Emeline Cros-Perrial

Steve Saulnier

Muhammad Zawwad Raza

Rémi Charmelot

David Egron

Charles Dumontet

Laurent Chaloin

Suzanne Peyrottes

Lars Petter Jordheim

Cytotoxic and antitumoral activity of N-(9H-purin-6-yl) benzamide derivatives and related watersoluble prodrugs Running title: Cytotoxic purine derivatives

Keywords: Cell death, cancer cells, prodrug, in vivo

The development of small molecules as cancer treatments is still of both interest and importance.

Having synthesized and identified initial cytotoxic activity of a series of chemically related N-(9H-purin-6-yl) benzamide derivatives, we continued their evaluation on cancer cell models. We also synthesized water-soluble prodrugs of the main compound and performed in vivo experiments. Our results show activities on cancer cell lines ranging from 3-39 µM for the best compounds, with both induction of apoptosis and decrease in cell proliferation. Two compounds evaluated in vivo showed weak antitumoral activity. In addition, the lead compound and its prodrug had synergistic activity with the nucleoside analogue fludarabine in vitro and in vivo.

Introduction

Despite tremendous advances in the treatment options for cancer patients, in particular due to the development of immunotherapy and monoclonal antibodies, the search for new cytotoxic compounds is still of main importance in cancer research. Among the existing therapeutic arsenal, the purine scaffold is well represented [START_REF] Jordheim | Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases[END_REF][START_REF] Savic | Purine nucleoside analogs in the therapy of cancer and neuroinflammation[END_REF] (Figure 1). Purine nucleobases, such as 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well known drugs used in the treatment of childhood ALL [START_REF] Bhatia | Systemic Exposure to Thiopurines and Risk of Relapse in Children With Acute Lymphoblastic Leukemia: A Children's Oncology Group Study[END_REF][START_REF] Bostrom | Cellular pharmacology of 6-mercaptopurine in acute lymphoblastic leukemia[END_REF], and fludarabine, cladribine and clofarabine among others are purine containing nucleoside analogues commonly used in clinic [START_REF] Robak | Older and new purine nucleoside analogs for patients with acute leukemias[END_REF]. In addition, N-6-benzoyl adenine derivatives and some substituted 6phenyl purine analogues (Figure 2) have recently been identified as potential anticancer agents, respectively as BRD4 inhibitors [START_REF] Amemiya | Structure-Activity Relationship Study of N(6)-Benzoyladenine-Type BRD4 Inhibitors and Their Effects on Cell Differentiation and TNF-alpha Production[END_REF][START_REF] Noguchi-Yachide | Discovery and structure-activity relationship studies of N6-benzoyladenine derivatives as novel BRD4 inhibitors[END_REF] and as cytotoxic agents on a panel of hepatocellular cancer lines [START_REF] Kucukdumlu | Synthesis of Some Substituted 6-Phenyl Purine Analogues and Their Biological Evaluation as Cytotoxic Agents[END_REF].

Within a research program aiming at discovering cytosolic 5'-nucleotidase II inhibitors using a fragment-based drug design approach, [START_REF] Marton | Identification of Noncompetitive Inhibitors of Cytosolic 5'-Nucleotidase II Using a Fragment-Based Approach[END_REF][START_REF] Guillon | Lead optimization and biological evaluation of fragment-based cN-II inhibitors[END_REF] we identified a series of N-(9H-purin-6-yl)benzamide derivatives with cytotoxic effects in the micromolar range on human lymphoma cells. Some of these compounds showed moderate to good inhibition of the 5'-nucleotidase cN-II in vitro and synergy with nucleoside analogues on cancer cell lines. Based on the structural analogy of these series with the previously cited 6-substituted purine analogues (Figure 2) and the identified anticancer activity in the initial study, we continued herein the evaluation of the biological activity of these compounds. To this purpose, we included additional cell lines and performed various cell biology experiments on proliferation, cell cycle arrest and cell death induction. In order to perform in vivo experiments of the hit compound and due to its very low aqueous solubility, we also designed and synthesized watersoluble prodrugs that were evaluated both on cell models and in mice. These were obtained by the introduction of biolabile hydrophilic substituents on the N-9-position of the purine.

Materials and Methods

Synthesis of derivatives 1-9 and water-soluble prodrugs of compound 1 (derivatives 10-15) Synthesis of N-(9H-purin-6-yl)benzamide derivatives (1-9) has been previously reported [START_REF] Marton | Identification of Noncompetitive Inhibitors of Cytosolic 5'-Nucleotidase II Using a Fragment-Based Approach[END_REF].

Experimental details for the preparation of water-soluble prodrugs 10-15 and corresponding NMR spectra are available as SI.

Cell lines and culture

Human follicular lymphoma cells (RL), human acute promyelocytic leukemia cell (HL-60), human acute lymphoblastic leukemia (ALL) cells (CCRF-CEM), human acute T cell lymphoma cells (Jurkat), human B cell precursor leukemia cells (REH and RS4;11) and murine T cell lymphoma cells (L1210) were grown in RPMI media (Life technologies) containing L-glutamine, penicillin (200 IU/mL, Life technologies), streptomycin (200 µg/mL, Life technologies) and 10% feral bovine serum at 37 °C in a humidified atmosphere and in the presence of 5% of CO2.

Cell survival assay and synergy experiments

The sensitivity to nucleoside analogues was assessed by cell survival after 72 hours' exposure to molecules using the MTT assay and 20,000 cells per well in 96 well plates. Inhibitory concentrations 50 (IC50) and combination index 95 (CI95) were calculated with CompuSyn software 1.0 (ComboSyn, Inc., USA) as indicated earlier [START_REF] Marton | Identification of Noncompetitive Inhibitors of Cytosolic 5'-Nucleotidase II Using a Fragment-Based Approach[END_REF][START_REF] Guillon | Lead optimization and biological evaluation of fragment-based cN-II inhibitors[END_REF]. Synergy was defined as CI95 < 0.9, additivity as 0.9 < CI95 < 1.1, and antagonism as CI95 > 1.1 [START_REF] Chou | Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors[END_REF].

Apoptosis

Cell death was determined on cells incubated for 48 hours in absence or presence of compounds, by AnnexinV-positivity assessment and propidium iodide (PI) incorporation into DNA by flow cytometry using a FACS Calibur in conditions earlier described [START_REF] Jordheim | Identification and characterization of inhibitors of cytoplasmic 5'-nucleotidase cN-II issued from virtual screening[END_REF] using 5 x 10 5 cells per well in 24-well plates and a final volume of 0.5 mL. Dead or dying cells were identified as AnnexinV and/or PI positive cells.

Cell proliferation

Cell proliferation in absence or presence of selected compounds was determined by quantification of CellTrace CFSE in dividing cells by flow cytometry on a FACScalibur as indicated earlier [START_REF] Jordheim | Determination of the enzymatic activity of cytosolic 5'-nucleotidase cN-II in cancer cells: development of a simple analytical method and related cell line models[END_REF], using 0.5 x 10 6 cells per well in 6-well plates and a final volume of 3 mL. Results were analyzed with FlowJo and expressed as percentage of MFI of CFSE as compared to the signal on day 1.

Cell cycle assessment

Cell cycle distribution was assessed in cells incubated for 24 hours in absence or presence of selected compounds using an LSR-II and conditions described earlier [START_REF] Jordheim | Determination of the enzymatic activity of cytosolic 5'-nucleotidase cN-II in cancer cells: development of a simple analytical method and related cell line models[END_REF], using 5 x 10 5 cells per well in 24-well plates and a final volume of 0.5 mL. Results were analyzed using FlowJo and expressed as percentage of cells in each phase of the cell cycle.

Animal studies

For in vivo studies, 4 weeks old and acclimated female scid CB17 mice (Charles River) were injected subcutaneously with 5 x 10 6 RL cells. When tumors reached approximately 100 mm 3 (calculated by the formula 4/3 × π × [(length + width)/2] 3 ), mice received two intraperitoneal treatments with seven days interval. Body weight and tumor volume were determined 2-3 times per week and mice sacrificed when it reached 1500 mm 3 . All mice were raised in SPF environment with free access to standard food and water. This study was approved by the Animal Ethics Committee of the University Claude Bernard of Lyon.

Results

Studied compounds are cytotoxic

Compounds 1-9 were first evaluated for their intrinsic cytotoxic activity on a panel of three cancer cell lines (RL, HL-60, L1210) using the MTT assay (Table 1). We obtained IC50-values from 3.4 µM (8 on L1210 cells) to more than 300 µM (in particular on L1210 cells). Comparable toxicity was observed on all tested cell lines for several compounds (1, 2, 3, 6 and 8), whereas others had cell line specific toxicity with a better sensitivity for RL cells (4, 7 and 9).

In an initial work, and linked to its inhibitory effect on the 5'-nucleotidase cN-II [START_REF] Marton | Identification of Noncompetitive Inhibitors of Cytosolic 5'-Nucleotidase II Using a Fragment-Based Approach[END_REF], 1 was found to act synergistically with cladribine and clofarabine but not with fludarabine, in RL cells. Thus, we extended this work to other cell lines (CCRF-CEM, HL-60, Jurkat, REH, RS4;11 and L1210) as well as to the purine nucleobase 6-mercaptopurine (Table 2). No synergy was observed between 1 and the purine nucleoside analogues cladribine, clofarabine and fludarabine in human or murine cells of haematological origin, whereas there was a very good synergy with 6-mercaptopurine in REH and Jurkat cells, and a weak synergy in HL-60 and L1210 cells.

Different induction of apoptosis by compounds 1-9

In order to better describe the biological activities of these N-(9H-purin-6-yl)benzamide derivatives, we evaluated their capacity to induce apoptosis in RL and HL-60. We used high concentration of cisplatin as a positive control for induction of cell death, and two or three concentrations for fludarabine (control compound) and our compounds. These concentrations were selected based on MTT results as approximatively IC50/2, IC50 and IC50x5. As shown in Figure 3A, a dosedependent induction of apoptosis was observed for fludarabine, 2, 3, 6, 7, 8 and 9, but not for 1 and 4 on RL cells. For HL-60 cells (Figure 3B), we observed a dose-dependent induction of cell death with all tested compounds (fludarabine, 1, 2, 3 and 8).

Antiproliferative activity of the studied compounds

As the decreased MTT reducing activity was not always associated with increased cell death as shown by apoptosis, we evaluated whether some of the compounds had antiproliferative effects using CFSE staining on RL and HL-60 cells (Figures 4 and5). For fludarabine, even low concentrations not related to cell death induction, were associated with decreased cell proliferation. Compound 1 decreased proliferation of RL cells at a low concentration that was not associated with cell death, but not on HL-60 cells. Higher concentrations (125 µM for RL and 100 µM for HL-60, data not shown) were also evaluated but cells did not survive, in line with the results on apoptosis. Compounds 2 and 3 did not decrease cell proliferation even at high doses associated with cell death induction. Finally, 8 at IC50x5 slowed down proliferation of RL cells but not HL-60 cells even though this concentration induced cell death in both cell lines. 10

Cell cycle arrest of compounds 1-9

We completed the in vitro evaluation of our N-(9H-purin-6-yl)benzamide derivatives with their effect on cell cycle distribution. For RL cells (Figure 6A), we observed a dose-dependent increase in G1phase for 1, 8 and 9, as well as an important increase at all concentrations tested for 4, 6 and 7. For 3, the increase was only observed at the concentration corresponding to IC50, whereas no modification was seen with the three concentrations of 2. For HL-60 cells (Figure 6B), we did not observe any main modification in cell cycle distribution, except for with the highest concentration of 1 inducing an important increased in pre-G0 phase, consistent with AnnexinV results.

