Mass spectrometric studies on 4-aryl-1-cyclopropyl-1,2-dihydropyridinyl derivatives: An examination of a novel fragmentation pathway

Article abstract of DOI:10.1002/jms.1135

Examination of the electrospray ionization product ion spectra of 1,2-dihydropyridinyl and 4-aryl-1,2-dihydropyridinyl derivatives bearing a 1-cyclopropyl or 1-trans-2-phenylcyclopropyl group has led to the characterization of unexpected fragment ions. For example, the base peak at m/z 156 present in the product ion spectrum of trans-1-(2-phenylcyclopropyl)-4- phenyl-1,2-dihydropyridine proved not to be the expected 4-phenylpyridinium species but rather the isomeric 3-phenyl-5-azoniafulvenyl species. The results of studies with a series of structural and isotopically labeled analogs require a novel fragmentation pathway to account for the formation of this and related fragment ions. One possible pathway is based on an initial 1,5-sigmatropic shift of a cyclopropylmethylene hydrogen atom that is accompanied by opening of the cyclopropyl ring. The resulting eniminium intermediates then fragment to yield the 5-azoniafulvenyl species. Copyright

Full text of DOI:10.1002/jms.1135

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 2006; 41: 1643–1653
Published online in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/jms.1135
Mass spectrometric studies on
4-aryl-1-cyclopropyl-1,2-dihydropyridinyl derivatives:
an examination of a novel fragmentation pathway
Philippe Bissel,1 Skye Geherin,1 Kazuo Igarashi,1 Richard D. Gandour,1 Iulia M. Lazar2 and
Neal Castagnoli Jr1∗
1
Department of Chemistry, Virginia Tech, Blacksburg, VA 24061-0212, USA
Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24061-0212, USA
2
Received 14 July 2006; Accepted 6 October 2006
Examination of the electrospray ionization product ion spectra of 1,2-dihydropyridinyl and 4-aryl-1,2-
dihydropyridinyl derivatives bearing a 1-cyclopropyl or 1-trans-2-phenylcyclopropyl group has led to
the characterization of unexpected fragment ions. For example, the base peak at m/z 156 present in the
product ion spectrum of trans-1-(2-phenylcyclopropyl)-4-phenyl-1,2-dihydropyridine proved not to be
the expected 4-phenylpyridinium species but rather the isomeric 3-phenyl-5-azoniafulvenyl species. The
results of studies with a series of structural and isotopically labeled analogs require a novel fragmentation
pathway to account for the formation of this and related fragment ions. One possible pathway is based on
an initial 1,5-sigmatropic shift of a cyclopropylmethylene hydrogen atom that is accompanied by opening
of the cyclopropyl ring. The resulting eniminium intermediates then fragment to yield the 5-azoniafulvenyl
species. Copyright  2006 John Wiley & Sons, Ltd.
KEYWORDS: 1-cyclopropyl-4-aryl-1,2-dihydropyridines; fragmentation mechanism; 5-azoniafulvenyl species; stable
isotopes; 1,5-sigmatropic hydrogen migration
ionization-mass spectrometric (ESI/MS) assay.10 The fates of
the EC-generated 1-cyclopropyltetrahydropyridinyl radical
cations have been particularly informative. For example,
MHC of the first product detected by ESI/MS following the
EC 1-electron oxidation of trans-1-(2-phenylcyclopropyl)-4-
phenyl-1,2,3,6-tetrahydropyridine (5) at an EC cell potential
of 100 mV has m/z 274. It should be clear that the ion at
m/z 274 is detected only after the substrate molecule 5 has
undergone EC oxidation. The full triple-quadrupole tandem
mass spectrum of unoxidized 5 displayed an ion at 276 (5HC,
100%) and only one significant fragment ion at m/z 146
(10%). No ion was detected at m/z 275 (corresponding to
5C). Consequently, products observed in the EC-ESI/MS
experiment are not formed by oxidation of the analyte
molecule 5 during the course of the ESI/MS analysis. The full
MS1 spectrum of this species shows MHC at m/z 274 (100%)
and in-source fragment ions at m/z 272 (10%) and 117 (5%).
One possible structure for this ion is 6H+, a product
that would be expected to form enzymatically by the HAT
pathway.11 In the EC chemical model, the formation of 6H+
would proceed via ring ˛-carbon deprotonation of the EC-
generated cyclopropylaminyl radical cation 5až+ to form 6ž
followed by a second 1-electron oxidation. A second possible
structure of the product detected at m/z 274 in the EC-
ESI/MS experiment is the ring-opened eniminium species
7+ (one or more diastereomers) that would be generated via
ring opening of 5až+ and subsequent 1-electron oxidation
INTRODUCTION
Interest in 1,4-disubstituted-1,2,3,6-tetrahydropyridines is
derived in part from the potent and selective neurodegen-
erative properties of the parkinsonian-inducing ‘street drug’
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP (1)].1,2
The neurotoxicity of MPTP is reported to be mediated by the
corresponding pyridinium metabolite 4+, which is formed
via monoamine oxidase3- and cytochrome P4504-catalyzed
oxidations that generate the 2,3-dihydropyridinium interme-
diate 3H+ (Scheme 1). One mechanism proposed to account
for this 2-electron ˛-carbon oxidation proceeds via an initial
single electron transfer (SET) step to form the corresponding
aminyl radical cation 1ž+. Ring ˛-carbon deprotonation of 1ž+
yields the neutral radical 2ž that undergoes further oxidation
to the 2,3-dihydropyridinium product 3H+.5,6 An alternative
proposal assumes an initial hydrogen atom transfer (HAT)
step to yield 2ž directly.7–9 The second 2-electron oxidation
(3 ! 4+) has not been characterized fully.
As part of our efforts to characterize the pathway of
these enzyme catalyzed reactions, we have investigated the
chemical fate of tetrahydropyridinyl radical cations in a
model of the SET reaction that involves the electrochemical
(EC) 1-electron oxidation of the substrate molecules. The
reaction products were detected by an on-line electrospray
ŁCorrespondence to: Neal Castagnoli Jr, Department of Chemistry,
Virginia Tech, Blacksburg, VA, USA. E-mail: ncastagn@vt.edu
Copyright  2006 John Wiley & Sons, Ltd.

