PBR 28 FOR INJECTION: PRECURSOR AND REFERENCE
COMPOUND − ACCEPTANCE TESTING AND FORMS
PET Radiopharmaceutical Sciences Section,
Molecular Imaging Branch,
National Institute of Mental Health,
National Institutes of Health,
Bldg. 10, Rm. B3 C338,
Bethesda, MD 20892
Date of review: 12/01/05
1. Overview
The radiopharmaceutical production of [C-11]-PBR 28 for the National Institute of Mental Health
requires the use of desmethyl PBR 28 ( 3-[N-Acetyl-N-(2-hydroxybenzyl)amino]-4-phenoxypyridine)
as a precursor for C-11 methylation. The standard operating procedure (SOP) for acceptance testing
of desmethyl PBR 28 is described below. Acceptance testing is required to validate chemical purity
and chemical identity of desmethyl PBR 28. Upon acceptance of desmethyl PBR 28, the desmethyl
PBR 28 may be released for the production of [C-11]-PBR 28. Upon release, the desmethyl-PBR 28
is to be tested annually thereafter for chemical purity (via HPLC, section 3.1) and chemical identity
(via LC-MS, section 3.2).
This SOP also contains testing methods and acceptance criteria for a standard sample of PBR 28
(section 5). This non-radioactive material is utilized in the quality control testing of [C-11]-PBR 28 for
the determination of specific radioactivity and chemical identity.
2. Synthetic Schemes for Preparation of Reference Standard, PBR 28 and precursor compound,
Desmethyl PBR 28
PBR 28 is synthesized according to Nakazoto A. et al. Japaneses Patent, 2000/001476, as outlined
in Scheme A. Desmethyl PBR 28 is produced in six steps from commercially available
salicylaldehyde, 4-chloro-5-nitro-pyridine and phenol, according to Scheme B.
Scheme A. Synthesis of PBR 28 from 4 and commercial material (10).
OMe
CHO
4
10
Toluene, reflux 24h
NaBH4, MeOH, 0oC
RT, 1h
N
H
N
O
N
OMe
Ac2O/AcOH
Ac
N
O
OMe
RT, 2h, reflux
11
PBR 28
Document 8. [11C]PBR 28 for Injection: Precursor and Reference Compound − Acceptance Testing and Form
Page 1 of 15
PBR 28 FOR INJECTION: PRECURSOR AND REFERENCE
COMPOUND − ACCEPTANCE TESTING AND FORMS
PET Radiopharmaceutical Sciences Section,
Molecular Imaging Branch,
National Institute of Mental Health,
National Institutes of Health,
Bldg. 10, Rm. B3 C338,
Bethesda, MD 20892
Date of review: 12/01/05
Scheme B. Synthesis of desmethyl PBR 28 from commercial materials (1,2 and 5)
NO2
N
OH
Cl
N
2
NO2
Fe/HCl
O
t.BuOK
THF
RT,15 h
O
3
CHO
4
CHO
AcO
Ac2O
NH2
EtOH
Reflux, 3 h
1
HO
N
4
N
H
N
OH
O
Pyridine
RT, 15 h
1. Toluene, reflux 24 h,
2. NaBH4, MeOH, 0oC,
3. RT, reflux 3 h
5
7
6
Ac2O
Reflux
N
Ac
N
O
5% KOH
OH
MeOH, RT, 1 h
9
N
Ac
N
O
OAc
8
Desmethyl PBR 028
Document 8. [11C]PBR 28 for Injection: Precursor and Reference Compound − Acceptance Testing and Form
Page 2 of 15
PBR 28 FOR INJECTION: PRECURSOR AND REFERENCE
COMPOUND − ACCEPTANCE TESTING AND FORMS
PET Radiopharmaceutical Sciences Section,
Molecular Imaging Branch,
National Institute of Mental Health,
National Institutes of Health,
Bldg. 10, Rm. B3 C338,
Bethesda, MD 20892
Date of review: 12/01/05
3. Acceptance criteria for precursor and reference compound
(A) Precursor
Source: the starting material, desmethyl PBR 28 is obtained from this laboratory.
Acceptance criteria: the acceptance criteria for precursor are as follows:

1H-NMR

LC-MS of desmethyl PBR 28 performed in the PET Radiopharmaceutical Sciences Section on
spectrum of desmethyl PBR 28 from this laboratory to verify chemical structure.
the Finnigan LCQDeca LC/MS (ThermoQuest, Germany) to confirm chemical structure and purity

HPLC in the PET Radiopharmaceutical Sciences Section showing the chemical purity of
precursor to be ≥ 98%.
(B) Reference compound
Source: the reference compound (PBR 28) is obtained from this laboratory.
Acceptance criteria: the acceptance criteria for reference compound are as follows:

