Synthesis of Imidazolo Analogues of the Oxidation-Reduction Cofactor Pyrroloquinoline Quinone (PQQ)

Article abstract of DOI:10.1021/jo035390x

Parallel syntheses of 2-hydro-, 2-methyl-, and 2-methoxycarbonylimidazo-7,9-dimethoxycarbonyl analogues of the oxidation-reduction cofactor pyrroloquinoline quinone [4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid] have been developed. The properties of the imidazolo analogues in relation to the corresponding pyrrole analogues will be important in assessing the origins of catalysis and biological activity in the cofactor, which has recently been shown to be a vitamin.

Full text of DOI:10.1021/jo035390x

Syn th esis of Im id a zolo An a logu es of th e
Oxid a tion -Red u ction Cofa ctor
P yr r oloqu in olin e Qu in on e (P QQ)
David M. D. Fouchard,1,† L. M. V. Tillekeratne,†,‡ and
Richard A. Hudson*,†,‡
Department of Medicinal and Biological Chemistry,
College of Pharmacy, and Department of Chemistry,
College of Arts and Sciences, University of Toledo,
Toledo, Ohio 43606
richard.hudson@utoledo.edu
Received September 23, 2003
Abstr a ct: Parallel syntheses of 2-hydro-, 2-methyl-, and
2-methoxycarbonylimidazo-7,9-dimethoxycarbonyl analogues
of the oxidation-reduction cofactor pyrroloquinoline quinone
[4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tri-
carboxylic acid] have been developed. The properties of the
imidazolo analogues in relation to the corresponding pyrrole
analogues will be important in assessing the origins of
catalysis and biological activity in the cofactor, which has
recently been shown to be a vitamin.
F IGURE 1. Structure of PQQ and several isomers.
basis for the determination of their presence or absence
in representative biological systems.9 We have synthe-
sized and report here imidazolo analogues of PQQ, where
the pyrrole ring is replaced by imidazole, to assess the
importance of the pyrrole ring in the catalytic function
of PQQ (5-7, Figure 2). As a mechanistically relevant
equilibration of the tautomeric forms10 of the imidazole
ring during the course of catalysis may likely depend on
the nature of the substituent at C-2, the syntheses have
been developed to allow incorporation of variable func-
tional groups in position 2.
In our plan for the synthesis of the targeted imidazole
derivatives of pyrroloquinoline quinone (5-7), we started
by assembling an appropriately substituted benzimida-
zole moiety and then constructed the quinoline ring
system. This approach was analogous to the one taken
by Corey and Tramontano in the synthesis of PQQ11 and
subsequently adapted by others to the synthesis of
several isomeric8 and nonisomeric analogues12 of PQQ.
In the synthesis of the 2-methyl analogue 6 (Scheme
1) compound 8 was prepared by directly nitrating com-
mercially available 2-methoxy-4-nitroaniline followed by
selective reduction of the o-nitro group using conditions
analogous to those reported to be favorable to the
reduction of nitro groups ortho to amino and hydroxy
Pyrroloquinoline quinone (1, PQQ, methoxatin, 4,5-
dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricar-
boxylic acid) is an oxidation-reduction cofactor found in
methylotropic bacteria. The structure of the cofactor was
elucidated in 1979, when it was isolated from Psuedomo-
nas.2 PQQ as well as other quinonoid cofactors have now
been found in association with many enzymes from a
variety of organisms.3 In bacteria, in addition to being a
redox cofactor, PQQ was shown to be a growth factor.4
Important physiological roles have also been suggested
for PQQ in eukaryotes.5 Indeed, the documented role of
PQQ as a micronutrient in mammals,5 the absence of
biosynthesis pathways for PQQ in eukaryotes, and the
recent demonstration that PQQ is a required cofactor in
a mammalian enzyme6 have established PQQ as a
vitamin.
Preparation of key analogues of this vitamin will be
important in understanding the mechanism of action of
PQQ. Isomeric analogues of PQQ as well as analogues
with simple isoelectronic substitutions may be particu-
larly interesting in that regard. In addition, PQQ ana-
logues, particularly those with altered redox potentials,
may be usefully integrated into microsensors.7 Previ-
ously, we prepared several PQQ isomers8 (2-4, Figure
1), allowing the establishment of their properties and the
(7) Inoue, T.; Kirchhoff, J . R. Anal. Chem. 2000, 72, 5755. For further
applications, see: Katz, E.; Willner, I. J . Am. Chem. Soc. 2003, 125,
6803 and Katz, E.; Buckmann, A. F.; Willner, I. J . Am. Chem Soc.
