Tuning the structure of aminoferrocene-based anticancer prodrugs to prevent their aggregation in aqueous solution

Accepted Manuscript Tuning the structure of aminoferrocene-based anticancer prodrugs to prevent their aggregation in aqueous solution

Steffen Daum, Svetlana Babiy, Helen Konovalova, Walter Hofer, Alexander Shtemenko, Natalia Shtemenko, Christina Janko, Christoph Alexiou, Andriy Mokhir PII: DOI: Reference:

S0162-0134(17)30193-9 doi: 10.1016/j.jinorgbio.2017.08.038 JIB 10332

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

28 March 2017 24 August 2017 30 August 2017

Please cite this article as: Steffen Daum, Svetlana Babiy, Helen Konovalova, Walter Hofer, Alexander Shtemenko, Natalia Shtemenko, Christina Janko, Christoph Alexiou, Andriy Mokhir , Tuning the structure of aminoferrocene-based anticancer prodrugs to prevent their aggregation in aqueous solution, Journal of Inorganic Biochemistry (2017), doi: 10.1016/j.jinorgbio.2017.08.038

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ACCEPTED MANUSCRIPT

Tuning the Structure of Aminoferrocene-Based

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Aqueous Solution

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Anticancer Prodrugs to Prevent Their Aggregation in

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Steffen Daum,a Svetlana Babiy,b Helen Konovalova,c Walter Hofer,a Alexander Shtemenko,d

a

P b

9

“

M

M str. 9, 49044 Dnipro, Ukraine

U

”, V. Vernadsky

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Department of Inorganic Chemistry, Ukrainian State University of Chemical Technology, Gagarin Av. 8, 49005 Dnipro, Ukraine

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Leontovich str. 9, 01601 Kyiv, Ukraine f

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e

-

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d

-U

Institute of Gastroenterology AMSU, Slobozansky Av. 96, 49074 Dnipro, Ukraine

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S

O

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M

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c

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Natalia Shtemenko,e Christina Janko,f Christoph Alexiou,f and Andriy Mokhira*

Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine (SEON), University Hospital Erlangen, Glückstraße 10a, 91054 Erlangen, Germany

KEYWORDS Cancer, Prodrug, Aminoferrocene, Reactive Oxygen Species, Lipophilicity * Corresponding author: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT. Aminoferrocene-based prodrugs are activated in cancer cells by reactive oxygen species (ROS). They were shown to exhibit high cytotoxicity towards a variety of cancer cell lines and primary cancer cells, but remain not toxic towards non-malignant cells. However, these prodrugs have rather high lipophilicity leading to relatively low water solubility. In particular, an

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n-octanol/water partition coefficient for the best aminoferrocene-based prodrug (2) was found to

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be 4.51 + 0.03. Though the approaches for decreasing lipophilicity are straightforward and

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include the addition of polar residues to the drug structure, these modifications also lead to dramatic decrease of cell permeability and, correspondingly, lower the activity of the drug.

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Therefore, a delicate balance of polar and unpolar groups should be found to reduce lipophilicity

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without compromising the useful drug properties. In this study we optimized an N-alkyl substituent, which is a key element responsible for the stabilization of the aminoferrocene drug

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released in cancer cells from prodrug 2. We found that an N-propargyl residue is an optimal

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replacement for the N-benzyl fragment. In particular, such a substitution (prodrug 7a) leads to reduction of prodrug lipophilicity down to logP= 3.78 + 0.05, improvement of its water

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solubility, decrease of its propensity towards aggregation and dramatic increase of its ROS-

u

’

(T8) in vivo at the dose of 30 mg/kg.

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w

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generating properties. Finally, we demonstrated that the optimized prodrug strongly suppresses

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ACCEPTED MANUSCRIPT INTRODUCTION Prodrugs activated at cancer specific conditions are excellent alternatives to usual anticancer agents, since they are less toxic and retain the activity of the parent drug. This allows using higher prodrug doses thereby potentiating the antitumor effect. One can make use of e.g.

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overexpressed enzymes [1,2], protons [3-5], hypoxic conditions [6-7] and reactive oxygen species (ROS) [8-21] as cancer specific triggers. The latter trigger is especially attractive, since

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most cancer cells in both isolated form and in tissue produce large amounts of ROS [22-27]. As a

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consequence, they function at higher ROS concentrations, which can exceed that of nonmalignant cells by over 10 fold [28-30]. Therefore, ROS-responsive prodrugs can potentially be

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used for the treatment of many different cancer types. Examples of such prodrugs include

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hydroxyferrocifen, its analogues as well as adducts with glutathione [8-11], organochalcogenes [12], pro-alkylating agents [13, 14], H2O2-responsive prodrugs releasing SN-38 [15] and

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aminoferrocene-based prodrugs, developed in our group [16-21]. The mechanism of activation of

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the latter prodrugs is explained in Figure 1 on the example of 4-(N-benzyl-Nferrocenylaminocarbonyloxymethyl)-phenylboronic acid pinacol ester 2, which up to date was

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our most potent anticancer agent active against a wide range of human cancer cell lines including

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promyelocytic leukemia HL-60, Burkitt lymphoma RAJI and BL-2, mantle cell lymphoma JVM2, prostate cancer LNCaP and DU-145, cervix carcinoma HeLa, glioblastoma U-373, pancreatic cancer PANC-1 as well as primary cancer cells (chronic lymphocytic leukemia CLL cells) [1621]. Importantly, the latter prodrug remains non-toxic to non-malignant cells including fibroblasts (<

µM)

u

MN ’ (<10 µM) [19,20]. In the presence

of cancer specific amounts of H2O2 prodrug 2 generates two active products. One of them is quinone methide 3 (Figure 1), which inhibits the antioxidant system of the cell. Another one is

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ACCEPTED MANUSCRIPT N-benzylaminoferrocene 4, which induces catalytic generation of ROS. Both products 3 and 4 act synergistically by increasing the intracellular ROS amount that leads to cell death [16-21]. The role of the N-benzyl substituent is critical, since it stabilizes the aminoferrocene against the oxidative decomposition with formation of iron ions and enhances cell membrane permeability

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of the parent prodrug [19,20]. Therefore, in cell culture assays prodrug 2 is a more potent

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anticancer agent than unsubstituted (1) and N-alkylated analogues [19-21]. However, in our

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previous studies we observed that 2 generates low amounts of ROS both in cell free settings and in cells that does not correlate with its high activity in cellular assays and in vivo. In this paper

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we conducted a series of experiments to understand reasons of this behavior. In particular, we

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found that prodrug 2 aggregates in aqueous solution that strongly inhibits its ROS-generating catalytic properties. The aggregate formation is a marked disadvantage, since it may humper or

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prevent obtaining a stable drug formulation and can potentially cause poor reproducibility of

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anticancer effects in clinical trials. We approached a solution of this problem by replacing an Nbenzyl fragment in prodrug 2 for a series of related fragments: N-allyl or N-propargyl and their

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C3-methylated analogues. All these groups are electronically similar, since they have a

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methylene group connected to an unsaturated fragment. Therefore, we assumed that this substitution will not change strongly the oxidative stability of aminoferrocenes. However, since

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ethinyl and vinyl fragments are less hydrophobic than phenyl, the new prodrugs were expected to have decreased lipophilicity and, correspondingly, be less prone to aggregation. Herein we investigated solubility of these prodrugs in the aqueous buffer, their proneness to aggregation, lipophilicity (logP), the ROS-generating ability in cell free settings and in representative cancer cells (BL-2) and their anticancer activity towards selected cancer cell lines (human Burkitt lymphoma BL-2 cells, human T lymphocyte Jurkat cells, human ovarian carcinoma A-2780

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ACCEPTED MANUSCRIPT cells) and non-malignant cells (human dermal fibroblasts-adult, HDFa). Finally, we studied the w

u

’

(T8) in Wistar rats upon systemic (intraperitoneal)

delivery of solutions of the optimized aminoferrocene-based prodrug 7a.

