Description:FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patent Rules 2003
COMPLETE SPECIFICATION
(see sections 10 & rule 13)
1. TITLE OF THE INVENTION
“A METHOD FOR SYNTHESISING DEUTERATED ENRICHED TOLUENE”
2. APPLICANT (S)
NAME NATIONALITY ADDRESS
CLEARSYNTH LABS LIMITED Indian 17th Floor, Lotus Nilkamal Business Park, New Link Road, Andheri [West], Mumbai - 400053, Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION

COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it is to be performed

FIELD OF INVENTION
The present invention relates to a method for synthesising deuterated enriched toluene of formula I. Most particularly, it relates to method for synthesising deuterated enriched toluene-d5 and toluene-d8. The synthesised deuterated enriched toluene is useful as a raw material for pharmaceuticals, agricultural chemicals, functional materials, analytical tracers and the like.

BACKGROUND OF INVENTION
Deuterated compounds, in which hydrogen is replaced with deuterium, have significant applications in scientific research, drug development, industrial processes, and environmental studies. They are widely used in NMR solvents, mass spectrometry standards, kinetic isotope effect (KIE) studies, deuterated drugs, isotope tracers, neutron scattering, and hydrology research.
The synthesis of deuterated compounds presents several challenges. Conventional methods involve multiple steps and require expensive reagents, making the production process costly, often amounting to tens of thousands of USD per gram. Additionally, the availability of diverse deuterated compounds is limited due to the complexity of existing synthesis routes.
Prior art methods exhibit several limitations. JP2020070291 describes a process using an aliphatic hydrocarbon solvent, a deuterium source, and a metal catalyst. However, the method also requires an alcohol additive and decalin, making purification difficult.
WO2016073425A2 discloses a synthesis process at high pressure and temperature using D₂O, but this approach is energy-intensive and costly.
Deuterated toluene (toluene-d₈) is widely used as a solvent in NMR spectroscopy, as an internal standard in mass spectrometry, and for isotopic labelling. It plays a crucial role in selective deuteration, kinetic isotope studies, neutron scattering, polymer research, and drug development.
The need for improved synthesis methods arises due to the inefficiencies of traditional approaches, which rely on H/D exchange or metal catalysts that are expensive, inefficient, and require harsh conditions. A new synthesis method should provide high isotopic purity (greater than 99%), improved yield, enhanced safety, and sustainability while minimizing side reactions and degradation. The increasing demand for deuterated compounds in pharmaceuticals, material science, and isotope labeling necessitates eco-friendly and scalable processes.
In the pharmaceutical industry, deuterium incorporation modifies drug absorption, distribution, and toxicity while retaining therapeutic effects and increasing drug stability. Since toluene is widely used in drug synthesis, toluene-d8 is expected to contribute to the development of high-value therapeutic drugs.
Existing synthesis methods pose several challenges. Metal-catalyzed or acid/base-promoted H/D exchange is commonly used but requires multiple exchange processes to achieve high deuterium incorporation. Homogeneous noble metal catalysts are expensive and complex to synthesize. Supercritical deuterium oxide exchange offers advantages such as short reaction times and selective deuteration; however, it requires high pressure and temperature, which limits large-scale application.
A more efficient process is essential, as the current domestic supply of toluene-d8 is mainly imported, making it expensive and logistically complex to transport.
The new method aims to achieve high isotope utilization, high product purity, and an easy, scalable process for cost-effective and efficient deuterated toluene production.
The present invention addresses these challenges by providing a cost-effective and step-efficient method for synthesizing deuterated enriched toluene without the need for expensive reagents. This method enhances accessibility and enables broader applications of deuterated enriched toluene.

The present invention provides a method for synthesising a deuterated enriched toluene compound, which requires neither a number of steps nor an expensive reagent, and thus is considerably economical and can be widely used for general-purposes.

