Design and Synthesis of Poly(ADP-ribose) Polymerase Inhibitors: Impact of Adenosine Pocket-Binding Motif Appendage to the 3-Oxo-2,3-dihydrobenzofuran-7-carboxamide on Potency and Selectivity

Article abstract of DOI:10.1021/acs.jmedchem.8b01709

Poly(adenosine 5′-diphosphate-ribose) polymerase (PARP) inhibitors are a class of anticancer drugs that block the catalytic activity of PARP proteins. Optimization of our lead compound 1 ((Z)-2-benzylidene-3-oxo-2,3-dihydrobenzofuran-7-carboxamide; PARP-1 IC₅₀ = 434 nM) led to a tetrazolyl analogue (51, IC₅₀ = 35 nM) with improved inhibition. Isosteric replacement of the tetrazole ring with a carboxyl group (60, IC₅₀ = 68 nM) gave a promising new lead, which was subsequently optimized to obtain analogues with potent PARP-1 IC₅₀ values (4-197 nM). PARP enzyme profiling revealed that the majority of compounds are selective toward PARP-2 with IC₅₀ values comparable to clinical inhibitors. X-ray crystal structures of the key inhibitors bound to PARP-1 illustrated the mode of interaction with analogue appendages extending toward the PARP-1 adenosine-binding pocket. Compound 81, an isoform-selective PARP-1/-2 (IC₅₀ = 30 nM/2 nM) inhibitor, demonstrated selective cytotoxic effect toward breast cancer gene 1 (BRCA1)-deficient cells compared to isogenic BRCA1-proficient cells.

Full text of DOI:10.1021/acs.jmedchem.8b01709

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Article
Design and Synthesis of Poly(ADP-ribose) polymerase Inhibitors:
Impact of Adenosine Pocket-Binding Motif Appendage to the 3-
Oxo-2,3-dihydrobenzofuran-7-carboxamide on Potency and Selectivity
Uday Kiran Velagapudi, Marie-France Langelier, Cristina Delgado-Martin, Morgan E Diolaiti,
Sietske Bakker, Alan Ashworth, Bhargav A Patel, Xuwei Shao, John M. Pascal, and Tanaji T Talele
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01709 • Publication Date (Web): 01 May 2019
Downloaded from http://pubs.acs.org on May 2, 2019
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Design and Synthesis of Poly(ADP-ribose)
polymerase Inhibitors: Impact of Adenosine Pocket-
Binding Motif Appendage to the 3-Oxo-2,3-
dihydrobenzofuran-7-carboxamide on Potency and
Selectivity
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†
‡
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Uday Kiran Velagapudi , Marie-France Langelier , Cristina Delgado-Martin , Morgan E.
┴
┴
┴,$
†§
†
‡
Diolaiti , Sietske Bakker , Alan Ashworth , Bhargav A. Patel , Xuwei Shao , John M. Pascal ,
Tanaji T. Talele†*
†Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's
University, Queens, New York 11439, United States
‡Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal H3T
1
J4 Canada
┴UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San
Francisco, California 94158, United States
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$Department of Medicine, University of California, San Francisco, California 94158, United
States
ABSTRACT: Poly(ADP-ribose) polymerase (PARP) inhibitors are a class of anticancer drugs
that block the catalytic activity of PARP proteins. Optimization of our lead compound 1 ((Z)-2-
benzylidene-3-oxo-2,3-dihydrobenzofuran-7-carboxamide; PARP-1 IC50 = 434 nM) led to a
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tetrazolyl analogue (51, IC = 35 nM) with improved inhibition. Isosteric replacement of the
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tetrazole ring with a carboxyl group (60, IC = 68 nM) gave a promising new lead, which was
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subsequently optimized to obtain analogues with potent PARP-1 IC values (4 nM – 200 nM).
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PARP enzyme profiling revealed that the majority of compounds are selective toward PARP-2
with IC values comparable to clinical inhibitors. X-ray crystal structures of the key inhibitors
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bound to PARP-1 illustrated the mode of interaction with analogue appendages extending toward
the PARP-1 adenosine-binding pocket. Compound 81, an isoform-selective PARP-1/-2 (IC = 30
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nM/2 nM) inhibitor, demonstrated selective cytotoxic effect toward BRCA1-deficient cells
compared to isogenic BRCA1–proficient cells.
INTRODUCTION
Exogenous and endogenous genotoxic injuries lead to DNA single strand breaks (SSBs) and
DNA double strand breaks (DSBs), which collectively trigger the DNA damage response (DDR)
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in cells. In cancer cells, SSBs occur at a frequency of up to 10,000 per cell each day. While SSBs
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are repaired by base excision repair (BER), repair of DSBs requires a functional homologous
recombination (HR) or non-homologous end joining (NHEJ) repair mechanism.3, 4 Computational
analyses indicated the involvement of approximately 400 proteins in the regulation of the DDR
process.5, 6 Poly(ADP-ribose) polymerase-1 (PARP-1) plays an important role in BER-mediated
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DNA damage repair as well as other pathways by binding to the damaged DNA through the
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coordinated action of its N-terminal zinc finger motifs. The C-terminal catalytic site of PARP-1
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hydrolyzes NAD substrate into ADP-ribose and nicotinamide (NI). Branched and linear chains
of ADP-ribose units are covalently transferred onto a wide range of target proteins such as DNA
polymerases, histones, DNA ligases, p53 and topoisomerase I/II (heteromodification), and onto
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PARP itself (automodification). Thus, PARP-1 acts as a “writer” of poly (ADP-ribosylation)
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(
PARylation). PARylation has been shown to play a role in cellular processes such as DNA
damage repair, maintaining genomic stability, regulation of transcription, and cell death.10, 11
PARylation of PARP-1 is necessary for non-covalent recruitment of DNA repair proteins,
including DNA ligase III, DNA polymerase β (pol β) and XRCC1 to the sites of DNA breaks.12-14
PARylation of PARP-1 is also thought to promote its dissociation from DNA damage sites to allow
repair.15, 16 Therefore, targeting PARP-1 with small molecule inhibitors is an attractive strategy to
enhance antitumor effect.17-23
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Synthetic lethality is a strategy that exploits gene defects in cancer for therapeutic benefit. The
foremost example of synthetic lethality as a targeted cancer therapy is the use of PARP inhibitors
in the treatment of cancer in individuals with germline mutations in BRCA1 or BRCA2. In addition
to blocking the catalytic activity of PARP proteins, some PARP inhibitors (niraparib, olaparib,
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rucaparib and talazoparib) act at least in part by trapping PARP on damaged DNA. This trapping
interferes with DNA replication causing double stranded breaks that cannot be repaired in HR-
defective tumor cells. PARP-1 inhibitors as single agents are, therefore, efficacious in treating
tumors deficient in HR components, including BRCA1/2, but are of limited utility in tumors with
normal or restored function of HR repair mechanism.25-29 Consequently, the use of FDA approved
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PARP-1 inhibitors such as olaparib, niraparib, rucaparib and talazoparib has mainly focused
on their therapeutic role as a monotherapy to treat BRCA-deficient tumors based on the concept of
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synthetic lethality. These drugs and another PARP-1 inhibitor, veliparib, are also currently
undergoing advanced clinical trials as combination and/or single agents in cancer therapy (Figure
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1). Clinical PARP-1 inhibitors are also useful for the treatment of other cancers with DNA DSB
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repair deficiency such as those with BRCAness. Taking these factors into consideration, PARP-1
remains an attractive target for anticancer drug development.
In this article, we report the design, synthesis, structure-activity relationship (SAR), and in vitro
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evaluation of our previously published lead compound 1, thereby leading to the identification of
several unique PARP-1 inhibitors (Figure 2A and 2B). Compound 1 binds to the NI pocket of the
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PARP-1 catalytic fold. Based on previous structural data, we hypothesized that installation of a
4’-carboxyl group in compound 1 is an ideal vector to facilitate the incorporation of a wide range
of substituents (pyridine, pyrimidine, pyrazine, 1,3,5-triazine, 1,3,4-thiadiazole, 5,6,7,8-
tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine (THTP), and benzimidazole) directed toward engaging
the adenosine-binding pocket (ABP) of PARP-1 with the goal of developing PARP-1 inhibitors
with improved potency and a unique mode of engaging the PARP-1 active site. Indeed, we show
that these new compounds act as potent inhibitors of PARP-1 and PARP-2 with desirable (low
nM) IC values. Key target compounds showed high selectivity toward PARP-1 and PARP-2 over
50
other catalytic PARP isoforms and specifically inhibited growth of BRCA1-mutant cells, thus
providing refined leads for further optimization to produce preclinical candidates.
