Synthesis of 5-methyl phenanthridium derivatives: A new class of human DOPA decarboxylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.04.047

DOPA decarboxylase (DDC) is responsible for the decarboxylation of l-DOPA and related aromatic amino acids and correlates closely with a number of clinical disorders. Sanguinarine, a natural quaternary benzophenanthridine alkaloid (QBA), was reported to b

Full text of DOI:10.1016/j.bmcl.2014.04.047

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2712–2716
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 5-methyl phenanthridium derivatives: A new class
of human DOPA decarboxylase inhibitors
Pi Cheng a,b, Jie Zhou c, Zhixing Qing c, Weisong Kang c, Sheng Liu b, Wei Liu a, Hongqi Xie a,
,
⇑
Jianguo Zeng a,b,c,
⇑
a Pre-State Key Laboratory for Germplasm Innovation and Utilization of Crop, Hunan Agricultural University, Changsha, Hunan 410128, China
b National Research Center of Engineering Technology for Utilization of Functional Ingredients from Botanicals, Hunan Agricultural University, Changsha, Hunan 410128, China
c School of Pharmaceutical, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China
a r t i c l e i n f o
a b s t r a c t
Article history:
DOPA decarboxylase (DDC) is responsible for the decarboxylation of L-DOPA and related aromatic amino
Received 3 December 2013
Revised 21 March 2014
Accepted 11 April 2014
Available online 19 April 2014
acids and correlates closely with a number of clinical disorders. Sanguinarine, a natural quaternary
benzophenanthridine alkaloid (QBA), was reported to be inhibitor of rat DDC and possessed a different
inhibitory mechanism. In this study, several natural QBAs were assayed as human DDC inhibitors for
the first time. A series of 5-methyl phenanthridium derivatives that contain the basic core structure of
QBAs were also synthesized and evaluated as human DDC inhibitors. The title compounds still possessed
DDC inhibitory potential. Among the synthesized compounds, 2-hydroxyl-8-methoxy-5-methylphe-
nanthridinium chloride (11k) showed good inhibitory activity with an IC50 value of 0.12 mM. Preliminary
structure–activity relationship indicated that DDC inhibitory potential of 5-methyl phenanthridium
Keywords:
Quaternary benzophenanthridine alkaloids
Phenanthridium
Synthesis
Human DOPA decarboxylase
Inhibitor
derivatives correlated with the p-electro densities on C@N double bond of iminium cation. The hydroxyl
group on compound 11k possibly contributed to the formation of hydrogen bond between DDC and the
inhibitor.
Ó 2014 Elsevier Ltd. All rights reserved.
Dopa decarboxylase (DDC; EC 4.1.1.28) is a pyridoxal 50-phos-
phate (PLP)-dependent enzyme that possesses broad substrate
nausea and vomiting.5 As a result,
DDC inhibitor to prevent side effects and reduce the catabolism
of -DOPA by the peripheral dopaminergic system.
CarbiDOPA6 (1, Scheme 1) and benserazide7 (2), the most com-
monly used DDC inhibitor, are analogues of -DOPA. These two
L-DOPA is administered with a
specificity.1 DDC recognized substrates, such as
L
-3,4-dihydroxy-
L
phenylalanine
(L-DOPA) and
L
-5-hydroxytryptophan -5-HTP),
(L
are decarboxylated to form the important neurotransmitters
dopamine and serotonin, respectively. In mammalian tissue,
L
commercialized DDC inhibitors possess two characteristic struc-
ture motifs: catechol unit and hydrazine group. The crystal struc-
tures of DDC complex with carbiDOPA8 has indicated that the
catechol unit generated a hydrogen bonding interaction between
the inhibitor and the active side of DDC. Meanwhile, the hydrazine
group reacted with the coenzyme PLP and formed an imine adduct.
Based on these findings, a series of synthetic catechin derivatives
such as the analogues of compound 3 have been synthesized and
evaluated for their DDC inhibitory activities.9 As to the natural
DDC inhibitors, epigallocatechin-3-gallate (EGCG) (4, Scheme 1),
the most abundant catechin in green tea, was also proved to be
the inhibitor of DDC.10 Docking experiments11 have shown that
EGCG bound inside the DDC active site and formed important
hydrogen bonds between the catechin motif and several residues
in the binding pocket.
