Certificate of Analysis

Name: Ozanimod (R)-enantiomer Impurity

Synonyms: (R)-4-(3-(1-((2-Hydroxyethyl)amino)-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-
isopropoxybenzonitrile

Structure:

Product Information :
Product code: OZN403 Batch Number : A3-523
Molecular Formula : C23H24N4O3: HCl Molecular Weight:404.5 : 36.45
Analysis Date : Aug-2022 Retest Date : Aug-2026
Long term Storage Condition: Store at 2°C to 8°C
Shipping Condition: This product is stable to be shipped at room temperature
Analytical Information :
Sr.No Test Results
1 Appearance Off-White Solid
2 Identification
1) H1NMR Conforms to the structure
2) C13NMR Conforms to the structure
3) IR Conforms to the structure
4) Mass Conforms to the structure
3 Purity (By HPLC) 99.49%
4 TGA (Weight Loss: 30°C to 105°C) 1.23%
5 ROI (800°C) 6.24%
6 DEFINED POTENCY 92.05%
7 UV 231nm
Prepared By: Reviewed By:

Dr. Amit Nagare Dr. Amol Taywade

Date: 01.06.2023 Date: 01.06.2023
Note: This product is not for human or animal consumption
 02-Aug-22 4:19:46 PM Page 1 / 1

Analysis Report
<Sample Information>
Sample Name : Diluent
Sample ID : Blank
Data Filename : QC14-220802-001.lcd
Method Filename : 0.1%OPA METHOD.lcm
Batch Filename : 02.08.2022.lcb
Vial # : 1-1 Sample Type : Unknown
Injection Volume : 10 uL
Date Acquired : 02-Aug-22 2:51:36 PM Acquired by : B Swapna
Date Processed : 02-Aug-22 4:06:52 PM Processed by : B Swapna

<Chromatogram>
mAU
1000
PDA Multi 1 254nm,4nm

750

500

250

0 5 10 15 20
min
<Peak Table>
PDA Ch1 254nm
Peak# Ret. Time Area Conc. Area%
Total

AUG-2022 - 2-1/2-1 - QC14-220802-001.lcd

 02-Aug-22 4:08:12 PM Page 1 / 1

Analysis Report
<Sample Information>
Sample Name : A3-523
Sample ID : A3-523
Data Filename : QC14-220802-003.lcd
Method Filename : 0.1%OPA METHOD.lcm
Batch Filename : 02.08.2022.lcb
Vial # : 1-33 Sample Type : Unknown
Injection Volume : 10 uL
Date Acquired : 02-Aug-22 3:42:22 PM Acquired by : B Swapna
Date Processed : 02-Aug-22 4:07:24 PM Processed by : B Swapna

<Chromatogram>
mAU

PDA Multi 1 254nm,4nm

7.387

1000

750

500

250
8.221

0 5 10 15 20
min
<Peak Table>
PDA Ch1 254nm
Peak# Ret. Time Area Height Area%
1 7.387 9988869 1045965 99.491
2 8.221 51098 5720 0.509
Total 10039967 1051685 100.000

AUG-2022 - 2-1/2-3 - QC14-220802-003.lcd

KARPSCHEM LABORATORIES PVT LTD
A3-523
1H-NMR/DMSO
01-AUG-2022

Current Data Parameters

NAME QC11220801009
9.117
8.543
8.538
8.434
8.428
8.411
8.406
8.179
8.161
7.938
7.919
7.599
7.584
7.580
7.561
5.305
5.292
5.280
5.002
4.987
4.972
4.917
3.719
3.706
3.693
3.680
3.507
3.485
3.467
3.453
3.447
3.303
3.293
3.282
3.270
3.259
3.248
3.060
3.006
2.588
2.565
2.304
2.293
2.281
1.392
1.377
EXPNO 1
PROCNO 1

