Discovery of quinazoline-2,4(1: H,3 H)-dione derivatives as novel PARP-1/2 inhibitors: Design, synthesis and their antitumor activity

Article abstract of DOI:10.1039/c8ob00286j

Novel quinazoline-2,4(1H,3H)-dione derivatives bearing a 3-amino pyrrolidine moiety were designed and synthesized as PARP-1/2 inhibitors. Structure-activity relationships were examined which revealed a number of potent PARP-1/2 inhibitors with moderate se

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ISSN 1477-0520
COMMUNICATION
Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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Discovery of quinazoline-2,4(1H,3H)-dione derivatives as novel
PARP-1/2 inhibitions: design, synthesis and their antitumor
activity
Received 00th January 20xx,
Accepted 00th January 20xx
Jie Zhou,a,# Ming Ji, b,# Haiping Yao,a Ran Cao,a Hailong Zhao,a Xiaoyu Wang,a
DOI: 10.1039/x0xx00000x
Xiaoguang Chen,b,* and Bailing Xu a,*
www.rsc.org/
Abstract
Novel quinazoline-2,4(1H,3H)-dione derivatives bearing 3-amino pyrrolidine moiety were designed and synthesized as
PARP-1/2 inhibitors. Structure-activity relationships were conducted and led to a number of potent PARP-1/2 inhibitors
with moderate selectivity toward PARP-1 over PARP-2. These compounds had IC50 values against PARP-1 at 10-9 M level
and against PARP-2 at 10-8 M level. Among all the synthesized compounds, compounds 10 and 11 displayed strong
cytotoxicities either used as a single agent or in combination with temozolomide (TMZ) in MX-1 cells (10, IC50 < 3.12 μM,
PF50 >10; 11, IC50 = 3.02 μM, PF50 ≈ 10). In vivo tumor growth inhibition was investigated using compound 11 in
combination with TMZ, and it was demonstrated that compound 11 could strongly potentiate the cytotoxicity of TMZ in
MX-1 xenograft tumor model. The co-crystal structure of compound 11 complexed with PARP-1 was achieved and
demonstrated a unique binding mode.
Key words: PARP-1 inhibitor; PARP-2 inhibitor; Quinazoline-2,4(1H,3H)-dione; Anti-tumor agents.
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1. Introduction
In our efforts to discover novel PARP-1/2 inhibitors, a series of
quinazoline-2,4(1H,3H)-dione derivatives was designed based on
the structure-activity relationships (SARs) of known PARP inhibitors
and their binding features in the catalytic domain of PARP-1. The
Genomic instability is a hallmark of cancer，which is largely driven
by abnormalities in the robust DNA repair mechanisms of human
cells[1]. Therefore, targeting DNA damage repair pathway has
become an appealing therapeutic strategy in cancer therapy.
Poly(ADP-ribose) polymerase-1/2 (PARP-1/2) played a key role in
DNA breaks repair and have been extensively studied as targets for
cancer treatments in preclinical and clinical models[2-4]. Both of
them catalyzed the cleavage of nicotinamide adenine dinucleotides
(NAD+) into nicotinamide and ADP-ribose units, which were
transferred to proteins participating in DNA damage repair
processes including histone and PARP-1/2 and formed ADP-ribose
polymers (PAR) [5,6]. While both PARP-1 and PARP-2 were involved
in the DNA breaks repair, their specific roles in the repair pathway
have not been well understood [7-9]. PARP-1 and PARP-2 were the
closest homologs in this 17-member family and they possessed 69%
similarity in the catalytic domain, therefore, most of initially known
PARP-1 inhibitors such as AZD-2281, ABT-888, AG014699, MK-4827
and BMN673 also bound PARP-2 with comparable binding affinity
[10-16].
quinazoline-2,4(1H,3H)-dione was chosen as
a key structural
fragment since it has been identified as a suitable subunit to occupy
the nicotinamide-ribose binding site (NI site) in the catalytic domain
and formed characteristic hydrogen bonds with residues Ser904
and Gly863; the substituted benzyl group not only could generate
π-π stacking interaction with Tyr907, but also served as a linker to
direct 1-substituted 3-amino pyrrolidine motifs into the adenine-
ribose binding site (AD site)[29, 30], which is rather large and
usually used to search for a wide variety of novel inhibitors with the
improved potency and pharmacokinetic properties. In this work, we
explored the SARs of this series of quinazoline-2,4(1H,3H)-diones by
variation of the substituents on the nitrogen of 3-amino pyrrolidine
moiety. Herein, the enzymatic activities against PARP-1/2 and the
chemical synthesis of these new quinazoline-2,4(1H,3H)-dione
derivatives were described. The in vitro and in vivo anti-tumor
activities of selected compounds were also evaluated. The co-
crystal structure of compound 11 with PARP-1 was presented as
well to elicit the action mode of the designed molecules.
It has been demonstrated that PARP-1/2 inhibitors were useful for
the treatment of cancers either as a single agent or in the
combination therapy. PARP-1/2 inhibitors could potentiate the
cytotoxicity of chemotherapeutics such as temozolomide (TMZ) and
cisplatin by blocking the repair of DNA breaks resulted from DNA
alkylating agents. Specifically, cancer patients with BRCA1/2
mutations benefited from PARP-1/2 inhibitors treatment even more
greatly since PARP-1/2 were synthetic lethal with BRCA1/2, which
are involved in DNA double strand breaks repair through
homologous recombination pathway [17-20].
2. Results and discussion
2.1 Chemical synthesis
The quinazoline-2,4(1H,3H)-dione derivatives were prepared
according to the synthetic route as outlined in Scheme 1 and their
chemical structures were presented in Tables 1-2. Upon treatment
of quinazoline-2,4(1H,3H)-dione with methyl 5-(bromomethyl)-2-
fluorobenzoate or methyl 5-(bromomethyl)-2-chlorobenzoate and
HMDS, the substituted benzyl groups were selectively introduced
onto the N-1 position of quinazoline-2,4(1H,3H)-dione, giving rise to
compounds 1 and 2 in 42% and 83% yields, respectively. In the
presence of lithium hydroxide, compounds 1 and 2 were hydrolyzed
into the corresponding carboxylic acids 3 and 4 in 92% and 62%
yields, respectively, which were the key intermediates for the
preparation of various designed target molecules. The target
molecules 5-15 were prepared by direct coupling of compounds 3
and 4 with 1-substituted pyrrolidin-3-amines in moderate to good
yields, while target compounds 18-26 were synthesized in two steps
starting from compounds 14 and 15 including removal of Boc group
Since nicotinamide and 3-aminobenzamide were identified as PARP-
1 inhibitors in 1980’s, tremendous efforts have been devoted and
many structurally diverse PARP-1/2 inhibitors were discovered [21-
24]. Presently, a number of inhibitors have been advanced into
clinical trials [25-28]. Among them, Olaparib (AZD-2281), Rucaparib
(AG014699) and Niraparib (MK4827) have been approved by FDA
for the treatment of ovarian cancer in patients with or without
BRCA mutations during the period of 2014-2017. It has been
demonstrated that targeting on PARP-1/2 would be a viable way in
developing cancer drugs.
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and acylation or alkylation of pyrrolidine ring. The amide nitrogen yield. The Boc group was removed from compounds 31-33 giving
substituted derivatives 27-33 were prepared by reaction of rise to compounds 34-36 in the presence of TFA.
compound 3 with N,1-disubstituted pyrrolidine-3-amines in 26-63%
Scheme 1. Reagents and conditions: a) HMDS, conc. H2SO4, toluene, reflux; substituted bromomethylbenzene, 130 °C; MeOH, dioxane, 70
°C; b) LiOH, THF, MeOH, H2O, r.t.; c) N-substitued 3-amino pyrrolidine, EDC, HOBt, Et3N, DMF, r.t. or HBTU, HOBt, DIEA, DMF, r.t. or HATU,
HOBt, DIEA, DMF, r.t.; d) TFA, DCM, rt; e) Acyl chlorides, DCM, Et3N, or aldehydes or ketones, DIEA, THF, then NaBH(OAc)3; (f) 2N HCl,
acetone, r.t.; g) N,1-disubstitued pyrrolidin-3-amine, EDC, HOBt, Et3N, DMF, r.t.
difluorocyclohexyl and cyclohexanone on the N-1 position of
pyrrolidine ring led to pronounced reduction in inhibitory activity
2.2 Enzymatic activity against PARP-1 and PARP-2
toward both PARP-1 and PARP-2 (compounds 13 and 19-21).
Presumably, the increased rigidity and the shape of these cyclo
The inhibitory activities against PARP-1 and PARP-2 of all target
fragments are not appropriate for binding in AD pocket of PARP-
compounds (5-36) were evaluated. AZD2281 was used as the
1/2. While compounds 22-24 with benzyl and substituted benzyl
reference molecule. The corresponding results were expressed as
groups on the N-1 position exhibited significant inhibition effects on
IC50 values and presented in Tables 1-2.
PARP-1 and favorable binding for PARP-1 than PARP-2. Together
with alky substituted compounds, these compounds further
demonstrated that a large hydrophobic binding pocket was existed
in the AD site and a variety of moieties could be introduced to
improve the potency and physicalchemical properties.
Initially, we incorporated a number of alky groups on the N-1
position of pyrrolidine ring to explore the structure-activity
relationships. Compounds 5-11 bearing ethyl, propyl, butyl, iso-
butyl, iso-pentyl, pentan-3-yl and cyclopropyl methyl group
strongly inhibited PARP-1 enzymatic activity with IC50 values ranging
from 3.8 nM to 15.1 nM, which were comparable with that of non-
substituted compound 16 (IC50 = 5.9 nM). Noticeably, compounds 6-
11 showed preferable binding to PARP-1 than PARP-2, giving a
selectivity of 5-16 folds, while compounds 5 and 16 displayed little
preference. These data suggested that bulky alky groups were
tolerated on the N-1 position of pyrrolidine ring and even more
favorable for PARP-1 binding in comparison with PARP-2 binding. In
contrast, the placement of cycloalkyl groups such as
Substitution on the nitrogen with Boc and cyclopropanecarbonyl
fragments resulted in compounds 14 and 18, which possessed
comparable activity against both PARP-1 and PARP-2 with IC50
values at double digit nanomolar level. Compared with compound
11, compound 18 with an acyl group showed over 2-fold reduction
in potency toward PARP-1, suggesting that alkylation on the
nitrogen was a little more beneficial to the PARP-1 binding affinity
than acylation. In addition, we replaced the fluoro group on the
benzyl linker with a chloro atom to give rise to compounds 12, 15,
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17, 25 and 26. In comparison with the corresponding fluoro results also indicated that the binding features of the benzyl linker
substituted counterparts, these chloro substituted compounds were somewhat different in the binding site of PARP-1 and PARP-2.
exhibited markedly decreased potency toward PARP-1, while this And this distinct feature might be used to design iso-form selective
decrease was not so pronounced toward PARP-2. Therefore, inhibitors.
grafting a fluoro atom on the benzyl linker is essential in terms of
PARP-1 binding as demonstrated in the literatures [11, 29]. These
Table 1
The chemical structures and inhibitory activities against PARP-1 and PARP-2 of compounds 5-26a
Compd. R1 R2
PARP-1
PARP-2
Compd.
