Small molecule-drug conjugates: A novel strategy for cancer-targeted treatment

Accepted Manuscript Small molecule-drug conjugates: A novel strategy for cancer-targeted treatment Chunlin Zhuang, Xianghong Guan, Hao Ma, Hui Cong, Wannian Zhang, Zhenyuan Miao PII:

S0223-5234(18)31072-9

DOI:

https://doi.org/10.1016/j.ejmech.2018.12.035

Reference:

EJMECH 10968

To appear in:

European Journal of Medicinal Chemistry

Received Date: 24 July 2018 Revised Date:

14 December 2018

Accepted Date: 14 December 2018

Please cite this article as: C. Zhuang, X. Guan, H. Ma, H. Cong, W. Zhang, Z. Miao, Small moleculedrug conjugates: A novel strategy for cancer-targeted treatment, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2018.12.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Small Molecule-Drug Conjugates: A Novel Strategy for Cancer-Targeted Treatment Chunlin Zhuang, †‡§* Xianghong Guan,ǁ‡ Hao Ma,†§ Hui Cong,§ Wannian Zhang,†§ Zhenyuan Miao†* †

School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China.

‖

Department of Medicinal Chemistry, Institute for Therapeutics Discovery and Development, University of Minnesota,

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717 Delaware Street SE, Minneapolis, MN, 55414, USA. §

School of Pharmacy, Ningxia Medical University, 1160 Shengli Street, Yinchuan 750004, China

Abstract

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Targeted therapy has become an effective strategy of precision medicine for improving cancer treatment. Selectivity improvement is always popular in modern oncology because of decreased side effects in conventional cancer

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chemotherapy. The use of antibody-drug conjugates (ADC), a robust strategy for targeted therapy, applies antibodies to selectively deliver a potent cytotoxic compound to tumor cells and thus improve the therapeutic efficacy of the chemotherapeutic agents. Three ADC products (trastuzumab emtansine, brentuximab vedotin and inotuzumab ozogamicin) are already on the market, and several compounds are in clinical trials. Compared with ADCs, small

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molecule-drug conjugates (SMDCs) provide a new, less established perspective for targeted delivery. Nevertheless, SMDCs have several strengths: they have 1) a non-immunogenic nature, 2) much more manageable synthesis, 3) lower molecular weights, which confer a high potential for good cell penetration in solid tumors. SMDCs might therefore be a promising alternative with similar efficacy to ADCs. In this article, we highlight the medicinal chemistry aspects of

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SMDC design. SMDC targeting ligands, linkers and small-molecule payloads will be discussed. Successful cases of

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SMDCs used as therapeutic agents and other applications of SMDC will also be included.

ACCEPTED MANUSCRIPT 1. INTRODUCTION Comparison of ADC and SMDC cancer-targeted therapy and conventional therapy Precision medicine is one of the hottest terms in the age of big health, and cancer treatment is not an exception [1]. In the past century, chemotherapy and treatments with chemicals play a dominant role in cancer treatment [2]. Nevertheless, the

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limitation of traditional anticancer drugs, such as cytotoxic compounds, cannot be ignored in clinical therapy [3]. Lack of selectivity and specificity are a major drawback because these agents typically destroy all cells [2-4]. A narrow safety profile of these non-selective cytotoxic compounds limits their further application in clinics [3]. In other words, most small molecule therapeutic agents are not preferentially localized at the tumor site [5, 6]. The development of resistance or

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failure of therapeutic regimens to induce objective response leads to a final failure of conventional therapy [7]. In recent years and as an important part of precision medicine, targeted therapy, including kinase inhibitors, monoclonal antibodies,

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antibody-drug conjugates (ADCs), small molecule-drug conjugates (SMDCs) and antisense/siRNA approaches, has become an effective strategy in anticancer therapy [3, 4, 8]. Among these approaches, ADCs have achieved great successes using antibodies to selectively deliver potent cytotoxic compounds to tumor cells and improve the therapeutic efficacy of these chemotherapeutic agents. Three ADC-based products (trastuzumab emtansine, brentuximab vedotin and inotuzumab ozogamicin) have been launched, and several compounds have been processed to reach clinical trials.[4]

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SMDCs, an alternative approach to antibodies for drug delivery and tumor targeting applications, are attracting increasing interest [2, 4]. Compared to the accuracy value of the small molecule targeting ligand and payload of SMDCs, the ratio of payload and antibodies is uncertain in the preparation of ADCs. Therefore, SMDCs have their own strengths such as

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manageable synthesis and a non-immunogenic nature [9, 10]. The molecular weights of SMDCs are potentially much lower, leading to better cell permeability in solid tumors and better in vitro and in vivo stability [11-13]. Similar with the

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ADCs, almost all the SMDC are administrated by injection, which is an inconvenient drawback in clinical use. Overview of SMDCs

Typically, an SMDC contains a targeting ligand, a spacer, a cleavable bridge and a therapeutic payload, or warhead (Figure 1). The antibodies of the ADCs are replaced by these ligands. The cleavable bridges are stable in circulation and release the payload after they penetrate into the tumor cells.

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Figure 1. Schematic of a typical SMDC.

