RGD – DMXAA chemical

Conjugates of cytotoxic agents with RGD peptides (Arg-Gly-Asp) addressed to ανβ
3

Sotirios Katsamakas1,

α β1 and ανβ6 integrin receptors overexpressed by cancer cells, have recently gained
5
attention as potential selective anticancer chemotherapeutics. In this review, the design and the development of RGD conjugates coupled to different small molecules including known cytotoxic drugs and natural products will be discussed.

First draft submitted: 16 January 2017; Accepted for publication: 14 February 2017; Published online: 10 April 2017

Keywords: angiogenesis • integrin avβ3 • RGD conjugates • RGD peptide • targeted drug delivery • tumor therapy

Before the end of the 19th century, the members of integrin, cadherin, selectin and
founder of chemotherapy Paul Ehrlich devel- immunoglobulin families [5]. Integrins are a
oped a theory about an ideal therapy for dis- family of heterodimeric, transmembrane gly-
ease described as the magic bullets; the arti- coproteins that are involved in cell–cell and
ficial antibodies that would precisely attack cell–extracellular matrix interactions [6]. So
pathogens without damaging the rest of the far, 18 α and eight β subunits are known to
body [1]. He imagined that such a therapeutic assemble into 24 integrins. Each subunit con-
approach would have the potential to treat a tains three regions, an extracellular domain,
wide range of diseases, including cancer. Since a single pass transmembrane region and a
then, important steps have been made toward cytoplasmic region [7]. Integrins typically
the realization of targeted cancer therapy and regulate the adhesion of epithelial cells to the
personalized medicine. In 1971, Judah Folk- basement membrane; however, they can also
man formed a hypothesis that tumor growth contribute to migration, proliferation, differ-
and metastasis are angiogenesis-dependent entiation and survival of tumor cells. Most
processes and inhibition of tumor angiogene- commonly, ανβ3, α5β1 and ανβ6 integrins
sis could be an advantageous therapy against are expressed in adult epithelia, but are also
cancer [2]. Angiogenesis is referred to a physi- upregulated in some tumors [8]. Evidence
ological process that leads to the formation has long accumulated to show an essential
of new blood vessels from pre-existing ones role of ανβ3 integrin in bone [9–11], lymph
during fetal development, ovulation, wound node [12,13] , liver metastasis [14] and decreased
repair as well as growth and development [3]. patient survival [15,16] . Overexpression of this
These blood vessels provide the developing integrin has also been associated with vari-
and healing tissues with vital nutrients and ous types of cancer including melanoma [17],
oxygen [4]. Hence, targeting tumor angiogen- glioblastoma [18–20], breast [21], prostate [22],
esis evolved to a promising approach in the pancreatic [13], ovarian [23–25], cervical [26]
battle against cancer. and colon cancer [14]. Based on these data, it
In tumor angiogenesis, various classes of is not surprising that ανβ3 integrin receptor
Theodora Chatzisideri2, Savvas Thysiadis2
& Vasiliki Sarli*,2
1Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, University Campus, 54124, Thessaloniki, Greece 2Department of Chemistry, Aristotle University of Thessaloniki, University Campus, 54124, Thessaloniki, Greece *Author for correspondence:
Tel.: +30 231 099 7840 [email protected]

adhesion molecules are implicated including has been targeted for anticancer therapy.
part of

HN NH2

H2N
NH
NH

N
H

NH HN
NH HN

R O
O

O
O

O
OH
O

1, R = OH, c(RGDyK) 2, R = H, c(RGDfK)
3, c(RGDf[N-Me]V) or Cilengitide

Figure 1. Chemical structures of c(RGDyK), c(RGDfK) and (c(RGDf[N-Me]V) or cilengitide.

Presently, a variety of monoclonal antibodies, small molecules, peptides, peptidomimetics and peptide conjugates have been identified as ανβ3 integrin antag- onists [27]. In the early 1970s, E Ruoslahti discovered that the RGD sequence (the tripeptide Arg-Gly-Asp) serves as a cell attachment site in fibronectin (an extra- cellular matrix protein) [28]. Thereof, this sequence has been recognized as the minimal integrin recognition sequence present in many natural ligands that bind to ανβ3 receptor [29,30] . Subsequently, linear as well as cyclic RGD peptides have been developed as ανβ integrin ligands [31,32] . Among these RGD peptides,3 c(RGDfK), c(RGDyK) and cilengitide have showed high binding affinity to integrin ανβ3 and are most thoroughly studied (Figure 1). Up until now, cilengit- ide represents the most advanced integrin inhibitor in clinical development and has been extensively tested against glioblastoma [30].
Classical chemotherapy does not present specificity for tumor cells and is based on the assumption that a cytotoxic agent will target the most rapidly prolif- erating cancer cells. Thus, commonly used cytotoxic agents are toxic to normal cells leading to systemic toxicity and causing severe side effects such as hair loss, nausea, vomiting, damages to liver, kidney and bone marrow [25] . An approach to overcome the limi- tations of classical chemotherapy involves the targeted (cellular or tissue) delivery of drugs. To increase selec- tivity, a tumor-targeting drug delivery system must consist of a tumor recognition unit and a cytotoxic drug linked to an appropriate linker [26] . In the last few years, among the various developed delivery systems, antibodies and peptides have been widely applied as carriers and recognition units for cancer cells. RGD peptide conjugates with cytotoxic drugs have attracted particular attention and are intensively investigated. These delivery systems may specifically address drugs to angiogenic endothelial cells and cancer cells by the binding of the RGD peptide to
ανβ3 integrins, which are overexpressed by these cells (Figure 2). This strategy is especially appealing, since integrin receptors and its ligands have been shown to be efficiently internalized via receptor-mediated endo- cytosis [33–37] . Thus, upon internalization the RGD containing conjugate may release the active cytotoxic drug into the cancer cells [38] .
The therapeutic efficiency of RGD conjugates is dependent on the properties of the conjugate (specific- ity and activity), the toxicity of the parent drug and the chemistry of the linker. Existing linkers, are divided into two categories, the noncleavable and the cleavable, that is hydrazone, disulphide, ester and peptide linkers. A thorough review covering the different linkers employed in RGD drug delivery systems has recently been pub- lished [39]. At present, RGD-based drug delivery systems have mainly gained attention as potential selective anti- cancer chemotherapeutics and antithrombotic drugs. RGD–drug conjugates that contain both a cytotoxic drug and a fluorophore have also been developed in order to study the uptake and delivery mechanisms by which the active drug is delivered to a cell. These con- jugates combine both therapeutic and diagnostic capa- bilities enabling the diagnosis and treatment of a dis- ease (theragnostics) [40–42]. Herein, the development of RGD-based drug conjugates coupled to different small molecules including known cytotoxic drugs and natural products will be discussed (for other aspects of RGD- based therapy, the reader is referred to excellent recent reviews [43–45]).

RGD–paclitaxel conjugates
Taxol (generic name paclitaxel, PTX) is a taxane diter- pene, isolated from Taxus brevifolia in 1971 [46]. It is an antimitotic agent that stabilizes cytoskeletal micro- tubules against depolymerization [47,48] . PTX displays remarkable antitumor activity and is used for the treat- ment of ovarian, breast, head and neck cancer and Kaposi’s sarcoma [49]. Due to its nonselective mode of

action its applications are limited by its clinical tox- icities such as acute myelosuppression and peripheral neurotoxicity. Other drawbacks to its uses are its low aqueous solubility, short half-life and poor bioavail- ability [50]. To improve treatment outcomes, various drug delivery systems are currently being developed including liposomes, prodrugs, micelles, nanoparticles and peptide conjugates [51–53]. The first PTX conju- gate with an RGD peptide was developed by Chen and coworkers in 2005 (Figure 3) [54]. Based on previ- ous findings that dimeric and multimeric cyclic RGD peptides have higher receptor binding affinity in vitro and better tumor retention in vivo, the bicyclic peptide E[c(RGDyK)]2 was employed [55,56] . A hydrolyzable ester bond was used to release PTX from the conjugate inside the cell.
The synthesis of E[c(RGDyK)]2-PTX (7) was accomplished as depicted in Figure 3. The PTX hemisuccinate ester 5 was activated using EDC and NHS, followed by coupling with E[c(RGDyK)]2 pep- tide glutamate amino group. The cytotoxic effect of conjugate 7 compared with PTX and E[c(RGDyK)]2 was assessed by the 3-(4,5-dimethylthiazole-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT) assay in MDA-MB-435 breast cancer cells. PTX conjugate 7 was found less cytotoxic (IC50 = 134 ± 28 nM) than PTX (IC50 = 34 ± 5 nM). E[c(RGDyK)]2 alone had an