F2 - Acquisition Parameters
Date_ 20220801
Time 12.40 h
INSTRUM spect
PROBHD Z108618_0828 (
PULPROG zg30
TD 65536
SOLVENT DMSO
NS 16
DS 2
SWH 8012.820 Hz
FIDRES 0.244532 Hz
AQ 4.0894465 sec
RG 195.98
DW 62.400 usec
DE 16.75 usec
TE 0 K
D1 1.00000000 sec
TD0 1
SFO1 400.0904705 MHz
NUC1 1H
P0 5.33 usec
P1 16.00 usec
PLW1 12.89900017 W

F2 - Processing parameters
SI 65536
SF 400.0880045 MHz
WDW EM
SSB 0
LB 0.30 Hz
GB 0
PC 1.00

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm
1.97
1.00
1.04
1.06
1.07
2.12

1.03
1.09
1.08

2.11
1.39
1.39
2.26
1.13
0.89

6.10
KARPSCHEM LABORATORIES PVT LTD
A3-523
1H-NMR/DMSO
01-AUG-2022

Current Data Parameters

NAME QC11220801009
9.117

8.543
8.538
8.434
8.428
8.411
8.406

8.179
8.161

7.938
7.919

7.599
7.584
7.580
7.561
EXPNO 1
PROCNO 1

F2 - Acquisition Parameters
Date_ 20220801
Time 12.40 h
INSTRUM spect
PROBHD Z108618_0828 (
PULPROG zg30
TD 65536
SOLVENT DMSO
NS 16
DS 2
SWH 8012.820 Hz
FIDRES 0.244532 Hz
AQ 4.0894465 sec
RG 195.98
DW 62.400 usec
DE 16.75 usec
TE 0 K
D1 1.00000000 sec
TD0 1
SFO1 400.0904705 MHz
NUC1 1H
P0 5.33 usec
P1 16.00 usec
PLW1 12.89900017 W

F2 - Processing parameters
SI 65536
SF 400.0880045 MHz
WDW EM
SSB 0
LB 0.30 Hz
GB 0
PC 1.00

9.3 9.2 9.1 9.0 8.9 8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 ppm
1.97

1.00

1.04

1.06

1.07

2.12
KARPSCHEM LABORATORIES PVT LTD
A3-523
1H-NMR/DMSO
01-AUG-2022

Current Data Parameters

NAME QC11220801009
5.305
5.292
5.280
5.017
5.002
4.987
4.972
4.958
4.917

3.719
3.706
3.693
3.680
3.530
3.507
3.485
3.467
3.453
3.447
3.303
3.293
3.282
3.270
3.259
3.248
3.237
3.224
3.060
3.006
2.588
2.565
2.304
2.293
2.281
2.269
2.258
EXPNO 1
PROCNO 1

F2 - Acquisition Parameters
Date_ 20220801
Time 12.40 h
INSTRUM spect
PROBHD Z108618_0828 (
PULPROG zg30
TD 65536
SOLVENT DMSO
NS 16
DS 2
SWH 8012.820 Hz
FIDRES 0.244532 Hz
AQ 4.0894465 sec
RG 195.98
DW 62.400 usec
DE 16.75 usec
TE 0 K
D1 1.00000000 sec
TD0 1
SFO1 400.0904705 MHz
NUC1 1H
P0 5.33 usec
P1 16.00 usec
PLW1 12.89900017 W

F2 - Processing parameters
SI 65536
SF 400.0880045 MHz
WDW EM
SSB 0
LB 0.30 Hz
GB 0
PC 1.00

