Synthesis of a 13C labeled N-cyclopropylamine tetrahydropyridine derivative

Article abstract of DOI:10.1002/jlcr.605

The synthesis of 1-(2-¹³C)-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine (8) is reported. Attempts were first made to prepare labeled cyclopropylamine via a cyclopropanation/Curtius rearrangement sequence, but the yields were too modest to be suitable for the synthesis of a labeled compound. The preparation of 8 was achieved via cyclopropanation of the N-formyl tetrahydropyridine derivative 21 using the Grignard reagent of ethyl bromide and Ti(O-iPr)₄ as a catalyst. The synthesis proceeded in high yield (82%). The method has a wide potential for the synthesis of other cyclopropyl ring labeled and substituted cyclopropyl ring labeled tetrahydropyridine dervatives which can be used in Monoamine Oxidase (MAO) and Cyt P₄₅₀ enzymes mechanistic studies. Copyright

Full text of DOI:10.1002/jlcr.605

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 813–822.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.605
Research Article
13
Synthesis of a C labeled
N-cyclopropylamine tetrahydropyridine
derivative
Simon Kuttab*,1,2 and Stephan Mabic2
1 Department of Chemistry, Birzeit University, Birzeit, West Bank, Via Israel
2 Department of Chemistry, Peters Center, Virginia Tech, Blacksburg,
VA 24012–0212, USA
Summary
The synthesis of 1-(2-13C)-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine (8)
is reported. Attempts were first made to prepare labeled cyclopropylamine via
a cyclopropanation/Curtius rearrangement sequence, but the yields were too
modest to be suitable for the synthesis of a labeled compound. The preparation
of 8 was achieved via cyclopropanation of the N-formyl tetrahydropyridine
derivative 21 using the Grignard reagent of ethyl bromide and Ti(O-iPr)4 as a
catalyst. The synthesis proceeded in high yield (82%). The method has a wide
potential for the synthesis of other cyclopropyl ring labeled and substituted
cyclopropyl ring labeled tetrahydropyridine dervatives which can be used
in Monoamine Oxidase (MAO) and Cyt P450 enzymes mechanistic studies.
Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: N-formyl tetrahydropyridine; N-cyclopropyltetrahydropyridine;
13C-label; 1-(2-13C)-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine; monoa-
mine oxidase
*Correspondence to: Dr. S. Kuttab, P.O. Box 19684, Jerusalem, via Israel 91196. E-mail:
skuttab@birzeit.edu.
Contract/grant sponsor: National Institute of Neurological Disorders and stroke (NS 28792),
Contract/grant sponsor: Harvey W. Peters Research Center
Copyright # 2002 John Wiley & Sons, Ltd.
Received 19 February 2002
Revised 22 March 2002
Accepted 4 April 2002

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S. KUTTAB AND S. MABIC
Introduction
In our ongoing efforts to investigate the enzymatic mechanism and
inactivation of the flavin (FAD) containing enzymes monoamine
oxidases A and B (MAO-A and MAO-B)1–4, we have focused on the
use of 1,4-disubstituted-1, 2, 3, 6-tetrahydropyridines as useful probes.5–9
Two principal pathways have been proposed to account for the MAO-B
catalytic pathway. The first involves an a-hydrogen atom transfer
(HAT) in the initial step to directly generate 3 from 1 which
subsequently goes to 4.10–12 The alternative pathway involves an initial
single electron transfer (SET) leading to the aminyl radical cation 2
followed by deprotonation and a second electron transfer to yield 4
(Scheme 1).13–16
Scheme 1. SET and HAT pathways proposed for the MAO-B catalysed
Oxidation of Amines
Studies with various cyclopropylamines have been offered as evidence
in support of the SET pathway for the inactivation of both the
Cytochrome P-450 family of enzymes17–21 and the flavoenzymes
Monoamine Oxidases (MAO) A and B.22,23
The inactivating properties of 1-cyclopropyl-4-phenyl-1,2,3,6 tetra-
hydropyridine 5 and other analogs6,8,9,24 are consistent with the SET
pathway. The inactivation may be considered to proceed via the
cyclopropylaminyl radical cation 6 and the subsequent bioalkylation of
the enzyme active site by the ring opened carbon centered radical 7
(Scheme 2), leading to loss of catalytic activity.