1644
P. Bissel et al.
Scheme 1. Proposed SET and HAT pathways for the enzyme catalyzed oxidation of the parkinsonian-inducing neurotoxin MPTP (1).
Scheme 2. Pathways leading to the proposed oxidation products 6H+ and/or 7+ following the EC 1-electron oxidation of 5.
of the resulting distonic radical cation 5bž+ (Scheme 2).12
The characterization of the product(s) obtained from the
1-electron oxidation of 5 required product ion analyses by
direct infusion into the ESI source of authentic samples
of 6H+ and 7+. This report describes the MS2 obtained by
ESI/MS analysis of synthetic dihydropyridinyl compounds,
including 6. These studies have led to the identification of
unexpected fragment ions and to the proposal of a possible
fragmentation pathway to account for their formation.
1-Cyclopropyl-4-phenyl-−1,2-dihydropyridine (24)
A mixture of 25+ClO4 (5.0 g, 17 mmol) and NaBH4
(3.2 g. 85 mmol) in 100 ml MeOH containing 2 g KOH
was shaken vigorously for 5 min. Short path distillation
(60 C/10ꢀ5 mmHg) of the organic isolate gave 2.7 g (81%) of
°
pure 24 as an orange oil: UV (MeOH) ꢀmax 249 nm (ε 13 200);
1H NMR (500 MHz, C6D6) υ 0.16 (m, 2H), 0.28 (m, 2H), 1.85
(m, 1H), 3.85 (dd, J D 1.0, 4.0 Hz, 2H), 5.24 (dd, J D 2.0,
7.5 Hz, 1H), 5.31 (dt, 1.0, 4.0 Hz, 1H), 6.14 (dd, J D 1.0,
7.5 Hz, 1H), 7.12 (m, 1H), 7.18 (m, 2H), 7.45 (m, 2H); 13C
NMR (125.8 MHz, C6D6) υ 5.8, 34.5, 49.3, 97.6, 109.1, 125.7,
EXPERIMENTAL
127.1, 128.4, 137.0, 139.3, 140.5. Anal. Calcd for C14 H15
N
(197.28): C, 85.24; H, 7.66; N, 7.10. Found: C, 85.07; H, 7.71;
N, 7.17. The corresponding hydrochloride salt, prepared in
methanolic HCl, proved to be identical to an authentic sample
of 1-cyclopropyl-4-phenyl-1,2-dihydropyridinium chloride
(24H+Cl−).14
Important notice: Some 1,4-disubstituted-1,2,3,6-tetrahydro-
pyridines are known neurotoxins. Compounds of this
general type should be handled with disposable gloves
in a properly ventilated hood following good laboratory
practices. Detailed procedures for the safe handling of MPTP
have been reported.13
4-Phenyl-1-(2-trans-phenylcyclopropyl)pyridinium
perchlorate (12CClO4
)
ꢀ
Chemistry
The known,12 hygroscopic 12+−Cl− was converted into its
stable perchorate salt 12+ClO4 in methanolic HClO4. The
solid that separated upon addition of ether was analytically
1-Cyclopropyl-4-phenylpyridinium perchlorate
(25CClO4
)
ꢀ
The known14 hydrochloride salt 25+Cl− was converted into
the more stable perchlorate salt 25+ClO4 in methanolic
−
1
°
pure: mp 167–168 C; H NMR (500 MHz, DMSO-d6) υ 1.85
1
°
(m, 1H), 2.21 (m, 1H), 3.07 (m, 1H), 4.65 (m, 1H), 7.34 (m,
5H), 7.66 (m, 3H), 8.12 (m, 2H), 8.49 (d, J D 6.5 Hz, 2H), 9.17
(d, J D 6.5 Hz, 2H); 13C NMR (125.8 MHz, DMSO-d6) υ 16.4,
26.0, 49.6, 124.7, 127.3, 127.5, 128.8, 129.0, 130.3, 132.8, 134.0,
138.8, 145.4, 155.7. Anal. Calcd for C20H18ClNO4 (371.81): C,
64.61; H, 4.88; N, 3.77. Found: C, 64.40; H, 4.63; N, 3.82.
HClO4: mp 131–132 C; H NMR (500 MHz, CD3OD) υ 1.42
(m, 4H), 4.35 (m, 1H), 7.62 (m, 3H), 7.97 (m, 2H), 8.32
(d, J D 7.0 Hz, 2H), 8.97 (d, J D 7.0 Hz, 2H); 13C NMR
(125.8 MHz, CD3OD) υ 6.8, 42.1, 124.5, 127.9, 129.6, 132.1,
134.0, 145.3, 157.1. Anal. Calcd for C14H14ClNO4 (295.72): C,
56.86; H, 4.77; N, 4.74. Found: C, 56.75; H, 4.70; N, 4.87.
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1643–1653
DOI: 10.1002/jms

ESI-MS/MS Studies on 1,2-Dihydropyridines
1645
4-Phenyl-1-(2-trans-phenylcyclopropyl)-1,2-
dihydropyridine (6)
4-(4-Methylphenyl)-1-(2-trans-phenylcyclopropyl)-1,2-
dihydropyridine (19)
−
−
The pyridinium perchlorate 12+ClO4 was converted into
Reduction of 18+ClO4 in methanolic KOH with NaBH4
the oily 6 in the same manner as described above for the
preparation of 24: UV (MeOH) ꢀmax 249 nm (ε 16 900); 1H
NMR (500 MHz, DMSO-d6) υ 1.15 (m, 1H), 1.26 (m, 1H),
2.08 (m, 1H), 2.46 (m, 1H), 4.02 (m, 2H), 5.05 (dd, J D 2.0,
7.5 Hz, 1H), 5.46 (m, 1H), 6.35 (dd, J D 0.5, 7.5 Hz, 1H),
7.27 (m, 10H); 13C NMR (125.8 MHz, DMSO-d6) υ 16.1, 24.3,
45.1, 49.1, 96.8, 109.6, 125.5, 126.3, 126.5, 127.7, 128.8, 129.0,
135.9, 139.4, 139.5, 141.6. FAB-HRMS: Calcd for C20H20NC:
274.1596. Found: 274. 1577.
gave 19 as a dark yellow oil (53 mg, 71%): UV (MeOH) ꢀmax
253 nm (ε 11 200), 319 nm (ε 5800); 1H NMR (500 MHz, C6D6)
υ 0.72 (m, 1H), 0.93 (m, 1H), 1.83 (m, 1H), 2.14 (s, 3H), 2.15
(m, 1H), 3.87 (m, 2H), 5.32 (dd, J D 7.5, 2.0 Hz, 1H), 5.38 (m,
1H), 6.17 (d, J D 3.0 Hz, 1H), 6.83 (d, J D 8.0 Hz, 2H), 7.10 (m,
5H), 7.43 (d, J D 8.0 Hz, 2H); 13C NMR (125.8 MHz, C6D6) υ
15.9, 20.9, 24.5, 44.5, 49.1, 98.2, 108.7, 125.6, 125.9, 126.1, 128.4,
129.2, 136.6, 136.8, 137.6, 138.5, 141.1. FAB-HRMS: Calcd for
C21H22NC: 288.1752. Found: 288.1729.
1-Formyl-4-phenyl-1,2,3,6-tetrahydropyridine-1-13C
(21-13C)16
4-Phenyl-1-(2-trans-phenylcyclopropyl)-1,2-
dihydropyridine-d1 (6-d1)
−
In a similar manner, 12+ClO4 was converted into the oily
A mixture of 4-phenyl-1,2,3,6-tetrahydropyridine (20, 3.3 g,
diastereomeric mixture 6-d1: 1H NMR (500 MHz, C6D6) υ 0.70
(m, 1H), 0.91 (m, 1H), 1.81 (m, 1H), 2.13 (m, 1H), 3.83 (m, 1H),
5.28 (dd, J D 2.5, 7.0 Hz, 1H), 5.33 (m, 1H), 6.16 (m, 1H), 7.81
(m, 2H), 7.12 (m, 6H), 7.48 (m, 2H); 13C NMR (125.8 MHz,
C6D6) υ 15.9, 16.0, 44.4, 48.6 (t), 48.7 (t), 97.9, 109.2, 125.7,
125.9, 126.1, 127.2, 128.4, 128.5, 137.0, 138.6, 138.7, 140.4,
141.1. FAB-HRMS: Calcd for C20H19DNC: 275.1659. Found:
275.1646.
21 mmol), H13COOEt (2.0 g, 27 mmol) in CH3CN (30 ml) was
13
°
heated at 60 C for 36 h to give 21- C (mixture of rotomers)
as a pale yellow waxy solid (3.55 g, 90%): 1H NMR (500 MHz,
CDCl3) υ 2.55 (m, 4H), 3.61 (m, 4H), 3.77 (m, 1H), 4.04 (m,
1H), 4.16 (m, 4H), 6.02 (m, 2H), 7.31 (m, 10H), 8.14 (d,
1JCH D 182 Hz, 1H), 8.20 (d, JCH D 182 Hz, 1H); 13C NMR
1
(125.8 MHz, CDCl3) υ 26.8, 28.1, 37.2, 40.4, 42.8, 42.9, 45.2,
45.3, 119.4, 119.5, 119.7, 125.0, 125,1, 127.7, 127.8, 128.6, 135.4,
136.8, 140.3, 140.4, 161.2 (13C), 161.6 (13C). FAB-HRMS: Calcd
for C1113CH14NOC: 189.1109. Found: 189.1101.
4-(4-Methylphenyl)pyridine (16)15
The published procedure was followed except that [Pd
1-Cyclopropyl-1-13C-2,2,3,3-d4-4-phenyl-1,2,3,6-tetra-
hydropyridinium oxalate salt (22-13C-d4 Ð C2H2O4)16
Ethyl bromide-d5 (3.6 ml, 48 mmol) was added dropwise to
a suspension of magnesium turnings in THF (50 ml). The
mixture was heated under reflux for 3 h and, after cooling,
was added slowly via a cannula to 50 ml THF containing
21-13C (3.55 g, 19 mmol) and Ti(iOPr)4 (6.1 ml, 21 mmol). By
12
°
°
ꢁPPh3ꢂ4] was used as catalyst: mp 86–87 C (lit. 88–90 C).
1-(2,4-Dinitrophenyl)-4-(4-methylphenyl)pyridinium
chloride (17CClꢀ)12
A solution of 4-(4-methylphenyl)pyridine15 (6.2 g, 37 mmol)
and 2,4-DNCB (11.2 g, 55 mmol) in dry acetone (160 ml)
was heated under reflux for 48 h. The insoluble product
was collected and recrystallized from MeOH to give
17+Cl− as hygroscopic light yellow crystals (9.2 g, 67%):
°
stirring at 60 C for 18 h, the reaction mixture was worked up
to give 3.0 g (54%) of the oxalate salt 22-13C-d4 Ð C2H2O4: mp
13
1
1
°
°
°
mp 197–199 C; H NMR (500 MHz, DMSO-d6) υ 2.45 (s, 3H),
181–182 C (lit mp 185–186 C for 22 Ð C2H2O4); H NMR
(500 MHz, DMSO-d6) υ 2.43 (d, 1JCH D 178 Hz, 1H), 2.65 (m,
2H), 3.25 (m, 2H), 3.67 (m, 2H), 6.16 (m, 1H), 7.28 (m, 1H),
7.36 (m, 2H), 7.45 (m, 2H); 13C NMR (125.8 MHz, DMSO-d6)
υ 25.0, 37.9 (13C), 49.8, 51.6, 118.4, 125.3, 128.2, 129.1, 134.6,
139.5, 164.8. FAB-HRMS: Calcd for C1313CH14D4NC: 205.1724.
Found: 205.1724.
7.51 (d, J D 8.0 Hz, 2H), 8.21 (d, J D 8.0 Hz, 2H), 8.52 (d,
J D 8.5 Hz, 1H), 8.86 (d, J D 7.0 Hz, 2H), 8.99 (dd, J D 2.5,
8.5 Hz, 1H), 9.12 (d, J D 2.4 Hz, 1H), 9.48 (d, J D 7.0 Hz, 2H);
13C NMR (125.8 MHz, DMSO-d6) υ 21.7, 122.0, 124.1, 129.3,
130.7, 130.8, 131.1, 132.8, 139.2, 143.8, 144.6, 146.4, 149.5, 157.6.