1H-NMR

LC-MS of PBR 28 performed in the PET Radiopharmaceutical Sciences Section on the Finnigan
spectrum of PBR 28 from this laboratory to verify chemical structure.
LCQDeca LC/MS (ThermoQuest, Germany) to confirm chemical structure and purity

HPLC in the PET Radiopharmaceutical Sciences (MIB, NIMH, NIH) showing the chemical purity
of PBR 28 to be ≥ 98%.
Document 8. [11C]PBR 28 for Injection: Precursor and Reference Compound − Acceptance Testing and Form
Page 3 of 15
PBR 28 FOR INJECTION: PRECURSOR AND REFERENCE
COMPOUND − ACCEPTANCE TESTING AND FORMS
PET Radiopharmaceutical Sciences Section,
Molecular Imaging Branch,
National Institute of Mental Health,
National Institutes of Health,
Bldg. 10, Rm. B3 C338,
Bethesda, MD 20892
Date of review: 12/01/05
4. Precursor and reference compounds acceptance forms
Approved by: __________________________ Initials___________
Date:__________
Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences, NIMH
Desmethyl PBR 28 Acceptance Form
Receipt of compound:
Compound name: Desmethyl PBR 28
Lot #
Supplier: PET Radiopharmaceutical Sciences
Received by:
Section (PRSS), Molecular Imaging Branch (MIB)
National Institute of Mental Health
National Institutes of Health (NIH)
Building 10, Room B3C338
10 Center Drive, MSC 1003
Bethesda, MD 20892
Date received:
Quantity:
Molecular formula: C20H18N2O3
Molecular weight: 334.37
1
H-NMR spectrum
Record weight of desmethyl PBR 28 _________ dissolved in ca. 0.8 mL of 0.03% TMS (v/v) in CDCl3
Proton Assignment
Expected Resonance (ppm)
Observed Resonance
Acceptance?
(ppm)
CO-CH3
methylene
Aromatic protons
2.03 (s, 3H)
Y or N
4.82 (dd, 2H)
Y or N
8.44 (d, 1H, ), 8.39 (s,1H),
7.40-7.42 (m, 2 H), 7.30-
Y or N
7.35 ( m, 1H), 7.28
(Solvent, CDCl3), 7.25-7.27
(m, 1H), 6.95-6.92 (d, 1H),
6.65-6.77 (m, 5H)
Document 8. [11C]PBR 28 for Injection: Precursor and Reference Compound − Acceptance Testing and Form
Page 4 of 15
PBR 28 FOR INJECTION: PRECURSOR AND REFERENCE
COMPOUND − ACCEPTANCE TESTING AND FORMS
PET Radiopharmaceutical Sciences Section,
Molecular Imaging Branch,
National Institute of Mental Health,
National Institutes of Health,
Bldg. 10, Rm. B3 C338,
Bethesda, MD 20892
Date of review: 12/01/05
Desmethyl PBR 28 Acceptance Form
HPLC
Record weight _____________ mg in 10 ml of 20/80 Ethanol/saline.
Column:
Eluent:
Flow rate:
UV Detector:
Onyx (monolithic C18; 4.6 mm x 100 mm; Phenomenex; part # CH0 7643)
Isocratic: 0.01 M ammonium formate in water-acetonitrile (70: 30 v/v)
3 mL/min
220 nm
Record Serial # of this column: __________
Criterion
Acceptance? (circle
Date
one)
Retention time 4.525 1.0
Y or N
min
Peak area ≥ 98 %
Y or N
LC/MS
Criterion (expected m/z,
Observed ion, M + 1
Acceptance (circle one)?
M + 1)
335.13
Y or N
Attached documents, acceptance criteria and test results:
1.
1H-NMR
2.
LC-MS data from this laboratory?
YES_________ NO__________.
3.
HPLC chromatogram from this laboratory?
YES_________ NO__________.
spectrum from this laboratory?
YES_________ NO__________.
(An example of a set of satisfactory attached documents is included in Document 8).
Document 8. [11C]PBR 28 for Injection: Precursor and Reference Compound − Acceptance Testing and Form
Page 5 of 15
PBR 28 FOR INJECTION: PRECURSOR AND REFERENCE
COMPOUND − ACCEPTANCE TESTING AND FORMS
PET Radiopharmaceutical Sciences Section,
Molecular Imaging Branch,
National Institute of Mental Health,
National Institutes of Health,
Bldg. 10, Rm. B3 C338,
Bethesda, MD 20892
Date of review: 12/01/05
Desmethyl PBR 28 Acceptance Form
Chemical structure:
Acceptance criteria:
1. Identity: confirmed by 1H-NMR and LC/MS
O
2. Purity: ≥ 98 % by HPLC
N
N
3. Identity: confirmed by LC/MS
OH
O
Test Results
Test
Results
Procedure
1. NMR ( This laboratory)
SOP # QA308(2.1)
2. LC-MS (This laboratory)
