New Synthesis, Structure and Analgesic Properties of Methyl 1-R-4-Methyl-2,2-Dioxo-1H-2λ6,1-Benzothiazine-3-Carboxylates

According to the principles of the methodology of bioisosteric replacements a series of methyl 1-R-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylates has been obtained as potential analgesics. In addition, a fundamentally new strategy for the synthesis of compounds of this chemical class involving the introduction of N-alkyl substituent at the final stage in 2,1-benzothiazine nucleus already formed has been proposed. Using nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and X-ray diffraction analysis it has been proven that in the DMSO/K2CO3 system the reaction of methyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate and alkyl halides leads to formation of N-substituted derivatives with good yields regardless of the structure of the alkylating agent. The peculiarities of NMR (1Н and 13С) spectra of the compounds synthesized, their mass spectrometric behavior and the spatial structure are discussed. In N-benzyl derivative the ability to form a monosolvate with methanol has been found. According to the results of the pharmacological testing conducted on the model of the thermal tail-flick it has been determined that replacement of 4-ОН-group in methyl 1-R-4-hydroxy-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylates for the methyl group is actually bioisosteric since all methyl 1-R-4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylates synthesized demonstrated a statistically significant analgesic effect. The majority of the substances can inhibit the thermal pain response much more effective than piroxicam in the same dose. Under the same conditions as an analgesic the N-methyl-substituted analog exceeds not only piroxicam, but more active meloxicam as well. Therefore, it deserves in-depth biological studies on other experimental models.


Introduction
The topical issues of pharmaceutical and medical chemistry are the search of lead compounds with the high analgesic activity, their subsequent optimization and creation of new pain killers, which with the high analgesic activity, their subsequent optimization and creation of new pain killers, which can meet the current requirements for efficiency and safety of medicines. The concept of bioisosteric replacements has proven to be one of the successful and fruitful methods for solving problems of this type [1,2]. By now it has become a powerful tool for purposeful synthesis of biologically active substances not only with the desired pharmacological activity, but with improved characteristics compared to the structures-prototypes [3][4][5][6][7][8][9].
The interesting objects of study in this respect are various derivatives 4-hydroxy-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylic acids. We have chosen these compounds for a number of reasons. First, because of synthetic difficulties, they still remain chemically and pharmacologically understudied, and it makes them particularly attractive from the point of view of novelty that is important for any innovation project. Secondly, having synthesized and studied only approximately 200 compounds of this chemical class we succeeded in demonstrating convincingly that esters (especially methyl esters) [10,11], arylalkylamides [12][13][14], anilides [15][16][17] and hetarylamides [18,19] of 1-R-4-hydroxy-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylic acids have real prospects for developing highly effective analgesics. Finally, another important factor is that derivatives of 4-hydroxy-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylic acids and, first of all, their esters of the general formula I (Figure 1), possess a wide synthetic potential allowing to make a variety of chemical transformations to the basic molecule virtually unrestrictedly and thereby to change its pharmaceutical and (or) pharmacological properties in the right direction.
Even a cursory analysis of the structure of esters I shows that here the replacement of 4-ОН-group by one of its classic isosteres suggests itself as a most obvious variant of the methodology of bioisosteric replacements [1,2], for example by the methyl group. However, only experimental pharmacological testing will show whether transfer from 4-ОН-esters I to their 4-methyl-substituted analogs II ( Figure 1) will be really bioisosteric and how exactly this transformation will affect the analgesic properties of the compounds under study. The potentially bioisosteric replacement [1,2] from methyl 1-R-4-hydroxy-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylates I to their 4-methyl-substituted analogs II.
It may happen that the above transformation of the ОН-group into the methyl one can be performed via the intermediate formation of the corresponding 4-Cl-derivatives with their subsequent treatment with methylene active compounds followed by alkaline hydrolysis. At least, in the case of structurally similar alkyl 1-R-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylates this method has shown excellent results [20,21]. Nevertheless, a completely different principle of formation of molecular systems of type II is known (Scheme 1). It is acylation of ortho-aminoacetophenones III with ethyl (chlorosulfonyl)acetate, after that ortho-acetylsulfonamides IV formed are subjected to intramolecular cyclization in the target benzothiazines II under the influence of bases [22,23].  [1,2] from methyl 1-R-4-hydroxy-2,2-dioxo-1H-2λ 6 , 1-benzothiazine-3-carboxylates I to their 4-methyl-substituted analogs II.
Even a cursory analysis of the structure of esters I shows that here the replacement of 4-OH-group by one of its classic isosteres suggests itself as a most obvious variant of the methodology of bioisosteric replacements [1,2], for example by the methyl group. However, only experimental pharmacological testing will show whether transfer from 4-OH-esters I to their 4-methyl-substituted analogs II (Figure 1) will be really bioisosteric and how exactly this transformation will affect the analgesic properties of the compounds under study.
It may happen that the above transformation of the OH-group into the methyl one can be performed via the intermediate formation of the corresponding 4-Cl-derivatives with their subsequent treatment with methylene active compounds followed by alkaline hydrolysis. At least, in the case of structurally similar alkyl 1-R-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylates this method has shown excellent results [20,21]. Nevertheless, a completely different principle of formation of molecular systems of type II is known (Scheme 1). It is acylation of ortho-aminoacetophenones III with ethyl (chlorosulfonyl)acetate, after that ortho-acetylsulfonamides IV formed are subjected to intramolecular cyclization in the target benzothiazines II under the influence of bases [22,23]. Known method for the synthesis of ethyl 1-R-4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylates II [22,23].
Undoubtedly, the weakest link in this synthetic chain is the use of N-substituted ortho-aminoacetophenones III. Basically, this approach is entirely justified when obtaining esters II (R = H) that are unsubstituted in position 1, in their synthesis the use of commercially available acetophenones III with a free amino group is expected. However, introduction of N-alkyl substituents directly to the molecule of ortho-aminoacetophenone is associated with rather serious difficulties conditioned by both its extreme chemical instability and the secondary processes that are inevitable for such reactions (formation of N,N-dialkylation products, etc.). Hence, it is not surprising to make attempts to obtain 1-substituted esters II (R = alkyl) in some other manner that excludes the need for alkylation of ortho-aminoacetophenones. Although, in general, there are no complaints to the sequence of chemical transformations proposed in the method under consideration since the time-tested strategic rule of organic synthesis recommending all controversial and difficult stages to transfer to the beginning of the synthetic scheme has been fully complied [24]. In light of this, the possibility of obtaining 1-alkyl-substituted esters II by the fundamentally different way has been studied in the present work when the desired N-alkyl substituent is not introduced at first stage, but it is introduced at the final stage of the synthesis, i.e., by alkylation of the 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine nucleus previously formed.

