Julien Couvidat
email: julien.couvidat@insa-lyon.fr

Cécile Diliberto
email: cecile.diliberto@univ-lorraine.fr

Eric Meux
email: eric.meux@univ-lorraine.frl.izoret@atilh.fr

Laurent Izoret

André Lecomte
email: andre.lecomte@univ-lorraine.fr

Greening effect of concrete containing granulated blast-furnace slag composite cement: is there an environmental impact?

Keywords: blast-furnace slag, greening effect, sulfide, fair faced concrete, foundation, demolition

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Greening effect of concrete containing granulated blast-furnace slag composite cement: Is there an environmental impact?

Introduction

Granulated Blast-Furnace Slag (GBFS) is a common by-product of iron and steel production. While ground GBFS has a long history of varied uses, in the late 19 th century, it began being used as a supplementary cementitious material (SCM) to partially replace clinker in composite cements and strengthen durability. The blue-green coloration or "greening" effect occurs when GBFS or GGBFS are hydrated and will fade within days or weeks when exposed to air. This greening effect is a well-documented phenomenon seen across a range of construction applications that rely on CEM III based or slag based concretes, i.e. (a) civil engineering, (b) precast concrete use in small-and large-scale construction (c) flooring and (d) masonry (Fig. 1). The issue has been addressed by several professional journals and white papers [START_REF] Euroslag | Technical leaflet n°1[END_REF][START_REF]Slag Cement Association, Slag Cement in Concrete N°10[END_REF]. In France, while CEM III cement types (cement containing from 36 to 95% GGBFS) represent roughly 10% of overall production (16.5 M metric tons in 2018), the full range of overhead applications and those using fair-faced concrete (excluding foundation work) that rely on CEM III and slag based binders (CEM I type with Slag as addition) are potentially at stake. The origin of this coloration effect was for a long time poorly understood, but more recent investigations have pointed to the potential causal role of sulfur compounds in concrete aggregates.

One of the first studies to show how sulfides potentially contribute to coloration analyzed the cement hydrates containing the sulfide ion into hydrated mortar, and found that hexagonal aluminates hydrates contained a solid solution of sulfides and sulfates that displayed a characteristic green color [START_REF] Vernet | Comportement de l'ion S--au cours de l'hydratation des ciments riche en laitier (CLK)[END_REF]. More recent studies have explored the origin of this coloration in GGBFS-containing materials by using UV-Visible [START_REF] Le Cornec | Greening effect in slag cement materials[END_REF] or XANES spectroscopy [START_REF] Chaouche | On the origin of the blue/green color of blast-furnace slag-based materials: Sulfur K-edge XANES investigation[END_REF] and concluded that radical polysulfides

anions are primarily responsible for the greening effect. In cement materials containing slag, low porosity acts to prevent important oxygen penetration, and soluble sulfides are most likely consumed by precipitation with cement hydrates rather than by an oxidative reaction [START_REF] Vernet | Comportement de l'ion S--au cours de l'hydratation des ciments riche en laitier (CLK)[END_REF]. Cement hydration will then likely form a protective surrounding to the chromophore species, allowing the metastable blue-green color to occur for hours, days or weeks.

To date, few studies have examined this phenomenon, despite a wide and historical use in civil engineering and the cement industry. To the best of our knowledge, no studies have focused on the potential environmental impacts whereas CEM III cement customers regularly question producers and resellers about the safety of 'blue-green concrete'. The concern is that this effect is consistently present through several key construction phases including form removal, but also during deconstruction, given that the blue-green color remains visible when a GBFS-containing concrete is demolished.

The objective of this study was to assess the environmental impact of the greening effect occurring in GBFS-rich material and CEM III based concretes. For this purpose, leaching tests were conducted on materials made with commercially available GBFS-composite cements. These tests were performed on cement Portland paste, for example, to study transport mechanisms of leached components, or long-term leaching of hazardous trace metals [START_REF] Hillier | Long-term leaching of toxic trace metals from Portland cement concrete[END_REF][START_REF] Haga | Effects of porosity on leaching of Ca from hardened ordinary Portland cement paste[END_REF]. Other authors have studied the leaching characteristics of standard cement mortars during their whole life cycle, from first use to reuse or recycling purposes [START_REF] Van Der Sloot | Comparison of the characteristic leaching behavior of cements using standard (EN 196-1) cement mortar and an assessment of their long-term environmental behavior in construction products during service life and recycling[END_REF]. Van der Sloot advised situating leaching tests as closely as possible to the field conditions to be assessed. Studies that have focused on slag-based materials have emphasized the intrinsic properties of the structure and its geochemical behavior [START_REF] Faucon | Leaching of cement: Study of the surface layer[END_REF].

Our understanding of the potential environmental impact of this phenomenon is surprisingly limited. The fact that GBFS is widely used as a construction material merits a closer look at its geoenvironmental characteristics. The leaching tests were conducted on a standardized basis (monolithic and crushed samples) in order to simulate the behavior of foundation concrete (monolithic) and during a recycling process (crushed). The impact of rainfall on fair-faced (green) concrete was assessed by means of a configuration (sprinkling test) developed by the authors for this specific purpose. In addition, H2S emissions during demolding were determined using a second specific configuration designed the authors.

2 Materials and methods

Materials

Four industrial containing slag cements were used to formulate the concretes and cement pastes. As our primary focus was studying the greening effect and environmental implications of using cementitious materials containing various amounts of GBFS, only commercially available CEM III cements were used composed mainly of clinker and GBFS. This cement type is available in three classes differentiated by the amount of incorporated GBFS: CEM III/A (36-65% of GBFS), CEM III/B (66-80% of GBFS), and CEM III/C (81-95% of GBFS). For comparison purposes, we determined that the best reference material would be CEM I cement, which is mostly composed of clinker and minor additives, but contains no GBFS. The fineness of cements, given as Blaine specific surface area, was not modified, and ranged between 3600 and 5400 cm²/g (Table 1). Sulfides content in cement powders was quantified accordingly to the French cement norm, by acid distillation of sulfides and recovery of gaseous H2S in an alkaline antioxidant solution (NF EN 196-2, 2013). A notable modification of the recovery method and analytic technic was conducted. The outlet of the distillation apparatus was connected on the bubble jar which was filled with a Sulfides Antioxidant Buffer (SAOB) solution. The SAOB solution was prepared with 80 g of NaOH, 35 g of ascorbic acid and 67 g of EDTA for one liter. This solution was then analyzed using a Silver/Sulfide ion-selective electrode. All leachates were analyzed using the same method (see section II.4.d). Major composition of cements was determined by X-Ray Fluorescence (XRF) on a Bruker S4 Explorer spectrometer using SpectraPlus software (Table 1). Samples were prepared as melted beads by mixing 1 g of cement or GBFS with 8 g of Lithium Tetraborate (LiB4O7) in a Pt-Au crucible (95-5 % w/w). Melting was performed on an Autofluxer Breitlander fusion bead machine. Concretes were made with siliceous alluvial (Moselle river) aggregates used for producing concrete in northeastern France. The cement paste was made solely with CEM III/C, to assess the most concentrated sample in GBFS. Throughout the rest of this article, concrete samples will be referred to by the cements used (i.e. CEM I, CEM III/A, B or C) and the cement paste sample will be referred to as "P CEM III/C". Blaine surface area is given as cm²/g, GBFS content of cement is given as wt.%, sulfides content is given as g of sulfur for 100 g of cement, and major elements are given as oxides in wt.%.

Concrete and cement paste preparation and conditioning

Concrete and cement paste samples were prepared by mixing the required amounts of components in a concrete mixer (Controlab Perrier). Concrete was made with a water/cement (w/c) ratio of 0.5, with 420 kg/m 3 of cement, 210 kg/m 3 of water, 885 kg/m 3 of 0-5 mm sand, and 750 kg/m 3 of 4-8 mm gravel. Cement paste was made solely with water and cement with a w/c = 0.5. The mixing procedure was performed according to the NF EN 196-1 (09/2016) standard for concrete samples, and the NF EN 196-3 (09/2017) for cement pastes. Immediately after mixing, the paste was poured into cylindrical hermetically sealed HDPE bottles (12.5 cm height and 7.2 cm diameter) at 3 time intervals, each vibrated for 30 s on a vibrating table to remove air pockets within the sample. Samples were cured in the dark for 3 days in a tank at 25°C and with a relative humidity greater than 90%.

Color analysis

Coloration of the samples was examined using a portable spectrophotometer Konica Minolta CM-700d. This device allows a precise characterization of the color in each color space. We used a common color space defined in 1976 by the International Commission on Illumination known as CIE 1976 (L*a*b*) or CIELAB, according to the NF EN ISO/CIE 11664-4 (07/2019) standard. The L* parameter quantifies the brightness of the color (from darkest to lightest on a scale of 0 to 100), whereas a* and b* quantify the chromaticity. On a diagram, the color on the a* axis ranges between -60 (green) and +60 (red), and on the b* axis between -60 (blue) and +60 (yellow). All concrete and cement paste samples subjected to the different leaching tests were assessed for their color just before the test. The bottom face, being smaller, was analyzed for at least 3 different points, whereas the larger side curved face was analyzed for at least 5 different points. Values were averaged for each sample and each face, and then averaged for different samples.

Leaching tests

In the present study, we constructed three separate scenarios to determine where the greening effect might have an impact on the surrounding environment. The rainfall leaching of a freshly unmolded facing wall made with GBFS-rich cement is the scenario that is most visible by workers and outside observers. To assess potential leaching of hazardous substances, we used a custom designed test that involved sprinkling the block surface. The second scenario considered involved leaching by immersion of freshly unmolded building foundations, largely because GBFS-rich cements are frequently used in more aggressive wet environments. In this context, we used a standard monolith leaching test. The last scenario considered rainfall leaching in deconstruction of materials containing GBFS-rich cement, which constitutes the final step of building construction. In this case, a standard batch leaching test on crushed samples was used.

Sprinkling leaching test

On working sites, after removal of formwork, "green" surfaces of slag cement-based concretes may be subjected to rainy weather, especially heavy rainfall. To assess the effect of rainfall events on concrete surfaces, an innovative leaching test was designed (Fig. 2). Its process consists of sprinkling deionized water on a concrete or cement paste block and collecting it in a tank connected in a close circuit to the sprinkler using a peristaltic pump.

Fig. 2. Experimental device of the sprinkling leaching test -A block of sample is fixed above a

container filled with water. Water is pumped with a peristaltic pump, and sprinkled on the sample by a perforated tubing.

After a curing time of 3 days, a block of about 21.5 x 15.2 x 3.5 cm was fixed to the device above a container filled with about 5 L of water, with a tilt angle of 40°. A peristaltic pump (Watson-Marlow 323E) at the speed of 100 RPM sprinkled the water over the surface of the block on a mean surface of 370 cm 2 through a regularly perforated PVC tubing (one hole of a rate of roughly 1 mm every 4 mm). The mean liquid/area ratio (L/A) obtained was close to 13.5 cm 3 /cm 2 . The water flowed on the surface of the block at a rate of about 125 mL/min, and dropped into the container, to circulate again for a total of 4 hours. Marprene tubing was used between the container and the top PVC tubing.

Blue-green concrete

Water container Water circulation

Peristaltic pump

Before the first use, and between each use, the sprinkling device was cleaned with soap, HNO3 0.1M, and deionized water.

Monolith leaching test

In civil engineering, GBFS-based cements are commonly used in building foundations. In this case, the concrete can be subjected to water flooding, or leaching by infiltrated rainwater. A tank monolith leaching test following the XP CEN/TS 15862 (10/2012) standard has been used to assess the environmental impact of GBFS-based cement applied in a flooded environment. The principle is to immerse a solidified monolith in deionized water for 24h, without water renewal. The L/A ratio for all samples was defined to 12 cm 3 /cm² of exposed solid. Exposed samples were used directly after demolding for an average dimension of 12.5 cm height and 7.2 cm diameter. To ensure proper leaching on every faces, with a minimal 2 cm height of water above and below the sample, all samples were placed on a cut HDPE mesh at the bottom of an HDPE container of 5 L. A magnetic stirrer ensured the homogenization of the solution during the test.

