Design, synthesis and biological evaluation of quinazoline–phosphoramidate mustard conjugates as anticancer drugs

Article abstract of DOI:10.1016/j.ejmech.2016.12.055

A series of novel compounds with phosphoramide mustard functionality incorporated into the quinazoline scaffold of EGFR/HER2 inhibitors were designed and synthesized as multi-target-directed ligands against tumor cells. In?vitro assays showed that tumor cell lines with high HER2 level were more sensitive to the compounds than tumor cells with low HER2 level. Compound 10d (EMB-3) was one of the most potent inhibitors with IC₅₀of 7.4?nM and 82?nM against EGFR and HER2, respectively. The mechanism studies were also supported by the effect of 10d-induced DNA damage in MDA-MB-468?cells. In?vivo efficacy study showed that 10d could significantly inhibit H522 tumor xenograft model with a TGI of 68% at dose of 100?mg/kg (QDx28, p.o.) and no significant body weight loss was observed. MTD study indicated that compound 10d had no acute toxicity to mice at doses up to 900?mg/kg (single dose).

Full text of DOI:10.1016/j.ejmech.2016.12.055

Accepted Manuscript
Design, synthesis and biological evaluation of quinazoline–phosphoramidate mustard
conjugates as anticancer drugs
Songwen Lin, Yingbo Li, Yufen Zheng, Laichun Luo, Qi Sun, Zemei Ge, Tieming
Cheng, Runtao Li
PII:
S0223-5234(16)31061-3
10.1016/j.ejmech.2016.12.055
EJMECH 9146
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 October 2016
Revised Date: 26 December 2016
Accepted Date: 26 December 2016
Please cite this article as: S. Lin, Y. Li, Y. Zheng, L. Luo, Q. Sun, Z. Ge, T. Cheng, R. Li, Design,
synthesis and biological evaluation of quinazoline–phosphoramidate mustard conjugates as anticancer
drugs, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.055.
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ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of
quinazoline–phosphoramidate mustard conjugates as
anticancer drugs
†
†
*
Songwen Lin , Yingbo Li , Yufen Zheng, Laichun Luo, Qi Sun , Zemei Ge, Tieming Cheng,
Runtao Li*
State Key Lab of Natural and Mimetic Drugs, School of Pharmaceutical Sciences, Peking
University, Beijing, P. R. China, 100191
Corresponding Authors
*
*
Qi Sun: e-mail sunqi@bjmu.edu.cn; fax 86-10-82801571; tel 86-10-82801504.
Runtao Li: e-mail lirt@ bjmu.edu.cn; fax 86-10-82805954; tel 86-10-82805954.
Author Contributions
S. Lin and Y. Li contributed equally to this work.
†
Research highlight: 1) Phosphoramide mustard functionality incorporated into the quinazoline scaffold as
EGFR/HER2 inhibitors were proposed. 2) The mechanism studies were supported on DNA damage. 3)
Compound 10d is a potential candidate for treatment of lung cancer. 4) MTD study indicated that compound
1
0d had no obvious acute toxicity.
1

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ABSTRACT: A series of novel compounds with phosphoramide mustard functionality incorporated into the
quinazoline scaffold of EGFR/HER2 inhibitors were designed and synthesized as multi-target-directed ligands
against tumor cells. In vitro assays showed that tumor cell lines with high HER2 level were more sensitive to
the compounds than tumor cells with low HER2 level. Compound 10d (EMB-3) was one of the most potent
inhibitors with IC of 7.4 nM and 82 nM against EGFR and HER2, respectively. The mechanism studies were
5
0
also supported by the effect of 10d-induced DNA damage in MDA-MB-468 cells. In vivo efficacy study
showed that 10d could significantly inhibit H522 tumor xenograft model with a TGI of 68% at dose of 100
mg/kg (QDx28, p.o.) and no significant body weight loss was observed. MTD study indicated that compound
1
0d had no acute toxicity to mice at doses up to 900 mg/kg (single dose).
KEYWORDS: Multi-target-directed ligands, anticancer drugs, EGFR/HER2 inhibitors, DNA alkylating agents,
phosphoramide mustard.
Table of Contents graphic
R
2
Cl
O
HN
N
N
P
O
2
X
n
N
NH
Cl
R
1
1
1 2
0d (EMB-3): n = 4, X = O, R = H, R = 3-Br
EGFR: IC50 = 7.4 nM
HER2: IC50 = 82 nM
T1/2 = 1.9 h (po), F = 72 %
H522 tumor xenograft model: TGI=68% (100mg/kg, ig, qd, 28d)
MTD study: no obvious toxicities up to 900 mg/kg
2