Chemistry
1 Н-and 13 С-NMR spectra were acquired on a Varian Mercury-400 (Varian Inc., Palo Alto, CA, USA) instrument (400 and 100 MHz, respectively) in dimethyl sulfoxide-d6 (DMSO-d6), chloroform-d (CDCl3) or their mixtures in the ratio of 1:1 with tetramethylsilane as internal standard. The chemical shift values were recorded on a δ scale and the coupling constants (J) in hertz. The following abbreviations were used in reporting spectra: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. The electron impact mass spectra were recorded on a Varian 1200 L (Varian Inc.) mass spectrometer with complete scanning in the m/z range from 35 to 700 and direct sample inlet. The electron impact ionization was at 70 eV. Elemental analysis was performed on a Euro Vector EA-3000 (Eurovector SPA, Redavalle, Italy) microanalyzer. Melting points were determined in a capillary using a Electrothermal IA9100X1 (Bibby Scientific Limited, Stone, UK) digital melting point apparatus. Monitoring of alkylation and purity of the compounds obtained was done by thin-layer chromatography (TLC) on Silica gel 60 F254 aluminium sheets (Merck KGaA, Darmstadt, Germany); the eluent was CH2Cl2-MeOH-hexane, 5:1:5; visualization of the chromatograms was with iodine vapor. In the synthesis of methyl 1-R-4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylates 3 and 5 described in this article the commercial ortho-aminoacetophenone and the corresponding alkyl halides of Sigma Aldrich company (St. Louis, MO, USA) were used.
Undoubtedly, the weakest link in this synthetic chain is the use of N-substituted ortho-aminoacetophenones III. Basically, this approach is entirely justified when obtaining esters II (R = H) that are unsubstituted in position 1, in their synthesis the use of commercially available acetophenones III with a free amino group is expected. However, introduction of N-alkyl substituents directly to the molecule of ortho-aminoacetophenone is associated with rather serious difficulties conditioned by both its extreme chemical instability and the secondary processes that are inevitable for such reactions (formation of N,N-dialkylation products, etc.). Hence, it is not surprising to make attempts to obtain 1-substituted esters II (R = alkyl) in some other manner that excludes the need for alkylation of ortho-aminoacetophenones. Although, in general, there are no complaints to the sequence of chemical transformations proposed in the method under consideration since the time-tested strategic rule of organic synthesis recommending all controversial and difficult stages to transfer to the beginning of the synthetic scheme has been fully complied [24]. In light of this, the possibility of obtaining 1-alkyl-substituted esters II by the fundamentally different way has been studied in the present work when the desired N-alkyl substituent is not introduced at first stage, but it is introduced at the final stage of the synthesis, i.e., by alkylation of the 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine nucleus previously formed.