SOP # QA308(2.3)
3. HPLC (This laboratory)
SOP # QA308(2.2)
Checked by:
Accepted by:
Storage condition:
Date
Date
Initials
Document 8. [11C]PBR 28 for Injection: Precursor and Reference Compound − Acceptance Testing and Form
Page 6 of 15
PBR 28 FOR INJECTION: PRECURSOR AND REFERENCE
COMPOUND − ACCEPTANCE TESTING AND FORMS
PET Radiopharmaceutical Sciences Section,
Molecular Imaging Branch,
National Institute of Mental Health,
National Institutes of Health,
Bldg. 10, Rm. B3 C338,
Bethesda, MD 20892
Date of review: 12/01/05
PBR 28 Reference Acceptance Form
Approved by: __________________________ Initials___________
Date:__________
Victor W Pike, Ph.D., Chief, PET Radiopharmaceutical Sciences, NIMH
Receipt of compound:
Compound name: PBR 28
Lot #
Supplier Name: PET Radiopharmaceutical
Received by:
Sciences Section (PRSS), Molecular Imaging
Branch (MIB)
National Institute of Mental Health
National Institutes of Health (NIH)
Building 10, Room B3C338
10 Center Drive, MSC 1003
Bethesda, MD 20892
Date received:
Quantity:
Molecular formula: C21H20N2O3
Molecular weight: 348.4
1
H-NMR spectrum
Record the weight __________of PBR 28 dissolved in ca. 0.8 mL of 0.03% TMS (v/v) in CDCl3
Proton Assignment
Expected Resonance (ppm)
Observed Resonance
Acceptance?
(ppm)
-CO- CH3
2.01 (S, 3H)
Y or N
-OCH3
3.60 (S, 3H)
Y or N
-CH2
5.18- 5.14 (dd, 2H)
Y or N
Aromatic protons
8.30 (d, 1H), 8.23 (S, 1H),
Y or N
7.28 (Solvent, CDCl3),
7.46~7.22 (m, 5H),
6.59~6.93 ( m, 5H),
Document 8. [11C]PBR 28 for Injection: Precursor and Reference Compound − Acceptance Testing and Form
Page 7 of 15
PBR 28 FOR INJECTION: PRECURSOR AND REFERENCE
COMPOUND − ACCEPTANCE TESTING AND FORMS
PET Radiopharmaceutical Sciences Section,
Molecular Imaging Branch,
National Institute of Mental Health,
National Institutes of Health,
Bldg. 10, Rm. B3 C338,
Bethesda, MD 20892
Date of review: 12/01/05
PBR 28 Reference Acceptance Form
HPLC
Record weight of reference compound _____ mg in 10 ml of 20/80 USP grade ethanol/ saline.
Column:
Eluent:
Flow rate:
UV Detector:
Onyx (monolithic C18; 4.6 mm x 100 mm; Phenomenex; part # CH0 7643)
Isocratic: 0.01 M ammonium formate in water-acetonitrile (70: 30 v/v)
3 mL/min
220 nm
Record Serial # of this column: __________
Criterion
Acceptance? (circle
Date
one)
Retention time 6.683 min
Y or N
Peak area ≥ 98%
Y or N
LC/MS
Criterion (expected
Observed ion, M + 1
Acceptance (circle one)?
m/z, M + 1)
349.15
Y or N
Attached documents, acceptance criteria and test results:
1.
1H-NMR
2.
LC-MS data from this laboratory?
YES_________ NO__________.
3.
HPLC chromatogram from this laboratory?
YES_________ NO__________.
spectrum from this laboratory attached?
YES_______NO__________.
(An example of a set of satisfactory attached documents is included in Document 8).
Document 8. [11C]PBR 28 for Injection: Precursor and Reference Compound − Acceptance Testing and Form
Page 8 of 15
PBR 28 FOR INJECTION: PRECURSOR AND REFERENCE
COMPOUND − ACCEPTANCE TESTING AND FORMS
PET Radiopharmaceutical Sciences Section,
Molecular Imaging Branch,
National Institute of Mental Health,
National Institutes of Health,
Bldg. 10, Rm. B3 C338,
Bethesda, MD 20892
Date of review: 12/01/05
PBR 28 Reference Acceptance Form
Chemical structure:
Acceptance criteria:
1. Identity: confirmed by 1H-NMR
O
2. Purity: ≥ 98 % by HPLC
N
3. Identity: confirmed by LC/MS
N
O
O
Test Results
Test
Results
Procedure
1. NMR ( This laboratory)
SOP # QA308(3.1)
2. LC-MS (This laboratory)
SOP # QA308(3.3)
3. HPLC (This laboratory)
SOP # QA308(3.2)
Checked by:
Accepted by:
Storage condition:
Date
Date
Initials
Chemist_____________________________Initials_________________Date________________
Document 8. [11C]PBR 28 for Injection: Precursor and Reference Compound − Acceptance Testing and Form
Page 9 of 15