Continuous-flow synthesis of amine-sulfonic acid buffers

Abstract

The instant invention discloses a continuous flow synthesis of amine-sulfonic acid buffers, specifically biological buffers such as MOPS, MOBS, CAPS, CABS by the reaction of amines such as morpholine, N-cyclohexylamine with corresponding sulfo-alkylating agents or alkane- sultones, in a solvent-free method wherein, an agitated thin film dryer (AFTD) is used to remove the excess of the amine-reagents such as morpholine, cyclohexylamine, so as to obtain the final products namely the buffers in a dried condition. The invention also discloses an economically viable, environmentally friendly process for obtaining biological buffers such as MOPS, MOBS, CAPS, CABS wherein the unreacted reagent / solvent is recycled to the continuous- flow-reactor.

Description

CONTINUOUS-FLOW SYNTHESIS OF AMINE-SULFONIC ACID BUFFERS RELATED DOCUMETS This invention claims priority to the provisional application filed at the Indian Patent Office on 01-03-2024 with the same title” CONTINUOUS-FLOW SYNTHESIS OF AMINE- SULFONIC ACID BUFFERS” bearing the application no202441015423 , TEMP / E- 1 / 18320 / 2024- CHE and it is hereby incorporated in its entirety for reference sake. FIELD OF THE INVENTION

[0001] The instant invention relates to the preparation of amine-sulfonic acid buffers and more particularly to the continuous-flow synthesis of biological buffers namely morpholine buffers such as MOPS and MOBS and cyclohexylamine-buffers such as CAPS and CABS that can be employed to maintain the pH in biological reactions. BACKGROUND OF THE INVENTION

[0002] Zwitterionic addition compounds based on morpholine and sultone’ such as MOPS (3- morpholinopropane-1-sulfonic acid), MOBS (3-morpholinobutane-1-sulfonic acid), etc and those based on cyclohexylamine and sultone’ namely CAPS (N-cyclohexyl-3- aminopropanesulfonic acid), CABS (N-cyclohexyl-3-aminobutanesulfonic acid), are widely used as buffering agents in biological and biochemistry experiments. Good and coworkers came out with MOPS in mid-sixties as a buffering agent for biology and biochemistry experiments. It is an excellent buffer (pH range is 6.5-7.9) having pH nearing to neutral, which is the pH range of many biological systems. In addition to this, MOPS has exceptional chemical and enzymatic stability, very high-water solubility, minimal solubility in other solvents, minimal or no absorption in UV-Visible region and storage stability of solid.

[0003] The range of pH offered by these buffers and also their inertness in biological systems make MOPS, MOBS, CAPS and CABS a class of versatile buffering agents that can be employed in many of the pharmaceutical compositions. Currently there is a huge demand for the aforementioned buffers as they find extensive applications in the preparation of oncologicals, targeted delivery therapeutics, maintenance of pH for preservation and avoidance of damage to the nuclear materials such as DNA, RNA, nucleotides etc. Not only this they can also prevent the formation of reactive oxygen species (ROS) through sacrificial electron donation in case of photo-biocatalytic processes.

[0004] These buffers have a defined-narrow pH range. The chemical and enzymatic stability, very high-water solubility, minimal solubility in other solvents, minimal or no absorption in UV-Visible region and storage stability of solid of these buffers, make them the most preferred candidates to act as buffering agents with a huge demand.

[0005] Similarly the 3-cyclohexylamine-1-propanesulfonic acid is also a biological buffer with huge demand in biochemical diagnostic kits, DNA / RNA extraction kits and PCR diagnostic kits. It is used as a buffer solution for separating alkaline drugs by enzyme chemistry and HPLC, and is also used for purifying fibronectin after the pH is adjusted to 11.0 on dissolution in deionized wate.

[0006] Prior art search has revealed many reports on the synthesis of this class of buffers. These buffers are generally synthesized by the reaction between suitable amine and sultone dissolved in prescribed organic solvents. Patent CN109836397 disclosed the reaction between these two reagents in ethanol solvent at low temperature for longer reaction times (more than 10 hours).

[0007] CN110981831A disclosed the synthesis of MOPS, by the reaction between morpholine and 1,3- propane sultone dissolved in suitable solvents in a continuous flow microreactor.

[0008] Patent CN109836397A disclosed biological buffer-morpholinopropane sulfonic acid (MOPS) preparation method, wherein, morpholine and 1,3- propane sultone (cheaper relative to using 1,3- dibromopropane) reaction generates morpholinopropane sulfonic acid. Preparation process of the invention is supposed to be simple, easy to operate, practicability with higher yields.

