Synthesis and Anti-Angiogenic Activity of Novel c(RGDyK) Peptide-Based JH-VII-139-1 Conjugates

Peptide–drug conjugates are delivery systems for selective delivery of cytotoxic agents to target cancer cells. In this work, the optimized synthesis of JH-VII-139-1 and its c(RGDyK) peptide conjugates is presented. The low nanomolar SRPK1 inhibitor, JH-VII-139-1, which is an analogue of Alectinib, was linked to the ανβ3 targeting oligopeptide c(RGDyK) through amide, carbamate and urea linkers. The chemostability, cytotoxic and antiangiogenic properties of the synthesized hybrids were thoroughly studied. All conjugates retained mid nanomolar-level inhibitory activity against SRPK1 kinase and two out of four conjugates, geo75 and geo77 exhibited antiproliferative effects with low micromolar IC50 values against HeLa, K562, MDA-MB231 and MCF7 cancer cells. The activities were strongly related to the stability of the linkers and the release of JH-VII-139-1. In vivo zebrafish screening assays demonstrated the ability of the synthesized conjugates to inhibit the length or width of intersegmental vessels (ISVs). Flow cytometry experiments were used to test the cellular uptake of a fluorescein tagged hybrid in MCF7 and MDA-MB231 cells that revealed a receptor-mediated endocytosis process. In conclusion, most conjugates retained the inhibitory potency against SRPK1 as JH-VII-139-1 and demonstrated antiproliferative and antiangiogenic activities. Further animal model experiments are needed to uncover the full potential of such peptide conjugates in cancer therapy and angiogenesis-related diseases.


Introduction
Angiogenesis is described as the formation of new blood vessels from pre-existing vessels [1,2]. It is a vital physiological process for growth, development, and wound repair by which various materials, oxygen and nutrients are delivered to tissues and cellular wastes are removed. In cancer, abnormal angiogenesis is related to cell growth, tumorigenesis, and metastases [3][4][5]. However, recent findings implicate angiogenesis in many other diseases [6], such as diabetic retinopathy, neovascular glaucoma [7], autoimmune diseases, multiple sclerosis [8], rheumatoid arthritis [9], cardiovascular diseases [10], atherosclerosis [11], and cerebral ischemia. Angiogenesis is regulated by many pathways and signaling molecules such as the vascular endothelial growth factor (VEGF), the transforming growth factor (TGF)-β, the angiopoietin-1 and 2, the placental growth factor (PGF), α v β 3 and α v β 5 integrins [12], the Notch, Akt, Jak/STAT, ephrin/Eph signaling pathways and others [13,14]. VEGF binds to receptor VEGFR and this interaction predominantly regulates pathological and physiological angiogenesis [15][16][17]. The VEGFR signaling is monitored at multiple levels [18]. For example, serine-arginine (SR) protein kinases (SRPKs) regulate the splicing of proangiogenic VEGF165 by phosphorylation of the serine/arginine splicing factor 1 (SRSF1) [19]. Therefore, targeting SRPK1 kinase to regulate angiogenesis in angiogenic disorders has been proposed by several research groups. Bates et al. has used the SRPK1 inhibitor SRPIN340 to reduce proangiogenic VEGF165 and inhibit melanoma tumor growth in vivo [20]. Oltean et al. demonstrated that SPHINX and SRPIN340 treatment changed the expression of VEGF165 towards the anti-angiogenic splice isoform VEGF165b and decreased tumor growth in orthotopic PC3 models in mice [21].
In previous research efforts by our group, the cyclic pentapeptide c(RGDyK), which is a potent integrin α v β 3 ligand, was employed for the targeted delivery of various anticancer drugs and bioactive molecules such as gemcitabine [22], the triterpenoids cucurbitacins [23], and platinum complexes [24]. c(RGDyK) peptide potently binds α v β 3 integrins (IC 50 = 3.8 ± 0.42 nM) and, less potently, α v β 5 (IC 50 = 503 ± 55 nM), α v β 6 (IC 50 = 86 ± 7 nM), and α 5 β 1 integrins (IC 50 = 236 ± 45 nM) [25]. In earlier studies, the SRPIN803 inhibitor which binds to SRPK1 and CK2 kinases was coupled to c(RGDyK) peptide aiming to form a new conjugate with improved antiangiogenic activities compared to both the peptide and the SRPIN803 (Figure 1) [26]. Although the antiangiogenic activities of the synthesized SRPIN803-c(RGDyK) hybrids and SRPIN803 were evident in zebrafish embryos, an unfavorable stability profile of SRPIN803 was observed as SR-PIN803 and its derivatives undergo a retro-Knoevenagel reaction. As a next step, we intended to develop new anti-angiogenic compounds based on the more potent SRPK1 inhibitor, JH-VII-139-1 [27]. JH-VII-139-1 came up from the optimization of FDA-approved ALK inhibitor Alectinib [28], which previously was shown to bind to SRPK1. Gray and coworkers demonstrated that JH-VII-139-1 potently inhibits SRPK1 with an IC 50 value of 1.1 nM and blocks angiogenesis in an age-related macular degeneration (AMD) animal model [27]. In this work, the synthesis of JH-VII-139-1 peptide conjugates with c(RGDyK) and the properties of the new hybrid compounds against SRPK1, cancer cell growth and angiogenesis are reported. protein kinases (SRPKs) regulate the splicing of proangiogenic VEGF165 by phosphorylation of the serine/arginine splicing factor 1 (SRSF1) [19]. Therefore, targeting SRPK1 kinase to regulate angiogenesis in angiogenic disorders has been proposed by several research groups. Bates et al. has used the SRPK1 inhibitor SRPIN340 to reduce proangiogenic VEGF165 and inhibit melanoma tumor growth in vivo [20]. Oltean et al. demonstrated that SPHINX and SRPIN340 treatment changed the expression of VEGF165 towards the anti-angiogenic splice isoform VEGF165b and decreased tumor growth in orthotopic PC3 models in mice [21]. In previous research efforts by our group, the cyclic pentapeptide c(RGDyK), which is a potent integrin αvβ3 ligand, was employed for the targeted delivery of various anticancer drugs and bioactive molecules such as gemcitabine [22], the triterpenoids cucurbitacins [23], and platinum complexes [24]. c(RGDyK) peptide potently binds αvβ3 integrins (IC50 = 3.8 ± 0.42 nM) and, less potently, αvβ5 (IC50 = 503 ± 55 nM), αvβ6 (IC50 = 86 ± 7 nM), and α5β1 integrins (IC50 = 236 ± 45 nM) [25]. In earlier studies, the SRPIN803 inhibitor which binds to SRPK1 and CK2 kinases was coupled to c(RGDyK) peptide aiming to form a new conjugate with improved antiangiogenic activities compared to both the peptide and the SRPIN803 (Figure 1) [26]. Although the antiangiogenic activities of the synthesized SRPIN803-c(RGDyK) hybrids and SRPIN803 were evident in zebrafish embryos, an unfavorable stability profile of SRPIN803 was observed as SRPIN803 and its derivatives undergo a retro-Knoevenagel reaction. As a next step, we intended to develop new anti-angiogenic compounds based on the more potent SRPK1 inhibitor, JH-VII-139-1 [27]. JH-VII-139-1 came up from the optimization of FDA-approved ALK inhibitor Alectinib [28], which previously was shown to bind to SRPK1. Gray and coworkers demonstrated that JH-VII-139-1 potently inhibits SRPK1 with an IC50 value of 1.1 nM and blocks angiogenesis in an age-related macular degeneration (AMD) animal model [27]. In this work, the synthesis of JH-VII-139-1 peptide conjugates with c(RGDyK) and the properties of the new hybrid compounds against SRPK1, cancer cell growth and angiogenesis are reported.

