Amani Toumi

Sarra Boudriga

Khaled Hamden

Mansour Sobeh

Mohammed Cheurfa

Moheddine Askri
email: moheddine.askri@fsm.rnu.tn.

Michael Knorr
email: michael.knorr@univ-fcomte.fr.

Carsten Strohmann

Lukas Brieger

Synthesis, antidiabetic activity and molecular docking study of rhodanine-substitued spirooxindole pyrrolidine derivatives as novel α-amylase inhibitors

Keywords: Spirooxindole pyrrolidine, Rhodanine, Antidiabetic, α-Amylase, 1,3-Dipolar cycloaddition, Structure-activity relationship (SAR), Molecular docking

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

Introduction

Type 2 diabetes mellitus (T2DM) is a serious chronic metabolic disorder manifested by elevated blood glucose levels caused by inadequate insulin secretion or insulin resistance [1].

With the rapid increase of people suffering from T2DM, it has become a major health problem worldwide. It affects approximately 425 million of the global population in 2017 and has been estimated to increase by 629 million in 2045. Twelve percent of global healthcare expenditure, 727 billion dollars, is spent on diabetes [2]. Chronic hyperglycemic sufferers have to live with a high risk of micro-and macrovascular complications. Cardiovascular diseases, retinopathy, nephropathy, and neuropathy, are the main important characters of T2DMand represent a major risk for the development of diabetes [3-5]. These chronic complications may lead to coronary heart disease, stroke and other blood vessel diseases, nerve damage, kidney failure, and ophthalmic infections with time [START_REF]American Diabetes Association, Diagnosis and classification of diabetes mellitus[END_REF][START_REF] Rubin | Rubin's Pathology: Clinicopathologic Foundations of Medicine[END_REF][START_REF] Michael | Microvascular and macrovascular complications of diabetes[END_REF].

One of the relevant therapeutic approaches introduced for prevention of T2DM is the inhibition of the activity of the α-amylase enzyme to reduce the absorption of glucose in intestines [START_REF] Date | Pancreatic α-Amylase Controls Glucose Assimilation by Duodenal Retrieval through N-Glycan-specific Binding, Endocytosis, and Degradation[END_REF].α-Amylase (α-1,4-d-glucan glucanohydrolases) is a amylolytic enzyme, secreted from salivary gland and pancreas participating in the hydrolysis of the insoluble starch complex to oligosaccharides in the intestinal mucosa. For this purpose, inhibition of this enzyme can effectively delay the liberation of glucose into the bloodstream and thus the management of degenerative health problems such as T2DM [START_REF] De Souza | Application of microbial α-amylase in industry-A review[END_REF].

To date, multiple drugs have been introduced for the treatment of T2DM including metformin, sulfonylureas and thiazolidinediones (TZDs) [11][START_REF] Chaudhury | Clinical Review of Antidiabetic Drugs: Implications for Type 2 Diabetes Mellitus Management[END_REF][13]. These medicines are used alone as monotherapy or in combination if the goal of achieving glycemic control is not achieved [START_REF] Rhee | Monotherapy in patients with type 2 diabetes mellitus[END_REF]. TZDs (rosiglitazone, pioglitazone) are insulin-sensitizing drugs used in T2DM that bind the peroxisome proliferator-activated receptor-gamma (PPAR-γ), which is an important regulator of blood glucose level and dyslipidemia with minimal risk of hypoglycemia [START_REF] Thangavel | Targeting peroxisome proliferator-activated receptors using thiazolidinediones: Strategy for design of novel antidiabetic drugs[END_REF][START_REF] Sawant | Targeting PPAR-γ to design and synthesize antidiabetic thiazolidines[END_REF][START_REF] Chigurupati | A step ahead of PPARγ full agonists to PPARγ partial agonists: therapeutic perspectives in the management of diabetic insulin resistance[END_REF].

As one of the thiazolidines subtypes, rhodanines (2-thioxothiazolidin-4-ones) have been attracting much attention in medicinal and pharmaceutical chemistry [START_REF] Kaminskyy | Recent developments with rhodanine as a scaffold for drug discovery[END_REF][START_REF] Mousavi | A conceptual review of rhodanine: current applications of antiviral drugs, anticancer and antimicrobial activities[END_REF]. These moleculeshave been reported as, e.g.,HIV-1 Integrase Inhibitors [START_REF] Ramkumar | Design, Synthesis and Structure-activity Studies of Rhodanine Derivatives as HIV-1 Integrase Inhibitors[END_REF],topoisomerase II inhibition potency [21], anticancer activity against MCF-7 breast cancer [START_REF] Mandal | Novel rhodanines with anticancer activity: design, synthesis and CoMSIA study[END_REF]and potential 1,3-dipolar cycloaddition of azomethine ylides across electron-deficient dipolarophiles is established as a powerful strategy for the synthesis of structurally prominent spiroxindole pyrrolidine derivatives that make part of many pharmaceutical and natural products [START_REF] Barakat | Substituted spirooxindole derivatives as potent anticancer agents through inhibition of phosphodiesterase 1[END_REF][START_REF] Haddad | Synthesis of novel dispiropyrrolothiazoles by three-component 1,3-dipolar cycloaddition and evaluation of their antimycobacterial activity[END_REF][START_REF] Garcia Prado | Antiproliferative effects of mitraphylline, a pentacyclic oxindole alkaloid of Uncaria tomentosa on human glioma and neuroblastoma cell lines[END_REF]. In particular, have emerged as attractive synthetic targets, since their widespread panoply of significant bioactivities covers antimicrobial [START_REF] Haddad | A strategic approach to the synthesis of functionalized spirooxindole pyrrolidine derivatives: in vitro antibacterial, antifungal, antimalarial and antitubercular studies[END_REF], anticancer [START_REF] Arun | Novel Spirooxindole-Pyrrolidine Compounds: Synthesis, Anticancer and Molecular Docking Studies[END_REF],

cholinesterase inhibition activities [START_REF] Almansour | A facile ionic liquid promoted synthesis, cholinesterase inhibitory activity and molecular modeling study of novel highly functionalized spiropyrrolidines[END_REF] and antidiabetic ones [START_REF] Murugan | Synthesis and in vivo antidiabetic activity of novel dispiropyrrolidines through [3+2] cycloaddition reactions with thiazolidinedione and rhodanine derivatives[END_REF] (Fig. 1).Their prevalence in privileged structures has attracted a salient synthetic interest. Up to now, only a few methods have been described in literature for the synthesis of rhodanine-substituted-spiro[pyrrolidineoxindole] derivatives by 1,3-dipolar cycloaddition reactions [35-37]. Despite these recent advances, a 1,3-dipolar cycloaddition of stabilized azomethine ylides generated in situ by thermal [1,[START_REF] Jheng | Dietary low-fat soy milk powder retards diabetic nephropathy progression via inhibition of renal fibrosis and renal inflammation[END_REF]-prototropy with rhodanine has yet not been reported for the synthesis of the related rhodanine-linked-spirooxindole compounds (Scheme 1). In our continuous effort on the design of new bioactive heterocyclic scaffolds for biological screenings [START_REF] Haddad | Synthesis of novel dispiropyrrolothiazoles by three-component 1,3-dipolar cycloaddition and evaluation of their antimycobacterial activity[END_REF][START_REF] Haddad | A strategic approach to the synthesis of functionalized spirooxindole pyrrolidine derivatives: in vitro antibacterial, antifungal, antimalarial and antitubercular studies[END_REF][START_REF] Boudriga | Three-Component Access to Functionalized Spiropyrrolidine Heterocyclic Scaffolds and Their Cholinesterase Inhibitory Activity[END_REF], we highlight herein the synthesis of an extended series of nineteen Furthermore, an in vivo antidiabetic study was conducted. For this purpose, compounds 5g, 5k, 5s and 5l were orally administered to alloxan-induced diabetic rats.

Results and discussion

2.1.Synthetic Chemistry

We started our investigation by carrying out the reaction of (Z)-5-arylidene-2thioxothiazolidin-4-one 1a with stabilized azomethine ylides, generated in situ by thermal [1,[START_REF] Jheng | Dietary low-fat soy milk powder retards diabetic nephropathy progression via inhibition of renal fibrosis and renal inflammation[END_REF]-prototropy of isatin 2a with glycine methyl ester chloride 3, as a model substrate (Scheme 2). The 5-arylidenerhodanines 1a-f have been prepared via base-catalyzed Knoevenagel condensation reaction between rhodanines and diverse aromatic aldehydes [START_REF] Gouveia | Synthesis, antimicrobial and cytotoxic activities of some 5-arylidene-4-thioxo-thiazolidine-2-ones[END_REF].

