Structure–activity relationships and biological evaluation of 7-substituted harmine analogs for human β-cell proliferation

Article abstract of DOI:10.3390/molecules25081983

Recently, we have shown that harmine induces β-cell proliferation both in vitro and in vivo, mediated via the DYRK1A-NFAT pathway. We explore structure–activity relationships of the 7-position of harmine for both DYRK1A kinase inhibition and β-cell proliferation based on our related previous structure–activity relationship studies of harmine in the context of diabetes and β-cell specific targeting strategies. 33 harmine analogs of the 7-position substituent were synthesized and evaluated for biological activity. Two novel inhibitors were identified which showed DYRK1A inhibition and human β-cell proliferation capability. The DYRK1A inhibitor, compound 1-2b, induced β-cell proliferation half that of harmine at three times higher concentration. From these studies we can draw the inference that 7-position modification is limited for further harmine optimization focused on β-cell proliferation and cell-specific targeting approach for diabetes therapeutics.

Full text of DOI:10.3390/molecules25081983

molecules
Article
Structure–Activity Relationships and Biological
Evaluation of 7-Substituted Harmine Analogs for
Human β-Cell Proliferation
Kunal Kumar 1,2,†, Peng Wang 3,†, Ethan A. Swartz 3, Susmita Khamrui 2, Cody Secor 2,
Michael B. Lazarus 1,2 , Roberto Sanchez 1,2, Andrew F. Stewart 3 and Robert J. DeVita 1,2,
*
1
Drug Discovery Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA;
kunal.kumar@mssm.edu (K.K.); Michael.Lazarus@mssm.edu (M.B.L.); Roberto.Sanchez@mssm.edu (R.S.)
Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai,
New York, NY 10029, USA; Susmita.Khamrui@mssm.edu (S.K.); Cody.Secor@mssm.edu (C.S.)
Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai,
New York, NY 10029, USA; Peng.wang@mssm.edu (P.W.); ethan.swartz@mssm.edu (E.A.S.);
Andrew.Stewart@mssm.edu (A.F.S.)
2
3
*
Correspondence: robert.devita@mssm.edu; Tel.: +212-659-5542
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 25 March 2020; Accepted: 20 April 2020; Published: 23 April 2020
Abstract: Recently, we have shown that harmine induces
in vivo, mediated via the DYRK1A-NFAT pathway. We explore structure–activity relationships of
the 7-position of harmine for both DYRK1A kinase inhibition and -cell proliferation based on our
related previous structure–activity relationship studies of harmine in the context of diabetes and
-cell specific targeting strategies. 33 harmine analogs of the 7-position substituent were synthesized
and evaluated for biological activity. Two novel inhibitors were identified which showed DYRK1A
inhibition and human -cell proliferation capability. The DYRK1A inhibitor, compound 1-2b, induced
-cell proliferation half that of harmine at three times higher concentration. From these studies
β-cell proliferation both in vitro and
β
β
β
β
we can draw the inference that 7-position modification is limited for further harmine optimization
focused on β-cell proliferation and cell-specific targeting approach for diabetes therapeutics.
Keywords: dual-specificity tyrosine-regulated kinases (DYRKs); harmine; DYRK1A inhibitor;
structure–activity relationship study; β-cell proliferation; diabetes
1. Introduction
The dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is a eukaryotic
protein kinase that has been shown to play important roles in biological processes related to various
diseases [
-cells [5–8]. Numerous studies have previously reported the development of DYRK1A inhibitor
scaffolds for a variety of therapeutic applications including diabetes [ 33]. Among all the DYRK1A
inhibitors, harmine and its analogues ( -carbolines) are the most commonly studied, and remain one
of the most potent and orally bioavailable class of inhibitors known to date [2,10].
Recently, our group found that harmine is able to induce human -cell proliferation and
DYRK1A-NFAT pathway as being the major pathway for this cell proliferation [ ]. These results have
been confirmed in other labs with other DYRK1A inhibitors unrelated to the harmine scaffold, including
from our own lab [ 34 37]. Since, modification of harmine has not been explored previously in
context of -cell proliferation, we therefore decided to carry out structure–activity relationship studies
1–4] and more recently, as a major regulator of human insulin-producing pancreatic
β
2–6,8–
β
β
5
6,8, –
β
of harmine for both DYRK1A inhibition and
β-cell proliferation. Based on the known binding pose
Molecules 2020, 25, 1983; doi:10.3390/molecules25081983
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1983
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of harmine, we surmised that there are three positions, 1-methyl,7-methoxy and 9-N indole, where
rational modifications of harmine can be carried out (Figure 1) without disrupting key DYRK1A
binding contacts. Previously, we reported the optimization of the 1-position of harmine which led
to two compounds with robust human β-cell proliferation at doses of 3–30 µM and one of those,
compound I (R = CH2OH), showing improved kinase selectivity as compared to harmine (Figure 1) [38].
Next, we recently reported the optimization of the N-9 position to identify 2-2c, a highly optimized
in vivo active harmine based DYRK1A inhibitor (Figure 1) [39]. To extend this systematic medicinal
chemistry strategy, we last focused our effort on the harmine 7-position to understand the impact of
modifications on DYRK1A inhibition and
was to explore the potential for 7-substituents to link to putative targeting molecules for cell specific
delivery to the -cells, without negatively affecting DYRK1A and -cell proliferative biological activity.
One such prototype targeting technology for cells that highly express GLP1-receptor, including -cells,
was reported by DiMarchi and colleagues [40 41]. Herein, we report the results of our 7-position of
β-cell proliferation (Figure 1). An additional consideration
β
β
β
,
harmine medicinal chemistry studies. We first determined the effect of these modifications on DYRK1A
inhibition, measured by FRET-based LanthaScreen® Eu Kinase Binding Assay [42]. Since the overall
goal of the project is to develop DYRK1A inhibitors that can induce
β-cells to proliferate, we also
assayed potent 7-substituted harmine analogs for human
immunolabeling in beta cells [5,39,43–45].
β-cell proliferation by quantifying Ki67
Figure 1. Optimization of the 7-position of harmine.
2. Results and Discussion
2.1. Synthesis
Modification of the harmine 7-position was carried out by several standard reaction sequences
outlined in Scheme 1. Harmine underwent demethylation in the presence of HBr/AcOH to give
harmol 1-1 in 99% yield [46]. Alkylation of harmol 1-1 with various alkyl bromides in the presence
of cesium carbonate as base generated, in 40–68% yield, 7-ether harmine analogs 1-2a
a terminal methyl ester, t-butyl ester and BOC-protected amino group with carbon chain lengths
of 1–5 carbons [46]. Harmine analogs 1-2a 1-2e with terminal methyl esters were refluxed in
excess methanolic ammonia solution to provide the corresponding terminal carboxamide analogs
1-3a 1-3e in 84–95% yield. t-Butyl ester harmine analogs 1-2f 1-2j were treated with hydrochloric
acid at room temperature to give corresponding acids 1-4a 1-4e in excellent yield. Compounds
1-2k 1-2n underwent N-BOC deprotection to generate harmine analogs 1-5a 1-5d with terminal
amino functional group which then, underwent acylation using acetic anhydride to afford harmine
7-alkoxy acetamide analogs 1-6a 1-6d. Harmol (1-1) was also treated with trifluoromethanesulfonic
–1-2n having
–
–
–
–
–
–
–
anhydride to form O-trifluoromethanesulfonyl harmine analog 1-8 which underwent palladium
catalyzed nitration reaction, developed by Buchwald lab [47], followed by nitro group reduction to
generate 7-amino harmine analog 1-9 in 77% yield. Derivatization of 7-amino functional group of
1-9 with acetic anhydride and benzoyl chloride in presence of triethyl amine afforded carboxamides
1-10 and 1-11, respectively. Harmine analogs 1-2o and 1-2p, bearing alkyl ether substituents at the
7-positions, were synthesized by standard basic alkylation of harmol 1-1. Compound 1-12 containing
the carbamate functional group at the 7-position was synthesized in 32% yield by treating harmol 1-1