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Introduction
Like other multigenic diseases, cancer arises from complicated network of interdependent pathological
changes. Traditional cytotoxic drugs have encountered many limitations such as lack of efficacy, severe
toxicity and drug resistance development, which prompts the interests in developing more efficacious and safer
targeted therapies. Numerous studies have shown that the dysregulations of signaling pathways play an
important role in the development of cancers. Tyrosine kinases, such as ERBB receptor family, VEGFR, c-
Met, and Src-Abl, have been validated as targets of cancer molecular targeted therapies.
The ERBB receptor family consists of four members including EGFR/erbB-1/HER1, erbB-2/ HER2, erbB-
3
/HER3, and erbB-4/HER4. Activation of HER2 and EGFR induces transphosphorylation of ERBB dimer
partner and stimulates intracellular pathways, such as RAS/RAF/MEK/ERK, PI3K/AKT/TOR, Src kinases,
and STAT transcription factors [1]. These pathways in turn regulate a variety of other proteins that are
involved in cell differentiation, proliferation, motility, and metastasis. In recent years, FDA has approved
several quinazoline derivatives as anticancer drugs, such as lapatinib [2] (1, Figure 1), gefitinib [3] (2, Figure
1
), and erlotinib [4] (3, Figure 1). However, the efficacy of receptor tyrosine kinase (RTK) inhibitors is limited
by the drug resistance that frequently emerges following the treatment [5, 6]. Direct stimulation of the
pathways by activated oncogene leads to a compensatory signaling mechanism that frees cell proliferation and
survival from EGFR control. HER2s harboring exon 20 insertions are not inhibited by lapatinib and the EGFR
T790M gatekeeper mutation leads to acquired resistance to first and second generation EGFR inhibitors [7].
These mutations make EGFR and HER2 inhibitors ineffective, but effectively inhibited by osimertinib, a third-
generation EGFR inhibitor [8] (4, Figure 1). Such a resistance can manifest intrinsic or primary resistance, or
after the selective pressure imposed on tumor subclones by EGFR therapy itself (“acquired” [9] or “secondary”
[10] resistance). Due to molecular heterogeneity among and within tumors, their efficacy is restricted to only a
small subset of patients [11].
Fig. 1
When a single medicine is not sufficient to effectively treat a disease, a multiple-medication therapy (MMT)
also referred to as a “cocktail” or “combination of drugs”) may be used. But this approach might be
(
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disadvantageous for patients with compliance problems. Another natural evolution of medication is call
multiple-compound medication (MCM). A third medication approach, named multi-target-directed ligands
(MTDLs), has been also developed to overcome undesirable clinical effects [12]. Several MTDLs have been
developed for the treatment of multifaceted neurodegenerative disease by academia and industry in recent
years [13]. One strategy of MTDLs is to incorporate the pharmacophores of drugs via stable chemical bonds to
generate one multifunctional drug that works as one unit. Another strategy is to incorporate the
pharmacophores via chemically degradable bonds to form molecules that can be converted to two or more
active ingredients after administration.
Various strategies of MTDLs have also been developed by medicinal chemists for cancer theraophies [14].
Evidences show that the combination of MET inhibitors can foster cancer stem cell eradication and durable
tumor regression for the patients sensitive to EGFR therapy [15]. CUDC-101 is a histone deacetylase inhibitor
developed by Qian’s research group. It can synergistically block key regulators of EGFR/HER2 signaling
pathways and attenuate multiple compensatory pathways, such as AKT, HER3, and MET, which enables
cancer cells to escape from the inhibition of conventional EGFR/HER2 inhibitors [16]. However, these MTDL
methods need to be tested in clinic trials.
DNA alkylating agents have been widely used due to their potent antitumor effects with broad anti-cancer cell
spectrum. The combinations of targeted therapies with cytotoxic chemotherapy may overcome the EGFR-
resistant mechanisms [17]. For example, EGFR/DNA dual targeting combi-molecules can directly inhibit
EGFR TK or fragment into another EGFR inhibitor and an alkylating agent. Jean-Claude reported another
design of EGFR/DNA dual targeting combi-molecules, which could directly inhibit EGFR [18-24]. For
example, combi-molecule 5 (Figure 2) could block EGF induced EGFR autophosphorylation in A431 cells in a
dose-dependent manner. A dose-dependent DNA damage in A431 cells was induced by 5 within a 30-min
drug exposure. Qi designed and synthesized a series of novel quinazoline nitrogen mustard derivatives and
evaluated their anticancer activities in vitro and in vivo [25]. Cytotoxicity assays indicated that compound 6
(
Figure 2) had an IC50 of 3.06 µM to HepG2, lower than that of sorafenib. In addition, compound 6 could
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block cell cycle at S and G2/M phase and induced cell apoptosis. Su reported a series of N-mustard
pharmacophore attached at the C-6 of 4-anilinoquinazolines via a urea linker [26]. Compounds 7a (R = 2-F, 3-
Cl, Figure 2), and 7b (R = 3-Cl, 4-F, Figure 2) were selected for further antitumor activity evaluation against
prostate PC-3 (7a, 69%) and human breast carcinoma MX-1 (7b, 72%) xenograft in animal model. Although
these strategies have demonstrated novel potencies against tumors in vivo, the toxicity of aryl mustard motif
has still not well resolved.
Fig. 2
Cyclophosphamide (8, CP, Figure 3) is a widely used anti-cancer agent metabolized by cytochrome P450 for
its therapeutic effectiveness. CP releases two important metabolites including phosphoramide mustard (PM)
and acrolein. PM is then transported into cells to cross-link DNA to realize its anti-tumor activities. In contrast,
acrolein is responsible for hemorrhagic cystitis, a side effect observed during CP therapy. To reduce the side
effect and discover more anticancer candidates, numerous modifications of CP have been reported in the past
two decades. The study of phosphoramide mustard derivatives is mainly focused on targeting tumor cells and
five different types of prodrugs have been well designed (Figure 4) including nitroreductase-activated cyclic
9
a [27] /acyclic [28] nitroheteroaryl phosphoramide mustards, novel DT-diaphorase targeted quinone
phosphorodiamide prodrugs 9b [29], aldophosphamide analogues 9c [30], hydrogen peroxide inducible DNA
cross-linking agents 9d [31] and heterocyclic pharmacophores and 9e [32] with high affinity to tumor cells. All
these compounds can release phosphoramide mustard that can induce the DNA interstrand crosslinks and/or
DNA alkylations and thus show potent antitumor activities via different activation mechanism. Therefore, the
design of combi-molecules targeting EGFR/HER2/DNA without acrolein-induced side-effect is highly
attractive to the medicinal chemists.
Fig. 3
Fig. 4
Based on our previous work on the modification of cyclophosphamide [33] and the synthesis of dithiocarbamic
acid esters derived EGFR inhibitors [34], we designed and synthesized quinazoline–phosphoramidate mustard
conjugate 10 (Figure 5) and determined its structure-activity relationship (SAR) in the present work. These
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compounds structurally featured a N,N-bis(chloroethyl)phosphoramide functionality incorporated into
quinazoline scaffolds via ether and amide linkers. Previous theoretical studies on molecular modeling indicate
that bulky substituents are tolerated at the 6-position of quinazoline [35]. The flexible ether linker could
specifically project the N,N-bis(chloroethyl)phosphoramide functionality in to the pocket, forming additional
interaction to increase affinity. The N,N-bis(chloroethyl)phosphoramide group could exhibit additional DNA
alkylating ability. Structural elaboration indicated that subtle change of the linker component leads to
significant difference in the potency and selectivity for EGFR. For example, 10d (EMB–3, n = 4, R = H, R =
1
2
3
-Br) effectively suppressed the progression of a lung cancer typed H522 in both in vitro anti-proliferative
assay and in vivo xenograft models. Mechanism studies indicate that 10d (EMB–3) can directly inhibit both
EGFR and HER2 signaling pathways and attenuate the cell circle as an alkylating agent.
Fig. 5
Chemistry
The preparation of the analogues with ether linkers, except 10f and 10i, is outlined in Scheme 1. Quinazoline-
4
,6-diol 11 was used as the starting material that was acetylated and treated sequentially with thionyl chloride
and substituted anilines to afford compound 12. Deacetylation of compound 12 afforded compound 13 that
was further treated with haloalcohol to yield the key intermediate 14. Alternatively, compound 14 was also
prepared from compound 15 via nucleophilic substitution with different anilines, followed by copper-catalyzed
Ullman coupling with different alkyl diols without additional ligands [36]. Compound 13 and 14 were treated
with bis(2-chloroethyl)phosphoramidic dichloride to afford the desired compound 10a–n in 12–55% yields.
Scheme 1
Compounds 10f and 10i were prepared with an alternative method (showed in Scheme 2). Protected of ethynyl
group of 10b–c using palladium-catalyzed trimethylsilanyl reaction produced key intermediates 10h and 10k.
After the phosphorodiamidate was formed, trimethylsilanyl group was treated with potassium carbonate (2
o
equiv.) in a THF/MeOH mixed solvent at 0 C to provide the desired products 10f and 10i.
Scheme 2
Similarly, the preparation of analogues with amide linker is outlined in Scheme 3. Compound 17 was treated
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sequentially with thionyl chloride and substituted anilines to afford compound 18, followed by the reduction of
the nitro group to yield compound 19. Compound 19 was treated with freshly prepared 4-
(benzoyloxy)butanoyl chloride 20 [37] and then its benzoyl group was removed to yield compound 21 in 50–
7
9% yields. Compounds 10o–q were prepared in 26–32% yields by treating compound 21 with similar
procedures as previously described procedure for compound 10.
Scheme 3
Results and discussion
Enzymatic assay.
4
-arylamino-quinazoline scaffold has long been recognized as a classical kinase inhibitor motif against EGFR
and HER2. Therefore, all synthetic compounds are usually tested for their inhibitory effects on both EGFR and
HER2 and compounds with high potency and selectivity for EGFR and/or HER2 are selected for further
profiling. As shown in Table 1, compound 10a (EMA-1) with bis(chloroethyl)phosphoramide group linked
directly on the 6-position of quinazoline scaffold via ether bond showed significant inhibitory activity for
EGFR ( IC =9.2 nM) but low inhibitory activity for HER2 ( IC =1200 nM). Increasing the linker length from
50
50
2
to 5 carbon atoms improved their inhibitory activities for both EGFR and HER2. The selectivity of all
compounds except compound 10c for EGFR versus HER2 was also improved with 130-fold for 10a, 40-fold
for 10b, 290-fold for 10c, 10-fold for 10d and 70-fold for 10e. It is interesting that the inhibitory activity for
HER2 was more sensitive to linker length. For example, compound 10d (IC =82 nM) was 15-fold more
5
0
potent than compound 10a (IC =1200 nM) without losing potency for EGFR, which could be attributed to the
5
0
structural difference between EGFR and HER2 binding pockets. As a result, 3 carbon atoms length (10c) and 4
carbon atoms length (10d) were both considered in the further structural modification for their contrary
selectivity.
Table 1
SAR study was then focused on the substituents of phenylamino group on the 4-position of quinazoline ring.
For the 4-carbon atoms length, it was found that substituted groups (10f: IC =11 nM, R = 3-ethynyl; 10g:
5
0
2
IC =6.0 nM, R = 3-chloro-4-fluoro; 10h: IC =27 nM, R = 3-chloro-4-(3’-fluorobenzyloxyl)) were well
5
0
2
50
2
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tolerated in the inhibitory activities for EGFR, but led to a considerable decrease in the inhibitory potency for
HER2. For the 3-carbon atoms length, all compounds, except the compound with 3-alkynyl aniline (10i:
IC =190 nM for HER2) that demonstrated moderate activity, with both small and large substituents showed
5
0
low activities. However, its inhibitory activity for EGFR was slightly decreased. Compound 10k showed the
lowest inhibitory activities for both EGFR and HER2.
The introduction of methoxyl group onto the 7-position of qunazoline ring resulted in a significant
improvement of the inhibitory activity for EGFR in one order of magnitude (10l: IC =0.3 nM vs 10c: IC =3.0
5
0
50
nM; 10m: IC =0.5 nM vs 10j: IC =5.7 nM). However, no obvious improvement was observed on the
5
0
50
inhibitory activity for HER2 (10l: IC =6000 nM vs 10c: IC =890 nM; 10m: IC =1300 nM vs 10j: IC =
5
0
50
50
50
2
700 nM).
Inspired by the high potency of afatinib 4 with an amide group on 6-position, we also explored the effect 4-
carbon atoms amide linker on the inhibitory activities of qunazoline derivatives against EGFR and HER2.
Compounds 10o–q were designed and screened with EGFR and HER2 assay. Similar to ether linker, small
substituted groups were well tolerated in the inhibitory activities for EGFR and large groups led to an 8-fold
decreased inhibitory activity. In contrast, the introduction of substituent groups onto the 4-position of aniline
decreased the inhibitory activity for HER2. As can be seen from Table 1, compound 10o showed better
inhibitory activities for both EGFR and HER2 with an IC of 3.3 nM and 240 nM, respectively.
5
0
Cellular inhibition study.
Based on the results of enzymatic assays, compounds 10b, 10c, 10d, 10i, 10l, 10m and 10o were selected for
further cellular assay.
Anti-proliferative activities of the selected compounds were evaluated in four cancer cell lines with different
EGFR and HER-2 expression levels, including MDA-MB-468 (high EGFR level and low HER-2 level),
HCT116 (low EGFR level and low HER-2 level), SK-BR-3 (low EGFR level and high HER-2 level) and H522
(low EGFR level and high HER-2 level) cell lines. Lapatinib, a clinically used EGFR/HER-2 targeted agent,
was used as a reference drug. The results are summarized in Table 2.
As can be seen, SKBR3 and H522 cell lines with high HER-2 level were more sensitive to the target
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compounds than HCT116 and MDA-MB-468 cell lines with low HER-2 level, indicating that the inhibitory
effects might be achieved through the regulation of HER-2. In contrast, the inhibitory effects of each
compound on HCT116 (low EGFR level) and MDA-MB-468 (high EGFR level) cell line were similar,
suggesting a relatively less contribution of EGFR to the cytotoxic effects of target compounds.
All selected compounds except 10c and 10l showed enzymatic dependent anti-proliferative activity for SK-
BR-3 tumor cell line. Compound 10d bearing 4-carbon atoms linker showed higher cellular potency with an
IC50 value of 2.8 µM than the corresponding compounds with shorter linkers. 10b with 2-carbon linker
exhibited moderate potency. Increasing the linker to three carbon atoms led to significantly increased potency
by approximate one order of magnitude. Further increasing the linker length to four carbon atoms slightly
increased the inhibitory activity. Amide linker resulted in about 3-fold decrease in the inhibition of SK-BR-3
cells (10d vs 10o). Compared with their enzymatic activities, compounds 10c and 10l bearing 3-carbon-atom
ether linkers and 4-(3-bromo-phenyl)-amino substituted groups showed much higher cellular potency.
Consistent with the results of HER2 enzymatic assays, most of the selected compounds displayed high to
moderate anti-proliferative activities for H522 cells. Compounds 10d, 10b and 10o showed an IC of 2.5, 3.0
5
0
and 2.6 µM, respectively. Compounds 10e and 10i displayed moderate anti-tumor activities. Surprisingly,
compounds 10c, 10l and 10m showed potent anti-tumor activity, which might be attributed to the induced
DNA damage in cells.
Table 2
SAR assays indicate that 10d (EMB-3) has high inhibitory activities with an IC50 of 7.4 nM and 82 nM for
EGFR and HER2, respectively. Notably, 10d stood out with higher anti-proliferative activities in both H522
and SKBR3 tumor cell lines. Therefore, it was selected for further profiling with more cell lines. As shown in
Table 3, H522 cell line was most sensitive to 10d.
Table 3
1
0d (EMB-3) induced DNA damage.
N,N-bis(chloroethyl)phosphoramide is an active metabolite of cyclophosphamide. Therefore, the DNA
alkylating ability of 10d was assessed with alkaline comet assay. Cancer cells were exposed to 10d at
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concentrations 1, 3 and 10 times of its IC , respectively. As shown in Figure 6, 10d significantly increased
50
comet tail length and Tail DNA/Head DNA ratio of MDA-MB-468 cells at all test concentrations, indicating
its DNA damaging activity to the cell line. In addition, 10d also showed DNA damaging activities in HCT116
and Sk-Br-3 cells, indicating that 10d-induced DNA damage was not cell type-specific.
Fig. 6
Preliminary pharmacokinetic study
The pharmacokinetic parameters of 10d in rats following per os administration are summarized in Table 4. The
iv administration of 10d at a single dose of 1 mg/kg led to a CL of 76 mL/min/kg. The po administration of
1
0d at a single dose of 10 mg/kg resulted in a moderate t1/2 of 1.9 h. In addition, compound 10d was rapidly
absorbed and its maximum plasma concentration Cmax = 1987 ng/mL was reached in 0.5 h. Notably,
compound 10d demonstrated high oral bioavailability (F=72 %) in rats.
Table 4.
In vivo efficacy study
Based on its high in vitro activities and favorable in vivo pharmacokinetic properties, compound 10d was
selected for in vivo anti-tumor efficacy study. A tumor xenograft model was established by injecting H522
3
cells into nude mice. When tumors reached to 200–500 mm , the mice were i.g. administrated daily with 10d
at a dose of 50 and 100 mg/kg, respectively. Lapatinib (100 mg/kg) and Cyclophosphamide (CTX, 50 mg/kg)
treated mice were included as positive controls. Established tumors were harvested and weighted at day 28. As
shown in Figure 7, compared with that of the control group, the tumor volume was significantly reduced by the
treatments with 10d, lapatinib and CTX. Tumor volume in the vehicle control group increased 4.77 fold in 28
days. In addition, 10d demonstrated good dose-dependent inhibition on the tumor (Figure 8A). The treatments
with 50 and 100 mg/kg 10d resulted in a TGI of 59% and 68 %, respectively, in 28 days (Figure 8B).
Fig. 7
In vivo toxic study
Maximum tolerant dose (MTD) of compound 10d was also determined. Mice were randomized into vehicle
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control and four treatment groups (n=3). The treatment groups were orally administrated at a single dose of
1
00, 300, 600, and 900 mg/kg 10d, respectively. The mice were monitored continuously for the first 4 h for
any abnormal behavior and mortality change and observed intermittently in the next 24 h and occasionally
thereafter for 14 days for any delayed onset effect. All mice were sacrificed on day 14 after the administration
and examined under a macroscopy for any possible damage in the heart, liver, and kidneys. The result
indicates that compound 10d has no acute toxicity and mortality to mice either immediately or during the post-
treatment period. In addition, no significant abnormal change or morbidity was observed during the
experimental period in terms of water and food consumption and body weight. In all, compound 10d is well
tolerated by mice at doses up to 900 mg/kg and no obvious toxicities were detected (Figure 8).
Fig. 8
■
CONCLUSION
In summary, we have designed and synthesized a series of novel compounds by incorporating phosphoramide
mustard functionality into the quinazoline scaffold of EGFR/HER2 inhibitors. These compounds were
demonstrated as multi-target-directed ligands against tumor cells. The designed compounds were screened
with the in vitro enzymatic assay. The results indicate that tumor cell lines with high HER2 level are more
sensitive to the compounds than tumor cells with low HER2 level. Compound 10d (EMB-3), one of the most
potent inhibitors with IC50 of 7.4 nM and 82 nM against EGFR and HER2, respectively, was selected for the
antitumor screen on H522 cell line. It showed an excellent inhibitory activity in H522 tumor xenograft model
with a TGI of 68% at a dose of 100mg/kg daily for 28 days without causing any significant body weight loss.
In addition, compound 10d showed no observed acute toxicity to mice at single doses up to 900 mg/kg. These
results indicate that compound 10d is a potential lead compound for the treatment of lung cancer. Further
investigations of the therapeutic candidate among these compounds are in progress.
Supporting Information
All reagents and solvents were from commercial sources. EGFR and HER2 were purchased from Invitrogen.
1
All the tumor cell lines were purchased from ATCC. Melting points were determined on X4 microscope. H
1
3
31
Ⅲ
NMR, C NMR and P NMR spectra were recorded on JOEL AL 300 (300 MHz) and Bruker AVANCE
4
00 (400 MHz) instruments. Elemental analyses were performed on a Vario ELIII (Germany) instrument.
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HRMS (ESI+) spectra were performed on a Bruker Apex IV FTMS.
4
-((3-Bromophenyl)amino)quinazolin-6-yl acetate (12a)
A mixture of quinazoline-4,6-diol 11a (23.55 g, 145.24 mmol), pyridine (27 mL) in acetic anhydride (220
mL) was stirred at 100 °C for 2 h. After cooling to room temperature, the reaction mixture was poured into ice
water. The resulting solid was collected by suck filtration, washed with water, and dried at 50 °C to afford 4-
1
hydroxyquinazolin-6-yl acetate as an off-white solid (22.40 g, 76% yield). mp > 300 °C [34b]. H NMR (300
MHz, d -DMSO) δ 12.35 (s, 1H), 8.11 (s, 1H), 7.83 (d, J = 2.7 Hz, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.60 (dd, J =
6
2
.7, 8.7 Hz, 1H), 2.32 (s, 3H).
A mixture of 4-hydroxyquinazolin-6-yl acetate (0.62 g, 3 mmol) and DMF (0.1 mL) in thionyl chloride
(10 mL) was stirred under reflux for 6 h and then thionyl chloride was removed under reduced pressure. To the
residue was added isopropanol (20 mL), 3-bromoaniline (0.62 g, 3.6 mmol) and triethylamine (0.36 g, 3.6
mmol). The resulting reaction mixture was stirred at room temperature for 6 h and then at reflux for another 3
h. After cooling to room temperature, the yellow solid was collected by suck filtration, wash with isopropanol,
water and ether sequentially, and dried at 50 °C to afford compound 12a as a yellow solid (0.74 g, 69% yield).
1
mp 238–239 °C [34b]. H NMR (300 MHz, d -DMSO) δ 11.39 (s, 1H), 8.99 (s, 1H), 8.67(s, 1H), 7.93–8.09
6
(m, 3H), 7.78 (d, J = 7.5 Hz, 1H), 7.44–7.54 (m, 2H), 2.40 (s, 3H).
4
-((3-Bromophenyl)amino)-7-methoxy-quinazolin-6-yl acetate (12b)
4
-hydroxy-7-methoxyquinazolin-6-yl acetate was prepared from 7-methoxyquinazoline-4,6-diol (11b) in a
1
yield of 87%, according to the procedure of compound 12a. mp 290–292 °C (Lit.: 293 °C [38]). H NMR (400
MHz, d -DMSO) δ 11.19 (br s, 1H), 8.94 (s, 1H), 8.67 (d, J = 4.8 Hz, 1H), 8.06 (s, 1H), 7.76 (d, J = 8 Hz, 1H),
6
7
.42–7.48 (m, 3H), 4.01 (s, 3H), 2.39 (s, 3H).
4
-((3-Chloro-4-fluorophenyl)amino)-7-methoxy-quinazolin-6-yl acetate (12c)
Compound 12c was prepared from 11b in a yield of 78%, according to the procedure of compound 12a, mp
1
1
2
84–186 °C [34c]. H NMR (400 MHz, d -DMSO) δ 11.12 (br s, 1H), 8.94 (s, 1H), 8.62 (s, 1H), 8.07 (dd, J =
6
.4, 6.8 Hz, 1H), 7.71–7.75 (m, 1H), 7.54 (t, J = 8.8 Hz, 1H), 7.46 (s, 1H), 4.01 (s, 3H), 2.39 (s, 3H).
4
-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-7-methoxyquinazolin-6-yl acetate (12d)
Compound 12d was prepared from 11b in a yield of 52%, according to the procedure of compound 12a, mp
1
1
94–196 °C [34c]. H NMR (400 MHz, d -DMSO) δ 9.69 (s, 1H), 8.58 (s, 1H), 8.30 (s, 1H), 8.03 (s, 1H),
6
7
.70–7.72 (m, 1H), 7.46–7.48 (m, 1H), 7.25–7.34 (m, 4H), 7.16–7.21 (m, 1H), 5.25 (s, 2H), 3.95 (s, 3H), 2.37
12

