Floriane Gibault

Prof Fabrice P Bailly

Mathieu Corvaisier

Mathilde Coevoet

Dr Guillemette Huet

Patricia Melnyk

Philippe Cotelle
email: philippe.cotelle@univ-lille1.fr

Matthieu Corvaisier

Dr Ms F Gibault

ORCID Melnyk

Prof P Cotelle

Dr M Corvaisier

Molecular Features of the YAP Inhibitor Verteporfin: Synthesis of Hexasubstituted Dipyrrins as Potential Inhibitors of YAP/TAZ, the Downstream Effectors of the Hippo Pathway

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

Introduction

YAP(TAZ), the downstream effectors of the Hippo pathway, interact with TEAD to regulate stem cell proliferation, tissue growth and organ size [4]. Oncogenic YAP/TAZ are overexpressed in many cancers [5][6][7][8] causing overgrowth phenotypes and metastasis. Liu-Chittenden and colleagues [9] were the first to demonstrate to feasibility to disrupt YAP/TEAD complex as a pertinent therapeutic strategy in cancer therapy and identified three members of the porphyrin family, namely VP, protoporphyrin IX (PPIX) and hematoporphyrin (HP), as inhibitors of YAP/TEAD-dependent transcription. VP was found to be the most effective compound to inhibit the complex between YAP and TEAD2 and was used VP for in vivo validation. Indeed, VP treatment efficiently suppressed liver overgrowth induced by YAP overexpression in mice models. Verteporfin, is a photosensitizer initially designed for cancer therapy, which presents high cytotoxicity for tumor cells only under light stimulation [1]. Rapidly VP was repositioned in the field of ophthalmology and photodynamic therapy for the treatment of age-related macular degeneration [2] (VP was FDA-approved in 2000 and marketed by Novartis under the trade name: visudyne ® ).

VP was recently reported to exert different biological properties without light activation [3 and references cited therein]. However, the use of VP as an inhibitor of YAP-induced overgrowth presents several drawbacks: (i) it is a photosensitizer and therefore is highly phototoxic; (ii) it has been identified as an inhibitor of autophagy via a low-level production of singlet oxygen [10]; (iii) it induces the oligomerization of up to 250 proteins including 80 mitochondrial proteins [11]; (iv) it has also been reported that VP up-regulates 14-3-3 (a protein responsible for the cytoplasmic retention of YAP) [12]. So, several questions therefore arise on how VP interacts with YAP. In this paper, we first focused our attention on the structural features that confer to VP its ability to control the YAP/TEAD interaction and decrease the induction of the main target genes. For this purpose, we first compared the biological properties of VP, PPIX and its dimethyl ester (Figure 1) in MDA-MB-231 breast cancer cell line. In a second round, we synthesized the hexasubstituted dipyrrin 1 corresponding to the western part of PPIX dimethyl ester (PPIX-DME). We report herein the first convergent synthesis of this dipyrrin derivative starting from the known 2-formylpyrrole 2 (Scheme 1) [13]. Additionally, we synthesized two other symmetric hexasubstituted dipyrrins 18 and 19 and report their cellular activities on the YAP(TAZ)/TEAD complexes. Chemistry of dipyrrins [14] is directly connected to the synthesis of porphyrins and the access to dipyrrinato complexes possessing remarkable optical properties, however nothing is known about the biological properties of this class of molecules. The biological evaluation has been made in two distinct steps: firstly, as a reference, we measured the effect of the inhibition of expression YAP and/or TAZ by siRNA in MDA-MB-231 cells on the expression of some relevant target genes (Cyr61, CTGF and AXL). In a second time, we measured the biological effect of VP and related protoporphyrins and dipyrrins on by using two different methods: 1-measurement of TEAD transcriptional activity using 8xGTIIC-luciferase reporter assay [15], 2evaluation of the protein levels of Cyr61, CTGF and AXL.

Results and Discussion

Chemistry

Our first strategy to obtain the dipyrrin 1 (Scheme 1) was inspired from the total synthesis of hematoporphyrin published by Martin et al. [16].

Scheme 1. (a) HBr 33% in AcOH (8 eq.), MeOH, rt, 1 h, 89%; (b) CeCl3.7H2O (1 eq.), NaBH4 (2 eq.), dioxane/propan-2-ol, 100 °C, 20 h, 75%.

The condensation of the two pyrrolo units 2 [17] and 3 [18] using HBr in acetic acid gave the dipyrrin 4 with high yield (89%) but the reduction of the acetyl group in presence of NaBH4 [15] alone or with CeCl3 [19] yielded the dipyrromethane 5' instead of the expected alcohol 5.

In a second strategy (Scheme 2), we planned to obtain the target compound 1 through the similar condensation of the pyrrolo units 2 and 8. Firstly the vinylpyrrole 8 was obtained via the Stille vinylation [20] of the iodopyrrole 6. Unfortunately, vinylpyrrole 8 was found to be unstable under the acidic conditions required for the dipyrrin synthesis.

The third strategy (Scheme 3) consisted in the ultimate Stille vinylation of the iododipyrrin 10 which could be issued from the condensation of the formylpyrrole 2 and the iodopyrrole 9 [21,22]. Unfortunately, the condensation of the two pyrrolo units 2 and 7 yielded the unexpected deiododipyrrin 11. Scheme 2. (a) Vinyltributyltin (2 eq.), Pd(PPh3)2Cl2 (0.05 eq.), toluene, 110 °C, 2 h, 48%; (b) LiOH (8 eq.), EtOH/H2O, 90 °C, 8 h, 32%; (c) HBr 33% in AcOH (8 eq.), MeOH, rt, 1h. Scheme 3. (a) HBr 33% in AcOH (8 eq.), MeOH, rt, 1 h, 75%; (b) Vinyltributyltin (2 eq.), Pd(PPh3)2Cl2 (0.05 eq.), toluene, 110 °C, 2 h. Our fourth strategy (Scheme 4) was based on the condensation of the formylpyrrole 2 and the iodoethylpyrrole 13 (synthesized from the known hydroxyethylpyrrole 12 [23]). Base-induced dehydroiodation of the yielded iodoethyldipyrrin 14 gave successfully the target dipyrrin 1 with a moderate yield (47%). Symmetric tetramethyldi(3-methoxy-3-oxopropyl)dipyrrin 18 and di(2-iodoethyl)tetramethyldipyrrin 17 were also synthesized using the same strategy than for the synthesis of 14 (Scheme 5). 18 was obtained with the same yield as recently reported by Lund and Thompson, who synthesized symmetric dipyrrin hydrobromides from 2-formylpyrroles [24]. The double dehydroiodation of di(2-iodoethyl)tetramethyldipyrrin 17 using DBU gave tetramethyldivinyldipyrrin 19 with a modest 36% yield. (Scheme 5). Scheme 4. (a) PPh3 (2 eq.), Imidazole (2 eq.), I2 (2 eq.), DCM, rt, 20 h, 83%; (b) i) TFA (44 eq.), rt, 30min ii) HBr 33% in AcOH (8 eq.), MeOH, rt, 1 h, 63%; (c) DBU (2.5 eq.), DCM, rt, 20 h, 47%. Scheme 5. (a) HBr 33% in AcOH (8 eq.), MeOH, rt, 1 h, 65% for 17 and 68% for 18; (b) DBU (4 eq.), DCM, rt, 20 h, 36%. Biology YAP and TAZ are transcriptional coactivators that function as effectors of the Hippo pathway. To maintain Hippo pathway homeostasis, TAZ accumulation can be negatively regulated by YAP abundance [25]. More recently, Moroishi et al. [26] reported a reciprocal negative regulation between these two coactivators in MCF10A cells, through TEAD-dependent transcription. Submitted to oxidative stress, differences were found between YAP and TAZ. TAZ was less sensitive to oxidative stress than YAP [27]. For these reasons, we focused our attention on the effects of VP, PPIX and dipyrrins on the two Hippo pathway effectors (YAP and TAZ) and on the YAP(TAZ)/TEAD gene targets (Axl, Cyr61 and CTGF). Depending on the cell lines, the Hippo pathway effectors can be differently expressed. This has been clearly shown on different colon cancer cell lines of us [15] that HT-29 cells expressed mainly YAP whereas HCT-116 or RKO cells expressed both YAP and TAZ. Moreover, the relative TEAD activity is also cell line-dependent. We selected MDA-MB-231 breast cancer cell line because it similarly expressed YAP and TAZ co-activators, presented a high TEAD transcriptional activity and yielded a high expression of gene targets (Axl, Cyr61 and CTGF).