4-(4-Methylphenyl)-1-(trans-2-phenylcyclopropyl)-
ꢀ 12
pyridinium perchlorate (18CClO4
)
1-Cyclopropyl-1-13C-2,2,3,3-d4-4-phenyl-2ꢀ,3-1d4ihydro-
A solution of crude 17+Cl− (2.7 g, 7.5 mmol) and trans-2-
phenylcyclopropylamine (2.0 g, 15 mmol) in anhydrous 1-
butanol (90 ml) was heated under reflux for 12 h. The solvent
was removed under reduced pressure and the residue was
partitioned between water and CH2Cl2. Treatment of the
residue obtained after removing the water under reduced
pyridinium perchlorate (24C-13C-d4-ClO4
)
A mixture of 70% m-chloroperoxobenzoic acid m-CPBA (m-
CPBA, 157 mg, 0.64 mmol) 22-13C-d4 (100 mg, 0.49 mmol) in
°
CH2Cl2 (5 ml) was stirred at 0 C for 20 min. The product
was passed through basic alumina [CH2Cl2 followed by
CH2Cl2: MeOH (97 : 3)] to give 98 mg (91%) of crude 23-
1
pressure with ethereal methanoli−c HClO4 gave 920 mg (32%)
13C-d4: H NMR (500 MHz, CDCl3) υ 2.79 (m, 1H), 2.99 (d,
1
of the white, crystalline 18+ClO4 : mp 135–137 C; H NMR
1JCH D 173 Hz, 1H), 3.05 (m, 1H), 3.50 (m, 2H), 4.03 (m, 2H),
6.00 (m, 1H), 7.30 (m, 1H), 7.35 (m, 2H), 7.42 (m 2H). TFAA
(0.29 ml, 2.1 mmol) was added dropwise to a solution of
°
(500 MHz, CD3OD) υ 1.86 (m, 1H), 2.12 (m, 1H), 2.45 (s, 3H),
3.01 (m, 1H), 4.52 (m, 1H), 7.34 (m, 5H), 7.44 (d, J D 8.0 Hz,
2H), 7.91 (d, J D 6.5 Hz, 2H), 8.33 (d, J D 6.5 Hz, 2H), 8.96 (d,
J D 6.5 Hz, 2H); 13C NMR (125.8 MHz, CD3OD) υ 15.4, 20.2,
25.3, 49.2, 124.0, 126.4, 127.0, 127.9, 128.5, 130.4, 131.0, 137.8,
143.6, 144.8, 157.1. Anal. Calcd for C21H20ClNO4 (385.84): C,
65.37; H, 5.22; N, 3.63. Found: C, 65.29; H, 5.12; N, 3.66.
13
°
23- C-d4 (90 mg, 0.41 mmol) in CH2Cl2 (5 ml) at 0 C. After
°
15 min at 0 C, a methanolic solution of 70% HClO4 (3 ml,
0.3 M) was added and the mixture was stirred for 1 h. The
solvent was removed under reduced pressure. Addition of
a few drops of ether to a methanolic solution of the residue
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1643–1653
DOI: 10.1002/jms

1646
P. Bissel et al.
−
gave 80 mg (65%) of 24+-13C-d4-ClO4 : mp 125–127 C (dec.)
37.2, 108.9, 114.6, 117.5, 122.1, 125.4, 126.2, 128.7, 128.8, 134.9.
FAB-HRMS: Calcd for C12H11N2C: 183.0922. Found: 183.0916.
°
(lit.11 mp 138–139 C for 24 ClO4−); 1H NMR (500 MHz,
+
°
CD3OD) υ 3.32 (m, 2H), 3.60 (d, 1JCH D 187 Hz, 1H), 4.12 (m,
2H), 6.95 (m, 1H), 7.55 (m, 3H), 7.84 (m, 2H), 8.51 (m, 1H);
13C NMR (125.8 MHz, CD3OD) υ 24.9, 40.5 (13C), 47.3, 113.3,
127.2, 129.1, 132.6, 134.8,160.2, 163.4. FAB-HRMS: Calcd for
C1313CH12D4NC: 203.1567. Found: 203.1562.
Mass spectrometry
A TSQ equipped with an ESI source (Thermo Electron,
San Jose, CA, USA) was used for the MS1 and MS2
analyses. Sample solutions (final analyte concentration 5 µM)
in acetonitrile containing 1% formic acid were infused into
the TSQ ion source at a total flow rate of 28 µl/ min using
a syringe pump (Harvard Apparatus, Holliston, MA). The
following ESI operating conditions were used: The spray
voltage was 4 kV, the temperature of the capillary inlet
1-Cyclopropyl-4-phenyl-2,6-13C2-1,2,3,6-tetrahydropy-
ridinium oxalate salt (22-13C2 Ð C2H2O4)17
A 50/50 mixture of 13C-labeled and unlabeled formaldehyde
was used. Mass selection allowed us to examine specifically
the fragmentation properties of the 2,6-13C2 product.
A mixture of cyclopropylamine hydrochloride (486 mg,
5.2 mmol), H13CHO (200 mg, 6.5 mmol) and H12CHO
°
was 270 C and the nitrogen sheath gas pressure was 8 psi.
Collision induced dissociation (CID) studies were run at an
isolation width of m/z 0.7 with argon as the collision gas at
a pressure of approximately 1 mTorr. Data acquisition was
performed with Xcalibur 1.2 software (Thermo Electron).
MS3 spectra were acquired on a LTQ mass spectrometer
(Thermo Electron, San Jose, CA) equipped with an in-house
built nano-ESI source (LTQ). Samples, prepared in CH3CN
acidified with acetic acid (0.1%), were infused at 0.3 µl/ min
with the aid of a syringe pump (Harvard Apparatus,
Holliston, MA) and electrosprayed at 1700 V. The ion source
°
(200 mg, 6.5 mmol) was heated at 60 C for 10 min at which
time ˛-methylstyrene (0.35 ml, 2.6 mmol) was added. After
°
8 h at 60 C, MeOH (5 ml) was added and the reaction mix-
ture was stirred overnight at room temperature. The solvent
was removed under reduced pressure and the residue in
37% HCl (5 ml) was heated under reflux for 1 h. The reac-
tion mixture was poured slowly into a saturated aqueous
solution of K2CO3; the crude CH2Cl2 soluble product was
purified on basic alumina chromatography (hexanes : ethyl
°
capillary inlet temperature was set at 200 C. CID parameters
acetate-9 : 1). The oxalate salt 22-13C2 Ð C2H2O4 was obtained
were set at isolation width 1.4 m/z, normalized collision
energy 50%, activation Q 0.25 and activation time 30 ms.
