Accepted Manuscript

Design, synthesis and biological evaluation of quinazolinephosphoramidate 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
DOI: 10.1016/j.ejmech.2016.12.055
Reference: EJMECH 9146

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 quinazolinephosphoramidate 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

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quinazolinephosphoramidate mustard conjugates as

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anticancer drugs

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Songwen Lin, Yingbo Li, Yufen Zheng, Laichun Luo, Qi Sun*, Zemei Ge, Tieming Cheng,

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Runtao Li*

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State Key Lab of Natural and Mimetic Drugs, School of Pharmaceutical Sciences, Peking

University, Beijing, P. R. China, 100191

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Corresponding Authors
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*Qi Sun: e-mail sunqi@bjmu.edu.cn; fax 86-10-82801571; tel 86-10-82801504.

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* Runtao Li: e-mail lirt@ bjmu.edu.cn; fax 86-10-82805954; tel 86-10-82805954.

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Author Contributions

S. Lin and Y. Li contributed equally to this work.

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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

10d had no obvious acute toxicity.

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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

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inhibitors with IC50 of 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

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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

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10d had no acute toxicity to mice at doses up to 900 mg/kg (single dose).

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KEYWORDS: Multi-target-directed ligands, anticancer drugs, EGFR/HER2 inhibitors, DNA alkylating agents,

phosphoramide mustard.
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Table of Contents graphic

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Cl R2
O HN
N P O X
n N
NH2
Cl R1 N

10d (EMB-3): n = 4, X = O, R1 = H, R2 = 3-Br

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EGFR: IC50 = 7.4 nM

HER2: IC50 = 82 nM
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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

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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

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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

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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.

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The ERBB receptor family consists of four members including EGFR/erbB-1/HER1, erbB-2/ HER2, erbB-

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3/HER3, and erbB-4/HER4. Activation of HER2 and EGFR induces transphosphorylation of ERBB dimer

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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
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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
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1), and erlotinib [4] (3, Figure 1). However, the efficacy of receptor tyrosine kinase (RTK) inhibitors is limited
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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
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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].
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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
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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

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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

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pharmacophores via chemically degradable bonds to form molecules that can be converted to two or more

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active ingredients after administration.

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Various strategies of MTDLs have also been developed by medicinal chemists for cancer theraophies [14].

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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
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developed by Qians 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
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cancer cells to escape from the inhibition of conventional EGFR/HER2 inhibitors [16]. However, these MTDL
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methods need to be tested in clinic trials.

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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-
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resistant mechanisms [17]. For example, EGFR/DNA dual targeting combi-molecules can directly inhibit
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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 M 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

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these strategies have demonstrated novel potencies against tumors in vivo, the toxicity of aryl mustard motif

has still not well resolved.

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Fig. 2
Cyclophosphamide (8, CP, Figure 3) is a widely used anti-cancer agent metabolized by cytochrome P450 for

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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,

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acrolein is responsible for hemorrhagic cystitis, a side effect observed during CP therapy. To reduce the side
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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
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five different types of prodrugs have been well designed (Figure 4) including nitroreductase-activated cyclic

9a [27] /acyclic [28] nitroheteroaryl phosphoramide mustards, novel DT-diaphorase targeted quinone
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phosphorodiamide prodrugs 9b [29], aldophosphamide analogues 9c [30], hydrogen peroxide inducible DNA
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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
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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

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attractive to the medicinal chemists.

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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 quinazolinephosphoramidate 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

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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

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significant difference in the potency and selectivity for EGFR. For example, 10d (EMB3, n = 4, R1 = H, R2 =

3-Br) effectively suppressed the progression of a lung cancer typed H522 in both in vitro anti-proliferative

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assay and in vivo xenograft models. Mechanism studies indicate that 10d (EMB3) can directly inhibit both

EGFR and HER2 signaling pathways and attenuate the cell circle as an alkylating agent.

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Chemistry
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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
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and substituted anilines to afford compound 12. Deacetylation of compound 12 afforded compound 13 that
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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
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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 10an in 1255% yields.
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Scheme 1
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Compounds 10f and 10i were prepared with an alternative method (showed in Scheme 2). Protected of ethynyl

group of 10bc using palladium-catalyzed trimethylsilanyl reaction produced key intermediates 10h and 10k.

After the phosphorodiamidate was formed, trimethylsilanyl group was treated with potassium carbonate (2

equiv.) in a THF/MeOH mixed solvent at 0 oC 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

79% yields. Compounds 10oq were prepared in 2632% yields by treating compound 21 with similar

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procedures as previously described procedure for compound 10.

Scheme 3

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Results and discussion

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Enzymatic assay.

4-arylamino-quinazoline scaffold has long been recognized as a classical kinase inhibitor motif against EGFR

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and HER2. Therefore, all synthetic compounds are usually tested for their inhibitory effects on both EGFR and
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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
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directly on the 6-position of quinazoline scaffold via ether bond showed significant inhibitory activity for

EGFR ( IC50=9.2 nM) but low inhibitory activity for HER2 ( IC50=1200 nM). Increasing the linker length from
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2 to 5 carbon atoms improved their inhibitory activities for both EGFR and HER2. The selectivity of all
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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
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HER2 was more sensitive to linker length. For example, compound 10d (IC50=82 nM) was 15-fold more

potent than compound 10a (IC50=1200 nM) without losing potency for EGFR, which could be attributed to the
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structural difference between EGFR and HER2 binding pockets. As a result, 3 carbon atoms length (10c) and 4
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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: IC50=11 nM, R2 = 3-ethynyl; 10g:

IC50=6.0 nM, R2 = 3-chloro-4-fluoro; 10h: IC50=27 nM, R2 = 3-chloro-4-(3-fluorobenzyloxyl)) were well

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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:

IC50=190 nM for HER2) that demonstrated moderate activity, with both small and large substituents showed

low activities. However, its inhibitory activity for EGFR was slightly decreased. Compound 10k showed the

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lowest inhibitory activities for both EGFR and HER2.

The introduction of methoxyl group onto the 7-position of qunazoline ring resulted in a significant

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improvement of the inhibitory activity for EGFR in one order of magnitude (10l: IC50=0.3 nM vs 10c: IC50=3.0

nM; 10m: IC50=0.5 nM vs 10j: IC50=5.7 nM). However, no obvious improvement was observed on the

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inhibitory activity for HER2 (10l: IC50=6000 nM vs 10c: IC50=890 nM; 10m: IC50=1300 nM vs 10j: IC50=

2700 nM).

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Inspired by the high potency of afatinib 4 with an amide group on 6-position, we also explored the effect 4-
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carbon atoms amide linker on the inhibitory activities of qunazoline derivatives against EGFR and HER2.

Compounds 10oq were designed and screened with EGFR and HER2 assay. Similar to ether linker, small
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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
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decreased the inhibitory activity for HER2. As can be seen from Table 1, compound 10o showed better
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inhibitory activities for both EGFR and HER2 with an IC50 of 3.3 nM and 240 nM, respectively.

Cellular inhibition study.

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Based on the results of enzymatic assays, compounds 10b, 10c, 10d, 10i, 10l, 10m and 10o were selected for

further cellular assay.

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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.

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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

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IC50 value of 2.8 M 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

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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

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cells (10d vs 10o). Compared with their enzymatic activities, compounds 10c and 10l bearing 3-carbon-atom
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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
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moderate anti-proliferative activities for H522 cells. Compounds 10d, 10b and 10o showed an IC50 of 2.5, 3.0
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and 2.6 M, 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
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DNA damage in cells.

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Table 2

SAR assays indicate that 10d (EMB-3) has high inhibitory activities with an IC50 of 7.4 nM and 82 nM for
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EGFR and HER2, respectively. Notably, 10d stood out with higher anti-proliferative activities in both H522
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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

10d (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 IC50, respectively. As shown in Figure 6, 10d significantly increased

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.

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Fig. 6

Preliminary pharmacokinetic study

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The pharmacokinetic parameters of 10d in rats following per os administration are summarized in Table 4. The

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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

10d at a single dose of 10 mg/kg resulted in a moderate t1/2 of 1.9 h. In addition, compound 10d was rapidly

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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.

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In vivo efficacy study
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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
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cells into nude mice. When tumors reached to 200500 mm3, 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)
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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
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treatments with 10d, lapatinib and CTX. Tumor volume in the vehicle control group increased 4.77 fold in 28
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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

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

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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-

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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

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tolerated by mice at doses up to 900 mg/kg and no obvious toxicities were detected (Figure 8).

