Novel Amination and 1,2-Amino,hydro-Elimination between 2,3-Diketopyrido[4,3,2-de]quinolines and Primary Amino Compounds

Article abstract of DOI:10.1021/jo9910132

A novel animation and 1,2-amino,hydro-elimination reaction occurs between 2,3-diketopyrido[4,3,2-de]quinoline (1, 2) and amino compounds which include α-amino acids and peptides which contain a primary amino group. The amino group undergoes nucleophilic addition to the double bond between C3a and C4 in 2,3-diketopyrido[4,3,2-de]quinoline to form a 3a,4-dihydro-2,3-diketopyrido-[4,3,2-de]quinoline. This dihydro intermediate is immediately oxidized by ambient air to produce the more stable aromatic system, 4-(N-alkyl or aryl)-2,3-diketopyrido[4,3,2-de]quinoline (5-12). In the cases of aliphatic amines bearing a β-proton in THF or chloroform, this 4-(N-alkyl)-2,3-diketopyrido[4,3,2-de]quinoline undergoes a 1,2-amino,hydro-elimination reaction to eliminate an alkene and produce the 4-amino-2,3-diketopyrido[4,3,2-de]quinoline (13, 14). In the cases of α-amino acids in aqueous solution, the 4-(N-alkyl)-2,3-diketopyrido[4,3,2-de]quinoline undergoes an amino-transferring reaction, via a mechanism similar to the action of pyridoxal, to form the 4-amino-2,3-diketopyrido[4,3,2-de]quinolme (13) and the α-keto acid. 2,3-Diketopyrido[4,3,2-de]quinoline (1) can also react with the peptide which contains a primary amino group to form the 2,3-diketopyrido-[4,3,2-de]quinoline-peptide conjugate. This novel amination-elimination reaction may underlie the marked cytotoxic potency of the 2,3-diketopyrido[4,3,2-de]quinolines (1 and 2). As inorganic amino compounds, hydroxylamine and hydrazine can also undergo the nucleophilic addition to 2,3-diketopyrido[4,3,2-de]quinoline (1, 2) to produce 4-amino-2,3-diketopyrido[4,3,2-de]quinoline (13, 14) which includes a elimination reaction between C4 and α-nitrogen.

Full text of DOI:10.1021/jo9910132

J . Org. Chem. 1999, 64, 7965-7972
7965
Novel Am in a tion a n d 1,2-Am in o,h yd r o-Elim in a tion betw een
2
,3-Dik etop yr id o[4,3,2-d e]qu in olin es a n d P r im a r y Am in o
Com p ou n d s
Qizhu Ding and J . William Lown*
Department of Chemistry,University of Alberta, Edmonton, Alberta, Canada, T6G 2G2
Received J une 24, 1999
A novel amination and 1,2-amino,hydro-elimination reaction occurs between 2,3-diketopyrido[4,3,2-
de]quinoline (1, 2) and amino compounds which include R-amino acids and peptides which contain
a primary amino group. The amino group undergoes nucleophilic addition to the double bond
between C3a and C4 in 2,3-diketopyrido[4,3,2-de]quinoline to form a 3a,4-dihydro-2,3-diketopyrido-
[4,3,2-de]quinoline. This dihydro intermediate is immediately oxidized by ambient air to produce
the more stable aromatic system, 4-(N-alkyl or aryl)-2,3-diketopyrido[4,3,2-de]quinoline (5-12).
In the cases of aliphatic amines bearing a â-proton in THF or chloroform, this 4-(N-alkyl)-2,3-
diketopyrido[4,3,2-de]quinoline undergoes a 1,2-amino,hydro-elimination reaction to eliminate an
alkene and produce the 4-amino-2,3-diketopyrido[4,3,2-de]quinoline (13, 14). In the cases of R-amino
acids in aqueous solution, the 4-(N-alkyl)-2,3-diketopyrido[4,3,2-de]quinoline undergoes an amino-
transferring reaction, via a mechanism similar to the action of pyridoxal, to form the 4-amino-2,3-
diketopyrido[4,3,2-de]quinoline (13) and the R-keto acid. 2,3-Diketopyrido[4,3,2-de]quinoline (1) can
also react with the peptide which contains a primary amino group to form the 2,3-diketopyrido-
[
4,3,2-de]quinoline-peptide conjugate. This novel amination-elimination reaction may underlie
the marked cytotoxic potency of the 2,3-diketopyrido[4,3,2-de]quinolines (1 and 2). As inorganic
amino compounds, hydroxylamine and hydrazine can also undergo the nucleophilic addition to
2
1
,3-diketopyrido[4,3,2-de]quinoline (1, 2) to produce 4-amino-2,3-diketopyrido[4,3,2-de]quinoline (13,
4) which includes a elimination reaction between C4 and R-nitrogen.
In tr od u ction
Amphimedine was isolated from a pacific sponge
amphimedon sp.) as a cytotoxic compound in 1983 and
was the first example of the pyridoacridine alkaloids
(
The physical and chemical properties of the marine
environment are quite different from those of the ter-
1
restrial environment. During the last 30 years, organic
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chemists have intensively studied the chemistry of
marine organisms and begun to investigate the molecular
structure and the biomedical potential of the metabolites
from marine sources.1 With researchers developing an
increasing interest in marine research, many marine
products have been examined in anticipation of new
chemistry or desirable physiological activity. For ex-
ample, the pyrido[4,3,2-de]quinoline core structure has
been found in many sponges and tunicates (Chart 1). The
chemistry and pharmacological properties of these ma-
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rine natural products have been the subject of much
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structure elucidation4 and synthesis,
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as well as for
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(20) McDonald, L. A.; Eldredge, G. S.; Barrows, L. R.; Ireland, C.
*
Corresponding author. Tel: 780-492-3646; fax: 780-492-8231.
M. J . Nat. Prod. 1998, 61, 301-305.
(
1) Scheuer, P. J . Science 1990, 248, 173.
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Lett. 1988, 29, 6655-6656.
(
2) (a) Davidson, B. S. Chem. Rev. 1993, 93, 1771-1791. (b) Molinski,
T. F. Chem. Rev. 1993, 93, 1825-1838.
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Y.; Sasaki, T.; Ohizomi, Y. J . Org. Chem. 1988, 52, 1800.
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Y.; Faulkner, D. J . J . Org. Chem. 1992, 57, 1523-1526.