General Experimental Details
All reactions were carried out under an atmosphere of Argon unless otherwise specified. Commercial reagents were used without further purification. Reactions were monitored by TLC and using UV light as a visualizing agent and aqueous ceric sul-

General Experimental Details
All reactions were carried out under an atmosphere of Argon unless otherwise specified. Commercial reagents were used without further purification. Reactions were monitored by TLC and using UV light as a visualizing agent and aqueous ceric sulfate/phosphomolybdic acid, ethanolic p-anisaldehyde solution, potassium permanganate solution, and heat as developing agents. The 1 H and 13 C NMR spectra were recorded at  13 C 29.84, 206.26). LC-MS analysis was performed on a LC-20AD Shimadzu connected to Shimadzu LCMS-2010EV (Shimadzu Kyoto, Japan) equipped with C18 analytical column (Supelco discovery C18, 5 µm 250 × 4.6 mm).

Stability in Buffer Solutions
The stability of the conjugates was examined by performing chemostability experiments at two different buffer solutions (pH = 5.2 and 7.4). c(RGDyK)-based conjugates were dissolved in 5 µL DMSO and transferred to 0.5 mL of the relevant buffer solution (acetate or phosphate aquatic buffer solutions). The mixture was then incubated at 37 • C, samples were collected at predetermined time points (0, 1, 2, 3, 4, 5, 24 and 48 h), and analyzed by LC-MS. Results are presented as the mean ± of SD after repeating the experiment three times.

Stability in Dulbecco's Modified Eagle Medium
Conjugates geo75, geo77, geo85 and geo107 (200 µg in 5 µL DMSO) were diluted in 0.5 mL DMEM (+10% fetal bovine serum) mixture and incubated at 37 • C. At predetermined time intervals, 50 µL of the mixture was removed and quenched with 150 µL water/acetonitrile/formic acid solution (100/100/0.1 volume ratio) and the sample was analyzed by LC-MS. Three independent experiments were carried out and the results are presented as the mean ± standard deviation.

Stability in Human Plasma
First, 250 µg of (JH-VII-139-1)-c(RGDyK)-based conjugates were diluted in 5 µL DMSO and the mixture was added to 0.5 mL of human plasma. The mixtures were incubated at 37 • C. 50 µL of aliquot was removed at predetermined time points and quenched with 150 µL ice-cold acetonitrile (+0.1% formic acid). Then, the mixture was centrifuged at 10,000 rpm for 10 min. Then, 50 µL of supernatant was added to 50 µL of ultrapure water (+0.1% formic acid) and analyzed by LC-MS. Results are presented as the mean ± SD of three independent experiments.

In Vitro Kinase Assays
The pGEX-2T bacterial expression vector (Amersham Biosciences GmbH, Freiburg, Germany) was used to construct plasmids that encode human SRPK1 and a fragment of the N-terminal domain of turkey LBR comprising amino acids 62-92 (LBRNt(62-92)) [36]. The GST-fusion proteins were produced in bacteria and purified using glutathione-Sepharose (Amersham Biosciences) according to the manufacturer's instructions. Kinase assays were carried out in a total volume of 25 µL containing 0.5 µg GST-SRPK1, 1.5 µg GST-LBRNt(62-92) as substrate, 12 mM Hepes pH 7.5, 10 mM MgCl 2 , 25 µM ATP, and increasing concentrations of the inhibitors as indicated. In all assays, the final concentration of dimethyl sulfoxide (DMSO) was adjusted to 4%. Phosphorylated GST-LBRNt(62-92) was detected by autoradiography using Super RX (Fuji medical X-ray film,(Fujifilm Holdings Corporation, Tokyo, Japan)). Incorporated radioactivity was quantified by excising the radioactive bands from the SDS-PAGE gel and scintillation counting.

MTT Assays
HeLa, MCF7, MDA-MB-231 and K562 cells were seeded in 96-well plates (3 × 10 3 cells per well) and grown in medium supplemented with 10% FBS as monolayers at 37 • C in a 5% CO 2 incubator. One day after seeding, cells were exposed to increasing concentrations of the inhibitors for 48 h. The viability of the cells was estimated by an (3-(4,5-imethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolic assay as described previously [37], and the percentage growth inhibition (GI 50 , TGI, IC 50 ) was calculated according to National Cancer Institute recommendations as referred to Leonidis et al. [26]. Values shown represent the means ± SE of three independent experiments.