Synthesis of water-soluble prodrugs of compound 1

Due to the low water solubility of the studied compounds, that is an issue commonly encountered for large aromatic and heterocyclic derivatives, we envisaged to obtain prodrugs [START_REF] Jornada | The Prodrug Approach: A Successful Tool for Improving Drug Solubility[END_REF][START_REF] Rautio | Prodrugs: design and clinical applications[END_REF][START_REF] Rautio | The expanding role of prodrugs in contemporary drug design and development[END_REF] of our best characterized compound, 1. Thus, synthesis of acyl-oxymethyl (11a and 11b) and phosphor-oxymethyl (13, 15a, 15b and 15c) derivatives was performed and they also included different type of salts (Table 3 and Supplemental Information).

The aqueous solubilities of 11a and 15c have been determined by HPLC to be respectively 47 mg and 83 mg per mL, which is an increase of more than 1000-fold compared to the parent compound 1 (22.6 μg per mL) [START_REF] Guillon | Lead optimization and biological evaluation of fragment-based cN-II inhibitors[END_REF].

In vitro activity of soluble prodrugs

In vitro assays with RL and L1210 cells (Table 3) showed that four out of six compounds (15c, 15a, 11a and 11b) had similar cytotoxicity as the parent compound 1 (IC50 = 16-28 µM), whereas the two others had weaker (IC50 = 72 µM for 15b) or no toxicity (IC50 > 300 µM for 13). When assessed together with nucleoside analogues and 6-mercaptopurine, we observed only synergy for 1 with fludarabine on RL cells and with 6-mercaptopurine on L1210 cells, for 15a with cladribine on RL cells, and for 11b with fludarabine and 6-mercaptopurine on RL cells (Table 4).

In vivo antitumoral activity of soluble prodrugs

Based on availability, we designed an in vivo experiment with 15c and 11a alone and in combination with fludarabine using immunodeficient mice with tumors from RL cells. We used fludarabine as all prodrugs are supposed to deliver compound 1 in vivo, and that this compound shows synergy with fludarabine in RL cell model. As shown in Figure 7, treatment with 100 mg/kg fludarabine and 15 mg/kg 15c delayed tumor growth. For 21 mg/kg 11a, only the combination with 100 mg/kg fludarabine resulted in important tumor growth inhibition. None of these treatments was associated with weight loss or any other macroscopic sign of toxicity. The combination of 100 mg/kg fludarabine with 15 mg/kg 15c was however lethal with 2 of 5 mice dying three days after the first treatment.

Discussion

The first part of our study concerned the additional biological evaluation of selected N-(9Hpurin-6-yl)benzamide derivatives on cancer cells. We used various techniques to determine the activity of the compounds on metabolic activity (MTT assay), cell death induction (AnnexinV/PI staining), cell proliferation (CFSE staining) and cell cycle arrest (PI staining).

Three compounds (2, 3, and 8) out of nine clearly showed the best cytotoxicity in MTT assay with IC50 values below 15 µM, and compounds 3 and 8 exhibited significant cytotoxic activity against all three cell lines tested. For comparison, IC50 values on RL cells are approximately 40 nM for clofarabine, 100 nM for cladribine, 7 µM for fludarabine, 0.8 µM for 6-mercaptopurine and 1.2 µM for 6-thioguanine (raw data from [START_REF] Jordheim | Determination of the enzymatic activity of cytosolic 5'-nucleotidase cN-II in cancer cells: development of a simple analytical method and related cell line models[END_REF]), whereas other chemically related compounds had IC50 of around 5 µM on liver cancer cells [START_REF] Kucukdumlu | Synthesis of Some Substituted 6-Phenyl Purine Analogues and Their Biological Evaluation as Cytotoxic Agents[END_REF]. In addition, these compounds were also able to induce apoptosis in RL cells at comparable levels as with fludarabine (more than 50% dead cells at 50 µM). These results suggest that the second phenyl group should rather be in the meta position (R1=H) and a pyrrolidine substituent (compound 3) for a better activity. When, the imidazolyl substituent is attached through the nitrogen atoms (compounds 6 and 7), derivatives are lacking biological activity as determined by our MTT assay, whereas if it is linked through the C-4 atom and in the para orientation (compound 8) some cytotoxic activity is recovered. Finally, compounds 1 and 8 slowed down cell proliferation, in particular in RL cells.

Our goal was not to determine the mechanism of action of these compounds, but to better describe their activity. Therefore, we can only hypothesize on how they induce cell death in our models. Other compounds belonging to similar chemical series have been reported to be inhibitors of BRD4, an enzyme of the bromodomain and extra-terminal domain protein family involved in gene regulations and cancer cell survival, but whether our compounds do the same is unknown [START_REF] Amemiya | Structure-Activity Relationship Study of N(6)-Benzoyladenine-Type BRD4 Inhibitors and Their Effects on Cell Differentiation and TNF-alpha Production[END_REF][START_REF] Noguchi-Yachide | Discovery and structure-activity relationship studies of N6-benzoyladenine derivatives as novel BRD4 inhibitors[END_REF]. The initial target for these compounds, the 5'-nucleotidase cN-II is expressed in both RL and HL-60 cell lines, with an approximately 2-fold higher expression and activity in HL-60 cells [START_REF] Jordheim | Determination of the enzymatic activity of cytosolic 5'-nucleotidase cN-II in cancer cells: development of a simple analytical method and related cell line models[END_REF]. If the mechanism of action is based upon cN-II inhibition, we would expect a higher sensitivity in RL cells. This is the case for compounds 2, 4, 7, 8 and 9 inhibiting cN-II activity in vitro by 28, 73, 39 and 26% respectively but not for compounds 1 and 3 inhibiting by 68 and 56% respectively [START_REF] Marton | Identification of Noncompetitive Inhibitors of Cytosolic 5'-Nucleotidase II Using a Fragment-Based Approach[END_REF]. This could therefore suggest that this is not the main mechanism, or that the cN-II inhibition in cells is different from and not proportional to our previously used in vitro conditions. Indeed, we do not know whether RL and HL-60 cells express wild-type or hyperactive mutants [START_REF] Meyer | Relapsespecific mutations in NT5C2 in childhood acute lymphoblastic leukemia[END_REF][START_REF] Tzoneva | Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL[END_REF] or how cN-II is regulated in these cells by allosteric regulators or by energy status [START_REF] Itoh | Enzymatic properties and physiological roles of cytosolic 5'-nucleotidase II[END_REF][START_REF] Pesi | Cytosolic 5'-Nucleotidase II Is a Sensor of Energy Charge and Oxidative Stress: A Possible Function as Metabolic Regulator[END_REF].

The extensive study of compound 1 revealed that its moderate cytotoxic activity (25-50 µM in RL, HL-60 and L1210) is neither associated with the induction of cell death in RL cells or significant antiproliferative effect in HL-60. However, this compound clearly showed synergic effect with 6mercaptopurine in four out of six of the studied cell lines. Taking into account the mechanism of action of 6-mercaptopurine and its cellular metabolization (involving hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and its catabolic conversion into xanthine-5'-monophoshpate (from 6thioguanosine-5'-monophosphate) and that compound 1 is one of the best cN-II inhibitors within the series of N-(9H-purin-6-yl)benzamide derivatives, we could hypothesize that the observed synergy may be associated with the inhibition of cN-II, thus limiting the catabolism of xanthine-5'-monophosphate [START_REF] Brouwer | Role of 5'-nucleotidase in thiopurine metabolism: enzyme kinetic profile and association with thio-GMP levels in patients with acute lymphoblastic leukemia during 6-mercaptopurine treatment[END_REF][START_REF] Tzoneva | Clonal evolution mechanisms in NT5C2 mutant-relapsed acute lymphoblastic leukaemia[END_REF].

The second part of the study concerned the in vivo evaluation of our compounds. Common features of N-(9H-purin-6-yl)benzamide derivatives are their polycyclic aromatic structures, and as a consequence thereof, many of them have poor aqueous solubility. Despite its promising in vitro cytotoxic activity and synergistic effects with nucleoside analogues, 1 shows very low solubility in water (22.6 µg/mL), and this represents a major obstacle for its further development and study in experimental animal models. As initial attempts to formulate parenteral 1 using co-solvents (PEG…) or cyclodextrin-based agents were not successful, we designed prodrugs to improve physicochemical properties (such as water solubility) of our bioactive derivative [START_REF] Jornada | The Prodrug Approach: A Successful Tool for Improving Drug Solubility[END_REF]. Recent data from the literature [START_REF] Meanwell | Inhibitors of HIV-1 Attachment: The Discovery and Development of Temsavir and its Prodrug Fostemsavir[END_REF][START_REF] Oslob | Water-soluble prodrugs of an Aurora kinase inhibitor[END_REF] demonstrate that the incorporation of a phosphate or amino group, either directly attached to the compound or introduced via a linker, enhance the solubility of compounds to be administered intravenously or orally. We explored two series of water-soluble prodrugs of 1 which are expected to release the parent drug through different two-step enzyme-mediated hydrolytic activation (Figure 8).

The first series involved the introduction of L-valine through an oxymethyl linker. According to literature data, valyl ester prodrugs are expected to be substrates of plasma esterase leading to the labile heminal intermediate that would rapidly degraded into the parent drug and formaldehyde. The second approach involved the introduction of an oxymethylphosphate moiety that is sensitive to an initial alkaline phosphatase hydrolysis leading to the same heminal intermediate as before and in fine to 1.

Due to the potential effect of the nature of the salts on the physicochemical properties (hygroscopicity, physical form, solubility, stability…) of the resulting prodrugs, we studied five different salts (sodium, lysine and tromethamine for phosphate derivatives, trifluoroacetate and mesylate for valyl ester prodrugs) in order to select the best candidate for in vivo investigations. The water solubility, as determined for 11a and 15c, was 1000-fold increased as compared to 1. MTT-based in vitro assay showed similar activity (22-28 µM and 72 µM for 15b) of all compounds except for 13, strongly suggesting intracellular decomposition into compound 1 as shown in Figure 8. The in vivo evaluation of these drugs validated the prodrug approach, and showed weak antitumoral activity for 15c and a weak potentiation of fludarabine with compound 11a.

In conclusion, our study offers extended biological evaluation of selected N-(9H-purin-6yl)benzamide derivatives on cancer cell lines, and we also describe water-soluble prodrugs for such compounds that allow in vivo experiments. For compound 1, we can conclude on the induction of a decreased proliferation without cell cycle modifications or induction of AnnexinV-related cell death RL cells, and the induction of apoptosis in HL-60 cells. For the other compounds, the cellular modifications responsible for the initially observed cytotoxicity is less clear. Further studies would be warranted to determine the mechanism of action as well as the potential toxicity on healthy cells as this would be of main importance in an eventual further development of these compounds as anticancer drugs.

Figure 1 .

 1 Figure 1. Examples of purine and purine nucleoside analogues used as anti-cancer drugs. 6-MP: 6-

Figure 2 .