Fig. 8

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CONCLUSION
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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
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demonstrated as multi-target-directed ligands against tumor cells. The designed compounds were screened
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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
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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
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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
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results indicate that compound 10d is a potential lead compound for the treatment of lung cancer. Further
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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.
All the tumor cell lines were purchased from ATCC. Melting points were determined on X4 microscope. 1H
13
NMR, C NMR and 31P NMR spectra were recorded on JOEL AL 300 (300 MHz) and Bruker AVANCE
400 (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

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water. The resulting solid was collected by suck filtration, washed with water, and dried at 50 C to afford 4-
hydroxyquinazolin-6-yl acetate as an off-white solid (22.40 g, 76% yield). mp > 300 C [34b]. 1H NMR (300

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MHz, d6-DMSO) 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 =
2.7, 8.7 Hz, 1H), 2.32 (s, 3H).

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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

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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,
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water and ether sequentially, and dried at 50 C to afford compound 12a as a yellow solid (0.74 g, 69% yield).
mp 238239 C [34b]. 1H NMR (300 MHz, d6-DMSO) 11.39 (s, 1H), 8.99 (s, 1H), 8.67(s, 1H), 7.938.09
(m, 3H), 7.78 (d, J = 7.5 Hz, 1H), 7.447.54 (m, 2H), 2.40 (s, 3H).
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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
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yield of 87%, according to the procedure of compound 12a. mp 290292 C (Lit.: 293 C [38]). 1H NMR (400
MHz, d6-DMSO) 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),
7.427.48 (m, 3H), 4.01 (s, 3H), 2.39 (s, 3H).
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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
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184186 C [34c]. 1H NMR (400 MHz, d6-DMSO) 11.12 (br s, 1H), 8.94 (s, 1H), 8.62 (s, 1H), 8.07 (dd, J =
2.4, 6.8 Hz, 1H), 7.717.75 (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
194196 C [34c]. 1H NMR (400 MHz, d6-DMSO) 9.69 (s, 1H), 8.58 (s, 1H), 8.30 (s, 1H), 8.03 (s, 1H),
7.707.72 (m, 1H), 7.467.48 (m, 1H), 7.257.34 (m, 4H), 7.167.21 (m, 1H), 5.25 (s, 2H), 3.95 (s, 3H), 2.37

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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

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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
[34b]. 1H NMR (300 MHz, d6-DMSO) 10.11 (s, 1H), 9.60 (s, 1H), 8.52 (s, 1H),8.27 (s, 1H), 7.80 (s, 1H),

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7.71 (d, J = 9.0 Hz, 1H), 7.46 (d, J = 9.0 Hz, 1H), 7.287.37 (m, 1H).

SC
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.

U
mp 280282 C (Lit.: 272 C [39]). 1H NMR (400 MHz, d6-DMSO) 9.50 (br s, 1H), 9.44 (s, 1H), 8.50 (s,

AN
1H), 8.25 (s, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.81 (s, 1H), 7.227.34 (m, 3H), 3.98 (s, 3H).
M
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,
mp 285288 C (Lit.: 285 C [39]). 1H NMR (400 MHz, d6-DMSO) 9.67 (s, 1H), 9.46 (s, 1H), 8.48 (s, 1H),
D

8.21 (dd, J = 2.4, 6.8 Hz, 1H), 7.827.86 (m, 1H),7.78 (s, 1H), 7.41 (t, J = 9.2 Hz, 1H), 7.22 (s, 1H), 3.98 (s,
TE

3H).
EP

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,
mp 239241 C [34c]. 1H NMR (400 MHz, d6-DMSO) 8.43 (s, 1H), 8.04 (d, J = 2.4 Hz, 1H), 7.76 (s, 1H),
C

7.73 (dd, J = 2.4, 8.8 Hz, 1H), 7.457.50 (m, 1H), 7.307.34 (m, 2H), 7.167.25 (m, 3H), 5.24 (s, 2H), 3.97 (s,
AC

3H).

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
15 (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
50 C to afford compound 12a as a yellow solid (0.74 g, 69% yield), mp 238239 C [36]. 1H NMR (400
MHz, d6-DMSO) 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

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). 13C NMR (100 MHz, d6-
DMSO) 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,
93.69.

4-((3-Chloro-4-fluorophenyl)amino)-6-iodo-quinazoline (16b)

PT
Compound 16b was prepared in a yield of 95% according to the procedure of compound 16a as yellow
solid, mp 162165 C [36]. 1H NMR (400 MHz, DMSO-d6) 10.77 (s, 1H), 9.15 (s, 1H), 8.80 (s, 1H), 8.22

RI
(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
(t, J = 9.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) 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),

SC
116.76, 101.28.

U
4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-7-methoxyquinazoline (16c)

AN
Compound 16c was prepared in a yield of 93% according to the procedure of compound 16a as white
solid, mp 221224 C [36]. 1H NMR (400 MHz, d6-DMSO) 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,
M
1H), 7.38 7.24 (m, 3H), 7.19 (t, J = 8.5 Hz, 1H), 5.26 (s, 2H). 13C NMR (100 MHz, d6-DMSO) 163.65,
161.23, 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),
130.01, 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,
D

91.74, 69.63.
TE

2-((4-((3-Bromophenyl)amino)quinazolin-6-yl)oxy)ethanol (14a)
EP

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
C

with ethyl acetate (30 mL2). The combined organic layers was washed with brine (30 mL2), dried over
AC

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
yellow solid (0.22 g, 60% yield), mp 213215 C [34c]. 1H NMR (300 MHz, d6-DMSO) 8.57 (s, 1H), 8.21
(s, 1H), 7.927.94 (m, 2H), 7.79 (d, J = 9.0 Hz, 1H), 7.54 (dd, J = 2.4, 9.0 Hz, 1H), 7.307.43 (m, 2H), 4.21 (t,
J = 4.8 Hz, 2H), 3.85 (t, J = 4.8 Hz, 2H), 3.57 (br s, 1H). 13C NMR (75 MHz, d6-DMSO) 157.08, 156.72,
152.22, 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 C16H14BrN3O2: C, 53.35; H, 3.92; N, 11.67. Found: C, 53.36; H, 3.95; N, 11.69.

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
184186 C [36]. 1H NMR (300 MHz, d6-DMSO) 9.70 (s, 1H), 8.57 (s, 1H), 8.20 (t, J = 1.8 Hz 1H), 7.93
7.94 (m, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.52 (dd, J = 2.4, 9.0 Hz, 1H), 7.307.40 (m, 2H), 4.68 (t, J = 5.1 Hz,
1H), 4.24 (t, J = 6.3 Hz, 2H), 3.623.68 (m, 2H), 1.942.02 (m, 2H). 13C NMR (75 MHz, d6-DMSO) 157.12,

PT
156.71, 152.16, 145.12, 141.08, 130.52, 129.56, 126.05, 124.73, 124.26, 121.36, 120.80, 115.77, 102.81,
65.63, 57.31, 32.13. Anal. Calcd for C17H16BrN3O2: C, 54.56; H, 4.31; N, 11.23. Found: C, 54.63; H, 4.37; N,
11.41.

RI
SC
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
202204 C [36]. 1H NMR (300 MHz, d6-DMSO) 9.65 (s, 1H),8.56 (s, 1H), 8.20 (s, 1H), 7.917.94 (m, 2H),

U
7.75 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 9.0 Hz, 1H), 7.297.40 (m, 2H), 4.50 (t, J = 4.8 Hz, 1H), 4.18 (t, J = 6.0
Hz, 2H), 3.48-3.53 (m, 2H), 1.821.91 (m, 2H), 1.601.69 (m, 2H). 13C NMR (100 MHz, d6-DMSO) 157.06,
AN
156.69, 152.13, 145.07, 141.04, 130.50, 129.56, 126.04, 124.62, 124.24, 121.32, 120.79, 115.76, 102.92,
68.43, 60.42, 29.05, 25.50. Anal. Calcd for C18H18BrN3O2: C, 55.68; H, 4.67; N, 10.82. Found: C, 55.68; H,
M
4.77; N, 10.84.
D

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
TE

251253 C [34c]. 1H NMR (400 MHz, d6-DMSO) 9.53 (br s, 1H), 8.52 (s, 1H), 8.15 (s, 1H), 7.88 (d, J = 8.4
Hz, 1H), 7.85 (s, 1H), 7.217.37 (m, 3H), 4.61 (t, J = 4.8 Hz, 1H), 4.23 (t, J = 6.4 Hz, 2H), 3.95 (s, 3H), 3.61
EP

13
3.66 (m, 2H), 1.962.02 (m, 2H). C NMR (75 MHz, d6-DMSO) 156.00, 154.58, 152.66, 148.59, 147.00,
141.32, 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 C18H18BrN3O3: C, 53.48; H, 4.49; N, 10.39. Found: C, 53.31; H, 4.62; N, 10.09.
C
AC

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.
mp 280282 C [34c]. 1H NMR (400 MHz, d6-DMSO) 9.56 (s, 1H), 8.50 (s, 1 H), 8.12 (dd, J = 2.4, 6.8 Hz,
1H), 7.787.83 (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),
3.94 (s, 3H), 3.613.65 (m, 2H), 1.952.03 (m, 2H). 13C NMR (75 MHz, d6-DMSO) 156.05, 154.59, 153.27,
152.66, 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,
32.07. Anal. Calcd for C18H17ClFN3O3: C, 57.22; H, 4.54; N, 11.12. Found: C, 56.97; H, 4.70; N, 10.95.

