Piperazin-1-ylpyridazine Derivatives Are a Novel Class of Human dCTP Pyrophosphatase 1 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b00182

The dCTP pyrophosphatase 1 (dCTPase) is a nucleotide pool “housekeeping” enzyme responsible for the catabolism of canonical and noncanonical nucleoside triphosphates (dNTPs) and has been associated with cancer progression and cancer cell stemness. We have identified a series of piperazin-1-ylpyridazines as a new class of potent dCTPase inhibitors. Lead compounds increase dCTPase thermal and protease stability, display outstanding selectivity over related enzymes and synergize with a cytidine analogue against leukemic cells. This new class of dCTPase inhibitors lays the first stone toward the development of drug-like probes for the dCTPase enzyme.

Full text of DOI:10.1021/acs.jmedchem.7b00182

Article
pubs.acs.org/jmc
Piperazin-1-ylpyridazine Derivatives Are a Novel Class of Human
dCTP Pyrophosphatase 1 Inhibitors
,
†,#
†,#,∇
†,○
†
Sabin Llona-Minguez,*
Jose Manuel Calderon-Montan
Ingrid Almlof, Tobias Koolmeister, Andre
Andreas Ho
̈
glund,
Artin Ghassemian, Matthieu Desroses,
†
,◆
†,◆
Nicholas C. K. Valerie, Elisee Wiita,†
†
́
́
̃
o,
Estefanía Burgos Moron
́
,
†
†
†
⊥,¶
†
†
̈
́
Mateus, Cindy Cazares-Ko
̈
rner, Kumar Sanjiv,
†
⊥
§
§
Evert Homan, Olga Loseva, Pawel Baranczewski, Masoud Darabi, Amir Mehdizadeh,
∥
†
†
†,‡,+
Shabnam Fayezi, Ann-Sofie Jemth, Ulrika Warpman Berglund, Kristmundur Sigmundsson,
†
,‡,●
†,‡
⊥
†
Thomas Lundbac
̈
k,
,†Annika Jenmalm Jensen, Per Artursson, Martin Scobie,
and Thomas Helleday*
†Division of Translational Medicine and Chemical Biology, Science for Life Laboratory, Department of Medical Biochemistry and
Biophysics, Karolinska Institutet, Stockholm 171 65, Sweden
‡
Chemical Biology Consortium Sweden, Science for Life Laboratory, Division of Translational Medicine and Chemical Biology,
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm 171 65, Sweden
§
Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz
5
165665931, Iran
∥
Department of Biology and Anatomical Sciences, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran
1
⊥
983969411, Iran
Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Department of Pharmacy, Science for Life
Laboratory, Uppsala University, Uppsala 752 37, Sweden
*
S Supporting Information
ABSTRACT: The dCTP pyrophosphatase 1 (dCTPase) is a nucleotide pool “housekeeping” enzyme responsible for the
catabolism of canonical and noncanonical nucleoside triphosphates (dNTPs) and has been associated with cancer progression
and cancer cell stemness. We have identified a series of piperazin-1-ylpyridazines as a new class of potent dCTPase inhibitors.
Lead compounds increase dCTPase thermal and protease stability, display outstanding selectivity over related enzymes and
synergize with a cytidine analogue against leukemic cells. This new class of dCTPase inhibitors lays the first stone toward the
development of drug-like probes for the dCTPase enzyme.
INTRODUCTION
up-regulation of the multidrug resistance protein 1 (MDR1)
and degradation of the nucleoside’s active form.
Only a handful of dCTPase inhibitors have been reported to
■
2
,6
The human dCTPase, also known as DCTPP1 or XTP3-
transactivated protein A (XTP3TPA), regulates the cellular
nucleotide pool through catabolism of canonical and non-
date (Figure 1): triptolide 1, a terpenoid epoxide with a broad
7
poly pharmacological profile; the photo-cross-linking agent 2,
1
,2
8
canonical dNTPs. Recent evidence suggests that dCTPase is
implicated in cancer development and progression through
with no reported binding affinity toward dCTPase; benzimi-
dazole 3 and triazolothiadiazole 4, the first potent dCTPase
3
9,10
nuclear accumulation in multiple cancer histological subtypes,
inhibitors.
Herein, we report on the discovery and
up-regulation and promotion of cancer cell growth and
4
,5
stemness, association with poor clinical prognosis, decreased
response to anticancer nucleoside analogues through epigenetic
Received: February 3, 2017
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.7b00182
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Article
Figure 1. Structures of known dCTPase inhibitors/binders.
structure−activity relationships (SAR) of a series of piperazin-
condition c), or as a final step with section B + RHS (8, 9, 10,
14, 17, 23, 24; Scheme 2, condition c). Once the tert-
butyloxycarbonyl (BOC)-protected LHS region (32, 35−38)
and the BOC-protected section B + RHS region (53−56) were
assembled, the BOC groups were removed using HCl in 1,4-
dioxane conditions (Scheme 1, condition b, and Scheme 2,
condition b). The resulting amine hydrochlorides 33 and 39−
42 were coupled with the required RHS regions via an aromatic
nucleophilic substitution, to afford compounds 5, 6, 7, 13, 15,
1
-ylpyridazine derivatives as novel and potent dCTPase
inhibitors.
CHEMISTRY
■
Compounds in this series (5−30) can be conveniently divided
into left-hand side (LHS) and right-hand side (RHS) regions
Figure 2). The LHS region contains sections A (aromatic ring)
(
16, 19, and 20 (Scheme 1, condition d), and the amine
hydrochlorides 59−62 were coupled with the required sulfonyl
chlorides to afford compounds 8, 9, 10, 14, 17, 23, and 24
(
Scheme 2, condition c).
The LHS regions containing an amino-substituted benzene
rings (38, 11, and 12) were prepared via an aromatic
nucleophilic substitution on the parent fluoro-benzene
precursors 37 (Scheme 1, condition e) and 10 (Scheme 2,
condition d). The methyl-substituted pyridine derivative 18 was
prepared via a Suzuki coupling on the parent chloro-aryl
precursor 17 with methyl boronic acid (Scheme 2, condition e).
Compounds 21 and 22 were synthesized following Scheme
3
. Hydroxy-piperidine 57 was converted to mesylate 63 and
treated with 2-methylbenzene-1-thiol to yield thioether 64. A
subsequent mild meta-chloroperoxybenzoic acid (m-CPBA)-
mediated oxidation delivered a mixture of sulfoxide 21 and
sulfone 22.
Amine 25 was prepared following Scheme 4. Methyl ester 58
was reduced with NaBH4 to alcohol 65, followed by
chlorination with SOCl to afford 66 and subsequently reacted
with benzylamine to give 25.
2
Figure 2. Structure−activity relationship (SAR) summary. Color code:
green > yellow > red, inhibitory potency.
Sulfone 26 was prepared in three synthetic steps as depicted
in Scheme 5. 3,6-Dichloropyridazine 67 was treated with
phenylethane-1-thiol, and the resulting thioether 68 was fully
oxidized with m-CPBA to the sulfone 69. Compound 26 was
obtained after aromatic nucleophilic substitution of 39 with 69.
The synthesis of the amide replacement derivatives 27−30 is
presented in Scheme 6. Chloropyridazine-3-carboxylic acid 43
was converted to the corresponding acid chloride 70 and
reacted with the organocuprate derivate from 4-fluorophenethyl
bromide to afford 71. The chloro-aryl intermediate 71 was then
reacted with amine 39 via an aromatic nucleophilic substitution
to obtain 27. A Wittig reaction between ketone 27 and
(methoxymethyl)triphenylphosphonium chloride provided
and B (piperazine derivative) connected by an amide or a
sulfonamide bond. The RHS region contains sections C
(
pyridazine) and D (aromatic ring) connected by an amide
or an alternative bond. The bond between sections A and B
amide 32) and C and D (amides 44−49; esters 50 and 51)
(
was established via a propylphoshonic anhydride-mediated
coupling between the corresponding carboxylic acids and
amines/alcohols (Scheme 1, condition a). The sulfonamide
bond between sections A and B was formed from the
corresponding sulfonyl chlorides and amines, either as a first
synthetic step to assemble the LHS region (35−37, Scheme 1,
B
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Article
a
Scheme 1. Synthesis of Compounds 5, 6, 7, 13, 15, 16, 19, and 20
a
Reagents and conditions: (a) amine or alcohol (1.1 equiv), propylphoshonic anhydride (2.2 equiv), Et N (2.5 equiv), CH Cl , 0 °C to rt; 18 h; (b)
3
2
2
4
M HCl in 1,4-dioxane, rt, 18 h; (c) sulfonyl chloride (1.1 equiv), Et N (2.2 equiv), rt, 18 h; (d) amine (1 equiv), Et N (2 equiv), 1,4-dioxane, 100
3
3
°C, 18 h; (e) amine (5 equiv), DMF, 150 °C, 18 h.
a
Scheme 2. Synthesis of Compounds 8, 9, 10, 11, 12, 14, 17, 18, 23, and 24
a
Reagents and conditions: (a) amine (1 equiv), Et N (2 equiv), 1,4-dioxane, 100 °C, 18 h; (b) 4 M HCl in 1,4-dioxane, rt, 18 h; (c) sulfonyl chloride
3
(
(
1.1 equiv), Et N (2.2 equiv), rt, 18 h; (d) amine (5 equiv), DMF, 150 °C, 18 h; (e) MeB(OH) (3 equiv), Pd(dppf)Cl ·CH Cl (0.1 equiv), P(Cy)
3
2
2
2
2
3
0.4 equiv), K PO (3.5 equiv), toluene/water, 100 °C, 5 h.
3
4
C
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Article
a
Scheme 3. Synthesis of Compounds 21 and 22
a
Reagents and conditions: (a) mesyl chloride (1.2 equiv), pyridine (1 equiv), CH Cl , rt, 18 h; (b) 2-methylbenzene-1-thiol (1.1 equiv), K CO (1.1
2
2
2
3
equiv), DMF, 60 °C, 18 h; (c) m-CPBA (3 equiv), CH Cl , 0 °C to rt, 18 h.
2
2
a
Scheme 4. Synthesis of Compound 25
a
Reagents and conditions: (a) NaBH (14 equiv), EtOH, 0−40 °C, 1 h; (b) SOCl (28 equiv), CH Cl , 0 °C to rt, 18 h; (c) benzylamine (11 equiv),
3
4
2
2
2
Et N (3 equiv), acetonitrile, rt, 18 h.
a
Scheme 5. Synthesis of Compound 26
a
Reagents and conditions: (a) phenylethane-1-thiol (1 equiv), Et N (1 equiv), DMF, 70 °C, 18 h; (b) m-CPBA (3 equiv), CH Cl , 0 °C to rt, 18 h;
3
2
2
(
c) 39 (1 equiv), Et N (2 equiv), 1,4-dioxane, 100 °C, 18 h.
3
enol ether 72, which was subsequently hydrolyzed to aldehyde
3. A Cannizzaro reaction on 73 delivered diol 28, which after
tosylation and deprotonation cyclized to oxetane 29, with
concomitant formation of the Grob fragmentation product 30,
in a ratio of 1:3.
established that shortening the linker between sections C and D
to a one carbon unit brought potency down to the
submicromolar range (1-C > 2-C > 0-C) (6, 5, 7; Table 1).
Replacement of the carboxamide bond between sections A and
B in 6 with a sulfonamide in 8 retained potency but decreased
binding efficiency (6 BEI = 14.1 vs 8 BEI = 13.0). This could be
improved by changing the trifluoromethyl group to a methyl
group in 9 (BEI = 15.0). The RHS region of the molecule was
able to accommodate benzene surrogates such as 2-thiophene
7
RESULTS AND DISCUSSION
■
To identify novel dCTPase inhibitors, we screened a
proprietary library (5500 molecules tested at 10 μM compound
concentration at the Chemical Biology Consortium Sweden)
against the full-length human dCTPase protein using a HTS-
(
13), 4-fluorobenzene (14), or acetylene (16), delivering
improved or equivalent potency when compared with their
respective matched pairs. Altering the LHS benzene ring with a
fluorine (10) or an amine substituent (11, 12) at the 2-position
was less tolerated but improved aqueous solubility (visual
inspection of biochemical assay buffer solutions, data not
shown). An additional lipophilic group at the benzene ring’s 3-
position (15, 16) or an exchange for a pyridine nitrogen lone
pair (17, 18) were favorable structural modifications. Minor
modifications to the piperazine ring in section B resulted in a
significant drop in activity (carbon/nitrogen swap at the 4-
position, either as a sulfoxide (21) or a sulfone (22), bridged
piperazine (23), or ring expansion (24) (Table 2). The
carboxamide motif linking sections C and D could be replaced
by a carboxylate (19, 20) to give more potent compounds than
their respective matched pairs (9, 12) and, to a lesser extent by
9
,11
adapted malachite green assay.
There were 110 preliminary
hits identified (2% hit rate, μ + 3σ at 36.9% inhibition, Figure
S1). Among the initial hits, compound 5 (Figure 1) inhibited
90% of dCTPase activity at 10 μM and was confirmed to be a
low micromolar inhibitor (IC50 = 2.8 μM). Despite the low
12
binding efficiency index (BEI = pIC /MW) of 5, we were
5
0
attracted by the drug-like properties and modular nature of the
13,14
piperazin-1-ylpyridazine scaffold
and decided to pursue the
SAR study presented in this manuscript (Figure 2 for SAR
overview and Tables 1−3 for detailed SAR).