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(s, 3H).
4
-((3-Bromophenyl)amino)quinazolin-6-ol (13a)
A mixture of compound 12a (1.40 g, 3.91 mmol) and ammonium hydroxide (8 mL) in methanol (30 mL) was
stirred at room temperature for 2 h. After removing the volatile under reduced pressure, the residue was diluted
with water (30 mL), and stirred for 10 min. The resulting solid was collected by suck filtration, washed with
water, and dried at 50 °C to afford compound 13a as an off-white solid (1.16 g, 94% yield), mp > 300 °C
1
[
34b]. H NMR (300 MHz, d -DMSO) δ 10.11 (s, 1H), 9.60 (s, 1H), 8.52 (s, 1H),8.27 (s, 1H), 7.80 (s, 1H),
6
7
.71 (d, J = 9.0 Hz, 1H), 7.46 (d, J = 9.0 Hz, 1H), 7.28–7.37 (m, 1H).
4
-((3-Bromophenyl)amino)-7-methoxyquinazolin-6-ol (13b)
Compound 13b was prepared from 12b in a yield of 79%, according to the procedure of compound 13a.
1
mp 280–282 °C (Lit.: 272 °C [39]). H NMR (400 MHz, d -DMSO) δ 9.50 (br s, 1H), 9.44 (s, 1H), 8.50 (s,
6
1
H), 8.25 (s, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.81 (s, 1H), 7.22–7.34 (m, 3H), 3.98 (s, 3H).
4
-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-ol (13c)
Compound 13c was prepared from 12c in a yield of 85%, according to the procedure of compound 13a,
1
mp 285–288 °C (Lit.: 285 °C [39]). H NMR (400 MHz, d -DMSO) δ 9.67 (s, 1H), 9.46 (s, 1H), 8.48 (s, 1H),
6
8
.21 (dd, J = 2.4, 6.8 Hz, 1H), 7.82–7.86 (m, 1H),7.78 (s, 1H), 7.41 (t, J = 9.2 Hz, 1H), 7.22 (s, 1H), 3.98 (s,
3
H).
4
-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-7-methoxyquinazolin-6-ol (13d)
Compound 13d was prepared from 12d in a yield of 86%, according to the procedure of compound 13a,
1
mp 239–241 °C [34c]. H NMR (400 MHz, d -DMSO) δ 8.43 (s, 1H), 8.04 (d, J = 2.4 Hz, 1H), 7.76 (s, 1H),
6
7
3
.73 (dd, J = 2.4, 8.8 Hz, 1H), 7.45–7.50 (m, 1H), 7.30–7.34 (m, 2H), 7.16–7.25 (m, 3H), 5.24 (s, 2H), 3.97 (s,
H).
4
-((3-Bromophenyl)amino)-6-iodo-quinazoline (16a)
To a round bottle, isopropanol (20 mL), 3-bromoaniline (0.62 g, 3.6 mmol), 4-cholo-6-iodo-quinazoline
1
5 (0.87 g, 3 mmol) and triethylamine (0.36 g, 3.6 mmol) was added. The resulting reaction mixture was
stirred at room temperature for 6 h and then at reflux for another 3 h. After cooling to room temperature, the
yellow solid was collected by suck filtration, wash with isopropanol, water and ether sequentially, and dried at
1
5
0 °C to afford compound 12a as a yellow solid (0.74 g, 69% yield), mp 238–239 °C [36]. H NMR (400
MHz, d6-DMSO) δ 10.90 (s, 1H), 9.22 (s, 1H), 8.84 (s, 1H), 8.23 (dd, J = 8.2, 3.4 Hz, 1H), 8.13 (s, 1H),
13

ACCEPTED MANUSCRIPT
1
3
7
.85 (dd, J = 8.2, 3.4 Hz, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.40 (t, J = 9.0 Hz, 1H). C NMR (100 MHz, d6-
DMSO) δ 157.84, 152.05, 143.47, 141.3, 138.94, 132.65, 130.52, 128.31, 126.16, 123.9, 122.6, 121.12, 115.6,
9
3.69.
4
-((3-Chloro-4-fluorophenyl)amino)-6-iodo-quinazoline (16b)
Compound 16b was prepared in a yield of 95% according to the procedure of compound 16a as yellow
1
solid, mp 162–165 °C [36]. H NMR (400 MHz, DMSO-d6) δ 10.77 (s, 1H), 9.15 (s, 1H), 8.80 (s, 1H), 8.22
(
(
(
d, J = 8.7 Hz, 1H), 8.15 (dd, J = 6.7, 2.3 Hz, 1H), 7.84 (dd, J = 8.2, 3.4 Hz, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.49
1
3
t, J = 9.0 Hz, 1H). C NMR (100 MHz, DMSO-d6) δ 156.18, 154.52, 152.11, 150.02, 147.86, 143.16, 137.78
d, J = 2.9 Hz), 129.2, 124.3, 123.24, 122.17 (d, J = 6.6 Hz), 119.14 (d, J = 18.3 Hz), 117.01 (d, J = 7.4 Hz),
1
16.76, 101.28.
4
-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-7-methoxyquinazoline (16c)
Compound 16c was prepared in a yield of 93% according to the procedure of compound 16a as white
1
solid, mp 221–224 °C [36]. H NMR (400 MHz, d6-DMSO) δ 9.84 (s, 1H), 8.95 (s, 1H), 8.62 (s, 1H), 8.10 (d,
J = 8.6 Hz, 1H), 8.05 (s, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 8.7 Hz, 1H), 7.48 (dd, J = 13.9, 7.8 Hz,
1
3
1
1
1
9
H), 7.38 – 7.24 (m, 3H), 7.19 (t, J = 8.5 Hz, 1H), 5.26 (s, 2H). C NMR (100 MHz, d6-DMSO) δ 163.65,
61.23, 156.53, 155.03, 149.98, 148.97, 141.52, 139.87 (d, J = 7.7 Hz), 133.23, 131.59, 130.77 (d, J = 8.0 Hz),
30.01, 124.16, 123.53, 122.30, 121.30, 117.05, 114.92 (d, J = 20.8 Hz), 114.42 (d, J = 13.6 Hz), 114.14,
1.74, 69.63.
2
-((4-((3-Bromophenyl)amino)quinazolin-6-yl)oxy)ethanol (14a)
A mixture of 13a (0.32 g, 1.00 mmol), 2-chloroethanol (0.10 g, 1.25 mmol), potassium carbonate (0.28 g, 2.00
mmol) and potassium iodide (0.017 g, 0.10 mmol) in acetonitrile (10 mL) was stirred under reflux for 12 h.
After removing the solvent under reduced pressure, the residue was diluted with water (20 mL) and extracted
with ethyl acetate (30 mL×2). The combined organic layers was washed with brine (30 mL×2), dried over
anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column
chromatography (silica gel, petroleum ether/ethyl acetate = 1:2 then 1:5) to afford compound 14a as a pale
1
yellow solid (0.22 g, 60% yield), mp 213–215 °C [34c]. H NMR (300 MHz, d -DMSO) δ 8.57 (s, 1H), 8.21
6
(s, 1H), 7.92–7.94 (m, 2H), 7.79 (d, J = 9.0 Hz, 1H), 7.54 (dd, J = 2.4, 9.0 Hz, 1H), 7.30–7.43 (m, 2H), 4.21 (t,
13
J = 4.8 Hz, 2H), 3.85 (t, J = 4.8 Hz, 2H), 3.57 (br s, 1H). C NMR (75 MHz, d -DMSO) δ 157.08, 156.72,
6
1
52.22, 145.17, 141.05, 130.56, 129.62, 126.10, 124.86, 124.25, 121.36, 120.81, 115.74, 102.80, 70.41, 59.52.
Anal. Calcd for C H BrN O : C, 53.35; H, 3.92; N, 11.67. Found: C, 53.36; H, 3.95; N, 11.69.
1
6
14
3
2
14