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.
mp 212213 C [34c]. 1H NMR (400 MHz, d6-DMSO) 8.46 (s, 1H), 7.97(d, J = 2.4 Hz, 1H), 7.81 (s, 1H),
7.72 (dd, J = 2.4, 8.8 Hz, 1H), 7.457.51 (m, 1H), 7.187.35 (m, 5H), 5.25 (s, 2H), 4.22 (t, J = 6.4 Hz, 2H),
3.94 (s, 3H), 3.65 (t, J = 6.0 Hz, 2H), 1.972.03 (m, 2H). 13C NMR (100 MHz, d6-DMSO) 162.33, 156.24,

PT
154.46, 152.84, 149.51, 148.45, 146.73, 139.79, 133.55, 130.68, 124.02, 123.45, 122.19, 121.19, 114.82,
114.43, 114.14, 108.80, 107.19, 102.48, 69.54, 66.03, 57.36, 55.91, 32.08. Anal. Calcd for C25H23ClFN3O4: C,
62.05; H, 4.79; N, 8.68. Found: C, 61.79; H, 4.77; N, 8.65.

RI
5-((4-((3-Bromophenyl)amino)-quinazolin-6-yl)oxy)pentan-1-ol (14g)

SC
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)

U
and N,N-dimethylformamide (25 mL) were then injected into the flask and the mixture was stirred at 80 C for
18h. After cooling to room temperature, 100 mL water was added to the reaction mixture. After filtration, the
AN
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 7879 C [36].
M
1
H NMR (400 MHz, d6-DMSO) 9.68 (s, 1H), 8.61 (s, 1H), 8.23 (s, 1H), 7.96 (d, J = 7.5 Hz, 2H), 7.78 (s,
1H), 7.40 (t, J = 8.0 Hz, 1H), 7.35 (t, J = 10.8 Hz, 1H), 4.46 (t, J = 5.0 Hz, 1H), 4.264.08 (m, 2H), 3.48 (d, J
D

= 5.0 Hz, 2H), 1.86 (s, 2H), 1.56 (s, 4H).13C NMR (100 MHz, d6-DMSO) 157.85, 157.50, 152.77, 146.00,
142.07, 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,
TE

23.21.
EP

4-((4-((3-Trimethylsilanylethynyl-phenyl)amino)-quinazolin-6-yl)oxy)butan-1-ol (14h)
C

A mixture of 14c (8.25 g, 21.3 mmol), trimethyl silyl acetylene (20g, 213.6mmol), Pd(PPh3)2Cl2 (1.65 g,
AC

2.35 mmol), cuprous iodide (1.43g, 7.5 mmol) and dimethylformamide (40 mL) was stirred at 90C under
nitrogen for 36h. The reaction mixture was purified by flash column chromatography (silica gel,
dichloromethane /methanol) to afford compound 14h as a yellow solid (28% yield), mp: 138139 oC. 1H NMR
(400 MHz, d6-DMSO) 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), 1.941.77 (m, 2H), 1.751.54 (m, 2H), 0.25
(s, 9H). 13C NMR (100 MHz, d6-DMSO) 157.82, 157.61, 153.04, 145.95, 140.56, 130.42, 129.88, 127.28,
125.63, 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 C23H27N3O2Si [M+H]+ 406.19453; found 406.19381.

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

PT
14g. Grey solid, mp 210211 C [36]. 1H NMR (400 MHz, d6-DMSO) 9.70 (s, 1H), 8.62 (s, 1H), 8.20 (s,
1H), 8.077.62 (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
(s, 2H). 13C NMR (100 MHz, d6-DMSO) 157.83, 157.55, 155.42, 153.01, 137.54, 137.51, 130.48, 125.28,

RI
124.55, 123.42, 123.35, 119.83, 119.64, 117.57, 117.36, 103.82, 69.22, 61.31, 29.93, 26.34.

SC
4-((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

U
14g. Grey solid, mp 156157 C [36]. 1H NMR (400 MHz, d6-DMSO) 9.61 (s, 1H), 8.57 (s, 1H), 8.04 (d, J

AN
= 2.5 Hz, 1H), 7.92 (s, 1H), 7.77 (dd, J = 8.9, 2.5 Hz, 2H), 7.607.44 (m, 2H), 7.447.28 (m, 3H), 7.22 (td, J =
8.6, 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),
1.971.79 (m, 2H), 1.67 (dt, J = 9.7, 6.5 Hz, 2H). 13C NMR (100 MHz, d6-DMSO) 164.37, 161.94, 157.72,
M
157.65, 150.47, 140.61, 140.54, 134.33, 131.43, 131.35, 125.01, 124.95, 124.17, 124.14, 123.07, 122.04,
115.66, 115.46, 115.13, 115.02, 114.80, 103.78, 70.34, 69.19, 61.37, 29.97, 26.38.
D
TE

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
14h. Grey solid, mp 145146 C. 1H NMR (400 MHz, d6-DMSO) 9.65 (s, 1H), 8.61 (s, 1H), 8.05 (d, J = 8.3
EP

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 =
7.6 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,
13
C

9H). C NMR (100 MHz, d6-DMSO) 157.87, 157.57, 152.95, 146.08, 140.62, 130.40, 129.78, 127.23,
125.63, 125.34, 123.49, 123.16, 106.14, 103.56, 94.83, 66.39, 58.19, 33.06, 0.82.
AC

3-((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
14g. Grey solid, mp 183184 C [36]. 1H NMR (400 MHz, d6-DMSO) 9.73 (s, 1H), 8.59 (s, 1H), 8.20 (dd, J
= 6.9, 2.6 Hz, 1H), 8.047.68 (m, 3H), 7.667.40 (m, 2H), 4.69 (t, J = 5.0 Hz, 1H), 4.26 (t, J = 6.3 Hz, 2H),
3.67 (dd, J = 11.2, 6.0 Hz, 2H), 2.01 (p, J = 6.2 Hz, 2H). 13C NMR (100 MHz, d6-DMSO) 157.88, 157.58,
155.42, 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

103.68, 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

PT
14g. Grey solid, mp 8788 C [36]. 1H NMR (400 MHz, d6-DMSO) 9.64 (s, 1H), 8.67 (s, 1H), 8.04 (d, J =
2.5 Hz, 1H), 7.96 (s, 1H), 7.897.68 (m, 2H), 7.587.43 (m, 2H), 7.437.27 (m, 3H), 7.22 (td, J = 8.6, 2.1 Hz,
1H), 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

RI
Hz, 2H). 13C NMR (100 MHz, d6-DMSO) 164.39, 161.97, 157.81, 157.66, 150.49, 140.64, 140.56, 134.35,
133.97, 132.31, 131.46, 131.38, 125.13, 124.98, 124.83, 124.21, 124.18, 123.10, 122.93, 122.06, 115.69,

SC
115.49, 115.16, 115.05, 114.83, 103.70, 92.39, 70.36, 66.42, 58.24, 49.56, 33.07.