OBJECTS OF THE INVENTION
One of the objects of the present invention is to provide a method for synthesising deuterated enriched toluene of formula I.
Another object of the present invention is to provide a method for synthesising deuterated enriched toluene (e.g. toluene-d5 and toluene-d8).
Another object of the present invention is to provide solvent-free reaction in which involves elimination of solvents minimizes waste and improves sustainability.
Yet another object of the present invention is to provide recovery and recyclability of acid catalyst for 4 to 5 cycles without loss of reactivity for continuous production of deuterated enriched toluene.
Yet another object of the present invention is to provide acid catalyst that can direct reactions toward specific products, reducing by-products as well as use of mild reaction conditions such as lower temperature and pressures which save energy.

SUMMARY OF THE INVENTION
One of the aspects of the present invention provides a method for synthesising deuterated enriched toluene of formula I

the method comprising:
a. contacting toluene with a base in presence of a solvent at a temperature in the range of 50oC to 100oC for a time period in the range of 10hrs to 14 hrs to obtain a deuterated toluene;
b. treating the toluene or deuterated toluene with an acid catalyst at a temperature in the range of 10oC to 60oC for a period in the range of 10 hrs to 15 hrs to obtain a reaction mass comprising deuterated enriched toluene;
c. recovering the acid catalyst from the reaction mass obtained from step (b) and recycling the recovered catalyst in the step (b) for continuous production of reaction mass comprising deuterated enriched toluene; and
d. isolating the deuterated enriched toluene from the reaction mass obtained from step (b) to obtain a deuterated enriched toluene of formula I;
wherein R1 to R8 are independently selected from hydrogen (H), deuterium (-D) and combinations thereof.

DETAILED DESCRIPTION OF THE INVENTION
In the specification different terms are used to describe the invention. The definitions of the terms are provided below.
The term ‘Isotopic enrichment’ refers to the process of increasing the relative abundance of a specific isotope of an element (H) within a deuterated toluene.
The term ‘deuterated enriched toluene of formula I’ or ‘formula I’ or ‘toluene-d5’ or toluene-d8’ used herein refers to a compound represented by a particular chemical structure containing indicated deuterium atoms, will also contain lesser amounts of isotopologues (species in which the chemical structure differs from a specific compound of this invention only in the isotopic composition thereof.) having hydrogen atoms at one or more of the designated deuterium positions in that structure. The terms ‘deuterated enriched toluene of formula I’, ‘formula I’, ‘toluene-d5’, ‘toluene-d8’ can be used interchangeably in the specification.
The term ‘deuterated toluene’ used herein refers to deuterium-labelled toluene compounds which is made by selectively replacing hydrogen atoms with deuterium atoms. In the present invention deuterated toluene may include such as but is not limited to toluene-d3.
A deuterated enriched toluene is a deuterated toluene in which the proportion of deuterium (D) has been increased beyond its natural abundance through isotopic enrichment processes.
The term ‘reaction mass’ used herein refers to mixtures formed during chemical processes where individual components are not isolated but exist together as a final product. In the present invention, reaction mass may contain solvent, catalyst deuterated enriched toluene (toluene-d5/toluene-d8).
The term ‘reaction mass comprising deuterated enriched toluene’ used herein refers to reaction mass comprising toluene-d5 and/or toluene-d8.
In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I

the method comprising:
a. contacting toluene with a base in presence of a solvent at a temperature in the range of 50oC to 100oC for a time period in the range of 10hrs to 14 hrs to obtain a deuterated toluene;
b. treating the toluene or deuterated toluene with an acid catalyst at a temperature in the range of 10oC to 60oC for a period in the range of 10 hrs to 15 hrs to obtain a reaction mass comprising deuterated enriched toluene;
c. recovering the acid catalyst from the reaction mass obtained from step (b) and recycling the recovered catalyst in the step (b) for continuous production of reaction mass comprising deuterated enriched toluene; and
d. isolating the deuterated toluene from the reaction mass obtained from step (b) to obtain deuterated enriched toluene of formula I;
wherein R1 to R8 are independently selected from hydrogen (H), deuterium (D) and combinations thereof.
In another embodiment of the present invention provides a method for synthesising deuterated enriched toluene of formula I, wherein the deuterated toluene obtained from step (a) is toluene-d3 which is represented by following formula

