Discovery of 1-substituted benzyl-quinazoline-2,4(1H,3H)-dione derivatives as novel poly(ADP-ribose)polymerase-1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.12.071

Poly(ADP-ribose)polymerase-1 (PARP-1) has emerged as a promising anticancer drug target due to its key role in the DNA repair process. In this work, a novel series of 1-benzyl-quinazoline-2,4(1H,3H)-dione derivatives were designed and synthesized as human PARP-1 inhibitors, structure-activity relationships were conducted and led to a number of potent PARP-1 inhibitors having IC₅₀ values of single or double digit nanomolar level. Compound 7j was a potent PARP-1 and PARP-2 inhibitor and it could selectively kill the breast cancer cells MX-1 and MDA-MB-468 with mutated BRCA1/2 and PTEN, respectively, in comparison with homologous recombination proficient cell types such as breast cancer cells MDA-MB-231. In addition, compound 7j displayed the strongest potentiation effect on temozolomide in MX-1 cells (PF₅₀ = 3.77) in this series of PARP-1 inhibitors.

Full text of DOI:10.1016/j.bmc.2014.12.071

Accepted Manuscript
Discovery of 1-substituted benzyl-quinazoline-2,4(1H,3H)-dione derivatives as
novel poly(ADP-ribose)polymerase-1 inhibitors
Haiping Yao, Ming Ji, Zhixiang Zhu, Jie zhou, Ran Cao, Xiaoguang Chen,
Bailing Xu
PII:
S0968-0896(15)00003-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.12.071
BMC 12003
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 October 2014
31 December 2014
31 December 2014
Please cite this article as: Yao, H., Ji, M., Zhu, Z., zhou, J., Cao, R., Chen, X., Xu, B., Discovery of 1-substituted
benzyl-quinazoline-2,4(1H,3H)-dione derivatives as novel poly(ADP-ribose)polymerase-1 inhibitors, Bioorganic
& Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2014.12.071
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Discovery of 1-substituted benzyl-quinazoline-2,4(1H,3H)-dione
derivatives as novel poly(ADP-ribose)polymerase-1 inhibitors
Haiping Yaob,#, Ming Jia,#, Zhixiang Zhua, Jie zhoub, Ran Caob,
Xiaoguang Chena,*, Bailing Xub,*
aState Key Laboratory of Bioactive Substances and Functions of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences＆Peking Union
Medical College, Beijing, 100050, China
bBeijing Key Laboratory of Active Substance Discovery and Druggability Evaluation,
Institute of Materia Medica, Chinese Academy of Medical Sciences＆Peking Union
Medical College, Beijing, 100050, China
Abstract
Poly(ADP-ribose) polymerase-1 (PARP-1) has emerged as a promising anticancer drug target
due to its key role in the DNA repair process. In this work, a novel series of
1-benzyl-quinazoline-2,4(1H,3H)-dione derivatives were designed and synthesized as human
PARP-1 inhibitors, structure-activity relationships were conducted and led to a number of potent
PARP-1 inhibitors having IC50 values of single or double digit nanomolar level. Compound 7j was
a potent PARP-1 and PARP-2 inhibitor and it could selectively kill the breast cancer cells MX-1
and MDA-MB-468 with mutated BRCA1/2 and PTEN respectively, in comparison with
homologous recombination proficient cell types such as breast cancer cells MDA-MB-231. In
addition, compound 7j displayed the strongest potentiation effect on temozolomide in MX-1 cells
(PF50 = 3.77) in this series of PARP-1 inhibitors.
Key words: PARP-1 inhibitor; PARP-2 inhibitor; quinazoline-2,4(1H,3H)-dione; antitumor
activity.
1. Introduction
Poly (ADP-ribose) polymerase-1 (PARP-1) belongs to the PARP nuclear enzyme superfamily
containing 17 members.1-3 It has three domains including an N-terminal DNA-binding domain
with three zinc fingers, a central auto-modification domain and a C-terminal catalytic domain.4, 5
When the damaged DNA was recognized by PARP-1 through its DNA-binding domain, the
enzymatic activity of PARP-1 was rapidly increased to about 500-fold. The activated PARP-1 can
# The first two authors contributed equally.
* Corresponding author. B. Xu, Tel: +86 10 63166764; X. Chen, Tel: +86 10 63165207
E-mail address: xubl@imm.ac.cn (B. Xu); chxg@imm.ac.cn (X. Chen)

bind to nicotinamide adenine dinucleotides (NAD+) through its catalytic binding site, and cleave
the NAD+ to generate nicotinamide and ADP-ribose units, the latter is successively transferred
onto the nuclear DNA-binding proteins and PARP-1 itself to form poly(ADP-ribose) (PAR).6, 7
The poly(ADP-ribosyl)ation of various proteins is believed to be an essential post-translational
modification in modulating a plethora of cellular functions, including DNA repair.3, 8
It has been demonstrated that PARP-1 plays a pivotal role in the repair of single strand breaks
of DNA by the base excision repair (BER) pathway.9-11 Therefore, the inhibition of PARP-1
activity can augment the cytotoxic effects of the DNA damaging agents (e.g. temozolomide,
cyclophosphamide and camptothecin) which cause lesions normally repaired by BER
pathway.12-16 Moreover, given the key role of PARP-1 in DNA repair, PARP-1 inhibitors can
induce synthetic lethality in cells defective in homologous recombination (HR) repair, such as
tumor cells with mutations in BRCA1 and BRCA2.17-19 Thus PARP-1 inhibitors are expected to
kill BRCA defective tumor cells effectively and selectively. Taken together, these promising
results have further sparked tremendous research works to exploit PARP-1 inhibition as a
therapeutic approach in cancer treatment.20, 21
Nicotinamide and 3-aminobenzamide were identified as PARP-1 inhibitors more than 30
years ago,22 and since then many structural diversified PARP-1 inhibitors have been
investigated.23-26 The devoted efforts have resulted in a number of drug candidates undergoing
clinical trials including AZD-2281, ABT-888, AG014699, MK-4827 and BMN673 (Figure 1).27-31
These candidates were used either as a single agent or in combination with chemotherapy for the
treatment of a wide variety of cancers including breast, ovarian, pancreatic and melanoma tumors
with or without BRCA1/2 deficiency. Recently, it has been reported that AZD-2281 was most
likely to be beneficial to the patients with platinum-sensitive recurrent serous ovarian cancer with
BRCA1/2 mutations.32 Nonetheless, so far there are no PARP-1 inhibitors as anticancer drugs that
have reached the market. It is still a great challenge to develop PARP-1 inhibitors with desired
pharmacodynamic and pharmacokinetic properties as novel therapeutic agents in cancer
treatment.33
The NAD+ binding site of PARP-1 contains two characteristic binding regions, namely, a
nicotinamide-ribose binding site (NI site) and an adenine-ribose binding site (AD site).34 All of the
known PARP-1 inhibitors interacted with the NI site through three key hydrogen bonds formed
between the lactam or the carboxamide group of the small molecules and residues Ser904 and

Gly863.35, 36 In addition, a π-π stacking interaction between the aromatic scaffold of PARP-1
inhibitors and Tyr907 of the NI site was generally observed as another key feature. Compared with
the relatively narrow NI site, the AD site of PARP-1 is rather large and may be used to search for a
wide variety of novel inhibitors with the improved potency and pharmacokinetic properties. In fact,
some of the reported PARP-1 inhibitors such as AZD-2281 and PJ-34 took advantage of this AD
site to further improve their binding affinities.37 In this work,
a
series of
quinazoline-2,4(1H,3H)-dione derivatives (7a-7s, 8a-8t) were designed based on the
structure–activity relationships (SAR) of the known PARP inhibitors and their binding features in
the PARP active site.26 The 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 NI site;34 a 3-amino and
3-amino-4-fluoro benzyl groups were used as a linker to direct the amino acid motifs into the AD
site. We investigated the SARs of this series of quinazoline-2,4(1H,3H)-dione derivatives by
tentatively utilizing various amino acid building blocks to explore the AD site. Herein, the
chemical synthesis of these new quinazoline-2,4(1H,3H)-dione derivatives is described in details.
The PARP-1inhibitory activities of these compounds are presented along with their SAR analysis.
Some of the potent PARP-1 inhibitors were further evaluated in terms of their PARP-2 inhibitory
activities, growth inhibition and potentiation effect on temozolomide (TMZ) in cancer cells.
2. Chemistry
The quinazoline-2,4(1H,3H)-dione derivatives (8a–8t) were prepared according to the
synthetic route as outlined in Scheme 1. The substituted 2-aminobenzoic acids were condensed
with urea in a solvent-free condition providing the corresponding quinazoline-2,4(1H,3H)-dione
scaffolds (2a-2e).38 In the presence of hexamethyldisilazane (HMDS) and concentric sulfuric acid,
compounds 2a-2e in toluene were first transformed into silylated compounds 3a-3e, which were
then reacted with 3-nitro benzyl bromide or 4-fluoro-3-nitro benzyl bromide to generate the N-1
substituted intermediates 4a-4f. Upon treatment of the reaction with methanol, compounds 5a-5f
were obtained in 25-77% yields.39 Compounds 6a-6f, which were obtained by reducing
compounds 5a-5f, were converted into compounds 7a-7s and 8t by coupling with a range of
N-Boc protected amino acids in the presence of HBTU/HOBt/DIEA, HATU/HOBt/TEA or
EDCI/HOBt/TEA in moderate to good yields. The deprotection of Boc group in compounds 7a-7s
using TFA offered the target compounds 8a-8s in good yields.
3. Results and discussion

3.1 Enzymatic activity against PARP-1
The inhibitory activities against PARP-1 of all target compounds (8a-8t) and their Boc
protected derivatives (7a-7s) were evaluated. Several potent PARP-1 inhibitors were chosen to
further test their inhibition effect on PARP-2. The clinical drug candidates ABT888 and AZD2281
were used as reference molecules. The corresponding results were expressed as IC50 values and
presented in Tables 1–4.
Initially, a number of α-amino acids were introduced onto 3-position of the phenyl ring of the
benzyl group by coupling with amines 6a and 6b. As shown in Table 1, the target compounds
(8a-8g and 8t) exhibited greatly varied inhibitory activities against PARP-1 with IC50 values
ranging from 48.2 nM to 5.29 µM. Compound 8a containing a glycine moiety showed moderate
inhibitory activity against PARP-1 with an IC50 value of 171 nM. Mono- or dimethylation on the
glycine nitrogen atom (compounds 8b and 8t) led to a decrease in activity, and N,N-dimethyl
compound 8t showed a much weaker inhibition, which was about 25 times less potent than
compound 8a. It was indicated that a tertiary amine was not favorable presumably due to the steric
hinderance caused by dimethyl group. Compound 8c (IC50: 5.29 µM) was the least active of all the
target compounds, which indicated that bulky groups such as an isopropyl group substituted at C-2
position of the glycine moiety could be detrimental to the enzymatic potency. When a proline
fragment was introduced, the (R)- enantiomer (8d) displayed comparable activity to compound 8a,
while the S-enantiomer (8f) was 16 times less potent than 8a and 5 times less potent than the (R)-
enantiomer. It was demonstrated that the (R)-enantiomer was preferred to the binding interactions.
Noticeably, the placement of a fluoro atom at para-position of the benzyl linker resulted in a
significant improvement in potency, as compounds 8e and 8g had >10 times stronger inhibition
than the corresponding des-fluoro derivatives 8d and 8f, respectively. Compound 8e was the most
potent inhibitor in this series and displayed inhibitory activity at double digit nanomolar level.
This result was consistent with the structure-activity relationships of the known PARP-1
inhibitors.40 As for the Boc-protected derivatives 7a-7g, they all displayed dramatic decreases in
potency in comparison with their counterparts 8a-8g. It was indicated that the large bulky group
on the nitrogen atom was not beneficial to the binding affinity, which was consistent with the
result demonstrated by compound 8t.
Based on the preliminary SAR results from α-amino acids as depicted above, we then turned
our attention to the cyclic β-amino acids such as β-proline or piperidine-3-carboxylic acid as the
key pharmacophoric groups (Table 2). These β-amino acids were coupled with compounds 6a and