[0009] Seldom et al reported the earliest synthetic method about MOPS [The synthetic method of MOPS ( Anal. Chen.37 (1) .156-81965 (Eng]).

[0010] WO2001085678 revealed the method for synthesizing MOPS with morpholine and 1,3- dibromopropane, ( supposed to be an expensive method since bromide is too expensive).

[0011] Patent CN110981831A discloses a preparation method of 3-morpholine propane sulfonic acid, which comprises the steps of dissolving 1, 3-propane sultone by using an organic solvent A to obtain an organic solution containing 1, 3-propane sultone, and dissolving morpholine by using an organic solvent B to obtain an organic solution containing morpholine; the mol ratio of the 1, 3-propane sultone to the morpholine is 1: 1; respectively and simultaneously pumping an organic solution containing 1, 3-propane sultone and an organic solution containing morpholine into a microchannel reactor for reaction to obtain reaction liquid, performing freeze crystallization on the obtained reaction liquid to separate out a crude product of the 3-morpholine propane sulfonic acid, and then introducing nitrogen to perform filter pressing to obtain a finished product of the 3-morpholine propane sulfonic acid.

[0012] Patent CN106117163A discloses a kind of 3 (N morpholine) 2 hydroxy-propane sulfonic acid synthesis technique, disclose the preparation method of a kind of N substituted morpholines organic compounds, comprised the following steps: wherein, cyclodehydration obtains morpholine, again through neutralizing, filtration, rectification, synthesis obtains 3 (N morpholine) 2 hydroxy-propane sulfonic acids, eventually through chromatography and purification obtained final products.

[0013] Patent CN110885299A disclosed a preparation method of 3-cyclohexylamine-1- propanesulfonic acid, which comprises the following steps: dissolving 1, 3-propane sultone by using an organic solvent A to obtain an organic solution containing 1, 3-propane sultone, and dissolving cyclohexylamine by using an organic solvent B to obtain an organic solution containing cyclohexylamine; respectively and simultaneously pumping the organic solution containing 1, 3-propane sultone and the organic solution containing cyclohexylamine into a microchannel reactor for reaction to obtain reaction liquid; performing freeze crystallization on the obtained reaction liquid to separate out a crude product of the 3-cyclohexylamine-1- propanesulfonic acid, and then introducing nitrogen to perform filter pressing to obtain a finished product of the 3-cyclohexylamine-1-propanesulfonic acid. The method adopts amicrochannel reactor by taking 1, 3-propane sultone and cyclohexylamine as raw materials, and sets reaction conditions, so that the yield of the product can be greatly improved. The invention further unexpectedly finds that the organic solvent a is ethyl acetate, the organic solvent B is dichloroethane, and when the mass ratio of ethyl acetate to dichloroethane is 5: the yield can reach 99%.

[0014] AU2005326962 which has dealt with the - METHODS AND COMPOSITIONS FOR TREATING AMYLOID-RELATED DISEASES disclosed Preparation of 4-(cyclohexylamino)-2- butanesulfonic acid wherein 2,4-butane sultone (2.04 g, 14.4 mmol) was added to a solution of cyclohexylamine (1.50 g, 15.1 mmol) in tetrahydrofuran (15 mL) The solution stirred at reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration and dried in vacuo. Yield: was 59%. Similarly, Preparation of 4-(cyclohexylamino)-1- butanesulfonic acid consisted of addition of 1,4-butane sultone (2.61 g, 19.2 mmol) to a solution of cyclohexylamine (2.0 g, 20.2 mmol) in 1,4-dioxane (13 mL). The solution was heated to reflux for 2 hours. The reaction was cooled to room temperature. The solid was collected by filtration, washed with acetone (2 x 20 mL) and dried in vacuo (Yield: 52%).

[0015] LETICIA C. P. GONÇALVES et al, reported Morpholine-based buffers activate aerobic photobiocatalysis via spin correlated ion pair formation[(Catal Sci Technol.2019 Mar 21; 9(6): 1365–1371) Published online 2019 Feb 11. doi: 10.1039 / c8cy02524j; PMCID: PMC6468414; PMID: 31131076] . They discussed the use of enzymes for synthetic applications as a powerful and environmentally-benign approach to increase molecular complexity. Oxidoreductases selectively introduce oxygen and hydrogen atoms into myriad substrates, catalysing the synthesis of chemical and pharmaceutical building blocks for chemical production. However, broader application of this class of enzymes is limited by the requirements of expensive cofactors and low operational stability. They showed that morpholine-based buffers, especially 3-(N-morpholino)propanesulfonic acid (MOPS), promote photoinduced flavoenzyme-catalyzed asymmetric redox transformations by regenerating the flavin cofactor via sacrificial electron donation and by increasing the operational stability of flavin- dependent oxidoreductases. The stabilization of the active forms of flavin by MOPS via formation of the spin correlated ion pair3[flavin˙––MOPS˙+] ensemble reduced theformation of hydrogen peroxide, circumventing the oxygen dilemma under aerobic conditions detrimental to fragile enzymes.