RESULTS AND DISCUSSION
Chemistry.
Benzaldehyde derivatives and the other intermediates as precursors to the synthesis of target
compounds were prepared according to Schemes 1-4.
Synthesis of Benzaldehyde Intermediates 2-8 (Scheme 1).
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Scheme 1 represents the synthesis of substituted benzaldehydes. For the synthesis of 4-phenyl
or 4-thiazol-2-yl benzaldehydes 2 and 3, reported Suzuki coupling conditions were utilized.38, 39
The [2+3] cycloaddition reaction of 4-cyanobenzaldehyde with sodium azide in the presence of
triethylamine produced tetrazolyl derivative 4.40 An alternate procedure (sodium
azide/diethylamine hydrochloride/toluene)41 was used for conversion of 4-cyano-3-
fluorobenzaldehyde and 4-cyano-2-methoxybenzaldehyde to corresponding tetrazolyl substituted
benzaldehydes 5 and 6 because the conditions used for the synthesis of 4 proved unsuccessful. N-
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Methyl derivative 7 was prepared from benzaldehyde 4 using iodomethane.42 Further, S Ar
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reaction of 4-fluorobenzaldehyde with pyrimidin-2-yl-piperazine yielded benzaldehyde derivative
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8. These benzaldehyde intermediates (2-8) and the other commercially available benzaldehydes
(see experimental) served as precursors for the synthesis of target compounds shown in Table 1.
Synthesis of Benzaldehyde Intermediates 9-17 and 20-28 (Scheme 2).
Benzaldehyde intermediates 9-17 were prepared by coupling commercially available N4-
substituted piperazines with commercially available 4-carboxybenzaldehyde in the presence of
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HCTU, HOBt and N,N-diisopropylethylamine (DIPEA). Ester 18 was synthesized by S Ar
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reaction on methyl 2-chloropyrimidinyl-5-carboxylate using N4-Boc protected piperazine as the
nucleophile.37, 45 The Boc protection was then removed with 4N HCl in dioxane to obtain the
hydrochloride salt 19. Piperazinyl derivative 19 was next coupled with 4-carboxybenzaldehyde in
the presence of HCTU, HOBt and DIPEA to produce benzaldehyde intermediate 20.
Benzaldehydes 21-24 were prepared using commercially available N4-substituted piperazines and
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- or 4-carboxybenzaldehyde in the presence of previously mentioned peptide coupling conditions.
Similarly, intermediates 25 and 26 were prepared by coupling commercially available piperidine
or aminopiperidine with 4-carboxybenzaldehyde. Intermediate 27 was obtained by reacting
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commercially available 4-formylbenzene sulfonyl chloride with pyrimidin-2-yl-piperazine in the
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presence of triethylamine. Intermediate 28 was obtained via an S 2 reaction using commercially
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available 4-bromomethyl benzaldehyde and pyrimidin-2-yl-piperazine.37 These benzaldehyde
intermediates were used for the preparation of target compounds shown in Table 2.
Synthesis of Benzaldehyde Intermediates 29-41 (Scheme 3).
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Scheme 3 depicts the preparation of benzaldehyde intermediates 29-41, which were utilized to
synthesize target compounds listed in Tables 3 and 4. The 3-carboxy or 4-carboxy benzaldehydes
were coupled with commercially available (un)substituted THTPs to obtain intermediates 29-36
or (un)substituted benzimidazole-2-yl-ethylamines for intermediates 37-41 in the presence of
HCTU/HOBt coupling conditions.44
Synthesis of Intermediate Amines 43, 43a and 47 (Scheme 4).
Scheme 4 represents the synthesis of key amine intermediates as precursors to obtain target
compounds shown in Table 4. The 2,3-diaminobenzamide or methyl 2,3-diaminobenzoate were
condensed with benzyl 3-oxopropylcarbamate leading to cyclized intermediates 42 or 42a, which
were subjected to hydrogenolysis to obtain amines 43 or 43a. To synthesize piperazine
intermediate 47, ortho-phenylenediamine was reacted with 1,1′-carbonyldiimidazole to generate a
cyclic urea compound 44, followed by a chlorination reaction using neat POCl to obtain 2-
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chlorobenzimidazole 45. The chloro group in compound 45 was then replaced via microwave
assisted S Ar reaction by N-Boc piperazine to obtain 46 and a subsequent Boc-deprotection using
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N HCl/dioxane mixture gave 47 as a di-hydrochloride salt.
Preparation of Target Compounds 48-105 (Scheme 5).
Target compounds were synthesized using the key intermediate 3-oxo-2,3-dihydrobenzofuran-
7-carboxamide (I) (Supporting Information, Scheme S1 for details regarding synthesis of I).
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Scheme 5 depicts the synthesis of target compounds 48-84 and 91-97, using modified Knoevenagel
condensation reaction of intermediate I with various synthesized or commercially obtained
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benzaldehydes. Target compounds 85-90, 98, 100-102 and 104 were prepared by coupling 60
with commercially obtained amines whereas target compounds 99, 103 and 105 were respectively
prepared using synthesized amines 43, 47 and 43a in the presence of HCTU/HOBt coupling
conditions.
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Structure-Activity Relationship.
The present SAR study is based on our lead compound 1 ((Z)-2-benzylidene-3-oxo-2,3-
dihydrobenzofuran-7-carboxamide), which showed PARP-1 inhibitory activity at a sub-
micromolar concentration (IC = 434 nM) in PARP-1 enzyme assay. We began optimizing lead
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to obtain a new and potent PARP-1 inhibitory series of dihydrobenzofuran-7-carboxamide
(DHBF) compounds. This lead optimization program led to four innovative SAR phases as
outlined in Tables 1-4. All newly synthesized compounds, along with positive controls, olaparib
and veliparib, were tested using PARP-1 and PARP-2 chemiluminescence assay to obtain IC and
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pIC values (Supporting Information Figure S1 and S2 indicate PARP-1 and PARP-2 IC dose-
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response curves of representative compounds). Our IC values for the positive controls, olaparib
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and veliparib, were comparable to the reported values.30, 34
From each of the four SAR studies, X-ray crystal structures were determined for key target
compounds in complex with the minimal ADP-ribosyltransferase (ART) fold of human PARP-1,
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representing a constitutively active form of PARP-1. ART interaction with NAD is primarily
mediated through a NI-binding site and ABP. Attached to the ART fold is an autoinhibitory helical
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domain (HD) that acts to selectively block access to substrate NAD by interfering with the ABP
(Figure 3A).46, 47 Upon binding to damaged DNA, the N-terminal regulatory domains of PARP-1
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assemble in a way that leads to unfolding of the HD, thus relieving autoinhibition and allowing for
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binding of NAD to the active site.
Our initial binding analysis of the newly synthesized
compounds by differential scanning fluorimetry (DSF) revealed that most of these compounds
were unable to bind to the catalytic domain (CAT) in the presence of a folded HD, but they
successfully bound to the CAT with the deleted helical domain (CATHD) (Figure 3B). These
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compounds thus behave similar to the NAD analogue benzamide adenine dinucleotide (BAD),
which can only bind to PARP-1 when the HD is deleted, or unfolded in the presence of DNA
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damage. This biophysical study suggests that our compounds may display increased efficacy and
cancer cell specificity while showing reduced cytotoxicity in normal cells. Moreover, the binding
analysis indicated that the designed extensions to compound 1 were likely to engage the ABP.
Based on these data, the CATHD construct was used for crystallographic analysis. Five structures
were determined using X-ray diffraction data extending to resolutions between 1.5 to 2.2 Å
(Supporting Information Table S1 and Figure S3 and Figure 4). Each of the bound inhibitors
exhibited hydrogen bonding interactions with key amino acid residues in the NI-binding site such
as Ser904 and Gly863 and a water-mediated hydrogen bond with the catalytic residue Glu988. The
crystal structures helped to understand the molecular basis of PARP-1 inhibition and allowed us
to rationalize the observed SAR trends, as described in the sections below.
Exploration of para-/meta-Aryl/Heteroaryl Substituted Benzylidene Analogues of Lead 1
(SAR 1).