We first analyzed the expression of mRNA (by RT-qPCR) and proteins (Western Blot) of YAP, TAZ, AXL, Cyr61 and CTGF in the presence of siYAP, siTAZ or both (Si Y-T). Knockdown of either YAP or TAZ decreased the expression of gene targets (Figure 2) but did not show any selectivity of one co-activator. Dual YAP/TAZ knockdown significantly decreased the protein levels of Cyr61, CTGF and Axl. As previously reported [10], VP is a photoactivable molecule and exposure to light must always be stringently avoided during cell treatment. However, even in the absence of light, we observed a decrease in YAP and TAZ levels in western blots. While the RNA expression of YAP is not affected (Figure 2A), its protein production decreases by 74%. This already reported proteotoxicity [11] was also observed TAZ (Figure 2B) even if TAZ could be considered to be less sensitive to oxidative stress conditions [27]. The reduction of YAP expression was previously reported in hepatocellular carcinoma cells treated by a 1,2dithiole-3-thione derivative [28], known to generate in vitro superoxide anion [29] and hydrogen peroxide [30]. VP (10 µM) efficiently decreased the mRNA expression of the downstream gene targets (Figure 3A). At the protein levels, AXL, and CTGF appeared mostly affected (Figure 3B). CTGF was already found to be more sensitive than Cyr61 in ovarian cancer cells treated by VP [31]. Inversely, VP affects more efficiently Cyr61 than CTGF in retinoblastoma cells [32]. It may be suggested that VP could also affect Axl, and CTGF levels through its proteotoxic properties. Using the TEAD luciferase assay, we measured the ability of VP and related compounds to inhibit the TEAD activity (Figure 3). We found that VP at a 10 µM concentration inhibited this activity by 50%. Tested protoporphyrins and dipyrrins did not show significant effect except dipyrrin 19. Conversely to the results obtained in HEK293 cells [9], we did not observe a significant decrease of the TEAD luciferase activity following PPIX (or PPIX-DME) treatment. We then analyzed the effect of PPIX, PPIX-DME and dipyrrin derivatives on the expression of YAP, TAZ and their target genes. Conversely to VP, PPIX, PPIX-DME did not induce a dramatic decrease in YAP and TAZ (Figure 4). This may be related to the expected weaker proteotoxicity of both compounds, which possess a significantly lowest singlet oxygen quantum yield than VP [3]. Dipyrrin 1, the western part of protoporphyrin IX dimethyl ester (PPIX-DME) did not show any significant changes. But interestingly, symmetric dipyrrin 19 containing two vinyl groups significantly decreased the level of YAP whereas TAZ level remained at a similar level. This result is well connected to the fact that dipyrrin 19 is the only dipyrrin derivative able to induce a decrease in TEAD transcriptional activity in the TEAD luciferase assay. This derivative also resulted in a decrease in AXL level.

Conclusion

We successfully synthesized the dipyrrin western part of protoporphyrin IX, dipyrrin 1, from the known tert-butyl 4-(2hydroxyethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate. The sensitive vinyl group was created at the final stage by a dehydroiodation reaction. We submitted dipyrrin 1 to Diels-Alder reaction conditions but failed to obtain the western part of VP due to the unstability of 1 to prolonged heat at toluene boiling point. This dipyrrin 1 could be useful to study other biological properties. Using the same strategy,the divinyl-dipyrrin 19 was synthesized. Independently to its mechanism of action, it has been wellestablished in several animal models that VP administration allows to control efficiently the organ growth via the Hippo pathway. VP has been firstly reported as a YAP/TEAD complex inhibitor whereas its ability to oligomerize several cellular proteins could correspond to a low protein selectivity [11]. We observed that VP significantly inhibits the TEAD luciferase activity in agreement to previously reported data [9]. However, we also observed that VP decreases the expression of YAP and TAZ, and such effect must result in a decrease in YAP/TEAD cotranscriptional activity. It has been reported that VP can induce the formation of high molecular weight oligomers through a crosslinking of proteins potentiated by oxidative stress [10]. The decrease in YAP and TAZ here observed after VP treatment might result from oligomerization of these proteins. In addition, we identified here a compound, dipyrrin 19, able to decrease YAP level and TEAD transcriptional activity. It may be suggested that such effect of dipyrrin 19 can be due the addition of two vinyl groups to the dipyrrin skeleton resulting in proteotoxicity. Altogether, these data suggest that in function of their chemical structure, porphyrin-and dipyrrin-related derivatives can directly target YAP and/or TAZ proteins and inhibits TEAD transcriptional activity. VP, which is mainly localized in mitochondria, might also affected the YAP and TAZ expression via the mitochondrial fusion/Hippo pathway cross-talking [33]. This link between Hippo pathway and mitochondria fusion could be of significance to cancer biology [34].

Experimental Section

Biology Human breast cancer cell line MDA-MB-231 cell line was purchased from the American Type Culture Collection and cultivated in DMEM media containing 10% of heat inactivated FBS, L-glutamine and penicillin/streptomycin. Western blotting Total extracts of cells were obtained with a RIPA based buffer containing protease and phosphatase inhibitors (Roche). Western blot were carried out using 20 μg of protein lysates with the NuPage Electrophoresis and Iblot transfer systems (Life Technologies). β-Actin was used as loading control for total extracts, TEAD transcriptional activity Cells at 50% confluence were transfected with the TEAD luciferase reporter plasmid 8XGTIIC-Luciferase (Addgene reference 34615) vs a control luciferase plasmid. After 48 hours post transfection, cells are lysed in Reporter Lysis Buffer (Promega) and luciferase activity was measured on the Mithras LB940 plate reader and normalized to protein concentration. mRNA expression mRNAs were extracted from cultured cells or human tumors with the NucleoSpin kit (Macherey-Nagel). Retrotranscription was done on 1 μg of mRNA accordingly to the Advantage RT-for-PCR Kit protocol (Clontech). PCR was performed using SsoFastTM Evagreen Supermix kit following the manufacturer's protocol using the CFX96 real time PCR system (Bio-Rad). To monitor any change in mRNA expression, we used the ΔΔCt method between one condition and a control condition after normalization with the housekeeper gene RPLP0. Each sample was done in triplicate.

Chemistry

Chemicals and solvents were purchased from various suppliers (Sigma-Aldrich, Alfa Aesar, Fisher, VWR) and used without purification. The reaction monitoring was performed by thin layer chromatography (TLC) on Macherey-Nagel Alugram® Sil 60/UV254 (thickness 0.2 mm). TLC were revealed by UV (λ = 254 nm) and/or the appropriate stain. Purification of the compounds was carried out by column chromatography (flash or manual). Manual chromatography was performed using Macherey-Nagel silica gel (0.04-0.063 mm of particule size). Flash chromatography was performed on a Reveleris® Flash Chromatography System using Macherey-Nagel Chromabond flash RS columns. NMR spectra were recorded on a Bruker DRX 300 spectrometer (operating at 300 MHz for 1 H and 75 MHz for 13 C). Chemical shifts are expressed in ppm relative to tetramethylsilane (TMS) or to residual proton signal in deuterated solvents. Chemical shifts are reported as position (δ in ppm), multiplicity (s = singulet, d = doublet, t = triplet, q = quartet, m = massif, br = broad), coupling constant (J in Hz), relative integral and assignment. The attributions of protons and carbons were achieved by analysis of 1D and 2D experiments ( 1 H, 13 C, COSY, HSQC and HMBC). LC-MS were performed on a Varian triple quadrupole 1200W mass spectrometer equipped with a non-polar C18 TSK-gel Super ODS (4.6 x 50 mm) column, using electrospray ionisation and a UV detector (diode array). Elution was performed at a flow rate of 2 mL/min with water-formic acid (pH = 3.8) as eluent A and ACN-formic acid (pH = 3.8) as eluent B, employing a 0.25 min plateau with 0 % B and a linear gradient from 0 % B to 98 % B in 3.25 min, followed by a 0.5 min plateau with 98 % B. Then, column re-equilibration was performed for 1 min. The injection duty cycle was 5 min, taking into account the column equilibration time. The melting point analyses were performed on Barnstead Electrothermel Melting Point Series IA9200.

The UV-vis spectra were recorded using a NanoDrop spectrometer (ThermoFisherScientific). Characteristic peaks are given in nm and classified according to their relative intensity (s strong, m medium, w weak).