For this concise SAR study, it was decided to modify sections
A and D while maintaining section C constant and introducing
discrete alterations to section B (Figure 2). First, it was
D
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a
Scheme 6. Synthesis of Compounds 27, 28, 29, and 30
a
Reagents and conditions: (a) (CO) Cl (2 equiv), DMF (cat.), CH Cl , 0 °C to rt, 2 h; (b) 4-fluorophenethyl bromide (1.2 equiv), Mg (1.2 equiv),
2
2
2
2
I (cat.), CuCN·2LiCl (1.3 equiv), THF, −40 °C, 1 h; (c) 39 (1 equiv), Et N (2 equiv), 1,4-dioxane, 100 °C, 18 h; (d) LDA (1.7 equiv),
2
3
(
methoxymethyl)triphenylphosphonium chloride (1.7 equiv), THF, −78 °C to rt, 5 h; (e) H SO /THF/water, 45 °C, 2 days; (f) aq CH O/aq
2
4
2
NaOH/THF, rt, 18 h; (g) NaH (1.1 equiv), p-tosyl chloride (2 equiv), THF, −78 °C to rt, 2 h, then NaH (20 equiv), −78 to 50 °C, THF, 1 h.
Table 1. Structure−Activity Relationships: LHS/RHS Variations
R1
W
X
R2
IC50 (μM)
a
BEI
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2-(trifluoromethyl)phenyl
2-(trifluoromethyl)phenyl
2-(trifluoromethyl)phenyl
2-(trifluoromethyl)phenyl
2-methylphenyl
CO
CO
CO
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
2-phenylethan-1-yl
benzyl
2.8
0.240
>10
11.5
14.1
phenyl
benzyl
0.250
0.170
0.350
0.502
0.260
0.051
0.065
0.019
0.034
0.025
0.036
0.038
0.041
13.0
15.0
14.2
13.1
12.3
15.9
15.3
15.7
17.2
15.5
15.8
16.4
15.2
benzyl
0
1
2
3
4
5
6
7
8
9
0
2-fluorophenyl
benzyl
2-(dimethylamino)phenyl
2-(4-hydroxypiperidin-1-yl)phenyl
2-methylphenyl
benzyl
benzyl
thiophen-2-ylmethanyl
4-fluoro-benzyl
thiophen-2-ylmethanyl
propargyl
2-methylphenyl
3-chloro-2-methylphenyl
3-chloro-2-methylphenyl
2-chloropyridin-3-yl
4-fluoro-benzyl
4-fluoro-benzyl
benzyl
2-methylpyridin-3-yl
2-methylphenyl
2-(4-hydroxypiperidin-1-yl)phenyl
O
propargyl
a
The 11-point IC50 curves were calculated based on the average of two replicates per data point with standard deviation.
a ketone (27, Table 3). Removal or replacement of the
carbonyl group completely abolished activity, as seen with the
amine (25), sulfone (26), bis-hydroxymethyl (28), oxetane
Compound 18 consistently docked with the RHS-benzene ring
wedged between Trp47(C) and Trp73(B), thus overlaying with
the cytosine base of dCTP, while the LHS-pyridine substituent
was oriented toward the solvent, consistent with the SAR
observations. Representative poses are shown for 18 (Figure
3B,C. The top-ranked pose suggested H-bond formation
between the carboxamide −NH and Tyr102(C), the
carboxamide carbonyl and Lys121(D), one of the pyridazine
ring nitrogens and Trp73(B), and one of the sulfonamide
oxygens and Arg87(B). The positioning and orientation of the
RHS 4-fluorophenyl suggested the possibility of face-to-face
π−π interactions with Tyr102(C), while additional edge-to-face
π−π interactions were possible between the central pyridazine
ring and Trp73(B) and between the LHS pyridine ring and
(29), or alkene (30) derivatives, highlighting the importance of
this structural feature.
To rationalize the observed SAR, we built a homology model
of the human dCTPase enzyme based on available crystal
structures of the mouse enzyme (see Molecular Docking,
Experimental Section). The resulting model contained four
active sites (dimer of dimers), each occupied by dCTP, and
residues from three different monomers contributing to each
active site. Because the human and mouse dCTPase active site
share identical residue sequence, this homology model was
deemed a good starting point for docking studies (Figure 3A).
E
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Table 2. Structure−Activity Relationships: Piperazine
a
Variations
a
*The 11-point IC50 curves were calculated based on the average of
two replicates per data point with standard deviation.
Table 3. Structure−Activity Relationships: RHS Amide
a
Replacements
a
*
The 11-point IC50 curves were calculated based on the average of
two replicates per data point with standard deviation
Figure 3. (A) Overall view of the human dCTPase homology model.
Trp73(B). Furthermore, the LHS pyridine might be involved in
a π−cation interactions with Arg87(B) (interaction summary in
Figure 3D).
(B,C) Representative poses of 18 docked into the dCTPase active site,
shown from different perspectives. (D) Two-dimensional residue
interaction diagram of 18 with the dCTPase active site.
Having discovered a number of potent inhibitors (IC50 < 50
nM) with reasonable BEI (≥15), we set to validate a diverse set
of compounds as suitable probes for cellular experiments. First,
we confirmed target engagement using thermal and proteolytic
stabilization assays. Potent inhibitors 13, 18, and 20 increased
the melting temperature of the purified dCTPase protein in a
differential scanning fluorimetry (DSF) assay, whereas the
concentration was available in the intracellular environment,
18
Supporting Information, Table S3), and the results observed
in the whole cell CETSA and DARTS assays, it was assumed
that this series of piperazin-1-ylpyridazines generally possessed
good cellular exposure. Key compounds were then assessed in a
9
cellular efficacy model. When leukemia-derived HL60 cells
15
inactive inhibitors 29 and 30 did not (Table S1). Next, we
demonstrated that the ligand-mediated thermal stabilization of
dCTPase translated to a whole cell thermal shift assay
were exposed to compounds 9, 13, or 15 in a combination
matrix with 5-azacytidine (5-AzaC) (Supporting Information,
Figure S2), a nucleoside analogue of clinical relevance, we
observed a synergistic decrease in cell viability (combination
index (CI) < 1) (Figure 5A), according to the Chou−Talalay
16
(
CETSA), as seen with compound 9 (Figure 4A). On the
basis of the principle of stabilization to protease digestion upon
ligand binding, we also observed that 20 protected dCTPase
from Pronase digestion using a whole cell drug affinity
19
method for synergy quantification. Moreover, the combina-
tion of a low-cytotoxic dose of 5-AzaC with a nontoxic dose of
compounds 14, 18, or 27 caused synergistic lethality in the
leukemic cells, as determined by fluorescence-activated cell
sorting (FACS) analysis (Figure 5B). Furthermore, compounds
9, 18, and 20 did not show a cytotoxic effect on BJ hTERT
normal cells (Supporting Information, Table S4).
17
responsive target stability (DARTS) assay (Figure 4B).
Next, we assessed the pharmacological profile of the
inhibitors within the dNTPase/nucleoside diphosphate linked
to X (NUDIX) protein family. Compounds 9, 18, and 20
displayed >1000-fold selectivity against a panel of relevant
enzymes (Supporting Information, Table S2).
It is worth noting that compounds 5, 6, and 7 are potent
inhibitors of the stearoyl-CoA desaturase-1 (SCD1) published
On the basis of the encouraging intracellular bioavailability
Fic) of all the inhibitors tested (>50% of the compound
13
(
by Zhang et al. at Xenon Pharmaceuticals. SCD1 is a key
F
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Figure 4. Target engagement validation: cellular assays. (A) Western blot images from the CETSA assay. MCF7 cells were incubated for 2 h with 9
0, 5, 20 μM) and subsequently heated at different temperatures. (B) Western blot images from the DARTS assay. HL60 cells were incubated for 4 h
(
with DMSO (0.1%) or 20 (10 μM) followed by pronase digestion. ND = nondigested sample.
Figure 5. (A) Combination index (CI) plots of compounds 9, 13, and 15 in combination with 5-AzaC. HL60 leukemia-derived cells were incubated
for 72 h with different concentrations of the inhibitors and 5-AzaC. Cytotoxic effect was determined by Resazurin cell viability assay. (B) Inhibitors
1
4, 18, and 27 show synergystic lethality with 5-AzaC. HL60 leukemia-derived cells were incubated for 72 h with the inhibitor (10 μM) and 5-AzaC
(
2.5 μM). Cell death was determined by flow cytometry analysis.
enzyme in the synthesis of monounsaturated fatty acids, and
SCD1 inhibitors have received a great deal of attention as a
therapeutic option against diabetes, cancer, and some skin
(74 IC50 = 0.67 μM vs 6 IC50 = 0.75 μM). In comparison,
compound 9 was >20-fold less active (IC50 = 19.74 μM),
indicating that introducing a sulfonamide motif in this
chemotype can reduce SCD1 inhibitory activity.
2
0,21
disorders.
Selected dCTPase inhibitors were profiled for
their effect on SCD1 activity by examining the ratio of cellular
SCD1 products over substrates (16:1/16:0)/(18:1/18:0) (16:0
CONCLUSION
■
=
=
palmitic acid, 16:1 = palmitoleic acid, 18:0 = stearic acid, 18:1
oleic acid), referred as the cumulative SCD1 desaturation
In summary, we have reported on a new class of potent
inhibitors of dCTPase, a novel biological target with clear links
to cancer progression. Here we have profiled a diverse set of
compounds in biochemical and cellular assays in order to
validate this class of piperazin-1-ylpyridazines as dCTPase
inhibitors. Several compounds presented in this study inhibit
dCTPase enzymatic activity in the low nanomolar range,
engage with the biological target in the intracellular environ-
ment, and do not interfere with related enzymes. Moreover, this
class of inhibitors enhances the cytotoxic effect of the cytidine
index. Unlike kinetic inhibition data obtained using labeled
substrates, estimation of SCD1 activity based on the cell’s
endogenous desaturation index offers a realistic assessment of
inhibitor potency in the cellular context. We confirmed that 6
was equipotent to the Abbott compound 74 (3-[4-(2-chloro-5-
fluorophenoxy)-1-piperidinyl]-6-(5-methyl-1,3,4-oxadiazol-2-
yl)-pyridazine, CAY10566, Supporting Information, Figure
2
2
S2) in decreasing the SCD1 activity index in HepG2 cells
G
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analogue 5-AzaC in leukemia-derived HL60 cells. Concomitant
SCD1 antagonism is to be expected in some piperazin-1-
ylpyridazines given their structural similarity to known SCD1
inhibitors, although SCD1 affinity can be reduced through
subtle structural changes, as seen in 9. The utility of dual
nucleotide/lipid metabolism modulators is unknown, and
additional work will be required to elucidate the relevance of
this polypharmacological profile and will dictate the optimal
target potency balance. Currently, there is a need for chemical
probes to explore dNTPases/NUDIX hydrolase biology, and
the compounds presented in this article represent an exciting
starting point toward the development of novel and well-
validated probes of the dCTPase enzyme.
General Procedure D for the Synthesis of 32 and 44−51. A
propylphoshonic anhydride (T3P) solution (≥50 wt % in EtOAc, 23.1
mmol, 2.2 equiv) was added to a solution of carboxylic acid (2 g, 10.52
mmol, 1equiv), amine (11.57 mmol, 1.1 equiv), and Et N (26.3 mmol,
3
2
.5 equiv) in CH Cl (10 mL) at 0 °C. The reaction was allowed to
2 2
warm to room temperature overnight. The mixture was subsequently
diluted with CH Cl , and the organic layer was sequentially washed
2
2
with saturated aqueous NaHCO and aqueous 2 M HCl and finally
3
dried over Na SO . After evaporation, the product was obtained and
2
4
used in the next step without any further purification.
General Procedure E for the Synthesis of 33, 39−42, and 59−62.
A 4 M solution of HCl in 1,4-dioxane (10 mL) was added to a solution
of BOC-protected amine (3.50 g, 9.86 mmol) in 1,4-dioxane (10 mL)
and stirred at room temperature overnight. The solvent was removed
by filtration and the residue triturated with diethyl ether to afford the
title product. Used in the next step without any further purification.
N-Phenethyl-6-(4-(2-(trifluoromethyl)benzoyl)-piperazin-1-yl)-
pyridazine-3-carboxamide (5). General procedure A. Prepared from
EXPERIMENTAL SECTION
■
Chemistry: General Information for Synthetic Procedures.
All commercial reagents and solvents were used without further
purification. Compounds 31, 34, 43, 52, and 67 are commercial
reagents. Analytical thin-layer chromatography was performed on silica
gel 60 F-254 plates (Merck) and visualized under a UV lamp. Flash
column chromatography was performed in a Biotage SP4MPLC
4
4 (0.1 mmol) and 33. The crude product was purified by flash
chromatography (CH Cl /MeOH 95/5) to afford the title compound.