Molecules 2020, 25, 1983
3 of 18
with isopropylisocyanate and trimethylamine [48]. HNMR spectra of synthesized compounds are
included in Supplementary Material.
N
N
N
a
b
RO
N
N
N
HO
O
MeO
H
n
H
H
O
R = Me, n = 1-5, 1-2a - 1-2e
R = t-Bu, n = 1-5, 1-2f - 1-2j
1-1
N
N
N
c
MeO
H2N
N
N
H
O
O
H
n
n
O
O
O
1-2a - 1-2e
1-3, n = 1-5
d
N.HCl
t-BuO
HO
N
H
N
H
O
O
n
n
O
1-2f - 1-2j
1-4, n = 1-5
b
e
N
N
N
O
N
H
N
N
H
HO
t-BuO
N
H
O
ClH3N
O
H
n
n
1-5
1-1
1-2k - 1-2n, n = 2-4
N
O
f
N
N
H
O
H
n
1-6
b
N
N
O
N
H
N
HO
R
O
H
1-1
1-2o, R = Me
1-2p, R = Et
h
N
N
g
N
N
N
H
N
H
O2N
HO
TfO
H
1-7
1-1
1-8
N
N
j or k
i
O
N
H
N
R
N
H
H2N
H
1-9
1-10, R = Ph
1-11, R = Me
N
l
N
O
N
N
H
1-12
HO
N
O
H
H
1-1
Scheme 1. Synthesis of 7-Substituted Harmine Analogs. Reagents and conditions: (
reflux, 12 h, 99%; (
in MeOH (20 eq.), 90 ◦C, 12 h, 84–97%; (
dioxane, rt, 12 h, 90–99%; ( ) Acetic anhydride (1 eq.), Et3N (2.2eq.), DCM, rt, 12 h, 49–79%; (
(1.2 eq.), Pyridine, DCM, 0 ◦C-rt, 12h, 92%; (
) NaNO2 (2 eq.), Pd2(dba)3 (5 mol%), BrettPhos (12 mol%),
TDA (5 mol%), t-BuOH, 150 ◦C, 24 h, 77%; (
) 10% wt Pd on C, N2H4.H2O (20 eq.), MeOH, reflux, 2 h,
98%; ( ) Acetic anhydride (1.2 eq.),
) Benzoyl chloride (1.05 eq.), Et3N (2 eq.), THF, 0 ◦C-rt, 12 h, 75%; (
a) AcOH, 48% HBr,
b
) Cs2CO3 (1.5 eq.), Br(CH2)nCO2R (1.5 eq.), DMF, 50 ◦C, 12 h, 32–68%; (
c
) 7N NH3
) 4N HCl in dioxane,
) Tf2O
d) 4N HCl in dioxane, rt, 12 h, 82–98%; (e
f
g
h
i
j
k
Et3N (2 eq.), THF, rt, 24 h, 77%; (l) Isopropylisocyanate (1.1 eq.), DMF, rt, 12 h, 32%.
2.2. Structure–Activity Relationships of 7-Substituted Harmine Analogs
As outlined in Figure 1 and Scheme 1, modification of the harmine 7-position was carried out to
identify linkable harmine analogs by first introducing substituents bearing a terminal methyl ester (1-2a

Molecules 2020, 25, 1983
4 of 18
to 1-2e), carboxamide (1-3a to 1-3e), carboxylic acid (1-4a to 1-4e) and amino/substituted amino (1-2k
to 1-2n 1-5a to 1-5d, and 1-6a to 1-6d) functional groups with chain length varying from 1–5 carbons.
,
We first studied the effect of introducing these modifications on DYRK1A inhibition, measured by
FRET-based LanthaScreen® Eu Kinase Binding Assay [42]. Among the 33 harmine analogs, only
carboxamide and methyl ester functional groups were well tolerated to provide DYRK1A inhibition
IC500s less than 100 nM. 7-substituted methyl ester harmine analog (1-2b) and related 3-carbon analog
(1-3b) with ethylene (2-carbon) chain between harmine 7-oxygen and the carboxamide, showed the
most potent DYRK1A inhibitory activity of this set of analogs with DYRK1A IC50 in the range of
89–90 nM (Figure 2). These compounds are ~3-fold less potent inhibitors of DYRK1A kinase in vitro as
compared to harmine itself (27 nM).
N
N
R
N
N
R
O
H
H
1-2, 1-3, 1-4, 1-5, 1-6, 1-12
1-9, 1-10, 1-11
R
% DYRK1A Inhibition
% DYRK1A Inhibition
Compound
IC50 (nM)a
IC50 (nM)a
R
Compound
1000 nM
300 nM
1000 nM
300 nM
O
O
-
-
*
1-4b
HO
O
1810±100.7
1-2a
1-2b
85
-
58
-
194±11.7
*
MeO
O
*
89.7±7.68
1-4c
1-4d
19
44
6
-
-
HO
O
*
*
MeO
O
20
1-2c
1-2d
1-2e
78
67
81
47
35
56
-
*
*
HO
MeO
O
O
1-4e
1-5a
1-5b
74
25
28
43
12
8
-
-
-
-
HO
*
*
MeO
O
ClH3N
*
134±4.55
MeO
H
O
O
N
*
*
*
1-2k
1-2l
61
50
58
35
24
31
-
-
-
ClH3N
O
1-5c
1-5d
1-6a
42
32
49
15
15
24
-
-
-
*
*
O
O
N
H
ClH3N
1-2m
O
ClH3N
O
O
N
H
*
*
O
1-2n
1-2o
1-2p
1-3a
1-3b
O
48
48
37
30
-
27
23
19
10
-
-
O
N
H
*
*
N
H
O
O
O
-
1-6b
1-6c
1-6d
1-9
49
69
41
21
65
22
45
17
8
-
-
-
-
-
*
*
N
H
O
*
*
-
N
H
O
O
-
H2N
O
O
N
H
*
*
*
91±9.38
*
*
H2N
O
H2N
O
1-10
37
N
1-3c
H2N
58
32
-
H
O
O
*
1-11
1-12
77
69
48
42
389±32.59
1-3d
1-3e
88
67
67
39
407±28.13
*
*
H2N
O
Ph
N
H
O
-
-
H2N
*
N
H
O
*
1-4a
6
3
-
Harmine
-
-
27±2.04
HO
a = IC50 values are determined using ten serial three fold dilutions (in duplicate).
Figure 2. Dual-specificity Tyrosine-phosphorylation-Regulated Kinase A (DYRK1A) inhibition of 7-C
harmine analogs a.

Molecules 2020, 25, 1983
5 of 18
Interestingly, other analogs with longer carbon lengths did not provide any improvement for
DYRK1A inhibition, a result that did not align with our original docking hypothesis, which indicated
that extension into solvent would be possible. Subsequent docking studies of these novel 7-C harmine
analogs based on the crystal structure of DYRK1A bound to harmine analog 2-2c [39] were performed
and suggested that the loss of activity with longer substituents at the 7-position could be due to interplay
of several factors that diminish interactions with the DYRK1A back bone H-bond at Leu 241. These
include steric hindrance, the entropic effects of longer, more flexible side chains, combined with the
possibility of partially compensating additional new contacts (Figure 3A, subpanel A–F). The shorter
chains (1-2b (Figure 3A, subpanel B), 1-3b (Figure 3A, subpanel F)) are expected to have a reduced
entropic effect and therefore, the activity is similar to that of harmine. In the case of carboxamide 1-3b
there is also the potential for a compensatory new contact with Ser 242 (Figure 3A, subpanel F). Several
analogs with longer chains are accommodated as a result of their flexibility, which allows them to fold
back in the case of 1-2c (Figure 3A, subpanel C) or expand into unoccupied space, as the case of 1-2l
(Figure 3A, subpanel E) based on this model. These data suggest that analogs that do not contribute
new contacts to DYRK1A, result in a loss of potency, likely due to of the increased entropic cost of more
flexible 7-position side chains. These docking studies also indicate that the chain in 1-2e (Figure 3A,
subpanel D) folds back to make a hydrogen bond with Asn 244 (one of the crucial contacts made by
the 9-N harmine carboxamide analog, Figure 3D) partially offsetting this entropic penalty.
Figure 3. Docking of selected 7-substituted harmine analogs. A. Docking of 1-2a (subpanel A), 1-2b
(subpanel B), 1-2c (subpanel C), 1-2e (subpanel D), 1-2l (subpanel E), 1-3b (subpanel F). Ligands
are shown in green, the protein surface in gray, and selected residues in light blue. B. Docking of
compounds 1-10 (subpanel A,D), 1-11 (subpanel B,E), and 1-12 (subpanel C,F). Subpanel A-C stick
model of the ligand docked into the ATP-binding pocket of DYRK1A. Subpanel D–F, space filling
models of the same structures shown in A–C. All three compounds are unable to hydrogen bond
with the backbone of Leu 241 (used by harmine, panel D) due to the unfavorable orientation of their
carbonyl oxygen. The hydrophobic substituents however interact with a hydrophobic cleft formed by
the side chain of Ile 165 and Met 240 (shown in yellow). C. Docking of 7-substituted Harmine analogs
containing carboxylic acid groups. Compound 1-4c is shown in green and compounds 1-4e in yellow.
D. Harmine bound to DYRK1A (PDB 3ANR). The surface of the protein is colored according to the
electrostatic potential (red for negative and blue for positive).
It was also observed that carboxylic acid and amino functional groups at the 7-position, regardless
of their chain length, did not exhibit DYRK1A inhibition in vitro at the concentrations tested. Docking

