the binding affinity was due
2
+
to ionic interactions of the mꢀcarboxylate group with the Zn atom and Trp191 and Arg47 residues.
Meanwhile the 1,6ꢀdihydropyrimidine nucleus engages hydrogen bond interactions with Lys41 and
Lys44 (Figure 5).
Figure 4. hACMSD 2ꢀthiopyrimidone screening hit, compound 10.
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hACMSD IC50 = 0.045 µM
Figure 5. Docking model of 10 (pink) in the hACMSD active site (light green). The Zinc ion is
displayed as a grey ball. The hydrogen bonds engaged with Lys41, Lys44, Arg47, and Trp191
residues are also shown with yellow dashes.
Based on these observations, medicinal chemistry efforts were focused on hit expansion with the
primary goal of exploring the structureꢀactivity relationships (SAR) and corroborating the proposed
binding hypothesis.
To this end, we first investigated the role of the carboxylate functionality on the right hand phenyl
5
0
ring. Hydroxamic acid and 3ꢀmethylꢀ5ꢀtrifluoromethylꢀ[1,2,4]oxadiazole (TFMO) were selected
2+
as the most representative functional groups to probe the Zn binding hypothesis of the carboxylate
moiety of the starting compound 10. Whilst the 2Hꢀtetrazole nucleus was also investigated as
classical acid surrogate concluding with the homologation of the meta carboxylate group of 10.
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We then moved our attention to a short exploration of the 2ꢀthiopyrimidoneꢀ5ꢀcarbonitrile
nucleus producing SAR through systematic replacements at position Cꢀ4, Cꢀ5, and Cꢀ6 respectively
(Table 1).
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Chemistry. Final compounds 10, 12-13, 16-18, and 20-22 were easily prepared by regioꢀselective
Sꢀalkylation reactions between a series of 5,6 disubstitutedꢀ2ꢀthiopyrimidinꢀ4ꢀones (23, 26-29) and
commercially available 3ꢀ(chloromethyl)benzoic acid (24) and benzyl bromide (25) or the
appropriate chloroꢀ or bromoꢀmethyl phenyl intermediate (30ꢀ33) (Scheme 1ꢀ3).
a
Scheme 1. Synthetic route to 4ꢀphenylꢀ5ꢀcyanoꢀꢀ2ꢀthiopyrimidone derivatives 10-12
b
NC
O
NC
X
N
N
CO H
2
CO H
2
N
H
S
N
S
NC
O
a
10
NH
1
1: X = Cl
N
H
S
23
NC
N
H
O
N
S
H
1
2
a
2 3 3
Reagents and conditions: (a) K CO , CH CN, 3ꢀ(chloromethyl)benzoic acid (24) or benzylbromide
(
25), reflux; (b) POCl , 70 °C.
3
Chlorination in position Cꢀ6 of the 2ꢀthiopyrimidone nucleus of compound 10 was accomplished by
using POCl thus affording derivative 11 (Scheme 1), Halogen replacements in the Cꢀ5 position,
compounds 14 and 15, were obtained by reacting a suspension of compound 13 in AcOH with
bromine or Nꢀchlorosuccinimide respectively in the presence of PbO (Scheme 2).
3
2
a
Scheme 2. Synthetic route to 4ꢀarylꢀ5ꢀmodifiedꢀ2ꢀthiopyrimidone derivatives 13ꢀ15
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1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
b
H
O
H
O
R
1
NH
N
N
CO H
CO H
2
2
N
H
S
N
H
S
O
N
H
S
2
6
13
14: R1 = Br
15: R1 = Cl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
2 3 3 2 2
Reagents and conditions: (a) K CO , CH CN, 24, reflux; (b) AcOH, PbO , Br or NCS.
The synthesis of the hydroxamic acid derivative 19 was described in Scheme 3. SꢀAlkylation of 4ꢀ
oxoꢀ6ꢀ(thiophenꢀ2ꢀyl)ꢀ2ꢀthioxoꢀpyrimidineꢀ5ꢀcarbonitrile (29) with 3ꢀchloromethylꢀNꢀ(tetrahydroꢀ
pyranꢀ2ꢀyloxy)ꢀbenzamide (33) gave intermediate 34 which readily provided the desired
hydroxamate compound 19 after acidic removal of the tetrahydropyran acetal protecting group.
a
Scheme 3. Synthetic route to substituted 5ꢀcyanoꢀ2ꢀthiopyrimidone derivatives 16-22
R2
NC
N
Y
O
N
H
S
R2
NC
O
16-18, 20-22
NH
Y
a
Z
+
N
H
S
S
N
O
2
2
2
7: R2 = methyl
24: Z = Cl, Y =
NC
O
OH
O
O
8: R2 = cyclohexyl
9: R2 = 2-thienyl
N
N
OTHP
CF
3
N
S
N
H
3
3
3
3
0: Z = Cl, Y =
1: Z = Br, Y =
2: Z = Br, Y =
3: Z = Cl, Y =
N
H
N
N
34
N
H
b
OH
O
S
O
OTHP
N
H
NC
O
N
O
OH
N
S
N
H
H
1
9
a
2 3 3
Reagents and conditions: (a) K CO , CH CN, reflux or DIPEA, DMSO, r.t.; (b) TFA, DCM, r.t.