Fluorescence Microscopy
MCF7 and MDA-MB-231 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37 • C in 5% CO 2 . Cells were seeded in 24-well plates with 5 × 10 4 cells per well on coverslips, and after 24 h they were treated with JH-VII-139-1-c(RGDyK) hybrid compound, Geo106 (5 µM), for 2, 24 and 48 h. To test the effect of okadaic acid (endocytosis inhibitor), cells were preincubated, prior to addition of Geo106, with okadaic acid (100 nM) for 30 min. After the incubation period, the cells coverslips were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Following the quenching of paraformaldehyde (100 mM Tris-HCl pH 7.5), the cells were washed with PBS and DNA was stained with DAPI. The coverslips were mounted with mounting medium (0.01% p-phenylenediamine and 50% glycerol in PBS) and visualized with Zeiss LSM 780 confocal microscope, using the Zen 2011 software.

Zebrafish Screening Assays
The Tg(kdrl:gfp) s843 [38] was used to measure angiogenesis. Zebrafish breeding was carried out according to the European Directive 2010/63 and the Recommended Guidelines for Zebrafish Husbandry Conditions [39].
Zebrafish Tg(kdrl:gfp) s843 embryos were raised in E3 medium up to 24 hpf; then dechorinated and finally transferred to a 12-well plate (10 embryos per well). Each compound was dissolved in DMSO and then added in the wells at the following final concentrations: 10 µM JH-VII-139-1 and c(RGDyK), and 1.3 µM geo75, geo77, geo85, geo107. Treated embryos and control embryos (at the corresponding DMSO concentration) were kept at 28 • C and imaged at 72 hpf.

Zebrafish Imaging Analysis
After anesthetizing embryos with 0.4% tricaine methanesulfonate (MS222), a fluorescent image was acquired using a Hamamatsu ORCA-Flash4.0LT digital camera. We used the total length of 8 intersegmental vessels (ISVs) per embryo and the width from each of the above ISVs was measured at three different points, i.e., the top, the middle, and the bottom point of each ISV, and then the average width was estimated. We measured the 8 ISVs that flank the end of the yolk extension. Length and widths were measured using the open-source software ImageJ 1.53a (http://imagej.nih.gov/ij (accessed on 30 May 2022)). A total of 30 embryos (4-7 per group) from independent experiments were measured. Statistically significant differences in total width, or in the length of ISVs were calculated using ordinary one-way ANOVA between the average lengths or widths for each embryo in GraphPad Prism 9. The synthesis of the Alectinib core structure that eventually leads to JH-VII-139-1 is presented in Scheme 1. Most Alectinib analogues, including JH-VII-139-1 are fused polycyclic indoles that combine the indole moiety with a polysubstituted tetralone. To synthesize the polycyclic scaffold 6a, Fischer indole synthesis was implemented based on Kinoshita et al. [30]. Initially, 7-methoxy-2-tetralone 1 was emethylated with MeI to produce 2, which was brominated with NBS to give the desired tetralone 3. Then, 3aminobenzonitrile was converted to 3-hydrazinylbenzonitrile 5, which reacted with 3 in a Fischer indole synthesis reaction. After removing the unwanted regioisomer 6b by filtration, 6a was isolated by column chromatography in satisfying yield (50%) compared to similar indole synthesis of the literature [30]. Oxidation of 6a with DDQ afforded 7, which was subsequently alkylated with Et 3 B, Pd(dppf)Cl 2 and Cs 2 CO 3 in a Suzuki-Miyaura reaction, resulting in 8. To modify the scaffold and insert the required pyrazole moiety that will lead to the synthesis of JH-VII-139-1, Zeisel-Prey ether cleavage with pyridinum chloride at high temperatures was performed to form phenol 9. Finally, 9 reacted with Nphenyl-bis(trifluoromethanesulfonimide) to produce the triflate 10, which was subsequently coupled to 1H-pyrazole-3-boronic acid 13 (Scheme 2).