 2 Figure 2. Generic structures of purine derivatives investigated in the literature as anticancer agents [6-

Figure 3 .

 3 Figure 3. Induction of cell death by studied compounds in RL (A) and HL-60 (B) cells. Cell death was

Figure 5 .

 5 Figure 5. Proliferation of HL-60 cells in absence (NT) or presence of molecules at indicated

Figure 6 .

 6 Figure 6. Cell cycle distribution of RL (A) and HL-60 (B) cells exposed 24 hours to indicated

Figure 7 .

 7 Figure 7. Tumor growth of RL cells in mice treated with PBC prodrugs. Mice with RL-tumors were

Figure 8 .

 8 Figure 8. Structures and expected bioactivation of the two series of water-soluble prodrugs of 1.

Table 1 . Cytotoxic activity of compounds on cancer cell lines

 1 . IC50 values (µM) are mean values of at least three independent experiments ± SEM. Results on RL cells are from[START_REF] Marton | Identification of Noncompetitive Inhibitors of Cytosolic 5'-Nucleotidase II Using a Fragment-Based Approach[END_REF].

				RL a	HL-60	L1210
		R1=	R2=
	1	H		25 ± 4	18 ± 3	39 ± 5
	2	H		8.0 ± 2.5	49 ± 11	n.d.
	3	H		11 ± 2	4.1 ± 0.4	8.3 ± 0.2
	4		H	23 ± 9	>300	>300
	5		H	35 ± 3	n.d.	n.d.
	6	H		203 ± 20	91 ± 21	>300
	7	H		34 ± 5	285 ± 8	>300
	8	H		4.7 ± 0.7 12.9 ± 0.6 3.4 ± 0.2
	9	H		30 ± 3	204 ± 7	>300

Table 2 . Antiproliferative activity and synergy with nucleoside analogues of compound 1 on additional cell lines

 2 . IC50 values (µM) are mean values of at least three independent experiments ± SEM. CI95 values were calculated with the CompuSyn software and expressed as a range from

	10^(mean(logCI95))-SEM to 10^(mean(logCI95))+SEM from at least three independent experiments.
	Synergy and additivity are indicated in bold.
		CCRF-CEM	HL-60	Jurkat	REH	RS4,11	L1210
	IC50	20 ± 3	18 ± 3	20 ± 3	25 ± 7	28 ± 4	39 ± 5
	CI95 fludarabine	1.0-1.4	1.5-2.9	0.8-2.5	1.0-4.8	1.1-1.9	2.2-4.5
	CI95 cladribine	1.2-1.6	1.6-2.0	n.d.	n.d.	n.d.	n.d.
	CI95 clofarabine	1.1-1.9	0.9-2.2	n.d.	n.d.	n.d.	n.d.
	CI95 6-MP	0.9-1.8	0.6-1.0	0.4-0.6	0.1-0.4	3.4-11.9	0.8-1.0

Table 3 . Cytotoxic activity on RL and L1210 cancer cell lines of compounds for in vivo administration.

 3 IC50 values (µM) are mean values of at least three independent experiments ± SEM. n.d: not determined.

	Structure	RL	L1210
	1	22 ± 5	16 ± 3
	11a	28 ± 5	26 ± 3
	11b	26 ± 3 24 ± 11
	13	>300	>300
	15a	26 ± 5	20 ± 3
	15b	72 ± 28	n.d.

Table 4 . Synergy between nucleoside analogues and studied compounds on RL and L1210 cells.

 4 CI95values were calculated with the CompuSyn software and expressed as a range from 10^(mean(logCI95))-SEM to 10^(mean(logCI95))+SEM from at least three independent experiments.

	Synergy and additivity are indicated in bold.
			1	11a	11b	15a	15c
		CI95 fludarabine	0.49-0.74	0.77-1.47 0.78-0.95 2.18-7.22 0.91-1.13
	RL	CI95 cladribine CI95 clofarabine	0.89-1.58 0.63-1.20	0.77-1.45 0.99-1.14 0.15-0.83 0.64-1.44 0.92-1.38 1.09-1.43 0.52-1.05 1.05-1.69
		CI95 6MP	1.02-1.43	1.24-2.72 0.95-0.95 1.76-4.16 0.54-1.01
		CI95 fludarabine	1.54-2.38	1.67-2.12 1.41-1.74 1.56-2.91 1.90-2.87
	L1210	CI95 cladribine CI95 clofarabine	0.94-1.24 1.52-3.46	1.26-1.51 1.44-1.65 1.35-1.94 1.19-2.01 1.64-2.20 0.91-1.19 1.29-2.22 2.22-2.96
		CI95 6MP	0.47-0.53	2.01-4.44 1.47-2.32 1.17-2.57 0.99-2.92

This study was supported by the Agence Nationale de la Recherche (ANR grant 11-BS07-032) and by Institut National du Cancer (INCA_11560). LPJ received funding from Olav Raagholt og Gerd Meidel Raagholts stiftelse for forskning.


Gabriel S Vignoli Muniz

Aditya N Agnihotri

Basile Augé

Rafael Martinez

Christian F Mejía

Hermann Rothard

Alicja Domaracka

Philippe Boduch

Radiolysis of cytosine at cryogenic temperatures by swift heavy ion bombardments

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Nucleobases are aromatic heterocyclic compounds that contain nitrogen. Nucleobases are divided into two groups: purines, which include two heterocyclic fused rings, and pyrimidines, which contain a single heterocyclic ring such as cytosine (Figure 1). Nucleobases are crucial to the biosphere since they are building blocks of biomolecules such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). These nucleic acids are responsible for the storage and transmission of genetic and hereditary information and responsible for the control of enzymatic cellular machinery. Nucleobases also play an Figure 1. Schematic structure of cytosine molecules. important role in cell energy processing and cell signalization. 1 Moreover, they are components of daily human nutrition, for example, caffeine and vitamins B3 and B4; additionally, they are part of important medications. [START_REF] Girke | Nucleobase and Nucleoside Transport and Integration into Plant Metabolism[END_REF][START_REF] Yssel | Repurposing of Nucleoside-and Nucleobase-Derivative Drugs as Antibiotics and Biofilm Inhibitors[END_REF] Thus, a better understanding of nucleobase radiolysis can have relevance in medicine [START_REF] Jäkel | Heavy Ion Therapy: Status and Perspectives[END_REF][START_REF] Pompos | Heavy Ions in Cancer Therapy[END_REF] and the areas of radiation processing of food and drugs. [START_REF] Silindir | The Effect of Radiation on a Variety of Pharmaceuticals and Materials Containing Polymers[END_REF][START_REF] Diehl | Food Irradiation Past, Present and Future[END_REF] The presence of nucleobases in the meteorites found on Earth strongly indicates that this class of molecules can exist in outer space. [START_REF] Shimoyama | Search for Nucleic Acid Bases in Carbonaceous Chondrites from Antarctica[END_REF][START_REF] Martins | Extraterrestrial Nucleobases in the Murchison Meteorite[END_REF][START_REF] Callahan | Carbonaceous Meteorites Contain a Wide Range of Extraterrestrial Nucleobases[END_REF][START_REF] Smith | Investigation of Pyridine Carboxylic Acids in CM2 Carbonaceous Chondrites: Potential Precursor Molecules for Ancient Coenzymes[END_REF] Albeit attempts to detect nucleobases in outer space by radio spectroscopy were fruitless. [START_REF] Kuan | A Search for Interstellar Pyrimidine[END_REF][START_REF] Kuan | Searches for Interstellar Molecules of Potential Prebiotic Importance[END_REF][START_REF] Brunken | Improved Line Frequencies for the Nucleic Acid Base Uracil for a Radioastronomical Search[END_REF] Several chemical routes were proposed to form nucleobases from simple species present in outer space under astronomical conditions of cryogenic temperature and ionizing radia-tion. 15 -19 Furthermore, experimental works mimicking astro-nomical environments revealed the formation of complex organic molecules including nucleobases. [START_REF] Saladino | Formamide and the Origin of Life[END_REF][START_REF] Horst | Formation of Amino Acids and Nucleotide Bases in a Titan Atmosphere Simulation Experiment[END_REF][START_REF] Nuevo | Irradiation of Pyrimidine in Pure H 2 O Ice with High-Energy Ultraviolet Photons[END_REF][START_REF] Bibang | Ion Radiation in Icy Space Environments: Synthesis and Radioresistance of Complex Organic Molecules[END_REF][START_REF] Cottin | Astrobiology and the Possibility of Life on Earth and Elsewhere[END_REF][START_REF] Materese | The Formation of Nucleobases from the Ultraviolet Photoirradiation of Purine in Simple Astrophysical Ice Analogues[END_REF][START_REF] Pilling | DNA Nucleobase Synthesis at Titan Atmosphere Analog by Soft X-Rays[END_REF] The omnipresent ionizing radiation field in space raises an important question if nucleobases are formed in astronomical environments, can they survive the harsh conditions of outer space? Studying the radioresistance of nucleobases can give clues about their existence in outer space and even the possibility of their detection. Therefore, information about the radioresistance of nucleobases has pivotal importance in astrochemistry and astrobiology. Particularly, this could be relevant for the hypothesis of the RNA world; see more in. [START_REF] Higgs | The RNA World: Molecular Cooperation at the Origins of Life[END_REF] In astrophysical environments, nucleobases are expected to exist in a low-temperature solid molecular matrix of water, CO2, and NH3. [START_REF] Oba | Nucleobase Synthesis in Interstellar Ices[END_REF] As far as we know, a few experiments were performed under astrophysical conditions, i.e., low temperature and solid phase. Adenine in a noble gas matrix was irradiated by 5 keV electrons. [START_REF] Evans | On the interaction of adenine with ionizing radiation: mechanistical studies and astrobiological implications[END_REF] Thymine with and without different water-ice contents was exposed to swift protons (0.8-1.0 MeV). [START_REF] Materese | The Radiation Stability of Thymine in Solid H2O[END_REF] Uracil with different contents of water or CO2 was irradiated by 0.9 MeV protons. [START_REF] Gerakines | Radiolytic Destruction of Uracil in Interstellar and Solar System Ices[END_REF] Nucleobases in a matrix of noble gas [START_REF] Peeters | Formation and Photostability of N-Heterocycles in Space: I. The Effect of Nitrogen on the Photostability of Small Aromatic Molecules[END_REF][START_REF] Peeters | The Astrobiology of Nucleobases[END_REF] and the pure solid phase (thin-film or solid samples) [START_REF] Poch | Laboratory Insights into the Chemical and Kinetic Evolution of Several Organic Molecules under Simulated Mars Surface UV Radiation Conditions[END_REF][START_REF] Pilling | Photostability of Gas-and Solid-Phase Biomolecules within Dense Molecular Clouds Due to Soft X-Rays: Photostability of Biomolecules in ISM[END_REF][START_REF] Guan | Compared Photochemistry of Prebiotic Organic Compounds in Low Earth Orbit and in the Laboratory[END_REF][START_REF] Saiagh | VUV and Mid-UV Photoabsorption Cross Sections of Thin Films of Guanine and Uracil: Application on Their Photochemistry in the Solar System[END_REF][START_REF] Saiagh | VUV and Mid-UV Photoabsorption Cross Sections of Thin Films of Adenine: Application on Its Photochemistry in the Solar System[END_REF][START_REF] Rouquette | Dimerization of Uracil in a Simulated Mars-like UV Radiation Environment[END_REF] were exposed to electromagnetic radiation.

