Synthesis of deuterated 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP) and its N-hydroxy derivative

Article abstract of DOI:10.1002/jlcr.468

The syntheses of the deuterium-labeled food mutagen 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP) and its N-hydroxy metabolite are described. Unlabeled PhIP is deuterated using a boron trifluoride phosphoric acid complex in one step. Labeled

Full text of DOI:10.1002/jlcr.468

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 405–411.
DOI: 10.1002/jlcr.468
Synthesis of deuterated 2-amino-1-methyl-6-
phenyl-1H-imidazo[4,5-b]pyridine (PhIP)
and its N-hydroxy derivative
Mary J. Tanga*, James E. Bupp and Wallace W. Bradford
Pharmaceutical Discovery Division, SRI International, 333 Ravenswood Avenue,
Menlo Park, CA 94025, USA
Summary
The syntheses of the deuterium-labeled food mutagen 2-amino-1-methyl-6-
phenyl-1H-imidazo[4,5-b]pyridine (PhIP) and its N-hydroxy metabolite are
described. Unlabeled PhIP is deuterated using a boron trifluoride phosphoric
acid complex in one step. Labeled PhIP–2H5 is nitrosated to give nitro-PhIP,
which is then reduced to N-hydroxy–PhIP–2H5. Copyright # 2001 John
Wiley & Sons, Ltd.
Key Words: heterocyclic amines; food mutagens; PhIP; nitro-PhIP;
N-hydroxy–PhIP; boron trifluoride phosphoric–2H3 acid complex
Introduction
Heterocyclic amines are mutagenic and carcinogenic compounds
formed during the cooking of beef, pork, lamb, poultry, and fish at
high temperature. The consumption of well-done meats, and thus
exposure to heterocyclic amines formed during cooking, may play a
significant role in the risk of breast cancer, colon cancer, and other
cancers in humans.1–4 Among the known food mutagens, 2-amino-1-
methyl-6-phenyl-1H-imidazo[4,5-B]pyridine (PhIP) is considered very
important because of its high content in various foods.1,4–7 Exposure to
PhIP is widespread; PhIP has been found in home-cooked and
*Correspondence to: M. J. Tanga, Pharmaceutical Discovery Division, SRI International, 333
Ravenswood Avenue, Menlo Park, CA 94025, USA. E-mail: mary.tanga@sri.com
Contract/grant sponsor: National Cancer Institute; contract/grant number: N02-CB-67257.
Copyright # 2001 John Wiley & Sons, Ltd.
Received 17 March 2000
Revised 25 January 2001
Accepted 26 January 2001
Published online \ \ \ \

406
M. J. TANGA ET AL.
Figure 1. Structure of guanosine–C8–PhIP nucleoside adduct
restaurant foods at levels up to hundreds of parts per billion. It has
recently been identified in human urine2 and as DNA and protein
adducts in the colon and blood.1 Metabolic activation of PhIP leads to
N-hydroxy–PhIP, which can attack DNA to form adducts (Figure 1).8,9
An accurate assessment of the risk posed by consumption of hetero-
cyclic amines formed during cooking needs to be determined. Thus, it is
essential to have analytically pure standards and labeled analogues of
PhIP to assess the risk it poses to humans. A versatile synthesis of PhIP
and its N-hydroxy metabolite labeled with deuterium is presented.
Results and discussion
Convenient syntheses of deuterium-labeled PhIP (2) and its metabolite,
N-hydroxy–PhIP (4) have been developed (Scheme 1). Unlabeled PhIP
(1) is deuterated using a boron trifluoride phosphoric–2H3 acid complex.
The reagent, BF3 ꢀ 2H3PO4, is made in quantitative yield at 08C from
deuterium oxide and phosphorus pentoxide, into which is bubbled
boron trifluoride gas.10 The reagent is then added to PhIP (1) in
anhydrous liquid SO2 cooled to ꢁ788C. The reaction mixture is heated
in a sealed flask at 908C for 4 days. Subsequent workup and purification
of the reaction mixture give a 40% yield of labeled PhIP–2H5 (2). Mass
spectroscopy showed the deuterium incorporation was 96.6% 2H
isotopically pure material labeled exclusively in the phenyl group.
2
2
2
Product 2 contains no H0, H1, or H2 species by mass spectroscopy.
NMR studies confirmed this result showing no phenyl ring protons.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 405–411

