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Targeting Tumor Associated Phosphatidylserine with New Zinc Dipicolylamine-Based Drug Conjugates. Yu-Wei Liu, Kak-Shan Shia, Chien-Huang Wu, Kuan-Liang Liu, Yu-Cheng Yeh, CHEN-FU Lo, ChiungTong Chen, Yun-Yu Chen, Teng-Kuang Yeh, Wei-Han Chen, Jiing-Jyh Jan, Yu-Chen Huang, Chen-Lung Huang, Ming-Yu Fang, Brian D. Gray, Koon Y. Pak, Tsu-An Hsu, Kuan-Hsun Huang, and Lun Kelvin Tsou Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 05 Jun 2017 Downloaded from http://pubs.acs.org on June 8, 2017

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Bioconjugate Chemistry

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Targeting Tumor Associated Phosphatidylserine with New Zinc Dipicolylamine-Based Drug Conjugates

Yu-Wei Liu,†♯ Kak-Shan Shia,†♯ Chien-Huang Wu,†♯ Kuan-Liang Liu,† Yu-Cheng Yeh,† Chen-Fu Lo,† Chiung-Tong Chen,† Yun-Yu Chen,† Teng-Kuang Yeh,† Wei-Han Chen,† Jiing-Jyh Jan,† Yu-Chen Huang,† Chen-Lung Huang,† Ming-Yu Fang,† Brian D. Gray, § Koon Y. Pak, § Tsu-An Hsu,† Kuan-Hsun Huang,† and Lun K. Tsou*† †

Institute of Biotechnology and Pharmaceutical Research, National Health Research

Institutes, Miaoli 35053, Taiwan, ROC §

Molecular Targeting Technologies, Inc. West Chester, PA 19380, USA

KEYWORDS Zinc dipicolylamine, small molecule drug conjugates, phosphatidylserine, tumor microenvironment

Correspondence and

requests for materials should be addressed to

([email protected])

ACS Paragon Plus Environment

L.K.T.

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ABSTRACT A series of zinc(II) dipicolylamine (ZnDPA)-based drug conjugates have been synthesized to probe the potential of phosphatidylserine (PS) as a new antigen for small molecule drug conjugate (SMDC) development. Using in vitro cytotoxicity and plasma stability studies, PS-binding assay, in vivo pharmacokinetic studies and maximum tolerated dose profiles, we provided a roadmap and the key parameters required for the development of the ZnDPA based drug conjugate. In particular, conjugate 24 induced tumor regression in the COLO 205 xenograft model and exhibited a more potent antitumor effect with a 70% reduction of cytotoxic payload compared to that of the marketed irinotecan when dosed at the same regimen. In addition to the validation of PS as an effective pharmacodelivery target for SMDC, our work also provided the foundation that if applicable, a variety of therapeutic agents could be conjugated in the same manner to treat other PS-associated diseases.



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INTRODUCTION Ligand-targeted cancer therapeutics represent a viable strategy to deliver potent cytotoxic agents to the disease site.1 In particular, conjugation of a therapeutic moiety via a suitable linker to antibodies, capable of specific binding to tumor antigens, has facilitated selective accumulation and increased the effective drug concentration in situ while alleviating adverse side effects systemically.2 Indeed, with recent approvals of two Antibody Drug Conjugates (ADC) in oncology, brentuximab vedotin3 and adotrastuzumab ematasine4, more than forty ADCs are being evaluated in the clinical trials.5 In spite of clinical success of the ADCs, daunting challenges associated with the development of ADCs have led to the emerging strategy of using small molecule drug conjugates (SMDC).6 Contrary to limitations of ADC-based development, SMDC can be advantageous with lower cost of development, better penetration of tumors, easier optimization of pharmacokinetic properties with distinct drug ratio, and lower immunogenicity.7-11 Some of the promising SMDCs target prostate specific membrane antigen,12 carbonic anhydrase IV,13 and somatostatin derivatives for neuroendocrine tumors.14 Moreover, folate-based conjugates have entered Phase II/III clinical trials for treating multiple cancer diseases.15, 16 However, there are only a limited number of small ligands to tumor-associated antigens that have been assessed systemically for pharmacodelivery applications. Thus, it is of great importance to validate new tumorassociated ligands for the development of novel SMDC in cancer therapy. Cancer progression involves a complex interplay between tumor cells and their microenvironment.

To validate the use of SMDC in targeting tumor-associated

phosphatidylserine (PS) in the tumor microenvironment, herein, we have designed and


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synthesized zinc(II) dipicolylamine (ZnDPA)-based drug conjugates and evaluated their anti-tumor activities.

Under normal cellular state, PS is tightly regulated to be

asymmetrically segregated in the inner leaflet of the normal cells.17 Apoptosis induced PS exposure is readily cleared by macrophages under normal conditions, however, tumor cells can evade surveillance by the macrophages.18 Indeed, PS was found in many tumor cell lines and primary tumors.19-21 Not only does the basal level of apoptosis occurs in the neoplastic masses owing to lack of blood supply, but abundant PS is found on viable cells at the tumor vasculatures.22,

23

More importantly, recent studies with a PS-targeting

antibody Bavituximab in Phase III clinical trail has provided a significant insight into the expansion of tumor-associated macrophages with PS-containing vacuoles in the tumor microenvironment.24 Thus, PS at the tumor microenvironment has become a novel biomarker.21 While Bavituximab has demonstrated the proof-of-concept of targeting PS selectively, the drug conjugate development for Bavituximab remains challenging. To circumvent these challenges, we envisioned that a PS-targeted small molecule in conjugation with a potent cytotoxic agent via a suitable linker could be developed as a novel SMDC. Synthetic ZnDPA, initially described for the selective molecular recognition of multi-anionic phosphorylated biomolecules,25 has been used to construct optical imaging probes that target the outer surfaces of anionic PS-containing cell membranes.26,

27

Pioneered by Smith and coworkers, positively charged ZnDPA-derived fluorescent ligands were shown to accumulate specifically at the tumor site in vivo through its interaction with negatively charged PS.28,

29

However, ZnDPA based cancer drug

conjugates (ZnDPADCs) have not been described for cancer therapy. We reasoned that



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spatial- and temporal-delivery of cancer therapeutics could be achieved through engineering suitable linkers for the controlled release of the drugs using ZnDPA as a probe. In particular, these designed conjugates should allow anchoring of ZnDPADC in the tumor tissues via PS binding, uncaging of cytotoxic payload via enzymatic cleavage, and killing the cancer cells by the released bioactive cytotoxic. In the current report, we wish to 1) describe the facile SMDC synthesis of the first ZnDPA linked to 1 (SN-38)30; 2) determine structure activity, plasma stability, and PS-binding relationships between different linker types; and 3) provide insight into optimization of conjugates’ pharmacokinetic properties with respect to anti-tumor efficacy in vivo.

CHEMISTRY For the development of PS-targeting SMDCs, we have designed ZnDPA and cytotoxic payload 1 conjugates to address the unmet medical need in colorectal cancer (CRC) as it is the third most common cancer worldwide and is mainly a disease of developed countries.31 Therapeutic strategies that target new molecular features of CRC would provide alternative treatment options. Camptothecins comprise an important class of FDA approved anticancer drugs for the treatment of colorectal cancer. Moreover, nanoliposomal irinotecan was approved in 2015 for treating pancreatic cancer patients who failed the first-line treatment.32 The potent topoisomerase-1 inhibitor 1, is the active metabolite of 2 (CPT-11).30 We hypothesize that 1 can be delivered by a ZnDPA-based SMDC and specifically accumulate in the tumor microenvironment. Behaving as a prodrug, 1’s bioactivity is shielded during systemic circulation while conjugated to ZnDPA, but activated via enzymatic cleavage upon approaching the tumor site.


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Conjugation to the 10-hydroxyl position of 1 with a linker was experimentally realized via facile synthesis to generate the eventual conjugates.

Scheme 1. Synthetic procedures for compound 10. Reagents and conditions: (1) 2,2o

o

dimethoxypropane, p-TsOH, MeOH, 60 C, 12h, 96%. (2) LiAlH4, THF, 40 C. (3) o

BrCH2(CH2)5CO2Me, K2CO3, DMF, 85 C, 12h, 73%. (4) CBr4, PPh3, DCM, overnight, 85%. (5) bis(pyridin-2-ylmethyl)amine, K2CO3, DMF, 3h, 82%. (6) LiOH(aq), MeOH. (7) SN-38 (1), EDCl, HOBt, N-Methylmorpholine, DMF, 15h, 42%. (8) 2 equiv. Zn(NO3)2, DCM/MeOH.

One of the many advantages over ADC lies in the cost-down of SMDC, hence, we looked for simple linker modifications and facile synthesis for the construction of these conjugates. As shown in scheme 1, synthesis of 10 was performed via eight steps in our first example. Treatment of 5-hydroxyisophthalic acid 3 with 2,2-dimethoxypropane and p-toluenesulfonic acid in methanol at 60 °C gave the dimethyl 5-hydroxyisophthalate 4 in good yield (96%). Reduction of 4 with lithium aluminum hydride in dry THF at 40 °C to furnish the fully reduced (5-hydroxy-1,3-phenylene)dimethanol 5, which after work-up



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and without purification, alkylation of 5 was performed in the presence of potassium carbonate and methyl 7-bromoheptanoate to afford the corresponding diol compound 6 in 73% yield. This was followed by the addition of an excess of CBr4 and PPh3 to afford the methyl 7-(3,5-bis(bromomethyl)phenoxy)heptanoate 7 in 85% yield. Dibromo intermediate 7 thus obtained was then substituted with bis(pyridin-2ylmethyl)amine in DMF with potassium carbonate to generate methyl 7-(3,5bis((bis(pyridin-2-ylmethyl)amino)methyl)phenoxy)heptanoate, which was subsequently hydrolyzed with lithium hydroxide to yield the desired product 8 in 82% over two steps. Activation of acid 8 in the presence of EDCI and HOBt allowed for the conjugation with 1 to afford 9 in moderate yield (42%), which was then treated with two equivalents of zinc nitrate to furnish 10 in quantitative yield, the first SMDC designed in our laboratories. In scheme 2, synthesis of the starting material 11 has been described in the literature.33 The primary amine functional group in 11 was reacted with reagents in conditions (1a)-(1e) to generate various types of DPA-linkers, designated as the linker type R, with a terminal ester functional group in 12a-e (Scheme 2). Hydrolysis of methyl ester 12a-e with LiOH (0.5 N) at room temperature proceeded smoothly to afford the corresponding intermediate acid 13a-e in moderate to high yields (83-97%). Activation of the carboxylic acids in DPA-linkers 13a-e with EDCl and HOBt in dry N,Ndimethylformamide followed by addition of 1.1 equivalent of 1 gave rise to DPA-drug conjugate 14a-e in 38-80% yields. Incubating each of the DPA-drug conjugates 14a-e with two equivalents of zinc nitrate in dichloromethane/methanol (1:1) at room


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temperature for 1h resulted in the formation of ZnDPADCs 15-19, respectively, in quantitative yield.

Scheme 2. Synthetic procedures for compound 15-19. Reagents and conditions: (1) (a) methyl

4-chloro-4-oxobutanoate,

DCM,

TEA,

2h,

83%.