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RESULTS AND DISCUSSION

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Preliminary studies of aggregation of known prodrug 2 in aqueous solution

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We observed that the solution of prodrug 2 (50 µM) in Dulbecco's Phosphate-Buffered Saline (DPBS) containing traces of dimethylsulfoxide (DMSO, 1 %, v/v), which we usually use for

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assays conducted both in cell free settings and with cell cultures [16-21], appears slightly cloudy.

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This indicates the presence of larger particles able to reflect light, e.g. prodrug aggregates. Since the aggregation can potentially affect permeability and activity of the prodrugs, we decided to

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quantify this effect. Neither aminoferrocene-based prodrugs nor buffer components absorb light

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at > 800 nm. Therefore, increase of the absorbance at > 800 nm can be caused only by the light reflection on aggregated prodrugs. In this study, we evaluated the turbidity of the solutions

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by measuring the absorbance at 900 nm (A900nm, Table 1). A900nm of the buffer/DMSO mixture

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alone (A900nm(buffer)) was found to be 0.003 + 0.001. This value corresponds to the absorbance of the true solution. A900nm values for prodrug solutions/suspensions (A900nm(prodrug)) given in

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Table 1 were determined using A900nm(buffer) as a background value. For the solution of prodrug 2 substantial A900nm equal to 0.126 + 0.008 was determined. The latter parameter remained constant for at least 8 h after the dissolution of 2 (longer incubation times were not tested), which indicated that the aggregates of 2 are relatively stable. The propensity of prodrug 2 to aggregation in aqueous solution correlates with its high lipophilicity: logP= 4.51 + 0.03. Furthermore, we observed that addition of less polar than water solvent dimethylformamide

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ACCEPTED MANUSCRIPT (DMF, Relative polarity of DMF with respect to that of water is 0.386 [31]) to the aqueous buffered solution of 2 (1/1, v/v) induces dissociation of the aggregates as evidenced by

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Synthesis of new prodrugs and their properties in cell free settings

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substantially reduced A900nm~0 of the resulting solutions.

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To solve the problem of aggregation of prodrug 2 [19,20] in aqueous solution, we prepared a series of its analogues (6a, 6b, 7a and 7b), in which the N-phenyl fragment was replaced for less

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lipophilic residues. These new compounds were obtained by alkylation of compound 1 with

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electrophiles 8a-d in N,N-dimethylformamide (DMF) in the presence of excess Cs2CO3 and (NBu4)I. The target compounds were obtained with isolated yields between 40 and 76 % as

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described in detail in the experimental part of the paper (Scheme 1). We observed that allyl

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derivative 6a (logP= 4.16 + 0.05, p<0.001) and especially propargyl derivative 7a (logP= 3.78 + 0.05, p<0.001) exhibit siginificantly lower lipophilicity than benzyl analogue 2 (logP= 4.51 +

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0.03, Table 1). The replacement of 3-H for 3-CH3 as in 6b and 7b increased lipophilicity of the

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prodrugs, which, however, did not exceed that of 2. Importantly, all new compounds studied were found to be soluble in DPBS buffer containing traces of dimethylsulfoxide (DMSO, 1 %,

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v/v) at concentrations <50 µM, which is an important prerequisite for their possible application as prodrugs. As expected, we observed that A900nm values obtained for the new prodrugs and the parent prodrug 2 correlate with their logP values (Table 1). For example, the least lipophilic prodrug 7a (50 µM) is only weakly aggregated in solution (A900nm= 0.019 + 0.003), whereas the most lipophilic prodrug 2 (50 µM) is strongly aggregated as evidenced by ~7 fold larger A900nm= 0.126 + 0.008. By using the assay based on

’ 7’-dichlorofluorescin (DCFH)

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ACCEPTED MANUSCRIPT [19,20], we observed that the efficiency of ROS-generation in cell free settings is lower for highly lipophilic prodrugs exhibiting enhanced propensity for the formation of aggregates, e.g. 2 (Figure 2A). This is a logical result, since in the aggregates each individual catalyst is less exposed to the components of the medium, e.g. to H2O2, than molecular, non-aggregated

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catalysts. For example, addition of 50 % DMF to the solution 2 in aqueous buffer, which causes

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the aggregate decomposition as discussed above, was found to restore the ROS generating ability

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generating ability of aminoferrocene-based prodrugs.

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of 2 (Figure 2B). These data confirm the negative effect of the aggregation on the ROS-

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Prodrug-induced generation of reactive oxygen species in cells, cell membrane permeability and cytotoxicity of new aminoferrocene-based prodrugs

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Since 7a has the highest catalytic activity in the reaction of ROS generation among all studied

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here and previously reported aminoferrocene-based prodrugs and was least prone to aggregation, it was selected for further studies in cell lines and in vivo. We observed that incubation of Burkitt

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lymphoma BL-2 cells in OptiMEM medium with prodrug 7a for 2 h followed by their loading

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with an ROS indicator 5(6)-chloromethyl- ′ 7′-dichlorodihydrofluorescein diacetate (CMDCFH-DA) causes 15.5 + 0.4

u

(λex = 88

λem = 530

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nm) of the cells with respect to that of the cells not treated with any prodrug. In contrast, previously reported prodrug 2 induces only 2.1 + 0.1 fold increase of the fluorescence at the same conditions. However, replacing the initially used, protein free OptiMEM medium for RPMI 1640 medium supplemented with fetal calf serum (FCS) led to the dramatic increase of the intracellular oxidative stress in the presence of prodrug 2 (Table 1). Additionally, we observed that in RPMI 1640 medium supplemented with fetal calf serum (FCS) the 50 µMolar solution of

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ACCEPTED MANUSCRIPT prodrug 2 is substantially less turbid (A900nm= 0.028 + 0.008) than that in the DPBS buffer (A900nm= 0.126 + 0.008) indicating that prodrug aggregates are destabilized at these conditions. These data may explain why prodrug 2 exhibits high cytotoxicity in cellular assays, in which FCS is usually added to the medium, despite the fact that it releases low amount of ROS in cells

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under standard experimental conditions including incubation of cells in the protein free

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OptiMEM medium [16-21]. It should be also mentioned that albumin, which a major component

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of FCS, contains hydrophobic pockets potentially capable of binding prodrug 7a and facilitating its uptake by the cell. Because of this possibility, we cannot definitely conclude on the prodrug

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7a state (molecular form or albumin-bound form or both) after its de-aggregation in the presence

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of FCS.

