Exploring kinase inhibition properties of 9hpyrimido[5,4-b]- and [4,5-b]indol-4-amine derivatives

Article abstract of DOI:10.3390/ph13050089

We previously highlighted the interest in 6,5,6-fused tricyclic analogues of 4-aminoquinazolines as kinase inhibitors in the micromolar to the nanomolar range of IC50 values. For the generation of chemical libraries, the formamide-mediated cyclization of the cyanoamidine precursors was carried out under microwave irradiation in an eco-friendly approach. In order to explore more in-depth the pharmacological interest in such tricyclic skeletons, the central five member ring, i.e., thiophène or furan, was replaced by a pyrrole to afford 9H-pyrimido[5,4-b]- and [4,5-b]indol-4-amine derivatives inspired from harmine. The inhibitory potency of the final products was determined against four protein kinases (CDK5/p25, CK1δ/ε, GSK3α/β and DYRK1A). As a result, we have identified promising compounds targeting CK1δ/ε and DYRK1A and displaying micromolar and submicromolar IC₅₀ values.

Full text of DOI:10.3390/ph13050089

Communication
Exploring Kinase Inhibition Properties of 9H-
pyrimido[5,4-b]- and [4,5-b]indol-4-amine Derivatives
1
1
2
3
Yvonnick Loidreau , Carole Dubouilh-Benard , Marie-Renée Nourrisson , Nadège Loaëc ,
3,4
1,
2,
Laurent Meijer , Thierry Besson * and Pascal Marchand *
1
Normandie Université, UNIROUEN, INSA Rouen, CNRS, COBRA UMR 6014, F-76000 Rouen, France;
yvonnick.loidreau@gmail.com (Y.L.); carole.dubouilh@univ-rouen.fr (C.D.-B.)
Université de Nantes, Cibles et Médicaments des Infections et du Cancer, IICiMed, EA 1155, F-44000
2
Nantes, France; marie-renee.nourrisson@univ-nantes.fr
Station Biologique de Roscoff, Protein Phosphorylation & Human Disease group, 29680 Roscoff, France;
3
nadege.loaec@univ-brest.fr (N.L.); meijer@perha-pharma.com (L.M.)
Perha Pharmaceuticals, Perharidy Peninsula, 29680 Roscoff, France
4
* Correspondence: thierry.besson@univ-rouen.fr (T.B.), pascal.marchand@univ-nantes.fr (P.M.);
Tel.: +33 235-522-904 (T.B.); +33 253 009 155 (P.M.)
Received: 10 April 2020; Accepted: 8 May 2020; Published: 9 May 2020
Abstract: We previously highlighted the interest in 6,5,6-fused tricyclic analogues of 4-
aminoquinazolines as kinase inhibitors in the micromolar to the nanomolar range of IC50 values. For
the generation of chemical libraries, the formamide-mediated cyclization of the cyanoamidine
precursors was carried out under microwave irradiation in an eco-friendly approach. In order to
explore more in-depth the pharmacological interest in such tricyclic skeletons, the central five
member ring, i.e., thiophène or furan, was replaced by a pyrrole to afford 9H-pyrimido[5,4-b]- and
[4,5-b]indol-4-amine derivatives inspired from harmine. The inhibitory potency of the final products
was determined against four protein kinases (CDK5/p25, CK1δ/ε, GSK3α/β, and DYRK1A). As a
result, we have identified promising compounds targeting CK1δ/ε and DYRK1A and displaying
micromolar and submicromolar IC50 values.
Keywords: microwave-assisted chemistry; protein kinases; CK1; DYRK1A; CDK5; GSK-3
1. Introduction
Protein kinases are an important family of enzymes able to phosphorylate tyrosine (Tyr) and
serine (Ser)/threonine (Thr) residues present in various proteins [1]. Abnormal protein kinase
regulation and phosphorylation are now associated with numerous diseases including cancer [2,3],
and neurodegenerative disorders [4–6]. In the last decade, about 300 protein kinase inhibitors were
involved in clinical trials and 49 have been recently approved by the US Food and Drug
Administration (FDA), mostly tyrosine kinase inhibitors, and mainly for cancer therapy [7]. In the
same period, our groups have been particularly invested in the development of efficient and eco-
compatible chemical methodologies allowing rapid access to libraries of potent bioactive arenes and
their heteroarenes analogues. Studying ancestral thermal-sensitive reactions for which usual
methods require forcing conditions or prolonged reaction times (e.g., Niementowski reaction [8,9]
and Dimroth transposition [10]), microwave-assisted syntheses of novel benzo[b]thieno[3,2-
d]pyrimidin-4-amines (series A) [11,12] and their pyrido (series B) and pyrazino analogues (series C)
[13,14] have been successfully described (Figure 1). In this context, more than one hundred
derivatives of the heterocyclic systems have been studied. Among them, the pyrido[b]thieno[3,2-
d]pyrimidin-4-amine derivatives (series B) have shown the most interesting selectivity and inhibitory
potency towards CK1δ/ε over the other tested enzymes (CDK5/p25, GSK3α/β, and Dyrk1A) [14].
Pharmaceuticals 2020, 13, 89; doi:10.3390/ph13050089
www.mdpi.com/journal/pharmaceuticals

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Figure 1. Previous benzo[b]thieno[3,2-d]pyrimidin-4-amines (A) and their pyrido (B) and pyrazino
(C) analogues (left). The 9H-pyrimido[5,4-b]indol-4-amine derivatives (1 and 2) and their
9Hpyrimido[4,5-b]indole isomers (3 and 4) described in this work (right).
