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

Article abstract of DOI:10.1021/acs.bioconjchem.7b00225

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.

Full text of DOI:10.1021/acs.bioconjchem.7b00225

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Article
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 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 is published by the American Chemical Society. 1155
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Targeting Tumor Associated Phosphatidylserine with New
Zinc DipicolylamineꢀBased Drug Conjugates
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YuꢀWei Liu, KakꢀShan Shia, ChienꢀHuang Wu, KuanꢀLiang Liu, YuꢀCheng Yeh,
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ChenꢀFu Lo, ChiungꢀTong Chen, YunꢀYu Chen, TengꢀKuang Yeh, WeiꢀHan Chen,
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JiingꢀJyh Jan, YuꢀChen Huang, ChenꢀLung Huang, MingꢀYu Fang, Brian D. Gray,
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Koon Y. Pak, TsuꢀAn Hsu, KuanꢀHsun 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 L.K.T.
(kelvintsou@nhri.org.tw)
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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, PSꢀbinding 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 antiꢀ
tumor 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 PSꢀassociated diseases.
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INTRODUCTION
Ligandꢀtargeted cancer therapeutics represent a viable strategy to deliver potent
1
cytotoxic agents to the disease site. In particular, conjugation of a therapeutic moiety via
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a suitable linker to antibodies, capable of specific binding to tumor antigens, has
facilitated selective accumulation and increased the effective drug concentration in situ
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while alleviating adverse side effects systemically. Indeed, with recent approvals of two
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Antibody Drug Conjugates (ADC) in oncology, brentuximab vedotin and adoꢀ
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trastuzumab ematasine , more than forty ADCs are being evaluated in the clinical trials.
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
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conjugates (SMDC). Contrary to limitations of ADCꢀbased 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
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immunogenicity.
Some of the promising SMDCs target prostate specific membrane
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antigen, carbonic anhydrase IV, and somatostatin derivatives for neuroendocrine
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tumors. Moreover, folateꢀbased conjugates have entered Phase II/III clinical trials for
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treating multiple cancer diseases.
However, there are only a limited number of small
ligands to tumorꢀassociated antigens that have been assessed systemically for
pharmacodelivery applications. Thus, it is of great importance to validate new tumorꢀ
associated 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 tumorꢀassociated
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
antiꢀtumor activities. Under normal cellular state, PS is tightly regulated to be
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asymmetrically segregated in the inner leaflet of the normal cells. Apoptosis induced PS
exposure is readily cleared by macrophages under normal conditions, however, tumor
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cells can evade surveillance by the macrophages. Indeed, PS was found in many tumor
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cell lines and primary tumors.
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
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at the tumor vasculatures.
More importantly, recent studies with a PSꢀtargeting
antibody Bavituximab in Phase III clinical trail has provided a significant insight into the
expansion of tumorꢀassociated macrophages with PSꢀcontaining vacuoles in the tumor
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microenvironment. Thus, PS at the tumor microenvironment has become a novel
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biomarker. While Bavituximab has demonstrated the proofꢀofꢀconcept of targeting PS
selectively, the drug conjugate development for Bavituximab remains challenging. To
circumvent these challenges, we envisioned that a PSꢀtargeted 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
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multiꢀanionic phosphorylated biomolecules, has been used to construct optical imaging
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probes that target the outer surfaces of anionic PSꢀcontaining cell membranes.
Pioneered by Smith and coworkers, positively charged ZnDPAꢀderived fluorescent
ligands were shown to accumulate specifically at the tumor site in vivo through its
28, 29
interaction with negatively charged PS.
However, ZnDPA based cancer drug
conjugates (ZnDPADCs) have not been described for cancer therapy. We reasoned that
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spatialꢀ and temporalꢀdelivery 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
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wish to 1) describe the facile SMDC synthesis of the first ZnDPA linked to 1 (SNꢀ38) ;
) determine structure activity, plasma stability, and PSꢀbinding relationships between
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different linker types; and 3) provide insight into optimization of conjugates’
pharmacokinetic properties with respect to antiꢀtumor efficacy in vivo.