Leaching test on crushed samples

The greening effect is often still visible when a building is demolished. Even after several decades, a concrete's interior can display a vivid blue-green color when exposed. This indicates that the demolition debris might be subjected to water leaching, either by rainfall or manual sprinkling. In this case scenario, a simple batch of crushed leaching was tested following the NF EN 12457-4 (12/2002) standard to assess the environmental impact of GBFS-based cement after deconstruction. The monolith was crushed with a jaw crusher to ensure 95% w/w of passing to 10 mm sieve. Once crushed, the sample was weighted for about 90g (dry weight) and poured into a HDPE bottle with the leaching water to ensure a L/S ratio of 10 L/kg. Preliminary tests allowed the determination of a mean water content, considering that the leaching test has to be carried out immediately after demolding and could not allow the proper case-by-case determination of the water content of each sample. After 3 days of curing, the tested concretes showed a mean water content of about 6.5%, and the CEM III/C cement paste showed roughly 25% water content. Real water content was determined for each sample to evaluate the deviation to the mean value used and adjust the L/S and L/A ratio. When water and the sample were mixed, the bottle was deoxygenated with N2 for about 8 min to limit the oxidation of solubilized sulfides. The bottles were then agitated on a turnaround shaker at 7 RPM for 24 hours.

Leachates analysis

Immediately after each test, leachates were vacuum filtrated on a 0.45 µm Sartorius cellulose acetate filter. The filtrates were analyzed for pH and conductivity on a portable multimeter (Knick Portavo® 907 Multi), and divided into 3 subsamples: one subsample without other treatment for the anion analysis, one acidified with 2 mL HNO3 65% analytical grade (Merck) for elemental analysis, and one mixed at a 1:1 ratio with SAOB for sulfides analysis. We focused on anion analysis of the leachates on the sulfur compounds, since the presence of several sulfur anions, i.e. S 2-, S2O3 2-and SO4 2-, has been regularly reported in leachates of hydrated GBFS and cements containing GBFS [START_REF] Lothenbach | Hydration of a low-alkali CEM III/B-SiO2 cement (LAC)[END_REF][START_REF] Gruskovnjak | Hydration of alkali-activated slag: comparison with ordinary Portland cement[END_REF][START_REF] Glasser | Modification of cement pore fluid compositions by pozzolanic additives[END_REF]. These species have been shown to be thermodynamically stable considering the alkaline and mildly reductive environment occurring in the leachates [START_REF] Pourbaix | Potential-pH equilibrium diagrams for the system S-H2O from 25 to 150° C: Influence of access of oxygen in sulphide solutions[END_REF]. Sulfides analysis was carried out accordingly to a modified USEPA method 9215 (12/1996). The SAOB solution was prepared with 80 g NaOH, 35 g of ascorbic acid and 67 g of EDTA for one liter. An alkaline solution (pH > 10) favored the trapping of sulfides under the S 2-form, protected from oxygen oxidation by a reducing agent as the ascorbic acid and by trapping of metals with EDTA. Sulfides were then analyzed with an ion-selective electrode Thermo Scientific Orion Silver/Sulfide 9616BNWP, plugged on an HP multimeter (model 3478A). An electrode was calibrated with a serial dilution of a stock solution of Na2S. The stock solution was sulfide ISE standard of 1000 mg/L bought at Mettler-Toledo and prepared daily with Roth analytical grade Na2S in 0.4M NaOH. The stock solution was standardized with an AgNO3 solution, calibrated beforehand with a KCl solution, using a Tacussel TT-Processeur 2 automated titrator equipped with the ion-selective silver/sulfide electrode. The subsamples for anion analysis (SO4 2-, S2O3 2-, Cl -) were filtrated on 0.22 µm Sartorius filter and stored in the dark at 4°C. The analysis was conducted on an ion chromatography instrument (Metrohm 882 Compact IC) with a chemical suppressor and conductimetric detection, equipped with a Metrosep A Supp 4 column (250 x 4.0 mm) and a Metrosep A Supp 5 Guard/4.0 precolumn (5 x 4.0 mm). A mix Na2CO3 1.8 mM / NaHCO3 1.7 mM / 2% acetone was used as a mobile phase with a 1 mL/min flow rate and an injection volume of 20 µL. Standard solutions for calibration were prepared in the laboratory from a certified Alfa Aesar multi-anions commercial solution (ref. 041693) at 1000 µg/mL. Concentrations were determined from peak areas. Major element analyses (Si, Al, Fe, Mn, Mg, Ca, Na, K, Cr) were carried out by ICP-OES (Thermo Scientific iCAP 6500) on acidified subsamples.

H2S assessment

A custom designed device was used to assess the potential release of H2S during the demolding of concretes containing GBFS (Fig. 3). This instrument is composed of a cell with a cylindrical section with transparent PMMA (approx. 10.1 cm diameter and 25.4 cm height), and top and bottom sections made of PTFE with an O-ring to ensure water and air tightness. Openings are situated on the lower section and underneath to allow for inlet and outlet of fluids. The bottom cap is pierced and a cable gland is inserted to allow for vertical movements of a metal rod equipped with a PTFE bucket.

Concrete is poured into the bucket, with a metal hook fixed at the top cap that dips into the concrete. After 3 days, once the concrete has hardened, the hook becomes firmly secure and pulling the metal rod allows the concrete cylindrical faces to be exposed. During the demolding process, a constant argon flow is injected by the inlet valve at the bottom of the cell, and a pipe is connected to a washing bottle filled with 200 mL of SAOB solution to trap H2S gas. The sulfide analysis of the SAOB solution was performed with the silver/sulfide ion-selective electrode following the protocol described previously (section II.4.d.).

Blue-green concrete

Results

The greening effect of GBFS-containing concrete has gained increasing attention as a public health concern, in part due to its visibility. From the demolding stage through to building deconstruction, a number of known environmental events can cause deleterious interactions with green materials and release potentially harmful compounds into the environment that could pose a risk to human health.

Analysis of the greening effect

The color of GBFS-containing materials was evaluated just prior to setting-up the leaching tests. The effect of the GBFS concentration in the different samples was highlighted by the shifting of the color to the green-blue side of the color space. Ranging from -0.24 for CEM I to -4.43 for the cement paste of CEM III/C, a* decreased while the GBFS content increased in the mixture (Fig. 4a). This indicates that the color turning more green corresponded with increased levels of GBFS (see Fig. 4b for example). For the blue-yellow axis b*, the overall trend showed a decrease from 3.29 for CEM I to -1.88 for the cement paste of CEM III/C. The color then shifted toward blue with increased GBFS content. However, in this case, standard deviation between samples was considerably higher than for a*, and ranged between 0.32 for CEM I sample and 0.9 for the cement paste of CEM III/C (for a* it was contained in a 0.12-0.32 range). On an a*-b* plot, the samples are more scattered along the b* axis than the a* axis (Fig. 4c). Samples with the same mixture appear gathered in clusters, and those clusters are fairly widely spaced vertically. Results for L* are not shown, as variation of the brightness for the different samples is close in range (between 47.5 and 58.3 with SD from 2.65 to 7.77), without any identifiable trend.

Rainfall leaching of building faces: sprinkling test

The first leaching event on a freshly unmolded wall normally comes from rainfall. Given that classical leaching tests for assessing environmental impact are less practical for gauging rainfall leaching on horizontal concrete surfaces, we relied on our own test designed specifically for this purpose, as described previously (cf. section II.4.a and Fig. 2).

Concrete and cement pastes are alkaline mineral materials, as shown in our pH analysis following leaching (Fig. 5). Concretes made with CEM III/A and CEM III/B are the most alkaline when subjected to a surface dynamic leaching, with a pH value of about 11. For leaching tests based on a leached surface, concentrations in eluates as mg/L are normalized with the L/A (L/m 2 ) ratio as mg/m 2 . Concentrations of sulfur anions into the leachates increased when the GBFS ratio in concretes/paste also increased (Fig. 6). Sulfides were observed to be the least released anions and varied from 10.6 mg/m² for CEM III/A, the least concentrated with GBFS, to 25.8 mg/m² for the CEM III/C cement paste. On the other hand, sulfates were the measured anion that showed the highest release levels and followed the same pattern, from 524 mg/m² for CEM III/A concrete to 918 mg/m² for the CEM III/C cement paste. Thiosulfates were released at roughly the same concentration for all samples, between 180 and 230 mg/m². Overall, sulfur was predominantly under its higher oxidation degree in leachates, with more than 70% as sulfates, and 15 to 25% in thiosulfates form.

Building foundations flooded by water: tank monolith leaching test

One of the primary uses of cement blended with GBFS is in building foundations. In this case, the concrete is most likely to be subjected to immersion by groundwater infiltration, as well as leaching by water percolating from the surface. We conducted a tank monolith leaching test to assess this specific situation.

Alkalinity of leachate solutions showed to be higher in this test (Fig. 7). The pH ranged from 11.33 for CEM III/C, to 11.9 for CEM I, and evolved in the same way as observed previously for the sprinkling test. The global increase may be explained by the longer test duration (24 hr. versus 4 hr. for the sprinkling test). The CEM III/C cement paste was more alkaline than the concrete, due to the higher concentration of binder in the absence of the concrete's aggregate skeleton. Conductivity followed the same scheme, showing ranges from 0.46 mS/cm for CEM III/C concrete to 1.14 mS/cm for CEM I concrete. Conductivity was also higher for the CEM III/C cement paste than for the concrete, with 0.64 mS/cm. Sulfur speciation leaching between all samples was significantly more pronounced under these leaching conditions (Fig. 8). The concrete made with CEM I only exhibited release of sulfates, for 108 mg/m². Increasing amounts of sulfates were released in samples that positively correlated with higher GBFS content, as observed previously in the sprinkling test, from 132 mg/m² for CEM III/A concrete to 960 mg/m² for the CEM III/C cement paste. The proportion of thiosulfates released was roughly 20-30% of the total amount of sulfur anions, except for CEM III/A concrete, and ranged from 192 mg/m² for CEM III/A concrete to 312 mg/m² for CEM III/C cement paste. Furthermore, sulfides were also detected and quantified in leachates and, similarly, corresponded with the GBFS content, up to 52.7 mg/m² for the CEM III/C cement paste.

Fig. 8. Concentration of sulfides S 2-, thiosulfates S2O3

Deconstruction products leaching by rainfalls: leaching test on crushed samples

Deconstruction is the other aspect to take into account when it comes to common uses of slagcontaining cements. A leaching test on crushed samples was used to assess the potential release from deconstruction debris subjected to both natural rainfalls and artificial sprinkling.

Leachates from the crushed leaching tests showed to be the most alkaline of the three tests (Fig. 9). Regardless of the type of cement used, all leachates exceeded 12 pH units, and ranged from 12.10 for CEM III/C concrete to 12.49 for CEM I concrete. Conductivity was also higher, with 1.92 mS/cm for the CEM III/C concrete to 7.36 mS/cm for the CEM I concrete. For leaching tests dependent on the mass of the assessed materials, units in mg/L were normalized with the L/S (L/kg of dry weight material) ratio as mg/kg dry weight (DW). High releases of sulfur anions were observed in the crushed leaching test (Fig. 10). The CEM I concrete released a negligible amount of thiosulfates, about 11 mg/kg DW, but no sulfates were found in the leachates. As such, thiosulfates represented from one to two thirds of the total sulfur anions found in other concretes and cement paste, ranging from 58 to 154 mg/kg DW. The other form of sulfur was sulfates, with 31 and 25 mg/kg DW respectively for CEM III/A and B concretes leachates, and increased to 204 mg/kg DW for CEM III/C concrete, and 112 mg/kg DW for CEM III/C cement paste. Sulfur content was noticeably well divided between the 3 forms analyzed for the CEM III/C cement paste, given that close to 165 mg/kg DW of sulfides was recovered in the leachates.