ACCEPTED MANUSCRIPT
3
-((4-((3-Bromophenyl)amino)quinazolin-6-yl)oxy)propan-1-ol (14b)
Compound 14b was prepared from 13a in a yield of 60%, according to the procedure of compound 14a. mp
1
1
7
1
1
6
1
84–186 °C [36]. H NMR (300 MHz, d -DMSO) δ 9.70 (s, 1H), 8.57 (s, 1H), 8.20 (t, J = 1.8 Hz 1H), 7.93–
6
.94 (m, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.52 (dd, J = 2.4, 9.0 Hz, 1H), 7.30–7.40 (m, 2H), 4.68 (t, J = 5.1 Hz,
1
3
H), 4.24 (t, J = 6.3 Hz, 2H), 3.62–3.68 (m, 2H), 1.94–2.02 (m, 2H). C NMR (75 MHz, d -DMSO) δ 157.12,
6
56.71, 152.16, 145.12, 141.08, 130.52, 129.56, 126.05, 124.73, 124.26, 121.36, 120.80, 115.77, 102.81,
5.63, 57.31, 32.13. Anal. Calcd for C H BrN O : C, 54.56; H, 4.31; N, 11.23. Found: C, 54.63; H, 4.37; N,
17
16
3
2
1.41.
4
-((4-((3-Bromophenyl)amino)quinazolin-6-yl)oxy)butan-1-ol (14c)
Compound 14c was prepared from 13a in a yield of 68%, according to the procedure of compound 14a. mp
1
2
02–204 °C [36]. H NMR (300 MHz, d -DMSO) δ 9.65 (s, 1H),8.56 (s, 1H), 8.20 (s, 1H), 7.91–7.94 (m, 2H),
6
7
.75 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 9.0 Hz, 1H), 7.29–7.40 (m, 2H), 4.50 (t, J = 4.8 Hz, 1H), 4.18 (t, J = 6.0
1
3
Hz, 2H), 3.48-3.53 (m, 2H), 1.82–1.91 (m, 2H), 1.60–1.69 (m, 2H). C NMR (100 MHz, d -DMSO) δ 157.06,
6
1
6
4
56.69, 152.13, 145.07, 141.04, 130.50, 129.56, 126.04, 124.62, 124.24, 121.32, 120.79, 115.76, 102.92,
8.43, 60.42, 29.05, 25.50. Anal. Calcd for C H BrN O : C, 55.68; H, 4.67; N, 10.82. Found: C, 55.68; H,
1
8
18
3
2
.77; N, 10.84.
3
-((4-((3-Bromophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propan-1-ol (14d)
Compound 14d was prepared from 13b in a yield of 78%, according to the procedure of compound 14a.mp
1
2
51–253 °C [34c]. H NMR (400 MHz, d -DMSO) δ 9.53 (br s, 1H), 8.52 (s, 1H), 8.15 (s, 1H), 7.88 (d, J = 8.4
6
Hz, 1H), 7.85 (s, 1H), 7.21–7.37 (m, 3H), 4.61 (t, J = 4.8 Hz, 1H), 4.23 (t, J = 6.4 Hz, 2H), 3.95 (s, 3H), 3.61–
1
3
3
.66 (m, 2H), 1.96–2.02 (m, 2H). C NMR (75 MHz, d -DMSO) δ 156.00, 154.58, 152.66, 148.59, 147.00,
6
1
41.32, 130.45, 125.71, 124.08, 121.33, 120.63, 109.03, 107.21, 102.38, 66.05, 57.37, 55.94, 32.10. Anal.
Calcd for C H BrN O : C, 53.48; H, 4.49; N, 10.39. Found: C, 53.31; H, 4.62; N, 10.09.
1
8
18
3
3
3
-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propan-1-ol (14e)
Compound 14e was prepared from 13c in a yield of 91%, according to the procedure of compound 14a.
1
mp 280–282 °C [34c]. H NMR (400 MHz, d -DMSO) δ 9.56 (s, 1H), 8.50 (s, 1 H), 8.12 (dd, J = 2.4, 6.8 Hz,
6
1
3
1
3
H), 7.78–7.83 (m, 2H), 7.44 (t, J = 8.8 Hz, 1H), 7.21 (s, 1H), 4.62 (t, J = 4.8 Hz, 1H), 4.23 (t, J = 6.0 Hz, 2H),
1
3
.94 (s, 3H), 3.61–3.65 (m, 2H), 1.95–2.03 (m, 2H). C NMR (75 MHz, d -DMSO) δ 156.05, 154.59, 153.27,
6
52.66, 148.58, 146.92, 136.84, 123.50, 122.34, 118.91, 116.61, 108.86, 107.23, 102.39, 66.05, 57.34, 55.96,
2.07. Anal. Calcd for C H ClFN O : C, 57.22; H, 4.54; N, 11.12. Found: C, 56.97; H, 4.70; N, 10.95.
1
8
17
3
3
15

ACCEPTED MANUSCRIPT
3
-((4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propan-1-ol (14f)
Compound 14f was prepared from 13d in a yield of 83%, according to the procedure of compound 14a.
1
mp 212–213 °C [34c]. H NMR (400 MHz, d -DMSO) δ 8.46 (s, 1H), 7.97(d, J = 2.4 Hz, 1H), 7.81 (s, 1H),
6
7
3
1
1
6
.72 (dd, J = 2.4, 8.8 Hz, 1H), 7.45–7.51 (m, 1H), 7.18–7.35 (m, 5H), 5.25 (s, 2H), 4.22 (t, J = 6.4 Hz, 2H),
1
3
.94 (s, 3H), 3.65 (t, J = 6.0 Hz, 2H), 1.97–2.03 (m, 2H). C NMR (100 MHz, d -DMSO) δ 162.33, 156.24,
6
54.46, 152.84, 149.51, 148.45, 146.73, 139.79, 133.55, 130.68, 124.02, 123.45, 122.19, 121.19, 114.82,
14.43, 114.14, 108.80, 107.19, 102.48, 69.54, 66.03, 57.36, 55.91, 32.08. Anal. Calcd for C H ClFN O : C,
2
5
23
3
4
2.05; H, 4.79; N, 8.68. Found: C, 61.79; H, 4.77; N, 8.65.
5
-((4-((3-Bromophenyl)amino)-quinazolin-6-yl)oxy)pentan-1-ol (14g)
A mixture of 16a (3.80 g, 8.9 mmol), sodium tert-butoxide (2.50 g, 26.7 mmol), cuprous iodide
170mg) was added to a 100-mL flask with three necks under nitrogen. 1, 5-pentylene glycol (2.4g, 26.7 mmol)
and N,N-dimethylformamide (25 mL) were then injected into the flask and the mixture was stirred at 80 °C for
8h. After cooling to room temperature, 100 mL water was added to the reaction mixture. After filtration, the
(
1
resulting solid was dissolved in mixed solution of DCM: MeOH (50:1). The filtrate was concentrated under
reduced pressure to afford compound 14g as a white solid (48% yield), mp 78–79 °C [36].
1
H NMR (400 MHz, d6-DMSO) δ 9.68 (s, 1H), 8.61 (s, 1H), 8.23 (s, 1H), 7.96 (d, J = 7.5 Hz, 2H), 7.78 (s,
1
H), 7.40 (t, J = 8.0 Hz, 1H), 7.35 (t, J = 10.8 Hz, 1H), 4.46 (t, J = 5.0 Hz, 1H), 4.26–4.08 (m, 2H), 3.48 (d, J
1
3
=
5.0 Hz, 2H), 1.86 (s, 2H), 1.56 (s, 4H). C NMR (100 MHz, d6-DMSO) δ 157.85, 157.50, 152.77, 146.00,
1
2
42.07, 131.20, 130.45, 127.04, 126.73, 125.17, 125.11, 122.17, 121.61, 103.76, 69.29, 61.64, 33.24, 29.57,
3.21.
4
-((4-((3-Trimethylsilanylethynyl-phenyl)amino)-quinazolin-6-yl)oxy)butan-1-ol (14h)
A mixture of 14c (8.25 g, 21.3 mmol), trimethyl silyl acetylene (20g, 213.6mmol), Pd(PPh ) Cl (1.65 g,
3
2
2
2
.35 mmol), cuprous iodide (1.43g, 7.5 mmol) and dimethylformamide (40 mL) was stirred at 90°C under
nitrogen for 36h. The reaction mixture was purified by flash column chromatography (silica gel,
o
1
dichloromethane /methanol) to afford compound 14h as a yellow solid (28% yield), mp: 138–139 C. H NMR
400 MHz, d6-DMSO) δ 9.58 (s, 1H), 8.54 (s, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.97 (s, 1H), 7.91 (s, 1H), 7.73 (d,
(
J = 9.1 Hz, 1H), 7.50 (d, J = 11.6 Hz, 1H), 7.40 (t, J = 6.3 Hz, 1H), 7.20 (d, J = 7.7 Hz, 1H), 4.51 (t, J = 5.1
Hz, 1H), 4.16 (t, J = 6.5 Hz, 2H), 3.50 (dd, J = 11.6, 6.3 Hz, 2H), 1.94–1.77 (m, 2H), 1.75–1.54 (m, 2H), 0.25
1
3
(
s, 9H). C NMR (100 MHz, d6-DMSO) δ 157.82, 157.61, 153.04, 145.95, 140.56, 130.42, 129.88, 127.28,
1
25.63, 125.36, 123.55, 123.12, 116.61, 106.08, 103.70, 94.92, 69.22, 61.31, 29.95, 26.42, 0.84. HRMS: m/z
16