Next, we evaluated effects of new aminoferrocene prodrugs on the viability of three

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representative cancer cell lines - Burkitt lymphoma BL-2, human T lymphocyte Jurkat and

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human ovarian carcinoma A2780 as well as on human dermal fibroblasts-adult (HDFa, Table 2). The first two cell lines were selected, since previously reported aminoferrocene-based prodrugs

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exhibited substantial activity against blood cancers [16-21]. A2780 was selected as a

u

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representative carcinoma cell line, since we planned to study the in vivo antitumor activity of the u

u

u

’

was

prodrugs.

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selected as a representative normal cell line to evaluate the toxicity of new aminoferrocene-based

We observed that prodrugs 6a, 6b and 7a exhibit intermediate activity towards BL-2 cells (IC50= 31-36 µM), whereas prodrug 7b was most active (IC50= 8 + 1 µM). The latter prodrug is also the most lipophilic compound among the newly prepared aminoferrocene-based prodrugs. However, lipophilicity seems to be not a single determinant of the cellular effects of these prodrug, since

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ACCEPTED MANUSCRIPT more lipophilic parent prodrug 2 was found to be less toxic than 7b under our experimental conditions (IC50= 23 + 2 µM). This complex behavior can be caused by the dependence of the ROS-releasing ability and cell membrane permeability of aminoferrocene-based prodrugs from a variety of factors including lipophilicity and binding affinity of the prodrugs to albumin from

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FCS,

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Further, cytotoxicity of both prodrugs 2 and 7a towards A2780 cells was found to be practically

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identical (IC50= 14-15 µM), whereas 7a was significantly more toxic towards Jurkat cells than parent prodrug 2 (IC50= 25 + 2 µM versus 44 + 2 µM for 2). Importantly, 7a as well as known

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before 2 exhibits lower toxicity towards non-malignant cells: IC50 exceeds 50 µM (Table 2).

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Unfortunately, the effect of prodrug 7a at the concentration >50 µM could not be studied due to its poor solubility in aqueous buffered solutions. Therefore, only rough estimation of cancer cell

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specificity of 7a is possible using the data obtained with the cell lines: >1.6-3.3 fold depending

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on which cancer cell line is used as a reference. These data confirm that the cancer-cell specificity is retained upon substitution of an N-benzyl residue in 2 for an N-propargyl residue in

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7a.

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Based on these in vitro data we can conclude that lipophilicity is one of the crucial factors defining cell membrane permeability, aggregation state and activity of aminoferrocene-based

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prodrugs. Though, effects of its fine-tuning on prodrug cytotoxicity are complex and difficult to predict, optimization of lipophilicity is certainly advantageous for preventing aggregation of aminoferrocene-based prodrugs in aqueous solution.

Antitumor activity of prodrug 7a in vivo

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ACCEPTED MANUSCRIPT Though many of prodrugs inducing oxidative stress exhibit strong in vitro effects, e.g. in cell cultures and primary cells, attempts to demonstrate their activity in vivo led to only limited success. In particular, Maiti, Hong, Kim and co-workers have observed small prolongation of survival time of mice with metastatic lung tumor upon intratracheal administration of SN-38-

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prodrug (0.25 mg/kg, 4 times per day every 2 days) locally to the lung tissues [15]. Furthermore,

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Passirani and colleagues have observed significant tumor growth inhibition in 9L tumor-bearing

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fischer rats upon systemic delivery of a hydroxyferrocifen derivative ansa-FcdiOH encapsulated within stealth lipid nanocapsules (20 mg/kg) via repeated intravenous injections: 10 injections

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over 14 days [32]. Furthermore, D. Lee and co-workers have developed a hybrid prodrug

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releasing quinone methide and cinnamaldehyde in the presence of H2O2, which was found to strongly inhibit growth of SW620 and DU-145 tumor xenografts when applied intravenously

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every 3 days at doses of 2-3 mg/kg [33]. Finally, our group has observed limited in vivo

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antitumor effects of aminoferrocene-based prodrugs in mice carrying human prostate cancer xenografts [18] and in hybrid male mice BDF1 carrying L1210 leukemia [17]. In the latter case

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prolongation of survival time of treated animals from 13.7 + 0.6 to 17.5 + 0.7 days was observed

µg/kg.

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in the result of 6 daily intraperitoneal injections of the best prodrug 2 (Figure 1) at a dose of 26

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One of the key challenges associated with the in vivo applications of prodrugs, whose mechanism of action relies on the interaction with intracellular targets, is finding a fine balance between high lipophilicity, required for crossing cellular membrane, and water solubility. Encouraged by the positive results for 7a in cell free settings and in cellular assays, which confirmed that this prodrug seems to have optimal lipophilicity, we investigated its effects in vivo. First, we treated healthy Wistar rats with single doses of prodrug 7a in the range of 0-250

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ACCEPTED MANUSCRIPT mg/kg administered via intraperitoneal (i.p.) injections. After the injections, we monitored the animals for the following 14 days. All of them survived this treatment and their growth was not suppressed in comparison to the control treated with the solvent. We observed no signs of acute toxicity of 7a. Furthermore, we studied the antitumor activity of 7a in Wistar rats having ’

(T8) (

u

3) O

u

(T8)

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u

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single intraperitoneal administration of the prodrug dissolved in DMSO (carrier) was conducted.

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We observed substantial tumor growth inhibition upon injection of 7a in the dose of 30 mg/kg (0.25 mL injection volume, N=6, p < 0.001). The antitumor effect was not enhanced at higher

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doses applied (60, 125 mg/kg). In contrast the dose of 10 mg/kg (0.1 mL injection volume) was

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found to be inactive (Figure 3A, B). Importantly, we confirmed that injection of the carrier only in volumes between 0.1 and 0.25 mL does not affect the tumor growth. On day 21 of the

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experiment blood of healthy, tumor-bearing non-treated and tumor-bearing treated (7a, 10 and 30

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mg/kg doses) rats was analyzed. We observed that tumor growth leads to reduction of erythrocytes and the concentration of haemoglobin in blood and the treatment with the carrier

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only (0.1-0.25 mL) as well as with the inactive 7a dose of 10 mg/kg did not affect these

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parameters. In contrast, the treatment with the active 7a dose of 30 mg/kg led to their recovery

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back to the normal values (Figure 4).