R1 R2
PARP-1
PARP-2
IC50(nM)b± IC50(nM)b±
IC50(nM)b± IC50(nM)b±
SDc
SDc
SDc
SDc
5
6
7
F
F
F
15.1±1.8
15.5±0.9
16
17
18
F
H
H
5.9±0.5
15.2±1.2
6.9±0.8
5.8±0.9
40.1±3.6
>100
Cl
F
>100
39.0±2.1
40.1±3.1
31.5±4.5
8
9
F
F
3.8±0.6
5.7±0.8
17.2±4.1
>100
19
20
F
F
21.8±1.6
90.1±11.2
46.4±5.1
51.0±11.1
10
11
F
F
14.2±1.6
13.3±1.4
>100
21
22
F
F
>100
43.0±9.9
28.5±2.1
67.8±10.4
16.0±1.8
12
Cl
38.1±3.2
>100
23
F
4.7±0.6
28.0±0..6
13
14
F
F
>100
45.8±5.7
35.0±8.1
24
25
F
7.1±1.0
17.1±3.2
>100
18.7±3.1
Cl
92.5±9.8
15
Cl
>100
>100
26
Cl
98.2±11.2
65.2±10.3
a The ability of compounds to inhibit PARP-1 enzyme activity were tested using ELISA method and AZD2281 was used as a positive control.
The measured IC50 against PARP-1 was 2.09 nM, the measured IC50 against PARP-2 was 2.26 nM .
b Concentrations for 50% inhibition in PARP-1 enzyme assay (IC50).
c Standard Deviation
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The effects of substitution on the amide nitrogen with R3 group deteriorated activity toward PARP-1 and PARP-2 with IC50 values of
were also investigated primarily (Table 2). Generally, grafting a >100 nM, as exemplified by compounds 30, 33 and 36, regardless of
methyl or an ethyl group onto the nitrogen was tolerated and the substituents on the nitrogen of pyrrolindine ring. It is evidently
produced comparable activity with the non-substituted that bulky alkyl groups were not tolerated to this amide position.
counterparts, while placement of
a propyl group greatly
Table 2
The chemical structures and inhibitory activities against PARP-1 and PARP-2 of compounds 27-36a
O
NH
N
O
O
F
N
R2
N
R3
Com
pd.
R2
R3
PARP-1
IC50(nM) b
±SDc
PARP-2
IC50(nM)b
±SDc
Comp
d.
R2
R3
PARP-1
IC50(nM)b
±SDc
PARP-2
IC50(nM)b ±SDc
27
28
CH3
CH3
6.4±0.1
87.5±8.8
32
33
38.8±3.3
30.2±1.3
>100
32.6±5.6
14.5±2.2
91.5±14.3
29
30
31
67.2±9.3
>100
28.1±2.3
>100
34
35
36
H
H
H
CH3
5.2±0.8
4.6±0.6
>100
53.6±4.0
63.0±6.7
>100
CH3
8.8±3.0
43.8±6.3
a The ability of compounds to inhibit PARP-1 enzyme activity were tested using ELISA method and AZD2281 was used as a positive control.
The measured IC50 against PARP-1 was 2.09 nM, the measured IC50 against PARP-2 was 2.26 nM .
b Concentrations for 50% inhibition in PARP-1 enzyme assay (IC50).
c Standard Deviation
compounds had relatively weak cytotoxicities with IC50 values >10
μM. The potentiation effects on the DNA damaging agent
2.3 Cellular potency
temozolomide (TMZ) were evaluated at 10 μM concentration using
MX-1 cell lines. Compounds 9-11 could strongly potentiate TMZ
The cellular potency of 12 compounds, which acted as highly potent
cytotoxicities in comparison with other tested compounds, the
PARP-1 inhibitors with IC50 values of 3.8-15.1 nM, was evaluated for
cytotoxicity of TMZ was increased to more than 10 fold. Of note,
their cytotoxicities as single agents and their potentiation effects on
although all of the tested compounds possessed strong enzymatic
TMZ-induced cytotoxicity in MX-1 breast cancer cells with BRCA1
inhibition, most of them did not show potent cellular activity. In
deficiency [15]. As presented in Table 3, among the tested
particular, compound 16 without substituents on both nitrogens
derivatives, compounds 10 and 11 demonstrated strong cytotoxities
showed poor cytotoxicity. Therefore, these data demonstrated that
with IC50 values of < 3.12 μM and 3.02 μM, respectively, and other
variation of fragments on the nitrogen of pyrrolidine ring would be
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facilitated to improve the cellular activity, presumably, due to the inhibitors.
improvement of physicochemical properties of this series of
Table 3
Cytotoxicities and potentiation effects (PF50) of selected compounds on MX-1 cells
b
Compd.
R2
R3
H
IC50 (μM)a
91.7
PF50
1.3
Compd.
R2
H
R3
H
IC50 (μM)a
3.02
PF50
~10
5
11
6
7
8
H
H
H
42.2
52.6
61.3
1.1
1.3
1.5
16
27
31
H
>100
58.8
27.6
ND c
2.5
CH3
CH3
5.3
9
H
H
13.1
>10
>10
34
35
H
H
CH3
41.5
92.3
ND
ND
10
<3.12
a Cytotoxicity (IC50): the concentration required to reduce cell proliferation and growth by 50% in single-agent cytotoxicity assay. AZD2281
was used as a positive control and its IC50 value was 8.26 μM.
b Potentiation factor (PF50): The fold of potentiation was calculated as the ratio of the IC50 for TMZ divided by the IC50 of TMZ + PARP-1
inhibitor, the test compounds were used at a fixed concentration of 10 μM.
c ND: Not Determined.
2.4 In vivo antitumor activity
of compound 11 administered at the same dose. Remarkably, when compound 11
As compound 11 showed pronounced potentiation effect on TMZ- was used with TMZ in combination, both dosing at 50 mg/kg, the
induced cytotoxicity and had potent cytotoxicity (IC50 = 3.02 μM) as tumors weight decreased markedly and 91% tumor growth
a single agent in MX-1 cells, we further evaluated its anti-cancer inhibition was achieved. When TMZ treated group was used as the
activity as a sensitizer of TMZ using MX-1 xenograft tumor model. control, the combination dosing still produced 76% inhibition effect.
The results were shown in Table 4. When TMZ (50 mg/kg) was It was demonstrated that compound 11 was an effective chemo-
administered orally to BalB/c nude mice bearing MX-1 xenograft sensitizing agent.
tumors, 62% tumor growth inhibition was observed. In contrast,
only 13% inhibition was produced when compound 11 was
Table 4
In vivo tumor growth inhibition of compound 11
Dose
Number
Weight Change
(g)
Tumor Weight
(g±SE)
% T/Cveh
(%TGI)
%T/CTMZ
(%TGI)
Group
(mg/kg×times)
（Start/End）
Vehicle
TMZ
6/6
6/6
6/6
5.32
0.02
4.38
4.09±0.64
1.57±0.64
3.57±0.90
NA
NA
NA
NA
50×5
50×5
38 (62)
87 (13)
Compd. 11 (50)
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Compd. 11 (25)/TMZ
Compd. 11 (50)/TMZ
NA: not applicable.
25×5/50×5
50×5/50×5
6/6
6/6
-0.78
0.10
0.50±0.14
0.38±0.13
12 (88)
9 (91)
32 (68)
24 (76)
2.5. The crystal structure of compound 11 complexed with PARP-1
Hydrogen bonds are represented with green lines. The 2Fo–Fc
electron density map (contoured at 1σ) around the inhibitor is
shown as pink mesh (PDB: 5WRY). The image was generated with
Pymol [33].
In order to explore the binding feature of the designed quinazoline-
2,4(1H,3H)-dione derivatives, we obtained and analyzed the co-
crystal structure of compound 11 within the catalytic site of PARP-1.
As shown in Figure 1, as we expected, the quinazolinone scaffold
occupied the NI-site and interacted with Ser904, Gly863 and Tyr907
through characteristic hydrogen bonds and π-π stacking interaction.
Interestingly, the 2-fluorobenzamide linker formed a π-π interaction
with Tyr896 and a hydrogen bond with the backbone of Tyr896.
These binding interactions presumably made positive contributions
greatly to the binding affinity. The nitrogen on the pyrrolidine ring
formed an H-bond with the carbonyl group of Arg878, suggesting
that the basic nitrogen was beneficial to the binding interaction.
The cyclopropylmethyl group on the pyrrolidine ring was nicely
inserted into a deep hydrophobic pocket lined with the side chain of
Asp766, Leu769, Asp770 and Arg878 within AD site. This suggested
that variation of the size and shape of substituents on the
pyrrolidine ring could further increase the binding affinity of this
series of inhibitors with PARP-1, and therefore leading to potent
PARP-1 selective inhibitors which are pursued by many research
groups [31,32].
3. Conclusions
In summary,
a series of novel quinazoline-2,4(1H,3H)-dione
derivatives were designed and synthesized as PARP-1/2 inhibitors
based on the characteristics of the catalytic domain in PARP-1. The
SARs were conducted by varying the substituents on the nitrogen of
pyrrolidine ring and on the amide nitrogen. The enzymatic
inhibition results demonstrated that various alkyl and aromatic
hydrophobic groups were well tolerated on the ring nitrogen and
bulky alkyl substituents on the amide nitrogen were not tolerated
at all. The bulky hydrophobic side chain on the ring nitrogen
improved the physicochemical properties and resulted in potent
compounds 9-11 in cytotoxicity assay. Furthermore, compound 11
demonstrated high efficacy as a sensitizer of TMZ in MX-1 xenograft
tumor model, suggesting that these quinazoline-2,4(1H,3H)-dione
based PARP-1/2 inhibitors which contained 1-substituted 3-amino
pyrrolidine moiety could be developed into useful anti-tumor
agents. The co-crystal structure of compound 11 located in the
catalytic domain of PARP-1 was solved. It has been shown that
quinazoline-2,4(1H,3H)-dione scaffold was situated in the NI site
and the benzyl spacer greatly contributed to the binding affinity by
forming H-bond and π-π interaction. More importantly, the
hydrophobic side chain on the pyrrolidine ring could extend into a
deep hydrophobic pocket formed by Asp766, Leu769 and Arg878
and contributed to the binding affinity by Van der Waals
interaction. And this distinct binding feature could be used to
develop PARP-1 selective inhibitors.