(Z)-4-Acetyl-2-((4-(3-methoxy-3-oxopropyl)-3,5-dimethyl-1H-pyrrol-2yl)methylene)-3,5-dimethyl-2H-pyrrolium bromide (4)

A solution of 33% HBr in AcOH (2.7 eq., 497 mg, 0.3 mL, 2.9 mmol) was added dropwise to a solution of methyl 3-(5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)propanoate [17] (1 eq., 228 mg, 1.1 mmol) and 3-acetyl-2,4dimethylpyrrole [18] (1 eq., 150 mg, 1.1 mmol) in MeOH (3 mL) at room temperature under nitrogen atmosphere. The mixture was stirred at room temperature for 4 h and the product was collected by filtration. The residue was used without further purification in the next step. The title compound (400 mg, 0.97 mmol, 89%) was obtained as an orange solid. 1

Methyl 3-(5-((4-acetyl-3,5-dimethyl-1H-pyrrol-2-yl)methyl)-2,4-dimethyl-1H-pyrrol-3-yl)propanoate (5')

Dipyrrin 4 (1 eq., 0.19 g, 0.46 mmol) was dissolved in a mixture of DCM (28 mL) and MeOH (7 mL). Sodium borohydride (5.0 eq., 88 mg, 2.3 mmol) was added in one portion and the mixture was stirred at room temperature for 2 h. Then the solution was washed with saturated aqueous bicarbonate solution and brine, dried over sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography (DCM/MeOH, 95:5 (v/v), NH3g sat).The title compound (115 mg, 0.35 mmol, 75%) was obtained as an orange solid. 1

Ethyl 4-iodo-3,5-dimethyl-1H-pyrrole-2-carboxylate (6)

To a solution of N-chlorosuccinimide (1.2 eq., 0.96 g, 7.2 mmol) in DMF (20 mL) was added NaI (1.2 eq., 1.08 g, 7.2 mmol) in small portions. The resulting brown solution was stirred at room temperature for 1 h before the slow addition at 0 °C of ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate (1 eq., 1 g, 6.0 mmol) in DMF (20 mL). The reaction was stirred at room temperature for 1 h. Aqueous 10% sodium thiosulfate solution (20 mL) was added and the mixture was stirred for 1 h. The solid was collected by filtration. The residue was used without further purification in the next step. The title compound (1.61 g, 5.5 mmol, 92%) was obtained as a white solid. 1 H NMR (300 MHz), δ (ppm, CDCl3): 1.36 (t, J = 1.7 Hz, 3 H, CH3), 2.29 (s, 3 H, CH3), 2.29 (s, 3 H, CH3), 4.33 (q, J = 1.7 Hz, 2 H, CH2), 8.96 (s, 1 H, NH). 13 C NMR (75 MHz), δ (ppm, CDCl3): 14.3 (CH3), 14.5 (CH3), 14.

Ethyl 3,5-dimethyl-4-vinyl-1H-pyrrole-2-carboxylate (7)

A mixture of ethyl 4-iodo-3,5-dimethyl-1H-pyrrole-2-carboxylate (1 eq., 1.00 g, 3.4 mmol) , vinyltributyltin (2 eq., 2.16 g, 2.0 mL, 6.8 mmol) and bis(triphenylphosphine)palladium(II) dichloride (5 %, 0.12 g, 0.17 mmol) in toluene (50 mL) was refluxed for 2 h under a nitrogen atmosphere. During the course of the reaction, the color changed from yellow to black as Pd° was formed. The reaction was cooled to room temperature and DCM (50 mL) was added. The resulting solution was washed with satured aqueous NaCl (50 mL) and water (50 mL). The organic phase was separated, dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash chromatography (DCM/MeOH, 95:5 (v/v)). The title compound (317 mg, 1.6 mmol, 48%) was obtained as a dark brown solid. 1 H NMR (300 MHz), δ (ppm, CDCl3): 1.36 (t, J = 7.1 Hz, 3 H, CH3), 2.32 (s, 3 H, CH3), 2.39 (s, 3 H, CH3), 4.31 (q, J = 7.1 Hz, 2 H, CH2), 5.17 (dd, J = 1.7 Hz, Jcis = 11.6 Hz, 1 H, CH2), 5.28 (dd, J = 1.7 Hz, Jtrans = 17.9 Hz, 1 H, CH2), 6.59 (dd, Jtrans = 17.9 Hz, Jcis = 11.6 Hz, 1 H, CH), 8.62 (s, 1 H, NH).

3,5-Dimethyl-4-vinyl-1H-pyrrole-2-carboxylic acid (8)

Ethyl 4-ethenyl-3,5-dimethyl-1H-pyrrole-2-carboxylate (1 eq., 158 mg, 0.82 mmol) and LiOH (8 eq., 156 mg, 6.5 mmol) were dissolved in EtOH (2 mL) and H2O (2 mL) . The solution was heated at 90 °C for 1 h. The solvent was evaporated under vacuum. The residue was taken up in EtOAc (5 mL) and water (5 mL). The aqueous layer was acidified until pH 5.0 with H3BO3 (pH 5.1) or a few drops of concentrate H2SO4 before being extracted with EtOAc. Organics layers were dried over Na2SO4 and concentrated under vacuum. The residue was used without further purification in the next step. The title compound (43 mg, 0.3 mmol, 32%) was obtained as a dark brown solid. 1 H NMR (300 MHz), δ (ppm, Acetone-d6): 2.32 (s, 3 H, CH3), 2.36 (s, 3 H, CH3), 5.07 (dd, J = 1.8 Hz, Jcis = 11.6 Hz, 1 H, CH2), 5.25 (dd, J = 1.8 Hz, Jtrans = 17.9 Hz, 1 H, CH2), 6.61 (dd, Jtrans = 17.9 Hz, Jcis = 11.6 Hz, 1 H, CH), 10.24 (s, 1 H, NH). 13

4-iodo-3,5-dimethyl-1H-pyrrole-2-carboxylic acid (9)

Ethyl 4-iodo-3,5-dimethyl-1H-pyrrole-2-carboxylate (1 eq., 500 mg, 1.7 mmol) and LiOH (8 eq., 326 mg, 13.6 mmol) were dissolved in EtOH (5 mL) and H2O (5 mL). The solution was heated at 90 °C for 1 h. The solvent was evaporated under vacuum. The residue was taken up in EtOAc (50 mL) and water (50 mL). The aqueous layer was acidified to pH 5.0 with H3BO3 (pH 5.1) or few drops of concentrate H2SO4 before being extracted with EtOAc. Organics layers were dried over sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography (DCM/MeOH, 95:5 (v/v)). The title compound (399 mg, 1.5 mmol, 88%) was obtained as a dark pink solid. 1 H NMR (300 MHz), δ (ppm, Acetone-d6): 2.25 (s, 3 H, CH3), 2.27 (s, 3 H, CH3), 10.82 (s, 1 H, OH). 13 C NMR (75 MHz), δ (ppm, CDCl3): 13.1 (CH3), 13.7 (CH3), 70.

(Z)-3-((4-(3-methoxy-3-oxopropyl)-3,5-dimethyl-1H-pyrrol-2yl)methylene)-2,4-dimethyl-3H-pyrrol-1-ium bromide (11)

A solution of 48% HBr in water (2.7 eq., 0.18 mL, 1.6 mmol) was added dropwise to a solution of pyrrole 2 (1 eq., 0.12 g, 0.6 mmol) and pyrrole 9 (1 eq., 0.16 g, 0.6 mmol) in methanol (1.5 mL).The mixture was stirred for 1 h at room temperature and concentrated under vacuum. The residue was purified by flash chromatography (DCM/MeOH, 95:5 (v/v)). The title compound (65 mg, 0.2 mmol, 30%) was obtained as a dark brown solid. 1 H NMR (300 MHz), δ (ppm, CDCl3): 2.25 (s, 3 H, CH3), 2.29 (s, 3 H, CH3), 2.38 (t, J = 7.3 Hz, 2 H, CH2), 2.58 (bs, 6 H, 2 CH3), 2.68 (t, J = 7.3 Hz, 2 H, 3.60 (s, 3 H, 6.08 (s, 1 H, CH), 7.01 (s, 1 H, CH), 13.00 (bs, 1 H, NH).

