1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-, ethyl ester

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 21 July 2016 Experimental completion date: 27 October 2016

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

BMS-589152-01

IUPAC Name:

BMS-589152-01

Test material form:

solid: particulate/powder

Details on test material:

Identification: BMS-589152-01
Physical state/Appearance: Tan-colored powder
Batch: 2G72631N
Purity: 99.5%
Expiry date: 19 June 2014
Storage conditions: Room temperature in the dark
Intended use/Application: Chemical intermediate

Specific details on test material used for the study:

Identification: BMS-589152-01
Batch: AAG8999N
Purity: 100.1% w/w
Physical state / Appearance: beige powder
Expiry Date: 27 December 2017
Storage Conditions: room temperature, in the dark
Safety Precautions: The safety precautions documented in the relevant COSHH form and approved by the Study Director must be followed.

The test item was protected from light.

Study design

Details on sampling:

Analysis of the Sample Solutions
Duplicate vessels of test item sample solutions were removed from the waterbath at each timepoint and the pH of each solution recorded.
The concentration of test item in the sample solution was determined by high performance liquid chromatography (HPLC).
Samples

Solid phase extraction cartridges (Phenomenex Strata X 33 µm, 60 mg/3 mL) were primed with approximately 5 mL of methanol, which was then displaced with approximately 5 mL of relevant, undosed buffer solution. An aliquot of the sample solution (50 mL) was then passed through the cartridge followed by approximately 5 mL of water to remove any buffer salts. The cartridges were then allowed to run dry and dried under vacuum before a final drying with nitrogen. Each cartridge was then eluted with 3 mL of acidified acetonitrile , employing a 1 minute soak time on initial wetting of the sorbent. An aliquot (2 mL) of purified water was then added to each sample extract prior to analysis.

Standards

Duplicate standard solutions of test item were prepared in acidified acetonitrile* :water (60:40 v/v) at a nominal concentration of 0.40 mg/L.
Sample matrix blanks
Aliquots (50 mL) of un-dosed buffer solutions were extracted as detailed for the samples.
Standard matrix blank
Acidified acetonitrile :water (60:40 v/v).

Buffers:

The test system consisted of sterile buffer solutions at pH’s 4, 7 and 9.

Specification of Buffer Solutions

Buffer solution
(pH) Components Concentration (mol dm-3)
4 Citric acid 0.06
Sodium chloride 0.04
Sodium hydroxide 0.07
7Disodium hydrogen orthophosphate (anhydrous) 0.03
Potassium dihydrogen orthophosphate 0.02
Sodium chloride 0.02
9 Disodium tetraborate 0.01
Sodium chloride 0.02

These solutions were subjected to ultrasonication and degassing with nitrogen to minimize dissolved oxygen content.

Details on test conditions:

Performance of the Test
Preparation of the Test Solutions
Sample solutions were prepared in stoppered glass flasks at a nominal concentration of 4.0 x 10-5 g/L in the three buffer solutions. A 1% co-solvent of acetonitrile was used to aid solubility.
The test solutions were split into individual vessels for each data point.
The solutions were shielded from light whilst maintained at the test temperature.

Preliminary Test/Tier 1
Sample solutions at pH 4 and 7 were maintained at 50.0 ± 0.5 °C for a maximum period of 120 hours.
Sample solutions at pH 9 were maintained at 50.0 ± 0.5 °C for a maximum period of 24 hours.

Tier 2
Results from the Preliminary test/Tier 1 showed it was necessary to undertake further testing at pH 7 and pH 9. For testing at pH 7, test item solutions were maintained at temperatures of 50.0 ± 0.5 °C, 60.0 ± 0.5 °C and 70.0 ± 0.5 °C for maximum periods of 30 days, 15 days and 5 days respectively. For testing at pH 9, test item solutions were maintained at temperatures of 20.0 ± 0.5 °C, 30.0 ± 0.5 °C and 50.0 ± 0.5 °C for maximum periods of 21 days, 5 days and 5.0 hours respectively. Samples were removed periodically for analysis at the selected intermediate timepoints.

Tier 3 – Identification of Hydrolysis Products
A solution of test item was prepared in pH 9 buffer solution at a nominal concentration of 5 mg/L with the aid of 5% v/v acetonitrile co-solvent to achieve dissolution. This solution was maintained at 50.0 ± 0.5 °C overnight before vialling directly for analysis. The sample solution was analysed against a test item marker standard prepared at a nominal concentration of 5 mg/L in a matrix of water: acetonitrile (95:5 v/v) by high performance liquid chromatography mass spectrometry (HPLC-MS) using parameters as detailed in Section 4.1.2.5. Appropriate hydrolyzed sample solution and standard solution matrix blanks of pH 9 buffer solution: acetonitrile (95:5 v/v) and water: acetonitrile (95:5 v/v) were also prepared and analysed.

