Structure-Based Design and Synthesis of Harmine Derivatives with Different Selectivity Profiles in Kinase versus Monoamine Oxidase Inhibition

Article abstract of DOI:10.1002/cmdc.201600539

Dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is an emerging biological target with implications in diverse therapeutic areas such as neurological disorders (Down syndrome, in particular), metabolism, and oncology. Harmine, a natural product that selectively inhibits DYRK1A amongst kinases, could serve as a tool compound to better understand the biological processes that arise from DYRK1A inhibition. On the other hand, harmine is also a potent inhibitor of monoamine oxidase A (MAO-A). Using structure-based design, we synthesized a collection of harmine analogues with tunable selectivity toward these two enzymes. Modifications at the 7-position typically decreased affinity for DYRK1A, whereas substitution at the 9-position had a similar effect on MAO-A inhibition but DYRK1A inhibition was maintained. The resulting collection of compounds can help to understand the biological role of DYRK1A and also to assess the interference in the biological effect originating in MAO-A inhibition.

Full text of DOI:10.1002/cmdc.201600539

Accepted Article
Title: Structure-based design and synthesis of Harmine derivatives
with different selectivity profiles in kinase vs monoamine oxidase
inhibition
Authors: Balazs Balint, Csaba Weber, Francisco Cruzalegui, Mike
Burbridge, and Andras Kotschy
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201600539
Link to VoR: http://dx.doi.org/10.1002/cmdc.201600539
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Structure-based design and synthesis of Harmine derivatives
with different selectivity profiles in kinase vs monoamine oxidase
inhibition
Balázs Bálint,a Dr.Csaba Wéber,a Dr.Francisco Cruzalegui,b,# Dr. Mike Burbridge,b and Dr. Andras
Kotschya,*
[a]
Andras Kotschy, Csaba Wéber, Balázs Bálint
Servier Research Institute of Medicinal Chemistry
Záhony u. 7., H-1031 Budapest, Hungary
E-mail: andras.kotschy@servier.com
[b]
[#]
Francisco Cruzalegui, Mike Burbridge
Institute de Recherche Servier
50 Rue Carnot, Suresnes, France
Present address: Pierre Fabre R&D Centre
3 Av Hubert Curien, 31035 Toulouse Cedex 1, France
E-mail: francisco.cruzalegui@pierre-fabre.com
Abstract: DYRK1A is an emerging biological target with implications
in diverse therapeutic areas such as neurological disorders (Down
syndrome in particular), metabolism, and oncology. Harmine, a
natural product that selectively inhibits DYRK1A amongst kinases
could serve as a tool compound to better understand the biological
processes that arise from DYRK1A inhibition. On the other hand
harmine is also a potent inhibitor of monoamine oxidase A (MAO-A).
Using structure-based design we have synthesized a collection of
with more than 10.000-fold specificity for MAO-A (0.5 nM) over
MAO-B (6.4 µM).[12] Due to the pharmacological interest in both
selective DYRK1A and MAO-A inhibitors, harmine is positioned
ideally as a starting point for the structure-based design of such
harmine derivatives that maintain the parent structure’s DYRK1A
or MAO-A inhibition potency while being devoid of inhibiting the
other target. Some recent papers describe benzothiazole
derivatives showing a comparable inhibitory potency and kinase
harmine analogs with tunable selectivity towards these two enzymes. selectivity with harmine without inhibiting MAO-A.[13],[14] The
Modifications in position 7 typically decreased affinity for DYRK1A
while substitution in position 9 had a similar effect on MAO-A
inhibition while maintaining DYRK1A inhibition. The resulting
collection of compounds can help to understand the biological role of
DYRK1A and also to assess the interference in the biological effect
originating in MAO-A inhibition.
decrease of MAO-A inhibition through the N-substitution of β-
carbolines while maintaining DYRK1A inhibition has also been
elegantly demonstrated by Becker.[15]
Results and Discussion
The X-ray structures of the harmine(1)-MAO-A[16] and
harmine(1)-DYRK1A[13] complexes have been published recently.
The design of selective inhibitors described here relied on the
assessment of the principal interactions between harmine and
the proteins as well as the exploration of the unoccupied space
around the ligand. In MAO (Figure 1, left) there are two
hydrogen bonds between residual water molecules and the ring
N-atoms. The water molecule interacting with the ring NH group
is held in position through hydrogen bonds with the side chain
carbonyl group of Asn187 and the backbone carbonyl group of
Ile207 (not shown). On the other hand the pyridine N-atom is
connected to a chain of water molecules occupying a fairly open
pocket and connecting both the co-factor FAD and some side
chains. The methoxy group of harmine only partially fills the
available space and doesn’t participate in any significant
interaction with MAO-A. On the other hand in the kinase pocket
of DYRK1A the same methoxy group interacts with the
backbone NH-group of the hinge Leu241 residue fitting nicely to
the available space. The pyridine nitrogen forms also a strong
hydrogen bond with the side chain of the salt bridge Leu188
residue. The ATP pocket of DYRK1A is closed from three
directions while the central ring NH group points towards the
open outer pocket and doesn’t participate in any apparent
secondary interaction.
Introduction
DYRK1A (dual-specificity tyrosine-phosphorylation-regulated
kinase 1A) is essential for maintaining the normal development
and function of the brain phosphorylating several proteins and
thereby regulating their functions.[1] DYRK1A has also been
implicated in the pathogenesis of Down Syndrome, one of the
most frequent genetic disorders.[2] In addition to its function in
neuronal development,[3] recent evidence has also implicated
DYRK1A in the pathology of neurodegenerative disorders such
as Alzheimer’s disease,[4],[5] dementia with Lewy bodies and
Parkinson’s disease,[6] and in pancreatic β cell mass expansion
leading to improved glucose tolerance.[7], [8]
The monoamine oxidases (MAO-A and B) catalyze the
degradation of neuroactive and vasoactive amines in the central
nervous system and in peripheral tissues.[9] MAOs play a key
role in psychiatric and neurological disorders such as depression
and Parkinson’s disease. The development of selective MAO
inhibitors is important both from the standpoint of treating these
diseases and because of their neuroprotective effects through
the prevention or delay of neurodegeneration itself.[10]
Harmine (1), a β-carboline alkaloid found in several plants, was
identified as
a very potent inhibitor of DYRK1A with a
Comparison of the binding modes of harmine in MAO-A and
DYRK1A and the assessment of the shape of the binding
remarkable selectivity over other kinases.[11] At the same time it
was reported that harmine is a strong reversible MAO inhibitor
1
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cavities left us with three sites to confer selectivity against one
protein or the other: i) due to its pivotal role in DYRK1A binding,
chemical modifications of the methoxy group should have a
more pronounced effect in this system than on MAO-A binding;
ii) decoration of the ring NH group should be tolerated in
DYRK1A while they should lead to a decreased affinity towards
MAO-A; iii) It is less trivial that how does the functionalization of
the pyridine methyl group effects binding but on the basis of
spatial effects we might expect MAO-A binding being more
influenced than DYRK1A binding. As the structural variations
outlined in points i)-iii) are complementary we expected to be
able to fine tune the selectivity of the novel harmine derivatives
with respect to their DYRK1A and MAO-A binding.