6b to furnish the target compounds 8h-8m. Surprisingly, compounds 8h-8k containing either
(R)-isomer or (S)-isomer of β-proline, all were highly active against PARP-1 with IC50 values of
double digit nanomolar. The fluoro group substituted compounds (8i and 8k) displayed similar
activities to the unsubstituted derivatives (8h and 8j) as well. In contrast, when
piperidine-3-carboxylic amide was installed at 3-position of benzyl linker, compound 8m with
fluoro atom was >10 times more potent than the unsubstituted derivative 8l. Of note, all the
Boc-protected derivatives (7h-7m) also exhibited potent inhibitory activities , with an exception of
compound 7l. It was suggested that the nitrogen atom on pyrrolidine or piperidine ring in this
series was allowed to modify, and therefore it provided a chance for further developing more
potent and druggable candidates. Taken together, these results demonstrated that β-proline and
piperidine-3-carboxylic amide moieties were more favorable than α-proline subunit for PARP-1
inhibitory activity.
The cyclic γ-amino acid, as a key structural fragment, was also primarily explored (Table 3).
The piperidine-4-carboxylic acid was incorporated through amide bond at 3-position of benzyl
group. The introduction of a fluoro atom at para position of the benzyl group greatly increased the
inhibitory activity (compound 8n vs 8o) and as a result compound 8o had an IC50 value of 23.1
nM. The substitution with a fluoro atom on the quinazoline-2,4(1H,3H)-dione scaffold was also
investigated. The 5-, 6- and 8-fluoro substituted analogs (8p, 8q and 8s) displayed comparable
activities to the unsubstituted compound 8o, and 7-fluoro analog (8r) showed about 10 times
reduction in potency in comparison with compound 8o. The corresponding Boc protected
derivatives (7n-7s) were tested as well. In general, the inhibitory potency was decreased to some
extent. Surprisingly, compound 7p showed a 3-fold enhancement in potency (compound 7p vs 8p),
and had an IC50 value of 9.51 nM, which was the most potent PARP-1 inhibitor of all the tested
compounds.
3.2 Enzymatic activity against PARP-2
Among the numerous members of the PARP superfamily, in addition to PARP-1, PARP-2 is
another enzyme that has been characterized to be involved in DNA repair.41 Due to the high
homology of the catalytic domain between PARP-1 and PARP-2, PARP-1 inhibitors usually bind
to PARP-2 as well.42 In fact, many of the reported PARP-1 inhibitors displayed comparable
inhibitory activities against PARP-2. In this work, some potent PARP-1 inhibitors were selected to
test their inhibition effects on PARP-2 (Table 4). As expected, most of them had similar inhibitory
activities (less than 10-fold difference) against both PARP-1 and PARP-2. However, compound 7k

showed its strong preference for binding to PARP-2. It presented the most potent inhibition effect
on PARP-2 (IC50 = 1.47 nM) and had high selectivity over PARP-1 (IC50: PARP1/PARP2 = 15/1).
Further molecular modeling study on compound 7k may help to develop more selective PARP-2
inhibitors, which had been reported in a very few literatures.34, 43
3.3 Cellular Potency
The cellular potency of 18 compounds, which acted as potent PARP-1 inhibitors with IC50
values of 10-100 nM, were evaluated for their cytotoxicities as single agents in MX-1 breast
cancer cells with mutated BRCA1 and BRCA2. Among them, 10 compounds had cytotoxicities
with IC50 values lower than 50 µM, which were more potent than ABT-888 (Table 5). Others were
inactive (IC50, >100 µM) as single agents. The potentiation effects on the DNA damaging agent
temozolomide (TMZ) of 9 compounds were evaluated at 5 µM concentration using MX-1 cell
lines. Compounds 7j and 8e could strongly potentiate TMZ cytotoxicities in comparison with
other tested compounds, although their potentiation effects were weaker than that of ABT-888 at
this concentration.
It has been reported that tumor cells with defective homologous recombination (HR) repair
are highly sensitive to PARP-1 inhibitors. In addition to the well known tumor-suppressor genes
BRCA1 and BRCA2, other genes such as PTEN and PIK3CA, which were involved in DNA
damage repair, displayed synthetic lethality with PARP-1 as well.44-46 Accordingly, we further
evaluated the cytotoxicities of compound 7j using a number of cancer cell lines with or without
deficient and mutated genes in DNA HR repair. As summarized in Table 6, compound 7j was
highly active in MX-1, MDA-MB-468, MDA-MB-453 cancer cells with mutated genes of
BRCA1/2, PTEN and PIK3CA, respectively, whereas it was less cytotoxic in BRCA2 deficient
pancreatic cancer cell line (Capan-1). In contrast, it was much less cytotoxic in MDA-MB-231
breast cancer cells without deficiency in HR repair, and this result is consistent with that of the
known PARP-1 inhibitors. In addition, compound 7j was more cytotoxic than reference molecule
AZD-2281 in all tested cell lines. Surprisingly, compound 7j showed high growth inhibition in
ovarian cancer cells A2780, although this cell line did not carry the mutant gene of HR.
Presumably, the off-target effect might exist, and that led to the A2780 cancer cells to be sensitive
to compound 7j.
3.4 Prediction of the binding mode
To gain insights into the binding mode of the designed compounds, compound 7j which was
highly potent in enzymatic activity as well as in cell assay, was docked into the co-crystal

structure of BMN673 in PARP-1 (PDB code, 4PJT). As we expected, the
quinazoline-2,4(1H,3H)-dione scaffold bound to the NI site and the N-Boc-pyrrolidin-3-yl
fragment extended into the AD site (Figure 2A). As shown in Figure 2B, three characteristic
hydrogen bonds were formed between the lactam group in compound 7j and Gly863 as well as
Ser904. In addition, another hydrogen bond was also formed between amide nitrogen on the
phenyl ring and Gly894, which was not observed in the known PARP-1 inhibitors. Two key π-π
stacking interactions were built up between the quinazolinone and 2-fluorophenyl motif in
compound 7j and the amino acid residues Tyr907 and Tyr896, respectively. These π-π stacking
interactions were believed to make significant contributions to the binding affinity as they were
frequently exploited by the known PARP-1 inhibitors.27-31 Furthermore, the carbonyl group in Boc
motif was surrounded by the positive charged side chain of Arg878, which could contribute the
binding affinity through the charge-dipole interaction. It was noted that the Boc group on the
pyrrolidine ring was situated in a spacious subpocket and extended into a solvent exposed surface.
Therefore, modifications on the nitrogen of the pyrrolidine ring would offer more chances to
further improve the binding affinity and drug-like properties of this series of PARP-1 inhibitors.
4. Conclusion
In summary, a novel series of quinazoline-2,4(1H,3H)-dione derivatives were designed and
synthesized by utilizing an array of amino acid building blocks as key pharmacophoric groups.
The SAR studies demonstrated that the β-proline and piperidine-4-carboxylic acid would be the
favorable structural fragments in this series of PARP-1 inhibitors because most of compounds with
these two subunits demonstrated inhibitory activities with IC50 values of single or double digit
nanomolar. Compound 7j showed high inhibitory activity in both enzymatic and cellular level,
which could serve as a new structure template for further optimization to explore novel potent
PARP-1 inhibitors.
5. Experimental section
5.1. General
Melting points were measured on a Yanaco micro melting point apparatus and are
uncorrected.1H NMR (300 MHz or 400 MHz) on a Varian Mercury 300 or 400 spectrometer was
recorded in DMSO-d6, acetone-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 chemicals and solvents used
were of reagent grade without purified or dried before use. All the reactions were monitored by

thin-layer chromatography (TLC) on pre-coated silica gel G plates at 254 nm under a UV lamp.
Column chromatography separations were performed with silica gel(200–300 mesh).
5.2. Synthesis of compounds 8a–8t
5.2.1 Synthesis of quinazoline-2,4(1H,3H)-diones (2a-2e)
5.2.1.1 Quinazoline-2,4(1H,3H)-dione (2a) The mixture of 2-amino-benzoic acid (5 g, 36.46
mmol) and urea (50 g, 83.25 mmol) was stirred at 150℃ for 7 h. The reaction mixture was cooled
to 100℃ and then water (50 mL) was added to quench the reaction. The crude product was
obtained by filtration, then dissolved in NaOH aq. (6 M, 500 mL). The pH was adjusted to 3 and a
precipitate was formed. After filtration and dried under vacuum condition, compound 2a was
1
obtained as white solid (4.5 g, 76.1%); m.p. >250℃; H NMR (400 MHz, DMSO-d6) δ (ppm):
11.26 (s, 1H), 11.12 (s, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.14-7.19 (m, 2H);
HR-MS (ESI): m/z, calcd. for C8H7N2O2 [M+H]+ 163.0502, Found: 163.0500.
5.2.1.2 5-Fluoroquinazoline-2,4(1H,3H)-dione (2b) Following the preparation protocol of Section
5.2.1.1, starting from 2-amino-6-fluorobenzoic acid (2 g, 12.9 mmol), the title compound 2b was
1
obtained as white solid (738 mg, 31.8%); m.p.>250℃; H NMR (300 MHz, DMSO-d6) δ (ppm):
11.27 (s, 1H), 11.25 (s, 1H), 7.60 (dt, J1 = 8.4 Hz, J2 = 5.7 Hz, 1H), 6.88-6.99 (m, 2H); HR-MS
(ESI): m/z, calcd. for C8H6N2O2F [M+H]+ 181.0408, Found: 181.0404.
5.2.1.3 6-Fluoroquinazoline-2,4(1H,3H)-dione (2c) Following the preparation protocol of Section
5.2.1.1, starting from 2-amino-5-fluorobenzoic acid (2 g, 12.9 mmol), the title compound 2c was
obtained as white solid (1.76 g, 75.8%); m.p.235-237℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm):
11.18 (brs, 2H), 7.47-7.58 (m, 2H), 7.18 (dd, J1 = 8.7 Hz, J2 = 4.2 Hz, 1H); HR-MS (ESI): m/z,
calcd. for C8H6N2O2F [M+H]+ 181.0408, Found: 181.0404.
5.2.1.4 7-Fluoroquinazoline-2,4(1H,3H)-dione (2d) Following the preparation protocol of Section
5.2.1.1, starting from 2-amino-4-fluorobenzoic acid (1.5 g, 9.67 mmol), the title compound 2d was
obtained as yellow solid (1.13 g, 61.9%); m.p.139-141℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm):
11.35 (s, 1H), 11.26 (s, 1H), 7.95 (dd, J1 = 8.7 Hz, J2 = 6.6 Hz, 1H), 7.00-7.06 (m, 1H), 6.88-6.92
(m, 1H); HR-MS (ESI): m/z, calcd. for C8H6N2O2F [M+H]+ 181.0408, Found: 181.0404.
5.2.1.5 8-Fluoroquinazoline-2,4(1H,3H)-dione (2e) Following the preparation protocol of Section
5.2.1.1, starting from 2-amino-3-fluorobenzoic acid (500 mg, 3.22 mmol), the title compound 2e
was obtained as yellow solid (350 mg, 60.3%); m.p.241-243℃; 1H NMR (300 MHz, DMSO-d6) δ

(ppm): 11.43 (s, 1H), 11.28 (s, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.56 (ddd, J1 = 10.8 Hz, J2 = 8.4 Hz ,
J3 = 1.2 Hz , 1H), 7.16 (td, J1 = 8.1 Hz, J2 = 4.8 Hz, 1H) ; HR-MS (ESI): m/z, calcd. for
C8H6N2O2F [M+H]+ 181.0408, Found: 181.0405.
5.2.2 Synthesis of 1-benzyl-quinazoline-2,4(1H,3H)-diones (5a-5f)
5.2.2.1 1-(3-Nitrobenzyl)quinazoline-2,4(1H,3H)-dione (5a) To a suspension of 2a (140 mg, 0.86
mmol) in toluene (4 mL) and hexamethyldisilazane (HMDS; 347 mg, 2.15 mmol), three drops of
sulfuric acid were added with caution. The mixture was heated to reflux and stirred under
refluxing for 8 h untill clear solution was obtained. After the removal of toluene and excess
HMDS under vacuum distillation, 1-(bromomethyl)-3-nitrobenzene (744 mg, 3.44 mmol) was
added to the residue. The reaction mixture was heated to 130°C and was stirred at this temperature
for 3 h, the reaction mixture was diluted with 1,4-dioxane (2 mL) at 100°C, and then methanol (3
mL) was added at 70°C for 30 min. The suspension was cooled below 5°C and precipitates were
collected by filtriation. After washing with methanol (5 mL) and water (5 mL), the crude product
was dried under vacuum condition to afford 5a as white solid (200 mg, 77.3%); m.p. > 250 ℃; 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 11.79 (s, 1H), 8.25 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.04 (d,
J = 8.0 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.60-7.68 (m, 2H), 7.31 (d, J = 8.8 Hz, 1H), 7.26 (t, J =
7.6 Hz, 1H), 5.46 (s, 2H); HR-MS (ESI): m/z, calcd. for C15H12N3O4 [M+H]+ 298.0822, Found:
298.0816.
5.2.2.2 1-(4-Fluoro-3-nitrobenzyl)quinazoline-2,4(1H,3H)-dione (5b) Following the preparation
protocol of Section 5.2.2.1, starting from quinazoline-2,4(1H,3H)-dione (500 mg, 3.08 mmol), the
1
title compound 5b was obtained as yellow solid (644 mg, 66.2%); m.p. > 250 ℃; H NMR (400
MHz, DMSO-d6) δ (ppm): 11.75 (s, 1H), 8.18 (d, J = 6.4 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.75
(m, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.51-7.57 (m, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.26 (t, J = 7.6 Hz,
1H), 5.38 (s, 2H); HR-MS (ESI): m/z, calcd. for C15H11N3O4F [M+H]+ 316.0728, Found:
316.0722.
5.2.2.3 5-Fluoro-1-(4-fluoro-3-nitrobenzyl)quinazoline-2,4(1H,3H)-dione (5c) Following the
preparation protocol of Section 5.2.2.1, starting from 5-fluoroquinazoline-2,4(1H,3H)-dione (320
mg, 1.78 mmol), the title compound 5c was obtained as gray solid (350 mg, 59.2%); m.p. >
1
250 ℃; H NMR (300 MHz, DMSO-d6) δ (ppm): 11.71 (s, 1H), 8.18 (d, J = 5.4 Hz, 1H),
7.73-7.77 (m, 1H), 7.62 (dt, J1 = 8.4 Hz, J2 = 5.7 Hz, 1H), 7.54 (dd, J1 = 11.1 Hz, J2 = 8.4 Hz, 1H),
7.00-7.10 (m, 2H), 5.37 (s, 2H); HR-MS (ESI): m/z, calcd. for C15H10N3O4F2 [M+H]+ 334.0634,