(b)

methyl

3-

(chlorocarbonyl)benzoate, DCM, TEA, 2h, 55%. (c) methyl 4-formylbenzoate, DCM; then NaBH4, MeOH, 15h, 66%. (d) methyl 4-(chlorosulfonyl)benzoate, DCM, TEA, 2h, 55%. (e) methyl 3-isocyanatobenzoate, DCM, TEA, 1h, 61%. (2) 0.5N LiOH(aq), MeOH, 15h, 83-97%. (3) SN-38, EDCl, HOBt, N-Methylmorpholine, DMF, 38-80%. (4) 2 equiv. Zn(NO3)2, DCM/MeOH.



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Scheme 3.

Synthetic procedures for compound 24-25 and 27-28. Reagents and

conditions: (1) (a) [1,1'-biphenyl]-4-carbaldehyde, DCM; then NaBH4, MeOH, 80%. (b) methyl 4-formylbenzoate, DCM; then NaBH4, MeOH, 15h, 66%. (2) (a) methyl 3isocyanatobenzoate, DCM, TEA, 1h, 73%. (b) [1,1'-biphenyl]-4-carboxylic acid, EDCl, HOBt, N-Methylmorpholine, DMF, 30%. (3) 0.5N LiOH(aq), MeOH, 15h. (4) SN-38, EDCl, HOBt, N-Methylmorpholine, DMF, 40-48%. (5) 2 equiv. Zn(NO3)2, DCM/MeOH. (6) 26, TEA, THF, 40-90%.

In scheme 3, reductive amination of the primary amine in 11 with NaBH4 and [1,1'-biphenyl]-4-carbaldehyde generated the corresponding DPA-linker 20a in 80% yield. Conjugate 27 was prepared by coupling of 11 with 26 followed by incubation with 2 equivalents of zinc nitrate in good yield over two steps (90%). In a similar manner,


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carbamate-containing 28 was achieved in 40% yield by conjugation of intermediate 20a and 26 (4-nitrophenyl carbonate activated 1)34 under basic conditions in THF followed by incubation with two equivalents of zinc nitrate. On the other hand, compound 12c was coupled with [1,1'-biphenyl]-4-carboxylic acid under EDCl and HOBt to install amidetype linker 21b (30%) or compound 20a was coupled with isocyanatobenzoate to establish urea-type linker 21a (70%). Subsequently, the terminal ester group of 21a and 21b was hydrolyzed by LiOH (0.5 N) to generate the corresponding acids 22a and 22b, which without purification, was coupled with 1 under EDCl and HOBt followed by incubation with Zn(NO3)2 to afford final products 24 (48%) and 25 (40%) over three steps.

Scheme 4. Synthetic procedures for compound 31. Reagents and conditions: (1) 1(tert-butoxycarbonyl)piperidine-4-carboxylic acid, EDCl, HOBt, N-Methylmorpholine, DMF, 12h, 81%. (2) TFA, DCM, 82%. (3) 26, TEA, urea, THF, 2h, 96%. (4) 2 equiv. Zn(NO3)2, DCM/MeOH.

In scheme 4, 11 was coupled with 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid under activation with EDCI and HOBt to form intermediate 29 in 81% yield, which was followed by treatment with TFA to remove the Boc-protecting group to afford intermediate 30 (82%), with which the amino group of was subsequently reacted with



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prepared 26 under basic conditions followed by incubation with zinc nitrate to yield conjugate 31 in 96% over two steps.

RESULTS AND DISCUSSION In vitro plasma stability and cytotoxicities of the conjugates Plasma stability of the synthesized conjugates is one of the important parameters for the design of SMDC. We envisioned that linker modifications play an important role in governing the stability of the intact conjugates. To examine the plasma stability of ZnDPADCs, we have monitored the time-dependent release of 1 from these conjugates with an HPLC assay after incubation in mouse plasma (Figure 1A). For example, the release of 1 from conjugate 16 was less then 10% after 3 h incubation with mouse plasma, while it was > 90% for that of conjugate 27 (Figure 1A). When 1 was conjugated to ZnDPA via a simple alkyl chain with an ester bond (conjugate 10) or with a carbamate moiety (conjugate 27), unfavorable plasma stabilities resulted as only 37% and 0% of the intact conjugates were observed after 24 h incubation with the mouse plasma at 37 oC, respectively (Figure 1B). As such, conjugates 15-18 with intramolecular amido- or benzoic esters linkages were synthesized to further diversify the linker options. In an attempt to increase the solubility of 17, sulfonamide-containing 18 was synthesized. However, there was no improvement from these two conjugates but with rapid hydrolysis to 1 (Figure 1B). Compound 16 exhibited moderate stability with a ratio of 72:28 (conjugate: 1) after 24 h incubation with mouse plasma at 37 oC. Further modification of the carbamate linker via installation of a biphenyl group at the nitrogen has provided the most stable linker in 28. Similar to 2, we reasoned that the ZnDPADCs would behave as


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Figure 1. In vitro plasma stability and cytotoxicities studies. (A) Representative HPLC data on the retention of 16 and 27 in comparison to 1 at time=0 and after 3h incubation with mouse plasma. (B) Structures and cytotoxic effects of 10, 15-18, 27, 28, 1, and 2 on COLO 205 human colorectal adenocarcinoma and Detroit 551 normal skin fibroblast



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from three separate experiments. The cells were incubated with conjugates or drugs continuously for 72 h, and the cytotoxic effects were determined by reduction of tetrazolium compound by viable cells.

prodrugs and exhibit cellular cytotoxicity profiles. Since 1 is poorly soluble, modification at the C10-phenolic hydroxyl group not only increases its bioavailability, but also shields its potent bioactivity with reduced toxicity in systemic circulation. Cytotoxicities of ZnDPADCs were assayed against COLO 205 colorectal cancer cells and Detroit 551 normal skin fibroblast cells (Figure 1B). As 1 is the bioactive metabolite of FDA approved anticancer drug for the treatment of colorectal cancer and to address the unmet medical need in colorectal cancer with these ZnDPADCs, we therefore chose COLO205 for the cellular studies. Detroit 551 was chosen as the normal cell control. With a similar trend to that of 2, conjugates 10, 15-18, 27 and 28 showed 4- to 10-fold decrease in potencies towards COLO 205 and modest 3-fold decrease in cytotoxicities for Detroit 551 as compared to that of cytotoxic component 1.

Interestingly, the most stable

conjugate 28, with IC50 at 7 µM against COLO 205 and >20 µM against Detroit 551, exhibited a similar cytotoxicity profile to the prodrug 2. Taken together, we identified two linker types in 16 and 28 that conferred improved stabilities in mouse plasma with prodrug-like properties.

Binding ability of the conjugate to PS To investigate whether the PS-targeting ZnDPA motif retains its association property with the linker-drug modification, we conducted in vitro binding studies


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between conjugate 16 and a DOPS-containing supported lipid bilayer by surface plasmon resonance (SPR) measurements (Figure 2).35 Uniform-sized liposomes with 100 % DOPC were prepared as a non-specific binding control (Figure S1A). On the other hand, PS-coated liposomes (DOPC/ DOPS (3:1,v/v)) were prepared according to procedures reported in literature (Figure S1A).36 Compound 38, ZnDPA without linker-drug modification, exhibited a association rate constant (ka) of 303.3 (1/Ms) and a dissociation rate constant (kd) of 9.95 x 10-3 (1/s) in a 1:1 complex binding model.33 Moreover, the equilibrium dissociation constant (KD) of compound 38 was determined to be 3.28 x 10-5 M, which was in good agreement from the fluorophore-linked ZnDPA sensor binding studies reported by Smith and co-workers and showed that the SPR experiments could be used for the PS association studies (Figure S1B).33 While compound 38 might act as a single molecular unit and exhibited mono-exponential behavior in binding, the binding kinetic plot for conjugate 16 showed to be multi-exponential and the dissociation kinetic plot for the compound exhibited at least two dissociation events (Figure 2).

Figure 2. In vitro SPR PS-association studies. Sensorgrams generated using a Biacore T200. Conjugate 16 was analyzed across a two-fold concentration series descending from 62.5µM. Compound 38 showed association and the negative controls, 37 and 1 showed



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no apparent association. The association was monitored for 1 min, and the dissociation time was 1 min. Liposome (DOPC/ DOPS (3:1,v/v)) was immobilized on a L1 chip at 5000 RU, where liposome of (DOPC 100%) was used as nonspecific binding control.

The complicated kinetics of conjugate 16 may be due to many factors and selfaggregation of the conjugate-PS complexes within the membrane after association could also contribute to the differences in the curve.37 The sensograms of conjugate 16 were hard to fit in a quantitative manner, nonetheless, our results showed that conjugate 16 could readily associate with the PS-containing liposome. Conjugate 37, containing the same linker-drug modification yet differing in zinc-coordinated nitrogen atoms from those in 16, was synthesized as the control. As suggested from the association studies of the conjugate 37, the presence of the dipicolylamine species are the important factors. Moreover, results from conjugate 37 and 1 also indicated that the linker-drug or drug itself did not contribute to the direct association with PS. Taken together, conjugate 16’s association with PS was driven by the two dipicolylamine units. These data strongly suggested that: 1) the ZnDPA motif in the conjugates played an important role for PS recognition in these conjugates; 2) linker-drug modifications in ZnDPADCs did not interfere with ability of recognition between the ZnDPA and PS-coated liposomes.

In vivo anti-tumor activities of conjugates 16 and 28 To examine PS expression in the COLO 205 xenograft model, we have intravenously injected PSVue®794 to demonstrate that the PS-seeking ZnDPA probe could efficiently accumulate to the tumor site (Figure 3). In addition to the findings by


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Smith and co-workers who had showed ZnDPA possessed high affinity for the exposed anionic surfaces of apoptotic cells in breast or prostate tumors,29 our data also showed enrichment of PS at the COLO 205 tumor site, thus providing an important insight for a therapeutic delivery strategy using ZnDPA.

Figure 3. In vivo detection of PS-expression in COLO 205 tumor xenograft model. Representative IVIS images of PSVue®794 fluorescence probe in mice.

From our initial efforts, we have identified 28 with a stable linker modification in plasma, prodrug-like in vitro cytotoxicity, and maximum tolerated dose at 40 mg/kg (mpk). To our surprise, we did not observe potent anti-tumor activity with 28 in the COLO 205 xenograft model (Figure S2). We reasoned that PS, a membrane associated lipid, in vast contrast to the many established ADC or other SMDC targeted receptors,



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might not trigger the internalization of the conjugate for efficient cleavage to release the cytotoxic payload 1. Even though 28 exhibited a seemingly similar profile to that of 2, it’s not suitable for the development as a ZnDPA based conjugate. However, these data provided insight into the design and synthesis of new ZnDPADCs, wherein conjugates readily releasing drug in the acidic tumor microenvironment and/or by locally higher protease activities (released from the dead cells) and exhibiting moderate stability in blood circulation should be desirable. On the other hand, 16 showed potent anti-tumor activity in the COLO 205 xenograft model (Figure 4). In addition, further studies are required to identify the enzyme that cleaves the payload from these ZnDPADCs and its role in the tumor microenvironment. Compound 39, conjugate 16 without the addition of the cytotoxic 1, was synthesized and showed no anti-tumor activities, demonstrating the in vivo anti-tumor activity was dependent on 1 (Figure 4).