4. Experimental Section
4.1 Chemical Synthesis General
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. All
reactions involving air- or moisture-sensitive reagents were
performed under an argon atmosphere. Melting points were
measured on a Yanaco micro melting point apparatus and are
uncorrected. 1H NMR (300 MHz and 400 MHz) on a Varian Mercury
spectrometer was recorded in DMSO-d6 or CDCl3. Chemical shifts
are reported in δ (ppm) units relative to the internal standard
tetramethylsilane (TMS). High resolution mass spectra (HRMS) were
obtained on an Agilent Technologies LC/MSD TOF spectrometer. All
Figure 1. The co-crystal structure of PARP-1 in complex with 11.
Protein and ligand are colored in tan and sky blue, respectively.
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chemicals and solvents used were of reagent grade without purified After the removal of solvent under vacuum distillation, H2O (10 mL)
or dried before use. All the reactions were monitored by thin-layer was added to the residue. The resulting mixture was washed with
chromatography (TLC) on pre-coated silica gel G plates at 254 nm ethyl acetate (5 mL), and the pH of aqueous layer was adjusted to 2
under a UV lamp. Column chromatography separations were with aq. HCl (2N). After filtration, the title compound 3 was
obtained as a white solid (176 mg, 91.9%); m.p.>250℃; 1H-NMR
performed with silica gel (200 - 300 mesh).
(300 MHz, DMSO-d6) δ (ppm) : 13.31 (s, 1H), 11.76 (s, 1H), 8.02 (d, J
= 7.6 Hz, 1H), 7.82 (d, J = 5.6 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.55-
7.57 (m, 1H), 7.23-7.31 (m, 3H), 5.33 (s, 2H); HR-MS (ESI): m/z,
4.2 Synthesis of compounds
calcd. for C16H12N2O4F [M+H]+ 315.0776, Found: 315.0768.
Methyl
5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-
fluorobenzoate (1)
2-Chloro-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-
To a suspension of quinazoline-2,4(1H,3H)-dione (411 mg, 2.54
mmol) in toluene (4 mL) and hexamethyldisilazane (HMDS; 819 mg,
5.07 mmol), four drops of sulfuric acid were added with caution.
The mixture was heated to reflux and stirred under refluxing for 8 h
until clear solution was obtained. After the removal of toluene and
excess HMDS under vacuum distillation, methyl 5-(bromomethyl)-2-
fluorobenzoate (938 mg, 3.80 mmol) was added to the residue. The
reaction mixture was heated to 140 °C and was stirred at this
temperature for 3 h, the reaction mixture was diluted with 1,4-
dioxane (3 mL) at 100 °C, and then methanol (2 mL) was added at
70 °C for 30 min. The suspension was cooled below 5 °C and
precipitates were collected by filtration. After washing with
methanol (5 mL) and water (5 mL) the crude product was dried
under vacuum condition to afford compound 1 as a white solid (413
mg, 41.6%); m.p. 212-214°C; 1H-NMR (400 MHz, DMSO-d6) δ (ppm) :
11.47 (brs, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 6.0 Hz, 1H), 7.66
(t, J = 7.6 Hz, 1H), 7.58-7.61 (m, 1H), 7.23-7.34 (m, 3H), 5.34 (s, 2H),
3.83 (s, 3H); HR-MS (ESI): m/z, calcd. for C17H14N2O4F [M+H]+
329.0932, Found: 329.0929.
yl)methyl)benzoic acid (4)
Following the preparation protocol of compound 3, starting from
methyl
2-chloro-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-
yl)methyl)benzoate (1.5 g, 4.35 mmol), the title compound 4 was
obtained as a yellow solid (900 mg, 62.5%); m.p.>250 °C; 1H-NMR
(500 MHz, DMSO-d6) δ (ppm): 13.51 (brs, 1H), 11.76 (s, 1H), 8.04 (d,
J = 7.5 Hz, 1H), 7.76 (d, J = 2.0 Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.49
(d, J = 8.5 Hz, 1H), 7.44 (dd, J1 = 8.5 Hz, J2 = 2.5 Hz, 1H), 7.24-7.28 (m,
2H), 5.34 (s, 2H); HR-MS (ESI): m/z, calcd. for C16H12N2O4Cl [M+H]+
331.0480, Found: 331.0476.
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)
-N-(1-
ethylpyrrolidin-3-yl)-2-fluorobenzamide (5)
A mixture of 5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-
2-fluorobenzoic acid (120 mg, 0.38 mmol), HBTU ( 286 mg, 0.76
mmol), HOBt (103 mg, 0.76 mmol), DIEA (0.33 mL, 1.9 mmol) and 1-
ethylpyrrolidin-3-amine 2,2,2-trifluoroacetate (260 mg, 0.76 mmol)
in DMF (15 mL) was stirred at room temperature for 20 h and then
H2O was added to the mixture. The solution was extracted with the
solvent mixture (methylene chloride/methanol = 10:1) (50 mL × 3)
and then the organic layer was washed with brine (20 mL). The
combined organic layer was dried over anhydrous MgSO4. After
filtration and concentration, the crude product was obtained and
purified with column chromatography (methylene chloride
/methanol/Et3N = 50:1:0.3) to give compound 5 as a white solid (29
mg, 18.7%); m.p. 152-154℃; 1H-NMR (400 MHz, CDCl3) δ (ppm):
8.19 (d, J = 7.6 Hz, 1H), 7.97 (d, J = 6.8 Hz, 1H), 7.70 (brs, 1H), 7.59
(t, J = 8.4 Hz, 1H), 7.21-7.30 (m, 2H), 7.10 (d, J = 8.8 Hz, 1H), 7.04 (t,
J = 8.8 Hz, 1H), 5.30 (s, 2H), 4.83 (br s, 1H), 3.37 (brs, 1H), 3.20 (brs,
1H), 2.73-2.85 (m, 3H), 2.60 (brs, 1H), 2.51 (brs, 1H), 2.04 (brs, 1H),
1.28 (t, J = 7.2 Hz, 3H); HR-MS (ESI): m/z, calcd. for C22H24N4O3F
[M+H]+ 411.1827, Found: 411.1832.
Methyl
2-chloro-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-
yl)methyl)benzoate (2)
Following the preparation protocol of compound 1, starting from
methyl 5-(bromomethyl)-2-chlorobenzoate, the title compound 2
was obtained as a white solid (2.3 g, 83.3%); m.p.238-240 °C; 1H-
NMR (500 MHz, DMSO-d6) δ (ppm): 11.75 (s, 1H), 8.03 (dd, J1 = 8.0
Hz, J2 = 1.5 Hz, 1H), 7.78 (d, J = 2.0 Hz, 1H), 7.66 (td, J1 = 9.0 Hz, J2 =
1.5 Hz, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.48 (dd, J1 = 8.5 Hz, J2 = 2.5 Hz,
1H), 7.26 (t, J = 7.0 Hz, 2H), 5.35 (s, 2H), 3.84 (s, 3H); HR-MS (ESI):
m/z, calcd. for C17H14N2O4Cl [M+H]+ 345.0637, Found: 345.0633.
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)
-2-
fluorobenzoic acid (3)
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -2-fluoro-N-
(1-propylpyrrolidin-3-yl)benzamide (6)
To
a
solution
of
methyl
2-chloro-5-((2,4-dioxo-3,4-
dihydroquinazolin-1(2H)-yl)methyl)benzoate (200 mg, 0.61 mmol)
in H2O (2 mL), THF (2 mL) and MeOH (4 mL), LiOH (82 mg, 1.94
mmol) was added, and the mixture was stirred at 55°C for 55 min.
Following the preparation protocol of compound 5, starting from
compound 3 and 1-propylpyrrolidin-3-amine 2,2,2-trifluoroacetate,
the title compound 6 was obtained as a white solid (97 mg, 60.2%);
8 | J. Name., 2012, 00, 1-3
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m.p. 187-189 °C; 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.19 (d, J = 7.6 mmol), HOBt (103 mg, 0.76 mmol), DIEA (0.33 mL, 1.90 mmol) and
Hz, 1H), 7.98 (d, J = 6.8 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.43 (t, J = 1- iso-pentylpyrrolidin-3-amine 2,2,2-trifluoroacetate (189 mg, 0.57
8.0 Hz, 1H),7.19-7.26 (m, 2H), 7.05 (d, J = 8.4 Hz, 1H), 6.99 (t, J = 9.2 mmol) in DMF (10 mL) was stirred at room temperature for 24 h
Hz, 1H), 5.27 (s, 2H), 4.68 (s, 1H), 3.05-3.15 (m, 1H), 2.90 (d, J = 10.0 and then H2O was added to the mixture. The solution was extracted
Hz, 1H), 2.55-2.70 (m, 2H), 2.30-2.45 (m, 3H), 1.77 (brs, 1H), 1.53- with the solvent mixture (methylene chloride/methanol = 10:1) (30
1.61 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H); 13C-NMR (125 MHz, DMSO-d6) mL × 3) and then the organic layer was washed with brine (20 mL ×
δ (ppm): 163.29, 161.81, 158.13 (d, J = 246.5 Hz), 150.73, 140.69, 2). The combined organic layer was dried over anhydrous MgSO4.
135.25, 132.62 (d, J = 2.9 Hz), 130.19 (d, J = 8.3 Hz), 128.17 (d, J = After filtration and concentration, the crude product was obtained
2.9 Hz), 127.68, 124.58 (d, J = 15.3 Hz), 122.81, 116.31 (d, J = 22.6 and purified with column chromatography (methylene chloride
Hz), 115.99, 115.021, 59.67, 57.28, 52.55, 48.55, 44.35, 30.73, /methanol = 40:1 to 20:1) to give compound 9 as a white solid (90
21.10, 11.84;HR-MS (ESI): m/z, calcd. for C23H26N4O3F [M+H]+ mg, 52.6%); m.p. 175-177 °C; 1H-NMR (400 MHz, CDCl3) δ (ppm):
425.1984, Found: 425.1985.
8.19 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 6.0 Hz, 1H), 7.57 (t, J = 7.2 Hz,
1H), 7.42 (brs, 1H), 7.21-7.27 (m, 2H), 7.06 (d, J = 8.4 Hz, 1H), 7.01
(t, J = 9.2 Hz, 1H), 5.29 (s, 2H), 4.71 (s, 1H), 3.10 (brs, 1H), 2.92 (brs,
1H), 2.65 (brs, 2H), 2.53-2.70 (m, 3H), 1.80 (brs, 1H), 1.52-1.62 (m,
1H), 1.42-1.46 (m, 2H), 0.90 (t, J = 6.4 Hz, 6H); 13C-NMR (125 MHz,
DMSO-d6) δ (ppm): 163.28, 161.81, 158.12 (d, J = 246.5 Hz), 150.73,
140.69, 135.25, 132.62 (d, J = 2.9 Hz), 130.18 (d, J = 8.5 Hz), 128.17
(d, J = 2.8 Hz), 127.67, 124.57 (d, J = 15.5 Hz), 122.80, 116.31 (d, J =
22.9 Hz), 115.98, 115.02, 59.75, 53.57, 52.61, 48.54, 44.35, 36.81,
30.74, 25.76, 22.57; HR-MS (ESI): m/z, calcd. for C25H30N4O3F [M+H]+
453.2296, Found: 453.2294.