Tert-butyl 4-(2-iodoethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (13)

Tert-butyl 4-(2-hydroxyethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (1 eq., 100 mg, 0.42 mmol) , imidazole (2 eq., 56.9 mg, 0.84 mmol) and triphenylphosphine (2 eq., 219 mg, 0.84 mmol) were dissolved in DCM (5 mL) under a nitrogen atmosphere at room temperature. After complete dissolution, iodine (2 eq., 212 mg, 0.84 mmol) was added portionwise at 0 °C. The mixture was stirred overnight at room temperature. The reaction was quenched with aqueous 10% Na2S2O3 solution (10 mL). The aqueous layer was extracted with DCM (3 x 10 mL), dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash chromatography (Cyclohexane/EtOAc, 7:3 (v/v)). The title compound (138 mg, 0.4 mmol, 95%) was obtained as a white solid. 1 H NMR (300 MHz), δ (ppm, CDCl3): 1.56 (s, 9 H, 3 CH3), 2.22 (s, 3 H, CH3), 2.24 (s, 3 H, CH3), 2.95 (t, J = 7.9 Hz, 2 H, CH2), 3.14 (t, J = 8.3 Hz, 2 H, CH2), 8.64 (s, 1 H, NH). 13 14) TFA (44.4 eq., 1.15 mL, 25.5 mmol) was added dropwise to a solution of tert-butyl 4-(2-iodoethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (1 eq., 200 mg, 0.57 mmol) at room temperature under a nitrogen atmosphere. The solution was stirred for 30 min. Then, a solution of methyl 3-(5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)propanoate (1 eq., 119 mg, 0.57 mmol) in MeOH (2.3 mL) was added dropwise, followed by 33% HBr solution in AcOH (8.99 eq., 1263 mg, 0.86 mL, 5.1 mmol). The mixture was stirred at room temperature for 1 h and the product was collected by filtration. The residue was used without further purification in the next step. The title compound (200 mg, 0.38 mmol, 67%) was obtained as an orange solid. 1 H NMR (300 MHz), δ (ppm, CDCl3): 2.31 (s, 6 H, 2 CH3), 2.47 (t, J = 7.4 Hz, 2 H, CH2), 2.71 (s, 6 H, 2 CH3), 2.76 (t, J = 7.3 Hz, 2 H, CH2), 3.02 (t, J = 6.9 Hz, 2 H, CH2), 3.19 (t, J = 6.9 Hz, 2 H, CH2), 3.67 (s, 3 H, CH3), 7.08 (s, 1 H, CH), 13.11 (s, 1 H, NH). 13 -yl]methylidene}-3,5-dimethyl-2H-pyrrol-1-ium bromide (1 eq., 510 mg, 0.98 mmol) was dissolved in dry DCM (10.9 mL) under nitrogen atmosphere. DBU (2.5 eq., 0.36 mL, 2.4 mmol) was added dropwise at room temperature and the mixture was heated at 50 °C. After complete reaction, the solvent was evaporated under vacuum and the residue was purified by flash column chromatography with DCM/MeOH (95:5). The title compound (143 mg, 0.45 mmol, 47%) was obtained as a dark brown solid. UV-vis (Acetonitrile) max (nm) 490 (s), 370 (w), 291 (w), 224 (m). 1 H NMR (300 MHz), δ (ppm, CDCl3): 2.32 (s, 3 H, CH3), 2.37 (s, 3 H, CH3), 2.47 (t, J = 1.5 Hz, 2 H, CH2), 2.72 (s, 3 H, CH3), 2.76 (t, J = 1.5 Hz, 2 H, CH2), 2.80 (s, 3 H, CH3), 5.40 (dd, J = 1.1 Hz, Jcis = 7.7 Hz, 1 H, CH2), 5.45 (dd, J = 1.1 Hz, Jtrans = 14.1 Hz, 1 H, CH2), 6.54 (dd, Jcis = 11.6 Hz, Jtrans = 17.9 Hz, 1 H, CH), 7.12 (s, 1 H, CH), 12.92 (s, 1 H, NH). 16) TFA (9 eq., 0.59 mL, 7.7 mmol) was added dropwise to a solution of tertbutyl 4-(2-iodoethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (1 eq., 0.3 g, 0.9 mmol) in DCM (4 mL) at 0 °C under a nitrogen atmosphere. Trimethyl orthoformate (5 eq., 0.47 mL, 4.3 mmol) was added dropwise and the reaction was stirred under N2 for 20 min at 0 °C. The solution was warmed to room temperature and stirred for 30 min. The mixture was neutralized carefully with aqueous 10% NaHCO3 solution and extracted with DCM (3 x 20 mL). The combined organic layers dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash column chromatography with Cyclohexane/EtOAc (8:2). The title compound (150 mg, 0.5 mmol, 63 %) was obtained as light brown solid.

1 H NMR (300 MHz), δ (ppm, CDCl3): 2.29 (s, 3 H, 3 CH3), 2.30 (s, 3 H, CH3), 2.97 (t, J = 7.9 Hz, 2 H, CH2), 3.17 (t, J = 7.4 Hz, 2 H, CH2), 9.47 (s, 1 H, CHO), 10.25 (s, 1 H, NH). 13 C NMR (75 MHz), δ (ppm, CDCl3): 4.9 (CH2), 9.07 (CH3), 11.8 (CH3), 28.7 (CH2), 117.6 (Cquat), 122.0 (Cquat), 128.0 (Cquat), 139.5 (Cquat), 176.3 (CHO).

(Z)-4-(2-Iodoethyl)-2-((4-(2-iodoethyl)-3,5-dimethyl-1H-pyrrol-2yl)methylene)-3,5-dimethyl-2H-pyrrolium bromide (17)

TFA (27 eq., 1.1 mL, 14.5 mmol) was added dropwise to a solution of tertbutyl 4-(2-iodoethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (1 eq., 0.19 g, 0.5 mmol) at room temperature under a nitrogen atmosphere. The solution was stirred for 30 min. Then a solution of 4-(2-iodoethyl)-3,5-dimethyl-1Hpyrrole-2-carbaldehyde (1 eq., 0.15 g, 0.5 mmol) in MeOH (2 mL) was added dropwise, followed by 33% HBr in AcOH (8.6 eq., 0.81 mL, 4.6 mmol). The mixture was stirred at room temperature for 1 h and the product was collected by filtration. The residue was used without further purification in the next step.

The title compound (0.2 g, 0.3 mmol, 65%) was obtained as a dark red solid. 1 H NMR (300 MHz), δ (ppm, CDCl3): 2.31 (s, 3 H, 3 CH3), 2.70 (s, 3 H, CH3), 3.02 (t, J = 7.1 Hz, 2 H, CH2), 3.19 (t, J = 7.4 Hz, 2 H, CH2), 7.10 (s, 1 H, CH), 13.12 (s, 1 H, NH). 13 yl]methylidene}-3,5-dimethyl-2H-pyrrol-1-ium bromide (1 eq., 170 mg, 0.3 mmol) was dissolved in dry DCM (3 mL) under a nitrogen atmosphere. DBU (4 eq., 0.172 mL, 1.2 mmol) was added dropwise to the solution at room temperature and the mixture was heated at 50 °C. After complete reaction, the solvent was evaporated under vacuum and the solid residue was purified by flash column chromatography with DCM/MeOH (95:5). The title compound (26 mg, 0.1 mmol, 36%) was obtained as a brown solid. UV-vis (Acetonitrile) max (nm) 506 (s), 366 (s), 292 (s), 230 (s). 1 TFA (44 eq., 1.56 mL, 21.1 mmol) was added dropwise to a solution of 4-(3-methoxy-3-oxopropyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic acid (1 eq., 110 mg, 0.50 mmol) at room temperature under a nitrogen atmosphere. The solution was stirred for 30 min. Then, a solution of methyl 3-(5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)propanoate (1 eq., 100 mg, 0.50 mmol) in MeOH (2.0 mL) was added dropwise, followed by 33% HBr solution in AcOH (8.6 eq., 1.13 g, 0.81 mL, 4.6 mmol). The mixture was stirred at room temperature for 1 h and the product was collected by filtration. The residue was used without further purification in the next step. The title compound (192 mg, 0.40 mmol, 63%) was obtained as a brown solid. UV-vis (Acetonitrile) max (nm) 480 (s), 392 (w), 226 (m).