2
2
1
Yield: 7 mg, 15%. H NMR (400 MHz, MeOD ) δ = 7.94 (d, J = 9.5
4
Hz, 1 H), 7.83 (d, J = 8.0 Hz, 1 H), 7.79−7.72 (m, 1 H), 7.72−7.64
(
m, 1 H), 7.52 (d, J = 7.5 Hz, 1 H), 7.32 (d, J = 9.5 Hz, 1 H), 7.30−
7
3
2
.23 (m, 4 H), 7.22−7.15 (m, 1 H), 4.56 (s, 1 H), 3.99−3.87 (m, 4 H),
1
system using Merck silica gel 60 Å (40−63 mm mesh). H NMR
.76−3.71 (m, 2 H), 3.66 (d, J = 14.8 Hz, 2 H), 3.46−3.33 (m, 2 H),
NMR spectra were recorded on a Bruker DRX-400. Chemical shifts
are expressed in parts per million (ppm) and referenced to the residual
solvent peak. Analytical HPLC-MS was performed on an Agilent MSD
mass spectrometer connected to an Agilent 1100 system with: method
+
.92 (t, J = 7.4 Hz, 2 H). LC-MS (ESI+) m/z = 484 [M + H] .
N-Benzyl-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-yl)-
pyridazine-3-carboxamide (6). General procedure A. Prepared from
4
5 (0.08 mmol) and 33. The crude product was purified by flash
acidic pH, Column ACE 3 C8 (50 mm × 3.0 mm), H O (+0.1%
2
chromatography (5% MeOH in DCM) to afford the title compound.
TFA), and MeCN were used as mobile phases at a flow rate of 1 mL/
min, with a gradient time of 3.0 min; or Method basic pH, Column X-
1
Yield: 6 mg, 20%. H NMR (400 MHz, CDCl ) δ = 8.20 (t, J = 5.8 Hz,
3
1
7
H), 8.08 (d, J = 9.3 Hz, 1 H), 7.77−7.71 (m, 1 H), 7.64 (s, 1 H),
Terra MSC18 (50 mm × 3.0 mm), H O (containing 10 mM
2
.60−7.53 (m, 1 H), 7.39−7.31 (m, 6 H), 7.31−7.26 (m, 1 H), 7.00
NH HCO ; pH = 10), and MeCN were used as mobile phases at a
4
3
(
3
d, J = 9.5 Hz, 1 H), 4.66 (d, J = 6.0 Hz, 2 H), 4.09−4.00 (m, 1 H),
flow rate of 1 mL/min, with a gradient time of 3.0 min. Preparative
HPLC was performed on a Gilson HPLC system. Basic pH: column
.93−3.83 (m, 2 H), 3.83−3.69 (m, 3 H), 3.37−3.31 (m, 2 H). LC-
+
MS (ESI+) m/z = 470 [M + H] .
Xbridge Prep C18, 5 μM CBD (30 mm × 75 mm), H O (containing
2
N-Phenyl-6-(4-(2-(trifluoromethyl)benzoyl)piperazin-1-yl)-
pyridazine-3-carboxamide (7). General procedure A. Prepared from
5
0 mM NH HCO ; pH = 10), and MeCN were used as mobile phases
4 3
at a flow rate of 45 mL/min, with a gradient time of 9 min. Acidic pH:
4
6 (0.08 mmol) and 33. The crude product was purified by flash
column ACE 5 C8 (150 mm × 30 mm), H O (containing 0.1% TFA),
2
chromatography (2−5% MeOH in DCM) to afford the title
and MeCN were used as mobile phases at a flow rate of 45 mL/min,
with a gradient time of 9 min. For HPLC-MS, detection was made by
UV using the 180−305 nM range and MS (ESI+). For preparative
HPLC, detection was made by UV at 254 or 214 nM. All final
compounds were assessed to be >95% pure by HPLC-MS analysis.
General Synthetic Procedures. General Procedure A for the
Synthesis of 5, 6, 7, 13, 15, 16, 19, 20, and 53−58. Aryl halide (30
1
compound. Yield: 9 mg, 26%. H NMR (400 MHz, CDCl ) δ =
3
9
=
.82 (s, 1 H), 8.16 (d, J = 9.4 Hz, 1 H), 7.80−7.69 (m, 3 H), 7.65 (d, J
14.4 Hz, 1 H), 7.58 (d, J = 15.2 Hz, 1 H), 7.36 (s, 1 H), 7.40 (s, 2
H), 7.19−7.10 (m, 1 H), 7.05 (d, J = 9.5 Hz, 1 H), 4.15−4.03 (m, 1
H), 3.98−3.87 (m, 2 H), 3.87−3.71 (m, 3 H), 3.42−3.32 (m, 2 H).
+
LC-MS (ESI+) m/z = 456 [M + H] .
N-Benzyl-6-(4-((2-(trifluoromethyl)phenyl)sulfonyl)-piperazin-1-
yl)pyridazine-3-carboxamide (8). General procedure B. Prepared
from 59 (0.12 mmol) and 2-(trifluoromethyl)benzene-1-sulfonyl
mg, 0.1 mmol, 1 equiv), amine (0.1 mmol, 1 equiv), and Et N (0.2
3
mmol, 2 equiv) were heated in 1,4-dioxane (1 mL) at 100 °C for 18 h.
The resulting solution was concentrated in vacuo, dissolved in CH Cl ,
2
2
chloride. Product purified by flash chromatography (5−80% EtOAc
1
and washed with brine. The organic layer was concentrated in vacuo,
and the desired compound was purified by chromatography (final
compounds) or used in the next step without any further purification
in pentane). Yield: 38 mg, 75%. H NMR (400 MHz, CDCl ) δ =
3
8
7
4
.21−8.15 (m, 2 H), 8.06 (d, J = 9.5 Hz, 1 H), 7.94−7.90 (m, 1 H),
.77−7.70 (m, 2 H), 7.37−7.27 (m, 5 H), 6.98 (d, J = 9.5 Hz, 1 H),
(
intermediate compounds).
.66 (d, J = 6.0 Hz, 2 H), 3.90−3.81 (m, 4 H), 3.44−3.37 (m, 4 H).
+
General Procedure B for the Synthesis of 8, 9, 10, 14, 17, 23, 24,
LC-MS (ESI+) m/z = 506 [M + H] .
and 35−37. A solution amine (3 mmol, 1 equiv) and Et N (3.6 mmol,
3
N-Benzyl-6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazine-3-car-
boxamide (9). General procedure B. Prepared from 59 (0.12 mmol)
and o-tolylsulfonyl chloride. Product purified by flash chromatography
1
.2 equiv) in CH Cl (10 mL) was added to a solution of sulfonyl
2 2
chloride (570 mg, 3 mmol, 1 equiv) in CH Cl (5 mL) at 0 °C, and
2
2
1
the reaction was stirred at room temperature overnight. The resulting
(5−80% EtOAc in pentane). Yield: 30 mg, 66%. H NMR (400 MHz,
mixture was filtered, washed with water, dried with MgSO , and then
CDCl ) δ = 8.18 (t, J = 5.8 Hz, 1 H), 8.06 (d, J = 9.5 Hz, 1 H), 7.92
4
3
passed through a short silica pad. After evaporation of the solvent in
vacuo, the desired compound was purified by chromatography (final
compounds) or used in the next step without any further purification
(dd, J = 8.4, 1.4 Hz, 1 H), 7.52−7.45 (m, 1 H), 7.37−7.28 (m, 6 H),
6.97 (d, J = 9.5 Hz, 1 H), 4.66 (d, J = 6.0 Hz, 2 H), 3.88−3.82 (m, 4
H), 3.34−3.32 (m, 4 H), 2.66 (s, 3 H). LC-MS (ESI+) m/z = 452 [M
+
(
intermediate compounds).
+ H] .
General Procedure C for the Synthesis of 11, 12, and 38. Aryl
N-Benzyl-6-(4-((2-fluorophenyl)sulfonyl)piperazin-1-yl)-
pyridazine-3-carboxamide (10). General procedure B. Prepared from
59 (0.4 mmol) and 2-fluorobenzene-1-sulfonyl chloride. Product
halide (15 mg, 0.033 mmol, 1 equiv) and amine (0.165 mmol, 5 equiv)
were dissolved in DMF (0.5 mL) and heated at 150 °C overnight in a
sealed tube. The desired compound was purified by chromatography
purified by flash chromatography (20−100% EtOAc in hexane). Yield:
1
(final compounds) or used in the next step without any further
115 mg, 63%. H NMR (400 MHz, CDCl ) δ = 8.20 (t, J = 5.9 Hz,
3
purification (intermediate compounds).
1H), 8.05 (d, J = 9.5 Hz, 1H), 7.89−7.82 (m, 1H), 7.63−7.55 (m,
H
DOI: 10.1021/acs.jmedchem.7b00182
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
1
2
4
H), 7.40−7.17 (m, 7H), 7.00 (d, J = 9.5 Hz, 1H), 4.65 (d, J = 5.9 Hz,
in hexane). Yield: 45 mg, 76%. H NMR (400 MHz, CDCl ) δ = 8.59
3
H), 3.92−3.84 (m, 4H), 3.40−3.31 (m, 4H). LC-MS (ESI+) m/z =
(dd, J = 4.8, 1.8 Hz, 1 H), 8.43 (dd, J = 7.8, 2.0 Hz, 1 H), 8.19 (br. s, 1
H), 8.08 (d, J = 9.3 Hz, 1 H), 7.45 (dd, J = 7.8, 4.8 Hz, 1 H), 7.29−
7.35 (m, 2 H), 6.95−7.07 (m, 3 H), 4.63 (d, J = 6.1 Hz, 2 H), 3.83−
3.94 (m, 4 H), 3.49−3.57 (m, 4 H). LC-MS (ESI+) m/z = 491 [M +
+
56 [M + H] .
N-Benzyl-6-(4-((2-(dimethylamino)phenyl)sulfonyl)piperazin-1-
yl)pyridazine-3-carboxamide (11). General procedure C. Prepared
from 10 (0.033 mmol) and dimethylamine (5.6 M solution in EtOH).
+
H] .
The product was purified by preparative HPLC basic pH to afford the
N-(4-Fluorobenzyl)-6-(4-((2-methylpyridin-3-yl)sulfonyl)-
piperazin-1-yl)pyridazine-3-carboxamide (18). 17 (36 mg, 0.07
mmol, 1 equiv), methyl boronic acid (0.22 mmol, 3 equiv),
1
desired product. Yield: 4 mg, 24%. H NMR (400 MHz, CDCl ) δ =
3
8.18 (t, J = 5.8 Hz, 1H), 8.04 (d, J = 9.5 Hz, 1H), 7.98 (dd, J = 7.9, 1.6
Hz, 1H), 7.54−7.48 (m, 1H), 7.35−7.27 (m, 6H), 7.24−7.18 (m, 1H),
Pd(dppf)Cl ·CH Cl complex (0.1 equiv), tricyclohexylphosphine
2 2 2
6
3
H] .
.95 (d, J = 9.5 Hz, 1H), 4.65 (d, J = 5.8 Hz, 2H), 3.85−3.78 (m, 4H),
(0.03 mmol, 0.4 equiv), and tripotassium phosphate (0.26 mmol, 3.5
equiv) were suspended in toluene/water mixture (95:5, 5 mL),
.36−3.30 (m, 4H), 2.73 (s, 6H). LC-MS (ESI+) m/z = 481 [M +
+
degassed with N , and heated at 100 °C for 2 h. Additional amounts of
2
all reagents were added and stirred at 100 °C for 5 h. The reaction
mixture was purified by flash chromatography (0−3% MeOH in
N-Benzyl-6-(4-((2-(4-hydroxypiperidin-1-yl)phenyl)sulfonyl)-
piperazin-1-yl)pyridazine-3-carboxamide (12). General procedure C.
Prepared from 10 (0.04 mmol) and 4-hydroxypiperidine. The product
CH Cl ). The product was further purified by preparative HPLC
2 2
1
was purified by preparative HPLC basic pH to afford the desired
acidic pH to yield the title product. Yield: 19 mg, 47%. H NMR (400
MHz, DMSO-d ) δ = 9.40 (t, J = 6.4 Hz, 1 H), 8.71 (dd, J = 1.8, 4.8
1
product. Yield: 11 mg, 47%. H NMR (400 MHz, CDCl ) δ = 8.17 (t,
6
3
J = 5.8 Hz, 1H), 8.03 (d, J = 9.5 Hz, 1H), 7.95 (dd, J = 8.0, 1.8 Hz,
Hz, 1 H), 8.19 (dd, J = 8.1, 1.8 Hz, 1 H), 7.86 (d, J = 9.6 Hz, 1 H),
7.52−7.46 (m, 1 H), 7.38−7.31 (m, 3 H), 7.16−7.08 (m, 2 H), 4.45
1
6
3
2
H), 7.55−7.48 (m, 1H), 7.35−7.26 (m, 6H), 7.25−7.20 (m, 1H),
.93 (d, J = 9.5 Hz, 1H), 4.64 (d, J = 5.8 Hz, 2H), 3.93−3.84 (m, 1H),
.83−3.76 (m, 4H), 3.32−3.25 (m, 4H), 3.25−3.16 (m, 2H), 2.86−
(d, J = 6.4 Hz, 2 H), 3.83 (app dd, J = 5.9, 4.4 Hz, 5 H), 3.26 (app dd,
J = 5.9, 4.4 Hz, 4 H), 2.79 (s, 3 H). LC-MS (ESI+) m/z = 471 [M +
+
H] .