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ACCEPTED MANUSCRIPT

(1) Targeting ligand

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The targeting ligand plays the role that the antibody plays in ADCs. Binding affinity, target selectivity, and the size of the conjugates need to be taken into consideration for the SMDC design. Increased binding affinities of the targeting ligand reasonably reduce the dose of the drug that is needed to achieve high efficacy. Selectivity or specificity is of great importance in the design of drug conjugates as the original intention is to decrease the toxicity of the payload toward

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normal cells [4]. The cell targets could be easily bound by the SMDC with an affinity in the nanomolar range. In normal cells, the minimum systematic toxicity from non-specific binding is significantly lower than in cancer cells [14]. A criterion raised by Low et al. is that the Kd values of targeting ligands should be less than or equal 10 nM. Ligands with extremely high affinity are challenging to find. Indeed, a multivalent ligand, comprising multiple copies of ligands

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conjugated to the same therapeutic payload, is an alternative approach for improving affinity [15, 16]. As mentioned in the previous section, its large molecular size is an ADC drawback and influences cell penetration into solid tumors. The size

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of SMDCs cannot also be ignored because the targeting ligands and the linker are adjusted to make conjugates with the desired pharmacokinetics [15, 17]. Size will generally influence drug delivery into solid tumors via different mechanisms, including permeability, retention effects and excretion through the kidneys. Low-molecular-weight therapeutic cargo is much easier to release and let diffuse independently into tumors [18]. Although low-molecular-weight drug conjugates do not accumulate in solid tumors owing to the enhanced permeability and retention (EPR) effect, they nevertheless passively perfuse a cancer mass more thoroughly and rapidly than macromolecular drug carriers. Moreover, molecules smaller than 40 kDa are extracted from the blood by the glomerulus and rapidly excreted from the body [19]. Off-target lowmolecular-weight drug conjugates are commonly excreted from the body, reducing undesired toxicity in normal cells.

ACCEPTED MANUSCRIPT SMDCs are extracted markedly faster than the larger conjugates [20, 21]. Molecules that selectively block novel pathways and proteins or homing ligands that specifically bind to a receptor are chosen to be targeting ligands.[22] For instance, Gleevec (imatinib) was designed to target the fusion protein BCR/ABL [23], and folic acid works as a ligand of the folate receptor [22, 24]. Both are representative applications of these two strategies. In addition, several targets of SMDCs, such

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as glucose transporter 1 (GLUT1) [25], aminopeptidase N (APN) [26], low-density lipoprotein receptor-related protein 1 (LRP1) [27], prostate-specific membrane antigen (PSMA) [28], αvβ3, the bombesin receptor, and the somatostatin receptor (SSTR), have been used in clinical trials of SMDCs [4].

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(2) Linker

The linker is the structure between the targeting ligand and therapeutic payload, which contains a spacer and a cleavage

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bridge (Figure 1). To conserve the activity of the targeting ligand and the payload efficacy, the linker should be meticulously designed. The linker is also modified to optimize drug release, pharmacokinetic and pharmacodynamic properties and the function of the targeting ligand or payload [4, 22, 24]. a. Spacer

The spacer typically serves as a connection between the targeting ligand and the cleavable bridge and is important to

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SMDCs for maintaining the receptor binding. The unsuitable spacer can result the decrease of the binding affinity for the unwanted intramolecular association. However, the spacer feature is unclear and the rigid spacer is better than flexible in some cases. [4, 22, 29]. The targeting ligand, close to the drug payload, may influence the binding affinity and non-

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specific adsorption of the conjugates to off-target cells. Leamon et al. reported the relative affinities of several folic acid drug conjugates for the folate receptor [29]. The relative affinity (RA) of folate (Figure 2) for the folate receptor was set to

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1.000. Cell-based relative affinity measurements showed the low RA of compound EC216 (lacking a spacer), while EC145 (vintafolide), with an amino acid linkage as the spacer, showed 10-fold higher potency (RA = 0.470). Firstgeneration spacers contained carbohydrate units, repeating acidic residues and saccharo-amino acid residues. Secondgeneration spacers used glutamic acid and glutamine as epimerization-inert modules (See Table 3) [30]. Recently, valinecitrulline and valine-alanine spacers used in acetazolamide-drug conjugates exhibited greater serum stability and superior therapeutic activity [31].

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ACCEPTED MANUSCRIPT

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Figure 2. Chemical structure of folate and representative folate-targeted drug conjugates (EC216 and EC145) with their relative affinities (RA).

Another function of the spacer is to improve the hydrophilicity of the SMDCs [4]. Targeting ligands and therapeutic payloads are usually hydrophobic to maximize membrane permeability and receptor affinity. This property may lead to undesired non-specific associations with lipoproteins, scavenger receptors, lipid bilayers and membranes. Using water-

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soluble spacers, such as polysaccharides, hydrophilic amino acids, PEGs and peptidoglycans, thus has the potential to confer the hydrophilicity of the final SMDCs [4, 24, 32]. Moreover, the design of spacers also contributes to decreasing the cytotoxicity of the payload because the penetration of polar conjugates through cell membranes is more difficult

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b. Cleavage bridge

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without receptor-based endocytosis [4, 32].