This three-component reaction was conducted under various conditions in order to optimize the reaction parameters ( 1, entries 1, 3 and4). Among all probed bases, NEt3 was found to be the most suitable one for this reaction. (Table 1, entry 1). When the reaction temperature was lowered from reflux to room temperature, no reaction occurred even after 24h (Table 1, entry 2). Subsequently, a survey of various solvents, using NEt3 as base, was performed and revealed acetonitrile to be the best reaction medium, providing higher yields and shorter reaction time (Table 1, entry 8). No significant increase in yield could be achieved when increasing the reaction time from 2 h to 8 h (65% vs. 64% yields, respectively, entries 1 and 5). We also noticed that the use of only a 0.05 mmol of the organic base (NEt3) was not beneficial for the reaction yield ( Once the optimal reaction conditions established (Table 1, entry 8), we examined the scope of the cycloaddition reaction with regard to the diverse dipolarophiles 1 and cyclic ketones 2 (Table 2). Both electron-donating and electron-withdrawing groups at the aryl units of the Knoevenagel adduct 1(H, CH3, OCH3, F, Cl and Br) were well tolerated. Results revealed that the desired pyrrolidine-fused spirooxindole cycloadducts can be obtained in relatively high yields and selectivities (dr = 100%) from different sets of substrates (Table 2). Notably, substituted isatins with electron-withdrawing functional groups including Cl, Br and NO2 (5substitution) exhibited good performance and delivered the corresponding products in good yields along with excellent diastereoselectivities (Table 2). Furthermore, when isatin 2a was replaced with N-benzyl-substituted isatin 2e, a significant positive influence on the reaction was observed affording considerably improved yields (Table 2). The structure and the relative configuration of the isolated spiropyrrolidines 5a-s were elucidated by analyzing their spectroscopic data ( 1 H NMR and 13 C NMR) and confirmed by an X-ray diffraction study performed on cycloadduct 5c.

Entry

Spectroscopic and crystallographic characterization of the isomeric cycloadducts

The specific regioisomer was determined on the basis of the 1 H NMR chemical shifts of the H-4' and H-5' protons (Fig. 2). The 1 H NMR spectrum of 5g shows two mutually coupled doublets at δ 4.39 ppm and 5.11 ppm with a 3 J coupling of 9.6 Hz, corresponding to the pyrrolidine ring and corroborating the formation of regioisomer 5g. In the case of the hypothetical other regioisomer 6g (Scheme 3), one would expect in the 1 H NMR spectrum the occurrence of two singlets for the pyrrolidinyl protons instead of two doublets. Furthermore, the methoxy protons give rise to a sharp singlet resonating 3.71 ppm. The aromatic protons found in the region between δ 6.82to 7.58 ppm appear as a multiplet. In addition, there are two broad singlets at δ 8.21 and δ 9.35 assigned to the NH protons of oxindole and rhodanine, respectively. The off-resonance decoupled 13 C-NMR spectrum of 5g reveals the presence of two spiro carbons resonating at δ80.8 and 74.0 ppm, corresponding to C-2'and C-3', respectively (Fig. 2). The two carbons C-4' and C-5' of the pyrrolidine ring are resonating at δ 64.8 and 60.8 ppm. In addition, the resonances at δ 176.7 ppm and δ 171.3 ppm are due to the oxindole and ester carbonyl groups, respectively. The signals at δ 178.2 ppm and δ197.2 ppm of 5g are attributed to the ketonic carbonyl and thiocarbonyl carbons of rhodanine, respectively, corroborating further our structural proposal.

It is worth noting that in all cases, the reactions were found to be highly regioselective leading to the generation of only one regioisomer 5. No indication for the copresence of isomer 6, even in smaller amounts, was found (Scheme 2).

Crystal structure determination of cycloadduct 5c

The stereochemical outcome of the reaction was confirmed by single-crystal X-ray diffraction analysis of the cycloadduct 5c. The molecular structure of 5c is shown in Fig. 3. Noteworthy, an intramolecular C-H1 … O hydrogen bonding between H15 attached on the p-tolyl cycle and the O atom of the imide-type C27-O1 carbonyl function occurs. In the crystal, the individual molecules are associated through weak intermolecular N1-H1 … O3 1 , N3-H3 … O3 2 bonding, giving rise to a supramolecular network (see Fig. S40 and Tables S1 andS2 in the SI). This structural characterization reveals that the cycloaddition reaction proceeds through an exotransition state between the (Z)-5-arylidine-2-thioxothiazolidin-4-one and the Z,E-dipole affording only one diastereoisomer. Based on this finding, we suppose, also in line with the NMR data (see above), that the same plausible mechanism operates also for the other derivatives (see below) and that in consequence the stereochemistry of the other cycloadducts can be assigned by analogy.

Discussion of the regio-and stereochemical route of the reaction

Based on the experimental results and similar previous reports [START_REF] Boudriga | Highly diastereoselective construction of novel dispiropyrrolo[2,1-a]isoquinoline derivatives via multicomponent 1,3-dipolar cycloaddition of cyclic diketones-based tetrahydroisoquinolinium Nylides[END_REF][START_REF] Gazzeh | Stoichiometry-controlled cycloaddition of nitrilimines with unsymmetrical exocyclic dienones: microwave-assisted synthesis of novel mono-and dispiropyrazoline derivatives[END_REF][START_REF] Haddad | Regio-and Stereoselective Synthesis of Spiropyrrolizidines and Piperazines through Azomethine Ylide Cycloaddition Reaction[END_REF], a plausible mechanism for the regio-and diasterioselective spiropyrrolidines formation represented in Scheme 4 can be proposed. The reaction proceeds through the generation of stabilized azomethine ylide 4 by condensation of isatin 2 with a primary amine 3 by thermal [1,[START_REF] Jheng | Dietary low-fat soy milk powder retards diabetic nephropathy progression via inhibition of renal fibrosis and renal inflammation[END_REF]-prototropy of the isatinderived iminoester. The dipolarophile 1 undergoes then a subsequent 1,3-dipolar cycloaddition across the azomethine ylides, affording the desired products in a completely regio-and stereoselective manner. Four possible stereoisomers, related to two regioisomeric and two stereoisomeric approaches, might à priori be present in the thus formed spiro[pyrrolidine-3,2'-oxindoles] (Scheme 4). The cycloadducts were labelled exo -5, endo-5, exo-6, endo-6 and their transition states (TS) TS-exo-5, TS-endo-5, TS-exo-6 and TS-endo-6. As depicted in Scheme 3, a high regio-and diasterioselectivity was encountered in the formation of cycloadduct exo-5, which may be directed by the presence of stabilizing secondary orbital interactions (SOI) [START_REF] Houk | The Role of Secondary Orbital Interactions in Cycloaddition Reactions[END_REF][START_REF] Boudriga | Straightforward and Highly Diastereoselective Synthesis of a New Set of Functionalized Dispiropyrrolidines Involving Multicomponent 1,3-Dipolar Cycloaddition with Azomethine Ylides[END_REF][START_REF] Ess | Distortion/Interaction Energy Control of 1,3-Dipolar Cycloaddition Reactivity[END_REF] between the oxygen atom of the carbonyl of the indole ring and the carbonyl carbon atom of the dipolarophile residue. The alternative possible transition states depicted in Scheme 3 appear to be less favorable due to steric crowding (TS-exo-6), and/or electronic repulsion occurred between the cis carbonyls of the arylidene and indole part, in case of TS-endo-5 andTS-endo-6.

In-vitro α-amylase inhibitory activity

One of the therapeutic approaches for decreasing postprandial hyperglycaemia is to delay glucose absorption by the inhibition of the α-amylase activity [START_REF] Kaleshkumar | Muscle extract of Arothron immaculatus regulates the blood glucose level and the antioxidant system in high-fat diet and streptozotocin induced diabetic rats[END_REF][START_REF] Nguyen | Biosynthesis of α-glucosidase inhibitors by a newly isolated bacterium, Paenibacillus sp. TKU042 and Its effect on reducing plasma glucose in a mouse model[END_REF]. Once in hand, the newly synthesized compounds were subjected to a preliminary evaluation of their in vitro αamylase enzyme inhibition activity according to the standard method [START_REF] Hansawasdi | α-Amylase Inhibitors from Roselle (Hibiscus sabdariffa Linn.) Tea[END_REF]. The anti-diabetic drug Acarbose served as a standard in this investigation for comparison. The half maximal inhibitory concentration (IC50) values of our compounds are reported in Table 3. We found that all compounds exhibit promising activities against α-amylase enzyme when used in µM concentration. It is notable that compounds 5g, 5k, and 5s (IC50, 1.49± 0.10, 1.50± 0.07, 1.57± 0.10 µM, respectively), followed by 5l,5q and 5n(IC50, 1.59± 0.08, 1.63± 0.09and 1.67± 0.09 µM respectively) exhibited the highest inhibition potential, while 5c, 5d and 5j showed moderate activity (IC50, 1.72± 0.08, 1.73± 0.07, 1.75± 0.07 µM, respectively).