Synthesis of Compounds
presented in Scheme 1. Most Alectinib analogues, including JH-VII-139-1 are fused polycyclic indoles that combine the indole moiety with a polysubstituted tetralone. To synthesize the polycyclic scaffold 6a, Fischer indole synthesis was implemented based on Kinoshita et al. [30]. Initially, 7-methoxy-2-tetralone 1 was 12emethylated with MeI to produce 2, which was brominated with NBS to give the desired tetralone 3. Then, 3-aminobenzonitrile was converted to 3-hydrazinylbenzonitrile 5, which reacted with 3 in a Fischer indole synthesis reaction. After removing the unwanted regioisomer 6b by filtration, 6a was isolated by column chromatography in satisfying yield (50%) compared to similar indole synthesis of the literature [30]. Oxidation of 6a with DDQ afforded 7, which was subsequently alkylated with Et3B, Pd(dppf)Cl2 and Cs2CO3 in a Suzuki-Miyaura reaction, resulting in 8. To modify the scaffold and insert the required pyrazole moiety that will lead to the synthesis of JH-VII-139-1, Zeisel-Prey ether cleavage with pyridinum chloride at high temperatures was performed to form phenol 9. Finally, 9 reacted with N-phenyl-bis(trifluoromethanesulfonimide) to produce the triflate 10, which was subsequently coupled to 1H-pyrazole-3-boronic acid 13 (Scheme 2). The synthesis of 1H-pyrazole-3-boronic acid 13, as well as the synthesis of JH-VII-139-1, are outlined in Scheme 2. Pyrazole was protected with a DHP group leading to the formation of 12, which was then selectively borylated with (MeO)3B and n-BuLi [29,30]. The DHP-protected boronic acid was then deprotected under acidic conditions to give 13, which reacted with triflate 10. The handling of 10 proved to be difficult compared to the iodide analogue used in the JH-VII-139-1 synthesis proposed by Gray et al. [27], since it was easily hydrolyzed in H2O and heat. To avoid the hydrolysis of triflate 10, dioxane was used as the sole solvent, which unfortunately led to no reaction. The presence of H2O in the reaction proved to be crucial; therefore, the reaction was examined at different temperatures. The reaction was also tested under different conditions and solvents, where in general the starting material 10 was consumed, leading to the hydrolyzed The synthesis of 1H-pyrazole-3-boronic acid 13, as well as the synthesis of JH-VII-139-1, are outlined in Scheme 2. Pyrazole was protected with a DHP group leading to the formation of 12, which was then selectively borylated with (MeO) 3 B and n-BuLi [29,30]. The DHP-protected boronic acid was then deprotected under acidic conditions to give 13, which reacted with triflate 10. The handling of 10 proved to be difficult compared to the iodide analogue used in the JH-VII-139-1 synthesis proposed by Gray et al. [27], since it was easily hydrolyzed in H 2 O and heat. To avoid the hydrolysis of triflate 10, dioxane was used as the sole solvent, which unfortunately led to no reaction. The presence of H 2 O in the reaction proved to be crucial; therefore, the reaction was examined at different temperatures. The reaction was also tested under different conditions and solvents, where in general the starting material 10 was consumed, leading to the hydrolyzed byproduct 9 and the byproduct 14, as depicted in Table 1. After several attempts, microwave irradiation improved the yield of JH-VII-139-1 to 80% and reduced the formation of 9 to 12%. It should be noted that the yield 80% for JH-VII-139-1 is significantly improved compared to the 48% yield reported by Gray et al. using a similar methodology [27]. Table 1. Suzuki-Miyaura coupling conditions for JH-VII-139-1. Reaction conditions: all reactions were carried out using triflate 10 (1.2 equiv), boronic acid 13 (1 equiv), Pd(dppf)Cl 2 as catalyst (5%) and tBuXPhos (10%).

Synthesis of (JH-VII-139-1)-c(RGDyK) Peptide-Drug Conjugates
Our first efforts focused on the conjugation of JH-VII-139-1 with c(RGDyK) through a double carbamate linker. The pentapeptide c(RGDyK) was prepared using solid-phase peptide synthesis (SPPS) and head-to-tail cyclization based on the methodology of Davis et al. followed by semipreparative HPLC purification [40]. The synthesis of geo75 conjugate is summarized in Scheme 3.
Pharmaceutics 2023, 15,381 et al. followed by semipreparative HPLC purification [40]. The synthesis of geo7 jugate is summarized in Scheme 3. Starting from triethylene glycol 15, the highly reactive dinitrophenyl carbonate 16 was obtained after reaction with 4-nitrophenyl chloroformate, Et 3 N in DCM (Scheme 3). Then, dropwise addition of JH-VII-139-1 solution was performed to a solution of 16, TEA and DMF to give the monosubstituted 17 in 55% yield. Careful handling and low concentrations of reactants reduced the unwanted double nucleophilic acyl substitution of 16 by JH-VII-139-1. Finally, 17 was coupled to the ε-amino group of lysine on c(RGDyK) under basic DIPEA conditions to afford conjugate geo75 in 56% yield. Conjugate geo75 was purified by semipreparative reversed-phase HPLC and its purity was examined by LC-MS, and it was found greater than 98%.
After achieving conjugation of JH-VII-139-1 and c(RGDyK) through a dicarbamate linker, linkers with different length or greater chemical stability were used for the preparation of new derivatives such as the diamide conjugate geo77 (Scheme 4). The synthesis started with the commercially available glutaric acid 18 which reacted with NHS and EDCi to quantitatively form the activated NHS glutarate 19, which then reacted with JH-VII-139-1 in basic conditions and afforded the JH-VII-139-1 derivative in 47% yield. The subsequent reaction of 20 with oligopeptide c(RGDyK) and DIPEA eventually produced conjugate geo77 in 45% yield, which was purified by HPLC with purity higher than 98%.
Starting from triethylene glycol 15, the highly reactive dinitrophen was obtained after reaction with 4-nitrophenyl chloroformate, Et3N in D Then, dropwise addition of JH-VII-139-1 solution was performed to a solu and DMF to give the monosubstituted 17 in 55% yield. Careful handlin centrations of reactants reduced the unwanted double nucleophilic acyl su by JH-VII-139-1. Finally, 17 was coupled to the ε-amino group of lysin under basic DIPEA conditions to afford conjugate geo75 in 56% yield. C was purified by semipreparative reversed-phase HPLC and its purity w LC-MS, and it was found greater than 98%.
After achieving conjugation of JH-VII-139-1 and c(RGDyK) through linker, linkers with different length or greater chemical stability were us aration of new derivatives such as the diamide conjugate geo77 (Scheme 4 started with the commercially available glutaric acid 18 which reacted EDCi to quantitatively form the activated NHS glutarate 19, which the JH-VII-139-1 in basic conditions and afforded the JH-VII-139-1 derivativ The subsequent reaction of 20 with oligopeptide c(RGDyK) and DIPEA duced conjugate geo77 in 45% yield, which was purified by HPLC wit than 98%. In an attempt to increase the chemical stability, triethylene glycol in was replaced by 2,2′-(ethylenedioxy)bis(ethylamine), which could lea In an attempt to increase the chemical stability, triethylene glycol in geo75 synthesis was replaced by 2,2 -(ethylenedioxy)bis(ethylamine), which could lead to a bis-urea linker. The synthetic procedure is outlined in Scheme 5. In detail, highly reactive dicarbamate 22 was formed in a similar fashion by reacting 2,2 -(ethylenedioxy)bis(ethylamine) with 4nitrophenyl chloroformate and EDCi. Then, JH-VII-139-1 reacted with 22 in basic conditions, and after careful handling, 23 was obtained in good yield (66%). Compound 23 was then coupled to c(RGDyK) by forming a second urea moiety, providing geo85 in 88% yield. Conjugate geo85 was easier to handle, and was isolated in greater overall yield compared to geo75, as urea derivatives were more stable and did not hydrolyze as readily as their carbamate counterparts. Conjugate geo85 was isolated after semipreparative HPLC with purity higher than 97%.
Afterwards, a linker that would increase the distance between JH-VII-139-1 and c(RGDyK) and could carry another bioactive moiety was employed. Lysine was a good candidate, as it could extend the triethylene glycol chain and bear both a carboxylic and an amino group. This approach that eventually led to the synthesis of conjugate geo107 is highlighted in Scheme 6. The synthesis starts with a nucleophilic attack by N ε -Boc-L-lysine to the highly reactive 4-nitrophenyl carbamate of 23 that produces Boc-protected 24 in 63% yield. The carboxyl group is then subjected to in situ NHS activation and direct amidation with c(RGDyK) oligopeptide and DIPEA to form Boc-protected 25 in 30% yield over two steps. The final conjugate geo107 was obtained after a rapid Boc-deprotection with TFA and triethylsilane (TESH) followed by semi-preparative reversed-phase HPLC purification (purity of geo107 > 97%).
and was isolated in greater overall yield compared to geo75, as urea derivatives were more stable and did not hydrolyze as readily as their carbamate counterparts. Conjugate geo85 was isolated after semipreparative HPLC with purity higher than 97%.
Afterwards, a linker that would increase the distance between JH-VII-139-1 and c(RGDyK) and could carry another bioactive moiety was employed. Lysine was a good candidate, as it could extend the triethylene glycol chain and bear both a carboxylic and an amino group. This approach that eventually led to the synthesis of conjugate geo107 is highlighted in Scheme 6. The synthesis starts with a nucleophilic attack by Nε-Boc-L-lysine to the highly reactive 4-nitrophenyl carbamate of 23 that produces Boc-protected 24 in 63% yield. The carboxyl group is then subjected to in situ NHS activation and direct amidation with c(RGDyK) oligopeptide and DIPEA to form Boc-protected 25 in 30% yield over two steps. The final conjugate geo107 was obtained after a rapid Boc-deprotection with TFA and triethylsilane (TESH) followed by semi-preparative reversed-phase HPLC purification (purity of geo107 > 97%). and was isolated in greater overall yield compared to geo75, as urea derivatives were more stable and did not hydrolyze as readily as their carbamate counterparts. Conjugate geo85 was isolated after semipreparative HPLC with purity higher than 97%.
Afterwards, a linker that would increase the distance between JH-VII-139-1 and c(RGDyK) and could carry another bioactive moiety was employed. Lysine was a good candidate, as it could extend the triethylene glycol chain and bear both a carboxylic and an amino group. This approach that eventually led to the synthesis of conjugate geo107 is highlighted in Scheme 6. The synthesis starts with a nucleophilic attack by Nε-Boc-L-lysine to the highly reactive 4-nitrophenyl carbamate of 23 that produces Boc-protected 24 in 63% yield. The carboxyl group is then subjected to in situ NHS activation and direct amidation with c(RGDyK) oligopeptide and DIPEA to form Boc-protected 25 in 30% yield over two steps. The final conjugate geo107 was obtained after a rapid Boc-deprotection with TFA and triethylsilane (TESH) followed by semi-preparative reversed-phase HPLC purification (purity of geo107 > 97%).