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ACCEPTED MANUSCRIPT

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Figure 3. Four kinds of self-cleaving mechanisms for drug release of SMDCs. (a, b) Disulfide-bond-based linker; (c) Acetal linker; and (d) Hydrazine linker. X=N or O; TL = targeting ligand. The activity of highly potent SMDCs always relies on the ability of the cleavable bridge to release the parent drug from

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the SMDC at a predictable site and reliable rate after penetration into targeted cells. The cleavable bridge should thus be stable during the transport from the vasculature to the tumor; otherwise, the SMDC will not exhibit low toxicity to normal

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cells [33]. Two classes of triggering methods are used in SMDCs. First, the use of disulfide-based linkers is one of the most successful approaches; their reduction from their oxidized counterparts by intracellular excesses of glutathione (GSH), thioredoxin, peroxiredoxins, and nicotinamide adenine dinucleotides (NADH and NADPH) assures their cleavage in cancer cells. Self-immolative linkers using disulfide bonds were reduced by 1,2-elimination (Figure 3A) and 1,6elimination mechanisms (Figure 3B) in the presence of GSH at 37 °C and pH 7.4 in phosphate-buffered saline (PBS). Second, since most endosomal compartments are acidic, SMDCs are also designed to be sensitive to the pH value in target cells. For instance, acetals (Figure 3C) and hydrazones (Figure 3D) can be hydrolyzed in the acidic environments found in endosomes [33, 34].

ACCEPTED MANUSCRIPT (3) Small molecule payload

A small molecule payload or active drug is the central part of an SMDC. A good cytotoxic payload needs to meet some criteria (Table 1): (1) have a high efficiency of releasing the therapeutic payload, (2) cause fewer multidrug interactions and less intracellular metabolism after its release, and most importantly, (3) have a high binding affinity. A carefully

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selected therapeutic small molecule payload is critical for further evaluation and clinical success [4]. First, the potency of the therapeutic agent needs to be considered. An IC50 value of 10 nM against the proteins for the therapeutic drug is typically required when the number of targeted receptors exceeds 1 million per cancer cell. If the receptors number more than 100 million per cancer cell, an IC50 of just 1 µM is sufficient [35]. Similar to that of the targeting ligand, the potency

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of a small molecule payload can be complemented by attaching more warheads to the same conjugate. However, developing multivalent therapeutic agents with high potency is challenging. The IC50 values of almost all SMDC drugs in

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clinical trials such as desacetyl vinblastine, mitomycin C, tubulysin and epothilone are in the low nanomolar or submicromolar ranges [24]. Structurally, cytotoxic drugs must have modifiable functional groups, such as amines, sulfhydryl, carboxyl, and aldehyde groups [22, 24]. For their mechanisms of action, most of the therapeutic drug payloads inhibit fundamental cell processes (e.g., cytokinesis, DNA-replication, anti-apoptotic process, and protein synthesis) and metabolic processes (e.g., glycolysis, glutaminolysis, (Na+, K+)-ATPase function, and sugar transport). As mentioned

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above, the self-cleaving linker must release the drug payload in the cancer cells with the low pH environment, reducing environment or some other specific environment. After escaping from the encapsulating endosome, drug molecules will accumulate in cancer cells. Although most well-established chemotherapeutic agents have adequate membrane

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permeability, endosome escaping strategies require further improvement to reach the full therapeutic potential of the agents [4].

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Table 1. Criteria for payload selection. Criteria

Details

Examples

Receptor numbers per cancer cell

Required IC50 (nM)

Desacetyl vinblastine

≥ 1 million

≤ 10

Mitomycin C

≥ 100 million

≤ 1000

Potency

Tubulysin Epothilone −NH2, −NH−, −OH, −SH, −CHO,

Vinblastine,, Mitomycin C, Tubulysin

R−C(O)−R’, −COOH

Epothilone Paclitaxel, Camptothecin

Structure

Cytokinesis Mechanism of action

(1) Inhibit fundamental cell processes DNA-replication

ACCEPTED MANUSCRIPT Anti-apoptotic process Protein synthesis

Glycolysis, Glutaminolysis (Na+, K+)-ATPase

(2) Disrupt metabolic processes

(1) Drug releases and locates inside the cancer cell. Drug release and metabolism

/

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(2) Escape the encapsulating endosome

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Sugar transport

2. SUCCESSFUL CASES OF SMDCS

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Given that the main part of the efficacy is due to the therapeutic warhead, some successful SMDCs are classified by their drug payloads. Several highly potent cytotoxic agents such as vinblastine, paclitaxel, mitomycin C, epothilone, tubulysin

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and camptothecin derivative SN-38 have been utilized in the design of SMDCs (Figure 4).

Figure 4. Chemical structures of representative cytotoxic agents used in SMDCs.

ACCEPTED MANUSCRIPT (1) Vinblastine

Vinblastine was first isolated from Catharanthus roseus in 1958 and was used to treat cancers such as non-small cell lung, bladder and brain cancers [36]. Mechanistically, vinblastine is a microtubule-destabilizing agent (MDA) and inhibits the assembly of microtubules and mitosis. High selectivity and lower toxicity against normal tissues are benefits from new

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drug delivery methodsusing an SMDC strategy. A series of folate-vinca alkaloid SMDCs containing the same cleavable linker system was published; this series was constructed with DAVLBH (vintafolide), vincristine (EC0275), vindesine (EC192), vinorelbine (EC1041), and vinflunine (EC1044). Among them, DAVLBH-based SMDCs are the most potent folate-vinca alkaloid against FR-expressing KB cells [30, 32]. Vintafolide, also known as EC145 and MK-8109 (Figure 2

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and Table 3) and developed by Endocyte and Merck & Co., consists of folic acid ligand and desacetyl vinblastine cytotoxic payload connected through a disulfide linker for parent drug release [37]. A Phase IIb study in non-small-cell

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lung carcinoma (NSCLC) using EC145 alone, an EC145/docetaxel combination and docetaxel alone was completed in 2015 (CTI: NCT01577654). The combination of EC145 and docetaxel improved the progression-free survival of 7.1 months and the overall survival of 10.9 months. A Phase III study evaluating EC145 in combination with Doxil (doxorubicin hydrochloride liposome injection) for the treatment of platinum-resistant ovarian cancer has been suspended in 2014. A potential reason was its failure to improve progression-free survival. In EC145, the parent drug desacetyl

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vinblastine was released from the conjugate by a common reducing condition (1 mM EC145 in PBS, pH = 7.4, with 20 mM GSH, 37 °C). The complete cleavage of the disulfide bond occurred within 6 h (t1/2 = 1 h) through the mechanism in Figure 3A. Similar release profiles were achieved using the reducing agent (2S,3S)-dithiothreitol (DTT), which cleaved

more than 24 h [24],[38].