DDC was found to decarboxylate other aromatic
L
-amino acids,
so it is also known as aromatic
L-amino acid decarboxylase
(AADC).2
DDC correlates closely with a number of clinical disorders such
as Parkinson’s disease (PD) and hypertension.3 PD is reported to be
the result of degeneration of dopamine-producing cells in the sub-
stantia nigra of the brain.4 Dopamine, the decarboxylated product
of
not administered as a drug in the treatment of PD independently.
On the other hand, -DOPA is rapidly converted to dopamine in
L-DOPA, cannot pass the blood–brain barrier. Thus, dopamine is
L
the blood stream when administered as a drug, and only a small
percentage of a given dose reaches central nervous system. Periph-
eral decarboxylation of L-DOPA to dopamine causes prominent
Quaternary benzophenanthridine alkaloids (QBAs) are a small
family of natural N-containing compounds that are widely distrib-
uted in papaveraceous and rutaceous plants and display obvious
⇑
Corresponding authors. Tel.: +86 731 84686560 (H.X.), +86 731 84673824 (J.Z.).
E-mail addresses: xiehongqi2006@126.com (H. Xie), ginkgo@world-way.net
(J. Zeng).
http://dx.doi.org/10.1016/j.bmcl.2014.04.047
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

P. Cheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2712–2716
2713
OH
HO
HO
O
OH
O
OH
N
HO
HO
CH3
COOH
O
OH
N
HO
HO
HO
HO
HO
HO
N
HN
HN
NH2
NH2
OH
benserazide
EGCG
4
carbiDOPA
2
3
1
Scheme 1. DDC inhibitors with catechin motif.
antitumor12 and antibacterial activities.13 In previous study,14
QBAs such as sanguinarine (5, Scheme 2) and chelerythrine (6)
were also reported to be inhibitors of rat DDC. In this study, san-
guinarine, chelerythrine and nitidine (7) were selected for human
DDC inhibition experiments. For comparison, dihydrosanguinarine
(8) and berberine (9) were also assayed for their inhibitory activi-
ties. QBAs showed obvious inhibitory activities under our experi-
mental conditions. When the C@N double bond was reduced,
dihydrosanguinarine lost inhibitory activity, which suggested that
the positive charge on QBAs was crucial to the inhibitory potential.
Nevertheless, berberine, an analogue of QBAs, was inactive
although that berberine possessed a positive charge and conju-
gated planar structure similar to QBAs. The preliminary findings
gave us an impetus to synthesize 5-methyl phenanthridium (10,
Scheme 2) and related derivatives (11) which possessed the core
structure of QBAs.
As shown in Scheme 3, commercially available phenanthridine
(12) was used as the starting material to synthesize 5-methyl phe-
nanthridium chloride (10). Compound 12 underwent N-methyla-
tion with dimethyl sulphate to form 5-methyl phenanthridium
sulphate salt (13). Without further purification, compounds 13
was refluxed in basic alcoholic solution to provide 6-ethoxyl-5,6-
dihydrophenanthridine (14), which lost a ethanol molecule in
dilute HCl to give target compound 10.
anilines to provide the benzamides 17 in high yields. Before the
intramolecular Heck-type cyclization of the benzamides to the cor-
responding phenanthridinones, it was necessary to convert the
secondary amides to the tertiary amides. Direct intramolecular
Heck coupling of benzamides 17 gave corresponding phenanthrid-
inones in poor yields. Thus, the nitrogen atom in benzamides 17
was protected by methoxylmethyl group (MOM) firstly by reacting
the benzamides with NaH and MOMBr in dry THF to give the cor-
responding N-MOM protected benzamides. The N-MOM benzam-
ides (18) were cyclized to the phenanthridinones (19)
successively via an intramolecular Heck reaction using Pd(OAc)2/
PPh3/Na2CO3/DMF catalytic system.15
5-Methyl-5,6-dihydrophenanthridin-6-ols (20) were obtained
through diisobutylaluminium hydride (DIBAL) mediated reduction
of compound 19. Without purification, treatment of compound 20
with dilute HCl afforded the desired phenanthridinium chloride
salts (11) in moderate yields.
