text:
One-pot synthesis of an indole-substituted 7,8-dicarba- nido -dodecahydroundecaborate( − 1) †

Carbaboranes are increasingly used as pharmacophores to replace phenyl substituents in established drug molecules. In contrast to traditional organic chemistry, elaborate procedures to introduce functionality frequently fail in the case of carbaboranes and their chemistry is often hampered by degradation of the cluster. Herein, the development of a one-pot synthesis of a water-soluble N - nido -dicarbaborato indole is presented, including a proposed mechanism for the reaction sequence. These studies provide useful synthetic tools for the conjugation of two important pharmacophores, indoles and carbaboranes.

Introduction
Indole is one of the most abundant heterocycles in nature (e.g., in tryptophan, serotonin, melatonin) and is of great relevance as a pharmacophore in medical applications, which is reflected by the product scope of more than thirty indolebased pharmaceuticals with various applications including anti-cancer, anti-depressive, anti-hypertensive, anti-inflammatory and anti-HIV agents. 1 Besides their proposed applications as boron-delivery agents in boron neutron capture therapy, 2 carbaboranes have also received pronounced attention as pharmacophores in the last decade. 3 1,2-, 1,7-and 1,12-dicarbacloso-dodecaboranes(12) (i.e., ortho-, meta-and para-carboranes) are icosahedral boron clusters composed of ten BH and two CH vertices. These cluster compounds offer several advantages compared to other pharmacophores, including non-toxicity, remarkably high lipophilicity, and high metabolic stability, and their predominantly hydridic periphery facilitates unprecedented interactions with biomolecules (e.g., enzymes and receptors). 3,4 Due to delocalisation of the cluster electrons and a volume similar to that of a rotating phenyl ring, carbaboranes are also considered as three-dimensional aromatic systems and are increasingly used as phenyl mimetics. We could recently demonstrate their unique pharmacophoric properties when introduced into established inhibitors of cyclooxygenase (COX), such as aspirin and indomethacin. 5 The replacement of the 4-chlorophenyl ring in indomethacin (an established anti-inflammatory drug to relieve pain, stiffness and swelling) by an ortho-carbaborane generates a highly potent and selective COX-2 inhibitor 1 (Scheme 1). 5c The amide bond in indomethacin and its analogues, however, is rather labile and can be easily hydrolysed, especially under basic conditions. Upon the introduction of an ortho-carbaborane, analogue 1 becomes even more prone to cleavage due to the high electron deficiency of the adjacent cluster. Thus, the replacement of the amide bond by a methylene group should generate a more stable analogue 2 (Scheme 1). The pronounced lipophilicity of carbaboranes is of advantage for biochemical interactions (e.g., penetration of cell membranes), but insolubility in water may hamper the bioavailability of the compound. Directed deboronation (decapping) of the carbaborane cluster to yield the respective anionic nido-dicarbaborate analogue 3 (Scheme 1) thus would contribute to a better water solubility.
The preparation of indoles is well studied, and various synthetic procedures have been reported, although some involve numerous steps or apply rather harsh reaction conditions. However, when carbaboranes are present, the reaction conditions must be chosen carefully, as the cluster tends to undergo deboronation, especially in the presence of bases and nucleophiles, such as amines. 4b, 6 Thus, mild or acidic conditions are preferred for the introduction of carbaboranes. In general, C-substituted carbaboranes can be prepared by a reaction of decaborane with substituted acetylenes. 4b,7 This method, however, is limited to the preparation of ortho-carbaboranes, preventing the application of a developed synthetic method for the other carbaborane isomers. Furthermore, reactive groups have to be protected during this reaction and bulky substituents at the carbon atoms of the acetylene derivative lead to only low yields.
A variety of aminoalkyl carbaboranes has been synthesised. 4b,8 However, to the best of our knowledge, carbaboranes bearing indolyl substituents at the carbon vertices of the cluster have not yet been reported, except from our own investigations. 5c,d This might be due to complications associated with the synthesis of such compounds, as discussed below. But as both indoles and carbaboranes are important pharmacophores, the development of suitable synthetic routes is of increasing interest.

Results and discussion
Reduction of N-acylindole A first synthetic approach towards 2 aimed at the reduction of the amide bond in 1 by using borane as a reducing agent (Scheme 2), which has been successfully applied to the reduction of other acylindoles. 9 However, only the respective semi-aminal was formed (monitored by TLC), which decomposed to the corresponding indole and carbaboranyl aldehyde upon isolation (characterised by NMR). The high electron deficiency of the carbaborane cluster may decrease the nucleophilicity of the carbonyl oxygen atom and thereby impede the attack of borane. 9b Application of sodium borohydride also resulted in cleavage of the C-N bond. 8a Due to the low basicity of the indole nitrogen atom [ pK a (indole-NH): 20.95 in DMSO], 10 acylindoles are generally prone to C-N cleavage, and this effect is enhanced by the electron-withdrawing carbaboranyl substituent.

Fischer indole synthesis
Fischer indole synthesis is one of the most studied procedures for generating indole systems from arylhydrazones and aldehydes or ketones in the presence of acid catalysts. 11 For the introduction of the carbaboranyl substituent, acetone phenylhydrazone was treated with bromomethyl carbaborane, followed by a reaction with levulinic acid methyl ester for indole formation (Scheme 3). 12 However, the substitution reaction was not successful. Although halomethyl carbaboranes in general show low reactivity in nucleophilic substitutions, 13 this is unusual, since dialkylamines were reported to readily replace the halogen, as they reduce the electron deficiency of the cluster by coordination at boron. 14 Furthermore, even the application of the usually more reactive triflate analogue did not result in a substitution. 15

Nenitzescu indole synthesis
Therefore, in another approach the nitrogen atom was directly introduced at the carbaborane cluster as an aminomethyl group before synthetic steps towards the formation of the heterocycle by Nenitzescu indole synthesis were attempted. Aminoalkyl carbaboranes are highly reactive and versatile building blocks. 4b,8 Reaction of the carbaboranyl amine with methyl acetoacetate resulted in a tautomeric mixture of the enamine and the respective imine despite employing different acids [Brønstedt ( para-TsOH) 12b, 16 and Lewis acids (CF 3 COOCa) 17 ] as catalysts (Scheme 4). However, the following reaction with para-benzoquinone 12b,16 did not give the desired heterocycle. Although the Nenitzescu reaction is an important regioselective route to substituted indoles, its efficiency is highly dependent on substituent effects. The cyclisation proceeds via a Michael addition and nucleophilic attack by the Scheme 2 Attempted reduction of 1, which resulted in the C-N bond cleavage.
Scheme 3 Attempted Fischer indole synthesis towards compound 2. enamine at the quinone. 18 The highly electron-withdrawing carbaborane cluster probably reduces the electron density, especially at the nitrogen atom, and together with the bulkiness of the cluster may thus restrict the reactivity of the enamine.

Nucleophilic substitution
Further approaches were carried out with the indole derivative 4 as the starting material for nucleophilic substitution reactions. First, the introduction of a methylene group with a terminal leaving group [Br, OH (to be further functionalised)] at the indole nitrogen atom was attempted (Scheme 5). Reaction of this indole derivative with a C-lithiated carbaborane could then readily generate the desired compound 2 in a salt elimination, often used for the substitution of carbaboranes. 4b However, the reaction of 4 with dibromomethane or paraformaldehyde in the presence of a strong base almost exclusively afforded the methylene-bridged dimer of 4, 19 even when the reaction was carried out under high-dilution conditions and in the presence of a large excess of the reactants [CH 2 Br 2 , (CHO) n ]. A general method for the synthesis of N-benzyl derivatives of indomethacin is benzylation of unsubstituted indoles with benzyl halides in the presence of a strong base. 12b Therefore, 1-bromomethyl-or 1-trifluoromethanesulfonylmethyl-orthocarbaborane was treated with indole 4 under various reaction conditions (Scheme 6). 15a Furthermore, Mitsunobu conditions, which have also been reported for the alkylation of indoles, 20 were tested with the respective carbaboranyl methanol. 21 However, while none of these reactions gave the desired indomethacin analogue 2, most of them resulted in prompt deboronation of the carbaborane cluster (monitored by TLC). Substitution reactions at the indole nitrogen atom usually require strong bases due to the very low acidity of the NH group. 10 The deprotonated indole in turn is a strong, nucleophilic base, which tends to attack the carbaborane cluster resulting in decapping. However, when 4 was used in excess in the reaction with bromomethyl-ortho-carbaborane, the respective nido-carbaborane derivative 3 was obtained (Scheme 6). This suggests a reaction mechanism in which the bromomethyl-ortho-carbaborane is first decapped by a deprotonated indole molecule, followed by a nucleophilic substitution with a second indolate anion yielding 3. The increased electron density of the negatively charged nido-cluster enhances the reactivity compared to the charge-neutral halomethyl carbaborane and thereby enables a substitution reaction at the carbaborane cluster. Thus, this protocol presents a direct route towards indole-substituted nido-dicarbaborates. In contrast to compound 1, indole derivative 3 is water-soluble and the bond linking both pharmacophores is stable under various conditions. Scheme 4 Attempted Nenitzescu indole synthesis towards an analogue of compound 2.
Scheme 5 Attempted introduction of a bromo-or hydroxymethyl group at indole derivative 4, which resulted in the formation of a methylene-bridged dimer (TBAF: tetrabutylammonium fluoride trihydrate).