*: p < 0.05 vs. vehicle control by one-way ANOVA analysis and Newman-Keuls multiple comparison test.


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Figure 4. Anti-tumor activities of 16 and 1 against COLO 205 tumor growth and comparison of the amount of 1 used in each regimen. SN-38 proportion is the molecular weight ratio of 1 to its conjugates. 16 (5 and 20 mg/kg) effectively suppressed COLO 205 growth in mice. Compound 39, similar to conjugate 16 but lacked the drug component 1, showed no antitumor activity. Dots at the bottom of the graph indicate dosing frequencies.

Notably, 16 exerted similar tumor growth inhibition with only 13% of the cytotoxic payload 1 delivered in comparison to the treatment with 1 given at the dosing schedule of once per day (qd) for 5 days in a row, two-day rest and another 5 days in a row (5+5) at 10 mpk. Dose-dependent in vivo anti-tumor efficacy (complete tumor growth inhibition) of 16 was observed when a total of 42 mg of the cytotoxic payload 1 was delivered by the conjugate. Taken together, in contrast to the most stable conjugate 28, potent tumor growth inhibition by 16 with much less cytotoxic payload 1 was achieved than treatment with 1 alone, suggesting spatiotemporal release of the cytotoxic payload is a key parameter for efficacy in the design of ZnDPADCs. To prepare other new ZnDPADCs, we hybridized the plasma stability-enhanced biphenyl group with the cleavable ester-modified cytotoxic payload 1 to generate 19, 24, and 25 (Scheme 2). Moreover, a urea-containing linker extended via the amino functionality in 11 was readily prepared to afford 19. Modifications on the urea-linker could be accomplished through reductive amination from 11 as alkyl-biphenyl (24) and aryl-biphenyl (25) functionalities were installed in the linkers (Scheme 2). On the other hand, linker



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modification to mimic the carbamate moiety in 2 was also carried out for the synthesis of 31 (Scheme 4).

In vitro properties and pharmacokinetic studies of 19, 24, 25, and 31 Conjugates 24 and 25 with biphenyl functionalities and 2-mimicking linker moiety in 31 exhibited improved in vitro plasma stabilities and retained pro-drug properties when compared to the previous conjugates (Table 1A). Moreover, conjugate 24 also showed good stability in human plasma with an intact conjugate to 1 ratio of 94:6 after 24hr of incubation (Figure S3). Maximum tolerated dose (MTD) studies revealed that ICR mice survived the regimen when 19, 24, and 25 were given at >30 mpk per day for five consecutive days (Table 1A). In particular, 24 and 25 exhibited a MTD at 40 mpk. Based on these MTD profiles, 19, 24, and 25 were further selected for pharmacokinetic (PK) studies to assess their in vivo stability profiles (Table 1B). Consistent with the in vitro profile, 19 had the least favorable PK profile as the area of drug concentration under curve (AUC) ratio between the intact conjugate and the released cytotoxic payload 1 was 0.7 with high volume of distribution (Vss) at 86.2 (L/kg). These results might contribute to the lower MTD values of 30 mpk when compared to 24 and 25. On the other hand, the AUC ratio of the intact conjugate to cytotoxic payload 1 was higher for 24, indicating that the alkyl-biphenyl linker possessed better in vivo stability than the aryl-biphenyl moiety in 25. Taken together, we identified 24 with improved pharmacokinetic profiles (Table 1B), increased MTD, and retained pro-drug properties among the designed ZnDPADCs.


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Table 1. In vitro plasma stability, cytotoxicities, maximum tolerated dose, and in vivo pharmacokinetics studies of 19, 24, 25, and 31. (A) Chemical structures, mouse plasma stability, cytotoxic effects on COLO 205 human colorectal adenocarcinoma and Detroit 551 normal skin fibroblast from three separate experiments, and MTD studies of 19, 24, 25, and 31. (B) Pharmacokinetic profiles of 19, 24, and 25 in male ICR mice (n=3) at 5 mg/kg with intravenous administration. CL, clearance; Vss, apparent volume of distribution at steady state; AUC, area of drug concentration under curve; mpk, mg/kg.



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In vivo anti-tumor activities of 24 We finally evaluated the therapeutic efficacy of conjugate 24 in mice bearing COLO 205 human colorectal tumor (Figure 5). We observed potent dose-dependent tumor growth inhibition effects with intravenous administration of 24 at the qd (5+5) dosing schedule. With the same dosing regimen, 24 exhibited similar tumor growth inhibition to that of 1, albeit the ZnDPA based conjugate only loaded with 23% of cytotoxic payload 1. Tumor shrinkage was observed in the treatment group of 24 given at 20 mpk with the qd (5+5) schedule. These data provided evidence for the tumor targeting ability of the ZnDPA based conjugate to increase the cytotoxic payload 1 concentration in situ for improved efficacy. In comparison to the clinical drug 2, we observed greater anti-tumor effect after 40 mpk was given twice per week after the first week (Figure 5).

*: p < 0.05 vs. vehicle control by one-way ANOVA analysis and Newman-Keuls multiple comparison test.


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Figure 5. Anti-tumor activities of 24, 1, and 2 against COLO 205 tumor growth and comparison of the amount of 1 used in each regimen. Dots at the bottom of the graph indicate dosing frequencies.

Moreover, we found that 24 only deployed a total of 28 mg of cytotoxic payload 1 in the 40 mpk (2+1) regimen, yet it achieved better anti-tumor efficacy than the CPT-11 (40 mpk (2+2)) treatment group that deployed 93 mg of 1, indicating that a 70% reduction of cytotoxic payload 1 in 24 relative to prodrug 2. Thus, 24 provided a useful platform for spatial- and temporal- delivery of 1 to the targeted tumor sites, and also augment the therapeutic index of 1 by significantly reducing its payload as compared to that of the marketed prodrug 2.

CONCLUSION To the best of our knowledge, this is the first chemically distinct ZnDPA conjugated to an anti-cancer drug via a covalent linkage in the form of a SMDC. In a separate study to demonstrate the useful strategy of deploying the DPA motif, recent development of a dipicolyl-vancomycin conjugate was shown to exhibit enhanced antibacterial activities against vancomycin-resistant bacteria.38 Inspired by the important role of PS in tumor progression and the PS-targeting ability of ZnDPA probes, we devised ZnDPA-based drug conjugates to expand the repertoire of cancer therapeutic targets for the development of SMDC. In addition to the stable linkage provided in the ZnDPAbased probe developed by Smith and co-workers, ZnDPADCs should employ a linker with moderate in vivo plasma stability and favorable MTD profile, yet, with an



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enzymatically cleavable bond to the drug that allows delivery of the cytotoxic payload once engaged in the tumor microenvironment. Not only did our studies underscore the important parameters, such as in vitro cytotoxicity, PS-binding and plasma stability profiles, in vivo pharmacokinetics properties, and requirement of MTD, in the design of such conjugates, but also validate PS at the tumor microenvironment as a potential target for the SMDC-based cancer therapy.

Moreover, in contrast to the complications

encountered in the development of ADC, our work highlighted the integration of a ZnDPA warhead with a cytotoxic payload 1 via a simple yet plasma stable linker to achieve potent anti-tumor efficacy.

Taken together, this work should motivate the

conjugation of a wide array of therapeutic agents to ZnDPA for the treatment of cell death associated diseases.

EXPERIMENTAL SECTION 1. General. Unless otherwise stated, all materials used were commercially obtained and used as supplied. Reactions requiring anhydrous conditions were performed in flamedried glassware and cooled under an argon or nitrogen atmosphere. Unless otherwise stated, reactions were carried out under argon or nitrogen and monitored by analytical thin layer chromatography performed on glass-backed plates (5 × 10 cm) precoated with silica gel 60 F254 as supplied by Merck (Merck & Co., Inc., Whitehouse Station in Readington Township, NJ). Visualization of the resulting chromatograms was performed by looking under an ultraviolet lamp (λ = 254 nm) followed by dipping in an ethanol solution of vanillin (5% w/v) containing sulfuric acid (3% v/v) or phosphomolybdic acid


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(2.5% w/v) and charring with a heat gun. Solvents for reactions were dried and distilled under an argon or nitrogen atmosphere prior to use as follows: THF, diethyl ether (ether), and DMF from a dark blue solution of sodium benzophenone ketyl; toluene, dichromethane, and pyridine from calcium hydride. Flash chromatography was used routinely for purification and separation of product mixtures using silica gel 60 of 230−400 mesh size as supplied by Merck. Eluent systems are given in volume/volume concentrations. 1H and

13

C NMR spectra were recorded on a Varian Mercury-300 (300

MHz), a Varian Mercury-400 (400 MHz), Bruker DMX-600 (600 MHz) and a Varian Vnmr-700 (700 MHz). Chloroform-d or dimethyl sulfoxide-d6 was used as the solvent and TMS (δ 0.00 ppm) as an internal standard. Chemical shift values are reported in ppm relative to the TMS in delta (δ) units. Multiplicities are recorded as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), and m (multiplet). Coupling constants (J) are expressed in hertz. Electrospray mass spectra (ESMS) were recorded as m/z values using an Agilent 1100 MSD mass spectrometer. High resolution liquid chromatography tamden mass spectrometer by Bruker (Impact HD) was used for obtaining HRMS. All test compounds displayed more than 95% purity as determined by Agilent 1100 series HPLC system using a C18 column (Thermo Golden, 4.6 mm × 250) and the Agilent 1100 series variable wavelength detector. The gradient system for HPLC separation was composed of MeOH (mobile phase A) and H2O solution containing 0.1% trifluoro-acetic acid (mobile phase B). The starting flow rate was 0.5 mL/min and the injection volume was 10 µL. During first 2 min the percentage of phase A was 10%. At 6 min, the percentage of phase A was increased to 50%. At 16 min, the percentage of phase A was increased to 90% over 9 min. The



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system was operated at 25 oC. Peaks were detected at 254 nm. IUPAC nomenclature of compounds was determined with ACD/Name Pro software. The animal use protocol was approved by The Institutional Care and Use Committee of National Health Research Institutes (Protocol No: NHRI-IACUC-103061). 2. Chemistry. Synthetic procedures for 12b, 13b, 14b, 16, 20a, 21a, 22a, 23a, and 24: 2.1. Methyl 3-((4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)phenoxy)butyl) carbamoyl)benzoate (12b). To a stirred solution of 11 (400 mg, 0.68 mmol) was dissolved

in

CH2Cl2 (40

ml),

and

triethylamine

(2

ml)

and

methyl

3-

(chlorocarbonyl)benzoate were added at 0 oC. The mixture was stirred at 0 oC for 2 hr. After completion of reaction, the mixture was extracted with CH2Cl2 (100 ml) and saturated aqueous NH4Cl (2 x 100 ml). The combined organic layers were dried over MgSO4, concentrated under reduced pressure. The resulting residues were purified by column chromatography (MeOH: CH2Cl2 = 1: 13) to yield product (280 mg, 55%). 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 4.4 Hz, 4H, H-4, H-10, H-20, H-26, -CH=N-), 8.36 (s, 1H), 8.12 (d, J = 8 Hz, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.63-7.56 (m, 8H), 7.507.46 (m, 1H), 7.13-7.10 (m, 4H), 7.05 (s, 1H, H-35), 6.85 (s, 2H, H-33, H-37), 4.02 (t, J = 5.2 Hz, 2H, H-40, -OCH2-), 3.91 (s, 3H, CH3OCO), 3.78 (s, 8H, H-13, H-14, H-29, H30, NCH2C=N), 3.63 (s, 4H, H-16, H-32, NCH2C=C), 3.58-3.54 (m, 2H, H-43, -NCH2-), 1.91-1.84 (m, 4H, H-41, H-42, -CH2CH2CH2CH2-). 2.2. 3-((4-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)phenoxy)butyl)carbamoyl) benzoic acid (13b). To a stirred solution of 12b (280 mg, 0.37 mmol) was dissolved in MeOH (3 ml) and aqueous LiOH (3 ml, 0.5 N). The mixture was stirred at room temperature for 15 hr. After completion of reaction, the MeOH was removed under