5.5 5.0 4.5 4.0 3.5 3.0 2.5 ppm

1.03

1.09

1.08

2.11

1.39

1.39

2.26

1.13

0.89
KARPSCHEM LABORATORIES PVT LTD
A3-523
1H-NMR/DMSO
01-AUG-2022

Current Data Parameters

NAME QC11220801009

1.392
1.377
EXPNO 1
PROCNO 1

F2 - Acquisition Parameters
Date_ 20220801
Time 12.40 h
INSTRUM spect
PROBHD Z108618_0828 (
PULPROG zg30
TD 65536
SOLVENT DMSO
NS 16
DS 2
SWH 8012.820 Hz
FIDRES 0.244532 Hz
AQ 4.0894465 sec
RG 195.98
DW 62.400 usec
DE 16.75 usec
TE 0 K
D1 1.00000000 sec
TD0 1
SFO1 400.0904705 MHz
NUC1 1H
P0 5.33 usec
P1 16.00 usec
PLW1 12.89900017 W

F2 - Processing parameters
SI 65536
SF 400.0880045 MHz
WDW EM
SSB 0
LB 0.30 Hz
GB 0
PC 1.00

1.7 1.6 1.5 1.4 1.3 1.2 1.1 ppm

6.10
KARPSCHEM LABORATORIES PVT LTD
A3-523
1H-NMR/DMSO
01-AUG-2022

# ADDRESS FREQUENCY INTENSITY

[Hz] [PPM]
1 23104.5 3647.519 9.1168 0.04
2 24980.6 3418.137 8.5435 0.15
3 24998.9 3415.907 8.5379 0.16
4 25339.5 3374.258 8.4338 0.07
5 25357.9 3372.004 8.4282 0.07
6 25413.2 3365.250 8.4113 0.08
7 25431.5 3363.005 8.4057 0.07
8 26172.6 3272.403 8.1792 0.08
9 26232.6 3265.063 8.1609 0.09
10 26960.7 3176.044 7.9384 0.07
11 27022.8 3168.444 7.9194 0.08
12 28072.5 3040.108 7.5986 0.05
13 28119.3 3034.377 7.5843 0.12
14 28134.3 3032.545 7.5797 0.11
15 28195.3 3025.086 7.5611 0.13
16 35577.9 2122.445 5.3049 0.04
17 35619.3 2117.388 5.2923 0.10
18 35659.6 2112.457 5.2800 0.04
19 36518.6 2007.433 5.0175 0.02
20 36568.3 2001.359 5.0023 0.05
21 36618.0 1995.278 4.9871 0.06
22 36666.7 1989.332 4.9722 0.04
23 36714.9 1983.439 4.9575 0.02
24 36847.3 1967.250 4.9170 0.03
25 40766.9 1488.011 3.7192 0.04
26 40809.8 1482.767 3.7061 0.10
27 40852.0 1477.603 3.6932 0.10
28 40894.3 1472.437 3.6803 0.04
29 41387.4 1412.145 3.5296 0.02
30 41461.5 1403.087 3.5069 0.02
31 41534.2 1394.204 3.4847 0.03
32 41592.9 1387.018 3.4668 0.04
33 41639.1 1381.367 3.4527 0.03
34 41658.1 1379.054 3.4469 0.03
35 42128.4 1321.547 3.3031 0.03
36 42163.1 1317.309 3.2925 0.03
37 42198.6 1312.968 3.2817 0.03
38 42237.6 1308.195 3.2698 0.03
39 42274.5 1303.687 3.2585 0.02
40 42308.7 1299.500 3.2480 0.02
41 42345.1 1295.058 3.2369 0.02
42 42386.4 1289.999 3.2243 0.02
43 42925.2 1224.123 3.0596 0.03
44 43101.7 1202.551 3.0057 0.03
45 44470.1 1035.235 2.5875 0.03
46 44542.9 1026.333 2.5653 0.05
47 45397.5 921.852 2.3041 0.02
48 45433.7 917.423 2.2931 0.03
49 45473.3 912.579 2.2809 0.02
50 45510.9 907.989 2.2695 0.02
51 45548.0 903.446 2.2581 0.01
52 48380.6 557.117 1.3925 0.73
53 48429.9 551.088 1.3774 0.72
KARPSCHEM LABORATORIES PVT LTD
A3-523
13C-NMR/DMSO
13-MAY-2023