Scheme 6
Nucleophilic substitution at indole 4 with functionalised carbaboranes under basic or Mitsunobu conditions (DIAD: diisopropyl azodicarboxylate; TMAD: tetramethyl azodicarboxamide) and the proposed mechanism of sequential decapping and nucleophilic substitution leading to the formation of derivative 3.

Conclusions
Different attempts to synthesise N-dicarbaboranyl indoles were described including discussions of probable interferences, which seem to result mainly from the high electron deficiency of the carbaborane cluster. A one-pot synthesis of N-nidodicarbaborato indoles was presented and a possible reaction mechanism was proposed. This synthesis avoids the additional synthetic step of decapping the carbaborane cluster, which often involves rather harsh conditions (e.g., alkoxides), and thus minimises interferences with other substituents at the indole systems. The procedure is generally applicable for N-substitution of indoles bearing moieties which are stable in the presence of strong bases. As indoles and carbaboranes are important pharmacophores, the development of further synthetic methods for linking them is of increasing interest. The presented synthetic procedure provides ready access to watersoluble and chiral nido-dicarbaborato indoles.

Experimental
General Reactions were carried out under a nitrogen atmosphere using anhydrous solvents which were purified using an MBRAUN Solvent Purification System MB SPS-800. Chemicals were used as purchased. The synthesis of the starting materials is described in the ESI. † Thin-layer chromatography (TLC) was performed on pre-coated glass plates (0.25 mm, silica gel 60 F 254 ); visualisation of carbaborane compounds on TLC plates was achieved by treatment with a solution of PdCl 2 (1% in MeOH) and gentle heating. Column chromatography was carried out with silica gel (0.035-0.070 mm, 60 Å). 1 H, 11 B and 13 C NMR spectra were recorded on a Bruker AVANCE DRX 400 (400 MHz) with tetramethylsilane as an internal standard and referencing to the unified scale. FTIR spectra were recorded on a Perkin-Elmer System 2000 FTIR spectrometer, scanning between 400 and 4000 cm −1 . Mass spectra (HR-MS) were recorded on an ESI-FT-ICR Bruker-Daltonics spectrometer (APEX II, 7 T). Melting points were measured in sealed tubes.

Reduction of N-acylindole
General procedure. 1 was dissolved in THF before the addition of a reducing agent (BH 3 (THF), 9b NaBH 4 , BH 3 (THF)/ BF 3 (OEt 2 ) or BH 3 (THF)/NaBH 4 ) and the reaction mixture was stirred (at room temperature, heating at reflux or in a microwave reactor). The progress of the reactions was monitored by TLC. The reaction was quenched upon formation of the semiaminal by addition of methanol, and the product was isolated by extraction. 1 H NMR spectra of the crude product showed a mixture of 4 and the carbaboranyl aldehyde (1-H(O)C-1,2-C 2 B 10 H 11 ). 22 Fischer indole synthesis General procedure. 1-Bromomethyl-1,2-dicarba-closo-dodecaborane(12) or 1-trifluoromethanesulfonylmethyl-1,2-dicarba-closo-dodecaborane(12) was added to a solution of acetone (4-methoxyphenyl)hydrazone and a base, and the reaction mixture was stirred for several hours. For the formation of the heterocycle, trifluoroacetic acid (TFA) and levulinic acid methyl ester were added and the mixture was heated at reflux. 12c,d The progress of the reaction was monitored by TLC.

Nenitzescu indole synthesis
Synthesis of carbaboranyl enamine. 1-Aminomethyl-1,2dicarba-closo-dodecaborane(12) hydrochloride was deprotonated with NEt 3 in THF, filtered and the solvent was removed. 23 The free amine (1 eq.) was then refluxed with methyl acetoacetate (1 eq.) and a catalytic amount of p-TsOH in toluene, 12b,16 yielding a mixture of enamine and imine. 11  General procedure for indole synthesis. A solution of carbaboranyl enamine (in CH 2 Cl 2 , CH 3 CN or CH 3 NO 2 ) was added dropwise to a solution of para-benzoquinone and the reaction mixture was stirred (at room temperature or reflux). 12b, 16 The reaction was monitored by TLC.

Nucleophilic substitution
General procedure for substitution at 4 with CH 2 Br 2 . 4 was dissolved in DMF and deprotonated with NaH at 0°C. The deprotonated indole was then added dropwise to a solution of CH 2 Br 2 (excess) and the reaction was stirred for several hours at room temperature. The solvent was removed and purification was carried out by column chromatography.
General procedure for substitution at 4 with (CHO) n . 19,24 4 and paraformaldehyde (excess) were dissolved in DMSO. TBAF (tetrabutylammonium fluoride) and some drops of water were added and the reaction was stirred for 2 h at room temperature. The product was extracted with EtOAc and purification was carried out by column chromatography.


Investigation on New Innovation in Natural Gas Dehydration Based on Supersonic Nozzle Technology

According to growing of demand for natural gas in global markets, it causes to modify and intensify its process. Indisputably, the existance of water in natural gas has so long been a controversial issue in gas industry and removing water from NG becomes a big deal, indeed. Gas dehydration is the most essntial and important way to solve this problem and there are several approaches to attain this aim. Among these methods, dehydration of natural gas with supersonic nozzle is an effictive method which is more efficient way than other methods. The main advantages of this method are its small size that is big deal for off-shore units because of limitation of location and also low operating costs with no deleterious environmental impact. In this study at first we examined some important methods for gas dehydration and then we proceed the details of supersonic nozzle method, and finally we compare it with other methods and conclude its unique advantages as abest method for gas dehydration. *Author for correspondence Indian Journal of Science and Technology, Vol 8(S9), 450–454, May 2015 ISSN (Print) : 0974-6846 ISSN (Online) : 0974-5645 Investigation on New Innovation in Natural Gas Dehydration Based on Supersonic Nozzle Technology Saeed Jamali Ashtiani1*, Athareh Haghnejat2, Mehdi Sharif2 and Ali Fazli1 1Department of Polymer Engineering, Sahand University of Technology, Tabriz, Iran; Ashtiani.saeed@yahoo.com m 2Department of Polymer Engineering, Shiraz Branch, Islamic Azad University, Shiraz, Iran; a_fazli@sut.ac.ir

Introduction
Dehydration process is an essential part of gas processing in offshore units which avoid several important problems in gas processing. The problems encompass erosion, fouling, gas hydrate and such like. There are the varity of methods for gas dehydration in gas industry but some them are most important compare to others like adsobrtion, adsorbtion and direct cooling for instance, due to absorption preocess the amin part is uing di-ethylene and glycoles that are used for withdrawing water content in gas. One of the modern high technology intersted to developed target ingredients from natural gases supersonic-technology. If we using a convergentdivergent Laval nozzle, the supersonic flow is produced. The components of C3+ are heavy and when mixed with condensed drops, they are separated from the natural gas flow in the supersonic nozzle.
Under action of centrifugal forces, we can separated the condensate drops with target ingredients condensed in the supersonic nozzle is carry out. This means that flow swirling at the supersonic nozzle entry.
The supersonic-high technology have many advantages as compared with old technologies. Some of these differences are list in below: • Small size of the hydrocarbons separation from natural gas.

Gas Dehydration
Natural gas can carry certain amounts of water in the vapour form. Water is a contaminant in natural gas and its concentration increases with temperature or equally with reduced pressure. Physical appearance of gas hydrates is like packed snow. The conditions at which hydrates start to solidify, and become a cause for trouble, depend on many factors including but not limited to gas temperature, pressure, composition, and the water content. Therefore it is very important to develop techniques to prevent hydrate formation. The following methods have been conventionally used for hydrate prevention like identifying the temperature at which hydrates formation and keeping the gas above this temperature, reducing pressure and therefore reducing the possibility of hydrate formation, adding hydrate inhibitors to the gas, which in tum will either reduce the temperature at which hydrates are likely to form or the rate at which they form and reducing the water content of the gas and therefore reducing the possibility of hydrate formation. This process is called gas dehydration which is reported by Michalnetusil et al 2 in their article.

Problems with Water in the Gas
As mentioned before there are some amount of water in gas which is named water content and in the cold region as the ambient temprature of fell down and decrease under the dew point temprature of water, the small molculars of water which are in vapor phase in pip,beging to condence and aggregate to big one and cause to form methane hydrate that is in solid form and this small solid particle which are crystal, beging to make large partile and if they be in the way of natural gas which consist of H 2 S and CO 2 can cause several serious problems like corrosion and erosion.