N-(1-Butylpyrrolidin-3-yl)-5-((2,4-dioxo-3,4-dihydro
1(2H)-yl)methyl)-2-fluorobenzamide (7)
quinazolin-
Following the preparation protocol of compound 5, starting from
compound 3 and 1-butylpyrrolidin-3-amine 2,2,2-trifluoroacetate ,
the title compound 7 was obtained as a white solid (160 mg, 83%);
m.p. 134-136 °C; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.75 (s,
1H), 8.55 (s, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 7.2 Hz, 1H),
7.54-7.56 (m, 1H), 7.43-7.47 (m, 1H), 7.22-7.29 (m, 3H), 5.31 (s, 2H),
4.41 (s, 1H), 3.28 (s, 1H), 2.71-3.07 (m, 5H), 2.20 (brs, 1H), 1.81 (brs,
1H), 1.45-1.55 (m, 2H), 1.26-1.35 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H);
13C-NMR (100 MHz, DMSO-d6) δ (ppm): 163.39, 161.77, 158.13 (d, J
= 246.7 Hz), 150.69, 140.66, 135.23, 132.69 (d, J = 3.1 Hz), 130.45
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -2-fluoro-N-
(1-(pentan-3-yl)pyrrolidin-3-yl)benzamide (10)
(d, J = 8.4 Hz), 128.16 (d, J = 2.9 Hz), 127.65, 124.18 (d, J = 15.0 Hz), Following the preparation protocol of compound 9, starting from
122.78, 116.35 (d, J = 22.7 Hz), 115.96, 114.97, 58.69, 54.69, 52.40, compound and 1-(pentan-3-yl)pyrrolidin-3-amine 2,2,2-
48.24, 44.34, 30.18, 28.74, 19.73, 13.70; HR-MS (ESI): m/z, calcd. for trifluoroacetate, the title compound 10 was obtained as a white
3
C
24H28N4O3F [M+H]+ 439.2140, Found: 439.2154.
solid (50 mg, 29.2%); m.p. 143-145 °C; 1H-NMR (400 MHz, CDCl3) δ
(ppm): 12.02 (s, 1H), 8.88 (s, 0.4H), 8.77 (s, 0.6H), 8.18 (d, J = 7.6 Hz,
1H), 7.93 (d, J = 6.0 Hz, 1H), 7.67 (t, J = 7.2 Hz, 1H), 7.33 (brs, 1H),
7.23-7.26 (m, 2H), 7.07 (t, J = 9.6 Hz, 1H), 5.33 (s, 2H), 5.13 (s, 1H),
3.96 (brs, 1H), 3.73 (brs, 1H), 3.24 (brs, 1H), 2.94 (brs, 2H), 2.55 (s,
1H), 2.49 (s, 1H), 1.87 (brs, 4H), 1.09 (d, J = 5.2 Hz, 6H); HR-MS (ESI):
m/z, calcd. for C25H30N4O3F [M+H]+ 453.2296, Found: 453.2297.
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -2-fluoro-N-
(1-isobutylpyrrolidin-3-yl)benzamide (8)
Following the preparation protocol of compound 5, starting from
compound
3
and
1-isobutylpyrrolidin-3-amine
2,2,2-
trifluoroacetate, the title compound 8 was obtained as a white solid
(67 mg, 40.3%); m.p. 174-176 °C; 1H-NMR (400 MHz, CDCl3) δ (ppm):
8.20 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 6.4 Hz, 1H), 7.58 (t, J = 7.2 Hz,
1H), 7.20-7.35 (m, 2H), 7.10 (d, J = 8.4 Hz, 1H), 7.04 (t, J = 8.8 Hz,
N-(1-(Cyclopropylmethyl)pyrrolidin-3-yl)-5-((2,4-dioxo-3,4-
dihydroquinazolin-1(2H)-yl)methyl)-2-fluorobenzamide (11)
1H), 5.32 (s, 2H), 4.73 (s, 1H), 3.10 (brs, 1H), 2.93 (brs, 1H), 2.68 Following the preparation protocol of compound 9, starting from
(brs, 1H), 2.39 (brs, 4H), 1.83 (brs, 2H), 0.96 (d, J = 6.4 Hz, 6H); HR- compound 3 and 1-(cyclopropylmethyl)pyrrolidin-3-amine 2,2,2-
MS (ESI): m/z, calcd. for C24H28N4O3F [M+H]+ 439.2140, Found: trifluoroacetate, the title compound 11 was obtained as a white
439.2156.
solid (40 mg, 24.0%); m.p. 113-115 °C; 1H NMR (400 MHz, CDCl3) δ
(ppm): 8.19 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 6.8 Hz, 1H), 7.50-7.57 (m,
2H), 7.23 (t, J = 7.6 Hz, 1H), 7.15-7.17 (m, 1H), 7.03 (d, J = 8.4 Hz,
1H), 6.96 (t, J = 8.8 Hz, 1H), 5.26 (s, 2H), 4.71 (brs, 1H), 3.11-3.15 (m,
1H), , 3.01 (d, J = 10.0 Hz, 1H), 2.68-2.77 (m, 2H), 2.54-2.59 (m, 1H),
2.27-2.45 (m, 3H), 1.75-1.78 (m, 1H), 0.90-0.95 (m, 1H), 0.50-0.53
(m, 2H), 0.14-0.15(m, 2H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm):
163.29, 161.76, 158.11 (d, J = 246.7 Hz), 150.69, 140.66, 135.21,
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -2-fluoro-N-
(1-isopentylpyrrolidin-3-yl)benzamide (9)
A
mixture of 2-fluoro-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-
yl)methyl)benzoic acid (120 mg, 0.38 mmol), EDC ( 146 mg, 0.76
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132.59 (d, J = 3.0 Hz), 130.18 (d, J = 8.6 Hz), 128.16 (d, J = 2.7 Hz), stirred at room temperature for 20 h and then H2O was added to
127.64, 124.50 (d, J = 15.4 Hz), 122.77, 116.28 (d, J = 22.6 Hz), the mixture. The solution was extracted with the solvent mixture
115.96, 114.98, 59.88, 59.54, 52.52, 48.48, 44.33, 30.63, 9.22, 3.65; (ethyl acetate/methanol = 10:1) (30 mL × 3) and then the organic
HR-MS (ESI): m/z, calcd. for C24H26N4O3F [M+H]+ 437.1983, Found: layer was washed with brine (20 mL × 2). The combined organic
437.1976.
layer was dried over anhydrous MgSO4. After filtration and
concentration, the crude product was obtained and purified with
column chromatography to give compound 14 as a white solid (189
mg, 61.7%); m.p. 171-173°C; 1H-NMR (400 MHz, DMSO-d6) δ (ppm):
11.73 (s, 1H), 8.57 (d, J = 6.4 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.66 (t,
J = 7.2 Hz, 1H), 7.54 (d, J = 5.6 Hz, 1H), 7.42 (brs, 1H), 7.20-7.29 (m,
3H), 5.31 (s, 2H), 4.34-4.39 (m, 1H), 3.45-3.55 (m, 1H), 3.25-3.39 (m,
2H), 3.12-3.19 (m, 1H), 2.02-2.11 (m, 1H), 1.78-1.87 (m, 1H), 1.39 (s,
9H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 163.66, 161.75, 158.03
(d, J = 246.7 Hz), 153.46, 150.69, 140.66, 135.20, 132.63 (d, J = 3.1
Hz), 130.18 (d, J = 7.9 Hz), 128.14, 127.65, 124.57 (d, J = 15.8 Hz),
122.76, 116.28 (d, J = 22.5 Hz), 115.97, 114.96, 78.30, 50.82 (50.48),
49.40 (48.64), 44.32, 43.98 (43.72), 30.84 (29.86), 28.15; HR-MS
(ESI): m/z, calcd. for C25H27N4O5FNa [M+Na]+ 505.1858, Found:
505.1853.
2-Chloro-N-(1-(cyclopropylmethyl)pyrrolidin-3-yl)-5-((2,4-dioxo-
3,4-dihydroquinazolin-1(2H)-yl)methyl) benzamide (12)
Following the preparation protocol of compound 9, starting from
compound 4 and 1-(cyclopropylmethyl)pyrrolidin-3-amine 2,2,2-
trifluoroacetate, the title compound 12 was obtained as a light
yellow solid (92 mg, 61.3%); m.p. 105-107 °C; 1H NMR (500 MHz,
CDCl3) δ (ppm): 8.25 (brs, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.50-7.53 (m,
2H), 7.19-7.24 (m, 2H), 6.90 (d, J = 8.5 Hz, 1H), 6.81 (d, J = 9.0 Hz,
1H), 5.19 (d, J = 17.0 Hz, 1H), 4.92 (d, J = 16.0 Hz, 1H), 4.84 (brs, 1H),
3.38-3.44 (m, 2H), 2.94 (dd, J1 = 12.5 Hz, J2 = 6.5 Hz, 1H), 2.68 (t, J =
9.0 Hz, 1H), 2.66-2.71 (m, 1H), 2.48-2.55 (m, 1H), 2.36-2.39 (m, 1H),
2.29-2.33 (m, 1H), 1.88-1.90 (m, 1H), 0.97-1.02 (m, 1H), 0.52-0.61
(m, 2H), 0.18-0.23 (m, 2H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm):
165.78, 161.77, 150.69, 140.66, 137.11, 135.62, 135.22, 129.69,
128.69, 128.54, 127.64, 127.07, 122.78, 115.99, 114.96, 59.87,
59.44, 52.52, 48.38, 44.42, 30.50, 9.22, 3.70, 3.62; HR-MS (ESI): m/z,
calcd. for C24H26N4O3Cl [M+H]+ 453.1688, Found: 453.1675.
tert-Butyl 3-(2-chloro-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-
yl)methyl)benzamido)
pyrrolidine-1-carboxylate (15)
Following the preparation protocol of compound 9, starting from
compound 4 and N-Boc-3-aminopyrrolidine, the title compound 15
was obtained as a white solid(1.06 g, 85.5%); m.p. 197-199°C; 1H-
NMR (500 MHz, DMSO-d6) δ (ppm): 11.74 (s, 1H), 8.71 (brs, 1H),
8.03 (d, J = 7.0 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.42-7.44 (m, 2H),
7.33 (d, J = 8.5 Hz, 1H), 7.23-7.28 (m, 2H), 5.31 (s, 2H), 4.32-4.36 (m,
1H), 3.42-3.52 (m, 1H), 3.32-3.40 (m, 1H), 3.28 (brs, 1H), 3.15-3.24
(m, 1H), 2.01-2.11 (m, 1H), 1.84 (brs, 1H), 1.39 (s, 9H); 13C-NMR (125
MHz, DMSO-d6) δ (ppm): 166.07, 161.82, 153.49, 150.72, 140.67,
137.01, 135.72, 135.28, 129.72, 128.70, 128.63, 127.66, 127.13,
122.83, 116.00, 115.04, 78.28, 50.92 (50.57), 49.39 (48.58), 44.42,
44.02 (43.74), 30.68 (29.68), 28.19; HR-MS (ESI): m/z, calcd. for
C25H28N4O5Cl [M+H]+ 499.1743, Found: 499.1757.