.75 (m, 2H), 2.08−1.97 (m, 2H), 1.82−1.70 (m, 2H). LC-MS (ESI+)
+
Benzyl 6-(4-(o-Tolylsulfonyl)piperazin-1-yl)pyridazine-3-carboxy-
late (19). General procedure A. Prepared from 50 (0.08 mmol) and
m/z = 537 [M + H] .
N-(Thiophen-2-ylmethyl)-6-(4-(o-tolylsulfonyl)piperazin-1-yl)-
pyridazine-3-carboxamide (13). General procedure A. Prepared from
3
9. Reaction was run in acetonitrile (0.5 mL) and was heated in the
microwave oven at 190 °C for 15 min. The resulting solution was
4
8 (0.08 mmol) and 39. The crude product was purified by flash
concentrated in vacuo and purified by flash chromatography (5−80%
chromatography (20−80% EtOAc in pentane) to afford the title
1
1
EtOAc in hexane) to afford the title compound. Yield: 17 mg, 46%. H
compound. Yield: 18 mg, 50%. H NMR (400 MHz, CDCl ) δ =
3
NMR (400 MHz, CDCl ) δ = 7.93−7.88 (m, 2H), 7.51−7.44 (m,
8
.23−8.16 (m, 1 H), 8.04 (d, J = 9.5 Hz, 1 H), 7.92 (d, J = 7.5 Hz, 1
3
3
3
4
H), 7.39−7.29 (m, 5H), 6.85 (d, J = 9.8 Hz, 1H), 5.43 (s, 2H), 3.93−
H), 7.52−7.45 (m, 1 H), 7.38−7.31 (m, 2 H), 7.22 (d, J = 5.0 Hz, 1
H), 7.03 (d, J = 3.3 Hz, 1 H), 6.99−6.92 (m, 2 H), 4.82 (d, J = 5.8 Hz,
2
.84 (m, 4H), 3.36−3.26 (m, 4H), 2.65 (s, 3H). LC-MS (ESI+) m/z =
+
53 [M + H] .
H), 3.89−3.80 (m, 4 H), 3.36−3.29 (m, 4 H), 2.66 (s, 3 H). LC-MS
+
Prop-2-yn-1-yl 6-(4-((2-(4-Hydroxypiperidin-1-yl)phenyl)-
(
ESI+) m/z = 458 [M + H] .
sulfonyl)piperazin-1-yl)pyridazine-3-carboxylate (20). General pro-
cedure A. Prepared from 51 (0.08 mmol) and 42. Reaction was run in
acetonitrile (0.5 mL) and was heated in the microwave oven at 190 °C
for 15 min. The resulting solution was concentrated in vacuo and
purified by flash chromatography (50−100% EtOAc in hexane) to
N-(4-Fluorobenzyl)-6-(4-(o-tolylsulfonyl)piperazin-1-yl)-
pyridazine-3-carboxamide (14). General procedure B. Prepared from
6
2 (0.12 mmol) and o-tolylsulfonyl chloride. Purified by preparative
1
HPLC acidic pH. Yield: 15 mg, 26%. H NMR (400 MHz, CDCl ) δ =
3
8
1
4
.18 (t, J = 5.8 Hz, 1 H), 8.05 (d, J = 9.6 Hz, 1 H), 7.92 (d, J = 8.1 Hz,
H), 7.43−7.55 (m, 1 H), 7.28−7.39 (m, 4 H), 7.02−6.94 (m, 3 H),
1
afford the title compound. Yield: 11 mg, 30%. H NMR (400 MHz,
CDCl ) δ = 7.95 (dd, J = 8.0, 1.8 Hz, 1 H), 7.89 (d, J = 9.5 Hz, 1 H),
3
.62 (d, J = 5.8 Hz, 2 H), 3.85 (m, 4 H), 3.33 (m, 4 H), 2.66 (s, 3 H).
+
7.55−7.49 (m, 1 H), 7.34−7.30 (m, 1 H), 7.25−7.19 (m, 1 H), 6.83
LC-MS (ESI+) m/z = 470 [M + H] .
(
d, J = 9.5 Hz, 1 H), 4.97 (d, J = 2.5 Hz, 2 H), 3.91−3.83 (m, 5 H),
6
-(4-((3-Chloro-2-methylphenyl)sulfonyl)piperazin-1-yl)-N-(thio-
3
.31−3.25 (m, 4 H), 3.25−3.18 (m, 2 H), 2.85−2.76 (m, 2 H), 2.50 (t,
phen-2-ylmethyl)pyridazine-3-carboxamide (15). General procedure
A. Prepared from 48 (0.08 mmol) and 40. Reaction was refluxed in n-
BuOH overnight. The resulting solution was concentrated in vacuo
and purified by flash chromatography (20−75% EtOAc in hexane).
Fractions containing the product were pooled, concentrated in vacuo,
J = 2.5 Hz, 1 H), 2.04−1.97 (m, 2 H), 1.82−1.71 (m, 2 H). LC-MS
+
(ESI+) m/z = 486 [M + H] .
N-Benzyl-6-(4-(o-tolylsulfinyl)piperidin-1-yl)pyridazine-3-carbox-
amide (21) and Benzyl-6-(4-(o-tolylsulfonyl)piperidin-1-yl)-
pyridazine-3-carboxamide (22). 1-(6-(Benzylcarbamoyl)pyridazin-
3-yl)piperidin-4-yl Methanesulfonate (63). A solution of mesyl
chloride (0.576 mmol, 1.2 equiv) in CH Cl (0.5 mL) was added
and triturated with Et O to afford the title compound. Yield: 34 mg,
2
1
8
7
7
7%. H NMR (400 MHz, DMSO-d ) δ = 9.41 (t, J = 6.3 Hz, 1 H),
6
2
2
.92−7.84 (m, 2 H), 7.82−7.75 (m, 1 H), 7.46 (t, J = 8.0 Hz, 1 H),
dropwise to a stirred solution of 57 (150 mg, 0.480 mmol, 1 equiv)
and pyridine (0.480 mmol, 1 equiv) in CH Cl (1.5 mL) at 0 °C. The
.41−7.33 (m, 2 H), 7.02−6.98 (m, 1 H), 6.94 (dd, J = 3.4, 5.1 Hz, 1
2
2
H), 4.63 (d, J = 6.3 Hz, 2 H), 3.86−3.79 (m, 4 H), 3.29−3.19 (m, 4
H), 2.63 (s, 3 H). LC-MS (ESI+) m/z = 492 [M + H] .
mixture was left to warm to rt overnight, then diluted with CH Cl .
2
2
+
The organic layer was washed with brine, dried over MgSO , and
4
6
-(4-((3-Chloro-2-methylphenyl)sulfonyl)piperazin-1-yl)-N-(prop-
concentrated in vacuo. The crude was used in the next step without
2
-yn-1-yl)pyridazine-3-carboxamide (16). General procedure A.
+
any further purification. LC-MS (ESI+) m/z = 391 [M + H] .
Prepared from 49 (0.08 mmol) and 40. Reaction was refluxed in n-
BuOH overnight. The resulting solution was concentrated in vacuo
and purified by flash chromatography (20−75% EtOAc in hexane).
Fractions containing the product were pooled, concentrated in vacuo,
N-Benzyl-6-(4-(o-tolylthio)piperidin-1-yl)pyridazine-3-carboxa-
mide (64). 63 (81 mg, 0.207 mmol, 1 equiv), 2-methylbenzene-1-thiol
(0.228 mmol, 1.1 equiv), and potassium carbonate (0.228 mmol, 1.1
equiv) were suspended in DMF (1 mL) and stirred overnight at 60 °C.
The reaction was purified by flash chromatography (20−50% EtOAc
and triturated with Et O to afford the title compound. Yield: 20 mg,
2
1
1
5
9
1
5
2
7%. H NMR (400 MHz, CDCl ) δ = 8.03 (br s, 1 H), 8.02 (d, J =
in hexane) to yield the title compound. Yield: 80 mg, 92%. H NMR
3
.3 Hz, 1 H), 7.90 (dd, J = 1.3, 8.0 Hz, 1 H), 7.62 (dd, J = 1.3, 8.0 Hz,
H), 7.32−7.26 (m, 1 H), 6.97 (d, J = 9.5 Hz, 1 H), 4.26 (dd, J = 2.5,
.5 Hz, 2 H), 3.89−3.83 (m, 4 H), 3.38−3.32 (m, 4 H), 2.70 (s, 3 H),
(400 MHz, acetone-d ) δ = 8.77−8.65 (m, 1 H), 7.96−7.90 (m, 1 H),
6
7.80−7.74 (m, 1 H), 7.48−7.38 (m, 4 H), 7.36−7.21 (m, 5 H), 4.77−
4.70 (m, 1 H), 4.69−4.63 (m, 3 H), 3.22−3.03 (m, 3 H), 2.45−2.39
(m, 3 H), 1.95−1.73 (m, 2 H), 1.60−1.51 (m, 1 H). LC-MS (ESI+)
+
.26 (t, J = 2.5 Hz, 1 H). LC-MS (ESI+) m/z = 434 [M + H] .
+
6
-(4-((2-Chloropyridin-3-yl)sulfonyl)piperazin-1-yl)-N-(4-
m/z = 419 [M + H] .
fluorobenzyl)pyridazine-3-carboxamide (17). General procedure B.
Prepared from 62 (0.12 mmol) and 2-chloropyridine-3-sulfonyl
chloride. Product purified by flash chromatography (40−95% EtOAc
N-Benzyl-6-(4-(o-tolylsulfinyl)piperidin-1-yl)pyridazine-3-carbox-
amide (21). To a cooled solution (ice-bath) of 64 (80 mg, 0.191
mmol, 1 equiv) in CH Cl (3 mL) was added m-CPBA (0.382 mmol,
2
2
I
DOI: 10.1021/acs.jmedchem.7b00182
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Journal of Medicinal Chemistry
Article
2
equiv), and the mixture was let to warm up overnight. The mixture
4.39 (s, 2 H), 4.28−4.34 (m, 2 H), 3.70−3.77 (m, 4 H), 3.26−3.33
+
was concentrated in vacuo, and after purification by preparative HPLC
basic pH, the title compound was isolated. Yield: 29 mg, 35%. H
NMR (400 MHz, acetone-d ) δ = 8.73 (t, J = 6.1 Hz, 1 H), 7.91 (d, J =
(m, 4 H), 2.64 (s, 3 H). LC-MS (ESI+) m/z = 438 [M + H] .
1
3-(Phenethylsulfonyl)-6-(4-(o-tolylsulfonyl)piperazin-1-yl)-
pyridazine (26). 3-Chloro-6-[(2-phenylethyl)sulfanyl]pyridazine
6
(
68). 67 (200 mg, 1.3 mmol, 1 equiv), Et N (1.3 mmol, 1 equiv),
9
2
4
1
.5 Hz, 1 H), 7.77−7.73 (m, 1 H), 7.47−7.43 (m, 2 H), 7.42−7.37 (m,
H), 7.34−7.28 (m, 4 H), 7.25−7.19 (m, 1 H), 4.75−4.62 (m, 2 H),
.66 (d, J = 6.3 Hz, 2 H), 3.20−3.01 (m, 3 H), 2.40 (s, 3 H), 1.92−
3
and 2-phenylethane-1-thiol (1.3 mmol, 1 equiv) were dissolved in
DMF (5 mL) and heated at 70C overnight. Silica was added to the
reaction mixture and concentrated in vacuo for flash chromatography
purification (0−8% EtOAc in hexane) to obtain the title product.
.72 (m, 3 H), 1.57−1.49 (m, 1 H). LC-MS (ESI+) m/z = 435 [M +
+
H] .
+
Yield. 337 mg, 82%. LC-MS (ESI+) m/z = 251 [M + H] .
N-Benzyl-6-(4-(o-Tolylsulfonyl)piperidin-1-yl)pyridazine-3-car-
3
-Chloro-6-(phenethylsulfonyl)pyridazine (69). m-CPBA (3.30
boxamide (22). After preparative HPLC basic pH purification of the
above-mentioned crude mixture, the title compound was also isolated.
Yield: 15 mg, 17%. H NMR (400 MHz, acetone-d ) δ = 8.70 (t, J =
mmol, 3 equiv) was added portionwise to an ice-bath cooled solution
1
of 68 (276 mg, 1.10 mmol, 1 equiv) in CH Cl (15 mL), and the
2
2
6
reaction was allowed to warm up to rt overnight. The reaction mixture
was diluted with CH Cl and washed with satd aq NaHCO . The
6
2
4
.1 Hz, 1 H), 7.96−7.89 (m, 2 H), 7.67−7.60 (m, 1 H), 7.51−7.47 (m,
H), 7.44−7.37 (m, 2 H), 7.37−7.30 (m, 3 H), 7.28−7.22 (m, 1 H),
.77−4.69 (m, 2 H), 4.66 (d, J = 6.3 Hz, 2 H), 3.68 (tt, J = 3.9, 11.8
2
2
3
organic layer was dry-loaded for flash chromatography (0−30% EtOAc
in hexane) to afford the title product. Yield: 230 mg, 74%. LC-MS
Hz, 1 H), 3.20−3.10 (m, 2 H), 2.73 (s, 3 H), 2.04−1.96 (m, 2 H),
+
+
(ESI+) m/z = 283 [M + H] .