bodiimide and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS). It was reported that the conjugation reaction was strongly pH-dependent. The best yields (60–70%) were obtained by maintaining the pH at
7.3.The ability of the cyclo[DKP-RGD]–PTX conju- gates 12–15 to compete with biotinylated vitronectin for binding to the purified αVβ3 and αVβ5 receptors was confirmed and the in vitro cytotoxic activities were evaluated in six human cancer cell lines in comparison with PTX. It was found that the conjugates displayed a cytotoxic activity similar to that of PTX, while they exhibited a cytostatic effect in normal human diploid fibroblast-like cell (HDFC) fibroblasts. In stability assays, cyclo[DKP-f 3-RGD]-PTX 13 displayed suf- ficient stability in physiological solution and in both human and murine plasma. Thus, the in vivo anti- tumor effect of 13 was examined on the αvβ3-rich IGROV-1/Pt1 carcinoma xenotransplanted in nude mice. Compound 13 was more effective than PTX despite the lower molar dosage used. It should be noted that the conjugate as well as PTX were formulated for intravenous administration in a mixture of Cremophor EL and ethanol. In addition, compound 13 led to the disappearance of two out of eight tumors in treated animals. The histopathological analysis in tumors from untreated mice and from mice treated with 13 revealed the presence of aberrant mitotic cells, which is

IC
50
of 2.9 ± 0.1 μM and 5 had an IC50 of 67 ± 9 nM.
consistent with the mechanism of action of PTX.

By using a flow cytometric analysis it was found that 7 exhibited a G2/M cell cycle arrest and apoptosis as observed with PTX. In 2008, the same research group reported the in vivo testing of 7 in nude mice bearing MDA-MB-435 tumor [57] . Their findings demonstrated the selective delivery of PTX to inte-
In 2012, Zanardi and coworkers reported the synthe- sis and evaluation of nine conjugates of PTX covalently attached to cyclic AbaRGD (Azabicycloalkane–RGD)

grin-expressing tumor cells. It was also shown that 7 was more effective than the combination of PTX and E[c(RGDyK)]2 ; however, the tumor volume still increased after treatment with E[c(RGDyK)]2-PTX or PTX and E[c(RGDyK)]2 even with multiple dose administrations. The authors attributed the observed activities to the limited bioavailability and poor solu- bility of E[c(RGDyK)]2-PTX due to its high lipo- philicity. Biodistribution studies demonstrated that 3H-E[c(RGDyK)]2-PTX exhibited higher initial tumor exposure dose and prolonged tumor reten- tion compared with that of 3H–PTX. Furthermore,

Cell
Integrin Drug-linker peptide receptor

Endocytosis

Nucleus

in vivo 18F-FLT/PET imaging revealed reduced tumor metabolism after PTX and E[c(RGDyK)]2-PTX treatment.
In 2012, Gennari and coworkers reported the syn- thesis of cyclic RGD conjugates containing a diketo- piperazine with PTX, as shown in Figure 4 [58]. In this

Drug release

Cell death

case, the conjugates were synthesized after the activa- tion of PTX hemisuccinate ester 6 by diisopropylcar-
Figure 2. Schematic representation of drug delivery mediated by integrin endocytosis.

AcO O OH

Ph
O
O
O O

O
Ph
NH

OH
O

H
O

OAc

pyridine
PTX1
O

O
5
OH

PTX1
EDC, CH3CN H2O, NHS

4
O Ph O

O
O

PTX1
O

O
N
E[c(RGDyK)]2
O

O O c(RGDyK) c(RGDyK)
6
O O
7

NH O
AcO
O

c(RGDyK)
NH2

c(RGDyK)

PTX1 = Ph O
O O

HO
BzO
H

O
E[c(RGDyK)]2

AcO

Figure 3. Synthesis of E[c(RGDyK)]2-PTX conjugate 7.

II and AmproRGD (Aminoproline–RGD) III peptides by an amide or a triazolyl link (Figure 5) [59]. The tri- azolyl conjugates were prepared by a standard Huisgen 1,3-dipolar cycloaddition reaction. An ester bond was also used for the conjugation of PTX to the linkers.
Conjugates 18–26 were tested for their ability and selectivity to bind to ανβ3 and ανβ5 integrin receptors in vitro [59]. Generally, the results varied with respect to the parent cyclopeptides 16 and 17 (Figure 6). The diglycolic group between the RGD moiety and PTX was favorable for binding activity. The ability of PTX conjugates 18–26 to inhibit growth of tumor cells was measured using a growth inhibition assay on a panel of αvβ3/αvβ5-overexpressing human tumor cell lines. The synthesized derivatives showed antiproliferative activi- ties with low nanomolar to micromolar IC50 values. Interestingly, compound 20 exerted a more potent anti- proliferative activity on ovarian carcinoma IGROV-1 cells compared with free PTX (IC50 = 1.04 nM for 20 vs IC50 = 23.4 nM for PTX). Derivative 20 was selected for in vivo studies in an ovarian carcinoma model xeno- grafted in immunodeficient mice. The compound was
conjugates (29–31 and 32) constructed on glutamic acid dendrons (Figure 6) [60] . The synthetic strategy employed was again based on the copper catalyzed Huisgen 1,3-dipolar cycloaddition. In general, the bivalent conjugates compared with the previous syn- thesized monovalent counterparts, have displayed an improvement of the αvβ3-receptor affinity and had potent antiproliferative activity against dif- ferent human tumor cell lines. In vivo evaluation of 30 revealed potent inhibition of tumor growth of the IGROV-1/Pt1 carcinoma similar to that of PTX and a favorable toxicity profile. Compound 30 was administered intravenously at a dose level of 30 mg/kg every 4 days for four times and induced a tumor volume inhibition of 86% similarly to PTX.
The same year, Gennari and coworkers reported the development of cyclic RGD–PTX conjugates bearing lysosomally cleavable linkers [61]. The conjugates 33 and 34 have the Val–Ala and Phe–Lys peptide sequences and conjugate 35 that served as a negative control has an ‘uncleavable’ nonpeptide linker (Figure 7). The abil- ity of cyclo[DKP–RGD]–PTX conjugates to inhibit

well tolerated and was administered intravenously (36
biotinylated vitronectin binding to the a
β3 and a
v
β
v

mg/kg) every 4 days for four times. The tumor vol- ume inhibition introduced by 20 was 98%, while for PTX was 81%. Furthermore, the histological analysis showed induction of cells in mitotic arrest, which is consistent with the mechanism of action of PTX. This study clearly demonstrates the successful and selective delivery of PTX by an RGD peptide.
In 2015, the same research group reported the syn- thesis and evaluation of four multivalent RGD–PTX
receptors was comparable to that of cyclo[DKP–RGD]. Conjugate 34 showed the worst inhibition of biotinyl- ated vitronectin binding to integrin avβ3 and low solu- bility in the medium. Compounds 33–35 were stable at different pH conditions, while treatment of the conjugates 33 and 34 with lysosome extract resulted to cleavage of the peptide linkers and release of PTX. The ability of the conjugates to inhibit the cell prolifera- tion of CCRF-CEM avβ3 acute lymphoblastic leukemia

cell line was tested. Conjugates 33 and 34 inhibited the proliferation of CCRF-CEM (IC50 values of 77 ± 20 nM and 34 ± 2 nM, respectively) with slightly less potency than PTX (IC50 = 21 ± 2 nM) [44].