U
N-(3-Bromophenyl)-6-nitroquinazolin-4-amine (18a)

AN
A mixture of compound 17 (4.4 g, 23 mmol) and DMF (0.1 mL) in SOCl2 (30 mL) was reflux until the
soli disappeared. After removing SOCl2 under reduce pressure, i-PrOH (140 mL), 3-bromoaniline (3.96 g, 23
mmol) and triethylamine (2.33 g, 23 mmol) were added to the residue and the resulting mixture was refluxed
M
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
269 C (Lit.: 269271 C [40]). 1H NMR (300 MHz, d6-DMSO) 10.48 (s, 1H), 9.65 (s, 1H), 8.78 (s, 1H),
D

8.57 (dd, J = 1.2, 9.0 Hz, 1H), 8.20 (s, 1H), 7.907.97 (m, 2H), 7.367.43 (m, 2H).
TE

N-(3-Chloro-4-fluorophenyl)-6-nitroquinazolin-4-amine (18b)
EP

Compound 18b was prepared from 17 in a yield of 65%, according to the procedure of compound 18a,
using 3-chloro-4-fluoroaniline in place of 3-bromoaniline. mp 278280 C (Lit.: 274.5277 C [41]). 1H NMR
(400 MHz, d6-DMSO) 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
C

= 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).
AC

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 279281 C (Lit.: 246248 C
[42]). 1H NMR (400 MHz, d6-DMSO) 11.19 (br s, 1H), 9.71 (d, J = 6.0 Hz, 1H), 8.88 (d, J = 10.0 Hz, 1H),
8.68 (t, J = 6.8Hz, 1H), 7.988.04 (m, 2H), 7.71 (d, J = 8.8 Hz, 1H), 7.467.51 (m, 1H), 7.317.36 (m, 3H),
7.187.22 (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 mL3).The

PT
combined organic layers were washed brine (50 mL3), dried over anhydrous sodium sulfate, filtered and
concentrated to afford compound 19a as a yellow solid (2.52g, 94% yield), mp 263265 C (Lit.: 267270 C

RI
[23]. 1H NMR (300 MHz, d6-DMSO) 9.46 (br s, 1H), 8.39 (s, 1H), 8.24 (t, J = 1.8 Hz, 1H), 7.90 (d, J = 8.4
Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.227.35 (m, 4H), 5.64 (br s, 2H).

SC
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,

U
mp 253255 C (Lit.: 244 C [43]). 1H NMR (400 MHz, d6-DMSO) 9.48 (s, 1H), 8.37 (s, 1 H), 8.21 (dd, J =
AN
2.4, 6.8 Hz, 1H), 7.817.85 (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,
1H), 7.26 (dd, J = 2.0, 8.8 Hz, 1H), 5.63 (s, 2H).
M
4-(3-Chloro-4-(3-fluorobenzyloxyl)phenylamino)-6-aminoquinazoline (19c)
D

Compound 19c was prepared from 18c in a yield of 97%, according to the procedure of compound 19a,
mp 190192 C (Lit.: 186188 C [43]). 1H NMR (400 MHz, d6-DMSO) 8.52 (s, 1 H), 7.94 (d, J = 2.4 Hz, 1
TE

H), 7.65 (dd, J = 2.0, 8.8 Hz, 1 H), 7.59 (d, J = 9.2 Hz, 1 H), 7.457.51 (m, 1 H), 7.287.39 (m, 5 H), 7.17
7.21 (m, 1 H), 5.28 (s, 2 H).
EP

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
C

compound 19a (1.58 g, 5 mmol) and Na2CO3 (0.53 g, 5 mmol) in anhydrous THF (50 mL). The resulting
AC

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 mL3). The combined organic layers were
washed brine (50 mL2), 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
afford compound 21a in a yield of 50%. mp 175176 C. 1H NMR (300 MHz, d6-DMSO) 10.28 (s, 1H), 9.92
(s, 1H), 8.76 (s, 1H), 8.59 (s, 1H), 8.18 (s, 1H), 7.777.88 (m, 3H), 7.287.38 (m, 2H), 4.59 (t, J = 4.8 Hz, 1H),
3.473.53 (m, 2H), 2.47 (t, J = 7.5 Hz, 2H), 1.771.86 (m, 2H). 13C NMR (100 MHz, d6-DMSO) 171.81,
19
 ACCEPTED MANUSCRIPT

157.29, 153.04, 146.53, 141.14, 137.16, 130.41, 128.45, 127.21, 125.99, 124.34, 121.27, 120.89, 115.50,
111.65, 60.23, 33.08, 28.38. Anal. Cald for C18H17BrN4O2: C, 53.88; H, 4.27; N, 13.96; Found: C, 53.58; H,
4.35; N, 13.68.

N-(4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)-4-hydroxybutanamide (21b)

PT
Compound 21b was prepared from 19b in a yield of 79%, according to the procedure of compound 21a,
mp 208209 C. 1H NMR (400 MHz, d6-DMSO) 10.29 (s, 1H), 8.70 (d, J = 1.6 Hz, 1H), 8.55 (s, 1H), 8.13

RI
(dd, J = 2.4, 6.8 Hz, 1H), 7.777.86 (m, 3H), 7.44 (t, J = 8.4 Hz, 1H), 3.48 (t, J = 6.4 Hz, 2H), 2.46 (t, J = 7.6
Hz, 2H), 1.771.84 (m, 2H). 13C NMR (75 MHz, d6-DMSO) 171.74, 157.34, 153.48, 153.05, 146.47, 137.08,

SC
136.68, 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
C18H16ClFN4O2: C, 57.68; H, 4.30; N, 14.95; Found: C, 57.48; H, 4.30; N, 14.90.

U
N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)-4-hydroxybutanamide (21c)
AN
Compound 21c was prepared from 19c in a yield of 51%, according to the procedure of compound 21a,
mp 209211 C. 1H NMR (400 MHz, d6-DMSO) 8.68 (s, 1H), 8.51 (s, 1H), 7.98 (d, J = 2.0 Hz, 1H), 7.69
7.85 (m, 3H), 7.457.51 (m, 1H), 7.177.35 (m, 4H), 5.26 (s, 2H), 3.49 (t, J = 7.2 Hz, 2H), 2.46 (t, J = 7.2 Hz,
M
13
2H), 1.771.84 (m, 2H). C NMR (75 MHz, d6-DMSO) 171.70, 162.35, 157.45, 153.27, 149.69, 146.40,
139.80, 136.93, 133.41, 130.70, 128.41, 127.05, 124.26, 123.45, 122.47, 121.15, 115.36, 114.83, 114.34,
D

114.15, 111.57, 69.52, 60.24, 33.09, 28.41. Anal. Cald for C25H22ClFN4O3: C, 62.44; H, 4.61; N, 11.65;
Found: C, 62.17; H, 4.68; N, 11.38.
TE

4-((3-Bromophenyl)amino)quinazolin-6-yl N,N-bis(2-chloroethyl)phosphorodiamidate (10a)

EP

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-
C

chloroethyl)phosphoramidic dichloride (0.31 g, 1.2 mmol) in anhydrous THF (10 mL) at 0 C under nitrogen
AC

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 mL2). 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
(0.22 g, 42% yield), mp 200202 C. 1H NMR (400 MHz, d6-DMSO) 8.64 (s, 1H), 8.27 (s, 1H), 8.21 (s, 1H),
13
7.857.91 (m, 3H), 7.327.40 (m, 2H), 3.663.71 (m, 4H), 3.413.46 (m, 4H). C NMR (100 MHz, d6-
DMSO) 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

115.59, 113.28, 48.92, 42.44. 31P NMR (122 MHz, d6-DMSO) 15.59. Anal. Cald for C18H19BrCl2N5O2P: C,
41.64; 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)

PT
Compound 10b was prepared from 14a in a yield of 37%, according to the procedure of compound 10a,
mp 143144 C. 1H NMR (400 MHz, CDCl3) 9.08 (br s, 1H), 8.70 (s, 1 H), 8.25 (s, 1H), 7.988.01 (m, 2H),

RI
7.79 (d, J = 9.2 Hz, 1H), 7.35 (d, J = 2.0, 8.8 Hz, 1H), 7.227.25 (m, 2H), 4.204.47 (m, 4H), 3.383.64 (m,
13
8H), 3.07 (br s, 2H). C NMR (100 MHz, d6-DMSO) 156.75, 156.55, 152.26, 145.10, 140.98, 130.49,
31

SC
129.55, 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, d6-DMSO) 18.68. Anal. Cald for C20H23BrCl2N5O3P: C, 42.65; H, 4.12; N, 12.43; Found: C, 42.67; H,
4.16; N, 12.45.