Number of replicates:

3

Positive controls:

no

Negative controls:

no

Results and discussion

Transformation products:

not measured

Dissipation DT50 of parent compound

Dissipation DT50 of parent compound 1

Key result

pH:

4

Temp.:

25 °C

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

Dissipation DT50 of parent compound 2

Key result

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0 s-1

DT50:

244 d

Type:

(pseudo-)first order (= half-life)

Dissipation DT50 of parent compound 3

Key result

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0 s-1

DT50:

111 h

Type:

(pseudo-)first order (= half-life)

Any other information on results incl. tables

Results

Examples of typical quantitative chromatography are presented in Appendix 1 (See Attachment Section of this Summary).

Examples of typical chromatography and spectra relating to the identification of the hydrolysis products are presented in Appendix 3, Attached to this Summary).

Preliminary Test/Tier 1

The mean peak areas relating to the standard and sample solutions are shown Appendix 2 (Please see Attachment Section of this Summary).

The test item concentrations at the given time points are shown in the following tables:

Table 2            pH 4 at 50.0 ± 0.5 ºC

Time (Hours)	Sample Replicate	Concentration  (g/L)	% of mean initial concentration
0	A	3.91x 10^-5	99.9
	B	3.92x 10^-5	100
24	A	3.78x 10^-5	96.6
	B	3.77x 10^-5	96.3
120	A	3.86x 10^-5	98.6
	B	3.85x 10^-5	98.4

 Result:            Less than 10% hydrolysis after 5 days at 50 °C, equivalent to a half-life greater than 1 year at 25 °C. No further testing was therefore required as the test item was confirmed to be hydrolytically stable at pH 4.

Table 3            pH 7 at 50.0 ± 0.5 ºC

Time (Hours)	Sample Replicate	Concentration  (g/L)	% of mean initial concentration
0	A	3.92 x 10^-5	100
	B	3.90x 10^-5	99.7
24	A	3.59x 10^-5	91.8
	B	3.56x 10^-5	91.1
120	A	2.86x 10^-5	73.1
	B	2.86x 10^-5	73.1

Result:            The extent of hydrolysis after120hours indicated that a further test (Tier 2) was required to estimate the rate constant and half-life at 25 °C.

Table 4            pH 9 at 50.0 ± 0.5 ºC

Time (Hours)	Sample Replicate	Concentration  (g/L)	% of mean initial concentration
0	A	3.90 x 10^-5	99.8
	B	3.92x 10^-5	100
24	A	1.43 x 10^-6	3.66
	B	1.66x 10^-6	4.25

 Result:            The extent of hydrolysis after 24hours indicated that a further test (Tier 2) was required to estimate the rate constant and half-life at 25 °C.

Tier 2

The mean peak areas relating to the standard and sample solutions are shown Appendix 2 (Please see Attachment Section of this Summary).

The test item concentrations at the given time points are shown in the following tables:

Table 5            pH 7 at 50.0 ± 0.5 ºC

Time (Hours)	Sample Replicate	Concentration  (g/L)	Log10Concentration (g/L)	% of mean initial concentration
0	A	3.80 x 10^-5	-4.421	100
	B	3.80x 10^-5	-4.421	100
72	A	3.13x 10^-5	-4.504	82.5
	B	3.12x 10^-5	-4.506	82.3
144	A	2.74x 10^-5	-4.563	72.1
	B	2.72x 10^-5	-4.565	71.8
216	A	2.30x 10^-5	-4.638	60.7
	B	2.30x 10^-5	-4.638	60.6
360	A	1.58x 10^-5	-4.801	41.7
	B	1.62x 10^-5	-4.791	42.6
480	A	1.27x 10^-5	-4.898	33.3
	B	1.28x 10^-5	-4.893	33.7
720	A	7.17 x 10^-6	-5.144	18.9
	B	7.11x 10^-6	-5.148	18.8

Result:           Slope = -1.00 x 10^-3 (see Figure 1 - Attached to this Summary)
                      k_obs = 2.30 x 10^-3hour^-1
                      t_½ = 301 hours
                                  