Supersonic Separation
We can use the 3S technology to exploit target component from natural gases. Basis of this high technology, is realted to the cooling of natural gas in a supersonic swirling gas flow. Following thermodynamics devices is similar to the supersonic separator: • Turbo expander.
• Combining expansion. • Cyclonic gas/liquid separation. • Tubular device. For transforing the pressure to shaft power, we can used the turbo expander. Also, using of the supersonic systems in industry is one of the best methods to remove compressible vapours like as natural gas liquids or water from a gas stream.
In the 3S-technology, under action of centrifugal forces, separation of condensate drops with target ingredients condensed in the supersonic nozzle is carry outed. The two phase mixture continues its supersonic swirling now and under the influence of the strong inertial force, the droplets will collide with the wall and merge. A thin film of water will form around the swirling gas which then will be separated by means of a vortex finder as in cyclones. The gas is now dry, and will slow down in the diverging part of the nozzle, regaining almost 70% of its original pressure.
The concept of supersonic separators was introduced to the oil and gas industry in 1990s. These separators were immediately identified as reliable devices with no rotating pans that required no chemicals and were capable of unmanned operation. It has been the purpose of a few studies since then to optimize the performance of supersonic separators and to expand their functionality from a dehydration device to a more sophisticated hydrocarbon dew•pointing and NGL recovery device. The following is a quick review of the achievements of these studies. Their study of supersonic separators is tracked back to 1997 and their first full scalc tcst unit became operational in 1998 and in 2003, supersonic separator used in gas conditioning technology. Their proposed design consisted of a supersonic nozzle that incorporated a small blade (supersonic wing) in the supersonic region to create the swirling motion of thc gas and henee benefit from the centrifugal separation of the heavier particles. This design was later improved to include a swirl generator (ring wing) upstream of the nozzle and in the subsonic region. Another group known for their extensive work on the supersonic separators is a group of Russian specialists who named their separator 3-S.
The design of the 3-S separator was similar to the improved Twister design, incorporating a swirl generator upstream of the nozzle. A third Chinese group has also performed studies on supersonic separators. They built a pilot scale test which used wet gas as process fluid which was capable of attaining a dewpoint depression of about 20°C. Their design used a cyclonic swirl generator in the supersonic region. Another study was performed by a group in Newfoundland, Canada on which the current study is based. They performed CFD based predictions of the flow characteristics inside a converging. Diverging nozzle and showed that this method is a valid tool for this type of study by comparing their results with similar published experimental data. They moreover developed a software that linked to a process simulator (HYSYS), was capable of predicting the performance of a supersonic separator under certain operating conditions with much less computational resources than a CFD package which is reported by Bart prast et al 3 in their article. A supersonic dehydration unit may be consisted of several parts, the most important of which is a converging -diverging supersonic nozzle. It is in this nozzle where the condensa-tion or as it may be in some cases, solidification occurs as well as the separation itself. Since the gas mixture is flowing at supersonic velocities, residence time is extremely low in this type of separators and hydrate has no time to deposit along the device. The unit is considerably smaller than conventional dehydration units and therefore suitable for offshore applications. Moreover, the nozzle has no moving parts and is simple to operate which makes it a very good candidate for unmanned operations for subsea application is also important to note that the gas temperature is reduced based on gas expansion principles and requires no external refrigerant. This brings another major advantage over conventional dehydration units in that intensive water dew points, down to 60°C, can be achieved without any use of external cooling. The selectivity of separation is another asset when water is removed  without the removal of hydrocarbons. This is beneficial in that presence of heavier hydrocarbons increases the gas gravity and reduces the compressibility factor, which results in increased pipeline mass flow capacity. Malyshkina developed mathematical simulations for velocity components, pressure and other parameters as functions of radius for flow within the supersonic separator using the two dimensional Euler model. Qingfcn, Depang, and other colleagues investigated the performance of supersonic separators incorporating a method of particle enlargement to reduce the length of the device. Brouwer, J. M. et al 4,5 used air-ethanol as their medium and water droplets as nucleation centers. Figure 1 display the shematic design of a supersonic nozzle and Figure 2 shows the schematic of the super sonic nozzle body 6 . These examples which is offered Qingfen Maa et al 7 the probability of increasing the profitability of gas processing plants by means of an cheap modernization figure.
The picture of a supersonic dehydration line working on the essential described which is reported by Wen C et al 8 and also with Okimoto Fa and Brouwer J. M. 9 here is pictured in Figure 3.

Conclusions
There are several processes involved in processing the reservoir fluid into oil, gas andwater. One of the most important processes offshore is gas dehydration, because wet gas increases corrosion and can course plugs from ice or gas hydrate.

Acknowledgement
I would like to thank of Dr. Khosravi Niko for his invaluable support and guidance throughout this article, my parents and my friends at the Petroleum University of Technology for making my stay, a wonderful and memorable experience.


extfieldsofstudy: Chemistry Medicine

text:
Gd-Complexes of New Arylpiperazinyl Conjugates of DTPA-Bis(amides): Synthesis, Characterization and Magnetic Relaxation Properties

Two new DTPA-bis(amide) based ligands conjugated with the arylpiperazinyl moiety were synthesized and subsequently transformed into their corresponding Gd(III) complexes 1 and 2 of the type [Gd(L)H2O] nH2O. The relaxivity (R1) of these complexes was measured, which turned out to be comparable with that of Omniscan®, a commercially available MRI contrast agent. The cytotoxicity studies of these complexes indicated that they are non-toxic, which reveals their potential and physiological suitability as MRI contrast agents. All the synthesized ligands and complexes were characterized with the aid of analytical and spectroscopic methods, including elemental analysis, 1H-NMR, FT-IR, XPS and fast atom bombardment (FAB) mass spectrometry.

Introduction
Magnetic Resonance Imaging (MRI) is at present one of the most powerful and efficient non-invasive imaging modalities available for clinical diagnosis. MRI is considered the safest diagnostic technique compared to competing radio-diagnostic methods due to the fact it does use harmful high-energy radiation [1][2][3]. With an enormous diagnostic potential, MRI can be used to assess anatomical changes and for monitoring of organ functions, for instance following functions of the human brain on a real time-scale by functional-MRI (fMRI). In cranial abnormalities or multiple sclerosis, MRI is considered the only reliable diagnostic method [4,5]. MRI contrast agents (CAs) usually involve low molecular weight Gd(III) chelates with an acyclic or macrocyclic ligand [6]. They are diagnostic magneto-pharmaceuticals used to enhance the image contrast by increasing the water proton relaxation rate in the body. The efficacy, known as relaxivity, of a CA is measured by its ability to transmit the paramagnetic properties into the bulk water proton and thereby shorten the longitudinal relaxation (T1) time of water protons, which in turn provides impressive anatomical information [2]. Some representative advantages of employing the Gd(III) ion in most MRI CAs are due to its favorable combination of a large magnetic moment (spin-only μeff = ¼ 7.94 BM, from seven half-filled f orbitals) and long electron spin relaxation time (10 −8 to 10 −9 s, from symmetric S electronic state) [7].
In general, anionic Gd-complexes, for instance Gd(DTPA) 2− , suffer from limitations such as hyperosmolality under physiological conditions and limited utility in focal lesion detection, leading to adverse effects [8]. To overcome the inherent limitations of anionic Gd-complexes and to improve the tissue and/or organ-specificity, the preparation of neutral Gd-macrocyclic analogues for the development of efficient ("optimized") CAs is highly desirable [9][10][11][12]. Earlier reports have suggested that incorporation of alkyl and aromatic groups in the side arm of diethylenetriamine pentaacetic acid (DTPA) rendered excellent relaxivity and water solubility [13][14][15]. In the light of the above, we have designed novel ligands from the reaction of DTPA-bis(anhydride) with suitably modified arylpiperazines and subsequently transformed them into their corresponding Gd(III)-complexes 1 and 2. Herein, we wish to disclose the synthesis, characterization, relaxivity measurements and cytotoxic studies of these new complexes.