It was worth noting that when ortho-substituted anilines were
used for synthesis of 4-substituted phenanthridium 22 (Scheme 5),
intramolecular Heck cyclization could not be accomplished effec-
tively (yield <5%). It was suggested that the interaction between
group R3 and bulky N-MOM group could affect the formation of
transition state in intramolecular Heck coupling. A newly devel-
oped KOtBu promoted intramolecular homolytic aromatic substi-
tution (HAS) methodology16,17 was also applied for synthesis of
compound 22, but the target compound was isolated with very
low yield (<5%) in present study.
The general synthetic methodology for the synthesis of substi-
tuted phenanthridium chloride (11) was shown in Scheme 4. The
bromobenzoic acids (15) were converted to corresponding acid
chlorides (16) in situ by refluxing in SOCl2. After evaporation of
SOCl2, the acid chlorides were directly reacted with the various
In previous literature, DDC inhibitory activity was determined
by measuring the production of dopamine with a spectrophotometric
1
O
2
O
O
OMe
MeO
MeO
O
OMe
3
4
5
N
N
N
MeO
MeO
O
6
O
nitidine
chelerythrine
sanguinarine
7
6
5
O
O
R2
O
O
2
1
3
C
N
10
7
R1
R2
N
9
8
4
N
O
MeO
MeO
A
B
5
N
O
6
dihydrosanguinarine
phenanthridiums
berberine
5-methyl phenanthridium
11
10
8
9
Scheme 2. Structures of QBAs (5–7), dihydrosanguinarine (8), berberine (9) and synthetic quaternary phenanthridine derivatives (10–11).

2714
P. Cheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2712–2716
(i)
(ii)
(iii)
N
N
N
N
Cl-
-
MeOSO3
OEt
14
phenanthridium
13
10
12
Scheme 3. Reagents and conditions: (i) Me2SO4, 160 °C, 1 h; (ii) NaOEt, EtOH, reflux, 2 h; (iii) 2 M HCl, 30 min.
R3
R3
N
Br
Br
R1
R2
R1
R2
R1
R2
Br
R1
R2
Br
17a-20a: R1 = OMe, R2 = OMe, R3 = H
(i)
(ii)
(iii)
17b-20b: R1 = OMe, R2 = OMe, R3 = F
NH
COOH
MOM
COCl
1
: R = OMe, R2 = OMe, R3 = Cl
17c-20c
17d-20d
O
O
: R1 = OMe, R2 = OMe, R3 = Me
18
15
16
17
R3
17e-20e: R1 = OMe, R2 = OMe, R3 = OMe
17f-20f: R1 = H, R2 = OMe, R3 = H
17g-20g: R1 = H, R2 = OMe, R3 = F
R3
R3
N
: R1 = H, R2 = OMe, R3 = Cl
: R1 =H, R2 = OMe, R3 = Me
17h-20h
17i-20i
(iv)
(v)
R1
R2
R1
R2
(vi)
R1
R2
17j-20j: R1 =H, R2 = OMe, R3 = OMe
N
N
Cl-
MOM
MOM
OH
20
O
19
11
Scheme 4. Reagents and conditions: (i) SOCl2, reflux, 2 h; (ii) phenyl anilines, TEA, DCM, rt, 3 h, 80–95%; (iii) NaH, THF, 0 °C–rt, 78–90%; (iv) Pd(OAc)2, PPh3, Na2CO3, dry DMF,
reflux 7 h, 65–85%; (v) DIBAL, 0°C–rt; (vi) 2 M HCl, 20 min, 42–61% in two steps.