2001, 123, 10753. For a review, see: Ikeda, T.; Kano, K. Biochim.
Biophys. Acta 2003, 1647, 121.
(8) Zhang, Z. P.; Tillekeratne, L. M. V.; Hudson, R. A. Synthesis
1996, 377. Martin, P.; Winkler, T. Helv. Chim. Acta 1994, 77, 100.
(9) Zhang, Z. P.; Tillekeratne, L. M. V.; Hudson, R. A. Biochem.
Biophys. Res. Commun. 1995, 212, 41. Smith, A. R.; Kirchhoff, J . R.;
Tillekeratne, L. M. V.; Hudson, R. A. Anal. Commun. 1999, 136, 371.
Smith, A. R.; Kirchhoff, J . R.; Tillekeratne, L. M. V.; Hudson, R. A. J .
Chromatogr. A 2000, 816, 193.
(10) Itoh, S.; Fukui, Y.; Haranou, S.; Ogino, M.; Komatsu, M.; Oshiro,
Y. J . Org. Chem. 1992, 57, 4452. Only a single tautomeric form of each
of the final imidazole analogues 5-7 is shown for each isomer. While
the spectroscopic properties are consistent with the presence of a single
tautomeric form, we cannot specify which form is present. In Figure 2
and in Schemes 1-3, only a single tautomeric form of the imidazole is
shown by convention.
(11) Corey, E. J .; Tramontano, A. J . Am. Chem. Soc. 1981, 103, 5599.
(12) Itoh, S.; Kato, J .; Onoue, T.; Kitamura, Y.; Komatsu, M.; Oshiro,
T. Synthesis 1987, 1067. J ongejan, J . A.; Bezemer, R. P.; Duine, J . A
Tetrahedron Lett. 1988, 29, 3709. Itoh, S.; Fukui, Y.; Ogino, M.;
Haramou, S.; Komatsu, M.; Ohshiro, T. J . Org. Chem 1992, 57, 2788.
† College of Arts and Sciences.
‡ College of Pharmacy.
(1) Current address: Department of Chemistry and Biochemistry,
Montana State University (Gaines Hall, #328), Bozeman, MT 59715.
(2) Salisbury, S. A.; Forest, H. S.; Cruse, W. B.; Kennard, O. A.
Nature (London) 1979, 280, 843.
(3) For a review, see: Klinman, J . P.; Mu, D. Annu. Rev. Biochem.
1994, 63, 299. Anthony, C.; Ghosh, M. Curr. Sci. 1997, 72, 716.
Anthony, C. Antioxid. Redox. Signaling 2001, 3, 757.
(4) Ameyama, M.; Shinagawa; E.; Matsushita, K.; Adachi, O. Agric.
Biol. Chem. 1984, 48, 2909.
(5) For a review, see: McIntire, W. S. Annu. Rev. Nutr. 1998, 18,
145. Stites, T. E.; Mitchell, A. E.; Rucker, R. B. J . Nutr. 2000, 130,
719.
(6) Kasahara, T.; Kato, T. A. Nature (London) 2003, 422, 832.
10.1021/jo035390x CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/05/2004
2626
J . Org. Chem. 2004, 69, 2626-2629

F IGURE 2. Structures of imidazole analogues of PQQ.
SCHEME 1
SCHEME 2
substituents.13 Compound 8 was previously synthesized
in three steps beginning with 3,5-dinitroanisole.14
The treatment of 2-amino-3-methoxy-5-nitroaniline 8
with glacial acetic acid produced the nitrobenzimidazole
9, which was subsequently reduced by catalytic hydro-
genation to yield the amine 10. Coupling of 10 with
dimethyl trans-2-ketoglutaconate under Doebner-von
Miller conditions11 resulted in the imidazoquinoline 11
in good yield. Demethylation of 11 was carried out in
quantitative yield using HBr in glacial acetic acid. During
this process, the two ester groups were also cleaved and
re-esterification was acomplished quantitatively without
intermediate purification to yield 12. This product was
then treated with Fremy’s salt in aqueous acetonitrile
to generate the target 6.