Table 1 represents various substituents at C2-position of DHBF scaffold (I, see Scheme 5). The
initial optimization efforts were mainly focused on exploring simplified substitution of various
aryl/heteroaryl ring systems at the para-position of the benzylidene moiety present in lead 1. A
biphenyl analogue 48 failed to show any PARP-1 inhibition at the tested concentration of 50 nM.
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Therefore, we replaced the para-phenyl ring with various saturated and unsaturated heterocyclic
rings with the intention of capturing hydrogen bonding and/or ionic interactions within the ABP
of PARP-1. Amongst the unsaturated heterocycles (49-52) such as thiazole, 1,2,4-triazole, 2H-
tetrazole, and 1H-pyrazole, only the tetrazolyl analogue, 51, gave a promising enzyme inhibition
with an IC value of 35 nM. Encouragingly, 51 showed ~12-fold improvement in inhibition as
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compared to lead 1. The 4-position was optimal for the tetrazole ring as moving it to the meta-
position of the benzylidene moiety (53) proved detrimental to the activity. Next, the distal
aromatic/heteroaromatic ring in the above-mentioned analogues was replaced with basic saturated
heterocycle such as substituted piperazine. The N-methylpiperazine analogue 54, showed loss of
activity as compared to 51. Replacing the N-methylpiperazine of 54 with bulky and hydrophobic
N-benzylpiperazine and pyrimidin-2-yl-piperazine yielded compounds 55 and 56; however, both
proved inferior to 51. Having established 51 as the best inhibitor from this series, we conducted a
limited SAR on 51 by introducing electron-withdrawing 3-fluoro (57, IC = 56 nM) and electron-
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donating 2-methoxy (58, IC = 47 nM) substituents, both of which gave a degree of PARP-1
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inhibition comparable to that of 51. X-ray crystallographic analysis revealed favorable localization
of the tetrazole moiety of a representative analogue 57 in the vicinity of active site residue Arg865
as shown in Figure 4A. This observation was also corroborated by the detrimental result obtained
by replacing the acidic tetrazole proton with a methyl group as observed in 59.
Since the tetrazole ring is not amenable for further chemical optimization, we sought to
determine if the tetrazole ring can be isosterically replaced with a carboxyl group, because the
carboxyl derivative could further be efficiently derivatized to improve potency similar to what has
been done during the development of olaparib.30 Toward this goal, we prepared a 4-
carboxybenzylidene analogue (60, IC50 = 68 nM) and noted appreciable inhibitory potency,
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suggesting this compound would serve as a refined lead for subsequent SAR studies. To further
confirm the role of acidic group at the para-position of a benzylidene moiety, we replaced the
carboxyl group with a cyano substituent and as expected, it showed decreased activity as compared
to 60 (data not shown). We found that replacement of a carboxyl group in 60 with a primary
carboxamide group resulted in the retention of activity (data not shown), which prompted us to
explore further SAR using various alicyclic amines such as N4-heteroaryl substituted piperazines,
un(substituted) THTPs and un(substituted) benzimidazoles with various linkers.
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Exploration of the Vector Addressing ABP of PARP-1 with Heteroaryl
Piperazine/Piperidine Motifs (SAR 2).
Compounds displayed in Table 2 were designed to extend carboxyl group of the newly identified
lead 60 to capture additional interactions within the ABP of PARP-1. The activities of analogues
in this series were also compared to compound 51, which was the best compound from SAR 1. To
obtain more potent compounds, first we coupled the pendant carboxyl group in 60 with an N-
phenylpiperazine moiety to obtain 61, which showed a detrimental effect on activity compared to
6
0. We next replaced the hydrophobic phenyl ring with polar isosteric heteroaromatic rings to
obtain potent inhibitors. For example, pyridin-2-yl (62), pyrimidin-2-yl (63), and pyrazin-2-yl (64)
derivatives showed tolerance for heteroaryl-piperazine extensions with IC s ranging from 55 nM
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to 77 nM. X-ray crystal structure of 63 bound to CATHD PARP-1 demonstrated extension of
pyrimidine moiety in the vicinity of Asn868 and the ABP residue Arg878 as shown in Figure 4B.
The 1,3,5-triazin-2-yl (65) and 5-trifluoromethyl-1,3,4-thiadiazol-2-yl (66) analogues were found
inferior compared to 63. Because pyridin-2-yl (62) and pyrimidin-2-yl (63) analogues gave
appreciable enzyme inhibition, we decided to determine the influence of electron-withdrawing
groups (67, 68, 70, and 71) or electron-donating group (69) on pyridine or pyrimidine rings. While
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-trifluoromethylpyridin-2-yl analogue (67) was inferior, the 3-cyanopyridin-2-yl analogue (68,
IC = 66 nM) was as active as unsubstituted analogue 62. Amongst small to large substituents, 4-
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methoxy substitution on pyrimidine ring (IC50 = 66 nM) showed appreciable inhibition as
evidenced from analogue 69, whereas, analogues 70 and 71 with an ester and methoxymethyl
oxadiazolyl substitutions led to a moderate inhibition. Having established a favorable role of
heteroaryl substituted piperazine motifs at the para-position of the benzylidene, we next
determined whether these motifs could be tolerated at the meta-position. Toward this objective,
we made meta-counterparts of 63, 69, and 64 to obtain 72-74, which were not well tolerated except
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for analogue 72 (IC = 58 nM). To investigate the significance of a carbonyl group in the
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disposition of a pyrimidin-2-yl-piperazine moiety in 63, compounds 75 and 76 were prepared.
Compound 75, a non-classical rigid sulfone isostere, showed ~3.5-fold decreased enzyme
inhibition as compared to 63. Similarly, compound 76, a non-classical flexible methylene isostere,
also led to decreased activity. These results underscore the contribution of a carbonyl group in
directing the pyrimidin-2-yl-piperazine moiety toward the amino acid residues located within ABP
(Figure 4B). Next, we sought to explore the role of the piperazine ring in 63 by replacing it with
a 4-aminopiperidine ring (77, IC = 112 nM) and found that this substitution resulted in two-fold
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loss of activity. We replaced the piperazine linker in 63 with a piperidine linker to obtain 78, which
also showed a detrimental effect on potency as compared to 63 indicating the requirement of
terminal piperazine ring nitrogen for an improved inhibition. Because substitutions on pyrimidin-
2-yl- or pyridin-2-yl-piperazines failed to give us better enzyme inhibition than analogue 51, we
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decided to generate a new series with a fused bicyclic ring system containing sp N atoms with the
expectation of obtaining potent inhibitors.
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Exploration of THTP Amides Linked to the meta- or para-Position of Benzylidene Moiety
SAR 3).
(
SAR 3 optimization involved extension of carboxyl group toward ABP of PARP-1 by coupling
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with THTP as a rigid isostere of pyrimidinylpiperazine moiety as shown in Table 3. Unsubstituted
THTP analogue (79) gave appreciable enzyme inhibition (IC = 97 nM). To enhance productive
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interactions with residues from ABP of PARP-1, we inserted functional groups at the 3-position
of THTP ring with varying molecular size and electronic properties such as electron neutral
(methyl, isopropyl, cyclopropylmethyl, cyclopentyl and -CH OH), electron-withdrawing (-CHF ,
2 2
-
CF , -COOEt, 3-flurobenzyl, and N-methyl imidazole) and pi-electron donor (cyclopropyl). These
3
efforts led to a series of target compounds (80-90, and 92) with improved inhibitory profile as
compared to SAR 2 analogues. The C3-methyl substitution on THTP (80) resulted in a slight
improvement in enzyme inhibition as compared to 79. Similarly, C3-trifluoromethyl analogue (81)
gave a 3-fold improvement in the activity as compared to unsubstituted analogue 79 and methyl
analogue 80. The C3-ethyl ester analogue (82, IC = 40 nM) was also well tolerated, which
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indicates the favorable contribution of moderately sized electron-withdrawing groups at C3-
position of THTP. Based on the recent review on versatile role of a cyclopropyl ring in medicinal
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chemistry, next we added a cyclopropyl group at C3-position to obtain analogue 83 (IC = 27
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nM) with the best enzyme inhibition of the THTP series. X-ray structure of 83 bound to CATHD
PARP-1 revealed localization of a cyclopropyl ring in the vicinity of Asn868 and ABP residue
Arg878 (Figure 4C). Increasing the steric bulk and hydrophobicity at C3-position of THTP with
meta-fluorobenzyl substituent yielded 84 with unfavorable enzyme inhibition. Replacing the
trifluoromethyl group at C3-position of THTP in 81 by a difluoromethyl group (85, IC = 30 nM)
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was well tolerated. Insertion of a methylene bridge between the C3 of a THTP ring and a
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cyclopropyl ring in 83 gave 86 (IC = 47 nM) with considerable retention of the activity of 83. A
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polar C3-hydroxymethyl substituent (87) proved to be a weak inhibitor. Substitution of an
isopropyl group, noncyclic isostere of a cyclopropyl ring, at C3-position of THTP (88, IC = 42
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nM) was well tolerated. Replacement of a cyclopropyl ring in 83 with cyclopentyl (89, IC = 37
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nM) or 1-methylimidazol-4-yl (90, IC50 = 32 nM) also showed comparable potency to that
observed for 83. Based on the inhibition profile of various substitutions at C3-position of THTP,
it is evident that smaller substituents with electronegative property improve inhibition by
interacting with polar residues in ABP of PARP-1.