Synthesis of Fluorescein-Tagged (JH-VII-139-1)-c(RGDyK) Conjugate
To gain insight into the cellular entry mechanism and the cellular biodistribution of (JH-VII-139-1)-c(RGDyK) conjugates, fluorescence labeling of geo107 with fluorescein was undertaken (Scheme 7). Two molecules of rescorcin reacted with trimellitic anhydride to produce a mixture of 5-and 6-carboxy-fluoresceins 28 in quantitative conversion [41]. Activation of 5(6) carboxyl group with NHS and DIPEA led to the formation of NHS ester 29 in 48% yield [42], which subsequently reacted with geo107 under basic conditions and formed the 5(6)-carboxyfluorescein-tagged conjugate geo106 in 63% yield. The crude photosensitive material was then subjected to semi-preparative HPLC and geo106 was isolated with 96% purity.

In Vitro Stability Studies
The stability of JH-VII-139-1 and its c(RGDyK) conjugates geo75, geo77, geo85 and geo107 was studied at pH = 5.2 and 7.4, in cell medium and human plasma. All experiments were carried out at 37 • C, and samples were analyzed with LC-ESI-MS at predetermined time intervals. Starting with JH-VII-139-1, the compound was found to be very stable under all conditions tested as presented in Figure 2 (Figures S77 and S78). The c(RGDyK) conjugates exhibit different stability profiles as analyzed below. Hydrolysis and JH-VII-139-1 release were the main reactions, with the half-lives of the conjugates ranging from minutes to hours.
To gain insight into the cellular entry mechanism and the cellular biodistribution of (JH-VII-139-1)-c(RGDyK) conjugates, fluorescence labeling of geo107 with fluorescein was undertaken (Scheme 7). Two molecules of rescorcin reacted with trimellitic anhydride to produce a mixture of 5-and 6-carboxy-fluoresceins 28 in quantitative conversion [41]. Activation of 5(6) carboxyl group with NHS and DIPEA led to the formation of NHS ester 29 in 48% yield [42], which subsequently reacted with geo107 under basic conditions and formed the 5(6)-carboxyfluorescein-tagged conjugate geo106 in 63% yield. The crude photosensitive material was then subjected to semi-preparative HPLC and geo106 was isolated with 96% purity. Scheme 7. Synthesis of multifunctional geo106 conjugates.

In Vitro Stability Studies
The stability of JH-VII-139-1 and its c(RGDyK) conjugates geo75, geo77, geo85 and geo107 was studied at pH = 5.2 and 7.4, in cell medium and human plasma. All experiments were carried out at 37 °C, and samples were analyzed with LC-ESI-MS at predetermined time intervals. Starting with JH-VII-139-1, the compound was found to be very stable under all conditions tested as presented in Figure 2 (Figures S77, S78). The c(RGDyK) conjugates exhibit different stability profiles as analyzed below. Hydrolysis and JH-VII-139-1 release were the main reactions, with the half-lives of the conjugates ranging from minutes to hours.