Figure 1. The binding pocket of MAO-A (left, pdb:2ZGX) and of DYRK1A (right, pdb:3ANR) in their respective harmine complexes highlighting the principal
secondary interactions and the unoccupied space in the binding pocket.
d
a-c
R= Me 3
R= H 4
R= H 5
R= Me 6
2
c
e-f
g
h
d
R= OMe 12
R= OH 13
R= NH2 14
R= Me 7, Pr 8, iPr 9
CH2CO2Et 10, Bn 11
1
c
15
i-j
l-m
k
1
16
17
18
o
n
19
20
21
Scheme 1. The preparation of harmine derivatives. a) (CF3SO2)2O, pyridine, CH2Cl2, -20°C, 16h, 80%. b) CO, MeOH, Pd(OAc)2, dppp, TEA, H2O, DMF, 70°C, 7h,
80%. c) LiOH, H2O, MeOH, rt, 6-16h. d) LiAlH4, THF, 0°C, 2h. e) SOCl2, CH2Cl2, rt, 3h. f) NaOMe, MeOH, rt, 16h, 30% (for 2 steps). g) NaH, DMF than RX, rt, 16h.
h) methyl acrylate, DBU, MeCN, 60°C, 16h, 72%. i) (COCl)2, CH2Cl2, rt, 1h. j) NH3, MeOH, rt, 6h, 21% (for 2 steps). k) CF3CH(OEt)OH, DDQ, CH2Cl2, 110°C, 5h,
47%. l) mCPBA, CH2Cl2, rt, 2h, 61%. m) (CF3SO2)2O, DMF , 0°C, 16h, 20%. n) POCl3, reflux, 5d, 50%. o) MeNHC2H4OH, Pd2dba3, tBu-XPhos, NaOtBu, PhMe, -
80°C, 16h, 37%.
2
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Synthesis
triptamine with trifluoroacetaldehyde hemiacetal gave 17, while
the N-oxidation, triflation, thermal rearrangement sequence gave
the hydroxymethyl analog 18. 20 was prepared from the oxo
compound 19, and its palladium catalyzed reaction with 2-
methylamino-ethanol gave 21.
To vary the methoxy group of harmine, harmol (2) was
converted to the corresponding ester (3) in a two-step sequence
through its triflate derivative (Scheme 1). Hydrolysis of 3 under
standard conditions gave the appropriate acid (4) while
reduction by LiAlH4 resulted in alcohol 5. The methylated analog
6 was prepared from 5 through a chlorination, nucleophilic
substitution sequence.
Variations around the methoxy group
The DYRK1A and MAO-A inhibition of the prepared compounds
were assessed in vitro and the results are summarized in Table
1. The simplest transformation – demethylation of the methoxy
group giving harmol (2) – had only a negligible effect on
DYRK1A inhibition while its MAO-A inhibition was compromised.
One could argue that the observed effect is a combination of
increased desolvation affecting both targets and retained or
increased hydrogen bond acceptor property effecting only
DYRK1A affinity. The swapping of the oxygen and carbon atoms
in harmine’s methoxy group (5) has led to a near 100-fold drop
in DYRK1A binding, as expected for the demolition of harmine’s
key hinge interaction.
The N-functionalized compounds were prepared from harmine
(1). A series of alkyl/benzyl substituents were introduced through
the deprotonation of harmine by sodium hydride followed by the
addition of the appropriate alkyl/benzyl halide. The homolog of
10 was prepared in a Michael addition with methyl acrylate in the
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) that was
superior to the alternate alkylation by methyl 3-chloropropionate.
Ester hydrolysis gave the corresponding acid (13) that was
converted to the 14 amide through the ammonolysis of the acid
chloride. Reduction of 12 gave the N-hydroxypropyl-harmine
(15) in good yield.
The harmine analogs modified on the pyridine ring were
prepared by different routes. Oxidative ring closure of methoxy-
N
R3
R1
N
R2
R3
DYRK1A
IC50 (nM)a
MAO-A
IC50 (nM)b
No
R1
R2
1
2
OCH3
OH
H
CH3
CH3
31.5 3.3
44.0 7.4
26.9 6.4
8240c
5.3
530
H
3
COOCH3
COOH
CH2OH
CH2OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
CH3
2700
4
H
CH3
inactive
40
5
H
CH3
2000 240
9808 4293
47.8 2.1
34.6 6.4
71.6 5.9
52.4 2.2
262 3.0
59.0 22.9
1063 42
199 77
6
H
CH3
CH3
30
7
CH3
11
8
(CH2)2CH3
CH(CH3)2
CH2COOC2H5
CH2C6H5
(CH2)2COOCH3
(CH2)2COOH
(CH2)2COONH2
(CH2)3OH
H
CH3
690
9
CH3
740
10
11
12
13
14
15
17
18
20
21
CH3
>10000
>10000
>10000
>10000
inactive
>10000
inactive
1900
CH3
CH3
CH3
CH3
CH3
83.2 8.4
77.1 0.6
106 4.0
56.0 26
144 19
CF3
H
CH2OH
Cl
H
inactive
3000
H
O(CH2)2NHCH3
a
b
an average of at least 2 independent measurements. values obtained from 2 parallel measurements averaging data points before mathematical evaluation.
>10000 denotes less than 50% inhibition at 10 µM, inactive denotes less than 25% inhibition at 10 µM. c less than 50% inhibition observed at 10 µM in the parallel
measurement.
Table 1. In vitro DYRK1A and MAO-A inhibition of the harmine analogs 1-21.
3
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On the other hand the MAO-A inhibition remained practically
unchanged further supporting the absence of strong
1-position of the β-carboline core one might retain potent
DYRK1A inhibition while demolish activity towards MAO-A.
a
interaction for this moiety in MAO-A. Further support for this
argument was provided by observing the same effect for the
methyl ether analogue 6. DYRK1A binding was further
compromised without significant deterioration of MAO-A binding.