In previous work, we investigated the radioresistance of solid adenine, a purine nucleobase, at low temperature exposed to swift heavy ions mimicking galactic cosmic rays (GCRs). [START_REF] Vignoli Muniz | Radioresistance of Adenine to Cosmic Rays[END_REF] In the present work, we investigated the radiolysis of solid cytosine at low temperatures (∼12 K) exposed to swift heavy ions. Cytosine was prepared in two different ways for which induced effects are found to differ at low doses, mostly correlated with structural rearrangement. From experiments using several heavy ion beams provided by Grand Accelerateur National d'Ions Lourds (GANIL) in Caen, France, and by Helmholtzzentrum fur Schwerionenforschung (FAIR-GSI) in Darmstadt, Germany, we studied radiolysis as a function of the electronic stopping power of the ionic projectiles.

EXPERIMENTAL METHODOLOGY

2.1. Sample Preparation. Cytosine (≥98% pure) was purchased from Sigma-Aldrich. Cytosine powder was dissolved in a solution of ethanol and water (60.8% ethanol in water v/v) at a concentration of 1.5 mg mL -1 . The solution was exposed to ultrasound until all solid particles had visibly been dissolved. Then, drops of these solutions were applied directly onto windows of ZnSe (13 mm diameter, thickness 2 mm). The windows were heated up to 100 °C until the entire evaporation of the solvent, thus obtaining a solid sample. Hereafter, samples prepared in this manner will be referred to as "grainy" because of their grainy aspect under optical microscopy (see Figure S1 in the Supporting Information).

The second method was the preparation of uniform films from the sublimation of cytosine powder at low pressure (10 -5 mbar) using an oven at 150 °C. A similar methodology was adopted by. [START_REF] Guan | Compared Photochemistry of Prebiotic Organic Compounds in Low Earth Orbit and in the Laboratory[END_REF][START_REF] Saiagh | VUV and Mid-UV Photoabsorption Cross Sections of Thin Films of Guanine and Uracil: Application on Their Photochemistry in the Solar System[END_REF][START_REF] Saiagh | VUV and Mid-UV Photoabsorption Cross Sections of Thin Films of Adenine: Application on Its Photochemistry in the Solar System[END_REF] Optical microscopy and profilometry confirm that these samples are uniform thin films, henceforth for the sake of simplicity, we will refer to these samples as "films". The spectra of the nucleobase films are in good agreement with those reported in the literature, see Section 3.1, demonstrating that the process of heating does not destroy these molecules.

The preparation of nucleobase films from sublimation typically takes 3 or 4 h to produce a nucleobase film of a few hundred nanometers, whereas the production of grainy nucleobase samples is much faster, not taking longer than 20 min.

2.2. Sample Irradiation. Solid cytosine samples were irradiated at the beamline IRRSUD (17 MeV Ni 8+ , 92 MeV Xe 23+ , and 116 MeV U 32+ ) at GANIL (Caen, France) and the M-Branch of UNILAC (230 MeV Ca 10+ ) at FAIR-GSI (Darmstadt, Germany) in high vacuum chambers (10 -8 mbar). The experiments at GANIL were performed in the CASIMIR setup described in detail elsewhere. [START_REF] Mejia | Compaction of Porous Ices Rich in Water by Swift Heavy Ions[END_REF] Each target was mounted on a cold finger connected to a closed cycle helium cold head equipped with a target heater, and the samples were irradiated at 12 K. The setup used in GSI is very similar, but it allows to mount of three samples inside the vacuum chamber; the irradiation temperature was slightly higher (≈20 K). For more details, see. [START_REF] Mejia | Compaction of Porous Ices Rich in Water by Swift Heavy Ions[END_REF][START_REF] Severin | The MBranch, a New UNILAC Irradiation Facility with in-Situ Analytical Techniques for Materials Research GSI Sci[END_REF] The average flux was about 10 9 ions cm -2 s -1 at IRRSUD and SME and 2 × 10 8 ions cm -2 s -1 at UNILAC.

The infrared (IR) absorption spectra were obtained in situ before and after irradiation with a Fourier transform infrared (FTIR) spectrometer (Nicolet Magna 550, Mercury Cadmium Telluride (MCT) detector cooled with liquid nitrogen), operating in transmission mode. The spectra were acquired as an average of 128 scans with wavenumbers ranging from 4000 to 700 cm -1 with a resolution of 2 cm -1 . The background of the bare window was taken at 12 K (20 K for GSI) and 300 K and used according to the temperature of the samples, thus avoiding thermal optics effects.

The projectile and target properties are summarized in Table 1. The electronic (Se) and nuclear stopping (Sn) powers of the ionic projectiles in the solid nucleobase samples were calculated with the "Stopping and Ranges of Ions in Matter" (SRIM) software. [START_REF] Ziegler | [END_REF] Note that for the swift projectiles used in this work, we are in the electronic regime of deposition of energy, Se ≫ Sn. Furthermore, the ion penetration depth is much larger than the sample thickness; therefore, there is no ion implantation in the solid nucleobase samples.

RESULTS AND DISCUSSION

3.1. Cytosine IR Absorption Spectra. Grainy and film cytosine samples display similar absorption spectra, Figure 2, in terms of band position but not in absorbance values (Table S1). Moreover, the absorption bands of the cytosine film are narrower than those of grainy cytosine samples. These minor MM of the cytosine molecules. Table 2 summarizes the absorption bands of grainy and film cytosine spectra, including the bands reported by [START_REF] Mathlouthi | Laser-Raman Spectra of Cytosine and Cytidine[END_REF] for cytosine in KBr pellets.

All of the nucleobases present a large IR absorption band between 3500 and 2000 cm -1 (the region containing characteristic vibration modes of C-H, N-H, and O-H). In the literature, this later region is called the α-band or βband. [START_REF] Saiagh | VUV and Mid-UV Photoabsorption Cross Sections of Thin Films of Guanine and Uracil: Application on Their Photochemistry in the Solar System[END_REF][START_REF] Saiagh | VUV and Mid-UV Photoabsorption Cross Sections of Thin Films of Adenine: Application on Its Photochemistry in the Solar System[END_REF] In this work, we will refer to this cytosine absorption band (3500-2000 cm -1 ) as the "α-band". The grainy samples do not present a smooth surface (Figure S1). the cytosine density (1.5 g cm -3 ) and S is the area of the ZnSe window (see Table 1).

Cytosine Film Thickness.

Using a "Dektak 150 surfacer" profilometer from the Veeco brand in the GREYC laboratory (ENSICAEN, Caen, France), the thickness (d) and the roughness of cytosine film samples were measured. The samples were positioned in the center of the profilometer; using the microscope coupled to the profilometer, the profilometry tip was positioned onto a naked part of the ZnSe window. Then, the tip was moved throughout the sample, to determine the height between the ZnSe window and the edge of the film, thus giving us the thickness of the film. The thickness of each sample was determined as an average of four measurements (Table 1) corresponding to different scanned positions of the sample. A picture of a cytosine sample (film on the substrate window) and the corresponding measured profile are shown in Figure S2.

The cytosine column density (N) was determined as follows: N = d NA × 10 4 [molecules cm -2 ], where d is the film thickness, in μm, ρ is its density here assumed to be 1.5 g cm -3 , NA is the Avogadro number, and MM is the cytosine Here, A is the integrated absorbance of a given IR feature and N is the column density. IR absorbance is measured on a log base-10 scale, therefore it is necessary to multiply the ratio between A and N by ln 10 to obtain the A-value. To evaluate the band strength, we used the determined thickness of three different samples. Thus, we estimate the A-value for the α-band as the (8.8 ± 0.7) × 10 -16 cm molecule -1 . Additionally, we estimated the A-value for several cytosine absorption bands (Table S2). We would like to point out that the A-values estimated in this work are only valid for thin cytosine films with a thickness in the order of few hundred of nanometers.

Film Compaction.

IR absorption spectroscopy is a suitable technique to follow the sample evolution during ion bombardment. It allows assessing the radiolytic destruction of cytosine, such as solid-phase transitions, radiolysis, sputtering, and formation of new chemical species.

Two cytosine samples were irradiated with the same projectile, 17 MeV Ni 8+ (Table 1). Cytosine films (Figure 3a) present two different regimes with respect to target fluences. Note that the integrated absorbance at 1539 cm -1 increases at low fluences, F < 6.0 × 10 10 cm -2 ; however, at molecular weight, 111.10 g mol -1 . To estimate the band strength (A-value) of a given IR absorption band, we used eq 1 higher fluences, the integrated absorbance decreases exponentially.

A value = ln 10A N (1)
The increase of integrated absorbance is correlated with the compaction process of cytosine films, i.e., structural rearrangement of cytosine molecules by the irradiation, and the subsequent changes in the band strength. [START_REF] Mejia | Compaction of Porous Ices Rich in Water by Swift Heavy Ions[END_REF][START_REF] Costa | Valine Radiolysis by H + , He + , N + , and S 15+ MeV Ions[END_REF] Assuming that the cytosine column density does not change much at low fluences, it is feasible to estimate the variation in the band strength induced by the film compaction. Under this assumption, the variation of the band strength at 1539 cm -1 is 8%. Interestingly, the compaction effects are unobserved for grainy samples, and that cytosine decay is very similar in both film and grainy samples (Figure 3b).

Cytosine Radiolysis.

Henceforth, we studied cytosine radiolysis at cryogenic temperatures with the electronic energy loss ranging from 2.9 to 13.4 MeV μm -1 . For this task, we used two facilities with two similar setups and three different ion beamlines (GANIL and GSI).

We observe that for doses higher than 0.35 eV per molecule, the overall IR absorption intensity decreases exponentially with the accumulated number of projectiles (fluence). For example, The decrease of the IR absorbance values is due to the disappearance of the initial molecule caused by either destruction or sputtering. Both effects, destruction and sputtering, can be quantified by introducing the apparent destruction cross section (σd); σd is defined as a sum between the molecule destruction cross section and the sputtering per incoming ion. For more details, see. [START_REF] Vignoli Muniz | Radioresistance of Adenine to Cosmic Rays[END_REF][START_REF] De Barros | Radiolysis of Astrophysical Ices by Heavy Ion Irradiation: Destruction Cross Section Measurement[END_REF][START_REF] Mejia | Cosmic Ray-Ice Interaction Studied by Radiolysis of 15 K Methane Ice with MeV O, Fe and Zn Ions[END_REF] The integrated absorbance (A) evolution as an ion fluence function can be written according to eq 2 per square centimeter. This figure shows that the projectiles with higher electronic energy loss (Table 1) cause a faster and stronger decay of the IR integrated absorbance.

The evolution of several absorption bands was monitored during the heavy ion bombardment, and the respective destruction cross sections were determined by fitting eq 2(see Table 3). The IR feature at 1539 cm -1 yielded the highest cross section values (Table S3). It is important to mention that, in principle, all of the bands should yield the same cross section values. We speculate that the different cross section values are due to the presence of IR absorption features from the radioproducts and/or the changes in the band strength induced by the irradiation and the subsequent changes in cytosine's local chemical environment. Therefore, for our calculations, we will use the highest cross section values, that is, the cross sections determined from the decay of the IR absorption band at 1539 cm -1 (Table 3). Note that grainy and film cytosine samples were irradiated by the same projectiles (17 MeV Ni 8+ ) leading to very similar cross sections (Tables 3 andS3).