U
AN
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,
M
mp 158159 C. 1H NMR (400 MHz, d6-DMSO) 8.56 (s, 1H), 8.21 (s, 1 H), 7.96 (s, 1H), 7.93 (d, J = 8.0 Hz,
1H), 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
(t, J = 5.6 Hz, 2H), 4.034.08 (m, 2H), 3.65 (t, J = 7.2 Hz, 4H), 3.263.33 (m, 4H), 2.122.18 (m, 2H). 13C
D

NMR (100 MHz, d6-DMSO) 156.87, 156.75, 152.22, 145.18, 141.04, 130.52, 129.61, 126.07, 124.67,
TE

124.25, 121.32, 120.80, 115.76, 103.10, 65.05, 61.28, 48.86, 42.56, 29.85. 31P NMR (122 MHz, d6-DMSO)
18.12. Anal. Cald for C21H25BrCl2N5O3P: C, 43.70; H, 4.37; N, 12.13; Found: C, 43.51; H, 4.65; N, 11.84.
EP

4-((4-((3-Bromophenyl)amino)quinazolin-6-yl)oxy)butyl N,N-bis(2-chloroethyl)phosphorodiamidate
(10d)
C

Compound 10d was prepared from 14c in a yield of 36%, according to the procedure of compound 10a,
AC

mp 7071 C. 1H NMR (400 MHz, CDCl3) 9.40 (br s, 1H), 8.64 (s, 1H), 8.03 (s, 1H), 7.847.85 (m, 1H),
7.727.77 (m, 2H), 7.33 (dd, J = 2.0, 4.8 Hz, 1H), 7.187.22 (m, 2H), 3.974.12 (m, 4H), 3.59 (t, J = 6.4 Hz,
13
4H), 3.393.45 (m, 4H), 3.13 (s, 2H), 1.801.93 (m, 4H). C NMR (100 MHz, CDCl3) 157.28, 156.84,
152.36, 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,
31
48.73, 42.26, 26.70, 24.79. P NMR (122 MHz, d6-DMSO) 17.98. Anal. Cald for C22H27BrCl2N5O3P: C,
44.69; 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.
Yellow solid, mp 6465oC. 1H NMR (400 MHz, d6-DMSO) 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), 7.447.31 (m, 2H), 4.44 (d, J = 5.1 Hz,
2H), 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,

PT
4H), 1.941.83 (m, 2H), 1.771.66 (m, 2H), 1.58 (dt, J = 14.5, 7.2 Hz, 2H). 13C NMR (100 MHz, d6-DMSO)
157.89, 157.70, 152.78, 145.41, 141.91, 131.27, 130.00, 126.93, 125.51, 125.27, 122.09, 121.81, 116.60,
103.98, 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

RI
calcd for C23H29BrCl2N5O3P[M+H]+ 604.06412; found 604.06372. HPLC:tR 5.70 min, 99.2% (214 nm), 98.7%
(254 nm).

SC
4-((4-((3-Ethynylphenyl)amino)quinazolin-6-yl)oxy)butyl N,N-bis(2-chloroethyl)phosphorodiamidate

U
(10f)

AN
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
(15 mL) at 0 oC for 1h. After concentrated the reaction solution, the reaction solution was diluted with water
M
(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
7071oC.
D

1
H NMR (400 MHz, d6-DMSO) 9.81 (s, 1H), 8.59 (s, 1H), 8.08 (s, 1H), 7.99 (dd, J = 16.0, 5.9 Hz, 2H),
TE

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,
7.5 Hz, 4H), 1.92 (dd, J = 13.9, 6.2 Hz, 2H), 1.83 (dt, J = 12.6, 6.1 Hz, 2H). 13C NMR (100 MHz, d6-DMSO)
EP

157.82, 157.76, 152.94, 145.49, 140.46, 130.05, 129.84, 127.67, 126.03, 125.45, 123.82, 122.69, 116.60,
104.02, 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 C24H28N5O3PCl2 [M+H]+ 536.13796; found 536.13736. HPLC:tR 3.54 min, 95.4% (214
C

nm), 95.2% (254 nm).

AC

4-((4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)oxy)butyl N,N-bis(2-
chloroethyl)phosphorodiamidate (10g)
Compound 10g 300mg was prepared from 14i in a yield of 13 %, according to the procedure of
compound 10a. Yellow solid, mp 7273oC. 1H NMR (400 MHz, d6-DMSO) 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

13
14.6, 7.4 Hz, 4H), 2.01 1.74 (m, 4H). C NMR (100 MHz, d6-DMSO) 157.76, 157.70, 155.47, 153.05,
152.84, 145.53, 137.48, 130.11, 125.44, 124.65, 123.52, 123.45, 119.81, 119.62, 117.53, 117.32, 116.50,
104.01, 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 C22H26N5O3PCl3F [M+H]+ 564.08956; found 564.08913. HPLC:tR 4.02 min, 96.9% (214 nm), 96.8%
(254 nm).

PT
4-((4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)oxy)butyl N,N-bis(2-
chloroethyl)phosphorodiamidate (10h)

RI
Compound 10h 400mg was prepared from 14j in a yield of 28 %, according to the procedure of
compound 10a. Yellow solid, mp 6768oC.1H NMR (400 MHz, d6-DMSO) 9.78 (s, 1H), 8.54 (s, 1H), 8.04

SC
(d, J = 2.6 Hz, 1H), 7.98 (d, J = 2.4 Hz, 1H), 7.827.72 (m, 2H), 7.587.47 (m, 2H), 7.407.28 (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,

U
13
2H), 3.70 (t, J = 7.4 Hz, 4H), 3.44 3.23 (m, 5H), 1.991.76 (m, 4H). C NMR (100 MHz, d6-DMSO)
164.34, 161.92, 157.87, 157.71, 152.99, 150.60, 145.18, 140.63, 140.55, 134.12, 131.52, 131.44, 129.87,
AN
125.33, 125.15, 124.26, 124.23, 123.33, 121.97, 116.45, 115.72, 115.51, 115.21, 115.06, 114.84, 104.00,
70.32, 68.91, 64.84, 64.79, 49.77, 49.73, 43.42, 27.74, 27.67, 26.04. LCMS: 669.6 [M+1]+. HRMS: m/z
calcd. for C29H32N5O4PCl3F [M+H]+ 670.13143; found 670.13171. HPLC:tR 4.78 min, 98.6% (214 nm), 98.6%
M
(254 nm).
D

3-((4-((3-Ethynylphenyl)amino)quinazolin-6-yl)oxy)propyl N,N-bis(2-chloroethyl)phosphorodiamidate
TE

(10i)

Compound 10i was prepared from 14k in a yield of 55 %, according to the procedure of compound
10f. Yellow solid, mp 159160oC. 1H NMR (400 MHz, d6-DMSO) 9.73 (s, 1H), 8.58 (s, 1H), 8.09 (s, 1H),
EP

7.99 (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,
1H), 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,
C

2H), 3.69 (t, J = 7.3 Hz, 4H), 3.32 (dt, J = 11.7, 7.4 Hz, 4H), 2.25 2.13 (m, 2H). 13C NMR (100 MHz, d6-
DMSO) 157.73, 157.59, 153.10, 145.98, 140.54, 130.37, 129.81, 127.55, 125.89, 125.30, 123.67, 122.69,
AC

116.61, 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 C23H26N5O3PCl2 [M+H]+ 522.12231; found 522.12126. HPLC:tR 3.41 min, 99.2% (214
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
compound 10a. Yellow solid, mp 145146 oC. 1H NMR (400 MHz, d6-DMSO) 9.87 (s, 1H), 8.58 (s, 1H),
23
 ACCEPTED MANUSCRIPT

8.23 (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),
4.31 (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). 13C NMR (100 MHz,
d6-DMSO) 157.67, 157.63, 155.48, 153.01, 152.97, 145.84, 137.53, 130.31, 125.39, 124.57, 123.44, 123.37,
119.80, 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:
551.9 [M+1]+. HRMS: m/z calcd. for C21H24N5O3PCl3F [M+H]+ 550.07391; found 550.07268. HPLC:tR 3.84

PT
min, 98.7% (214 nm), 98.4% (254 nm).