Compounds 5a, 5e and 5f (IC50, 2.08± 0.11, 2.33± 0.13, 3.06± 0.17 µM, respectively) were the least active ones, but showed nevertheless satisfactory results in preliminary experiments, as compared to the hypoglycemic control drug acarbose (IC50, 1.56± 0.07 µM) (Table 3).

Structure Activity Relationship -SAR.

It is evident that the inhibitory activity of the synthesized derivatives depends in a crucial manner on structural factors such as the nature of the substituents on the phenyl rings and the N-substituted group attached at the isatin core. As shown in Table 3, non-substituted spiropyrrolidine 5e (Ar= Ph, R1= H, R2= H) reveal a modest inhibition activity compared to the other compounds (IC50= 2.33± 0.13µM). The incorporation of a bromo atom at the pposition of the isatin ring enhances extremely the inhibitory activity, rendering5g (Ar= Ph, R1= Br, R2= H) the most active compound (IC50= 1.49± 0.10 µM). This result demonstrates further that the nature of the substituent on the phenyl ring of the isatin exerts a noticeable effect on the inhibition activity of spiropyrrolidine derivatives. The same improvement was noticed for series 5 derivatives incorporating a bromo-substituted isatin moiety such as 5k, 5s, 5c and 5i (Fig. 4). Double-halogenated compounds that contain also a p-halogen-substitution on the aromatic group of the dipolarophile (such as 5k and 5s) display somewhat superior activity compared to compounds 5c and 5i, the order of activity being (Br>Cl>Me>OMe).

This difference of activity between all these derivatives may be explained by the inductive effect (-I) and the donor mesomeric effect (+M) of the substituent groups attached to the aromatic ring of the dipolarophile. This observation was evidenced from the screening of derivatives 5p, 5aand 5e (Fig. 5).

Compound 5p that is chloro-substituted on the aromatic group of the dipolarophile is among these latter three representatives still the most potent one (activity Cl>Me>H). Interestingly, compounds of series 5 bearing a chloro-substituent on the isatin nucleus (Fig. 6), in particular compound 5d bearing also a methyl group (Ar = p-CH3C6H4, R1= Cl, R2= H),

showed a superior activity compared to compounds 5j, 5o, 5r and 5f, the order of activity being Me> Br>Cl >F> H, respectively. In this case, the activity was considerably affected by introducing a methyl group at the 4-position of the aromatic cycle of the dipolarophile, most probably due to stronger interactions of 5d with the active enzyme site. Furthermore, the incorporation of a benzyl group at the N-position of the isatin core causes a considerable increase in the α-amylase inhibition (Fig. 7). For instance, compound 5q (Ar= p-ClC6H4, R1= H, R2= Bn) is obviously more active than 5p (Ar= p-ClC6H4, R1= H, R2= H).

The p-halogen-substituted aryl derivatives of dipolarophile 5q and 5n also showed a better inhibitory activity in the order Cl> F> OMe> Me. Worth mentioning is also compound 5l (IC50 = 1.59± 0.08 μM) (Table 3) bearing a p-nitro aryl cycle on the of isatin core, which displayed a good activity. This might be due to the electron-withdrawing nature of the NO2 group. Limited inhibition activity of otherwise substituted spiropyrrolidine derivatives like 5f suggest that the α-amylase inhibitory activity depends in a sensitive manner both on substitution on the dipolarophile and isatin core. In addition, compounds containing halogen substituents attached to the phenyl ring of (Z)-5arylidine-2-thioxothiazolidin-4-ones exhibit generally a higher inhibitory potency against the amylase enzyme than derivatives incorporating an electron-donating group bonded to the same phenyl ring.

The inhibition potency of our heterocyclic systems may be compared favorably with those of other heterocycles compounds investigated by other research groups such as arylhydrazidethiazolidinone derivatives (IC50 values ranging between 4.10 and 38.20μM as compared to the standard acarbose 1.70 µM) [START_REF] Rahim | Synthesis of new arylhydrazide bearing Schiff bases/thiazolidinone: α-Amylase, urease activities and their molecular docking studies[END_REF], thiazolidine-2,4-dione-pyrazole conjugates (IC50= 4.08->200 μg/mL as compared to acarbose 8.0 μg/mL [START_REF] Bansala | Synthesis and biological evaluation of thiazolidine-2,4-dione-pyrazole conjugates as antidiabetic, anti-inflammatory and antioxidant agents[END_REF], or spirooxindoles and benzo[b]furan analogues (IC50= 22.61-779.08 µM in comparison with acarbose 0.75 µM) [START_REF] Altowyan | Spiroindolone Analogues as Potential Hypoglycemic with Dual Inhibitory Activity on α-Amylase and α-Glucosidase[END_REF].

Molecular docking studies

In order to get a deeper insight into the binding interactions of the spiropyrrolidine derivatives, molecular docking study of five selected compounds was performed against active site of pig pancreatic α-amylase. The docked compounds 5d, 5g, 5k, 5s and 5p fitted well in the binding site of the α-amylase and gave rise to different polar and non-polar interactions with the embedded amino acid residues, most importantly the ionic interaction between the protonated secondary cyclic amine group in the ligand and the carboxylate moiety of Asp300 and/or Glu233. Compound 5d and 5g showed the lowest binding energy reverberated by a docking score of -19.78 and -18.45kcal/mol followed by 5k and 5s that showed comparable binding energies of -18.29 and -17.55 kcal/mol, and finally came compound 5p with a docking score of -16.91 kcal/mol.

Ligand interactions of compound 5d

The molecular docking study of compound 5d revealed various interactions with the binding site residues. The His305 residue showed a hydrogen bonding interaction with the negatively charged nitrogen of the thiazolidine ring, as shown in Fig. 8. The carbonyl groups of the thiazolidine and indoline rings are also able to hydrogen bonding with the His305 and Gly306 residues, respectively. Leu162 is involved in a σ-π-interaction with the π-system of the indole ring. Moreover, the protonated pyrrolidine nitrogen forms an ionic interaction (salt bridge) with the anionic carboxylate group of Asp300, which is an essential interaction afforded by the reference inhibitor acarbose. The chloro-substituent of the indoline ring can enter in contact with Val163. The positive electrostatic potential on the outer lobe of such a covalent bond could interact with electron cloud of the alkyl group of Val163. This noncovalent interaction might contribute greatly to the activity of the compound. In addition, the methyl group of the p-tolyl moiety does not seem to contribute to the binding since it is exposed to the exterior of the binding site and is not coming in contact to any of the amino acid residues lining the binding site of the enzyme.

Ligand interactions of compound 5g (the most potent inhibitor)

His305 of active site residue forms hydrogen bonding interactions with the negatively charged nitrogen and the carbonyl group of the thiazolidine core. The π-system of the indole ring and the π-system of the 4'-phenyl cycle on the pyrolidine ring gives rise to a σ-π-interaction with the Leu162 and Trp59 residues, respectively. Furthermore, the protonated pyrrolidine nitrogen is involved in anionic interaction (salt bridge) with Asp300. The covalently bonded bromine atom at the indoline ring comes in contact with Val163. Again, the positive electrostatic potential on the outer lobe of this covalent bond could interact with the electron cloud of the alkyl group of Val163. As in the case of 5d, this non-covalent interaction might contribute significantly to the inhibitory activity of this derivative (Fig. 8).

Ligand interactions of compound 5k

For the docked 5k derivative, the negatively charged nitrogen and the carbonyl group of the thiazolidine ring interact through hydrogen bonding with His305. The π-system of the indole ring forms a σ-pi-interaction with Leu162. In addition, the protonated pyrrolidine nitrogen shows an ionic interaction (salt bridge) with the negatively charged carboxylate group of Asp300. The covalently bonded bromine atom of the indoline ring enters in contact with Val163. As in the previous cases, the positive electrostatic potential on the outer lobe of this covalent bond could interact with the electron cloud of the alkyl group of Val163. In contrast, the bromine atom at the para-position of the 4'-phenyl cycle on the pyrrolidine ring does not seem to contribute to the binding of the compound, since it is exposed to the exterior of the binding site and therefore not entering in contact to any of the amino acid residues lining the binding site of the enzyme (Fig. 8).