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EC145 faster than GSH (t1/2 = 5 min). EC145 was stable (pH = 7.4, 37 °C) in PBS in the absence of reducing agents for

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Several desacetyl vinblastine-based SMDCs exist in addition to EC145 (Table 3). Their linkers vary but include different self-immolative linker systems and peptide-based spacer units. A recent SAR study also demonstrated that a cleavable linker is important for biological activity in folate-DAVLBH SMDCs [39]. For instance, the potency of EC1142 and EC1177, which contain noncleavable thioether-based linkers, is significantly lower than related SMDCs with cleavable linkers. Disulfide bonds have been widely applied to many drug delivery designs. However, its instability cannot be ignored because human serum contains an abundance of free thiol compounds such as GSH, cysteine, and homocysteine. Albumin readily attacks disulfide bonds. Folate-based SMDCs are eliminated quickly, with half-lives of approximately 26 min, and

ACCEPTED MANUSCRIPT no free drug has been observed in EC145-treated patients’ serum samples. An SN2 mechanism is theoretically preferred when an external thiol group substitutes into the disulfide moiety. Increasing steric blockage will thus diminish the rate of disulfide cleavage. Based on the above analyses, compounds EC0265, EC0272, and EC0276 were generated by introducing methyl groups near the disulfide bond regions. The stabilities of the compounds were significantly greater

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than those of the unmodified compounds, and the rate of linker cleavage decreased. The half-lives of EC0265 and EC0272 (8 mM DTT, pH = 7.4, 37 °C) were t1/2, EC0265 = 1 h and t1/2, EC0272 = 0.5 h under these reducing conditions. The half-life of the four methyl group-substituted disulfide derivative (EC0276) was improved to more than 8 h, and it was extremely stable for more than 3 days in PBS buffer. However, the cytotoxicity will be influenced by the increased stability. EC195

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was proposed to release the parent drug by thioquinone-methide-based 1,6-elimination (Figure 3B). The parent drug was released slightly faster with 1,2-elimination (e.g., EC145) than 1,6-elimination. The half-lives in GSH and DTT were 45

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min and less than 5 min, respectively, and the compound was stable in PBS for over 24 h (pH = 7.4, 37 °C). As the endosomal milieu was more acidic, another release approach was used in SMDCs such as EC193 and EC140; in this approach, the compounds were meant to be stable at neutral pH and reactive under mildly acidic pH conditions (pH 5~6), exactly, as low as 6.2 [33, 40]. EC193, which had an acetal-based linker, was pH sensitive and easily hydrolyzed because of the electron-donating effect of the p-alkoxy substituent in the aromatic ring (Figure 3C). The parent drug of EC193, N-

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hydroxyethylvindesine, was released at pH 5.0 (37 °C) with a hydrolysis rate of t1/2 = 25 min and was very stably at pH 7.4, 37 °C (t1/2 = 22 h). However, the compound was inactive in vitro for unknown reasons. EC140, containing the acyl

5.5 h.

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hydrazone linker, was labile at pH = 5.0, 37 °C. Its hydrazone bond was cleaved to release the parent drug at a rate of t1/2 =

As mentioned above, moderately long linker is required to maintain ligand affinity. Table 2 shows that EC216 (lacking a

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spacer) was less potent than EC145. The following systematic studies of spacers were performed to avoid steric obstruction and unwanted intramolecular associations and improve hydrophilicity. First-generation spacers contained carbohydrate units, repeating acidic residues (Asp) and saccharo-amino acid residues (e.g., EC140, EC145, EC193, EC195, EC0265, EC0272, and EC0276). These units were easy to assemble using standard Fmoc-solid peptide synthesis and resulted in highly water-soluble SMDCs. However, these SMDC designs ignored problems associated with the spacers, which could be epimerized via aspartimide formation and have their lactone forms stabilized. These problems were addressed in the second-generation spacers (e.g., EC0489 and EC0492). Glutamic acid and glutamine, as epimerization-inert modules, were connected using an amide bond, and the configurations of their carbon-hydrogen bonds

ACCEPTED MANUSCRIPT were not easily changed. For instance, EC0489 was cleaved in the presence of DTT in PBS (pH = 7.4, 37 °C) within 15 min. The first strength of this SMDC, compared with EC145, was its lower toxicity. Its maximum tolerated dose is better than that of EC145 by 70%. In addition, this compound had a short elimination half-life in a tumor mouse model, an ∼70% decrease in bile clearance, a 4-fold increase in urinary excretion, and improved tolerability in rodents.[30] A Phase I study

Table 2. Vinblastine-based SMDCs. Release

Current

Profile

stage

Structure

Indications

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Compound

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(CTI: NCT00852189) for the treatment of refractory or metastatic tumors has been completed.