RI
3-((4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)oxy)propyl N,N-bis(2-
chloroethyl)phosphorodiamidate (10k)

SC
Compound 10k 200 mg was prepared from 14m in a yield of 14 %, according to the procedure of
compound 10a. Yellow solid, mp 7778oC. 1H NMR (400 MHz, d6-DMSO) 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),

U
4.51 (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
(m, 4H), 2017 (m, 2H). 13C NMR (100 MHz, d6-DMSO) 164.35, 161.93, 157.86, 157.58, 153.11, 150.56,
AN
145.46, 140.64, 140.57, 134.20, 131.52, 131.44, 130.04, 125.29, 125.07, 124.27, 124.24, 123.25, 121.97,
116.48, 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,
30.67. LC-MS: 655.9. HRMS: m/z calcd. for C28H30Cl3FN5O4P [M+H]+ 656.11578; found 656.11483.
M
HPLC:tR 4.61 min, 99.4% (214 nm), 99.8% (254 nm).
D

3-((4-((3-Bromophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl N,N-bis(2-
TE

chloroethyl)phosphorodiamidate (10l)
Compound 10l was prepared from 14d in a yield of 30%, according to the procedure of compound
1
10a. H NMR (400 MHz, CDCl3) 9.27 (s, 1H), 8.63 (s, 1H), 8.04 (s, 1H), 7.897.92 (m, 2H), 7.187.21 (m,
EP

3H), 4.174.37 (m, 4H), 3.96 (s, 3H), 3.62 (t, J = 6.0 Hz, 4H), 3.423.49 (m, 4H), 3.09 (d, J = 4.8 Hz, 2H),
13
2.202.33 (m, 2H). C NMR (100 MHz, CDCl3) 156.83, 155.20, 153.65, 148.08, 147.60, 141.07, 129.93,
31
126.18, 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
C

(122 MHz, CDCl3) 16.07. Anal. Calcd for C22H27BrCl2N5O4P: C, 43.51; H, 4.48; N, 11.53. Found: C, 43.24;
AC

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
10a, mp 150-152 C. 1H NMR (400 MHz, CDCl3) 9.30 (br s, 1H), 8.59 (s, 1H), 7.96 (dd, J = 2.4, 6.4 Hz,
1H), 7.84 (s, 1H), 7.727.76 (m, 1H), 7.15 (s, 1H), 7.09 (t, J = 8.8 Hz, 1H), 4.164.36 (m, 4H), 3.93 (s, 3H),
3.61 (t, J = 6.0 Hz, 4H), 3.393.50 (m, 4H), 3.19 (d, J = 4.4 Hz, 2H), 2.192.29 (m, 2H). 13C NMR (100 MHz,

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 ACCEPTED MANUSCRIPT

CDCl3) 156.86, 155.19, 154.24, 153.57, 148.08, 147.42, 136.26, 124.02, 121.84, 120.30, 116.12, 109.47,
107.43, 105.10, 66.98, 62.89, 56.00, 48.65, 42.27, 29.45. 31P NMR (122 MHz, CDCl3) 15.79. Anal. Calcd for
C22H26Cl3FN5O4P: C, 45.49; H, 4.51; N, 12.06. Found: C, 45.72; H, 4.64; N, 11.89.

3-((4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl N,N-bis(2-

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chloroethyl)phosphorodiamidate (10n)
Compound 10n was prepared from 14f in a yield of 29%, according to the procedure of compound 10a,

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mp 137138 C. 1H NMR (400 MHz, CDCl3) 9.21 (s, 1H), 8.57 (s, 1H), 7.85 (d, J = 2.8 Hz, 1H), 7.79 (s,
1H), 7.65 (d, J = 2.4, 8.8 Hz, 1H), 7.317.36 (m, 1H), 7.147.22 (m, 3H), 6.987.02 (m, 1H), 6.88 (d, J = 8.8

SC
Hz, 1H), 4.134.33 (m, 4H), 3.91 (s, 3H), 3.57 (t, J = 6.4 Hz, 4H), 3.383.45 (m, 4H), 3.20 (d, J = 4.8 Hz, 2H),
13
2.192.25 (m, 2H). C NMR (100 MHz, CDCl3) 162.91, 157.00, 154.99, 153.71, 150.20, 147.96, 147.27,
139.22, 133.67, 130.06, 124.57, 122.90, 122.41, 121.87, 114.75, 114.24, 113.90, 109.45, 107.40, 104.77,

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70.35, 66.77, 62.84, 55.96, 48.74, 42.32, 29.46. P NMR (122 MHz, CDCl3) 15.75. Anal. Calcd for
C29H32Cl3FN5O5P: C, 50.71; H, 4.70; N, 10.20. Found: C, 50.56; H, 4.88; N, 9.97.
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4-oxo-4-((4-((3-Bromophenyl)amino)quinazolin-6-yl)amino)butyl N,N-bis(2-
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chloroethyl)phosphorodiamidate (10o)
Compound 10o was prepared from 21a in a yield of 44%, according to the procedure of compound
D

10a, using NaH in place of n-butyl lithium. 1H NMR (300 MHz, CDCl3) 2.012.07 (m, 2H), 2.52 (d, J = 5.4
Hz, 2H), 3.323.46 (m, 6H), 3.59 (t, J = 8.4 Hz, 4H), 4.014.11 (m, 2H), 7.147.22 (m, 2H), 7.627.67 (m,
TE

13
3H), 7.97(s, 1H), 8.508.61(m, 3H), 9.95 (s, 1H). C NMR (100 MHz, CDCl3) 171.98, 157.38, 153.32,
146.21, 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,
33.07, 26.41. 31P NMR (122 MHz, d6-DMSO) 18.08. Anal. Cald for C22H26BrCl2N6O3P: C, 43.73; H, 4.34;
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N, 13.91; Found: C, 43.81; H, 4.55; N, 14.07.

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4-oxo-4-((4-((3-Chloro-4-fluorophenyl)amino)quinazolin-6-yl)amino)butyl N,N-bis(2-
AC

chloroethyl)phosphorodiamidate (10p)
Compound 10p was prepared from 21b in a yield of 32%, according to the procedure of compound 10a,
using NaH in place of n-butyl lithium. 1H NMR (400 MHz, CDCl3) 10.03 (s, 1H), 8.73 (s, 1H), 8.54 (s, 1H),
8.52 (s, 1H), 7.83 (dd, J = 2.4, 6.4 Hz, 1H), 7.527.67 (m, 3H), 7.05 (t, J = 8.8 Hz, 1H), 3.994.09 (m, 2H),
13
3.403.63 (m, 10H), 2.462.58(m, 2H), 2.002.06 (m, 2H). C NMR (100 MHz, CDCl3) 171.98, 157.39,
154.65, 153.36, 146.44, 136.52, 135.32, 128.45, 126.77, 124.25, 121.99, 120.52, 116.25, 115.27, 111.03,
64.50, 48.74, 42.26, 33.02, 26.56. 31P NMR (122 MHz, CDCl3) 18.00. Anal. Cald for C22H25Cl3FN6O3P: C,
45.73; H, 4.36; N, 14.54; Found: C, 45.79; H, 4.66; N, 14.43.

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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
10a, using NaH in place of n-butyl lithium. 1H NMR (400 MHz, CDCl3) 10.01 (br s,1H), 8.67 (br s,1H), 8.52

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(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,
1H), 7.297.35 (m, 1H), 7.167.20 (m, 2H), 6.977.01 (m, 1H), 6.83 (d, J = 9.2 Hz, 1H), 5.04 (s, 2 H), 3.97

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4.06 (m, 2H), 3.523.60 (m, 6 H), 3.363.43 (m, 4H), 2.48 (d, J = 4.8 Hz, 2H), 1.952.01 (m, 2H). 13C NMR
(100 MHz, CDCl3) 171.89, 162.88, 157.57, 153.58, 150.65, 146.22, 139.06, 136.46, 132.62, 130.08, 128.21,
126.65, 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,

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32.97, 26.44. 31P NMR (122 MHz, CDCl3) 18.10. HRMS (ESI+) m/z calcd for C29H32Cl3FN6O4P (M + H)+,
683.12668, found 683.12562 (M + H)+.

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Cell proliferation assay
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MDA-MB-468, SK-Br-3, HCT-116 and H522 cells were seeded in 96-well plates with 23.5104 cells/well
and incubated for 24 h. Cells were then exposed to agents for 72 h. Cells in control group were treated with a
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medium containing 0.5% DMSO. After the treatments, cells were then incubated with 100 L medium
containing 0.5 mg/mL MTT at 37 C for 4 h. Cell supernatants were discarded. MTT crystals were dissolved
D

in 200 L 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
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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.
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Tyrosine kinase assay

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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 l kinase reaction buffer (40 mM
AC

Tris (pH 7.4), 10 mM MgCl2, 0.1 mg/ml BSA, 1 mM DTT, 10 M ATP, 0.2 mg/ml Poly (Glu, Tyr)) for 40
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
(1104 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,
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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
50 randomly selected cells in each group with the Komet 5.5 software.