As pointed out by, [START_REF] Da Costa | Infrared Analysis of Glycine Dissociation by MeV Ions and KeV Electrons[END_REF] it has been reported, for more than four decades, that the destruction cross sections for swift ions follow a power law on the electronic stopping power under the form σd = C S n , where C is a constant and n is a dimensionless quantity. Additionally, it has been observed that the parameter n ranges from 1.0 to 1.5 for different molecules; for more details, see [START_REF] Da Costa | Infrared Analysis of Glycine Dissociation by MeV Ions and KeV Electrons[END_REF][START_REF] Rothard | Modification of Ices by Cosmic Rays and Solar Wind[END_REF] and the references therein.

Figure 6 shows the destruction cross section of cytosine as a function of the electronic stopping power by using the following power law: σd = C S n . Figure 6. Apparent destruction cross section of cytosine as a function of the electronic stopping power.

Cytosine displays a very complex vibrational spectrum, making it hard to identify the molecules responsible for the new absorption features unequivocally. It is nevertheless possible to propose that some candidates of new molecular species are responsible for the observed absorption. We observe that new absorption features emerge from all nucleobase spectra due to sample radio degradation.

Grainy and film samples exhibit the same new absorption features for the same deposited local dose. Figure 7 displays the evolution of the IR absorption spectra of cytosine before and during the bombardment by 116 MeV U 32+ at different fluences. The IR spectra were shifted for better visualization.

The energetic processing of cytosine samples produced new absorption bands in the region of 2300-2000 cm -1 (Figure 7a). Note that cytosine does not display absorption peaks in that region; this region is characteristic of nitrile and isonitrile vibrations. [START_REF] Gunzler | IR Spectroscopy: An Introduction[END_REF] At lower wavenumbers (Figure 7b), small peaks emerge between 1720 and 1500 cm -1 , which can be attributed to H2CO, [START_REF] De Barros | Radiolysis of Astrophysical Ices by Heavy Ion Irradiation: Destruction Cross Section Measurement[END_REF][START_REF] Gerakines | Ultraviolet Processing of Interstellar Ice Analogs[END_REF] as indicated in Figure 7b.

To identify the radioproducts, the absorption band between 2300 and 2000 cm -1 was deconvoluted (Figure 8c). The following absorption peaks are observed: 2077, 2138, 2164, 2227, and 2256 cm -1 . The FWHH of the absorption peak at 2139 cm -1 is 4.4 cm -1 ; accordingly, this seems to be the IR absorption peak of CO. [START_REF] Seperuelo Duarte | Laboratory Simulation of Heavy-Ion Cosmic-Ray Interaction with Condensed CO[END_REF] The absorption band at 2165 cm -1 may be assigned to OCN -.52 Indeed, the formation of OCN - from the destruction of the cytosine ring is plausible (see Figure 1). The absorption band at 2258 cm -1 is attributed to HNCO. [START_REF] Peeters | The Astrobiology of Nucleobases[END_REF][START_REF] Lowenthal | Infrared Spectrum of Solid Isocyanic Acid (HNCO): Vibrational Assignments and Integrated Band Intensities[END_REF]

ASTROPHYSICAL IMPLICATIONS

From the astrophysical point of view, it is important to estimate the half-life (τ1/2) of molecules exposed to ionizing radiation fields. This can indicate the probability of detecting a given molecule in an astronomical environment and the possibility of a molecule composing the inventory of molecules in a planetary disk.

To estimate the half-life of solid cytosine exposed to galactic cosmic rays (GCRs), we calculate the cytosine destruction rate, i.e., the product of the destruction cross section, and the flux of cosmic rays. We selected the 10 most-abundant ions that compose the GCRs and determined the destruction cross section as a function of the energy per nucleon (E) using the code SRIM [START_REF] Ziegler | Stopping of Energetic Light Ions in Elemental Matter[END_REF] and the observed power law (see Section 3.4 (Figure S3)). As expected, heavier elements display higher destruction cross sections since, for swift ions, the stopping power is proportional to the square of the projectile atomic number and approximately inversely proportional to the square of the projectile velocity. [START_REF] Bethe | Zur Theorie des Durchgangs schneller Korpuskularstrahlen durch Materie[END_REF][START_REF] Ziegler | Stopping of Energetic Light Ions in Elemental Matter[END_REF] The distribution of cosmic rays (Φ(Z, E)) is welldetermined at energies above 1 GeV per nucleon (GeV/n). The best fitting processes yielded σ = (3 ± 1) × 10 -17

Though, the differential flux at low energies is less accurate, due to the interaction between the charged particles, the Se with Se in keV μm for σd in cm . The fitting heliosphere, and the planetary magnetic fields. [START_REF] Shen | Cosmic Ray Induced Explosive Chemical Desorption in Dense Clouds[END_REF] In this work, yielded a χ 2 = 0.97.

3.5. Radioproducts: New Molecules from Cytosine Radiolysis. Although the goal of this paper is not to the GCR differential flux was calculated using the model proposed by [START_REF] Shen | Cosmic Ray Induced Explosive Chemical Desorption in Dense Clouds[END_REF][START_REF] Webber | A Measurement of the Energy Spectra and Relative Abundance of the Cosmic-Ray H and He Isotopes over a Broad Energy Range[END_REF] according to eq 3. investigate the radioproducts from cytosine degradation, we observed that swift ion irradiation not only leads to the (Z, E) = C(Z)E 0.3 /(E0 + E) [START_REF] Yssel | Repurposing of Nucleoside-and Nucleobase-Derivative Drugs as Antibiotics and Biofilm Inhibitors[END_REF] (3) decrease in nucleobase IR absorption lines, but new absorption bands arise in the IR spectra. We will highlight the new absorption bands and possible candidates for these IR bands.

C(Z) is a normalization constant. The numerical values of C(Z) were adapted from [START_REF] Portugal | Radiolysis of Amino Acids by Heavy and Energetic Cosmic Ray Analogues in Simulated Space Environments: α-Glycine Zwitterion Form[END_REF] (see Table S4). In this model, E0 is a parameter that modulates the differential fluxes only at low orders of magnitude higher than hydrogen (Figure S3), thus iron compensates for its low abundance and becomes the most efficient cosmic ray element to destroy cytosine. The half-life of cytosine can be calculated by τ1/2(Z) = ln 2/ (4 π ∑Z ∫ σd (E,Z)Φ (E,Z) dE), and the integral was performed from 0.1 to 1000 MeV/n. The factor 4π is due to the fact that the GCR flux is isotropic; however, this value is small in comparison with the uncertainties of the GCR distribution (see Figure S4). For the total half-life, it is necessary to perform a summation of each element. He/ 4 He measured ratio. [START_REF] Webber | A Measurement of the Energy Spectra and Relative Abundance of the Cosmic-Ray H and He Isotopes over a Broad Energy Range[END_REF] Here, we decided to investigate how different E0 values, namely, 200, 400, and 600 MeV/n, would impact the cytosine half-live. Therefore, here we adopted these three values for the E0 parameter to simulate the GCR fluxes and estimate the half-live of cytosine exposed to GCRs. Figure S4 shows the GCRs calculated using these three values of the parameter E0.

The cytosine destruction rate (σd(Z, E) × Φ(Z, E)) is exhibited in Figure 8. Interestingly, at energies higher than 4 MeV/n, iron is the most effective element to destroy cytosine (see the red dashed line in Figure 8). Note that in the energy range of 4-1000 MeV/n, iron displays cross sections about 4 displays the half-life of cytosine exposed to cosmic rays for distinct values of E0. It is important to point out that the values here are only an estimation given the uncertainties of the GCR flux.

Taking into consideration the same GCR distributions, cytosine was found slightly more radio susceptible than adenine. [START_REF] Vignoli Muniz | Radioresistance of Adenine to Cosmic Rays[END_REF] Indeed, more experimental and theoretical approaches are necessary to better understand the susceptibility of purine and pyrimidine nucleobases to ionizing radiation.

CONCLUSIONS

In this work, the processing of solid cytosine at cryogenic temperatures by swift heavy ions was investigated. Cytosine targets were produced by two different methods: (1) by dropping a water-ethanol nucleobase solution on ZnSe windows followed by heating and solvent evaporation and e ■ (2) by sublimation of the nucleobase and condensation on a ZnSe window. This latter method produces a thin smooth solid nucleobase film. By using profilometry and combining with IR data, band strengths for several IR absorption bands of the cytosine film were estimated at room temperature, with the α-band (3500-2000 cm -1 ) presenting an A-value that equals (8.8 ± 0.7) × 10 -16 cm molecule -1 .

At low fluences (F < 1 × 10 11 cm -2 ), cytosine films present an increase in the IR absorbance; this is probably correlated with molecular rearrangement and changes in their band strength. For both types of samples, film and grainy, the evolution of the IR spectra under heavy ion bombardment is very similar, yielding destruction cross sections equal within error bars. Furthermore, as observed for many organic molecules, cytosine destruction cross section follows an electronic stopping (Se) power law under the form: (3 ± 1) × 10 -17 S (1.25±0.06) . The half-life of cytosine exposed to GCRs was estimated using different GCR fluxes; the values estimated here are in order of Mega years. The results here suggest that the search for nucleobases by radio spectroscopy should be performed.

Figure 2 .

 2 Figure 2. Normalized absorption spectra of grainy (red line) and film (black line) cytosine samples at room temperature. The grainy cytosine spectrum was shifted for clarity. The α-band (3500-2000 cm _1 ) is indicated in the figure.

  We estimated their thicknesses (d) considering the deposited mass (m) from the previously prepared cytosine solution. d = m/ρS, where ρ is

Figure 4 . 2 )Figure 3 .

 423 Figure 4. Spectra of grainy (a) and film (b) cytosine samples at 12 K. The samples were irradiated by 17 MeV Ni 8+ at different fluences. A = A0e d F

Figure 5 .

 5 Figure 5. Normalized integrated absorbance at 1539 cm -1 as a function of the projectile fluence. The full lines correspond to the fitting of the data with eq 2.

Figure 4

 4 Figure 4 displays the evolution of grainy and film cytosine samples bombarded by 17 MeV Ni 8+ at 12 K.The decrease of the IR absorbance values is due to the disappearance of the initial molecule caused by either destruction or sputtering. Both effects, destruction and sputtering, can be quantified by introducing the apparent destruction cross section (σd); σd is defined as a sum between the molecule destruction cross section and the sputtering per incoming ion. For more details, see.[START_REF] Vignoli Muniz | Radioresistance of Adenine to Cosmic Rays[END_REF][START_REF] De Barros | Radiolysis of Astrophysical Ices by Heavy Ion Irradiation: Destruction Cross Section Measurement[END_REF][START_REF] Mejia | Cosmic Ray-Ice Interaction Studied by Radiolysis of 15 K Methane Ice with MeV O, Fe and Zn Ions[END_REF] The integrated absorbance (A) evolution as an ion fluence function can be written according to eq 2

  e e

Figure 7 .

 7 Figure 7. IR spectra of the cytosine film irradiated by 116 MeV U 32+ at different fluences. The sample was irradiated by 116 MeV U 32+ at different fluences, within wavenumbers ranging from 2300 to 2050 cm -1 (a) and from 1900 to 1200 cm -1 (b). Deconvolution of the band between 2300-2050 cm -1 of the cytosine film irradiated by 116 MeV U 32+ at the fluence of 1.44 × 10 12 ions cm -2 (19.6 eV per molecule) at 12 K (c).