4. Ligand interactions of compound 5s

Compound 5s displays two interactions, one hydrogen bonding between the negatively charged nitrogen and the carbonyl group of the thiazolidine ring with His305 and another σπ-interaction between the π-system of the 4'-phenyl cycle with Val163 and Trp59. The protonated pyrrolidine nitrogen shows also an ionic interaction (salt bridge) with an anionic carboxylate function of Asp300. The covalently bonded bromine atom at the indoline ring comes in contact with Gly306. The positive electrostatic potential on the outer lobe of such covalent bond allows again an interaction with the electron cloud of the alkyl group of Gly306. The chloro-substituent at the p-position of the 4'-phenyl cycle attached on the pyrrolidine ring does not seem to contribute to the binding of the compound, as it is exposed to the exterior of the binding site and is thus not coming in contact to any of the amino acid residues lining the binding site of the enzyme (Fig. 8).

4. Ligand interactions of compound 5p

The His305 residue was found to form a hydrogen bond interaction with the negatively charged nitrogen of the thiazolidine ring, while Asp300 interacts via hydrogen bonding with the protonated pyrrolidine nitrogen. Compound 5p exhibits also an ionic interaction (salt bridge) between the protonated pyrrolidine nitrogen and the anionic carboxylate of Glu233.

Moving the halogen atom from the indoline ring to the p-position of the 4'-phenyl on the pyrrolidine ring results in a diminished activity. In this case, the chlorine atom is situated outside the binding site and is therefore not in contact to any of the amino acid residues lining the active site of the enzyme. Accordingly, it was not able to form any kind of halogen interaction, which could be the reason behind the poor activity of the drug. It also forces to push the indoline ring a little bit outside the binding site, which in turn causes to lose the σ-πinteraction with Leu162. This feature could be another explanation for the lower activity of compound 5p (Fig. 8).

Docking the reference inhibitor Acarbose using the same docking protocol showed that Acarbose iterated most of the amino acid interactions reported between the X-ray crystallized protein and the co-crystallized acarbose, such as Asp197, Val163, Trp59, and most importantly the polar interactions with the negatively charged Asp300 and Glu233 (Fig. 9).

The binding energy was estimated to be -24.67 kcal/mol.

Anti-hyperglycemic effect of spiropyrrolidines 5 on diabetic rats

In vivo studies were carried out to evaluate the blood glucose lowering effect of our spiropyrrolidines in diabetic rats. As shown in Fig. 10, the blood glucose level increased significantly by 83% in the diabetic group (Group II) in comparison to normal rats (Group I).

However, the daily administration of compounds 5g, 5s, 5k and 5l for one month to surviving diabetic rats was found to cause a significantly decrease of the serum glucose level by 6.8, 9.9, 12.3 and 5.5% respectively as compared to untreated diabetic rats. Fig. 10. Effect of spiropyrrolidines supplements on blood glucose level of diabetic rats. Values are given as mean ± SD for groups of 8 animals each. Values are statistically presented as follows: * P < 0.05 significant differences compared to control group; # P < 0.05 significant differences compared to diabetic rats; @ P < 0.05 significant differences compared to acarbose-treated diabetic rats.

Oral glucose tolerance test in diabetic rats treated with spiropyrrolidines

The blood glucose levels were checked as per standard methods at 0, 30, 60, 90, 120 min intervals of the commencement of the experiment. The principal observation is that oral administration of all these heterocycles caused a significant decrease in blood glucose level as compared to the untreated diabetic control group. The blood glucose levels of 5g, 5s, 5k and 5l were lowered after 1h to 251, 269, 281 and 267 mg/dl, respectively, as compared to untreated diabetic rats (315 mg/dl) (Fig. 11). In fact, 1 hour after oral administration of 5g, 5s, 5k and 5l to diabetic rats, the blood glucose levels were lowered significantly by 20, 14.6, 10.7 and 15.2%. Therefore, these compounds deserve further exploration for the development of antidiabetic drugs with minimal side effects (Fig. 11). Limited structure-activity relationship (SAR) suggested that the nature and the electronic effect of the substituents present on the isatin moiety and the phenyl part of (Z)-5-arylidine-2thioxothiazolidin-4-ones exert a significant influence on the inhibitory activity. It was noticed that substituted spiropyrrolidine derivatives exhibit a superior inhibitory activity than their unsubstituted congeners. This investigation furthermore evidences that compounds 5 bearing a halogen substituent on the phenyl rings feature a more potent inhibitory activity against αamylase than derivatives bearing electron-donating groups.

Molecular docking study allowed to predict several important binding interactions of the spiropyrrolidine derivatives with the active site of α-amylase enzyme. Furthermore, an in vivo antidiabetic activity study using alloxan-induced diabetic rats indicated that compound 5k presents significant hypoglycemic properties. Thus, these rhodanine-substitued spiropyrrolidine derivatives are valuable candidates deserving further exploration for future development of potential antidiabetic agents.

3.

Experimental section

Materials and methods

1 H and 13 C NMR were recorded using a Bruker spectrometer operating at 400 and 100 MHz, respectively. The chemical shifts were recorded in ppm relative to tetramethylsilane and with the solvent resonance as the internal standard. Data were reported as follows: chemical shift, multiplicity (bs = broad singlet, s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m = multiplet), coupling constants (Hz), integration. 13 C NMR data were collected with complete proton decoupling. Chemical shifts were reported in ppm with respect to TMS with the solvent resonance as internal standard. All chemicals were obtained from commercial sources and were used without further purification. Elemental analyses were performed on a Perkin Elmer 2400 Series II Elemental CHNS analyzer. Column chromatography was carried out on silica gel (300-400 mesh, Qingdao Marine Chemical Ltd., Qingdao, China). Thin layer chromatography (TLC) was performed on TLC silica gel 60 F254 plates 0.2 mm 200x200 nm;

The spots were visualized using UV light at 254 nm and 360 nm.

General procedure for the synthesis of products 5a-s

A mixture of (Z)-5-arylidine-2-thioxothiazolidin-4-ones 1, (1 mmol), glycine methyl ester chloride 3 (1 mmol), and isatin 2(1 mmol) in acetonitrile (5 mL) was stirred for 2h at 80 °C.

After completion of the reaction (TLC), the solvent was removed under vacuum. The crude product was subjected to column chromatography on silica gel using cyclohexane/ethyl acetate (4:1 v/v) as eluent to give the pure products 5. 4.2.1. Methyl(3S,3'S,4'S,[2][3] 2,52.6,60.6,64.7,73.7,79.5,110.5,119.0,123.5,124.0,124.8,129.9,130.2,130.7,135.7,141.5,147.0,152.1,171.6,176.7,178.0,201.3;Anal. Calcd. For C22H19N3O4S2: C,58.36;H,4.22;N,9.27;found: C,58.46;H,4.23;N,9.30. 4.2.2. Methyl(3S,3'S,4'S,[2][3] 2,43.8,52.6,60.6,65.2,73.9,77.7,110.1,123.6,125.3,127.0,127.3,127.7,128.9,129.0,129.4,129.5,129.7,129.8,130.2,131.1,133.2,134.5,138.1,144.1,171.6,175.7,178.3,197.4;Anal. Calcd. For C29H25N3O4S2: C,64.07;H,4.64;N,7.73;found: C,64.21;H,4.62;N,7.71. 4.2.3. Methyl(3S,3'S,4'S,[2][3] MicroMountfrom MiTeGen. For the data collection, the programs APEX 3 Suite (v.2018.7-2) with the integrated programs SAINT (integration) and SADABS (adsorption correction) by

Bruker AXS GmbH were used. The processing and finalization of the crystal structure was done with the program Olex2 [START_REF] Dolomanov | OLEX2: a complete structure solution, refinement and analysis program[END_REF]. The crystal structure was solved with the ShelXT [START_REF] Sheldrick | SHELXT -Integrated space-group and crystal-structure determination[END_REF] structure solution program using Intrinsic Phasing and refined with the ShelXL [START_REF] Sheldrick | Crystal structure refinement with SHELXL[END_REF] refinement package using Least Squares minimization. The non-hydrogen atoms were refined anisotropically. The C-bound H atoms were placed in geometrically calculated positions and each was assigned a fixed isotropic displacement parameter based on a riding-model: C-H = 0.95-1.00 Å with Uiso(H) = 1.5Ueq(CH3) and 1.2Ueq( CH) for other hydrogen atoms. The Nbound hydrogen atoms were located in the difference-Fourier-map and refined independently.

Crystallographic data for the structure of 5c have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number 1994835. Copy of these data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 IEZ, UK, fax: 144-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk.

Experimental method for the in vitro α-amylase assay

The in vitro α-amylase inhibition activity of all drugs was determined based on the spectrophotometric assay using acarbose as the reference compound. The heterocycles 5 were dissolved in DMSO to give concentrations ranging from 1, 4, 8 and 16 mg/mL. Appropriate dilutions (1-16 mg/mL) of the heterocycles 5 and 500 µl of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) containing porcine pancreatic α-amylase (EC 3.2.1.1) (0.5 mg/mL) were incubated at 25°C for 10 min. Then, 500 µl of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) was added to the reacting mixture.