Molecules 2020, 25, 1983
6 of 18
of the harmine analogs that possess negatively charged substituents, such as carboxylic acid or
positively charged protonated amines, indicate that they have a detrimental effect on binding due
to the electrostatic environment of the DYRK1A binding pocket, irrespective of the orientation of
the chain (Figure 3C). For these substituents the expectation is that, the longer the chain the lower
the electrostatic effect (because it can extend out of the negatively charged binding pocket toward
solvent), resulting in greater entropic cost. Thus, based on these data and our analysis of subsequent
structural docking studies, it is now difficult to expect compounds with a carboxylic acid or amino
containing substituents at the harmine 7-position to be potent DYRK1A inhibitors suitable for linkage
to cell-specific targeting molecules.
Simple 7-position alkyl ether substitutions, for example, 7-oxo-(methoxyethyl) (1-2o) and
7-oxo-(ethoxyethyl) (1-2p), were also not favorable for the DYRK1A inhibition. We speculate that these
modifications hinder the hydrogen bond from Leu241 backbone N-H with the 7-oxygen atom that
is more effectively made with the sterically less demanding 7-OMe group of harmine. Additionally,
we also investigated the effect of replacing 7-methoxy group with amino and substituted amino groups.
Unfortunately, 7-amino harmine analog 1-9 completely lost its DYRK1A inhibitory activity. Docking
results indicate that due to the orientation of the Leu241 carbonyl oxygen, hydrogen bonding with
Leu241 residue seems unlikely (Figure 3B, subpanel A–F. DYRK1A inhibitory activity was partially
recovered when the 7-amino group was modified to benzamido (1-11) group (Figure 3B, subpanel B,E).
From modeling studies, it appears that favorable packing of the benzyl ring between the sidechains
of Met240 and Ile165 may contribute to the DYRK1A binding of this compound. These two side
chains form a small hydrophobic cleft (shown in yellow in Figure 3B, subpanel B,E). The 7-carbamate
harmine analog 1-12 was also found to be moderately active for DYRK1A inhibition. The majority
of 7-substituted analogs demonstrate that these harmine modifications do not result in DYRK1A
inhibitors superior in potency to harmine, though some retain potency in the 90–100 nM range.
2.3. Effects of Harmine Analogs on Human β-Cell Proliferation
After assessing the effect of these modifications on DYRK1A inhibition, we next explored
the structure–activity relationships for in vitro β-cell proliferation activity of the most potent
7-O-substituted DYRK1A inhibitor harmine analogs. Compounds 1-2b and 1-3b, with DYRK1A
inhibitory activities (IC50) of 89 and 91 nM, respectively, were studied as previously reported [
for their ability to induce human -cell proliferation in vitro as assessed by Ki-67-insulin
co-immunolabeling after 4 days, our standard assay protocol [49] (Figure 4A). Both the harmine
analogues, 1-2b and 1-3b exhibited human -cell proliferation at 10 M (Figure 4A). However,
the extent of -cell proliferation was significantly lower than that of harmine itself at that concentration,
with analog 1-2b trending slightly better than 1-3b -cell proliferation dose-response experiments
indicated that 1-2b induced proliferation in a dose-dependent manner. Compound 1-2b induced
proliferation half that of harmine (10 M) at the highest tested concentration of 30 M (Figure 4B).
5,39,43–45],
β
β
µ
β
.
β
µ
µ
This could be attributed to the fact that compound 1-2b is 4-fold less potent in DYRK1A inhibition as
compared to harmine, though it can be inferred that it effectively engages with the target DYRK1A
in the live
β-cell. Note that the harmine dose-response curve is identical to that reported earlier [
5].
Additionally, we can also speculate that slightly lower
β-cell proliferation activity of 1-3b as compared
to 1-2B could be caused by inhibition of kinases which could have anti-proliferative effects. Collectively,
these data indicate that modification at 7-position of harmine does provide us with compounds which
show real and comparable increases in β-cell proliferation but less than that of harmine itself.

Molecules 2020, 25, 1983
7 of 18
Figure 4. Effects of harmine analogues on human
analogues on human -cell proliferation at 10 M. DMSO was used as a negative control, and harmine
was used as a positive control (n = 3). ( ) Dose–response curves for human -cell proliferation for
1-2b in human -cells (n = 3 human islet donors for each dose; harmine (10 M) is the positive control.
) A representative example from ( ) of Ki67-insulin double-positive cells induced by 1-2b and
harmine. Error bars indicate SEM and * indicates p < 0.05. A minimum of 1000 -cells was counted for
β-cell proliferation. (A) Initial screening of harmine
β
µ
B
β
β
µ
(
C
B
β
each graph. In all relevant panels, error bars indicate SEM and, * indicates p < 0.05. A minimum of
1000 β-cells was counted for each graph.
3. Materials and Methods
1H-NMR were acquired on a Bruker DRX-600 spectrometer at 600 MHz. (Brucker Corporation,
Billerica, MA, USA). TLC was performed on silica coated aluminum sheets (thickness 200
µm) or
alumina coated (thickness 200 m) aluminum sheets supplied by Sorbent Technologies (Sorbent
µ
Technologies, Inc., Norcross, GA, USA) and column chromatography was carried out on Teledyne
ISCO combiflash (Teledyne ISCO, Lincoln, NE, USA) equipped with a variable wavelength detector
and a fraction collector using a RediSep Rf high performance silica flash columns by Teledyne ISCO.
LCMS/HPLC analysis for purity and HRMS was conducted on an Agilent Technologies G1969A
high-resolution API-TOF mass spectrometer attached to an Agilent Technologies 1200 HPLC system
(Agilent Technologies, Santa Clara, CA, USA). Samples were ionized by electrospray ionization (ESI) in
positive mode. Chromatography was performed on a 2.1
with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid
×
150 mm Zorbax 300SB-C18 5-µm column
as solvent B at a flow rate of 0.4 mL/min. The gradient program was as follows: 1% B (0
−
1 min),
◦
1
−
99% B (1 4 min), and 99% B (4
−
−
8 min). The temperature of the column was held at 50 C for
the entire analysis. The chemicals and reagents were purchased from Sigma Aldrich Co. (Sigma
Aldrich Corp., St. Louis, MO, USA) Alfa Aesar (Alfa Aesar, Tewksbury, MA, USA), Enamine (Enamine
Ltd, Monmouth Jct. NJ, USA), TCI America (TCI America, Seekonk, MA, USA). All solvents were