H2S assessment

A typical phenomenon during formwork removal blue-green concrete is the presence of a characteristic odor resembling the distinct smell commonly associated with hydrogen sulfide. The main issue with use of this particular compound is one of safety; its high toxicity may increase health and environmental risks. According to WHO guidelines, the recommended safe exposure limit is 0.15 mg/m 3 over a 24 hour period [START_REF]Hydrogen Sulfide[END_REF].

The purpose of our customized instrument was to detect and quantify the potential emission of H2S during the unmolding process. Argon was set to flow for 30 minutes after demolding, and bubbled in 200mL SAOB solution to trap emitted H2S. No sulfides were recovered with this method. The limit of detection of the ISE silver/sulfide electrode is about 3.10 -3 mg/L. Under the experimental conditions, this equated a minimum of 6.10 -4 mg emitted by the concrete that could be quantified in the final solution.

4 Discussion

Trace and major elements releases in the different scenarios

In this study, three scenarios involving the potential impact of the greening effect of GBFS-rich cement were considered. Trace and major elements were analyzed and quantified in the leachates. Despite relatively recent investigations pointing to the potentially important role of sulfur compounds in the greening effect process, notably under the sulfide form, it was previously thought that other compounds might also be involved. This was the case for iron and manganese compounds [START_REF] Sioulas | The coloration phenomenon associated with slag blended cements[END_REF][START_REF] Schwab | Characteristics of Blast Furnace Slag Leachate Produced Under Reduced and Oxidized Conditions[END_REF][START_REF] Mansfeldt | Identification of a Crystalline Cyanide-Containing Compound in Blast Furnace Sludge Deposits R. Dohrmann, present address: Bundesanstalt für Geowissenschaften und Rohstoffe (BGR), D-30655[END_REF], and even chromium salts such as CrCl3.6H2O. Accordingly, we included these elements for analysis in the leachates, and paid particularly close attention to chromium content for the known health hazards associated with its exposure. In the three cases, no Cr element was found in the leachates (detection limit was 0.06 mg/L), and Fe and Mn elements were under the detection limit, or showed statistical uncertainty near the detection limit (respectively 0.01 mg/L and 0.001 mg/L). This is consistent with the literature on the use of cement (either with or without GBFS) in cementitious solidification/stabilization (S/S) treatment processes of hazardous wastes, including those contaminated with chromium [START_REF] Rha | Investigation of the stability of hardened slag paste for the stabilization/solidification of wastes containing heavy metal ions[END_REF]. Moreover, Fe and Mn were not expected to be leached considering their significantly low solubility in the alkaline conditions of cementitious materials [START_REF] Scrivener | Hydration of cementitious materials, present and future[END_REF].

We observed Na, K, Ca, Al, and Si among the major elements released by cement and GBFS leaching, although their release through rainfall leaching or solubilization and diffusion into groundwater does not typically represent an environmental hazard. While cement and blast furnace slag have similar raw composition, mainly composed of CaO, SiO2 and Al2O3, proportions of oxides varied. Compared with Portland cement, GBFS is richer in SiO2 and Al2O3, and depleted in CaO [START_REF] Scrivener | Hydration of cementitious materials, present and future[END_REF]. In fact, CEM I is composed of nearly 62.9% of CaO, 5.4% of Al2O3, and 20.7% of SiO2 (Table 1 .04 < L.D.: result is lower than the limit of detection Description: Concentrations of major elements (Na, Ca, K, Al, Si) and sulfur anions (S 2-, SO4 2-and S2O3 2-) are given as mmol/m² for the sprinkling leaching test, mmol/m² for the monolith tank leaching test and mmol/kg DW for the crushed leaching test. Ca/Si ratio is displayed, as well as the calculation of a ratio between the sum of the 5 major elements (Na, Ca, K, Al, Si) over the sum of the three sulfur anions (S 2-, S2O3 2-and SO4 2-), indicated as ∑cations/∑anions ratio for simplification. increased levels of Al2O3 and SiO2. The lack of available Ca in GBFS high substituted systems leads to the consumption of Portlandite hydrates to allow precipitation of C-S-H, and the enrichment in Al2O3 of C-S-H to form C-A-S-H [START_REF] Escalante | Reactivity of blastfurnace slag in Portland cement blends hydrated under different conditions[END_REF][START_REF] Kolani | Hydration of slagblended cements[END_REF]. This behavior can be observed in the leachates chemistry and matches our own results of the Ca/Si ratio of leachates (Table 2). We observed an overarching trend in Ca and Si releases of a decreased Ca/Si ratio that appeared to correlate with increased GBFS content, presumably due to the fact that the Calcium is consumed by C-S-H incorporated into GBFS containing pastes. This was particularly the case for the crushed test and the monolith tank test, where CEM I concrete showed a Ca/Si ratio 5 to 30 times higher than the other concretes and cement paste. Consequently, Ca/Si decreased in the following order: CEM III/A > CEM III/B > CEM III/C. Calcium leaching mostly comes from the dissolution of Portlandite and interstitial solutions, whereas Si comes from the incongruent dissolution of C-S-H [START_REF] Faucon | Leaching of cement: Study of the surface layer[END_REF]. In the crushed test, the cement paste had a higher ratio than CEM III/C, given that the overall concentration was not diluted by aggregates. This was not observed for the other tests specific to surface leaching, unlike the crushed test.

The second effect of portlandite dissolution is that the alkalinity of the leachates increases. Cement containing more GBFS contains less clinker and consequently lowers CaO levels. If less portlandite forms when the concrete or cement paste is leached, less portlandite is dissolved which induces a lower pH. The pH ranges obtained for each test differed: the sprinkling test showed a pH range of 10.88-11.26, the monolith tank test showed a pH range of 11.33-11.90, and the crushed test pH range was observed a 12.10-12.49 (Fig. 5, 7 and 9). These differences depend, to a certain degree, on the testing method itself. The sprinkling test is carried out over a relatively short time duration and only leaches the surface of the block, often a very thin superficial layer. In the monolith tank test, the block was immersed for 24 hours, wherein water could penetrate the first millimeters of the surface, allowing a deeper leaching by diffusion. Finally, the crushed leaching test involved a highly specific surface due to block crushing, which allowed a high dissolution of hydrates and access to inner portions of the block.

Examining electrical conductivity of leachates is a particularly reliable way to assess the behavior of concrete and cement blocks subjected to various leaching test conditions. Conductivity ranges for the sprinkling test were between 0.17 and 0.36 mS/cm, for the monolith test between 0.46 and 1.14 mS/cm, and for the crushed test between 1.92 and 7.36 mS/cm (Fig. 5, 7 and9). The crushed test proved by far to be the most "extractive" method, followed by the monolith tank test, and then the sprinkling test. Moreover, the same observations were made for a similar leaching test which showed a decrease of conductivity in leachates when GBFS content increased. In addition to the availability of the more soluble species (i.e. Ca(OH)2 in particular), the porosity might also affect the dissolution, diffusion, and subsequent leaching of the more soluble species. In a solidified cement paste, and by extension in concrete, the mass transport is controled by the diffusion mechanism, and depends on the matrix' porosity [START_REF] Haga | Effects of porosity on leaching of Ca from hardened ordinary Portland cement paste[END_REF]. Importantly, GBFS impacts porosity, and effectively will lower the overall porosity of concretes and cement paste [START_REF] Ramezanianpour | Effect of curing on the compressive strength, resistance to chloride-ion penetration and porosity of concretes incorporating slag, fly ash or silica fume[END_REF]. Thus, blocks with a high level of GBFS not only have less Portlandite, but are also less likely to diffuse and release elements and ions due to lower permeability. The amounts of the major elements (Na, Ca, and K) released in leachates decrease when GBFS increases (Table 2). Compared to those elements, the trend of Al is different. Between CEM III/A, B, and C concretes, Al slightly varies in a close range, 4.31-6.25 mmol/m² for sprinkling test, 4.24-5.23 mmol/m² for monolith tank test, and 0.74-0.84 for crushed test, with CEM III/B being lower than for CEM III/A and CEM III/C. This is consistent with the cement composition, as CEM III/B has a lower Al2O3 content than the two others with 9.56% versus 9.91 and 10.54% respectively for CEM III/A and CEM III/C (Table 1). Furthermore, despite the higher content of Al2O3 in GBFS-rich concretes, Al leaching is most likely limited by the solubility of the incorporated hydrates, or Al dissolution may be controlled by precipitation of secondary Al-oxyhydroxides phases [START_REF] Piatak | Characteristics and environmental aspects of slag: A review[END_REF].

The leaching tests used in this study were designed to represent selected scenarios where a bluegreen material is subjected to bad weather or environmental situations leading to surface or mass leaching. The choice of the monolith tank test and the crushed leaching test was made to follow as close as possible the related standard (i.e. NF EN 14957-4 12/2002 and XP CEN/TS 15862, 10/2012). For future environmental assessment of construction materials, these initial conditions could be improved. One European standard currently in development aims specifically to normalize the ecotoxicological assessment of construction materials and suggests reducing the L/S or L/A to concentrate the released compounds (draft standard "Construction Products -Assessment of release of dangerous substances -Determination of ecotoxicity of construction product eluates" FprCEN/TS, 2019). This could be specifically pertinent when applied to the monolith test, which by design concentrates on gauging minimal quantities of leaching solution required to surround the sample. Regarding the released concentrations in the crushed test, a L/S of 10 might be sufficient. This particular test, however, is possibly the most distant approximation of a real-life scenario, more so than the other two, in that it simulates a more aggressive environment than what would typically be associated with construction debris. It is highly unlikely than such debris would remain immersed in a large amount of water. It is more likely to be sporadically sprinkled with water by workers or rainfall. In this case, an innovative test could be developped, such as test that might account for column leaching but with a transient water regime, including dry phases, similar to those used in other fields as for marine sediments [START_REF] Couvidat | An innovative coupling between column leaching and oxygen consumption tests to assess behavior of contaminated marine dredged sediments[END_REF]. In the same way, the sprinkling leaching test was designed to fill a gap, since no surface dynamic test was found in literature. Even if the water regime is continuous, this test is probably the most realistic in terms of releases, although the L/A is the highest with about 13.5 cm 3 /cm 2 . This L/A is calculated as the ratio of the total volume of leaching solution (5000 cm 3 ) by the leached area of the block (about 370 cm 2 , representing the top surface and one of the small sides). In this case, the L/S might be reduced, by decreasing the volume of leaching solution. Moreover, this test accounts for a virtual L/A, by taking into account the total volume of 30 liters sprinkled, because the pump is set to a speed of 125 mL/min, and the test duration is 4 hrs. On a mean surface of 370 cm² during the duration of the test, a mean L/A of 79 cm 3 /cm 2 is obtained. This is effectively close to an annual rainfall of about 800 mm, which is equal to 80 cm 3 /cm 2 .

Sulfur speciation in leachates

Unlike major elements, sulfur anions tend to increase in leachates when GBFS concentrations are higher in concretes and cement paste (Table 2). However, major elements and sulfur anions are not released in the same proportions. The sum of the 3 sulfur anions (S 2-, S2O3 2-and SO4 2-) is always at least 10 times lower than the sum of the 5 major elements released (Na, K, Ca, Si, Al). For the sprinkling test, the ∑cations/∑anions ratio increased from 12 for the CEM III/C cement paste to 27 for CEM III/A concrete. For the monolith tank test, this ratio expanded from 17.2 for P CEM III/C to 108.7 for CEM III/A concrete, and even 617.6 for CEM I concrete. Finally, for the crushed leaching test, the ratio went from 14.0 for P CEM III/C to 149.8 for CEM III/A, and 1859.4 for CEM I.