Animal experiment

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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.
Treatments were initiated when the tumor volume reached 200-500 mm3. Mice were randomized into vehicle

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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: H2O=12.5:12.5:75 was intragastrically (i.g.)

SC
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
with the formula of Size (mm3)= 1/2(LW2). All mice were sacrificed at day 28 after administration and

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tumors were harvested and weighted.
In vivo toxicity study AN
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
M
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
D

day 14 after the administration and examined under a macroscopy for any possible damage in the heart, liver,
and kidneys.
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Preclinical PK study of compound 10d

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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, 200L of whole blood was
C

collected at preset time (2min, 5min, 15min, 30min, 1h, 2h, 4h, 8h, 24h). The blood samples were centrifuged
AC

(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.

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* Supporting Information

Copies of the 1H and 13C 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

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

This research was supported by the grant (Grant 2012ZX09103101-042) from Ministry of

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Science and Technology of China. We thank Professor Heng Xu (Institute of Materia Medica,
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Chinese Academy of Medical Sciences & Peking Union Medical College) for his review and
comments on the manuscript.
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D

ABBREVIATIONS

EGFR/HER2, epidermal growth factor receptor/ human epidermal growth factor receptor-2; TGI,
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tumor growth inhibition; MTD, maximum tolerated dose; VEGFR, Vascular Endothelial Growth
Factor Receptor; Abl, abelson-related proto-oncogene; RAF, rapidly accelerated fibrosarcoma;
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MAPK, Mitogen-activated protein kinase; ERK, extracellular regulated protein kinases; PI3K,
phosphatidylinositol 3-kinase; PKB, protein kinase B; TOR, target of rapamycin; STAT, signal
C

transduction and transcription factors; FDA, Food and Drug Administration; RTK, receptor
tyrosine kinase; MMT, multiple-medication therapy; MCM, multiple-compound medication;
AC

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.

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aldophosphamide analogues as novel anticancer prodrugs targeted against cyclophosphamide-resistant tumor
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agents: targeted anticancer prodrugs. J. Am. Chem. Soc. 133 (2011) 1927819281; (b) Chen, W.; Han, Y.;
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Peng, X. Aromatic nitrogen mustard-based prodrugs: activity, selectivity, and the mechanism of DNA cross-
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phosphoramidates as floate receptor-targeted alkylating agents. J. Med. Chem. 44 (2001) 6973; (b) Jiang, Y.;
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[33] Sun, Q.; Li, R.; Guo, W.; Cui, J.; Cheng, T.; Ge, Z. Novel class of cyclophosphamide prodrug:
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EGFR inhibitors. Arch. der Pharm. 346 (2013) 4452; (b) Li, R.; Zhang, X.; Li, Q.; Ge, Z.; Li, R. Novel EGFR
inhibitors prepared by combination of dithiocarbamic acid esters and 4-anilinoquinazolines. Bioorg. Med.
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[36] Zheng, Y.; Zou, W.; Luo, L.; Chen, J.; Lin, S.; Sun, Q. Ligand-free Cu-catalyzed O-arylation of aliphatic
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[37] Luniewski, W.; Wietrzyk, J.; Godlewska, J.; Switalska, M.; Piskozub, M.; Peczynska-Czoch, W.;
Kaczmarek, L. New derivatives of 11-methyl-6-[2-(dimethylamino)ethyl]-6H-indolo[2,3-b]quinoline as

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cytotoxic DNA topoisomerase II inhibitors. Bioorg. Med. Chem. Lett. 22 (2012) 61036107.
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[39] Chandregowda, V.; Kush, A.K.; Reddy, G. C. Synthesis and in vitro antitumor activities of novel 4-
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[40] Saad, S. M.; Ghouri, N.; Perveen, S.; Khan, K. M.; Choudhary, M. I. 4-Arylamino-6-nitroquinazolines:
Synthesis and their activities against neglected disease leishmaniasis. Eur. J. Med. Chem. 108 (2016) 1320.
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Bridges, A. J.; Zhou , H.; Showalter, H. D. H.; Winters, R. T.; Leopold, W. R.; Fry, D. W.; Nelson, J. M.;
Slintak, V.; Elliot, W. L.; Roberts, B. J.; Vincent, P. W.; Patmore, S. J. (Phenylamino)pyrido[d]pyrimidine
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acrylamides as irreversible inhibitors of the ATP binding site of the epidermal growth factor receptor. J. Med.
Chem. 42 (1999) 18031815.
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Roh, E. J. Targeting EGFR/HER2 tyrosine kinases with a new potent series of 6-substituted 4-
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Med. Chem. Lett. 25 (2015) 51475154.

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[43] Albuschat, R.; Loewe, W.; Weber, M.; Luger, P.; Jendrossek, V. 4-Anilinoquinazolines with Lavendustin
A subunit as inhibitors of epidermal growth factor receptor tyrosine kinase: syntheses, chemical and
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Legend for Figures

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Figure 1. FDA approved EGFR and/or HER2 inhibitors

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Figure 2. EGFR/DNA dual targeting combi-molecules

Figure 3. Cyclophosphamide 8 and its metabolites

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Figure 4. Prodrugs of phosphoramide mustard derivatives

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Figure 5. Quinazolinephosphoramidate mustardconjugate 10

Figure 6. Selected images (A) and statistical analysis (B) of 10d (EMB-3)-induced DNA damage in MDA-

MB-468 cells.
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Figure 7. Inhibition of tumor growth in vivo by 10d (EMB-3).
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Figure 8. MTD study of compound 10d in mice

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Figure 1. FDA approved EGFR and/or HER2 inhibitors

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O Cl
N Me HN Cl O HN N
N N N O
Cl N N
O Cl
N O N

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5 6

R
HN
H H
N N
N

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Cl O
N N

7a R = 2-F, 3-Cl
Cl 7b R = 3-Cl, 4-F

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Figure 2. EGFR/DNA dual targeting combi-molecules

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Cl Cl
OO O

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N P H
N P O +
HN NH 2 O
Cl Cl
8 (CP) PM acrolein

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Figure 3. Cyclophosphamide 8 and its metabolites

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Figure 4. Prodrugs of phosphoramide mustard derivatives
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R2

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Cl
O HN
N P O X
n N
NH2
Cl

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R1 N
10
when n = 0, X = single bond;
when n = 2-5, X = O or CONH;

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R1 = H, MeO;
R2 = 3-Br, 3-Cl-4-F, 3-ethynyl,
3-Cl-4-((3-F-benzyl)oxo)phenyl

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Figure 5. Quinazolinephosphoramidate mustard conjugate 10

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Figure 6. Selected images (A) and statistical analysis (B) of 10d (EMB-3)-induced DNA damage in MDA-
MB-468 cells. MDA-MB-468 cells were exposed to 10d for 2 h and the DNA damage was then determined
using alkaline comet assay. IC50 means the IC50 value of 10d against MDA-MB-468 cell line Data are reported
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as the mean SD of at least 50 random select cells. #P<0.01.

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Figure 7. Inhibition of tumor growth in vivo by 10d (EMB-3). Human lung cancer H522 xenografts were
treated with vehicle control, lapatinib (100 mg/kg/d), CTX (50 mg/kg/d) and 10d (50 and 100 mg/kg/d) for 28
days. All mice were sacrificed at day 28 after administration and tumors were harvested and weighted. RTV

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(A) and tumor weight (% of control) (B) were then analyzed. *P<0.05, **P<0.01. Days in X-axis (A) were the
days after the start of the treatment.
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Figure 8. MTD study of compound 10d in mice (n=3). The treatment groups were orally administrated at a
single dose of 100, 300, 600, and 900 mg/kg 10d, respectively. Body weights were monitored for 5 days after
administration
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Legend for Schemes

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Scheme 1:

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R2 R2
OH HN HN Cl

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HO AcO d HO g-i O HN Br
N a, b, c
N N N P O
N
R1 N R1 N R1 N NH2
Cl N
11a: R1 = H 12 13 10a
11b: R1 = OMe 12a, 13a: R1 = H, R2 = 3-Br

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12b, 13b: R1 = OMe, R2 = 3-Br
12c, 13c: R1 = OMe, R2 = 3-Cl-4-F e
12d, 13d: R1 = OMe, R2 = 3-Cl-4-((3-F-benzyl)oxo)phenyl