1.86−1.76 (m, 2 H). LC-MS (ESI+) m/z = 451 [M + H] .
3
-(Phenethylsulfonyl)-6-(4-(o-tolylsulfonyl)piperazin-1-yl)-
pyridazine (26). 69 (20 mg, 0.070 mmol, 1 equiv), 39 (0.085 mmol,
.2 equiv), and Et N (0.15 mmol, 2 equiv) were heated in DMF at 130
N-Benzyl-6-(5-(o-tolylsulfonyl)-2,5-diazabicyclo[2.2.1]heptan-2-
yl)pyridazine-3-carboxamide (23). General procedure B. Prepared
from 60 (0.12 mmol) and o-tolylsulfonyl chloride. Product purified by
1
3
°
C for 3 h. The reaction mixture was dry-loaded on silica for flash
flash chromatography (20−80% EtOAc in hexane). Yield: 42 mg, 68%.
1
chromatography (25−70% EtOAc in hexane). The resulting solid was
H NMR (400 MHz, CDCl ) δ = 8.18 (t, J = 6.0 Hz, 1 H), 8.06 (d, J =
3
1
triturated with Et O to afford the title product. Yield: 22 mg, 64%. H
2
9
7
.5 Hz, 1 H), 7.97−7.91 (m, 1 H), 7.50−7.43 (m, 1 H), 7.39−7.27 (m,
H), 6.68 (d, J = 9.3 Hz, 1 H), 5.20−5.18 (m, 1 H), 4.69−4.66 (m, 3
NMR (400 MHz, CDCl ) δ = 7.95−7.90 (m, 1 H), 7.79 (d, J = 9.7 Hz,
3
1
7
3
H), 7.53−7.47 (m, 1 H), 7.39−7.32 (m, 2 H), 7.26−7.20 (m, 2 H),
.20−7.12 (m, 3 H), 6.90 (d, J = 9.7 Hz, 1 H), 3.94−3.87 (m, 4 H),
H), 3.69−3.56 (m, 2 H), 3.48−3.39 (m, 2 H), 2.62 (s, 3 H), 2.06 (s, 2
H). LC-MS (ESI+) m/z = 464 [M + H] .
+
.81−3.74 (m, 2 H), 3.36−3.30 (m, 4 H), 3.14−3.07 (m, 2 H), 2.66
N-Benzyl-6-(4-(o-tolylsulfonyl)-1,4-diazepan-1-yl)pyridazine-3-
carboxamide (24). General procedure B. Prepared from 61 (0.12
mmol) and o-tolylsulfonyl chloride. Product purified by flash
+
(s, 3 H). LC-MS (ESI+) m/z = 487 [M + H] .
Procedures for the Synthesis of Compounds 27−30. 6-
Chloropyridazine-3-carbonyl Chloride (70). 43 (200 mg, 1 mmol, 1
chromatography (20−80% EtOAc in hexane). Yield: 31 mg, 49%.
equiv) was suspended in CH Cl (10 mL) and cooled to 0 °C. Oxalyl
1
2
2
H NMR (400 MHz, CDCl ) δ = 8.18 (t, J = 6.0 Hz, 1 H), 8.04 (d, J =
3
chloride (2 mmol, 2 equiv) was added, followed by addition of one
drop of DMF. The reaction was allowed to warm to room temperature
and stirred for 2 days. The clear solution was concentrated in vacuo
and azeotropped with toluene. Used in the next step without any
9
7
4
3
.5 Hz, 1 H), 7.81 (d, J = 8.5 Hz, 1 H), 7.46−7.39 (m, 1 H), 7.38−
.25 (m, 7 H), 6.88 (d, J = 9.5 Hz, 1 H), 4.67 (d, J = 6.0 Hz, 2 H),
.10−4.07 (m, 2 H), 3.99−3.88 (m, 2 H), 3.59−3.50 (m, 2 H), 3.34−
+
.26 (m, 2 H), 2.55 (s, 3 H), 2.19−2.07 (m, 2 H). LC-MS (ESI+) m/z
further purification. LC-MS (ESI+) m/z = 173 [M + H] (methyl
+
=
466 [M + H] .
ester).
N-Benzyl-1-(6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazin-3-yl)-
1
-(6-Chloropyridazin-3-yl)-3-(4-fluorophenyl)propan-1-one (71).
methanamine 2,2,2-trifluoroacetate Salt (25). (6-(4-(o-
Tolylsulfonyl)piperazin-1-yl)pyridazin-3-yl)methanol (65). 58 (130
mg, 0.34 mmol, 1 equiv) was dissolved in THF (20 mL) under N2
atmosphere and cooled to 0 °C. NaBH (4.8 mmol, 14 equiv) was
dissolved absolute EtOH (5 mL) and slowly added to the ester
solution at 0 °C. The mixture was stirred at 0 °C for 10 min and then
stirred at 40 °C for 1 h. The reaction was quenched by addition of 2 M
HCl until H gas evolution ceased. The solution was then brought to
pH ∼ 8 by addition of satd NaHCO . Water was added, and the
resulting mixture was extracted with EtOAc and dried with MgSO .
A solution of 4-fluorophenethyl bromide (1.51 mmol, 1.2 equiv) in
anhydrous THF (2 mL) was added dropwise to the degassed
suspension of magnesium turnings (1.51 mmol. 1.2 equiv) and a single
crystal of iodine in anhydrous THF (3 mL) at room temperature.
Once all the alkyl bromide was added, the suspension was refluxed at
4
1
00 °C under N for 1 h. The solution was allowed to cool to RT, then
2
cooled to −40 °C on a dry ice/acetonitrile bath. An anhydrous 1 M
2
solution of CuCN ·LiCl (1.64 mmol, 1.3 equiv) was then added
2
3
dropwise to the cooled Grignard solution for transmetalation. The
mixture was allowed to stir for 30 min. 70 (223 mg, 1.26 mmol, 1
equiv) was dissolved in anhydrous THF (5 mL) and added dropwise
to the organometallic suspension at −40 °C over 5 min and allowed to
stir for 1 h. The reaction mixture was then quenched by addition of
4
The organic layer was dried in vacuo to afford the title product. Yield:
114 mg, 94%. Used in the next step without any further purification.
+
LC-MS (ESI+) m/z = 349 [M + H] .
-(Chloromethyl)-6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazine
66). 65 (34 mg, 0.09 mmol, 1 equiv) was suspended in CH Cl (5
3
satd NH Cl. The mixture was then diluted with EtOAc, and a thick
4
(
2
2
precipitate was formed. The mixture was filtered via vacuum filtration,
and the residue was washed thoroughly with EtOAc. The filtrate was
then washed with water and brine. The organic layer was dried with
mL) and cooled to 0 °C, to which SOCl (2.7 mmol, 28 equiv) was
added. The mixture was stirred overnight at room temperature. The
resulting solution was washed with satd NaHCO and brine and then
dried over MgSO . The organic layer was concentrated in vacuo and
azeotroped with toluene to yield the title product. Yield: 36 mg, 87%.
Used in the next step without any further purification. LC-MS (ESI+)
m/z = 367 [M + H] .
2
3
MgSO , filtered, and loaded on to silica for purification by flash
4
4
chromatography (0−15% EtOAc in cyclohexane) to obtain the desired
1
product. Yield: 185 mg, 56%. H NMR (400 MHz, CDCl ): δ = 8.09
3
(d, J = 8.8 Hz, 1 H), 7.67 (d, J = 8.8 Hz, 1 H), 7.19−7.26 (m, 2 H),
+
6.93−7.02 (m, 2 H), 3.70 (t, J = 7.6 Hz, 2 H), 3.09 (t, J = 7.6 Hz, 2 H).
+
N-Benzyl-1-(6-(4-(o-tolylsulfonyl)piperazin-1-yl)pyridazin-3-yl)-
LC-MS (ESI+) m/z = 265 [M + H] .
methanamine 2,2,2-Trifluoroacetate Salt (25). 66 (31 mg, 0.084
mmol, 1 equiv), benzylamine (0.93 mmol, 11 equiv), and Et N (0.25
3
-(4-Fluorophenyl)-1-(6-(4-(o-tolylsulfonyl)piperazin-1-yl)-
3
pyridazin-3-yl)propan-1-one (27). 27 was prepared from 71 and 39,
mmol, 3 equiv) were suspended in acetonitrile (5 mL) and stirred at
room temperature overnight. The mixture was concentrated in vacuo
and purified by preparative HPLC acidic pH to obtain the title product
following general procedure A. The reaction mixture was diluted with
EtOAc and washed with satd aq NaHCO . The organic layer was dry-
3
loaded for flash chromatography (10−50% EtOAc in cyclohexane) to
1
1
as the trifluoroacetate salt. Yield: 8 mg, 17%. H NMR (400 MHz,
afford the title product. Yield: 68 mg, 79%. H NMR (CDCl , 400
3
CDCl ): δ = 7.90 (dd, J = 8.3, 1.3 Hz, 1 H), 7.63 (d, J = 9.6 Hz, 1 H),
MHz) δ = 7.86−7.97 (m, 2 H), 7.46−7.53 (m, 1 H), 7.31−7.40 (m, 2
H), 7.17−7.25 (m, 2 H), 6.87−6.99 (m, 3 H), 3.87−3.95 (m, 4 H),
3
7.47−7.53 (m, 1 H), 7.32−7.45 (m, 7 H), 7.16 (d, J = 9.1 Hz, 1 H),
J
DOI: 10.1021/acs.jmedchem.7b00182
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
3
2
.55−3.64 (t, J = 7.6 Hz, 2 H), 3.29−3.37 (m, 4 H), 3.05 (t, J = 7.6 Hz,
−78 °C for 15 min, warmed to RT, and stirred for 30 min, then stirred
at 50 °C for 30 min. The mixture was concentrated in vacuo, taken up
in CH Cl , and washed with satd NaHCO . The organic layer was
+
H), 2.66 (s, 3 H). LC-MS (ESI+) m/z = 469 [M + H] .
3
-(4-(4-Fluorophenyl)-1-methoxybut-1-en-2-yl)-6-(4-(o-
2
2
3
tolylsulfonyl)piperazin-1-yl)pyridazine (72). A 1.8 M solution of
lithium diisopropylamide (0.675 mmol, 1.7 equiv) in anhydrous THF
was added dropwise under N2 atmosphere to a solution of
then dry loaded on to silica and purified by flash chromatography
(30−85% EtOAc in cyclohexane) to obtain the product. Yield: 6 mg,
1
19%. H NMR (400 MHz, CDCl ) δ = 7.90−7.98 (m, 1 H), 7.46−
3
(
methoxymethyl)triphenylphosphanium chloride (0.675 mmol, 1.7
7.52 (m, 1 H), 7.32−7.40 (m, 2 H), 7.13 (d, J = 9.6 Hz, 1 H), 7.01−
7.08 (m, 2 H), 6.87−6.96 (m, 3 H), 5.06 (d, J = 6.1 Hz, 2 H), 4.70 (d,
J = 6.1 Hz, 2 H), 3.72−3.81 (m, 4 H), 3.30−3.39 (m, 4 H), 2.68 (s, 3
equiv) in anhydrous THF (5 mL) cooled to −78 °C. The mixture was
stirred at −78 °C for 30 min, then allowed to warm to room
temperature and stir for a further 30 min. The bright-orange mixture
was then cooled back down to −78 °C, and a solution of 27 (186 mg,
+
H), 2.40−2.52 (m, 4 H). LC-MS (ESI+) m/z = 497 [M + H] .
3-(4-(4-Fluorophenyl)but-1-en-2-yl)-6-(4-(o-tolylsulfonyl)-pipera-
0.40 mmol, 1 equiv) in anhydrous THF (5 mL) was added dropwise.
zin-1-yl)pyridazine (30). After flash chromatography purification of
The mixture was stirred at −78 °C for 1 h, allowed to warm to room
temperature, and stirred at room temperature for a further 4 h. The
solution was dry loaded on to silica for purification by flash
chromatography (20−50% EtOAc in cyclohexane), and two isomers
the above-mentioned crude mixture, the title compound was also
1
isolated; 12 mg, 42% yield. H NMR (400 MHz, CDCl
) δ = 7.93 (d, J
3
= 7.8 Hz, 1 H), 7.45−7.53 (m, 2 H), 7.31−7.40 (m, 2 H), 7.07−7.18
(m, 2 H), 6.85−6.98 (m, 3 H), 5.53 (s, 1 H), 5.20 (s, 1 H), 3.73−3.84
(m, 4 H), 3.33 (t, J = 4.7 Hz, 4 H), 2.97−3.07 (m, 2 H), 2.81−2.91
were isolated (161 mg, 82% combined yield of two isomers). Isomer A
1
+
(
less polar by TLC): H NMR (400 MHz, CDCl ): δ = 7.89−7.95 (m,
(m, 2 H), 2.67 (s, 3 H). LC-MS (ESI+) m/z = 467 [M + H] .