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Furthermore, 2ꢀthiopyrimidinꢀ4ꢀone intermediates (23, 28, and 29) were prepared as
described in Scheme 4. In particular, 5ꢀcyanoꢀ6ꢀarylꢀ2ꢀthiopyrimidinones 23, 28, and 29 were
prepared following literature procedures by oneꢀpot multicomponent condensation between the
corresponding aldehyde 35a,c ethyl cyanoacetate 36, and thiourea 37 in the presence of potassium
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
51
carbonate as base. In a similar manner, reaction between commercially available ethylbenzoyl
acetate (38) or 2ꢀcyanoꢀ3ꢀethoxyꢀbutꢀ2ꢀenoic acid ethyl esters (39) and 37 in basic conditions
furnished the corresponding 6ꢀphenylꢀ4ꢀoxoꢀ2ꢀthioxoꢀ2,3ꢀdihydroꢀ1Hꢀpyrimidinꢀ4ꢀone (26) and 6ꢀ
5
2
methylꢀ4ꢀoxoꢀ2ꢀthioxoꢀpyrimidineꢀ5ꢀcarbonitrile (27) in acceptable yields (Scheme 4).
a
Scheme 4. Synthetic route to 5,6ꢀdisubstitutedꢀ2ꢀthiopyrimidone derivatives 23, 26-29
R2
O
NC
O
a
NH
+
NC
CO Et
2
R2
H
N
H
S
35a: R2 = phenyl
36
23, 28-29
3
5b: R2 = cyclohexyl
3
5c: R2 = 2-thienyl
O
O
b
OEt
H
O
NH
N
H
S
3
8
26
Me
NH
NC
CO Et
2
b
NC
O
OEt
N
H
S
39
27
a
2 3
Reagents and conditions: (a) Thiourea (37), K CO or piperidine, EtOH, reflux; (b) 37, EtONa,
EtOH, reflux.
Meanwhile bromoꢀ or chloromethyl phenyl intermediates 30ꢀ33 were prepared using the chemistry
described in Scheme 5. Treatment of metaꢀtolunitrile (40) with NH OH•HCl yielded quantitatively
2
the corresponding hydroxyl amidine 41. Intramolecular cyclization to give the trifluoromethyl
oxadiazole (TFMO) intermediate (42) was accomplished by treating 41 with trifluoroacetic
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
anhydride using pyridine as solvent. Finally, benzylic bromination of 42 using standard condition
such as NBS/DPO provided the target compound 30. 5ꢀmꢀtolylꢀ2Hꢀtetrazole (43) was easily
prepared by 1,3ꢀdipolar cycloaddition reaction within the nitrile 40 and sodium azide and further
derivatized in the benzylic position to provide intermediate 31 in satisfactory yield. In the same
context bromination of commercially available phenylacetic acid 44 cleanly gave intermediate 32.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Attempts for direct coupling of 24 with NH OH failed even using various promoting agents such as
2
COCl , 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide (EDCI), or hydroxybenzotriazole (HOBT)
2
2 2
(data not shown). Alternatively, NH OH was protected as the tetrahydropyran acetal (NH OTHP)
5
3
following literature procedure and the amide forming reaction with 24 proceeded smoothly via
acyl chloride formation to give the desired intermediate 33.
a
Scheme 5. Synthesis of intermediates 30-33
NH
N
O
Br
N
O
a
b
c
OH
CF3
CF3
N
N
N
H
42
CN
41
30
N
N
N
N
Br
4
0
d
e
NH
NH
N
N
43
31
Br
CO H
2
f
CO2H
4
2
4
4
32
Cl
Cl
O
O
O
g
CO2H
H2N
O
O
+
N
H
45
33
a
Reagents and conditions: (a) NH
NBS, DPO, CH CN (d) NaN , Et
NBS, AIBN, CCl , reflux; (g) CO Cl , DMF, DCM, Et N.
2
OH•HCl, NaHCO
3
, EtOH; (b) (CF
3
CO)
2
O, pyridine, reflux; (c)
+
ꢀ
3
3
3 4 3
NH Cl , toluene, reflux; (e) NBS, AIBN, CH CN, reflux; (f)
4
2
2
3
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Biochemical and SAR Evaluation. The newly synthesized compounds were evaluated for their
ability to inhibit the catalytic activity of hACMSD. IC50 values for tested compounds were
calculated by assaying the enzyme activity at 10 µM concentration of the substrate, which is very
close to the K
m
value (6.5 µM, as reported in ref. 39)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Table 1. Biochemical activity of hACMSD Inhibitors
NC
N
CO H
2
X
N
S
m
#
11
X
ꢀCl
IC50 ± SD (µM)
0.316 ± 0.019
R2
R1
N
Y
O
N
H
S
m
#
R
1
R
2
Y
IC50 ± SD (µM)
0.045 ± 0.005
10
12
13
14
15
ꢀCN
ꢀCN
ꢀH
ꢀCOOH
ꢀH
4.45 ± 0.81
1.055 ± 0.287
0.099 ± 0.032
ꢀCOOH
ꢀCOOH
ꢀBr
ꢀCl
ꢀCOOH
ꢀCOOH
ꢀCOOH
0.151 ± 0.013
1.899 ± 0.383
0.089 ± 0.042
Me
16
ꢀCN
ꢀCN
17
S
S
18
ꢀCN
ꢀCN
ꢀCOOH
0.020 ± 0.006
1.746 ± 0.123
19
ꢀCONHOH
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N O
S
20
21
22
ꢀCN
ꢀCN
ꢀCN
3.35 ± 0.68
0.003 ± 0.001
0.013 ± 0.003
CF3
N
N
N
S
S
N
N
H
2
ꢀCH COOH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
ACMSD enzymatic assay is described in the Experimental Section. IC50 were calculated with
concentrationꢀresponse curves, and shown values are the mean ± SD from three independent
experiments.
As summarized in Table 1 we investigated variations around the functional groups presented by
compound 10, starting with an investigation of variations at the Cꢀ6 position of the pyrimidone
nucleus and the meta carboxylic acid group as the two key pharmacophoric elements of our starting
hit 10. Chlorination of the carbonyl group at position Cꢀ6 resulted in detrimental effects on
compound potency (derivative 11). The same trend was observed with removal of the carboxylate at
position meta. Indeed, compound 12 lacking the acid moiety was approximately 100ꢀfold less
potent than hit compound 10, thus supporting our initial binding hypothesis and design strategy.
Having established a SAR at the mꢀposition of the right hand phenyl ring, we turned our attention to
the nitrile group at position Cꢀ5 of the 2ꢀthio pyrimidinone scaffold. Interestingly, whilst bromine
and chlorine in position Cꢀ5, 14 and 15 respectively, worked as nitrile bioisosteres maintaining low
IC50 values, complete removal of the nitrile group drastically reduced the inhibitory potency as
shown in derivative 13. Continuing to probe the minimal requirements for ACMSD inhibitory
activity of our starting chemotype 10, we next set out to survey SAR at the Cꢀ4 position of the 2ꢀ
thiopyrimidinone nucleus through alkyl, cycloalkyl, and bioisosteric replacement of the phenyl ring.