After that, 1.0 mL of dinitrosalicylic acid (DNSA) was added to the mixture which was incubated at 37 °C for 5 min. The reaction was stopped by incubating in a boiling water bath for 5 min and subsequent cooling to room temperature. The absorbance was measured at 540 nm. The α-amylase inhibitory activity was expressed as percentage inhibition [55].

Experimental induction of diabetes

Inhibition (%) = [(Absref -Abssample)/=Absref]* 100

The assays of the present study were conducted on adult male Wistar rats, weighing 170 ± 10 g, which were obtained from the local Central Pharmacy, Tunisia. All rats were kept in an environmentally controlled breeding room (temperature: 24 ± 2°C, humidity: 60 ± 5%, 12-h dark/light cycle) where they had standard diets and free access to tap water. The animal studies conducted in this work followed the International Guidelines for Animal Care Directive 86/609/EEC, and was approved by the Tunisian Ethics Committee of the University of Monastir (Monastir, Tunisia) for the care and use of laboratory animals.

Diabetes was induced in rats by a single intraperitoneal injection of freshly prepared alloxan solution in normal saline at a dose of 150 mg/kg body weight [55]. After three days, the rats whose blood glucose was higher 280 mg/dl were chosen for the experiment. The rats were divided into eight groups of eight animals, each as follows: Group I: normal animal; Group II: untreated diabetic rats; Group III: diabetic animals treated with standard drug as acarbose at a dose of 10 mg/kg; and Group IV: the diabetic rats treated with the spiropyrrolidine derivatives at a dose of 20 mg/kg. The spiropyrrolidine derivatives and acarbose were dissolved in DMSO at a concertation of 4% in water. All groups of rats were given a dose of DMSO of 4% in water by gastric gavage route to standardize the conditions of the experiment. One month later and 12 h after last feeding, the rats were sacrificed by decapitation, and their trunk blood collected. The serum was prepared by centrifugation (1,500 ×g, 15 min, 4°C). The serum glucose level was measured by measuring the amount of red quinoneimine released from oxidation of glucose substrate (Kits, Biolabo, France, ref

80009).

For oral glucose tolerance test (OGTT), 8 normal and 36 surviving diabetic rats obtained 3 days after alloxan injection were used. Rats of the experimental groups received 20 mg/kg of the test substances or acarbose in 4% DMSO by gastric gavage route. After 30 min, all animals were administered intragastrically with 2 g/kg sucrose in 0.9% sodium chloride solution. Blood glucose levels of all rats were tested initially before the glucose administration and 0, 30, 60, 90 and 120 min of post-glucose administration [START_REF] Stalin | Synthesis of a 1,2,3-bistriazole derivative of embelin and evaluation of its effect on high-fat diet fed-streptozotocin-induced type 2 diabetes in rats and molecular docking studies[END_REF][START_REF] Liu | Bioactivity-based analysis and chemical characterization of hypoglycemic and antioxidant components from Artemisia argyi[END_REF].

Docking protocol

The X-ray crystallographic structure of the procine α-amylase in complex with acarbose (PDB code: 1OSE) was downloaded from the protein data bank (www.rcsb.org). The docking protocol was applied using Molecular Operating Environment (2010.10;Chemical Computing Group Inc., Montreal, Canada) software. The downloaded protein was protonated to add the hydrogen atoms that were not detected during the crystallization process. Compounds selected for docking were drawn using MOE builder tool. The compounds were then washed to set their ionization state. The MMFF94x force field was used to compute the energy minimization for the compounds. Docking was done applying the default settings of placement and scoring [START_REF] Sobeh | Albizia harveyi: phytochemical profiling, antioxidant, antidiabetic and hepatoprotective activities of the bark extract[END_REF].

Fig. 1 .

 1 Fig.1. Examples of bioactive molecules containing rhodanine and spiro[pyrrolidine-oxindole] units.

Scheme 1 .

 1 Scheme 1. Strategy for the construction of novel rhodanine-fused spiro[pyrrolidine-2,3'-oxindoles].

  spiro[pyrrolidine-2,3'-oxindole] derivatives via one-pot three-component 1,3-dipolar cycloaddition reactions of stabilized azomethine ylides across rhodanine. The current protocol provides a facile and efficient approach for the incorporation of rhodanine and spiro[pyrrolidine-2,3'-oxindole] scaffolds, allowing the formation of several new covalent bonds in only one single step, effectively well adapted for combinatorial synthesis.The synthetic work presented in our contribution is completed by the in vitro investigation of the antidiabetic activity of this novel family of spiro[pyrrolidine-oxindole] derivatives against the α-amylase enzyme. The structure-activity relationships (SAR) of designed compounds is also discussed. A molecular docking study was performed in order to understand the binding interactions of the most active analogs with the active site of the α-amylase enzyme.

Scheme 2 . 1 , 3 -

 213 Scheme 2. 1,3-Dipolar cycloaddition leading to spirooxindole pyrrolidine 5a.

2a: R 1 =

 1 H,R 2 = H, 2b: R 1 = Cl,R 2 = H, 2c: R 1 = Br,R 2 = H, 2d: R 1 = NO 2 , R 2 = H, 2e: R 1 = H,R 2 = Bn a All reactions were carried out with 1 (1 mmol), 2 (1 mmol), 3 (1 mmol), NEt3 (1 mmol) in CH3CN (5.0 mL) at reflux for 2 h. Yields of the isolated cycloadducts are given.

Fig. 2 .

 2 Fig. 2. Selected 1 H (black)and 13 C NMR (red) chemical shifts (ppm)of 5g.

Fig. 3 .

 3 Fig. 3. Ball and sticks presentation of the molecular structure of 5c recorded at 100 K. Only relevant H atoms are shown.

Scheme 3 .

 3 Scheme 3. Plausible mechanism for the regio-and stereoselective formation of spiropyrrolidines 5.

Fig. 4 .

 4 Fig. 4. Structure-activity relationship of compounds 5k, 5s, 5c, 5i, and 5g.

Fig. 5 .

 5 Fig. 5. Structure-activity relationship of compounds 5a, 5e and 5p.

Fig. 6 .

 6 Fig. 6. Structure-activity relationship of compounds 5d, 5j, 5o, 5r and 5f.

Fig. 7 .

 7 Fig. 7. Structure-activity relationship of compounds 5n, 5q, 5b and 5h.

Fig. 8 .

 8 Fig. 8. Docking poses (2D, 3D) of compounds 5d(a), 5g(b), 5k(c), 5s(d) and 5p(e) docked within the porcine α-amylase binding site.

Fig. 9 .

 9 Fig. 9. Docking of the reference inhibitor acarbose at the porcine α-amylase binding site showing that it has iterated most of the reported amino acid interactions.

Fig. 11 .

 11 Fig. 11.Oral glucose tolerance test in diabetic rats treated with of spiropyrrolidines (5g, 5s, 5k, 5l) and standard Acarbose. Group I: Normal rats with normal diet; Group II: diabetic control group; Group III: diabetic animals orally fed with acarbose at a dose of 10 mg/kg p.c daily by the gastric gavage method.

Table

  ). Reaction temperature, time, solvents with varying polarity and different bases were screened, and the results are summarized in Table1. Initially, the reaction was conducted in MeOH in the presence of different bases such as NEt3, K2CO3, DABCO, and NH4OAc (Table

Table

Table 1 .

 1 Optimization of the reaction conditions a .

	a Unless otherwise noted, all reactions were carried out with 1a(0.1 mmol), 2a(0.1 mmol), 3 (0.1 mmol), and base (0.1 mmol) in solvent (1.0 mL) at specified temperature. b Isolated yield. c No reaction. d Reaction was
	carried out with 0.05 mmol of NEt3.

Table 2 .