Found: 334.0633.
5.2.2.4 6-Fluoro-1-(4-fluoro-3-nitrobenzyl)quinazoline-2,4(1H,3H)-dione (5d) Following the
preparation protocol of Section 5.2.2.1, starting from 6-fluoroquinazoline-2,4(1H,3H)-dione (500
mg, 2.78 mmol), the title compound 5d was obtained as white solid (450 mg, 48.6%); m.p. >
250 ℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.88 (s, 1H), 8.18 (dd, J1 = 7.2 Hz, J2 = 1.8 Hz,
1H), 7.70-7.76 (m, 2H), 7.50-7.59 (m, 2H), 7.34 (dd, J1 = 9.0 Hz, J2 = 3.9 Hz, 1H), 5.38 (s, 2H);
HR-MS (ESI): m/z, calcd. for C15H10N3O4F2 [M+H]+ 334.0634, Found: 334.0632.
5.2.2.5 7-Fluoro-1-(4-fluoro-3-nitrobenzyl)quinazoline-2,4(1H,3H)-dione (5e) Following the
preparation protocol of Section 5.2.2.1, starting from 7-fluoroquinazoline-2,4(1H,3H)-dione (150
mg, 0.84 mmol), the title compound 5e was obtained as white solid (153 mg, 55.1%); m.p. >
250 ℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.81 (s, 1H), 8.18 (dd, J1 = 6.9 Hz, J2 = 1.8 Hz,
1H), 8.07 (dd, J1 = 8.4 Hz, J2 = 6.3 Hz, 1H), 7.75-7.78 (m, 1H), 7.54 (dd, J1 = 11.1 Hz, J2 = 8.7 Hz,
1H), 7.24 (dd, J1 = 11.4 Hz, J2 = 2.1 Hz, 1H), 7.11 (dt, J1 = 8.4 Hz, J2 = 2.1 Hz, 1H), 5.36 (s, 2H);
HR-MS (ESI): m/z, calcd. for C15H10N3O4F2 [M+H]+ 334.0634, Found: 334.0634.
5.2.2.6 8-Fluoro-1-(4-fluoro-3-nitrobenzyl)quinazoline-2,4(1H,3H)-dione (5f) Following the
preparation protocol of Section 5.2.2.1, starting from 8-fluoroquinazoline-2,4(1H,3H)-dione (700
mg, 3.89 mmol), the title compound 5f was obtained as yellow solid (317 mg, 24.6%); m.p. >
250 ℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.92 (s, 1H), 8.10 (dd, J1 = 7.2 Hz, J2 = 1.8 Hz,
1H), 7.90 (dd, J1 = 7.8 Hz, J2 = 0.9 Hz , 1H), 7.73-7.76 (m, 1H), 7.50-7.61 (m, 2H), 7.27 (td, J1 =
8.1 Hz, J2 = 4.2 Hz, 1H), 5.38 (s, 2H); HR-MS (ESI): m/z, calcd. for C15H10N3O4F2 [M+H]+
334.0634, Found: 334.0631.
5.2.3 Synthesis of 1-benzyl-quinazoline-2,4(1H,3H)-diones (6a-6f)
5.2.3.1 1-(3-Aminobenzyl)quinazoline-2,4(1H,3H)-dione (6a) The mixture of compound 5a (120
mg, 0.40 mmol) and 10% Pd-C (60 mg) in methanol (25 mL) and ethanol (15 mL) was
hydrogenated at room temperature and 1 atm for 30 min. Pd-C was filtered, The crude product
obtained after concentration was purified with column chromatography to afford compound 6a as
1
white solid (106 mg, 98.2%); m.p. > 250 ℃; H NMR (400 MHz, DMSO-d6) δ (ppm): 11.70 (s,
1H), 8.01 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.18-7.25 (m, 2H), 6.95 (t, J = 7.6 Hz, 1H),
6.39-6.44 (m, 2H), 6.36 (s, 1H), 5.15 (s, 2H), 5.05 (s, 2H); HR-MS (ESI): m/z, calcd. for
C15H14N3O2 [M+H]+ 268.1080, Found: 268.1076.

5.2.3.2 1-(3-Amino-4-fluorobenzyl)quinazoline-2,4(1H,3H)-dione (6b) Following the preparation
protocol of Section 5.2.3.1, starting from 5b (454 mg, 1.44 mmol), the title compound 6b was
obtained as white solid (290 mg, 70.6%); m.p. > 250 ℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm):
11.76 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H), 6.88-7.33 (m, 7H), 5.23 (s, 2H);
HR-MS (ESI): m/z, calcd. for C15H13N3O2F [M+H]+ 286.0986, Found: 286.0980.
5.2.3.3 1-(3-Amino-4-fluorobenzyl)-5-fluoroquinazoline-2,4(1H,3H)-dione (6c) Following the
preparation protocol of Section 5.2.3.1, starting from 5c (270 mg, 0.81 mmol), the title compound
6c was obtained as white solid (220 mg, 89.6%); m.p. > 250 ℃; 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 11.74 (s, 1H), 8.10 (dd, J1 = 14.0 Hz, J2 = 8.0 Hz, 1H), 7.12 (t, J = 10.0 Hz, 1H),
7.00-7.05 (m, 2H), 6.86-6.93 (m, 2H), 6.43 (brs, 2H), 5.21 (s, 2H); HR-MS (ESI): m/z, calcd. for
C15H12N3O2F2 [M+H]+ 304.0892, Found: 304.0886.
5.2.3.4 1-(3-Amino-4-fluorobenzyl)-6-fluoroquinazoline-2,4(1H,3H)-dione (6d) Following the
preparation protocol of Section 5.2.3.1, starting from 5d (300 mg, 0.90 mmol), the title compound
6d was obtained as white solid (230 mg, 84.3%); m.p. > 250 ℃; 1H NMR (300 MHz, DMSO-d6)
δ (ppm): 11.87 (s, 1H), 7.72 (dd, J1 = 8.4 Hz, J2 = 3.0 Hz, 1H), 7.56 (dt, J1 = 8.7 Hz, J2 = 3.0 Hz,
1H), 7.25 (dd, J1 = 9.0 Hz, J2 = 3.9 Hz, 1H), 6.92 (dd, J1 = 11.4 Hz, J2 = 8.4 Hz, 1H), 6.58 (d, J =
9.0 Hz, 1H), 6.46-6.48 (m, 1H), 5.15 (s, 2H), 5.12 (s, 2H); HR-MS (ESI): m/z, calcd. for
C15H12N3O2F2 [M+H]+ 304.0892, Found: 304.0886.
5.2.3.5 1-(3-Amino-4-fluorobenzyl)-7-fluoroquinazoline-2,4(1H,3H)-dione (6e) Following the
preparation protocol of Section 5.2.3.1, starting from 5e (150 mg, 0.45 mmol), the title compound
6e was obtained as yellow solid (120 mg, 87.9%); m.p. > 250 ℃; 1H NMR (300 MHz, DMSO-d6)
δ (ppm): 11.82 (s, 1H), 8.07 (dd, J1 = 8.7 Hz, J2 = 6.6 Hz, 1H), 7.04-7.14 (m, 3H), 6.77-6.85 (m,
5H), 5.19 (s, 2H); HR-MS (ESI): m/z, calcd. for C15H12N3O2F2 [M+H]+ 304.0892, Found:
304.0885.
5.2.3.6 1-(3-Amino-4-fluorobenzyl)-8-fluoroquinazoline-2,4(1H,3H)-dione (6f) Following the
preparation protocol of Section 5.2.3.1, starting from 5f (30 mg, 0.09 mmol), the title compound
1
6f was obtained as yellow solid (20 mg, 73.3%); m.p. > 250 ℃; H NMR (400 MHz, DMSO-d6)
δ (ppm): 11.92 (s, 1H), 7.90 (d, J = 7.2 Hz, 1H), 7.56 (dd, J1 = 14.0 Hz, J2 = 8.0 Hz, 1H), 7.26 (m,
1H), 6.89 (t, J = 9.6 Hz, 1H), 6.56 (d, J = 8.4 Hz, 1H), 6.35 (m, 1H), 5.24 (s, 2H); HR-MS (ESI):

m/z, calcd. for C15H12N3O2F2 [M+H]+ 304.0892, Found: 304.0886.
5.2.4 Synthesis of compounds 7a-7s and 8t
5.2.4.1 tert-Butyl (2-((3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)amino)-
2-oxoethyl)carbamate (7a) To a stired solution of (tert-butoxycarbonyl)glycine (162 mg, 0.92
mmol) in DMF was added EDC (176 mg, 0.92 mmol)、HOBt (125 mg, 0.92 mmol) and DMAP
(10 mg, 0.077 mmol), then TEA (156 mg, 1.54 mmol) was added dropwise. The mixture was
stired at room temperature for 30 min, then 6a (205 mg, 0.77 mmol) was added, and the reaction
mixture was continuously stired at room temperature overnight. The solution was evaporated to
dryness and DCM (2 mL) was added to the residue, a crude product was obtained after filtration,
1
and recrystallized with DCM to afford 7a as white solid (200 mg, 61.2%); m.p. 209-211℃; H
NMR (400 MHz, DMSO-d6) δ (ppm): 11.75 (s, 1H), 9.87 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.63 (t,
J = 7.6 Hz, 1H ), 7.56 (d, J = 8.0 Hz, 1H), 7.18-7.34 (m, 4H), 6.96-7.02 (m, 2H), 5.28 (s, 2H),
3.65 (d, J = 6.0 Hz, 2H), 1.36 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 168.22, 161.81,
155.86, 150.59, 140.82, 139.38, 136.97, 135.23, 129.14, 127.57, 122.70, 121.27, 117.82, 116.30,
115.82, 115.11, 77.80, 45.09, 43.71, 28.17; HR-MS (ESI): m/z, calcd. For C22H24N4NaO5
447.1639 [M+Na]+, Found: 447.1652.
5.2.4.2 tert-Butyl (2-((3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)amino)-2-
oxoethyl)(methyl)carbamate (7b) To
a
stirred solution of N-(tert-butoxycarbonyl)-N-
methylglycine (188 mg, 0.99 mmol) in DMF was added HBTU (372 mg, 0.98 mmol)、HOBt (133
mg, 0.98 mmol) and DMAP (6 mg, 0.049 mmol), then DIEA (127 mg, 0.98 mmol) was added
dropwise. The mixture was stirred at room temperature for 5 min, then 6a (130 mg, 0.49 mmol)
was added, and the reaction mixture was continuously stirred at room temperature overnight. The
solution was evaporated to dryness and DCM (2 mL) was added to the residue. After filtration a
crude product was obtained, and recrystallized with DCM to afford 7b as white solid (115 mg,
1
53.6%); m.p. 226-227 ℃; H NMR (400 MHz, DMSO-d6) δ (ppm): 11.77 (s, 1H), 9.93 (s, 1H ×
0.45), 9.87 (s, 1H × 0.55), 8.03 (d, J = 7.6 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 7.6 Hz,
1H), 7.19-7.35 (m, 4H), 7.00-7.05 (m, 1H), 5.29 (s, 2H), 3.92 (s, 2H × 0.45), 3.86 (s, 2H × 0.55),
2.85 (s, 3H × 0.45), 2.82 (s, 3H × 0.55), 1.40 (s, 9H × 0.45), 1.26 (s, 9H × 0.55); 13C NMR (100
MHz, DMSO-d6) δ (ppm): (167.71, 167.53), 161.82, (155.38, 155.08), 150.60, 140.82, 139.32,
137.02, 135.20, 129.17, 127.58, 122.72, (121.44, 121.26), (118.00, 117.76), (116.41, 116.26),
115.83, 115.12, (78.83, 78.61), (52.20, 51.36), 45.09, (35.70, 35.63), (28.02, 27.85); HR-MS (ESI):
m/z, calcd. For C23H26N4NaO5 461.1795 [M+Na]+, Found: 461.1798.