Table 6            pH 7 at 60.0 ± 0.5 ºC

Time (Hours)	Sample Replicate	Concentration  (g/L)	Log10Concentration (g/L)	% of mean initial concentration
0	A	3.80 x 10^-5	-4.421	100
	B	3.80x 10^-5	-4.421	100
48	A	2.78x 10^-5	-4.556	73.2
	B	2.79x 10^-5	-4.555	73.4
96	A	2.02x 10^-5	-4.695	53.1
	B	2.03x 10^-5	-4.693	53.4
144	A	1.45x 10^-5	-4.840	38.1
	B	1.45x 10^-5	-4.839	38.1
192	A	9.87x 10^-6	-5.006	26.0
	B	9.94x 10^-6	-5.003	26.2
264	A	6.94x 10^-6	-5.159	18.3
	B	6.84x 10^-4	-5.165	18.0
360	A	3.44x 10^-6	-5.464	9.06
	B	3.60x 10^-6	-5.443	9.49

 Result:           Slope  = 2.87 x 10^-3 (see Figure 2 - Attached to this Summary)
                       k_obs =   6.60 x 10^-3hour^-1
                       t_½ = 105 hours

Table 7            pH 7 at 70.0 ± 0.5 ºC

Time (Hours)	Sample Replicate	Concentration  (g/L)	Log10Concentration (g/L)	% of mean initial concentration
0	A	3.84 x 10^-5	-4.416	100
	B	3.84x 10^-5	-4.416	100
2.5	A	3.79x 10^-5	-4.421	98.8
	B	3.79x 10^-5	-4.422	98.7
23	A	2.56x 10^-5	-4.592	66.7
	B	2.57x 10^-5	-4.590	67.0
27	A	2.28x 10^-5	-4.643	59.3
	B	2.28x 10^-5	-4.643	59.4
49	A	1.45x 10^-5	-4.838	37.8
	B	1.45x 10^-5	-4.839	37.7
72	A	1.08x 10^-5	-4.967	28.1
	B	1.08x 10^-5	-4.968	28.0
96	A	6.68x 10^-6	-5.175	17.4
	B	6.61x 10^-6	-5.180	17.2
120	A	4.43x 10^-6	5.353	11.6
	B	4.43x 10^-6	-5.353	11.6

 

Result:           Slope =  -7.87 x 10^-3 (see Figure 3 - Attached to this Summary)
                       k_obs =   1.81 x 10^-2hour^-1
                       t_½    =    38.3 hours
                                  

Table 8            pH 9 at 20.0 ± 0.5 ºC

Time (Hours)	Sample Replicate	Concentration  (g/L)	Log10Concentration (g/L)	% of mean initial concentration
0	A	3.87 x 10^-5	-4.412	100
	B	3.86x 10^-5	-4.414	99.8
48	A	3.20 x 10^-5	-4.495	82.7
	B	3.20 x 10^-5	-4.495	82.9
96	A	2.90 x 10^-5	-4.538	75.0
	B	2.92 x 10^-5	-4.535	75.6
144	A	2.37 x 10^-5	-4.625	61.3
	B	2.38 x 10^-5	-4.624	61.5
216	A	1.89 x 10^-5	-4.724	48.9
	B	1.88 x 10^-5	-4.726	48.6
312	A	1.52 x 10^-5	-4.819	39.3
	B	1.49 x 10^-5	-4.827	38.6
504	A	8.71 x 10^-6	-5.060	22.6
	B	8.53 x 10^-6	-5.069	22.1

Result:           Slope  = -1.28 x 10^-3 (see Figure 4 - Attached to this Summary)
                       k_obs  =   2.95 x 10^-3hour^-1
                       t_½     =   235 hours
                                  

Table 9            pH 9 at 30.0 ± 0.5 ºC

Time (Hours)	Sample Replicate	Concentration  (g/L)	Log10Concentration (g/L)	% of mean initial concentration
0	A	3.79 x 10^-5	-4.421	99.7
	B	3.81x 10^-5	-4.419	100
2.5	A	3.73x 10^-5	-4.429	98.1
	B	3.72x 10^-5	-4.430	97.8
23	A	2.95x 10^-5	-4.530	77.6
	B	2.93x 10^-5	-4.533	77.2
27	A	2.73x 10^-5	-4.564	71.8
	B	2.72x 10^-5	-4.565	71.7
49	A	2.03x 10^-5	-4.693	53.4
	B	2.03x 10^-5	-4.692	53.5
72	A	1.56x 10^-5	-4.807	41.1
	B	1.56x 10^-5	-4.808	40.9
96	A	1.03x 10^-5	-4.986	27.2
	B	1.04x 10^-5	-4.985	27.3
120	A	7.80x 10^-6	-5.108	20.5
	B	7.84x 10^-6	-5.106	20.6