In an effort to expand the chemical space and to highlight efficient kinase inhibitors, the
synthesis of indole counterparts of the previously-described series has been envisaged. Such
compounds are considered as analogues of harmine (see 11a in scheme 2), a natural alkaloid that still
generates a lot of work in the hope of developing therapies for Alzheimer’s disease (AD) and Down
syndrome (DS) [15,16]. This paper describes simple and convenient synthetic routes to 9H-
pyrimido[5,4-b]indol-4-amine derivatives (series 1 and 2) and their 9Hpyrimido[4,5-b]indole isomers
(series 3 and 4). The chemistry described in this paper was mainly carried out under microwave
irradiation in an eco-friendly approach. Kinase inhibition of the products obtained was evaluated on
an array of four Ser/Thr kinases (CDK5/p25, CK1δ/ε, DYRK1A, and GSK3α/β), all members of the
CMGC kinase family, chosen for their strong implication in various cellular regulation processes [17–
25].
2. Results
2.1. Chemistry
Synthesis of series 1, 2, 3, and 4 was inspired from our previous works on various benzo-, pyrido-,
and pyrazino[b]thieno[3,2-d] pyrimidines (see series A, B and C in Figure 1) [11–14]. The nitrogen
ring in pyridine and in pyrazine may be considered to be deactivated compared with benzene. It is
sometimes compared with nitrobenzene as the molecules behave similarly in relation to the deficit of
1
electron density. This consideration guided the choice of the nitro substitution (R ) on the starting
indole derivatives for completion of the structure-activity relationship (SAR) study. The N’-(cyano-
1H-indolyl)-N,N-dimethyl formimidamide precursors (7a–d, 8a–d, 9a–d, and 10a–d) were heated at
170–200 °C under microwaves in the presence of an excess of formamide (40 equiv.) (for reaction
times and yields see Table 1).
Functionalized indoles (7a–d, 8a–d, 9a–d, and 10a–d) were previously obtained from 3-amino-
1H-indole-2-carbonitriles (5a,b) or their 5-nitro-3-amino-1H-indole-2-carbonitrile isomers (6a,b)
which were condensed with 10 equiv. of DMF-dialkylacetals like N,N-dimethylformamide dimethyl
acetal (DMF-DMA), N,N-dimethylformamide diethyl acetal (DMF-DEA), and N,N-
dimethylformamide dibenzyl acetal (DMF-DBA), respectively (Scheme 1). A strict control of the
experimental conditions in terms of temperature and reaction time allowed access to the expected
compounds in good to excellent yields (for details on suggested mechanism and experimental details
see ref. [26]).

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Scheme 1. Synthesis of 9H-pyrimido[5,4-b]indol-4-amines (series 1a–d and 2a–d) and their 9H-
pyrimido[4,5-b]indoles isomers (series 3a–d and 4a–d).
Table 1. Synthesis of series 1, 2, 3, and 4: temperature, reaction time, and isolated yields.
1
2
Starting Indole
Temperature (°C)
Time (min)
Product
1a
R
R
Yield (%)
81
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
10a
10b
10c
10d
170
170
170
170
170
170
170
170
200
200
200
200
200
200
200
200
30
30
30
40
60
40
30
30
40
30
30
30
30
30
30
30
H
H
H
H
Me
Et
Bn
H
Me
Et
Bn
H
Me
Et
Bn
H
Me
Et
Bn
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
58
64
52
65
75
72
79
71
85
96
67
67
80
63
67
H
NO2
NO2
NO2
NO2
H
H
H
H
NO2
NO2
NO2
NO2
4a
4b
4c
4d
To compare the biological results of harmine (11a) and some of its N9-alkylated derivatives (11b–
d) with the pyrimidoindoles prepared in this work (series 1, 2, 3, and 4, Table 1), we decided to
explore the capacity of DMF-dialkylacetals to transfer an alkyl group to nucleophilic atoms, as an
interesting alternative to previous methods [27–30]. Then, harmine was heated under controlled
microwave-assisted heating in sealed vials (10 mL) in the presence of 10 equiv. of DMF-DMA, DMF-
DEA, or DMF-DBA. The corresponding N9-alkylated 7-methoxy-1-methyl-β-carbolines (11b–d) were
obtained in good yields (Scheme 2, Table 2).

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Scheme 2. Microwave-assisted synthesis of N-alkylated harmine derivatives (compounds 11b–d).
Table 2. Synthesis of compounds 11b–d: experimental conditions and isolated yields.
N,N-dimethylformamide (DMF)-dialkylacetal (R)
DMF-DMA (Me)
Temperature (°C) Product Yield (%)
140
160
160
11b
11c
11d
79
80
62
DMF-DEA (Et)
DMF-DBA (Bn)
2.2. Biological Evaluation
The inhibitory potency of the synthesized pyrimido[4,5-b]indol-4-amines and pyrimido[5,4-
b]indol-4-amines towards CDK5/p25, CK1δ/ε, DYRK1A, and GSK-3α/β was investigated according
preceding procedures [11–14]. Data are listed in Table 3 including results obtained with harmine (11a)
and its congeners (11b–d).
Table 3. Kinase inhibitory potencies (IC50 in μM) 1 for compounds of the series 1, 2, 3, and 4.