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C. E.; Frobenius, W.; Herderich, M.; van Soest, R. W. M. J . Nat. Prod.
1998, 61, 301-305.
(
1
(
(27) Muller, C. E.; Hide, I.; Daly, J . W.; Rothenhausler, K.; Eger,
K. J . Med. Chem. 1990, 33, 2822-2828.
(
(28) Muller, C. E.; Geis, U.; Grahner, B.; Lanzner, W.; Egger, K. J .
Med. Chem. 1996, 39, 2482-2491.
1
0.1021/jo9910132 CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/30/1999

7
966 J . Org. Chem., Vol. 64, No. 21, 1999
Ding and Lown
Ch a r t 1. Th e Sk eleton of P yr id o[4,3,2-d e]qu in olin e
a n d Str u ctu r es of Am p h im ed in e, Cystod ytin ,
Ar oa m in e, Ku a n on ia m in e, Dip la m in e, a n d
Ar oa m in e
Ch a r t 2. Str u ctu r es of P yr id o[4,3,2-d e]qu in olin e
1 a n d 2
4,3,2-de]quinolines3
3-36
and pyrido[4,3,2-de]quinolines.37-39
[
The biological evaluation data shows that pyrido[4,3,2-
de]quinoline 1 and 2 (Chart 2) exhibit significant cyto-
toxic potency. Compound 2 exhibits a wide spectrum of
anticancer activities against 22 cell lines in seven cancer
panels with LC50 values less than 9 µM, especially in
nonsmall cell lung cancer, melanoma cancer, renal
cancer, prostate cancer, and breast cancer. Compounds
1
selectively affects the cell growth against nonsmall cell
lung cancer HOP-92 cell line, breast cancer MDA-MB-
35, and MDA-N cell lines with LC50 values less than 8
4
µM. The promising results of these compounds have
aroused our interest in investigating the chemistry and
the biochemistry of the novel 2,3-diketopyrido[4,3,2-de]-
quinoline alkaloids. We herein report a novel amination
and elimination reaction between 2,3-diketopyrido[4,3,2-
de]quinolines and amino compounds which include R-ami-
no acids and peptides and that may be related to their
observed cytotoxic potency.
Resu lts a n d Discu ssion
1
. Am in a tion a n d 1,2-Am in o,h yd r o-Elim in a tion
which contain a pyrido[4,3,2-de]quinoline core struc-
ture.29 Cystodytin J and diplamine inhibit HCT replica-
betw een 2,3-Dik etop yr id o[4,3,2-d e]qu in olin e a n d
P r im a r y Alip h a tic Am in es. In our previous studies,
the C3 position in 2,3-diketopyrido[4,3,2-de]quinoline 1
and 2 was observed to be a highly active position toward
nucleophilic addition. In the presence of organic bases,
the C3 carbonyl group undergoes nucleophilic addition
by acetone and acetophenone.3 For example, compound
tion with IC50 values less than 2 µM.30 The pharmaco-
logical effects of kuanoniamine, kuanoniamine C, and
kuanoniamine D were also studied.31 The IC50 values of
all these compounds against human cell lines HeLa and
MONO-MAC-6 are less than 5.5 µM. Arnoamine A and
B are proposed to be the first members of a new class of
8,39
1
reacts with acetone in the present of triethylamine to
pentacyclic pyridoacridine alkaloids possessing a pyrrole
ring fused to the pyridoacridine ring system.32 Arnoamine
form the O-hydro,C-acetonyl-addition product in high
yield. In the present study, it was found that primary
amino nucleophiles react with 2,3-diketopyrido[4,3,2-de]-
quinoline 1 and 2 at the C4 position.
The n-propylamine can easily react with 2,3-diketopy-
rido[4,3,2-de]quinoline in THF to form a purple interme-
diate. This intermediate then forms a final product with
red color. The HRMS results show that this red product
has a composition which contains only one extra NH
compared with the starting 2,3-diketopyrido[4,3,2-de]-
A exhibits selective cytotoxicity against the MCF breast
cancer cell line with a GI50 value of 0.3 µg/mL versus
GI50s of 2 and 4 µg/mL against A-549 and HT-29 cell
lines, respectively. Arnoamine B exhibits GI50 of 5.0, 2.0,
and 3.0 µg/mL against the MCF-7, A-549, and HT-29 cell
lines, respectively. Initial assays showed differential
toxicity between the DNA repair-deficient CHO cell line
xrs-6 and the DNA repair-proficient CHO cell line BR1,
suggesting a mechanism of action involving topo-
isomerase II inhibition. The new alkaloids provide useful
lead structures for the development of new types of
antitumor agents, a goal that would certainly be facili-
tated by the clarification of the mode of action of
pyridoquinoline.
1
quinoline. The H NMR shows that there are two extra
exchangeable protons contained in this product with
different chemical shifts (9.85 and 6.33 ppm for the
product from 2,3-diketopyrido[4,3,2-de]quinoline 2 and
3
n-propylamine in DCCl ). To determine where the nitro-
In previous papers, we have reported the synthesis,
cytotoxic potency and anticancer activities of some pyrrolo-
(
(
33) Zhao, R. L.; Lown, J . W. Synth. Commun. 1997, 27, 2103-2110.
34) Wang, H. Y.; Al-Said, N. H.; Lown, J . W. Tetrahedron Lett. 1994,
3
5, 4085-4086.
(
29) Schmitz, F. J .; Agarwal, S. K.; Gunasekera, S. P.; Schmidt, P.
G.; Shoolery, J . N. J . Am. Chem. Soc. 1983, 105, 4835-4836.
30) McDonald, L. A.; Eldredge, G. S.; Barrows, L. R.; Ireland, C.
M. J . Med. Chem. 1994, 37, 3819-3827.
31) Ede, C.; Schupp, P.; Proksch, P.; Wray, V.; Steube, K.; Muller,
C. E.; Frobenius, W.; Herderich, M.; Van Soest, R. W. M. J . Nat. Prod.
(35) Zhao, R. L.; Oreski, B.; Lown, J . W. Bioorg. Med. Chem. Lett.
1996, 6, 2169-2172.
(
(36) Ding, Q. Z.; Zhao, R. L.; Lown, J . W. Heterocycl. Commun. 1997,
3, 489-492.
(
(37) Ding, Q. Z.; Lown, J . W. Heterocycl. Commun. 1998, 4, 125-
129.
1
998, 61, 301-305.