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -2-fluoro-N-
(1-(oxetan-3-yl)pyrrolidin-3-yl)benzamide (13)
Following the preparation protocol of compound 9, starting from
compound
3
and 1-(oxetan-3-yl)pyrrolidin-3-amine 2,2,2-
trifluoroacetate, the title compound 13 was obtained as a light
yellow solid (25 mg, 41.6%); m.p. 117-119 °C; 1H-NMR (400 MHz,
CDCl3) δ (ppm): 9.02 (s, 1H), 8.21 (d, J = 7.6 Hz, 1H), 8.05 (d, J = 7.2
Hz, 1H), 7.57 (t, J = 7.2 Hz, 1H), 7.34-7.38 (m, 1H), 7.24 (t, J = 7.6 Hz,
1H), 7.03-7.10 (m, 3H), 5.33 (s, 2H), 4.61-4.73 (m, 5H), 3.65-3.72 (m,
1H), 2.90-2.98 (m, 1H), 2.63-2.75 (m, 2H), 2.32-2.43 (m, 2H), 1.75-
1,80 (m, 1H); 13C NMR (125 MHz, DMSO-d6) δ (ppm): 163.36,
161.81, 158.10 (d, J = 246.5 Hz), 150.73, 140.69, 135.25, 132.63 (d, J
= 2.8 Hz), 130.16 (d, J = 8.8 Hz), 128.14 (d, J = 2.8 Hz), 127.68,
124.67 (d, J = 15.6 Hz), 122.81, 116.30 (d, J = 22.5 Hz), 115.99, 5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -2-fluoro-N-
115.02, 74.64, 57.11, 56.26, 48.99, 48.63, 44.35, 30.81; HR-MS (ESI): (pyrrolidin-3-yl)benzamide (16)
m/z, calcd. for C23H24N4O4F [M+H]+ 439.1776, Found: 439.1761.
To a solution of compound 14 (500 mg, 1.04 mmol) in DCM (15.0
mL), TFA (2 mL, 31.3 mmol) was added and then the reaction
mixture was stirred at room temperature for 2.5 h. After removal of
tert-Butyl 3-(5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-
the solvent under vacuum distillation, H2O (10 mL) was added to
2-fluorobenzamido) pyrrolidine -1-carboxylate (14)
the residue. The resulting mixture was washed with ethyl acetate (5
mL), and the pH of aqueous layer was adjusted to 8 with aqueous
A mixture of 5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-
ammonia. The solution was extracted with the solvent mixture
2-fluorobenzoic acid (200 mg, 0.64 mmol), HATU ( 487 mg, 1.28
(methylene chloride/methanol = 10:1) (20 mL × 3). The combined
mmol), HOBt (173 mg, 1.28 mmol), DIEA (0.22 mL, 1.28 mmol) and
organic layer was dried over anhydrous MgSO4. After filtration and
N-Boc-3-aminopyrrolidine (178 mg, 0.96 mmol) in DMF (15 mL) was
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concentration, the title compound 16 was obtained as a white solid To a solution of 16 (100 mg, 0.40 mmol) in dichloromethane (4.0
(380 mg, 96%); m.p. 170-172 °C; 1H-NMR (400 MHz, DMSO-d6) δ mL) and methanol (1 mL), 4,4-difluorocyclohexan-1-one (110 mg,
(ppm): 8.31 (d, J = 7.2 Hz, 1H), 8.03 (dd, J1 = 7.6 Hz, J2 = 2.0 Hz, 1H), 0.78 mmol) and sodium acetate (87 mg, 1.04 mmol) were added.
7.63-7.68 (m, 1H), 7.54 (dd, J1 = 6.8 Hz, J2 = 2.0 Hz, 1H), 7.38-7.43 The resulting solution was stirred at 37 °C for 2.5 h. Then sodium
(m, 1H), 7.19-7.29 (m, 3H), 5.31 (s, 2H), 4.23-4.28 (m, 1H), 2.92-2.97 cyanoborohydride (52 mg, 0.78 mmol) was added and the mixture
(m, 1H), 2.83-2.89 (m, 1H), 2.70-2.76 (m, 1H), 2.59-2.63 (m, 1H), was stirred for 2 d. The dichloromethane (40 mL) and methanol (4
1.91-2.00 (m, 1H), 1.55-1.63 (m, 1H); HR-MS (ESI): m/z, calcd. for mL) were added and washed with saturated aqueous sodium
C20H20N4O3F [M+H]+ 383.1514, Found: 383.1508.
bicarbonate (10 mL×2) and brine (10 mL×2), dried over anhydrous
magnesium sulfate. After filtration and concentration, the crude
product was obtained and purified with column chromatography
(methylene chloride /methanol = 50:1 to 30:1) to give compound 19
as a white solid (95 mg, 72.5%); m.p. 211-213°C; 1H-NMR (500 MHz,
DMSO-d6) δ (ppm): 11.74 (s, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.03 (dd, J1
= 8.0 Hz, J2 = 2.0 Hz, 1H), 7.66 (td, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H), 7.52
(dd, J1 = 6.5 Hz, J2 = 2.5 Hz, 1H), 7.40-7.43 (m, 1H), 7.20-7.29 (m, 3H),
5.31 (s, 2H), 4.30-4.35 (m, 1H), 2.76-2.81 (m, 1H), 2.65-2.71 (m, 1H),
2.43-2.48 (m, 2H), 1.96-2.24 (m, 4H), 1.55-1.82 (m, 7H); 13C-NMR
(125 MHz, DMSO-d6) δ (ppm): 163.23, 161.80, 158.10 (d, J = 246.3
Hz), 150.72, 140.68, 135.24, 132.63 (d, J = 3.0 Hz), 130.14 (d, J = 7.8
Hz), 128.14 (d, J = 2.8 Hz), 127.67, 124.70 (d, J = 15.1 Hz), 124.09 (t,
J = 243.3 Hz), 122.80, 116.30 (d, J = 22.6 Hz), 115.98, 115.01, 58.47,
57.53, 49.98, 48.58, 44.34, 30.78, 30.41 (t, J = 23.6 Hz) (30.35 (t, J =
23.9 Hz)), 26.95; HR-MS (ESI): m/z, calcd. for C26H28N4O3F3 [M+H]+
501.2108, Found: 501.2096.
2-Chloro-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-N-
(pyrrolidin-3-yl)benzamide (17)
Following the preparation protocol of compound 16, starting from
compound 15, the title compound 17 was obtained as a white solid
1
(620 mg, 86.4%); m.p. 162-164 °C; H-NMR (500 MHz, DMSO-d6) δ
(ppm): 11.79 (brs, 1H), 8.90 (brs, 1H), 8.83 (d, J = 6.5 Hz, 1H), 8.06
(d, J = 7.5 Hz, 1H), 7.69 (t, J = 7.0 Hz, 1H), 7.45-7.52 (m, 2H), 7.41
(dd, J1 = 8.5 Hz, J2 = 1.5 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.27 (d, J =
8.5 Hz, 1H), 5.35 (s, 2H), 4.46-4.51 (m, 1H), 3.51 (dd, J1 = 12.0 Hz, J2
= 7.0 Hz, 1H), 3.24-3.36 (m, 3H), 3.12 (dd, J1 = 12.0 Hz, J2 = 5.5 Hz,
1H), 2.17-2.25 (m, 1H), 1.92-1.99 (m, 1H); HR-MS (ESI): m/z, calcd.
for C20H20N4O3Cl [M+H]+ 399.1218, Found: 399.1212.
N-(1-(Cyclopropanecarbonyl)pyrrolidin-3-yl)-5-((2,4-dioxo-3,4-
dihydroquinazolin-1(2H)-yl)methyl)-2-fluorobenzamide (18)
N-(1-(1,4-Dioxaspiro[4.5]decan-8-yl)pyrrolidin-3-yl)-5-((2,4-dioxo-
3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorobenzamide (20)
Following the preparation protocol of compound 14, starting from
compound 16 and cyclopropanecarboxylic acid, the title compound
18 was obtained as a light yellow solid (55 mg, 73.0%); m.p. 136-
138 °C; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.75 (s, 1H), 8.66 (d,
J = 6.8 Hz, 0.5H), 8.60 (d, J = 6.8 Hz, 0.5H), 8.03 (d, J = 8.0 Hz, 1H),
7.66 (t, J = 8.0 Hz, 1H), 7.53-7.56 (m, 1H), 7.39-7.45 (m, 1H), 7.21-
7.29 (m, 3H), 5.32 (s, 2H), 4.46-4.52 (m, 0.5H), 4.36-4.43 (m, 0.5H),
3.88-3.93 (m, 0.5H), 3.65-3.74 (m, 1H), 3.24-3.58 (m, 2.5H), 2.16-
2.23 (m, 0.5H), 2.04-2.12 (m, 0.5H), 1.93-1.99 (m, 0.5H), 1.83-1.88
(m, 0.5H), 1.69-1.77 (m, 1H), 0.69-0.73 (m, 4H); 13C-NMR (125 MHz,
DMSO-d6) δ (ppm): 170.95 (170.87), 163.73 (163.71), 161.81,
158.05 (d, J = 246.6 Hz), 150.73, 140.69, 135.25, 132.69 (d, J = 3.9
Hz) (132.67 (d, J = 3.1 Hz)), 130.29 (d, J = 9.1 Hz) (130.20 (d, J = 9.4
Hz)), 128.21 (d, J = 2.3 Hz) (128.12 (d, J = 2.8 Hz)), 127.68, 124.63 (d,
J = 16.4 Hz) (124.58 (d, J = 15.8 Hz)), 122.81, 116.34 (d, J = 22.3 Hz)
(116.32 (d, J = 21.9 Hz)), 116.00, 115.01, 51.15 (50.59), 49.69
(48.19), 44.34 (44.32), 43.78, 31.16 (29.43), 12.03 (11.72), 7.10
(7.00), 6.98; HR-MS (ESI): m/z, calcd. for C24H24N4O4F [M+H]+
451.1776, Found: 451.1796.
Following the preparation protocol of compound 19, starting from
compound 16 and 1,4-dioxaspiro[4.5]decan-8-one, the title
compound 20 was obtained as a white solid (240 mg, 58.7%); m.p.