Pd(OAc)2, PPh3, Na2CO3
Br
R1
DMF, reflux, 7 h
R1
R2
R3
R3
N
R2
MOM
N
MOM
O
22
O
KOtBu, DMEDA
21
yield < 5%
mesitylene, 100 C, 6 h
Scheme 5.
assay outlined by Sherald et al.18 and modified by Charteris and
John.19 The decarboxylased product dopamine reacted with chro-
mogenic agent trinitrobenzenesulphonic acid (TNBS) to yield a
condensation product which could be detected by UV spectroscopy
at the wavelength of 340 nm. In present study, QBAs and the syn-
thetic phenanthridium salts had a maximum UV absorption at
340 nm. To overcome the perturbation, we developed an HPLC
Table 1
Human DDC inhibitory activities of compounds 5–10 and 11a–11k
R1
R2
R3
IC50 mM
dH-6 (ppm)
a
Compounds
5 (Sanguinarine)
6 (Chelerythrine)
7 (Nitidine)
8 (Dihyrosanguinarine)
9 (Berberine)
10
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
—
—
—
—
—
H
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
—
—
—
—
—
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
—
—
—
—
—
H
H
F
0.20
1.28
1.34
n.a.b
n.a.
0.40
3.50
2.69
1.39
1.49
1.17
0.95
0.57
0.93
0.60
0.19
0.12
—
—
—
—
method to analyze the conversion rate of L
-DOPA in this study.20
—
10.10
9.71
9.73
9.71
9.55
9.52
10.00
10.10
10.10
9.87
9.80
9.68
The natural QBAs (5–7), dihrdrosanguinarine (8), berberine (9)
and all synthesized analogues (10, 11a–11j) were evaluated for
their human DDC inhibitory activities. An overview of the results
of all compounds was given in Table 1. The lead compound
sanguinarine chloride (5) showed strong inhibitory activity
(IC50 = 0.20 mM) to recombinant human DDC under the experi-
mental conditions. Chelerythrine (6) and nitidine (7), the ana-
logues of sanguinarine, exhibited approximately 6-times
decreased activities with IC50 values of 1.28 and 1.34 mM respec-
tively. Dihydrosanguinarine (8) lost the inhibitory activity to
DDC. It was suggested that the C@N+ double bond or the positive
charge on the quaternary N-atom was crucial to inhibitory
potential.
Cl
Me
OMe
H
F
Cl
Me
OMe
OH
11k
H
a
IC50 was the mean value of two separated experiments.
n.a.: not active.
b
It was worth mentioning that berberine (9) was inactive to inhi-
bit DDC. Thus, 5-methyl phenanthridium (10), the basic core struc-
ture of QBAs, was synthesized and included for a comparison
purpose. Interestingly, compound 10 showed better inhibitory
activity (IC50 = 0.40 mM) than chelerythrine and nitidine.
The inhibitory activities of ten synthesized quaternary phenan-
thridiums (11a–11e) were assayed and outlined in Table 1. Com-
pounds 11a–11e were synthesized with 2-bromo-4,5-dimethoxyl
benzoic acid as the starting material and carried two methoxyl

P. Cheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2712–2716
2715
groups at position 8 and 9 on ring A of phenanthridine core. The
inhibitory activities of compounds 11a–11e were obviously lower
than compound 10. For example, the IC50 value of compound 11a
was 9-times higher than compound 10, which indicated that two
methoxyl groups on ring A of phenanthridine core generated neg-
ative effect on inhibitory activity. Different substituents on ring C
of phenanthridine core also affected the inhibitory potential. The
electro-donating groups such as methyl and methoxyl at position
2 of ring C could increase the inhibitory activity. For example, com-
pounds 11d (R3 = Me) and 11e (R3 = OMe) showed increased activ-
ities compared with compound 11a.