Figure 8 .

 8 Figure 8. Cytosine destruction rate for the 10 most-abundant

Table 1 .

 1 Summary of Targets and Projectile Characteristics

	sample	ion	energy (MeV)	electronic stopping power (MeV μm -1 )	nuclear stopping power (10 -3 MeV μm -1 )	penetration depth (μm)	temperature (K)	thickness (μm)	column density (10 17 cm -2 )
	cytosine	Xe 23+	92	11.3	73	15.5	12	0.8	6.5
	grainy
	cytosine	U 32+	116	13.4	250	16.7	12	0.34	2.8
	film
	cytosine	Ni 8+	17	4.8	44	9.2	12	0.28	2.3
	film
	cytosine	Ni 8+	17	4.8	44	9.2	12	0.20	1.6
	grainy
	cytosine	Ca 10+	230	2.9	1.9	63	20	1.1	9.1
	grainy

Table 2 .

 2 Comparison between Our Results and Those of[START_REF] Mathlouthi | Laser-Raman Spectra of Cytosine and Cytidine[END_REF]

				position (cm -1 )	position (cm -1 )
	assignment	vibration mode	position (cm -1 ) 43	grainy cytosine	film cytosine
	NH2	antisymmetric	3384	3381	3381
		stretching
				3351	3349
	NH2	symmetric	3180	3170	3173
		stretching
			2840	2840	2863
	CH	stretching	2796	2793	2794
	CH	stretching	2690	2686	2688
	NH2	bending	1705	1703
	C2O	stretching	1667	1660	1658
					1627
	C5=C6	stretching	1616	1614	1615
	NH	bending in plane	1540	1539	1538

Table 3 .

 3 Destruction Cross Sections of Cytosine for Different Projectiles

	projectile	σd [10 -12 cm 2 ]	sample
	116 MeV U 32+	(3.50 ± 0.09)	film
	92 MeV Xe 23+	(3.0 ± 0.3)	grainy
	17 MeV Ni 8+	(1.17 ± 0.03)	grainy
	17 MeV Ni 8+	(1.31 ± 0.06)	film
	230 MeV Ca 10+	(0.51 ± 0.01)	grainy

Table 4 Table 4 .

 44 Half-life of Cytosine Exposed to Cosmic Rays for Different Values of the Parameter E0

	E0 (MeV/n)	τ1/2 (Myears)
	200	1.2
	400	7.3

ACKNOWLEDGMENTS

This work was supported by Brazilian agencies CNPq (INEspaco and Science without Borders) and FAPERJ, as well as the CAPES-COFECUB French-Brazilian exchange program, the European Commission, FP7 for RTD Capacities Programme (Contract No. 262010, ENSAR), and the EU's Horizon 2020 Research and Innovation Programme (grant agreement no. 654002 ENSAR2). It is a pleasure to thank Thierry Been, Jean-Marc Ramillon, Toiammou Madi, and Dr. Clara Grygiel for their invaluable support. The authors wish to acknowledge the staff of GANIL and GSI for their invaluable help throughout the course of the experiments. The authors are particularly thankful to Prof. Dr. Christina Trautmann, Dr. Daniel Severin, Dr. Markus Bender, Alexander Warth, and Arne Siegmund at GSI. The experiments were performed at Grand Accélérateur National d'Ions Lourds (GANIL) Caen, France, and at (GSI) Helmholtzzentrum fur Schwerionenforschung, Darmstadt, Germany. The authors also thank the reviewers for their constructive remarks.


Dr Minh-Duc Hoang

Farah Savina

Dr Philippe Durand

Pr Rachel Méallet-Renault

Dr Gilles Clavier

Dr Arnaud Chevalier
email: arnaud.chevalier@cnrs.fr

Tunable Naphthalimide/Cinnoline-Fused (CinNapht) Hybrid Dyes for Fluorescence Imaging in Living Cells

This paper presents the synthesis, photophysical characterization and use for cell imaging of 14 flurophores derived from a hybrid Naphthalimide/Cinnoline fused backbone called "CinNapht". Photophysical properties of these fluorophores including absorbance, excitation and emission spectra have been recorded in CHCl 3 , DMSO and PBS + 5%BSA. They exhibit red emission associated to a large Stokes Shift and fluorescence quantum yields up to 52%. Theoretical calculations have been undertaken in order to provide elements of rationalization for a major part of the results. Some of these analogues have been tuned to enable imaging of cell organelles such as mitochondria, lysosome or lipid droplets. This study comes to confirm that CinNapht dyes can be considered as relevant alternatives to existing tools for cell imaging.

Introduction

The synthesis of new organic fluorophores and the optimization of their photophysical properties has enabled the emergence of scaffolds endowed with a wide range of performances, contributing to the development of fluorescence imaging. The motivations connected to this can be multiple. It can be to modulate the absorption and emission wavelengths of the fluorophores, [1] to improve its solubility in biological medium, [START_REF] Li | [END_REF] to enhance its photostability [3] or to address a given cell compartment. [4] The increasing number of fluorophores that have been, and are still being proposed, demonstrates that the perfect fluorophore does not exist. Despite the wide variety of existing fluorophores, there is still a need for new tools to solve specific problems of fluorescence imaging. [5] The knowledge acquired since decades on the structure/photophysical properties relationship now enables to anticipate the consequences of functional adjustments to obtain fluorophores with optimized properties. For example, the nature of the amine substituents carried by the fluorophores significantly influences their emission properties and the fluorescence efficiency. This made possible the fine tuning of fluorophores photophysical properties, whether they are xanthene [6] or ICT involving structures. [7] In this context, combining a stokes shift enhancement with a redshift of the absorption and emission wavelengths is one of the most addressed topics. [8] Multiple approaches have been implemented for this purpose, as clearly explains by Yuan's group. [9] Possible approaches consist of either introducing alternating vibronic structures or extending the polyaromatic system's conjugation of existing structures, for example, by elaborating fused hybrid fluorophores. The latter strategy has already been implemented on multiple fluorophores including coumarins, [10] BODIPY's [11] or rhodamines [6a] being fused with either coumarin moiety [12] or cyanine dyes. [13] Efforts to develop fused hybrid fluorophores based on a naphthalimide structure have also been reported (Figure 1). [14] Inspired by work describing "covalent-assembly" probes [15] resulting in the formation of cinnoline incorporating fluorophores, [16] we recently published the study of a fused hybrid structure involving a naphthalimide and a cinnoline, called CinNapht [17] . We showed that the extended conjugation of the Naphthalimides resulted in a bathochromic shift of the absorption and emission wavelengths of the fluorophores while preserving a high Stokes shift. In the present article, we describe a synthetic method that allowed structural adjustments on the CinNapht backbone (Figure 1). The specificity of these fluorophores lies in their easy modification at two distinct positions. We show that photophysical properties of the fluorophore can be modulated by changing the nature of a donor group (shown in blue, Scheme 1). Furthermore, we illustrate the utility of varying the substituent of the phthalimide moiety (Red R, Scheme 1) to provide cell organelles targeting ability to the fluorophore. This convergent synthesis, involving coupling of functionalized naphthalimide and cinnoline moieties in the final steps, gives easy access to multiple combinations and therefore to a high diversity of fluorophores.

Results and Discussion

Synthesis of CinNapht Analogues.

The designed synthetic strategy for CinNapht 8 is based on Suzuki cross-coupling reaction between boronate 6 and 3-bromo-4-amino-N-substituted 1,8-naphthalimide 3 followed by intramolecular diazotization of the formed ortho amino biphenyl. derivatives 7. The N-alkylated phthalimides 3 were synthesized in a two steps process from 4-amino-1,8-naphthyl anhydride as previously reported. [17] Scheme 1. Synthesis route and structure of CinNapht analogues.

The brominated intermediates 2 are easily isolated by precipitation and can react with various amines (Scheme 1), allowing the introduction of alkyl group R carrying alcohol (3e), Boc protected amine (3d) or triphenylphosphonium groups (3c) The obtained isolated yields vary from 68 to 99% (See Supporting Information for detailed protocols,). Note that these two steps can be interchanged if necessary, but resulting in lower overall yield. The boronic acid pinacol ester 6a-g, second partners in the Suzuki cross-coupling reaction, were synthesized by palladium catalyzed coupling reaction of bis(pinacolato)diboron with either 3-bromo dialkylamino aryl 5d-f or 3-dialkylamino aryl triflate 5a-c. The latter were obtained from the corresponding phenol under standard conditions with good yields. These two possible precursors for 6 give access to CinNapht with various donor moieties, allowing their emission wavelength tuning. After Pdcatalyzed Suzuki coupling between boronate 6a-d and brominated naphthalimide 3a-f, the bisaryl adduct 7a-n can be obtained in up to 93% yield. At this step, purification on silica gel was necessary to provide a compound pure enough to ensure its easy and good yielding transformation into the CinNapht fluorophore 8a-n. This last step consists in the formation of a diazonium salt using nitrosyl tetrafluoroborate that undergoes the internal addition of the aromatic ring through a SE Ar type reaction.

It must be noted here that this last step leads to a mixture of regioisomers resulting from para and ortho addition. Fortunately, under these conditions, the fluorescent para-substitution isomer was systematically found to be the major product. A set of 14 fluorophores was obtained through this synthetic route with multiple structures presented in Scheme 1. Brightness [d] 8a

CHCl3 489 [e] 15 700 [e] 566 [e] 2 782 [e] 0.25 [e] 3 925 [e] DMSO 496 [e] 15 500 [e] 682 [e] 5 499 [e] 0.06 [e] 930 [e] PBS [c] Relative QY determined at 25 °C using [Ru(bpy)3]Cl2 (QY = 0.04 in air Saturated H2O), All compounds QYFl were determined using excitation wavelength at 470 nm except for 8c (Exc = 490 nm) and compound 8d (Exc = 450 nm) [18] [d] Brightness was calculated using the formula: Brightness = QYFl × max, [e] Previously reported values [17] , [f]. Poor solubility could not enable to obtain an accurate value.

Their chemical structure, as well as the synthetic intermediates compounds, were unambiguously confirmed by detailed measurements, including ESI-HRMS and NMR analyses (see Supporting Information). The CinNaphts from 8a to 8h constitute a set of fluorophores for which the N-butylphthalimide moiety was fixed and for which the substituents on the cinnoline moiety (blue in scheme 1) were modulated to allow photophysical tuning. On the other hand, for fluorophores 8i to 8n, the dimethylaniline donor moiety is kept unchanged to study the impact of a modification of the phthalimide moiety.