platinum-resistant

Phase III

A

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EC145

EC216

H2N

OH N N H

N H OH O

O

O N

O

HO2C

O

N N H OH H H

S

O

S

N H

EC193

N H OH O

O

S

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O

O

EC195

S

O

S HO2C

N

Phase IIb

NSCLC

NCT01577654

A

/

Cancer

[33, 40]

A

/

Cancer

[33, 40]

A

/

Cancer

[33, 40]

A

/

Cancer

[33, 40]

B

/

Cancer

[30]

C

/

Cancer

[30]

Folate

HO2C

O

H N

NH

O

N N H OH H H

O

S

HO2C H N

CO2HO

O

HO 2C

O

N H HO 2C

O

H N

N H

H2 N

OH

N HO

HO2C

O

N N N H OH H H

O

EC0276

O

HO 2C

H N

NCT01170650 ovarian cancer

HN

N H

O

N

N H

H2N

OH O O

O

H N

N H

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EC0272

O

H N

CO2HO

OH N

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EC0265

NH

HN

Refs

N H

H N

Folate

HO2C

NH

HN O

H N O

N H HO2C

H N

Folate

ACCEPTED MANUSCRIPT EC140

C

/

EC0489

A

Phase I

Cancer

[30]

refractory or metastatic NCT00852189

A

/

Cancer

[30]

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EC0492

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tumors

A: 1,2-Elimination; B: 1,6-Elimination; C: pH-dependent cleavage

(2) Tubulysin

Tubulysins are a family of myxobacterial antibiotic products that function as anticancer, anti-angiogenic, and anti-

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proliferative agents targeting microtubules [41, 42]. They exhibit very potent cytostatic effects on various tumor cell lines with IC50 values in the picomolar range [43]. However, their severe toxicity is a double-edged sword with regard to their therapeutic potentials. Targeted therapy using tubulysin is an alternative strategy to address the drawbacks of off-target

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binding (Table 4). EC0305, a folate conjugate of tubulysin B hydrazide (TubBH), contained that same linker as EC145 and showed an IC50 value of 7 nM against KB cells [44]. EC0510, a folate conjugate of tubulysin A, showed an IC50 value

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similar to that of EC0305. These two conjugates exhibited different efficacies in KB tumor mouse models. EC0510 displayed excellent antitumor activity, and 80% of animals showed a complete reversion at a dose of 0.5 µmol/kg. EC0305 showed only moderate tumor regression at the same dose level. When the dose was elevated to 1.0 µmol/kg, EC0305 generated a remarkable antitumor effect and tumors of all mice disappeared throughout the 80-day duration of the study. EC0317, a folate conjugate of a methyl ether analog of tubulysin B, was less active than EC0305 against cells in vitro and a mouse model in vivo, even using a dose of 2.0 µmol/kg. EC0305 showed better antitumor effects than EC145 against FR-positive M109 and 4T1-cl2 models [45]. A variety of spacers were explored in the modification of the tubulysin-folate conjugates. A conjugate of TubBH, EC1456, using a second-generation spacer, has been moved to Phase I clinical trials for advanced solid cancer [46].

ACCEPTED MANUSCRIPT 2-[3-(1,3-Dicarboxypropyl)ureido] pentanedioic acid (DUPA) is a ligand of PSMA that can selectively attach imaging and therapeutic agents to prostate cancer cells without off-target binding [47]. PSMA is known to be highly expressed on the majority of prostate cancers with limited expression on normal tissues. A PSMA-targeted chemotherapeutic agent, DUPA-TubBH (Table 3), was designed to kill PSMA-positive LNCaP cells (IC50 = 3 nM) and to eliminate established

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tumor xenografts in nude mice with no detectable loss in body weight. EC1169, a derivative of DUPA-TubBH with an undisclosed structure and constructed of a high affinity PSMA-targeting ligand conjugated through a releasable linker system to TubBH, has been moved to Phase I clinical trials for advanced prostate cancer [48]. Tubulysin B has also been conjugated with a ligand of the cholecystokinin 2 receptor (CCK2R). Conjugate CRL-L1-TubBH selectively inhibits

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CCK2R-positive tumors and generates a dramatic receptor-specific antitumor effect with negligible toxicity in healthy

Table 3. Tubulysin-based SMDCs. Compound

Structure

EC1456

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EC0317

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EC0510

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EC0305

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tissues [49].

Release

Current

Profile

stage

A

Indications

Refs

/

Cancer

[44]

A

/

Cancer

[45]

A

/

Cancer

[45]

A

Phase I

Advanced Solid NCT01999738 Tumors

Recurrent Metastatic, CastrationA

EC1169

Phase I

NCT02202447 Resistant Prostate Cancer

DUPA-TubBH

(MCRPC)

ACCEPTED MANUSCRIPT CCK2R-

CRL-L1A

[49]

/ positive tumors

TubBH

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A: 1,2-Elimination

(3) Paclitaxel

Paclitaxel (PTX) is a first-line chemotherapeutic medication in clinics that treats several types of cancer and promotes

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tubulin assembly into microtubules [50, 51]. However, severe drawbacks, such as low selectivity, poor water solubility, and serious side effects, including hypersensitivity, neurotoxicity, nephrotoxicity, and cardiotoxicity, limit its clinical

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applications [52]. The folic acid strategy is also used for targeted therapy involving PTX conjugates. Folic acid-5aminofluorescein-glutamic-paclitaxel (FA-5AF-Glu-PTX) was designed and exhibits high antitumor activity by folate receptor-mediated endocytosis [53]. Octreotide, which selectively binds to SSTR2 and SSTR5, is used as a ligand to address the above drawbacks and achieve a high efficacy against cancer cells (Table 4). Octreotide(Phe)–polyethene glycol–paclitaxel [OCT(Phe)–PEG–PTX] was synthesized and used for targeted cancer therapy [52]. This conjugate

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exhibited a pH-dependent profile and had significant cytotoxicity against NCI-H446 cells (SSTR overexpression) but low cytotoxicity against WI-38 cells (normal cells without SSTR expression). In vivo experiments demonstrated that OCT(Phe)–PEG–PTX had better antitumor efficacy (Tumor weight inhibition: 66.3%) and lower systemic toxicity than a

mouse

model.