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reduced pressure. The resulting residues were extracted with CH2Cl2 (100 ml) and saturated aqueous NH4Cl (2 x 100 ml). The combined organic layers were dried over MgSO4, concentrated under reduced pressure to yield product (240 mg, 88%). 1H NMR (300 MHz, CDCl3) δ 8.56 (d, J = 4.8 Hz, 4H, H-4, H-10, H-20, H-26, -CH=N-), 8.47 (s, 1H), 8.21 (d, J = 7.2 Hz, 2H), 7.62-7.51 (m, 9H), 7.20-7.12 (m, 7H), 7.08 (s, 2H), 4.14 (m, 2H, 2H, H-40, -OCH2CH2-), 3.80 (s, 8H, H-13, H-14, H-29, H-30, NCH2C=N), 3.64 (m, 6H), 1.97-1.86 (m, 4H, H-41, H-42, -CH2CH2CH2CH2-). ESI-MS, calcd for C44H45N7O4 735, found 736 (M+H)+, 758 (M + Na)+. 2.3.

(4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-

pyrano[3',4':6,7]indolizino

[1,2-b]quinolin-9-yl3-{[4-(3,5-bis{[bis(pyridin-2-

ylmethyl)amino]methyl}phenoxy)butyl] carbamoyl}benzoate (14b). To a stirred solution of acid 13b (100 mg, 0.14 mmol) was dissolved in DMF (10 ml), and 4,11diethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14

(4H,12H)-

dione (SN-38, 83 mg, 0.21 mmol) and hydroxybenzotriazole (HOBt, 57 mg, 0.42 mmol) and N-(3-Dimethylaminopropyl)-N´-ethylcarbodiimide hydrochloride (EDCl, 80 mg, 0.42 mmol, 3 equiv) and N-methylmorpholine (NMM, 85 mg, 0.84 mmol) were added. The mixture was stirred at room temperture for 15 hr. After completion of reaction, the mixture was extract with CH2Cl2 (300 ml) and saturated aqueous NaHCO3 (300 ml) and H2O (5 x 300 ml). The combined organic layers were dried over MgSO4, concentrated under reduced pressure. The resulting residues were purified by column chromatography (MeOH : CH2Cl2 = 1 : 13) to yield product (60 mg, 40%). 1H NMR (400 MHz, CD3OD) δ 8.69 (d, J = 4 Hz, 4H, H-4, H-10, H-20, H-26, -CH=N-), 8.39 (d, J = 7.2 Hz, 1H), 8.228.11 (m, 8H), 7.76-7.67 (m, 8H), 7.65 (s, 1H), 7.60 (d, J = 7.6 Hz, 4H), 6.81 (s, 2H, H-



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33, H-37), 6.72 (s, 1H), 5.58 (d, J = 16.4 Hz, 1H, H-70), 5.38 (d, J = 16.4 Hz, 1H, H-70), 5.32 (s, 2H, H-62), 4.34 (d, J = 16 Hz, 4H), 4.11 (m, 2H), 3.96 (d, J = 16 Hz, 4H), 3.78 (s, 4H, H-16, H-32, NCH2C=C), 3.57 (m, 2H), 3.23-3.21 (m, 2H), 1.98-1.93 (m, 6H, H41, H-42, H-75), 1.39 (t, J = 7.6 Hz, 3H, H-61), 1.02 (t, J = 7.6 Hz, 3H, H-77). ESI-MS, calcd for C66H63N9O8: 1110, found 556 (M + 2H+)2+. 2.4. (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7]indolizino[1,2-b]quinolin-9-yl 3-{[4-(3,5-bis{[bis(pyridin-2-ylmethyl)amino] methyl}phenoxy)butyl]carbamoyl}benzoate·2[Zn(NO3)2] (16). To a stirred solution of compound 14b (110.9 mg, 0.10 mmol) in 2 mL DCM was added 1 mL of Zn(NO3)2 (75.7 mg, 0.20 mmol) in MeOH at room temperature. After 1hr, the mixture was concentrated under reduced pressure. 1H NMR (500 MHz, DMSO-d6) δ 8.91 (t, J = 5.1 Hz, 1H), 8.75-8.57 (m, 4H), 8.37 (d, J = 7.9 Hz, 1H), 8.33-8.18 (m, 3H), 8.09 (t, J = 7.3 Hz, 4H), 7.84 (dd, J = 9.1, 2.5 Hz, 1H), 7.77 (t, J = 7.8 Hz, 1H), 7.71-7.49 (m, 7H), 7.36 (s, 1H), 7.03 (s, 2H), 6.89 (s, 1H), 6.53 (s, 1H), 5.45 (s, 2H), 5.36 (s, 2H), 4.36 (d, J = 15.9 Hz, 4H), 4.16 (s, 2H), 3.97-3.63 (m, 8H), 3.45 (d, J = 6.0 Hz, 2H), 3.21 (d, J = 7.6 Hz, 2H), 1.85 (dd, J = 32.0, 5.1 Hz, 6H), 1.30 (t, J = 7.6 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 172.95, 165.64, 164.85, 159.34, 157.31, 154.81, 152.63, 150.51, 149.75, 148.36, 147.17, 146.35, 145.99, 141.24, 135.72, 134.18, 133.14, 132.94, 131.90, 129.73, 129.55, 129.17, 127.65, 127.31, 126.09, 125.32, 125.10, 119.57, 118.13, 116.19, 97.19, 72.86, 67.89, 65.75, 57.44, 56.19, 50.07, 38.56, 30.79, 30.27, 28.83, 26.75, 26.39, 23.73, 22.86, 22.74, 14.36, 11.27, 11.15, 8.24. ESI-MS C66H63N9O8Zn22+: 586.7040, found: 586.6975 (M+Zn2+)2+. Purity: 95%


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2.5.

1,1'-(5-(4-(([1,1'-biphenyl]-4-ylmethyl)amino)butoxy)-1,3-phenylene)bis(N,N-

bis(pyridin-2-ylmethyl)methanamine) (20a). To a stirred solution of compound 11 (2130 mg, 3.6 mmol) in 36 mL DCM was added aldehyde (1312 mg, 7.2 mmol) at room temperature. After 1.0 hour, NaBH4 (410 mg, 10.8 mmol) in 6 mL MeOH was added to the above solution at 0 oC. And then the mixture was warmed to room temperature. After 4 h, the volatile material was removed under reduced pressure. The mixture was diluted with DCM (20 mL), washed with 1M HCl (50 mL × 3) and brine (50 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to flash column chromatography (CH2Cl2 : MeOH = 9 : 1) to give compound 20a in 80% yield (2.17 g). 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 4.0 Hz, 4H, H-4, H-10, H-20, H-26, -CH=N-), 7.67-7.45 (m, 14H), 7.39 (t, J = 8.0 Hz, 3H), 7.11 (t, J = 8.0 Hz, 4H), 7.03 (s, 1H), 6.80 (s, 2H), 3.94 (s, 4H), 3.77 (s, 8H, H-13, H-14, H-29, H-30, NCH2C=N), 3.62 (s, 4H), 3.47 (s, 1H), 2.84 (t, J = 7.3 Hz, 2H), 1.98-1.74 (m, 4H, H-41, H-42). ESIMS, calcd for C49H51N7O 753, found 776 (M + Na)+. 2.6.

Methyl

3-(3-([1,1'-biphenyl]-4-ylmethyl)-3-(4-(3,5-bis((bis(pyridin-2-

ylmethyl)amino)methyl)phenoxy)butyl)ureido)benzoate (21a). To a stirred solution of compound 20a (230 mg, 0.31mmol) in 4.7 mL DCM were added isocyanatobenzoate (115 mg, 0.465mmol) and TEA (81g, 0.62 mmol) at 0 oC. The mixture was warmed to room temperature. After 1.0 h, the mixture was diluted with DCM (10.0 mL), washed with saturated NH4Cl (5 mL x 3) and brine (5 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to flash column chromatography (CH2Cl2 : MeOH = 19 : 1) to give compound 21a in 73% yield (210.5 mg). 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 4.8 Hz, 4H, H-4, H-10, H-20, H-26, -



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CH=N-), 7.87 (d, J = 2.1 Hz, 1H), 7.67-7.51 (m, 14H), 7.46-7.33 (m, 5H), 7.10 (t, J = 6.8, 4.8, 1.5 Hz, 4H), 7.06 (s, 1H), 6.86 (d, J = 1.4 Hz, 2H), 6.79 (s, 1H), 4.65 (s, 2H, H85), 4.04 (s, 2H, H-40), 3.82 (s, 3H, CH3OCO), 3.78 (s, 8H, H-13, H-14, H-29, H-30, NCH2C=N), 3.63 (s, 4H, H-16, H-32, NCH2C=C), 3.54 (br, 2H, H-43, -NCH2-), 1.901.83 (m, 5H). ESI-MS, calcd for C58H58N8O4: 930, found 931 (M + H)+, 953 (M + Na)+. 2.7.

3-(3-([1,1'-biphenyl]-4-ylmethyl)-3-(4-(3,5-bis((bis(pyridin-2-

ylmethyl)amino)methyl)phenoxy)butyl)ureido)benzoic acid (22a). To a stirred solution of urea 21a (213 mg, 0.23 mmol) in 10 mL MeOH were added 0.5N LiOH (3.5 ml) at 0 oC. The mixture was heated to 35 oC. After 3.0 h, the mixture was diluted with DCM (10.0 mL), washed with saturated NH4Cl (5 mL x 3) and brine (5 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to flash column chromatography (CH2Cl2 : MeOH = 5 : 1) to give compound 22a in 98% yield (206.5 mg). 1H NMR (300 MHz, CDCl3) δ 8.51 (d, J = 4.9 Hz, 4H), 8.08 (d, J = 8.1 Hz, 1H), 7.70-7.63 (m, 2H), 7.63-7.47 (m, 13H), 7.46-7.38 (m, 4H), 7.38-7.28 (m, 3H), 7.11 (dt, J = 7.0, 3.2 Hz, 4H), 6.96 (s, 2H), 6.92 (s, 1H), 4.67 (s, 2H), 4.10 (brs, 2H), 3.79 (s, 8H), 3.61 (s, 4H), 3.51 (d, J = 7.4 Hz, 2H), 1.90-1.86 (m, 4H). ESI-MS, calcd for C57H56N8O4: 916, found 917 (M + H)+, 939 (M + Na)+. 2.8.