While the cyclohexyl substituent maintained the starting inhibitory potency with a slight decrease of
the IC50 value (compound 17), a methyl replacement of the phenyl drastically reduced the starting
potency as shown in compound 16. Interestingly, phenyl to 2ꢀthienyl bioisosteric group
manipulation at position Cꢀ4 of the 2ꢀthiopyrimidoneꢀ5ꢀcarbonitrile nucleus provided compound 18,
that exhibited a 2ꢀfold improvement in the ACMSD inhibitory potency (Table 1) with respect to the
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starting hit 10. Interesting results were obtained by the two zinc binding groups selected to work as
carboxylate equivalents. Surprisingly, both the hydroxamic acid and the TFMO derivatives
(compound 19 and 20, respectively Table 1) recorded a dramatic drop in the inhibitory potency thus
pointing out the importance of the ionic characteristics of the interaction established between the
carboxylate function with the zinc atom and key amino acid residues.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Instead canonical replacement of the carboxylate group of 10 with an acid surrogate, such as the
2
Hꢀtetrazole motif, provided the 1,6ꢀdihydroꢀ6ꢀoxoꢀ2ꢀ[[[3ꢀ(2Hꢀtetrazolꢀ5ꢀyl)phenyl]methyl]thio]ꢀ4ꢀ
(2ꢀthienyl)ꢀ5ꢀpyrimidinecarbonitrile derivative, 21 (TESꢀ991) which stands out as the most potent
analog of the series, suggesting an optimal fit of the tetrazole moiety within the catalytic binding
site of the ACMSD enzyme (Figure 6a). These findings were confirmed by the homologation of the
mꢀcarboxylate group of compound 10 which enabled the discovery of 3ꢀ[[(5ꢀcyanoꢀ1,6ꢀdihydroꢀ6ꢀ
5
4
oxoꢀ4ꢀ(2ꢀthienyl)ꢀ2ꢀpyrimidinyl)thio] methyl] phenylacetic acid, 22 (TESꢀ1025) , (Table 1) as a
low nanomolar inhibitor of hACMSD suitable for future development.
Furthermore, analysis of the docking pose of both 21 and 22 (Figure 6a,b) in our hACMSD model
suggested that the excellent potency shown by the inhibitors may arise in part from the 2ꢀthiophene
ring fitting better the hydrophobic cleft defined by the Trp191 and Met180 residues, combined with
orientation of the 2Hꢀtetrazole or carboxymethylene motif respectively, which optimize the
interaction geometry.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Docking models of 21 (grey, 6a) and 22 (green, 6b) in the hACMSD active site. The Zinc
ion is displayed by a grey ball. The hydrogen bonds engaged by the two compounds with Lys41,
Lys44, Arg47 and Trp191 are shown with yellow dashes.
In Vitro Studies. To further validate the newly synthesized derivatives, we investigated whether
+
the most potent compounds (21 and 22) were able to affect NAD levels in primary human
+
hepatocytes. Inhibition of the ACMSD enzyme is expected to increase the rate of the de novo NAD
+
biosynthesis, and should be reflected by an increase in intracellular NAD levels. The expression of
ACMSD in these cells was confirmed by RTꢀPCR (not shown). Notably, compounds 21 and 22
+
were both able to significantly increase intracellular NAD levels, which represents further proof of
their mechanism of action (Figure 7).
1
.5
.0
*
*
1
0.5
0.0
NT
21
22
5
µM
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+
Figure 7. NAD levels in primary cultured human hepatocytes incubated in the absence (NT) and in
the presence of 21 or 22. One way Anova: Dunnett’s test: * p < 0.01.
These results establish for the first time that ACMSD inhibitor compounds are able to increase
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
NAD content in a cellular setting.
As our initial purpose was to develop a lead compound suitable for in vivo studies, solubility and
passive cellular permeability of the two compounds 21 and 22 were evaluated and are reported in
Table 2. Furthermore, they profiled over a panel of cytochromes P450 (CYP 3A4, 2D6, 2C19 and
3
A1). Compound safety was evaluated in an in vitro human ERG potassium channel assay (hERG)
for cardiotoxicity and in selected cellular lines (murine AMLꢀ12 and human HepG2) for hepatic
toxicity. Selectivity to the target was initially assessed toward an inꢀhouse nuclear receptor panel
and then extended to a larger safety panel of about 126 offꢀtargets including kinases, phosphatases,
ion channels, and GPCRs (for details see the SI). As shown in Table 2, both compounds 21 and 22
exhibited good solubility with low permeability and a safe profile in a cellular setting, being devoid
of any additional offꢀtarget activities (Selectivity). Whilst the free acid compound 22 was
completely clean over all cytochromes P450, compound 21 showed an inhibition of cytochrome
P450 2C19 suggesting a possible involvement of the 2Hꢀtetrazole motif in this interaction.