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corresponding mPEG–PTX conjugate (43%) and commercial PTX (Taxol, 54.3%) in an NCI-H446 cancer xenograft Degarelix

[Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2],

an

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antagonist of the gonadotropin-releasing hormone receptor (GnRH-R), was employed as a targeted moiety for PTX [54]. A tetrameric peptide named H2009.1, targeting the integrin αvβ6, was conjugated with PTX and developed to treat αvβ6expressed NSCLC in vitro and in vivo [55]. Table 4. Paclitaxel-based SMDCs.

Release Compound

Structure

Receptor

Indications

Refs

Profile murine H22

FA-5AF/ Glu-PTX

/

FR metastasis

[53]

ACCEPTED MANUSCRIPT SSTROCT(Phe)– C

SSTR

overexpressing

[52]

PEG–PTX cancer

/

Gonadotropin-

MCF-7 human

releasing

breast cancer/

hormone

HT-29 human

receptor

colon cancer

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Paclitaxel– /

degarelix

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conjugate

Peptide–

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paclitaxel conjugate

(4) Mitomycin C

avβ6-expressing avβ6

[55]

NSCLC

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C: pH-dependent cleavage

C

[54]

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Mitomycin, usually referring to mitomycin C (MMC), is an antibiotic isolated from the broth of Streptomyces caespitosus [56, 57]. It has an excellent wide clinical antitumor spectrum, including effectiveness against gastric cancer, pancreatic

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cancer, breast cancer, NSCLC, cervical cancer, head and neck cancer and bladder cancer [58]. However, clinical use of MMC is limited due to its fast clearance and toxicity [59]. As shown in Table 5, EC72, using folic acid as targeting ligand, produced dose-response in vitro activity against a panel of FR-positive cell lines and good antitumor effects in animals without apparent toxicity or pathological degeneration. A non-releasable conjugate (EC110) was obtained by changing the disulfide linker of EC72 to a carbon chain, but it failed to exhibit potent antitumor activity at the same dose and schedule as EC72, indicating the importance of the disulfide bond for drug delivery. However, the therapeutic potential of EC72 decreased with increasing subcutaneous tumor size. An advanced folate-MMC conjugate (EC118) that was synthesized to improve the activity was constructed with both a reducible disulfide bond and an acid-labile hydrazone bond in the linker region. This conjugate was more potent in subcutaneous M109 tumors than EC72.

ACCEPTED MANUSCRIPT A combination of MMC and paclitaxel, compared with either agent alone, significantly improved antitumor efficacy [60]. Given that the multidrug targeted approach has a distinct therapeutic advantage in facilitating greater drug deposition within the tumor mass, EC0225 contained PTX and MMC tethered to a single folate unit. This conjugate was highly active and specific against FR-expressing tumors.[61] This “first-in-class” folate-targeted multidrug conjugate exhibited

refractory or metastatic tumors who have exhausted standard therapy. Table 5. Mitomycin C-based SMDCs. Structure

Release

Current

Profile

stage

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Compound

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impressive preclinical performance and was initiated into Phase I clinical trials (CTI: NCT00441870) for patients with

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EC72

TE D

EC110

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EC0225

A: 1,2-Elimination

Refs

A

/

Cancer

[60]

A

/

Cancer

[60]

A

/

Cancer

[60]

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EC118

Indications

solid tumors A

Phase I

(refractory or metastatic)

NCT00441870

ACCEPTED MANUSCRIPT (5) Others

The epothilones are a class of anticancer drugs targeting microtubules [62]. One of their analogs, ixabepilone (Ixempra), was approved by the FDA in 2007 for aggressive metastatic or locally advanced breast cancers that no longer respond to standard chemotherapies [63]. Epofolate (Table 6), also termed BMS-753493, was developed by Endocyte Inc. and

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Bristol Myers Squibb together. Epofolate is a folate conjugate constructed with a semi-synthetic analog of epothilone A [64]. Phase II clinical trials of this compound were terminated for unknown reasons (CTI: NCT00550017). Camptothecin (CPT), a topoisomerase I inhibitor, was conjugated with folic acid using a hydrophilic peptide spacer and a releasable disulfide carbonate linker. This conjugate exhibited high affinity against folate receptor-expressing cells and inhibited cell

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proliferation in human KB cells with an IC50 of 10 nM [65]. A zinc(II) dipicolylamine (ZnDPA)-SN38 conjugate was developed to validate the use of SMDC in targeting tumor-associated phosphatidylserine (PS) in the tumor

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microenvironment [66]. EC131 was a conjugate with folic acid attached to an anti-microtubule agent, maytansinoid, via a cleavable disulfide bond [67]. A preclinical investigation found that this compound had a high affinity for FR-positive cells with an IC50 in the low nanomolar range. This conjugate exhibited significant antitumor efficacy in subcutaneous FR-positive M109 BALB/c mice and human KB models. An HDAC inhibitor (NCH-31) was attached to folic acid via a disulfide bond. The resulting compound displayed good growth-inhibitory activity against FR-positive MCF-7 breast

to treat cancers [69].