RGD–doxorubicin conjugates
Doxorubicin (Adriamycin, DOX, 36) is an anthracy- cline, originally isolated from fungus Streptomyces peu- cetius in the 1970s and one of the most widely applied chemotherapeutic agents (Figure 8). It is used either as a single agent or in combination chemotherapy for the treatment of cervical, endometrial, breast, ovarian, pancreatic, prostate, lung, thyroid, multiple myeloma, sarcoma and pediatric cancers [62] . However, its clini- cal application is limited by its side effects such as nausea, vomiting, diarrhea, myelosuppression and car- diotoxicity [63,64] . DOX operates in the cancer cell pri- marily by intercalation into the DNA and disruption of topoisomerase-II and secondly by generation of free radicals causing damage to cellular membranes, DNA and proteins. DOX is oxidized to semiquinone (in ring

B), an unstable metabolite, which is converted back to quinone with generation of reactive oxygen species (ROS) leading to cell death [65–67] .
In 2004, Burhart and coworkers reported the syn- thesis of two RGD conjugates with doxaliform (38, DOXSF), a derivative of DOX with formaldehyde and salicylamide [68], which is used to overcome resistance to DOX (Figure 8). DOXSF has an N-Mannich base which upon hydrolysis releases the doxorubicin active metabolite. DOXSF have been coupled with two pep- tides, the acyclic RGD4C and the cilengitide, 3. Both conjugates had an oxime group on the linker to attach 5′-formyldoxaliform to the peptide. Additionally, D-phenylalanine was substituted by D-4-aminophe- nylalanine in c(RGDf[N-Me]V), providing a connec- tion position for DOXSF (Figure 8). The conjugates displayed good binding affinity for ανβ3 integrin in the vitronectin cell adhesion assay, with an IC50 of 10 ± 1 nM for 40 and 5 ± 1 nM for 41. The antiprolifera- tive properties of the conjugates have been evaluated in MDA-MB-435 cells. After 4 h of treatment, both

HN HN
NH2 NH2
O NH O NH
O O

HN
NNH

O
HN
NNH

O
HN
O
HN

H2N
NH
H
N

O
O
OH
O
O
OH

O O
8, c(DKP-f2-RGD) 9, c(DKP-f3-RGD)

H2N HN
O
NH
H2N HN
O
NH

N
H
O
N
H
O

N
O
HN
N
O
HN

H2N
O
NH

O
OH
H2N
O
NH

O
OH

10, c(DKP-f4-RGD)
O

O11, c(DKP-f6-RGD)
O

O O

PTX1
O
c(DKP-f2-RGD)
O
PTX1
c(DKP-f3-RGD)

O O

12
13

O O

PTX1
O

14
c(DKP-f4-RGD)
PTX
1
O

15
c(DKP-f6-RGD)

Figure 4. Molecular structures of cyclo[DKP-RGD] peptides and cyclo[DKP-RGD]–PTX conjugates.

HN OH
NH2

N
O
HN
O

O
NH

O
O

H
N
N

N
H

HN
N
H

NH2

O
16, AbaRGD II
HO
17, AmproRGD III

Figure 5. Structures of AbaRGD II and AmproRGD III peptides.

40 and 41 were more cytotoxic than DOX with IC50 values of 50 ± 10 nM and 90 ± 20 nM, respectively. DOXSF had an IC50 of 50 ± 10.4 nM and DOX an
observed for 44. The conjugates have been tested for their ability to inhibit HUVEC proliferation. Free DOX (IC50 = 700 fM) inhibited proliferation more

IC
50
of 120 ± 30 nM. The uptake of 40 by MDA-
efficiently than the four peptide conjugates. The IC50

MB-435 cells was measured by flow cytometry and showed that the conjugate does not significantly pen- etrate the cell membrane. This result, together with the growth inhibition experiments, implies that the release of the DOX active metabolite occurred extracellularly.
During the same time, Kim and Lee published the synthesis and activity of a DOX conjugate with the cyclic RGD4C. The conjugate was synthesized using an activating ester of DOX according to Figure 9 [69]. DOX–cRGD4C conjugate 43 was less cytotoxic than free DOX against hepatocellular carcinoma cells (MH134) with IC50 values of 16.5 and 0.14 μM, respectively. However, the in vivo results showed that 43 inhibited the growth of an orthotopic mouse hepa- toma (C3H/He) more effectively than free DOX, at a dosage of 20 μg of DOX equivalent per admin- istration. In addition, histological analysis demon- strated complete tumor cell death in two of the five cases treated with 43. It should be noted that MH134 do not express integrin αν receptors in vitro and the authors attributed the enhanced in vivo activity to the disruption of integrin αν -expressing tumor vessels.
In 2008, Ryppa and coworkers have published the synthesis and evaluation of DOX conjugates with E-[c(RGDfK)]2 [70]. Two maleimide derivatives of DOX to E-[c(RGDfK)]2 have been developed. In the first conjugate 44, DOX is attached to the peptide via an amide bond and in the second 45, an MMP2/MM9 cleavable octapeptide was used as linker between DOX and the peptide (Figure 9). This octapeptide was selected because the matrix metalloproteases MMP2 and MMP9 are usually overexpressed in tumor vas- culature. In addition, two analogous DOX conjugates have been prepared bearing the peptide c(RADfK), which does not bind to ανβ3 and served as negative control. As expected, 45 was cleaved by MMP2 and MMP9 in OVCAR-3 tumor homogenates releasing DOX or Ile-Ala-Gly-Gln-DOX, while this was not
values was 20 nM for 44, 3 nM for 45, 5 nM for 47 and 30 nM for 46. These encouraging in vitro data prompted in vivo experiments in OVCAR-3 xenograft mice. However, the conjugates exhibited no or moder- ate antitumor efficacy at doses of 3 × 24 mg/kg DOX equivalents compared with the antitumor effect of DOX (2 × 8 mg/kg). The authors describe a number of explanations for this result. One explanation could be that the conjugates are taken up rapidly by endocyto- sis and that would prevent the release of DOX by the MMP2 and MMP9 metalloproteases.
This concept of a DOX conjugate linked to an RGD peptide via a peptide linker, which would be cleaved by a protease was earlier applied by Groot’s group [71] . They studied the synthesis and activities of a conju- gate (48), in which a cyclic RGD4C peptide carries DOX and a D-Ala-Phe-Lys tripeptide linker (Figure 9). This tripeptide is selectively recognized by the tumor- associated protease plasmin. Compound 48 inhibited the binding of the cells to vitronectin on HUVEC cells and could be cleaved by plasmin, though only 30% of prodrug were easily converted to DOX. In the presence of plasmin, 48 was almost as toxic as DOX against HUVEC and HT1080 cells (IC50 = 0.75 μM and IC50 = 0.28 μM). Therefore, although a number of RGD– DOX conjugates have been prepared providing impor- tant insights for the delivery of DOX, further devel- opment of new RGD delivery systems with different linkers is clearly necessary for the selective targeting of tumor cells with DOX.

RGD–platinum conjugates
Platinum(II) complexes, such as cisplatin (49), carbo- platin (50) and oxaliplatin (51) are widely used antitu- mor agents for the treatment of lung, colorectal, ovar- ian, breast, head/neck, bladder and testicular cancers (Figure 10) [72]. The mechanism of action of platinum complexes has been associated to their ability to inter-

act with DNA, cause DNA damage and subsequently induce apoptosis in cancer cells. Major limitations to their clinical efficacy are associated with resistance development and increased toxicity. In recent years, attempts to overcome these disadvantages have been focused on the discovery of targeted drug delivery systems such as peptide platinum conjugates ana-

lyzed in this section [73] or the development of stable in blood platinum(IV) complexes, which are used as prodrugs [74,75] . The most noticeable representative of the second case is satraplatin (52), which is the first orally bioavailable platinum(IV) compound currently undergoing Phase 3 clinical trials as single and combi- nation therapy for different cancer types [76]. It should

O O O O

N
N

m
OPTX1
N
H
O
O
PTX1
N
N
O
PTX
1

N
O
O N

Aba
18, m = 1 19, m = 7
3
N
H
N
N
N
20
n
Ampro
21, n = 1 22, n = 5

O O

Aba
N
O
O
PTX1

HN
O

O
O

PTX1

HN
O

O
O

PTX1
HN
H
O

Ampro
24 Aba 23

Aba

H
N

PTX1
N
N
N
O
O
N
N
N

PTX1
O

26
Ampro

PTX1
Ampro

O
PTX
1
O
O
O

HN
O

N
H

N
N
N
Aba PTX1

N
N
N
Aba
28
Aba
N
N
N

H
N

29, n = 0 30, n = 1

O
O
H
N
Aba
N
N
N

Aba

N
N

O
O

N
N
Aba
PTX1
O
O

N
N

O
N

H
N

O
O
H
N

O
N

N
N

HN
N

O
N
N

Aba

H
N

Aba
N
N

3
N

Aba
31
Aba 32

HN
NH2

N
O

HN
NH
N

Aba =

O
O
O
HN

NH
O
Ampro =
O
O

HO
HN

H
N

O
HN

N
H

O
N
H
NH2

Figure 6. AbaRGD– and AmproRGD–PTX conjugates.