Table 2. Measured properties for compound 21 and 22.
a
b
c
d
e
f
g
#
MW
393.4
383.4
S
P
app
CYPs
hERG
Cytotoxicity
NRs
Selectivity
k
j
k
21
22
1.0
0.8
5.84
4.56
0.1 µM, 2C19
>10 µM
NA
NT
NA
NA
NA<10µM
NA<10µM
k
j
k
NA
NT
a
b
Solubility: mg/mL, PBS, pH 7.4. Passive membrane Permeability in MDCKꢀMDR1 cells: nm/sec
(A>B). Cytochrome Pꢀ450 (1A2, 3A4, 2D6, 2C19). Human ERG assay kit used. AMLꢀ12, Hepꢀ
G2 cell lines used. Inꢀhouse nuclear receptor selectivity panel used (PXR, PPARs, LXR, VDR, GR,
RXR, TR, FXR). Eurofin Safety Panel used, 126 offꢀtargets (for details see Figure 1Sꢀ3S of SI).
c
d
e
f
g
k
j
NA: Not Active. NT: Not Toxic.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In Vivo Studies. Compounds 21 and 22 were then subjected to in vivo pharmacokinetic studies,
following intravenous (IV) and oral (PO) dosing on male CDꢀ1 mice (Table 3). Since ACMSD is
reported to be mostly expressed in kidney and liver, penetration of both compounds in these two
organs was evaluated.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. PK Profile of Compound 21 and 22 Following a Single IV or Oral Dose.
a
b
iv
po
#
AUClast
CL
Vdss
t1/2
(h)
Cmax
AUClast(h*ng/mL)
Tmax
(h)
%
F
(
h*ng/mL)
(mL/min/kg)
(L/kg)
(ng/mL)
Heart blood
Liver
4290
Kidney
2830
2
1
6900
1.20
0.78
0.306
0.273
4.02
5.33
255
1620
2.0
2.0
2.3
12
22
10700
2570
12700
19200
36600
a
Mean iv 0.5 mg/kg. Vehicle: saline water containing 5% (v/v) DMSO and 20% (v/v) PEG400.
Mean po 5 mg/kg. Vehicle: HPMC 0.5% (w/v) in water, containing 5% (v/v) DMSO.
b
As shown in Table 3 comparable results were obtained for both the tetrazole and the acid
compounds. After intravenous administration at 0.5 mg/kg, 21 and 22 showed low blood clearance,
with a low volume of distribution and a halfꢀlife (t ) of about 4.0 and 5.0 hours, respectively.
½
Whilst, after oral administration at 5 mg/kg, the blood concentrations of both compounds were
quantifiable up to 8 h (see Table 2S of SI for full PK profile). A moderate systemic exposure was
observed for the 2Hꢀtetrazole analog 21 (Cmax of 255 ng/mL, Table 3), and a good systemic
exposure was recorded for the free acid 22 with a Cmax of 2570 ng/mL reached at 2 h after dosing
(Table 3). The greater oral exposure of compound 22 was further confirmed in liver and kidney
with AUC0ꢀ8h of 19200 hr*ng/mL and 36600 hr*ng/mL, respectively (Table 3).
Liver to blood AUC ratio for compound 22 was 1.5 and kidney to blood AUC ratio was 2.9
suggesting a peculiar tissue distribution for this derivative. The CNS penetration for both
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compounds was negligible and did not change in presence of Elacridar (a known inhibitor of Pꢀgp
efflux transporters) suggesting no Pꢀgp interactions for our compounds (see Table 2S of SI) as
demonstrated through in vitro assay, and evidencing a restricted peripheral distribution of 21 and
22. According to their cellular permeability profile, the fraction absorbed for the 2Hꢀtetrazole
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analog 21 was low (estimated %F of 2.3%, Table 3) and that for the free acid compound 22 was
moderate resulting in an estimated oral bioavailability of 12% (Table 3).
Based on the better oral exposure of 22, this compound was chosen as chemical tool in a target
engagement study, in order to validate in vivo its mechanism of action. To this end, ACMSD
activity was measured in kidney and liver extracts prepared from mice at different times after PO
dosing at 5 mg/kg.
16
1
1
1
4
2
0
**
**
**
Time after PO dosing 5 mg/kg
Figure 8. ACMSD enzyme activity in kidneys of mice at different time points after PO dosing at 5
mg/kg of compound 22. Statistical oneꢀway analysis of variance ANOVA: Dunnett’s test:
**p<0.01.
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As shown in Figure 8, the enzyme activity was significantly reduced in kidneys from 0.5 up to 2
hours after dosing with compound 22, thus proving target engagement of ACMSD by 22 in vivo in
the kidney.
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In the livers of control and treated mice, the ACMSD activity was under the detection limit of the
+
enzymatic assay, therefore a pharmacodynamic validation of the enhancement of de novo NAD
+
biosynthesis with compound 22 was performed by evaluating the NAD levels in the liver. Thus,
male C57BL/6 mice were fed for 10 days with compound 22 as a diet supplement at 15 and 30
+
mg/kg/day. At the end of the treatment, changes in NAD levels were measured in liver extracts and
compared to those of control mice (Figure 9).
6
*
*
4
2
0
Vehicle
15mg/kg
30mg/kg
22
+
Figure 9. NAD levels in liver extracts of mice fed 10 days with compound 22 at 15 and 30
mg/kg/day. Vehicle 5% DMSO, 20% PEG. Oneꢀway analysis of variance ANOVA: Dunnett’s test:
*p<0.05, **p<0.01.
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+
As illustrated in Figure 9, liver NAD levels were significantly increased over control animals at
both doses of compound 22, giving us proof of concept of the in vivo efficacy of our compound in
+
the enhancement of de novo NAD biosynthesis via ACMSD inhibition.
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CONCLUSIONS
In summary, we report here the discovery, synthesis, and biological evaluation of a series of 2ꢀ
thiopyrimidoneꢀ5ꢀcarbonitriles as the first class of small molecule drugꢀlike ACMSD inhibitors.
SAR investigation around the screening hit 10 lead to the discovery of compound 22 as the first low
54
nanomolar inhibitor of human ACMSD with a suitable overall balance of physicochemical
properties and in vitro safety profile. Proof of concept studies demonstrate for the first time that
+
inhibition of ACMSD by this class of compounds leads to the modulation of intracellular NAD
+
levels with consequent in vivo enhancement of de novo NAD biosynthesis via ACMSD target
engagement. Based on the discovery of compound 22 and related analogs we have established
valuable tools for a better understanding of the therapeutic applications of ACMSD inhibitors for
disorders, such as mitochondrial dysfunctions, metabolic, and renal diseases, associated with
+
dysregulation of ACMSD activity or reduced NAD levels. The efficacy of compound 22 and
+
compound 21, in restoring depleted NAD content in the context of animal models of AKI and
NAFLD is now being tested (to be reported separately).