ACCEPTED MANUSCRIPT
+
calcd. for C H N O Si [M+H] 406.19453; found 406.19381.
2
3
27
3
2
4
-((4-((3-Chloro-4-fluoro-phenyl)amino)-quinazolin-6-yl)oxy)butan-1-ol (14i)
Compound 14i was prepared from 16b in a yield of 65%, according to the procedure of compound
1
1
4g. Grey solid, mp 210–211 °C [36]. H NMR (400 MHz, d6-DMSO) δ 9.70 (s, 1H), 8.62 (s, 1H), 8.20 (s,
1
H), 8.07–7.62 (m, 3H), 7.52 (d, J = 25.4 Hz, 2H), 4.55 (s, 1H), 4.19 (s, 2H), 3.54 (s, 2H), 1.89 (s, 2H), 1.67
1
3
(
s, 2H). C NMR (100 MHz, d6-DMSO) δ 157.83, 157.55, 155.42, 153.01, 137.54, 137.51, 130.48, 125.28,
1
24.55, 123.42, 123.35, 119.83, 119.64, 117.57, 117.36, 103.82, 69.22, 61.31, 29.93, 26.34.
4
1
-((4-((3-Chloro-4-((3-fluorobenzyl)oxy)-phenyl)amino)-quinazolin-6-yl)oxy)butan-1-ol (14j)
Compound 14j was prepared from 16c in a yield of 72%, according to the procedure of compound
1
4g. Grey solid, mp 156–157 °C [36]. H NMR (400 MHz, d6-DMSO) δ 9.61 (s, 1H), 8.57 (s, 1H), 8.04 (d, J
=
2.5 Hz, 1H), 7.92 (s, 1H), 7.77 (dd, J = 8.9, 2.5 Hz, 2H), 7.60–7.44 (m, 2H), 7.44–7.28 (m, 3H), 7.22 (td, J =
8
1
1
1
.6, 2.2 Hz, 1H), 5.29 (s, 2H), 4.54 (t, J = 5.1 Hz, 1H), 4.19 (t, J = 6.5 Hz, 2H), 3.54 (dd, J = 11.5, 6.3 Hz, 2H),
1
3
.97–1.79 (m, 2H), 1.67 (dt, J = 9.7, 6.5 Hz, 2H). C NMR (100 MHz, d6-DMSO) δ 164.37, 161.94, 157.72,
57.65, 150.47, 140.61, 140.54, 134.33, 131.43, 131.35, 125.01, 124.95, 124.17, 124.14, 123.07, 122.04,
15.66, 115.46, 115.13, 115.02, 114.80, 103.78, 70.34, 69.19, 61.37, 29.97, 26.38.
3
-((4-((3-Trimethylsilanylethynyl-phenyl)amino)-quinazolin-6-yl)oxy)propan-1-ol (14k)
Compound 14k was prepared from 14b in a yield of 21%, according to the procedure of compound
1
1
4h. Grey solid, mp 145–146 °C. H NMR (400 MHz, d6-DMSO) δ 9.65 (s, 1H), 8.61 (s, 1H), 8.05 (d, J = 8.3
Hz, 1H), 8.01 (s, 1H), 7.97 (s, 1H), 7.78 (s, 1H), 7.54 (d, J = 6.6 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.24 (d, J =
7
9
1
.6 Hz, 1H), 4.69 (s, 1H), 4.27 (t, J = 6.3 Hz, 2H), 3.68 (t, J = 6.1 Hz, 2H), 2.01 (p, J = 6.1 Hz, 2H), 0.29 (s,
1
3
H). C NMR (100 MHz, d6-DMSO) δ 157.87, 157.57, 152.95, 146.08, 140.62, 130.40, 129.78, 127.23,
25.63, 125.34, 123.49, 123.16, 106.14, 103.56, 94.83, 66.39, 58.19, 33.06, 0.82.
3
1
-((4-((3-Chloro-4-fluoro-phenyl)amino)-quinazolin-6-yl)oxy)propan-1-ol (14l)
Compound 14l was prepared from 16b in a yield of 60%, according to the procedure of compound
1
4g. Grey solid, mp 183–184 °C [36]. H NMR (400 MHz, d6-DMSO) δ 9.73 (s, 1H), 8.59 (s, 1H), 8.20 (dd, J
=
6.9, 2.6 Hz, 1H), 8.04–7.68 (m, 3H), 7.66–7.40 (m, 2H), 4.69 (t, J = 5.0 Hz, 1H), 4.26 (t, J = 6.3 Hz, 2H),
1
3
3
1
.67 (dd, J = 11.2, 6.0 Hz, 2H), 2.01 (p, J = 6.2 Hz, 2H). C NMR (100 MHz, d6-DMSO) δ 157.88, 157.58,
55.42, 153.01, 152.93, 137.53, 130.44, 125.39, 124.54, 123.40, 123.34, 119.83, 119.64, 117.57, 117.35,
17

ACCEPTED MANUSCRIPT
1
03.68, 66.43, 58.16, 33.02.
3
-((4-((3-Chloro-4-((3-fluorobenzyl)oxy)-phenyl)amino)-quinazolin-6-yl)oxy)propan-1-ol (14m)
Compound 14m was prepared from 16c in a yield of 77%, according to the procedure of compound
1
1
2
1
4g. Grey solid, mp 87–88 °C [36]. H NMR (400 MHz, d6-DMSO) δ 9.64 (s, 1H), 8.67 (s, 1H), 8.04 (d, J =
.5 Hz, 1H), 7.96 (s, 1H), 7.89–7.68 (m, 2H), 7.58–7.43 (m, 2H), 7.43–7.27 (m, 3H), 7.22 (td, J = 8.6, 2.1 Hz,
H), 5.29 (s, 2H), 4.69 (t, J = 5.0 Hz, 1H), 4.26 (t, J = 6.3 Hz, 2H), 3.67 (q, J = 6.0 Hz, 2H), 2.01 (p, J = 6.2
1
3
Hz, 2H). C NMR (100 MHz, d6-DMSO) δ 164.39, 161.97, 157.81, 157.66, 150.49, 140.64, 140.56, 134.35,
1
33.97, 132.31, 131.46, 131.38, 125.13, 124.98, 124.83, 124.21, 124.18, 123.10, 122.93, 122.06, 115.69,
15.49, 115.16, 115.05, 114.83, 103.70, 92.39, 70.36, 66.42, 58.24, 49.56, 33.07.
1
N-(3-Bromophenyl)-6-nitroquinazolin-4-amine (18a)
A mixture of compound 17 (4.4 g, 23 mmol) and DMF (0.1 mL) in SOCl (30 mL) was reflux until the
2
soli disappeared. After removing SOCl under reduce pressure, i-PrOH (140 mL), 3-bromoaniline (3.96 g, 23
2
mmol) and triethylamine (2.33 g, 23 mmol) were added to the residue and the resulting mixture was refluxed
for 6 h. After cooling to room temperature, the yellow solid was collected by suck filtration, washed with i-
PrOH, water and ether, dried at 50 °C to afford compound 18a as a yellow solid (5.88 g, 74% yield), mp 268–
1
2
8
69 °C (Lit.: 269–271 °C [40]). H NMR (300 MHz, d -DMSO) δ 10.48 (s, 1H), 9.65 (s, 1H), 8.78 (s, 1H),
6
.57 (dd, J = 1.2, 9.0 Hz, 1H), 8.20 (s, 1H), 7.90–7.97 (m, 2H), 7.36–7.43 (m, 2H).
N-(3-Chloro-4-fluorophenyl)-6-nitroquinazolin-4-amine (18b)
Compound 18b was prepared from 17 in a yield of 65%, according to the procedure of compound 18a,
1
using 3-chloro-4-fluoroaniline in place of 3-bromoaniline. mp 278–280 °C (Lit.: 274.5–277 °C [41]). H NMR
(
400 MHz, d -DMSO) δ 10.56 (br s, 1 H), 9.64 (s, 1 H), 8.79 (s, 1 H), 8.59 (dd, J = 2.4, 9.2 Hz, 1 H), 8.17 (d, J
6
=
4.8 Hz, 1 H), 7.97 (d, J = 8.8 Hz, 1 H), 7.84 (d, J = 8.4 Hz, 1 H), 7.50 (t, J = 9.2 Hz, 1 H).
N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-nitroquinazolin-4-amine (18c)
Compound 18c was prepared from 17 in a yield of 97%, according to the procedure of compound 18a,
using 3-chloro-4-((3-fluorobenzyl)oxy)anilinein place of 3-bromoaniline. mp 279–281 °C (Lit.: 246–248 °C
1
[
42]). H NMR (400 MHz, d -DMSO) δ 11.19 (br s, 1H), 9.71 (d, J = 6.0 Hz, 1H), 8.88 (d, J = 10.0 Hz, 1H),
6
8
.68 (t, J = 6.8Hz, 1H), 7.98–8.04 (m, 2H), 7.71 (d, J = 8.8 Hz, 1H), 7.46–7.51 (m, 1H), 7.31–7.36 (m, 3H),
7
.18–7.22 (m, 1H), 5.30 (s, 2H).
18

ACCEPTED MANUSCRIPT
4
-(3-Bromophenylamino)-6-aminoquinazoline (19a)
A mixture of Sn powder (5.07 g, 42.75 mmol) and compound 18a (2.95 g, 8.55 mmol) in concentrated
hydrochloride solution (22 mL) was stirred at room temperature for 4 hours. The mixture was diluted with
water (200 mL), alkalized with NaOH solution to pH = 9, and extracted with ethyl acetate (100 mL×3).The
combined organic layers were washed brine (50 mL×3), dried over anhydrous sodium sulfate, filtered and
concentrated to afford compound 19a as a yellow solid (2.52g, 94% yield), mp 263–265 °C (Lit.: 267–270 °C
1
[
23]. H NMR (300 MHz, d -DMSO) δ 9.46 (br s, 1H), 8.39 (s, 1H), 8.24 (t, J = 1.8 Hz, 1H), 7.90 (d, J = 8.4
6
Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.22–7.35 (m, 4H), 5.64 (br s, 2H).
4
-(3-Chloro-4-fluorophenylamino)-6-aminoquinazoline (19b)
Compound 19b was prepared from 18b in a yield of 92%, according to the procedure of compound 19a,
1
mp 253–255 °C (Lit.: 244 °C [43]). H NMR (400 MHz, d -DMSO) δ 9.48 (s, 1H), 8.37 (s, 1 H), 8.21 (dd, J =
6
2
.4, 6.8 Hz, 1H), 7.81–7.85 (m, 1H), 7.55 (d, J = 9.2 Hz, 1H), 7.41 (t, J = 8.4 Hz, 1H), 7.13 (d, J = 2.0 Hz,
H), 7.26 (dd, J = 2.0, 8.8 Hz, 1H), 5.63 (s, 2H).
1
4
-(3-Chloro-4-(3-fluorobenzyloxyl)phenylamino)-6-aminoquinazoline (19c)
Compound 19c was prepared from 18c in a yield of 97%, according to the procedure of compound 19a,
1
mp 190–192 °C (Lit.: 186–188 °C [43]). H NMR (400 MHz, d -DMSO) δ 8.52 (s, 1 H), 7.94 (d, J = 2.4 Hz, 1
6
H), 7.65 (dd, J = 2.0, 8.8 Hz, 1 H), 7.59 (d, J = 9.2 Hz, 1 H), 7.45–7.51 (m, 1 H), 7.28–7.39 (m, 5 H), 7.17–
7
.21 (m, 1 H), 5.28 (s, 2 H).
N-(4-((3-Bromophenyl)amino)quinazolin-6-yl)-4-hydroxybutanamide (21a)
The freshly prepared 4-(benzoyloxy)butanoyl chloride 20 was added dropwise to a stirred mixture of
compound 19a (1.58 g, 5 mmol) and Na CO (0.53 g, 5 mmol) in anhydrous THF (50 mL). The resulting
2
3
mixture was stirred at room temperature for 12 hours. The volatiles were removed under reduced pressure. To
the residue was added methanol (50 mL), water (10 mL) and NaOH (0.80 g, 20 mmol), and the resulting
mixture was stirred for 4 hours. The volatiles were removed under reduced pressure and the residue was
diluted with water (50 mL) and extracted with ethyl acetate (30 mL×3). The combined organic layers were
washed brine (50 mL×2), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was
purified by flash column chromatography (silica gel, petroleum ether/ethyl acetate = 1:6 then ethyl acetate) to
1
afford compound 21a in a yield of 50%. mp 175–176 °C. H NMR (300 MHz, d -DMSO) δ 10.28 (s, 1H), 9.92
6
(s, 1H), 8.76 (s, 1H), 8.59 (s, 1H), 8.18 (s, 1H), 7.77–7.88 (m, 3H), 7.28–7.38 (m, 2H), 4.59 (t, J = 4.8 Hz, 1H),
1
3
3
.47–3.53 (m, 2H), 2.47 (t, J = 7.5 Hz, 2H), 1.77–1.86 (m, 2H). C NMR (100 MHz, d -DMSO) δ 171.81,
6
19