SYNTHESIS OF DEUTERATED PhIP
407
Scheme 1
The synthesis of deuterated 4 is achieved as follows: Labeled
PhIP–2H5 (2) in HOAc and H2O is treated with an aqueous solution
of sodium nitrite to give 2-nitro–PhIP–2H5 (3). This intermediate is
reduced to the N-hydroxy derivative 4 with hydrazine in the presence of
Pd/C catalyst. Compound 4 has shown good stability when stored as a
solid at ꢁ808C underArbut it is extermely sensitive to oxygen,
especially when dissolved in organic solvents. All solvents used must be
thoroughly degassed with argon or nitrogen before coming in contact
with N-hydroxy–PhIP–2H5 (4). Dilute solutions (0.0001 M) in degassed
methanol have been observed to slowly turn pink due to the formation
of the azoxy derivative (Figure 2). The reactivity of N-hydroxy–
PhIP–2H5 (4) is consistent with the fact that PhIP (1) is metabolically
activated to form N-hydroxy–PhIP, which on solvolysis gives a
nitrenium ion that is attacked by DNA (Figure 2).11
Experimental
Infrared spectra were recorded on a Perkin Elmer 1600 FTIR
spectrophotomer. Ultraviolet spectra were recorded on a Varian
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 405–411

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M. J. TANGA ET AL.
Figure 2. Proposed active-2-nitrenium ion and azoxy derivative
DMS-90. NMR spectra were recorded on a Varian Gemini 300
spectrophotometer using tetramethylsilane as the internal standard.
The following abbreviations are used in reporting the NMR multi-
plicities: apt, apparent; s, singlet; d, doublet; t, triplet, q, quartet; m,
multiplet; and br, broad. The mass spectra were obtained on a
Ribermag R 10-10 GC/MS. Column chromatography was performed
using E. Merck silica gel 40 (70–230 mesh ASTM). All solvents were
dried over molecular sieves.
Caution should be taken when manipulating these corrosive liquids.
The reactions should be done in a well-ventilated hood with proper
shielding.
Boron trifluoride phosphoric–2H3 acid complex
Under Ar in a dry 100 ml round-bottomed flask, a sample of 25.0 g
(1.25 mol) of deuterium oxide (99.9 at % D) at 08C was slowly treated
with phosphorus pentoxide 58.0 g, 0.409 mol) over 30 min, using a
Teflon1-screw solid feeder. The mixture was stirred overnight at RT.
To the reaction solution, a stream trifluoride gas was introduced
below the surface at 08C slowly over 1 h. The resulting product
was 138 g (100%) of BF3 ꢀ 2H3PO4.10
2-amino-1-methyl-6-phenyl-(2,3,4,5,6-[2H]5)-1H-imidazo-
[4,5-b]pyridine-d5[PhIP-2H5(2)]
Under Ar in a dry Pyrex pressure reaction flask, a sample of 2-amino-1-
methyl-6-phenyl-1H-imidazo[4,5-b]pyridine hydrochloride [PhIP (1)]
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 405–411