(4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H


[1,2-b]quinolin-9-yl

3-({(biphenyl-4-ylmethyl)[4-(3,5-

bis{[bis(pyridin-2-ylmethyl)amino]methyl} phenoxy)butyl]carbamoyl}amino)benzoate (23a). To a stirred solution of compound 22a (204 mg, 0.22 mmol) and EDCl (253 mg, 1.32 mmol), HOBt (178 mg, 1.32 mmol) in 7.3 mL DMF was added N-Methylmorpholine (267 L, 1.32 mmol) at 0 oC. After 10


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min, SN-38 (130 mg, 0.33 mmol) in 2 mL DMF was added to the above solution and then the mixture was warmed to room temperature. After 4 h, the mixture was diluted with DCM (10 mL), washed with saturated NaHCO3 (50 mL × 3) and brine (5.0 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was subjected to flash column chromatography (CH2Cl2 : MeOH = 15 : 1) to give compound 23a in 48.0% yield (136.2 mg). 1H NMR (300 MHz, CDCl3) δ 8.49 (dt, J = 4.9, 1.3 Hz, 4H, H-4, H-10, H-20, H-26, -CH=N-), 8.25-8.16 (m, 2H, H-51, H-82), 7.89-7.81 (m, 2H), 7.68-7.50 (m, 13H), 7.45-7.30 (m, 6H), 7.16-7.02 (m, 6H), 6.86 (s, 2H, H-33, H-37), 5.72 (d, J = 16.4 Hz, 1H, H-70a), 5.35-5.18 (m, 3H, , H-62, H-70b), 4.69 (s, 2H, H-85), 4.08 (d, J = 17.7 Hz, 2H, H-40), 3.77 (s, 8H, H-13, H-14, H-29, H-30), 3.62 (s, 4H, H-16, H32), 3.58-3.55 (m, 2H, H-43), 3.10 (q, J = 7.6 Hz, 2H, H-60), 2.19 (brs, 1H), 1.99-1.80 (m, 6H, H-41, H-42, H-75), 1.36 (t, J = 7.6 Hz, 3H, H-61), 1.03 (t, J = 7.3 Hz, 3H, H-77). ESI-MS, calcd for C79H74N10O8: 1290, found 646 (M + 2H+)2+. 2.9. (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7]indolizino[1,2-b]quinolin-9-yl

3-({(biphenyl-4-ylmethyl)[4-(3,5-

bis{[bis(pyridin-2 ylmethyl)amino]methyl}phenoxy)butyl]carbamoyl}amino)benzoate·2[Zn(NO3)2] (24). To a stirred solution of compound 23a (150 mg, 0.116 mmol) in 2 mL DCM was added 1 mL of Zn(NO3)2 (87.9 mg, 0.232 mmol) in MeOH at room temperature. After 1hr, the mixture was concentrated under reduced pressure. 1H NMR (700 MHz, DMSOd6): δ 8.84 (br, 1H), 8.63 (d, J = 5.6 Hz, 4H, H-4, H-10, H-20, H-26, -CH=N-), 8.42 (s, 1H), 8.20 (d, J = 5.6 Hz, 1H), 8.03 (t, J = 7.7 Hz, 4H), 7.93 (d, J = 9.1 Hz, 1H), 7.76 (t, J = 7.7 Hz, 2H), 7.61 (d, J = 7.7 Hz, 2H), 7.58-7.62 (m, 6H), 7.52 (d, J = 7.7 Hz, 4H), 7.47



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(t, J = 7.7 Hz, 2H), 7.40 (t, J = 7.7 Hz, 4H), 7.37 (d, J = 7.7 Hz, 2H), 7.31-7.29 (m, 1H), 6.98 (s, 2H, H-33, H-37), 6.86 (s, 1H, H-35), 6.52 (s, 1H), 5.41 (s, 2H, H-70), 5.32 (s, 2H, H-62), 4.73 (s, 2H, H-85), 4.32 (d, J = 16.1 Hz, 4H, H-13, , H-14), 4.10 (br, 1H, H-40), 3.81 (br, 4H, H-16, , H-32), 3.73 (d, J = 16.1 Hz, 4H, H-29, , H-30), 3.48 (s, 2H, H-43), 3.16 (q, J = 7.7 Hz, 2H, H-60), 1.88-1.79 (m, 6H, H-41, , H-42), 1.25 (t, J = 7.7 Hz, 3H, H-61), 0.86 (t, J = 7.0 Hz, 3H, H-77). 13C NMR (176 MHz, DMSO-d6): δ 172.50 (C-72, C=O, 4o), 164.90 (C-48, C=O, 4o), 158.86 (C-65, 4o), 156.84 (C-38, 4o), 155.25 (C-45, 4o), 154.30, 152.08 (C-53, 4o), 150.05, 149.37, 147.87 (3o), 146.64, 145.89, 145.50, 141.28, 140.73 (3o), 139.79, 138.93, 137.84, 133.70, 131.38, 129.17, 129.15, 129.02, 128.95, 128.84, 128.66, 127.65, 127.42, 127.19, 126.82, 126.55, 126.36, 125.72, 125.18, 124.83, 124.60, 124.43, 124.42, 124.33, 123.30, 120.93 (3o), 119.07, 117.64 (3o), 115.68 (3o), 96.72 (C-68, 3o), 72.41 (C-73, 4o), 67.60 (C-70, 2o), 65.28 (C-16, C-32, 2o), 56.94 (C-40, 2o), 55.70 (C-13, C-14, C-29, C-30, 2o), 49.60 (C-62, 2o), 49.06 (C-85, 2o), 46.16 (C-43, 2o), 30.30 (C-75, 2o), 26.17 (C-42, 2o), 24.59 (C-41, 2o), 22.26 (C-60, 2o), 13.91 (C-61, 1o), 7.79 (C-77, 1o). ESI-MS C79H72N10O8Zn22+: 709.2038, found: 709.1964 (M+2Zn2+-2H+)2+. Purity: 95% 2.10.

(4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-


[1,2-b]quinolin-9-yl

7-(3,5-bis{[bis(pyridin-2-

ylmethyl)amino]methyl}phenoxy)heptanoate·2[Zn(NO3)2] (10).

1

H NMR (700 MHz,

DMSO-d6): δ 8.63 (s, 4H), 8.13 (d, J = 7.0 Hz, 1H), 8.06 (t, J = 7.7 Hz, 4H), 7.96 (s, 1H), 7.61-7.52 (m, 8H), 7.28 (s, 1H), 7.01 (s, 2H), 6.86 (s, 1H), 6.50 (s, 1H), 5.40 (s, 2H), 5.30 (s, 2H), 4.33 (d, J = 15.4 Hz, 2H), 4.01-3.67 (m, 10H), 3.15-3.11 (m, 2H), 3.06-3.04 (m, 2H), 2.71 (t, J = 7.0 Hz, 2H), 1.86-1.50 (m, 11H), 1.25 (t, J = 7.7 Hz, 3H), 0.85 (t, J = 7.7


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Hz, 3H).

13

C NMR (176 MHz, DMSO-d6) δ172.49 (C=O), 156.88, 156.82, 156.75,

154.31, 151.98, 150.02, 149.20, 148.87, 147.86, 146.49, 145.87, 145.29, 143.66, 142.79, 140.74, 131.28, 128.61, 127.10, 125.65, 124.84, 124.60, 119.05, 115.29, 96.64 (3o), 72.39 (C-O, 4o), 65.27 (2o), 55.75 (2o ), 49.58 (2o), 30.30 (2o), 28.29 (2o), 25.41 (2o), 24.26 (2o), 22.25 (2o), 13.84 (1o), 7.78 (1o). ESI-MS C61H62N8O7Zn2+: 541.2011, found: 541.1942 (M+Zn2+)2+. Purity: 98% 2.11. (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7]indolizino[1,2-b]quinolin-9-yl 4-{[4-(3,5-bis{[bis(pyridin-2-ylmethyl)amino] methyl}phenoxy)butyl]amino}-4-oxobutanoate·2[Zn(NO3)2] (15). 1H NMR (700 MHz, DMSO-d6): δ 8.63 (s, 4H), 8.02 (q, J = 7.7, 16.1 Hz, 4H), 7.89 (s, 1H), 7.59-7.34 (m, 8H), 7.29 (s, 1H), 6.88 (br, 2H), 6.82 (s, 1H), 6.80 (br, 1H), 5.40-5.38 (m, 1H), 5.32 (t, J = 16.8 Hz, 1H), 5.23 (s, 1H), 5.18 (s, 1H), 4.29-4.25 (m, 2H), 4.01-3.64 (m, 8H), 3.43 (t, J = 7.0 Hz, 2H), 3.18 (t, J = 7.0 Hz, 2H), 3.15-3.11 (m, 2H), 3.08 (br, 2H), 3.02 (br, 2H), 2.85 (t, J = 7.0 Hz, 2H), 2.54 (t, J = 7.0 Hz, 2H), 1.85-1.57 (m, 7H), 1.23 (t, J = 7.7 Hz, 3H), 0.84 (t, J = 7.0 Hz, 3H). 13C NMR (176 MHz, DMSO-d6) δ178.21 (C=O, 4o), 172.91 (C=O, 4o), 161.58, 159.20, 157.33, 157.24, 154.75, 152.19, 150.53, 150.42, 149.67, 148.24, 146.86, 146.81, 146.23, 145.58, 141.08, 134.01, 131.72, 128.66, 128.13, 127.39, 126.82, 125.74, 125.07, 124.87, 117.84, 115.31, 104.98, 97.10(3o), 72.71(C-O, 4o), 65.50, 55.93, 49.60, 30.57 (2o), 29.05 (2o), 28.20 (2o), 26.34 (2o), 22.55 (2o), 13.32 (1o), 7.75 (1o). ESI-MS C62H63N9O8Zn2+: 562.7040, found: 562 (M+Zn2+)2+. Purity: 96% 2.12. (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7]indolizino[1,2-b]quinolin-9-yl 4-({[4-(3,5-bis{[bis(pyridin-2-ylmethyl)amino]