32) Plubrukarn, A.; Davidson, B. S. J . Org. Chem. 1998, 63, 1657-
659.
(38) Ding, Q. Z.; Lown, J . W. Heterocycl. Commun. 1998, 4, 535-
(
545.
1
(39) Ding, Q. Z.; Lown, J . W. J . Med. Chem., in press.

Novel Amination and Deamination
J . Org. Chem., Vol. 64, No. 21, 1999 7967
F igu r e 1. Structures of 15N-labeled 4-amino-2,3-diketopyrido-
F igu r e 2. Structures of 15N-labeled 4-(N-propyl)amino-2,3-
diketopyrido[4,3,2-de]quinoline 5 and 6.
[4,3,2-de]quinoline 3 and 4.
gen atom is located, an 15N-labeled-n-propylamine was
used for a similar reaction. The results of the isotope-
labeling studies show that the red product contains one
the O atom at the C3 position to form a hydroxy group.
In the presence of base this hydroxy group is deproto-
nated to produce a negative oxygen at the C3 position.
The â-proton in the n-propyl group is located very close
to this negative charged oxygen, and it is possible to
transfer this â-proton onto the oxygen at the C3 position.
Both n-heptylamine and tyramine possess a â-proton
and can also undergo a similar reaction in THF and
generate the identical red product as was formed with
n-propylamine. In case of tyramine a byproduct, p-
hydroxystyrene, was identified by GC/MS technology as
well as GC/IR technology. These results demonstrate that
an 1,2-amino,hydro-elimination reaction is involved in
the formation of p-hydroxystyrene. A novel amination-
elimination mechanism was proposed for these reactions
1
5
15
N atom incorporated from the N labeled n-propy-
1
lamine. The H NMR spectrum of the final product shows
that both of the two exchangeable protons are attached
to the 15N atom with the same coupling constant but
1
5
different chemical shifts, 9.88 ppm (d, 1 H, N-Ha, J
1
1
5
5
15
N-Ha ) 92.5 Hz) and 6.34 ppm (d, 1 H, N-Hb, J
N-Hb ) 92.5 Hz) for the red product from compound
in DCCl . NOE interactions reveal that the proton at
3
2
9
.88 ppm (proton a) was affected by the proton at 6.34
1
5
ppm (proton b) at a level of 91%. The N-hmqc (hetero-
nuclear multiple quantum correlation) spectrum also
confirms that both of these two protons are coupled with
the 15N atom. In addition, all of the NMR studies show
that the red product lacks a aromatic proton compared
with the starting 2,3-diketopyrido[4,3,2-de]quinoline.
From these results, the structure of the red product was
proposed as shown in structure 4 (Figure 1).
(Scheme 1).
The double bond between C3a and C4 is subject to
nucleophilic addition by an amino group first to form a
C3a,C4-dihydro intermediate (I). This dihydro intermedi-
ate is then air-oxidized to form the more stable aromatic
system, 2,3-diketo-4-(N-alkyl)aminopyrido[4,3,2-de]quin-
oline (7-12). It was demonstrated that an oxidation
reaction was involved in this amination reaction (see
below). An 1,2-amino,hydro-elimination reaction was
involved in this amination-elimination reaction which
may be catalyzed by the 3-oxido ion. After the elimination
reaction, a 2-keto-3-hydroxy-4-iminopyrido[4,3,2-de]quin-
oline (III) is formed and transformed immediately into
the final product 4-amino-2,3-diketopyrido[4,3,2-de]-
quinoline (13, 14). The results of 15N-labeling experi-
ments (compound 3 and 4) show the evidence favoring
tautomer 13 and 14 over tautomer III.
A hydrogen bond exists between the 15NH and the
3
-carbonyl group. This is presumably why these two
protons on the nitrogen atom have different chemical
shifts. The 15N atom is derived from the 15N labeled
n-propylamine, and the n-propyl group has been removed
from the 15N atom. The overall result of the reaction
between 2,3-diketopyrido[4,3,2-de]quinoline and n-pro-
pylamine is therefore that 2,3-diketopyrido [4,3,2-de]-
quinoline extracted the amino group from n-propylamine.
To understand the mechanism of this novel reaction,
an intermediate was isolated during the reaction of 2,3-
1
5
diketopyrido[4,3,2-de]quinoline 2 with N labeled n-
propylamine. This intermediate is a purple product which
is not very stable in THF at room temperature and is
slowly transformed into the red product. Electrospray
mass spectrum (ESMS) shows that the composition of
this intermediate is only two protons less than the
combination of starting 2,3-diketopyrido[4,3,2-de]quino-
2. Am in a tion of 2,3-Dik etop yr id o[4,3,2-d e]qu in o-
lin e w ith Ar om a tic Am in es. Aromatic amines such as
aniline and 3,4-dimethoxyaniline can also react with 2,3-
diketopyrido[4,3,2-de]quinoline to form a more stable
purple product than that of aliphatic amines. For the
purple product from 3,4-dimethoxyaniline and 1-(ethoxy-
carbonylmethyl)-2,3-diketopyrido[4,3,2-de]quinoline (2),
the HRMS shows that the composition is only two protons
less than the combination of compound 2 and 3,4-
1
15
line and n-propylamine. The H NMR studies of the
N
labeled intermediate show that a proton and a n-propyl
1
5
group are attached to the N atom. This proton was
coupled with 15N atom with J N-H ) 92.5 Hz. The 1N-
15
15
1
dimethoxyaniline. H NMR spectrum shows a exchange-
5
hmqc spectrum also confirms the coupling between
N
able proton (12.27 ppm, NH) is contained in this product
which indicates that this proton is involved in a hydrogen
bond. To identify its structure, an X-ray diffraction
analysis was carried out on a Bruker P4/RA/SMART 1000
CCD diffractometer. The result of the X-ray diffraction
analysis shows that the 3,4-dimethoxyaniline group is
covalently bonded to C4 in the 2,3-diketopyrido[4,3,2-de]-
quinoline moiety (see Figure 3). A hydrogen bond exists
between the N-H and the 3-carbonyl group as was
previously deduced from NMR evidence. The plane of the
benzene ring in 3,4-dimethoxyaniline group is rotated out
off the pyrido[4,3,2-de]quinoline plane with a torsional
and this proton. The results of 13C NMR of this interme-
1
5
diate show that an aliphatic carbon (49.70 ppm, J N,
1
3
C ) 8.5 Hz) and an aromatic carbon (146.15 ppm, J
1
5
13
15
N, C ) 15.8 Hz) are directly linked to the N atom.