Results and Discussion
The development of an optimum MRI contrast agent would necessitate consideration of the high relaxivity, non-cytotoxicity and high water solubility of the targeted complexes. As suggested by earlier reports, modification of the ligand, for instance by the introduction of polar groups on the alkyl substituents of the amide N-atoms of DTPA-bis(amide), can lead to the formation of water soluble Gd-complexes [16]. Therefore, we intended to synthesize Gd-complexes with different arylpiperazine ligands bearing different functionalities in the aryl moiety.
To this end, we first planned to synthesize ligand 7 (Scheme 1), possessing a nitrile group in the aryl moiety. Condensation of benzonitrile 3 [17] with piperazine 4 in DMF generated the coupled product 5 in excellent yield. Reduction of the nitro group of intermediate 5, using Pd-C or Ra-Ni as catalysts, proved problematic and led to the formation of complex mixtures of products, which were difficult to resolve by column chromatography purification. Hence, the reduction of nitro group via transfer hydrogenation with ammonium formate in methanol, using Pd-C as catalyst was employed to produce the corresponding intermediate 6 in high yield. Finally, reaction of aryl amine 6 with diethylene triamine pentaacetic acid dianhydride (DTPAA) [18] in DMF produced the desired ligand 7. With 7 available, we turned to the synthesis of Gd-complex formation. Unfortunately, the reaction of 7 either with GdCl3 or Gd(OAc)3 in pyridine was unsuccessful, unreacted 7 being recovered from these reactions. The decreased reactivity of 7 towards complexation could be attributed to both steric and electronic factors. The substitution position and electron withdrawing ability of nitrile group on the aryl moiety generated congestion and reduced the nucleophilicity of the amine moiety (Scheme 1).  Consequently, we decided to synthesize ligands 16 and 17, which bear methoxy and methoxymethyl substituents in the aryl moiety (Scheme 2). Thus, phenol 9 was reacted with the appropriate alkyl halide in DMF to produce known compounds 10 [19] and 11 [20], respectively. Treatment of intermediates 10 and 11 with piperazine 4 produced 12 and 13 in good yields (Scheme 2). Reduction of the nitro group of intermediates 12 and 13 with ammonium formate in methnaol, using Pd-C as catalyst, generated the corresponding aryl amines 14 and 15 in high yields. Condensation of amines 14 and 15 with DTPAA produced the desired ligands 16 and 17, respectively. Finally, heating of ligands 16 and 17 with GdCl3 in pyridine produced the desired complexes 1 and 2 in good yield (Scheme 2). The structures of complexes 1 and 2 were established by their infrared (IR) spectra, elemental analysis, fast atomic bombardment (FAB) mass spectrometry and X-ray photoelectron spectroscopy (XPS) measurements. Complexes 1 and 2 are highly hygroscopic and were isolated as hydrated solids. The appearance of the ν (OH) band from the water of crystallization at 3426 and 3480 cm −1 , supported this observation [13]. The disappearance of the carbonyl stretching bands of the free ligands at 1728 and 1733 cm −1 in both complexes 1 and 2 indicated the participation of the carbonyl groups in coordination [21]. The chemical composition of compounds 1 and 2 was further confirmed by XPS ( Figure 1). In compound 1, the C 1s region showed three peaks (eV) at 284.8 (C-C), 285.8 (C-N, C-O) and 287.7 (C=C, C=O) [4], whereas the Gd 4d region had four peaks, showing a multiplet structure, the N 1s region displayed one peak at 399.7 eV. Likewise, the O 1s region has peaks (eV) at 531.0 (C-O) and 532.7 (C=O) (Figure 1) [22]. The XPS measurement of compound 2 showed a similar peak pattern as 1 ( Figure 1).   After structural characterization of 1 and 2, we next moved on to measure their relaxvities. Whereas the commercially available MRI contrast agent Omniscan ® is freely soluble in water and methanol; compounds 1 and 2 have moderate water solubility. Relaxvities were calculated as the inverse of the relaxation times per mM based on the earlier report [23]. The R1 data (Table 1) revealed that 2 had a higher relaxivity than 1, but lower than Omniscan ® . This relatively lower relaxivity of 1 and 2 compared Omniscan ® could be attributed to their lower solubility in water. In vitro cell toxicity of compounds 1 and 2 was studied on adherent 3T3 mouse embryonic fibroblastic cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [25]. This assay is used as a quantitative colorimetric method to measure cytotoxicity as well as cell viability. The study revealed that compounds 1 and 2 were non-toxic (Table 1), which warrants their physiological suitability as potential contrast agents for MRI.

CN
In conclusion, two new Gd(III) complexes 1 and 2 of the type [Gd(L)H2O]·nH2O have been synthesized The relaxivity of these complexes was slightly lower compared to Omniscan ® , a commercially available MRI contrast agent. The lower relaxivity of 1 and 2 was attributed to their lower solubility in water. The cytotoxicity studies of these complexes revealed that they are non-toxic which warrants their potential and physiological suitability as MRI contrast agents. The water solubility of these compounds could be increased by introducing polar functions in the aromatic ring, which in turn, may improve their relaxivity. Hence, these compounds may serve as a starting point to obtain optimized structures to produce more efficient MRI contrast agents.

General Information
Melting points were determined on a Büchi apparatus (Büchi, Flawil, Switzerland) and were uncorrected. Elemental analysis was carried out on a Perkin Elmer Elemental Analyzer Series 11 Model 2400 (PerkinElmer, Waltham, MA, USA). IR spectra were recorded on a Perkin Elmer 16F PC FTIR spectrophotometer. 1 H-and 13 C-NMR spectra were measured in CDCl3 and DMSO-d6 using TMS as internal standard on a LA 500 MHz spectrometer (JEOL, Peabody, MA, USA). Mass spectra were recorded on a 6890 N GC-MS system (Agilent Technologies, Santa Clara, CA, USA). Analytical TLC was carried out on silica gel 60 F254 plates (catalog #-5554-7, Merck, Darmstadt, Germany); column chromatography was carried out on Merck silica gel (200-400 mesh, catalog # 61860805001730). All chemicals and reagents were obtained from Sigma-Aldrich (Buchs, Switzerland) in reagent grade and were used without further purification.

Relaxivity Measurements
Longitudinal relaxation times measurements were performed in a 3T MRI machine (3T Imaging, Morton Grove, IL, USA) The magnetic resonance images were taken at 17 different TR values ranging from 20 to 1500 ms and the T1 were obtained from non-linear least square fit measured at each T1 values. R1 were calculated as an inverse of relaxation times per mM.

In Vitro Cell Toxicity
Toxicity for compounds 1 and 2 was analyzed on adherent 3T3 mouse embryonic fibroblastic cells by MTT assay following Mesaik et al. [25] with some modification. In brief, cells were incubated at 6 × 104 mL −1 concentration in 96 well flat bottom plate in 5% CO2 and 37 °C for 24 h. After adherence of cells, compounds were added at various concentrations for further 48 h incubation. On day 3 the tetrazolium dye MTT was added. After 4 h of incubation media was removed and organic solvent DMSO was added to dissolve insoluble purple formazan. Absorbance was taken at 540 nm using a spectrophotometer and the % viability of the cells was calculated.

Conclusions
In conclusion, two new Gd(III) complexes 1 and 2 of the type [Gd(L)H2O]·nH2O have been synthesized The relaxivity of these complexes were slightly lower compare to Omniscan ® , a commercially available MRI contrast agent. The lower relaxivity of 1 and 2 was attributed to their lower solubility in water. The cytotoxicity studies of these complexes revealed that they are non-toxic which warrant their potential and physiological suitability as MRI contrast agents. The water solubility of these complexes may be increased by introducing polar functions in the aromatic ring, which in turn, may improve their relaxivity. Hence, these complexes may serve as a starting point to get optimized compound to achieve more efficient MRI contrast agent.


s2fieldsofstudy: Chemistry Biology

text:
Original 2-(3-Alkoxy-1H-pyrazol-1-yl)azines Inhibitors of Human Dihydroorotate Dehydrogenase (DHODH)

Following our discovery of human dihydroorotate dehydrogenase (DHODH) inhibition by 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidine derivatives as well as 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)-5-methylpyridine, we describe here the syntheses and evaluation of an array of azine-bearing analogues. As in our previous report, the structure–activity study of this series of human DHODH inhibitors was based on a phenotypic assay measuring measles virus replication. Among other inhibitors, this round of syntheses and biological evaluation iteration led to the highly active 5-cyclopropyl-2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-3-fluoropyridine. Inhibition of DHODH by this compound was confirmed in an array of in vitro assays, including enzymatic tests and cell-based assays for viral replication and cellular growth. This molecule was found to be more active than the known inhibitors of DHODH, brequinar and teriflunomide, thus opening perspectives for its use as a tool or for the design of an original series of immunosuppressive agent. Moreover, because other series of inhibitors of human DHODH have been found to also affect Plasmodium falciparum DHODH, all the compounds were assayed for their effect on P. falciparum growth. However, the modest in vitro inhibition solely observed for two compounds did not correlate with their inhibition of P. falciparum DHODH.

■ INTRODUCTION
As explained in more detail in our previous report, 1 in the course of a screening campaign of new chemical entities 2−11 against infectious agents, compounds 1 and 2 were found active on our whole cell measles virus replication assay ( Figure 1). 12 An initial structure−activity study led to confirmation of the potential of this original chemotype and to greatly improved antiviral compounds, including the 2-(3-isopropyloxy-1H-pyrazol-1-yl)pyrimidine derivative 3, which displayed a subnanomolar MIC 50 on this measles virus replication assay ( Figure 1). Moreover, a search for the biochemical mechanism of action of this series pointed out that, as for other recently reported compounds, 13−18 the inhibition of the cellular dihydroorotate dehydrogenase (DHODH) is at the origin of the antiviral effect. 1