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methyl}phenoxy)butyl]amino}methyl)benzoate·2[Zn(NO3)2] (17). 1H NMR (600 MHz, DMSO-d6) δ 8.67 (d, J = 4.6 Hz, 3H), 8.25-8.18 (m, 4H), 8.09 (t, J = 7.5 Hz, 4H), 7.79 (d, J = 8.6 Hz, 1H), 7.64 (t, J = 7.2 Hz, 4H), 7.57 (d, J = 7.2 Hz, 5H), 7.51 (d, J = 8.0 Hz, 2H), 7.34 (s, 1H), 7.02 (s, 2H), 6.91 (s, 1H), 6.54 (s, 1H), 5.43 (s, 2H), 5.34 (s, 2H), 4.57 (s, 2H), 4.36 (d, J = 15.9 Hz, 4H), 4.11 (s, 2H), 3.93-3.73 (m, 8H), 3.18 (d, J = 7.3 Hz, 2H), 1.87-1.75 (m, 7H), 1.28 (t, J = 7.6 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 172.47, 164.55, 158.85, 156.83, 154.33, 152.07, 150.03, 149.33, 147.90, 146.62, 145.89, 145.46, 140.76, 133.73, 131.34, 130.24, 128.63, 127.47, 127.28, 127.15, 126.83, 125.66, 124.85, 124.61, 119.04, 117.63, 115.60, 96.69, 79.10, 77.93, 72.38, 69.08, 67.44, 65.27, 56.95, 55.72, 49.57, 46.72, 39.92, 39.78, 39.64, 39.50, 39.36, 39.22, 39.08, 30.31, 28.02, 26.14, 24.54, 22.25, 13.87, 7.76. ESI-MS C66H65N9O7Zn2+: 580.2160, found: 580 (M+Zn2+)2+. Purity: 95% 2.13. (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7]indolizino[1,2-b]quinolin-9-yl 4-{[4-(3,5-bis{[bis(pyridin-2-ylmethyl)amino] methyl}phenoxy)butyl]sulfamoyl}benzoate·2[Zn(NO3)2] (18). 1H NMR (700 MHz, DMSO-d6): δ 8.64 (s, 4H), 8.40 (s, 1H), 8.39 (s, 1H), 8.24 (br, 1H), 8.22 (s, 2H), 8.077.53 (m, 16H), 6.99 (s, 2H), 6.88 (s, 1H), 6.52 (s, 1H), 5.41 (s, 2H), 5.32 (s, 2H), 4.33 (d, J = 2.1 Hz, 2H) , 4.07-3.74 (m, 12H), 3.17-3.16 (m, 2H), 2.91-2.83 (m, 2H), 1.88-1.59 (m, 6H), 1.26 (t, J = 7.7 Hz, 3H), 0.85 (t, J = 7.7 Hz, 3H). 13C NMR (176 MHz, DMSO-d6) δ172.49, 163.76, 158.80, 156.83, 154.31, 152.20, 150.04, 149.15, 147.86, 146.71, 145.86, 145.52, 145.31, 140.74, 133.73, 132.17, 131.43, 130.98, 128.71, 127.16, 127.03, 126.87, 126.68, 125.49, 124.85, 124.61, 119.11, 117.64, 115.72, 96.74, 72.40, 67.25, 65.28, 55.75,


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49.61, 30.30, 26.07, 25.94, 22.30, 13.90, 7.78. ESI-MS C65H61N9O9SZn22+: 636.6427, found: 636.6375 (M+2Zn2+-2H+)2+. Purity: 97%. 2.14. (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7]indolizino[1,2-b]quinolin-9-yl 3-({[4-(3,5-bis{[bis(pyridin-2-ylmethyl)amino] methyl}phenoxy)butyl]carbamoyl}amino)benzoate·2[Zn(NO3)2] (19). 1H NMR (700 MHz, DMSO-d6) : δ 8.95 (s, 1H), 8.63 (d, J = 5.6 Hz, 3H), 8.37 (s, 1H), 8.21 (d, J = 9.1 Hz, 1H), 8.14 (s, 1H), 8.05 (t, J = 7.7 Hz, 4H), 7.65 (dd, J = 9.1, 2.1 Hz, 2H), 7.71 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.0 Hz, 4H), 7.54 (d, J = 7.7 Hz, 4H), 7.44 (t, J = 7.7 Hz, 1H), 7.02 (s, 1H), 6.99 (s, 2H), 6.86 (s, 1H), 6.51 (s, 1H), 6.48 (br, 1H), 5.41 (s, 2H), 5.31 (s, 2H), 4.33 (d, J = 16.1 Hz, 4H), 4.11 (br, 2H), 3.84 (s, 4H), 3.75 (d, J = 16.1 Hz, 4H), 3.20 (q, J = 7.7 Hz, 2H), 3.16 (q, J = 7.7 Hz, 2H), 1.89-1.79 (m, 4H), 1.66(p, J = 7.7 Hz, 2H), 1.25 (t, J = 7.7 Hz, 3H), 0.86 (t, J = 7.0 Hz, 3H). 13C NMR (176 MHz, DMSO-d6)：δ 172.50, 164.87, 158.87, 156.84, 155.28, 154.32, 152.05, 150.05, 149.38, 147.86, 146.62, 145.90, 145.52, 141.28, 140.74, 133.72, 131.35, 129.33, 129.13, 128.63, 127.18, 125.73, 124.82, 124.62, 122.95, 122.48, 119.0, 118.61, 117.64, 115.66, 96.72, 72.41, 67.49, 65.29, 56.93, 55.72, 49.60, 30.31, 26.65, 26.19, 22.27, 13.91, 7.79. ESI-MS C66H62N10O8Zn22+: 626.1646, found: 626.1571 (M+2Zn2+-2H+)2+. Purity: 95% 2.15. (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7]indolizino[1,2-b]quinolin-9-yl

4-({(biphenyl-4-ylcarbonyl)[4-(3,5-

bis{[bis(pyridin-2ylmethyl)amino]methyl}phenoxy)butyl]amino}methyl)benzoate·2[Zn(NO3)2] 1

(25).

H NMR (700 MHz, DMSO-d6): δ 8.64 (d, J = 5.6 Hz, 4H), 8.21-8.18 (m, 3H), 8.15 (s,

1H), 8.05 (t, J = 7.7 Hz, 4H), 7.75 (d, J = 9.1 Hz, 1H), 7.68 (d, J = 7.7 Hz, 2H), 7.64-7.30



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(m, 17H), 7.02 (s, 1H), 6.93 (s, 1H), 6.88-6.54 (m, 2H), 6.51 (s, 1H), 5.40 (s, 2H), 5.30 (s, 2H), 4.88 (s, 1H), 4.73 (s, 1H), 4.38-4.30 (m, 4H), 4.16 (br, 1H), 3.94 (br, 1H), 3.83-3.69 (m, 8H), 3.53 (s, 1H), 3.15 (q, J = 7.7 Hz, 3H), 1.88-1.74 (m, 6H), 1.25 (t, J = 7.7 Hz, 3H), 0.85 (t, J = 7.0 Hz, 3H). 13C NMR (176 MHz, DMSO-d6) δ 172.49, 156.82, 154.31, 152.04, 150.04, 149.31, 147.90, 146.60, 145.88, 145.46, 140.76, 139.10, 135.34, 133.71, 131.35, 130.48, 130.35, 129.04, 128.60, 127.96, 127.30, 127.14, 126.70, 125.65, 124.85, 124.61, 119.03, 117.58, 115.61, 96.72, 72.40, 65.28, 56.89, 55.68, 49.58, 30.31, 22.28, 13.88, 7.79. ESI-MS C79H73N8O8Zn2+: 669.7431, found: 669.7321 (M+Zn2+)2+. Purity: 96% 2.16. (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7]indolizino[1,2-b]quinolin-9-yl [4-(3,5-bis{[bis(pyridin-2-ylmethyl)amino] methyl}phenoxy)butyl]carbamate·2[Zn(NO3)2] (27). 1H NMR (700 MHz, DMSO-d6): δ 8.64 (s, 4H), 8.13 (d, J = 7.0 Hz, 1H), 8.06 (t, J = 7.0 Hz, 4H), 7.91 (s, 1H), 7.61-7.54 (m, 8H), 7.28 (s, 1H), 7.00 (s, 2H), 6.88 (s, 1H), 6.50 (s, 1H), 5.41 (s, 2H), 5.29 (s, 2H), 4.33 (br, 4H), 4.13 (br, 2H), 3.83-3.75 (m, 8H), 3.24-3.23 (m, 2H), 3.14-3.13 (m, 2H), 1.88-1.73 (m, 6H), 1.24 (t, J = 7.7 Hz, 3H), 0.85 (t, J = 7.7 Hz, 3H). 13C NMR (176 MHz, DMSO-d6) δ172.50, 158.86, 156.83, 154.30, 151.60, 150.04, 149.84, 147.89, 146.11, 145.96, 145.07, 140.75, 133.73, 131.04, 128.47, 127.10, 125.85, 124.83, 124.62, 118.91, 117.64, 114.61, 96.56, 72.40, 67.38, 65.28, 56.94, 55.72, 49.58, 30.29, 26.12, 26.09, 22.25, 13.80, 7.79. ESI-MS C59H59N9O7Zn2+: 534.6909, found: 534.6835 (M+Zn2+)2+. Purity: 95% 2.17. (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano


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[3',4':6,7]indolizino[1,2-b]quinolin-9-yl

(biphenyl-4-ylmethyl)[4-(3,5-

bis{[bis(pyridin-2-ylmethyl)amino]methyl}phenoxy)butyl]carbamate·2[Zn(NO3)2] (28). 1H NMR (700 MHz, DMSO-d6): δ 8.63 (d, J = 4.9 Hz, 4H), 8.13 (t, J = 7.7 Hz, 1H), 8.03 (t, J = 7.7 Hz, 3H), 7.72 (d, J = 7.7 Hz, 1H), 7.72-7.26 (m, 20H), 6.99 (s, 2H), 6.86 (s, 1H), 6.51 (s, 1H), 5.41 (s, 2H), 5.29 (s, 2H), 4.82 (s, 1H), 4.65 (s, 1H), 4.31 (t, J = 14.7 Hz, 4H), 4.14-4.12 (m, 2H), 3.81 (s, 4H), 3.71 (t, J = 17.5 Hz, 4H), 3.60 (br, 1H), 3.50 (br, 1H), 3.15-3.07 (m, 2H), 1.84-1.82 (m, 6H), 1.24 (t, J = 7.7 Hz, 1H), 1.19 (t, J = 7.0 Hz, 2H), 0.85 (t, J = 7.0 Hz, 3H). 13C NMR (176 MHz, DMSO-d6) δ 172.52, 158.86, 156.82, 154.28, 153.96, 151.76, 150.01, 149.88, 147.89, 146.26, 145.89, 145.21, 145.10, 140.73, 139.76, 139.70, 139.30, 139.20, 137.30, 136.87, 133.70, 131.08, 128.99, 128.54, 128.31, 127.85, 127.51, 127.01, 126.95, 126.92, 126.58, 125.87, 124.84, 124.60, 118.98, 117.64, 114.98, 114.79, 96.60, 72.40, 67.38, 65.28, 56.89, 55.67, 50.12, 49.58, 30.30, 26.18, 26.04, 24.81, 24.09, 22.23, 13.85, 13.77, 7.78. ESI-MS C72H69N9O7Zn2+: 617.7300, found: 617.7207 (M+Zn2+)2+. Purity: 97% 2.18. (4S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7]indolizino[1,2-b]quinolin-9-yl 4-{[4-(3,5-bis{[bis(pyridin-2-ylmethyl)amino] methyl}phenoxy)butyl]carbamoyl}piperidine-1-carboxylate·2[Zn(NO3)2]

(31).