In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein the deuterated toluene obtained from step (a), toluene-d3 is having isotopic enrichment ranging from 95% to 98%.
In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein the deuterated enriched toluene obtained from step (d) is toluene-d5 which is represented by following formula

In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein the deuterated enriched toluene obtained from step (d) is toluene-d8 which is represented by following formula

In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein toluene-d5 is having isotopic enrichment ranging from 95% to 98%.
In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein toluene-d8 is having isotopic enrichment ranging from 95% to 98%.
In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein the acid catalyst is a deuterated acid catalyst selected from trifluoromethanesulfonic acid-d (Triflic-d acid, CF₃SO₃D), methanesulfonic acid-d (CH₃SO₃D), p-toluenesulfonic acid-d (TsOD, C₆H₄CD₃SO₃D), formic acid-d (DCO₂D), acetic acid-d (CD₃CO₂D), trifluoroacetic acid-d (CF₃CO₂D), benzoic acid-d (C₆D₅CO₂D), fluorosulfonic acid-d (FSO₃D), perchloric acid-d (DClO₄), hexafluorophosphoric acid-d (DHPF₆), hexafluoroantimonic acid-d (DHSbF₆) and combinations thereof.
In most preferred embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein deuterated acid catalyst is trifluoromethanesulfonic acid-d (Triflic-d acid, CF₃SO₃D).
In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein the solvent is used in 1volume (Vol) to 4 vol to the total vol of toluene to carry out the step (a) of the method.
In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein acid catalyst is used in 1vol to 6vol to the total vol of toluene or deuterated toluene in the step (b) of the method.
In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein solvent is polar and/or non-polar solvent.
In yet another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein solvent is a deuterated solvent selected from benzene-d6, cyclohexane-d12, n-Hexane-d14, chloroform-d, dichloromethane-d2, chlorobenzene-d5, 1,1,2,2-tetrachloroethane-d2, tetrahydrofuran-d8, methanol-d4, ethanol-d6, isopropanol-d8, acetone-d6, acetonitrile-d3, dimethyl sulfoxide-d6, N, N-dimethylformamide-d7, n-methylpyrrolidone-d9 and combinations thereof.
In most preferred embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein solvent is dimethyl sulfoxide-d6.
In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein the base used in step (a) is selected from sodium hydroxide (NaOH) , potassium hydroxide (KOH), lithium hydroxide (LiOH), calcium hydroxide (Ca(OH)₂, barium hydroxide (Ba(OH)₂), sodium carbonate (Na₂CO₃), potassium carbonate (K₂CO₃), sodium bicarbonate (NaHCO₃), sodium tert-butoxide (NaOtBu), potassium tert-butoxide (KOtBu), lithium tert-butoxide (LiOtBu), cesium tert-butoxide (CsOtBu), aluminum tert-butoxide (Al(OtBu)₃), titanium(IV) tert-butoxide (Ti(OtBu)₄), zinc tert-butoxide (Zn(OtBu)₂) and combinations thereof.
In most preferred embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein the base used in step (a) is potassium tert-butoxide (KOtBu).
In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein isolating the deuterated toluene from the reaction mass obtained from step (b) to obtain a pure deuterated toluene is carried out by extracting with a solvent followed by concentrating solvent layer.
In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein the deuterated toluene is isolated by distillation with or without vacuum to obtain toluene-d3 having isotopic enrichment ranging from 95% to 98%.
In another embodiment of the present invention there is provided a method for synthesising deuterated enriched toluene of formula I, wherein recovering and recycling triflic acid-d from the reaction mass obtained from step (c) is used for 4 to 5 cycles without loss of reactivity for continuous production of pure deuterated toluene (toluene-d5 or toluene-d8).
The details of the present invention are provided in the examples given below to illustrate the invention only and therefore they should not be construed to limit the scope of invention.