Ion accumulation was allowed for 500 ms with enabled
automatic gain control. Ten microscans were averaged to
generate a mass spectrum. Data acquisition was performed
with Xcalibur 1.2 software (Thermo Electron).
14
°
°
from methanol: mp 183–184 C (lit mp 185–186 C for
1
22 Ð C2H2O4); H NMR (500 MHz, CD3OD) υ 1.01 (m, 4H),
2.88 (m, 3H), 3.64 (dt, J D 10 Hz, 1JCH D 146 Hz, ¾1H), 3.65 (t,
J D 10 Hz, ¾1H), 4.03 (m, ¾1H), 4.11 (d, 1JCH D 146 Hz, ¾1H),
6.14 (m, 1H), 7.35 (m, 3H), 7.46 (m, 2H); 13C NMR (125.8 MHz,
CD3OD) υ 3.3, 24.5, 38.3, 50.1 (13C), 51.6 (13C), 115.3, 124.7,
128.0, 128.4, 135.6, 138.7, 163.7. Calcd for C1213C2H18NC:
202.1506. Found: 202.1510.
RESULTS AND DISCUSSION
1-Cyclopropyl-4-phenyl-2,6-13C2-2,3-dihydropyridinium
Initial attempts to prepare the dihydropyridinium target
compound 6HC by treatment of the diastereomeric N-
oxides 8/9 with trifluoroacetic anhydride (TFAA), a reaction
that has been employed successfully in the synthesis of
other 1,2-dihydropyridinium analogs,20 led unexpectedly
and exclusively to the eniminium isomers 10C and 11C
(Scheme 3).10
perchlorate (24HC-13C2-ClO4
)
ꢀ
The procedure described for the synthesis of 24-13C-d4
−
gave 24H+-13C2 Ð ClO4 in 60% yield: mp 128–130 C (dec.)
°
(lit11 mp 138–139 C for 24H Ð ClO4 ); 1H NMR (500 MHz,
−
+
°
CD3OD) υ 1.16 (m, 2H), 1.27 (m, 2H), 3.33 (m, 2H), 3.63
1
(m, 1H), 4.12 (dt, J D 10 Hz, JCH D 146 Hz, ¾1H), 4.13 (t,
An alternative approach to this system, involving treat-
ment of 4-phenyl-tra−ns-1-(2-phenylcyclopropyl)pyridinium
perchlorate (12+ClO4 ) with NaBH4 in methanolic KOH,21
J D 10 Hz, ¾1H), 6.94 (m, 1H), 7.57 (m, 3H), 7.85 (m, 2H), 8.52
(m, 0.5H), 8.61 (m, 0.5H); 13C NMR (125.8 MHz, CD3OD) υ
5.4, 24.9, 40.8, 47.5 (13C), 113.2, 127.2, 129.1, 132.7, 134.8,
160.2, 163.4 (13C). Calcd for C1213C2H16NC: 200.1350. Found:
200.1353.
gave the free base 6 in good yield (Scheme 4). The 1H and 13
C
NMR spectra are fully consistent with the assigned structure.
The TSQ MS2 of 6 obtained at a collision induced
dissociation energy (CIDE) of 23 V is shown in Fig. 1;
the proposed rationalizations for the formation of the
observed product ions are presented in Schemes 5 and 6.
C-Protonation of 6 gives the thermodynamically preferred
2,3-dihydropyridinium species 6aH+ (Scheme 5), which
rearranges to yield the fused bicyclo system 13H+. Loss
of H2 from 13H+ gives i+, the structure assigned to
the ion of low abundance at m/z 272. Alternatively,
cleavage of the N–C bond of the cyclopropylaminyl group,
accompanied by opening of the cyclopropyl ring, yields the
phenyl vinyl carbocation ii+ (m/z 117) with 4-phenyl-2,3-
dihydropyridine (A) as the neutral loss species. Cyclization
of ii+ followed by dehydrogenation of the resulting fused
1-Cyanomethyl-3-phenylpyrrole (30)18
Small pieces of potassium (168 mg, 4.3 mmol) were added
to 3-phenylpyrrole19 (29, 614 mg, 4.3 mmol). After heating at
°
50 C for 3 h, toluene (2 ml) was added, which was followed,
under reflux, by the dropwise addition of chloroacetonitrile
(1.4 ml, 21 mmol). After 48 h, the reaction mixture was
partitioned between water and ethyl acetate. Work up
of the organic phase gave an oil that was subjected to
chromatography on silicagel (hexanes : ethyl acetate 7 : 3) to
1
°
give 15 mg (2%) of 30 as a white solid: mp 92–93 C; H NMR
(500 MHz, CDCl3) υ 4.82 (s, 2H), 6.54 (dd, J D 3.0, 1.5 Hz,
1H), 6.74 (t, J D 2.5 Hz, 1H), 7.00 (t, J D 2.0 Hz, 1H), 7.20 (m,
1H), 7.35 (m, 2H), 7.49 (m, 2H); 13C NMR (125.8 MHz, CDCl3)
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1643–1653
DOI: 10.1002/jms

ESI-MS/MS Studies on 1,2-Dihydropyridines
1647
Scheme 3. Reaction of N-oxides 8/9 with TFAA.
Scheme 4. Synthesis and NMR characterization of 6.
Figure 1. TSQ MS2 of 6H+ obtained at a CIDE of 23 V.
bicyclo intermediate iii+ generates iv+ (m/z 115). Fragment
ii+ also may lose acetylene to form the benzylcarbocation
v+ (m/z 91). The formation of the fragment ion at m/z 129
is visualized as proceeding via 6aꢀH+ to yield vi+ with the
iminocyclopropanyl species B as the neutral loss.