According to these results, the structure of this interme-
diate can therefore be proposed as shown in structure 6
(
Figure 2).
A hydrogen bond also exists between the 15N-H and
the 3-carbonyl group. It shifts this proton to lower field
1
5
(δ
NH ) 11.6 ppm for compound 6 in DCCl
3
). Evidently,
this proton can be easily transferred from the N atom to

7
968 J . Org. Chem., Vol. 64, No. 21, 1999
Ding and Lown
Sch em e 1. Rea ction of 2,3-Dik etop yr id o[4,3,2-d e]qu in olin es w ith Alip h a tic Am in es
Sch em e 2. Rea ction of
,3-Dik etop yr id o[4,3,2-d e]qu in olin es w ith Ar om a tic
Am in es
2
F igu r e 3. Perspective view of 4-(N-3,4-dimethoxyphenyl)-
amino-2,3-diketopyrido[4,3,2-de]quinoline 16 showing the atom
labeling scheme. Non-hydrogen atoms are represented by
Gaussian ellipsoids at the 20% probability level.
oxidant to oxidize the dihydro intermediate to form
compound 15 and water.
3
. Am in a tion of 2,3-Dik etop yr id o[4,3,2-d e]qu in o-
lin e w ith NH OH a n d NH NH . Both hydroxylamine
2
2
2
and hydrazine react with 2,3-diketopyrido[4,3,2-de]-
quinoline 2 to produce a red product. This red product
was identified by mp, H NMR, and HRMS. All of these
analytical results demonstrated that this red compound
is 4-amino-2,3-diketopyrido[4,3,2-de]quinoline 14, which
is the same as that produced from the reaction of 2,3-
diketopyrido[4,3,2-de]quinoline 2 with n-propylamine.
Thus there is no doubt that the nitrogen atom at the 4
position in the red product originates from hydroxylamine
or hydrazine. During the reaction, the OH group or the
angles of 58.3° (angle-C2N3C18C23 ) 58.3°, angle-
C2N3C18C19 ) -123.9°). The hydrogen bond stabilizes
the carbonyl group at C3 position and inhibits the
reaction of amino compound with this carbonyl group to
form the Schiff base.
1
We propose a mechanism in which the nucleophilic
addition by amino group takes place at the position-4 first
to form the dihydro intermediate. This intermediate is
then air-oxidized to form a more stable aromatic system
(
Scheme 2). The ratio of the oxygen involved in this
reaction was determined as 1.0 mol of 2,3-diketopyrido-
4,3,2-de]quinoline to 0.5 mol of molecular oxygen in
2
NH group is eliminated to afford the red compound 14.
Compound 1 reacts with hydroxylamine or hydrazine in
the manner similar to afford compound 13. The amino
group in hydroxylamine or hydrazine first undergoes a
nucleophilic addition to the double bond between C3a and
C4 to form a 3a,4-dihydro intermediate (V). This inter-
[
chloroform (see compound 15 in the Experimental Sec-
tion). Hydrogen peroxide may be formed during the
oxidation. This hydrogen peroxide can be used as an

Novel Amination and Deamination
J . Org. Chem., Vol. 64, No. 21, 1999 7969
Sch em e 3. Rea ction of
,3-Dik etop yr id o[4,3,2-d e]qu in olin es w ith
Hyd r oxyla m in e a n d Hyd r a zin e
Sch em e 4. Rea ction of
2,3-Dik etop yr id o[4,3,2-d e]qu in olin e 1 w ith r-a m in o
Acid s
2
mediate then undergoes an elimination reaction between
C4 and the amino group to form an imino group at the
4
-position (VI). The proton at the C3a is then transferred
to the oxygen at 3-position and finally transferred to the
nitrogen at 4-position to afford the red product, 4-amino-
2
,3-diketopyrido[4,3,2-de]quinoline (13, 14) (Scheme 3).
. Am in a tion a n d Elim in a tion betw een 2,3-Dik e-
4
top yr id o[4,3,2-d e]qu in olin e a n d r-Am in o Acid s. Gly-
cine can react with 2,3-diketopyrido[4,3,2-de]quinoline (1)
in aqueous solution to form the same product, 4-amino-
Sch em e 5. Rea ction of P yr id oxa l w ith r-Am in o
Acid s
2
,3-diketopyrido[4,3,2-de]quinoline (13), as was formed
with n-propylamine. Because glycine lacks a â-proton, the
mechanism cannot follow that elucidated with n-propy-
lamine, but resembles that with pyridoxal. Alanine,
phenylanaline, and tryptophan can also react in a similar
manner with 2,3-diketopyrido[4,3,2-de]quinoline (1) to
produce 4-amino-2,3-diketopyrido[4,3,2-de]quinoline (13).
The R-amino group undergoes nucleophilic addition to the
double bond between C3a and C4 to form a dihydro
intermediate (VIII) which is then oxidized to 4-(N-
carboxymethyl)amino-2,3-diketopyrido[4,3,2-de]quino-
line (IX) (Scheme 4). In analogy with pyridoxal (17)
(Scheme 5), in the presence of base, 4-(N-carboxymethyl)-
amino-2,3-diketopyrido[4,3,2-de]quinoline (IX) can be
transformed into 3-hydroxy-6-hydropyrido[4,3,2-de]quin-
oline (XI) which contains a carboxymethyleneamino
group at the 4-position. The NdC double bond formed in
this intermediate is then hydrolyzed by water to form
an amino group at 4-position affording a product (XII)
which is the reduced form of the final product of 4-amino-
group in methionine and an ω-amino group in lysine.
After reaction with 2,3-diketopyrido[4,3,2-de]quinoline 1,
the 1:1 conjugate (19) and its hydrolysis product (20) were
produced as the major products (Scheme 6). The 1:2
conjugate was also determined in the reaction solution
by using the MALDI MS analysis technology. It is
demonstrated that both of the amino groups in lysine and
in methionine can react with 2,3-diketopyrido[4,3,2-de]-
2
,3-diketopyrido[4,3,2-de]quinoline (13).