PTX
1

c(DKP-f2-RGD)

PTX
1

H
R2 O
H

NO N
N
NO
N
c(DKP-f2-RGD)

O O
H
R1

33, R1 = CH(CH3)2, R2 = CH3 34, R1 = Bn, R2 = (CH2)4NH2

Figure 7. Structures of cyclo[DKP-f2-RGD]-Val-Ala-PTX (33), cyclo[DKP-f2-RGD]-Phe-Lys-PTX (34) and cyclo[DKP- f2-RGD]-uncleavable-PTX (35).

be noted that platinum(IV) complexes have to be acti- vated after reduction to the active platinum(II) species in order to exert their chemotherapeutic activity.
In 2007, Lippard, Barnés and coworkers have reported the synthesis and the biological evaluation of platinum(IV) conjugates with RGD, c(CRGDC), c(RGDfK) or NGR peptides [77]. In total, six mono- functionalized and five difunctionalized analogs were synthesized as shown in Figure 10. The conjugates were tested for their inhibitory activities against different primary endothelial cell and tumor cell lines. Cisplatin was used as positive control and the compounds 53, 56a, 56b, 57a and 57b as negative controls. In bovine endothelial cells, cisplatin was the most efficient in inhibiting cell growth with an IC of 1.1 ± 0.11
50
μM. In general, the monofunctionalized and difunc- tionalized conjugates showed similar effects. Among them, compounds 54a, 54b and 59a, 59b were the most potent with IC50≈2.1–3.4 μM. A similar inhibi- tory activity was witnessed when the conjugates were tested against human microvascular endothelial cells. In this case, 58, 59a and 59b were the most active with IC ≈2.7–3.4 μM. Therefore, the RGD-Pt(IV)
50
complexes were the most potent inhibitors of cellular proliferation in this study compared with nontargeted platinum(IV) compounds (56, 57), to the uncon- jugated RGD tri- and penta-peptides and to NGR conjugates.
Conjugates of a Pt(IV) derivative of picoplatin with monomeric and tetrameric RGD-containing peptides, Pt-c(RGDfK), 63 and Pt-RAFT-{c(RGDfK)}4, 64 were recently reported by Marchán and coworkers [78]. After their synthesis, the antiproliferative activity of the conjugates was determined in SK-MEL-28, CAPAN-1 and 1BR3G cell lines using the MTT assay. It was found that the cytotoxic effect of picoplatin was superior by conjugation to both RGD peptides. Conjugate 63 dis- played an IC50 value of 12.8 ± 2.1 μM, while picoplatin showed an IC50 of 33.6 ± 6.6 μM (Figure 10). Conju- gate 64 was more active with an IC50 of 1.7 ± 0.7 μM. The authors further demonstrated the activation of the
Pt(IV)–peptide conjugate by reduction using ascor- bate as reducing agent, which leads to the generation of Pt(II) species. These species have the capacity to react with DNA nucleobases, as indicated by the formation of 5′-guanosine monophosphate adducts.
In 2015, the same research group developed a pho- toactivable RGD-Pt(IV) conjugate as a selective anti- cancer prodrug [79]. The succinylated complex 66 was coupled to c(RGDfK) by using 1-[bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate in the presence of N,N- diisopropylethylamine in anhydrous DMF to produce 68 in 54% yield (Figure 11). The photoactivation prop- erties of the Pt-c(RGDfK) conjugate were investigated by irradiation of the complex (λmax = 420 nm, 11 mW cm-2, 45 min, 37°C) in the presence of 5′-GMP (5′-guanosine monophosphate). This experiment showed the disappearance of 68 and the formation of the adduct Pt(II)-GMP, 70. After showing the pho- toactivation of conjugate 68, the toxicity toward dif- ferent cancer cell lines in the presence of visible light was assessed. Compound 68 had an IC50 value of 19.5 μM in SK-MEL-28, similar to that of the parent com- plex 66 (IC50 = 15.5 μM), whereas the IC50 values in DU-145 cells were different, IC50 = 20 μM for 66 and 54 μM for 68.
Fluorogens with aggregation-induced emission (AIE) characteristics have attracted particular atten- tion in biomedical applications and in photody- namic therapy because they are nonemissive in the molecularly dissolved state and highly emissive in the aggregated state [80]. Liu and coworkers synthesized a platinum(IV) prodrug, 71 conjugated with a pep- tide and a photosensitizer with AIE characteristics for selective and real-time monitoring of drug activation in situ, as well as for combinatorial photodynamic chemotherapy against cisplatin resistant cancer cells (Figure 11) [81]. The authors showed that upon glutathi- one (GSH) treatment in vitro 71 was activated with the fluorescence turn-on from the released photosensitizer. Furthermore, they proved the formation of ROS spe-

cies upon light irradiation. The drug release and the generation of ROS species were verified in vivo upon reduction by intracellular GSH. The antiproliferative properties of 71 were evaluated in MDA-MB-231 cells,

U87-MG cells, MCF-7 cells and 293T cells. Conju- gate 71 was found equally cytotoxic to cisplatin (IC50 = 33.4 μM) against MDA-MB-231 cells under dark con- ditions, but its cytotoxicity was enhanced significantly

8a
OOH O

OH NH2
O OH O

DC B A OH
OH 37
OH

O O OH O O
HCHO
O O OH O O

38, DOXSF

36, DOX
NH2 NH

Release
H2O

OH
O
H
O
2′
3′

1′

4′
NH

6′

5′

pH 7.4

37°C
O O

39, DOX
OH
O
O

active metabolite
NH
OH

8b
OH

O
NH
NH
O
NH2
HS

HS
O
O
N
H
HS
H
N

H
N
O
HS

N
H
O

O
OO
N
H
HN
H
N

DOX1
NH

acyclic-RGD4C
OH

HN
NH
2

O O

DOX1
N
H
HO
N
O
N
H

40
O
acyclic-RGD4C

HN NH2
NH
O O

O
N

DOX1

N
H

N
O

N
H
c(RGDf[N-Me]V)

O
N
H
HN HO

NH HN
O
O
O
OH
c(RGDf[N-Me]V)

Figure 8. RGD conjugates with DOX. (A) Synthesis of DOXSF and the mechanism by which it releases the proposed DOX active metabolite. (B) Structures of c(RGDf[N-Me]V)–DOXSF and acyclic-RGD4C–DOXSF conjugates.

NH2

36
O

O
O

O DOX1

H2N
O

N
H

H
N
HO

O
O

O
NH

EDCI O
42
O

NH
S S

DOX1
O

43
cRGD4C

cRGD4C

H
N

N
H

H
N
S

O
O

NH
NH

cRGD4C

O
Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-DOX1

4 DOX1
O
O 3

N N
HN NH2

O
HN
NH

44 45
N
O

NH
HN

NH
O
NH
H
HN

c(RGDfK) c(RGDfK) c(RGDfK) c(RGDfK) NH HN
O

O O O O
O

O
Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-DOX1 O

OH
O c(RADfK)

O
4 DOX1
O
O 3

S
N

NH2 NH2

46
47
c(RGDfK) c(RGDfK) c(RADfK) c(RADfK)

HN
HN O O O O

c(RADfK)
NH

c(RADfK) c(RADfK)
NH

c(RADfK)
E-[c(RGDfK)2]
E-[c(RADfK)2]

O O
O O

NH2

HO NH O

cRGD4C1

H
N

N
H

H
N

CH2

N
H
4

DOX1
H2N

N
H

O
H
N
HN

O
O
NH

S
HN OHN O
O

48
NH2
O

H
N

O
S

N
H
O

H
N

NH
O

cRGD4C1

Figure 9. Conjugates of DOX with RGD4C, E-[c(RGDfK)]2, E-[c(RADfK)]2 and cRGD4C (see facing page).