■ RESULTS AND DISCUSSION
From these results, in order to increase the chemical diversity possible in this series, we initially replaced the pyrimidine ring by a pyridine moiety because its additional carbon would provide another position for structure−activity studies. Accordingly, as in our first report, 1 we are describing here a succession of synthesis campaigns followed by comments on the antiviral effect obtained. Most of these results are presented in tables in which the first result column (%) provides the yield of the reaction depicted and the second column describes the observed antiviral effect expressed as pMIC 50 values. 19 This corresponds to the negative log of the minimum compound concentration required to inhibit viral growth by 50% when using a recombinant measles virus strain expressing a luciferase as a reporter. 20 We first undertook the preparation of the 2-pyridyl derivatives 6a−t depicted in Table 1. As compound 2, these analogues were prepared from the 3ethoxypyrazole 4 10 and commercially available 2-halogenopyr-idines 5a−p. From 2-halogenopyridines featuring electronattracting substituents, the use of cesium carbonate in dry dimethylformamide or acetonitrile and heating at 150°C with a microwave oven for 1 h efficiently gave the N-pyridyl derivatives. On the other hand, from 2-fluoropyridines featuring electron donating substituents, a temperature of 180°C (under pressure) was found necessary and, because of its decomposition into dimethylamine, dimethylformamide was replaced by acetonitrile. For the preparation of compound 6a 7 or 6p, we used 2bromopyridine 5a or 5p and Taillefer and Cristau coppercatalyzed arylation conditions. 21 Moreover, as for compound 1, the regioselectivity of these reactions was unambiguously checked in a couple of instances. 7 The 5-cyclopropyl bearing derivative 6q was prepared in 48% yield via a Suzuki−Miyaura reaction between cyclopropyl boronic acid and the 5-bromo derivatives 6j. Alternatively, as described in the Experimental Section, the 2fluoropyridines 5q−s were prepared via Suzuki−Miyaura reactions between cyclopropyl boronic acid and the corresponding 5-bromo precursors. From them, the analogues 6q−s were then obtained by N-arylation of 4. The N-arylation of compound 4 with 2-(6-fluoropyridin-3-yl)propan-2-ol (5t) gave the hydroxylated analogue 6t, and a reduction of its alcohol function led to the 5-isopropyl derivative 6u. Interestingly, the palladium-catalyzed hydrogenation at room temperature of 6t was mostly inefficient. To avoid the use of high hydrogen pressure, which would probably have also resulted in the hydrogenation of the pyridine ring, 7 we used triethylsilane in the presence of trifluoromethanesulfonic acid and achieved its hydrogenation into 6u in an unoptimized 24% yield. Concerning the antiviral effects, as seen in Table 1, the "methyl scan" leading to compounds 2 and 6b−d, pointed out the importance of occupying the position 5 of the pyridine ring for a tangible antiviral effect (pMIC 50 = 6.2 for compound 2). The lack of substituents or the introduction of a small fluorine atom seen in 6a, 6e, and 6f also gave almost inactive compounds although some improvement can be observed when comparing the antiviral effect of compound 6a (pMIC 50 < 5) and the 3-fluorinated derivative 6e (pMIC 50 = 5.2). The recourse to a trifluoromethyl (compound 6g) or a chlorine atom (compound 6i) on position 5 gave slightly more active analogues (both pMIC 50 of 5.5), but the bigger bromine present in compound 6j did not improve this further (pMIC 50 = 5.3). On the other hand, the combination of the 3-fluoro and 5bromo substituents seen in compound 6k appeared to be the cause of slight synergic effect (pMIC 50 = 5.9) when considering the pMIC 50 of the parent monosubstituted compounds 6e and 6j (respectively 5.2 and 5.3). In contrast to a fluorine group, the 3methyl substituent of 5-brominated derivative 6l led only to a weak antiviral effect. A loss of effect was also seen for the three derivatives 6m, 6n, and 6o featuring respectively a cyano, an ester, or a methyl ketone on position 5. To a lesser extent, a similar phenomenon was observed with the methoxy of compound 6p (pMIC 50 = 5.5). It is only the introduction of the cyclopropyl group featured by compound 6q that led to an improved inhibition of the virus replication (pMIC 50 = 7.0 nM). Combination of this group with the fluorine of compound 6r led to an unchanged antiviral effect (pMIC 50 7.0), whereas the nitrile of compound 6s replacing this fluorine caused a loss (pMIC 50 = 6.6). Interestingly, the branched isopropyl derivative 6u turns out to be an order of magnitude less active (pMIC 50 = 5.9) than the cyclopropyl analogue 6q. Finally, the polar hydroxyl moiety of compound 6t (pMIC 50 5.3) led to an even bigger loss of effect in comparison with the hydrogen-bearing isopropyl group of compound 6u.
As for the pyrimidine-containing series, 1 by arylation of the 4aryloxypyrazoles 7a−q with the 2-fluoropyridine 5q, we prepared  the 4-aryloxy derivatives 8a−q depicted in Table 2. Later on, we also prepared the corresponding pyridazine homologues 10b−q from 3-chloro-6-cyclopropylpyridazine (9). For comparison purposes, Table 2 provides the pMIC 50 for these two series.
With few variations, the pattern of antiviral effect for analogues 8a−q featuring a 5-cyclopylpyridine is somehow mirroring the one seen for the 5-ethylpyrimidyl bearing homologues. 1 In comparison with the phenoxy-bearing compound 8a (pMIC 50 = 7.0), ortho-substitution with a fluorine, a chlorine of a bromine atom improved the antiviral effect (pMIC 50 of respectively 7.7, 7.9, and 7.8). A trifluoromethyl group instead of these halogens had a lesser effect (compound 8e, pMIC 50 = 7.3). Shifting this trifluoromethyl group on the meta position of the phenoxy ring did not alter much the antiviral effect (pMIC 50 = 7.1 for 8f), whereas placing this group on the para position led to a substantial loss (pMIC 50 = 5.3 for 8g). A similar trend is observed with the fluorine atom of compounds 8k−m, the ortho-fluoro derivative 8k being the most active (pMIC 50 = 7.4) and the para-fluorinated analogue 8m far less effective (pMIC 50 = 5.9). Trends in the polyhalogenated derivatives are somehow less clear-cut although ortho-substituted derivatives are the most active but, as in the pyrimidine series, 1 with a pMIC 50 of 9.0, the 2,6-difluorophenoxy derivative 8q is the best antiviral of this group of analogues. Very similar comments can be made on the antiviral effects of compounds 10b−q, and the differences between any given pair of analogues is always less than an order of magnitude. As depicted in Scheme 1, we also explored the preparation and use of precursors featuring a 5-cyclopropylpyridine moiety to which additional chemistry would allow the introduction of various group on the carbon 4 of the pyrazole ring. The N-arylation of the 4-iodopyrazole 11a 5 with 5-cyclopropyl-2-fluoropyridine (5q) had to be conducted at 180°C for up to 12 h and only led to a rather small amount (13%) of the 4-iodinated target compound 12a along with some (9%) of the reduced derivative 12b. This is quite in contrast with the 55% yield we reported for the N-arylation of compound 11a with 2-fluoropyridine at the same temperature for 3 h. 7 Extensive analysis of the reaction mixture pointed out the presence of a large amount of 3-ethoxy-5-methylpyrazole (11b) resulting from the reduction of the 4-iodopyrazole 11a under this long reaction time. Although an explanation for this reduction is not obvious (a carbene occurrence?), we suggest that the much decreased reactivity of the electron rich 5-cyclopropyl-2fluoropyridine (5q) toward nucleophilic reaction in comparison with 2-fluoropyridine is allowing the necessary time for this side reaction to proceed to a large extent. Somehow related to such behavior is the quick evolution of iodine, or iodochloride, upon heating 3-ethoxy-4-iodopyrazole in hydrochloric acid. 5 In any case, the iodinated target compound 12b was treated with butyllithium followed by the addition of benzoyl chloride to give the 4-benzoyl derivative 13 in 44% yield. In an attempt to improve these results, we undertook the N-arylation of the 4-bromopyr- azole 11c. Under similar conditions, we could obtain the 4brominated precursor 12c in a much improved 48% yield. The sodium borohydride reduction of ketone 13 led to the secondary alcohol 14a, whereas the addition of methyl lithium to ketone 13 provided an access to the tertiary alcohol 14b. From compound 12c, palladium-catalyzed reactions allowed the preparation of few analogues. A Suzuki−Miyaura reaction with phenylboronic acid gave 33% of compound 15, whereas Negishi reactions with benzylzinc bromide or (1-phenylethyl)zinc chloride gave compounds 6q and 16 in, respectively, 71 and 42%. Of note in this part is an optimization of the preparation of 1-phenylethylzinc chloride using the lithium chloride, dibromoethane, and trimethysilyl chloride-based protocol 22−24 as well as the use of CPhos instead of XPhos in an attempt to lessen the extent of βelimination possible with (1-phenylethyl)zinc chloride. 25 Concerning the antiviral effects of these compound, as for the pyrimidine-containing series, 1 the benzoyl derivative 13 (pMIC 50 = 6.9) was slightly less effective than the corresponding methylene analogue 6q (pMIC 50 = 7.0). On the other hand, the alcohol 14a retained a more sizable antiviral effect (pMIC 50 = 7.4) but the gain is far from what we could observe in the case of the pyrimidinecontaining series. 1 Introducing an additional methyl on this carbon led the tertiary alcohol 14b and to a much reduced antiviral activity (pMIC 50 = 6.1). A similar pattern was seen when adding a methyl to compound 6q (pMIC 50 = 7.0), leading to the much less effective analogue 16 (pMIC 50 = 5.2). Finally, the low antiviral effect of the 4-phenyl derivative 15 (pMIC 50 = 6.0) pointed out the importance of a bridge between the pyrazole and the aromatic ring. As depicted in Table 3, we then retained the 2,6difluorophenoxy component of compound 8q and prepared the 2-pyridyl derivatives 18a−s. Analogues 18a−n were obtained by the N-arylation of the pyrazole 7q by the corresponding 2halogenopyridines 5a, 5g, 5j−o, and 5q−t and the 2fluoropyridines 17l−n (prepared as described in the Experimental Section). In the case of the methoxy-bearing analogue 18p, we used again the 2-bromo-5-methoxypyridine (5p) and Taillefer− Cristau copper-catalyzed arylation conditions. 