The increase of sulfur anions in the leachates of GBFS-rich materials is not surprising, given that GBFS includes noticeable quantities of reduced sulfur compounds which promptly react when GBFS is hydrated. This mechanism is probably at the origin of the blue-green color. Unlike clinker, essentially all sulfur in GBFS is present in sulfide form due to the reducing conditions in the blast furnace [START_REF] Glasser | Modification of cement pore fluid compositions by pozzolanic additives[END_REF][START_REF] Roy | Sulfur speciation in granulated blast furnace slag: An X-ray absorption spectroscopic investigation[END_REF]. Cement containing up to 90% GBFS can reach a redox potential of -250 to 350 mV (versus calomel electrode) [START_REF] Macphee | Phase Development and Pore Solution Chemistry in Ageing Blast Furnace Slag-Portland Cement Blends[END_REF][START_REF] Angus | The chemical environment in cement matrices[END_REF]. Thus, sulfur speciation might take different forms in interstitial water, from sulfides (oxidation degree of -II) to sulfates (+VI) [START_REF] Lothenbach | Hydration of a low-alkali CEM III/B-SiO2 cement (LAC)[END_REF][START_REF] Gruskovnjak | Hydration of alkali-activated slag: comparison with ordinary Portland cement[END_REF]. When GBFS-rich material is subjected to leaching, those sulfur species can be easily leached, and most oxidizable species, i.e. sulfides, might be readily oxidized when leached, depending on the oxygen diffusion during the leaching test. The three tests performed in this study showed not only the presence of sulfates in leachates, but also thiosulfates and sulfides. Up to 0.80 mmol/m² (0.19 mg/L measured in the solution) of sulfides were quantified in the sprinkling test for the CEM III/C cement paste. Sulfides concentration reached 1.64 mmol/m² (0.44 mg/L in the solution) for the same material in the monolith tank test, and 5.12 mmol /kg DW (16.55 mg/L) in the crushed leaching test. In the same way, thiosulfates in the CEM III/C cement paste was quantified to 2.08 mmol/m², 2.78 mmol/m² and 1.37 mmol/kg DW respectively in the sprinkling test, monolith test, and the crushed leaching test. Thermodynamically, sulfides are oxidized in sulfates. However, it has been shown than the main products of sulfides' oxidation by molecular oxygen at pH > 8.5 are thiosulfates, and oxidation to the most stable product can take weeks [START_REF] Chen | Kinetics of oxidation of aqueous sulfide by oxygen[END_REF][START_REF] Kleinjan | Kinetics of the chemical oxidation of polysulfide anions in aqueous solution[END_REF][START_REF] O'brien | Kinetics of oxygenation of reduced sulfur species in aqueous solution[END_REF]. From the three leaching tests used in this study, the sprinkling test was estimated to favor oxygen diffusion by creating a thin moving water slide upon a large area, unlike the monolith tank test for example. Yet, despite the bottles in the crushed test being purged with N2, it is likely that oxygen concentrations were lowered, but not removed, as a function of the short duration time of the bubbling. Thus, even with reduced oxygen content, the crushed leaching test is more likely highly oxidative for released sulfides because of the 24 hours of turnaround shaking. Our results show a relatively low recovery of sulfides, for example for CEM III/B concrete, in the crushed samples test compared to the monolith test (0.02 mmol/kg DW versus 0.63 mmol/m², or 0.06 mg/L versus 0.17 mg/L in the leachates solutions) (Table 2). Moreover, CEM I only released about 1.12 mmol/m² of sulfates in the monolith test, or 0.10 mmol/kg DW of thiosulfates for the crushed test, meaning that for other samples, the majority of sulfur anions leached comes from the sulfides leaching and oxidation originated from GBFS. Finally, it is not surprising not to find gaseous H2S during the unmolding process. With a pKa1 = 7.04 and a pKa2 = 11.96, and considering the strong alkaline pH, it seems unlikely that sulfides turn into H2S.

The most striking aspect of the sulfur chemistry occurring in GBFS-rich materials is the origin of the blue-green color. In fact, the intensity of the color effect is basically a slag conditional mechanism and is related to sulfides content. In particular, the association of the three chromophorous radical ions S4 •-, S3 •-, S2 •-is responsible for the blue-green color of the hydrated slag [START_REF]Slag Cement Association, Slag Cement in Concrete N°10[END_REF]. In Lapis Lazulis, the mix of chromophores S2 •-(yellow) and S3 •-(blue) in zeolithe-type structures explains the ultramarine colors ranging from deep blue to green [START_REF] Steudel | Inorganic Polysulfides S n 2-and Radical Anions S n[END_REF]. For cement, the appearance of the greening color depends more highly on the replacement ratio of clinker by GBFS (Fig. 4). From almost 100% of clinker (for CEM I) to 85% replacement by GBFS (CEM III/C), the color shifts to green, toward -a*, and blue, toward -b*. Vernet proposed that the color is concentrated in a fraction containing a solid solution of sulfides and sulfates in an aluminate hydrate [START_REF] Vernet | Comportement de l'ion S--au cours de l'hydratation des ciments riche en laitier (CLK)[END_REF]. The precise chemistry governing the appearance and disappearance of the blue-green color has not yet been determined. However, the disappearance of the color probably involves an oxidation of the sulfides by molecular oxygen penetrating in the superficial surface. Preliminary experiments showed that the color took some weeks to disappear. Below 20 cm of water, the color does not visually change for months.

Finally, concerning the human health impact of such leachates, we detected no hazardous substances, except dissolved sulfides. Regarding these dissolved sulfides, there is no threshold value in any recommendation from Word Health Organization.

Gas emission from use of hydrogen sulfides is unlikely to occur, but depending on the GBFS content, sulfides might be released during leaching by rainfall or groundwater. As chemical oxidation of sulfides to sulfates by molecular oxygen is a slow process, sulfides might be spilled into groundwater [START_REF] Luther | Thermodynamics and Kinetics of Sulfide Oxidation by Oxygen: A Look at Inorganically Controlled Reactions and Biologically Mediated Processes in the Environment[END_REF]. However, two processes will quickly prevent any accumulation or damages to the environment. Firstly, even if the chemical oxidation is slow, some ionic species act as catalysts for this reaction, such as Mn 2+ , Fe 2+ or Cu 2+ , and reduce the meta-stability of sulfides from one or two orders of magnitude [START_REF] Dohnalek | The chemistry of reduced sulfur species and their removal from groundwater supplies[END_REF][START_REF] Zhang | The products from the oxidation of H2S in seawater[END_REF]. Biotic oxidation with sulfo-oxidant bacteria is also highly effective for sulfides oxidation [START_REF] Luther | Thermodynamics and Kinetics of Sulfide Oxidation by Oxygen: A Look at Inorganically Controlled Reactions and Biologically Mediated Processes in the Environment[END_REF]. Additionally, precipitation with trace metals is another means of removing sulfides from the leaching solution, since metals such as Zn (pKs = 23 for Sphalerite), Cu (pKs = 47.6 for Cu2S) or Pb (pKs = 27 for PbS) form insoluble sulfides [START_REF] Lewis | Review of metal sulphide precipitation[END_REF]. Incidentally, no threshold was determined for soluble sulfides by WHO [START_REF]Hydrogen Sulfide[END_REF]. Concerning the environmental impact, sulfate is the only compound which can be potentially problematic. For comparison purposes, the values obtained in this study can be compared to French legislation, concerning the admission of inert wastes into classified installation [START_REF]des déchets inertes dans les installations relevant des rubriques 2515, 2516, 2517 et dans les installations de stockage de déchets inertes relevant de la rubrique 2760 de la nomenclature des installations classes[END_REF]. Concrete is included in the category of recognized inert wastes. For this purpose, the waste is submitted for leach testing and needs to comply with threshold values for 18 parameters. Sulfates are one of the parameters, with a threshold value of 6000 mg/kg DW for L/S = 10 ml/g DW. Also, a more severe threshold is observed for the reuse of slags in road construction, with a threshold value for sulfates of 1300 mg/kg DW for type 3 use [START_REF] Chateau | Guide méthodologique -Acceptabilité de matériaux alternatifs en technique routière -Evaluation environnementale[END_REF]. The highest value obtained in this study was 204 mg/kg DW for CEM III/C concrete, almost 20 times lower than the legislation threshold (Fig. 10).

For a more thorough environmental study of construction materials, additional aspects might well be considered. In particular, beyond the chemical leaching behavior, it could be useful to assess the ecotoxicological aspects [START_REF] Bandow | Evaluation of the impact of construction products on the environment by leaching of possibly hazardous substances[END_REF].

Conclusions

Greening effect occurs either during removal of formwork of blast furnace cement (CEM III) based concrete, or demolishing CEM III based old concrete, and has raised question about its environmental and health safety. Three scenarios were studied, with a specific leaching test for each:

-Freshly demolded fair faced blue-green concrete subjected to leaching by rainfall was assessed with an innovative leaching test designed to sprinkle water on the surface of a concrete block; -CEM III based concrete of building foundations immersed in groundwater was assessed with a standard monolith leaching test; -Demolition debris of old concrete blocks containing GBFS, sprinkled with water by workers or by rainfall was assessed with a standard batch leaching test on crushed samples.

Three cements with GBFS were selected (CEM III/A, CEM III/B and CEM III/C) to form concrete blocks and one cement paste (with CEM III/C cement), and one reference (CEM I, clinker only) as concrete.

The greening effect was observed to correlate with concentrations of GBFS, wherein the color shifted to green and blue with the increase of GBFS content. Slag content in concretes and cement paste was also linked to a higher release of sulfur anions in the leachates, in the form of sulfides, thiosulfates and sulfates. Compared to the very low releases of sulfur anions in CEM I leachates, 108 mg/m 2 of sulfates in tank monolith test and 11 mg/kg of Dry Weight (DW) of thiosulfates in a crushed leaching test, it appears that most of these anions came from GBFS, where sulfur is mainly under sulfides form. As such, thiosulfates and sulfates are the main oxidation products of sulfides included in GBFS. For example, sulfates release in the monolith tank test for CEM III/C concrete is 588 mg/m 2 , with 30.1 mg/m 2 of sulfides and 234 mg/m 2 of thiosulfates. Nevertheless, sulfates levels reported here are lower than thresholds for inert wastes or for reuse of alternate materials in road construction. Moreover, no chromium, or other hazardous metals, were detected in leachates. The major elements released were Na, K and Ca.

Fig. 1 .

 1 Fig. 1. Examples of the "greening effect" in various sectors of construction -Photographs illustrating the "greening effect" in different sectors of construction: a) civil engineering, b) pre-cast industry, c) Flooring works, d) masonry works.

Fig. 3 .

 3 Fig. 3. Experimental device for H2S emission assessment -A closed chamber contained a concrete sample in a mold, maintained by a screw to the cap of the device. A metal rod can move vertically in the bottom of the device to remove the mold and expose the sample to an Ar flux bubbling in an alkaline protective solution to trap H2S.

Fig. 4 .

 4 Fig. 4. Color analysis of concrete and cement paste samples -Average values for a* and b* parameters of concrete and cement paste samples (a), pictures of concretes and cement paste (b), and dispersion of samples on a* and b* axis depicted on the chromatic circle representing the L*a*b* CIE 1976 color space, modified from www.colorimax.com (c).

Fig. 6 .

 6 Fig. 6. Concentration of sulfides S 2-, thiosulfates S2O3 2-, and sulfates SO4 2-(as mg/m 2 ) in leachates of the sprinkling test from concretes and cement pastes -Sulfates (blue diagonal up bars) are the most released anions with concentrations ranging from 524 mg/m² for CEM III/A to 918 mg/m² for P CEM III/C. Thiosulfates (green dotted bars) releases are close between the four samples, with concentrations between 180 and 230 mg/m². Sulfides (orange plain bars) releases are very low, from 10.6 mg/m² for CEM III/A to 25.8 mg/m² for P CEM III/C.

Fig. 7 .

 7 Fig. 7. pH and conductivity in leachates of the tank monolith leaching test from concretes and cement pastes -pH in leachates (blue plain bars) ranges from 11.33 for CEM III/C sample to 11.90 for CEM I reference sample. Conductivity (blue dotted bars) ranges from 0.46 mS/cm for CEM III/C sample to 1.14 mS/cm for CEM I reference sample.