upon light irradiation (IC50 = 4.2 μM).
A theragnostic prodrug cRGD-TPE-Pt-DOX com- prising of a cyclic RGD peptide, an AIE fluorogen and two anticancer agents, DOX and a platinum(IV) prodrug has also been developed (Figure 11) [82]. Upon intracellular reduction the two drugs are released from the conjugate, while the AIE fluorogen shows intense fluorescence, allowing prodrug tracking and real-time monitoring of drug activation. The targeted delivery of the cRGD-TPE-Pt-DOX was studied in MDA- MB-231 (overexpressed αvβ3 integrin), MCF-7 breast cancer cells (low αvβ3 integrin expression) and normal 293T cells (low αvβ3 integrin expression) by confocal microscopy. It was shown that the fluorescence from cRGD-TPE-Pt-DOX in MDA-MB-231 cells is 4.7 and 5.2-fold higher than that in MCF-7 and 293T cells, respectively. The cytotoxicity of cisplatin, DOX and 72 to MDA-MB-231 cells was assessed by MTT assays. Conjugate 72 was more cytotoxic than DOX with IC50 value of 0.69 μM. DOX and cisplatin had an
less cytotoxic than CPT in A2780 ovarian carcinoma, A498 renal carcinoma and PC3 prostate carcinoma cells. The reduced activity was attributed to the nature of the amide bond, which is too stable to release the drug. Therefore, the authors proceeded to the develop- ment of conjugates 78 and 79, which carry the acid- labile hydrazone linker. Both 78 and 79 showed high cytotoxic activity, but poor solubility and stability even at pH 7.4. In the cytotoxicity assays, 78 showed similar cytotoxicity to CPT in all cell lines tested. The antitu- mor effect of conjugate 77 was further investigated on A2780 human ovarian carcinoma xenograft in nude mice. The compound was administered bis in die intra- peritoneally or subcutaneously and caused a tumor vol- ume inhibition of about 40% at a dose of 48 mg/Kg. Pharmacokinetic studies with 77 using the same xeno- graft model agreed with the in vitro experiments, and showed that free CPT is very slowly released from the conjugate, thus confirming in vivo its stability.
During the same year, Huang and coworkers

IC
50
of 1.6 μM and IC50 of 18.1 μM, respectively. Fur-
reported the synthesis of an RGD–CPT conjugate

thermore, it was proven that the combination of DOX with cisplatin in 72 resulted in a synergistic anticancer effect.

RGD–camptothecin conjugates
Camptothecin (CPT) is an anticancer pentacyclic qui- nolone alkaloid with a terminal α-hydroxy-δ-lactone ring, isolated in 1966 by Wall and Wani, from the bark of a Chinese tree Camptotheca acuminate [83]. CPT inhibits DNA topoisomerase I and stabilizes the topoisomerase–DNA covalent complex, preventing DNA replication, and thus causing apoptosis [84,85] . The integrity of the lactone ring appears essential for activity. Depending on the pH of the aqueous solution, CPT exists at equilibrium between the active closed lactone and inactive open carboxylate (Figure 12) [86]. To overcome the solubility and poor stability sev- eral CPT derivatives have been developed including irinotecan (CPT-11, SN38), 9-nitrocamptothecin, 9-aminocamptothecin, tomotecan, belotecan, which have been approved for the treatment of metastatic colorectal cancer, ovarian cancer, cervical cancer and small-cell-lung cancer [87,88] .
In 2010, Dal Pozzo and coworkers have reported the synthesis and the evaluation of CPT conjugates with cyclic peptide analogs of c(RGDfV) [89]. CPT was attached to the cyclopeptides via an amide bond in conjugates 75–77, while cRGD1 was attached to CPT via hydrazone-based bond in conjugates 78 and 79 (Figure 12). All the conjugates showed good recep- tor affinity. However, conjugates 75–77 were found
using the CPT derivative, SN38 [90] (Figure 12). The prodrug consists of three parts: the cytotoxic com- pound, the indolequinone moiety and the peptide c(RGDyK). The release of SN38 from the prodrug takes place in the presence of DT-diaphorase, which is a reductase that catalyzes the two-electron reduc- tion of quinones to hydroquinones. Preliminary stud- ies verified the drug release from the conjugate, which was monitored via HPLC analysis. Thus, the conju- gate alone was noncytotoxic at ≈300 nM, whereas in the presence of DT-diaphorase there was a 50–70% reduction of cell growth.
Dal Pozzo and coworkers have reported the synthe- sis and the biological evaluation of seven RGD conju- gates of the CPT derivative, namitecan. These conju- gates differ to their linkers, consisting of glycol and/or peptide moieties in order to release the drug into tumor cells after enzymatic hydrolysis [91]. The first conju- gate 81 had the Phe-Lys dipeptide in the linker and exhibited rapid lysosomal hydrolysis and high instabil- ity in the murine blood. The other conjugates had the more stable dipeptide Ala-Cit (Figure 13). To improve the solubility, different poly(ethylene glycol) moieties were introduced in the linker. Among the compounds tested, conjugates 86 and 87 showed acceptable stabil- ity in murine blood and high cytotoxic activity with IC50 values of 8 nM in A2780 cells and IC50 values of 1 μM in PC3 cells.
Lee and coworkers developed an RGD–CPT con- jugate for theragnostic applications [92] . Conjugate 88 consists of c(RGDyK) peptide, CPT, a disulphide

H2N

O
O
H
NO

H2N
O

Pt Pt Pt Pt

H2N
Cl
H2N
O

O
N
H
O
O
N
H
O
Cl

49, cisplatin 50, carboplatin 51, oxaliplatin 52, satraplatin

O O O

O
OH
O
OR
O
OR

O
H2N H2N

Pt
Cl
Cl
O

+ R
EDC/NHS H2O

O
H2N H2N

Pt
Cl
Cl
O

O
H2N H2N

Pt
Cl
Cl
O

O
O

53
HO

O
O
RO

O
O

R = RGD R = NGR R = AGR R = Gly
R = c(CRGDC) R = c(RGDfK)
54a
55a
56a
57a
58
59a
54b
55b
56b
57b
not observed 59b

Cl
Cl

NH2

Cl
Cl
OH
Pt
OH

NH2

Cl
Cl
O
Pt
OH

NH2

O
OH

N
H

c(RGDfK)

60, picoplatin 61
62
63

NH2 NH

c(RGDKf) O

c(RGDfK)
O
N
N
N

N
N
N

c(RGDfK)

N
N
N

c(RGDfK)

N
N

N
O N

O
NH
H
HN
O
OO O

NH HN

O
Lys Lys

O Gly
Ala
Pro

O Lys Lys

OH
c(RGDfK)
Pro
Lys
Gly

Cl
Cl
O
O
NH2
Pt
N
OH

H
N

RGD-containing
peptide
GMP Ascorbate

N7-GMP N7-GMP

NH2 N

H
N

RGD-containing
peptide

Figure 10. Structures of anticancer platinum complexes and conjugates of Pt(IV) complexes.

linker cleavable by thiols existing in tumor cells and a naphthalimide fluorophore. Figure 14 presents the mechanism for the release of CPT from the conjugate upon GSH treatment. GSH cleaves the disulfide bond,
which leads to intramolecular cyclization and cleav- age of the carbamate group followed by release of the fluorescent compound 89. Confocal microscopic stud- ies revealed the selective uptake of the conjugate by

U87 glioma cell lines, which express the avβ3 integrin receptors. Competition experiments with the inhibitor of endocytosis, okadaic acid, revealed that the uptake of the conjugate takes place via an RGD-dependent endocytosis mechanism. The cytotoxicity of 88 was assessed in the same cell line with conjugate 88 to show 49% cell viability at 1 μM within 48 h after treatment.
Gellerman’s group had a great contribution on the identification and characterization of bioactive con- jugates of CPT and the alkylating anticancer agent Chlorabucil (CLB) with c(RGDfK) pentapeptide and c(RGDfS), which resulted by substitution of the amino acid Lys with Ser (Figure 14) [93]. Amide and

ester bonds were selected for CLB and a carbamate bond for CPT for the attachment of the cytotoxic moi- ety to the core peptides. The synthesized conjugates were evaluated for their biostability using murine liver homogenate. The biodegradable ester (conjugate 93) and carbamate (conjugate 94) released a maximal amount of drug in 10 min and 45 min, respectively, whereas the amide conjugate 92 did not release CPT. Chemostability tests have been performed in pH 5.2 and 7.4, which showed a relative fast decomposition of 92 and 93, without any release of the drug. Conju- gate 94 displayed relative stability at both pH values, but released CPT only at pH 7.4. The cytotoxicity of the conjugates was measured in human non-small-cell

OH
N N3
Pt
N N
3 OH
O
N N3
Pt
N N
3 OH

O
OH

H2N

O
65

c(RGDfK)
66

66
HATU, DIPEA

N
H

c(RGDfK)

67 DMF 68

O
N3

Pt
N
O

68
5′-GMP hv
HO

O
N
H
O

69
O

O
c(RGDfK)

O
P
O

O
NN
N
O

HO OH

N
NH
NH2 70

O O

H
O

ON
O

O
H2N H2N

Pt
Cl
Cl

O
COOH

NC
CN

71
O

O
N
H

O
c(RGDfK)

N N
N

N
H
HN

N
N

N
H

H2N H2N
O
O
Pt
O

Cl
Cl

O
DOX1

HO
NH HN

O
N
H

NH2

Figure 11. Structures of photoactivable RGD-Pt(IV) conjugate 68 and platinum(IV) prodrugs 71 and 72.