The evaluation of the effect of moving THTP from para- to the meta-position led to two
representative analogues. For example, meta-version of 79 led to a loss of activity as exemplified
by 91. However, meta-version of 81 produced 92 (IC = 42 nM) with retention of inhibitory
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activity. In summary, this SAR study revealed favorable impact of THTP scaffold on PARP-1
inhibition as compared to the lead compounds 51 and 60.
Exploration of Benzimidazoles with Various Linkers as ABP Motifs, Coupled to the meta-
or para-Position of Benzylidene Moiety (SAR 4).
Table 4 shows the extension of a carboxyl group of 60 for ligand occupancy within the ABP of
PARP-1. Carboxyl group of 60 was subjected to coupling with various (un)substituted
benzimidazolyl ethylamines as novel ABP-motifs. An initial lead from this series, compound 93
(
IC = 36 nM) with no substituent on the benzimidazole ring, had set the stage for obtaining potent
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PARP-1 inhibitors. Further SAR work on lead 93 involved exploration of different substituents on
the benzimidazole ring with electronic properties such as electron neutral (CH ) and electron
3
withdrawing (F) groups as exemplified by 94 (IC = 22 nM) and 95 (IC = 51 nM), respectively.
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As anticipated, moving benzimidazole ethylamine moiety in 93 and 95 to the meta-position
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produced 96 (IC = 88 nM) and 97 (IC = 97 nM) with a 2-fold decreased potency. Introducing
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electron donor methoxy group at 5-position of the benzimidazole moiety led to 98 (IC = 28 nM)
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with comparable activity to analogue 93. These results conclude that 4-position of the benzylidene
moiety is the ideal vector to access and produce productive interactions within ABP of PARP-1.
Replacement of the benzimidazole ring in 93 with benzimidazole-4-carboxamide led to 99 (IC =
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nM) with 9-fold improvement in potency compared to 93. Further SAR involved modification
of the ethyl linker and, toward this goal, we synthesized analogues with a gem-dimethyl50
substitution (100) which led to a slight decrease in activity. Replacement of the ethyl linker in 93
with the propyl linker produced 101 with marginally decreased activity compared to 93.
Finally, we explored the impact of replacing flexible ethylamine linker with azetidine, piperazine
or piperidine linkers to limit the conformational flexibility and allow for entropically favorable
binding within the ABP of PARP-1. These efforts led to the synthesis of azetidine analogue 102
(IC = 30 nM), piperazine analogue 103 (IC = 18 nM) and piperidine analogue 104 (IC = 58
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nM) amongst which 103 exhibited the best inhibition. Since benzimidazole-4-carboxamide in 99
serves as an excellent NI mimic, we decided to elucidate whether DHBF-7-carboxamide or
benzimidazole-4-carboxamide binds to the NI site. Toward this objective, we synthesized a methyl
ester analogue 105 (PARP-1 IC50 = 98 nM) and observed a 25-fold decrease in activity as
compared to 99, and thus, validated the switched positioning of benzimidazole-4-carboxamide and
DHBF-7-carboxamide, respectively, within NI and ABP of PARP-1 active site. Further
biophysical characterization of 99 and 105 will confirm above-mentioned observation. In
summary, SAR 4 revealed potent analogues such as 99 and 103 with 108- and 24-fold increase in
activity, respectively, as compared to lead 1.
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X-ray crystal structures of 93 and 103 in complex with CATHD PARP-1 revealed that the
benzimidazole portion was directed toward Arg878 of ABP (Figure 4D and 4E). Additionally,
compound 93 exhibited pi-pi stacking and hydrogen bonding interactions with the side chain of
Tyr889.
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Investigation of PARP-2 Enzyme Inhibition by Selected Target Compounds.
PARP-2, via physical interaction or PARylation of various target proteins, plays an important
51
-/-
role in a wide range of cellular processes that are dysregulated in tumorigenesis. PARP-2 mice
are highly sensitive to alkylating agents as well as ionizing radiation.52, 53 Further, both PARP-1
and PARP-2 are required for efficient BER as evidenced from global decrease in PARP activity
54
upon PARP-2 depletion. Because the C-terminal catalytic domains of PARP-1 and PARP-2
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exhibit ~69% homology, it is not surprising that clinically utilized PARP inhibitors potently
inhibit both PARP-1 and PARP-2 (Figure 1). We, therefore, conducted a screen of highly active
PARP-1 inhibitors against PARP-2 as shown in Tables 1-4. The tetrazolyl and piperazinyl
analogues 51, 53, and 56-58 from SAR 1 (Table 1) demonstrated potent PARP-2 inhibition (IC50
=
2.1 nM, 76%, 56%, 100% at 50 nM and IC = 1.6 nM, respectively). Compound 58 showed a
50
29-fold greater potency against PARP-2 as compared to PARP-1 and its PARP-2 IC , was
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comparable to that observed for olaparib (PARP-2 IC = 0.5 nM). PARP-2 inhibition profiles of
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representative compounds 63, 64, 69, 71-74 and 76 from SAR 2 (Table 2) also showed greater
selectivity toward PARP-2 compared to PARP-1 as evidenced by 51-98% inhibition of PARP-2
at 50 nM concentration. We further investigated the inhibition profile of THTP analogues, from
SAR 3 (Table 3), in PARP-2 enzyme assay at 10 nM concentration. Compound 51 displayed potent
PARP-2 inhibition (IC = 2.1 nM) and it was used for comparison in Tables 3 and 4. Compounds
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81 (IC = 2 nM) and 83 (IC = 1.9 nM) both displayed high potency against PARP-2 with IC s
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comparable to that of olaparib and with a 15- and 14-fold selectivity, respectively, as compared to
PARP-1 inhibition. Compounds 80, 84 and 87 inhibited PARP-2 by 55-61% at 10 nM
concentration and thus demonstrate higher potency toward PARP-2 as compared to PARP-1.
Analogues 82, 85, 86, and 88-90 also exhibited potent inhibition of PARP-2 with IC values
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ranging from 3 - 4.6 nM. PARP-2 screening of compounds 93, 98, and 101 from Table 4 at 10 nM
concentration showed 44-50% inhibition. Compounds 94 (IC = 5 nM), 99 (IC = 0.7 nM) and
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03 (IC = 4 nM) also exhibited potent PARP-2 inhibition and moderate selectivity toward PARP-
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over PARP-1. Modest PARP-2 inhibitory activity was observed for the meta-analogues 96 and
7. Overall, most of these compounds exhibited selectivity toward PARP-2, which is a common
trend for the FDA approved drugs olaparib, niraparib, rucaparib and talazoparib. However, the
extent of the selectivity for the compounds toward PARP-2, in the current study, is greater than
that observed for the clinically used PARP inhibitors.
PARP-Isoform Profiling for Selected Target Compounds.
Discovery of isoform-selective inhibitors is at the forefront of medicinal chemistry and chemical
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biology research. Because the majority of clinically validated PARP-1 inhibitors show a wide
spectrum of inhibitory activity toward catalytically active PARP-isoforms,57, 58 we obtained
selectivity profiles of our four best PARP-1 inhibitors (81, 83, 99 and 103) at 500 nM concentration
against a panel of six catalytic PARP-isoforms (Figure 5). Based on this data, compounds 81 and
8
3 will serve as high affinity chemical probes for PARP-1 and PARP-2 without interfering with
other catalytic PARP-isoforms (PARP-3, TNKS1, TNKS2, PARP-8, PARP-10 and PARP-14).