[0016] Patent CN110790687A discloses a method for producing 3- (cyclohexylamine) -1- propanesulfonic acid, which comprises the following steps: dissolving 100g of propane sultone in 1L of N, N-dimethylformamide according to the weight ratio; adding 81.2g of cyclohexylamine at a preset speed, and controlling the reaction temperature at 30 ℃ through the preset speed of feeding; after the addition, the reaction is continued for N hours under heat preservation, and then the crude product of the 3- (cyclohexylamine) -1-propanesulfonic acid is obtained after filtration and drying; and dissolving the crude 3- (cyclohexylamine) -1- propanesulfonic acid product in 100g of hot water, adding 500g of ethanol, cooling to 0 ℃, filtering, and drying to obtain 156 g of a finished 3- (cyclohexylamine) -1-propanesulfonic acid product. Compared with the prior art, the production method of the 3- (cyclohexylamine) -1- propanesulfonic acid has high product yield and good quality.

[0017] Debra M. Hausladen et al Organic buffers act as reductants of abiotic and biogenic manganese oxides (Scientific reports volume 13, Article number : 6498 (2023) showed that organic buffers, including morpholinic and piperazinic Good’s buffers and TRIS, should be avoided for pH control in Mn oxide systems due to their ability to transfer electrons to Mn, which modifies the composition and reactivity of these redox-active minerals. To control pH, laboratory studies involving redox-sensitive minerals like manganese (Mn) oxides frequently use organic buffers (typically Good’s buffers); however, two Good’s buffers, HEPES and MES, have been shown to reduce Mn(IV) to Mn(III).

[0018] Patent CN115466201A discloses sulfonic acid modified polyisocyanate and a preparation method thereof, wherein the reaction rate of sulfamic acid and polyisocyanate is accelerated by controlling the content of cyclohexylamine in raw materials, so that the prepared product has the advantages of light colour, low turbidity and good storage stability.

[0019] Though the prior art search revealed processes of obtaining biological buffers such as MOPS, MOBS, CAPS and CABS etc, there is an urgent necessity to come out with a solvent free, continuous-flow process for obtaining these biological buffers such as MOPS, MOBS, CAPS and CABS etc, with high purity, high yield percentage at an affordable price as there is a huge demand for such pure buffers. Solvent-free processes are always desirable since they arefree from down-stream processing and also are environmentally friendly. Keeping these points under consideration, the instant invention of “CONTINUOUS-FLOW SYNTHESIS OF AMINE - SULFONIC ACID BUFFERS” is taken up.

[0020] The exemplary aspects of various embodiments of the invention are disclosed in the summary of the invention and all the essential aspects related to various embodiments of the invention are described in a detailed manner in the following paragraphs with specific references towards the corresponding figures as given hereunder. All the prior art references are incorporated hereby in their entirety for reference-sake and in no way taking away the novelty of the instant invention. The various aspects of the instant invention disclosed here are definitely an improvement over the existing prior art and further stress upon the inventorship, novelty and applicability of the instant invention.

[0021] OBJECTIVES OF THE INVENTION 1. To come out with a continuous-flow process for preparing amine-sulfonic acid buffers based on morpholine and cyclohexylamine, such as MOPS, MOBS, CAPS, CABS etc. 2. To come out with a solventless process for obtaining amine-sulfonic acid buffers based on the reaction between amines such as morpholine, cyclohexylamine and corresponding sulfo-alkylating agents or alkane-sultones. 3. To come out with a continuous-flow process of obtaining amine-sulfonic acid buffers based on morpholine and cyclohexylamine wherein the excess of the amine reagents namely morpholine and cyclohexylamine are removed using an agitated thin film dryer (ATFD), so that the respective buffers can be obtained as dry crude powders, and the removed amine - reagents can be recycled to flow reactor thereby ensuring high purity and yields of the crude buffers thus obtained. SUMMARY OF THE INVENTION

[0022] The exemplary aspect of the instant invention discloses a continuous flow synthesis of biological buffers such as MOPS, MOBS, CAPS, CABS by the reaction of amines such as morpholine, cyclohexylamine with corresponding sulfo-alkylating agents or alkane-sultones in a solvent-free method wherein, wherein an agitated thin film dryer (AFTD) is used to removethe excess of the amine reagents of morpholine, cyclohexylamine, so as to obtain the final products namely the buffers in a dried condition.