As part of our mechanistic studies, we have initiated efforts to
synthesize various labeled cyclopropyl tetrahydropyridrine derivatives
to enable us to characterize the inactivation pathway of MAO-B using
various techniques and model reactions. To this aim, the synthesis of the
13C labeled analog 8 was initially targeted with the labeling at the C2
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 813–822

SYNTHESIS OF A 13C LABELED
815
Scheme 2. SET pathway proposed for the inactivation of MAO-B by 5
carbon of the cyclopropyl ring in an effort to characterize the
inactivation pathway of MAO-B by 8 using NMR spectroscopy.
Results and discussion
Our initial synthetic approach, described by the retrosynthetic path
shown in Scheme 3, was to proceed via reaction of labeled
cyclopropylamine 9 with the Zincke salt 1016,24 to produce the
N-cyclopropylpyridinium intermediate 11. Reduction of 11 with NaBH4
in MeOH25,26 yields 8.16,27,28
Scheme 3. Retrosynthesis to 8
A Simon–Smith type cyclopropanation of allylic alcohol 12 with
labeled diiodomethane 13, and a Curtius rearrangement of the acid 14
were the key steps of the synthesis of labeled cyclopropylamine 9. The
Curtius rearrangement first was studied in detail (Table 1). Because of
the low boiling point (508C) of 9, the t-Boc derivative 15 was prepared
first (Scheme 4). The carbamate 15 was then efficiently (>98% yield)
deprotected using trifluoroacetic acid (TFA), and cyclopropylamine was
isolated as its non volatile TFA salt.29
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S. KUTTAB AND S. MABIC
Table 1 Curtius rearrangement of Cylopropanecarboxylic acid 14
Entry
Conditions
DPPA, Net3, t-BuOH, D, 5 h
DPPA, Net3, t-BuOH, CuCl, D, 3h
DPPA, NET3, t-BuOH, Toluene, D, 6 h
1- DPPA, NET3, Toluene, D, 2-t-BuOH, D, 5 h
1-NEt3, ClO2Et, H2O/Acetone, 08C; 2-NaN3; 3-t-BuOH, D, 14 h
1-SOCl2, D, 1 h; 2-NaN3, H2O/Acetone, 08C; 3-t-BuOH, D, 8 h
Yield (%)
1
2
3
4
5
6
73
5
81
47
22
7
Scheme 4. Synthesis of Cyclopropylamine 9 from Cylopropane- carboxylic
acid 14
The in situ formation of the isocyanate intermediate 16 using
diphenylphosphoryl azide (DPPA), and reaction with t-BuOH gave
the best yields (Table 1. Entries 1 and 3). 1,3-Dicyclopropylurea was
obtained as a side product when the reaction was not performed under
rigorously anhydrous conditions.29,30 A slightly higher yield was
obtained using toluene as a solvent (Entry 3). The higher temperature
reached in toluene might favor the rearrangement. The use of CuCl
(Entry 2) as well as running the two steps of the reaction sequentially
(Entry 4)31 results in a dramatic decrease in yield. Other methods
(Entries 532,33, and 634) gave much poorer yields presumably due to the
low boiling point of the intermediates.