CHEMISTRY
For the development of PSꢀtargeting 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
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developed countries. 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
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who failed the firstꢀline treatment. The potent topoisomeraseꢀ1 inhibitor 1, is the active
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metabolite of 2 (CPTꢀ11). We hypothesize that 1 can be delivered by a ZnDPAꢀbased
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 10ꢀhydroxyl position of 1 with a linker was experimentally realized
via facile synthesis to generate the eventual conjugates.
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Scheme 1. Synthetic procedures for compound 10. Reagents and conditions: (1) 2,2ꢀ
o
o
dimethoxypropane, pꢀTsOH, MeOH, 60 C, 12h, 96%. (2) LiAlH , THF, 40 C. (3)
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BrCH (CH ) CO Me, K CO , DMF, 85 C, 12h, 73%. (4) CBr , PPh , DCM, overnight,
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5%. (5) bis(pyridinꢀ2ꢀylmethyl)amine, K CO , DMF, 3h, 82%. (6) LiOH , MeOH. (7)
2 3 (aq)
SNꢀ38 (1), EDCl, HOBt, NꢀMethylmorpholine, DMF, 15h, 42%. (8) 2 equiv. Zn(NO ) ,
3
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DCM/MeOH.
One of the many advantages over ADC lies in the costꢀdown 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 5ꢀhydroxyisophthalic acid 3 with 2,2ꢀdimethoxypropane and
pꢀtoluenesulfonic acid in methanol at 60 °C gave the dimethyl 5ꢀhydroxyisophthalate 4 in
good yield (96%). Reduction of 4 with lithium aluminum hydride in dry THF at 40 °C to
furnish the fully reduced (5ꢀhydroxyꢀ1,3ꢀphenylene)dimethanol 5, which after workꢀup
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and without purification, alkylation of 5 was performed in the presence of potassium
carbonate and methyl 7ꢀbromoheptanoate to afford the corresponding diol compound 6 in
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3% yield. This was followed by the addition of an excess of CBr and PPh to afford the
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methyl 7ꢀ(3,5ꢀbis(bromomethyl)phenoxy)heptanoate 7 in 85% yield.
Dibromo intermediate 7 thus obtained was then substituted with bis(pyridinꢀ2ꢀ
ylmethyl)amine in DMF with potassium carbonate to generate methyl 7ꢀ(3,5ꢀ
bis((bis(pyridinꢀ2ꢀylmethyl)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
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literature. The primary amine functional group in 11 was reacted with reagents in
conditions (1a)ꢀ(1e) to generate various types of DPAꢀlinkers, designated as the linker
type R, with a terminal ester functional group in 12aꢀe (Scheme 2). Hydrolysis of methyl
ester 12aꢀe with LiOH (0.5 N) at room temperature proceeded smoothly to afford the
corresponding intermediate acid 13aꢀe in moderate to high yields (83ꢀ97%). Activation
of the carboxylic acids in DPAꢀlinkers 13aꢀe with EDCl and HOBt in dry N,Nꢀ
dimethylformamide followed by addition of 1.1 equivalent of 1 gave rise to DPAꢀdrug
conjugate 14aꢀe in 38ꢀ80% yields. Incubating each of the DPAꢀdrug conjugates 14aꢀe
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 15ꢀ19, respectively, in
quantitative yield.
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Scheme 2. Synthetic procedures for compound 15ꢀ19. Reagents and conditions: (1) (a)
methyl 4ꢀchloroꢀ4ꢀoxobutanoate, DCM, TEA, 2h, 83%. (b) methyl 3ꢀ
(chlorocarbonyl)benzoate, DCM, TEA, 2h, 55%. (c) methyl 4ꢀformylbenzoate, DCM;
then NaBH , MeOH, 15h, 66%. (d) methyl 4ꢀ(chlorosulfonyl)benzoate, DCM, TEA, 2h,
4
55%. (e) methyl 3ꢀisocyanatobenzoate, DCM, TEA, 1h, 61%. (2) 0.5N LiOH , MeOH,
(aq)
1
5h, 83ꢀ97%. (3) SNꢀ38, EDCl, HOBt, NꢀMethylmorpholine, DMF, 38ꢀ80%. (4) 2 equiv.
Zn(NO ) , DCM/MeOH.