Current Data Parameters

NAME QC11230513081
173.30
167.95
162.55
144.62
139.26
134.62
133.84
131.50
129.35
129.09
128.64
127.51
122.87
115.86
115.25
114.91
102.46
EXPNO 2

72.54
61.31
61.09
56.60
46.50

31.65
27.79
21.47
13.86
PROCNO 1

F2 - Acquisition Parameters
Date_ 20230513
Time 23.15 h
INSTRUM spect
PROBHD Z108618_0828 (
PULPROG zgpg30
TD 65536
SOLVENT DMSO
NS 3000
DS 4
SWH 29761.904 Hz
FIDRES 0.908261 Hz
AQ 1.1010048 sec
RG 195.98
DW 16.800 usec
DE 6.50 usec
TE 0 K
D1 2.00000000 sec
D11 0.03000000 sec
TD0 1
SFO1 100.6122678 MHz
NUC1 13C
P0 3.67 usec
P1 11.00 usec
PLW1 40.78400040 W
SFO2 400.0896004 MHz
NUC2 1H
CPDPRG[2 waltz65
PCPD2 90.00 usec
PLW2 12.89900017 W
PLW12 0.40766999 W
PLW13 0.20506001 W

F2 - Processing parameters
SI 32768
SF 100.6022568 MHz
WDW EM
SSB 0
LB 1.00 Hz
GB 0
PC 1.40

200 180 160 140 120 100 80 60 40 20 0 ppm

KARPSCHEM LABORATORIES PVT LTD
A3-523
13C-NMR/DMSO
13-MAY-2023

Current Data Parameters

NAME QC11230513081
173.30

167.95

162.55

144.62

139.26
134.62
133.84
131.50
129.35
129.09
128.64
127.51
122.87

115.86
115.25
114.91

102.46
EXPNO 2
PROCNO 1

F2 - Acquisition Parameters
Date_ 20230513
Time 23.15 h
INSTRUM spect
PROBHD Z108618_0828 (
PULPROG zgpg30
TD 65536
SOLVENT DMSO
NS 3000
DS 4
SWH 29761.904 Hz
FIDRES 0.908261 Hz
AQ 1.1010048 sec
RG 195.98
DW 16.800 usec
DE 6.50 usec
TE 0 K
D1 2.00000000 sec
D11 0.03000000 sec
TD0 1
SFO1 100.6122678 MHz
NUC1 13C
P0 3.67 usec
P1 11.00 usec
PLW1 40.78400040 W
SFO2 400.0896004 MHz
NUC2 1H
CPDPRG[2 waltz65
PCPD2 90.00 usec
PLW2 12.89900017 W
PLW12 0.40766999 W
PLW13 0.20506001 W

F2 - Processing parameters
SI 32768
SF 100.6022568 MHz
WDW EM
SSB 0
LB 1.00 Hz
GB 0
PC 1.40

180 170 160 150 140 130 120 110 100 ppm
KARPSCHEM LABORATORIES PVT LTD
A3-523
13C-NMR/DMSO
13-MAY-2023

Current Data Parameters

NAME QC11230513081
EXPNO 2

72.54

61.31
61.09
56.60

46.50

31.65

27.79

21.47

13.86
PROCNO 1

F2 - Acquisition Parameters
Date_ 20230513
Time 23.15 h
INSTRUM spect
PROBHD Z108618_0828 (
PULPROG zgpg30
TD 65536
SOLVENT DMSO
NS 3000
DS 4
SWH 29761.904 Hz
FIDRES 0.908261 Hz
AQ 1.1010048 sec
RG 195.98
DW 16.800 usec
DE 6.50 usec
TE 0 K
D1 2.00000000 sec
D11 0.03000000 sec
TD0 1
SFO1 100.6122678 MHz
NUC1 13C
P0 3.67 usec
P1 11.00 usec
PLW1 40.78400040 W
SFO2 400.0896004 MHz
NUC2 1H
CPDPRG[2 waltz65
PCPD2 90.00 usec
PLW2 12.89900017 W
PLW12 0.40766999 W
PLW13 0.20506001 W