and 0.646, respectively.24 Thus, the double bond C@N+ of sanguin-
arine is polar and sensitive to the attack by nucleophiles.22 In pres-
ent study, an overall comparison of the inhibitory activities for
phenanthridiums 11a–11j has indicated that the
p-electro densi-
ties on ring A could influence the DDC inhibitory potential. The
chemical shift values of H-6 in 1H NMR spectroscopy of the phe-
nanthridium derivatives reflected the
p-electro densities. As out-
lined in Table 1, chemical shift values of compounds 11g–11j
were higher than the corresponding compounds 11b–11f. It was
suggested that low p
-electron densities of C@N+ of QBAs and phe-
nanthridiums could facilitate the neuleophilic attack from SH
Compounds 11f–11j possessed one methoxyl group at position
8 on ring A. An overall comparison of inhibitory activities between
compounds 11f–11j and compounds 11a–11e strongly suggested
that electro-donating group on ring A could decrease the inhibitory
potential. IC50 values of compounds 11f–11j were lower than com-
pounds 11a–11e. Substituents on ring C of compounds 11f–11j
exhibited similar effect on the activities: electro-donating group,
for example methoxyl, on ring C could increase the inhibitory
potential. For example, IC50 value of compound 11j was 5-times
lower than compound 11f. Compound 11j (IC50 = 0.19 mM) exhib-
ited good inhibition potential as sanguinarine.
To further confirm the effect of substituent on ring C of com-
pound 11j, the 2-demethylated derivative 11k was also synthe-
sized as outlined in Scheme 6. An O-TBDMS temporary protected
4-animophenol (24) was synthesized21 and reacted with corre-
sponding acid chloride to afford O-TBDMS protected benzamide
(25) which was further transferred to N-MOM protected benzam-
ide 26. Intramolecular Heck cyclization of compound 26 afforded
the desired phenanthridinone 27 accompanied with the O-depro-
tection process. In the same condition, compound 27 was trans-
ferred to 11k in two steps. DDC inhibitory activity of compound
11k slightly increased compared with compound 11j and pos-
sessed IC50 value of 0.12 mM.
groups of enzyme.
DDC contains 11–13 cysteine (Cys) residues according to the
species of animals. The crystal structures of DDC complex8 with
carbiDOPA has indicated that Ile 1010 residue in active side of
human DDC belongs to the substrate binding pocket and is in
Van der Waals contact with the catechol ring of the inhibitor car-
biDOPA. The SH-group of Cys 1000, the residue preceding Ile 1010,
is only 4.1 Å apart from that of Cys 1110. Replacement of Cys
1110 by an Ala residue or formation of a disulfide bridge between
these residues would perturb the active site geometry and reduce
or even abolish enzyme activity.25 In this study, it was assumed
that QBAs and synthetic phenanthridiums could block the SH-
groups of Cys 1000 or Cys 1110 and change the geometry of DDC
active side. Compound 11k was more active than compound 11j
which indicated that free hydroxyl group on the phenanthridium
core could possibly contribute to the formation of hydrogen bond
with residue of DDC active side. Based on above analysis, the pos-
sible binding mode of compound 11k with DDC was provided as
shown in Scheme 7. However, the acurate binding mode of QBAs
and phenanthridiums as DDC inhibitors needs to be further
investigated.
formation of
hydrogen bond?
The biological results in this study suggested a different DDC
inhibitory mechanism of QBAs and quaternary phenanthridines.
In previous literature,22 the iminium cation C@N+ of the QBAs
was reported to interact with nucleophilic groups, especially SH
groups in enzymes and other proteins. For example, it inhibited
Na+/K+ and Ca2+-ATPases by blocking the SH-groups essential for
their activities.23 According to quantum chemistry calculation,
OH
DDC
OH
N
DDC-Cys-SH
N
MeO
MeO
S
formation of
covalent bond
Cys DDC
11k
p-electron densities on the heterocyclic nitrogen atom at position
5, and on the carbon atom at position 6 in sanguinarine, are 1.660
Scheme 7. Possible binding mode of active compound 11k with DDC.
TBDMSO
HO
(i)
OTBDMS
OTBDMS
NH2
Br
NH2
Br
Br
23
24
16
(iii)
(iv)
NH
N
MeO
MeO
MOM
Br
O
O
25
26
(ii)
Cl
OH
(v)
MeO
MeO
O
O
15
OH
OH
OH
(vii)
(vi)
N
N
N
MeO
MOM
MeO
MOM
MeO
-
Cl
O
OH
28
11k
27
Scheme 6. Reagents and conditions: (i) TBDMSCl, imidazole, THF, rt, 89%; (ii) SOCl2, reflux, 2 h; (iii) TEA, DCM, rt, 3 h, 85%; (iv) NaH, THF, 0 °C–rt, 77%; (v) Pd(OAc)2, PPh3,
Na2CO3, dry DMF, reflux 7 h, 44%; (vi) DIBAL, 0 °C–rt; (vii) 2 M HCl, 20 min, 31% in two steps.