. Am in a tion of 2,3-Dik etop yr id o[4,3,2-d e]qu in o-
lin e w ith P ep tid es. 2,3-Diketopyrido[4,3,2-de]quinoline
can react with peptides which contain an R-amino
5
1
group or lysine residue to form 2,3-diketopyrido[4,3,2-
de]quinoline-peptide conjugates. Substance P (18) is
composed of 11 amino acids and contains an R-amino
quinoline to form the conjugates. The CH
3
S group was
oxidized during this amination reaction. This may be
caused by hydrogen peroxide which was produced by the

7
970 J . Org. Chem., Vol. 64, No. 21, 1999
Ding and Lown
Sch em e 6. Rea ction of
,3-Dik etop yr id o[4,3,2-d e]qu in olin e 1 w ith
diketopyrido[4,3,2-de]quinoline undergoes a 1,2-amino,-
hydro-elimination reaction to eliminate an alkene and
produce the 4-amino-2,3-diketopyrido[4,3,2-de]quinolines.
In the cases of R-amino acids and compound 1 in aqueous
solution, the 4-(N-alkyl)-2,3-diketopyrido[4,3,2-de]quino-
line undergoes another deamination reaction, via a
mechanism similar to the action of pyridoxal, to form the
2
Su bsta n ce P
4
-amino-2,3-diketopyrido[4,3,2-de]quinoline 13 and the
R-keto acid. 2,3-Diketopyrido[4,3,2-de]quinoline (1) can
also react with peptides which contain primary amino
groups to form the 2,3-diketopyrido[4,3,2-de]quinoline-
peptide conjugates. This novel amination-elimination
reaction may underlie the marked cytotoxic potency of
the 2,3-diketopyrido[4,3,2-de]quinolines (1 and 2). As
inorganic amino compounds, hydroxylamine and hydra-
zine can also undergo the nucleophilic addition to 2,3-
diketopyrido[4,3,2-de]quinoline (1, 2) to produce 4-amino-
2
,3-diketopyrido[4,3,2-de]quinoline (13, 14), which includes
an elimination reaction between C4 and R-nitrogen. The
amination reaction between 2,3-diketopyrido[4,3,2-de]-
quinoline and nucleotides as well as nucleic acids are
ongoing.
Exp er im en ta l Section
The hmqc 15N spectra were recorded on the Varian Unity
5
00 MHz spectrometer. High-resolution mass spectra (HRMS)
were recorded on a modified KRATOS MS-50 mass spectrom-
eter equipped with a VG11-250J data system. Electrospray
mass spectra were recorded on a Micromass ZabSpec spec-
trometer. MALDI MS analysis was performed on a Model
G2025A time-of-flight system (Hewlett-Packard, Reno, NV).
The X-ray diffraction analysis was carried out on a Bruker
P4/RA/SMART 1000 CCD diffractometer. The programs were
used in this analysis are DIRDIF-96 program system (Crystal-
lography Laboratory, University of Nijmegen, The Nether-
lands) and SHELXL-93 program for crystal structure deter-
mination (University of G o¨ ttingen, Germany). Preparative
separations were performed by flash chromatography on silica
gel (Merck, 70-230 or 230-400 mesh). Substance P was
purchased from Sigma and stored under -20 °C. Tetrahydro-
furan was dried by distillation from sodium benzophenone
ketyl. Triethylamine was dried over molecular sieves (3 Å)
before use. All other solvents were used as received and were
reagent grade where available.
1
5
1
H-2,3-Dik eto-4-a m in o( N)-5-ch lor o-7,8-d im eth oxyp y-
r id o[4,3,2-d e]qu in olin e (3). A solution of 29 mg (0.10 mmol)
of 1H-2,3-diketopyrido[4,3,2-de]quinoline (1) in 20 mL of THF
1
5
was mixed with 100 mg of N-labeled-n-propylamine hydro-
1
5
+
-
3 2 2 3
chloride (CH CH CH N H Cl ) and 10 drops of triethy-
lamine. The mixture was stirred at room temperature for 24
h. The resulting solution was then subjected to column
chromatography (5:1 of ethyl acetate to hexane) to give 25 mg
1
5
air-oxidation of the 3a,4-dihydro intermediate and will
be examined further in a separate study.
of the red product, 1H-2,3-diketo-4-amino( N)-5-chloro-7,8-
dimethoxypyrido[4,3,2-de]quinoline (3). Yield 83%, mp > 300
1
4
15
°
3
9
C. HRMS: calcd for C13
H
10
N
2
N
1
O
4
Cl 308.0360, found
+
1
08.0365 (M , 100%); H NMR (DMSO-d ): δ 11.92 (s, 1 H),
6
Con clu sion s
1
5
15
15
.57 (d, 1 H, N-Ha, J N-Ha ) 92 Hz), 8.26 (d, 1 H, N-
1
5
Hb, J N-Hb ) 92 Hz), 7.20 (s, 1 H), 3.90 (s, 3 H), 3.88 (s, 3
The double bond between C3a and C4 in 2,3-diketo-
pyrido[4,3,2-de]quinoline (1 or 2) is highly active site for
nucleophilic addition. A novel amination-elimination
reaction occurs between 2,3-diketopyrido[4,3,2-de]quino-
line (1, 2) and amino compounds which include R-amino
acids and peptides. The amino group undergoes nucleo-
philic addition to the double bond between C3a and C4
to form a 3a,4-dihydro-2,3-diketopyrido[4,3,2-de]quino-
line. This dihydro intermediate is immediately oxidized
by ambient air to produce the more stable aromatic
system, 4-(N-alkyl or aryl)-2,3-diketopyrido[4,3,2-de]-
quinoline. In the cases of aliphatic amines bearing a
â-proton in THF or chloroform, this 4-(N-alkyl)-2,3-
1
4
15
H). Anal. Calcd for C13
10 2 1 4
H N N O Cl: C, 50.58; H, 3.27; N,
1
3.93; Cl, 11.50. Found C, 50.26; H, 3.42; N, 13.67; Cl, 11.28.