149-151 °C; 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 11.73 (s, 1H),
8.40 (d, J = 5.0 Hz, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.66 (t, J = 8.0 Hz,
1H), 7.53 (d, J = 5.5 Hz, 1H), 7.41 (brs, 1H), 7.18-7.33 (m, 3H), 5.31
(s, 2H), 4.31 (s, 1H), 3.83 (s, 4H), 2.80 (brs, 1H), 2.65 (brs, 1H), 2.46
(brs, 2H), 2.10 (brs, 2H), 1.64-1.79 (m, 5H), 1.42-1.49 (m, 4H); 13C-
NMR (100 MHz, DMSO-d6) δ (ppm): 163.19, 161.76, 158.10 (d, J =
246.3 Hz), 150.69, 140.66, 135.21, 132.57 (d, J = 3.2 Hz), 130.08 (d, J
= 8.3 Hz), 128.17 (d, J = 2.9 Hz), 127.64, 124.64 (d, J = 15.7 Hz),
122.76, 116.26 (d, J = 22.6 Hz), 115.96, 114.98, 107.79, 63.60, 63.56,
60.13, 57.50, 49.95, 48.55, 44.33, 31.91, 30.72, 28.04; HR-MS (ESI):
m/z, calcd. for C28H32N4O5F [M+H]+ 523.2351, Found: 523.2335.
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -2-fluoro-N-
(1-(4-oxocyclohexyl)pyrrolidin-3-yl)benzamide (21)
To a solution of compound 20 (100 mg, 0.19 mmol) in acetone (10.0
mL), 2N HCl (15 mL) was added and then the reaction mixture was
stirred at room temperature for 24 h. After removal of the solvent
under vacuum distillation, the solvent mixture (methylene
chloride/methanol = 10:1) (30 mL) was added to the residue. Then
N-(1-(4,4-Difluorocyclohexyl)pyrrolidin-3-yl)-5-((2,4-dioxo-3,4-
dihydroquinazolin-1(2H)-yl)methyl)-2-fluorobenzamide (19)
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the pH of mixture was adjusted to alkaline with saturated aqueous 4H), 7.03-7.14 (m, 3H), 5.30 (s, 2H), 4.31 (brs, 1H), 3.54 (brs, 2H),
sodium bicarbonate. The solution was extracted with the solvent 2.76 (brs, 1H), 2.58 (brs, 1H), 2.47 (brs, 1H), 2.38 (brs, 1H), 2.29 (s,
mixture (methylene chloride/methanol = 10:1) (20 mL × 3) and then 3H), 2.09-2.19 (m, 1H), 1.64-1.74 (m, 1H); 13C-NMR (100 MHz,
the organic layer was washed with brine (20 mL). The combined DMSO-d6) δ (ppm): 163.26, 161.75, 158.09 (d, J = 246.4 Hz), 150.68,
organic layer was dried over anhydrous MgSO4. After filtration and 140.66, 138.77, 137.17, 135.20, 132.58 (d, J = 3.2 Hz), 130.10 (d, J =
concentration, the title compound 21 was obtained as a light yellow 8.3 Hz), 129.16, 128.15 (d, J = 3.0 Hz), 128.03, 127.64, 127.51,
solid (52 mg, 57.1%); m.p. 201-203 °C;1H-NMR (500 MHz, DMSO-d6) 125.62, 124.59 (d, J = 15.6 Hz), 122.76, 116.26 (d, J = 22.6 Hz),
δ (ppm): 11.79 (s, 1H), 8.49 (d, J = 7.0 Hz, 1H), 8.05 (d, J = 7.5 Hz, 115.96, 114.97, 59.64, 59.19, 52.37, 48.66, 44.33, 30.70, 20.98; HR-
1H), 7.68 (t, J = 7.5 Hz, 1H), 7.55 (d, J = 5.0 Hz, 1H), 7.44 (brs, 1H), MS (ESI): m/z, calcd. for C28H28N4O3F [M+H]+ 487.2140, Found:
7.21-7.33 (m, 3H), 5.34 (s, 2H), 4.39 (brs, 1H), 2.87 (brs, 1H), 2.73- 487.2141.
2.83 (m, 1H), 2.51-2.57 (m, 2H), 2.37-2.49 (m, 3H), 2.13-2.28 (m,
3H), 1.94 (br, 2H), 1.79-1.89 (m, 2H), 1.69-1.78 (m, 1H); HR-MS (ESI):
m/z, calcd. for C26H28N4O4F [M+H]+ 479.2089, Found: 479.2088.
2-Chloro-N-(1-(4,4-difluorocyclohexyl)pyrrolidin-3-yl)-5-((2,4-
dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)benzamide (25)
Following the preparation protocol of compound 19, starting from
N-(1-Benzylpyrrolidin-3-yl)-5-((2,4-dioxo-3,4-dihydroquinazolin-
compound 17 and 4,4-difluorocyclohexan-1-one, the title
1(2H)-yl)methyl)-2-fluorobenzamide (22)
compound 25 was obtained as a white solid (110 mg, 56.7%); m.p.
Following the preparation protocol of compound 19, starting from 164-166 °C; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.74 (s, 1H),
compound 16 and benzaldehyde, the title compound 22 was 8.61 (d, J = 7.2 Hz, 1H), 8.03 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz, 1H), 7.63-
obtained as a white solid (65 mg, 52.8%); m.p. 175-177 °C; 1H-NMR 7.68 (m, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.32
(500 MHz, DMSO-d6) δ (ppm): 11.74 (s, 1H), 8.44 (d, J = 7.0 Hz, 1H), (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H),7.24-7.28 (m, 2H), 5.31 (s, 2H), 4.29
8.02-8.04 (m, 1H), 7.64-7.68 (m, 1H), 7.51-7.53 (m, 1H), 7.40-7.42 (brs, 1H), 2.79 (brs, 1H), 2.68 (brs, 1H), 2.53 (brs, 1H), 2.45 (brs, 1H),
(m, 1H), 7.19-7.31 (m, 8H), 5.30 (s, 2H), 4.32 (brs, 1H), 3.54-3.61 (m, 1.97-2.22 (m, 4H), 1.65-1.87 (m, 5H), 1.51-1.64 (m, 2H); HR-MS
2H), 2.76 (brs, 1H), 2.60 (brs, 1H), 2.45 (brs, 1H), 2.39 (brs, 1H), (ESI): m/z, calcd. for C26H28N4O3ClF2 [M+H]+ 517.1812, Found:
2.12-2.16 (m, 1H), 1.70 (brs, 1H); HR-MS (ESI): m/z, calcd. for 517.1803.
C27H26N4O3F [M+H]+ 473.1984, Found: 473.1973.
N-(1-Benzylpyrrolidin-3-yl)-2-chloro-5-((2,4-dioxo-3,4-
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -2-fluoro-N- dihydroquinazolin-1(2H)-yl)methyl)benzamide (26)
(1-(4-methoxybenzyl) pyrrolidin-3-yl)benzamide (23)
Following the preparation protocol of compound 19, starting from
Following the preparation protocol of compound 19, starting from compound 17 and benzaldehyde, the title compound 26 was
compound 16 and 4-methoxybenzaldehyde, the title compound 23 obtained as a white solid (97 mg, 52.7%); m.p. 188-190 °C; 1H-NMR
was obtained as a white solid (82 mg, 62.1%); m.p. 171-173 °C; 1H- (400 MHz, DMSO-d6) δ (ppm): 11.74 (s, 1H), 8.62 (d, J = 6.8 Hz, 1H),
NMR (400 MHz, DMSO-d6) δ (ppm):11.75 (s, 1H), 8.43 (d, J = 6.8 Hz, 8.03 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz, 1H), 7.63-7.68 (m, 1H), 7.38-7.43
1H), 8.02 (d, J = 8.0 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H), 7.50-7.54 (m, (m, 2H), 7.30-7.33 (m, 5H), 7.22-7.28 (m, 3H), 5.30 (s, 2H), 4.29 (brs,
1H), 7.37-7.43 (m, 1H), 7.18-7.30 (m, 5H), 6.87 (d, J = 8.4 Hz, 2H), 1H), 3.62 (d, J = 12.4 Hz, 1H), 3.54 (d, J = 13.2 Hz, 1H), 2.76 (brs, 1H),
5.30 (s, 2H), 4.25-4.35 (m, 1H), 3.73 (s, 3H), 3.51 (brs, 2H), 2.75 (brs, 2.58 (brs, 1H), 2.40-2.48 (m, 2H), 2.08-2.19 (m, 1H), 1.65-1.76 (m,
1H), 2.56 (brs, 1H), 2.45 (brs, 1H), 2.36 (brs, 1H), 2.08-2.18 (m, 1H), 1H); 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 166.78, 161.81, 150.72,
1.67-1.71 (m, 1H); HR-MS (ESI): m/z, calcd. for C28H28N4O4F [M+H]+ 140.69, 139.04, 137.13, 135.64, 135.24, 129.69, 128.70, 128.52,
503.2089, Found: 503.2080.
128.16, 127.67, 127.08, 126.86, 122.81, 116.01, 114.99, 59.60,
59.23, 52.47, 48.60, 44.42, 30.58; HR-MS (ESI): m/z, calcd. for
C
27H26N4O3Cl [M+H]+ 489.1688, Found: 489.1692.
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -2-fluoro-N-
(1-(3-methylbenzyl)pyrrolidin-3-yl)benzamide (24)
N-(1-Butylpyrrolidin-3-yl)-5-((2,4-dioxo-3,4-dihydroquinazolin-
1(2H)-yl)methyl)-2-fluoro-N-methylbenzamide (27)
Following the preparation protocol of compound 19, starting from
compound 16 and 3-methylbenzaldehyde, the title compound 24
was obtained as a white solid (72 mg, 48%); m.p. 185-187 °C; 1H- Following the preparation protocol of compound 9, starting from
NMR (400 MHz, DMSO-d6) δ (ppm): 11.74 (s, 1H), 8.44 (d, J = 6.8 Hz, compound and 1-butyl-N-methylpyrrolidin-3-amine, the title
3
1H), 8.02 (dd, J1 = 7.6 Hz, J2 = 1.2 Hz, 1H), 7.61-7.70 (m, 1H), 7.52 compound 27 was obtained as a white solid (60 mg, 41.9%); m.p.
(dd, J1 = 6.8 Hz, J2 = 2.4 Hz, 1H), 7.38-7.43 (m, 1H), 7.17-7.30 (m, 104-106 °C; 1H-NMR (400 MHz, CDCl3) δ (ppm): 9.05 (brs, 1H), 8.23
12 | J. Name., 2012, 00, 1-3
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(s, 1H), 7.60 (s, 1H), 7.27 (brs, 2H), 7.08 (brs, 2H), 5.32 (s, 2H), 4.18 Following the preparation protocol of compound 9, starting from
(s, 1H), 3.08 (s, 2H), 2.92 (s, 1H), 2.30-2.73 (m, 6H), 2.00-2.50 (m, compound and 1-ethyl-N-propylpyrrolidin-3-amine, the title
3
4H), 1.87 (brs, 1H), 1.61 (brs, 1H), 1.20-1.50 (m, 4H), 0.91 (brs, 3H); compound 30 was obtained as a light yellow solid (45 mg, 26%);
HR-MS (ESI): m/z, calcd. for C25H30N4O3F [M+H]+ 453.2297, Found: m.p. 105-107 °C; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.75 (s,
453.2310.