Fig. 9 .

 9 Fig. 9. pH and conductivity in leachates of the crushed leaching test from concretes and cement pastes -pH in leachates (orange plain bars) ranges from 12.10 for CEM III/C sample to 11.49 for CEM I reference sample. Conductivity (orange dotted bars) ranges from 1.92 mS/cm for CEM III/C to 7.36 mS/cm for CEM I reference sample.

Table 1 . Composition of cements used for concretes and cement pastes preparation

 1

		Blaine
		surface	GBFS	S 2-	Al2O3 CaO Fe2O3 K2O	MgO MnO Na2O SiO2	SO3
		area
		cm 2 /g wt.% g(S)/100g			wt.%
	CEM I	4370	0	-	5.44 62.91 3.03	0.81	1.89	0.08	0.20 20.69 3.33
	CEM III/A 5400	62	0.61	9.91 48.91 1.84	0.55	4.64	0.39	0.35 29.74 2.72
	CEM III/B 3643	71	0.55	9.56 47.62 1.15	0.55	4.99	0.24	0.22 31.32 3.52
	CEM III/C 4662	85	0.74	10.54 44.87 0.79	0.34	5.80	0.28	0.31 33.74 3.00

Description: Composition of CEM I, CEM III/A, CEM III/B and CEM III/C cements is given in this table.

. pH and conductivity in leachates of the sprinkling test from concretes and cement pastes

  [START_REF] Vernet | Comportement de l'ion S--au cours de l'hydratation des ciments riche en laitier (CLK)[END_REF]. For concrete with CEM III/C and cement paste of CEM III/C, the pH is lower, around 10.9. Conductivity of the samples decreased from 0.36 mS/cm for CEM III/A concrete to about 0.18 mS/cm for CEM III/C concrete and cement paste. Conductivity in this case appeared to increase with the Portland clinker content. -pH in leachates (green plain bars) ranges from 10.88 for P CEM III/C sample to 11.27 for CEM III/B sample. Conductivity (green dotted bars) ranges from 0.17 mS/cm for CEM III/C sample to 0.36 mS/cm for CEM III/A sample.

	Fig. 5
				Conductivity (mS/cm)
			1.00	0.50		0.00
	P CEM III/C		10.88		0.19
	CEM III/C		10.92	pH	0.17
	CEM III/B		11.27	Conductivity	0.29
	CEM III/A		11.26	0.36
	10	pH	11	12	13	14

2-, and sulfates SO4 2-(as mg/m 2 ) in leachates of the tank monolith leaching test from concretes and cement pastes

						Conductivity (mS/cm)
	3		2		1	0
	P CEM III/C		11.54			0.64
	CEM III/C		11.33		pH	0.46
	CEM III/B		11.61	Conductivity	0.75
	CEM III/A		11.73	0.86
	CEM I			11.90	1.14
	10	pH	11	12	13	14
	P CEM III/C
	CEM III/C
	CEM III/B					Sulfides
	CEM III/A					Thiosulfates
	CEM I					Sulfates
	0	200 400 600 800 1000 1200 1400
			Concentrations (mg/m²)
					-Sulfates (blue diagonal up bars)
	are released with concentrations ranging from 108 mg/m² for CEM I reference sample to 960 mg/m²
	for P CEM III/C sample. Thiosulfates (green dotted bars) releases range from 192 mg/m² for CEM III/A
	sample to 312 mg/m² for P CEM III/C. Sulfides (orange plain bars) releases are low, between 6.58
	mg/m² for CEM III/A sample to 52.67 mg/m² for P CEM III/C sample. Thiosulfates and sulfides were
	not detected in CEM I leachates.

. Concentration of anions sulfides S 2-, thiosulfates S2O3 2-, and sulfates SO4 2-(in mg/kg dry weight) in leachates of the crushed leaching test from concretes and cement pastes

	Fig. 10-Sulfates (blue
	diagonal up bars) are released with concentrations ranging from 25 mg/kg DW for CEM III/B sample
	to 204 mg/kg DW for CEM III/C sample. Thiosulfates (green dotted bars) releases range from 11
	mg/kg DW for CEM I reference sample to 154 mg/kg DW for P CEM III/C. Sulfides (orange plain bars)
	releases range between 6.58 mg/m² for CEM III/A sample to 52.67 mg/m² for P CEM III/C sample.
	Thiosulfates and sulfides were not detected in CEM I leachates.
						Conductivity (mS/cm)
	20		15	10		5	0
	P CEM III/C			12.38		3.63
	CEM III/C		12.10		pH		1.92
	CEM III/B			12.36	Conductivity	3.36
	CEM III/A			12.33			3.47
	CEM I			12.49	7.36
	11	pH	12	13	14
	P CEM III/C
	CEM III/C
	CEM III/B					Sulfides
	CEM III/A					Thiosulfates
	CEM I					Sulfates
	0	100	200	300	400	500
			Concentrations (mg/kg)

Table 2 Concentrations of major elements and sulfur anions in the leachates of the three leaching tests

 2

				Na	Ca	K	Al	Si	S 2-	SO4 2-S2O3 2-Ca/Si ∑cations/∑anions
			CEM III/A	65.43	91.52 32.39	6.25	9.11	0.33	5.46	1.80	10.05	26.98
	Sprinkling (mmol/m²)	CEM III/B CEM III/C	35.95 52.12	67.42 21.52 73.57 9.73	4.61 4.88	11.70 10.15	0.40 0.39	6.80 9.05	1.63 1.64	5.76 7.25	15.99 13.57
			P CEM III/C	68.38	44.86 19.64	5.28	12.00	0.80	9.56	2.08	3.74	12.07
			CEM I	80.76 524.19 82.43	1.63	5.48	< L.D.	1.12	< L.D.	95.61	617.58
			CEM III/A	118.24 178.79 45.89	4.92	9.89	0.21	1.37	1.71	18.08	108.68
	Monolith (mmol/m²)	CEM III/B	57.34 172.92 42.80	4.24	11.89	0.63	3.69	1.77	14.55	47.53
			CEM III/C	35.68 105.37 13.49	5.23	12.90	0.94	6.12	2.09	8.17	18.88
			P CEM III/C	38.78 154.48 27.00	8.39	19.61	1.64	9.99	2.78	7.88	17.22
			CEM I	17.93 135.73 23.84	0.28	0.20	< L.D. < L.D.	0.10	681.77	1859.41
	Crushed	(mmol/kg DW)	CEM III/A CEM III/B CEM III/C	17.11 6.95 5.40	96.39 13.48 71.42 10.23 41.88 5.93	0.84 0.74 0.84	0.65 0.56 1.90	0.01 0.02 0.11	0.33 0.26 2.13	0.52 0.56 0.86	148.18 127.95 22.09	149.76 106.36 18.08
			P CEM III/C	14.63	66.03 25.29	0.98	0.53	5.12	1.17	1.37	123.56	14

Acknowledgments

The authors wish to thank the LCPME Laboratory (Villers-lès-Nancy, France) for its contribution to the leachates analysis of sulfur anions, and the SARM Laboratory (CRPG, Vandoeuvre-lès-Nancy, France) for its contribution to the element analysis. The authors also want to acknowledge Vincent Chatain from the DEEP laboratory (INSA Lyon, Villeurbanne, France) for the material support that allowed us to develop the H2S analysis device, and José Manuel-Lopez (ICPM, University of Lorraine, Metz, France) for building the sprinkling device. The authors also wish to thank Dimitri Loschi for his help designing our artwork. Finally, the authors are particularly grateful for invaluable financial backing from the ATILH Professional Association (Paris-La-Defense, France), and especially Horacio Colina for offering constant support throughout the project.

7 References


Dabbugoddu Brahmaiah

A K D Bhavani

Pasula Aparna

Nangunoori Sampath Kumar

Hélène Solhi

Blandine Baratte

Sandrine Ruchaud

Stéphane Bach

Paul Mosset

René Grée
email: rene.gree@univ-rennes1.fr

Anagani Kanaka

Durga Bhavani

Rémy Le Guevel

Synthesis and biological studies of new quinazolines with ether functions in position 2

Keywords: Quinazolines, triazoles, cytotoxicity, kinases

published or not. The documents may come

Introduction

Many quinazoline alkaloids and derivatives have been isolated already, and the antimalarial (+)-Febrifugine 1 and the cytotoxic Luotonin A 2 are representative examples of this family of natural products (Figure 1). 1 From a structural point of view, almost all of them possess a quinazoline-4(3H)-one ring system. On the other hand, the quinazoline nucleus is also a privilege scaffold found in the basic skeleton of various types of drugs and/or bioactive molecules. [2][3][4] Representative examples are indicated in Figure 1 and it is worth noting that all compounds 3-8, which are potent kinase inhibitors used as anticancer drugs, have an aniline-type substituent in position 4 of this structure. Some other quinazolines, like Doxasocine 9 and Prazosin 10 (these two derivatives are used against hypertension) have both a primary amine in position 4 and a substituted piperazine in position 2. Further, extensive literature search shows that much less is known about type-A molecules with X-R 1 chains in position 2 (X being heteroatom like O, S and N and R 1 = alkyl or substituted alkyl chains, aromatics, heteroaromatics…..) (Figure 2). Therefore, we embarked on a program involving the synthesis and biological evaluation of such derivatives. The goal of this paper is to report the first part of this research, dealing with the synthesis of a focused library of oxygen-linked molecules 11 with a short linker and triazoles in terminal position. The choice of the triazole nucleus is based upon the fact that this core structure is found also in many biologically active molecules. 5

Results and Discussion

Chemical synthesis

Various strategies and disconnections have been reported for the synthesis of quinazolines. [START_REF] Yamamoto | Science of Synthesis[END_REF][START_REF] Aitken | Science of Synthesis: Knowledge Updates[END_REF] In our case we favored the approach through two C-N bond disconnections and starting from an ortho-aminobenzaldehyde derivative 13 (Scheme 1). Reaction with urea gave in good yield quinazoline 14 which was further treated by POCl3 to give chloroquinazoline 14a. This intermediate was reacted with propargyl alcohol to isolate in 65% yield propargyl ether 15. Final click-type reactions, [START_REF] Kolb | [END_REF][9][10][11] with various azido derivatives 16a-j, 12 afforded the target molecules 11a-j in good yields (Table1). All intermediates and final products have spectral and analytical data in agreement with their structures.

Biological studies

Based on the known biological properties of molecules with quinazoline scaffolds, we performed a few primary biological screenings of the ten molecules 11a-11j. First, their cytotoxicity has been checked on seven representative cancer cell lines (HuH7, CaCo-2, MDA-MB231, HCT116, PC3, NCI-H727, MCF7). All compounds were found to be devoid of any significant cytotoxicity at 10 M concentration (See Supplementary Material).

Their activity was also screened against a panel of eight representative kinases (HsCDK5/p25, HsCDK9/CyclinT, HsPIM1, MmCLK1, RnDYRK1A, HsHASPIN, HsGSK3β, HsCK1ε) and the results are reported in Table 2. Interestingly only one of these kinases (MmCLK1) was found to be responding to these derivatives. A few molecules (11a, 11c, 11d, 11e, 11f, 11j) exhibited a moderate inhibition of this kinase at 10M. On the other hand, these molecules were found to be devoid of any significant activity at 10M concentration on the other kinases. Percentages of residual kinase activity were determined at 1 and 10 M concentration for each compound. Kinase activities were assayed in duplicate.

Conclusions

In summary, we designed a short and convergent strategy toward the desired quinazolines linked to triazoles through an ether chain in position 2 of the quinazoline. Some of these derivatives exhibit a moderate, but significant and selective, activity against the MmCLK1 kinase. Extension of this approach to other quinazolinederived derivatives is under active study and will be reported in due course.