Further evaluation of compound 81 at 1 µM concentration against above mentioned catalytic
PARP-isoforms led to minimal inhibition (12%, 5%, 16%, 34%, 4% and 9%, respectively). Thus,
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compound 81 demonstrated >33 and 500-fold selectivity toward PARP-1 and PARP-2 compared
to the other catalytic PARP-isoforms.
Since compounds 99 and 103 showed significant inhibition of anticancer targets TNKS1 and
TNKS2 at 500 nM concentration, we obtained their IC values (Table 5). Compound 99 inhibited
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TNKS1 and TNKS2 with IC values of 6.3 nM and 8.8 nM, respectively, which was comparable
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to TNKS-selective analogue XAV939. Compound 99 thus inhibits clinically significant isoforms
of PARP (PARP-1, PARP-2, TNKS1 and TNKS2) with low nM IC values (Table 5). Compound
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03, however, moderately inhibited TNKS1 and TNKS2 with 131 nM and 198 nM IC values,
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respectively (Supporting Information, dose-response curves of 99 and 103 toward TNKS1 and
TNKS2). It may be concluded that a bicyclic ring system attached to a flexible linker is important
for the inhibition of TNKS1 and TNKS2 as evidenced from flexible analogue 99 and rigid
analogues 81 and 103. Inhibition data of 81 against PARP-1, PARP-2, TNKS1 and TNKS2 is also
shown in Table 5 for comparison.
Investigation of the Cellular Activity of PARP Inhibitors in BRCA1-mutant Cells.
Extensive preclinical and clinical data have established that loss of BRCA1 or BRCA2 is
associated with increased sensitivity to small molecule inhibitors targeting PARP-1/-2 (PARPi).23
To determine whether compounds 81 and 83, the most selective PARP-1/-2 inhibitors of the series,
demonstrate specific cytotoxicity, we tested them in a pair of isogenic BRCA1-deficient and -
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proficient SUM149 breast cancer cell lines. We found that BRCA1-mutant cells are >10-fold
more sensitive to both compounds 81 and 83 when compared to BRCA1-proficient cells (Figure
6
A, 6B and Supporting Information Figure S4). This differential sensitivity was similar to
talazoparib or olaparib treatment (Figure 6C and 6D). This finding suggests that compounds 81
and 83 have PARP-specific cytotoxicity in the context of BRCA1 loss.
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CONCLUSIONS
A series of dihydrobenzofuran-7-carboxamides was designed, starting from the X-ray crystal
structure of moderately active lead 1 (Z-2-benzylidene-3-oxo-2,3-dihydrobenzofuran-7-
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37
carboxamide, PARP-1 IC = 434 nM) in complex with a full length multi-domain PARP-1. In
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this study, four different SARs were explored at the meta- or para-position of the benzylidene
portion of lead 1 to identify effective adenosine-binding motifs. For example, the 4-tetrazole motif
yielded analogues with PARP-1 IC50 values of 35 nM - 56 nM. The pyridinyl/pyrimidinyl
piperazine motifs displayed IC values ranging from 55 nM - 197 nM. Modifications on THTP
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motif demonstrated IC values of 27 nM - 97 nM and benzimidazolyl ethylamine/piperazine
50
/azetidine/piperidine motifs also gave desirable IC50s in the range of 4 nM - 98 nM. Additionally,
most of the compounds in the series were PARP-2 selective and their IC s were similar to
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clinically utilized PARP inhibitors as exemplified by compounds with <5 nM IC s against PARP-
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2. Differential scanning fluorimetry (DSF) of selected compounds from each of the SARs revealed
that these compounds are unable to bind to the CAT domain in the presence of a folded helical
domain; however, they efficiently bound to the CAT with the helical domain deleted (CATHD).
Therefore, we propose that these compounds can bind to PARP-1 either with HD deleted or
unfolded potentially as a result of DNA damage. X-ray crystal structures of selected compounds
from four different SARs in complex with CATΔHD PARP-1 provided insights into the binding
mechanism and will form the basis for optimization efforts in the future. Compounds 81 and 83
showed selective inhibition of PARP-1 and PARP-2 over other catalytic PARP-isoforms such as
PARP-3, TNKS1, TNKS2, PARP-8, PARP-10, and PARP-14. Compound 99 exhibited single digit
nM IC values against clinically significant PARP-isoforms (PARP-1, PARP-2, TNKS1 and
50
TNKS2). PARP-isoform selective compounds 81 and 83 demonstrated BRCA1-dependent
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cytotoxic effect in the SUM149 cell line suggesting that they are promising lead compounds for
further optimization.
EXPERIMENTAL
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Chemical Synthesis.
Materials and Instrumentation. All chemicals were procured from Accela Chembio (San
Diego, CA), Aldrich Chemical Co. (Milwaukee, WI), Alfa Aesar (Ward Hill, MA), Arkpharm,
Inc. (Arlington Heights, IL), Chem-Impex Int. Inc. (Wood Dale, IL), Combi-Blocks Inc. (San
Diego, CA), Enamine LLC (Monmouth Jct., NJ), Oakwood Products (West Columbia, SC),
Oxchem Corporation (Wood Dale, IL), Synthonix (Wake Forest, NC) and were used without
additional purification. Qualitative analysis of reactions was performed by thin layer
chromatography (TLC) with silica gel G as the adsorbent (250 microns) on aluminum backed
plates (Agela Technologies) and Ultraviolet (UV) light at 254 nm or 365 nm for visualization
1
TM
purposes. H NMR experiments were performed using a Bruker 400 Ultrashield spectrometer
(1
H at 400 MHz and C at 100 MHz) equipped with a z-axis gradient probe. H NMR chemical
13
1
shifts were reported downfield from tetramethylsilane (TMS as an internal standard) in parts per
1
million (δ ppm) for majority of the intermediates and all the target compounds. The H NMR data
are depicted as: chemical shift (multiplicity s (singlet), bs (broad singlet), d (doublet), t (triplet),
dd (doublet of doublets), ddd (doublet of doublets of doublets), dq (doublet of quartets), dt (doublet
of triplets), tt (triplet of triplets), td (triplet of doublets), h (hextate), m (multiplet), qd (quartet of
doublets), number of protons and coupling constant). Column chromatography purifications were
performed using silica gel (40-63 µm) purchased from Silicycle Inc. (Quebec City, CANADA)
and flash chromatography was conducted using Reveleris® X2 flash chromatography system
(BUCHI Corporation, New Castle, DE). Preparative TLC was performed using Silica Gel GF 1000
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µm 20x20 cm glass backed plates procured from Analtech (Miles Scientific, Newark, DE). Purity
analysis for target compounds 48-78 and mass analysis of all the target compounds was performed
on an Agilent 1260 infinity series liquid chromatography (LC) system connected with Agilent
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120 quadrupole mass spectrometer (MS) (Agilent, Santa Clara, CA). Purity analysis of
compounds 79-105 were carried out using Agilent 1260 infinity series HPLC system (Agilent,
Santa Clara, CA). Purity and mass analysis was performed using Agilent Eclipse plus C18, 3.5
μm, 4.6 mm × 100 mm column and the runs were monitored at 254 nm. All target compounds were
analyzed to be ≥ 95% pure (based on major peak area/total area of combined peaks). Acetonitrile
(ACN) and water (0.1% formic acid) mixtures were used as mobile phase for purity analysis of
compounds 48-78. For analogues 48 and 52, a 12 min gradient run was performed with 30 – 70%
ACN in water. For analogue 50, a gradient run was performed with 60 – 40% ACN in water over
8
min. For analogues 51, 56, 57, 60, 61, 63 – 75, 77 and 78, a gradient run was performed with 40
– 60% ACN in water over 8 min. For analogue 54, an 8 min isocratic run was performed with 60%
ACN in water. The flow rate was 0.5 mL/min for analysis of all the above-mentioned target
compounds. For mass analysis of compounds 79-92, an 8 min gradient run of 70-90% ACN in
water was used with a flow rate of 0.5 mL/min and for compounds 93-105, the flow rate was
increased to 1 mL/min. For the purity analysis of compounds 49, 53, 58, 59, 62, 76, 79-105, ACN
(0.1% DEA) and water (0.1% DEA) combination was used as the mobile phase. A gradient run
with 10% ACN to 90% ACN in water over 8 min (flow rate of 1 mL/min) was used as the mobile
phase. The elemental analyses (C, H, and N) were carried out by Atlantic Microlabs, Inc.,
(Norcross, GA), and the observed values were within ± 0.4% of the calculated values.