1H), 8.02 (dd, J1 = 7.6 Hz, J2 = 1.6 Hz, 1H), 7.61-7.68 (m, 1H), 7.40-
7.52 (m, 1H), 7.23-7.34 (m, 4H), 5.33 (s, 2H), 4.51 (brs, 0.5H), 4.06
(brs, 0.5H), 3.20-4.00 (m, 4H), 2.98-3.09 (m, 3H), 2.27-2.34 (m,
0.5H), 2.10-2.18 (m, 0.5H), 1.50-1.90 (m, 2H), 1.25-1.31 (m, 2H),
1.17-1.21 (m, 1.5H), 1.06 (brs, 1.5H), 0.89 (t, J = 7.2 Hz, 1.5H), 0.47
(t, J = 7.2 Hz, 1.5H); HR-MS (ESI): m/z, calcd. for C25H30N4O3F [M+H]+
453.2296, Found: 453.2283.
N-(1-(Cyclopropylmethyl)pyrrolidin-3-yl)-5-((2,4-dioxo-3,4-
dihydroquinazolin-1(2H)-yl)methyl)-2-fluoro-N-methylbenzamide
(28)
Following the preparation protocol of compound 9, starting from
compound
3
and 1-(cyclopropylmethyl)-N-methylpyrrolidin-3-
amine, the title compound 28 was obtained as a white solid (95 mg,
55.5%); m.p. 114-116 °C; 1H-NMR (400 MHz, DMSO-d6) δ (ppm):
11.73 (s, 1H), 8.01-8.03 (m, 1H), 7.62-7.68 (m, 1H), 7.37-7.47 (m,
tert-Butyl 3-(5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-
2-fluoro-N-methylbenzamido) pyrrolidine-1-carboxylate (31)
1H), 7.23-7.30 (m, 4H), 5.31 (s, 2H), 5.07-5.13 (m, 0.4H), 3.96 (brs, Following the preparation protocol of compound 9, starting from
0.6H), 2.93 (s, 3H), 2.60-2.80 (m, 2H), 1.60-2.40 (m, 6H), 0.76-0.89 compound and tert-butyl 3-(methylamino)pyrrolidine-1-
3
(m, 1H), 0.42-0.49 (m, 2H), 0.06-0.14 (m, 2H); 13C-NMR (125 MHz, carboxylate, the title compound 31 was obtained as a white solid
DMSO-d6) δ (ppm): 165.48 (164.87), 161.82, 158.68 (d, J = 243.0 Hz) (37 mg, 46.8%); m.p. 107-109 °C; 1H-NMR (400 MHz, DMSO-d6) δ
(156.55 (d, J = 245.4 Hz)), 150.72, 140.72 (140.67), 135.21 (135.15), (ppm): 11.73 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.61-7.68 (m, 1H), 7.23-
132.20 (d, J = 3.0 Hz) (132.11 (d, J = 2.6 Hz)), 129.40 (d, J = 7.8 Hz), 7.45 (m, 5H), 5.25-5.39 (m, 2H), 4.95-5.01 (m, 0.4H), 4.02 (brs, 0.6H)
127.66, 126.82 (d, J = 4.0 Hz), 124.82 (d, J = 18.4 Hz), 122.76, 116.25 3.39-3.53(m, 1H), 3.17-3.35 (m, 3H), 2.88 (s, 1.8H), 2.71(s, 1.2H),
(d, J = 21.0 Hz), 116.06 (116.04), 115.01, 59.72, 56.86, 53.16 (52.93), 1.80-2.10 (m, 2H), 1.38-1.41 (m, 9H); 13C-NMR (100 MHz, DMSO-d6)
51.59, 44.41 (44.35), 28.04, 27.67, 9.35, 3.78 (3.73), 3.58 (3.51); HR- δ (ppm): 165.81 (165.30), 161.78, 156.67 (d, J = 242.9 Hz) (156.55
MS (ESI): m/z, calcd. for C25H28N4O3F [M+H]+ 451.2140, Found: (d, J = 243.3 Hz)), 153.43 (153.23), 150.69, 140.69 (140.64), 135.16
451.2133.
(135.01), 132.18 (d, J = 3.1 Hz), 129.47 (129.39), 127.62, 126.93,
124.87 (d, J = 18.9 Hz) (124.48 (d, J = 18.5 Hz)) , 122.73 (122.66),
116.30 (d, J = 21.5 Hz), 116.06 (115.98), 114.98 (114.95), 78.50,
57.06 (56.29), 52.44 (51.71), 46.71 (46.36), 44.40 (44.34), 44.02
(43.82), 31.38 (29.31), 28.09; HR-MS (ESI): m/z, calcd. for
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -N-ethyl-N-
(1-ethylpyrrolidin-3-yl)-2-fluorobenzamide (29)
C
26H29N4O5FNa [M+Na]+ 519.2014, Found: 519.2033.
Following the preparation protocol of compound 9, starting from
compound 3 and N,1-diethylpyrrolidin-3-amine, the title compound
30 was obtained as a white solid (45 mg, 26%); m.p. 64-66 °C; 1H-
NMR (400 MHz, CDCl3) δ (ppm): 8.91 (brs, 1H), 8.23 (d, J = 7.6 Hz,
1H), 7.58 (t, J = 7.2 Hz, 1H), 7.36 (brs, 1H), 7.22-7.30 (m, 2H), 7.02-
tert-Butyl 3-(5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-
N-ethyl-2-fluorobenzamido) pyrrolidine-1-carboxylate (32)
7.14 (m, 2H), 5.33 (s, 2H), 4.17-4.40 (m, 1H), 3.76 (brs, 1H), 3.40- Following the preparation protocol of compound 9, starting from
3.58 (m, 3H), 3.08-3.22 (m, 3H), 2.43-2.76 (m, 3H), 1.64-1.94 (m, compound and tert-butyl 3-(ethylamino)pyrrolidine-1-
3
2H), 1.46 (t, J = 7.2 Hz, 2H), 1.33 (t, J = 6.0 Hz, 1H), 1.14 (brs, 1H), carboxylate, the title compound 32 was obtained as a light yellow
1.06 (t, J = 6.4 Hz, 3H), 13C-NMR (125 MHz, DMSO-d6) δ (ppm): solid (37 mg, 46.8%); m.p. 107-109 °C; 1H-NMR (400 MHz, DMSO-d6)
165.95 (165.05), 161.85, 156.71 (d, J = 242.9 Hz) (156.65 (d, J = δ (ppm): 11.73 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.64 (t, J = 7.2 Hz,
243.0 Hz)), 150.74, 140.69 (140.66), 135.23 (135.16), 133.18 1H), 7.22-7.42 (m, 5H), 5.23-5.40 (m, 2H), 4.53-4.62 (m, 0.4H), 3.96
(133.16), 129.58 (d, J = 7.5 Hz) (129.37 (d, J = 7.0 Hz)), 127.66, (brs, 0.6H), 3.07-3.59 (m, 6H), 1.68-2.23 (m, 2H), 1.37-1.41 (m, 9H),
126.37 (126.07), 124.89 (d, J = 18.1 Hz), 122.77, 116.29 (d, J = 21.4 1.14 (t, J = 6.8 Hz, 1.2H), 0.83 (t, J = 7.2 Hz, 1.8H); 13C-NMR (100
Hz), 116.06, 115.09 (114.98), 56.34, 53.81, 52.46 (51.42), 49.16 MHz, DMSO-d6) δ (ppm): 165.89 (165.11), 161.77, 156.66 (d, J =
(48.98), 44.42 (43.15), 36.59, 27.65, 14.80 (14.33), 10.92; HR-MS 242.6 Hz) (156.39 (d, J = 242.8 Hz)), 153.45 (153.25), 150.72,
(ESI): m/z, calcd. for
439.2155.
C
24H28N4O3F [M+H]+ 439.2140, Found: 140.64, 135.07, 133.21 (133.10), 129.34 (129.12), 127.62, 126.21,
125.37 (d, J = 18.9 Hz) (124.75 (d, J = 19.6 Hz)) , 122.71 (122.67),
116.29 (d, J = 21.2 Hz), 116.05, 114.98, 78.56 (78.46), 57.20 (56.42),
54.20 (53.50), 47.17 (46.81), 44.33 (43.97), 41.28, 35.94, 28.13
(28.09), 15.18 (14.33); HR-MS (ESI): m/z, calcd. for C27H31N4O5FNa
[M+Na]+ 533.2171, Found: 533.2194.
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)
ethylpyrrolidin-3-yl)-2-fluoro-N-propylbenzamide (30)
-N-(1-
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ARTICLE
Journal Name
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -2-fluoro-N-
propyl-N-(pyrrolidin-3-yl)benzamide 2,2,2-trifluoroacetate (36)
tert-Butyl 3-(5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-
2-fluoro-N-propylbenzamido) pyrrolidine-1-carboxylate (33)
Following the preparation protocol of compound 34, starting from
compound 33, the title compound 36 was obtained as a white solid
(85 mg, 93.4%); m.p. 114-116 °C; 1H-NMR (400 MHz, DMSO-d6) δ
(ppm): 11.72 (s, 1H), 8.98 (brs, 1H), 8.60 (brs, 1H), 8.01 (d, J = 7.6
Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.49 (s, 1H), 7.21-7.41 (m, 4H), 5.31
(s, 2H), 4.02-4.33 (m, 1H), 3.50-3.65 (m, 3H), 2.97-3.25(m, 3H), 2.25-
2.35 (m, 1H), 2.10-2.18 (m, 1H), 1.56 (brs, 0.5H), 1.29 (q, J = 7.2 Hz,
1.5H), 0.87 (t, J = 7.2 Hz, 1H), 0.47 (t, J = 7.2 Hz, 2H); HR-MS (ESI):
m/z, calcd. for C23H26N4O3F [M+H]+ 425.1983, Found: 425.1968.