Table of Contents

We herein report the efficient convergent synthesis of the hexasubstituted dipyrrins structurally related to verteporfin where the sensitive vinyl group was created at the final stage by a dehydroiodation reaction. VP showed a direct effect on YAP and TAZ levels whereas dipyrrin 19 showed a direct effect only on YAP level. Altogether, these data suggests that dipyrrin-related compounds may directly affect the expression of YAP and/or TAZ.

Figure 1 .

 1 Figure 1. Structures of VP, HP, PPIX and related dipyrrins

Figure 2 .

 2 Figure 2. Effects of VP (10 µM), YAP, TAZ or YAP/TAZ-silencing on mRNA expression (A) or protein production (B) of Cyr61, CTGF or Axl in MDA-MB-231 cells. For the mRNA expression, results are the average of three independent experiments. Data are presented as mean SEM (n = 3, *p < 0.1, ** p < 0.01, *** p < 0.001). For protein production, data are representative of 3 independent experiments.

Figure 3 .

 3 Figure 3. Inhibition of YAP(TAZ)/TEAD interaction by VP, protoporphyrins and dipyrrins. MDA-MB-231 cells expressing 8xGTIIC luciferase were incubated for 48h with tested drugs (10 µM). Data are representative of three independent experiments in duplicate (Data are presented as mean SEM (n = 6, *p < 0.1).

Figure 4 .

 4 Figure 4. Effects of porphyrins (left) and dipyrrins (right) on YAP, TAZ, Cyr61, CTGF and Axl levels. MDA-MB-231 cells were incubated in the presence of the drug (10 µM) for 48h. Actin was used as loading control. Data are representative of 3 independent experiments.

  H NMR (300 MHz), δ (ppm, CDCl3): 2.37 (s, 3 H, CH3), 2.48-2.53 (m, 5 H, CH2 + CH3), 2.59 (s, 3 H, CH3), 2.77-2.82 (m, 5 H, CH2 + CH3), 2.98 (s, 3 H, CH3), 3.68 (s, 3 H, OCH3), 7.26 (s, 1 H, CH), 13.22 (s, 1 H, NH), 13.69 (s, 1 H, NH). 13 C NMR (75 MHz), δ (ppm, CDCl3): 10.4 (CH3), 12.7 (CH3), 13.5 (CH3), 15.8 (CH3), 19.2 (CH2), 31.6 (CH3), 33.4 (CH2), 51.9 (OCH3), 120.6 (CH), 126.8, 127.8, 129.4, 132.7, 143.4, 144.8, 154.3, 160.5, 172.5 (CO), 203.5 (CO). LC-MS (ESI) m/z Calculated: 408.10, Found: 329.16 [M-HBr+H] + ; tR = 2.15

  13 C NMR (75 MHz), δ (ppm, CDCl3): 9.5 (CH3), 11.3 (CH3), 12.3 (CH3), 15.5 (CH3), 20.6 (CH2), 22.0 (CH2), 31.2 (CH3), 35.9 (CH2), 51.9 (OCH3), 113.8, 115.7, 117.0, 121.6, 122.5, 122.8, 125.3, 135.0, 174.5 (CO), 196.5 (CO).

  7 (CH3), 60.3 (CH2), 72.0 (C), 118.3 (C), 130.7 (C), 134.6 (C), 161.4 (CO). LC-MS (ESI) m/z Calculated: 292.99, Found: 291.99 [M-H] -; tR = 3.14 min

  13 C NMR (75 MHz), δ (ppm, CDCl3): 11.2 (CH3), 12.8 (CH3), 14.5 (CH3), 59.8 (CH2), 112.7 (CH2), 125.3 (CH), 128.2 (C), 129.0 (C), 133.6 (C), 139.3 (C), 161.6 (CO).

  C NMR (75 MHz), δ (ppm, CDCl3): 10.5 (CH3), 11.5 (CH3), 111.0 (CH2), 117.7 (C), 126.0 (C) 129.4 (CH), 131.2 (C), 132.6 (C), 164.1 (CO).

  8 (C), 129.7 (C), 134.3 (C), 134.3 (C), 161.0 (CO). LC-MS (ESI) m/z Calculated: 264.96, Found: 263.92 [M-H] -; tR = 2.50 min

13 C

 13 NMR (75 MHz), δ (ppm, CDCl3): 10.2 (CH3), 12.1 (CH3), 12.8 (CH3), 14.4 (CH3), 14.2 (CH2), 33.7 (CH2), 51.8 (OCH3), 117.2 (CH), 119.6 (CH), 126.2 (C), 126.7 (C), 127.0 (C), 142.7 (C), 145.6 (C), 154.5 (C), 154.7 (C), 172.6 (CO).

  C NMR (75 MHz), δ (ppm, CDCl3): 5.6 (CH2), 10.6 (CH3), 11.6 (CH3), 28.5 (3 CH3), 29.3 (CH2), 80.4 (C), 118.5 (C), 121.0 (C), 125.7 (C), 129.1 (C), 161.1 (CO). LC-MS (ESI) m/z Calculated: 349.05, Found: 295.07 [M-tBu+H] + , tR = 3.39 min (Z)-4-(2-Iodoethyl)-2-((4-(3-methoxy-3-oxopropyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3,5-dimethyl-2H-pyrrolium bromide (

  C NMR (75 MHz), δ (ppm, CDCl3): 3.4 (CH2), 10.2 (CH3), 10.4 (CH3), 13.0 (2 CH3), 19.3 (CH2), 28.3 (CH2), 33.7 (CH2), 51.8 (OCH3), 119.4 (CH), 126.0 (C), 126.4 (C), 127.3 (C), 127.4 (C), 142.1 (C), 142.9 (C), 153.4 (C), 155.0 (C), 172.6 (CO). LC-MS (ESI) m/z Calculated: 520.02, Found: 441.03 [M-HBr+H] + , tR = 2.73 min (Z)-Methyl 3-(5-((3,5-dimethyl-4-vinyl-2H-pyrrol-2-ylidene)methyl)-2,4dimethyl-1H-pyrrol-3-yl)propanoate (1) (Z)-4-(2-Iodoethyl)-2-{[4-(3-methoxy-3-oxopropyl)-3,5-dimethyl-1H-pyrrol-2

13 C

 13 NMR (75 MHz), δ (ppm, CDCl3): 10.3 (CH3), 11.0 (CH3), 13.0 (CH3), 14.0 (CH3), 19.3 (CH2), 33.7 (CH2), 51.8 (OCH3), 117.4 (CH2), 119.4 (CH), 125.8 (C), 126.1 (C), 126.5 (C), 126.7 (C), 127.4 (CH), 141.0 (C), 142.9 (C), 154.0 (C), 155.1, (C) 172.6 (CO). LC-MS (ESI) m/z Calculated: 312.18, Found: 313.14 [M+H] + , tR =2.65 min 4-(2-Iodoethyl)-3,5-dimethyl-1H-pyrrole-2-carbaldehyde (

  C NMR (75 MHz), δ (ppm, CDCl3): 3.3 (2 CH2), 10.5 (2 CH3), 13.1 (2 CH3), 28.3 (2 CH2), 119.7 (CH), 126.3 (2 C), 127.7 (2 C), 142.6 (2 C), 154.3 (2 C). (Z)-2-((3,5-Dimethyl-4-vinyl-2H-pyrrol-2-ylidene)methyl)-3,5-dimethyl-4vinyl-1H-pyrrole (19) (Z)-4-(2-Iodoethyl)-2-{[4-(2-iodoethyl)-3,5-dimethyl-1H-pyrrol-2

  13 C NMR (75 MHz), δ (ppm, CDCl3): 11.1 (2 CH3), 14.2 (2 CH3), 117.8 (2 CH2), 119.7 (CH), 126.5 (2 CH), 137.5 (2 C), 141.8 (2 C), 154.0 (2 C), 154.9 (2 C). LC-MS (ESI) m/z Calculated: 252.16, Found: 253.14 [M+H] + , tR =3.31 min(Z)-4-(3-Methoxy-3-oxopropyl)-2-((4-(3-methoxy-3-oxopropyl)-3,5dimethyl-1H-pyrrol-2-yl)methylene)-3,5-dimethyl-2H-pyrrolium bromide (18)

1 H

 1 NMR (300 MHz), δ (ppm, CDCl3): 2.29 (s, 6 H, 2 CH3), 2.47 (t, J = 7.5 Hz, 2 H, CH2), 2.69 (s, 6 H, 2 CH3), 2.76 (t, J = 7.5 Hz, 2 H, CH2), 3.69 (s, 6 H, 2 OCH3), 7.05 (s, 1 H, CH), 13.05 (bs, 1 H, NH). 13 C NMR (75 MHz), δ (ppm, CDCl3): 10.2 (2 CH3), 12.9 (2 CH3), 19.3 (2 CH2), 33.8 (2 CH2), 51.8 (2 OCH3), 119.2 (CH), 126.1 (2 C), 126.9 (2 C), 143.2 (2 C), 154.2 (2 C), 172.7 (2 CO). LC-MS (ESI) m/z Calculated: 452.13, Found: 373.19 [M-HBr+H] + , tR = 2.52 min.