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Scheme 3. Synthetic procedures for compound 24ꢀ25 and 27ꢀ28. Reagents and
conditions: (1) (a) [1,1'ꢀbiphenyl]ꢀ4ꢀcarbaldehyde, DCM; then NaBH , MeOH, 80%. (b)
4
methyl 4ꢀformylbenzoate, DCM; then NaBH , MeOH, 15h, 66%. (2) (a) methyl 3ꢀ
4
isocyanatobenzoate, DCM, TEA, 1h, 73%. (b) [1,1'ꢀbiphenyl]ꢀ4ꢀcarboxylic acid, EDCl,
HOBt, NꢀMethylmorpholine, DMF, 30%. (3) 0.5N LiOH , MeOH, 15h. (4) SNꢀ38,
(aq)
EDCl, HOBt, NꢀMethylmorpholine, DMF, 40ꢀ48%. (5) 2 equiv. Zn(NO ) , DCM/MeOH.
3 2
(6) 26, TEA, THF, 40ꢀ90%.
In scheme 3, reductive amination of the primary amine in 11 with NaBH and
4
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1,1'ꢀbiphenyl]ꢀ4ꢀcarbaldehyde generated the corresponding DPAꢀlinker 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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carbamateꢀcontaining 28 was achieved in 40% yield by conjugation of intermediate 20a
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and 26 (4ꢀnitrophenyl carbonate activated 1) 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]ꢀ4ꢀcarboxylic acid under EDCl and HOBt to install amideꢀ
type linker 21b (30%) or compound 20a was coupled with isocyanatobenzoate to
establish ureaꢀtype linker 21a (70%). Subsequently, the terminal ester group of 21a and
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1b 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(NO ) to afford final products 24 (48%) and 25 (40%) over three
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steps.
Scheme 4. Synthetic procedures for compound 31. Reagents and conditions: (1) 1ꢀ
tertꢀbutoxycarbonyl)piperidineꢀ4ꢀcarboxylic acid, EDCl, HOBt, NꢀMethylmorpholine,
(
DMF, 12h, 81%. (2) TFA, DCM, 82%. (3) 26, TEA, urea, THF, 2h, 96%. (4) 2 equiv.
Zn(NO ) , DCM/MeOH.
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In scheme 4, 11 was coupled with 1ꢀ(tertꢀbutoxycarbonyl)piperidineꢀ4ꢀcarboxylic
acid under activation with EDCI and HOBt to form intermediate 29 in 81% yield, which
was followed by treatment with TFA to remove the Bocꢀprotecting 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.
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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 timeꢀdependent 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
o
intact conjugates were observed after 24 h incubation with the mouse plasma at 37 C,
respectively (Figure 1B). As such, conjugates 15ꢀ18 with intramolecular amidoꢀ or
benzoic esters linkages were synthesized to further diversify the linker options. In an
attempt to increase the solubility of 17, sulfonamideꢀcontaining 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
o
(
conjugate: 1) after 24 h incubation with mouse plasma at 37 C. 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, 15ꢀ18, 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.
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prodrugs and exhibit cellular cytotoxicity profiles. Since 1 is poorly soluble, modification
at the C10ꢀphenolic 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, 15ꢀ18, 27 and 28 showed 4ꢀ to 10ꢀfold decrease in
potencies towards COLO 205 and modest 3ꢀfold decrease in cytotoxicities for Detroit
5
51 as compared to that of cytotoxic component 1. Interestingly, the most stable
conjugate 28, with IC at 7 ꢁM against COLO 205 and >20 ꢁM against Detroit 551,
5
0
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
prodrugꢀlike properties.
Binding ability of the conjugate to PS
To investigate whether the PSꢀtargeting ZnDPA motif retains its association
property with the linkerꢀdrug modification, we conducted in vitro binding studies
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between conjugate 16 and a DOPSꢀcontaining supported lipid bilayer by surface plasmon
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resonance (SPR) measurements (Figure 2). Uniformꢀsized liposomes with 100 %
DOPC were prepared as a nonꢀspecific binding control (Figure S1A). On the other hand,
PSꢀcoated liposomes (DOPC/ DOPS (3:1,v/v)) were prepared according to procedures
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reported in literature (Figure S1A). Compound 38, ZnDPA without linkerꢀdrug
modification, exhibited a association rate constant (k ) of 303.3 (1/Ms) and a dissociation
a
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rate constant (k ) of 9.95 x 10 (1/s) in a 1:1 complex binding model. Moreover, the
d
ꢀ5
equilibrium dissociation constant (K ) of compound 38 was determined to be 3.28 x 10
D
M, which was in good agreement from the fluorophoreꢀlinked ZnDPA sensor binding
studies reported by Smith and coꢀworkers and showed that the SPR experiments could be
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used for the PS association studies (Figure S1B). While compound 38 might act as a
single molecular unit and exhibited monoꢀexponential behavior in binding, the binding
kinetic plot for conjugate 16 showed to be multiꢀexponential and the dissociation kinetic
plot for the compound exhibited at least two dissociation events (Figure 2).