CONCLUSIONS

We optimized lipophilicity of the aminoferrocene-based prodrug 2 (logP= 4.51 + 0.03) by replacing the N-benzyl with a variety of related N-substituents including N-allyl, N-(2-propenyl), N-propargyl and N-(2-propinyl) residues. The lowest lipophilicity was found for the N-propargyl derivative 7a (logP= 3.78 + 0.05). The latter prodrug exhibited the best solubility in the aqueous

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ACCEPTED MANUSCRIPT buffer and the highest activity as a catalyst for the generation of reactive oxygen species both in cell free settings and in cells. We observed that 7a was toxic to a series of human cancer cells including human Burkitt lymphoma (BL-2), human ovarian carcinoma A2780 and human T lymphocyte cell line with IC50 values ranging between 15 and 31 µM, but was not toxic towards

u

u

w

u

’

(T8)

W

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7a w

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non-malignant cells (human dermal fibroblasts-adult, HDFa) up to 50 µM. Importantly, prodrug

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of 30 mg/kg. Moreover, the latter treatment increased the number of erythrocytes and amount of haemoglobin in blood to normal values, whereas in tumor-bearing and untreated animals these

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values were substantially lowered. This is the first observation of the strong antitumor in vivo

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effect for aminoferrocene-based prodrugs.

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EXPERIMENTAL SECTION

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General Information

Commercially available chemicals of the best quality from Sigma-Aldrich (Germany) were

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obtained and used without purification. NMR spectra were acquired on either a Bruker Avance

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300 or a Bruker Avance 400 or a Bruker Avance III 600 spectrometer. ESI mass spectra were recorded on a Bruker ESI MicroTOF II mass spectrometer. C, H, and N analysis was performed

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by the microanalytical laboratory of the chemical institutes of the Friedrich-AlexanderUniversity of Erlangen-Nürnberg. UV-Vis spectra were measured on either a Lambda Bio+ UV/Vis spectrophotometer (Perkin Elmer) or a Cary 100 UV-Vis Spectrophotometer (Agilent Technologies) by using quartz glass cuvettes (Hellma GmbH, Germany) with a sample volume of 1 mL or micro-cuvettes with a sample volume of 100 µL (BRAND GmbH, Germany). The fluorescence of live cells was quantified using a Guava easyCyteTM 6-2L Flow cytometer from

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ACCEPTED MANUSCRIPT Merck Millipore. The data were processed using the inCyteTM software package from Merck Millipore and the ModFIT LTTM software from Verity Software House. The purity of the new compounds was determined by C, H, and N analysis. According to these data, their purity was greater than 95%.

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Synthesis

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N-Allyl-4-(ferrocenylcarbamatmethyl)phenyl boronic acid pinacol ester (prodrug 6a)

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Compound 1 (350 mg, 759 µmol) was dissolved in anhydrous DMF (20 mL) under nitrogen atmosphere. Then Cs2CO3 (750 mg, 2.23 mmol) and tetrabutylammonium iodide (822 mg,

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2.23 mmol) were added to the solution. After stirring for 30 minutes at 22 °C, allyl chloride

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(61.0 mg, 60.0 µL, 800 µmol) was added. The mixture was stirred for 18 h at 22 °C. Then, the solvent was removed in vacuum (0.01 mbar) and the product was purified by column

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chromatography on silica gel using petroleum ether/acetone (10/1, v/v) as eluent yielding an

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orange oil. Yield: 150 mg, 305 µmol (40%). TLC (SiO2, eluent petroleum ether/acetone, 4:1, v/v) Rf = 0.28; 1H-NMR (aceton-d6 3

M z) δ (

)77 (

3

J = 7.8 Hz, 2 H), 7.42 (d, 3J =

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7.6 Hz, 2 H), 5.95 (m, 1H), 5.19 (s, 2 H), 5.18 (m, 2 H) 4.49 (s, 2 H), 4.36 (s, 2 H), 4.12 (s, 5 H), 13

C-NMR (75 MHz, CDCl3): δ (

)

39

3

9

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3.97 (s, 2 H), 1.31 (s, 12 H).

134.31, 127.30, 116.10, 114.95, 84.00, 77.36, 69.10, 67.56, 64.54, 62.56, 52.85, 25.01; HR-MS

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(ESI+), m/z: calculated for C27H32BFeNO4 [M]+ 501.17732, found 501.17870; C, H, N analysis: calculated for C27H32BFeNO4 (%) – C 64.70, H 6.44, N 2.79; found – C 64.55, H 6.25, N 2.88.

N-(But-2-en-1-yl)-4-(ferrocenylcarbamatmethyl)phenyl boronic acid pinacol ester (prodrug 6b) This prodrug was prepared analogously to 6a except that trans-crotyl-bromide was used in place of allyl chloride and the product was purified by preparative TLC on silica gel using

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ACCEPTED MANUSCRIPT petroleum ether/acetone (4/1, v/v) as eluent yielding an orange oil. Yield: 147 mg, 434 µmol (66 %) of the E and Z isomer. TLC (SiO2, eluent petroleum ether/acetone, 4:1, v/v) Rf = 0.47; 1HNMR (aceton-d6 3

M z) δ (

)77 (

3

J = 8.1 Hz, 2 H), 7.42 (d, 3J = 8.1 Hz, 2 H), 5.63

(m, 2 H), 5.19 (s, 2 H), 4.50 (s, 2 H), 4.26 (s, 2 H) 4.11 (s, 5 H), 3.96 (s, 2 H), 1.66 (m, 3 H), 1.31 (s, 12 H). 13C-NMR (75 MHz, CDCl3): δ (

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) 154.52, 139.60, 135.07, 127.70, 127.25, 127.04,

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126.11, 83.97, 77.36, 69.08, 67.46, 64.49, 62.44, 51.98, 24.99, 17.91; HR-MS (ESI+), m/z:

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calculated for C28H34BFeNO4 [M]+ 515.19299, found 515.19396; C, H, N analysis: calculated for

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C28H34BFeNO4 (%) – C 65.27, H 6.65, N 2.72; found – C 65.35, H 6.64, N 2.92.

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N-Propargyl-4-(ferrocenylcarbamatmeBrBrthyl)phenyl boronic acid pinacol ester (prodrug 7a) This prodrug was prepared analogously to 6a except that propargyl chloride was used in place of

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allyl chloride and the product was purified by column chromatography on silica gel using

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petroleum ether/acetone (6/1, v/v) as eluent yielding an orange oil. Yield: 270 mg, 0.54 mmol (50%). TLC (SiO2, eluent petroleum ether/acetone, 4:1, v/v) Rf = 0.47; 1H-NMR (aceton-d6, 300 ) 7 73 (

3

J = 7.9 Hz, 2 H), 7.45 (d, 3J = 7.7 Hz, 2 H), 5.22 (s, 2 H), 4.56 (s, 2 H),

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M z) δ (

aceton-d6): δ (

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4.54 (s, 2 H), 4.17 (s, 5 H), 4.01 (s, 2 H), 2.87 (s, 1 H), 1.31 (s, 12 H). )

8

3

3

3

9

7 38

99 8

13

C-NMR (101 MHz,

3 8 97 73 3

69

AC

67.50, 64.85, 62.91, 39.84, 24.88; HR-MS (ESI+), m/z: calculated for C27H30BFeNO4 [M]+ 499.1617, found 499.1634; C, H, N analysis: calculated for C27H30BFeNO4 (%) – C 64.96, H 6.06, N 2.81; found – C 65.14, H 6.26, N 2.76.