In the synthesis of the 2-hydroimidazolo-PQQ analogue
7 (Scheme 2), we employed the known benzimidazole
13,15 which was prepared from compound 8 and subse-
quently treated with formic acid in the presence of
palladium on carbon followed by subsequent base hy-
drolysis of the intermediate formanilide to generate the
amine 13. Compound 13 also has been previously syn-
thesized by an alternative five-step method.14 When
treated with dimethyl trans-2-ketoglutaconate, 13 un-
derwent cyclization to form the quinoline derivative 14
in modest yield. Removal of the methoxy group of 14 was
accomplished quantitatively using HBr in glacial acetic
acid, followed by quantitative re-esterification of the two
cleaved carboxylic acid groups to yield 15. Oxidation of
15 using Fremy’s salt led to the quinone 7.
The synthesis of the imidazolo-PQQ analogue 5 (Scheme
(13) Terpko, M. O.; Heck, R. F. J . Org. Chem. 1980, 45, 4992.
(14) Gillespie, H. B.; Engelman, M.; Graff, S. J . Am. Chem. Soc.
1954, 76, 3531
(15) Tsuji, J .; Mandai, T. Synthesis 1996, 1,
J . Org. Chem, Vol. 69, No. 7, 2004 2627

SCHEME 3
3) proved initially to be quite challenging. Several ap-
proaches were undertaken. Alternative routes to com-
pound 5 were considered starting from 11 and 14. We
attempted without success to selectively oxidize the
2-methyl group in 11.16 A lithiation-carboxylation se-
quence at the 2-position of 14 was also attempted without
success.17 Despite the use of a variety of protecting
conditions such as carbon dioxide,18 pyrrolidine methy-
lation,19 and formaldehyde,20 the benzimidazole nitrogen
could not be protected effectively, rendering lithiation at
C-2 impractical.
Compound 5 was finally synthesized starting from the
diamine 8 using procedures analogous to those employed
for the previous two analogues (6 and 7). However, the
initial formation of the key benzimidazole required a
special condensing agent. The benzimidazole 16 was
obtained in good yield by condensation of 8 with ethyl
triethoxyacetate.21 The nitrobenzimidazole 16 was quan-
titatively reduced to the amine 17 by palladium-catalyzed
hydrogenation, and the pyridine ring was assembled
using Doebner-von Miller conditions to yield 18. Depro-
tection of methoxy in 18 was achieved by prolonged
treatment in HBr in acetic acid with simultaneous
hydrolysis of all three ester groups. Re-esterification
without purification led to the phenolic trimethyl ester
19 in excellent overall yield. Oxidation of 19 with Fremy’s
salt led to the quinone 5 in modest yield.
reactions of the native coenzyme. Substituting an imi-
dazole ring for the pyrrole ring of PQQ should have
interesting effects on the catalytic power of these quino-
nes, and we are expecting these molecules to have
improved catalytic potential over the corresponding PQQ
analogues 2-4. The catalytic properties of analogues 5-7
are currently under investigation and will be reported
elsewhere. The ester analogues (5-7) have been targeted
as final products rather than the carboxylic acids, as they
are more conveniently used in the analysis of the model
catalytic properties of the cofactor analogues. The dem-
ethylated forms of 5-7 may be useful eventually in
studies with reconstituted enzymes and in studies of
vitamin function.
Exp er im en ta l Section
2-Eth oxyca r bon yl-4-m eth oxy-6-n itr oben zim id a zole (16).
A mixture of compound 8 (500 mg, 2.73 mmol) and ethyl
triethoxyacetate (3.60 g, 16.4 mmol) was heated under nitrogen
at 100 °C for 18 h producing a paste too thick to be stirred. The
reaction mixture was then cooled to room temperature and
washed with diethyl ether to give a yellowish powder (500 mg,
1.89 mmol, 69%). An analytical sample was recrystalized from
methylene chloride-acetone to produce a pale yellow solid: mp
298-299 °C; 1H NMR (DMSO-d6 δ) 1.38 (t, 3H, J ) 3.2 Hz),
3.99 (s, 3H), 4.41 (q, 2H, J ) 3.2 Hz), 7.20 (d, 1H, J ) 2 Hz),
7.90 (d, 1H, J ) 2 Hz), 12.34 (br, 1H); 13C NMR (DMSO-d6 δ)
13.86, 56.72, 63.27, 102.46, 114.02, 125.92, 129.66, 142.40,
146.05, 150.23, 156.41; TLC Rf ) 0.50 (5% CH3OH in CH2Cl2).