Molecules 2020, 25, 1983
8 of 18
purchased in anhydrous from Acros Organics (Thermo Fisher Scientific, Washington, USA) and used
without further purification.
3.1. Chemistry
1-Methyl-9H-pyrido [3,4-b]indol-7-ol (1-1), a solution of harmine (1.0 g, 4.02 mmol) in glacial acetic acid
(16 mL) and 48% hydrobromic acid solution (20 mL) was heated at reflux for 10 h. After cooling to
room temperature, the mixture was adjusted to pH 8 with a saturated aqueous solution of NaHCO3.
The yellow slurry was filtered, and the cake was washed with water to afford harmol 1-1 as a white
1
solid (0.91 g, 99%). H-NMR (600 MHz, d6-DMSO):
δ 10.46 (s, 1H, NH), 8.39 (d, J = 6.6 Hz, 1H, ArH3),
8.35 (d, J = 6 Hz, 1H, ArH4), 8.26 (d, J = 8.4 Hz, 1H, ArH5), 7.00 (s, 1H, ArH8), 6.90 (m,1H, ArH6), 2.93
(s, 3H, CH3); MS (ESI) m/z 199.08 [M + H]+.
General procedure for the synthesis of (1-2), a solution of harmalol 1-1 (2.02 mmol) and cesium
◦
carbonate (1.5 eq.) in DMF (7 mL) was stirred at 60 C for 1 h. To this solution was added alkyl
bromide (1.5 eq.) and stirred at 50 ◦C for 12 h. After completion of the reaction confirmed by TLC,
the reaction mixture was diluted with water, transferred to separatory funnel and extracted with
ethyl acetate (50 mL
×
2). The organic layer was washed with water, dried over magnesium sulfate,
filtered, evaporated and purified by flash column chromatography to yield the desired product 1-2 as
white solid.
2-(7-Oxy-1-methyl-9-H-b-carbolin)acetic acid methyl ester (1-2a), white solid. Yield 59%. 1H-NMR
(600 MHz, CD3OD): δ 8.07 (d, J = 5.4 Hz, 1H, ArH3), 7.96 (d, J = 9 Hz, 1H, ArH5), 7.76 (d, J = 5.4 Hz, 1H,
ArH4), 6.96 (d, J = 2.4 Hz, 1H, ArH8), 6.87 (m, 1H, ArH6), 4.79 (s, 2H, CH2), 3.80 (s, 3H, CH3), 2.73 (s,
3H, CH3); HRMS (ESI): m/z [M + H]+ calcd for C15H15N2O3+: 271.1077, found: 271.1071; Purity >95%.
1
3-(7-Oxy-1-methyl-9-H-b-carbolin)propionic acid methyl ester (1-2b), white solid. Yield 44%. H-NMR
(600 MHz, CD3OD):
δ 8.09 (d, J = 5.4 Hz, 1H, ArH3), 7.95 (d, J = 8.4 Hz, 1H, ArH5), 7.80 (d, J = 5.4 Hz,
1H, ArH4), 6.92 (s, 1H, ArH8), 6.79 (m, 1H, ArH6), 4.84 (t, J = 7.2 Hz, 2H, CH2), 3.57 (s, 3H, CH3), 2.98
(s, 3H, CH3), 2.80 (t, J = 7.2 Hz, 2H, CH2); HRMS (ESI): m/z [M + H]+ calcd for C16H17N2O3+: 285.1234,
found: 285.1235; Purity >95%.
4-(7-Oxy-1-methyl-9-H-b-carbolin)butnoic acid methyl ester (1-2c), white solid. Yield 68%. 1H-NMR
(600 MHz, CD3OD):
δ 8.08 (d, J = 5.4 Hz, 1H, ArH3), 7.98 (d, J = 8.4 Hz, 1H, ArH5), 7.79 (d, J = 5.4 Hz,
1H, ArH4), 7.02 (s, 1H, ArH8), 6.84 (d, J = 9 Hz, 1H, ArH6), 4.11 (t, J = 6 Hz, 2H, CH2), 3.68 (s, 3H,
CH3), 2.75 (s, 3H, CH3), 2.56 (t, J = 7.2 Hz, 2H, CH2), 2.13 (t, J = 6.6 Hz, 2H, CH2); HRMS (ESI): m/z
[M + H]+ calcd for C17H19N2O3+: 299.1390, found: 299.1393; Purity >95%.
1
5-(7-Oxy-1-methyl-9-H-b-carbolin)pentanoic acid methyl ester (1-2d), white solid. Yield 50%. H-NMR
(600 MHz, CD3OD): δ 8.08 (d, J = 5.4 Hz, 1H, ArH3), 7.96 (d, J = 8.4 Hz, 1H, ArH5), 7.79 (d, J = 6 Hz,
1H, ArH4), 7.02 (d, J = 1.8 Hz, 1H, ArH8), 6.84 (m, 1H, ArH6), 4.10 (t, J = 6 Hz, 2H, CH2), 3.65 (s, 3H,
CH3), 2.75 (s, 3H, CH3), 2.45 (t, J = 7.2 Hz, 2H, CH2), 1.85 (m, 4H, CH2); HRMS (ESI): m/z [M + H]+
calcd for C18H21N2O3+: 313.1547, found: 313.1554; Purity >95%.
6-(7-Oxy-1-methyl-9-H-b-carbolin)hexanoic acid methyl ester (1-2e), white solid. Yield 50%. 1H-NMR
(600 MHz, CD3OD): δ 8.08 (d, J = 5.4 Hz, 1H, ArH3), 7.97 (d, J = 9 Hz, 1H, ArH5), 7.78 (d, J = 5.4 Hz,
1H, ArH4), 7.00 (d, J = 2.4 Hz, 1H, ArH8), 6.84 (m, 1H, ArH6), 4.06 (t, J = 6 Hz, 2H, CH2), 3.65 (s, 3H,
CH3), 2.75 (s, 3H, CH3), 2.37 (t, J = 7.8 Hz, 2H, CH2), 1.84 (m, 2H, CH2), 1.70 (m, 2H, CH2), 1.54 (m, 2H,
CH2); HRMS (ESI): m/z [M + H]+ calcd for C19H23N2O3+: 327.1709, found: 327.1703; Purity >95%.
2-(7-Oxy-1-methyl-9-H-b-carbolin)acetic acid tert-butyl ester (1-2f), white solid. Yield 42%. 1H-NMR
(600 MHz, d6-DMSO):
δ 11.49 (s, 1H, NH), 8.16 (d, J = 5.4 Hz, 1H, ArH3), 8.07 (d, J = 8.4 Hz, 1H, ArH5),
7.84 (d, J = 5.4 Hz, 1H, ArH4), 6.93 (d, J = 2.4 Hz, 1H, ArH8), 6.84 (m, 1H, ArH6), 4.77 (s, 2H, CH2), 2.73
(s, 3H, CH3), 1.45 (s, 9H, (CH3)3); MS (ESI) m/z 313.81 [M + H]+.