21 The volatile 2bromo-5-(1,1-difluoroethyl)pyridine (17n) was obtained from 2bromo-5-acetylpyridine (5o). The following N-arylation of compound 7q with this 2-bromopyridine 17n had to be conducted at a relatively lower temperature to avoid extensive decomposition, and the pure difluoromethylene-bearing analogue 18n was thus obtained in a modest 12% yield. A rather slow catalytic hydrogenation of the cyclopropyl ring of compound 18d enabled an access to the propyl analogue 18o in a 30% yield. The preparation of analogues 18p−r turned out to be possible as, under basic conditions, the fluorine atom on the pyridine ring of 18d could be displaced by methanol, dimethylamine, or benzylalcohol using high temperature and a microwave oven. In the last case, the catalytic hydrogenation of the resulting 3-benzyloxyderivative 18r led to the 3-hydroxypyridinyl derivative 18s. Unexpectedly, despite few trials, the direct hydrolysis of compound 18d into 18s, using sodium hydroxide in THF, was not successful. As seen in Table 3, with these compounds we could assess the effectof varying the pyridine substituents while retaining a 2,6-difluorophenoxy moiety. Going from the hydrogen of the N-pyridine derivative 18a (pMIC 50 = 4.9) to the 5-methyl homologue 18b (pMIC 50 = 7.6), a 400-fold improvement of the antiviral effect was observed. From then, the 5-cyclopropyl group of compound 8q (pMIC 50 = 9) provided another order of magnitude. On the other hand, the ethyl group of compound 18l (pMIC 50 = 7.1) caused a loss of antiviral effect. This is actually reminiscent of what was observed for the 5isopropyl bearing compound 6u (pMIC 50 = 5.9) in comparison with the 5-cyclopropyl bearing analogue 6q (pMIC 50 = 7.0) described above. Moreover, it is contrasting with what we previously reported for the 5-ethyl and 5-cyclopropyl pyrimidine analogues which had mostly the same antiviral effect(pMIC 50 =9.2 and 8.9). 1 Replacement of the methyl group of compound 18b (pMIC 50 = 7.6) by the trifluoromethyl of compound 18c (pMIC 50 = 6.5) led to more than a 10-fold loss. If adding a fluorine atom on carbon 3 of the pyridine ring has very little effect when comparing the 5-ethyl bearing analogues 18l and 18m (pMIC 50 both of 7.1), a 2-fold loss was seen in comparison with the 5-cyclopropyl pair 8q and 18d (pMIC 50 = 9 and 8.6). On the other hand, as for the pair of analogues 6j and 6k, a fluorine on the same position is improving the antiviral effect when comparing the activity of the 5-bromo derivatives 18f and 18g (pMIC 50 = 6.3 and 6.9). Attempts to introduce various polar groups on carbon 5 of the pyridine ring such as the one seen in compound 18i−k did not improve the antiviral effect, and the two fluorine atoms of compound 18n (pMIC 50 = 7.1) had no effect in comparison with the 5-ethyl analogue 18l (pMIC 50 = 7.1). Only relatively small losses were observed for the bis-substitued derivatives 18p−s featuring a cyclopropyl group. Interestingly, the influence of the methoxy moiety of compound 18p (pMIC 50 = 7.5) is comparable to the dimethylaminated derivative 18q (pMIC 50 = 7.5), whereas the hydroxyl-bearing analogue 18o (pMIC 50  relatively stronger activity. Somehow unexpectedly, the larger benzyloxy group of compound 18r (pMIC 50 = 7) only caused a relatively small loss of antiviral effect. In any case, none of the analogues described in Table 3 displayed an antiviral effect better than the nanomolar level of compound 8q. As depicted in Figure 2 and fully described in the Experimental Section, from the 2,6-difluorophenoxy pyrazole 7q, some other possible azines-bearing compounds such as 3-pyridyl and 5pyrimidyl, the 2-pyrazinyl and the 1,2,4-triazine derivatives were also prepared. The 3-pyridyl bearing compounds 19a−c were made by the Taillefer−Cristau 21 copper catalyzed N-arylation of 7q with the commercially available 5-bromo-2-methyl, 5-bromo-2-ethyl, and 5-bromo-2-methoxypyridines. The 5-pyrimidyl derivative 20a was also made with the copper catalyzed reaction between 7q and 5-bromo-2-tert-butylpyrimidine. For the preparation of the 5-cyclopropyl analogue 20b, we had recourse to the N-arylation of compound 7q with a small sample of 2cyclopropylpyrimidin-5-ylboronic acid using Lam and Chan reaction conditions. 26 The N-arylation of 2,6-difluorophenoxy pyrazole 7q with 2,5-dibromopyrazine led to the 5-bromopyrazine analogue 21a. Similarly, from 2-chloro-5-(trifluoromethyl)pyrazine, the 5-trifluoromethyl analogue 21b was obtained. The methoxy-bearing analogue 21c was then made from 21a by displacement of the bromine atom with methanol under basic conditions. As described in the Experimental Section, the preparation of 2-bromo-5-cyclopropylpyrazine allowed the synthesis of the cyclopropyl-bearing analogue 21d by N-arylation. In similar ways, the pyridazine-bearing analogues 10q and 22a−b were prepared from the corresponding chloropyridazines. The methoxy or ethoxy-bearing analogues 22c−d were made by displacement of the chlorine atom of compound 22b with the corresponding alcohols because an N-arylation trials using 3chloro-6-methoxypyridazine led to extensive side reactions, including, as seen by LC/MS, a methylation of compound 7q. The 1,2,4-triazine-bearing analogue 23 was prepared in a modest 10% yield by the N-arylation of compound 7q with 6-ethyl-3-(methylthio)-1,2,4-triazin-5-ol (preparation given) at 200°C. Finally, as fully described in the Experimental Section, we also prepared the N-ethyl imidazole-bearing analogues 24 by the copper catalyzed N-arylation of compound 7q with 1-ethyl-4iodo-1H-imidazole. In view of the antiviral effect of the azinebearing analogues 8q, 10q, 19b, and 21d, one fact clearly stands out. As for the 2-pyrimidyl analogues previously studied, an ethyl or a cyclopropyl group para to the pyrazole ring often provides a low nanomolar antiviral effect. However, this is not true for the 2cyclopropylpyrimidine derivative 20b (pMIC 50 = 6.8) or the triazine 23 (pMIC 50 = 7.6), which is in this case featuring an additional hydroxyl group. Attempt to replace such alkyl groups by side chains of similar size but featuring more polar groups mostly failed, as seen for the 2-methoxypyridine derivative 19c (pMIC 50 = 6.0), the 2-methoxypyrazine 21c (pMIC 50 = 6.1), or the 3methoxypyridazine 22c (pMIC 50 = 7.1). The same trend was observed for the halogen-bearing analogues 21a−b (pMIC 50 = 5.5 and 5.3) or 22b (pMIC 50 = 6.8). Finally, the relatively low antiviral effect of the N-ethylimidazole derivative 23 may be another example of the trend which points at a detrimental effect of polar atoms (a nitrogen in this case) in the vicinity of this alkyl side chain.
The elucidation 1 of the biochemical target of our series has led us to publish a survey of all the reported inhibitors of DHODH along with their uses. 27 This review pointed out that teriflunomide (25) depicted in Figure 3 is the only human DHODH inhibitor used in medicine against autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Interestingly, in our cellular assay, teriflunomide (25) displayed an antiviral effect with an MIC 50 of 5 μM, which is reflected in the previously reported IC 50 of 1 μM on recombinant human DHODH. 28,29 This relatively modest effect of teriflunomide (25) on the enzyme had actually triggered the search and the discovery of some off-target inhibitions in the past. 30−32 This value can also be compared to the enzymatic IC 50 of 10 nM reported for brequinar (26), 29 a stronger inhibitor of DHODH which underwent disappointing phase II trials against solid tumors. 33−37 To assess the potential of our series in comparison with these compounds, we undertook an array of biological assays using compound 18d. In cellulo, as depicted in Figure 4, we could point out that compound 18d is affecting pyrimidine nucleoside  biosynthesis. Indeed, while adding 18d at concentration varying from 4 to 100 nM blocked the measles virus replication in cells, the addition of the pyrimidine-containing nucleoside uridine at 10 μg/ mL ( Figure 4A) restored its replication. On the other hand, the addition of the purine-containing nucleoside guanosine at 10 μg/ mL did not restore this ( Figure 4B). Moreover, a restored virus replication was seen with the addition of orotic acid at 3 mM ( Figure 4C) while, as seen in Figure 4D, dihydroorotic acid at 3 mM had no such effect. These last results thus narrowed down the biochemical target of compound 18d to DHODH. Accordingly, as reported, 1 we produced recombinant human DHODH and compound 18d was indeed found to be an inhibitor of this enzyme with an IC 50 of 25 ± 5 nM.
By using a metabolite analysis protocol, 38 the HEK-293 T cells content in adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP) treated for 24 h with various concentration of compound 18d could be determined. As seen in Table 4, intracellular concentrations of uridine and cytidine collapsed in cells treated with 18d, whereas purine nucleotides concentrations were slightly increased, likely as a consequence of the control loops connecting purine and pyrimidine metabolic pathways. This strongly demonstrates in cell cultures the inhibition of de novo pyrimidine biosynthesis by 18d.
We recently reported that the inhibition of pyrimidine biosynthesis amplifies cellular response to pathogen-associated molecular patterns such as exogenous RNA molecules. 17 When transfecting cells with small synthetic RNA molecules (ssRNA) that mimic viral RNA genomes or transcripts, activation of the interferon-stimulated response element (ISRE) that drives innate immunity genes was enhanced by DHODH inhibition. As depicted in Figure 5, we thus monitored that compound 18d increased the expression of an ISRE-luciferase reporter gene when transfecting cells with ssRNA molecules. This adds to the panel of cellular assays that support the inhibition of pyrimidine biosynthesis by compound 18d.
Aside from their antiviral effect, pyrimidine biosynthesis inhibitors are also well-known for blocking the proliferation of lymphocytes, and this probably accounts for their immunosuppressive property in vivo. 39 Accordingly, we also determined the effect of compound 18d on the growth of Jurkat T lymphocyte cell line, which has been demonstrated to be sensitive to DHODH inhibitors. 40,41 As shown in Figure 6, compound 18d strongly inhibited Jurkat cells proliferation (IC 50 = 0.02 μM) and its level of inhibition compared favorably with brequinar (IC 50 = 0.2 μM) or teriflunomide (IC 50 close to 60 μM, data not shown).
Also of much interest is the recent demonstration that Plasmodium falciparum DHODH is a valid target for the treatment of Malaria 42−46 and that a dual inhibition of human and P. falciparum DHODH was noticed for some series. 47−50 Accordingly, we screened all these antiviral compounds for a potential inhibition of P. falciparum growth. No growth inhibition was seen in a cellular assay at the concentration of 8.66 μg/mL (data not shown) for all the compounds aside from a modest effect for the pyridazine-bearing analogues 10q and 22a (IC 50 of 0.33 and 0.47 μM). Of course, these values led us to prepare many more pyridazine-bearing analogues, including compound 10b−p, but all of them turned out to be at least an order of magnitude less effective on the parasite growth. Just in case, compound 10q and 22a were also assayed for their eventual inhibition of recombinant P. falciparum DHODH but they turned out to be completely devoid of effect on this biochemical assay.