Chemostability Profile of Conjugate geo75
Dicarbamate geo75 was tested for its stability to buffer solutions and it was completely stable at pH = 5.2 for more than 48 h, while after 24 h its residual concentration at pH = 7.4 retained more than 51% of its initial concentration. In addition, the conjugate Scheme 7. Synthesis of multifunctional geo106 conjugates.

In Vitro Stability Studies
The stability of JH-VII-139-1 and its c(RGDyK) conjugates geo75, geo77, geo85 and geo107 was studied at pH = 5.2 and 7.4, in cell medium and human plasma. All experiments were carried out at 37 °C, and samples were analyzed with LC-ESI-MS at predetermined time intervals. Starting with JH-VII-139-1, the compound was found to be very stable under all conditions tested as presented in Figure 2 (Figures S77, S78). The c(RGDyK) conjugates exhibit different stability profiles as analyzed below. Hydrolysis and JH-VII-139-1 release were the main reactions, with the half-lives of the conjugates ranging from minutes to hours.

Chemostability Profile of Conjugate geo75
Dicarbamate geo75 was tested for its stability to buffer solutions and it was completely stable at pH = 5.2 for more than 48 h, while after 24 h its residual concentration at pH = 7.4 retained more than 51% of its initial concentration. In addition, the conjugate

Chemostability Profile of Conjugate geo75
Dicarbamate geo75 was tested for its stability to buffer solutions and it was completely stable at pH = 5.2 for more than 48 h, while after 24 h its residual concentration at pH = 7.4 retained more than 51% of its initial concentration. In addition, the conjugate had a declining stability pattern in Dulbecco's modified eagle medium (DMEM) and human plasma, where it was almost completely hydrolyzed after 9-and 1-h respectively, exhibiting half-lives t 1/2 = 2 h 36 min and t 1/2 = 11 min (Figures 3 and S61-S64).

Chemostability Profile of Conjugate geo77
The stability of the conjugate geo77 was also examined in the same conditions (Figures 4 and S65-S68). In detail, geo77 was less stable at pH = 5.2 buffer, having a halflife t 1/2 = 14 h 42 min compared to the t 1/2 > 48 h of geo75. At neutral pH and in DMEM solutions, the conjugate was readily hydrolyzed leading to half-lives t 1/2 = 1 h 44 min and t 1/2 = 0 h 50 min, respectively. In human plasma, the conjugate was also unstable, and the half-life time was less than ten minutes.

Chemostability Profile of Conjugate geo85
Since both carbamate and amide conjugates, geo75 and geo77 exhibited short half-lives during their incubation in cell media and human plasma, our efforts were focused on the urea derivative geo85 (Figures 5 and S69-S72). Conjugate geo85 proved to be completely stable in both buffer media having half-lives that exceeded 48 h. Geo85 maintained its stability in DMEM and human plasma, although a 10% hydrolysis was observed after 24 h. Therefore, geo85 was the most stable conjugate and proved to be an excellent candidate for fluorescent tagging and mechanistic studies.

Chemostability Profile of Conjugate geo77
The stability of the conjugate geo77 was also examined in the same conditions ure 4, Figures S65-S68). In detail, geo77 was less stable at pH = 5.2 buffer, havi half-life t1/2 = 14 h 42 min compared to the t1/2 > 48 h of geo75. At neutral pH and in DM solutions, the conjugate was readily hydrolyzed leading to half-lives t1/2 = 1 h 44 min t1/2 = 0 h 50 min, respectively. In human plasma, the conjugate was also unstable, and half-life time was less than ten minutes. had a declining stability pattern in Dulbecco's modified eagle medium (DMEM) human plasma, where it was almost completely hydrolyzed after 9-and 1-h respecti exhibiting half-lives t1/2 = 2h 36 min and t1/2 = 11 min (Figure 3, Figures S61-S64).

Chemostability Profile of Conjugate geo77
The stability of the conjugate geo77 was also examined in the same conditions ure 4, Figures S65-S68). In detail, geo77 was less stable at pH = 5.2 buffer, havi half-life t1/2 = 14 h 42 min compared to the t1/2 > 48 h of geo75. At neutral pH and in DM solutions, the conjugate was readily hydrolyzed leading to half-lives t1/2 = 1 h 44 min t1/2 = 0 h 50 min, respectively. In human plasma, the conjugate was also unstable, an half-life time was less than ten minutes.

Chemostability Profile of Conjugate geo85
Since both carbamate and amide conjugates, geo75 and geo77 exhibited half-lives during their incubation in cell media and human plasma, our efforts wer cused on the urea derivative geo85 ( Figure 5, Figures S69-S72). Conjugate geo85 pr to be completely stable in both buffer media having half-lives that exceeded 48

Chemostability Profile of geo107
The stability profile of geo107 was analogous to geo85, as it maintained half-lives t 1/2 > 48 h in all stability experiments (Figures 6 and S73-S76). Hydrolysis and JH-VII-139-1 was also detected. Residual concentrations of the conjugate dropped no further than 80% of its initial concentration even in cell media or human plasma.
Pharmaceutics 2023, 15,381 Geo85 maintained its stability in DMEM and human plasma, although a 10% was observed after 24 h. Therefore, geo85 was the most stable conjugate and p an excellent candidate for fluorescent tagging and mechanistic studies.

Chemostability Profile of geo107
The stability profile of geo107 was analogous to geo85, as it maintained h > 48 h in all stability experiments ( Figure 6, Figures S73-S76). Hydr JH-VII-139-1 was also detected. Residual concentrations of the conjugate d further than 80% of its initial concentration even in cell media or human plasm  Geo85 maintained its stability in DMEM and human plasma, although a 10% h was observed after 24 h. Therefore, geo85 was the most stable conjugate and pro an excellent candidate for fluorescent tagging and mechanistic studies.