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1
3-(7-Oxy-1-methyl-9-H-b-carbolin)propionic acid tert-butyl ester (1-2g), white solid. Yield 32%. H-NMR
(600 MHz, d6-DMSO): 9.82 (s, 1H, NH), 8.13 (d, J = 4.8 Hz, 1H, ArH3), 7.98 (d, J = 8.4 Hz, 1H, ArH5),
δ
7.80 (d, J = 4.8 Hz, 1H, ArH4), 6.93 (d, J = 1.2 Hz, 1H, ArH8), 6.74 (m, 1H, ArH6), 4.72 (t, J = 7.2 Hz, 2H,
CH2), 2.92 (s, 3H, CH3), 2.67 (t, J = 7.2 Hz, 2H, CH2), 1.24 (s, 9H, (CH3)3); MS (ESI) m/z 327.22 [M + H]+.
1
4-(7-Oxy-1-methyl-9-H-b-carbolin)butnoic acid tert-butyl ester (1-2h), White solid. Yield 55%. H-NMR
(600 MHz, d6-DMSO):
δ 11.37 (s, 1H, NH), 8.13 (d, J = 5.4 Hz, 1H, ArH3), 8.03 (d, J = 9 Hz, 1H, ArH5),
7.80 (d, J = 5.4 Hz, 1H, ArH4), 6.98 (d, J = 1.8 Hz, 1H, ArH8), 6.83 (m, 1H, ArH6), 4.08 (t, J = 6.6 Hz, 2H,
CH2), 2.71 (s, 3H, CH3), 2.41 (t, J = 7.2 Hz, 2H, CH2), 1.99 (m, 2H, CH2), 1.41 (s, 9H, (CH3)3); MS (ESI)
m/z 341.17 [M + H]+.
1
5-(7-Oxy-1-methyl-9-H-b-carbolin)pentanoic acid tert-butyl ester (1-2i), white solid. Yield 40%. H-NMR
(600 MHz, CDCl3):
δ 9.40 (s, 1H, NH), 8.31 (d, J = 4.8 Hz, 1H, ArH3), 7.95 (d, J = 8.4 Hz, 1H, ArH5),
7.72 (d, J = 5.4 Hz, 1H, ArH4), 6.89 (d, J = 1.8 Hz, 1H, ArH8), 6.86 (m, 1H, ArH6), 4.00 (t, J = 6 Hz, 2H,
CH2), 2.79 (s, 3H, CH3), 2.31 (t, J = 6.6 Hz, 2H, CH2), 1.81 (m, 4H, CH2), 1.45 (s, 9H, (CH3)3); MS (ESI)
m/z 355.63 [M + H]+.
1
6-(7-Oxy-1-methyl-9-H-b-carbolin)hexanoic acid tert-butyl ester (1-2j), white solid. Yield 39%. H-NMR
(600 MHz, CDCl3):
δ 9.75 (s, 1H, NH), 8.24 (d, J = 5.4 Hz, 1H, ArH3), 7.92 (d, J = 8.4 Hz, 1H, ArH5),
7.73 (d, J = 5.4 Hz, 1H, ArH4), 7.00 (s, 1H, ArH8), 6.85 (d, J = 8.4 Hz, 1H, ArH6), 3.99 (t, J = 6.6 Hz, 2H,
CH2), 2.84 (s, 3H, CH3), 2.25 (t, J = 7.8 Hz, 2H, CH2), 1.81 (m, 2H, CH2), 1.66 (m, 2H, CH2), 1.49 (m, 2H,
CH2), 1.44 (s, 9H, (CH3)3); MS (ESI) m/z 369.77 [M + H]+.
2-(7-Oxy-1-methyl-9-H-b-carbolin)ethyl tert-butyl carbamate (1-2k), white solid. Yield 67%. 1H-NMR
(600 MHz, CD3OD): δ 8.08 (d, J = 5.4 Hz, 1H, ArH3), 7.98 (d, J = 9 Hz, 1H, ArH5), 7.80 (d, J = 5.4 Hz, 1H,
ArH4), 7.05 (d, J = 1.8 Hz, 1H, ArH8), 6.88 (m, 1H, ArH6), 4.10 (t, J = 5.4 Hz, 2H, CH2), 3.48 (t, J = 6 Hz,
+
2H, CH2), 2.75 (s, 3H, CH3), 1.44 (s, 9H, (CH3)3); HRMS (ESI): m/z [M + H]+ calcd for C19H24N3O3
342.1812, found: 342.1822; Purity >95%.
:
1
3-(7-Oxy-1-methyl-9-H-b-carbolin)propyl tert-butyl carbamate (1-2l), white solid. Yield 57%. H-NMR
(600 MHz, d6-DMSO): δ 8.13 (d, J = 4.8 Hz, 1H, ArH3), 8.04 (d, J = 8.4 Hz, 1H, ArH5), 7.79 (d, J = 4.8 Hz,
1H, ArH4), 6.97 (d, J = 1.8 Hz, 1H, ArH8), 6.94 (t, J = 5.4 Hz, 1H, NH), 6.82 (m, 1H, ArH6), 4.07 (t,
J = 6.6 Hz, 2H, CH2), 3.12 (t, J = 6.6 Hz, 2H, CH2), 2.71 (s, 3H, CH3), 1.89 (m, 2 H, CH2), 1.38 (s, 9H,
(CH3)3); HRMS (ESI): m/z [M + H]+ calcd for C20H26N3O3+: 356.1969, found: 356.1982; Purity >95%.
1
4-(7-Oxy-1-methyl-9-H-b-carbolin)butyl tert-butyl carbamate (1-2m), white solid. Yield 67%. H-NMR
(600 MHz, d6-DMSO):
δ 8.13 (d, J = 4.8 Hz, 1H, ArH3), 8.04 (d, J = 9 Hz, 1H, ArH5), 7.81 (d, J = 5.4 Hz,
1H, ArH4), 6.97 (d, J = 2.4 Hz, 1H, ArH8), 6.87 (t, J = 5.4 Hz, 1H, NH), 6.82 (m, 1H, ArH6), 4.06
(t, J = 6 Hz, 2H, CH2), 2.99 (m, 2H, CH2), 2.71 (s, 3H, CH3), 1.75 (m, 2H, CH2), 1.56 (m, 2H, CH2),
1.37 (s, 9H, (CH3)3); HRMS (ESI): m/z [M + H]+ calcd for C21H28N3O3+: 370.2125, found: 370.2189;
Purity >95%.
2-((7-Oxy-1-methyl-9-H-b-carbolin)ethoxy)ethyl tert-butyl carbamate (1-2n), white solid. Yield 46%.
1H-NMR (600 MHz, d6-DMSO):
δ 8.13 (d, J = 4.8 Hz, 1H, ArH3), 8.04 (d, J = 8.4 Hz, 1H, ArH5), 7.80 (d,
J = 5.4 Hz, 1H, ArH4), 7.00 (d, J = 1.8 Hz, 1H, ArH8), 6.84 (m, 1H, ArH6), 6.81 (t, J = 5.4 Hz, 1H, NH),
4.19 (t, J = 4.8 Hz, 2H, CH2), 3.78 (t, J = 4.2 Hz, 2H, CH2), 3.48 (t, J = 6 Hz, 2H, CH2), 3.12 (m, 2H, CH2),
2.71 (s, 3H, CH3), 1.37 (s, 9H, (CH3)3); HRMS (ESI): m/z [M + H]+ calcd for C21H28N3O4+: 386.2074,
found: 386.2110; Purity >95%.
2-(7-oxy-1-methyl-9-H-b-carbolin)methoxyethyl (1-2o), white solid. Yield 38%. 1H-NMR (600 MHz,
CD3OD): δ 8.10 (d, J = 6 Hz, 1H, ArH3), 8.01 (d, J = 8.4 Hz, 1H, ArH5), 7.83 (d, J = 5.4 Hz, 1H, ArH4),
7.05 (d, J = 2.4 Hz, 1H, ArH8), 6.89 (m, 1H, ArH6), 4.22 (t, J = 4.8 Hz, 2H, CH2), 3.80 (t, J = 4.2 Hz, 2H,
CH2), 3.45 (s, 3H, CH3), 2.77 (s, 3H, CH3); HRMS (ESI): m/z [M + H]+ calcd for C15H17N2O2+: 257.1285,
found: 257.1291; Purity >95%.

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3-(7-Oxy-1-methyl-9-H-b-carbolin)ethoxyethyl (1-2p), white solid. Yield 48%. 1H-NMR (600 MHz,
CD3OD): 8.10 (d, J = 5.4 Hz, 1H, ArH3), 8.0 (d, J = 8.4 Hz, 1H, ArH5), 7.80 (d, J = 5.4 Hz, 1H, ArH4),
δ
7.06 (d, J = 1.2 Hz, 1H, ArH8), 6.90 (m, 1H, ArH7), 4.22 (t, J = 4.8 Hz, 2H, CH2), 3.84 (t, J = 4.2 Hz, 2H,
CH2), 3.63 (m, 2H, CH2), 3.45 (s, 3H, CH3), 2.76 (s, 3H, CH3), 1.23 (t, J = 6.6 Hz, 2H, CH2); HRMS (ESI):
m/z [M + H]+ calcd for C16H19N2O2+: 271.1441, found: 271.1429; Purity >95%.
General procedure for the synthesis of (1-3), a solution of 1-2a to 1-2e◦(0.175 mmol) and 7N
ammonia in methanol (4 mL) in a sealed pressure vessel was stirred at 90 C for 12 h. After the
completion of the reaction, the mixture was evaporated and purified by flash column chromatography
using DCM/MeOH/Ammonia (90/9/1) as eluent to give the desired final compound 1-3 as white solid.
2-(7-Oxy-1-methyl-9-H-b-carbolin)acetamide (1-3a), white solid. Yield 91%. 1H-NMR (600 MHz,
d6-DMSO):
δ 8.14 (d, J = 5.4 Hz, 1H, ArH3), 8.07 (d, J = 8.4 Hz, 1H, ArH5), 7.80 (d, J = 5.4 Hz,
1H, ArH4), 7.63 (s, 1H, NH), 7.46 (s, 1H, NH), 7.0 (d, J = 1.8 Hz, 1H, ArH8), 6.90 (m, 1H, ArH6), 4.53
(s, 2H, CH2), 2.71 (s, 3H, CH3); HRMS (ESI): m/z [M + H]+ calcd for C14H14N3O2+: 256.1081, found:
256.1084; Purity >95%.
3-(7-Oxy-1-methyl-9-H-b-carbolin)propionamide (1-3b), white solid. Yield 97%. 1H-NMR (600 MHz,
d6-DMSO):
δ 8.13 (d, J = 5.4 Hz, 1H, ArH3), 7.98 (d, J = 8.4 Hz, 1H, ArH5), 7.81 (d, J = 5.4 Hz, 1H,
ArH4), 7.44 (s, 1H, NH), 6.96 (s, 2H, ArH8, NH), 6.73 (m, 1H, ArH6), 4.67 (t, J = 7.8 Hz, 2H, CH2), 2.95
(s, 3H, CH3), 2.54 (t, J = 7.8 Hz, 2H, CH2); HRMS (ESI): m/z [M + H]+ calcd for C15H16N3O2+: 270.1237,
found: 270.1245; Purity >95%.
4-(7-Oxy-1-methyl-9-H-b-carbolin)butanamide (1-3c), white solid. Yield 84%. 1H-NMR (600 MHz,
d6-DMSO):
δ 8.13 (d, J = 5.4 Hz, 1H, ArH3), 8.03 333e3(d, J = 8.4 Hz, 1H, ArH5), 7.80 (d, J = 5.4 Hz,
1H, ArH4), 7.35 (s, 1H, NH), 6.98 (s, 1H, ArH8), 6.82 (m, 2H, ArH6, NH), 4.06 (t, J = 6 Hz, 2H, CH2),
2.71 (s, 3H, CH3), 2.27 (t, J = 7.8 Hz, 2H, CH2), 1.98 (m, 2H, CH2); HRMS (ESI): m/z [M + H]+ calcd for
C16H18N3O2+: 284.1394, found: 284.1396; Purity >95%.
5-(7-Oxy-1-methyl-9-H-b-carbolin)pentanamide (1-3d), white solid. Yield 85%. 1H-NMR (600 MHz,
d6-DMSO):
δ 8.13 (d, J = 5.4 Hz, 1H, ArH3), 8.03 (d, J = 8.4 Hz, 1H, ArH5), 7.80 (d, J = 5.4 Hz, 1H,
ArH4), 7.29 (s, 1H, NH), 6.98 (d, J = 2.4 Hz,1H, ArH8), 6.82 (m, 1H, ArH6), 6.75 (s, 1H, NH), 4.07 (t,
J = 6.6 Hz, 2H, CH2), 2.71 (s, 3H, CH3), 2.13 (t, J = 7.2 Hz, 2H, CH2), 1.76 (m, 2H, CH2), 1.69 (m, 2H,
CH2); HRMS (ESI): m/z [M + H]+ calcd for C17H20N3O2+: 298.1550, found: 298.1560; Purity >95%.
6-(7-Oxy-1-methyl-9-H-b-carbolin)hexanamide (1-3e), white solid. Yield 94%. 1H-NMR (600 MHz,
d6-DMSO): δ 8.13 (d, J = 5.4 Hz, 1H, ArH3), 8.03 (d, J = 8.4 Hz, 1H, ArH5), 7.80 (d, J = 4.8 Hz, 1H, ArH4),
7.26 (s, 1H, NH), 6.98 (d, J = 2.4 Hz,1H, ArH8), 6.82 (m, 1H, ArH6), 6.27 (s, 1H, NH), 4.06 (t, J = 6.6 Hz,
2H, CH2), 2.71 (s, 3H, CH3), 2.09 (t, J = 7.2 Hz, 2H, CH2), 1.77 (m, 2H, CH2), 1.57 (m, 2H, CH2), 1.45 (m,
2H, CH2); HRMS (ESI): m/z [M + H]+ calcd for C18H22N3O2+: 312.1707, found: 312.1728; Purity >95%.
General procedure for the synthesis of (1-4), a solution of 1-2f to 1-2j (0.18 mmol) and 4N
hydrochloric acid in dioxane (4 mL) was stirred at room temperature for 24 h. The reaction mixture
was evaporated and triturated with diethyl ether to get the desired acid 1-4 as white solid.
2-(7-Oxy-1-methyl-9-H-b-carbolin)acetic acid (1-4a), white solid. Yield 89%. 1H-NMR (600 MHz,
d6-DMSO): δ 8.48 (d, J = 6 Hz, 1H, ArH3), 8.43 (d, J = 6 Hz, 1H, ArH4), 8.38 (d, J = 9 Hz, 1H, ArH5),
7.09 (m, 2H, ArH6, ArH8), 4.90 (s, 2H, CH2), 2.97 (s, 3H, CH3); HRMS (ESI): m/z [M + H]+ calcd for
C14H13N2O3+: 257.0921, found: 257.0927; Purity >95%.
3-(7-Oxy-1-methyl-9-H-b-carbolin)propionic acid (1-4b), white solid. Yield 98%. 1H-NMR (600 MHz,
d6-DMSO):
δ 8.45 (d, J = 6 Hz, 1H, ArH3), 8.40 (d, J = 6.6 Hz, 1H, ArH4), 8.30 (d, J = 9 Hz, 1H, ArH5),
7.14 (s, 1H, ArH8), 6.97 (d, J = 9 Hz, 1H, ArH6), 4.81 (t, J = 7.8 Hz, 2H, CH2), 3.16 (s, 3H, CH3), 2.82 (t,
J = 7.2 Hz, 2H, CH2); HRMS (ESI): m/z [M + H]+ calcd for C15H15N2O3+: 271.1077, found: 271.1075;
Purity >95%.