Interestingly, the replacement of the methylene group of 6 by a
carbonyl group led to a compound (3) whose DYRK1A binding
was on-scale with harmine. In the meantime we also observed
the erosion of its MAO-A binding. The acid derivative 4 was
devoid of DYRK1A or MAO-A inhibition, which was attributed to
the fact that in the absence of the possibility to form strong
secondary interaction in any of the binding pockets, the
desolvation penalty of the carboxylate function cannot be
compensated on binding. In summary by transforming the
methoxy group in harmine one can abolish the DYRK1A binding
while maintaining the affinity for MAO-A.
Conclusions
Evaluation of the characteristic features of the MAO-A and
DYRK1A binding pockets as well as the principal interactions
formed on binding between harmine, a potent inhibitor of natural
origin of both proteins, has allowed us to identify the sites on
harmine that are crucial either for MAO-A or for DYRK1A binding.
We have also identified regions where chemical modifications
were expected to result in discrimination between the two target
proteins. By variation of the methoxy group of harmine that is
tightly bound to DYRK1A and sits in an enlarged sub pocket of
MAO-A without forming strong secondary interactions, we were
able to identify such molecules (e.g. 5, 6) that inhibit MAO-A but
have a significantly decreased affinity towards DYRK1A. On the
other hand the functionalization of the ring NH moiety of harmine
or the 1-position of the β-carboline skeleton, both facing the
solvent exposed area in DYRK1A while enclosed into the
binding pocket of MAO-A, led to molecules (e.g. 8, 10, 15, 17,
and 20), which bind efficiently to DYRK1A but lost most of their
potency on MAO-A. As a consequence of this work a number of
potent inhibitors of DYRK1A and MAO-A are now available with
different selectivity profiles, which should facilitate the
establishment of the biological relevance of these targets and
the pharmacological relevance of their inhibition.
Variations of the ring NH group
Modification of the ring NH group was achieved by the
introduction of differently functionalised alkyl chains. The N-
methyl derivative (7) bearing the smallest substituent in this
position retains harmine’s activity against both targets. By
increasing the size of the substituent (8-15) we observed a
significant drop in MAO-A inhibition while the affinity towards
DYRK1A has remained alike. The propyl (8) and isopropyl (9)
substitution was equally tolerated as expected on the basis of
DYRK1A’s structure since these substituents are projected
towards the solvent exposed area. Variation of the polarity of the
N-substituent in the analogues of 8, as expected, did not restore
the MAO-A binding but led to some variations in the DYRK1A
affinity. The ester bearing compound (10) remained equipotent
while the 11 benzyl derivative as well as the amide analogue
(14) were 6-8 fold less potent. The least potent compound in this
series was the acid derivative 13. The loss of potency for 13 and
14 might be attributed to desolvation (c.f. compound 4), while
the benzyl group of 11 might clash with the glycine loop of
DYRK1A (omitted from Figure 1 for clarity). These results
demonstrate that by the proper functionalisation of the ring NH
group of harmine one might obtain potent DYRK1A inhibitors
that are devoid of MAO-A inhibition.[17]
Experimental Section
Chemistry
1H, 13C and 19F NMR spectra were recorded on a Bruker Avance III 500
or Bruker Avance III 400 instrument. Chemical shifts are given in parts
per million (ppm) and spectra were obtained as DMSO-d6 or CDCl3
solutions (reported in ppm), using the residual signals of chloroform (7.26
ppm) or DMSO (2.50 ppm) as the reference. The following abbreviations
are used to denote signal multiplicities: s = singlet, d = doublet, t = triplet,
m = multiplet, and br = broadened. All coupling constants (J) are given in
hertz (Hz). Analytical HPLC was performed on Agilent 1200 Series
utilizing Waters Acquity UPLC BEH C18, 2.1×30mm, 1.7µm column
(solvent A: 100:2:0.1 H2O/CH3CN/MeSO3H, solvent B: 100:2:0.1
CH3CN/H2O/MeSO3H, gradient: 0min 0%B, 6min 100%, 7min 100%,
7.5min 0%, 9min 0%, flow: 0,8 mL/min) and UV detection at 210 nm.
High resolution mass spectrometric identification of compounds was
performed using SHIMADZU LCMS-IT-TOF ESI, WATERS SUNFIRE
C18, 2.1×50mm, 2.5µm column (solvent A: 100:3 H2O/propan-2-ol +
0.05% HCOOH, solvent B: 95:5:3 H2O/CH3CN/propan-2-ol + 0.05%
HCOOH, gradient: 0min 0%B, 9min 100%, 11min 100%, 11.1min 0%,
15min 0%, flow: 0.7ml/min). Flash chromatography was performed on
Teledyne ISCO CombiFlashRF instrument, using RediSept RF pre-
packed silica gel columns and UV detection at 254 or 210 nm. Reagents
and solvents were used as obtained from commercial suppliers without
further purification. Yields refer to purified products and are not optimized.
All tested compounds were >95% pure as assessed by LCMS and 1H
NMR.
Variations around the substitution of the pyridine ring
The first modification to harmine in this area was the
replacement of the methyl group by trifluoromethyl (17). This
change had only a minor effect on the DYRK1A inhibition – a
slight drop was observed – while the MAO-A inhibition was
completely abolished due probably to steric reasons.
Interestingly the chloro analogue 20 showed a similar behaviour:
efficient inhibition of DYRK1A and absence of activity on MAO-A.
The hydroxylation of the methyl group on the pyridine (18) was
again better tolerated by DYRK1A where a threefold drop in
inhibition was observed while MAO-A inhibition was nearly
abolished (1.9 µM instead of 5.3 nM). The harmine derivative
bearing a polar flexible substituent in position 1 (21) showed an
analogous inhibition profile to 18 verifying that this substituent is
projected towards the solvent exposed area in DYRK1A. These
results also demonstrate that by changing the substituent in the
Synthesis of 7-substituted-1-methyl-9H-β-carbolines
O-Trifluoromethanesulfonyl 1-methyl-9H-β-carbolin-7-ol
4
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To a suspension of harmol (10.0 g, 50.4 mmol) and pyridine (40 mL,
504.0 mmol) in anhydrous CH2Cl2 (220 mL) trifluoromethanesulfonic
anhydride (9.3 mL, 50.4 mmol) was added dropwise at -20°C. The
mixture was stirred overnight while allowed to warm up to room
temperature. The reaction mixture was poured onto saturated aqueous
NH4Cl (300 mL), the organic layer was separated and washed with water
(2×200 mL), dried over MgSO4 and concentrated to dryness. The crude
product was purified by crystallization from ethyl acetate to give O-
trifluoromethanesulfonyl-1-methyl-9H-β-carbolin-7-ol (13.3 g, 40.3 mmol,
80% yield) as an off-white solid.