Result:           Slope  = -5.81 x 10^-3 (see Figure 5 - Attached to this Summary).
                       k_obs      =         1.34 x 10^-2hour^-1
                       t_½       =         51.8hours
                                

Table 10          pH 9 at 50.0 ± 0.5 ºC

Time (Hours)	Sample Replicate	Concentration  (g/L)	Log10Concentration (g/L)	% of mean initial concentration
0	A	3.90 x 10^-5	-4.409	99.5
	B	3.94x 10^-5	-4.405	101
0.5	A	3.52x 10^-5	-4.454	89.8
	B	3.53x 10^-5	-4.452	90.1
1.0	A	3.29x 10^-5	-4.483	83.9
	B	3.29x 10^-5	-4.483	84.0
2.0	A	2.84x 10^-5	-4.547	72.5
	B	2.85x 10^-5	-4.545	72.8
3.0	A	2.46x 10^-5	-4.610	62.7
	B	2.44x 10^-5	-4.612	62.4
4.0	A	2.15x 10^-5	-4.668	54.9
	B	2.14x 10^-5	-4.669	54.7
5.0	A	1.83x 10^-5	-4.737	46.8
	B	1.84x 10^-5	-4.736	46.9

Result:           Slope  =         -6.42 x 10^-2 (seeFigure 6 - Attached to this Summary)
                       k_obs      =         0.148hour^-1
                       t_½       =         4.69hours
                                  

The Arrhenius plot was constructed using the data shown in the following tables:

Table 11          pH 7 Arrhenius Data

T (ºC)	T (K)	1/T (K)	kobs(hr^-1)	Ln kobs
50	323	3.10 x 10^-3	2.30 x 10^-3	-6.074
60	333	3.00 x 10^-3	6.60 x 10^-3	-5.021
70	343	2.91 x 10^-3	1.81 x 10^-2	-4.011

From the graph (see Figure 7 - Please see Attachment Section of this Summary) of the above data, the rate constant and half-life at 25 °C have been estimated to be as follows:

k_obs      =         1.18 x 10^-4 hour^-1

=         3.28 x 10^-8s^-1
t_½        =         5.87 x 10³ hours
           =         244 days

Table 12          pH 9 Arrhenius Data

T (ºC)	T (K)	1/T (K)	kobs(hr^-1)	Ln kobs
20.0	293	3.41 x 10^-3	2.95 x 10^-3	-5.826
30.0	303	3.30 x 10^-3	1.34 x 10^-2	-4.315
50.0	323	3.10 x 10^-3	0.148	-1.912

From the graph (see Figure 8 - Please see Attachment Section of this Summary) of the above data, the rate constant and half-life at 25 °C have been estimated to be as follows:

k_obs      =         6.27 x 10^-3 hour^-1

                               1.74 x 10^-6s^-1

t_½        =         111 hours


                                  

                                  

                                

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The estimated rate constant and half-life at 25 °C of the test item are shown in the following table:

pH Rate constant (s-1) Estimated half-life at 25 °C
4 not applicable >1 year
7 3.28 x 10-8 244 days
9 1.74 x 10-6 111 hours

High performance liquid chromatography mass spectrometry (HPLC-MS) analysis of the hydrolysis product fully supported the proposed hydrolysis pathway to be base catalyzed cleavage of the ethyl ester functional group resulting in generation of the free carboxylic acid and ethanol as shown within the Attachment Section of this Summary.

Executive summary:

Hydrolysis as a Function of pH

The determination was carried out using a procedure designed to be compatible with Method C.7 Abiotic Degradation, Hydrolysis as a Function of pH of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 111 of the OECD Guidelines for Testing of Chemicals, 13 April 2004.

Conclusion

The estimated rate constant and half-life at 25 °C of the test item are shown in the following table:

pH	Rate constant (s^-1)	Estimated half-life at 25 °C
4	not applicable	>1 year
7	3.28 x 10^-8	244 days
9	1.74 x 10^-6	111 hours

 

High performance liquid chromatography mass spectrometry (HPLC-MS) analysis of the hydrolysis product fully supported the proposed hydrolysis pathway to be base catalyzed cleavage of the ethyl ester functional group resulting in generation of the free carboxylic acid and ethanol as shown in the Attachment Section of this Summary.