■ CONCLUSION
In conclusion, along with our previous report, 1 this study made good use of the alkoxypyrazole chemistry we previously reported, 2−11 which somehow cleared a sort of "chemical blind spot" existing in pyrazole chemistry. The ensuing screenings of the resulting new chemical entities led us to extensively explore the structure−activity relationship of a new series of human DHODH inhibitors. Of note is a cellular antiviral assay which greatly simplified the evaluation of the compounds prepared and probably filtered out analogues of low cell membrane permeability. Concerning the potential of this series of inhibitors against immune diseases, the current success of teriflunomide (25) 51,52 has led to renewed interest in the search for better inhibitors of   (26), are featuring a carboxylic acid function, we are currently working on the selection of compounds displaying optimal preclinical properties in order to secure a proof of effect on an animal model of autoimmune disease. This endeavor is based on the reasonable assumption that our carboxylic acid-free series of inhibitors could display a different and beneficial pharmacological profile in comparison with the inhibitors currently in preclinical or clinical studies. However, so far, our best inhibitors can be only considered as tools as a too short microsomal stability has been a recurrent feature for this series. Indeed, low half-life values were observed for compounds such as 8k (t 1/2 = 4 min) or 8q (t 1/2 = 6 min) on human microsomes and only modest improvements were seen for the "fluorine-protected" compound 18d (t 1/2 = 27−41 min), the "hydroxy-protected" compound 18s (t 1/2 = 38 min), or the less active analogues 22c (t 1/2 = 22 min) and 18g (t 1/2 = 60 min). Related to this aspect is a report mentioning similar difficulties for another type of N-arylated pyrazoles. 62 Accordingly, additional work will be required to improve this and the X-ray based determination of the binding mode of this series of inhibitors to human DHODH could be very useful in this regard. Finally, these results are probably an illustration of the interest of designing whole cell/phenotypic assays of sufficient sensitivity (a bioluminescent virus in the present case). 12 This led us to detect an unexpected biological effect and provided a simple biological mean not only to undertake further structure−activity study but also to find the mode of action of this series of compounds.