1
2
Compound
R
R
CDK5/p25
>10
>10
>10
>10
>10
>10
>10
>10
6
CK1δ/ε
2.0
DYRK1A
2.2
GSK-3α/β
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
H
H
H
H
Me
Et
Bn
H
Me
Et
Bn
H
Me
Et
Bn
H
Me
Et
Bn
4.0
2.8
0.6
>10
>10
>10
>10
0.7
2.5
1.6
2.7
3.5
2.8
1.6
>10
1.5
5.8
4.1
>10
7.6
>10
>10
>10
3.1
>10
9.8
>10
7.6
>10
5.9
>10
H
NO2
NO2
NO2
NO2
H
H
H
H
NO2
NO2
NO2
NO2
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4d
11a (Harmine)
11b
R = H
0.029
0.13
0.037
0.059
R = Me
R = Et
R = Bn
>10
>10
>10
11c
11d
1 Average of triplicate determination (<10% variation among values).

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All the tested compounds were inactive against CDK-5/p25 and GSK3α/β. 8-Nitro-5H-
pyrimido[5,4-b]indol-4-amines (2a–d) were also inactive against the two other tested kinases, except
compound 2a which exhibited a micromolar range IC50 value (7.6 μM) against DYRK1A. General
comparison of series 1, 3, and 4 revealed a similar inhibitory activity against the array of four kinases.
Values were mainly in the micromolar range against CK1δ/ε, except compounds 1d and 3a which
disclosed submicromolar IC50 values (0.6 and 0.7 μM, respectively). It can be noted that 1d seemed
more specific for CK1δ/ε in view of its lack of activity against DYRK1A, whilst 3a maintained a
micromolar affinity (IC50 = 3.1 μM) against this biological target.
Considering CK1δ/ε kinase inhibition, the introduction of a nitro group was deleterious for
series 1 vs. series 2, whereas inhibitory activity remained for series 3 vs. series 4, depending of the
orientation of the aminopyrimidine ring in the fused system and its relative position to the nitro
group. On the other hand, for the unsubstituted tricyclic derivatives on indole (series 1 and 3) the
position of the amino group does not seem critical since micromolar CK1 inhibition was conserved
within the two series.
Interestingly, a loss of DYRK1A inhibitory activity was observed for the compounds bearing a
N-methyl or a N-benzyl chain compared to their NH or N-ethyl analogues, except for the inactive
series 2 and the very active compounds 11a–d.
Tested as a positive control under the same conditions as series 1–4, harmine (11a) was definitely
inactive against CDK5/p25 and GSK3α/β. This natural product shows interesting activity against
DYRK1A and a weak inhibition of CK1δ/ε (IC50 = 1.5 μM) as previously mentioned in various papers
relating the use of harmine for treatment of neurodegenerative diseases [31,32]. The N-methyl, N-
ethyl, and N-benzyl derivatives (11b, 11c, and 11d) were totally inactive against the three kinases
CDK5/p25, CK1δ/ε, and GSK3α/β. Their affinity was focused on DYRK1A with interesting IC50 values,
quite close to the nanomolar range IC50 obtained for the lead harmine (11a) (see Table 3), confirming
recently-published results [29].
3. Discussion
Results described above confirm the interest in developing chemical methods that allow easy
access to libraries of various potentially bioactive molecules. This is particularly noticeable in the case
of compounds 1a and 1b, which are the only derivatives of these new series that were already
synthesized via multistep processes (2–4 steps), in long time reactions (6–20 h) and sometimes
difficult operating conditions, using toxic reagents (e.g., POCl3) [33]. The combination of microwave-
assisted heating and the use of DMF-dialkylacetals provided in all cases short reaction times and
comfortable operating conditions.
The two unsubstituted isomeric forms (series 1a–d and 3a–d) expressed a similar average
activity against the two kinases CK1δ/ε and DYRK1A. Moreover, the data obtained for the two series
of pyrimidoindoles bearing a nitro group, demonstrate that the pyrimido[4,5-b]indol-4-amines (series
4a–d) are more active than their pyrimido[5,4-b]indol-4-amine isomers (series 2a–d). In case of the
first series (1a–d and 2a–d), the data suggest that addition of a nitro group significantly decreases the
inhibitory activity. In contrast, the two sets of pyrimido[4,5-b]indol-4-amines (3a–d and 4a–d) exhibit
similar micromolar range IC50 values which are independent of the substitution pattern on the
benzene moiety of the indole ring.
Figure 2 focuses on the two kinases mainly inhibited in this study and the IC50 values towards
CK1δ/ε and DYRK1A protein kinases are reported for selected pyrimido[4,5-b or 5,4-b]indol-4-amines
(1a, 2a, 3a, and 4a). Inhibitory activity is compared with the biological data already published for
thieno[2,3-d]pyrimidin-4-amine analogues described as potential Ser/Thr kinases inhibitors [12–14].

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Figure 2. Comparison between selected pyrimido[4,5-b or 5,4-b]indol-4-amines described in this work
and some of their thieno[2,3-d]pyrimidin-4-amine congeners (12, 13, 14 and 15) described in preceding
work.
It can be observed that the replacement of the sulfur atom, in the structure of compound 12, by
a nitrogen atom (NH, more exactly, in compound 1a) resulted in increasing DYRK1A inhibitory
potency (from 33 to 2.2 µM) and keeping CK1 activity. The major difference between the two
compounds lies in the nature of the hydrogen bonding (acceptor for S vs. donor for NH) that would
allow a particular binding mode and interaction with the target protein, explaining this trend. When
comparing the activity of nitroindole derivative 2a with the deactivated analogues 13, 14, and 15, due
to the presence of a pyrazine or a pyridine ring, opposite selectivity towards CK1δ/ε and DYRK1A
was observed. Indeed, the last ones displayed micromolar and submicromolar activity against CK1
and no activity on DYRK1A, whereas compound 2a exhibited no inhibition against CK1δ/ε and low
micromolar activity towards DYRK1A. Again, the four compounds behave identically in terms of
electronic deficiency but, for three of them, we assumed that the presence of a nitrogen atom could
bring an additional possibility of interaction with the protein through the lone pair of electrons.