1H NMR (400 MHz, DMSO-d6) δ 2.84 (s, 3H), 7.19 (dd, J = 2.2, 8.6 Hz,
1H), 7.44 (d, J = 2.2 Hz, 1H), 7.83 (d, J = 5.4 Hz, 1H), 8.14 (d, J = 8.6 Hz,
1H), 8.43 (d, J = 5.4 Hz, 1H), 9.39 (br s, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 20.3, 104.9, 113.1, 113.4, 121.9, 123.3, 129.4, 135.6, 139.3,
140.1, 142.3, 149.2; 19F NMR (40.5 MHz, DMSO-d6) δ -72.68; HRMS
m/z 331.0338 [M+H]+ (C13H9N2O3F3S + H+ requires 331.0364; ∆f = -
6.34 ppm).
1H), 11.45 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 20.9, 63.8, 109.8,
112.9, 118.6, 120.3, 121.7, 127.3, 135.1, 138.0, 141.1, 142.4, 143.4;
HRMS m/z 213.1015 [M+H]+ (C13H12N2O + H+ requires 213.1028; ∆f = -
3.28 ppm).
7-Methoxymethyl-1-methyl-9H-β-carboline (6)
To a stirred suspension of 7-hydroxymethyl-1-methyl-9H-β-carboline (212
mg, 1.0 mmol) in CH2Cl2 (3 mL) SOCl2 (220 µL, 3.0 mmol) was added
dropwise. After 3 hours the mixture was concentrated in vacuo to
dryness and the resulting residue was dissolved in a methanolic solution
of Na (230 mg, 10.0 mmol). The mixture was allowed to stir overnight at
room temperature. The reaction was quenched by addition of water (2
mL) and concentrated to dryness in vacuo. The crude product was
purified by silica gel chromatography, eluting with CH2Cl2/methanol (95:5)
to give 7-methoxymethyl-1-methyl-9H-β-carboline (70 mg, 0.3 mmol,
30% yield) as an off-white solid.
1H NMR (400 MHz, DMSO-d6) δ 2.75 (s, 3H), 3.35 (s, 3H), 4.60 (s, 2H),
7.17 (dd, J = 1.4, 8.1 Hz, 1H), 7.54 (d, J = 1.4 Hz, 1H), 7.90 (d, J = 5.3
Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 8.19 (d, J = 5.3 Hz, 1H), 11.51 (s, 1H);
13C NMR (100 MHz, DMSO-d6) δ 20.9, 58.0, 74.5, 111.0, 113.0, 119.4,
120.9, 121.9, 127.2, 135.2, 138.0, 138.8, 140.9, 142.5; HRMS m/z
227.1180 [M+H]+ (C14H14N2O + H+ requires 227.1184; ∆f = 0.44 ppm).
1-Methyl-9H-β-carboline-7-carboxylic acid methyl ester (3)
O-trifluoromethanesulfonyl-1-methyl-9H-β-carbolin-7-ol (10.0 g, 30.3
mmol), triethylamine (8.4 ml, 60.6 mmol), Pd(OAc)2 (67 mg, 0.3 mmol)
and 1,3-bis(diphenylphosphino)propane (dppp) (240 mg, 0.6 mmol) were
dissolved in the mixture of methanol (150 mL), DMF (50 mL) and water
(50 mL). The mixture vas stirred at 70°C under CO atmosphere (ambient
pressure) for 7 hours. The reaction mixture was filtered, concentrated to
100 mL and cooled to 0°C. The formed precipitate was filtered off,
washed with water and dried under reduced pressure to give 1-methyl-
Synthesis of 9-substituted-7-methoxy-1-methyl-9H-β-carbolines
Typical procedure
9H-β-carboline-7-carboxylic acid methyl ester (5.8 g, 24.2 mmol, 80%
To a stirred solution of harmine (300 mg, 1.4 mmol) in anhydrous DMF (9
mL) NaH (56 mg, 1.4 mmol) was added at room temperature. After 10
minutes of stirring, alkylating agent (1.1 eq) was added and allowed to
stir overnight. The mixture then was poured onto water (50 mL). The
formed precipitate was collected by filtration and it was purified by
crystallization from acetonitrile or by silica gel column chromatography
(CH2Cl2/methanol) to give the desired product.
yield) as an off-white solid.
1H NMR (500 MHz, DMSO-d6) δ 2.79 (s, 3H), 3.92 (s, 3H), 7.82 (dd, J =
1.4, 8.2 Hz, 1H), 8.00 (d, J = 5.3 Hz, 1H), 8.19 (d, J = 1.4 Hz, 1H), 8.26
(d, J = 5.3 Hz, 1H), 8.32 (d, J = 8.2 Hz, 1H), 11.83 (s, 1H); 13C NMR (125
MHz, DMSO-d6) δ 21.0, 52.7, 113.6, 113.8, 120.0, 122.4, 125.2, 126.4,
129.0, 136.1, 138.4, 140.0, 143.3, 167.1; HRMS m/z 241.0979 [M+H]+
(C14H12N2O2 + H+ requires 241.0977; ∆f = 2.90 ppm).
7-Methoxy-1,9-dimethyl-9H-β-carboline (7)[18]
1-Methyl-9H- β -carboline-7-carboxylic acid (4)
Using methyl iodide as alkylating agent we obtained 7 (210 mg, 0.93
mmol, 66% yield) as an off-white solid.
A solution of 1-methyl-9H-β-carboline-7-carboxylic acid methyl ester (200
mg, 0.83 mmol) and LiOH×H2O (70 mg, 1.66 mmol) in a mixture of
methanol (5 mL) and water (5 mL) was stirred overnight at room
temperature. The solvent was removed in vacuo, water (10 mL) was
added and the resulting mixture was neutralized with 1N aqueous HCl
(500 µL). The resulting solid was filtered off and washed with water to
give 1-methyl-9H- β -carboline-7-carboxylic acid (141 mg, 0.62 mmol,
75% yield) as an off-white solid.
1H NMR (500 MHz, DMSO-d6) δ 2.99 (s, 3H), 3.91 (s, 3H), 4.10 (s, 3H),
6.85 (dd, J = 2.2, 8.6 Hz, 1H), 7.17 (d, J = 2.2 Hz, 1H), 7.84 (d, J = 5.2
Hz, 1H), 8.09 (d, J = 8.6 Hz, 1H), 8.14 (d, J = 5.2 Hz, 1H) ; 13C NMR (125
MHz, DMSO-d6) δ 23.6, 32.5, 56.0, 93.8, 109.5, 112.6, 114.4, 122.7,
128.4, 135.8, 138.1, 141.6, 143.7, 161.0 ; HRMS m/z 227.1160 [M+H]+
(C14H14N2O + H+ requires 227.1184; ∆f = -8.37 ppm).