Experimental Section

Chemical Synthesis

General. All anhydrous reactions were performed in heat gun-dried round-bottomed flasks under a dry argon or nitrogen atmosphere. Air and moisture-sensitive compounds were introduced via syringes or cannula, using Synthesis of 8-methoxyquinazolin-2-ol (14). 2-Amino-3-methoxybenzaldehyde (13) (5 g, 33.1 mmol), urea (20 g, 336.3 mmol) and cat. NH4OAc (10 mg) were thoroughly mixed together in a round bottom flask. The solid mixture was heated to 160 o C and the solids quickly melted and stirring was continued for 15 min. A solid started to precipitate from the hot solution, NMP (25 mL) was added to dissolve the solids, the reaction was heated with stirring for an additional 2 h at 155-160 o C. The hot reaction mixture was poured into vigorously stirred ice water (150 mL).The reaction mixture was filtered, washed with water (50 mL) and ethyl acetate (15 mL) giving a brown color solid which was dried to give compound 14 (4.9 g, 82% yield) as a off white solid. 1 General procedure for preparation of compounds, 11a-j. To the stirred solution of compound 15 (0.1 mmol, 1.0 eq.) and compound 16a-j [prepared using general protocol, 12 (0.1 mmol, 1 eq.) in a 1:1 mixture of t-butanol and water (0.56 mL) was added copper sulfate pentahydrate (0.01 mmol, 0.1 eq.) and sodium ascorbate (0.03 mmol, 0.3 eq.) at room temperature. The resulting mixture was stirred for overnight. After completion of the reaction, it was diluted with a mixture of ethyl acetate and water (7.5 ml in a 1:1 ratio), the organic layer was separated and washed with 5% ammonium hydroxide solution (0.5 mL) and followed by brine (0.5 mL). The combined organic layers were dried over Na2SO4. The solvents were evaporated under reduced pressure to get the crude product, which was purified by chromatography on silica gel affording target compounds 11a-j (65-75% yield) as light yellow to brown solids.

Protein kinase assays

Kinase enzymatic activities were assayed in 384-well plates using the ADP-Glo assay kit (Promega, Madison, WI) according to the recommendations of the manufacturer. This assay is a luminescent ADP detection assay that provides a homogeneous and high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during a kinase reaction. Briefly, the reactions were carried out in a © ARKAT USA, Inc final volume of 6 µl for 30 min at 30°C in the following buffer: 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5, 50 µg/ml heparin; with either protein or peptide as substrate in the presence of 10 µM ATP. After stopping the kinase reaction, Kinase Detection Reagent was added for one hour at RT. The transmitted signal was then measured using an Envision microplate luminometer (PerkinElmer, Waltham, MA) and expressed in Relative Light Unit (RLU). HsCDK5/p25 (human, recombinant, expressed in bacteria) was assayed on 0.8 µg/µl of histone H1 as substrate. HsCDK9/CyclinT (human, recombinant, expressed by baculovirus in Sf9 insect cells) was assayed on 0.27 µg/µl of the following peptide: YSPTSPSYSPTSPSYSPTSPSKKKK, as substrate.

HsGSK3β (human, recombinant, expressed by baculovirus in Sf9 insect cells) was assayed on 0.010 µg/µl of GS-1 peptide, a GSK-3-selective substrate (YRRAAVPPSPSLSRHSSPHQSpEDEEE). HsCK1ε (human, recombinant, expressed by baculovirus in Sf9 insect cells) was assayed on 0.022 µg/µl of the following peptide: RRKHAAIGSpAYSITA ("Sp" stands for phosphorylated serine) as CK1-specific substrate. RnDYRK1A-kd (Rattus norvegicus, kinase domain aa 1 to 499, expressed in bacteria, DNA vector kindly provided by Dr. W. Becker, Aachen, Germany) was assayed on 0.033 μg/µl of the following peptide: KKISGRLSPIMTEQ as substrate.

MmCLK1 (from Mus musculus, recombinant, expressed in bacteria) was assayed on 0.027 μg/µl of the following peptide: GRSRSRSRSRSR as substrate. HsPim-1 (human proto-oncogene, recombinant, expressed in bacteria) was assayed on 0.8 µg/µl of histone H1 (Sigma #H5505) as substrate. HsHaspin-kd (human, kinase domain, amino acids 470 to 798, recombinant, expressed in bacteria) was assayed on 0.007 µg/µl of Histone H3 (1-21) peptide (ARTKQTARKSTGGKAPRKQLA) as substrate. Peptide substrates were obtained from Proteogenix (Schiltigheim, France).

Figure 1 .

 1 Figure 1. Representative examples of bioactive natural products and drugs with quinazoline cores.

Figure 2 .

 2 Figure 2. Our target molecules.

Scheme 1 .

 1 Scheme 1. Synthesis of quinazoline-based triazoles.

Table 1 .

 1 Synthesis of our target molecules

	Entry	n	R 2	Molecule	Yield %
	1	0	4-MeO	11a	75
	2	0	4-Cl	11b	65
	3	0	2-Cl	11c	64
	4	0	2-MeO	11d	68
	5	0	3-MeO	11e	71
	6	0	4-Br	11f	64
	7	0	H	11g	74
	8	1	H	11h	74
	9	1	4-NO2	11i	50
	10	1	2,6-di-Cl	11j	60

Synthesis of 2-amino-3-methoxybenzaldehyde (13). To

	standard inert atmosphere techniques. In addition, the gas stream was passed through glass cylinder filled
	with P2O5 to remove any traces of residual moisture. Reactions were monitored by thin layer chromatography
	(TLC) using E. Merck silica gel plates and components were visualized by illumination with short wavelength
	UV light and/or staining (Ninhydrin or basic KMnO4). THF and Et2O were dried over sodium-benzophenone and
	distilled prior to use. Anhydrous CH2Cl2 was prepared by refluxing in the presence of CaH2 and distilled right
	before use unless otherwise noted. Infrared spectra have been recorded on a Bruker alpha II FTIR
	spectrometer. 1 H NMR spectra were recorded at 300 and 400 MHz, and 13 C NMR spectra at 75 and 100 MHz,in
	CDCl3 or DMSO-d6 using tetramethylsilane (TMS) as an internal standard on Bruker spectrometers (Avance
	300III and Avance 300I and Avance III 400). Assignments were made using standard 2D NMR techniques (COSY,
	HMQC/HSQC, HMBC). High resolution mass spectra were performed using a time of flight Maxis 4G (Bruker
	Daltonik Gmbh, Bremen, Germany) in Electrospray positive ionization mode. LC-MS analyses were carried out
	on a Shimadzu [LCMS-2020], SHIMPAK, XR ODS-II column (50 x 2 mm) utilizing the following method. Solvent A
	= Acetonitrile, B = 0.1% TFA in water; Initial 95% of solvent B, then run gradient, which should reach 90%
	solvent A within 10 min and hold 90% solvent A for another 10 min. Flow Rate: 0.2 ml/min.
	a stirred solution of 3-methoxy-2-nitrobenzaldehyde
	(12) (10 g, 54 mmol) in a mixture of methanol (100 mL) and glacial acetic acid (100 mL) and water (50 mL) was
	added reduced iron powder (16 g, 144 mmol) in lot wise manner. The resulting suspension was stirred at 30-
	35 o C for 2h. The reaction mixture was filtered on Celite bed and washed with methanol (50 mL). The filtrate
	was concentrated to remove volatiles and it was partitioned between sat. NaHCO3 (150 mL) and ethyl acetate
	(200 mL). The basic layer was further extracted with ethyl acetate (100 mL). The combined organic extracts
	were washed with brine (50 mL), dried over Na2SO4 and
	ARKAT USA, Inc

concentrated to dryness and purified by chromatography on silica gel eluting with 20% EtOAc in hexane giving a compound 13 (6.5 g, 78% Yield) as a off white solid.

1

H NMR (400 MHz, CDCl3) δ ppm: 9.93 (s, 1H), 8.45 (d, 1H), 7.82 (d, 1H), 6.80 (t, 1H); Mass (m/z): 165.2 (M+H).

  Propargyl alcohol (518 mg, 9.24 mmol) was added to a stirred suspension of Cs2CO3 (3g, 9.24 mmol) and DMF (10 mL), after stirring 15 minutes at 30 o C compound 14a (600 mg, 3.08 mmol) was added. After stirring at 100 o C for 16h, the reaction mixture was quenched with water (30 mL) and extracted with DCM (2x25 mL). The combined organic extracts were washed with brine (5%, 15 mL), dried over Na2SO4 and concentrated to dryness. Purification by chromatography on silica gel (elution with EtOAc in hexane) gave compound 15 (450 mg, 65% yield) as a off white solid.

	1 H NMR
	(400 MHz, CDCl3) δ ppm: 9.23 (s, 1H), 7.46-7.38 (m, 2H), 7.21 (d, J 7.6, 1.6 Hz, 1H), 5.21 (s, 2H), 4.06 (s, 3H),
	2.49 (t, J 2.4 Hz, 1H); 13 C NMR (400 MHz, CDCl3): δ 163.62, 161.11, 153.84, 143.67, 125.43, 122.96, 118.90,
	113.06, 78.55, 74.63, 56.33, 55.18. Mass (m/z): 215.1 (M+H).

H NMR (500 MHz, DMSO-d6) δ ppm: 11.3 (br, 1H), 9.23 (s, 1H), 7.43 (t, 1H), 7.34 (d, 2H), 3.90 (s, 3H); Mass (m/z): 177.2 (M+H) Synthesis of 2-chloro-8-methoxyquinazoline (14a). A mixture of compound 14 (4 g, 22 mmol) and 2,3,5collidine (5 mL, 38.8 mmol) in acetonitrile (80 mL) was stirred at room temperature for 10 min. POCl3 (22 mL, 222 mmol) was added to reaction mixture at 0 o C and the reaction mixture was heated for 10 h at reflux. The mixture was concentrated to dryness. The obtained residue was partitioned between EtOAc (200 mL) and sat. NaHCO3 (200 mL). The mixture was stirred cautiously watching gas evolution until the pH reached to 8. The layers were separated and the organic layer was washed with sat. NaHCO3 (75 ml), brine (50ml), dried (Na2SO4), and concentrated to dryness, giving compound 14a (2.8 g, 63.5% yield) as a pale yellow solid.

1

H NMR (400 MHz, CDCl3) δ ppm: 9.25 (s, 1H), 7.61-7.57 (t, J 16.0 Hz, 1H), 7.51-7.48 (t, J 8.4, 1H), 7.28 (dd, J 8.0, 0.8 Hz, 1H), 4.07 (s, 3H); 13 C NMR (400 MHz, CDCl3): δ 162.72, 156.93, 154.08, 143.80, 128.59, 124.33, 118.41, 113.08, 56.26. Mass (m/z): 195.1 (M+H). ARKAT USA, Inc Synthesis of 8-methoxy-2-(prop-2-yn-1-yloxy)quinazoline (15).