Figure 2. In vitro SPR PSꢀassociation studies. Sensorgrams generated using a Biacore
T200. Conjugate 16 was analyzed across a twoꢀfold concentration series descending from
6
2.5ꢁM. 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
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000 RU, where liposome of (DOPC 100%) was used as nonspecific binding control.
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The complicated kinetics of conjugate 16 may be due to many factors and selfꢀ
aggregation of the conjugateꢀPS complexes within the membrane after association could
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also contribute to the differences in the curve. 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 PSꢀcontaining liposome. Conjugate 37, containing the
same linkerꢀdrug modification yet differing in zincꢀcoordinated 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 linkerꢀdrug 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) linkerꢀdrug modifications in ZnDPADCs did not
interfere with ability of recognition between the ZnDPA and PSꢀcoated liposomes.
In vivo antiꢀtumor 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 PSꢀseeking ZnDPA probe
could efficiently accumulate to the tumor site (Figure 3). In addition to the findings by
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Smith and coꢀworkers who had showed ZnDPA possessed high affinity for the exposed
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anionic surfaces of apoptotic cells in breast or prostate tumors, 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.
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Figure 3. In vivo detection of PSꢀexpression 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, prodrugꢀlike in vitro cytotoxicity, and maximum tolerated dose at 40 mg/kg
(mpk). To our surprise, we did not observe potent antiꢀtumor 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 antiꢀtumor
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 antiꢀtumor activities, demonstrating the
in vivo antiꢀtumor activity was dependent on 1 (Figure 4).
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*: p < 0.05 vs. vehicle control by oneꢀway ANOVA analysis and NewmanꢀKeuls multiple comparison test.
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Figure 4. Antiꢀtumor activities of 16 and 1 against COLO 205 tumor growth and
comparison of the amount of 1 used in each regimen. SNꢀ38 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.
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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, twoꢀday rest and another 5 days in a
row (5+5) at 10 mpk. Doseꢀdependent in vivo antiꢀtumor 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
2
8, 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 stabilityꢀenhanced biphenyl group with the
cleavable esterꢀmodified cytotoxic payload 1 to generate 19, 24, and 25 (Scheme 2).
Moreover, a ureaꢀcontaining linker extended via the amino functionality in 11 was
readily prepared to afford 19. Modifications on the ureaꢀlinker could be accomplished
through reductive amination from 11 as alkylꢀbiphenyl (24) and arylꢀbiphenyl (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
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In vitro properties and pharmacokinetic studies of 19, 24, 25, and 31
Conjugates 24 and 25 with biphenyl functionalities and 2ꢀmimicking linker
moiety in 31 exhibited improved in vitro plasma stabilities and retained proꢀdrug
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 (V ) at 86.2 (L/kg). These
ss
results might contribute to the lower MTD values of 30 mpk when compared to 24 and
2
5. On the other hand, the AUC ratio of the intact conjugate to cytotoxic payload 1 was
higher for 24, indicating that the alkylꢀbiphenyl linker possessed better in vivo stability
than the arylꢀbiphenyl moiety in 25. Taken together, we identified 24 with improved
pharmacokinetic profiles (Table 1B), increased MTD, and retained proꢀdrug 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
5
51 normal skin fibroblast from three separate experiments, and MTD studies of 19, 24,
5, and 31. (B) Pharmacokinetic profiles of 19, 24, and 25 in male ICR mice (n=3) at 5
2
mg/kg with intravenous administration. CL, clearance; V , apparent volume of
ss
distribution at steady state; AUC, area of drug concentration under curve; mpk, mg/kg.
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In vivo antiꢀtumor 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 doseꢀdependent
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
antiꢀtumor effect after 40 mpk was given twice per week after the first week (Figure 5).