  H NMR (300 MHz), δ (ppm, CDCl3): 2.40 (s, 6 H, 2 CH3), 2.83 (s, 6 H, 2 CH3), 5.36-5.49 (m, 4 H, 2 CH2), 6.53 (dd, Jcis = 11.9 Hz, Jtrans = 17.6 Hz, 2 H, 2 CH) 7.19 (s, 1 H, CH), 12.66 (s, 1 H, NH).

Acknowledgements

The work was financially supported by grants from la Région Nord Pas-de-Calais and CHRU of Lille (M. Corvaisier) and le Ministère de l'Education et de la Recherche (F. Gibault) and fundings from SIRIC OncoLille and le Cancéropôle Nord-Ouest.

Keywords: Dipyrrins • verteporfin • non-photoinduced therapy • Hippo pathway • cancer


Anna Carla Araujo

Guillaume Fromentin

Investigation of tool deflection during milling of thread in Cr-Co dental implant

Keywords: Thread milling, Dental alloy, Thread accuracy, Chrome-cobalt, Tool deflection Nomenclature

Milling is a good option for manufacturing internal threads in hard-to-cut dental components due to lower cutting forces. In the case of a very small drilled diameter, the tool cannot be large enough to reduce tool deflection nor sufficiently small to avoid the influence of the tool penetration. As a consequence, both situations need to be considered and no other research study dealt with this aspect for the modeling of thread milling forces. This article deals with the analysis of forces, deflection, and undercutting during machining of one typical internal thread geometry used for implants in a chrome-cobalt dental alloy. The geometry is analyzed considering the influence of tool penetration and it is presented new equations to identify the regions where it occurs. Machining experiments are conducted acquiring cutting forces and tool axis position in order to calculate the tool radial forces and estimate tool deflection. Manufactured threads geometry is measured to evaluate dimension quality. It can be claimed that the tool trajectory should consider one extra revolution around the drilled hole in order to machine the undercut material due to tool deflection for this small tool diameter.

F 0 = [F x , F y , F z ] Force components in R 0 O Drilled hole position, R 0 Referential Frame Origin R 1
Moving reference Frame centered in O and tangential to workpiece surface

F 1 = [F rad , F tan , F z ] Force components in R 1 R 2
Moving reference frame centered in tool axis and fixed in one point of the cutting flute F 2 = [F r , F t , F z ] Force components in R 2

Introduction

Titanium and chrome-cobalt alloys are used for implants because it presents high corrosion resistance and strengthto-weight ratio and per its bio-compatibility [START_REF] Elias | Mechanical properties, surface morphology and stability of a modified commercially pure high strength titanium alloy for dental implants[END_REF]. Although titanium is mostly used by dentist industry, is increasing the use of chrome-cobalt alloys because it has sufficient strength to withstand the occlusal forces applied to partial denture frameworks [START_REF] Mccracken | Dental implant materials: commercially pure titanium and titanium alloys[END_REF]. Two-part implant systems having internal machined connection seem to offer an advantage considering failure compared to other systems [START_REF] Schwitalla | Maximum insertion torque of a novel implant-abutment-interface design for peek dental implants[END_REF] but it is important to optimize implant geometry to avoid part breakage during extraction by pullout procedure [START_REF] Rittel | Modelling dental implant extraction by pullout and torque procedures[END_REF].

Different manufacturing processes can be used to produce external threads in implants: grinding, quoted as slow and inefficient, electric discharge machining, using fabricated graphite die, external whirling, with several inserts having the cutting edge geometry of the thread envelop profile. and form and cut tapping, used for internal threads. Even if the implant is produced by additive manufacturing, the internal thread should be produced by subtractive processes [START_REF] Rysava | Micro-drilling and threading of the Ti6Al4V titanium alloy produced through additive manufacturing[END_REF]. The typical manufacturing process used for an internal thread is forming or cut tapping [START_REF] Kuhn | Bone deformation by thread-cutting and thread-forming cortex screws[END_REF], but there are other two options: internal whirling and thread milling. Internal whirling depends on precision multi-axis machine, with a synchronized rotation of tool and workpiece [START_REF] Zanger | Comparison of modeling methods to determine cutting tool profile for conventional and synchronized whirling[END_REF]. In tapping, residual stresses could be much higher if compared to thread milling or whirling. Hernandez [START_REF] Hernandez-Rodriguez | Failure analysis in a dental implant[END_REF] studied failure in threads due to the high-stress rate for screw manufactured by deformation processes that could produce weak regions for the structure. The internal threads produced by deformation failed catastrophically after 6 months of service due to a fracture presented at the inner screw used for fixation of the abutment. Some articles studied the optimization of the workpiece geometries, as Wu et al. [START_REF] Wu | The effects of flute shape and thread profile on the insertion torque and primary stability of dental implants[END_REF], that presented the effects of thread geometry and profile on the stability of dental implants finding an optimal abutment design, with a lower resistance to initial insertion and higher stability, for final instrumentation. Also, Hasan et al. [START_REF] Hasan | Influence of abutment design on the success of immediately loaded dental implants: experimental and numerical studies[END_REF] analyzed fine threads and the influence of the design on loading immediately dental implants. If the geometry cannot be adapted, the manufacturing process could change to reduce the problem.

Thread milling could produce internal and external threads, especially on machining hard materials, as titanium alloys, because its feed rate could be reduced with no impact on cutting speed. In thread turning, the feed per revolution should be equal to the thread pitch and cannot be reduced [START_REF] Khoshdarregi | Generalized modeling of chip geometry and cutting forces in multi-point thread turning[END_REF]. Comparing to tapping, in case of a breakage, thread milling is safer because it can be removed without damage to the workpiece. Deep analysis of geometry and kinematic should be done in thread milling because the tool geometry and the helical tool trajectory are complex [START_REF] Fromentin | Computerized simulation of interference in thread milling of non-symmetric thread profiles[END_REF]. A geometrical model and an analytical formulation to define the local rake face on the cutting edge by Fromentin et al. [START_REF] Fromentin | Geometrical analysis of thread milling-part 1: evaluation of tool angles[END_REF] and it is used for calculating uncut chip thickness during internal thread milling [START_REF] Fromentin | Geometrical analysis of thread milling-part 2: calculation of uncut chip thickness[END_REF]. Araujo et al. [START_REF] Araujo | Analytical and experimental investigations on thread milling forces in titanium alloy[END_REF] used the local rake angle approach to study geometrical analysis of thread milling parameters in Ti-6Al-4V with metric threads, and an analysis of variance for determining the influence parameters and bring a physical understanding of the process and focused in finding the optimum flute angle. Wan and Altintas [START_REF] Wan | Mechanics and dynamics of thread milling process[END_REF] developed a detailed analysis of the dynamics of thread milling.

A previous experimental study compared forces on the thread milling of mini-threads in titanium and chromecobalt alloy [START_REF] Araujo | Modeling thread milling forces in mini-hole in dental metallic materials[END_REF]. In the present article, it is done a geometrical analysis of thread milling and a tool deflection analysis in the case that the drilled hole is very close to the milling tool diameter, which was not studied before. The standard M2 thread is produced in a Chrome-cobalt commercial dental alloy Romur 400. The geometry is predicted considering the influence of tool penetration and experiments are conducted acquiring cutting forces and tool axis position in order to estimate tool deflection.

Analytical formulation of influence of tool penetration in thread milling

In thread milling, the tool travels inside a drilled hole describing a helical trajectory to manufacture the thread profile with a nominal diameter D. The basic analysis of the thread milling process is extensively presented by many authors [START_REF] Araujo | Analytical and experimental investigations on thread milling forces in titanium alloy[END_REF][START_REF] Araujo | A model for thread milling cutting forces[END_REF][START_REF] Fromentin | Effect of thread milling penetration strategies on the dimensional accuracy[END_REF]. The milling tool diameter is D t and the drilled diameter is D 1 . The tool path is described by the CNC coordinates (x(t), y(t), z(t)) in the machine-tool reference frame R 0 .