1

H

NMR (700 MHz, DMSO-d6): δ 8.64 (s, 4H), 8.11-8.05 (m, 4H), 7.98 (br, 1H), 7.92 (s, 1H), 7.62-7.55 (m, 8H), 7.28 (s, 1H), 6.97 (s, 2H), 6.86 (s, 1H), 6.52 (br, 1H), 6.51 (s, 1H), 5.39 (s, 2H), 5.27 (s, 2H), 4.33 (br, 4H), 4.24 (br, 1H), 4.08-4.02 (m, 4H), 3.82 (s, 4H), 3.77 (d, J = 2.1 Hz, 4H), 3.16-3.12 (m, 5H), 2.92 (br, 2H), 2.41 (br, 2H), 1.87-1.53 (m, 10H), 1.24 (t, J = 7.7 Hz, 3H), 0.84 (t, J = 7.7 Hz, 3H). 13C NMR (176 MHz, DMSOd6)：δ173.88, 172.49, 158.86, 156.82, 154.34, 152.70, 151.63, 150.03, 149.94, 147.90,



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146.22, 145.93, 145.18, 140.76, 133.72, 130.94, 128.42, 128.03, 127.03, 126.80, 126.41, 125.96, 124.84, 124.63, 118.89, 117.62, 114.93, 96.60, 72.40, 67.39, 65.27, 56.89, 55.71, 49.54, 44.05, 43.57, 41.46, 38.10, 30.30, 28.50, 28.17, 26.15, 25.97, 22.23, 13.85, 7.78. ESI-MS C65H68N10O8Zn2+: 590.2251, found: 590.2165 (M+Zn2+)2+. Purity: 95% 3. Cell culture viability assay and data analysis. COLO 205 cells were grown in RPMI 1640 (Roswell Park Memorial Institute) medium (RPMI; Gibco). Detroit 551 cells were grown in Dulbecco's modified Eagle's medium (DMEM). Detroit 551 media were supplemented with 10% fetal bovine serum (FBS; Gibco), 50 U/mL of penicillin and streptomycin, and 1% Nonessential amino acids (NEAA; Gibco). COLO 205 media were supplemented with 10% fetal bovine serum (FBS; Gibco). Cell viability was examined by the MTS assay (Promega, Madison, WI, USA). In brief, cells were grown (2500~3000 cells/well) in flat bottomed 96-well plates for 24 h. The medium was replaced with that containing serial diluted compound and the cells were further incubated for 72 h. At the end of the 72 h incubation period with the test chemicals, the culture medium were removed and added 100 µl including MTS and PMS mixture solution. The cells were incubated for 1.5 h at 37 °C in a humidified incubator with 5% CO2 to allow viable cells to convert the tetrazolium salt into formazan. The conversion to formazan was determined by measuring the absorbance at 490 nm using a BioTek PowerWave-X Absorbance Microplate Reader. For MTS assays, the measured data were normalized using DMSO-treated controls (100% viability) and background controls (0% viability) to verify growth inhibition. The IC50 value was defined as the amount of compound that induced a 50% reduction in cell viability in comparison with DMSO-treated controls and was calculated using GraphPad Prism version 4 software (San Diego, CA, USA).


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4. Liposome preparation. Phospholipids ， 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS) ， were purchased from Avanti Polar Lipids (Alabaster, AL) as chloroform solutions. These stock solutions were admixed to the desired ratios according to the experimental design. A 0.4-mL aliquot of lipid solution at concentration of 10 mg/mL was transferred to a round bottom flask and the solvent was evaporated under a N2 gas stream to obtain a thin lipid film. Lipid films were subsequently hydrated for at least 1 h at room temperature in PBS buffer. Lipid suspensions were extruded through a 100-nm polycarbonate filter using an Avanti MiniExtruder (Avanti Polar Lipids, Alabaster, AL) according to the manufacturer’s instructions. The lipid suspension was passed though the membrane 21 times to yield a clear solution of uniformly sized liposomes. Particle size distributions and zeta potential (ZP) of liposome were further measured by dynamic light scattering (DLS) and microelectrophoresis using Zetasizer Nano ZS instrument (Malvern Instruments, Worcestershire, UK). 5. Biacore SPR binding assay. The binding kinetics between ZnDPADCs and the liposome were performed at 25 °C using a Biacore T200 biosensor equipped with an L1 sensor chip (GE Healthcare). Running buffer was 5% DMSO in phosphate-buffered saline (PBS) with final pH 7.4. Two consecutive 30-s pulses of 2:3 v/v 50 mM HCl / isopropanol were applied at a flow rate of 30 µL/min to precondition new sensor chips. For each binding cycle a fresh liposome capture was made. Liposomes were diluted in PBS buffer to 0.5-1 mM and captured to saturation (30-150 sec) across isolated flow cells at 2-5 µL/min. Drugs were diluted with running buffer and injected over lipid surfaces in a single injection. Association and dissociation phases were monitored for 60s at 30



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µL/min. At the end of each binding cycle the surface was regenerated to the original matrix by injecting 2:3 v/v 50 mM HCl / isopropanol and equilibrated with running buffer before the next injection of test compound. Unspecific binding was removed by subtracting SPR signals from a reference flow cell (DOPC immobilized surface) and additional blank injections. Sensograms were fit (1:1 or two-state) with BIAcore T200 evaluation software 3.0 using 1:1 complex analyte model. 6. In vivo maximum tolerated dose determination studies. The acute toxicity studies were conducted with ICR male mice. In general, the studies were conducted using the dose of 10-50 mpk for dosing 5 consecutive days; survival and body weight was observed for 14 days after 1st dosing. 7. Pharmacokinietic studies of 24. Male ICR mice were purchased from the Biolasco, Taiwan. Six-week-old ICR mice were divided into groups of three and were given 100 µL of dosing solution (2 mg/mL) intravenously. At 0.003, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after dosing, the animals were sacrificed and the blood sample was drawn from each animal via cardiac puncture and stored on ice (0-4 oC). Plasma was separated from the blood by centrifugation (3000 rpm for 15 min at 4 oC in a Beckman Model Allegra 6R centrifuge) and stored in frozen (-20 oC). The plasma samples were analyzed by liquid chromatography with tandem mass spectrometry (LC/MS/MS). The chromatographic system consisted of an Agilent 1200 series LC system and an Agilent ZORBAX Eclipse XDB-C8 column (5 µm, 3.0 × 150 mm) interfaced to an MDS Sciex API4000 tandem mass spectrometer, equipped with an ESI in the positive scanning mode at 600 oC. Data acquisition was via multiple reactions monitoring (MRM). A gradient system was employed for the separation of analyte and IS. Mobile phase A was 10 mM ammonium


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acetate aqueous solution containing 0.1% formic acid. Mobile phase B was acetonitrile. The gradient profile was as follows: 0.0-1.1 min, 50%B; 1.2-3.7 min, 55%B-90%B; 3.85.0 min, 90%B-50%B.

The flow rate was 1.5 mL/min.

The autosampler was

programmed to inject 15 µL of sample every 5 min. 8. Materials for the in vivo studies. Sources of the mice, cell lines and materials used for the in vivo anti-tumor studies were as follows: 8 weeks old athymic NU-Fox1nu mice, male (BioLASCO, Ilan, Taiwan), CPT-11 (2) (Herocan®, Lot. 1B3130, Nang Kuang Pharmaceutical Co., Ltd., Tainan, Taiwan), dimethyl sulfoxide, DMSO (D1435, SigmaAldrich, St. Louis, MO, USA), cremophor EL (C5135, Sigma-Aldrich, St. Louis, MO, USA), dextrose Injection 5% (Tai Yu Chemical & Pharmaceutical, Hsinchu, Taiwan), sodium Chloride 0.9% inj., saline (Tai Yu Chemical & Pharmaceutical, Hsinchu, Taiwan), Matrigel™ (356237, BD Biosciences, San Jose CA, USA), RPMI 1640 Medium (31800-022, Thermo Fisher Scientific Inc., Waltham, MA, USA), RPMI 1640 Medium, no phenol red (11835-030, Thermo Fisher Scientific Inc., Waltham, MA, USA), fetal Bovine Serum (04-001-1A-US, Biological Industries, Beit Haemek, Israel), 1 mL Syringe (Terumo Medical Corp., Laguna, Philippines), needle 24G × 1/2 inch (Terumo Medical Corp., Yamanashi Prefecture, Japan), needle 27G × 1/2 inch (Terumo Medical Corp., Yamanashi Prefecture, Japan), digital caliper (FOW54-200-777, PRO-MAX, Newton, Massachusetts, USA), animal holder (mouse, Yeong Jyi Chemical Apparatus Co., Ltd., Sanchong, Taiwan), Attane Isoflurane (Panion & BF Biotech Inc., Taipei, Taiwan), D-Luciferin potassium salt (PerkinElmer, Waltham, MA, USA), IVIS spectrum system (Caliper Life Sciences, Inc., Hopkinton, MA, USA ), 100% CO2 gas (Sinda Gases, Hsinchu, Taiwan).



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9. Preparation and inoculation of tumor cells. COLO 205 cells were cultured and maintained in flasks with RPMI-1640 Media. The culture medium was supplemented with 10% fetal bovine serum. The cells were incubated at 37• in a humidified atmosphere containing 5% CO2. On the day of tumor cell inoculation, the number of viable cells was counted by using a hemocytometer with trypan blue staining under a light microscope. Cells were suspended with phenol red free RPMI-1640 and matrigel in 1:1 ratio. The COLO 205 suspension cells (1×106 cells) were implanted subcutaneously into the left flank of the nude mice using a 1 mL syringe (needle 24G × 1 in., 0.55×25 mm; TERUMO). Tumor-bearing mice were randomized (n=7-8 per group) when the mean tumor volume was approximately at 170 mm3. Tumor dimensions were measured with a digital caliper, and the tumor volume in mm3 was calculated by the formula: Volume = (length × width ^2)/2. 10. Treatment of animals with 24. The mice were divided into 5 groups of 7-8 animals each, and the treatment was initiated. The reference drugs CPT-11 (2) was intravenously (i.v.) administered at various doses and frequencies were as follows: CPT-11 (2), 40 mg/kg, twice/wk for two weeks; SN-38 (10 mg/kg, 10% DMSO/20% Cremophor/10% Na2CO3/60% dextrose) was i.v. administered at once daily for five consecutive days for two weeks. Various regimens of test compound 24 (10% DMSO/20% Cremophor/70% dextrose) were i.v. administered to nude mice as follows: 24, 40 mg/kg, twice/wk for one weeks then once/week; 24, 20 mg/kg, qd(5+5); 24, 10 mg/kg, qd(5+5). Body weight and tumor size were measured twice weekly. 11.

IVIS

imaging

of

PS

associated

tumor

with

PSVue®794.

The COLO 205 suspension cells (1×106 cells) were implanted subcutaneously into the


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left flank of the nude mice (7-week old athymic NU-Fox1nu mice) using a 1 mL syringe (needle 24G × 1 in., 0.55×25 mm; TERUMO). The mice were imaged by using an IVIS spectrum system at 24 hr after the intravenous injection of PSVue®794 (2 mg/kg) on Day 12 after tumor cell inoculation. PSVue®794 as a near-infrared fluorescent probe for detection of PS-exposed tumor and apoptotic cells was prepared according to the manufacturer’s instructions. Briefly, apo-PSS-794 was dissolved in Diluent X solution and then mixed with an equal volume of 4.2 mM zinc nitrate solution. Finally, a clear solution of 1 mM PSVue®794 was obtained. The solution was kept in a water bath at 37°C to 40°C before use. The mice were anesthetized by 2.5% isoflurane inhalation and placed on stage of IVIS apparatus in the right lateral decubitus position. Imaging conditions were set as follows: excitation filter, 745 nm; emission filter, 820 nm; exposure time, 1 s; bin, 8 (medium); f/stop, 2; field of view, 22.7 cm. The fluorescence image processing was done by using Living Image Software 4.0 (PerkinElmer, Alameda, CA, USA).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS publication website. Synthetic procedures, analytical characterization data for key conjugates, copies of NMR spectra, and Molecular formula strings are included.