Chemostability Profile of geo107
The stability profile of geo107 was analogous to geo85, as it maintained hal > 48 h in all stability experiments ( Figure 6, Figures S73-S76). Hydrol JH-VII-139-1 was also detected. Residual concentrations of the conjugate dro further than 80% of its initial concentration even in cell media or human plasma

Inhibition of Kinase Activity by JH-VII-139-1-c(RGDyK) Conjugates
The inhibitory activity of JH-VII-139-1 and RGD-conjugated compounds w uated by in vitro kinase assay. As shown in Table 2, both JH-VII-139-1 and the co compounds had a significant effect on SRPK1 activity, with IC50 values at the ( Figure S79). Therefore, the conjugation of JH-VII-139-1 to the peptide did not The inhibitory activity of JH-VII-139-1 and RGD-conjugated compounds was evaluated by in vitro kinase assay. As shown in Table 2, both JH-VII-139-1 and the conjugated compounds had a significant effect on SRPK1 activity, with IC 50 values at the nM level ( Figure S79). Therefore, the conjugation of JH-VII-139-1 to the peptide did not affect the inhibitory activity against SRPK1. The cytotoxic activity of JH-VII-139-1 and JH-VII-139-1-c(RGDyK) hybrid compounds was evaluated at different concentrations (0.5-50 µM) over a panel of cell lines including HeLa cervical cancer, MCF7 mammary carcinoma, the triple-negative breast cancer MDA-MB-231 and K562 lymphoblast cells. Integrin receptors are highly overexpressed on the surface of many types of cancer [43]. The metastatic breast cancer cell lines MDA-MB-435 [44,45] and MCF-7 [46,47], as well as HeLa cells [48], express high levels of α V β 3 integrins. On the other hand, K562 cells express very low levels of α V β 3 integrins [22].
The cytotoxic and cytostatic activities of the JH-VII-139-1-c(RGDyK) hybrid compounds were estimated by three concentration-dependent parameters: GI 50 (concentration that results in 50% growth inhibition), TGI (concentration that results in total growth inhibition or cytostatic effect), and IC 50 (concentration that results in 50% growth cytotoxic effect) ( Table 3)

Cellular Uptake of c(RGDyK) Conjugate geo106
The cellular uptake of geo106, a c(RGDyK) conjugate carrying a fluorescent label, was examined by fluorescent microscopy in MCF7 and MDA-MB-231 cancer cell lines (Figure 7). Confocal image data showed efficient cellular uptake of geo106 even after a short time of incubation (2 h) in MDA-MB-231 cells, with the most intense fluorescent signal obtained in 48 h in both cancer cell lines.

Cellular Uptake of c(RGDyK) Conjugate geo106
The cellular uptake of geo106, a c(RGDyK) conjugate carrying a fluorescent label, was examined by fluorescent microscopy in MCF7 and MDA-MB-231 cancer cell lines (Figure 7). Confocal image data showed efficient cellular uptake of geo106 even after a short time of incubation (2 h) in MDA-MB-231 cells, with the most intense fluorescent signal obtained in 48 h in both cancer cell lines.
To demonstrate the role of the RGD moiety in targeting the conjugate to avβ3 integrin-rich tumor cells, MDA-MB-231 cells were preincubated with okadaic acid (an inhibitor of endocytosis) prior to addition of geo106 [49]. Confocal image data showed a decreased fluorescence signal, confirming that the cellular uptake of the conjugates was mediated by endocytosis ( Figure 8).   To demonstrate the role of the RGD moiety in targeting the conjugate to a v β 3 integrinrich tumor cells, MDA-MB-231 cells were preincubated with okadaic acid (an inhibitor of endocytosis) prior to addition of geo106 [49]. Confocal image data showed a decreased fluorescence signal, confirming that the cellular uptake of the conjugates was mediated by endocytosis ( Figure 8). The cellular uptake of geo106, a c(RGDyK) conjugate carrying a fluorescent label, was examined by fluorescent microscopy in MCF7 and MDA-MB-231 cancer cell lines (Figure 7). Confocal image data showed efficient cellular uptake of geo106 even after a short time of incubation (2 h) in MDA-MB-231 cells, with the most intense fluorescent signal obtained in 48 h in both cancer cell lines.
To demonstrate the role of the RGD moiety in targeting the conjugate to avβ3 integrin-rich tumor cells, MDA-MB-231 cells were preincubated with okadaic acid (an inhibitor of endocytosis) prior to addition of geo106 [49]. Confocal image data showed a decreased fluorescence signal, confirming that the cellular uptake of the conjugates was mediated by endocytosis ( Figure 8).

In Vivo Zebrafish Angiogenesis Studies
To determine if compounds c(RGDyK), JH-VII-139-1, geo75, geo77, geo85, and geo107 affect angiogenesis, we used the transgenic zebrafish line Tg(kdrl:gfp). The compounds were added in the embryo medium at 24 h post fertilization (hpf) and angiogenesis was monitored by imaging the intersegmental vessels (ISVs) at 72 hpf. The length as well as the width of the ISVs were used as measurements to quantify angiogenesis.
Our results show that the conjugates significantly inhibit the width of ISVs (Figure 9), while exerting a milder effect on their length ( Figure 10). More specifically, geo75, geo77, geo85, and geo107 significantly inhibited width angiogenesis at concentrations of 1.3 µM, while JH-VII-139-1 at 10 µM and c(RGDyK) showed a non-significant trend. On the other hand, geo77 also exhibited an effect on ISV length inhibition, while the other compounds showed a non-significant trend at the concentrations tested. In this study, no other additional morphological phenotypic changes were observed.