 2 Substrate Scope. a

	1a: Ar= p-MeC 6 H 5 , 1b: Ar= p-OMeC 6 H 5 , 1c: Ar= p-BrC 6 H 5 , S S HN O N O O H 3 CO O R 1 Ar R 2 1 2	3	NH 3, Cl	CH 3 CN, 80°C Et 3 N	S	HN	O	S	N H Ar N R 2 O 5a-s OCH 3 H	R 1
	1d: Ar= p-ClC 6 H 5 , 1e: Ar= C 6 H 5 , 1f: Ar= p-FC 6 H 5
																				Me													Me	Me	Me
														O	H																O	H	O	H	O	H
	S	HN	O	N 5a , 80% N H S	CO 2 Me H	S	HN	O	N 5b , 95% CO 2 Me N S H Bn	S	HN	O	S	N 5c , 86% CO 2 Me H Br N H	S	HN	O	CO 2 Me H Cl 5d , 83% N S N H
																																	OMe
														O	H																O	H	O	H	O	H
				S	HN	O	S			N H	N		CO 2 Me H			S	HN	O	S	N H	N	CO 2 Me H Cl	S	HN	O	S	H N	N	CO 2 Me H Br	S	HN	O	S	N Bn	N	CO 2 Me H
													5e , 85 %													5f , 88%	5g , 91 %	5h , 75%
																				OMe													Br	Br	Br
															O	H																O	H	O	H	O	H
				S	HN	O	S			N H	N	CO 2 Me H Br			S	HN	O	S	N H	N	CO 2 Me H Cl	S	HN	O	S	N H	N	CO 2 Me H Br	S	HN	O	S	H N	N	CO 2 Me H NO 2
														5i, 66%	F													5j, 81 %	F	5k , 85%	F	5l, 83%	Cl
	S	HN	O		S	O	H N H	N	CO 2 Me H Br	S	HN	O		S	O	H N Bn	N	CO 2 Me H	S	HN	O	S	O	H H N	N	CO 2 Me H Cl	S	HN	O	S	O	N H H	N	CO 2 Me H
														5m , 84%													5n , 94%	5o , 78%	5p , 80%
																				Cl													Cl	Cl
													O		H																O	H	O	H
	S	HN	O		S			N Bn	N		CO 2 Me H	S	HN	O	S	N H	N	CO 2 Me H Cl	S	HN	O	S	H N	N	CO 2 Me H Br
														5q , 96%													5r , 90%	5s , 86%

Table 3 .

 3 In vitro α-amylase inhibition assay of spiropyrrolidines 5.

		Compound	Ar	R1	R2	IC50(µM± SD)
	1	5a	Me	H	H	2.08± 0.11
	2	5b	Me	H	Bn	1.81± 0.09
	3	5c	Me	Br	H	1.72± 0.08
	4	5d	Me	Cl	H	1.73± 0.07
	5	5e	H	H	H	2.33± 0.13
	6	5f	H	Cl	H	3.06± 0.17
	7	5g	H	Br	H	1.49± 0.10
	8	5h	OMe	H	Bn	1.77± 0.08
	9	5i	OMe	Br	H	1.97± 0.07
	10	5j	Br	Cl	H	1.75± 0.07
	11	5k	Br	Br	H	1.50± 0.07
	12	5l	Br	NO2	H	1.59± 0.08
	13	5m	F	Br	H	Not tested
	14	5n	F	H	Bn	1.67± 0.09
	15	5o	F	Cl	H	2.00± 0.10
	16	5p	Cl	H	H	1.83± 0.09
	17	5q	Cl	H	Bn	1.63± 0.09
	18	5r	Cl	Cl	H	1.97± 0.11
	19	5s	Cl	Br	H	1.57± 0.10
	20	Acarbose				1.56± 0.07
	Mean value ± SD (n = 3).

Acknowledgments

The authors would like to thank Dr. R. Rachid for her help in collecting the molecular docking data.

Notes

The authors declare no competing financial interest.


Dr Hamza Sakhi
email: sakhi.hamza@gmail.com

MD Olivier Join-Lambert

PhD Anna Goujon

MD Thibault Culty

PhD Paul Loubet

PhD Julien Dang

MD Sylvain Drouot

PharmD Hubert De Bayser

MD Christophe Michaud

MD, PhD Louise Ghislain

MD Thomas Stehlé

MD Christophe Legendre

MD Dominique Joly

PhD Paul Meria

PhD Mohamad Zaidan
email: mohamad.zaidan@aphp.fr

Encrusted urinary tract infections due to Corynebacteria species

Keywords: Corynebacterium, Corynebacterium urealyticum, encrusted cystitis, encrusted pyelitis, encrusted urinary tract infections

Encrusted urinary tract infections are rare, characterized by a severe renal and overall prognosis in the absence of appropriate treatment. Topical urinary acidification and appropriate antibiotherapy are efficient but may be burdened by significant adverse events.

Introduction

Encrusted pyelitis and cystitis are peculiar disorders characterized by the calcification of the vesical, the pyelic and/or the ureteral walls. These calcifications are composed of struvite and calcium carbonate-apatite due to the presence of Corynebacterium urealyticum.

Methods

We reported the clinical features and outcomes of 17 patients with encrusted pyelitis (n=15) or encrusted cystitis (n=2). Diagnosis was based on CT scan and sonography including thickening and calcified lesions of the urinary tract.

Results

Main clinical presentation was suggestive of sub-acute urinary tract infection with fever and urological symptoms, mostly gross hematuria. Biological features were characterized by the presence of struvite crystals and alkaline urine. Acute kidney injury (AKI) was reported in 70.6% of cases. Predisposing factors were mostly due to urological background (82.4%) with a history of urological procedure (71%) and prior exposure to antibiotics (59%). All patients received appropriate antibiotherapy and 15 were treated with topical urinary acidification. A significant reduction of encrusted calcifications was observed in 88% of cases. Renal function improved in 71% of patients. Nevertheless, poor tolerance of the treatment and side effects were common, affecting 71% of patients with gram-negative bacilli urinary tract infections (53%) being the most frequent. At last follow-up, 4 patients (23.5%) progressed to end-stage renal disease and only one had a clinical relapse.

Conclusion

INTRODUCTION

Encrusted urinary tract infections are rare disorders characterized by the calcification of the bladder (cystitis), the ureteral, and/or the pyelic (pyelitis) walls. [START_REF] Meria | Encrusted pyelitis: an underdiagnosed condition?[END_REF] Similarly to infectious-related nephrolithiasis, these calcifications are usually composed of struvite (ammonium magnesium phosphate) and calcium carbonate-apatite crystals. [START_REF] Nadler | Corynebacterium urealyticum (CDC Group D2) associated with staghorn calculus: treatment by percutaneous debulking and chemolysis[END_REF][START_REF] Meria | Encrusted cystitis and pyelitis[END_REF][START_REF] Soriano | In vitro and in vivo study of stone formation by Corynebacterium group D2 (Corynebacterium urealyticum)[END_REF] Struvite stones represent about 10% of nephrolithiasis, and are due to urinary infections with ureasplitting bacteria. [START_REF] Bichler | Urinary infection stones[END_REF] The expression of urease activity allows the conversion of urea and water into ammonium and bicarbonate leading to urine alkalization (urinary pH is > 7.2).

Subsequent co-precipitation of ammonium with magnesium and phosphate then results in struvite crystals and urinary encrustations. [START_REF] Flannigan | Renal struvite stones-pathogenesis, microbiology and management strategies[END_REF] Urea-splitting agents include a wide range of pathogens depending on the level of urease activity. 7 Struvite stones are most often related to upper urinary tract infections due to Klebsiella sp and Proteus sp. Encrusted pyelitis and cystitis are almost invariably due to infections by Corynebacterium urealyticum, a gram positive slow-growing bacillus, characterized by a major urease activity that colonizes the skin. [START_REF] Soriano | Microbiological and clinical features of Corynebacterium urealyticum: urinary tract stones and genomics as the Rosetta Stone[END_REF][START_REF] Tauch | The lifestyle of Corynebacterium urealyticum derived from its complete genome sequence established by pyrosequencing[END_REF] C.urealyticum is identified in up to 30% of hospitalized patients, [START_REF] Soriano | Skin colonization by Corynebacterium groups D2 and JK in hospitalized patients[END_REF] and infection usually develops in a nosocomial context, particularly after exposure to antibiotics. [START_REF] López-Medrano | Urinary tract infection due to Corynebacterium urealyticum in kidney transplant recipients: an underdiagnosed etiology for obstructive uropathy and graft dysfunction-results of a prospective cohort study[END_REF] Encrusted pyelitis and cystitis were initially reported in renal transplant patients. [START_REF] Aguado | Encrusted pyelitis and cystitis by Corynebacterium urealyticum (CDC group D2): a new and threatening complication following renal transplant[END_REF] However, cases involving native kidneys were also reported. [START_REF] Hertig | Encrusted pyelitis of native kidneys[END_REF][START_REF] Giannakopoulos | Encrusted cystitis and pyelitis[END_REF] In the present retrospective study, we addressed the spectrum and outcome of patients with encrusted urinary tract infections, focusing more specifically on management and renal outcome.