The tool axis position angle is calculated by θ 1 (t) = arctan y (t) x(t) . The tool revolution angle θ 2 (t) is driven by the spindle speed N: θ2 = 2πN/60. The tool rotates clock-wise, so θ 2 has opposite sense for θ 1 , in the case of down milling.

Feed per tooth f t is defined on the trajectory direction f t = V f .N f .N, where V f is feed velocity. The feed per tooth is decomposed in xy plane and in z direction, as described in Eq. 1:

f t = f 2 tz + f 2 txy (1)
Thread milling process presents three principal stages: tool penetration, full machining (FM), and tool exit. The two first stages are presented in Fig. 1.

(i) Tool penetration: the tool moves in a half revolution penetration strategy (HRPS) [START_REF] Sharma | Investigation of tool geometry effect and penetration strategies on cutting forces during thread milling[END_REF] from the center O until point A. The radial penetration increases until the desired tool engagement r max p . The vertical displacement during tool penetration is proportional to the thread pitch: P /4. For reference, the time range, from 0 to t 1 , and θ 1 range in tool penetration is defined below:

t = [0, t 1 ] → θ 1 (t) = π t 1 t -π → θ 1 (t) ∈ {-π < θ 1 < 0}
(ii) Full machining: the tool is fully engaged. The nominal radial penetration is constant until there is no influence of tool penetration. The tool travels a helical trajectory where D tt is its diameter and the tool vertical displacement is the thread pitch P , if the number of threads n P to be machined is lower than the number of threads on the cutting edge n max P . If not, the tool continues the helical trajectory for more then one pitch. In the case n P < n max P , the depth of cut is described by a doc (t) = (n P -θ 1 (t) 2π )P and the time range, from t 1 to t 2 , and θ 1 range in the case is:

t = [t 1 , t 2 ] → θ 1 (t) = 2π (t 2 -t 1 ) .(t -t 1 ) → θ 1 (t) ∈ {0 < θ 1 < 2π } (iii)
Tool exit: the tool goes back to the initial hole axis position as the radial penetration reduces to zero. The vertical displacement is P /4. The time range, from t 2 to t 3 , and θ 1 range in tool exit is defined by:

t = [t 2 , t 3 ] → θ 1 (t) = π (t 3 -t 2 -t 1 ) .(t -t 2 ) → θ 1 (t) ∈ {0 < θ 1 < π}

Geometrical analysis during tool penetration

As it is shown in Fig. 1, the tool penetration trajectory is developed from O to A. But, during this path, the tool envelope only meet the workpiece surface in point P 1 when its axis is centered in Q (Fig. 2a). In this position, if one point the cutting edge is engaged and aligned with the line OP 1 , the first chip is removed. This position is calculated by Eq. 2:

θ 1 (Q) = arccos D 1 -D t D tt (2)
After this point, the tool removes material until its axis arrives at A. The area of the material removed during this stage is identified on Fig. 2a by the limit points P 2 and P 3 . In order to locate the position of P 2 , which it is important to analyze penetration influence on thread milling process, it is identified in Fig. 2 the angle β between OP 2 and OA. Equation 3 calculates the value of β:

β = arccos D 2 1 -D 2 t + D t .D tt D tt .D 1 (3)

Geometrical analysis during full machining

The tool is in full machining after passing the point A.

During this period, it can be identified two regions: in the beginning when the nominal radial penetration is constant without any influence of tool penetration, so-called steady state, and the region where the material has already been taken by tool penetration. The maximum uncut chip thickness t cmax achieved by the front cutting edge (FCE) in steady state [START_REF] Fromentin | Geometrical analysis of thread milling-part 1: evaluation of tool angles[END_REF] is identified Fig. 2b. In this figure, it is presented the regular radial depth The feed per tooth projected in xy plane is f txy = f t /( ( 2P πD tt ) 2 + 1). The number of revolutions that the tool spins around its axis (θ 2 ) is D tt π/f txy .

The depth of cut a doc (t) varies, so the flute engagement angle δ(t) and cutting continuity c(t) [START_REF] Araujo | Modeling thread milling forces in mini-hole in dental metallic materials[END_REF] are described by Eq. 4, where λ st is the tool helix angle and θ f the angle between flutes [START_REF] Araujo | Analytical and experimental investigations on thread milling forces in titanium alloy[END_REF].

δ(t) 2.a doc (t). tan(λ st )/D t (4) c(t) = (δ(t) + θ tw )/θ f (5)
The region influenced by tool penetration begins when the tool axis reaches the point B, as it can be observed in Fig. 2c. The angle that locates the tool axis position on the end of steady state, θ 1 (B), is calculated by Eq. 6.

θ 1 (B) = 2π -β -arccos D 2 tt -D 2 t + D 2 1 2.D 1 .D tt ( 6
)
3 The mini-thread and tool geometry

The analysis of tool penetration described by Section 2 is specially important in the case of mini-threads, in which there is not substantial gap between D 1 and D t . In Fig. 1, the gap is shown in initial position when the tool and drilled hole are centralized in point O. For small threads, the percentage of engagement e% is higher than regular cases:

e% = D -D 1 D -D t . ( 7
)
This study is focused on the manufacturing of M2 metric thread, the specific dimension used in internal threads for dental implants. Figure 3 shows the tool geometry for thread milling and Table 1 presents a complete panorama of geometrical parameters for the this case and it is detailed in this section:

-The value of D t is experimentally taken with Alicona Infinite Focus Measurement (IFM): D t = 1.53 ± 0.05, slightly smaller then catalog dimension. -Tool diameter is measured on the front cutting edge (Fig. 3a andb).

Cutting conditions and experimental set-up

This study includes a batch of thread milling experiments that it is the base of the methodology that is described in the next section. During experiments, the acquisition of cutting forces, tool spin angle, and tool position were developed while the thread are manufactured. The machined material is a Cr-Co-based commercial dental alloy Romur 400, having the following chemical composition (% weight): 62.5% Co, 28.5% Cr, 6.1% Mo, 0.55% Mn, and typical mechanical properties: 705 MPa yield stress (0.2%), 2.6% elongation in fracture, and 185 GPa elastic modulus.

Machining tests were performed in a 3-axis milling center CNC DMC65V using water based emulsion. The drilling tool reference is MWS0160SB (Mitshubishi) and cutting parameters are: V c =10 m/min and f t =0.01 mm/th. It was drilled 4 mm in depth. The thread milling tool is a H5087006-M2 (Walter Co) in solid carbide coated (TiCN) and cutting parameters were a constant cutting speed V c =20 m/min (N = 4110 rpm) and two levels for feed per tooth: f t =0.025 mm/th/rev and f t = 0.035 mm/th. Three cases of depth of cut were used: a max doc = 0.4, 0.8, and 1.2 mm, meaning 1, 2 or 3 machined pitches in the workpiece.

The acquisition set-up (Fig. 4a) is composed of (1) a derivative electronic interface Heidenhain IBV 606 needed to acquire tool angular position from spindle encoder in a high band width, (2) a sensitive Kistler mini-dynamometer 9256C2 with (3) a signal amplifier 5019A, (4) the CNC machine analog output to acquire tool axis position (x, y and z) and ( 5) a data acquisition card DAC 9188 (National Instruments -NI) using two modules 9215 for analogical signals and a trigger coupled to a digital module 9401 (NI) for encoder signals. The synchronous sampling is ensured thanks to this configuration. The sampling rate used in this case is 50 kHz. Dasylab software is used for data acquisition and Matlab software is used for data treatment and analysis.

The local interaction between the tool and the machined surface is described by the local forces F rad and F tan , radial and tangent to the drilled surface. These components are calculated using θ 1 and the dynamometer components F x , F y and F z , using Eq. 9. The resultant force F res is also calculated.

⎡ ⎣ F rad F tan F z ⎤ ⎦ = ⎡ ⎣ cos(-θ 1 ) -sin(-θ 1 ) 0 sin(-θ 1 ) cos(-θ 1 ) 0 0 0 1 ⎤ ⎦ ⎡ ⎣ F x F y F z ⎤ ⎦ (8)

Methodology for calculating tool tip position using experimental data

In order to calculate the tool tip trajectory considering tool deflection, it is proposed an hybrid procedure, illustrated in Fig. 5.