5.2.4.3 tert-Butyl (S)-(1-((3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)amino)-3-
methyl-1-oxobutan-2-yl)carbamate (7c) To a stirred solution of (tert-butoxycarbonyl)-(S)-valine
(242 mg, 1.13 mmol) in DMF was added HATU (571 mg, 1.50 mmol)、HOBt (203 mg, 1.50 mmol)
and DMAP (10 mg, 0.075 mmol) , then TEA (152 mg, 1.50 mmol) was added dropwise. The
mixture was stirred at room temperature for 5 min, then 6a (200 mg, 0.75 mmol) was added, and
the reaction mixture was continuously stirred at room temperature overnight. The solution was
evaporated to dryness and DCM (20 mL) was added to the residue. The mixture was then washed
with NaHCO3 (25 mL×2) and H2O (25 mL). The organic layer was dried over anhydrous Na2SO4.
After filtration and concentration, the crude product was obtained and purified with column
chromatography on silica gel to afford compound 7c as white solid (310 mg, 88.7%);
m.p.178-180 ℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.76 (s, 1H), 9.94 (s, 1H), 8.03 (d, J =
7.6 Hz, 1H), 7.59-7.66 (m, 2H), 7.38 (s, 1H), 7.20-7.29 (m, 3H), 7.01 (d, J = 6.8 Hz, 1H), 6.83 (d,
J = 8.0 Hz, 1H), 5.29 (s, 2H), 3.85-3.88 (m, 1H), 1.92-1.95 (m, 1H), 1.21-1.36 (m, 9H), 0.84-0.86
(d, J = 5.6 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.73, 161.80, 155.52, 150.58,
140.83, 139.30, 136.97, 135.22, 129.10, 127.57, 122.70, 121.34, 117.95, 116.48, 115.80, 115.10,
77.98, 60.53, 45.10, 30.26, 28.15, 19.18, 18.39; HR-MS (ESI): m/z, calcd. For C25H31O5N4
467.2289 [M+H]+, Found: 467.2287.
5.2.4.4 tert-Butyl (R)-2-((3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)carbamoyl)
pyrrolidine-1-carboxylate (7d) Following the preparation protocol of Section 5.2.4.3, starting from
6a (150 mg, 0.56 mmol) and (tert-butoxycarbonyl)-(R)-proline (181 mg, 0.84 mmol), the title
compound 7d was obtained as white solid (40 mg, 15.3%); m.p. 128-130 ℃; 1H NMR (400 MHz,
CDCl3) δ (ppm):9.56 (brs, 1H), 8.75 (brs, 1H), 8.20 (d, J = 7.6 Hz, 1H), 7.54-7.56 (m, 2H), 7.39
(m, 1H), 7.20-7.27 (m, 2H), 7.12 (d, J = 7.6 Hz, 1H), 6.94 (m, 1H), 5.32 (s, 2H), 4.44 (m, 1H),
3.45-3.51 (m, 2H), 2.51 (m, 1H), 1.93 (m, 3H), 1.46 (s, 9H); 13C NMR (100 MHz, CDCl3) δ (ppm):
170.14, 161.78, 156.55, 150.68, 140.99, 139.08, 136.33, 135.55, 129.62, 128.66, 123.27, 121.54,
118.95, 117.63, 116.05, 114.98, 80.97, 60.54, 47.21, 46.51, 28.32, 27.10, 24.62; HR-MS (ESI):
m/z, calcd. For C25H28N4O5Na 487.1952 [M+Na]+, Found: 487.1945.
5.2.4.5 tert-Butyl (R)-2-((5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)
carbamoyl)pyrrolidine-1-carboxylate (7e) Following the preparation protocol of Section 5.2.4.2,
starting from 6b (200 mg, 0.70 mmol) and (tert-butoxycarbonyl)-(R)-proline (227 mg, 1.05 mmol),
the title compound 7e was obtained as yelow solid (75 mg, 22.2%); m.p.215-216℃; 1H NMR (300

MHz, CDCl3) δ (ppm): 9.55 (brs, 1H), 8.71 (s, 1H), 8.45 (d, J = 6.0 Hz, 1H), 8.21 (d, J = 7.5 Hz,
1H), 7.58 (t, J = 7.5 Hz, 1H), 7.21-7.27 (m, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.01 (t, J = 9.3 Hz, 1H),
6.87 (m, 1H), 5.30 (s, 2H), 4.50 (m, 1H), 3.45 (m, 2H), 2.53 (m, 1H), 1.95 (m, 3H), 1.49 (s, 9H);
13C NMR (100 MHz, CDCl3) δ (ppm): 170.59, 161.75, 156.05, 151.71 (d, JCF = 243.4 Hz), 150.64,
140.91, 135.59, 131.77, 128.76, 127.27, 123.33, 121.61, 119.87, 116.08, 115.40 (d, JCF = 19.80
Hz), 114.88, 81.18, 60.61, 47.17, 46.19, 28.28, 27.21, 24.56; HR-MS (ESI): m/z, calcd. For
C25H27FN4NaO5 505.1858 [M+Na]+, Found: 505.1860.
5.2.4.6 tert-Butyl (S)-2-((3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)carbamoyl)
pyrrolidine-1-carboxylate (7f) Following the preparation protocol of Section 5.2.4.3, starting from
6a (200 mg, 0.75 mmol) and (tert-butoxycarbonyl)-(S)-proline (242 mg, 1.13 mmol), the title
compound 7f was obtained as white solid (110 mg, 31.7%); m.p. 124-126 ℃; 1H NMR (400 MHz,
DMSO-d6) δ (ppm): 11.76 (s, 1H), 9.94 (s, 1H), 8.02 (s, 1H), 7.52-7.63 (m, 2H), 7.20-7.37 (m,
4H), 7.01-7.06 (m, 1H), 5.31 (s, 2H), 4.10-4.20 (m, 1H), 3.37 (m, 2H), 2.14 (m, 1H), 1.84 (m, 3H).
1.14-1.38 (m, 9H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): (171.43, 171.10); 161.82, 153.07,
150.58, 140.82, 139.44, 136.95, (135.27, 135.16), 129.08, 127.57, 122.70, (121.52, 121.18),
(118.18, 117.84), (116.56, 116.30), 115.83, 115.12, 78.35, 60.26, 51.97, 46.52, 45.13, 30.90, 27.80;
HR-MS (ESI): m/z, calcd. For C25H28N4O5Na 487.1952 [M+Na]+, Found: 487.1944.
5.2.4.7 tert-Butyl (S)-2-((5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)
carbamoyl)pyrrolidine-1-carboxylate (7g) Following the preparation protocol of Section 5.2.4.2,
starting from 6b (200 mg, 0.70 mmol) and (tert-butoxycarbonyl)-(S)-proline (227 mg, 1.05 mmol),
1
the title compound 7g was obtained as yellow solid (35 mg, 10.4%); m.p. 211-212℃; H NMR
(400 MHz, CDCl3) δ (ppm): 9.56 (brs, 1H), 8.64 (s, 1H), 8.44 (m, 1H), 8.21 (d, J = 7.2 Hz, 1H),
7.56-7.59 (m, 1H), 7.23-7.27 (m, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.01 (m, 1H), 6.87 (m, 1H), 5.30
(s, 2H), 4.50 (m, 1H), 3.46 (m, 2H), 2.53 (m, 1H), 1.95 (m, 3H), 1.49 (s, 9H); 13C NMR (150 MHz,
CDCl3) δ (ppm): 170.44, 161.43, 151.71 (d, JCF = 243.5 Hz), 150.39, 140.92, 135.65, 131.60,
128.78, 127.22, 123.38, 121.50, 119.82, 116.06, 115.42 (d, JCF = 21.1 Hz), 114.90, 81.20, 60.66,
47.19, 46.26, 28.30, 27.15, 24.59; HR-MS (ESI): m/z, calcd. For C25H27FN4NaO5 505.1858
[M+Na]+, Found: 505.1849.
5.2.4.8 tert-Butyl (R)-3-((3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)carbamoyl)
pyrrolidine-1-carboxylate (7h) Following the preparation protocol of Section 5.2.4.2, starting from
6a (186 mg, 0.70 mmol) and (R)-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid (227 mg,

1.05 mmol), the title compound 7h was obtained as white solid (125 mg, 65.2%); m.p.
244-245 ℃; 1H NMR (300 MHz, CDCl3) δ (ppm): 9.66 (brs, 1H), 8.07-8.13 (m, 2H), 7.74 (m,
1H), 7.60 (t, J = 7.5 Hz, 1H), 7.41 (s, 1H), 7.22-7.32 (m, 3H), 7.04 (d, J = 7.8 Hz, 1H), 5.30 (s,
2H), 3.55-3.67 (m, 3H), 3.31-3.35 (m, 1H), 3.04-3.08 (m, 1H), 2.04-2.15 (m, 2H), 1.44 (s, 9H);
13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.99, 161.80, 153.33, 150.58, 140.82, 139.51, 136.97,
135.22, 129.09, 127.57, 122.70, 121.40, 117.97, 116.43, 115.82, 115.11, 78.27, 48.27, 45.37, 45.11,
44.27, 43.37, 28.13; HR-MS (ESI): m/z, calcd. For C25H28N4O5Na 487.1952 [M+Na]+, Found:
487.1946.
5.2.4.9 tert-Butyl (R)-3-((5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)
carbamoyl)pyrrolidine-1-carboxylate (7i) Following the preparation protocol of Section 5.2.4.3,
starting from 6b (170 mg, 0.53 mmol) and (R)-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic
acid (173 mg, 0.84 mmol), the title compound 7i was obtained as white solid (153 mg, 68.0%);
m.p. 188-190℃; 1H NMR (400 MHz, CDCl3) δ (ppm): 8.74 (s, 1H), 8.44 (d, J = 6.8 Hz, 1H), 8.21
(d, J = 8.0 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.52 (s, 1H), 7.23 (d, J = 7.2 Hz, 1H), 7.13 (d, J = 8.4
Hz, 1H), 7.00-7.06 (m, 1H), 6.91 (m, 1H), 5.30 (s, 2H), 3.69-3.75 (m, 1H), 3.58-3.64 (m, 2H),
3.37-3.44 (m, 1H), 3.03-3.11 (m, 1H), 2.17-2.30 (m, 2H), 1.47 (s, 9H); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): (171.52, 171.36), 161.77, 153.34, 152.90 (d, JCF = 243.8 Hz), 150.64, 140.73,
135.22, 132.52 (d, JCF = 3.1 Hz), 127.62, 126.16 (d, JCF = 12.1 Hz), 123.49, 122.75, 122.39,
115.90, 115.67 (d, JCF = 19.9 Hz), 115.04, 78.31, (48.34, 48.25), (45.38, 45.20), 44.60, (43.70,
42.80), (29.21, 28.38), 28.15; HR-MS (ESI): m/z, calcd. For C25H27FN4NaO5 505.1858 [M+Na]+,
Found: 505.1847.
5.2.4.10 tert-Butyl (S)-3-((3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)-
carbamoyl)pyrrolidine-1-carboxylate (7j) Following the preparation protocol of Section 5.2.4.2,
starting from 6a (186 mg, 0.70 mmol) and (S)-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic
acid (227 mg, 1.05 mmol), the title compound 7j was obtained as white solid (124 mg, 65.2%);
m.p. 227-228 ℃; 1H NMR (300 MHz, CDCl3) δ (ppm): 9.74 (s, 1H), 8.16 (s, 1H), 8.10 (d, J = 7.2
Hz, 1H), 7.74 (m, 1H), 7.59 (m, 1H), 7.40 (s, 1H), 7.20-7.26 (m, 3H), 7.03 (m, 1H), 5.28 (s, 2H),
3.05-3.65 (m, 5H), 2.02-2.14 (m, 2H), 1.37-1.43 (m, 9H); 13C NMR (150 MHz, CDCl3) δ (ppm):
170.82, 161.89, 154.37, 151.27, 140.91, 138.88, 136.20, 135.77, 129.68, 128.58, 123.55, 122.52,
119.43, 118.02, 116.08, 114.94, 79.53, 48.69, 46.71, 46.49, 28.48; HR-MS (ESI): m/z, calcd. For
C25H28N4O5Na 487.1952 [M+Na]+, Found: 487.1944.

5.2.4.11 tert-Butyl (S)-3-((5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)
carbamoyl)pyrrolidine-1-carboxylate (7k) Following the preparation protocol of Section 5.2.4.3,
starting from 6b (170 mg, 0.53 mmol) and (S)-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic
acid (173 mg, 0.84 mmol), the title compound 7k was obtained as yellow solid (145 mg, 64.4%);
m.p. 209-211℃; 1H NMR (400 MHz, CDCl3) δ (ppm): 8.76 (s, 1H), 8.43 (d, J = 6.8 Hz, 1H), 8.21
(d, J = 7.6 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.53 (s, 1H), 7.22-7.27 (m, 1H), 7.14 (d, J = 8.4 Hz,
1H), 7.04 (t, J = 9.2 Hz, 1H), 6.91 (m, 1H), 5.30 (s, 2H), 3.59-3.72 (m, 3H), 3.37-3.45 (m, 1H),
3.05-3.10 (m, 1H), 2.21-2.25 (m, 2H), 1.48 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ (ppm):
(171.52, 171.36), 161.77, 153.34, 152.93 (d, JCF = 243.8 Hz), 150.64, 140.73, 135.22, 132.52 (d,
JCF = 3.0 Hz), 127.62, 126.16 (d, JCF = 12.1 Hz), 123.48, 122.75, 122.40, 115.90, 115.68 (d, JCF
=
19.9 Hz), 115.04, 78.31, (48.34, 48.25), (45.20, 44.95), 43.70, (42.79, 42.41), (29.21, 28.38),
28.15; HR-MS (ESI): m/z, calcd. For C25H27FN4NaO5 505.1858 [M+Na]+, Found: 505.1850.
5.2.4.12 tert-Butyl 3-((3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl) carbamoyl)
piperidine-1-carboxylate (7l) Following the preparation protocol of Section 5.2.4.3, starting from
6a (200 mg, 0.75 mmol) and 1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid (260 mg, 1.13
mmol), the title compound 7l was obtained as white solid (255 mg, 71.0%); m.p. 136-138 ℃;1H
NMR (300 MHz, DMSO-d6) δ (ppm): 11.77 (s, 1H), 9.95 (s, 1H), 8.03 (d, J = 7.5 Hz, 1H),
7.56-7.67 (m, 2H), 7.38 (s, 1H), 7.19-7.27 (m, 3H), 7.01 (d, J = 6.9 Hz, 1H), 5.28 (s, 2H),
3.81-3.95 (m, 2H), 2.69-2.77 (m, 2H), 2.38 (m, 1H), 1.85-1.90 (m, 1H), 1.50-1.65 (m, 2H), 1.37
(m, 10H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.55, 162.51, 154.48, 151.29, 141.53,
140.33, 137.62, 135.92, 129.74, 128.27, 123.40, 121.96, 118.58, 117.07, 116.52, 115.81, 79.37,
46.00, 45.83, 43.61, 28.71, 28.18; HR-MS (ESI): m/z, calcd. For C26H30N4O5Na 501.2108
[M+Na]+, Found: 501.2101.
5.2.4.13 tert-Butyl 3-((5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)
carbamoyl)piperidine-1-carboxylate (7m) Following the preparation protocol of Section 5.2.4.3,
starting from 6b (180 mg, 0.56 mmol) and 1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid
(193 mg, 0.84 mmol), the title compound 7m was obtained as white solid (190 mg, 60.6%); m.p.
188-190℃; 1H NMR (300 MHz, CDCl3) δ (ppm): 8.85 (s, 1H), 8.41 (d, J = 5.7 Hz, 1H), 8.21 (d, J
= 7.2 Hz, 1H), 7.56-7.62 (m, 1H), 7.22-7.27 (m, 1H), 7.13 (d, J = 8.4 Hz, 1H), 6.98-7.05 (m, 1H),
6.88 (m, 1H), 5.29 (s, 2H), 4.12-4.18 (m, 1H), 3.88-3.93 (m, 1H), 3.14-3.22 (m, 1H), 2.95 (m, 1H),
2.52 (m, 1H), 1.67-2.00 (m, 3H), 1.46-1.55 (m, 10H); 13C NMR (100 MHz, CDCl3) δ (ppm):
171.87, 161.77, 154.89, 151.79 (d, JCF = 242.8 Hz), 150.69, 140.87, 135.62, 131.84 (d, JCF = 2.6