Chemistry
1 H-and 13 C-NMR spectra were acquired on a Varian Mercury-400 (Varian Inc., Palo Alto, CA, USA) instrument (400 and 100 MHz, respectively) in dimethyl sulfoxide-d 6 (DMSO-d 6 ), chloroform-d (CDCl 3 ) or their mixtures in the ratio of 1:1 with tetramethylsilane as internal standard. The chemical shift values were recorded on a δ scale and the coupling constants (J) in hertz. The following abbreviations were used in reporting spectra: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. The electron impact mass spectra were recorded on a Varian 1200 L (Varian Inc.) mass spectrometer with complete scanning in the m/z range from 35 to 700 and direct sample inlet. The electron impact ionization was at 70 eV. Elemental analysis was performed on a Euro Vector EA-3000 (Eurovector SPA, Redavalle, Italy) microanalyzer. Melting points were determined in a capillary using a Electrothermal IA9100X1 (Bibby Scientific Limited, Stone, UK) digital melting point apparatus. Monitoring of alkylation and purity of the compounds obtained was done by thin-layer chromatography (TLC) on Silica gel 60 F 254 aluminium sheets (Merck KGaA, Darmstadt, Germany); the eluent was CH 2 Cl 2 -MeOH-hexane, 5:1:5; visualization of the chromatograms was with iodine vapor. In the synthesis of methyl 1-R-4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylates 3 and 5 described in this article the commercial ortho-aminoacetophenone and the corresponding alkyl halides of Sigma Aldrich company (St. Louis, MO, USA) were used.  (20 mL) and cool (−5 to 0 • C). After 10 h add water (50 mL) to the reaction mixture, then acidify it to pH 4 with 1 N HCl and mix thoroughly. Separate the organic layer, dry over anhydrous CaCl 2 , and distil the solvent (at reduced pressure at the end). Subject the resulting anilide 2 to heterocyclization without purification. Add the solution of sodium methylate in anhydrous methanol (from metallic sodium (0.69 g, 0.030 mol) and absolute methanol (15 mL)), boil the mixture and store for 15 h at room temperature. Dilute the reaction mixture with cold water and acidify with 1 N HCl to pH 4.

General Procedure for the Synthesis of 1-Alkyl-Substituted
To the solution of 2.53 g (0.01 mol) of methyl 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3carboxylate (3) in 15 mL of DMSO add 2.07 g (0.015 mol) of K 2 CO 3 and mix for 30 min. Then add 0.015 mol of the corresponding alkyl halide, and mix within the time specified in Table 1 at 25 • C. Dilute the mixture with cold water and acidify with diluted HCl to pH 4. Extract CH 2 Cl 2 (3 × 10 mL). Combine the extracts, remove the solvent by distillation (at reduced pressure at the end). Dissolve the residue in 15-20 mL of a hot methanol, purify with activated charcoal and filter. Purify the resulting solution with coal, then place in the freezer (−20 • C) for 24 h. Filter the crystals of methyl 1-alkyl-substituted 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylate (5a-g) obtained and dry in the air, and recrystallized from the methanol (esters 5a-f) or acetone (ester 5g). Esters 5a-g were colorless or white with yellowish crystals.   graphite monochromator, and ω-scanning to 2θ max 60 • . The structure was solved by the direct method using the SHELXTL program package (Institute of Inorganic Chemistry, Göttingen, Germany) [25]. The restrictions on the bond lengths were applied for the disordered isopropyl (1.54 Å). The positions of the hydrogen atoms were found from the electron density difference map and refined using the "rider" model with U iso = nU eq for the non-hydrogen atom bonded to a given hydrogen atom (n = 1.5 for methyl, and n = 1.2 for the other hydrogen atoms). The structure was refined using F 2 full-matrix least-squares analysis in the anisotropic approximation for non-hydrogen atoms to wR 2 0.229 for 4,218 reflections (R 1 0.072 for 2386 reflections with F > 4σ (F), S 1.035). CCDC 1518396 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Center [26].