Following the preparation protocol of compound 5, starting from
compound
3
and tert-butyl 3-(propylamino)pyrrolidine-1-
carboxylate, the title compound 33 was obtained as a white solid
1
(160 mg, 63.7%); m.p. 117-119 °C; H-NMR (400 MHz, CDCl3) δ
(ppm): 8.80 (s, 1H), 8.23 (d, J = 8.0 Hz, 1H), 755-7.62 (m, 1H), 7.25-
7.32 (m, 2H), 7.06-7.10 (m, 2H), 5.33 (s, 2H), 4.74-4.78 (m, 0.5H),
4.09 -4.12 (m, 1H), 3.72-3.76 (m,0.5H), 3.04-3.59 (m, 5H), 1.63-2.18
(m, 4H), 1.38-1.46 (m, 9H), 0.96 (t, J = 5.6 Hz, 2H), 0.60 (t, J = 6.4 Hz,
1H); 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 165.98 (165.40),
161.76, 156.62 (d, J = 248.2 Hz) (156.39 (d, J = 242.7 Hz)), 153.40
(153.20), 150.72, 140.63 (140.58), 135.04, 133.21 (133.10), 129.40
(129.33), 127.62, 126.35, 125.33 (d, J = 18.9 Hz) (125.78 (d, J = 18.8
Hz)) , 122.71 (122.68), 116.31 (d, J = 19.1 Hz), 116.04, 114.99
(114.91), 78.54 (78.50), 57.19 (56.43), 54.56 (53.83), 48.39, 47.08
(46.88), 44.30 (43.96), 43.51 (42.80), 28.12 (28.09), 22.92 (21.86),
Note: Except for target compounds, the synthesis and
characterization of all other compounds mentioned in the Scheme 1
were described in the supporting information.
11.26 (10.65); HR-MS (ESI): m/z, calcd. for
[M+Na]+547.2215, Found: 547.2231.
C28H33N4O5F
4.3 Biological evaluation
4.3.1 The assay for PARP-1 and PARP-2 inhibition
Plasmid pET32a-PARP1 was a gift from Prof. Satoh (Canada). Human
recombinant PARP1/2 were expressed and purified as described
[34]. The ability of compounds to inhibit PARP1/2 enzyme activity
were tested using ELISA method as described [35,36]. IC50 values
were calculated using GraphPad Prism 5 software.
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluoro-N-
methyl-N-(pyrrolidin-3-yl)benzamide 2,2,2-trifluoroacetate (34)
To a solution of compound 31 (45 mg, 0.12 mmol) in DCM (2.0 mL),
TFA (2 mL) was added and then the reaction mixture was stirred at
room temperature for 5 h. Ether (20 mL) was added into the
mixture. After filtration, the title compound 34 was afforded as a
white solid (40 mg, 86.5%); m.p. 116-118 °C; 1H-NMR (400 MHz,
DMSO-d6) δ (ppm): 11.74 (s, 1H), 9.00 (brs, 0.5H), 8.91 (brs, 1H),
8.67 (brs, 0.5H), 8.03 (d, J = 8.0 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.46
(brs, 1H), 7.24-7.38 (m, 4H), 5.32 (s, 2H), 4.89-4.97 (m, 0.5H), 4.26
(brs, 0.5H), 3.59 (brs, 2H), 3.17-3.24 (m, 2H), 2.90 (s, 1.5H), 2.76 (s,
1.5H), 2.00-2.10 (m, 2H); HR-MS (ESI): m/z, calcd. for C21H22N4O3F
[M+H]+ 397.1671, Found: 397.1699.
4.3.2 The cytotoxicity assay
MX-1 were purchased from National Infrastruture of Cell Line
Resources, Cells were seeded in
a density in 96-well plates
(2,000cells/well). Cells were treated in their recommended growth
media containing increasing concentrations of PARP inhibitors 24 h
later. After 72 h treatment, cell survival was determined by MTT
assay. IC50 values were calculated using GraphPad Prism 5 software.
5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl) -N-ethyl-2-
fluoro-N-(pyrrolidin-3-yl)benzamide 2,2,2-trifluoroacetate (35)
4.3.3 The PF50 assay in MX-1 cells
In chemosensitization assays, MX-1 cells were seeded in a density
(2,000 cells/well) in 96-well plates. Cells were treated with PARP
inhibitors at a fixed concentration of 10 μmol/L and temozolomide
Following the preparation protocol of compound 34, starting from
compound 32, the title compound 35 was obtained as a white solid
(40 mg, 86.5%); m.p. 111-112 °C; 1H-NMR (400 MHz, DMSO-d6) δ
(ppm): 11.74 (s, 1H), 9.01 (brs, 1H), 8.64 (brs, 1H), 8.04 (s, 1H), 7.64
(s, 1H), 7.25-7.48 (m, 4H), 5.33 (s, 2H), 4.20-4.34 (m, 1H), 2.90-3.54
(m, 6H), 2.32 (s, 1H), 2.16 (s, 1H), 0.88-1.18 (m, 3H); HR-MS (ESI):
m/z, calcd. for C22H24N4O3F [M+H]+ 411.1827, Found: 411.1838.
(TMZ) at different concentration (0-0.5 mmol/L). After 72
h
treatment, cell survival was determined by MTT assay. IC50 values
were calculated using GraphPad Prism5 software. Potentiation
factor (PF50) was calculated as the ratio of the IC50 for TMZ divided
by the IC50 of combination (TMZ + PARP inhibitor).
14 | J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
9. L. Virág and C. Szabó, The Therapeutic Potential of Poly(ADP-
Ribose)Polymerase Inhibitors, Pharmacol. Rev., 2002, 54, 375-
429.
10. J.C. Amé, V. Rolli, V. Schreiber, C. Niedergang, F. Apiou, P.
Decker, S. Muller, T. Höger, J. Ménissierde Murcia and G. de
Murcia, PARP-2, A novel mammalian DNA damage-dependent
poly(ADPribose) polymerase, J. Biol. Chem., 1999, 274, 17860-
17868.
11. K.A. Menear, C. Adcock, R. Boulter, X. Cockcroft, L. Copsey, A.
Cranston, K.J. Dillon, J. Drzewiecki, S. Garman, S. Gomez, H.
Javaid, F. Kerrigan, C. Knights, A. Lau, V.M.L Jr, I.T.W. Matthews,
S. Moore, M.J. O’Connor, G.C.M. Smith and N.M.B. Martin, 4-[3-
4.3.4 Xenograft experiment
The anti-cancer activity as a sensitizer of TMZ using MX-1 xenograft
tumor model was tested as described in our previous work [35].
TMZ and compound 11 were orally administrated respectively or
combined for 5 days. %T/C is calculated as (tumor weight of
treatment group/Vehicle or TMZ treatment group) ×100, while
%TGI is calculated as (100-%T/C). Statistical analyses were
performed by GraphPad Prism5 software and the significance levels
were evaluated using one-way ANOVA model.
(4-Cyclopropanecarbonylpiperazine-1-
fluorobenzyl]-2H-phthalazin-1-one:
carbonyl)-4-
novel bioavailable
A
All animal experiments were approved by the Ethics Committee for
Animal Experiments of the Institute of Materia Medica, Chinese
Academy of Medical Sciences & Peking Union Medical College (No:
00001207) and conducted in accordance with the Guidelines for
Animal Experiments of Peking Union Medical College.
inhibitor of poly(ADP-ribose) polymerase-1, J. Med. Chem.,
2008, 51, 6581-6591.
12. T.D. Penning, G.D. Zhu, J. Gong, V.B. Gandhi, Y. Luo, X. Liu, Y.
Shi, V. Klinghofer, E.F. Johnson, D. Frost, C. Donawho, L.
Rodriguez, G. Bukofzer, K. Jarvis, J. Bouska, D.J. Osterling, A.
Olson, K.C. Marsh, S.H. Rosenberg and V. Giranda, Discovery of
the poly(ADP-ribose) polymerase (PARP) inhibitor 2-[(R)-2-
methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide (ABT-
888) for the treatment of cancer, J. Med. Chem., 2009, 52, 514-
523.
4.4 The X-ray crystallographic experiment
13. S.S. Canan Koch, L.H. Thoresen, J.G. Tikhe, K.A. Maegley, R.J.
Almassy, J. Li, X.H. Yu, S.E. Zook, R.A. Kumpf, C. Zhang, T.J.
Boritzki, R.N. Mansour, K.E. Zhang, A. Ekker, C.R. Calabrese, N.J.
Curtin, S. Kyle, H.D. Thomas, L.Zh. Wang, A. Hilary Calvert, B.T.
Golding, R.J. Griffin, D.R. Newell, S.E. Webber and Z. Hostomsky,
Novel Tricyclic Poly(ADP-ribose) Polymerase-1 Inhibitors with
Potent Anticancer Chemopotentiating Activity: Design,
Synthesis, and X-ray Cocrystal Structure, J. Med. Chem., 2002,
45, 4961-4974.
The protein expression, purification and the crystallographic
experiment were performed as described in our previous work [36].
Acknowledgements
14. P. Jones, S. Altamura, J. Boueres, F. Ferrigno, M. Fonsi, C.
Giomini, S. Lamartina, E. Monteagudo, J.M. Ontoria, M.V.
Orsale, M.C. Palumbi,; S. Pesci, G. Roscilli, R. Scarpelli, C.
Schultz-Fademrecht, C. Toniatti and M. Rowley, Discovery of 2-
{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide (MK-
This work is supported by National Natural Science Foundation of
China (No. 81673300) and CAMS Initiative for Innovative Medicine
(CAMS-I2M-2-004). We greatly appreciate Dr. Niu Huang’s lab
(National Institute of Biological Sciences, Beijing) and Shanghai
Synchrotron Radiation Facility for their support in X-ray
crystallographic experiment.
4827):
A novel oral poly(ADP-ribose)polymerase (PARP)
inhibitor efficacious in BRCA-1 and -2 mutant tumors, J. Med.
Chem., 2009, 52, 7170-7185.
15. Y. Shen, F.L. Rehman, Y. Feng, J. Boshuizen, I. Bajrami, R. Elliott,
B. Wang, C.J. Lord, L.E. Post and A. Ashworth, BMN 673, a Novel
and Highly Potent PARP1/2 Inhibitor for the Treatment of
Human Cancers with DNA Repair Deficiency, Clin. Cancer Res.,
2013, 19, 5003-5015.
16. B. Wang, D. Chu, Y. Feng, Y.Q. Shen, M. Aoyagi-Scharber and
L.E. Post, Discovery and Characterization of (8S,9R)-5-Fluoro-8-
(4-fluorophenyl)-9-(1-methyl 1H 1,2,4-triazol-5-yl)-2,7,8,9-
tetrahydro 3H pyrido[4,3,2-de]phthalazin-3-one (BMN 673,
Talazoparib), a Novel, Highly Potent, and Orally Efficacious
Poly(ADP-ribose) Polymerase-1/2 Inhibitor, as an Anticancer
Agent, J. Med. Chem., 2016, 59, 335-357.
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Discovery of quinazoline-2,4(1H,3H)-dione derivatives as novel PARP-1/2
inhibitions: design, synthesis and their antitumor activity
Jie Zhou, Ming Ji, Haiping Yao, Ran Cao, Hailong Zhao, Xiaoyu Wang, Xiaoguang
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