But-2-yn-1-yl-tosylat (8d) But-2-yn-1-ol (160 µL, 150 mg, 2.14 mmol) was dissolved in 5 mL diethyl ether. NaOH (840 mg, 21.0 mmol) was added in portions at 0 °C. Afterwards, 4-

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ACCEPTED MANUSCRIPT toluenesulfonyl chloride was added (407 mg, 2,14 mmol) After stirring for 48 h at 22 °C, the reaction was quenched with 20 mL water. The aqueous phase was extracted three times with 10 mL ethylacetate and the combined organic layers were dried over MgSO4. Then, the solvent was removed in vacuum (0.01 mbar) and the product was purified by column chromatography on

M z) δ (

); 7

(

);

8

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(s, 2H); 2.30 (s, 2H); 1.76 (s, 3H).

) 7 66 (

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mg, 892 µmol (42%). 1H-NMR (aceton-d6 3

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silica gel using petroleum ether/ethyl acetate (7/1, v/v) as eluent yielding colorless oil. Yield: 200

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N-(But-2-yn)-4-(ferrocenylcarbamatmethyl)phenyl boronic acid pinacol ester (prodrug 7b)

AN

This prodrug was prepared analogously to 6a except that but-2-yn-1-yl-tosylat was used in place of allyl chloride and the product was purified by preparative TLC on silica gel using petroleum

M

ether/acetone (4/1, v/v) as eluent yielding an orange oil. Yield: 170 mg, 434 µmol (76 %). TLC

ED

(SiO2, eluent petroleum ether/acetone, 4:1, v/v) Rf = 0.41; 1H-NMR (aceton-d6 3

M z) δ

(ppm) 7.75 (d, 3J = 8.0 Hz, 2 H), 7.46 (d, 3J = 7.8 Hz, 2 H), 5.23 (s, 2 H), 4.57 (s, 2 H), 4.48 (s, 2

PT

H), 4.17 (s, 5 H), 4.01 (s, 2 H), 1.83 (s, 3 H), 1.31 (s, 12 H).

13

C-NMR (75 MHz, aceton-d6): δ

CE

(ppm) 154.45, 140.93, 135.60, 130.46, 127.77, 101.59, 84.54, 80.09, 76.53, 69.73, 67.73, 65.10, 63.14, 40.42, 25.18, 3.37.; HR-MS (ESI+), m/z: calculated for C28H32BFeNO4 [M]+ 513.17734,

AC

found 513.17773; C, H, N analysis: calculated for C28H32BFeNO4 (%) – C 65.53, H 6.28, N 2.73; found – C 65.86, H 5.88, N 2.55.

Assays in cell free settings Determination of logP-values Octanol/water partition coefficients (LogP values) were determined by using reversed phase - thin layer chromatography (RP-TLC) on TLC plates [34,

15

ACCEPTED MANUSCRIPT 35] from Macherey-Nagel (Germany): Alugram RP-18W/UV254, stationary phase thickness: 0.15 mm. The spots of prodrugs and reference compounds were monitored by using UVimaging. A mixture of DMF/aqueous MOPS buffer (100 mM, pH 7.4) (1/1, v/v) was used as an eluent. A set of reference compounds was used, for which logP-values were reported:

T

benzylalcohol (1.1) [36], 8-hydroxyquinoline (1.9) [37], benzophenone (3.18) [36] and

IP

anthracene (4.5) [36]. A calibration plot of Rf versus logP, which was obtained based on these

CR

data, was used to determine logP values of new prodrugs. Data obtained are provided in Table 1.

μL) w

DPBS (99 μL)

°

u

R u

u

AN

MSO

US

Determination of turbidity of solutions of prodrugs at 50 µM A solution of a prodrug (5 mM in

(the final prodrug concentration 50 µM) appeared as slightly cloudy, but stable for at least over 8

ED

absorbance was measured at 900 nm.

M

h solutions. After 8 h standing at 22 °C these solutions were mixed thoroughly and the

μL)

w

qu u N O

CE

(

PT

ROS formation in aqueous MOPS buffer [16-21] DCFH-DA (4.9 mg) was dissolved in DMF (

M 9

μL) T

u

u

w

u

for 30 min at 22 °C in the dark to obtain a stock solution of DCFH (10 mM). Next, a solution (1

AC

L)

mM), and H2O2 ( 3

)

u

(

μM) MOPS u

(100 mM, pH 7.5), EDTA (10 mM), GSH (5

M) w w

u u

(

(λex = M

MSO

λem = μL) w

added, and the fluorescence monitoring was continued until the fluorescence signal growth was stalled. Data obtained are given in table 1 of the main text of the paper. A representative kinetics of ROS release in the presence of prodrugs 2 and 7a is given in Figure 2A.

16

ACCEPTED MANUSCRIPT

ROS formation in DMF/MOPS buffer 1:1 A stock solution of DCFH (10 mM) was prepared as N

u

(

L)

(

μM), MOPS buffer (100 mM,

pH 7.5), EDTA (10 mM), GSH (5 mM), and H2O2 (10 mM) was prepared and mixed with DMF u u

(

(λex = M

λem = 531 nm) of this solution was

MSO

μL) w

T

L) M

added, and the fluorescence

IP

(

CR

monitoring was continued until the fluorescence signal growth was stalled. A representative

US

kinetics of ROS release in the presence of prodrugs 2 and 7a is given in Figure 2B.

AN

Cellular Assays

Cells and Cell Cultures Burkitt lymphoma cell line BL2 and the T cell leukemia cell line Jurkat

M

were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

ED

(DSMZ, Germany). Human ovarian carcinoma cell line A2780 and Human Dermal Fibroblasts adult (HDFa) cell line were obtained from Sigma-Aldrich. Cells were cultured according to

PT

recommendations of DSMZ. In particular, BL-2 cells were grown in RPMI 1640 medium

CE

supplemented with 20% FCS, 1% L-glutamine, and 1% penicillin/streptomycin. The cells were grown to 0.5−1.5×106 cells/mL and diluted as required. Jurkat cells were cultivated in RPMI

AC

1640 medium supplemented with 10% FCS and 1% glutamine (ThermoFisher Scientific, Waltham, MA, USA) to 1x105 – 1x106 cells/ml. A2780 cells were grown in 1640 RPMI medium and HDFa cells were grown in DMEM medium all supplemented with 10% FCS, 1% Lu

%

/

u

7 −8 %

u

.