The preparation of cyclopropanecarboxylic acid 14 was examined
next. Cyclopropanation of the TBDMS derivative 17 of allylic alcohol
12 is reported to proceed efficiently with ClCH2I to yield the
silyloxymethylcyclopropane 18, (Scheme 5).35,36 We examined the
deprotection/oxidation sequence from 18 to the carboxylic acid 14. As
expected, the silyl group could be easily removed using TBAF (91%
yield). The intermediate alcohol 19 was oxidized to 14 with RuO2 and
NaIO4 in 48% yield32,35 and with RuCl3 and NaIO4 in 62% yield.37 It
occurred to us, however, that the conditions used for the oxidation
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF A 13C LABELED
817
Scheme 5. Cyclopropanation of 17
should also allow for the deprotection of the silyloxy group to the
alcohol. The compound 18 indeed was converted in one step and 78%
yield to 14 using RuO2 and NaIO4. The efficient cyclopropanation
reagent ClCH2I is not commercially available as a labeled compound,
but 13CH2I2 can be purchased. Catalysis with ZnEt2 was shown to be an
efficient method to prepare a variety of cyclopropyl derivatives.38–42
However, using one equivalent of CH2I2 only (instead of the usual 5–10
equivalents relative to the alkene) and an excess of alkene 17 led to
modest yields of 18 and therefore this pathway was considered
unsuitable for the synthesis of labeled 9.
An alternative synthetic approach was considered based on a recent
literature report describing the synthesis of N-cyclopropyl dervatives
from amides and Grignard reagents with a Ti(O-i-Pr)4 catalyst.43 The
synthetic scheme (Scheme 6) is reduced to two steps from the
commercially available tetrahydropyridine 20, and offers the advantage
of being able to efficiently introduce the label in the last step.
Scheme 6. Synthesis of 8 from the Tetrahydropyridine 20
Various reagents were used to prepare the key intermediate 1-formyl-
4-phenyl-1,2,3,6-tetrahydropyridine 21: ZrO2, DMF (47% yield),44
silicic acid, DMF (18%)44 (The major product of this reaction was
the 1-methyl-4-plenyl-1,2,3,6-tetrahydropyridine(52%)), carboxydiimi-
dazole, formic acid (72% yield),45 and ethyl formate in CH3CN (93%
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818
S. KUTTAB AND S. MABIC
yield). The last method proved to be the simplest and the most efficient
1
in our hands. Two H NMR signals, in a ratio 2:1 are observed for the
formyl hydrogen of 21. The major isomer exhibited a higher chemical
shift (4.19 vs 4.07 for the minor rotamer) for the signals of hydrogen
atoms at the C6 position, and a lower chemical shift (3.63 vs 3.79 for the
minor rotamer) for the signals of the hydrogen atoms at the C2 position.
This trend is consistent with an arrangement of the carbonyl group syn
to the C6 position. The upfield chemical shifts are due to the deshielding
effect of the carbonyl group.46 The 13C labeled 1-cyclopropyl-4-phenyl-
1,2,3,6-tertrahydropyridine (8) was obtained in 82% yield by reaction of
21 with the ethyl Grignard reagent prepared from the labeled ethyl
bromide, using Ti(O-i-Pr)4 as a catalyst. The tetrahydropyridine 8 was
isolated as its stable oxalate salt. The synthesis of 21 and its subsequent
relatively facile conversion to the cyclopropyl ring substituted tetra-
hydropyridine dervative as described above broadens the range of
synthetically accessible cyclopropyl ring labeled and substituted
cyclopropyl ring labeled tetrahydropyridine derivatives at more than
one position which can be used in Monoamine Oxidase (MAO) or
Cyt P450 enzyme mechanistic studies. Efforts in this direction are
currently underway.
Experimental
General procedures can be found in Reference 16. The spectroscopic
data of the following compounds were consistent with literature reports:
88, 1147, 1548, 1749, and 1850. The 13C labeled ethyl bromide (99%
enrichment) was purchased from Cambridge Isotope Laboratories.