I
Cl

N c
I
HN

N
R2

f
AN HO
n
O
HN

N
R2

g-i
Cl
O
N P O O
HN
R2

n N
N NH2
R1 N Cl
N
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R1 N
15 16a: R2 = 3-Br
16b: R2 = 3-Cl-4-F 14a-g, 14i-j, 14l-m 10b-e, 10g-h, 10j-n
16c: R2 = 3-Cl-4-((3-F-benzyl)oxo)phenyl
14a, 10b: n = 2, R1 = H, R2 = 3-Br
14b, 10c: n = 3, R1 = H, R2 = 3-Br
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14c, 10d: n = 4, R1 = H, R2 = 3-Br

14g, 10e: n = 5, R1 = H, R2 = 3-Br
14i, 10g: n = 4, R1 = H, R2 = 3-Cl-4-F
14j, 10h: n = 4, R1 = H, R2 = 3-Cl-4-((3-F-benzyl)oxo)phenyl
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14l, 10j: n = 3, R1 = H, R2 = 3-Cl-4-F

14m, 10k: n = 3, R1 = H,
14d, 10l: n = 3, R1 = OMe,
14e, 10m: n = 3, R1 = OMe,
14f, 10n: n = 3, R1 = OMe,
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R2 = 3-Cl-4-((3-F-benzyl)oxo)phenyl
R2 = 3-Br
R2 = 3-Cl-4-F
R2 = 3-Cl-4-((3-F-benzyl)oxo)phenyl

Reagents and conditions: (a) Ac2O, pyridine, 100 oC; (b) DMF (cat.), SOCl2, reflux; (c) substituted aniline, Et3N, i-PrOH, reflux; (d) NH4OH, MeOH, rt; (e) K2CO3,
CH3CN or DMF, reflux; (f) alkyldiol, CuI(10mol%), NaOBut, DMF (dry), 80o C; (g) n-BuLi, THF, 0 oC, N2; (h) Cl2P(O)N(CH2 CH2Cl)2 , THF, 0 oC; (i) NH3(gas), 0 oC.
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Scheme 2:

HN Br Si HN
O O Si
HO N HO n N
n

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N a) N
14b: n = 0 14h: n = 1
14c: n = 1 14k: n = 0

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Cl HN
O
b) O
N P O n N
NH2
N

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Cl
10f: n = 1
10i: n = 0

Reagents and conditions: a) CuI, Pd(PPh3)2Cl2, Et3N, DMF, N2, 90oC; b) BuLi,THF,N2,0oC; then
Cl2P(O)N(CH2CH2Cl)2, THF, 0oC; then NH3(gas), 0oC; then K2CO3,THF, MeOH.

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Scheme 3:

R R
OH HN HN
O2N a, b c
N O 2N H2N
N N

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N N N
17 18 19
18a: R = 3-Bromo 19a: R = 3-Bromo
18b: R = 3-chloro-4-fluoro 19b: R = 3-chloro-4-fluoro

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18c : R = 3-chloro-4-((3-fluoro-benzyl)oxy) 19c: R = 3-chloro-4-((3-fluoro-benzyl)oxy)

R R
HN Cl HN
d, e H O H
f

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N N
HO N N P O N
O NH 2 O
N Cl N
21 10o-q
21a: R = 3-Bromo 10o: R = 3-Bromo
21b: R = 3-chloro-4-fluoro

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10p: R = 3-chloro-4-fluoro
21c: R = 3-chloro-4-((3-fluoro-benzyl)oxy) 10q: R = 3-chloro-4-((3-f luoro-benzyl)oxy)

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Reagents and conditions: (a) SOCl2, DMF (cat.), reflux; (b) substituted aniline, Et3 N, i-PrOH, reflux; (c) Sn powder, conc. HCl, rt;
(d) 4-(benzoyloxy)butanoyl chloride 20, Na 2CO3 , THF, rt; (e) NaOH, MeOH, H2O, rt; (f) NaH, THF, 0 o C, N 2; then
Cl2P(O)N(CH 2CH 2Cl)2 , THF, 0 o C; then NH3 (gas), 0 oC.
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Legend for Tables

Table 1. Compounds tested for their inhibitory effects on both EGFR and HER2

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Table 2. Inhibitory activities of potent compounds on cell proliferation

Table 3. Effects of compound 10d on cell proliferation of different cell lines

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Table 4. Preliminary PK profile of 10d

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Table 1. Compounds tested for their inhibitory effects on both EGFR and HER2

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EGFR HER2
Compd Structure
(nM) (nM)

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10a 9.2 1.0 1200 100
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10b 4.3 1.0 180 120
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10c 3.0 0.4 890 330

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Cl HN Br
O
10d O 7.4 0.4 82 2
N P O N
NH2
Cl N
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Cl
O HN Br
10e N P O O 2.3 1.4 160 10
N
NH 2
Cl N
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Cl HN
O
10f O 11 1.8 2100 900
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N P O N
NH2
Cl N

Cl HN Cl
10g O
O 6.0 1.2 2400 100
N P O N
NH2
Cl N

O
F
Cl
10h O
HN Cl 27 7.4 190 110
O
N P O N
NH2
Cl N

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Cl
O HN
10i N P O O 7.2 0.8 190 30
N
NH 2
Cl N

F
Cl
O HN Cl
10j N P O
5.7 1.7 2700 500
O
N

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NH 2
Cl N

O
F
Cl

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10k O HN Cl 75 2.5 1700 500
N P O O
N
NH 2
Cl N

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10l 0.3 0.1 6000 2000

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10m 0.5 0.2 1300 900

Cl
AN O
F

10n O HN Cl 22 8.9 1100 500

N P O O
N
M
NH2
Cl H3 CO N
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10o 3.3 0.9 240 20

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10p 8.7 0.4 2500 300

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10q 26 0.8 3200 800

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Staurosporine - 68 16 34 12

IC50 was calculated by Logit method from the results of at least two independent tests with eight concentrations each and expressed as meanSD.

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Table 2. Inhibitory activities of selected compounds on cell proliferation

MDA-MB-468 HCT116 SK-BR-3 H522

Compd

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(M) (M) (M) (M)

10a 61 38 22 3.4

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10b 66 39 26 3.0

10c 30 34 1.4 5.6

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10d 19 11 2.8 2.5

10e >100 25 6.3 20

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10f >100 66 7.9 42

10i >100 46 4.6 16

10l 20
AN 45 4.9 4.6

10m 9.6 26 11 2.5

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10o 31 23 8.0 2.6
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Lapatinib 20 6.2 4.4 6.4

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* For each tumor cell line, one experiment was performed with eight concentrations in triplicate.
IC50 values were expressed as mean values. MDA-MB-468, human breast cancer cell line.
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HCT116, human colorectal cancer. SK-BR-3, human breast cancer cell line. H522, human lung
cell line.
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Table 3. Effects of compound 10d on cell proliferation of different cell lines

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Cellular IC50 (M)
Compound
H460 H522 COLO205 Bel7402 AS431NS Calu-3

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10d 9.8 2.5 5.0 13 10 5.7

lapatinib 8.4 6.4 4.6

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* For each tumor cell line, one experiment was performed with eight concentrations in triplicate. IC50 values
were expressed as mean values. H460, human lung cell line. H522, human lung cell line. COLO205, human
colorectal cell line. Bel7402, human hepatocellular carcinoma cell line. AS431NS is human skin cancer cell line.
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Calu-3, human lung cell line.

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Table 4. Preliminary PK profile of 10d

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Mean(SD) Mean(SD)
Animal (rats, n = 3)
(iv, 1 mg/kg) (po, 10 mg/kg)
t1/2 (hr) 0.52 (0.023) 1.9 (1.5)

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C0 (ng/mL) 2059 (309) -
tmax (hr) - 0.500 (0.00)
Cmax (ng/mL) - 1987 (1075)

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AUClast (hr*ng/mL) 659 (84) 4780 (1620)
AUCInf (hr*ng/mL) 661 (85) 5150 (1334)
AUC Extr (%) 0.30 (0.088) 7.9(13)

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Vz (L/kg) 1.2 (0.11) -
Vss (L/kg) 0.79 (0.035) -
CL (mL/min/kg)
MRT (hr)
76 (8.5)
0.52 (0.046)
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2.8 (1.9)
AUC/D (hr*kg*ng/mL/mg) - 515 (133)
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F (%) - 73 (25)
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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 10d had no obvious acute toxicity.

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