(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)-8-methoxyquinazoline (11h). Reaction

  CH2), 56.23 (CH3, OMe). LC-MS: 367.79 [M]. HRMS-ESI (m/z) [M+Na] + calcd for C18H14 35 ClN5NaO2: 390.07282, found 390.0726, [M+K] + calcd for C18H14 35 ClKN5O2: 406.04676, found 406.0460. Reaction of 15 (20 mg, 0.093 mmol) with 16c (14.28 mg, 0.11 mmol) gave 11c: 22.1 mg, 64% yield. mp 134-136 °C; FT-IR (KBr, cm -1 ): 3146, 3004, 1580, 1486, 1417, 1382, 1288, 1168, 1119, 1022, 757, 665. 1 H NMR (300 MHz, CDCl3): δ 9.23 (s, C8a of quinazoline), 136.13 (C, δ to Br), 132.91 (2CH, ortho to Br of 4-bromophenyl), 125.36 (CH, C6 of quinazoline), 122.91 (C, C4a of quinazoline), 122.73 (CH of triazole), 122.34 (C, α to Br), 121.88 (2CH, meta to Br of 4-bromophenyl), 119.07 (CH, C7 of quinazoline), 112.92 (CH, C5 of quinazoline), 61.17 (CH2), 56.23 (CH3, OMe). LC-MS: 412.24 [M]. HRMS-ESI (m/z) [M+Na] + calcd for C18H14 79 BrN5NaO2: 434.02231, found 434.0225, [M+K] + calcd for C18H14 79 BrKN5O2: 449.99624, found 449.9959. Chemicals are solubilized in DMSO at a concentration of 10 mM (stock solution) and diluted in culture medium to the desired final concentrations. The dose effect cytotoxic assay of chemical is performed at 25 µM. Cells are plated in 96 wells plates (4000 cells/well). Twenty-four hours after seeding, cells are exposed to chemicals. After 48h of treatment, cells are washed in PBS and fixed in cooled 90% ethanol/5% acetic acid for 20 minutes and the nuclei are stained with Hoechst 33342 (B2261 Sigma). Image acquisition and analysis are performed using a Cellomics ArrayScan VTI/HCS Reader (ThermoScientific). The survival percentages are calculated as the percentage of cell number after compound treatment over cell number after DMSO treatment.

	quinazoline), 112.33 (CH, C6 of 3-methoxyphenyl), 106.31 (CH, C2 of 3-methoxyphenyl), 61.27 (CH2), 56.24 9.43 (s, 1H, H-quinazoline), 8.41 (s, 1H, H-triazole), 8.21 (half part of an A2X2 system, 2H ortho to NO2 of 4-
	(CH3, OMe on quinazoline), 55.64 (CH3, OMe of 3-methoxyphenyl). LC-MS: 363.13 [M]. HRMS-ESI (m/z) nitrobenzyl), 7.62 (dd, J 8.1, 1.3 Hz, 1H, H-C7 of quinazoline), 7.53 (half part of an A2X2 system with small
	[M+Na] + calcd for C19H17N5NaO3: 386.12236, found 386.1277, [M+K] + calcd for C19H17KN5O3: 402.09630, found coupling with CH2, 2H meta to NO2 of 4-nitrobenzyl), 7.48 (dd, J 8.0, 7.9 Hz, 1H, H-C6 of quinazoline), 7.40 (dd,
	402.0962. J 7.9, 1.2 Hz, 1H, H-C5 of quinazoline), 5.81 (s, 2H, CH2 of 4-nitrobenzyl), 5.55 (s, 2H, CH2 between O and
	2-((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-8-methoxyquinazoline (11f):. Reaction of 15 (25 mg, triazole), 3.96 (s, 3H, CH3, OMe). 13 C NMR (100 MHz, DMSO-d6): δ 164.22 (CH, α to N of quinazoline), 160.86
	1H, H-quinazoline), 8.48 (br t, J 0.5 Hz, 1H, H-triazole), 7.66-7.59 (m, 1H, H-4 of 2-chlorophenyl), 7.59-7.53 (m, 0.11 mmol) with 16f (23.1 mg, 0.11 mmol) gave 11f: 31 mg, 64% yield. mp 144-146 °C; FT-IR (KBr, cm -1 ): 3061, (C, C2 of quinazoline), 153.11 (C, C8 of quinazoline), 147.14 (C, α to NO2), 143.28 (C para to NO2 of 4-
	1H, H-5 of 2-chlorophenyl), 7.48-7.40 (m, 3H, H-3 and H-6 of 2-chlorophenyl and H-C7 of quinazoline), 7.40 3007, 1581, 1491, 1418, 1291, 1172, 1024, 829, 762. 1 H NMR (400 MHz, CDCl3): δ 9.24 (s, 1H, H-quinazoline), nitrobenzyl), 142.70 (C of triazole), 142.32 (C, C8a of quinazoline), 128.94 (2CH, meta to NO2 of 4-nitrobenzyl),
	(dd, J 8.1, 7.4 Hz, 1H, H-C6 of quinazoline), 7.20 (dd, J 7.4, 1.6 Hz, 1H, H-C5 of quinazoline), 5.84 (d, J 0.5 Hz, 8.47 (br t, J 0.6 Hz, 1H, H-triazole), 7.67-7.60 (symmetrical m, 4H of 4-bromophenyl), 7.47 (dd, J 8.1, 1.4 Hz, 1H, 125.73 (CH of triazole), 125.39 (CH, C6 of quinazoline), 123.79 (2CH, ortho to NO2 of 4-nitrobenzyl), 122.35 (C,
	2H, CH2), 4.06 (s, 3H, CH3, OMe). 13 C NMR (75 MHz, CDCl3): δ 163.74 (CH, α to N of quinazoline), 161.54 (C, C2 H-C7 of quinazoline), 7.42 (dd, J 8.1, 7.5 Hz, 1H, H-C6 of quinazoline), 7.23 (dd, J 7.6, 1.3 Hz, 1H, H-C5 of C4a of quinazoline), 118.96 (CH, C7 of quinazoline), 113.54 (CH, C5 of quinazoline), 60.09 (CH2 between O and
	of quinazoline), 153.73 (C, C8 of quinazoline), 143.42 (C, C8a of quinazoline), 143.32 (C of triazole), 134.93 (C, quinazoline), 5.80 (br d, J 0.6 Hz, 2H, CH2), 4.10 (s, 3H, CH3, OMe). 13 C NMR (100 MHz, CDCl3): δ 163.78 (CH, α triazole), 55.84 (CH3, OMe), 51.84 (CH2 of 4-nitrobenzyl). LC-MS: 392.37 [M]. HRMS-ESI (m/z) [M+Na] + calcd
	α to Cl), 130.75 (CH, C5 of 2-chlorophenyl), 130.62 (CH, C6 of 2-chlorophenyl), 128.29 (C, C1 of 2-to N of quinazoline), 161.57 (C, C2 of quinazoline), 153.79 (C, C8 of quinazoline), 144.70 (C of triazole), 143.51 for C19H16N6NaO4: 415.11252, found 415.1126, [M+K] + calcd for C19H16KN6O4: 431.08646, found 431.0861.
	chlorophenyl), 127.94 (CH, C3 of 2-chlorophenyl), 127.76 (CH, C4 of 2-chlorophenyl), 126.89 (CH of triazole), 125.29 (CH, C6 of quinazoline), 122.80 (C, C4a of quinazoline), 118.90 (CH, C7 of quinazoline), 112.70 (CH, C5 of quinazoline), 61.03 (CH2), 56.13 (CH3, OMe). LC-MS: 367.79 [M]. HRMS-ESI (m/z) [M+Na] + calcd for C18H14 35 ClN5NaO2: 390.07282, found 390.0729, [M+K] + calcd for C18H14 35 ClKN5O2: 406.04676, found 406.0462, [M+H] + calcd for C18H15 35 ClN5O2: 368.09088, found 368.0904. 2-((1-(2,6-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-8-methoxyquinazoline (11j). Reaction of 15 (25 mg, 0.11 mmol) with 16j (23.57 mg, 0.11 mmol) gave 11j: 29.3 mg, 60% yield. mp 186-188 °C; FT-IR (KBr, cm -1 ): 3002, 1578, 1426, 1268, 1166, 1114, 1016, 758. 1 H NMR (300 MHz, CDCl3): δ 9.19 (s, 1H, H-quinazoline), 7.85 (br s, 1H, H-triazole), 7.44 (dd, J 8.2, 1.7 Hz, 1H, H-C7 of quinazoline), 7.42-7.36 (m, 3H, 2H ortho to Cl at 7.38 ppm and H-C6 of quinazoline at 7.40 ppm), 7.29 (dd, J 9.1, 6.8 Hz, 1H, H meta to Cl), 7.19 (dd, J 7.3, 1.7 Hz, 1H, H-C5 of quinazoline), 5.85 (s, 2H, CH2 of 2,6-dichlorobenzyl), 5.70 (d, J 0.6 Hz, 2H, CH2 between O and triazole), 4.03 (s, 3H, CH3, OMe). : (C, 40 (C of 416.26 [M]. HRMS-ESI (m/z) [M+Na] + calcd for C19H15 35 Cl2N5NaO2: 438.04950, found 438.0495, [M+K] + calcd
	triazole), 143.57 (C, C8a of quinazoline), 137.20 (C ipso of Ph), 129.74 (2CH, meta of Ph), 128.69 (CH, para of for C19H15 35 Cl2KN5O2: 454.02344, found 454.0229.
	Ph), 125.30 (CH, C6 of quinazoline), 122.90 (CH of triazole), 122.85 (C, C4a of quinazoline), 120.52 (2CH, ortho
	of Ph), 119.01 (CH, C7 of quinazoline), 112.88 (CH, C5 of quinazoline), 61.28 (CH2), 56.22 (CH3, OMe). LC-MS: Cytotoxicity studies
	333.3 [M]. HRMS-ESI (m/z) [M+Na] + calcd for C18H15N5NaO2: 356.11179, found 356.1121, [M+K] + calcd for Cell culture. Skin normal fibroblastic cells are purchased from Lonza (Basel, Switzerland), HuH7, Caco-2, MDA-
	C18H15KN5O2: 372.08573, found 372.0856. MB-231, HCT116, PC3, MCF7 and NCI-H727 cancer cell lines were obtained from the ECACC collection (Porton
	27 (CH, C3 of 2-(of 15 (20 mg, 0.093 mmol) Down, UK). Cells are grown at 37°C, 5% CO2 in ECACC recommended media: DMEM for HuH7, MDA-MB-231
	33 (CH2), 2-methoxyphenyl), 61.38 (CH2), 56.23 (CH3, OMe on quinazoline), 55.94 (CH3, OMe of 2-methoxyphenyl). LC-with 16h (12.38 mg, 0.093 mmol) gave 11h: 24.2 mg, 74% yield. mp 109-111 °C; FT-IR (KBr, cm -1 ): 3006, 1579, and fibroblast, EMEM for MCF7 and CaCo-2, McCoy's for HCT116 and RPMI for PC3 and NCI-H727. All culture
	56.23 (CH3, OMe on quinazoline), 55.64 (CH3, OMe of 4-methoxyphenyl). LC-MS: 363.13 [M]. HRMS-ESI (m/z) MS: 363.13 [M]. HRMS-ESI (m/z) [M+Na] + calcd for C19H17N5NaO3: 386.12236, found 386.1228, [M+K] + calcd 1468, 1414, 1280, 1166, 1117, 1002, 816, 742. 1 H NMR (300 MHz, CDCl3): δ 9.19 (s, 1H, H-quinazoline), 7.84 (s, media are supplemented by 10% of FBS, 1% of penicillin-streptomycin and 2 mM glutamine.
	[M+Na] + calcd for C19H17N5NaO3: 386.12236, found 386.1222, [M+K] + calcd for C19H17KN5O3: 402.09630, found for C19H17KN5O3: 402.09630, found 402.0963, [M+H] + calcd for C19H18N5O3: 364.14041, found 364.1404. 1H, H-triazole), 7.43 (dd, J 8.1, 1.4 Hz, 1H, H-C7 of quinazoline), 7.39 (dd, J 8.1, 7.5 Hz, 1H, H-C6 of quinazoline), Cytotoxic assay.
	402.0957. 8-Methoxy-2-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)quinazoline (11e). Reaction of 15 (25 mg, 7.37-7.32 (m, 3H, 2H meta and 1H para of benzyl), 7.29-7.23 (m, 2H, 2H ortho of benzyl), 7.18 (dd, J 7.5, 1.3
	2-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-8-methoxyquinazoline (11b). Reaction of 15 (25 mg, 0.11 mmol) with 16e (17.4 mg, 0.11 mmol) gave 11e: 30 mg, 71% yield. mp 137-139 °C; FT-IR (KBr, cm -1 ): 3144, Hz, 1H, H-C5 of quinazoline), 5.71 (s, 2H, CH2 between O and triazole), 5.52 (s, 2H, CH2 of benzyl), 3.99 (s, 3H,
	0.11 mmol) with 16b (17.92 mg, 0.11 mmol) gave 11b: 28 mg, 65% yield. mp 130 °C (dec); FT-IR (KBr, cm -1 ): 3002, 1594, 1483, 1420, 1289, 1165, 1015, 1031, 855, 764. 1 H NMR (300 MHz, CDCl3): δ 9.23 (s, 1H, H-CH3, OMe). 13 C NMR (75 MHz, CDCl3): δ 163.63 (CH, α to N of quinazoline), 161.52 (C, C2 of quinazoline),
	3339, 3332, 2999, 1581, 1493, 1421, 1382, 1293, 1171, 1108, 1029, 830, 756. 1 H NMR (400 MHz, CDCl3): δ quinazoline), 8.48 (br t, J 0.6 Hz, 1H, H-triazole), 7.46 (dd, J 8.2, 1.6 Hz, 1H, H-C7 of quinazoline), 7.44-7.36 (m, 153.71 (C, C8 of quinazoline), 144.07 (C of triazole), 143.49 (C, C8a of quinazoline), 134.54 (C ipso of Ph),
	9.24 (s, 1H, H-quinazoline), 8.47 (s, 1H, H-triazole), 7.68 (half part of an A2X2 system, 2H meta to Cl), 7.49 (half 2H, H-C6 of quinazoline at 7.43 ppm and H5 of 3-methoxyphenyl at 7.39 ppm), 7.34 (dd, J 2.4, 2.1 Hz, 1H, H2 of 129.09 (2CH, meta of benzyl), 128.73 (CH, para of benzyl), 128.15 (2CH, ortho of benzyl), 125.24 (CH, C6 of
	part of an A2X2 system, 2H ortho to Cl), 7.46 (dd, J 8.2, 1.4 Hz, 1H, H-C7 of quinazoline), 7.42 (dd, J 8.2, 7.5 Hz, 3-methoxyphenyl), 7.26 (ddd, J 8.0, 2.1, 1.0 Hz, 1H, H6 of 3-methoxyphenyl), 7.23 (br dd, J 7.5, 1.6 Hz, 1H, H-C5 quinazoline), 124.19 (CH of triazole), 122.77 (C, C4a of quinazoline), 118.89 (CH, C7 of quinazoline), 112.72
	(CH, C5 of quinazoline), 61.32 (CH2 between O and triazole), 56.12 (CH3, OMe), 54.19 (CH2 of benzyl). LC-MS:
	347.37 [M]. HRMS-ESI (m/z) [M+Na] + calcd for C19H17N5NaO2: 370.12744, found 370.1277, [M+K] + calcd for
	C19H17KN5O2: 386.10138, found 386.1013.
	8-Methoxy-2-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)quinazoline (11i). Reaction of 15 (20 mg,
	0.093 mmol) with 16i (16.56 mg, 0.093 mmol) gave 11i: 18.4 mg, 50% yield. mp 179-181 °C; FT-IR (KBr, cm -1 ):
	3132, 3008, 1692, 1586, 1519, 1471, 1416, 1342, 1290, 1169, 1117, 1016, 756. 1 H NMR (400 MHz, DMSO-d6): δ
	ARKAT USA, Inc