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Figure 5. Antiꢀtumor 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.
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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 antiꢀtumor efficacy than the CPTꢀ11
(
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 spatialꢀ and temporalꢀ 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 antiꢀcancer 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 dipicolylꢀvancomycin conjugate was shown to exhibit enhanced antiꢀ
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bacterial activities against vancomycinꢀresistant bacteria. Inspired by the important role
of PS in tumor progression and the PSꢀtargeting ability of ZnDPA probes, we devised
ZnDPAꢀbased drug conjugates to expand the repertoire of cancer therapeutic targets for
the development of SMDC. In addition to the stable linkage provided in the ZnDPAꢀ
based probe developed by Smith and coꢀworkers, 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, PSꢀbinding 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 SMDCꢀbased 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 antiꢀtumor 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.
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EXPERIMENTAL SECTION
1. General. Unless otherwise stated, all materials used were commercially obtained and
used as supplied. Reactions requiring anhydrous conditions were performed in flameꢀ
dried 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 glassꢀbacked 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
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2
30−400 mesh size as supplied by Merck. Eluent systems are given in volume/volume
1
13
concentrations. H and C NMR spectra were recorded on a Varian Mercuryꢀ300 (300
MHz), a Varian Mercuryꢀ400 (400 MHz), Bruker DMXꢀ600 (600 MHz) and a Varian
Vnmrꢀ700 (700 MHz). Chloroformꢀd or dimethyl sulfoxideꢀd6 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 H O solution containing 0.1% trifluoroꢀacetic acid (mobile phase B). The
2
starting flow rate was 0.5 mL/min and the injection volume was 10 ꢁL. 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 C. 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: NHRIꢀIACUCꢀ103061).
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2. Chemistry. Synthetic procedures for 12b, 13b, 14b, 16, 20a, 21a, 22a, 23a, and 24:
2
.1. Methyl 3ꢀ((4ꢀ(3,5ꢀbis((bis(pyridinꢀ2ꢀylmethyl)amino)methyl)phenoxy)butyl)
carbamoyl)benzoate (12b). To a stirred solution of 11 (400 mg, 0.68 mmol) was
dissolved in CH Cl (40 ml), and triethylamine (2 ml) and methyl 3ꢀ
2
2
o
o
(
chlorocarbonyl)benzoate were added at 0 C. The mixture was stirred at 0 C for 2 hr.
After completion of reaction, the mixture was extracted with CH Cl (100 ml) and
2
2
saturated aqueous NH Cl (2 x 100 ml). The combined organic layers were dried over
4
MgSO concentrated under reduced pressure. The resulting residues were purified by
4
,
1
column chromatography (MeOH: CH Cl = 1: 13) to yield product (280 mg, 55%). H
2
2
NMR (400 MHz, CDCl ) δ 8.49 (d, J = 4.4 Hz, 4H, Hꢀ4, Hꢀ10, Hꢀ20, Hꢀ26, ꢀCH=Nꢀ),
3
8
.36 (s, 1H), 8.12 (d, J = 8 Hz, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.63ꢀ7.56 (m, 8H), 7.50ꢀ
7.46 (m, 1H), 7.13ꢀ7.10 (m, 4H), 7.05 (s, 1H, Hꢀ35), 6.85 (s, 2H, Hꢀ33, Hꢀ37), 4.02 (t, J
5.2 Hz, 2H, Hꢀ40, ꢀOCH ꢀ), 3.91 (s, 3H, CH OCO), 3.78 (s, 8H, Hꢀ13, Hꢀ14, Hꢀ29, Hꢀ
=
2
3
3
0, NCH C=N), 3.63 (s, 4H, Hꢀ16, Hꢀ32, NCH C=C), 3.58ꢀ3.54 (m, 2H, Hꢀ43, ꢀNCH ꢀ),
2 2 2
1.91ꢀ1.84 (m, 4H, Hꢀ41, Hꢀ42, ꢀCH CH CH CH ꢀ).