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EXPERIMENTAL SECTION
Chemistry. All commercial chemicals and solvents are reagent grade and were used without
1
13
further treatment unless specified. H NMR spectra were recorded at 200 and 400 MHz and
NMR spectra were recorded at 100.6 and 50.3 MHz using the solvents indicated below. Chemical
shifts are reported in parts per million (ppm) calibrated against solvent peaks δ 2.50 (DMSOꢀd ) or
C
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δ 7.24 (CDCl ). All the reactions were performed under nitrogen atmosphere using distilled solvent.
3
All tested compounds were found to have > 95% purity determined either by HPLC or by HRMS
(HPLC/QꢀTOF) analyses. The HPLC analytical scale measurements were carried out on a
Shimadzu LC Workstation Class LCꢀ10 equipped with SCLꢀ10 A VP system controller, a LCꢀ
1
0AT VP high pressure binary gradient delivery system, an SPDꢀ10A VP variableꢀwavelength UVꢀ
vis detector, and a Rheodyne 7725i injector with a 20 µL stainless steel loop. The chromatographic
profile was obtained with EZ Start software. All analytical runs were performed using a
2 3
H O/CH CN/TFA (60/40/0.1 v/v/v) solution as the mobile phase. HPLC grade water was obtained
from a tandem MilliꢀRo / MilliꢀQ apparatus. A Grace Smart C18 250 x 4.6 mm i.d. 5 µm 100 Å
analytical column was used after previous conditioning by passing through the column the selected
mobile phase for at least 30 min. The UV detection wavelengths were set at 254 and 230 nm.
Samples for analyticalꢀscale analyses were prepared in approximate concentration between 0.1 and
0
.5 mg/mL in filtered mobile phase components with auxiliary DMSO, if necessary, and sonicated
until completely dissolved. The HRMS (HPLC/QꢀTOF) analyses were assessed by the method
detailed below. The LC system was an Agilent 1290 Infinity module equipped with an autosampler,
a binary pump, a thermostated column compartment and a diodeꢀarray detector. The analytical
column was a Zorbax Eclipse Plus (2.1x50mm, 1.8µm). The column temperature was maintained at
4
0 °C. The mobile phase consisted of an eluent A (water containing 0.1% formic acid) and an
eluent B (acetonitrile plus 0. 1% formic acid). A 0 min (B= 20%) started a linear gradient at B 80%
within 4 min, this mobile phase was maintained for 1 min, at the end of run (5min.) returned back to
2
0% B. The flow rate was of 0.25 mL/min. The LC system was connected to a detector Agilent
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540 UHD AccurateꢀMass QꢀTOF/MS system equipped with a source dual Jet Stream. The mass
spectrometer operated with positive acquisition, Gas Temp. 300°C, gas flow 6.6L/min, nebulizer
pressure 16 psi, sheath gas temp. 290°C, fragmentor 200V, Skimmer 65V, Octapole RFPeaks 750,
Capillary voltage 4000V and Nozzle 0V and Reference masses 121.05087 and 922.009798. The
analysis were performed on a Mass Hunter workstation.
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General Procedure A. To a stirred suspension of the appropriate intermediate 2ꢀthiopyrimidone
2 3 3
23, 26ꢀ29 (1 eq), and K CO (3 eq) in CH CN was added 1 eq of the corresponding chloroꢀ or
bromomethyl derivative 24ꢀ25 or 30. Stirring was continued overnight at reflux. The volatiles were
removed under vacuo. The crude was taken up with water, acidified to pH 5 by diluted
hydrochloride acid (3M) and washed with EtOAc. The pH was then adjusted to pH 3 and the
mixture was extracted with EtOAc (3x 50 mL). The organic layers were combined, and dried over
sodium sulfate. Final compounds were obtained with high purity after trituration with hot acetone.
General Procedure B. To a stirred suspension of intermediate 29 (1 eq), and DIPEA (1 eq) in
DMSO was added the corresponding chloro or bromomethyl derivative 31ꢀ33 (1 eq). Stirring was
continued overnight at room temperature. The crude was poured in water, washed with EtOAc then
acidified to pH 3 and extracted with EtOAc (3 x 50 mL). The organic layers were combined, and
dried over sodium sulfate. The pure product was obtained by flash column chromatography on
silica gel eluting with DCM/MeOH (from 5% to 10% for product) and after washing with a mixture
of EtO /acetone if required.
2
3-[[(5-Cyano-1,6-dihydro-6-oxo-4-phenyl-2-pyrimidinyl)thio]methyl]benzoic acid (10).
Following the general procedure A and starting from 23 (1.6 g, 6.98 mmol), K CO (2.88 g, 20.9
2
3
mmol) and 3ꢀ(chloromethyl)benzoic acid (24) (1.19 g, 6.98 mmol) in CH
3
CN (80 mL) the title
1
compound was obtained in 40% (936 mg, 2.78 mmol) yield as yellowish solid. mp 270ꢀ272 °C. H
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6
NMR (400 MHz, DMSOꢀd ): δ 4.58 (s, 2H), 7.44 (t, J = 7.5 Hz, 1H), 7.54ꢀ7.61 (m, 3H), 7.67 (d, J
6
13
= 7.1 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.91 (d, J = 7.2 Hz, 2H), 8.04 (s, 1H), 13 (s, 1H). C NMR
100 MHz, DMSOꢀd ): δ 33.5, 93.2, 115.6, 128.2, 128.4, 128.4, 128.5, 128.5, 128.6, 129.7, 130.8,
31.5, 133.3, 135.1, 137.4, 165.4, 166.8, 167.3. HPLC: 96.3%. HRMS (m/z): [MH]+ calcd for
(
6
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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C
19
H
13
N
3
O
3
S: 364.0686, found: 364.07575.
3
-(6-Chloro-5-cyano-4-phenyl-pyrimidin-2-ylsulfanylmethyl)-benzoic acid (11).
(3 mL) was heated at 70 °C
for 6 h. The reaction crude was poured in crushed ice. The mixture was extracted with EtOAc (3 x
0 mL). The collected organic phase were washed with brine, dried over Na SO and evaporated.