1H NMR (500 MHz, DMSO-d6) δ 2.79 (s, 3H), 7.81 (dd, J = 1.1, 8.2 Hz,
1H), 7.99 (d, J = 5.4 Hz, 1H), 8.19 (brs, 1H), 8.25 (d, J = 5.4 Hz, 1H),
8.29 (d, J = 8.2 Hz, 1H), 11.79 (s, 1H), 12,9 (br, 1H); 13C NMR (125 MHz,
DMSO-d6) δ 20,9, 113.4, 113.9, 120.3, 121.9, 124.7, 126.3, 130.6, 136.0,
138.2, 140.0, 143.1, 168.1; HRMS m/z 227.0804 [M+H]+ (C13H10N2O2+
H+ requires 227.0821; ∆f = -4.84 ppm).
7-Methoxy-1-methyl-9-propyl-9H-β-carboline (8)[18]
Using 1-iodopropane as alkylating agent we obtained 8 (132 mg, 0,52
mmol, 37% yield) as an off-white solid.
1H NMR (500 MHz, DMSO-d6) δ 0.95 (t, J = 7.4 Hz, 3H), 1.76-1.88 (m,
2H), 3.21 (s, 3H), 3.98 (s, 3H), 4.62 (t, J = 7.6 Hz, 2H), 7.06 (dd, J = 2.0,
8.8 Hz, 1H), 7.39 (d, J = 2.0 Hz, 1H), 8.35 (d, J = 6.2 Hz, 1H), 8.37 (d, J =
8.8 Hz, 1H), 8.50 (d, J = 6.2 Hz, 1H), 15.81 (br s, 1H); 13C NMR (125
MHz, DMSO-d6) δ 11.3, 17.8, 23.9, 46.2, 56.4, 94.1, 112.9, 114.7, 124.8,
129.3; HRMS m/z 255.1482 [M+H]+ (C16H18N2O + H+ requires 255.1497;
∆f = -3.92 ppm).
7-Hydroxymethyl-1-methyl-9H-β-carboline (5)
To a solution of 1-methyl-9H-β-carboline-7-carboxylic acid methyl ester
(3.6 g, 15.0 mmol) in anhydrous THF (150 mL) LiAlH4 (1.4 g, 37 mmol)
was added portion wise at 0°C. The reaction mixture was stirred for 2
hours at 0°C and quenched by successive addition of water and
saturated aq. NH4Cl. The reaction mixture was filtered through Celite®
and the filter cake was rinsed with THF (500 mL). The filtrate was
concentrated in vacuo until a precipitate was formed that was removed
by filtration, dried in vacuo to give 7-hydroxymethyl-1-methyl-9H-β-
carboline (3.1 g , 14.6 mmol, 98% yield) as an off-white solid.
9-Isopropyl-7-methoxy-1-methyl-9H-β-carboline (9)
Using 2-iodopropane as alkylating agent we obtained 9 (67 mg, 0.26
mmol, 19% yield) as an off-white solid.
1H NMR (400 MHz, DMSO-d6) δ 1.68 (d, J = 7.0 Hz, 6H), 2.95 (s, 3H),
3.91 (s, 3H), 5.49 (sept, J = 7.0 Hz, 1H), 6.88 (dd, J = 2.1, 8.6 Hz, 1H),
7.18 (d, J = 2.1 Hz, 1H), 7.86 (d, J = 5.2 Hz, 1H), 8.11 (d, J = 8.6 Hz, 1H),
8.15 (d, J = 5.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 21.2, 24.9,
1H NMR (500 MHz, DMSO-d6) δ 2.75 (s, 3H), 4.69 (d, J = 5.7 Hz, 2H),
5.31 (t, J = 5.7 Hz, 1H), 7.16 (dd, J = 1.2, 8.2 Hz, 1H), 7.55 (d, J = 1.2 Hz,
1H), 7.87 (d, J = 5.3 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 8.18 (d, J = 5.3 Hz,
5
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48.0, 55.9, 97.2, 108.9, 112.4, 116.1, 122.9, 128.5, 135.6, 138.0, 140.6,
H+
stir for 60 minutes. NH3 was then passed through the reaction mixture for
6 hours. The formed precipitate was filtered off and washed with CH2Cl2.
The crude product was purified by silica gel chromatography, eluting with
CH2Cl2/methanol (95:5) to give 3-(7-methoxy-1-methyl-β-carbolin-9-yl)-
propionamide (100 mg, 0.35 mmol, 21% yield) as an off-white solid.
1H NMR (500 MHz, DMSO-d6) δ 2.58 (t, J = 7.2 Hz, 2H), 2.97 (s, 3H),
3.91 (s, 3H), 4.76 (t, J = 7.2 Hz, 2H), 6.87 (dd, J = 1.2, 8.5 Hz, 1H), 6.93
(br s, 1H), 7.23 (d, J = 1.2 Hz, 1H), 7.39 (br s, 1H), 7.86 (d, J = 5.1 Hz,
1H), 8.07 (d, J = 8.5 Hz, 1H), 8.16 (d, J = 5.1 Hz, 1H); 13C NMR (125
MHz, DMSO-d6) δ 23.5, 36.3, 41.2, 56.0, 94.4, 109.6, 112.6, 114.6,
122.7, 128.8, 134.9, 138.3, 141.1, 143.0, 160.9; HRMS m/z 284.1404
[M+H]+ (C16H17N3O2 + H+ requires 284.1399; ∆f = 3.52 ppm).
141.1, 159.9; HRMS m/z 255.1490 [M+H]+ (C16H18N2O
+
requires 255.1497; ∆f = -0.78 ppm).
(7-Methoxy-1-methyl-β-carbolin-9-yl)-acetic acid ethyl ester (10)[19]
Using ethyl 2-chloroacetate as alkylating agent we obtained 10 (338 mg,
1.13 mmol, 80% yield) as an off-white solid.
1H NMR (500 MHz, DMSO-d6) δ 1.22 (t, J = 7.0 Hz, 3H), 2.82 (s, 1H),
3.88 (s, 1H), 4.20 (q, J = 7.0 Hz, 2H), 5.51 (s, 2H), 6.89 (dd, J = 1.8, 8.6
Hz, 1H), 7.28 (d, J = 1.8 Hz, 1H), 7.88 (d, J = 5.2 Hz, 1H), 8.09 (d, J = 8.6
Hz, 1H), 8.18 (d, J = 5.2 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 14.4,
22.8, 46.7, 56.1, 61.6, 94.2, 110.0, 112.7, 114.6, 122.7, 129.1, 135.4,
138.8, 141.0, 143.8, 161.1, 170.0; HRMS m/z 299.1391 [M+H]+
(C17H18N2O3 + H+ requires 299.1395; ∆f = 0.33 ppm).