2
2
2
2
2
.2. 3ꢀ((4ꢀ(3,5ꢀbis((bis(pyridinꢀ2ꢀylmethyl)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 CH Cl (100 ml) and
2
2
saturated aqueous NH Cl (2 x 100 ml). The combined organic layers were dried over
4
1
MgSO , concentrated under reduced pressure to yield product (240 mg, 88%). H NMR
4
10
11
12
13
14
15
16
17
18
19
20
21
22
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24
25
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(
300 MHz, CDCl ) δ 8.56 (d, J = 4.8 Hz, 4H, Hꢀ4, Hꢀ10, Hꢀ20, Hꢀ26, ꢀCH=Nꢀ), 8.47 (s,
3
1H), 8.21 (d, J = 7.2 Hz, 2H), 7.62ꢀ7.51 (m, 9H), 7.20ꢀ7.12 (m, 7H), 7.08 (s, 2H), 4.14
(m, 2H, 2H, Hꢀ40, ꢀOCH CH ꢀ), 3.80 (s, 8H, Hꢀ13, Hꢀ14, Hꢀ29, Hꢀ30, NCH C=N), 3.64
2 2 2
(m, 6H), 1.97ꢀ1.86 (m, 4H, Hꢀ41, Hꢀ42, ꢀCH CH CH CH ꢀ). ESIꢀMS, calcd for
2
2
2
2
+
+
C H N O 735, found 736 (M+H) , 758 (M + Na) .
44
45
7
4
2
.3.
(4S)ꢀ4,11ꢀdiethylꢀ4ꢀhydroxyꢀ3,14ꢀdioxoꢀ3,4,12,14ꢀtetrahydroꢀ1Hꢀ
[1,2ꢀb]quinolinꢀ9ꢀyl3ꢀ{[4ꢀ(3,5ꢀbis{[bis(pyridinꢀ2ꢀ
pyrano[3',4':6,7]indolizino
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 4,11ꢀ
diethylꢀ4,9ꢀdihydroxyꢀ1Hꢀpyrano[3',4':6,7]indolizino[1,2ꢀb]quinolineꢀ3,14
(4H,12H)ꢀ
dione (SNꢀ38, 83 mg, 0.21 mmol) and hydroxybenzotriazole (HOBt, 57 mg, 0.42 mmol)
and Nꢀ(3ꢀDimethylaminopropyl)ꢀN´ꢀethylcarbodiimide hydrochloride (EDCl, 80 mg,
0.42 mmol, 3 equiv) and Nꢀmethylmorpholine (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 CH Cl (300 ml) and saturated aqueous NaHCO (300 ml) and
2
2
3
H O (5 x 300 ml). The combined organic layers were dried over MgSO , concentrated
2
4
under reduced pressure. The resulting residues were purified by column chromatography
1
(MeOH : CH Cl = 1 : 13) to yield product (60 mg, 40%). H NMR (400 MHz, CD OD)
2
2
3
δ 8.69 (d, J = 4 Hz, 4H, Hꢀ4, Hꢀ10, Hꢀ20, Hꢀ26, ꢀCH=Nꢀ), 8.39 (d, J = 7.2 Hz, 1H), 8.22ꢀ
8
.11 (m, 8H), 7.76ꢀ7.67 (m, 8H), 7.65 (s, 1H), 7.60 (d, J = 7.6 Hz, 4H), 6.81 (s, 2H, Hꢀ
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3, Hꢀ37), 6.72 (s, 1H), 5.58 (d, J = 16.4 Hz, 1H, Hꢀ70), 5.38 (d, J = 16.4 Hz, 1H, Hꢀ70),
.32 (s, 2H, Hꢀ62), 4.34 (d, J = 16 Hz, 4H), 4.11 (m, 2H), 3.96 (d, J = 16 Hz, 4H), 3.78
5
(s, 4H, Hꢀ16, Hꢀ32, NCH C=C), 3.57 (m, 2H), 3.23ꢀ3.21 (m, 2H), 1.98ꢀ1.93 (m, 6H, Hꢀ
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1, Hꢀ42, Hꢀ75), 1.39 (t, J = 7.6 Hz, 3H, Hꢀ61), 1.02 (t, J = 7.6 Hz, 3H, Hꢀ77). ESIꢀMS,
+
2+
calcd for C H N O : 1110, found 556 (M + 2H ) .