(a) In the first step, the CNC tool holder position (x(t), y(t)) is taken from data file for each experiment. The trajectory is verified and θ 1 is calculated as it is shown in Fig. 5a. (b) On the second step, the tool stiffness is estimated by simulation, independent of the experimental results. The tool geometry is imported from experimental data (using STL technology) from IFM into a CAD software, Catia Software (Dassault systems).

A chosen force is applied on the cutting edge perpendicular to the tool axis. No vertical force is applied. The mechanical properties of Tungsten Carbide are used as parameter on the software. It is considered the static deflection model, as the natural frequency is not included in harmonics of the experimental forces.

Five different values of the applied force (10 to 50 N) are used on the simulations. Triangular linear elements are used on the simulations. For each cutting force, the tool tip displacement is registered and the global stiffness is calculated. Using this procedure, tool stiffness is 833 N/mm (Fig. 5b). (c) On the third step, the radial and tangential cutting forces are calculated (Eq. 9) using acquired cutting forces (F x , F y ) and θ 1 , based on (x(t), y(t)) measurements, for each experiment (Fig. 5c).

(d) The last step calculates the tool tip trajectory considering the tool holder position, the forces applied on the tool and the tool stiffness. In real time, the displacement is calculated, as shown in Fig. 5d. The maximum tool deflection per tool spin revolution is analyzed.

Results and discussions

An extract of the results is presented in this section: cutting force results, tool tip deflection, and a comparison of the measured thread dimension with experimental results.

Cutting force results

Figure 6a presents the evolution of the resultant force along the variation of θ 1 . In that situation, the tool turns 20 revolutions during helical trajectory, meaning 60 teeth cuts as there are 3 flutes in this case. It can be seen the three stages of the average evolution of the forces: from 0 to t 1 , it can be identified that tool penetration from θ 1 (Q) < θ 1 < 0 and the increasing of forces; from 0 < θ 1 < θ 1 (B) it is the part of full machining without influence of tool penetration, with constant force peaks and from θ 1 (B) < θ 1 < 360 • , the influence of the tool penetration on the reduction of forces. It would be usual to have a linear decrease from 0 • to 360 • , as the depth of cut reduces linearly. It is not the case, there is strong difference between the two stages after tool penetration. Specially on the last 15 revolutions, the amplitude reduces significantly due to tool penetration influence. In Fig. 6b, the same evolution can be seen in tangential and radial components. These forces are important to analyze the deflection, specially in in radial direction because it reduces the engagement. In feed direction (tangential force) the component does not affect the thread geometry. Figure 6c presents a complete panorama of all the experiments, the force values show the average of thee peaks per revolution in each experiment.

Tool tip deflection results

The maximum tool deflection is calculated in each tool revolution using the described procedure. Figure 7 presents the average of the maximum deflection for all experiments and the error bars represent the dispersion of the data. Introducing a constant radial correction on the tool trajectory (correction in D tt ), would not solve the problem because deflection is not constant. From Fig. 6, it can be noted that there is a big difference of the deflection for machining only one pitch which contribute to the recommendation of second pass for mini thread milling trajectory.

Analysis of workpiece dimensions

The workpiece is cut using wire-EDM process in order to measure the produced thread profiles in two θ 1 positions: 8a shows the cut workpiece and a image of the IFM measurement aligned with x direction. It is indicated the location machined when the tool is centered in A, the end of tool penetration θ 1 = 0 in one half of the workpiece. In the other half, θ 1 = 180 • is measured. The thread angle was measured in all threads (tool and workpiece) confirming the desired angle: 60 ± 1 • (Fig. 8b).

The height of thread profile on the tool h p is measured h p = 0.227 ± 0.028 mm and it is confirmed that h p > r max p (r max p = 200 μm). It allows to conclude that the thread milling tool did not machine the drilled surface D 1 (Fig. 8a) and that it can be taken as a reference in thread profiles on the workpiece to measure h i wp in order to calculate the i front cutting edge (FCE) deflection r i exp :

r i exp = r max p -h i wp ( 9
)
Figure 8b presents the workpiece profile taken on the thread manufactured using n P = 3 and f t = 0.025 mm/th/rev experiment as well the tool profile for reference. From the thread height on the workpiece profile for the three FCE: h 1 wp , h 2 wp and h 3 wp in position θ 1 = 0 and θ 1 = 180 • . The results are presented in Table 2.

The results taken in position θ 1 = 0 present lower values for the difference 1 exp and 2 exp (* values in bold in Table 2). In this specific point, one cannot claim that this is the deflection because the tool machines twice this surface as the deflection reduces from t 1 to t 2 , as it is represented in Fig. 9a andb. For all other values r i = 200h i wp (μm). Considering ISO 965-1 standard, for M2x0.4 mm thread, to achieve tolerance 6H in the internal thread, the thread diameter could vary 90 μm, which means the r exp ≤ 45 μm and for tolerance 5H, r exp ≤ 35 μm. It can be claimed, as per results presented in Table 2, that manufactured threads are under tolerance 6H. Values on the lower thread (n P = 3) were neglected as the screw could not achieve this region as it is not completely machined.

Conclusions

This article deals with milling process to manufacture mini-thread M2 in two dental materials. The geometry of the cutting related to the engagement between tool and workpiece was completely studied. The tool stages of tool penetration, steady state, and the region influenced by tool penetration were modeled analytically for any tool and thread dimension.

Experiments were developed in chrome-cobalt used in dental industry with: constant cutting speed and two levels for feed per tooth. Three levels for depth of cut were used machining at maximum 1, 2, or 3 thread at the same time, which means that the tool travels only one round in full machining as the tool have three pitches in each flute. During experiments, tool axis position and revolution was acquired to allow the computation of radial and tangential forces based on dynamometer signals. Deflection was estimated using tool stiffness and experimental cutting forces for all data points. The workpiece was cut and the thread height was measured in order to evaluate the quality of produced threads. It was noted that in the beginning of full machining, at θ 1 = 0, in the first and second threads (from the top surface), the difference between the radial penetration and the measured thread height was lower because the tool re-machine the thread surface at this point. At θ 1 = 180 • it can be calculate the tool deflection influence on the thread height and determine the tolerance achieved.

Tool deflection is a consequence of forces proportional to geometrical tool engagement but, as it is not constant during the full thread machining, it cannot be compensated by a correction of the tool path radius. Future work could focus on the study of the tool geometry to prevent tool deflection. Also, micro surface quality of the thread could be introduced in the future analysis.

Fig. 1 Fig. 2

 12 Fig. 1 Tool penetration and full machining in thread milling

  -The drilled diameter D 1 is also measured by IFM: D 1 = 1.61 ± 0.05 mm. -The percentage of engagement is calculated as 85.1%, which means the tool is very close to the drilled surface. -The radial penetration when the tool is fully engaged during thread milling is r max p = (D -D 1 )/2 = 0.2 mm. -The relation between pitch and tool diameter affects the radial depth of cut, r doc = 0.822 mm, higher then milling tool radius, as a consequence, the tooth working angle θ tw is 94.2 • (θ tw > 90 • ) [15]. -The thread milling tool helix angle (Fig. 3c) is λ st = 15 • with three threaded cutting edges (N f = 3 and

Fig. 3

 3 Fig. 3 Mini-thread

Fig. 4

 4 Fig. 4 set-up

Fig. 5

 5 Fig. 5 Procedure to tool tip trajectory considering tool deflection

Fig. 6 Fig. 7

 67 Fig. 6 cutting forces

Figure

Fig. 9

 9 Fig. 9 of the thread height and deflection

Table 1

 1

	Geometrical parameters calculated for M2	D	P	D t	D tt	r max p	r doc	θ 1 (Q)	β	θ 1 (B)
	Dental Implants	2 mm	0.4 mm	1.53 mm	0.24 mm	0.2 mm	0.82 mm	-81.6 •	132.1 •	154.3 •

Table 2

 2 Results from IFM measurements: r i

			exp = r max p	-h i wp
	Number of pitches cut/position	r 1 exp	r 2 exp	r 3 exp
	n P = 1 / 0 deg	12.8 μm	-	-
	n P = 1 / 180 deg	14.5 μm	-	-
	n P = 2 / 0 deg	2.0 μm*	29.9 μm	-
	n P = 2 / 180 deg	29.3 μm	35.0 μm	-
	n P = 3 / 0 deg	13.6 μm* 15.3 μm* 50.6 μm
	n P = 3 / 180 deg	33.0 μm	38.7 μm	48.7 μm

(*) two tool passes -see Fig.

8b

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