3-(7-Methoxy-1-methyl-β-carbolin-9-yl)-propan-1-ol (15)
To a solution of 3-(7-methoxy-1-methyl-β-carbolin-9-yl)-propionic acid
methyl ester (800 mg, 2.7 mmol) in anhydrous THF (10 mL) LiAlH4 (201
mg, 5.3 mmol) was added portion wise at 0°C. The reaction mixture was
stirred for 2 hours at the same temperature, and then it was allowed to
warm up to room temperature. The mixture was quenched by successive
addition of water and 2N aqueous NaOH, filtered through a plug of
Celite® and the filter cake was rinsed with THF (50 mL). The filtrate was
concentrated in vacuo and the crude product was purified by
crystallization from acetonitrile to give 3-(7-methoxy-1-methyl-β-carbolin-
9-yl)-propan-1-ol (531 mg , 2.0 mmol, 73% yield) as an off-white solid.
1H NMR (400 MHz, DMSO-d6) δ 1.85-1.95 (m, 2H), 2.97 (s, 3H), 3.45-
3.51 (m, 2H), 3.90 (s, 3H), 4.59-4.66 (m, 2H), 4.72 (t, J = 5.0 Hz, 1H),
6.86 (dd, J = 2.2, 8.6 Hz, 1H), 7.20 (d, J = 2.2 Hz, 1H), 7.86 (d, J = 5.2
Hz, 1H), 8.07 (d, J = 8.6 Hz, 1H), 8.16 (d, J = 5.2 Hz, 1H); 13C NMR (100
MHz, DMSO-d6) δ 23.5, 33.9, 41.7, 56.0, 58.3, 94.1, 109.6, 112.6, 114.3,
122.8, 128.9, 135.1, 138.2, 141.2, 143.3, 160.9; HRMS m/z 271.1434
[M+H]+ (C16H18N2O2 + H+ requires 271.1446; ∆f = -2.58 ppm).
9-Benzyl-7-methoxy-1-methyl-9H-β-carboline (11)[20]
Using benzyl bromide as alkylating agent we obtained 11 (197 mg, 0.65
mmol, 46% yield) as an off-white solid.
1H NMR (400 MHz, DMSO-d6) δ 2.97 (s, 3H), 3.90 (s, 3H), 6.03 (s, 2H),
7.00 (d, J = 7.5 Hz, 2H), 7.11 (dd, J = 2.0, 8.8 Hz, 1H), 7.27 (t, J = 7.5 Hz,
1H), 7.32 (t, J = 7.5 Hz, 2H), 7.41 (d, J = 2.0 Hz, 1H), 8.41 (d, J = 6.2 Hz,
1H), 8.45 (d, J = 8.8 Hz, 1H), 8.58 (d, J = 6.2 Hz, 1H), 15.47 (br s, 1H);
13C NMR (100 MHz, DMSO-d6) δ 17.7, 47.9, 56.4, 94.0, 113.2, 114.9,
125.0, 125.8, 128.0, 129.4, 130.1; HRMS m/z 303.1487 [M+H]+
(C20H18N2O + H+ requires 303.1497; ∆f = -1.65 ppm).
3-(7-Methoxy-1-methyl-β-carbolin-9-yl)-propionic acid methyl ester (12)
To a stirred suspension of harmine (2.13 g, 10.0 mmol) and DBU (1.76
mL, 12.0 mmol) in acetonitrile (20 mL) acrylic acid methyl ester (2.1 mL,
25 mmol) was added and the mixture was stirred at 60°C overnight. The
resulting solid was filtered off and washed with acetonitrile (2×10 mL) to
give 3-(7-methoxy-1-methyl-β-carbolin-9-yl)-propionic acid methyl ester
(2.15 g, 7.2 mmol, 72% yield) as an off-white solid.
Synthesis of 1-substituted-7-Methoxy-9H-β-carbolines
7-Methoxy-1-trifluoromethyl-9H-β-carboline (17)[22]
1H NMR (500 MHz, DMSO-d6) δ 2.81-2.86 (m, 2H), 2.95 (s, 3H), 3.55 (s,
3H), 3.91 (s, 3H), 4.82-4.88 (m, 2H), 6.87 (dd, J = 2.2, 8.6 Hz, 1H), 7.22
(d, J = 2.2 Hz, 1H), 7.86 (d, J = 5.2 Hz, 1H), 8.07 (d, J = 8.6 Hz, 1H), 8.17
(d, J = 5.2 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 23.4, 34.5, 40.4,
51.9, 56.0, 94.1, 109.9, 112.6, 114.7, 122.8, 129.0, 134.8, 138.4, 141.1,
142.7, 161.0, 171.6; HRMS m/z 299.1397 [M+H]+ (C17H18N2O3 + H+
requires 299.1395; ∆f 2.34 ppm).
A
suspension of 6-methoxytryptamine (500 mg, 2.63 mmol) in
trifluoroacetaldehyde ethyl hemiacetal (455 mg, 3.15 mmol) was heated
at reflux (110°C) under argon for 5 hours. The solvent was removed in
vacuo. The residue was dissolved in CH2Cl2 (15 mL), DDQ (1.5 g, 6.57
mmol) was added and the mixture was stirred for 1 hour at room
temperature. The mixture was concentrated to dryness in vacuo and the
crude product was purified by reverse phase chromatography, eluting
with water/ acetonitrile to give 7-methoxy-1-trifluoromethyl-9H-β-carboline
(328 mg, 1.23 mmol, 47% yield) as a white solid.
3-(7-Methoxy-1-methyl-β-carbolin-9-yl)-propionic acid (13)[21]
1H NMR (400 MHz, DMSO-d6) δ 3.89 (s, 3H), 6.95 (dd, J = 2.2, 8.7 Hz,
1H), 7.13 (d, J = 2.2 Hz, 1H), 8.20 (d, J = 8.7 Hz, 1H), 8.32 (d, J = 5.2 Hz,
1H), 8.42 (d, J = 5.2 Hz, 1H), 11.7 (s, 1H); 13C NMR (100 MHz, DMSO-
d6) δ 55.9, 95.6, 110.7, 114.1, 118.0, 123.5, 129.1, 132.0, 132.1, 138.2,
143.8, 161.7; 19F NMR (376.5 MHz, DMSO-d6) δ -63.2;HRMS m/z
267.0730 [M+H]+ (C13H9N2OF3 + H+ requires 267.0745; ∆f = -3.74 ppm).
A
solution of 3-(7-methoxy-1-methyl-β-carbolin-9-yl)-propionic acid
methyl ester (1.0 g, 3.3 mmol) and LiOH×H2O (277 mg, 6.6 mmol) in a
mixture of methanol (10 mL) and water (2 mL) was stirred for 6 hours at
room temperature. The solvent was then removed in vacuo, water (10
mL) was added and the resulting mixture was neutralized with 1N aq.