8-Methoxy-2-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)quinazoline (11a).

Reaction of 15 (25 mg, 0.11 mmol) with 16a (17.4 mg, 0.11 mmol) gave 11a: 31.6 mg, 75% yield. mp 179-181 °C; FT-IR (KBr, cm -1 ): 3137, 2958, 2841, 1584, 1516, 1470, 1419, 1292, 1261, 1175, 1117, 1034, 836, 763.

1

H NMR (300 MHz, CDCl3): δ 9.23 (s, 1H, H-quinazoline), 8.37 (br t, J 0.5 Hz, 1H, H-triazole), 7.62 (half part of an A2X2 system, 2H meta to OMe), 7.46 (dd, J 8.1, 1.6 Hz, 1H, H-C7 of quinazoline), 7.40 (dd, J 8.1, 7.4 Hz, 1H, H-C6 of quinazoline), 7.22 (ddd, J 7.4, 1.6, 0.2 Hz, 1H, H-C5 of quinazoline), 7.01 (half part of an A2X2 system, 2H ortho to OMe), 5.80 (d, J 0.5 Hz, 2H, CH2), 4.09 (s, 3H, CH3, OMe on quinazoline), 3.86 (s, 3H, CH3, OMe of 4-methoxyphenyl).

13

C NMR (75 MHz, CDCl3): δ 163.72 (CH, α to N of quinazoline), 161.65 (C, C2 of quinazoline), 159.81 (C, α to OMe), 153.83 (C, C8 of quinazoline), 144.19 (C of triazole), 143.59 (C, C8a of quinazoline), 130.67 (C, δ to OMe), 125.28 (CH, C6 of quinazoline), 122.92 (CH of triazole), 122.89 (C, C4a of quinazoline), 122.15 (2CH, meta to OMe), 118.99 (CH, C7 of quinazoline), 114.78 (2CH, ortho to OMe), 112.87 (CH, C5 of quinazoline), 61.1H, H-C6 of quinazoline), 7.23 (dd, J 7.6, 1.

4 Hz, 1H, H-C5 of quinazoline), 5.80 (d, J 0.6 Hz, 2H, CH2), 4.10 (s, 3H, CH3, OMe), 13 C NMR (100 MHz, CDCl3): δ 163.77 (CH, α to N of quinazoline), 161.58 (C, C2 of quinazoline), 153.80 (C, C8 of quinazoline), 144.68 (C of triazole), 143.53 (C, C8a of quinazoline), 135.66 (C, δ to Cl), 134.50 (C, α to Cl), 129.94 (2CH, ortho to Cl), 125.36 (CH, C6 of quinazoline), 122.91 (C, C4a of quinazoline), 122.77 (CH of triazole), 121.65 (2CH, meta to Cl), 119.04 (CH, C7 of quinazoline), 112.93 (CH, C5 of quinazoline), 61.17

(2-((1-(2-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-8-methoxyquinazoline (11c). 8-Methoxy-2-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)quinazoline (11d). Reaction of 15 (25 mg, 0.11 mmol) with 16d (17.4 mg, 0.11 mmol) gave 11d: 29.4 mg, 68% yield. mp 147-149 °C; FT-IR (KBr, cm -1 ): 3157, 2963, 2838, 1580, 1470, 1417, 1286, 1170, 1119, 1017, 753, 668. 1 H NMR (300 MHz, CDCl3): δ 9.23 (s, 1H, H-quinazoline), 8.44 (br t, J 0.6 Hz, 1H, H-triazole), 7.77 (ddd, J 7.8, 1.7, 0.3 Hz, 1H, H6 of 2methoxyphenyl), 7.45 (dd, J 8.2, 1.5 Hz, 1H, H-C7 of quinazoline), 7.41 (ddd, J 8.2, 7.6, 1.7 Hz, 1H, H-C6 of quinazoline), 7.39 (dd, J 8.1, 7.4 Hz, 1H, H4 of 2-methoxyphenyl), 7.20 (br dd, J 7.5, 1.5 Hz, 1H, H-C5 of quinazoline), 7.09 (td, J 7.7, 1.3 Hz, 1H, H5 of 2-methoxyphenyl), 7.07 (br dd, J 8.3, 1.1 Hz, 1H, H3 of 2methoxyphenyl), 5.84 (d, J 0.6 Hz, 2H, CH2), 4.07 (s, 3H, CH3, OMe on quinazoline), 3.84 (s, 3H, CH3, OMe of 2methoxyphenyl). 13 C NMR (75 MHz, CDCl3): δ 163.63 (CH, α to N of quinazoline), 161.70 (C, C2 of quinazoline), 153.85 (C, C8 of quinazoline), 151.23 (C α to OMe of 2-methoxyphenyl), 143.69 (C, C8a of quinazoline), 143.03 (C of triazole), 130.04 (CH, C4 of 2-methoxyphenyl), 126.47 (CH of triazole), 126.41 (C, C1 of 2-methoxyphenyl), 125.62 (CH, C6 of 2-methoxyphenyl), 125.20 (CH, C6 of quinazoline), 122.85 (C, C4a of quinazoline), 121.22 (CH, C5 of 2-methoxyphenyl), 118.94 (CH, C7 of quinazoline), 112.83 (CH, C5 of quinazoline), 112.of quinazoline), 6.96 (ddd, J 8.3, 2.5, 1.0 Hz, 1H, H4 of 3-methoxyphenyl), 5.81 (d, J 0.5 Hz, 2H, CH2), 4.10 (s, 3H, CH3, OMe on quinazoline), 3.87 (s, 3H, CH3, OMe of 3-methoxyphenyl). 13 C NMR (75 MHz, CDCl3): δ 163.74 (CH, α to N of quinazoline), 161.63 (C, C2 of quinazoline), 160.64 (C, α to OMe of 3-methoxyphenyl), 153.83 (C, C8 of quinazoline), 144.34 (C of triazole), 143.56 (C, C8a of quinazoline), 138.20 (C, C1 of 3-methoxyphenyl), 130.49 (CH, C5 of 3-methoxyphenyl), 125.30 (CH, C6 of quinazoline), 122.96 (CH of triazole), 122.90 (C, C4a of quinazoline), 119.00 (CH, C7 of quinazoline), 114.60 (CH, C4 of 3-methoxyphenyl), 112.88 (CH, C5 of 8-Methoxy-2-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)quinazoline (11g). Reaction of 15 (25 mg, 0.11 mmol) with 16g (13.90 mg, 0.11 mmol) gave 11g: 29.1 mg, 74% yield. mp 168-170 °C; FT-IR (KBr, cm -1 ): 3143, 3070, 3003, 1585, 1484, 1418, 1292, 1171, 1117, 1030, 764, 690. 1 H NMR (400 MHz, CDCl3): δ 9.24 (s, 1H, H-quinazoline), 8.48 (s, 1H, H-triazole), 7.76-7.71 (m, 2H, H ortho of Ph), 7.55-7.49 (m, 2H, H meta of Ph), 7.48-7.38 (m, 3H, H-C6 of quinazoline, H-C7 of quinazoline and H para of Ph), 7.23 (dd, J 7.6, 1.3 Hz, 1H, H-C5 of quinazoline), 5.82 (s, 2H, CH2), 4.10 (s, 3H, CH3, OMe). 13 C NMR (75 MHz, CDCl3): δ 163.74 (CH, α to N of quinazoline), 161.64 (C, C2 of quinazoline), 153.83 (C, C8 of quinazoline), 144.13 C NMR (75 MHz, CDCl3): δ 163.61 (CH, α to N of quinazoline), 161.50 (C, C2 of quinazoline), 153.73 (C, C8 of quinazoline), 143.62 (C of triazole), 143.53 (C, C8a of quinazoline), 136.85 (2C, α to Cl), 131.05 (CH, meta to Cl), 130.08 (C, β to Cl), 128.87 (2CH, ortho to Cl), 125.23 (CH, C6 of quinazoline), 123.76 (CH of triazole), 122.78 (C, C4a of quinazoline), 118.89 (CH, C7 of quinazoline), 112.71 (CH, C5 of quinazoline), 61.39 (CH2 between O and triazole), 56.19 (CH3, OMe), 49.03 (CH2 of 2,6-dichlorobenzyl). LC-MS

Acknowledgements

We thank Chemveda Life Sciences for providing laboratory facility for carrying these research experiments. This research has been performed also as part of the Indo-French "Joint Laboratory for Natural Products and Synthesis towards Affordable Health". We thank CSIR, CNRS and University of Rennes 1 for their support. AKDB and DB thank Osmania University for financial support through the OU-DST-PURSE-II Programme (2017-2021). We thank CRMPO (University of Rennes 1) for the high resolution mass spectra analysis. Financial support by the "Ligue contre le Cancer, Conseil Interrégional Grand Ouest" is gratefully acknowledged. The authors thank the Cancéropôle Grand Ouest (axis: natural sea products in cancer treatment), IBiSA (French Infrastructures en sciences du vivant) and Biogenouest (Western France life science and environment core facility network) for supporting KISSf screening facility.

Supplementary Material

Electronic Supplementary Information (ESI) available: cytotoxic studies of the quinazolines 11 plus copies of the 1 H and 13 C NMR spectra for all new compounds