Photophysical properties

The photophysical properties of all these dyes were firstly evaluated in solution in chloroform and the results are presented in Table 1. No significant variation was observed in the absorption maxima by modifying the R substituent while keeping the donor part unchanged. Thus, CinNapht 8i-n show similar absorption maxima fluctuating between 485 and 495 nm. On the other hand, the increased donor character of the cinnoline susbtituents causes a bathochromic shift of the absorption maxima. Thus compounds 8h, 8d, 8a, 8b, and 8c, ranked according to the donor strength of their substituents, show an increasing trend of absorption maxima with values of 398, 463, 489, 506 and 535 nm respectively. More surprisingly, the substituent ring size in the case of CinNaphts 8e, 8f and 8g also seems to impact the absorption maxima, although the donor character is not supposed to be taken into consideration in this case. The most remarkable impact on the absorbance was observed for the molar extinction coefficient. Among all the cyclic amines 8e-g used as substituent, the azetidine provide the strongest increase of the The same behavior was observed for the emission maxima with values of 560, 566, 575 and 615 nm for CinNaphts 8d, 8a, 8b, and 8c respectively. A surprisingly high emission wavelength was measured for the compound 8m associated to a shoulder shaped emission spectrum (See Figure S12). We suppose that this might be due to some aggregation phenomenon, but no definitive explanation can be given at this stage. Note that for all the synthesized fluorophores, a large Stokes shift was observed with a lowest value of 2371 cm -1 (8b) which rises to 5196 cm -1 for CinNapht 8m. This is a common consequence when fluorophores excited state is of an intramolecular charge transfer (ICT) nature. [17] Despite our efforts in trying to bring an explanation, in particular by calculation, we could not provide any element allowing to link the Stokes Shift values to the structure of the compounds in a rational way. Concerning the fluorescence quantum yield (QY Fl ), significant variations are to be noted. The use of a diethyl amine unit in CinNapht 8b in place of the dimethyl amine analogue (8a) only results in a slight variation. In contrast, the introduction of the julolidine unit leads to a significant increase in the quantum yield to 52%. This can be explained by the prohibition of the rotation of the C-N bond, which could generate twisted forms that are often non-fluorescent. [19] Another meaningful result is the almost complete fluorescence extinction for compound 8h with an alkoxy donor group, (QY Fl < 0.1%).

Although a partial decrease in fluorescence is widely reported in the literature, with this substituent, its complete disappearance is unusual. The graphical representation reported in Figure 2 clearly illustrates how modifying the donor part affects CinNaphts properties. For example, the introduction of a particularly donor group, such as julolidine, induces a pronounced bathochromic shift in both the absorption and emission spectra. This result is consistent with the literature, which has consistently supported the relevance of this group for shifting wavelengths to the red zone. Fluorescence quantum yields were measured around 20% to 30% and found homogenous for CinNaphts 8i-n regardless of the nature of R substituting phthalimide moiety and the emission wavelength (Figure 2A). By contrast, the impact of the structural modifications on the brightness value was more evident (Figure 2B). Considering the poor variations observed in the QY Fl values, we can assert that these variations are mostly related to molar extinction coefficients heterogeneity. Thus the most efficient fluorophore finally seems to be CinNapht 8e which confirms once again the benefit of introducing an azetidine motif to improve the fluorophore's performance. [6c, 20] The Judolidine motif seems also convenient as proved by the acceptable brightness of CinNapht 8c associated with a red-shifted emission. In order to confirm the solvatochromism effect observed in the previous study [17] , the photophysical properties of the compounds have also been investigated more polar solvents. The measurements in DMSO (Table 1) confirmed a systematic bathochromic shift of the emission wavelengths characterizing the ICT fluorophores showing a marked solvatochromism. Given the A.

B.

small shift in absorbance wavelengths, this is also manifested by a significant increase in the Stokes shift which reaches high values up to 5 998 cm-1. Likewise in the previous study, we noticed a significant drop in the quantum yield. However, we could observe quite good fluorescence efficiency for some of the dyes including CinNapht 8b (QY Fl = 0.17) for the CinNapht 8d (QY Fl = 0.14) for which fluorescence quantum yields values were found to be two to three times higher than for the reference compound 8a [17] . We also studied the fluorescence of these compounds in an aqueous medium. The fluorescence was extremely low in PBS buffer (data not shown), but a supplementation of the buffer with 5% BSA, in order to limit aggregation, allowed to observe more reliable fluorescence. The values presented in Table 1 show nevertheless a rather low fluorescence intensity that are probably resulting from a lack of solubility. Note that the epsilon values are very variable with respect to the solvent employed, with significantly lower values in PBS buffer. We have no concrete explanation at this stage of the study, although an aggregation phenomenon may be at the origin of these observed discrepancies.

TD-DFT Calculation

In order to rationalize the obtained experimental results, a series of quantum mechanical calculations has been undertaken. The very slight variation in absorption when the dimethylaniline donor moiety is kept unchanged is supported by theoretical calculation.

We could indeed notice a quite constant HOMO-LUMO gap values observed for CinNaphts 8i to 8h (Figure S19). Note that the red-shifted absorption  Max of the CinNapht 8c can also be explained by a smaller calculated value of HOMO-LUMO gap (Figure S19) as well as the increase of HOMO-LUMO gap calculated for compound 8d can also explain the hypsochromic shift its absorbance wavelength. All the structures have been optimized in their ground and first excited states by DFT followed by a TDDFT analysis of both states to obtain calculated transitions corresponding to absorption and emission respectively (Table 2).The calculated absorbance values are in good agreement with the experimentally generated data. We can observe only very small deviations of up to only 6%. These percentages tend to be higher when considering the emission. Nevertheless, consistency is observed in the trend of the bathochromic shift resulting from the increase of the donating character of the amine of CinNapht. Oscillator strengths vary between 0.3 and 0.4 which is quite consistent with transitions having intermediate molar absorption coefficients around 10 000-15 000. All the compounds bearing a nitrogen substituent on the cinnoline moiety display a HOMO-LUMO π-π* transition that has a certain charge transfer character as can be seen from the orbitals representation (Figure 3 left). The calculations made on CinNapht 8h, with a methoxy substituent, indicate that the relaxed S1 state is of a different nature since it corresponds to a n-π* transition (note the nature of the HOMO that is constituted by the lone pairs of the nitrogen atoms of the cinnoline in Figure 3 right). This major difference explains its poor emission properties. A set of selected fluorophores were incubated in the presence of A549 lung cancer cells to evaluate their potential for fluorescence microscopy. We first examined the intracellular distribution of the fluorescent CinNaphts 8c, 8e, 8i and 8m. The compounds were incubated for 3h at a concentration of 5 µM in live A549 lung cancer cell and then imaged with confocal microscopy (Figure 4). At first glance, we could rapidly notice that the intracellular biodistribution of these four fluorophores was significantly different. While compound 8c does not seem to display any particular compartmentalization, compounds 8e, 8i, and 8m are visibly localized in cellular compartments, suggesting respectively lipid droplets, lysosome, and mitochondria. These results were expected for CinNaphts 8i and 8m, carrying a morpholine and moiety triphenylphosphonium [21] moieties known to target lysosome [22] and mitochondria [23] respectively when attached to fluorophores including naphthalimides. On the other hand, the preferential accumulation of 8c in lipid droplets was not initially expected. This being said, taking into account the fact that the solvatochromism usually characterizes ICT fluorophores, including our CinNaphts, is one of the prerequisites for the design of probes targeting these lipid droplets, it finally appears plausible to be capable of targeting these compartments with some of our analogs. [24] In order to confirm the observed subcellular compartmentalization, colocalization experiments were conducted. The CinNapht 8i was incubated for 3h at 5µM in A549 cells in the presence of a lysosome marker (LysoView TM 488) to validate its lysosomal localization (Figure 5). Results reveal a pronounced correlation between the red and green signals characterized by the appearance of yellow spots on the overlay shot. If it is evident that the signal peaks observed in the case of CinNapht 8i match the signal of the lysosome, the noisy aspect of the signal highlight the imperfect lysosomal labeling. The cytofluorogram (Figure S20A) and the plot profile (Figure S20B) supported these preliminary observations. In addition, the control experiment performed with a mitochondrial marker (MitoView TM Green) shows a mismatch between the green and red signals (Figure S21). Together, these results confirms that the morpholine moiety enabeled the accumulation of the CinNapht 8i in the lysosome. Similarly, we studied the mitochondrial localization of CinNapht 8m. For this experiment, a MitoView TM Green was used, and the control experiment was performed with a LysoView TM 488. The images depicted in Figure 6 indicate a much more evident localization. These observations are validated by a cytofluorogramm (Figure S22A) showing pixels following the correlation line (Paearson Correlation Coefficient : 0.84) and a plot profile (Figure S22B) revealing a high degree of correlation between the green and red signals. By contrast, the control using the lysosome marker displayed a mismatch of the two signals (Figure S23). All together, these results demonstrate that the functionalization of CinNapht with a triphenylphosphonium motif allows a very efficient targeting of the mitochondria.To complete the study, we also checked the localization of CinNapht 8e. The fluorophore was incubated at 5 µM in A549 cells and a lipid droplet marker was added. The pictures displayed in Figure 7 show a strong correlation between the signal of 8e and LipidSpot 610 thus confirming the preferential accumulation of CinNapht 8e in lipid droplets. However, it is still evident that this fluorophore does not allow a specific visualization of these compartments because of the high signal observed in the rest of the cell. The CinNapht 8e can however constitute an alternative tool for the visualization of these hydrophobic compartments in living cells. We evaluated the toxicity of the 4 compounds studied by microscopy (Figure S24). The CinNaphts 8c, 8i and 8e displayed no obvious toxicity after 72h of incubation, even at higher concentrations up to 10 times the concentration used for the microscopy experiments. On the other hand, compound 8m turned out to be quite toxic at 50 µM after 72h. Considering its low toxicity at 5µM after 72h of incubation, we can still consider it as compatible with imaging experiments for which the incubation time is much shorter.

Fluorescence Microscopy

Conclusion

To summarize, the synthetic route presented here allows to functionalize independently the two parts of the CinNapht fluorophores in order to link them together in a late stage of the synthesis. This method offers multiple combinations as illustrated by these 14 different fluorophores presented in this article including 13 undescribed analogues. Photophysical measurments supported by theoretical calculation demonstrated the interest of structural adjustment of these CinNaphts in order to tune their optical properties. Fluorescence microscopy experiments revealed that the introduction of specific functions on the phthalimide moiety allows to vectorize these fluorophores in a given cell compartment such as mitochondria or the lysosome. This set of fluorophores, although not exhaustive, demonstrates the modularity of CinNapht dyes and how they can represent plausible alternative to existing tolls in both photophysical and biological purposes.

Experimental Section

Organic synthesis protocols

General procedure A (synthesis of brominated naphthalimides molecules 3a-f)

To a suspension of compound 1 (0.17 mmol, 1.00 eq.) in absolute ethanol (1.00 mL) was added the appropriate amine partner (0.34 mmol, 2.00 eq.). The reaction mixture was refluxed overnight.

After the reaction was completed, the mixture was cooled down to room temperature and the resulting solid was isolated by filtration then washed with diethyl ether to furnish the desired brominated naphthalimide. The complete description of each compound including copy of NMR and MS spectra can be found in Supplementary Material.

General procedure B (synthesis of triflates 5a-c)

Trifluoromethanesulfonic anhydride (244 µL, 1.45 mmol, 1.10 eq.) was slowly added to a solution of appropriate phenol (1.32 mmol, 1.00 eq.) and triethylamine (220 µL, 1.58 mmol, 1.20 eq.) in anhydrous DCM (5.00 mL) at 0 °C. The reacting mixture was stirred under an argon atmosphere at 0 °C for 2 h. After the reaction was completed, 5.00 mL of a saturated NH 4 Cl solution was added to quench the reaction. The reaction mixture was transferred to a separating funnel and the aqueous phase was removed. The organic phase was dried (MgSO 4 ), filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel using an appropriate elution system to give the phenolic triflates 5a-c. The complete description of each compound including NMR and MS spectra can be found in Supplementary Material.