A stirred suspension of compound 10 (160 mg, 0.44 mmol) and POCl
3
2
2
4
The title compound was obtained in 36% (60 mg, 0.16 mmol) yield as white solid after flash
1
chromatography purification (eluting with CH Cl /MeOH). mp 170ꢀ174 °C. H NMR (400 MHz,
2
2
DMSOꢀd
6
) δ 4.58 (s, 2H), 7.44 (t, J = 7.7 Hz, 1H), 7.58ꢀ7.62 (m, 2H), 7.66 (d, J = 7.1 Hz, 1H), 7.70
(d, J = 7.7 Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.94 (d, J = 7.1 Hz, 2H), 8.08 (s, 1H), 12.98 (s, 1H).
1
3
C NMR (100 MHz, DMSOꢀd
31.4, 132.7, 133.9, 134.7, 138.0, 162.9, 167.5, 169.0, 174.3. HPLC: 98.8%. HRMS (m/z): [MH]+
calcd for C H ClN O S: 382.03524, found: 382.042495.
6
) δ 34.9, 102.5, 115.2, 128.7, 129.2, 129.2, 129.6, 129.6, 130.4,
1
19
12
3
2
1,6-Dihydro-6-oxo-4-phenyl-2-[(phenylmethyl)thio]-5-pyrimidinecarbonitrile (12).
Following general procedure A and starting from 23 (300 mg, 1.3 mmol), K CO (542 mg, 3.93
2
3
mmol) and benzyl bromide (25) (0.15 mL, 1.3 mmol) in CH CN (15 mL) the title compound was
3
1
obtained in 41% (172 mg, 0.53 mmol) as yellowish solid. mp 194ꢀ196°C. H NMR (400 MHz,
DMSOꢀd
6
): δ 4.53 (s, 2H), 7.25ꢀ7.32 m, (3H), 7.40 (d, J = 6.9 Hz, 2H), 7.56ꢀ7.61 (m, 3H), 7.92 (d,
13
J = 6.9 Hz, 2H). C NMR (100 MHz, DMSOꢀd
6
): δ 36.1, 95.1, 117.6, 129.3, 130.3, 130.3, 130.4,
130.4, 130.5, 130.5, 130.8, 130.8, 133.6, 137.1, 138.2, 162.9, 167.5, 169.1. HPLC: 98.3%. HRMS
(
m/z): [MH]+ calcd for C H N OS: 320.07846, found: 320.08548.
18 13
3
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3-(6-Oxo-4-phenyl-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-benzoic acid (13).
2 3
Following general procedure A and starting from intermediate 26 (100 mg, 0.43 mmol), K CO
3
(178 mg, 1.2 mmol) and 24 (74 mg, 0.43 mmol) in CH CN (15 mL) the title compound was
1
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2
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2
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0
1
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0
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0
obtained in 21% (30 mg, 0.08 mmol) yield as white solid after washing with a mixture of
1
Et O/acetone. mp 270ꢀ275 °C. H NMR (400 MHz, DMSOꢀd ) δ 4.59 (s, 2H), 6.69 (s, 1H), 7.41
2
6
(m, 1H), 7.46 (m, 3H), 7.71 (d, J = 7.5 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 8.06 (m, 3H), 12.85 (s,
1
3
2H). C NMR (100 MHz, DMSOꢀd
6
) δ 33.8, 127.3, 127.3, 128.5, 129.1, 129.1, 129.2, 130.1,
131.0, 131.3, 131.5, 133.6, 136.3, 138.8, 167.5. HPLC: 99.6%. HRMS (m/z): [MH]+ calcd for
C
18
H
14
N
2
O
3
S: 339.07337, found: 339.08044.
3-(5-Bromo-6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-benzoic acid (14).
To a stirred solution of compound 13 (100 mg, 0.29 mmol) in acetic acid (5 mL) was added lead
dioxide (77.2 mg, 0.32 mmol) and bromine (0.02 mL, 0.32 mmol). Stirring was continued for 6 hrs
at room temperature. The mixture was poured in a solution of Na
2 2 5
S O and was extracted with
EtOAc (3 x 20 mL). The collected organic phases were washed with water and brine and then were
dried over Na
2
SO
4
. Washing with a mixture of Et
2
O/acetone afforded the title compound in 33%
1
(
40 mg, 0.09 mmol) yield as white solid. mp 246ꢀ250 °C. H NMR (400 MHz, DMSOꢀd ) δ 4.44 (s,
6
2H), 7.42 (t, J = 7.6 Hz, 1H), 7.48 (m, 3H), 7.61ꢀ7.65 (m, 3H), 7.82 (d, J = 7.5 Hz, 1H), 8.0 (s, 1H),
1
3
13.1 (m, 2H). C NMR (100 MHz, DMSOꢀd
130.1, 130.2, 131.3, 133.9, 138.1, 138.4, 167.5. HPLC: 96.8%. HRMS (m/z): [MH]+ calcd for
S: 418.98357, found: 418.98886.
6
) δ 33.9, 128.4, 128.4, 128.6, 129.14, 129.3, 129.3,
18 2 3
C H13BrN O
3-(5-Chloro-6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-benzoic acid (15).
To a stirred solution of compound 13 (100 mg, 0.29 mmol) in acetic acid (5 mL) was added lead
dioxide (55.8 mg, 0.35 mmol) and Nꢀchlorosuccinimide (47 mg, 0.35 mmol). Stirring was
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continued for 6 hrs at room temperature. The mixture was poured in water and it was extracted with
EtOAc (3 x 20 mL). The collected organic phases were washed with water and brine and dried over
Na
2
SO
4
. Washing with a mixture of Et
2
O/acetone afforded the title compound in 37% (40 mg, 0.1
1
mmol) yield as white solid. mp 278ꢀ280 °C. H NMR (400 MHz, DMSOꢀd
6
) δ 4.47 (s, 2H), 7.43 (t,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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0
J = 7.7 Hz, 1H), 7.49 (m, 3H), 7.64 (d, J = 7.2 Hz, 1H), 7.71 (m, 2H), 7.83 (d, J = 7.5 Hz, 1H), 8.02
13
(
s, 1H), 13.1 (s, 1H), 13.25 (s, 1H). C NMR (100 MHz, DMSOꢀd ) δ 33.9, 128.4, 128.4, 128.4,
6
128.6, 129.1, 129.4, 129.4, 129.4, 130.2, 131.3, 133.8, 136.5, 138.3, 156.1, 167.5. HPLC: 95.3%.