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cancer cells [68]. Another DNA-reactive cytotoxic agent (EC1788) with unknown structure was developed by Endocyte

Epofolate (BMS-753493)

Release

Current

Profile

stage

Structure

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Compound

EP

Table 6. Epothilone-, Camptothecin-, and Maytansine-based SMDCs and Other SMDCs. Indications

Advanced A

Phase II

Solid

NCT00550017

Tumors

CamptothecinA based SMDC

Refs

/

Cancer

[65]

ACCEPTED MANUSCRIPT

D

/

Cancer

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ZnDPA-SN38

A

Preclinical

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EC131

A

/

EC1788

A: 1,2-Elimination; D: an enzymatic cleavage

3. CONCLUSIONS AND PERSPECTIVES

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Folate-NCH-31

/

Cancer

MCF-7 /

Preclinical

[66]

[67]

[68]

cancer

Cancer

[69]

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Targeted therapy has become an effective strategy of precision medicine for better cancer treatments [1]. SMDCs have many advantages as an important part of targeted therapy [4]. The first advantage of SMDCs over traditional chemotherapeutic agents is the minimization of undesirable toxicities by

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specifically delivering the drug payloads to pathological cells, thus reducing the exposure of healthy cells to the cytotoxic agents. Second, water solubility is a key bottleneck that limited the activity of several first-line anticancer agents, such as

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PTX and CPT. Hydrophilicity could be significantly improved via the introduction of spacers or linkers such as polysaccharides, hydrophilic amino acids, PEGs and peptidoglycans. Conjugates with high hydrophilicities have less potential to penetrate through cell membranes by passive diffusion. Folate receptor-mediated endocytosis of cancer cells facilitates transmembrane transport, which further improves selectivity for carcinomas. Third, the SMDCs are also more flexible for drug optimization than non-targeted therapeutics. If adequate drug efficacy is not achieved with an initial payload, the drug payload can be optimized. The targeting ligand can be changed until adequate tumor specificity is achieved to minimize toxic side effects. Linkers or spacers could be optimized to achieve the desired pharmacokinetics and pharmacodynamics.

ACCEPTED MANUSCRIPT The SMDC strategy has already been used in inflammatory and kidney diseases, in addition to in cancers. For instance, the mTOR kinase inhibitor (EC0371) for polycystic kidney disease (PKD) and an anti-inflammatory therapy (EC1669) for the activation of macrophages expressing folate receptors have been reported, although their structures are undisclosed. This strategy has also been extended into the field of diagnosis using companion imaging agents. These agents are very

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similar to SMDCs and are created by replacing the potent drug with an imaging agent [4, 6, 40]. These agents are designed to identify the overexpressed targeted receptor of specific patients and to treat patients with the customized prescription.

However, there are only a limited number of targeting ligands that have been assessed systemically. Thus, it is of great

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importance to validate new targets for the development of novel SMDC in human diseases.

In conclusion, the SMDC field is attracting increasing interest as a bridge between cytotoxic compounds and targeted

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therapy. More targeted receptors will be used for active targeting, and more targeting ligands including peptides [70] will be developed. Moreover, the application of SMDCs to other diseases will also be extended in the future.

AUTHOR INFORMATION Corresponding Author *

*

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For Z.M.: Phone/Fax, 86-21-81871241; E-mail, [email protected] For C.Z.: Phone/Fax, 86-21-81871258; E-mail, [email protected]

Author Contribution ‡

Notes

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These two authors contributed equally to this work.

Biographies

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The authors declare no competing financial interest.

Chunlin Zhuang is currently a faculty member at the School of Pharmacy, Second Military Medical University, China. He received his Ph.D. in medicinal chemistry from the research group of Professor Wannian Zhang in 2014. In 2012, he was sponsored by the China Scholarship Council’s Ph.D. Abroad Training Plan to work under the supervision of Prof. Chengguo Xing at the University of Minnesota. His research interests focus on drug design and medicinal chemistry. Xianghong Guan is currently a graduate student at the School of Pharmacy, University of Minnesota Twin Cities. He joined the research group of Prof. Gunda I. Georg and has been sponsored by the medicinal chemistry Ph.D. graduate

ACCEPTED MANUSCRIPT program since 2014. His research focuses on the discovery of bioactive agents and fluorescence tools in the field of epigenetics. Hao Ma is currently a graduate student in the School of Pharmacy, Ningxia Medical University, China. He joined the research group of Prof. Wannian Zhang at the Second Military Medical University in 2015. His research focuses on

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antitumor agents. Hui Cong is currently a graduate student in the School of Pharmacy, Ningxia Medical University, China. She joined the research group of Prof. Chengguo Xing at the Ningxia Medical University in 2017. Her research focuses on antitumor conjugates.

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Wannian Zhang received his bachelor’s degree in pharmacy (1968) and MS degree in medicinal chemistry (1981) from the Second Military Medical University. He has worked as a professor of Medicinal Chemistry since 1992 and was the

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Director of the Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, from 1992 to 1994. From 1994 to 2001, he served as the Dean of the School of Pharmacy. Currently, Professor Zhang is the Chief of the State’s Key Discipline of Medicinal Chemistry, Second Military Medical University. He has held a joint professorship with Ningxia Medical University and served as the Dean of the School of Pharmacy since 2011. His research interests are mainly focused on anti-fungal and antitumor drug design and development.