Tert-butoxycarbonyl aminocyclopropane 15
The acid 14 (860 mg, 10 mmol) was dissolved in anhydrous t-BuOH and
anhydrous toluene (20 ml). The NEt3 (2 ml, 10.5 mmol) and diphenyl-
phosphoryl azide (2.5 ml, 11.5 mmol) were added and the reaction
mixture was heated up to reflux for 6 h. After cooling, the solvents were
evaporated under reduced pressure and the crude residue was taken up
in CHCl3 (50 ml), washed with a solution of citric acid (10%, 40 ml),
a solution of NaOH (2%, 25 ml), and water. After drying over MgSO4
and evaporation of the solvents under reduced pressure, the crude
product was purified by Al2O3 column chromatography (CHCl3) to
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF A 13C LABELED
819
give 15 as a white solid (1.25 g, 8.1 mmol, 81% yield), which was further
purified by recrystallization from Hex/Et2O at ꢀ208C. 1H NMR
(CDCl3, 360 MHz) d 4.7 (1H, bs) 2.53 (1H, m), 1.44 (9H, s), 0.67
(2H, m); 13C NMR (CDCl3, 90 MHz) d 79.4, 28.4, 22.9, 6.7; EIMS m/z
(rel intensities %) 157 (M+2), 142(2), 101(17), 57(100), 41 (62). Anal.
Calcd for C8H15NO2; C, 61.12; H, 9.62; N, 8.91. Found: C, 61.21;
H, 9.58; N, 8.97.
1-Formyl-4-phenyl-1,2,3,6-tetrahydropyridine 21
The freshly prepared free base 20 (2.54 g, 16 mmol) was dissolved in dry
CH3CN in a 50 ml round bottomed flask fitted with a reflux condenser.
Ethyl formate (1.6 g, 21.8 mmol) was added and the reaction mixture
was heated under nitrogen at 65–708C for 48 h. The solvent was
removed in vacuo and the thick, yellowish oily residue was dissolved in
CH2Cl2 (30 ml). This solution was washed with dilute HCl (10%,
3 ꢁ 20 ml), dried over Na2SO4 and the solvent removed in vacuo to yield
a yellowish solid, which was purified by SiO2 column chromatography
(CH2Cl2/MeOH 95:5) to give 21 as pale yellow crystals (2.6 g, 14 mmol,
1
88% yield), mp: 77–788C. H NMR (CDCl3, 360 MHz, mixture of the
two rotamers) d 8.21 (1H, s), 8.15 (1H, s, ratio 1:2), 7.34 (5H, m),
6.05 (1H, m), 4.2 (2H, m), 4.06 (2H, m, ratio 2:1), 3.79 (2H,
t, j ¼ 6:2 Hz), 3.63 (2H, t, j ¼ 6:2 Hz, ratio 1:2), 2.62 (2H, m),
1
2.57(2H, m, ratio 2:1); H NMR (DMSO-d6, 360 MHz, mixture of the
two rotamers, ratio 1:2), d 8.17 (1H, s), 8.12 (1H, s, ratio 1:2)), 7.30
(5H, m), 6.19 (1H, m), 6.14 (1H, m, ratio 1:2) 4.08 (2H, m), 4.04 (2H, m,
ratio 1:2), 3.62 (2H, m) 2.52 (2H, m); 13C NMR (DMSO-d6, 90 MHz,
mixture of the two rotamers)d 161.4, 160.9, 139.8, 134.8, 134.6, 128.4,
127.6, 124.7, 120.6, 119.9, 44.2, 41.7, 36.1, 27.2, 25.9; GC-EIMS m/z
(rel intensities %) 187 (M+100), 158 (20), 143 (17), 130(24), 115(30),
91(26); UV (nm, MeOH) 208, 245. Anal. Calcd for C12H13NO: C, 76.96;
H, 7.00; N, 7.48. Found: C, 76.89; H, 7.08; N, 7.55.