F2 - Processing parameters
SI 32768
SF 100.6022568 MHz
WDW EM
SSB 0
LB 1.00 Hz
GB 0
PC 1.40

90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 ppm
KARPSCHEM LABORATORIES PVT LTD
A3-523
13C-NMR/DMSO
13-MAY-2023

# ADDRESS FREQUENCY INTENSITY

[Hz] [PPM]
1 8210.4 17434.829 173.3046 0.15
2 8803.6 16896.043 167.9489 0.14
3 9402.1 16352.397 162.5450 0.14
4 11387.4 14549.281 144.6218 0.19
5 11981.2 14009.937 139.2607 0.15
6 12495.4 13542.925 134.6185 0.16
7 12581.4 13464.810 133.8420 0.16
8 12840.8 13229.206 131.5001 0.07
9 13079.3 13012.585 129.3468 0.12
10 13107.7 12986.820 129.0907 0.11
11 13157.7 12941.375 128.6390 0.08
12 13283.3 12827.323 127.5053 0.16
13 13796.5 12361.168 122.8717 0.14
14 14572.9 11655.966 115.8619 0.13
15 14640.6 11594.534 115.2512 0.13
16 14678.4 11560.167 114.9096 0.17
17 16057.6 10307.515 102.4581 0.17
18 19371.7 7297.388 72.5370 0.22
19 20615.3 6167.911 61.3099 0.11
20 20639.6 6145.826 61.0903 0.19
21 21136.7 5694.333 56.6024 0.22
22 22256.1 4677.659 46.4966 0.16
23 23901.0 3183.636 31.6458 0.15
24 24328.3 2795.576 27.7884 0.16
25 25028.5 2159.611 21.4668 0.76
26 25870.9 1394.474 13.8613 0.10
Print of window 80: MS Spectrum
Data File : D:\DATA_MS\AUG-2022\01-08-2022-001 2022-08-01 17-25-20\QC01-2208-0003.D
Sample Name : A3-523
=====================================================================
Acq. Operator : SYSTEM Seq. Line : 3
Acq. Instrument : ALR-QC-LCMS Location : 9
Injection Date : 8/1/2022 5:34:33 PM Inj : 1
Inj Volume : 10.000 µl
Acq. Method : D:\data_ms\AUG-2022\01-08-2022-001 2022-08-01 17-25-20\MASS FA.M
Last changed : 8/1/2022 5:25:20 PM by SYSTEM
Analysis Method : C:\CHEM32\1\METHODS\MASS FA..M
Last changed : 7/30/2022 6:34:18 PM by SYSTEM
(modified after loading)
Method Info : Mobile phase: Mobile Phase:1ml of Formic Acid in 1000ml Water:ACN (50:50
) Flow:0.5 mlFragmentor-110

Sample Info : Diluent:Methanol Expected Mas:404.5

MS Spectrum
*MSD1 SPC, time=0.194 of D:\DATA_MS\AUG-2022\01-08-2022-001 2022-08-01 17-25-20\QC01-2208-0003.D
405.2 ES-API, Pos, Scan,

100 Max: 223328

80

+ve

60

40

20
406.2

0
100 200 300 400 500 600 700 800 m/z
ALR-QC-LCMS 8/1/2022 5:37:37 PM SYSTEM Page 1 of 1
 TGA
Sample: A3-523

Sample Size: 4.10010 mg

Operator:

Date of Experiment: 2023-05-13

File Path: \\Ta-pc\ta\Data\SDT\MAY 2023\ALR\ALR-

64.001

Method Log:

1: Ramp 10.00°C/min to 840.00°C

2: End of method
120

35.80°C
100.0% 105.00°C
100 98.76%
1.238%
92.52%
(3.669mg)
80
Time:9.08 min
Temperature:105°C Delta Y = 1.238%
Weight loss : 1.238% Loss on drying
From RT to 105C Time:80.24 min
60
Weight (%)