[0023] One important embodiment of the invention discloses a method of obtaining amine- sulfonic acid based morpholine and cyclohexylamine buffers without encountering precipitation / high viscous mass in flow reactors.

[0024] Another exemplary embodiment of the invention discloses synthesis of the buffers based on morpholine and cyclohexylamine or any other primary or secondary amine wherein the alkane-sultone and corresponding amine pairs are miscible with each other and also the buffers are miscible in the corresponding amines.

[0025] One more embodiment of the invention discloses the inline isolation of crude buffer product and recycling of the recovered amines.

[0026] Yet another embodiment of the invention discloses an improved purification method for the crude buffer product in batch mode.

[0027] The various embodiments of the instant invention are described in detail in the following paragraphs and the best methods of practicing the same are described in detail in the following paragraphs.. DETAILED DESCRIPTION OF THE INVENTION

[0028] The examples of the reactants, constituents, reagents, apparatus discussed herein are not limited in application to the details of the reactions, products thus obtained, construction and the arrangement of components set forth in the following description or illustrated in the accompanying drawings. It will be understood by one of skill in the art that the invention is capable of implementation in other embodiments and of being practiced or carried out in various ways. Examples of specific embodiments are provided herein for illustrative purposes only and are not intended to be limiting. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0029] Any references to examples, embodiments, components, elements or acts of the apparatus herein referred to in the singular may also embrace embodiments including aplurality, and any references in plural to any embodiment, component, element, or act herein may also embrace embodiments including only a singularity (or unitary structure). References in the singular or plural form are not intended to limit the presently disclosed apparatus, its components, acts, or elements.

[0030] The use herein of “including,” “comprising,” “having,” “containing,” “involving,” and variations thereof is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. References to “or” may be construed as inclusive so that any terms described using “or” may indicate any of a single, more than one, and all of the described terms. The various embodiments of invention are described in detail herein and the various aspects of the invention are disclosed below.

[0031] DEFINITIONS MOPS is (3-morpholinopropane-1-sulfonic acid). MOBS is (3-morpholinobutane-1-sulfonic acid). CAPS is (N-cyclohexyl-3-aminopropanesulfonic acid). CABS is (N-cyclohexyl-3-aminobutanesulfonic acid). ATFD is agitated thin film dryer. TLC is thin layer chromatography. LCMS is Liquid Chromatography Mass Spectroscopy Sultone is a cyclic sulfonate ester which acts as a sulfo-alkylating agent. 1,4-butane sultone is a six-membered δ-sultone and the cyclic ester of 4- hydroxybutanesulfonic acid that is used as a sulfo-alkylating agent. 1,3-Propane sultone is the organosulfur compound with the formula (CH2)3SO3. It is a cyclic sulfonate ester belonging to a class of compounds called sultones.GENERAL PROCEDURE FOR PREPARING AMINE-SULFONIC ACID BUFFERS OF MORPHOLINE AND CYCLOHEXYLAMINE

[0032] The general preparation method for producing morpholine-sulfonic acid and cyclohexylamine-sulfonic acid buffers comprised of the following steps:

[0033] Step-1: Neat sultone and amines were made to react in a continuous flow reactor by maintaining their mole ratio in such a way that, the reaction mixture remains homogeneous. The temperature of the reactor was maintained at 60̊C, owing to the superior heat transfer abilities of continuous flow reactor despite the process being exothermic. The reaction time was found to be nearly 2-5 minutes for the complete conversion of the sultone, which is confirmed by thin layer chromatography (TLC).

[0034] Step-2: The excess morpholine or cyclohexylamine used in the reaction was evaporated by agitated thin film dryer (ATFD) and was reused for the next reaction. Solid crude buffer was collected from the ATFD in the powder-form.

[0035] Step-3: The crude buffer was purified by improved antisolvent recrystallization process utilizing methanol-water mixture. The colour of the product was removed by employing carbon-powder during recrystallization process so as to obtain the buffer as a pure white or nearly white final product. The obtained final product was pure enough to meet the set standard pharmaceutical specifications.