17

ACCEPTED MANUSCRIPT Estimation of oxidative stress in live BL-2 cells An aliquot of the BL-2 cells was taken from the cultivation medium (RPMI 1640 supplemented with 20% FCS, 1% L-glutamine, and 1% penicillin/ streptomycin). The medium was replaced with PBS buffer to obtain a cell suspension containing 106 cells/mL. CM-DCFH-

u

(8 7 μL

M

MSO) w

T

suspension (8.7 mL) and incubated in the dark chamber filled with CO2 (5%) at 37 °C for 15

IP

min. Then the cells were washed with phosphate buffered saline (PBS), re-suspended either in

CR

Opti-MEM medium or in RPMI 1640 medium (supplemented with 5% FCS, 1% L-glutamine, and 1% penicillin/ streptomycin)

μL

( μL

US

solutions, solvent DMSO, different concentrations) were added. After 120 min of incubation in (λex = 488

AN

the dark chamber filled with CO2 (5%) at 37 °C, the mean fluoresce

λem = 530 nm) in the suspensions was determined by using the flow cytometer. Data

ED

M

obtained are given in Table 1 of the main text of the paper.

Determination of cell permeability of the prodrugs BL-2 cells grown in RPMI 1640 medium

PT

supplemented with 20% FCS, 1% glutamine, and 1% penicillin/streptomycin were centrifuged,

CE

and the medium was replaced with RPMI 1640 medium (5% FCS, 1% L-glutamine, 1% penicillin/streptomycin) to obtain suspensions containing 106 cells/mL. Solutions of prodrugs (10 MSO) w

AC

μL

w

PBS u

u

u (3 ×

(

u μL)

w w

L)

u

μM

T

w

ntrated H2O2

u

w

w

(

μL

M)

30 min, and all volatiles were removed by lyophilization. Dry, lysed cells were washed with w

(

μL)

qu u

u

this solution was extracted with 2-ethyl-1,3-

w

w (

μL

( %

μL

M) T 3,

v/v), and a

18

ACCEPTED MANUSCRIPT (7 μL) w / )

u u

u

u

w (

2SO4/CH3CO2

μL

(

μL

L

/

)w

added and allowed to react for 2 h. The reaction was quenched by addition of water (1 mL). The light absorbance at 550 and 780 nm of the organic phase was measured. The former value

T

corresponds to absorbance of curcumin−boron complex, whereas the second one is taken as a

IP

baseline. The baseline corrected absorbance at 550 nm (A(550 nm) − A(780 nm)) was

CR

proportional to the concentration of boron in the mixture. The baseline corrected absorbance obtained for prodrug 2 was used as a reference – 1.0. Relative to prodrug 2 cell permeability of

AN

US

prodrug 7a was found to be 0.200 + 0.003.

Determination of the viability of BL-2 cells The cells were centrifuged, the medium was

M

removed, and the cells were washed two times with PBS buffer and resuspended in RPMI 1640

ED

medium containing 5% FBS, 1% L-glutamine, and 1% penicillin/ streptomycin. This suspension was spread in the wells of a 96-w u

μM) w

μL) S

MSO

w

CE

wells w

w

( μL

PT

u

(

u

8

37 ° u

5% CO2. Four experiments were conducted for each concentration of the prodrug. Finally, MTT μL w

u

AC

(

u

3

MTT ( w

u

)

PBS u u

(S S)

( mL)) was added to u

(9 μL

%

solution in 0.01 M aqueous HCl), and incubated overnight. Afterward, the intensity of the absorbance at 590 nm was measured. MTT is converted in live cells to blue dye with the u

λmax at 590 nm. The absorbance at 690 nm was taken as a baseline value.

The baseline corrected absorbance at 590 nm (A(590 nm) − A(690 nm)) was applied to calculate

19

ACCEPTED MANUSCRIPT the relative number of viable cells. IC50 values were determined by fitting the experimental data expressing the number of viable cells (%, OY-axis) versus drug concentration (OX axis) with a sigmoidal curve using a curve fitting system for Windows: CurveExpert 1.4.

T

Determination of the viability of A2780 and HDFa. The medium was removed, and the cells

IP

were washed two times with PBS buffer, trypsinated, and resuspended in RPMI 1640 medium

CR

containing 5% FCS, 1% L-glutamine, and 1% penicillin/streptomycin (for A2780 cell line) or in DMEM supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin (for

US

HDFa cell line). This suspension was spread in the wells of a 96-well microtiter plate containing

AN

either 12.500 cells (for A2780) or 10.000 cells (for HDFa)

w

μL

standing

at 37 °C in the chamber filled with CO2 (5 %) for 18 h. Stock solutions of prodrugs of different MSO

w

M

( μL

w

μM)

ED

were added to the wells and incubated for specific periods of time. Four experiments were conducted for each concentration of the prodrug. Finally, MTT (

μL

u

S S

u

(9

CE

w

PT

by dissolving MTT (5 mg) in PBS buffer (1 mL)) was added to each well, incubated for 3 h, μL

%

lution in 0.01 M aqueous HCl), and incubated

AC

overnight. Afterward, the intensity of the absorbance at 590 nm was measured. MTT is u

w

u

λmax at 590 nm. The absorbance

at 690 nm was taken as a baseline value. The baseline corrected absorbance at 590 nm (A(590 nm) − A(690 nm)) was applied to calculate the relative number of viable cells. IC 50 values were determined by fitting the experimental data expressing the number of viable cells (%, OY-axis) versus drug concentration (OX axis) with a sigmoidal curve using a curve fitting system for Windows: CurveExpert 1.4.

20

ACCEPTED MANUSCRIPT

Determination of the viability of Jurkat cells Jurkat cells were counted in MUSE Cell Analyzer using MUSE® Count &Viability Assay Kit (Merck-Millipore, Billerica, MA, USA) and adjusted to a density of 1.0 x 105/ml in RPMI 1640

T

medium (with 10% FCS and 1% glutamine). 9.000 cells per well per 90 µl were seeded into 96

IP

well culture plates. The 50 mM prodrug stock solutions were diluted to 500 µM, 250 µM, 125

CR

µM and 62.5 µM in cell culture medium and 10 µl of the dilutions was pipetted to the cells, receiving final test concentrations of 50 µM, 25 µM, 12.5 µM and 6.25 µM. Every concentration

US

was tested in triplicates. Cells with corresponding amounts of DMSO served as negative

AN

controls. After 48 h cells were mixed and 25 µl of the cell suspension was incubated with 200 µL of freshly prepared staining solution, consisting of 1 µl AnnexinA5-Fitc, 0.4 µl 1,1′,3,3,3′,3′-

M

hexamethylindodicarbocyanine iodide (DiIC1(5)) (both from Thermo Fisher Scientific,

ED

Waltham, MA, USA) and 66.6 ng propidium iodide (PI) (Sigma-Aldrich, Taufkirchen, Germany) per 1 ml Ringer’s solution (Fresenius Kabi AG, Bad Homburg, Germany). Cells were incubated

PT

for 20 min at 4°C and then analyzed in Gallios cytofluorometer™ (Beckman Coulter, Fullerton,

CE

CA, USA). Fitc and propidium iodide were excited at 488 nm; Fitc fluorescence was recorded on the FL1 sensor (525/38 nm band pass, BP) and propidium iodide fluorescence was detected on

AC

FL3 sensor (620/30 nm BP). DiIC1(5) fluorescence was excited at 638 nm and detected at 675/20 nm BP. To eliminate any fluorescence bleed-through, electronic compensation was used. Data were analyzed employing Kaluza™ software Version 1.2 (Beckman Coulter, Fullerton, CA, USA) and processed in Microsoft Excel. Cells negative for Annexin A5-Fitc and propidium iodide were considered viable. IC50 values were determined by fitting the % values of Ax-PIcells versus drug concentration with a sigmoidal curve using GraphPad Prism software.