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Zhenyuan Miao is currently an associate professor at the School of Pharmacy, Second Military Medical University, China. He received his Ph.D. degree in medicinal chemistry from the research group of Professor Wannian Zhang in 2006. From 2010 to 2012, he worked with Chinese Academy Member Prof. Fener Chen as a postdoctoral associate at Fudan

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University. In 2015, he joined the research group of Professor Gunda I. Georg as a visiting scholar. His research interests

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are mainly focused on antitumor drug discovery.

ACKNOWLEDGMENTS

This research was funded by grants from the National Natural Science Foundation of China (81502978, 81872791 to C.Z. and 81673352 to Z.M.), the Shanghai Municipal commission of Health and Family Planning (2017YQ052 to C.Z.), the Shanghai ‘‘ChenGuang’’ Project (16CG42 to C.Z.), the Bio-Pharmaceutical Project of Science and Technology of Shanghai (15431901700 to Z.M.), the Young Elite Scientists Sponsorship Program by the China Association for Science and Technology (2017QNRC061 to C.Z.); the Key Research and Development Program of Ningxia (2018BFH02001 to W. Z. and 2018BFH02001-01 to C.Z.).

ACCEPTED MANUSCRIPT ABBREVIATIONS USED ADC, antibody-drug conjugate; SMDC, small molecule-drug conjugate; EPR, enhanced permeability and retention; GLUT1, glucose transporter 1; APN, aminopeptidase N; LRP1, low density lipoprotein receptor-related protein 1; PSMA,

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prostate-specific membrane antigen; SSTR, somatostatin receptor; RA, relative affinity; GSH, glutathione; NADPH, nicotinamide adenine dinucleotide; PBS, phosphate-buffered saline; MDA, microtubule-destabilizing agent; NSCLC, nonsmall-cell lung carcinoma; TubBH, tubulysin B hydrazide; DUPA, 2-[3-(1,3-dicarboxypropyl)ureido] pentanedioic acid; PTX, Paclitaxel; OCT, octreotide; GnRH-R, gonadotropin-releasing hormone receptor; MMC, mitomycin C; CPT,

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camptothecin; ZnDPA, zinc(II) dipicolylamine.

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peptide camptothecin prodrug, Bioorg. Med. Chem. Lett., 16 (2006) 5350-5355. [66] Y.W. Liu, K.S. Shia, C.H. Wu, K.L. Liu, Y.C. Yeh, C.F. Lo, C.T. Chen, Y.Y. Chen, T.K. Yeh, W.H. Chen, J.J. Jan, Y.C. Huang, C.L. Huang, M.Y. Fang, B.D. Gray, K.Y. Pak, T.A. Hsu, K.H. Huang, L.K. Tsou, Targeting Tumor Associated Phosphatidylserine with New Zinc Dipicolylamine-Based Drug Conjugates, Bioconjug. Chem., 28 (2017) 1878-1892. [67] J.A. Reddy, E. Westrick, H.K. Santhapuram, S.J. Howard, M.L. Miller, M. Vetzel, I. Vlahov, R.V. Chari, V.S. Goldmacher, C.P. Leamon, Folate receptor-specific antitumor activity of EC131, a folate-maytansinoid conjugate, Cancer Res., 67 (2007) 6376-6382.

ACCEPTED MANUSCRIPT [68] T. Suzuki, S. Hisakawa, Y. Itoh, N. Suzuki, K. Takahashi, M. Kawahata, K. Yamaguchi, H. Nakagawa, N. Miyata, Design, synthesis, and biological activity of folate receptor-targeted prodrugs of thiolate histone deacetylase inhibitors, Bioorg. Med. Chem. Lett., 17 (2007) 4208-4212. [69] Product Profile, in, http://bciq.biocentury.com/products/ec1788, 2018.

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[70] L. Ma, C. Wang, Z. He, B. Cheng, L. Zheng, K. Huang, Peptide-Drug Conjugate: A Novel Drug Design Approach,

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Curr. Med. Chem., 24 (2017) 3373-3396.

ACCEPTED MANUSCRIPT Figure Legends Figure 1. Schematic of a typical SMDC. Figure 2. Chemical structure of folate and representative folate-targeted drug conjugates (EC216 and EC145) with their relative affinities (RA).

Acetal linker; and (d) Hydrazine linker. X=N or O; TL = targeting ligand. Figure 4. Chemical structures of representative cytotoxic agents used in SMDCs. Table 1. Criteria for payload selection.

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Table 2. Vinblastine-based SMDCs.

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Figure 3. Four kinds of self-cleaving mechanisms for drug release of SMDCs. (a, b) Disulfide-bond-based linker; (c)

Table 4. Paclitaxel-based SMDCs. Table 5. Mitomycin C-based SMDCs.

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Table 3. Tubulysin-based SMDCs.

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Table 6. Epothilone-, Camptothecin-, and Maytansine-based SMDCs and Other SMDCs.

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Graphic Abstract

ACCEPTED MANUSCRIPT Research Highlights 1. Small molecule-drug conjugates (SMDCs) provide a new, less established perspective for targeted delivery. 2. The medicinal chemistry aspects of SMDC design have been described.

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3. Successful cases of SMDCs used as therapeutic agents have been presented.

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