[0036] The following general procedures clearly describe the reaction procedures adopted to obtain individual morpholine-sulfonic acid and cyclohexylamine-sulfonic acid buffers. PROCESS FOR OBTAINING MOPS

[0037] For comparison sake MOPS synthesis was also carried out in Batch mode as per the procedure and schemes given hereunder so as to establish the necessity for taking up the reaction in a solvent-free continuous-flow mode to obtain MOPS in high yields with high purity. BATCH PROCESS FOR MOPS WITH ACETONE AS THE SOLVENTProcedure For Obtaining MOPS in Batch-Process

[0038] 393 g (0.0032 mol) of 1,3 – propane sultone was charged with 15 V of dry acetone. 284 g (0.0032 mol) morpholine dissolved in 5 V of dry acetone was added dropwise under N2 atmosphere to the sultone solution. The mixture was heated to 56 ⁰C and the reaction was continued till all the 1,3 – propane sultone is consumed (based on the TLC). Then the reaction mixture is cooled to room temperature, filtered, and the solid thus obtained was washed with 5 V dry acetone; dried in an oven at 65 ⁰C to yield crude solid. (644 g; 94.3 %). The solid was subjected to HPLC analysis for obtaining the purity of the product and the purity was found to be 99.7 %. BATCH PROCESS TO OBTAIN MOPS WITHOUT SOLVENTProcedure for obtaining MOPS without solvent in a batch process

[0039] Morpholine (20 ml, 4 volumes) was taken in a round bottomed flask, cooled to 0-10 ⁰C, 5 g of molten 1,3-propane sultone was added dropwise to this (during which, heat liberation was observed). The mixture was maintained at room temperature for 30 minutes and was subjected to TLC analysis which confirmed the absence of sultone. After the reaction, the morpholine was removed under reduced pressure and the crude obtained was recrystallized to obtain pure MOPS.

[0040] Since the reaction is exothermic, heat management at a large scale is difficult to achieve under batch conditions. This could be overcome by adopting the continuous-flow process for obtaining MOPS. At the same time the reaction time is considerably reduced in the continuous-flow process, compared to the batch process, since the batch process needed longer time periods for the slow-addition of the reagents in order to avoid excess heat- generation. CONTINUOUS FLOW PROCESS FOR THE SYNTHESIS OF CRUDE MOPSScheme 1: The schematic of continuous flow process for the production of MOPS buffer Procedure for obtaining MOPS in a solvent-free Continuous-flow process

[0041] MOPS is prepared in a solvent-free continuous-flow method as per the schematic (scheme 1) given above in a continuous-flow static-mixer reactor. 75 g (0.614 mol) 1,3 – propane sultone was pumped using pump 1 and morpholine (3.5 mol) was pumped using pump 2 into a static mixer reactor. The reactor was maintained at 60 ⁰C. A residence time of 2 -5 min was allowed to achieve desired conversion. The sample collected under steady state showed the absence of 1,3 propane sultone and formation of MOPS as determined by TLC and HPLC analyses.

[0042] The steady-state-exit-mass of the static mixer reactor was collected is a vessel and continuously fed to an ATFD for the removal of morpholine in telescopic way which then separated crude solid and excess morpholine. The recovered morpholine is recycled to morpholine feed tank for reuse in the reaction.

[0043] The crude MOPS (99.6% purity, off-white colour) as obtained from ATFD was recrystallized as per the recrystallization method given hereunder. RECRYSTALLIZATION OF MOPS

[0044] The crude solid (MOPS) as obtained from ATFD was dissolved in 0.7 volume of water and heated to 65 ⁰C. To the clear solution, 7 wt. % charcoal was added and stirred for 1 hr. Hot filtration of this solution was done to remove the charcoal, the hot filtrate was cooled slowly to 0-10 ⁰C. To this, 6 volumes of methanol was added in a dropwise manner which resulted in solidification of MOPS, slurry was stirred for 1 hr and filtered, washed with 2 V cold methanol; Dried in oven at 65 ⁰C, and the purity was checked by HPLC (99.9 %) [enhanced purity can be achieved by repeating this step to meet more stringent colour specifications, if any].

[0045] The instant invention produces MOBS with morpholine and 1,4-butanesultone, CAPS with N-cyclohexylamine and 1,3-propanesultone, and CABS with N-cyclohexylamine and 1,4- butanesultone. The schematic 1 given for MOPS production is the general schematic for producing all the above said buffers namely MOBS, CAPS and CABS wherein pump 1 pumps respective sulfo-alkylating agent or alkane-sultone and pump 2 pumps the corresponding amine at suitable experimental conditions to produce corresponding buffers.

[0046] Thus by varying the conditions governing the reactions, amounts of the reactants, employing different sulfo-alkylating agent or alkane-sultone, different amines such as substituted morpholines, substituted cyclohexylamines and other suitable amines, the instant invention can produce various biologically useful amine-sulfonic acid buffers in a continuous-flow manner.