3
tert-Butyl-4-(2-(trifluoromethyl)benzoyl)-piperazine-1-carboxy-
2
6
3
H), 7.45−7.51 (m, 1 H), 7.31−7.37 (m, 2 H), 7.04−7.10 (m, 2 H),
.88−6.95 (m, 2 H), 6.85 (d, J = 9.6 Hz, 1 H), 6.08 (s, 1 H), 3.72−
late (32). General procedure D. Prepared from 31 (10.52 mmol) and
1
3
4. Yield: 3.5 g, 94%. H NMR (400 MHz, CDCl ) δ = 7.74−7.68 (m,
.78 (m, 4 H), 3.63 (s, 3 H), 3.28−3.35 (m, 4 H), 2.79 (apparent s, 4
3
+
1H), 7.63−7.57 (m, 1H), 7.56−7.50 (m, 1H), 7.35−7.30 (m, 1H),
3.86−3.79 (m, 1H), 3.76−3.68 (m, 1H), 3.57−3.46 (m, 2H), 3.37−
.31 (m, 2H), 3.18−3.11 (m, 2H), 1.46 (s, 9H). LC-MS (ESI+) m/z =
H), 2.67 (s, 3 H). LC-MS (ESI+) m/z = 497 [M + H] . Isomer B
1
(
more polar by TLC): H NMR (400 MHz, CDCl ) δ = 7.93 (dd, J =
3
3
3
8
.6, 1.3 Hz, 1 H), 7.46−7.52 (m, 1 H), 7.32−7.38 (m, 2 H), 7.11−7.19
+
59 [M + H] .
(
m, 3 H), 6.91 (t, J = 8.8 Hz, 2 H), 6.80−6.85 (m, 2 H), 3.69−3.75
Piperazin-1-yl(2-(trifluoromethyl)phenyl)-methanone Hydro-
(
m, 4 H), 3.67 (s, 3 H), 3.29−3.35 (m, 4 H), 2.84−2.92 (m, 2 H),
chloride (33). General procedure E. Prepared from 32 (9.86 mmol).
2
.71−2.77 (m, 2 H), 2.67 (s, 3 H). LC-MS (ESI+) m/z = 469 [M +
1
+
Yield: 2.81 g, 97%. H NMR (400 MHz, DMSO-d ) δ = 9.63 (br s,
6
H] .
2
7
3
1
H), 7.87−7.82 (m, 1H), 7.81−7.76 (m, 1H), 7.72−7.66 (m, 1H),
4
-(4-Fluorophenyl)-2-(6-(4-(o-tolylsulfonyl)piperazin-1-yl)-
.65−7.60 (m, 1H), 4.07−3.97 (m, 1H), 3.79−3.69 (m, 1H), 3.41−
.29 (m, 2H), 3.27−3.17 (m, 1H), 3.16−3.03 (m, 2H), 2.96−2.85 (m,
pyridazin-3-yl)butanal (73). 72 (mixture of isomers) (161 mg, 0.324
mmol, 1 equiv) was dissolved in THF (8 mL) and diluted with water
+
H). LC-MS (ESI+) m/z = 259 [M + H] .
(
11 mL). The solution was cooled to 0 °C, and conc H SO (5 mL)
2 4
tert-Butyl 4-(o-Tolylsulfonyl)piperazine-1-carboxylate (35). Gen-
was added slowly. The solution was allowed to stir at 45 °C for 2 days.
The mixture was then neutralized to pH 8 with 6 M and then satd
NaHCO . The mixture was then extracted with CH Cl , and the
eral procedure B. Prepared from 34 and o-tolylsulfonyl chloride. Yield:
1
9
7
3
30 mg, 91%. H NMR (400 MHz, CDCl ) δ = 7.94−7.86 (m, 1 H),
3
3
2
2
.52−7.44 (m, 1 H), 7.38−7.31 (m, 2 H), 3.55−3.43 (m, 4 H), 3.19−
.06 (m, 4 H), 2.64 (s, 3 H), 1.44 (s, 9 H). LC-MS (ESI+) m/z = 341
combined organic layer was dried over MgSO and concentrated in
4
vacuo to yield the title compound. Yield: 156 mg, 99%. Used in the
+
[
M + H] .
next step without any further purification. LC-MS (ESI+) m/z = 487
+
+
tert-Butyl 4-((3-Chloro-2-methylphenyl)sulfonyl)piperazine-1-
[
M + H] (aldehyde). LC-MS (ESI+) m/z = 501 [M + H] (aldehyde
carboxylate (36). General procedure B. Prepared from 34 and 3-
hydrate).
1
chloro-2-methylbenzene-1-sulfonyl chloride. Yield: 830 mg, 96%. H
2
-(4-Fluorophenethyl)-2-(6-(4-(o-tolylsulfonyl)piperazin-1-yl)-
NMR (400 MHz, CDCl ) δ = 7.88 (dd, J = 8.0, 1.0 Hz, 1 H), 7.61 (dd,
3
pyridazin-3-yl)propane-1,3-diol (28). 73 (156 mg, 0.323 mmol, 1
equiv) was dissolved in THF (10 mL). To this was added 10 mL of
J = 8.0, 1.0 Hz, 1 H), 7.31−7.25 (m, 1 H), 3.55−3.39 (m, 4 H), 3.23−
3
.08 (m, 4 H), 2.69 (s, 3 H), 1.45 (s, 9 H). LC-MS (ESI+) m/z = 376
37% aqueous formaldehyde followed by 6 M NaOH (2.5 mL). The
+
[
M + H] .
reaction was stirred at room temperature overnight and subsequently
tert-Butyl 4-((2-Fluorophenyl)sulfonyl)piperazine-1-carboxylate
37). General procedure B. Prepared from 34 and 2-fluorobenzene-
quenched with 2 M HCl and extracted with CH Cl . The organic layer
2
2
(
was concentrated under vacuum, redissolved in THF:H O:6 M NaOH
2
1
+
-sulfonyl chloride. Yield: 900 mg, 89%. LC-MS (ESI+) m/z = 345 [M
H] .
(
10 mL:10 mL:2.5 mL), and stirred at 40 °C for 30 min. The mixture
was diluted with methanol and brine and extracted repeatedly with
0:90 MeOH:CH Cl . The combined organic layer was dried over
+
tert-Butyl 4-((2-(4-Hydroxypiperidin-1-yl)phenyl)sulfonyl)-
1
2
2
piperazine-1-carboxylate (38). General procedure C. Prepared from
37 (0.3 mmol) and 4-hydroxypiperidine. The reaction mixture was
concentrated in vacuo, and the resulting solid was triturated with water
to yield to title compound. Yield: 117 mg, 95%. Used in the next step
MgSO , filtered, dry loaded on silica, and purified by flash
4
chromatography (60% EtOAc in cyclohexane to 4% MeOH in
1
EtOAc) to afford the title product; yield 112 mg, 67%). H NMR (400
MHz, CDCl ) δ = 7.90−7.95 (m, 1 H), 7.46−7.52 (m, 1 H), 7.31−
+
3
without further purification. LC-MS (ESI+) m/z = 426 [M + H] .
-(o-Tolylsulfonyl)piperazine Hydrochloride (39). General proce-
7
1
.39 (m, 3 H), 6.92−6.99 (m, 3 H), 6.83−6.91 (m, 2 H), 4.30 (d, J =
1.1 Hz, 2 H), 3.97 (d, J = 11.4 Hz, 2 H), 3.69−3.78 (m, 4 H), 3.59
1
1
dure E. Prepared from 35. Yield: 1.45 g, 59%. H NMR (400 MHz,
(
br s, 2 H), 3.27−3.36 (m, 4 H), 2.67 (s, 3 H), 2.28−2.39 (m, 2 H),
DMSO-d ) δ = 9.45 (br s, 2 H), 7.82 (d, J = 7.8 Hz, 1 H), 7.63 (app t,
6
+
1
.79−1.88 (m, 2 H). LC-MS (ESI+) m/z = 515 [M + H] .
-(3-(4-Fluorophenethyl)oxetan-3-yl)-6-(4-(o-tolylsulfonyl)-
piperazin-1-yl)pyridazine (29). A solution of 28 (30 mg, 0.058 mmol,
equiv) in 1 mL of THF was added dropwise to a suspension of NaH
0.064 mmol, 1.1 equiv) in 1 mL o THF at 0 °C. The mixture was
J = 7.5 Hz, 1 H), 7.53−7.40 (m, 2 H), 3.37−3.26 (m, 4 H), 3.16−3.10
+
3
(
m, 4 H). LC-MS (ESI+) m/z = 241 [M + H] .
1
-((3-Chloro-2-methylphenyl)sulfonyl)piperazine Hydrochloride
1
(
(
40). General procedure E. Prepared from 36. Yield: 2.20 g, 88%.
+
LC-MS (ESI+) m/z = 275 [M + H] .
-((2-Fluorophenyl)sulfonyl)piperazine Hydrochloride (41). Gen-
stirred at 0 °C for 5 min, then allowed to warm to room temperature
and stirred for 30 min. The mixture was then cooled down to −78 °C,
and a solution of p-tosyl chloride (0.058 mmol 1 equiv) in 1 mL of
THF was added dropwise. The mixture was stirred at −78 °C for 5
min and allowed to warm to R, and stirred for 2 h. Additional p-tosyl
chloride and NaH were added at −78 °C. After 5 h, the reaction
mixture was diluted with 20 mL of dry THF, cooled to −78 °C, and
NaH (1.16 mmol, 20 equiv) was added. The mixture was stirred at
1
eral procedure E. Prepared from 37. Yield: 2.28 g, 93%. LC-MS (ESI+)
+
m/z = 245 [M + H] .
1-[2-(Piperazine-1-sulfonyl)phenyl]piperidin-4-ol Hydrochloride
(42). General procedure E. Prepared from 38 (0.27 mmol). Yield:
+
95 mg, 96%. LC-MS (ESI+) m/z = 326 [M + H] .
6-Chloro-N-phenethylpyridazine-3-carboxamide (44). General
procedure D. Prepared from 43 (3.17 mmol) and 2-phenylethan-1-
K
DOI: 10.1021/acs.jmedchem.7b00182
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Journal of Medicinal Chemistry
Article
1
amine. Yield: 145 mg, 17%. H NMR (400 MHz, CDCl ) δ = 8.27 (d,
H), 3.88−3.68 (m, 4 H), 3.57 (app t, J = 5.4 Hz, 2 H), 3.32 (app t, J =
5.5 Hz, 1 H), 3.23 (app t, J = 5.9 Hz, 1 H), 1.97−1.88 (m, 2 H), 1.34
3
J = 8.8 Hz, 1 H), 8.13−8.05 (m, 1 H), 7.67 (d, J = 8.8 Hz, 1 H), 7.35−
.28 (m, 2 H), 7.26−7.22 (m, 3 H), 3.82−3.75 (m, 2 H), 2.96 (t, J =
.2 Hz, 2 H).
N-Benzyl-6-chloropyridazine-3-carboxamide (45). General proce-
dure D. Prepared from 43 (3.17 mmol) and benzylamine. Yield: 210
+
7
7
(s, 9 H). LC-MS (ESI+) m/z = 412 [M + H] .
tert-Butyl 4-(6-((4-Fluorobenzyl)carbamoyl)pyridazin-3-yl)-
piperazine-1-carboxylate (56). General procedure A. Prepared from
47 (1 mmol) and 34. The crude product was triturated with Et O to
2
1
mg, 26%. H NMR (400 MHz, CDCl ) δ = 8.37 (br s, 1 H), 8.30 (d, J
obtain the title compound (yield: 504 mg, 100%). Used in the next
3
+
=
8.8 Hz, 1 H), 7.69 (d, J = 8.8 Hz, 1 H), 7.40−7.26 (m, 6 H), 4.70 (d,
step without further purification. LC-MS (ESI+) m/z = 416 [M + H] .
+
J = 6.3 Hz, 2 H). LC-MS (ESI+) m/z = 248 [M + H] .
-Chloro-N-phenylpyridazine-3-carboxamide (46). General pro-
cedure D. Prepared from 43 (3.17 mmol) and aniline. Yield: 460 mg,
N-Benzyl-6-(4-hydroxypiperidin-1-yl)pyridazine-3-carboxamide
57). General procedure A. Prepared from 45 (0.56 mmol) and
(
6
piperidin-4-ol. Reaction was refluxed in n-BuOH overnight. The
resulting solution was concentrated in vacuo, dissolved in CH Cl ,
1
6
2%. H NMR (400 MHz, CDCl ) δ = 8.39 (br s, 1H), 8.31 (d, J = 8.8
Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.38−7.26 (m, 5H), 4.70 (d, J = 6.0
2
2
3
washed with brine, and dried with Na SO . The organic layer was
2 4
Hz, 2H)
6
concentrated in vacuo to afford the title compound. Yield: 147 mg,
83%. Used in the next step without further purification. LC-MS (ESI+)
-Chloro-N-(4-fluorobenzyl)pyridazine-3-carboxamide (47). Gen-
+
eral procedure D. Prepared from 43 (3.17 mmol) and 4-fluorobenzyl-
m/z = 313 [M + H] .
1
amine. Yield: 460 mg, 55%. H NMR (400 MHz, CDCl ) δ = 8.36 (br
Methyl 6-(4-(o-Tolylsulfonyl)piperazin-1-yl)pyridazine-3-carboxy-
late (58). General procedure A. Prepared from 52 (1 mmol) and 39.