Analgesic Test
The analgesic activity of the synthesized methyl 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3carboxylates (3) and its N-alkyl-substituted analogues 5a-g were studied compared to piroxicam (Jenapharm, Jena, Germany) and meloxicam (Boehringer Ingelheim, Ingelheim am Rhein, Germany) being similar to the structure on the model of the thermal tail-flick procedure in white male rats weighing 180-200 g (Tail Immersion Test) [28]. For this purpose, the rat's tail tip was immersed in a water bath heated to 54 • C, and the latent period of the tail withdrawal (immersion) expressed in seconds was determined. The analgesic effect (in %) was assessed by the change of the latent period 2 h after introduction of the test substances and the reference drugs. Seven experimental animals were involved to obtain statistically reliable results (the significance level of the confidence interval accepted in this work was p ≤ 0.05) in testing each of esters 3 and 5a-g, the reference drugs and control. All substances under research, piroxicam, and meloxicam were introduced orally in the form of fine aqueous suspensions stabilized with Tween-80 in a screening dose of 20 mg/kg. The animals of the control group received an equivalent amount of water with Tween-80.
All biological experiments were carried out in full accord with the European Convention on the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and the Ukrainian Law No. 3447-IV "On protection of animals from severe treatment" (2006) (project ID 3410U14 approved October 15, 2015).

Chemistry
The initial methyl 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylate (3) was obtained by the traditional linear scheme with a good yield and purity. This synthesis does not require special explanation. It should be only noted that treatment of type 2 anilides with alkali metal alcoholates is often accompanied by complete or partial transesterification [10]. Therefore, as a basic catalyst of heterocyclization it is necessary to use the alcoholate prepared from the same alcohol, which fragment is already contained in alkyl (chlorosulfonyl)acetate: it is clear that in our case it is sodium methylate (Scheme 2). When meeting this simple condition the target methyl ester 3 is formed, but not its mixtures that are difficult to separate with other 3-alkoxycarbonyl analogues in various ratios.
In the interaction of alkyl halides and salts that are structurally close to methyl 4-hydroxy-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylates, as it is known, the isomeric products of 3-C-alkylation are formed rather easily and with high yields in addition to 4-O-alkyl-substituted derivatives being typical for such reactions [29]. The similar electrophilic attack of position 3 in the anion of the potassium salt of methyl 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylate (4) is unlikely. However, at least theoretically, a possibility of occurrence of other side chemical processes in the course of the reaction studied is not excluded. Consequently, one of the key aspects of our study is confirmation of the purity of the resulting compounds and unambiguous identification of their structure.
A series of experiments conducted with TLC-monitoring has shown that the reaction of the potassium salt anion of methyl 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylate (4) and alkyl halides proceeds rather quickly already at room temperature in the DMSO/K 2 CO 3 system. For example, in the experiment with a highly reactive allyl bromide the initial ester 3 is not chromatographically detectable in the reaction mixture already in 0.5 h after the start of alkylation. With the less reactive alkyl halides the reaction duration naturally increases, but it must not significantly exceed the time limits experimentally found and recommended by us ( Table 1). The cause for this restriction is simple-as it turned out, the initially formed alkyl-substituted esters 5a-g do not differ in chemical stability, and according to TLC-monitoring they gradually undergo further deeper structural changes under the synthetic conditions, therefore, these esters require a separate and more detailed study.
The initial methyl 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylate (3) was obtained by the traditional linear scheme with a good yield and purity. This synthesis does not require special explanation. It should be only noted that treatment of type 2 anilides with alkali metal alcoholates is often accompanied by complete or partial transesterification [10]. Therefore, as a basic catalyst of heterocyclization it is necessary to use the alcoholate prepared from the same alcohol, which fragment is already contained in alkyl (chlorosulfonyl)acetate: it is clear that in our case it is sodium methylate (Scheme 2). When meeting this simple condition the target methyl ester 3 is formed, but not its mixtures that are difficult to separate with other 3-alkoxycarbonyl analogues in various ratios. In the interaction of alkyl halides and salts that are structurally close to methyl 4-hydroxy-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylates, as it is known, the isomeric products of 3-C-alkylation are formed rather easily and with high yields in addition to 4-О-alkyl-substituted derivatives being typical for such reactions [29]. The similar electrophilic attack of position 3 in the anion of the potassium salt of methyl 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylate (4) is unlikely. However, at least theoretically, a possibility of occurrence of other side chemical processes in the course of the reaction studied is not excluded. Consequently, one of the key aspects of our study is confirmation of the purity of the resulting compounds and unambiguous identification of their structure.
A series of experiments conducted with TLC-monitoring has shown that the reaction of the potassium salt anion of methyl 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylate (4) and alkyl halides proceeds rather quickly already at room temperature in the DMSO/K2CO3 system. For example, in the experiment with a highly reactive allyl bromide the initial ester 3 is not chromatographically detectable in the reaction mixture already in 0.5 h after the start of alkylation. With the less reactive alkyl halides the reaction duration naturally increases, but it must not significantly exceed the time limits experimentally found and recommended by us ( Table 1). The cause for this restriction is simple-as it turned out, the initially formed alkyl-substituted esters 5a-g do not differ in chemical stability, and according to TLC-monitoring they gradually undergo 1 H-and 13 C-NMR spectra of all esters of benzothiazine-3-carboxylic acids 3 and 5 synthesized fully comply with the structure attributed to them (see Materials and Methods). Essentially, a simple comparison of the spectral data obtained allows to solve the main structural analytical task of this study-to determine the true direction of alkylation-even without the use of special methods of NMR spectroscopy. In particular, the chemical shifts of alkyl carbon atoms in 13 C-NMR spectra suggest that, in general, the substituent is either at the atom of nitrogen or oxygen (the last variant is possible as a result of the exchange of alkyl groups in the ester fragment). A comparative analysis of the "aromatic" region of the 1 H-NMR spectra of the starting ester 3 and its alkyl-substituted derivatives 5a-g allows excluding the oxygen atom of the potential reaction centers. The basis for this conclusion was the fact that after introduction of the alkyl substituent the spectral pattern changes significantly ( Figure 2). The resonance signals of all aromatic protons undergo a paramagnetic shift, but in this respect, the signal of H-8 proton is particularly illustrative, it shifts downfield by 0.3 ppm in the spectrum of methyl-substituted ester 5a compared to unalkylated analog 3. With the increase in size of the alkyl substituent the shift of the specified signal also increases-it reaches 0.4 ppm in ethyl derivative 5b. Subsequently the signal of H-8 proton remains approximately in the same position irrespective of the spatial structure of the alkyl substituent. Even the isopropyl fragment of ester 5e does not change the situation. However, this substituent being the most volumetric sterically brings its own corrections, it noticeably shifts downfield the signals not only the neighboring H-8, but also protons, which are more remote in space, in particular H-6 proton ( Figure 2). In our opinion, the strong interaction observed in the experimental 1 H-NMR spectra between H-8 proton and alkyl substituents introduced is a typical manifestation of the van der Waals effect [30]. Consequently, these two fragments are close together in space.  Mass spectrometry also gives important and useful information about the structure of the compounds synthesized. For example, in the case of the lower homologs-esters 3 and 5a,b-the molecular mass of each test sample can be determined. Other esters do not possess stability in the conditions of electron impact ionization, which is sufficient for formation of molecular cation-radicals, but there are peaks that are close to the mass of fragmented ions [M − H] + in their mass spectra. The common feature, which characterizes the mass spectrometric behavior of all the compounds studied, is release of the methanol molecule from their molecular ions. This is evidenced by the appearance of peaks of the corresponding fragmentation ions [M − CH 3 OH] + or [M − H − CH 3 OH] + in the mass spectra.