SYNTHESIS OF DEUTERATED PhIP
409
(440 mg, 1.69 mmol) 1 was cooled to ꢁ788C and liquid anhydrous SO2
(16 ml) added. The resulting solution was treated with BF3 ꢀ 2H3PO4
(20 ml, 40.0 g, 237 mmol) and the flask sealed with a Teflon1 stopper.
The reaction mixture D/H ratio was 60/1. The solution was heated at
908C for4 days. Aftercooling to ꢁ788C, the ampoule was opened and
the SO2 removed with a stream of Ar at RT. The mixture was cooled
and treated with D2O (7 ml) and then poured over ice and treated
with 20% NaOH to pH 10. The mixture was extracted with CHCl3 and
evaporated to give 182 mg of crude material, which was triturated
with Et2O and then MeOH to give 155 mg (40%) of pure product 2.
The deuterium incorporation in the phenyl group was found to be
128.14, 137.51, 137.76, 152.20, 157.06; MS m=z (rel. intensity) 230
(M+H, d5, 100), 229 (d4, 17), 228 (d3, 1); FTIR (KBr) 3340, 3163, 1636,
1545, 1472, 1433, 1383, 1274, 1095 cmꢁ1; UV (CH3OH)lmax 222 nm
(e 21,735), 269 (8,598), 315 (16,779), 373 (3,821); TLC System 1:
(Bakerflex silica gel IB2-F plates) (CHCl3/CH3OH/Et3N, 89.9/10/0.1),
Rf ¼ 0:35; TLC System 2: (Bakerflex silica gel IB2-F plates) (CH2Cl2/
CH3OH/Et3N, 79.9/20/0.1), Rf ¼ 0:45; HPLC System 1: Brownlee
Spheri-5 RP 8 Column, flow rate 1.00 ml/min, detection 320 nm,
solvent CH3OH/0.5% Et3N in H2O (60/40), retention time 2.60 min,
purity 99%; HPLC System 2: Brownlee Spheri-5 Phenyl Column,
flow rate 1.00 ml/min, detection 320 nm, solvent CH3OH/0.5%
Et3N in H2O (50/50), retention time 2.81 min, purity 99%; HRMS
(DCI, NH3 in CH4) calculated forC 13H8D5N4 [M+H]: 230.1449;
Found: 230.1472.
1-methy-2-nitro-6-phenyl-(2,3,4,5,6-[2H]5)-1H-imidazo[4,5-b]
pyridine [2-nitro-PhIP-2H5 (3)]
To a solution of PhIP-2H5 2 (90.0 mg, 0.393 mmol) in HOAc/H2O (50/
50) (4 ml) underArat 0 8C was added a solution of NaNO2 (1.20 g,
17.4 mmol) in H2O (2 ml) slowly over a 6 min period with intermittent
stirring. A precipitate formed, and the resulting mixture was allowed to
stand at 08C for15 min and then at RT for1 h. The mixture was cooled
to 08C, filtered, rinsed with cold H2O (2 ꢂ 1 ml), and dried under high
vacuum to give 53 mg of crude material, which was chromatographed
on Et3N deactivated silica gel (12 g) equilibrated with CH3OH/CH2Cl2
(1/100) (40 ml) and eluted with the same solvent. The first 12 ml of
solvent was eluted, and then the next 18 ml was collected and
evaporated to give 27.5 mg (28%) of product 3.
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M. J. TANGA ET AL.
2-hydroxylamino-1-methyl-6-phenyl-(2,3,4,5,6-[2H]5)-1H-imidazo
[4,5-b]pyridine (N-hydroxy-PhIP-2H5) (4)
A sample of 10% Pd/C (27.5 mg) was placed in a 100 ml round bottom
flask and the flask was evacuated and treated with Ar repeatedly to
remove O2. To the flask was added CH3OH (10 ml) degassed by purging
with Ar, and the mixture was purged with Ar for 5 min. After cooling
the mixture with an ice bath, 64% hydrazine hydrate (104 ml) was added
2
and the mixture stirred at 08C for30 min. A solution of nitro-PhIP- H5
3 (27.5 mg, 0.108 mmol) in degassed CH3OH (20 ml) was added over
2 min. The mixture was stirred for 10 min CH3OH/H2O (80/20)
(3 ꢂ 1 ml) and evaporated to give 5.5 mg (21%) of pure product 4. MS
m=z (rel. intensity) (DCI) 246 (M+H, 55), 230 (100), 215 (47). The mass
spectrum indicates that the compound contains 95.6% 2H in the phenyl
ring. UV (CH3OH) lmax 230 nm (e 20,495), 318 (13,627): HPLC:
Brownlee Spheri-5 RP-18 Column, flow rate 1.50 ml/min, detection
240 nm, solvent CH3OH/2% ammonium acetate buffer, pH 5.85 (45/55),
retention time 5.38 min, purity 97%; HRMS (DCI, NH3 in CH4)
calculated forC 13H6D5N4O; 245.1320; found: 245.1466.
Conclusions
This method allows convenient syntheses of deuterium-labeled PhIP
and its metabolite N-hydroxy–PhIP. Labeled PhIP–2H5 and N-
hydroxy–PhIP–2H5 are available as reference materials from
Dr Harold Seifried, National Cancer Institute, 6130 Executive
Boulevard, Room 212F, Bethesda, MD 20892.
Acknowledgements
This work was supported by the National Cancer Institute under
contract number N02-CB-67257.
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J Labelled Cpd Radiopharm 2001; 44: 405–411