ACCEPTED MANUSCRIPT
1
1
4
57.29, 153.04, 146.53, 141.14, 137.16, 130.41, 128.45, 127.21, 125.99, 124.34, 121.27, 120.89, 115.50,
11.65, 60.23, 33.08, 28.38. Anal. Cald for C H BrN O : C, 53.88; H, 4.27; N, 13.96; Found: C, 53.58; H,
1
8
17
4
2
.35; N, 13.68.
N-(4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)-4-hydroxybutanamide (21b)
Compound 21b was prepared from 19b in a yield of 79%, according to the procedure of compound 21a,
1
mp 208–209 °C. H NMR (400 MHz, d -DMSO) δ 10.29 (s, 1H), 8.70 (d, J = 1.6 Hz, 1H), 8.55 (s, 1H), 8.13
6
(dd, J = 2.4, 6.8 Hz, 1H), 7.77–7.86 (m, 3H), 7.44 (t, J = 8.4 Hz, 1H), 3.48 (t, J = 6.4 Hz, 2H), 2.46 (t, J = 7.6
1
3
Hz, 2H), 1.77–1.84 (m, 2H). C NMR (75 MHz, d -DMSO) δ 171.74, 157.34, 153.48, 153.05, 146.47, 137.08,
6
1
36.68, 128.49, 127.18, 123.83, 122.70, 118.91, 116.61, 115.37, 111.44, 60.22, 33.08, 28.38. Anal. Cald for
C H ClFN O : C, 57.68; H, 4.30; N, 14.95; Found: C, 57.48; H, 4.30; N, 14.90.
18
16
4
2
N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)-4-hydroxybutanamide (21c)
Compound 21c was prepared from 19c in a yield of 51%, according to the procedure of compound 21a,
1
mp 209–211 °C. H NMR (400 MHz, d -DMSO) δ 8.68 (s, 1H), 8.51 (s, 1H), 7.98 (d, J = 2.0 Hz, 1H), 7.69–
6
7
.85 (m, 3H), 7.45–7.51 (m, 1H), 7.17–7.35 (m, 4H), 5.26 (s, 2H), 3.49 (t, J = 7.2 Hz, 2H), 2.46 (t, J = 7.2 Hz,
1
3
2
H), 1.77–1.84 (m, 2H). C NMR (75 MHz, d -DMSO) δ 171.70, 162.35, 157.45, 153.27, 149.69, 146.40,
6
1
39.80, 136.93, 133.41, 130.70, 128.41, 127.05, 124.26, 123.45, 122.47, 121.15, 115.36, 114.83, 114.34,
14.15, 111.57, 69.52, 60.24, 33.09, 28.41. Anal. Cald for C H ClFN O : C, 62.44; H, 4.61; N, 11.65;
1
2
5
22
4
3
Found: C, 62.17; H, 4.68; N, 11.38.
4
-((3-Bromophenyl)amino)quinazolin-6-yl N,N-bis(2-chloroethyl)phosphorodiamidate (10a)
To a mixture of compound 13a (0.32 g, 1 mmol) in anhydrous THF (40 mL) at 0 °C under nitrogen
atmosphere was added dropwise n-butyl lithium solution (1.6 M in hexane, 0.63 mL, 1.0 mmol). The reaction
mixture was stirred at 0 °C for 1 h then transferred dropwise into a solution of bis(2-
chloroethyl)phosphoramidic dichloride (0.31 g, 1.2 mmol) in anhydrous THF (10 mL) at 0 °C under nitrogen
atmosphere. After stirring for 3 h at 0 °C under nitrogen atmosphere, the reaction mixture was bubbled with
ammonium gas for 30 min and then diluted with water (25 mL). The resulting mixture was neutralized with 1N
HCl solution and extracted with ethyl acetate (25 mL×2). The combined organic layers were washed brine (25
mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column
chromatography (silica gel, ethyl acetate/methanol = 25:1） to afford compound 10a as an off-white solid
1
(
0.22 g, 42% yield), mp 200–202 °C. H NMR (400 MHz, d -DMSO) δ 8.64 (s, 1H), 8.27 (s, 1H), 8.21 (s, 1H),
6
1
3
7
.85–7.91 (m, 3H), 7.32–7.40 (m, 2H), 3.66–3.71 (m, 4H), 3.41–3.46 (m, 4H). C NMR (100 MHz, d6-
DMSO) δ 157.25, 153.62, 149.02, 146.83, 140.83, 130.49, 129.44, 127.86, 126.25, 124.35, 121.33, 120.87,
20

ACCEPTED MANUSCRIPT
31
1
15.59, 113.28, 48.92, 42.44. P NMR (122 MHz, d -DMSO) δ 15.59. Anal. Cald for C H BrCl N O P: C,
6
18 19
2
5
2
4
1.64; H, 3.69; N, 13.49; Found: C, 41.70; H, 3.83; N, 13.53.
2
-(6-(4-((3-Bromophenyl)amino)quinazolin-6-yl)oxy)ethyl
N,N-bis(2-chloroethyl)phosphorodiamidate
(10b)
Compound 10b was prepared from 14a in a yield of 37%, according to the procedure of compound 10a,
1
mp 143–144 °C. H NMR (400 MHz, CDCl ) δ 9.08 (br s, 1H), 8.70 (s, 1 H), 8.25 (s, 1H), 7.98–8.01 (m, 2H),
3
7
8
1
.79 (d, J = 9.2 Hz, 1H), 7.35 (d, J = 2.0, 8.8 Hz, 1H), 7.22–7.25 (m, 2H), 4.20–4.47 (m, 4H), 3.38–3.64 (m,
1
3
H), 3.07 (br s, 2H). C NMR (100 MHz, d -DMSO) δ 156.75, 156.55, 152.26, 145.10, 140.98, 130.49,
6
3
1
29.55, 126.08, 124.71, 124.20, 121.34, 120.73, 115.67, 102.98, 68.05, 62.87, 48.93, 42.54. P NMR (122
MHz, d -DMSO) δ 18.68. Anal. Cald for C H BrCl N O P: C, 42.65; H, 4.12; N, 12.43; Found: C, 42.67; H,
6
20 23
2
5
3
4
.16; N, 12.45.
3
-(6-(4-((3-Bromophenyl)amino)quinazolin-6-yl)oxy)propyl N,N-bis(2-chloroethyl)phosphorodiamidate
(10c)
Compound 10c was prepared from 14b in a yield of 31%, according to the procedure of compound 10a,
1
mp 158–159 °C. H NMR (400 MHz, d -DMSO) δ 8.56 (s, 1H), 8.21 (s, 1 H), 7.96 (s, 1H), 7.93 (d, J = 8.0 Hz,
6
1
H), 7.77 (d, J = 9.2 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 4.28
13
(t, J = 5.6 Hz, 2H), 4.03–4.08 (m, 2H), 3.65 (t, J = 7.2 Hz, 4H), 3.26–3.33 (m, 4H), 2.12–2.18 (m, 2H).
C
NMR (100 MHz, d -DMSO) δ 156.87, 156.75, 152.22, 145.18, 141.04, 130.52, 129.61, 126.07, 124.67,
6
3
1
1
1
24.25, 121.32, 120.80, 115.76, 103.10, 65.05, 61.28, 48.86, 42.56, 29.85. P NMR (122 MHz, d -DMSO) δ
6
8.12. Anal. Cald for C H BrCl N O P: C, 43.70; H, 4.37; N, 12.13; Found: C, 43.51; H, 4.65; N, 11.84.
2
1
25
2
5
3
4
-((4-((3-Bromophenyl)amino)quinazolin-6-yl)oxy)butyl
N,N-bis(2-chloroethyl)phosphorodiamidate
(10d)
Compound 10d was prepared from 14c in a yield of 36%, according to the procedure of compound 10a,
1
mp 70–71 °C. H NMR (400 MHz, CDCl ) δ 9.40 (br s, 1H), 8.64 (s, 1H), 8.03 (s, 1H), 7.84–7.85 (m, 1H),
3
7
4
1
4
4
.72–7.77 (m, 2H), 7.33 (dd, J = 2.0, 4.8 Hz, 1H), 7.18–7.22 (m, 2H), 3.97–4.12 (m, 4H), 3.59 (t, J = 6.4 Hz,
1
3
H), 3.39–3.45 (m, 4H), 3.13 (s, 2H), 1.80–1.93 (m, 4H). C NMR (100 MHz, CDCl ) δ 157.28, 156.84,
3
52.36, 144.85, 140.49, 129.81, 129.10, 126.62, 125.03, 124.53, 121.94, 120.88, 116.05, 102.72, 67.63, 64.69,
3
1
8.73, 42.26, 26.70, 24.79. P NMR (122 MHz, d -DMSO) δ 17.98. Anal. Cald for C H BrCl N O P: C,
6
22 27
2
5
3
4.69; H, 4.60; N, 11.84; Found: C, 44.63; H, 4.72; N, 11.68.
5
-((4-((3-Bromophenyl)amino)quinazolin-6-yl)oxy)pentyl
N,N-bis(2-chloroethyl)phosphorodiamidate
(10e)
21

ACCEPTED MANUSCRIPT
Compound 10e 380mg was prepared from 14g in a yield of 25%, according to the procedure of compound 10a.
o
1
Yellow solid, mp 64–65 C. H NMR (400 MHz, d6-DMSO) δ 9.84 (s, 1H), 8.60 (s, 1H), 8.24 (s, 1H), 7.98
dd, J = 14.8, 5.3 Hz, 2H), 7.79 (s, 1H), 7.56 (dd, J = 9.1, 2.5 Hz, 1H), 7.44–7.31 (m, 2H), 4.44 (d, J = 5.1 Hz,
(
2
4
1
1
H), 4.21 (t, J = 6.3 Hz, 2H), 3.88 (dt, J = 6.0, 4.1 Hz, 2H), 3.69 (t, J = 7.4 Hz, 4H), 3.32 (dt, J = 11.7, 7.5 Hz,
1
3
H), 1.94–1.83 (m, 2H), 1.77–1.66 (m, 2H), 1.58 (dt, J = 14.5, 7.2 Hz, 2H). C NMR (100 MHz, d6-DMSO) δ
57.89, 157.70, 152.78, 145.41, 141.91, 131.27, 130.00, 126.93, 125.51, 125.27, 122.09, 121.81, 116.60,
+
03.98, 69.26, 64.98, 64.92, 43.44, 30.67, 30.60, 29.17, 22.87. LC-MS: 605.9 [M+1] . HRMS (ESI+) m/z
+
calcd for C H BrCl N O P[M+H] 604.06412; found 604.06372. HPLC: 5.70 min, 99.2% (214 nm), 98.7%
2
3
29
2
5
3
tR
(254 nm).
4
-((4-((3-Ethynylphenyl)amino)quinazolin-6-yl)oxy)butyl
N,N-bis(2-chloroethyl)phosphorodiamidate
(10f)
Compound 10f was prepared from 14h according to the procedure of compound 10a, then the formed
intermediate (1.15g) was treated with potassium carbonate (2 equiv.) in tetrahydrofuran (15 mL) and methanol
o
(
15 mL) at 0 C for 1h. After concentrated the reaction solution, the reaction solution was diluted with water
10 mL) and extracted with ethyl acetate (20 mL). The combined organic layers was dried over anhydrous
(
sodium sulfate, filtered and concentrated to afford compound 10f as a yellow solid (350 mg, 40% yield), mp
o
7
0–71 C.
1
H NMR (400 MHz, d -DMSO) δ 9.81 (s, 1H), 8.59 (s, 1H), 8.08 (s, 1H), 7.99 (dd, J = 16.0, 5.9 Hz, 2H),
6
7
.78 (d, J = 9.1 Hz, 1H), 7.56 (dd, J = 9.1, 2.5 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.28 (d, J = 7.7 Hz, 1H), 4.47
(d, J = 5.3 Hz, 2H), 4.29 – 4.20 (m, 3H), 3.94 (d, J = 4.9 Hz, 2H), 3.69 (t, J = 7.4 Hz, 4H), 3.33 (dt, J = 11.7,
1
3
7
1
1
.5 Hz, 4H), 1.92 (dd, J = 13.9, 6.2 Hz, 2H), 1.83 (dt, J = 12.6, 6.1 Hz, 2H). C NMR (100 MHz, d -DMSO) δ
6
57.82, 157.76, 152.94, 145.49, 140.46, 130.05, 129.84, 127.67, 126.03, 125.45, 123.82, 122.69, 116.60,
+
04.02, 84.39, 81.56, 68.93, 64.85, 64.80, 49.77, 49.73, 43.42, 27.74, 27.67, 26.05. LC-MS: 536.0 [M+1] .
+
HRMS: m/z calcd. for C H N O PCl [M+H] 536.13796; found 536.13736. HPLC: 3.54 min, 95.4% (214
2
4
28
5
3
2
tR
nm), 95.2% (254 nm).
4
-((4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)oxy)butyl
chloroethyl)phosphorodiamidate (10g)
Compound 10g 300mg was prepared from 14i in a yield of 13 %, according to the procedure of
N,N-bis(2-
o
1
compound 10a. Yellow solid, mp 72–73 C. H NMR (400 MHz, d6-DMSO) δ 9.88 (s, 1H), 8.58 (s, 1H), 8.22
(
d, J = 6.6 Hz, 1H), 7.99 (s, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 9.1 Hz, 1H), 7.62 – 7.41 (m, 2H), 4.48
d, J = 4.9 Hz, 2H), 4.24 (t, J = 5.9 Hz, 2H), 3.94 (d, J = 5.1 Hz, 2H), 3.69 (t, J = 7.3 Hz, 4H), 3.33 (dt, J =
(
22