EXAMPLES
A. process for preparation of deuterated enriched toluene (formula I):
Trifluoromethanesulfonic acid-d (Triflic acid-D) was prepared using reported literature process.
I. Synthesis of deuterated toluene (toluene-d3):
100 ml Toluene was taken in 1 lit reactor followed by addition of DMSO-d6 (1vol. to 4 vol.) and potassium tert-butoxide (1 to 2 eq.). The resulted mixture was heated at a temperature varies from 60oC to 90oC for 12 hours under stirring. After completion of reaction (12 hrs.), the product was isolated by distillation as pure Toluene-d3 which was having isotopic enrichment ranging from 95% to 99%.
Reaction scheme:

II. Synthesis of deuterated enriched toluene (toluene-d5):
100ml Toluene was taken in 1 lit reactor. Trifluoromethanesulfonic acid-d (Triflic acid-d) was added. The quantity of Triflic acid-d was varies from 1 vol., 3 vol. and 6 vol. The resulted mixture was heated at a temperature ranging from 10oC to 60oC. After completion of reaction (after 12 hrs.). The recovered Triflic acid-d was recovered and reused for 4 to 5 cycles without loss of reactivity. The reaction mixture was diluted with dichloromethane (DCM). The organic layer was separated. DCM layer (containing Toluene-d5) was concentrated to remove DCM. The pure Toulene-d8 isolated was having isotopic enrichment ranging from 95% to 99%.
Reaction scheme:

III. Synthesis of deuterated enriched toluene (toluene-d8):
100ml Toluene-d3 was taken in 1 lit reactor. Trifluoromethanesulfonic acid-d (Triflic acid-d) was added. The quantity of Triflic acid-d was varies from 1 vol., 3 vol. and 6 vol. The resulted mixture was heated at a temperature ranging from 10oC to 60oC. After completion of reaction (after 12 hrs.). The recovered Triflic acid-d was recovered and reused for 4 to 5 cycles without loss of reactivity. The reaction mixture was diluted with dichloromethane (DCM). The organic layer was separated. DCM layer (containing Toluene-d8) was concentrated to remove DCM. The pure Toulene-d8 isolated was having isotopic enrichment ranging from 95% to 99%.
Reaction scheme:


We claim:
1. A method for synthesising deuterated enriched toluene of formula I

the method comprising:
a. contacting toluene with a base in presence of a solvent at a temperature in the range of 50oC to 100oC for a time period in the range of 10hrs to 14hrs to obtain a deuterated toluene;
b. treating the toluene or deuterated toluene with an acid catalyst at a temperature in the range of 10oC to 60oC for a period in the range of 10hrs to 15hrs to obtain a reaction mass comprising deuterated enriched toluene;
c. recovering the acid catalyst from the reaction mass obtained from step (b) and recycling the recovered catalyst in the step (b) for continuous production of reaction mass comprising deuterated enriched toluene; and
d. isolating the deuterated toluene from the reaction mass obtained from step (b) to obtain deuterated enriched toluene of formula I;
wherein R1 to R8 are independently selected from hydrogen (H), deuterium (-D) and combinations thereof.
2. The method as claimed in claim 1, wherein the deuterated toluene obtained from step (a) is toluene-d3,

and is having isotopic enrichment ranging from 95% to 98%.
3. The method as claimed in claim 1, wherein the deuterated enriched toluene of formula I obtained from step (d) is toluene-d5,