Hz), 128.76, 126.59 (d, JCF = 10.8 Hz), 123.37, 122.07 (d, JCF = 7.8 Hz), 120.40, 116.10, 115.43
(d, JCF = 19.8 Hz), 114.81, 80.21, 77.20, 46.14, 46.02, 44.06, 28.37, 27.70, 24.05; HR-MS (ESI):
m/z, calcd. For C26H29FN4NaO5 519.2014 [M+Na]+, Found: 519.2007.
5.2.4.14 tert-Butyl 4-((3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)carbamoyl)
piperidine-1-carboxylate (7n) Following the preparation protocol of Section 5.2.4.2, starting from
6a (200 mg, 0.75 mmol) and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (260 mg, 1.13
mmol), the title compound 7n was obtained as white solid (215 mg, 66.6%); m.p. 224-226 ℃;1H
NMR (400 MHz, DMSO-d6) δ (ppm): 11.77 (s, 1H), 9.87 (s, 1H), 8.03 (d, J = 7.2 Hz, 1H),
7.58-7.65 (m, 2H), 7.37 (s, 1H), 7.19-7.25 (m, 3H), 7.00 (d, J = 6.8 Hz, 1H), 5.27 (s, 2H),
3.95-3.98 (m, 2H), 2.72 (m, 2H), 2.44-2.50 (m, 1H), 1.70-1.74 (m, 2H), 1.40 (m, 11H); 13C NMR
(100 MHz, DMSO-d6) δ (ppm): 173.01, 161.81, 153.81, 150.58, 140.82, 139.74, 136.90, 135.21,
129.03, 127.60, 122.69, 121.19, 117.86, 116.31, 115.82, 115.10, 78.60, 45.72, 45.12, 42.60, 28.04;
HR-MS (ESI): m/z, calcd. For C26H30N4O5Na 501.2108 [M+Na]+, Found: 501.2100.
5.2.4.15 tert-Butyl 4-((5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)
carbamoyl)piperidine-1-carboxylate (7o) Following the preparation protocol of Section 5.2.4.3,
starting from 6b (170 mg, 0.53 mmol)and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid
(214 mg, 0.93 mmol), the title compound 7o was obtained as white solid (200 mg, 65.0%); m.p.
189-191℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.75 (s, 1H), 9.68 (s, 1H), 8.02 (d, J = 7.6
Hz, 1H), 7.82 (d, J = 6.8 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.16-7.27 (m, 3H), 7.07 (m, 1H), 5.26
(s, 2H), 3.95-3.98 (m, 2H), 2.60-2.74 (m, 3H), 1.73-1.77 (m, 2H), 1.40-1.48 (m, 11H); 13C NMR
(100 MHz, DMSO-d6) δ (ppm): 173.37, 161.77, 153.83, 153.23 (d, JCF = 243.6 Hz), 150.63,
140.73, 135.23, 132.44 (d, JCF = 3.0 Hz), 127.61, 126.38 (d, JCF = 12.1 Hz), 123.25 (d, JCF = 7.3
Hz), 122.75, 122.22, 115.88, 115.60 (d, JCF = 20.0 Hz), 115.05, 78.61, 44.61, 40.13, 28.18, 28.06;
HR-MS (ESI): m/z, calcd. For C26H29FN4NaO5 519.2014 [M+Na]+, Found: 519.2007.
5.2.4.16 tert-Butyl 4-((2-fluoro-5-((5-fluoro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)
phenyl)carbamoyl)piperidine-1-carboxylate (7p) Following the preparation protocol of Section
5.2.4.3, starting from 6c (220 mg, 0.66 mmol) and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic
acid (227 mg, 0.99 mmol), the title compound 7p was obtained as white solid (90 mg, 24.1%);
m.p. 233-235 ℃; 1H NMR (300 MHz, CDCl3) δ (ppm): 8.43-8.46 (m, 2H), 7.51 (dt, J1 = 8.1 Hz,
J2 = 6.0 Hz, 1H), 7.46 (m, 1H), 7.04 (dd, J1 = 10.5 Hz, J2 = 8.4 Hz, 1H), 6.87-6.95 (m, 3H), 5.27
(s, 2H), 4.18 (m, 2H), 2.76-2.85 (m, 2H), 2.41-2.49 (m, 1H), 1.90-1.94 (m, 2H), 1.67-1.81 (m, 2H),

1.47 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 173.38, 161.72 (d, JCF = 260.1 Hz), 158.97
(d, JCF = 3.1 Hz), 153.84, 152.89 (d, JCF = 243.6 Hz), 150.36, 142.52 (d, JCF = 2.5 Hz), 135.92 (d,
JCF = 11.2 Hz), 132.14 (d, JCF = 3.2 Hz), 126.38 (d, JCF = 12.1 Hz), 123.17 (d, JCF = 7.6 Hz),
122.16, 115.61 (d, JCF = 19.9 Hz), 111.17, 110.12 (d, JCF = 20.6 Hz), 105.50 (d, JCF = 8.7 Hz),
78.62, 45.25, 41.87, 28.18, 28.06; HR-MS (ESI): m/z, calcd. For C26H28F2N4NaO5 537.1920
[M+Na]+, Found: 537.1914.
5.2.4.17 tert-Butyl 4-((2-fluoro-5-((6-fluoro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)
phenyl)carbamoyl)piperidine-1-carboxylate (7q) Following the preparation protocol of Section
5.2.4.3, starting from 6d (400 mg, 1.32 mmol) and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic
acid (454 mg, 1.98 mmol), the title compound 7q was obtained as white solid (210 mg, 30.9%);
m.p. 182-184 ℃; 1H NMR (300 MHz, CDCl3) δ (ppm): 8.89 (s, 1H), 8.44 (d, J = 6.6 Hz, 1H),
7.87 (dd, J1 = 7.8 Hz, J2 = 2.7 Hz, 1H), 7.26-7.34 (m, 1H), 7.12 (dd, J1 = 9.0 Hz, J2 = 3.6 Hz, 1H),
7.00-7.08 (m, 1H), 6.88 (m, 1H), 5.28 (s, 2H), 4.17-4.22 (m, 2H), 2.76-2.86 (m, 2H), 2.42-2.50 (m,
1H), 1.90-1.95 (m, 2H), 1.68-1.81 (m, 2H), 1.47 (m, 9H); 13C NMR (100 MHz, DMSO-d6) δ
(ppm): 173.40, 160.98 (d, JCF = 2.5 Hz), 157.51 (d, JCF = 240.1 Hz), 153.85, 152.91 (d, JCF
243.8 Hz), 150.41, 137.47, 132.23 (d, JCF = 3.0 Hz), 126.41 (d, JCF = 12.0 Hz), 123.23 (d, JCF
=
=
7.4 Hz), 122.73 (d, JCF = 20.0 Hz), 122.22, 117.54 (d, JCF = 7.6 Hz), 117.31 (d, JCF = 7.6 Hz),
115.63 (d, JCF = 19.9 Hz), 112.82 (d, JCF = 23.7 Hz), 78.64, 44.88, 41.88, 28.19, 28.07; HR-MS
(ESI): m/z, calcd. For C26H28F2N4NaO5 537.1920 [M+Na]+, Found: 537.1913.
5.2.4.18 tert-Butyl 4-((2-fluoro-5-((7-fluoro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)
phenyl)carbamoyl)piperidine-1-carboxylate (7r) Following the preparation protocol of Section
5.2.4.3, starting from 6e (350 mg, 1.16 mmol) and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic
acid (399 mg, 1.74 mmol), the title compound 7r was obtained as white solid (200 mg, 33.7%);
m.p. 115-117 ℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.82 (s, 1H), 9.69 (s, 1H), 8.04-8.10
(m, 1H), 7.83 (d, J1 = 7.5 Hz, 1H), 7.07-7.24 (m, 4H), 5.26 (s, 2H), 3.95-3.99 (m, 2H), 2.64-2.74
(m, 3H), 1.73-1.78 (m, 2H), 1.40-1.46 (m, 11H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 173.38,
166.09 (d, JCF = 249.3 Hz), 160.97, 153.83, 152.93 (d, JCF = 243.8 Hz), 150.67, 142.92 (d, JCF
=
12.3 Hz), 132.06 (d, JCF = 3.1 Hz), 130.73 (d, JCF = 11.2 Hz), 126.38 (d, JCF = 12.1 Hz), 123.38 (d,
JCF = 7.5 Hz), 122.39, 115.63 (d, JCF = 19.8 Hz), 112.80, 110.48 (d, JCF = 22.8 Hz), 102.26 (d, JCF
= 27.7 Hz), 79.09, 44.88, 41.88, 28.18, 28.06; HR-MS (ESI): m/z, calcd. For C26H28F2N4NaO5
537.1920 [M+Na]+, Found: 537.1912.

5.2.4.19 tert-Butyl 4-((2-fluoro-5-((8-fluoro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)
phenyl)carbamoyl)piperidine-1-carboxylate (7s) Following the preparation protocol of Section
5.2.4.3, starting from 6f (300 mg, 0.99 mmol) and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic
acid (340 mg, 1.49 mmol), the title compound 7s was obtained as white solid (150 mg, 29.5%);
1
m.p. 143-145 ℃; H NMR (300 MHz, CDCl3) δ (ppm): 8.56 (s, 1H), 8.37 (d, J = 6.9 Hz, 1H),
8.05 (d, J = 8.1 Hz, 1H), 7.32-7.41 (m, 2H), 7.20 (td, J1 = 7.8 Hz, J2 = 3.6 Hz, 1H), 6.99-7.06 (m,
1H), 6.91 (m, 1H), 5.47 (s, 2H), 4.16-4.21 (m, 2H), 2.75-2.84 (m, 2H), 2.38-2.46 (m, 1H),
1.88-1.93 (m, 2H), 1.67-1.80 (m, 2H), 1.47 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ (ppm):
173.38, 160.97 (d, JCF = 2.6 Hz), 153.83, 151.43, 150.73, 150.33, 149.10 (d, JCF = 245.4 Hz),
133.81, 129.68 (d, JCF = 6.9 Hz), 126.14 (d, JCF = 12.2 Hz), 123.95 (d, JCF = 2.7 Hz), 123.87 (d,
JCF = 8.1 Hz), 122.91 (d, JCF = 23.4 Hz), 122.36 (d, JCF = 7.4 Hz), 121.47, 119.08, 115.37 (d, JCF
=
19.8 Hz), 78.61, 47.98, 47.85, 41.88, 28.19, 28.06; HR-MS (ESI): m/z, calcd. For
C26H28F2N4NaO5 537.1920 [M+Na]+, Found: 537.1912.
5.2.4.20
2-(Dimethylamino)-N-(3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)
acetamide (8t) Following the preparation protocol of Section 5.2.4.2, starting from 6a (200 mg,
0.75 mmol) and dimethylglycine (116 mg, 1.12 mmol), the title compound 8t was obtained as
yellow solid (210 mg, 79.6%); m.p. 251-252℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.79 (s,
1H), 10.47 (s, 1H), 9.71 (brs, 1H), 8.04 (d, J = 7.5 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.52 (d, J =
8.4 Hz, 1H), 7.16-7.39 (m, 5H), 5.32 (s, 2H), 4.04 (s, 2H), 2.82 (s, 6H); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 163.13, 161.85, 150.60, 140.79, 138.32, 137.36, 135.30, 129.44, 127.64,
122.81, 122.67, 118.25, 116.50, 115.82, 115.10, 58.03, 44.99, 43.45; HR-MS (ESI): m/z, calcd.
For C19H21N4O3 353.1608 [M+Na]+, Found: 353.1603.
5.2.5 Synthesis of compunds 8a-8s
5.2.5.1
2-Amino-N-(3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)acetamide
2,2,2-trifluoroacetate (8a) To a stired solution of 7a (100 mg, 0.24 mmol) in DCM (2 mL) was
added TFA (2 mL) dropwise, the reaction mixture was then allowed to stir at room temperature
overnight. DCM and excessive TFA was then removed under reduced pressure, ethyl ether (2 mL)
was then added to the residue. After filtration and dried under vacuum condition, compound 8a
1
was obtained as white solid without further purification (83 mg, 80.4%); m.p. 216-218 ℃; H
NMR (400 MHz, DMSO-d6) δ (ppm): 11.78 (s, 1H), 10.40 (s, 1H), 8.10 (brs, 3H), 8.04 (d, J = 7.6
Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.39 (s, 1H), 7.35 (t, J = 7.6 Hz, 1H),
7.21-7.28 (m, 2H), 7.15 (d, J = 7.6 Hz, 1H), 5.32 (s, 2H), 3.71 (s, 2H); 13C NMR (100 MHz,