1-[2-13C]-Cyclopropyl-4-phenyl-1,2,3, 6-tertrahydropyridine 8
Ethyl bromide 2-13C (1.0 g, 9.17 mmol) was added slowly via a dropping
funnel to magnesium turnings in dry THF under nitrogen. The reaction
was heated under reflux for 3 h. Into a flame dried three-necked round
bottomed flask fitted with a reflux condenser, a dropping funnel,
a septum, and a magnetic stirrer, was added a solution of 21(0.68 g,
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S. KUTTAB AND S. MABIC
3.67 mmol) in dry THF. Titanium isopropoxide (1.14 g, 4.03 mmol) was
added via the dropping funnel and the resulting ethyl magnesium
bromide was immediately transferred via a syringe. The reaction
mixture, which slowly turned black, was warmed (oil bath 608C) and left
to stir overnight (18 h). The reaction was cooled to 258C and a saturated
aqueous solution of ammonium chloride was added with stirring.
A precipitate of titanium hydroxide formed. The reaction mixture was
filtered through a sintered glass funnel and the filtrate extracted with
ether (3 ꢁ 20 ml). The ether layer was washed with brine (2 ꢁ 20 ml),
dried over Na2SO4 and evaporated to yield a yellowish solid. After
column chromatography on Al2O3 (Hex/AcOEt 97:3) the desired
product 8 was obtained as a white crystalline solid (82%): mp
1
63–648C; H NMR (CDCl3) d 7.30 (5H, m), 6.05 (1H, m), 3.31 (2H,
m), 2.87 (2H, m), 2.55 (2H, bs), 1.73 (1H, m), 0.74 (1H, m), 0.49 (1H,
m), 0.29 (1H, m); 13C NMR (CDCl3) d 128, 126, 124, 122.53, 51.37, 29,
22, 20, 5.5; EIMS (m/z, rel int) 200 (71), 185 (100), 184 (76), 156 (15),
128(45), 115(61), 91(22), 69(36). The free base was then converted to the
oxalate salt and recrystallized from MeOH/Et2O(mp 175–1768C).
Acknowledgements
This work was supported by the National Institute of Neurological
Disorders and Stroke (NS 28792) and the Harvey W. Peters Research
Center.
References
1. Dosert P, Strolin-Benedetti M, Tipton KF. Med Res Rev 1989; 9: 45–89.
2. Langston JW, Ballard P, Tetrud JW, Irwin I. Science 1983; 219: 979.
3. Langston JW, Irwin I, Langston EB, Frono LS. Neurosci Lett 1984; 48: 87.
4. Walker MC, Edmonson DE. Biochemistry 1994; 33: 7088.
5. Kalkutgar AS, Castagnoli K, Hall A, Castagnoli Jr N. J Med Chem 1994;
37: 944–951.
6. Kuttab S, Kalkutgar A, Castagnoli Jr N. Chem Res Toxicol 1994; 6:
740–744.
7. Franot C, Mabic S, Castagnoli Jr N. Bioorg Med Chem 1998; 6: 283–291.
8. Hall L, Murray S, Castagnoli K, Castognoli Jr N. Chem Res Toxicol 1992;
5: 625–633.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 813–822

SYNTHESIS OF A 13C LABELED
821
9. Rimoldi JM, Wang YX, Nimkar SK, et al. Chem Res Toxicol 1995; 8:
705–710.
10. Miller RJ, Edmonson DE, Grisson CB. J Am Chem Soc 1995; 117:
7830–7831.
11. Silverman RB, Zelechonok Y. J Org Chem 1992; 35: 6379–6384.
12. Silverman RB. In Advances in Electron Transfer Chemistry, vol. 2,
Mariano PS (ed.). JAI Press: Greenwich, 1992; 177–213.
13. Silverman RB. Accounts Chem Res 1995; 28: 335–342.
14. Silverman RB, Yamasaki RB. Biochemistry 1984; 23: 1322–1331.
15. Silverman RB, Zhow JP, Eaton PE. J Am Chem Soc 1993; 115: 8841–8842.
16. Nimkar SK, Anderson A, Rimoldi JM, et al. Chem Res Toxicol 1996; 9:
1013–1022.
17. Bondon A, Macdonald TL, Harris TM, Guengerich FP. J Biol Chem 1989;
264: 1988–1997.