[0047] Various other embodiments of the instant invention can be realized within the scope of the inventive aspect as disclosed by the invention as given hereunder: 1. Under continuous flow process the residence time of the reaction is found to be 2- 5 minutes and various other embodiments of the instant invention are possible by varying the residence time of the reaction anywhere between 30 seconds to 10 minutes. 2. Under continuous flow process, the mole equivalent of morpholine is found to be equal to 5.7 and hence other embodiments of the invention can be realized by varying the mole equivalents of the respective amine between 3 -8. 3. Under continuous flow process, the temperature of the process is maintained at 60 C̊ and the temperature can be varied between 30 C̊ and 80 C̊. 4. Similarly the ratio of methanol : water can be varied between 3-6 volumes of methanol (as antisolvent) to 0.5 – 1 volumes of water (to dissolve the crude buffer) during the crude buffer purification stage of in place of the ratio of 6:0.7 of methanol : water as given in the general procedure for obtaining the suitable buffer of high purity with respect to the combination of the chosen sultone and corresponding amine. 5. In a similar fashion various other devices that can bring about similar action as that exhibited by the ATFD that is used in the instant invention for drying of the amines namely morpholine / cyclohexylamine from the reaction mixture and recovery of the same. 6. Another important additional embodiment of the invention is the complete automation of the continuous-flow synthesis of the amine-sulfonic acid buffers from respective amines and corresponding alkane-sultones is by the continuous end to end telescoping of the process with automated process controls without humanintervention. The fully automatic synthesis of amine-sulfonic acid buffers involves the following steps: 1. The amine and alkane-sultone reagents can be fed to the continuous flow reactor and the exit of the reactor can be directly connected to ATFD for the telescoping amine-reagent evaporation and solid buffer generation. 2. The solid buffers thus formed in step 1 are continuously transported for dissolving in water (solvent) with the help of a conveyor belt (as and when they are formed). The formed aqueous buffer solution can be continuously passed through a carbon cartridge for the colour improvement. 3. The aqueous-buffer solution from the step 2 is directly fed to the connected continuous crystallizer where it meets with methanol (antisolvent), while the reactor is being maintained at low temperature. The formed slurry is continuously filtered with the help of rotary pressure filters, the solid pure amino-sulfonic acid buffers thus obtained can be continuously dried using a fluidized-bed-dryer (FBD) which results in the formation of dried pure white buffers such as MOPS, MOBS, etc. All the unit operations can be remotely controlled without human intervention thereby making the entire continuous- flow process of producing amino-sulfonic acid buffers completely automatic.

[0048] Those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for designing other products without departing from the spirit and scope of the invention. Therefore, the different aspects of the invention disclosed herein are not limited to the specific examples depicted herein. For example, the features of one example disclosed above can be used with the features of another example.

[0049] Furthermore, various modifications and rearrangements of the parts may be made without departing from the spirit and scope of the underlying inventive concept. For example, the reactants and conditions disclosed herein may be altered depending upon the application, as may be the material selection for the components. Thus, the details of these components as set forth in the above-described examples, should not limit the scope of the claims.

[0050] The exemplary embodiments of the instant invention can be realized with the help of the following examples of preparation of different cyclohexylamine, morpholine based buffers from different sulfo-alkylating agents / alkane-sultones and substituted morpholines and cyclohexylamines which can be clearly understood in the light of the detailed procedures as disclosed by the instant invention of “CONTINUOUS-FLOW SYNTHESIS OF AMINE-SULFONIC ACID BUFFERS”. . EXAMPLES EXAMPLE 1: CONTINUOUS FLOW PROCESS FOR THE SYNTHESIS OF MOPS

[0051] 1,3 – propane sultone 75 g (0.61 mol) was pumped using pump 1 at a flow rate of (3.88 ml / min) and morpholine (3.5 mol) was pumped using pump 2 at a flow rate of (21.6 ml / min) into a static mixer reactor. The reactor was maintained at 60 ⁰C. A residence time of 2 min was allowed to achieve desired conversion. The sample collected under steady state showed the absence of 1,3 propane sultone, and formation of MOPS as determined by TLC and HPLC analyses. The crude MOPS (99.6% purity, off-white colour) as obtained from ATFD was recrystallized as per the recrystallization procedures mentioned above. EXAMPLE 2: CONTINUOUS FLOW PROCESS FOR THE SYNTHESIS OF MOBSPROCEDURE FOR PRODUCING MOBS

[0052] 1,4 – butane sultone 33.13 g (0.24 mol) was pumped using pump 1 at a flow rate of (1.34 ml / min) and morpholine (120.9 g, 1.4 mole) was pumped using pump 2 at a flow rate of (14.31 ml / min) into a static mixer reactor. The morpholine was preheated to reactiontemperature before it meets with 1,4-butane sultone. The total volume of the reactor was near 17 ml which was maintained at 60 ⁰C. A residence time of 1 min was allowed to achieve desired conversion. The sample collected under steady state showed the absence of 1, 4 – butane sultone and formation of MOBS as determined by TLC, LCMS and HPLC (99.9 A%) analyses. EXAMPLE 3: CONTINUOUS FLOW PROCESS FOR THE SYNTHESIS OF CAPS