AUTHOR INFORMATION Corresponding Authors



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* Telephone: +886-37-246-166 ext. 35769. Fax: + 886-37-586-456. E-mail: [email protected] Author Contributions ♯

These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We are grateful to the National Health Research Institutes and Ministry of Economic Affairs of the Republic of China (MOEA 103-EC-17-A-22-1099) for financial support.

ABBREVIATIONS USED ZnDPA, zinc dipicolylamine; ADC, antibody drug conjugates; SMDC, small molecule drug conjugates; PS, phosphatidylserine.

REFERENCES (1) Srinivasarao, M., Galliford, C. V., and Low, P. S. (2015) Principles in the Design of Ligand-Targeted Cancer Therapeutics and Imaging Agents. Nat Rev Drug Discovery 14, 203-219. (2) Allen, T. M., and Cullis, P. R. (2004) Drug Delivery Systems: Entering the Mainstream. Science 303, 1818-1822. (3) Perini, G. F., and Pro, B. (2013) Brentuximab Vedotin in CD30+ Lymphomas. Biol Ther. 3, 15-23.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(4) Haddley, K. (2013) Trastuzumab Emtansine for the Treatment of HER2-positive Metastatic Breast Cancer. Drugs Today (Barc) 49, 701-715. (5) Deonarain, M. P., Yahioglu, G., Stamati, I., and Marklew, J. (2015) Emerging Formats for Next-Generation Antibody Drug Conjugates. Expert Opin. Drug Discovery 10, 463-481. (6) Casi, G., and Neri, D. (2015) Antibody-Drug Conjugates and Small MoleculeDrug Conjugates: Opportunities and Challenges for the Development of Selective Anticancer Cytotoxic Agents. J. Med. Chem. 58, 8751-8761. (7) Jeannin, P., Delneste, Y., Buisine, E., Le Mao, J., Didierlaurent, A., Stewart, G. A., Tartar, A., Tonnel, A. B., and Pestel, J. (1993) Immunogenicity and Antigenicity of Synthetic Peptides Derived from the Mite Allergen Der p I. Mol. Immunol. 30, 15111518. (8) Kurzrock, R., Gabrail, N., Chandhasin, C., Moulder, S., Smith, C., Brenner, A., Sankhala, K., Mita, A., Elian, K., Bouchard, D., et al. (2012) Pharmacokinetics, and Activity of GRN1005, a Novel Conjugate of Angiopep-2, a Peptide Facilitating Brain Penetration, and Paclitaxel, in Patients with Advanced Solid Tumors. Mol. Cancer Ther. 11, 308-316. (9) Firer, M. A., and Gellerman, G. (2012) Targeted Drug Delivery for Cancer Therapy: the Other Side of Antibodies. J. Hematol. Oncol. 5, 70. (10) Jain, R. K., and Stylianopoulos, T. (2010) Delivering Nanomedicine to Solid Tumors. Nat. Rev. Clin. Oncol. 7, 653-664.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(11) Yuan, F., Dellian, M., Fukumura, D., Leunig, M., Berk, D. A., Torchilin, V. P., and Jain, R. K. (1995) Vascular Permeability in a Human Tumor Xenograft: Molecular Size Dependence and Cutoff Size. Cancer Res. 55, 3752-3756. (12) Sanna, V., and Sechi, M. (2012) Nanoparticle Therapeutics for Prostate Cancer Treatment. Maturitas 73, 27-32. (13) Krall, N., Pretto, F., Decurtins, W., Bernardes, G. J., Supuran, C. T., and Neri, D. A. (2014) Small-Molecule Drug Conjugate for the Treatment of Carbonic Anhydrase IX Expressing Tumors. Angew. Chem., Int. Ed. Engl. 53, 4231-4235. (14) Sun, L. C., Mackey, L. V., Luo, J., Fuselier, J. A., and Coy, D. H. (2008) Targeted Chemotherapy Using a Cytotoxic Somatostatin Conjugate to Inhibit Tumor Growth and Metastasis in Nude Mice. Clin. Med.: Oncol. 2, 491-499. (15) Leamon, C. P., Reddy, J. A., Vlahov, I. R., Westrick, E., Dawson, A., Dorton, R., Vetzel, M., Santhapuram, H. K., and Wang, Y. (2007) Preclinical Antitumor Activity of a Novel Folate-Targeted Dual Drug Conjugate. Mol. Pharm. 4, 659-667. (16) Lorusso, P. M., Edelman, M. J., Bever, S. L., Forman, K. M., Pilat, M., Quinn, M. F., Li, J., Heath, E. I., Malburg, L. M., Klein, P. J., Leamon, C. P., et al. (2012) Phase I Study of Folate Conjugate EC145 (Vintafolide) in Patients with Refractory Solid Tumors. J. Clin. Oncol. 30, 4011-4016. (17) Op den Kamp, J. A. (1979) Lipid Asymmetry in Membranes. Annu. Rev. Biochem. 48, 47-71. (18) Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L., and Henson, P. M. (1992) Exposure of Phosphatidylserine on the Surface of Apoptotic


Page 46 of 49

Page 47 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Lymphocytes Triggers Specific Recognition and Removal by Macrophages. J. Immunol. 148, 2207-2216. (19) Utsugi, T., Schroit, A. J., Connor, J., Bucana, C. D., and Fidler, I. J. (1991) Elevated Expression of Phosphatidylserine in the Outer Membrane Leaflet of Human Tumor Cells and Recognition by Activated Human Blood Monocytes. Cancer Res. 51, 3062-3066. (20) Connor, J., Bucana, C., Fidler, I. J., and Schroit, A. J. (1989) DifferentiationDependent Expression of Phosphatidylserine in Mammalian Plasma Membranes: Quantitative Assessment of Outer-Leaflet Lipid by Prothrombinase Complex Formation. Proc. Natl. Acad. Sci. U. S. A. 86, 3184-3188. (21) Riedl, S., Rinner, B., Asslaber, M., Schaider, H., Walzer, S., Novak, A., Lohner, K., and Zweytick, D. (2011) In Search of a Novel Target - Phosphatidylserine Exposed by Non-Apoptotic Tumor Cells and Metastases of Malignancies with Poor Treatment Efficacy. Biochim. Biophys. Acta 1808, 2638-2645. (22) Thorpe, P. E. (2010) Targeting Anionic Phospholipids on Tumor Blood Vessels and Tumor Cells. Thromb. Res. 125 Suppl 2, S134-137. (23) Ran, S., He, J., Huang, X., Soares, M., Scothorn, D., and Thorpe, P. E. (2005) Antitumor Effects of a Monoclonal Antibody that Binds Anionic Phospholipids on the Surface of Tumor Blood Vessels in Mice. Clin. Cancer Res. 11, 1551-1562. (24) Yin, Y., Huang, X., Lynn, K. D., and Thorpe, P. E. (2013) PhosphatidylserineTargeting Antibody Induces M1 Macrophage Polarization and Promotes MyeloidDerived Suppressor Cell Differentiation. Cancer Immunol. Res. 1, 256-268.



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(25) Ojida, A., Mito-Oka, Y., Inoue, M. A., and Hamachi, I. (2002) First Artificial Receptors and Chemosensors Toward Phosphorylated Peptide in Aqueous Solution. J. Am. Chem. Soc. 124, 6256-6258. (26) Rice, D. R., Clear, K. J., and Smith, B. D. (2016) Imaging and Therapeutic Applications of Zinc(ii)-Dipicolylamine Molecular Probes for Anionic Biomembranes. Chem. Commun. (Camb) 52, 8787-8801. (27) Koulov, A. V., Stucker, K. A., Lakshmi, C., Robinson, J. P., and Smith, B. D. (2003) Detection of Apoptotic Cells Using a Synthetic Fluorescent Sensor for Membrane Surfaces that Contain Phosphatidylserine. Cell Death Differ. 10, 1357-1359. (28) Leevy, W. M., Gammon, S. T., Jiang, H., Johnson, J. R., Maxwell, D. J., Jackson, E. N., Marquez, M., Piwnica-Worms, D., and Smith, B. D. (2006) Optical Imaging of Bacterial Infection in Living Mice Using a Fluorescent Near-Infrared Molecular Probe. J. Am. Chem. Soc. 128, 16476-16477. (29) Smith, B. A., Akers, W. J., Leevy, W. M., Lampkins, A. J., Xiao, S., Wolter, W., Suckow, M. A., Achilefu, S., and Smith, B. D. (2010) Optical Imaging of Mammary and Prostate Tumors in Living Animals Using a Synthetic Near Infrared Zinc(II)Dipicolylamine Probe for Anionic Cell Surfaces. J. Am. Chem. Soc. 132, 67-69. (30) Kawato, Y., Aonuma, M., Hirota, Y., Kuga, H., and Sato, K. (1991) Intracellular Roles of SN-38, a Metabolite of the Camptothecin Derivative CPT-11, in the Antitumor Effect of CPT-11. Cancer Res. 51, 4187-4191. (31) Haggar, F. A., and Boushey, R. P. (2009) Colorectal Cancer Epidemiology: Incidence, Mortality, Survival, and Risk Factors. Clin. Colon Rectal Surg. 22, 191-197.


Page 48 of 49

Page 49 of 49

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(32) Chiang, N. J., Chang, J. Y., Shan, Y. S., and Chen, L. T. (2016) Development of Nanoliposomal Irinotecan (nal-IRI, MM-398, PEP02) in the Management of Metastatic Pancreatic Cancer. Expert Opin. Pharmacother. 17, 1413-1420. (33) Lakshmi, C., Hanshaw, R. G., and Smith, B. D. (2004) Fluorophore-Linked Zinc(II)-Dipicolylamine Coordination Complexes as Sensors for PhosphatidylserineContaining Membranes. Tetrahedron 60, 11307-11315. (34) Zhang, X., Tang, K., Wang, H., Liu, Y., Bao, B., Fang, Y., Zhang, X., and Lu, W. (2016) Design, Synthesis, and Biological Evaluation of New Cathepsin B-Sensitive Camptothecin Nanoparticles Equipped with a Novel Multifuctional Linker. Bioconjugate Chem. 27, 1267-1275. (35) Richter, R. P., Berat, R., and Brisson, A. R. (2006) Formation of Solid-Supported Lipid Bilayers: an Integrated View. Langmuir 22, 3497-3505. (36) Murugova, T. N., and Balgavy, P. (2014) Molecular Volumes of DOPC and DOPS in Mixed Bilayers of Multilamellar Vesicles. Phys. Chem. Chem. Phys. 16, 1821118216. (37) Peck, E. M., Battles, P. M., Rice, D. R., Roland, F. M., Norquest, K. A., and Smith, B. D. (2016) Pre-Assembly of Near-Infrared Fluorescent Multivalent Molecular Probes for Biological Imaging. Bioconjugate Chem. 27, 1400-1410. (38) Yarlagadda, V., Sarkar, P., Samaddar, S., and Haldar, J. (2016) A Vancomycin Derivative with a Pyrophosphate-Binding Group: A Strategy to Combat VancomycinResistant Bacteria. Angew. Chem., Int. Ed. Engl. 55, 7836-7840.



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Graphical abstract


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Targeting Tumor Associated Phosphatidylserine ... - ACS Publications

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