18. Macdonald TL, Zirvi K, Burka LT, Peyman P, Guengerich FP. J Am
Chem Soc 1982; 104: 2050–2052.
19. Guengerich FP, Willard RJ, Shea JP, Richard LE, Macdonald TL. J Am
Chem Soc 1984; 106: 6446–6447.
20. Guengerich FP, Macdonald TL. In Advances in Electron-Transfer
Chemistry, vol 3, Mariano PS (ed.), JAI Press: Greenwich, Connecticut,
1993; 191–241.
21. Hanzlik RP, Tullman RH. J Am Chem Soc 1982; 104: 2048–2050.
22. Vasquez ML, Silverman RB. Biochemistry 1985; 24: 6538–6543.
23. Mitchell DJ, Nikolic D, Rivera E, et al. Biochemistry 2001; 40: 5447–5456.
24. Zincke T. Chem Ber 1904; 37: 361–375.
25. Lyle RF, Nelson DA, Anderson PS. Tetrahedron Lett 1962; 4: 553–556.
26. Anderson PS, Lyle RE. Tetrahedron Lett 1964; 6: 153–156.
27. Wang XY, Castagnoli Jr N. J Med Chem 1995; 38: 1904–1910.
28. Nimkar SK, Mabic S, Anderson AM, et al. J Med Chem 1999; 42:
1828–1835.
29. Kimura Y, Atarashi S, Kawakami K, Sato K, Hayakawa I. J Med Chem
1994; 37: 3344–3352.
30. Thornton TJ, Jarman MJ. Synthesis 1990; 295–299.
31. Vallgarda J, Appelberg U, Ardvisson IE, et al. J Med Chem 1996; 39:
1485–1493.
32. Zhao Y, Yang T, Lee M, Lee D, Newton MG, Chu CK. J Org Chem 1995;
60: 5236–5242.
33. Vangveravong S, Nichols DE. J Org Chem 1995; 60: 3409–3413.
34. Rewcastle GW, Denny WA. Synthesis 1985; 220–223.
35. Zhao Y, Yang T, Lee M, et al. Tetrahedron Lett 1994; 35: 5405–5408.
36. Denmark SE, Edwards JP. J Org Chem 1991; 56: 6974–6981.
37. Wipf P, Xu W. J Org Chem 1996; 61: 6556–6562.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 813–822

822
S. KUTTAB AND S. MABIC
38. Furukawa J, Kawabata N, Nishimura J. Tetrahedron 1968; 24: 53–58.
39. Furukawa J, Kawabata N, Nishimura J. Tetrahedron 1968; 31: 3495–3498.
40. Morikawa T, Sasaki M, Hanai R, Shibuya A, Taguchi T. J Org Chem
1994; 59: 97–103.
41. Charette AB, Prescott S, Brochu CJ. J Org Chem 1995; 60: 1081–1083.
42. Denmark SE, O’Connor SP. J Org Chem 1997; 62: 584–594.
43. Chaplinski V, de Meijere A. Angew Chem Int Ed Engl 1996; 35: 413–414.
44. Takahashi K, Slibagaki M, Matsushita H. Agric Biol Chem 1988; 52:
853–854.
45. Katigawa T, Aria J, Nagahata A. Chem Pharm Bull 1994; 42: 1655–1657.
46. Rubiralta M, Giralt E, Diez A. In Piperidine: Studies in Organic Chemistry,
vol. 43, Elsevier: Amsterdam, Oxford, New York, Tokyo, 1991.
47. Genisson Y, Marazano C, Mehmandoust M, Gnecco D, Das BC. Synlett
1992; 431–434.
48. Bridges AJ. J Heterocycl Chem 1990; 27: 1527–1536.
49. Ali AS, Khan JM, Wazeer MIM. Tetrahedron 1988; 44: 5911–5920.
50. Nonhebel DC, Suckling CJ, Walton JC. Tetrahedron Lett 1982; 22:
4477–4480.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 813–822