ACCEPTED MANUSCRIPT
1
3
1
1
1
4.6, 7.4 Hz, 4H), 2.01 – 1.74 (m, 4H). C NMR (100 MHz, d6-DMSO) δ 157.76, 157.70, 155.47, 153.05,
52.84, 145.53, 137.48, 130.11, 125.44, 124.65, 123.52, 123.45, 119.81, 119.62, 117.53, 117.32, 116.50,
+
04.01, 68.94, 64.85, 64.80, 49.77, 49.73, 43.42, 27.73, 27.66, 26.04. LC-MS: 563.9 [M+1] . HRMS: m/z
+
calcd. for C H N O PCl F[M+H] 564.08956; found 564.08913. HPLC: 4.02 min, 96.9% (214 nm), 96.8%
2
2
26
5
3
3
tR
(254 nm).
4
-((4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)oxy)butyl
N,N-bis(2-
chloroethyl)phosphorodiamidate (10h)
Compound 10h 400mg was prepared from 14j in a yield of 28 %, according to the procedure of
o
1
compound 10a. Yellow solid, mp 67–68 C. H NMR (400 MHz, d6-DMSO) δ 9.78 (s, 1H), 8.54 (s, 1H), 8.04
(d, J = 2.6 Hz, 1H), 7.98 (d, J = 2.4 Hz, 1H), 7.82–7.72 (m, 2H), 7.58–7.47 (m, 2H), 7.40–7.28 (m, 3H), 7.22
(td, J = 8.7, 2.3 Hz, 1H), 5.29 (s, 2H), 4.47 (d, J = 5.4 Hz, 2H), 4.23 (t, J = 6.2 Hz, 2H), 3.95 (d, J = 6.6 Hz,
1
3
2
1
1
7
H), 3.70 (t, J = 7.4 Hz, 4H), 3.44 – 3.23 (m, 5H), 1.99–1.76 (m, 4H). C NMR (100 MHz, d6-DMSO) δ
64.34, 161.92, 157.87, 157.71, 152.99, 150.60, 145.18, 140.63, 140.55, 134.12, 131.52, 131.44, 129.87,
25.33, 125.15, 124.26, 124.23, 123.33, 121.97, 116.45, 115.72, 115.51, 115.21, 115.06, 114.84, 104.00,
+
0.32, 68.91, 64.84, 64.79, 49.77, 49.73, 43.42, 27.74, 27.67, 26.04. LC–MS: 669.6 [M+1] . HRMS: m/z
+
calcd. for C H N O PCl F[M+H] 670.13143; found 670.13171. HPLC: 4.78 min, 98.6% (214 nm), 98.6%
2
9
32
5
4
3
tR
(254 nm).
3
-((4-((3-Ethynylphenyl)amino)quinazolin-6-yl)oxy)propyl N,N-bis(2-chloroethyl)phosphorodiamidate
(10i)
Compound 10i was prepared from 14k in a yield of 55 %, according to the procedure of compound
o
1
1
7
1
2
0f. Yellow solid, mp 159–160 C. H NMR (400 MHz, d6-DMSO) δ 9.73 (s, 1H), 8.58 (s, 1H), 8.09 (s, 1H),
.99 (dd, J = 12.1, 5.2 Hz, 2H), 7.78 (d, J = 9.1 Hz, 1H), 7.56 (dd, J = 9.1, 2.2 Hz, 1H), 7.45 (t, J = 7.9 Hz,
H), 7.27 (d, J = 7.6 Hz, 1H), 4.51 (d, J = 5.3 Hz, 2H), 4.31 (t, J = 6.0 Hz, 2H), 4.25 (s, 1H), 4.16 – 4.02 (m,
13
H), 3.69 (t, J = 7.3 Hz, 4H), 3.32 (dt, J = 11.7, 7.4 Hz, 4H), 2.25 – 2.13 (m, 2H). C NMR (100 MHz, d6-
DMSO) δ 157.73, 157.59, 153.10, 145.98, 140.54, 130.37, 129.81, 127.55, 125.89, 125.30, 123.67, 122.69,
+
1
16.61, 104.02, 84.40, 81.49, 65.85, 62.04, 61.99, 49.73, 49.69, 43.37, 30.74, 30.67. LC-MS: 521.9 [M+1] .
+
HRMS: m/z calcd. for C H N O PCl [M+H] 522.12231; found 522.12126. HPLC: 3.41 min, 99.2% (214
2
3
26
5
3
2
tR
nm), 99.5% (254 nm).
3
-((4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)oxy)propyl
N,N-bis(2-
chloroethyl)phosphorodiamidate (10j)
Compound 10j 230mg was prepared from 14l in a yield of 12 %, according to the procedure of
o
1
compound 10a. Yellow solid, mp 145–146 C. H NMR (400 MHz, d6-DMSO) δ 9.87 (s, 1H), 8.58 (s, 1H),
23

ACCEPTED MANUSCRIPT
8
4
.23 (d, J = 4.4 Hz, 1H), 8.02 (s, 1H), 7.90 (s, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.62 – 7.41 (m, 2H), 4.52 (s, 2H),
1
3
.31 (s, 2H), 4.07 (d, J = 5.7 Hz, 2H), 3.68 (t, J = 6.4 Hz, 4H), 3.33 (s, 4H), 2.17 (s, 2H). C NMR (100 MHz,
d6-DMSO) δ 157.67, 157.63, 155.48, 153.01, 152.97, 145.84, 137.53, 130.31, 125.39, 124.57, 123.44, 123.37,
1
19.80, 119.62, 117.54, 117.32, 116.51, 104.09, 65.91, 62.06, 62.01, 49.72, 49.68, 43.38, 30.72, 30.65. LC-MS:
+
+
5
51.9 [M+1] . HRMS: m/z calcd. for C H N O PCl F [M+H] 550.07391; found 550.07268. HPLC: 3.84
2
1
24
5
3
3
tR
min, 98.7% (214 nm), 98.4% (254 nm).
3
-((4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)oxy)propyl
chloroethyl)phosphorodiamidate (10k)
Compound 10k 200 mg was prepared from 14m in a yield of 14 %, according to the procedure of
N,N-bis(2-
o
1
compound 10a. Yellow solid, mp 77–78 C. H NMR (400 MHz, d6-DMSO) δ 9.79 (s, 1H), 8.54 (s, 1H), 8.05
t, J = 8.7 Hz, 2H), 7.74 (m, 2H), 7.48 – 7.55 (m, 2H), 7.30 – 7.36 (m, 2H), 7.22 (t, J = 8.7, 1H), 5.29 (s, 2H),
.51 (d, J = 5.4 Hz, 2H), 4.30 (t, J = 6.2 Hz, 2H), 4.07 (d, J = 6.6 Hz, 2H), 3.67 (t, J = 7.4 Hz, 4H), 3.29 – 3.35
(
4
1
3
(
m, 4H), 2017 (m, 2H). C NMR (100 MHz, d6-DMSO) δ 164.35, 161.93, 157.86, 157.58, 153.11, 150.56,
1
1
3
45.46, 140.64, 140.57, 134.20, 131.52, 131.44, 130.04, 125.29, 125.07, 124.27, 124.24, 123.25, 121.97,
16.48, 115.72, 115.52, 115.22, 115.07, 114.85, 104.08, 70.33, 65.87, 62.06, 62.01, 49.73, 49.69, 43.39, 30.74,
+
0.67. LC-MS: 655.9. HRMS: m/z calcd. for C H Cl FN O P [M+H] 656.11578; found 656.11483.
2
8
30
3
5
4
HPLC: 4.61 min, 99.4% (214 nm), 99.8% (254 nm).
tR
3
-((4-((3-Bromophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl
chloroethyl)phosphorodiamidate (10l)
Compound 10l was prepared from 14d in a yield of 30%, according to the procedure of compound
N,N-bis(2-
1
1
3
2
1
0a. H NMR (400 MHz, CDCl ) δ 9.27 (s, 1H), 8.63 (s, 1H), 8.04 (s, 1H), 7.89–7.92 (m, 2H), 7.18–7.21 (m,
3
H), 4.17–4.37 (m, 4H), 3.96 (s, 3H), 3.62 (t, J = 6.0 Hz, 4H), 3.42–3.49 (m, 4H), 3.09 (d, J = 4.8 Hz, 2H),
1
3
.20–2.33 (m, 2H). C NMR (100 MHz, CDCl ) δ 156.83, 155.20, 153.65, 148.08, 147.60, 141.07, 129.93,
3
3
1
26.18, 124.58, 122.04, 120.40, 109.70, 107.54, 105.14, 67.00, 62.93, 56.03, 48.76, 42.39, 29.46. P NMR
(
122 MHz, CDCl ) δ 16.07. Anal. Calcd for C H BrCl N O P: C, 43.51; H, 4.48; N, 11.53. Found: C, 43.24;
3 22 27 2 5 4
H, 4.34; N, 11.62.
3
-((4-((3-Chloro-4-fluorophenyl)amino))-7-methoxyquinazolin-6-yl)oxy)propyl
N,N-bis(2-
chloroethyl)phosphorodiamidate (10m)
Compound 10m was prepared from 14e in a yield of 46%, according to the procedure of compound
1
1
1
3
0a, mp 150-152 °C. H NMR (400 MHz, CDCl ) δ 9.30 (br s, 1H), 8.59 (s, 1H), 7.96 (dd, J = 2.4, 6.4 Hz,
3
H), 7.84 (s, 1H), 7.72–7.76 (m, 1H), 7.15 (s, 1H), 7.09 (t, J = 8.8 Hz, 1H), 4.16–4.36 (m, 4H), 3.93 (s, 3H),
1
3
.61 (t, J = 6.0 Hz, 4H), 3.39–3.50 (m, 4H), 3.19 (d, J = 4.4 Hz, 2H), 2.19–2.29 (m, 2H). C NMR (100 MHz,
24