[0053] 1,3 – Propane Sultone 64.73 g (0.53 mol) was pumped using pump 1 at a flow rate of (0.71 ml / min) and cyclohexylamine (311.7 g, 3.1 mole) was pumped using pump 2 at a flow rate of (14.86 ml / min) into a static mixer reactor. The cyclohexylamine was preheated to reaction temperature before it meets with 1,3 – Propane Sultone. The total volume of the reactor was near 17 ml which was maintained at 60 ⁰C. A residence time of 1.1 min was allowed inside the reactor to achieve desired conversion. The sample collected under steady state showed the absence of 1,3 – Propane Sultone and formation of CAPS as determined by TLC, LCMS and HPLC (99.9 A%) analyses. EXAMPLE 4: CONTINUOUS FLOW SYNTHESIS OF CABSPROCEDURE FOR PRODUCING CABS

[0054] 1,4 – butane sultone 35.43 g (0.26 mol) was pumped using pump 1 at a flow rate of (1.12 ml / min) and cyclohexylamine (153.2 g, 1.55 mole) was pumped using pump 2 at a flow rate of (15.96 ml / min) into a static mixer reactor. The cyclohexylamine was preheated to reaction temperature before it meets with 1,4-butane sultone. The total volume of the reactor was near 17 ml which was maintained at 60 ⁰C. A residence time of 1 min was allowed to achieve desired conversion. The sample collected under steady state showed the absence of 1, 4 – butane sultone and formation of CABS as determined by TLC and LCMS analyses. ADVANTAGES OF THE INVENTION

[0055] The instant invention of “CONTINUOUS-FLOW SYNTHESIS OF AMINE-SULFONIC ACID BUFFERS” has the following advantages: 1. The instant invention discloses a continuous-flow process for producing amine-sulfonic acid buffers based on the reaction between alkane-sultones and corresponding amines such as morpholine- cyclohexylamine that find extensive application as biological buffers with minimal carbon footprint. 2. The continuous process as disclosed by the instant invention is coupled with ATFD (dryer) to continuously separate product buffer and unreacted amine reagent, and recycles the unreacted amine-reagent back to the continuous flow reactor, thus making the process more economically viable compared to the existing prior art processes. 3. The continuous flow process is solvent-free. ANALYSIS OF NOVELTY, INVENTIVENESS / NON-OBVIOUSNESS AND UTILITY / INDUSTRIAL APPLICABILITY ASPECTS OF THE INVENTION

[0056] The instant invention of “Continuous-flow synthesis of amine-sulfonic acid buffers” is novel in the light of the prior art cited, with considerable inventiveness and tremendous utility since it provides a continuous-flow method of producing biological buffers such asMOPS, MOBS, CAPS, CABS, that are in huge demand in biological applications and does not appear obvious to a person skilled in the art. NOVELTY OF THE INVENTION

[0057] The instant invention of “Continuous-flow synthesis of amine-sulfonic acid buffers” is novel in the light of the prior art cited and it cannot be anticipated from the existing prior art since it offers a continuous-flow process of producing amine-sulfonic acid buffers from amines and sulfo-alkylating agents / alkane-sultones in a solvent-free mode wherein an agitated thin film dryer is employed to completely remove the amine reagent from the crude buffer that is produced and recycles the removed amine reagent to the reaction medium for further use. INVENTIVENESS / NON-OBVIOUSNESS OF THE INVENTION

[0058] The instant invention of “Continuous-flow synthesis of amine-sulfonic acid buffers” is having inventiveness and does not appear obvious to a person skilled in art for the following reasons: 1. The invention produces biological buffers such as MOPS, MOBS, CAPS and CABS that find extensive application to maintain pH in biological reactions, as stabilizers, as preservatives and also form a part of many biological formulations, pharmaceutical compositions especially oncological formulations in a solvent-free continuous-flow mode in an economically viable and environmentally friendly manner. 2. The invention produces important biological buffers such as MOPS etc with high purity in higher amounts since purity of such buffers plays a vital role in biological applications. 3. Since the instant invention offers a solvent-free mode of producing important amine- sulfonic acid biological buffers, it is devoid of the risks associated with down-stream purification and precipitation to obtain the said biological buffers in highly pure state. UTILITY / INDUSTRIAL APPLICABILITY OF THE INVENTION

[0059] The instant invention of “Continuous-flow synthesis of amine-sulfonic acid buffers” has tremendous industrial applicability and utility value since it produces important biological buffers comprising of amine-sulfonic acid nucleus, that find extensive applicationsand are in huge demand in biological reactions for maintaining the pH, as constituents of pharmaceutical formulations and also as important constituents of certain oncological drugs.