21

ACCEPTED MANUSCRIPT

In vivo experiments Wistar rats weighing 70-110 g were obtained from the vivarium of Dnepropetrovsk Agricultural University. All manipulations with animals have been carried out under narcosis in

T

accordance with the EU Directive 2010/63/EU for animal experiments and Permission of the

CR

IP

Ministry of Education and Science of Ukraine.

Study of the anti-tumor activity of prodrug 7a Tumor transplantation was performed by u

j

u

’

u

US

u u

(

% n PBS, v/v) in the thigh

AN

area of the animals as described previously [38-40]. A single intraperitoneal administration of prodrug 7a at doses of either 10 (0.1 mL injection volume) or 30 mg/kg (0.25 mL injection

M

volume) was made on the ninth day after tumor inoculation. In control groups of tumor-bearing

ED

animals the same quantities of the solvents (carrier – DMSO, 0.1 and 0.25 mL) were introduced. Each group contained 4-6 animals. The volume of tumors was measured during this in vivo

’

-test. On day 21, the animals were sacrificed under chloroform narcosis

CE

Su

PT

experiment as previously described [41]. The data were compared with each other using

according to the rules of the Ethics Committee and the tumors were isolated and weighed.

AC

Quantities of erythrocytes and amount of haemoglobin in blood were measured according to commonly accepted methods [42].

ABBREVIATIONS BL-2, Burkitt lymphoma; CM-DCFH-DA, 5(6)-chloromethyl- ′ 7′-dichlorodihydrofluorescein diacetate;

’ 7’-dichlorofluorescin;

M M

u

’

u ; DMF,

22

ACCEPTED MANUSCRIPT N,N-dimethylformamide; DMSO, dimethylsulfoxide; DPBS, Dulbecco's Phosphate-Buffered Saline; DU-145, human prostate carcinoma; EDTA, N,N,N’,N’-ethylenediaminetetraacetic acid FCS, fetal calf serum; GSH, glutathione; Hb, haemoglobin; IC50, the concentration, at which half of the cells remains viable; i.p., intraperitoneal; JVM-2, human mantle cell lymphoma; L1210,

T

mouse lymphocytic leukemia; LogP, n-octanol/water partition coefficient; MOPS, 3-(N-

IP

morpholino)propanesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

CR

bromide; Opti-MEM, reduced serum medium; PBS, phosphate buffered saline; RAJI, Burkitt lymphoma; RBC, red blood cells; Rf, retention factor (for TLC); ROS, reactive oxygen species;

u

; T8

u

’

;

AN

SDS, sodium dodecyl sulfate; SW6

US

RPMI, Roswell Park Memorial Institute; RP-TLC, reversed phase thin layer chromatography;

M

TLC, thin layer chromatography

ED

ACKNOWLEDGMENT

Erlangen-Nürnberg /

PT

AM thanks German Research Council (MO 1418/7-1) Friedrich-Alexander-University of (

j

“

L

”)

CE

Hertha und Helmut Schmauser-Stiftung (H4-812-01) for financial support. AM and NS thanks German Academic Exchange Service (DAAD) for funding a short term visit of N.S. to the

AC

laboratory of A.M.

23

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T

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ACCEPTED MANUSCRIPT 13. Y. Kuang, K. Balakrishnan, V. Gandhi, X. Peng, J. Am. Chem. Soc. 133(48) (2011), 1927819281. 14. S. Cao, Y. Wang, X. Peng, Chem. Eur. J. 18(13) (2012), 3850-3854. 15. E. J. Kim, S. Bhuniya, H. Lee, H. M. Kim, C. Cheong, S. Maiti, K. S. Hong, J. S. Kim, J.

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ACCEPTED MANUSCRIPT 28. F. Antunes, R. Cadenas, FEBS Lett. 475 (2000), 121-126. 29. T. P. Szatrowski, C. F. Nathan, Cancer Res. 51 (1991), 794-798. 30. J O’

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31. C. Reichardt, Solvents and Solvent Effects in Organic Chemistry, Wiley-VCH Publishers, 3rd

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ed., 2003.

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32. A.-L. Laine, A. Clavreul, A. Rousseau, C. Tètaud, A. Vessières, E. Garcion, G. Jaouen, L.

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Aubert, M. Guilbert, J.-P. Benoit, R.-A. Toillon, C. Passirani, Nanomed. 10(8) (2014), 16671677.

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33. J. Noh, B. Kwon, E. Han, M. Park, W. Yang, W. Cho, W. Yoo, G. Khang, D. Lee, Nature

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Comm. 6 (2015), 1-9.

34. G. L. Biagi, A. M. Barbaro, M. C. Guerra, M. F. Gamba, J. Chromatography 41 (1969), 371-

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35. G. L. Biagi, A. M. Barbaro, M. C. Guerra, M. F. Gamba, J. Chromatography 44 (1969), 195198.

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36. J. Sangster, J. Phys. Chem. Ref. Data 18 (1989), 1111-1227.

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37. K. A. Lewis, J. Tzilivakis, D. Warner, A. Green, Hum. Ecol. Risk Assess. 22(4) (2016), 1050-1064.

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38. N. Shtemenko, P. Collery, A. Shtemenko, Anticancer Res. 27(4B) (2007), 2487-2492. 39. A. V. Shtemenko, P. Collery, N. I. Shtemenko, K. V. Domasevitch, E. D. Zabitskaya, A. A. Golichenko, J. Chem. Soc., Dalton Trans. 26 (2009), 5132-5136. 40. N. Shtemenko, K. Domasevitch, A. Golichenko, S. Babiy, Z. Li, K. Paramonova, A. Shtemenko, H. Chifotides, K. Dunbar, J. Inorg. Biochem. 129 (2013), 127–134. 41. L.-Q. Sun, Y.-X. Li, L. Guillou, P. A. Coucke, Cancer Res. 58 (1998), 5411-5417.

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ACCEPTED MANUSCRIPT 42. C. A. Lugovskaya, B. T. Morozova, M. E. Pochtar, V. V. Dolgov, Laboratory hematology

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CE

PT

ED

M

AN

US

CR

IP

T

(Russ). – “Triada” Tver, Russia, 2006 – 223 pp, ISBN 5-94789-156-5.