General procedure C (synthesis of molecules 6a-f)

Compound 5 (1.00 eq.), bis(pinacolato)diboron (1.50 eq.) and KOAc (3.00 eq.) were dissolved in toluene (0.10M) in a Schlenk tube. The resulting mixture was deoxygenated with freeze-pumpthaw cycling before addition of Pd(dppf)Cl 2 .DCM (0.10 eq.). The reaction was heated to 90 °C with constant stirring under argon for 17 h. The mixture was then cooled to room temperature, diluted with CH 2 Cl 2 and washed with water. The organic phase was dried over MgSO 4 , filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel using an appropriate elution system to furnish pure pinacol borane esters 6a-f. The complete description of each compound including copy of NMR and MS spectra can be found in Supplementary Material.

General procedure D (synthesis of molecules 7a-f):

Brominated naphthalimide analogue 3 (1.00 eq.), appropriate pinacol borane ester 6 (1.20 eq.), and Na 2 CO 3 (3.00 eq.) were dissolved in a tertiary mixt of solvent composed of H 2 O, EtOH, and toluene (v/v 3:3:10, 0.15M) in a Schlenk tube. The resulting mixture was deoxygenated with freeze-pump-thaw cycling before addition of Pd(dppf)Cl 2 (0.10 eq.). The reaction was heated to 80 °C with constant stirring under argon atmosphere for 17 h. The mixture was then cooled to room temperature, diluted with CH 2 Cl 2 and washed with water. The organic layer was collected, dried over MgSO 4 , and concentrated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel using an appropriate elution system to give biaryl compounds 7a-f. The complete description of each compound including NMR and MS spectra can be found in Supplementary Material.

General procedure E (synthesis of CinNaphts 8a-m): A solution of compound 7 (1.00 eq.) in dry acetonitrile (0.01M) was cooled in an ice bath to 0 °C. Nitrosyl tetrafluoroborate (1.1 eq.) was added and the resulting reaction mixture was kept under stirring at 0 °C under and Argon atmosphere for 1 h. After the reaction was completed, the mixture was neutralized with as saturated NaHCO 3 solution and extracted with CH 2 Cl 2 . The organic layer was dried over MgSO 4 and concentrated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel using an appropriate elution system. The complete description of each compound including copy of NMR and MS spectra can be found in Supplementary Material.

Photophysical Studies

UV-vis absorption measurements (scan mode) were conducted on a Shimadzu UV-visible spectrophotometer UV-2600 using rectangular 10 mm path length quartz cuvettes from Thuet, at 25 °C. Fluorescence spectroscopic studies (scan mode) were performed with a Fluoromax-4 spectrofluorometer (Jobin-Yvon; Horiba) at 25 °C, with rectangular 10 mm path length quartz cuvettes from Thuet. The absorption spectra were recorded with concentrations in the range 47-2.4 μM (total volume = 10 mL volumetric flask, six distinct dilutions for the accurate determination of molar extinction coefficients). The emission and excitation spectra were recorded with a diluted solution lower than 0.1 absorbance values. For the excitation and emission spectra the slits width were adjusted between 2-5 nm, integration time = 0.1 s, 1 nm step, HV (S1) = 950 V). All fluorescence spectra were corrected from the lamp fluctuations and the apparatus response fluctuations. Fluorescence quantum yields were measured at 25 °C, using three diluted solution (A<0.1) and three diluted solution of Ru(bpy) 3 2+ in air-saturated miliQ water (A<0.1). For each QY measurements, slidth width, excitation wavelength, scan rate, integration time and emission range were keep the same for the reference and the sample. The fluorescence quantum yield was determined thanks to the following formula:

F(x) = Sx/Ss(nX/nS) 2 F(s)
Where S is the slope of the linear plot of the integrated fluorescence intensity in function of the absorbance value, n is the refractive index of the solvents (at 25 °C) used in measurements, and the subscripts s and x represent standard and unknown, respectively.

The fluorescence quantum yield for the reference, Ru(bpy) 3 2+ in air-saturated miliQ water, was measured to be 0.040 in aerated water with the Horiba K-sphere accessory, in good correlation with the literature values. [18] DFT Calculation

Ground state geometries were optimized at the B3LYP/6-31+g(d) level of calculation followed by a frequency calculation to confirm the convergence to a local minimum. TDDFT calculations were then done at the PBE0/6-311+g(d,p) level of theory to estimate absorption energies. Then first singlet excited state geometries were optimized at the PBE0/6-311+g(d,p) level followed by a frequency calculation at the same level. Finally, a TDDFT calculation was done on this S1 optimized geometries at the same level of theory to estimate the fluorescence emission wavelength. All calculations were done with Gaussian 16 (Revision B.01) software. Data were analyzed with GaussView 6.0 software

Cell culture and fluorescence Imaging

Cell lines were obtained from the American type Culture Collection (Rockville, USA) and were cultured according to the supplier's instructions. A459 cells were grown in RPMI 1640 containing 10% FCS and 1% glutamine. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO 2 and were split every 3 or 4 days at a time when enough confluence was obtained. An Ibidi® µ-Slide 8 Well high Glass Bottom plate was seeded with 10 000 cells/well and then maintained at 37 °C in a humidified atmosphere containing 5% CO 2 for 24h to 48h. After adequate confluence was obtained, the medium was removed and replaced by a solution of fluorophore at 5 μM in the appropriate medium, prepared from a stock solution of fluorophore in DMSO, final concentration in DMSO = 1.0%, please note that no solubility issue was noted). The cells were incubated for 3h at 37 °C in a humidified atmosphere containing 5% CO2. The fluorophofore solution was then removed and the cells were washed twice with pre-warmed PBS (1X). Finally, RPMI medium was added before imaging. (Procedures for organelle staining aiming colocalization experiments are given in Supporting Information)

Confocal fluorescence microscopy

Fluorescence images were acquired using a Leica SP8-X inverted confocal microscope with a 40× oil immersion objective (HC PL APO CS2 Leica). Excitation was performed using a White laser pulsed at 80MHz set at the desired excitation wavelength. Detection was carried out by using PMT detector (Hamamatsu 6357) collecting photons over the appropriate emission wavelength window. Recorded image size was 1024x1024 pixels and a pixel size of 65nm was reach using the zoom factor according sampling laws

Figure 1 .

 1 Figure 1. Previous work relating to hybride fused dyes incorporating a naphthalimide moiety.

  [a] Values corresponding to S0-S1 transition but strong S0-S2 transition is also observed (see Supporting Information Fig S1 to S17), [b] Molar extinction coefficient related to S0-S1 transition.

Figure 2 .

 2 Figure 2. Graphical representation of photophysical properties heterogeneity of CinNapht 8a-n at 25 °C in CHCl3. A: Quantum Yield and Emission wavelength plot. B: Brightness and Emission wavelength plot.

Figure 3 .

 3 Figure 3. Left: frontiers molecular orbitals of 8c which are representative of all compounds substituted by a nitrogen; right: frontiers molecular orbitals of 8h in its first excited state geometry. Note the nature of the HOMO that is constituted by the lone pairs of the nitrogen atoms of the cinnoline.

Figure 4 .

 4 Figure 4. Confocal microscopic images of live A549 Cancer cells treated with compounds 8c, 8e, 8i and 8m (Exc: 490 nm, Em: 520-750 nm) 5 µM for 3 h at 37 °C using a 40x oil immersion objective.

Figure 5 .

 5 Figure 5. Confocal Mircoscopy Colocalization experiment with CinNapht 8i (5µM) and LysoView TM 488 on A549 live cells. Left : LysoView TM 488 (Exc: 480 nm, Em: 500 to 530 nm), Middle: CinNapht 8i (Exc 488 nm, Em: 600 to 700 nm), Right: Overlay.

Figure 6 .

 6 Figure 6. Confocal Mircoscopy Colocalization experiment with CinNapht 8m (5µM) on A549 live cells and MitoView TM Green. Left : MitoView TM Green (Exc: 480 nm, Em: 500 to 530 nm), Middle: CinNapht 8m (Exc 488 nm, Em: 600 to 700 nm), Right: Overlay.

Figure 7 .

 7 Figure 7. Confocal Mircoscopy Colocalization experiment with CinNapht 8e (5µM) and LipidSpot TM 610 on A549 live cells. Left : CinNapht 8e (Exc 480 nm, Em: 550 to 600 nm), Middle:, LipidSpot TM 610 (Exc: 610 nm, Em: 650 to 750 nm) Right: Overlay.

Table 1 . Photophysical properties of CinNaphts 8a-n in different solvents at 25 °C CinNapht Dye Solvent λmax Abs [a] (nm) max [b] (M -1 .cm -1 ) λmax Em (nm) Stokes Shift (cm -1 ) QYFL [c]

 1

Table 2 .

 2 Comparison between experimental and theoretical results including oscillator strength (f)

CinNapht Dye Absorption Emission λmax Calc (nm)  (Exp- Calc) f λmax Calc (nm)  (Exp- Calc)

	8a	497	1,6 %	0.310	544	3,9 %
	8b	491	3,0 %	0.321	549	4,5 %
	8c	522	2,4 %	0.372	580	5,7 %
	8d	470	1,5 %	0.335	521	7,0 %
	8e	492	1,7 %	0.332	555	3,8 %
	8f	506	0,8 %	0.322	555	3,0 %
	8g	476	1,9 %	0.331	566	4,4 %
	8h	396	0,5 %	0.401	/	/
	8i	499	2,5 %	0.309	547	4,4 %
	8j	499	0,8 %	0.307	563	3,9 %
	8k	501	3,7 %	0.309	548	3,5 %
	8l	501	1,2 %	0.305	548	6,3 %
	8m	516	6,0 %	0.329	562	13,8 %
	8n	498	0,4 %	0.310	546	9,3 %

Acknowledgements

This project has received funding by the French National Research Agency under the program CHARMMMAT ANR-11-LABX-0039-grant. This work was granted access to HPC resources from the "Mésocentre" computing center of CentraleSupélec and École Normale Supérieure Paris-Saclay supported by CNRS and Région Île-de-France (http://mesocentre.centralesupelec.fr/). We also thank the Institut de Chimie des Subtances Naturelles for their financial support. We also thank the pharamaceutical compunds screening plateform (CIBI Plateform) for their advices and technical support. The present work has benefited from Imagerie-Gif light microscopy core facility supported by French National Research Agency (ANR-11-EQPX-0029/Morphoscope, ANR-10-INBS-04/ FranceBioImaging; ANR-11-IDEX-0003-02/Saclay Plant Sciences). We thanks the Région Ile-de-France and DIM NanoK for financial support. The University Paris-Saclay and the CNRS are acknowledged for funding. Keywords: Fluorophore • Naphthalimide • Cinnoline • Fluorescence Imaging • TD-DFT •

Entry for the Table of Contents

The versatile synthesis, photophysical characterization and use of multiple Cinnoline/Naphthalimide fused hybrid fluorophores for live cell imaging provides a comprehensive overview of "CinNapht" potential as bio-imaging agent.

CinNapht Dyes

Fluorescence Tuning

Organelle Targeting