Temperature: 814.76°C
Weight loss : 92.52%

40

20
814.76°C
6.248%

-20
25 225 425 625 825
Temperature (°C) Universal V4.5A TA Instruments
 QC-08 ALR LABS PVT. LTD. UV SPECTRA

Scan Spectrum Curve

2.000

1.475

231.00,1.007
276.00,0.867
Abs

0.950 213.50,0.775

0.425

-0.100
200.00 250.00 300.00 350.00 400.00
Wavelength(nm)

Instrument Performance
Model : UV-VIS Spectrophotometer
Number : 23-1950-01-0032
Spectral Bandwidth : 2.00 nm
Scan Spectrum Performance
Scan Range : 200.00 to 400.00 nm
Measure Mode : Abs
Interval : 0.50 nm
Speed : Medium
Data File : QC18-2305-007.spd
Create Date/Time : Monday, May 15, 2023 3:35:14 PM
Data Type : Original
Method File:
Analyse Note
Analyser : Administrator
Sample Name : A3-523
Comment : A3-523
No. P/V Wavelength(nm) Abs Comment
1 Peak 276.00 0.867

1
QC-08 ALR LABS PVT. LTD. UV SPECTRA

2 Peak 231.00 1.007

3 Peak 213.50 0.775

2
Structure Elucidation Report (SER): Ozanimod (R)-
enantiomer Impurity (batch number A3-523)

NMR Study: The NMR experiments were performed in Bruker’s 400 MHZ FT-NMR
Spectrometer using DMSO-d6 Solvent. Chemical shifts were reported in delta (δ) units, parts
per million (ppm) with respect to the solvent DMSO-d6 2.50 ppm for proton and 39.5 ppm for
carbon. Coupling constants were reported in hertz (Hz). All assignments of Ozanimod (R)-
enantiomer Impurity were shown in the table below.

N
27
15
22 23 14
O N
16
13
9
O 18 8
24 21 N 10
19 17
H3 C 20
7
25 CH3 11
12
26 6
4
2
5
NH OH
3 1
HCl

1 13
Atom Label H chemical shift δ Number of Coupling constant C chemical shift
(ppm) protons (Hz) δ (ppm)
1 3.70 2 q, J=4.0 56.60
1-OH 5.29 1 t, J=4.8
2 3.01,3.06 2 m 46.50
3NH2+ 9.12 2 m
4 4.92 1 m 61.31
5 2.28,2.57 2 m 27.79
6 3.27,3.48 1 31.65
7C 144.62
8C 122.87
 9 8.17 1 d, J=7.6 129.35
10 7.58 1 m 127.51
11 7.92 1 d, J=7.6 129.09
12C 139.26
13C 167.95
14N
15O
16C 173.30
17N
18C 115.86
19 8.42 1 dd, J=2.0,8.8 134.62
20 7.58 1 m 114.91
21C 162.55
22C 102.46
23 8.54 1 d, J=2.0 133.84
24 4.99 1 Sept, J=6.1 72.54
25,26 1.38 6 d, J=6.0 21.47
27C 115.25
d indicates doublet; dd-doublet of doublet; t-triplet, q-quartet; sept-septet and m, multiplet.

IR study: IR Assignment (Make:Bruker Model: Alpha +)

Wave number (Cm-1) Assignment
3410.81 -OH stretching
2928.10 Aliphatic -CH stretching
1275.09 -C-O stretching

Mass analysis: Mass data was acquired by using instrument details and mode (Make:Agilent
Model: 3120b)
Mass data of Ozanimod (R)-enantiomer impurity showed peak of protonated molecular ion at m/z
405.2 daltons.
Ozanimod (R)-enantiomer impurity
m/z Assignment
405.2 [M+H]+

Ozanimod (R)-enantiomer Impurity:- This impurity has been synthesized

using chiral starting material. The obtained product is having desired R-stereo center.