HCl (6.6 mL). The resulting solid was filtered off and washed with water
to give 3-(7-methoxy-1-methyl-β-carbolin-9-yl)-propionic acid (650 mg,
2.9 mmol, 69% yield) as an off-white solid.
7-Methoxy-1-methyl-9H-β-carboline 2-oxide
1H NMR (500 MHz, DMSO-d6) δ 2.74 (t, J = 7.5 Hz, 2H), 2.96 (s, 3H),
3.91 (s, 3H), 4.82 (t, J = 7.5 Hz, 2H), 6.87 (dd, J = 1.6, 8.6 Hz, 1H), 7.24
(d, J = 1.6 Hz, 1H), 7.86 (d, J = 5.0 Hz, 1H), 8.07 (d, J = 8.6 Hz, 1H), 8.17
(d, J = 5.0 Hz, 1H), 12.52 (br s, 1H); 13C NMR (125 MHz, DMSO-d6) δ
23.4, 35.0, 40.6, 56.0, 94.2, 109.8, 112.6, 115.0, 122.7, 128.8, 134.8,
138.3, 141.0, 142.4, 160.9, 172.7; HRMS m/z 285.1229 [M+H]+
(C16H16N2O3 + H+ requires 285.1239; ∆f = -1.75 ppm).
To a suspension of harmine (3.18 g, 15.0 mmol) in CH2Cl2 (300 mL) 3-
chloroperbenzoic acid (4.2 g, 24.3 mmol) was added portion wise and
allowed to stir for 2 hours. The mixture was concentrated to dryness in
vacuo and it was purified by silica gel chromatography, eluting with
CH2Cl2/ methanol (9:1) to give 7-methoxy-1-methyl-9H-β-carboline 2-
oxide (2.1 g, 9.2 mmol, 61% yield) as a white solid.
1H NMR (500 MHz, DMSO-d6) δ 2.62 (s, 3H), 3.86 (s, 3H), 6.85 (dd, J =
2.1, 8.6 Hz, 1H), 6.97 (d, J = 2.1 Hz, 1H), 7.85 (d, J = 6.7 Hz, 1H), 7.99
(d, J = 8.6 Hz, 1H), 8.04 (d, J = 6.7 Hz, 1H), 11.58 (s, 1H); 13C NMR (125
MHz, DMSO-d6) δ 13.0, 55.8, 95.3, 110.0, 113.7, 115.7, 119.1, 122.4,
131.4, 132.8, 136.6, 143.4, 159.9; 15N NMR (40.5 MHz, DMSO-d6) δ
281.6; HRMS m/ 229.0967 [M+H]+ (C13H12N2O2 + H+ requires 229.0977;
∆f = -2.18 ppm).
3-(7-Methoxy-1-methyl-β-carbolin-9-yl)-propionamide (14)[21]
To a stirred solution of 3-(7-methoxy-1-methyl-β-carbolin-9-yl)-propionic
acid (500 mg, 1.68 mmol) and a drop of DMF in CH2Cl2 (5 mL) oxalyl
chloride (505 µL, 8.4 mmol) was added dropwise and it was allowed to
6
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1-Hydroxymethyl-7-methoxy-9H-β-carbolinium trifluoroacetate (18)
A solution of 1-chloro-7-methoxy-9H-β-carboline (232 mg, 1.0 mmol), 2-
(methylamino)ethanol (120 µL, 1.5 mmol), Pd2(dba)3 (23 mg, 0.025
mmol), t-butylXPhos (43 mg, 0.1 mmol) and sodium tert-butoxide (288
mg, 3.0 mmol) in toluene (10 mL) was stirred at 80°C under argon
atmosphere overnight. The reaction mixture was filtered through Celite®
and the filter cake was rinsed with toluene. The filtrate was concentrated
to dryness and the residue was purified by silica gel chromatography
eluted with CH2Cl2/methanol to give [2-(7-methoxy-9H-β-carbolin-1-
yloxy)-ethyl]-methyl-amine as colourless oil, which was treated with
hydrochloric acid in diethyl ether to give [2-(7-methoxy-9H-β-carbolin-1-
yloxy)-ethyl]-methyl-amine monohydrochloride (113 mg, 0.37 mmol, 37%
yield) as an off-white solid.
1H NMR (500 MHz, DMSO-d6) δ 2.68 (t, J = 5.4 Hz, 3H), 3.40-3.49 (m,
2H), 3.87 (s, 3H), 4.73 (t, J = 5.0 Hz, 2H), 6.87 (dd, J = 2.2, 8.7 Hz, 1H),
7.00 (d, J = 2.2 Hz, 1H), 7.74 (d, J = 5.6 Hz, 1H), 7.83 (d, J = 5.6 Hz, 1H),
8.06 (d, J = 8.7 Hz, 1H), 9.36-9.56 (m, 2H), 12.28 (s, 1H); 13C NMR (125
MHz, DMSO-d6) δ 32.9, 47.7, 55.8, 62.1, 94.8, 110.2, 110.6, 115.3,
123.2, 123.8, 130.1, 133.5, 142.3, 149.2, 160.7; HRMS m/z 272.1400
[M+H]+ (C15H17N3O2 + H+ requires 272.1399; ∆f = -2.25 ppm).
To a solution of 7-methoxy-1-methyl-9H-β-carboline 2-oxide (700 mg, 3.1
mmol) in DMF (30 mL) trifluoroacetic anhydride (1.51 mL, 6.2 mmol) was
added dropwise at 0°C and allowed to stir overnight. The formed
precipitate was filtered off. The filtrate was concentrated to dryness in
vacuo and the crude product was purified by crystallization from
acetonitrile to give the trifluoroacetic acid salt of (7-methoxy-9H-β-
carbolin-1-yl)-methanol (190 mg, 0.56 mmol, 20% yield) as an off-white
solid.
1H NMR (500 MHz, DMSO-d6) δ 3.94 (s, 3H), 5.23 (s, 2H), 6.48 (br s,
1H), 7.06 (dd, J = 2.2, 8.8 Hz, 1H), 7.17 (d, J = 2.2 Hz, 1H), 8.37 (d, J =
8.8 Hz, 1H), 8.39 (d, J = 6.2 Hz, 1H), 8.5 (d, J = 6.2 Hz, 1H), 12.58 (s,
1H), 14.78 (br s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 56.2, 58.4, 95.0,
112.9, 113.9, 115.3, 125.0, 129.6, 132.0, 133.2, 140.1, 146.0, 163.2;
HRMS m/z 229.0967 [M+H]+ (C13H12N2O2 + H+ requires 229.0977; ∆f = -
1.31 ppm).