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2
.4. (4S)ꢀ4,11ꢀdiethylꢀ4ꢀhydroxyꢀ3,14ꢀdioxoꢀ3,4,12,14ꢀtetrahydroꢀ1Hꢀpyrano
3',4':6,7]indolizino[1,2ꢀb]quinolinꢀ9ꢀyl 3ꢀ{[4ꢀ(3,5ꢀbis{[bis(pyridinꢀ2ꢀylmethyl)amino]
methyl}phenoxy)butyl]carbamoyl}benzoate·2[Zn(NO ) ] (16).
[
3
2
To a stirred solution of compound 14b (110.9 mg, 0.10 mmol) in 2 mL DCM was added
1
mL of Zn(NO ) (75.7 mg, 0.20 mmol) in MeOH at room temperature. After 1hr, the
3 2
1
mixture was concentrated under reduced pressure. H NMR (500 MHz, DMSOꢀd ) δ 8.91
6
(
t, J = 5.1 Hz, 1H), 8.75ꢀ8.57 (m, 4H), 8.37 (d, J = 7.9 Hz, 1H), 8.33ꢀ8.18 (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), 7.71ꢀ7.49 (m,
(
7
H), 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), 3.97ꢀ3.63 (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,
(
1
3
3
1
H). C NMR (125 MHz, DMSOꢀd ) δ 172.95, 165.64, 164.85, 159.34, 157.31, 154.81,
6
52.63, 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,
1
18.13, 116.19, 97.19, 72.86, 67.89, 65.75, 57.44, 56.19, 50.07, 38.56, 30.79, 30.27,
8.83, 26.75, 26.39, 23.73, 22.86, 22.74, 14.36, 11.27, 11.15, 8.24. ESIꢀMS
2
2
+
2+ 2+
C H N O Zn : 586.7040, found: 586.6975 (M+Zn ) . Purity: 95%
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.5.
1,1'ꢀ(5ꢀ(4ꢀ(([1,1'ꢀbiphenyl]ꢀ4ꢀylmethyl)amino)butoxy)ꢀ1,3ꢀphenylene)bis(N,Nꢀ
bis(pyridinꢀ2ꢀylmethyl)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
(
10
11
12
13
14
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temperature. After 1.0 hour, NaBH (410 mg, 10.8 mmol) in 6 mL MeOH was added to
4
o
the above solution at 0 C. 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 Na SO , and concentrated under reduced pressure. The residue was subjected
2
4
to flash column chromatography (CH Cl : MeOH = 9 : 1) to give compound 20a in 80%
2
2
1
yield (2.17 g). H NMR (400 MHz, CDCl ) δ 8.48 (d, J = 4.0 Hz, 4H, Hꢀ4, Hꢀ10, Hꢀ20,
3
Hꢀ26, ꢀCH=Nꢀ), 7.67ꢀ7.45 (m, 14H), 7.39 (t, J = 8.0 Hz, 3H), 7.11 (t, J = 8.0 Hz, 4H),
7
3
.03 (s, 1H), 6.80 (s, 2H), 3.94 (s, 4H), 3.77 (s, 8H, Hꢀ13, Hꢀ14, Hꢀ29, Hꢀ30, NCH C=N),
2
.62 (s, 4H), 3.47 (s, 1H), 2.84 (t, J = 7.3 Hz, 2H), 1.98ꢀ1.74 (m, 4H, Hꢀ41, Hꢀ42). ESIꢀ
+
MS, calcd for C H N O 753, found 776 (M + Na) .
4
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.6.
Methyl
3ꢀ(3ꢀ([1,1'ꢀbiphenyl]ꢀ4ꢀylmethyl)ꢀ3ꢀ(4ꢀ(3,5ꢀbis((bis(pyridinꢀ2ꢀ
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
o
(
115 mg, 0.465mmol) and TEA (81 g, 0.62 mmol) at 0 C. The mixture was warmed to
room temperature. After 1.0 h, the mixture was diluted with DCM (10.0 mL), washed
with saturated NH Cl (5 mL x 3) and brine (5 mL), dried over anhydrous Na SO , and
4
2
4
concentrated under reduced pressure. The residue was subjected to flash column
chromatography (CH Cl : MeOH = 19 : 1) to give compound 21a in 73% yield (210.5
2
2
1
mg). H NMR (400 MHz, CDCl ) δ 8.48 (d, J = 4.8 Hz, 4H, Hꢀ4, Hꢀ10, Hꢀ20, Hꢀ26, ꢀ
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