15
1
-(Eth oxyca r bon ylm eth yl)-2,3-d ik eto-4-a m in o( N)-5-
ch lor o-7,8-d im eth oxyp yr id o[4,3,2-d e]qu in olin e (4). A so-
lution of 38 mg (0.10 mmol) of 1-(ethoxycarbonylmethyl)-2,3-
diketopyrido[4,3,2-de]quinoline (2) in 20 mL of THF was mixed
1
5
with 100 mg of N labeled n-propylamine hydrochloride
1
5
+
-
(
3 2 2 3
CH CH CH N H Cl ) and 10 drops of triethylamine. The
mixture was stirred at room temperature for 24 h. The
resulting solution was then subjected to column chromatog-
raphy (1:1 of ethyl acetate to hexane then 4:1 of ethyl acetate
to hexane) to give 35 mg of the red product, 1-(ethoxycarbo-
15
nylmethyl)-2,3-diketo-4-amino( N)-5-chloro-7,8-dimethoxy-
py-

Novel Amination and Deamination
J . Org. Chem., Vol. 64, No. 21, 1999 7971
rido[4,3,2-de]quinoline (4). Yield 88%, mp 265-7 °C, HRMS:
4.06 (m, 2 H), 4.10 (s, 3 H), 3.97 (s, 3 H), 1.84 (m, 2 H), 1.26 (t,
3 H, J ) 7.2 Hz), 1.12 (t, 3 H, J ) 7.2 Hz).
1
4
15
+
calcd for C17
16 2 1 6
H N N O Cl 394.0698, found 394.0698 (M ,
1
15
15
1
)
9
3
C
00%); H NMR (CDCl
3
): δ 9.88 (d, 1 H, N-Ha, J N-Ha
The syntheses of compound 9-12 were carried out by the
similar manner of compound 7 and 8. These compounds were
isolated from the reaction solution as soon as possible and
1
5
15
92.5 Hz), 6.89 (s, 1 H), 6.34 (d, 1 H, N-Hb, J N-Hb )
2.5 Hz), 5.10 (s, 2 H), 4.26 (q, 2 H, J ) 7.2 Hz), 4.13 (s, 3 H),
1
.99 (s, 3 H), 1.28 (t, 3 H, J ) 7.2 Hz). Anal. Calcd for
identified by H NMR spectra and electrospray mass spectra.
1
4
15
17
H
16
N
2
N
1
O
6
Cl: C, 51.72; H, 4.09; N, 10.89; Cl, 8.99.
They are not stable in the solution especially in the basic
solution.
1H-2,3-Dik eto-4-a m in o-5-ch lor o-7,8-d im eth oxyp yr id o-
Found C, 51.48; H, 3.86; N, 10.96; Cl, 9.35.
H -2,3-D i k e t o -4-(1 5 N -p r o p y l)a m i n o -5-c h lo r o -7,8-
1
[
1
4,3,2-d e]qu in olin e (13). A solution of 29 mg (0.10 mmol) of
H-2,3-diketopyrido[4,3,2-de]quinoline (1) in 20 mL of THF
d im eth oxyp yr id o[4,3,2-d e]qu in olin e (5). A solution of 29
mg (0.10 mmol) of 1H-2,3-diketopyrido[4,3,2-de]quinoline (1)
1
5
was mixed with 50 mg of n-propylamine and 5 drops of
triethylamine. The mixture was stirred at room temperature
for 24 h. The resulting solution was then subjected to column
chromatography (5:1 of ethyl acetate to methanol) to give 26
mg of the red product, 1H-2,3-diketo-4-amino-5-chloro-7,8-
dimethoxypyrido[4,3,2-de]quinoline (13). Yield 85%, mp > 300
in 20 mL of THF was mixed with 100 mg of N labeled
1
5
+
-
3 2 2 3
n-propylamine hydrochloride (CH CH CH N H Cl ) and 10
drops of triethylamine. The mixture was stirred at room
temperature for 3 h. The purple intermediate formed during
the reaction was isolated immediately by flash chromatogra-
phy (4:1 of ethyl acetate to hexane and then ethyl acetate) to
1
5
°C. HRMS: calcd for C13
H
10
N
3
O
4
Cl 307.0360, found 307.0361
): δ 11.92 (s, 1 H), 9.57 (s, 1
H), 8.28 (s, 1 H), 7.20 (s, 1 H), 3.90 (s, 3 H), 3.88 (s, 3 H). Anal.
Calcd for C13 Cl: C, 50.75; H, 3.28; N, 13.66; Cl, 11.52.
give 20 mg of the purple 1H-2,3-diketo-4-( N-propyl)amino-
+
1
(M , 100%); H NMR (DMSO-d
6
5
-chloro-7,8-dimethoxypyrido[4,3,2-de]quinoline (5). Yield 57%.
1
4
15
+
ESMS: calcd for C16
H
16
N
2
N
1
O
4
Cl 350.1, found 351.1 (MH ,
1
10 3 4
H N O
1
00%); H NMR (DMSO-d
N-Ha, J N-Ha ) 92 Hz), 7.23 (s, 1 H), 3.90 (s, 3 H), 3.88
6
): δ 11.95 (s, 1 H), 10.85 (d, 1 H,
1
5
15
Found C, 51.03; H, 3.43; N, 13.78; Cl, 11.83.
1-(Eth oxycar bon ylm eth yl)-2,3-diketo-4-am in o-5-ch lor o-
1
5
(
)
s, 3 H), 3.84 (m, 2 H, NCH
7.2 Hz).
-(E t h oxyca r b on ylm et h yl)-2,3-d ik et o-4-(15N-p r op yl)-
2
), 1.72 (m, 2 H), 1.02 (t, 3 H, J
7
,8-d im eth oxyp yr id o[4,3,2-d e]qu in olin e (14). A solution of
3
8 mg (0.10 mmol) of 1-(ethoxycarbonylmethyl)-2,3-diketopy-
1
rido[4,3,2-de]quinoline (2) in 20 mL of THF was mixed with
0 mg of n-propylamine and 5 drops of triethylamine. The
a m in o-5-ch lor o-7,8-d im e t h oxyp yr id o[4,3,2-d e]q u in o-
lin e (6). A solution of 38 mg (0.10 mmol) of 1-(ethoxycarbon-
ylmethyl)-2,3-diketopyrido[4,3,2-de]quinoline (2) in 20 mL of
5
mixture was stirred at room temperature for 24 h. The
resulting solution was then subjected to column chromatog-
raphy (1:1 of ethyl acetate to hexane and then 4:1 of ethyl
acetate to hexane) to give 36 mg of the red product, 1-(ethoxy-
carbonylmethyl)-2,3-diketo-4-amino-5-chloro-7,8-dimethoxypy-
1
5
THF was mixed with 100 mg of N labeled n-propylamine
1
5
+
-
hydrochloride (CH
ethylamine. The mixture was stirred at room temperature for
.5 h. The purple intermediate formed during the reaction was
3 2 2 3
CH CH N H Cl ) and 10 drops of tri-
0
rido[4,3,2-de]quinoline (14). Yield 91%, mp 266-7 °C. HRMS:
isolated immediately by flash chromatography (1:1 of ethyl
acetate to hexane and then 4:1 of ethyl acetate to hexane) to
give 32 mg of the purple 1-(ethoxycarbonylmethyl)-2,3-diketo-
+
calcd for C17
H
16
N
3
3
O
6
Cl 393.0727, found 393.0726 (M , 100%);
1
H NMR (CDCl
.10 (s, 2 H), 4.27 (q, 2 H, J ) 7.2 Hz), 4.13 (s, 3 H), 3.99 (s, 3
Cl:
): δ 9.83 (s, 1 H), 6.90 (s, 1 H), 6.33 (s, 1 H),
5
1
5
4
-( N-propyl)amino-5-chloro-7,8-dimethoxypyrido[4,3,2-de]-
H), 1.28 (t, 3 H, J ) 7.2 Hz). Anal. Calcd for C17
16 3 6
H N O
1
4
15
quinoline (6). Yield 73%. ESMS: calcd for C20
H
22
N
2
N
1
O
6
Cl
): δ 11.62
d, 1 H, N-H, J N-H ) 92 Hz), 6.84 (s, 1 H), 5.10 (s, 2 H),
C, 51.85; H, 4.06; N, 10.67; Cl, 9.02. Found C, 52.18; H, 4.34;
N, 10.78; Cl, 9.20.