Formation of the base peak fragment ion at m/z
156 is proposed to proceed via the kinetically pre-
ferred N-protonated species 6bH+ (Scheme 6). Cleavage
of the N-cyclopropyl bond, this time with transfer of a
proton from C(2) of the 1,2-dihydropyridinium moiety,
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1643–1653
DOI: 10.1002/jms

1648
P. Bissel et al.
Scheme 5. Proposed scheme to account for the fragment ions observed in the TSQ MS2 of 6.
yields vii+ and 1-phenylpropene (C) as the neutral loss
species.
of 25%. These results forced us to consider an alternative
structure for the m/z 156 fragment ion.
The C(2)-d1 analog 6-d1 was prepared by reaction of 12+
with NaBD4. The TSQ MS2 (CIDE 25 V) of 6-d1 [6H+-d1 m/z
275 ꢁ8%ꢂ ! 273 (2%), 157 (100%), 130 (3%), 117 (70%), 115
(35%) and 91 (12%)] was consistent with the pathways shown
in Scheme 5. However, the fragmentation pathway proposed
for the formation of vii+ [6 ! 6bH+ ! vii+] (Scheme 6)
should lead, in the case of 6-d1, to a mixture of undeuterated
and monodeuterated fragment ions at m/z 156 and m/z 157,
respectively (Scheme 7). The ion current intensity at m/z 156
in the TSQ MS2 of 6-d1, however, was below the limits of
detection of the triple quadrupole instrument. Furthermore,
the MHC ion of a synthetic sample of 4-phenylpyridinium
chloride (m/z 156) was stable when exposed in the LTQ
to a normalized collision energy of 40%, whereas an MS3
experiment established that the fragment ion in the LTQ MS2
of 6 at m/z 156 fragmented at a normalized collision energy
The presence of a phenyl substituent on both the dihy-
dropyridinyl and cyclopropyl groups of 6 introduced some
ambiguity with respect to the identity of the phenyl group
present in the m/z 156 fragment ion. Consequently we
examined the TSQ MS2 of 4-(4-methylphenyl)-1-(2-trans-
phenylcyclopropyl)-1,2-dihydropyridine (19). The synthesis
of 19 (Scheme 8) was achieved via a reaction sequence
involving the coupling of 4-methylphenylboronic acid (14)
and 4-bromopyridine (15) in the presence of PdꢁPPh3ꢂ4 to
give 4-(4-methylphenyl)pyridine (16).15 Reaction of 16 with
2,4-dinitrochlorobenzene (2,4-DNCB) led to the pyridinium
intermediate 17+Cl−, which formed, upon heating with trans-
2-phenylcyclopropylamine (t-2-PCA) in BuOH, the phenyl-
cyclopropylpyridinium product 18+ and 2,4-dinitroaniline
(2,4-DNA).12 The perchlorate salt of 18+ underwent smooth
reduction with NaBH4 in methanolic KOH to give the desired
dihydropyridine 19.
The base peak present in the TSQ MS2 of 19 (CIDE 23 V)
[19H+ m/z 288 ꢁ10%ꢂ ! 286 (2%), 156 (100%), 129 (2%) 117
(45%), 115 (8%) and 91 (2%)] again appeared at m/z 156.
Therefore, the phenyl group present in this fragment ion is
derived from the phenyl group of the cyclopropyl moiety.
This outcome ruled out the pathway 6 ! 6bH+ ! vii+
(Scheme 6).
Additional studies were undertaken with the aid of
the known14 1-cyclopropyl-4-phenyl-2,3-dihydropyridinium
species 24H+ and two stable isotopically labeled analogs,
namely, 24H+-13C2 and 24H+-13C-d4. The synthetic sequence
Scheme 6. Pathway leading to the proposed fragment ion vii+.
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1643–1653
DOI: 10.1002/jms

ESI-MS/MS Studies on 1,2-Dihydropyridines
1649
Scheme 7. Fragmentation of the putative MHC ions 6bH+ and 6bH+−d1 to yield vii+ and vii+−d1.
Scheme 8. Synthesis of the 4-(4-methylphenyl)dihydropyridinyl analog 19.
to 24H+-13C2 started with 22-13C2, which was obtained from
respectively, which is fully consistent with the proposed
ring expansion-dehydrogenation pathway.
the condensation of ˛-methylstyrene, cyclopropylamine and
13C-labeled formaldehyde (Scheme 9).17 The preparation
of 24H+-13C-d4 required 22-13C-d4, which was obtained
by treatment of 4-phenyl-1,2,3,6-tetrahydropyridine (21)
with H13COOEt followed by construction of the cyclo-
propyl ring from the resulting formamide 22-13C by reac-
tion with C2D5MgBr in the presence of titanium tetra-
isopropoxide [TiꢁOPriꢂ4].16 Treatment of these tetrahydropy-
ridinyl intermediates with m-CPBA gave the correspond-
ing N-oxides 23, 23-13C2 and 23-13C-d4, which were con-
verted into the desired N-cyclopropyldihydropyridinium
products 24H+, 24H+-13C2 and 24H+-13C-d4 with TFAA.
The unlabeled dihydropyridinyl analog 24 was prepared
by reduction of the known14 pyridinium species 25+
with NaBH4 in methanolic KOH. Treatment of 24 with
methanolic HCl gave a good yield of 24H+Cl−. The
conversion of 24 into 24H+ documents that the proto-
nation of 1-cyclopropyl-1,2-dihydropyridines in methanol
yields the thermodynamically preferred C-protonated prod-
ucts.
The molecular composition of the base peak in the TSQ
MS2 of 24H+ at m/z 80 could be assigned tentatively to
C5H6NC. The mass of this ion shifted to m/z 82 in the
corresponding spectrum of 24H+-13C2, establishing that both
C(2) and C(6) of the dihydropyridinyl ring are retained in
the base fragment ion peaks in this series. The corresponding
fragmentation in the TSQ MS2 of 24H+-13C-d4 led to a 1 : 1
doublet at m/z 83 and 84 (Fig. 2).