Claims

CONTINUOUS-FLOW SYNTHESIS OF AMINE-SULFONIC ACID BUFFERS What is Claimed:

1. A solvent-free continuous-flow process of synthesizing amine-sulfonic acid buffers from sulfo-alkylating agents or alkane-sultones and amines, in a continuous-flow reactor, at preferred conditions of temperature, residence-time, and concentration of the amine and the sulfo-alkylating agent or the alkane-sultone, to yield amine-sulfonic acid buffers wherein, the continuous-flow process comprises use of a device that facilitates the evaporation of the amine reagent and recycling of the amine reagent to the reaction medium, resulting in the formation of the buffer as a crude dry powder, which is followed by the purification of the crude buffer using a combination of an anti- solvent and a solvent in a preferred ratio.

2. The amine-sulfonic acid buffers as claimed in claim 1 are synthesized from amines wherein, the amine is selected from a group comprising of morpholine, N- cyclohexylamine, alkyl amines, cycloalkyl amines, heterocyclic amines, heteroalkyl amines, primary amines, aryl amines, substituted-aryl amines, heteroaryl amines, heteroarylalkyl amines, heterocycloalkyl amines ,N-substituted amines, secondary amines, N-methyl cyclohexylamine, hexylamine, cyclopentylamine, N-methyl piperazine, N-methyl cyclopentylamine, more preferably from morpholine, N- cyclohexylamine.

3. The amine-sulfonic acid buffers as claimed in claim 1 wherein, the alkane-sultone - amine pairs are miscible with each other and the buffers thus synthesized from the alkane-sultone and amine pairs are miscible in the corresponding amines.

4. The amine-sulfonic acid buffers as claimed in claim 1 wherein, the sulfo-alkylating agent or the alkane-sultone is selected from a group comprising of sulfo-alkylating agents or alkane -sultones, more preferably from 1,3-propane-sultone, 1-4-butane- sultone.

5. The amine-sulfonic acid buffers as claimed in claim 1 are biological buffers that can be employed in pharmaceutical compositions or stabilizing compositions, or as pH maintainers in biological reactions.

6. The continuous-flow process as claimed in claim 1, wherein the device that facilitates the evaporation of the excess amine reagent is selected from a group consisting of 1devices that facilitate evaporation of the excess reagent / solvent and recycle of the same to the reaction medium and more preferably the device is selected from Agitated Thin Film Dryer.

7. The residence time of the reaction as claimed in claim 1 is maintained anywhere between 30 seconds to 10 minutes, more preferably the residence time of reaction is maintained at 2 to 5 minutes.

8. The concentration of the amine as claimed in claim 1 is maintained anywhere between 3 -8 mole equivalents of the corresponding amine, more preferably the amine reagent is used in excess with respect to the corresponding alkane-sultone.

9. The concentration of the amine as claimed in claim 1 is 5.7 mole equivalents when morpholine is employed as the amine for obtaining the corresponding amine-sulfonic acid buffer.

10. The temperature of the continuous-flow process as claimed in claim 1 is preferably maintained between 30̊C and 80̊C, more preferably maintained at 60̊C.

11. The continuous-flow process as claimed in claim 1 employs methanol and water as anti-solvent and solvent respectively wherein, the ratio of methanol to water is preferably varied between 3-6 volumes of methanol (antisolvent) to 0.5 – 1 volumes of water (to dissolve the crude buffer) during the crude buffer purification stage, more preferably maintained in the ratio of 6:0.7 of methanol: water.

12. The continuous-flow process as claimed in claim 1 produces 3-morpholinopropane-1- sulfonic acid (MOPS) from 1,3-propanesultone and morpholine.

13. The continuous-flow process as claimed in claim1 produces 3-morpholinobutane-1- sulfonic acid (MOBS) from 1,4 – butanesultone and morpholine.

14. The continuous-flow process as claimed in claim 1 produces N-cyclohexyl-3- aminopropanesulfonic acid (CAPS) from 1,3-propanesultone and N-cyclohexylamine.

15. The continuous-flow process as claimed in claim1 produces N-cyclohexyl-3- aminobutanesulfonic acid (CABS) from 1,4 – butanesultone and N-cyclohexylamine.

16. The continuous-flow process as claimed in claim 1 is devoid of precipitation of the product and the risks associated with down-stream purification. 2

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