7-Methoxy-2,9-dihydro-β-carbolin-1-one (19)[23]
Pharmacology
To a suspension of 6-methoxytryptamine (3.0 g, 15.8 mmol) in a mixture
of CH2Cl2 (45 mL) and 5% aqueous NaHCO3 (30 mL) phenyl
chloroformate (2.0 mL, 15.8 mmol) was added dropwise at 0°C and it
was allowed to stir for 2 hours at the same temperature. The mixture was
diluted with CH2Cl2 (200 mL), the organic layer was separated, dried over
MgSO4 and concentrated to dryness. This crude product was purified by
silica gel chromatography, eluting with heptane/ethyl acetate to give the
desired carbamate, which was used in the following step. To a stirred
solution of the previously synthesized carbamate in toluene (225 mL)
BF3-Et2O (2.2 mL, 17.34 mmol) was added dropwise at 0°C and the
mixture was stirred under reflux for 5 hours under argon atmosphere.
The reaction was quenched by successive addition of water and
saturated aqueous NaHCO3. The mixture was extracted with ethyl
acetate, the organic layer was separated, dried over MgSO4 and
concentrated to dryness. The remaining material was dissolved in 1,4-
dioxane (200 mL), treated with DDQ (3.9 g, 17.34 mmol) and stirred for
60 minutes. The mixture was diluted with CH2Cl2, washed with water, the
separated organic layer was, dried over MgSO4 and concentrated to
dryness to give 7-methoxy-2,9-dihydro- β -carbolin-1-one ( 1.7 g, 7.9
mmol, 50% yield).
DYRK1A inhibition assay
Inhibition of the enzymatic activity of DYRK1A was evaluated in a Time-
Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay in
384-well reaction plates. In this assay, full-length DYRK1A from Carna
Biosciences (NM_001396, ref. 04-130; 2.0 ng/µl) was incubated for 40
minutes at room temperature with ATP (Sigma A2383, 10 µM) and a
ULightTM-labelled human Myelin Basic Protein (MBP) peptide substrate
(Perkin Elmer TRF0109, 100 nM) in a reaction buffer composed of 50
mM HEPES pH7.4, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT and 0.01%
Tween20. Test compounds were added in reaction buffer at a range of
concentrations from 0.1 nM to 30 µM. Following addition of EDTA (Sigma
E7889, 10 mM) to stop the reaction, Europium-labeled mouse
monoclonal antibody recognizing phospho-Thr232 in MBP (Perkin Elmer
TRF0201, 1 nM) was added. After one hour, the reaction plates were
read using a fluorescence reader (EnVision®, Perkin Elmer) at 620nm
and 665 nm (excitation at 340 nm).
1H NMR (500 MHz, DMSO-d6) δ 3.82 (s, 3H), 6.80 (dd, J = 2.2, 8.7 Hz,
1H), 6,90 (d, J = 6,8 Hz, 1H), 6,93 (d, J = 2,2 Hz, 1H), 7,05 (br, 1H), 7,88
(d, J = 8.7 Hz, 1H), 11.29 (s, 1H), 11.79 (s, 1H); 13C NMR (125 MHz,
DMSO-d6) δ 55.7, 94.7, 99.9, 110.8, 116.4, 122.6, 125.2, 125.2, 127.8,
140.8, 155.8, 159.4; HRMS m/z 215.0807 [M+H]+ (C12H10N2O2+ H+
requires 215.0820; ∆f = -4.29 ppm).
MAO-A inhibition assay
Microsomes from insect cells containing recombinant human MAO-A (5
mg/mL) were obtained from Sigma–Aldrich, prealiquoted and stored at -
70°C. All enzymatic reactions were carried out in potassium phosphate
buffer (100 mM, pH 7.4, made isotonic with KCl) containing MAO-A (7.5
µg/mL), various concentrations of the test inhibitor (0–10 µM) and
kynuramine (0.15 mM) in 500 µL of final volume that also contained 4%
(v/v) DMSO. The reactions were incubated for 30 min at 30°C. After
termination of the reaction and centrifugation the concentration of 4-
hydroxyquinoline was determined by fluorescence spectroscopy. IC50
values were determined by plotting the initial rate of oxidation versus the
logarithm of the inhibitor concentration to obtain a sigmoidal dose–
response curve. Compounds not reaching 25% inhibition at the top
concentration were classified inactive.
1-Chloro-7-methoxy-9H-β-carboline (20)
The solution of 7-methoxy-2,9-dihydro-β-carbolin-1-one (1.52 g, 7.1
mmol) in POCl3 (60 mL) was stirred under reflux for 5 days in argon
atmosphere. The reaction mixture was concentrated to approximately 10
mL volume and poured onto ice (500 mL), neutralized with addition of
K2CO3 and the formed precipitate was removed by filtration. The crude
product was purified by silica gel chromatography, eluting with CH2Cl2/
methanol (9:1) to give 1-chloro-7-methoxy-9H-β-carboline (1.3 g, 5.6
mmol, 35% yield) as a solid.
Keywords: DYRK1A • MAO-A • Harmine • β-carbolines •
1H NMR (500 MHz, DMSO-d6) δ 3.88 (s, 3H), 6.91 (dd, J = 2.2, 8.8 Hz,
1H), 7.05 (d, J = 2.2 Hz, 1H), 8.04 (d, J = 5.2 Hz, 1H), 8.09 (d, J = 5.2 Hz,
1H), 8.13 (d, J = 8.8 Hz, 1H), 11.82 (s, 1H); 13C NMR (125 MHz, DMSO-
d6) δ 55.9, 95.3, 110.7, 114.5, 115.1, 123.6, 130.8, 132.8, 133.0, 138.2,
selectivity
142.9, 161.2; HRMS m/z 233.0466 [M+H]+ (C12H9N2OCl
+
H+
requires 233.0481; ∆f = -4.29 ppm).
[2-(7-Methoxy-9H-β-carbolin-1-yloxy)-ethyl]-methyl-amine
monohydrochloride (21)
7
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Becker , W.; Weber, Y.; Wetzel, K.; Eirmbter, K.; Tejedor, F.J.; Joost,
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10.1002/cmdc.201600539
ChemMedChem
FULL PAPER
Entry for the Table of Contents
MAO-A
selective
DYRK1A
selective
The different orientations of harmine in the X-ray structures of this natural product bound to DYRK1A and MAO-A suggested
chemical ways to fine-tune the selectivity profile of this compound class. These structure guided transformations led to the abolition of
DYRK1A and/or MAO-A inhibition in harmine derivatives as well as to the synthesis of dual inhibitors of mixed inhibition ratio.
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