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ACCEPTED MANUSCRIPT Tables

Table 1. Properties of new and previously reported aminoferrocene-based anticancer prodrugs. Prodrug

LogPi

A900 nm(prodrug)ii

ROS in vitroiii

ROS in cellsiv

24 + 15

6a

4.16 + 0.05

0.032 + 0.011

42 + 12

6b

4.42 + 0.04

0.097 + 0.008

30 + 7

7a

3.78 + 0.05

0.019 + 0.003

7b

4.18 + 0.03

0.085 + 0.020

IP

0.126 + 0.008

2.1 + 0.1 / 14.7 + 0.5 -

AN

CR

4.51 + 0.03

-

53 + 6

15.5 + 0.4 / 23 + 1

28 + 14

-

ii

M

LogP – n-octanol/water partition coefficients.

A900 nm – absorbance at 900 nm of solutions of prodrugs (50 µM) in DPBS buffer containing

ED

i

2

US

T

-FCS / +FCS

These values correspond to F/F0 w u

(λex =

λem = 531 nm) of a

’ 7’-dichlorofluorescin (DCFH), N,N,N’,N’-ethylenediaminetetraacetic acid

CE

iii

PT

DMSO (1 %, v/v). A900 nm of the buffer containing DMSO (1 %, v/v) was used as a baseline.

AC

(EDTA, 10 mM), GSH (5 mM) and H2O2 (10 mM) in 3-(N-morpholino)propanesulfonic acid buffer (MOPS, 100 mM, pH 7.5) treated with corresponding prodrugs (50 µM) for 145 min; F0 is the emission of same mixture lacking the prodrugs. iv

These values correspond to F/F0, where F is the mean fluorescence of BL-

(λex = 488

λem = 530 nm) loaded with 5(6)-chloromethyl- ′ 7′-dichlorodihydrofluorescein diacetate (CM-DCFH-DA) and incubated with corresponding prodrugs (20 µM) for 2 h. F0 is the mean

28

ACCEPTED MANUSCRIPT fluorescence of BLw

u ; “-

“+

S” – 5 %

S” – no protein was added to the medium.

CE

PT

ED

M

AN

US

CR

IP

T

u

u

AC

fetal b

u

29

ACCEPTED MANUSCRIPT Table 2. Cytotoxicity of new prodrugs towards selected cancer and normal cell lines. IC50 (µM)ii

ii

A2780

Jurkat

HDFa

2

23 + 2

14 + 1

44 + 2

>50

6a

36 + 5

-

-

-

6b

31 + 1

-

7a

31 + 1

15 + 2

7b

8+1

-

IP CR

-

-

25 + 2

>50

-

-

US

Structures of prodrugs are shown in Figure 1.

T

BL-2

AN

i

Prodrugi

IC50 is the concentration, at which half of the cells remains viable; BL-2: Burkitt lymphoma

M

cell line; A2780: human ovarian carcinoma cell line; Jurkat: human T lymphocyte cell line;

AC

CE

PT

ED

HDFa: human dermal fibroblasts-adult (representative non-malignant cells).

30

ACCEPTED MANUSCRIPT Figure/Scheme captions

Figure 1. Structures of known aminoferrocene-based prodrugs 1 and 2 and their modifications studied in this paper (prodrugs 6a, 6b, 7a and 7b). A mechanism of activation of prodrug 2 in u

;

R’S

u

;

T

cancer cells is given in an inset. GS =

IP

ROS= reactive oxygen species. The formal charge of Fe in the ferrocene units was shown to

CR

explain the charge balance in the given equation. However, in the real system the charge is

u

(λex = 501 nm) of solutions containing (plot A) -

AN

Figure 2

US

delocalized over the whole ferrocene system rather than being localized on the metal center.

DCFH (10 µM), MOPS buffer (100 mM, pH 7.5), EDTA (10 mM), GSH (5 mM), corresponding

M

prodrugs (50 µM) and H2O2 (10 mM) or (plot B) - DCFH (5 µM), MOPS buffer (50 mM, pH

ED

7.5), EDTA (5 mM), GSH (2.5 mM), DMF (50 %, v/v) and H2O2 (5 mM). Corresponding

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prodrugs (50 µM) (as shown in legends) were added on the 5th minute to initiate the reaction.

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Figure 3. A: Change of tumor (T8) volume in non-treated rats () and rats treated by a single intraperitoneal administration of either a carrier only (DMSO, 0.1 mL,) or prodrug 7a in a

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carrier (10 mg/kg, ). B: the same as A except that carrier volume was increased to 0.25 mL and prodrug 7a dose – to 30 mg/kg. Starting from 9th day tumor volume in treated animals (N=6) is lower than that in animals, which received only the carrier (N=4, p < 0.001).

Figure 4. A: Amount of red blood cells (RBC) in non-treated rats (11.7 + 1.6 x 1012) was taken “

” R

u

RBS (

[RB ]

B:

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ACCEPTED MANUSCRIPT Concentration of haemoglobin (Hb) in non-treated rats (164 + 12 g/L) was taken as a reference “

” R

[

]

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Scheme 1. Synthesis of new N-substituted aminoferrocene-based prodrugs 6a, 6b, 7a and 7b.

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Figures and schemes

Figure 1.

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ACCEPTED MANUSCRIPT

A

B

20

Fluorescnece (a.u.)

no prodrug prodrug 2 prodrug 7a

100

0

no prodrug

10

prodrug 2 prodrug 7a

180 Time (min)

360

0

180 Time (min)

0

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80

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80

B

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160 Tumor volume (mm3)

0

9 11 13 15 17 19 21 Time (days)

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Figure 3.

9 11 13 15 17 19 21 Time (days)

0.2 T8 Carrier, mL 0 7a, mg/kg 0

B

A

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relative [RBC]

1

0.6

7

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7

relative [Hb]

Tumor volume (mm3)

160

T8 T8+carrier_0.25mL T8+7a_30 mg/kg

A

M

T8 T8+carrier_0.1mL T8+7a_10 mg/kg

360

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Figure 2.

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0 0

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Fluorescnece (a.u.)

200

1

0.9

0.8 + 0 0

+ + + + 0.1 0.1 0.25 0.25 0 10 0 30

0 0

+ 0 0

+ + + + 0.1 0.1 0.25 0.25 0 10 0 30

Figure 4.

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ACCEPTED MANUSCRIPT

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Scheme 1.

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Graphical abstract

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Synopsis

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Substitution of N-benzyl for an N-propargyl residue in the anticancer prodrug 4-(N-benzyl-Nferrocenylaminocarbonyloxymethyl)phenylboronic acid pinacol ester led to reduction of its

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lipophilicity, improvement of its water solubility, decrease of its propensity towards aggregation and dramatic increase of its ROS-generating properties without any negative effect on its anticancer activity.

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ACCEPTED MANUSCRIPT Highlights A series of N-alkylaminoferrocene-based prodrugs were prepared



A derivative with an N-propargyl residue was found to have the optimal properties



The optimized prodrug exhibited relatively low lipophilicity and high water solubility

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The activity of the prodrug in selected cancer cell lines was in the range of 15-31 µM

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The optimized prodrug exhibited high antitumor activity in vivo (rat, T8 carcinoma).

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

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