DMSO-d6) δ (ppm): 164.83, 161.86, 150.60, 140.81, 138.73, 137.29, 135.30, 129.41, 127.63,
122.79, 122.23, 117.86, 116.19, 115.82, 115.12, 45.01, 40.96; HR-MS (ESI): m/z, calcd. For
C17H17N4O3 325.1295 [M+H]+, Found: 325.1290.
5.2.5.2 N-(3-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)-2-(methylamino)-
acetamide 2,2,2-trifluoroacetate (8b) Following the preparation protocol of Section 5.2.5.1,
starting from 7b (40 mg, 0.09 mmol), the title compound 8b was obtained as white solid (33 mg,
1
83.1%); m.p.> 250 ℃; H NMR (400 MHz, DMSO-d6) δ (ppm): 11.78 (s, 1H), 10.46 (s, 1H),
8.82 (brs, 2H), 8.04 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H),
7.33-7.38 (m, 2H), 7.21-7.28 (m, 2H), 7.16 (d, J = 7.2 Hz, 1H), 5.32 (s, 2H), 3.86 (s, 2H), 2.59 (s,
3H) ; 13C NMR (100 MHz, DMSO-d6) δ (ppm): 163.96, 161.85, 150.60, 140.79, 138.52, 137.32,
135.29, 129.42, 127.63, 122.79, 122.41, 117.98, 116.30, 115.81, 115.12, 49.64, 44.97, 32.67;
HR-MS (ESI): m/z, calcd. For C18H19N4O3 339.1452 [M+H]+, Found: 339.1440.
5.2.5.3 (S)-2-Amino-N-(3-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)-3-methyl-
butanamide 2,2,2-trifluoroacetate (8c) Following the preparation protocol of Section 5.2.5.1,
starting from 7c (160 mg, 0.34 mmol), the title compound 8c was obtained as white solid (156 mg,
94.7%); m.p. 167-169 ℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.78 (s, 1H), 10.44 (s, 1H),
8.20 (brs, 3H), 8.03 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.42 (s,
1H), 7.35 (t, J = 7.6 Hz, 1H), 7.22-7.28 (m, 2H), 7.15 (d, J = 7.6 Hz, 1H), 5.32 (s, 2H), 3.68 (m,
1H), 2.11-2.16 (m, 1H), 0.94 (t, J = 7.6 Hz, 6H) ; 13C NMR (100 MHz, DMSO-d6) δ (ppm):
166.93, 161.85, 150.61, 140.83, 138.48, 137.32, 135.29, 129.37, 127.64, 122.80, 122.47, 118.35,
116.71, 115.82, 115.12, 58.23, 45.05, 29.86, 18.43, 17.60; HR-MS (ESI): m/z, calcd. For
C20H23O3N4 367.1765 [M+H]+, Found: 367.1761.
5.2.5.4 (R)-N-(3-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)pyrrolidine-2-
carboxamide 2,2,2-trifluoroacetate (8d) Following the preparation protocol of Section 5.2.5.1,
starting from 7d (65 mg, 0.14 mmol), the title compound 8d was obtained as white solid (50 mg,
74.7%); m.p. 145-147 ℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.77 (brs, 1H), 10.50 (s, 1H),
8.96 (brs, 2H), 8.03 (d, J = 7.6 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.51 (d, J = 8.4Hz, 1H), 7.43 (s,
1H), 7.35 (t, J = 7.6 Hz, 1H), 7.20-7.27 (m, 2H), 7.17 (d, J = 7.6 Hz, 1H), 5.31 (s, 2H), 4.23-4.27
(m, 1H), 3.17-3.29 (m, 2H), 2.29-2.37 (m, 1H), 1.85-1.97 (m, 3H); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 167.35, 162.32, 151.07, 141.28, 139.10, 137.83, 135.77, 129.86, 128.10,
123.27, 123.05, 118.68, 116.95, 116.29, 115.58, 60.17, 46.23, 45.53, 30.00, 24.05; HR-MS (ESI):

m/z, calcd. For C20H21N4O3 365.1608 [M+H]+, Found: 365.1599.
5.2.5.5 (R)-N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)pyrrolidine-
2-carboxamide 2,2,2-trifluoroacetate (8e) Following the preparation protocol of Section 5.2.5.1,
starting from 7e (40 mg, 0.08 mmol), the title compound 8e was obtained as white solid (30 mg,
1
92.4%); m.p. 208-210℃; H NMR (300 MHz, DMSO-d6) δ (ppm): 11.76 (s, 1H), 10.37 (s, 1H),
9.29 (brs, 1H), 8.65 (brs, 1H), 8.03 (d, J = 7.2 Hz, 1H), 7.79 (d, J = 6.3 Hz, 1H), 7.63-7.66 (m,
1H), 7.27-7.32 (m, 4H), 5.30 (s, 2H), 4.40 (m, 1H), 3.23 (m, 2H), 2.35 (m, 1H), 1.90 (m, 3H); 13
C
NMR (100 MHz, DMSO-d6) δ (ppm): 167.56, 161.82, 152.73 (d, JCF = 244.0 Hz), 150.60, 140.71,
135.30, 132.83 (d, JCF = 3.2 Hz), 127.66, 125.37 (d, JCF = 12.0 Hz), 124.59 (d, JCF = 7.6 Hz),
122.83, 121.48, 116.05, 115.85, 115.04, 59.51, 45.82, 44.57, 29.70, 23.56; HR-MS (ESI): m/z,
calcd. For C20H20N4O3F 383.1514 [M+H]+, Found: 383.1507.
5.2.5.6 (S)-N-(3-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)pyrrolidine-2-
carboxamide 2,2,2-trifluoroacetate (8f) Following the preparation protocol of Section 5.2.5.1,
starting from 7f (65 mg, 0.14 mmol), the title compound 8f was obtained as white solid (50 mg,
74.7%); m.p. 138-140 ℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.77 (s, 1H), 10.52 (s, 1H),
9.40 (brs, 1H), 8.65 (brs, 1H), 8.04 (d, J = 7.2 Hz, 1H), 7.65 (t, J = 7.2 Hz, 1H), 7.51 (d, J = 7.2
Hz, 1H), 7.43 (s, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.21-7.28 (m, 2H), 7.17 (d, J = 7.2 Hz, 1H), 5.32 (s,
2H), 4.27 (m, 1H), 3.25 (m, 2H), 2.32-2.34 (m, 1H), 1.91 (m, 3H); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 166.88, 161.85, 150.61, 140.81, 138.64, 137.35, 135.30, 129.38, 127.63,
122.80, 122.56, 118.21, 116.49, 115.82, 115.11, 59.69, 45.75, 45.07, 29.54, 23.58; HR-MS (ESI):
m/z, calcd. For C20H21N4O3 365.1608 [M+H]+, Found: 365.1604.
5.2.5.7 (S)-N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)pyrrolidine-
2-carboxamide 2,2,2-trifluoroacetate (8g) Following the preparation protocol of Section 5.2.5.1,
starting from 7g (20 mg, 0.04 mmol), the title compound 8g was obtained as white solid (18 mg,
87.4%); m.p. 209-211℃; 1H NMR (400 MHz, CD3OD) δ (ppm): 8.06 (d, J = 8.0 Hz, 1H), 7.90 (d,
J = 7.2 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.19-7.23 (m, 2H), 7.10-7.15 (m, 2H), 5.30 (s, 2H),
4.37-4.41 (m, 1H), 3.25-3.41 (m, 2H), 2.41-2.49 (m, 1H), 1.98-2.11 (m, 3H); 13C NMR (150 MHz,
CD3OD) δ (ppm): 168.36, 163.88, 154.34 (d, JCF = 244.8 Hz), 152.44, 142.23, 136.41, 133.81 (d,
JCF = 3.5 Hz), 128.93, 126.68 (d, JCF = 12.0 Hz), 125.44 (d, JCF = 7.8 Hz), 124.12, 122.80, 117.22,
116.76 (d, JCF = 20.1 Hz), 116.12, 61.46, 47.29, 46.18, 30.94, 24.86; HR-MS (ESI): m/z, calcd.
For C20H20N4O3F 383.1514 [M+H]+, Found: 383.1504.

5.2.5.8 (R)-N-(3-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)pyrrolidine-3-
carboxamide 2,2,2-trifluoroacetate (8h) Following the preparation protocol of Section 5.2.5.1,
starting from 7h (80 mg, 0.17 mmol), the title compound 8h was obtained as white solid (72 mg,
87.4%); m.p. 133-135 ℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.78 (s, 1H), 10.20 (s, 1H),
8.95 (brs, 2H), 8.03 (d, J = 7.5 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.57 (d, J = 7.5 Hz, 1H), 7.39 (s,
1H), 7.19-7.33 (m, 3H), 7.09 (d, J = 6.9 Hz, 1H), 5.29 (s, 2H), 3.20-3.42 (m, 5H), 2.17-2.24 (m,
1H), 1.98-2.05 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.40, 161.86, 150.63, 140.84,
139.37, 137.10, 135.27, 129.19, 127.62, 122.77, 121.83, 118.11, 116.48, 115.85, 115.12, 46.88,
45.15, 44.88, 43.02, 28.89; HR-MS (ESI): m/z, calcd. For C20H21N4O3 365.1608 [M+H]+, Found:
365.1603.
5.2.5.9 (R)-N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)pyrrolidine-
3-carboxamide 2,2,2-trifluoroacetate (8i) Following the preparation protocol of Section 5.2.5.1,
starting from 7i (40 mg, 0.08 mmol), the title compound 8i was obtained as yellow solid (35 mg,
1
85.0%); m.p. 100-102℃; H NMR (400 MHz, DMSO-d6) δ (ppm): 11.75 (s, 1H), 10.01 (s, 1H),
8.85 (brs, 2H), 8.02 (m, 1H), 7.79 (m, 1H), 7.65 (m, 1H), 7.18-7.25 (m, 4H), 5.28 (s, 2H),
3.19-3.36 (m, 5H), 2.22 (m, 1H), 2.02 (m, 1H); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 170.90,
161.78, 152.84 (d, JCF = 244.2 Hz), 150.60, 140.72, 135.24, 132.59, 127.63, 125.96 (d, JCF = 11.9
Hz), 123.97 (d, JCF = 7.7 Hz), 122.79, 121.92, 115.86, 115.75 (d, JCF = 20.0 Hz), 115.02, 46.89,
44.95, 42.39, 41.45, 28.89; HR-MS (ESI): m/z, calcd. For C20H20N4O3F 383.1514 [M+H]+, Found:
383.1505.
5.2.5.10 (S)-N-(3-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)pyrrolidine-3-
carboxamide 2,2,2-trifluoroacetate (8j) Following the preparation protocol of Section 5.2.5.1,
starting from 7j (67 mg, 0.14 mmol), the title compound 8j was obtained as white solid (65 mg,
94.2%); m.p. 132-134 ℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.78 (s, 1H), 10.20 (s, 1H),
8.90 (brs, 2H), 8.04 (d, J = 7.2 Hz, 1H), 7.56-7.65 (m, 2H), 7.39 (s, 1H), 7.20-7.31 (m, 3H), 7.10
(m, 1H), 5.30 (s, 2H), 3.20-3.36 (m, 5H), 2.02-2.20 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ
(ppm): 170.41, 161.87, 150.63, 140.84, 139.37, 137.11, 135.28, 129.19, 127.62, 122.77, 121.83,
118.10, 116.46, 115.85, 115.13, 46.88, 45.14, 44.88, 43.02, 28.90; HR-MS (ESI): m/z, calcd. For
C20H21N4O3 365.1608 [M+H]+, Found: 365.1597.
5.2.5.11 (S)-N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)pyrroli-