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4-(7-Oxy-1-methyl-9-H-b-carbolin)butnoic acid (1-4c), white solid. Yield 82%. 1H-NMR (600 MHz,
d6-DMSO): 8.45 (d, J = 6 Hz, 1H, ArH3), 8.40 (d, J = 6 Hz, 1H, ArH4), 8.35 (d, J = 9 Hz, 1H, ArH5),
δ
7.12 (s, 1H, ArH8), 7.05 (d, J = 9 Hz, 1H, ArH6), 4.17 (t, J = 6 Hz, 2H, CH2), 2.97 (s, 3H, CH3), 2.45 (t,
J = 7.2 Hz, 2H, CH2), 2.03 (m, 2H, CH2); HRMS (ESI): m/z [M + H]+ calcd for C16H17N2O3+: 285.1234,
found: 285.1236; Purity >95%.
5-(7-Oxy-1-methyl-9-H-b-carbolin)pentanoic acid (1-4d), white solid. Yield 85%. 1H-NMR (600 MHz,
d6-DMSO):
δ 8.45 (d, J = 6.6 Hz, 1H, ArH3), 8.40 (d, J = 6 Hz, 1H, ArH4), 8.35 (d, J = 9 Hz, 1H, ArH5),
7.11 (s, 1H, ArH8), 7.05 (d, J = 9 Hz, 1H, ArH6), 4.16 (t, J = 6.6 Hz, 2H, CH2), 2.97 (s, 3H, CH3), 2.32
(t, J = 7.2 Hz, 2H, CH2), 1.83 (m, 2H, CH2), 1.72 (m, 2H, CH2); HRMS (ESI): m/z [M + H]+ calcd for
C17H19N2O3+: 299.1390, found: 299.1401; Purity >95%.
6-(7-Oxy-1-methyl-9-H-b-carbolin)hexanoic acid (1-4e), White solid. Yield 91%. 1H-NMR (600 MHz,
d6-DMSO):
δ 8.46 (d, J = 6.6 Hz, 1H, ArH3), 8.40 (d, J = 6 Hz, 1H, ArH4), 8.35 (d, J = 9 Hz, 1H, ArH5),
7.11 (s, 1H, ArH8), 7.05 (d, J = 9 Hz, 1H, ArH6), 4.14 (t, J = 6.6 Hz, 2H, CH2), 2.97 (s, 3H, CH3), 2.26
(t, J = 7.8 Hz, 2H, CH2), 1.80 (m, 2H, CH2), 1.59 (m, 2H, CH2), 1.49 (m, 2H, CH2); HRMS (ESI): m/z
[M + H]+ calcd for C18H21N2O3+: 313.1547, found: 313.1557; Purity >95%.
General procedure for the synthesis of (1-5), to a solution of 1-2k to 1-2n (1.93 mmol) in 1,4-dioxane
(4 mL) was added 4N HCl in dioxane (4 eq.) and stirred at room temperature for 24 h. After the
completion of the reaction as monitored by LCMS, the mixture was evaporated to give the desired
product 10-5 as white solid.
1
2-(7-Oxy-1-methyl-9-H-b-carbolin)ethylamine hydrochloric acid (1-5a), white solid. Yield 90%. H-NMR
(600 MHz, d6-DMSO): δ 8.47 (d, J = 6.6 Hz, 1H, ArH3), 8.38 (m, 2H, ArH4, ArH5), 8.33 (bs, 2H, NH2),
7.19 (d, J = 1.8 Hz, 1H, ArH8), 7.10 (m, 1H, ArH6), 4.37 (t, J = 5.4 Hz, 2H, CH2), 3.30 (t, J = 4.8 Hz, 2H,
CH2), 3.03 (s, 3H, CH3); HRMS (ESI): m/z [M + H]+ calcd for C14H16N3O+: 242.1288, found: 242.1301;
Purity >95%.
1
3-(7-Oxy-1-methyl-9-H-b-carbolin)propylamine hydrochloric acid (1-5b), white solid. Yield 98%. H-NMR
(600 MHz, d6-DMSO): δ 8.45 (d, J = 6 Hz, 1H, ArH3), 8.38 (m, 2H, ArH4, ArH5), 8.09 (bs, 2H,NH2), 7.15
(d, J = 2.4 Hz, 1H, ArH8), 7.06 (m, 1H, ArH6), 4.25 (t, J = 6 Hz, 2H, CH2), 3.03 (m, 5H, CH2, CH3), 2.12
(t, J = 6.6 Hz, 2H, CH2); HRMS (ESI): m/z [M + H]+ calcd for C15H18N3O+: 256.1444, found: 256.1433;
Purity >95%.
1
4-(7-Oxy-1-methyl-9-H-b-carbolin)butylamine hydrochloric acid (1-5c), white solid. Yield 99%. H-NMR
(600 MHz, d6-DMSO): δ 8.45 (d, J = 6 Hz, 1H, ArH3), 8.38 (d, J = 6 Hz, 1H, ArH4), 8.35 (d, J = 8.4 Hz,
1H, ArH5), 7.97 (bs, 2H, NH2), 7.14 (d, J = 1.8 Hz, 1H, ArH8), 7.05 (d, J = 9 Hz, 1H, ArH6), 4.17 (t,
J = 6 Hz, 2H, CH2), 3.02 (s, 3H, CH3), 2.87 (t, J = 6.6 Hz, 2H, CH2), 1.87 (m, 2H, CH2), 1.77 (m, 2H,
CH2); HRMS (ESI): m/z [M + H]+ calcd for C16H20N3O+: 270.1601, found: 270.1594; Purity >95%.
2-((7-Oxy-1-methyl-9-H-b-carbolin)ethoxy)ethylamine hydrochloric acid (1-5d), white solid. Yield 97%.
1H-NMR (600 MHz, d6-DMSO):
δ 8.45 (d, J = 6 Hz, 1H, ArH3), 8.36 (m, 2H, ArH4, ArH5), 8.10 (bs, 2H,
NH2), 7.18 (d, J = 1.8 Hz, 1H, ArH8), 7.06 (d, J = 9 Hz,1H, ArH6), 4.31 (t, J = 4.8 Hz, 2H, CH2), 3.88 (t,
J = 4.2 Hz, 2H, CH2), 3.73 (t, J = 5.4 Hz, 2H, CH2), 3.03 (s, 3H, CH3), 3.0 (t, J = 5.4 Hz, 2H, CH2); HRMS
(ESI): m/z [M + H]+ calcd for C16H20N3O+: 286.1550, found: 286.1552; Purity >95%.
General procedure for the synthesis of (1-6), to a solution of 1-5 (0.36 mmol) and triethylamine
(2.2 eq.) in DCM was added acetic anhydride dropwise and the reaction mixture was stirred at room
temperature for 1 h. Upon completion of the reaction, the mixture was diluted with DCM, transferred
to separatory funnel and washed with water. The organic layer was collected, dried over magnesium
sulfate, filtered and evaporated to get the desired product 1-6 as white solid.
1
2-(7-Oxy-1-methyl-9-H-b-carbolin)-2-acetyl-ethylamine (1-6a), white solid. Yield 74%. H-NMR (600 MHz,
d6-DMSO):
δ 8.17 (s, 1H, NH), 8.15 (d, J = 5.4 Hz, 1H, ArH3), 8.06 (d, J = 8.4 Hz, 1H, ArH5), 7.82