O O

A B C N
N
D
OH

N
N
OH

EO
H
O

HO O
74

HO O

H
cRGD N
2

CPT1

cRGD1

H
N

O CPT1
75 76 O
N
N

CPT1 N

N
O

cRGD1

CPT1
O

H 2
O
OH O

O
cRGD1

O
H
N

CPT1
O
cRGD1

O
H
N

CPT1

2
O
78
2

79
O

SN38

N
O

N
O
O
R

O
O
O N
O
N

O
O
NH
N
H
HN
O

O
N
H

H
N

N
H

O
OH O

O
cRGDyK
HO

cRGD1
NH H N
O

H2
R = C

NH NH2

cRGD2 R = -(CH2)3CO-

O OR OH OR
OH

O
2e-
O
-RO
O

N N
N

O OH
OH

Figure 12. RGD conjugates with CPT. (A) Structures of CPT–cyclopeptide conjugates; (B) the reductive mechanism by which SN38 is released.
CPT: Camptothecin.

lung carcinoma cell line (H1299) and murine mela- noma cell line (B16F10), while human embryonic kid- ney 293 (HEK293) has been used as negative control. It was found that H1299 and B16F10 cells were less sensitive to 93 with comparison to 92, while the nega- tive control cells (HEK293) were slightly more affected by the treatment with 93. CPT conjugate 94 was less cytotoxic than the free drug, in all tested cell lines up to a concentration of 50 μM, while all three conjugates
affect the negative control as well. This can probably be explained, as the author suggests, by the premature release of the drug from the conjugates, which leads to the nonspecific mode of action similarly to the parent drug.
Gellerman and coworkers also developed biloaded conjugates of CLB and CPT on c(RGDfK) (Figure 14) [94]. Lys residue of c(RGDfK) was modified by coupling with a sixth aminoacid (Lys or Ser) result-

ing in two functional sites and enabling the loading of two drug equivalents onto a single peptide carrier. Amide, ester and carbamate bonds were used for the coupling of the drugs to the peptide. The chemosta-

whereas 97 showed a moderate release of CPT at pH
7.4.As observed in the previous study, the biodegrad- able ester and carbamate bonds released the drugs in biostability assays. On the other hand, the stable amide

bility of the conjugates 95–98 at neutral and acidic
bond led to the decomposition of 95 (t
1/2
<7 min), pH and the biostability in murine liver homogenate were studied. Conjugates 95 and 96 were decomposed without releasing the drug at both pH 7.4 and pH 5.2, without the release of the DNA alkylator. The cyto- toxicity of the biloaded conjugates compared with the monoloaded conjugates of Figure 14 was also studied. NMT O O O N H O H N O NH2 N H O 81 O O 2 O cRGD1 NMT O N O H N N O O O cRGD1 O H O H 2 O NMT O O H O NH N H N O N H N N N H2N O 82 NH O H2N O O O NMT O N O H N N O N N 83 2 O cRGD1 H O H N O O H N O H2N NH O 2 O N H O O O Q 2 84a, Q = cRGD1 84b, Q = cRGD3 NH O H2N O NMT O N H O H N O N H O N N N NH O O H N O H2N O 85 2 O N H O O O cRGD3 2 NH 2 H2N O H2N O NH O H N O O cRGD3 NMT O H N O N H O H N O O O O O H N N N N O N H O H N O 2 O O 2 O cRGD3 O 86 O cRGD3 NMT O N H O H N O NH H2N O N H O O 87 O N N N HN O O H N O N H N N N O O O O 2 2 O cRGD3 O O 11 O N H N N N O Figure 13. Conjugates of cyclic RGD peptides with namitecan (cont. from previous page). Conjugates 95, 96 and 98 were more cytotoxic than the monoloaded counterparts (92–94) and 95 was more effective than its amide-ester analog 96. Gellerman’s group further reported the synthesis of a linear RGD-NGR peptide–CPT conjugate 99, two cyclic RGD conjugates of diacetyl colchicine 100, amonafide 101 and the known 92 and 94 [95]. In this work, the cytotoxicity of the linear 99 compared with its cyclic counterpart 94 was tested in H1299, PC3 cells in HEK293 cells. CPT displayed a nonspecific dose-dependent manner of action toward all the tested cell lines. The best results were obtained with conju- gate 94, which was more potent and selective than 99 against the integrin overexpressing H1299 and PC3 cells. Remarkably, 94 was more toxic than free CPT on PC3 in all concentrations tested. The biological evaluation of 100 and 101 remains to be explored. RGD–SMAC mimetic conjugates Gennary and coworkers have recently reported the syn- thesis and in vitro biological evaluation of dual action conjugates 102–104 (Figure 15), containing the previ- ously described c(DKP-f 3-RGD) and Aba peptides with a proapoptotic AVPI/SMAC mimetic unit (cyclo-RGD/ SMAC mimetic conjugates) [96]. The conjugates were obtained via an ester or an amide linker among the two units. SMAC mimetics have recently received particu- lar attention and are currently undergoing clinical tri- als [97]. This class of drugs mimic SMAC and sensitize cells to apoptosis by binding the Inhibitor of Apoptosis Proteins [98]. Thus, the synthesized conjugates aimed to target both the apoptosis and angiogenesis of tumor cells. After their synthesis, it was shown that 102–104 were able to inhibit biotinylated vitronectin binding to the purified αvβ3 and αvβ5 receptors. The cytotoxic effects of the compounds were tested in ovarian IGROV-1 carci- noma cells and MDA-MB-231 cells. Conjugate 102 and the parent SMAC mimetic were similarly potent against MDA-MB-231 cells with IC50 = 9.7 ± 1.6 μM, while con- jugate 103 and 104 were less active with IC50 >25 μM
and IC50 = 20.5 ± 2.2 μM, respectively. Thus, the conju- gation of an RGD peptide to a SMAC mimetic proved not particularly favorable for antiproliferative activity.

RGD–peptide conjugates
Early studies have reported the preparation and evalu- ation of a number of conjugates of RGD with peptides and proteins, which are thoroughly analyzed by Kok and coworkers [44]. Herein, a recent representative exam- ple from this category is given, which involves an RGD conjugate with a peptidyl VEGF receptor (VEGFR) antagonist. This dual action conjugate contains the previously described cyclo[DKP-RGD]-CH2NH2 pep- tide and targets both integrin avβ3 and VEGFRs [99]. It is known that the VEGF and its receptor are playing important roles in vasculogenesis and angiogenesis [100]. Studies of many groups have demonstrated that the cooperative interaction between integrin αvβ3 receptors and VEGFR2 seems to be particularly important pro- cess during angiogenesis [101]. Based on these findings a conjugate that targets both αvβ3 and VEGFR receptors could prove beneficial for targeting tumor angiogen- esis. As VEGFR ligand, a previously reported α-helical decapentapeptide was employed [102]. Conjugate 105 has been shown to bind to both receptors although with lower potency and have potent antiangiogenic activity in VEGF stimulated morphogenesis assays on HUVEC (Figure 15). However, its efficiency in preventing angio- genesis was similar to that of the starting peptides; cyclo[DKP-RGD]-CH2NH2 and the VEGFR antago- nist, and no significant benefit could be observed by their conjugation.