4. Materials and Methods
4.1. General Information
All reagents were purchased from commercial suppliers and were used without further
purification, except for DMF, which was stored under argon and activated molecular sieves. All
reactions were monitored by thin-layer chromatography with silica gel 60 F254 (Merck KGaA,
Darmstadt, Germany) precoated aluminium plates (0.25 mm). Visualization was performed with a
UV light at wavelengths of 254 nm. Purifications were conducted with a flash column
chromatography system equipped with a dual UV/vis spectrophotometer (200–600 nm), a fraction
collector (176 tubes), a dual piston pump (1 to 200 mL/min, Pmax = 15 bar), which allowed quaternary
gradients, and an additional inlet for air purge ((Puriflash, Interchim, Montluçon, France). Melting
points of solid compounds were measured with a SMP3 melting point instrument (STUART, Bibby
Scientific Ltd., Roissy, France) with a precision of 1.5 °C. IR spectra were recorded with a Spectrum
1
13
100 Series FTIR spectrometer (PerkinElmer, Villebon S/Yvette, France). NMR spectra ( H and C)
were acquired at 295 K using an AVANCE 300 MHz spectrometer (Bruker, Wissembourg, France) at
300 and 75.4 MHz, using trimethylsilane (TMS) as an internal standard. Coupling constants J are in
Hz, and chemical shifts are given in ppm. Mass spectrometry was performed by the Mass
Spectrometry Laboratory of the University of Rouen. The mass spectra electrospray ionization (ESI),
electron impact ionization (EI), and field desorption (FD) were recorded with an LCP 1er XR
spectrometer (WATERS, Guyancourt, France). Microwave experiments were carried out at
atmospheric pressure in 50–250 mL round bottom flasks fitted with a reflux condenser, in a
TM
RotoSYNTH (Milestone S.r.l., Milano, Italy), a multi-mode cavity microwave reactor designed for
synthetic chemistry (0–1200 W). Microwave reactions in sealed tubes (10 mL) were performed with a
TM
Initiator microwave synthesis instrument (Biotage, Uppsala, Sweden) (0–400W). Temperatures of
the reactions were monitored via IR-sensors. The percentage of purity of all tested products was more

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1
13
than 95% determined by high pressure liquid chromatography (HPLC) analysis. H-NMR and C-
NMR spectra of new compounds are available in supplementary materials section Figures S1–16.
4.2. Chemistry
All
details
conerning
the
synthesis
of
N’-(3-Cyano-1-alkyl-1H-indol-2-yl)-N,N-
dimethylformimidamide intermediates (7a–d, 8a–d, 9a–d, and 10a–d) are described in a preceding
work [26].
Compounds 1a and 1b were already described and data given in the corresponding patent are
1
only H NMR spectra [33]. Harmine derivatives 11b, 11c, and 11d were described in a preceding work
[28] cited in some recent studies [29,30].
4.2.1. General Procedure for the Synthesis of 5H-Pyrimido[5,4-b]indol-4-amines (Series 1 and 2) and
5H-Pyrimido[4,5-b]indol-4-amines (Series 3 and 4).
Formamide (40 equiv.) was added to the formimidamide precursor (7b–d, 8b–d, 9b–d, or 10b–
d) (1 mmol) and the mixture was heated under microwave irradiation (200 W). On completion, the
reaction was cooled to room temperature and water was added. The solid was filtered off, washed
with water, and dried. The crude solid was purified by silica gel column chromatography using
PE/EtOAc (100:0 to 0:100, v/v) as the eluent to give the desired compounds.
5H-Pyrimido[5,4-b]indol-4-amine (1a): brown powder (0.149 g, 81%) obtained from 7a after 30 min
-1
of irradiation at 170 °C according to the general procedure; mp > 320 °C; IR (neat) νmax(cm ): 3047,
1
1616, 1600, 1559, 1498, 1462, 1428, 1354, 1343, 1316, 1295, 1234, 1207, 1119, 756, 745; H NMR (300
MHz, DMSO-d6): δ 10.98 (br s, 1H, NH), 8.29 (s, 1H, H-2), 8.05 (d, 1H, J = 8 Hz, H-8), 7.63 (d, 1H, J = 8
Hz, H-6), 7.50 (td, 1H, J1 = 2 Hz, J2 = 8 Hz, H-7), 7.22 (td, 1H, J1 = 2 Hz, J2 = 8 Hz, H-6), 6.95 (s, 2H, NH2);
13
C NMR (75 MHz, DMSO-d6): δ 150.8, 149.9, 141.9, 138.8, 127.7, 120.9, 120.4, 119.5, 117.4, 112.5; HRMS
+
calcd for C10H9N4 [M + H] 185.0827 found 185.0822
5-Methyl-5H-pyrimido[5,4-b]indol-4-amine (1b): brown powder (0.115 g, 58%) obtained from 7b
after 30 min of irradiation at 170 °C according to the general procedure; mp 201–202 °C; IR (neat)
-1
1
νmax(cm ): 3074, 1657, 1623, 1590, 1537, 1493, 1462, 1431, 1400, 1352, 1329, 1244, 962, 842, 789, 737; H
NMR (300 MHz, DMSO-d6): δ 8.29 (s, 1H, H-2), 8.06 (d, 1H, J = 8 Hz, H-9), 7.69 (d, 1H, J = 8 Hz, H-6),
7.58 (td, 1H, J1 = 2 Hz, J2 = 8 Hz, H-7), 7.24 (td, 1H, J1 = 2 Hz, J2 = 8 Hz, H-8), 6.93 (s, 2H, NH2), 4.08 (s,
13
3H, CH3); C NMR (75 MHz, DMSO-d6): δ 151.4, 149.7, 140.9, 128.0, 120.4, 120.3, 119.5, 114.2, 110.4,
106.3, 31.7; HRMS calcd for C11H11N4 [M + H] 199.0984 found 199.0981.