ACCEPTED MANUSCRIPT
CDCl ) δ 156.86, 155.19, 154.24, 153.57, 148.08, 147.42, 136.26, 124.02, 121.84, 120.30, 116.12, 109.47,
3
3
1
1
07.43, 105.10, 66.98, 62.89, 56.00, 48.65, 42.27, 29.45. P NMR (122 MHz, CDCl ) δ 15.79. Anal. Calcd for
3
C H Cl FN O P: C, 45.49; H, 4.51; N, 12.06. Found: C, 45.72; H, 4.64; N, 11.89.
22
26
3
5
4
3
-((4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl N,N-bis(2-
chloroethyl)phosphorodiamidate (10n)
Compound 10n was prepared from 14f in a yield of 29%, according to the procedure of compound 10a,
1
mp 137–138 °C. H NMR (400 MHz, CDCl ) δ 9.21 (s, 1H), 8.57 (s, 1H), 7.85 (d, J = 2.8 Hz, 1H), 7.79 (s,
3
1
H), 7.65 (d, J = 2.4, 8.8 Hz, 1H), 7.31–7.36 (m, 1H), 7.14–7.22 (m, 3H), 6.98–7.02 (m, 1H), 6.88 (d, J = 8.8
Hz, 1H), 4.13–4.33 (m, 4H), 3.91 (s, 3H), 3.57 (t, J = 6.4 Hz, 4H), 3.38–3.45 (m, 4H), 3.20 (d, J = 4.8 Hz, 2H),
1
3
2
1
7
.19–2.25 (m, 2H). C NMR (100 MHz, CDCl ) δ 162.91, 157.00, 154.99, 153.71, 150.20, 147.96, 147.27,
3
39.22, 133.67, 130.06, 124.57, 122.90, 122.41, 121.87, 114.75, 114.24, 113.90, 109.45, 107.40, 104.77,
3
1
0.35, 66.77, 62.84, 55.96, 48.74, 42.32, 29.46. P NMR (122 MHz, CDCl ) δ 15.75. Anal. Calcd for
3
C H Cl FN O P: C, 50.71; H, 4.70; N, 10.20. Found: C, 50.56; H, 4.88; N, 9.97.
29
32
3
5
5
4
-oxo-4-((4-((3-Bromophenyl)amino)quinazolin-6-yl)amino)butyl
N,N-bis(2-
chloroethyl)phosphorodiamidate (10o)
Compound 10o was prepared from 21a in a yield of 44%, according to the procedure of compound
1
1
0a, using NaH in place of n-butyl lithium. H NMR (300 MHz, CDCl ) δ 2.01–2.07 (m, 2H), 2.52 (d, J = 5.4
3
Hz, 2H), 3.32–3.46 (m, 6H), 3.59 (t, J = 8.4 Hz, 4H), 4.01–4.11 (m, 2H), 7.14–7.22 (m, 2H), 7.62–7.67 (m,
1
3
3
1
3
H), 7.97(s, 1H), 8.50–8.61(m, 3H), 9.95 (s, 1H). C NMR (100 MHz, CDCl ) δ 171.98, 157.38, 153.32,
3
46.21, 140.14, 136.69, 129.93, 128.24, 126.86, 124.86, 122.11, 120.67, 115.40, 111.08, 64.57, 48.81, 42.55,
3
1
3.07, 26.41. P NMR (122 MHz, d -DMSO) δ 18.08. Anal. Cald for C H BrCl N O P: C, 43.73; H, 4.34;
6
22 26
2
6
3
N, 13.91; Found: C, 43.81; H, 4.55; N, 14.07.
4
-oxo-4-((4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)amino)butyl
N,N-bis(2-
chloroethyl)phosphorodiamidate (10p)
Compound 10p was prepared from 21b in a yield of 32%, according to the procedure of compound 10a,
1
using NaH in place of n-butyl lithium. H NMR (400 MHz, CDCl ) δ 10.03 (s, 1H), 8.73 (s, 1H), 8.54 (s, 1H),
3
8
3
1
6
4
.52 (s, 1H), 7.83 (dd, J = 2.4, 6.4 Hz, 1H), 7.52–7.67 (m, 3H), 7.05 (t, J = 8.8 Hz, 1H), 3.99–4.09 (m, 2H),
1
3
.40–3.63 (m, 10H), 2.46–2.58(m, 2H), 2.00–2.06 (m, 2H). C NMR (100 MHz, CDCl ) δ 171.98, 157.39,
3
54.65, 153.36, 146.44, 136.52, 135.32, 128.45, 126.77, 124.25, 121.99, 120.52, 116.25, 115.27, 111.03,
3
1
4.50, 48.74, 42.26, 33.02, 26.56. P NMR (122 MHz, CDCl ) δ 18.00. Anal. Cald for C H C FN O P: C,
3
22 25 l3
6
3
5.73; H, 4.36; N, 14.54; Found: C, 45.79; H, 4.66; N, 14.43.
25

ACCEPTED MANUSCRIPT
4
-Oxo-4-((4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)amino)butyl
N,N-bis(2-
chloroethyl)phosphorodiamidate (10q)
Compound 10q was prepared from 21c in a yield of 26%, according to the procedure of compound
1
1
0a, using NaH in place of n-butyl lithium. H NMR (400 MHz, CDCl ) δ 10.01 (br s,1H), 8.67 (br s,1H), 8.52
3
(s, 2H), 7.73 (d, J = 2.0 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.46 (dd, J = 1.2, 8.4 Hz,
1
4
H), 7.29–7.35 (m, 1H), 7.16–7.20 (m, 2H), 6.97–7.01 (m, 1H), 6.83 (d, J = 9.2 Hz, 1H), 5.04 (s, 2 H), 3.97–
1
3
.06 (m, 2H), 3.52–3.60 (m, 6 H), 3.36–3.43 (m, 4H), 2.48 (d, J = 4.8 Hz, 2H), 1.95–2.01 (m, 2H). C NMR
(
100 MHz, CDCl ) δ 171.89, 162.88, 157.57, 153.58, 150.65, 146.22, 139.06, 136.46, 132.62, 130.08, 128.21,
3
1
3
6
26.65, 124.81, 122.88, 122.40, 122.10, 115.25, 114.77, 113.97, 113.86, 111.09, 70.22, 64.46, 48.76, 42.27,
3
1
+
2.97, 26.44. P NMR (122 MHz, CDCl ) δ 18.10. HRMS (ESI+) m/z calcd for C H Cl FN O P (M + H) ,
3
29 32
3
6
4
+
83.12668, found 683.12562 (M + H) .
Cell proliferation assay
4
MDA-MB-468, SK-Br-3, HCT-116 and H522 cells were seeded in 96-well plates with 2–3.5×10 cells/well
and incubated for 24 h. Cells were then exposed to agents for 72 h. Cells in control group were treated with a
medium containing 0.5% DMSO. After the treatments, cells were then incubated with 100 µL medium
containing 0.5 mg/mL MTT at 37 °C for 4 h. Cell supernatants were discarded. MTT crystals were dissolved
in 200 µL DMSO and measured with a Multilabel counter at 620 nm. Average OD (optical density) value of a
given compound concentration was calculated from the triplicate wells. Then percent inhibiton was calculated
according to the following equation “percent inhibition=(average ODvehicle –average ODsample)/ average OD
vehicle”. IC50 was calculated by plotting percent inhibition to drug concentration in nonlinear curves in
GraphPad Prism software.
Tyrosine kinase assay
Compounds were respectively dissolved in DMSO to prepare 10 mM stock solutions. EGFR (25 ng) or HER2
(150 ng) were incubated with different concentrations of compounds in 50 µl kinase reaction buffer (40 mM
Tris (pH 7.4), 10 mM MgCl , 0.1 mg/ml BSA, 1 mM DTT, 10 µM ATP, 0.2 mg/ml Poly (Glu, Tyr)) for 40
2
min at 30 °C. ATP content was determined by measuring the luminescence of the reaction mixture with a MD-
SpectraMax M5 Multilabel counter.
Alkaline comet assay
Alkaline comet assay was performed to evaluate the DNA damage as previously described.[23] Briefly, cells
4
(
1×10 cells) treated with 10d (EMB-3) for 2 h were harvested, mixed with 0.7% low melting point agarose,
and then mounted on a microscope slide precoated with normal melting point agarose. After solidification,
26

ACCEPTED MANUSCRIPT
lysis, electrophoresis and equilibration, slides were stained with SYBR Green and examined under a
fluorescence microscope. DNA damage was quantified by measuring the tail lengths and cell bodies of at least
5
0 randomly selected cells in each group with the Komet 5.5 software.
Animal experiment
Animal procedures were approved by the Peking University Health Science Center Institutional Animal Care
and Use Committee. H522 cells were injected subcutaneously into the right flanks of female BALB/c-nu mice.
3
Treatments were initiated when the tumor volume reached 200-500 mm . Mice were randomized into vehicle
control and treatment groups (n = 5). Lapatinib (100 mg/kg/d), CTX (50 mg/kg/d) and 10d (50 and 100
mg/kg/d) in a solution containing Cremoph EL: 95% EtOH: H O=12.5:12.5:75 was intragastrically (i.g.)
2
administrated to the treatment group for 28 days. Meanwhile, the mice in control group were intragastrically
(i.g.) administrated with an equal volume of vehicle. Tumor sizes were measured using calipers and calculated
3
with the formula of Size (mm )= 1/2×(L×W2). All mice were sacrificed at day 28 after administration and
tumors were harvested and weighted.
In vivo toxicity study
Mice were randomized into vehicle control and four treatment groups (n=3). The treatment groups were orally
administrated at a single dose of 100, 300, 600, and 900 mg/kg 10d, respectively. The mice were monitored
continuously for the first 4 h for any abnormal behavior and mortality change and observed intermittently in
the next 24 h and occasionally thereafter for 14 days for any delayed onset effect. All mice were sacrificed on
day 14 after the administration and examined under a macroscopy for any possible damage in the heart, liver,
and kidneys.
Preclinical PK study of compound 10d
The pharmacokinetics of compound 10d was studied following single intravenous and oral administration to
male Sprague Dawley rats (n=3). The IV and PO clear solution formulation for mice was prepared in
DMA:PEG400:30% Cyclodextrin (SBECD) =5:25:70. After administration, 200µL of whole blood was
collected at preset time (2min, 5min, 15min, 30min, 1h, 2h, 4h, 8h, 24h). The blood samples were centrifuged
(6,000 rpm, 5min) and the obtained plasma samples were assayed for compound 10d using protein
precipitation with acetonitrile/methanol (1:1, v/v) solution followed by LC-MS/MS analysis employing
positive-ion electrospray ionization. The pharmacokinetic parameters were determined using
noncompartmental analysis with WinNonlin software.
.
27

ACCEPTED MANUSCRIPT
*
Supporting Information
1
13
Copies of the H and C spectra of all new compounds and the docking studies are shown in
SI. These material are available free of charge via the Internet at http://pubs.acs.org.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This research was supported by the grant (Grant 2012ZX09103101-042) from Ministry of
Science and Technology of China. We thank Professor Heng Xu (Institute of Materia Medica,
Chinese Academy of Medical Sciences & Peking Union Medical College) for his review and
comments on the manuscript.
■
ABBREVIATIONS
EGFR/HER2, epidermal growth factor receptor/ human epidermal growth factor receptor-2; TGI,
tumor growth inhibition; MTD, maximum tolerated dose; VEGFR, Vascular Endothelial Growth
Factor Receptor; Abl, abelson-related proto-oncogene; RAF, rapidly accelerated fibrosarcoma;
MAPK, Mitogen-activated protein kinase; ERK, extracellular regulated protein kinases; PI K,
3
phosphatidylinositol 3-kinase; PKB, protein kinase B; TOR, target of rapamycin; STAT, signal
transduction and transcription factors; FDA, Food and Drug Administration; RTK, receptor
tyrosine kinase; MMT, multiple-medication therapy; MCM, multiple-compound medication;
MTDLs, multi-target-directed ligands; DNA, deoxyribonucleic acid; RTV, relative tumor
volume; DMA, dimethyl acetamide; PEG, polyethelyne glycol; PM, phosphoramide mustard;
SAR, structure-activity relationship; CTX, cyclophosphamide; NMR, nuclear magnetic
resonance; HRMS, high resolution mass spectrometer; DMSO, dimethyl sulfoxide; DMF, N, N-
dimethylformamide; DCM, dichloromethane; THF, tetrahydrofuran; DTT, DL-dithiothreitol;
ATP, adenosine triphosphate; SYBR Green, SYBR Green I nucleic acid gel stain.
28

ACCEPTED MANUSCRIPT
■
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[
[
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29