Synthesis. Procedures for synthesizing the key intermediate I and conditions for Knoevenagel
condensation to obtain the target compounds were adapted from our previously reported work.37
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Target compounds obtained via Knoevenagel condensation were either washed with methanol and
water, thereby resulting in pure compounds or were purified using chromatographic techniques
such as preparative TLC or flash chromatography.
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General Procedure for Suzuki Coupling Reaction (A). Reactions were performed using
conditions reported in previously published studies.38, 39
[1,1'-Biphenyl]-4-carbaldehyde (2). Intermediate 2 was obtained by Suzuki coupling
(procedure A) of 4-formylphenylboronic acid (500 mg, 3.34 mmol) with bromobenzene (0.35 mL,
3
.34 mmol), as a pale yellow solid (485 mg, 80% yield).61 1H NMR (400 MHz, CDCl ; TMS) δ
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.93 (s, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 8.2 Hz, 2H), 7.54 – 7.48 (m, 5H).
4-(Thiazol-2-yl)benzaldehyde (3). Intermediate 3 was synthesized using procedure A and 4-
formylphenylboronic acid (500 mg, 3.34 mmol) and 2-chlorothiazole (0.29 mL, 3.34 mmol), as a
1
pale brown solid (424 mg, 67% yield); H NMR (400 MHz, DMSO-d ; TMS) δ 10.11 (s, 1H), 8.13
6
(d, J = 7.8 Hz, 2H), 8.01 (d, J = 3.1 Hz, 1H), 7.97 (d, J = 7.6 Hz, 2H), 7.90 (d, J = 3.2 Hz, 1H).
4
-(2H-Tetrazol-5-yl)benzaldehyde (4). To a solution of 4-formylbenzonitrile (1 g, 7.63 mmol)
dissolved in N,N-dimethylformamide (DMF), triethylamine (2.13 mL, 15.25 mmol) was added
with subsequent addition of sodium azide (1.49 g, 22.88 mmol) and ensuing reaction mixture was
heated to 180ºC for overnight. The reaction mixture was then vacuum dried on a rotary evaporator
to remove majority of DMF. The resulting crude mixture was then partitioned between 1N aqueous
HCl and ethyl acetate and the organic layer was collected and further extracted 3X with brine to
remove the residual DMF from the organic layer. Later, ethyl acetate layer was dried over MgSO4,
filtered and evaporated to obtain brown solid, which was suspended in ethyl acetate and washed
1
with ethyl acetate to yield pure compound 4 as a cream colored solid (897 mg, 67% yield). H
NMR (400 MHz, DMSO-d ) δ 10.11 (s, 1H), 8.31 (d, J = 7.3 Hz, 2H), 8.13 (d, J = 7.3 Hz, 2H).
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General Procedure for Preparation of Substituted 2H-tetrazol-5-yl benzaldehyde
Intermediates (B). These intermediates were prepared by slightly modifying reported
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1
procedure, wherein sodium azide and diethylamine hydrochloride were added to a solution of
the appropriate benzonitrile in toluene. The reaction mixture was then allowed to reflux in an inert
condition for a period of 24 h. Thereafter, toluene was evaporated and subsequently extracted with
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X 1N aqueous HCl and ethyl acetate. Ethyl acetate layer was then dried over MgSO and
4
evaporated to obtain the crude benzaldehyde derivative that was purified by flash chromatography.
-Fluoro-4-(2H-tetrazol-5-yl)benzaldehyde (5). Intermediate 5 was obtained using the general
3
procedure B, by reacting 2-fluoro-4-formylbenzonitrile (500 mg, 3.35 mmol) with sodium azide
(371 mg, 5.7 mmol) and diethylamine hydrochloride (625mg, 5.7 mmol), as a white solid (173
1
mg, 27% yield). H NMR (400 MHz, DMSO-d ) δ 10.09 (s, 1H), 8.32 (t, J = 7.4 Hz, 1H), 8.03 –
6
7
.95 (m, 2H).
2-Methoxy-4-(2H-tetrazol-5-yl)benzaldehyde (6). Intermediate 6 was prepared using the
general procedure B, by reacting 3-methoxy-4-formylbenzonitrile (500 mg, 3.10 mmol), sodium
azide (343 mg, 5.27 mmol) and diethylamine hydrochloride (578 mg, 5.27 mmol) as a white solid
1
(
520 mg, 82% yield); H NMR (400 MHz, DMSO-d ) δ 10.40 (s, 1H), 7.93 – 7.83 (m, 2H), 7.75
6
(dt, J = 8.0, 1.1 Hz, 1H), 4.04 (s, 3H).
4
-(2-Methyl-2H-tetrazol-5-yl)benzaldehyde (7). Intermediate 7 was obtained by reacting
4
2
tetrazole intermediate 4 (250 mg, 0.57 mmol), as described in a reported procedure, as a yellow
1
solid (196 mg, 73% yield). H NMR (400 MHz, DMSO-d ; TMS) δ 10.10 (s, 1H), 8.29 (d, J = 8.3
6
Hz, 2H), 8.10 (d, J = 8.0 Hz, 2H), 4.47 (s, 3H).
4
-(4-(Pyrimidin-2-yl)piperazin-1-yl)benzaldehyde (8). Intermediate 8 was obtained using a
4
3
reported procedure by reacting 4-fluorobenzaldehyde (1 g, 8.06 mmol) with pyrimidin-2-yl-
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piperazine (2.16 g, 8.06 mmol) and potassium carbonate (2.23 g, 16.11 mmol), as a white solid
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(
1.575 g, 73% yield). H NMR (400 MHz, CDCl ; TMS) δ 10.05 (s, 1H), 8.32 (d, J = 4.8 Hz, 2H),
3
7.95 (d, J = 8.2 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 6.55 (t, J = 4.7 Hz, 1H), 4.00 – 3.75 (m, 6H),
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.53 – 3.39 (m, 2H)
General Procedure for Peptide Coupling Reactions (C). To a suspension of appropriate
carboxylic acid [(3-carboxy or 4-carboxy benzaldehyde or 60) 1 eq] in dichloromethane, HCTU
1.5 eq) and HOBt (1.5 eq) were added and the temperature was brought down to 0ºC while the
(
reaction was stirring. To this mixture, DIPEA was added (2 eq) and the resultant mixture was left
stirring at 0ºC for 15 min. Subsequently, the amine (1.1 eq) was added as such or by dissolving in
a minimum volume of dichloromethane (for amines which were liquids at rt) to the reaction
mixture and the reaction was stirred at rt for overnight. The reaction was then diluted with DCM
and washed 3X with small portions of water. Resultant organic phase was dried over MgSO4,
filtered and evaporated to yield crude coupled products, which were either used as obtained or
purified by flash chromatography using gradient DCM and methanol combinations as the mobile
phase, wherein the concentration of methanol in dichloromethane was varied from 1-8% based on
the nature of product to be purified.
4
-(4-Phenylpiperazine-1-carbonyl)benzaldehyde (9). Intermediate 9 was prepared using the
general procedure C, where 4-formylbenzoic acid (500 mg, 3.33 mmol) was reacted with 1-
1
phenylpiperazine (594 mg, 3.66 mmol), as a brown solid (724 mg, 74% yield). H NMR (400
MHz, CDCl ; TMS) δ 10.01 (s, 1H), 7.92 (d, J = 7.9 Hz, 2H), 7.57 (d, J = 7.8 Hz, 2H), 7.27 (t, J
3
=
7.8 Hz, 2H), 6.96 – 6.81 (m, 3H), 4.01 – 3.86 (m, 2H), 3.60 – 3.46 (m, 2H), 3.31 – 3.18 (m, 2H),
3
.18 – 3.03 (m, 2H).
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-(4-(Pyridin-2-yl)piperazine-1-carbonyl)benzaldehyde (10). Intermediate 10 was
synthesized using general procedure C, by reacting 4-formylbenzoic acid (500 mg, 3.33 mmol)
with pyridin-2-yl-piperazine (598 mg, 3.66 mmol), as a brown oil (638 mg, 65% yield) that was
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used as such in the next step; H NMR (400 MHz, CDCl ; TMS) δ 10.02 (s, 1H), 8.20 – 8.13 (m,
3
1
1
H), 7.92 (dd, J = 8.2, 2.1 Hz, 2H), 7.57 (dd, J = 8.3, 2.0 Hz, 2H), 7.49 (ddd, J = 10.6, 6.5, 2.0 Hz,
H), 6.65 (dd, J = 7.5, 4.8 Hz, 2H), 4.02 – 3.82 (m, 2H), 3.66 – 3.43 (m, 6H).