+
1
4
(
36.1, found 437.1 (MH , 100%); H NMR (CDCl
3
1
5
15
1
-(Eth oxycar bon ylm eth yl)-2,3-diketo-4-(N-ph en yl)am i-
1
5
4
3
3
4
6
1
1
.23 (q, 2 H, J ) 7.2 Hz), 4.08 (m, 2 H, NCH
2
), 4.13 (s, 3 H),
n o-5-ch lor o-7,8-d im eth oxyp yr id o[4,3,2-d e]qu in olin e (15)-
.97 (s, 3 H), 1.85 (m, 2 H), 1.27 (t, 3 H, J ) 7.2 Hz), 1.12 (t,
.
A solution of 175 mg (0.60 mol) of 1-(ethoxycarbonylmethyl)-
1
3
H, J ) 7.2 Hz). C NMR (CDCl ): δ 12.012, 14.894, 24.756,
15 13
3
2
,3-diketopyrido[4,3,2-de]quinoline (2) in 50 mL of chloroform
1
5
2 2 3
5.661, 49.696 ( NCH CH CH , J N, C ) 8.5 Hz), 58.490,
was placed in a three-neck reaction flask which was connected
with a volumeter system. The reaction system was filled with
air and sealed with water in a U-type tube. Under atmospheric
pressure (745 mmHg) at 23 °C, the solution was injected with
2.793, 63.579, 104.534, 109.045, 111.629, 126.585, 132.946,
41.167, 142.376, 146.149 (4-C, J 15N, C ) 15.8 Hz), 149.966,
58.071, 168.151, 175.159.
13
1
H-2,3-Diketo-4-(N-pr opyl)am in o-5-ch lor o-7,8-dim eth ox-
0
.5 mL of triethylamine followed by 0.5 mL of aniline. The
yp yr id o[4,3,2-d e]qu in olin e (7). A solution of 29 mg (0.10
mmol) of 1H-2,3-diketopyrido[4,3,2-de]quinoline (1) in 20 mL
of THF was mixed with 50 mg of n-propylamine and 5 drops
of triethylamine. The mixture was stirred at room temperature
for 3 h. The purple intermediate formed during the reaction
was isolated immediately by flash chromatography (4:1 of ethyl
acetate to hexane and then ethyl acetate) to give 22 mg of the
purple 1H-2,3-diketo-4-(N-propyl)amino-5-chloro-7,8-dimethox-
ypyrido[4,3,2-de]quinoline (7). Yield 63%. ESMS: calcd for
resulting mixture was stirred at 23 °C under atmospheric
pressure for 24 h, and the difference of the volume in the
reaction atmosphere was measured as 7.4 mL which is
2
equivalent to 0.299 mol of oxygen (O ). The reaction solution
was washed with 1 N of hydrochloric acid (50 mL × 2) and
then subjected to column chromatography (1:1 of ethyl acetate
to hexane and then 4:1 of ethyl acetate to hexane) to give 160
mg of the purple product, 1-(ethoxycarbonylmethyl)-2,3-diketo-
4
-(N-phenyl)amino-5-chloro-7,8-dimethoxypyrido[4,3,2-de]quin-
+
1
C
16
H
16
N
3
O
6
4
Cl 349.1, found 350.1 (MH , 100%); H NMR
): δ 11.95 (s, 1 H), 10.85 (s, 1 H), 7.23 (s, 1 H), 3.90
s, 3 H), 3.88 (s, 3 H), 3.84 (m, 2 H), 1.72 (m, 2 H), 1.02 (t, 3 H,
oline (15). Yield 57%, mp 205-7 °C. HRMS: calcd for
(DMSO-d
+
1
C
23
H
20
N
3
O
6
Cl 469.1041, found 469.1041 (M , 100%); H NMR
(
(
CDCl ): δ 12.15 (s, 1 H), 7.41 (t, 2 H, J ) 7.4 Hz), 7.30 (t, 1
3
J ) 7.2 Hz).
H, J ) 7.4 Hz), 7.18 (d, 2 H, J ) 7.4 Hz), 6.92 (s, 1 H), 5.15 (s,
1
-(Eth oxyca r bon ylm eth yl)-2,3-d ik eto-4-(N-p r op yl)a m i-
2 H), 4.32 (q, 2 H, J ) 7.2 Hz), 4.16 (s, 3 H), 4.02 (s, 3 H), 1.34
n o-5-ch lor o-7,8-d im eth oxyp yr id o[4,3,2-d e]qu in olin e (8)-
20 3 6
(t, 3 H, J ) 7.2 Hz). Anal. Calcd for C23H N O Cl: C, 58.79;
.
A solution of 38 mg (0.10 mmol) of 1-(ethoxycarbonylmethyl)-
H, 4.29; N, 8.94; Cl, 7.55. Found C, 58.68; H, 4.41; N, 9.21; Cl,
7.88.