HPLC: 95.2 %. HRMS (m/z): [MH]+ calcd for C18 S: 373.03454, found: 373.04157.
H
2 3
13ClN O
3
-(5-Cyano-4-methyl-6-oxo-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-benzoic acid (16).
Following general procedure A and starting from intermediate 27 (145 mg, 0.86 mmol), K
599 mg, 4.33 mmol) and 24 (148 mg, 0.86 mmol) in CH CN (15 mL) the title compound was
2
CO
3
(
3
obtained in 23% (60 mg, 0.2 mmol) yield as white solid after flash chromatography purification
1
eluting with (CH
Cl
2 2
/ MeOH + AcOH 3%). mp 245ꢀ250 °C. H NMR (400 MHz, DMSOꢀd
6
) δ 2.44
(s, 3H), 4.49 (s, 2H), 7.45 (t, J = 7.6 Hz, 1H), 7.68 (d, J = 7.4 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H),
13
8
.05 (m, 1H), 13.1 (s, 1H). C NMR (100 MHz, DMSOꢀd
6
) δ 23.3, 33.9, 95.3, 115.6, 128.7, 129.1,
130.6, 131.2, 134.1, 138.0, 161.1, 165.7, 167.4, 170.9. HPLC 96.5%. HRMS (m/z): [MH]+ calcd
for C H N O S: 302.05282, found: 302.05998.
14
11
3
3
3-(5-Cyano-4-cyclohexyl-6-oxo-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-benzoic
17).
Following general procedure A and starting from intermediate 28 (250 mg, 1.18 mmol), K
acid
(
2
CO
3
(495 mg, 3.56 mmol) and 24 (202 mg, 1.18 mmol) the title compound was obtained in 21% (90 mg,
1
0.24 mmol) yield as white solid after washing with hot Et
2
O. mp 255ꢀ260 °C. H NMR (400 MHz,
DMSOꢀd
6
) δ 1.24 (m, 3H), 1.60 (m, 7H), 2.74 (m, 1H), 4.52 (s, 2H), 7.45 (t, J = 7.1 Hz, 1H), 7.67
1
3
(d, J = 6.8 Hz, 1H), 7.82 (d, J = 7.1 Hz, 1H), 8.04 (s, 1H), 13.0 (s, 1H). C NMR (100 MHz,
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DMSOꢀd ) δ 25.4, 25.7, 25.7, 30.3, 30.3, 33.8, 44.9, 94.1, 115.3, 128.6, 129.2, 130.1, 131.3, 133.6,
6
138.5, 161.1, 166.2, 167.4, 177.9. HPLC: 98.1%. HRMS (m/z): [MH]+ calcd for C H N O S:
19
19
3
3
370.11564, found: 370.12271.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
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3
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5
6
7
8
9
0
3-[[[5-Cyano-1,6-dihydro-6-oxo-4-(2-thienyl)-2-pyrimidinyl]thio]methyl]benzoic acid (18).
Following general procedure A and starting from 29 (250 mg, 1.06 mmol), K CO (440 mg, 3.18
2
3
mmol) and 24 (180 mg, 1.06 mmol) in CH CN (15 mL) the title compound was obtained in 12%
3
1
(45 mg, 0.12 mmol) yield as yellowish solid. mp 267ꢀ270 °C. H NMR (400 MHz, DMSOꢀd
6
): δ
4.62 (s, 2H), 7.33 (t, J = 4.3 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.82 (d, J =
13
7
.5 Hz, 1H), 8.05 (m, 2H), 8.26 (d, J = 3.8 Hz, 1H), 12.99 (s, 1H). C NMR (100 MHz, DMSOꢀd
6
):
δ 33.9, 88.7, 116.5, 128.8, 129.3, 129.9, 130.2, 131.5, 132.1, 133.7, 135.4, 137.9, 139.7, 159.0,
1
61.2, 165.3, 167.4. HPLC: 97.2%. HRMS (m/z): [MH]+ calcd for C H N O S : 370.02472,
17 11 3 3 2
found: 370.03188.
3
-(5-Cyano-6-oxo-4-thiophen-2-yl-1,6-dihydro-pyrimidin-2-ylsulfanylmethyl)-N-hydroxy-
benzamide (19)
To a stirred suspension of intermediate 34 (103 mg, 0.21 mmol) in DCM (5 mL), was added TFA
0.14 mL, 2.1 mmoL). Stirring was continued at room temperature overnight. The volatile were
(
removed under vacuo. The crude was purified by flash chromatography eluting with (DCM/MeOH,
10% for product). The title compound was obtained in 40% (30 mg, 0.07 mmol) yield as yellowish
1
solid. mp 240ꢀ245 °C. H NMR (400 MHz, DMSOꢀd
6
): δ 4.50 (s, 2H), 7.28 (t, J = 3.7 Hz, 1H), 7.38
(t, J = 7.6 Hz, 1H), 7.60 (d, J = 7.3 Hz, 2H), 7.84 (s, 1H), 7.93 (d, J = 4.3 Hz, 1H), 8.19 (d, J = 2.9
13
Hz, 1H), 9.02 (brs, 1H), 11.2 (s, 1H). C NMR (100 MHz, DMSOꢀd ): 34.1, 87.4, 118.4, 126,
6
128.1, 128.9, 129.4, 130.1, 130.7, 132.0, 133.5, 138.6, 140.9, 159.1, 164.5, 168. HPLC: 95.1%.
HRMS (m/z): [MH]+ calcd for C17 : 385.03608, found: 385.04334.