Reaction was refluxed in DMF for 2 h. Solid material crashed out of
solution upon cooling to 0 °C and was subsequently triturated with
water to provide the title compound. Yield: 287 mg, 77%. Used in the
next step without further purification. LC-MS (ESI+) m/z = 377 [M +
3
s, 1 H), 8.31 (d, J = 8.8 Hz, 1 H), 7.71 (d, J = 8.8 Hz, 1 H), 7.31−7.39
(
+
m, 2 H), 7.01−7.09 (m, 2 H), 4.68 (d, J = 6.1 Hz, 2 H). LC-MS (ESI
+
) m/z = 266 [M + H] .
-Chloro-N-(thiophen-2-ylmethyl)pyridazine-3-carboxamide
48). General procedure D. Prepared from 43 (3.17 mmol) and
6
(
1
+
thiophen-2-ylmethanamine. Yield: 650 mg, 81%. H NMR (400 MHz,
H] .
CDCl ) δ = 9.09−8.98 (br s, 1 H), 8.60−8.53 (m, 1 H), 8.28 (d, J =
N-Benzyl-6-(piperazin-1-yl)pyridazine-3-carboxamide Hydro-
3
chloride (59). General procedure E. Prepared from 53 (7 mmol).
8
7
.8 Hz, 1 H), 7.72−7.64 (m, 2 H), 7.32 (d, J = 8.0 Hz, 1 H), 7.24−
1
Yield: 2.25 g, 96%. H NMR (400 MHz, DMSO-d ) δ = 9.61 (br s,
.17 (m, 1 H), 4.82 (d, J = 5.5 Hz, 2 H). LC-MS (ESI+) m/z = 254
6
+
2
H), 9.43 (t, J = 6.3 Hz, 1H), 7.95 (d, J = 9.5 Hz, 1H), 7.49 (d, J = 9.5
[
M + H] .
Hz, 1H), 7.36−7.28 (m, 4H), 7.27−7.20 (m, 1H), 4.50 (d, J = 6.3 Hz,
6
-Chloro-N-(prop-2-yn-1-yl)pyridazine-3-carboxamide (49). Gen-
eral procedure D. Prepared from 43 (3.17 mmol) and propargylamine.
Yield: 466 mg, 75%. H NMR (400 MHz, CDCl ) δ = 8.29 (d, J = 8.8
Hz, 1 H), 8.25−8.17 (br s, 1 H), 7.71 (d, J = 8.8 Hz, 1 H), 4.33 (dd, J
5.5, 2.5 Hz, 2 H), 2.31 (t, J = 2.5 Hz, 1 H). LC-MS (ESI+) m/z =
2
2
H), 4.04−3.93 (m, 4H), 3.27−3.16 (m, 4H). LC-MS (ESI+) m/z =
+
1
98 [M + H] .
3
(
1S,4S)-N-Benzyl-6-(2,5-diazabicyclo[2.2.1]heptan-2-yl)-
pyridazine-3-carboxamide Hydrochloride (60). General procedure E.
=
1
+
Prepared from 54 (0.8 mmol). Yield: 229 mg, 86%. H NMR (400
196 [M + H] .
MHz, DMSO-d ) δ = 9.36 (app t, J = 6.5 Hz, 2 H), 7.97 (d, J = 9.4 Hz,
Benzyl 6-Chloropyridazine-3-carboxylate (50). General procedure
D. Prepared from 43 (3.17 mmol) and benzyl alcohol. Reaction was
run in EtOAc and heated at 100 °C for 30 min in the microwave oven.
Same workup as in general procedure. Yield: 270 mg, 34%. LC-MS
ESI+) m/z = 249 [M + H] .
6
1
4
3
H), 7.39−7.13 (m, 6 H), 5.20−5.08 (m, 1 H), 4.57−4.53 (m, 1 H),
.51 (d, J = 6.4 Hz, 2 H), 3.94−3.81 (m, 1 H), 3.76−3.67 (m, 1 H),
.39−3.20 (m, 2 H), 2.24−2.13 (m, 1 H), 2.08−1.99 (m, 1 H). LC-
+
+
MS (ESI+) m/z = 310 [M + H] .
(
N-Benzyl-6-(1,4-diazepan-1-yl)pyridazine-3-carboxamide Hydro-
Prop-2-yn-1-yl 6-chloropyridazine-3-carboxylate (51). General
chloride (61). General procedure E. Prepared from 55 (0.8 mmol).
procedure D. Prepared from 43 (3.17 mmol) and propargyl alcohol.
Reaction was run in EtOAc and heated at 100 °C for 30 min in the
microwave oven. Same workup as in general procedure. Yield: 370 mg,
0%. LC-MS (ESI+) m/z = 197 [M + H] .
tert-Butyl 4-(6-(Benzylcarbamoyl)pyridazin-3-yl)piperazine-1-
carboxylate (53). General procedure A. Prepared from and 45 (8
mmol) and 34. The crude product was triturated with Et O to obtain
the title compound. Yield: 2.8 g, 87%. Used in the next step without
further purification. H NMR (400 MHz, CDCl ) δ = 8.24 (t, J = 5.7
1
Yield: 238 mg, 89%. H NMR (400 MHz, DMSO-d ) δ = 9.38 (app t,
6
J = 6.3 Hz, 2 H), 7.92 (d, J = 9.5 Hz, 1 H), 7.38 (d, J = 9.5 Hz, 1 H),
7.34−7.28 (m, 4 H), 7.26−7.18 (m, 1 H), 4.50 (d, J = 6.3 Hz, 2 H),
+
6
4
3
H] .
.15−4.05 (m, 2 H), 3.87−3.77 (m, 2 H), 3.33−3.24 (m, 2 H), 3.22−
.12 (m, 2 H), 2.17−2.08 (m, 2 H). LC-MS (ESI+) m/z = 312 [M +
+
2
N-(4-Fluorobenzyl)-6-(piperazin-1-yl)pyridazine-3-carboxamide
1
Hydrochloride (62). General procedure E. Prepared from 56 (1.2
3
+
mmol). Yield: 426 mg, 100%. LC-MS (ESI+) m/z = 316 [M + H] .
Hz, 1 H), 8.07 (d, J = 9.5 Hz, 1 H), 7.39−7.24 (m, 6 H), 6.99 (d, J =
Differential Scanning Fluorimetry (DSF). The assay buffer in
which dCTPase, inhibitors, and dye were diluted consisted of 100 mM
Tris-acetate pH 8.0, 100 mM KCl, 0.005% Tween 20, and 1 mM DTT.
The assay concentrations used were 33 μM inhibitor, 20 μM
recombinant human dCTPase, and Sypro Orange 5×; 1% final
concentration of DMSO. The assay volumes were 20 μL in 96-well Q-
PCR plates. A BioRad Q-PCR instrument was used to ramp the
temperature from 20 to 100 °C at 1 °C/min and analyzing
fluorescence intensity at each step. The Bio-Rad software was used
9
3
H] .
.5 Hz, 1 H), 4.68 (d, J = 6.0 Hz, 2 H), 3.80−3.71 (m, 4 H), 3.63−
.54 (m, 4 H), 1.52−1.47 (m, 9 H). LC-MS (ESI+) m/z = 398 [M +
+
(
1S,4S)-tert-Butyl-5-(6-(benzylcarbamoyl)pyridazin-3-yl)-2,5-
diazabicyclo[2.2.1]heptane-2-carboxylate (54). General procedure
A. Prepared from 45 (2 mmol) and (1S,4S)-(−)-2-Boc-2,5-
diazabicyclo[2.2.1]heptane. The crude product was triturated with
Et O to obtain the title compound. Yield: 362 mg, 44%. Used in the
next step without further purification. H NMR (400 MHz, CDCl ) δ
8.20 (t, J = 5.9 Hz, 1 H), 8.04 (d, J = 9.3 Hz, 1 H), 7.40−7.20 (m, 5
2
1
3
=
to calculate the T (negative) of each well of the plate.
m
17
DARTS Assay. HL60 cells were treated with 0.1% DMSO or 10
μM inhibitor in 0.1% DMSO for 4 h, media was aspirated, and the cells
were washed with ice-cold PBS. The cells were lysed in the
mammalian protein extraction lysis buffer M-PER (Thermo Scientific)
supplemented with 1× complete protease inhibitor cocktail (Roche)
and 1× Halt phosphatase inhibitor cocktail (Thermo Scientific) on ice
for 10 min. After centrifugation at 16000g at 4 °C, the supernatant
(cell lysate) was transferred to a new tube and kept on ice. Then 10×
TN buffer (500 mM Tris-HCl, pH 8.0, 500 mM NaCl) was added to
the lysate to make a final concentration of 1× TN buffer. Protein
concentration of the cell lysate was determined by Bradford method
H), 6.69 (d, J = 9.3 Hz, 1 H), 4.75−4.72 (m, 0.5 H), 4.67 (d, J = 6.0
Hz, 2 H), 4.62−4.60 (m, 0.5 H), 3.70−3.54 (m, 1 H), 3.54−3.34 (m, 3
H), 2.06−1.95 (m, 2 H), 1.47 (s, 4 H), 1.42 (s, 9 H). LC-MS (ESI+)
+
m/z = 410 [M + H] .
tert-Butyl 4-(6-(Benzylcarbamoyl)pyridazin-3-yl)-1,4-diazepane-
-carboxylate (55). General procedure A. Prepared from 45 (2
1
mmol) and tert-butyl 1,4-diazepane-1-carboxylate. The crude product
was triturated with Et O to obtain the title compound. Yield: 391 mg,
2
1
4
7%. Used in the next step without further purification. H NMR (400
MHz, CDCl ) δ = 8.16 (t, J = 5.9 Hz, 1 H), 7.97 (d, J = 9.5 Hz, 1 H),
3
7.33−7.17 (m, 5 H), 6.83 (d, J = 9.5 Hz, 1 H), 4.61 (d, J = 5.9 Hz, 2
L
DOI: 10.1021/acs.jmedchem.7b00182
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
with Coomassie Plus protein assay reagent and BSA as a protein
standard (Thermo Scientific). Pronase stock solution (10 mg/mL)
was diluted in TN buffer to achieve the final ratio of enzyme to total
protein 1:200 and 1:100. The cell lysate was split into aliquots, and 1
μL of the range of Pronase solutions was added and incubated with
shaking at room temperature for 30 min. For the nondigested (ND)
sample, 1 μL of TN buffer was added instead of protease. Each
digestion reaction was stopped by adding of 4× Laemmli loading
buffer with 200 mM DTT and heating at 95° for 5 min. The samples
were separated by SDS-PAGE and blotted onto nitrocellulose
membranes (Bio-Rad) using Trans-Blot Turbo transfer system (Bio-
Rad). A blot was first probed with rabbit anti-dCTPase antibody
*For T.H.: phone, 0046852480000; E-mail, thomas.helleday@
scilifelab.se.
ORCID
Sabin Llona-Minguez: 0000-0003-3187-722X
Nicholas C. K. Valerie: 0000-0002-9423-964X
Present Addresses
∇
̈
For A. Hoglund: Sprint BioScience AB, Huddinge, Sweden.
○
For A. Ghassemian: Faculty of Medicine, McGill University,
Montreal, Canada.
◆
̃
́ ́
For J. M. Calderon-Montano, E. B. Moron: Faculty of
(
produced in house), followed by incubation with antirabbit-HRP
Pharmacy, University of Seville, Seville, Spain.
¶
secondary antibody, and protein bands were visualized using
SuperSignal West Femto chemiluminescence substrate (Thermo
Scientific). Next, the blot was stripped with Restore Plus Western
Blot Stripping buffer (Thermo Scientific) and probed with rabbit anti-
GAPDH Ab (sc 257782), followed by incubation with donkey
antirabbit IRDye 800CW Ab (Li-COR), and images were taken with
Odyssey Fc imager (Li-COR). GAPDH was used as a loading and
digestion control.
For A. Mateus: European Molecular Biology Laboratory,
Heidelberg, Germany.
+
For K. Sigmundsson: Duke-NUS Graduate Medical School,
Singapore.
For T. Lundbac
and Early Development Biotech Unit, AstraZeneca, Mo
Sweden.
●
̈
k: Discovery Sciences, Innovative Medicines
lndal,
̈
SCD1 Index Assay. HepG2 cell line were grown in L-glutamine
rich RPMI-1640 (Life Technologies) supplemented with 10% FBS
Author Contributions
S.Ll.-M. and A.H. contributed equally to this work.
Notes
#
(
Gibco). Cultures were maintained at 37 °C in a humidified
atmosphere of 5% CO :air. Cells were seeded in duplicate sets of 6-
2
6
The authors declare no competing financial interest.
well plates at 1 × 10 per well. The cells were treated with either
vehicle DMSO (0.1%) or 74 (Cayman Chemicals) or inhibitors in 2%
FBS-supplemented media. Following 72 h incubation, culture medium
was removed and the cell monolayer was washed three times with cold
PBS and collected for cellular fatty acid measurement. Cells were
collected into glass vials, and total lipids were esterified with MeOH
ACKNOWLEDGMENTS
■
We acknowledge Dr. Adam Throup and Maghsod Shaaker for
insightful manuscript and scientific discussions. This project is
primarily supported by The Knut and Alice Wallenberg
Foundation. Further support was received from the Felix
Mindus Foundation for leukemia research, the Swedish
Research Council, the European Research Council, Goran
̈
Gustafsson Foundation, Swedish Cancer Society, the Swedish
23
catalyzed by acetyl chloride. Fatty acid methyl ester derivatives
formed from isolated cellular lipids were separated on a highly polar
biscyanopropyl polysiloxane capillary column (TR-CN100 60 mm ×
0
.25 mm × 0.2 μm film thickness, Teknokroma). The gas
chromatograph was a Buck Scientific model 610 equipped with a
split flow injector and a hydrogen flame ionization detector. Helium
was employed as the carrier gas. The oven temperature program was
from 190 to 210 °C with 1 °C/min, then isothermal continued for 10
min. Tridecanoic acid (13:0) (Sigma Chemicals) was used as internal
standard. Peak retention times were identified by injecting known fatty
acid methyl ester standards (Sigma Chemicals). PeakSimple software
was used for data acquisition and processing.