2
,3-diketopyrido[4,3,2-de]quinoline (2) in 20 mL of THF was
mixed with 50 mg of n-propylamine and 5 drops of triethy-
lamine. The mixture was stirred at room temperature for 0.5
h. The purple intermediate formed during the reaction was
isolated immediately by flash chromatography (1:1 of ethyl
acetate to hexane and then 4:1 of ethyl acetate to hexane) to
give 35 mg of the purple compound, 1-(ethoxycarbonylmethyl)-
1-(E t h o x y c a r b o n y lm e t h y l)-2,3-d i k e t o -4-[N -(3,4-
d im eth oxy)p h en yl]a m in o-5-ch lor o-7,8-d im eth oxyp yr id o-
[4,3,2-d e]qu in olin e (16). A solution of 38 mg (0.10 mmol) of
1-(ethoxycarbonylmethyl)-2,3-diketopyrido[4,3,2-de]quino-
line (2) in 20 mL of chloroform was mixed with 100 mg of 3,4-
dimethoxyaniline and 10 drops of triethylamine. The mixture
was stirred at room temperature for 24 h. The resulting
solution was washed with 1 N of hydrochloric acid (50 mL ×
2) and then subjected to column chromatography (1:1 of ethyl
acetate to hexane and then 4:1 of ethyl acetate to hexane) to
2
,3-diketo-4-(N-propyl)amino-5-chloro-7,8-dimethoxypyrido-
[
4,3,2-de]quinoline (8). Yield 80%. ESMS: calcd for C20H N O -
22 3 6
+
1
Cl 435.1, found 436.1 (MH , 100%); H NMR (CDCl
(
3
): δ 11.58
s, 1 H), 6.82 (s, 1 H), 5.08 (s, 2 H), 4.234 (q, 2 H, J ) 7.2 Hz),

7
972 J . Org. Chem., Vol. 64, No. 21, 1999
Ding and Lown
1
give 40 mg of the purple product, 1-(ethoxycarbonylmethyl)-
,3-diketo-4-[N-(3,4-dimethoxy)phenyl]amino-5-chloro-7,8-
dimethoxypyrido[4,3,2-de]quinoline (16). Yield 75%, mp 225-7
C. HRMS: calcd for C25 Cl 529.1252, found 529.1229
): δ 12.27 (s, 1 H), 6.94 (s, 1 H),
.90 (d, 1 H, J ) 6.3 Hz), 6.70-6.80 (m, 2 H), 5.12 (s, 2 H),
.30 (q, 2 H, J ) 7.2 Hz), 4.14 (s, 3 H), 4.01 (s, 3 H), 3.93 (s, 3
results of H NMR and HRMS show that this precipitate is
the pure compound of 4-amino-2,3-diketopyrido[4,3,2-de]quino-
line (13).
2
°
H
24
N
3
O
8
Rea ction of Su bsta n ce P w ith 2,3-Dik etop yr id o[4,3,2-
d e]qu in olin e 1. A solution of 10 µL of 1.2 mM Substance P
and 10 µL of 10 mM of 1H-2,3-diketopyrido[4,3,2-de]quinoline
1 in DMSO were mixed in 1 mL of water whose pH value was
adjusted to 9 with triethylamine. The resulting mixture was
stirred at 37 °C and subjected to MALDI mass and electrospray
mass analysis at 10 min, 3.5 h, and 3 days. Samples were
typically prepared by using the two-layer method. The first
layer was formed by depositing 1 µL of 12 mg/mL matrix
material in acetone on the probe,41 followed by addition of 1
µL of standards or sample mixed with matrix. MALDI MS:
+
1
(M , 100%); H NMR (CDCl
3
6
4
H), 3.89 (s, 3 H), 1.31 (t, 3 H, J ) 7.2 Hz). Anal. Calcd for
25 24 3 8
C H N O
Cl: C, 56.66; H, 4.56; N, 7.93; Cl, 6.69. Found C,
5
6.68; H, 4.38; N, 8.21; Cl, 6.94.
4
0
Compound 16 can be crystallized from 4:1 of ethyl acetate
to hexane to give single crystals.
Rea ction of Hyd r oxyla m in e or Hyd r a zin e w ith 2,3-
Dik etop yr id o[4,3,2-d e]qu in olin es. A solution of 38 mg (0.10
mmol) of 1-(ethoxycarbonylmethyl)-2,3-diketopyrido[4,3,2-de]-
quinoline (2) in 20 mL of THF was mixed with 100 mg of
hydroxylamine hydrochloride (or hydrazine monohydrate) and
+
calcd for 19 1654.3, found 1655.1 (MH ). ESMS: calcd for 19
1654.3, found 1655.7 (MH ).
+
1
0 drops of triethylamine. The mixture was stirred at room
Ack n ow led gm en t. We gratefully thank the Natural
Sciences and Engineering Research Council of Canada
for financial support of this work (to J .W.L.).
temperature for 24 h. A red product formed in the solution.
After removing the solvent, the residue was extracted with
chloroform (30 mL × 2) and subjected to the column chroma-
tography (1:1 of ethyl acetate to hexane and then 4:1 of ethyl
acetate to hexane and then ethyl acetate). The red product
was identified by mp, H NMR, and HRMS. All of these
analytical results demonstrate that this red product is identical
with compound 14.
Rea ction of <bold>R-Am in o Acid s w ith 2,3-Dik etop y-
r id o[4,3,2-d e]qu in olin e 1. A solution of 29 mg (0.10 mmol)
of 1H-2,3-diketopyrido[4,3,2-de]quinoline (1) was suspended
in 30 mL of water whose pH value was adjusted to 10 with
triethylamine, and 76 mg (10 mmol) of glycine was added. The
resulting mixture was stirred at room temperature for 72 h.
After neutralizing with diluted hydrochloric acid, the precipi-
tate was collected by filtration and washed with water. The
Su p p or tin g In for m a tion Ava ila ble: ORTEP drawings
for 16. Crystallographic experimental details, atomic coordi-
nates and equivalent isotropic displacement parameters, bond
lengths and angles, torsional angles, anisotropic displacement
parameters, derived atomic coordinates, and displacement
1
1
15
parameters for hydrogen atoms. Two H NMR spectra for N-
13
15
labeled compound 4 and 6 and one C NMR spectrum for N-
labeled compound 6.
J O9910132
(
40) Dai, Y. Q.; Whittal, R. M.; Li, L. Anal. Chem. 1996, 68, 2494.
(41) Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281.