or
toluene-d8,

respectively; toluene-d5 & toluene-d8 both are having isotopic enrichment ranging from 95% to 98%.
4. The method as claimed in claim 1, wherein the acid catalyst is a deuterated acid catalyst selected from trifluoromethanesulfonic acid-d (Triflic-d acid, CF₃SO₃D), methanesulfonic acid-d (CH₃SO₃D), p-toluenesulfonic acid-d (TsOD, C₆H₄CD₃SO₃D), formic acid-d (DCO₂D), acetic acid-d (CD₃CO₂D), trifluoroacetic acid-d (CF₃CO₂D), benzoic acid-d (C₆D₅CO₂D), fluorosulfonic acid-d (FSO₃D), perchloric acid-d (DClO₄), hexafluorophosphoric acid-d (DHPF₆), hexafluoroantimonic acid-d (DHSbF₆) and combinations thereof.
5. The method as claimed in claim 1, wherein the solvent is used in 1volume (Vol) to 4 vol to the total vol of toluene; and wherein acid catalyst is used in 1vol to 6vol to the total vol of toluene or deuterated toluene.
6. The method as claimed in claim 1, wherein solvent is a deuterated solvent selected from benzene-d6, cyclohexane-d12, n-Hexane-d14, chloroform-d, dichloromethane-d2, chlorobenzene-d5, 1,1,2,2-tetrachloroethane-d2, tetrahydrofuran-d8, methanol-d4, ethanol-d6, isopropanol-d8, acetone-d6, acetonitrile-d3, dimethyl sulfoxide-d6, N, N-dimethylformamide-d7, n-methylpyrrolidone-d9 and combinations thereof, preferably dimethyl sulfoxide-d6.
7. The method as claimed in claim 1, wherein the base is selected from sodium hydroxide (NaOH) , potassium hydroxide (KOH), lithium hydroxide (LiOH), calcium hydroxide (Ca(OH)₂, barium hydroxide (Ba(OH)₂), sodium carbonate (Na₂CO₃), potassium carbonate (K₂CO₃), sodium bicarbonate (NaHCO₃), sodium tert-butoxide (NaOtBu), potassium tert-butoxide (KOtBu), lithium tert-butoxide (LiOtBu), cesium tert-butoxide (CsOtBu), aluminum tert-butoxide (Al(OtBu)₃), titanium(IV) tert-butoxide (Ti(OtBu)₄), zinc tert-butoxide (Zn(OtBu)₂) and combinations thereof, preferably potassium tert-butoxide (KOtBu).
8. The method as claimed in claim 1, wherein isolating the deuterated enriched toluene from the reaction mass obtained from step (b) to obtain deuterated enriched toluene is carried out by extracting with a solvent followed by concentrating solvent layer.
9. The method as claimed in claim 1, wherein the deuterated toluene is isolated by distillation with or without vacuum to obtain toluene-d3 having isotopic enrichment ranging from 95% to 98%.
10. The method as claimed in claim 1, wherein recycling the recovered catalyst in the step (b) for continuous production of reaction mass comprising deuterated enriched toluene (toluene-d5 or toluene-d8) for 4 to 5 cycles without loss of reactivity.

Dated this: 27th day of March 2025

Vijaykumar Shivpuje
IN/PA-1096
Agent for the Applicant(s)
To
The Controller of Patents,
The Patent Office, Mumbai

ABSTRACT

“A METHOD FOR SYNTHESISING DEUTERATED ENRICHED TOLUENE”

The present invention provides a method for synthesising deuterated enriched toluene of formula I

wherein R1 to R8 are independently selected from hydrogen (H), deuterium (-D) and combinations thereof. The method is carried out by: (a) contacting toluene with a base in presence of a solvent at a temperature in the range of 50oC to 100oC for a time period in the range of 10hrs to 14 hrs to obtain a deuterated toluene; (b)treating the toluene or deuterated toluene with an acid catalyst at a temperature in the range of 10oC to 60oC for a period in the range of 10 hrs to 15 hrs to obtain a reaction mass comprising deuterated enriched toluene; (c) recovering the acid catalyst from the reaction mass obtained from step (b) and recycling the recovered catalyst in the step (b) for continuous production of reaction mass comprising deuterated enriched toluene; and (d) isolating the deuterated enriched toluene from the reaction mass obtained from step (b) to obtain deuterated enriched toluene of formula I.