dine-3-carboxamide 2,2,2-trifluoroacetate (8k) Following the preparation protocol of Section
5.2.5.1, starting from 7k (50 mg, 0.10 mmol), the title compound 8k was obtained as white solid
(48 mg, 93.3%); m.p. 130-132℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.75 (s, 1H), 10.02 (s,
1H), 8.92 (brs, 2H), 8.02 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 6.4 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H),
7.17-7.27 (m, 4H), 5.28 (s, 2H), 3.37 (m, 3H), 3.20 (m, 2H), 2.22-2.24 (m, 1H), 2.01-2.03 (m, 1H);
13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.91, 161.81, 152.88 (d, JCF = 244.0 Hz), 150.63,
140.74, 135.27, 132.61 (d, JCF = 3.1 Hz), 127.64, 126.01 (d, JCF = 12.0 Hz), 123.95 (d, JCF = 7.5
Hz), 122.80, 122.01, 115.88, 115.67, 115.05, 46.85, 44.93, 44.62, 42.44, 28.93; HR-MS (ESI): m/z,
calcd. For C20H20N4O3F 383.1514 [M+H]+, Found: 383.1508.
5.2.5.12 N-(3-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)piperidine-3-
carboxamide 2,2,2-trifluoroacetate (8l) Following the preparation protocol of Section 5.2.5.1,
starting from 7l (100 mg, 0.21 mmol), the title compound 8l was obtained as white solid (90 mg,
87.4%); m.p. 135-137 ℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.77 (s, 1H), 10.14 (s, 1H),
8.69 (brs, 2H), 8.03 (d, J = 7.6 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.38 (s,
1H), 7.19-7.31 (m, 3H), 7.06 (d, J = 7.6 Hz, 1H), 5.28 (s, 2H), 3.26-3.30 (m, 1H), 3.02-3.19 (m,
2H), 2.89 (m, 1H), 2.76 (m, 1H), 1.96-1.99 (m, 1H), 1.79 (m, 1H), 1.58-1.66 (m, 2H); 13C NMR
(100 MHz, DMSO-d6) δ (ppm): 170.77, 161.84, 150.61, 140.84, 139.31, 137.07, 135.26, 129.16,
127.61, 122.76, 121.73, 118.03, 116.42, 115.83, 115.13, 45.66, 45.11, 44.05, 42.95, 26.14, 21.13;
HR-MS (ESI): m/z, calcd. For C21H23N4O3 379.1765 [M+H]+, Found: 379.1757.
5.2.5.13 N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)piperidine-3-
carboxamide 2,2,2-trifluoroacetate (8m) Following the preparation protocol of Section 5.2.5.1,
starting from 7m (130 mg, 0.26 mmol), the title compound 8m was obtained as white solid (125
mg, 93.5%); m.p. 131-133℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.75 (s, 1H), 9.97 (s, 1H),
8.62 (brs, 2H), 8.03 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 6.8 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H),
7.15-7.27 (m, 4H), 5.28 (s, 2H), 3.27-3.31 (m, 1H), 3.15-3.19 (m, 1H), 2.91-3.05 (m, 3H),
1.99-2.02 (m, 1H), 1.80 (m, 1H), 1.53-1.67 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ (ppm):
171.18, 161.81, 153.04 (d, JCF = 244.1 Hz), 150.65, 140.74, 135.27, 132.57 (d, JCF = 3.0 Hz),
127.65, 125.92 (d, JCF = 12.1 Hz), 123.92 (d, JCF = 7.4 Hz), 122.80, 122.34, 115.90, 115.67,
115.05, 44.61, 44.01, 42.93, 26.31, 21.17; HR-MS (ESI): m/z, calcd. For C21H22N4O3F 397.1670
[M+H]+, Found: 397.1664.
5.2.5.14 N-(3-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)piperidine-4-

carboxamide 2,2,2-trifluoroacetate (8n) Following the preparation protocol of Section 5.2.5.1,
starting from 7n (110 mg, 0.23 mmol), the title compound 8n was obtained as white solid (77 mg,
68.0%); m.p. 134-136 ℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.78 (s, 1H), 11.01 (s, 1H),
8.68 (brs, 1H), 8.39 (brs, 1H), 8.04 (d, J = 6.9 Hz, 1H), 7.58-7.65 (m, 2H), 7.37 (s, 1H), 7.20-7.28
(m, 3H), 7.04 (d, J = 6.6 Hz, 1H), 5.29 (s, 2H), 3.30-3.40 (m, 2H), 2.88-2.92 (m, 2H), 2.50-2.58
(m, 1H), 1.74-1.94 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.10, 161.85, 150.63,
140.85, 139.56, 137.00, 135.27, 129.12, 127.61, 122.76, 121.44, 118.02, 116.47, 115.85, 115.14,
45.64, 45.13, 42.45, 25.08; HR-MS (ESI): m/z, calcd. For C21H23N4O3 379.1765 [M+H]+, Found:
379.1758.
5.2.5.15 N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluorophenyl)piperidine-
4-carboxamide 2,2,2-trifluoroacetate (8o) Following the preparation protocol of Section 5.2.5.1,
starting from 7o (50 mg, 0.10 mmol), the title compound 8o was obtained as white solid (59 mg,
1
114.8%); m.p. 151-153℃; H NMR (400 MHz, DMSO-d6) δ (ppm): 11.75 (s, 1H), 9.78 (s, 1H),
8.58 (brs, 2H), 8.32 (brs, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.78 (d, J = 6.4 Hz, 1H), 7.65 (t, J = 7.6
Hz, 1H), 7.18-7.27 (m, 3H), 7.12 (m, 1H), 5.27 (s, 2H), 3.30-3.36 (m, 2H), 2.88-2.91 (m, 2H),
2.74 (m, 1H), 1.91-1.96 (m, 2H), 1.71-1.80 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ (ppm):
172.43, 161.78, 153.02 (d, JCF = 243.9 Hz), 150.65, 140.74, 135.25, 132.52, 127.63, 126.15 (d, JCF
= 12.1 Hz), 123.62 (d, JCF = 6.2 Hz), 122.78, 122.38, 115.89, 115.69 (d, JCF = 19.9 Hz), 115.05,
44.59, 42.47, 25.12; HR-MS (ESI): m/z, calcd. For C21H22N4O3F 397.1670 [M+H]+, Found:
397.1664.
5.2.5.16
N-(2-Fluoro-5-((5-fluoro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)
piperidine-4-carboxamide 2,2,2-trifluoroacetate (8p) Following the preparation protocol of
Section 5.2.5.1, starting from 7p (20 mg, 0.04 mmol), the title compound 8p was obtained as
brown solid (14 mg, 68.1%); m.p. 181.5-183.5 ℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm):
11.72 (s, 1H), 9.80 (s, 1H), 8.62 (brs, 1H), 8.34 (brs, 1H), 7.77 (d, J = 6.3 Hz, 1H), 7.61-7.64 (m,
1H), 6.99-7.24 (m, 4H), 5.25 (s, 2H), 3.30-3.35 (m, 2H), 2.74-3.02 (m, 3H), 1.91-2.04 (m, 2H),
1.70-1.78 (m, 2H); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 172.40, 161.71 (d, JCF = 260.1 Hz),
158.95, 153.04 (d, JCF = 243.8 Hz), 150.35, 142.50, 135.92 (d, JCF = 11.2 Hz), 132.20, 126.12 (d,
JCF = 12.2 Hz), 123.54 (d, JCF = 7.5 Hz), 122.31, 115.68 (d, JCF = 19.8 Hz), 111.13, 110.13 (d, JCF
= 20.4 Hz), 105.50 (d, JCF = 8.6 Hz), 45.20, 42.44, 25.08; HR-MS (ESI): m/z, calcd. For
C21H21N4O3F2 415.1576 [M+H]+, Found: 415.1568.

5.2.5.17
N-(2-Fluoro-5-((6-fluoro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)
piperidine-4-carboxamide 2,2,2-trifluoroacetate (8q) Following the preparation protocol of
Section 5.2.5.1, starting from 7q (45 mg, 0.09 mmol), the title compound 8q was obtained as
white solid (35 mg, 75.7%); m.p. 62-64 ℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.90 (s,
1H), 9.82 (s, 1H), 8.65 (brs, 1H), 8.38 (brs, 1H), 7.78 (d, J = 7.2 Hz, 1H), 7.73 (dd, J1 = 8.1 Hz, J2
= 2.7 Hz, 1H), 7.54-7.61 (m, 1H), 7.29 (dd, J1 = 9.3 Hz, J2 = 3.6 Hz, 1H), 7.17-7.24 (m, 1H), 7.11
(m, 1H), 5.27 (s, 2H), 3.30-3.35 (m, 2H), 2.73-2.92 (m, 3H), 1.91-1.96 (m, 2H), 1.69-1.81 (m, 2H);
13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.43, 160.98 (d, JCF = 2.3 Hz), 157.51 (d, JCF = 239.9
Hz), 153.04 (d, JCF = 243.7 Hz), 150.41, 137.46, 132.32 (d, JCF = 3.0 Hz), 126.17 (d, JCF = 12.2
Hz), 123.59 (d, JCF = 7.7 Hz), 122.73 (d, JCF = 23.4 Hz), 122.36, 117.54 (d, JCF = 7.6 Hz), 117.32
(d, JCF = 7.5 Hz), 115.71 (d, JCF = 19.9 Hz), 112.84 (d, JCF = 23.9 Hz), 44.84, 42.50, 25.12;
HR-MS (ESI): m/z, calcd. For C21H21N4O3F2 415.1576 [M+H]+, Found: 415.1570.
5.2.5.18
N-(2-Fluoro-5-((7-fluoro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)
piperidine-4-carboxamide 2,2,2-trifluoroacetate (8r) Following the preparation protocol of Section
5.2.5.1, starting from 7r (40 mg, 0.08 mmol), the title compound 8r was obtained as white solid
(35 mg, 85.1%); m.p. 232-233.5 ℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.82 (brs, 1H),
9.81 (s, 1H), 8.04-8.10 (m, 1H), 7.79 (d, J = 6.6 Hz, 1H), 7.10-7.25 (m, 5H), 5.26 (s, 2H),
3.29-3.34 (m, 2H), 2.74-2.94 (m, 3H), 1.91-1.96 (m, 2H), 1.73-1.82 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 172.45, 166.12 (d, JCF = 249.3 Hz), 160.99, 153.10 (d, JCF = 243.9 Hz),
150.69, 142.94 (d, JCF = 12.4 Hz), 132.14 (d, JCF = 2.9 Hz), 130.76 (d, JCF = 11.3 Hz), 126.15 (d,
JCF = 12.0 Hz), 123.73 (d, JCF = 7.3 Hz), 122.59, 115.72 (d, JCF = 19.9 Hz), 112.82, 110.52 (d, JCF
= 22.8 Hz), 102.28 (d, JCF = 27.9 Hz), 44.72, 42.46, 25.12; HR-MS (ESI): m/z, calcd. For
C21H21N4O3F2 415.1576 [M+H]+, Found: 415.1568.
5.2.5.19
N-(2-Fluoro-5-((8-fluoro-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)
piperidine-4-carboxamide 2,2,2-trifluoroacetate (8s) Following the preparation protocol of Section
5.2.5.1, starting from 7s (45 mg, 0.09 mmol), the title compound 8s was obtained as white solid
(35 mg, 74.4%); m.p. 159-161 ℃; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.93 (s, 1H), 9.78 (s,
1H), 8.68 (brs, 1H), 8.39 (brs, 1H), 7.89-7.92 (m, 1H), 7.72 (m, 1H), 7.52-7.57 (m, 1H), 7.16-7.34
(m, 2H), 6.99 (m, 1H), 5.31 (s, 2H), 3.36-3.39 (m, 2H), 2.74-2.92 (m, 3H), 1.77-1.93 (m, 4H); 13
C
NMR (150 MHz, DMSO-d6) δ (ppm): 172.38, 160.95 (d, JCF = 2.6 Hz), 152.76 (d, JCF = 243.0
Hz), 150.71, 149.09 (d, JCF = 245.2 Hz), 133.87, 129.67 (d, JCF = 6.9 Hz), 125.86 (d, JCF = 12.0
Hz), 123.95, 123.78 (d, JCF = 8.0 Hz), 122.90 (d, JCF = 23.4 Hz), 122.68 (d, JCF = 7.1 Hz), 121.60,

1
119.07, 115.44 (d, JCF = 19.7 Hz), 47.93, 47.85, 42.48, 25.09; HR-MS (ESI): m/z, calcd. For
C21H21N4O3F2 415.1576 [M+H]+, Found: 415.1569.
5.3 Computational modeling
Crystal structure of PARP1 in complex with BMN-673(PDB ID: 4PJT) was selected for the
molecular modeling. Molecular docking was performed with DOCK 3.5.54.48 The target
compounds were prepared in db format with ZINC protocol.49 The amino acid residues in binding
site were identified within 12 Å of native ligand (BMN673) from the cocrystal structure, and the
solvent-accessible molecular surface was calculated with the program DMS using a probe radius
of 1.4 Å. Receptor-derived spheres were generated with the program SPHGEN while the
ligand-derived spheres were created based on the positions of the heavy atoms of ligands. The
PARP1 was kept intact, and the small molecules were subjected to 25 steps of rigid-body
minimization process. The obtained ligand conformations were scored on the basis of the total
energy, which is the sum of electrostatic and van der Waals interaction energies, corrected by the
partial ligand desolvation energy. The refinement of the complex structure in implicit solvent
model was performed using the Protein Local Optimization Program (PLOP).50 In summary, the
docked PARP1−ligand complex was submitted to multiscale truncated Newton energy
minimization using all-atom OPLS force field and Generalized Born solvent model. The whole
binding site defined with residues within 5 Å of the docked ligand was energetically minimized
with MM-GB/SA method as described previously.51, 52
5.4 Biological evaluation
5.4.1 The assay for PARP1 and PARP2 inhibition
Plasmid pET32a-PARP1 was a gift from Prof. Satoh (Canada). Human recombinant PARP1/2
were expressed and purified as described.53 The ability of compounds to inhibit PARP1/2 enzyme
activity were tested using ELISA method as described.53 IC50 values were calculated using
GraphPad Prism 5 software.
5.4.2 The cytotoxicity assay
A2780, Capan-1, MX-1, MDA-MB-231, MDA-MB-453 and MDA-MB-468 were purchased from
National Infrastruture of Cell Line Resources, Cells were seeded in a density in 96-well plates
(2,000–5,000 cells/well). Cells were treated in their recommended growth media containing
increasing concentrations of PARP inhibitors 24 h later. After 72 treatment, cell survival was
determined by MTT assay. IC50 values were calculated using GraphPad Prism 5 software.