Evaluation of the Analgesic Activity
The pharmacological tests conducted have fully confirmed the expediency of our studies undertaken and shown that the transfer from methyl 1-R-4-hydroxy-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylates I to their 4-methyl-substituted analogs II presented in Figure 1 is really bioisosteric. The proof of this is the fact that all methyl 1-R-4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylates 3 and 5a-g synthesized showed pronounced analgesic properties (Table 2). Moreover, by the ability to inhibit the thermal pain response of the animals tested most of them are not inferior and even significantly superior to piroxicam, which was the first in the group of oxicams-new non-steroidal anti-inflammatory drugs with a good analgesic effect, and to date has become a classic standard for biological research.

Conclusions
The present study describes methyl 1-R-4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylates synthesized as potential bioisosteres of analgesics from a series of 4-hydroxy-2,1-benzothiazines. The complex use of X-ray diffraction analysis, NMR ( 1 H and 13 C) spectroscopy and mass spectrometry has allowed to prove convincingly that methyl 4-methyl-2,2-dioxo-1H-2λ 6 ,1-benzothiazine-3-carboxylate is alkylated by alkyl halides on the nitrogen atom in the DMSO/K 2 CO 3 system. Simultaneously on the example of N-benzyl derivative the ability of 4-methyl-2,2-dioxo-2,1-benzothiazines to form solvates with methanol has been found, and it is useful to remember when working with substances of this chemical class. The pharmacological screening has revealed highly active analgesics being of interest for more detailed study among the compounds synthesized.