In Vivo Zebrafish Angiogenesis Studies
To determine if compounds c(RGDyK), JH-VII-139-1, geo75, geo77, geo85, and geo107 affect angiogenesis, we used the transgenic zebrafish line Tg(kdrl:gfp). The com pounds were added in the embryo medium at 24 h post fertilization (hpf) and angio genesis was monitored by imaging the intersegmental vessels (ISVs) at 72 hpf. The length as well as the width of the ISVs were used as measurements to quantify angiogenesis.
Our results show that the conjugates significantly inhibit the width of ISVs ( Figure  9), while exerting a milder effect on their length ( Figure 10). More specifically, geo75 geo77, geo85, and geo107 significantly inhibited width angiogenesis at concentrations o 1.3 μM, while JΗ-VII-139-1 at 10 μΜ and c(RGDyK) showed a non-significant trend. On the other hand, geo77 also exhibited an effect on ISV length inhibition, while the othe compounds showed a non-significant trend at the concentrations tested. In this study, no other additional morphological phenotypic changes were observed.

Discussion
In the present study, we disclose the synthesis and biological evaluation of several JH-VII-139-1-c(RGDyK) conjugates, which inhibit SRPK1 kinase. To develop novel antiangiogenic compounds JH-VII-139-1, a potent SRPK1 inhibitor, was coupled to c(RGDyK) peptide using different linkers. Four c(RGDyK) conjugates geo75, geo77, geo85 and geo107 were synthesized, which were examined for their stability at pH = 5.2 and 7.4 buffer solutions, cell medium and human plasma. Conjugates geo75 and geo77 demonstrated limited stability and were rapidly hydrolyzed releasing JH-VII-139-1. In human plasma, the same conjugates had short half-lives of 10-50 min. On the other hand, geo85 and geo107 showed lasting stability in buffer solutions, cell medium and human plasma. In vitro kinase assay against SRPK1 showed that all the tested JH-VII-139-1-c(RGDyK) conjugates retained the inhibitory activity against SRPK1 in the nanomolar range (30-42 nM). The cytotoxic activity of the synthesized compounds was tested against the cancer cell lines HeLa, MCF7, MDA-MB-231 and K562. Among them, MCF7 proved to be the most sensitive (lower IC50 values) to JH-VII-139-1 and its conjugates. Overall, the conjugates geo75 and geo77 bearing the cleavable linkers slightly improved the antiproliferative activity compared to JH-VII-139-1 against integrin αvβ3 overexpressing cells (HeLa, MDA-MB-231, MCF7). However, the observed activities are

Discussion
In the present study, we disclose the synthesis and biological evaluation of several JH-VII-139-1-c(RGDyK) conjugates, which inhibit SRPK1 kinase. To develop novel antiangiogenic compounds JH-VII-139-1, a potent SRPK1 inhibitor, was coupled to c(RGDyK) peptide using different linkers. Four c(RGDyK) conjugates geo75, geo77, geo85 and geo107 were synthesized, which were examined for their stability at pH = 5.2 and 7.4 buffer solutions, cell medium and human plasma. Conjugates geo75 and geo77 demonstrated limited stability and were rapidly hydrolyzed releasing JH-VII-139-1. In human plasma, the same conjugates had short half-lives of 10-50 min. On the other hand, geo85 and geo107 showed lasting stability in buffer solutions, cell medium and human plasma. In vitro kinase assay against SRPK1 showed that all the tested JH-VII-139-1-c(RGDyK) conjugates retained the inhibitory activity against SRPK1 in the nanomolar range (30-42 nM). The cytotoxic activity of the synthesized compounds was tested against the cancer cell lines HeLa, MCF7, MDA-MB-231 and K562. Among them, MCF7 proved to be the most sensitive (lower IC 50 values) to JH-VII-139-1 and its conjugates. Overall, the conjugates geo75 and geo77 bearing the cleavable linkers slightly improved the antiproliferative activity compared to JH-VII-139-1 against integrin α v β 3 overexpressing cells (HeLa, MDA-MB-231, MCF7). However, the observed activities are in part attributed to an early JH-VII-139-1 release as the stability studies indicate. The stable conjugate geo85 induced selective cytotoxicity towards MCF7 cancer cells in the mid-micromolar range, whereas it was significantly less active against HeLa and MDA-MB-231 cells. The cellular uptake of the conjugate geo106, which carries a fluorescent label, was examined by fluorescent microscopy in MCF7 and MDA-MB-231 cancer cell lines. Confocal image data showed efficient cellular uptake of geo106 even after a short time probably by a mechanism of integrin mediated endocytosis. Then, the effect of (JH-VII-139-1)-c(RGDyK) conjugates on angiogenesis was examined in vivo in zebrafish embryos. Notably, the new conjugates significantly inhibit zebrafish width angiogenesis and exert a milder effect in length angiogenesis. Among the tested conjugates, the stable geo85 was the most potent inhibitor of zebrafish width angiogenesis.

Conclusions
The synthesized (JH-VII-139-1)-c(RGDyK) conjugates retained the inhibitory activity of JH-VII-139-1 against SRPK1, while exhibiting different cytotoxicity profile against cancer cells expressing integrins to various extend. The activities were strongly related to the stability of the linkers and the early release of JH-VII-139-1. The stable conjugate geo85 induced increased targeting ability against MCF7 cancer cells; however it was mildly cytotoxic with GI 50 = 33 ± 0.9 µM, TGI = 64 ± 0.9 µM, IC 50 = 45 ± 1.0 µM. Remarkably, all (JH-VII-139-1)-c(RGDyK) conjugates displayed in vivo antiangiogenic effects, although a more significant effect was observed for geo85 against width angiogenesis in zebrafish embryos. Since all the synthesized conjugates inhibit SRPK1 with similar potency, the observed difference in biological activities related to cytotoxicity and anti-angiogenesis, depend essentially on the different drug release and endosomal escape mechanisms, as well as the stability of the conjugates. In addition, a v β 3 integrin and SRPK1 signaling may defer considerably in different cancer cell lines. Further experiments, in particular in vivo, are clearly necessary to reveal the full potential of such peptide conjugates in cancer therapy and angiogenesis-related diseases.  Informed Consent Statement: Not applicable.

Conflicts of Interest:
The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.