Children’s Cancer Foundation, the Swedish Pain Relief
Foundation, and the Torsten and Ragnar Soderberg
̈
Foundation. Chemical Biology Consortium Sweden was
supported by the Swedish Research Council. We also
acknowledge the Faculty of Medicine at Tabriz University of
Medical Sciences for providing research facilities and
ChemAxon (http://www.chemaxon.com) for technical sup-
port. We are grateful to the Protein Science Facility at
Karolinska Institutet for purification of proteins.
Other Assays. Antibodies, purification of human recombinant
dCTPase, malachite green screening assay, NTPase/NUDIX hydro-
lases selectivity assays, cell culture, CETSA assay, viability assays/
combination index, and flow cytometry analysis were performed as
9
,24
described previously.
Intracellular bioavailability (F ) and molec-
ABBREVIATIONS USED
-AzaC, 5-azacytidine; dCTP, deoxycytidine triphosphate;
dNTP, deoxynucleoside triphosphate; NUDIX, nucleoside
diphosphate linked to X; XTP3TPA, XTP3-transactivated
protein A
ic
0,25
■
1
ular docking were performed as described previously.
5
ASSOCIATED CONTENT
■
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.7b00182.
REFERENCES
■
(1) Requena, C. E.; Perez-Moreno, G.; Ruiz-Perez, L. M.; Vidal, A.
E.; Gonzalez-Pacanowska, D. The NTP pyrophosphatase DCTPP1
contributes to the homoeostasis and cleansing of the dNTP pool in
human cells. Biochem. J. 2014, 459, 171−180.
HTS output overview, chemical structures of 5-Aza and
74, DSF assay data, NTPase/NUDIX selectivity data,
intracellular bioavailability data, BJ hTERT cell viability
data (PDF)
Homology model (PDB)
(2) Requena, C. E.; Perez-Moreno, G.; Horvath, A.; Vertessy, B. G.;
Ruiz-Perez, L. M.; Gonzalez-Pacanowska, D.; Vidal, A. E. The
nucleotidohydrolases DCTPP1 and dUTPase are involved in the
cellular response to decitabine. Biochem. J. 2016, 473, 2635−2643.
Molecular formula strings (CSV)
(
3) Zhang, Y.; Ye, W. Y.; Wang, J. Q.; Wang, S. J.; Ji, P.; Zhou, G. Y.;
Zhao, G. P.; Ge, H. L.; Wang, Y. dCTP pyrophosphohydrase exhibits
nucleic accumulation in multiple carcinomas. Eur. J. Histochem. 2013,
AUTHOR INFORMATION
■
*
5
(
7, 29.
4) Morisaki, T.; Yashiro, M.; Kakehashi, A.; Inagaki, A.; Kinoshita,
Corresponding Authors
For S.LL.-M.: phone, 0046852480000; E-mail, sabin.llona.
minguez@scilifelab.se.
H.; Fukuoka, T.; Kasashima, H.; Masuda, G.; Sakurai, K.; Kubo, N.;
Muguruma, K.; Ohira, M.; Wanibuchi, H.; Hirakawa, K. Comparative
M
DOI: 10.1021/acs.jmedchem.7b00182
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
proteomics analysis of gastric cancer stem cells. PLoS One 2014, 9,
e110736.
affinity responsive target stability (DARTS). Proc. Natl. Acad. Sci. U. S.
A. 2009, 106, 21984−21989.
(
5) Song, F. F.; Xia, L. L.; Ji, P.; Tang, Y. B.; Huang, Z. M.; Zhu, L.;
(18) Mateus, A.; Matsson, P.; Artursson, P. Rapid measurement of
intracellular unbound drug concentrations. Mol. Pharmaceutics 2013,
10, 2467−2478.
Zhang, J.; Wang, J. Q.; Zhao, G. P.; Ge, H. L.; Zhang, Y.; Wang, Y.
Human dCTP pyrophosphatase 1 promotes breast cancer cell growth
and stemness through the modulation on 5-methyl-dCTP metabolism
and global hypomethylation. Oncogenesis 2015, 4, e159.
(19) Chou, T. C. Drug combination studies and their synergy
quantification using the Chou-Talalay method. Cancer Res. 2010, 70,
440−446.
(
6) Xia, L. L.; Tang, Y. B.; Song, F. F.; Xu, L.; Ji, P.; Wang, S. J.; Zhu,
J. M.; Zhang, Y.; Zhao, G. P.; Wang, Y.; Liu, T. T. DCTPP1 attenuates
the sensitivity of human gastric cancer cells to 5-fluorouracil by up-
regulating MDR1 expression epigenetically. Oncotarget 2016, 7,
(20) Meingassner, J. G.; Aschauer, H.; Winiski, A. P.; Dales, N.;
Yowe, D.; Winther, M. D.; Zhang, Z. H.; Stutz, A.; Billich, A.
Pharmacological inhibition of stearoyl CoA desaturase in the skin
induces atrophy of the sebaceous glands. J. Invest. Dermatol. 2013, 133,
2091−2094.
(21) Igal, R. A. Stearoyl CoA desaturase-1: new insights into a central
regulator of cancer metabolism. Biochim. Biophys. Acta, Mol. Cell Biol.
Lipids 2016, 1861, 1865−1880.
6
8623−68627.
7) Corson, T. W.; Cavga, H.; Aberle, N.; Crews, C. M. Triptolide
directly inhibits dCTP pyrophosphatase. ChemBioChem 2011, 12,
767−1773.
8) Kambe, T.; Correia, B. E.; Niphakis, M. J.; Cravatt, B. F. Mapping
(
1
(
the protein interaction landscape for fully functionalized small-
molecule probes in human cells. J. Am. Chem. Soc. 2014, 136,
(22) Liu, G.; Lynch, J. K.; Freeman, J.; Liu, B.; Xin, Z.; Zhao, H.;
Serby, M. D.; Kym, P. R.; Suhar, T. S.; Smith, H. T.; Cao, N.; Yang, R.;
Janis, R. S.; Krauser, J. A.; Cepa, S. P.; Beno, D. W.; Sham, H. L.;
Collins, C. A.; Surowy, T. K.; Camp, H. S. Discovery of potent,
selective, orally bioavailable stearoyl-CoA desaturase 1 inhibitors. J.
Med. Chem. 2007, 50, 3086−3100.
1
(
0777−10782.
9) Llona-Minguez, S.; Hoglund, A.; Jacques, S. A.; Johansson, L.;
Calderon-Montano, J. M.; Claesson, M.; Loseva, O.; Valerie, N. C.;
Lundback, T.; Piedrafita, J.; Maga, G.; Crespan, E.; Meijer, L.; Burgos
Moron, E.; Baranczewski, P.; Hagbjork, A. L.; Svensson, R.; Wiita, E.;
Almlof, I.; Visnes, T.; Jeppsson, F.; Sigmundsson, K.; Jensen, A. J.;
Artursson, P.; Jemth, A. S.; Stenmark, P.; Warpman Berglund, U.;
Scobie, M.; Helleday, T. Discovery of the first potent and selective
inhibitors of human dCTP pyrophosphatase 1. J. Med. Chem. 2016, 59,
(
23) Lepage, G.; Roy, C. C. Direct transesterification of all classes of
lipids in a one-step reaction. J. Lipid Res. 1986, 27, 114−120.
24) Gad, H.; Koolmeister, T.; Jemth, A. S.; Eshtad, S.; Jacques, S. A.;
(
Strom, C. E.; Svensson, L. M.; Schultz, N.; Lundback, T.; Einarsdottir,
B. O.; Saleh, A.; Gokturk, C.; Baranczewski, P.; Svensson, R.;
Berntsson, R. P. A.; Gustafsson, R.; Stromberg, K.; Sanjiv, K.; Jacques-
Cordonnier, M. C.; Desroses, M.; Gustavsson, A. L.; Olofsson, R.;
Johansson, F.; Homan, E. J.; Loseva, O.; Brautigam, L.; Johansson, L.;
Hoglund, A.; Hagenkort, A.; Pham, T.; Altun, M.; Gaugaz, F. Z.;
Vikingsson, S.; Evers, B.; Henriksson, M.; Vallin, K. S. A.; Wallner, O.
A.; Hammarstrom, L. G. J.; Wiita, E.; Almlof, I.; Kalderen, C.;
Axelsson, H.; Djureinovic, T.; Puigvert, J. C.; Haggblad, M.; Jeppsson,
F.; Martens, U.; Lundin, C.; Lundgren, B.; Granelli, I.; Jensen, A. J.;
Artursson, P.; Nilsson, J. A.; Stenmark, P.; Scobie, M.; Berglund, U.
W.; Helleday, T. MTH1 inhibition eradicates cancer by preventing
sanitation of the dNTP pool. Nature 2014, 508, 215−221.
1
(
140−1148.
10) Llona-Minguez, S.; Hoglund, A.; Wiita, E.; Almlof, I.; Mateus,
A.; Calderon-Montano, J. M.; Cazares-Korner, C.; Homan, E.; Loseva,
O.; Baranczewski, P.; Jemth, A. S.; Haggblad, M.; Martens, U.;
Lundgren, B.; Artursson, P.; Lundback, T.; Jenmalm Jensen, A.;
Warpman Berglund, U.; Scobie, M.; Helleday, T. Identification of
triazolothiadiazoles as potent inhibitors of the dCTP pyrophosphatase
1
(
. J. Med. Chem. 2017, 60, 2148−2154.
11) Itaya, K.; Ui, M. A new micromethod for the colorimetric
determination of inorganic phosphate. Clin. Chim. Acta 1966, 14,
61−366.
12) Abad-Zapatero, C.; Metz, J. T. Ligand efficiency indices as
guideposts for drug discovery. Drug Discovery Today 2005, 10, 464−
69.
13) Zhang, Z. H.; Sun, S. Y.; Kodumuru, V.; Hou, D. J.; Liu, S. F.;
3
(
(25) Mateus, A.; Matsson, P.; Artursson, P. A high-throughput cell-
based method to predict the unbound drug fraction in the brain. J.
Med. Chem. 2014, 57, 3005−3010.
4
(
Chakka, N.; Sviridov, S.; Chowdhury, S.; McLaren, D. G.; Ratkay, L.
G.; Khakh, K.; Cheng, X.; Gschwend, H. W.; Kamboj, R.; Fu, J. M.;
Winther, M. D. Discovery of piperazin-1-ylpyridazine-based potent
and selective stearoyl-CoA desaturase-1 inhibitors for the treatment of
obesity and metabolic syndrome. J. Med. Chem. 2013, 56, 568−583.
(
14) Carron, C. P.; Trujillo, J. I.; Olson, K. L.; Huang, W.; Hamper,
B. C.; Dice, T.; Neal, B. E.; Pelc, M. J.; Day, J. E.; Rohrer, D. C.; Kiefer,
J. R.; Moon, J. B.; Schweitzer, B. A.; Blake, T. D.; Turner, S. R.;
Woerndle, R.; Case, B. L.; Bono, C. P.; Dilworth, V. M.; Funckes-
Shippy, C. L.; Hood, B. L.; Jerome, G. M.; Kornmeier, C. M.;
Radabaugh, M. R.; Williams, M. L.; Davies, M. S.; Wegner, C. D.;
Welsch, D. J.; Abraham, W. M.; Warren, C. J.; Dowty, M. E.; Hua, F.
M.; Zutshi, A.; Yang, J. Z.; Thorarensen, A. Discovery of an oral potent
selective inhibitor of hematopoietic prostaglandin D synthase
(
HPGDS). ACS Med. Chem. Lett. 2010, 1, 59−63.
(
15) Lo, M.-C.; Aulabaugh, A.; Jin, G.; Cowling, R.; Bard, J.;
Malamas, M.; Ellestad, G. Evaluation of fluorescence-based thermal
shift assays for hit identification in drug discovery. Anal. Biochem.
2
(
004, 332, 153−159.
16) Molina, D. M.; Jafari, R.; Ignatushchenko, M.; Seki, T.; Larsson,
E. A.; Dan, C.; Sreekumar, L.; Cao, Y.; Nordlund, P. Monitoring drug
target engagement in cells and tissues using the cellular thermal shift
assay. Science 2013, 341, 84−87.
(
17) Lomenick, B.; Hao, R.; Jonai, N.; Chin, R. M.; Aghajan, M.;
Warburton, S.; Wang, J.; Wu, R. P.; Gomez, F.; Loo, J. A.;
Wohlschlegel, J. A.; Vondriska, T. M.; Pelletier, J.; Herschman, H.
R.; Clardy, J.; Clarke, C. F.; Huang, J. Target identification using drug
N
DOI: 10.1021/acs.jmedchem.7b00182
J. Med. Chem. XXXX, XXX, XXX−XXX