Molecules 2020, 25, 1983
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(d, J = 5.4 Hz, 1H, ArH4), 7.00 (s, 1H, ArH8), 6.84 (m, 1H, ArH6), 4.09 (t, J = 6 Hz, 2H, CH2), 3.48
(t, J = 5.4 Hz, 2H, CH2), 2.72 (s, 3H, CH3), 1.84 (s, 3H, CH3); HRMS (ESI): m/z [M + H]+ calcd for
C16H18N3O2+: 284.1394, found: 284.1419; Purity >95%.
3-(7-Oxy-1-methyl-9-H-b-carbolin) -2-acetyl-propylamine (1-6b), white solid. Yield 79%. 1H-NMR
(600 MHz, d6-DMSO):
δ 8.14 (d, J = 5.4 Hz, 1H, ArH3), 8.06 (d, J = 8.4 Hz, 1H, ArH5), 7.95 (s, 1H, NH),
7.80 (t, J = 5.4 Hz, 1H, ArH4), 6.99 (d, J = 2.4 Hz, 1H, ArH8), 6.82 (m, 1H, ArH6), 4.08 (t, J = 6 Hz, 2H,
CH2), 3.23 (m, 2H, CH2), 2.71 (s, 3H, CH3), 1.90 (m, 2H, CH2), 1.81 (s, 3H, CH3); HRMS (ESI): m/z
[M + H]+ calcd for C17H20N3O2+: 298.1550, found: 298.1552; Purity >95%.
1
4-(7-Oxy-1-methyl-9-H-b-carbolin) -2-acetyl-butylamine (1-6c), white solid. Yield 70%. H-NMR (600 MHz,
d6-DMSO): δ 8.16 (d, J = 5.4 Hz, 1H, ArH3), 8.05 (d, J = 8.4 Hz, 1H, ArH5), 7.87 (bs, 1H, NH), 7.83 (d,
J = 5.4 Hz, 1H, ArH4), 6.99 (d, J = 1.8 Hz, 1H, ArH8), 6.84 (m, 1H, ArH6), 4.08 (t, J = 5.4 Hz, 2H, CH2),
3.10 (m, 2H, CH2), 2.73 (s, 3H, CH3), 1.80 (m, 5H, CH2, CH3), 1.59 (m, 2H, CH2); HRMS (ESI): m/z
[M + H]+ calcd for C18H22N3O2+: 312.1707, found: 312.1724; Purity >95%.
1
2-((7-Oxy-1-methyl-9-H-b-carbolin)ethoxy)-2-acetyl-ethylamine (1-6d), white solid. Yield 49%. H-NMR
(600 MHz, d6-DMSO):
δ 8.14 (d, J = 5.4 Hz, 1H, ArH3), 8.06 (d, J = 9.0 Hz, 1H, ArH5), 7.93 (m, 1H, NH),
7.80 (d, J = 5.4 Hz, 1H, ArH4), 7.01 (s, 1H, ArH8), 6.85 (m, 1H, ArH6), 4.20 (t, J = 4.8 Hz, 2H, CH2), 3.79
(m, 2H, CH2), 3.50 (t, J = 6 Hz, 2H, CH2), 3.55 (m, 2H, CH2), 2.71 (s, 3H CH3), 1.79 (s, 3H, CH3); HRMS
(ESI): m/z [M + H]+ calcd for C18H22N3O3+: 328.1656, found: 328.1669; Purity >95%.
O-Trifluoromethanesulfonyl-1-methyl-9 H -b-carbolin-7-ol (1-7), to a solution of 1-1 (500 mg, 2.52 mmol)
and pyridine (25.2 mmol) in DCM (4 mL) was added trifluoromethanesulfonic anhydride (1.2 eq.)
at 0◦C dropwise. The reaction mixture was allowed to warm to room temperature and stirred for
12 h. Upon completion of the reaction, the mixture was evaporated and purified by flash column
chromatography using DCM/MeOH (95/5) as eluent to get the desired product 1-7 as white solid
1
(875 mg, 92%). H-NMR (600 MHz, d6-DMSO):
δ
13.01 (s, 1H, NH), 8.67 (d, J = 9 Hz, 1H, ArH3), 8.65
(d, J = 6 Hz, 1H, ArH4), 8.54 (d, J = 6 Hz, 1H, ArH5), 7.89 (m, 1H, ArH8), 7.54 (d, J = 9 Hz, 1H, ArH6),
3.01 (s, 3H, CH3); MS (ESI) m/z 331.5 [M + H]+.
7-Nitro-1-methyl-9 H -b-carboline (1-8), a pressure vessel was charged with 1-7 (100 mg, 0.30 mmol),
Pd2(dba)3 (5 mol%), BrettPhos (6 mol%) and sodium nitrite (42 mg, 0.6 mmol) and evacuated and back
filled with argon three times. To the mixture was added tert-butanol (0.6 mL) and TDA (5 mol%) under
argon. The pressure was sealed and heated to 150 ◦C for 24 h. Upon completion of the reaction, the
mixture was evaporated and purified by column chromatography to get 1-8 as yellow solid (80 mg,
77%). 1H-NMR (600 MHz, CD3OD):
δ 8.41 (m, 1H, ArH3), 8.30 (d, J = 8.4 Hz, 1H, ArH5), 8.23 (d,
J = 5.4 Hz, 1H, ArH4), 8.08 (m, 1H, ArH8), 8.01 (d, J = 5.4 Hz, 1H, ArH6), 2.82 (s, 3H, CH3); MS (ESI)
m/z 228.31 [M + H]+.
7-Amino-1-methyl-9 H -b-carboline (1-9), to a solution of 1-8 (80 mg, 0.35 mmol) and palladium on carbon
(10% by wt, 100 mg) in methanol (4 mL) was added hydrazine monohydrate (0.34 mL, 7 mmol) and
heated to 85 ◦C for 1 h. Upon completion of the reaction, catalyst was filtered over Celite and filtrate
was evaporated and purified by column chromatography using DCM/MeOH (90/10) as eluent to
1
provide the desired amino compound 1-9 as brown solid (65 mg, 98%). H-NMR (600 MHz, CD3OD):
δ
8.02 (d, J = 5.4 Hz, 1H, ArH3), 7.82 (d, J = 8.4 Hz, 1H, ArH5), 7.69 (d, J = 4.8 Hz, 1H, ArH4), 6.80
(s, 1H, ArH8), 6.68 (d, J = 8.4 Hz, 1H, ArH9), 2.71 (s, 3H, CH3); HRMS (ESI): m/z [M + H]+ calcd for
C12H12N3+: 198.1026, found: 198.1050; Purity >95%.
7-Acetamide-1-methyl-9 H -b-carboline (1-10), to a solution of 1-9 (20 mg, 0.10 mmol) and triethylamine
(0.028 mL, 0.20 mmol) in THF (2 mL) was added acetic anhydride dropwise at 0 ◦C and the reaction
mixture was stirred at room temperature for 12 h. Upon completion of the reaction, the mixture was
diluted with DCM, transferred to separatory funnel and washed with water. The organic layer was
collected, dried over magnesium sulfate, filtered and evaporated to get the desired product 1-10 as