■ EXPERIMENTAL SECTION
Measles Virus Inhibition Assay. HEK-293T cells (ATCC) were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco-Invitrogen) containing 10% fetal calf serum (FCS), penicillin, and streptomycin at 37°C and 5% CO 2 . Antiviral activity of compounds was determined using a recombinant vaccine strain of measles virus expressing firefly luciferase (rMV2/Luc) from an additional transcription unit. 20 To determine the MIC 50 , HEK-293T cells were infected with rMV2/Luc (MOI = 0.1) and incubated in 96-well culture plates at 3 × 10 +4 cells/well with increasing concentrations of compounds or DMSO alone. After 24 h, luciferase expression was determined. The MIC 50 corresponds to the concentration of a compound inhibiting luciferase activity by 50%.
P. falciparum Growth Assay. Compounds were screened against P. falciparum 3D7 strain parasites with a starting parasitemia of 0.8% at 2% hematocrit in 96-well plates. Compound concentrations were 8.66 μg/ mL final in the assay. Parasite growth and proliferation was measured using SYBR Green I. 63 Human DHODHs. The assay was performed as described previously. 1 P. falciparum DHODHs. This enzyme was expressed as recombinant proteins in Escherichia coli and purified as previously described. 44,64 Steady-state kinetic analysis was performed using the 2,5-dichloroindophenol (DCIP)-based spectrophotometric method as described. 44 Enzyme and substrate concentrations were: DHODH (E = 5−10 nM) and substrates (0.2 mM L-dihydroorotate and 0.02 mM CoQd). The 100× compound stock solutions were made in DMSO by covering a 3-fold dilution series such that final concentrations in the assay for inhibition ranged from 0.001−100 μM.   (26). Jurkat cells were incubated with increasing doses of 18d or brequinar. As a control, cells were treated with DMSO alone. At t = 0 and after 72 h of culture, the number of living cells was determined using the CellTiter-Glo reagent. The inhibition of cellular proliferation is expressed as a percentage relative to DMSO-treated control wells. The results presented correspond to the mean ± SD of two independent experiments.
Metabolite Analyses. HEK-293T cells were plated in 6-well plates (10 6 cells per well). One day later, culture medium was supplemented with increasing doses of compound 18d or DMSO alone. After an additional 24 h of culture, cells were harvested, carefully counted, and monitored for viability by trypan blue exclusion. Cellular nucleotides were quantified as previously described. 17 ISRE-Luciferase Reporter Assay. STING-37 cell line was previously described and corresponds to HEK-293 T cells that express luciferase under control of five interferon-stimulated response elements (ISRE). 17 Cells were transfected with increasing amounts of ssRNA molecules using JetPrime PEI reagents (Polyplus transfection) and dispensed in 96-well plates at 35000 cells/well in 100 μL of Dulbecco's Modified Eagle's Medium (DMEM; Gibco-Invitrogen) containing 10% fetal calf serum (FCS), penicillin, and streptomycin. DMSO or compound 18d was added to culture medium, and after mixing, cells were cultured for 24 h at 37°C and 5% CO 2 . Finally, firefly luciferase activity was determined using the Bright-Glo reagent following manufacturer's recommendations (Promega).
Inhibition of Jurkat Cells Proliferation. Jurkat cells were cultured in at 5 × 10 4 cells per well in flat-bottom 96-well culture dishes. Cells were maintained at 37°C and 5% CO 2 in RPMI (Gibco-Invitrogen) containing 10% fetal calf serum (FCS), pyruvate sodium, nonessential amino acids, penicillin, and streptomycin. Number of living cells was determined by quantification of adenosine triphosphate (ATP) in culture wells using the CellTiter-Glo Assay (Promega) following manufacturer's recommendations. This luciferase-based assay evaluates by ATP quantification the number of metabolically active cells in culture wells.
Human Microsome Stability. The microsomal stability assessments were subcontracted to Oroxcell, Romainville, France.
Chemistry. A Biotage Initiator 2 microwave oven was used for reactions mentioning such heating method. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance 400 spectrometer at 400 and 100 MHz, respectively. Shifts (δ)are givenin ppmwith respect to the TMS signal, and coupling constants (J) are given in hertz. Column chromatography were performed either on Merck silica gel 60 (0.035− 0.070 mm) or neutral alumina containing 1.5% of added water using a solvent pump and an automated collecting system driven by a UV detector set to 254 nm unless required otherwise. Sample deposition was carried out by adsorption of the mixture to be purified on a small amount of the solid phase followed by its deposition of the top of the column. The low resolution mass spectra were obtained on an Agilent 1100 series LC/MSD system using an atmospheric electrospray ionization system and the high resolution mass spectra (HRMS) were obtained using a Waters Micromass Q-Tof with an electrospray ion source. Unless stated otherwise, a purity of at least 95% was obtained for all the compounds by means of chromatography, recrystallization, or distillation and this level of purity was established by TLC, LC/MS and NMR spectroscopy Preparations of All the Commercially Unavailable Halogenoazines Used in This Work. 5-Cyclopropyl-2-fluoropyridine (5q). Under an inert atmosphere, 5-bromo-2-fluoropyridine (5j) (17 g, 0.096 mol) and cesium carbonate (114 g, 0.35 mol) were dispersed in a 95/5 vv mixture of toluene and water (500 mL). This was degassed by gently bubbling argon in the reaction and cyclopropyl boronic acid (10 g, 0.11 mol) and [1,1′-bis(diphenyl phosphino)ferrocene] dichloropalladium complexed with dichloromethane (1.45 g, 0.0018 mol) was added. The flask was heated to reflux for 40 min; upon cooling, the suspension was diluted in ethyl acetate, and the filtered organic layer was washed with water and brine, dried over magnesium sulfate, and concentrated to dryness. The residue was purified by a distillation at ambient pressure (E 760 mm = 206°C) to give a volatile oil (8.06 g, 62%). A lesser pure fraction (about 7%) was also collected. 1  5-Cyclopropyl-2,3-difluoropyridine (5r). By using the procedure described for the preparation of 5-cyclopropyl-2-fluoropyridine (5q), this compound was obtained from 5-bromo-2,3-difluoropyrimidine (5k) in 63% yield, as a volatile oil, after a chromatography over silica gel (cyclohexane−dichloromethane 2/1). 1  2-Chloro-5-cyclopropylnicotinonitrile (5s). By using the procedure described for the preparation of 5-cyclopropyl-2-fluoropyridine (5q), this compound was obtained from 5-bromo-2-chloronicotinonitrile in 53% yield, as a solid, after a chromatography over silica gel (cyclohexane− dichloromethane 1/1). 1  2-(6-Fluoropyridin-3-yl)propan-2-ol (5t). Under an inert atmosphere, 5-bromo-2-fluoropyridine (5j) (3.12 g, 17.7 mmol) was dissolved in dry ether (100 mL) and the solution cooled to −78°C. Butyl lithium (9.3 mL, 18.6 mmol, 2N in cyclohexane) was added. The resulting precipitate was stirred at −78°C for 15 mn, acetone (0.4 mL, 90 mmol, dried over 4 Å molecular sieves) was added, and the reaction was allowed to warm back to room temperature for 30 mn. This was diluted with a saturated solution of ammonium chloride and extracted with ethyl acetate. The organic layer was washed with a saturated solution of ammonium chloride and brine, dried over magnesium sulfate, and concentrated to dryness. The residue was further purified by a chromatography over silica gel (dichloromethane−ethanol 98.5/1.5) to yield compound 5t as an oil (0.57 g, 20%). 1  2-Fluoro-5-ethylpyridine (17l). Under an inert atmosphere, 5-bromo-2-fluoropyridine (5j) (5.35 g, 30.39 mmol) and potassium carbonate (16.8 g, 121.59 mmol) were dissolved in dry dimethylformamide (75 mL, dried over 4 Å molecular sieves). Oxygen was removed from this solution by a slow stream of argon, and [1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium complexed with dichloromethane (0.62 g, 1.52 mmol) was added, followed by a 1 M solution of triethylborane (40 mL, 40.4 mmol) which was slowly added with a syringe. This darkening suspension was heatedat 85°C for 4 husing an oil bath. The resulting black suspension was diluted in diethyl ether and water, the organic layer was washed with water 5 times and brine, dried over magnesium sulfate, and cautiously concentrated to dryness to take into account the volatility of the reaction product. The residue was purified by a chromatography over silica gel (cyclohexane−dichloromethane from 2/3 to 1/4) to yield the 5-ethyl derivative 17l as a volatile oil (1.52 g, 40%). 1  2,3-Difluoro-5-ethylpyridine (17k). By using the protocol described for the synthesis of 2-fluoro-5-ethylpyridine (17l), compound 17k was obtained in 17% yield from 5-bromo-2,3-difluoropyridine (5q) as a volatile oil after a chromatography over silica gel (cyclohexane− dichloromethane from 1/1 to 1/4). 1  Another fraction of this chromatography led, after a recrystallization in cyclohexane, to 5,5′,6,6′-tetrafluoro-3,3′-bipyridine in a 25% yield as fine needles. 1  2-Bromo-5-(1,1-difluoroethyl)pyridine (17n). In a closed Teflon flask, 1-(6-bromopyridin-3-yl)ethanone (5o) (0.95 g, 4.75 mmol) and bis(2-methoxyethyl)aminosulfur trifluoride (3.6 g, 11.4 mmol) were stirred for 7 days (as only a 78% conversion was monitored by 1 H NMR, a longer reaction time may lead to an even better yield). This was diluted in water, solid calcium chloride was added, and the solution was extracted with ethyl acetate. The organic layer was washed with water and brine, dried over magnesium sulfate, and concentrated to dryness (not for too long, this compound is volatile). The residue was purified by a chromatography over silica gel (cyclohexane −dichloromethane 1/1) to yield the difluorinated pyridine 17n as a colorless and volatile oil (0.57 g, 54%). 1  2-Bromo-5-cyclopropylpyrazine. Under an inert atmosphere, 2,5dibromopyrazine (0.21 g, 0.88 mmol) and cesium carbonate (1.12 g, 3.43 mmol) were dispersed in a 95/5 v/v mixture of toluene and water (5 mL). Note: potassium carbonate works as well. This was degassed by gently bubbling argon in the reaction, and [1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium complexed with dichloromethane (0.018 g, 0.022 mmol) and cyclopropyl boronic acid (0.098 g, 1. 14 mmol) was added. The flask was heated to reflux for 30 min; upon cooling, the suspension was diluted in ethyl acetate, the filtered organic layer was washed with water and brine, dried over magnesium sulfate, and concentrated to dryness. This was residue was purified by a chromatography over silica gel (cyclohexane−ethyl acetate 97/3 → 9/1 to yield the 2-bromo-5-cyclopropylpyrazine (0.08 g, 45%) as a solid. 1  3-Chloro-6-cyclopropylpyridazine (9). By using the same procedure used for the preparation of 2-bromo-5-cyclopropylpyrazine, this compound was obtained in 40% yield as a solid (on a much larger scale), from 3,6-dichloropyridazine, after two consecutive chromatography processes over silica gel (dichloromethane−ethanol 99/1) and the second using only dichloromethane to yield the 2-bromo-5-cyclopropylpyrazine. 1  6-Ethyl-3-(methylthio)-1,2,4-triazin-5-ol. First step. Preparation of 2-(2-carbamothioylhydrazono)butanoic acid: As previously described, 66 2oxobutyric acid (3.15 g, 0.0308 mol) and thiosemicarbazide (2.81 g, 0.0308 mol) were stirred in water (60 mL) at 70°C for 10 min. This was left to cool, and the precipitate was filtered, washed with water, and dried at 50°C under vacuum to give the hydrazone (4.47 g, 82%) as 4/17 mixture of isomers. 1  Preparation of 6-ethyl-3-mercapto-1,2,4-triazin-5-ol: As previously described, 66 the hydrazone (4.24 g, 0.0269 mol) and sodium carbonate (2.56 g, 0.0269 mol) were heated to reflux in water (300 mL) for 3 h. The solution was made acid with acetic acid and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, and concentrated to dryness to yield the 1,2,4-triazin-5-ol as a white powder (3.09 g, 86%). 1  Methyl iodide (1.07 mL, 0.017 mmol), diluted in tetrahydrofuran (2 mL), was then slowly added. The solution was stirred at room temperature for 4 h; this was diluted with water, made acid with acetic acid, and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, and concentrated to dryness to yield the pure thioether as a white powder (2.28 g, 85%). 1  1-Ethyl-4-iodo-1H-imidazole.
Step 1: Under a moisture-protected atmosphere, a 50/50 mixture of 4,5-diiodo-1H-imidazole and 2,4,5triiodo-1H-imidazole, obtained when iodinating imidazole using the described procedure, 67 and ethyl iodide (2.14 g, 0.0137 mol) potassium carbonate (3.97 g, 0.0286 mol) were stirred in dimethylformamide (50 mL, dried over 4A molecular sieve) for 22 h. This was diluted in water, extracted with ethyl acetate, and the organic layer was washed with water and brine, dried over magnesium sulfate, and concentrated to dryness. The residue was purified by a chromatography over silica gel (dichloromethane−ethanol 99/1 to 97/3) to give the 1-ethyl-4,5diiodo-1H-imidazole (1.79 g, 37% from imidazole). 1  Step 2: Under argon, 1-ethyl-4,5-diiodo-1H-imidazole (1.8 g, 0.0051 mol) was dissolved in dry tetrahydrofuran (20 mL). The solution was cooled to 0°C, and ethyl magnesium (1.8 mL, 0.0054 mol, 3 M solution in ether) was added. The resulting suspension was stirred for 30 min, quenched with an excess of ammonium chloride in water, and extracted with ethyl acetate. The organic layer was washed with water and brine, dried over magnesium sulfate, and concentrated to dryness. The residue was purified by a chromatography over silica gel (dichloromethane−ethanol 98/2) to give the 1-ethyl-4-iodo-1H-imidazole (0.78 g, 68%) as an oil. 1  N-Arylation of 3-Alkoxypyrazoles without Copper Catalyst, General Method. In a reaction vial designed for microwave heating, the considered alkoxypyrazole (2 mmol), the considered halogenated heteroaryl (2.2 mmol), and cesium carbonate (2.8 mmol) were stirred in dimethylformamide or acetonitrile (3 mL) as specified. This was heated using a microwave at a temperature between 120 and 180°C for the specified duration. The resulting suspension was diluted in water and extracted with ethyl acetate, and the organic layer was washed with brine, dried over magnesium sulfate, and concentrated to dryness. The residue was further purified as specified below.