12 4 3 2
H N O S
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6
-Oxo-4-thiophen-2-yl-2-[3-(5-trifluoromethyl-[1,2,4]oxadiazol-3-yl)-benzylsulfanyl]-1,6-
dihydro-pyrimidine-5-carbonitrile (20).
2 3
Following general procedure A and starting from intermediate 29 (146 mg, 0.62 mmol), K CO
(
3
128 mg, 0.93 mmol) and intermediate 30 (200 mg, 0.65 mmol) in CH CN (20 mL) the title
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
compound was obtained in 39% (110 mg, 0.24 mmol) yield as white solid after flash
1
chromatography purification eluting with DCM/MeOH (5% for product). mp 244ꢀ248 °C. H NMR
(
400 MHz, DMSOꢀd ) δ 4.68 (s, 2H), 7.36 (t, J = 4.2 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1 H), 7.8 (d, J =
6
7.7 Hz, 1H), 7.96 (d, J = 7.7 Hz, 1H), 8.07 (d, J = 4.9 Hz, 1H), 8.24 (s, 1H), 8.29 (d, J = 3.9 Hz,
13
1H). C NMR (100 MHz, DMSOꢀd
30.3, 132.1, 133.4, 135.4, 139.2, 139.6, 159, 161.2, 165.2, 168.6. HPLC > 97.9%. HRMS (m/z):
MH]+ calcd for C H F N O S : 462.02393, found: 462.03109.
6
) δ 33.7, 88.8, 114.8, 116.5, 117.5, 125.1, 126.8, 128.2, 129.9,
1
[
19
10
3
5
2 2
1
,6-Dihydro-6-oxo-2-[[[3-(2H-tetrazol-5-yl)phenyl]methyl]thio]-4-(2-thienyl)-5-
pyrimidinecarbonitrile (21).
Following general procedure B and starting from 29 (160 mg, 0.66 mmol), DIPEA (0.09 mL, 0.55
mmol) and intermediate 31 (171 mg, 0.55 mmol) in DMSO (3 mL) the title compound was obtained
1
in 23% (90 mg, 0.22 mmol) yield as white solid. mp 251ꢀ254 °C. H NMR (400 MHz, DMSOꢀd ):
6
δ 4.67 (s, 2H), 7.33 (t, J = 6.3 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.7 (d, J = 7.7 Hz, 1H), 7.93 (d, J =
1
3
7
.7 Hz, 1H), 8.04 (d, 5 Hz, 1H), 8.18 (s, 1H), 8.26 (d, J = 3.9 Hz, 1H), 13.9 (brs, 1H). C NMR
(100 MHz, DMSOꢀd ): δ 33.9, 88.7, 116.5, 124.9, 126.4, 127.8, 129.9, 130.1, 132, 132.1, 135.4,
6
138.7, 139.7, 157, 159.1, 161.2, 165.3. HPLC: 99.2%. HRMS (m/z): [MH]+ calcd for
C H N OS : 394.04736, found: 393.07436.
17
11
7
2
3
-[[[5-Cyano-1,6-dihydro-6-oxo-4-(2-thienyl)-2-pyrimidinyl]thio]methyl]-phenylacetic
acid
(
22).
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Following general procedure B and starting from 29 (500 mg, 2.12 mmol), DIPEA (0.4 mL, 2.12
mmol) and intermediate 32 (487 mg, 2.12 mmol) in DMSO (5 mL) the title compound was obtained
1
in 25% (200 mg, 0.52 mmol) yield as pure yellowish solid. mp 237ꢀ239 °C. H NMR (400 MHz,
DMSOꢀd
6
): δ 3.54 (s, 2H), 4.53 (s, 2H), 7.17 (d, J = 7.1 Hz, 1H), 7.36ꢀ7.37 (m, 3H), 8.06 (d, J = 4.4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
Hz, 1H), 8.27 (d, J = 3 Hz, 1H), 12.5 (brs, 1H). C NMR (100 MHz, DMSOꢀd
6
): δ 34.2, 40.9, 88.4,
116.9, 127.6, 128.9, 129.1, 129.8, 130.4, 131.8, 135.2, 135.7, 137.1, 140, 159, 162, 165.8, 172.9.
HPLC: 97.1 %. HRMS (m/z): [MH]+ calcd for C H N O S : 384.03953, found: 384.04668
18 13
3
3 2
General procedure C for the synthesis of 4-Oxo-6-substituted-2-thioxo-1,2,3,4-tetrahydro-
pyrimidines-5-carbonitrile. To a stirred solution of the corresponding aldehyde 35a-c (1 eq),
ethylcyano acetate 36 (1 eq) and thiourea 37 (1 eq) in ethanol, was added K CO (1.2 eq) or
2
3
piperidine (2 eq). Stirring was continued at reflux overnight. The pale yellow solid was collected
after cooling, taken up with boiling water and filtered again. The aqueous phase was acidified to pH
5 with AcOH, the precipitate was filtered and dried under vacuum to afford the desired compound
in acceptable yields.
4
-Oxo-6-phenyl-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5-carbonitrile (23).
Following general procedure C and starting from benzaldehyde 35a (0.5 mL, 0.83 mL, 4.9 mmol),
6 (0.52 ml, 4.9 mmol), 37 (372 mg, 4.9 mmol) and K CO (812 mg, 5.88 mmol) in ethanol (25
3
2
3
mL) the title compound was obtained in 44% (500 mg, 2.18 mmol) yield as yellow solid. mp 286ꢀ
1
288 °C. H NMR (400 MHz, DMSOꢀd ): δ 7.48ꢀ7.61 (m, 3H), 7.70 (d, J = 7.6 Hz, 1H), 8.0 (d, J =
6
1
3
7
1
.3 Hz, 1H), 8.27 (s, 1H), 12.1 (brs, 1H). C NMR (100 MHz, DMSOꢀd ): δ 88.1, 117.3, 128.6,
6
28.8, 129.4, 130.7, 131.4, 133.1, 153.6, 161.1, 163.6.
6-Phenyl-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one (26).
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