It is apparent from the available evidence that the carbon
atoms identified in structures 24 and 6 are retained in the
fragment ions at m/z 80 and 156, respectively. Furthermore,
in order to accommodate the fragment ion at m/z 84, three
of the four deuterium atoms colored blue in 24-13C-d4 must
be retained. As this fragment ion also appears at m/z 83, the
fragmentation pathway also must include a mechanism to
account for loss of one of these three deuterons. Structures
As expected, the TSQ MS2 of synthetic 24 and synthetic
24H+ obtained at a CIDE of 24 V were identical – 24H+ m/z
198 ꢁ10%ꢂ ! 196 (5%), 156 (6%), 141 (6%), 128 (6%), 115 (7%),
91 (10%) and 80 (100%). Therefore, protonation of 24 under
ESI conditions also takes place on carbon. Structure viii+ was
assigned to the ion of low abundance at m/z 196 by analogy
with the sequence 6aH+ ! 13H+ ! i+ C H2 (Scheme 4).
Confirmation of this assignment was obtained with the
spectra of 24H+-13C2 (MHC at m/z 200) and 24H+-13C-d4
(MHC at m/z 203) that displayed the corresponding weak
signals at m/z 198 (viii+-13C2) and 200 (m/z viii+-13C-d3),
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1643–1653
DOI: 10.1002/jms

1650
P. Bissel et al.
Scheme 9. Synthetic pathways to 24H+, 24H+-13C2 and 24H+-13C-d4.
Figure 2. The TSQ MS2 of 24aH+-13C-d4 obtained at a collision energy of 20 V.
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1643–1653
DOI: 10.1002/jms

ESI-MS/MS Studies on 1,2-Dihydropyridines
1651
Scheme 10. Synthetic pathway to 1-cyanomethyl-3-phenylpyrrole and the convergent fragmentation of 26H+ and 6aH+ to x+ and of
6aH+-d1 to x+-d1.
Scheme 11. Pathway to account for the LTQ MS3 fragmentation of 5-azoniafulvenyl ions x+ and x+-d1.
Scheme 12. Proposed pathway to account for the formation of azoniafulvenyl fragment ions x+-13C-d3 and x+-13C-d2 observed in
the TSQ MS2 of 24aH+-13C-d4.
3-phenylpyrrole (25)19 with chloroacetonitrile (Scheme 10).18
As expected, the TSQ MS2 spectrum of 26 displayed a major
fragment ion at m/z 156 for the 5-azoniafulvenyl species
x+. We reasoned that identical MS3 fragmentation patterns
of the m/z 156 ions derived from 6 and 26 would provide
convincing evidence supporting the structure of x+.22
The LTQ MS3 tracings for m/z 156 derived from the LTQ
MS2 of 26 and 6 are presented in Fig. 3 together with the
corresponding MS3 tracing of m/z 157 present in the MS2 of
6-d1 (discussed below). The only significant MS3 fragment
ion of m/z 156 derived from 26 appears at m/z 129. The
MS3 of m/z 156 derived from 6 also displays m/z 129 as the
only significant fragment ion. We conclude, therefore, that
the m/z 156 fragment ions derived from 26 and from 6 must
be the same, i.e. x+!
that are consistent with these data are the 5-azoniafulvenyl
species shown below, right.
The fragmentation m/z 156 ! m/z 129 may proceed via
the fused bicyclo species xi+, which ring expands to xii+.
Loss of HCN from xii+ gives xiii+ (Scheme 11). This analysis
is consistent with the LTQ MS3 features of the fragment ion
x+-d1 [x+-d1 ! xi+-d1 ! xii+-d1 ! xiii+-d1] generated in the
LTQ MSC of 6-d1 and is analogous to the pathway proposed
for the formation of the cyclobutenyl cation observed in the
EI-MS spectrum of 1-butylpyrrole.23
Evidence supporting the 5-azoniafulvenyl assignment
was sought with the aid of 1-cyanomethyl-3-phenylpyrrole
(26), a compound that could be prepared, albeit in poor
yield, by treatment of the potassium salt of the known
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1643–1653
DOI: 10.1002/jms

1652
P. Bissel et al.
Figure 3. Full LTQ MS3 tracings of ions at m/z 156 derived from 26H+ (top) and 6H+ (middle) and of m/z 157 derived from
6H+-d1 (bottom).
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1643–1653
DOI: 10.1002/jms

ESI-MS/MS Studies on 1,2-Dihydropyridines
1653
5. Hanzlik RP, Kishore V, Tullman R. Cyclopropylamines as
suicide substrates for cytochromes P-450. J. Med. Chem. 1979;
22: 759.
6. Vazquez ML, Silverman RG. Revised mechanism for
inactivation of mitochondrial monoamine oxidase by N-
cyclopropylbenzylamine. Biochemistry 1985; 24: 6538.
7. Bissel P, Penich SE, Castagnoli N Jr. Studies on the cytochrome
P450 catalyzed oxidation of 13C labeled 1-cyclopropyl-4-phenyl-
1,2,3,6-tetrahydropyridine by 13C NMR. Bioorg. Med. Chem. 2005;
13: 2975.
8. Bhakta MN, Wimalasena K. Microsomal P450-catalyzed N-
dealkylation of N,N-dialkylanilines: evidence for a C(alpha)-H
abstraction mechanism. J. Am. Chem. Soc. 2002; 214: 1844.
9. Shaffer CL, Harriman S, Koen YM, Hanzlik RP. Formation
of Cyclopropanone during Cytochrome P450-catalyzed N-
Dealkylation of a Cyclopropylamine. J. Am. Chem. Soc. 2002;
124: 8268.
An attempt to devise a pathway to accommodate the
CID fragmentation of these dihydropyridinium species
is presented in Scheme 12 with 24aH+-13C-d4. The initial
reaction involves deuteride transfer from the methylene of
the cyclopropyl group to C(6) of the dihydropyridinium
species with concomitant ring opening to give the eniminium
species 27H+. This intermediate then fragments to give
xiv+-13C-d4 with 1-phenylcyclopropene (D) as the neutral
loss species. Loss of D2 from xiv+-13C-d4 leads to ix+-d2 (m/z
83) whereas loss of HD leads to ix+-d3 (m/z 84). This analysis
predicts structure x+ for the m/z 156 fragment ion observed
in the TSQ MS2 of trans-(2-phenylcyclopropyl)-4-phenyl-1,2-
dihydropyridine (6) and structure x+-d1 for the m/z 157
fragment ion observed in the TSQ MS2 of 6-d1 (Scheme 10).