4
-(4-(Pyrimidin-2-yl)piperazine-1-carbonyl)benzaldehyde (11). Intermediate 11 was
obtained by using general procedure C, where 4-formylbenzoic acid (500 mg, 3.33 mmol) was
treated with pyrimidin-2-yl-piperazine (602 mg, 3.66 mmol), as an off-white solid after flash
1
purification (838 mg, 85% yield); H NMR (400 MHz, DMSO-d ; TMS) δ 10.08 (s, 1H), 8.39 (d,
6
J = 4.8 Hz, 2H), 8.00 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 7.9 Hz, 2H), 6.68 (t, J = 4.7 Hz, 1H), 3.96 –
3
.64 (m, 6H), 3.45 – 3.30 (m, 2H).
4-(4-(Pyrazin-2-yl)piperazine-1-carbonyl)benzaldehyde (12). 12 was synthesized by using 4-
formylbenzoic acid (500 mg, 3.33 mmol), pyrazin-2-yl-piperazine (602 mg, 3.66 mmol) and the
general procedure C, as a brown oil that was used as such without any purification for subsequent
1
synthesis; H NMR (400 MHz, CDCl ; TMS) δ 10.04 (s, 1H), 8.20 – 8.14 (m, 1H), 8.13 – 8.05 (m,
3
1
2
H), 7.96 (d, J = 7.9 Hz, 2H), 7.87 (d, J = 2.7 Hz, 1H), 7.61 (d, J = 7.9 Hz, 2H), 3.98 – 3.83 (m,
H), 3.78 – 3.66 (m, 2H), 3.65 – 3.49 (m, 4H).
4
-(4-(1,3,5-Triazin-2-yl)piperazine-1-carbonyl)benzaldehyde (13). Aldehyde 13 was
prepared using the general procedure C, where 4-formylbenzoic acid (500 mg, 3.33 mmol) was
reacted with triazin-2-yl-piperazine (605 mg, 3.66 mmol), as a brown oil (738 mg, 75% yield) that
1
was as such subjected to the next step; H NMR (400 MHz, CDCl ; TMS) δ 10.04 (s, 1H), 8.52 (s,
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H), 7.98 (d, J = 7.9 Hz, 2H), 7.63 (d, J = 7.9 Hz, 2H), 4.05 – 3.94 (m, 2H), 3.92 – 3.80 (m, 4H),
.55 – 3.44 (m, 2H).
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4-(4-(5-(Trifluoromethyl)-1,3,4-thiadiazol-2-yl)piperazine-1-carbonyl)benzaldehyde (14).
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Aldehyde 14 was prepared by using general procedure C, where 4-formylbenzoic acid (500 mg,
3
3
.33 mmol) was treated with 2-(piperazin-1-yl)-5-(trifluoromethyl)-1,3,4-thiadiazole (503 mg,
.66 mmol), as a pale yellow solid (838 mg, 85% yield), after evaporating the organic layer and
1
washing the crude solid with ethyl acetate; H NMR (400 MHz, DMSO-d ; TMS) δ 10.08 (s, 1H),
6
8
.02 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.1 Hz, 2H), 3.89 – 3.41 (m, 8H).
-(4-(3-(Trifluoromethyl)pyridin-2-yl)piperazine-1-carbonyl)benzaldehyde
Intermediate 15 was prepared via general procedure C by using 4-formylbenzoic acid (500 mg,
4
(15).
3
.33 mmol) and 1-(3-(trifluoromethyl)pyridin-2-yl)piperazine (847 mg, 3.66 mmol) as a dark
1
brown oil (757 mg, 63% yield), which was directly used as obtained for subsequent synthesis; H
NMR (400 MHz, CDCl ; TMS) δ 10.06 (s, 1H), 8.48 (dd, J = 5.0, 1.9 Hz, 1H), 8.00 – 7.96 (m,
3
2
H), 7.93 (dd, J = 7.8, 1.9 Hz, 1H), 7.65 – 7.58 (m, 2H), 7.18 – 7.10 (m, 1H), 3.99 – 3.90 (m, 2H),
3
.60 – 3.52 (m, 2H), 3.39 – 3.31 (m, 2H), 3.26 – 3.21 (m, 2H).
2
-(4-(4-Formylbenzoyl)piperazin-1-yl)nicotinonitrile (16). Aldehyde 16 was prepared using
the general procedure C, where 4-formylbenzoic acid (500 mg, 3.33 mmol) was allowed to react
with 2-(piperazin-1-yl)nicotinonitrile (689 mg, 3.66 mmol) as a brown oil (638 mg, 60% yield)
1
that was directly used as obtained for subsequent synthesis; H NMR (400 MHz, CDCl ; TMS) δ
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10.06 (s, 1H), 8.38 (dd, J = 4.9, 2.0 Hz, 1H), 7.97 (d, J = 7.8 Hz, 2H), 7.84 (dd, J = 7.7, 2.1 Hz,
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H), 7.60 (d, J = 7.8 Hz, 2H), 6.89 (dd, J = 7.6, 4.9 Hz, 1H), 4.01 – 3.92 (m, 2H), 3.85 – 3.77 (m,
H), 3.68 – 3.62 (m, 2H), 3.62 – 3.52 (m, 2H).
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-(4-(4-Methoxypyrimidin-2-yl)piperazine-1-carbonyl)benzaldehyde (17). Aldehyde 17
was synthesized by using 4-formylbenzoic acid (250 mg, 1.67 mmol) and 4-methoxy-2-(piperazin-
1-yl)pyrimidine (356 mg, 1.83 mmol) as per the general procedure C as a dark yellow oil, which
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was used as obtained for subsequent synthesis; H NMR (400 MHz, CDCl ; TMS) δ 10.04 (s, 1H),
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.00 (dd, J = 23.9, 6.7 Hz, 3H), 7.63 (d, J = 7.8 Hz, 2H), 6.03 (d, J = 5.7 Hz, 1H), 3.99 – 3.90 (m,
2
H), 3.90 – 3.77 (m, 7H), 3.52 – 3.43 (m, 2H).
Methyl
2-(4-(tert-butoxycarbonyl)piperazin-1-yl)pyrimidine-5-carboxylate
(18).
Intermediate 18 was prepared according to reported procedures.37, 45 To a solution of N-Boc
piperazine (270 mg, 1.45 mmol) in acetonitrile, potassium carbonate (400 mg, 2.9 mmol) and
methyl 2-chloropyrimidine-5-carboxylate (250 mg, 1.45 mmol) were added and the suspension
was allowed to reflux for overnight. Subsequently, the solvent was evaporated and the residue was
subjected to extraction with ethyl acetate and water. The organic phase was then dried over MgSO4
and was further vacuum dried to obtain the N-Boc intermediate 18 as an off-white solid (413 mg,
1
8
8% yield). H NMR (400 MHz, CDCl ; TMS) δ 8.86 (s, 2H), 3.99 – 3.91 (m, 4H), 3.90 (s, 3H),
3
3
.59 – 3.46 (m, 4H), 1.51 (s, 9H).
Methyl 2-(piperazin-1-yl)pyrimidine-5-carboxylate hydrochloride (19). Intermediate 19 was
prepared by using the N-Boc piperazine 18 (413 mg, 1.28 mmol) and dissolving it in dioxane,
followed by lowering the temperature of the reaction to 0ºC, using ice. Further, 4N aqueous
solution of HCl (4.7 mL, 12.81 mmol) was added drop wise and the reaction mixture was stirred
at rt for overnight. The solvent was then evaporated and the resultant semi-solid mass was triturated
with a small amount of methanol to obtain a white suspension, which was filtered and dried to
1
obtain 19 (278 mg, 88% yield) as a hydrochloride salt. H NMR (400 MHz, D O) δ 8.70 (s, 2H),
2
4.05 – 3.98 (m, 4H), 3.77 (s, 3H), 3.30 – 3.24 (m, 4H).
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Methyl 2-(4-(4-formylbenzoyl)piperazin-1-yl)pyrimidine-5-carboxylate (20). Intermediate
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0 was synthesized using general procedure C and by reacting 4-formylbenzoic acid (125 mg, 0.83
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mmol) with intermediate 19 (237 mg, 0.92 mmol) as a white solid (185 mg, 63% yield); H NMR
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(
400 MHz, DMSO-d ; TMS) δ 10.08 (s, 1H), 8.82 (s, 2H), 8.01 (d, J = 7.9 Hz, 2H), 7.68 (d, J =
6
7
.9 Hz, 2H), 4.10 – 3.68 (m, 9H), 3.51 – 3.37 (m, 2H).