RGD–dexamethasone conjugates Dexamethasone (Dex) is a synthetic glucocorticoid used in the treatment of autoimmune, allergic and inflammatory diseases [103]. Its clinical efficacy is restricted by a series of side effects including osteopo- rosis, darkening or lightening of skin color, diarrhea, nausea, indigestion, dizziness, swelling and vomiting,

14a

H
N

c(RGDyK) N O O

O O
88
O
N

O
GSH

c(RGDyK)
O

N
H
N

O
O

N
O

O
O O 89 90

HS
O O O

O
O NH2
c(RGDyK) N + CPT
O O
91

14b

O
O
O
O
O
O
O Cl

n CLB
HO NHHN X
NH
H HN O
O
N
HO

O
NHHN NH H
HN
N
n

O
N
H
CPT2

O O CLB
HN NH HN NH

92, n = 4, X = NH 93, n = 1, X = O

O CLB
X
n
c(RGDfK) CLB
N
H
O
NH2

c(RGDfK)

X
n
N
H
94, n = 4

CPT2 O
c(RGDfK)
CPT2
NH2

X
n
N
H

CPT
2

CLB

95, n = 4, X = NH 96, n = 1, X = O
97, n = 4, X = NH
98, n = 4, X = NH

O
14c CPT2

H-Asn-Gly-Arg-NH

NGR O
99

NH
O

Arg-Gly-Asp-OH

RGD

O
O
O
O
n Q
HO NHHN X
NH
H HN O
O
N
O
HN NH

O
O

N
NH2

100, n = 2, X = CO2NH, Q = Q1 101, n = 2, X = CO2NH, Q = Q2

Q1, Deacetyl colchicine Q2, Amonafide

Figure 14. RGD–CPT conjugates (cont. from previous page). (A) Structure of the theragnostic prodrug 88 and
the mechanism for the release of CPT upon GSH treatment. (B) Structures of the conjugates of CPT and CLB with c(RGDfK). (C) Linear RGD-NGR peptide–CPT conjugate and two cyclic RGD conjugates of diacetyl colchicine and amonafide.
CLB: Chlorabucil; CPT: Camptothecin; GSH: Glutathione.

among others [104]. The development of RGD conju- gates in order to enhance the anti-inflammatory effi- cacy and limit the osteoporotic risk of Dex has recently been reported. Three conjugates of RGDV, RGDS and RGDF and Dex 106–108 with succinic linker have been synthesized (Figure 15) [105]. It was found that the conjugates have nanoproperties and are form- ing trimers and nanoparticles. The anti-inflammatory effects of Dex and its conjugates were evaluated with xylene-induced ear edema assay. It was shown that 106–108 exhibit increased anti-inflammatory activi- ties compared with Dex and lower osteoporotic risk.
The immunosuppressive activities, thrombosis risks and toxicities have also been investigated [106]. Among the compounds tested, RGDF-Dex 108 was the most potent inhibitor of ConA-induced spleen lymphocyte proliferation with IC50 = 0.031 μM (Dex has an IC of
50
0.233 μM). In addition, the rejection reaction was stud- ied with the survival time of implanted myocardium of the mice to be increased after treatment with the conju- gates. In vivo experiments showed that 106–108 possess no liver, kidney or systemic toxicity (at a single dose of 14.3 μmol/kg) and exhibit better antithrombotic activity compared with the parent drug.

H
N

c(DKP-f3-RGD)
O

X
N
H

HN
O
O
O

O
N

102, X = O 103, X = NH

H
N

O
H

Aba

N
H

104

HN
O
O

N
H
O
N
H
10

H
O

O N
O
O
N
c(DKP-f3-RGD)

NH
N
N
7
O

Leu-Thr-Trp-Met-Glu-Leu-Tyr-Gln-Leu-Ala-Tyr-Lys(Ac)-Gly-Ile-amide O 105
O
O

Dex1
O
RGDV
HO OH

O Dex1 =
H

106 F H
O

O O

Dex
1
O

RGDG
Dex1
O
RGDF

O O

107
108

Figure 15. Conjugates of c(DKP-f3-RGD) and Aba with a SMAC mimetic (102–104), conjugate of cyclo[DKP-RGD]- CH2NH2 with a VEGF receptor antagonist 105 and conjugates of RGD with DEX (106–108).

c(RGDyK)

N
H

H
N

H
NO

H
N

N
H

H
NO

H
N

109, X = CH2 110, X = CH2CH2
111, X = CH2CH2CH2 112, X = CH2CH2CH2CH2 113, X = CH2OCH2CH2

114, P = dimer-cRGD 115, P = RGD-PEG4 116, P = c(RADyK)

O
H
NO

c(RGDyK) OH N
PD0325901

O
F I

117
PD0325901
F

O O

c(RGDyK)

O
O

4
N
H

O
PD0325901
c(RGDyK)

O
N
H

O
PD0325901

118
119

O O

O
OO

O
HN Cl
O
OO HN Cl

R1 R2
N
H
O
O

O
O
HN
O
O
HN

NH
2

122
NH

120, R1 = CH3, R2 = H, cryptophycin 1

121, R1 = R2 = CH3, cryptophycin 52
N
N
N

O
N

O
H
NH
NH

O
HN
HN

OHN O O
O c(RGDfK)
O
OH

123
N

O
OH O

N
NO
HO
RGD(OX1)-NH2

X2
124, X1 = Bn, X2 = Br

O
125, X1 = H, X2 = Cl 126, X1 = Bn, X2 = Cl 127, X1 = Bn, X2 = NO2

Figure 16. RGD–PD0325901 conjugates (109–119), cRGDfK–cryptophycin conjugates (120–123) and RGD–salicylic acid conjugates (124–127).

RGD–PD0325901 conjugates
In 2013, Chen and coworkers reported the development of RGD–MEK1 kinase inhibitor conjugates based on the PD0325901 structure (Figure 16) [107]. PD0325901 is a potent allosteric inhibitor of mitogen-activated pro- tein kinases MEK1 and MEK2 with potent anticancer activity [108]. The dual specific threonine/tyrosine kinase MEK is a key component of the RAS/RAF/MEK/
ERK signaling pathway that is often dysregulated in human cancers [109]. PD0325901 inhibitor was coupled with monomeric c(RGDyk), dimeric E[c(RGDyK)2], cRGD–PEG4 and [c(RADyK)] peptides. The conju- gates were tested for their kinase-inhibiting activity, which was not significantly affected by the different length carbon chain. The IC50 values in the BRAF– MEK1 assay were 2.6 nM for PD0325901 and 47.9 nM

for 110. The cytotoxic potency and selectivity of the conjugates were measured against three different tumor cell lines, including the U87 (glioblastoma), the A549 (NSCLC) and the BRAF-V600E mutant A375 cell line. 114, 110 and 111 exhibited selective cytotoxicity against the tested carcinoma cell lines (A375 > U87 > A549) and similar potency compared with PD0325901. Conjugate 117, which has a succinyl linker with an ester bond for the attachment of PD0325901 exhibited

side effect [113]. In attempt to optimize the pharmaco- logical profile of cryptophycins, Sewald and coworkers synthesized a number of cryptophycin derivatives and an RGD–cryptophycin conjugate [114]. The cytotoxicity of cryptophycin analogs was evaluated using a cell-based resazurin assay in the human cervix carcinoma cell lines KB-3–1 and KB-V1. Conjugate 122 exhibited a very low cytotoxicity compared with the parent drug with IC50 = 55.8 nM in KB-3–1 cells and IC50 = 1.8 nM in KB-V1

potent antiproliferative activity with IC
50
= 17.6 nM,
cells (IC50 = 15.5 pM in KB-3-1 cells and IC50 = 0.26 nM

while PD0325901 had IC
50
= 0.47 nM against A375
in KB-V1 cells for 121). A fluorescent cyclic RGD–cryp-

cells. It should be noted that the antiproliferative effect of 117 was higher than PD0325901 in U87 glioblas- toma cells with an IC50 value of 2.38 μM. Furthermore, it was shown that conjugate 117 inhibited p-ERK-1/2 and DNA replication in a dose-dependent manner.
The same research group extended their studies on 117 against glioblastoma [110]. Two more conjugates with a PEG and a peptide linker were synthesized (Figure 16). In all cases PD0325901 was connected to the linker with a succinyl group and a hydrolysable ester bond. The anti- cancer efficacy of 117 was screened against seven cancer
tophycin conjugate 123 was also synthesized and was used for the visualization studies of internalization and final localization of 123. It was found that within 15 min the conjugate undergone endocytosis and was localized in the lysosomes of WM-115 melanoma cells.