Molecules 2020, 25, 1983
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1
white solid (18 mg, 75%). H-NMR (600 MHz, d6-DMSO): δ 11.46 (s, 1H, NH), 10.16 (s, 1H, NH), 8.19
(s, 1H, ArH3), 8.15 (d, J = 5.4 Hz, 1H, ArH4), 8.06 (d, J = 8.4 Hz, 1H, ArH5), 7.81 (s, 1H, ArH8), 7.23
(d, J = 8.4 Hz, 1H, ArH6), 2.71 (s, 3H, CH3), 2.10 (s, 3H, CH3); HRMS (ESI): m/z [M + H]+ calcd for
C14H14N3+: 240.1131, found: 240.1135; Purity >95%.
7-Benzamide-1-methyl-9 H -b-carboline (1-11), to a solution of 1-9 (20 mg, 0.10 mmol) and triethylamine
◦
(0.028 mL, 0.20 mmol) in THF (1 mL) was added benzoyl chloride at 0 C and stirred at room
temperature for 2 h. Upon completion of the reaction monitored by LCMS, the mixture was evaporated
and purified by column chromatography using DCM/MeOH (95/5) as eluent to get the desired product
1
1-11 as white solid (23 mg, 77%). H-NMR (600 MHz, d6-DMSO):
δ 8.32 (s, 1H, NH), 8.17 (d, J = 4.8 Hz,
1H, ArH3), 8.13 (d, J = 8.4 Hz, 1H, ArH5), 8.00 (d, J = 7.2 Hz, 2H, ArH (benzamido)), 7.85 (d, J = 5.4 Hz,
1H, ArH4), 7.61 (m, 1H, ArH8), 7.57 (m, 1H, ArH6); 7.54 (m, 3H, ArH (benzamido)), 2.74 (s, 3H, CH3);
HRMS (ESI): m/z [M + H]+ calcd for C19H16N3+: 302.1288, found: 302.1296; Purity >95%.
1-Methyl-9H-pyrido[3,4-b]indol-7-yl isopropylcarbamate (1-12), to a solution of harmalol 1-1 (50 mg,
0.25 mmol) in DMF (2 mL) was added isopropyl isocyanate (0.030 mL, 0.3 mmol) and stirred at room
temperature for 12 h. The reaction mixture was evaporated and purified by column chromatography
1
using DCM/MeOH (95/5) as eluent to get the desired product 1-12 as white solid (23 mg, 32%). H-NMR
(600 MHz, CD3OD): δ 8.15 (d, J = 5.4 Hz, 1H, ArH3), 8.12 (d, J = 8.4 Hz, 1H, ArH5), 7.90 (d, J = 5.4 Hz, 1H,
ArH4), 7.31 (m, 1H, ArH8), 7.09 (m, 1H, ArH6), 3.80 (m, 1H, CH), 2.79 (s, 3H, CH3), 1.23 (d, J = 6.6 Hz,
6H); HRMS (ESI): m/z [M + H]+ calcd for C16H18N3O2+: 284.1394, found: 284.1416; Purity >95%.
3.2. DYRK1A Binding Assays
Compounds were tested for DYRK1A binding activity by a commercial kinase profiling services,
Life Technologies which uses the FRET-based LanthaScreen® Eu Kinase Binding Assay. [38] Compounds
were screened for DYRK1A activity at concentrations of 1000 nM and 300 nM in duplicates. The IC50
was determined by 10 point LanthaScreen® Eu Kinase Binding Assay [38] in duplicates.
3.3. β-Cell Proliferation Assay
Human pancreatic islets were obtained from the NIH/NIDDK-supported Integrated Islet
Distribution Program (IIDP) Duarte, CA, USA (https://iidp.coh.org) and studied using protocols
that have been published in detail [5]. Briefly, islets were first dispersed with Accutase (Sigma, St. Louis,
MO, USA) onto coverslips, and treated with vehicle (0.1% DMSO) or with harmine-related compounds
in RPMI1640 complete medium for 96 h. Then the cells were then, fixed with formaldehyde and
immunolabeled for insulin and Ki67 as described below. Total insulin-positive cells and cells double
positive Ki67 and insulin positive cells were imaged and counted. Islets from 3–5 different adult donors
were tested as described in Figure 4. At least 1000 β-cells were counted for human islet donor.
3.4. Docking
All docking calculations and molecule preparations were carried out using tools in the Schrödinger
Small Molecule Drug Discovery suite release 2018-4. Proteins were prepared using the protein
preparation wizard and ligands were prepared using ligprep. The target structure for all docking
calculations was the crystal structure of DYRK1A in complex with compound 2-2c. Compound were
docked using Glide with XP precision, restricting the Harmine core to its reference position in the 2-2c
complex [50–52].
3.5. Electrostatic Potential
Electrostatic potential calculation was carried out on the DYRK1A crystal structure in complex
with compound 2-2c using the poisson–boltzmann method as implemented in APBS within Maestro
(Schrödinger, LLC). The potential was mapped on the protein surface [53,54].

Molecules 2020, 25, 1983
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4. Conclusions
In these translational studies, we have reported our efforts to study the harmine 7-position with the
objective to identify novel harmine-based DYRK1A inhibitors with human -cell proliferation activity
β
and chemical potential for targeted delivery. We developed chemistry to carry out modifications of
the 7-position of harmine by introducing substituents bearing terminal methyl ester, carboxamide,
carboxylic acid and amino/substituted amino groups with chain lengths varying from 1–5 carbons.
Two compounds were active at less than 100 nM in the biochemical DYRK1A inhibition assay (1-2b
(89 nM) and 1-3b (91nM), respectively), ~3–4-fold less than that of harmine (27 nM). However,
the majority of 7-substituted analogs regardless of carbon chain lengths did not show any improvement
in DYRK1A inhibitory activity. It can be inferred from modeling studies that harmine 7-position
analogs result in disruption of a key binding contact of harmine (H-bond to Leu241) or introduce
unproductive electrostatic interactions with the harmine binding pocket. These results indicate that
these 7-position modifications are not well tolerated for further optimization of harmine scaffold
for β-cell proliferation and conjugation for cell-specific targeted delivery. We did however observe
that both of the potent 7-O DYRK1A inhibitor harmine analogues, 1-2b and 1-3b did exhibit human
β-cell proliferation at screening concentration of 10 µM in vitro, with analog 1-2b performing slightly
better than 1-3b. Dose-response experiments further indicated that 1-2b induced proliferation in
a dose-dependent manner, but required 3-fold higher concentration (30
µM) to induce maximal
proliferation, 66% that of harmine (10 M). The reduced -cell proliferation could be a result of the
µ
β
following: a) its reduced potency for inhibition of DYRK1A as compared to harmine; b) reduced cell
permeability; c) reduced DYRK1A cell target engagement; d) off-target kinase activity; e) a combination
of these. Collectively, these data indicate that modifications of 7-position of harmine are less promising
for retaining potent DYRK1A inhibition for this scaffold as compared to those at the harmine 1-
and 9-positions. We did observe that two 7-O harmine analogs 1-2b and 1-3b showed DYRK1A
inhibitory and β-cell proliferation assay activity in vitro only three-fold less that of harmine. It is
important to note that this study provides deeper insights into the effects of 7-position modifications
of harmine on DYRK1A inhibition in context of diabetes than previously reported, with two novel
analogs compounds showing robust
β-cell proliferation activity. In conclusion, this study, based on the
structure–activity relationships that have been developed, demonstrates that the harmine 7-position is
less than optimal to develop a β-cell proliferative and cell-specific targeted diabetes therapeutic from
this heterocyclic scaffold.
Supplementary Materials: NMR spectral data are available in the Supplementary Material.
Author Contributions: The manuscript was written through contributions of all authors (K.K., P.W., E.A.S., S.K.,
C.S., M.B.L., R.S., A.F.S., R.J.D.). K.K. designed and synthesized harmine analogs. P.W. designed experiments and
performed β-cell proliferation assay and immunocytochemistry. E.A.S. helped P.W. perform β-cell proliferation
assay and immunocytochemistry. R.S. performed computational studies. A.F.S. and R.J.D. conceived the project,
designed translational strategy: biology (A.F.S.); medicinal chemistry, drug discovery (R.J.D.) and supervised the
project. All authors have given approval to the final version of the manuscript.
Funding: This research was supported by grant DK015015, DK116904 (R.J.D., K.K., P.W., A.F.S., E.A.S.), UC4
DK104211, P-30 DK020541, JDRF 2-SRA-2017 514-S-B (A.F.S., P.W., E.A.S.). This work was supported in part
through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of
Medicine at Mount Sinai, the Human Islet and Adenoviral Core of the Einstein-Sinai Diabetes Research Center,
and the NIDDK Human Islet Research Network (HIRN).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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