10. Jurva U, Bissel P, Isin EM, Igarashi K, Kuttab S, Castagnoli N Jr.
Model electrochemical-mass spectrometric studies of the
cytochrome P450-catalyzed oxidations of cyclic tertiary
allylamines. J. Am. Chem. Soc. 2005; 127: 12368.
11. Nimkar SK, Anderson A, Rimoldi JM, Stanton M, Castag-
noli KP Jr, Mabic S, Wang Y-X, Castagnoli N. Synthesis and
monoamine oxidase-B catalyzed oxidation of C-4 heteroaro-
matic substituted 1,2,3,6-tetrahydropyridine derivatives. Chem.
Res. Toxicol. 1996; 9: 1013.
12. Kuttab S, Shang J, Castagnoli N Jr. Rat liver microsomal enzyme
catalyzed oxidation of 4-phenyl-trans-1-(2-phenylcyclopropyl)-
1,2,3,6-tetrahydropyridine. Bioorg. Med. Chem. 2001; 9: 1685.
13. Pitts SM, Markey SP, Murphy DL, Weiss A. In Recommended
Practices for the Safe Handling of MPTP, Markey SP,
Castagnoli N Jr, Trevor AJ, Kopin IJ (eds). Academic Press: New
York, 1995; 703.
14. Zhao Z, Mabic S, Kuttab S, Franot C, Castagnoli K, Castag-
noli N Jr. Rat liver microsomal enzyme catalyzed oxidation of
1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine. Bioorg. Med.
Chem. 1998; 6: 2531.
CONCLUSIONS
The structures of the base peaks observed in the TSQ MS2 of
various 1-cyclopropyl-1,2-dihydropyridines proved not to be
the expected pyridinium species. Instead, a rather complex
fragmentation process appears to take place leading to 5-
azoniafulvenyl products. With the aid of structural analogs
and a comparison of the MS3 of the m/z 156 base peak present
in the LTQ MS2 of the dihydropyridinyl analog 6 with the
corresponding MS3 of the m/z 156 base peak present in
the LTQ MS2 of the 1-cyanomethylpyrrolyl compound 26,
it was possible to assign the structure of this fragment ion
as x+. Analogous 5-azoniafulvenyl structures account for
the corresponding base peaks observed in the TSQ MS2
of the other 1,2-dihydropyridines reported in this paper.
A possible pathway to account for the formation of these
fragment ions is presented in Scheme 12. This pathway, while
consistent with the results obtained with various structural
and isotopically labeled substrates, may not be exclusive.
A critical step involves an initial 1,5-sigmatropic hydrogen
migration from one of the cyclopropyl methylene groups to
C(6) of the protonated dihydropyridine, a proposal that may
merit further exploration.
15. Casalnuovo AL, Calabrese JC. Palladium-catalyzed alkylations
in aqueous media. J. Am. Chem. Soc. 1990; 112: 4324.
16. Kuttab S, Mabic S. Synthesis of
a
13C labeled N-
cyclopropylamine tetrahydropyridine derivative. J. Labelled
Comp. Radiopharm. 2002; 45: 813.
17. Hall L, Murry S, Castagnoli K, Castagnoli N Jr. Studies on
1,2,3,6-tetrahydropyridine derivatives as potential monoamine
oxidase inactivators. Chem. Res. Toxicol. 1992; 5: 625.
18. Herz W, Raden DS, Murty DRK. Synthesis of 1- and 2-
pyrrolealkylamines. J. Org. Chem. 1956; 21: 896.
19. Smith ND, Huang D, Cosford NDP. One-Step Synthesis of 3-
aryl- and 3,4-diaryl-(1H)-pyrroles using tosylmethyl isocyanide
(TOSMIC). Org. Lett. 2002; 4: 3537.
20. Zhao Z, Dalvie D, Naiman N, Castagnoli K, Castagnoli N Jr.
Design, synthesis, and biological evaluation of novel 4-
substituted 1-methyl-1,2,3,6-tetrahydropyridine analogs of
MPTP. J. Med. Chem. 1992; 35: 4473.
Acknowledgements
The authors thank Dr Mehdi Ashraf-Khorassani for the TSQ spectra.
This work was supported by the Harvey W. Peters Center for the
Study of Parkinson’s Disease (Virginia Tech).
REFERENCES
21. Saunders M,
Gold EH.
Preparation
of
N-phenyl-1,2-
1. Royland JE, Langston JW. Highly Selective Neurotoxins. Basic and
Clinical Applications. Humana Press: New Jersey, 1998; 141.
2. Youdim MBH. Brain monoamine oxidase (MAO) B: a unique
neurotoxin- and neurotransmitter-producing enzyme. Prog.
Neuropsychopharmacol. Biol. Psychiatry 1989; 13: 363.
3. Chiba K, Trevor AJ, Castagnoli N Jr. Metabolism of the
neurotoxic tertiary amine, MPTP, by brain monoamine oxidase.
Biochem. Biophys. Res. Commun. 1984; 120: 574.
4. Ottoboni S, Carlson TJ, Trager WF, Castagnoli K, Castag-
noli N Jr. Studies on the cytochrome P-450 catalyzed ring
alpha-carbon oxidation of the nigrostriatal toxin 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP). Chem. Res. Toxicol.
1990; 3: 423.
dihydropyridine. J. Org. Chem. 1962; 27: 1439.
22. Burger U, Bringhen AO, Wirthner PJ, Scha¨rer J-C. The
unsubstituted 5-azoniafulvenyl ion has been generated from
the N-pyrrolidinylmethyl precursor and trapped as C-arylated
products. Helv. Chim. Acta 1985; 68: 2275.
23. Duffield AM, Beugelmans R, Budzikiewicz H, Lightner DA,
Williams DH, Djerassi C. Mass spectrometry in structural and
stereochemical problems. LXIII. Hydrogen rearrangements
induced by electron impact on N-n-butyl- and N-n-
pentylpyrroles. J. Am. Chem. Soc. 1965; 87: 805.
Copyright  2006 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2006; 41: 1643–1653
DOI: 10.1002/jms