5.4.3 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 5 µmol/L and
temozolomide (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).
Acknowledgment
This work is supported by the Special Funds for National Public Benefit Research Institutes
(No. 2013CHX16).
References and notes
1. Do, K.; Chen, A. P. Clin. Cancer Res. 2013, 19, 977.
2. Rouleau, M.; Patel, A.; Hendzel, M. J.; Kaufmann, S. H.; Poirier, G. G. Nat. Rev. Cancer 2010, 10,
293.
3. Gibson, B. A.; Kraus, W. L. Nat. Rev. Mol. Cell Biol. 2012, 13, 411.
4. Langelier, M. F.; Planck, J. L.; Roy, S.; Pascal, J. M. Science 2012, 336, 728.
5. Langelier, M. F.; Planck, J. L.; Roy, S.; Pascal, J. M. J. Biol. Chem. 2011, 286, 10690.
6. de Murcia, G.; Schreiber, V.; Molinete, M.; Saulier, B.; Poch, O.; Masson, M.; Niedergang, C.;
Ménissier de Murcia, J. Mol. Cell. Biochem. 1994, 138, 15.
7. Hottiger, M. O.; Hassa, P. O.; Lüscher, B.; Schüler, H.; Koch-Nolte, F. Trends Biochem. Sci.35, 208.
8. Gagné, J. P.; Isabelle, M.; Lo, K. S.; Bourassa, S.; Hendzel, M. J.; Dawson, V. L.; Dawson, T. M.;
Poirier, G. G. Nucleic Acids Res. 2008, 36, 6959.
9. De Vos, M.; Schreiber, V.; Dantzer, F. Biochem. Pharmacol. 2012, 84, 137.
10. Durkacz, B. W.; Omidiji, O.; Gray, D. A.; Shall, S. Nature 1980, 283, 593.
11. Javle, M.; Curtin, N. J. Br. J. Cancer 2011, 105, 1114.
12. Delaney, C. A.; Wang, L. Z.; Kyle, S.; White, A. W.; Calvert, A. H.; Curtin, N. J.; Durkacz, B. W.;
Hostomsky, Z.; Newell, D. R. Clin. Cancer Res. 2000, 6, 2860.
13. Daniel, R. A.; Rozanska, A. L.; Mulligan, E. A.; Drew, Y.; Thomas, H. D.; Castelbuono, D. J.;
Hostomsky, Z.; Plummer, E. R.; Tweddle, D. A.; Boddy, A. V.; Clifford, S. C.; Curtin, N. J. Br. J. Cancer
2010, 103, 1588.
14. Kummar, S.; Ji, J.; Morgan, R.; Lenz, H. J.; Puhalla, S. L.; Belani, C. P.; Gandara, D. R.; Allen, D.;
Kiesel, B.; Beumer, J. H.; Newman, E. M.; Rubinstein, L.; Chen, A.; Zhang, Y.; Wang, L.; Kinders, R. J.;
Parchment, R. E.; Tomaszewski, J. E.; Doroshow, J. H. Clin. Cancer Res. 2012, 18, 1726.
15. Evers, B.; Drost, R.; Schut, E.; de Bruin, M.; van der Burg, E.; Derksen, P. W.; Holstege, H.; Liu, X.;
van Drunen, E.; Beverloo, H. B.; Smith, G. C.; Martin, N. M.; Lau, A.; O'Connor, M. J.; Jonkers, J. Clin.
Cancer Res. 2008, 14, 3916.
16. Donawho, C. K.; Luo, Y.; Luo, Y.; Penning, T. D.; Bauch, J. L.; Bouska, J. J.; Bontcheva-Diaz, V. D.; Cox,
B. F.; DeWeese, T. L.; Dillehay, L. E.; Ferguson, D. C.; Ghoreishi-Haack, N. S.; Grimm, D. R.; Guan, R.; Han,
E. K.; Holley-Shanks, R. R.; Hristov, B.; Idler, K. B.; Jarvis, K.; Johnson, E. F.; Kleinberg, L. R.; Klinghofer, V.;
Lasko, L. M.; Liu, X.; Marsh, K. C.; McGonigal, T. P.; Meulbroek, J. A.; Olson, A. M.; Palma, J. P.;

Rodriguez, L. E.; Shi, Y.; Stavropoulos, J. A.; Tsurutani, A. C.; Zhu, G. D.; Rosenberg, S. H.; Giranda, V. L.;
Frost, D. J. Clin. Cancer Res. 2007, 13, 2728.
17. Farmer, H.; McCabe, N.; Lord, C. J.; Tutt, A. N.; Johnson, D. A.; Richardson, T. B.; Santarosa, M.;
Dillon, K. J.; Hickson, I.; Knights, C.; Martin, N. M.; Jackson, S. P.; Smith, G. C.; Ashworth, A. Nature
2005, 434, 917.
18. Bryant, H. E.; Schultz, N.; Thomas, H. D.; Parker, K. M.; Flower, D.; Lopez, E.; Kyle, S.; Meuth, M.;
Curtin, N. J.; Helleday, T. Nature 2005, 434, 913.
19. Fong, P. C.; Boss, D. S.; Yap, T. A.; Tutt, A.; Wu, P.; Mergui-Roelvink, M.; Mortimer, P.; Swaisland, H.;
Lau, A.; O'Connor, M. J.; Ashworth, A.; Carmichael, J.; Kaye, S. B.; Schellens, J. H.; de Bono, J. S. N. Engl.
J. Med. 2009, 361, 123.
20. Mason, K. A.; Raju, U.; Buchholz, T. A.; Wang, L.; Milas, Z. L.; Milas, L. American Journal of Clinical
Oncology 2014, 37, 90.
21. Liu, J. F.; Konstantinopoulos, P. A.; Matulonis, U. A. Gynecol. Oncol. 2014, 133, 362.
22. Purnell, M. R.; Whish, W. J. Biochem. J. 1980, 185, 775.
23. Patel, M. R.; Bhatt, A.; Steffen, J. D.; Chergui, A.; Murai, J.; Pommier, Y.; Pascal, J. M.; Trombetta, L.
D.; Fronczek, F. R.; Talele, T. T. J. Med. Chem. 2014, 57, 5579.
24. Ye, N.; Chen, C. H.; Chen, T.; Song, Z.; He, J. X.; Huan, X. J.; Song, S. S.; Liu, Q.; Chen, Y.; Ding, J.; Xu,
Y.; Miao, Z. H.; Zhang, A. J. Med. Chem. 2013, 56, 2885.
25. Zhu, G. D.; Gong, J.; Gandhi, V. B.; Liu, X.; Shi, Y.; Johnson, E. F.; Donawho, C. K.; Ellis, P. A.; Bouska,
J. J.; Osterling, D. J.; Olson, A. M.; Park, C.; Luo, Y.; Shoemaker, A.; Giranda, V. L.; Penning, T. D. Bioorg.
Med. Chem. 2012, 20, 4635.
26. Ferraris, D. V. J. Med. Chem. 2010, 53, 4561.
27. Menear, K. A.; Adcock, C.; Boulter, R.; Cockcroft, X. L.; Copsey, L.; Cranston, A.; Dillon, K. J.;
Drzewiecki, J.; Garman, S.; Gomez, S.; Javaid, H.; Kerrigan, F.; Knights, C.; Lau, A.; Loh, V. M., Jr.;
Matthews, I. T.; Moore, S.; O'Connor, M. J.; Smith, G. C.; Martin, N. M. J. Med. Chem. 2008, 51, 6581.
28. Penning, T. D.; Zhu, G. D.; Gandhi, V. B.; Gong, J.; Liu, X.; Shi, Y.; Klinghofer, V.; Johnson, E. F.;
Donawho, C. K.; Frost, D. J.; Bontcheva-Diaz, V.; Bouska, J. J.; Osterling, D. J.; Olson, A. M.; Marsh, K. C.;
Luo, Y.; Giranda, V. L. J. Med. Chem. 2009, 52, 514.
29. Plummer, R.; Lorigan, P.; Steven, N.; Scott, L.; Middleton, M. R.; Wilson, R. H.; Mulligan, E.; Curtin,
N.; Wang, D.; Dewji, R.; Abbattista, A.; Gallo, J.; Calvert, H. Cancer Chemother. Pharmacol. 2013, 71,
1191.
30. Jones, P.; Altamura, S.; Boueres, J.; Ferrigno, F.; Fonsi, M.; Giomini, C.; Lamartina, S.; Monteagudo,
E.; Ontoria, J. M.; Orsale, M. V.; Palumbi, M. C.; Pesci, S.; Roscilli, G.; Scarpelli, R.; Schultz-Fademrecht,
C.; Toniatti, C.; Rowley, M. J. Med. Chem. 2009, 52, 7170.
31. Shen, Y.; Rehman, F. L.; Feng, Y.; Boshuizen, J.; Bajrami, I.; Elliott, R.; Wang, B.; Lord, C. J.; Post, L.
E.; Ashworth, A. Clin. Cancer Res. 2013, 19, 5003.
32. Ledermann, J.; Harter, P.; Gourley, C.; Friedlander, M.; Vergote, I.; Rustin, G.; Scott, C. L.; Meier, W.;
Shapira-Frommer, R.; Safra, T.; Matei, D.; Fielding, A.; Spencer, S.; Dougherty, B.; Orr, M.; Hodgson, D.;
Barrett, J. C.; Matulonis, U. Lancet Oncol. 2014, 15, 852.
33. Curtin, N. J. Drug Discov. Today Dis. Models 2012, 9, e51.
34. Ishida, J.; Yamamoto, H.; Kido, Y.; Kamijo, K.; Murano, K.; Miyake, H.; Ohkubo, M.; Kinoshita, T.;
Warizaya, M.; Iwashita, A.; Mihara, K.; Matsuoka, N.; Hattori, K. Bioorg. Med. Chem. 2006, 14, 1378.
35. Ruf, A.; Ménnissier de Murcia, J.; de Murcia, G.; Schulz, G. E. Proceedings of the National
Academy of Sciences of the United States of America 1996, 93, 7481.

36. Ruf, A.; de Murcia, G.; Schulz, G. E. Biochemistry 1998, 37, 3893.
37. Lehtiö, L.; Jemth, A. S.; Collins, R.; Loseva, O.; Johansson, A.; Markova, N.; Hammarström, M.;
Flores, A.; Holmberg-Schiavone, L.; Weigelt, J.; Helleday, T.; Schüler, H.; Karlberg, T. J. Med. Chem. 2009,
52, 3108.
38. Zhu, L.; Jin, J.; Liu, C.; Zhang, C.; Sun, Y.; Guo, Y.; Fu, D.; Chen, X.; Xu, B. Bioorg. Med. Chem. 2011,
19, 2797.
39. Goto, S.; Tsuboi, H.; Kanoda, M.; Mukai, K.; Kagara, K. Org. Proc. Res. Dev. 2003, 7, 700.
40. Menear, K. A.; Adcock, C.; Boulter, R.; Cockcroft, X. L.; Copsey, L.; Cranston, A.; Dillon, K. J.;
Drzewiecki, J.; Garman, S.; Gomez, S.; Javaid, H.; Kerrigan, F.; Knights, C.; Lau, A.; Loh, V. M., Jr.;
Matthews, I. T.; Moore, S.; O'Connor, M. J.; Smith, G. C.; Martin, N. M. J. Med. Chem. 2008, 51, 6581.
41. Yélamos, J.; Schreiber, V.; Dantzer, F. Trends Mol. Med. 2008, 14, 169.
42. Amé, J. C.; Rolli, V.; Schreiber, V.; Niedergang, C.; Apiou, F.; Decker, P.; Muller, S.; Höger, T.;
Ménissier-de Murcia, J.; de Murcia, G. J. Biol. Chem. 1999, 274, 17860.
43. Sunderland, P. T.; Woon, E. C. Y.; Dhami, A.; Bergin, A. B.; Mahon, M. F.; Wood, P. J.; Jones, L. A.;
Tully, S. R.; Lloyd, M. D.; Thompson, A. S.; Javaid, H.; Martin, N. M. B.; Threadgill, M. D. J. Med. Chem.
2011, 54, 2049.
44. Narod, S. A.; Foulkes, W. D. Nat. Rev. Cancer 2004, 4, 665.
45. Mendes-Pereira, A. M.; Martin, S. A.; Brough, R.; McCarthy, A.; Taylor, J. R.; Kim, J. S.; Waldman,
T.; Lord, C. J.; Ashworth, A. EMBO Mol. Med. 2009, 1, 315.
46. Gil del Alcazar, C. R.; Hardebeck, M. C.; Mukherjee, B.; Tomimatsu, N.; Gao, X.; Yan, J.; Xie, X. J.;
Bachoo, R.; Li, L.; Habib, A. A.; Burma, S. Clin. Cancer Res. 2014, 20, 1235.
47. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T.
E. J. Comput. Chem. 2004, 25, 1605.
48. Meng, E. C.; Shoichet, B. K.; Kuntz, I. D. J. Comput. Chem. 1992, 13, 505.
49. Irwin, J. J.; Shoichet, B. K. J. Chem. Inf. Model. 2004, 45, 177.
50. Jacobson, M. P.; Kaminski, G. A.; Friesner, R. A.; Rapp, C. S. J. Phys. Chem. B 2002, 106, 11673.
51. Cao, R.; Liu, M.; Yin, M.; Liu, Q.; Wang, Y.; Huang, N. J. Chem. Inf. Model. 2012, 52, 2730.
52. Cao, R.; Huang, N.; Wang, Y. J. Chem. Inf. Model. 2014, 54, 1987.
53. Zhu, Z.; Jin, J.; Xue, N.; Song, X.; Chen, X. Anal. Biochem. 2014, 449, 188.