2716
P. Cheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2712–2716
8. Burkhard, P.; Dominici, P.; Borri-Voltattorni, C.; Jansonius, J. N.; Malashkevich,
In summary, several natural QBAs and synthetic quaternary
V. N. Nat. Struct. Biol. 2001, 8, 963.
9. Daidone, F.; Montioli, R.; Paiardini, A.; Cellini, B.; Macchiarulo, A.; Giardina, G.;
Bossa, F.; Voltattorni, C. B. PLoS ONE 2013, 7, e31610.
10. Bertoldi, M.; Gonsalvi, M.; Voltattorni, C. B. Biochem. Biophys. Res. Commun.
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11. Ruiz-Perez, M. V.; Pino-Angeles, A.; Medina, M. A.; Sanchez-Jimenez, F.; Moya-
Garcia, A. A. J. Chem. Inf. Model. 2012, 52, 1113.
phenanthridines were assayed for the human DDC inhibitory activ-
ities for the first time. QBAs and phenanthridium salts were sug-
gested to possess the same DDC inhibitory mechanism by
blocking the SH-groups close to the active side of enzyme. DDC
inhibitory potential of 5-methyl phenanthridium derivatives corre-
12. Lynch, M. A.; Duval, O.; Sukhanova, A.; Devy, J.; MacKay, S. P.; Waigh, R. D.;
Nabiev, I. Bioorg. Med. Chem. Lett. 2001, 11, 2643.
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2012, 22, 7080.
lated with the p-electro densities on C@N double bond of iminium
cation. Further modification of quaternary phenanthridium is
under way in our lab.
14. Drsata, J.; Ulrichova, J.; Walterova, D. J. Enzyme Inhib. Med. Chem. 1996, 10, 231.
15. Bernardo, P. H.; Wan, K. F.; Sivaraman, T.; Xu, J.; Moore, F. K.; Hung, A. W.; Mok,
H. Y. K.; Yu, V. C.; Chai, C. L. L. J. Med. Chem. 2008, 51, 6699.
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Acknowledgments
This research was financially supported by Natural Science
Foundation of Hunan Province (No. 13JJ5044), Research Fund for
the Doctoral Program of Higher Education, Ministry of Education
of China (No. 20124320120006) and the Important Science & Tech-
nology Specific Projects of Hunan province (No. 2012FJ1004).
(pH = 6.8). The final concentration of DDC,
L-Dopa and PLP were 0.1 lM, 1 mM
and 5 M respectively. DDC activity was assayed in the presence and in the
l
absence of a fixed amount of each compound. The reaction was carried at 37 °C
for 30 min and stopped by inactivation of the enzyme at 100 °C for 2 min.
Enzyme activity was determined by measuring the production of dopamine
through an HPLC method developed in our lab. HPLC conditions: The
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
04.047. These data include MOL files and InChiKeys of the most
important compounds described in this article.
chromatographic column was Unitary-C18 (250 ꢀ 4.6 mm, 5
lm, Acchrom
Technologies, China). The elution system was consisted with 0.2% formic acid
aqueous solution and methanol (9:1, V/V), flow rate was 1 mL/min, and the UV
detection wavelength was set at 280 nm. Doseresponse curves were generated
to determine the concentration required to inhibit 50% of decarboxylase
activity (IC50). Compounds were evaluated at 6 concentrations, and each in
duplicate. IC50 values were derived by nonlinear regression analysis.
21. Knaggs, S.; Malkin, H.; Osborn, H. M. I.; Williams, N. A. O.; Yaqoob, P. Org.
Biomol. Chem. 2005, 3, 4002.
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