METHODS

Study population

Patients were retrospectively identified through a French nationwide survey. The diagnosis of encrusted cystitis and pyelitis was based on imaging features including thickening and calcified lesions of the bladder and/or renal calyces and pelvis. Diagnosis of isolated encrusted cystitis was made by ultrasonography while diagnosis of encrusted pyelitis with or without cystitis was based on CT-scan. The identification of Corynebacteria in the urine was a major diagnostic criterion. The presence of gram positive bacilli, struvite crystals and/or alkaline urinary pH (≥ 8) was considered as an alternative criterion in the absence of Corynebacteria. [START_REF] Meria | Encrusted cystitis and pyelitis[END_REF]

Data Collection

Demographic, clinical and biological data were recorded retrospectively at diagnosis, during and at the end of follow-up for all patients. Systemic symptoms, signs of cystitis or pyelonephritis, serum creatinine level, urine culture, urine pH, and crystalluria were collected. AKI was defined according to clinical practice guidelines. [START_REF]Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group. KDIGO Clinical Practice Guideline for Acute Kidney Injury[END_REF] Estimated glomerular filtration rate (eGFR) was estimated using the simplified Modification of Diet in Renal Disease equation, and chronic kidney disease (CKD) was defined according to the Kidney Disease Outcomes Quality Initiative classification. [START_REF] Inker | KDOQI US commentary on the 2012 KDIGO clinical practice guideline for the evaluation and management of CKD[END_REF] Patients were evaluated by ultrasonography and/or CT-scan at diagnosis and during follow-up.

Treatment modalities and outcome

Treatment modalities included surgical resection of encrusted calcifications, use of topical urine acidification, and nature of the antibiotics at diagnosis and in case of relapse.

Treatment efficacy was based on clinical assessment, kidney function improvement, imaging studies, and urine culture. The regression of the calcifications was evaluated by CT-scan (encrusted pyelitis) and/or ultrasonography (encrusted cystitis) performed at different time points during follow-up.

Statistical analysis

Descriptive statistics were used to summarize the data using STATA 11.2 software (StataCorp). Continuous variables were expressed using median values with interquartile range, as appropriate for non-parametric data. Categorical variables were expressed as counts and percentages for categorical variables.

RESULTS

Demographic characteristics

Over the last two decades, 17 patients with encrusted pyelitis and/or cystitis, including 7 males and 10 females, were identified from seven different French Hospitals. Two patients had isolated encrusted cystitis based on ultrasonography findings, and 15 displayed encrusted pyelitis, associated with encrusted cystitis in three, as assessed by CT-scan. Four patients had bilateral encrusted pyelitis, and four had encrusted pyelitis on the renal allograft (Table 1 and Figure 1).

Past medical history included type 2 diabetes mellitus (23.5%), untreated HIV infection (one patient), and autoimmune hepatitis in 4 (one patient). Ten (58.9%) patients had preexisting CKD with a median eGFR at baseline of 39 [30.5-71.5] ml/min/1.73m 2 (Table 2 and Supplementary Table S1), but none had been previously referred to a Nephrologist. Four patients had undergone a renal transplantation. Overall, 14 patients (82.4%) had a "urological background", including urological cancer (n=4), former neurogenic bladder (n=3), urological complication after renal transplantation (n=2) which were lymphocele and ureteral anastomosis necrosis, extrinsic chronic ureteral obstruction (n=2), and other causes (n=3). Interestingly, 12 patients (70.6 %) had undergone some kind of urological procedure, including continent skin diversion or cutaneous ureterostomy in five patients, endoscopic procedures in three cases, and post-transplantation re-intervention in two renal transplant patients. Two patients had non urological cancer with non-Hodgkin B-cell lymphoma and colonic cancer with peritoneal carcinomatosis. Finally, 10 patients (59%) had been exposed to antibiotics prior to diagnosis, most often (n=9) due to urinary tract infections with gramnegative bacilli (Table 2 and Supplementary table S1).

Diagnosis

Presenting features are summarized in Table 1 and detailed in Supplementary Table S1. The delay between initial symptoms and diagnosis was variable with a median of 2 months IQR [1-6]. Median age at diagnosis was 67.5 years IQR[56-68]. Eleven patients (64.7%) had systemic manifestations including weight loss and fever. Urological signs were reported in more than 70% of cases, including dysuria in two patients with encrusted cystitis. Flank pain was observed in three cases (20%). Hematuria was present in all but one patient, with gross hematuria in 64.7% of cases. Inflammatory syndrome was reported in 10 patients, all with encrusted pyelitis. Corynebacterium was identified in 14 patients, but initially missed in 4 cases. C. urealyticum was involved in 13 cases and C. striatum in one patient. Three patients had a negative urine culture on standard media, albeit gram positive bacilli were initially detected on direct urine examination. Alkali urinary pH ≥ 8 was a constant finding.

Crystalluria showed struvite crystals in all cases. Acute kidney injury was observed in 12 (70.6%) patients, all with encrusted pyelitis, and dilatation of the collecting duct system was observed in 11 patients (64.7%). At diagnosis, median serum creatinine was 315 µmol/L IQR[209. 8-395].

Treatment and outcome

All patients were treated with appropriate antibiotherapy for a median period of 9 weeks IQR [4.5-12.5]. Intra-venous glycopeptides (Vancomycin or Teicoplanin) were administered in 15 patients, and Linezolid in one.

Conservative therapy consisted in topical urine acidification using Thomas' solution (27 g each of sodium gluconate, citric acid and malic acid, in 1 L of distilled water) or Suby's solution (citric acid 32.3 g, sodium carbonate 4.4 g, magnesium oxide 3.8 g, distilled water 1 L). Recommended parameters of irrigation were a flow rate of 10-20 ml/h increasing to 50-100 ml/h with an intrarenal or pelvic pressure < 25 cmH2O, but treatment was tailored under patient's doctor supervision. Treatment was administered through a percutaneous nephrostomy placed at the initiation of the treatment. All the patients, except two, received topical acidification, which was administered for a median time of 3 weeks IQR [2-6]. When available, urine pH acidification (urinary pH < 4) was obtained in all treated patients. Acetohydroxamic acid was given for one month after topical acidification in two patients, but was well-tolerated in only one case (Supplementary Table S2). Among the two patients who did not receive acidification therapy, one underwent a surgical resection of the encrusted material and the other was given Linezolid for only two weeks. The availability of the topical acidification at the time of management in these two cases was unknown.

Another patient with encrusted pyelitis and struvite stones also underwent a pyelotomy to extract the stones (Supplementary Table S2).

All the patients had a favorable clinical and biological response, including the two patients with isolated encrusted cystitis (Supplementary table S3). Nevertheless, poor clinical tolerance of the topical acidification was reported in four (23.5%) patients due to pain and malnutrition. Metabolic acidosis was observed in five (29.4%) patients (Table 3), whereas infections were observed in 11 (73.3%) patients. Nine patients developed urinary tract infection with a septic shock in two, one had a cutaneous fistula near the nephrostomy, and seven displayed urinary candidosis in the course of topical acidification. Urine cultures finally returned negative for Corynebacteria in all cases. Complete or partial regression of encrusted calcifications was observed in 15 patients, including 14 who were treated by acidification (Figure 2). Persistent calcifications were described in 10 (67%) patients treated by acidification. The non-responder patient was actually treated for only five days, and before kidney function worsened, leading to treatment discontinuation (Table 3). At the end of the treatment, renal function improved in almost all cases, whereas it remains stable in 5 patients, 3 with initially normal baseline value, and 2 with CKD.

Nevertheless, return to baseline value was observed in only 6 patients (35.3%), and median serum creatinine was 165 µmol/L IQR[105-210] (Table 3).

Only one of the treated patients with acidification presented a new symptomatic flare. The patient had been treated less than one week and rapidly progressed to end-stage renal disease requiring hemodialysis. He finally died from a septic shock. One patient presented a progression of the encrustations one month after treatment discontinuation, and finally underwent a surgical resection. Two patients had recurrence of struvite crystals in the urine, including one with a positive urine culture for C. urealyticum, but none displayed de novo renal calcifications or symptomatic encrusted pyelitis. Finally, two patients presented new struvite stones one of which resulted in obstructive AKI (Table 3).

At last follow-up, and after a median follow-up time of 15 [10-30] months, median eGFR was 34 mL/min/1.73m 2 IQR [16-70], eight (47%) patients had severe CKD, and four (23.5%) had reached end-stage renal disease. Four patients died (23.5%) because of urothelial carcinoma (n=1), progressive lymphoma (n=1), septic shock (n=2) (Table 3 and Supplementary table S4).