-(4-(4-(5-(Methoxymethyl)-1,2,4-oxadiazol-3-yl)pyrimidin-2-yl)piperazine-1-
4
carbonyl)benzaldehyde (21). Aldehyde 21 was prepared using the general procedure C, where 4-
formylbenzoic acid (130 mg, 0.87 mmol) was allowed to react with 5-(methoxymethyl)-3-(2-
(
piperazin-1-yl)pyrimidin-4-yl)-1,2,4-oxadiazole (250 mg, 0.95 mmol) as a cream colored solid
1
(189 mg, 53% yield), by washing the crude solid with ethyl acetate; H NMR (400 MHz, CDCl ;
3
TMS) δ 10.07 (s, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 8.1 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H),
7
.46 (d, J = 8.4 Hz, 2H), 4.79 (s, 2H), 4.12 – 3.97 (m, 2H), 3.97 – 3.79 (m, 4H), 3.60 – 3.47 (m,
5H).
3
-(4-(Pyrimidin-2-yl)piperazine-1-carbonyl)benzaldehyde (22). Intermediate 22 was
prepared by using general procedure C, where 3-formylbenzoic acid (500 mg, 3.33 mmol) was
treated with pyrimidin-2-yl-piperazine (602 mg, 3.66 mmol) as a dark yellow oil (812 mg, 82%
1
yield) that was directly used for subsequent synthesis without additional purification; H NMR
(
400 MHz, CDCl ; TMS) δ 10.05 (s, 1H), 8.33 (d, J = 4.7 Hz, 2H), 8.03 – 7.92 (m, 2H), 7.74 (d, J
3
=
7.6 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 6.58 (t, J = 4.7 Hz, 1H), 4.02 – 3.76 (m, 6H), 3.61 – 3.46
(m, 2H).
3
-(4-(4-Methoxypyrimidin-2-yl)piperazine-1-carbonyl)benzaldehyde (23). Aldehyde 23
was synthesized by using 4-formylbenzoic acid (250 mg, 1.67 mmol) and 4-methoxy-2-(piperazin-
1-yl)pyrimidine (356 mg, 1.83 mmol) and as per the general procedure C as a brown oil (338 mg,
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2% yield) that was directly used in the subsequent step without additional purification; H NMR
(
400 MHz, CDCl ; TMS) δ 10.03 (s, 1H), 8.03 (d, J = 5.7 Hz, 1H), 7.97 – 7.92 (m, 2H), 7.70 (dt,
3
J = 7.6, 1.5 Hz, 1H), 7.61 (t, J = 7.9 Hz, 1H), 6.02 (d, J = 5.6 Hz, 1H), 3.97 – 3.74 (m, 9H), 3.57
3.41 (m, 2H).
-(4-(Pyrazin-2-yl)piperazine-1-carbonyl)benzaldehyde (24). Intermediate 24 was prepared
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using the general procedure C, where 3-formylbenzoic acid (500 mg, 3.33 mmol) was allowed to
react with pyrazin-2-yl-piperazine (602 mg, 3.66 mmol) as a brown oil (753 mg, 76% yield), which
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was used as obtained in the subsequent step; H NMR (400 MHz, CDCl ; TMS) δ 10.03 (d, J =
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1
.3 Hz, 1H), 8.15 (d, J = 1.6 Hz, 1H), 8.07 (dt, J = 3.2, 1.6 Hz, 1H), 7.98 – 7.92 (m, 2H), 7.89 (dd,
J = 2.8, 1.3 Hz, 1H), 7.71 (dq, J = 7.7, 1.6 Hz, 1H), 7.62 (t, J = 7.4 Hz, 1H), 3.99 – 3.81 (m, 2H),
.76 – 3.48 (m, 6H).
-(4-(Pyrimidin-2-yl)piperidine-1-carbonyl)benzaldehyde (25). Aldehyde 25 was prepared
by using general procedure C, where 4-formylbenzoic acid (250 mg, 1.67 mmol) was treated with
3
4
2
-(piperidin-4-yl)pyrimidine (299 mg, 1.83 mmol) as a yellow oil (364 mg, 74% yield), which was
1
used in the subsequent synthesis without additional purification; H NMR (400 MHz, CDCl ;
3
TMS) δ 10.05 (s, 1H), 8.72 (d, J = 4.9 Hz, 2H), 7.95 (d, J = 7.7 Hz, 2H), 7.58 (d, J = 7.9 Hz, 2H),
7
.25 (t, J = 5.0 Hz, 1H), 4.85 – 4.71 (m, 1H), 3.82 – 3.63 (m, 4H), 3.27 – 3.16 (m, 4H).
-Formyl-N-(1-(pyrimidin-2-yl)piperidin-4-yl)benzamide (26). Intermediate 26 was
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synthesized by using 4-formylbenzoic acid (500 mg, 3.33 mmol) and 1-(pyrimidin-2-yl)piperidin-
4-amine (653 mg, 3.66 mmol) as per general procedure C as a pale yellow solid (666 mg, 64%
1
yield); H NMR (400 MHz, CDCl ; TMS) δ 10.02 (s, 1H), 8.27 (d, J = 4.8 Hz, 2H), 7.95 (d, J =
3
8
.3 Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.04 (d, J = 7.9 Hz, 1H), 6.46 (t, J = 4.8 Hz, 1H), 4.78 –
4.66 (m, 2H), 4.31 – 4.19 (m, 1H), 3.98 – 3.68 (m, 2H), 3.10 – 2.99 (m, 2H), 2.11 – 2.02 (m, 2H).
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-((4-(Pyrimidin-2-yl)piperazin-1-yl)sulfonyl)benzaldehyde (27). Intermediate 27 was
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prepared using procedure from our previously published work, where pyrimidin-2-yl-piperazine
(201 mg, 1.22 mmol) was reacted with triethylamine (0.34 mL, 2.44 mmol) in dichloromethane,
followed by drop wise addition of 4-formylphenyl sulfonyl chloride (dissolved in
dichloromethane) under 0ºC and brought to rt after which it was left stirring for a period of 12 h.
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The solvent was later evaporated, and the mixture was purified by flash chromatography to yield
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7 as a white solid (286 mg, 70% yield). H NMR (400 MHz, DMSO-d ) δ 10.11 (s, 1H), 8.34 (d,
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J = 4.7 Hz, 2H), 8.14 (d, J = 7.9 Hz, 2H), 7.97 (d, J = 7.9 Hz, 2H), 6.64 (t, J = 4.7 Hz, 1H), 3.88 –
3
.78 (m, 4H), 3.04 – 2.96 (m, 4H).
4-((4-(Pyrimidin-2-yl)piperazin-1-yl)methyl)benzaldehyde (28). Intermediate 28 was
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prepared according to our previous report. To a solution of pyrimidin-2-yl-piperazine (454 mg,
2
.76 mmol) in acetonitrile, potassium carbonate (694 mg, 5.02 mmol) and 4-bromomethyl
benzaldehyde (500 mg, 2.51 mmol) were added and the suspension was allowed to reflux for
overnight. Subsequently, the solvent was evaporated followed by extraction of the reaction mass
with ethyl acetate and water. Ethyl acetate layer was then dried over MgSO and was further
4
1
purified using flash chromatography to obtain 28 as a yellow solid (536 mg, 76% yield). H NMR
(
400 MHz, CDCl ; TMS) δ 10.00 (s, 1H), 8.30 (dd, J = 4.8, 0.7 Hz, 2H), 7.86 (d, J = 8.0 Hz, 2H),
3
7
2
.55 (d, J = 8.0 Hz, 2H), 6.48 (td, J = 4.7, 0.7 Hz, 1H), 3.88 – 3.79 (m, 4H), 3.62 (s, 2H), 2.56 –
.45 (m, 4H).
4-(5,6,7,8-Tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine-7-carbonyl)benzaldehyde
(29).
Intermediate 29 was synthesized using general procedure C, by reacting 4-formylbenzoic acid (275
mg, 1.83 mmol) with 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine (250 mg, 2.01 mmol) as a
white solid (213 mg, 45% yield), after purification using flash chromatography with DCM and
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