+
5-Ethyl-5H-pyrimido[5,4-b]indol-4-amine (1c): brown powder (0.136 g, 64%) obtained from 7c after
-
30 min of irradiation at 170 °C according to the general procedure; mp 161–162 °C; IR (neat) νmax(cm
1
1
): 3349, 1641, 1589, 1532, 1402, 1382, 1334, 1226, 1046, 1024, 988, 827, 729; H NMR (300 MHz, DMSO-
d6): δ 8.31 (s, 1H, H-2), 8.08 (d, 1H, J = 8 Hz, H-9), 7.72 (d, 1H, J = 8 Hz, H-6), 7.58 (td, 1H, J1 = 2 Hz, J2 =
8 Hz, H-7), 7.22 (td, 1H, J1 = 2 Hz, J2 = 8 Hz, H-8), 6.90 (s, 2H, NH2), 4.61 (q, 2H, J = 7 Hz, CH2), 1.21 (t,
13
3H, J = 7 Hz, CH3); C NMR (75 MHz, DMSO-d6): δ 150.9, 149.7, 140.1, 128.1, 120.7, 120.5, 119.6, 118.2,
+
112.1, 110.4, 39.7, 16.0; HRMS calcd for C12H13N4 [M + H] 213.1140 found 213.1133.
5-Benzyl-5H-pyrimido[5,4-b]indol-4-amine (1d): yellow powder (0.143 g, 52%) obtained from 7d
after 40 min of irradiation at 170 °C according to the general procedure; mp 219–220 °C; IR (neat)
-1
1
νmax(cm ): 3062, 1641, 1619, 1581, 1530, 1494, 1453, 1403, 1378, 1330, 1254, 1207, 1193, 954, 754, 734; H
NMR (300 MHz, DMSO-d6): δ 8.33 (s, 1H, H-2), 8.10 (d, 1H, J = 8 Hz, H-9), 7.79 (d, 1H, J = 8 Hz, H-6),
7.55 (td, 1H, J1 = 2 Hz, J2 = 8 Hz, H-7), 7.28-7.17 (m, 4H, H-8, and H-ar), 7.00 (dd, 2H, J1 = 1 Hz, J2 = 8
13
Hz, H-ar), 6.90 (s, 2H, NH2), 5.87 (s, 2H, CH2); C NMR (75 MHz, DMSO-d6): δ 151.0, 150.0, 143.8,
141.0, 138.3, 128.5 (2C), 128.3, 127.2, 126.2 (2C), 120.9, 120.5, 120.0, 118.0, 111.1, 46.9; HRMS calcd for
+
C17H15N4 [M + H] 275.1297 found 275.1295.
8-Nitro-5H-pyrimido[5,4-b]indol-4-amine (2a): brown powder (0.149 g, 65%) obtained from 8a after
-1
60 min of irradiation at 170 °C according to the general procedure; mp > 320 °C; IR (neat) νmax(cm ):
1
3102, 1692, 1627, 1597, 1548, 1512, 1474, 1453, 1377, 1321, 1303, 1233, 1131, 1044, 816, 733; H NMR (300
MHz, DMSO-d6): δ 11.73 (br s, 1H, NH), 8.89 (d, 1H, J = 1 Hz, H-9), 8.40 (s, 1H, H-2), 8.35 (dd, 1H, J1 =
13
1 Hz, J2 = 8 Hz, H-7), 7.85 (d, 1H, J = 8 Hz, H-6), 7.22 (s, 2H, NH2); C NMR (75 MHz, DMSO-d6): δ

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+
151.4, 151.3, 142.6, 141.4, 140.5, 122.5, 120.4, 119.4, 117.2, 113.3; HRMS calcd for C10H8N5O2 [M + H]
230.0678 found 230.0682.
5-Methyl-8-nitro-5H-pyrimido[5,4-b]indol-4-amine (2b): yellow powder (0.182 g, 75%) obtained
from 8b after 40 min of irradiation at 170 °C according to the general procedure; mp > 320 °C; IR
-1
1
(neat) νmax(cm ): 3087, 1617, 1585, 1510, 1472, 1327, 1302, 1259, 1235, 1083, 1026, 915, 844, 791, 729; H
NMR (300 MHz, DMSO-d6): δ 8.89 (d, 1H, J = 1 Hz, H-9), 8.40 (dd, 1H, J1 = 1 Hz, J2 = 8 Hz, H-7), 8.39
13
(s, 1H, H-2), 7.90 (d, 1H, J = 8 Hz, H-6), 7.21 (s, 2H, NH2), 4.14 (s, 3H, CH3); C NMR (75 MHz, DMSO-
d6): δ 151.9, 151.1, 143.5, 143.0, 140.3, 122.7, 120.5, 119.7, 117.1, 111.3, 32.4; HRMS calcd for C11H10N5O2
+
[M + H] 244.0834 found 244.0827.