, Claims:We claim:
1. A method for synthesising deuterated enriched toluene of formula I

the method comprising:
a. contacting toluene with a base in presence of a solvent at a temperature in the range of 50oC to 100oC for a time period in the range of 10hrs to 14hrs to obtain a deuterated toluene;
b. treating the toluene or deuterated toluene with an acid catalyst at a temperature in the range of 10oC to 60oC for a period in the range of 10hrs to 15hrs to obtain a reaction mass comprising deuterated enriched toluene;
c. recovering the acid catalyst from the reaction mass obtained from step (b) and recycling the recovered catalyst in the step (b) for continuous production of reaction mass comprising deuterated enriched toluene; and
d. isolating the deuterated toluene from the reaction mass obtained from step (b) to obtain deuterated enriched toluene of formula I;
wherein R1 to R8 are independently selected from hydrogen (H), deuterium (-D) and combinations thereof.
2. The method as claimed in claim 1, wherein the deuterated toluene obtained from step (a) is toluene-d3,

and is having isotopic enrichment ranging from 95% to 98%.
3. The method as claimed in claim 1, wherein the deuterated enriched toluene of formula I obtained from step (d) is toluene-d5,

or
toluene-d8,

respectively; toluene-d5 & toluene-d8 both are having isotopic enrichment ranging from 95% to 98%.
4. The method as claimed in claim 1, wherein the acid catalyst is a deuterated acid catalyst selected from trifluoromethanesulfonic acid-d (Triflic-d acid, CF₃SO₃D), methanesulfonic acid-d (CH₃SO₃D), p-toluenesulfonic acid-d (TsOD, C₆H₄CD₃SO₃D), formic acid-d (DCO₂D), acetic acid-d (CD₃CO₂D), trifluoroacetic acid-d (CF₃CO₂D), benzoic acid-d (C₆D₅CO₂D), fluorosulfonic acid-d (FSO₃D), perchloric acid-d (DClO₄), hexafluorophosphoric acid-d (DHPF₆), hexafluoroantimonic acid-d (DHSbF₆) and combinations thereof.
5. The method as claimed in claim 1, wherein the solvent is used in 1volume (Vol) to 4 vol to the total vol of toluene; and wherein acid catalyst is used in 1vol to 6vol to the total vol of toluene or deuterated toluene.
6. The method as claimed in claim 1, wherein solvent is a deuterated solvent selected from benzene-d6, cyclohexane-d12, n-Hexane-d14, chloroform-d, dichloromethane-d2, chlorobenzene-d5, 1,1,2,2-tetrachloroethane-d2, tetrahydrofuran-d8, methanol-d4, ethanol-d6, isopropanol-d8, acetone-d6, acetonitrile-d3, dimethyl sulfoxide-d6, N, N-dimethylformamide-d7, n-methylpyrrolidone-d9 and combinations thereof, preferably dimethyl sulfoxide-d6.
7. The method as claimed in claim 1, wherein the base is selected from sodium hydroxide (NaOH) , potassium hydroxide (KOH), lithium hydroxide (LiOH), calcium hydroxide (Ca(OH)₂, barium hydroxide (Ba(OH)₂), sodium carbonate (Na₂CO₃), potassium carbonate (K₂CO₃), sodium bicarbonate (NaHCO₃), sodium tert-butoxide (NaOtBu), potassium tert-butoxide (KOtBu), lithium tert-butoxide (LiOtBu), cesium tert-butoxide (CsOtBu), aluminum tert-butoxide (Al(OtBu)₃), titanium(IV) tert-butoxide (Ti(OtBu)₄), zinc tert-butoxide (Zn(OtBu)₂) and combinations thereof, preferably potassium tert-butoxide (KOtBu).
8. The method as claimed in claim 1, wherein isolating the deuterated enriched toluene from the reaction mass obtained from step (b) to obtain deuterated enriched toluene is carried out by extracting with a solvent followed by concentrating solvent layer.
9. The method as claimed in claim 1, wherein the deuterated toluene is isolated by distillation with or without vacuum to obtain toluene-d3 having isotopic enrichment ranging from 95% to 98%.
10. The method as claimed in claim 1, wherein recycling the recovered catalyst in the step (b) for continuous production of reaction mass comprising deuterated enriched toluene (toluene-d5 or toluene-d8) for 4 to 5 cycles without loss of reactivity.