Anal. Calcd for C11H11N3O5: C, 49.81; H, 4.18; N, 15.84. Found:
C, 50.07; H, 4.26; N, 15.71.
In summary, several imidazolo analogues of pyrrolo-
quinoline quinone were synthesized to further investigate
the importance of the pyrrole ring in the catalytic
2-Eth oxycar bon yl-6-am in o-4-m eth oxyben zim idazole (17).
Compound 16 (300 mg, 1.13 mmol) was suspended in absolute
ethanol (50 mL). To the mixture was added 10% Pd/C (54 mg).
The mixture was stirred at room temperature under 80 psi of
hydrogen for 4 h. The catalyst was removed by filtration and
rinsed with ethanol. The ethanol was then removed in vacuo to
yield a light brown powder (285 mg, 1.13 mmol, 100%): mp 233-
236 °C; 1H NMR (CD3OD δ) 1.43 (t, 3H, J ) 3.2 Hz), 3.90 (s,
3H), 4.41 (q, 2H, J ) 3.2 Hz), 6.46 (d, 1H, J ) 2 Hz), 6.48 (d,
1H, J ) 2 Hz); 13C NMR (CD3OD δ) 14.60, 56.64, 64.36, 99.52,
104.21, 113.10, 134.11, 146.18, 148.39, 152.10, 156.44; TLC Rf
) 0.65 (10% CH3OH in CH2Cl2). Anal. Calcd for C11H13N3O3‚
0.5H2O: C, 54.08; H, 5.79; N, 17.19. Found: C, 54.28; H, 5.89;
N, 17.20.
(16) Bamberger, E.; Berle, B. Ann. 1893, 273, 315; Bamberger, E.;
Berle, B. Ann. 1893, 273, 323.
(17) Alley, P. W.; Shirley, D. A. J . Org. Chem. 1958, 23, 1791.
Hudkins, R. L.; Diebold, J . L.; Marsh, F. D. J . Org. Chem. 1995. 60,
6218. Whitten, J . P.; Matthews, D. P.; McCarthy, J . R. J . Org. Chem.
1986, 51, 1891. Piffl, M.; Weston, J .; Anders, E. Eur. J . Org. Chem.
2000, 2851.)
(18) Katritzky, A. R.; Akutagawa, K. Tetrahedron. Lett. 1985, 26,
5935.
(19) Katritzky, A. R.; Lue, P.; Yannakopoulou, K. Terahedron 1990,
46, 641.
(20) Katritzky, A. R.; Akutagawa, K. J . Org. Chem. 1989, 54, 2949.
(21) Musser, J . H.; Bailey, H. T. T. Synth. Commun. 1984, 14, 947.
J ones, R. G. J . Am. Chem. Soc. 1951, 73, 5168.
2628 J . Org. Chem., Vol. 69, No. 7, 2004

2-E t h oxyca r b on yl-4-m et h oxy-7,9-d im et h oxyca r b on yl-
1H-im id a zolo[5,4-f]qu in olin e (18). A mixture of compound 17
(90 mg, 0.383 mmol), dimethyl trans-2-ketoglutaconate (91 mg,
0.528 mmol), and dry methylene chloride (10 mL) was stirred
under nitrogen at room temperature for 90 h. Methylene chloride
(40 mL) was added, and dry hydrogen chloride was bubbled
through the solution for 3 h. Oxygen and dry hydrogen chloride
were then bubbled through the solution for 4 h. The solvent was
removed in vacuo, and the resulting solid was dissolved in
methylene chloride (40 mL). The methylene chloride solution
was washed with 0.2 M sodium bicarbonate and with brine. The
methylene chloride extract was dried over anhydrous sodium
sulfate, evaporated to dryness, and subsequently recrystallized
from methylene chloride-hexane to yield a pale yellow powder
118.18, 118.94, 119.52, 119.67, 125.06, 125.15, 125.26, 142.41,
144.23, 146.63, 152.48, 154.27, 157.76, 157.90, 163.20, 170.18;
TLC Rf ) 0.06 (5% CH3OH in CH2Cl2). HRMS-FAB (m/z): [M
+ Na]+ calcd for C16H13N3O7H 360.0831; found 360.0806.