5-Ethyl-8-nitro-5H-pyrimido[5,4-b]indol-4-amine (2c): yellow powder (0.185 g, 72%) obtained from
8c after 30 min of irradiation at 170 °C according to the general procedure; mp 299–300 °C; IR (neat)
-1
1
νmax(cm ): 3072, 1617, 1586, 1507, 1475, 1456, 1329, 1302, 1257, 1227, 1090, 944, 805, 753, 735; H NMR
(300 MHz, DMSO-d6): δ 8.91 (d, 1H, J = 1 Hz, H-9), 8.42 (s, 1H, H-2), 8.40 (dd, 1H, J1 = 1 Hz, J2 = 8 Hz,
H-7), 7.97 (d, 1H, J = 8 Hz, H-6), 7.23 (s, 2H, NH2), 4.70 (q, 2H, J = 7 Hz, CH2), 1.27 (t, 3H, J = 7 Hz, CH3);
13
C NMR (75 MHz, DMSO-d6): δ 152.4, 152.3, 144.1, 142.3, 140.5, 122.9, 120.1, 119.3, 117.2, 111.3, 40.1,
16.1; HRMS calcd for C12H12N5O2 [M + H] 258.0991 found 258.0986.
+
5-Benzyl-8-nitro-5H-pyrimido[5,4-b]indol-4-amine (2d): yellow powder (0.252 g, 79%) obtained
from 8d after 30 min of irradiation at 170 °C according to the general procedure; mp > 320 °C; IR
-1
(neat) νmax(cm ): 3052, 1643, 1620, 1586, 1513, 1473, 1451, 1400, 1331, 1315, 1203, 1076, 1045, 939, 804,
1
791, 732; H NMR (300 MHz, DMSO-d6): δ 8.94 (d, 1H, J = 1 Hz, H-9), 8.44 (s, 1H, H-2), 8.41 (dd, 1H, J1
= 1 Hz, J2 = 8 Hz, H-7), 8.03 (d, 1H, J = 8 Hz, H-6), 7.28-7.18 (m, 5H, NH2, and H-ar), 7.01 (dd, 2H, J1 = 1
13
Hz, J2 = 8 Hz, H-ar), 5.99 (s, 2H, CH2); C NMR (75 MHz, DMSO-d6): δ 151.5 (2C), 144.3, 143.3, 140.9,
137.4, 128.7 (2C), 127.5, 126.1 (2C), 123.2, 120.4, 119.7, 117.2, 111.9, 47.4; HRMS calcd for C17H14N5O2
+
[M + H] 320.1147 found 320.1145.
9H-Pyrimido[4,5-b]indol-4-amine (3a): cream-coloured powder (0.131 g, 71%) obtained from N’-
(3-cyano-1H-indol-2-yl)-N,N-dimethylformimidamide 9a after 40 min of irradiation at 200 °C
-1
according to the general procedure; mp > 320 °C; IR (neat) νmax(cm ): 3068, 1637, 1624, 1604, 1580,
1
1569, 1302, 1255, 981, 798, 750, 705; H NMR (300 MHz, DMSO-d6): δ 11.81 (br s, 1H, NH), 8.27 (d, 1H,
J = 8 Hz, H-5), 8.24 (s, 1H, H-2), 7.43 (d, 1H, J = 8 Hz, H-8), 7.34 (td, 1H, J1 = 1 Hz, J2 = 8 Hz, H-7), 7.22
13
(td, 1H, J1 = 1 Hz, J2 = 8 Hz, H-6), 7.13 (s, 2H, NH2); C NMR (75 MHz, DMSO-d6): δ 157.6, 155.6, 154.8,
+
136.2, 124.4, 121.2, 120.0, 119.8, 110.8, 95.2; HRMS calcd for C10H9N4 [M + H] 185.0827 found 185.0820.
9-Methyl-9H-pyrimido[4,5-b]indol-4-amine (3b): brown powder (0.168 g, 85%) obtained from 9b
after 30 min of irradiation at 200 °C according to the general procedure; mp 189–190 °C; IR (neat)
-1
1
νmax(cm ): 3059, 1622, 1588, 1556, 1507, 1463, 1449, 1326, 1309, 1300, 1194, 978, 795, 731; H NMR (300
MHz, DMSO-d6): δ 8.32 (d, 1H, J = 8 Hz, H-5), 8.31 (s, 1H, H-2), 7.59 (d, 1H, J = 8 Hz, H-8), 7.43 (td, 1H,
13
J1 = 1 Hz, J2 = 8 Hz, H-7), 7.29 (td, 1H, J1 = 1 Hz, J2 = 8 Hz, H-6), 7.22 (s, 2H, NH2), 3.82 (s, 3H, CH3); C
NMR (75 MHz, DMSO-d6): δ 157.7, 155.2, 154.9, 137.5, 124.7, 124.4, 121.2, 120.3, 119.3, 109.2, 28.1;
+
HRMS calcd for C11H11N4 [M + H] 199.0984 found 199.0974.