RGD–salicylic acid conjugates
In order to develop potent inhibitors of platelet aggre- gation Liakopoulou and coworkers have synthesized RGD and KGD peptide conjugates with resveratrol or salicylic acid derivatives [115,116]. Antagonists of platelet

cell lines (including U87MG, U251MG, A549, MDA-
glycoprotein (GPIIb/IIIa or integrin αIIb
β
3
) efficiently

MB231, A375, HT29 and HCT116) by using the SRB method and comparing to the free PD0325901. All the conjugates demonstrated similar antiproliferative effects as PD0325901 on MDA-MB231, A375, HT29 and HCT116 cell lines at 10 μM, but higher against GBM cell lines (U87MG and U251MG). The IC50 values of 117–119 against U87 cells were 4.9, 3.24 and 2.16 μM, respectively. The synergistic effect of PD0325901 with the RGD peptide in U87MG cells and in vivo was also shown. The biostability of conjugates was tested by incu- bating 117, 118 or 119 with 50% human serum. It was found that the conjugates 118 and 117 were much more stable than 119. In vivo, conjugate 118 (5 mg/kg, seven times) was more effective in inhibiting glioblastoma xenograft U87MG than the parent drug, PD0325901.

RGD–cryptophycin conjugates
Cryptophycins are a group of 16-membered macrocyclic depsipeptides with potent antiproliferative properties against drug-resistant tumors [111]. Cryptophycin-1 120 was originally isolated from the cyanobacteria Nostoc sp. ATCC 53789 in 1990 (Figure 16) [112]. Treatment of cells with cryptophycins causes mitotic arrest with the formation of abnormal mitotic spindles by interacting with tubulin. Thus, they have a similar mechanism of action to microtubule destabilizing agents such as vinca alkaloids, colchicine and combretastatins. Among the natural cryptophycins and their numerous synthetic analogs cryptophycin-52 (121) was the first synthetic derivative that entered clinical trials (by Eli Lilly). The results were moderate with neurotoxicity to be a serious
reduce morbidity and mortality during percutaneous coronary intervention by preventing platelet aggrega- tion and thrombus formation. They act by antagoniz- ing GPIIb/IIIa receptors on the platelet surface and prevent the binding of fibrinogen that forms bridges between adjacent platelets. This binding is mediated by the sequence RGD in fibrinogen [117]. Four RGD deriva- tives with salicylic acid at their N-terminal amino group have been synthesized and evaluated for inhibitory activ- ity on human platelet aggregation induced by collagen in vitro (Figure 16). Compounds 124 and 126 were the most potent inhibitors exhibiting 67 and 75% inhibitory activity, respectively.

RGD–monomethylauristatin E conjugates Monomethyl auristatin E (MMAE, vedotin) is a very potent antineoplastic agent that inhibits tubulin polym- erization, and thus induces apoptosis [118]. Due to its toxicity, the clinical application of MMAE is limited; however, several antibody–MMAE conjugates are under clinical trials, and one of them, brentuximab vedotin, has been approved for cancer therapy [119]. In 2014, Tsien’s group reported the development of a complex conjugate of MMAE with a cyclic RGD peptide for theragnostic applications, cyclic-RGD-PLGC(Me)AG- MMAE-ACPP, 128 [120]. The conjugate consists of an activable cell penetrating peptide, a far-red fluorescent dye (Cy5) and MMAE. These basic units are con- nected with PEG and maleimide linkers as depicted in Figure 17. ACPPs are generally comprised of a polyca- tionic peptide, a polyanion and a protease (MMP2/9)

cleavable linker (PLGC(Me)AC). The conjugate showed better cellular uptake compared with the correspond- ing cyclic-RAD-PLGC(Me)AG-MMAE-ACPP in U87MG glioblastoma cells in vitro and better tumor volume inhibition compared with the corresponding cyclic-RAD-PEG6-MMAE and MMAE in vivo with orthotopic MDA-MB-231 mammary tumors. For the in vivo experiments, the conjugate was administered at a therapeutic dose of 0.2 mg/kg MMAE (≈6.5 nano- moles of the peptide–MMAE conjugate) and was well- tolerated. The tumor homing of the conjugate was also shown with fluorescence imaging.

Conclusion
In recent years, a variety of targeted drug delivery systems have been developed in order to improve the efficacy of currently available antitumor agents. Peptides, proteins and antibodies have been employed as carriers to target

receptors overexpressed in several tumors with great suc- cess. In particular, peptides have gained higher attention due to their higher stability and easy availability. Peptides bearing the RGD sequence target the integrin recep- tor and have been conjugated with different anticancer drugs. Presently, there is accumulating data indicating the efficacy of these RGD conjugates for selective tumor- targeted delivery both in vitro and in vivo. The scientific creativity drives research to elegant delivery systems that actualize the real time monitoring of drug delivery. These systems will advance our understanding for the mechanisms of drug release and have the potential to play an important role in cancer diagnosis and treatment. Efforts are currently underway for the development of new effective delivery systems. The use of RGD conju- gates is a promising approach in tumor-targeted therapy, however more studies are clearly necessary to investigate their potential clinical utility.

HN NH2

H
N

O
NH

S
(D-Glu) 12 9
PLGC(Me)AG O
HN
(D-Arg)9

O
N
O
S

O
O
N
NH H HN
NH HN

N
O
HN
S
Cy5
O

H2N
O
N

O
O
NH
2

H NH O

O
128

OH
N
H

N
N
N

O O O

MMAE

O
MMAE

SO3

N
H

HN
O

ONH

H
O

O
N
O

Cy5

N
N
O

PLGC(Me)AG

S
N
H

O
N
O
N
H

O3S

Figure 17. Structure of the cyclic-RGD-PLGC(Me)AG-MMAE-ACPP conjugate.

Future perspective
In the field of cancer imaging and therapy the overex- pression of integrin receptors on the surface of various malignant tumor cells has increasingly become of inter- est. RGD containing conjugates have attracted particu- lar attention and are intensively investigated as means to address drugs to angiogenic endothelial cells and cancer cells by their binding to ανβ3 integrins. Currently, new delivery systems with other chemotherapeutics coupled to integrin ligands with suitable linkers are strongly sought. Furthermore, the use of other cancer targeting peptides would allow the delivery of cytotoxic drugs to specific receptors of other types of tumor cells. Research on cancer is thriving with numerous novel drug candi- dates reported annually aiming specific protein mod- ules, enzymes, mRNAs, DNA and/or surrounding environment. Combination of these factors on a single

conjugate could prove beneficial. Hence, endless pos- sibilities emerge on the field of targeted drug delivery systems that need to be investigated on the upcoming years.

Financial & competing interests disclosure
This program was implemented within the framework of the IKY/SIEMENS Excellence Research Grants 2016 (synthesis of anticancer peptide derivatives). S Katsamakas is thankful to Aristotle University of Thessaloniki Research Committee for financial support through an excellence Postdoctoral Scholar- ship. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or ma- terials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Executive summary
Integrins & RGD peptides
•Integrins play a crucial role in vivo by regulating epithelial cell adhesion and contributing to migration, proliferation, differentiation and survival of tumor cells. Overexpression of integrins ανβ3, α5β1 and ανβ6 has been associated with various types of cancers.
•RGD sequence was identified to be a repeated motif of many naturally occurring ligands binding to ανβ3 receptor.
RGD containing conjugates
•Conjugates of RGD peptides linked with various linkers to a cytotoxic agent selectively target tumor cells. All the progress on the field of RGD conjugates in combination with cytotoxic drugs and natural products is being reviewed.
•RGD–paclitaxel conjugates.
•RGD–doxorubicin conjugates.
•RGD–platinum conjugates.
•RGD–camptothecin conjugates.
•RGD–SMAC mimetic conjugates.
•RGD–peptide conjugates.
•RGD–dexamethasone conjugates.
•RGD–PD0325901 conjugates.
•RGD–cryptophycin conjugates.
•RGD–salicylic acid conjugates.
•RGD–monomethylauristatin E conjugates.

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