2,7,9-Tr im eth oxyca r bon yl-1H-im id a zolo[5,4-f]qu in olin e-
4,5-d ion e (5). A mixture of compound 19 (100 mg, 0.332 mmol),
KH2PO4 (70 mg), and K2HPO4 (100 mg) was stirred in 5:1
acetonitrile-water (60 mL) for 1 h. The pH was adjusted to 7
by addition of dilute aqueous HCl. A solution of Fremy’s salt
(178 mg, 0.664 mmol) and K2HPO4 (67 mg) in water (7 mL) was
added in fractions, and the mixture was stirred at room
temperature for 12 h. The solution was brought to pH 7 by
addition of dilute NaOH. A solution of Fremy’s salt (150 mg,
0.559 mmol) and K2HPO4 (67 mg) in water (7 mL) was added in
fractions. The mixture was stirred at room temperature for an
additional 24 h. The mixture was extracted with methylene
chloride, and the combined organic fractions were dried over Na2-
SO4. The solvent was removed in vacuo, and the residue was
recrystallized from methylene chloride-hexane to yield an
orange powder (37 mg, 30%): mp 290-293 °C; 1H NMR (CD3OD
δ) 3.99 (s, 3H), 4.03 (s, 3H), 4.08 (s, 3H), 8.13 (s, 1H); 13C NMR
(DMSO-d6 δ) 52.83, 52.88, 55.67, 120.04, 126.59, 129.84, 139.22,
145.92, 146.72, 149.41, 163.82, 167.09, 171.31, 173.77; TLC Rf
) 0.42 (5% CH3OH in CH2Cl2). HRMS-FAB (m/z): [M + Na]+
calcd for C16H11N3O8Na 396.0444; found 396.0439.
1
(115 mg, 0.297 mmol, 78%): mp > 300 °C; H NMR (DMSO-d6
δ) 1.42 (t, 3H, J ) 7.2 Hz), 3.96 (s, 3H), 3.97 (s, 3H), 4.09 (s,
3H), 4.37 (q, 2H, J ) 7.2 Hz), 7.65 (s, 1H), 8.02 (s, 1H), 12.52
(br, 1H); 13C NMR (DMSO-d6 δ) 13.97, 52.59, 52.63, 56.65, 63.41,
106.18, 116.40, 117.98, 122.30, 122.87, 138.16, 145.61, 145.83,
149.96, 150.19, 154.96, 164.62, 168.21; TLC Rf ) 0.48 (5%
CH3OH in CH2Cl2). Anal. Calcd for C18H17N3O7: C, 55.81; H,
4.42; N, 10.85. Found: C, 55.52; H, 4.56; N, 10.72.
4-Hyd r oxy-2,7,9-tr im eth oxyca r bon yl-1H-im id a zolo[5,4-
f]qu in olin e (19). A mixture of compound 18 (500 mg, 1.29
mmol) and 33% HBr in glacial acetic acid (50 mL) was placed
in a round-bottom flask equipped with a condenser and an argon-
filled balloon. The mixture was heated under vigorous reflux for
5 days. Each day, the acid mixture was removed under vacuum
and replaced with 33% HBr in glacial acetic acid (50 mL), and
a new argon-filled balloon was applied. The solvent was then
removed in vacuo. Dry methanol (80 mL) and thionyl chloride
(1 mL) were added to the flask. The resulting solution was
refluxed under nitrogen overnight. The solvent was removed in
vacuo to yield a brown powder (417 mg, 1.90 mmol, 90%). An
analytical sample was recrystalized from methanol-methylene
chloride-hexane to give a yellow powder: mp > 300 °C; 1H NMR
(CD3OD δ) 4.11 (s, 3H), 4.14 (s, 3H), 4.16 (s, 3H), 7.68 (s, 1H),
8.15 (s, 1H); 13C NMR (DMSO-d6 δ) 53.81, 54.38, 54.58, 56.48,
Ack n ow led gm en t. We wish to thank the DeArce
Foundation for financial support.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails are given for general procedures and for the preparation
of the imidazolo analogues 6 and 7 as well as for the synthetic
intermediates described in the preparation of compounds 6 and
7. This material is available free of charge via the Internet at
http://pubs.acs.org.
J O035390X
J . Org. Chem, Vol. 69, No. 7, 2004 2629