DISCUSSION

The present study illustrates the diagnostic challenge, management modalities, and renal outcome of patients with encrusted pyelitis and/or cystitis. So far, only a few series have characterized the spectrum of encrusted urinary tract infections due to Corynebacteria species (Supplementary table S5). [START_REF] López-Medrano | Urinary tract infection due to Corynebacterium urealyticum in kidney transplant recipients: an underdiagnosed etiology for obstructive uropathy and graft dysfunction-results of a prospective cohort study[END_REF][START_REF] Aguado | Encrusted pyelitis and cystitis by Corynebacterium urealyticum (CDC group D2): a new and threatening complication following renal transplant[END_REF][START_REF] Hertig | Encrusted pyelitis of native kidneys[END_REF][START_REF] Giannakopoulos | Encrusted cystitis and pyelitis[END_REF][START_REF] Aguado | Bacteriuria with a multiply resistant species of Corynebacterium (Corynebacterium group D2): an unnoticed cause of urinary tract infection[END_REF][START_REF] Soriano | Urinary tract infection caused by Corynebacterium group D2: report of 82 cases and review[END_REF][START_REF] Meria | The conservative management of corynebacterium group D2 encrusted pyelitis[END_REF][START_REF] Moreno Arcas | Encrusted pyelitis. Lithiasic disease with infectious etiology[END_REF][START_REF] Audard | Fatal septic shock caused by Corynebacterium D2[END_REF][START_REF] Vázquez | Corynebacterium urealyticum in renal trasplantation. CT and sonography imaging characteristics of encrusted cistitis and pielitis[END_REF][START_REF] Khallouk | Conservative management of Corynebacterium urealyticum encrusted cystitis[END_REF][START_REF] Cappuccino | Nephrolithiasis by Corynebacterium urealyticum infection: literature review and case report[END_REF][START_REF] Sánchez-Martín | Corinebacterium urealyticum: increased incidence of infection and encrusted uropathy[END_REF][START_REF] Saljoghi | Encrusted Uretero-pyelitis: Case Report[END_REF] Despite suggestive imaging features, the diagnosis of encrusted urinary tract infections is challenging given the need for specific culture media to identify Corynebacteria species and the important delay between first symptoms and established diagnosis. [START_REF] Sakhi | The Case | Acute kidney injury, flank pain, and kidney calcifications in an 80-year-old woman[END_REF] Of note, most patients present with obstructive renal failure, and most have preexisting chronic kidney disease at the time of diagnosis, suggesting a chronic unrecognized process. Although urinary tract obstruction is likely the main cause of chronic kidney disease, local inflammation due to encrustations, and pyelonephritis may also contribute to renal function deterioration. Recent studies indicate an increase in the incidence of Corynebacterium urinary infections, [START_REF] Sánchez-Martín | Corinebacterium urealyticum: increased incidence of infection and encrusted uropathy[END_REF] suggesting a potential underestimation of encrusted pyelitis and cystitis. Corynebacterium urinary colonization is observed in less than 1% of patients, mostly in those who had been hospitalized and had previous antibiotic treatment and urological gestures, as observed in our study. [START_REF] Nebreda-Mayoral | Incidence and characteristics of urinary tract infections caused by Corynebacterium urealyticum (Corynebacterium group D2)[END_REF] At-risk patients include immunocompromised subjects, [START_REF] Aguado | Encrusted pyelitis and cystitis by Corynebacterium urealyticum (CDC group D2): a new and threatening complication following renal transplant[END_REF][START_REF] Soriano | Urinary tract infection caused by Corynebacterium group D2: report of 82 cases and review[END_REF] and those with a heavy urological background, including malignancy and other urological disorders.

Urological gestures associated with a mucosal breach, particularly transurethral prostate interventions or percutaneous stone surgery, may favor bacterial inoculation within the urinary tract leading to infection in 1-5% of cases. [START_REF] Grabe | Preoperative assessment of the patient and risk factors for infectious complications and tentative classification of surgical field contamination of urological procedures[END_REF] Thus, Corynebacterium, which usually colonizes the skin, 10 may be inoculated within the urinary tract during such procedures.

Given the risk of incomplete recovery of renal function after urine diversion and septic shock with pyelocaliceal microabcesses, [START_REF] Meria | The conservative management of corynebacterium group D2 encrusted pyelitis[END_REF] an earlier diagnosis would improve renal and overall outcome. The detection of alkali urine (especially pH ≥ 8), and struvite crystals in at-risk patients may thus be helpful and cost-effective to make an early diagnosis of Corynebacteria infections. CT-scan may also show typical encrusted calcifications. Differential diagnosis includes other infectious diseases such as schistosomiasis and tuberculosis, cytotoxic chemotherapy, and underlying neoplastic lesions. Nevertheless, it should be stressed that the presence of linear calcifications along the pyelic wall is strongly evocative [START_REF] Vázquez | Corynebacterium urealyticum in renal trasplantation. CT and sonography imaging characteristics of encrusted cistitis and pielitis[END_REF] , and the presence of Corynebacteria in urine culture make the diagnosis. However because Corynebacteria are slow-growing germs, and the gram positive rods initially identified on direct examination may be missed by standard urine culture, the diagnosis should not be ruled out on the basis of negative standard cultures, and the bacteriologist should be aware of the suspicion of Corynebacterium infection to adapt the medium culture and the diagnostic procedure. [START_REF] Salem | Corynebacterium urealyticum: a comprehensive review of an understated organism[END_REF] The broader use of modern techniques, such as MaldiTOF, will certainly allow a more accurate recognition of such species [START_REF] Salem | Corynebacterium urealyticum: a comprehensive review of an understated organism[END_REF] and should be carried out in these cases.

Although Corynebacterium seem to be less virulent than other urinary tract bacteria, [START_REF] Soriano | In vitro and in vivo study of stone formation by Corynebacterium group D2 (Corynebacterium urealyticum)[END_REF] the germs are resistant to most of the commonly used antibiotics for urinary tract infections. [START_REF] López-Medrano | Urinary tract infection due to Corynebacterium urealyticum in kidney transplant recipients: an underdiagnosed etiology for obstructive uropathy and graft dysfunction-results of a prospective cohort study[END_REF] Because ineffective treatment is associated with poor outcome, [START_REF] Aguado | Encrusted pyelitis and cystitis by Corynebacterium urealyticum (CDC group D2): a new and threatening complication following renal transplant[END_REF] it should be underscored that glycopeptides with vancomycin represent the most commonly used antibiotics for the treatment of Corynebacteria. Given the risk of nephrotoxicity associated with glycopetids, particularly when prescribed for a prolonged period, the use of linezolid, as reported in one of our patients, may represent an interesting alternative, but require further investigations.

The present series also illustrates the different options to treat encrusted urinary tract infections due to Corynebacteria species. The management was historically based on the surgical resection of the encrusted material, which is burdened by a significant risk of hemorrhage. [START_REF] Meria | Encrusted pyelitis: an underdiagnosed condition?[END_REF][START_REF] Aguado | Encrusted pyelitis and cystitis by Corynebacterium urealyticum (CDC group D2): a new and threatening complication following renal transplant[END_REF][START_REF] Hertig | Encrusted pyelitis of native kidneys[END_REF] Oral acetohydroxamic acid was also proposed as a complementary treatment, albeit its use was limited because of a poor tolerance [START_REF] Griffith | A randomized trial of acetohydroxamic acid for the treatment and prevention of infection-induced urinary stones in spinal cord injury patients[END_REF] and important delay before significant improvement. [START_REF] Meria | Encrusted pyelitis: an underdiagnosed condition?[END_REF] As suggested by our study, the combination of topical urine acidification and antibiotherapy represent a good alternative to control the infection and improve immediate renal function.

Nevertheless, a prolonged hospitalization is most often necessary, exposing to iatrogenic complications in elderly and frail patients. Acidification may be poorly tolerated and may require reducing the flow rate of instillation. Patients are also exposed to a high risk of de novo urinary tract infections and candiduria, which may convert into septic shock.

Altogether, such adverse events may prompt the interruption of the treatment before the initially defined duration of 4 weeks. Surprisingly, the rate of recurrence seems to be very low, despite the potential persistence of calcifications in some cases. Indeed, complete regression of encrustations was observed in only 7 (41.2%) patients. The eradication of calcifications at any cost should thus be discussed in the light of treatment tolerance and overall prognosis.

In conclusion, encrusted pyelitis and cystitis affect frail patients with a long-standing urological history and comorbid conditions. Early diagnosis is mandatory to initiate appropriate treatment. The therapeutic strategy is unique compared to other urinary tract infections. The combination of topical urine acidification and antibiotherapy represents an alternative to surgery. Nevertheless, treatment tolerance and potential complications should also be considered. Further studies would help to better clarify treatment modalities, including the duration of urine acidification and antibiotherapy. A better monitoring of atrisk patients would also improve renal and overall outcomes.

Figure 1 .

 1 Figure 1. Abdominal CT-scan findings at diagnosis showing bilateral encrusted pyelitis of

Figure 2 .

 2 Figure 2. Comparison of CT-Scan findings before (left panels) and after (right panels)

Table 1 . Patients' characteristics at diagnosis.

 1

	All Patients
	(n =17)

ACKNOWLEDGMENTS

We would to thank Dr Eugénie Koumakis for her helpful editing advice.

DISCLOSURES

The authors have nothing to disclose

CONCLUSION:

Encrusted urinary tract infections (UTI) due to Corynebacteria species Encrusted urinary tract infections primarily affect patients with heavy urological background.s Topical acidification is an effective treatment.