9-Ethyl-9H-pyrimido[4,5-b]indol-4-amine (3c): brown powder (0.204 g, 96%) obtained from 9c after
-
30 min of irradiation at 200 °C according to the general procedure; mp 137–138 °C; IR (neat) νmax(cm
1
1
): 3062, 1623, 1558, 1448, 1464, 1329, 1113, 798, 736, 708; H NMR (300 MHz, DMSO-d6): δ 8.34 (d, 1H,
J = 8 Hz, H-5), 8.32 (s, 1H, H-2), 7.63 (d, 1H, J = 8 Hz, H-8), 7.45 (td, 1H, J1 = 1 Hz, J2 = 8 Hz, H-7), 7.26
(td, 1H, J1 = 1 Hz, J2 = 8 Hz, H-6), 7.21 (s, 2H, NH2), 4.41 (q, 2H, J = 7 Hz, CH2), 1.31 (t, 3H, J = 7 Hz,
13
CH3); C NMR (75 MHz, DMSO-d6): δ 157.5, 154.8, 154.5, 136.3, 124.5, 121.4, 120.3, 119.6, 109.3, 94.8,
+
35.7, 14.0; HRMS calcd for C12H13N4 [M + H] 213.1140 found 213.1130.
9-Benzyl-9H-pyrimido[4,5-b]indol-4-amine (3d): cream-coloured powder (0.184 g, 67%) obtained
from 9d after 30 min of irradiation at 200 °C according to the general procedure; mp 217–218 °C; IR
-1
1
(neat) νmax(cm ): 3051, 1620, 1585, 1568, 1556, 1458, 1445, 1428, 1297, 799, 753, 740; H NMR (300 MHz,
DMSO-d6): δ 8.34 (d, 1H, J = 8 Hz, H-5), 8.33 (s, 1H, H-2), 7.55 (d, 1H, J = 8 Hz, H-8), 7.37 (td, 1H, J1 = 1
13
Hz, J2 = 8 Hz, H-7), 7.29-7.21 (m, 8H, H-6, NH2, and H-ar), 5.60 (s, 2H, CH2); C NMR (75 MHz, DMSO-
d6): δ 157.6, 155.0, 137.5, 136.6, 128.5 (2C), 127.3, 127.1 (2C), 124.6, 121.4, 120.6, 119.6, 117.2, 109.8, 94.8,
+
44.0; HRMS calcd for C17H15N4 [M + H] 275.1297 found 275.1292.

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7-Nitro-9H-pyrimido[4,5-b]indol-4-amine (4a): brown powder (0.153 g, 67%) obtained from 10a
-
after 30 min of irradiation at 200 °C according to the general procedure; mp > 320 °C; IR (neat) νmax(cm
1
1
): 3052, 1621, 1529, 1500, 1471, 1375, 1304, 1287, 1257, 1204, 1124, 1071, 884, 820, 753, 734; H NMR
(300 MHz, DMSO-d6): δ 12.42 (br s, 1H, NH), 8.53 (d, 1H, J = 9 Hz, H-5), 8.34 (s, 1H, H-2), 8.25 (d, 1H,
13
J = 2 Hz, H-8), 8.11 (dd, 1H, J1 = 2 Hz, J2 = 9 Hz, H-6), 7.62 (s, 2H, NH2); C NMR (75 MHz, DMSO-d6):
+
δ 158.4, 157.9, 156.8, 143.8, 135.4, 125.5, 121.2, 115.2, 106.5, 94.9; HRMS calcd for C10H8N5O2 [M + H]
230.0678 found 230.0689.
9-Methyl-7-nitro-9H-pyrimido[4,5-b]indol-4-amine (4b): yellow powder (0.195 g, 80%) obtained
from 10b after 30 min of irradiation at 200 °C according to the general procedure G; mp 309–310 °C;
-1
1
IR (neat) νmax(cm ): 3031, 1568, 1513, 1482, 1331, 1309, 1284, 1273, 1193, 987, 875, 853, 811, 726; H NMR
(300 MHz, DMSO-d6): δ 8.55 (d, 1H, J = 9 Hz, H-5), 8.51 (d, 1H, J = 2 Hz, H-8), 8.39 (s, 1H, H-2), 8.12
13
(dd, 1H, J1 = 2 Hz, J2 = 9 Hz, H-6), 7.67 (s, 2H, NH2), 3.91 (s, 3H, CH3); C NMR (75 MHz, DMSO-d6): δ
158.2, 157.3, 156.7, 144.0, 136.7, 124.9, 121.1, 115.5, 105.5, 94.5, 27.9; HRMS calcd for C11H10N5O2 [M +
+
H] 244.0834 found 244.0831.
9-Ethyl-7-nitro-9H-pyrimido[4,5-b]indol-4-amine (4c): brown powder (0.162 g, 63%) obtained from
10c after 30 min of irradiation at 200 °C according to the general procedure; mp 285–286 °C; IR (neat)
-1
1
νmax(cm ): 3109, 1618, 1582, 1563, 1509, 1485, 1448, 1322, 1276, 1184, 1135, 820, 743, 732; H NMR (300
MHz, DMSO-d6): δ 8.58 (d, 1H, J = 2 Hz, H-8), 8.55 (d, 1H, J = 9 Hz, H-5), 8.39 (s, 1H, H-2), 8.12 (dd,
1H, J1 = 2 Hz, J2 = 9 Hz, H-6), 7.69 (s, 2H, NH2), 4.53 (q, 2H, J = 7 Hz, CH2), 1.32 (t, 3H, J = 7 Hz, CH3);
13
C NMR (75 MHz, DMSO-d6): δ 158.3, 156.8, 156.7, 144.1, 135.6, 125.1, 121.3, 115.4, 105.4, 94.5, 36.2,
14.1; HRMS calcd for C12H12N5O2 [M + H] 258.0991 found 258.0999.
+
9-Benzyl-7-nitro-9H-pyrimido[4,5-b]indol-4-amine (4d): yellow powder (0.214 g, 67%) obtained
from 10d after 30 min of irradiation at 200 °C according to the general procedure; mp 241–242 °C; IR
-1
1
(neat) νmax(cm ): 3031, 1558, 1508, 1477, 1448, 1319, 1268, 1170, 1086, 801, 733; H NMR (300 MHz,
DMSO-d6): δ 8.60 (d, 1H, J = 2 Hz, H-8), 8.49 (d, 1H, J = 9 Hz, H-5), 8.43 (s, 1H, H-2), 8.13 (dd, 1H, J1 =
13
2 Hz, J2 = 9 Hz, H-6), 7.76 (s, 2H, NH2), 7.32-7.21 (m, 5H, H-ar), 4.53 (s, 2H, CH2); C NMR (75 MHz,
DMSO-d6): δ 158.4, 157.4, 156.9, 144.1, 137.0, 135.9, 128.7 (2C), 127.5, 127.0 (2C), 125.3, 121.4, 115.8,
+
105.7, 94.5, 44.2; HRMS calcd for C17H14N5O2 [M + H] 320.1147 found 320.1133.
4.2.2. General Procedure for the Synthesis of N-alkylated Harmine Derivatives (Compounds 11b–d).
A mixture of starting harmine (11a, 212 mg, 1 mmol) and corresponding DMF-dialkylacetal
(DMF-DMA, DMF-DEA, or DMF-DBA) (10 mmol) in DMF (10 mmol) was irradiated (atmospheric
pressure) at various temperatures (800W). On completion, the solution was cooled to room
temperature and crude products were extracted with ethylacetate. The organic layers were washed
with cold water, dried over Na2SO4, filtered, and evaporated in vacuo. Purification by silica gel
column chromatography using a gradient of dichloromethane/ethylacetate (100:0 to 0:100, v/v) as the
eluent gave the desired products.
7-Methoxy-1,9-dimethyl-β-carboline (11b): Yield: 79%, obtained from DMF-DMA after 60 min of
1
irradiation at 140 °C according to the general procedure; mp 125–126 °C; H NMR (300 MHz, DMSO-
d6): δ 8.14 (d, 1H, J = 5 Hz, H-3), 8.09 (d, 1H, J = 8 Hz, H-5), 7.86 (d, 1H, J = 5 Hz, H-4), 7.20 (d, 1H, J = 2
Hz, H-8), 6.86 (dd, 1H, J1 = 2 Hz, J2 = 8 Hz, H-6), 4.13 (s, 3H, NCH3), 3.92 (s, 3H, OCH3), 3.01 (s, 3H,
+
CH3); HRMS calcd for C14H15N2O [M + H] 227.1184 found 227.1180.
9-Ethyl-7-methoxy-1-methyl-β-carboline (11c): Yield: 80%, obtained from DMF-DEA after 60 min of
1
irradiation at 160 °C according to the general procedure; mp 109–110 °C; H NMR (300 MHz, DMSO-
d6): δ 8.16 (d, 1H, J = 5 Hz, H-3), 8.09 (d, 1H, J = 8 Hz, H-5), 7.87 (d, 1H, J = 5 Hz, H-4), 7.20 (d, 1H, J = 2
Hz, H-8), 6.87 (dd, 1H, J1 = 2 Hz, J2 = 8 Hz, H-6), 4.62 (q, 2H, J = 7 Hz, CH2), 3.92 (s, 3H, OCH3), 2.96 (s,
+
3H, CH3), 1.35 (t, 3H, J = 7 Hz, CH2CH3); HRMS calcd for C15H17N2O [M + H] 241.1341 found 241.1338.
9-Benzyl-7-methoxy-1-methyl-β-carboline (11d): Yield: 62%, obtained from DMF-DBA after 60 min
1
of irradiation at 160 °C according to the general procedure; mp 131–132 °C; H NMR (300 MHz,
DMSO-d6): δ 8.18 (d, 1H, J = 5 Hz, H-3), 8.14 (d, 1H, J = 8 Hz, H-5), 7.94 (d, 1H, J = 5 Hz, H-4), 7.38-7.19
(m, 5H, H-8, and H-ar), 6.94-6.89 (m, 2H, H-6, and H-ar), 5.90 (s, 2H, CH2), 3.82 (s, 3H, OCH3), 2.74 (s,
+
3H, CH3); HRMS calcd for C20H19N2O [M + H] 303.1497 found 303.1499.

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4.3. In Vitro Kinase Preparation and Assays
Buffers, kinase preparations, and assays were performed as described in ref. [12–14] according
methods initially published in previous works [34–36].
Supplementary Materials: The following materials are available online at www.mdpi.com/1424-
8247/13/5/89/s1. Figures S1–16. 1H- & 13C-NMR Spectra for Compounds 1a-1d, 2a-2d, 3a-3d and 4a-4d.
Author Contributions: P.M. and T.B. conceived the project and designed the experiments; Y.L. performed the
experimental work, accompanied by C.D.-B. and M.-R.N.; L.M. designed and supervised the overall aspect of
biological experiments; N.L. ran the kinase assays; and P.M. wrote the manuscript with the cooperation of T.B.
All authors discussed the results and commented on the manuscript.
Funding: T.B. and his co-workers (Y.L. and C.D.-B.) thank the LABEX SynOrg (ANR-11-LABX-0029) for
financial support. L.M. was supported by the Fondation J. Lejeune.
Acknowledgments: Financial support from the MESR (Ministère de l’Enseignement Supérieur & de la
Recherche) is gratefully acknowledged for the doctoral fellowships to Y.L. T.B. and his co-workers thank the
University of Rouen-Normandie for its support.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the
decision to publish the results.
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